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WO2025062293A1 - Methods and materials for scalable 3d cellular redifferentiation - Google Patents

Methods and materials for scalable 3d cellular redifferentiation Download PDF

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Publication number
WO2025062293A1
WO2025062293A1 PCT/IB2024/059028 IB2024059028W WO2025062293A1 WO 2025062293 A1 WO2025062293 A1 WO 2025062293A1 IB 2024059028 W IB2024059028 W IB 2024059028W WO 2025062293 A1 WO2025062293 A1 WO 2025062293A1
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medium
cell
cells
cell suspension
tissue culture
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PCT/IB2024/059028
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French (fr)
Inventor
Richard Lloyd YOUNGBLOOD
Aparna SUMANTH
Chiamin Liao BULLAUGHEY
Sandra HAYES
Naman Patel
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Janssen Biotech, Inc.
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Publication of WO2025062293A1 publication Critical patent/WO2025062293A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/11—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from blood or immune system cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention teaches methods and materials for the cellular redifferentiation of induced pluripotent stem cell (iPSC)-derived hematopoietic stem cells (e.g., iHSCs), which are useful in, inter alia, generating gamma delta ( ⁇ ) T cells, derived from such iPSCs.
  • iPSC induced pluripotent stem cell
  • iHSCs induced pluripotent stem cell
  • ⁇ gamma delta
  • Immune cell therapy such as T cell immunotherapy, including autologous chimeric antigen receptor (CAR) T cell therapies, has been reported to be an efficacious therapy for the treatment of some diseases, such as some cancers, including some hematologic malignancies.
  • CAR autologous chimeric antigen receptor
  • applications of cell therapy to treat multiple cancer types has met numerous and varied challenges (see, e.g., Advances and challenges of CAR T therapy and suitability of animal models (Review), Authors: Xavier E. Ramos-Cardona Weichuan Luo Sulma I. Mohammed; Published online on: July 12, 2022).
  • an iPSC-derived cell therapy platform in accordance with the present invention is based on production of ⁇ T cells, which possess intrinsic antitumor activity and whose cellular tumor infiltration is associated with more favorable outcomes.
  • iPSCs iPSC-derived HSCs
  • Such iHSCs are of sufficient purity and fold expansion, and as iHSCs, exhibit high T cell redifferentiation potential for use for T cell-based products, as well as ⁇ T cells that have superior increased purity and fold expansion properties and are more readily made and used.
  • the present invention is directed to such improved methods and materials.
  • the inventors address a need in the art for methods of generating iHSCs from iPSCs with a scalable method using a 3D vertical wheel (3D-VW) bioreactor and without the use of, for example, serum or additional cells (e.g., feeder or stromal cells).
  • This method results in iHSCs with high purity, viability, fold expansion, and potential to redifferentiate into immune effector cells, including lymphocytes such as ⁇ T cells, specifically iPSC-derived ⁇ (i ⁇ ) T cells.
  • lymphocytes such as ⁇ T cells, specifically iPSC-derived ⁇ (i ⁇ ) T cells.
  • a method of de novo generation of hematopoietic stem cells derived from human ⁇ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue culture vessel, said method comprising: seeding human ⁇ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; redifferentiating the iPSCs to iHSCs by the timely addition of key growth factors and small molecules and by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; retaining all cells in the culture on day 8 of culture while removing the medium from the culture for about 2 days; and harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction.
  • iHSCs hematopoietic stem cells
  • iPSCs hematopoietic stem cells
  • the tissue culture vessel has non-traditional bioreactor geometries.
  • the iPSCs are seeded at a density of about 1.0 x 10 5 cells/mL to about 1.8 x 10 5 cells/mL.
  • the tissue culture vessel comprising a vertical wheel further comprises a first medium comprising StemFit® Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, Emricasan, Polyamines, and Trans ⁇ ISRIB (CEPT) cocktail, wherein the BMP4 and the rhVEGF are present in a ratio of 1:1, and wherein the first medium does not comprise antibiotics.
  • a first medium comprising StemFit® Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman
  • the method further comprises exchanging spent medium in the tissue culture vessel comprising a vertical wheel on each of days 2, 4, and 6 of culture for the about 8 days, wherein the step of exchanging spent medium comprises: removing the spent medium from the tissue culture vessel comprising a vertical wheel using a 40 ⁇ m cell retention filter and adding a wash medium at a pump flow rate of 30 mL/min, the wash medium comprising Advanced DMEM/F12 medium and StemFit ® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio; exchanging about 3 L to about 3.5 L of the wash medium in the bioreactor and removing 0.1 L of the wash medium from the tissue culture vessel comprising a vertical wheel using the 40 ⁇ m cell retention filter; and adding about 50 mL of the wash medium and about 50 mL of a medium to from the tissue culture vessel comprising the vertical wheel.
  • the iPSCs are dissociated into single cells on day 7 of culture.
  • the step of retaining all cells comprises retaining clusters, semi- clusters, and single cells.
  • the step of retaining all cells comprises removing spent medium from the tissue culture vessel comprising a vertical wheel using a 4 ⁇ m cell retention filter and adding fresh medium to the bioreactor over about 1.8 days to about 2.2 days, preferably about 2 days, at a rate of about 0.69 mL/min (1 L/day), until 2 L of media have been exchanged, wherein the fifth medium comprises Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, IL-6, TPO, and one of (i) L-glutamine and (ii) GlutaMax TM , wherein the fresh medium does not comprise antibiotics.
  • the vertical wheel spins at a rate of about 10 rpm to about 60 rpm, preferably about 23 rpm.
  • the step of harvesting comprises: a) removing an entire cell volume comprising EBs and iHSCs from the tissue culture vessel comprising a vertical wheel and centrifuging the entire cell volume, thereby generating a first supernatant and a first cell pellet; b) centrifuging the first supernatant, thereby generating a second supernatant and a second cell pellet, removing the second supernatant, and resuspending the second cell pellet in Advanced DMEM/F12 medium and StemFit ® For Differentiation medium without cytokines, growth factors, or antibiotics; c) adding a first volume of 20 mg/mL collagenase II to the first cell pellet and the second cell pellet, combining the resuspended first cell pellet and the resuspended second cell pellet and adding a second volume of 20 mg/mL collagenase II, thereby generating a combined cell suspension; d) adding a volume of Advanced DMEM/F
  • the cells are cultured under normoxic conditions.
  • the method further comprises further purifying and/or isolating the redifferentiated iHSCs.
  • the redifferentiated iHSCs are isolated differentiated cells or are capable of being further purified and/or isolated.
  • determining purity of the redifferentiated iHSCs comprises assaying for one or more of lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a via flow cytometry or an equivalent thereof.
  • the lineage markers comprise all of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a.
  • a method of de novo generation of hematopoietic stem cells derived from human ⁇ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue culture vessel, said method comprising: seeding human ⁇ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction.
  • iHSCs hematopoietic stem cells
  • iPSCs hematopoietic stem cells derived from human ⁇ T cell-derived induced pluripotent stem cells
  • the tissue culture vessel has non-traditional bioreactor geometries.
  • the iPSCs are seeded at a density of about 1.0 x 10 5 cells/mL to about 1.8 x 10 5 cells/mL.
  • the tissue culture vessel comprising a vertical wheel further comprises a first medium comprising StemFit® Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, Emricasan, Polyamines, and Trans ⁇ ISRIB (CEPT) cocktail, wherein the BMP4 and the rhVEGF are present in a ratio of 1:1, and wherein the first medium does not comprise antibiotics.
  • a first medium comprising StemFit® Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman
  • the method further comprises exchanging spent medium in the tissue culture vessel comprising a vertical wheel on each of days 2, 4, 6, and 8 of culture for the about 8 days, wherein the step of exchanging the spent medium comprises: a) allowing cell aggregates to settle in the tissue culture vessel comprising a vertical wheel for about 3 to about 5 minutes; b) collecting 80% of the spent medium from the tissue culture vessel comprising a vertical wheel; c) centrifuging the collected medium, thereby obtaining a cell pellet; d) resuspending the cell pellet in StemFit® for Differentiation, thereby obtaining a cell suspension; and e) adding the cell suspension and fresh medium to the tissue culture vessel comprising a vertical wheel.
  • the step of exchanging the spent medium on days 2, 4, and 6 of culture comprises: i) allowing cell aggregates to settle in the tissue culture vessel comprising a vertical wheel for about 3 to about 5 minutes; collecting 80% of the spent medium from the tissue culture vessel comprising a vertical wheel; centrifuging the collected medium, thereby obtaining a cell pellet; resuspending the cell pellet in StemFit® for Differentiation medium, thereby obtaining a cell suspension; and adding the cell suspension and fresh medium to the tissue culture vessel comprising a vertical wheel, or ii) allowing cell aggregates to settle in the tissue culture vessel comprising a vertical wheel for about 3 to about 5 minutes; collecting 80% of the spent medium from the tissue culture vessel comprising a vertical wheel; centrifuging the collected medium, thereby obtaining a first cell pellet and a first supernatant; resuspending the first cell pellet in Ste
  • the fresh medium added on day 2 of culture comprises: i) Advanced DMEM/F12 medium and StemFit ® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SB431542 (inhibitor of Activin/BMP/TGF ⁇ pathway), recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), and one of L-glutamine and GlutaMax TM supplement, or ii) StemPro TM -34 Serum-free Medium (SFM) and StemProTM-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, Insulin-Transferrin-Selenium (ITS-G), monothioglycerol, SB431542, recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (SFM) and StemProTM
  • the iPSCs are dissociated into single cells on day 7 of culture.
  • the step of harvesting redifferentiated iHSCs comprises retaining clusters, semi-clusters, and single cells.
  • the step of harvesting comprises retaining all cells in the tissue culture vessel comprising a vertical wheel. 6 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 [0031]
  • the vertical wheel spins at a rate of about 10 rpm to about 60 rpm, preferably about 40 rpm.
  • the step of harvesting redifferentiated iHSCs comprises: a) removing an entire cell volume comprising embryoid bodies (EBs) and iHSCs from the tissue culture vessel comprising a vertical wheel and centrifuging the entire cell volume, thereby generating a first supernatant and a first cell pellet; b) centrifuging the first supernatant, thereby generating a second supernatant and a second cell pellet and removing the second supernatant; c) resuspending the first cell pellet and the second cell pellet in Advanced DMEM/F12 medium without cytokines, growth factors, or antibiotics; d) adding a volume of 20 mg/mL collagenase II to the first cell pellet, thereby generating a first cell suspension; e) adding a volume of Advanced DMEM/F12 medium without cytokines, growth factors, or antibiotics equal to the volume of the first cell suspension to the first cell suspension; f) centrifuging the
  • the cells are cultured under normoxic conditions.
  • the method further comprises further purifying and/or isolating the redifferentiated iHSCs.
  • the redifferentiated iHSCs are isolated differentiated cells or are capable of being further purified and/or isolated.
  • determining purity of the redifferentiated iHSCs comprises assaying for one or more of lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a via flow cytometry or an equivalent thereof.
  • the lineage markers comprise all of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a.
  • iHSCs hematopoietic stem cells
  • iPSCs human ⁇ T cell-derived induced pluripotent stem cells
  • iHSCs hematopoietic stem cells
  • iPSCs human ⁇ T cell-derived induced pluripotent stem cells
  • said method comprising: seeding human ⁇ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; retaining all cells in the culture on day 8 of culture while removing the medium from the culture for about 2 days; and harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction.
  • the tissue culture vessel has non-traditional bioreactor geometries.
  • the iPSCs are seeded at a density of about 1.0 x 10 5 cells/mL to about 1.8 x 10 5 cells/mL.
  • the tissue culture vessel comprising a vertical wheel further comprises a first medium comprising StemFit® Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, Emricasan, Polyamines, and Trans ⁇ ISRIB (CEPT) cocktail, wherein the BMP4 and the rhVEGF are present in a ratio of 1:1, and wherein the first medium does not comprise antibiotics.
  • a first medium comprising StemFit® Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman
  • the method further comprises exchanging spent medium in the tissue culture vessel comprising a vertical wheel on each of days 2, 4, and 6 of culture for the about 8 days, wherein the step of exchanging spent medium comprises: removing the spent medium from the tissue culture vessel comprising a vertical wheel using a 40 ⁇ m cell retention filter and adding a wash medium at a pump flow rate of 30 mL/min, the wash medium comprising Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio; exchanging about 3 L to about 3.5 L of the wash medium in 8 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 the bioreactor and removing 0.1 L of the wash medium from the tissue culture vessel comprising a vertical wheel using the 40 ⁇ m cell retention filter; and adding about 50 mL of the wash medium and about 50 mL of a medium to from the tissue culture vessel
  • the iPSCs are dissociated into single cells on day 7 of culture.
  • the step of retaining all cells comprises retaining clusters, semi- clusters, and single cells.
  • the step of retaining all cells comprises removing spent medium from the tissue culture vessel comprising a vertical wheel using a 4 ⁇ m cell retention filter and adding fresh medium to the bioreactor over about 1.8 days to about 2.2 days, preferably about 2 days, at a rate of about 0.69 mL/min (1 L/day), until 2 L of media have been exchanged, wherein the fifth medium comprises Advanced DMEM/F12 medium and StemFit ® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, IL-6, TPO, and one of (i) L-glutamine and (ii) GlutaMax TM , wherein the fresh medium does not comprise antibiotics.
  • the vertical wheel spins at a rate of about 10 rpm to about 60 rpm, preferably about 23 rpm.
  • the step of harvesting comprises: a) removing an entire cell volume comprising EBs and iHSCs from the tissue culture vessel comprising a vertical wheel and centrifuging the entire cell volume, thereby generating a first supernatant and a first cell pellet; b) centrifuging the first supernatant, thereby generating a second supernatant and a second cell pellet, removing the second supernatant, and resuspending the second cell pellet in Advanced DMEM/F12 medium and StemFit ® For Differentiation medium without cytokines, growth factors, or antibiotics; c) adding a first volume of 20 mg/mL collagenase II to the first cell pellet and the second cell pellet, combining the resuspended first cell pellet and the resuspended second cell pellet and adding
  • the cells are cultured under normoxic conditions.
  • the method further comprises further purifying and/or isolating the redifferentiated iHSCs.
  • the redifferentiated iHSCs are isolated differentiated cells or are capable of being further purified and/or isolated.
  • determining purity of the redifferentiated iHSCs comprises assaying for one or more of lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a via flow cytometry or an equivalent thereof.
  • the lineage markers comprise all of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a.
  • iHSCs hematopoietic stem cells
  • iPSCs hematopoietic stem cells
  • iPSCs hematopoietic stem cells
  • a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days
  • redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days
  • the tissue culture vessel has non-traditional bioreactor geometries.
  • the iPSCs are seeded at a density of about 1.0 x 10 5 cells/mL to about 1.8 x 10 5 cells/mL.
  • the tissue culture vessel comprising a vertical wheel further comprises a first medium comprising StemFit ® Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, Emricasan, Polyamines, and Trans ⁇ ISRIB (CEPT) cocktail, wherein the BMP4 and the rhVEGF are present in a ratio of 1:1, and wherein the first medium does not comprise antibiotics.
  • a first medium comprising StemFit ® Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chr
  • the method further comprises exchanging spent medium in the tissue culture vessel comprising a vertical wheel on each of days 2, 4, 6, and 8 of culture for the about 8 days, wherein the step of exchanging the spent medium comprises: a) allowing cell aggregates to settle in the tissue culture vessel comprising a vertical wheel for about 3 to about 5 minutes; b) collecting 80% of the spent medium from the tissue culture vessel comprising a vertical wheel; c) centrifuging the collected medium, thereby obtaining a cell pellet; d) resuspending the cell pellet in StemFit® for Differentiation, thereby obtaining a cell suspension; and e) adding the cell suspension and fresh medium to the tissue culture vessel comprising a vertical wheel.
  • the step of exchanging the spent medium on days 2, 4, and 6 of culture comprises: i) allowing cell aggregates to settle in the tissue culture vessel comprising a vertical wheel for about 3 to about 5 minutes; collecting 80% of the spent medium from the tissue culture vessel comprising a vertical wheel; centrifuging the collected medium, thereby obtaining a cell pellet; resuspending the cell pellet in StemFit® for Differentiation medium, thereby obtaining a cell suspension; and adding the cell suspension and fresh medium to the tissue culture vessel comprising a vertical wheel, or ii) allowing cell aggregates to settle in the tissue culture vessel comprising a vertical wheel for about 3 to about 5 minutes; collecting 80% of the spent medium from the tissue culture vessel comprising a vertical wheel; centrifuging the collected medium, thereby obtaining a first cell pellet and a first supernatant; resuspending the first cell pellet in StemFit® for Differentiation medium and adding the resuspended first cell pellet to the tissue culture vessel comprising
  • the fresh medium added on day 2 of culture comprises: i) Advanced DMEM/F12 medium and StemFit ® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SB431542 (inhibitor of Activin/BMP/TGF ⁇ pathway), recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), and one of L-glutamine and GlutaMax TM supplement, or ii) StemPro TM -34 Serum-free Medium (SFM) and StemProTM-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, Insulin-Transferrin-Selenium (ITS-G), monothioglycerol, SB431542,
  • the iPSCs are dissociated into single cells on day 7 of culture.
  • the step of harvesting redifferentiated iHSCs comprises retaining clusters, semi-clusters, and single cells.
  • the step of retaining all cells comprises removing spent medium from the tissue culture vessel comprising a vertical wheel using a 4 ⁇ m cell retention filter and adding fresh medium to the bioreactor over about 1.8 days to about 2.2 days, preferably about 2 days, at a rate of about 0.69 mL/min (1 L/day), until 2 L of media have been exchanged, wherein the fifth medium comprises Advanced DMEM/F12 medium and StemFit ® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, IL-6, TPO, and one of (i) L-glutamine and (ii) GlutaMax TM , wherein the fresh medium does not comprise antibiotics.
  • the vertical wheel spins at a rate of about 10 rpm to about 60 rpm, preferably about 40 rpm.
  • the step of harvesting redifferentiated iHSCs comprises: a) removing an entire cell volume comprising embryoid bodies (EBs) and iHSCs from the tissue culture vessel comprising a vertical wheel and centrifuging the entire cell volume, thereby generating a first supernatant and a first cell pellet; 12 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 b) centrifuging the first supernatant, thereby generating a second supernatant and a second cell pellet and removing the second supernatant; c) resuspending the first cell pellet and the second cell pellet in Advanced DMEM/F12 medium without cytokines, growth factors, or antibiotics; d) adding a volume of 20 mg/mL collagenase II to
  • the cells are cultured under normoxic conditions.
  • the method further comprises further purifying and/or isolating the redifferentiated iHSCs.
  • the redifferentiated iHSCs are isolated differentiated cells or are capable of being further purified and/or isolated.
  • the one or more cells have high purity, viability, fold expansion, and potential to redifferentiate into immune effectors, including iPSC-derived ⁇ (i ⁇ ) T cells.
  • the step of determining purity of the iHSCs comprises assaying for one or more, optionally all, of the lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a via flow cytometry or an equivalent thereof.
  • the lineage markers comprise one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a.
  • a composition comprising the one or more cells of any one of the embodiments described herein.
  • a system for performing a method of de novo generation of hematopoietic stem cells (iHSCs) derived from human ⁇ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions comprising the steps of any one of the embodiments described herein, wherein the vertical wheel is either perpendicular or non-perpendicular to the vessel.
  • iHSCs hematopoietic stem cells
  • iPSCs human ⁇ T cell-derived induced pluripotent stem cells
  • a method of de novo generation of hematopoietic stem cells (iHSCs) derived from human ⁇ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue culture vessel comprising: a means for seeding human ⁇ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; a means for redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; a means for retaining all cells in the culture on day 8 of culture while removing the medium from the culture for about 2 days; and a means for harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction.
  • iHSCs hematopoietic stem cells
  • a method of de novo generation of hematopoietic stem cells derived from human ⁇ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue culture vessel, said method comprising: a means for seeding human ⁇ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; a means for redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; a means for harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction.
  • iHSCs hematopoietic stem cells
  • iPSCs hematopoietic stem cells
  • an iHSC produced according to any of the methods described herein.
  • one or more cells obtained from a method of de novo generation of hematopoietic stem cells (iHSCs) derived from human ⁇ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue 14 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 culture vessel, said method comprising: a means for seeding human ⁇ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; a means for redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; a means for retaining all cells in the culture on day 8 of culture while removing the medium from
  • iHSCs hematopoietic stem cells
  • iPSCs hematopoietic stem cells
  • said method comprising: a means for seeding human ⁇ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; a means for redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; a means for harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction.
  • a method of de novo generation of induced pluripotent stem cell (iPSC)-derived hematopoietic stem cells (iHSCs) from ⁇ T cell-derived iPSCs ( ⁇ T- iPSCs) under serum-free, feeder-free culture conditions comprising one or more of the following steps: i) obtaining one or more human induced pluripotent stem cells (iPSCs), and wherein the method further comprises one or more of the following steps: a) after incubating the iPSCs in a tissue culture vessel comprising a vertical wheel in a first medium with agitation at about 10-60 rpm, preferably 40 rpm, for about 1.8 to about 2.2 days, preferably about 2 days, removing an entire cell volume from the tissue culture vessel comprising a vertical wheel and centrifuging the entire cell volume, wherein the first medium comprises StemFit ® Basic 04 medium, CHIR99021 (glycogen synthase
  • iPSC induced pluripotent stem cell
  • iHSCs induced pluripotent stem cell-derived hematopoietic stem cells
  • iPSCs human induced pluripotent stem cells
  • the method further comprises one or more of the following steps: a) seeding the iPSCs, wherein the seeding is performed by suspending the iPSCs in a first medium into a tissue culture vessel comprising a vertical wheel, thereby producing a first cell suspension, wherein the first medium comprises one of StemFit ® Basic 04 medium and StemFit ® Basic 03 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human
  • iPSC induced pluripotent stem cell
  • iHSCs induced pluripotent stem cell-derived hematopoietic stem cells
  • ⁇ T cell-derived iPSCs ⁇ T- iPSCs
  • the method further comprises one or more of the following steps: 22 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 a.
  • the seeding is performed by suspending the iPSCs in a first medium into a bioreactor, wherein the bioreactor is a vertical-wheel bioreactor, thereby producing a first cell suspension
  • the first medium comprises one of StemFit ® Basic 04 medium and StemFit ® Basic 03 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, Emricasan, Polyamines, and Trans ⁇ ISRIB (CEPT) cocktail, and wherein the first medium does not comprise antibiotics; b.
  • the first cell suspension in the bioreactor at about 10-60 rpm, preferably 40 rpm, wherein the first cell suspension is cultured and agitated for about 1.8 to about 2.2 days, preferably about 2 days, wherein embryoid bodies (EBs) are generated; c. centrifuging the first cell suspension and thereby generating a first cell pellet; d. resuspending the first cell pellet into a second medium to form a second cell suspension, wherein the second medium comprises: i.
  • Advanced DMEM/F12 medium and StemFit ® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SB431542 (inhibitor of Activin/BMP/TGF ⁇ pathway), rhVEGF, basic fibroblast growth factor (bFGF), stem cell factor (SCF), and one of L-glutamine and GlutaMax TM supplement, or ii.
  • SB431542 inhibitor of Activin/BMP/TGF ⁇ pathway
  • rhVEGF basic fibroblast growth factor
  • bFGF basic fibroblast growth factor
  • SCF stem cell factor
  • L-glutamine and GlutaMax TM supplement or ii.
  • Advanced DMEM/F12 medium and StemFit ® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), interleukin- 3 (IL-3), interleukin-6 (IL-6), one of (i) thrombopoietin (TPO) and (ii) erythropoietin (EPO), and one of (i) L-glutamine and (ii) GlutaMax TM , or ii.
  • SCF SCF
  • rhVEGF FMS-like tyrosine kinase 3 ligand
  • Flt3L FMS-like tyrosine kinase 3 ligand
  • IL-3 interleukin- 3
  • IL-6 interleukin-6
  • TPO thrombopoie
  • StemProTM-34 SFM and StemProTM-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, rhVEGF, IL-3, IL-6, Flt3L, one of (i) TPO and (ii) EPO, and one of (i) L-glutamine and (ii) GlutaMax TM , and wherein the third medium does not comprise antibiotics; j. placing the third cell suspension into the bioreactor with remaining second cell suspension forming a fourth cell suspension; k. culturing and agitating the fourth cell suspension in the bioreactor for about 1.8 to about 2.2 days, preferably about 2 days; l.
  • Advanced DMEM/F12 medium and StemFit ® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, IL-6, one of (i) TPO and (ii) EPO, and one of (i) L-glutamine and (ii) GlutaMax TM , or ii.
  • StemProTM-34 SFM and StemProTM-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, IL-6, one of (i) TPO and (ii) EPO, and one of (i) L-glutamine and (ii) GlutaMax TM , and wherein the fourth medium does not comprise antibiotics; o. placing the fifth cell suspension into the bioreactor with the remaining fourth cell suspension thereby forming a sixth cell suspension; p. culturing and agitating the sixth cell suspension in the bioreactor for about 1.8 to about 2.2 days, preferably about 2 days; q. repeating steps l) to p); r.
  • the fifth medium comprises Advanced DMEM/F12 medium, wherein the fifth medium does not 25 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 comprise cytokines, antibiotics or growth factors, and wherein cells of the ninth cell suspension comprise iHSCs, which are defined as CD34+ and lineage marker-negative, where the lineage markers comprise one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a; u.
  • iHSCs wherein the cell strainer is 70 ⁇ m mesh size
  • aa determining the purity of the iHSCs from the first supernatant of step t) and the iHSCs from step z), said purity defined as CD34+ and lineage marker-negative, where the lineage markers comprise one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a
  • the EB and ⁇ T cell-derived iPSCs are from a tissue culture vessel.
  • the first, second, fourth, sixth, and/or eighth cell suspensions are cultured under normoxic conditions. 26 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1
  • the method further comprises further purifying and/or isolating the iHSCs.
  • the obtained iHSCs are isolated redifferentiated cells or are capable of being further purified and/or isolated.
  • the method further comprises assaying for all of the lineage markers CD3, CD19, CD14, CD11b, CD56, CD20, CD16, CD2, and CD235a via flow cytometry or an equivalent thereof.
  • any one of steps a)-cc) comprises gently dispersing the cells using a pipet or equivalent thereof.
  • cells of the first medium are at a concentration of about 80,000 to about 180,000 cells/mL in a volume of about 50 to about 70 mL.
  • the cells of the first medium are at a concentration of about 150,000 cells/mL in a volume of about 60 mL.
  • the medium of step a) further comprises a ROCK inhibitor.
  • the medium of step a) further comprises a Chroman 1, Emricasan, Polyamines, and Trans ⁇ ISRIB (CEPT) cocktail.
  • all the cell volume of the first cell suspension is removed from the bioreactor prior to centrifugation.
  • the cell morphology is imaged via microscopy, e.g., bright field microscopy.
  • the medium in steps d), i), n), or t) comprises L-glutamine.
  • the medium in steps d), i), n), or t) comprises GlutaMax TM .
  • the medium in steps i) or n) comprises TPO.
  • the medium in steps i) or n) comprises EPO.
  • the centrifugation of any one of the steps is performed at 100g for 1 minute at brake 6.
  • the centrifugation of any one of the steps is performed at 300g for 5 minutes.
  • the centrifugation of any one of the steps is performed at 300g for 3 minutes.
  • the centrifugation in step r) is performed twice, first at 100g for 27 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 1 minute at brake 6 and second at 300g for 5 minutes.
  • the tenth cell suspension of step u) is incubated with collagenase for 20 minutes at 37 °C.
  • the eleventh cell suspension of step w) is incubated with TrypLE for 5 minutes at 37 °C.
  • passing of the twelfth cell suspension in step z) further comprises passing the twelfth cell suspension through a second cell strainer with a 40 ⁇ m mesh size.
  • the culturing of the iPSC cells, EBs, and iHSCs is performed at 37 °C and 5% CO 2 .
  • the bioreactor wheel is rotating at 60 rpm.
  • the bioreactor wheel is rotating at 30 rpm.
  • the bioreactor wheel is rotating at 24 rpm.
  • the bioreactor wheel is rotating at 15 rpm.
  • the bioreactor wheel is perpendicular to the bioreactor vessel.
  • the bioreactor wheel is not perpendicular to the bioreactor vessel.
  • iPSCs in the first medium are at a concentration of about 80,000 to about 180,000 cells/mL in a volume of about 90 to about 110 mL.
  • the iPSCs in the first medium are at a concentration of about 150,000 cells/mL in a volume of about 100 mL.
  • iPSCs in the first medium are at a concentration of about 5,000 to about 180,000 cells/mL in a volume of about 0.45 to about 0.55 L. [0116] In some embodiments, the iPSCs in the first medium are at a concentration of about 20,000 cells/mL in a volume of about 0.5 L. [0117] In some embodiments, iPSCs in the first medium are at a concentration of about 5,000 to about 180,000 cells/mL in a volume of about 2 to about 5 L. [0118] In some embodiments, iPSCs in the first medium are at a concentration of about 5,000 to about 180,000 cells/mL in a volume of about 10 to about 20 L.
  • iHSCs under serum-free and feeder-free culture conditions
  • said method 28 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 comprises: i. obtaining at least one human iPSC clone, and wherein the method further comprises one or more of the following steps: a.
  • redifferentiating the at least one human iPSC clone by culturing the at least one human iPSC clone in a first cell culture medium generating a first cell suspension
  • said first cell culture medium comprises one of StemFit ® Basic 04 medium and StemFit ® Basic 03 medium supplemented with basic fibroblast growth factor (bFGF) to a final concentration of about 100 ng/mL bFGF
  • said first cell culture medium further comprises CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, Emricasan, Polyamines, and Trans ⁇ ISRIB (CEPT) cocktail, wherein culturing is performed in a vertical wheel bioreactor at about 10-60 rpm, preferably 40 rpm, for about
  • the second cell culture medium comprises: i. Advanced DMEM/F12 medium and StemFit ® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises one of L-glutamine and GlutaMax TM , SB431542 (inhibitor of Activin/BMP/TGF ⁇ pathway), rhVEGF, bFGF, and stem cell factor (SCF), or ii.
  • Advanced DMEM/F12 medium and StemFit ® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises one of L-glutamine and GlutaMax TM , SB431542 (inhibitor of Activin/BMP/TGF ⁇ pathway), rhVEGF, bFGF, and stem cell factor (SCF), or ii.
  • the second cell suspension comprising iHSCs in the vertical wheel bioreactor at about 10-60 rpm, preferably 40 rpm, for about 1.8 to about 2.2 days, preferably about 2 days; d. stopping the agitation of the vertical wheel bioreactor for about 5 minutes and collecting about 80% of the second cell suspension; e. centrifuging the 80% of the second cell suspension, thereby generating a second cell pellet; f. suspending the second pellet in a third cell culture medium forming a third cell suspension, wherein the third cell culture medium comprises: i.
  • Advanced DMEM/F12 medium and StemFit ® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, FMS-like tyrosine kinase 3 ligand (Flt3L), interleukin-6 (IL-6), rhVEGF, IL-3, one of (i) thrombopoietin (TPO) and (ii) erythropoietin (EPO), and one of (i) L-glutamine and (ii) GlutaMax TM , or ii.
  • FMS-like tyrosine kinase 3 ligand FMS-like tyrosine kinase 3 ligand
  • IL-6 interleukin-6
  • rhVEGF interleukin-6
  • IL-3 interleukin-6
  • TPO thrombopoietin
  • EPO erythropoie
  • StemProTM-34 SFM and StemProTM-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, rhVEGF, IL-3, IL-6, Flt3L, one of (i) TPO and (ii) EPO, and one of (i) L-glutamine and (ii) GlutaMax TM , and wherein the third medium does not comprise antibiotics, and placing the third cell suspension into the vertical wheel bioreactor with remaining second cell suspension, thereby forming a fourth cell suspension; g. culturing fourth cell suspension in the vertical wheel bioreactor for an additional about 1.8 to about 2.2 days, preferably about 2 days; h.
  • Advanced DMEM/F12 medium and StemFit ® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, IL-6, one of (i) TPO and (ii) EPO, and one of (i) L-glutamine and (ii) GlutaMax TM , or ii.
  • StemProTM-34 SFM and StemProTM-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, IL-6, one of (i) TPO and (ii) EPO, and one of (i) L-glutamine and (ii) GlutaMax TM , and adding the fifth cell suspension into remaining fourth cell suspension in the vertical wheel bioreactor, thereby generating a sixth cell suspension; k. culturing the sixth cell suspension for about 1.8 to about 2.2 days, preferably about 2 days in the vertical wheel bioreactor; l. repeating steps h) to k); m.
  • the eighth cell suspension generating a first supernatant and a fourth cell pellet; n. collecting and centrifuging the first supernatant, thereby generating a fifth cell pellet and resuspending the fifth cell pellet in a fifth medium thereby generating a ninth cell suspension
  • the fifth medium comprises Advanced DMEM/F12 medium, wherein the fifth medium does not comprise cytokines, antibiotics or growth factors
  • cells of the ninth cell suspension comprises iHSCs, which are defined as CD34+ and lineage marker-negative, where the lineage 31 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 markers comprise one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a; o.
  • step m resuspending the fourth cell pellet of step m) in a volume of about 20 mg/mL collagenase type II, thereby generating a tenth cell suspension; p. adding the fifth medium to the tenth cell suspension; q. centrifuging the tenth cell suspension, thereby generating a sixth cell pellet, and adding TrypLE to the sixth cell pellet, thereby generating an eleventh cell suspension; r. adding additional fifth medium to the eleventh cell suspension and mixing; s. centrifuging the eleventh cell suspension to form a seventh cell pellet and resuspending the seventh cell pellet in additional fifth medium to form a twelfth cell suspension; t.
  • iHSCs wherein the cell strainer is 70 ⁇ m mesh size; u. determining the purity of the iHSCs from the first supernatant of step n) and the iHSCs from step t), said purity defined as CD34+ and lineage marker-negative, where the lineage markers comprise one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a; v. not introducing serum in any of steps a)-u); w. not introducing any additional cells, including feeder cells or stromal cells, in any of steps a)-v); and x.
  • the one or more cells have high purity, viability, fold expansion, and potential to redifferentiate into immune effectors, including iPSC-derived ⁇ (i ⁇ ) T cells.
  • the EB and ⁇ T cell-derived iPSCs are from a tissue culture vessel.
  • the first, second, fourth, sixth, and/or eighth cell suspensions are cultured under normoxic conditions.
  • the method further comprising further purifying and/or isolating 32 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 the iHSCs.
  • the obtained iHSCs are isolated redifferentiated cells or are capable of being further purified and/or isolated.
  • the method further comprises assaying for all of the lineage markers CD3, CD19, CD14, CD11b, CD56, CD20, CD16, CD2, and CD235a via flow cytometry or an equivalent thereof.
  • any one of steps a)-w) comprises gently dispersing the cells using a pipet or equivalent thereof.
  • cells of the first medium are at a concentration of about 80,000 to about 180,000 cells/mL in a volume of about 50 to about 70 mL.
  • the cells of the first medium are at a concentration of about 150,000 cells/mL in a volume of about 60 mL.
  • the medium of step a) further comprises a ROCK inhibitor.
  • the medium of step a) further comprises a Chroman 1, Emricasan, Polyamines, and Trans ⁇ ISRIB (CEPT) cocktail.
  • all the cell volume of the first cell suspension is removed from the bioreactor prior to centrifugation.
  • the cell morphology is imaged via microscopy, e.g., bright field microscopy.
  • the medium in steps b), f), j), or n) comprises L-glutamine. [0134] In some embodiments, the medium in steps b), f), j), or n) comprises GlutaMax TM . [0135] In some embodiments, the medium in steps f) or j) comprises TPO. [0136] In some embodiments, the medium in steps f) or j) comprises EPO. [0137] In some embodiments, the centrifugation of any one of the steps is performed at 100g for 1 minute at brake 6. [0138] In some embodiments, the centrifugation of any one of the steps is performed at 300g for 5 minutes.
  • the centrifugation of any one of the steps is performed at 300g for 3 minutes.
  • the centrifugation in step l) is performed twice, first at 100g for 1 minute at brake 6 and second at 300g for 5 minutes.
  • 33 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1
  • the tenth cell suspension of step o) is incubated with collagenase for 20 minutes at 37 °C.
  • the eleventh cell suspension of step q) is incubated with TrypLE for 5 minutes at 37 °C.
  • passing of the twelfth cell suspension in step t) further comprises passing the twelfth cell suspension through a second cell strainer with a 40 ⁇ m mesh size.
  • the culturing of the iPSC cells, EBs, and iHSCs is performed at 37 °C and 5% CO2.
  • the bioreactor wheel is rotating at 60 rpm.
  • the bioreactor wheel is rotating at 30 rpm.
  • the bioreactor wheel is rotating at 24 rpm.
  • the bioreactor wheel is rotating at 15 rpm.
  • the bioreactor wheel is perpendicular to the bioreactor vessel.
  • the bioreactor wheel is not perpendicular to the bioreactor vessel.
  • iPSCs of the first medium are at a concentration of about 80,000 to about 180,000 cells/mL in a volume of about 90 to about 110 mL.
  • the iPSCs of the first medium are at a concentration of about 150,000 cells/mL in a volume of about 100 mL.
  • iPSCs of the first medium are at a concentration of about 5,000 to about 180,000 cells/mL in a volume of about 0.45 to about 0.55 L. [0154] In some embodiments, the iPSCs of the first medium are at a concentration of about 20,000 cells/mL in a volume of about 0.5 L. [0155] In some embodiments, iPSCs of the first medium are at a concentration of about 5,000 to about 180,000 cells/mL in a volume of about 2 to about 5 L. [0156] In some embodiments, iPSCs of the first medium are at a concentration of about 5,000 to about 180,000 cells/mL in a volume of about 10 to about 20 L.
  • composition comprising the one or more cells as described herein.
  • Figures 1A-1B show the iHSC redifferentiation process.
  • Figure 1A shows the process flow of the iHSC redifferentiation process.
  • Figure 1B shows an exemplary bioreactor system schematic of the present invention, comprising a waste member, medium reservoir member, pump member, reactor chamber member including a vertical wheel member (black circle), cell inlet member (not shown), cell release member (not shown) and connector members.
  • Figure 2 shows the step-by-step iHSC redifferentiation process in a 3D-VW bioreactor.
  • Figure 3 shows the purity, total yield, viability, and fold expansion of iHSCs redifferentiated from a single human ⁇ T cell-derived iPSC clone in a 3D-VW bioreactor culture, where iHSCs were defined as lineage marker-negative CD34+ cells. Both thrombopoietin (TPO) and erythropoietin (EPO) can support iHSC redifferentiation. iHSCs were detected as single cells and in dissociated cell clusters. This novel 3D iHSC protocol met the exemplary acceptance criteria for iHSC+ purity.
  • TPO thrombopoietin
  • EPO erythropoietin
  • FIG 4 shows the 3D-vertical wheel (VW) process reproducibly generated iHSCs with high purity and yield and with sufficient fold expansion.
  • iHSCs were redifferentiated from two different iPSC clones in a research-scale 2D culture system and in the 3D-VW culture system.
  • the initial exemplary success criterion for iHSC differentiation was 70% iHSC cells (Lin-CD34+) and 0.5-fold iHSC expansion. Purity and cell viability were comparable between the 3D and 2D processes for these two iPSC clones.
  • Figure 5 shows iHSCs generated in the 3D-VW process possess i ⁇ T cell redifferentiation potential.
  • iHSC cells generated in the 3D-VW process yielded i ⁇ T cells at a purity that is consistent with iHSC cells generated with the 2D process.
  • the iHSC to i ⁇ T cell redifferentiation process was carried out in a 2D culture system following a basic protocol and utilizing iHSCs redifferentiated in 2D and 3D culture systems.
  • the 3D-derived iHSCs used in this experiment were generated by seeding iPSCs at 1.5x standard protocol seeding density.
  • iHSC differentiation 50% of the harvested single cell population was treated with the same enzymes used to dissociate clusters to examine the effects of such enzymes on T cell differentiation potential.
  • the initial exemplary success criterion for iHSC differentiation was 70% iHSC cells (defined as Lin-CD34 + ) and 0.5-fold iHSC expansion. Three independent cell clones, Clone B, Clone C, and Clone E, possessed high T cell differentiation potential.
  • treating refers to any success or indicia of success in the attenuation or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement, remission, diminishing of symptoms or making the condition more tolerable to the patient, slowing in the rate of degeneration or decline, making the final point of degeneration less debilitating, improving a subject’s physical or mental well-being, or prolonging the length of survival.
  • iPSC induced pluripotent stem cell
  • HSC hematopoietic stem cell
  • the iPSC is preferably derived from a primate (e.g., monkey, orangutan, chimpanzee, human), more preferably human.
  • a primate e.g., monkey, orangutan, chimpanzee, human
  • iHSC refers to hematopoietic stem cells that are derived from iPSCs.
  • non-traditional geometries or non-traditional bioreactors refer to those geometries or bioreactors that do not fall under the definitions of “traditional geometries” or “traditional bioreactors”, respectively, i.e., geometries that pre-date this invention.
  • Non- traditional geometries or “non-traditional bioreactors” may refer to geometries or bioreactors that combine radial and axial flow components.
  • the vertical-wheel bioreactor is a non-limiting example of a non-traditional bioreactor.
  • “Traditional geometries” or “traditional bioreactors” may refer to stirred-tank geometries or stirred-tank bioreactors, respectively.
  • “Traditional geometries” or “traditional bioreactors” may refer to geometries or bioreactors that have radial flow components.
  • Horizontal-blade bioreactors and turbine impellers are non-limiting examples of traditional bioreactors.
  • ⁇ T cells were selected as the iPSC cell of origin because it is known that the epigenetic and transcriptional memory of the starting cell influences and enhances iPSC redifferentiation potential to that cell type.
  • An optimized three-dimensional (3D) serum-free, feeder-free (SFFF) protocol was used to redifferentiate ⁇ T-iPSCs into iHSCs and then into i ⁇ T cells as shown in Figures 1A, 1B, and 2.
  • Purity and fold expansion were determined from two different iPSC clones to iHSCs and from iHSCs to i ⁇ T cells. Images of the cells were recorded. Exemplary success criteria were set for the purity and fold expansion of redifferentiated iHSCs and i ⁇ T cells based on results from a 3D redifferentiation protocol and from a 2D redifferentiation protocol.
  • the 3D-VW bioreactor protocol for the redifferentiation of iPSCs to iHSCs resulted in high purity, high cell viability, high yield, and sufficient fold expansion as shown in Figures 3-6.
  • the protocol also yielded i ⁇ T cells at a purity that met the exemplary success criterion and with a fold expansion that is consistent with iHSCs generated with the 2D process.
  • These data demonstrate the efficiency and validity of the 3D-VW bioreactor protocol for the generation of iHSCs from ⁇ T-iPSCs as shown in Figures 3-6. Maintaining iPSCs Prior to Initiating Redifferentiation [0176] As is known in the art, iPSC colonies generally exhibit circular colonies, with densely packed cells, higher nucleus to cytoplasmic ratio, well defined edges, and distinct borders.
  • the iPSCs can be thawed from a frozen stock, and a desired number of live cells can be utilized in the methods described herein. Cell counting, size, and viability determination can be performed by any method known in the art, e.g., by using a NucleoCounter NC-200. Live cells having a preferred size, e.g., between 10 ⁇ m and 25 ⁇ m, can be used in the steps described herein. [0178] The iPSCs can be seeded at a cell density of 1.0 x 10 5 to 1.8 x 10 5 .
  • the iPSCs are maintained in an appropriate tissue or cell culture vessel, such as for example and not 38 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 limitation, a tissue culture vessel comprising a vertical wheel.
  • the tissue or cell culture vessels are incubated at 37 °C, 5% CO2.
  • the iPSCs are grown in StemFit ® Basic 04 medium further comprising basic fibroblast growth factor (bFGF) at a final concentration of about 90 ng/mL to about 110 ng/mL bFGF, preferably about 100 ng/mL bFGF.
  • bFGF basic fibroblast growth factor
  • the medium contains either 10 ⁇ M Y-27632 ROCK inhibitor or a Chroman 1, Emricasan, Polyamines, and Trans ⁇ ISRIB (CEPT) cocktail.
  • StemFit ® Basic 04 cell culture medium (Ajinomoto) is an animal-origin free, defined medium for human pluripotent stem cell culture. This medium is a complete medium and does not require any other supplements.
  • StemFit ® Basic 03 cell culture medium (Ajinomoto) is an animal-origin free, defined medium for human pluripotent stem cell culture.
  • StemPro TM -34 serum-free medium (SFM) (Gibco TM ) is a serum-free medium specifically formulated to support the development of human hematopoietic cells in culture, including HSC and progenitor cells isolated from bone marrow, neonatal cord blood, and peripheral blood.
  • StemPro TM -34 SFM is a flexible cell culture media that can be used for a wide variety of applications.
  • the iPSCs can be examined at regular intervals, e.g., daily, for cell morphology. Cell morphology can be determined by microscopy, e.g., bright field microscopy.
  • the colonies can be redifferentiated to iHSCs as discussed herein.
  • the medium can be changed at regular intervals as known in the art.
  • Redifferentiation of the iPSCs into iHSCs with a small volume bioreactor comprising a vertical wheel [0182]
  • the invention provides methods of redifferentiating iPSCs into iHSCs using a small volume bioreactor or tissue culture vessel comprising a vertical wheel.
  • Nonlimiting exemplary volumes include a 0.1 L bioreactor or tissue culture vessel.
  • a method of de novo generation of hematopoietic stem cells derived from human ⁇ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue culture vessel, said method comprising: seeding human ⁇ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction.
  • iHSCs hematopoietic stem cells
  • iPSCs hematopoietic stem cells derived from human ⁇ T cell-derived induced pluripotent stem cells
  • the tissue culture vessel has non-traditional bioreactor geometries as discussed herein.
  • the iPSCs are seeded at a density of about 1.0 x 10 5 cells/mL to about 1.8 x 10 5 cells/mL.
  • the tissue culture vessel comprising a vertical wheel further comprises a first medium comprising StemFit ® Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, Emricasan, Polyamines, and Trans ⁇ ISRIB (CEPT) cocktail, wherein the BMP4 and the rhVEGF are present in a ratio of 1:1, and wherein the first medium does not comprise antibiotics.
  • a first medium comprising StemFit ® Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chr
  • the method further comprises exchanging spent medium in the tissue culture vessel comprising a vertical wheel on each of days 2, 4, 6, and 8 of culture for the about 8 days, wherein the step of exchanging the spent medium comprises: a) allowing cell aggregates to settle in the tissue culture vessel comprising a vertical wheel for about 3 to about 5 minutes; b) collecting 80% of the spent medium from the tissue culture vessel comprising a vertical wheel; c) centrifuging the collected medium, thereby obtaining a cell pellet; d) resuspending the cell pellet in StemFit® for Differentiation, thereby obtaining a cell suspension; and e) adding the cell suspension and fresh medium to the tissue culture vessel comprising a vertical wheel.
  • the step of exchanging the spent medium on days 2, 4, and 6 of culture comprises: i) allowing cell aggregates to settle in the tissue culture vessel comprising a vertical wheel for about 3 to about 5 minutes; collecting 80% of the spent medium from the tissue culture vessel comprising a vertical wheel; centrifuging the collected medium, thereby obtaining a cell pellet; resuspending the cell pellet in StemFit® for Differentiation medium, thereby obtaining a cell suspension; and adding the cell suspension and fresh medium to the tissue culture vessel comprising a vertical wheel, or ii) allowing cell aggregates to settle in the tissue culture vessel comprising a vertical wheel for about 3 to about 5 minutes; collecting 80% of the spent medium from the tissue culture vessel comprising a vertical wheel; centrifuging the collected medium, thereby obtaining a first cell pellet and a first supernatant; resuspending the first cell pellet in StemFit® for Differentiation medium and adding the resuspended first cell pellet to the tissue culture vessel comprising
  • the fresh medium added on day 2 of culture comprises: i) Advanced DMEM/F12 medium and StemFit ® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SB431542 (inhibitor of Activin/BMP/TGF ⁇ pathway), recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), and one of L-glutamine and GlutaMax TM supplement, or ii) StemPro TM -34 Serum-free Medium (SFM) and StemProTM-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, Insulin-Transferrin-Selenium (ITS-G), monothioglycerol, SB431542, recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (SFM) and StemProTM
  • the iPSCs are dissociated into single cells on day 7 of culture.
  • the step of harvesting redifferentiated iHSCs comprises retaining clusters, semi-clusters, and single cells.
  • the step of harvesting redifferentiated iHSCs comprises retaining all cells in the tissue culture vessel comprising a vertical wheel.
  • the vertical wheel spins at a rate of about 10 rpm to about 60 rpm, preferably about 40 rpm.
  • the step of harvesting redifferentiated iHSCs comprises: a) removing an entire cell volume comprising embryoid bodies (EBs) and iHSCs from the tissue culture vessel comprising a vertical wheel and centrifuging the entire cell volume, thereby generating a first supernatant and a first cell pellet; b) centrifuging the first supernatant, thereby generating a second supernatant and a second cell pellet and removing the second supernatant; c) resuspending the first cell pellet and the second cell pellet in Advanced DMEM/F12 medium without cytokines, growth factors, or antibiotics; 41 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 d) adding a volume of 20 mg/mL collagenase II to the first cell pellet, thereby generating a first cell suspension; e) adding a volume of Advanced DMEM/F12 medium without cytokines, growth factors
  • the cells are cultured under normoxic conditions.
  • the method further comprises further purifying and/or isolating the redifferentiated iHSCs.
  • the redifferentiated iHSCs are isolated differentiated cells or are capable of being further purified and/or isolated.
  • determining purity of the redifferentiated iHSCs comprises assaying for one or more of lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a via flow cytometry or an equivalent thereof.
  • the lineage markers comprise all of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a.
  • a method of de novo generation of hematopoietic stem cells (iHSCs) derived from human ⁇ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue culture vessel comprising: a means for seeding human ⁇ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; a means for redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; a means for harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction
  • iPSC induced pluripotent stem cell
  • iHSCs induced pluripotent stem cell-derived hematopoietic stem cells
  • iPSCs human induced pluripotent stem cells
  • the method further comprises one or more of the following steps: a) after incubating the iPSCs in a tissue culture vessel comprising a vertical wheel in a first medium with agitation at about 10-60 rpm, preferably 40 rpm, for about 1.8 to about 2.2 days, preferably about 2 days, removing an entire cell volume from the tissue culture vessel comprising a vertical wheel and centrifuging the entire cell volume, wherein
  • a method of de novo generation of hematopoietic stem cells (iHSCs) derived from human ⁇ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue culture vessel comprising: 46 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 a) a means for seeding human ⁇ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; b) a means for redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; c) a means for harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction.
  • iHSCs hematopoietic stem cells
  • a method of de novo generation of induced pluripotent stem cell (iPSC)-derived hematopoietic stem cells (iHSCs) from ⁇ T cell-derived iPSCs ( ⁇ T- iPSCs) under serum-free, feeder-free culture conditions comprising one or more of the following steps: 1. obtaining one or more human induced pluripotent stem cells (iPSCs), and wherein the method further comprises one or more of the following steps: a.
  • the seeding is performed by suspending the iPSCs in a first medium into a bioreactor, wherein the bioreactor is a vertical-wheel bioreactor, thereby producing a first cell suspension
  • the first medium comprises one of StemFit ® Basic 04 medium and StemFit ® Basic 03 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, Emricasan, Polyamines, and Trans ⁇ ISRIB (CEPT) cocktail, and wherein the first medium does not comprise antibiotics; b.
  • the first cell suspension in the bioreactor at about 10-60 rpm, preferably 40 rpm, wherein the first cell suspension is cultured and agitated for about 1.8 to about 2.2 days, preferably about 2 days, wherein embryoid bodies (EBs) are generated; c. centrifuging the first cell suspension and thereby generating a first cell pellet; d. resuspending the first cell pellet into a second medium to form a second cell suspension, wherein the second medium comprises: i.
  • Advanced DMEM/F12 medium and StemFit ® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SB431542 (inhibitor of Activin/BMP/TGF ⁇ pathway), 47 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 rhVEGF, basic fibroblast growth factor (bFGF), stem cell factor (SCF), and one of L-glutamine and GlutaMax TM supplement, or ii.
  • SB431542 inhibitor of Activin/BMP/TGF ⁇ pathway
  • 47 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 rhVEGF, basic fibroblast growth factor (bFGF), stem cell factor (SCF), and one of L-glutamine and GlutaMax TM supplement, or ii.
  • the third medium comprises: i.
  • Advanced DMEM/F12 medium and StemFit ® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, IL-6, one of (i) TPO and (ii) EPO, and one of (i) L- glutamine and (ii) GlutaMax TM , or ii.
  • StemProTM-34 SFM and StemProTM-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, IL-6, one of (i) TPO and (ii) EPO, and one of (i) L-glutamine and (ii) GlutaMax TM , and wherein the fourth medium does not comprise antibiotics; o. placing the fifth cell suspension into the bioreactor with the remaining fourth cell suspension thereby forming a sixth cell suspension; p. culturing and agitating the sixth cell suspension in the bioreactor for about 1.8 to about 2.2 days, preferably about 2 days; q. repeating steps l) to p); r.
  • the fifth medium comprises Advanced DMEM/F12 medium, wherein the fifth medium does not comprise cytokines, antibiotics or growth factors, and wherein cells of the ninth cell suspension comprise iHSCs, which are defined as CD34+ and lineage marker-negative, where the lineage markers comprise one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a; u.
  • step r resuspending the fourth cell pellet of step r), in a volume of about 20 mg/mL collagenase type II, thereby generating a tenth cell suspension; v. adding the fifth medium to the tenth cell suspension; w. centrifuging the tenth cell suspension, thereby generating a sixth cell pellet, and adding TrypLE to the sixth cell pellet, thereby generating an eleventh cell suspension; x. adding additional fifth medium to the eleventh cell suspension and mixing; y. centrifuging the eleventh cell suspension to form a seventh cell pellet and resuspending the seventh cell pellet in additional fifth medium to form a twelfth cell suspension; z.
  • iHSCs wherein the cell strainer is 70 ⁇ m mesh size
  • aa determining the purity of the iHSCs from the first supernatant of step t) and the iHSCs from step z), said purity defined as CD34+ and lineage marker-negative, where the lineage markers comprise one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a
  • the iPSCs can be cultured and agitated at about 10 rpm to 60 rpm, preferably about 40 rpm, for about 1.8 days to about 2.2 days, preferably approximately about two days, at 37 o C, 5% CO2 in a first culture medium comprising StemFit ® Basic 04 medium or StemFit ® Basic 03 medium further comprising CHIR99021 (glycogen synthase kinase inhibitor), a Rho kinase inhibitor (ROCK inhibitor) or a Chroman 1, Emricasan, Polyamines, and 50 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 Trans ⁇ ISRIB (CEPT) cocktail, bone morphogenetic protein 4 (BMP4), and recombinant human vascular endothelial growth factor (rhVEGF).
  • a first culture medium comprising StemFit ® Basic 04 medium or StemFit ® Basic 03 medium further comprising CHIR99021 (gly
  • the morphology of the redifferentiating iPSCs can be determined at the beginning of the culture period and/or at regular intervals by microscopy, e.g., bright field microscopy. Culturing the iPSCs in this medium can induce mesoderm formation, which is a prerequisite for hematopoietic lineage specification and differentiation.
  • the iPSC-derived cell intermediates can be collected and subsequently cultured and agitated at about 10 rpm to 60 rpm, preferably about 40 rpm, in a second culture medium to begin onset of hematopoietic differentiation, for approximately about 1.8 days to about 2.2 days, preferably approximately about two days, at 37 °C with 5% CO 2 .
  • the second cell culture medium comprises Advanced DMEM/F12 medium (Thermo Fisher Scientific Catalog #12634) and StemFit ® For Differentiation medium (Ajinomoto) present in about a 3.9 to 0.9 ratio to about a 4.1-1.1 ratio, preferably about a 4:1 ratio, and further comprises SB431542 (inhibitor of Activin/BMP/TGF ⁇ pathway), rhVEGF, bFGF, and stem cell factor (SCF), and one of L-glutamine and GlutaMax TM .
  • SB431542 inhibitor of Activin/BMP/TGF ⁇ pathway
  • rhVEGF rhVEGF
  • bFGF bFGF
  • SCF stem cell factor
  • the second cell culture medium may comprise StemProTM-34 SFM + StemProTM-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, Insulin-Transferrin-Selenium (ITS-G), monothioglycerol, and further comprises, SB431542, rhVEGF, bFGF, SCF, and one of L-glutamine and GlutaMax TM .
  • SB431542 is diluted from a stock concentration of 5 mM to achieve a final concentration of about 2 ⁇ M in order to use a minimal amount of the diluent dimethylsulfoxide (DMSO).
  • the morphology of the cells can be determined at the beginning of the culture period and/or at regular intervals by microscopy, e.g., bright field microscopy.
  • Advanced DMEM/F12 medium includes amino acids, vitamins, inorganic salts, proteins, reducing elements, trace elements, dextrose, ethanolamine, hypoxanthine Na, linoleic acid, lipoic acid, phenol red, putrescine 2HCl, sodium pyruvate, and thymidine.
  • StemFit ® For Differentiation medium is a chemically defined & animal-origin free (CD-AOF) supplement for differentiation of human embryonic stem (ES) cells and iPSCs.
  • the iPSC-derived cell intermediates can be collected and subsequently cultured and agitated at about 10 rpm to 60 rpm, preferably about 40 rpm, in a third culture medium to induce hemangioblast formation, for approximately about 1.8 days to about 2.2 days, preferably approximately about two days, at 37 °C with 5% CO 2 .
  • the third culture medium comprises 51 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 Advanced DMEM/F12 medium and StemFit ® For Differentiation medium present in about a 3.9 to 0.9 ratio to about a 4.1-1.1 ratio, preferably about a 4:1 ratio, and further comprises, SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), , interleukin-3 (IL-3), interleukin-6 (IL-6), one L-glutamine and GlutaMax TM , and one of thrombopoietin (TPO) and erythropoietin (EPO).
  • SCF SCF
  • rhVEGF FMS-like tyrosine kinase 3 ligand
  • IL-3 interleukin-3
  • IL-6 interleukin-6
  • TPO thrombopoietin
  • EPO erythro
  • the third cell culture medium may comprise StemProTM-34 SFM + StemProTM-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, Insulin-Transferrin-Selenium (ITS-G), monothioglycerol, and further comprises IL-3, IL-6, SCF, rhVEGF, Flt3L, one of L- glutamine and GlutaMax TM , and one of TPO and EPO.
  • the TPO is prepared from a stock concentration of 100 ⁇ g/mL to a final concentration of 30 ng/mL.
  • the EPO is prepared from a stock concentration of 500 IU/mL to a final concentration of 10 IU/mL.
  • the morphology of the cells can be determined at the beginning of the culture period and/or at regular intervals by microscopy, e.g., bright field microscopy.
  • the iHSCs can be collected and subsequently cultured and agitated at about 10 rpm to 60 rpm, preferably about 40 rpm, in a fourth culture medium for about 1.8 days to about 4.2 days, preferably about two days to about four days, at 37 °C with 5% CO2 for the endothelial to hematopoietic transition phase.
  • the fourth culture medium comprises Advanced DMEM/F12 medium and StemFit ® For Differentiation medium present in about a 3.9 to 0.9 ratio to about a 4.1-1.1 ratio, preferably about a 4:1 ratio, and further comprises, SCF, IL-6, one of L-glutamine and GlutaMax TM , and one of TPO and EPO.
  • the fourth cell culture medium may comprise StemProTM-34 SFM + StemProTM-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, Insulin-Transferrin-Selenium (ITS-G), monothioglycerol, and further comprises SCF, IL-6, one of L-glutamine and GlutaMax TM , and one of TPO and EPO.
  • the TPO is prepared from a stock concentration of 100 ug/mL to a final concentration of 30 ng/mL.
  • the EPO is prepared from a stock concentration of 500 IU/mL to a final concentration of 10 IU/mL.
  • the morphology of the cells can be determined at the beginning of the culture period and/or at regular intervals by microscopy, e.g., bright field microscopy.
  • microscopy e.g., bright field microscopy.
  • non-adherent iHSCs in the medium can be collected, centrifuged, resuspended in the fourth cell culture medium, and then added back to the culture.
  • the single cell fraction of iHSCs from the 3D-VW bioreactor protocol can be harvested by centrifugation and resuspended in a fifth cell culture medium, the fifth cell culture medium comprising Advanced DMEM/F12 medium, but does not have cytokines or growth factors.
  • the iHSCs can be counted as described herein.
  • the cell cluster fraction of iHSCs from the 3D-VW bioreactor protocol can be resuspended in collagenase type II and the fifth cell culture medium, followed by centrifugation and resuspension in TrypLE and the fifth cell culture medium, followed by further centrifugation and resuspension in the fifth cell culture medium.
  • the dispersed cells can then be passed through a cell strainer, having a mesh size of 70 ⁇ m. The cells can be counted as described herein.
  • the cells can be assessed for purity, e.g., using flow cytometry or an equivalent, to analyze the presence or absence of certain markers.
  • the purity can be defined by the absence of expression of one or more lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a and by the presence of CD34 expression (CD34+).
  • the harvested cells are lineage-marker negative and CD34+.
  • the redifferentiated iHSCs can be cryopreserved.
  • the EB and ⁇ T cell-derived iPSCs are from a tissue culture vessel. [0214] In some embodiments, any of the cell suspensions are cultured under normoxic conditions. [0215] In some embodiments, the method further comprises further purifying and/or isolating the iHSCs. [0216] In some embodiments, the obtained iHSCs are isolated redifferentiated cells or are capable of being further purified and/or isolated. [0217] In some embodiments, the method further comprises assaying for all of the lineage markers CD3, CD19, CD14, CD11b, CD56, CD20, CD16, CD2, and CD235a via flow cytometry or an equivalent thereof.
  • any one of the steps comprises gently dispersing the cells using 53 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 a pipet or equivalent thereof.
  • cells of the first medium are at a concentration of about 80,000 to about 180,000 cells/mL in a volume of about 50 to about 70 mL.
  • the cells of the first medium are at a concentration of about 150,000 cells/mL in a volume of about 60 mL.
  • the medium of step a) further comprises a ROCK inhibitor.
  • the medium of step a) further comprises a Chroman 1, Emricasan, Polyamines, and Trans ⁇ ISRIB (CEPT) cocktail.
  • all the cell volume of the first cell suspension is removed from the bioreactor prior to centrifugation.
  • the cell morphology is imaged via microscopy, e.g., bright field microscopy.
  • the medium of any one of the steps comprises L-glutamine.
  • the medium of any one of the steps comprises GlutaMax TM .
  • the medium of any one of the steps comprises TPO.
  • the medium of any one of the steps comprises EPO.
  • the centrifugation of any one of the steps is performed at 100g for 1 minute at brake 6.
  • the centrifugation of any one of the steps is performed at 300g for 5 minutes.
  • the centrifugation of any one of the steps is performed at 300g for 3 minutes.
  • the centrifugation of any one of the steps is performed twice, first at 100g for 1 minute at brake 6 and second at 300g for 5 minutes.
  • the tenth cell suspension is incubated with collagenase for 20 minutes at 37 °C.
  • the eleventh cell suspension is incubated with TrypLE for 5 minutes at 37 °C.
  • passing of the twelfth cell suspension further comprises passing the twelfth cell suspension through a second cell strainer with a 40 ⁇ m mesh size.
  • the culturing of the iPSC cells, EBs, and iHSCs is performed at 54 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 37 °C and 5% CO 2 .
  • the bioreactor wheel is rotating at 60 rpm.
  • the iPSCs in the first medium are at a concentration of about 150,000 cells/mL in a volume of about 100 mL. [0245] In some embodiments, iPSCs in the first medium are at a concentration of about 5,000 to about 180,000 cells/mL in a volume of about 0.45 to about 0.55 L. [0246] In some embodiments, the iPSCs in the first medium are at a concentration of about 20,000 cells/mL in a volume of about 0.5 L. [0247] In some embodiments, iPSCs in the first medium are at a concentration of about 5,000 to about 180,000 cells/mL in a volume of about 2 to about 5 L.
  • a method of de novo generation of hematopoietic stem cells derived from human ⁇ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue culture vessel, said method comprising: seeding human ⁇ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; retaining all 55 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 cells in the culture on day 8 of culture while removing the medium from the culture for about 2 days; and harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction.
  • iHSCs hematopoietic stem cells
  • the tissue culture vessel has non-traditional bioreactor geometries.
  • the iPSCs are seeded at a density of about 1.0 x 10 5 cells/mL to about 1.8 x 10 5 cells/mL.
  • the tissue culture vessel comprising a vertical wheel further comprises a first medium comprising StemFit ® Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, Emricasan, Polyamines, and Trans ⁇ ISRIB (CEPT) cocktail, wherein the BMP4 and the rhVEGF are present in a ratio of 1:1, and wherein the first medium does not comprise antibiotics.
  • a first medium comprising StemFit ® Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chr
  • the method further comprises exchanging spent medium in the tissue culture vessel comprising a vertical wheel on each of days 2, 4, and 6 of culture for the about 8 days, wherein the step of exchanging spent medium comprises: removing the spent medium from the tissue culture vessel comprising a vertical wheel using a 40 ⁇ m cell retention filter and adding a wash medium at a pump flow rate of 30 mL/min, the wash medium comprising Advanced DMEM/F12 medium and StemFit ® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio; exchanging about 3 L to about 3.5 L of the wash medium in the bioreactor and removing 0.1 L of the wash medium from the tissue culture vessel comprising a vertical wheel using the 40 ⁇ m cell retention filter; and adding about 50 mL of the wash medium and about 50 mL of a medium to from the tissue culture vessel comprising the vertical wheel.
  • the iPSCs are dissociated into single cells on day 7 of culture.
  • the step of retaining all cells comprises retaining clusters, semi- clusters, and single cells.
  • the step of retaining all cells comprises removing spent medium from the tissue culture vessel comprising a vertical wheel using a 4 ⁇ m cell retention filter and adding fresh medium to the bioreactor over about 1.8 days to about 2.2 days, preferably about 2 days, at a rate of about 0.69 mL/min (1 L/day), until 2 L of media have been exchanged, wherein the fifth medium comprises Advanced DMEM/F12 medium and StemFit ® For Differentiation 56 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, IL-6, TPO, and one of (i) L-
  • the vertical wheel spins at a rate of about 10 rpm to about 60 rpm, preferably about 23 rpm.
  • the step of harvesting comprises: a) removing an entire cell volume comprising EBs and iHSCs from the tissue culture vessel comprising a vertical wheel and centrifuging the entire cell volume, thereby generating a first supernatant and a first cell pellet; b) centrifuging the first supernatant, thereby generating a second supernatant and a second cell pellet, removing the second supernatant, and resuspending the second cell pellet in Advanced DMEM/F12 medium and StemFit ® For Differentiation medium without cytokines, growth factors, or antibiotics; c) adding a first volume of 20 mg/mL collagenase II to the first cell pellet and the second cell pellet, combining the resuspended first cell pellet and the resuspended second cell pellet and adding
  • iPSC induced pluripotent stem cell
  • iHSCs induced pluripotent stem cell-derived hematopoietic stem cells
  • said method comprising one or more of the following steps: i) obtaining one or more human induced pluripotent stem cells (iPSCs), and wherein the method further comprises one or more of the following steps: a) seeding the iPSCs, wherein the seeding is performed by suspending the iPSCs in a first medium into a tissue culture vessel comprising a vertical wheel, thereby producing a first cell suspension, wherein the first medium comprises one of StemFit ® Basic 04 medium and StemFit ® Basic 03 medium, CHIR99021 (glycogen synthe
  • the cells are cultured under normoxic conditions.
  • the method further comprises further purifying and/or isolating the redifferentiated iHSCs.
  • the redifferentiated iHSCs are isolated differentiated cells or are capable of being further purified and/or isolated.
  • determining purity of the redifferentiated iHSCs comprises assaying for one or more of lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a via flow cytometry or an equivalent thereof.
  • the lineage markers comprise all of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a.
  • a method of de novo generation of hematopoietic stem cells (iHSCs) derived from human ⁇ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue culture vessel comprising: a means for seeding human ⁇ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; a means for redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; a means for retaining all cells in the culture on day 8 of culture while removing the medium from the culture for about 2 days; and a means for harvest
  • iHSCs hematopoietic stem cells
  • iPSCs human ⁇ T cell-derived induced pluripotent stem cells
  • said method comprising: seeding human ⁇ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; retaining all cells in the culture on day 8 of culture while removing the medium from the culture for about 2 days; and harvesting redifferentiated iHSCs at about day 10 of culture into two different
  • the tissue culture vessel has non-traditional bioreactor geometries.
  • the iPSCs are seeded at a density of about 1.0 x 10 5 cells/mL to about 1.8 x 10 5 cells/mL.
  • the tissue culture vessel comprising a vertical wheel further comprises a first medium comprising StemFit® Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, Emricasan, Polyamines, and Trans ⁇ ISRIB (CEPT) cocktail, wherein the BMP4 and the rhVEGF are present in a ratio of 1:1, and wherein the first medium does not comprise antibiotics.
  • a first medium comprising StemFit® Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman
  • the method further comprises exchanging spent medium in the tissue culture vessel comprising a vertical wheel on each of days 2, 4, and 6 of culture for the about 8 days, wherein the step of exchanging spent medium comprises: removing the spent medium from the tissue culture vessel comprising a vertical wheel using a 40 ⁇ m cell retention filter and adding a wash medium at a pump flow rate of 30 mL/min, the wash medium comprising Advanced DMEM/F12 medium and StemFit ® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio; exchanging about 3 L to about 3.5 L of the wash medium in the bioreactor and removing 0.1 L of the wash medium from the tissue culture vessel comprising a vertical wheel using the 40 ⁇ m cell retention filter; and adding about 50 mL of the wash medium and about 50 mL of a medium to from the tissue culture vessel comprising the vertical wheel.
  • the iPSCs are dissociated into single cells on day 7 of culture.
  • 62 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1
  • the step of retaining all cells comprises retaining clusters, semi- clusters, and single cells.
  • the step of retaining all cells comprises removing spent medium from the tissue culture vessel comprising a vertical wheel using a 4 ⁇ m cell retention filter and adding fresh medium to the bioreactor over about 1.8 days to about 2.2 days, preferably about 2 days, at a rate of about 0.69 mL/min (1 L/day), until 2 L of media have been exchanged, wherein the fifth medium comprises Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, IL-6, TPO, and one of (i) L-glutamine and (ii) GlutaMax TM , wherein the fresh medium does not comprise antibiotics.
  • the fifth medium comprises Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, IL-6, TPO, and one of (i
  • the vertical wheel spins at a rate of about 10 rpm to about 60 rpm, preferably about 23 rpm.
  • the step of harvesting comprises: a) removing an entire cell volume comprising EBs and iHSCs from the tissue culture vessel comprising a vertical wheel and centrifuging the entire cell volume, thereby generating a first supernatant and a first cell pellet; b) centrifuging the first supernatant, thereby generating a second supernatant and a second cell pellet, removing the second supernatant, and resuspending the second cell pellet in Advanced DMEM/F12 medium and StemFit ® For Differentiation medium without cytokines, growth factors, or antibiotics; c) adding a first volume of 20 mg/mL collagenase II to the first cell pellet and the second cell pellet, combining the resuspended first cell pellet and the resuspended second cell pellet and adding
  • the cells are cultured under normoxic conditions.
  • the method further comprises further purifying and/or isolating the redifferentiated iHSCs.
  • the redifferentiated iHSCs are isolated differentiated cells or are capable of being further purified and/or isolated.
  • determining purity of the redifferentiated iHSCs comprises assaying for one or more of lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a via flow cytometry or an equivalent thereof.
  • the lineage markers comprise all of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a.
  • iHSCs hematopoietic stem cells
  • iPSCs hematopoietic stem cells
  • iPSCs hematopoietic stem cells
  • a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days
  • redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days
  • the tissue culture vessel has non-traditional bioreactor geometries.
  • the iPSCs are seeded at a density of about 1.0 x 10 5 cells/mL to about 1.8 x 10 5 cells/mL.
  • the tissue culture vessel comprising a vertical wheel further comprises a first medium comprising StemFit ® Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, 64 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 Emricasan, Polyamines, and Trans ⁇ ISRIB (CEPT) cocktail, wherein the BMP4 and the rhVEGF are present in a ratio of 1:1, and wherein the first medium does not comprise antibiotics.
  • a first medium comprising StemFit ® Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (
  • the method further comprises exchanging spent medium in the tissue culture vessel comprising a vertical wheel on each of days 2, 4, 6, and 8 of culture for the about 8 days, wherein the step of exchanging the spent medium comprises: a) allowing cell aggregates to settle in the tissue culture vessel comprising a vertical wheel for about 3 to about 5 minutes; b) collecting 80% of the spent medium from the tissue culture vessel comprising a vertical wheel; c) centrifuging the collected medium, thereby obtaining a cell pellet; d) resuspending the cell pellet in StemFit® for Differentiation, thereby obtaining a cell suspension; and e) adding the cell suspension and fresh medium to the tissue culture vessel comprising a vertical wheel.
  • the step of exchanging the spent medium on days 2, 4, and 6 of culture comprises: i) allowing cell aggregates to settle in the tissue culture vessel comprising a vertical wheel for about 3 to about 5 minutes; collecting 80% of the spent medium from the tissue culture vessel comprising a vertical wheel; centrifuging the collected medium, thereby obtaining a cell pellet; resuspending the cell pellet in StemFit® for Differentiation medium, thereby obtaining a cell suspension; and adding the cell suspension and fresh medium to the tissue culture vessel comprising a vertical wheel, or ii) allowing cell aggregates to settle in the tissue culture vessel comprising a vertical wheel for about 3 to about 5 minutes; collecting 80% of the spent medium from the tissue culture vessel comprising a vertical wheel; centrifuging the collected medium, thereby obtaining a first cell pellet and a first supernatant; resuspending the first cell pellet in StemFit® for Differentiation medium and adding the resuspended first cell pellet to the tissue culture vessel comprising
  • the fresh medium added on day 2 of culture comprises: i) Advanced DMEM/F12 medium and StemFit ® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SB431542 (inhibitor of Activin/BMP/TGF ⁇ pathway), recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), and one of L-glutamine and GlutaMax TM supplement, or ii) StemPro TM -34 Serum-free Medium (SFM) and StemProTM-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, Insulin-Transferrin-Selenium (ITS-G), monothioglycerol, SB431542, recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (SFM) and StemProTM
  • the iPSCs are dissociated into single cells on day 7 of culture.
  • the step of harvesting redifferentiated iHSCs comprises retaining clusters, semi-clusters, and single cells.
  • the step of harvesting comprises retaining all cells in the tissue culture vessel comprising a vertical wheel.
  • the vertical wheel spins at a rate of about 10 rpm to about 60 rpm, preferably about 40 rpm.
  • the step of harvesting redifferentiated iHSCs comprises: a) removing an entire cell volume comprising embryoid bodies (EBs) and iHSCs from the tissue culture vessel comprising a vertical wheel and centrifuging the entire cell volume, thereby generating a first supernatant and a first cell pellet; b) centrifuging the first supernatant, thereby generating a second supernatant and a second cell pellet and removing the second supernatant; c) resuspending the first cell pellet and the second cell pellet in Advanced DMEM/F12 medium without cytokines, growth factors, or antibiotics; d) adding a volume of 20 mg/mL collagenase II to the first cell pellet, thereby generating a first cell suspension; e) adding a volume of Advanced DMEM/F12 medium without cytokines, growth factors, 66 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOP
  • the cells are cultured under normoxic conditions.
  • the method further comprises further purifying and/or isolating the redifferentiated iHSCs.
  • the redifferentiated iHSCs are isolated differentiated cells or are capable of being further purified and/or isolated.
  • the one or more cells have high purity, viability, fold expansion, and potential to redifferentiate into immune effectors, including iPSC-derived ⁇ (i ⁇ ) T cells.
  • the step of determining purity of the iHSCs comprises assaying for one or more, optionally all, of the lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a via flow cytometry or an equivalent thereof.
  • the lineage markers comprise one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a.
  • iHSCs hematopoietic stem cells
  • iPSCs human ⁇ T cell-derived induced pluripotent stem cells
  • said method comprising: a means for seeding human ⁇ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; a means for redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; a means for retaining all cells in the culture on day 8 of culture while removing the medium from the culture for about 2 days; and a means for harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a 67 301689646v1 Attorney Docket No: 25
  • iHSCs hematopoietic stem cells
  • iPSCs hematopoietic stem cells
  • said method comprising: a means for seeding human ⁇ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; a means for redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; a means for harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction.
  • iHSCs under serum-free and feeder-free culture conditions
  • said method comprises: i. obtaining at least one human iPSC clone, and wherein the method further comprises one or more of the following steps: a.
  • redifferentiating the at least one human iPSC clone by culturing the at least one human iPSC clone in a first cell culture medium generating a first cell suspension
  • said first cell culture medium comprises one of StemFit ® Basic 04 medium and StemFit ® Basic 03 medium supplemented with basic fibroblast growth factor (bFGF) to a final concentration of about 100 ng/mL bFGF
  • said first cell culture medium further comprises CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, Emricasan, Polyamines, and Trans ⁇ ISRIB (CEPT) cocktail, wherein culturing is performed in a vertical wheel bioreactor at about 10-60 rpm, preferably 40 rpm, for about
  • Advanced DMEM/F12 medium and StemFit ® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises one of L-glutamine and GlutaMax TM , SB431542 (inhibitor of Activin/BMP/TGF ⁇ pathway), rhVEGF, bFGF, and stem cell factor (SCF), or ii.
  • the third cell culture medium comprises: i.
  • Advanced DMEM/F12 medium and StemFit ® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, FMS-like tyrosine kinase 3 ligand (Flt3L), interleukin-6 (IL-6), rhVEGF, IL-3, one of (i) thrombopoietin (TPO) and (ii) erythropoietin (EPO), and one of (i) L-glutamine and (ii) GlutaMax TM , or 69 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 ii.
  • Flt3L FMS-like tyrosine kinase 3 ligand
  • IL-6 interleukin-6
  • rhVEGF interleukin-6
  • IL-3 interleukin-6
  • TPO thrombopo
  • StemProTM-34 SFM and StemProTM-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, rhVEGF, IL-3, IL-6, Flt3L, one of (i) TPO and (ii) EPO, and one of (i) L-glutamine and (ii) GlutaMax TM , and wherein the third medium does not comprise antibiotics, and placing the third cell suspension into the vertical wheel bioreactor with remaining second cell suspension, thereby forming a fourth cell suspension; g. culturing fourth cell suspension in the vertical wheel bioreactor for an additional about 1.8 to about 2.2 days, preferably about 2 days; h.
  • the fourth cell culture medium comprises: i. Advanced DMEM/F12 medium and StemFit ® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, IL-6, one of (i) TPO and (ii) EPO, and one of (i) L-glutamine and (ii) GlutaMax TM , or ii.
  • StemProTM-34 SFM and StemProTM-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, IL-6, one of (i) TPO and (ii) EPO, and one of (i) L-glutamine and (ii) GlutaMax TM , and adding the fifth cell suspension into remaining fourth cell suspension in the vertical wheel bioreactor, thereby generating a sixth cell suspension; 70 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 k. culturing the sixth cell suspension for about 1.8 to about 2.2 days, preferably about 2 days in the vertical wheel bioreactor; l.
  • m centrifuging the eighth cell suspension generating a first supernatant and a fourth cell pellet
  • n collecting and centrifuging the first supernatant, thereby generating a fifth cell pellet and resuspending the fifth cell pellet in a fifth medium thereby generating a ninth cell suspension
  • the fifth medium comprises Advanced DMEM/F12 medium, wherein the fifth medium does not comprise cytokines, antibiotics or growth factors
  • cells of the ninth cell suspension comprises iHSCs, which are defined as CD34+ and lineage marker-negative, where the lineage markers comprise one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a; o.
  • step m resuspending the fourth cell pellet of step m) in a volume of about 20 mg/mL collagenase type II, thereby generating a tenth cell suspension; p. adding the fifth medium to the tenth cell suspension; q. centrifuging the tenth cell suspension, thereby generating a sixth cell pellet, and adding TrypLE to the sixth cell pellet, thereby generating an eleventh cell suspension; r. adding additional fifth medium to the eleventh cell suspension and mixing; s. centrifuging the eleventh cell suspension to form a seventh cell pellet and resuspending the seventh cell pellet in additional fifth medium to form a twelfth cell suspension; t.
  • iHSCs wherein the cell strainer is 70 ⁇ m mesh size; u. determining the purity of the iHSCs from the first supernatant of step n) and the iHSCs from step t), said purity defined as CD34+ and lineage marker-negative, where the lineage markers comprise one or 71 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a; v. not introducing serum in any of steps a)-u); w.
  • the one or more cells have high purity, viability, fold expansion, and potential to redifferentiate into immune effectors, including iPSC-derived ⁇ (i ⁇ ) T cells.
  • the EB and ⁇ T cell-derived iPSCs are from a tissue culture vessel.
  • the first, second, fourth, sixth, and/or eighth cell suspensions are cultured under normoxic conditions.
  • the method further comprising further purifying and/or isolating the iHSCs.
  • the obtained iHSCs are isolated redifferentiated cells or are capable of being further purified and/or isolated.
  • the method further comprises assaying for all of the lineage markers CD3, CD19, CD14, CD11b, CD56, CD20, CD16, CD2, and CD235a via flow cytometry or an equivalent thereof.
  • any one of the comprises gently dispersing the cells using a pipet or equivalent thereof.
  • cells of the first medium are at a concentration of about 80,000 to about 180,000 cells/mL in a volume of about 50 to about 70 mL.
  • the cells of the first medium are at a concentration of about 150,000 cells/mL in a volume of about 60 mL.
  • the medium of step a) further comprises a ROCK inhibitor.
  • the medium of step a) further comprises a Chroman 1, Emricasan, Polyamines, and Trans ⁇ ISRIB (CEPT) cocktail.
  • step a) all the cell volume of the first cell suspension is removed from the bioreactor prior to centrifugation.
  • the cell morphology is imaged via microscopy, e.g., bright field 72 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 microscopy.
  • the medium in any one of the steps comprises L-glutamine.
  • the medium in any one of the steps comprises GlutaMax TM .
  • the medium in any one of the steps comprises TPO.
  • the medium in any one of the steps comprises EPO.
  • the centrifugation of any one of the steps is performed at 100g for 1 minute at brake 6.
  • the centrifugation of any one of the steps is performed at 300g for 5 minutes. [0322] In some embodiments, the centrifugation of any one of the steps is performed at 300g for 3 minutes. [0323] In some embodiments, the centrifugation in any one of the steps is performed twice, first at 100g for 1 minute at brake 6 and second at 300g for 5 minutes. [0324] In some embodiments, the tenth cell suspension is incubated with collagenase for 20 minutes at 37 °C. [0325] In some embodiments, the eleventh cell suspension is incubated with TrypLE for 5 minutes at 37 °C.
  • passing of the twelfth cell suspension further comprises passing the twelfth cell suspension through a second cell strainer with a 40 ⁇ m mesh size.
  • the culturing of the iPSC cells, EBs, and iHSCs is performed at 37 °C and 5% CO 2 .
  • the bioreactor wheel is rotating at 60 rpm.
  • the bioreactor wheel is rotating at 30 rpm.
  • the bioreactor wheel is rotating at 24 rpm.
  • the bioreactor wheel is rotating at 15 rpm.
  • the bioreactor wheel is perpendicular to the bioreactor vessel.
  • the bioreactor wheel is not perpendicular to the bioreactor vessel.
  • iPSCs of the first medium are at a concentration of about 80,000 to about 180,000 cells/mL in a volume of about 90 to about 110 mL.
  • the iPSCs of the first medium are at a concentration of about 73 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 150,000 cells/mL in a volume of about 100 mL.
  • iPSCs of the first medium are at a concentration of about 5,000 to about 180,000 cells/mL in a volume of about 0.45 to about 0.55 L. [0337] In some embodiments, the iPSCs of the first medium are at a concentration of about 20,000 cells/mL in a volume of about 0.5 L. [0338] In some embodiments, iPSCs of the first medium are at a concentration of about 5,000 to about 180,000 cells/mL in a volume of about 2 to about 5 L. [0339] In some embodiments, iPSCs of the first medium are at a concentration of about 5,000 to about 180,000 cells/mL in a volume of about 10 to about 20 L.
  • provided herein is a composition comprising the one or more cells as described herein.
  • Cell viability decreases have been reported due to single-cell enzymatic dissociation of hPSCs. Thus, typically, hPSCs are grown as colonies or aggregates and clump seeding is used as the inoculation strategy for bioreactor culture.
  • the invention herein overcomes challenges by maintaining single cells throughout the processes described herein by using, for example, a cell retention filter that does not remove single cells, but rather preserves them.
  • a cell retention filter that does not remove single cells, but rather preserves them.
  • the present invention therefore positively impacts bioecosystems and the environment by driving advancements that help reduce human resources, power, energy consumption, carbon emissions, and overall environmental impact.
  • the present invention provides automation and efficiency features and provides more efficient processes and systems, reducing the need for extensive manual labor, while minimizing the environmental impact associated with resource- intensive activities.
  • the automation of the present invention optimizes energy consumption, 74 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 streamlines operations, and reduces waste, resulting in improved efficiency and reduced carbon emissions.
  • 3D vertical-wheel bioreactor As described herein, the iPSCs suspended in a medium are cultured in a 3D vertical- wheel bioreactor, also referred to herein as a tissue culture vessel comprising a vertical wheel.
  • the vertical-wheel bioreactor is used to manufacture cells via rotation of a vertical-wheel impeller within a U-shaped vessel, combining radial and axial flow components. This allows for suspension and gentle agitation of the cells.
  • the use of a vertical- wheel bioreactor is detailed within Croughan, M. S., et al. Initial power measurements for a family of novel vertical-wheel bioreactors. Can. J. Chem. Eng.2022, 1-12; Dang, T., et al. Computational fluid dynamic characterization of vertical-wheel bioreactors used for effective scale-up of human induced pluripotent stem cell aggregate culture. Can. J. Chem. Eng. 2021, 1-18; Borys, B.
  • the hydrodynamics of the 3D vertical-wheel bioreactor are different than that of a typical stirred tank bioreactor as the 3D vertical-wheel bioreactor provides a reduced cell shear.
  • the parts of the vertical-wheel bioreactor, including the vertical-wheel impeller and the U-shaped vessel can vary in size, as a person having ordinary skill in the art would understand.
  • a 0.5 L bioreactor may use a vertical-wheel impeller with a diameter of about 73 mm and a width of about 25 mm and a U-shaped vessel with a radius of about 43 mm.
  • the impeller radius may be about 85% of the U-shaped vessel radius.
  • a 3 75 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 L bioreactor may use a vertical-wheel impeller with a diameter of about 137 mm and a width of about 37 mm and a U-shaped vessel with a radius of about 83 mm.
  • the impeller radius may be about 83% of the U-shaped vessel radius.
  • a 15 L bioreactor may use a vertical-wheel impeller with a diameter of about 227 mm and a width of about 113 mm and a U- shaped vessel with a radius of about 138 mm.
  • the impeller radius may be about 83% of the U- shaped vessel radius.
  • EDR energy dissipation rate
  • the use of traditional bioreactors has exhibited hydrodynamic environments that are unfavorable to iPSC maintenance and growth.
  • the traditional bioreactor may provide an environment wherein the high shear stress may damage cells by tearing apart cells and cell aggregates, lowering cell yield and quality.
  • the shear stress increases with scale, and during the expansion phase in traditional bioreactors, only moderate cell fold increases have been shown.
  • the use of traditional bioreactors has limited the ability to scale the inoculation and harvesting phases.
  • the computational fluid dynamics model provides confirmation that the vertical-wheel bioreactor, by combining radial and axial flow components, creates hydrodynamic forces that are more uniformly distributed.
  • the vertical-wheel bioreactor has lemniscate velocity streamlines whereas the traditional bioreactor has circular velocity streamlines.
  • the vertical-wheel bioreactor has decreased dissipation energy gradients, lower shear effects, and more uniform particle suspension compared to traditional bioreactors, supporting the vertical-wheel bioreactor environment to better scale compared to the environment of traditional bioreactors.
  • the cells of the first medium are at a concentration of about 80,000 to about 180,000 cells/mL in a volume of about 50 to about 70 mL. In one embodiment, the cells of the first medium are at a concentration of about 150,000 cells/mL in a volume of about 60 mL. In some embodiments, the cells of the first medium are at a concentration of about 80,000 to about 120,000 cells/mL in a volume of about 0.09 to about 0.11 L. In one embodiment, the cells of the first medium are at a concentration of about 100,000 cells/mL in a volume of about 0.1 L.
  • the cells of the first medium are at a concentration of about 5,000 to about 180,000 cells/mL in a volume of about 0.45 L to about 0.55 L. In one embodiment, the cells of the first medium are at a concentration of about 20,000 cells/mL in a volume of about 0.5 L.
  • the vertical wheel is perpendicular to the bioreactor vessel. In some embodiments, the vertical wheel is tilted at about a 5° angle to about a 60° angle relative to the bioreactor vessel. In one embodiment, the vertical wheel is tilted at about a 20° angle relative to the bioreactor vessel. In one embodiment, the vertical wheel is tilted at about a 40° angle relative to the bioreactor vessel.
  • the bioreactor may be a fed-batch bioreactor.
  • the bioreactor may be perfused or perfusable. Perfusion is a method wherein the cells are kept within a bioreactor while the media is continuously exchanged. Fresh media is added to replenish carbon sources and other nutrients. Depleted media and cellular waste are removed. 77 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 In some embodiments, perfusion is performed via cell binding, wherein the cells are kept in the bioreactor by binding to capillary membranes or fibers. In some embodiments, the bioreactor contains capillary membranes or fibers.
  • perfusion is performed via filtration, wherein the spent medium flows through a membrane with pores that are too small to accomodate the cells.
  • the bioreactor contains membranes with pores that are too small to accomodate the cells.
  • perfusion is performed via settling, wherein the cells are allowed to settle and the rest of the suspension is removed as spent medium.
  • the cells settle within the bioreactor.
  • the bioreactor may contain multiple bioreactor wheels.
  • the bioreactor contains 2 to 6 bioreactor wheels.
  • the bioreactor contains 2, 3, 4, 5, or 6 bioreactor wheels.
  • one or more of the bioreactor wheels are perpendicular to the bioreactor vessel. In some embodiments, one or more of the bioreactor wheels are not perpendicular to the bioreactor vessel. In some embodiments, one or more of the bioreactor wheels are rotating between about 10 and about 60 rpm. In some embodiments, one or more of the bioreactor wheels are rotating at about 60 rpm. In some embodiments, one or more of the bioreactor wheels are rotating at about 30 rpm. In some embodiments, one or more of the bioreactor wheels are rotating at about 24 rpm. In some embodiments, one or more of the bioreactor wheels are rotating at about 15 rpm.
  • Example 1 Serum-free, feeder-free, three-dimensional redifferentiation of ⁇ T cell-derived iPSCs into i ⁇ T cells.
  • Example 2 contains instructions to maintain and redifferentiate ⁇ T- iPSCs. Materials [0357] The following abbreviations are used herein (Table 1). 78 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 Table 1. Abbreviations.
  • Reagents needed for hematopoietic redifferentiation (listed by day of redifferentiation).
  • Reagent Product No. Day 0 Reagents StemFit ® Basic 04 Complete Amsbio, cat.# SFB-504-CT CHIR99021 TOCRIS, cat.# 4423 rh BMP4 R&D, cat.# 314-BP rh VEGF R&D, cat.# 293-VE Day 2 Reagents Advanced DMEM/F12 Gibco, cat.# 12634-10 StemFit ® For Differentiation Ajinomoto, cat.# 20170228A L-Glutamine Gibco, cat.# 25030-081 Penicillin-Streptomycin (P/S) Gibco, cat.# 15140-122 SB431542 Fujifilm Wako pure chemical corporation, cat.# 033-24631 rh VEGF R&D, cat.# 293-VE rh bFGF Amsbio, cat.# AMS-FGF-100
  • Reagents for i ⁇ T cell redifferentiation Reagent Product No. StemSpanTM SFEM II StemCell Technologies, cat.# 09655 Lymphoid Progenitor Expansion Supplement (10X) StemCell Technologies, cat.# 09915 rh VCAM-1-Fc R&D, cat.# 862-VC-100 rh DLL4-Fc R&D, cat.# 10185-D4-050 81 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 Table 5. Supplies and consumables needed for cell culture. Item Product No.
  • Y-27632 ROCK inhibitor stock solution was prepared as follows: a. Y-27632 ROCK inhibitor was received as a crystalline solid. b. Inhibitor was stored at -20 °C for up to 12 months from date of receipt. c. A 10 mM stock solution was prepared in 1X DPBS. i. 312 ⁇ L of 1X DPS was added to 1 mg Y-27632 and solution was resuspended completely. ii. 25 ⁇ L of stock solution was aliquoted into Eppendorf tubes labeled as Y-27632 stock. iii. Aliquots were stable at -20 °C for up to 6 months. iv.
  • T-iPSC vial was thawed as follows: a. A vial of frozen ⁇ T-iPSCs was thawed in a 37 °C water bath for 1 minute or until small pieces of frozen floating cells were visible. b.
  • Plate was imaged with EVOS M7000 and images were recorded at 4X and 10X magnification.
  • e Three images of different regions on the plate were taken to record the morphology and landscape of the culture. There were single cells and two-celled colonies present. Cells had a spiky morphology due to culturing in the presence of the ROCK inhibitor. i. Note: as cells divide, borders became more defined. 87 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 ii. At 10X magnification, the cells appeared with more defined borders (data not shown).
  • iMatrix 511-coated 6-well plate(s) were prepared, as many as were required, as in Step 1.3, to passage iPSCs.
  • b. Passaging iPSCs was performed as follows: i. Medium was aspirated from cells plated in Step 1.7. ii. Wells were rinsed with 1 mL 1X DPBS. iii. 1X DPBS was aspirated from wells. iv. 1 mL TrypLE was added. v. Plates were incubated at 37 °C for 6-8 minutes. vi. Wells were examined under a microscope. Cells appeared bright with clear cell borders (data not shown). vii. TrypLE was gently removed. viii.
  • Exemplary acceptance criteria for pluripotency were: >85% Oct4+, Sox2+, SSEA3+, SSEA4+ ⁇ 1% SSEA1+ ⁇ 5% CD34+ Procedure to redifferentiate ⁇ T-iPSC lines into hematopoietic stem cells (HSCs) using a serum- free, feeder-free (SFFF) three-dimensional (3D) culture method.
  • SFFF serum- free, feeder-free
  • Step 1.15.b Add 8 mL STEP 4 base medium without added cytokines or growth factors (Step 1.15.b) and mix solution. v. Pass cells through 70 ⁇ m filter and centrifuge at 300g for 5 min at brake 9. vi. Resuspend in 10 mL of STEP 4 base medium without added cytokines or growth factors (Step 1.15.b), and perform cell count using NC-200. e. 2.0x10 6 cells were aliquoted for flow cytometric analysis using the iHSC panel. f. iHSC flow panel is shown in Table 6. g. Exemplary success criteria for iHSC redifferentiation step: purity of >70% Lineage- CD34+ cells and >5 fold expansion per input iPSC.
  • the protocol enabled redifferentiation of ⁇ T-iPSC lines into hematopoietic stem cells (HSCs) at a variety of RPM in a 0.1L vertical wheel bioreactor using a serum-free, feeder- free (SFFF) three-dimensional (3D) culture method, the results including cell cluster formation, morphology, cell growth (e.g., metabolism), purity, etc. This was true with perfusion in the bioreactor system.
  • Example 2 Serum-free, feeder-free redifferentiation of ⁇ T cell-derived iPSCs into i ⁇ T cells.
  • the example provided contains instructions to redifferentiate ⁇ T-iPSCs, using GMP-compliant or GMP-like reagents.
  • Reagents for iPSC culture and maintenance Reagent Product No. Storage Condition StemFit ® Basic 04 Amsbio, cat.# SFB-504- -20 °C, upon thaw use within two Complete Type CT weeks and store at 4 °C. rh bFGF Peprotech, cat.# AF-100- Store lyophilized at 18B -20 to -80 °C. Upon reconstitution store at 4 °C for one week. Y-27632 – ROCK Tocris, cat.# 1254 Desiccate at RT, upon reconstitution Inhibitor, 1 mg store aliquots at -20 °C or below.
  • VEGF-GMP Upon PBS) VEGF-GMP reconstitution store at 4 °C for one month.
  • 98 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 rhbFGF (Sterile PBS) Peprotech, cat.# AF- Store lyophilized at -20 to -80 °C.
  • rhSCF Step PBS
  • R&D cat.# BT-SCF
  • Day 4 Reagents Advanced Gibco, cat.# 12634-10 Store at 4 °C.
  • StemFit ® For Ajinomoto, cat.# -20 °C, upon thaw use within two weeks Differentiation 20170228A and store at 4 °C.
  • L-Glutamine Gibco cat.# 25030- -20 °C, upon thaw use within two weeks 081 and store at 4 °C.
  • rhSCF Step PBS
  • R&D cat.# BT-SCF Store lyophilized at -20 °C.
  • rhVEGF (Sterile R&D, cat.# BT- Store lyophilized at -20 °C.
  • VEGF-GMP Upon PBS) VEGF-GMP reconstitution store at 4 °C for one month.
  • rhIL-3 (Sterile PBS) PeproTech, cat.# AF- Store lyophilized at -20 °C.
  • rhIL-6 (Sterile PBS) R&D, cat.# 206-IL- Store lyophilized at -20 °C.
  • rhFlt3L (Sterile PBS) R&D, cat.# BT-FT3L- Store lyophilized at -20 °C.
  • AFL reconstitution store at 4 °C for one month.
  • rh TPO (Sterile PBS) Peprotech, cat.# AF- Store lyophilized at -20 °C. Upon 300-18 reconstitution store at 4 °C for one month. Days 6-10 Reagents Advanced Gibco, cat.# 12634-10 Store at 4 °C. Stable until 12 months from DMEM/F12 date of manufacture. StemFit ® For Ajinomoto, cat.# -20 °C, upon thaw use within two weeks Differentiation 20170228A and store at 4 °C. L-Glutamine Gibco, cat.# 25030- -20 °C, upon thaw use within two weeks 081 and store at 4 °C.
  • Reagents for flow cytometry Reagent Vendor Product No. Dilution Storage Flow staining Reagents Stain Buffer (BSA) BD Biosciences 554657 1X 4 °C Brilliant Stain Buffer BD Biosciences 361651 1:10 4 °C Stabilizing Fixative 3X BD Biosciences 338036 1:3 4 °C Concentrate Human TruStain FcXTM (Fc Receptor Blocking BioLegend 422302 1:20 4 °C Solution) ICS Reagents BD Cytofix/Cytoperm kit 1:10 for Perm/ (Perm/Wash buffer and Wash Buffer, BD Biosciences 554714 4 °C Fixation/Permeabilization 1X for Fix/Perm solution) solution Staining Control Kasumi-1 (myeloblast cell line used as positive ATCC CRL-2724 N/A control for iCD34 flow panel) Table 20.
  • BSA Stain Buffer
  • Antibodies for Pluripotency Flow Panels Fluorochrome Marker Vendor Cat# Pluripotency BV711 SSEA-1 BioLegend 323050 PE-Cy7 SSEA-3 BioLegend 330326 PerCP/Cy5.5 CD34+ BioLegend 343522 PE SSEA-4 BioLegend 330406 AF647 OCT 3/4 BioLegend 653710 BV421 SOX2 BioLegend 656114 101 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 Table 21. Antibodies for iCD34 Flow Panels. Fluorochrome Marker Clone Vendor Product No.
  • iCD34 Panel PE CD34 581 BioLegend 343506 APC CD43 1G10 BD 560198 BUV395 CD45 HI30 BD 563792 BV421 CD38 HIT2 BioLegend 303526 CD3 HIT3a BioLegend 300306 CD235a HI264 BioLegend 349104 CD11c Bu15 BioLegend 337214 CD11b ICRF44 BioLegend 301330 FITC CD14 HCD14 BioLegend 325604 Dump Channel CD56 HCD56 BioLegend 318304 CD19 HIB19 BioLegend 302206 CD20 2H7 BioLegend 302304 CD16 3G8 BioLegend 302006 CD2 RPA-2.10 BioLegend 300206 Table 22.
  • StemFit ® Basic 04 Complete Type Medium was supplemented with an additional 20 ng/mL of bFGF to achieve a final concentration of 100 ng/mL bFGF in the supplemented medium.
  • Supplemented medium was stored up to two (2) weeks at 4 °C.
  • Medium was brought to RT for at least one (1) hour prior to use.
  • 2.210 mM Y-27632 ROCK inhibitor stock solution was prepared as follows: a. Y-27632 ROCK inhibitor (ROCKi) was received as a crystalline solid. b. Inhibitor was stored at -20 °C for up to 12 months from date of receipt. c. A 10 mM stock solution was prepared in 1X DPBS. i.
  • the cryotube was washed with 1 mL of warmed media and added to the 15 mL tube. Thawed cell solution in the 15 mL tube was topped off to reach a total of 10 mL and was gently mixed. g. The iPSC cells were centrifuged at 300 g for 5 minutes at room temperature. The supernatant was removed and cells were resuspended in 5 mL of warmed media. 2 x 200 ⁇ L samples were taken in Eppendorf tubes and cell count was performed on NC200. h. Using the NC200 count, cell volume needed was calculated in order to reach a 20,000 cells/mL seeding density. i. Cells were collected from Eppendorf tube in Via1-Cassette. ii.
  • Via1-Cassette was inserted into NC-200 cell counter and cells were counted. iii. Note: Only the total number of live cells was used for cell seeding purposes. i. The calculated cell volume was transferred to prewarmed media in a vessel. The vessel was topped off to reach 500 mL total cell volume and was put back on the base in the incubator to incubate at 37 °C for three days. [0381] 2.4 Exchanging the cell media in 3D cultures was performed as follows: 106 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 a. 50% media exchanges were performed on Days 4, 5, and 6. Media changes did not contain ROCKi. b.
  • Step 2.6 for iCD34 differentiation, to seed cells into new 0.5 L vessels at 20,000 cells/mL for further iPSC expansion, or to freeze down the sample for flow analysis by centrifuging the required number of cells at 300 g for 5 minutes, removing the supernatant, and resuspending the cells in 1 mL of ⁇ CS10, then storing overnight in -80 °C. i.
  • the reactor was removed from the base and aggregates were allowed to settle in the reactor for approximately 5 minutes or long enough that the clusters in suspension were no longer seen visibly moving to the bottom of the reactor. About 2 minutes in, the 0.1 L reactor was gently rocked a few times to ensure clusters that settled on the top of the vertical wheel were moved off. Then, media was pulled from the vertical wheel leaving 20% volume left in the reactor. The pulled supernatant was spun down at 100 g for 1 minute at brake 6. d. While cells were in the centrifuge, 38.4 mL DMEM/F12 + 8.6 mL StemFit for Differentiation media were added to the reactor. The reactor was placed back on the base to turn at 40 rpm. e.
  • Reagents for STEP 3 medium for Days 4-6 were thawed. Table 28. Step 3 Medium for Days 4-6.
  • the reactor was removed from the base and aggregates were allowed to settle in the reactor for approximately 5 minutes or long enough that the clusters in suspension were no longer seen visibly moving to the bottom of the reactor. About 2 minutes in, the 0.1 L reactor was gently rocked a few times to ensure clusters that settled on the top of the vertical wheel were moved off. Then, media was pulled from the vertical wheel leaving 20% volume left in the reactor. The pulled supernatant was spun down at 100 g for 1 minute at brake 6. k. While cells were in the centrifuge, 38.4 mL DMEM/F12 + 8.6 mL StemFit for Differentiation media were added to the reactor. The reactor was placed back on the base to turn at 40 rpm. l.
  • the supernatant containing single cells was transferred to a 50 mL conical tube.
  • the pellet from the first conical tube was resuspended in 500 ⁇ L StemFit for Differentiation medium and added to reactor.
  • m Supernatant transferred to the second 50 mL conical tube was spun at 300 g for 5 minutes at brake 9. After centrifuging, 500 ⁇ L from the cell supernatant was collected for a metabolic sample. The rest of the supernatant was removed, and the pellet was resuspended with 500 ⁇ L of StemFit for Differentiation medium and added to reactor. n.
  • Reagents for STEP 4 medium for Days 8-10 were thawed (similarly to Step 2.9e) and added to the reactor. o. Cells were incubated and cultured at 37 °C, 5% CO 2 , and 40 rpm for another two days. 113 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 [0387] 2.10 Day 10: Harvesting hematopoietic stem cells (iCD34 cells) and preparing cells for flow cytometric analysis and cryopreservation was performed as follows: a. Images were recorded at Day 10 as in Step 2.7b. b. 21 mL of STEP 4 base medium without cytokines and growth factors were prepared. c.
  • Single cell fraction was processed as follows: i. Pull entire cell volume out of reactor, transfer 30 mL to each of 2x 50 mL conicals, and spin down at 100 g for 1 minute at brake 6. Pellets were kept for dissociation in Step 2.10d and supernatant containing single cells was dissociated in the following step. ii. Transfer supernatant, containing single cells, to new 50 mL conical tubes. Spin down at 300 g for 5 minutes at brake 9. iii. After centrifuging, collect 500 ⁇ L from cell supernatant for metabolic sample. Remove rest of supernatant and resuspend pellet in 5 mL of fresh Advanced DMEM/F12 base medium without added cytokines or growth factors. iv.
  • iCD34 flow panel is shown in Table 21.
  • Exemplary success criteria for iCD34 redifferentiation step purity [defined as Lineage marker-negative (Lin ⁇ ) CD34+] is >70% and fold expansion per input iPSC is >0.5-folds.
  • the protocol enabled redifferentiation of ⁇ T-iPSC lines into hematopoietic stem cells (HSCs) at a variety of RPM in a 0.1L vertical wheel bioreactor using a serum-free, feeder- free (SFFF) three-dimensional (3D) culture method, the results including cell cluster formation, morphology, cell growth (e.g., metabolism), purity, etc. This was true with perfusion in the bioreactor system.
  • Example 3 Serum-free, feeder-free, three-dimensional redifferentiation of ⁇ T cell-derived iPSCs into iCD34s in a 3 L bioreactor scale.
  • the example provided contains instructions to redifferentiate ⁇ T-iPSCs.
  • Reagents needed for hematopoietic redifferentiation (listed by day of redifferentiation).
  • Reagent (Reconstitution Solution) Product No. Day 0 Reagents StemFit Basic 04 Complete Amsbio, cat.# SFB-504-CT CHIR99021 (DMSO) TOCRIS, cat.# 4423 116 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 rh BMP4 (4 mM HCl) R&D, cat.# AFL314E rh VEGF (Sterile PBS) R&D, cat.# BT-VEGF-GMP Day 2 Reagents Advanced DMEM/F12 Gibco, cat.# 12634-10 StemFit ® For Differentiation Ajinomoto, cat.# 20170228A L-Glutamine Gibco, cat.# 25030-081 SB431542 (DMSO) Tocris, cat.# 1614 rh VEGF (Ste
  • Item Product No. 117 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 CorningTM CostarTM 6-well Clear TC-treated Multiple Corning, cat.# 3516 Well Plates, Individually Wrapped, Sterile FisherbrandTM Sterile Polystyrene Disposable ThermoFisher, Serological Pipets with Magnifier Stripe (various cat.# 13-676-10H, sizes) cat.# 13-678-11E, cat.# 13-678-11, cat.# 13-678-11F Pipette Tips RT LTS 20 ⁇ L F 960A/10 (P20) Rainin, cat.# 30389225 Pipette Tips RT LTS 200 ⁇ L F 960A/10 (P200) Rainin, cat.# 30389239 Pipette Tips RT LTS 1000 ⁇ L F 768A/8 (P1000) Rainin, cat.# 30389212 50 mL conical tubes Corning, cat.
  • Y-27632 ROCK inhibitor stock solution was prepared as follows: a. Y-27632 ROCK inhibitor (ROCKi) was received as a crystalline solid. b. Inhibitor was stored at -20 °C for up to 12 months from date of receipt. c. A 10 mM stock solution was prepared in 1X DPBS. i. 312 mL of 1X DPS was added to 10 mg Y-27632 and solution was resuspended completely. ii. 500 ⁇ L of stock solution was aliquoted into Eppendorf tubes labeled as ROCKi stock. iii. Aliquots were stable at -20 °C for up to 6 months. iv.
  • 3.3 iPSCs were thawed directly into 3D cultures as follows: a. One ROCKi aliquot was obtained from -20 °C and thawed to room temperature. b. 500 mL of StemFit Basic 04 Complete Type Medium was warmed to 37 °C. c. For the 0.5 L vertical-wheel platform, 490 mL of complete media and 500 ⁇ L of ROCKi were transferred into the vessel and placed on a magnetic base in the incubator at 30 rpm agitation. The vessel sat in the incubator for at least an hour before cells were added.
  • the supernatant was removed and cells were resuspended in 5 mL of warmed media. 2 x 200 ⁇ L samples were taken in Eppendorf tubes and cell count was performed on NC200. h. Using the NC200 count, cell volume needed was calculated in order to reach a 20,000 cells/mL seeding density. i. Cells were collected from Eppendorf tube in Via1-Cassette. ii. Via1-Cassette was inserted into NC-200 cell counter and cells were counted. iii. Note: Only the total number of live cells was used for cell seeding purposes. i. The calculated cell volume was transferred to prewarmed media in a vessel.
  • the vessel was topped off to reach 500 mL total cell volume and was put back on the base in the incubator to incubate at 37 °C for three days.
  • 3.4 Exchanging the cell media in 3D cultures was performed as follows: a. 50% media exchanges were performed on Days 4, 5, and 6. Media changes did not contain ROCKi. b. Aliquots were made for exchanging half of the total media volume and were put in a water bath at 37 °C for ⁇ 30 minutes. c. The vessel was removed from the incubator and was placed in the hood to let the cells settle inside the vessel. d. During the cell settling time, the vessels were not disturbed.
  • the vessel was installed onto ⁇ its base inside the incubator with agitation speed of 50 rpm ⁇ for enzymatic cell dissociation ⁇ for 20 minutes. g. The vessel was removed once dissociation ⁇ time was completed and 25 mL volumes were transferred to 50 mL centrifuge tubes so that four conical tubes each had approximately 25 mL of cell solution. If there was extra volume left in the reactor, this was distributed across the conical tubes. The solution in each conical tube was mixed with a 10 mL pipette until no clusters were visible (approximately 5-10 122 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 mixes).
  • One ⁇ 150 ⁇ L ⁇ sample ⁇ was taken from the conical tube and added into a microtube ⁇ for counting.
  • m. 150 ⁇ L ⁇ of ⁇ PBS ⁇ was added into the microtube containing the cell sample for a 2x dilution.
  • n. The cell number was counted using NC-200. ⁇ Viable cells/mL, cell viability, ⁇ cell diameter, ⁇ and % in aggregates remaining were recorded. o.
  • the next step was either to proceed to Step 3.6 to begin the iCD34 differentiation process in a 3 L reactor, to seed cells into new 0.5 L vessels at 20,000 cells/mL for further iPSC expansion, or to freeze down the sample for flow analysis by centrifuging the required number of cells at 300 g for 5 minutes, removing the supernatant, and resuspending the cells in 1 mL of ⁇ CS10, then storing overnight in -80 °C.
  • i. 5x10 6 cells were used for pluripotency flow analysis.
  • Pluripotency flow panel is shown in Table 34.
  • Acceptance criteria were met before proceeding to redifferentiation steps.
  • Exemplary acceptance criteria for pluripotency were: >85% Oct4+, Sox2+, SSEA3+, SSEA4+ ⁇ 1% SSEA1+ ⁇ 5% CD34+ 123 301689646v1
  • SFFF serum-free, feeder-free
  • the PBS 3MAG was brought into the BSC and the shrink-wrapped plastic around it was carefully removed.
  • c The PBS 3MAG vessel was placed upright in the BSC and all lines were clamped.
  • d Autoclaved pouches of dip tubes were brought into the BSC.
  • the unused port cover cap front right port
  • the 4 ⁇ m dip tube was unpacked from the autoclave bag and placed gently into the vessel carefully as not to touch the outside of the vessel.
  • the dip tube was lowered to where the glass frit was positioned at half the height of the wheel from the bottom of the vessel.
  • the dip tube was threaded into the port.
  • the pH probe port (back right port) was carefully unscrewed and was placed to the side.
  • the 40 ⁇ m dip tube was unpacked from the autoclave bag and placed gently into the vessel carefully as not to touch the outside of the vessel.
  • the dip tube was lowered to where the glass frit was positioned above the bridge that holds the wheel, at 3/4 the height of the wheel from the bottom of the vessel.
  • the dip tube was threaded into the port.
  • the vessel was transferred out of the BSC and moved towards the PBS 3MAG control tower.
  • the PBS 3MAG vessel was inserted into the holder in the front and the vessel bottom was carefully ensured to be in contact with the heating pad on the bottom.
  • the temperature probe was inserted into the thermocouple holder.
  • the dissolved oxygen (DO) cable was connected to the probe. k.
  • the main gas and O2 gas lines from the vessel were attached to the 3MAG connectors.
  • 124 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 l.
  • the exhaust filter was installed to the filter oven and the condensation bag was placed appropriately.
  • m. A 2 L bottle was welded to each dip tube line to collect waste.
  • a 2 L bottle of DMEM media was welded to the harvest line.
  • a masterflex pump was used to drain or pump in media.
  • the harvest line was clamped once the media from the bag was completely transferred. q.
  • the volume was split up into a first bottle or bag containing 1600 mL DMEM/F12 + 400 mL StemFit for Differentiation media (2 L total volume) and a second bottle or bag containing 1120 mL DMEM/F12 + 280 mL StemFit for Differentiation media (1.4 L total volume).
  • the empty waste bottle was welded onto the 40 ⁇ m dip tube waste line.
  • media was removed from the reactor through the 40 ⁇ m dip tube line and media was added to the reactor via the harvest line at a rate of 30 mL/min, until 3.4 L of media had been exchanged. i.
  • the exchange was tracked by ensuring the level indicated on the vessel screen remained consistent.
  • the pump was temporarily paused for the harvest line or the dip tube line until the level returned to the original value.
  • approximately 50 mL of STEP 2 medium for days 2-4 was prepared, and the bottle or bag was welded onto the harvest line. Table 37. STEP 2 medium for Days 2-4.
  • Step 4 Medium for Days 6-8.
  • v. Cell count was performed using NC-200.
  • iHSC flow panel is shown in Table 34.
  • Exemplary success criteria for iHSC redifferentiation step purity of >70% Lineage- CD34+ cells and >5 fold expansion per input iPSC.
  • the protocol enabled redifferentiation of ⁇ T-iPSC lines into hematopoietic stem cells (HSCs) at a variety of RPM in a 3L vertical wheel bioreactor using a serum-free, feeder- free (SFFF) three-dimensional (3D) culture method, the results including cell cluster formation, morphology, cell growth (e.g., metabolism), purity, etc. This was true with perfusion in the bioreactor system.
  • SFFF serum-free, feeder- free
  • Figures 3-6 show that the 3D-VW bioreactor protocol for the redifferentiation of iPSCs to iHSCs resulted in high purity, high cell viability, high yield, and sufficient fold expansion.
  • the protocol also yielded i ⁇ T cells at a purity that met the exemplary success criterion and with a fold expansion that is consistent with iHSCs generated with the 2D process.
  • These data demonstrate the efficiency and validity of the 3D-VW bioreactor protocol for the generation of iHSCs from ⁇ T-iPSCs as shown in Figures 3-6.
  • List of Embodiments [0406] A nonexhaustive list of embodiments encompassed by the present invention follows.
  • a method of de novo generation of hematopoietic stem cells (iHSCs) derived from human ⁇ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue culture vessel comprising: seeding human ⁇ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; retaining all cells in the culture on day 8 of culture while removing the medium from the culture for about 2 days; and harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction.
  • A2 The method of item A1, wherein the tissue culture vessel has non-traditional bioreactor geometries.
  • A3 The method of item A1 or item A2, wherein the iPSCs are seeded at a density of about 1.0 x 10 5 cells/mL to about 1.8 x 10 5 cells/mL.
  • the tissue culture vessel comprising a vertical wheel further comprises a first medium comprising StemFit ® Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, Emricasan, Polyamines, and Trans ⁇ ISRIB (CEPT) cocktail, wherein the BMP4 and the rhVEGF are present in a ratio of 1:1, and wherein the first medium does not comprise antibiotics.
  • a first medium comprising StemFit ® Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chr
  • A6 The method of any one of items A1-A5, wherein the iPSCs are dissociated into single cells on day 7 of culture.
  • A7 The method of any one of items A1-A6, wherein the step of retaining all cells comprises retaining clusters, semi-clusters, and single cells.
  • the step of retaining all cells comprises removing spent medium from the tissue culture vessel comprising a vertical wheel using a 4 ⁇ m cell retention filter and adding fresh medium to the bioreactor over about 1.8 days to about 2.2 days, preferably about 2 days, at a rate of about 0.69 mL/min (1 L/day), until 2 L of media have been exchanged, wherein the fifth medium comprises Advanced DMEM/F12 medium and StemFit ® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, IL-6, TPO, and one of (i) L-glutamine and (ii) GlutaMax TM , wherein the fresh medium does not comprise antibiotics.
  • A9 The method of any one of items A1-A8, wherein the vertical wheel spins at a rate of about 10 rpm to about 60 rpm, preferably about 23 rpm.
  • the step of harvesting comprises: a) removing an entire cell volume comprising EBs and iHSCs from the tissue culture vessel comprising a vertical wheel and centrifuging the entire cell volume, thereby generating a first supernatant and a first cell pellet; b) centrifuging the first supernatant, thereby generating a second supernatant and a second cell pellet, removing the second supernatant, and resuspending the second cell pellet in Advanced DMEM/F12 medium and StemFit ® For Differentiation medium without cytokines, growth factors, or antibiotics; 134 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 c) adding a first volume of 20 mg/m
  • A11 The method of any one of items A1-A10, wherein the cells are cultured under normoxic conditions.
  • A12 The method of any one of items A1-A11, further comprising further purifying and/or isolating the redifferentiated iHSCs.
  • A13 The method of any one of items A1-A12, wherein the redifferentiated iHSCs are isolated differentiated cells or are capable of being further purified and/or isolated.
  • A14 The method of any one of items A1-A10, wherein the cells are cultured under normoxic conditions.
  • determining purity of the redifferentiated iHSCs comprises assaying for one or more of lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a via flow cytometry or an equivalent thereof.
  • lineage markers comprise all of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a.
  • a method of de novo generation of hematopoietic stem cells (iHSCs) derived from human ⁇ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue culture vessel comprising: seeding human ⁇ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction.
  • iHSCs hematopoietic stem cells derived from human ⁇ T cell-derived induced pluripotent stem cells
  • the method of item B1 wherein the tissue culture vessel has non-traditional bioreactor geometries.
  • B3 The method of item B1 or B2, wherein the iPSCs are seeded at a density of about 1.0 x 10 5 cells/mL to about 1.8 x 10 5 cells/mL.
  • the tissue culture vessel comprising a vertical wheel further comprises a first medium comprising StemFit ® Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, Emricasan, Polyamines, and Trans ⁇ ISRIB (CEPT) cocktail, wherein the BMP4 and the rhVEGF are present in a ratio of 1:1, and wherein the first medium does not comprise antibiotics.
  • any one of items B1-B5, wherein the step of exchanging the spent medium on days 2, 4, and 6 of culture comprises: i) allowing cell aggregates to settle in the tissue culture vessel comprising a vertical wheel for about 3 to about 5 minutes; collecting 80% of the spent medium from the tissue culture vessel comprising a vertical wheel; centrifuging the collected medium, thereby obtaining a cell pellet; resuspending the cell pellet in StemFit® for Differentiation medium, thereby obtaining a cell suspension; and adding the cell suspension and fresh medium to the tissue culture vessel comprising a vertical wheel, or ii) allowing cell aggregates to settle in the tissue culture vessel comprising a vertical wheel for about 3 to about 5 minutes; collecting 80% of the spent medium from the tissue culture vessel comprising a vertical wheel; centrifuging the collected medium, thereby obtaining a first cell pellet and a first supernatant; resuspending the first cell pellet in StemFit® for Differentiation medium and adding the resuspended first
  • any one of items B1-B6, wherein the fresh medium added on day 2 of culture comprises: i) Advanced DMEM/F12 medium and StemFit ® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SB431542 (inhibitor of Activin/BMP/TGF ⁇ pathway), recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), and one of L-glutamine and GlutaMax TM supplement, or ii) StemPro TM -34 Serum-free Medium (SFM) and StemProTM-34 Nutrient Supplement in 137 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 a 500:13 ratio, ascorbic acid 2-phosphate, Insulin-Transferrin-Selenium (ITS-G), monothiog
  • B8 The method of any one of items B1-B7, wherein the iPSCs are dissociated into single cells on day 7 of culture.
  • B9. The method of any one of items B1-B8, wherein the step of harvesting redifferentiated iHSCs comprises retaining clusters, semi-clusters, and single cells.
  • B10. The method of any one of items B1-B9, wherein the step of harvesting comprises retaining all cells in the tissue culture vessel comprising a vertical wheel.
  • B11 The method of any one of items B1-B10, wherein the vertical wheel spins at a rate of about 10 rpm to about 60 rpm, preferably about 40 rpm.
  • the step of harvesting redifferentiated iHSCs comprises: a) removing an entire cell volume comprising embryoid bodies (EBs) and iHSCs from the tissue culture vessel comprising a vertical wheel and centrifuging the entire cell volume, thereby generating a first supernatant and a first cell pellet; b) centrifuging the first supernatant, thereby generating a second supernatant and a second cell pellet and removing the second supernatant; c) resuspending the first cell pellet and the second cell pellet in Advanced DMEM/F12 medium without cytokines, growth factors, or antibiotics; d) adding a volume of 20 mg/mL collagenase II to the first cell pellet, thereby generating a first cell suspension; e) adding a volume of Advanced DMEM/F12 medium without cytokines, growth factors, or antibiotics equal to the volume of the first cell suspension to the first cell suspension; f
  • B13 The method of any one of items B1-B12, wherein the cells are cultured under normoxic conditions.
  • B14. The method of any one of items B1-B13, further comprising further purifying and/or isolating the redifferentiated iHSCs.
  • B15. The method of any one of items B1-B14, wherein the redifferentiated iHSCs are isolated differentiated cells or are capable of being further purified and/or isolated.
  • B16 The method of any one of items B1-B12, wherein the cells are cultured under normoxic conditions.
  • determining purity of the redifferentiated iHSCs comprises assaying for one or more of lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a via flow cytometry or an equivalent thereof.
  • lineage markers comprise all of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a.
  • iHSCs hematopoietic stem cells
  • iPSCs human ⁇ T cell-derived induced pluripotent stem cells
  • One or more cells obtained from a method of de novo generation of hematopoietic stem cells (iHSCs) derived from human ⁇ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue culture vessel comprising: seeding human ⁇ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; 139 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; retaining all cells in the culture on day 8 of culture while removing the medium from the culture for about 2 days; and harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction.
  • iHSCs
  • tissue culture vessel comprising a vertical wheel further comprises a first medium comprising StemFit ® Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, Emricasan, Polyamines, and Trans ⁇ ISRIB (CEPT) cocktail, wherein the BMP4 and the rhVEGF are present in a ratio of 1:1, and wherein the first medium does not comprise antibiotics.
  • a first medium comprising StemFit ® Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chro
  • the method further comprises exchanging spent medium in the tissue culture vessel comprising a vertical wheel on each of days 2, 4, and 6 of culture for the about 8 days, wherein the step of exchanging spent medium comprises: removing the spent medium from the tissue culture vessel comprising a vertical wheel using a 40 ⁇ m cell retention filter and adding a wash medium at a pump flow rate of 30 mL/min, the wash medium comprising Advanced DMEM/F12 medium and StemFit ® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio; exchanging about 3 L to about 3.5 L of the wash medium in the bioreactor and removing 0.1 L of the wash medium from the tissue culture vessel comprising a vertical wheel using the 40 ⁇ m cell retention filter; and 140 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 adding about 50 mL of the wash medium and about 50 mL of
  • C6 The one or more cells of any one of items C1-C5, wherein the iPSCs are dissociated into single cells on day 7 of culture.
  • C7 The one or more cells of any one of items C1-C6, wherein the step of retaining all cells comprises retaining clusters, semi-clusters, and single cells.
  • the one or more cells of any one of items C1-C7 wherein the step of retaining all cells comprises removing spent medium from the tissue culture vessel comprising a vertical wheel using a 4 ⁇ m cell retention filter and adding fresh medium to the bioreactor over about 1.8 days to about 2.2 days, preferably about 2 days, at a rate of about 0.69 mL/min (1 L/day), until 2 L of media have been exchanged, wherein the fifth medium comprises Advanced DMEM/F12 medium and StemFit ® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, IL-6, TPO, and one of (i) L-glutamine and (ii) GlutaMax TM , wherein the fresh medium does not comprise antibiotics.
  • C11 The one or more cells of any one of items C1-C10, wherein the cells are cultured under normoxic conditions.
  • C12 The one or more cells of any one of items C1-C11, further comprising further purifying and/or isolating the redifferentiated iHSCs.
  • C13 The one or more cells of any one of items C1-C12, wherein the redifferentiated iHSCs are isolated differentiated cells or are capable of being further purified and/or isolated.
  • determining purity of the redifferentiated iHSCs comprises assaying for one or more of lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a via flow cytometry or an equivalent thereof.
  • the one or more cells of item C14, wherein the lineage markers comprise all of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a.
  • One or more cells obtained from a method of de novo generation of hematopoietic stem cells (iHSCs) derived from human ⁇ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue culture vessel comprising: seeding human ⁇ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; 142 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction.
  • iHSCs hematopoietic stem cells
  • iPSCs hematopoietic stem cells
  • D2 The one or more cells of item D1, wherein the tissue culture vessel has non-traditional bioreactor geometries.
  • D3. The one or more cells of item D1 or D2, wherein the iPSCs are seeded at a density of about 1.0 x 10 5 cells/mL to about 1.8 x 10 5 cells/mL. D4.
  • tissue culture vessel comprising a vertical wheel further comprises a first medium comprising StemFit ® Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, Emricasan, Polyamines, and Trans ⁇ ISRIB (CEPT) cocktail, wherein the BMP4 and the rhVEGF are present in a ratio of 1:1, and wherein the first medium does not comprise antibiotics.
  • a first medium comprising StemFit ® Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chro
  • the method further comprises exchanging spent medium in the tissue culture vessel comprising a vertical wheel on each of days 2, 4, 6, and 8 of culture for the about 8 days, wherein the step of exchanging the spent medium comprises: a) allowing cell aggregates to settle in the tissue culture vessel comprising a vertical wheel for about 3 to about 5 minutes; b) collecting 80% of the spent medium from the tissue culture vessel comprising a vertical wheel; c) centrifuging the collected medium, thereby obtaining a cell pellet; d) resuspending the cell pellet in StemFit® for Differentiation, thereby obtaining a cell suspension; and e) adding the cell suspension and fresh medium to the tissue culture vessel comprising 143 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 a vertical wheel.
  • the one or more cells of any one of items D1-D5, wherein the step of exchanging the spent medium on days 2, 4, and 6 of culture comprises: i) allowing cell aggregates to settle in the tissue culture vessel comprising a vertical wheel for about 3 to about 5 minutes; collecting 80% of the spent medium from the tissue culture vessel comprising a vertical wheel; centrifuging the collected medium, thereby obtaining a cell pellet; resuspending the cell pellet in StemFit® for Differentiation medium, thereby obtaining a cell suspension; and adding the cell suspension and fresh medium to the tissue culture vessel comprising a vertical wheel, or ii) allowing cell aggregates to settle in the tissue culture vessel comprising a vertical wheel for about 3 to about 5 minutes; collecting 80% of the spent medium from the tissue culture vessel comprising a vertical wheel; centrifuging the collected medium, thereby obtaining a first cell pellet and a first supernatant; resuspending the first cell pellet in StemFit® for Differentiation medium and adding the resus
  • the one or more cells of any one of items D1-D6, wherein the fresh medium added on day 2 of culture comprises: i) Advanced DMEM/F12 medium and StemFit ® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SB431542 (inhibitor of Activin/BMP/TGF ⁇ pathway), recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), and one of L-glutamine and GlutaMax TM supplement, or ii) StemPro TM -34 Serum-free Medium (SFM) and StemProTM-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, Insulin-Transferrin-Selenium (ITS-G), monothioglycerol, SB431542, recombinant human vascular endothelial growth factor (r
  • D8 The one or more cells of any one of items D1-D7, wherein the iPSCs are dissociated into single cells on day 7 of culture.
  • D9 The one or more cells of any one of items D1-D8, wherein the step of harvesting redifferentiated iHSCs comprises retaining clusters, semi-clusters, and single cells.
  • the one or more cells of any one of D1-D9 wherein the step of retaining all cells comprises removing spent medium from the tissue culture vessel comprising a vertical wheel using a 4 ⁇ m cell retention filter and adding fresh medium to the bioreactor over about 1.8 days to about 2.2 days, preferably about 2 days, at a rate of about 0.69 mL/min (1 L/day), until 2 L of media have been exchanged, wherein the fifth medium comprises Advanced DMEM/F12 medium and StemFit ® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, IL-6, TPO, and one of (i) L-glutamine and (ii) GlutaMax TM , wherein the fresh medium does not comprise antibiotics.
  • D13 The one or more cells of any one of items D1-D12, wherein the cells are cultured under normoxic conditions.
  • D14 The one or more cells of any one of items D1-D13, further comprising further purifying and/or isolating the redifferentiated iHSCs.
  • D15 The one or more cells of any one of claims D1-D14, wherein the redifferentiated iHSCs are isolated differentiated cells or are capable of being further purified and/or isolated.
  • D16 The one or more cells of any one of items D1-D15, wherein the one or more cells have high purity, viability, fold expansion, and potential to redifferentiate into immune effectors, including iPSC-derived ⁇ (i ⁇ ) T cells.
  • D17 The one or more cells of any one of items D1-D16, wherein the step of determining purity of the iHSCs comprises assaying for one or more, optionally all, of the lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a via flow cytometry or an equivalent thereof.
  • D18 The one or more cells of any one of items D1-D17, wherein the lineage markers comprise one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a.
  • E1. A composition comprising the one or more cells of any one of items C1-D18.
  • a method of de novo generation of hematopoietic stem cells (iHSCs) derived from human ⁇ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue culture vessel comprising: a means for seeding human ⁇ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; a means for redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; a means for retaining all cells in the culture on day 8 of culture while removing the medium from the culture for about 2 days; and a means for harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction.
  • iHSCs hematopoietic stem cells derived from human ⁇ T cell-derived
  • a method of de novo generation of hematopoietic stem cells (iHSCs) derived from human ⁇ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue culture vessel comprising: a means for seeding human ⁇ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; a means for redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; a means for harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction.
  • iHSCs hematopoietic stem cells derived from human ⁇ T cell-derived induced pluripotent stem cells
  • iHSCs hematopoietic stem cells
  • iPSCs human ⁇ T cell-derived induced pluripotent stem cells
  • One or more cells obtained from a method of de novo generation of hematopoietic stem cells (iHSCs) derived from human ⁇ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue culture vessel comprising: a means for seeding human ⁇ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; a means for redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; a means for harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction.
  • iHSCs hematopoietic stem cells
  • iPSCs hematopoietic stem cells
  • a method of de novo generation of induced pluripotent stem cell (iPSC)-derived hematopoietic stem cells (iHSCs) from ⁇ T cell-derived iPSCs ( ⁇ T-iPSCs) under serum-free, feeder-free culture conditions comprising one or more of the following steps: i) obtaining one or more human induced pluripotent stem cells (iPSCs), and wherein the method further comprises one or more of the following steps: a) after incubating the iPSCs in a tissue culture vessel comprising a vertical wheel in a first medium with agitation at about 10-60 rpm, preferably 40 rpm, for about 1.8 to about 2.2 days, preferably about 2 days, removing an entire cell volume from the tissue 148 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 culture vessel comprising a vertical wheel and centrifuging the entire cell volume, wherein the first medium comprises StemFit ® Basic
  • a method of de novo generation of induced pluripotent stem cell (iPSC)-derived hematopoietic stem cells (iHSCs) from ⁇ T cell-derived iPSCs ( ⁇ T-iPSCs) under serum-free, feeder-free culture conditions comprising one or more of the following steps: i) obtaining one or more human induced pluripotent stem cells (iPSCs), and wherein the method further comprises one or more of the following steps: a) seeding the iPSCs, wherein the seeding is performed by suspending the iPSCs in a first medium into a tissue culture vessel comprising a vertical wheel, thereby producing a first cell suspension, wherein the first medium comprises one of StemFit ® Basic 04 medium and StemFit ® Basic 03 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rh
  • a method of de novo generation of induced pluripotent stem cell (iPSC)-derived hematopoietic stem cells (iHSCs) from ⁇ T cell-derived iPSCs ( ⁇ T-iPSCs) under serum-free, feeder-free culture conditions comprising one or more of the following steps: i. obtaining one or more human induced pluripotent stem cells (iPSCs), and wherein the method further comprises one or more of the following steps: a.
  • the seeding is performed by suspending the iPSCs in a first medium into a bioreactor, wherein the bioreactor is a vertical-wheel bioreactor, thereby producing a first cell suspension
  • the first medium comprises one of StemFit ® Basic 04 medium and StemFit ® Basic 03 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, Emricasan, Polyamines, and Trans ⁇ ISRIB (CEPT) cocktail, and wherein the first medium does not comprise antibiotics; b.
  • the first cell suspension in the bioreactor at about 10-60 rpm, preferably 40 rpm, wherein the first cell suspension is cultured and agitated for about 1.8 to about 2.2 days, preferably about 2 days, wherein embryoid bodies (EBs) are generated; c. centrifuging the first cell suspension and thereby generating a first cell pellet; d. resuspending the first cell pellet into a second medium to form a second cell suspension, wherein the second medium comprises: i.
  • Advanced DMEM/F12 medium and StemFit ® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SB431542 (inhibitor of Activin/BMP/TGF ⁇ pathway), rhVEGF, basic fibroblast growth factor (bFGF), stem cell factor (SCF), and one of L-glutamine and GlutaMax TM supplement, or ii.
  • SB431542 inhibitor of Activin/BMP/TGF ⁇ pathway
  • rhVEGF basic fibroblast growth factor
  • bFGF basic fibroblast growth factor
  • SCF stem cell factor
  • L-glutamine and GlutaMax TM supplement or ii.
  • StemPro TM -34 Serum-free Medium (SFM) and StemProTM-34 156 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2- phosphate, Insulin-Transferrin-Selenium (ITS-G), monothioglycerol, SB431542, rhVEGF, bFGF, SCF, and one of L-glutamine and GlutaMax TM , and wherein the second medium does not comprise antibiotics; e. culturing and agitating the second cell suspension in the bioreactor for about 1.8 to about 2.2 days, preferably about 2 days; f.
  • Advanced DMEM/F12 medium and StemFit ® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), interleukin- 3 (IL-3), interleukin-6 (IL-6), one of (i) thrombopoietin (TPO) and (ii) erythropoietin (EPO), and one of (i) L-glutamine and (ii) GlutaMax TM , or ii.
  • SCF SCF
  • rhVEGF FMS-like tyrosine kinase 3 ligand
  • Flt3L FMS-like tyrosine kinase 3 ligand
  • IL-3 interleukin- 3
  • IL-6 interleukin-6
  • TPO thrombopoie
  • Advanced DMEM/F12 medium and StemFit ® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, IL-6, one of (i) TPO and (ii) EPO, and one of (i) L-glutamine and (ii) GlutaMax TM , or ii.
  • StemProTM-34 SFM and StemProTM-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, IL-6, one of (i) TPO and (ii) EPO, and one of (i) L-glutamine and (ii) GlutaMax TM , and wherein the fourth medium does not comprise antibiotics; o. placing the fifth cell suspension into the bioreactor with the remaining fourth cell suspension thereby forming a sixth cell suspension; p. culturing and agitating the sixth cell suspension in the bioreactor for about 1.8 to about 2.2 days, preferably about 2 days; q. repeating steps l) to p); r.
  • the fifth medium comprises Advanced DMEM/F12 medium, wherein the fifth medium does not comprise cytokines, antibiotics or growth factors, and wherein cells of the ninth cell suspension comprise iHSCs, which are defined as CD34+ and lineage marker-negative, where the lineage markers comprise one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a; u.
  • step r resuspending the fourth cell pellet of step r), in a volume of about 20 mg/mL collagenase type II, thereby generating a tenth cell suspension; v. adding the fifth medium to the tenth cell suspension; w. centrifuging the tenth cell suspension, thereby generating a sixth cell pellet, and adding TrypLE to the sixth cell pellet, thereby generating an eleventh cell suspension; x. adding additional fifth medium to the eleventh cell suspension and mixing; y. centrifuging the eleventh cell suspension to form a seventh cell pellet and resuspending the seventh cell pellet in additional fifth medium to form a twelfth cell suspension; z.
  • iHSCs wherein the cell strainer is 70 ⁇ m mesh size; aa. determining the purity of the iHSCs from the first supernatant of step t) and the iHSCs from step z), said purity defined as CD34+ and lineage marker-negative, where the lineage markers comprise one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a; bb. not introducing serum in any of steps a)-aa); cc.
  • O7 The method of any one of embodiments O1-O6, wherein any one of steps a)-cc) comprises gently dispersing the cells using a pipet or equivalent thereof.
  • the method of any one of claims O1-O13, wherein the medium in steps d), i), n), or t) comprises GlutaMax TM . O16.
  • the method of any one of embodiments O1-O15, wherein the medium in steps i) or n) comprises TPO.
  • the method of any one of embodiments O1-O15, wherein the medium in steps i) or n) comprises EPO.
  • O18 The method of any one of embodiments O1-O17, wherein the centrifugation of any one of the steps is performed at 100g for 1 minute at brake 6. O19.
  • the method of any one of embodiments O1-O18, wherein the centrifugation of any one of the steps is performed at 300g for 5 minutes. O20.
  • the method of any one of embodiments O1-O20, wherein the centrifugation in step r) is performed twice, first at 100g for 1 minute at brake 6 and second at 300g for 5 minutes.
  • the method of any one of embodiments O1-O21, wherein the tenth cell suspension of step u) is incubated with collagenase for 20 minutes at 37 °C. O23.
  • the method of any one of embodiments O1-O22, wherein the eleventh cell suspension of step w) is incubated with TrypLE for 5 minutes at 37 °C. O24.
  • passing of the twelfth cell suspension in step z) further comprises passing the twelfth cell suspension through a second cell strainer with a 40 ⁇ m mesh size.
  • the method of any one of embodiments O1-O24, wherein the culturing of the iPSC cells, EBs, and iHSCs is performed at 37 °C and 5% CO2.
  • O26. The method of any one of embodiments O1-O25, wherein the bioreactor wheel is rotating at 60 rpm.
  • iPSCs in the first medium are at a concentration of about 80,000 to about 180,000 cells/mL in a volume of about 90 to about 110 mL. O33.
  • O34 The method of any one of embodiments O1-O7, and O10-O31, wherein iPSCs in the first medium are at a concentration of about 5,000 to about 180,000 cells/mL in a volume of about 0.45 to about 0.55 L. O35.
  • One or more cells obtained from a method of de novo generation of iHSCs under serum-free and feeder-free culture conditions comprising: i. obtaining at least one human iPSC clone, and wherein the method further comprises one or more of the following steps: a.
  • redifferentiating the at least one human iPSC clone by culturing the at least one human iPSC clone in a first cell culture medium generating a first cell suspension
  • said first cell culture medium comprises one of StemFit ® Basic 04 medium and StemFit ® Basic 03 medium supplemented with basic fibroblast growth factor (bFGF) to a final concentration of about 100 ng/mL bFGF
  • said first cell culture medium further comprises CHIR99021 (glycogen synthase kinase 162 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, Emricasan, Polyamines, and Trans ⁇ ISRIB (CEPT) cocktail, wherein culturing
  • the second cell culture medium comprises: i. Advanced DMEM/F12 medium and StemFit ® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises one of L-glutamine and GlutaMax TM , SB431542 (inhibitor of Activin/BMP/TGF ⁇ pathway), rhVEGF, bFGF, and stem cell factor (SCF), or ii.
  • Advanced DMEM/F12 medium and StemFit ® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises one of L-glutamine and GlutaMax TM , SB431542 (inhibitor of Activin/BMP/TGF ⁇ pathway), rhVEGF, bFGF, and stem cell factor (SCF), or ii.
  • Advanced DMEM/F12 medium and StemFit ® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, FMS-like tyrosine kinase 3 ligand (Flt3L), interleukin-6 (IL-6), rhVEGF, IL-3, one of (i) thrombopoietin (TPO) and (ii) erythropoietin (EPO), and one of (i) L-glutamine and (ii) GlutaMax TM , or ii.
  • FMS-like tyrosine kinase 3 ligand FMS-like tyrosine kinase 3 ligand
  • IL-6 interleukin-6
  • rhVEGF interleukin-6
  • IL-3 interleukin-6
  • TPO thrombopoietin
  • EPO erythropoie
  • StemProTM-34 SFM and StemProTM-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, rhVEGF, IL-3, IL-6, Flt3L, one of (i) TPO and (ii) EPO, and one of (i) L-glutamine and (ii) GlutaMax TM , and wherein the third medium does not comprise antibiotics, and placing the third cell suspension into the vertical wheel bioreactor with remaining second cell suspension, thereby forming a fourth cell suspension; g. culturing fourth cell suspension in the vertical wheel bioreactor for an additional about 1.8 to about 2.2 days, preferably about 2 days; h.
  • the fourth cell culture medium comprises: i.
  • Advanced DMEM/F12 medium and StemFit ® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, 164 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 IL-6, one of (i) TPO and (ii) EPO, and one of (i) L-glutamine and (ii) GlutaMax TM , or ii.
  • StemProTM-34 SFM and StemProTM-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, IL-6, one of (i) TPO and (ii) EPO, and one of (i) L-glutamine and (ii) GlutaMax TM , and adding the fifth cell suspension into remaining fourth cell suspension in the vertical wheel bioreactor, thereby generating a sixth cell suspension; k. culturing the sixth cell suspension for about 1.8 to about 2.2 days, preferably about 2 days in the vertical wheel bioreactor; l. repeating steps h) to k); m.
  • centrifuging the eighth cell suspension generating a first supernatant and a fourth cell pellet; n. collecting and centrifuging the first supernatant, thereby generating a fifth cell pellet and resuspending the fifth cell pellet in a fifth medium thereby generating a ninth cell suspension
  • the fifth medium comprises Advanced DMEM/F12 medium, wherein the fifth medium does not comprise cytokines, antibiotics or growth factors
  • cells of the ninth cell suspension comprises iHSCs, which are defined as CD34+ and lineage marker-negative, where the lineage markers comprise one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a; o.
  • step m) resuspending the fourth cell pellet of step m) in a volume of about 20 mg/mL collagenase type II, thereby generating a tenth cell suspension; p. adding the fifth medium to the tenth cell suspension; q. centrifuging the tenth cell suspension, thereby generating a sixth cell pellet, and adding TrypLE to the sixth cell pellet, thereby generating an eleventh cell suspension; r. adding additional fifth medium to the eleventh cell suspension and mixing; 165 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 s.
  • P5. The one or more cells of embodiments P1-P4, further comprising further purifying and/or isolating the iHSCs.
  • P6. The one or more cells of any one of embodiments P1-P5, wherein the obtained iHSCs are isolated redifferentiated cells or are capable of being further purified and/or isolated.
  • 166 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 P9.
  • the one or more cells of any one of embodiments P1-P10, wherein the medium of step a) further comprises a ROCK inhibitor.
  • the one or more cells of any one of embodiments P1-P14, wherein the medium in steps b), f), j), or n) comprises L-glutamine.
  • the one or more cells of any one of embodiments P1-P14, wherein the medium in steps b), f), j), or n) comprises GlutaMax TM .
  • the one or more cells of any one of embodiments P1-P16, wherein the medium in steps f) or j) comprises TPO.
  • the one or more cells of any one of embodiments P1-P16, wherein the medium in steps f) or j) comprises EPO. P19.
  • the one or more cells of any one of embodiments P1-P18, wherein the centrifugation of any one of the steps is performed at 100g for 1 minute at brake 6.
  • P20 The one or more cells of any one of embodiments P1-P19, wherein the centrifugation of any one of the steps is performed at 300g for 5 minutes.
  • P21 The one or more cells of any one of embodiments P1-P20, wherein the centrifugation of any one of the steps is performed at 300g for 3 minutes.
  • the one or more cells of any one of embodiments P1-P21, wherein the centrifugation in step l) is performed twice, first at 100g for 1 minute at brake 6 and second at 300g for 5 minutes. P23.
  • the one or more cells of any one of embodiments P1-P24, wherein passing of the twelfth cell suspension in step t) further comprises passing the twelfth cell suspension through a second cell strainer with a 40 ⁇ m mesh size.
  • P26 The one or more cells of any one of embodiments P1-P25, wherein the culturing of the iPSC cells, EBs, and iHSCs is performed at 37 °C and 5% CO 2 . P27. The one or more cells of any one of embodiments P1-P26, wherein the bioreactor wheel is rotating at 60 rpm. P28. The one or more cells of any one of embodiments P1-P26, wherein the bioreactor wheel is rotating at 30 rpm. P29. The one or more cells of any one of embodiments P1-P26, wherein the bioreactor wheel is rotating at 24 rpm. P30.
  • P33. The one or more cells of any one of embodiments P1-P8, and P11-P32, wherein iPSCs of the first medium are at a concentration of about 80,000 to about 180,000 cells/mL in a volume of about 90 to about 110 mL. P34.
  • 168 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 P37.
  • the one or more cells of any one of embodiments P1-P8, and P11-P32, wherein iPSCs of the first medium are at a concentration of about 5,000 to about 180,000 cells/mL in a volume of about 10 to about 20 L.
  • Q1. A composition comprising the one or more cells of any one of embodiments P1-P38. R1.

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Abstract

Methods and materials for the cellular redifferentiation of induced pluripotent stem cell (iPSC)-derived hematopoietic stem cells (iHSCs) comprising a scalable, 3D method using a vertical-wheel bioreactor.

Description

Attorney Docket No: 253505.000510 / JBI6830WOPCT1 METHODS AND MATERIALS FOR SCALABLE 3D CELLULAR REDIFFERENTIATION CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to U.S. Provisional Application No. 63/685,394, filed on August 21, 2024; U.S. Provisional Application No. 63/650,039, filed on July 29, 2024; and U.S. Provisional Application No. 63/539,993, filed on September 22, 2023, the disclosures of all of which are herein incorporated by reference in their entirety. FIELD OF THE INVENTION [0002] The present invention teaches methods and materials for the cellular redifferentiation of induced pluripotent stem cell (iPSC)-derived hematopoietic stem cells (e.g., iHSCs), which are useful in, inter alia, generating gamma delta (γδ) T cells, derived from such iPSCs. The methods and materials of the present invention comprise a scalable, three-dimensional (3D) method using a vertical-wheel bioreactor to redifferentiate iHSCs for manufacture of iPSC-derived cell therapy products. BACKGROUND [0003] Immune cell therapy, such as T cell immunotherapy, including autologous chimeric antigen receptor (CAR) T cell therapies, has been reported to be an efficacious therapy for the treatment of some diseases, such as some cancers, including some hematologic malignancies. However, applications of cell therapy to treat multiple cancer types has met numerous and varied challenges (see, e.g., Advances and challenges of CAR T therapy and suitability of animal models (Review), Authors: Xavier E. Ramos-Cardona Weichuan Luo Sulma I. Mohammed; Published online on: July 12, 2022). SUMMARY OF THE INVENTION [0004] Advances in T cell immunotherapy have been reported, such as in autologous CAR T cell therapy; however, applications of cell therapy to treat multiple cancer types has unmet challenges and the present invention, against this backdrop, meets many of the challenges that impede access to therapeutic cell products, such as for example prohibitive, high manufacturing 1 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 costs, increased complexity, lack of a consistent and scalable manufacturing process, etc. For example, the inventors of the present invention use iPSCs, which can undergo inter alia self- renewal, gene editing, multilineage differentiation, etc., to advance off-the-shelf, allogeneic, CAR- engineered T cell therapy platforms with more commercially viable, automated, energy efficient, and resource sparing manufacturing processes. For example, an iPSC-derived cell therapy platform in accordance with the present invention is based on production of γδ T cells, which possess intrinsic antitumor activity and whose cellular tumor infiltration is associated with more favorable outcomes. There is therefore a need in the art for methods of efficiently redifferentiating iPSCs, derived from γδ T cells, into iPSC-derived HSCs (iHSCs) as a source for manufacturing robust allogeneic γδ T cells in sufficient numbers. Such iHSCs are of sufficient purity and fold expansion, and as iHSCs, exhibit high T cell redifferentiation potential for use for T cell-based products, as well as γδ T cells that have superior increased purity and fold expansion properties and are more readily made and used. The present invention is directed to such improved methods and materials. [0005] The inventors address a need in the art for methods of generating iHSCs from iPSCs with a scalable method using a 3D vertical wheel (3D-VW) bioreactor and without the use of, for example, serum or additional cells (e.g., feeder or stromal cells). This method results in iHSCs with high purity, viability, fold expansion, and potential to redifferentiate into immune effector cells, including lymphocytes such as γδ T cells, specifically iPSC-derived γδ (iγδ) T cells. These unexpected results met or exceeded the estimated outcomes for yield, purity, viability, fold expansion, and potential to redifferentiate based on a research-scale 2D culture system. The present application addresses these and other needs. [0006] In an aspect, provided herein is a method of de novo generation of hematopoietic stem cells (iHSCs) derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue culture vessel, said method comprising: seeding human γδ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; redifferentiating the iPSCs to iHSCs by the timely addition of key growth factors and small molecules and by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; retaining all cells in the culture on day 8 of culture while removing the medium from the culture for about 2 days; and harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction. 2 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 [0007] In some embodiments, the tissue culture vessel has non-traditional bioreactor geometries. [0008] In some embodiments, the iPSCs are seeded at a density of about 1.0 x 105 cells/mL to about 1.8 x 105 cells/mL. [0009] In some embodiments, the tissue culture vessel comprising a vertical wheel further comprises a first medium comprising StemFit® Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, Emricasan, Polyamines, and Trans‑ISRIB (CEPT) cocktail, wherein the BMP4 and the rhVEGF are present in a ratio of 1:1, and wherein the first medium does not comprise antibiotics. [0010] In some embodiments, the method further comprises exchanging spent medium in the tissue culture vessel comprising a vertical wheel on each of days 2, 4, and 6 of culture for the about 8 days, wherein the step of exchanging spent medium comprises: removing the spent medium from the tissue culture vessel comprising a vertical wheel using a 40 µm cell retention filter and adding a wash medium at a pump flow rate of 30 mL/min, the wash medium comprising Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio; exchanging about 3 L to about 3.5 L of the wash medium in the bioreactor and removing 0.1 L of the wash medium from the tissue culture vessel comprising a vertical wheel using the 40 µm cell retention filter; and adding about 50 mL of the wash medium and about 50 mL of a medium to from the tissue culture vessel comprising the vertical wheel. [0011] In some embodiments, the iPSCs are dissociated into single cells on day 7 of culture. [0012] In some embodiments, the step of retaining all cells comprises retaining clusters, semi- clusters, and single cells. [0013] In some embodiments, the step of retaining all cells comprises removing spent medium from the tissue culture vessel comprising a vertical wheel using a 4 µm cell retention filter and adding fresh medium to the bioreactor over about 1.8 days to about 2.2 days, preferably about 2 days, at a rate of about 0.69 mL/min (1 L/day), until 2 L of media have been exchanged, wherein the fifth medium comprises Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, IL-6, TPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, wherein the fresh medium does not comprise antibiotics. 3 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 [0014] In some embodiments, the vertical wheel spins at a rate of about 10 rpm to about 60 rpm, preferably about 23 rpm. [0015] In some embodiments, the step of harvesting comprises: a) removing an entire cell volume comprising EBs and iHSCs from the tissue culture vessel comprising a vertical wheel and centrifuging the entire cell volume, thereby generating a first supernatant and a first cell pellet; b) centrifuging the first supernatant, thereby generating a second supernatant and a second cell pellet, removing the second supernatant, and resuspending the second cell pellet in Advanced DMEM/F12 medium and StemFit® For Differentiation medium without cytokines, growth factors, or antibiotics; c) adding a first volume of 20 mg/mL collagenase II to the first cell pellet and the second cell pellet, combining the resuspended first cell pellet and the resuspended second cell pellet and adding a second volume of 20 mg/mL collagenase II, thereby generating a combined cell suspension; d) adding a volume of Advanced DMEM/F12 medium and StemFit® For Differentiation medium without cytokines, growth factors, or antibiotics equal to the volume of the combined cell suspension to the combined cell suspension; e) centrifuging the combined cell suspension, thereby generating a third cell pellet; f) resuspending the third cell pellet in a volume of TrypLE and adding a volume of Advanced DMEM/F12 medium and StemFit® For Differentiation medium without cytokines, growth factors, or antibiotics, thereby generating a fourth cell suspension; g) mixing the fourth cell suspension until clusters of cells are no longer visible; h) optionally passing the fourth cell suspension through a cell strainer to obtain iHSCs, wherein the cell strainer is 70 µm mesh size, if clusters of cells are visible after the mixing step; and i) centrifuging the fourth cell suspension. [0016] In some embodiments, the cells are cultured under normoxic conditions. [0017] In some embodiments, the method further comprises further purifying and/or isolating the redifferentiated iHSCs. [0018] In some embodiments, the redifferentiated iHSCs are isolated differentiated cells or are capable of being further purified and/or isolated. 4 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 [0019] In some embodiments, determining purity of the redifferentiated iHSCs comprises assaying for one or more of lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a via flow cytometry or an equivalent thereof. [0020] In some embodiments, the lineage markers comprise all of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a. [0021] In an aspect, provided herein is a method of de novo generation of hematopoietic stem cells (iHSCs) derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue culture vessel, said method comprising: seeding human γδ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction. [0022] In some embodiments, the tissue culture vessel has non-traditional bioreactor geometries. [0023] In some embodiments, the iPSCs are seeded at a density of about 1.0 x 105 cells/mL to about 1.8 x 105 cells/mL. [0024] In some embodiments, the tissue culture vessel comprising a vertical wheel further comprises a first medium comprising StemFit® Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, Emricasan, Polyamines, and Trans‑ISRIB (CEPT) cocktail, wherein the BMP4 and the rhVEGF are present in a ratio of 1:1, and wherein the first medium does not comprise antibiotics. [0025] In some embodiments, the method further comprises exchanging spent medium in the tissue culture vessel comprising a vertical wheel on each of days 2, 4, 6, and 8 of culture for the about 8 days, wherein the step of exchanging the spent medium comprises: a) allowing cell aggregates to settle in the tissue culture vessel comprising a vertical wheel for about 3 to about 5 minutes; b) collecting 80% of the spent medium from the tissue culture vessel comprising a vertical wheel; c) centrifuging the collected medium, thereby obtaining a cell pellet; d) resuspending the cell pellet in StemFit® for Differentiation, thereby obtaining a cell suspension; and e) adding the cell suspension and fresh medium to the tissue culture vessel comprising a vertical wheel. 5 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 [0026] In some embodiments, the step of exchanging the spent medium on days 2, 4, and 6 of culture comprises: i) allowing cell aggregates to settle in the tissue culture vessel comprising a vertical wheel for about 3 to about 5 minutes; collecting 80% of the spent medium from the tissue culture vessel comprising a vertical wheel; centrifuging the collected medium, thereby obtaining a cell pellet; resuspending the cell pellet in StemFit® for Differentiation medium, thereby obtaining a cell suspension; and adding the cell suspension and fresh medium to the tissue culture vessel comprising a vertical wheel, or ii) allowing cell aggregates to settle in the tissue culture vessel comprising a vertical wheel for about 3 to about 5 minutes; collecting 80% of the spent medium from the tissue culture vessel comprising a vertical wheel; centrifuging the collected medium, thereby obtaining a first cell pellet and a first supernatant; resuspending the first cell pellet in StemFit® for Differentiation medium and adding the resuspended first cell pellet to the tissue culture vessel comprising a vertical wheel; centrifuging the supernatant, thereby obtaining a second cell pellet and a second supernatant; and resuspending the second cell pellet in StemFit® for Differentiation medium and adding the resuspended second cell pellet and fresh medium to the tissue culture vessel comprising a vertical wheel. [0027] In some embodiments, the fresh medium added on day 2 of culture comprises: i) Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SB431542 (inhibitor of Activin/BMP/TGFβ pathway), recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), and one of L-glutamine and GlutaMaxTM supplement, or ii) StemProTM-34 Serum-free Medium (SFM) and StemPro™-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, Insulin-Transferrin-Selenium (ITS-G), monothioglycerol, SB431542, recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), and one of L-glutamine and GlutaMaxTM, wherein the VEGF, bFGF, and SCF are present in a ratio of 1:0.625:0.625, and wherein the second medium does not comprise antibiotics. [0028] In some embodiments, the iPSCs are dissociated into single cells on day 7 of culture. [0029] In some embodiments, the step of harvesting redifferentiated iHSCs comprises retaining clusters, semi-clusters, and single cells. [0030] In some embodiments, the step of harvesting comprises retaining all cells in the tissue culture vessel comprising a vertical wheel. 6 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 [0031] In some embodiments, the vertical wheel spins at a rate of about 10 rpm to about 60 rpm, preferably about 40 rpm. [0032] In some embodiments, the step of harvesting redifferentiated iHSCs comprises: a) removing an entire cell volume comprising embryoid bodies (EBs) and iHSCs from the tissue culture vessel comprising a vertical wheel and centrifuging the entire cell volume, thereby generating a first supernatant and a first cell pellet; b) centrifuging the first supernatant, thereby generating a second supernatant and a second cell pellet and removing the second supernatant; c) resuspending the first cell pellet and the second cell pellet in Advanced DMEM/F12 medium without cytokines, growth factors, or antibiotics; d) adding a volume of 20 mg/mL collagenase II to the first cell pellet, thereby generating a first cell suspension; e) adding a volume of Advanced DMEM/F12 medium without cytokines, growth factors, or antibiotics equal to the volume of the first cell suspension to the first cell suspension; f) centrifuging the first cell suspension, thereby generating a third cell pellet; g) resuspending the third cell pellet in a volume of TrypLE and adding a volume of Advanced DMEM/F12 medium without cytokines, growth factors, or antibiotics, thereby generating a second cell suspension; h) mixing the second cell suspension until clusters of cells are no longer visible; i) optionally passing the second cell suspension through a cell strainer to obtain iHSCs, wherein the cell strainer is 70 µm mesh size, if clusters of cells are visible after the mixing step; and j) centrifuging the second cell suspension. [0033] In some embodiments, the cells are cultured under normoxic conditions. [0034] In some embodiments, the method further comprises further purifying and/or isolating the redifferentiated iHSCs. [0035] In some embodiments, the redifferentiated iHSCs are isolated differentiated cells or are capable of being further purified and/or isolated. [0036] In some embodiments, determining purity of the redifferentiated iHSCs comprises assaying for one or more of lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a via flow cytometry or an equivalent thereof. 7 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 [0037] In some embodiments, the lineage markers comprise all of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a. [0038] In an aspect, provided herein is one or more cells obtained from a method of de novo generation of hematopoietic stem cells (iHSCs) derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions, said method comprising the steps of any of the methods described herein. [0039] In an aspect, provided herein is one or more cells obtained from a method of de novo generation of hematopoietic stem cells (iHSCs) derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue culture vessel, said method comprising: seeding human γδ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; retaining all cells in the culture on day 8 of culture while removing the medium from the culture for about 2 days; and harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction. [0040] In some embodiments, the tissue culture vessel has non-traditional bioreactor geometries. [0041] In some embodiments, the iPSCs are seeded at a density of about 1.0 x 105 cells/mL to about 1.8 x 105 cells/mL. [0042] In some embodiments, the tissue culture vessel comprising a vertical wheel further comprises a first medium comprising StemFit® Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, Emricasan, Polyamines, and Trans‑ISRIB (CEPT) cocktail, wherein the BMP4 and the rhVEGF are present in a ratio of 1:1, and wherein the first medium does not comprise antibiotics. [0043] In some embodiments, the method further comprises exchanging spent medium in the tissue culture vessel comprising a vertical wheel on each of days 2, 4, and 6 of culture for the about 8 days, wherein the step of exchanging spent medium comprises: removing the spent medium from the tissue culture vessel comprising a vertical wheel using a 40 µm cell retention filter and adding a wash medium at a pump flow rate of 30 mL/min, the wash medium comprising Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio; exchanging about 3 L to about 3.5 L of the wash medium in 8 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 the bioreactor and removing 0.1 L of the wash medium from the tissue culture vessel comprising a vertical wheel using the 40 µm cell retention filter; and adding about 50 mL of the wash medium and about 50 mL of a medium to from the tissue culture vessel comprising the vertical wheel. [0044] In some embodiments, the iPSCs are dissociated into single cells on day 7 of culture. [0045] In some embodiments, the step of retaining all cells comprises retaining clusters, semi- clusters, and single cells. [0046] In some embodiments, the step of retaining all cells comprises removing spent medium from the tissue culture vessel comprising a vertical wheel using a 4 µm cell retention filter and adding fresh medium to the bioreactor over about 1.8 days to about 2.2 days, preferably about 2 days, at a rate of about 0.69 mL/min (1 L/day), until 2 L of media have been exchanged, wherein the fifth medium comprises Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, IL-6, TPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, wherein the fresh medium does not comprise antibiotics. [0047] In some embodiments, the vertical wheel spins at a rate of about 10 rpm to about 60 rpm, preferably about 23 rpm. [0048] In some embodiments, the step of harvesting comprises: a) removing an entire cell volume comprising EBs and iHSCs from the tissue culture vessel comprising a vertical wheel and centrifuging the entire cell volume, thereby generating a first supernatant and a first cell pellet; b) centrifuging the first supernatant, thereby generating a second supernatant and a second cell pellet, removing the second supernatant, and resuspending the second cell pellet in Advanced DMEM/F12 medium and StemFit® For Differentiation medium without cytokines, growth factors, or antibiotics; c) adding a first volume of 20 mg/mL collagenase II to the first cell pellet and the second cell pellet, combining the resuspended first cell pellet and the resuspended second cell pellet and adding a second volume of 20 mg/mL collagenase II, thereby generating a combined cell suspension; d) adding a volume of Advanced DMEM/F12 medium and StemFit® For Differentiation medium without cytokines, growth factors, or antibiotics equal to the volume of the combined cell suspension to the combined cell suspension; 9 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 e) centrifuging the combined cell suspension, thereby generating a third cell pellet; f) resuspending the third cell pellet in a volume of TrypLE and adding a volume of Advanced DMEM/F12 medium and StemFit® For Differentiation medium without cytokines, growth factors, or antibiotics, thereby generating a fourth cell suspension; g) mixing the fourth cell suspension until clusters of cells are no longer visible; h) optionally passing the fourth cell suspension through a cell strainer to obtain iHSCs, wherein the cell strainer is 70 µm mesh size, if clusters of cells are visible after the mixing step; and i) centrifuging the fourth cell suspension. [0049] In some embodiments, the cells are cultured under normoxic conditions. [0050] In some embodiments, the method further comprises further purifying and/or isolating the redifferentiated iHSCs. [0051] In some embodiments, the redifferentiated iHSCs are isolated differentiated cells or are capable of being further purified and/or isolated. [0052] In some embodiments, determining purity of the redifferentiated iHSCs comprises assaying for one or more of lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a via flow cytometry or an equivalent thereof. [0053] In some embodiments, the lineage markers comprise all of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a. [0054] In an aspect, provided herein is one or more cells obtained from a method of de novo generation of hematopoietic stem cells (iHSCs) derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue culture vessel, said method comprising: seeding human γδ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction. [0055] In some embodiments, the tissue culture vessel has non-traditional bioreactor geometries. [0056] In some embodiments, the iPSCs are seeded at a density of about 1.0 x 105 cells/mL to about 1.8 x 105 cells/mL. 10 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 [0057] In some embodiments, the tissue culture vessel comprising a vertical wheel further comprises a first medium comprising StemFit® Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, Emricasan, Polyamines, and Trans‑ISRIB (CEPT) cocktail, wherein the BMP4 and the rhVEGF are present in a ratio of 1:1, and wherein the first medium does not comprise antibiotics. [0058] In some embodiments, the method further comprises exchanging spent medium in the tissue culture vessel comprising a vertical wheel on each of days 2, 4, 6, and 8 of culture for the about 8 days, wherein the step of exchanging the spent medium comprises: a) allowing cell aggregates to settle in the tissue culture vessel comprising a vertical wheel for about 3 to about 5 minutes; b) collecting 80% of the spent medium from the tissue culture vessel comprising a vertical wheel; c) centrifuging the collected medium, thereby obtaining a cell pellet; d) resuspending the cell pellet in StemFit® for Differentiation, thereby obtaining a cell suspension; and e) adding the cell suspension and fresh medium to the tissue culture vessel comprising a vertical wheel. [0059] In some embodiments, the step of exchanging the spent medium on days 2, 4, and 6 of culture comprises: i) allowing cell aggregates to settle in the tissue culture vessel comprising a vertical wheel for about 3 to about 5 minutes; collecting 80% of the spent medium from the tissue culture vessel comprising a vertical wheel; centrifuging the collected medium, thereby obtaining a cell pellet; resuspending the cell pellet in StemFit® for Differentiation medium, thereby obtaining a cell suspension; and adding the cell suspension and fresh medium to the tissue culture vessel comprising a vertical wheel, or ii) allowing cell aggregates to settle in the tissue culture vessel comprising a vertical wheel for about 3 to about 5 minutes; collecting 80% of the spent medium from the tissue culture vessel comprising a vertical wheel; centrifuging the collected medium, thereby obtaining a first cell pellet and a first supernatant; resuspending the first cell pellet in StemFit® for Differentiation medium and adding the resuspended first cell pellet to the tissue culture vessel comprising a vertical wheel; centrifuging the supernatant, thereby obtaining a second cell pellet and a second supernatant; and resuspending the second cell pellet in StemFit® for Differentiation medium and adding the resuspended second cell pellet and fresh medium to the tissue culture vessel comprising a vertical wheel. 11 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 [0060] In some embodiments, the fresh medium added on day 2 of culture comprises: i) Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SB431542 (inhibitor of Activin/BMP/TGFβ pathway), recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), and one of L-glutamine and GlutaMaxTM supplement, or ii) StemProTM-34 Serum-free Medium (SFM) and StemPro™-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, Insulin-Transferrin-Selenium (ITS-G), monothioglycerol, SB431542, recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), and one of L- glutamine and GlutaMaxTM, wherein the VEGF, bFGF, and SCF are present in a ratio of 1:0.625:0.625, and wherein the second medium does not comprise antibiotics. [0061] In some embodiments, the iPSCs are dissociated into single cells on day 7 of culture. [0062] In some embodiments, the step of harvesting redifferentiated iHSCs comprises retaining clusters, semi-clusters, and single cells. [0063] In some embodiments, the step of retaining all cells comprises removing spent medium from the tissue culture vessel comprising a vertical wheel using a 4 µm cell retention filter and adding fresh medium to the bioreactor over about 1.8 days to about 2.2 days, preferably about 2 days, at a rate of about 0.69 mL/min (1 L/day), until 2 L of media have been exchanged, wherein the fifth medium comprises Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, IL-6, TPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, wherein the fresh medium does not comprise antibiotics. [0064] In some embodiments, the vertical wheel spins at a rate of about 10 rpm to about 60 rpm, preferably about 40 rpm. [0065] In some embodiments, the step of harvesting redifferentiated iHSCs comprises: a) removing an entire cell volume comprising embryoid bodies (EBs) and iHSCs from the tissue culture vessel comprising a vertical wheel and centrifuging the entire cell volume, thereby generating a first supernatant and a first cell pellet; 12 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 b) centrifuging the first supernatant, thereby generating a second supernatant and a second cell pellet and removing the second supernatant; c) resuspending the first cell pellet and the second cell pellet in Advanced DMEM/F12 medium without cytokines, growth factors, or antibiotics; d) adding a volume of 20 mg/mL collagenase II to the first cell pellet, thereby generating a first cell suspension; e) adding a volume of Advanced DMEM/F12 medium without cytokines, growth factors, or antibiotics equal to the volume of the first cell suspension to the first cell suspension; f) centrifuging the first cell suspension, thereby generating a third cell pellet; g) resuspending the third cell pellet in a volume of TrypLE and adding a volume of Advanced DMEM/F12 medium without cytokines, growth factors, or antibiotics, thereby generating a second cell suspension; h) mixing the second cell suspension until clusters of cells are no longer visible; i) optionally passing the second cell suspension through a cell strainer to obtain iHSCs, wherein the cell strainer is 70 µm mesh size, if clusters of cells are visible after the mixing step; and j) centrifuging the second cell suspension. [0066] In some embodiments, the cells are cultured under normoxic conditions. [0067] In some embodiments, the method further comprises further purifying and/or isolating the redifferentiated iHSCs. [0068] In some embodiments, the redifferentiated iHSCs are isolated differentiated cells or are capable of being further purified and/or isolated. [0069] In some embodiments, the one or more cells have high purity, viability, fold expansion, and potential to redifferentiate into immune effectors, including iPSC-derived γδ (iγδ) T cells. [0070] In some embodiments, the step of determining purity of the iHSCs comprises assaying for one or more, optionally all, of the lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a via flow cytometry or an equivalent thereof. [0071] In some embodiments, the lineage markers comprise one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a. [0072] In an aspect, provided herein is a composition comprising the one or more cells of any one of the embodiments described herein. 13 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 [0073] In an aspect, provided herein is a use of the one or more cells of any one of the embodiments described herein, in preparation of cells for treating a pathology, disease(s), in preparation of lymphocytes, in a bioreactor, in tissue engineering or in vitro drug screening for diseases. [0074] In an aspect, provided herein is a system for performing a method of de novo generation of hematopoietic stem cells (iHSCs) derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions, said method comprising the steps of any one of the embodiments described herein, wherein the vertical wheel is either perpendicular or non-perpendicular to the vessel. [0075] In an aspect, provided herein is a method of de novo generation of hematopoietic stem cells (iHSCs) derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue culture vessel, said method comprising: a means for seeding human γδ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; a means for redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; a means for retaining all cells in the culture on day 8 of culture while removing the medium from the culture for about 2 days; and a means for harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction. [0076] In an aspect, provided herein is a method of de novo generation of hematopoietic stem cells (iHSCs) derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue culture vessel, said method comprising: a means for seeding human γδ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; a means for redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; a means for harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction. [0077] In an aspect, provided herein is an iHSC produced according to any of the methods described herein. [0078] In an aspect, provided herein is one or more cells obtained from a method of de novo generation of hematopoietic stem cells (iHSCs) derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue 14 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 culture vessel, said method comprising: a means for seeding human γδ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; a means for redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; a means for retaining all cells in the culture on day 8 of culture while removing the medium from the culture for about 2 days; and a means for harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction. [0079] In an aspect, provided herein is one or more cells obtained from a method of de novo generation of hematopoietic stem cells (iHSCs) derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue culture vessel, said method comprising: a means for seeding human γδ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; a means for redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; a means for harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction. [0080] In an aspect, provided herein is a method of de novo generation of induced pluripotent stem cell (iPSC)-derived hematopoietic stem cells (iHSCs) from γδ T cell-derived iPSCs (γδ T- iPSCs) under serum-free, feeder-free culture conditions, said method comprising one or more of the following steps: i) obtaining one or more human induced pluripotent stem cells (iPSCs), and wherein the method further comprises one or more of the following steps: a) after incubating the iPSCs in a tissue culture vessel comprising a vertical wheel in a first medium with agitation at about 10-60 rpm, preferably 40 rpm, for about 1.8 to about 2.2 days, preferably about 2 days, removing an entire cell volume from the tissue culture vessel comprising a vertical wheel and centrifuging the entire cell volume, wherein the first medium comprises StemFit® Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF) and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, Emricasan, Polyamines, and Trans‑ISRIB (CEPT) cocktail, wherein the first medium does not comprise antibiotics; 15 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 b) centrifuging the first cell suspension and thereby generating a first cell pellet; c) resuspending the first cell pellet into a second medium to form a second cell suspension, wherein the second medium comprises: i) Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SB431542 (inhibitor of Activin/BMP/TGFβ pathway), recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), and one of L-glutamine and GlutaMaxTM supplement, or ii) StemProTM-34 Serum-free Medium (SFM) and StemPro™-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, Insulin-Transferrin- Selenium (ITS-G), monothioglycerol, SB431542, recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), and one of L-glutamine and GlutaMaxTM, wherein the VEGF, bFGF, and SCF are present in a ratio of 1:0.625:0.625, and wherein the second medium does not comprise antibiotics; d) culturing and agitating the second cell suspension in the tissue culture vessel comprising a vertical wheel at about 10-60 rpm, preferably 40 rpm, for about 1.8 to about 2.2 days, preferably about 2 days; e) stopping the agitating for about 5 minutes, wherein cell aggregates settle in the second cell suspension; f) collecting about 80% of the second cell suspension from a top volume of the second cell suspension, from the tissue culture vessel comprising a vertical wheel; g) centrifuging the collected second cell suspension, thereby forming a second cell pellet and resuspending the second pellet in about 1 mL of one of StemFit® For Differentiation solution and/or StemPro™-34 SFM; h) adding the resuspended second cell pellet into a third medium forming a third cell suspension, wherein the third medium comprises: i) Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), interleukin- 16 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 3 (IL-3), interleukin-6 (IL-6), one of (i) thrombopoietin (TPO) and (ii) erythropoietin (EPO), and one of (i) L-glutamine and (ii) GlutaMaxTM, or ii) StemPro™-34 SFM and StemPro™-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, rhVEGF, IL-3, IL-6, Flt3L, one of (i) TPO and (ii) EPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, and wherein the third medium does not comprise antibiotics; i) placing the third cell suspension into the tissue culture vessel comprising a vertical wheel with remaining second cell suspension forming a fourth cell suspension; j) culturing and agitating the fourth cell suspension in the tissue culture vessel comprising a vertical wheel at about 10-60 rpm, preferably 40 rpm, for about 1.8 to about 2.2 days, preferably about 2 days; k) stopping the agitating for about 5 minutes; wherein cell aggregates settle in the cell suspension; l) collecting about 80% of the fourth cell suspension and centrifuging the collected fourth cell suspension forming a third cell pellet, and resuspending the third cell pellet in about 1 mL of one of StemFit® For Differentiation solution and StemPro™-34 SFM; m) suspending the third cell pellet dissolved in the about 1 mL of one of StemFit® For Differentiation solution and StemPro™-34 SFM into a fourth medium forming a fifth cell suspension, wherein the fourth medium comprises: i) Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, IL-6, one of (i) TPO and (ii) EPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, or ii) StemPro™-34 SFM and StemPro™-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, IL-6, one of (i) TPO and (ii) EPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, and wherein the fourth medium does not comprise antibiotics; n) placing the fifth cell suspension into the tissue culture vessel comprising a vertical wheel with the remaining fourth cell suspension thereby forming a sixth cell suspension; 17 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 o) culturing and agitating the sixth cell suspension in the tissue culture vessel comprising a vertical wheel at about 10-60 rpm, preferably 40 rpm, for about 1.8 to about 2.2 days, preferably about 2 days; p) repeating steps k) to o); optionally wherein the sixth cell suspension is centrifuged and a supernatant from the centrifuged sixth cell suspension is centrifuged and the resulting cell pellet is resuspended in about 1 mL of one of StemFit® For Differentiation solution and StemPro™-34 SFM and combined with a seventh cell suspension obtained by resuspending a cell pellet from the first centrifugation to form an eighth cell suspension before culturing and agitating the eighth cell suspension in the tissue culture vessel comprising a vertical wheel at about 10-60 rpm, preferably 40 rpm, for about 1.8 to about 2.2 days, preferably about 2 days; q) removing all the eighth cell suspension from the tissue culture vessel comprising a vertical wheel and harvesting EBs and iHSCs in the eighth cell suspension by centrifugation, thereby generating a fourth cell pellet and a first supernatant; r) collecting the first supernatant; s) centrifuging the first supernatant, thereby generating a fifth cell pellet and resuspending the fifth cell pellet in a fifth medium thereby generating a ninth cell suspension, wherein the fifth medium comprises Advanced DMEM/F12 medium, wherein the fifth medium does not comprise cytokines, antibiotics, or growth factors, and wherein cells of the ninth cell suspension comprise iHSCs, which are defined as CD34+ and lineage marker-negative, where the lineage markers comprise one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a; t) resuspending the fourth cell pellet of step q), in a volume of about 20 mg/mL collagenase type II, thereby generating a tenth cell suspension; u) adding the fifth medium to the tenth cell suspension; v) centrifuging the tenth cell suspension, thereby generating a sixth cell pellet, and adding TrypLE to the sixth cell pellet, thereby generating an eleventh cell suspension; w) adding additional fifth medium to the eleventh cell suspension and mixing; x) centrifuging the eleventh cell suspension to form a seventh cell pellet and resuspending the seventh cell pellet in additional fifth medium to form a twelfth cell suspension; y) optionally passing the twelfth cell suspension through a cell strainer to obtain iHSCs, 18 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 wherein the cell strainer is 70 µm mesh size, if cell clumps are present in the twelfth cell suspension; z) centrifuging the twelfth cell suspension; aa) not introducing serum in any of steps a)-z); bb) not introducing any additional cells, including feeder cells or stromal cells, in any of steps a)-aa); cc) determining the purity of the iHSCs from the first supernatant of step s) and the iHSCs from step y), said purity defined as CD34+ and lineage marker-negative, where the lineage markers comprise one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a; and dd) obtaining de novo generated iHSCs. [0081] In an aspect, provided herein is a method of de novo generation of induced pluripotent stem cell (iPSC)-derived hematopoietic stem cells (iHSCs) from γδ T cell-derived iPSCs (γδ T- iPSCs) under serum-free, feeder-free culture conditions, said method comprising one or more of the following steps: i) obtaining one or more human induced pluripotent stem cells (iPSCs), and wherein the method further comprises one or more of the following steps: a) seeding the iPSCs, wherein the seeding is performed by suspending the iPSCs in a first medium into a tissue culture vessel comprising a vertical wheel, thereby producing a first cell suspension, wherein the first medium comprises one of StemFit® Basic 04 medium and StemFit® Basic 03 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, Emricasan, Polyamines, and Trans‑ISRIB (CEPT) cocktail, and wherein the first medium does not comprise antibiotics; b) culturing and agitating the cells in the tissue culture vessel comprising a vertical wheel at about 10-60 rpm, preferably 23 rpm, for about 1.8 to about 2.2 days, preferably about 2 days, wherein embryoid bodies (EBs) are generated; c) removing the first medium from the tissue culture vessel comprising a vertical wheel and adding a wash medium comprising Advanced DMEM/F12 medium and StemFit® 19 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, at a rate of about 30 mL/min; d) exchanging about 3L to about 3.5 L of the wash medium in the tissue culture vessel comprising a vertical wheel using a 40 µm cell retention filter at a pump flow rate of 30 mL/min and removing 0.1 L of the wash medium from the tissue culture vessel comprising a vertical wheel; e) adding about 50 mL of the wash medium and about 50 mL of a second medium to the tissue culture vessel comprising a vertical wheel, wherein the second medium comprises Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SB431542 (inhibitor of Activin/BMP/TGFβ pathway), recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), and one of L-glutamine and GlutaMaxTM supplement, wherein the second medium does not comprise antibiotics; f) culturing and agitating the cells in the tissue culture vessel comprising a vertical wheel at about 10-60 rpm, preferably 23 rpm, for about 1.8 to about 2.2 days, preferably about 2 days; g) removing the second medium from the tissue culture vessel comprising a vertical wheel and adding the wash medium at a rate of about 30 mL/min; h) exchanging about 3L to about 3.5 L of the wash medium in the tissue culture vessel comprising a vertical wheel using a 40 µm cell retention filter at a pump flow rate of 30 mL/min and removing 0.1 L of the wash medium from the tissue culture vessel comprising a vertical wheel; i) adding about 50 mL of the wash medium and about 50 mL of a third medium to the tissue culture vessel comprising a vertical wheel, wherein the third medium comprises Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises and further comprises SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), interleukin-3 (IL-3), interleukin-6 (IL-6), thrombopoietin (TPO), and one of (i) L- glutamine and (ii) GlutaMaxTM, wherein the third medium does not comprise antibiotics; 20 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 j) culturing and agitating the cells in the tissue culture vessel comprising a vertical wheel at about 10-60 rpm, preferably 23 rpm, for about 1.8 to about 2.2 days, preferably about 2 days; k) removing the third medium from the tissue culture vessel comprising a vertical wheel and adding the wash medium at a rate of about 30 mL/min; l) exchanging about 3L to about 3.5 L of the wash medium in the tissue culture vessel comprising a vertical wheel using a 40 µm cell retention filter at a pump flow rate of 30 mL/min and removing 0.1 L of the wash medium from the tissue culture vessel comprising a vertical wheel; m) adding about 50 mL of the wash medium and about 50 mL of a fourth medium to the tissue culture vessel comprising a vertical wheel, wherein the fourth medium comprises Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, IL-6, TPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, wherein the fourth medium does not comprise antibiotics; n) culturing and agitating the cells in the tissue culture vessel comprising a vertical wheel at about 10-60 rpm, preferably 23 rpm, for about 1.8 to about 2.2 days, preferably about 2 days; o) simultaneously removing the fourth medium from the tissue culture vessel comprising a vertical wheel and adding a fifth medium to the tissue culture vessel comprising a vertical wheel over about 1.8 days to about 2.2 days, preferably about 2 days, using a 4 µm cell retention filter at a pump flow rate of about 0.69 mL/min (1 L/day), until 2 L of media have been exchanged, wherein the fifth medium comprises Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, IL- 6, TPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, wherein the fifth medium does not comprise antibiotics; p) removing an entire cell volume comprising EBs and iHSCs from the tissue culture vessel comprising a vertical wheel and centrifuging the entire cell volume, thereby generating a first supernatant and a first cell pellet; q) centrifuging the first supernatant, thereby generating a second supernatant and a 21 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 second cell pellet, removing the second supernatant, and resuspending the second cell pellet in Advanced DMEM/F12 medium and StemFit® For Differentiation medium without cytokines, growth factors, or antibiotics; r) adding a first volume of 20 mg/mL collagenase II to the first cell pellet, resuspending the first cell pellet, and adding a second volume of 20 mg/mL collagenase II, thereby generating a first cell suspension; s) adding a volume of Advanced DMEM/F12 medium and StemFit® For Differentiation medium without cytokines, growth factors, or antibiotics equal to the volume of the first cell suspension to the first cell suspension; t) centrifuging the cell suspension, thereby generating a third cell pellet; u) resuspending the third cell pellet in a volume of TrypLE and adding a volume of Advanced DMEM/F12 medium and StemFit® For Differentiation medium without cytokines, growth factors, or antibiotics, thereby generating a second cell suspension; v) mixing the second cell suspension until clusters of cells are no longer visible; w) optionally passing the second cell suspension through a cell strainer to obtain iHSCs, wherein the cell strainer is 70 µm mesh size, if clusters of cells are visible after the mixing step; x) centrifuging the second cell suspension; y) not introducing serum in any of steps a)-x); z) not introducing any additional cells, including feeder cells or stromal cells, in any of steps a)-y); aa) determining the purity of the iHSCs from step w), said purity defined as CD34+ and lineage marker-negative, where the lineage markers comprise one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a; and bb) obtaining de novo generated iHSCs. [0082] In one aspect, provided herein is a method of de novo generation of induced pluripotent stem cell (iPSC)-derived hematopoietic stem cells (iHSCs) from γδ T cell-derived iPSCs (γδ T- iPSCs) under serum-free, feeder-free culture conditions, said method comprising one or more of the following steps: i. obtaining one or more human induced pluripotent stem cells (iPSCs), and wherein the method further comprises one or more of the following steps: 22 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 a. seeding the iPSCs, wherein the seeding is performed by suspending the iPSCs in a first medium into a bioreactor, wherein the bioreactor is a vertical-wheel bioreactor, thereby producing a first cell suspension, wherein the first medium comprises one of StemFit® Basic 04 medium and StemFit® Basic 03 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, Emricasan, Polyamines, and Trans‑ISRIB (CEPT) cocktail, and wherein the first medium does not comprise antibiotics; b. culturing and agitating the first cell suspension in the bioreactor at about 10-60 rpm, preferably 40 rpm, wherein the first cell suspension is cultured and agitated for about 1.8 to about 2.2 days, preferably about 2 days, wherein embryoid bodies (EBs) are generated; c. centrifuging the first cell suspension and thereby generating a first cell pellet; d. resuspending the first cell pellet into a second medium to form a second cell suspension, wherein the second medium comprises: i. Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SB431542 (inhibitor of Activin/BMP/TGFβ pathway), rhVEGF, basic fibroblast growth factor (bFGF), stem cell factor (SCF), and one of L-glutamine and GlutaMaxTM supplement, or ii. StemProTM-34 Serum-free Medium (SFM) and StemPro™-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2- phosphate, Insulin-Transferrin-Selenium (ITS-G), monothioglycerol, SB431542, rhVEGF, bFGF, SCF, and one of L-glutamine and GlutaMaxTM, and wherein the second medium does not comprise antibiotics; e. culturing and agitating the second cell suspension in the bioreactor for 23 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 about 1.8 to about 2.2 days, preferably about 2 days; f. stopping the agitating for about 5 minutes, wherein cell aggregates settle in the second cell suspension; g. collecting about 80% of the second cell suspension from a top volume of the second cell suspension, from the bioreactor; h. centrifuging collected second cell suspension forming a second cell pellet and resuspending the second pellet in about 1 mL of one of StemFit® For Differentiation solution and StemPro™-34 SFM; i. suspending the second cell pellet in the about 1 mL of one of StemFit® For Differentiation solution and or StemPro™-34 SFM into a third medium forming a third cell suspension, wherein the third medium comprises: i. Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), interleukin- 3 (IL-3), interleukin-6 (IL-6), one of (i) thrombopoietin (TPO) and (ii) erythropoietin (EPO), and one of (i) L-glutamine and (ii) GlutaMaxTM, or ii. StemPro™-34 SFM and StemPro™-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, rhVEGF, IL-3, IL-6, Flt3L, one of (i) TPO and (ii) EPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, and wherein the third medium does not comprise antibiotics; j. placing the third cell suspension into the bioreactor with remaining second cell suspension forming a fourth cell suspension; k. culturing and agitating the fourth cell suspension in the bioreactor for about 1.8 to about 2.2 days, preferably about 2 days; l. stopping the agitating for about 5 minutes; wherein cell aggregates settle in the cell suspension; 24 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 m. collecting about 80% of the fourth cell suspension and centrifuging the collected fourth cell suspension forming a third cell pellet, and resuspending the third cell pellet in about 1 mL of one of StemFit® For Differentiation solution and StemPro™-34 SFM; n. suspending the third cell pellet dissolved in the about 1 mL of one of StemFit® For Differentiation solution and StemPro™-34 SFM into a fourth medium forming a fifth cell suspension, wherein the fourth medium comprises: i. Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, IL-6, one of (i) TPO and (ii) EPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, or ii. StemPro™-34 SFM and StemPro™-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, IL-6, one of (i) TPO and (ii) EPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, and wherein the fourth medium does not comprise antibiotics; o. placing the fifth cell suspension into the bioreactor with the remaining fourth cell suspension thereby forming a sixth cell suspension; p. culturing and agitating the sixth cell suspension in the bioreactor for about 1.8 to about 2.2 days, preferably about 2 days; q. repeating steps l) to p); r. removing all the eighth cell suspension from the bioreactor and harvesting EBs and iHSCs in the cell suspension by centrifugation, thereby generating a fourth cell pellet and a first supernatant; s. collecting the first supernatant; t. centrifuging the first supernatant, thereby generating a fifth cell pellet and resuspending the fifth cell pellet in a fifth medium thereby generating a ninth cell suspension, wherein the fifth medium comprises Advanced DMEM/F12 medium, wherein the fifth medium does not 25 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 comprise cytokines, antibiotics or growth factors, and wherein cells of the ninth cell suspension comprise iHSCs, which are defined as CD34+ and lineage marker-negative, where the lineage markers comprise one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a; u. resuspending the fourth cell pellet of step r), in a volume of about 20 mg/mL collagenase type II, thereby generating a tenth cell suspension; v. adding the fifth medium to the tenth cell suspension; w. centrifuging the tenth cell suspension, thereby generating a sixth cell pellet, and adding TrypLE to the sixth cell pellet, thereby generating an eleventh cell suspension; x. adding additional fifth medium to the eleventh cell suspension and mixing; y. centrifuging the eleventh cell suspension to form a seventh cell pellet and resuspending the seventh cell pellet in additional fifth medium to form a twelfth cell suspension; z. passing the twelfth cell suspension through a cell strainer to obtain iHSCs, wherein the cell strainer is 70 µm mesh size; aa. determining the purity of the iHSCs from the first supernatant of step t) and the iHSCs from step z), said purity defined as CD34+ and lineage marker-negative, where the lineage markers comprise one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a; bb. not introducing serum in any of steps a)-aa); cc. not introducing any additional cells, including feeder cells or stromal cells, in any of steps a)-bb); and dd. obtaining de novo generated iHSCs. [0083] In some embodiments, the EB and γδ T cell-derived iPSCs are from a tissue culture vessel. [0084] In some embodiments, the first, second, fourth, sixth, and/or eighth cell suspensions are cultured under normoxic conditions. 26 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 [0085] In some embodiments, the method further comprises further purifying and/or isolating the iHSCs. [0086] In some embodiments, the obtained iHSCs are isolated redifferentiated cells or are capable of being further purified and/or isolated. [0087] In some embodiments, the method further comprises assaying for all of the lineage markers CD3, CD19, CD14, CD11b, CD56, CD20, CD16, CD2, and CD235a via flow cytometry or an equivalent thereof. [0088] In some embodiments, any one of steps a)-cc) comprises gently dispersing the cells using a pipet or equivalent thereof. [0089] In some embodiments, cells of the first medium are at a concentration of about 80,000 to about 180,000 cells/mL in a volume of about 50 to about 70 mL. [0090] In some embodiments, the cells of the first medium are at a concentration of about 150,000 cells/mL in a volume of about 60 mL. [0091] In some embodiments, the medium of step a) further comprises a ROCK inhibitor. [0092] In some embodiments, the medium of step a) further comprises a Chroman 1, Emricasan, Polyamines, and Trans‑ISRIB (CEPT) cocktail. [0093] In some embodiments, after step b) all the cell volume of the first cell suspension is removed from the bioreactor prior to centrifugation. [0094] In some embodiments, the cell morphology is imaged via microscopy, e.g., bright field microscopy. [0095] In some embodiments, the medium in steps d), i), n), or t) comprises L-glutamine. [0096] In some embodiments, the medium in steps d), i), n), or t) comprises GlutaMaxTM. [0097] In some embodiments, the medium in steps i) or n) comprises TPO. [0098] In some embodiments, the medium in steps i) or n) comprises EPO. [0099] In some embodiments, the centrifugation of any one of the steps is performed at 100g for 1 minute at brake 6. [0100] In some embodiments, the centrifugation of any one of the steps is performed at 300g for 5 minutes. [0101] In some embodiments, the centrifugation of any one of the steps is performed at 300g for 3 minutes. [0102] In some embodiments, the centrifugation in step r) is performed twice, first at 100g for 27 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 1 minute at brake 6 and second at 300g for 5 minutes. [0103] In some embodiments, the tenth cell suspension of step u) is incubated with collagenase for 20 minutes at 37 °C. [0104] In some embodiments, the eleventh cell suspension of step w) is incubated with TrypLE for 5 minutes at 37 °C. [0105] In some embodiments, passing of the twelfth cell suspension in step z) further comprises passing the twelfth cell suspension through a second cell strainer with a 40 µm mesh size. [0106] In some embodiments, the culturing of the iPSC cells, EBs, and iHSCs is performed at 37 °C and 5% CO2. [0107] In some embodiments, the bioreactor wheel is rotating at 60 rpm. [0108] In some embodiments, the bioreactor wheel is rotating at 30 rpm. [0109] In some embodiments, the bioreactor wheel is rotating at 24 rpm. [0110] In some embodiments, the bioreactor wheel is rotating at 15 rpm. [0111] In some embodiments, the bioreactor wheel is perpendicular to the bioreactor vessel. [0112] In some embodiments, the bioreactor wheel is not perpendicular to the bioreactor vessel. [0113] In some embodiments, iPSCs in the first medium are at a concentration of about 80,000 to about 180,000 cells/mL in a volume of about 90 to about 110 mL. [0114] In some embodiments, the iPSCs in the first medium are at a concentration of about 150,000 cells/mL in a volume of about 100 mL. [0115] In some embodiments, iPSCs in the first medium are at a concentration of about 5,000 to about 180,000 cells/mL in a volume of about 0.45 to about 0.55 L. [0116] In some embodiments, the iPSCs in the first medium are at a concentration of about 20,000 cells/mL in a volume of about 0.5 L. [0117] In some embodiments, iPSCs in the first medium are at a concentration of about 5,000 to about 180,000 cells/mL in a volume of about 2 to about 5 L. [0118] In some embodiments, iPSCs in the first medium are at a concentration of about 5,000 to about 180,000 cells/mL in a volume of about 10 to about 20 L. [0119] In one aspect, provided herein is one or more cells obtained from a method of de novo generation of iHSCs under serum-free and feeder-free culture conditions, wherein said method 28 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 comprises: i. obtaining at least one human iPSC clone, and wherein the method further comprises one or more of the following steps: a. redifferentiating the at least one human iPSC clone by culturing the at least one human iPSC clone in a first cell culture medium generating a first cell suspension, wherein said first cell culture medium comprises one of StemFit® Basic 04 medium and StemFit® Basic 03 medium supplemented with basic fibroblast growth factor (bFGF) to a final concentration of about 100 ng/mL bFGF, wherein said first cell culture medium further comprises CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, Emricasan, Polyamines, and Trans‑ISRIB (CEPT) cocktail, wherein culturing is performed in a vertical wheel bioreactor at about 10-60 rpm, preferably 40 rpm, for about 1.8 to about 2.2 days, preferably about 2 days; b. centrifuging the first cell suspension comprising iPSCs of step a) to form a first pellet and resuspending the first pellet into a second cell culture medium forming a second cell suspension, wherein said second cell culture medium comprises: i. Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises one of L-glutamine and GlutaMaxTM, SB431542 (inhibitor of Activin/BMP/TGFβ pathway), rhVEGF, bFGF, and stem cell factor (SCF), or ii. StemProTM-34 Serum-free Medium (SFM) and StemPro™-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2- phosphate, Insulin-Transferrin-Selenium (ITS-G), monothioglycerol, SB431542, rhVEGF, bFGF, SCF, and one of L-glutamine and GlutaMaxTM, 29 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 and wherein the second cell culture medium does not comprise antibiotics; c. culturing the second cell suspension comprising iHSCs in the vertical wheel bioreactor at about 10-60 rpm, preferably 40 rpm, for about 1.8 to about 2.2 days, preferably about 2 days; d. stopping the agitation of the vertical wheel bioreactor for about 5 minutes and collecting about 80% of the second cell suspension; e. centrifuging the 80% of the second cell suspension, thereby generating a second cell pellet; f. suspending the second pellet in a third cell culture medium forming a third cell suspension, wherein the third cell culture medium comprises: i. Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, FMS-like tyrosine kinase 3 ligand (Flt3L), interleukin-6 (IL-6), rhVEGF, IL-3, one of (i) thrombopoietin (TPO) and (ii) erythropoietin (EPO), and one of (i) L-glutamine and (ii) GlutaMaxTM, or ii. StemPro™-34 SFM and StemPro™-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, rhVEGF, IL-3, IL-6, Flt3L, one of (i) TPO and (ii) EPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, and wherein the third medium does not comprise antibiotics, and placing the third cell suspension into the vertical wheel bioreactor with remaining second cell suspension, thereby forming a fourth cell suspension; g. culturing fourth cell suspension in the vertical wheel bioreactor for an additional about 1.8 to about 2.2 days, preferably about 2 days; h. stopping the vertical wheel bioreactor for about 5 minutes; 30 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 i. collecting about 80% of the fourth cell suspension and centrifuging the about 80% of the fourth suspension, thereby generating a third cell pellet; j. resuspending the third cell pellet in a fourth cell culture medium forming a fifth cell suspension, wherein the fourth cell culture medium comprises: i. Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, IL-6, one of (i) TPO and (ii) EPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, or ii. StemPro™-34 SFM and StemPro™-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, IL-6, one of (i) TPO and (ii) EPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, and adding the fifth cell suspension into remaining fourth cell suspension in the vertical wheel bioreactor, thereby generating a sixth cell suspension; k. culturing the sixth cell suspension for about 1.8 to about 2.2 days, preferably about 2 days in the vertical wheel bioreactor; l. repeating steps h) to k); m. centrifuging the eighth cell suspension generating a first supernatant and a fourth cell pellet; n. collecting and centrifuging the first supernatant, thereby generating a fifth cell pellet and resuspending the fifth cell pellet in a fifth medium thereby generating a ninth cell suspension, wherein the fifth medium comprises Advanced DMEM/F12 medium, wherein the fifth medium does not comprise cytokines, antibiotics or growth factors, and wherein cells of the ninth cell suspension comprises iHSCs, which are defined as CD34+ and lineage marker-negative, where the lineage 31 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 markers comprise one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a; o. resuspending the fourth cell pellet of step m) in a volume of about 20 mg/mL collagenase type II, thereby generating a tenth cell suspension; p. adding the fifth medium to the tenth cell suspension; q. centrifuging the tenth cell suspension, thereby generating a sixth cell pellet, and adding TrypLE to the sixth cell pellet, thereby generating an eleventh cell suspension; r. adding additional fifth medium to the eleventh cell suspension and mixing; s. centrifuging the eleventh cell suspension to form a seventh cell pellet and resuspending the seventh cell pellet in additional fifth medium to form a twelfth cell suspension; t. passing the twelfth cell suspension through a cell strainer to obtain iHSCs, wherein the cell strainer is 70 µm mesh size; u. determining the purity of the iHSCs from the first supernatant of step n) and the iHSCs from step t), said purity defined as CD34+ and lineage marker-negative, where the lineage markers comprise one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a; v. not introducing serum in any of steps a)-u); w. not introducing any additional cells, including feeder cells or stromal cells, in any of steps a)-v); and x. obtaining de novo generated iHSCs. [0120] In some embodiments, the one or more cells have high purity, viability, fold expansion, and potential to redifferentiate into immune effectors, including iPSC-derived γδ (iγδ) T cells. [0121] In some embodiments, the EB and γδ T cell-derived iPSCs are from a tissue culture vessel. [0122] In some embodiments, the first, second, fourth, sixth, and/or eighth cell suspensions are cultured under normoxic conditions. [0123] In some embodiments, the method further comprising further purifying and/or isolating 32 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 the iHSCs. [0124] In some embodiments, the obtained iHSCs are isolated redifferentiated cells or are capable of being further purified and/or isolated. [0125] In some embodiments, the method further comprises assaying for all of the lineage markers CD3, CD19, CD14, CD11b, CD56, CD20, CD16, CD2, and CD235a via flow cytometry or an equivalent thereof. [0126] In some embodiments, any one of steps a)-w) comprises gently dispersing the cells using a pipet or equivalent thereof. [0127] In some embodiments, cells of the first medium are at a concentration of about 80,000 to about 180,000 cells/mL in a volume of about 50 to about 70 mL. [0128] In some embodiments, the cells of the first medium are at a concentration of about 150,000 cells/mL in a volume of about 60 mL. [0129] In some embodiments, the medium of step a) further comprises a ROCK inhibitor. [0130] In one embodiment, the medium of step a) further comprises a Chroman 1, Emricasan, Polyamines, and Trans‑ISRIB (CEPT) cocktail. [0131] In some embodiments, after step a) all the cell volume of the first cell suspension is removed from the bioreactor prior to centrifugation. [0132] In some embodiments, the cell morphology is imaged via microscopy, e.g., bright field microscopy. [0133] In some embodiments, the medium in steps b), f), j), or n) comprises L-glutamine. [0134] In some embodiments, the medium in steps b), f), j), or n) comprises GlutaMaxTM. [0135] In some embodiments, the medium in steps f) or j) comprises TPO. [0136] In some embodiments, the medium in steps f) or j) comprises EPO. [0137] In some embodiments, the centrifugation of any one of the steps is performed at 100g for 1 minute at brake 6. [0138] In some embodiments, the centrifugation of any one of the steps is performed at 300g for 5 minutes. [0139] In some embodiments, the centrifugation of any one of the steps is performed at 300g for 3 minutes. [0140] In some embodiments, the centrifugation in step l) is performed twice, first at 100g for 1 minute at brake 6 and second at 300g for 5 minutes. 33 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 [0141] In some embodiments, the tenth cell suspension of step o) is incubated with collagenase for 20 minutes at 37 °C. [0142] In some embodiments, the eleventh cell suspension of step q) is incubated with TrypLE for 5 minutes at 37 °C. [0143] In some embodiments, passing of the twelfth cell suspension in step t) further comprises passing the twelfth cell suspension through a second cell strainer with a 40 µm mesh size. [0144] In some embodiments, the culturing of the iPSC cells, EBs, and iHSCs is performed at 37 °C and 5% CO2. [0145] In some embodiments, the bioreactor wheel is rotating at 60 rpm. [0146] In some embodiments, the bioreactor wheel is rotating at 30 rpm. [0147] In some embodiments, the bioreactor wheel is rotating at 24 rpm. [0148] In some embodiments, the bioreactor wheel is rotating at 15 rpm. [0149] In some embodiments, the bioreactor wheel is perpendicular to the bioreactor vessel. [0150] In some embodiments, the bioreactor wheel is not perpendicular to the bioreactor vessel. [0151] In some embodiments, iPSCs of the first medium are at a concentration of about 80,000 to about 180,000 cells/mL in a volume of about 90 to about 110 mL. [0152] In some embodiments, the iPSCs of the first medium are at a concentration of about 150,000 cells/mL in a volume of about 100 mL. [0153] In some embodiments, iPSCs of the first medium are at a concentration of about 5,000 to about 180,000 cells/mL in a volume of about 0.45 to about 0.55 L. [0154] In some embodiments, the iPSCs of the first medium are at a concentration of about 20,000 cells/mL in a volume of about 0.5 L. [0155] In some embodiments, iPSCs of the first medium are at a concentration of about 5,000 to about 180,000 cells/mL in a volume of about 2 to about 5 L. [0156] In some embodiments, iPSCs of the first medium are at a concentration of about 5,000 to about 180,000 cells/mL in a volume of about 10 to about 20 L. [0157] In one aspect, provided herein is a composition comprising the one or more cells as described herein. [0158] In one aspect, provided herein is a use of the one or more cells described herein, in 34 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 preparation of cells for treating diseases, in preparation of lymphocytes, in a bioreactor, in tissue engineering or in vitro drug screening for diseases. BRIEF DESCRIPTION OF THE DRAWINGS [0159] Figures 1A-1B show the iHSC redifferentiation process. Figure 1A shows the process flow of the iHSC redifferentiation process. Figure 1B shows an exemplary bioreactor system schematic of the present invention, comprising a waste member, medium reservoir member, pump member, reactor chamber member including a vertical wheel member (black circle), cell inlet member (not shown), cell release member (not shown) and connector members. Adapted from Bioreactor Systems for Bone Tissue Engineering Article in Tissue Engineering Part B Reviews, 2011, DOI: 10.1089/ten.TEB.2010.0612. [0160] Figure 2 shows the step-by-step iHSC redifferentiation process in a 3D-VW bioreactor. [0161] Figure 3 shows the purity, total yield, viability, and fold expansion of iHSCs redifferentiated from a single human γδ T cell-derived iPSC clone in a 3D-VW bioreactor culture, where iHSCs were defined as lineage marker-negative CD34+ cells. Both thrombopoietin (TPO) and erythropoietin (EPO) can support iHSC redifferentiation. iHSCs were detected as single cells and in dissociated cell clusters. This novel 3D iHSC protocol met the exemplary acceptance criteria for iHSC+ purity. [0162] Figure 4 shows the 3D-vertical wheel (VW) process reproducibly generated iHSCs with high purity and yield and with sufficient fold expansion. iHSCs were redifferentiated from two different iPSC clones in a research-scale 2D culture system and in the 3D-VW culture system. The initial exemplary success criterion for iHSC differentiation was 70% iHSC cells (Lin-CD34+) and 0.5-fold iHSC expansion. Purity and cell viability were comparable between the 3D and 2D processes for these two iPSC clones. [0163] Figure 5 shows iHSCs generated in the 3D-VW process possess iγδ T cell redifferentiation potential. iHSC cells generated in the 3D-VW process yielded iγδ T cells at a purity that is consistent with iHSC cells generated with the 2D process. The iHSC to iγδ T cell redifferentiation process was carried out in a 2D culture system following a basic protocol and utilizing iHSCs redifferentiated in 2D and 3D culture systems. The 3D-derived iHSCs used in this experiment were generated by seeding iPSCs at 1.5x standard protocol seeding density. The initial 35 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 exemplary success criterion for iγδ T cell redifferentiation was 20-40% iγδ T cells (CD3+TCRγδ+) and 5-fold expansion. [0164] Together, these data indicated that the 3D-VW process generated iHSC cells with T cell differentiation potential. [0165] Figure 6 shows historical data of T cell differentiation potential across multiple clones for iCD34 redifferentiation in an exemplary 3D-VW reactor as measured in terms of purity, fold expansion, and yield. For the dissociated single cells, 50% of the harvested single cell population was treated with the same enzymes used to dissociate clusters to examine the effects of such enzymes on T cell differentiation potential. The initial exemplary success criterion for iHSC differentiation was 70% iHSC cells (defined as Lin-CD34+) and 0.5-fold iHSC expansion. Three independent cell clones, Clone B, Clone C, and Clone E, possessed high T cell differentiation potential. DETAILED DESCRIPTION Definitions [0166] Various terms relating to aspects of the description are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definitions provided herein. [0167] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like. [0168] The term “about” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of up to ±10% from the specified value, as such variations are appropriate to perform the disclosed methods. Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine 36 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. [0169] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. [0170] The terms “treating” or “treatment” refer to any success or indicia of success in the attenuation or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement, remission, diminishing of symptoms or making the condition more tolerable to the patient, slowing in the rate of degeneration or decline, making the final point of degeneration less debilitating, improving a subject’s physical or mental well-being, or prolonging the length of survival. The treatment may be assessed by objective or subjective parameters; including the results of a physical examination, neurological examination, or psychiatric evaluations. [0171] The term “induced pluripotent stem cell” (hereinafter “iPSC”) means a stem cell that is established by introducing reprogramming factors into a somatic cell, has pluripotency permitting differentiation into any cell types present in living organisms, and also has proliferation (i.e., self-renewal) capacity. It encompasses any cell that can be redifferentiated into a hematopoietic stem cell (HSC) to be used in the present invention. The iPSC is preferably derived from a primate (e.g., monkey, orangutan, chimpanzee, human), more preferably human. [0172] The term “iHSC” refers to hematopoietic stem cells that are derived from iPSCs. [0173] The terms “non-traditional geometries” or “non-traditional bioreactors” refer to those geometries or bioreactors that do not fall under the definitions of “traditional geometries” or “traditional bioreactors”, respectively, i.e., geometries that pre-date this invention. “Non- traditional geometries” or “non-traditional bioreactors” may refer to geometries or bioreactors that combine radial and axial flow components. The vertical-wheel bioreactor is a non-limiting example of a non-traditional bioreactor. “Traditional geometries” or “traditional bioreactors” may refer to stirred-tank geometries or stirred-tank bioreactors, respectively. “Traditional geometries” or “traditional bioreactors” may refer to geometries or bioreactors that have radial flow components. Horizontal-blade bioreactors and turbine impellers are non-limiting examples of traditional bioreactors. “Traditional geometries” and “traditional bioreactors” are further defined 37 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 in Borys, B. S., et al. Overcoming bioprocess bottlenecks in the large-scale expansion of high- quality hiPSC aggregates in vertical-wheel stirred suspension bioreactors. Stem Cell Research & Therapy. 2021, 12:55, which is incorporated herein by reference in its entirety. The terms “3D vertical-wheel bioreactor” and “tissue culture vessel comprising a vertical wheel” and the like are used interchangeably herein. [0174] The embodiments described herein are not limited to particular methods, reagents, compounds, compositions, or biological systems, which can, of course, vary. Methods of Generating iHSCs [0175] γδ T cells were selected as the iPSC cell of origin because it is known that the epigenetic and transcriptional memory of the starting cell influences and enhances iPSC redifferentiation potential to that cell type. An optimized three-dimensional (3D) serum-free, feeder-free (SFFF) protocol was used to redifferentiate γδ T-iPSCs into iHSCs and then into iγδ T cells as shown in Figures 1A, 1B, and 2. Purity and fold expansion were determined from two different iPSC clones to iHSCs and from iHSCs to iγδ T cells. Images of the cells were recorded. Exemplary success criteria were set for the purity and fold expansion of redifferentiated iHSCs and iγδ T cells based on results from a 3D redifferentiation protocol and from a 2D redifferentiation protocol. The 3D-VW bioreactor protocol for the redifferentiation of iPSCs to iHSCs resulted in high purity, high cell viability, high yield, and sufficient fold expansion as shown in Figures 3-6. The protocol also yielded iγδ T cells at a purity that met the exemplary success criterion and with a fold expansion that is consistent with iHSCs generated with the 2D process. These data demonstrate the efficiency and validity of the 3D-VW bioreactor protocol for the generation of iHSCs from γδ T-iPSCs as shown in Figures 3-6. Maintaining iPSCs Prior to Initiating Redifferentiation [0176] As is known in the art, iPSC colonies generally exhibit circular colonies, with densely packed cells, higher nucleus to cytoplasmic ratio, well defined edges, and distinct borders. [0177] The iPSCs can be thawed from a frozen stock, and a desired number of live cells can be utilized in the methods described herein. Cell counting, size, and viability determination can be performed by any method known in the art, e.g., by using a NucleoCounter NC-200. Live cells having a preferred size, e.g., between 10 µm and 25 µm, can be used in the steps described herein. [0178] The iPSCs can be seeded at a cell density of 1.0 x 105 to 1.8 x 105. After seeding, the iPSCs are maintained in an appropriate tissue or cell culture vessel, such as for example and not 38 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 limitation, a tissue culture vessel comprising a vertical wheel. The tissue or cell culture vessels are incubated at 37 °C, 5% CO2. [0179] The iPSCs are grown in StemFit® Basic 04 medium further comprising basic fibroblast growth factor (bFGF) at a final concentration of about 90 ng/mL to about 110 ng/mL bFGF, preferably about 100 ng/mL bFGF. The medium contains either 10 µM Y-27632 ROCK inhibitor or a Chroman 1, Emricasan, Polyamines, and Trans‑ISRIB (CEPT) cocktail. StemFit® Basic 04 cell culture medium (Ajinomoto) is an animal-origin free, defined medium for human pluripotent stem cell culture. This medium is a complete medium and does not require any other supplements. [0180] StemFit® Basic 03 cell culture medium (Ajinomoto) is an animal-origin free, defined medium for human pluripotent stem cell culture. StemProTM-34 serum-free medium (SFM) (GibcoTM) is a serum-free medium specifically formulated to support the development of human hematopoietic cells in culture, including HSC and progenitor cells isolated from bone marrow, neonatal cord blood, and peripheral blood. StemProTM-34 SFM is a flexible cell culture media that can be used for a wide variety of applications. [0181] During the maintenance period, the iPSCs can be examined at regular intervals, e.g., daily, for cell morphology. Cell morphology can be determined by microscopy, e.g., bright field microscopy. Once the cells are bright and have distinct borders, the colonies can be redifferentiated to iHSCs as discussed herein. The medium can be changed at regular intervals as known in the art. Redifferentiation of the iPSCs into iHSCs with a small volume bioreactor comprising a vertical wheel [0182] The invention provides methods of redifferentiating iPSCs into iHSCs using a small volume bioreactor or tissue culture vessel comprising a vertical wheel. Nonlimiting exemplary volumes include a 0.1 L bioreactor or tissue culture vessel. [0183] In one aspect, provided herein is a method of de novo generation of hematopoietic stem cells (iHSCs) derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue culture vessel, said method comprising: seeding human γδ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction. 39 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 [0184] In some embodiments, the tissue culture vessel has non-traditional bioreactor geometries as discussed herein. [0185] In some embodiments, the iPSCs are seeded at a density of about 1.0 x 105 cells/mL to about 1.8 x 105 cells/mL. [0186] In some embodiments, the tissue culture vessel comprising a vertical wheel further comprises a first medium comprising StemFit® Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, Emricasan, Polyamines, and Trans‑ISRIB (CEPT) cocktail, wherein the BMP4 and the rhVEGF are present in a ratio of 1:1, and wherein the first medium does not comprise antibiotics. [0187] In some embodiments, the method further comprises exchanging spent medium in the tissue culture vessel comprising a vertical wheel on each of days 2, 4, 6, and 8 of culture for the about 8 days, wherein the step of exchanging the spent medium comprises: a) allowing cell aggregates to settle in the tissue culture vessel comprising a vertical wheel for about 3 to about 5 minutes; b) collecting 80% of the spent medium from the tissue culture vessel comprising a vertical wheel; c) centrifuging the collected medium, thereby obtaining a cell pellet; d) resuspending the cell pellet in StemFit® for Differentiation, thereby obtaining a cell suspension; and e) adding the cell suspension and fresh medium to the tissue culture vessel comprising a vertical wheel. [0188] In some embodiments, the step of exchanging the spent medium on days 2, 4, and 6 of culture comprises: i) allowing cell aggregates to settle in the tissue culture vessel comprising a vertical wheel for about 3 to about 5 minutes; collecting 80% of the spent medium from the tissue culture vessel comprising a vertical wheel; centrifuging the collected medium, thereby obtaining a cell pellet; resuspending the cell pellet in StemFit® for Differentiation medium, thereby obtaining a cell suspension; and adding the cell suspension and fresh medium to the tissue culture vessel comprising a vertical wheel, or ii) allowing cell aggregates to settle in the tissue culture vessel comprising a vertical wheel for about 3 to about 5 minutes; collecting 80% of the spent medium from the tissue culture vessel comprising a vertical wheel; centrifuging the collected medium, thereby obtaining a first cell pellet and a first supernatant; resuspending the first cell pellet in StemFit® for Differentiation medium and adding the resuspended first cell pellet to the tissue culture vessel comprising a vertical wheel; centrifuging the supernatant, thereby obtaining a second cell pellet and a second supernatant; and resuspending the second cell pellet in StemFit® 40 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 for Differentiation medium and adding the resuspended second cell pellet and fresh medium to the tissue culture vessel comprising a vertical wheel. [0189] In some embodiments, the fresh medium added on day 2 of culture comprises: i) Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SB431542 (inhibitor of Activin/BMP/TGFβ pathway), recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), and one of L-glutamine and GlutaMaxTM supplement, or ii) StemProTM-34 Serum-free Medium (SFM) and StemPro™-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, Insulin-Transferrin-Selenium (ITS-G), monothioglycerol, SB431542, recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), and one of L- glutamine and GlutaMaxTM, wherein the VEGF, bFGF, and SCF are present in a ratio of 1:0.625:0.625, and wherein the second medium does not comprise antibiotics. [0190] In some embodiments, the iPSCs are dissociated into single cells on day 7 of culture. [0191] In some embodiments, the step of harvesting redifferentiated iHSCs comprises retaining clusters, semi-clusters, and single cells. [0192] In some embodiments, the step of harvesting redifferentiated iHSCs comprises retaining all cells in the tissue culture vessel comprising a vertical wheel. [0193] In some embodiments, the vertical wheel spins at a rate of about 10 rpm to about 60 rpm, preferably about 40 rpm. [0194] In some embodiments, the step of harvesting redifferentiated iHSCs comprises: a) removing an entire cell volume comprising embryoid bodies (EBs) and iHSCs from the tissue culture vessel comprising a vertical wheel and centrifuging the entire cell volume, thereby generating a first supernatant and a first cell pellet; b) centrifuging the first supernatant, thereby generating a second supernatant and a second cell pellet and removing the second supernatant; c) resuspending the first cell pellet and the second cell pellet in Advanced DMEM/F12 medium without cytokines, growth factors, or antibiotics; 41 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 d) adding a volume of 20 mg/mL collagenase II to the first cell pellet, thereby generating a first cell suspension; e) adding a volume of Advanced DMEM/F12 medium without cytokines, growth factors, or antibiotics equal to the volume of the first cell suspension to the first cell suspension; f) centrifuging the first cell suspension, thereby generating a third cell pellet; g) resuspending the third cell pellet in a volume of TrypLE and adding a volume of Advanced DMEM/F12 medium without cytokines, growth factors, or antibiotics, thereby generating a second cell suspension; h) mixing the second cell suspension until clusters of cells are no longer visible; i) optionally passing the second cell suspension through a cell strainer to obtain iHSCs, wherein the cell strainer is 70 µm mesh size, if clusters of cells are visible after the mixing step; and j) centrifuging the second cell suspension. [0195] In some embodiments, the cells are cultured under normoxic conditions. [0196] In some embodiments, the method further comprises further purifying and/or isolating the redifferentiated iHSCs. [0197] In some embodiments, the redifferentiated iHSCs are isolated differentiated cells or are capable of being further purified and/or isolated. [0198] In some embodiments, determining purity of the redifferentiated iHSCs comprises assaying for one or more of lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a via flow cytometry or an equivalent thereof. [0199] In some embodiments, the lineage markers comprise all of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a. [0200] In an aspect, provided herein is a method of de novo generation of hematopoietic stem cells (iHSCs) derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue culture vessel, said method comprising: a means for seeding human γδ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; a means for redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; a means for harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction. 42 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 [0201] In an aspect, provided herein is a method of de novo generation of induced pluripotent stem cell (iPSC)-derived hematopoietic stem cells (iHSCs) from γδ T cell-derived iPSCs (γδ T- iPSCs) under serum-free, feeder-free culture conditions, said method comprising one or more of the following steps: i) obtaining one or more human induced pluripotent stem cells (iPSCs), and wherein the method further comprises one or more of the following steps: a) after incubating the iPSCs in a tissue culture vessel comprising a vertical wheel in a first medium with agitation at about 10-60 rpm, preferably 40 rpm, for about 1.8 to about 2.2 days, preferably about 2 days, removing an entire cell volume from the tissue culture vessel comprising a vertical wheel and centrifuging the entire cell volume, wherein the first medium comprises StemFit® Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF) and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, Emricasan, Polyamines, and Trans‑ISRIB (CEPT) cocktail, wherein the first medium does not comprise antibiotics; b) centrifuging the first cell suspension and thereby generating a first cell pellet; c) resuspending the first cell pellet into a second medium to form a second cell suspension, wherein the second medium comprises: i) Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SB431542 (inhibitor of Activin/BMP/TGFβ pathway), recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), and one of L-glutamine and GlutaMaxTM supplement, or ii) StemProTM-34 Serum-free Medium (SFM) and StemPro™-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, Insulin-Transferrin- Selenium (ITS-G), monothioglycerol, SB431542, recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), and one of L-glutamine and GlutaMaxTM, wherein the VEGF, bFGF, and SCF are present in a ratio of 1:0.625:0.625, and wherein 43 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 the second medium does not comprise antibiotics; d) culturing and agitating the second cell suspension in the tissue culture vessel comprising a vertical wheel at about 10-60 rpm, preferably 40 rpm, for about 1.8 to about 2.2 days, preferably about 2 days; e) stopping the agitating for about 5 minutes, wherein cell aggregates settle in the second cell suspension; f) collecting about 80% of the second cell suspension from a top volume of the second cell suspension, from the tissue culture vessel comprising a vertical wheel; g) centrifuging the collected second cell suspension, thereby forming a second cell pellet and resuspending the second pellet in about 1 mL of one of StemFit® For Differentiation solution and/or StemPro™-34 SFM; h) adding the resuspended second cell pellet into a third medium forming a third cell suspension, wherein the third medium comprises: i) Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), interleukin- 3 (IL-3), interleukin-6 (IL-6), one of (i) thrombopoietin (TPO) and (ii) erythropoietin (EPO), and one of (i) L-glutamine and (ii) GlutaMaxTM, or ii) StemPro™-34 SFM and StemPro™-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, rhVEGF, IL-3, IL-6, Flt3L, one of (i) TPO and (ii) EPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, and wherein the third medium does not comprise antibiotics; i) placing the third cell suspension into the tissue culture vessel comprising a vertical wheel with remaining second cell suspension forming a fourth cell suspension; j) culturing and agitating the fourth cell suspension in the tissue culture vessel comprising a vertical wheel at about 10-60 rpm, preferably 40 rpm, for about 1.8 to about 2.2 days, preferably about 2 days; k) stopping the agitating for about 5 minutes; wherein cell aggregates settle in the cell suspension; l) collecting about 80% of the fourth cell suspension and centrifuging the collected fourth 44 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 cell suspension forming a third cell pellet, and resuspending the third cell pellet in about 1 mL of one of StemFit® For Differentiation solution and StemPro™-34 SFM; m) suspending the third cell pellet dissolved in the about 1 mL of one of StemFit® For Differentiation solution and StemPro™-34 SFM into a fourth medium forming a fifth cell suspension, wherein the fourth medium comprises: i) Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, IL-6, one of (i) TPO and (ii) EPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, or ii) StemPro™-34 SFM and StemPro™-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, IL-6, one of (i) TPO and (ii) EPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, and wherein the fourth medium does not comprise antibiotics; n) placing the fifth cell suspension into the tissue culture vessel comprising a vertical wheel with the remaining fourth cell suspension thereby forming a sixth cell suspension; o) culturing and agitating the sixth cell suspension in the tissue culture vessel comprising a vertical wheel at about 10-60 rpm, preferably 40 rpm, for about 1.8 to about 2.2 days, preferably about 2 days; p) repeating steps k) to o); optionally wherein the sixth cell suspension is centrifuged and a supernatant from the centrifuged sixth cell suspension is centrifuged and the resulting cell pellet is resuspended in about 1 mL of one of StemFit® For Differentiation solution and StemPro™-34 SFM and combined with a seventh cell suspension obtained by resuspending a cell pellet from the first centrifugation to form an eighth cell suspension before culturing and agitating the eighth cell suspension in the tissue culture vessel comprising a vertical wheel at about 10-60 rpm, preferably 40 rpm, for about 1.8 to about 2.2 days, preferably about 2 days; q) removing all the eighth cell suspension from the tissue culture vessel comprising a vertical wheel and harvesting EBs and iHSCs in the eighth cell suspension by centrifugation, thereby generating a fourth cell pellet and a first supernatant; 45 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 r) collecting the first supernatant; s) centrifuging the first supernatant, thereby generating a fifth cell pellet and resuspending the fifth cell pellet in a fifth medium thereby generating a ninth cell suspension, wherein the fifth medium comprises Advanced DMEM/F12 medium, wherein the fifth medium does not comprise cytokines, antibiotics, or growth factors, and wherein cells of the ninth cell suspension comprise iHSCs, which are defined as CD34+ and lineage marker-negative, where the lineage markers comprise one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a; t) resuspending the fourth cell pellet of step q), in a volume of about 20 mg/mL collagenase type II, thereby generating a tenth cell suspension; u) adding the fifth medium to the tenth cell suspension; v) centrifuging the tenth cell suspension, thereby generating a sixth cell pellet, and adding TrypLE to the sixth cell pellet, thereby generating an eleventh cell suspension; w) adding additional fifth medium to the eleventh cell suspension and mixing; x) centrifuging the eleventh cell suspension to form a seventh cell pellet and resuspending the seventh cell pellet in additional fifth medium to form a twelfth cell suspension; y) optionally passing the twelfth cell suspension through a cell strainer to obtain iHSCs, wherein the cell strainer is 70 µm mesh size, if cell clumps are present in the twelfth cell suspension; z) centrifuging the twelfth cell suspension; aa) not introducing serum in any of steps a)-z); bb) not introducing any additional cells, including feeder cells or stromal cells, in any of steps a)-aa); cc) determining the purity of the iHSCs from the first supernatant of step s) and the iHSCs from step y), said purity defined as CD34+ and lineage marker-negative, where the lineage markers comprise one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a; and dd) obtaining de novo generated iHSCs. [0202] In an aspect, provided herein is a method of de novo generation of hematopoietic stem cells (iHSCs) derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue culture vessel, said method comprising: 46 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 a) a means for seeding human γδ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; b) a means for redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; c) a means for harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction. [0203] In one aspect, provided herein is a method of de novo generation of induced pluripotent stem cell (iPSC)-derived hematopoietic stem cells (iHSCs) from γδ T cell-derived iPSCs (γδ T- iPSCs) under serum-free, feeder-free culture conditions, said method comprising one or more of the following steps: 1. obtaining one or more human induced pluripotent stem cells (iPSCs), and wherein the method further comprises one or more of the following steps: a. seeding the iPSCs, wherein the seeding is performed by suspending the iPSCs in a first medium into a bioreactor, wherein the bioreactor is a vertical-wheel bioreactor, thereby producing a first cell suspension, wherein the first medium comprises one of StemFit® Basic 04 medium and StemFit® Basic 03 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, Emricasan, Polyamines, and Trans‑ISRIB (CEPT) cocktail, and wherein the first medium does not comprise antibiotics; b. culturing and agitating the first cell suspension in the bioreactor at about 10-60 rpm, preferably 40 rpm, wherein the first cell suspension is cultured and agitated for about 1.8 to about 2.2 days, preferably about 2 days, wherein embryoid bodies (EBs) are generated; c. centrifuging the first cell suspension and thereby generating a first cell pellet; d. resuspending the first cell pellet into a second medium to form a second cell suspension, wherein the second medium comprises: i. Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SB431542 (inhibitor of Activin/BMP/TGFβ pathway), 47 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 rhVEGF, basic fibroblast growth factor (bFGF), stem cell factor (SCF), and one of L-glutamine and GlutaMaxTM supplement, or ii. StemProTM-34 Serum-free Medium (SFM) and StemPro™-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, Insulin- Transferrin-Selenium (ITS-G), monothioglycerol, SB431542, rhVEGF, bFGF, SCF, and one of L-glutamine and GlutaMaxTM, and wherein the second medium does not comprise antibiotics; e. culturing and agitating the second cell suspension in the bioreactor for about 1.8 to about 2.2 days, preferably about 2 days; f. stopping the agitating for about 5 minutes, wherein cell aggregates settle in the second cell suspension; g. collecting about 80% of the second cell suspension from a top volume of the second cell suspension, from the bioreactor; h. centrifuging collected second cell suspension forming a second cell pellet and resuspending the second pellet in about 1 mL of one of StemFit® For Differentiation solution and StemPro™-34 SFM; i. suspending the second cell pellet in the about 1 mL of one of StemFit® For Differentiation solution and or StemPro™-34 SFM into a third medium forming a third cell suspension, wherein the third medium comprises: i. Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), interleukin-3 (IL-3), interleukin-6 (IL-6), one of (i) thrombopoietin (TPO) and (ii) erythropoietin (EPO), and one of (i) L-glutamine and (ii) GlutaMaxTM, or ii. StemPro™-34 SFM and StemPro™-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, rhVEGF, IL-3, IL-6, Flt3L, one of (i) TPO and (ii) EPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, and wherein the third medium does not comprise antibiotics; j. placing the third cell suspension into the bioreactor with remaining second cell 48 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 suspension forming a fourth cell suspension; k. culturing and agitating the fourth cell suspension in the bioreactor for about 1.8 to about 2.2 days, preferably about 2 days; l. stopping the agitating for about 5 minutes; wherein cell aggregates settle in the cell suspension; m. collecting about 80% of the fourth cell suspension and centrifuging the collected fourth cell suspension forming a third cell pellet, and resuspending the third cell pellet in about 1 mL of one of StemFit® For Differentiation solution and StemPro™-34 SFM; n. suspending the third cell pellet dissolved in the about 1 mL of one of StemFit® For Differentiation solution and StemPro™-34 SFM into a fourth medium forming a fifth cell suspension, wherein the fourth medium comprises: i. Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, IL-6, one of (i) TPO and (ii) EPO, and one of (i) L- glutamine and (ii) GlutaMaxTM, or ii. StemPro™-34 SFM and StemPro™-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, IL-6, one of (i) TPO and (ii) EPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, and wherein the fourth medium does not comprise antibiotics; o. placing the fifth cell suspension into the bioreactor with the remaining fourth cell suspension thereby forming a sixth cell suspension; p. culturing and agitating the sixth cell suspension in the bioreactor for about 1.8 to about 2.2 days, preferably about 2 days; q. repeating steps l) to p); r. removing all the eighth cell suspension from the bioreactor and harvesting EBs and iHSCs in the cell suspension by centrifugation, thereby generating a fourth cell pellet and a first supernatant; s. collecting the first supernatant; t. centrifuging the first supernatant, thereby generating a fifth cell pellet and resuspending the fifth cell pellet in a fifth medium thereby generating a ninth cell 49 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 suspension, wherein the fifth medium comprises Advanced DMEM/F12 medium, wherein the fifth medium does not comprise cytokines, antibiotics or growth factors, and wherein cells of the ninth cell suspension comprise iHSCs, which are defined as CD34+ and lineage marker-negative, where the lineage markers comprise one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a; u. resuspending the fourth cell pellet of step r), in a volume of about 20 mg/mL collagenase type II, thereby generating a tenth cell suspension; v. adding the fifth medium to the tenth cell suspension; w. centrifuging the tenth cell suspension, thereby generating a sixth cell pellet, and adding TrypLE to the sixth cell pellet, thereby generating an eleventh cell suspension; x. adding additional fifth medium to the eleventh cell suspension and mixing; y. centrifuging the eleventh cell suspension to form a seventh cell pellet and resuspending the seventh cell pellet in additional fifth medium to form a twelfth cell suspension; z. passing the twelfth cell suspension through a cell strainer to obtain iHSCs, wherein the cell strainer is 70 µm mesh size; aa. determining the purity of the iHSCs from the first supernatant of step t) and the iHSCs from step z), said purity defined as CD34+ and lineage marker-negative, where the lineage markers comprise one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a; bb. not introducing serum in any of steps a)-aa); cc. not introducing any additional cells, including feeder cells or stromal cells, in any of steps a)-bb); and dd. obtaining de novo generated iHSCs. [0204] In the 3D protocol, the iPSCs can be cultured and agitated at about 10 rpm to 60 rpm, preferably about 40 rpm, for about 1.8 days to about 2.2 days, preferably approximately about two days, at 37oC, 5% CO2 in a first culture medium comprising StemFit® Basic 04 medium or StemFit® Basic 03 medium further comprising CHIR99021 (glycogen synthase kinase inhibitor), a Rho kinase inhibitor (ROCK inhibitor) or a Chroman 1, Emricasan, Polyamines, and 50 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 Trans‑ISRIB (CEPT) cocktail, bone morphogenetic protein 4 (BMP4), and recombinant human vascular endothelial growth factor (rhVEGF). The morphology of the redifferentiating iPSCs can be determined at the beginning of the culture period and/or at regular intervals by microscopy, e.g., bright field microscopy. Culturing the iPSCs in this medium can induce mesoderm formation, which is a prerequisite for hematopoietic lineage specification and differentiation. [0205] After the culture period for mesoderm induction in the 3D-VW bioreactor protocol, the iPSC-derived cell intermediates can be collected and subsequently cultured and agitated at about 10 rpm to 60 rpm, preferably about 40 rpm, in a second culture medium to begin onset of hematopoietic differentiation, for approximately about 1.8 days to about 2.2 days, preferably approximately about two days, at 37 °C with 5% CO2. The second cell culture medium comprises Advanced DMEM/F12 medium (Thermo Fisher Scientific Catalog #12634) and StemFit® For Differentiation medium (Ajinomoto) present in about a 3.9 to 0.9 ratio to about a 4.1-1.1 ratio, preferably about a 4:1 ratio, and further comprises SB431542 (inhibitor of Activin/BMP/TGFβ pathway), rhVEGF, bFGF, and stem cell factor (SCF), and one of L-glutamine and GlutaMaxTM. Alternatively, the second cell culture medium may comprise StemPro™-34 SFM + StemPro™-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, Insulin-Transferrin-Selenium (ITS-G), monothioglycerol, and further comprises, SB431542, rhVEGF, bFGF, SCF, and one of L-glutamine and GlutaMaxTM. SB431542 is diluted from a stock concentration of 5 mM to achieve a final concentration of about 2 µM in order to use a minimal amount of the diluent dimethylsulfoxide (DMSO). The morphology of the cells can be determined at the beginning of the culture period and/or at regular intervals by microscopy, e.g., bright field microscopy. Advanced DMEM/F12 medium includes amino acids, vitamins, inorganic salts, proteins, reducing elements, trace elements, dextrose, ethanolamine, hypoxanthine Na, linoleic acid, lipoic acid, phenol red, putrescine 2HCl, sodium pyruvate, and thymidine. StemFit® For Differentiation medium is a chemically defined & animal-origin free (CD-AOF) supplement for differentiation of human embryonic stem (ES) cells and iPSCs. [0206] After the culture period to begin onset of hematopoietic differentiation in the 3D-VW bioreactor protocol, the iPSC-derived cell intermediates can be collected and subsequently cultured and agitated at about 10 rpm to 60 rpm, preferably about 40 rpm, in a third culture medium to induce hemangioblast formation, for approximately about 1.8 days to about 2.2 days, preferably approximately about two days, at 37 °C with 5% CO2. The third culture medium comprises 51 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9 to 0.9 ratio to about a 4.1-1.1 ratio, preferably about a 4:1 ratio, and further comprises, SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), , interleukin-3 (IL-3), interleukin-6 (IL-6), one L-glutamine and GlutaMaxTM, and one of thrombopoietin (TPO) and erythropoietin (EPO). Alternatively, the third cell culture medium may comprise StemPro™-34 SFM + StemPro™-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, Insulin-Transferrin-Selenium (ITS-G), monothioglycerol, and further comprises IL-3, IL-6, SCF, rhVEGF, Flt3L, one of L- glutamine and GlutaMaxTM, and one of TPO and EPO. The TPO is prepared from a stock concentration of 100 μg/mL to a final concentration of 30 ng/mL. The EPO is prepared from a stock concentration of 500 IU/mL to a final concentration of 10 IU/mL. The morphology of the cells can be determined at the beginning of the culture period and/or at regular intervals by microscopy, e.g., bright field microscopy. [0207] After the culture period to induce hemangioblast formation in the 3D-VW bioreactor protocol, the iHSCs can be collected and subsequently cultured and agitated at about 10 rpm to 60 rpm, preferably about 40 rpm, in a fourth culture medium for about 1.8 days to about 4.2 days, preferably about two days to about four days, at 37 °C with 5% CO2 for the endothelial to hematopoietic transition phase. The fourth culture medium comprises Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9 to 0.9 ratio to about a 4.1-1.1 ratio, preferably about a 4:1 ratio, and further comprises, SCF, IL-6, one of L-glutamine and GlutaMaxTM, and one of TPO and EPO. Alternatively, the fourth cell culture medium may comprise StemPro™-34 SFM + StemPro™-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, Insulin-Transferrin-Selenium (ITS-G), monothioglycerol, and further comprises SCF, IL-6, one of L-glutamine and GlutaMaxTM, and one of TPO and EPO. The TPO is prepared from a stock concentration of 100 ug/mL to a final concentration of 30 ng/mL. The EPO is prepared from a stock concentration of 500 IU/mL to a final concentration of 10 IU/mL. The morphology of the cells can be determined at the beginning of the culture period and/or at regular intervals by microscopy, e.g., bright field microscopy. [0208] In the 3D-VW bioreactor protocol, after about 7.8 days to about 8.2 days, preferably about 8 days, of redifferentiation, non-adherent iHSCs in the medium can be collected, centrifuged, resuspended in the fourth cell culture medium, and then added back to the culture. 52 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 [0209] After about 10 days of redifferentiation in the 3D-VW bioreactor protocol, the appearance and morphology of the iHSCs can be monitored by microscopy, e.g., bright field microscopy. [0210] The single cell fraction of iHSCs from the 3D-VW bioreactor protocol can be harvested by centrifugation and resuspended in a fifth cell culture medium, the fifth cell culture medium comprising Advanced DMEM/F12 medium, but does not have cytokines or growth factors. The iHSCs can be counted as described herein. [0211] The cell cluster fraction of iHSCs from the 3D-VW bioreactor protocol can be resuspended in collagenase type II and the fifth cell culture medium, followed by centrifugation and resuspension in TrypLE and the fifth cell culture medium, followed by further centrifugation and resuspension in the fifth cell culture medium. The dispersed cells can then be passed through a cell strainer, having a mesh size of 70 µm. The cells can be counted as described herein. [0212] After the two fractions of iHSCs from the 3D-VW bioreactor protocol are harvested in the fifth culture medium, the cells can be assessed for purity, e.g., using flow cytometry or an equivalent, to analyze the presence or absence of certain markers. The purity can be defined by the absence of expression of one or more lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a and by the presence of CD34 expression (CD34+). In other words, the harvested cells are lineage-marker negative and CD34+. Following the purity analysis, the redifferentiated iHSCs can be cryopreserved. [0213] In some embodiments, the EB and γδ T cell-derived iPSCs are from a tissue culture vessel. [0214] In some embodiments, any of the cell suspensions are cultured under normoxic conditions. [0215] In some embodiments, the method further comprises further purifying and/or isolating the iHSCs. [0216] In some embodiments, the obtained iHSCs are isolated redifferentiated cells or are capable of being further purified and/or isolated. [0217] In some embodiments, the method further comprises assaying for all of the lineage markers CD3, CD19, CD14, CD11b, CD56, CD20, CD16, CD2, and CD235a via flow cytometry or an equivalent thereof. [0218] In some embodiments, any one of the steps comprises gently dispersing the cells using 53 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 a pipet or equivalent thereof. [0219] In some embodiments, cells of the first medium are at a concentration of about 80,000 to about 180,000 cells/mL in a volume of about 50 to about 70 mL. [0220] In some embodiments, the cells of the first medium are at a concentration of about 150,000 cells/mL in a volume of about 60 mL. [0221] In some embodiments, the medium of step a) further comprises a ROCK inhibitor. [0222] In some embodiments, the medium of step a) further comprises a Chroman 1, Emricasan, Polyamines, and Trans‑ISRIB (CEPT) cocktail. [0223] In some embodiments, after step b) all the cell volume of the first cell suspension is removed from the bioreactor prior to centrifugation. [0224] In some embodiments, the cell morphology is imaged via microscopy, e.g., bright field microscopy. [0225] In some embodiments, the medium of any one of the steps comprises L-glutamine. [0226] In some embodiments, the medium of any one of the steps comprises GlutaMaxTM. [0227] In some embodiments, the medium of any one of the steps comprises TPO. [0228] In some embodiments, the medium of any one of the steps comprises EPO. [0229] In some embodiments, the centrifugation of any one of the steps is performed at 100g for 1 minute at brake 6. [0230] In some embodiments, the centrifugation of any one of the steps is performed at 300g for 5 minutes. [0231] In some embodiments, the centrifugation of any one of the steps is performed at 300g for 3 minutes. [0232] In some embodiments, the centrifugation of any one of the steps is performed twice, first at 100g for 1 minute at brake 6 and second at 300g for 5 minutes. [0233] In some embodiments, the tenth cell suspension is incubated with collagenase for 20 minutes at 37 °C. [0234] In some embodiments, the eleventh cell suspension is incubated with TrypLE for 5 minutes at 37 °C. [0235] In some embodiments, passing of the twelfth cell suspension further comprises passing the twelfth cell suspension through a second cell strainer with a 40 µm mesh size. [0236] In some embodiments, the culturing of the iPSC cells, EBs, and iHSCs is performed at 54 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 37 °C and 5% CO2. [0237] In some embodiments, the bioreactor wheel is rotating at 60 rpm. [0238] In some embodiments, the bioreactor wheel is rotating at 30 rpm. [0239] In some embodiments, the bioreactor wheel is rotating at 24 rpm. [0240] In some embodiments, the bioreactor wheel is rotating at 15 rpm. [0241] In some embodiments, the bioreactor wheel is perpendicular to the bioreactor vessel. [0242] In some embodiments, the bioreactor wheel is not perpendicular to the bioreactor vessel. [0243] In some embodiments, iPSCs in the first medium are at a concentration of about 80,000 to about 180,000 cells/mL in a volume of about 90 to about 110 mL. [0244] In some embodiments, the iPSCs in the first medium are at a concentration of about 150,000 cells/mL in a volume of about 100 mL. [0245] In some embodiments, iPSCs in the first medium are at a concentration of about 5,000 to about 180,000 cells/mL in a volume of about 0.45 to about 0.55 L. [0246] In some embodiments, the iPSCs in the first medium are at a concentration of about 20,000 cells/mL in a volume of about 0.5 L. [0247] In some embodiments, iPSCs in the first medium are at a concentration of about 5,000 to about 180,000 cells/mL in a volume of about 2 to about 5 L. [0248] In some embodiments, iPSCs in the first medium are at a concentration of about 5,000 to about 180,000 cells/mL in a volume of about 10 to about 20 L. Redifferentiation of the iPSCs into iHSCs with a large volume bioreactor comprising a vertical wheel [0249] The invention provides methods of redifferentiating iPSCs into iHSCs using a large volume bioreactor or tissue culture vessel comprising a vertical wheel. Nonlimiting exemplary volumes include a 3 L bioreactor or tissue culture vessel. [0250] In one aspect, provided herein is a method of de novo generation of hematopoietic stem cells (iHSCs) derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue culture vessel, said method comprising: seeding human γδ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; retaining all 55 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 cells in the culture on day 8 of culture while removing the medium from the culture for about 2 days; and harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction. [0251] In some embodiments, the tissue culture vessel has non-traditional bioreactor geometries. [0252] In some embodiments, the iPSCs are seeded at a density of about 1.0 x 105 cells/mL to about 1.8 x 105 cells/mL. [0253] In some embodiments, the tissue culture vessel comprising a vertical wheel further comprises a first medium comprising StemFit® Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, Emricasan, Polyamines, and Trans‑ISRIB (CEPT) cocktail, wherein the BMP4 and the rhVEGF are present in a ratio of 1:1, and wherein the first medium does not comprise antibiotics. [0254] In some embodiments, the method further comprises exchanging spent medium in the tissue culture vessel comprising a vertical wheel on each of days 2, 4, and 6 of culture for the about 8 days, wherein the step of exchanging spent medium comprises: removing the spent medium from the tissue culture vessel comprising a vertical wheel using a 40 µm cell retention filter and adding a wash medium at a pump flow rate of 30 mL/min, the wash medium comprising Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio; exchanging about 3 L to about 3.5 L of the wash medium in the bioreactor and removing 0.1 L of the wash medium from the tissue culture vessel comprising a vertical wheel using the 40 µm cell retention filter; and adding about 50 mL of the wash medium and about 50 mL of a medium to from the tissue culture vessel comprising the vertical wheel. [0255] In some embodiments, the iPSCs are dissociated into single cells on day 7 of culture. [0256] In some embodiments, the step of retaining all cells comprises retaining clusters, semi- clusters, and single cells. [0257] In some embodiments, the step of retaining all cells comprises removing spent medium from the tissue culture vessel comprising a vertical wheel using a 4 µm cell retention filter and adding fresh medium to the bioreactor over about 1.8 days to about 2.2 days, preferably about 2 days, at a rate of about 0.69 mL/min (1 L/day), until 2 L of media have been exchanged, wherein the fifth medium comprises Advanced DMEM/F12 medium and StemFit® For Differentiation 56 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, IL-6, TPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, wherein the fresh medium does not comprise antibiotics. [0258] In some embodiments, the vertical wheel spins at a rate of about 10 rpm to about 60 rpm, preferably about 23 rpm. [0259] In some embodiments, the step of harvesting comprises: a) removing an entire cell volume comprising EBs and iHSCs from the tissue culture vessel comprising a vertical wheel and centrifuging the entire cell volume, thereby generating a first supernatant and a first cell pellet; b) centrifuging the first supernatant, thereby generating a second supernatant and a second cell pellet, removing the second supernatant, and resuspending the second cell pellet in Advanced DMEM/F12 medium and StemFit® For Differentiation medium without cytokines, growth factors, or antibiotics; c) adding a first volume of 20 mg/mL collagenase II to the first cell pellet and the second cell pellet, combining the resuspended first cell pellet and the resuspended second cell pellet and adding a second volume of 20 mg/mL collagenase II, thereby generating a combined cell suspension; d) adding a volume of Advanced DMEM/F12 medium and StemFit® For Differentiation medium without cytokines, growth factors, or antibiotics equal to the volume of the combined cell suspension to the combined cell suspension; e) centrifuging the combined cell suspension, thereby generating a third cell pellet; f) resuspending the third cell pellet in a volume of TrypLE and adding a volume of Advanced DMEM/F12 medium and StemFit® For Differentiation medium without cytokines, growth factors, or antibiotics, thereby generating a fourth cell suspension; g) mixing the fourth cell suspension until clusters of cells are no longer visible; h) optionally passing the fourth cell suspension through a cell strainer to obtain iHSCs, wherein the cell strainer is 70 µm mesh size, if clusters of cells are visible after the mixing step; and i) centrifuging the fourth cell suspension. [0260] In an aspect, provided herein is a method of de novo generation of induced pluripotent stem cell (iPSC)-derived hematopoietic stem cells (iHSCs) from γδ T cell-derived iPSCs (γδ T- 57 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 iPSCs) under serum-free, feeder-free culture conditions, said method comprising one or more of the following steps: i) obtaining one or more human induced pluripotent stem cells (iPSCs), and wherein the method further comprises one or more of the following steps: a) seeding the iPSCs, wherein the seeding is performed by suspending the iPSCs in a first medium into a tissue culture vessel comprising a vertical wheel, thereby producing a first cell suspension, wherein the first medium comprises one of StemFit® Basic 04 medium and StemFit® Basic 03 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, Emricasan, Polyamines, and Trans‑ISRIB (CEPT) cocktail, and wherein the first medium does not comprise antibiotics; b) culturing and agitating the cells in the tissue culture vessel comprising a vertical wheel at about 10-60 rpm, preferably 23 rpm, for about 1.8 to about 2.2 days, preferably about 2 days, wherein embryoid bodies (EBs) are generated; c) removing the first medium from the tissue culture vessel comprising a vertical wheel and adding a wash medium comprising Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, at a rate of about 30 mL/min; d) exchanging about 3L to about 3.5 L of the wash medium in the tissue culture vessel comprising a vertical wheel using a 40 µm cell retention filter at a pump flow rate of 30 mL/min and removing 0.1 L of the wash medium from the tissue culture vessel comprising a vertical wheel; e) adding about 50 mL of the wash medium and about 50 mL of a second medium to the tissue culture vessel comprising a vertical wheel, wherein the second medium comprises Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SB431542 (inhibitor of Activin/BMP/TGFβ pathway), recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), and one of L-glutamine and GlutaMaxTM supplement, wherein 58 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 the second medium does not comprise antibiotics; f) culturing and agitating the cells in the tissue culture vessel comprising a vertical wheel at about 10-60 rpm, preferably 23 rpm, for about 1.8 to about 2.2 days, preferably about 2 days; g) removing the second medium from the tissue culture vessel comprising a vertical wheel and adding the wash medium at a rate of about 30 mL/min; h) exchanging about 3L to about 3.5 L of the wash medium in the tissue culture vessel comprising a vertical wheel using a 40 µm cell retention filter at a pump flow rate of 30 mL/min and removing 0.1 L of the wash medium from the tissue culture vessel comprising a vertical wheel; i) adding about 50 mL of the wash medium and about 50 mL of a third medium to the tissue culture vessel comprising a vertical wheel, wherein the third medium comprises Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises and further comprises SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), interleukin-3 (IL-3), interleukin-6 (IL-6), thrombopoietin (TPO), and one of (i) L- glutamine and (ii) GlutaMaxTM, wherein the third medium does not comprise antibiotics; j) culturing and agitating the cells in the tissue culture vessel comprising a vertical wheel at about 10-60 rpm, preferably 23 rpm, for about 1.8 to about 2.2 days, preferably about 2 days; k) removing the third medium from the tissue culture vessel comprising a vertical wheel and adding the wash medium at a rate of about 30 mL/min; l) exchanging about 3L to about 3.5 L of the wash medium in the tissue culture vessel comprising a vertical wheel using a 40 µm cell retention filter at a pump flow rate of 30 mL/min and removing 0.1 L of the wash medium from the tissue culture vessel comprising a vertical wheel; m) adding about 50 mL of the wash medium and about 50 mL of a fourth medium to the tissue culture vessel comprising a vertical wheel, wherein the fourth medium comprises Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further 59 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 comprises SCF, IL-6, TPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, wherein the fourth medium does not comprise antibiotics; n) culturing and agitating the cells in the tissue culture vessel comprising a vertical wheel at about 10-60 rpm, preferably 23 rpm, for about 1.8 to about 2.2 days, preferably about 2 days; o) simultaneously removing the fourth medium from the tissue culture vessel comprising a vertical wheel and adding a fifth medium to the tissue culture vessel comprising a vertical wheel over about 1.8 days to about 2.2 days, preferably about 2 days, using a 4 µm cell retention filter at a pump flow rate of about 0.69 mL/min (1 L/day), until 2 L of media have been exchanged, wherein the fifth medium comprises Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, IL- 6, TPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, wherein the fifth medium does not comprise antibiotics; p) removing an entire cell volume comprising EBs and iHSCs from the tissue culture vessel comprising a vertical wheel and centrifuging the entire cell volume, thereby generating a first supernatant and a first cell pellet; q) centrifuging the first supernatant, thereby generating a second supernatant and a second cell pellet, removing the second supernatant, and resuspending the second cell pellet in Advanced DMEM/F12 medium and StemFit® For Differentiation medium without cytokines, growth factors, or antibiotics; r) adding a first volume of 20 mg/mL collagenase II to the first cell pellet, resuspending the first cell pellet, and adding a second volume of 20 mg/mL collagenase II, thereby generating a first cell suspension; s) adding a volume of Advanced DMEM/F12 medium and StemFit® For Differentiation medium without cytokines, growth factors, or antibiotics equal to the volume of the first cell suspension to the first cell suspension; t) centrifuging the cell suspension, thereby generating a third cell pellet; u) resuspending the third cell pellet in a volume of TrypLE and adding a volume of Advanced DMEM/F12 medium and StemFit® For Differentiation medium without cytokines, growth factors, or antibiotics, thereby generating a second cell suspension; 60 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 v) mixing the second cell suspension until clusters of cells are no longer visible; w) optionally passing the second cell suspension through a cell strainer to obtain iHSCs, wherein the cell strainer is 70 µm mesh size, if clusters of cells are visible after the mixing step; x) centrifuging the second cell suspension; y) not introducing serum in any of steps a)-x); z) not introducing any additional cells, including feeder cells or stromal cells, in any of steps a)-y); aa) determining the purity of the iHSCs from step w), said purity defined as CD34+ and lineage marker-negative, where the lineage markers comprise one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a; and bb) obtaining de novo generated iHSCs. [0261] In some embodiments, the cells are cultured under normoxic conditions. [0262] In some embodiments, the method further comprises further purifying and/or isolating the redifferentiated iHSCs. [0263] In some embodiments, the redifferentiated iHSCs are isolated differentiated cells or are capable of being further purified and/or isolated. [0264] In some embodiments, determining purity of the redifferentiated iHSCs comprises assaying for one or more of lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a via flow cytometry or an equivalent thereof. [0265] In some embodiments, the lineage markers comprise all of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a. [0266] In an aspect, provided herein is a method of de novo generation of hematopoietic stem cells (iHSCs) derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue culture vessel, said method comprising: a means for seeding human γδ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; a means for redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; a means for retaining all cells in the culture on day 8 of culture while removing the medium from the culture for about 2 days; and a means for harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction. 61 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 Cells Produced by the Methods [0267] In an aspect, provided herein is one or more cells obtained from a method of de novo generation of hematopoietic stem cells (iHSCs) derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue culture vessel, said method comprising: seeding human γδ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; retaining all cells in the culture on day 8 of culture while removing the medium from the culture for about 2 days; and harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction. [0268] In some embodiments, the tissue culture vessel has non-traditional bioreactor geometries. [0269] In some embodiments, the iPSCs are seeded at a density of about 1.0 x 105 cells/mL to about 1.8 x 105 cells/mL. [0270] In some embodiments, the tissue culture vessel comprising a vertical wheel further comprises a first medium comprising StemFit® Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, Emricasan, Polyamines, and Trans‑ISRIB (CEPT) cocktail, wherein the BMP4 and the rhVEGF are present in a ratio of 1:1, and wherein the first medium does not comprise antibiotics. [0271] In some embodiments, the method further comprises exchanging spent medium in the tissue culture vessel comprising a vertical wheel on each of days 2, 4, and 6 of culture for the about 8 days, wherein the step of exchanging spent medium comprises: removing the spent medium from the tissue culture vessel comprising a vertical wheel using a 40 µm cell retention filter and adding a wash medium at a pump flow rate of 30 mL/min, the wash medium comprising Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio; exchanging about 3 L to about 3.5 L of the wash medium in the bioreactor and removing 0.1 L of the wash medium from the tissue culture vessel comprising a vertical wheel using the 40 µm cell retention filter; and adding about 50 mL of the wash medium and about 50 mL of a medium to from the tissue culture vessel comprising the vertical wheel. [0272] In some embodiments, the iPSCs are dissociated into single cells on day 7 of culture. 62 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 [0273] In some embodiments, the step of retaining all cells comprises retaining clusters, semi- clusters, and single cells. [0274] In some embodiments, the step of retaining all cells comprises removing spent medium from the tissue culture vessel comprising a vertical wheel using a 4 µm cell retention filter and adding fresh medium to the bioreactor over about 1.8 days to about 2.2 days, preferably about 2 days, at a rate of about 0.69 mL/min (1 L/day), until 2 L of media have been exchanged, wherein the fifth medium comprises Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, IL-6, TPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, wherein the fresh medium does not comprise antibiotics. [0275] In some embodiments, the vertical wheel spins at a rate of about 10 rpm to about 60 rpm, preferably about 23 rpm. [0276] In some embodiments, the step of harvesting comprises: a) removing an entire cell volume comprising EBs and iHSCs from the tissue culture vessel comprising a vertical wheel and centrifuging the entire cell volume, thereby generating a first supernatant and a first cell pellet; b) centrifuging the first supernatant, thereby generating a second supernatant and a second cell pellet, removing the second supernatant, and resuspending the second cell pellet in Advanced DMEM/F12 medium and StemFit® For Differentiation medium without cytokines, growth factors, or antibiotics; c) adding a first volume of 20 mg/mL collagenase II to the first cell pellet and the second cell pellet, combining the resuspended first cell pellet and the resuspended second cell pellet and adding a second volume of 20 mg/mL collagenase II, thereby generating a combined cell suspension; d) adding a volume of Advanced DMEM/F12 medium and StemFit® For Differentiation medium without cytokines, growth factors, or antibiotics equal to the volume of the combined cell suspension to the combined cell suspension; e) centrifuging the combined cell suspension, thereby generating a third cell pellet; f) resuspending the third cell pellet in a volume of TrypLE and adding a volume of Advanced DMEM/F12 medium and StemFit® For Differentiation medium without cytokines, growth factors, or antibiotics, thereby generating a fourth cell suspension; 63 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 g) mixing the fourth cell suspension until clusters of cells are no longer visible; h) optionally passing the fourth cell suspension through a cell strainer to obtain iHSCs, wherein the cell strainer is 70 µm mesh size, if clusters of cells are visible after the mixing step; and i) centrifuging the fourth cell suspension. [0277] In some embodiments, the cells are cultured under normoxic conditions. [0278] In some embodiments, the method further comprises further purifying and/or isolating the redifferentiated iHSCs. [0279] In some embodiments, the redifferentiated iHSCs are isolated differentiated cells or are capable of being further purified and/or isolated. [0280] In some embodiments, determining purity of the redifferentiated iHSCs comprises assaying for one or more of lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a via flow cytometry or an equivalent thereof. [0281] In some embodiments, the lineage markers comprise all of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a. [0282] In an aspect, provided herein is one or more cells obtained from a method of de novo generation of hematopoietic stem cells (iHSCs) derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue culture vessel, said method comprising: seeding human γδ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction. [0283] In some embodiments, the tissue culture vessel has non-traditional bioreactor geometries. [0284] In some embodiments, the iPSCs are seeded at a density of about 1.0 x 105 cells/mL to about 1.8 x 105 cells/mL. [0285] In some embodiments, the tissue culture vessel comprising a vertical wheel further comprises a first medium comprising StemFit® Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, 64 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 Emricasan, Polyamines, and Trans‑ISRIB (CEPT) cocktail, wherein the BMP4 and the rhVEGF are present in a ratio of 1:1, and wherein the first medium does not comprise antibiotics. [0286] In some embodiments, the method further comprises exchanging spent medium in the tissue culture vessel comprising a vertical wheel on each of days 2, 4, 6, and 8 of culture for the about 8 days, wherein the step of exchanging the spent medium comprises: a) allowing cell aggregates to settle in the tissue culture vessel comprising a vertical wheel for about 3 to about 5 minutes; b) collecting 80% of the spent medium from the tissue culture vessel comprising a vertical wheel; c) centrifuging the collected medium, thereby obtaining a cell pellet; d) resuspending the cell pellet in StemFit® for Differentiation, thereby obtaining a cell suspension; and e) adding the cell suspension and fresh medium to the tissue culture vessel comprising a vertical wheel. [0287] In some embodiments, the step of exchanging the spent medium on days 2, 4, and 6 of culture comprises: i) allowing cell aggregates to settle in the tissue culture vessel comprising a vertical wheel for about 3 to about 5 minutes; collecting 80% of the spent medium from the tissue culture vessel comprising a vertical wheel; centrifuging the collected medium, thereby obtaining a cell pellet; resuspending the cell pellet in StemFit® for Differentiation medium, thereby obtaining a cell suspension; and adding the cell suspension and fresh medium to the tissue culture vessel comprising a vertical wheel, or ii) allowing cell aggregates to settle in the tissue culture vessel comprising a vertical wheel for about 3 to about 5 minutes; collecting 80% of the spent medium from the tissue culture vessel comprising a vertical wheel; centrifuging the collected medium, thereby obtaining a first cell pellet and a first supernatant; resuspending the first cell pellet in StemFit® for Differentiation medium and adding the resuspended first cell pellet to the tissue culture vessel comprising a vertical wheel; centrifuging the supernatant, thereby obtaining a second cell pellet and a second supernatant; and resuspending the second cell pellet in StemFit® for Differentiation medium and 65 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 adding the resuspended second cell pellet and fresh medium to the tissue culture vessel comprising a vertical wheel. [0288] In some embodiments, the fresh medium added on day 2 of culture comprises: i) Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SB431542 (inhibitor of Activin/BMP/TGFβ pathway), recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), and one of L-glutamine and GlutaMaxTM supplement, or ii) StemProTM-34 Serum-free Medium (SFM) and StemPro™-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, Insulin-Transferrin-Selenium (ITS-G), monothioglycerol, SB431542, recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), and one of L-glutamine and GlutaMaxTM, wherein the VEGF, bFGF, and SCF are present in a ratio of 1:0.625:0.625, and wherein the second medium does not comprise antibiotics. [0289] In some embodiments, the iPSCs are dissociated into single cells on day 7 of culture. [0290] In some embodiments, the step of harvesting redifferentiated iHSCs comprises retaining clusters, semi-clusters, and single cells. [0291] In some embodiments, the step of harvesting comprises retaining all cells in the tissue culture vessel comprising a vertical wheel. [0292] In some embodiments, the vertical wheel spins at a rate of about 10 rpm to about 60 rpm, preferably about 40 rpm. [0293] In some embodiments, the step of harvesting redifferentiated iHSCs comprises: a) removing an entire cell volume comprising embryoid bodies (EBs) and iHSCs from the tissue culture vessel comprising a vertical wheel and centrifuging the entire cell volume, thereby generating a first supernatant and a first cell pellet; b) centrifuging the first supernatant, thereby generating a second supernatant and a second cell pellet and removing the second supernatant; c) resuspending the first cell pellet and the second cell pellet in Advanced DMEM/F12 medium without cytokines, growth factors, or antibiotics; d) adding a volume of 20 mg/mL collagenase II to the first cell pellet, thereby generating a first cell suspension; e) adding a volume of Advanced DMEM/F12 medium without cytokines, growth factors, 66 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 or antibiotics equal to the volume of the first cell suspension to the first cell suspension; f) centrifuging the first cell suspension, thereby generating a third cell pellet; g) resuspending the third cell pellet in a volume of TrypLE and adding a volume of Advanced DMEM/F12 medium without cytokines, growth factors, or antibiotics, thereby generating a second cell suspension; h) mixing the second cell suspension until clusters of cells are no longer visible; i) optionally passing the second cell suspension through a cell strainer to obtain iHSCs, wherein the cell strainer is 70 µm mesh size, if clusters of cells are visible after the mixing step; and j) centrifuging the second cell suspension. [0294] In some embodiments, the cells are cultured under normoxic conditions. [0295] In some embodiments, the method further comprises further purifying and/or isolating the redifferentiated iHSCs. [0296] In some embodiments, the redifferentiated iHSCs are isolated differentiated cells or are capable of being further purified and/or isolated. [0297] In some embodiments, the one or more cells have high purity, viability, fold expansion, and potential to redifferentiate into immune effectors, including iPSC-derived γδ (iγδ) T cells. [0298] In some embodiments, the step of determining purity of the iHSCs comprises assaying for one or more, optionally all, of the lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a via flow cytometry or an equivalent thereof. [0299] In some embodiments, the lineage markers comprise one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a. [0300] In an aspect, provided herein is one or more cells obtained from a method of de novo generation of hematopoietic stem cells (iHSCs) derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue culture vessel, said method comprising: a means for seeding human γδ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; a means for redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; a means for retaining all cells in the culture on day 8 of culture while removing the medium from the culture for about 2 days; and a means for harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a 67 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 single cell fraction and a multicellular fraction. [0301] In one aspect, provided herein is one or more cells obtained from a method of de novo generation of hematopoietic stem cells (iHSCs) derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue culture vessel, said method comprising: a means for seeding human γδ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; a means for redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; a means for harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction. [0302] In one aspect, provided herein is one or more cells obtained from a method of de novo generation of iHSCs under serum-free and feeder-free culture conditions, wherein said method comprises: i. obtaining at least one human iPSC clone, and wherein the method further comprises one or more of the following steps: a. redifferentiating the at least one human iPSC clone by culturing the at least one human iPSC clone in a first cell culture medium generating a first cell suspension, wherein said first cell culture medium comprises one of StemFit® Basic 04 medium and StemFit® Basic 03 medium supplemented with basic fibroblast growth factor (bFGF) to a final concentration of about 100 ng/mL bFGF, wherein said first cell culture medium further comprises CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, Emricasan, Polyamines, and Trans‑ISRIB (CEPT) cocktail, wherein culturing is performed in a vertical wheel bioreactor at about 10-60 rpm, preferably 40 rpm, for about 1.8 to about 2.2 days, preferably about 2 days; b. centrifuging the first cell suspension comprising iPSCs of step a) to form a first pellet and resuspending the first pellet into a second cell 68 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 culture medium forming a second cell suspension, wherein said second cell culture medium comprises: i. Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises one of L-glutamine and GlutaMaxTM, SB431542 (inhibitor of Activin/BMP/TGFβ pathway), rhVEGF, bFGF, and stem cell factor (SCF), or ii. StemProTM-34 Serum-free Medium (SFM) and StemPro™-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2- phosphate, Insulin-Transferrin-Selenium (ITS-G), monothioglycerol, SB431542, rhVEGF, bFGF, SCF, and one of L-glutamine and GlutaMaxTM, and wherein the second cell culture medium does not comprise antibiotics; c. culturing the second cell suspension comprising iHSCs in the vertical wheel bioreactor at about 10-60 rpm, preferably 40 rpm, for about 1.8 to about 2.2 days, preferably about 2 days; d. stopping the agitation of the vertical wheel bioreactor for about 5 minutes and collecting about 80% of the second cell suspension; e. centrifuging the 80% of the second cell suspension, thereby generating a second cell pellet; f. suspending the second pellet in a third cell culture medium forming a third cell suspension, wherein the third cell culture medium comprises: i. Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, FMS-like tyrosine kinase 3 ligand (Flt3L), interleukin-6 (IL-6), rhVEGF, IL-3, one of (i) thrombopoietin (TPO) and (ii) erythropoietin (EPO), and one of (i) L-glutamine and (ii) GlutaMaxTM, or 69 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 ii. StemPro™-34 SFM and StemPro™-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, rhVEGF, IL-3, IL-6, Flt3L, one of (i) TPO and (ii) EPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, and wherein the third medium does not comprise antibiotics, and placing the third cell suspension into the vertical wheel bioreactor with remaining second cell suspension, thereby forming a fourth cell suspension; g. culturing fourth cell suspension in the vertical wheel bioreactor for an additional about 1.8 to about 2.2 days, preferably about 2 days; h. stopping the vertical wheel bioreactor for about 5 minutes; i. collecting about 80% of the fourth cell suspension and centrifuging the about 80% of the fourth suspension, thereby generating a third cell pellet; j. resuspending the third cell pellet in a fourth cell culture medium forming a fifth cell suspension, wherein the fourth cell culture medium comprises: i. Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, IL-6, one of (i) TPO and (ii) EPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, or ii. StemPro™-34 SFM and StemPro™-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, IL-6, one of (i) TPO and (ii) EPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, and adding the fifth cell suspension into remaining fourth cell suspension in the vertical wheel bioreactor, thereby generating a sixth cell suspension; 70 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 k. culturing the sixth cell suspension for about 1.8 to about 2.2 days, preferably about 2 days in the vertical wheel bioreactor; l. repeating steps h) to k); m. centrifuging the eighth cell suspension generating a first supernatant and a fourth cell pellet; n. collecting and centrifuging the first supernatant, thereby generating a fifth cell pellet and resuspending the fifth cell pellet in a fifth medium thereby generating a ninth cell suspension, wherein the fifth medium comprises Advanced DMEM/F12 medium, wherein the fifth medium does not comprise cytokines, antibiotics or growth factors, and wherein cells of the ninth cell suspension comprises iHSCs, which are defined as CD34+ and lineage marker-negative, where the lineage markers comprise one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a; o. resuspending the fourth cell pellet of step m) in a volume of about 20 mg/mL collagenase type II, thereby generating a tenth cell suspension; p. adding the fifth medium to the tenth cell suspension; q. centrifuging the tenth cell suspension, thereby generating a sixth cell pellet, and adding TrypLE to the sixth cell pellet, thereby generating an eleventh cell suspension; r. adding additional fifth medium to the eleventh cell suspension and mixing; s. centrifuging the eleventh cell suspension to form a seventh cell pellet and resuspending the seventh cell pellet in additional fifth medium to form a twelfth cell suspension; t. passing the twelfth cell suspension through a cell strainer to obtain iHSCs, wherein the cell strainer is 70 µm mesh size; u. determining the purity of the iHSCs from the first supernatant of step n) and the iHSCs from step t), said purity defined as CD34+ and lineage marker-negative, where the lineage markers comprise one or 71 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a; v. not introducing serum in any of steps a)-u); w. not introducing any additional cells, including feeder cells or stromal cells, in any of steps a)-v); and x. obtaining de novo generated iHSCs. [0303] In some embodiments, the one or more cells have high purity, viability, fold expansion, and potential to redifferentiate into immune effectors, including iPSC-derived γδ (iγδ) T cells. [0304] In some embodiments, the EB and γδ T cell-derived iPSCs are from a tissue culture vessel. [0305] In some embodiments, the first, second, fourth, sixth, and/or eighth cell suspensions are cultured under normoxic conditions. [0306] In some embodiments, the method further comprising further purifying and/or isolating the iHSCs. [0307] In some embodiments, the obtained iHSCs are isolated redifferentiated cells or are capable of being further purified and/or isolated. [0308] In some embodiments, the method further comprises assaying for all of the lineage markers CD3, CD19, CD14, CD11b, CD56, CD20, CD16, CD2, and CD235a via flow cytometry or an equivalent thereof. [0309] In some embodiments, any one of the comprises gently dispersing the cells using a pipet or equivalent thereof. [0310] In some embodiments, cells of the first medium are at a concentration of about 80,000 to about 180,000 cells/mL in a volume of about 50 to about 70 mL. [0311] In some embodiments, the cells of the first medium are at a concentration of about 150,000 cells/mL in a volume of about 60 mL. [0312] In some embodiments, the medium of step a) further comprises a ROCK inhibitor. [0313] In some embodiments, the medium of step a) further comprises a Chroman 1, Emricasan, Polyamines, and Trans‑ISRIB (CEPT) cocktail. [0314] In some embodiments, after step a) all the cell volume of the first cell suspension is removed from the bioreactor prior to centrifugation. [0315] In some embodiments, the cell morphology is imaged via microscopy, e.g., bright field 72 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 microscopy. [0316] In some embodiments, the medium in any one of the steps comprises L-glutamine. [0317] In some embodiments, the medium in any one of the steps comprises GlutaMaxTM. [0318] In some embodiments, the medium in any one of the steps comprises TPO. [0319] In some embodiments, the medium in any one of the steps comprises EPO. [0320] In some embodiments, the centrifugation of any one of the steps is performed at 100g for 1 minute at brake 6. [0321] In some embodiments, the centrifugation of any one of the steps is performed at 300g for 5 minutes. [0322] In some embodiments, the centrifugation of any one of the steps is performed at 300g for 3 minutes. [0323] In some embodiments, the centrifugation in any one of the steps is performed twice, first at 100g for 1 minute at brake 6 and second at 300g for 5 minutes. [0324] In some embodiments, the tenth cell suspension is incubated with collagenase for 20 minutes at 37 °C. [0325] In some embodiments, the eleventh cell suspension is incubated with TrypLE for 5 minutes at 37 °C. [0326] In some embodiments, passing of the twelfth cell suspension further comprises passing the twelfth cell suspension through a second cell strainer with a 40 µm mesh size. [0327] In some embodiments, the culturing of the iPSC cells, EBs, and iHSCs is performed at 37 °C and 5% CO2. [0328] In some embodiments, the bioreactor wheel is rotating at 60 rpm. [0329] In some embodiments, the bioreactor wheel is rotating at 30 rpm. [0330] In some embodiments, the bioreactor wheel is rotating at 24 rpm. [0331] In some embodiments, the bioreactor wheel is rotating at 15 rpm. [0332] In some embodiments, the bioreactor wheel is perpendicular to the bioreactor vessel. [0333] In some embodiments, the bioreactor wheel is not perpendicular to the bioreactor vessel. [0334] In some embodiments, iPSCs of the first medium are at a concentration of about 80,000 to about 180,000 cells/mL in a volume of about 90 to about 110 mL. [0335] In some embodiments, the iPSCs of the first medium are at a concentration of about 73 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 150,000 cells/mL in a volume of about 100 mL. [0336] In some embodiments, iPSCs of the first medium are at a concentration of about 5,000 to about 180,000 cells/mL in a volume of about 0.45 to about 0.55 L. [0337] In some embodiments, the iPSCs of the first medium are at a concentration of about 20,000 cells/mL in a volume of about 0.5 L. [0338] In some embodiments, iPSCs of the first medium are at a concentration of about 5,000 to about 180,000 cells/mL in a volume of about 2 to about 5 L. [0339] In some embodiments, iPSCs of the first medium are at a concentration of about 5,000 to about 180,000 cells/mL in a volume of about 10 to about 20 L. [0340] In one aspect, provided herein is a composition comprising the one or more cells as described herein. [0341] In one aspect, provided herein is a use of the one or more cells described herein, in preparation of cells for treating diseases, in preparation of lymphocytes, in a bioreactor, in tissue engineering or in vitro drug screening for diseases. [0342] Cell viability decreases have been reported due to single-cell enzymatic dissociation of hPSCs. Thus, typically, hPSCs are grown as colonies or aggregates and clump seeding is used as the inoculation strategy for bioreactor culture. This strategy has resulted in limitations to scalability, as for example clump sizes are difficult to control, leading to apoptosis and differentiation due to heterogeneous aggregate sizes. Studies using single-cell seeding as the inoculation strategy with traditional bioreactors have required large cell seeding densities and led to low cell production. Surprisingly, the invention herein overcomes challenges by maintaining single cells throughout the processes described herein by using, for example, a cell retention filter that does not remove single cells, but rather preserves them. The presence of single cells and thus a heterogeneous environment made of single cells and cell aggregates has been unexpectedly found to advance such processes, benefiting cell yield and quality. [0343] The present invention therefore positively impacts bioecosystems and the environment by driving advancements that help reduce human resources, power, energy consumption, carbon emissions, and overall environmental impact. The present invention provides automation and efficiency features and provides more efficient processes and systems, reducing the need for extensive manual labor, while minimizing the environmental impact associated with resource- intensive activities. The automation of the present invention optimizes energy consumption, 74 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 streamlines operations, and reduces waste, resulting in improved efficiency and reduced carbon emissions. The present invention enables more sustainable production, including the development of cleaner and more resource-efficient technologies and the adoption of circular economy practices, such as recycling and waste reduction as it minimizes material waste, including biomass loss, optimizes supply chains, and implements greener manufacturing practices. The present invention thereby brings about disruptive changes that drive resource efficiency, reduce waste and associated carbon emissions, and promotes sustainability. 3D vertical-wheel bioreactor [0344] As described herein, the iPSCs suspended in a medium are cultured in a 3D vertical- wheel bioreactor, also referred to herein as a tissue culture vessel comprising a vertical wheel. Without wishing to be bound by theory, the vertical-wheel bioreactor is used to manufacture cells via rotation of a vertical-wheel impeller within a U-shaped vessel, combining radial and axial flow components. This allows for suspension and gentle agitation of the cells. The use of a vertical- wheel bioreactor is detailed within Croughan, M. S., et al. Initial power measurements for a family of novel vertical-wheel bioreactors. Can. J. Chem. Eng.2022, 1-12; Dang, T., et al. Computational fluid dynamic characterization of vertical-wheel bioreactors used for effective scale-up of human induced pluripotent stem cell aggregate culture. Can. J. Chem. Eng. 2021, 1-18; Borys, B. S., et al. Overcoming bioprocess bottlenecks in the large-scale expansion of high-quality hiPSC aggregates in vertical-wheel stirred suspension bioreactors. Stem Cell Research & Therapy. 2021, 12:55; and Cuesta-Gomez, N., et al. Suspension culture improves iPSC expansion and pluripotency phenotype. Stem Cell Research & Therapy. 2023, 14:154; all of which are incorporated herein by reference in their entirety. The 3D vertical-wheel bioreactor is surprising for its adaptation to redifferentiation of iPSC-derived intermediates, as it has been used primarily for iPSC maintenance up to this point and has faced challenges in its uses. The hydrodynamics of the 3D vertical-wheel bioreactor are different than that of a typical stirred tank bioreactor as the 3D vertical-wheel bioreactor provides a reduced cell shear. [0345] The parts of the vertical-wheel bioreactor, including the vertical-wheel impeller and the U-shaped vessel can vary in size, as a person having ordinary skill in the art would understand. In some embodiments, a 0.5 L bioreactor may use a vertical-wheel impeller with a diameter of about 73 mm and a width of about 25 mm and a U-shaped vessel with a radius of about 43 mm. The impeller radius may be about 85% of the U-shaped vessel radius. In some embodiments, a 3 75 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 L bioreactor may use a vertical-wheel impeller with a diameter of about 137 mm and a width of about 37 mm and a U-shaped vessel with a radius of about 83 mm. The impeller radius may be about 83% of the U-shaped vessel radius. In some embodiments, a 15 L bioreactor may use a vertical-wheel impeller with a diameter of about 227 mm and a width of about 113 mm and a U- shaped vessel with a radius of about 138 mm. The impeller radius may be about 83% of the U- shaped vessel radius. [0346] Computational fluid dynamics modelling has supported the use of models across scales by producing scale-up equations and calculating volume average hydrodynamic values. The k- epsilon turbulence model is a commonly used model to simulate flow characteristics in suspension bioreactors. This model uses the following two equations to represent the conservation of momentum and mass: ^^ + ∇ ∙ ρu = 0 ^^ ^ ^ ^^^ + ∇ ^ρu × u^ = −∇P + μ∇^^ + ρg ^^ wherein ρ is the density, u is the
Figure imgf000078_0001
P is pressure, µ is viscosity, and g is the gravity vector. [0347] This model can showcase the impact of changing scale and agitation rate on the velocity and energy dissipation rate (EDR) within the bioreactor. Uniform volume average hydrodynamic force parameters are desired. If the fluid velocity is not great enough for effective mixing, it can lead to fluid dead zones, where mixing does not occur. EDR is commonly used as an alternative to shear stress in the characterization of flow conditions. If the EDR is too low, the aggregates may become too large and clump together and settle. If the EDR is too high, the aggregates may be sheared apart leaving single cells unable to form aggregates. These situations lead to reduced cell proliferation, increased cell death, and aggregates lacking uniformity in shape and size. [0348] The use of traditional bioreactors has exhibited hydrodynamic environments that are unfavorable to iPSC maintenance and growth. The traditional bioreactor may provide an environment wherein the high shear stress may damage cells by tearing apart cells and cell aggregates, lowering cell yield and quality. The shear stress increases with scale, and during the expansion phase in traditional bioreactors, only moderate cell fold increases have been shown. Thus, the use of traditional bioreactors has limited the ability to scale the inoculation and harvesting phases. 76 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 [0349] The computational fluid dynamics model provides confirmation that the vertical-wheel bioreactor, by combining radial and axial flow components, creates hydrodynamic forces that are more uniformly distributed. The vertical-wheel bioreactor has lemniscate velocity streamlines whereas the traditional bioreactor has circular velocity streamlines. The vertical-wheel bioreactor has decreased dissipation energy gradients, lower shear effects, and more uniform particle suspension compared to traditional bioreactors, supporting the vertical-wheel bioreactor environment to better scale compared to the environment of traditional bioreactors. [0350] High cell-fold expansion and morphologically healthy and uniform aggregate cell growth has been shown at a scale of 0.1 L with an agitation rate of 60 rpm. Using the correlation equations, to maintain the same volume average EDR through scale-up, these values correlate with a scale of 0.5 L with an agitation rate of 30 rpm, a scale of 3 L with an agitation rate of 24 rpm, and a scale of 15 L with an agitation rate of 15 rpm. A working range of volume average EDRs for culturing human iPSCs has been suggested to be between 3.0E-4 and 1.5E-3 m2/s2. [0351] In some embodiments, the cells of the first medium are at a concentration of about 80,000 to about 180,000 cells/mL in a volume of about 50 to about 70 mL. In one embodiment, the cells of the first medium are at a concentration of about 150,000 cells/mL in a volume of about 60 mL. In some embodiments, the cells of the first medium are at a concentration of about 80,000 to about 120,000 cells/mL in a volume of about 0.09 to about 0.11 L. In one embodiment, the cells of the first medium are at a concentration of about 100,000 cells/mL in a volume of about 0.1 L. In some embodiments, the cells of the first medium are at a concentration of about 5,000 to about 180,000 cells/mL in a volume of about 0.45 L to about 0.55 L. In one embodiment, the cells of the first medium are at a concentration of about 20,000 cells/mL in a volume of about 0.5 L. [0352] In some embodiments, the vertical wheel is perpendicular to the bioreactor vessel. In some embodiments, the vertical wheel is tilted at about a 5° angle to about a 60° angle relative to the bioreactor vessel. In one embodiment, the vertical wheel is tilted at about a 20° angle relative to the bioreactor vessel. In one embodiment, the vertical wheel is tilted at about a 40° angle relative to the bioreactor vessel. [0353] In some embodiments, the bioreactor may be a fed-batch bioreactor. In some embodiments, the bioreactor may be perfused or perfusable. Perfusion is a method wherein the cells are kept within a bioreactor while the media is continuously exchanged. Fresh media is added to replenish carbon sources and other nutrients. Depleted media and cellular waste are removed. 77 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 In some embodiments, perfusion is performed via cell binding, wherein the cells are kept in the bioreactor by binding to capillary membranes or fibers. In some embodiments, the bioreactor contains capillary membranes or fibers. In some embodiments, perfusion is performed via filtration, wherein the spent medium flows through a membrane with pores that are too small to accomodate the cells. In some embodiments, the bioreactor contains membranes with pores that are too small to accomodate the cells. In some embodiments, perfusion is performed via settling, wherein the cells are allowed to settle and the rest of the suspension is removed as spent medium. In some embodiments, the the cells settle within the bioreactor. [0354] In some embodiments, the bioreactor may contain multiple bioreactor wheels. In some embodiments, the bioreactor contains 2 to 6 bioreactor wheels. In some embodiments, the bioreactor contains 2, 3, 4, 5, or 6 bioreactor wheels. In some embodiments, one or more of the bioreactor wheels are perpendicular to the bioreactor vessel. In some embodiments, one or more of the bioreactor wheels are not perpendicular to the bioreactor vessel. In some embodiments, one or more of the bioreactor wheels are rotating between about 10 and about 60 rpm. In some embodiments, one or more of the bioreactor wheels are rotating at about 60 rpm. In some embodiments, one or more of the bioreactor wheels are rotating at about 30 rpm. In some embodiments, one or more of the bioreactor wheels are rotating at about 24 rpm. In some embodiments, one or more of the bioreactor wheels are rotating at about 15 rpm. EXAMPLES [0355] The following examples are provided to further describe some of the embodiments disclosed herein. The examples are intended to illustrate, not to limit, the disclosed embodiments. Example 1: Serum-free, feeder-free, three-dimensional redifferentiation of γδ T cell-derived iPSCs into iγδ T cells. [0356] The example provided contains instructions to maintain and redifferentiate γδ T- iPSCs. Materials [0357] The following abbreviations are used herein (Table 1). 78 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 Table 1. Abbreviations. Abbreviation Definition 2D Two-dimensional bFGF Basic Fibroblast Growth Factor BMP-4 Bone Morphogenetic Protein 4 DLL4-Fc Delta-like Canonical Notch Ligand 4 – Fc chimera DPBS Dulbecco’s Phosphate-buffered Saline EPO Erythropoietin Flt3L FMS-like tyrosine kinase 3 Ligand γδ T-iPSC γδ T cell-derived iPSC HSC Hematopoietic stem cell iγδ T cell iPSC-derived γδ T cell IL-3 Interleukin-3 IL-6 Interleukin-6 iPSC induced pluripotent stem cell IU International units P/S Penicillin-Streptomycin rh Recombinant human ROCK Rho-associated, Coiled-coil Containing Protein Kinase RT Room temperature SCF Stem Cell Factor SFFF Serum-free, feeder-free TC Tissue culture VCAM-1-Fc Vascular Cell Adhesion Molecule-1 – Fc chimera VEGF Vascular Endothelial Growth Factor 79 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 [0358] The materials needed herein include γδ T-iPSC lines and the reagents necessary to perform cell culture (Tables 2-6). Table 2. Reagents for iPSC culture and maintenance. Reagent Product No. γδ T-iPSC clones Academic collaborator, contract research organization iMatrix 511 Amsbio, cat.# AMS.892012 StemFit® Basic 04 Complete Type Amsbio, cat.# SFB-504-CT rh bFGF Amsbio, cat.# AMS-FGF-100 Y-27632 – ROCK Inhibitor, 1 mg StemCell Technologies, cat# 72302 TryPLETM Select 1X ThermoFisher, cat.# 12563011 1X DPBS ThermoFisher, cat.# 14190144 CryoStor CS10 StemCell Technologies, cat.# 07930 Table 3. Reagents needed for hematopoietic redifferentiation (listed by day of redifferentiation). Reagent Product No. Day 0 Reagents StemFit® Basic 04 Complete Amsbio, cat.# SFB-504-CT CHIR99021 TOCRIS, cat.# 4423 rh BMP4 R&D, cat.# 314-BP rh VEGF R&D, cat.# 293-VE Day 2 Reagents Advanced DMEM/F12 Gibco, cat.# 12634-10 StemFit® For Differentiation Ajinomoto, cat.# 20170228A L-Glutamine Gibco, cat.# 25030-081 Penicillin-Streptomycin (P/S) Gibco, cat.# 15140-122 SB431542 Fujifilm Wako pure chemical corporation, cat.# 033-24631 rh VEGF R&D, cat.# 293-VE rh bFGF Amsbio, cat.# AMS-FGF-100 rh SCF R&D, cat.# 255-SC Day 4 Reagents 80 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 Advanced DMEM/F12 Gibco, cat.# 12634-10 StemFit® For Differentiation Ajinomoto, cat.# 20170228A L-Glutamine Gibco, cat.# 25030-081 P/S Gibco, cat.# 15140-122 rh SCF R&D, cat.# 255-SC rh VEGF R&D, cat.# 293-VE rh IL-3 PeproTech, cat.# AF-200-03 rh IL-6 R&D, cat.# 206-IL-050 rh Flt3L R&D, cat.# 308-FK-025 rh EPO R&D, cat.# 287-TC-500 Days 6-10 Reagents Advanced DMEM/F12 Gibco, cat.# 12634-10 StemFit® For Differentiation Ajinomoto, cat.# 20170228A L-Glutamine Gibco, cat.# 25030-081 P/S Gibco, cat.# 15140-122 rh SCF R&D, cat.# 255-SC rh IL-6 R&D, cat.# 206-IL-050 rh EPO R&D, cat.# 287-TC-500 Table 4. Reagents for iγδ T cell redifferentiation. Reagent Product No. StemSpan™ SFEM II StemCell Technologies, cat.# 09655 Lymphoid Progenitor Expansion Supplement (10X) StemCell Technologies, cat.# 09915 rh VCAM-1-Fc R&D, cat.# 862-VC-100 rh DLL4-Fc R&D, cat.# 10185-D4-050 81 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 Table 5. Supplies and consumables needed for cell culture. Item Product No. Corning™ Costar™ 6-well Clear TC-treated Multiple Corning, cat.# 3516 Well Plates, Individually Wrapped, Sterile Corning™ Costar™ 48-well Clear TC-treated Corning, cat.# 3548 Multiple Well Plates, Individually Wrapped, Sterile Fisherbrand™ Sterile Polystyrene Disposable ThermoFisher, Serological Pipets with Magnifier Stripe (various cat.# 13-676-10H, sizes) cat.# 13-678-11E, cat.# 13-678-11, cat.# 13-678-11F Pipette Tips RT LTS 20 µL F 960A/10 (P20) Rainin, cat.# 30389225 Pipette Tips RT LTS 200 µL F 960A/10 (P200) Rainin, cat.# 30389239 Pipette Tips RT LTS 1000 µL F 768A/8 (P1000) Rainin, cat.# 30389212 50 mL conical tubes Corning, cat.# 352098 Falcon® 15 mL Polystyrene Centrifuge Tube, Corning, cat.# 352095 Conical Bottom, with Dome Seal Screw Cap, Sterile 1.8 mL cryovials Thermofisher, cat.# 375418 1.5 mL Eppendorf tubes Greiner-bio-one cat.# 616261 Cell lifters Corning, cat.# 3008 Corning CoolCellTM Freezing container Corning, cat.# 432006 Via-1-Cassette Chemometec, cat.# 941-0012 82 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 Table 6. Antibodies for Flow Panels. Fluorochrome Marker Vendor Cat# Pluripotency BV711 SSEA-1 BioLegend 323050 PE-Cy7 SSEA-3 BioLegend 330326 PerCP/Cy5.5 CD34+ BioLegend 343522 PE SSEA-4 BioLegend 330406 AF647 OCT 3/4 BioLegend 653710 BV421 SOX2 BioLegend 656114 iHSC Panel PE CD34 BioLegend 343506 APC CD43 BD 560198 BUV395 CD45 BD 563792 BV421 CD38 BioLegend 303526 APC-Cy7 Live/dead Near IR ThermoFisher L10119 FITC CD3 BioLegend 300306 FITC CD235a BioLegend 349104 FITC CD11c BioLegend 337214 FITC CD11b BioLegend 301330 FITC CD14 BioLegend 325604 FITC CD56 BioLegend 318304 FITC CD19 BioLegend 302206 FITC CD20 BioLegend 302304 FITC CD16 BioLegend 302006 FITC CD2 BioLegend 300206 iγδ T cell redifferentiation BUV395 CD3 BD 740283 BUV496 CD25 BD 741144 BUV563 CD45RA BD 612926 BUV615 CD2 BD 751450 83 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 BUV661 CD277 (BTN3) BD 750227 BUV737 CD56 BD 612766 BUV805 CD11a BD 748572 BV421 CD197 (CCR7) BioLegend 353208 BV480 CD95 BD 746675 BV605 CD5 BioLegend 364020 sBV650 CD16 BioLegend 302042 BV711 HLA-ABC BD 565333 BV786 CD122 BD 743118 FITC Vγ9 TCR BioLegend 331306 PerCP-Cy5.5, BB700 CD215 (IL-15Rα) Invitrogen 46-7149-82 PE BTN2A1 LSBio LS-C649249-100 PE-Texas Red NKG2D BD 562498 PE-Cy5 CD1a BioLegend 300108 PE-Cy7 CD27 BioLegend 356412 APC, Alexa 647 Vδ2 TCR BioLegend 331418 Alexa 700 CD127 BD 565185 APC-Cy7 Live/dead Near IR ThermoFisher L10119 Table 7. Equipment. NucleoCounter NC-200 EVOS M7000 imager CO2 Incubator 4 °C Refrigerator -20 °C Freezer -80 °C Freezer Centrifuge 84 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 Procedure to maintain γδ T cell-derived induced pluripotent stem cell (γδ T-iPSC) lines. [0359] 1.1 Supplemented StemFit® Basic 04 Complete Type Medium was prepared as follows: a. The StemFit® Basic 04 Complete Type Medium was provided frozen and was stored at or below -20 °C until use. Sterile techniques were used to prepare Supplemented StemFit® Complete Type Medium. b. Frozen StemFit® Basic 04 Complete Type Medium was thawed with occasional mixing at room temperature (15-25 °C) or in a refrigerator (2-8 °C). c. Note: Medium was not thawed at 37 °C, as it accelerates the degradation of the medium ingredients. d. StemFit® Basic 04 Complete Type Medium was supplemented with an additional 20 ng/mL of bFGF to achieve a final concentration of 100 ng/mL bFGF in the supplemented medium. e. Supplemented medium was stored up to two (2) weeks at 4 °C. f. Medium was brought to room temperature (15-25 °C) for at least one (1) hour prior to use. [0360] 1.210 mM Y-27632 ROCK inhibitor stock solution was prepared as follows: a. Y-27632 ROCK inhibitor was received as a crystalline solid. b. Inhibitor was stored at -20 °C for up to 12 months from date of receipt. c. A 10 mM stock solution was prepared in 1X DPBS. i. 312 μL of 1X DPS was added to 1 mg Y-27632 and solution was resuspended completely. ii. 25 μL of stock solution was aliquoted into Eppendorf tubes labeled as Y-27632 stock. iii. Aliquots were stable at -20 °C for up to 6 months. iv. Working volumes were aliquoted to avoid repeated freeze-thaw cycles. [0361] 1.3 One 6-well TC-treated plate coated with iMatrix 511 was prepared as follows: a. 12 mL of 1X DPBS was pipetted into a 15 mL conical tube. 85 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 b. 30 µL of iMatrix 511 was added to 1X DPBS and immediately mixed well (coating concentration: 0.25 μg/cm2). c. 2 mL of iMatrix solution was added to each well in the 6-well plate. d. Plates were incubated at 37 °C, 5% CO2, for at least 60 minutes (plates could be left overnight at 37 °C). e. Note: After coating step, plates could be stored at 4 °C for one week. [0362] 1.4 Supplemented StemFit® Basic 04 Complete Type Medium (from Step 1.1) with 10 µM Y-27632 ROCK inhibitor was prepared as follows: a. Aliquot of 10 mM Y-27632 ROCK inhibitor was thawed prior to use. b. 25 mL of Supplemented StemFit® Basic 04 Complete Type Medium (from Step 1.1) was aliquoted into a 50 mL conical tube. c. 25 µL of 10 mM Y-27632 ROCK inhibitor was added to Supplemented StemFit® Basic 04 Complete Type Medium to achieve a final concentration of 10 µM Y- 27632 ROCK inhibitor. [0363] 1.5 γδ T-iPSC vial was thawed as follows: a. A vial of frozen γδ T-iPSCs was thawed in a 37 °C water bath for 1 minute or until small pieces of frozen floating cells were visible. b. The thawed γδ T-iPSCs were transferred from the vial slowly in a drop-wise manner into a 15 mL conical tube containing 9 mL of room temperature Supplemented StemFit® 04 Basic Complete Type Medium with 10 μM Y-27632 ROCK inhibitor (from Step 1.4). c. Note: Total volume was 10 mL. [0364] 1.6 Cell count was measured as follows: a. The NucleoCounter NC-200 instrument was used for cell counting purposes. b. 150 µL aliquot was removed from cell suspension (Step 1.5b) to 1.5 mL Eppendorf tube. c. Cells were collected from Eppendorf tube in Via1-Cassette. d. Via1-Cassette was inserted to NC-200 cell counter and cells were counted. e. Note: Only the total number of live cells was used for cell seeding purposes. [0365] 1.7 Washing and plating thawed γδ T-iPSCs was performed as follows: 86 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 a. 15 mL conical tube containing thawed γδ T-iPSCs (Step 1.5b) was centrifuged at 300 x g for 3 minutes at room temperature. b. Supernatant was aspirated without disturbing the cell pellet by leaving behind 0.5 mL of medium. c. Conical tube was gently tapped to loosen pellet. d. 1 mL of Supplemented StemFit® Basic 04 Complete Type Medium with 10 μM Y- 27632 ROCK inhibitor (Step 1.4) was added with P1000. e. Additional Supplemented StemFit® Basic 04 Basic Complete Type Medium with 10 μM Y-27632 ROCK inhibitor (Step 1.4) was added to achieve a final cell concentration of 1x104 cells/mL. f. iMatrix 511 solution was removed from the 6-well plate by aspiration. i. Note: Leaving plate to dry out for extended period (e.g., more than 10 minutes) was avoided. Wells were not allowed to dry. g. Cells were thoroughly mixed and then 2 mL/well were plated. h. Cells were distributed evenly on the bottom of the well by manually rocking the plate gently back and forth and side to side. i. Plate was incubated overnight at 37 °C in 5% CO2. [0366] 1.8 Day 1: Medium change was performed as follows: a. Supplemented StemFit® Basic 04 Complete Type Medium (from Step 1.1) was brought from 4 °C to room temperature. b. Medium was gently aspirated from 6-well plate. c. 2 mL/well of fresh Supplemented StemFit® Basic 04 Complete Type Medium (without ROCK inhibitor) was added to 6-well plate. d. Plate was imaged with EVOS M7000 and images were recorded at 4X and 10X magnification. e. Three images of different regions on the plate were taken to record the morphology and landscape of the culture. There were single cells and two-celled colonies present. Cells had a spiky morphology due to culturing in the presence of the ROCK inhibitor. i. Note: as cells divide, borders became more defined. 87 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 ii. At 10X magnification, the cells appeared with more defined borders (data not shown). [0367] 1.9 Cells continued to be cultured until they reached typical morphology, exhibiting all iPSC characteristics including circular colonies with densely packed cells, high nucleus to cytoplasmic ratio, well-defined edges, and distinct borders. iPSC colonies were at ~70-80% confluency at this stage. a. Note: It could take 7 days +/- 1 day for the colonies to exhibit this morphology. b. iPSC colonies had defined borders and compact cells. i. At 10X magnification, the iPSC colonies appeared to have defined borders and compact cells (data not shown). [0368] 1.10 Cells were passaged and pluripotency was confirmed by flow cytometry before initiating redifferentiation: a. iMatrix 511-coated 6-well plate(s) were prepared, as many as were required, as in Step 1.3, to passage iPSCs. b. Passaging iPSCs was performed as follows: i. Medium was aspirated from cells plated in Step 1.7. ii. Wells were rinsed with 1 mL 1X DPBS. iii. 1X DPBS was aspirated from wells. iv. 1 mL TrypLE was added. v. Plates were incubated at 37 °C for 6-8 minutes. vi. Wells were examined under a microscope. Cells appeared bright with clear cell borders (data not shown). vii. TrypLE was gently removed. viii. 1 mL of Supplemented StemFit® Basic 04 Complete Type Medium with 10 μM Y-27632 ROCK inhibitor (from Step 1.4) was added with P1000 and then pipetted gently to remove cells from plate. Forming bubbles was avoided. c. Wells were examined with microscope to ensure removal of 95% of the cells from wells. 88 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 i. If residual cells were still adherent to the plate, then 1 mL of StemFit® Basic 04 Complete Type Medium with 10 μM Y-27632 ROCK inhibitor (from Step 1.4) was added. Cell lifter was used to scrape the well, and solution was pipetted with P1000 three (3) times to ensure uniformed, single cell suspension. d. Wells were collected into a 15 mL conical tube. e. Cell count was performed as in Step 1.6. f. 1x106 cells were used for pluripotency flow analysis. g. Pluripotency flow panel is shown in Table 6. h. Acceptance criteria were met before proceeding to redifferentiation steps. Exemplary acceptance criteria for pluripotency were: >85% Oct4+, Sox2+, SSEA3+, SSEA4+ <1% SSEA1+ <5% CD34+ Procedure to redifferentiate γδ T-iPSC lines into hematopoietic stem cells (HSCs) using a serum- free, feeder-free (SFFF) three-dimensional (3D) culture method. [0369] 1.11 Day 0: Onset of hematopoietic redifferentiation – Mesoderm induction phase. a. StemFit® Basic 04 Complete Type Medium (Step 1.1) or StemFit® Basic 03 medium was pre-warmed at room temperature and STEP 1 medium (Table 8) was prepared for Days 0-2. Table 8. STEP 1 medium Reagent Stock Final Volume for 60mL Concentration Concentration Bioreactor StemFit® Basic 04 60 mL Complete Type Medium (Step 1.1) CHIR99021 20 mM 4 μM 12 µL Rock Inhibitor 10 mM 10 µM 60 µL rh BMP4 100 μg/mL 80 ng/mL 48 µL rh VEGF 100 μg/mL 80 ng/mL 48 µL b. Images were recorded at Day 0. 89 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 c. Media exchange was performed. The entire cell suspension was spun down at 100g for 1min at brake 6. Reagents were taken out to thaw.500 uL metabolic sample was taken and the rest of the supernatant was removed. The cells were resuspended in fresh STEP 1 media (Table 8) and added to the vertical-wheel bioreactor. Growth factors were then added to the reactor. d. Cells were cultured at 37 °C, 5% CO2. [0370] 1.12 Day 2: Onset of hematopoietic redifferentiation – Mesoderm to definitive hematopoietic lineage specification. a. At Day 2, Advanced DMEM/F12 medium was pre-warmed at room temperature and STEP 2 medium (Table 9) or Alternative STEP 2 medium (Table 10) was prepared for Days 2-4. 90 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 Table 9. STEP 2 medium Reagent Stock Final Volume for 60mL Concentration Concentration Bioreactor Advanced DMEM/F12 100% 48 mL StemFit® For Differentiation 100% 20% 12 mL L-Glutamine 100X 1X 600 µL SB431542 5 mM 2 μM 24 µL rh VEGF 100 μg/mL 80 ng/mL 48 µL bFGF 100 μg/mL 50 ng/mL 30 µL SCF 500 μg/mL 50 ng/mL 6.0 µL Table 10. Alternative STEP 2 medium Reagent Stock Final Volume for 60 mL Concentration Concentration Bioreactor StemPro™-34 SFM + 100% 60 mL StemPro™-34 Nutrient Supplement Ascorbic Acid 2-phosphate 32.2 mg/mL 50 μg /mL 93 µL Insulin-Transferrin-Selenium 100x 1x 600 µL (ITS-G) Monothioglycerol 11.5 M 0.4 mM 2 µL L-Glutamine 100X 1X 600 µL SB431542 5 mM 2 μM 24 µL rh VEGF 100 μg/mL 80 ng/mL 48 µL bFGF 100 μg/mL 50 ng/mL 30 µL SCF 500 μg/mL 50 ng/mL 6 µL b. Images were recorded at Day 2 as in Step 1.11b. c. Media exchange was performed. Aggregates were allowed to settle in reactor for approx. 5 minutes. Then, media was pulled from the vertical wheel leaving 20% volume left in the reactor. The cell suspension was spun down at 100g for 1min at 91 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 brake 6. Reagents were taken out to thaw. 500 uL metabolic sample was taken and the rest of the supernatant was removed. The cells were resuspended in fresh STEP 2 media (Table 9) or fresh Alternative STEP 2 media (Table 10) and added back to the reactor. Growth factors were then added to the reactor. d. Cells were incubated and cultured at 37 °C, 5% CO2 for two days. [0371] 1.13 Day 4: Onset of hematopoietic redifferentiation – Hemangioblast induction phase. a. At Day 4, Advanced DMEM/F12 medium was pre-warmed at room temperature and STEP 3 medium (Table 11) or Alternative STEP 3 medium (Table 12) was prepared for Days 4-6. Table 11. STEP 3 medium Reagent Stock Final Volume for 60mL Concentration Concentration Bioreactor Advanced DMEM/F12 100% 38.4 mL StemFit® For Differentiation 100% 20% 9.6 mL L-Glutamine 100X 1X 480 µL SCF 500 μg/mL 50 ng/mL 4.8 µL rh VEGF 100 μg/mL 20 ng/mL 9.6 µL rh IL-3 100 μg/mL 50 ng/mL 24 µL rh IL-6 100 μg/mL 50 ng/mL 24 µL rh Flt3L 100 μg/mL 50 ng/mL 24 µL rh EPO 500 IU/mL 10 IU/mL 960 µL Alternative to rh EPO: rh TPO 100 ug/mL 30 ng/mL 14.4 µL 92 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 Table 12. Alternative STEP 3 medium Reagent Stock Final Volume for 60mL Concentration Concentration Bioreactor StemPro™-34 SFM + StemPro™-34 100% 48 mL Nutrient Supplement Ascorbic Acid 2-phosphate 32.2 mg/mL 50 μg/mL 74.4 µL Insulin-Transferrin-Selenium (ITS-G) 100x 1x 480 µL Monothioglycerol 11.5 M 0.4 mM 1.6 µL L-Glutamine 100X 1X 480 µL SCF 500 μg/mL 50 ng/mL 4.8 µL rh VEGF 100 μg/mL 20 ng/mL 9.6 µL rh IL-3 100 μg/mL 50 ng/mL 24 µL rh IL-6 100 μg/mL 50 ng/mL 24 µL rh Flt3L 100 μg/mL 50 ng/mL 24 µL rh EPO 500 IU/mL 10 IU/mL 960 uL Alternative to rh EPO: rh TPO 100 ug/mL 30 ng/mL 14.4 µL b. Images were recorded at Day 4 as in Step 1.11b. c. Media exchange was performed. Aggregates were allowed to settle in reactor for approx. 5 minutes. Then, media was pulled from the vertical wheel leaving 20% volume left in the reactor. Supernatant was spun down at 100g for 1min at brake 6. Reagents were taken out to thaw. 500 uL metabolic sample was taken and the rest of the supernatant was removed. The cells were resuspended with fresh STEP 3 media (Table 11) or Alternative STEP 3 media (Table 12). Solution was added back to the reactor. Growth factors were then added to the reactor. d. Cells were incubated and cultured at 37 °C, 5% CO2 for two days. [0372] 1.14 Day 6: Onset of hematopoietic redifferentiation – Endothelial to hematopoietic transition phase. a. On Days 6 and 8, Advanced DMEM/F12 medium was pre-warmed at room temperature and fresh STEP 4 medium (Table 13) or Alternative STEP 4 medium (Table 14) was prepared. 93 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 Table 13. STEP 4 medium Reagent Stock Final Volume for 60mL Concentration Concentration Bioreactor Advanced DMEM/F12 100% 38.4 mL StemFit® For Differentiation 100% 20% 9.6 mL L-Glutamine 100X 1X 480 µL rh SCF 500 μg/mL 50 ng/mL 4.8 µL rh IL-6 100 μg/mL 50 ng/mL 24 µL rh EPO 500 IU/mL 10 IU/mL 960 µL Alternative to rh EPO: rh TPO 100 ug/mL 30 ng/mL 14.4 µL Table 14. Alternative STEP 4 medium Reagent Stock Final Volume for Concentration Concentration 60mL Bioreactor StemPro™-34 SFM + StemPro™-34 100% 48 mL Nutrient Supplement Ascorbic Acid 2-phosphate 32.2 mg/mL 50 μg /mL 74.4 µL Insulin-Transferrin-Selenium (ITS-G) 100x 1x 480 µL Monothioglycerol 11.5 M 0.4 mM 1.6 µL L-Glutamine 100X 1X 480 µL rh SCF 500 μg/mL 50 ng/mL 4.8 µL rh IL-6 100 μg/mL 50 ng/mL 24 µL rh EPO 500 IU/mL 10 IU/mL 960 uL Alternative to rh EPO: rh TPO 100 ug/mL 30 ng/mL 14.4 µL b. Images were recorded at Day 6 as in Step 1.11b. c. Media exchange was performed. Aggregates were allowed to settle in reactor for approx. 5 minutes. Then, media was pulled from the vertical wheel leaving 20% volume left in the reactor. Supernatant was spun down at 100g for 1min at brake 6. Reagents were taken out to thaw. 500 uL metabolic sample was taken and the rest of the supernatant was removed. The cells were resuspended with fresh STEP 4 94 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 media (Table 13) or Alternative STEP 4 media (Table 14). Solution was added back to the reactor. Growth factors were then added to the reactor. d. Cells were incubated and cultured at 37 °C, 5% CO2 for two days. e. On Day 8, 48 mL of STEP 4 medium (Table 13) or Alternative STEP 4 medium (Table 14) were prepared. f. Aggregates were allowed to settle in reactor for approx. 5 minutes. Then, media was pulled from the vertical wheel leaving 20% volume left in the reactor. Supernatant was spun down at 300g for 5min. Reagents were taken out to thaw. 500 uL metabolic sample was taken and the rest of the supernatant was removed. The cells were resuspended with fresh STEP 4 media (Table 13) or Alternative STEP 4 media (Table 14). Solution was added back to the reactor. Growth factors were then added to the reactor. g. Cells were incubated and cultured at 37 °C, 5% CO2 for another two days. [0373] 1.15 Day 10: Harvesting hematopoietic stem cells (iHSC/iHSCs) and preparing cells for flow cytometric analysis and cryopreservation was performed as follows: a. Images were recorded at Day 10 as in Step 1.11b. b. 21 mL of STEP 4 base medium (Step 1.14a) without cytokines and growth factors were prepared. c. Single cell fraction was processed as follows: i. Pull entire cell volume out of reactor, transfer 30 mL to each of 2x 50 mL conicals, and spin down at 100g for 1 min at brake 6. Pellets will contain clusters, and single cells will remain in supernatant. The supernatant was discarded, and cells were resuspended in 2 mL of Collagenase II. Cells was incubated for 20 minutes at 37 °C. ii. Transfer supernatant, containing single cells, to new 50 mL conicals. Spin down at 300g for 5 min at brake 9. iii. After centrifuging, collect 500 uL from cell supernatant for metabolic sample. Remove rest of supernatant and resuspend pellet in 5 mL of the STEP 4 base medium without added cytokines or growth factors (Step 1.15b). iv. Cell count was performed using NC-200. d. Cluster cell fraction was processed as follows: 95 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 i. Add 1 mL collagenase to each pellet from Step 1.15c.i. Resuspend and combine into one 50 mL conical (total volume of collagenase = 2 mL). Incubate for 20 minutes at 37 °C. ii. Add equal volume (2 mL) STEP 4 base medium without added cytokines or growth factors (Step 1.15.b). Centrifuge at 300g for 5 min at brake 9. iii. Remove supernatant, add 2 mL TrypLE, and mix solution. Incubate for 5 minutes at 37 °C. iv. Add 8 mL STEP 4 base medium without added cytokines or growth factors (Step 1.15.b) and mix solution. v. Pass cells through 70 μm filter and centrifuge at 300g for 5 min at brake 9. vi. Resuspend in 10 mL of STEP 4 base medium without added cytokines or growth factors (Step 1.15.b), and perform cell count using NC-200. e. 2.0x106 cells were aliquoted for flow cytometric analysis using the iHSC panel. f. iHSC flow panel is shown in Table 6. g. Exemplary success criteria for iHSC redifferentiation step: purity of >70% Lineage- CD34+ cells and >5 fold expansion per input iPSC. [0374] The protocol enabled redifferentiation of γδ T-iPSC lines into hematopoietic stem cells (HSCs) at a variety of RPM in a 0.1L vertical wheel bioreactor using a serum-free, feeder- free (SFFF) three-dimensional (3D) culture method, the results including cell cluster formation, morphology, cell growth (e.g., metabolism), purity, etc. This was true with perfusion in the bioreactor system. Example 2: Serum-free, feeder-free redifferentiation of γδ T cell-derived iPSCs into iγδ T cells. [0375] The example provided contains instructions to redifferentiate γδ T-iPSCs, using GMP-compliant or GMP-like reagents. Materials [0376] The following abbreviations are used herein (Table 15). Table 15. Abbreviations. Abbreviation Definition 3D Three-dimensional 96 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 bFGF Basic Fibroblast Growth Factor BMP-4 Bone Morphogenetic Protein 4 DPBS Dulbecco’s Phosphate-buffered Saline Flt3L FMS-like tyrosine kinase 3 Ligand γδ T-iPSC γδ T cell-derived iPSC HSC Hematopoietic stem cell IL-3 Interleukin-3 IL-6 Interleukin-6 iPSC induced pluripotent stem cell IU International units rh Recombinant human ROCK Rho-associated, Coiled-coil Containing Protein Kinase RT Room temperature SCF Stem Cell Factor SFFF Serum-free, feeder-free TC Tissue culture TPO Thrombopoietin VEGF Vascular Endothelial Growth Factor [0377] The materials needed herein include γδ T-iPSC lines and the reagents necessary to perform each cell culture step (Tables 16-21). Table 16. Reagents for iPSC culture and maintenance. Reagent Product No. Storage Condition StemFit® Basic 04 Amsbio, cat.# SFB-504- -20 °C, upon thaw use within two Complete Type CT weeks and store at 4 °C. rh bFGF Peprotech, cat.# AF-100- Store lyophilized at 18B -20 to -80 °C. Upon reconstitution store at 4 °C for one week. Y-27632 – ROCK Tocris, cat.# 1254 Desiccate at RT, upon reconstitution Inhibitor, 1 mg store aliquots at -20 °C or below. 97 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 TryPLETM Select 1X ThermoFisher, cat.# 15–30 °C 12563011 1X DPBS ThermoFisher, cat.# 15–30 °C 14190144 CryoStor CS10 StemCell Technologies, Store at 4 °C. Stable until expiration cat.# 07930 date. Table 17. Reagents needed for iCD34 redifferentiation (listed by day of redifferentiation). Reagent Product No. Storage Condition (Reconstitution Solution) Day 0 Reagents StemFit Basic 04 Amsbio, cat.# SFB- -20 °C, upon thaw use within two weeks Complete 504-CT and store at 4 °C. CHIR99021 (DMSO) TOCRIS, cat.# 4423 -20 °C rh BMP4 (4 mM R&D, cat.# AFL314E Store lyophilized at -20 °C. Upon HCl) reconstitution store at 4 °C for one month. rhVEGF (Sterile R&D, cat.# BT- Store lyophilized at -20 °C. Upon PBS) VEGF-GMP reconstitution store at 4 °C for one month. Day 2 Reagents Advanced Gibco, cat.# 12634-10 Store at 4 °C. Stable until 12 months from DMEM/F12 date of manufacture. StemFit® For Ajinomoto, cat.# -20 °C, upon thaw use within two weeks Differentiation 20170228A and store at 4 °C. L-Glutamine Gibco, cat.# 25030- -20 °C, upon thaw use within two weeks 081 and store at 4 °C. SB431542 (DMSO) Tocris, cat.# 1614 Store at RT, stable until expiration date. rhVEGF (Sterile R&D, cat.# BT- Store lyophilized at -20 °C. Upon PBS) VEGF-GMP reconstitution store at 4 °C for one month. 98 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 rhbFGF (Sterile PBS) Peprotech, cat.# AF- Store lyophilized at -20 to -80 °C. Upon 100-18B reconstitution store at 4 °C for one week. rhSCF (Sterile PBS) R&D, cat.# BT-SCF Store lyophilized at -20 °C. Upon reconstitution store at 4 °C for one month. Day 4 Reagents Advanced Gibco, cat.# 12634-10 Store at 4 °C. Stable until 12 months from DMEM/F12 date of manufacture. StemFit® For Ajinomoto, cat.# -20 °C, upon thaw use within two weeks Differentiation 20170228A and store at 4 °C. L-Glutamine Gibco, cat.# 25030- -20 °C, upon thaw use within two weeks 081 and store at 4 °C. rhSCF (Sterile PBS) R&D, cat.# BT-SCF Store lyophilized at -20 °C. Upon reconstitution store at 4 °C for one month. rhVEGF (Sterile R&D, cat.# BT- Store lyophilized at -20 °C. Upon PBS) VEGF-GMP reconstitution store at 4 °C for one month. rhIL-3 (Sterile PBS) PeproTech, cat.# AF- Store lyophilized at -20 °C. Upon 200-03 reconstitution store at 4 °C for one month. rhIL-6 (Sterile PBS) R&D, cat.# 206-IL- Store lyophilized at -20 °C. Upon 001MG/CF reconstitution store at 4 °C for one month. rhFlt3L (Sterile PBS) R&D, cat.# BT-FT3L- Store lyophilized at -20 °C. Upon AFL reconstitution store at 4 °C for one month. rh TPO (Sterile PBS) Peprotech, cat.# AF- Store lyophilized at -20 °C. Upon 300-18 reconstitution store at 4 °C for one month. Days 6-10 Reagents Advanced Gibco, cat.# 12634-10 Store at 4 °C. Stable until 12 months from DMEM/F12 date of manufacture. StemFit® For Ajinomoto, cat.# -20 °C, upon thaw use within two weeks Differentiation 20170228A and store at 4 °C. L-Glutamine Gibco, cat.# 25030- -20 °C, upon thaw use within two weeks 081 and store at 4 °C. 99 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 rhSCF (Sterile PBS) R&D, cat.# BT-SCF Store lyophilized at -20 °C. Upon reconstitution store at 4 °C for one month. rh IL-6 (Sterile PBS) R&D, cat.# 206-IL- Store lyophilized at -20 °C. Upon 050001MG/CF reconstitution store at 4 °C for one month. rh TPO (Sterile PBS) Peprotech, cat.# AF- Store lyophilized at -20 °C. Upon 300-18 reconstitution store at 4 °C for one month. Table 18. Supplies and consumables needed for cell culture. Item Product No. Corning™ Costar™ 6-well Clear TC-treated Multiple Corning, cat.# 3516 Well Plates, Individually Wrapped, Sterile Fisherbrand™ Sterile Polystyrene Disposable ThermoFisher, Serological Pipets with Magnifier Stripe (various cat.# 13-676-10H, sizes) cat.# 13-678-11E, cat.# 13-678-11, cat.# 13-678-11F Pipette Tips RT LTS 20 µL F 960A/10 (P20) Rainin, cat.# 30389225 Pipette Tips RT LTS 200 µL F 960A/10 (P200) Rainin, cat.# 30389239 Pipette Tips RT LTS 1000 µL F 768A/8 (P1000) Rainin, cat.# 30389212 50 mL conical tubes Corning, cat.# 352098 Falcon® 15 mL Polystyrene Centrifuge Tube, Corning, cat.# 352095 Conical Bottom, with Dome Seal Screw Cap, Sterile 1.8 mL cryovials Thermofisher, cat.# 375418 1.5 mL Eppendorf tubes Greiner-bio-one cat.# 616261 PBS Biotech 0.1L Vertical Wheel Reactor PBS Biotech, cat.# FA-0.1-D-001 Corning CoolCellTM Freezing container Corning, cat.# 432006 Via-1-Cassette Chemometec, cat.# 941-0012 Collagenase Type II StemCell Technologies, cat# 07418 100 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 Table 19. Reagents for flow cytometry. Reagent Vendor Product No. Dilution Storage Flow staining Reagents Stain Buffer (BSA) BD Biosciences 554657 1X 4 °C Brilliant Stain Buffer BD Biosciences 361651 1:10 4 °C Stabilizing Fixative 3X BD Biosciences 338036 1:3 4 °C Concentrate Human TruStain FcX™ (Fc Receptor Blocking BioLegend 422302 1:20 4 °C Solution) ICS Reagents BD Cytofix/Cytoperm kit 1:10 for Perm/ (Perm/Wash buffer and Wash Buffer, BD Biosciences 554714 4 °C Fixation/Permeabilization 1X for Fix/Perm solution) solution Staining Control Kasumi-1 (myeloblast cell line used as positive ATCC CRL-2724 N/A control for iCD34 flow panel) Table 20. Antibodies for Pluripotency Flow Panels. Fluorochrome Marker Vendor Cat# Pluripotency BV711 SSEA-1 BioLegend 323050 PE-Cy7 SSEA-3 BioLegend 330326 PerCP/Cy5.5 CD34+ BioLegend 343522 PE SSEA-4 BioLegend 330406 AF647 OCT 3/4 BioLegend 653710 BV421 SOX2 BioLegend 656114 101 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 Table 21. Antibodies for iCD34 Flow Panels. Fluorochrome Marker Clone Vendor Product No. iCD34 Panel PE CD34 581 BioLegend 343506 APC CD43 1G10 BD 560198 BUV395 CD45 HI30 BD 563792 BV421 CD38 HIT2 BioLegend 303526 CD3 HIT3a BioLegend 300306 CD235a HI264 BioLegend 349104 CD11c Bu15 BioLegend 337214 CD11b ICRF44 BioLegend 301330 FITC CD14 HCD14 BioLegend 325604 Dump Channel CD56 HCD56 BioLegend 318304 CD19 HIB19 BioLegend 302206 CD20 2H7 BioLegend 302304 CD16 3G8 BioLegend 302006 CD2 RPA-2.10 BioLegend 300206 Table 22. Software. Software Vendor Version FlowJoTM BD 10.10 Cytobank Beckman Coulter 10.3 Prism GraphPad Software 8.3.0 Table 23. Equipment. NucleoCounter NC-200
Figure imgf000104_0001
301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 -80 °C Freezer Centrifuge PBS-Mini Vertical Wheel Platform BD Symphony A5 Inverted microscope Biosafety cabinet (BSC) Water bath Table 24. Configuration for BD LSRFortessaTM cytometer. Lasers Filter Set Fluorochrome 379/28 BUV395 354 (UV) 690LP, 740/35 BUV737 450LP, 515/30 Indo-1, BUV496 410LP, 431/28 BV421 505LP, 525/50 BV510 600LP, 610/20 BV605 405 (violet) 635LP, 670/30 BV650 690LP, 710/50 BV711 750LP, 780/60 BV786 505LP, 530/30 FITC, Alexa 488, BB515 488 (blue) 690P, 710/50 PerCP-Cy5.5 Additional FSC-A, H, W and SSC-A, H, W 586/15 PE 600LP, 610/20 PE-Texas Red 561 (green) 650LP, 670/30 PE-Cy5 750LP, 780/60 PE-Cy7 650LP, 670/30 APC, Alexa 647 640 (red) 690LP, 730/45 Alexa 700 750LP, 780/60 APC-Cy7 103 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 Table 25. Configuration for FACSymphonyTM A5 cytometer. Lasers Filter Set Fluorochrome 379/28 BUV395 450LP, 515/30 BUV496 550LP, 586/15 BUV563 600LP, 610/20 BUV615 635LP, 670/30 BUV661 354 (UV) 100 mW 690LP, 740/35 BUV737 770LP, 820/60 BUV805 410LP, 431/28 BV421 450LP, 470/14 BV480 550LP, 586/15 BV570 600LP, 610/20 BV605 405 (violet) 200 mW 630LP, 670/30 BV650 685LP, 710/50 BV711 710LP, 740/35 BV750 750LP, 780/60 BV786 505LP, 515/20 BB515 600LP, 610/20 BB630 635LP, 670/30 BB660 488 (blue) 100 mW 690LP, 710/50 PerCP-Cy5.5, BB700 750LP, 780/60 BB790 FSC-A, H, W and SSC-A, Additional H, W 586/15 PE 561 (green) 150 mW 600LP, 610/20 PE-Texas Red 635LP, 670/30 PE-Cy5 104 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 690LP, 710/50 PE-Cy5.5 750LP, 780/60 PE-Cy7 670/30 APC, Alexa 647 640 (red) 140 mW 690LP, 710/50 Alexa 700 750LP, 780/60 APC-Cy7 Procedure to maintain iPSCs. [0378] 2.1 Supplemented StemFit® Basic 04 Complete Type Medium was prepared as follows: a. The StemFit® Basic 04 Complete Type Medium was provided frozen and was stored at or below -20 °C until use. Sterile techniques were used to prepare Supplemented StemFit® Complete Type Medium. b. Frozen StemFit® Basic 04 Complete Type Medium was thawed with occasional mixing at room temperature (RT; 15-25 °C) or in a refrigerator (2-8 °C) overnight. c. Note: Medium was not thawed at 37 °C, as it accelerates the degradation of the medium ingredients. d. StemFit® Basic 04 Complete Type Medium was supplemented with an additional 20 ng/mL of bFGF to achieve a final concentration of 100 ng/mL bFGF in the supplemented medium. e. Supplemented medium was stored up to two (2) weeks at 4 °C. f. Medium was brought to RT for at least one (1) hour prior to use. [0379] 2.210 mM Y-27632 ROCK inhibitor stock solution was prepared as follows: a. Y-27632 ROCK inhibitor (ROCKi) was received as a crystalline solid. b. Inhibitor was stored at -20 °C for up to 12 months from date of receipt. c. A 10 mM stock solution was prepared in 1X DPBS. i. 312 μL of 1X DPS was added to 1 mg Y-27632 and solution was resuspended completely. ii. 25 μL of stock solution was aliquoted into Eppendorf tubes labeled as Y-27632 stock. iii. Aliquots were stable at -20 °C for up to 6 months. 105 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 iv. Working volumes were aliquoted to avoid repeated freeze-thaw cycles. [0380] 2.3 iPSCs were thawed directly into 3D cultures as follows: a. One ROCKi aliquot was obtained from -20 °C and thawed to room temperature. b. 500 mL of StemFit Basic 04 Complete Type Medium was warmed to 37 °C. c. For the 0.5 L vertical-wheel platform, 490 mL of complete media and 500 µL of ROCKi were transferred into the vessel and placed on a magnetic base in the incubator at 30 rpm agitation. The vessel sat in the incubator for at least an hour before cells were added. d. 25 mL of fresh media was warmed in a water bath. This media did not contain ROCKi. e. Frozen iPSC vial(s) were pulled and thawed in a 37 °C water bath. f. iPSC cell solution was transferred to a 15 mL tube. The cryotube was washed with 1 mL of warmed media and added to the 15 mL tube. Thawed cell solution in the 15 mL tube was topped off to reach a total of 10 mL and was gently mixed. g. The iPSC cells were centrifuged at 300 g for 5 minutes at room temperature. The supernatant was removed and cells were resuspended in 5 mL of warmed media. 2 x 200 µL samples were taken in Eppendorf tubes and cell count was performed on NC200. h. Using the NC200 count, cell volume needed was calculated in order to reach a 20,000 cells/mL seeding density. i. Cells were collected from Eppendorf tube in Via1-Cassette. ii. Via1-Cassette was inserted into NC-200 cell counter and cells were counted. iii. Note: Only the total number of live cells was used for cell seeding purposes. i. The calculated cell volume was transferred to prewarmed media in a vessel. The vessel was topped off to reach 500 mL total cell volume and was put back on the base in the incubator to incubate at 37 °C for three days. [0381] 2.4 Exchanging the cell media in 3D cultures was performed as follows: 106 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 a. 50% media exchanges were performed on Days 4, 5, and 6. Media changes did not contain ROCKi. b. Aliquots were made for exchanging half of the total media volume and were put in a water bath at 37 °C for ≥ 30 minutes. c. The vessel was removed from the incubator and was placed in the hood to let the cells settle inside the vessel. d. During the cell settling time, the vessels were not disturbed. e. The aggregates were not over-settled. Big aggregates took approximately 5 minutes and smaller aggregates took approximately 10 minutes to settle. f. 50% of the media in the vessel was removed while the wheel was kept still and the supernatant was discarded. g. Fresh media was added to replace the volume removed in the vessel. h. The reactor was placed back on the base in the 37 °C incubator and this cell media exchange process was repeated each day until harvest on Day 7. [0382] 2.5 Harvesting the iPSCs from the VW culture was performed as follows: a. On Day 7 of the iPSC 3D culture, 100 mL of^TrypLE was aliquoted and 1 μL of^ROCKi^ was added per 1 mL of^TrypLE^aliquots to result in a concentration of 10^μM. b. The vessel was removed from its base and was set aside^in the BSC to settle aggregates for 3–5 minutes^(3 minutes was used for larger aggregates). c. The vessel was not disturbed during^the settle period. d. After settling,^spent medium^was carefully removed from the vessel into a^container^using a serological pipette until less than 10^mL^of^the spent medium^remained^in the vessel. i. First, a^50 or 25 mL^serological pipette was used to remove the majority of spent medium. ii. Next, a 10 mL serological pipette was used to remove the remaining medium leaving <10^mL (medium and cells) in the vessel. e. The supernatant was discarded and 100 mL^TrypLE +^ROCKi^was added to the vessel for in-vessel cell dissociation. 107 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 f. The vessel was installed onto^its base inside the incubator with agitation speed of 50 rpm^for enzymatic cell dissociation^for 20 minutes. g. The vessel was removed once dissociation^time was completed and 25 mL volumes were transferred to 50 mL centrifuge tubes so that four conical tubes each had approximately 25 mL of cell solution. If there was extra volume left in the reactor, this was distributed across the conical tubes. The solution in each conical tube was mixed with a 10 mL pipette until no clusters were visible (approximately 5-10 mixes). Once complete dissociation was confirmed, PBS buffer was added until 50 mL total volume was reached.^The solution was then mixed. h. The cells were centrifuged at 300 g^for 5 minutes. i. Once centrifugation was done, the supernatant was discarded using a serological pipette^(or aspirator). j. 2 mL of^fresh media^were added with a micropipette to each tube and the cell pellet was gently resuspended. The cell solutions were combined into one conical tube reaching a total of 8 mL cell solution. k. One^150^μL^sample^was taken from the vessel and added into a microtube^for counting. l. 150^μL^of^PBS^was added into the microtube containing the cell sample for a 2x dilution. m. The cell number was counted using NC-200.^Viable cells/mL, cell viability,^cell diameter,^and % in aggregates remaining were recorded. n. The next step was either to proceed to Step 2.6 for iCD34 differentiation, to seed cells into new 0.5 L vessels at 20,000 cells/mL for further iPSC expansion, or to freeze down the sample for flow analysis by centrifuging the required number of cells at 300 g for 5 minutes, removing the supernatant, and resuspending the cells in 1 mL of^CS10, then storing overnight in -80 °C. i. 5x106 cells were used for pluripotency flow analysis. ii. Pluripotency flow panel is shown in Table 20. iii. Acceptance criteria were met before proceeding to redifferentiation steps. Exemplary acceptance criteria for pluripotency were: >85% Oct4+, Sox2+, SSEA3+, SSEA4+ 108 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 <1% SSEA1+ <5% CD34+ Procedure to redifferentiate γδ T-iPSC clones into iPSC-derived CD34 (iCD34) cells in vertical wheel bioreactors using a serum-free, feeder-free (SFFF) three-dimensional (3D) culture method. [0383] 2.6 Day 0: Onset of hematopoietic redifferentiation – Mesoderm induction phase. a. StemFit® Basic 04 Complete Type Medium (Step 2.1) was pre-warmed at RT and STEP 1 medium (Table 26) was prepared for Days 0-2. Table 26. STEP 1 medium for Days 0-2. Reagent Stock Final Volume for 60 Concentration Concentration mL Bioreactor StemFit® Basic 04 60 mL Complete Type Medium (Step 2.1) CHIR99021 20 mM 4 μM 12 µL ROCK inhibitor 10 mM 10 µM 60 µL rhBMP4 100 μg/mL 80 ng/mL 48 µL rhVEGF 500 μg/mL 80 ng/mL 9.6 µL b. Cells were harvested from plates and cell count was taken as in Steps 2.10b-e. c. 60 mL STEP 1 medium was added at a 150,000 cells/mL seeding density to the reactor. d. Cells were cultured at 37 °C, 5% CO2, with the reactor wheel turning at 40 rpm. [0384] 2.7 Day 2: Onset of hematopoietic redifferentiation – Mesoderm to definitive hematopoietic lineage specification. a. At Day 2, Advanced DMEM/F12 medium was pre-warmed at RT. b. At Day 2, images were recorded. i. The reactor was transferred to the spare reactor base inside BSC. ii. While the reactor wheel was turning at 40 rpm, 4 mL of cell suspension was removed and transferred to 1 well of an ultra-low attachment 6-well plate. iii. The plate was gently swirled to collect cells in the middle of the well, then cells were imaged. 109 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 iv. Once images were recorded, cells were transferred back to the reactor. The plate was swirled to collect cells in the middle of the well, 1-2 mL were pulled from the center of the well, then the plate was tilted forward and backward to remove cells from the top of the well. Starting from top of medium level with plate tilted forward, the rest of the cells were pipetted up by moving the pipette back and forth slowly. Cells were transferred to the reactor. c. Reactor was removed from the base and aggregates were allowed to settle in the reactor for approximately 5 minutes or long enough that the clusters in the suspension were no longer seen visibly moving to the bottom of the reactor. About 2 minutes in, the 0.1 L reactor was gently rocked a few times to ensure clusters that settled on the top of the vertical wheel were moved off. Then, media was pulled from the vertical wheel leaving 20% volume left in the reactor. Pulled supernatant was spun down at 100 g for 1 minute at brake 6. d. While cells were in the centrifuge, 38.4 mL DMEM/F12 and 8.6 mL StemFit for Differentiation media were added to the empty reactor. The reactor was placed back on the base to turn at 40 rpm. e. Reagents for STEP 2 medium for days 2-4 as listed below were thawed. Table 27. STEP 2 medium for Days 2-4. Reagent Stock Final Volume for 60 Concentration Concentration mL Bioreactor Advanced DMEM/F12 100% 38.4 mL StemFit® For Differentiation 100% 20% 9.6 mL L-Glutamine 100X 1X 480 µL SB431542 5 mM 2 μM 19.2 µL rhVEGF 100 μg/mL 80 ng/mL 38.4 µL rhbFGF 100 μg/mL 50 ng/mL 24 µL rhSCF 500 μg/mL 50 ng/mL 4.8 µL f. After centrifuging, 500 μL were collected from the cell supernatant for a metabolic sample. The rest of the supernatant was removed. 110 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 g. The pellet was resuspended with 1 mL of StemFit® For Differentiation medium and was added to the reactor. h. Other STEP 2 reagents were added to the reactor. i. Cells were incubated and cultured at 37 °C, 5% CO2, and 40 rpm for two days. [0385] 2.8 Day 4: Onset of hematopoietic redifferentiation – Hemangioblast induction phase. a. At Day 4, Advanced DMEM/F12 medium was pre-warmed at RT. b. Images at Day 4 were recorded as in Step 2.7b. c. The reactor was removed from the base and aggregates were allowed to settle in the reactor for approximately 5 minutes or long enough that the clusters in suspension were no longer seen visibly moving to the bottom of the reactor. About 2 minutes in, the 0.1 L reactor was gently rocked a few times to ensure clusters that settled on the top of the vertical wheel were moved off. Then, media was pulled from the vertical wheel leaving 20% volume left in the reactor. The pulled supernatant was spun down at 100 g for 1 minute at brake 6. d. While cells were in the centrifuge, 38.4 mL DMEM/F12 + 8.6 mL StemFit for Differentiation media were added to the reactor. The reactor was placed back on the base to turn at 40 rpm. e. Reagents for STEP 3 medium for Days 4-6 were thawed. Table 28. Step 3 Medium for Days 4-6. Reagent Stock Final Volume for 60mL Concentration Concentration Bioreactor Advanced DMEM/F12 100% 38.4 mL StemFit® For Differentiation 100% 20% 9.6 mL L-Glutamine 100X 1X 480 µL SCF 500 μg/mL 50 ng/mL 4.8 µL rh VEGF 100 μg/mL 20 ng/mL 9.6 µL rh IL-3 100 μg/mL 50 ng/mL 24 µL rh IL-6 100 μg/mL 50 ng/mL 24 µL rh Flt3L 100 μg/mL 50 ng/mL 24 µL rh EPO 500 IU/mL 10 IU/mL 111 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 f. After centrifuging, 500 μL were collected from the cell supernatant for a metabolic sample. The rest of the supernatant was removed. g. The pellet was resuspended with 1 mL of StemFit® For Differentiation medium and was added to the reactor. h. Other STEP 3 reagents were added to the reactor. i. Cells were incubated and cultured at 37 °C, 5% CO2, and 40 rpm for two days. [0386] 2.9 Day 6: Onset of hematopoietic redifferentiation – Endothelial to hematopoietic transition phase. a. On Day 6, Advanced DMEM/F12 medium was pre-warmed at RT. b. Images at Day 6 were recorded as in Step 2.7b. c. The reactor was removed from the base and aggregates were allowed to settle in the reactor for approximately 5 minutes or long enough that the clusters in suspension were no longer seen visibly moving to the bottom of the reactor. About 2 minutes in, the 0.1 L reactor was gently rocked a few times to ensure clusters that settled on the top of the vertical wheel were moved off. Then, media was pulled from the vertical wheel leaving 20% volume left in the reactor. The pulled supernatant was spun down at 100 g for 1 minute at brake 6. d. While cells were in the centrifuge, 38.4 mL DMEM/F12 + 8.6 mL StemFit for Differentiation media were added to the reactor. The reactor was placed back on the base to turn at 40 rpm. e. Reagents for STEP 4 medium for Days 6-8 were thawed. Table 29. Step 4 Medium for Days 6-10. Reagent Stock Final Volume for 60mL Concentration Concentration Bioreactor Advanced DMEM/F12 100% 38.4 mL StemFit® For Differentiation 100% 20% 9.6 mL L-Glutamine 100X 1X 480 µL rhSCF 500 μg/mL 50 ng/mL 4.8 µL rhIL-6 100 μg/mL 50 ng/mL 24 µL rhEPO 500 IU/mL 10 IU/mL 112 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 f. After centrifuging, 500 μL were collected from the cell supernatant for a metabolic sample. The rest of the supernatant was removed. The pellet was resuspended with 1 mL of StemFit® For Differentiation medium and was added to the reactor. g. Other STEP 4 reagents were added to the reactor. h. Cells were incubated and cultured at 37 °C, 5% CO2, and 40 rpm for two days. i. On Day 8, Advanced DMEM/F12 medium was pre-warmed at RT and images were recorded as in Step 2.7b. j. The reactor was removed from the base and aggregates were allowed to settle in the reactor for approximately 5 minutes or long enough that the clusters in suspension were no longer seen visibly moving to the bottom of the reactor. About 2 minutes in, the 0.1 L reactor was gently rocked a few times to ensure clusters that settled on the top of the vertical wheel were moved off. Then, media was pulled from the vertical wheel leaving 20% volume left in the reactor. The pulled supernatant was spun down at 100 g for 1 minute at brake 6. k. While cells were in the centrifuge, 38.4 mL DMEM/F12 + 8.6 mL StemFit for Differentiation media were added to the reactor. The reactor was placed back on the base to turn at 40 rpm. l. After centrifugation, the supernatant containing single cells was transferred to a 50 mL conical tube. The pellet from the first conical tube was resuspended in 500 µL StemFit for Differentiation medium and added to reactor. m. Supernatant transferred to the second 50 mL conical tube was spun at 300 g for 5 minutes at brake 9. After centrifuging, 500 µL from the cell supernatant was collected for a metabolic sample. The rest of the supernatant was removed, and the pellet was resuspended with 500 µL of StemFit for Differentiation medium and added to reactor. n. Reagents for STEP 4 medium for Days 8-10 were thawed (similarly to Step 2.9e) and added to the reactor. o. Cells were incubated and cultured at 37 °C, 5% CO2, and 40 rpm for another two days. 113 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 [0387] 2.10 Day 10: Harvesting hematopoietic stem cells (iCD34 cells) and preparing cells for flow cytometric analysis and cryopreservation was performed as follows: a. Images were recorded at Day 10 as in Step 2.7b. b. 21 mL of STEP 4 base medium without cytokines and growth factors were prepared. c. Single cell fraction was processed as follows: i. Pull entire cell volume out of reactor, transfer 30 mL to each of 2x 50 mL conicals, and spin down at 100 g for 1 minute at brake 6. Pellets were kept for dissociation in Step 2.10d and supernatant containing single cells was dissociated in the following step. ii. Transfer supernatant, containing single cells, to new 50 mL conical tubes. Spin down at 300 g for 5 minutes at brake 9. iii. After centrifuging, collect 500 µL from cell supernatant for metabolic sample. Remove rest of supernatant and resuspend pellet in 5 mL of fresh Advanced DMEM/F12 base medium without added cytokines or growth factors. iv. Cell count was performed using NC-200 as in Step 2.3h. d. Cluster cell fraction was processed as follows: i. Add 1 mL collagenase to each pellet from Step 2.10c.i. Resuspend and combine into one 50 mL conical tube (total volume of collagenase = 2 mL). Incubate for 20 minutes at 37 °C. ii. Add equal volume (2 mL) of Advanced DMEM/F12 base medium without added cytokines or growth factors (Step 2.15b). Centrifuge at 300 g for 5 minutes at brake 9. iii. Remove supernatant, add 2 mL TrypLE, and mix solution. Incubate for 5 minutes at 37 °C. iv. Add 8 mL Advanced DMEM/F12 base medium without added cytokines or growth factors and mix solution. v. Pass cells through 70 μm filter if clumping is observed after dissociation and centrifuge at 300 x g for 5 minutes at brake 9. 114 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 vi. Resuspend in 10 mL of Advanced DMEM/F12 base medium without added cytokines or growth factors and perform cell count using NC-200 as in Step 2.7b. e. Each cell fraction, single cells and clusters, was frozen down by spinning down cell suspensions at 300 g for 5 minutes, removing the supernatant, and resuspending in CS10 formulation. 2.0x106 cells / mL of CS10 were aliquoted for flow cytometric analysis and downstream processing. f. iCD34 flow panel is shown in Table 21. g. Exemplary success criteria for iCD34 redifferentiation step: purity [defined as Lineage marker-negative (Lin‒) CD34+] is >70% and fold expansion per input iPSC is >0.5-folds. [0388] The protocol enabled redifferentiation of γδ T-iPSC lines into hematopoietic stem cells (HSCs) at a variety of RPM in a 0.1L vertical wheel bioreactor using a serum-free, feeder- free (SFFF) three-dimensional (3D) culture method, the results including cell cluster formation, morphology, cell growth (e.g., metabolism), purity, etc. This was true with perfusion in the bioreactor system. Example 3: Serum-free, feeder-free, three-dimensional redifferentiation of γδ T cell-derived iPSCs into iCD34s in a 3 L bioreactor scale. [0389] The example provided contains instructions to redifferentiate γδ T-iPSCs. Materials [0390] The following abbreviations are used herein (Table 30). Table 30. Abbreviations. Abbreviation Definition 3D Three-dimensional bFGF Basic Fibroblast Growth Factor BMP-4 Bone Morphogenetic Protein 4 DPBS Dulbecco’s Phosphate-buffered Saline Flt3L FMS-like tyrosine kinase 3 Ligand γδ T-iPSC γδ T cell-derived iPSC 115 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 HSC Hematopoietic stem cell IL-3 Interleukin-3 IL-6 Interleukin-6 iPSC induced pluripotent stem cell IU International units rh Recombinant human ROCK Rho-associated, Coiled-coil Containing Protein Kinase RT Room temperature SCF Stem Cell Factor SFFF Serum-free, feeder-free TC Tissue culture TPO Thrombopoietin VEGF Vascular Endothelial Growth Factor [0391] The materials needed herein include γδ T-iPSC lines and the reagents necessary to perform cell culture (Tables 31-34). Table 31. Reagents for iPSC culture and maintenance. Reagent Product No. γδ T-iPSC clones Academic collaborator, contract research organization iMatrix 511 Amsbio, cat.# AMS.892012 StemFit® Basic 04 Complete Type Amsbio, cat.# SFB-504-CT rh bFGF Amsbio, cat.# AMS-FGF-100 Y-27632 – ROCK Inhibitor, 1 mg Tocris, cat# 1254 TryPLETM Select 1X ThermoFisher, cat.# 12563011 1X DPBS ThermoFisher, cat.# 14190144 CryoStor CS10 StemCell Technologies, cat.# 07930 Table 32. Reagents needed for hematopoietic redifferentiation (listed by day of redifferentiation). Reagent (Reconstitution Solution) Product No. Day 0 Reagents StemFit Basic 04 Complete Amsbio, cat.# SFB-504-CT CHIR99021 (DMSO) TOCRIS, cat.# 4423 116 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 rh BMP4 (4 mM HCl) R&D, cat.# AFL314E rh VEGF (Sterile PBS) R&D, cat.# BT-VEGF-GMP Day 2 Reagents Advanced DMEM/F12 Gibco, cat.# 12634-10 StemFit® For Differentiation Ajinomoto, cat.# 20170228A L-Glutamine Gibco, cat.# 25030-081 SB431542 (DMSO) Tocris, cat.# 1614 rh VEGF (Sterile PBS) R&D, cat.# BT-VEGF-GMP rh bFGF (Sterile PBS) Peprotech, cat.# AF-100-18B rh SCF (Sterile PBS) R&D, cat.# BT-SCF Day 4 Reagents Advanced DMEM/F12 Gibco, cat.# 12634-10 StemFit® For Differentiation Ajinomoto, cat.# 20170228A L-Glutamine Gibco, cat.# 25030-081 rh SCF (Sterile PBS) R&D, cat.# BT-SCF rh VEGF (Sterile PBS) R&D, cat.# BT-VEGF-GMP rh IL-3 (Sterile PBS) PeproTech, cat.# AF-200-03 rh IL-6 (Sterile PBS) R&D, cat.# 206-IL-001MG/CF rh Flt3L (Sterile PBS) R&D, cat.# 308-FK-025/CF rh TPO (Sterile PBS) Peprotech, cat.# AF-300-18 Days 6-10 Reagents Advanced DMEM/F12 Gibco, cat.# 12634-10 StemFit® For Differentiation Ajinomoto, cat.# 20170228A L-Glutamine Gibco, cat.# 25030-081 rh SCF (PBS) R&D, cat.# BT-SCF rh IL-6 (PBS) R&D, cat.# 206-IL-001MG/CF rh TPO (Sterile PBS) Peprotech, cat.# AF-300-18 Table 33. Supplies and consumables needed for cell culture. Item Product No. 117 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 Corning™ Costar™ 6-well Clear TC-treated Multiple Corning, cat.# 3516 Well Plates, Individually Wrapped, Sterile Fisherbrand™ Sterile Polystyrene Disposable ThermoFisher, Serological Pipets with Magnifier Stripe (various cat.# 13-676-10H, sizes) cat.# 13-678-11E, cat.# 13-678-11, cat.# 13-678-11F Pipette Tips RT LTS 20 µL F 960A/10 (P20) Rainin, cat.# 30389225 Pipette Tips RT LTS 200 µL F 960A/10 (P200) Rainin, cat.# 30389239 Pipette Tips RT LTS 1000 µL F 768A/8 (P1000) Rainin, cat.# 30389212 50 mL conical tubes Corning, cat.# 352098 Falcon® 15 mL Polystyrene Centrifuge Tube, Conical Corning, cat.# 352095 Bottom, with Dome Seal Screw Cap, Sterile 1.8 mL cryovials Thermofisher, cat.# 375418 1.5 mL Eppendorf tubes Greiner-bio-one cat.# 616261 PBS Biotech 0.1 L Vertical Wheel Reactor PBS Biotech, cat.# FA-0.1-D-001 Corning CoolCellTM Freezing container Corning, cat.# 432006 Via-1-Cassette Chemometec, cat.# 941-0012 Collagenase Type II StemCell Technologies, cat# 07418 Table 34. Antibodies for Flow Panels. Fluorochrome Marker Vendor Cat# Pluripotency BV711 SSEA-1 BioLegend 323050 PE-Cy7 SSEA-3 BioLegend 330326 PerCP/Cy5.5 CD34+ BioLegend 343522 PE SSEA-4 BioLegend 330406 AF647 OCT 3/4 BioLegend 653710 BV421 SOX2 BioLegend 656114 iHSC Panel 118 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 PE CD34 BioLegend 343506 APC CD43 BD 560198 BUV395 CD45 BD 563792 BV421 CD38 BioLegend 303526 APC-Cy7 Live/dead Near IR ThermoFisher L10119 FITC CD3 BioLegend 300306 FITC CD235a BioLegend 349104 FITC CD11c BioLegend 337214 FITC CD11b BioLegend 301330 FITC CD14 BioLegend 325604 FITC CD56 BioLegend 318304 FITC CD19 BioLegend 302206 FITC CD20 BioLegend 302304 FITC CD16 BioLegend 302006 FITC CD2 BioLegend 300206 Table 35. Equipment. NucleoCounter NC-200 EVOS M7000 imager CO2 Incubator 4 °C Refrigerator -20 °C Freezer -80 °C Freezer Centrifuge PBS-Mini Base Platform Procedure to maintain iPSCs. [0392] 3.1 Supplemented StemFit® Basic 04 Complete Type Medium was prepared as follows: 119 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 a. The StemFit® Basic 04 Complete Type Medium was provided frozen and was stored at or below -20 °C until use. Sterile techniques were used to prepare Supplemented StemFit® Complete Type Medium. b. Frozen StemFit® Basic 04 Complete Type Medium was thawed with occasional mixing at room temperature (RT; 15-25 °C) or in a refrigerator (2-8 °C). c. Note: Medium was not thawed at 37 °C, as it accelerates the degradation of the medium ingredients. d. StemFit® Basic 04 Complete Type Medium was supplemented with an additional 20 ng/mL of bFGF to achieve a final concentration of 100 ng/mL bFGF in the supplemented medium. e. Supplemented medium was stored up to two (2) weeks at 4 °C. f. Medium was brought to RT for at least one (1) hour prior to use. [0393] 3.210 mM Y-27632 ROCK inhibitor stock solution was prepared as follows: a. Y-27632 ROCK inhibitor (ROCKi) was received as a crystalline solid. b. Inhibitor was stored at -20 °C for up to 12 months from date of receipt. c. A 10 mM stock solution was prepared in 1X DPBS. i. 312 mL of 1X DPS was added to 10 mg Y-27632 and solution was resuspended completely. ii. 500 μL of stock solution was aliquoted into Eppendorf tubes labeled as ROCKi stock. iii. Aliquots were stable at -20 °C for up to 6 months. iv. Working volumes were aliquoted to avoid repeated freeze-thaw cycles. [0394] 3.3 iPSCs were thawed directly into 3D cultures as follows: a. One ROCKi aliquot was obtained from -20 °C and thawed to room temperature. b. 500 mL of StemFit Basic 04 Complete Type Medium was warmed to 37 °C. c. For the 0.5 L vertical-wheel platform, 490 mL of complete media and 500 µL of ROCKi were transferred into the vessel and placed on a magnetic base in the incubator at 30 rpm agitation. The vessel sat in the incubator for at least an hour before cells were added. 120 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 d. 25 mL of fresh media was warmed in a water bath. This media did not contain ROCKi. e. Frozen iPSC vial(s) were pulled and thawed in a 37 °C water bath. f. iPSC cell solution was transferred to a 15 mL tube. The cryotube was washed with 1 mL of warmed media and added to the 15 mL tube. Thawed cell solution in the 15 mL tube was topped off to reach a total of 10 mL and was gently mixed. g. The iPSC cells were centrifuged at 300 g for 5 minutes at room temperature. The supernatant was removed and cells were resuspended in 5 mL of warmed media. 2 x 200 µL samples were taken in Eppendorf tubes and cell count was performed on NC200. h. Using the NC200 count, cell volume needed was calculated in order to reach a 20,000 cells/mL seeding density. i. Cells were collected from Eppendorf tube in Via1-Cassette. ii. Via1-Cassette was inserted into NC-200 cell counter and cells were counted. iii. Note: Only the total number of live cells was used for cell seeding purposes. i. The calculated cell volume was transferred to prewarmed media in a vessel. The vessel was topped off to reach 500 mL total cell volume and was put back on the base in the incubator to incubate at 37 °C for three days. [0395] 3.4 Exchanging the cell media in 3D cultures was performed as follows: a. 50% media exchanges were performed on Days 4, 5, and 6. Media changes did not contain ROCKi. b. Aliquots were made for exchanging half of the total media volume and were put in a water bath at 37 °C for ≥ 30 minutes. c. The vessel was removed from the incubator and was placed in the hood to let the cells settle inside the vessel. d. During the cell settling time, the vessels were not disturbed. About 2 minutes in, the reactor was gently rocked a few times to ensure clusters that settled on the top of the vertical wheel were moved off. 121 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 e. The aggregates were not over-settled. Big aggregates took approximately 5 minutes and smaller aggregates took approximately 10 minutes to settle. f. 50% of the media in the vessel was removed while the wheel was kept still and the supernatant was discarded. g. Fresh media was added to replace the volume removed in the vessel. h. The reactor was placed back on the base in the 37 °C incubator and this cell media exchange process was repeated each day until harvest on Day 7. [0396] 3.5 Harvesting the iPSCs from the VW culture was performed as follows: a. On Day 7 of the iPSC 3D culture, 100 mL of^TrypLE was aliquoted and 1 μL of^ROCKi^ was added per 1 mL of^TrypLE^aliquots to result in a concentration of 10^μM. b. The vessel was removed from its base and was set aside^in the BSC to settle aggregates for 3–5 minutes^(3 minutes was used for larger aggregates). c. The vessel was not disturbed during^the settle period. d. After settling,^spent medium^was carefully removed from the vessel into a^container^using a serological pipette until less than 10^mL^of^the spent medium^remained^in the vessel. i. First, a^50 or 25 mL^serological pipette was used to remove the majority of spent medium. ii. Next, a 10 mL serological pipette was used to remove the remaining medium leaving <10^mL (medium and cells) in the vessel. e. The supernatant was discarded and 100 mL^TrypLE +^ROCKi^was added to the vessel for in-vessel cell dissociation. f. The vessel was installed onto^its base inside the incubator with agitation speed of 50 rpm^for enzymatic cell dissociation^for 20 minutes. g. The vessel was removed once dissociation^time was completed and 25 mL volumes were transferred to 50 mL centrifuge tubes so that four conical tubes each had approximately 25 mL of cell solution. If there was extra volume left in the reactor, this was distributed across the conical tubes. The solution in each conical tube was mixed with a 10 mL pipette until no clusters were visible (approximately 5-10 122 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 mixes). Once complete dissociation was confirmed, PBS buffer was added until 50 mL total volume was reached.^The solution was then mixed 2-3 times. h. The cells were centrifuged at 300 g^for 5 minutes. i. Once centrifugation was done, the supernatant was discarded using a serological pipette^(or aspirator). j. 2 mL of^fresh media^were added with a micropipette to each tube and the cell pellet was gently resuspended. The cell solutions were combined into one conical tube reaching a total of 8 mL cell solution. k. Cells were filtered with a 70 µm filter into a new conical tube. l. One^150^μL^sample^was taken from the conical tube and added into a microtube^for counting. m. 150^μL^of^PBS^was added into the microtube containing the cell sample for a 2x dilution. n. The cell number was counted using NC-200.^Viable cells/mL, cell viability,^cell diameter,^and % in aggregates remaining were recorded. o. The next step was either to proceed to Step 3.6 to begin the iCD34 differentiation process in a 3 L reactor, to seed cells into new 0.5 L vessels at 20,000 cells/mL for further iPSC expansion, or to freeze down the sample for flow analysis by centrifuging the required number of cells at 300 g for 5 minutes, removing the supernatant, and resuspending the cells in 1 mL of^CS10, then storing overnight in -80 °C. i. 5x106 cells were used for pluripotency flow analysis. ii. Pluripotency flow panel is shown in Table 34. iii. Acceptance criteria were met before proceeding to redifferentiation steps. Exemplary acceptance criteria for pluripotency were: >85% Oct4+, Sox2+, SSEA3+, SSEA4+ <1% SSEA1+ <5% CD34+ 123 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 Procedure to redifferentiate γδ T-iPSC lines into hematopoietic stem cells (HSCs) in a 3 L vertical wheel bioreactor using a serum-free, feeder-free (SFFF) three-dimensional (3D) culture method. [0397] 3.6 Day -3: Setup of PBS 3 L Reactor (Sterility Hold). a. Three days prior to inoculation, the PBS 3MAG vessel was removed from packaging b. The PBS 3MAG was brought into the BSC and the shrink-wrapped plastic around it was carefully removed. c. The PBS 3MAG vessel was placed upright in the BSC and all lines were clamped. d. Autoclaved pouches of dip tubes were brought into the BSC. e. The unused port cover cap (front right port) was carefully unscrewed and was placed to the side. The 4 μm dip tube was unpacked from the autoclave bag and placed gently into the vessel carefully as not to touch the outside of the vessel. The dip tube was lowered to where the glass frit was positioned at half the height of the wheel from the bottom of the vessel. The dip tube was threaded into the port. f. The pH probe port (back right port) was carefully unscrewed and was placed to the side. The 40 μm dip tube was unpacked from the autoclave bag and placed gently into the vessel carefully as not to touch the outside of the vessel. The dip tube was lowered to where the glass frit was positioned above the bridge that holds the wheel, at 3/4 the height of the wheel from the bottom of the vessel. The dip tube was threaded into the port. g. The vessel was transferred out of the BSC and moved towards the PBS 3MAG control tower. h. The PBS 3MAG vessel was inserted into the holder in the front and the vessel bottom was carefully ensured to be in contact with the heating pad on the bottom. i. The temperature probe was inserted into the thermocouple holder. j. The dissolved oxygen (DO) cable was connected to the probe. k. The main gas and O2 gas lines from the vessel were attached to the 3MAG connectors. 124 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 l. The exhaust filter was installed to the filter oven and the condensation bag was placed appropriately. m. A 2 L bottle was welded to each dip tube line to collect waste. n. A 2 L bottle of DMEM media was welded to the harvest line. o. A masterflex pump was used to drain or pump in media. p. The harvest line was clamped once the media from the bag was completely transferred. q. Media was run through the dip tubes by pumping media through each dip tube until it reaches the waste bottle and then pumping this media back into the vessel. r. After the media was completely transferred, the following parameters were set and the reactor was monitored for sterility over three days: i. Agitation = 23 RPM ii. Temperature = 37 °C / 5% CO2 iii. DO = Off iv. Main Gas = 0.20 L/min v. pH = Off s. Sterility was confirmed after 72 hours. t. Sample was removed for offline assessment via Metaflex or Novaflex. [0398] 3.7 Day 0: Onset of hematopoietic redifferentiation – Mesoderm induction phase. a. 2.4 L of StemFit Basic 04 Complete Type Medium was transferred to a weldable bottle or bag and the media was transferred to the empty 3 L vessel. i. Once the media level reached a volume of 0.5 L in the vessel, the temperature was set to 37 °C and the agitation was set to 23 rpm to allow the media to pre-warm. Additionally, the gas was set to 0.2 L/min. b. In another weldable bottle or bag, approximately 50 mL of STEP 1 medium for Days 0-2 were prepared with cells at a seeding density of 150,000 cells/mL of total working volume in the reactor (i.e., 375x106 cells for 2500 mL working volume). 125 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 Table 36. STEP 1 medium for Days 0-2. Reagent Stock Concentration Final Concentration Volume for 2500 mL Bioreactor StemFit Basic 04 50 mL Complete Type (2500 mL total Medium (Step 3.1) working volume) CHIR99021 20 mM 4 μM 0.5 mL Rock Inhibitor 10 mM 10 µM 2.5 mL rh BMP4 100 μg/mL 80 ng/mL 2.0 mL rh VEGF 500 μg/mL 80 ng/mL 0.4 mL c. The approximately 50 mL of cells + STEP 1 medium were transferred to the reactor. d. A third weldable bottle was prepared with 50 mL StemFit Basic 04 Complete Type Medium with no cytokines and the media was transferred into the reactor to wash the harvest line. e. The harvest line and both dip tube lines were clamped. f. Cells were cultured at 37 °C, 5% CO2, with the reactor wheel turning at 23 rpm. [0399] 3.8 Day 2: Onset of hematopoietic redifferentiation – Mesoderm to definitive hematopoietic lineage specification. a. At Day 2, Advanced DMEM/F12 and StemFit for Differentiation media were pre- warmed at RT. b. At Day 2, images were recorded. i. 4 mL of cell suspension was removed through the sample line and was transferred to 1 well of an ultra-low attachment 6-well plate. ii. The plate was gently swirled to collect cells in the middle of the well, then cells were imaged. iii. Once images were recorded, the plate was discarded. c. 2720 mL Advanced DMEM/F12 + 680 mL StemFit for Differentiation media were transferred into a weldable bottle or bag, and the bottle or bag was connected to the reactor harvest line. 126 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 i. Alternatively, the volume was split up into a first bottle or bag containing 1600 mL DMEM/F12 + 400 mL StemFit for Differentiation media (2 L total volume) and a second bottle or bag containing 1120 mL DMEM/F12 + 280 mL StemFit for Differentiation media (1.4 L total volume). d. The empty waste bottle was welded onto the 40 µm dip tube waste line. e. Simultaneously, media was removed from the reactor through the 40 µm dip tube line and media was added to the reactor via the harvest line at a rate of 30 mL/min, until 3.4 L of media had been exchanged. i. The exchange was tracked by ensuring the level indicated on the vessel screen remained consistent. If the level did not remain consistent, the pump was temporarily paused for the harvest line or the dip tube line until the level returned to the original value. f. In another weldable bottle or bag, approximately 50 mL of STEP 2 medium for days 2-4 was prepared, and the bottle or bag was welded onto the harvest line. Table 37. STEP 2 medium for Days 2-4. Reagent Stock Final Volume for 2500 mL Concentration Concentration Bioreactor Advanced DMEM/F12 100% 24 mL StemFit® For 100% 20% 6 mL Differentiation L-Glutamine 100X 1X 20 mL SB431542 5 mM 2 μM 800 µL rh VEGF 100 μg/mL 80 ng/mL 320 µL bFGF 100 μg/mL 50 ng/mL 1 mL SCF 500 μg/mL 50 ng/mL 200 µL g. Once 3.4 L of media were exchanged in the vessel, 0.1 L of media were removed through the 40 µm dip tube line. The volume removed was tracked by monitoring the level on the vessel screen. h. 50 mL of STEP 2 medium were added to the reactor. 127 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 i. Another weldable bottle or bag was prepared with 40 mL Advanced DMEM/F12 + 10 mL StemFit for Differentiation media, and the bottle or bag was welded onto the harvest line. j. The 50 mL of DMEM/F12 + StemFit for Differentiation media were transferred into the reactor. k. The harvest line and dip tube line were clamped. l. Cells were incubated and cultured at 37 °C, 5% CO2, and 23 rpm for two days. [0400] 3.9 Day 4: Onset of hematopoietic redifferentiation – Hemangioblast induction phase. a. At Day 4, Advanced DMEM/F12 and StemFit for Differentiation media were pre- warmed at RT. b. At Day 4, images were recorded as in Step 3.8b. c. 2720 mL Advanced DMEM/F12 + 680 mL StemFit for Differentiation media were transferred into a weldable bottle or bag. Alternatively, the volume was split up across two bottles or bags as in Step 3.8c.i. Media was removed from the reactor through the 40 µm dip tube line and fresh media was added to the reactor via the harvest line as in Steps 3.8d-e. d. In another weldable bottle or bag, 50 mL of STEP 3 medium for days 4-6 was prepared, and the bottle or bag was welded onto the harvest line. Table 38. Step 3 Medium for Days 4-6. Reagent Stock Final Concentration Volume for 2500 Concentration mL Bioreactor Advanced DMEM/F12 100% 24 mL StemFit® For 100% 20% 6 mL Differentiation L-Glutamine 100X 1X 20 mL SCF 500 μg/mL 50 ng/mL 200 µL rh VEGF 100 μg/mL 20 ng/mL 80 µL rh IL-3 100 μg/mL 50 ng/mL 1 mL rh IL-6 100 μg/mL 50 ng/mL 1 mL rh Flt3L 100 μg/mL 50 ng/mL 1 mL rh TPO 100 μg/mL 30 ng/mL 600 µL 128 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 e. Once 3.4 L of media were exchanged in the vessel, 0.1 L of media were removed through the 40 µm dip tube line. The volume removed was tracked by monitoring the level on the vessel screen. f. 50 mL of STEP 3 medium were added to the reactor. g. Another weldable bottle or bag was prepared with 40 mL Advanced DMEM/F12 + 10 mL StemFit for Differentiation media, and the bottle or bag was welded onto the harvest line. h. The 50 mL of DMEM/F12 + StemFit for Differentiation media were transferred into the reactor. i. The harvest line and dip tube line were clamped. j. Cells were incubated and cultured at 37 °C, 5% CO2, and 23 rpm for two days. [0401] 3.10 Day 6: Onset of hematopoietic redifferentiation – Endothelial to hematopoietic transition phase. a. At Day 6, Advanced DMEM/F12 and StemFit for Differentiation media were pre- warmed at RT. b. At Day 6, images were recorded as in Step 3.8b. c. 2720 mL Advanced DMEM/F12 + 680 mL StemFit for Differentiation media were transferred into a weldable bottle or bag. Alternatively, the volume was split up across two bottles or bags as in Step 3.8c.i. Media was removed from the reactor through the 40 µm dip tube line and fresh media was added to the reactor via the harvest line as in Steps 3.8d-e. d. In another weldable bottle or bag, 50 mL of STEP 4 medium for days 6-8 was prepared, and the bottle or bag was welded onto the harvest line. Table 39. Step 4 Medium for Days 6-8. Reagent Stock Final Volume for 2500 Concentration Concentration mL Bioreactor Advanced DMEM/F12 100% 24 mL StemFit® For 100% 20% 6 mL Differentiation L-Glutamine 100X 1X 20 mL 129 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 rh SCF 500 μg/mL 50 ng/mL 200 µL rh IL-6 100 μg/mL 50 ng/mL 1 mL rh TPO 100 μg/mL 30 ng/mL 600 µL e. Once 3.4 L of media were exchanged in the vessel, 0.1 L of media were removed through the 40 µm dip tube line. The volume removed was tracked by monitoring the level on the vessel screen. f. 50 mL of STEP 4 medium were added to the reactor. g. Another weldable bottle or bag was prepared with 40 mL Advanced DMEM/F12 + 10 mL StemFit for Differentiation media, and the bottle or bag was welded onto the harvest line. h. The 50 mL of DMEM/F12 + StemFit for Differentiation media were transferred into the reactor. i. The harvest line and dip tube line were clamped. j. Cells were incubated and cultured at 37 °C, 5% CO2, and 23 rpm for two days. [0402] 3.11 Day 8: Continued onset of hematopoietic redifferentiation – Endothelial to hematopoietic transition phase. a. At Day 8, Advanced DMEM/F12 and StemFit for Differentiation media were pre- warmed at RT and images were recorded as in Step 3.8b. b. In another weldable bottle or bag, approximately 2 L of STEP 5 medium for days 8-10 was prepared. Table 40. Step 5 Medium for Days 8-10. Reagent Stock Final Volume for 2500 Concentration Concentration mL Bioreactor Advanced DMEM/F12 100% 1584 mL StemFit® For 100% 20% 396 mL Differentiation L-Glutamine 100X 1X 20 mL rh SCF 500 μg/mL 50 ng/mL 200 µL rh IL-6 100 μg/mL 50 ng/mL 1 mL 130 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 rh TPO 100 μg/mL 30 ng/mL 600 µL c. The bottle or bag of STEP 5 medium was connected to the reactor harvest line. d. The empty 2 L waste bottle was welded onto the 4 µm dip tube waste line. e. Over the course of 2 days, simultaneously media was removed from the reactor through the 4 µm dip tube line and media was added to the reactor via the harvest line at a rate of 0.69 mL/min (1 L/day), until 2 L of media were exchanged. [0403] 3.12 Day 10: Harvesting hematopoietic stem cells (iCD34/iHSCs) and preparing cells for flow cytometric analysis and cryopreservation was performed as follows: a. Images were recorded at Day 10 as in Step 3.8b. b. 40 mL of Advanced DMEM/F12 medium without cytokines and growth factors were warmed. c. Single cell fraction was processed as follows: i. Pull entire 2.5 L cell volume out of reactor using harvest line pump. ii. Transfer entire cell volume over 500 mL aliquots to 500 mL centrifuge containers, and spin down at 100 g for 1 minute at brake 6. Pellets were kept for dissociation in Step 3.12d and supernatant containing single cells was dissociated in the following step. iii. Transfer supernatant, containing single cells, to new 500 mL centrifuge containers. Spin down at 300 g for 5 minutes at brake 9. iv. After centrifuging, collect 500 µL from cell supernatant for metabolic sample. Remove rest of supernatant and resuspend single cell pellet in 5 mL of fresh Advanced DMEM/F12 base medium without added cytokines or growth factors. v. Cell count was performed using NC-200. d. Cluster cell fraction was processed as follows: i. Add 1 mL collagenase to each pellet from Step 3.12c.ii. Resuspend and combine into one 50 mL conical tube (total volume of collagenase = 5 mL). Add an additional 5 mL of collagenase to solution and mix 5-10 times. Incubate for 20 minutes at 37 °C. 131 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 ii. After incubation, add equal volume of Advanced DMEM/F12 medium without added cytokines or growth factors to tube. Centrifuge at 300 g for 5 minutes at brake 9. iii. Remove supernatant, add 5 mL TrypLE, and mix solution. Incubate for 5 minutes at 37 °C. iv. Add 8 mL Advanced DMEM/F12 medium without added cytokines or growth factors and mix solution 5-10 times or until clusters are no longer visible. v. Pass cells through 70 μm filter if clumping is observed after dissociation. vi. Centrifuge at 300 g for 5 minutes at brake 9. vii. Resuspend in 10 mL of Advanced DMEM/F12 medium without added cytokines or growth factors and perform cell count using NC-200. e. 5.0x106 cells were aliquoted for flow cytometric analysis using the iHSC panel. f. iHSC flow panel is shown in Table 34. g. Exemplary success criteria for iHSC redifferentiation step: purity of >70% Lineage- CD34+ cells and >5 fold expansion per input iPSC. [0404] The protocol enabled redifferentiation of γδ T-iPSC lines into hematopoietic stem cells (HSCs) at a variety of RPM in a 3L vertical wheel bioreactor using a serum-free, feeder- free (SFFF) three-dimensional (3D) culture method, the results including cell cluster formation, morphology, cell growth (e.g., metabolism), purity, etc. This was true with perfusion in the bioreactor system. [0405] Figures 3-6 show that the 3D-VW bioreactor protocol for the redifferentiation of iPSCs to iHSCs resulted in high purity, high cell viability, high yield, and sufficient fold expansion. The protocol also yielded iγδ T cells at a purity that met the exemplary success criterion and with a fold expansion that is consistent with iHSCs generated with the 2D process. These data demonstrate the efficiency and validity of the 3D-VW bioreactor protocol for the generation of iHSCs from γδ T-iPSCs as shown in Figures 3-6. List of Embodiments [0406] A nonexhaustive list of embodiments encompassed by the present invention follows. 132 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 A1. A method of de novo generation of hematopoietic stem cells (iHSCs) derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue culture vessel, said method comprising: seeding human γδ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; retaining all cells in the culture on day 8 of culture while removing the medium from the culture for about 2 days; and harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction. A2. The method of item A1, wherein the tissue culture vessel has non-traditional bioreactor geometries. A3. The method of item A1 or item A2, wherein the iPSCs are seeded at a density of about 1.0 x 105 cells/mL to about 1.8 x 105 cells/mL. A4. The method of any one of items A1-A3, wherein the tissue culture vessel comprising a vertical wheel further comprises a first medium comprising StemFit® Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, Emricasan, Polyamines, and Trans‑ISRIB (CEPT) cocktail, wherein the BMP4 and the rhVEGF are present in a ratio of 1:1, and wherein the first medium does not comprise antibiotics. A5. The method of any one of items A1-A4, wherein the method further comprises exchanging spent medium in the tissue culture vessel comprising a vertical wheel on each of days 2, 4, and 6 of culture for the about 8 days, wherein the step of exchanging spent medium comprises: removing the spent medium from the tissue culture vessel comprising a vertical wheel using a 40 µm cell retention filter and adding a wash medium at a pump flow rate of 30 mL/min, the wash medium comprising Advanced DMEM/F12 medium and StemFit® For Differentiation 133 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio; exchanging about 3 L to about 3.5 L of the wash medium in the bioreactor and removing 0.1 L of the wash medium from the tissue culture vessel comprising a vertical wheel using the 40 µm cell retention filter; and adding about 50 mL of the wash medium and about 50 mL of a medium to from the tissue culture vessel comprising the vertical wheel. A6. The method of any one of items A1-A5, wherein the iPSCs are dissociated into single cells on day 7 of culture. A7. The method of any one of items A1-A6, wherein the step of retaining all cells comprises retaining clusters, semi-clusters, and single cells. A8. The method of any one of items A1-A7, wherein the step of retaining all cells comprises removing spent medium from the tissue culture vessel comprising a vertical wheel using a 4 µm cell retention filter and adding fresh medium to the bioreactor over about 1.8 days to about 2.2 days, preferably about 2 days, at a rate of about 0.69 mL/min (1 L/day), until 2 L of media have been exchanged, wherein the fifth medium comprises Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, IL-6, TPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, wherein the fresh medium does not comprise antibiotics. A9. The method of any one of items A1-A8, wherein the vertical wheel spins at a rate of about 10 rpm to about 60 rpm, preferably about 23 rpm. A10. The method of any one of items A1-A9, wherein the step of harvesting comprises: a) removing an entire cell volume comprising EBs and iHSCs from the tissue culture vessel comprising a vertical wheel and centrifuging the entire cell volume, thereby generating a first supernatant and a first cell pellet; b) centrifuging the first supernatant, thereby generating a second supernatant and a second cell pellet, removing the second supernatant, and resuspending the second cell pellet in Advanced DMEM/F12 medium and StemFit® For Differentiation medium without cytokines, growth factors, or antibiotics; 134 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 c) adding a first volume of 20 mg/mL collagenase II to the first cell pellet and the second cell pellet, combining the resuspended first cell pellet and the resuspended second cell pellet and adding a second volume of 20 mg/mL collagenase II, thereby generating a combined cell suspension; d) adding a volume of Advanced DMEM/F12 medium and StemFit® For Differentiation medium without cytokines, growth factors, or antibiotics equal to the volume of the combined cell suspension to the combined cell suspension; e) centrifuging the combined cell suspension, thereby generating a third cell pellet; f) resuspending the third cell pellet in a volume of TrypLE and adding a volume of Advanced DMEM/F12 medium and StemFit® For Differentiation medium without cytokines, growth factors, or antibiotics, thereby generating a fourth cell suspension; g) mixing the fourth cell suspension until clusters of cells are no longer visible; h) optionally passing the fourth cell suspension through a cell strainer to obtain iHSCs, wherein the cell strainer is 70 µm mesh size, if clusters of cells are visible after the mixing step; and i) centrifuging the fourth cell suspension. A11. The method of any one of items A1-A10, wherein the cells are cultured under normoxic conditions. A12. The method of any one of items A1-A11, further comprising further purifying and/or isolating the redifferentiated iHSCs. A13. The method of any one of items A1-A12, wherein the redifferentiated iHSCs are isolated differentiated cells or are capable of being further purified and/or isolated. A14. The method of any one of items A1-A13, wherein determining purity of the redifferentiated iHSCs comprises assaying for one or more of lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a via flow cytometry or an equivalent thereof. A15. The method of item A14, wherein the lineage markers comprise all of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a. 135 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 B1. A method of de novo generation of hematopoietic stem cells (iHSCs) derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue culture vessel, said method comprising: seeding human γδ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction. B2. The method of item B1, wherein the tissue culture vessel has non-traditional bioreactor geometries. B3. The method of item B1 or B2, wherein the iPSCs are seeded at a density of about 1.0 x 105 cells/mL to about 1.8 x 105 cells/mL. B4. The method of any one of items B1-B3, wherein the tissue culture vessel comprising a vertical wheel further comprises a first medium comprising StemFit® Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, Emricasan, Polyamines, and Trans‑ISRIB (CEPT) cocktail, wherein the BMP4 and the rhVEGF are present in a ratio of 1:1, and wherein the first medium does not comprise antibiotics. B5. The method of any one of items B1-B4, wherein the method further comprises exchanging spent medium in the tissue culture vessel comprising a vertical wheel on each of days 2, 4, 6, and 8 of culture for the about 8 days, wherein the step of exchanging the spent medium comprises: a) allowing cell aggregates to settle in the tissue culture vessel comprising a vertical wheel for about 3 to about 5 minutes; b) collecting 80% of the spent medium from the tissue culture vessel comprising a vertical wheel; c) centrifuging the collected medium, thereby obtaining a cell pellet; 136 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 d) resuspending the cell pellet in StemFit® for Differentiation, thereby obtaining a cell suspension; and e) adding the cell suspension and fresh medium to the tissue culture vessel comprising a vertical wheel. B6. The method of any one of items B1-B5, wherein the step of exchanging the spent medium on days 2, 4, and 6 of culture comprises: i) allowing cell aggregates to settle in the tissue culture vessel comprising a vertical wheel for about 3 to about 5 minutes; collecting 80% of the spent medium from the tissue culture vessel comprising a vertical wheel; centrifuging the collected medium, thereby obtaining a cell pellet; resuspending the cell pellet in StemFit® for Differentiation medium, thereby obtaining a cell suspension; and adding the cell suspension and fresh medium to the tissue culture vessel comprising a vertical wheel, or ii) allowing cell aggregates to settle in the tissue culture vessel comprising a vertical wheel for about 3 to about 5 minutes; collecting 80% of the spent medium from the tissue culture vessel comprising a vertical wheel; centrifuging the collected medium, thereby obtaining a first cell pellet and a first supernatant; resuspending the first cell pellet in StemFit® for Differentiation medium and adding the resuspended first cell pellet to the tissue culture vessel comprising a vertical wheel; centrifuging the supernatant, thereby obtaining a second cell pellet and a second supernatant; and resuspending the second cell pellet in StemFit® for Differentiation medium and adding the resuspended second cell pellet and fresh medium to the tissue culture vessel comprising a vertical wheel. B7. The method of any one of items B1-B6, wherein the fresh medium added on day 2 of culture comprises: i) Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SB431542 (inhibitor of Activin/BMP/TGFβ pathway), recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), and one of L-glutamine and GlutaMaxTM supplement, or ii) StemProTM-34 Serum-free Medium (SFM) and StemPro™-34 Nutrient Supplement in 137 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 a 500:13 ratio, ascorbic acid 2-phosphate, Insulin-Transferrin-Selenium (ITS-G), monothioglycerol, SB431542, recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), and one of L- glutamine and GlutaMaxTM, wherein the VEGF, bFGF, and SCF are present in a ratio of 1:0.625:0.625, and wherein the second medium does not comprise antibiotics. B8. The method of any one of items B1-B7, wherein the iPSCs are dissociated into single cells on day 7 of culture. B9. The method of any one of items B1-B8, wherein the step of harvesting redifferentiated iHSCs comprises retaining clusters, semi-clusters, and single cells. B10. The method of any one of items B1-B9, wherein the step of harvesting comprises retaining all cells in the tissue culture vessel comprising a vertical wheel. B11. The method of any one of items B1-B10, wherein the vertical wheel spins at a rate of about 10 rpm to about 60 rpm, preferably about 40 rpm. B12. The method of any one of items B1-B11, wherein the step of harvesting redifferentiated iHSCs comprises: a) removing an entire cell volume comprising embryoid bodies (EBs) and iHSCs from the tissue culture vessel comprising a vertical wheel and centrifuging the entire cell volume, thereby generating a first supernatant and a first cell pellet; b) centrifuging the first supernatant, thereby generating a second supernatant and a second cell pellet and removing the second supernatant; c) resuspending the first cell pellet and the second cell pellet in Advanced DMEM/F12 medium without cytokines, growth factors, or antibiotics; d) adding a volume of 20 mg/mL collagenase II to the first cell pellet, thereby generating a first cell suspension; e) adding a volume of Advanced DMEM/F12 medium without cytokines, growth factors, or antibiotics equal to the volume of the first cell suspension to the first cell suspension; f) centrifuging the first cell suspension, thereby generating a third cell pellet; g) resuspending the third cell pellet in a volume of TrypLE and adding a volume of 138 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 Advanced DMEM/F12 medium without cytokines, growth factors, or antibiotics, thereby generating a second cell suspension; h) mixing the second cell suspension until clusters of cells are no longer visible; i) optionally passing the second cell suspension through a cell strainer to obtain iHSCs, wherein the cell strainer is 70 µm mesh size, if clusters of cells are visible after the mixing step; and j) centrifuging the second cell suspension. B13. The method of any one of items B1-B12, wherein the cells are cultured under normoxic conditions. B14. The method of any one of items B1-B13, further comprising further purifying and/or isolating the redifferentiated iHSCs. B15. The method of any one of items B1-B14, wherein the redifferentiated iHSCs are isolated differentiated cells or are capable of being further purified and/or isolated. B16. The method of any one of items B1-B15, wherein determining purity of the redifferentiated iHSCs comprises assaying for one or more of lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a via flow cytometry or an equivalent thereof. B17. The method of item B16, wherein the lineage markers comprise all of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a. B18. One or more cells obtained from a method of de novo generation of hematopoietic stem cells (iHSCs) derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions, said method comprising the steps of any one of items A1-B17. C1. One or more cells obtained from a method of de novo generation of hematopoietic stem cells (iHSCs) derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue culture vessel, said method comprising: seeding human γδ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; 139 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; retaining all cells in the culture on day 8 of culture while removing the medium from the culture for about 2 days; and harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction. C2. The one or more cells of item C1, wherein the tissue culture vessel has non-traditional bioreactor geometries. C3. The one or more cells of item C1 or C2, wherein the iPSCs are seeded at a density of about 1.0 x 105 cells/mL to about 1.8 x 105 cells/mL. C4. The one or more cells of any one of items C1-C3, wherein the tissue culture vessel comprising a vertical wheel further comprises a first medium comprising StemFit® Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, Emricasan, Polyamines, and Trans‑ISRIB (CEPT) cocktail, wherein the BMP4 and the rhVEGF are present in a ratio of 1:1, and wherein the first medium does not comprise antibiotics. C5. The one or more cells of any one of items C1-C4, wherein the method further comprises exchanging spent medium in the tissue culture vessel comprising a vertical wheel on each of days 2, 4, and 6 of culture for the about 8 days, wherein the step of exchanging spent medium comprises: removing the spent medium from the tissue culture vessel comprising a vertical wheel using a 40 µm cell retention filter and adding a wash medium at a pump flow rate of 30 mL/min, the wash medium comprising Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio; exchanging about 3 L to about 3.5 L of the wash medium in the bioreactor and removing 0.1 L of the wash medium from the tissue culture vessel comprising a vertical wheel using the 40 µm cell retention filter; and 140 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 adding about 50 mL of the wash medium and about 50 mL of a medium to from the tissue culture vessel comprising the vertical wheel. C6. The one or more cells of any one of items C1-C5, wherein the iPSCs are dissociated into single cells on day 7 of culture. C7. The one or more cells of any one of items C1-C6, wherein the step of retaining all cells comprises retaining clusters, semi-clusters, and single cells. C8. The one or more cells of any one of items C1-C7, wherein the step of retaining all cells comprises removing spent medium from the tissue culture vessel comprising a vertical wheel using a 4 µm cell retention filter and adding fresh medium to the bioreactor over about 1.8 days to about 2.2 days, preferably about 2 days, at a rate of about 0.69 mL/min (1 L/day), until 2 L of media have been exchanged, wherein the fifth medium comprises Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, IL-6, TPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, wherein the fresh medium does not comprise antibiotics. C9. The one or more cells of any one of items C1-C8, wherein the vertical wheel spins at a rate of about 10 rpm to about 60 rpm, preferably about 23 rpm. C10. The one or more cells of any one of items C1-C9, wherein the step of harvesting comprises: a) removing an entire cell volume comprising EBs and iHSCs from the tissue culture vessel comprising a vertical wheel and centrifuging the entire cell volume, thereby generating a first supernatant and a first cell pellet; b) centrifuging the first supernatant, thereby generating a second supernatant and a second cell pellet, removing the second supernatant, and resuspending the second cell pellet in Advanced DMEM/F12 medium and StemFit® For Differentiation medium without cytokines, growth factors, or antibiotics; c) adding a first volume of 20 mg/mL collagenase II to the first cell pellet and the second cell pellet, combining the resuspended first cell pellet and the resuspended second cell pellet and adding a second volume of 20 mg/mL collagenase II, thereby generating a combined cell suspension; d) adding a volume of Advanced DMEM/F12 medium and StemFit® For Differentiation 141 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 medium without cytokines, growth factors, or antibiotics equal to the volume of the combined cell suspension to the combined cell suspension; e) centrifuging the combined cell suspension, thereby generating a third cell pellet; f) resuspending the third cell pellet in a volume of TrypLE and adding a volume of Advanced DMEM/F12 medium and StemFit® For Differentiation medium without cytokines, growth factors, or antibiotics, thereby generating a fourth cell suspension; g) mixing the fourth cell suspension until clusters of cells are no longer visible; h) optionally passing the fourth cell suspension through a cell strainer to obtain iHSCs, wherein the cell strainer is 70 µm mesh size, if clusters of cells are visible after the mixing step; and i) centrifuging the fourth cell suspension. C11. The one or more cells of any one of items C1-C10, wherein the cells are cultured under normoxic conditions. C12. The one or more cells of any one of items C1-C11, further comprising further purifying and/or isolating the redifferentiated iHSCs. C13. The one or more cells of any one of items C1-C12, wherein the redifferentiated iHSCs are isolated differentiated cells or are capable of being further purified and/or isolated. C14. The one or more cells of any one of items C1-C13, wherein determining purity of the redifferentiated iHSCs comprises assaying for one or more of lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a via flow cytometry or an equivalent thereof. C15. The one or more cells of item C14, wherein the lineage markers comprise all of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a. D1. One or more cells obtained from a method of de novo generation of hematopoietic stem cells (iHSCs) derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue culture vessel, said method comprising: seeding human γδ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; 142 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction. D2. The one or more cells of item D1, wherein the tissue culture vessel has non-traditional bioreactor geometries. D3. The one or more cells of item D1 or D2, wherein the iPSCs are seeded at a density of about 1.0 x 105 cells/mL to about 1.8 x 105 cells/mL. D4. The one or more cells of any one of items D1-D3, wherein the tissue culture vessel comprising a vertical wheel further comprises a first medium comprising StemFit® Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, Emricasan, Polyamines, and Trans‑ISRIB (CEPT) cocktail, wherein the BMP4 and the rhVEGF are present in a ratio of 1:1, and wherein the first medium does not comprise antibiotics. D5. The one or more cells of any one of items D1-D4, wherein the method further comprises exchanging spent medium in the tissue culture vessel comprising a vertical wheel on each of days 2, 4, 6, and 8 of culture for the about 8 days, wherein the step of exchanging the spent medium comprises: a) allowing cell aggregates to settle in the tissue culture vessel comprising a vertical wheel for about 3 to about 5 minutes; b) collecting 80% of the spent medium from the tissue culture vessel comprising a vertical wheel; c) centrifuging the collected medium, thereby obtaining a cell pellet; d) resuspending the cell pellet in StemFit® for Differentiation, thereby obtaining a cell suspension; and e) adding the cell suspension and fresh medium to the tissue culture vessel comprising 143 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 a vertical wheel. D6. The one or more cells of any one of items D1-D5, wherein the step of exchanging the spent medium on days 2, 4, and 6 of culture comprises: i) allowing cell aggregates to settle in the tissue culture vessel comprising a vertical wheel for about 3 to about 5 minutes; collecting 80% of the spent medium from the tissue culture vessel comprising a vertical wheel; centrifuging the collected medium, thereby obtaining a cell pellet; resuspending the cell pellet in StemFit® for Differentiation medium, thereby obtaining a cell suspension; and adding the cell suspension and fresh medium to the tissue culture vessel comprising a vertical wheel, or ii) allowing cell aggregates to settle in the tissue culture vessel comprising a vertical wheel for about 3 to about 5 minutes; collecting 80% of the spent medium from the tissue culture vessel comprising a vertical wheel; centrifuging the collected medium, thereby obtaining a first cell pellet and a first supernatant; resuspending the first cell pellet in StemFit® for Differentiation medium and adding the resuspended first cell pellet to the tissue culture vessel comprising a vertical wheel; centrifuging the supernatant, thereby obtaining a second cell pellet and a second supernatant; and resuspending the second cell pellet in StemFit® for Differentiation medium and adding the resuspended second cell pellet and fresh medium to the tissue culture vessel comprising a vertical wheel. D7. The one or more cells of any one of items D1-D6, wherein the fresh medium added on day 2 of culture comprises: i) Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SB431542 (inhibitor of Activin/BMP/TGFβ pathway), recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), and one of L-glutamine and GlutaMaxTM supplement, or ii) StemProTM-34 Serum-free Medium (SFM) and StemPro™-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, Insulin-Transferrin-Selenium (ITS-G), monothioglycerol, SB431542, recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), and one of L- 144 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 glutamine and GlutaMaxTM, wherein the VEGF, bFGF, and SCF are present in a ratio of 1:0.625:0.625, and wherein the second medium does not comprise antibiotics. D8. The one or more cells of any one of items D1-D7, wherein the iPSCs are dissociated into single cells on day 7 of culture. D9. The one or more cells of any one of items D1-D8, wherein the step of harvesting redifferentiated iHSCs comprises retaining clusters, semi-clusters, and single cells. D10. The one or more cells of any one of D1-D9, wherein the step of retaining all cells comprises removing spent medium from the tissue culture vessel comprising a vertical wheel using a 4 µm cell retention filter and adding fresh medium to the bioreactor over about 1.8 days to about 2.2 days, preferably about 2 days, at a rate of about 0.69 mL/min (1 L/day), until 2 L of media have been exchanged, wherein the fifth medium comprises Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, IL-6, TPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, wherein the fresh medium does not comprise antibiotics. D11. The one or more cells of any one of items D1-D10, wherein the vertical wheel spins at a rate of about 10 rpm to about 60 rpm, preferably about 40 rpm. D12. The one or more cells of any one of items D1-D11, wherein the step of harvesting redifferentiated iHSCs comprises: a) removing an entire cell volume comprising embryoid bodies (EBs) and iHSCs from the tissue culture vessel comprising a vertical wheel and centrifuging the entire cell volume, thereby generating a first supernatant and a first cell pellet; b) centrifuging the first supernatant, thereby generating a second supernatant and a second cell pellet and removing the second supernatant; c) resuspending the first cell pellet and the second cell pellet in Advanced DMEM/F12 medium without cytokines, growth factors, or antibiotics; d) adding a volume of 20 mg/mL collagenase II to the first cell pellet, thereby generating a first cell suspension; e) adding a volume of Advanced DMEM/F12 medium without cytokines, growth factors, 145 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 or antibiotics equal to the volume of the first cell suspension to the first cell suspension; f) centrifuging the first cell suspension, thereby generating a third cell pellet; g) resuspending the third cell pellet in a volume of TrypLE and adding a volume of Advanced DMEM/F12 medium without cytokines, growth factors, or antibiotics, thereby generating a second cell suspension; h) mixing the second cell suspension until clusters of cells are no longer visible; i) optionally passing the second cell suspension through a cell strainer to obtain iHSCs, wherein the cell strainer is 70 µm mesh size, if clusters of cells are visible after the mixing step; and j) centrifuging the second cell suspension. D13. The one or more cells of any one of items D1-D12, wherein the cells are cultured under normoxic conditions. D14. The one or more cells of any one of items D1-D13, further comprising further purifying and/or isolating the redifferentiated iHSCs. D15. The one or more cells of any one of claims D1-D14, wherein the redifferentiated iHSCs are isolated differentiated cells or are capable of being further purified and/or isolated. D16. The one or more cells of any one of items D1-D15, wherein the one or more cells have high purity, viability, fold expansion, and potential to redifferentiate into immune effectors, including iPSC-derived γδ (iγδ) T cells. D17. The one or more cells of any one of items D1-D16, wherein the step of determining purity of the iHSCs comprises assaying for one or more, optionally all, of the lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a via flow cytometry or an equivalent thereof. D18. The one or more cells of any one of items D1-D17, wherein the lineage markers comprise one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a. E1. A composition comprising the one or more cells of any one of items C1-D18. F1. Use of the one or more cells of any one of items C1-D18, in preparation of cells for treating a pathology, disease(s), in preparation of lymphocytes, in a bioreactor, in tissue engineering or in vitro drug screening for diseases. 146 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 G1. A system for performing a method of de novo generation of hematopoietic stem cells (iHSCs) derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions, said method comprising the steps of any one of claims A1-B17, wherein the vertical wheel is either perpendicular or non-perpendicular to the vessel. H1. A method of de novo generation of hematopoietic stem cells (iHSCs) derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue culture vessel, said method comprising: a means for seeding human γδ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; a means for redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; a means for retaining all cells in the culture on day 8 of culture while removing the medium from the culture for about 2 days; and a means for harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction. I1. A method of de novo generation of hematopoietic stem cells (iHSCs) derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue culture vessel, said method comprising: a means for seeding human γδ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; a means for redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; a means for harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction. J1. An iHSC produced according to the method of item H1 or item I1. 147 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 K1. One or more cells obtained from a method of de novo generation of hematopoietic stem cells (iHSCs) derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue culture vessel, said method comprising: a means for seeding human γδ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; a means for redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; a means for retaining all cells in the culture on day 8 of culture while removing the medium from the culture for about 2 days; and a means for harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction. L1. One or more cells obtained from a method of de novo generation of hematopoietic stem cells (iHSCs) derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue culture vessel, said method comprising: a means for seeding human γδ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; a means for redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; a means for harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction. M1. A method of de novo generation of induced pluripotent stem cell (iPSC)-derived hematopoietic stem cells (iHSCs) from γδ T cell-derived iPSCs (γδ T-iPSCs) under serum-free, feeder-free culture conditions, said method comprising one or more of the following steps: i) obtaining one or more human induced pluripotent stem cells (iPSCs), and wherein the method further comprises one or more of the following steps: a) after incubating the iPSCs in a tissue culture vessel comprising a vertical wheel in a first medium with agitation at about 10-60 rpm, preferably 40 rpm, for about 1.8 to about 2.2 days, preferably about 2 days, removing an entire cell volume from the tissue 148 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 culture vessel comprising a vertical wheel and centrifuging the entire cell volume, wherein the first medium comprises StemFit® Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF) and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, Emricasan, Polyamines, and Trans‑ISRIB (CEPT) cocktail, wherein the first medium does not comprise antibiotics; b) centrifuging the first cell suspension and thereby generating a first cell pellet; c) resuspending the first cell pellet into a second medium to form a second cell suspension, wherein the second medium comprises: i) Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SB431542 (inhibitor of Activin/BMP/TGFβ pathway), recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), and one of L-glutamine and GlutaMaxTM supplement, or ii) StemProTM-34 Serum-free Medium (SFM) and StemPro™-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, Insulin-Transferrin- Selenium (ITS-G), monothioglycerol, SB431542, recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), and one of L-glutamine and GlutaMaxTM, wherein the VEGF, bFGF, and SCF are present in a ratio of 1:0.625:0.625, and wherein the second medium does not comprise antibiotics; d) culturing and agitating the second cell suspension in the tissue culture vessel comprising a vertical wheel at about 10-60 rpm, preferably 40 rpm, for about 1.8 to about 2.2 days, preferably about 2 days; e) stopping the agitating for about 5 minutes, wherein cell aggregates settle in the second cell suspension; f) collecting about 80% of the second cell suspension from a top volume of the second cell suspension, from the tissue culture vessel comprising a vertical wheel; g) centrifuging the collected second cell suspension, thereby forming a second cell pellet and resuspending the second pellet in about 1 mL of one of StemFit® For 149 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 Differentiation solution and/or StemPro™-34 SFM; h) adding the resuspended second cell pellet into a third medium forming a third cell suspension, wherein the third medium comprises: i) Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), interleukin- 3 (IL-3), interleukin-6 (IL-6), one of (i) thrombopoietin (TPO) and (ii) erythropoietin (EPO), and one of (i) L-glutamine and (ii) GlutaMaxTM, or ii) StemPro™-34 SFM and StemPro™-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, rhVEGF, IL-3, IL-6, Flt3L, one of (i) TPO and (ii) EPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, and wherein the third medium does not comprise antibiotics; i) placing the third cell suspension into the tissue culture vessel comprising a vertical wheel with remaining second cell suspension forming a fourth cell suspension; j) culturing and agitating the fourth cell suspension in the tissue culture vessel comprising a vertical wheel at about 10-60 rpm, preferably 40 rpm, for about 1.8 to about 2.2 days, preferably about 2 days; k) stopping the agitating for about 5 minutes; wherein cell aggregates settle in the cell suspension; l) collecting about 80% of the fourth cell suspension and centrifuging the collected fourth cell suspension forming a third cell pellet, and resuspending the third cell pellet in about 1 mL of one of StemFit® For Differentiation solution and StemPro™-34 SFM; m) suspending the third cell pellet dissolved in the about 1 mL of one of StemFit® For Differentiation solution and StemPro™-34 SFM into a fourth medium forming a fifth cell suspension, wherein the fourth medium comprises: i) Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, IL-6, one of (i) TPO and (ii) EPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, or ii) StemPro™-34 SFM and StemPro™-34 Nutrient Supplement in a 500:13 150 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 ratio, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, IL-6, one of (i) TPO and (ii) EPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, and wherein the fourth medium does not comprise antibiotics; n) placing the fifth cell suspension into the tissue culture vessel comprising a vertical wheel with the remaining fourth cell suspension thereby forming a sixth cell suspension; o) culturing and agitating the sixth cell suspension in the tissue culture vessel comprising a vertical wheel at about 10-60 rpm, preferably 40 rpm, for about 1.8 to about 2.2 days, preferably about 2 days; p) repeating steps k) to o); optionally wherein the sixth cell suspension is centrifuged and a supernatant from the centrifuged sixth cell suspension is centrifuged and the resulting cell pellet is resuspended in about 1 mL of one of StemFit® For Differentiation solution and StemPro™-34 SFM and combined with a seventh cell suspension obtained by resuspending a cell pellet from the first centrifugation to form an eighth cell suspension before culturing and agitating the eighth cell suspension in the tissue culture vessel comprising a vertical wheel at about 10-60 rpm, preferably 40 rpm, for about 1.8 to about 2.2 days, preferably about 2 days; q) removing all the eighth cell suspension from the tissue culture vessel comprising a vertical wheel and harvesting EBs and iHSCs in the eighth cell suspension by centrifugation, thereby generating a fourth cell pellet and a first supernatant; r) collecting the first supernatant; s) centrifuging the first supernatant, thereby generating a fifth cell pellet and resuspending the fifth cell pellet in a fifth medium thereby generating a ninth cell suspension, wherein the fifth medium comprises Advanced DMEM/F12 medium, wherein the fifth medium does not comprise cytokines, antibiotics, or growth factors, and wherein cells of the ninth cell suspension comprise iHSCs, which are defined as CD34+ and lineage marker-negative, where the lineage markers comprise one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a; t) resuspending the fourth cell pellet of step q), in a volume of about 20 mg/mL collagenase type II, thereby generating a tenth cell suspension; u) adding the fifth medium to the tenth cell suspension; 151 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 v) centrifuging the tenth cell suspension, thereby generating a sixth cell pellet, and adding TrypLE to the sixth cell pellet, thereby generating an eleventh cell suspension; w) adding additional fifth medium to the eleventh cell suspension and mixing; x) centrifuging the eleventh cell suspension to form a seventh cell pellet and resuspending the seventh cell pellet in additional fifth medium to form a twelfth cell suspension; y) optionally passing the twelfth cell suspension through a cell strainer to obtain iHSCs, wherein the cell strainer is 70 µm mesh size, if cell clumps are present in the twelfth cell suspension; z) centrifuging the twelfth cell suspension; aa) not introducing serum in any of steps a)-z); bb) not introducing any additional cells, including feeder cells or stromal cells, in any of steps a)-aa); cc) determining the purity of the iHSCs from the first supernatant of step s) and the iHSCs from step y), said purity defined as CD34+ and lineage marker-negative, where the lineage markers comprise one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a;and dd) obtaining de novo generated iHSCs. N1. A method of de novo generation of induced pluripotent stem cell (iPSC)-derived hematopoietic stem cells (iHSCs) from γδ T cell-derived iPSCs (γδ T-iPSCs) under serum-free, feeder-free culture conditions, said method comprising one or more of the following steps: i) obtaining one or more human induced pluripotent stem cells (iPSCs), and wherein the method further comprises one or more of the following steps: a) seeding the iPSCs, wherein the seeding is performed by suspending the iPSCs in a first medium into a tissue culture vessel comprising a vertical wheel, thereby producing a first cell suspension, wherein the first medium comprises one of StemFit® Basic 04 medium and StemFit® Basic 03 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, Emricasan, Polyamines, and Trans‑ISRIB (CEPT) cocktail, 152 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 and wherein the first medium does not comprise antibiotics; b) culturing and agitating the cells in the tissue culture vessel comprising a vertical wheel at about 10-60 rpm, preferably 23 rpm, for about 1.8 to about 2.2 days, preferably about 2 days, wherein embryoid bodies (EBs) are generated; c) removing the first medium from the tissue culture vessel comprising a vertical wheel and adding a wash medium comprising Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, at a rate of about 30 mL/min; d) exchanging about 3L to about 3.5 L of the wash medium in the tissue culture vessel comprising a vertical wheel using a 40 µm cell retention filter at a pump flow rate of 30 mL/min and removing 0.1 L of the wash medium from the tissue culture vessel comprising a vertical wheel; e) adding about 50 mL of the wash medium and about 50 mL of a second medium to the tissue culture vessel comprising a vertical wheel, wherein the second medium comprises Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SB431542 (inhibitor of Activin/BMP/TGFβ pathway), recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), and one of L-glutamine and GlutaMaxTM supplement, wherein the second medium does not comprise antibiotics; f) culturing and agitating the cells in the tissue culture vessel comprising a vertical wheel at about 10-60 rpm, preferably 23 rpm, for about 1.8 to about 2.2 days, preferably about 2 days; g) removing the second medium from the tissue culture vessel comprising a vertical wheel and adding the wash medium at a rate of about 30 mL/min; h) exchanging about 3L to about 3.5 L of the wash medium in the tissue culture vessel comprising a vertical wheel using a 40 µm cell retention filter at a pump flow rate of 30 mL/min and removing 0.1 L of the wash medium from the tissue culture vessel comprising a vertical wheel; i) adding about 50 mL of the wash medium and about 50 mL of a third medium to the tissue culture vessel comprising a vertical wheel, wherein the third medium comprises 153 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises and further comprises SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), interleukin-3 (IL-3), interleukin-6 (IL-6), thrombopoietin (TPO), and one of (i) L- glutamine and (ii) GlutaMaxTM, wherein the third medium does not comprise antibiotics; j) culturing and agitating the cells in the tissue culture vessel comprising a vertical wheel at about 10-60 rpm, preferably 23 rpm, for about 1.8 to about 2.2 days, preferably about 2 days; k) removing the third medium from the tissue culture vessel comprising a vertical wheel and adding the wash medium at a rate of about 30 mL/min; l) exchanging about 3L to about 3.5 L of the wash medium in the tissue culture vessel comprising a vertical wheel using a 40 µm cell retention filter at a pump flow rate of 30 mL/min and removing 0.1 L of the wash medium from the tissue culture vessel comprising a vertical wheel; m) adding about 50 mL of the wash medium and about 50 mL of a fourth medium to the tissue culture vessel comprising a vertical wheel, wherein the fourth medium comprises Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, IL-6, TPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, wherein the fourth medium does not comprise antibiotics; n) culturing and agitating the cells in the tissue culture vessel comprising a vertical wheel at about 10-60 rpm, preferably 23 rpm, for about 1.8 to about 2.2 days, preferably about 2 days; o) simultaneously removing the fourth medium from the tissue culture vessel comprising a vertical wheel and adding a fifth medium to the tissue culture vessel comprising a vertical wheel over about 1.8 days to about 2.2 days, preferably about 2 days, using a 4 µm cell retention filter at a pump flow rate of about 0.69 mL/min (1 L/day), until 2 L of media have been exchanged, wherein the fifth medium comprises Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, IL- 154 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 6, TPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, wherein the fifth medium does not comprise antibiotics; p) removing an entire cell volume comprising EBs and iHSCs from the tissue culture vessel comprising a vertical wheel and centrifuging the entire cell volume, thereby generating a first supernatant and a first cell pellet; q) centrifuging the first supernatant, thereby generating a second supernatant and a second cell pellet, removing the second supernatant, and resuspending the second cell pellet in Advanced DMEM/F12 medium and StemFit® For Differentiation medium without cytokines, growth factors, or antibiotics; r) adding a first volume of 20 mg/mL collagenase II to the first cell pellet, resuspending the first cell pellet, and adding a second volume of 20 mg/mL collagenase II, thereby generating a first cell suspension; s) adding a volume of Advanced DMEM/F12 medium and StemFit® For Differentiation medium without cytokines, growth factors, or antibiotics equal to the volume of the first cell suspension to the first cell suspension; t) centrifuging the cell suspension, thereby generating a third cell pellet; u) resuspending the third cell pellet in a volume of TrypLE and adding a volume of Advanced DMEM/F12 medium and StemFit® For Differentiation medium without cytokines, growth factors, or antibiotics, thereby generating a second cell suspension; v) mixing the second cell suspension until clusters of cells are no longer visible; w) optionally passing the second cell suspension through a cell strainer to obtain iHSCs, wherein the cell strainer is 70 µm mesh size, if clusters of cells are visible after the mixing step; x) centrifuging the second cell suspension; y) not introducing serum in any of steps a)-x); z) not introducing any additional cells, including feeder cells or stromal cells, in any of steps a)-y); aa) determining the purity of the iHSCs from step w), said purity defined as CD34+ and lineage marker-negative, where the lineage markers comprise one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a; and bb) obtaining de novo generated iHSCs. 155 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 O1. A method of de novo generation of induced pluripotent stem cell (iPSC)-derived hematopoietic stem cells (iHSCs) from γδ T cell-derived iPSCs (γδ T-iPSCs) under serum-free, feeder-free culture conditions, said method comprising one or more of the following steps: i. obtaining one or more human induced pluripotent stem cells (iPSCs), and wherein the method further comprises one or more of the following steps: a. seeding the iPSCs, wherein the seeding is performed by suspending the iPSCs in a first medium into a bioreactor, wherein the bioreactor is a vertical-wheel bioreactor, thereby producing a first cell suspension, wherein the first medium comprises one of StemFit® Basic 04 medium and StemFit® Basic 03 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, Emricasan, Polyamines, and Trans‑ISRIB (CEPT) cocktail, and wherein the first medium does not comprise antibiotics; b. culturing and agitating the first cell suspension in the bioreactor at about 10-60 rpm, preferably 40 rpm, wherein the first cell suspension is cultured and agitated for about 1.8 to about 2.2 days, preferably about 2 days, wherein embryoid bodies (EBs) are generated; c. centrifuging the first cell suspension and thereby generating a first cell pellet; d. resuspending the first cell pellet into a second medium to form a second cell suspension, wherein the second medium comprises: i. Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SB431542 (inhibitor of Activin/BMP/TGFβ pathway), rhVEGF, basic fibroblast growth factor (bFGF), stem cell factor (SCF), and one of L-glutamine and GlutaMaxTM supplement, or ii. StemProTM-34 Serum-free Medium (SFM) and StemPro™-34 156 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2- phosphate, Insulin-Transferrin-Selenium (ITS-G), monothioglycerol, SB431542, rhVEGF, bFGF, SCF, and one of L-glutamine and GlutaMaxTM, and wherein the second medium does not comprise antibiotics; e. culturing and agitating the second cell suspension in the bioreactor for about 1.8 to about 2.2 days, preferably about 2 days; f. stopping the agitating for about 5 minutes, wherein cell aggregates settle in the second cell suspension; g. collecting about 80% of the second cell suspension from a top volume of the second cell suspension, from the bioreactor; h. centrifuging collected second cell suspension forming a second cell pellet and resuspending the second pellet in about 1 mL of one of StemFit® For Differentiation solution and StemPro™-34 SFM; i. suspending the second cell pellet in the about 1 mL of one of StemFit® For Differentiation solution and or StemPro™-34 SFM into a third medium forming a third cell suspension, wherein the third medium comprises: i. Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), interleukin- 3 (IL-3), interleukin-6 (IL-6), one of (i) thrombopoietin (TPO) and (ii) erythropoietin (EPO), and one of (i) L-glutamine and (ii) GlutaMaxTM, or ii. StemPro™-34 SFM and StemPro™-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, rhVEGF, IL-3, IL-6, Flt3L, one of (i) TPO and (ii) EPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, and wherein the third medium does not comprise antibiotics; 157 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 j. placing the third cell suspension into the bioreactor with remaining second cell suspension forming a fourth cell suspension; k. culturing and agitating the fourth cell suspension in the bioreactor for about 1.8 to about 2.2 days, preferably about 2 days; l. stopping the agitating for about 5 minutes; wherein cell aggregates settle in the cell suspension; m. collecting about 80% of the fourth cell suspension and centrifuging the collected fourth cell suspension forming a third cell pellet, and resuspending the third cell pellet in about 1 mL of one of StemFit® For Differentiation solution and StemPro™-34 SFM; n. suspending the third cell pellet dissolved in the about 1 mL of one of StemFit® For Differentiation solution and StemPro™-34 SFM into a fourth medium forming a fifth cell suspension, wherein the fourth medium comprises: i. Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, IL-6, one of (i) TPO and (ii) EPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, or ii. StemPro™-34 SFM and StemPro™-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, IL-6, one of (i) TPO and (ii) EPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, and wherein the fourth medium does not comprise antibiotics; o. placing the fifth cell suspension into the bioreactor with the remaining fourth cell suspension thereby forming a sixth cell suspension; p. culturing and agitating the sixth cell suspension in the bioreactor for about 1.8 to about 2.2 days, preferably about 2 days; q. repeating steps l) to p); r. removing all the eighth cell suspension from the bioreactor and harvesting EBs and iHSCs in the cell suspension by centrifugation, 158 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 thereby generating a fourth cell pellet and a first supernatant; s. collecting the first supernatant; t. centrifuging the first supernatant, thereby generating a fifth cell pellet and resuspending the fifth cell pellet in a fifth medium thereby generating a ninth cell suspension, wherein the fifth medium comprises Advanced DMEM/F12 medium, wherein the fifth medium does not comprise cytokines, antibiotics or growth factors, and wherein cells of the ninth cell suspension comprise iHSCs, which are defined as CD34+ and lineage marker-negative, where the lineage markers comprise one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a; u. resuspending the fourth cell pellet of step r), in a volume of about 20 mg/mL collagenase type II, thereby generating a tenth cell suspension; v. adding the fifth medium to the tenth cell suspension; w. centrifuging the tenth cell suspension, thereby generating a sixth cell pellet, and adding TrypLE to the sixth cell pellet, thereby generating an eleventh cell suspension; x. adding additional fifth medium to the eleventh cell suspension and mixing; y. centrifuging the eleventh cell suspension to form a seventh cell pellet and resuspending the seventh cell pellet in additional fifth medium to form a twelfth cell suspension; z. passing the twelfth cell suspension through a cell strainer to obtain iHSCs, wherein the cell strainer is 70 µm mesh size; aa. determining the purity of the iHSCs from the first supernatant of step t) and the iHSCs from step z), said purity defined as CD34+ and lineage marker-negative, where the lineage markers comprise one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a; bb. not introducing serum in any of steps a)-aa); cc. not introducing any additional cells, including feeder cells or stromal 159 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 cells, in any of steps a)-bb); and dd. obtaining de novo generated iHSCs. O2. The method of embodiment O1, wherein the EB and γδ T cell-derived iPSCs are from a tissue culture vessel. O3. The method of embodiment O1 or O2, wherein the first, second, fourth, sixth, and/or eighth cell suspensions are cultured under normoxic conditions. O4. The method of any one of embodiments O1-O3, further comprising further purifying and/or isolating the iHSCs. O5. The method of any one of embodiments O1- O4, wherein the obtained iHSCs are isolated redifferentiated cells or are capable of being further purified and/or isolated. O6. The method of any one of embodiments O1-O5, wherein the method further comprises assaying for all of the lineage markers CD3, CD19, CD14, CD11b, CD56, CD20, CD16, CD2, and CD235a via flow cytometry or an equivalent thereof. O7. The method of any one of embodiments O1-O6, wherein any one of steps a)-cc) comprises gently dispersing the cells using a pipet or equivalent thereof. O8. The method of any one of embodiments O1 – O7, wherein cells of the first medium are at a concentration of about 80,000 to about 180,000 cells/mL in a volume of about 50 to about 70 mL. O9. The method of any one of embodiments O1 – O8, wherein the cells of the first medium are at a concentration of about 150,000 cells/mL in a volume of about 60 mL. O10. The method of any one of claims O1-O9, wherein the medium of step a) further comprises a ROCK inhibitor. O11. The method of any one of embodiments O1-O9, wherein the medium of step a) further comprises a Chroman 1, Emricasan, Polyamines, and Trans‑ISRIB (CEPT) cocktail. O12. The method of any one of embodiments O1-O11, wherein after step b) all the cell volume of the first cell suspension is removed from the bioreactor prior to centrifugation. O13. The method of any one of embodiments O1-O12, wherein the cell morphology is imaged via microscopy, e.g., bright field microscopy. O14. The method of any one of embodiments O1-O13, wherein the medium in steps d), i), n), or t) comprises L-glutamine. 160 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 O15. The method of any one of claims O1-O13, wherein the medium in steps d), i), n), or t) comprises GlutaMaxTM. O16. The method of any one of embodiments O1-O15, wherein the medium in steps i) or n) comprises TPO. O17. The method of any one of embodiments O1-O15, wherein the medium in steps i) or n) comprises EPO. O18. The method of any one of embodiments O1-O17, wherein the centrifugation of any one of the steps is performed at 100g for 1 minute at brake 6. O19. The method of any one of embodiments O1-O18, wherein the centrifugation of any one of the steps is performed at 300g for 5 minutes. O20. The method of any one of embodiments O1-O19, wherein the centrifugation of any one of the steps is performed at 300g for 3 minutes. O21. The method of any one of embodiments O1-O20, wherein the centrifugation in step r) is performed twice, first at 100g for 1 minute at brake 6 and second at 300g for 5 minutes. O22. The method of any one of embodiments O1-O21, wherein the tenth cell suspension of step u) is incubated with collagenase for 20 minutes at 37 °C. O23. The method of any one of embodiments O1-O22, wherein the eleventh cell suspension of step w) is incubated with TrypLE for 5 minutes at 37 °C. O24. The method of any one of embodiments O1-O23, wherein passing of the twelfth cell suspension in step z) further comprises passing the twelfth cell suspension through a second cell strainer with a 40 µm mesh size. O25. The method of any one of embodiments O1-O24, wherein the culturing of the iPSC cells, EBs, and iHSCs is performed at 37 °C and 5% CO2. O26. The method of any one of embodiments O1-O25, wherein the bioreactor wheel is rotating at 60 rpm. O27. The method of any one of embodiments O1-O25, wherein the bioreactor wheel is rotating at 30 rpm. O28. The method of any one of embodiments O1-O25, wherein the bioreactor wheel is rotating at 24 rpm. O29. The method of any one of embodiments O1-O25, wherein the bioreactor wheel is rotating at 15 rpm. 161 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 O30. The method of any one of embodiments O1-O29, wherein the bioreactor wheel is perpendicular to the bioreactor vessel. O31. The method of any one of embodiments O1-O29, wherein the bioreactor wheel is not perpendicular to the bioreactor vessel. O32. The method of any one of embodiments O1-O7, and O10-O31, wherein iPSCs in the first medium are at a concentration of about 80,000 to about 180,000 cells/mL in a volume of about 90 to about 110 mL. O33. The method of any one of embodiments O1-A7, and O10-A32, wherein the iPSCs in the first medium are at a concentration of about 150,000 cells/mL in a volume of about 100 mL. O34. The method of any one of embodiments O1-O7, and O10-O31, wherein iPSCs in the first medium are at a concentration of about 5,000 to about 180,000 cells/mL in a volume of about 0.45 to about 0.55 L. O35. The method of any one of embodiments O1-O7, O10-O31, and O34, wherein the iPSCs in the first medium are at a concentration of about 20,000 cells/mL in a volume of about 0.5 L. O36. The method of any one of embodiments O1-O7, and O10-O31, wherein iPSCs in the first medium are at a concentration of about 5,000 to about 180,000 cells/mL in a volume of about 2 to about 5 L. O37. The method of any one of embodiments O1-O7, and O10-O31, wherein iPSCs in the first medium are at a concentration of about 5,000 to about 180,000 cells/mL in a volume of about 10 to about 20 L. P1. One or more cells obtained from a method of de novo generation of iHSCs under serum-free and feeder-free culture conditions, wherein said method comprises: i. obtaining at least one human iPSC clone, and wherein the method further comprises one or more of the following steps: a. redifferentiating the at least one human iPSC clone by culturing the at least one human iPSC clone in a first cell culture medium generating a first cell suspension, wherein said first cell culture medium comprises one of StemFit® Basic 04 medium and StemFit® Basic 03 medium supplemented with basic fibroblast growth factor (bFGF) to a final concentration of about 100 ng/mL bFGF, wherein said first cell culture medium further comprises CHIR99021 (glycogen synthase kinase 162 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, Emricasan, Polyamines, and Trans‑ISRIB (CEPT) cocktail, wherein culturing is performed in a vertical wheel bioreactor at about 10-60 rpm, preferably 40 rpm, for about 1.8 to about 2.2 days, preferably about 2 days; b. centrifuging the first cell suspension comprising iPSCs of step a) to form a first pellet and resuspending the first pellet into a second cell culture medium forming a second cell suspension, wherein said second cell culture medium comprises: i. Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises one of L-glutamine and GlutaMaxTM, SB431542 (inhibitor of Activin/BMP/TGFβ pathway), rhVEGF, bFGF, and stem cell factor (SCF), or ii. StemProTM-34 Serum-free Medium (SFM) and StemPro™-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2- phosphate, Insulin-Transferrin-Selenium (ITS-G), monothioglycerol, SB431542, rhVEGF, bFGF, SCF, and one of L-glutamine and GlutaMaxTM, and wherein the second cell culture medium does not comprise antibiotics; c. culturing the second cell suspension comprising iHSCs in the vertical wheel bioreactor at about 10-60 rpm, preferably 40 rpm, for about 1.8 to about 2.2 days, preferably about 2 days; d. stopping the agitation of the vertical wheel bioreactor for about 5 minutes and collecting about 80% of the second cell suspension; e. centrifuging the 80% of the second cell suspension, thereby generating a second cell pellet; 163 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 f. suspending the second pellet in a third cell culture medium forming a third cell suspension, wherein the third cell culture medium comprises: i. Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, FMS-like tyrosine kinase 3 ligand (Flt3L), interleukin-6 (IL-6), rhVEGF, IL-3, one of (i) thrombopoietin (TPO) and (ii) erythropoietin (EPO), and one of (i) L-glutamine and (ii) GlutaMaxTM, or ii. StemPro™-34 SFM and StemPro™-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, rhVEGF, IL-3, IL-6, Flt3L, one of (i) TPO and (ii) EPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, and wherein the third medium does not comprise antibiotics, and placing the third cell suspension into the vertical wheel bioreactor with remaining second cell suspension, thereby forming a fourth cell suspension; g. culturing fourth cell suspension in the vertical wheel bioreactor for an additional about 1.8 to about 2.2 days, preferably about 2 days; h. stopping the vertical wheel bioreactor for about 5 minutes; i. collecting about 80% of the fourth cell suspension and centrifuging the about 80% of the fourth suspension, thereby generating a third cell pellet; j. resuspending the third cell pellet in a fourth cell culture medium forming a fifth cell suspension, wherein the fourth cell culture medium comprises: i. Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, 164 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 IL-6, one of (i) TPO and (ii) EPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, or ii. StemPro™-34 SFM and StemPro™-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, IL-6, one of (i) TPO and (ii) EPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, and adding the fifth cell suspension into remaining fourth cell suspension in the vertical wheel bioreactor, thereby generating a sixth cell suspension; k. culturing the sixth cell suspension for about 1.8 to about 2.2 days, preferably about 2 days in the vertical wheel bioreactor; l. repeating steps h) to k); m. centrifuging the eighth cell suspension generating a first supernatant and a fourth cell pellet; n. collecting and centrifuging the first supernatant, thereby generating a fifth cell pellet and resuspending the fifth cell pellet in a fifth medium thereby generating a ninth cell suspension, wherein the fifth medium comprises Advanced DMEM/F12 medium, wherein the fifth medium does not comprise cytokines, antibiotics or growth factors, and wherein cells of the ninth cell suspension comprises iHSCs, which are defined as CD34+ and lineage marker-negative, where the lineage markers comprise one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a; o. resuspending the fourth cell pellet of step m) in a volume of about 20 mg/mL collagenase type II, thereby generating a tenth cell suspension; p. adding the fifth medium to the tenth cell suspension; q. centrifuging the tenth cell suspension, thereby generating a sixth cell pellet, and adding TrypLE to the sixth cell pellet, thereby generating an eleventh cell suspension; r. adding additional fifth medium to the eleventh cell suspension and mixing; 165 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 s. centrifuging the eleventh cell suspension to form a seventh cell pellet and resuspending the seventh cell pellet in additional fifth medium to form a twelfth cell suspension; t. passing the twelfth cell suspension through a cell strainer to obtain iHSCs, wherein the cell strainer is 70 µm mesh size; u. determining the purity of the iHSCs from the first supernatant of step n) and the iHSCs from step t), said purity defined as CD34+ and lineage marker-negative, where the lineage markers comprise one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a; v. not introducing serum in any of steps a)-u); w. not introducing any additional cells, including feeder cells or stromal cells, in any of steps a)-v); and x. obtaining de novo generated iHSCs. P2. The one or more cells of embodiment P2, wherein the one or more cells have high purity, viability, fold expansion, and potential to redifferentiate into immune effectors, including iPSC- derived γδ (iγδ) T cells. P3. The one or more cells of embodiment P1 or P2, wherein the EB and γδ T cell-derived iPSCs are from a tissue culture vessel. P4. The one or more cells of any one of embodiments P1-P3, wherein the first, second, fourth, sixth, and/or eighth cell suspensions are cultured under normoxic conditions. P5. The one or more cells of embodiments P1-P4, further comprising further purifying and/or isolating the iHSCs. P6. The one or more cells of any one of embodiments P1-P5, wherein the obtained iHSCs are isolated redifferentiated cells or are capable of being further purified and/or isolated. P7. The one or more cells of any one of embodiments P1-P6, wherein the method further comprises assaying for all of the lineage markers CD3, CD19, CD14, CD11b, CD56, CD20, CD16, CD2, and CD235a via flow cytometry or an equivalent thereof. P8. The one or more cells of any one of embodiments P1-P7, wherein any one of steps a)-w) comprises gently dispersing the cells using a pipet or equivalent thereof. 166 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 P9. The one or more cells of any one of embodiments P1-P8, wherein cells of the first medium are at a concentration of about 80,000 to about 180,000 cells/mL in a volume of about 50 to about 70 mL. P10. The one or more cells of any one of embodiments P1-P9, wherein the cells of the first medium are at a concentration of about 150,000 cells/mL in a volume of about 60 mL. P11. The one or more cells of any one of embodiments P1-P10, wherein the medium of step a) further comprises a ROCK inhibitor. P12. The one or more cells of any one of embodiments P1-P11, wherein the medium of step a) further comprises a Chroman 1, Emricasan, Polyamines, and Trans‑ISRIB (CEPT) cocktail. P13. The one or more cells of any one of embodiments P1-P12, wherein after step a) all the cell volume of the first cell suspension is removed from the bioreactor prior to centrifugation. P14. The one or more cells of any one of embodiments P1-P13, wherein the cell morphology is imaged via microscopy, e.g., bright field microscopy. P15. The one or more cells of any one of embodiments P1-P14, wherein the medium in steps b), f), j), or n) comprises L-glutamine. P16. The one or more cells of any one of embodiments P1-P14, wherein the medium in steps b), f), j), or n) comprises GlutaMaxTM. P17. The one or more cells of any one of embodiments P1-P16, wherein the medium in steps f) or j) comprises TPO. P18. The one or more cells of any one of embodiments P1-P16, wherein the medium in steps f) or j) comprises EPO. P19. The one or more cells of any one of embodiments P1-P18, wherein the centrifugation of any one of the steps is performed at 100g for 1 minute at brake 6. P20. The one or more cells of any one of embodiments P1-P19, wherein the centrifugation of any one of the steps is performed at 300g for 5 minutes. P21. The one or more cells of any one of embodiments P1-P20, wherein the centrifugation of any one of the steps is performed at 300g for 3 minutes. P22. The one or more cells of any one of embodiments P1-P21, wherein the centrifugation in step l) is performed twice, first at 100g for 1 minute at brake 6 and second at 300g for 5 minutes. P23. The method of any one of any one of embodiments P1-P22, wherein the tenth cell suspension of step o) is incubated with collagenase for 20 minutes at 37 °C. 167 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 P24. The method of any one of any one of embodiments P1-P23, wherein the eleventh cell suspension of step q) is incubated with TrypLE for 5 minutes at 37 °C. P25. The one or more cells of any one of embodiments P1-P24, wherein passing of the twelfth cell suspension in step t) further comprises passing the twelfth cell suspension through a second cell strainer with a 40 µm mesh size. P26. The one or more cells of any one of embodiments P1-P25, wherein the culturing of the iPSC cells, EBs, and iHSCs is performed at 37 °C and 5% CO2. P27. The one or more cells of any one of embodiments P1-P26, wherein the bioreactor wheel is rotating at 60 rpm. P28. The one or more cells of any one of embodiments P1-P26, wherein the bioreactor wheel is rotating at 30 rpm. P29. The one or more cells of any one of embodiments P1-P26, wherein the bioreactor wheel is rotating at 24 rpm. P30. The one or more cells of any one of embodiments P1-P26, wherein the bioreactor wheel is rotating at 15 rpm. P31. The one or more cells of any one of embodiments P1-P30, wherein the bioreactor wheel is perpendicular to the bioreactor vessel. P32. The one or more cells of any one of embodiments P1-P30, wherein the bioreactor wheel is not perpendicular to the bioreactor vessel. P33. The one or more cells of any one of embodiments P1-P8, and P11-P32, wherein iPSCs of the first medium are at a concentration of about 80,000 to about 180,000 cells/mL in a volume of about 90 to about 110 mL. P34. The one or more cells of any one of embodiments P1-P8, and P11-P33, wherein the iPSCs of the first medium are at a concentration of about 150,000 cells/mL in a volume of about 100 mL. P35. The one or more cells of any one of embodiments P1-P8, and P11-P32, wherein iPSCs of the first medium are at a concentration of about 5,000 to about 180,000 cells/mL in a volume of about 0.45 to about 0.55 L. P36. The one or more cells of any one of embodiments P1-P8, P11-P32, and P35, wherein the iPSCs of the first medium are at a concentration of about 20,000 cells/mL in a volume of about 0.5 L. 168 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 P37. The one or more cells of any one of embodiments P1-P8, and P11-P32, wherein iPSCs of the first medium are at a concentration of about 5,000 to about 180,000 cells/mL in a volume of about 2 to about 5 L. P38. The one or more cells of any one of embodiments P1-P8, and P11-P32, wherein iPSCs of the first medium are at a concentration of about 5,000 to about 180,000 cells/mL in a volume of about 10 to about 20 L. Q1. A composition comprising the one or more cells of any one of embodiments P1-P38. R1. Use of the one or more cells of any one of embodiments P1-P38, in preparation of cells for treating diseases, in preparation of lymphocytes, in a bioreactor, in tissue engineering or in vitro drug screening for diseases. References 1. Croughan, M. S., et al. Initial power measurements for a family of novel vertical-wheel bioreactors. Can. J. Chem. Eng. 2022, 1-12. 2. Dang, T., et al. Computational fluid dynamic characterization of vertical-wheel bioreactors used for effective scale-up of human induced pluripotent stem cell aggregate culture. Can. J. Chem. Eng. 2021, 1-18. 3. Cuesta-Gomez, N., et al. Suspension culture improves iPSC expansion and pluripotency phenotype. Stem Cell Research & Therapy. 2023, 14:154. 4. Borys, B. S., et al. Overcoming bioprocess bottlenecks in the large-scale expansion of high- quality hiPSC aggregates in vertical-wheel stirred suspension bioreactors. Stem Cell Research & Therapy. 2021, 12:55. * * * [0407] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims. [0408] All patents, applications, publications, test methods, literature, and other materials cited herein are hereby incorporated by reference in their entirety as if physically present in this specification. 169 301689646v1

Claims

Attorney Docket No: 253505.000510 / JBI6830WOPCT1 CLAIMS 1. A method of de novo generation of hematopoietic stem cells (iHSCs) derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue culture vessel, said method comprising: seeding human γδ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; retaining all cells in the culture on day 8 of culture while removing the medium from the culture for about 2 days; and harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction. 2. The method of claim 1, wherein the tissue culture vessel has non-traditional bioreactor geometries. 3. The method of claim 1 or claim 2, wherein the iPSCs are seeded at a density of about 1.0 x 105 cells/mL to about 1.8 x 105 cells/mL. 4. The method of any one of claims 1-3, wherein the tissue culture vessel comprising a vertical wheel further comprises a first medium comprising StemFit® Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, Emricasan, Polyamines, and Trans‑ISRIB (CEPT) cocktail, wherein the BMP4 and the rhVEGF are present in a ratio of 1:1, and wherein the first medium does not comprise antibiotics. 5. The method of any one of claims 1-4, wherein the method further comprises exchanging spent medium in the tissue culture vessel comprising a vertical wheel on each of days 2, 4, and 6 of culture for the about 8 days, wherein the step of exchanging spent medium comprises: removing the spent medium from the tissue culture vessel comprising a vertical wheel using a 40 µm cell retention filter and adding a wash medium at a pump flow rate of 30 mL/min, 170 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 the wash medium comprising Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio; exchanging about 3 L to about 3.5 L of the wash medium in the bioreactor and removing 0.1 L of the wash medium from the tissue culture vessel comprising a vertical wheel using the 40 µm cell retention filter; and adding about 50 mL of the wash medium and about 50 mL of a medium to from the tissue culture vessel comprising the vertical wheel. 6. The method of any one of claims 1-5, wherein the iPSCs are dissociated into single cells on day 7 of culture. 7. The method of any one of claims 1-6, wherein the step of retaining all cells comprises retaining clusters, semi-clusters, and single cells. 8. The method of any one of claims 1-7, wherein the step of retaining all cells comprises removing spent medium from the tissue culture vessel comprising a vertical wheel using a 4 µm cell retention filter and adding fresh medium to the bioreactor over about 1.8 days to about 2.2 days, preferably about 2 days, at a rate of about 0.69 mL/min (1 L/day), until 2 L of media have been exchanged, wherein the fifth medium comprises Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, IL-6, TPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, wherein the fresh medium does not comprise antibiotics. 9. The method of any one of claims 1-8, wherein the vertical wheel spins at a rate of about 10 rpm to about 60 rpm, preferably about 23 rpm. 10. The method of any one of claims 1-9, wherein the step of harvesting comprises: a) removing an entire cell volume comprising EBs and iHSCs from the tissue culture vessel comprising a vertical wheel and centrifuging the entire cell volume, thereby generating a first supernatant and a first cell pellet; b) centrifuging the first supernatant, thereby generating a second supernatant and a second cell pellet, removing the second supernatant, and resuspending the second cell pellet in Advanced DMEM/F12 medium and StemFit® For Differentiation medium without 171 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 cytokines, growth factors, or antibiotics; c) adding a first volume of 20 mg/mL collagenase II to the first cell pellet and the second cell pellet, combining the resuspended first cell pellet and the resuspended second cell pellet and adding a second volume of 20 mg/mL collagenase II, thereby generating a combined cell suspension; d) adding a volume of Advanced DMEM/F12 medium and StemFit® For Differentiation medium without cytokines, growth factors, or antibiotics equal to the volume of the combined cell suspension to the combined cell suspension; e) centrifuging the combined cell suspension, thereby generating a third cell pellet; f) resuspending the third cell pellet in a volume of TrypLE and adding a volume of Advanced DMEM/F12 medium and StemFit® For Differentiation medium without cytokines, growth factors, or antibiotics, thereby generating a fourth cell suspension; g) mixing the fourth cell suspension until clusters of cells are no longer visible; h) optionally passing the fourth cell suspension through a cell strainer to obtain iHSCs, wherein the cell strainer is 70 µm mesh size, if clusters of cells are visible after the mixing step; and i) centrifuging the fourth cell suspension. 11. The method of any one of claims 1-10, wherein the cells are cultured under normoxic conditions. 12. The method of any one of claims 1-11, further comprising further purifying and/or isolating the redifferentiated iHSCs. 13. The method of any one of claims 1-12, wherein the redifferentiated iHSCs are isolated differentiated cells or are capable of being further purified and/or isolated. 14. The method of any one of claims 1-13, wherein determining purity of the redifferentiated iHSCs comprises assaying for one or more of lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a via flow cytometry or an equivalent thereof. 15. The method of claim 14, wherein the lineage markers comprise all of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a. 172 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 16. A method of de novo generation of hematopoietic stem cells (iHSCs) derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue culture vessel, said method comprising: seeding human γδ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction. 17. The method of claim 16, wherein the tissue culture vessel has non-traditional bioreactor geometries. 18. The method of claim 16 or claim 17, wherein the iPSCs are seeded at a density of about 1.0 x 105 cells/mL to about 1.8 x 105 cells/mL. 19. The method of any one of claims 16-18, wherein the tissue culture vessel comprising a vertical wheel further comprises a first medium comprising StemFit® Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, Emricasan, Polyamines, and Trans‑ISRIB (CEPT) cocktail, wherein the BMP4 and the rhVEGF are present in a ratio of 1:1, and wherein the first medium does not comprise antibiotics. 20. The method of any one of claims 16-19, wherein the method further comprises exchanging spent medium in the tissue culture vessel comprising a vertical wheel on each of days 2, 4, 6, and 8 of culture for the about 8 days, wherein the step of exchanging the spent medium comprises: a) allowing cell aggregates to settle in the tissue culture vessel comprising a vertical wheel for about 3 to about 5 minutes; b) collecting 80% of the spent medium from the tissue culture vessel comprising a vertical wheel; c) centrifuging the collected medium, thereby obtaining a cell pellet; 173 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 d) resuspending the cell pellet in StemFit® for Differentiation, thereby obtaining a cell suspension; and e) adding the cell suspension and fresh medium to the tissue culture vessel comprising a vertical wheel. 21. The method of any one of claims 15-20, wherein the step of exchanging the spent medium on days 2, 4, and 6 of culture comprises: i) allowing cell aggregates to settle in the tissue culture vessel comprising a vertical wheel for about 3 to about 5 minutes; collecting 80% of the spent medium from the tissue culture vessel comprising a vertical wheel; centrifuging the collected medium, thereby obtaining a cell pellet; resuspending the cell pellet in StemFit® for Differentiation medium, thereby obtaining a cell suspension; and adding the cell suspension and fresh medium to the tissue culture vessel comprising a vertical wheel, or ii) allowing cell aggregates to settle in the tissue culture vessel comprising a vertical wheel for about 3 to about 5 minutes; collecting 80% of the spent medium from the tissue culture vessel comprising a vertical wheel; centrifuging the collected medium, thereby obtaining a first cell pellet and a first supernatant; resuspending the first cell pellet in StemFit® for Differentiation medium and adding the resuspended first cell pellet to the tissue culture vessel comprising a vertical wheel; centrifuging the supernatant, thereby obtaining a second cell pellet and a second supernatant; and resuspending the second cell pellet in StemFit® for Differentiation medium and adding the resuspended second cell pellet and fresh medium to the tissue culture vessel comprising a vertical wheel. 22. The method of any one of claims 15-22, wherein the fresh medium added on day 2 of culture comprises: i) Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SB431542 (inhibitor of Activin/BMP/TGFβ pathway), recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), and one of L-glutamine and GlutaMaxTM supplement, or ii) StemProTM-34 Serum-free Medium (SFM) and StemPro™-34 Nutrient Supplement in 174 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 a 500:13 ratio, ascorbic acid 2-phosphate, Insulin-Transferrin-Selenium (ITS-G), monothioglycerol, SB431542, recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), and one of L- glutamine and GlutaMaxTM, wherein the VEGF, bFGF, and SCF are present in a ratio of 1:0.625:0.625, and wherein the second medium does not comprise antibiotics. 23. The method of any one of claims 16-22, wherein the iPSCs are dissociated into single cells on day 7 of culture. 24. The method of any one of claims 16-23, wherein the step of harvesting redifferentiated iHSCs comprises retaining clusters, semi-clusters, and single cells. 25. The method of any one of claims 16-24, wherein the step of harvesting comprises retaining all cells in the tissue culture vessel comprising a vertical wheel. 26. The method of any one of claims 16-25, wherein the vertical wheel spins at a rate of about 10 rpm to about 60 rpm, preferably about 40 rpm. 27. The method of any one of claims 16-26, wherein the step of harvesting redifferentiated iHSCs comprises: a) removing an entire cell volume comprising embryoid bodies (EBs) and iHSCs from the tissue culture vessel comprising a vertical wheel and centrifuging the entire cell volume, thereby generating a first supernatant and a first cell pellet; b) centrifuging the first supernatant, thereby generating a second supernatant and a second cell pellet and removing the second supernatant; c) resuspending the first cell pellet and the second cell pellet in Advanced DMEM/F12 medium without cytokines, growth factors, or antibiotics; d) adding a volume of 20 mg/mL collagenase II to the first cell pellet, thereby generating a first cell suspension; e) adding a volume of Advanced DMEM/F12 medium without cytokines, growth factors, or antibiotics equal to the volume of the first cell suspension to the first cell suspension; f) centrifuging the first cell suspension, thereby generating a third cell pellet; g) resuspending the third cell pellet in a volume of TrypLE and adding a volume of 175 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 Advanced DMEM/F12 medium without cytokines, growth factors, or antibiotics, thereby generating a second cell suspension; h) mixing the second cell suspension until clusters of cells are no longer visible; i) optionally passing the second cell suspension through a cell strainer to obtain iHSCs, wherein the cell strainer is 70 µm mesh size, if clusters of cells are visible after the mixing step; and j) centrifuging the second cell suspension. 28. The method of any one of claims 16-27, wherein the cells are cultured under normoxic conditions. 29. The method of any one of claims 16-28, further comprising further purifying and/or isolating the redifferentiated iHSCs. 30. The method of any one of claims 16-29, wherein the redifferentiated iHSCs are isolated differentiated cells or are capable of being further purified and/or isolated. 31. The method of any one of claims 16-30, wherein determining purity of the redifferentiated iHSCs comprises assaying for one or more of lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a via flow cytometry or an equivalent thereof. 32. The method of claim 31, wherein the lineage markers comprise all of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a. 33. One or more cells obtained from a method of de novo generation of hematopoietic stem cells (iHSCs) derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions, said method comprising the steps of any one of claims 1-32. 34. One or more cells obtained from a method of de novo generation of hematopoietic stem cells (iHSCs) derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue culture vessel, said method comprising: seeding human γδ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; 176 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; retaining all cells in the culture on day 8 of culture while removing the medium from the culture for about 2 days; and harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction. 35. The one or more cells of claim 34, wherein the tissue culture vessel has non-traditional bioreactor geometries. 36. The one or more cells of claim 34 or claim 35, wherein the iPSCs are seeded at a density of about 1.0 x 105 cells/mL to about 1.8 x 105 cells/mL. 37. The one or more cells of any one of claims 34-36, wherein the tissue culture vessel comprising a vertical wheel further comprises a first medium comprising StemFit® Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, Emricasan, Polyamines, and Trans‑ISRIB (CEPT) cocktail, wherein the BMP4 and the rhVEGF are present in a ratio of 1:1, and wherein the first medium does not comprise antibiotics. 38. The one or more cells of any one of claims 34-37, wherein the method further comprises exchanging spent medium in the tissue culture vessel comprising a vertical wheel on each of days 2, 4, and 6 of culture for the about 8 days, wherein the step of exchanging spent medium comprises: removing the spent medium from the tissue culture vessel comprising a vertical wheel using a 40 µm cell retention filter and adding a wash medium at a pump flow rate of 30 mL/min, the wash medium comprising Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio; exchanging about 3 L to about 3.5 L of the wash medium in the bioreactor and removing 0.1 L of the wash medium from the tissue culture vessel comprising a vertical wheel using the 40 µm cell retention filter; and 177 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 adding about 50 mL of the wash medium and about 50 mL of a medium to from the tissue culture vessel comprising the vertical wheel. 39. The one or more cells of any one of claims 34-38, wherein the iPSCs are dissociated into single cells on day 7 of culture. 40. The one or more cells of any one of claims 34-39, wherein the step of retaining all cells comprises retaining clusters, semi-clusters, and single cells. 41. The one or more cells of any one of claims 34-40, wherein the step of retaining all cells comprises removing spent medium from the tissue culture vessel comprising a vertical wheel using a 4 µm cell retention filter and adding fresh medium to the bioreactor over about 1.8 days to about 2.2 days, preferably about 2 days, at a rate of about 0.69 mL/min (1 L/day), until 2 L of media have been exchanged, wherein the fifth medium comprises Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, IL-6, TPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, wherein the fresh medium does not comprise antibiotics. 42. The one or more cells of any one of claims 34-41, wherein the vertical wheel spins at a rate of about 10 rpm to about 60 rpm, preferably about 23 rpm. 43. The one or more cells of any one of claims 34-42, wherein the step of harvesting comprises: a) removing an entire cell volume comprising EBs and iHSCs from the tissue culture vessel comprising a vertical wheel and centrifuging the entire cell volume, thereby generating a first supernatant and a first cell pellet; b) centrifuging the first supernatant, thereby generating a second supernatant and a second cell pellet, removing the second supernatant, and resuspending the second cell pellet in Advanced DMEM/F12 medium and StemFit® For Differentiation medium without cytokines, growth factors, or antibiotics; c) adding a first volume of 20 mg/mL collagenase II to the first cell pellet and the second cell pellet, combining the resuspended first cell pellet and the resuspended second cell pellet and adding a second volume of 20 mg/mL collagenase II, thereby generating a combined cell suspension; d) adding a volume of Advanced DMEM/F12 medium and StemFit® For Differentiation 178 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 medium without cytokines, growth factors, or antibiotics equal to the volume of the combined cell suspension to the combined cell suspension; e) centrifuging the combined cell suspension, thereby generating a third cell pellet; f) resuspending the third cell pellet in a volume of TrypLE and adding a volume of Advanced DMEM/F12 medium and StemFit® For Differentiation medium without cytokines, growth factors, or antibiotics, thereby generating a fourth cell suspension; g) mixing the fourth cell suspension until clusters of cells are no longer visible; h) optionally passing the fourth cell suspension through a cell strainer to obtain iHSCs, wherein the cell strainer is 70 µm mesh size, if clusters of cells are visible after the mixing step; and i) centrifuging the fourth cell suspension. 44. The one or more cells of any one of claims 34-43, wherein the cells are cultured under normoxic conditions. 45. The one or more cells of any one of claims 34-44, further comprising further purifying and/or isolating the redifferentiated iHSCs. 46. The one or more cells of any one of claims 34-45, wherein the redifferentiated iHSCs are isolated differentiated cells or are capable of being further purified and/or isolated. 47. The one or more cells of any one of claims 34-46, wherein determining purity of the redifferentiated iHSCs comprises assaying for one or more of lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a via flow cytometry or an equivalent thereof. 48. The one or more cells of claim 47, wherein the lineage markers comprise all of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a. 49. One or more cells obtained from a method of de novo generation of hematopoietic stem cells (iHSCs) derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue culture vessel, said method comprising: seeding human γδ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; 179 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction. 50. The one or more cells of claim 49, wherein the tissue culture vessel has non-traditional bioreactor geometries. 51. The one or more cells of claim 49 or claim 50, wherein the iPSCs are seeded at a density of about 1.0 x 105 cells/mL to about 1.8 x 105 cells/mL. 52. The one or more cells of any one of claims 49-51, wherein the tissue culture vessel comprising a vertical wheel further comprises a first medium comprising StemFit® Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, Emricasan, Polyamines, and Trans‑ISRIB (CEPT) cocktail, wherein the BMP4 and the rhVEGF are present in a ratio of 1:1, and wherein the first medium does not comprise antibiotics. 53. The one or more cells of any one of claims 49-52, wherein the method further comprises exchanging spent medium in the tissue culture vessel comprising a vertical wheel on each of days 2, 4, 6, and 8 of culture for the about 8 days, wherein the step of exchanging the spent medium comprises: a) allowing cell aggregates to settle in the tissue culture vessel comprising a vertical wheel for about 3 to about 5 minutes; b) collecting 80% of the spent medium from the tissue culture vessel comprising a vertical wheel; c) centrifuging the collected medium, thereby obtaining a cell pellet; d) resuspending the cell pellet in StemFit® for Differentiation, thereby obtaining a cell suspension; and e) adding the cell suspension and fresh medium to the tissue culture vessel comprising 180 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 a vertical wheel. 54. The one or more cells of any one of claims 49-52, wherein the step of exchanging the spent medium on days 2, 4, and 6 of culture comprises: i) allowing cell aggregates to settle in the tissue culture vessel comprising a vertical wheel for about 3 to about 5 minutes; collecting 80% of the spent medium from the tissue culture vessel comprising a vertical wheel; centrifuging the collected medium, thereby obtaining a cell pellet; resuspending the cell pellet in StemFit® for Differentiation medium, thereby obtaining a cell suspension; and adding the cell suspension and fresh medium to the tissue culture vessel comprising a vertical wheel, or ii) allowing cell aggregates to settle in the tissue culture vessel comprising a vertical wheel for about 3 to about 5 minutes; collecting 80% of the spent medium from the tissue culture vessel comprising a vertical wheel; centrifuging the collected medium, thereby obtaining a first cell pellet and a first supernatant; resuspending the first cell pellet in StemFit® for Differentiation medium and adding the resuspended first cell pellet to the tissue culture vessel comprising a vertical wheel; centrifuging the supernatant, thereby obtaining a second cell pellet and a second supernatant; and resuspending the second cell pellet in StemFit® for Differentiation medium and adding the resuspended second cell pellet and fresh medium to the tissue culture vessel comprising a vertical wheel. 55. The one or more cells of any one of claims 49-54, wherein the fresh medium added on day 2 of culture comprises: i) Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SB431542 (inhibitor of Activin/BMP/TGFβ pathway), recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), and one of L-glutamine and GlutaMaxTM supplement, or ii) StemProTM-34 Serum-free Medium (SFM) and StemPro™-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, Insulin-Transferrin-Selenium (ITS-G), monothioglycerol, SB431542, recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), and one of L- 181 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 glutamine and GlutaMaxTM, wherein the VEGF, bFGF, and SCF are present in a ratio of 1:0.625:0.625, and wherein the second medium does not comprise antibiotics. 56. The one or more cells of any one of claims 49-55, wherein the iPSCs are dissociated into single cells on day 7 of culture. 57. The one or more cells of any one of claims 49-56, wherein the step of harvesting redifferentiated iHSCs comprises retaining clusters, semi-clusters, and single cells. 58. The one or more cells of any one of claims 49-57, wherein the step of retaining all cells comprises removing spent medium from the tissue culture vessel comprising a vertical wheel using a 4 µm cell retention filter and adding fresh medium to the bioreactor over about 1.8 days to about 2.2 days, preferably about 2 days, at a rate of about 0.69 mL/min (1 L/day), until 2 L of media have been exchanged, wherein the fifth medium comprises Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, IL-6, TPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, wherein the fresh medium does not comprise antibiotics. 59. The one or more cells of any one of claims 49-58, wherein the vertical wheel spins at a rate of about 10 rpm to about 60 rpm, preferably about 40 rpm. 60. The one or more cells of any one of claims 49-59, wherein the step of harvesting redifferentiated iHSCs comprises: a) removing an entire cell volume comprising embryoid bodies (EBs) and iHSCs from the tissue culture vessel comprising a vertical wheel and centrifuging the entire cell volume, thereby generating a first supernatant and a first cell pellet; b) centrifuging the first supernatant, thereby generating a second supernatant and a second cell pellet and removing the second supernatant; c) resuspending the first cell pellet and the second cell pellet in Advanced DMEM/F12 medium without cytokines, growth factors, or antibiotics; d) adding a volume of 20 mg/mL collagenase II to the first cell pellet, thereby generating a first cell suspension; e) adding a volume of Advanced DMEM/F12 medium without cytokines, growth factors, 182 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 or antibiotics equal to the volume of the first cell suspension to the first cell suspension; f) centrifuging the first cell suspension, thereby generating a third cell pellet; g) resuspending the third cell pellet in a volume of TrypLE and adding a volume of Advanced DMEM/F12 medium without cytokines, growth factors, or antibiotics, thereby generating a second cell suspension; h) mixing the second cell suspension until clusters of cells are no longer visible; i) optionally passing the second cell suspension through a cell strainer to obtain iHSCs, wherein the cell strainer is 70 µm mesh size, if clusters of cells are visible after the mixing step; and j) centrifuging the second cell suspension. 61. The one or more cells of any one of claims 49-60, wherein the cells are cultured under normoxic conditions. 62. The one or more cells of any one of claims 49-61, further comprising further purifying and/or isolating the redifferentiated iHSCs. 63. The one or more cells of any one of claims 49-62, wherein the redifferentiated iHSCs are isolated differentiated cells or are capable of being further purified and/or isolated. 64. The one or more cells of any one of claims 49-63, wherein the one or more cells have high purity, viability, fold expansion, and potential to redifferentiate into immune effectors, including iPSC-derived γδ (iγδ) T cells. 65. The one or more cells of any one of claims 49-64, wherein the step of determining purity of the iHSCs comprises assaying for one or more, optionally all, of the lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a via flow cytometry or an equivalent thereof. 66. The one or more cells of any one of claims 49-65, wherein the lineage markers comprise one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a. 67. A composition comprising the one or more cells of any one of claims 33-66. 68. Use of the one or more cells of any one of claims 38-66, in preparation of cells for treating a pathology, disease(s), in preparation of lymphocytes, in a bioreactor, in tissue engineering or in vitro drug screening for diseases. 183 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 69. A system for performing a method of de novo generation of hematopoietic stem cells (iHSCs) derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions, said method comprising the steps of any one of claims 1-32, wherein the vertical wheel is either perpendicular or non-perpendicular to the vessel. 70. A method of de novo generation of hematopoietic stem cells (iHSCs) derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue culture vessel, said method comprising: a means for seeding human γδ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; a means for redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; a means for retaining all cells in the culture on day 8 of culture while removing the medium from the culture for about 2 days; and a means for harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction. 71. A method of de novo generation of hematopoietic stem cells (iHSCs) derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue culture vessel, said method comprising: a means for seeding human γδ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; a means for redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; a means for harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction. 72. An iHSC produced according to the method of claim 38 or claim 39. 184 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 73. One or more cells obtained from a method of de novo generation of hematopoietic stem cells (iHSCs) derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue culture vessel, said method comprising: a means for seeding human γδ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; a means for redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; a means for retaining all cells in the culture on day 8 of culture while removing the medium from the culture for about 2 days; and a means for harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction. 74. One or more cells obtained from a method of de novo generation of hematopoietic stem cells (iHSCs) derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions in a tissue culture vessel, said method comprising: a means for seeding human γδ T cell-derived iPSCs in a tissue culture vessel comprising a vertical wheel, in which the iPSCs are maintained for up to about 7 days; a means for redifferentiating the iPSCs to iHSCs by culturing in the tissue culture vessel comprising a vertical wheel for about 8 days; a means for harvesting redifferentiated iHSCs at about day 10 of culture into two different cellular fractions, a single cell fraction and a multicellular fraction. 75. A method of de novo generation of induced pluripotent stem cell (iPSC)-derived hematopoietic stem cells (iHSCs) from γδ T cell-derived iPSCs (γδ T-iPSCs) under serum-free, feeder-free culture conditions, said method comprising one or more of the following steps: i) obtaining one or more human induced pluripotent stem cells (iPSCs), and wherein the method further comprises one or more of the following steps: a) after incubating the iPSCs in a tissue culture vessel comprising a vertical wheel in a first medium with agitation at about 10-60 rpm, preferably 40 rpm, for about 1.8 to about 2.2 days, preferably about 2 days, removing an entire cell volume from the tissue 185 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 culture vessel comprising a vertical wheel and centrifuging the entire cell volume, wherein the first medium comprises StemFit® Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF) and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, Emricasan, Polyamines, and Trans‑ISRIB (CEPT) cocktail, wherein the first medium does not comprise antibiotics; b) centrifuging the first cell suspension and thereby generating a first cell pellet; c) resuspending the first cell pellet into a second medium to form a second cell suspension, wherein the second medium comprises: i) Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SB431542 (inhibitor of Activin/BMP/TGFβ pathway), recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), and one of L-glutamine and GlutaMaxTM supplement, or ii) StemProTM-34 Serum-free Medium (SFM) and StemPro™-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, Insulin-Transferrin- Selenium (ITS-G), monothioglycerol, SB431542, recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), and one of L-glutamine and GlutaMaxTM, wherein the VEGF, bFGF, and SCF are present in a ratio of 1:0.625:0.625, and wherein the second medium does not comprise antibiotics; d) culturing and agitating the second cell suspension in the tissue culture vessel comprising a vertical wheel at about 10-60 rpm, preferably 40 rpm, for about 1.8 to about 2.2 days, preferably about 2 days; e) stopping the agitating for about 5 minutes, wherein cell aggregates settle in the second cell suspension; f) collecting about 80% of the second cell suspension from a top volume of the second cell suspension, from the tissue culture vessel comprising a vertical wheel; g) centrifuging the collected second cell suspension, thereby forming a second cell pellet and resuspending the second pellet in about 1 mL of one of StemFit® For 186 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 Differentiation solution and/or StemPro™-34 SFM; h) adding the resuspended second cell pellet into a third medium forming a third cell suspension, wherein the third medium comprises: i) Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), interleukin- 3 (IL-3), interleukin-6 (IL-6), one of (i) thrombopoietin (TPO) and (ii) erythropoietin (EPO), and one of (i) L-glutamine and (ii) GlutaMaxTM, or ii) StemPro™-34 SFM and StemPro™-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, rhVEGF, IL-3, IL-6, Flt3L, one of (i) TPO and (ii) EPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, and wherein the third medium does not comprise antibiotics; i) placing the third cell suspension into the tissue culture vessel comprising a vertical wheel with remaining second cell suspension forming a fourth cell suspension; j) culturing and agitating the fourth cell suspension in the tissue culture vessel comprising a vertical wheel at about 10-60 rpm, preferably 40 rpm, for about 1.8 to about 2.2 days, preferably about 2 days; k) stopping the agitating for about 5 minutes; wherein cell aggregates settle in the cell suspension; l) collecting about 80% of the fourth cell suspension and centrifuging the collected fourth cell suspension forming a third cell pellet, and resuspending the third cell pellet in about 1 mL of one of StemFit® For Differentiation solution and StemPro™-34 SFM; m) suspending the third cell pellet dissolved in the about 1 mL of one of StemFit® For Differentiation solution and StemPro™-34 SFM into a fourth medium forming a fifth cell suspension, wherein the fourth medium comprises: i) Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, IL-6, one of (i) TPO and (ii) EPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, or ii) StemPro™-34 SFM and StemPro™-34 Nutrient Supplement in a 500:13 187 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 ratio, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, IL-6, one of (i) TPO and (ii) EPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, and wherein the fourth medium does not comprise antibiotics; n) placing the fifth cell suspension into the tissue culture vessel comprising a vertical wheel with the remaining fourth cell suspension thereby forming a sixth cell suspension; o) culturing and agitating the sixth cell suspension in the tissue culture vessel comprising a vertical wheel at about 10-60 rpm, preferably 40 rpm, for about 1.8 to about 2.2 days, preferably about 2 days; p) repeating steps k) to o); optionally wherein the sixth cell suspension is centrifuged and a supernatant from the centrifuged sixth cell suspension is centrifuged and the resulting cell pellet is resuspended in about 1 mL of one of StemFit® For Differentiation solution and StemPro™-34 SFM and combined with a seventh cell suspension obtained by resuspending a cell pellet from the first centrifugation to form an eighth cell suspension before culturing and agitating the eighth cell suspension in the tissue culture vessel comprising a vertical wheel at about 10-60 rpm, preferably 40 rpm, for about 1.8 to about 2.2 days, preferably about 2 days; q) removing all the eighth cell suspension from the tissue culture vessel comprising a vertical wheel and harvesting EBs and iHSCs in the eighth cell suspension by centrifugation, thereby generating a fourth cell pellet and a first supernatant; r) collecting the first supernatant; s) centrifuging the first supernatant, thereby generating a fifth cell pellet and resuspending the fifth cell pellet in a fifth medium thereby generating a ninth cell suspension, wherein the fifth medium comprises Advanced DMEM/F12 medium, wherein the fifth medium does not comprise cytokines, antibiotics, or growth factors, and wherein cells of the ninth cell suspension comprise iHSCs, which are defined as CD34+ and lineage marker-negative, where the lineage markers comprise one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a; t) resuspending the fourth cell pellet of step q), in a volume of about 20 mg/mL collagenase type II, thereby generating a tenth cell suspension; u) adding the fifth medium to the tenth cell suspension; 188 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 v) centrifuging the tenth cell suspension, thereby generating a sixth cell pellet, and adding TrypLE to the sixth cell pellet, thereby generating an eleventh cell suspension; w) adding additional fifth medium to the eleventh cell suspension and mixing; x) centrifuging the eleventh cell suspension to form a seventh cell pellet and resuspending the seventh cell pellet in additional fifth medium to form a twelfth cell suspension; y) optionally passing the twelfth cell suspension through a cell strainer to obtain iHSCs, wherein the cell strainer is 70 µm mesh size, if cell clumps are present in the twelfth cell suspension; z) centrifuging the twelfth cell suspension; aa) not introducing serum in any of steps a)-z); bb) not introducing any additional cells, including feeder cells or stromal cells, in any of steps a)-aa); cc) determining the purity of the iHSCs from the first supernatant of step s) and the iHSCs from step y), said purity defined as CD34+ and lineage marker-negative, where the lineage markers comprise one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a; and dd) obtaining de novo generated iHSCs. 76. A method of de novo generation of induced pluripotent stem cell (iPSC)-derived hematopoietic stem cells (iHSCs) from γδ T cell-derived iPSCs (γδ T-iPSCs) under serum-free, feeder-free culture conditions, said method comprising one or more of the following steps: i) obtaining one or more human induced pluripotent stem cells (iPSCs), and wherein the method further comprises one or more of the following steps: a) seeding the iPSCs, wherein the seeding is performed by suspending the iPSCs in a first medium into a tissue culture vessel comprising a vertical wheel, thereby producing a first cell suspension, wherein the first medium comprises one of StemFit® Basic 04 medium and StemFit® Basic 03 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, Emricasan, Polyamines, and Trans‑ISRIB (CEPT) cocktail, 189 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 and wherein the first medium does not comprise antibiotics; b) culturing and agitating the cells in the tissue culture vessel comprising a vertical wheel at about 10-60 rpm, preferably 23 rpm, for about 1.8 to about 2.2 days, preferably about 2 days, wherein embryoid bodies (EBs) are generated; c) removing the first medium from the tissue culture vessel comprising a vertical wheel and adding a wash medium comprising Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, at a rate of about 30 mL/min; d) exchanging about 3L to about 3.5 L of the wash medium in the tissue culture vessel comprising a vertical wheel using a 40 µm cell retention filter at a pump flow rate of 30 mL/min and removing 0.1 L of the wash medium from the tissue culture vessel comprising a vertical wheel; e) adding about 50 mL of the wash medium and about 50 mL of a second medium to the tissue culture vessel comprising a vertical wheel, wherein the second medium comprises Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SB431542 (inhibitor of Activin/BMP/TGFβ pathway), recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), and one of L-glutamine and GlutaMaxTM supplement, wherein the second medium does not comprise antibiotics; f) culturing and agitating the cells in the tissue culture vessel comprising a vertical wheel at about 10-60 rpm, preferably 23 rpm, for about 1.8 to about 2.2 days, preferably about 2 days; g) removing the second medium from the tissue culture vessel comprising a vertical wheel and adding the wash medium at a rate of about 30 mL/min; h) exchanging about 3L to about 3.5 L of the wash medium in the tissue culture vessel comprising a vertical wheel using a 40 µm cell retention filter at a pump flow rate of 30 mL/min and removing 0.1 L of the wash medium from the tissue culture vessel comprising a vertical wheel; i) adding about 50 mL of the wash medium and about 50 mL of a third medium to the tissue culture vessel comprising a vertical wheel, wherein the third medium comprises 190 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises and further comprises SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), interleukin-3 (IL-3), interleukin-6 (IL-6), thrombopoietin (TPO), and one of (i) L- glutamine and (ii) GlutaMaxTM, wherein the third medium does not comprise antibiotics; j) culturing and agitating the cells in the tissue culture vessel comprising a vertical wheel at about 10-60 rpm, preferably 23 rpm, for about 1.8 to about 2.2 days, preferably about 2 days; k) removing the third medium from the tissue culture vessel comprising a vertical wheel and adding the wash medium at a rate of about 30 mL/min; l) exchanging about 3L to about 3.5 L of the wash medium in the tissue culture vessel comprising a vertical wheel using a 40 µm cell retention filter at a pump flow rate of 30 mL/min and removing 0.1 L of the wash medium from the tissue culture vessel comprising a vertical wheel; m) adding about 50 mL of the wash medium and about 50 mL of a fourth medium to the tissue culture vessel comprising a vertical wheel, wherein the fourth medium comprises Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, IL-6, TPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, wherein the fourth medium does not comprise antibiotics; n) culturing and agitating the cells in the tissue culture vessel comprising a vertical wheel at about 10-60 rpm, preferably 23 rpm, for about 1.8 to about 2.2 days, preferably about 2 days; o) simultaneously removing the fourth medium from the tissue culture vessel comprising a vertical wheel and adding a fifth medium to the tissue culture vessel comprising a vertical wheel over about 1.8 days to about 2.2 days, preferably about 2 days, using a 4 µm cell retention filter at a pump flow rate of about 0.69 mL/min (1 L/day), until 2 L of media have been exchanged, wherein the fifth medium comprises Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, IL- 191 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 6, TPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, wherein the fifth medium does not comprise antibiotics; p) removing an entire cell volume comprising EBs and iHSCs from the tissue culture vessel comprising a vertical wheel and centrifuging the entire cell volume, thereby generating a first supernatant and a first cell pellet; q) centrifuging the first supernatant, thereby generating a second supernatant and a second cell pellet, removing the second supernatant, and resuspending the second cell pellet in Advanced DMEM/F12 medium and StemFit® For Differentiation medium without cytokines, growth factors, or antibiotics; r) adding a first volume of 20 mg/mL collagenase II to the first cell pellet, resuspending the first cell pellet, and adding a second volume of 20 mg/mL collagenase II, thereby generating a first cell suspension; s) adding a volume of Advanced DMEM/F12 medium and StemFit® For Differentiation medium without cytokines, growth factors, or antibiotics equal to the volume of the first cell suspension to the first cell suspension; t) centrifuging the cell suspension, thereby generating a third cell pellet; u) resuspending the third cell pellet in a volume of TrypLE and adding a volume of Advanced DMEM/F12 medium and StemFit® For Differentiation medium without cytokines, growth factors, or antibiotics, thereby generating a second cell suspension; v) mixing the second cell suspension until clusters of cells are no longer visible; w) optionally passing the second cell suspension through a cell strainer to obtain iHSCs, wherein the cell strainer is 70 µm mesh size, if clusters of cells are visible after the mixing step; x) centrifuging the second cell suspension; y) not introducing serum in any of steps a)-x); z) not introducing any additional cells, including feeder cells or stromal cells, in any of steps a)-y); aa) determining the purity of the iHSCs from step w), said purity defined as CD34+ and lineage marker-negative, where the lineage markers comprise one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a; and bb) obtaining de novo generated iHSCs. 192 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 77. A method of de novo generation of induced pluripotent stem cell (iPSC)-derived hematopoietic stem cells (iHSCs) from γδ T cell-derived iPSCs (γδ T-iPSCs) under serum-free, feeder-free culture conditions, said method comprising one or more of the following steps: i) obtaining one or more human induced pluripotent stem cells (iPSCs), and wherein the method further comprises one or more of the following steps: a) seeding the iPSCs, wherein the seeding is performed by suspending the iPSCs in a first medium into a bioreactor, wherein the bioreactor is a vertical-wheel bioreactor, thereby producing a first cell suspension, wherein the first medium comprises one of StemFit® Basic 04 medium and StemFit® Basic 03 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a Chroman 1, Emricasan, Polyamines, and Trans‑ISRIB (CEPT) cocktail, and wherein the first medium does not comprise antibiotics; b) culturing and agitating the first cell suspension in the bioreactor, wherein the first cell suspension is cultured and agitated for about 1.8 to about 2.2 days, preferably about 2 days, wherein embryoid bodies (EBs) are generated; c) centrifuging the first cell suspension and thereby generating a first cell pellet; d) resuspending the first cell pellet into a second medium to form a second cell suspension, wherein the second medium comprises: i) Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SB431542 (inhibitor of Activin/BMP/TGFβ pathway), rhVEGF, basic fibroblast growth factor (bFGF), stem cell factor (SCF), and one of L-glutamine and GlutaMaxTM supplement, or ii) StemProTM-34 Serum-free Medium (SFM) and StemPro™-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, Insulin-Transferrin- Selenium (ITS-G), monothioglycerol, SB431542, rhVEGF, bFGF, SCF, and one of L-glutamine and GlutaMaxTM, 193 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 and wherein the second medium does not comprise antibiotics; e) culturing and agitating the second cell suspension in the bioreactor for about 1.8 to about 2.2 days, preferably about 2 days; f) stopping the agitating for about 5 minutes, wherein cell aggregates settle in the second cell suspension; g) collecting about 80% of the second cell suspension from a top volume of the second cell suspension, from the bioreactor; h) centrifuging collected second cell suspension forming a second cell pellet and resuspending the second pellet in about 1 mL of one of StemFit® For Differentiation solution and StemPro™-34 SFM; i) suspending the second cell pellet in the about 1 mL of one of StemFit® For Differentiation solution and or StemPro™-34 SFM into a third medium forming a third cell suspension, wherein the third medium comprises: i) Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), interleukin-3 (IL-3), interleukin-6 (IL-6), one of (i) thrombopoietin (TPO) and (ii) erythropoietin (EPO), and one of (i) L-glutamine and (ii) GlutaMaxTM, or ii) StemPro™-34 SFM and StemPro™-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, rhVEGF, IL-3, IL-6, Flt3L, one of (i) TPO and (ii) EPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, and wherein the third medium does not comprise antibiotics; j) placing the third cell suspension into the bioreactor with remaining second cell suspension forming a fourth cell suspension; k) culturing and agitating the fourth cell suspension in the bioreactor for about 1.8 to about 2.2 days, preferably about 2 days; l) stopping the agitating for about 5 minutes; wherein cell aggregates settle in the cell suspension; 194 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 m) collecting about 80% of the fourth cell suspension and centrifuging the collected fourth cell suspension forming a third cell pellet, and resuspending the third cell pellet in about 1 mL of one of StemFit® For Differentiation solution and StemPro™-34 SFM; n) suspending the third cell pellet dissolved in the about 1 mL of one of StemFit® For Differentiation solution and StemPro™-34 SFM into a fourth medium forming a fifth cell suspension, wherein the fourth medium comprises: i) Advanced DMEM/F12 medium and StemFit® For Differentiation medium present in about a 3.9:0.9 ratio to a 4.1:1.1 ratio, preferably a 4:1 ratio, and further comprises SCF, IL-6, one of (i) TPO and (ii) EPO, and one of (i) L- glutamine and (ii) GlutaMaxTM, or ii) StemPro™-34 SFM and StemPro™-34 Nutrient Supplement in a 500:13 ratio, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, IL-6, one of (i) TPO and (ii) EPO, and one of (i) L-glutamine and (ii) GlutaMaxTM, and wherein the fourth medium does not comprise antibiotics; o) placing the fifth cell suspension into the bioreactor with the remaining fourth cell suspension thereby forming a sixth cell suspension; p) culturing and agitating the sixth cell suspension in the bioreactor for about 1.8 to about 2.2 days, preferably about 2 days; q) repeating steps l) to p); r) removing all the eighth cell suspension from the bioreactor and harvesting EBs and iHSCs in the cell suspension by centrifugation, thereby generating a fourth cell pellet and a first supernatant; s) collecting the first supernatant; t) centrifuging the first supernatant, thereby generating a fifth cell pellet and resuspending the fifth cell pellet in a fifth medium thereby generating a ninth cell suspension, wherein the fifth medium comprises Advanced DMEM/F12 medium, wherein the fifth medium does not comprise cytokines, antibiotics or growth factors, and wherein cells of the ninth cell suspension comprise iHSCs, which are defined as CD34+ and lineage marker-negative, where the lineage 195 301689646v1 Attorney Docket No: 253505.000510 / JBI6830WOPCT1 markers comprise one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a; u) resuspending the fourth cell pellet of step r), in a volume of about 20 mg/mL collagenase type II, thereby generating a tenth cell suspension; v) adding the fifth medium to the tenth cell suspension; w) centrifuging the tenth cell suspension, thereby generating a sixth cell pellet, and adding TrypLE to the sixth cell pellet, thereby generating an eleventh cell suspension; x) adding additional fifth medium to the eleventh cell suspension and mixing; y) centrifuging the eleventh cell suspension to form a seventh cell pellet and resuspending the seventh cell pellet in additional fifth medium to form a twelfth cell suspension; z) passing the twelfth cell suspension through a cell strainer to obtain iHSCs, wherein the cell strainer is 70 µm mesh size; aa) determining the purity of the iHSCs from the first supernatant of step t) and the iHSCs from step z), said purity defined as CD34+ and lineage marker-negative, where the lineage markers comprise one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a; bb) not introducing serum in any of steps a)-aa); cc) not introducing any additional cells, including feeder cells or stromal cells, in any of steps a)-bb); and dd) obtaining de novo generated iHSCs. 196 301689646v1
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180320137A1 (en) * 2015-11-04 2018-11-08 Fate Therapeutics, Inc. Methods and compositions for inducing hematopoietic cell differentiation
KR20200099099A (en) * 2019-02-13 2020-08-21 주식회사 온코인사이트 Method of preparing hematopoietic stem cells derived from induced pluripotent stem cells

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180320137A1 (en) * 2015-11-04 2018-11-08 Fate Therapeutics, Inc. Methods and compositions for inducing hematopoietic cell differentiation
KR20200099099A (en) * 2019-02-13 2020-08-21 주식회사 온코인사이트 Method of preparing hematopoietic stem cells derived from induced pluripotent stem cells

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BORYS BREANNA S., SO TANIA, COLTER JAMES, DANG TIFFANY, ROBERTS ERIN L., REVAY TAMAS, LARIJANI LEILA, KRAWETZ ROMAN, LEWIS IAN, AR: "Optimized serial expansion of human induced pluripotent stem cells using low-density inoculation to generate clinically relevant quantities in vertical-wheel bioreactors", STEM CELLS TRANSLATIONAL MEDICINE, vol. 9, no. 9, 1 September 2020 (2020-09-01), US , pages 1036 - 1052, XP093293207, ISSN: 2157-6564, DOI: 10.1002/sctm.19-0406 *
MURAI NOBUYUKI, KOYANAGI-AOI MICHIYO; TERASHI HIROTO; AOI TAKASHI: "Re-generation of cytotoxic γδT cells with distinctive signatures from human γδT-derived iPSCs", STEM CELL REPORTS, vol. 18, no. 4, 1 April 2023 (2023-04-01), pages 853 - 868, XP093170218, DOI: 10.1016/j.stemcr.2023.02.010 *
NOGUEIRA DIOGO E., RODRIGUES CARLOS A. V., CARVALHO MARTA S., MIRANDA CLÁUDIA C., HASHIMURA YAS, JUNG SUNGHOON, LEE BRIAN, CABRAL : "Strategies for the expansion of human induced pluripotent stem cells as aggregates in single-use Vertical-Wheel™ bioreactors", JOURNAL OF BIOLOGICAL ENGINEERING, vol. 13, no. 1, 1 December 2019 (2019-12-01), Lo , pages 1 - 14, XP093293204, ISSN: 1754-1611, DOI: 10.1186/s13036-019-0204-1 *
WATANABE, D. ET AL.: "The generation of human γδT cell-derived induced pluripotent stem cells from whole peripheral blood mononuclear cell culture", STEM CELLS TRANSLATIONAL MEDICINE, vol. 7, 21 November 2017, pages 34 - 44, XP055534974, DOI: 10.1002/sctm.17-0021 *

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