[go: up one dir, main page]

WO2010059775A1 - Culture de cellules souches pluripotentes sur des micro-supports - Google Patents

Culture de cellules souches pluripotentes sur des micro-supports Download PDF

Info

Publication number
WO2010059775A1
WO2010059775A1 PCT/US2009/065062 US2009065062W WO2010059775A1 WO 2010059775 A1 WO2010059775 A1 WO 2010059775A1 US 2009065062 W US2009065062 W US 2009065062W WO 2010059775 A1 WO2010059775 A1 WO 2010059775A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
micro
carriers
cell
stem cells
Prior art date
Application number
PCT/US2009/065062
Other languages
English (en)
Inventor
Shelley Nelson
Original Assignee
Centocor Ortho Biotech Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to CA2744234A priority Critical patent/CA2744234C/fr
Priority to AU2009316580A priority patent/AU2009316580B2/en
Priority to BRPI0920956-5A priority patent/BRPI0920956A2/pt
Priority to PL09759851T priority patent/PL2366021T3/pl
Priority to MX2014000085A priority patent/MX356756B/es
Priority to EP09759851.0A priority patent/EP2366021B1/fr
Priority to RU2011124900/10A priority patent/RU2555538C2/ru
Priority to JP2011537603A priority patent/JP2012509085A/ja
Priority to KR1020177024255A priority patent/KR101837080B1/ko
Priority to ES09759851.0T priority patent/ES2642070T3/es
Application filed by Centocor Ortho Biotech Inc. filed Critical Centocor Ortho Biotech Inc.
Priority to KR1020117013824A priority patent/KR101774546B1/ko
Priority to CN200980155382.2A priority patent/CN102307991B/zh
Priority to MX2011005288A priority patent/MX2011005288A/es
Publication of WO2010059775A1 publication Critical patent/WO2010059775A1/fr
Priority to ZA2011/04506A priority patent/ZA201104506B/en
Priority to AU2016201220A priority patent/AU2016201220B2/en

Links

Classifications

    • 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
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • 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
    • 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
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • 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
    • 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
    • C12N5/0068—General culture methods using substrates
    • C12N5/0075—General culture methods using substrates using microcarriers
    • 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
    • C12N5/0676—Pancreatic cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/25—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving enzymes not classifiable in groups C12Q1/26 - C12Q1/66
    • 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
    • C12N2501/70—Enzymes
    • 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
    • C12N2501/70—Enzymes
    • C12N2501/72—Transferases [EC 2.]
    • C12N2501/727—Kinases (EC 2.7.)
    • 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
    • C12N2501/999—Small molecules not provided for elsewhere
    • 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
    • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic 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
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
    • 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
    • C12N2531/00—Microcarriers

Definitions

  • the present invention is directed to methods for the growth, expansion and differentiation of pluripotent stem cells on micro-carriers.
  • Pluripotent stem cells such as, for example, embryonic stem cells have the ability to differentiate into all adult cell types.
  • embryonic stem cells may be a source of replacement cells and tissue for organs that have been damaged as a result of disease, infection, or congenital abnormalities.
  • the potential for embryonic stem cells to be employed as a replacement cell source is hampered by the difficulty of propagating the cells in vitro while maintaining their pluripotency.
  • Embryonic stem cells provide a potential resource for research and drug screening. At present, large-scale culturing of human embryonic stem cell lines is problematic and provides substantial challenges. Current in vitro methods to propagate pluripotent stem cells are carried out in tissue flasks on planar surfaces pre-coated with extracellular matrix (ECM) proteins or feeder cells. Planar cultures also require frequent subculturing because their limited surface area cannot support long-term growth of pluripotent stem cells. Micro-carrier-based methods of pluripotent stem cell culture may provide a solution. Micro-carriers have a high surface-area-to-volume ratio and, therefore, eliminate the surface area restriction of growing pluripotent stem cells on planar surfaces.
  • ECM extracellular matrix
  • Fok et al disclose stirred-suspension culture systems for the propagation of undifferentiated ESC - micro-carrier and aggregate cultures (Stem Cells 2005;23:1333- 1342.)
  • Abranches et al disclose the testing of Cytodex 3® (GE Healthcare Life Sciences, NJ), a microporous micro-carrier made up of a dextran matrix with a collagen layer at the surface for its ability to support the expansion of the mouse S25 ES cell line in spinner flasks (Biotechnol. Bioeng. 96 (2007), pp. 1211-1221.)
  • US20070264713 disclose a process for cultivating undifferentiated stem cells in suspension and in particular to a method for cultivating stem cells on micro- carriers in vessels.
  • WO2006137787 disclose a screening tool which comprises particulate matter or micro-carriers, such as beads, attached to a solid support, such as a micro titer plate, for the cultivation of cells on said micro-carriers.
  • WO2008004990 disclose a method of promoting the attachment, survival and/or proliferation of a stem cell in culture, the method comprising culturing a stem cell on a positively-charged support surface.
  • WO2007012144 disclose a bioreactor, comprising: a support surface; and a synthetic attachment polypeptide bound to the support surface wherein the synthetic attachment polypeptide is characterized by a high binding affinity for an embryonic stem cell or a multipotent cell.
  • the present invention provides methods for the growth, expansion and differentiation of pluripotent stem cells on micro-carriers.
  • the present invention provides a method for the propagation of pluripotent stem cells, comprising the steps of:
  • FIG. 1 Rho-kinase inhibitor promotes attachment and growth of human embryonic stem cells to micro-carriers. Images of H9 cells grown in static culture for 2 days on HILLEX®II micro-carriers (Solohill, MI). The cells were cultured in mouse embryonic fibroblast conditioned medium (MEF-CM) with or without lO ⁇ M Rho Kinase inhibitor, Y27632 ((Sigma-Aldrich, MO) A and B, respectively).
  • MEF-CM mouse embryonic fibroblast conditioned medium
  • Y27632 (Sigma-Aldrich, MO) A and B, respectively).
  • FIG. 2 H9 cells grown on micro-carriers. H9 cells were allowed to attach to various micro-carriers and placed on a rocking platform at 37 0 C. Plastic micro-carriers, ProNectinF micro-carriers, HILLEX®II micro-carriers (Solohill, MI), and Plastic Plus micro-carriers, were used (A, B, C, D respectively). Growth after 3 days showed cells on HILLEX®II (Solohill, MI) with best cell attachment to the micro-carriers. Arrows identify cells forming aggregates without attachment to the micro-carriers.
  • FIG. 3 H9 cell proliferation on micro-carriers. H9 cells were attached to HILLEX®II micro-carriers, ProNectinF micro-carriers, Plastic Plus micro-carriers, and Plastic micro- carriers (Solohill, MI) and placed in a 6 well dish on a rocking platform at 37 0 C in the presence of lO ⁇ M Y27632 (Sigma-Aldrich, MO) and MEF-CM. The initial cell seeding density is the value at day 0. Day 3 and day 5 cell numbers are shown.
  • Figure 4 Hl cell images after attachment to micro-carriers.
  • FIG. 5 Hl cell images after attachment to micro-carriers. Images of cells at days 3, 5 and 7 are shown attached to Cytodex 1® micro-carriers, Cytodex 3® micro-carriers (GE Healthcare Life Sciences, NJ) and HILLEX®II micro-carriers (Solohill, MI). The cells were grown in MEF-CM with lO ⁇ M Y27632 (Sigma- Aldrich, MO) in a 12 well dish on a rocking platform at 37 0 C.
  • FIG. 6 Hl cell proliferation on micro-carriers.
  • Hl cells were allowed to attach to HILLEX®II micro-carriers (Solohill, MI), Cytodex 1® micro-carriers (GE Healthcare Life Sciences, NJ), Cytodex 3® micro-carriers (GE Healthcare Life Sciences, NJ), ProNectinF micro-carriers (Solohill, MI), Plastic Plus micro-carriers (Solohill, MI), and Plastic micro-carriers (Solohill, MI) and placed in a 12 well dish on a rocking platform at 37 0 C in the presence of lO ⁇ M Y27632 (Sigma-Aldrich, MO) and MEF-CM.
  • the initial cell seeding density is the value at day O. Day 3, 5, and 7 cell numbers are shown. The initial seeding density was 13,333 cells/cm 2 , as indicated by the line.
  • FIG. 7 H9 cell proliferation on micro-carriers in various concentrations of Rho kinase inhibitors.
  • Cells were grown in a 12 well plate on a rocking platform and counted at day 4 and 7 to determine attachment and proliferation rate.
  • A. H9 cells were grown in MEF- CM with 1, 2.5, 5, or lO ⁇ M Y27632 (Sigma-Aldrich, MO).
  • B. H9 cells were grown in MEF-CM with 0.5, 1, 2.5, or 5 ⁇ M Glycyl-H 1152 dihydrochloride (Tocris, MO).
  • Hl cells were grown in decreasing concentrations of Rho kinase inhibitors.
  • Hlp38 cells were grown in the presence of Y27632 (Sigma-Aldrich, MO) or Glycyl-H 1152 dihydrochloride (Tocris, MO) for two days at decreasing concentrations (10 ⁇ M /5 ⁇ M, 2.5 ⁇ M /0.5 ⁇ M or 1.0 ⁇ M /0.5 ⁇ M) or at 0.25 ⁇ M Glycyl-H 1152 dihydrochloride (Tocris, MO) continuously.
  • Y27632 Sigma-Aldrich, MO
  • Tocris, MO Glycyl-H 1152 dihydrochloride
  • HILLEX®II Solohill, MI
  • Cytodex 1® Cytodex 1®
  • Cytodex 3® (GE Healthcare Life Sciences, NJ) A, B, C, respectively). Cells were counted at 3, 5 and 7 days post seeding.
  • Figure 9 Determination of cell attachment to micro-carriers at different seeding densities in spinner flasks. Hl cells were seeded onto Cytodex 3® (GE Healthcare Life Sciences, NJ) micro-carriers at the densities listed on the left; Low (0.4 x 10 4 cells/cm 2 ), Mid (1.2 x 10 4 cells/cm 2 ) or High (3 x 10 4 cells/cm 2 ). At 3, 5 and 7 days the cells were imaged and the percentage of micro-carriers with cells attached was determined (embedded in image).
  • Cytodex 3® GE Healthcare Life Sciences, NJ
  • FIG. 10 Cell growth on micro-carriers in spinner flasks is affected by the initial seeding densities. Hl cells were seeded onto Cytodex 3® (GE Healthcare Life Sciences, NJ) micro-carriers at the densities listed on the left; Low (0.4 x 10 4 cells/cm 2 ), Mid (1.2 x 10 4 cells/cm 2 ) or High (3 x 10 4 cells/cm 2 ). At 3, 5 and 7 days the cells were dissociated from the micro-carriers and counted.
  • Cytodex 3® GE Healthcare Life Sciences, NJ
  • Figure 11 Determination of cell growth rate on micro-carriers at different seeding densities in spinner flasks. Hl cells were seeded onto Cytodex 3® (GE Healthcare Life Sciences, NJ) micro-carriers at different densities (day 0); Low (0.4 x 10 4 cells/cm 2 ), Mid (1.2 x 10 4 cells/cm 2 ) or High (3 x 10 4 cells/cm 2 ). At 3, 5 and 7 days the cells were dissociated from the micro-carriers and counted. The fold increase in cell number is shown versus initial seeding density.
  • Cytodex 3® GE Healthcare Life Sciences, NJ
  • FIG. 12 Hl cells grown on Cytodex 3® micro-carriers (GE Healthcare Life Sciences, NJ) were imaged after 7 days in culture. The cells received MEF-CM without Rho kinase inhibitor from day3 onward. The cells remained attached to the micro-carriers.
  • Figure 13 H9 cells growth and dissociation of H9 cells on HILLEX®II micro-carriers (Solohill, MI).
  • A B 10x and 2Ox images of H9 cells grown for 6 days on HILLEX®II micro-carriers (Solohill, MI).
  • C 20 x image of cells dissociated from HILLEX®II micro-carriers (Solohill, MI) for 10 minutes with 0.05% Trypsin/EDTA.
  • D 2Ox image of cells dissociated from HILLEX®II micro-carriers (Solohill, MI) for 10 minutes with TrypLETM Express.
  • Figure 14 Dissociation of H9 cells from micro-carriers.
  • H9 cells grown on HILLEX®II (Solohill, MI) on a rocking platform were dissociated with TrypLETM Express or 0.05% Trypsin/EDTA. The number of cells and their viability is shown, A and B respectively.
  • FIG. 15 Dissociation of Hl cells from micro-carriers.
  • Hl cells grown on Cytodex 3® GE Healthcare Life Sciences, NJ
  • TrypLETM Express Invitrogen, CA
  • AccutaseTM or Collagenase (lOmg/ml).
  • the number of cells and their viability is shown, A and B respectively.
  • FIG. 16 H9 cells grown on HILLEX®II (Solohill, MI) micro-carriers do not transfer between micro-carriers.
  • Figure 17 H9 at passage 43 were grown for 5 passages on HILLEX®II (Solohill, MI) micro-carriers in a spinner flask. Cells were counted every 2 to 3 days and passaged when cells reached 1-2 x 10 5 cells/cm 2 .
  • FIG. 18 H9 cells at passage 43 were grown for 5 passages on Cytodex 3® micro- carriers (GE Healthcare Life Sciences, NJ) in a spinner flask. Cells were counted every 2 to 3 days and passaged when cells reached 1-2 x 10 5 cells/cm 2 .
  • FIG 19 Fluorescent-activated cell sorting (FACS) shows pluripotency of H9 cells grown in spinner flasks.
  • A The majority of H9 p43 cells grown on HILLEX®II (Solohill, MI) micro-carriers express of pluripotency proteins. Passage 1 and 3 cells were not evaluated for TRA- 1-81.
  • B The majority of H9 p43 cells grown on Cytodex 3® (GE Healthcare Life Sciences, NJ) micro-carriers express of pluripotency proteins. Passage 1 cells were not evaluated for TRA- 1-81.
  • FIG. 20 Hl p49 cells were grown for 5 passages on Cytodex 1® micro-carriers (GE Healthcare Life Sciences, NJ) in a spinner flask. Cells were counted every 2 to 3 days and passaged when cells reached 4-8 x 10 4 cells/cm 2 .
  • FIG 21 Hl cells at passage 49 were grown for 5 passages on Cytodex 3® micro- carriers (GE Healthcare Life Sciences, NJ) in a spinner flask. Cells were counted every 2 to 3 days and passaged when cells reached 1-2 x 10 5 cells/cm 2 .
  • Figure 22 Fluorescence activated cell sorting (FACS) shows pluripotency of Hl cells grown in spinner flasks.
  • Figure 23 Population doublings of Hl and H9 cells on micro-carriers. Population doubling times were calculated from day 3 to the day of passaging (day 5,6 or 7).
  • FIG. 24 H9 cells cultured on micro-carriers in defined media.
  • the cells were cultured on HILLEX®II (HII, (Solohill, MI)) or Cytodex 3® (C3, (GE Healthcare Life Sciences, NJ)).
  • Cells were cultured on micro-carriers in one of the following media; mTESR (StemCell Technologies, Vancouver, Canada), StemPro or MEF-CM.
  • Hl cells at passage 38 were cultured on micro-carriers in defined media.
  • the cells were cultured on HILLEX®II (HII, (Solohill, MI)) or Cytodex 3® (C3, (GE Healthcare Life Sciences, NJ)) micro-carriers.
  • Cells were cultured on micro-carriers in one of the following medias; mTESR (StemCell Technologies, Vancouver, Canada), StemPro and MEF-CM. 10 ⁇ M Y27632 (Y, (Sigma- Aldrich, MO)) or 2.5 ⁇ M Glycyl-H 1152 dihydrochloride (H, (Tocris, MO)) was added to the media. Growth rate at 3, 5 and 7 days post seeding was determined.
  • FIG. 26 Hl cells at passage 50 were cultured on HILLEX®II (Solohill, MI)) micro- carriers with defined medium in a spinner flask.
  • A Images of Hl p50 cells grown in MEF-CM after 3, 7, or 9 days in a spinner flask.
  • B Images of Hl p50 cells grown in mTESR (StemCell Technologies, Vancouver, Canada) after 3, 7, or 9 days. Arrows identify cell clusters not attached to the micro-carriers.
  • FIG. 27 Differentiation of human embryonic stem cells passaged five times in spinner flasks.
  • A H9 cells at passage 43 were passaged five times on Cytodex 3® micro-carriers (GE Healthcare Life Sciences, NJ).
  • B Hl cells at passage 49 were passaged five times on Cytodex 1® micro-carriers (GE Healthcare Life Sciences, NJ). Both cell types were released from the micro-carriers and seeded onto MATRIGEL (BD Biosciences, CA) coated plates.
  • MATRIGEL BD Biosciences, CA coated plates.
  • the cells were exposed to a protocol that is capable of differentiating embryonic stem cells to definitive endoderm.
  • the cells were then analyzed by FACS for the percentage of cells expressing CXCR4, a definitive endoderm marker. The percent of CXCR4 positive cells is in the upper right corner of the plot.
  • Figure 28 Differentiation of Hl cells on micro-carriers to definitive endoderm.
  • FACS plots display the percentage of cells expressing the definitive endoderm marker CXCR4. Percent positive is in the upper right corner.
  • Cells were all expanded on micro- carriers in spinner flasks prior to treatment.
  • A Hl cells at passage 40 were grown on Cytodex 1® micro-carriers (GE Healthcare Life Sciences, NJ) for 6 days after passage 5 prior to differentiation.
  • B Hl cells at passage 40 were grown on Cytodex 3® micro- carriers (GE Healthcare Life Sciences, NJ) for 8 days after passage 1 prior to differentiation.
  • C Hl cells at passage 50 were grown on HILLEX®II micro-carriers (Solohill, MI) for 6 days after passage 1 prior to differentiation.
  • FIG. 29 Differentiation of Hl cells on Cytodex 3® micro-carriers (GE Healthcare Life Sciences, NJ) to definitive endoderm.
  • A Hl cells at passage 40 were grown on micro- carriers for eight days.
  • B Hl cells at passage 40 were grown on micro-carriers for 11 days. Both cell population were then differentiated to definitive endoderm on a rocking platform at 37°C.
  • FACS plots display the percentage of cells expressing the definitive endoderm marker CXCR4. Percent positive is in the upper right corner.
  • Figure 30 Differentiation of cells of the human embryonic stem cell line Hl, cultured on micro-carriers to definitive endoderm. FACS results for the percent positive CXCR4 cells are shown on the Y-axis. Hl cells were grown on HILLEX®II, Cytodex 1® or Cytodex 3® micro-carriers prior to and during differentiation.
  • FIG 31 Differentiation of cells of the human embryonic stem cell line Hl cultured on micro-carriers to pancreatic endoderm cells. CT values are shown on the Y-axis for pancreatic endodermal markers, Ngn3, Nkx ⁇ .l and Pdxl. Hl cells were differentiated on HILLEX®II (HII), Cytodex 1®(C1) or Cytodex 3®(C3) micro-carriers in either DMEM- High Glucose (HG) or DMEM-F 12 (F 12) media. The differentiation protocol lasted 13 days.
  • Figure 32 Differentiation of cells of the human embryonic stem cell line Hl cultured on micro-carriers to hormone producing pancreatic cells.
  • Percent positive cells were determined by FACS shown on the Y-axis for pancreatic hormone cell markers, Synaptophysin, Glucagon and Insulin. Hl cells were seeded at two different concentrations 10x10 5 (10) or 2OxIO 5 (20) onto Cytodex 3®(C-3) micro-carriers. The cells were differentiated in DMEM-High Glucose (HG) during days four to nine and further differentiated in either HG or DMEM-F 12 (F 12) media from days 10 through 24.
  • HG DMEM-High Glucose
  • FIG. 33 Differentiation of Hl cells on Cytodex 3® micro-carriers (GE Healthcare Life Sciences, NJ) to endocrine cells. Hl cells were differentiated to pancreatic endocrine cells through pancreatic endoderm (Day 14), pancreatic endocrine cells (Day 21) to insulin-expressing cells (Day28). Gene expression levels of Pdxl, Glucagon, and Insulin were measured (A,B,C respectively). Hl cells grown and differentiated on Cytodex 3® micro-carriers (GE Healthcare Life Sciences, NJ) (C3) were compared to those grown and differentiated on MATRIGEL (BD Biosciences, CA) coated 6 well dishes (planar). The gene expression values for cells grown on Cytodex 3® micro-carriers (GE Healthcare Life Sciences, NJ) was performed in triplicate.
  • FIG. 34 H9 cells were differentiated on Cytodex 3® micro-carriers (GE Healthcare Life Sciences, NJ) to definitive endoderm (DE). FACS plots of CXCR4 expression. Percent of definitive endoderm marker CXCR4 positive cells is stated in upper right corner.
  • A H9 cells at passage 39 were grown on a MATRIGEL (BD Biosciences, CA) coated 6 well dishes and differentiated to DE.
  • B C Duplicate samples of H9 cells on Cytodex 3® micro-carriers (GE Healthcare Life Sciences, NJ) from spinners were placed in a 12 well dish and incubated on a rocking platform.
  • FIG 35 Differentiation of H9 cells on Cytodex 3® micro-carriers (GE Healthcare Life Sciences, NJ) to insulin-expressing cells. H9 cells were differentiated to pancreatic endocrine cells through pancreatic endoderm (Day 14), Endocrine cells (Day 22) to Insulin-expressing cells (Day29). Gene expression level of Pdxl, Glucagon, and Insulin was measured (A, B, C respectively). H9 cells grown and differentiated on Cytodex 3® micro-carriers (GE Healthcare Life Sciences, NJ) (C3) were compared to those grown and differentiated on MATRIGEL (BD Biosciences, CA) coated 6 well dishes (planar).
  • MATRIGEL BD Biosciences, CA
  • Figure 36 Maintenance of pluripotency in human embryonic stem cells cultured for 5 passages on Cytodex 3® micro-carriers, then transferred and cultured on the planar substrates indicated and cultured in the presence of a Rho kinase inhibitor.
  • Panel A depicts the expression of the pluripotency markers CD9, SSEA3, SSEA4, Tra-160, and Tra-181 as detected by flow cytometry.
  • Panel B depicts the expression of the pluripotency markers Nanog, Pou5Fl, SOX2, and ZFP42 and markers of differentiation: FOXA2, FOXD3, GAT A2, GAT A4, and Brachyury as detected by real-time PCR.
  • Figure 37 Formation of definitive endoderm by human embryonic stem cells cultured for 5 passages on Cytodex 3® micro-carriers, then transferred and cultured on the planar substrates indicated and cultured in the presence of a Rho kinase inhibitor.
  • Panel A depicts the expression of CXCR4 as detected by flow cytometry.
  • Panel B depicts the expression of the markers indicated as detected by real-time PCR.
  • Figure 38 Formation of definitive endoderm by human embryonic stem cells cultured for 5 passages on Cytodex 3® micro-carriers, then transferred and cultured on a PRIMARIATM planar substrate. Expression of the genes indicated was determined by flow cytometry.
  • Figure 39 Human embryonic stem cells cultured on planar substrates maintain pluripotency. mRNA samples from TrypLE TM, AccutaseTM, or Collagenase passaged Hl human ES cells were collected and assayed for mRNA pluripotency gene expression. Cells were grown for either one passage for 4 days in culture on MATRIGEL in MEF conditioned media (A) or one passage on PrimariaTM in MEF conditioned media supplemented with Rock Inhibitor (B), or two passages on PrimariaTM in MEF conditioned media supplemented with Rock Inhibitor (C).
  • MATRIGEL MEF conditioned media
  • B Rock Inhibitor
  • C Rock Inhibitor
  • Figure 40 Hl human embryonic stem cells grown for greater than 7 passages on PRIMARIA (greater than p45) passaged with AccutaseTM or TrypLETM at 1 :4, 1 :8, or 1 :16 split ratios on PRIMARIA in the presence of Rho Kinase inhibitor Glycyl-H 1152 dihydrochloride were tested for pluripotency (A), and the ability to differentiate to Definitive Endoderm (B).
  • the control is Hlp48 human embryonic stem cells grown on 1 :30 MATRIGEL passaged with collagenase.
  • 1OmA passaged with 10 minute exposure to AccutaseTM.
  • P(X) indicate passage number since moving from MEF feeders to PrimariaTM plastic.
  • Figure 41 Hl human embryonic stem cells grown for greater than 7 passages on PRIMARIA (greater than p45) passaged with AccutaseTM or TrypLETM at 1 :4 ratio on PRIMARIA in the presence of Rho Kinase inhibitor Glycyl-H 1152 dihydrochloride were tested for mRNA expression of pluripotency and differentiation markers.
  • the control is the starting population of cells at passage 37.
  • lOmin AccutaseTM passaged with 10 minute exposure to AccutaseTM.
  • P(X) indicate passage number since moving from MEF feeders to PRIMARIATM plastic.
  • Figure 42 Hl human embryonic stem cells grown for greater than 7 passages on PRIMARIATM (greater than p45) passaged with AccutaseTM or TrypLETM at 1 :8 ratio on PRIMARIA in the presence of Rho Kinase inhibitor Glycyl-H 1152 dihydrochloride were tested for mRNA expression of pluripotency and differentiation markers.
  • the control is the starting population of cells at passage 37.
  • P(X) indicate passage number since moving from MEF feeders to PRIMARIATM plastic.
  • Figure 43 Hl human embryonic stem cells grown for greater than 7 passages on PRIMARIATM (greater than p45) passaged with AccutaseTM or TrypLETM at 1 : 16 ratio on PRIMARIA in the presence of Rho Kinase inhibitor Glycyl-H 1152 dihydrochloride were tested for mRNA expression of pluripotency and differentiation markers.
  • the control is the starting population of cells at passage 37.
  • lOmin AccutaseTM passaged with 10 minute exposure to AccutaseTM.
  • P(X) indicate passage number since moving from MEF feeders to PRIMARIATM plastic.
  • Figure 44 Images of Hl cells grown on PrimariaTM planar substrates (cat. no. 353846, Becton Dickinson, Franklin Lakes, NJ) then transferred to micro-carriers 3 days after seeding.
  • A-C Hl cells were seeded onto Cytodex 3® (GE Healthcare Life Sciences, NJ) micro-carriers.
  • D-F Cells were seeded onto HILLEXd)II micro-carriers (Solohill, MI).
  • A,D Hl cells were passaged on PrimariaTM planar substrate (cat. no. 353846, Becton Dickinson, Franklin Lakes, NJ) plates with 10 minutes of TrypLETM Express (Invitrogen, CA) treatment prior to transferring onto micro-carriers.
  • A,E Hl cells were passaged on PrimariaTM planar substrates (cat. no. 353846, Becton Dickinson, Franklin Lakes, NJ) plates with 10 minutes of AccutaseTM treatment prior to transferring onto micro-carriers.
  • C,F Hl cells at passage 46 were passaged on MATIRGEL (BD Biosciences, CA) coated plates with collagenase (lmg/ml) prior to transferring onto micro-carriers.
  • FIG. 45 Pluripotentency of Hl cells grown on PrimariaTM planar substrates (cat. no. 353846, Becton Dickinson, Franklin Lakes, NJ) then transferred to Cytodex 3® (GE Healthcare Life Sciences, NJ) and HILLEX®II micro-carriers. FACS analysis shows expression of pluripotent cell-surface proteins. Cells were treated with AccutaseTM or TrypLETM Express (Invitrogen, CA) for 3 to 10 minutes during passaging on PrimariaTM (cat. no. 353846, Becton Dickinson, Franklin Lakes, NJ).
  • FIG 46 Differentiation of Hl cells propagated on PrimariaTM (cat. no. 353846, Becton Dickinson, Franklin Lakes, NJ) then transferred to Cytodex 3® micro-carriers (GE Healthcare Life Sciences, NJ). FACS analysis of cell surface expression of CXCR4, definitive endoderm marker. Cells were treated with AccutaseTM or TrypLETM Express (Invitrogen, CA) for 3 to 10 minutes during passaging on PrimariaTM (cat. no. 353846, Becton Dickinson, Franklin Lakes, NJ).
  • Figure 47 FACS analysis of human embryonic stem cells cultured on planar substrates consisting of mixed cellulose esters prior to culture on micro-carriers.
  • Figure 48 FACS analysis of the expression of markers characteristic of the definitive endoderm lineage from human embryonic stem cells cultured on planar substrates consisting of mixed cellulose esters prior to culture and differentiation on micro-carriers.
  • Stem cells are undifferentiated cells defined by their ability at the single cell level to both self-renew and differentiate to produce progeny cells, including self-renewing progenitors, non-renewing progenitors, and terminally differentiated cells. Stem cells are also characterized by their ability to differentiate in vitro into functional cells of various cell lineages from multiple germ layers (endoderm, mesoderm and ectoderm), as well as to give rise to tissues of multiple germ layers following transplantation and to contribute substantially to most, if not all, tissues following injection into blastocysts.
  • Stem cells are classified by their developmental potential as: (1) totipotent, meaning able to give rise to all embryonic and extraembryonic cell types; (2) pluripotent, meaning able to give rise to all embryonic cell types; (3) multipotent, meaning able to give rise to a subset of cell lineages, but all within a particular tissue, organ, or physiological system (for example, hematopoietic stem cells (HSC) can produce progeny that include HSC (self- renewal), blood cell restricted oligopotent progenitors and all cell types and elements (e.g., platelets) that are normal components of the blood); (4) oligopotent, meaning able to give rise to a more restricted subset of cell lineages than multipotent stem cells; and (5) unipotent, meaning able to give rise to a single cell lineage (e.g. , spermatogenic stem cells).
  • HSC hematopoietic stem cells
  • Differentiation is the process by which an unspecialized ("uncommitted") or less specialized cell acquires the features of a specialized cell such as, for example, a nerve cell or a muscle cell.
  • a differentiated or differentiation-induced cell is one that has taken on a more specialized ("committed") position within the lineage of a cell.
  • the term "committed”, when applied to the process of differentiation, refers to a cell that has proceeded in the differentiation pathway to a point where, under normal circumstances, it will continue to differentiate into a specific cell type or subset of cell types, and cannot, under normal circumstances, differentiate into a different cell type or revert to a less differentiated cell type.
  • De-differentiation refers to the process by which a cell reverts to a less specialized (or committed) position within the lineage of a cell.
  • the lineage of a cell defines the heredity of the cell, i.e., which cells it came from and what cells it can give rise to.
  • the lineage of a cell places the cell within a hereditary scheme of development and differentiation.
  • a lineage-specific marker refers to a characteristic specifically associated with the phenotype of cells of a lineage of interest and can be used to assess the differentiation of an uncommitted cell to the lineage of interest.
  • Maintenance refers generally to cells placed in a growth medium under conditions that facilitate cell growth and/or division that may or may not result in a larger population of the cells.
  • Passaging refers to the process of removing the cells from one culture vessel and placing them in a second culture vessel under conditions that facilitate cell growth and/or division.
  • a specific population of cells, or a cell line is sometimes referred to or characterized by the number of times it has been passaged.
  • a cultured cell population that has been passaged ten times may be referred to as a PlO culture.
  • the primary culture i.e., the first culture following the isolation of cells from tissue, is designated PO.
  • the cells are described as a secondary culture (Pl or passage 1).
  • P2 or passage 2 After the second subculture, the cells become a tertiary culture (P2 or passage 2), and so on.
  • P2 or passage 2 tertiary culture
  • the expansion of cells (i.e., the number of population doublings) during the period between passaging depends on many factors, including but not limited to the seeding density, substrate, medium, growth conditions, and time between passaging.
  • ⁇ -cell lineage refer to cells with positive gene expression for the transcription factor PDX-I and at least one of the following transcription factors: NGN-3, Nkx2.2, Nkx ⁇ .l, NeuroD, IsI-I, FINF-3 beta, MAFA, Pax4, or Pax6.
  • Cells expressing markers characteristic of the ⁇ cell lineage include ⁇ cells.
  • Cells expressing markers characteristic of the definitive endoderm lineage refer to cells expressing at least one of the following markers: SOX- 17, GAT A-4, HNF-3 beta, GSC, Cerl, Nodal, FGF8, Brachyury, Mix-like homeobox protein, FGF4 CD48, eomesodermin (EOMES), DKK4, FGF 17, GAT A-6, CXCR4, C-Kit, CD99, or OTX2.
  • Cells expressing markers characteristic of the definitive endoderm lineage include primitive streak precursor cells, primitive streak cells, mesendoderm cells and definitive endoderm cells.
  • Cells expressing markers characteristic of the pancreatic endoderm lineage refer to cells expressing at least one of the following markers: PDX-I, FINF- lbeta, PTF-I alpha, FINF-6, or HB9.
  • Cells expressing markers characteristic of the pancreatic endoderm lineage include pancreatic endoderm cells.
  • Cells expressing markers characteristic of the pancreatic endocrine lineage refer to cells expressing at least one of the following markers: NGN-3, NeuroD, Islet-1, PDX-I, NKX6.1, Pax-4, Ngn-3, or PTF-I alpha.
  • Cells expressing markers characteristic of the pancreatic endocrine lineage include pancreatic endocrine cells, pancreatic hormone expressing cells, and pancreatic hormone secreting cells, and cells of the ⁇ -cell lineage.
  • Definitive endoderm refers to cells which bear the characteristics of cells arising from the epiblast during gastrulation and which form the gastrointestinal tract and its derivatives. Definitive endoderm cells express the following markers: CXCR4, HNF-3 beta, GATA-4, SOX- 17, Cerberus, 0TX2, goosecoid, c-Kit, CD99, and Mixll.
  • Extraembryonic endoderm refers to a population of cells expressing at least one of the following markers: SOX-7, AFP, or SPARC.
  • Markers are nucleic acid or polypeptide molecules that are differentially expressed in a cell of interest.
  • differential expression means an increased level for a positive marker and a decreased level for a negative marker.
  • the detectable level of the marker nucleic acid or polypeptide is sufficiently higher or lower in the cells of interest compared to other cells, such that the cell of interest can be identified and distinguished from other cells using any of a variety of methods known in the art.
  • Mesendoderm cell refers to a cell expressing at least one of the following markers: CD48, eomesodermin (EOMES), SOX- 17, DKK4, HNF-3 beta, GSC, FGF 17, or GAT A-6.
  • EOMES eomesodermin
  • SOX- 17, DKK4, HNF-3 beta a cell expressing at least one of the following markers: CD48, eomesodermin (EOMES), SOX- 17, DKK4, HNF-3 beta, GSC, FGF 17, or GAT A-6.
  • Pantendocrine cell or “pancreatic hormone expressing cell” as used herein refers to a cell capable of expressing at least one of the following hormones: insulin, glucagon, somatostatin, and pancreatic polypeptide.
  • Pantencreatic hormone secreting cell refers to a cell capable of secreting at least one of the following hormones: insulin, glucagon, somatostatin, and pancreatic polypeptide.
  • Pre-primitive streak cell refers to a cell expressing at least one of the following markers: Nodal, or FGF8.
  • Primary streak cell refers to a cell expressing at least one of the following markers: Brachyury, Mix-like homeobox protein, or FGF4.
  • Micro-carriers refers to particles, beads, or pellets useful for attachment and growth of anchorage dependent cells in culture.
  • the micro-carriers have the following properties: (a) They are small enough to allow them to be used in suspension cultures (with a stirring rate that does not cause significant shear damage to the micro-carriers or the cells); (b) They are solid, or have a solid core with a porous coating on the surface; and (c) Their surfaces (exterior and interior surface in case of porous carriers) may be positively or negatively charged.
  • the micro-carriers have an overall particle diameter between about 150 and 350 ⁇ m, and have a positive charge density of between about 0.8 and 2.0 meq/g.
  • micro-carriers include, without limitation, Cytodex 1®, Cytodex 2®, or Cytodex 3® (GE Healthcare Life Sciences, NJ).
  • the micro-carrier is a solid carrier. Solid carriers are particularly suitable for adhesion cells, e.g., anchorage-dependent cells.
  • the carrier particle can also be a porous micro-carrier.
  • Porous micro-carriers refers to particles useful for attachment and growth of anchorage-dependent cells in culture.
  • the porous micro-carriers have the following properties: (a) they are small enough to allow them to be used in suspension cultures (with a stirring rate that does not cause significant shear damage to the micro-carriers or the cells); (b) they have pores and interior spaces of sufficient size to allow cells to migrate into the interior spaces of the particle and (c) their surfaces (exterior and interior) may be positively or negatively charged.
  • the carriers (a) have an overall particle diameter between about 150 and 350 ⁇ m; (b) have pores having an average pore opening diameter of between about 15 and about 40 ⁇ m; and (c) have a positive charge density of between about 0.8 and 2.0 meq/g.
  • the positive charge is provided by DEAE (N,N,-diethylaminoethyl) groups.
  • Useful porous micro-carriers include, without limitation, Cytopore 1® and Cytopore 2® (GE Healthcare Life Sciences, Piscataway N. J.). Micro-carriers may be any shape, but are typically roughly spherical in shape, and can be either macro- or micro-porous, or solid.
  • micro-carriers Both porous and solid types of micro-particulate carriers are commercially available from suppliers. Examples of commercially available micro-carriers include Cytodex 1® and Cytodex 3® (GE Healthcare Life Sciences, NJ), which are both dextran-based micro- carriers from GE Healthcare Life Sciences. Porous micro-carriers on the market include Cytoline as well as Cytopore products also from GE Healthcare Life Sciences. Biosilon (NUNC) and Cultispher (Percell Biolytica) are also commercially available. In a further aspect, the micro-carriers can be comprised of, or coated with polycarbonate or mixed cellulose esters.
  • Micro-carriers suitable for use in the present invention can be comprised of natural or synthetically-derived materials. Examples include collagen-based micro-carriers, dextran-based micro-carriers, or cellulose-based micro-carriers, as well as glass, ceramics, polymers, or metals.
  • the micro-carrier can be protein-free or protein-coated, for example, with collagen.
  • the micro-carrier can be comprised of, or coated with, compounds that enhance binding of the cell to the micro-carrier and enhance release of the cell from the micro-carrier including, but not limited to, poly(monostearoylglyceride co-succinic acid), poly-D,L-lactide-co-glycolide, sodium hyaluronate, collagen, f ⁇ bronectin, laminin, elastin, lysine, n-isopropyl acrylamide, vitronectin.
  • compounds that enhance binding of the cell to the micro-carrier and enhance release of the cell from the micro-carrier including, but not limited to, poly(monostearoylglyceride co-succinic acid), poly-D,L-lactide-co-glycolide, sodium hyaluronate, collagen, f ⁇ bronectin, laminin, elastin, lysine, n-isopropyl acrylamide, vitronectin.
  • Micro-carrier culture is a technique, which makes possible the practical high yield culture of anchorage-dependent, cells, for example, human embryonic stem cells.
  • Micro-carriers have been specifically developed for the culture of cells, such as human embryonic stem cells, in culture volumes ranging from a few milliliters to greater than one thousand liters.
  • the micro-carrier is biologically inert and provides a strong but non-rigid substrate for stirred micro-carrier cultures.
  • the micro-carriers may be transparent, allowing microscopic examination of the attached cells.
  • Cytodex 3® GE Healthcare Life Sciences, NJ
  • the denatured collagen layer on Cytodex 3® (GE Healthcare Life Sciences, NJ) is susceptible to digestion by a variety of proteases, including trypsin and collagenase, and provides the ability to remove cells from the micro-carriers while maintaining maximum cell viability, function, and integrity.
  • Protein free micro -carriers can be used to culture human embryonic stem cells.
  • micro-carriers for use in manufacturing and laboratory or research use sold under the tradename HILLEX® are modified polystyrene beads with cationic trimethyl ammonium attached to the surface to provide a positively charged surface to the micro-carrier.
  • the bead diameter ranges from about 90 to about 200 microns in diameter.
  • Micro-carrier-based methods of cell culture provided many advantages including ease of downstream processing in many applications.
  • Micro-carriers are typically roughly spherical in shape, and can be either porous or solid.
  • the use of micro-carriers for cell attachment facilitates the use of stirred tank and related reactors for growth of anchorage- dependent cells.
  • the cells attach to the readily suspended micro-carriers.
  • the requirement for suspendability limits the physical parameters of the micro-carriers.
  • micro-carriers commonly have a mean diameter in the range of 50-2000 microns.
  • solid-type micro-carriers range from about 100 to about 250 microns whereas porous-type micro-carriers range from about 250 to about 2500 microns. These size ranges allow for selection of micro-carriers, which are large enough to accommodate many anchorage-dependent cells, while small enough to form suspensions with properties suitable for use in stirred reactors.
  • micro carriers Among the factors considered in using micro carriers and the like are: attachment efficiency, immunogenicity, biocompatibility, ability to biodegrade, time to reach confluence, the growth parameters of attached cells including maximum attainable density per unit surface area, detachment techniques where required, and the efficiency of the detachment, scalability of the culture conditions as well as homogeneity of the culture under scaled-up conditions, the ability to successfully scale-up detachment procedures, and whether the micro-carriers will be used for implantation. These considerations can be influenced by the surface properties of the micro-carrier, as well as by the porosity, diameter, density, and handling properties of the micro-carrier.
  • the density of the micro-carriers is a consideration. Excessive density may cause the micro-carriers to settle out of the suspension, or tend to remain completely towards the bottom of the culture vessel, and thus may result in poor bulk mixing of the cells, culture medium and gaseous phases in the reactor. On the other hand, a density that is too low may result in excessive floating of the micro-carrier. A density of 1.02 to 1.15 g/cm is typical of many micro-carriers.
  • micro-carriers and the volume of particles that can be added to a reactor allows the micro-carriers to contribute substantial surface area in vast excess to that found in roller bottles or other methods of growing anchorage-dependent cells, e.g. on plates.
  • Porous micro-carriers provide even greater surface area per unit volume or weight. These porous micro-carriers possess large cavities that are available for the growth of anchorage-dependent cells. These cavities increase the surface area greatly, and may protect cells from detrimental mechanical effects, such as shear stress, for example from mixing or from gas sparging.
  • the micro-carrier surface may be textured to enhance cell attachment and proliferation.
  • the micro-carrier surface texture be achieved by techniques including, but not limited to, molding, casting, leeching and etching.
  • the resolution of the features of the textured surface may be on the nanoscale.
  • the textured surface may be used to induce a specific cell alignment on the micro-carrier surface.
  • the surface of the pores within the porous micro-carriers may also be textured to enhance cell attachment and proliferation. Pore surface texture be achieved by techniques such as but not limited to molding, casting, leeching and etching.
  • the micro-carrier surface may be plasma-coated to impart a specific charge to micro- carrier surfaces. These charges may enhance cell attachment and proliferation.
  • the micro-carriers are composed of, or coated with, thermoresponsive polymers such as poly-N-isopropylacrylamide, or have electromechanical properties.
  • porous and solid types of microparticulate carriers are commercially available from suppliers.
  • solid micro-carriers include Cytodex 1® and Cytodex 3® (GE Healthcare Life Sciences, NJ), which are both dextran-based micro- carriers from GE Healthcare Life Sciences.
  • Porous micro-carriers on the market include Cytoline as well as Cytopore products also from GE Healthcare Life Sciences.
  • Biosilon (NUNC) and Cultispher (Percell Biolytica) are also commercially available.
  • the micro-carriers may also contain a bioactive agent.
  • the micro-carrier may also contain a bioactive agent that may regulate the growth or function of cells or the tissue milieu these factors may include but are not limited to fibroblast growth factors, erythropoietin, vascular endothelial cell growth factors, platelet derived growth factors, bone morphogenic proteins, transforming growth factors, tumor necrosis factors, epidermal growth factors, insulin-like growth factors. Complete factors, mimetics or active fragments thereof may be used.
  • the micro-carriers may be inoculated with a second cell type and co-cultured with the pluripotent stem cells.
  • the two (or more) cell types may be adherent to an individual micro-carrier in equal or un-equal proportions.
  • the two or more cell types can be inoculated onto the micro-carrier at the same time point or they may be inoculated at different times.
  • the micro-carriers can be treated in such a manner to preferentially adhere specific cell types onto specific regions of the micro-carrier.
  • the micro-carrier with adherent single or multiple cell types can be co-cultured in a culture vessel with a second cell type cultured in suspension.
  • Second cell types may include, for example, epithelial cells (e.g., cells of oral mucosa, gastrointestinal tract, nasal epithelium, respiratory tract epithelium, vaginal epithelium, corneal epithelium), bone marrow cells, adipocytes, stem cells, keratinocytes, melanocytes, dermal fibroblasts, keratinocytes, vascular endothelial cells (e.g., aortic endothelial cells, coronary artery endothelial cells, pulmonary artery endothelial cells, iliac artery endothelial cells, microvascular endothelial cells, umbilical artery endothelial cells, umbilical vein endothelial cells, and endothelial progenitors (e.g., CD34+, CD34+/CD117+ cells)), myoblasts, myocytes, hepatocytes, smooth muscle cells, striated muscle cells, stromal cells, and other
  • Pluripotent stem cells may express one or more of the stage-specific embryonic antigens (SSEA) 3 and 4, and markers detectable using antibodies designated Tra-1-60 and Tra-1- 81 (Thomson et al., Science 282:1145, 1998). Differentiation of pluripotent stem cells in vitro results in the loss of SSEA-4, Tra- 1-60, and Tra-1-81 expression (if present) and increased expression of SSEA-I .
  • SSEA stage-specific embryonic antigens
  • Undifferentiated pluripotent stem cells typically have alkaline phosphatase activity, which can be detected by fixing the cells with 4% paraformaldehyde and then developing with Vector Red as a substrate, as described by the manufacturer (Vector Laboratories, Burlingame Calif.) Undifferentiated pluripotent stem cells also typically express OCT4 and TERT, as detected by RT-PCR.
  • pluripotent stem cells Another desirable phenotype of propagated pluripotent stem cells is a potential to differentiate into cells of all three germinal layers: endoderm, mesoderm, and ectoderm tissues.
  • Pluripotency of stem cells can be confirmed, for example, by injecting cells into severe combined immunodef ⁇ cient (SCID) mice, fixing the teratomas that form using 4% paraformaldehyde, and then examining them histologically for evidence of cell types from the three germ layers.
  • SCID severe combined immunodef ⁇ cient
  • pluripotency may be determined by the creation of embryoid bodies and assessing the embryoid bodies for the presence of markers associated with the three germinal layers.
  • Propagated pluripotent stem cell lines may be karyotyped using a standard G-banding technique and compared to published karyotypes of the corresponding primate species. It is desirable to obtain cells that have a "normal karyotype,” which means that the cells are euploid, wherein all human chromosomes are present and not noticeably altered.
  • pluripotent stem cells include established lines of pluripotent cells derived from tissue formed after gestation, including pre-embryonic tissue (such as, for example, a blastocyst), embryonic tissue, or fetal tissue taken any time during gestation, typically but not necessarily before approximately 10-12 weeks gestation.
  • pre-embryonic tissue such as, for example, a blastocyst
  • embryonic tissue or fetal tissue taken any time during gestation, typically but not necessarily before approximately 10-12 weeks gestation.
  • Non- limiting examples are established lines of human embryonic stem cells or human embryonic germ cells, such as, for example the human embryonic stem cell lines Hl, H7, and H9 (WiCeIl).
  • Hl, H7, and H9 WiCeIl
  • Also contemplated is use of the compositions of this disclosure during the initial establishment or stabilization of such cells, in which case the source cells would be primary pluripotent cells taken directly from the source tissues.
  • pluripotent stem cell population already cultured in the absence of feeder cells.
  • mutant human embryonic stem cell lines such as, for example, BGOIv (BresaGen, Athens, GA).
  • pluripotent stem cells derived from non-pluripotent cells such as, for example, an adult somatic cells. Attaching Pluripotent Stem Cells to the Micro-Carriers Suitable for Use in the Present
  • Pluripotent stem cells may be cultured on a planar substrate by any method in the art, prior to attaching to micro-carriers.
  • pluripotent stem cells may be cultured on planar substrates, treated with an extracellular matrix protein (e.g. MATRIGEL).
  • pluripotent stem cells may be cultured on planar substrates seeded with a feeder cell layer.
  • the pluripotent stem cells are embryonic stem cells. In an alternate embodiment, the embryonic stem cells are human.
  • pluripotent stem cells are released from a planar substrate by treating the pluripotent stem cells with a protease that will release the cells from the planar substrate.
  • the protease may be, for example, collagenase, TrypLETM Express , AccutaseTM, trypsin, and the like.
  • the pluripotent stem cells are released from the micro-carrier substrate by treating the cells with AccutaseTM for about five to about ten minutes.
  • the pluripotent stem cells are released from the micro-carrier substrate by treating the cells with 0.05% trypsin/EDTA for about ten to about twenty minutes.
  • the pluripotent stem cells are released from the micro-carrier substrate by treating the cells with TrypLETM Express for about five to about twenty minutes.
  • the pluripotent stem cells are released from the micro-carrier substrate by treating the cells with 10 mg/ml Collagenase for about five to about ten minutes.
  • the released pluripotent cells are added to medium containing micro-carriers at a specific density.
  • the pluripotent stem cells were seeded at about 4,000 to about 30,000 cells per cm 2 of micro-carriers.
  • the released pluripotent cells are added to medium containing micro-carriers.
  • the attachment of the pluripotent stem cells is enhanced by treating the pluripotent stem cells with a Rho kinase inhibitor.
  • the Rho kinase inhibitor may be Y27632 (Sigma-Aldrich, MO).
  • the Rho kinase inhibitor is Glycyl-H 1152 dihydrochloride.
  • the pluripotent stem cells are treated with Y27632 at a concentration from about 1 ⁇ M to about 10 ⁇ M. In one embodiment, the pluripotent stem cells are treated with Y27632 at a concentration of about 10 ⁇ M.
  • the pluripotent stem cells are treated with Glycyl-H 1152 dihydrochloride at a concentration from about 0.25 ⁇ M to about 5 ⁇ M. In one embodiment, the pluripotent stem cells are treated with Glycyl-H 1152 dihydrochloride at a concentration of about 2.5 ⁇ M.
  • the medium containing the micro-carriers may be agitated. Agitation as used in the present invention may be the movement of the culture medium. Such agitation may be achieved manually, or, alternatively, by use of apparatus, such as, for example, a rocking platform, a spinner flask, and the like. In one embodiment, the medium containing the micro-carriers is agitated by the use of manual movement. The dish containing the micro-carriers and cells is moved back and forth for less than 30 seconds.
  • the medium containing the micro-carriers may be agitated.
  • the medium containing the micro-carriers is agitated by the use of a spinner flask.
  • the spinner flask (Corning, Lowell, MA) is placed on a stir plate at 30-70 RPM depending on bead type.
  • the medium containing the micro-carriers is agitated by the use of a rocking platform (Vari-mix, Barnstead, Dubuque, Iowa).
  • the rocking platform speed is about one rotation in 2 seconds.
  • the pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage on micro-carriers.
  • the pluripotent stem cells may be differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage on micro-carriers.
  • the pluripotent stem cells may be differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage on micro-carriers.
  • the pluripotent stem cells may be propagated on micro- carriers, then differentiated into cells expressing markers characteristic of the definitive endoderm lineage on planar surfaces.
  • the pluripotent stem cells may be propagated on micro-carriers, then differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage on planar surfaces.
  • the pluripotent stem cells may be propagated on micro-carriers, then differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage on planar surfaces.
  • Pluripotent stem cells treated in accordance with the methods of the present invention may be differentiated into a variety of other cell types by any suitable method in the art.
  • pluripotent stem cells treated in accordance with the methods of the present invention may be differentiated into neural cells, cardiac cells, hepatocytes, and the like.
  • pluripotent stem cells treated in accordance with the methods of the present invention may be differentiated into neural progenitors and cardiomyocytes according to the methods disclosed in WO2007030870.
  • pluripotent stem cells treated in accordance with the methods of the present invention may be differentiated into hepatocytes according to the methods disclosed in US patent 6,458,589.
  • Pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by any method in the art.
  • pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage according to the methods disclosed in D'Amour et al, Nature Biotechnology 23, 1534 - 1541 (2005).
  • pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage according to the methods disclosed in Shinozaki et al, Development 131, 1651 - 1662 (2004).
  • pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage according to the methods disclosed in McLean et al, Stem Cells 25, 29 - 38 (2007).
  • pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage according to the methods disclosed in D'Amour et al, Nature Biotechnology 24, 1392 - 1401 (2006).
  • Markers characteristic of the definitive endoderm lineage are selected from the group consisting of SOXl 7, GATA4, HNF-3 beta, GSC, CERl, Nodal, FGF8, Brachyury, Mix- like homeobox protein, FGF4, CD48, eomesodermin (EOMES), DKK4, FGF 17, GAT A6, CXCR4, C-Kit, CD99, and OTX2.
  • Suitable for use in the present invention is a cell that expresses at least one of the markers characteristic of the definitive endoderm lineage.
  • a cell expressing markers characteristic of the definitive endoderm lineage is a primitive streak precursor cell.
  • a cell expressing markers characteristic of the definitive endoderm lineage is a mesendoderm cell.
  • a cell expressing markers characteristic of the definitive endoderm lineage is a definitive endoderm cell.
  • pluripotent stem cells treated according to the methods of the present invention may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by culturing the pluripotent stem cells in medium containing activin A in the absence of serum, then culturing the cells with activin A and serum, and then culturing the cells with activin A and serum of a different concentration.
  • An example of this method is disclosed in Nature Biotechnology 23, 1534 - 1541 (2005).
  • pluripotent stem cells treated according to the methods of the present invention may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by culturing the pluripotent stem cells in medium containing activin A in the absence of serum, then culturing the cells with activin A with serum of another concentration.
  • An example of this method is disclosed in D' Amour et al, Nature Biotechnology, 2005.
  • pluripotent stem cells treated according to the methods of the present invention may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by culturing the pluripotent stem cells in medium containing activin A and a Wnt ligand in the absence of serum, then removing the Wnt ligand and culturing the cells with activin A with serum.
  • An example of this method is disclosed in Nature Biotechnology 24, 1392 - 1401 (2006).
  • pluripotent stem cells treated according to the methods of the present invention may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage according to the methods disclosed in US patent application Ser. No. 11/736,908, assigned to LifeScan, Inc.
  • pluripotent stem cells treated according to the methods of the present invention may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage according to the methods disclosed in US patent application Ser. No. 11/779,311, assigned to LifeScan, Inc.
  • Pluripotent stem cells may be differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage by any method in the art.
  • pluripotent stem cells may be differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage according to the methods disclosed in D'Amour et al, Nature Biotechnology 24, 1392 - 1401 (2006).
  • cells expressing markers characteristic of the definitive endoderm lineage obtained according to the methods of the present invention are further differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage, by treating the cells expressing markers characteristic of the definitive endoderm lineage with a fibroblast growth factor and the hedgehog signaling pathway inhibitor KAAD- cyclopamine, then removing the medium containing the fibroblast growth factor and KAAD-cyclopamine and subsequently culturing the cells in medium containing retinoic acid, a fibroblast growth factor and KAAD-cyclopamine.
  • An example of this method is disclosed in Nature Biotechnology 24, 1392 - 1401 (2006).
  • cells expressing markers characteristic of the definitive endoderm lineage obtained according to the methods of the present invention are further differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage, by treating the cells expressing markers characteristic of the definitive endoderm lineage with retinoic acid one fibroblast growth factor for a period of time, according to the methods disclosed in US patent application Ser. No. 11/736,908, assigned to LifeScan, Inc.
  • cells expressing markers characteristic of the definitive endoderm lineage obtained according to the methods of the present invention are further differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage, by treating the cells expressing markers characteristic of the definitive endoderm lineage with retinoic acid (Sigma- Aldrich, MO) and exendin 4, then removing the medium containing DAPT (Sigma- Aldrich, MO) and exendin 4 and subsequently culturing the cells in medium containing exendin 1, IGF-I and HGF.
  • retinoic acid Sigma- Aldrich, MO
  • DAPT Sigma- Aldrich, MO
  • cells expressing markers characteristic of the pancreatic endoderm lineage obtained according to the methods of the present invention are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by culturing the cells expressing markers characteristic of the pancreatic endoderm lineage in medium containing exendin 4, then removing the medium containing exendin 4 and subsequently culturing the cells in medium containing exendin 1, IGF-I and HGF.
  • An example of this method is disclosed in D' Amour et a ⁇ , Nature Biotechnology, 2006.
  • cells expressing markers characteristic of the pancreatic endoderm lineage obtained according to the methods of the present invention are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by culturing the cells expressing markers characteristic of the pancreatic endoderm lineage in medium containing DAPT (Sigma- Aldrich, MO) and exendin 4.
  • DAPT Sigma- Aldrich, MO
  • exendin 4 An example of this method is disclosed in D' Amour et al, Nature Biotechnology, 2006.
  • cells expressing markers characteristic of the pancreatic endoderm lineage obtained according to the methods of the present invention are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by culturing the cells expressing markers characteristic of the pancreatic endoderm lineage in medium containing exendin 4.
  • An example of this method is disclosed in D' Amour et al, Nature Biotechnology, 2006.
  • cells expressing markers characteristic of the pancreatic endoderm lineage obtained according to the methods of the present invention are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by treating the cells expressing markers characteristic of the pancreatic endoderm lineage with a factor that inhibits the Notch signaling pathway, according to the methods disclosed in US patent application Ser. No. 11/736,908, assigned to LifeScan, Inc.
  • cells expressing markers characteristic of the pancreatic endoderm lineage obtained according to the methods of the present invention are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by treating the cells expressing markers characteristic of the pancreatic endoderm lineage with a factor that inhibits the Notch signaling pathway, according to the methods disclosed in US patent application Ser. No. 11/779,311, assigned to LifeScan, Inc.
  • cells expressing markers characteristic of the pancreatic endoderm lineage obtained according to the methods of the present invention are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by treating the cells expressing markers characteristic of the pancreatic endoderm lineage with a factor that inhibits the Notch signaling pathway, according to the methods disclosed in US patent application Ser. No. 11/736,908, assigned to LifeScan, Inc.
  • cells expressing markers characteristic of the pancreatic endoderm lineage obtained according to the methods of the present invention are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by treating the cells expressing markers characteristic of the pancreatic endoderm lineage with a factor that inhibits the Notch signaling pathway, according to the methods disclosed in US patent application Ser. No. 11/779,311, assigned to LifeScan, Inc.
  • a pancreatic endocrine cell is capable of expressing at least one of the following hormones: insulin, glucagon, somatostatin, and pancreatic polypeptide.
  • Suitable for use in the present invention is a cell that expresses at least one of the markers characteristic of the pancreatic endocrine lineage.
  • a cell expressing markers characteristic of the pancreatic endocrine lineage is a pancreatic endocrine cell.
  • the pancreatic endocrine cell may be a pancreatic hormone-expressing cell.
  • the pancreatic endocrine cell may be a pancreatic hormone-secreting cell.
  • the pancreatic endocrine cell is a cell expressing markers characteristic of the ⁇ cell lineage.
  • a cell expressing markers characteristic of the ⁇ cell lineage expresses PDXl and at least one of the following transcription factors: NGN3, NKX2.2, NKX6.1, NEUROD, ISLl, HNF-3 beta, MAFA, PAX4, or PAX6.
  • a cell expressing markers characteristic of the ⁇ cell lineage is a ⁇ cell. The present invention is further illustrated, but not limited by, the following examples.
  • H9 cells passage 52 were released from MATRIGELTM (BD Biosciences, CA) coated plates with TrypLETM Express. They were then incubated with micro-carriers and MEF- CM. Suspensions of ProNectinF (PN), Plastic (P), PlasticPlus (PP), HILLEX®II (H), collagen (Col) and FACT III (SoIoHiIl, MI) micro-carriers were prepared according to manufacturer's instructions. After 2 days at 37°C, Table 1 describes the attachment and growth of the H9 cells on the micro-carriers based on daily images. Few cells attached and/or proliferated on most micro-carriers tested. H9 cells did attach and proliferate on HILLEX®II micro-carriers (Solohill, MI) but images showed fewer cell-bead aggregates after 2 days in static culture ( Figure IB).
  • Rho-associated coiled coil forming protein serine/threonine kinase Rho kinase inhibitor was added to the media.
  • Y27632, Y (Sigma- Aldrich, MO) was used.
  • MEF-CM plus 10 ⁇ M Y27632 (Sigma- Aldrich, MO) was changed daily.
  • 10 ⁇ M Y27632 (Sigma-Aldrich, MO) the H9 cells attached and formed aggregates with all micro-carriers tested (Table 2).
  • HILLEX®II micro- carriers Solohill, MI
  • H9 cells attached better to HILLEX®II (Solohill, MI) in the presence of the Rho kinase inhibitor ( Figure IA compared to IB).
  • Expansion of human embryonic stem cells for a cell therapy application is necessary to meet product demand.
  • the best techniques for expansion include spinner flasks and bioreactors. Both of these techniques require physical movement of the micro- carriers in suspension.
  • 6 or 12 well dishes were placed on a rocking platform in a 37° C incubator. After growth for 3 days, the cell aggregates began to release from some of the micro-carriers.
  • Figure 2 A, B, D illustrates that the cell aggregates disassociated from the Plastic Plus, Plastic, or Pronectin micro-carriers.
  • Example 4 describes the dissociation method used prior to cell counting in a Guava PCA-96 with Viacount Flex (Guava Technologies, Hayward, CA). Measuring the growth rate of the cells on micro-carriers reveals a dip in cell number at day 3 compared to the starting number at seeding. This is likely due to poor initial attachment of the cells to the micro-carriers followed by an expansion afterwards until the experiment was terminated at day 5.
  • H9 cells on HILLEX®II micro-carriers have the highest proliferation rate compared to the other bead types, likely due to better attachment of the cells to the HILLEX® II micro-carriers (Solohill, MI) ( Figures 2, 3).
  • the Hl human embryonic cell line was also tested for growth on micro-carriers for large- scale expansion. Because a Rho kinase inhibitor, Y27632 (Sigma-Aldrich, MO), was necessary for attachment of the H9 cell line, it was also assumed to be necessary for Hl cells. Cytodex 1®, Cytodex 3® (GE Healthcare Life Sciences, NJ), HILLEX®II, Plastic, ProNectinF, Plastic Plus micro-carriers (SoIoHiIl Ann Arbor, MI) were prepared according to the manufacturer's instructions.
  • the Hl human embryonic stem cells at passage 47 were seeded at about 13,333 cells/cm 2 of micro-carriers in MEF-CM plus lO ⁇ M Y27632 (Sigma-Aldrich, MO). Cells and micro-carriers were placed in a 12 well non-tissue culture treated dish at 15cm 2 per 12 well on a rocking platform at 37°C to allow movement of the micro-carriers and medium. After 3, 5 and 7 days, one well was imaged, harvested, and counted. The ability of cells to attach depended on the bead type. Similar results were observed with the Hl line as with the H9 line.
  • Example 2 Optimal Concentrations of Y27632 and Other Rho Kinase Inhibitors for Cell Attachment and Growth
  • Rho kinase inhibitor that best supports attachment and growth of the human embryonic stem cells on micro-carriers.
  • a starting aliquot of 13,333 cells/cm 2 H9 cells at passage 44 was seeded onto 15cm 2 of micro-carriers in a single well of a 12 well non-tissue culture treated plate.
  • the cells were placed at 37 0 C for at least 60 minutes before placing them onto a rocking platform at 37 0 C.
  • HILLEX®II Solohill, MI
  • Cytodex 3® GE Healthcare Life Sciences, NJ
  • Hl cells at passage 48 were dissociated from MATRIGELTM (BD Biosciences, CA) coated plates with TrypLETM Express. The cells were then seeded onto 15 cm 2 of micro-carriers into a single well of a 12 well non-tissue culture treated plate.
  • HILLEX®II Solohill, MI
  • Cytodex 1® Cytodex 3®
  • Rho kinase inhibitor 10 ⁇ M Y27632 (Sigma- Aldrich, MO) was used on day one followed by 0.5 ⁇ M on day two (Y 10/5 ⁇ M); 2.5 ⁇ M Glycyl-H 1152 dihydrochloride (Tocris, MO) was used on day one followed by 0.5 ⁇ M on day two (H2.5/0.5 ⁇ M); 1 ⁇ M Glycyl-H 1152 dihydrochloride (Tocris, MO) was used on day one followed by 0.5 ⁇ M Glycyl-H 1152 dihydrochloride (Tocris, MO) on day two (Hl/0.5 ⁇ M); or continuous addition of 0.25 ⁇ M Glycyl-H 1152 dihydrochloride (Tocris, MO) was applied daily in MEF-
  • Rho-kinase inhibitor maintains the cells in 0.25 ⁇ M Glycyl-H 1152 dihydrochloride (Tocris, MO) resulted in poor cell yield. Using a minimal amount of Rho-kinase inhibitor also helps reduce costs for the process and maybe beneficial to cell proliferation. These data also show that human embryonic stem cells did not require Rho kinase inhibitor in order to remain attached to micro-carriers and to proliferate.
  • Improving the seeding density is a method to reduce the total number of cells needed.
  • the number of micro-carriers per 4x objective field was counted. Hl cells were seeded at 0.4xl0 4 cells/cm 2 (low), 1.2 xlO 4 cells/cm 2 (mid), or 3 xlO 4 cells/cm 2 (high) densities into a 10 cm plate with Cytodex 3 micro-carriers (GE Healthcare Life Sciences, NJ) in MEF-CM plus lO ⁇ M Y27632 (Sigma-Aldrich, MO). The plate was then agitated every 45 minutes for 6 hours at 37 0 C.
  • the cells and micro- carriers were transferred to a spinner flask (described in Example 5) at 37 0 C at 30 rpm in 50ml MEF-CM plus 10 ⁇ M Y27632 (Sigma- Aldrich, MO). After 24 hours 25ml of MEF- CM with 5 ⁇ M Y27632 (Sigma-Aldrich, MO) was added. After 24 hours the speed of rotation was increased to 40 rpm. On day 3 and 5 of culture, 50ml of 75ml was removed and replaced with MEF-CM. Images were taken of an aliquot from the spinner flasks at 6 hours, 3 days, 5 days and 7days. The percentage of micro-carriers with cells attached is stated in the lower right corner in Figure 9 images.
  • the number of micro-carriers coated with cells corresponds to the original seeding density but at days 5 and 7 the number of micro-carriers coated with cells did not increase for the lower density seeded cells. This suggests that 0.4 xlO 4 cells/cm 2 is not a sufficient number of cells to allow incorporation of micro-carriers into the aggregates.
  • the number of micro-carriers with cells attached is similar to 1.2 xl O 4 cells/cm 2 seeded at days 5 and 7 ( Figure 9). When looking at the cell number, it is clear that more cells are attached to micro-carriers from high density cell seeding ( Figure 10).
  • H9 cells on HILLEXd)II micro-carriers were imaged at 1Ox and 2Ox magnification before dissociation of the cells from the micro-carriers ( Figure 13 A, B respectively).
  • Enzymatic treatment of the H9 cells on HILLEXd)II micro-carriers allowed for detachment of viable cells ( Figure 13 C, D and 14).
  • the H9 cells were grown for 6 days in a 6 well dish with HILLEXd)II micro-carriers (Solohill, MI) on a rocking platform at 37 0 C.
  • the cells attached to micro-carriers were placed in a 15ml conical tube and the medium was aspirated after allowing the micro-carriers to settle.
  • the settled micro-carriers were washed three times with 4 ml PBS (without magnesium and calcium ions) allowing the micro-carriers to settle by gravity sedimentation.
  • the PBS was aspirated and ImI of PBS was added.
  • the micro-carriers with cells were transferred into a single well of a 12 well non-tissue culture treated plate.
  • the plate was allowed to rest at an angle to allow the micro-carriers to settle.
  • the PBS was aspirated and 1 ml TrypLETM Express (Invitrogen, CA) or 0.05% Trypsin/EDTA was added to the well.
  • the plate was placed at 37 0 C on the rocking platform for 10 or 20 minutes.
  • the plate was removed and 3ml DMEM/F12 or MEF-CM was added to the well.
  • the medium was vigorously pipetted, releasing the cells ( Figure 13 C, D). Observation of the micro-carriers under a microscope determined detachment of the cells from the micro-carriers. The cells were then centrifuged at 200 x g for 5 minutes.
  • the medium was aspirated and the pellet was resuspended in ImI DMEM/F12 or MEF-CM medium.
  • the cells were then counted on a Guava PCA-96 (Guava Technologies, Hayward, CA) with Viacount dye. Specifically, a 200 ⁇ l volume of cells in appropriate dilution of medium, was incubated with 2 ⁇ l of Viacount for 10 minutes. The viability and cell number were determined ( Figure 14). Both TrypLETM Express and Trypsin/EDTA dissociated the cells effectively from micro-carriers.
  • TrypLETM Express released the cells from the micro-carriers and is available as a GMP product, it was tested against other possible dissociation agents, specifically Collagenase and AccutaseTM (Sigma-Aldrich, MO). Hl p48 cells were grown in a spinner flask (Example 5) for 10 days. The micro-carriers were then collected and transferred to a 50ml conical tube. The cells were washed in PBS as above and transferred to a 12 well plate. PBS was aspirated and ImI of TrypLETM Express, AccutaseTM or Collagenase (lOmg/ml) was added to the well and placed on a rocking platform at 37 0 C for 5 or 10 minutes.
  • the cells/micro-carriers were vigorously resuspended in DMEM/F12, and then the dissociated cells and micro-carriers were passed through a 40 ⁇ m cell strainer over a 50ml conical tube. The well was washed with an additional 2 ml medium, also added to the strainer before centrifuging at 200 x g for 5 minutes. The cells were then resuspended in ImI DMEM/F12 and diluted for cell counting, as above. Cell viability was similar with all tested enzymes. AccutaseTM and TrypLETM Express released similar cell numbers over 5 and 10 minute incubations ( Figure 15). This illustrates the suitability of AccutaseTM and TrypLETM Express as cell dissociation regents for human embryonic stem cells on micro-carriers.
  • H9 p43 cells were seeded onto HILLEXd)II micro-carriers (Solohill, MI) and incubated in a 125ml spinner flask (see below). Phenol red present in the medium and was taken up by the HILLEXd)II micro- carriers (Solohill, MI).
  • micro-carriers After 8 days of growth, a 10ml aliquot of the cells on micro- carriers was placed in a new spinner flask containing phenol red-free MEF-CM, 440mg OfHILLEXd ) II micro-carriers, and 5 ⁇ M Y27632 (Sigma-Aldrich, MO). After 5 days incubation at 37°C with 30rpm rotation, the micro-carriers were removed and images were acquired ( Figure 16). The dark micro-carriers shown are the micro-carriers covered with H9 cells grown in medium containing phenol red. The light micro-carriers are the newly added micro-carriers.
  • the H9 cells would detach and reattach to new micro-carriers, however, instead the cells formed aggregates with the new micro-carriers. No light micro-carriers had cells attached that are not also in aggregates with the dark micro-carriers, suggesting that the cells were not able to detach and reattach to micro-carriers. In order to propagate cells grown on micro-carriers, the cells must be enzymatically dissociated from the micro-carriers (see Example 4).
  • H9 passage 43 cells were seeded into 125ml spinner flasks. Cells were initially attached to the micro-carriers in a 10cm plate before transferring to the spinner flask. Specifically, H9 cells were released from the two six-well dishes by a five minute incubation with TrypLETM Express at 37°C.
  • the dish was placed at 37°C and gently rotated and agitated once every 45 minutes for 4.5 hours. Then the cells, micro- carriers and medium were transferred to a 125ml spinner flask. The spinner flask was then filled to 50ml with MEF-CM plus 10 ⁇ M Y27632 (Sigma-Aldrich, MO) and placed on a stir plate at 37°C at 40rpm. The following day the medium was changed and filled to 75ml with MEF-CM plus 5 ⁇ M Y27632 (Sigma-Aldrich, MO). The rate of stirring was increased to 70rpm. Medium was changed every other day without addition of Y27632 compound (Sigma-Aldrich, MO).
  • the cells were passaged according to the methods disclosed in Example 4, and 3xlO 6 cells were reseeded onto 250cm 2 of new micro- carriers. The cultures were passaged when they reached a confluency of 1-2x10 5 cells/cm 2 . This was conducted for 5 passages ( Figure 17). At each passage pluripotent marker expression was evaluated showing 80-95% of cells expressed the pluripotency markers CD9, SSEA4, SSEA3, TRA-1-60 and TRA-1-81 ( Figure 19A). A similar experiment was conducted with H9 p43 cells on Cytodex 3® micro-carriers ((GE Healthcare Life Sciences, NJ), Figure 18, 19B).
  • Cytodex 1® and Cytodex 3® over HILLEX®II micro-carriers is their larger surface area.
  • Figures 20 and 21 show the expansion of Hl cells on Cytodex 1® and Cytodex 3® respectively. The cells remained pluripotent over the five passages ( Figure 22).
  • Karyotype analysis of Hl cells on Cytodex 3® micro-carriers revealed duplication of the Y chromosome in 10% of the cells tested. These Hl cells were passaged onto micro- carriers at p48 and were analyzed 5 passages later.
  • Hlp55 cells grown on MATRI GELTM (BD Biosciences, CA) on a planar surface had a normal karyotype.
  • Example 6 Proliferation of Human Embryonic Stem Cells on Micro-Carriers in
  • human embryonic stem cells are maintained on MATRIGELTM (BD Biosciences, CA) in medium conditioned using mouse embryonic fibroblasts (MEF-CM). Both MATRIGELTM (BD Biosciences, CA) and MEF-CM are derived from mouse cells. Additionally, MEF-CM is an expensive and time-consuming medium to generate.
  • H9 cells were seeded onto Cytodex 3® (GE Healthcare Life Sciences, NJ) and HILLEX®II (Solohill, MI) micro-carriers in the presence of Rho kinase inhibitors, lO ⁇ M Y27632 (Sigma-Aldrich, MO) or 2.5 ⁇ M Glycyl-H 1152 dihydrochloride (Tocris, MO) in Stem Pro (Invitrogen, CA), mTESR (StemCell Technologies, Vancouver, Canada) or MEF-CM. The cells were placed in a 12 well dish on a rocking platform at 37 0 C.
  • H9 p39 cells grown in MEF-CM on both bead types showed typical expansion characteristics ( Figure 24). Similar cells grown in mTESR (StemCell Technologies, Vancouver, Canada) proliferated well on Cytodex 3® micro-carriers in the presence of 10 ⁇ M Y27632 (Sigma-Aldrich, MO) but exhibited a slow growth rate on HILLEX®II micro-carriers.
  • H9p64 Human embryonic stem cell line H9 at passage 64
  • HILLEX®II StemPro medium
  • Cytodex 3® Cells of the human embryonic stem cell line H9 at passage 64 (H9p64) cells that had been acclimated to StemPro medium for over 20 passages proliferated well on both HILLEX®II and Cytodex 3® in the presence of 10 ⁇ M Y27632 (Sigma-Aldrich, MO). Surprisingly, these cells did not proliferate well in the presence of 2.5 ⁇ M Glycyl- H 1152 dihydrochloride (Tocris, MO) on Cytodex 3® micro-carriers. Therefore the micro-carrier type, Rho kinase inhibitor, and medium all play a role in determining the ability of human embryonic stem cells to proliferate.
  • Hl human embryonic stem cells at passage 38 were seeded onto either Cytodex 3® or HILLEX®II micro-carriers in the presence of Rho kinase inhibitors, lO ⁇ M Y27632 (Sigma-Aldrich, MO) or 2.5 ⁇ M Glycyl-H 1152 dihydrochloride in mTESR (StemCell Technologies, Vancouver, Canada) or MEF-CM in a 12 well dish.
  • the cells were placed on a rocking platform at 37 0 C. The cells were counted at days 3, 5 and 7.
  • Hl p50 cells proliferated well in mTESR (StemCell Technologies, Vancouver, Canada) on HILLEXd)II micro-carriers 3xlO 6 cells were seeded onto 250 cm 2 HILLEXd)II micro-carriers. Cells were incubated at 37 0 C in a 10 cm 2 dish for 5 hours with agitation by hand every 45 minutes.
  • the differentiation potential of these cells must be determined.
  • Cells of the human embryonic stem cell line H9 at passage 43 were passaged five times on Cytodex 3d) micro-carriers (GE Healthcare Life Sciences, NJ). At passage 5, the cells were grown for 6 days on the micro-carriers before being dissociated from the micro-carriers with TrypLETM Express (see Example 4). The cells were then plated on 1 :30 MATRIGELTM: DMEM/ F12 coated plates. After the cells became 80 to 90% confluent on the plates they were exposed to differentiating agents.
  • Differentiation of the human embryonic stem cells to definitive endoderm was conducted by treating the cells for 2 days with 2% Albumin Bovine Fraction V Fatty Acid Free (FAF BSA, MP Biomedicals, Ohio) in RPMI plus lOOng/ml Activin A (PeproTech, NJ), 20ng/ml Wnt3a (R&D Biosciences, MN) and 8ng/ml bFGF (PeproTech, NJ).
  • the cells were treated for an additional 2 days in 2% FAF BSA in RPMI plus 100ng/ml Activin A (PeproTech, NJ) and 8ng/ml bFGF (PeproTech, NJ).
  • Medium was changed daily.
  • micro-carriers Three types of micro-carriers, Cytodex 1®, Cytodex 3® (GE Healthcare Life Sciences, NJ) and HILLEX®II (Solohill, MI), allow attachment and growth of Hl cells. Differentiation of Hl cells on these three micro-carriers was conducted. The cells were grown on these micro-carriers in spinner flasks (Example 5) for various passage numbers (1 to 5). Six to eight days after the last passage aliquots of the micro-carriers plus cells in suspension were transferred to 6 or 12 well plates. A total of 15cm 2 of micro-carriers plus cells per 12 well plate well or 30cm 2 micro-carriers plus cells per 6 well plate was transferred.
  • Differentiation medium was then added to the plate wells and the plate was placed on a rocking platform at 37°C.
  • Differentiation of the human embryonic stem cells to definitive endoderm was conducted by treating the cells for 2 days with 2% Albumin Bovine Fraction V Fatty Acid Free (MP Biomedicals, Ohio) in RPMI plus 100ng/ml Activin A (PeproTech, NJ), 20ng/ml Wnt3a (R&D Biosciences, MN) and 8ng/ml bFGF (PeproTech, NJ).
  • the cells were treated for an additional 2 days in 2% FAF BSA in RPMI plus lOOng/ml Activin A (PeproTech, NJ) and 8ng/ml bFGF (PeproTech, NJ). Medium was changed daily. FACS analysis was conducted for the definitive endoderm cell surface marker CXCR4 ( Figure 28). Cells grown on Cytodex 1®, and Cytodex 3® micro-carriers supported differentiation to definitive endoderm (87% and 92% respectively while HILLEX®II micro-carriers did not support differentiation as to the same extent as the other micro-carriers tested in this experiment (42%).
  • cells of the human embryonic stem cell line Hl at passage 40 were grown on Cytodex 3® micro-carriers in a spinner flask for either 8 days or 11 days. Then the equivalent of about 15 cm 2 of micro-carriers plus cells was placed in a 6 well dish and placed on a rocking platform. The cells were then incubated in definitive endoderm differentiating medium as above. After 4 days the cells were analyzed by FACS for CXCR4 expression. 87% of the cells grown for 6 days in spinner flask expressed CXCR4 while 56% of cells grown for 11 days in the spinner flask expressed CXCR4 ( Figure 29). This demonstrates that the number of days that the cells are in culture is important prior to differentiation, specifically, if the cell density is too high it may not allow the cells to efficiently differentiate.
  • human embryonic stem cells were differentiated into pancreatic endoderm cells on all three micro-carrier types determined sufficient for attachment and growth.
  • cells of the human embryonic stem cell line Hl at passage 41 (Hlp41) were seeded on to Cytodex 1®, Cytodex 3® micro-carriers (GE Healthcare Life Sciences, NJ) and HILLEX®II micro-carriers (Solohill, MI) (see Example 1).
  • Micro-carriers were prepared according to the manufactures instructions. 30cm 2 of micro-carriers were transferred to low attachment 6 well plates. The Hl cells were dissociated from two 10cm 2 plates with TrypLETM Express according to manufacturer's instructions. Cell were seeded at 5x10 5 cells per well.
  • Attachment of the cells to the beads was carried out according to the methods described in Example 3. Briefly, the cells and micro-carriers were incubated in MEF conditioned media with lO ⁇ M Y27632 at 37 0 C for four hours with brief agitation each hour. The cells on HILLEX®II and Cytodex 1® micro-carriers were placed on a rocking platform. The cells on Cytodex 3® micro-carriers were allowed to sit undisturbed overnight. The media was changed daily and no longer included Y27632.
  • the majority of cells in the Cytodex 1® plate were no longer attached to the micro-carriers. However, longer attachment time and/or slower rocking speed may improve the cell attachment.
  • the cells were differentiated to definitive endoderm with 2% fatty acid free (FAF) BSA (Proliant, IA) in RPMI and the following growth factors: bFGF (8ng/ml, (PeproTech, NJ)), Activin A (lOOng/ml, (PeproTech, NJ)), Wnt3a (20ng/ml, (R&D Biosciences, MN)).
  • FAF fatty acid free
  • FGF7 50ng/ml, (R&D Systems, MN)
  • KAAD-Cyclopamine (0.25 ⁇ M, (Calbiochem, NJ)
  • FAF BSA Proliant, IA
  • the cells were then differentiated for three days with Noggin (100ng/ml, (R&D Biosciences, MN)), DAPT (1 ⁇ M, (Sigma-Aldrich, MO)), Alk5 inhibitor II (1 ⁇ M, (Axxora, CA)) in DMEM/F12 or DMEM-HG with 1% B-27 supplement (day 13, pancreatic endoderm, (Invitrogen, CA)).
  • Figure 31 shows the expression level by Q-PCR for the pancreatic specific genes, NKX6.1, PDXl and NGN3.
  • CT values clearly show that cells differentiated on HILLEXd)II micro-carriers do not differentiate efficiently to express the necessary beta cell precursor cell markers. Although the cells differentiated efficiently to definitive endoderm on all three micro- carrier types further differentiation to pancreatic progenitors is not efficient on HILLEXd)II micro-carriers.
  • Hl p45 cells were grown on Cytodex 3d) micro-carriers (GE Healthcare Life Sciences, NJ) and differentiated similar to above. Briefly, Hl cells were dissociated from 10cm 2 plates with TrypLETM Express according to manufacturer's instructions. Cells were seeded at 1 or 2x10 6 cells per 6 well plate well. Attachment of the cells to the beads is described in Example 3. Briefly, the cells and micro-carriers were incubated in MEF conditioned media with lO ⁇ M Y27632 at 37 0 C for four hours with brief agitation each hour. The cells were then allowed to incubate overnight undisturbed.
  • the media was replaced with MEF conditioned media plus 5uM Y27632 and the plates were placed on a rocking platform. The media was changed each subsequent day without Y27632.
  • the media was replaced with definitive endoderm differentiation media, 2% fatty acid free (FAF) BSA (Proliant, IA) in RPMI with the following growth factors: : bFGF (8ng/ml, (PeproTech, NJ)), Activin A (100ng/ml, (PeproTech, NJ)), Wnt3a (20ng/ml, (R&D Biosciences, MN)).
  • FGF fatty acid free
  • the cells were then differentiated further for 2 days with FGF7 (50ng/ml, (R&D Systems, MN)), KAAD-Cyclopamine (0.25 ⁇ M, (Calbiochem, NJ)) in DMEM-High Glucose (HG) plus 2% FAF BSA (Proliant, IA).
  • Hl p44 cells were grown in a spinner flask for 7 days on Cytodex 3® micro-carriers (see Example 5). The cells plus micro-carriers were transferred to a 12 well plate at 15cm 2 /well and placed on a rocking platform at 37 0 C. The cells were differentiated to definitive endoderm as above but with DMEM/F12 instead of RMPI. FACS analysis after 4 days revealed CXCR4 levels of 75 to 77% positive cells in a triplicate analysis.
  • the cells were then differentiated further with 3 days of treatment with FGF7 (50ng/ml, (R&D Systems, MN)), KAAD-Cyclopamine (0.25 ⁇ M, (Calbiochem, NJ)) in DMEM/F12 plus 2% Albumin Bovine Fraction V Fatty Acid Free.
  • FGF7 50ng/ml, (R&D Systems, MN)
  • KAAD-Cyclopamine (0.25 ⁇ M, (Calbiochem, NJ)
  • DMEM/F12 plus 2% Albumin Bovine Fraction V Fatty Acid Free.
  • the cells were then differentiated for three days with Noggin (lOOng/ml, (R&D Biosciences, MN)), Netrin4 (lOOng/ml, (R&D Biosciences, MN)), DAPT (1 ⁇ M, (Sigma-Aldrich, MO)), Alk5 inhibitor II (1 ⁇ M, (Axxora, CA)) in DMEM/F12 with 1% B-27 supplement (day 15, pancreatic endoderm, (Invitrogen, CA)). This was followed by six days of treatment with Alk5 inhibitor II (1 ⁇ M, (Axxora, CA)) in DMEM/F12 with 1% B-27 supplement (day 21, pancreatic endocrine cells, (Invitrogen, CA)).
  • FIG. 33 shows the expression level by Q-PCR for the pancreatic specific genes, insulin, Pdxl and glucagon.
  • the data on micro-carriers is compared to previous data of Hl p42 cells differentiated on a MATRIGELTM (BD Biosciences, CA) coated planar surface.
  • the expression level of these pancreas specific genes is similar or better for micro-carrier differentiated cells compared to cells differentiated on planar surfaces.
  • pancreatic endoderm day 15
  • pancreatic- endocrine cells day 22
  • insulin-expressing cells day29
  • the medium components were identical to those listed for the above Hl differentiation experiment with one additional day in the endocrine cell differentiating components.
  • the insulin-expressing stage cells showed a surprising decrease in insulin expression compared to day 22. Since the decrease was noted in both micro-carrier and planar samples, it is likely not due to the attachment substrate. This shows that H9 cells can also be successfully differentiated to at least pancreatic endocrine cells on micro-carriers.
  • Example 8 Human embryonic stem cells passaged as single cells in a 3D micro- carrier based culture can be transferred to culture on an ECM free surface while maintaining pluripotency
  • Hl human embryonic stem cells were cultured on micro-carriers according to the methods described in Example 5.
  • Cells were removed from micro-carriers and plated to Nunc4, Nuncl3, CELLBINDTM, or PRIMARIATM tissue culture polystyrene (TCPS) planar surfaces with MEFCM 16 supplemented with 3 ⁇ M Glycyl-H 1152 dihydrochloride.
  • the cells were seeded at a density of 100,000 cells/cm 2 in six well plates and then cultured for one additional passage on the respective surface. Cells were then either lifted with TrypLE and tested by flow cytometry for pluripotency markers, or lysed in the well with RLT for mRNA purification and qRT-PCR, or differentiated to definitive endoderm.
  • Differentiation was induced by treating the cells with RPMI media supplemented with 2% BSA, lOOng/ml Activin A, 20ng/ml Wnt3a, 8ng/ml bFGF, and 3 ⁇ M Glycyl-H 1152 dihydrochloride for 24 hours. Media was then changed to RPMI media supplemented with 2% BSA, 100ng/ml Activin A, 8ng/ml bFGF, and 3 ⁇ M Glycyl- H 1152 dihydrochloride for an additional 48 hours with daily media change.
  • human embryonic stem cells can be passaged on micro-carriers and then subsequently cultured on another surface while maintaining pluripotency.
  • the cells may also be transferred to another surface and efficiently induced to differentiate.
  • Example 9 Human embryonic stem cells can be transferred directly from a cluster/colony style culture on mitotically inactivated fibroblast feeders to culture as single cells on ECM free surfaces for at least 10 passages without loss of pluripotency and without manual removal of fibroblast feeders.
  • Human embryonic stem cell lines are currently derived using a method that promotes a colony outgrowth of a single cell or a cluster of a few cells from a blastocyst. This colony outgrowth is then serially passaged and propagated until enough cluster/colonies of cells are available that they constitute a cell line.
  • the current best practice for large scale culture of mammalian cells is to use a 3- dimensional culture vessel that tightly maintains homeostatic, uniform conditions and can incorporate micro-carriers for support of adhesion dependent cells.
  • the current standard methods used for human embryonic stem cell culture- growth on fibroblast feeders or an ECM substrate and cluster/colony style culture pose a technical hurdle to successfully growing and maintaining a pluripotent human embryonic stem cell culture on micro-carriers, since these methods are not easily transferable to large scale culture on micro-carriers.
  • the human embryonic stem cell culture In order to effectively grow human embryonic stem cells on micro- carriers the human embryonic stem cell culture must be able to be passaged as single cells, and not as colonies or clusters, as is currently the standard in the art.
  • the human embryonic stem cells should be able to grow without a layer of feeder cells or ECM substrate.
  • Method Cells were routinely passaged by aspirating media, washing with PBS, and then treating the cells with a dissociation enzyme (collagenase, AccutaseTM,or TrypLE). Collagenase was used at lmg/ml concentration; AccutaseTM or TrypLE were used at IX stock concentration. All enzymes were used after reaching room temperature. A solution of 2% BSA in DMEM/F12 was added to each well and cells were uniformly suspended in the solution after treating the cells with enzyme.
  • a dissociation enzyme (collagenase, AccutaseTM,or TrypLE). Collagenase was used at lmg/ml concentration; AccutaseTM or TrypLE were used at IX stock concentration. All enzymes were used after reaching room temperature.
  • a solution of 2% BSA in DMEM/F12 was added to each well and cells were uniformly suspended in the solution after treating the cells with enzyme.
  • 2 nd passage Cells were passaged at a ratio of 1 to 4 using a 10 minutes exposure to TrypLETM or AccutaseTM. We also introduced a shorter enzyme exposure time that was determined empirically by treating the cells and monitoring for detachment. We observed that 3 minute exposure to TrypLE and 5 minute exposure to Accutase was sufficient to lift the cells. After treating the cells with enzyme, the cells were passaged as described above and aliquots of cell mRNA were taken for qRT-PCR at the time of passaging.
  • 3 rd passage Upon reaching confluence cells were washed with PBS, disrupted with enzyme for 3 or 10 minutes (TrypLETM) or 5 or 10 minutes (AccutaseTM), suspended in 2%BSA in DMEM/F12 and centrifuged, washed again with 2%BSA in DMEM/F12, centrifuged, and then resuspended and plated in their respective media. At this passage cells were plated at 1 :4 ratio and also at 2 additional split ratios- 1 :8 and 1 :16. Aliquots of cell mRNA were taken for qRT-PCR at each passage. 4 passages +: The conditions adopted for time of exposure to enzyme and passage ratio at passages 2 and 3 were maintained from passage 4 onward.
  • cells were assayed for pluripotency by flow cytometry for pluripotency surface markers ( Figure 40) and by qRT-PCR for pluripotency and differentiation markers ( Figures 41, 42, and 43).
  • Cells were also differentiated to definitive endoderm by treating the cells with RPMI media supplemented with 2% BSA, lOOng/ml Activin A, 20ng/ml Wnt3a, 8ng/ml bFGF, and 3uM Glycyl-H 1152 dihydrochloride for 24 hours.
  • Hl cells were cultured on PRIMARIATM (cat. no. 353846, Becton Dickinson, Franklin Lakes, NJ) tissue culture plates (method in Example 9) and released by treatment with TrypLETM Express for 3-5 minutes and seeded into 6 well non-tissue culture treated plates with Cytodex 3® (GE Healthcare Life Sciences, NJ) or HILLEX®II (Solohill, MI) micro-carriers in MEF-CM plus lO ⁇ M Y27632 (Sigma- Aldrich, MO).
  • PRIMARIATM catalog. no. 353846, Becton Dickinson, Franklin Lakes, NJ
  • TrypLETM Express for 3-5 minutes and seeded into 6 well non-tissue culture treated plates with Cytodex 3® (GE Healthcare Life Sciences, NJ) or HILLEX®II (Solohill, MI) micro-carriers in MEF-CM plus lO ⁇ M Y27632 (Sigma- Aldrich, MO).
  • Hlp46 cells grown on MATRIGEL (BD Biosciences, CA) coated plates and passaged with Collagenase (lmg/ml) were released and seeded onto micro-carriers in a similar manner.
  • the plates were incubated at 37°C for 5 hours, agitating by hand every 45 minutes.
  • the plates were then placed on a rocking platform at 37°C.
  • Medium was changed every day with MEF-CM plus lO ⁇ M Y27632 (Sigma- Aldrich, MO). Images show good attachment of cells to the micro-carriers at 3 days ( Figure 44).
  • Hl cells on the micro-carriers were differentiated to definitive endoderm.
  • the method is described in Example 7.
  • the Hl cells were released from the micro-carriers and underwent FACS analysis showing greater than 82% of the cells expressing CXCR4. See Figure 46.
  • the cells were efficiently differentiated into definitive endoderm regardless of the micro-carrier type or passaging enzyme on PRIMARIATM (cat. no. 353846, Becton Dickinson, Franklin Lakes, NJ). This proves the flexibility of the expansion system and allows for cells to be grown without matrix on plastic and micro-carriers.
  • Example 11 Human Embryonic Stem Cells Transferred from Planar Substrates Consisting of Mixed Cellulose Esters to Micro-Carriers
  • Hl cells were cultured on planar substrates consisting of mixed cellulose esters for 12 passages, according to the methods disclosed U.S. Patent Application No. 61/116,452.
  • the cells were released from the planar substrate by treatment with TrypLETM Express for 3-5 minutes and seeded into 6 well non-tissue culture treated plates with CYTODEX 3® (GE Healthcare Life Sciences, NJ) or HILLEX®II (Solohill, MI) micro-carriers in MEF-CM plus 1OmM Y27632 (Sigma-Aldrich, MO).
  • Hlp44 cells grown on MATRIGELTM coated plates (BD Biosciences, CA), passaged with Collagenase (lmg/ml) were released and seeded onto micro-carriers in a similar manner.
  • the plates were incubated at 37°C for 5 hours, agitating by hand every 45 minutes.
  • the plates were then placed on a rocking platform at 37°C. Media was changed daily. After 7 days the cells were released (described in Example 4) and analyzed by FACS for the pluripotency markers CD9, SSEA-4, SSEA-3, TRA-1-60, TRA-1-81 ( Figure 47). The majority of pluripotency markers were expressed on greater than 90% of the cells.
  • Hlp44 control cells were not tested for pluripotency after growth on HILLEX®II micro-carriers, since pluripotency had been confirmed by other experiments (see Example 5). Overall, the cells maintained pluripotency when transferred from planar substrates consisting of mixed cellulose esters onto micro-carriers.
  • Hl cells on the micro-carriers were differentiated to definitive endoderm, according to the methods described in Example 7. After 4 days of differentiation, the Hl cells were released from the micro-carriers and underwent FACS analysis showing greater than 65% of the cells expressing CXCR4 (Figure 48). The cells were efficiently differentiated into definitive endoderm regardless of the micro-carrier type. There appeared to be a lower number of cells differentiating into definitive endoderm on the HILLEX®II (Solohill, MI) micro-carriers. The ability of the cells to differentiate proves the flexibility of the expansion system. Additionally cells can be grown and differentiated directly on membranes and micro-carriers eliminating any need for an animal component matrix. Table 1: Attachment of H9 cells to micro-carrier beads in MEF-CM in static cultures.

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Gynecology & Obstetrics (AREA)
  • Reproductive Health (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Transplantation (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

La présente invention concerne des procédés pour la croissance, l'expansion et la différenciation de cellules souches pluripotentes sur des micro-supports.
PCT/US2009/065062 2008-11-20 2009-11-19 Culture de cellules souches pluripotentes sur des micro-supports WO2010059775A1 (fr)

Priority Applications (15)

Application Number Priority Date Filing Date Title
KR1020177024255A KR101837080B1 (ko) 2008-11-20 2009-11-19 마이크로-캐리어 상의 만능 줄기 세포 배양
BRPI0920956-5A BRPI0920956A2 (pt) 2008-11-20 2009-11-19 Cultura de células-tronco pluripotentes em microveículos
PL09759851T PL2366021T3 (pl) 2008-11-20 2009-11-19 Hodowla pluripotencjalnych komórek macierzystych na mikronośnikach
MX2014000085A MX356756B (es) 2008-11-20 2009-11-19 Células madre pluripotentes en microportadores.
EP09759851.0A EP2366021B1 (fr) 2008-11-20 2009-11-19 Culture de cellules souches pluripotentes sur des micro-supports
RU2011124900/10A RU2555538C2 (ru) 2008-11-20 2009-11-19 Культура плюрипотентных стволовых клеток на микроносителях
JP2011537603A JP2012509085A (ja) 2008-11-20 2009-11-19 マイクロキャリア上での多能性幹細胞の培養
CA2744234A CA2744234C (fr) 2008-11-20 2009-11-19 Culture de cellules souches pluripotentes sur des micro-supports
AU2009316580A AU2009316580B2 (en) 2008-11-20 2009-11-19 Pluripotent stem cell culture on micro-carriers
ES09759851.0T ES2642070T3 (es) 2008-11-20 2009-11-19 Cultivo de células madre pluripotentes en microportadores
KR1020117013824A KR101774546B1 (ko) 2008-11-20 2009-11-19 마이크로-캐리어 상의 만능 줄기 세포 배양
CN200980155382.2A CN102307991B (zh) 2008-11-20 2009-11-19 微载体上的多能干细胞培养
MX2011005288A MX2011005288A (es) 2008-11-20 2009-11-19 Celulas madre pluripotentes en microportadores.
ZA2011/04506A ZA201104506B (en) 2008-11-20 2011-06-17 Pluripotent stem cell culture on micro-carriers
AU2016201220A AU2016201220B2 (en) 2008-11-20 2016-02-26 Pluripotent stem cell culture on micro-carriers

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US11644708P 2008-11-20 2008-11-20
US61/116,447 2008-11-20

Publications (1)

Publication Number Publication Date
WO2010059775A1 true WO2010059775A1 (fr) 2010-05-27

Family

ID=41820296

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2009/065062 WO2010059775A1 (fr) 2008-11-20 2009-11-19 Culture de cellules souches pluripotentes sur des micro-supports

Country Status (15)

Country Link
US (3) US20100124781A1 (fr)
EP (2) EP3260534A1 (fr)
JP (1) JP2012509085A (fr)
KR (2) KR101837080B1 (fr)
CN (2) CN102307991B (fr)
AU (2) AU2009316580B2 (fr)
BR (1) BRPI0920956A2 (fr)
CA (1) CA2744234C (fr)
ES (1) ES2642070T3 (fr)
HK (1) HK1246356A1 (fr)
MX (2) MX2011005288A (fr)
PL (1) PL2366021T3 (fr)
RU (1) RU2555538C2 (fr)
WO (1) WO2010059775A1 (fr)
ZA (2) ZA201104506B (fr)

Cited By (43)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012044979A2 (fr) 2010-10-01 2012-04-05 The Goverment Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Manipulation de la fonction de cellules souches par des isoformes p53
US8637309B2 (en) 2008-03-17 2014-01-28 Agency For Science, Technology And Research Microcarriers for stem cell culture
WO2014017513A1 (fr) 2012-07-24 2014-01-30 日産化学工業株式会社 Composition de type milieu de culture, et procédé de culture d'une cellule ou d'un tissue l'utilisant
US8691569B2 (en) 2008-03-17 2014-04-08 Agency For Science, Technology And Research Microcarriers for stem cell culture
GB2484873B (en) * 2009-07-20 2014-04-30 Janssen Biotech Inc Differentiation of pancreatic endoderm cells.
JPWO2012056997A1 (ja) * 2010-10-28 2014-05-12 国立大学法人 熊本大学 多能性幹細胞の分化誘導効率を改善するための方法及び培地
US8828720B2 (en) 2008-03-17 2014-09-09 Agency For Science, Technology And Research Microcarriers for stem cell culture
WO2015111686A1 (fr) 2014-01-23 2015-07-30 日産化学工業株式会社 Composition de milieu de culture
WO2015111685A1 (fr) 2014-01-23 2015-07-30 日産化学工業株式会社 Procédé de production d'une composition pour milieu de culture
US9150829B2 (en) 2009-03-20 2015-10-06 Agency For Science, Technoloy And Research Culture of pluripotent and multipotent cells on microcarriers
US9181528B2 (en) 2010-08-31 2015-11-10 Janssen Biotech, Inc. Differentiation of pluripotent stem cells
CN105112361A (zh) * 2010-09-02 2015-12-02 生命技术公司 用于细胞的生物反应器放大的细胞培养系统
US9388387B2 (en) 2008-10-31 2016-07-12 Janssen Biotech, Inc. Differentiation of human embryonic stem cells
US9434920B2 (en) 2012-03-07 2016-09-06 Janssen Biotech, Inc. Defined media for expansion and maintenance of pluripotent stem cells
US9458431B2 (en) 2008-03-17 2016-10-04 Agency For Science, Technology And Research Microcarriers for stem cell culture
US9506036B2 (en) 2010-08-31 2016-11-29 Janssen Biotech, Inc. Differentiation of human embryonic stem cells
US9528090B2 (en) 2010-08-31 2016-12-27 Janssen Biotech, Inc. Differentiation of human embryonic stem cells
US9593305B2 (en) 2008-06-30 2017-03-14 Janssen Biotech, Inc. Differentiation of pluripotent stem cells
US9593310B2 (en) 2009-12-23 2017-03-14 Janssen Biotech, Inc. Differentiation of human embryonic stem cells
US9664671B2 (en) 2012-07-24 2017-05-30 Nissan Chemical Industries, Ltd. Culture medium composition and method of culturing cell or tissue using thereof
US9752125B2 (en) 2010-05-12 2017-09-05 Janssen Biotech, Inc. Differentiation of human embryonic stem cells
US9752126B2 (en) 2008-10-31 2017-09-05 Janssen Biotech, Inc. Differentiation of human pluripotent stem cells
US9969973B2 (en) 2008-11-20 2018-05-15 Janssen Biotech, Inc. Methods and compositions for cell attachment and cultivation on planar substrates
US9969972B2 (en) 2008-11-20 2018-05-15 Janssen Biotech, Inc. Pluripotent stem cell culture on micro-carriers
US9969981B2 (en) 2010-03-01 2018-05-15 Janssen Biotech, Inc. Methods for purifying cells derived from pluripotent stem cells
US9969982B2 (en) 2007-11-27 2018-05-15 Lifescan, Inc. Differentiation of human embryonic stem cells
US10006006B2 (en) 2014-05-16 2018-06-26 Janssen Biotech, Inc. Use of small molecules to enhance MAFA expression in pancreatic endocrine cells
US10017805B2 (en) 2012-08-23 2018-07-10 Nissan Chemical Industries, Ltd. Enhancing ingredients for protein production from various cells
US10066203B2 (en) 2008-02-21 2018-09-04 Janssen Biotech Inc. Methods, surface modified plates and compositions for cell attachment, cultivation and detachment
US10066210B2 (en) 2012-06-08 2018-09-04 Janssen Biotech, Inc. Differentiation of human embryonic stem cells into pancreatic endocrine cells
US10138465B2 (en) 2012-12-31 2018-11-27 Janssen Biotech, Inc. Differentiation of human embryonic stem cells into pancreatic endocrine cells using HB9 regulators
US10221392B2 (en) 2012-09-03 2019-03-05 Novo Nordisk A/S Generation of pancreatic endoderm from pluripotent stem cells using small molecules
US10316293B2 (en) 2007-07-01 2019-06-11 Janssen Biotech, Inc. Methods for producing single pluripotent stem cells and differentiation thereof
US10344264B2 (en) 2012-12-31 2019-07-09 Janssen Biotech, Inc. Culturing of human embryonic stem cells at the air-liquid interface for differentiation into pancreatic endocrine cells
US10358628B2 (en) 2011-12-22 2019-07-23 Janssen Biotech, Inc. Differentiation of human embryonic stem cells into single hormonal insulin positive cells
US10370644B2 (en) 2012-12-31 2019-08-06 Janssen Biotech, Inc. Method for making human pluripotent suspension cultures and cells derived therefrom
US10377989B2 (en) 2012-12-31 2019-08-13 Janssen Biotech, Inc. Methods for suspension cultures of human pluripotent stem cells
US10420803B2 (en) 2016-04-14 2019-09-24 Janssen Biotech, Inc. Differentiation of pluripotent stem cells to intestinal midgut endoderm cells
US10456424B2 (en) 2007-07-31 2019-10-29 Janssen Biotech, Inc. Pancreatic endocrine cells and methods thereof
CN110564675A (zh) * 2019-09-30 2019-12-13 广东华夏健康生命科学有限公司 一种毛囊干细胞的分离提取方法
EP3524672A3 (fr) * 2010-06-16 2019-12-18 Minerva Biotechnologies Corporation Reprogrammation de cellules cancéreuses
US11291691B2 (en) 2013-09-19 2022-04-05 The Board Of Trustees Of The Leland Stanford Junior University Methods and compositions for producing hepatocyte-like cells
JP7588097B2 (ja) 2019-06-13 2024-11-21 コーニング インコーポレイテッド 細胞を培養するための溶解性マイクロキャリア及び関連する方法

Families Citing this family (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100381768B1 (ko) * 2001-08-21 2003-04-26 김승환 무선 에이티엠망의 동적 자원할당 매체접속제어 프로토콜
US20110143433A1 (en) * 2008-03-17 2011-06-16 Agency For Science, Technology And Research Microcarriers for Stem Cell Culture
DK2638149T3 (da) 2010-11-12 2019-08-12 Univ Georgetown Immortalisering af epitelceller og fremgangsmåder til anvendelse
US9926523B2 (en) 2010-12-16 2018-03-27 General Electric Company Cell carriers and methods for culturing cells
US9453196B2 (en) 2010-12-16 2016-09-27 General Electric Company Cell carrier, methods of making and use
US9453197B2 (en) 2010-12-16 2016-09-27 General Electric Company Methods of making cell carrier
US9534206B2 (en) 2010-12-16 2017-01-03 General Electric Company Cell carrier, associated methods for making cell carrier and culturing cells using the same
US9518249B2 (en) 2010-12-16 2016-12-13 General Electric Company Cell carrier, associated methods for making cell carrier and culturing cells using the same
MX2014009132A (es) * 2012-03-07 2014-08-27 Hoffmann La Roche Metodo para determinar riesgo teratogenico.
US20150337267A1 (en) * 2013-01-04 2015-11-26 Kyoto University Reprogrammed stem cell
JP2016508726A (ja) * 2013-02-22 2016-03-24 セルラー ダイナミクス インターナショナル， インコーポレイテッド 組み合わせた遺伝的および化学的操作によるフォワードプログラミングを介した肝細胞の産生
CN104450604A (zh) * 2014-11-25 2015-03-25 成都威尔诺生物科技有限公司 用于制备疫苗工艺的微载体的清洁、灭菌的方法
US9944894B2 (en) 2015-01-16 2018-04-17 General Electric Company Pluripotent stem cell expansion and passage using a rocking platform bioreactor
AU2016344599B2 (en) * 2015-10-30 2020-12-17 Biolamina Ab Methods for producing hepatocytes
US20190127707A1 (en) * 2016-03-30 2019-05-02 Kyowa Hakko Bio Co., Ltd. Medium for culturing naive pluripotent stem cells and method for culturing pluripotent stem cells
US20200306299A9 (en) * 2016-10-21 2020-10-01 Georgia Tech Research Corporation Methods and Systems for T Cell Expansion
CN110892059A (zh) * 2017-05-19 2020-03-17 兴盛生物科技股份有限公司 细胞解离的系统和方法
WO2019102593A1 (fr) * 2017-11-24 2019-05-31 株式会社Ihi Dispositif de culture cellulaire
CN108753681B (zh) * 2018-04-28 2022-02-15 中山大学附属第一医院 一种鼻上皮干细胞培养方法及鼻上皮干细胞增殖培养基
KR102115360B1 (ko) * 2018-05-30 2020-05-26 주식회사 바이블리오테카 성체 줄기세포 배양액 및 그 제조방법
CN111040983A (zh) * 2019-12-25 2020-04-21 杭州原生生物科技有限公司 一种3d微载体细胞吸附培养的方法
CN111424011A (zh) * 2020-04-17 2020-07-17 深圳市旷逸生物科技有限公司 一种能够维持脐带间充质干细胞细胞形态的三维培养方法
CN111206015B (zh) * 2020-04-21 2020-07-31 广东省生物资源应用研究所 一种利用fact ⅲ微载体体外扩增精原干细胞的三维动态培养方法
CN113637635B (zh) * 2021-10-13 2022-01-25 北京华龛生物科技有限公司 微载体细胞培养体系中微载体聚团状态的解散方法
CN115089614A (zh) * 2022-06-28 2022-09-23 中国人民解放军军事科学院军事医学研究院 一种增强骨骼干细胞性能的方法及其在治疗骨关节炎中的应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007149182A2 (fr) * 2006-06-19 2007-12-27 Geron Corporation Différenciation et enrichissement de cellules de type cellules des îlots pancréatiques à partir de cellules souches pluripotentes humaines
WO2008004990A2 (fr) * 2006-07-06 2008-01-10 Es Cell International Pte Ltd Procédé de culture de cellules souches et cellules souches ainsi obtenues
WO2008035110A1 (fr) * 2006-09-22 2008-03-27 Riken Milieu de culture de cellules souches et procédé
WO2008102118A1 (fr) * 2007-02-19 2008-08-28 Stem Cell Sciences (Uk) Ltd Production à grande échelle de cellules souches
WO2009105570A2 (fr) * 2008-02-21 2009-08-27 Centocor Ortho Biotech Inc. Procédés, plaques à surface modifiée et compositions permettant la fixation, la culture et le détachement de cellules

Family Cites Families (245)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US264713A (en) * 1882-09-19 Neck-strap buckle and rein-ring
US3209652A (en) 1961-03-30 1965-10-05 Burgsmueller Karl Thread whirling method
AT326803B (de) 1968-08-26 1975-12-29 Binder Fa G Maschenware sowie verfahren zur herstellung derselben
US3935067A (en) 1974-11-22 1976-01-27 Wyo-Ben Products, Inc. Inorganic support for culture media
CA1201400A (fr) 1982-04-16 1986-03-04 Joel L. Williams Surfaces chimiquement specifiques pour influencer l'activite cellulaire pendant la culture
US4499802A (en) 1982-09-29 1985-02-19 Container Graphics Corporation Rotary cutting die with scrap ejection
US4537773A (en) 1983-12-05 1985-08-27 E. I. Du Pont De Nemours And Company α-Aminoboronic acid derivatives
US4557264A (en) 1984-04-09 1985-12-10 Ethicon Inc. Surgical filament from polypropylene blended with polyethylene
US5089396A (en) 1985-10-03 1992-02-18 Genentech, Inc. Nucleic acid encoding β chain prodomains of inhibin and method for synthesizing polypeptides using such nucleic acid
US5215893A (en) 1985-10-03 1993-06-01 Genentech, Inc. Nucleic acid encoding the ba chain prodomains of inhibin and method for synthesizing polypeptides using such nucleic acid
US4737578A (en) 1986-02-10 1988-04-12 The Salk Institute For Biological Studies Human inhibin
US5863531A (en) 1986-04-18 1999-01-26 Advanced Tissue Sciences, Inc. In vitro preparation of tubular tissue structures by stromal cell culture on a three-dimensional framework
US5567612A (en) 1986-11-20 1996-10-22 Massachusetts Institute Of Technology Genitourinary cell-matrix structure for implantation into a human and a method of making
US5759830A (en) 1986-11-20 1998-06-02 Massachusetts Institute Of Technology Three-dimensional fibrous scaffold containing attached cells for producing vascularized tissue in vivo
CA1340581C (fr) 1986-11-20 1999-06-08 Joseph P. Vacanti Neomorphogenese chimerique d'organes par implatation cellulaire controlee, utilisant des matrices artificielles
NZ229354A (en) 1988-07-01 1990-09-26 Becton Dickinson Co Treating polymer surfaces with a gas plasma and then applying a layer of endothelial cells to the surface
EP0363125A3 (fr) 1988-10-03 1990-08-16 Hana Biologics Inc. Produit contenant des cellules pancréatiques endocrines aptes à proliférer et procédé
US5837539A (en) 1990-11-16 1998-11-17 Osiris Therapeutics, Inc. Monoclonal antibodies for human mesenchymal stem cells
KR100249937B1 (ko) 1991-04-25 2000-04-01 나가야마 오사무 인간 인터루킨-6 수용체에 대한 재구성 인간 항체
US5449383A (en) 1992-03-18 1995-09-12 Chatelier; Ronald C. Cell growth substrates
GB9206861D0 (en) 1992-03-28 1992-05-13 Univ Manchester Wound healing and treatment of fibrotic disorders
CA2114282A1 (fr) 1993-01-28 1994-07-29 Lothar Schilder Implant multi-couches
JP3525221B2 (ja) 1993-02-17 2004-05-10 味の素株式会社 免疫抑制剤
CA2159804A1 (fr) 1993-04-08 1994-10-27 Hayden G. Coon Methode et milieu pour la culture de cellules
US5523226A (en) * 1993-05-14 1996-06-04 Biotechnology Research And Development Corp. Transgenic swine compositions and methods
GB9310557D0 (en) 1993-05-21 1993-07-07 Smithkline Beecham Plc Novel process and apparatus
TW257671B (fr) 1993-11-19 1995-09-21 Ciba Geigy
US6703017B1 (en) 1994-04-28 2004-03-09 Ixion Biotechnology, Inc. Reversal of insulin-dependent diabetes by islet-producing stem cells, islet progenitor cells and islet-like structures
US5834308A (en) 1994-04-28 1998-11-10 University Of Florida Research Foundation, Inc. In vitro growth of functional islets of Langerhans
US6001647A (en) 1994-04-28 1999-12-14 Ixion Biotechnology, Inc. In vitro growth of functional islets of Langerhans and in vivo uses thereof
US6083903A (en) 1994-10-28 2000-07-04 Leukosite, Inc. Boronic ester and acid compounds, synthesis and uses
ATE214602T1 (de) 1994-12-29 2002-04-15 Chugai Pharmaceutical Co Ltd Verstärker eines anti-tumoragens beinhaltend einen il-6 antagonisten
US5843780A (en) 1995-01-20 1998-12-01 Wisconsin Alumni Research Foundation Primate embryonic stem cells
US5718922A (en) 1995-05-31 1998-02-17 Schepens Eye Research Institute, Inc. Intravitreal microsphere drug delivery and method of preparation
US5908782A (en) 1995-06-05 1999-06-01 Osiris Therapeutics, Inc. Chemically defined medium for human mesenchymal stem cells
US5681561A (en) 1995-06-07 1997-10-28 Life Medical Sciences, Inc. Compositions and methods for improving autologous fat grafting
ES2202840T3 (es) 1997-04-24 2004-04-01 Ortho-Mcneil Pharmaceutical, Inc. Imidazoles sustituidos utiles en el tratamiento de enfermedades inflamatorias.
ATE358493T1 (de) 1997-07-03 2007-04-15 Osiris Therapeutics Inc Menschliche mesenchymale stammzellen aus peripherem blut
EP1538206B1 (fr) 1997-09-16 2010-03-24 Centocor, Inc. Synthèse chimique complète et synthèse de gènes et de génomes
US6670127B2 (en) 1997-09-16 2003-12-30 Egea Biosciences, Inc. Method for assembly of a polynucleotide encoding a target polypeptide
CA2307807C (fr) 1997-10-23 2008-09-02 Andrea G. Bodnar Procedes et matieres utiles pour la croissance de cellules souches primordiales de primate
ZA9811898B (en) 1997-12-29 2000-06-28 Ortho Mcneil Pharm Inc Anti-Inflammatory Compounds.
CA2320040C (fr) 1998-03-18 2007-05-22 Osiris Therapeutics, Inc. Cellules de souche mesenchymateuse servant a la prevention et au traitement des reponses immunitaires lors de la transplantation
MY132496A (en) 1998-05-11 2007-10-31 Vertex Pharma Inhibitors of p38
US6413773B1 (en) 1998-06-01 2002-07-02 The Regents Of The University Of California Phosphatidylinositol 3-kinase inhibitors as stimulators of endocrine differentiation
US6667176B1 (en) 2000-01-11 2003-12-23 Geron Corporation cDNA libraries reflecting gene expression during growth and differentiation of human pluripotent stem cells
US7410798B2 (en) 2001-01-10 2008-08-12 Geron Corporation Culture system for rapid expansion of human embryonic stem cells
US6610540B1 (en) 1998-11-18 2003-08-26 California Institute Of Technology Low oxygen culturing of central nervous system progenitor cells
US6413556B1 (en) 1999-01-08 2002-07-02 Sky High, Llc Aqueous anti-apoptotic compositions
NZ513598A (en) 1999-01-21 2001-09-28 Vitro Diagnostics Inc Immortalized cell lines and methods of making the same
US6815203B1 (en) 1999-06-23 2004-11-09 Joslin Diabetes Center, Inc. Methods of making pancreatic islet cells
US6306424B1 (en) 1999-06-30 2001-10-23 Ethicon, Inc. Foam composite for the repair or regeneration of tissue
US6333029B1 (en) 1999-06-30 2001-12-25 Ethicon, Inc. Porous tissue scaffoldings for the repair of regeneration of tissue
CA2385628A1 (fr) 1999-09-27 2001-04-05 Ammon B. Peck Inversion de diabetes dependant de l'insuline par des cellules souches insulaires, des cellules insulaires progenitrices et des structures de type insulaire
US6685936B2 (en) 1999-10-12 2004-02-03 Osiris Therapeutics, Inc. Suppressor cells induced by culture with mesenchymal stem cells for treatment of immune responses in transplantation
US20030082155A1 (en) 1999-12-06 2003-05-01 Habener Joel F. Stem cells of the islets of langerhans and their use in treating diabetes mellitus
EP1240518A4 (fr) 1999-12-13 2006-05-17 Scripps Research Inst Marqueurs pour identifier et isoler les progeniteurs des cellules alpha et beta d'ilots pancreatiques
US7005252B1 (en) 2000-03-09 2006-02-28 Wisconsin Alumni Research Foundation Serum free cultivation of primate embryonic stem cells
US7439064B2 (en) 2000-03-09 2008-10-21 Wicell Research Institute, Inc. Cultivation of human embryonic stem cells in the absence of feeder cells or without conditioned medium
US6436704B1 (en) 2000-04-10 2002-08-20 Raven Biotechnologies, Inc. Human pancreatic epithelial progenitor cells and methods of isolation and use thereof
US6458589B1 (en) 2000-04-27 2002-10-01 Geron Corporation Hepatocyte lineage cells derived from pluripotent stem cells
WO2002000849A1 (fr) 2000-06-26 2002-01-03 Renomedix Institute Inc. Fraction cellulaire contenant des cellules capables de se differencier en cellules du systeme nerveux
US7323472B2 (en) 2000-10-23 2008-01-29 Smithkline Beecham Corporation 2,4,8-trisubstituted-8H-pyrido[2,3-d]pyrimidin-7-one compounds
ES2245994T3 (es) 2000-12-08 2006-02-01 Ortho-Mcneil Pharmaceutical, Inc. Compuestos macro-heterociclicos utilizados como inhibidores de quinasa.
US6849643B2 (en) 2000-12-08 2005-02-01 Ortho-Mcneil Pharmaceutical, Inc. Indazolyl-substituted pyrroline compounds as kinase inhibitors
US6599323B2 (en) 2000-12-21 2003-07-29 Ethicon, Inc. Reinforced tissue implants and methods of manufacture and use
CA2435826A1 (fr) 2001-01-24 2002-08-01 The Government Of The United States Of America Differenciation de cellules souches par rapport aux ilots pancreatiques
PT3078667T (pt) 2001-01-25 2018-12-28 The United States Of America Represented By The Sec Dep Of Health And Human Services Formulação de compostos de ácido borónico
US6656488B2 (en) 2001-04-11 2003-12-02 Ethicon Endo-Surgery, Inc. Bioabsorbable bag containing bioabsorbable materials of different bioabsorption rates for tissue engineering
US20050054102A1 (en) 2001-04-19 2005-03-10 Anna Wobus Method for differentiating stem cells into insulin-producing cells
DE60231035D1 (de) 2001-04-24 2009-03-19 Ajinomoto Kk Stammzellen und verfahren zu deren trennung
CA2447015A1 (fr) 2001-05-15 2002-11-21 Rappaport Family Institute For Research In The Medical Sciences Cellules produisant de l'insuline derivees de cellules souches embryonnaires humaines
US6626950B2 (en) 2001-06-28 2003-09-30 Ethicon, Inc. Composite scaffold with post anchor for the repair and regeneration of tissue
KR100418195B1 (ko) 2001-07-05 2004-02-11 주식회사 우리기술 전력케이블의 다중절연진단장치 및 그 방법
GB0117583D0 (en) 2001-07-19 2001-09-12 Astrazeneca Ab Novel compounds
WO2003014313A2 (fr) 2001-08-06 2003-02-20 Bresagen, Ltd. Compositions et procedes de substitution pour la culture de cellules souches
US6617152B2 (en) 2001-09-04 2003-09-09 Corning Inc Method for creating a cell growth surface on a polymeric substrate
EP1298201A1 (fr) 2001-09-27 2003-04-02 Cardion AG Procédé de production de cellules présentant un état similaire à celui d'une cellule beta d'ilot pancréatique
US20030138951A1 (en) 2001-10-18 2003-07-24 Li Yin Conversion of liver stem and progenitor cells to pancreatic functional cells
EP1442115B9 (fr) 2001-11-15 2009-12-16 Children's Medical Center Corporation Techniques d'isolation, de developpement et de differenciation de cellules souches provenant de villosites choriales, de liquide amniotique ainsi que de placenta et applications therapeutiques
KR101083454B1 (ko) 2001-12-07 2011-11-16 사이토리 테라퓨틱스, 인크. 처리된 리포애스퍼레이트 세포로 환자를 치료하기 위한 시스템 및 방법
US7033831B2 (en) 2001-12-07 2006-04-25 Geron Corporation Islet cells from human embryonic stem cells
AU2002218893A1 (en) 2001-12-21 2003-07-09 Thromb-X Nv Compositions for the in vitro derivation and culture of embryonic stem (es) cell lines with germline transmission capability
IL162663A0 (en) 2001-12-28 2005-11-20 Cellartis Ab A method for the establishment of apluripotent human blastocyst-derived stem cell line
US20030162290A1 (en) 2002-01-25 2003-08-28 Kazutomo Inoue Method for inducing differentiation of embryonic stem cells into functioning cells
WO2003087349A1 (fr) 2002-04-17 2003-10-23 Otsuka Pharmaceutical Co., Ltd. Methode de formation de cellules pancreatiques $g(b) a partir de cellules mesenchymales
US20040161419A1 (en) 2002-04-19 2004-08-19 Strom Stephen C. Placental stem cells and uses thereof
JP2005529918A (ja) 2002-05-08 2005-10-06 ジヤンセン・フアーマシユーチカ・ナームローゼ・フエンノートシヤツプ 置換されたピロリンキナーゼ阻害剤
US20060003446A1 (en) 2002-05-17 2006-01-05 Gordon Keller Mesoderm and definitive endoderm cell populations
JP2006512046A (ja) 2002-05-28 2006-04-13 ベクトン・ディキンソン・アンド・カンパニー ｉｎｖｉｔｒｏにおけるヒト膵臓腺房細胞の増殖およびインスリン産生細胞への分化転換のための方法
CN1671694A (zh) 2002-06-05 2005-09-21 詹森药业有限公司 作为激酶抑制剂的二吲哚基－顺丁烯二酰亚胺衍生物
GB0212976D0 (en) 2002-06-06 2002-07-17 Tonejet Corp Pty Ltd Ejection method and apparatus
CN1171991C (zh) 2002-07-08 2004-10-20 徐如祥 人神经干细胞的培养方法
US6877147B2 (en) 2002-07-22 2005-04-05 Broadcom Corporation Technique to assess timing delay by use of layout quality analyzer comparison
US7838290B2 (en) 2002-07-25 2010-11-23 The Scripps Research Institute Hematopoietic stem cells and methods of treatment of neovascular eye diseases therewith
EP1539930A4 (fr) 2002-07-29 2006-08-09 Es Cell Int Pte Ltd Procede en plusieurs etapes de differenciation des cellules sensibles a l'insuline positive, au glucose
AU2003262628A1 (en) 2002-08-14 2004-03-03 University Of Florida Bone marrow cell differentiation
AU2003268534A1 (en) 2002-09-06 2004-03-29 Amcyte Inc. Cd56 positive human adult pancreatic endocrine progenitor cells
US9969977B2 (en) 2002-09-20 2018-05-15 Garnet Biotherapeutics Cell populations which co-express CD49c and CD90
US20040062753A1 (en) 2002-09-27 2004-04-01 Alireza Rezania Composite scaffolds seeded with mammalian cells
US20060252150A1 (en) 2002-11-08 2006-11-09 Linzhao Cheng Human embryonic stem cell cultures, and compositions and methods for growing same
US7144999B2 (en) 2002-11-23 2006-12-05 Isis Pharmaceuticals, Inc. Modulation of hypoxia-inducible factor 1 alpha expression
AU2003302702B2 (en) 2002-12-05 2008-08-07 Technion Research & Development Foundation Ltd. Cultured human pancreatic islets, and uses thereof
DK1572984T3 (en) 2002-12-16 2016-06-13 Technion Res & Dev Foundation FEEDER CELL-FREE, XENOPHRIC CULTIVATION SYSTEM FOR HUMAN EMBRYONAL STEM CELLS
RU2359671C2 (ru) 2003-01-29 2009-06-27 Такеда Фармасьютикал Компани Лимитед Способ получения препарата с покрытием
PL377403A1 (pl) 2003-01-29 2006-02-06 Takeda Pharmaceutical Company Limited Sposób wytwarzania preparatu powlekanego
WO2005045001A2 (fr) 2003-02-14 2005-05-19 The Board Of Trustees Of The Leland Stanford Junior University Cellules produisant de l'insuline derivees de cellules souches
WO2004073633A2 (fr) 2003-02-14 2004-09-02 The Board Of Trustees Of The Leland Stanford Junior University Methodes et compositions permettant de moduler le developpement de cellules souches
CA2520861A1 (fr) 2003-03-27 2004-10-14 Ixion Biotechnology, Inc. Methode de transdifferentiation de cellules souches non pancreatiques dans la voie de differentiation du pancreas
US20060194315A1 (en) 2003-03-31 2006-08-31 Condie Brian G Compositions and methods for the control, differentiaton and/or manipulation of pluripotent cells through a gamma-secretase signaling pathway
US20090203141A1 (en) 2003-05-15 2009-08-13 Shi-Lung Lin Generation of tumor-free embryonic stem-like pluripotent cells using inducible recombinant RNA agents
EP2338981B1 (fr) 2003-06-27 2015-04-22 DePuy Synthes Products, LLC Utilisation de cellules dérivées postpartum du cordon ombilical pour la régénération et la réparation de tissus oculaires
IL161903A0 (en) 2003-07-17 2005-11-20 Gamida Cell Ltd Ex vivo progenitor and stem cell expansion for usein the treatment of disease of endodermally- deri ved organs
ITRM20030395A1 (it) 2003-08-12 2005-02-13 Istituto Naz Per Le Malattie Infettive Lazz Terreno di coltura per il mantenimento, la proliferazione e il differenziamento di cellule di mammifero.
US7569385B2 (en) 2003-08-14 2009-08-04 The Regents Of The University Of California Multipotent amniotic fetal stem cells
US7157275B2 (en) 2003-08-15 2007-01-02 Becton, Dickinson And Company Peptides for enhanced cell attachment and growth
CA2536067A1 (fr) 2003-08-27 2005-03-10 Stemcells California, Inc. Populations de cellules progenitrices et de cellules souches pancreatiques enrichies, methodes d'identification, d'isolement et d'enrichissement de ces populations
US7468391B2 (en) 2003-12-17 2008-12-23 Allergan, Inc. Methods for treating retinoid responsive disorders using selective inhibitors of CYP26A and CYP26B
US20060030042A1 (en) 2003-12-19 2006-02-09 Ali Brivanlou Maintenance of embryonic stem cells by the GSK-3 inhibitor 6-bromoindirubin-3'-oxime
US20050266554A1 (en) 2004-04-27 2005-12-01 D Amour Kevin A PDX1 expressing endoderm
US7625753B2 (en) 2003-12-23 2009-12-01 Cythera, Inc. Expansion of definitive endoderm cells
CN109628371B (zh) 2003-12-23 2021-02-19 维亚希特公司 定形内胚层
NZ547920A (en) 2003-12-23 2009-06-26 Cythera Inc Definitive endoderm
US20050233446A1 (en) 2003-12-31 2005-10-20 Parsons Xuejun H Defined media for stem cell culture
TWI334443B (en) 2003-12-31 2010-12-11 Ind Tech Res Inst Method of single cell culture of undifferentiated human embryonic stem cells
US7794704B2 (en) 2004-01-23 2010-09-14 Advanced Cell Technology, Inc. Methods for producing enriched populations of human retinal pigment epithelium cells for treatment of retinal degeneration
WO2005071066A1 (fr) 2004-01-23 2005-08-04 Board Of Regents, The University Of Texas System Methodes et compositions de preparation de cellules de secretion de l'insuline pancreatique
WO2005080551A2 (fr) 2004-02-12 2005-09-01 University Of Newcastle Upon Tyne Cellules souches
JP4901471B2 (ja) 2004-02-19 2012-03-21 国立大学法人京都大学 体細胞核初期化物質のスクリーニング方法
WO2005086860A2 (fr) 2004-03-09 2005-09-22 Gang Xu Procedes d'obtention de cellules produisant de l'insuline
JP2007528226A (ja) 2004-03-10 2007-10-11 リージェンツ オブ ザ ユニヴァーシティ オブ カリフォルニア 胚幹細胞の増殖用組成物および方法
AU2005224569B2 (en) 2004-03-23 2011-07-14 Toshihiro Akaike Method of proliferating pluripotent stem cell
WO2005097980A2 (fr) 2004-03-26 2005-10-20 Geron Corporation Nouveau protocole de preparation d'hepatocytes a partir de cellules souches embryonnaires
EP1730268A2 (fr) 2004-04-01 2006-12-13 Wisconsin Alumni Research Foundation Differenciation de cellules souches en lignee endodermique et pancreatique
EP1740612B1 (fr) 2004-04-27 2019-08-07 Viacyte, Inc. Endoderme exprimant pdx1
WO2006016999A1 (fr) 2004-07-09 2006-02-16 Cythera, Inc. Methodes d'identification de facteurs pour differencier l'endoderme definitif
WO2006020919A2 (fr) 2004-08-13 2006-02-23 University Of Georgia Research Foundation, Inc. Compositions et procedes d'auto-renouvellement et de differentiation dans des cellules souches embryonnaires humaines
WO2006026473A2 (fr) 2004-08-25 2006-03-09 University Of Georgia Research Foundation, Inc. Procedes et compositions utilisant myc et gsk3$g(b) pour manipuler la nature multipotente de cellules souches embryonnaires
DE102004043256B4 (de) 2004-09-07 2013-09-19 Rheinische Friedrich-Wilhelms-Universität Bonn Skalierbarer Prozess zur Kultivierung undifferenzierter Stammzellen in Suspension
NZ553235A (en) 2004-09-08 2009-11-27 Wisconsin Alumni Res Found Culturing human pluripotent stem cells
BRPI0514778B1 (pt) 2004-09-08 2018-07-10 Wisconsin Alumni Research Foundation Meio para cultivar células tronco embrionárias
AU2006208944A1 (en) 2005-01-28 2006-08-03 Imperial College Innovations Limited Methods for embryonic stem cell culture
AU2006210955A1 (en) 2005-01-31 2006-08-10 Es Cell International Pte Ltd. Directed differentiation of embryonic stem cells and uses thereof
WO2006088867A2 (fr) 2005-02-15 2006-08-24 Medistem Laboratories, Incorporated Procede pour l'expansion de cellules souches
PL1860950T3 (pl) 2005-03-04 2017-09-29 Lifescan, Inc. Dojrzałe komórki podścieliska pochodzenia trzustkowego
GB0505970D0 (en) 2005-03-23 2005-04-27 Univ Edinburgh Culture medium containing kinase inhibitor, and uses thereof
WO2006113470A2 (fr) 2005-04-15 2006-10-26 Geron Corporation Procede pour traiter un cancer grace a l'inhibition combinee d'activites de proteasome et de telomerase
CN100425694C (zh) 2005-04-15 2008-10-15 北京大学 诱导胚胎干细胞向胰腺细胞分化的方法
US20080227656A1 (en) 2005-04-26 2008-09-18 Flemming Besenbacher Biosurface Structure Array
JP5092124B2 (ja) 2005-05-24 2012-12-05 国立大学法人 熊本大学 Ｅｓ細胞の分化誘導方法
AU2006202209B2 (en) 2005-05-27 2011-04-14 Lifescan, Inc. Amniotic fluid derived cells
BRPI0611733A2 (pt) 2005-06-10 2010-09-28 Irm Llc compostos que mantêm pluripotência de células-tronco embriÈnicas
WO2006138433A2 (fr) 2005-06-14 2006-12-28 The Regents Of The University Of California Induction de differenciation cellulaire par des polypeptides bhlh de classe i
EP1931764A1 (fr) 2005-06-21 2008-06-18 GE Healthcare Bio-Sciences AB Méthode de culture cellulaire
KR20150067394A (ko) 2005-06-22 2015-06-17 아스테리아스 바이오세라퓨틱스, 인크. 인간 배아 줄기 세포의 현탁 배양
US8778919B2 (en) 2005-06-30 2014-07-15 Janssen Pharmaceutica Nv Cyclic anilino—pyridinotriazines
WO2007016485A2 (fr) 2005-07-29 2007-02-08 Athersys, Inc. Emploi d'un inhibiteur de gsk-3 dans le but de conserver leur puissance a des cellules cultivees
US20090087907A1 (en) 2005-07-29 2009-04-02 Alice Pebay Compositions and Methods for Growth of Pluripotent Cells
WO2007025234A2 (fr) 2005-08-26 2007-03-01 The Trustees Of Columbia University In The City Of New York Generation de cellules endocrines pancreatiques a partir de cultures cellulaires de conduit primaire, et procedes d'utilisation pour le traitement de diabetes
EP1962719A4 (fr) 2005-08-29 2011-05-04 Technion Res And Dev Of Foundation Ltd Milieux de culture de cellules souches
WO2007027156A1 (fr) 2005-09-02 2007-03-08 Agency For Science, Technology And Research Procédé de dérivation de cellules souches mésenchymateuses
SG151259A1 (en) 2005-09-12 2009-04-30 Es Cell Int Pte Ltd Cardiomyocyte production
WO2008048671A1 (fr) 2006-10-18 2008-04-24 University Of Illinois Cellules souches de type embryonnaire dérivées du sang périphérique d'un être humain adulte et procédés d'utilisation associés
JP2009511061A (ja) 2005-10-14 2009-03-19 リージェンツ オブ ザ ユニバーシティ オブ ミネソタ 膵臓表現型を有する細胞への非胚性幹細胞の分化
US20070122905A1 (en) 2005-10-27 2007-05-31 D Amour Kevin A PDX1-expressing dorsal and ventral foregut endoderm
EP2206778B1 (fr) 2005-12-13 2018-08-01 Kyoto University Facteur de reprogrammation nucléaire
WO2007082963A1 (fr) 2006-01-18 2007-07-26 Fundación Instituto Valenciano De Infertilidad Lignées de cellules souches embryonnaires humaines et leurs méthodes d'utilisation
CA2643478C (fr) 2006-02-23 2019-06-18 Novocell, Inc. Compositions et procedes utiles pour la culture de cellules differenciables
US7695965B2 (en) 2006-03-02 2010-04-13 Cythera, Inc. Methods of producing pancreatic hormones
WO2007103282A2 (fr) 2006-03-02 2007-09-13 Cythera, Inc. Cellules precurseurs endocrines, cellules exprimant des hormones pancreatiques et procedes de productions associes
US8741643B2 (en) 2006-04-28 2014-06-03 Lifescan, Inc. Differentiation of pluripotent stem cells to definitive endoderm lineage
EP2021462B1 (fr) 2006-04-28 2019-01-09 Lifescan, Inc. Différentiation de cellules souches embryonnaires humaines
AU2007248609B2 (en) 2006-05-02 2012-11-01 Wisconsin Alumni Research Foundation Method of differentiating stem cells into cells of the endoderm and pancreatic lineage
US8685730B2 (en) 2006-05-02 2014-04-01 Wisconsin Alumni Research Foundation Methods and devices for differentiating pluripotent stem cells into cells of the pancreatic lineage
WO2007139929A2 (fr) 2006-05-25 2007-12-06 The Burnham Institute For Medical Research Procédés de culture et de production de populations de cellules isolées de cellules souches embryonnaires humaines
CN101541953A (zh) 2006-06-02 2009-09-23 佐治亚大学研究基金会 通过从人胚胎干细胞获得的定形内胚层细胞的分化得到胰和肝内胚层细胞及组织
US20090298169A1 (en) 2006-06-02 2009-12-03 The University Of Georgia Research Foundation Pancreatic and Liver Endoderm Cells and Tissue by Differentiation of Definitive Endoderm Cells Obtained from Human Embryonic Stems
CN100494359C (zh) * 2006-06-23 2009-06-03 中日友好医院 神经干细胞三维立体培养体外扩增的方法
PL2046946T3 (pl) 2006-06-26 2017-04-28 Lifescan, Inc. Hodowla pluripotencjalnych komórek macierzystych
US20080003676A1 (en) 2006-06-26 2008-01-03 Millipore Corporation Growth of embryonic stem cells
AU2007277364B2 (en) 2006-07-26 2010-08-12 Viacyte, Inc. Methods of producing pancreatic hormones
DK2733203T3 (en) 2006-08-02 2019-02-04 Technion Res & Dev Foundation PROCEDURES FOR EXPANSION OF EMBRYONAL STEM CELLS IN A SUSPENSION CULTURE
KR101331510B1 (ko) 2006-08-30 2013-11-20 재단법인서울대학교산학협력재단 저농도의 포도당을 함유하는 인간 배아줄기세포용 배지조성물 및 이를 이용한 인간 배아 줄기세포로부터 인슐린생산 세포 또는 세포괴로 분화시키는 방법, 그리고그로부터 유도된 인슐린 생산 세포 또는 세포괴
US20080091234A1 (en) 2006-09-26 2008-04-17 Kladakis Stephanie M Method for modifying a medical implant surface for promoting tissue growth
US20100323442A1 (en) 2006-10-17 2010-12-23 Emmanuel Edward Baetge Modulation of the phosphatidylinositol-3-kinase pathway in the differentiation of human embryonic stem cells
WO2008049027A1 (fr) 2006-10-17 2008-04-24 Stiefel Laboratories, Inc. Métabolites de talarazole
RU2323252C1 (ru) * 2006-10-25 2008-04-27 Антонина Ивановна Колесникова Способ культивирования мезенхимальных стволовых клеток человека ex vivo
EP2088190A4 (fr) 2006-11-09 2011-01-05 Japan Government Procédé destiné à la culture et au passage d'une cellule souche embryonnaire de primate, et procédé destiné à induire la différenciation de la cellule souche embryonnaire
TW200836749A (en) 2007-01-09 2008-09-16 Vioquest Pharmaceuticals Inc Compositions including triciribine and bortezomib and derivatives thereof and methods of use thereof
JP5419279B2 (ja) * 2007-01-17 2014-02-19 ウィスコンシン アラムニ リサーチ ファンデーション 改良された幹細胞の培養
CN103627671A (zh) 2007-01-30 2014-03-12 佐治亚大学研究基金会 产生中内胚层细胞及多能游走细胞的方法与细胞群及用途
US20090053182A1 (en) 2007-05-25 2009-02-26 Medistem Laboratories, Inc. Endometrial stem cells and methods of making and using same
DK3293256T3 (da) 2007-06-29 2019-08-12 Fujifilm Cellular Dynamics Inc Automatiseret fremgangsmåde og apparatur til dyrkning af embryonale stamceller
JP5738591B2 (ja) 2007-07-18 2015-06-24 ライフスキャン・インコーポレイテッドＬｉｆｅｓｃａｎ，Ｉｎｃ． ヒト胚幹細胞の分化
CN101952415B (zh) 2007-07-31 2017-06-27 生命扫描有限公司 人胚胎干细胞的分化
CA2694956C (fr) 2007-07-31 2017-12-19 Lifescan, Inc. Differenciation de cellules souches pluripotentes a l'aide de cellules d'alimentation humaines
CA2696622C (fr) 2007-08-24 2016-07-19 Stichting Het Nederlands Kanker Instituut Composition
US20110151447A1 (en) 2007-11-06 2011-06-23 Children's Medical Center Corporation Method to produce induced pluripotent stem (ips) cells from non-embryonic human cells
JP5710264B2 (ja) 2007-11-27 2015-04-30 ライフスキャン・インコーポレイテッドＬｉｆｅｓｃａｎ，Ｉｎｃ． ヒト胚性幹細胞の分化
SG154367A1 (en) 2008-01-31 2009-08-28 Es Cell Int Pte Ltd Method of differentiating stem cells
WO2009096049A1 (fr) 2008-02-01 2009-08-06 Kyoto University Cellules différenciées ayant pour origine des cellules souches pluripotentes artificielles
EP2250252A2 (fr) 2008-02-11 2010-11-17 Cambridge Enterprise Limited Reprogrammation perfectionnée de cellules de mammifère et cellules ainsi obtenues
WO2009110215A1 (fr) 2008-03-03 2009-09-11 独立行政法人 科学技術振興機構 Procédé d'induction de différentiation de cellules ciliées
SG188918A1 (fr) * 2008-03-17 2013-04-30 Agency Science Tech & Res
AU2008355123B2 (en) 2008-04-21 2014-12-04 Viacyte, Inc. Methods for purifying endoderm and pancreatic endoderm cells derived from human embryonic stem cells
US8338170B2 (en) 2008-04-21 2012-12-25 Viacyte, Inc. Methods for purifying endoderm and pancreatic endoderm cells derived from human embryonic stem cells
WO2009132083A2 (fr) 2008-04-22 2009-10-29 President And Fellows Of Harvard College Compositions et méthodes permettant de favoriser la production de cellules pancréatiques pdx1+
US7939322B2 (en) 2008-04-24 2011-05-10 Centocor Ortho Biotech Inc. Cells expressing pluripotency markers and expressing markers characteristic of the definitive endoderm
US8623648B2 (en) 2008-04-24 2014-01-07 Janssen Biotech, Inc. Treatment of pluripotent cells
WO2009154606A1 (fr) 2008-06-03 2009-12-23 Cythera, Inc. Facteurs de croissance pour la production de l'endoderme définitif
US20090298178A1 (en) 2008-06-03 2009-12-03 D Amour Kevin Allen Growth factors for production of definitive endoderm
DE102008032236A1 (de) 2008-06-30 2010-04-01 Eberhard-Karls-Universität Tübingen Isolierung und/oder Identifizierung von Stammzellen mit adipozytärem, chondrozytärem und pankreatischem Differenzierungspotential
CN102159703B (zh) 2008-06-30 2015-11-25 森托科尔奥索生物科技公司 多能干细胞的分化
US20100028307A1 (en) 2008-07-31 2010-02-04 O'neil John J Pluripotent stem cell differentiation
US9683215B2 (en) 2008-08-22 2017-06-20 President And Fellows Of Harvard College Methods of reprogramming cells
CN102272291B (zh) 2008-10-31 2018-01-16 詹森生物科技公司 人胚胎干细胞向胰腺内分泌谱系的分化
JP2012507289A (ja) 2008-10-31 2012-03-29 ヤンセン バイオテツク，インコーポレーテツド ヒト胚性幹細胞の膵内分泌系への分化
US8008075B2 (en) 2008-11-04 2011-08-30 Viacyte, Inc. Stem cell aggregate suspension compositions and methods of differentiation thereof
CN108342353B (zh) 2008-11-04 2022-01-11 韦尔赛特公司 干细胞聚集体悬浮组合物及其分化方法
WO2010057039A2 (fr) 2008-11-14 2010-05-20 Cythera, Inc. Encapsulation de cellules pancréatiques dérivées de cellules souches pluripotentes humaines
MX2011005288A (es) 2008-11-20 2011-06-01 Centocor Ortho Biotech Inc Celulas madre pluripotentes en microportadores.
DK2356218T3 (en) 2008-12-05 2017-08-21 Inserm (Institut Nat De La Santé Et De La Rech Médicale) METHOD AND MEDIUM FOR NEURAL DIFFERENTIZATION OF PLURIPOTENT CELLS
SG177416A1 (en) 2009-07-20 2012-02-28 Janssen Biotech Inc Differentiation of human embryonic stem cells
JP6219568B2 (ja) 2009-07-20 2017-10-25 ヤンセン バイオテツク，インコーポレーテツド ヒト胚性幹細胞の分化
WO2011059725A2 (fr) 2009-10-29 2011-05-19 Centocor Ortho Biotech Inc. Cellules souches pluripotentes
FI20096288A0 (fi) 2009-12-04 2009-12-04 Kristiina Rajala Formulations and methods for culturing stem cells
KR101773538B1 (ko) 2009-12-23 2017-08-31 얀센 바이오테크 인코포레이티드 인간 배아 줄기 세포의 분화
US9133439B2 (en) 2009-12-23 2015-09-15 Janssen Biotech, Inc. Differentiation of human embryonic stem cells
SG183400A1 (en) 2010-03-02 2012-09-27 Univ Singapore Culture additives to boost stem cell proliferation and differentiation response
EP3199623B1 (fr) 2010-03-31 2021-07-28 The Scripps Research Institute Nouvelle programmation de cellules
JP2013524836A (ja) 2010-04-25 2013-06-20 マウント・シナイ・スクール・オブ・メディスン 多能性細胞からの前部前腸内胚葉の生成
DK2569419T3 (da) 2010-05-12 2019-05-13 Janssen Biotech Inc Differentiering af humane embryoniske stamceller
US9279103B2 (en) 2010-08-05 2016-03-08 Wisconsin Alumni Research Foundation Simplified basic media for human pluripotent cell culture
BR112013004614A2 (pt) 2010-08-31 2024-01-16 Janssen Biotech Inc Diferenciação de células-tronco pluripotentes
MY177150A (en) 2011-02-28 2020-09-08 Stempeutics Res Malaysia Sdn Bhd Isolation and expansion of adult stem cells, their therapeutic composition and uses thereof
US20130274184A1 (en) 2011-10-11 2013-10-17 The Trustees Of Columbia University In The City Of New York Er stress relievers in beta cell protection
EP2766474B1 (fr) 2011-10-14 2020-10-07 Children's Medical Center Corporation Inhibition et amélioration de la re-programmation par des enzymes de modification de la chromatine
US9388386B2 (en) 2011-12-22 2016-07-12 Janssen Biotech, Inc. Differentiation of human embryonic stem cells into single hormonal insulin positive cells
US10519422B2 (en) 2012-02-29 2019-12-31 Riken Method of producing human retinal pigment epithelial cells
CN108103006A (zh) 2012-06-08 2018-06-01 詹森生物科技公司 人胚胎干细胞向胰腺内分泌细胞的分化
US20150247123A1 (en) 2012-09-03 2015-09-03 Novo Nordisk A/S Generation of pancreatic endoderm from Pluripotent Stem cells using small molecules
RU2684215C2 (ru) 2012-12-31 2019-04-04 Янссен Байотек, Инк. Способ получения панкреатических эндокринных клеток (варианты) и способ увеличения выхода бета-клеток
US8859286B2 (en) 2013-03-14 2014-10-14 Viacyte, Inc. In vitro differentiation of pluripotent stem cells to pancreatic endoderm cells (PEC) and endocrine cells
SG10201708332WA (en) 2013-03-15 2017-11-29 Jackson Lab Isolation of non-embryonic stem cells and uses thereof
US11645208B2 (en) 2021-03-29 2023-05-09 International Business Machines Corporation Translation bandwidth optimized prefetching strategy through multiple translation lookaside buffers

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007149182A2 (fr) * 2006-06-19 2007-12-27 Geron Corporation Différenciation et enrichissement de cellules de type cellules des îlots pancréatiques à partir de cellules souches pluripotentes humaines
WO2008004990A2 (fr) * 2006-07-06 2008-01-10 Es Cell International Pte Ltd Procédé de culture de cellules souches et cellules souches ainsi obtenues
WO2008035110A1 (fr) * 2006-09-22 2008-03-27 Riken Milieu de culture de cellules souches et procédé
WO2008102118A1 (fr) * 2007-02-19 2008-08-28 Stem Cell Sciences (Uk) Ltd Production à grande échelle de cellules souches
WO2009105570A2 (fr) * 2008-02-21 2009-08-27 Centocor Ortho Biotech Inc. Procédés, plaques à surface modifiée et compositions permettant la fixation, la culture et le détachement de cellules

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ABRANCHES ELSA ET AL: "Expansion of mouse embryonic stem cells on microcarriers", BIOTECHNOLOGY AND BIOENGINEERING, vol. 96, no. 6, April 2007 (2007-04-01), pages 1211 - 1221, XP002574145, ISSN: 0006-3592 *
FOK ELAINE Y L ET AL: "Shear-controlled single-step mouse embryonic stem cell expansion and embryoid body-based differentiation", STEM CELLS, ALPHAMED PRESS, DAYTON, OH, US, vol. 23, no. 9, 1 October 2005 (2005-10-01), pages 1333 - 1342, XP002445359, ISSN: 1066-5099 *
PHILLIPS B W ET AL: "Attachment and growth of human embryonic stem cells on microcarriers", JOURNAL OF BIOTECHNOLOGY, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 138, no. 1-2, 6 November 2008 (2008-11-06), pages 24 - 32, XP025532877, ISSN: 0168-1656, [retrieved on 20080814] *
WATANABE KIICHI ET AL: "A ROCK inhibitor permits survival of dissociated human embryonic stem cells", NATURE BIOTECHNOLOGY, NATURE PUBLISHING GROUP, NEW YORK, NY, US, vol. 25, no. 6, 1 June 2007 (2007-06-01), pages 681 - 686, XP002458303, ISSN: 1087-0156 *

Cited By (76)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10316293B2 (en) 2007-07-01 2019-06-11 Janssen Biotech, Inc. Methods for producing single pluripotent stem cells and differentiation thereof
US10456424B2 (en) 2007-07-31 2019-10-29 Janssen Biotech, Inc. Pancreatic endocrine cells and methods thereof
US9969982B2 (en) 2007-11-27 2018-05-15 Lifescan, Inc. Differentiation of human embryonic stem cells
US10066203B2 (en) 2008-02-21 2018-09-04 Janssen Biotech Inc. Methods, surface modified plates and compositions for cell attachment, cultivation and detachment
US11001802B2 (en) 2008-02-21 2021-05-11 Nunc A/S Surface of a vessel with polystyrene, nitrogen, oxygen and a static sessile contact angle for attachment and cultivation of cells
US9340770B2 (en) 2008-03-17 2016-05-17 Agency For Science, Technology And Research Microcarriers for stem cell culture
US8637309B2 (en) 2008-03-17 2014-01-28 Agency For Science, Technology And Research Microcarriers for stem cell culture
US8691569B2 (en) 2008-03-17 2014-04-08 Agency For Science, Technology And Research Microcarriers for stem cell culture
US8828720B2 (en) 2008-03-17 2014-09-09 Agency For Science, Technology And Research Microcarriers for stem cell culture
US9458431B2 (en) 2008-03-17 2016-10-04 Agency For Science, Technology And Research Microcarriers for stem cell culture
US9593305B2 (en) 2008-06-30 2017-03-14 Janssen Biotech, Inc. Differentiation of pluripotent stem cells
US10233421B2 (en) 2008-06-30 2019-03-19 Janssen Biotech, Inc. Differentiation of pluripotent stem cells
US10351820B2 (en) 2008-06-30 2019-07-16 Janssen Biotech, Inc. Methods for making definitive endoderm using at least GDF-8
US9593306B2 (en) 2008-06-30 2017-03-14 Janssen Biotech, Inc. Differentiation of pluripotent stem cells
US9388387B2 (en) 2008-10-31 2016-07-12 Janssen Biotech, Inc. Differentiation of human embryonic stem cells
US9752126B2 (en) 2008-10-31 2017-09-05 Janssen Biotech, Inc. Differentiation of human pluripotent stem cells
US9969972B2 (en) 2008-11-20 2018-05-15 Janssen Biotech, Inc. Pluripotent stem cell culture on micro-carriers
US9969973B2 (en) 2008-11-20 2018-05-15 Janssen Biotech, Inc. Methods and compositions for cell attachment and cultivation on planar substrates
US9150829B2 (en) 2009-03-20 2015-10-06 Agency For Science, Technoloy And Research Culture of pluripotent and multipotent cells on microcarriers
US10471104B2 (en) 2009-07-20 2019-11-12 Janssen Biotech, Inc. Lowering blood glucose
US10076544B2 (en) 2009-07-20 2018-09-18 Janssen Biotech, Inc. Differentiation of human embryonic stem cells
GB2484873B (en) * 2009-07-20 2014-04-30 Janssen Biotech Inc Differentiation of pancreatic endoderm cells.
US9593310B2 (en) 2009-12-23 2017-03-14 Janssen Biotech, Inc. Differentiation of human embryonic stem cells
US10704025B2 (en) 2009-12-23 2020-07-07 Janssen Biotech, Inc. Use of noggin, an ALK5 inhibitor and a protein kinase c activator to produce endocrine cells
US9969981B2 (en) 2010-03-01 2018-05-15 Janssen Biotech, Inc. Methods for purifying cells derived from pluripotent stem cells
US10329534B2 (en) 2010-03-01 2019-06-25 Janssen Biotech, Inc. Methods for purifying cells derived from pluripotent stem cells
US9752125B2 (en) 2010-05-12 2017-09-05 Janssen Biotech, Inc. Differentiation of human embryonic stem cells
EP3524672A3 (fr) * 2010-06-16 2019-12-18 Minerva Biotechnologies Corporation Reprogrammation de cellules cancéreuses
US9458430B2 (en) 2010-08-31 2016-10-04 Janssen Biotech, Inc. Differentiation of pluripotent stem cells
US9951314B2 (en) 2010-08-31 2018-04-24 Janssen Biotech, Inc. Differentiation of human embryonic stem cells
US9528090B2 (en) 2010-08-31 2016-12-27 Janssen Biotech, Inc. Differentiation of human embryonic stem cells
US9506036B2 (en) 2010-08-31 2016-11-29 Janssen Biotech, Inc. Differentiation of human embryonic stem cells
US9181528B2 (en) 2010-08-31 2015-11-10 Janssen Biotech, Inc. Differentiation of pluripotent stem cells
CN105112361A (zh) * 2010-09-02 2015-12-02 生命技术公司 用于细胞的生物反应器放大的细胞培养系统
CN105112361B (zh) * 2010-09-02 2018-11-16 生命技术公司 用于细胞的生物反应器放大的细胞培养系统
WO2012044979A2 (fr) 2010-10-01 2012-04-05 The Goverment Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Manipulation de la fonction de cellules souches par des isoformes p53
JP5938726B2 (ja) * 2010-10-28 2016-06-22 国立大学法人 熊本大学 多能性幹細胞の分化誘導効率を改善するための方法及び培地
JPWO2012056997A1 (ja) * 2010-10-28 2014-05-12 国立大学法人 熊本大学 多能性幹細胞の分化誘導効率を改善するための方法及び培地
US10000739B2 (en) 2010-10-28 2018-06-19 National University Corporation Kumamoto University Method and culture medium for improving pluripotent stem cell differentiation inducing efficiency
US10358628B2 (en) 2011-12-22 2019-07-23 Janssen Biotech, Inc. Differentiation of human embryonic stem cells into single hormonal insulin positive cells
US11377640B2 (en) 2011-12-22 2022-07-05 Janssen Biotech, Inc. Differentiation of human embryonic stem cells into single hormonal insulin positive cells
US12215354B2 (en) 2011-12-22 2025-02-04 Janssen Biotech, Inc. Differentiation of human embryonic stem cells into single hormonal insulin positive cells
US9434920B2 (en) 2012-03-07 2016-09-06 Janssen Biotech, Inc. Defined media for expansion and maintenance of pluripotent stem cells
US9593307B2 (en) 2012-03-07 2017-03-14 Janssen Biotech, Inc. Defined media for expansion and maintenance of pluripotent stem cells
US10066210B2 (en) 2012-06-08 2018-09-04 Janssen Biotech, Inc. Differentiation of human embryonic stem cells into pancreatic endocrine cells
US10208288B2 (en) 2012-06-08 2019-02-19 Janssen Biotech, Inc. Differentiation of human embryonic stem cells into pancreatic endocrine cells
KR20220147706A (ko) 2012-07-24 2022-11-03 닛산 가가쿠 가부시키가이샤 배지 조성물 및 당해 조성물을 사용한 세포 또는 조직의 배양 방법
KR20190133287A (ko) 2012-07-24 2019-12-02 닛산 가가쿠 가부시키가이샤 배지 조성물 및 당해 조성물을 사용한 세포 또는 조직의 배양 방법
KR20210022778A (ko) 2012-07-24 2021-03-03 닛산 가가쿠 가부시키가이샤 배지 조성물 및 당해 조성물을 사용한 세포 또는 조직의 배양 방법
KR20210118251A (ko) 2012-07-24 2021-09-29 닛산 가가쿠 가부시키가이샤 배지 조성물 및 당해 조성물을 사용한 세포 또는 조직의 배양 방법
RU2665793C2 (ru) * 2012-07-24 2018-09-04 Ниссан Кемикал Индастриз, Лтд. Композиция культуральной среды и способ культивирования клетки или ткани с использованием указанной композиции
WO2014017513A1 (fr) 2012-07-24 2014-01-30 日産化学工業株式会社 Composition de type milieu de culture, et procédé de culture d'une cellule ou d'un tissue l'utilisant
US11371013B2 (en) 2012-07-24 2022-06-28 Nissan Chemical Industries, Ltd. Culture medium composition and method of culturing cell or tissue using thereof
US10590380B2 (en) 2012-07-24 2020-03-17 Nissan Chemical Industries, Ltd. Culture medium composition and method of culturing cell or tissue using thereof
EP3409761A1 (fr) 2012-07-24 2018-12-05 Nissan Chemical Corporation Composition de milieu de culture et procédé de culture cellulaire ou tissulaire utilisant ladite composition
US9664671B2 (en) 2012-07-24 2017-05-30 Nissan Chemical Industries, Ltd. Culture medium composition and method of culturing cell or tissue using thereof
KR20150038190A (ko) 2012-07-24 2015-04-08 닛산 가가쿠 고교 가부시키 가이샤 배지 조성물 및 당해 조성물을 사용한 세포 또는 조직의 배양 방법
US10017805B2 (en) 2012-08-23 2018-07-10 Nissan Chemical Industries, Ltd. Enhancing ingredients for protein production from various cells
US10221392B2 (en) 2012-09-03 2019-03-05 Novo Nordisk A/S Generation of pancreatic endoderm from pluripotent stem cells using small molecules
US10344264B2 (en) 2012-12-31 2019-07-09 Janssen Biotech, Inc. Culturing of human embryonic stem cells at the air-liquid interface for differentiation into pancreatic endocrine cells
US10377989B2 (en) 2012-12-31 2019-08-13 Janssen Biotech, Inc. Methods for suspension cultures of human pluripotent stem cells
US10370644B2 (en) 2012-12-31 2019-08-06 Janssen Biotech, Inc. Method for making human pluripotent suspension cultures and cells derived therefrom
US10138465B2 (en) 2012-12-31 2018-11-27 Janssen Biotech, Inc. Differentiation of human embryonic stem cells into pancreatic endocrine cells using HB9 regulators
US10947511B2 (en) 2012-12-31 2021-03-16 Janssen Biotech, Inc. Differentiation of human embryonic stem cells into pancreatic endocrine cells using thyroid hormone and/or alk5, an inhibitor of tgf-beta type 1 receptor
US11291691B2 (en) 2013-09-19 2022-04-05 The Board Of Trustees Of The Leland Stanford Junior University Methods and compositions for producing hepatocyte-like cells
KR20160117496A (ko) 2014-01-23 2016-10-10 닛산 가가쿠 고교 가부시키 가이샤 배양 배지 조성물
WO2015111686A1 (fr) 2014-01-23 2015-07-30 日産化学工業株式会社 Composition de milieu de culture
KR20210034121A (ko) 2014-01-23 2021-03-29 닛산 가가쿠 가부시키가이샤 배양 배지 조성물
KR20220020418A (ko) 2014-01-23 2022-02-18 닛산 가가쿠 가부시키가이샤 배양 배지 조성물
KR20220088523A (ko) 2014-01-23 2022-06-27 닛산 가가쿠 가부시키가이샤 배양 배지 조성물
WO2015111685A1 (fr) 2014-01-23 2015-07-30 日産化学工業株式会社 Procédé de production d'une composition pour milieu de culture
US10870832B2 (en) 2014-05-16 2020-12-22 Janssen Biotech, Inc. Use of small molecules to enhance MAFA expression in pancreatic endocrine cells
US10006006B2 (en) 2014-05-16 2018-06-26 Janssen Biotech, Inc. Use of small molecules to enhance MAFA expression in pancreatic endocrine cells
US10420803B2 (en) 2016-04-14 2019-09-24 Janssen Biotech, Inc. Differentiation of pluripotent stem cells to intestinal midgut endoderm cells
JP7588097B2 (ja) 2019-06-13 2024-11-21 コーニング インコーポレイテッド 細胞を培養するための溶解性マイクロキャリア及び関連する方法
CN110564675A (zh) * 2019-09-30 2019-12-13 广东华夏健康生命科学有限公司 一种毛囊干细胞的分离提取方法

Also Published As

Publication number Publication date
US20150166950A1 (en) 2015-06-18
KR20110091768A (ko) 2011-08-12
HK1246356A1 (zh) 2018-09-07
AU2016201220A1 (en) 2016-03-17
BRPI0920956A2 (pt) 2015-08-18
EP2366021B1 (fr) 2017-08-02
ZA201606403B (en) 2018-05-30
US20100124781A1 (en) 2010-05-20
KR20170102075A (ko) 2017-09-06
ZA201104506B (en) 2018-11-28
CN102307991B (zh) 2017-08-15
CN102307991A (zh) 2012-01-04
AU2016201220B2 (en) 2017-09-28
EP3260534A1 (fr) 2017-12-27
ES2642070T3 (es) 2017-11-15
KR101774546B1 (ko) 2017-09-04
RU2011124900A (ru) 2012-12-27
US20180265842A1 (en) 2018-09-20
AU2009316580A1 (en) 2010-05-27
MX356756B (es) 2018-06-11
CA2744234A1 (fr) 2010-05-27
CA2744234C (fr) 2018-11-06
RU2555538C2 (ru) 2015-07-10
US9969972B2 (en) 2018-05-15
CN107267442A (zh) 2017-10-20
EP2366021A1 (fr) 2011-09-21
PL2366021T3 (pl) 2017-12-29
JP2012509085A (ja) 2012-04-19
MX2011005288A (es) 2011-06-01
KR101837080B1 (ko) 2018-03-09
AU2009316580B2 (en) 2016-04-14

Similar Documents

Publication Publication Date Title
AU2016201220B2 (en) Pluripotent stem cell culture on micro-carriers
CA2691975C (fr) Culture de cellule souche pluripotente unique
AU2009316583B2 (en) Methods and compositions for cell attachment and cultivation on planar substrates
US10316293B2 (en) Methods for producing single pluripotent stem cells and differentiation thereof

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 200980155382.2

Country of ref document: CN

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 09759851

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2009316580

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 12011500959

Country of ref document: PH

WWE Wipo information: entry into national phase

Ref document number: 2744234

Country of ref document: CA

Ref document number: MX/A/2011/005288

Country of ref document: MX

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 2011537603

Country of ref document: JP

WWE Wipo information: entry into national phase

Ref document number: 2294/KOLNP/2011

Country of ref document: IN

ENP Entry into the national phase

Ref document number: 2009316580

Country of ref document: AU

Date of ref document: 20091119

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 20117013824

Country of ref document: KR

Kind code of ref document: A

REEP Request for entry into the european phase

Ref document number: 2009759851

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 2009759851

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 2011124900

Country of ref document: RU

ENP Entry into the national phase

Ref document number: PI0920956

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20110519