[go: up one dir, main page]

US20150247123A1 - Generation of pancreatic endoderm from Pluripotent Stem cells using small molecules - Google Patents

Generation of pancreatic endoderm from Pluripotent Stem cells using small molecules Download PDF

Info

Publication number
US20150247123A1
US20150247123A1 US14/425,136 US201314425136A US2015247123A1 US 20150247123 A1 US20150247123 A1 US 20150247123A1 US 201314425136 A US201314425136 A US 201314425136A US 2015247123 A1 US2015247123 A1 US 2015247123A1
Authority
US
United States
Prior art keywords
cells
pancreatic
inhibitor
pancreatic cell
cell precursors
Prior art date
Legal status (The legal status 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 status listed.)
Abandoned
Application number
US14/425,136
Inventor
Jenny Ekberg
Mattias Hansson
Ulrik Doehn
Katja Hess
Nina Funa
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novo Nordisk AS
Cellectis SA
Takara Bio Europe AB
Original Assignee
Novo Nordisk AS
Cellectis SA
Takara Bio Europe AB
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
Application filed by Novo Nordisk AS, Cellectis SA, Takara Bio Europe AB filed Critical Novo Nordisk AS
Priority to US14/425,136 priority Critical patent/US20150247123A1/en
Assigned to NOVO NORDISK A/S reassignment NOVO NORDISK A/S ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: HESS, Katja, FUNA, Nina, HANSSON, MATTIAS, EKBERG, JENNY, DOEHN, ULRIK
Publication of US20150247123A1 publication Critical patent/US20150247123A1/en
Assigned to NOVO NORDISK A/S reassignment NOVO NORDISK A/S ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: HESS, Katja, DOEHN, ULRIK, FUNA, Nina, HANSSON, MATTIAS, EKBERG, JENNY
Assigned to CELLECTIS SA reassignment CELLECTIS SA ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: HESS, Katja, DOEHN, ULRIK, FUNA, Nina, HANSSON, MATTIAS, EKBERG, JENNY
Assigned to TAKARA BIO EUROPE AB reassignment TAKARA BIO EUROPE AB ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: CELLECTIS SA
Abandoned legal-status Critical Current

Links

Images

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
    • C12N5/0676—Pancreatic 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
    • C12N5/0676—Pancreatic cells
    • C12N5/0678—Stem cells; Progenitor cells; Precursor 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • 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
    • C12N2501/10—Growth factors
    • C12N2501/117—Keratinocyte growth factors (KGF-1, i.e. FGF-7; KGF-2, i.e. FGF-12)
    • 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
    • C12N2501/10—Growth factors
    • C12N2501/119—Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
    • 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
    • C12N2501/10—Growth factors
    • C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
    • 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
    • C12N2501/10—Growth factors
    • C12N2501/16—Activin; Inhibin; Mullerian inhibiting substance
    • 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
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/385—Hormones with nuclear receptors of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
    • 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
    • C12N2501/40—Regulators of development
    • C12N2501/41—Hedgehog proteins; Cyclopamine (inhibitor)
    • 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
    • C12N2501/40—Regulators of development
    • C12N2501/415—Wnt; Frizzeled
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening
    • 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells

Definitions

  • the present invention relates to methods of generating pancreatic endoderm from pluripotent stem (PS) cells, such as human definitive endoderm.
  • PS pluripotent stem
  • Beta cell transplantation potentially provides the ultimate cure for type I diabetes.
  • the limited availability of donor beta cells constrains the use of this treatment as a clinical therapy.
  • Pluripotent stem cells can proliferate infinitely and differentiate into many cell types; thus, PS cells are a promising source for beta cells.
  • PS cells before PS cells can be used to treat diabetes, they need to be efficiently and reproducibly differentiated to pancreatic cells.
  • a pluripotent cell gives rise to the three germ layers; ectoderm, mesoderm and endoderm.
  • Induction of definitive endoderm (DE) is the first step towards formation of endoderm derived tissues.
  • Generation of pancreatic endoderm (PE) from DE cells is necessary for the generation of insulin-producing beta cells.
  • PE cells with the potential to become endocrine progenitors (EP) are characterized by co-expression of two important transcription factors, PDX1 and NKX6.1.
  • Stepwise in vitro differentiation protocols have been established for generating pancreatic cells from PS cells. These protocols generally mimic the major events of pancreatic development, which includes several stages such as formation of the DE which co-expresses SOX17 and FOXA2, primitive gut, posterior foregut, PE, EP and ultimately the mature beta cells. To date, efficient DE differentiation of hES cells has been achieved by activin A treatment. The next major step in generating pancreatic beta cells is to generate PE that co-expresses PDX1 and NKX6.1.
  • the present invention relates to a method of producing pancreatic cells or pancreatic cell precursors where at least 5% of the cells co-express PDX1 and NKX6.1, comprising exposing definitive endoderm (DE) cells to an effective amount of at least one compound of the group consisting of:
  • the present invention further relates to a method for producing pancreatic cells or pancreatic cell precursors where at least 5% of the cells co-express PDX1 and NKX6.1, comprising exposing DE cells to an effective amount of at least one compound of the group consisting of:
  • the present invention further relates to a method for producing pancreatic cells or pancreatic cell precursors where at least 5% of the cells co-express PDX1 and NKX6.1, comprising exposing definitive endoderm cells to an effective amount of at least one compound of the group consisting of:
  • the present invention further relates to a method for generating pancreatic cells or pancreatic cell precursors where at least 5% of the cells co-express PDX1 and NKX6.1, comprising exposing definitive endoderm cells to an effective amount of the BMP inhibitor LDN-193189, to differentiate human DE cells into pancreatic or pancreatic cell precursors.
  • the present invention further relates to a method for generating pancreatic cells or pancreatic cell precursors where at least 5% of the cells co-express PDX1 and NKX6.1, comprising exposing DE cells to an effective amount of the BMP inhibitor LDN-193189, and subsequent exposure to one of the following molecules:
  • the present invention further relates to a method for generating pancreatic cells or pancreatic cell precursors where at least 5% of the cells co-express PDX1 and NKX6.1, comprising exposing DE cells to an effective amount of the BMP inhibitor LDN-193189, and subsequent exposure to a combination of JNK inhibitor II, retinoic acid or a retinoic acid derivative, bFGF and one of the following molecules:
  • the present invention further relates to a method for generating pancreatic cells or pancreatic cell precursors where at least 5% of the cells co-express PDX1 and NKX6.1, comprising exposing DE cells to an effective amount of the BMP inhibitor LDN-193189, and subsequent exposure to a combination of JNK inhibitor II in combination with retinoic acid or a retinoic acid derivative, bFGF and LDN-193189 to differentiate DE stem cells into pancreatic or pancreatic cell precursors.
  • any one of the retinoic acid receptor agonists or kinase inhibitors may be in combination with bFGF.
  • the present invention further relates to pancreatic cells or pancreatic cell precursors obtainable by the methods of the present invention.
  • the present invention relates to a pancreatic cell or pancreatic cell precursor produced by exposing a human pluripotent stem cell to at least one compound listed in tables 1 and 2.
  • the present invention relates to use of any one of the compounds of tables 1 and 2, to induce pancreatic cells or pancreatic cell precursors from stem cells.
  • the present invention relates to use of LDN-193189 to induce pancreatic cells or pancreatic cell precursors from stem cells.
  • the present invention relates to use of LDN-193189 followed by Cyclopamine or IWP2, to induce pancreatic cells or pancreatic cell precursors from stem cells.
  • the present invention takes an alternative approach to improve the efficiency of differentiating human PS cells toward mature beta cells, by providing a method to increase the fraction of NKX6.1/PDX1 double positive cells, a hallmark for PE cells committed to an endocrine cell fate.
  • the invention provides an improved pancreatic cell population, i.e. PE with increased fraction of NKX6.1/PDX1 double positive cells.
  • the present invention provides a more homogenous pancreatic cell population, which is important for the further development of these cells towards the endocrine lineage.
  • the present invention also provides a more synchronised pancreatic population to get to the next stage.
  • the present invention may also solve further problems that will be apparent from the disclosure of the exemplary embodiments.
  • FIG. 1 shows the PE screening approach—also referred to as the library screening approach—using small molecule libraries.
  • Pluripotent stem (PS) cells were differentiated into definitive endoderm (DE) according to the DE protocol (see general methods) and seeded in 96 well plates for screening.
  • the pancreatic endoderm (PE) screen was divided into an early and a late phase. In the early phase compounds were added on top of a published bFGF based protocol (Amen et al., 2010, cf. also WO/2010/136583) for the first seven days of PE differentiation and then continued for another six days without the compounds. In the late phase compounds were only added on top of the bFGF based protocol for the last six days.
  • FIG. 2 shows early phase hits for the library screening approach.
  • Definitive endoderm cells from human induced pluripotent stem cells (hiPSC) (black) or hESC (white) were seeded in 96 well optical plates and differentiated into pancreatic endoderm using a 14 day protocol based on bFGF.
  • Compounds were added on top of the bFGF based protocol for the first seven out of 14 days and analysed for NKX6.1/PDX1 double positive cells using the InCell analyzer 2000 (GE Healthcare).
  • the graph shows the % effect of the fraction of NKX6.1/PDX1 double positive cells compared to the Benchmark protocol.
  • FIG. 3 shows late phase hits for the library screening approach.
  • Definitive endoderm cells from hiPSC (black) or hESC (white) were seeded in 96 well optical plates and differentiated into pancreatic endoderm using a 14 day protocol based on bFGF.
  • Compounds were added on top of the bFGF based protocol for the last six days and analysed for NKX6.1/PDX1 double positive cells using the InCell analyzer 2000 (GE Healthcare).
  • the graph shows the % effect of the fraction of NKX6.1/PDX1 double positive cells compared to the Benchmark protocol.
  • FIG. 4 shows a second, candidate based PE screening approach.
  • Pluripotent stem (PS) cells were differentiated into definitive endoderm according to DE protocol (See general methods) and seeded in 96 well plates for screening.
  • the pancreatic endoderm screen was divided into two parts. In screen 1, compounds were added to a basal medium (RPMI1640+0.1% PEST+12% KOSR) the first eight days of PE differentiation. Compounds were tested in 4 different time windows having 2 day increments and then cells were left to continue differentiation for another six days in the bFGF based published protocol (Amen et al., 2010). In screen 2, cells were first differentiated for 4 days with the hit compounds from screen 1, then screening compounds were added the last 10 days to basal medium.
  • FIG. 5 shows hits from the candidate screen 1 and 2 compared to cells differentiated according to Amen et al, 2010 which was used as a benchmark protocol running in parallel with every screen.
  • one hit compound was identified (LDN-193189) and was found to be most effective when added for the first 4 days followed by 4 days basal medium.
  • two hit compounds were identified (Cyclopamine and IWP-2) when cells were first exposed to the hit compound from screen 1 for 4 days and hit compounds from screen 2 were added for the last 10 days of differentiation.
  • the graph shows the % effect of the fraction of NKX6.1/PDX1 double positive cells compared to the Benchmark protocol (Bars for hiPSC in black and hESC in white).
  • FIG. 6 shows the advantageous effect on the amount of PDX1/NKX6.1 double positive cells by the combination of hit compounds found in the two individual screens (small molecule libraries and candidate approach) compared to the benchmark protocol (Ameri et al. (2010)). Bars for hiPSC in black and hESC in white.
  • FGF basic Fibroblast Growth Factor
  • hBS human Blastocyst derived Stem hBSC; human Blastocyst-derived Stem Cells hES: human Embryonic Stem hESC: human Embryonic Stem Cells hiPSC: human induced Pluripotent Stem Cells hPSC: human Pluripotent Stem Cells
  • NKX6.1 NK6 homeobox 1
  • PDX1 Pancreatic and duodenal homeobox 1
  • PS Pluripotent Stem
  • the present invention related to methods of generating pancreatic endoderm from stem cells, such as human definitive endoderm cells and induced pluripotent stem cells.
  • the present invention takes an alternative approach to improve the efficiency of differentiating human PS cells toward mature beta cells, by providing a method to improve the percentage of NKX6.1/PDX1 double positive cells, which are markers for a PE cell population, one of the cell stages necessary to reach endocrine cell populations. Furthermore, the present invention provides a more homogenous and synchronised pancreatic cell population, which is important for the further development of these cells towards the endocrine lineage.
  • the present invention may also solve further problems that will be apparent from the disclosure of the exemplary embodiments.
  • the pancreatic endocrine cells obtainable by the method according to the invention are insulin producing cells, optionally together with cells differentiated towards glucagon, somatostatin, pancreatic polypeptide, and/or ghrelin producing cells.
  • insulin producing cells refers to cells that produce and store or secrete detectable amounts of insulin.
  • Insulin producing cells can be individual cells or collections of cells.
  • the cell population comprising pancreatic cells is obtained from a somatic cell population.
  • the somatic cell population has been induced to de-differentiate into an embryonic-like stem (ES, e.g., a pluripotent) cell.
  • ES embryonic-like stem
  • iPSC induced pluripotent stem cells
  • the cell population comprising pancreatic cells is obtained from embryonic stem (ES, e.g., pluripotent) cells.
  • ES embryonic stem
  • the cell population comprising pancreatic cells is pluripotent cells such as ES like-cells.
  • the cell population comprising pancreatic cells is embryonic differentiated stem (ES or pluripotent) cells. Differentiation takes place in embryoid bodies and/or in monolayer cell cultures or a combination thereof.
  • ES embryonic differentiated stem
  • the cell population is a population of stem cells.
  • the cell population is a population of stem cells differentiated to the pancreatic endocrine lineage.
  • 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) multi-potent, 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 multi-potent stem cells; and (5) unipotent, meaning able to give rise to a single cell lineage (e.g., spermatogenic stem cells).
  • HSC hematopoietic stem cells
  • a protocol for obtaining pancreatic cells from stem cells is exemplified by, but not limited to, the protocols described in D'Amour, K. A. et al. (2006); Jiang, J. et al. (2007); Kroon, E. et al. (2008).
  • a protocol for obtaining pancreatic cells from somatic cells or somatic cells induced to de-differentiate into pluripotent cells such as ES like-cells is exemplified by, but not limited to, the protocols described in Aoi, T. et al. (2008); D'Amour, K. A. et al. (2006); Jiang, J. et al. (2007); Kroon, E. et al. (2008); Takahashi, K. et al. (2007); Takahashi, K., and Yamanaka, S. (2006) and Wernig, M. et al. (2007).
  • differentiate refers to a process where cells progress from an undifferentiated state to a differentiated state, from an immature state to a less immature state or from an immature state to a mature state.
  • characteristics markers like PDX1, NKX6.1, and PTF1a.
  • Mature or differentiated pancreatic cells do not proliferate and do secrete high levels of pancreatic endocrine hormones or digestive enzymes.
  • fully differentiated beta cells secrete insulin at high levels in response to glucose. Changes in cell interaction and maturation occur as cells lose markers of undifferentiated cells or gain markers of differentiated cells.
  • differentiation factor refers to a compound added to pancreatic cells to enhance their differentiation to mature endocrine cells also containing insulin producing beta cells.
  • exemplary differentiation factors include hepatocyte growth factor, keratinocyte growth factor, exendin-4, basic fibroblast growth factor, insulin-like growth factor-1, nerve growth factor, epidermal growth factor platelet-derived growth factor, and glucagon-like peptide 1.
  • differentiation of the cells comprises culturing the cells in a medium comprising one or more differentiation factors.
  • human pluripotent stem cells refers to cells that may be derived from any source and that are capable, under appropriate conditions, of producing human progeny of different cell types that are derivatives of all of the 3 germinal layers (endoderm, mesoderm, and ectoderm). hPSC may have the ability to form a teratoma in 8-12 week old SCID mice and/or the ability to form identifiable cells of all three germ layers in tissue culture. Included in the definition of human pluripotent stem cells are embryonic cells of various types including human blastocyst derived stem (hBS) cells in 30 literature often denoted as human embryonic stem (hES) cells, (see, e.g., Thomson et al.
  • hBS human blastocyst derived stem
  • hES human embryonic stem
  • hPSC human induced pluripotent stem
  • hiPSC human induced pluripotent stem cells
  • ES cell lines can also be derived from single blastomeres without the destruction of ex utero embryos and without affecting the clinical outcome (Chung et al. (2006) and Klimanskaya et al. (2006)).
  • the term “blastocyst-derived stem cell” is denoted BS cell, and the human form is termed “hBS cells”.
  • the pluripotent stem cells used in the present invention can thus be embryonic stem cells prepared from blastocysts, as described in e.g. WO 03/055992 and WO 2007/042225, or be commercially available hBS cells or cell lines.
  • any human pluripotent stem cell can be used in the present invention, including differentiated adult cells which are reprogrammed to pluripotent cells by e.g. the treating adult cells with certain transcription factors, such as OCT4, SOX2, NANOG, and LIN28 as disclosed in Yu, et al. (2007); Takahashi et al. (2007) and Yu et al. (2009).
  • JNK inhibitor II includes isomers or tautomers of 1,9-pyrazoloanthrone with or without N-alkylation.
  • DEF medium or DEF-CS medium/system is a defined balanced culture medium for the establishment and propagation of human pluripotent stem cells, DEF-CS medium/system.
  • hES cells line SA121 and human induced pluripotent stem cells (hiPSC) chIPS4 (Cellectis) were grown in DEF-CS culture media (Cellectis) in T75 culture flasks.
  • Cells were single cell passaged with 5 ⁇ M Rock inhibitor Y-27632 (Sigma #Y0503) and seeded at a density of 40000 cells/cm2 for experiments.
  • Cells were cultured at 37° C. and 5% CO 2 in a humidified incubator (ThermoScientific Model 371).
  • hES cells line SA121 and hiPSC (chIPS4) were washed once in RPMI1640 (Gibco #61870) and treated with 3 ⁇ M CHIR99021 (Axon#1386) in RPMI1640. After 24 hours the cells were washed with RPMI1640 and treated with 100 ng/ml Activin A (Peprotech #120-14E) in RPMI1640. 24 hours later, 2% B27 (Invitrogen #17504-044) was added to the Activin A media for 2 days with daily media change. Cells were maintained at 37° C. and 5% CO 2 in a humidified incubator during the differentiation.
  • DAPI 4,6-diamidino-2-phenylindole, Applichem, A4099.0010
  • secondary antibodies Alexa Fluor 488 donkey anti-goat and Alexa Fluor 594 donkey anti-mouse (both Invitrogen) were diluted 1:1000 in 0.1% Triton X-100 in PBS and added to each well for 45 min. Cells were washed five times and left in 200 ⁇ L PBS for imaging.
  • Imaging was performed using the InCell Analyzer 2000 (GE Healthcare). 4 fields per well with 10 ⁇ objective were captured. The total cell number based in the DAPI counterstaining and the number of NKX6.1/PDX1 double positive cells was determined using InCell Developer Toolbox 1.8 (GE Healthcare). The fraction of NKX6.1/PDX1 double positive cells was normalized to the benchmark on each plate and the % effect was calculated. Values above 200% effect were categorized as hits.
  • Pancreatic endoderm is characterized by co-expression of two transcription factors, NKX6.1 and PDX1. Many of the published protocols for making PE are ineffective with low outcome of NKX6.1/PDX1 double positive cells. Enhancing the efficacy of the PE protocols is a desirable outcome. We therefore screened libraries of small molecules to identify novel compounds that would improve the existing PE protocols. This was done on the assumption that inhibitors, agonists or antagonists may regulate signaling pathways, or chromosomal accessibility, which would improve the fraction of NKX6.1/PDX1 double positive cells.
  • kinase inhibitor library (Calbiochem #539743), a bioactive lipid library (Enzo Life Sciences #BML-2800), a nuclear receptor ligand library (Enzo Life Sciences # BML-2802) and a phosphatase inhibitor library (Enzo Life Sciences #BML-2834).
  • the compounds within the bioactive library were tested at 1 uM and 0.1 uM.
  • Compounds from the other libraries were tested at 10 uM and 1 uM.
  • small molecules that target the main signalling pathways involved in pancreas development were included.
  • NKX6.1/PDX1 Screen The library compounds were screened on top of a bFGF based media formulation for making PE (Ameri et al. 2010) (RPMI1640, Gibco#61870; 12% KOSR, Gibco#10828; 0.1% PEST, Gibco#15140; 64 ng/mL bFGF, Peprotech #100-18B).
  • the library PE screening approach was divided into an early and a late phase ( FIG. 1 ).
  • DE cells were differentiated in the PE media for the first seven days. In the following six days compounds were tested on top of the PE media. 12 positive control wells (PE media) and 12 negative control wells (PE media without bFGF) were included in each 96 well plate. Media change was performed daily. Hits identified in the early phase screen are illustrated in FIG. 2 and listed in table 1. Hits identified in the late phase screen are illustrated in FIG. 3 and listed in table 2.
  • the compounds from the candidate approach were screened in basal medium (RPMI1640, Gibco#61870; 12% KOSR, Gibco#10828; 0.1% PEST, Gibco#15140) without the addition of bFGF.
  • This candidate approach screen was divided into two parts ( FIG. 4 ). In the first part, compounds were tested in time intervals with 2 day increments for the first eight days of PE differentiation (2 days exposure to compounds followed by 6 days basal medium or 4 days exposure to compounds followed by 4 days basal medium or 6 days exposure to compounds followed by 2 days basal medium or 8 days exposure to compounds).
  • DE cells were differentiated according to the hit compounds from the first part, the following 6-10 days compounds were tested in basal media.
  • Hits identified in the candidate screening approach are illustrated in FIG. 5 and also contained in Tables 1 and 2.
  • DE cells were exposed to 4 days 50 nM LDN-193189, followed by 8 days AM580 (AH Diagnostics, BML GF104 0025), JNK Inhibitor II (Calbiochem, 420119), 50 nM LDN-193189 and 64 ng/ml FGF2, or AM580, JNK Inhibitor II, 50 nM LDN-193189, 64 ng/ml FGF2 and IWP2, or AM580, JNK Inhibitor II, 50 nM LDN-193189, 64 ng/ml FGF2 and Cyclopamine ( FIG. 6 ). Media change was performed daily.
  • Hit compounds were screened on top of a bFGF based media formulation for making PE (Amen et al. 2010) (RPMI1640, Gibco#61870; 12% KOSR, Gibco#10828; 0.1% PEST, Gibco#15140; 64 ng/mL bFGF, Peprotech #100-18B).
  • the screen was divided into an early and a late phase ( FIG. 1 ).
  • the early phase compounds were tested on top of the PE media for the first seven days of PE differentiation, and then continued for additional six days using PE media without compounds.
  • the late phase DE cells were differentiated in the PE media for the first seven days. In the following six days compounds were tested on top of the PE media. Twelve positive control wells (PE media) and 12 negative control wells (PE media without bFGF) were included in each 96 well plate. Media change was performed daily.

Landscapes

  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

A method of producing pancreatic cells or pancreatic cell precursors expressing at least 5% PDX1/NKX6.1 double positive, comprising exposing definitive endoderm cells to an effective amount of one or more small molecules, to differentiate the human definitive endoderm cells into the pancreatic cells or pancreatic cell precursors. The present invention also relates to pancreatic endoderm cells produced by said methods and uses of said pancreatic endoderm cells.

Description

    TECHNICAL FIELD
  • The present invention relates to methods of generating pancreatic endoderm from pluripotent stem (PS) cells, such as human definitive endoderm.
    • BACKGROUND
  • Beta cell transplantation potentially provides the ultimate cure for type I diabetes. However, the limited availability of donor beta cells constrains the use of this treatment as a clinical therapy. Pluripotent stem cells can proliferate infinitely and differentiate into many cell types; thus, PS cells are a promising source for beta cells. However, before PS cells can be used to treat diabetes, they need to be efficiently and reproducibly differentiated to pancreatic cells.
  • During vertebrate embryonic development, a pluripotent cell gives rise to the three germ layers; ectoderm, mesoderm and endoderm. Induction of definitive endoderm (DE) is the first step towards formation of endoderm derived tissues. Generation of pancreatic endoderm (PE) from DE cells is necessary for the generation of insulin-producing beta cells. PE cells with the potential to become endocrine progenitors (EP) are characterized by co-expression of two important transcription factors, PDX1 and NKX6.1.
  • Stepwise in vitro differentiation protocols have been established for generating pancreatic cells from PS cells. These protocols generally mimic the major events of pancreatic development, which includes several stages such as formation of the DE which co-expresses SOX17 and FOXA2, primitive gut, posterior foregut, PE, EP and ultimately the mature beta cells. To date, efficient DE differentiation of hES cells has been achieved by activin A treatment. The next major step in generating pancreatic beta cells is to generate PE that co-expresses PDX1 and NKX6.1. Several groups have developed in vitro protocols that can differentiate PS cells into DE and PE, however they are only able to generate a modest fraction of NKX6.1/PDX1 double positive (db+ve) cells, and importantly none of them are able to generate fully mature beta cells in vitro (Cai et al. (2010); D'Amour et al. (2006); Kunisada et al. (2012); Schulz et al. (2012); Zhang et al. (2009); Amen et al. (2010)).
    • SUMMARY
  • The present invention relates to a method of producing pancreatic cells or pancreatic cell precursors where at least 5% of the cells co-express PDX1 and NKX6.1, comprising exposing definitive endoderm (DE) cells to an effective amount of at least one compound of the group consisting of:
      • a. BMP inhibitor LDN-193189 (listed in table 1)
      • b. Kinase inhibitors (listed in tables 1 and 2)
      • c. Retinoic acid receptor agonists (listed in table 2)
        to differentiate the human DE cells into the pancreatic cells or pancreatic cell precursors.
  • The present invention further relates to a method for producing pancreatic cells or pancreatic cell precursors where at least 5% of the cells co-express PDX1 and NKX6.1, comprising exposing DE cells to an effective amount of at least one compound of the group consisting of:
      • a. BMP inhibitor LDN-193189 (listed in table 1)
      • b. isomers of 1,9-pyrazoloanthrone with or without N-alkylation (listed in table 1 and 2)
      • c. Retinoic acid receptor agonists (listed in table 2)
        to differentiate the human DE cells into the pancreatic or pancreatic cell precursors.
  • The present invention further relates to a method for producing pancreatic cells or pancreatic cell precursors where at least 5% of the cells co-express PDX1 and NKX6.1, comprising exposing definitive endoderm cells to an effective amount of at least one compound of the group consisting of:
      • a. BMP inhibitor LDN-193189
      • b. JNK inhibitor II
      • c. AM580
        to differentiate the human DE cells into the pancreatic or pancreatic cell precursors.
  • The present invention further relates to a method for generating pancreatic cells or pancreatic cell precursors where at least 5% of the cells co-express PDX1 and NKX6.1, comprising exposing definitive endoderm cells to an effective amount of the BMP inhibitor LDN-193189, to differentiate human DE cells into pancreatic or pancreatic cell precursors. The present invention further relates to a method for generating pancreatic cells or pancreatic cell precursors where at least 5% of the cells co-express PDX1 and NKX6.1, comprising exposing DE cells to an effective amount of the BMP inhibitor LDN-193189, and subsequent exposure to one of the following molecules:
      • a. Wnt inhibitor IWP2
      • b. Hedgehog inhibitor Cyclopamine (Cyc)
        to differentiate human DE cells into pancreatic or pancreatic cell precursors.
  • The present invention further relates to a method for generating pancreatic cells or pancreatic cell precursors where at least 5% of the cells co-express PDX1 and NKX6.1, comprising exposing DE cells to an effective amount of the BMP inhibitor LDN-193189, and subsequent exposure to a combination of JNK inhibitor II, retinoic acid or a retinoic acid derivative, bFGF and one of the following molecules:
      • a. Wnt inhibitor IWP2
      • b. Hedgehog inhibitor Cyclopamine
        to differentiate human DE cells into pancreatic or pancreatic cell precursors.
  • The present invention further relates to a method for generating pancreatic cells or pancreatic cell precursors where at least 5% of the cells co-express PDX1 and NKX6.1, comprising exposing DE cells to an effective amount of the BMP inhibitor LDN-193189, and subsequent exposure to a combination of JNK inhibitor II in combination with retinoic acid or a retinoic acid derivative, bFGF and LDN-193189 to differentiate DE stem cells into pancreatic or pancreatic cell precursors.
  • In one embodiment of the present invention, any one of the retinoic acid receptor agonists or kinase inhibitors may be in combination with bFGF.
  • The present invention further relates to pancreatic cells or pancreatic cell precursors obtainable by the methods of the present invention.
  • The present invention relates to a pancreatic cell or pancreatic cell precursor produced by exposing a human pluripotent stem cell to at least one compound listed in tables 1 and 2.
  • The present invention relates to use of any one of the compounds of tables 1 and 2, to induce pancreatic cells or pancreatic cell precursors from stem cells.
  • The present invention relates to use of LDN-193189 to induce pancreatic cells or pancreatic cell precursors from stem cells.
  • The present invention relates to use of LDN-193189 followed by Cyclopamine or IWP2, to induce pancreatic cells or pancreatic cell precursors from stem cells.
  • The present invention takes an alternative approach to improve the efficiency of differentiating human PS cells toward mature beta cells, by providing a method to increase the fraction of NKX6.1/PDX1 double positive cells, a hallmark for PE cells committed to an endocrine cell fate.
  • In one aspect, the invention provides an improved pancreatic cell population, i.e. PE with increased fraction of NKX6.1/PDX1 double positive cells.
  • Furthermore, the present invention provides a more homogenous pancreatic cell population, which is important for the further development of these cells towards the endocrine lineage.
  • The present invention also provides a more synchronised pancreatic population to get to the next stage.
  • The present invention may also solve further problems that will be apparent from the disclosure of the exemplary embodiments.
    • BRIEF DESCRIPTION OF DRAWINGS
  • FIG. 1 shows the PE screening approach—also referred to as the library screening approach—using small molecule libraries. Pluripotent stem (PS) cells were differentiated into definitive endoderm (DE) according to the DE protocol (see general methods) and seeded in 96 well plates for screening. The pancreatic endoderm (PE) screen was divided into an early and a late phase. In the early phase compounds were added on top of a published bFGF based protocol (Amen et al., 2010, cf. also WO/2010/136583) for the first seven days of PE differentiation and then continued for another six days without the compounds. In the late phase compounds were only added on top of the bFGF based protocol for the last six days.
  • FIG. 2 shows early phase hits for the library screening approach. Definitive endoderm cells from human induced pluripotent stem cells (hiPSC) (black) or hESC (white) were seeded in 96 well optical plates and differentiated into pancreatic endoderm using a 14 day protocol based on bFGF. Compounds were added on top of the bFGF based protocol for the first seven out of 14 days and analysed for NKX6.1/PDX1 double positive cells using the InCell analyzer 2000 (GE Healthcare). The graph shows the % effect of the fraction of NKX6.1/PDX1 double positive cells compared to the Benchmark protocol.
  • FIG. 3 shows late phase hits for the library screening approach. Definitive endoderm cells from hiPSC (black) or hESC (white) were seeded in 96 well optical plates and differentiated into pancreatic endoderm using a 14 day protocol based on bFGF. Compounds were added on top of the bFGF based protocol for the last six days and analysed for NKX6.1/PDX1 double positive cells using the InCell analyzer 2000 (GE Healthcare). The graph shows the % effect of the fraction of NKX6.1/PDX1 double positive cells compared to the Benchmark protocol.
  • FIG. 4 shows a second, candidate based PE screening approach. Pluripotent stem (PS) cells were differentiated into definitive endoderm according to DE protocol (See general methods) and seeded in 96 well plates for screening. The pancreatic endoderm screen was divided into two parts. In screen 1, compounds were added to a basal medium (RPMI1640+0.1% PEST+12% KOSR) the first eight days of PE differentiation. Compounds were tested in 4 different time windows having 2 day increments and then cells were left to continue differentiation for another six days in the bFGF based published protocol (Amen et al., 2010). In screen 2, cells were first differentiated for 4 days with the hit compounds from screen 1, then screening compounds were added the last 10 days to basal medium.
  • FIG. 5 shows hits from the candidate screen 1 and 2 compared to cells differentiated according to Amen et al, 2010 which was used as a benchmark protocol running in parallel with every screen. In screen 1, one hit compound was identified (LDN-193189) and was found to be most effective when added for the first 4 days followed by 4 days basal medium. For screen 2, two hit compounds were identified (Cyclopamine and IWP-2) when cells were first exposed to the hit compound from screen 1 for 4 days and hit compounds from screen 2 were added for the last 10 days of differentiation. The graph shows the % effect of the fraction of NKX6.1/PDX1 double positive cells compared to the Benchmark protocol (Bars for hiPSC in black and hESC in white).
  • FIG. 6 shows the advantageous effect on the amount of PDX1/NKX6.1 double positive cells by the combination of hit compounds found in the two individual screens (small molecule libraries and candidate approach) compared to the benchmark protocol (Ameri et al. (2010)). Bars for hiPSC in black and hESC in white.
    •  LIST OF ABBREVIATIONS
  • +ve: positive
    bFGF: basic Fibroblast Growth Factor (FGF) (also known as FGF2)
    • Cyc: Cyclopamine
  • db: double positive
    • DE: Definitive Endoderm
  • hBS: human Blastocyst derived Stem
    hBSC; human Blastocyst-derived Stem Cells
    hES: human Embryonic Stem
    hESC: human Embryonic Stem Cells
    hiPSC: human induced Pluripotent Stem Cells
    hPSC: human Pluripotent Stem Cells
    • KOSR: Knock-out Serum Replacement
  • NKX6.1: NK6 homeobox 1
    PDX1: Pancreatic and duodenal homeobox 1
    • PEST: Penicillin Streptomycin PS: Pluripotent Stem Rockout: Rho Kinase Inhibitor III RT: Room Temperature DESCRIPTION
  • The present invention related to methods of generating pancreatic endoderm from stem cells, such as human definitive endoderm cells and induced pluripotent stem cells.
  • The present invention takes an alternative approach to improve the efficiency of differentiating human PS cells toward mature beta cells, by providing a method to improve the percentage of NKX6.1/PDX1 double positive cells, which are markers for a PE cell population, one of the cell stages necessary to reach endocrine cell populations. Furthermore, the present invention provides a more homogenous and synchronised pancreatic cell population, which is important for the further development of these cells towards the endocrine lineage.
  • The present invention may also solve further problems that will be apparent from the disclosure of the exemplary embodiments.
  • In one embodiment, the pancreatic endocrine cells obtainable by the method according to the invention are insulin producing cells, optionally together with cells differentiated towards glucagon, somatostatin, pancreatic polypeptide, and/or ghrelin producing cells. As used herein, “insulin producing cells” refers to cells that produce and store or secrete detectable amounts of insulin. “Insulin producing cells” can be individual cells or collections of cells.
  • In another embodiment, the cell population comprising pancreatic cells is obtained from a somatic cell population. In some aspects the somatic cell population has been induced to de-differentiate into an embryonic-like stem (ES, e.g., a pluripotent) cell. Such de-differentiated cells are also termed induced pluripotent stem cells (iPSC).
  • In another embodiment, the cell population comprising pancreatic cells is obtained from embryonic stem (ES, e.g., pluripotent) cells. In some aspects the cell population comprising pancreatic cells is pluripotent cells such as ES like-cells.
  • In another embodiment, the cell population comprising pancreatic cells is embryonic differentiated stem (ES or pluripotent) cells. Differentiation takes place in embryoid bodies and/or in monolayer cell cultures or a combination thereof.
  • In another embodiment, the cell population is a population of stem cells. In some aspects the cell population is a population of stem cells differentiated to the pancreatic endocrine lineage.
  • 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) multi-potent, 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 multi-potent stem cells; and (5) unipotent, meaning able to give rise to a single cell lineage (e.g., spermatogenic stem cells).
  • A protocol for obtaining pancreatic cells from stem cells is exemplified by, but not limited to, the protocols described in D'Amour, K. A. et al. (2006); Jiang, J. et al. (2007); Kroon, E. et al. (2008).
  • A protocol for obtaining pancreatic cells from somatic cells or somatic cells induced to de-differentiate into pluripotent cells such as ES like-cells is exemplified by, but not limited to, the protocols described in Aoi, T. et al. (2008); D'Amour, K. A. et al. (2006); Jiang, J. et al. (2007); Kroon, E. et al. (2008); Takahashi, K. et al. (2007); Takahashi, K., and Yamanaka, S. (2006) and Wernig, M. et al. (2007).
  • As used herein “differentiate” or “differentiation” refers to a process where cells progress from an undifferentiated state to a differentiated state, from an immature state to a less immature state or from an immature state to a mature state. For example, early undifferentiated embryonic pancreatic cells are able to proliferate and express characteristics markers, like PDX1, NKX6.1, and PTF1a. Mature or differentiated pancreatic cells do not proliferate and do secrete high levels of pancreatic endocrine hormones or digestive enzymes. E.g., fully differentiated beta cells secrete insulin at high levels in response to glucose. Changes in cell interaction and maturation occur as cells lose markers of undifferentiated cells or gain markers of differentiated cells. Loss or gain of a single marker can indicate that a cell has “matured or fully differentiated.” The term “differentiation factor” refers to a compound added to pancreatic cells to enhance their differentiation to mature endocrine cells also containing insulin producing beta cells. Exemplary differentiation factors include hepatocyte growth factor, keratinocyte growth factor, exendin-4, basic fibroblast growth factor, insulin-like growth factor-1, nerve growth factor, epidermal growth factor platelet-derived growth factor, and glucagon-like peptide 1. In some aspects differentiation of the cells comprises culturing the cells in a medium comprising one or more differentiation factors.
  • As used herein, “human pluripotent stem cells” (hPSC) refers to cells that may be derived from any source and that are capable, under appropriate conditions, of producing human progeny of different cell types that are derivatives of all of the 3 germinal layers (endoderm, mesoderm, and ectoderm). hPSC may have the ability to form a teratoma in 8-12 week old SCID mice and/or the ability to form identifiable cells of all three germ layers in tissue culture. Included in the definition of human pluripotent stem cells are embryonic cells of various types including human blastocyst derived stem (hBS) cells in 30 literature often denoted as human embryonic stem (hES) cells, (see, e.g., Thomson et al. (1998), Heins et al. (2004), as well as induced pluripotent stem cells (see, e.g. Yu et al. (2007); Takahashi et al. (2007)). The various methods and other embodiments described herein may require or utilise hPSC from a variety of sources. For example, hPSC suitable for use may be obtained from developing embryos. Additionally or alternatively, suitable hPSC may be obtained from established cell lines and/or human induced pluripotent stem (hiPS) cells.
  • As used herein “hiPSC” refers to human induced pluripotent stem cells.
  • ES cell lines can also be derived from single blastomeres without the destruction of ex utero embryos and without affecting the clinical outcome (Chung et al. (2006) and Klimanskaya et al. (2006)).
  • As used herein, the term “blastocyst-derived stem cell” is denoted BS cell, and the human form is termed “hBS cells”. In literature the cells are often referred to as embryonic stem cells, and more specifically human embryonic stem cells (hESC). The pluripotent stem cells used in the present invention can thus be embryonic stem cells prepared from blastocysts, as described in e.g. WO 03/055992 and WO 2007/042225, or be commercially available hBS cells or cell lines. However, it is further envisaged that any human pluripotent stem cell can be used in the present invention, including differentiated adult cells which are reprogrammed to pluripotent cells by e.g. the treating adult cells with certain transcription factors, such as OCT4, SOX2, NANOG, and LIN28 as disclosed in Yu, et al. (2007); Takahashi et al. (2007) and Yu et al. (2009).
  • As used herein JNK inhibitor II includes isomers or tautomers of 1,9-pyrazoloanthrone with or without N-alkylation. Where 1,9-pyrazoloanthrone can be defined as “SMILES: c1ccc2c(c1)-c3c4c(cccc4[nH]n3)C2=O” or “1,6-dihydrodibenzo[cd,g]indazol-6-one”.
  • DEF medium or DEF-CS medium/system is a defined balanced culture medium for the establishment and propagation of human pluripotent stem cells, DEF-CS medium/system.
    • EMBODIMENTS OF THE INVENTION
    • 1. A method of producing pancreatic cells or pancreatic cell precursors where at least 5% of the cells co-express PDX1 and NKX6.1, comprising exposing embryonic stem cells to an effective amount of at least one compound of the group consisting of:
      • a. BMP inhibitors
      • b. kinase inhibitors
      • c. retinoic acid receptor agonists
      • to differentiate the human embryonic stem cells into the pancreatic cells or pancreatic cell precursors.
    • 2. The method of embodiment 1, wherein said compounds are listed in tables 1 or 2.
    • 3. The method of embodiments 1 or 2, wherein the kinase inhibitor is an isomer of 1,9-pyrazoloanthrone with or without N-alkylation.
    • 4. The method of embodiments 1-3, wherein said kinase inhibitor is JNK inhibitor II.
    • 5. The method of embodiments 1-2, wherein said retinoic acid receptor agonist is AM580.
    • 6. The method of embodiments 1-5, wherein said JNK inhibitor II is in combination with AM580.
    • 7. The method of embodiments 3-6, wherein bFGF is present.
    • 8. The method of embodiments 7, wherein said bFGF is FGF2, FGF7 or FGF10.
    • 9. The method of embodiment 8, wherein said bFGF is FGF7.
    • 10. The method of embodiment 1, wherein said BMP inhibitor is LDN-193189.
    • 11. The method of embodiment 10, wherein said LDN-193189 is followed by exposure to a Wnt inhibitor or a hedgehog inhibitor.
    • 12. The method of embodiment 11, wherein said Wnt inhibitor is IWP2.
    • 13. The method of embodiment 12, wherein said hedgehog inhibitor is cyclopamine.
    • 14. The method of embodiments 1-13, wherein said pancreatic cells or pancreatic cell precursors are 10-20%, 10-30%, 10-40%, 5-20%, 5-30%, 5-40%, 5-50%, 5-60% or 5-70%, 5-80%, 40-80% or 5-90% PDX1/NKX6.1 double positive.
    • 15. The method of any one of embodiments 14, wherein said pancreatic cells or pancreatic cell precursors are 5-50% PDX1/NKX6.1 double positive.
    • 16. The method of any one of embodiments 1-9, wherein said pancreatic cells or pancreatic cell precursors are 40-80% PDX1/NKX6.1 double positive.
    • 17. A pancreatic cell or pancreatic cell precursors obtainable by the methods of embodiments 1-16.
    • 18. A pancreatic cell or pancreatic cell precursor produced by exposing a human pluripotent stem cell to at least one compound listed in Tables 1 or 2.
    • 19. The pancreatic cell or pancreatic cell precursor of embodiment 18, in which said compound is LDN-193189.
    • 20. The pancreatic cell or pancreatic cell precursor of embodiment 18, in which said compound is JNK inhibitor II.
    • 21. The pancreatic cell or pancreatic cell precursor of embodiment 18, in which said compound is AM580.
    • 22. The pancreatic cell or pancreatic cell precursor of embodiment 18, in which the pancreatic cell or pancreatic cell precursor is produced by exposing the stem cell to at least one compound in combination with at least one additional agent.
    • 23. The pancreatic cell or pancreatic cell precursor of embodiment 22, in which said LDN-193189 is in combination with JNK inhibitor II and AM580.
    • 24. The pancreatic cell or pancreatic cell precursor of embodiment 22, in which said LDN-193189 is followed by exposure to cyclopamine.
    • 25. The pancreatic cell or pancreatic cell precursor of embodiment 22, in which said LDN-193189 is followed by exposure to IWP2.
    • 26. The pancreatic cell or pancreatic cell precursor of embodiment 22, in which said compound is JNK inhibitor II in combination with a retinoic acid receptor agonist listed in Table 2.
    • 27. The pancreatic cell or pancreatic cell precursor of embodiment 22, wherein JNK inhibitor II is in combination with AM580.
    • 28. The pancreatic cell or pancreatic cell precursor of embodiment 22, wherein JNK inhibitor II is in combination with AM580 and bFGF.
    • 29. The pancreatic cell or pancreatic cell precursor of embodiment 28, wherein bFGF is FGF2, FGF7 or FGF10.
    • 30. The pancreatic cell or pancreatic cell precursor of embodiment 29, wherein bFGF is FGF7.
    • 31. Use of a compound of Tables 1 or 2 to induce pancreatic cells or pancreatic cell precursors from stem cells.
    • 32. Use of JNK inhibitor II to induce pancreatic cells or pancreatic cell precursors from stem cells.
    • 33. Use of JNK inhibitor II in combination with a retinoic acid receptor agonist to induce pancreatic cells or pancreatic cell precursors from stem cells.
    • 34. Use of JNK inhibitor II in combination with AM580 to induce pancreatic cells or pancreatic cell precursors from stem cells.
    • 35. Use of LDN-193189 to induce pancreatic cells or pancreatic cell precursors from stem cells.
    • 36. Use of LDN-193189 followed by Cyclopamine or IWP2 to induce pancreatic cells or pancreatic cell precursors from stem cells.
    • 37. Use of LDN-193189 followed by Cyclopamine to induce pancreatic cells or pancreatic cell precursors from stem cells.
    • 38. Use of LDN-193189 followed by IWP2 to induce pancreatic cells or pancreatic cell precursors from stem cells.
    • 39. Use of LDN-193189 followed by combination with JNK inhibitor II, AM580 and bFGF to induce pancreatic cells or pancreatic cell precursors from stem cells.
    • 40. Use of LDN-193189 followed by a combination of JNK inhibitor II, AM580, LDN-193189 and bFGF to induce pancreatic cells or pancreatic cell precursors from stem cells.
    • 41. Use of LDN-193189 followed by a combination of JNK inhibitor II, AM580, LDN-193189, bFGF and Cyclopamine or IWP2 to induce pancreatic cells or pancreatic cell precursors from stem cells.
    • 42. Use of LDN-193189 followed by a combination of JNK inhibitor II, AM580, LDN-193189, bFGF and Cyclopamine to induce pancreatic cells or pancreatic cell precursors from stem cells.
    • 43. Use of LDN-193189 followed by a combination of JNK inhibitor II, AM580, LDN-193189, bFGF and IWP2 to induce pancreatic cells or pancreatic cell precursors from stem cells.
    • 44. A method of producing pancreatic cells or pancreatic cell precursors expressing at least 5% PDX1/NKX6.1 double positive, comprising exposing definitive endoderm cells to an effective amount of at least one of the compounds from each of the following groups:
      • a. BMP inhibitor, and
      • b. Kinase inhibitors, and
      • c. Retinoic acid receptor agonists
      • to differentiate the definitive endoderm cells into the pancreatic cell or pancreatic cell precursors.
    • 45. The method of embodiment 44 wherein the BMP inhibitor is LDN-193189.
    • 46. The method of embodiment 44 wherein the retinoic acid receptor agonist is AM580.
    • 47. The method of embodiment 44 wherein the retinoic acid receptor agonist is a retinoic acid derivative.
    • 48. The method of embodiment 44 wherein the kinase inhibitor is an isomer of 1,9-pyrazoloanthrone with or without N-alkylation.
    • 49. The method of embodiment 44 wherein the kinase inhibitor is JNK inhibitor II and is in combination with AM580.
    • 50. The method of embodiment 44 wherein said kinase inhibitors and retinoic acid receptor agonists are in combination with bFGF.
    • 51. The method of embodiment 44 wherein said kinase inhibitors and retinoic acid receptor agonists are in combination with FGF7 or FGF10.
    • 52. The method of embodiments 44-46, comprising a first step of exposing definitive endoderm cells to an effective amount of LDN-193189, without bFGF, and a second step of exposure to JNK inhibitor II in combination with AM580, in the presence of bFGF.
    • 53. The method of embodiments 44-46, comprising exposing definitive endoderm cells to an effective amount of LDN-193189, following exposure to at least one compound of the group consisting of:
      • a wnt inhibitor, such as IWP2, and/or
      • a hedgehog inhibitor, such as cyclopamine
    • 54. The method of any one of embodiments 44-53, wherein said pancreatic cells or pancreatic cell precursors are at least 5%, at least 10%, 10-30%, 10-40%, 5-70%, 10-80% or 5-100% PDX1/NKX6.1 double positive.
    • 55. A pancreatic cell or pancreatic cell precursor obtainable by in vitro use of the methods of embodiments 44-54.
    • 56. A pancreatic cell or pancreatic cell precursor produced by in vitro exposing definitive endoderm cells to a kinase inhibitor targeting JNK1, 2 or 3, or Syc or Src or GSK-3 or P38 MAPK or P38 kinase or Rho kinase or MEK or Chk2 or VEGFR1, 2 or 3 or PDGFRb or KDR/Flk-1.
    • 57. A pancreatic cell or pancreatic cell precursor produced by the method of embodiment 56 wherein the kinase inhibitor is JNK inhibitor II and wherein the definitive endoderm cells are also exposed to at least one of the following compounds:
      • LDN-193189,
      • a Wnt inhibitor,
      • a hedgehog inhibitor,
      • a retinoic acid receptor agonist.
    • 58. The pancreatic cell or pancreatic cell precursor of embodiment 56 wherein the definitive endoderm cells are exposed to JNK inhibitor II in combination with a retinoic acid receptor agonist listed in Table 2.
    • 59. Use of a kinase inhibitor compound of Table 1 or 2 to induce pancreatic cells or pancreatic cell precursors from a pancreatic endoderm precursor.
    • 60. Use of JNK inhibitor II and LDN-193189 in combination to induce pancreatic cells or pancreatic cell precursors from a pancreatic endoderm precursor.
    • 61. Use of JNK inhibitor II in combination with a retinoic acid receptor agonist to induce pancreatic cells or pancreatic cell precursors from a pancreatic endoderm precursor.
    GENERAL METHODS In Vitro Culture of Pluripotent Stem Cells
  • Human embryonic stem (hES) cells line SA121 and human induced pluripotent stem cells (hiPSC) chIPS4 (Cellectis) were grown in DEF-CS culture media (Cellectis) in T75 culture flasks. Cells were single cell passaged with 5 μM Rock inhibitor Y-27632 (Sigma #Y0503) and seeded at a density of 40000 cells/cm2 for experiments. Cells were cultured at 37° C. and 5% CO2 in a humidified incubator (ThermoScientific Model 371).
  • In Vitro Differentiation of Pluripotent Stem Cells into Definitive Endoderm
  • Confluent cultures of hES cells (line SA121) and hiPSC (chIPS4) were washed once in RPMI1640 (Gibco #61870) and treated with 3 μM CHIR99021 (Axon#1386) in RPMI1640. After 24 hours the cells were washed with RPMI1640 and treated with 100 ng/ml Activin A (Peprotech #120-14E) in RPMI1640. 24 hours later, 2% B27 (Invitrogen #17504-044) was added to the Activin A media for 2 days with daily media change. Cells were maintained at 37° C. and 5% CO2 in a humidified incubator during the differentiation.
  • Seeding of hES and hiPS Cell Derived Definitive Endoderm.
  • Human ES cell derived DE and Human iPS cell derived DE cells were washed in PBS−/− and trypsinized for 5 min. using Tryple Select (Invitrogen, 12563-029). DE cells were carefully suspended in RPMI1640 and washed once before they were resuspended in DE seeding medium (Activin A 100 ng/ml, 2% B27, RPMI1640, 0.1% PEST (Gibco #15140)). The DE cells were seeded at 200 000/cm2 in 96 well optical plates (BD Bioscience), and PE differentiation using screening compounds was initiated the following day.
    • Analysis
  • At day 8 or 14 of PE differentiation, media were aspirated followed by fixation of the cells at room temperature for 30 min with 4% paraformaldehyde (VWR, 97.131.000). Cells were washed with PBS and permeabilized with 0.5% Triton X-100 (Sigma, 9002-93-1) for 10 min, washed and blocked in 0.5% TNB-buffer (Perkin Elmer) for 30 min at room temperature. Primary antibodies mouse anti-NKX6.1 (Abcore#A55F12) and goat anti-PDX1 (Abcam#47383) were diluted 1:500 and 1:8000, respectively, in 0.1% Triton X-100 in PBS and added to each well for overnight incubation at 4° C. Cells were washed three times with PBS. DAPI (4′,6-diamidino-2-phenylindole, Applichem, A4099.0010) and secondary antibodies, Alexa Fluor 488 donkey anti-goat and Alexa Fluor 594 donkey anti-mouse (both Invitrogen) were diluted 1:1000 in 0.1% Triton X-100 in PBS and added to each well for 45 min. Cells were washed five times and left in 200 μL PBS for imaging.
  • Imaging was performed using the InCell Analyzer 2000 (GE Healthcare). 4 fields per well with 10× objective were captured. The total cell number based in the DAPI counterstaining and the number of NKX6.1/PDX1 double positive cells was determined using InCell Developer Toolbox 1.8 (GE Healthcare). The fraction of NKX6.1/PDX1 double positive cells was normalized to the benchmark on each plate and the % effect was calculated. Values above 200% effect were categorized as hits.
    • Examples
  • Pancreatic endoderm is characterized by co-expression of two transcription factors, NKX6.1 and PDX1. Many of the published protocols for making PE are ineffective with low outcome of NKX6.1/PDX1 double positive cells. Enhancing the efficacy of the PE protocols is a desirable outcome. We therefore screened libraries of small molecules to identify novel compounds that would improve the existing PE protocols. This was done on the assumption that inhibitors, agonists or antagonists may regulate signaling pathways, or chromosomal accessibility, which would improve the fraction of NKX6.1/PDX1 double positive cells.
    • Example 1 Screening Small Molecules that Induce NKX6.1/PDX1 Co-Expression Small Molecules
  • Four different libraries were included in the screen; a kinase inhibitor library (Calbiochem #539743), a bioactive lipid library (Enzo Life Sciences #BML-2800), a nuclear receptor ligand library (Enzo Life Sciences # BML-2802) and a phosphatase inhibitor library (Enzo Life Sciences #BML-2834). The compounds within the bioactive library were tested at 1 uM and 0.1 uM. Compounds from the other libraries were tested at 10 uM and 1 uM. In a second candidate based screening approach, small molecules that target the main signalling pathways involved in pancreas development were included.
  • NKX6.1/PDX1 Screen The library compounds were screened on top of a bFGF based media formulation for making PE (Ameri et al. 2010) (RPMI1640, Gibco#61870; 12% KOSR, Gibco#10828; 0.1% PEST, Gibco#15140; 64 ng/mL bFGF, Peprotech #100-18B).
  • The library PE screening approach was divided into an early and a late phase (FIG. 1).
  • In the early phase, compounds were tested on top of the PE media for the first seven days of PE differentiation, and then the differentiation was continued for additional six days using PE media without compounds.
  • In the late phase, DE cells were differentiated in the PE media for the first seven days. In the following six days compounds were tested on top of the PE media. 12 positive control wells (PE media) and 12 negative control wells (PE media without bFGF) were included in each 96 well plate. Media change was performed daily. Hits identified in the early phase screen are illustrated in FIG. 2 and listed in table 1. Hits identified in the late phase screen are illustrated in FIG. 3 and listed in table 2.
  • The compounds from the candidate approach were screened in basal medium (RPMI1640, Gibco#61870; 12% KOSR, Gibco#10828; 0.1% PEST, Gibco#15140) without the addition of bFGF. This candidate approach screen was divided into two parts (FIG. 4). In the first part, compounds were tested in time intervals with 2 day increments for the first eight days of PE differentiation (2 days exposure to compounds followed by 6 days basal medium or 4 days exposure to compounds followed by 4 days basal medium or 6 days exposure to compounds followed by 2 days basal medium or 8 days exposure to compounds).
  • One plate was fixed after these 8 days and analysed for PDX1 and NKX6.1 expression. A second plate was further differentiated for additional six days using the published PE protocol (Amen et al. (2010)).
  • In the second part, DE cells were differentiated according to the hit compounds from the first part, the following 6-10 days compounds were tested in basal media.
  • The benchmark protocol (Amen et al. (2010)) served as a control.
  • Media change was performed daily in both first and second part experiments.
  • Hits identified in the candidate screening approach are illustrated in FIG. 5 and also contained in Tables 1 and 2.
    • Example 2 Combining Small Molecule Hits that Induce NKX6.1/PDX1 Co-Expression
  • Combining Hits from the Candidate Screening Approach with Hits from the Library Approach
  • DE cells were exposed to 4 days 50 nM LDN-193189, followed by 8 days AM580 (AH Diagnostics, BML GF104 0025), JNK Inhibitor II (Calbiochem, 420119), 50 nM LDN-193189 and 64 ng/ml FGF2, or AM580, JNK Inhibitor II, 50 nM LDN-193189, 64 ng/ml FGF2 and IWP2, or AM580, JNK Inhibitor II, 50 nM LDN-193189, 64 ng/ml FGF2 and Cyclopamine (FIG. 6). Media change was performed daily.
    • Example 3 Confirmation of Small Molecules that Induce NKX6.1/PDX1 Co-Expression in Human Induced Pluripotent Stem Cells
  • Hit compounds (Tables 1 and 2) were screened on top of a bFGF based media formulation for making PE (Amen et al. 2010) (RPMI1640, Gibco#61870; 12% KOSR, Gibco#10828; 0.1% PEST, Gibco#15140; 64 ng/mL bFGF, Peprotech #100-18B).
  • The screen was divided into an early and a late phase (FIG. 1). In the early phase, compounds were tested on top of the PE media for the first seven days of PE differentiation, and then continued for additional six days using PE media without compounds. In the late phase, DE cells were differentiated in the PE media for the first seven days. In the following six days compounds were tested on top of the PE media. Twelve positive control wells (PE media) and 12 negative control wells (PE media without bFGF) were included in each 96 well plate. Media change was performed daily.
  • Values above 200% effect were categorized as a hit (FIG. 2 and FIG. 3).
    • Table 1 Shows Early Hit Compounds.
  • Compounds that enhance the fraction of NKX6.1/PDX1 double positive cells more than 200% compared the PE media. Library, location of compound within the library, target, chemical structure, hit concentration and percentage of PDX1/NKX6.1 double positive cells are listed.
  • TABLE 1
    Early phase hits.
    Plate
    Library location Target Inhibitor Structure Concentration
    Kinase inhibitor B3 ATM kinase ATM Kinase Inhibitor
    Figure US20150247123A1-20150903-C00001
         		10 uM
    Kinase inhibitor D18 Chk2 Chk2 Inhibitor II
    Figure US20150247123A1-20150903-C00002
         		10 uM
    Kinase inhibitor N15 P38 MAPK SKF-86002
    Figure US20150247123A1-20150903-C00003
         		10 uM
    Kinase inhibitor P20 KDR/Flk-1, VEGGFR-2, PDGFRb, VEGFR-1, VEGFR-3 VEGF Receptor 2 Kinase Inhibitor IV
    Figure US20150247123A1-20150903-C00004
         		10 uM
    Kinase inhibitor H22 JNK1, JNK2, JNK3, JNK Inhibitor II
    Figure US20150247123A1-20150903-C00005
         		10 uM
    Kinase inhibitor H22 JNK1, JNK2, JNK3 JNK Inhibitor II
    Figure US20150247123A1-20150903-C00006
         		1 uM
    Candidate screen BMP inhibitor Candidate approach ALK2, ALK3, ALK6 LDN-193189
    Figure US20150247123A1-20150903-C00007
         		50 nM/100 nM
    • Table 2 Shows Late Hit Compounds.
  • Compounds that enhance the fraction of NKX6.1/PDX1 double positive cells more than 200% compared the PE media. Library, location of compound within the library, target, chemical structure, hit concentration and percentage of PDX1/NKX6.1 double positive cells are listed.
  • TABLE 2
    Late phase hits.
    Plate
    Library location Target Inhibitor Structure Concentration
    Kinase inhibitor H22 JNK1, JNK2, JNK3 JNK Inhibitor II
    Figure US20150247123A1-20150903-C00008
         		10 uM
    Kinase inhibitor J4 JNK Inhibitor, Negative Control
    Figure US20150247123A1-20150903-C00009
         		10 uM
    Kinase inhibitor P5 syc, src SYK Inhibitor III
    Figure US20150247123A1-20150903-C00010
         		10 uM
    Kinase inhibitor F20 GSK-3 GSK-3b Inhibitor II
    Figure US20150247123A1-20150903-C00011
         		10 uM
    Kinase inhibitor N15 P38 MAPK SKF-86002
    Figure US20150247123A1-20150903-C00012
         		10 uM
    Kinase inhibitor J14 PI3-kinase LY 294002
    Figure US20150247123A1-20150903-C00013
         		10 uM
    Kinase inhibitor N14 P38 MAPK SC-68376
    Figure US20150247123A1-20150903-C00014
         		10 uM
    Kinase inhibitor N6 Rho kinase Rho Kinase Inhibitor III, Rockout
    Figure US20150247123A1-20150903-C00015
         		10 uM
    Kinase inhibitor L5 MEK PD 98059
    Figure US20150247123A1-20150903-C00016
         		10 uM
    Kinase inhibitor N11 P38 MAPK SB203580
    Figure US20150247123A1-20150903-C00017
         		10 uM
    Bioactive lipids 2-E09 RAR AM-580
    Figure US20150247123A1-20150903-C00018
         		1 uM
    Bioactive lipids 2-E05 RAR RETINOIC ACID, ALL TRANS
    Figure US20150247123A1-20150903-C00019
         		1 uM
    Bioactive lipids 2-E07 RAR/RXR 13-CIS RETINOIC ACID
    Figure US20150247123A1-20150903-C00020
         		1 uM
    Bioactive lipids 2-E06 RAR/RXR 9-CIS RETINOIC ACID
    Figure US20150247123A1-20150903-C00021
         		1 uM
    Bioactive lipids 2-E10 RAR TTNPB
    Figure US20150247123A1-20150903-C00022
         		1 uM
    Kinase inhibitor H22 JNK1, JNK2, JNK3 JNK Inhibitor II
    Figure US20150247123A1-20150903-C00023
         		1 uM
    Kinase inhibitor J4 JNK Inhibitor, Negative Control
    Figure US20150247123A1-20150903-C00024
         		1 uM
    Bioactive lipids 2-E09 RAR AM-580
    Figure US20150247123A1-20150903-C00025
         		0.1 uM
    Bioactive liids 2-E07 RAR/RXR 13-CIS RETINOIC ACID
    Figure US20150247123A1-20150903-C00026
         		0.1 uM
    Bioactive lipids 2-E06 RAR/RXR 9-CIS RETINOIC ACID
    Figure US20150247123A1-20150903-C00027
         		0.1 uM
    Candidate screen Hedgehog pathway inhibitor Smoothened (SMO) Cydopamine
    Figure US20150247123A1-20150903-C00028
         		1 μM/5 μM
    Candidate screen Wnt pathway inhibitor O-acyltransferase Porcupine IWP2
    Figure US20150247123A1-20150903-C00029
         		1 μM/5 μM
  • While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.
    • REFERENCES
    • Amen et al. (2010) Stem Cells, 28:45-56
    • Cai J. et al. (2010). J Mol Cell Biol., February; 2(1):50-60
    • Chung Y. et al. (2006). Nature, January 12; 439(7073):216-9
    • D'Amour K. A. et al. (2006). Nat Biotechnol, 24: 1392-401
    • Heins N. et al. (2004). Stem Cells, 22(3):367-76
    • Jiang J. et al. (2007). Stem Cells, 25:1940-53
    • Kroon E. et al. (2008). Nat Biotechnol, 26:443-452
    • Klimanskaya I. et al. (2006). Nature, November 23; 444(7118):481-5
    • Kunisada Y. et al. (2012). Stem Cell Res, 8(2):274-84
    • Schulz T C. et al. (2012). PLoS One, 7(5):e37004
    • Takahashi K. et al. (2007). Cell, 131:861-72
    • Takahashi K. and Yamanaka S. (2006). Cell, 126(4):663-76
    • Thomson J A. et al. (1998). Science, November 6; 282(5391):1145-7
    • Wernig, M. et al. (2007). Nature, 448:318-24
    • Zhang D. et al. (2009). Cell Research, 19:429-438

Claims (16)

1. A method of producing pancreatic cells or pancreatic cell precursors, comprising exposing definitive endoderm cells to an effective amount of at least one BMP inhibitor, at least one kinase inhibitor, and at least one retinoic acid receptor agonist, wherein at least 5% of the resulting pancreatic cells or pancreatic cell precursors are PDX1/NKX6.1 double positive.
2. The method of claim 1, wherein the BMP inhibitor is LDN-193189.
3. The method of claim 1, wherein the retinoic acid receptor agonist is AM580.
4. The method of claim 1, wherein the retinoic acid receptor agonist is a retinoic acid derivative.
5. The method of claim 1, wherein the kinase inhibitor is an isomer of 1,9-pyrazoloanthrone with or without N-alkylation.
6. The method of claim 1, wherein kinase inhibitor is JNK inhibitor II and the retinoic acid receptor agonist is AM580.
7. The method of claim 1, further comprising exposing the definitive endoderm cells to bFGF.
8. The method of claim 1, further comprising exposing the definitive endoderm cells to FGF7 or FGF10.
9. The method of claim 1, wherein the exposing comprises a first exposure to an effective amount of LDN-193189 without bFGF and a second exposure to JNK inhibitor II, AM580, and bFGF.
10. The method of claim 2, further comprising exposing the definitive endoderm cells to at least one compound selected from the group consisting of a wnt inhibitor and a hedgehog inhibitor prior to exposing the definitive endoderm cells to the LDN-193189.
11. The method of claim 1, wherein said pancreatic cells or pancreatic cell precursors are at least 10% PDX1/NKX6.1 double positive.
12. An isolated population of pancreatic cells or pancreatic cell precursors produced by the method of claim 1.
13. An isolated population of pancreatic cells or pancreatic cell precursors produced by exposing definitive endoderm cells to a kinase inhibitor capable of inhibiting a target selected from the group consisting of JNK1, JNK2, JNK3, Syc, Src, GSK-3, P38, MAPK, P38 kinase, Rho kinase, MEK, Chk2, VEGFR1, VEGFR2, VEGFR3, PDGFRb, and KDR/Flk-1.
14. The isolated population of pancreatic cells or pancreatic cell precursors produced by the method of claim 13, wherein the kinase inhibitor is JNK inhibitor II, and wherein the definitive endoderm cells are also exposed to a compound selected from the group consisting of LDN-193189, a Wnt inhibitor, a hedgehog inhibitor, and a retinoic acid receptor agonist.
15. The isolated population of pancreatic cells or pancreatic cell precursors of claim 13, wherein the definitive endoderm cells are exposed to a retinoic acid receptor agonist.
16.-18. (canceled)
US14/425,136 2012-09-03 2013-09-03 Generation of pancreatic endoderm from Pluripotent Stem cells using small molecules Abandoned US20150247123A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US14/425,136 US20150247123A1 (en) 2012-09-03 2013-09-03 Generation of pancreatic endoderm from Pluripotent Stem cells using small molecules

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
EP12182747 2012-09-03
EP12182747.1 2012-09-03
US201261697970P 2012-09-07 2012-09-07
EP12198820 2012-12-21
EP12198820.8 2012-12-21
PCT/EP2013/068188 WO2014033322A1 (en) 2012-09-03 2013-09-03 Generation of pancreatic endoderm from pluripotent stem cells using small molecules
US14/425,136 US20150247123A1 (en) 2012-09-03 2013-09-03 Generation of pancreatic endoderm from Pluripotent Stem cells using small molecules

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2013/068188 A-371-Of-International WO2014033322A1 (en) 2012-09-03 2013-09-03 Generation of pancreatic endoderm from pluripotent stem cells using small molecules

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US15/215,807 Division US10221392B2 (en) 2012-09-03 2016-07-21 Generation of pancreatic endoderm from pluripotent stem cells using small molecules

Publications (1)

Publication Number Publication Date
US20150247123A1 true US20150247123A1 (en) 2015-09-03

Family

ID=50182554

Family Applications (3)

Application Number Title Priority Date Filing Date
US14/425,136 Abandoned US20150247123A1 (en) 2012-09-03 2013-09-03 Generation of pancreatic endoderm from Pluripotent Stem cells using small molecules
US15/215,807 Active US10221392B2 (en) 2012-09-03 2016-07-21 Generation of pancreatic endoderm from pluripotent stem cells using small molecules
US16/255,347 Abandoned US20190153394A1 (en) 2012-09-03 2019-01-23 Generation of pancreatic endoderm from pluripotent stem cells using small molecules

Family Applications After (2)

Application Number Title Priority Date Filing Date
US15/215,807 Active US10221392B2 (en) 2012-09-03 2016-07-21 Generation of pancreatic endoderm from pluripotent stem cells using small molecules
US16/255,347 Abandoned US20190153394A1 (en) 2012-09-03 2019-01-23 Generation of pancreatic endoderm from pluripotent stem cells using small molecules

Country Status (5)

Country Link
US (3) US20150247123A1 (en)
EP (1) EP2893000B1 (en)
JP (2) JP6470687B2 (en)
CN (1) CN104903440B (en)
WO (1) WO2014033322A1 (en)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160186143A1 (en) * 2014-12-18 2016-06-30 President And Fellows Of Harvard College Methods for generating stem cell-derived beta cells and uses thereof
US10030229B2 (en) 2013-06-11 2018-07-24 President And Fellows Of Harvard College SC-β cells and compositions and methods for generating the same
US10253298B2 (en) 2014-12-18 2019-04-09 President And Fellows Of Harvard College Methods for generating stem cell-derived beta cells and methods of use thereof
US10443042B2 (en) 2014-12-18 2019-10-15 President And Fellows Of Harvard College Serum-free in vitro directed differentiation protocol for generating stem cell-derived beta cells and uses thereof
WO2020041608A1 (en) * 2018-08-22 2020-02-27 The Penn State Research Foundation Generation of pancreatic progenitor cells and beta cells from human pluripotent stem cells
WO2021099532A1 (en) 2019-11-22 2021-05-27 Novo Nordisk A/S Spin-aggregated neural microspheres and the application thereof
US11466256B2 (en) 2018-08-10 2022-10-11 Vertex Pharmaceuticals Incorporated Stem cell derived islet differentiation
US11945795B2 (en) 2017-11-15 2024-04-02 Vertex Pharmaceuticals Incorporated Islet cell manufacturing compositions and methods of use

Families Citing this family (35)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9080145B2 (en) 2007-07-01 2015-07-14 Lifescan Corporation Single pluripotent stem cell culture
US9096832B2 (en) 2007-07-31 2015-08-04 Lifescan, Inc. Differentiation of human embryonic stem cells
CA2959401C (en) 2008-02-21 2021-12-07 Centocor Ortho Biotech Inc. Methods, surface modified plates and compositions for cell attachment, cultivation and detachment
KR20180018839A (en) 2008-06-30 2018-02-21 얀센 바이오테크 인코포레이티드 Differentiation of pluripotent stem cells
MX356756B (en) 2008-11-20 2018-06-11 Centocor Ortho Biotech Inc Pluripotent stem cell culture on micro-carriers.
ES2584053T3 (en) 2008-11-20 2016-09-23 Janssen Biotech, Inc. Methods and compositions for cell binding and culture in flat substrates
WO2011011300A2 (en) 2009-07-20 2011-01-27 Centocor Ortho Biotech Inc. Differentiation of human embryonic stem cells
KR101773538B1 (en) 2009-12-23 2017-08-31 얀센 바이오테크 인코포레이티드 Differentiation of human embryonic stem cells
PH12012501686A1 (en) 2010-03-01 2020-10-19 Janssen Biotech Inc Methods for purifying cells derived from pluripotent stem cells
RU2620938C2 (en) 2010-08-31 2017-05-30 Янссен Байотек, Инк. Differentiation of human embryonic stem cells
US9388386B2 (en) 2011-12-22 2016-07-12 Janssen Biotech, Inc. Differentiation of human embryonic stem cells into single hormonal insulin positive cells
CN108103005A (en) 2012-06-08 2018-06-01 詹森生物科技公司 Differentiation of the human embryo stem cell to pancreatic endocrine cell
US20150247123A1 (en) * 2012-09-03 2015-09-03 Novo Nordisk A/S Generation of pancreatic endoderm from Pluripotent Stem cells using small molecules
JP6529440B2 (en) 2012-12-31 2019-06-12 ヤンセン バイオテツク，インコーポレーテツド Suspension and clustering of human pluripotent cells for differentiation to pancreatic endocrine cells
ES2942484T3 (en) 2012-12-31 2023-06-01 Janssen Biotech Inc Differentiation of human embryonic stem cells into pancreatic endocrine cells using HB9 regulators
SG10201709338RA (en) 2012-12-31 2017-12-28 Janssen Biotech Inc Culturing of human embryonic stem cells at the air-liquid interface for differentiation into pancreatic endocrine cells
US10370644B2 (en) 2012-12-31 2019-08-06 Janssen Biotech, Inc. Method for making human pluripotent suspension cultures and cells derived therefrom
CN117821369A (en) * 2014-05-16 2024-04-05 詹森生物科技公司 Use of small molecules to enhance MAFA expression in pancreatic endocrine cells
CN108366987A (en) 2015-04-30 2018-08-03 河谷控股Ip有限责任公司 Patient is treated by teratogenic drug compound
CN108699515A (en) * 2016-02-24 2018-10-23 诺和诺德股份有限公司 The endocrine progenitor cells generating functionality β cells derived from human pluripotent stem cells
MA45479A (en) 2016-04-14 2019-02-20 Janssen Biotech Inc DIFFERENTIATION OF PLURIPOTENT STEM CELLS IN ENDODERMAL CELLS OF MIDDLE INTESTINE
MA45502A (en) 2016-06-21 2019-04-24 Janssen Biotech Inc GENERATION OF FUNCTIONAL BETA CELLS DERIVED FROM HUMAN PLURIPOTENT STEM CELLS WITH GLUCOSE-DEPENDENT MITOCHONDRIAL RESPIRATION AND TWO-PHASE INSULIN SECRETION RESPONSE
WO2018035454A1 (en) * 2016-08-19 2018-02-22 Memorial Sloan-Kettering Cancer Center Methods of differentiating stem cells into endoderm
EP3395942A1 (en) * 2017-04-25 2018-10-31 IMBA-Institut für Molekulare Biotechnologie GmbH Bi- or multi-differentiated organoid
CA3074910A1 (en) 2017-09-11 2019-03-14 Novo Nordisk A/S Enrichment of nkx6.1 and c-peptide co-expressing cells derived in vitro from stem cells
WO2020043292A1 (en) 2018-08-30 2020-03-05 Novo Nordisk A/S Generation of functional beta cells from human pluripotent stem cell-derived endocrine progenitors
CN113728090B (en) * 2019-04-08 2024-04-05 诺和诺德股份有限公司 Pancreatic endoderm generation from stem cell-derived definitive endoderm
EP4386081A3 (en) * 2019-05-21 2024-10-16 President and Fellows of Harvard College Endocrine differentiation-inducing molecule
US20230313145A1 (en) 2020-08-28 2023-10-05 Novo Nordisk A/S Method for screening in vitro population of stem cell derived beta like cells and novel markers thereof
WO2024008810A1 (en) 2022-07-06 2024-01-11 Novo Nordisk A/S Differentiation of stem cells to pancreatic endocrine cells
WO2024151541A1 (en) 2023-01-09 2024-07-18 Sana Biotechnology, Inc. Type-1 diabetes autoimmune mouse
WO2024243236A2 (en) 2023-05-22 2024-11-28 Sana Biotechnology, Inc. Methods of delivery of islet cells and related methods
WO2025093467A1 (en) 2023-10-31 2025-05-08 Novo Nordisk A/S Methods for preventing unwanted cells arising during differentiation of human pluripotent stem cells
EP4491712A1 (en) 2024-03-20 2025-01-15 Novo Nordisk A/S Method for detecting contaminant cells in a stem cell derived cell population
WO2025235403A1 (en) * 2024-05-06 2025-11-13 Vertex Pharmaceuticals Incorporated Differentiation of pancreatic islet cells

Family Cites Families (45)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1298201A1 (en) 2001-09-27 2003-04-02 Cardion AG Process for the production of cells exhibiting an islet-beta-cell-like state
US20040005301A1 (en) 2002-02-12 2004-01-08 Goldman Steven A. Identification and high-yield isolation of human pancreatic islet progenitor and stem cells
WO2005005608A2 (en) 2003-06-30 2005-01-20 Lifescan, Inc. Compositions and methods for differentiating adipose stromal cells into pancratic beta cells
AU2005221095A1 (en) 2004-03-09 2005-09-22 John J. O'neil Methods for generating insulin-producing cells
US20090053181A1 (en) 2005-06-13 2009-02-26 Novo Nordisk A/S Modulation of Cells
EP1999253B1 (en) 2006-03-02 2019-05-22 Viacyte, Inc. Endocrine precursor cells, pancreatic hormone-expressing cells and methods of production
ES2725601T3 (en) 2006-04-28 2019-09-25 Lifescan Inc Differentiation of human embryonic stem cells
SE534150C2 (en) 2006-05-02 2011-05-10 Wisconsin Alumni Res Found Procedure for differentiating stem cells into cells of the endodermal and pancreatic developmental line
CA2893679C (en) 2006-06-26 2018-10-30 Lifescan, Inc. Conditioned medium for pluripotent stem cell culture
CA2666789C (en) 2006-10-18 2016-11-22 Yong Zhao Embryonic-like stem cells derived from adult human peripheral blood and methods of use
DK3192865T3 (en) 2007-07-01 2021-06-28 Janssen Biotech Inc SINGLE PLURIPOTENT STEM CELL CULTURE
US9080145B2 (en) 2007-07-01 2015-07-14 Lifescan Corporation Single pluripotent stem cell culture
EP2185695B1 (en) 2007-07-18 2015-02-25 Lifescan, Inc. Differentiation of human embryonic stem cells
JP6087043B2 (en) 2007-07-31 2017-03-01 ライフスキャン・インコーポレイテッドＬｉｆｅｓｃａｎ，Ｉｎｃ． Differentiation of pluripotent stem cells using human feeder cells
US9096832B2 (en) 2007-07-31 2015-08-04 Lifescan, Inc. Differentiation of human embryonic stem cells
SG154367A1 (en) 2008-01-31 2009-08-28 Es Cell Int Pte Ltd Method of differentiating 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
DK2727998T3 (en) 2008-04-21 2019-08-26 Viacyte Inc Methods for purifying pancreatic endoderm cells derived from human embryonic stem cells
US8728812B2 (en) 2008-04-22 2014-05-20 President And Fellows Of Harvard College Compositions and methods for promoting the generation of PDX1+ pancreatic cells
US7939322B2 (en) 2008-04-24 2011-05-10 Centocor Ortho Biotech Inc. Cells expressing pluripotency markers and expressing markers characteristic of the definitive endoderm
KR20180018839A (en) 2008-06-30 2018-02-21 얀센 바이오테크 인코포레이티드 Differentiation of pluripotent stem cells
US20100028307A1 (en) 2008-07-31 2010-02-04 O'neil John J Pluripotent stem cell differentiation
US9234178B2 (en) * 2008-10-31 2016-01-12 Janssen Biotech, Inc. Differentiation of human pluripotent stem cells
CN102333862B (en) 2008-10-31 2018-04-27 詹森生物科技公司 Differentiation of human embryonic stem cells to the pancreatic endocrine lineage
EP3550012A1 (en) 2008-11-04 2019-10-09 ViaCyte, Inc. Stem cell aggregate suspension compositions and methods for differentiation thereof
US8008075B2 (en) 2008-11-04 2011-08-30 Viacyte, Inc. Stem cell aggregate suspension compositions and methods of differentiation thereof
EP2356227B1 (en) 2008-11-14 2018-03-28 Viacyte, Inc. Encapsulation of pancreatic cells derived from human pluripotent stem cells
MX356756B (en) 2008-11-20 2018-06-11 Centocor Ortho Biotech Inc Pluripotent stem cell culture on micro-carriers.
US8507274B2 (en) 2009-02-06 2013-08-13 President And Fellows Of Harvard College Compositions and methods for promoting the generation of definitive endoderm
WO2010096496A2 (en) * 2009-02-17 2010-08-26 Memorial Sloan-Kettering Cancer Center Methods of neural conversion of human embryonic stem cells
EP2233566A1 (en) 2009-03-17 2010-09-29 Vrije Universiteit Brussel Generation of pancreatic progenitor cells
EP3904505A1 (en) * 2009-04-22 2021-11-03 Viacyte, Inc. Cell compositions derived from dedifferentiated reprogrammed cells
JP2012527880A (en) * 2009-05-29 2012-11-12 ノヴォ ノルディスク アー／エス Derivation of specific endoderm from hPS cell-derived definitive endoderm
JP5819826B2 (en) 2009-07-20 2015-11-24 ヤンセン バイオテツク，インコーポレーテツド Differentiation of human embryonic stem cells
KR101785626B1 (en) 2009-07-20 2017-10-16 얀센 바이오테크 인코포레이티드 Differentiation of human embryonic stem cells
KR101773538B1 (en) * 2009-12-23 2017-08-31 얀센 바이오테크 인코포레이티드 Differentiation of human embryonic stem cells
AU2010333840B2 (en) 2009-12-23 2016-01-07 Janssen Biotech, Inc. Differentiation of human embryonic stem cells
EP2505639B1 (en) * 2009-12-29 2018-09-19 Takeda Pharmaceutical Company Limited Method for manufacturing pancreatic-hormone-producing cells
WO2011100291A1 (en) 2010-02-09 2011-08-18 The Regents Of The University Of California Method for identifying pancreatic progenitor cells
CN102884176B (en) * 2010-05-12 2017-09-05 詹森生物科技公司 Differentiation of human embryonic stem cells
EP2606122A1 (en) 2010-08-22 2013-06-26 Ramot at Tel Aviv University, Ltd. Induced pluripotent stem cells derived from human pancreatic beta cells
AU2011296383B2 (en) * 2010-08-31 2016-03-10 Janssen Biotech, Inc. Differentiation of pluripotent stem cells
ES2902650T3 (en) 2011-06-21 2022-03-29 Novo Nordisk As Efficient induction of definitive endoderm from pluripotent stem cells
JP6143268B2 (en) * 2011-12-19 2017-06-07 国立大学法人京都大学 Method for inducing differentiation from human pluripotent stem cells to intermediate mesoderm cells
US20150247123A1 (en) * 2012-09-03 2015-09-03 Novo Nordisk A/S Generation of pancreatic endoderm from Pluripotent Stem cells using small molecules

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Kim et al., Current Opinion in Genetics & Development 2002, 12:540–547 *

Cited By (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11078463B2 (en) 2013-06-11 2021-08-03 President And Fellows Of Harvard College SC-beta cells and compositions and methods for generating the same
US10030229B2 (en) 2013-06-11 2018-07-24 President And Fellows Of Harvard College SC-β cells and compositions and methods for generating the same
US11827905B2 (en) 2013-06-11 2023-11-28 President And Fellows Of Harvard College SC-beta cells and compositions and methods for generating the same
US12049646B2 (en) 2013-06-11 2024-07-30 President And Fellows Of Harvard College SC-beta cells and compositions and methods for generating the same
US11162078B2 (en) 2013-06-11 2021-11-02 President And Fellows Of Harvard College SC-beta cells and compositions and methods for generating the same
US12054745B2 (en) 2013-06-11 2024-08-06 President And Fellows Of Harvard College SC-beta cells and compositions and methods for generating the same
US10655106B2 (en) 2013-06-11 2020-05-19 President And Fellows Of Harvard College SC-beta cells and compositions and methods for generating the same
US11104883B2 (en) 2013-06-11 2021-08-31 President And Fellows Of Harvard College SC-beta cells and compositions and methods for generating the same
US11155787B2 (en) 2014-12-18 2021-10-26 President And Fellows Of Harvard College Methods for generating stem cell-derived beta cells and methods of use thereof
US12123023B2 (en) 2014-12-18 2024-10-22 President And Fellows Of Harvard College Methods for generating stem cell-derived β cells and methods of use thereof
US11085025B2 (en) 2014-12-18 2021-08-10 President And Fellows Of Harvard College Serum-free in vitro directed differentiation protocol for generating stem cell-derived beta cells and uses thereof
US11085027B2 (en) 2014-12-18 2021-08-10 President And Fellows Of Harvard College Serum-free in vitro directed differentiation protocol for generating stem cell-derived beta cells and uses thereof
US11085026B2 (en) 2014-12-18 2021-08-10 President And Fellows Of Harvard College Serum-free in vitro directed differentiation protocol for generating stem cell-derived beta cells and uses thereof
US10927350B2 (en) 2014-12-18 2021-02-23 President And Fellows Of Harvard College Methods for generating stem cell-derived beta cells and uses thereof
US10443042B2 (en) 2014-12-18 2019-10-15 President And Fellows Of Harvard College Serum-free in vitro directed differentiation protocol for generating stem cell-derived beta cells and uses thereof
US10253298B2 (en) 2014-12-18 2019-04-09 President And Fellows Of Harvard College Methods for generating stem cell-derived beta cells and methods of use thereof
US20160186143A1 (en) * 2014-12-18 2016-06-30 President And Fellows Of Harvard College Methods for generating stem cell-derived beta cells and uses thereof
US10190096B2 (en) * 2014-12-18 2019-01-29 President And Fellows Of Harvard College Methods for generating stem cell-derived β cells and uses thereof
US12304900B2 (en) 2017-11-15 2025-05-20 Vertex Pharmaceuticals Incorporated Islet cell manufacturing compositions and methods of use
US11945795B2 (en) 2017-11-15 2024-04-02 Vertex Pharmaceuticals Incorporated Islet cell manufacturing compositions and methods of use
US11999971B2 (en) 2018-08-10 2024-06-04 Vertex Pharmaceuticals Incorporated Stem cell derived islet differentiation
US11525120B2 (en) 2018-08-10 2022-12-13 Vertex Pharmaceuticals Incorporated Stem cell derived islet differentiation
US11466256B2 (en) 2018-08-10 2022-10-11 Vertex Pharmaceuticals Incorporated Stem cell derived islet differentiation
US12215355B2 (en) 2018-08-10 2025-02-04 Vertex Pharmaceuticals Incorporated Stem cell derived islet differentiation
US20210324337A1 (en) * 2018-08-22 2021-10-21 The Penn State Research Foundation Generation of pancreatic progenitor cells and beta cells from human pluripotent stem cells
WO2020041608A1 (en) * 2018-08-22 2020-02-27 The Penn State Research Foundation Generation of pancreatic progenitor cells and beta cells from human pluripotent stem cells
US12516295B2 (en) * 2018-08-22 2026-01-06 The Penn State Research Foundation Generation of pancreatic progenitor cells and beta cells from human pluripotent stem cells
WO2021099532A1 (en) 2019-11-22 2021-05-27 Novo Nordisk A/S Spin-aggregated neural microspheres and the application thereof

Also Published As

Publication number Publication date
JP2015528289A (en) 2015-09-28
US20190153394A1 (en) 2019-05-23
JP2019000126A (en) 2019-01-10
EP2893000B1 (en) 2019-04-10
EP2893000A1 (en) 2015-07-15
US20160326495A1 (en) 2016-11-10
JP6470687B2 (en) 2019-02-13
US10221392B2 (en) 2019-03-05
JP6954877B2 (en) 2021-10-27
CN104903440A (en) 2015-09-09
CN104903440B (en) 2018-04-06
WO2014033322A1 (en) 2014-03-06

Similar Documents

Publication Publication Date Title
US10221392B2 (en) Generation of pancreatic endoderm from pluripotent stem cells using small molecules
JP7420860B2 (en) Methods and systems for converting progenitor cells into gastric tissue by directed differentiation
US20200002670A1 (en) Generation of Endocrine Progenitor Cells from Human Pluripotent Stem Cells Using Small Molecules
US11274280B2 (en) Generation of functional beta cells from human pluripotent stem cell-derived endocrine progenitors
US10457916B2 (en) Method for inducing differentiation of insulin-producing cells
US20210230554A1 (en) Generation of functional beta cells from human pluripotent stem cell-derived endocrine progenitors
CN113728090B (en) Pancreatic endoderm generation from stem cell-derived definitive endoderm
ES2729776T3 (en) Generation of pancreatic endoderm from pluripotent stem cells through the use of small molecules
CN112119154A (en) Production of brown fat cells
Horst Activin-A and Bmp4 Levels Modulate Cell Type Specification during CHIR-Induced Cardiomyogenesis
ES2746232T3 (en) Generation of endocrine progenitor cells from human pluripotent stem cells using small molecules
Class et al. Patent application title: Generation of Endocrine Progenitor Cells from Human Pluripotent Stem Cells Using Small Molecules
Rotti 3D differentiation enhances the efficiency of differentiation of human induced pluripotent stem cells to insulin producing cells

Legal Events

Date Code Title Description
AS Assignment

Owner name: NOVO NORDISK A/S, DENMARK

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:EKBERG, JENNY;HANSSON, MATTIAS;DOEHN, ULRIK;AND OTHERS;SIGNING DATES FROM 20150325 TO 20150508;REEL/FRAME:035765/0626

AS Assignment

Owner name: NOVO NORDISK A/S, DENMARK

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:EKBERG, JENNY;HANSSON, MATTIAS;DOEHN, ULRIK;AND OTHERS;SIGNING DATES FROM 20150818 TO 20150823;REEL/FRAME:036749/0785

Owner name: CELLECTIS SA, FRANCE

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:EKBERG, JENNY;HANSSON, MATTIAS;DOEHN, ULRIK;AND OTHERS;SIGNING DATES FROM 20150818 TO 20150823;REEL/FRAME:036750/0059

AS Assignment

Owner name: TAKARA BIO EUROPE AB, SWEDEN

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:CELLECTIS SA;REEL/FRAME:037071/0840

Effective date: 20150416

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION