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
Other languages
English (en)
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)
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
EP12182747.1 2012-09-03
EP12182747 2012-09-03
US201261697970P 2012-09-07 2012-09-07
EP12198820.8 2012-12-21
EP12198820 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 Pending 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 Pending 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 (ja)
EP (1) EP2893000B1 (ja)
JP (2) JP6470687B2 (ja)
CN (1) CN104903440B (ja)
WO (1) WO2014033322A1 (ja)

Cited By (9)

* 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
US11999971B2 (en) 2022-11-14 2024-06-04 Vertex Pharmaceuticals Incorporated Stem cell derived islet differentiation

Families Citing this family (30)

* 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
KR101617243B1 (ko) 2007-07-31 2016-05-02 라이프스캔, 인코포레이티드 인간 배아 줄기 세포의 분화
JP5733986B2 (ja) 2008-02-21 2015-06-10 ヤンセン バイオテツク，インコーポレーテツド 細胞の付着、培養、及び剥離のための方法、表面改質されたプレート、並びに組成物
KR101651661B1 (ko) 2008-06-30 2016-08-26 얀센 바이오테크 인코포레이티드 만능 줄기 세포의 분화
JP5719305B2 (ja) 2008-11-20 2015-05-13 ヤンセン バイオテツク，インコーポレーテツド 平面支持体上での細胞付着及び培養のための方法及び組成物
KR101774546B1 (ko) 2008-11-20 2017-09-04 얀센 바이오테크 인코포레이티드 마이크로-캐리어 상의 만능 줄기 세포 배양
CN102482643B (zh) 2009-07-20 2016-06-29 詹森生物科技公司 人胚胎干细胞的分化
CN102741395B (zh) 2009-12-23 2016-03-16 詹森生物科技公司 人胚胎干细胞的分化
JP6013196B2 (ja) 2010-03-01 2016-10-25 ヤンセン バイオテツク，インコーポレーテツド 多能性幹細胞から誘導した細胞を精製するための方法
CA2809300A1 (en) 2010-08-31 2012-03-08 Janssen Biotech, Inc. Differentiation of human embryonic stem cells
SG10201608914WA (en) 2011-12-22 2016-12-29 Janssen Biotech Inc Differentiation of human embryonic stem cells into single hormonal insulin positive cells
US10066210B2 (en) 2012-06-08 2018-09-04 Janssen Biotech, Inc. Differentiation of human embryonic stem cells into pancreatic endocrine cells
CN104903440B (zh) * 2012-09-03 2018-04-06 诺和诺德股份有限公司 使用小分子从多能干细胞产生胰内胚层
KR101942769B1 (ko) 2012-12-31 2019-01-28 얀센 바이오테크 인코포레이티드 Hb9 조절제를 사용하는 인간 배아 줄기세포의 췌장 내분비 세포로의 분화
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
BR112015015714A2 (pt) 2012-12-31 2017-07-11 Janssen Biotech Inc suspensão e aglomeração de células pluripotentes humanas para diferenciação em célu-las endócrinas pancreáticas
US10370644B2 (en) 2012-12-31 2019-08-06 Janssen Biotech, Inc. Method for making human pluripotent suspension cultures and cells derived therefrom
CN117821369A (zh) * 2014-05-16 2024-04-05 詹森生物科技公司 小分子增强胰腺内分泌细胞中的mafa表达的用途
CA2990861A1 (en) * 2015-04-30 2016-11-03 Nant Holdings Ip, Llc Patient treatment via teratogenic pharmaceutical compounds
CN108699515A (zh) * 2016-02-24 2018-10-23 诺和诺德股份有限公司 由人多能干细胞衍生的内分泌祖细胞生成功能性β细胞
MA45479A (fr) 2016-04-14 2019-02-20 Janssen Biotech Inc Différenciation de cellules souches pluripotentes en cellules de l'endoderme de l'intestin moyen
MA45502A (fr) 2016-06-21 2019-04-24 Janssen Biotech Inc Génération de cellules bêta fonctionnelles dérivées de cellules souches pluripotentes humaines ayant une respiration mitochondriale glucose-dépendante et une réponse en sécrétion d'insuline en deux phases
AU2017313847A1 (en) * 2016-08-19 2019-02-28 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
EP3681992A1 (en) 2017-09-11 2020-07-22 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
JP2022528917A (ja) 2019-04-08 2022-06-16 ノヴォ ノルディスク アー／エス 幹細胞由来の胚体内胚葉からの膵臓内胚葉の生成
WO2020236985A1 (en) * 2019-05-21 2020-11-26 President And Fellows Of Harvard College Endocrine differentiation-inducing molecule
JP2023539215A (ja) 2020-08-28 2023-09-13 ノヴォ ノルディスク アー／エス 幹細胞由来ベータ様細胞のインビトロ集団およびその新規のマーカーをスクリーニングするための方法
WO2024008810A1 (en) 2022-07-06 2024-01-11 Novo Nordisk A/S Differentiation of stem cells to pancreatic endocrine 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
AU2003210959A1 (en) 2002-02-12 2003-09-04 Cornell Research Foundation, Inc. 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
WO2005086860A2 (en) 2004-03-09 2005-09-22 Gang Xu Methods for generating insulin-producing cells
WO2006134017A2 (en) 2005-06-13 2006-12-21 Novo Nordisk A/S Modulation of cells
CA3147112A1 (en) 2006-03-02 2007-09-13 Viacyte, Inc. Endocrine precursor cells, pancreatic hormone-expressing cells and methods of production
EP2021462B1 (en) 2006-04-28 2019-01-09 Lifescan, Inc. Differentiation of human embryonic stem cells
AU2007248609B2 (en) 2006-05-02 2012-11-01 Wisconsin Alumni Research Foundation Method of differentiating stem cells into cells of the endoderm and pancreatic lineage
AU2007297575A1 (en) 2006-06-26 2008-03-27 Lifescan, Inc. Pluripotent stem cell culture
US8835163B2 (en) 2006-10-18 2014-09-16 The Board Of Trustees Of The University Of Illinois Embryonic-like stem cells derived from adult human peripheral blood and methods of use
US9080145B2 (en) 2007-07-01 2015-07-14 Lifescan Corporation Single pluripotent stem cell culture
DK3192865T3 (da) 2007-07-01 2021-06-28 Janssen Biotech Inc Enkelt pluripotent stamcelle-kultur
CN101861386A (zh) 2007-07-18 2010-10-13 生命扫描有限公司 人胚胎干细胞的分化
EP2584034B8 (en) 2007-07-31 2017-12-06 Lifescan, Inc. Pluripotent stem cell differentiation by using human feeder cells
KR101617243B1 (ko) 2007-07-31 2016-05-02 라이프스캔, 인코포레이티드 인간 배아 줄기 세포의 분화
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
AU2008355123B2 (en) 2008-04-21 2014-12-04 Viacyte, Inc. Methods for purifying endoderm and pancreatic endoderm cells derived from human embryonic stem cells
WO2009132083A2 (en) 2008-04-22 2009-10-29 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
KR101651661B1 (ko) 2008-06-30 2016-08-26 얀센 바이오테크 인코포레이티드 만능 줄기 세포의 분화
US20100028307A1 (en) 2008-07-31 2010-02-04 O'neil John J Pluripotent stem cell differentiation
US9012218B2 (en) 2008-10-31 2015-04-21 Janssen Biotech, Inc. Differentiation of human embryonic stem cells
PL2346988T3 (pl) * 2008-10-31 2017-10-31 Janssen Biotech Inc Różnicowanie ludzkich zarodkowych komórek macierzystych do linii endokrynnej trzustki
WO2010053472A1 (en) 2008-11-04 2010-05-14 Novocell, 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
EP4176888A1 (en) 2008-11-14 2023-05-10 ViaCyte, Inc. Encapsulation of pancreatic cells derived from human pluripotent stem cells
KR101774546B1 (ko) 2008-11-20 2017-09-04 얀센 바이오테크 인코포레이티드 마이크로-캐리어 상의 만능 줄기 세포 배양
US8507274B2 (en) 2009-02-06 2013-08-13 President And Fellows Of Harvard College Compositions and methods for promoting the generation of definitive endoderm
US8642334B2 (en) * 2009-02-17 2014-02-04 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
EP2421957B1 (en) * 2009-04-22 2020-11-18 Viacyte, Inc. Cell compositions derived from dedifferentiated reprogrammed cells
WO2010136583A2 (en) * 2009-05-29 2010-12-02 Novo Nordisk A/S INDUCED DERIVATION OF SPECIFIC ENDODERM FROM hPS CELL-DERIVED DEFINITIVE ENDODERM
EP2456858B1 (en) 2009-07-20 2018-08-29 Janssen Biotech, Inc. Differentiation of human embryonic stem cells
EP2456859A4 (en) 2009-07-20 2015-03-18 Janssen Biotech Inc DIFFERENTIATION OF HUMAN EMBRYONIC STEM CELLS
EP2516626B1 (en) 2009-12-23 2017-05-10 Janssen Biotech, Inc. Differentiation of human embryonic stem cells
CN102741395B (zh) * 2009-12-23 2016-03-16 詹森生物科技公司 人胚胎干细胞的分化
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
AU2011250912A1 (en) * 2010-05-12 2012-11-22 Janssen Biotech, Inc. 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
EP2611907B1 (en) * 2010-08-31 2016-05-04 Janssen Biotech, Inc. Differentiation of pluripotent stem cells
CN103890167A (zh) 2011-06-21 2014-06-25 诺沃—诺迪斯克有限公司 自多潜能干细胞有效诱导定形内胚层
WO2013094771A1 (en) * 2011-12-19 2013-06-27 Kyoto University Method for inducing differentiation of human pluripotent stem cells into intermediate mesoderm cells
CN104903440B (zh) * 2012-09-03 2018-04-06 诺和诺德股份有限公司 使用小分子从多能干细胞产生胰内胚层

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 (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
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
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
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
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
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
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
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
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
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
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
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
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
US11945795B2 (en) 2017-11-15 2024-04-02 Vertex Pharmaceuticals Incorporated Islet cell manufacturing compositions and methods of use
US11466256B2 (en) 2018-08-10 2022-10-11 Vertex Pharmaceuticals Incorporated Stem cell derived islet differentiation
US11525120B2 (en) 2018-08-10 2022-12-13 Vertex Pharmaceuticals Incorporated Stem cell derived islet differentiation
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
US11999971B2 (en) 2022-11-14 2024-06-04 Vertex Pharmaceuticals Incorporated Stem cell derived islet differentiation

Also Published As

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

Similar Documents

Publication Publication Date Title
US10221392B2 (en) Generation of pancreatic endoderm from pluripotent stem cells using small molecules
JP7420860B2 (ja) 前駆細胞を指向性分化によって胃組織に変換するための方法及びシステム
US11274280B2 (en) Generation of functional beta cells from human pluripotent stem cell-derived endocrine progenitors
US20200002670A1 (en) Generation of Endocrine Progenitor Cells from Human Pluripotent Stem Cells Using Small Molecules
US10017734B2 (en) Method for producing dopaminergic neurons
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
Tan et al. Generation of clinical‐grade functional cardiomyocytes from human embryonic stem cells in chemically defined conditions
CN113728090B (zh) 从干细胞衍生的定形内胚层生成胰腺内胚层
ES2729776T3 (es) Generación de endodermo pancreático a partir de células madre pluripotentes mediante el uso de moléculas pequeñas
Hoelzl et al. Suppressor of Fused plays an important role in regulating mesodermal differentiation of murine embryonic stem cells In vivo
ES2746232T3 (es) Generación de células progenitoras endocrinas a partir de células madre pluripotentes humanas mediante el uso de moléculas pequeñas
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