SC-ß cells and compositions and methods for producing the cells

Abstract

An in vitro method for generating insulin-producing pancreatic β-cells (SC-β-cells) from precursor cells, the method comprising: Contacting PDX1-positive, NKX6-1-positive, or insulin-positive endocrine cells under conditions that promote cell clustering with a thyroid hormone signaling pathway activator, a transforming growth factor-β (TGF-β) signaling pathway inhibitor, and a γ-secretase inhibitor, thereby inducing the in vitro maturation of at least some PDX1-positive, NKX6-1-positive, or insulin-positive endocrine cells into SC-β cells, wherein the SC-β cells exhibit in vitro and / or in vivo glucose-stimulated insulin secretion (GSIS) similar to the GSIS of native pancreatic β cells.

Description

[0001] This application claims priority to U.S. Provisional Application No. 61 / 833,898, filed June 1, 2013, and U.S. Provisional Application No. 61 / 972,212, filed March 28, 2014, the contents of which are hereby incorporated by reference in their entirety. BACKGROUND OF THE INVENTION

[0002] Current research has so far only produced abnormally functioning insulin-expressing cells that do not secrete appropriate amounts of insulin in response to changing glucose levels, or pancreatic progenitor cells that only mature into functioning insulin-expressing cells after 3 months after transplantation into a host mouse (Cheng et al 2012; D'Amour et al, 2005; D'Amour et al, 2006; Kroon et al, 2008; Nostro et al, 2011; Rezania et al 2012; Schulz et al 2012; Xie et al, 2013).In contrast to normal islet cells or dispersed mature β-cells, which release high concentrations of insulin in response to high concentrations of glucose in the glucose-stimulated insulin secretion (GSIS) assay and can do so repeatedly, hPSC-derived insulin-expressing cells generated by existing methods are unable to adequately secrete insulin in response to the addition of various concentrations of glucose. Accordingly, there is a need for a method for deriving cells from hPSCs that exhibit a phenotype of normal islets or mature adult β-cells. WO2013095953 A1 discloses methods for promoting the differentiation of pluripotent stem cells, in particular, wherein more than 10% of the cells in the population express markers characteristic of individual hormonal pancreatic beta cells.US2007259421 A1 discloses cell cultures and enriched cell populations of endocrine progenitor cells, immature pancreatic hormone-expressing cells, and mature pancreatic hormone-expressing cells, as well as methods for their production. WO2011011302 A2 discloses methods for promoting the differentiation of pluripotent stem cells into insulin-producing cells. EP2505639 A1 discloses a method for more efficient production of pancreatic cells, in particular pancreatic hormone-producing cells, and applications thereof. SUMMARY OF THE INVENTION

[0003] In some aspects, the disclosure provides a stem cell-derived β-cell (SC-β).

[0004] In some embodiments, the cell is mature. In some embodiments, the cell exhibits an in vitro glucose-stimulated insulin secretion (GSIS) response. In some embodiments, the cell exhibits an in vivo GSIS response. In some embodiments, the cell exhibits in vitro and in vivo glucose-stimulated insulin secretion (GSIS) responses. In some embodiments, the cell exhibits a GSIS response to at least one glucose challenge. In some embodiments, the cell exhibits a GSIS response to at least two consecutive glucose challenges. In some embodiments, the cell exhibits a GSIS response to at least three consecutive glucose challenges. In some embodiments, the GSIS response is observed immediately after transplanting the cell into a human or animal.In some embodiments, the GSIS response is observed within about 24 hours after transplantation of the cell into a human or animal. In some embodiments, the GSIS response is observed within about two weeks of transplantation of the cell into a human or animal. In some embodiments, the stimulation index of the cell is determined by the ratio of insulin secreted in response to high glucose concentrations compared to low glucose concentrations, similar to the stimulation index of an endogenous mature β pancreatic cell. In some embodiments, the stimulation index is greater than or equal to 1, or greater than or equal to 1.1, or greater than or equal to 1.3, or greater than or equal to 2, or greater than or equal to 2.3, or greater than or equal to 2.6. In some embodiments, the cell exhibits cytokine-induced apoptosis in response to a cytokine.In some embodiments, the cytokine is selected from the group consisting of interleukin-β (IL-β), interferon-γ (INF-γ), tumor necrosis factor-α (TNF-α), and combinations thereof. In some embodiments, insulin secretion from the cell is enhanced in response to an antidiabetic agent. In some embodiments, the antidiabetic agent comprises a secretagogue selected from the group consisting of an incretin, a mimetic, a sulfonylurea, a meglitinide, and combinations thereof. In some embodiments, the cell is monohormonal. In some embodiments, the cell has a morphology similar to the morphology of an endogenous mature β pancreatic cell.

[0005] In some embodiments, the cell exhibits encapsulated crystalline insulin granules under electron microscopy that resemble insulin granules of an endogenous mature pancreatic β-cell. In some embodiments, the cell has a low rate of replication. In some embodiments, the cell exhibits a glucose stimulated Ca2+ flux (GSCF) that resembles the GSCF of an endogenous mature β-pancreatic cell. In some embodiments, the cell exhibits a GSCF response to at least one glucose challenge. In some embodiments, the cell exhibits a GSCF response to at least two glucose challenges. In some embodiments, the cell exhibits a GSCF response to at least three glucose challenges. In some embodiments, the cell exhibits increased calcium flux. In some embodiments, the increased calcium flux comprises an increased influx or a ratio of influx to low compared to high glucose concentrations.In some embodiments, the cell comprises at least one marker characteristic of an endogenous mature pancreatic β-cell selected from the group consisting of insulin, C-peptide, PDX 1 MAFA NKX6-1, PAX6, NEUROD1 glucokinase (GCK), SLC2A expressing 1, PCSK 1, KC J 1 1, ABCC8, SLC30A8, SNAP25, RAB3A, GAD2, PTPRN, NKX2-2, Pax4.

[0006] In some embodiments, the cell does not express at least one marker selected from the group consisting of a) a hormone selected from the group consisting of i) glucagon (GCG), and ii) somatostatin (SST); or b) an acinar cell tumor marker selected from the group consisting of i) amylase and ii) carboxypeptidase A (CPA 1); c) an a-cell marker selected from the group consisting of i) GCG, ii) Arx, iii) IRX1 and IRX2; and d) a ductal cell marker selected from the group consisting of i) CFTR and ii) Sox9. In some embodiments, the cell is differentiated in vitro from an insulin-positive endocrine cell or a precursor thereof selected from the group consisting of an NKX6-1 positive pancreatic progenitor cell, a Pdxl positive pancreatic progenitor cell, and a pluripotent stem cell.In some embodiments, the pluripotent stem cell is selected from the group consisting of an embryonic stem cell and pluripotent stem cells. In some embodiments, the cell is human. In some embodiments, the cell is not genetically modified. In some embodiments, the cell is genetically engineered. In some embodiments, the amount of insulin produced per cell is between 0.5 and 10 µIU per 1000 cells per 30-minute incubation at high glucose concentration. In some embodiments, the amount of insulin produced per cell is about 2.5 µIU per 1000 cells per 30-minute incubation at high glucose concentration. In some embodiments, the incubation is ex vivo.

[0007] In some aspects, the invention provides a cell line comprising an SC-β cell. In some embodiments, the cell line stably expresses insulin. In some embodiments, the cells can be frozen, thawed, and amplified to at least 30 passages with a doubling time of between about 24 and 44 hours without significant morphological changes.

[0008] In some aspects, the disclosure provides a method for generating insulin-producing pancreatic β-cells (SC-β-cells) from progenitor cells, the method comprising contacting PDX1-positive, NKX6-1-positive, or insulin-positive endocrine cells under conditions that promote cell clustering with a thyroid hormone signaling pathway activator, a transforming growth factor β (TGF-β) signaling pathway inhibitor, and a γ-secretase inhibitor, thereby inducing the in vitro maturation of at least some PDX1-positive, NKX6-1-positive, or insulin-positive endocrine cells. The SC-β-cells exhibit glucose-stimulated insulin secretion (GSIS) in vitro and / or in vivo that is similar to the GSIS of native pancreatic β-cells.

[0009] In some embodiments, the SC-β cell exhibits a response to at least two consecutive glucose challenges. In some embodiments, the SC-β cell exhibits a response to at least three consecutive glucose challenges. In some embodiments, the morphology of the SC-β cell resembles the morphology of an endogenous mature β cell. In some embodiments, the GSIS response is observed immediately after transplantation of the SC-β cell into an individual. In some embodiments, the GSIS response is observed within about 24 hours after transplantation of the SC-β cell into an individual. In some embodiments, the GSIS response is observed within about two weeks after transplantation of the SC-β cell into an individual. 100 µM - In some embodiments, the cell population is contacted with the TGF-β signaling pathway inhibitor at a concentration of between 100 nM.In some embodiments, the cell population is contacted with the TGF-β signaling pathway inhibitor at a concentration of 10 μM. In some embodiments, the TGF-β signaling pathway comprises the TGF-β receptor type I kinase signaling pathway.

[0010] In some embodiments, the TGF-β signaling pathway inhibitor is ALK5 Inhibitor II. In some embodiments, the TGF-β signaling pathway inhibitor comprises an analogue or derivative of ALK5 Inhibitor II. In some embodiments, the cell population is contacted with the thyroid hormone signaling pathway activator at a concentration of between 0.1 and 10 µM. In some embodiments, the cell population is contacted with the thyroid hormone signaling pathway activator at a concentration of 1 µM. In some embodiments, the thyroid hormone signaling pathway activator is triiodothyronine (T3). In some embodiments, the cell population is optionally contacted with a protein kinase inhibitor. In some embodiments, the cell population is not contacted with the protein kinase inhibitor. In some embodiments, the cell population is contacted with the protein kinase inhibitor.In some embodiments, the cell population is contacted with the protein kinase inhibitor at a concentration of between 10 nM and 1 µM. In some embodiments, the cell population is contacted with the protein kinase inhibitor at a concentration of 100 nM. In some embodiments, the protein kinase inhibitor comprises staurosporine. In some embodiments, the method comprises contacting the cell population with at least one additional β-cell maturation factor. In some embodiments, the at least one additional β-cell maturation factor comprises a cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor. In some embodiments, the cell population is contacted with the CFTR inhibitor at a concentration of between 100 nM and 100 µM. In some embodiments, the cell population is contacted with the CFTR inhibitor at a concentration of between 10 nM and 10 µM.In some embodiments, the CFTR inhibitor comprises Gly-H 011. In some embodiments, the at least one additional β-cell maturation factor comprises an O-GlcNAcase inhibitor. In some embodiments, the cell population is contacted with the O-GlcNAcase inhibitor at a concentration of 100 nM - 100 µM. In some embodiments, the cell population is contacted with the O-GlcNAcase inhibitor at a concentration of 10 nM - 10 µM. In some embodiments, the inhibitor of O-GlcNAcase comprises Thiamet G. In some embodiments, the cell population is grown in a suitable culture medium.

[0011] In some embodiments, the suitable culture medium comprises Connought Medical Research Laboratories 1066 Supplemented Islet Medium (CMRLS) or a component of CMRLS. In some embodiments, CMRLS is supplemented with serum. In some embodiments, CMRLS is supplemented with 10% fetal bovine serum. In some embodiments, the conditions that promote cell clustering are suspension culture. In some embodiments, the cell population is maintained in suspension culture for a period of time sufficient to induce in vitro maturation of at least one of the insulin-positive endocrine cells in the cell population into at least one SC-β cell. In some embodiments, the period of time is at least 7 days. In some embodiments, the period of time comprises from 7 days to 21 days. In some embodiments, the period of time can comprise between 7 and 14 days.In some embodiments, the period of time may be between 10 and 14 days. In some embodiments, the period of time may be 14 days. In some embodiments, the β-cell maturation factors are renewed every two days. In some embodiments, at least 1% of the insulin-positive cells in the endocrine cell population are induced to mature into SC-β cells. In some embodiments, at least 99% of the insulin-positive endocrine cells in the population are induced to mature into SC-β cells. In some embodiments, at least 30% of the resulting cells in the population comprise SC-β cells. In some embodiments, the SC-β cells express C-peptide, insulin, NKX6-1, Pdxl, and co-express NKX6-1 and C-peptide. In some embodiments, the insulin-positive endocrine cells also express Pdxl and NKX6-1.In some embodiments, the insulin-positive endocrine cells are produced from a population of pluripotent stem cells selected from the group consisting of embryonic stem cells and pluripotent stem cells. In some embodiments, the SC-β cells comprise human cells. In some embodiments, the generation of SC-β cells is scalable in vitro.

[0012] In some aspects, the disclosure provides an isolated population of SC-β cells prepared according to the methods described herein.

[0013] Also disclosed is a microcapsule comprising therein an isolated population of SC-β cells as prepared according to the methods described herein.

[0014] In some aspects, the disclosure provides a composition comprising a population of SC-β cells prepared according to a method described herein.

[0015] An assay is also disclosed comprising an isolated population of SC-β cells prepared according to a method described herein.

[0016] In some embodiments, the assay is for use in identifying one or more candidate agents to promote or inhibit a β-cell fate selected from the group consisting of β-cell proliferation, β-cell replication, β-cell death, β-cell function, β-cell susceptibility to immune attack, or β-cell susceptibility, dedifferentiation, or differentiation. In some embodiments, the assay is for use in identifying one or more candidate agents to promote the differentiation of at least one insulin-positive endocrine cell or precursor thereof into at least one SC β-cell.

[0017] In some aspects, the invention provides a composition for treating an individual in need thereof, for administering the composition to the patient comprising an isolated population of SC-β cells prepared according to a method described herein. In some embodiments, the SC-β cells are encapsulated in a microcapsule. In some embodiments, the SC-β cells are produced from a population of pluripotent stem cells derived from the same individual that the SC-β cells are administered. In some embodiments, the SC-β cells are produced from a population of iPS cells, wherein the iPS cells are derived from a cell from the same patient to whom the SC-β cells are to be administered. In some embodiments, the patient has an increased risk of developing diabetes or has diabetes. In some embodiments, the diabetes is from the group of type 1 diabetes, type 2 diabetes, type 1.5 diabetes and prediabetes. In some embodiments, the patient is at increased risk for developing or has a metabolic disorder.

[0018] In some embodiments, the isolated population of SC cells can be administered to the patient in microcapsules.

[0019] In some embodiments, the individual has diabetes or is at increased risk of developing it. In some embodiments, the diabetes is selected from the group of type I diabetes, type II diabetes, type 1 diabetes, and prediabetes. In some embodiments, the individual has or is at increased risk of developing a metabolic disorder.

[16] A culture medium is also disclosed, comprising a) ALK5 inhibitor, b) triiodothyronine (T3), optionally c) staurosporine, and optionally d) CMRLS or a component of CMRLS.

[0020] In some aspects of the disclosure, the use of the culture medium involves inducing in vitro maturation of insulin-positive endocrine cells in SC-β cells, wherein the SC-β cells exhibit both an in vitro and / or in vivo GSIS response.

[0021] A method for producing an NKX6-1 -positive pancreatic progenitor cell from a Pdxl -positive pancreatic progenitor cell is also disclosed, comprising contacting a cell population comprising Pdxl -positive pancreatic progenitor cells under conditions that promote cell clustering with at least two β-cell maturation factors containing a) at least one growth factor from the fibroblast growth factor (FGF) family, b) a sonic hedgehog signaling pathway inhibitor and optionally c) a low concentration of a retinoic acid (RA) signaling pathway activator for a period of at least five days to induce the differentiation of at least one Pdxl -positive pancreatic progenitor cell in the population into NKX6-1 -positive pancreatic progenitor cells, wherein the NKX6-1 -positive pancreatic progenitor cells express NKX6-1.

[0022] The cell population can be contacted with the at least one growth factor from the FGF family at a concentration of 1 ng / mL - 100 ng / mL. The cell population can be contacted with the at least one growth factor from the FGF family at a concentration of 50 ng / ml. The at least one growth factor from the FGF family can be keratinocyte growth factor (KGF). The at least one growth factor from the FGF family can be selected from the group consisting of FGF2, FGF8b, FGF10, FGF21. In some embodiments, the cell population is contacted with the RA signaling pathway activator. The cell population can be contacted with the RA signaling pathway activator at a concentration of between 0.01 µM and 1.0 mM. The cell population can be contacted with the RA signaling pathway activator at a concentration of 0.1 µM. The RA signaling pathway can comprise activator RA.The cell population can be contacted with the SHH signaling pathway inhibitor at a concentration of between 0 and 0.5 µM. The cell population can be contacted with the SHH signaling pathway inhibitor at a concentration of 0.25 µM. The SHH signaling pathway inhibitor can comprise Santl. The method can comprise exposing the cell population to at least one additional β-cell maturation factor. The at least one additional β-cell maturation factor can comprise at least one growth factor from the EGF family. The cell population can further be exposed to at least one growth factor from the EGF family at a concentration of 2 ng / ml - 200 ng / ml. The cell population can further be exposed to at least one growth factor from the EGF family at a concentration of 20 ng / ml. The one growth factor from the EGF family can be selected from the group consisting of EGF and betacellulin.The cell population can be grown in a suitable culture medium. Conditions that promote cell clustering include suspension culture. β-cell maturation factors can be replenished every other day. Protein kinase C activator can optionally be withheld from the suspension culture for 5 days.

[0023] In some embodiments, an activator of protein kinase C is removed from the suspension culture prior to the 5 days. The activator of protein kinase C may comprise PDBU. A BMP signaling pathway inhibitor may optionally not be added to the suspension culture for 5 days. A BMP signaling pathway inhibitor may be removed from the suspension culture prior to the 5 days. The BMP signaling pathway inhibitor may comprise LDN193 1 89. At least 10% of the Pdxl-positive pancreatic progenitor cells in the population can be induced to differentiate into NKX6-1-positive pancreatic progenitor cells. At least 95% of the Pdxl-positive pancreatic progenitor cells in the population can be induced to differentiate into NKX6-1-positive pancreatic progenitor cells. The NKX6-1 positive pancreatic progenitor cells can express Pdxl, NKX6-1, and Foxa2.The Pdxl-positive pancreatic progenitor cells produced from a population of pluripotent stem cells may be selected from the group consisting of embryonic stem cells and pluripotent stem cells.

[0024] In some aspects, the disclosure provides an isolated population comprising a plurality of SC-β cells obtained by a method described above. At least 10% of the cells in the population are SC-β cells.

[21] The disclosure further provides a microcapsule comprising the isolated population of SC-β cells encapsulated therein.

[0025] In some aspects, the disclosure provides a composition comprising a plurality of SC-β cells prepared by a method described herein. The SC-β cells comprise one or more crystalline insulin granules, or the SC-β cells express the genes: INS, PDX1, NKX6-1, and ZNT8, or the SC-β cells exhibit in vitro glucose-stimulated insulin secretion in response to an initial glucose challenge.

[0026] Also disclosed is an assay comprising an isolated population of NKX6-1 positive pancreatic progenitor cells prepared by a method described herein.

[0027] In some embodiments, the assay is for use in identifying one or more candidate substances that promote the differentiation of at least one Pdxl-positive pancreatic progenitor cell or a precursor thereof into NKX6-1-positive pancreatic progenitor cells.

[0028] In some aspects, the invention relates to the use of an isolated population of NKX6-1 positive pancreatic progenitor cells produced by a method according to any one of claims 1 to 13 to differentiate into SC-β cells.

[0029] In some aspects, the disclosure relates to the use of an isolated population of NKX6-1 positive pancreatic progenitor cells produced by a method described herein for administration to a patient in need thereof.

[0030] In some embodiments, the isolated population of NKX6-1-positive pancreatic progenitor cells can be administered to the individual in microcapsules. In some embodiments, the individual has or may have an increased risk of developing diabetes. In some embodiments, the diabetes is selected from the group comprising type 1 diabetes, type 2 diabetes, type 1 diabetes, and prediabetes. In some embodiments, the individual has an increased risk of developing a metabolic disorder.

[0031] In some aspects, the disclosure provides a culture medium comprising a) KGF, b) SANT1) and optionally c) RA, wherein the culture medium is substantially free of PDBu and LDN 193189.

[0032] In some aspects, the disclosure provides an in vitro method for producing an insulin-positive endocrine cell from an NKX6-1-positive pancreatic progenitor cell, comprising contacting a cell population comprising NKX6-1-positive pancreatic progenitor cells under conditions promoting cell clustering with at least two β-cell maturation factors comprising a) a TGF-β signaling pathway inhibitor and b) a thyroid hormone signaling pathway activator, to induce differentiation of at least one NKX6-1-positive pancreatic progenitor cell in the population into at least one insulin-positive endocrine cell, wherein the insulin-positive pancreatic progenitor cell expresses insulin. In some embodiments, the cell population is contacted with the TGF-β signaling pathway inhibitor at a concentration of between 100 nM.In some embodiments, the cell population is contacted with the TGF-β signaling pathway inhibitor at a concentration of 10 µM. In some embodiments, the TGF-β signaling pathway comprises the TGF-β receptor type I kinase signaling pathway. In some embodiments, the TGF-β signaling pathway inhibitor comprises the ALK5 inhibitor II. In some embodiments, the cell population is contacted with the thyroid hormone signaling pathway activator at a concentration of between 0.1 µ - 10 µM. In some embodiments, the cell population is contacted with the thyroid hormone signaling pathway activator at a concentration of 1 µM. In some embodiments, the thyroid hormone signaling pathway activator comprises triiodothyronine (T3). In some embodiments, the method comprises contacting the cell population with at least one additional β-cell maturation factor.In some embodiments, the at least one additional β-cell maturation factor comprises a γ-secretase inhibitor. In some embodiments, the cell population is contacted with the γ-secretase inhibitor at a concentration between 0.1 μM - 10 μM. In some embodiments, the cell population is contacted with the γ-secretase inhibitor at a concentration of 1 μM. In some embodiments, the γ-secretase inhibitor comprises XXL. In some embodiments, the γ-secretase inhibitor comprises DAPT. In some embodiments, the at least one additional β-cell maturation factor comprises at least one growth factor from the EGF family. In some embodiments, the cell population is contacted with the at least one growth factor from the EGF family at a concentration of 2 ng / ml - 200 ng / ml.In some embodiments, the cell population is contacted with at least one growth factor from the EGF family at a concentration of 20 ng / ml. In some embodiments, the at least one growth factor from the EGF family comprises betacellulin. In some embodiments, the at least one growth factor from the EGF family comprises EGF. In some embodiments, the at least one additional β-cell maturation factor comprises a low concentration of a retinoic acid (RA) pathway activator. In some embodiments, the cell population is contacted with the RA pathway activator at a concentration of between 0.01 µM. In some embodiments, the cell population is contacted with the RA pathway activator at a concentration of 0.1 µM - 1.0 µM. In some embodiments, the RA pathway activator comprises RA.In some embodiments, the at least one additional β-cell maturation factor comprises a Sonic Hedgehog (SHH) signaling pathway inhibitor. In some embodiments, the cell population is contacted with the SHH signaling pathway inhibitor at a concentration of between 0.1 and 0.5 µM. In some embodiments, the cell population is contacted with the SHH signaling pathway inhibitor at a concentration of 0.25 µM. In some embodiments, the SHH signaling pathway inhibitor comprises Sant 1. In some embodiments, the cell population is optionally contacted with a protein kinase inhibitor. In some embodiments, the cell population is not contacted with the protein kinase inhibitor. In some embodiments, the cell population is contacted with the protein kinase inhibitor. In some embodiments, the cell population is contacted with the protein kinase inhibitor at a concentration of between 10 nM - 1 µM.In some embodiments, the cell population is contacted with the protein kinase inhibitor at a concentration of 100 nM. In some embodiments, the protein kinase inhibitor comprises staurosporine. In some embodiments, the method comprises exposing the cell population to glucose. In some embodiments, the cell population is exposed to glucose at a concentration of 1 mM-50 mM. In some embodiments, the cell population is exposed to glucose at a concentration of 25 mM. In some embodiments, the conditions that promote cell clustering comprise suspension culture. In some embodiments, the cell population is maintained in suspension culture for a period of time sufficient to induce differentiation of at least one of the NKX61-positive pancreatic progenitor cells in the population into an insulin-positive endocrine cell. In some embodiments, the period is at least 7 days.In some embodiments, the β-cell maturation factors in the suspension culture are renewed every other day. In some embodiments, at least 15% of the NKX6-1-positive pancreatic progenitor cells in the population are induced to differentiate into insulin-positive endocrine cells. In some embodiments, at least 99% of the NKX6-1-positive pancreatic progenitor cells in the population are induced to differentiate into insulin-positive endocrine cells. In some embodiments, the insulin-positive endocrine cells express Pdxl, NKX6-1, NKX2-2, MAFB, glis3, Surl, Kir6.2, ZnT8, SLC2A1, SLC2A3, and / or insulin. In some embodiments, the NKX6-1 positive pancreatic progenitor cells are produced from a population of pluripotent stem cells selected from the group consisting of embryonic stem cells and pluripotent stem cells.

[0033] In some aspects, the disclosure relates to an isolated population of insulin-positive endocrine cells produced according to a method described herein.

[0034] In some aspects, the disclosure relates to a microcapsule comprising an isolated population of insulin-positive endocrine cells encapsulated therein. In some embodiments, the invention provides a composition comprising a population of insulin-positive endocrine cells prepared according to a method described herein.

[0035] In some aspects, the disclosure relates to a method of treating a patient in need thereof, the method comprising administering to an individual a composition comprising an isolated population of insulin-positive endocrine cells produced according to a method described herein.

[0036] In some embodiments, the insulin-positive endocrine cells are produced from a population of pluripotent stem cells obtained from the same individual to whom the insulin-positive endocrine cells are administered. In some embodiments, the insulin-positive cells are encapsulated in an endocrine microcapsule. In some embodiments, the individual has diabetes or an increased risk of developing it. In some embodiments, the diabetes is selected from the group of type 1 diabetes, type 2 diabetes, type 1 diabetes, and prediabetes. 5. In some embodiments, the individual has a metabolic disorder or an increased risk of developing it.

[0037] In some aspects, the disclosure relates to the use of an isolated population of insulin-positive endocrine cells produced according to a method described herein for differentiation into SC-β cells.

[0038] In some aspects, the disclosure relates to the use of an isolated population of insulin-positive endocrine cells produced according to a method described herein for administration to an individual in need thereof,

[0039] In some embodiments, the isolated population of insulin-positive endocrine cells is administered to the individual in microcapsules. In some embodiments, the individual has diabetes or is at increased risk of developing it. In some embodiments, the diabetes is selected from the group consisting of type 1 diabetes, type 2 diabetes, type 1 diabetes, and prediabetes. In some embodiments, the individual has a metabolic disorder or is at increased risk of developing it.

[0040] In some aspects, the disclosure relates to a culture medium comprising a) a TGF-β signaling pathway inhibitor, b) a TH signaling pathway activator, and at least one additional β-cell maturation factor selected from the group consisting of i) XXI, ii) betacellulin, iii) a low concentration of an RA signaling pathway activator, and iv) an SHH signaling pathway inhibitor.

[0041] In some aspects, the disclosure relates to the use of the culture medium to induce in vitro differentiation of NKX6-1-positive pancreatic progenitor cells into insulin-positive endocrine cells.

[0042] In some aspects, the invention relates to an in vitro method for generating SC-β cells, comprising: contacting Pdxl-positive, NKX6-1-positive or insulin-positive endocrine cells under conditions that promote cell clustering or cell aggregation with i) a transforming growth factor-β (TGF-β) signaling pathway inhibitor, ii) a thyroid hormone signaling pathway activator and optionally iii) a protein kinase inhibitor to induce the in vitro maturation of at least some of the Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells into SC-β cells, wherein the SC-β cells exhibit a GSIS response in vitro and / or in vivo.

[0043] In some embodiments, the GSIS response is recorded (i) immediately after transplantation of the SC-β cell into a subject; (ii) within about 24 hours after transplantation into a subject; or (iii) within about two weeks after transplantation into a subject. In some embodiments, the SC-β cells exhibit a response to (i) at least one glucose challenge; (ii) at least two consecutive glucose challenges; or (iii) at least three consecutive glucose challenges. In some embodiments, the morphology of the SC-β cells resembles the morphology of endogenous β cells. In some embodiments, the Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with the TGF-β signaling pathway inhibitor at a concentration of between 100 nm - 100 µm.In some embodiments, the Pdxl-positive, N X6-1-positive, insulin-positive endocrine cells are contacted with the TGF-β signaling pathway inhibitor at a concentration of 10 µM. In some embodiments, the TGF-β signaling pathway comprises the TGF-β receptor type I kinase signaling pathway.

[0044] In some embodiments, the TGF-β signaling pathway inhibitor comprises the ALK5 Inhibitor II. In some embodiments, the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with the thyroid hormone signaling pathway activator at a concentration of between 0.1 µM and 10 µM. In some embodiments, the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with the thyroid hormone signaling pathway activator at a concentration of 1 µM. In some embodiments, the thyroid hormone signaling pathway activator comprises triiodothyronine (T3). In some embodiments, the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are not contacted with the protein kinase inhibitor. In some embodiments, the Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with the protein kinase inhibitor.In some embodiments, the Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with the protein kinase inhibitor at a concentration between 10 nM - 1 µM. In some embodiments, the Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with the protein kinase inhibitor at a concentration of 100 nM. In some embodiments, the protein kinase inhibitor comprises staurosporine. In some embodiments, the method comprises contacting the Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells with a cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor. In some embodiments, the Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with the CFTR inhibitor at a concentration of between 100 nm and 100 µM.In some embodiments, the Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with the CFTR inhibitor at a concentration of 10 nM and 10 µM. In some embodiments, the CFTR inhibitor comprises Gly-H101. In some embodiments, the method comprises contacting the Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells with an O-GlcNAcase inhibitor. In some embodiments, the Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with the O-GlcNAcase inhibitor at a concentration of between 100 nM and 100 µM. In some embodiments, the Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with the O-GlcNAcase inhibitor at a concentration between 10 nM and 10 µg. In some embodiments, the O-GlcNAcase inhibitor comprises Thiamet G.In some embodiments, the Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells are grown in a suitable culture medium. In some embodiments, the suitable culture medium comprises Connought Medical Research Laboratories 1066 Supplemented Islet Medium (CMRLS) or a component of CMRLS. In some embodiments, the CMRLS is supplemented with serum. In some embodiments, the CMRLS is supplemented with 10% fetal bovine serum. In some embodiments, the conditions promoting cell clustering comprise suspension culture. In some embodiments, the Pdxl-positive, NX6-1-positive, insulin-positive endocrine cells are maintained in suspension culture for a period of time sufficient to induce in vitro maturation of at least some of the Pdxl-positive, X6-1-positive, insulin-positive endocrine cells into SC-β cells. In some embodiments, the period may be at least 7 days.In some embodiments, the period of time comprises 7 days to 21 days. In some embodiments, the period of time can be between 7 and 14 days. In some embodiments, the period of time comprises 14 days. In some embodiments, the suspension culture is renewed every other day. In some embodiments, at least 30% of the cells generated comprise SC-β cells. In some embodiments, the SC-β cells express C-peptide, insulin, NKX6-1, Pdx1, and co-express NKX6-1 and C-peptide. In some embodiments, the SC-β cells comprise human cells. In some embodiments, the generation of the SC-β cells is scalable in vitro.

[0045] In some embodiments, the insulin-positive, endocrine cells are obtained by contacting Pdx1-positive, NKX6-1-positive pancreatic progenitor cells under conditions that promote cell clusters with i) a TGF-β signaling pathway inhibitor and ii) a thyroid hormone signaling pathway activator, to effect differentiation of at least some of the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells into Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells, wherein the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells express Pdx1, NKX6-1, Nkx2-2, MafB, Glis3, Sur1, Kir6.2, ZnT8, Slc2a1, Slc2a3, and / or insulin.

[0046] In some embodiments, the Pdxl-positive, NKX6-1-positive pancreatic progenitor cells are contacted with the TGF-β pathway inhibitor at a concentration of between 100 μM - 100 μM. In some embodiments, the Pdxl-positive, NKX6-1-positive pancreatic progenitor cells are contacted with the TGF-β pathway inhibitor at a concentration of 10 μM. In some embodiments, the TGF-β pathway comprises the TGF-β receptor type I kinase pathway. In some embodiments, the TGF-β pathway inhibitor comprises the ALK5 inhibitor II. In some embodiments, the Pdxl-positive, NKX6-1-positive pancreatic progenitor cells are contacted with the thyroid hormone pathway activator at a concentration of between 0.1 μM - 10 μM. In some embodiments, the Pdxl-positive, NKX6-1-positive pancreatic progenitor cells are contacted with the thyroid hormone signaling pathway activator at a concentration of 1 µM.In some embodiments, the thyroid hormone signaling pathway activator triiodothyronine (T3). In some embodiments, the method comprises contacting the Pdxl-positive, NKX6-1-positive pancreatic progenitor cells with at least one of i) an SHH signaling pathway inhibitor, ii) an RA signaling pathway activator, iii) a γ-secretase inhibitor, iv) at least one growth factor from the epidermal growth factor (EGF) family, and optionally v) a protein kinase inhibitor. In some embodiments, the Pdxl-positive, NKX6-1-positive pancreatic progenitor cells are contacted with the SHH signaling pathway inhibitor at a concentration of between 0.1 µM and 0.5 µM. In some embodiments, the Pdxl-positive, NKX6-1-positive pancreatic progenitor cells are contacted with an SHH pathway inhibitor at a concentration of 0.25 µM. In some embodiments, the SHH pathway inhibitor comprises Sant 1.In some embodiments, the Pdxl-positive, NKX6-1-positive pancreatic progenitor cells are contacted with the RA signaling pathway activator at a concentration of between 0.01 µM and 1.0 µM. In some embodiments, the Pdxl-positive, NKX6-1-positive pancreatic progenitor cells are contacted with the RA signaling pathway activator at a concentration of 0.1 µM. In some embodiments, the RA signaling pathway activator comprises RA. In some embodiments, the Pdxl-positive, NKX6-1-positive pancreatic progenitor cells are contacted with the γ-secretase inhibitor at a concentration of between 0.1 µM and 10 µM. In some embodiments, the Pdxl-positive, NKX6-1-positive pancreatic progenitor cells are contacted with the γ-secretase inhibitor at a concentration of 1 μM. In some embodiments, the γ-secretase inhibitor comprises XXI. In some embodiments, the γ-secretase inhibitor comprises DAPT.In some embodiments, the Pdxl-positive, NKX6-1-positive pancreatic progenitor cells are contacted with the at least one growth factor from the EGF family at a concentration of 2 ng / ml - 200 ng / ml. In some embodiments, the Pdxl-positive, NKX6-1-positive pancreatic progenitor cells are contacted with the at least one growth factor from the EGF family at a concentration of 20 ng / ml. In some embodiments, the at least one growth factor from the EGF family comprises betacellulin. In some embodiments, the at least one growth factor from the EGF family comprises EGF. In some embodiments, the Pdxl-positive, NKX6-1-positive pancreatic progenitor cells are not contacted with the protein kinase inhibitor. In some embodiments, the Pdxl-positive, NKX6-1-positive pancreatic progenitor cells are contacted with the protein kinase inhibitor.In some embodiments, the Pdxl-positive, N X6-1-positive pancreatic progenitor cells are contacted with the protein kinase inhibitor at a concentration between 10 nM - 1 µM. In some embodiments, the Pdxl-positive, N X6-1-positive pancreatic progenitor cells are contacted with the protein kinase inhibitor at a concentration of 100 nM. In some embodiments, the protein kinase inhibitor comprises staurosporine. In some embodiments, the method comprises exposing the cell population to glucose. In some embodiments, the cell population is exposed to glucose at a concentration of 1 mM - 50 mM. In some embodiments, the cell population is exposed to glucose at a concentration of 25 mM. In some embodiments, the conditions that promote cell clustering comprise suspension culture.In some embodiments, the Pdxl-positive, NKX6-1-positive pancreatic progenitor cells are maintained in a suspension culture for a period of time sufficient to induce differentiation of at least some of the Pdxl-positive, NKX6-1-positive pancreatic progenitor cells into Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells. In some embodiments, the period is at least 7 days. In some embodiments, the suspension culture is renewed every other day. In some embodiments, differentiation of at least 15% of the Pdxl-positive, NKX6-1-positive pancreatic progenitor cells into Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells is induced. In some embodiments, differentiation of at least 99% of the Pdxl-positive, NKX6-1-positive pancreatic progenitor cells into Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells is induced.

[0047] In some embodiments, the Pdxl-positive, NKX6-1-positive pancreatic progenitor cells are obtained by contacting the Pdxl-positive pancreatic progenitor cells under conditions promoting cell clustering with i) at least one growth factor from the FGF family, ii) at least one SHH signaling pathway inhibitor, and optionally iii) low concentrations of an RA signaling pathway activator for a period of five days to induce differentiation of at least some of the Pdxl-positive pancreatic progenitor cells into Pdxl-positive, NKX6-1-positive pancreatic progenitor cells, wherein the Pdxl-positive, NKX6-1-positive pancreatic progenitor cells express Pdxl and NKX6-1.

[0048] In some embodiments, the Pdxl-positive pancreatic progenitor cells are contacted with the at least one growth factor from the FGF family at a concentration of 1 ng / ml - 100 ng / ml. In some embodiments, the Pdxl-positive pancreatic progenitor cells are contacted with the at least one growth factor from the FGF family at a concentration of 50 ng / ml. In some embodiments, the at least one growth factor from the FGF family comprises keratinocyte growth factor (GF). In some embodiments, the at least one growth factor from the FGF family is selected from the group consisting of FGF2, FGF8b, FGF10, and FGF21. In some embodiments, the Pdxl-positive pancreatic progenitor cells are contacted with the at least one SHH signaling pathway inhibitor at a concentration of between 0.1 and 0.5 µM.In some embodiments, the Pdxl-positive pancreatic progenitor cells are contacted with the at least one SHH signaling pathway inhibitor at a concentration of 0.25 µM. In some embodiments, the at least one SHH signaling pathway inhibitor comprises SantI 1. In some embodiments, the Pdxl-positive pancreatic progenitor cells are contacted with the RA signaling pathway activator at a concentration of between 0.01 µM - 1.0 µM. In some embodiments, the Pdxl-positive pancreatic progenitor cells are contacted with the RA signaling pathway activator at a concentration of 0.1 µM. In some embodiments, the RA signaling pathway activator comprises RA. In some embodiments, the method comprises contacting the Pdxl-positive pancreatic progenitor cells with at least one growth factor from the EGF family.In some embodiments, the Pdxl-positive pancreatic progenitor cells are contacted with the at least one growth factor from the EGF family at a concentration of 2 ng / ml - 200 ng / ml. In some embodiments, the Pdxl-positive pancreatic progenitor cells are contacted with the at least one growth factor from the EGF family at a concentration of 20 ng / ml. In some embodiments, the at least one growth factor from the EGF family comprises betacellulin. In some embodiments, the at least one growth factor from the EGF family comprises EGF. In some embodiments, the Pdxl-positive pancreatic progenitor cells are grown in a suitable culture medium. In some embodiments, the conditions that promote cell clustering comprise a suspension culture. In some embodiments, the suspension culture is renewed every other day.In some embodiments, no protein kinase C activator is added to the suspension culture for 5 days. In some embodiments, the protein kinase C activator is removed from the suspension culture prior to the 5 days. In some embodiments, the protein kinase C activator comprises PDBU. In some embodiments, no BMP signaling pathway inhibitor is added to the suspension culture for 5 days. In some embodiments, a BMP signaling pathway inhibitor is removed from the suspension culture prior to the 5 days. In some embodiments, the BMP signaling pathway inhibitor comprises LDN193189. In some embodiments, at least 10% of the Pdxl-positive pancreatic progenitor cells in the population are induced to differentiate into Pdxl-positive, NKX6-1-positive pancreatic progenitor cells.In some embodiments, at least 95% of the Pdxl-positive pancreatic progenitor cells are induced to differentiate into Pdxl-positive, NKX6-1-positive pancreatic progenitor cells.

[0049] In some aspects, the disclosure provides a method for generating SC-β cells from pluripotent cells, the method comprising: a) differentiating pluripotent stem cells in a population into Pdxl-positive pancreatic progenitor cells; b) differentiating at least some of the Pdxl-positive pancreatic progenitor cells into Pdxl-positive, NKX6-1-positive pancreatic progenitor cells by a method comprising contacting the Pdxl-positive pancreatic progenitor cells under conditions promoting cell clustering with i) at least one growth factor from the FGF family, ii) at least one SHH signaling pathway inhibitor, and optionally iii) an RA signaling pathway activator, every other day for a period of five days to induce differentiation of at least some of the Pdxl-positive pancreatic progenitor cells in the population into NKX6-1-positive pancreatic progenitor cells,where the NKX6-1-positive pancreatic progenitor cells express Pdxl and NKX6-1; c) differentiating at least a portion of the Pdxl-positive, NKX6-1-positive pancreatic progenitor cells into Pdxl-positive, N X6-1-positive, insulin-positive endocrine cells by a method comprising contacting the Pdxl-positive, NKX6-1-positive pancreatic progenitor cells under conditions that promote cell clustering with i) a TGF-β signaling pathway inhibitor, b) a TH signaling pathway activator and optionally c) at least one SHH signaling pathway inhibitor, ii) an RA signaling pathway activator, iii) a γ-secretase inhibitor, and vi) at least one growth factor from the epidermal growth factor (EGF) family, every other day for a period of five to seven days, in order to promote the differentiation of at least some of the Pdxl-positive, NKX6-1-positive pancreatic progenitor cells into Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells,wherein the Pdxl-positive, NKX6-1, insulin-positive endocrine cells express Pdxl, NKX6-1, NKX2-2, MAFB, glis3, Surl, Kir6.2, ZnT8, SLC2A 1, SLC2A3 and / or insulin; and d) differentiating at least a portion of the Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells into SC-β cells by a method comprising contacting the Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells under conditions that promote cell clustering with a transforming growth factor-β (TGF-β) signaling pathway inhibitor, ii) a thyroid hormone signaling pathway activator, and optionally iii) a protein kinase inhibitor, every other day for a period of seven to 14 days, to induce the in vitro maturation of at least some of the Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells into SC-β cells, wherein the SC-β cells exhibit a GSIS response in vitro and / or in vivo.

[0050] In some embodiments, the disclosure provides a method for generating SC-β cells from pluripotent cells, the method comprising: a) differentiating at least some pluripotent cells in a population into Pdxl-positive pancreatic progenitor cells; b) differentiating at least some of the Pdxl-positive pancreatic progenitor cells into Pdxl-positive, NKX6-1-positive pancreatic progenitor cells by a process of contacting the Pdxl-positive pancreatic progenitor cells under cell clustering-promoting conditions with i) KGF, ii) Santl and optionally iii) low concentrations of RA, every other day for a period of five days, to induce the differentiation of at least one Pdx1-positive pancreatic progenitor cell in the population into NKX6-1-positive pancreatic progenitor cells, wherein the NKX6-1-positive pancreatic progenitor cells express Pdxl and NKX6-1;c) differentiating at least a portion of the Pdxl-positive, NKX6-1-positive pancreatic progenitor cells into Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells by a process of contacting the Pdxl-positive, NKX6-1-positive pancreatic progenitor cells with i) the ALKS inhibitor II, ii) T3, and optionally iii) Santl, iv) RA, v) XXI and vi) betacellulin, every other day for a period of five to seven days, to induce the differentiation of at least some of the Pdxl-positive, NKX6-1-positive pancreatic progenitor cells into Pdxl-positive, NKX6-1, insulin-positive endocrine cells, wherein the Pdxl-positive, NKX6-1, insulin-positive endocrine cells are Pdxl, NKX6-1, NKX2-2, express MAFB, glis3, Sur 1, Kir6.2, ZnT8, SLC2A 1, SLC2A3 and / or insulin;and d) differentiating at least a portion of the Pdxl-positive, NX6-1-positive, insulin-positive endocrine cells into SC-β cells by a method of contacting the Pdxl-positive, NX6-1-positive, insulin-positive endocrine cells under cell clustering-promoting conditions with i) the ALK5 inhibitor II, ii) T3, and optionally iii) staurosporine, every other day for a period of seven to 14 days to induce the in vitro maturation of at least some of the Pdxl-positive, NX6-1-positive, insulin-producing endocrine cells into SC-β cells, wherein the SC-β cells exhibit a GSIS response in vitro and / or in vivo.;

[0051] In some aspects, the disclosure provides an artificial island containing SC-β cells differentiated in vitro from pluripotent stem cells.

[0052] In some aspects, the disclosure provides an artificial pancreas comprising SC-β cells differentiated in vitro from pluripotent stem cells.

[0053] To practice the present invention, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant nucleic acid (e.g., DNA) technology, immunology, and RNA interference (RNAi), which are within the skill of the art, are typically employed. Non-limiting descriptions of certain of these techniques are published in the following publications: Ausubel, F., et al., (eds.), Current Protocols in Molecular Biology, Current Protocols in Immunology, Current Protocols in Protein Science, and Current Protocols in Cell Biology, all John Wiley & Sons, NY, December 2008 edition; Sambrook, Russell, and Sambrook, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2001; Harlow, E. and Lane, D., Antibodies - A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1988; Freshney, RI, "Culture of Animal Cells, A Manual of Basic Technique," 5th ed., John Wiley & Sons, Hoboken, NJ 2005. Non-limiting information on therapeutic agents and human diseases is published in Goodman and Gilman: The pharmacological Basis of Therapeutics, 11th ed., McGraw Hill, 2005; Katzung, B. (ed.) Basic and Clinical Pharmacology, McGraw-Hill / Appleton & Lange; 10th ed. (2006) or 11th ed. (July 2009). Non-limiting information on genes and genetic disorders is published in McKusick, VA: Mendelian Inheritance in Man. A Catalogue of Human Genes and Genetic Disorders, Baltimore: Johns Hopkins University Press, 198 (12th edition) or in the newer online database: Online Mendelian Inheritance in Man, OMIM™.McKusick-Nathan Institute for Genetic Medicine, Johns Hopkins University (Baltimore, MD) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, MD), as of May 1, 2010, World Wide Web URL: http: / / www.ncbi.nlm.nih.gov / MIM / and in Online Mendelian Inheritance in Animals (OMIA), a database of genes, inherited diseases, and traits in animal species (excluding humans and mice), at http: / / omia.angis.org.au / contact.shtml. Unless otherwise indicated, art-recognized meanings of terms are used herein. Standard abbreviations for the various terms are used herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] The patent or application contains at least one drawing executed in color. The Fig. 1A and Fig. 1B show comparisons between a previously published controlled differentiation method and a new directed differentiation method. Fig. 1 A shows a schematic comparison of an exemplary directed differentiation method of the disclosure for generating INS + cells from hPSCs compared with a previously published controlled differentiation method. Fig. Figure 1B shows histological sections of HUES8 undifferentiated (top), differentiated to DE (middle), and differentiated to PP1 (bottom) and stained with OCT4, SOX17, and PDX1, each using a previously published guided differentiation method. Scale bar = 100 µM. The Fig. 2A, Fig. 2B and Fig. 2C show that stem cell-derived β (SC-β) cells generated in vitro secrete insulin in response to multiple consecutive high glucose challenges, like primary human β cells. Fig. 2A, Fig. 2B and Fig. 2C are representations showing ELISA measurements on secreted human insulin from SC-β- ( Fig. 2A), primary β-cells ( Fig. 2B) and PH cells ( Fig. 2C) sequentially challenged with 2, 20, 2, 20, 2, and 20 mM glucose. After successive high / low glucose challenges, the cells were depolarized with 30 mM KCl. The Fig. 3A, Fig. 3B and Fig. 3C show additional biological replicates of in vitro-derived SC-β cells that secrete insulin in response to multiple consecutive high glucose challenges like primary β cells. The left panels are the same as in Fig. 2. The cells, the SC-β cells (SC-β, Fig. 3A), the primary β-cells (1° β; Fig. 3B) and the PH cells ( Fig. 3C), were sequentially challenged with 2, 20, 2, 20, 2, and 20 mM glucose and 30 mM KCl, and human insulin was measured by ELISA. The Fig. 4A, Fig. 4B, Fig. 4C, Fig. 4D and Fig. 4E show that SC-β cells have a cytosolic Ca 2+ -Flux in response to several consecutive high glucose challenges as in primary β-cells. Fig. Figure 4A is a schematic representation of the detection of cytosolic Ca 2+ with Fluo 4.00 staining at the population level and single-cell level. Population-level measurements were performed on individual whole clusters (schematically marked with a large red circle), and individual cells within intact clusters (marked by small red circles) were analyzed for single-cell analysis. Fig. Figure 4B is a graphical representation of population measurements of dynamic normalized Fluo-4 fluorescence intensity for SC-β cells, primary β cells, and PH cells sequentially challenged with 2, 20, 2, 20, 2, and 20 mM glucose and 30 mM KCl. The Fig. Figure 4C shows fluorescence images of Fluo-4.00 staining in single-cell analysis. Fig. Figure 4D shows representative images of the location of individual cells responding to 3 (yellow), 2 (orange), 1 (blue), and 0 (red) glucose challenges. Fig. Figure 4E shows a graphical quantification of the frequency of SC β-cells (n = 156), primary β-cells (n = 114), and PH cells (n = 138) responding to 20 mM glucose. Scale bar = 100 µm. The Fig. 5A, Fig. 5B, Fig. 5C, Fig. 5D, Fig. 5E and Fig. 5F show that SC-β express human cell markers at the protein and gene expression level. Fig. Figure 5A shows the immunohistochemistry images of cells stained for C-peptide (green), NX6-1 (red), and somatostatin (gray). Fig. Figure 5B shows the immunohistochemistry images of cells stained for C-peptide (green) and Pdx1 (red). Fig. Figure 5C shows the immunohistochemistry images of cells stained for C-peptide (green) and glucagon (red) with the corresponding DAPI stain (blue). Fig. Figure 5D shows representative flow cytometry dot plots and population percentages of cells stained for C-peptide and NKX6-1. Fig. Figure 5E shows a hierarchical cluster analysis based on all microarrays of undifferentiated HUES8, PH cells, fetal β-cells, and adult primary β-cells sorted by INS (data from Hrvatins et al., Gene. (Hrvatins et al., 2014)), and SC-β-cells (SC-β) sorted for INS and NKX6-1. Fig. Figure 5F shows a heatmap of the 100 genes with the most variance across all samples. CP = C-peptide, SST = GCG = glucagon. Scale bar = 100 µm. The Fig. Figure 6 shows the histology of an SC β-cell cluster stained for DAPI (blue), insulin (green), and C-peptide (red). Scale bar = 100 µm. The Fig. 7A, Fig. 7B and Fig. 7C show further histological staining of SC-β cells. The Fig. Figure 7A shows staining for C-peptide (green) and ISL1 (red). Fig. Figure 7B shows staining for C-peptide (green) and MAFA (red). Fig. Figure 7C shows staining for C-peptide (green) and MAFB (red). Scale bar = 100 µm. The Fig. 8A, Fig. 8B and Fig. Figure 8C shows representative flow cytometry dot plots and population percentages of SC-β cells and PH cells stained for C-peptide and SST ( Fig. 8A), C-peptide and GCG ( Fig. 8B) and SST and GCG ( Fig. 8C). The Fig. 9A, Fig. 9B and Fig. 9C show that SC β-cell granules are structurally similar to primary human β-cell granules. Fig. Figure 9A shows electron microscopic images of granules, highlighting crystallized insulin granules (red), early insulin granules (yellow), and mixed endocrine granules (blue). Scale bar = 500 nm. Fig. Figure 9B shows higher magnification images of the granules in ( Fig. 9A). Scale bar = 500 nm. The Fig. Figure 9C shows electron micrographs of cells stained with immunogold, which can be used to stain granules containing insulin (black dots smaller than 5 nm) and / or glucagon (black dots larger than 15 nm). Representative immunogold particles are highlighted with red arrows (insulin) and blue arrows (glucagon). Scale bar = 100 nm. The Fig. 10A and Fig. Figure 10B shows that stem cell-derived β-cells (SC-β) generated from hiPSCs secrete insulin in vitro in response to multiple consecutive high glucose challenges, like primary human β-cells. Fig. 10A and Fig. 10B are representations showing ELISA measurements of secreted human insulin produced from non-diabetic SC-β cells ( Fig. 10A) and type 1 diabetic cells ( Fig. 10B) that were sequentially challenged with 2, 20, 2, 20, 2, and 20 mM glucose. The Fig. 11A, Fig. 11B, Fig. 11C, Fig. 11D, Fig. 11E and Fig. Figure 11F shows representative flow cytometry dot plots and population percentages of cells from several hiPSC lines stained for C-peptide and NKX6-1. Fig. 11A, Fig. 11B and Fig. Figure 11C shows representative flow cytometry dot plots and population percentages of cells from non-diabetic hiPSC lines stained for C-peptide and NX6-1. Fig. 1ID, Fig. 1IE and Fig. Figure 11F shows representative flow cytometry dot plots and population percentages of cells from type 1 diabetic hiPSC lines stained for C-peptide and NKX6-1. The Fig. 12A, Fig. 12B, Fig. 12C and Fig. 12D show that transplanted SC-β cells also function rapidly in vivo. Fig. Figure 12A shows ELISA measurements of human insulin from the serum of individual mice transplanted with SC-β cells (cultured in vitro for 1 week in the final stage), primary human β cells (β), or PH cells. The measurements were performed before (white bars) and 30 minutes after (black bars) a glucose injection of mice two weeks after transplantation. Fig. Figure 12B shows the immunohistochemistry images of the cells in ( Fig. 12A) transplanted cells were stained for C-peptide (green) and PDX 1 (red) to confirm the presence of the transplant. Fig. Figure 12C shows ELISA measurements of human insulin from the serum of individual mice transplanted with pancreatic progenitor cells. The measurements were performed before (white bars) and 30 minutes after (black bars) a glucose injection of mice two weeks after transplantation. Fig. Figure 12D shows ELISA measurements of human insulin from the serum of individual mice transplanted with SC-β cells grown in vitro for 2 weeks. Measurements were performed 30 min after (black bars) a glucose injection of mice two weeks after transplantation. nd = not determined, scale bar = 100 µm. The Fig. 13A and Fig. 13B show further histological sections of SC-β cells and PH cells in mice transplanted 2 weeks previously. Fig. Figure 13A shows a lower magnification of the images of grafts stained for DAPI (blue), C-peptide (green), and GCG (red). Scale bar = 200 µM. Fig. Figure 13B shows higher magnification images of grafts stained for C-peptide (green) and GCG (red). Scale bar = 100 µm. The Fig. 14A, Fig. 14B and Fig. 14C show the use of media at the final stage of differentiation to enable SC-β cells to secrete more insulin in vivo. The Fig. Figure 14A shows a schematic representation of the use of different media in the different steps of the differentiation process. Fig. Figure 14B shows that the addition of additional factors such as Santl, XXI, and SSP to CMRL media at the final stage of differentiation produces a better glucose-stimulated insulin secretion (GSIS) response in SC-β cells, as determined by the stimulation index between high and low glucose challenges. Fig. Figure 14C shows that addition of additional factors such as Santl, XXI, and SSP to CMRL media at the final stage of differentiation produces a better glucose-stimulated insulin secretion (GSIS) response of SC-β cells, as determined by the amount of insulin released. The Fig. 15A, Fig. 15B, Fig. Figures 15C, 15D, 15E, 15F, 15G, 15H, and 15I show modifications of the protocol that can improve the survival and quality of the generated SC-β cells. Fig. 15A is a schematic representation of the protocol. The Fig. Figure 15B shows how pure NKX6.1 + endocrine clusters can be generated using the modified protocol ( Fig. 15B). The Fig. Figure 15C shows how the use of a Rock inhibitor in steps 3-5 can improve cell survival. Fig. Figure 15D shows how the use of activin A with nicotinamide can downregulate SOX2 and improve cell survival. Fig. Figure 15E shows that SOX2 and NKX6-1 are mutually exclusive. Fig. Figure 15F shows how the use of staurospaurine in step 6 creates a nearly pure endocrine population and the Fig. Figure 15G shows that the use of staurospaurine in step 6 produces a higher percentage of NKX6-l / C-peptide + cells. Figure 15I shows how the use of XXI in combination with Alk5i and T3 in steps 5-6 increases the NeuroD + population compared to the use of only Alk5i and T3 ( Fig. 15H). The Fig. 16A, Fig. 16B, Fig. 16C, Fig. 16D, Fig. 16E, Fig. 16F, Fig. 16G, Fig. 16H and Fig. 161 demonstrate the clinical utility of SC-β cells for diabetes therapy or drug discovery platform. Fig. Figure 16A is a schematic representation of the utility of SC-β cells for treating diabetes or screening drugs to improve function or replication. Fig. Figure 16B is a table in which diabetic medications are examined and listed according to their general therapeutic category. Fig. Figure 16C is a representation of ELISA measurements of secreted human insulin from plated SC-β cells treated with the indicated drugs at 2 and 20 mM glucose. The indicated p-values ​​compare insulin values ​​at 20 mM glucose between the drug and the control. Fig. Figure 16D is an immunofluorescence image of dispersed and plated SC-β cells stained for DAPI (blue), C-peptide (green), and Ki67 (red) without treatment. Fig. Figure 16E is an immunofluorescence image of dispersed and plated SC-β cells treated with prolactin for 48 hours, stained for DAPI (blue), C-peptide (green), and Ki67 (red). Fig. Figure 16F shows a graphical quantification of the fraction of cells co-expressing C-peptide and Ki67. * p <0.05. The Fig. Figure 16G shows fasting blood glucose values ​​of Akita mice transplanted with SC-β cells (n = 6) or PH cells (n = 6). * p < 0.05 compares the two cell groups on the same day. Fig. Figure 16H is a representation of blood glucose measurements from progressively diabetic Akita mice transplanted with SC-β cells or PH cells. The measurements were taken before (white bars) and 20 minutes after (black bars) a glucose injection in mice transplanted 2 weeks previously. Glucose measurements were taken at 550 mg / dl saturation. *p <0.05 compares both cell groups simultaneously after glucose injection. Figure 16I is a representation of ELISA measurements of human insulin from the serum of Akita mice 20 minutes after glucose injection. The mice were challenged with glucose 2 weeks after transplantation. *p <0.05 compares both cell groups. Scale bar = 50 µm. The Fig. Figure 17 shows the body weight of Akita mice transplanted with SC-β cells (n = 6) or PH cells (n = 6). * p < 0.05 compares both cell groups at 18 and 28 days. DETAILED DESCRIPTION OF THE INVENTION

[0055] Aspects of the disclosure relate to compositions, methods, kits, and means for generating stem cell-derived β-cells (SC-β) (e.g., mature pancreatic β-cells) from at least one insulin-positive endocrine cell or precursor thereof (e.g., iPS cells, HES, definitive endoderm cells, archenteron cells, Pdxl-positive pancreatic progenitor cells, Pdxl-positive, NX6-1-positive pancreatic progenitor cells, Ngn3-positive endocrine progenitor cells, etc.), and SC-β cells produced from these compositions, methods, kits, and means for use in cell therapy assays (e.g., drug screening) and various treatment methods.

[0056] Furthermore, aspects of the disclosure relate to methods for identifying SC-β cells that are detectable based on morphological criteria without the need to use a selectable marker, as well as based on functional properties, such as the ability to express insulin and secrete insulin in response to one or more glucose challenges, to exhibit a mature GSIS response, and to organize into islets in vivo in the pancreas, typically having small spindle-like cells of about 9-15 µm in diameter.

[0057] Furthermore, aspects of the disclosure relate to methods for identifying β-cell maturation factors. Those of skill in the art will be aware of, or will readily be able to determine, using assays known in the art, whether a particular β-cell maturation factor is functional. For example, the ability of a β-cell maturation factor to convert at least one insulin-positive endocrine cell, or precursor thereof, into an SC β-cell can be assayed using the assays disclosed herein. Other suitable assays include determining the ability to activate transcription of a reporter construct having a β-cell marker binding site operably linked to a nucleic acid sequence encoding a detectable marker, such as luciferase.An assay involves determining whether the candidate β-cell maturation factor induces at least one insulin-positive endocrine cell to become an SC β-cell or to express markers of a β-cell or to exhibit functional properties of a mature β-cell, as disclosed herein. Such expressed β-cell markers can be determined by any suitable method, for example, immunoblotting. Such assays can be readily adapted to identify or confirm the activity of agents that directly convert at least one insulin-positive endocrine cell, or a precursor thereof, into an SC β-cell.

[0058] The in vitro matured SC-β cells (i.e., pancreatic β cells) produced according to the inventive methods described herein offer many advantages, e.g., they perform glucose-stimulated insulin secretion in vitro, resemble human islet β cells in gene expression and ultrastructure, secrete human insulin and ameliorate hyperglycemia when transplanted into mice, represent a new platform for cell therapy (e.g., transplantation into a patient in need of additional and / or functional β cells), for drug screening (e.g., for insulin production / secretion, survival, de-differentiation, etc.), for research (e.g., determining differences in function between normal and diabetic β cells), and for tissue engineering (e.g., using SC-β cells as the first cell type for islet cell reconstruction). DEFINITIONS

[0059] For convenience, certain terms used in the specification, examples, and appended claims are summarized. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0060] The term “differentiated cell” refers to any primary cell that is not pluripotent in its native form, as defined here. In other words, the term “differentiated cell” refers to a cell of a more specialized cell type derived from a cell of a less specialized cell type (e.g., a stem cell, such as a pluripotent stem cell) in a cellular differentiation process. Without being limited to any one theory, in the course of normal ontogeny, a pluripotent stem cell may first differentiate into an endoderm cell, which can give rise to pancreatic cells and other endoderm cell types. Further differentiation of an endoderm cell occurs via the pancreatic pathway, with ~98% of the cells becoming exocrine, ductular, or matrix cells and ~2% becoming endocrine cells. Early endocrine cells are islet precursor cells, which then further differentiate into insulin-producing cells (e.g.,functional endocrine cells) that secrete insulin, glucagon, somatostatin, or pancreatic polypeptide. Endoderm cells also differentiate into other cells of endodermal origin, e.g., lung, liver, intestine, thymus, etc.

[0061] As used herein, the term "somatic cell" refers to all cells that make up the body of an organism, as opposed to germline cells. In mammals, germline cells (also called "gametes") are sperm and egg cells that fuse at fertilization to form a zygote from which the entire mammalian embryo develops. Every other cell type in the mammalian body—with the exception of sperm and egg cells, the cells from which they are made (gametocytes), and undifferentiated stem cells—is a somatic cell: internal organs, skin, bone, blood, and connective tissue are all made from somatic cells. In some embodiments, the somatic cell is a "non-embryonic somatic cell," which is derived from a somatic cell that was not obtained in or from an embryo and was not obtained by proliferation of such a cell in vitro.In some embodiments, the somatic cell is an "adult somatic cell," which means a cell obtained in or from an organism other than an embryo or a fetus, or resulting from proliferation of such a cell in vitro. Unless otherwise stated, the methods for converting at least one insulin-positive endocrine cell or precursor thereof into an insulin-producing, glucose-responsive cell can be performed both in vivo and in vitro (where in vivo means that at least one insulin-positive endocrine cell or precursor thereof is present in an individual, and where in vitro means that at least one isolated insulin-positive endocrine cell or precursor thereof is maintained in culture).

[0062] The term “adult cell” used here refers to a cell that is present in the body after embryonic development is complete.

[0063] The term "endoderm cell," as used here, refers to a cell originating from one of the three primary germ cell layers in the very early embryo (the other two germ layers are the mesoderm and ectoderm). Endoderm is the innermost of the three layers. An endoderm cell differentiates first to form the embryonic gut and then the linings of the respiratory and digestive tracts (e.g., intestine), liver, and pancreas.

[0064] The term "a cell of endoderm origin," as used herein, refers to cells that have developed or differentiated from an endoderm cell. For example, cells of endoderm origin include cells of the liver, lung, pancreas, thymus, intestine, stomach, and thyroid. Without being bound by theory, liver and pancreatic precursors (also called pancreatic progenitor cells) develop from endoderm cells in the embryonic foregut. Shortly after their differentiation, liver and pancreatic precursors rapidly acquire distinctly different cellular functions and regenerative capacity. These changes are mediated by inductive signals and genetic regulatory factors that are highly conserved among vertebrates.Interest in organ development and regeneration has been fueled by the intense need for hepatocytes and pancreatic β-cells in the therapeutic treatment of liver failure and type 1 diabetes. Studies in various model organisms and humans have revealed evolutionarily conserved inductive signals and transcription factor networks that induce liver and pancreatic cell differentiation and provide clues as to how hepatocyte and β-cell differentiation is promoted from various stem and progenitor cell types.

[0065] The term "final endoderm," as used herein, refers to a cell that can be derived from an endoderm cell and differentiated into an SC β cell (e.g., a pancreatic β cell). A definitive endoderm cell expresses the marker Soxl 7. Other cell markers characteristic of definitive endoderm include, but are not limited to, MIXL2, GATA4, HNF3b, GSC, FGF 17, VWF, CALCR, FOXQ 1, CXCR4, Cerberus, OTX2, Goosecoid, C-Kit, CD99, CMKOR 1, and CRIP1. In particular, definitive endoderm cells express Soxl 7 and, in some embodiments, Soxl 7 and HNF3 B and do not express any appreciable amount of GATA4, SPARC, APF, or DAB. Definitive endoderm cells are not positive for the marker Pdxl (e.g., they are Pdxl-negative). Definitive endoderm cells have the ability to differentiate into cells, including liver, lung, pancreas, thymus, intestinal, gastric, and thyroid cells.The expression of Soxl 7 and other markers of the definitive endoderm can be detected by any method known to those skilled in the art, such as immunochemistry using an anti-Sox 17 antibody or quantitative RT-PCR.

[0066] The term “pancreatic endoderm” refers to a cell of endodermal origin that is capable of differentiating into multiple pancreatic lineages, including pancreatic β-cells, but no longer has the ability to differentiate into non-pancreatic lineages.

[0067] The term "archenteron cell" or "archenteron cell," as used herein, refers to a cell that has differentiated from the endoderm and that can differentiate into an SC-β cell (e.g., a pancreatic β cell). A archenteron cell expresses at least one of the following markers: HNF 1-β, HNP3-β, or HNF4-α. Archenteron cells have the ability to differentiate into cells, including lung, liver, pancreas, stomach, and intestinal cells. The expression of HNF 1-β and other archenteron markers can be detected by any method known to those skilled in the art, such as immunochemistry using an anti-HNF 1-β antibody.

[0068] The terms "pancreatic progenitor" and "pancreatic endocrine progenitor," "pancreatic precursor," or "pancreatic endocrine precursor" are used interchangeably herein and refer to a stem cell capable of giving rise to a pancreatic hormone-expressing pancreatic cell, pancreatic endocrine cells, pancreatic exocrine cells, or pancreatic duct cells. These cells are destined to differentiate into at least one type of pancreatic cell, e.g., beta cells that produce insulin; alpha cells that produce glucagon; delta cells (or D cells) that produce somatostatin; and / or F cells that produce pancreatic polypeptide. Such cells may express at least one of the following markers: Ngn3, NKX2.2, NeuroD, ISL-1, Pax4, Pax6, or ARX.

[0069] The term "Pdxl-positive pancreatic progenitor," as used herein, refers to a cell that is a pancreatic endoderm (PE) cell and has the ability to differentiate into SC β-cells, such as pancreatic β-cells. A Pdxl-positive pancreatic progenitor expresses the marker Pdxl. Other markers include, but are not limited to, Cdcpl or Ptfl or HNF6 or NRx2.2. Pdxl expression can be determined by any method known to those skilled in the art, such as immunohistochemistry using an anti-Pdxl antibody or quantitative RT-PCR.

[0070] The term "Pdxl-positive, NKX6-1-positive pancreatic progenitor," as used herein, refers to a cell that is a pancreatic endoderm (PE) cell and has the ability to differentiate into insulin-producing cells, such as pancreatic β-cells. A Pdxl-positive, NKX6-1-positive pancreatic progenitor expresses the markers Pdxl and NKX6-1. Other markers include, but are not limited to, CDCP 1 or Ptfl or HNF6 or NRx2.2. NKX6-1 expression can be determined by any method known to those skilled in the art, such as immunochemistry using an anti-NKX6-1 antibody or quantitative RT-PCR.

[0071] The term "Ngn3-positive endocrine progenitor" as used herein refers to precursors of endocrine pancreatic cells that express the transcription factor neurogenin-3 (Ngn3). Progenitor cells are more differentiated than multipotent stem cells and can differentiate into only a few cell types. In particular, Ngn3-positive endocrine progenitor cells have the ability to differentiate into the five endocrine pancreatic cell types (α, β, δ, ε, and PP). The expression of Ngn3 can be determined by any method known to those skilled in the art, such as immunochemistry using an anti-antibody or Ngn3 quantitative RT-PCR.

[0072] The terms “NeuroD” and “NeuroD1” are used interchangeably and refer to a protein expressed in pancreatic endocrine progenitor cells and the genes encoding it.

[0073] The terms "insulin-positive β-like cell" and "insulin-positive endocrine cell" refer to cells (e.g., pancreatic endocrine cells) that express at least one marker typical of a pancreatic β-cell and also express insulin, but do not exhibit a characteristic GSIS response of an endogenous β-cell.

[0074] A "precursor thereof", as the term refers to an insulin-positive endocrine cell, means any cell that can differentiate into an insulin-positive endocrine cell when grown under conditions suitable for the differentiation of precursor cells into insulin-positive endocrine cells, including, for example, a pluripotent stem cell, a definitive endoderm cell, an archenteron cell, a pancreatic precursor cell, or an endocrine precursor cell.

[0075] The terms "stem cell-derived β-cell," "SC β-cell," "functional β-cell," "functional pancreatic β-cell," and "mature SC β-cell" refer to cells (e.g., pancreatic β-cells) that have at least one marker typical of a pancreatic β-cell (e.g., PDX-1 or NKX6-1), express insulin, and exhibit a characteristic GSIS response of an endogenous mature β-cell. In some embodiments, the "SC β-cell" comprises mature pancreatic β-cells. It should be understood that the SC-β cells do not have to be derived from stem cells (e.g., directly), since with the methods disclosed herein, SC-β cells can be derived from any insulin-positive endocrine cell or a precursor thereof using any cell as a starting point (e.g., embryonic stem cells, induced pluripotent stem cells, precursor cells, partially reprogrammed somatic cells (e.g.,a somatic cell partially reprogrammed into an intermediate state between a pluripotent stem cell and the somatic cell from which it was derived), multipotent cells, totipotent cells, a trans-differentiated version of the foregoing cells, etc., as the invention is not limited in this regard). In some embodiments, the SC-β cells respond to multiple glucose challenges (e.g., at least one, at least two, or at least three or more consecutive glucose challenges). In some embodiments, the response resembles the response of endogenous islet cells (e.g., human islet cells) to multiple glucose challenges. In some embodiments, the morphology of the SC-β cell resembles the morphology of an endogenous β cell. In some embodiments, the SC-β cell exhibits an in vitro GSIS response that resembles the GSIS response of an endogenous β cell.In some embodiments, the SC-β cell exhibits an in vivo GSIS response that is similar to the GSIS response of an endogenous β cell. In some embodiments, the SC-β cell exhibits both an in vitro and an in vivo GSIS response that is similar to the GSIS response of an endogenous β cell. The GSIS response of the SC-β cell can be observed within two weeks of transplantation of the SC-β cell into a host (e.g., a human or an animal). In some embodiments, the SC-β cell packages insulin into secretory granules. In some embodiments, the SC-β cells have encapsulated crystalline insulin granules. In some embodiments, the SC-β cells have a stimulation index that is greater than 1. In some embodiments, the SC-β cell has a stimulation index that is greater than 1.1. In some embodiments, the SC-β cells have a stimulation index greater than 2.In some embodiments, the SC-β cells exhibit cytokine-induced apoptosis in response to cytokines. In some embodiments, insulin secretion of the SC-β cells is enhanced in response to known antidiabetic drugs (e.g., secretagogues). In some embodiments, the SC-β cells are monohormonal. In some embodiments, the SC-β cells do not abnormally co-express other hormones, such as glucagon, somatostatin, or pancreatic polypeptide. In some embodiments, the SC-β cells exhibit a low replication rate. In some embodiments, the SC-β cells increase intracellular Ca2+ in response to glucose.

[0076] The term "exocrine cell," as used herein, refers to a cell of an exocrine gland, i.e., a gland that secretes its secretion into an excretory duct. In particular embodiments, the term exocrine cell refers to an exocrine pancreatic cell, which is a pancreatic cell that produces enzymes secreted into the small intestine. These enzymes help digest food passing through the digestive tract. Pancreatic exocrine cells are also known as islets of Langerhans, which secrete two hormones, insulin and glucagon. A pancreatic exocrine cell can be one of several types: alpha-2 cells (which produce the hormone glucagon), or β-cells (which produce the hormone insulin), and alpha-1 cells (which produce the regulatory substance somatostatin). Exocrine cells that do not produce insulin are referred to herein as alpha-2 or alpha-1 cells.It should be noted that the term exocrine pancreatic cells includes “pancreatic endocrine cells,” which refers to a pancreatic cell that produces hormones (e.g., insulin (produced by β cells), glucagon (produced by alpha-2 cells), somatostatin (produced by delta cells), and pancreatic polypeptide secreted into the bloodstream (produced by F cells).

[0077] The term “insulin-producing cell” as used herein refers to a cell that has been differentiated from a pancreatic progenitor or precursor thereof that secretes insulin. An insulin-producing cell includes pancreatic β-cells, as that term is used herein, as well as pancreatic β-like cells (i.e., insulin-positive, endocrine cells) that synthesize (i.e., transcribe the insulin gene, translate the proinsulin mRNA, and modify the pro-insulin mRNA into the insulin protein), express (i.e., manifest phenotypic characteristics generated by the insulin gene), or secrete (release insulin into the extracellular space) insulin in a constitutive or inducible manner. A population of insulin-producing cells that, for example,by differentiating insulin-positive endocrine cells or a precursor thereof into SC-β cells according to the methods of the present invention may consist of pancreatic β cells or β-like cells (e.g., cells that have at least one or at least two properties of an endogenous β cell and exhibit a GSIS response similar to that of an endogenous adult β cell). The novelty of the present composition and methods is not negated by the presence in the population of cells that naturally produce insulin (e.g., (3-cells). It is also possible that the population of insulin-producing cells, e.g., as produced by the methods disclosed herein, may comprise mature pancreatic β cells or SC-β cells, and may also include non-insulin-producing cells (e.g., cells of the β-cell-like phenotype except that they do not produce or secrete insulin).

[0078] As used herein, the terms "endogenous β-cell," "endogenous mature pancreatic β-cell," or "endogenous pancreatic β-cell" refer to an insulin-producing pancreatic cell or a cell of a pancreatic β-cell (β-cell) phenotype. The phenotype of a pancreatic β-cell is well known to those of ordinary skill in the art and includes, for example, the secretion of insulin in response to an increase in blood glucose levels, the expression of markers such as C-peptide, Pdxl polypeptide, and Glut 2, and various morphological features, such as being organized in vivo into islets in the pancreas and typically having small spindle-like cells approximately 9-15 µm in diameter.

[0079] The term “SC-β-cell”, “pancreatic β-like cell” and “mature pancreatic β-cell” as used herein refers to cells produced by the methods disclosed herein and which express at least 15% of the amount of insulin expressed by an endogenous pancreatic β-cell, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100% or more than 100%, for example at least about 1.5 times, preferably at least 2 times, preferably at least 2.5 times, preferably at least 3 times, more preferably at least 4 times, or at least about 5 times or more than about 5 times the amount of insulin expressed by an endogenous pancreatic β-cell is secreted,or alternatively, exhibiting at least one or at least two properties of an endogenous pancreatic β-cell, for example, but not limited to, the secretion of insulin in response to glucose, and the expression of β-cell markers such as C-peptide Pdxl and Glut-2. In one embodiment, the SC-β-cell is a non-immortalized cell (e.g., proliferating indefinitely in culture). In one embodiment, the SC-β-cell is a non-transformed cell, for example, a cell exhibiting a transformation property, such as growing in soft agar, or the absence of contact inhibition.

[0080] The term "β-cell marker" refers to, but is not limited to, proteins, peptides, nucleic acids, polymorphisms of proteins and nucleic acids, splice variants, fragments of proteins or nucleic acids, elements, and other analytes that are specifically expressed or present in pancreatic β-cells. Exemplary β-cell markers include, but are not limited to, pancreatic and duodenal homeobox-1 (Pdxl) polypeptide, insulin, C-peptide, amylin, E-cadherin, Hηββ, PCI / 3, B2, Nkx2.2, NKX6-1, GLUT2, PC2, ZnT-8, Isll, Pax6, Pax4, NeuroD, Hnflb, HNF-6, HNF-3β, and MafA, and those described in Zhang et al., Diabetes. 50 (10): 2231 6 (2001). In some embodiments, the β-cell marker is a nuclear 3 cell marker. In some embodiments, the β-cell marker is Pdxl or PH3.

[0081] The term "pancreatic endocrine marker" refers to, but is not limited to, proteins, peptides, nucleic acids, polymorphisms of proteins and nucleic acids, splice variants, fragments of proteins or nucleic acids, elements, and other analytes that are specifically expressed or present in pancreatic endocrine cells. Exemplary pancreatic endocrine cell markers include, but are not limited to, Ngn-3, NeuroD, and Islet-1.

[0082] The term "non-insulin-producing cell," as used herein, refers to any cell of endodermal origin that does not naturally synthesize, express, or secrete insulin constitutively or by induction. Therefore, as used herein, the term "non-insulin-producing cells" includes pancreatic β-cells. Examples of non-insulin producing cells that can be used in the methods of the present invention include pancreatic cells other than β-cells, such as amylase producing acinar cells, cells of the ductal adenocarcinoma cell line (e.g., CD18, CD11, and Capan I cells (see Busik et al., 1997; Schaffert et al., 1997). Non-pancreatic cells of endodermal origin can also be used, such as non-pancreatic stem cells and cells of other endocrine or exocrine organs, such as liver cells, thymus cells, thyroid cells, intestinal cells, lung cells, and pituitary cells.In some embodiments, the non-insulin-producing endoderm cells may be mammalian cells or, more specifically, human cells. Examples of the present method using mammalian non-islet pancreatic cells, amylase-producing pancreatic cells, and pancreatic acinar cells are provided herein.

[0083] The term “phenotype” refers to one or a number of overall biological properties that define the cell or organism independently of the actual genotype under a specific set of environmental conditions and factors.

[0084] The term "pluripotent" refers here to a cell with the ability, under different conditions, to differentiate into more than one differentiated cell type, and preferably to differentiate into cell types that exhibit features of all three germ layers. Pluripotent cells are primarily characterized by their ability to differentiate into more than one cell type, preferably into cell types of all three germ layers, for example, using a nude mouse teratoma formation assay. Pluripotency is also demonstrated by the expression of embryonic stem cell (ES) markers, although the preferred test for pluripotency is the demonstration of the ability to differentiate into cells of any of the three germ layers. It should be noted that simply culturing such cells does not render them pluripotent themselves. Reprogrammed pluripotent cells (e.g.iPS, as defined here) also have the capacity for extended passage without loss of growth potential, relative to primary parent cells, which generally only have the capacity for a limited number of divisions in culture.

[0085] As used herein, the terms “iPS cell” and “induced pluripotent stem cell” are used interchangeably and refer to a pluripotent stem cell artificially derived (e.g., induced or by complete reversion) from a non-pluripotent cell, typically an adult somatic cell, for example, produced by inducing enforced expression of one or more genes.

[0086] The terms "precursor" or "progenitor" cell are used interchangeably here and refer to cells that exhibit a cellular phenotype that is more primitive (i.e., at an earlier stage in development or progression to a fully differentiated cell) than a cell into which it can differentiate. Progenitor cells also often exhibit substantial or very high proliferative potential. Progenitor cells can become several different differentiated cell types or a single differentiated cell type, depending on the developmental course and the environment in which the cells develop and differentiate.

[0087] The term "stem cell," as used herein, refers to an undifferentiated cell capable of proliferation and capable of giving rise to multiple progenitor cells, which have the capacity to generate a large number of parent cells that, in turn, can become differentiated, or differentiable, daughter cells. The daughter cells themselves can be induced to proliferate and produce offspring that subsequently differentiate into one or more mature cell types, while also retaining one or more cells with parental developmental potential. The term "stem cell" refers to a subset of progenitor cells that, under certain circumstances, have the capacity or potential to differentiate into a more specialized or differentiated phenotype and that retain the capacity to proliferate under certain circumstances without significantly differentiating.In one embodiment, the term stem cell generally refers to a naturally occurring parent cell whose derivatives (offspring) often specialize in various directions through differentiation, for example, by acquiring fully individualized characteristics, as occurs in progressive diversification from embryonic cells and tissues. Cell differentiation is a complex process that typically occurs over many cell divisions. A differentiated cell may be derived from a multipotent cell, which itself originated from a multipotent cell, and so on. Although each of these multipotent cells can be considered a stem cell, considerable variation can arise from the diversity of cell types. Some differentiated cells further exhibit the ability to develop into cells with greater developmental potential.Such an ability can be natural or artificially induced by treatment with various factors. In many biological cases, stem cells are also "multipotent" in that they can produce offspring of more than one specific cell type, although this is not required for stem cell trait. Self-renewal is the other classic part of the stem cell definition and, as used in this document, is essential. Self-renewal can theoretically occur by one of two main mechanisms. Stem cells can divide asymmetrically, with one daughter cell retaining stem cell trait and the other daughter cell expressing some other specific function and phenotype.Alternatively, some of the stem cells in a population may divide symmetrically into two lineages, preserving a certain number of stem cells in the overall population, while other cells in the population become only differentiated progeny. Formally, it is thus possible for cells that begin as stem cells to develop a differentiated phenotype, but then regress and return to the stem cell phenotype, a term often referred to by those skilled in the art as "dedifferentiation," "reprogramming," or "retrodifferentiation." As used herein, the term "pluripotent stem cell" includes embryonic stem cells, pluripotent stem cells, placental stem cells, etc.

[0088] In the context of cell ontogeny, the adjective "differentiated" or "differentiating" is a relative term for a "differentiated cell," which is a cell further downstream in the developmental chain than the cell to which it is compared. Thus, stem cells can differentiate into lineage-restricted progenitor cells (such as a mesodermal stem cell), which in turn can differentiate into other types of progenitor cells further downstream in the developmental chain (such as a cardiomyocyte progenitor), and ultimately into a terminally differentiated cell that performs a characteristic function in a particular tissue type and may or may not retain the capacity for proliferation.

[0089] The term "embryonic stem cell" is used to refer to pluripotent stem cells of the inner cell mass of the embryonic blastocyst (see US Pat. Nos. 5843780 and 6200806). Such cells can be derived in a comparable manner from the inner cell mass of blastocysts obtained by somatic cell nuclear transfer (see, for example, US Pat. Nos. 5945577 and 5994619, and 6235970). The defining characteristics of an embryonic stem cell define an embryonic stem cell phenotype. Accordingly, a cell exhibits an embryonic stem cell phenotype if it exhibits one or more of the unique properties of an embryonic stem cell, allowing that cell to be distinguished from other cells.Exemplary distinguishing characteristics of embryonic stem cells include, but are not limited to, gene expression profile, proliferative capacity, differentiation capacity, karyotype, responsiveness to specific culture conditions, and the like.

[0090] The term "adult stem cells" or "ASC" is used to refer to any multipotent stem cell derived from non-embryonic tissue, including fetal, juvenile, and adult tissue. Stem cells have been isolated from a variety of adult tissues, including blood, bone marrow, brain, olfactory epithelium, skin, pancreas, skeletal muscle, and cardiac muscle. Each of these stem cells can be characterized by gene expression, factor response, and morphology in culture. Exemplary adult stem cells include neural stem cells, neural crest stem cells, mesenchymal stem cells, hematopoietic stem cells, and pancreatic stem cells. As stated above, stem cells originating from virtually any tissue have been found. Accordingly, according to the present invention, stem cell populations can be isolated from virtually any animal tissue.

[0091] The term "pancreas" refers to a glandular organ that secretes digestive enzymes and hormones. In humans, the pancreas is a yellowish organ approximately 17.8 cm (7 inches) long and 3.8 cm (1.5 inches) wide. It lies beneath the stomach and small intestine, in a muscular tube-like portion of the gastrointestinal tract that extends from the lower end of the stomach (pylorus) to the anus. Most of the pancreatic tissue consists of grape-like clusters of cells that produce a clear fluid (pancreatic juice), which flows into the duodenum via a common duct with bile from the liver. Pancreatic juice contains three digestive enzymes: tryptase, amylase, and lipase, which, along with intestinal enzymes, complete the digestion of proteins, carbohydrates, and fats.Interspersed among the enzyme-producing cells of the pancreas are small groups of endocrine cells called islets of Langerhans, which secrete two hormones, insulin and glucagon. Pancreatic islets contain several types of cells: alpha-2 cells, which produce the hormone glucagon; β-cells (also referred to here as "pancreatic β-cells"), which produce the hormone insulin; and alpha-1 cells, which produce the regulatory substance somatostatin. These hormones are secreted into the bloodstream and together regulate blood glucose levels. Insulin lowers blood sugar levels and increases the amount of glycogen (carbohydrate) stored in the liver; glucagon has the opposite effect. Failure of the insulin-secreting cells to function properly causes diabetes, or diabetes mellitus.

[0092] The term "reprogramming," as used herein, refers to the process that changes or reverses the differentiation state of a somatic cell. The cell may be either partially or terminally differentiated prior to reprogramming. Reprogramming involves complete reversal of the differentiation state of a somatic cell to a pluripotent cell. Such complete reversal of differentiation produces an induced pluripotent (iPS) cell. Reprogramming, as used herein, also includes partial reversal of a cell's differentiation state, such as to a multipotent state or to a somatic cell that is neither pluripotent nor multipotent, but rather is a cell that has lost one or more specific properties of the differentiated cell from which it was derived, such as direct reprogramming of a differentiated cell to a different cell type.Reprogramming generally involves a change, such as a reversal, of at least some of the heritable patterns of nucleic acid modification (e.g., methylation), chromatin condensation, epigenetic changes, genomic imprinting, etc., that occur during cell differentiation of the zygote as it develops into an adult cell.

[0093] The term "agent," as used herein, refers to any compound or substance, such as, but not limited to, a small molecule, a nucleic acid, a polypeptide, a peptide, a drug, ions, etc. An "agent" can be any chemical group or moiety, including, but not limited to, synthetic and naturally occurring proteinaceous and non-proteinaceous substances. In some embodiments, an agent is a nucleic acid, nucleic acid analogs, proteins, antibodies, peptides, aptamers, oligomers of nucleic acids, amino acids, or carbohydrates, including, but not limited to, proteins, oligonucleotides, ribozymes, DNAzymes, glycoproteins, siRNAs, lipoproteins, aptamers, and modifications and combinations thereof, etc. In certain embodiments, agents are a small molecule that has a chemical group.For example, chemical groups containing unsubstituted or substituted alkyl, aromatic, or heterocyclyl residues, including macrolides, leptomycins, and related natural products or their analogues, may be known to possess a desired activity and / or property, or may be selected from a library of diverse compounds.

[0094] As used herein, the term "contacting" (i.e., contacting at least one insulin-positive endocrine cell or precursor thereof with a β-cell maturation factor or a combination of β-cell maturation factors) is intended to encompass incubating the β-cell maturation factor together with the cell in vitro (e.g., adding the β-cell maturation factors to cells in culture). In some embodiments, the term "contacting" is intended to encompass exposing cells in vivo to compounds as disclosed herein that may occur naturally in a subject (i.e., exposure that may occur as a result of a natural physiological process). The step of contacting at least one insulin-positive endocrine cell or precursor thereof with a β-cell maturation factor may be performed in any suitable manner, as in the embodiments relating to the production of SC-β cells.For example, the cells may be treated in adherent culture or in suspension culture. In some embodiments, the cells are treated under conditions that promote the formation of cell clusters. The disclosure encompasses any conditions that promote cell cluster formation. Examples of conditions that promote cell cluster formation include, but are not limited to, suspension culture in low-attachment tissue culture plates, spinner flasks, or Aggrewell plates. In some embodiments, the inventors found that clusters remained stable in media comprising 10% serum. In some embodiments, conditions that promote cluster formation include low-serum media.

[0095] It should be understood that cells that have been contacted with a β-cell maturation factor may also be contacted simultaneously or subsequently with another agent, such as a growth factor or other differentiation agents or environments to stabilize the cells or to further differentiate the cells.

[0096] Accordingly, at least one insulin-positive endocrine cell or precursor thereof can be contacted with at least one β-cell maturation factor and then contacted with at least one other β-cell maturation factor. In some embodiments, the cell is contacted with at least one β-cell maturation factor, wherein the contact is separated in time. In some embodiments, a cell is contacted with at least one β-cell maturation factor substantially simultaneously. In some embodiments, the cell is contacted with at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least 10 β-cell maturation factors.

[0097] The term "cell culture medium" (also "culture medium" or "medium"), as used herein, is a medium for growing cells that contains nutrients that maintain cell viability and support proliferation. The cell culture medium may contain any of the following substances in an appropriate combination: salt(s), buffers, amino acids, glucose or other sugars, antibiotics, serum or serum substitutes, and other components such as peptide growth factors, etc. Cell culture media used for specific cell types are usually known to those skilled in the art.

[0098] The term “cell line” refers to a population of largely or substantially identical cells, typically derived from an ancestral cell or from a defined and / or substantially identical ancestral cell population. The cell line may be capable of being maintained in culture for an extended period of time (e.g., months, years, or indefinitely). It may have undergone a spontaneous or induced transformation process that confers on the cells an unlimited lifespan in culture. Cell lines include all cell lines recognized as such in the art. It is understood that cells acquire mutations and possibly epigenetic changes over time, so that at least some properties of the individual cells of a cell line may differ from one another. In some embodiments, a cell line comprises an SC-β cell as described herein.

[0099] The term "exogenous" refers to a substance that may be present in a cell or organism outside of its natural source. For example, the terms "exogenous nucleic acid" or "exogenous protein" refer to a nucleic acid or protein that has been introduced by human manipulation into a biological system, such as a cell or organism, in which it is not normally found or in which it is present in smaller amounts. A substance is considered exogenous if it has been introduced into a cell or ancestor cell that inherits the substance. In contrast, the term "endogenous" refers to a substance that originates from the biological system.

[0100] The term "expression" refers to cellular processes involved in the production of RNA and proteins, and possibly protein secretion, including, but not limited to, transcription, translation, folding, modification, and processing. "Expression products" include RNA transcribed from a gene and polypeptides obtained by translation of mRNA transcribed from a gene.

[0101] The term "genetically modified" or "treated" cell, as used herein, refers to a cell into which an exogenous nucleic acid has been introduced by means of a technical manipulation process (or a derivative of such a cell that has inherited at least part of the nucleic acid). The nucleic acid may, for example, contain a sequence that is exogenous to the cell and may contain native sequences (i.e., sequences naturally present in the cells) but in a non-naturally occurring arrangement (e.g., a coding region linked to a promoter of another gene), or may contain altered versions of the native sequences, etc. The process of transferring the nucleic acid into the cell may be achieved by any suitable technique. Suitable methods include calcium phosphate- or lipid-mediated transfection, electroporation, and transduction or infection with a viral vector.In some embodiments, the polynucleotide or a portion thereof is integrated into the genome of the cell. The nucleic acid may subsequently have been removed or excised from the genome, provided that this removal or excision results in a detectable change in the cell relative to an unmodified but otherwise equivalent cell. It should be understood that the term "genetically modified" is intended to encompass the introduction of a modified RNA directly into a cell (e.g., a synthetic, modified RNA). Such synthetic, modified RNAs include changes to prevent rapid degradation by endo- and exo-nucleases and to avoid or reduce an innate or immune interferon response of the cell against the RNA. Modifications include, but are not limited to, (a) end modifications, e.g., 5'-end modifications (phosphorylation-dephosphorylation, conjugation, inverted bonds, etc.)), 3'-end modifications (conjugation, DNA nucleotides, inverted linkages, etc.), (b) base modifications, for example, replacement with modified bases, stabilizing bases, destabilizing bases, or bases that hybridize with an expanded repertoire of partners or conjugated bases, (c) sugar modifications (e.g., at the 2'-position or 4'-position) or replacement of the sugar, and (d) internucleoside bond modifications, including modification or replacement of phosphodiester bonds. If such modifications interfere with translation (i.e., result in a reduction in translation of 50% or more compared to no modification - e.g., in a rabbit reticulocyte in vitro translation assay), then the modification is not suitable for the methods and compositions described herein. In some embodiments, the SC-β cell is genetically engineered to express neurogenin 3.In some embodiments, genetically modifying the SC-β cell comprises introducing a synthetically modified mRNA encoding neurogenin 3. Genetically modifying SC-β cells with synthetically modified RNA encoding neurogenin 3 is believed to increase insulin production in the cells. Such genetic modification in any insulin-producing cell is expected to result in increased insulin production in the cell.

[0102] In some aspects, the disclosure provides a genetically engineered SC-β cell to introduce a detectable marker into the insulin locus. In some embodiments, the SC-β cell is modified to replace both alleles of the insulin locus with a detectable marker. In some embodiments, the SC-β cell is genetically modified to introduce the detectable marker into the insulin locus so that it is expressed with insulin in the SC-β cell in response to a glucose challenge. In some embodiments, the SC-β cell is genetically modified to introduce the detectable marker into the insulin locus in place of insulin so that it is expressed in place of insulin in the SC-β cell in response to a glucose challenge. Any detectable marker can be introduced into the insulin locus, including, for example, a nucleic acid encoding a fluorescent protein (e.g., GFP).It will be apparent to those skilled in the art that such genetically modified SC-β cells can be used in various screening methods, for example, to identify agents that stimulate insulin expression and / or secretion from β cells by determining the detectable marker in response to the agent. For example, a genetically modified SC-β cell in which the insulin gene has been replaced in both alleles (e.g., with GFP) can be contacted with a test agent and agents that cause the SC-β cells to fluoresce due to the expression of GFP, which are considered candidates to stimulate insulin gene expression in β cells. In other words, the detectable marker can be used as a surrogate marker for insulin expression in genetically modified SC-β cells.

[0103] The term "identity," as used herein, refers to the extent to which the sequence of two or more nucleic acids or polypeptides is the same. The percent identity between a sequence of interest and a second sequence over a scoring window, e.g., over the length of the sequence of interest, can be calculated by aligning the sequences, determining the number of residues (nucleotides or amino acids) within the scoring window that preclude an identical stretch to allow for the introduction of gaps to maximize identity, dividing by the total number of residues of the sequence of interest or the second sequence (whichever is larger) that lie within the window, and multiplying by 100. When calculating the number of identical residues required to achieve a particular percent identity, fractions are rounded up to the nearest whole number.Percent identity can be calculated using several computer programs known in the art. For example, computer programs such as BLAST2, BLASTN, BLASTP, Gapped BLAST, etc., generate alignments and provide percent identity between sequences of interest. The algorithm of Karlin and Altschul (Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87: 22264-2268, 1990), modified in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90: 5873-5877, 1993, is incorporated into the NBLAST and XBLAST programs of Altschul et al. (Altschul et al., J. Mol. Biol. 215: 403-410, 1990). To obtain gapped alignments for comparison purposes, Gapped BLAST is used, as described in Altschul et al. (Altschul et al. Nucleic Acids Res. 25: 3389-3402, 1997). When using BLAST and Gapped BLAST programs, the default parameters of the respective programs can be used. A PAM250 or BLOSUM62 matrix can be used.Software for performing BLAST analyses is publicly available from the National Center for Biotechnology Information (NCBI). Reference is made to the website for these programs at the URL address "ncbi.nlm nih.gov." In a specific embodiment, percent identity is calculated using BLAST2 with standard parameters, according to NCBI.

[0104] The term "isolated" or "partially purified," as used herein, in the case of a nucleic acid or polypeptide, means a nucleic acid or polypeptide that has been separated from at least one other component (e.g., nucleic acid or polypeptide) that is found with the nucleic acid or polypeptide in its natural source and / or that is present with the nucleic acid or polypeptide when expressed by a cell, or secreted in the case of secreted polypeptides. A chemically synthesized nucleic acid or polypeptide, or one synthesized using in vitro transcription / translation, is considered "isolated."

[0105] The term "isolated cell," as used herein, refers to a cell removed from an organism in which it originates, or a descendant of such a cell. The cell may have been cultured in vitro, e.g., in the presence of other cells. The cell may later be introduced into a second organism or reintroduced into the organism from which it (or the cell from which it originated) was isolated.

[0106] The term "isolated population" with respect to an isolated population of cells, as used herein, refers to a cell population that has been removed and separated from a mixed or heterogeneous cell population. In some embodiments, an isolated population is a substantially pure cell population compared to the heterogeneous population from which the cells were isolated or enriched.

[0107] The term "substantially pure" with reference to a particular cell population refers to a cell population that is at least about 75%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% pure, relative to the cells that make up the total cell population. This further means that the terms "substantially pure" or "substantially purified" with reference to a population of SC-β cells refer to a cell population that includes less than about 20%, more preferably less than about 15%, 10%, 8%, 7%, most preferably less than about 5%, 4%, 3%, 2%, 1%, or less than 1% cells that are not SC-β cells, as defined herein. The present disclosure also provides methods for expanding a population of SC-β cells, wherein the expanded population of SC-β cells is a substantially pure population of SC-β cells.

[0108] Accordingly, the term "substantially pure" or "substantially purified" population of insulin-positive endocrine cells refers to a cell population comprising less than about 20%, more preferably less than about 15%, 10%, 8%, 7%, most preferably less than about 5%, 4%, 3%, 2%, 1%, or less than 1% of the cells that are not insulin-positive endocrine cells, as defined by the terms herein. The present disclosure also provides methods for expanding a population of insulin-positive endocrine cells, wherein the expanded population of insulin-positive endocrine cells is a substantially pure population of insulin-positive endocrine cells.

[0109] Accordingly, the terms "substantially pure" or "substantially purified" population of Ngn3-positive endocrine progenitor cells refer to a cell population having less than about 20%, more preferably less than about 15%, 10%, 8%, 7%, most preferably less than about 5%, 4%, 3%, 2%, 1%, or less than 1% cells that are not Ngn3-positive endocrine progenitor cells or their progeny, as defined herein. Further disclosed are methods for expanding a population of Ngn3-positive endocrine progenitor cells, wherein the expanded population of Ngn3-positive endocrine progenitor cells is a substantially pure population of Ngn3-positive endocrine progenitor cells.

[0110] Accordingly, the terms "substantially pure" or "substantially purified" population of Pdxl-positive, NKX6-1-positive pancreatic progenitor cells refer to a cell population that includes less than about 20%, more preferably less than about 15%, 10%, 8%, 7%, most preferably less than about 5%, 4%, 3%, 2%, 1%, or less than 1% of the cells that are not Pdxl-positive, NKX6-1-positive pancreatic progenitor cells or their progeny, as defined herein. Also disclosed are methods for expanding a population of Pdxl-positive, NKX6-1-positive pancreatic progenitor cells, wherein the expanded population of Pdxl-positive, NKX6-1-positive pancreatic progenitor cells is a substantially pure population of Pdxl-positive, NKX6-1-positive pancreatic progenitor cells.

[0111] Accordingly, the terms "substantially pure" or "substantially purified" population of Pdxl-positive pancreatic progenitor cells refer to a cell population comprising less than about 20%, more preferably less than about 15%, 10%, 8%, 7%, most preferably less than about 5%, 4%, 3%, 2%, 1%, or less than 1% of the cells that are not Pdxl-positive pancreatic progenitor cells or their progeny, as defined herein. The present disclosure also provides methods for expanding a population of Pdxl-positive pancreatic progenitor cells, wherein the expanded population of Pdxl-positive pancreatic progenitor cells is a substantially pure population of Pdxl-positive pancreatic progenitor cells.

[0112] Accordingly, the terms "substantially pure" or "substantially purified" population of archenteron cells refer to a cell population comprising less than about 20%, more preferably less than about 15%, 10%, 8%, 7%, most preferably less than about 5%, 4%, 3%, 2%, 1%, or less than 1% of the cells that are not archenteron cells or their progeny, as defined herein. Also disclosed are methods for expanding a population of archenteron cells, wherein the expanded population of archenteron cells is a substantially pure population of archenteron cells.

[0113] Accordingly, the terms "substantially pure" or "substantially purified" population of definitive endoderm cells refer to a cell population comprising less than about 20%, more preferably less than about 15%, 10%, 8%, 7%, most preferably less than about 5%, 4%, 3%, 2%, 1%, or less than 1% of cells that are not definitive or differentiated endoderm cells or their progeny, as defined herein. Also disclosed are methods for expanding a population of definitive endoderm cells, wherein the expanded population of definitive or differentiated endoderm cells is a substantially pure population of definitive endoderm cells.

[0114] Accordingly, the terms "substantially pure" or "substantially purified" population of pluripotent cells refer to a cell population comprising less than about 20%, more preferably less than about 15%, 10%, 8%, 7%, most preferably less than about 5%, 4%, 3%, 2%, 1%, or less than 1% of cells that are non-pluripotent cells or their progeny, as defined herein. Also disclosed are methods for expanding a population of pluripotent cells, wherein the expanded population of pluripotent cells is a substantially pure population of pluripotent cells.

[0115] The terms “enriching” or “enriched” are used interchangeably here and mean that the yield (fraction) of cells of one type is increased by at least 10% compared to the proportion of cells of that type in the starting culture.

[0116] The terms "renewal," "self-renewal," or "proliferation" are used interchangeably here to refer to the ability of stem cells to renew themselves by dividing into the same non-specialized cell type over long periods of time and / or many months to years. In some cases, the term proliferation refers to the expansion of cells by repeated division of individual cells into two identical daughter cells.

[0117] The term "lineage," as used here, describes a cell sharing a common ancestor or cells sharing a common developmental fate. For example, in the context of a cell of endodermal origin or "endodermal lineage," this means that the cell is derived from an endodermal cell and can differentiate along pathways restricted to endodermal lineage, such as one or more developmental lineage pathways leading to terminal or fully differentiated endoderm cells, which can, in turn, differentiate into liver cells, thymus, pancreas, lung, and intestine.

[0118] As used here, the term “xenogeneic” refers to cells derived from different species.

[0119] A "marker," as used herein, is used to describe properties and / or the phenotype of a cell. Markers can be used to select cells that exhibit properties of interest. Markers will vary with specific cells. Markers are characteristics, whether morphological, functional, or biochemical (enzymatic) features of the cell of a particular cell type, or molecules expressed by the cell type. Such markers are preferably proteins, and more preferably have an epitope for antibodies or other molecules known in the art to bind thereto. However, a marker can consist of any molecule in a cell, including, but not limited to, proteins (peptides and polypeptides), lipids, polysaccharides, nucleic acids, and steroids. Examples of morphological properties or characteristics include, but are not limited to, shape, size, and the nucleus-to-cytoplasm ratio.Examples of functional properties or characteristics include, but are not limited to, the ability to adhere to specific substrates, the ability to uptake or exclude dyes, the ability to migrate under specific conditions, and the ability to differentiate along specific lineages. Markers can be detected by any method known to those skilled in the art. Markers can also be the lack of a morphological property or the absence of proteins, lipids, etc. Markers can be a combination of a group of unique properties, e.g., the presence or absence of polypeptides and other morphological features.

[0120] The term "modulate" is used in accordance with its prior art usage, meaning that it causes or facilitates a qualitative or quantitative change, variation, or modification in a process, pathway, or phenomenon of interest. Such changes may include, but are not limited to, an increase, decrease, or alteration in the relative strength or activity of various components or portions of the process, pathway, or phenomenon. A "modulator" is an agent that causes or facilitates a qualitative or quantitative change, variation, or modification in a process, pathway, or phenomenon of interest.

[0121] As used herein, the term “DNA” is defined as deoxyribonucleic acid.

[0122] The term "polynucleotide" is used interchangeably with "nucleic acid" to refer to a polymer of nucleosides. A polynucleotide of the present invention typically consists of nucleosides naturally found in DNA and RNA (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) linked by phosphodiester bonds. However, the term encompasses molecules, nucleosides, or nucleoside analogues that represent chemically or biologically modified bases, modified backbones, etc., that are also found in naturally occurring nucleic acids, and such molecules may be preferred for certain applications. Whenever this application refers to a polynucleotide, it should be understood that both DNA, RNA, and single- and double-stranded forms (and complements of a respective single-stranded molecule) are encompassed."Polynucleotide sequence," as used herein, may refer to the polynucleotide material itself and / or the sequence information (i.e., the sequence of letter abbreviations for bases) that biochemically characterizes a specific nucleic acid. A polynucleotide sequence referred to herein is given in a 5' to 3' direction unless otherwise indicated.

[0123] The term "polypeptide," as used herein, refers to a polymer of amino acids. The terms "protein" and "polypeptide" are used interchangeably herein. A peptide is a relatively short polypeptide, usually between about 2 and 60 amino acids in length. Polypeptides typically used herein contain amino acids, such as the 20 L-amino acids most commonly found in proteins. However, other amino acids and / or amino acid analogues known in the art may also be used. One or more of the amino acids in a polypeptide may be modified, for example, by the addition of a chemical moiety such as a carbohydrate group, a phosphate group, a fatty acid group, a linker for conjugation, functionalization, etc. A polypeptide that has a non-polypeptide moiety covalently or non-covalently attached to it is still referred to as a "polypeptide."Exemplary modifications include glycosylation and palmitoylation. Polypeptides can be purified from natural sources using recombinant DNA technology, with chemical synthesis means such as conventional solid-phase peptide synthesis, etc. The term "polypeptide sequence" or "amino acid sequence," as used herein, can refer to the polypeptide material itself and / or indicate the sequence information (i.e., the sequence of letters or three-letter codes as abbreviations for the amino acid names) that biochemically characterizes a polypeptide. A polypeptide sequence is presented herein in an N-terminal to C-terminal direction unless otherwise indicated.

[0124] The term "variant" with respect to a polypeptide may, for example, mean a polypeptide that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the full-length polypeptide. The variant may be a fragment of the full-length polypeptide. The variant may be a naturally occurring splice variant. The variant could be a polypeptide that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to a fragment of the polypeptide, wherein the fragment has a length of at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98% or 99% of the total length of the wild-type polypeptide or a domain thereof that includes an activity of interest, such as the ability to recognize the presence of an SC-β cell precursor or an insulin-positive endocrine cell or precursor thereof from which the SC-β cell is derived.The domain may be at least 100, 200, 300, or 400 amino acids long, starting at any of the amino acid positions in the sequence and extending toward the C-terminus. Variations known in the art to eliminate or substantially reduce the activity of the protein are preferably avoided.

[0125] The variant may lack an N- and / or C-terminal portion of the full-length polypeptide, for example, up to 10, 20, or 50 amino acids from each terminus. According to some embodiments, the polypeptide may comprise the sequence of a mature (full-length) polypeptide, i.e., a polypeptide having one or more regions, such as a signal peptide, removed during normal intracellular proteolytic processing (e.g., during co-translational or post-translational processing). In some embodiments, if the protein has not been purified from cells that naturally express it, it may be produced, wherein the protein is a chimeric polypeptide, meaning that it comprises portions of two or more different species.In some embodiments where a protein is produced without purification from cells that naturally produce it, the protein may be a derivative, by which it is meant that the protein comprises additional sequences that are not derived from the protein, as long as those sequences do not substantially reduce the biological activity of the protein.

[0126] The term "functional fragments," as used herein, refers to a polypeptide having an amino acid sequence that is less than but substantially homologous to the polypeptide of which it is a fragment, the functional fragment being a polypeptide sequence having a biological activity of at least 50%, or 60%, or 70%, or 80%, or 90%, or 100%, or more than 100%, for example, 1.5-fold, 2-fold, 3-fold, 4-fold, or more than 4-fold, compared to the polypeptide of which it is a fragment. Functional fragment polypeptides may have additional functions, which may include reduced antigenicity, increased DNA binding (as in transcription factors), or altered RNA binding (as in regulating RNA stability or degradation).

[0127] The term "vector" refers to a carrier DNA molecule into which a DNA sequence can be inserted for introduction into a host cell. Preferred vectors are those suitable for autonomous replication and / or expression of nucleic acids to which they are linked. Vectors suitable for inducing the expression of genes to which they are operably linked are referred to herein as "expression vectors." Thus, an "expression vector" is a specific vector that contains the necessary regulatory regions for the expression of a gene of interest in a host cell. The gene of interest may be operably linked to another sequence in the vector. Vectors can be viral vectors or non-viral vectors.When using viral vectors, it is preferred that the viral vectors be non-replicating, which can be achieved by removing all viral nucleic acids encoding replication. A replication-deficient viral vector will retain its infectious properties and enter cells in a similar manner to a replicating adenoviral vector, whereas a replication-defective viral vector will not replicate or replicate in the cell. Vectors also include liposomes, nanoparticles, and other means for delivering DNA molecules into a cell.

[0128] The term "operably linked" means that the regulatory sequences required for expression of the coding sequence are arranged in the DNA molecule in the appropriate positions relative to the coding sequence to effect expression of the coding sequence. The same definition is occasionally applied to arrangements of coding sequences and transcription control elements (e.g., promoters, enhancers, and termination signal elements) in an expression vector. The term "operably linked" includes a suitable initiation signal (e.g., ATG) in front of the polynucleotide sequence to be expressed and maintenance of the correct reading frame to enable expression of the polynucleotide sequence under the control of the expression control sequence to produce the desired polypeptide encoded by the polynucleotide sequence.

[0129] The term "viral vectors" refers to the use of viruses or virus-associated vectors to deliver a nucleic acid construct into a cell. Constructs can be introduced and packaged into non-replicating, defective viral genomes for infection or transduction into cells, such as adenovirus, adeno-associated virus (AAV), or herpes simplex virus (HSV), or others, including retroviral and lentiviral vectors. The vector may, but need not, be integrated into the cell's genome. If desired, the constructs may include viral sequences for transfection. Alternatively, the construct can be incorporated into vectors capable of episomal replication, e.g., EPV and EBV vectors.

[0130] The terms "regulatory sequence" and "promoter" are used interchangeably herein to refer to nucleic acid sequences, such as initiation signals, enhancers, and promoters, that initiate or direct transcription of protein-coding sequences to which they are operably linked. In some examples, transcription of a recombinant gene is under the control of a promoter sequence (or other transcriptional regulatory sequence) that directs expression of the recombinant gene in a cell type in which the gene is to be expressed. It is also understood that the recombinant gene may be under the control of transcriptional regulatory sequences that are the same as, or different from, those that direct transcription of the naturally occurring form of the protein.In some cases, the promoter sequence is recognized, introduced, or is a synthesis machinery required for the transcription of a specific gene by the cell's synthesis machinery.

[0131] As used herein, the term "transcription factor" refers to a protein that binds specific pieces of DNA via DNA-binding domains and is part of the system that controls the transfer (or transcription) of genetic information from DNA to RNA. As used herein, the terms "proliferate" and "proliferation" refer to an increase in the number of cells in a population (growth) by means of cell division. Cell proliferation is generally understood to result from the coordinated activation of multiple signal transduction pathways in response to the environment, including growth factors and other mitogens. Cell proliferation can also be promoted by overriding the activities of intra- or extracellular signals and mechanisms that block or negatively affect cell proliferation.

[0132] The term "selectable marker" refers to a gene, RNA, or protein that, when expressed, confers a selectable phenotype on cells, such as resistance to a cytotoxic or cytostatic agent (e.g., antibiotic resistance), nutritional prototrophy, or expression of a specific protein, the level of which can be used as a basis to distinguish cells that express the protein from cells that do not. Proteins whose expression can be easily detected, for example, a fluorescent or luminescent protein or an enzyme that acts on a substrate to produce a colored, fluorescent, or luminescent substance ("detectable marker"), constitute a subset of selectable markers.The presence of a selectable marker linked to native expression control elements for the gene normally expressed selectively or exclusively in pluripotent cells allows the identification and selection of somatic cells that have been reprogrammed to a pluripotent state. Various selectable marker genes can be used, such as the neomycin resistance gene (neo), the puromycin resistance gene (puro), guanine phosphoribosyltransferase (gpt), dihydrofolate reductase (DHFR), adenosine deaminase (ADA), puromycin N-acetyltransferase (PAC), the hygromycin resistance gene (hyg), the multidrug resistance gene (MDR), thymidine kinase (TK), hypoxanthine-guanine phosphoribosyltransferase (HPRT), and the hisD gene. Detectable markers include green fluorescent protein (GFP), blue, sapphire, yellow, red, orange, and cyan fluorescent proteins, and variants of any of these. Luminescent proteins, such as luciferase (e.g.,Firefly or Renilla luciferase) are also useful. It will be appreciated by one of ordinary skill in the art that the term "selectable marker," as used herein, may refer to a gene or an expression product of the gene, e.g., an encoded protein.

[0133] In some embodiments, the selectable marker confers a proliferation and / or survival advantage to cells that express it compared to cells that do not express it, or that express it at a significantly lower level. Such a proliferation and / or survival advantage typically occurs when the cells are maintained under specific conditions, i.e., "selective conditions." To ensure effective selection, a cell population may be maintained under conditions and for a sufficient period of time such that cells that do not express the marker do not proliferate and / or do not survive and are eliminated from the population, or their number is reduced to only a very small fraction of the population.The process of selecting cells that express a marker that confers a proliferation and / or survival advantage and, upon maintaining a cell population under selective conditions and for a period of time, completely or largely eliminating cells that do not express the marker, is referred to herein as "positive selection," and the marker is referred to as "useful for positive selection." Negative selection and markers suitable for negative selection are also of interest for certain of the methods described here. The expression of such markers confers a proliferation and / or survival disadvantage to cells that do not express the marker or express it at a significantly lower level (or, in other words, cells that do not express the marker have a proliferation and / or survival advantage over cells that do express the marker).Cells expressing the marker can thus be largely or completely eliminated from a cell population if they are kept under selective conditions for a sufficient period of time.

[0134] A "reporter gene," as used herein, includes any gene that has been genetically engineered into a cell and makes an additional contribution to the stem cell phenotype. Reporter genes, as disclosed herein, are intended to include fluorescent, luminescent, enzymatic, and resistance genes, as well as other genes readily recognized by one of ordinary skill in the art. In some embodiments of the invention, reporter genes are used as markers for identifying specific stem cells, cardiovascular stem cells, and their differentiated progeny. A reporter gene is generally operably linked to sequences that direct its expression in a manner dependent on one or more conditions monitored by determining the expression of the reporter gene. In some cases, the expression of the reporter gene can be determined in living cells.When live-cell reporter gene assays are used, reporter gene expression may be monitored at multiple time points, such as 2, 3, 4, 5, 6, 8, or 10 or more time points. In some cases where a live-cell reporter gene assay is used, reporter gene expression is monitored at a frequency of at least about 10 minutes to about 24 hours, such as 20 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 18 hours, or any other frequency from any integer between about 10 minutes to about 24 hours.

[0135] The terms "individual" and "subject" are used interchangeably herein and refer to an animal, for example a human, from which cells can be obtained and / or to which treatment, including prophylaxis, with the cells can be administered as described. For the treatment of such infections, conditions or disease states that are specific to a particular animal, such as a human subject, the term refers to the particular animal. The terms "non-human animals" and "non-human mammals", as used interchangeably herein, include mammals such as rats, mice, rabbits, sheep, cats, dogs, cows, pigs and non-human primates. The term "subject" also includes any vertebrate, including, but not limited to, mammals, reptiles, amphibians and fish. The subject is advantageously a mammal, such as a human, or another mammal, such as a domesticated mammal, e.g.a dog, a cat, a horse and the like or production mammals, e.g. a cow, a sheep, a pig, and the like.

[0136] The terms "diabetes" and "diabetes mellitus" are used interchangeably here. The World Health Organization defines the diagnostic value of fasting blood glucose concentration as 7.0 mmol / L (126 mg / dL) and above for diabetes mellitus (whole blood, 6.1 mmol / L or 110 mg / dL), or the 2-hour blood glucose level 11.1 mmol / L or higher (200 mg / dL or higher). Other values ​​that suggest or indicate an increased risk of diabetes mellitus include elevated arterial blood pressure 140 / 90 mm Hg or higher; elevated plasma triglycerides (1.7 mmol / L; 150 mg / dL) and / or low HDL cholesterol (less than 0.9 mmol / L, 35 mg / dL for men; less than 1.0 mmol / L, 39 mg / dL for women); general obesity (men: waist-to-hip ratio greater than 0.90; women: waist-to-hip ratio greater than 0.85) and / or a body mass index of more than 30 kg / m 2; microalbuminuria, where the urinary albumin excretion rate is 20 ng / min or higher or the albumin:xreatinine ratio is 30 mg / g or higher). The term diabetes includes all forms of diabetes, such as type I, type II, and type 1.5.

[0137] The terms "treat," "treated," "treatment," etc., as applied to an isolated cell, include subjecting the cell to any kind of process or condition, or any kind of manipulation or treatment of the cell. With respect to an individual, the terms mean providing medical or surgical attention, care, or treatment to the individual. The individual is usually ill or injured, or exhibits

[0138] As used herein, the terms "treating" and "treatment" refer to administering to an individual an effective amount of a composition such that the individual experiences a reduction in at least one symptom of the disease or an improvement in the disease, e.g., beneficial or desired clinical outcomes. For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, relief of one or more symptoms, reduction in the extent of the disease, stabilization (i.e., no worsening) of the disease state, delaying or slowing disease progression, improvement or alleviation of the disease state, and remission (whether partial or complete), whether detectable or undetectable. Treatment may refer to an extension of survival relative to the expected survival without treatment.Thus, it is clear to one of ordinary skill in the art that treatment may improve a disease state without being a complete cure. As used herein, the term "treatment" refers to prevention. Alternatively, treatment is "effective" if it reduces or stops the progression of a disease. "Treatment" can also mean an extension of survival compared to expected survival without treatment. Those in need of treatment include those already diagnosed with heart disease, as well as those likely to develop heart disease due to genetic predisposition or other factors, such as weight, diet, and health.

[0139] As used herein, the terms "administering," "introducing," and "transplanting" are used in connection with the delivery of the β-cells of the invention into a patient by a method or route that results, at least in part, in localizing the deployed cells to a desired location. For example, cells such as SC-β-cells (e.g., pancreatic β-cells or pancreatic β-like cells) may be implanted directly onto / into the pancreas, or alternatively, administered by any suitable route resulting in delivery to a desired location in the patient, wherein at least a portion of the implanted cells or cellular components remain viable. The period of viability of the cells after administration to a patient may be as short as a few hours, for example, about 24 hours, to a few days, and as long as several years.In some cases, the cells can also be administered at a non-pancreatic location (e.g., microcapsules) to maintain the implanted cells at the implantation site and prevent migration, such as in the liver or subcutaneously, for example, in a capsule, implanted cells.

[0140] The terms "parenteral administration" and "administered parenterally," as used herein, mean routes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion.The terms "systemic administration", "systemically administered", "peripheral administration" and "peripherally administered" as used herein mean the administration of cardiovascular stem cells and / or their progeny and / or compound and / or other material not directly into the central nervous system, such that they enter the animal's system and are thus subject to metabolism and other comparable processes, for example via subcutaneous administration.

[0141] The term "tissue" refers to a group or layer of specialized cells that work together to perform specific functions. The term "tissue-specific" refers to a source of cells from a specific tissue.

[0142] The terms "decrease," "reduced," "decrease," "lowering," or "inhibition" are all used herein generically to mean a decrease of a statistically significant amount. However, for the avoidance of any doubt, the terms "decreased," "decrease," or "decrease" or "inhibition" mean a decrease of at least 10% compared to a reference value, for example, a decrease of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including a decrease of 100% (i.e., no level compared to a reference sample), or any decrease between 10-100%, compared to a reference value.

[0143] The terms “increased,” “rise,” “increase,” or “activate” are all used here to denote a statistically significant increase. For the avoidance of doubt, the terms “increased”, “increase” or “increase” or “activate” mean an increase of at least 10% compared to a reference value, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including a 100% increase, or an increase between 10-100%, compared to a reference value, or at least about 2 times, preferably at least 3 times, more preferably at least 4 times, or at least about 5 times, or at least about 10 times, or an increase between 2 times and 10 times or more, compared to a reference value.

[0144] The term "statistically significant" or "significant" refers to statistical significance, and generally means a two-standard deviation (2SD) below the normal concentration of a marker or a small marker. This term indicates statistical data that there is a difference. It is defined as the probability that a decision can be made if the null hypothesis is actually true. The decision is often made using the p-value.

[0145] As used herein, the terms "comprising" and "containing" are used with reference to the compositions, methods, and respective component(s) thereof that are essential to the invention, but unstated elements, whether essential or not, may be further encompassed.

[0146] As used herein, the term "consisting essentially of" refers to those elements required for a particular embodiment. The term permits the presence of additional elements that do not substantially impair the basic and novel or functional characteristic(s) of that embodiment of the invention.

[0147] The term "consisting of" refers to compositions, methods, and corresponding components thereof as described herein that do not include elements specified in this description of the embodiment.

[0148] As used in this specification and the appended claims, the singular forms "a," "an," and "the" also include the plural forms, unless the context clearly dictates otherwise. For example, references to "the method" may include one or more methods and / or steps of the type described herein and / or what would be apparent to one of ordinary skill in the art upon reading this disclosure, and so on. Stem cells

[0149] Stem cells are cells that retain the ability to renew themselves by mitotic cell division and to differentiate into a wide range of specialized cell types. There are two major types of mammalian stem cells: embryonic stem cells (ES cells), which are found in blastocysts, and adult stem cells, which are found in adult tissues. In a developing embryo, stem cells can differentiate into all specialized embryonic tissues. In adult organisms, stem cells and progenitor cells act as a repair system for the body, supplying specialized cells, and also maintaining the normal turnover of regenerative organs such as blood, skin, or intestinal tissue. Pluripotent stem cells can differentiate into cells derived from any of the three germ layers.

[0150] Although certain embodiments are described below with reference to the use of stem cells to produce SC-β cells (e.g., mature pancreatic β cells or β-like cells) or precursors thereof, germ cells may also be used in place of or with stem cells to provide at least one SC-β cell using similar protocols to the illustrative protocols described herein. Suitable germ cells may, for example, be prepared from primordial germ cells present in human fetal material collected at approximately 8-11 weeks post-menstrual period. Illustrative germ cell preparation methods are described, for example, in Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998 and US-P. No. 6,090,622.

[0151] ES cells, such as human embryonic stem cells (hESCs) or mouse embryonic stem cells (mESCs), with a virtually unlimited replication capacity and the potential to differentiate into most cell types, essentially represent an unlimited starting material for generating differentiated cells for clinical therapy (http: / / stemcells.nih.gov / info / scireport / 2006report.htm, 2006). One potential application of ES cells is to generate new pancreatic β-cells for cell replacement therapy in type 1 diabetics. This involves first generating endoderm, e.g., definitive endoderm from hESCs, and then differentiating the definitive endoderm into at least one insulin-positive endocrine cell or a precursor thereof. This is followed by further differentiation of the at least one insulin-positive endocrine cell or precursor thereof into an SC-β-cell.

[0152] hES cells are described, for example, in Cowan et al. (N. Engl. J. Med. 350: 1353, 2004) and Thomson et al. (Science 282: 1145, 1998); embryonic stem cells from other primates, rhesus stem cells (Thomson et al., Proc. Natl. Acad. Sci. USA 92: 7844, 1995), marmoset stem cells (Thomson et al. Biol. Reprod. 55: 254, 1996), and human embryonic germ (hEG) cells (Shamblott et al., Proc. Natl. Acad. Sci. USA 95: 13726, 1998), which can also be used in the methods disclosed here. mESCs are described, for example, in Tremml et al. (Curr. Protoc. Stem Cell Biol. Chapter 1: Unit 1 C.4, 2008). The stem cells may, for example, be unipotent, totipotent, multipotent, or pluripotent. In some examples, any cells of primate origin capable of producing offspring that are derivatives of at least one germ layer or all three germ layers may be used in the methods disclosed herein.

[0153] In certain examples, ES cells can be isolated, for example, as described in Cowan et al. (N. Engl. J. Med. 350: 1353, 2004) and US Pat. No. 5,843,780 and Thomson et al., Proc. Natl. Acad. Sci. USA 92: 7844, 1995. For example, hESCs can be prepared from human blastocyst cells using the techniques described by Thomson et al. (US Pat. No. 6,200,806; Science 282: 1145, 1998; Curr. Top. Dev. Biol. 38: 133 ff, 1998) and by Reubinoff et al., Nature Biotech. 18: 399, 2000. Cell types equivalent to hESCs include their pluripotent derivatives, such as primitive ectoderm-like (EPL) cells, as shown, for example, in WO 01 / 51610 (BresaGen). hESCs can also be obtained from human preimplantation embryos. Alternatively, in vitro fertilized (IVF) embryos can be used, or a single-cell human embryo can be expanded to the blastocyst stage (Bongso et al., Hum Reprod. 4: 706, 1989).Embryos are cultured to the blastocyst stage in G1.2 and G2.2 medium (Gardner et al., Fertil. Steril. 69:84, 1998). The zona pellucida is removed from developed blastocysts by brief exposure to pronase (Sigma). The inner cell masses can be obtained by immunosurgery, in which blastocysts are exposed to rabbit anti-human spleen cell antiserum at a 1:50 dilution for 30 min, followed by three 5-minute washes in DMEM and subsequent exposure to guinea pig complement (Gibco) at a 1:5 dilution for 3 min (Solter et al., Proc. Natl. Acad. Sci. USA 72:5099, 1975). After two further washes in DMEM, lysed trophectoderm cells are removed from the intact inner cell mass (ICM) by careful pipetting and the ICM is plated on mEF feeder layers.After 9 to 15 days, growths derived from the inner cell mass can be dissociated into clumps, either by exposure to calcium- and magnesium-free phosphate-buffered saline (PBS) containing 1 mM EDTA, by exposure to dispase or trypsin, or by mechanical dissociation with a micropipette. They are then replated on mEF in fresh medium. Growing colonies with undifferentiated morphology can be individually selected by a micropipette, mechanically dissociated into clumps, and replated. An ES-like morphology is characterized as compact colonies with an apparently high nuclear-to-cytoplasmic ratio and prominent nucleoli. hESCs obtained in this way can then be routinely divided every 1 to 2 weeks, for example, by brief trypsinization, contact with Dulbecco's PBS (with 2 mM EDTA), exposure to type IV collagenase (200 U / ml; Gibco), or by selecting individual colonies using a micropipette.In some examples, clump sizes of approximately 50 to 100 cells are optimal. mESCs can be prepared using techniques described, for example, in Conner et al. (Curr. Prot. in Mol. Biol. Unit 23.4, 2003).

[0154] Embryonic stem cells can be isolated from blastocysts of members of primate species (U.S. Patent No. 5,843,780; Thomson et al., Proc. Natl. Acad. Sci. USA 92:7844, 1995). Human embryonic stem cells (hES cells) can be prepared from human blastocyst cells using the techniques described by Thomson et al. (U.S. Patent No. 6,200,806; Science 282:1145, 1998; Curr. Top. Dev. Biol. 38:133 ff, 1998) and Reubinoff et al., Nature Biotech. 18:399, 2000. Cell types equivalent to hES include their pluripotent derivatives, such as primitive ectoderm-like (EPL) cells, as described in WO 01 / 51610 (BresaGen).

[0155] Alternatively, in some embodiments, hES cells can be obtained from human preimplantation embryos. Alternatively, in vitro fertilized (IVF) embryos can be used, or a single-cell human embryo is grown to the blastocyst stage (Bongso et al., Hum Reprod. 4:706, 1989). Embryos are grown to the blastocyst stage in G1.2 and G2.2 medium (Gardner et al., Fertil. Steril. 69:84, 1998). The zona pellucida is removed from developed blastocysts by brief exposure to pronase (Sigma). The inner cell masses are isolated by immunosurgery, whereby blastocysts are exposed to rabbit anti-human spleen cell antiserum at a 1:50 dilution for 30 min, then washed three times in DMEM for 5 min and exposed to a 1:5 dilution of guinea pig complement (Gibco) for 3 min (Solter et al., Proc. Natl. Acad. Sci. USA 72: 5099, 1975).After two further washes in DMEM, lysed trophectoderm cells are removed from the intact inner cell mass (ICM) by careful pipetting and the ICM is plated on mEF feeder layers.

[0156] After 9–15 days, the outgrowths derived from the inner cell mass are dissociated into clumps either by exposure to calcium- and magnesium-free phosphate-buffered saline (PBS) containing 1 mM EDTA, by exposure to dispase or trypsin, or by mechanical dissociation using a micropipette. They are then replated on mEF in fresh medium. Growing colonies with undifferentiated morphology are individually selected using a micropipette, mechanically dissociated into clumps, and replated. ES-like morphology is characterized as compact colonies with a seemingly high nuclear-to-cytoplasmic ratio and prominent nucleoli. The resulting ES cells are then routinely divided every 1 to 2 weeks by brief trypsinization, contact with Dulbecco's PBS (containing 2 mM EDTA), exposure to type IV collagenase (approximately 200 U / ml; Gibco), or by selection of individual colonies using a micropipette.Clumps with a size of about 50 to 100 cells are optimal.

[0157] In some embodiments, human embryonic germ cells (hEG) are pluripotent stem cells that can be used in the methods disclosed herein to differentiate into primitive endoderm cells.

[0158] hEG cells can be prepared from primordial germ cells present in human fetal material collected approximately 8-11 weeks after the last menstrual period. Suitable preparation methods are disclosed in Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998, and U.S. Patent No. 6,090,622, which are incorporated herein by reference in their entirety.

[0159] In short, genital ridges are processed to form disaggregated cells. Growth medium for EG is DEM, 4500 mg / L D-glucose, 2200 mg / L m NaHCO 3; 15% ES qualified fetal calf serum (BRL); 2 mM glutamine (BRL); 1 mM sodium pyruvate (BRL); 1000-2000 U / ml human recombinant leukemia inhibitory factor (LIF, Genzyme); 1-2 ng / ml human recombinant bFGF (Genzyme); and 10 µM forskolin (in 10% DMSO). In 96-well tissue culture plates containing a subconfluent layer of feeder cells (e.g., STO cells, ATCC No. CRL 1503) cultured for 3 days in modified EG growth medium without LIF, bFGF, or forskolin and inactivated with 5000 rad of γ-irradiation, ~0.2 ml of a primary germ cell (PGC) suspension was added to each well. The first passage occurred after 7–10 days in EG growth medium, with each well being transferred to a well of a 24-well culture plate previously prepared with irradiated STO mouse fibroblasts.Cells were grown with daily medium replacement until cell morphology comparable to EG cells was observed, typically after 7–30 days or 1–4 passages.

[0160] In certain examples, the stem cells (e.g., a cell that is not committed to a particular lineage) may be undifferentiated prior to exposure to at least one β-cell maturation factor according to the methods disclosed herein, while in other examples, it may be desirable for the stem cells to differentiate into one or more intermediate cell types prior to exposure to the at least one β-cell maturation factor(s) described herein. For example, the stem cells may exhibit morphological, biological, or physical characteristics of undifferentiated cells that can be used to distinguish them from differentiated cells of embryonic or adult origin. In some examples, undifferentiated cells may appear in the two dimensions of a microscopic view in cell colonies with a high nuclear:cytoplasmic ratio and prominent nucleoli.The stem cells can be themselves (e.g., substantially without any undifferentiated cells present), or can be used in the presence of differentiated cells. In certain examples, the stem cells can be cultured in the presence of suitable nutrients, and optionally other cells, so that the stem cells can grow and optionally differentiate. For example, embryonic fibroblasts or fibroblast-like cells can be present in culture to support the growth of the stem cells. The fibroblasts can be present in some stages of stem cell growth, but not necessarily all. For example, fibroblasts can be added to the stem cell culture in an initial cultivation stage, but not in one or more subsequent cultivation stages.

[0161] Stem cells used according to the present disclosure can be any cells derived from any type of tissue (for example, embryonic tissue such as fetal or pre-fetal tissue or adult tissue), wherein the stem cells have the property of being capable, under suitable conditions, of producing progeny of various cell types, for example, derivatives of all of at least one of the three germ layers (endoderm, mesoderm and ectoderm). These cell types can be provided in the form of an established cell line, or can be obtained directly from a primary embryonic tissue and used immediately for differentiation. Included are cells listed in the NIH Human Embryonic Stem Cell Registry, e.g., hESBGN-01, hESBGN-02, hESBGN-03, hESBGN-04 (BresaGen, Inc.); Hes-1, HES-2, HES-3, HES-4, HES-5, HES-6 (ES Cell International); Miz-hES-1 (MizMedi Hospital-Seoul National University); HSF-1, HSF-6 (University of California at San Francisco); and HI, H7, H9, HI 3, HI 4 (Wisconsin Alumni Research Foundation (WiCell Research Institute)). In some embodiments, the source of the human stem cells or pluripotent stem cells used for chemically induced differentiation into mature, insulin-positive cells does not involve destruction of a human embryo.

[0162] In another embodiment, the stem cells can be isolated from tissue, including solid tissue. In some embodiments, the tissue is skin, adipose tissue (e.g., fat tissue), muscle tissue, heart, or cardiac tissue. In other embodiments, the tissue is, for example, but not limited to, umbilical cord blood, placenta, bone marrow, or cartilage.

[0163] Stem cells of interest further include embryonic cells of various types, as represented by human embryonic stem cells (hES cells) described by Thomson et al. (1998) Science 282: 1145; embryonic stem cells from other primates such as rhesus stem cells (Thomson et al. (1995) Proc. Natl. Acad. Sci. USA 92: 7844); marmoset stem cells (Thomson et al. (1996) Biol. Reprod, 55: 254); and human embryonic germ (hEG) cells (Shambloft et al., Proc. Natl. Acad. Sci. USA 95: 13726, 1998). Also of interest are lineage committed stem cells such as mesodermal stem cells and other early cardiogenic cells (see Reyes et al. (2001) Blood 98: 2615-2625; Eisenberg & Bader (1996) Circ Res. 78 (2): 205-16; etc.) The stem cells can be obtained from all mammalian species, e.g. human, horse, cattle, pig, dog, cat, rodents, e.g. mice, rats, hamsters, primates etc.In some embodiments, no human embryo has been destroyed for a source of the pluripotent cells used in the methods and compositions as disclosed herein.

[0164] ES cells are considered undifferentiated if they are not committed to development into a specifically differentiated lineage. Such cells exhibit morphological characteristics that distinguish them from differentiated cells of embryonic or adult origin. Undifferentiated ES cells are readily recognized by those skilled in the art and usually appear in the two dimensions of a microscopic view as cell colonies with a high nucleus:cytoplasm ratio and prominent nucleoli. Undifferentiated ES cells express genes used as markers to detect the presence of undifferentiated cells, and their polypeptide products can be used as markers for negative selection. See, for example, U.S. Patent Application Ser. No. 2003 / 0224411 A1; Bhattacharya (2004) Blood 103(8):2956-64; and Thomson (1998), supra, each of which is incorporated herein by reference.Human ES cell lines express cell surface markers that identify undifferentiated ES cells, human primates, and human EC cells, including phase-specific embryonic antigen (SSEA-3), SSEA-4, TRA-1-60, TRA-1-81, and alkaline phosphatase. The Globo-series glycolipid GL7, which carries the SSEA-4 epitope, is formed by the addition of sialic acid to the Globo-series glycolipid GBS, which carries the SSEA-3 epitope. Thus, GL7 reacts with antibodies against both SSEA-3 and SSEA-4. The undifferentiated human ES cell lines did not stain for SSEA-1, but differentiated cells stained strongly for SSEA-1. Methods for propagating hES cells in undifferentiated form are described in WO 99 / 20741, WO 01 / 51616 and WO 03 / 020920.

[0165] A mixture of cells derived from a suitable source of endothelial, muscle, and / or neural stem cells can be obtained from a mammalian donor by methods known in the art. One suitable source is the hematopoietic microenvironment. For example, circulating peripheral blood, preferably mobilized (i.e., recruited), can be removed from an individual. Alternatively, bone marrow can be obtained from a mammal, such as a human patient who has undergone an autologous transplant. In some embodiments, stem cells can be obtained from the individual's adipose tissue, for example, using Cytori's Celution™ System, as described in U.S. Pat. Nos. 7,390,484 and 7,429,488, which are incorporated herein by reference in their entirety.

[0166] In some embodiments, human umbilical cord blood cells (HUCBC) are useful in the methods disclosed herein. Human UBC cells have been recognized as a rich source of hematopoietic and mesenchymal progenitor cells (Broxmeyer et al., 1992 Proc. Natl. Acad Sci. USA 89: 4109-4113). Previously, umbilical cord and placental blood were typically discarded as a waste product at the birth of a child. Umbilical cord blood cells are used as a source of transplantable stem and progenitor cells and as a source of bone marrow repopulating cells for the treatment of malignant diseases (i.e., acute lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome, and neuroblastoma) and non-malignant diseases such as Fanconi anemia and aplastic anemia (Kohli-Kumar et al., 1993 Br. J. Haematol. 85: 419-422; Wagner et al. 1992, Blood 79, 1874-1881; Lu et al., 1996 Crit. Rev. Oncol. Hematol 22: 61-78; .Lu et al. 1995 Cell Transplantation 4: 493-503). A distinct advantage of HUCBC is the immature immunity of these cells, which is very similar to fetal cells, reducing the risk of host rejection (Taylor & Bryson, 1985 J. Immunol. 134: 1493-1497). Human umbilical cord blood contains mesenchymal and hematopoietic progenitor cells and endothelial progenitor cells that can be expanded in tissue culture (Broxmeyer et al., 1992 Proc. Natl. Acad. Sci. USA 89: 4109-4113; Kohli-Kumar et al., 1993 Br. J. Haematol. 85: 419-422; Wagner et al. 1992, Blood 79, 1874-1881; Lu et al., 1996 Crit. Rev. Oncol. Hematol 22: 61-78; Lu et al. 1995 Cell Transplantation 4: 493-503; Taylor & Bryson, 1985 J. Immunol. 134: 1493-1497 Broxmeyer 1995 Transfusion 35: 694-702; Chen et al. 2001 Stroke. 32: 2682-2688; Nieda et al., 1997 Br. J. Hematology 98: 775-777; Erices et al., 2000 Br. J. Hematology 109: 235-242).The total content of hematopoietic progenitor cells in cord blood is equal to or greater than in bone marrow, with the additional presence of highly proliferative hematopoietic cells being eightfold more present in HUCBC than in bone marrow and expressing hematopoietic markers such as CD14, CD34 and CD45 (Sanchez-Ramos et al. 2001 Exp. Neur. 171: 109-115; Bicknese et al., 2002 Cell Transplantation 11: 261-264; Lu et al., 1993, J. Exp Med. 178: 2089-2096).

[0167] In another embodiment, pluripotent cells are derived from a hematopoietic microenvironment, such as circulating peripheral blood, preferably from the mononuclear fraction of peripheral blood, umbilical cord blood, bone marrow, fetal liver, or the yolk sac of a mammal. The stem cells, particularly neural stem cells, can also be derived from the central nervous system, including the meninges.

[0168] In another embodiment, pluripotent cells present in embryonic bodies are obtained by harvesting ES cells by brief protease digestion and growing small clumps of undifferentiated human ESR in suspension culture. Differentiation is induced by removal of the conditioned medium. The resulting embryoid bodies are plated on semisolid substrates. Formation of differentiated cells can be observed after approximately 7 days to approximately 4 weeks. Viable differentiating cells from in vitro cultures of stem cells are selected by partial dissociation of embryoid bodies or similar structures to form cell aggregates. Aggregates comprising cells of interest are selected for phenotypic characteristics by methods that substantially maintain cell-cell contacts in the aggregates.

[0169] In an alternative embodiment, the stem cells may be reprogrammed stem cells, such as stem cells derived from somatic or differentiated cells. In such an embodiment, the dedifferentiated stem cells may be, for example, but not limited to, neoplastic cells, tumor cells, and cancer cells, or alternatively, induced reprogrammed cells such as pluripotent stem cells or iPS cells. Cloning and cell culture

[0170] Exemplary molecular genetics and gene engineering techniques that can be used in the technology described here can be found, for example, in current editions of Molecular Cloning: A Laboratory Manual (Sambrook et al., Cold Spring Harbor); Gene Transfer Vectors for Mammalian Cells (Miller & Calos eds.); and Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., Wiley & Sons). Cell biology, protein chemistry, and antibody techniques can be found in, for example, Current Protocols in Protein Science (J.E. Colligan et al. eds., Wiley & Sons); Current Protocols in Cell Biology (J.S. Bonifacino et al., Wiley & Sons); and Current Protocols in Immunology (J.E. Colligan et al., eds., Wiley & Sons). Exemplary reagents, cloning vectors, and genetic manipulation kits are commercially available from, for example, BioRad, Stratagene, Invitrogen, Clontech, and Sigma-Aldrich Co.

[0171] Suitable cell culture procedures can be found, for example, in the general descriptions in the current edition of Culture of Animal Cells: A Manual of Basic Technique (RI Freshney ed., Wiley & Sons); General Techniques of Cell Culture (MA Harrison & IF Rae, Cambridge Univ. Press); and Embryonic Stem Cells: Methods and Protocols (K. Turksen ed., Humana Press). Suitable tissue culture materials and reagents are commercially available, for example, from Gibco / BRL, Nalgene-Nunc International, Sigma Chemical Co., and ICN Biomedicals.

[0172] Pluripotent stem cells can be continuously propagated in culture by one skilled in the art using culture conditions that promote proliferation without promoting differentiation. Exemplary serum-containing ES medium is prepared with 80% DMEM (such as Knock-Out DMEM, Gibco), with 20% either defined fetal bovine serum (FBS, Hyclone) or serum substitute (WO 98 / 30679), with 1% non-essential amino acids, 1 mM L-glutamine, and 0.1 mM β-mercaptoethanol. Immediately before use, human bFGF is added to 4 ng / ml (WO 99 / 20741, Geron Corp.). Traditionally, the ES cells are grown on a layer of feeder cells, typically fibroblasts derived from embryonic or fetal tissue.

[0173] Scientists at Geron have found that pluripotent SCs can be maintained in an undifferentiated state even without feeder cells. The environment for feeder-free cultures includes a suitable culture substrate, specifically an extracellular matrix such as Matrigel® or laminin. Typically, enzymatic digestion is stopped before the cells are fully dispersed (i.e., approximately ~5 min with collagenase IV). Clumps of ~10 to 2000 cells are then plated directly onto the substrate without further spreading.

[0174] Feeder-free cultures are supported by a nutrient medium containing factors that promote cell proliferation without differentiation. Such factors can be introduced into the medium by culturing the medium with cells that secrete such factors, such as irradiated (~4,000 rad) primary mouse embryonic fibroblasts, telomerized mouse fibroblasts, or fibroblast-like cells derived from pPS cells. The medium can be supplemented by plating feeder cells at a density of ~5-6 × 10 4 cm -2 in a serum-free medium, such as KO DMEM supplemented with 20% serum replacement and 4 ng / ml bFGF. Medium conditioned for 1-2 days is supplemented with additional bFGF and used to support pluripotent SC culture for 1-2 days. Features of the feeder-free culture method are described in International Patent Publication WO 01 / 51616 and Xu et al., Nat. Biotechnol. 19:971 2001.

[0175] Under the microscope, ES cells appear with a high nuclear / cytoplasmic ratio, prominent nucleoli, and compact colony formation with poorly discernible cell junctions. Primate ES cells express stage-specific embryonic antigens (SSEA) 3 and 4, and markers detectable with the antibodies designated Tra-1-60 and Tra-1-81 (Thomson et al., Science 282:1, 145, 1998). Mouse ES cells are used as a positive control for SSEA-1 and as a negative control for SSEA-4, whereas Tra-1-60 and Tra-1-81 are constantly present in human embryonic carcinoma (hEC) cells. Differentiation of pluripotent SCs in vitro results in the loss of SSEA-4, Tra-1-60 and Tra-1-81 expression and increased expression of SSEA-1, which is also found on undifferentiated hEG cells. Stem cell-derived β-cell (SC-β)Stem cell-derived β-cell (SC-β)

[0176] In some aspects, the disclosure provides a stem cell-derived β-cell (SC-β). The SC-β cells disclosed herein share many distinguishing features of native β-cells, but differ in certain aspects (e.g., gene expression profiles). In some embodiments, the SC-β cell is non-native. As used herein, the term "non-native" refers to the SC-β cells differing significantly in certain aspects from β-cells found in nature, i.e., native β-cells. However, it should be understood that these distinct differences typically relate to structural features that cause the SC-β cells to exhibit certain functional differences, such that although the gene expression pattern of SC-β cells differs from native β-cells, the SC-β cells behave in a manner similar to native β-cells, but with certain functions altered compared to native β-cells (e.g.,improved). For example, react like . Fig. 2E, a larger number of SC-β cells at 20 m glucose compared to the number of native β cells. Further differences between the SC-β cells and native β cells will be apparent to one of skill in the art based on the data disclosed herein.

[0177] The SC β-cells of the disclosure exhibit many characteristic features of β-cells that are important for normal β-cell function. In some embodiments, the SC β-cell exhibits a glucose-stimulated insulin secretion (GSIS) response in vitro. In some embodiments, the SC cell exhibits a β-GSIS response in vivo. In some embodiments, the SC β-cell exhibits GSIS responses in vitro and in vivo. In some embodiments, the GSIS responses are similar to the GSIS responses of an endogenous mature pancreatic β-cell. In some embodiments, the SC β-cell exhibits a GSIS response to at least one glucose challenge. In some embodiments, the SC β-cell exhibits a GSIS response to at least two consecutive glucose challenges. In some embodiments, the SC-β cell exhibits a GSIS response to at least three consecutive glucose challenges.In some embodiments, the GSIS responses are similar to the GSIS responses of endogenous human islet cells to multiple glucose challenges. In some embodiments, the GSIS response is observed immediately after transplantation of the cell into a human or animal. In some embodiments, the GSIS response is observed within about 24 hours after transplantation of the cell into a human or animal. In some embodiments, the GSIS response is observed within about one week after transplantation of the cell into a human or animal. In some embodiments, the GSIS response is observed within about two weeks after transplantation of the cell into a human or animal.In some embodiments, the stimulation index of the cell, defined by the ratio of insulin secreted in response to high glucose concentrations compared to low glucose concentrations, is comparable to the stimulation index of an endogenous mature β pancreatic cell. In some embodiments, the SC β cell has a stimulation index greater than 1. In some embodiments, the SC β cell has a stimulation index greater than or equal to 1. In some embodiments, the SC β cell has a stimulation index greater than or equal to 1.1. In some embodiments, the SC β cell has a stimulation index greater than or equal to 1.1. In some embodiments, the SC β cell has a stimulation index greater than 2. In some embodiments, the SC β cell has a stimulation index greater than or equal to 2.In some embodiments, the SC-β cell has a stimulation index of at least 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5.0 or more.

[0178] In some embodiments, the SC-β cell exhibits cytokine-induced apoptosis in response to cytokines. In some embodiments, the SC-β cell exhibits cytokine-induced apoptosis in response to a cytokine selected from the group consisting of interleukin-ΐβ (IL-β), interferon-γ (INF-γ), tumor necrosis factor-α (TNF-α), and combinations thereof.

[0179] In some embodiments, SC β-cell insulin secretion is increased in response to known antidiabetic agents (e.g., antidiabetic drugs acting on β-cells ex vivo or in vitro and / or antidiabetic drugs generally in vivo). The disclosure relates to any known antidiabetic agent. In some embodiments, SC β-cell insulin secretion is enhanced in response to a secretagogue. In some embodiments, the secretagogue is selected from the group consisting of an incretin mimetic, a sulfonylurea, a meglitinide, and combinations thereof.

[0180] In some embodiments, the SC-β cell is monohormonal. In some embodiments, the SC-β cell has a morphology similar to an endogenous mature pancreatic β cell. In some embodiments, the SC-β cell has encapsulated crystalline insulin granules. In some embodiments, the SC-β cell has encapsulated crystalline insulin granules under electron microscopy that resemble insulin granules of an endogenous mature pancreatic β cell. In some embodiments, the SC-β cell has a low replication rate. In some embodiments, the SC-β cell has a low replication rate. In some embodiments, the SC-β cell has a low but increased replication rate as measured by staining for C-peptide and i67 in response to prolactin treatment.

[0181] In some embodiments, the SC cell β increases intracellular Ca 2+in response to glucose. In some embodiments, the SC-β cell has a glucose-stimulated Ca 2+ Current (GSCF) that resembles the GSCF of an endogenous mature pancreatic β-cell. In some embodiments, the SC-β-cell exhibits a GSCF response to at least three consecutive glucose challenges in a manner that resembles the GSCF response of an endogenous mature pancreatic β-cell.

[0182] In some embodiments, the SC-β-cell expresses at least one marker characteristic of an endogenous mature pancreatic β-cell selected from the group consisting of insulin, C-peptide, PDX 1, M AFA, NX6-1 PAX6, NeuroD 1, glucokinase (GCK), SLC2A 1, PCSK 1, KCNJ 1 1, ABCC8, SLC30A8, SNAP25, RAB3A, GAD2 and PTPRN.

[0183] In some embodiments, the SC β-cell does not express at least one marker (e.g., a marker not expressed by endogenous mature pancreatic β-cells) selected from the group consisting of a) a hormone selected from the group consisting of i) glucagon (GCG) and ii) somatostatin (SST); b) an acinar cell tumor marker selected from the group consisting of i) amylase and ii) carboxypeptidase A (CPA 1), c) a cell marker selected from the group consisting of i) GCG Arx, Irxl, and IRX2, d) a ductal cell marker selected from the group consisting of i) CFTR and ii) Sox9.

[0184] The SC-β cells are differentiated in vitro from any parental cell, as the invention is not intended to be limited to any parental cell from which the SC-β cells are derived. Exemplary parental cells include, but are not limited to, insulin-positive endocrine cells or a precursor thereof, such as an NKX6-1-positive pancreatic progenitor cell, a Pdxl-positive pancreatic progenitor cell, a pluripotent stem cell, an embryonic stem cell, and an induced pluripotent stem cell. In some embodiments, the SC-β cells are differentiated in vitro from a reprogrammed cell, a partially reprogrammed cell (i.e., a somatic cell, such as a fibroblast, that has been partially reprogrammed and exists between an induced pluripotent cell and the somatic cell from which it was derived), and a trans-differentiated cell.In some embodiments, the SC-β cells disclosed herein can be differentiated in vitro from an insulin-positive endocrine cell or a precursor thereof. In some embodiments, the SC-β cell is differentiated in vitro from a precursor selected from the group consisting of an NKX6-1-positive pancreatic progenitor cell, a Pdxl-positive pancreatic progenitor cell, and a pluripotent stem cell. In some embodiments, the pluripotent stem cell is selected from the group consisting of an embryonic stem cell and pluripotent stem cells. In some embodiments, the SC-β cell or the pluripotent stem cell from which the SC-β cell is derived is from a human. In some embodiments, the SC-β cell is human.

[0185] In some embodiments, the SC-β cell is not genetically engineered. In some embodiments, the SC-β cell acquires the properties shared by native β cells without genetic modification of the cells. In some embodiments, the SC-β cell is genetically engineered.

[0186] In some embodiments, the insulin produced by the SC-β cell is at least 0.5 µIU per 1000 cells per 30 minutes of incubation (e.g., ex vivo) at high glucose concentration.

[0187] In some embodiments, the insulin produced by the SC-β cell is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 µIU per 1000 cells per 30-minute incubation at high glucose concentration. In some embodiments, the insulin produced by the SC-β cell is between 0.5 and 10 µIU per 1000 cells per 30-minute incubation at high glucose concentration. In some embodiments, the insulin produced by the SC-β cell is about 2.5 µIU per 1000 cells per 30-minute incubation at high glucose concentration.

[0188] The present disclosure provides cell lines comprising an SC-β cell described herein. In some embodiments, the SC-β cell stably expresses insulin. In some embodiments, the SC-β cell can be frozen, thawed, and amplified with a doubling time of 24 to 44 hours without significant morphological changes for at least 30 passages. Generation of SC-β cells

[0189] Aspects of the disclosure relate to the generation of SC-β cells (e.g., pancreatic β cells). Thus, in general, the at least one SC-β cell or precursor thereof, for example, the pancreatic progenitor cells produced according to the methods disclosed herein, may comprise a mixture or combination of different cells, for example, a mixture of cells such as Pdxl-positive pancreatic progenitor cells, positive pancreatic progenitor cells co-expressing Pdxl and NKX6-1, Ngn3-positive endocrine progenitor cells, insulin-positive endocrine cells (e.g., β-like cells), and insulin-positive endocrine cells, and / or other pluripotent or stem cells.

[0190] The at least one SC-β cell or precursor thereof can be produced according to any suitable culture protocol for differentiating a stem cell or pluripotent cell to a desired stage of differentiation. In some embodiments, the at least one SC-β cell or precursor thereof is produced by culturing at least one pluripotent cell for a period of time and under conditions suitable for the at least one pluripotent cell to differentiate into the at least one SC-β cell or precursor thereof.

[0191] In some embodiments, the at least one SC-β cell or precursor thereof is a substantially pure population of SC-β cells or precursors thereof. In some embodiments, a population of SC-β cells or precursors thereof from a mixture of pluripotent cells or differentiated cells. In some embodiments, the population of SC-β cells or precursors thereof is substantially free of or without embryonic stem cells or pluripotent cells or iPS cells.

[0192] In some embodiments, a somatic cell, e.g., a fibroblast, can be isolated from a patient, e.g., as a tissue sample, such as a skin biopsy, and reprogrammed into a pluripotent stem cell for further differentiation to generate the at least one SC-β cell or a precursor thereof for use in the compositions and methods described herein. In some embodiments, a somatic cell, e.g., a fibroblast, is maintained in culture using methods known to one of ordinary skill in the art and, in some embodiments, expanded before being converted into SC-β cells using the methods disclosed herein.

[0193] In some embodiments, the at least one SC-β cell or precursor thereof is maintained in culture by methods known to one of ordinary skill in the art and, in some embodiments, expanded before being converted into SC-β cells by methods disclosed herein.

[0194] The at least one SC-β cell or a precursor thereof, for example, pancreatic precursor, may further be derived from any mammalian species, non-limiting examples being a mouse, bovine, monkey, porcine, equine, sheep, or human cell. For clarity and simplicity, the description of the methods of the at least one SC-β cell or a precursor thereof relates to a mammal, it being understood that all methods described herein can be readily applied to other cell types of the at least one SC-β cell or a precursor thereof. In some embodiments, the at least one SC-β cell or a precursor thereof is derived from a human individual. Inducing the differentiation of pluripotent stem cells into definitive endoderm cells

[0195] Aspects of the disclosure include definitive or final endoderm cells. Definitive endoderm cells used herein may be derived from any source or generated in accordance with any suitable protocol. In some aspects, pluripotent stem cells, for example, iPSCs or hESCs, are differentiated to generate endoderm cells. In some aspects, the endoderm cells are further differentiated, for example, into archenteron cells, Pdxl-positive pancreatic progenitor cells, NKX6-1-positive pancreatic progenitor cells, Ngn3-positive endocrine progenitor cells, or insulin-positive endocrine cells, followed by induction or maturation into SC-β cells.

[0196] In some embodiments, the stem cells can be plated onto a new substrate or the medium can be replaced to remove the extracellular matrix or soluble factors that inhibit differentiation. This is sometimes referred to as the "direct differentiation method" and is generally described in International Patent Publication WO 01 / 51616 and Published US Patent 2002 / 0019046, which are hereby incorporated by reference in their entirety. It is usually preferable to begin the direct differentiation method with a feeder-free culture of stem cells, thus avoiding potential complications in the differentiation process from residual feeder cells. Another approach is to maintain undifferentiated stem cells in suspension culture, which often results in them forming aggregates of differentiated and undifferentiated cells.For example, stem cells can be harvested by brief collagenase digestion, dissociated into clusters, and passaged in non-adherent cell culture plates. The aggregates can be fed with nutrients every few days and then harvested after a suitable period of time, typically 4–8 days. Depending on the conditions, the aggregates generally begin by forming a heterogeneous population of cell types, including a substantial amount of endoderm cells. The aggregates can then be spread and replated for the next step in the differentiation process on substrates such as laminin or fibronectin, or passaged in suspension culture on, for example, non-adherent plates and a suitable medium.

[0197] Direct differentiation or differentiation into aggregates can be monitored for the presence of endoderm cells using suitable markers, such as those listed in US Patent No. 7,326,572. In some preferred embodiments, differentiation into endoderm cells can be monitored by the presence of markers, such as Soxl 7. Once a sufficient proportion of endoderm is obtained, the cells can be replated or otherwise manipulated to begin another stage of differentiation. Under certain circumstances, differentiation or cell recovery can be enhanced if the cells are maintained in micromass clusters (e.g., 50 to 5000 cells). Further stages of differentiation encompassed by the disclosure are described in Fig. 1 shown.

[0198] In some embodiments, definitive endoderm cells are produced by contacting (e.g., culturing) a pluripotent stem cell with a compound of formula (I) as described in U.S. Pat. No. 8,507,274 ("the '274 patent"), which is incorporated herein by reference. A compound of formula (I) as described in the '274 patent are cell-permeable small molecules and can control cellular processes by modulating signal transduction pathways, gene expression, or metabolism, and have been effectively used in stem cell differentiation protocols. Small molecules can be produced in high quantity and purity, as well as conveniently delivered or delivered, thus having great potential for therapeutic applications. High-throughput screens have been conducted to identify novel small molecules that support ES cell self-renewal (Chen et al., 2006, Desbordes et al., 2008), cardiogenic specification of mouse ES cells (Wu et al., 2004) or neural progenitor cells (Diamandis et al., 2007) as well as induction of specific cell types, particularly neuronal and muscle cells (review (Ding and Schultz, 2004). It is expected that compounds of formula (I) from the '274 patent will be used to differentiate a pluripotent stem cell into a definitive cell endoderm.

[0199] In some embodiments, the compound of formula (I) from the '274 patent is comprised: where: R 1 and R 2 are independently H, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cyclyl or cyclyl, each of which may be optionally substituted and / or interrupted in the main chain by one or more of O, N, S, S(O) and C(O); R 3 and R 4are independently H, halogen, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, cyclyl or cyclyl, each of which may be optionally substituted, or R 3 and R 4 , together with the carbon to which they are attached, an optionally substituted cyclyl or heterocyclyl s; and LC 1 -C 10 Alkylenyl, C 2 -C 10 Alkenylenyl or C 2 -C 10 Alkynylenyl, each of which may be optionally substituted and / or interrupted in the main chain by one or more of O, N, S, S(O) and C(O).

[0200] In some embodiments, the compound of formula (I) of the '274 patent IDE1 comprises:

[0201] In some embodiments, the compound of formula (I) of the '274 patent IDE2 comprises:

[0202] The '274 patent describes methods for determining the identity of the endoderm cell derived in this manner, as well as methods for isolating and preserving, expanding, and further differentiating the final endoderm, all of which can be used with the compositions and methods described herein, as will be known to those skilled in the art.

[0203] In some embodiments, definitive endoderm cells can be obtained by differentiating at least some pluripotent cells in a population into definitive endoderm cells, e.g., by contacting a population of pluripotent cells with i) at least one growth factor from the TGF-β superfamily, and ii) a Wnt signaling pathway activator to induce differentiation of at least some of the pluripotent cells into definitive endoderm cells, wherein the definitive endoderm cells express at least one marker characteristic of definitive endoderm.

[0204] The invention relates to the use of any growth factor from the TGF-β superfamily that induces pluripotent stem cells to differentiate into definitive endoderm cells (e.g., alone or in combination with a Wnt signaling pathway activator). In some embodiments, the at least one growth factor from the TGF-β superfamily comprises activin A. In some embodiments, the at least one growth factor from the TGF-β superfamily comprises growth differentiation factor 8 (GDF8).

[0205] The disclosure relates to the use of any Wnt pathway activator to induce pluripotent stem cells to differentiate into definitive endoderm cells (e.g., alone or in combination with a growth factor from the TGF-β superfamily). In some embodiments, the Wnt pathway activator comprises CHIR99021. In some embodiments, the Wnt pathway activator is Wnt3a, a recombinant protein.

[0206] It will be appreciated by those skilled in the art that the concentrations of the agents used (e.g., growth factors) may vary. In some embodiments, the pluripotent cells are contacted with the at least one growth factor from the TGF-β superfamily at a concentration of 10 ng / ml - 1000 ng / ml. In some embodiments, the pluripotent cells are contacted with the at least one growth factor from the TGF-β superfamily at a concentration of 100 ng / ml. In some embodiments, the pluripotent cells are contacted with the at least one growth factor from the TGF-β superfamily at a concentration of 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, or 90 ng / ml.In some embodiments, the pluripotent cells are contacted with the at least one growth factor from the TGF-β superfamily at a concentration of 91 ng / ml, 92 ng / ml, 93 ng / ml, 94 ng / ml, 95 ng / ml, 96 ng / ml, 97 ng / ml, 98 ng / ml, or 99 ng / ml. In some embodiments, the pluripotent cells are contacted with the at least one growth factor from the TGF-β superfamily at a concentration of 1 to 10 ng / ml, 120 ng / ml, 130 ng / ml, 140 ng / ml, 150 ng / ml, 160 ng / ml, 170 ng / ml, 180 ng / ml, or 190 ng / ml. In some embodiments, the pluripotent cells are contacted with the at least one growth factor from the TGF-β superfamily at a concentration of 101 ng / ml, 102 ng / ml, 103 ng / ml, 104 ng / ml, 105 ng / ml, 106 ng / ml, 107 ng / ml, 108 ng / ml or 109 ng / ml.

[0207] In some embodiments, the pluripotent cells are contacted with the Wnt signaling pathway activator at a concentration of 1.4 µg / ml. In some embodiments, the pluripotent cells are contacted with the Wnt signaling pathway activator at a concentration of 14 µg / ml. In some embodiments, the pluripotent cells are contacted with the Wnt signaling pathway activator at a concentration of 2 µg / ml, 3 µg / ml, 4 µg / ml, 5 µg / ml, 6 µg / ml, 7 µg / ml, 8 µg / ml, 9 µg / ml, 10 µg / ml, 11 µg / ml, 12, or 13 µg / ml. In some embodiments, the pluripotent cells are contacted with the Wnt signaling pathway activator at a concentration of 15 µg / ml, 16 µg / ml, 17 µg / ml, 18 µg / ml, 19 µg / ml, 20 µg / ml, 21 µg / ml, 22 µg / ml, 23 µg / ml, 24 µg / ml, 25 µg / ml, 26 µg / ml, 27 µg / ml, 28 µg / ml, 29 µg / ml, or 30 µg / ml.In some embodiments, the pluripotent cells are contacted with the Wnt signaling pathway activator at a concentration of 13.1 µg / ml, 13.2 µg / ml, 13.3 µg / ml, 13.4 µg / ml, 13.5 µg / ml, 13.6 µg / ml, 13.7 µg / ml, 13.8 µg / ml, or 13.9 µg / ml. In some embodiments, the pluripotent cells are contacted with the Wnt signaling pathway activator at a concentration of 14.1 µg / ml, 14.2 µg / ml, 14.3 µg / ml, 14.4 µg / ml, 14.5 µg / ml, 14.6 µg / ml, 14.7 µg / ml, 14.8 µg / ml, or 14.9 µg / ml.

[0208] The pluripotent cells are generally maintained in a suitable culture medium (e.g., a suspension culture) for a period of time sufficient to induce differentiation of at least some of the pluripotent cells into definitive endoderm cells. An exemplary suitable culture medium is shown in Table 1 below. Table 1 Agenz Menge MCDB131 1 l Glukose 0,44 NaHCO3 2,46 g FAF-BSA 20 g IST-X 20 µl Glutamax 10 ml Vitamin C 0.044 g Heparin 0 g P / S 10 ml

[0209] In some embodiments, a suitable culture medium for differentiating pluripotent cells into definitive endoderm cells may comprise SI media.

[0210] In some embodiments, the contacting of the pluripotent cells is performed in suspension culture. In some embodiments, the suspension culture is maintained in a spinner flask. In some embodiments, the period is 3 days. In some embodiments, the at least one growth factor from the TGF-β superfamily and the Wnt signaling pathway activator are added to the suspension culture on the first day. In some embodiments, the at least one growth factor from the TGF-β superfamily is added to the suspension culture on the second day. In some embodiments, the Wnt signaling pathway activator is added to the suspension culture on the second day. In some embodiments, the Wnt signaling pathway activator is removed from the suspension culture on the second day.In some embodiments, the at least one growth factor from the TGF-β superfamily is added to the suspension culture on the second day, and the Wnt signaling pathway activator is removed from the suspension culture or not added to the suspension culture on the second day. In some embodiments, neither the at least one growth factor from the TGF-β superfamily nor the Wnt signaling pathway activator is added to the suspension culture on the third day. In some embodiments, both the at least one growth factor from the TGF-β superfamily and the Wnt signaling pathway activator are removed from the suspension culture on the third day.

[0211] The methods can induce differentiation of at least one pluripotent cell in a cell population into a definitive endoderm cell. Generally, any pluripotent cell can be differentiated into a definitive endoderm cell using a method described herein. In some embodiments, the pluripotent cells comprise pluripotent stem cells. In some embodiments, the pluripotent cells comprise embryonic stem cells. In some embodiments, the pluripotent cells comprise human cells.

[0212] In some embodiments, differentiation of at least some pluripotent cells in a population into definitive endoderm cells is achieved by a method of contacting a population of pluripotent cells with i) activin A, and ii) CHIR99021 to induce differentiation of at least some of the pluripotent cells in the population into definitive endoderm cells, wherein the definitive endoderm cells express at least one marker characteristic of definitive endoderm.

[0213] Other methods for producing definitive endoderm cells are known in the art, including, for example, the methods further disclosed in published U.S. applications US2006 / 0.003.446 to G. Keller, et al.; US2006 / 0.003.313 to K. D'Amour et al.; US2005 / 0.158.853 to K. D'Amour et al.; and US2005 / 0.260.749 to Jon Odorico, et al., the relevant portions of which are incorporated herein by reference.

[0214] In some embodiments, a definitive endoderm cell produced by the disclosed methods expressed at least one marker selected from the group consisting of: Nodal, TMPRSS2, Tmem30b, Stl 4 Spink3, Sh3g12, Ripk4, Rabl 5, NPNT, Clic6, Cldn8, Cacnal b, Bnipl, Anxa4, EMB FOXA 1, Soxl 7, and Rbm35a, wherein the expression of the at least one marker was statistically significantly upregulated in the definitive endoderm cell relative to the pluripotent stem cell from which it was derived. In some embodiments, a definitive endoderm cell produced by a method disclosed herein does not express a statistically significant amount of at least one marker selected from the group consisting of: Gata4, SPARC, AFP, and DAB2, relative to the pluripotent stem cell from which it was derived.In some embodiments, a final endoderm cell produced by the disclosed methods does not express a statistically significant amount of at least one marker selected from the group consisting of: Zicl, Pax6, Flkl, and CD31 relative to the pluripotent stem cell from which it was derived.

[0215] In some embodiments, a definitive endoderm cell produced by the methods disclosed herein has a statistically significantly higher level of phosphorylation of Smad2 than the pluripotent stem cell from which it was derived. In some embodiments, a definitive endoderm cell produced by the methods disclosed herein has the ability to form an archenteron in vivo. In some embodiments, a definitive endoderm cell produced by the methods disclosed herein can differentiate into a cell having characteristic morphology of an intestinal cell, wherein a cell having characteristic morphology of an intestinal cell expresses FOXA2 and / or Claudin6. In some embodiments, a definitive endoderm cell produced by the methods disclosed herein can be further differentiated into a cell of endoderm origin.

[0216] In some embodiments, a population of pluripotent stem cells is cultured prior to differentiation or during the first stage of differentiation in the presence of at least one β-cell maturation factor. Any pluripotent stem cell may be used, such as human pluripotent stem cells or human iPS cells or any of the pluripotent stem cells described herein, or other suitable pluripotent stem cells. In some embodiments, a β-cell maturation factor as described herein may be present in the culture medium of a population of pluripotent stem cells, or added as a bolus or periodically during growth (e.g., replication or expansion) of the population of pluripotent stem cells. In certain examples, a population of pluripotent stem cells may be exposed to at least one β-cell maturation factor prior to differentiation.In other examples, a population of pluripotent stem cells may be exposed to at least one β-cell maturation factor during the first stage of differentiation. Induction of differentiation of definitive endoderm cells to archenteron cells

[0217] Aspects of the disclosure include archenteron cells. Archenteron cells used herein may be derived from any source or generated in accordance with any suitable protocol. In some aspects, the definitive endoderm cells are differentiated into archenteron cells. In some aspects, the archenteron cells are further differentiated, e.g., into PDX-1-positive pancreatic progenitor cells, NKX6-1-positive pancreatic progenitor cells, Ngn3-positive endocrine progenitor cells, insulin-positive endocrine cells, followed by induction or maturation into SC-β cells.

[0218] In some embodiments, archenteron cells can be obtained by differentiating at least some definitive endoderm cells in a population into archenteron cells, for example by contacting definitive endoderm cells with at least one growth factor from the fibroblast growth factor (FGF) family to induce differentiation of at least some of the definitive endoderm cells into archenteron cells, wherein the archenteron cells express at least one marker characteristic of archenteron cells.

[0219] The disclosure relates to the use of any growth factor from the FGF family to induce differentiation of definitive endoderm cells into archaeal tube cells (e.g., alone or in combination with other factors). In some embodiments, the at least one growth factor from the FGF family comprises keratinocyte growth factor (KGF). In some embodiments, the at least one growth factor from the FGF family comprises FGF2. In some embodiments, the at least one growth factor from the FGF family comprises FGF8b. In some embodiments, the at least one growth factor from the FGF family comprises FGF10. In some embodiments, the at least one growth factor from the FGF family comprises FGF21.

[0220] It will be appreciated by those skilled in the art that the concentrations of the growth factors used may vary. In some embodiments, the final endoderm cells are contacted with the at least one growth factor from the FGF family at a concentration of between 5 ng / ml and 500 ng / ml. In some embodiments, the final endoderm cells are contacted with the at least one growth factor from the FGF family at a concentration of 10 ng / ml, 15 ng / ml, 20 ng / ml, 25 ng / ml, 30 ng / ml, 35 ng / ml, or 40 ng / ml. In some embodiments, the final endoderm cells are contacted with the at least one growth factor from the FGF family at a concentration of 60 ng / ml, 65 ng / ml, 70 ng / ml, 75 ng / ml, 80 ng / ml, 85 ng / ml, 90 ng / ml, 95 ng / ml or 100 ng / ml.In some embodiments, the final endoderm cells are contacted with the at least one growth factor from the FGF family at a concentration of 41 ng / ml, 42 ng / ml, 43 ng / ml, 44 ng / ml, 45 ng / ml, 46 ng / ml, 47 ng / ml, 48 ng / ml, or 49 ng / ml. In some embodiments, the final endoderm cells are contacted with the at least one growth factor from the FGF family at a concentration of 51 ng / ml, 52 ng / ml, 53 ng / ml, 54 ng / ml, 55 ng / ml, 56 ng / ml, 57 ng / ml, 58 ng / ml, or 59 ng / ml. In some embodiments, the final endoderm cells are contacted with the at least one growth factor from the FGF family at a concentration of 50 ng / ml.

[0221] In some embodiments, the final endoderm cells are grown in a suitable culture medium.

[0222] Generally, the definitive endoderm cells are maintained in a suitable culture medium (e.g., a suspension culture) for a period of time sufficient to induce differentiation of at least some of the definitive endoderm cells into archenteron cells. An exemplary suitable culture medium is shown in Table 2 below. Table 2 Agenz Menge MCDB131 1 l Glukose 0,44 g NaHCO3 1,23 g FAF-BSA 20 g IST-X 20 µl Glutamax 10 ml Vitamin C 0.044 g Heparin 0 g P / S 10 ml

[0223] In some embodiments, a suitable culture medium for the differentiation of definitive endoderm cells to archenteron cells may comprise S2 media.

[0224] In some embodiments, contacting the final endoderm cells is performed in suspension culture. In some embodiments, the suspension culture is maintained in a spinner flask. In some embodiments, the period is between 2 days and 5 days. In some embodiments, the period is 3 days. In some embodiments, the suspension culture is renewed every other day.

[0225] In some embodiments, definitive endoderm cells can be obtained by differentiating at least a portion of the definitive endoderm cells in a population into archenteron cells, for example, by contacting the definitive endoderm cells with KGF to induce differentiation of at least some of the definitive endoderm cells into archenteron cells, wherein the archenteron cells express at least one marker characteristic of definitive endoderm. Induction of differentiation of archenteron cells into Pdxl-positive pancreatic progenitor cells

[0226] Aspects of the disclosure include Pdxl-positive pancreatic progenitor cells. Pdxl-positive pancreatic progenitor cells as used herein may be derived from any source or generated in accordance with any suitable protocol. In some aspects, archenteron cells are differentiated into Pdxl-positive pancreatic progenitor cells. In some aspects, the Pdxl-positive pancreatic progenitor cells are further differentiated, e.g., into NKX6-1-positive pancreatic progenitor cells, Ngn3-positive endocrine progenitor cells, insulin-positive endocrine cells, followed by induction or maturation into SC-β cells.

[0227] In some aspects, Pdxl-positive pancreatic progenitor cells can be obtained by differentiating at least some of the archenteron cells in a population into Pdxl-positive pancreatic progenitor cells, for example, by contacting the archenteron cells with (i) at least one bone morphogenesis protein (BMP) pathway inhibitor, ii) at least one growth factor from the FGF family, iii) at least one SHH pathway inhibitor, iv) at least one retinoic acid (RA) pathway activator; and v) at least one protein kinase C activator, to induce the differentiation of at least some of the archenteron cells into Pdxl-positive pancreatic progenitor cells, wherein the Pdxl-positive pancreatic progenitor cells express.

[0228] The disclosure relates to the use of any BMP signaling pathway inhibitor capable of inducing differentiation of archaeal tubule cells into Pdxl-positive pancreatic progenitor cells (e.g., alone or with any combination of at least one growth factor from the FGF family, at least one SHH inhibitor, at least one retinoic acid signaling pathway activator, and at least one protein kinase C activator). In some embodiments, the BMP signaling pathway inhibitor comprises LDN 193 1 89.

[0229] The disclosure relates to the use of any growth factor from the FGF family with which differentiation of archenteron cells into Pdxl-positive pancreatic progenitor cells can be induced (for example, alone or with any combination of at least one BMP signaling pathway inhibitor, at least one SHH inhibitor, at least one retinoic acid signaling pathway activator, and at least one protein kinase C activator). In some embodiments, the at least one growth factor from the FGF family comprises keratinocyte growth factor (GF). In some embodiments, the at least one growth factor from the FGF family is selected from the group consisting of FGF2, FGF8b, FGF10, FGF21.

[0230] The disclosure relates to the use of any SHH pathway inhibitor that induces differentiation of archaeal tubule cells into Pdxl-positive pancreatic progenitor cells (e.g., alone or with any combination of at least one BMP pathway inhibitor, at least one growth factor from the FGF family, at least one retinoic acid pathway activator, and at least one protein kinase C activator). In some embodiments, the SFIH pathway inhibitor comprises Santl.

[0231] The disclosure relates to the use of any RA pathway activator to induce differentiation of archilecinal tubule cells into Pdxl-positive pancreatic progenitor cells (e.g., alone or with any combination of at least one BMP pathway inhibitor, at least one FGF family growth factor, at least one SHH pathway inhibitor, and at least one protein kinase C activator). In some embodiments, the RA pathway activator comprises retinoic acid.

[0232] The disclosure relates to the use of any PKC activator to induce differentiation of archilecinal tube cells into Pdxl-positive pancreatic progenitor cells (e.g., alone or with any combination of at least one BMP pathway inhibitor, at least one growth factor from the FGF family, at least one SHH pathway inhibitor, and at least one RA pathway activator). In some embodiments, the PKC activator comprises PDBU. In some embodiments, the PKC activator comprises TPB.

[0233] It will be appreciated by those skilled in the art that the concentrations of the agents used (e.g., growth factors) may vary. In some embodiments, the archenteron cells are contacted with the BMP pathway inhibitor at a concentration of 20 nM. In some embodiments, the archenteron cells are contacted with the BMP pathway inhibitor at a concentration of 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 100 nM, 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180 nM, or 190 nM. In some embodiments, the archenteron cells are contacted with the BMP pathway inhibitor at a concentration of 191 nM, 192 nM, 193 nM, 194 nM, 195 nM, 196 nM, 197 nM, 198 nM, or 199 nM.In some embodiments, the archenteron cells are contacted with the BMP pathway inhibitor at a concentration of 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1000 nM, 1200 nM, 1300 nM, 1400 nM, 1500 nM, 1600 nM, 1700 nM, 1800 nM, or 1900 nM. In some embodiments, the archenteron cells are contacted with the BMP pathway inhibitor at a concentration of 210 nM, 220 nM, 230 nM, 240 nM, 250 nM, 260 nM, 270 nM, 280 nM, or 290 nM. In some embodiments, the archenteron cells are contacted with the BMP pathway inhibitor at a concentration of 200 nM.

[0234] In some embodiments, the archenteron cells are contacted with the at least one growth factor from the FGF family at a concentration between 5 ng / ml - 500 ng / ml. In some embodiments, the archenteron cells are contacted with the at least one growth factor from the FGF family at a concentration of 10 ng / ml, 15 ng / ml, 20 ng / ml, 25 ng / ml, 30 ng / ml, 35 ng / ml or 40 ng / ml. In some embodiments, the archenteron cells are contacted with the at least one growth factor from the FGF family at a concentration of 60 ng / ml, 65 ng / ml, 70 ng / ml, 75 ng / ml, 80 ng / ml, 85 ng / ml, 90 ng / ml, 95 ng / ml or 100 ng / ml. In some embodiments, the archenteron cells are contacted with the at least one growth factor from the FGF family at a concentration of 41 ng / ml, 42 ng / ml, 43 ng / ml, 44 ng / ml, 45 ng / ml, 46 ng / ml, 47 ng / ml, 48 ng / ml or 49 ng / ml.In some embodiments, the archenteron cells are contacted with the at least one growth factor from the FGF family at a concentration of 51 ng / ml, 52 ng / ml, 53 ng / ml, 54 ng / ml, 55 ng / ml, 56 ng / ml, 57 ng / ml, 58 ng / ml, or 59 ng / ml. In some embodiments, the archenteron cells are contacted with the at least one growth factor from the FGF family at a concentration of 50 ng / ml.

[0235] In some embodiments, the archenteron cells are contacted with the at least one SHH signaling pathway inhibitor at a concentration of between 0.1 µM and 0.5 µM. In some embodiments, the archenteron cells are contacted with the at least one SHH signaling pathway inhibitor at a concentration of 0.11 µM, 0.12 µM, 0.13 µM, 0.14 µM, 0.15 µM, 0.16 µM, 0.17 µM, 0.18 µM, 0.19 µM, 0.2 µM, 0.21 µM, 0.22 µM, 0.23 µM, or 0.24 µM. In some embodiments, the archenteron cells are treated with the at least one SHH signaling pathway inhibitor at a concentration of 0.26 µM, 0.27 µM, 0.28 µM, 0.29 µM, 0.30 µM, 0.31 µM, 0.32 µM, 0.33 µM, 0.34 µM, 0.35 µM, 0.36 µM, 0.37 µM, 0.38 µM, 0.39 µM, 0.40 µM, 0.41 µM, 0.42 µM, 0.43 µM, 0.44 µM, 0.45 µM, 0.46 µM, 0.47 µM, 0.48 µM, 0.49 µM. In some embodiments, the archenteron cells are contacted with the at least one SHH signaling pathway inhibitor at a concentration of 0.25 µM.

[0236] In some embodiments, the archenteron cells are contacted with the RA pathway activator at a concentration of between 0.01 µM and 1.0 µM. In some embodiments, the archenteron cells are contacted with the RA pathway activator at a concentration of 0.02 µM, 0.03 µM, 0.04 µM, 0.05 µM, 0.06 µM, 0.07 µM, 0.08 µM, or 0.09 µM. In some embodiments, the archenteron cells are contacted with the RA signaling pathway activator at a concentration of 0.20 µM, 0.30 µM, 0.40 µM, 0.05 µM, 0.60 µM, 0.70 µM, 0.80 µM, or 0.90 µM. In some embodiments, the archenteron cells are contacted with the RA signaling pathway activator at a concentration of 0.1 µM.

[0237] In some embodiments, the archenteron cells are contacted with a PKC activator at a concentration of 50 nM. In some embodiments, the archenteron cells are contacted with the PKC activator at a concentration of 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 460 nM, 470 nM, 480 nM, or 490 nM. In some embodiments, the archenteron cells are contacted with the PKC activator at a concentration of 491 nM, 492 nM, 493 nM, 494 nM, 495 nM, 496 nM, 497 nM, 498 nM, or 499 nM. In some embodiments, the archenteron cells are contacted with the PKC activator at a concentration of 600 nM, 700 nM, 800 nM, 900 nM, 1000 nM, 1100 nM, 1200 nM, 1300 nM, 1400 nM, 1500 nM, 1600 nM, 1700 nM, 1800 nM, 1900 nM, or 2000 nM.In some embodiments, the archenteron cells are contacted with the PKC activator at a concentration of 501 nM, 502 nM, 503 nM, 504 nM, 505 nM, 506 nM, 507 nM, 508 nM, or 509 nM, 510 nM, 520 nM, 530 nM, 540 nM, 550 nM, 560 nM, 570 nM, 580 nM, or 590 nM, respectively. In some embodiments, the archenteron cells are contacted with the PKC activator at a concentration of 500 nM.

[0238] Generally, the archenteron cells are maintained in a suitable culture medium for a period of time sufficient to induce differentiation of at least some of the archenteron cells into Pdxl-positive pancreatic progenitor cells (e.g., in suspension culture). An exemplary suitable culture medium is shown in Table 3 below. Table 3 Agenz Menge MCDB131 1 l Glukose 0,44 NaHCO3 1,23 FAF-BSA 20 g IST-X 5 ml Glutamax 10 ml Vitamin C 0.044 g Heparin 0 g P / S 10 ml

[0239] In some embodiments, S3 media can be used as a suitable culture medium for differentiating archenteron cells into pancreatic progenitor cells.

[0240] In some embodiments, contacting the archenteron cells can be performed in suspension culture. In some embodiments, the suspension culture is maintained in a spinner flask. In some embodiments, the period is at least 2 days. In some embodiments, the suspension culture is replenished daily.

[0241] In some embodiments, archenteron cells can be obtained by differentiating at least some of the archenteron cells into a population of Pdxl-positive pancreatic progenitor cells, for example, by contacting the archenteron cells with i) LDN 193 189, ii) KGF, iii) Santl; iv) RA; and iv) PDBu, which induce differentiation of at least some of the archenteron cells into Pdxl-positive pancreatic progenitor cells, wherein the Pdxl-positive pancreatic progenitor cells express Pdxl. Induction of differentiation of Pdxl-positive pancreatic progenitor cells to NKX6-1 + pancreatic progenitor cells

[0242] Aspects of the disclosure include NKX6-1-positive pancreatic progenitor cells. NKX6-1-positive pancreatic progenitor cells as used herein may be derived from any source or generated in accordance with any suitable protocol. In some aspects, Pdxl-positive pancreatic progenitor cells are differentiated into NKX6-1-positive pancreatic progenitor cells. In some aspects, the NKX6-1-positive pancreatic progenitor cells are further differentiated, for example, into Ngn3-positive endocrine progenitor cells or insulin-positive endocrine cells, followed by induction or maturation into SC-β cells.

[0243] In some aspects, a method for producing an NKX6-1-positive pancreatic progenitor cell from a Pdxl-positive pancreatic progenitor cell comprises contacting a cell population (e.g., under conditions that promote the formation of cell clumps) comprising Pdxl-positive pancreatic progenitor cells with at least two β-cell maturation factors comprising a) at least one growth factor from the fibroblast growth factor (FGF) family, b) a sonic hedgehog signaling pathway inhibitor, and optionally c) a low concentration of retinoic acid (RA)-positive pancreatic progenitor cells, for a period of at least five days to induce the differentiation of at least one Pdxl-positive pancreatic progenitor cell in the population into NKX6-1-positive pancreatic progenitor cells, wherein the NKX6-1-positive pancreatic progenitor cell expresses NKX6-1.

[0244] In some embodiments, the Pdxl-positive, NKX6-1-positive pancreatic progenitor cells are obtained by contacting Pdxl-positive pancreatic progenitor cells under conditions that promote cell clump formation with i) at least one growth factor from the FGF family, ii) at least one SHH signaling pathway inhibitor, and optionally iii) a low concentration of an RA signaling pathway activator, for a period of five days to induce differentiation of at least some of the Pdxl-positive pancreatic progenitor cells into Pdxl-positive, NKX6-1-positive pancreatic progenitor cells, wherein the Pdxl-positive, NKX6-1-positive pancreatic progenitor cells express Pdxl and NX6-1.

[0245] In some embodiments, the Pdxl-positive, NX6-1-positive pancreatic progenitor cells are obtained by contacting the Pdxl-positive pancreatic progenitor cells with i) at least one growth factor from the FGF family, ii) at least one SHH signaling pathway inhibitor, and iii) an RA signaling pathway activator to induce differentiation of at least some of the Pdxl-positive pancreatic progenitor cells into Pdxl-positive, NKX6-1-positive pancreatic progenitor cells, wherein the Pdxl-positive, NKX6-1-positive pancreatic progenitor cells express Pdxl and NKX6-1.

[0246] In some embodiments, the Pdxl-positive pancreatic progenitor cells are obtained from a population of pluripotent cells. In some embodiments, the Pdxl-positive pancreatic progenitor cells are produced from a population of iPS cells. In some embodiments, the Pdxl-positive pancreatic progenitor cells are produced from a population of ESC cells. In some embodiments, the Pdxl-positive pancreatic progenitor cells are produced from a population of definitive endoderm cells. In some embodiments, the Pdxl-positive pancreatic progenitor cells are produced from a population of archenteron cells.

[0247] The disclosure relates to the use of any growth factor from the FGF family with which differentiation of Pdxl-positive pancreatic progenitor cells into NKX6-1-positive pancreatic progenitor cells can be induced (for example, alone or with any combination of at least one SHH pathway inhibitor or optionally at least one retinoic acid pathway activator). In some embodiments, the at least one growth factor from the FGF family comprises keratinocyte growth factor (KGF). In some embodiments, the at least one growth factor from the FGF family is selected from the group consisting of FGF2, FGF8b, FGF10, FGF21.

[0248] The disclosure relates to the use of any SHH signaling pathway inhibitor capable of inducing differentiation of Pdxl-positive pancreatic progenitor cells into NKX6-1-positive pancreatic progenitor cells (e.g., alone or with any combination of at least one growth factor from the FGF family or at least one retinoic acid signaling pathway activator). In some embodiments, the SHH signaling pathway inhibitor comprises Santl.

[0249] The disclosure relates to the use of any RA signaling pathway activator capable of inducing differentiation of Pdxl-positive pancreatic progenitor cells into NX6-1-positive pancreatic progenitor cells (e.g., alone or with any combination of at least one FGF-family growth factor and at least one SHH signaling pathway inhibitor). In some embodiments, the RA signaling pathway activator comprises retinoic acid.

[0250] In some embodiments, the method comprises contacting the cell population (e.g., Pdxl-positive pancreatic progenitor cells) with at least one additional β-cell maturation factor. In some embodiments, the at least one additional β-cell maturation factor comprises at least one growth factor from the EGF family. In some embodiments, the method comprises contacting the Pdxl-positive pancreatic progenitor cells with at least one growth factor from the EGF family. The disclosure relates to the use of any growth factors from the EGF family to enable differentiation of Pdxl-positive pancreatic progenitor cells into NX6-1-positive pancreatic progenitor cells (e.g., together with any combination of at least one growth factor from the FGF family, at least one SHH signaling pathway inhibitor, and optionally at least one RA signaling pathway activator).In some embodiments, the at least one growth factor from the EGF family comprises betacellulin. In some embodiments, the at least one growth factor from the EGF family comprises EGF.

[0251] It will be appreciated by those skilled in the art that the concentrations of the agents used (e.g., growth factors) may vary. In some embodiments, the Pdxl-positive pancreatic progenitor cells are contacted with the at least one growth factor from the FGF family at a concentration of 1 ng / ml - 100 ng / ml. In some embodiments, the Pdxl-positive pancreatic progenitor cells are contacted with the at least one growth factor from the FGF family at a concentration of 5 ng / ml, 10 ng / ml, 15 ng / ml, 20 ng / ml, 25 ng / ml, 30 ng / ml, 35 ng / ml, or 40 ng / ml. In some embodiments, the Pdxl-positive pancreatic progenitor cells are contacted with the at least one growth factor from the FGF family at a concentration of 60 ng / ml, 65 ng / ml, 70 ng / ml, 75 ng / ml, 80 ng / ml, 85 ng / ml, 90 ng / ml, 95 ng / ml or 100 ng / ml.In some embodiments, the Pdxl-positive pancreatic progenitor cells are contacted with the at least one growth factor from the FGF family at a concentration of 41 ng / ml, 42 ng / ml, 43 ng / ml, 44 ng / ml, 45 ng / ml, 46 ng / ml, 47 ng / ml, 48 ng / ml, or 49 ng / ml. In some embodiments, the Pdxl-positive pancreatic progenitor cells are contacted with the at least one growth factor from the FGF family at a concentration of 51 ng / ml, 52 ng / ml, 53 ng / ml, 54 ng / ml, 55 ng / ml, 56 ng / ml, 57 ng / ml, 58 ng / ml, or 59 ng / ml. In some embodiments, the Pdxl-positive pancreatic progenitor cells are contacted with the at least one growth factor from the FGF family at a concentration of 50 ng / ml.

[0252] In some embodiments, the Pdxl-positive pancreatic progenitor cells are contacted with the at least one SHH signaling pathway inhibitor at a concentration of between 0.1 µM and 0.5 µM. In some embodiments, the Pdxl-positive pancreatic progenitor cells are contacted with the at least one SHH signaling pathway inhibitor at a concentration of 0.11 µM, 0.12 µM, 0.13 µM, 0.14 µM, 0.15 µM, 0.16 µM, 0.17 µM, 0.18 µM, 0.19 µM, 0.2 µM, 0.21 µM, 0.22 µM, 0.23 µM, or 0.24 µM. In some embodiments, the Pdxl-positive pancreatic progenitor cells are treated with the at least one SHH signaling pathway inhibitor at a concentration of 0.26 µM, 0.27 µM, 0.28 µM, 0.29 µM, 0.30 µM, 0.31 µM, 0.32 µM, 0.33 µM, 0.34 µM, 0.35 µM, 0.36 µM, 0.37 µM, 0.38 µM, 0.39 µM, 0.40 µM, 0.41 µM, 0.42 µM, 0.43 µM, 0.44 µM, 0.45 µM, 0.46 µM, 0.47 µM, 0.48 µM, 0.49 µM.In some embodiments, the Pdxl-positive pancreatic progenitor cells are contacted with the at least one SHH signaling pathway inhibitor at a concentration of 0.25 µM.

[0253] In some embodiments, the Pdxl-positive pancreatic progenitor cells are contacted with the RA signaling pathway activator at a concentration of between 0.01 µM. In some embodiments, the Pdxl-positive pancreatic progenitor cells are contacted with the RA signaling pathway activator at a concentration of 0.02 µM, 0.03 µM, 0.04 µM, 0.05 µM, 0.06 µM, 0.07 µM, 0.08 µM, or 0.09 µM. In some embodiments, the Pdxl-positive pancreatic progenitor cells are contacted with the RA signaling pathway activator at a concentration of 0.20 µM, 0.30 µM, 0.40 µM, 0.05 µM, 0.60 µM, 0.70 µM, 0.80 µM, or 0.90 µM. In some embodiments, the Pdxl-positive pancreatic progenitor cells are contacted with the RA signaling pathway activator at a concentration of 0.1 µM.

[0254] In some embodiments, the Pdxl-positive pancreatic progenitor cells are contacted with the at least one growth factor from the EGF family at a concentration of between 2 ng / ml - 200 ng / ml. In some embodiments, the Pdxl-positive pancreatic progenitor cells are contacted with the at least one growth factor from the EGF family at a concentration of 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, 10 ng / ml, 11 ng / ml, 12 ng / ml, 13 ng / ml, 14 ng / ml, 16 ng / ml, 17 ng / ml, 18 ng / ml or 19 ng / ml. In some embodiments, the Pdxl-positive pancreatic progenitor cells are contacted with the at least one growth factor from the EGF family at a concentration of 30 ng / ml, 35 ng / ml, 40 ng / ml, 45 ng / ml, 50 ng / ml, 55 ng / ml, 60 ng / ml, 65 ng / ml, 70 ng / ml, 75 ng / ml, 80 ng / ml, 85 ng / ml, 90 ng / ml, 95 ng / ml, or 100 ng / ml.In some embodiments, the Pdxl-positive pancreatic progenitor cells are contacted with the at least one growth factor from the EGF family at a concentration of 21 ng / ml, 22 ng / ml, 23 ng / ml, 24 ng / ml, 25 ng / ml, 26 ng / ml, 27 ng / ml, 28 ng / ml, or 29 ng / ml. In some embodiments, the Pdxl-positive pancreatic progenitor cells are contacted with the at least one growth factor from the EGF family at a concentration of 20 ng / ml.

[0255] Generally, the Pdxl-positive pancreatic progenitor cells are maintained in a suitable culture medium for a period of time sufficient to induce differentiation of at least some of the Pdxl-positive pancreatic progenitor cells in the population into Pdxl-positive, NKX6-1-positive pancreatic progenitor cells. An exemplary suitable culture medium is shown in Table 3, above. In some embodiments, the conditions promoting cell aggregation comprise a suspension culture. In some embodiments, the suspension culture is maintained in a spinner flask. In some embodiments, the period is at least 5 days. In some embodiments, the suspension culture is renewed every other day. In some embodiments, the β-cell maturation factors are renewed every two days.

[0256] In some embodiments, no protein kinase C activator is added to the suspension culture for 5 days. In some embodiments, a protein kinase C activator is removed from the suspension culture prior to the 5 days. In some embodiments, the protein kinase C activator comprises PDBU. In some embodiments, no BMP signaling pathway inhibitor is added to the suspension culture for 5 days. In some embodiments, a BMP signaling pathway inhibitor is removed from the suspension culture prior to the 5 days. In some embodiments, the BMP signaling pathway inhibitor comprises LDN 193189.

[0257] In some embodiments, differentiation of at least 10% of the Pdxl-positive pancreatic progenitor cells in the population is induced to differentiate into Pdxl-positive, NKX6-1-positive pancreatic progenitor cells. In some embodiments, differentiation of at least 95% of the Pdxl-positive pancreatic progenitor cells is induced to differentiate into Pdxl-positive, NKX6-1-positive pancreatic progenitor cells.

[0258] In general, any Pdxl-positive pancreatic progenitor cells can be differentiated into a Pdxl-positive, NKX6-1-positive pancreatic progenitor cell. In some embodiments, the NKX6-1-positive pancreatic progenitor cells express Pdxl, NKX6-1, and / or FOXA2.

[0259] In some embodiments, the Pdxl-positive, NKX6-1-positive pancreatic progenitor cells can be obtained by contacting the Pdxl-positive pancreatic progenitor cells under conditions that promote the formation of cell clumps with i) at least one growth factor from the FGF family, ii) at least one SHH signaling pathway inhibitor, and optionally iii) a low concentration of an RA signaling pathway activator for a period of five days to induce the differentiation of at least some of the Pdxl-positive pancreatic progenitor cells into Pdxl-positive, NKX6-1-positive pancreatic progenitor cells, wherein the Pdxl-positive, NKX6-1-positive pancreatic progenitor cells express Pdxl and NKX6-1.

[0260] In some embodiments, the NKX6-1-positive pancreatic progenitor cells can be obtained by differentiating at least some of the Pdxl-positive pancreatic progenitor cells into Pdxl-positive, NKX6-1-positive pancreatic progenitor cells by a method of contacting the Pdxl-positive pancreatic progenitor cells under conditions that promote the formation of cell clumps with i) at least one growth factor of the FGF family, ii) at least one SHH signaling pathway inhibitor, and optionally iii) an RA signaling pathway activator, every other day for a period of five days to induce the differentiation of at least some of the Pdxl-positive pancreatic progenitor cells in the population into NKX6-1-positive pancreatic progenitor cells, wherein the NKX6-1-positive pancreatic progenitor cells express Pdxl and NKX6.

[0261] In some embodiments, the NX6-1-positive pancreatic progenitor cells can be obtained by differentiating at least some of the Pdxl-positive pancreatic progenitor cells in a population into Pdxl-positive, NKX6-1-positive pancreatic progenitor cells, e.g., by contacting the Pdxl-positive pancreatic progenitor cells with i) at least one growth factor from the FGF family, ii) at least one SHH signaling pathway inhibitor, and optionally iii) an RA signaling pathway activator, to induce differentiation of at least some of the Pdxl-positive pancreatic progenitor cells in the population into NX6-1-positive pancreatic progenitor cells, wherein the NKX6-1-positive pancreatic progenitor cells express Pdxl and NKX6-1. Induction of differentiation of NKX6-1 + pancreatic progenitor cells into insulin + endocrine cells

[0262] Aspects of the disclosure encompass insulin-positive endocrine cells. Insulin-positive cells as used herein may be derived from any source or generated in accordance with any suitable protocol. In some aspects, NKX6-1-positive pancreatic progenitor cells are differentiated into insulin-positive endocrine cells. In some aspects, the insulin-positive endocrine cells are further differentiated, for example, by induction or maturation into SC-β cells.

[0263] In some aspects, a method for producing an insulin-positive endocrine cell from an NKX6-1-positive pancreatic progenitor cell comprises contacting a cell population containing NKX6-1-positive pancreatic progenitor cells (e.g., under conditions that promote the formation of cell clumps) with at least two β-cell maturation factors comprising a) a TGF-β signaling pathway inhibitor and b) a thyroid hormone signaling pathway activator to induce differentiation of at least one NKX6-1-positive pancreatic progenitor cell in the population into an insulin-positive endocrine cell, wherein the insulin-positive pancreatic progenitor cell expresses insulin.

[0264] The invention relates to the use of any TGF-β signaling pathway inhibitor capable of inducing the differentiation of NKX6-1-positive pancreatic progenitor cells into insulin-positive endocrine cells (e.g., alone or in combination with other β-cell maturation factors, such as a thyroid hormone signaling pathway activator). In some embodiments, the TGF-β signaling pathway comprises the TGF-β receptor type I kinase signaling pathway. In some embodiments, the TGF-β signaling pathway inhibitor comprises the ALK5 inhibitor II.

[0265] The invention relates to the use of any thyroid hormone signaling pathway activator capable of inducing the differentiation of NKX6-1-positive pancreatic progenitor cells into insulin-positive endocrine cells (e.g., alone or in combination with other β-cell maturation factors, such as a TGF-β signaling pathway inhibitor). In some embodiments, the thyroid hormone signaling pathway activator comprises triiodothyronine (T3).

[0266] In some embodiments, the method comprises contacting the cell population (e.g., the NKX6-1-positive pancreatic progenitor cells) with at least one additional β-cell maturation factor. In some embodiments, the method comprises contacting the Pdxl-positive NKX6-1-positive pancreatic progenitor cells with at least i) an SHH signaling pathway inhibitor, ii) an RA signaling pathway activator, iii) a γ-secretase inhibitor, iv) at least one growth factor from the epidermal growth factor (EGF) family, and optionally v) a protein kinase inhibitor.

[0267] In some embodiments, the at least one additional β-cell maturation factor comprises a γ-secretase inhibitor. The disclosure provides the use of any γ-secretase inhibitor suitable for inducing the differentiation of NKX6-1-positive pancreatic progenitor cells in a population into insulin-positive endocrine cells (e.g., alone or in combination with any TGF-β signaling pathway inhibitor and / or a thyroid hormone signaling pathway activator). In some embodiments, the γ-secretase inhibitor comprises XXI. In some embodiments, the γ-secretase inhibitor comprises DAPT.

[0268] In some embodiments, the at least one additional β-cell maturation factor comprises at least one growth factor from the EGF family. The disclosure provides the use of any growth factors from the EGF family suitable for inducing the differentiation of NX6-1-positive pancreatic progenitor cells in a population into insulin-positive endocrine cells (e.g., alone or in combination with any of a TGF-β signaling pathway inhibitor and / or a thyroid hormone signaling pathway activator). In some embodiments, the at least one growth factor from the EGF family comprises betacellulin. In some embodiments, the one growth factor from the EGF family comprises EGF.

[0269] In some embodiments, the at least one additional β-cell maturation factor comprises a low concentration of a retinoic acid (RA) pathway activator. The disclosure provides the use of any RA pathway activator capable of inducing the differentiation of NKX6-1-positive pancreatic progenitor cells into insulin-positive endocrine cells (e.g., alone or in combination with one of a TGF-β pathway inhibitor and / or a thyroid hormone pathway activator). In some embodiments, the RA pathway activator comprises RA.

[0270] In some embodiments, the at least one additional β-cell maturation factor comprises a Sonic Hedgehog (SHH) signaling pathway inhibitor. The disclosure provides the use of any SHH inhibitor that induces the differentiation of NKX6-1-positive pancreatic progenitor cells into insulin-positive endocrine cells (e.g., alone or in combination with one of a TGF-β signaling pathway inhibitor and / or a thyroid hormone signaling pathway activator). In some embodiments, the SHH signaling pathway inhibitor comprises Santl.

[0271] In some embodiments, the cell population (e.g., NKX6-1 positive pancreatic progenitor cells) is exposed to glucose.

[0272] In some embodiments, the cell population is optionally contacted with a protein kinase inhibitor. In some embodiments, the cell population is not contacted with the protein kinase inhibitor. In some embodiments, the cell population is contacted with the protein kinase inhibitor. The disclosure encompasses the use of any protein kinase inhibitor capable of inducing the differentiation of pancreatic progenitor NKX6-1-positive cells in a population into insulin-positive endocrine cells (e.g., alone or in combination with one of a TGF-β signaling pathway inhibitor and / or a thyroid hormone signaling pathway activator). In some embodiments, the protein kinase inhibitor comprises staurosporine.

[0273] In some embodiments, the insulin-positive endocrine cells are obtained by contacting Pdxl-positive, NKX6-1-positive pancreatic progenitor cells with at least one SHH signaling pathway inhibitor, ii) an RA signaling pathway activator, iii) a γ-secretase inhibitor, iv) a TGF-β signaling pathway inhibitor, v) a TH signaling pathway activator, and vi) at least one growth factor from the EGF family of epidermal growth factor, to induce the differentiation of at least some of the Pdxl-positive, NKX6-1-positive pancreatic progenitor cells into Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells, wherein the Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells are Pdxl, NKX6-1, NKX2-2, MAFB, express glis3, Surl, ir6.2, ZnT8, SLC2A 1, SLC2A3 and / or insulin.

[0274] It is clear to the person skilled in the art that the concentrations of the agents used (e.g. growth factors) may vary.

[0275] In some embodiments, the NKX6-1-positive pancreatic progenitor cells are contacted with the at least one TGF-β signaling pathway inhibitor at a concentration of between 100 nM - 100 µM. In some embodiments, the NKX6-1-positive pancreatic progenitor cells are contacted with the at least one TGF-β signaling pathway inhibitor at a concentration of 10 µM. In some embodiments, the NKX6-1-positive pancreatic progenitor cells are contacted with the at least one TGF-β signaling pathway inhibitor at a concentration of 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, or 900 nM. In some embodiments, the NKX6-1 positive pancreatic progenitor cells are contacted with the at least one TGF-β signaling pathway inhibitor at a concentration of 2 µM, 3 µM, 4 µM, 5 µM, 6 µM, 7 µM, 8 µM or 9 µM.In some embodiments, the NKX6-1-positive pancreatic progenitor cells are contacted with the at least one TGF-β signaling pathway inhibitor at a concentration of 9.1 µM, 9.2 µM, 9.3 µM, 9.4 µM, 9.5 µM, 9.6 µM, 9.7 µM, 9.8 µM, or 9.9 µM. In some embodiments, the NX6-1-positive pancreatic progenitor cells are contacted with the at least one TGF-β signaling pathway inhibitor at a concentration of 11 µM, 12 µM, 13 µM, 14 µM, 15 µM, 16 µM, 17 µM, 18 µM, or 19 µM. In some embodiments, the NKX6-1 positive pancreatic progenitor cells are contacted with the at least one TGF-β signaling pathway inhibitor at a concentration of 10.1 µM, 10.2 µM, 10.3 µM, 10.4 µM, 10.5 µM, 10.6 µM, 10.7 µM, 10.8 µM or 10.9 µM.

[0276] In some embodiments, the NKX6-1-positive pancreatic progenitor cells are contacted with the thyroid hormone signaling pathway activator at a concentration of between 0.1 µM - 10 µM. In some embodiments, the NKX6-1-positive pancreatic progenitor cells are contacted with the thyroid hormone signaling pathway activator at a concentration of 1 µM. In some embodiments, the NKX6-1-positive pancreatic progenitor cells are contacted with the thyroid hormone signaling pathway activator at a concentration of 0.2 µM, 0.3 µM, 0.4 µM, 0.5 µM, 0.6 µM, 0.7 µM, 0.8 µM, or 0.9 µM. In some embodiments, the NKX6-1 positive pancreatic progenitor cells are contacted with the γ-thyroid hormone pathway activator at a concentration of 1.1 µM, 1.2 µM, 1.3 µM, 1.4 µM, 1.5 µM, 1.6 µM, 1.7 µM, 1.8 µM, or 1.9 µM.In some embodiments, the NKX6-1 positive pancreatic progenitor cells are contacted with the thyroid hormone signaling pathway activator at a concentration of 2 µM, 3 µM, 4 µM, 5 µM, 6 µM, 7 µM, 8 µM, or 9 µM.

[0277] In some embodiments, the NX6-1-positive pancreatic progenitor cells are contacted with the γ-secretase inhibitor at a concentration between 0.1 µM - 10 µM. In some embodiments, the NKX6-1-positive pancreatic progenitor cells are contacted with the γ-secretase inhibitor at a concentration of 1 µM. In some embodiments, the NKX6-1-positive pancreatic progenitor cells are contacted with the γ-secretase inhibitor at a concentration of 0.2 µM, 0.3 µM, 0.4 µM, 0.5 µM, 0.6 µM, 0.7 µM, 0.8 µM, or 0.9 µM. In some embodiments, the NKX6-1 positive pancreatic progenitor cells are contacted with the γ-secretase inhibitor at a concentration of 1.1 µM, 1.2 µM, 1.3 µM, 1.4 µM, 1.5 µM, 1.6 µM, 1.7 µM, 1.8 µM or 1.9 µM.In some embodiments, the NKX6-1 positive pancreatic progenitor cells are contacted with the γ-secretase inhibitor at a concentration of 2 µM, 3 µM, 4 µM, 5 µM, 6 µM, 7 µM, 8 µM or 9 µM.

[0278] In some embodiments, the NKX6-1-positive pancreatic progenitor cells are contacted with the at least one growth factor from the EGF family at a concentration of 2 ng / ml - 200 ng / ml. In some embodiments, the NKX6-1-positive pancreatic progenitor cells are contacted with the at least one growth factor from the EGF family at a concentration of 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, 10 ng / ml, 11 ng / ml, 12 ng / ml, 13 ng / ml, 14 ng / ml, 16 ng / ml, 17 ng / ml, 18 ng / ml, or 19 ng / ml. In some embodiments, the NKX6-1-positive pancreatic progenitor cells are contacted with the at least one growth factor from the EGF family at a concentration of 30 ng / ml, 35 ng / ml, 40 ng / ml, 45 ng / ml, 50 ng / ml, 55 ng / ml, 60 ng / ml, 65 ng / ml, 70 ng / ml, 75 ng / ml, 80 ng / ml, 85 ng / ml, 90 ng / ml, 95 ng / ml, or 100 ng / ml.In some embodiments, the NKX6-1-positive pancreatic progenitor cells are contacted with the at least one growth factor from the EGF family at a concentration of 21 ng / ml, 22 ng / ml, 23 ng / ml, 24 ng / ml, 25 ng / ml, 26 ng / ml, 27 ng / ml, 28 ng / ml, or 29 ng / ml. In some embodiments, the NKX6-1-positive pancreatic progenitor cells are contacted with the at least one growth factor from the EGF family at a concentration of 20 ng / ml.

[0279] In some embodiments, the NX6-1-positive pancreatic progenitor cells are contacted with the RA signaling pathway activator at a concentration of between 0.01 µM - 1.0 µM. In some embodiments, the NX6-1-positive pancreatic progenitor cells are contacted with the RA signaling pathway activator at a concentration of 0.02 µM, 0.03 µM, 0.04 µM, 0.05 µM, 0.06 µM, 0.07 µM, 0.08 µM, or 0.09 µM. In some embodiments, the NKX6-1-positive pancreatic progenitor cells are contacted with the RA signaling pathway activator at a concentration of 0.20 µM, 0.30 µM, 0.40 µM, 0.05 µM, 0.60 µM, 0.70 µM, 0.80 µM, or 0.90 µM. In some embodiments, the NKX6-1-positive pancreatic progenitor cells are contacted with the RA signaling pathway activator at a concentration of 0.1 µM.

[0280] In some embodiments, the NX6-1-positive pancreatic progenitor cells are contacted with a low concentration of an RA signaling pathway activator at a concentration of between 0.01 µM - 1.0 µM. In some embodiments, the NX6-1-positive pancreatic progenitor cells are contacted with a low concentration of the RA signaling pathway activator at a concentration of 0.02 µM, 0.03 µM, 0.04 µM, 0.05 µM, 0.06 µM, 0.07 µM, 0.08 µM, or 0.09 µM. In some embodiments, the NKX6-1-positive pancreatic progenitor cells are contacted with a low concentration of an RA signaling pathway activator at a concentration of 0.20 µM, 0.30 µM, 0.40 µM, 0.05 µM, 0.60 µM, 0.70 µM, 0.80 µM, or 0.90 µM. In some embodiments, the NX6-1-positive pancreatic progenitor cells are contacted with a low concentration of the RA signaling pathway activator at a concentration of 0.1 µM.

[0281] In some embodiments, the NKX6-1-positive pancreatic progenitor cells are contacted with the at least one SHH signaling pathway inhibitor at a concentration of between 0.1 µM and 0.5 µM. In some embodiments, the NKX6-1-positive pancreatic progenitor cells are contacted with the at least one SHH signaling pathway inhibitor at a concentration of 0.11 µM, 0.12 µM, 0.13 µM, 0.14 µM, 0.15 µM, 0.16 µM, 0.17 µM, 0.18 µM, 0.19 µM, 0.2 µM, 0.21 µM, 0.22 µM, 0.23 µM, or 0.24 µM. In some embodiments, the NKX6-1-positive pancreatic progenitor cells are treated with the at least one SHH signaling pathway inhibitor at a concentration of 0.26 µM, 0.27 µM, 0.28 µM, 0.29 µM, 0.30 µM, 0.31 µM, 0.32 µM, 0.33 µM, 0.34 µM, 0.35 µM, 0.36 µM, 0.37 µM, 0.38 µM, 0.39 µM, 0.40 µM, 0.41 µM, 0.42 µM, 0.43 µM, 0.44 µM, 0.45 µM, 0.46 µM, 0.47 µM, 0.48 µM, 0.49 µM.In some embodiments, the NX6-1 positive pancreatic progenitor cells are contacted with the at least one SHH signaling pathway inhibitor at a concentration of 0.25 µM.

[0282] In some embodiments, the NKX6-1-positive pancreatic progenitor cells are contacted with the protein kinase inhibitor at a concentration between 10 nM and 1 µM. In some embodiments, the NKX6-1-positive pancreatic progenitor cells are contacted with the protein kinase inhibitor at a concentration of 100 nM. In some embodiments, the NX6-1-positive pancreatic progenitor cells are contacted with the protein kinase inhibitor at a concentration of 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, or 90 nM. In some embodiments, the NX6-1 positive pancreatic progenitor cells are contacted with the protein kinase inhibitor at a concentration of 110 nM, 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180 nM, or 190 nM.In some embodiments, the NKX6-1 positive pancreatic progenitor cells are contacted with the protein kinase inhibitor at a concentration of 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, or 900 nM.

[0283] In some embodiments, the NX6-1-positive pancreatic progenitor cells are contacted with glucose at a concentration of 1 mM. In some embodiments, the NKX6-1-positive pancreatic progenitor cells are contacted with glucose at a concentration of 25 mM.

[0284] In some embodiments, the insulin-positive endocrine cells can be obtained by differentiating at least a portion of the Pdxl-positive, NX6-1-positive pancreatic progenitor cells into Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells by a method of contacting the Pdxl-positive, NKX6-1-positive pancreatic progenitor cells under conditions that promote the formation of cell clusters with i) a TGF-β signaling pathway inhibitor, b) a TH signaling pathway activator, and optionally c) at least one SHH signaling pathway inhibitor, ii) an RA signaling pathway activator, iii) a γ-secretase inhibitor, and vi) at least one growth factor from the epidermal growth factor (EGF) family, every other day for a period of five to seven days, to promote the differentiation of at least some of the Pdxl-positive, NX6-1-positive pancreatic progenitor cells. Pdxl-positive, NKX6-1, insulin-positive endocrine cells,wherein the Pdxl-positive, NKX6-1, insulin-positive endocrine cells express Pdxl, NX6-1, NKX2-2, MAFB, glis3, Surl, ir6.2, ZnT8, SLC2A 1, SLC2A3 and / or insulin.

[0285] In some embodiments, the insulin-positive endocrine cells can be obtained by differentiating at least some of the Pdxl-positive, NKX6-1-positive pancreatic progenitor cells in a population into Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells, e.g., by contacting the Pdxl-positive, NKX6-1-positive pancreatic progenitor cells with i) at least one SHH signaling pathway inhibitor, ii) an RA signaling pathway activator, iii) a γ-secretase inhibitor, iv) a TGF-β signaling pathway inhibitor, v) a TH signaling pathway activator, and vi) at least one growth factor from the epidermal growth factor (EGF) family, to promote the differentiation of at least some of the Pdxl-positive, NKX6-1-positive pancreatic progenitor cells into Pdxl-positive, NKX6-1, To induce insulin-positive endocrine cells, whereby the Pdxl-positive, NX6-1, insulin-positive endocrine cells Pdxl, NX6-1, NKX2-2, MAFB, glis3, Surl, Kir6.2, ZnT8, SLC2A 1, SLC2A3 and / or insulin.

[0286] Generally, the cell population is maintained in a suitable culture medium for a period of time sufficient to induce differentiation of at least one of the NX6-1-positive pancreatic progenitor cells in the population into an insulin-positive endocrine cell. An exemplary culture medium is shown in Table 4. Table 4 Agenz Konzentration MCDB131 1 l Glukose 3,6 g NaHCO3 1,754 g FAF-BSA 20 g IST-X 5 ml Glutamax 10 ml Vitamin C 0.044 g Heparin 10 mg P / S 10 ml

[0287] In some embodiments, BE5 media can be used as suitable culture media for differentiating NKX6-1-positive pancreatic progenitor cells into insulin-positive endocrine cells. In some embodiments, a suitable culture medium is shown in Table 5.

[0288] In some embodiments, conditions that promote the formation of cell clusters or cell aggregates comprise a suspension culture. In some embodiments, the period of time comprises a period of time sufficient to maximize the number of cells co-expressing the C-peptide and NKX6-1. In some embodiments, the period of time is at least 5 days. In some embodiments, the period of time is between 5 and 7 days. In some embodiments, the period of time is at least 7 days. In some embodiments, the suspension culture is replenished daily (e.g., with β-cell maturation factors). In some embodiments, a period of time between 5 and 7 days maximizes the number of cells co-expressing the C-peptide and NKX6-1.

[0289] In some embodiments, at least 15% of the NKX6-1-positive pancreatic progenitor cells in the population are induced to differentiate into insulin-positive endocrine cells.

[0290] In some embodiments, at least 99% of the NKX6-1-positive pancreatic progenitor cells in the population are induced to differentiate into insulin-positive endocrine cells. Induction of the maturation of insulin+ endocrine cells into SC-β cells

[0291] Aspects of the disclosure encompass SC-β cells. SC-β cells as used herein may be derived from any source or generated in accordance with any suitable protocol. In some aspects, insulin-positive endocrine cells are induced to mature in the SC-β cells.

[0292] In some aspects, the disclosure provides a method for generating mature, glucose-responsive β-cells from insulin-positive endocrine cells, the method comprising contacting a cell population containing the insulin-positive endocrine cells (e.g., under conditions that promote the formation of cell clumps) with at least two β-cell maturation factors comprising a) a transforming growth factor-β (TGF-β) signaling pathway inhibitor, b) a thyroid hormone (TH) signaling pathway activator, to induce the in vitro maturation of at least one insulin-positive endocrine cell in the population into an SC-β-cell.

[0293] Aspects of the disclosure include the generation of SC-β cells that resemble endogenous mature β cells in form and function, yet are distinct from native β cells. The SC-β cells can exhibit a response to at least one glucose challenge. In some embodiments, the SC-β cells exhibit a response to at least two consecutive glucose challenges. In some embodiments, the SC-β cells exhibit a response to at least three consecutive glucose challenges. In some embodiments, the SC-β cells exhibit a response to multiple (e.g., sequential) glucose challenges comparable to a response of endogenous human islet cells to multiple glucose challenges. In some embodiments, the SC-β cells can release or secrete insulin in response to two consecutive glucose challenges.In some embodiments, the SC-β cells may release or secrete insulin in response to three consecutive glucose challenges. In some embodiments, the SC-β cells may release or secrete insulin in response to four consecutive glucose challenges. In some embodiments, the SC-β cells may release or secrete insulin in response to five consecutive glucose challenges. In some embodiments, the SC-β cells may release or secrete insulin in response to constant consecutive glucose challenges. In some embodiments, the cells may be assayed to determine if they respond to sequential glucose challenges by determining if they produce intracellular Ca. 2+ increase several times, as described in the examples here.

[0294] In some embodiments, the morphology of the SC β-cells may resemble the morphology of endogenous β-cells. In some embodiments, the SC β-cell exhibits a glucose-stimulated insulin secretion (GSIS) response in vitro. In some embodiments, the SC cell exhibits a β-GSIS response in vivo. In some embodiments, the SC cell exhibits a β-GSIS response in vitro and in vivo. In some embodiments, the in vitro and / or in vitro GSIS response resembles the GSIS responses of endogenous mature β-cells. In some embodiments, the SC β-cell exhibits an in vitro (GSIS) response that resembles the GSIS response of endogenous β-cells. In some embodiments, the SC β-cell exhibits an in vivo GSIS response that resembles the GSIS response of endogenous β-cells. A GSIS response can be observed immediately after transplantation into a human or animal.In some embodiments, the GSIS response is observed within two weeks after transplantation of the SC-β cell into a human or animal subject. In some embodiments, the GSIS response is observed within two weeks after transplantation of the SC-β cell into a human or animal subject. In some embodiments, the GSIS response of the SC-β cell is observed up to three weeks, four weeks, five weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or up to 1 year or more after transplantation of the SC-β cell into a human or animal subject.

[0295] In some embodiments, the SC-β cells display at least one marker of mature endogenous pancreatic β cells. Example markers include, but are not limited to, Pdx1, HNF6, Ptf1, Sox9, Foxa2, Nkx2.2, Ngn3, and NKX6-1. In some embodiments, the expression of a marker selected from the group consisting of HNF6, Ptf1, Sox9, Foxa2, Nkx2.2, Ngn3, and NKX6-1 is upregulated by a statistically significant amount in the SC-β cells compared to the pluripotent stem cells (e.g., embryonic stem cells or pluripotent cells) from which the SC-β cells are derived.

[0296] The invention relates to the use of any TGF-β signaling pathway inhibitor to differentiate insulin-positive endocrine cells and / or mature them into SC-β cells (e.g., alone or with any combination of at least one thyroid hormone (TH) signaling pathway activator or, optionally, a protein kinase inhibitor). In some embodiments, the TGF-β signaling pathway comprises the TGF-β receptor type I kinase signaling pathway. In some embodiments, the TGF-β signaling pathway inhibitor comprises the ALK5 inhibitor II.

[0297] The invention relates to the use of any thyroid hormone pathway activator that can differentiate insulin-positive endocrine cells and / or mature into SC-β cells (e.g., alone or with any combination of at least one TGF-β signaling pathway inhibitor, or optionally a protein kinase inhibitor). In some embodiments, the thyroid hormone pathway activator comprises T3.

[0298] In some embodiments, the Pdxl-positive, NKX6-1-positive, insulin-positive cells are optionally contacted with a protein kinase inhibitor. In some embodiments, the Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells are not contacted with the protein kinase inhibitor. In some embodiments, the Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with the protein kinase inhibitor. The disclosure relates to the use of any protein kinase inhibitor to differentiate and / or mature insulin-positive endocrine cells into SC-β cells (e.g., alone or with any combination of at least one TGF-β signaling pathway inhibitor and / or thyroid hormone signaling pathway activator). In some embodiments, the protein kinase inhibitor comprises staurosporine.

[0299] In some embodiments, the method comprises contacting the cell population (e.g., insulin-positive endocrine cells) with at least one additional β-cell maturation factor.

[0300] In some embodiments, the at least one additional β-cell maturation factor comprises a cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor. In some embodiments, the method comprises contacting the Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells with a CFTR inhibitor. The disclosure relates to the use of any CFTR inhibitor to differentiate and / or mature insulin-positive endocrine cells into SC-β cells (e.g., alone or with any combination of at least one TGF-β signaling pathway inhibitor and / or a thyroid hormone signaling pathway activator, and optionally the protein kinase inhibitor). In some embodiments, the CFTR inhibitor comprises Gly-H 101.

[0301] In some embodiments, the at least one additional β-cell maturation factor comprises an O-GlcNAcase inhibitor. In some embodiments, the method comprises contacting the Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells with an O-GlcNAcase inhibitor. The disclosure relates to the use of any O-GlcNAcase inhibitor to differentiate and / or mature insulin-positive endocrine cells into SC-β cells (e.g., alone or with any combination of at least one TGF-β signaling pathway inhibitor and / or a thyroid hormone signaling pathway activator, and optionally a protein kinase inhibitor). In some embodiments, the inhibitor of O-GlcNAcase comprises Thiamet G.

[0302] It will be appreciated by those skilled in the art that the concentrations of the agents used (e.g., growth factors) may vary. In some embodiments, the insulin-positive endocrine cells are contacted with the at least one TGF-β signaling pathway inhibitor at a concentration of between 100 nM - 100 µM. In some embodiments, the insulin-positive endocrine cells are contacted with the at least one TGF-β signaling pathway inhibitor at a concentration of 10 µM. In some embodiments, the insulin-positive endocrine cells are contacted with the at least one TGF-β signaling pathway inhibitor at a concentration of 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, or 900 nM. In some embodiments, the insulin-positive endocrine cells are contacted with the at least one TGF-β signaling pathway inhibitor at a concentration of 2 µM, 3 µM, 4 µM, 5 µM, 6 µM, 7 µM, 8 µM, or 9 µM.In some embodiments, the insulin-positive endocrine cells are contacted with the at least one TGF-β signaling pathway inhibitor at a concentration of 9.1 µM, 9.2 µM, 9.3 µM, 9.4 µM, 9.5 µM, 9.6 µM, 9.7 µM, 9.8 µM, or 9.9 µM. In some embodiments, the insulin-positive endocrine cells are contacted with the at least one TGF-β signaling pathway inhibitor at a concentration of 11 µM, 12 µM, 13 µM, 14 µM, 15 µM, 16 µM, 17 µM, 18 µM, or 19 µM. In some embodiments, the insulin-positive endocrine cells are contacted with the at least one TGF-β signaling pathway inhibitor at a concentration of 10.1 µM, 10.2 µM, 10.3 µM, 10.4 µM, 10.5 µM, 10.6 µM, 10.7 µM, 10.8 µM, or 10.9 µM.

[0303] In some embodiments, the insulin-positive endocrine cells are contacted with the thyroid hormone signaling pathway activator at a concentration of between 0.1 µM. In some embodiments, the insulin-positive endocrine cells are contacted with the thyroid hormone signaling pathway activator at a concentration of 1 µM. In some embodiments, the insulin-positive endocrine cells are contacted with the thyroid hormone signaling pathway activator at a concentration of 0.2 µM, 0.3 µM, 0.4 µM, 0.5 µM, 0.6 µM, 0.7 µM, 0.8 µM, or 0.9 µM. In some embodiments, the insulin-positive cells are contacted with the endocrine γ-thyroid hormone signaling pathway activator at a concentration of 1 µM, 1.2 µM, 1.3 µM, 1.4 µM, 1.5 µM, 1.6 µM, 1.0.7 µM, 1.0.8 µM, or 1.9 µM.In some embodiments, the insulin-positive endocrine cells are contacted with the thyroid hormone signaling pathway activator at a concentration of 2 µM, 3 µM, 4 µM, 5 µM, 6 µM, 7 µM, 8 µM, or 9 µM.

[0304] In some embodiments, the insulin-positive endocrine cells are contacted with the protein kinase inhibitor at a concentration of 10 nM - 1 µM. In some embodiments, the insulin-positive endocrine cells are contacted with the protein kinase inhibitor at a concentration of 100 nM. In some embodiments, the insulin-positive endocrine cells are contacted with the protein kinase inhibitor at a concentration of 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, or 90 nM. In some embodiments, the insulin-positive endocrine cells are contacted with the protein kinase inhibitor at a concentration of 110 nM, 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180 nM, or 190 nM.In some embodiments, the insulin-positive endocrine cells are contacted with the protein kinase inhibitor at a concentration of 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, or 900 nM.

[0305] In some embodiments, the Pdxl-positive, NX6-1-positive, insulin-positive endocrine cells are contacted with the CFTR inhibitor at a concentration of between 100 nm and 100 µM. In some embodiments, the Pdxl-positive, NX6-1-positive, insulin-positive endocrine cells are contacted with the CFTR inhibitor at a concentration of between 10 nm and 10 µM.

[0306] In some embodiments, the Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with the O-GlcNAcase inhibitor at a concentration of 100 nM - 100 µM. In some embodiments, the Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with the O-GlcNAcase inhibitor at a concentration of 10 nM - 10 µM.

[0307] In some embodiments, an SC-β cell can be obtained by differentiating at least some of the Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells into SC-β cells by a method of contacting the Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells under conditions that promote the formation of cell clusters with i) a transforming growth factor-β (TGF-β) signaling pathway inhibitor, ii) a thyroid hormone signaling pathway activator, and optionally iii) a protein kinase inhibitor, every other day for a period of seven to 14 days to induce the in vitro maturation of at least some of the Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells into SC-β cells, wherein the SC-β cells exhibit a GSIS response both in vitro and / or in vivo. In some embodiments, the GSIS response resembles the GSIS response of an endogenous β-cell.

[0308] In some embodiments, an SC-β cell can be obtained by differentiating at least some of the Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells in a population into SC-β cells, for example, by contacting the Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells with i) a transforming growth factor β (TGF-β) signaling pathway inhibitor, ii) a thyroid hormone signaling pathway activator, and optionally iii) a protein kinase inhibitor to induce the in vitro maturation of at least some of the Pdxl-positive, NKX6-1-positive, insulin-producing endocrine cells into SC-β cells, wherein the SC-β cells exhibit a GSIS response both in vitro and / or in vivo that is similar to the GSIS response of an endogenous β cell.

[0309] In some aspects, the disclosure provides a method for generating SC-β cells, comprising: contacting Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells under conditions that promote the formation of cell clusters with i) a transforming growth factor-β (TGF-β) signaling pathway inhibitor, ii) a thyroid hormone signaling pathway activator, and optionally iii) a protein kinase inhibitor to induce the in vitro maturation of at least some of the Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells into SC-β cells, wherein the SC-β cells exhibit a GSIS response both in vitro and / or in vivo. In some embodiments, the GSIS response resembles the GSIS response of an endogenous β cell.

[0310] In some aspects, the disclosure provides a method for generating SC-β cells from pluripotent cells, the method comprising: a) differentiating pluripotent stem cells in a population into Pdxl-positive pancreatic progenitor cells; b) differentiating at least some of the Pdxl-positive pancreatic progenitor cells into Pdxl-positive, NKX6-1-positive pancreatic progenitor cells by a method comprising contacting the Pdxl-positive pancreatic progenitor cells under conditions promoting cell cluster formation with i) at least one growth factor from the FGF family, ii) at least one SHH signaling pathway inhibitor, and optionally iii) an RA signaling pathway activator, every other day for a period of five days to induce differentiation of at least some of the Pdxl-positive pancreatic progenitor cells in the population into NKX6-1-positive pancreatic progenitor cells,where the NKX6-1-positive pancreatic progenitor cells express Pdxl and NKX6-1; c) differentiating at least some of the Pdxl-positive, NKX6-1-positive pancreatic progenitor cells into Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells by a method which comprises contacting the Pdxl-positive, NKX6-1-positive pancreatic progenitor cells under conditions which promote the formation of cell clustering with i) a TGF-β signalling pathway inhibitor, b) a TH signalling pathway activator and optionally c) at least one SHH signalling pathway inhibitor, ii) an RA signalling pathway activator, iii) a γ-secretase inhibitor, and vi) at least one growth factor from the epidermal growth factor (EGF) family, every other day for a period of five to seven days, in order to promote the differentiation of at least some of the Pdxl-positive, NKX6-1-positive pancreatic progenitor cells. Pdxl-positive, NKX6-1, insulin-positive endocrine cells,wherein the Pdxl-positive, NKX6-1, insulin-positive endocrine cells express Pdxl, NX6-1, NKX2-2, afb, glis3, Surl, Kir6.2, ZnT8, SLC2A 1, SLC2A3 and / or insulin; and d) differentiating at least some of the Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells into SC-β cells by a method comprising contacting the Pdxl-positive, NX6-1-positive, insulin-positive endocrine cells under conditions that promote the formation of cell clusters with i) a transforming growth factor-β (TGF-β) signaling pathway inhibitor, ii) a thyroid hormone signaling pathway activator, and optionally iii) a protein kinase inhibitor, every other day for a period of seven to 14 days to induce the in vitro maturation of at least some of the Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells into SC-β cells,wherein the SC-β cells exhibit a GSIS response in vitro and / or in vivo. In some embodiments, the GSIS response resembles the GSIS response of an endogenous mature β cell.

[0311] In some aspects, the disclosure provides a method for generating SC-β cells from pluripotent cells, the method comprising: a) differentiating at least some of the pluripotent cells in a population into Pdxl-positive pancreatic progenitor cells; b) differentiating at least some of the Pdxl-positive pancreatic progenitor cells into Pdxl-positive, NKX6-1-positive pancreatic progenitor cells by a process of contacting the Pdxl-positive pancreatic progenitor cells under conditions promoting cell cluster formation with i) KGF, ii) Santl and optionally iii) a low concentration of RA, every other day for a period of five days, to induce the differentiation of at least some Pdxl-positive pancreatic progenitor cells in the population into NKX6-1-positive pancreatic progenitor cells, wherein the NKX6-1-positive pancreatic progenitor cells express Pdxl and NKX6-1;c) differentiating at least some of the Pdxl-positive, NX6-1-positive pancreatic progenitor cells into Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells by a process of contacting the Pdxl-positive, NKX6-1-positive pancreatic progenitor cells with i) ALK5 Inhibitor II, ii) T3, and optionally iii) Santl, iv) RA, v) XXI and vi) betacellulin, every other day for a period of five to seven days, to induce differentiation of at least some of the Pdxl-positive, NKX6-1-positive pancreatic progenitor cells into Pdxl-positive, NKX6-1, insulin-positive endocrine cells, wherein the Pdxl-positive, NKX6-1, insulin-positive endocrine cells express Pdxl, NX6-1, NKX2-2, MAFB, express glis3, Surl, Kir6.2, ZnT8, SLC2A 1, SLC2A3 and / or insulin;and d) differentiating at least some of the Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells into SC-β cells by a process of contacting the Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells under conditions promoting the formation of cell clusters with i) ALK5 Inhibitor II, ii) T3, and optionally iii) staurosporine, every other day for a period of seven to 14 days to induce in vitro maturation of at least some of the Pdxl-positive, NKX6-1-positive, insulin-producing endocrine cells into SC-β cells, wherein the SC-β cells exhibit a GSIS response in vitro and in vivo that is similar to the GSIS response of an endogenous β cell.;

[0312] Generally, the Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells are maintained in a suitable culture medium for a period of time sufficient to induce the in vitro maturation of at least some of the Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells into SC-β cells. Examples of suitable culture media are shown above in Table 4 and below in Table 5. Table 5 CMR Inselmedium („CMRLS") CMRL 1066 ergänzt cat#99-603-CV Mediatech mit 10% Hyclone ergänzt FBS

[0313] In some embodiments, suitable culture medium comprises Connought Medical Research Laboratories 1066 supplemented islet media (CMRLS). In some embodiments, suitable culture medium comprises a component of CMRLS (e.g., supplemented zinc). In some embodiments, suitable culture medium is shown in Table 3. In some embodiments, the CMRLS is supplemented with serum (e.g., human). In some embodiments, the CMRLS is supplemented with serum replacement (e.g., KOSR). In some embodiments, the CMRLS is supplemented with fetal bovine serum. In some embodiments, the CMRLS is supplemented with 10% fetal bovine serum. In some embodiments, suitable culture medium for differentiating insulin-positive endocrine cells into SC-β cells may comprise S3 media. In some embodiments, conditions that promote cell clustering include suspension culture.In some embodiments, the period may be at least 7 days. In some embodiments, the period may be from 7 days to 21 days. In some embodiments, the period may be between 7 and 14 days. In some embodiments, the period may be between 10 and 14 days. In some embodiments, the period may be 14 days. In some embodiments, the suspension culture is supplemented every other day (e.g., with the β-cell maturation factors).

[0314] In some embodiments, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50% of the Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells are induced to mature into SC β-cells. In some embodiments, at least 60%, at least 70%, at least 80%, at least 90%, at least 99% of the Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells are induced to mature into SC β-cells. In some embodiments, at least 30% of the cells generated comprise SC β-cells. In some embodiments, the SC-β cells express C-peptide, insulin, NKX6-1 Pdxl, and co-express NKX6-1 and the C-peptide.

[0315] In some embodiments, the SC-β cells comprise human cells. In some embodiments, the generation of the SC-β cells is scalable in vitro. Isolated cell populations

[0316] Aspects of the disclosure relate to isolated populations of cells as produced according to a method described herein. In some embodiments, a population of SC-β cells is produced by contacting at least one insulin-positive endocrine cell or precursor thereof with at least one β-cell maturation factor described herein. In some embodiments, a population of SC-β cells is produced by contacting at least one insulin-positive endocrine cell or precursor thereof with at least two of the β-cell maturation factors described herein. In some embodiments, a population of SC-β cells is produced by contacting at least one insulin-positive endocrine cell or precursor thereof with at least three β-cell maturation factors described herein.In some embodiments, a population of SC β-cells is prepared by contacting at least one insulin-positive endocrine cell or precursor thereof with at least four β-cell maturation factors described herein. In some embodiments, a population of SC β-cells is prepared by contacting at least one insulin-positive endocrine cell or precursor thereof with at least five β-cell maturation factors described herein. In some embodiments, a population of SC β-cells is prepared by contacting at least one insulin-positive endocrine cell or precursor thereof with at least six, at least seven, at least eight, at least nine, at least ten of the β-cell maturation factors described herein.

[0317] In some aspects, the disclosure provides an isolated population of definitive endoderm cells. An isolated population of definitive endoderm cells can be obtained by differentiating at least some pluripotent cells in a population into definitive endoderm cells, e.g., by a method of contacting a population of pluripotent cells with i) at least one growth factor from the TGF-β superfamily, and ii) a Wnt signaling pathway activator, to induce differentiation of at least some of the pluripotent cells in the population into definitive endoderm cells, wherein the definitive endoderm cells express at least one marker characteristic of definitive endoderm.

[0318] In some aspects, the disclosure provides an isolated population of archenteron cells. An isolated population of archenteron cells can be obtained by differentiating at least some of the definitive endoderm cells in a population into archenteron cells, for example, by a method of contacting the definitive endoderm cells with at least one growth factor from the fibroblast growth factor (FGF) family to induce differentiation of at least some of the definitive endoderm cells into archenteron cells, wherein the archenteron cells express at least one marker characteristic of definitive endoderm.

[0319] In some aspects, the disclosure provides an isolated population of Pdxl-positive pancreatic progenitor cells. An isolated population of Pdxl-positive pancreatic progenitor cells can be obtained by differentiating at least some of the archenteron cells in a population into Pdxl-positive pancreatic progenitor cells, for example, by a method of contacting the archenteron cells with i) at least one bone morphogenesis protein (BMP) pathway inhibitor, ii) at least one growth factor from the FGF family, iii) at least one SHH pathway inhibitor, iv) at least one retinoic acid (RA) pathway activator; and v) at least one protein kinase C activator, to induce differentiation of at least some of the archenteron cells into Pdxl-positive pancreatic progenitor cells, wherein the Pdxl-positive pancreatic progenitor cells express Pdxl.

[0320] In some aspects, the disclosure provides an isolated population of NKX6-1-positive pancreatic progenitor cells. An isolated population of NKX6-1-positive pancreatic progenitor cells can be obtained by differentiating at least some of the Pdxl-positive pancreatic progenitor cells in a population into Pdxl-positive, NKX6-1-positive pancreatic progenitor cells, for example, by a method of contacting the Pdxl-positive pancreatic progenitor cells with i) at least one growth factor from the FGF family, ii) at least one SHH signaling pathway inhibitor, and optionally iii) an RA signaling pathway activator, to induce differentiation of at least some Pdxl-positive pancreatic progenitor cells in the population into NKX6-1-positive pancreatic progenitor cells, wherein the NKX6-1-positive pancreatic progenitor cells express Pdx1 and NKX6-1.

[0321] In some aspects, the disclosure provides an isolated population of insulin-positive endocrine cells. An isolated population of insulin-positive endocrine cells can be obtained by differentiating at least some Pdxl-positive, NKX6-1-positive pancreatic progenitor cells in a population into Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells, for example by a method of contacting the Pdxl-positive, NKX6-1-positive pancreatic progenitor cells with i) a TGF-β) signaling pathway inhibitor, ii) a TH signaling pathway activator and optionally at least one additional β-cell maturation factor selected from the group consisting of i) at least one SHH signaling pathway inhibitor, ii) an RA signaling pathway activator iii) a γ-secretase inhibitor, iv) and vi) at least one growth factor from the epidermal growth factor (EGF) family, in order to promote the differentiation of at least some of the Pdxl-positive,To induce NKX6-1-positive pancreatic progenitor cells to Pdxl-positive, NKX6-1, insulin-positive endocrine cells, wherein the Pdxl-positive, NKX6-1, insulin-positive endocrine cells express Pdxl, NKX6-1, NKX2-2, MAFB, glis3, Surl, Kir6.2, ZnT8, SLC2A 1, SLC2A3 and / or insulin.

[0322] In some aspects, the disclosure provides an isolated population of SC-β cells. An isolated population of SC-β cells can be obtained by differentiating at least some of the Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells in a population into SC-β cells, for example, by a method of contacting the Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells with i) a transforming growth factor-β (TGF-β) signaling pathway inhibitor, ii) a thyroid hormone signaling pathway activator, and optionally iii) a protein kinase inhibitor, to induce in vitro maturation of at least some of the Pdxl-positive, NKX6-1-positive, insulin-positive endocrine cells into SC-β cells, wherein the SC-β cells exhibit a GSIS response in vitro and / or in vivo. In some embodiments, the GSIS response resembles the GSIS response of an endogenous β-cell.

[0323] Aspects of the disclosure include microcapsules containing isolated populations of the cells described herein (e.g., SC-β cells). Microcapsules are known in the art. Suitable examples of microcapsules are described in the literature (e.g., Jahansouz et al., “Evolution of β-Cell Replacement Therapy in Diabetes Mellitus: Islet Cell Transplantation”. Journal of Transplantation 2011, Volume 2011, Article ID 247959; Orive et al., “Application of Cell Encapsulation for Controlled Delivery of Biological Therapeutics”, Advanced Drug Delivery Reviews (2013), http: / / dx.doi.org / 10.1016 / j.addr.2013.07.009; Hernandez et al., “Microcapsules and Microcarriers for In Situ Cell Delivery”, Advanced Drug Delivery Reviews 2010; 62:711-730; Murua et al., “Cell Microencapsulation: Towards Clinical Application”., Journal of Controlled Release 2008; 132: 76-83; and Zanin et al., "The Development of Encapsulated Cell Technologies as Therapies for Neurological and Sensory Disorders," Journal of Controlled Release 2012; 160: 3-13). Microcapsules can be formulated in several ways. Exemplary capsules comprise an alginate core surrounded by a polycation layer covered by an outer alginate membrane. The polycation membrane is a semipermeable membrane that imparts stability and biocompatibility. Examples of polycations include, but are not limited to, poly-L-lysine, poly-L-ornithine, chitosan, lactose-modified chitosan, and photopolymerized biomaterials. In some embodiments, the core alginate is modified, for example, to form a scaffold comprising an alginate core with covalently conjugated oligopeptides having an RGD sequence (arginine, glycine, aspartic acid).In some embodiments, the core alginate is modified, for example, to produce a covalently reinforced microcapsule with a chemoenzymatic alginate of increased stability. In some embodiments, the core alginate is modified, for example, to produce membrane-mimetic films by in situ polymerization of acrylate-functionalized phospholipids. In some embodiments, the microcapsules consist of alginates enzymatically modified using epimerases. In some embodiments, the microcapsules comprise covalent links between adjacent layers of the microcapsule membrane. In some embodiments, the microcapsule contains a subsieve-sized capsule comprising alginate coupled to phenolic moieties. In some embodiments, the microcapsule comprises a scaffold comprising alginate-agarose. In some embodiments, the SC-β cell is modified with PEG before being encapsulated in the alginate.In some embodiments, the isolated populations of cells, for example, SC-β cells, are encapsulated in photoreactive liposomes and alginate. It should be understood that the alginate used in the microcapsules can be replaced with other suitable biomaterials, including, but not limited to, PEG, chitosan, PES hollow fibers, collagen, hyaluronic acid, dextran with RGD, EHD and PEGDA, PMBV and PVA, PGSAS agarose, agarose with gelatin, PLGA, and multilayered embodiments thereof.

[0324] In some embodiments, compositions containing the populations of cells according to the methods described herein can also be used as a functional component in a mechanical device to produce one or more of the pancreatic islet endocrine cell polypeptides. In its simplest form, the device contains a population of pancreatic β-cells (e.g., populations produced from insulin-positive endocrine cells or precursors thereof) behind a semipermeable membrane that prevents passage and retains the cell population within the device while allowing passage of insulin, glucagon, or somatostatin secreted by the cell population. This includes populations of pancreatic β-cells that are microencapsulated, usually in the form of cell clusters, allowing cell interaction that inhibits dedifferentiation. For example, US Pat. No.4,391,909 Islet cells encapsulated in a spherical semipermeable membrane composed of polysaccharide polymers > 3,000 mol wt. that are cross-linked to be permeable to insulin-sized proteins but impermeable to molecules larger than 100,000 mol wt. US Patent No. 6,023,009 describes islet cells encapsulated in a semipermeable membrane composed of agarose and agaropectin. Microcapsules of this type are adapted for administration into the body cavity of a diabetic patient and are believed to offer some advantages in reducing tissue compatibility problems or bacterial susceptibility.

[0325] More sophisticated devices are also contemplated for use containing a population of pancreatic β-cells prepared from insulin-positive endocrine cells or precursors thereof according to the methods described herein, whether for implantation in patients with diabetes or for extracorporeal therapy. U.S. Patent No. 4,378,016 describes an artificial endocrine gland comprising an extracorporeal portion, a subcutaneous portion, and a replaceable sheath containing the hormone-producing cells. U.S. Patent No. 5,674,289 describes a bioartificial pancreas having an islet chamber separated from one or more vessel-forming chambers by a semipermeable membrane and open to surrounding tissue.Suitable devices usually comprise a chamber containing islet cells and a chamber separated from the islet cells by a semipermeable membrane which collects the proteins secreted by the islet cells and also allows feedback to the islet cells, for example of the circulating blood glucose level.

[0326] Aspects of the disclosure include assays comprising isolated populations of the cells described herein (e.g., SC β-cells). In some embodiments, the assays can be used to identify one or more candidate agents that promote or inhibit a β-cell fate selected from the group consisting of β-cell proliferation, β-cell replication and death, β-cell function, β-cell susceptibility to immune attack, or β-cell susceptibility to dedifferentiation or differentiation. In some embodiments, the assays can be used to identify one or more candidate agents that promote the differentiation of at least one insulin-positive endocrine cell or precursor thereof into SC β-cells.In some embodiments, the assays can be used to identify one or more candidate agents that stimulate β-cells to produce insulin or increase the production or secretion of insulin.

[0327] The invention relates to methods in which SC-β cells are generated according to the methods described herein from iPS cells derived from cells extracted or isolated from individuals suffering from a disease (e.g., diabetes, obesity, or a β-cell disorder), wherein these SC-β cells are compared with normal β cells from healthy individuals who do not have the disease to identify differences between the SC-β cells and normal β cells that can serve as markers for the diseases (e.g., epigenetic and / or genetic). In some embodiments, β cells are obtained from a diabetic individual and compared with normal β cells, whereupon the β cells are reprogrammed to iPS, wherein the iPS cells are examined for genetic and / or epigenetic markers that are present in the β cells of the diabetic individual but not in the normal β cells, in order to identify the markers (e.g.,prediabetic). In some embodiments, the iPS cells and / or SC-β are derived from diabetic patients to screen for agents (e.g., agents capable of modulating genes that contribute to a diabetic phenotype). Method for differentiating insulin-positive endocrine cells into SC-β cells

[0328] The generation of SC-β cells by converting at least one insulin-positive endocrine cell or a precursor thereof using the methods disclosed herein has several advantages. First, the disclosed methods enable the production of autologous SC-β cells that are specific to and compatible with an individual. In general, autologous cells are less likely than non-autologous cells to undergo immunological rejection. The cells are derived from at least one insulin-positive endocrine cell or a precursor thereof, for example, a pancreatic progenitor, by reprogramming a somatic cell (e.g.a fibroblast) derived from the individual to a pluripotent state and then culturing the pluripotent cells to differentiate at least some of the pluripotent cells into at least one insulin-positive endocrine cell or a precursor thereof, followed by transplanting the at least one insulin-positive endocrine cell or a precursor thereof into the individual such that the at least one insulin-positive endocrine cell or a precursor thereof matures into an SC-β cell in vivo, or the maturation of the at least one insulin-positive endocrine cell into an SC cell is induced in vitro.

[0329] In some embodiments, an individual from whom at least one insulin-positive endocrine cell or precursor thereof was obtained is a mammal, such as a human. In some embodiments, the individual suffers from a β-cell disorder. In some embodiments, the individual suffers from diabetes. In some embodiments, the individual suffers from prediabetes. In such embodiments, the at least one insulin-positive endocrine cell or precursor thereof can be differentiated into an SC β-cell ex vivo using the methods described herein and then administered to the individual from whom the cells were obtained in a method as described above to treat the individual having the β-cell disorder (e.g., diabetes).

[0330] In some embodiments, there is at least one insulin-positive endocrine cell or precursor thereof in an individual (in vivo) that is converted to become an SC-β cell by the methods described herein. In some embodiments, the conversion of at least one insulin-positive endocrine cell or precursor thereof to an SC-β cell in vivo can be achieved by administering to an individual a composition containing at least one, at least two, at least three, at least four, at least five, at least six, or more β-cell maturation factors as described herein.In some embodiments, the conversion of at least one insulin-positive endocrine cell or precursor thereof to an SC-β cell in vivo can be achieved by administering to an individual a composition containing at least one, at least two, at least three, at least four, at least five, at least six, or β cell maturation factors as described herein.

[0331] In some embodiments, contacting may be accomplished by maintaining the at least one insulin-positive endocrine cell or precursor thereof in a culture medium containing the one or more β-cell maturation factors. In some embodiments, the at least one insulin-positive endocrine cell or precursor thereof may be genetically engineered. In some embodiments, at least one insulin-positive endocrine cell or precursor thereof may be genetically engineered to express one or more β-cell markers as disclosed herein, for example, to express a polypeptide selected from pancreatic and duodenal homeobox-1 (PDX-1) polypeptide, insulin, C-peptide, amylin, E-cadherin, Hηέ3(3, PC1 / 3, B2, Nkx2.2, NX6-1, GLUT2, PC2, ZnT-8, or an amino acid sequence substantially homologous thereto, or functional fragments or functional variants thereof.

[0332] If the at least one insulin-positive endocrine cell or precursor thereof is maintained under in vitro conditions, conventional tissue culture conditions and methods known to those skilled in the art can be used. Isolation and cultivation methods for various cells are within the general capabilities of those skilled in the art.

[0333] In the disclosed methods, the at least one insulin-positive endocrine cell or precursor thereof can generally be grown under standard temperature, pH, and other environmental conditions, for example, as adherent cells in tissue culture plates at 37°C in a 5 to 10% CO2 atmosphere. The cells and / or the culture medium are / is appropriately modified to achieve conversion to SC-β cells, as described herein. In certain embodiments, the at least one insulin-positive endocrine cell or precursor thereof, for example, a pancreatic precursor, can be grown on or in the presence of a material that mimics one or more properties of the extracellular matrix or comprises one or more extracellular matrices or basement membrane components. In some embodiments, Matrigel™ is used. Other materials include proteins or mixtures thereof, such as gelatin, collagen, fibronectin, etc.In certain embodiments of the invention, at least one insulin-positive endocrine cell or precursor thereof can be cultured in the presence of a feeder layer of cells. Such cells can be, for example, of murine or human origin. They can be further irradiated, chemically inactivated by treatment with a chemical inactivator such as mitomycin C, or otherwise treated to inhibit their proliferation as desired. In other embodiments, at least one insulin-positive endocrine cell or precursor thereof is cultured without feeder cells. In some embodiments, the insulin-positive endocrine cells or precursors thereof are cultured under conditions that promote cell cluster formation. As used herein, "conditions that promote cell cluster formation" means any condition that stimulates the clustering of cells during the differentiation of the cells toward SC-β cells.In some embodiments, the conditions that promote the formation of cell clusters or cell clumps or cell aggregates comprise suspension culture. Boretti and Gooch (Tissue Eng. 2006 April; 12 (4): 939-48) report that culture under minimally adherent conditions (medium with low serum concentration, substrate with low adherence) stimulated cell cluster formation during the transdifferentiation of adult pancreatic duct epithelial cells to β-cells in vitro. Therefore, without being bound by theory, in some embodiments, the conditions that promote the formation of cell clusters comprise minimally adherent conditions, for example, low serum concentration and a substrate with low adherence.

[0334] In certain examples, the β-cell maturation factors can be used to induce the differentiation of at least one insulin-positive endocrine cell or a precursor thereof by exposing or contacting at least one insulin-positive endocrine cell or a precursor thereof to or with an effective amount of a β-cell maturation factor described herein to differentiate the at least one insulin-positive endocrine cell or a precursor thereof into at least one SC β-cell (e.g., a mature pancreatic β-cell).

[0335] Accordingly, cells and compositions prepared by the methods described herein are encompassed. The exact amount and type of β-cell maturation factor may vary depending on the number of insulin-positive endocrine cells or a precursor thereof, the desired stage of differentiation, and the number of pre-differentiation stages performed.

[0336] In certain examples, a β-cell maturation factor is present in an effective amount. As used herein, "effective amount" means the amount of the compound that should be present to differentiate at least 10%, or at least 20%, or at least 30% of the cells in a population of insulin-positive endocrine cells or precursors thereof into SC β-cells.

[0337] In further examples, β-cell maturation factors may be present in the culture medium of the at least one insulin-positive endocrine cell or precursor thereof, or alternatively, the β-cell maturation factors may be added to the at least one insulin-positive endocrine cell or precursor thereof at any stage of growth. Confirmation of the presence and identification of cells, SC-β cells

[0338] To confirm the presence of an SC β-cell, for example a mature pancreatic β-cell produced by inducing the differentiation of at least one insulin-positive endocrine cell or a precursor thereof by the action of at least one β-cell maturation factor as described herein, any means known to those skilled in the art may be used.

[0339] In some embodiments, the presence of β-cell markers, e.g., chemically induced SC β-cells, can be determined by detecting the presence or absence of one or more markers indicative of an endogenous β-cell. In some embodiments, the method can comprise detecting positive expression (e.g., the presence) of a marker for mature β-cells. In some embodiments, the marker can be detected using a reagent, e.g., a reagent for detecting NKX6-1 and the C-peptide. For example, SC β-cells can express NKX6-1 and the C-peptide, but not significant amounts of other markers indicative of immature β-cells (e.g., MAFB). A reagent for a marker can, for example, be an antibody against the marker or a primer for an RT-PCR or PCR reaction, e.g., a semi-quantitative or quantitative RT-PCR or PCR reaction.Such markers can be used to assess whether an SC-β cell has been produced. The antibody or other detection reagent for a marker can be linked to a label, such as a radiological, fluorescent (GFP), or colorimetric marker, for use in detection.

[0340] The development of the at least one insulin-positive endocrine cell or a precursor thereof into an SC-β cell can be monitored by determining the expression of markers characteristic of mature β cells. In some methods, the expression of certain markers is determined by detecting the presence or absence of the marker. Alternatively, the expression of certain markers can be determined by measuring the amount of the marker present in the cells of the cell culture or cell population. In certain methods, the expression of markers characteristic of SC-β cells is determined, as well as the absence of significant expression of markers characteristic of insulin-positive endocrine cells or precursors thereof, e.g. pluripotent stem cells or pancreatic progenitor cells, from which they were derived.

[0341] As described in connection with monitoring the production of an SC β-cell (e.g., a mature pancreatic β-cell) from an insulin-positive endocrine cell, qualitative or semi-quantitative techniques, such as blot transfer methods and immunocytochemistry, can be used to determine the expression of the markers using methods known to those of ordinary skill in the art. Alternatively, the expression of markers can be accurately quantified using methods commonly known in the art, such as quantitative PCR. Furthermore, it is clear that, at the polypeptide level, many of the markers of pancreatic islet hormone-expressing cells are secreted proteins. Therefore, techniques for determining the amount of extracellular marker, such as ELISA, can be used.

[0342] SC-β cells can also be characterized by downregulation of markers characteristic of pluripotent stem cells from which the SC-β cell was induced. For example, SC-β cells derived from pluripotent stem cells can be characterized by statistically significant downregulation of the pluripotent stem cell markers alkaline phosphatase (AP), NANOG, OCT-4, SOX-2, SSEA4, TRA-1-60, or TRA-1-81 in the mature cell compared to expression in the pluripotent stem cell from which it was derived. Other markers expressed by pluripotent cells include, but are not limited to, alkaline phosphatase (AP); ABCG2; stage-specific embryonic antigen-1 (SSEA-1); SSEA-3; SSEA-4; TRA-1-60; TRA-1-81; Tra-2-49 / 6E; Eras / ECATS, E-cadherin; βIII-tubulin; smooth muscle actin (α-SMA); fibroblast growth factor 4 (FGF4), Cripto, Daxl; Zinc Finger Protein 296 (Zfp296);N-acetyltransferase-1 (National); (ES cell marker 1 (ECAT 1); ESG 1 / DPPAS / ECAT2; ECAT3; ECAT6; ECAT7; ECAT8; ECAT9; ECAT10; ECAT15-1; ECAT1 5-2; Fth 1 17; Sal 14; undifferentiated embryonic cell transcription factor (Utfl); Rexl; p53; G3PDH; telomerase, including TERT; silent X-chromosome gene; Dnmt3a; Dnmt3b; TRIM28; F-box-containing protein 15 (Fbxl 5); Nanog / ECAT4; Oct3 / 4; Sox2; Klf4; c-Myc; Esrrb; TDGF1; GABRB3; Zfp42, Foxd3; GDF3; CYP25A 1; developmental pluripotency-associated 2 (DPPA2); T-cell lymphoma breakpoint 1 (Tel 1); DPPA3 / Stella; DPPA4; Dnmt3L; Soxl 5; Stat3; Grb2; SV40 large T antigen; HPV 16 E6; HPV 16-E7, β-catenin, and BMI 1 and other general markers of pluripotency, etc., where at least one or more of these are regulated in a statistically significant amount in a mature cell compared to the pluripotent stem cell from which it was derived.;

[0343] It is clear that the present invention is not limited to these markers as markers for mature β-cells listed herein, and the present invention also encompasses markers such as cell surface markers, antigens and other gene products including ESTs, RNA (including microRNAs and antisense RNA), DNA (including genes and cDNAs) and portions thereof. Enrichment, isolation and purification of an SC-β cell

[0344] Another aspect of the present invention relates to the isolation of a population of SC-β cells from a heterogeneous cell population, such as a mixed cell population containing SC-β cells and insulin-positive endocrine cells or precursors thereof from which the SC-β cells were derived. A population of SC-β cells produced by any of the methods described above can be enriched, isolated, and / or purified by using cell surface markers present on the SC-β cells that are not present on the insulin-positive endocrine cells or precursors thereof from which they were derived. Such cell surface markers are also referred to as an affinity tag specific for an SC-β cell.Examples of SC-β cell-specific affinity tags are antibodies, ligands, or other binding agents specific for a marker molecule, such as a polypeptide, that is present on the cell surface of an SC-β cell but not present to a substantial extent on other cell types (e.g., insulin-positive endocrine cells or precursors thereof). In some methods, an antibody that binds to a cell surface antigen on an SC-β cell (e.g., a human SC-β cell) is used as an affinity tag for the enrichment, isolation, or purification of chemically induced (e.g., by contacting with at least one β-cell maturation factor described herein) SC-β cells by the methods described herein. Such antibodies are known and commercially available.

[0345] The method for using antibodies for the enrichment, isolation, and / or purification of SC-β cells will be readily apparent to those skilled in the art. For example, in some embodiments, the reagent, such as an antibody, can be incubated with a cell population comprising SC-β cells, wherein the cell population has been treated to reduce intercellular adhesion and adhesion to the substrate. The cell population is then washed, centrifuged, and resuspended. In some embodiments, if the antibody has not already been labeled, the cell suspension is then incubated w...

Claims

[1] An in vitro method for generating insulin-producing pancreatic β-cells (SC-β-cells) from precursor cells, the method comprising: Contacting PDX1-positive, NKX6-1-positive, or insulin-positive endocrine cells under conditions that promote cell clustering with a thyroid hormone signaling pathway activator, a transforming growth factor-β (TGF-β) signaling pathway inhibitor, and a γ-secretase inhibitor, thereby inducing the in vitro maturation of at least some PDX1-positive, NKX6-1-positive, or insulin-positive endocrine cells into SC-β cells, wherein the SC-β cells exhibit in vitro and / or in vivo glucose-stimulated insulin secretion (GSIS) similar to the GSIS of native pancreatic β cells. [2] The method of claim 1, wherein the thyroid hormone signaling pathway activator is triiodothyronine. [3] The method of claim 2, wherein the concentration of triiodothyronine is at least 0.1 µM. [4] The method of claim 1, wherein the TGF-β signaling pathway inhibitor is Alk5 Inhibitor II. [5] The method of claim 4, wherein the concentration of Alk5 Inhibitor II is at least 100 nM. [6] The method of claim 4, wherein the concentration of Alk5 inhibitor II is 100 µM or less. [7] The method of claim 1, wherein the γ-secretase inhibitor is XXI. [8] The method of claim 1, wherein the cultivation comprises: changing the culture medium at least every other day. [9] The method of claim 1, wherein the cultivation lasts at least seven days. [10] The method of claim 1, wherein the culture medium is a serum-free culture medium. [11] The method of claim 1, wherein the PDX1-positive, NKX6-1-positive, or insulin-positive endocrine cells are cultured in the presence of a low-adherent substrate. [12] The method of claim 1, wherein the PDX1-positive, NKX6-1-positive, or insulin-positive endocrine cells are human PDX1-positive, NKX6-1-positive, or insulin-positive endocrine cells. [13] The method of claim 1, wherein the SC-β cells exhibit in vitro glucose-stimulated insulin secretion in response to a first glucose challenge and a second glucose challenge when the first glucose challenge and the second glucose challenge are applied sequentially. [14] Composition comprising several SC-β cells, wherein the SC-β cells are obtainable by an in vitro method according to any one of claims 1-13, and where the SC-β cells (a) comprise one or more crystalline insulin granules; (b) express the following genes: INS, PDX1, NKX6-1 and ZNT8; and (c) exhibit in vitro glucose-stimulated insulin secretion in response to a first glucose challenge. [15] The composition of claim 14, wherein the SC-β cells stimulate in vitro glucose-stimulated insulin secretion in response to a first glucose challenge, a second glucose challenge, and a third glucose challenge when the first glucose challenge, the second glucose challenge, and the third glucose challenge are applied sequentially. [16] The composition of claim 14, wherein the SC-ß cells further express at least one gene selected from the group consisting of MAFA, PAX6 NEUROD1, GCK, SLC2A1, PCSK1, KCNJ11, ABCC8, SNAP25, RAB3A, GAD2, PTPRN, NKX2-2 and PAX4. [17] The composition of claim 14, wherein the SC-β cells secrete insulin in response to a first glucose concentration compared to a second glucose concentration in a ratio of at least 1:1, wherein the first glucose concentration is higher than the second glucose concentration. [18] The composition of claim 14, wherein the SC-β cells are monohormonal. [19] The composition of claim 14, wherein the insulin secreted by the SC-β cells comprises at least 0.5 µIU per 1000 cells per 30 minutes of incubation when the SC-β cells are exposed to at least 20 mM glucose. [20] The composition of claim 14, wherein insulin secretion from SC-β cells is enhanced in response to an anti-diabetes agent. [21] The composition of claim 14, wherein the SC-β cells exhibit cytokine-induced apoptosis in response to a cytokine. [22] The composition of claim 14, wherein the SC-β cells have a gene expression profile that differs from the gene expression profile of a native β cell. [23] A population of cells comprising a plurality of SC-β cells according to claim 14, wherein at least 10% of the cells in the population of cells are SC-β cells. [24] A population of cells according to claim 23, further comprising insulin-positive endocrine cells. [25] A population of cells according to claim 23, wherein the population of cells comprises one of the following: a. multiple C-peptide-negative / glucagon-positive cells; b. multiple C-peptide-negative / somatostatin-positive cells; c. multiple glucagon-positive / somatostatin-negative cells; d. multiple glucagon-negative / somatostatin-positive cells; or e. a combination of these. [26] A population of cells according to claim 23, wherein at least 3% of the population of cells are C-peptide negative / glucagon positive cells or glucagon positive / somatostatin negative cells. [27] An artificial islet organ or a pancreas comprising the population of cells according to claim 23.

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