SC-β cells and compositions and methods for producing same

Abstract

To provide SC-β cells and compositions, and methods for generating the same.SOLUTION: A composition comprises a population of cells, where the population comprises a plurality of non-native pancreatic β-cells. The non-native pancreatic β-cells (a) express INS, PDX1, NKX6.1, and one or more of genes PC2, MNX1 and ABCC8, (b) exhibit an in vitro glucose-stimulated insulin secretion response when subjected to a first glucose challenge, and (c) do not express one or any of somatostatin and glucagon.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 61 / 833,898, filed June 11, 2013, and U.S. Provisional Application No. 61 / 972,212, filed March 28, 2014, the contents of which are incorporated by reference in their entireties.

[0002] The present invention relates to SC-β cells and compositions and methods for their production. [Background technology]

[0003] To date, studies have generated either abnormally functioning insulin-expressing cells that do not secrete adequate amounts of insulin in response to continuously changing glucose levels, or pancreatic progenitor cells that can mature into functional insulin-expressing cells only three months after transplantation into a mouse host (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). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Cheng, X., Ying, L., Lu, L., Galvao, AM, Mills, JA, Lin, HC, Kotton, DN, Shen, SS, Nostro, MC, et al. (2012). Self-renewing endodermal progenitor lines generated from human pluripotent stem cells. Cell Stem Cell 10, 371-384. Summary of the Invention [Problem to be solved by the invention]

[0005] In contrast to normal pancreatic islets or scattered adult beta cells, which can repeatedly release high levels of insulin in response to high levels of glucose in a "glucose-stimulated insulin secretion" (GSIS) assay, hPSC-based insulin-expressing cells generated by existing methods fail to adequately secrete insulin in response to the addition of various concentrations of glucose. Thus, there is a need for methods to obtain hPSC-derived cells that exhibit the phenotype of normal pancreatic islets or mature adult beta cells. [Means for solving the problem]

[0006] In some aspects, the present disclosure provides stem cell-based beta cells (SC-β).

[0007] In some embodiments, the cells are mature. In some embodiments, the cells exhibit a glucose-stimulated insulin secretion (GSIS) response in vitro. In some embodiments, the cells exhibit a GSIS response in vivo. In some embodiments, the cells exhibit a glucose-stimulated insulin secretion (GSIS) response in vitro and in vivo. In some embodiments, the cells exhibit a GSIS response to at least one glucose challenge. In some embodiments, the cells exhibit a GSIS response to at least two consecutive glucose challenges. In some embodiments, the cells exhibit a GSIS response to at least three consecutive glucose challenges. In some embodiments, the GSIS response is observed immediately after transplantation of the cells into a human or animal. In some embodiments, the GSIS response is observed within about 24 hours after transplantation of the cells into a human or animal. In some embodiments, the GSIS response is observed within about two weeks after transplantation of the cells into a human or animal. In some embodiments, the stimulation index of the cells, characterized by the ratio of insulin secreted in response to a high glucose concentration compared to a low glucose concentration, is similar to the stimulation index of endogenous mature pancreatic beta cells. In some embodiments, the stimulation index is 1 or greater, or 1.1 or greater, or 1.3 or greater, or 2 or greater, or 2.3 or greater, or 2.6 or greater. In some embodiments, the cells exhibit cytokine-induced apoptosis in response to a cytokine. In some embodiments, the cytokine is selected from the group consisting of interleukin-1β (IL-β), interferon-γ (INF-γ), tumor necrosis factor-α (TNF-α), and combinations thereof. In some embodiments, insulin secretion from the cells 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 mimetic, a sulfonylurea, a meglitinide, and combinations thereof. In some embodiments, the cells are monohormonal. In some embodiments, the cells exhibit a morphology similar to that of endogenous mature pancreatic β cells.In some embodiments, the cells exhibit encapsulated crystalline insulin granules under electron microscopy similar to insulin granules in endogenous mature pancreatic beta cells. In some embodiments, the cells exhibit a slow rate of replication. In some embodiments, the cells exhibit glucose-stimulated Ca2+ expression similar to GSCF in endogenous mature pancreatic beta cells. 2+In some embodiments, the cells exhibit increased GSCF flux (GSCF) to at least one glucose challenge. In some embodiments, the cells exhibit a GSCF response to at least two glucose challenges. In some embodiments, the cells exhibit a GSCF response to at least three glucose challenges. In some embodiments, the cells exhibit increased calcium flux. In some embodiments, the increased calcium flux comprises an increased amount of influx or a reduced influx ratio relative to high glucose concentrations. In some embodiments, the cells express at least one marker characteristic of endogenous mature pancreatic beta cells selected from the group consisting of insulin, C-peptide, PDX1, MAFA, NKX6-1, PAX6, neuroD1, glucokinase (GCK), SLC2A1, PCSK1, KCNJ11, ABCC8, SLC30A8, SNAP25, RAB3A, GAD2, PTPRN, NKX2-2, and Pax4. In some embodiments, the cells do 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) a glandular cell marker selected from the group consisting of i) amylase and ii) carboxypeptidase A (CPAl); c) an alpha 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 cells are differentiated in vitro from insulin-positive endocrine cells or precursors thereof selected from the group consisting of Nkx6-1-positive pancreatic progenitor cells, Pdx1-positive pancreatic progenitor cells, and pluripotent stem cells. In some embodiments, the pluripotent stem cells are selected from the group consisting of embryonic stem cells and induced pluripotent stem cells. In some embodiments, the cells are human. In some embodiments, the cells are not genetically modified. In some embodiments, the cells are genetically modified. In some embodiments, the insulin produced per cell is between 0.5 and 10 μIU per 1000 cells after 30 minutes of incubation in high glucose concentration.In some embodiments, the insulin produced per cell is about 2.5 μIU per 1000 cells after 30 minutes of incubation in high glucose concentrations, hi some embodiments, the incubation occurs ex vivo.

[0008] In some aspects, the present disclosure provides cell lines comprising SC-β cells. In some embodiments, the cell lines stably express insulin. In some embodiments, the cells can be frozen, thawed, and expanded for at least 30 passages without obvious morphological changes, with a doubling time of between about 24 and 44 hours.

[0009] In some aspects, the present disclosure provides a method for generating SC-β cells from insulin-positive endocrine cells, comprising contacting a cell population comprising insulin-positive endocrine cells with at least two β cell maturation factors comprising a) a transforming growth factor β (TGF-β) signaling inhibitor and b) a thyroid hormone signaling pathway activator under conditions that promote cell cluster formation, thereby inducing in vitro maturation of at least one insulin-positive endocrine cell in the population to an SC-β cell.

[0010] In some embodiments, the SC-β cells 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 morphology of the SC-β cells resembles that of endogenous mature β cells. In some embodiments, the SC-β cells exhibit a glucose-stimulated insulin secretion (GSIS) response in vitro and / or in vivo. In some embodiments, the GSIS response is observed immediately after transplantation of the SC-β cells into a subject. In some embodiments, the GSIS response is observed within about 24 hours after transplantation of the SC-β cells into a subject. In some embodiments, the GSIS response is observed within about 2 weeks after transplantation of the SC-β cells into a subject. In some embodiments, the cell population is contacted with the TGF-β signaling pathway inhibitor at a concentration between 100 nM and 100 μM. 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 TGF-β receptor type I kinase signaling. In some embodiments, the TGF-β signaling pathway inhibitor comprises Alk5 inhibitor II. In some embodiments, the TGF-β signaling pathway inhibitor comprises an analog or derivative of Alk5 inhibitor II. In some embodiments, the cell population is contacted with the thyroid hormone signaling pathway activator at a concentration between 0.1 μM 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 comprises 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 a protein kinase inhibitor. In some embodiments, the cell population is contacted with a 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 beta cell maturation factor. In some embodiments, the at least one additional beta 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-H101. In some embodiments, the at least one additional beta cell maturation factor comprises an O-GlcNAcase inhibitor. In some embodiments, the cell population is contacted with the O-GlcNAcase inhibitor at a concentration between 100 nM and 100 μM. In some embodiments, the cell population is contacted with the O-GlcNAcase inhibitor at a concentration of 10 nM to 10 μM. In some embodiments, the O-GlcNAcase inhibitor comprises Thiamet G. In some embodiments, the cell population is cultured in a suitable culture medium. In some embodiments, the suitable culture medium comprises pancreatic islet culture medium (CMRLS) or Connought Medical Research Laboratories 1066 supplemented with components 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 that promote cell cluster formation comprise 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 insulin-positive endocrine cell in the cell population into at least one SC-β cell. In some embodiments, the period of time comprises at least 7 days, hi some embodiments, the period of time comprises between 7 and 21 days.In some embodiments, the period of time comprises between 7 and 14 days. In some embodiments, the period of time comprises between 10 and 14 days. In some embodiments, the period of time comprises 14 days. In some embodiments, the beta cell maturation factor is replenished every two days. In some embodiments, at least 1% of the insulin-positive endocrine cells in the cell population are induced to mature into SC-beta cells. In some embodiments, at least 99% of the insulin-positive endocrine cells in the population are induced to mature into SC-beta cells. In some embodiments, at least 30% of the resulting cells in the population comprise SC-beta cells. In some embodiments, the SC-beta cells express C-peptide, insulin, NKX6-1, and Pdx1, or co-express NKX6-1 and C-peptide. In some embodiments, the insulin-positive endocrine cells also express Pdx1 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 induced pluripotent stem cells. In some embodiments, the SC-β cells comprise human cells. In some embodiments, the generation of the SC-β cells in vitro is scalable.

[0011] In some aspects, the present disclosure provides an isolated population of SC-β cells produced according to the methods described herein.

[0012] In some aspects, the present disclosure provides microcapsules comprising an isolated population of SC-β cells encapsulated therein.

[0013] In some aspects, the present disclosure provides compositions comprising a population of SC-β cells produced according to the methods described herein.

[0014] In some aspects, the present disclosure provides assays comprising isolated populations of SC-β cells produced according to the methods described herein.

[0015] In some embodiments, the assay is for use in identifying 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, β-cell death, β-cell function, β-cell susceptibility to immune attack, or β-cell susceptibility to dedifferentiation or differentiation. In some embodiments, the assay is for use in identifying one or more candidate agents that promote the differentiation of at least one insulin-positive endocrine cell or precursor thereof into at least one SC-β cell.

[0016] In some aspects, the present disclosure provides methods of treating a subject in need thereof, comprising administering to the subject a composition comprising an isolated population of SC-β cells produced according to the methods described herein. In some embodiments, the SC-β cells are microencapsulated. In some embodiments, the SC-β cells are produced from a population of pluripotent stem cells obtained from the same subject to which the SC-β cells are administered. In some embodiments, the SC-β cells are produced from a population of iPS cells, which are obtained from cells obtained from the same subject to which the SC-β cells are administered. In some embodiments, the subject has diabetes or is at increased risk of developing diabetes. In some embodiments, the diabetes is selected from the group consisting of type 1 diabetes, type 2 diabetes, type 1.5 diabetes, and prediabetes. In some embodiments, the subject has a metabolic disorder or is at increased risk of developing a metabolic disorder.

[0017] In some aspects, the present disclosure relates to the use of an isolated population of SC-β cells produced by the method of any one of claims 41 to 102 for administration to a subject in need thereof.

[0018] In some embodiments, the isolated population of SC-β cells is microencapsulated and administered to the subject. In some embodiments, the subject has diabetes or is at increased risk of developing diabetes. In some embodiments, the diabetes is selected from the group consisting of type 1 diabetes, type 2 diabetes, type 1.5 diabetes, and prediabetes. In some embodiments, the subject has a metabolic disorder or is at increased risk of developing a metabolic disorder.

[0019] In some aspects, the present disclosure provides a culture medium comprising: a) an Alk5 inhibitor; b) triiodothyronine (T3); optionally, c) staurosporine; and optionally d) a CMRLS or a component of a CMRLS.

[0020] In some aspects, the present disclosure includes the use of the culture medium to induce in vitro maturation of insulin-positive endocrine cells into SC-β cells, wherein the SC-β cells exhibit a GSIS response both in vitro and / or in vivo.

[0021] In some aspects, the present disclosure provides a method for producing NKX6-1-positive pancreatic progenitor cells from Pdx1-positive pancreatic progenitor cells, comprising contacting a cell population comprising Pdx1-positive pancreatic progenitor cells with a) at least one growth factor from the fibroblast growth factor (FGF) family, b) at least two beta cell maturation factors including a sonic hedgehog pathway inhibitor, and optionally c) a low concentration of a retinoic acid (RA) signaling pathway activator for a period of at least 5 days under conditions that promote cell cluster formation, to induce differentiation of at least one Pdx1-positive pancreatic progenitor cell in the population into an NKX6-1-positive pancreatic progenitor cell, wherein the NKX6-1-positive pancreatic progenitor cell expresses NKX6-1.

[0022] In some embodiments, the cell population is contacted with the at least one growth factor from the FGF family at a concentration between 1 ng / mL and 100 ng / mL. In some embodiments, the cell population is 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 (KGF). 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 cell population is not contacted with the RA signaling pathway activator. In some embodiments, the cell population is contacted with the RA signaling pathway activator at a concentration between 0.01 μM and 1.0 μM. In some embodiments, the cell population is 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 cell population is contacted with the SHH pathway inhibitor at a concentration between 0.1 μM and 0.5 μM. In some embodiments, the cell population is contacted with the SHH pathway inhibitor at a concentration of 0.25 μM. In some embodiments, the SHH pathway inhibitor comprises Sant1. The method comprises exposing the cell population to at least one additional beta cell maturation factor. In some embodiments, the at least one additional beta cell maturation factor comprises at least one growth factor from the EGF family. In some embodiments, the cell population is exposed to the at least one growth factor from the EGF family at a concentration between 2 ng / mL and 200 ng / mL. In some embodiments, the cell population is exposed to 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 is selected from the group consisting of betacellulin and EGF. In some embodiments, the cell population is cultured in an appropriate culture medium. In some embodiments, the conditions promoting cell cluster formation comprise suspension culture. In some embodiments, the beta cell maturation factor is replenished every two days.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 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, the BMP signaling pathway inhibitor is removed from the suspension culture prior to 5 days. In some embodiments, the BMP signaling pathway inhibitor comprises LDN193189. In some embodiments, at least 10% of the Pdx1-positive pancreatic progenitor cells in the population are induced to differentiate into NKX6-1-positive pancreatic progenitor cells. In some embodiments, at least 95% of the Pdx1-positive pancreatic progenitor cells in the population are induced to differentiate into NKX6-1-positive pancreatic progenitor cells. In some embodiments, the NKX6-1-positive pancreatic progenitor cells express Pdx1, NKX6-1, and FoxA2. In some embodiments, the Pdx1-positive pancreatic progenitor cells are produced from a population of pluripotent stem cells selected from the group consisting of embryonic stem cells and induced pluripotent stem cells.

[0023] In some aspects, the present disclosure provides an isolated population of NKX6-1-positive pancreatic progenitor cells obtained by the methods described herein.

[0024] In some aspects, the present disclosure provides microcapsules comprising an isolated population of NKX6-1-positive pancreatic progenitor cells encapsulated therein.

[0025] In some aspects, the present disclosure provides compositions comprising an isolated population of NKX6-1-positive pancreatic progenitor cells produced according to the methods described herein.

[0026] In some aspects, the present disclosure provides assays comprising an isolated population of NKX6-1-positive pancreatic progenitor cells produced according to the methods described herein.

[0027] In some embodiments, the assay is for use in identifying one or more candidate agents that promote the differentiation of at least one Pdx1-positive pancreatic progenitor cell or precursor thereof into an NKX6-1-positive pancreatic progenitor cell.

[0028] In some aspects, the present disclosure provides a method of treating a subject in need thereof, comprising administering to the subject a composition comprising an isolated population of NKX6-1-positive pancreatic progenitor cells produced according to the methods described herein.

[0029] In some embodiments, the NKX6-1-positive pancreatic progenitor cells are produced from a population of pluripotent stem cells obtained from the same subject to which the NKX6-1-positive pancreatic progenitor cells are administered. In some embodiments, the NKX6-1-positive pancreatic progenitor cells are microencapsulated. In some embodiments, the subject has diabetes or is at increased risk of developing diabetes. In some embodiments, the diabetes is selected from the group consisting of type I diabetes, type II diabetes, type 1.5 diabetes, and prediabetes. In some embodiments, the subject has a metabolic disorder or is at increased risk of developing a metabolic disorder.

[0030] In some aspects, the present disclosure 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 113 to 142 for differentiation into SC-β cells.

[0031] In some aspects, the present disclosure includes the use of an isolated population of NKX6-1-positive pancreatic progenitor cells produced by the methods described herein for administration to a subject in need thereof.

[0032] In some embodiments, the isolated population of NKX6-1-positive pancreatic progenitor cells is microencapsulated and administered to the subject. In some embodiments, the subject has diabetes or is at increased risk of developing diabetes. In some embodiments, the diabetes is selected from the group consisting of type I diabetes, type II diabetes, type 1.5 diabetes, and prediabetes. In some embodiments, the subject has a metabolic disorder or is at increased risk of developing a metabolic disorder.

[0033] In some aspects, the present disclosure provides a culture medium comprising a) KGF, b) SANT1, and optionally c) RA, wherein the culture medium is substantially free of PdbU and LDN193189. In some embodiments, the present disclosure includes use of the culture medium of claim 160 for inducing in vitro differentiation of Pdx1-positive pancreatic progenitor cells into NKX6-1-positive pancreatic progenitor cells.

[0034] In some aspects, the present disclosure provides a method for producing insulin-positive endocrine cells from NKX6-1-positive pancreatic progenitor cells, comprising contacting a cell population comprising NKX6-1-positive pancreatic progenitor cells with at least two beta cell maturation factors, including a) a TGF-β signaling pathway inhibitor and b) a thyroid hormone signaling pathway activator, under conditions that promote cell cluster formation, 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 and 100 μM. In some embodiments, the cell population is contacted with the TGF-β signaling pathway inhibitor at a concentration of between 10 μM and 10 μM. In some embodiments, the TGF-β signaling pathway comprises TGF-β receptor type I kinase signaling. In some embodiments, the TGF-β signaling pathway inhibitor comprises Alk5 inhibitor II. In some embodiments, the cell population is contacted with the thyroid hormone signaling pathway activator at a concentration between 0.1 μM 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 comprises triiodothyronine (T3). In some embodiments, the method comprises contacting the cell population with at least one additional beta cell maturation factor. In some embodiments, the at least one additional beta cell maturation factor comprises a gamma-secretase inhibitor. In some embodiments, the cell population is contacted with the gamma-secretase inhibitor at a concentration between 0.1 μM and 10 μM. In some embodiments, the cell population is contacted with the gamma-secretase inhibitor at a concentration of 1 μM. In some embodiments, the gamma-secretase inhibitor comprises XXI. In some embodiments, the gamma-secretase inhibitor comprises DAPT. In some embodiments, the at least one additional beta 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 between 2 ng / mL and 200 ng / mL. In some embodiments, the cell population is 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 at least one additional beta cell maturation factor comprises a low concentration of a retinoic acid (RA) signaling pathway activator. In some embodiments, the cell population is contacted with the RA signaling pathway activator at a concentration between 0.01 μM and 1.0 μM. In some embodiments, the cell population is 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 at least one additional beta cell maturation factor comprises a sonic hedgehog (SHH) pathway inhibitor. In some embodiments, the cell population is contacted with the SHH pathway inhibitor at a concentration between 0.1 μM and 0.5 μM. In some embodiments, the cell population is contacted with the SHH pathway inhibitor at a concentration of 0.25 μM. In some embodiments, the SHH pathway inhibitor comprises Sant1. 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 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 between 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 promoting cell cluster formation include 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 NKX6-1-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 beta cell maturation factor is replenished to the suspension culture every two days. 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 Pdx1, NKX6-1, NKX2-2, Mafb, glis3, Sur1, 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 induced pluripotent stem cells.

[0035] In some aspects, the present disclosure provides an isolated population of insulin-positive endocrine cells produced according to the methods described herein.

[0036] In some aspects, the present disclosure provides microcapsules comprising an isolated population of insulin-positive endocrine cells encapsulated therein. In some embodiments, the present disclosure provides compositions comprising a population of insulin-positive endocrine cells produced according to the methods described herein.

[0037] In some aspects, the present disclosure provides methods of treating a subject in need thereof, comprising administering to the subject a composition comprising an isolated population of insulin-positive endocrine cells produced according to the methods described herein.

[0038] In some embodiments, the insulin-positive endocrine cells are produced from a population of pluripotent stem cells obtained from the same subject to which the insulin-positive endocrine cells are administered. In some embodiments, the insulin-positive endocrine cells are microencapsulated. In some embodiments, the subject has diabetes or is at increased risk of developing diabetes. In some embodiments, the diabetes is selected from the group consisting of type 1 diabetes, type 2 diabetes, type 1.5 diabetes, and pre-diabetes. In some embodiments, the subject has a metabolic disorder or is at increased risk of developing a metabolic disorder.

[0039] In some aspects, the present disclosure includes the use of an isolated population of insulin-positive endocrine cells produced by the methods described herein for differentiation into SC-β cells.

[0040] In some aspects, the present disclosure includes the use of an isolated population of insulin-positive endocrine cells produced by the methods described herein for administration to a subject in need thereof.

[0041] In some embodiments, the isolated population of insulin-positive endocrine cells is microencapsulated and administered to the subject. In some embodiments, the subject has diabetes or is at increased risk of developing diabetes. In some embodiments, the diabetes is selected from the group consisting of type 1 diabetes, type 2 diabetes, type 1.5 diabetes, and pre-diabetes. In some embodiments, the subject has a metabolic disorder or is at increased risk of developing a metabolic disorder.

[0042] In some aspects, the present disclosure provides a culture medium comprising a) a TGF-β signaling pathway inhibitor, b) a TH pathway activator, and at least one beta cell maturation factor selected from the group consisting of i) XXI, ii) betacellulin, iii) a low concentration of a RA signaling pathway activator, and iv) an SHH pathway inhibitor.

[0043] In some aspects, the disclosure includes the use of the culture medium of claim 221 for inducing in vitro differentiation of NKX6-1-positive pancreatic progenitor cells into insulin-positive endocrine cells.

[0044] In some aspects, the present disclosure provides a method for generating SC-β cells, comprising contacting Pdx1-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 under conditions that promote cell cluster formation, to induce in vitro maturation of at least some of the Pdx1-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.

[0045] In some embodiments, the GSIS response is observed (i) immediately after transplantation of the SC-β cells into a subject; (ii) within about 24 hours of transplantation into a subject; or (iii) within about 2 weeks of transplantation into a subject. In some embodiments, the SC-β cells respond 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 is similar to that of endogenous β cells. In some embodiments, the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with the TGF-β signaling pathway inhibitor at a concentration of 100 nM-100 μM. In some embodiments, the Pdx1-positive, NKX6-1-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 TGF-β receptor type I kinase signaling. In some embodiments, the TGF-β signaling pathway inhibitor comprises 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 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 Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with the protein kinase inhibitor. In some embodiments, the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with the protein kinase inhibitor at a concentration between 10 nM and 1 μM. In some embodiments, the Pdx1-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 Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells with a cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor. In some embodiments, the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with the CFTR inhibitor at a concentration between 100 nM and 100 μM. In some embodiments, the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with the CFTR inhibitor at a concentration between 10 nM and 10 μM. In some embodiments, the CFTR inhibitor comprises Gly-H101. In some embodiments, the method comprises contacting the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells with an O-GlcNAcase inhibitor. In some embodiments, the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with the O-GlcNAcase inhibitor at a concentration between 100 nM and 100 μM. In some embodiments, the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with the O-GlcNAcase inhibitor at a concentration between 10 nM and 10 μM. In some embodiments, the O-GlcNAcase inhibitor comprises Thiamet G. In some embodiments, the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are cultured in an appropriate culture medium. In some embodiments, the appropriate culture medium comprises pancreatic islet growth medium (CMRLS) or Connought Medical Research Laboratories 1066 supplemented with CMRLS components. 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 that promote cell cluster formation comprise suspension culture. In some embodiments, the Pdx1-positive, NKX6-1-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 Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells into SC-β cells.In some embodiments, the period of time comprises at least 7 days. In some embodiments, the period of time comprises between 7 and 21 days. In some embodiments, the period of time comprises between 7 and 14 days. In some embodiments, the period of time comprises 14 days. In some embodiments, the suspension culture is replenished every two days. In some embodiments, at least 30% of the generated cells comprise SC-β cells. In some embodiments, the SC-β cells express C-peptide, insulin, NKX6-1, Pdx1, or co-express NKX6-1 and C-peptide. In some embodiments, the SC-β cells comprise human cells. In some embodiments, the in vitro generation of SC-β cells is capable of being scaled up.

[0046] In some embodiments, the insulin-positive endocrine cells are obtained by contacting Pdx1-positive, NKX6-1-positive pancreatic progenitor cells with i) a TGF-β signaling pathway inhibitor and ii) a thyroid hormone signaling pathway activator under conditions that promote cell cluster formation, thereby inducing 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.

[0047] In some embodiments, the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells are contacted with the TGF-β signaling pathway inhibitor at a concentration of between 100 nM and 100 μM. In some embodiments, the Pdx1-positive, NKX6-1-positive pancreatic progenitor 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 TGF-β receptor type I kinase signaling. In some embodiments, the TGF-β signaling pathway inhibitor comprises Alk5 inhibitor II. In some embodiments, the Pdx1-positive, NKX6-1-positive pancreatic progenitor 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 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 comprises triiodothyronine (T3). In some embodiments, the method comprises contacting the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells with at least one of: i) an SHH 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 Pdx1-positive, NKX6-1-positive pancreatic progenitor cells are contacted with the SHH pathway inhibitor at a concentration between 0.1 μM and 0.5 μM. In some embodiments, the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells are contacted with the SHH pathway inhibitor at a concentration of 0.25 μM. In some embodiments, the SHH pathway inhibitor comprises Sant1. In some embodiments, the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells are contacted with the RA signaling pathway activator at a concentration between 0.01 μM and 1.0 μM. In some embodiments, the Pdx1-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 Pdx1-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 Pdx1-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 Pdx1-positive, NKX6-1-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 and 200 ng / mL. In some embodiments, the Pdx1-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 Pdx1-positive, NKX6-1-positive pancreatic progenitor cells are not contacted with the protein kinase inhibitor. In some embodiments, the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells are contacted with the protein kinase inhibitor. In some embodiments, the Pdx1-positive, 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 Pdx1-positive, NKX6-1-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 between 1 mM and 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 cluster formation comprise suspension culture.In some embodiments, the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells are maintained in suspension culture for a period of time sufficient to induce differentiation of at least a portion of the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells into Pdx1-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 replenished every two days. In some embodiments, at least 15% of the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells are induced to differentiate into Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells. In some embodiments, at least 99% of the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells are induced to differentiate into Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells.

[0048] In some embodiments, the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells are obtained by contacting Pdx1-positive pancreatic progenitor cells with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally iii) a low concentration of an RA signaling pathway activator for a period of 5 days under conditions that promote cell cluster formation, thereby inducing differentiation of at least some of the Pdx1-positive pancreatic progenitor cells into Pdx1-positive, NKX6-1-positive pancreatic progenitor cells, wherein the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells express Pdx1 and NKX6-1.

[0049] In some embodiments, the Pdx1-positive pancreatic progenitor cells are contacted with the at least one growth factor from the FGF family at a concentration between 1 ng / mL and 100 ng / mL. In some embodiments, the Pdx1-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 (KGF). 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 Pdx1-positive pancreatic progenitor cells are contacted with the at least one SHH pathway inhibitor at a concentration between 0.1 μM and 0.5 μM. In some embodiments, the Pdx1-positive pancreatic progenitor cells are contacted with the at least one SHH pathway inhibitor at a concentration of 0.25 μM. In some embodiments, the at least one SHH pathway inhibitor comprises Sant1. In some embodiments, the Pdx1-positive pancreatic progenitor cells are contacted with the RA signaling pathway activator at a concentration between 0.01 μM and 1.0 μM. In some embodiments, the Pdx1-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 Pdx1-positive pancreatic progenitor cells with at least one growth factor from the EGF family. In some embodiments, the Pdx1-positive pancreatic progenitor cells are contacted with the at least one growth factor from the EGF family at a concentration between 2 ng / mL and 200 ng / mL. In some embodiments, the Pdx1-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 Pdx1-positive pancreatic progenitor cells are cultured in a suitable culture medium.In some embodiments, the conditions promoting cell cluster formation include suspension culture. In some embodiments, the suspension culture is replenished every two days. In some embodiments, a protein kinase C activator is not added to the suspension culture for five days. In some embodiments, the protein kinase C activator is removed from the suspension culture prior to the fifth day. In some embodiments, the protein kinase C activator comprises PdbU. In some embodiments, a BMP signaling pathway inhibitor is not added to the suspension culture for five days. In some embodiments, the BMP signaling pathway inhibitor is removed from the suspension culture prior to the fifth day. In some embodiments, the BMP signaling pathway inhibitor comprises LDN193189. In some embodiments, at least 10% of the Pdx1-positive pancreatic progenitor cells in the population are induced to differentiate into Pdx1-positive, NKX6-1-positive pancreatic progenitor cells. In some embodiments, at least 95% of said Pdx1-positive pancreatic progenitor cells are induced to differentiate into Pdx1-positive, NKX6-1-positive pancreatic progenitor cells.

[0050] In some aspects, the disclosure provides a method for producing a method of differentiation comprising: a) differentiating pluripotent stem cells in a collection into Pdx1-positive pancreatic progenitor cells; b) differentiating at least some of the Pdx1-positive pancreatic progenitor cells into Pdx1-positive, NKX6-1-positive pancreatic progenitor cells by contacting the Pdx1-positive pancreatic progenitor cells with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally iii) an RA signaling pathway activator, once every two days for a period of five days under conditions that promote cell cluster formation, to induce differentiation of at least some of the Pdx1-positive pancreatic progenitor cells in the collection into NKX6-1-positive pancreatic progenitor cells, wherein the NKX6-1-positive pancreatic progenitor cells express Pdx1 and NKX6-1; c) differentiating the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells under conditions that promote cell cluster formation. and inducing 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 by contacting the cells with i) a TGF-β signaling pathway inhibitor, b) a TH signaling pathway activator, and optionally c) at least one SHH 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 once every two days for a period of between five and seven days, wherein the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells are differentiated into Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells, wherein the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are differentiated into Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells by a method comprising: contacting the cells with i) a TGF-β signaling pathway inhibitor, b) a TH signaling pathway activator, and optionally c) at least one SHH 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 once every two days for a period of between five and seven days.and d) differentiating at least some of the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells into SC-β cells by contacting the Pdx1-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 once every two days for a period of between 7 and 14 days under conditions that promote cell cluster formation, thereby inducing in vitro maturation of at least some of the Pdx1-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.

[0051] In some embodiments, the disclosure provides a method for differentiating at least some of the Pdx1-positive pancreatic progenitor cells into Pdx1-positive, NKX6-1-positive pancreatic progenitor cells by a method comprising: a) differentiating at least some of the pluripotent cells in the collection into Pdx1-positive pancreatic progenitor cells; b) contacting the Pdx1-positive pancreatic progenitor cells with i) KGF, ii) Sant1, and optionally iii) a low concentration of RA, once every two days for a period of five days under conditions that promote cell cluster formation, to induce differentiation of at least one Pdx1-positive pancreatic progenitor cell in the collection into an NKX6-1-positive pancreatic progenitor cell, wherein the NKX6-1-positive pancreatic progenitor cells express Pdx1 and NKX6-1; and c) differentiating at least some of the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells into Pdx1-positive, NKX6-1-positive pancreatic progenitor cells by a method comprising: and (v) XXI and vi) betacellulin, once every two days for a period of between five and seven days, to induce differentiation of at least a portion 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 are differentiated into Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells by a method comprising contacting Pdx1-positive, NKX6-1-positive pancreatic progenitor cells with i) Alk5 inhibitor II, ii) T3, and optionally iii) Sant1, iv) RA, v) XXI and vi) betacellulin once every two days for a period of between five and seven days.and d) differentiating at least some of the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells into SC-β cells by contacting the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells with i) Alk5 inhibitor II, ii) T3, and optionally iii) staurosporine once every two days for a period of between 7 and 14 days under conditions that promote cell cluster formation, thereby inducing in vitro maturation of at least some of the Pdx1-positive, NKX6-1-positive, insulin-producing endocrine cells into SC-β cells, wherein the SC-β cells exhibit a GSIS response in vitro and / or in vivo.

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

[0053] In some aspects, the present disclosure provides an artificial pancreas comprising SC-β cells differentiated in vitro from pluripotent stem cells.

[0054] Unless otherwise indicated, the practice of the present invention will typically employ conventional techniques of cell biology, cell culture, molecular biology, recombinant genetic biology, microbiology, recombinant nucleic acid (e.g., DNA) technology, immunology, and RNA interference (RNAi), which are within the skill of the art. Non-limiting descriptions of certain of these techniques can be found 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, edition as of December 2008; 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 regarding therapeutic agents and human diseases can be found in Goodman and Gilman's 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 edition (July 2009). Non-limiting information regarding genes and genetic disorders can be found in McKusick, VA: Mendelian Inheritance in Man. A Catalog of Human Genes and Genetic Disorders. Baltimore: Johns Hopkins University Press, 1998 (12th edition) or more up-to-date online databases: Online Mendelian Inheritance in Man, OMIM™, McKusick-Nathans Institute of 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 / omim / , and Online Mendelian Inheritance in Animals (OMIA), a database of genes, inherited disorders and traits in animal species (other than human and mouse), at http: / / omia.angis.org.au / contact.shtml.All patents, patent applications, and other publications (e.g., scientific articles, books, websites, and databases) mentioned herein are incorporated by reference in their entirety. In the event of a conflict between this specification and any of the incorporated references, the specification (e.g., including any amendments thereto, which may be based on the incorporated references) will control. Unless otherwise specified, standard, art-accepted meanings of terms are used herein. Standard abbreviations for various terms are used herein.

[0055] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0056] That is, the gist of the present invention relates to the following. Item 1 1. A composition comprising a population of cells, the population comprising a plurality of non-native pancreatic beta cells, the non-native pancreatic beta cells comprising: (a) expressing INS, PDX1, NKX6.1, and one or more of the following genes: PC2, MNX1, or ABCC8; (b) shows the in vitro glucose-stimulated insulin secretory response when subjected to a first glucose challenge; (c) A composition that does not express one or both of somatostatin and glucagon. Section 2 Item 1. The composition of paragraph 1, wherein the plurality of non-native pancreatic beta cells exhibits an in vitro glucose-stimulated insulin secretion response when subjected to a first glucose challenge, a second glucose challenge, and a third glucose challenge, where the first glucose challenge, the second glucose challenge, and the third glucose challenge are applied sequentially. Section 3 2. The composition of paragraph 1, wherein the plurality of non-native pancreatic beta cells is generated in vitro from human stem cells. Section 4 Item 4. The composition of item 3, wherein the stem cells are embryonic stem cells. Section 5 Item 4. The composition of item 3, wherein the stem cells are induced pluripotent cells. Section 6 2. The composition of paragraph 1, wherein the plurality of non-native pancreatic beta cells expresses PC2. Section 7 2. The composition of paragraph 1, wherein the plurality of non-native pancreatic beta cells expresses MNX1. Section 8 2. The composition of paragraph 1, wherein the plurality of non-native pancreatic beta cells expresses ABCC8. Section 9 2. The composition of paragraph 1, wherein the plurality of non-native pancreatic beta cells expresses chromogranin A. Section 10 2. The composition of paragraph 1, wherein the plurality of non-native pancreatic beta cells expresses C-peptide. Section 11 Item 1, wherein the plurality of non-native pancreatic beta cells expresses MAFB. Section 12 2. The composition of paragraph 1, wherein the plurality of non-naturally occurring pancreatic beta cells exhibits an in vitro glucose-stimulated insulin secretion response when subjected to a first glucose challenge at a first glucose concentration and a second glucose challenge at a second glucose concentration, where the first glucose challenge and the second glucose challenge are applied sequentially, wherein the first glucose concentration is greater than the second glucose concentration, and the plurality of non-naturally occurring pancreatic beta cells exhibits a stimulation index of at least 1.1, wherein the stimulation index is equal to the ratio of insulin secreted in response to the first glucose concentration to the second glucose concentration. Section 13 Item 10. The composition of paragraph 1, wherein the plurality of non-native pancreatic beta cells is monohormonal. Section 14 13. The composition of paragraph 12, wherein the insulin secreted by the plurality of non-naturally occurring pancreatic beta cells is at least 0.5 μIU per 1000 cells per 30 minute incubation when the plurality of non-naturally occurring pancreatic beta cells is exposed to at least 20 mM glucose. Section 15 2. The composition of paragraph 1, wherein the plurality of non-native pancreatic beta cells are genetically modified. Section 16 Item 10. The composition of paragraph 1, wherein at least 10% of the cells in the population of cells are a plurality of non-native pancreatic beta cells. Section 17 Item 10. The composition of claim 1, wherein the population of cells is a cluster of cells. Section 18 18. The composition of paragraph 17, wherein the composition comprises multiple clusters of cells. Section 19 The population of cells comprises: a) C-peptide negative / glucagon positive cells; b) C-peptide negative / somatostatin positive cells; c) glucagon-positive / somatostatin-negative cells; d) glucagon-negative / somatostatin-positive cells; or e) Any combination of these Item 1. The composition according to item 1, comprising one of the following: Section 20 Item 2. The composition according to Item 1, wherein at least 3% of the cell population is C-peptide negative / glucagon positive cells or glucagon positive / somatostatin negative cells. Section 21 the plurality of non-native pancreatic beta cells comprising: a) expresses MNX1; b) generated in vitro from human stem cells; c) The composition of paragraph 1, which has a gene expression profile that differs from the gene expression profile of a native beta cell. Section 22 22. The composition of paragraph 21, wherein the plurality of non-native pancreatic beta cells expresses MAFB. Section 23 22. The composition of paragraph 21, wherein the plurality of non-native pancreatic beta cells expresses chromogranin A and C peptide. Section 24 22. The composition of paragraph 21, wherein the plurality of non-native pancreatic beta cells exhibits an in vitro glucose-stimulated insulin secretion response when subjected to a first glucose challenge, a second glucose challenge, and a third glucose challenge, where the first glucose challenge, the second glucose challenge, and the third glucose challenge are applied sequentially. Section 25 22. The composition of claim 21, wherein the population of cells is a cluster of cells. Section 26 22. The composition of paragraph 21, wherein the plurality of non-native pancreatic beta cells are genetically modified. Section 27 22. The composition of paragraph 21, wherein the plurality of non-native pancreatic beta cells expresses PC2 and ABCC8. Section 28 the plurality of non-native pancreatic beta cells comprising: a) expressing PC2, ABCC8, MAFB, chromogranin A and C peptide; b) The composition according to item 21, which exhibits an in vitro glucose-stimulated insulin secretion response when subjected 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 consecutively. Section 29 29. The composition of paragraph 28, wherein the plurality of non-native pancreatic beta cells are genetically modified. Item 30 29. The composition of paragraph 28, wherein the population of cells is a cluster of cells. Item 31 Item 10. The composition of claim 1, wherein the population of cells further comprises a population of cells that express glucagon. [Effects of the Invention]

[0057] The present invention may provide SC-β cells and compositions, as well as methods for producing the same. [Brief explanation of the drawings]

[0058] [Figure 1] Figures 1A and 1B show a comparison between a previously published control differentiation method and the novel direct differentiation method. Figure 1A shows a schematic comparing the exemplary direct differentiation method of the present disclosure for generating INS+ cells from hPSCs compared to a previously published control differentiation method. Figure 1B illustrates histological sections of undifferentiated HUES8 (top row), differentiated to DE (aborted), and differentiated to PP1 (bottom row) using the previously published control differentiation method, stained with OCT4, SOX17, and PDX1, respectively. Scale bar = 100 μm. [Figure 2] Figures 2A, 2B, and 2C illustrate that in vitro generated stem cell-based β (SC-β) cells secrete insulin similarly to primary human β cells in response to multiple consecutive high glucose challenges. Figures 2A, 2B, and 2C are graphs showing ELISA measurements of human insulin secreted from SC-β (Figure 2A), primary β cells (Figure 2B), and PH cells (Figure 2C) consecutively challenged with 2, 20, 2, 20, 2, and 20 mM glucose. After consecutive low / high glucose challenges, cells were depolarized with 30 mM KCl. [Figure 3] Figures 3A, 3B, and 3C illustrate a further biological recapitulation of the in vitro SC-β cell system, which secretes insulin similarly to primary β cells in response to multiple successive high-glucose challenges. The left panel is the same as in Figure 2. Cells: SC-β cells (SC-β; Figure 3A), primary β cells (1°β; Figure 3B), and PH cells (Figure 3C) were sequentially loaded with 2, 20, 2, 20, 2, and 20 mM glucose and 30 mM KCl, and human insulin was measured by ELISA. [Figure 4]Figures 4A, 4B, 4C, 4D, and 4E illustrate that SC-β cells, like primary β cells, mobilize cytosolic Ca2+ in response to multiple consecutive high-glucose challenges. Figure 4A is a schematic diagram of ensemble-level and single-cell-level detection of cytosolic Ca2+ using Fluo-4 AM staining. Measurements at the ensemble level were performed on individual whole clusters (indicated by large red circles in the schematic), while individual cells within intact clusters (indicated by small red circles) were analyzed for single-cell analysis. Figure 4B is a graph showing ensemble 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. Figure 4C shows a fluorescent image of Fluo-4 AM staining used for single-cell analysis. Figure 4D shows representative images showing the location of single cells in response to 3 (yellow), 2 (orange), 1 (blue), and 0 (red) glucose challenges. Figure 4E shows graphical quantification of the frequency of SC-β cells (n = 156), primary β cells (n = 114), and PH cells (n = 138) in response to 20 mM glucose. Scale bar = 100 μm. [Figure 5]Figures 5A, 5B, 5C, 5D, 5E, and 5F illustrate that SC-β expresses human β cell markers at the protein and gene expression levels. Figure 5A shows immunohistochemical images of cells stained with C-peptide (green), NKX6-1 (red), and somatostatin (gray). Figure 5B shows immunohistochemical images of cells stained with C-peptide (green) and PDX1 (red). Figure 5C shows immunohistochemical images of cells stained with C-peptide (green) and glucagon (red) with corresponding DAPI staining (blue). Figure 5D shows representative flow cytometry dot plots and population percentages of cells stained with C-peptide and NKX6-1. Figure 5E shows a hierarchical clustering analysis based on all genes measured by microarray in undifferentiated HUES8, PH, fetal β-cells, and adult primary β-cells sorted for INS (data from Hrvatin et al. (Hrvatin et al., 2014)), and SC-β-cells (SC-β) sorted for INS and NKX6-1. Figure 5F shows a heatmap of the 100 genes with the highest variance across all samples. CP = C-peptide, SST = somatostatin, GCG = glucagon. Scale bar = 100 μm. [Figure 6] Figure 6 shows histology of SC-β cell clusters stained with DAPI (blue), insulin (green), and C-peptide (red). Scale bar = 100 μm. [Figure 7] Figures 7A, 7B, and 7C illustrate further histological staining of SC-β cells. Figure 7A illustrates staining with C-peptide (green) and ISL1 (red). Figure 7B illustrates staining with C-peptide (green) and MAFA (red). Figure 7C illustrates staining with C-peptide (green) and MAFB (red). Scale bar = 100 μm. [Figure 8] Figures 8A, 8B, and 8C show representative flow cytometry dot plots and population percentages of SC-β cells and PH cells stained with C-peptide and SST (Figure 8A), C-peptide and GCG (Figure 8B), and SST and GCG (Figure 8C). [Figure 9]Figures 9A, 9B, and 9C illustrate that SC-β cell granules are structurally similar to primary human β cell granules. Figure 9A shows an electron microscope image of granules highlighting representative crystallized insulin granules (red), early insulin granules (yellow), and mixed endocrine granules (blue). Scale bar = 500 nm. Figure 9B shows a higher magnification image of the granules highlighted in (Figure 9A). Scale bar = 500 nm. Figure 9C shows an electron microscope image of cells labeled with immunogold staining, showing granules containing insulin (smaller 5 nm black dots) and / or glucagon (larger 15 nm black dots). Representative immunogold particles are highlighted by red arrows (insulin) and blue arrows (glucagon). Scale bar = 100 nm. [Figure 10] Figures 10A and 10B illustrate that stem cell-based β (SC-β) cells generated in vitro from hiPSCs secrete insulin similar to primary human β cells in response to multiple sequential high-glucose challenges. Figures 10A and 10B are graphs showing ELISA measurements of human insulin secreted from SC-β generated from nondiabetic cells (Figure 10A) and type 1 diabetic cells (Figure 10B) sequentially challenged with 2, 20, 2, 20, 2, and 20 mM glucose. [Figure 11] Figures 11A, 11B, 11C, 11D, 11E, and 11F show representative flow cytometry dot plots and population percentages of cells stained with C-peptide and NKX6-1 from multiple hiPSC lines. Figures 11A, 11B, and 11C show representative flow cytometry dot plots and population percentages of cells stained with C-peptide and NKX6-1 from a non-diabetic hiPSC line. Figures 11D, 11E, and 11F show representative flow cytometry dot plots and population percentages of cells stained with C-peptide and NKX6-1 from a type 1 diabetic hiPSC line. [Figure 12]Figures 12A, 12B, 12C, and 12D illustrate that transplanted SC-β cells rapidly function in vivo. Figure 12A shows ELISA measurements of human insulin from serum of individual mice transplanted with SC-β cells (cultured for 1 week in the final in vitro step), primary human β cells (1°β), or PH cells. Measurements were performed before (white bars) and 30 minutes after (black bars) glucose injection in mice 2 weeks after transplantation. Figure 12B shows immunohistochemical images of transplanted cells (Figure 12A) stained with C-peptide (green) and PDX1 (red) to confirm the presence of the graft. Figure 12C shows ELISA measurements of human insulin from serum of individual mice transplanted with pancreatic progenitor cells. Measurements were performed before (white bars) and 30 minutes after (black bars) glucose injection in mice 2 weeks after transplantation. Figure 12D is a graph showing ELISA measurements of human insulin from the serum of individual mice transplanted with SC-β cells cultured for 2 weeks during the final in vitro step. Measurements were performed 30 minutes after glucose injection (black bar) in mice 2 weeks after transplantation. nd = not measured. Scale bar = 100 μm. [Figure 13] Figures 13A and 13B illustrate additional histological sections of SC-β cells and PH cells transplanted into mice two weeks earlier. Figure 13A shows a low-magnification image of the transplant stained with DAPI (blue), C-peptide (green), and GCG (red). Scale bar = 200 μM. Figure 13B shows a higher-magnification image of the transplant stained with C-peptide (green) and GCG (red). Scale bar = 100 μM. [Figure 14A]Figures 14A, 14B, and 14C illustrate the use of media at the final steps of differentiation to enable SC-β cells to secrete more insulin in vivo. Figure 14A is a schematic diagram showing the use of various media at various steps of the differentiation process. Figure 14B shows that adding additional factors, e.g., Sant1, XXI, and SSP, to CMRL medium at the final steps of differentiation results in a better glucose-stimulated insulin secretion (GSIS) response by SC-β cells, as measured by the stimulation index between high and low glucose challenges. Figure 14C shows that adding additional factors, e.g., Sant1, XXI, and SSP, to CMRL medium at the final steps of differentiation results in a better glucose-stimulated insulin secretion (GSIS) response by SC-β cells, as measured by the amount of insulin released. [Figure 14B-C] Figures 14A, 14B, and 14C illustrate the use of media at the final steps of differentiation to enable SC-β cells to secrete more insulin in vivo. Figure 14A is a schematic diagram showing the use of various media at various steps of the differentiation process. Figure 14B shows that adding additional factors, e.g., Sant1, XXI, and SSP, to CMRL medium at the final steps of differentiation results in a better glucose-stimulated insulin secretion (GSIS) response by SC-β cells, as measured by the stimulation index between high and low glucose challenges. Figure 14C shows that adding additional factors, e.g., Sant1, XXI, and SSP, to CMRL medium at the final steps of differentiation results in a better glucose-stimulated insulin secretion (GSIS) response by SC-β cells, as measured by the amount of insulin released. [Figure 15A-C]Figures 15A, 15B, 15C, 15D, 15E, 15F, 15G, 15H, and 15I illustrate improvements to the protocol that can improve the survival and quality of the resulting SC-β cells. Figure 15A is a schematic illustration of the protocol. Figure 15B shows how many pure NKX6.1+ endocrine clusters can be generated using the improved protocol (Figure 15B). Figure 15C illustrates how using a Rock inhibitor in steps 3-5 can improve cell survival. Figure 15D illustrates how using activin A with nicotinamide can downregulate SOX2 and improve cell survival. Figure 15E shows that SOX2 and NKX6-1 are mutually exclusive. Figure 15F illustrates how using staurosporine in step 6 results in a near-pure endocrine population. Figure 15G illustrates how using staurosporine in step 6 results in a higher percentage of NKX6-1 / C-peptide+ cells. Figure 15I illustrates how using XXI in combination with Alk5i and T3 in steps 5-6 increases the NeuroD+ population when compared to using Alk5i and T3 alone (Figure 15H). [Figure 15D-E]Figures 15A, 15B, 15C, 15D, 15E, 15F, 15G, 15H, and 15I illustrate improvements to the protocol that can improve the survival and quality of the resulting SC-β cells. Figure 15A is a schematic illustration of the protocol. Figure 15B shows how many pure NKX6.1+ endocrine clusters can be generated using the improved protocol (Figure 15B). Figure 15C illustrates how using a Rock inhibitor in steps 3-5 can improve cell survival. Figure 15D illustrates how using activin A with nicotinamide can downregulate SOX2 and improve cell survival. Figure 15E shows that SOX2 and NKX6-1 are mutually exclusive. Figure 15F illustrates how using staurosporine in step 6 results in a near-pure endocrine population. Figure 15G illustrates how using staurosporine in step 6 results in a higher percentage of NKX6-1 / C-peptide+ cells. Figure 15I illustrates how using XXI in combination with Alk5i and T3 in steps 5-6 increases the NeuroD+ population when compared to using Alk5i and T3 alone (Figure 15H). [Figure 15F-G]Figures 15A, 15B, 15C, 15D, 15E, 15F, 15G, 15H, and 15I illustrate improvements to the protocol that can improve the survival and quality of the resulting SC-β cells. Figure 15A is a schematic illustration of the protocol. Figure 15B shows how many pure NKX6.1+ endocrine clusters can be generated using the improved protocol (Figure 15B). Figure 15C illustrates how using a Rock inhibitor in steps 3-5 can improve cell survival. Figure 15D illustrates how using activin A with nicotinamide can downregulate SOX2 and improve cell survival. Figure 15E shows that SOX2 and NKX6-1 are mutually exclusive. Figure 15F illustrates how using staurosporine in step 6 results in a near-pure endocrine population. Figure 15G illustrates how using staurosporine in step 6 results in a higher percentage of NKX6-1 / C-peptide+ cells. Figure 15I illustrates how using XXI in combination with Alk5i and T3 in steps 5-6 increases the NeuroD+ population when compared to using Alk5i and T3 alone (Figure 15H). [Figure 15H-I]Figures 15A, 15B, 15C, 15D, 15E, 15F, 15G, 15H, and 15I illustrate improvements to the protocol that can improve the survival and quality of the resulting SC-β cells. Figure 15A is a schematic illustration of the protocol. Figure 15B shows how many pure NKX6.1+ endocrine clusters can be generated using the improved protocol (Figure 15B). Figure 15C illustrates how using a Rock inhibitor in steps 3-5 can improve cell survival. Figure 15D illustrates how using activin A with nicotinamide can downregulate SOX2 and improve cell survival. Figure 15E shows that SOX2 and NKX6-1 are mutually exclusive. Figure 15F illustrates how using staurosporine in step 6 results in a near-pure endocrine population. Figure 15G illustrates how using staurosporine in step 6 results in a higher percentage of NKX6-1 / C-peptide+ cells. Figure 15I illustrates how using XXI in combination with Alk5i and T3 in steps 5-6 increases the NeuroD+ population when compared to using Alk5i and T3 alone (Figure 15H). [Figure 16]Figures 16A, 16B, 16C, 16D, 16E, 16F, 16G, 16H, and 16I illustrate the clinical utility of SC-β cells as a diabetes treatment or drug discovery platform. Figure 16A is a schematic diagram of the utility of SC-β cells for treating diabetes or screening for drugs that improve function or replication. Figure 16B is a table listing the diabetes drugs tested and their general therapeutic categories. Figure 16C is a graph showing ELISA measurements of human insulin secreted from plated SC-β cells treated with the indicated drugs at 2 and 20 mM glucose. The p-values ​​shown compare insulin levels at 20 mM glucose between the drugs and the control. Figure 16D is an immunofluorescence image of dispersed and plated SC-β cells without treatment and stained with DAPI (blue), C-peptide (green), and Ki67 (red). Figure 16E shows immunofluorescence images of dispersed and seeded SC-β cells treated with prolactin for 48 hours, stained with DAPI (blue), C-peptide (green), and Ki67 (red). Figure 16F shows graphical quantification of the function of cells coexpressing C-peptide and Ki67. *p<0.05. Figure 16G shows graphs illustrating fasting blood glucose measurements from Akita mice transplanted with SC-β cells (n=6) or PH cells (n=6). *p<0.05 compared with the two cell groups on the same day. Figure 16H shows graphs illustrating blood glucose measurements from progressively diabetic Akita mice transplanted with SC-β cells or PH cells. Measurements were taken before (white bars) and 20 minutes after (black bars) glucose injection in mice transplanted 2 weeks prior. Glucose measurements were saturated at 550 mg / dL. *p<0.05 compared with the two cell groups at the same time point after glucose injection. Figure 16I is a graph showing ELISA measurement of human insulin from serum of Akita mice 20 minutes after glucose injection. Two weeks after transplantation, the mice were challenged with glucose. *p<0.05 compared with the two cell groups. Scale bar = 50 μm. [Figure 17]Figure 17 is a graph illustrating the body weight of Akita mice transplanted with SC-β cells (n=6) or PH cells (n=6). *p<0.05 compared with the two cell groups at 18 and 28 days. DETAILED DESCRIPTION OF THE INVENTION

[0059] Aspects of the present disclosure relate to compositions, methods, kits, and agents for generating stem cell-type beta (SC-β) cells (e.g., mature pancreatic beta cells) from at least one insulin-positive endocrine cell or its precursor (e.g., iPS cells, hESCs, definitive endoderm cells, primitive gut cells, Pdx1-positive pancreatic progenitor cells, Pdx1-positive, NKX6-1-positive pancreatic progenitor cells, Ngn3-positive endocrine progenitor cells, etc.), as well as SC-β cells produced by these compositions, methods, kits, and agents for use in cell therapy, assays (e.g., drug screening), and various treatment methods.

[0060] Furthermore, embodiments of the present disclosure relate to methods for identifying such SC-β cells, which are detectable without the use of a selection marker based on morphological criteria as well as functional characteristics, such as their ability to express insulin, secrete insulin in response to one or more glucose challenges, exhibit a mature GSIS response, organize into pancreatic islets in vivo in the pancreas, and typically have small, spindle-like cells approximately 9-15 μm in diameter.

[0061] Furthermore, aspects of the present disclosure relate to methods for identifying β-cell maturation factors. Those skilled in the art will recognize, or be able to readily ascertain, whether a particular β-cell maturation factor is functional using assays known in the art. For example, the ability of a β-cell maturation factor to convert at least one insulin-positive endocrine cell or its precursor into an SC-β cell can be assessed using the assays disclosed herein. Another convenient assay involves measuring the ability to activate transcription of a reporter construct comprising a β-cell marker binding site operably linked to a nucleic acid sequence encoding a detectable marker, e.g., luciferase. One assay involves determining whether a candidate β-cell maturation factor induces at least one insulin-positive endocrine cell to become an SC-β cell, express a β-cell marker, or exhibit functional characteristics of mature β cells as disclosed herein. Determining such expression of a β-cell marker can be determined using any suitable method, such as immunoblotting. Such assays can be readily employed to identify or confirm the activity of agents that directly convert at least one insulin-positive endocrine cell or its precursor into an SC-β cell.

[0062] The in vitro matured SC-β cells (i.e., pancreatic β cells) generated according to the methods of the present invention described herein demonstrate numerous advantages, including the fact that they undergo glucose-stimulated insulin secretion in vitro, resemble human pancreatic islet β cells in terms of gene expression and ultrastructure, secrete human insulin, and ameliorate hyperglycemia when transplanted into mice, providing a new platform for cell therapy (e.g., transplantation into subjects requiring additional and / or functional β cells), drug screening (e.g., for insulin production / secretion, survival, dedifferentiation, etc.), research (e.g., determining differences in function between normal and diabetic β cells), and regenerative medicine (e.g., using the SC-β cells as a primary cell type to restore pancreatic islets).

[0063] definition

[0064] For convenience, certain terms used herein in the specification, examples, and appended claims are collected here. 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 to which this invention belongs.

[0065] The term "differentiated cells," as used herein, refers to any primary cell that is not naturally pluripotent. That is, the term "differentiated cells" refers to cells of a more specialized cell type obtained from a less specialized cell type (e.g., stem cells, e.g., induced pluripotent stem cells) during the cell differentiation process. Without being bound by theory, during normal ontogeny, pluripotent stem cells can first differentiate into endoderm cells capable of forming pancreatic cells and other endodermal cell types. Further, differentiation of endodermal cells leads to the pancreatic pathway. In this case, approximately 98% of the cells become exocrine, ductular, or matrix cells, and approximately 2% become endocrine cells. Early endocrine cells are pancreatic islet precursors. These precursors can then further differentiate into insulin-producing cells (e.g., functional endocrine cells) that secrete insulin, glucagon, somatostatin, or pancreatic polypeptide. Endodermal cells can also differentiate into other cells of endodermal origin, such as lung, liver, intestine, thymus, etc.

[0066] As used herein, the term "somatic cell" refers to any cell that forms the body of an organ, as opposed to a germline cell. In mammals, germline cells (also known as "gametes") are sperm and eggs that fuse during fertilization to produce a cell called a zygote. From the zygote, the entire mammalian embryo develops. All other cell types in the mammalian body (except sperm and eggs, from which gametocytes and undifferentiated stem cells are formed) are somatic cells. Internal organs, skin, bone, blood, and connective tissue are all composed of somatic cells. In some embodiments, the somatic cell is a "non-embryonic somatic cell." The non-embryonic somatic cell refers to a somatic cell that is not present in an embryo, is derived from an embryo, and does not result in the proliferation of such cells in vitro. In some embodiments, the somatic cell is an "adult somatic cell." The adult somatic cell refers to a cell that is present in an organ or fetus other than an embryo, is derived from the embryo, or results in the proliferation of such cells in vitro. Unless otherwise specified, the method of converting at least one insulin-positive endocrine cell or precursor thereof to an insulin-producing, glucose-responsive cell can be performed both in vivo and in vitro (wherein in vivo is practical when the at least one insulin-positive endocrine cell or precursor thereof is present in a subject; in vitro is practical when using isolated at least one insulin-positive endocrine cell or precursor thereof maintained in culture).

[0067] As used herein, the term "adult cell" refers to a cell found throughout the body after embryonic development.

[0068] As used herein, the term "endodermal cells" refers to cells derived from one of the three main germ cell layers in the very early embryo (the other two being mesoderm and ectoderm). Endoderm is the innermost of the three layers. Endodermal cells differentiate to give rise first to the embryonic digestive tract, and then to the lining of the respiratory and digestive tracts (e.g., the intestine), the liver, and the pancreas.

[0069] As used herein, the term "cells of endodermal origin" refers to any cell that develops or differentiates from endodermal cells. For example, cells of endodermal origin include cells of the liver, lung, pancreas, thymus, gastrointestinal tract, stomach, and thyroid gland. Without being bound by theory, hepatic and pancreatic precursors (also called pancreatic precursors) develop from endodermal cells into the embryonic foregut. Shortly after their specification, hepatic and pancreatic precursors rapidly acquire distinct cellular functions and regenerative capabilities. These changes are driven by inductive signals and gene regulatory factors that are highly conserved among vertebrates. This organ development and regeneration is stimulated by the intense demand 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 demonstrated evolutionarily conserved inductive signal and transcription factor networks that drive hepatic and pancreatic cell differentiation and provide guidance for methods to promote hepatic and β cell differentiation from various stem and progenitor cell types.

[0070] As used herein, the term "definitive endoderm" refers to cells differentiated from endoderm cells. Such cells can differentiate into SC-β cells (e.g., pancreatic β cells). Definitive endoderm cells express the marker Sox17. Other markers specific to definitive endoderm cells include, but are not limited to, MIXL2, GATA4, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CXCR4, cerberus, OTX2, goosecoid protein, C-Kit, CD99, CMKOR1, and CRIP1. Specifically, definitive endoderm cells herein express Sox17, and in some embodiments, Sox17 and HNF3B, but do not express significant levels of GATA4, SPARC, APF, or DAB. Definitive endoderm cells are not positive for the marker Pdx1 (e.g., they are Pdx1 negative). Definitive endoderm cells have the potential to differentiate into cells including liver, lung, pancreas, thymus, intestine, stomach, and thyroid cells. The expression of Sox17 and other markers of the definitive endoderm can be assessed by any method known to those skilled in the art, such as immunochemistry or quantitative RT-PCR using anti-Sox17 antibodies.

[0071] The term "pancreatic endoderm" refers to cells of endodermal origin that can differentiate into multiple pancreatic lineages, e.g., pancreatic β cells, but no longer have the capacity to differentiate into non-pancreatic lineages.

[0072] As used herein, the term "primitive gut cells" or "gut cells" refers to cells differentiated from endoderm cells. These cells can differentiate into SC-β cells (e.g., pancreatic β cells). Primitive gut cells express at least one of the following markers: HNF1-β, HNF3-β, or HNF4-α. Primitive gut cells have the ability to differentiate into cells including lung, liver, pancreatic, stomach, and intestinal cells. Expression of HNF1-β and other markers of the primitive gut can be assessed by any method known to those skilled in the art, for example, immunochemistry using anti-HNF1-β antibodies.

[0073] The terms "pancreatic progenitor," "pancreatic endocrine progenitor," "pancreatic precursor," or "pancreatic endocrine progenitor" are used interchangeably herein to refer to stem cells capable of giving rise to pancreatic hormone-expressing cells that can form pancreatic endocrine cells, pancreatic exocrine cells, or pancreatic ductal cells. These cells are committed to differentiation into at least one type of pancreatic cell, e.g., beta cells, which produce insulin; alpha cells, which produce glucagon; delta cells (or D cells), which produce somatostatin; and / or F cells, which produce pancreatic polypeptide. Such cells may express at least one of the following markers: NGN3, NKX2.2, NeuroD, ISL-1, Pax4, Pax6, or ARX.

[0074] As used herein, the term "Pdx1-positive pancreatic progenitor" refers to a pancreatic endoderm (PE) cell that has the potential to differentiate into an SC-β cell, e.g., a pancreatic β cell. Pdx1-positive pancreatic progenitor expresses the marker Pdx1. Other markers include, but are not limited to, Cdcp1, Ptf1a, HNF6, or NRx2.2. The expression of Pdx1 can be assessed by any method known to those skilled in the art, such as immunochemistry or quantitative RT-PCR using an anti-Pdx1 antibody.

[0075] As used herein, the term "pdx1-positive, NKX6-1-positive pancreatic progenitors" refers to cells that are pancreatic endoderm (PE) cells and have the potential to differentiate into insulin-producing cells, e.g., pancreatic β cells. pdx1-positive, NKX6-1-positive pancreatic progenitors express the markers Pdx1 and NKX6-1. Other markers include, but are not limited to, Cdcp1, Ptf1a, HNF6, or NRx2.2. NKX6-1 expression can be assessed by any method known to those skilled in the art, such as immunochemistry or quantitative RT-PCR using an anti-NKX6-1 antibody.

[0076] As used herein, the term "Ngn3-positive endocrine progenitor" refers to a precursor of pancreatic endocrine cells that expresses the transcription factor neurogenin-3 (Ngn3). These progenitor cells are differentiated from pluripotent stem cells and can differentiate into only a few cell types. In particular, Ngn3-positive endocrine progenitor cells have the potential to differentiate into five pancreatic endocrine cell types (α, β, δ, ε, and PP). The expression of Ngn3 can be assessed by any method known to those skilled in the art, such as immunochemistry or quantitative RT-PCR using anti-Ngn3 antibodies.

[0077] The terms "neuroD" and "neuroD1" are used interchangeably to identify proteins and their encoding genes expressed in pancreatic endocrine cells.

[0078] The terms "insulin-positive beta-like cell" and "insulin-positive endocrine cell" refer to cells (e.g., pancreatic endocrine cells) that display at least one marker indicative of a pancreatic beta cell and express insulin, but lack the GSIS response characteristic of endogenous beta cells.

[0079] The term "precursor thereof" in relation to insulin-positive endocrine cells means any cell that can differentiate into an insulin-positive endocrine cell, such as a pluripotent stem cell, an adult endoderm cell, an primitive gut cell, a pancreatic progenitor cell or an endocrine progenitor cell, when cultured under conditions suitable for differentiating the precursor somatic cell into the insulin-positive endocrine cell.

[0080] The terms "stem cell-based β cells," "SC-β cells," "functional β cells," "functional pancreatic β cells," and "mature SC-β cells" refer to cells (e.g., pancreatic β cells) that display at least one marker indicative of pancreatic β cells (e.g., PDX-1 or NKX6-1), secrete insulin, and display a GSIS response characteristic of endogenous mature β cells. In some embodiments, the "SC-β cells" include mature pancreatic β cells. It should be understood that, if the method of the present disclosure can derive SC-β cells from any insulin-positive endocrine cell or its precursor using any cell as a starting point (e.g., embryonic stem cells, induced pluripotent stem cells, progenitor cells, partially reprogrammed somatic cells (e.g., somatic cells partially reprogrammed to an intermediate state between induced pluripotent stem cells and the somatic cells and the somatic cells from which they are derived), pluripotent cells, totipotent cells, transdifferentiated versions of any of the aforementioned cells, etc.), the SC-β cells need not be derived (e.g., directly) from stem cells, provided that the method of the present disclosure can derive SC-β cells from any insulin-positive endocrine cell or its precursor using any cell as a starting point (e.g., the present disclosure is not intended to be limited by this method, and may use, for example, embryonic stem cells, induced pluripotent stem cells, progenitor cells, partially reprogrammed somatic cells (e.g., somatic cells partially reprogrammed to an intermediate state between induced pluripotent stem cells and the somatic cells and the somatic cells from which they are derived), pluripotent cells, totipotent cells, transdifferentiated versions of any of the aforementioned cells, etc.). In some embodiments, the SC-β cells exhibit a response 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 is similar to the response of endogenous pancreatic islets (e.g., human pancreatic islets) to multiple glucose challenges. In some embodiments, the morphology of the SC-β cells is similar to that of endogenous β cells. In some embodiments, the SC-β cells exhibit an in vitro GSIS response similar to that of endogenous β cells. In some embodiments, the SC-β cells exhibit an in vivo GSIS response similar to that of endogenous β cells. In some embodiments, the SC-β cells exhibit a GSIS response both in vitro and in vivo similar to that of endogenous β cells. The GSIS response of the SC-β cells can be observed within two weeks of transplantation of the SC-β cells into a host (e.g., a human or animal). In some embodiments, the SC-β cells package insulin into secretory granules. In some embodiments, the SC-beta cells exhibit encapsulated crystalline insulin granules.In some embodiments, the SC-β cells exhibit a stimulation index greater than 1. In some embodiments, the SC-β cells exhibit a stimulation index greater than 1.1. In some embodiments, the SC-β cells exhibit a stimulation index greater than 2. In some embodiments, the SC-β cells exhibit cytokine-induced apoptosis in response to a cytokine. In some embodiments, insulin secretion from the SC-β cells is enhanced in response to a known antidiabetic agent (e.g., a secretagogue). In some embodiments, the SC-β cells are monohormonal. In some embodiments, the SC-β cells do not abnormally co-express other hormones, e.g., glucagon, somatostatin, or pancreatic polypeptide. In some embodiments, the SC-β cells exhibit a slow rate of replication. In some embodiments, the SC-β cells exhibit intracellular Ca in response to glucose. 2+ As used herein, the term "exocrine cells" refers to cells of an exocrine gland, i.e., a gland that excretes its secretions through a duct. In certain embodiments, exocrine cells refer to pancreatic exocrine cells. The pancreatic exocrine cells are pancreatic cells that produce enzymes secreted into the small intestine. These enzymes help digest food as it passes through the digestive tract. Pancreatic exocrine cells are also known as islets of Langerhans. The islets secrete two hormones, insulin and glucagon. Pancreatic exocrine cells can be one of several cell types: alpha-2 cells (which produce the hormone glucagon); beta cells (which produce the hormone insulin); and alpha-1 cells (which produce the modulator somatostatin). As used herein, non-insulin-producing exocrine cells refer to alpha-2 cells or alpha-1 cells. It should be noted that the term pancreatic exocrine cells encompasses "pancreatic endocrine cells," which refers to pancreatic cells that produce the hormones insulin (e.g., produced by beta cells), glucagon (produced by alpha-2 cells), somatostatin (produced by delta cells), and pancreatic polypeptide (produced by F cells), which are secreted into the bloodstream.

[0081] As used herein, the term "insulin-producing cells" refers to cells differentiated from pancreatic precursors or their precursors. Such cells secrete insulin. Insulin-producing cells, as that term is used herein, include pancreatic β cells and pancreatic β-like cells (i.e., insulin-positive endocrine cells) that synthesize (i.e., transcribe the insulin gene, translate proinsulin mRNA, and modify the proinsulin mRNA into insulin protein), express (i.e., express the trait conveyed by the insulin gene), or secrete (release insulin into the extracellular space) insulin in a constitutive or inducible manner. For example, a population of insulin-producing cells produced by differentiating insulin-positive endocrine cells or their precursors into SC-β cells according to the methods of the present invention are pancreatic β cells or β-like cells that are characteristic of endogenous β cells (e.g., cells with at least one or at least two β-cells) and exhibit a GSIS response similar to that of endogenous adult β cells. The novelty of the present compositions and methods is not negated by the presence of cells in the population that naturally produce insulin (e.g., β cells). For example, it is contemplated that the population of insulin-producing cells produced by the methods disclosed herein may include mature pancreatic β cells or SC-β cells, and may also include non-insulin-producing cells (i.e., cells of a β cell-like phenotype, except that they do not produce or secrete insulin).

[0082] As used herein, the terms "endogenous beta cells," "endogenous mature pancreatic beta cells," or "endogenous pancreatic beta cells" refer to insulin-producing cells of the pancreas or cells of the pancreatic beta cell (β cell) phenotype. The pancreatic beta cell phenotype is well known to those of skill in the art and includes, for example, insulin secretion in response to elevated glucose levels, expression of markers such as c-peptide, Pdx1 polypeptide, and Glut2, and distinctive morphological characteristics, such as organization into pancreatic islets in the pancreas in vivo, typically comprising small, spindle-like cells approximately 9-15 μm in diameter.

[0083] As used herein, the terms "SC-β cells," "pancreatic β-like cells," and "mature pancreatic β-like" refer to cells produced by the methods described herein that express at least 15% of the amount of insulin expressed by endogenous pancreatic β cells, 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%, e.g., at least about 1.5-fold, or at least about 2-fold, or at least about 2.5-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or more than about 5-fold the amount of insulin secreted by endogenous pancreatic β cells, or that exhibit at least one or at least two characteristics of endogenous pancreatic β cells, such as, but not limited to, insulin secretion in response to glucose and expression of β cell markers, e.g., c-peptide, Pdx1, and glut-2. In one embodiment, the SC-β cells are not immortalized cells (i.e., they grow indefinitely in culture). In one embodiment, the SC-β cells are not transformed cells, e.g., cells that exhibit transformation properties, e.g., the absence of growth in soft agar or contact inhibition.

[0084] The term "β cell marker" refers to, but is not limited to, proteins, peptides, nucleic acids, protein and nucleic acid polymorphisms, splice variants, protein or nucleic acid fragments, 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 (Pdx1) polypeptide, insulin, c-peptide, amylin, E-cadherin, Hnf3β, PCI / 3, B2, Nkx2.2, NKX6-1, GLUT2, PC2, ZnT-8, Isll, Pax6, Pax4, NeuroD, Hnflb, Hnf-6, Hnf-3beta, and MafA, as well as those described in Zhang et al., Diabetes. 50(10):2231-6 (2001). In some embodiments, the β cell marker is a nuclear β cell marker. In some embodiments, the beta cell marker is Pdx1 or PH3.

[0085] The term "pancreatic endocrine marker" refers to, but is not limited to, proteins, peptides, nucleic acids, protein and nucleic acid polymorphisms, splice variants, protein or nucleic acid fragments, 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.

[0086] As used herein, the term "non-insulin-producing cells" refers to any cell of endodermal origin that does not essentially synthesize, express, or secrete insulin, either constitutively or upon induction. Thus, as used herein, the term "non-insulin-producing cells" excludes pancreatic β cells. Examples of non-insulin-producing cells that can be used in the methods of the present invention include pancreatic non-β cells, such as amylase-producing cells, acinar cells, and cells of ductal adenocarcinoma cell lines (e.g., CD18, CD11, and Capan-I cells (see Busik et al., 1997; Schaffert et al., 1997)). Non-pancreatic cells of endodermal origin, such as non-pancreatic stem cells and cells of other endocrine and exocrine organs, such as liver cells, thymocytes, thyroid cells, intestinal cells, lung cells, and pituitary cells, can also be used. In some embodiments, the non-insulin-producing endodermal cells can be mammalian cells, or even more specifically, human cells. Examples of this method using mammalian pancreatic non-islets, pancreatic amylase-producing cells, and pancreatic acinar cells are provided herein.

[0087] The term "phenotype" refers to the collective biological characteristics, one or many, that define a cell or organism under a particular set of environmental conditions and factors, regardless of actual genotype.

[0088] As used herein, the term "pluripotent" refers to cells that have the ability to differentiate into two or more differentiated cell types, preferably into cell types specific to all three germ layers, under various conditions. Pluripotent cells are primarily characterized by their ability to differentiate into two or more cell types, preferably into all three germ layers, using, for example, a nude mouse teratoma formation assay. While pluripotency can also be demonstrated by the expression of embryonic stem (ES) cell markers, the preferred test for pluripotency is the ability to differentiate into cells of any of the three germ layers. It should be noted that simply clustering such cells does not, in and of itself, indicate their pluripotency. Reprogrammed pluripotent cells (e.g., iPS cells, as that term is defined herein) also possess the characteristic of long-term passage potential without losing the proliferation potential of the original parent cells. The parent cells generally have the ability to divide only a limited number of times in culture.

[0089] As used herein, the terms "iPS cells" and "induced pluripotent stem cells" are used interchangeably and refer to pluripotent stem cells that have been artificially derived (e.g., induced or fully restored) from non-pluripotent cells, typically adult somatic cells, for example, by inducing the forced expression of one or more genes.

[0090] The terms "progenitor" cell or "precursor" cell are used interchangeably herein to refer to cells that have a more primitive cellular phenotype (i.e., earlier along a developmental pathway or stage than a fully differentiated cell) relative to the cells that can result from differentiation. Progenitor cells often also have significant or very high proliferative potential. Progenitor cells can give rise to multiple distinct differentiated cell types or to a single differentiated cell type, depending on the developmental pathway and the environment in which the cells develop and differentiate.

[0091] As used herein, the term "stem cell" refers to an undifferentiated cell capable of proliferating and giving rise to a number of progenitor cells, which in turn can give rise to differentiated or differentiable daughter cells. The daughter cells themselves can be induced to proliferate and produce progeny that sequentially differentiate into one or more mature cell types, but can also maintain one or more cells with the developmental potential of the parent. The term "stem cell" refers to a subset of progenitors that, under certain circumstances, have the ability or potential to differentiate into a more specialized or differentiated phenotype and maintain the ability to proliferate without substantial differentiation under certain circumstances. In one embodiment, the term stem cell generally refers to a naturally occurring mother cell whose offspring (progeny) often specialize in various directions by differentiation, e.g., acquiring completely individual characteristics, as they arise in the progressive diversity of embryonic cells and tissues. Cell differentiation is a complex process that typically occurs through many cell divisions. Differentiated cells are derived from pluripotent cells, which themselves are derived from pluripotent cells, etc. Although all of these pluripotent cells may be considered stem cells, the range of cell types can vary considerably. Some differentiated cells also have the ability to give rise to cells of higher developmental potential. This ability may be naturally occurring or may be artificially induced based on treatment with various factors. In many biological cases, stem cells are also "pluripotent" because they can produce progeny of two or more distinct cell types. However, this cell type does not require "stemness." Self-renewal is another categorical part of the definition of stem cells, and as used in this document, it is essential. Theoretically, self-renewal can occur through either of two primary mechanisms. Stem cells can divide asymmetrically, with one daughter maintaining the stem cell state and the other daughter expressing some other distinct, specific function and phenotype. Alternatively, some stem cells in a population may divide symmetrically into two stem cells, thereby maintaining the stemness of the population as a whole, while other cells in the population give rise only to differentiated progeny.Formally, cells that begin as stem cells progress toward a differentiated phenotype, but may revert to and re-express 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 cells" includes embryonic stem cells, induced pluripotent stem cells, placental stem cells, and the like.

[0092] With respect to cellular ontogeny, the adjectives "differentiated" or "differentiating" are relative terms, meaning that a "differentiated cell" is a cell that has progressed further down the developmental pathway than the comparison cell. Thus, stem cells can differentiate into lineage-restricted progenitor cells (e.g., mesodermal stem cells). These progenitor cells can then differentiate further down the pathway into other types of progenitor cells (e.g., cardiomyocyte precursors) and then into terminally differentiated cells. These terminally differentiated cells fulfill characteristic roles in specific tissue types and may or may not retain the capacity for further proliferation.

[0093] The term "embryonic stem cells" is used to refer to pluripotent stem cells in the inner cell mass of blastocysts (see U.S. Patent Nos. 5,843,780 and 6,200,806). Such cells can also be obtained from the inner cell mass of blastocysts obtained from somatic cell nuclear transfer (see, e.g., U.S. Patent Nos. 5,945,577, 5,994,619, and 6,235,970). The distinguishing characteristics of embryonic stem cells define their phenotype. Thus, a cell has an embryonic stem cell phenotype if it possesses one or more unique features of an embryonic stem cell, which makes the cell distinguishable from other cells. Exemplary distinguishable embryonic stem cell characteristics include, but are not limited to, gene expression profile, proliferation potential, differentiation potential, karyotype, responsiveness to specific culture conditions, etc.

[0094] The term "adult stem cell" or "ASC" is used to refer to any pluripotent stem cell obtained from non-embryonic tissues, e.g., fetal, juvenile, and adult tissues. Stem cells have been isolated from a wide variety of adult tissues, e.g., blood, bone, bone marrow, brain, olfactory epithelium, skin, pancreas, skeletal muscle, and cardiac muscle. Each of these stem cells can be characterized based on gene expression, factor responsiveness, and morphology in culture. Exemplary adult stem cells include neural stem cells, neural crest stem cells, mesodermal stem cells, hematopoietic stem cells, and pancreatic stem cells. As noted above, stem cells have been found to be present in virtually all tissues. Accordingly, the present invention contemplates that stem cell populations may be isolated from virtually any animal tissue.

[0095] The term "pancreas" refers to a glandular organ that secretes digestive enzymes and hormones. In humans, the pancreas is a yellowish organ approximately 7 inches (17.8 cm) long and 1.5 inches (3.8 cm) wide. The pancreas lies below the stomach and is connected to the small intestine, a muscular, hose-like portion of the digestive tract that extends from the lower end of the stomach (pylorus) to the anal opening. Most pancreatic tissue consists of grape-like clusters of cells that produce a clear fluid (pancreatic juice), which, along with bile from the liver, flows through the common duct into the duodenum. Bile contains three digestive enzymes: tryptase, amylase, and lipase, which, along with intestinal enzymes, complete the digestion of proteins, carbohydrates, and fats, respectively. Dispersed among these enzyme-producing cells in the pancreas are small groups of endocrine cells, called the islets of Langerhans, that secrete two hormones: insulin and glucagon. Pancreatic islets contain several types of cells: alpha-2 cells, which produce the hormone glucagon; beta cells (also referred to herein as "pancreatic beta cells"), which produce the hormone insulin; and alpha-1 cells, which produce the regulator somatostatin. These hormones are secreted directly into the bloodstream, and together they regulate blood glucose levels. Insulin lowers blood sugar levels and increases the amount of glycogen (storage carbohydrate) in the liver. Glucagon has the opposite effect. Malfunction of normally functioning insulin-secreting cells leads to diabetes, or diabetes mellitus.

[0096] As used herein, the term "reprogramming" refers to the process of changing or returning a somatic cell to its differentiated state. The cell can be either partially or terminally differentiated prior to the reprogramming. Reprogramming encompasses the complete reversion of the differentiated state of a somatic cell to a pluripotent cell. Such complete reversion of differentiation produces induced pluripotent (iPS) cells. As used herein, reprogramming encompasses the partial reversion of the differentiated state of a cell, e.g., to a pluripotent state, or to a somatic cell that is neither pluripotent nor multipotent but has lost one or more specific characteristics of a differentiated cell, resulting, for example, in the direct reprogramming of the differentiated cell to a different somatic cell type. Reprogramming generally involves the conversion, e.g., reversion, of at least some inherited patterns of nucleic acid modifications (e.g., methylation), chromatin condensation, epigenetic changes, genomic imprinting, etc., which occur during cell differentiation as a zygote develops into an adult.

[0097] As used herein, the term "agent" refers to any compound or substance, including, but not limited to, small molecules, nucleic acids, polypeptides, peptides, drugs, ions, etc. An "agent" can be any chemical entity, entity, or moiety, including, but not limited to, synthetic and naturally occurring proteinaceous and non-proteinaceous entities. In some embodiments, the agent is a nucleic acid, nucleic acid analog, protein, antibody, peptide, aptamer, oligomer of nucleic acid, amino acid, or carbohydrate, including, but not limited to, protein, oligonucleotide, ribozyme, DNAzyme, glycoprotein, siRNA, lipoprotein, aptamer, and modifications and combinations thereof. In certain embodiments, the agent is a small molecule bearing a chemical moiety. For example, the chemical moiety includes substituted or unsubstituted alkyl, aromatic, or heterocyclyl moieties, such as macrolides, leptomycin, and related natural products or analogs thereof. The compound can be known to have the desired activity and / or properties, or can be selected from a library of diverse compounds.

[0098] As used herein, the term "contacting" (i.e., contacting at least one insulin-positive endocrine cell or its precursor with a β cell maturation factor or combination of β cell maturation factors) is intended to include incubating the β cell maturation factor and the cells together in vitro (e.g., adding the β cell maturation factor to the cells during culture). In some embodiments, the term "contacting" is not intended to include in vivo exposure of cells to a compound disclosed herein, which may occur inherently in a subject (i.e., exposure that may occur as a result of a natural physiological process). As in embodiments relating to the production of SC-β cells, contacting at least one insulin-positive endocrine cell or its precursor with a β cell maturation factor can be carried out in any suitable manner. For example, the cells can be treated in adherent or suspension culture. In some embodiments, the cells are treated under conditions that promote cell cluster formation. The present disclosure contemplates 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-adherent tissue culture plates, spinner flasks, and aggrewell plates. In some embodiments, the inventors have observed that clusters remain stable in medium containing 10% serum, hi some embodiments, the conditions that promote cluster formation include low serum medium.

[0099] It is understood that the cells contacted with a beta cell maturation factor can also be simultaneously or sequentially contacted with another agent, such as a growth factor or other differentiation agent, or an environment that stabilizes the cells or further differentiates them.

[0100] Similarly, at least one insulin-positive endocrine cell or its precursor can be contacted with at least one beta cell maturation factor and then with at least another beta cell maturation factor. In some embodiments, the cells are contacted with at least one beta cell maturation factor, and the contacting is temporally separated. In some embodiments, the cells are contacted with at least one beta cell maturation factor substantially simultaneously. In some embodiments, the cells are 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 ten beta cell maturation factors.

[0101] The term "cell culture medium" (also referred to herein as "culture medium" or "culture medium") refers to a medium for culturing cells, containing nutrients that maintain cell viability and support cell growth. The cell culture medium may contain any of the following in appropriate combinations: salts, buffers, amino acids, glucose or other sugars, antibiotics, serum or serum replacements, and other components, such as peptide growth factors. Cell culture media commonly used for specific cell types are known to those skilled in the art.

[0102] The term "cell line" refers to a population of primarily or substantially identical cells, typically derived from a single ancestral cell or a defined and / or substantially identical population of ancestral cells. The cell line may be maintained in culture for extended periods of time (e.g., months, years, or indefinitely). The cells may also undergo simultaneous or induced transformation methods that confer indefinite culture life span on the cells. It should be noted that cell line includes all cell lines recognized in the art. It is understood that cells acquire mutations and / or epigenetic changes over time. As a result, at least some characteristics of individual cells of a cell line may differ from one another. In some embodiments, the cell line comprises SC-β cells as described herein.

[0103] The term "exogenous" refers to a substance that is present in a cell or organism other than its natural source. For example, the terms "exogenous nucleic acid" or "exogenous protein" refer to a nucleic acid or protein that has been introduced into a biological system, e.g., a cell or organism, by man-made means, where the nucleic acid or protein is not normally found in the biological system, or is found in lower amounts. A substance would be considered exogenous if it was introduced into a cell or into an ancestor of the cell that inherited the substance. In contrast, the term "endogenous" refers to a substance that is native to a biological system.

[0104] The term "expression" refers to the cellular processes that produce RNA and protein, and optionally secrete the protein, including, but not limited to, transcription, translation, folding, modification, and processing, as applicable. "Expression product" includes RNA transcribed from a gene and polypeptides obtained by translation of mRNA transcribed from a gene.

[0105] As used herein, the terms "genetically modified" or "genetically engineered" cell refer to a cell into which exogenous nucleic acid has been introduced by human means (or to the progeny of such a cell that inherit at least a portion of the nucleic acid). The nucleic acid may, for example, include a sequence that is exogenous to the cell. The nucleic acid may be a native sequence (i.e., a sequence naturally found in the cell), but may also include a non-naturally occurring configuration (e.g., a coding region linked to a promoter from a different gene) or a variant of a native sequence. Delivery of the nucleic acid into the cell may be accomplished by any suitable technique. Suitable techniques include calcium phosphate or lipid-mediated transfection, electroporation, and transduction or infection using viral vectors. In some embodiments, a polynucleotide or portion thereof is integrated into the genome of the cell. The nucleic acid may then be removed or excised from the genome, provided that such removal or excision results in a detectable change in the cell relative to an unmodified but otherwise equivalent cell. The term "genetically modified" is intended to include the direct introduction of modified RNA (e.g., synthetically modified RNA) into cells. Such synthetically modified RNA includes modifications that prevent rapid degradation by endonucleases and exonucleases and that prevent or reduce the cell's natural immune or interferon response to the RNA. Modifications include, but are not limited to, (a) terminal modifications, such as 5'-end modifications (phosphorylation, dephosphorylation, splicing, inverted linkage, etc.), 3'-end modifications (splicing, DNA nucleotides, inverted linkage, etc.), (b) base modifications, such as modified bases, stabilizing bases, destabilizing bases, or bases with an extended repertoire of base-pair partners or conjugated bases, (c) sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions, and (d) internucleoside linkage modifications, such as modification or substitution of a phosphodiester bond.To the extent that such modifications interfere with translation (i.e., a 50% or greater reduction in translation relative to the absence of the modification—e.g., in an in vitro translation assay in rabbit reticulocytes), the modifications are not suitable for the methods and compositions described herein. In some embodiments, the SC-β cells are genetically engineered to express neurogenin 3. In some embodiments, genetic engineering of the SC-β cells comprises introducing a synthetic, modified mRNA encoding neurogenin 3. Genetic engineering of SC-β cells with a synthetic, modified RNA encoding neurogenin 3 is believed to increase insulin production from the cells. It is anticipated that such genetic engineering of any insulin-producing cell would be expected to increase gene production in that cell.

[0106] In some aspects, the present disclosure provides SC-β cells genetically engineered to contain a detectable marker at the insulin locus. In some embodiments, the SC-β cells are modified to replace both alleles of the insulin locus with a detectable marker. In some embodiments, the SC-β cells are genetically engineered to insert the detectable marker into the insulin locus, such that the detectable marker is expressed together with insulin in the SC-β cells in response to a glucose challenge. In some embodiments, the SC-β cells are genetically engineered to insert the detectable marker into the insulin locus in place of insulin, such that the detectable marker is expressed in the SC-β cells in place of insulin in response to a glucose challenge. It is contemplated that any detectable marker, such as a nucleic acid encoding a fluorescent protein (e.g., GFP), can be inserted into the insulin locus. Those skilled in the art will appreciate that such genetically modified SC-β cells can be used in a variety of screening methods to identify agents that stimulate insulin expression and / or secretion from β cells, e.g., by assaying for the detectable marker in response to the agent. For example, SC-β cells genetically modified to replace the insulin gene in both alleles (e.g., with GFP) can be contacted with test agents and those agents that cause the SC-β cells to fluoresce, since expression of GFP is considered a candidate agent that may activate insulin gene expression in β cells. That is, the detectable marker can be used as a surrogate marker for insulin expression in such genetically modified SC-β cells.

[0107] As used herein, the term "identity" refers to the degree to which two or more nucleic acid or polynucleotide sequences are the same. The percent identity between a desired sequence and a second sequence over a window, e.g., a desired sequence length, can be calculated by aligning the sequences, determining the number of residues (nucleotides or amino acids) within the window that will maximize identity by introducing gaps, dividing by the total number of residues in the desired sequence or the second sequence (the longer one) within the window, and multiplying by 100. When calculating the number of identical residues required to achieve a specific percent identity, decimals are rounded to the nearest integer. Percent identity can be calculated using various computer programs known in the art. For example, computer programs such as BLAST2, BLASTN, BLASTP, and gapped BLAST generate alignments and provide the percent identity between the desired sequences. The algorithm of Karlin and Altschul (Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:22264-2268, 1990), as modified in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993, has been 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 the BLAST and Gapped BLAST programs, the default parameters of each program may be used. PAM250 or BLOSUM62 matrices may be used. Software for performing BLAST analyses is publicly available from the National Center for Biotechnology Information (NCBI).For these programs, see the website having the URL World Wide Web address "ncbi.nlm nih.gov." In a specific embodiment, percent identity is calculated using BLAST2 with the default parameters provided by NCBI.

[0108] As used herein, the terms "isolated" or "partially purified," in the case of a nucleic acid or polynucleotide, mean a nucleic acid or polynucleotide that has been separated from at least one other component (e.g., a nucleic acid or polynucleotide) that is present with the nucleic acid or polynucleotide when found in its natural source and / or that, in the case of a secreted polypeptide, would be present with the nucleic acid or polynucleotide when expressed or secreted by a cell. Chemically synthesized nucleic acids or polynucleotides or those synthesized using in vitro transcription / translation are considered "isolated."

[0109] As used herein, the term "isolated cell" refers to a cell that has been removed from the organism in which it is originally found, or the progeny of such a cell. Optionally, the cell has been cultured in vitro, e.g., in the presence of other cells. Optionally, the cell is then introduced into a second organism or reintroduced into the organism from which it (or cells derived from it) was isolated.

[0110] As used herein, the term "isolated population," with respect to an isolated population of cells, refers to a population of cells that has been removed and separated from a mixed or heterogeneous population of cells. In some embodiments, an isolated population is a substantially pure population of cells compared to the heterogeneous population from which the cells are isolated or enriched.

[0111] The term "substantially pure" with respect 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 with respect to the cells that make up the total cell population. That is, with respect to a population of SC-β cells, the term "substantially pure" or "essentially purified" refers to a cell population that contains no more than about 20%, more preferably no more than about 15%, 10%, 8%, or 7%, and most preferably no more than about 5%, 4%, 3%, 2%, 1%, or less than 1% of cells that are not SC-β cells as defined herein by those terms. In some embodiments, the invention encompasses 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.

[0112] Similarly, by "substantially pure" or "essentially purified" population of insulin-positive endocrine cells, we mean a cell population that contains no more than about 20%, more preferably no more than about 15%, 10%, 8%, 7%, and most preferably no more than about 5%, 4%, 3%, 2%, 1%, or less than 1% of cells that are not insulin-positive endocrine cells as defined herein by that term. In some embodiments, the invention encompasses methods of 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.

[0113] Similarly, a "substantially pure" or "essentially purified" population of Ngn3-positive endocrine precursors refers to a cell population that contains no more than about 20%, more preferably no more than about 15%, 10%, 8%, 7%, and most preferably no more than about 5%, 4%, 3%, 2%, 1%, or less than 1% of cells that are not Ngn3-positive endocrine precursors or their progeny as defined herein by that term. In some embodiments, the invention encompasses methods of expanding a population of Ngn3-positive endocrine precursors, wherein the expanded population of Ngn3-positive endocrine precursors is a substantially pure population of Ngn3-positive endocrine precursors.

[0114] Similarly, a "substantially pure" or "essentially purified" population of Pdx1-positive, NKX6-1-positive pancreatic progenitors refers to a cell population that contains no more than about 20%, more preferably no more than about 15%, 10%, 8%, 7%, and most preferably no more than about 5%, 4%, 3%, 2%, 1%, or less than 1% of cells that are not Pdx1-positive, NKX6-1-positive pancreatic progenitors or their progeny as defined herein by that term. In some embodiments, the invention encompasses methods of expanding a population of Pdx1-positive, NKX6-1-positive pancreatic progenitors, wherein the expanded population of Pdx1-positive, NKX6-1-positive pancreatic progenitors is a substantially pure population of Pdx1-positive, NKX6-1-positive pancreatic progenitors.

[0115] Similarly, a "substantially pure" or "essentially purified" population of Pdx1-positive pancreatic progenitors refers to a cell population that contains no more than about 20%, more preferably no more than about 15%, 10%, 8%, 7%, and most preferably no more than about 5%, 4%, 3%, 2%, 1%, or less than 1% of cells that are not Pdx1-positive pancreatic progenitors or their progeny as defined herein by that term. In some embodiments, the invention encompasses methods of expanding a population of Pdx1-positive pancreatic progenitors, wherein the expanded population of Pdx1-positive pancreatic progenitors is a substantially pure population of Pdx1-positive pancreatic progenitors.

[0116] Similarly, by "substantially pure" or "essentially purified" population of primary gut cells, we mean a population of cells that contains no more than about 20%, more preferably no more than about 15%, 10%, 8%, 7%, and most preferably no more than about 5%, 4%, 3%, 2%, 1%, or less than 1% of cells that are not primary gut cells or their progeny as defined herein by that term. In some embodiments, the invention encompasses methods of expanding a population of primary gut cells, wherein the expanded population of primary gut cells is a substantially pure population of primary gut cells.

[0117] Similarly, a "substantially pure" or "essentially purified" population of definitive endoderm cells refers to a cell population that contains no more than about 20%, more preferably no more than about 15%, 10%, 8%, 7%, and most preferably no more than about 5%, 4%, 3%, 2%, 1%, or less than 1% of cells that are not definitive endoderm cells or their progeny as defined herein by that term. In some embodiments, the invention encompasses methods of expanding a population of definitive endoderm cells, wherein the expanded population of definitive endoderm cells is a substantially pure population of definitive endoderm cells.

[0118] Similarly, a "substantially pure" or "essentially purified" population of pluripotent cells refers to a cell population that contains no more than about 20%, more preferably no more than about 15%, 10%, 8%, 7%, and most preferably no more than about 5%, 4%, 3%, 2%, 1%, or less than 1% of cells that are not pluripotent cells or their progeny as defined herein by that term. In some embodiments, the invention encompasses methods of expanding a population of pluripotent cells, wherein the expanded population of pluripotent cells is a substantially pure population of pluripotent cells.

[0119] The terms "enriching" or "enriched" are used interchangeably herein and mean that the yield (proportion) of one type of cell is increased by at least 10% above the proportion of that type of cell in the starting culture or preparation.

[0120] The terms "replication" or "self-renewal" or "proliferation" are used interchangeably herein to refer to the ability of a stem cell to replicate itself by dividing into the same unspecialized cell type over extended periods of time and / or over periods of months to years. In some instances, proliferation refers to the multiplication of a cell by the repeated division of a single cell into two identical daughter cells.

[0121] As used herein, the term "lineage" describes cells that share a common ancestor or a common developmental fate. For example, for a cell of endodermal origin or of "endodermal lineage," this means that the cell is derived from an endoderm cell and can differentiate along one or more developmental lineage pathways that are restricted to the endodermal lineage, e.g., give rise to definitive endoderm cells, which can then differentiate into liver cells, thymus, pancreas, lungs, and the digestive tract.

[0122] As used herein, the term "xenogenic" refers to cells obtained from a different species.

[0123] As used herein, the term "marker" is used to describe the characteristics and / or phenotype of a cell. Markers can be used to select cells containing desired characteristics. Markers will vary with specific cells. A marker is unique to a particular cell type, regardless of the morphological, functional, or biochemical (enzymatic) characteristics of the cell or the molecules expressed by said cell type. Preferably, such a marker is a protein, more preferably having an epitope for an antibody or other binding molecule available in the art. However, a marker may consist of any molecule found in cells, including, but not limited to, proteins (peptides and polypeptides), lipids, polysaccharides, nucleic acids, and steroids. Examples of morphological characteristics or properties include, but are not limited to, shape, size, and nucleus / cytoplasm ratio. Examples of functional characteristics or properties include, but are not limited to, the ability to adhere to a particular substrate, the ability to incorporate or exclude a particular dye, the ability to migrate under particular conditions, and the ability to differentiate along a particular lineage. Markers may be detected by any method available to those skilled in the art. Markers may be absent from morphological characteristics or may be absent from proteins, lipids, etc. Markers can be a combination of a panel of unique features, the presence or absence of polypeptides and other morphological features.

[0124] The term "modulate" is used consistent with its use in the art, i.e., to cause or facilitate a qualitative or quantitative change, alteration, or modification in a desired process, pathway, or phenomenon. Such a change can be, but is not limited to, an increase, decrease, or change in the relative strength or activity of various components, or a divergence of the process, pathway, or phenomenon. A "modulator" is an agent that causes or facilitates a qualitative or quantitative change, alteration, or modification in a desired process, pathway, or phenomenon.

[0125] As used herein, the term "DNA" is defined as deoxyribonucleic acid.

[0126] The term "polynucleotide" is used interchangeably herein with "nucleic acid" to refer to a polymer of nucleosides. Typically, polynucleotides of this invention are composed of nucleosides naturally found in DNA or RNA (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) linked by phosphodiester bonds. However, the term also encompasses molecules containing nucleosides or nucleoside analogs containing chemically or biologically modified bases, modified backbones, etc., whether or not found in naturally occurring nucleic acids. Such molecules may be preferred for certain applications. In this application, when a polynucleotide is referred to, it is understood to provide both DNA and RNA, and both single-stranded and double-stranded forms (and the complementary strand of each single-stranded molecule). As used herein, "polynucleotide sequence" can refer to the polynucleotide material itself and / or the sequence information (i.e., the sequence of letters used as abbreviations for bases) that biochemically characterizes a particular nucleic acid. Polynucleotide sequences presented herein are presented in the 5' to 3' direction unless otherwise specified.

[0127] As used herein, the term "polypeptide" refers to a polymer of amino acids. The terms "protein" and "polypeptide" are used interchangeably herein. Peptides are relatively short polypeptides, typically between about 2 and 60 amino acids in length. Polypeptides, as typically used herein, contain the amino acids most commonly found in proteins, e.g., 20 L-amino acids. However, other amino acids and / or amino acid analogs known in the art may be used. One or more amino acids in a polypeptide may be modified, for example, by adding a chemical entity, e.g., a carbohydrate group, a phosphate group, a fatty acid group, a linker for conjugation, functionalization, etc. A polypeptide having non-polypeptide moieties covalently or non-covalently associated therewith is still considered a "polypeptide." Typical modifications include glycosylation and palmitoylation. Polypeptides may be purified from natural sources, produced by recombinant DNA technology, or synthesized by chemical means, e.g., conventional solid-phase peptide synthesis. As used herein, the terms "polypeptide sequence" or "amino acid sequence" can refer to the polypeptide material itself and / or the sequence information biochemically characterizing the polypeptide (i.e., the sequence of letters or three-letter codes used as an abbreviation for the amino acid names). Polypeptide sequences presented herein are presented from the N-terminus to the C-terminus unless otherwise specified.

[0128] The term "variant" in reference to a polypeptide can be, for example, a polypeptide at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the full-length polypeptide. The variant can be a fragment of the full-length polypeptide. The variant can be a naturally occurring splice variant. The variant can be a polypeptide at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the fragment of the polypeptide. In this case, the fragment can be at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the wild-type full-length polynucleotide or a domain thereof that has the desired activity, e.g., the ability to detect the presence of SC-β cells or insulin-positive endocrine cells or their precursors from which SC-β cells are derived. In some embodiments, the domain is at least 100, 200, 300, or 400 amino acids in length, starting at any amino acid position in the sequence and extending toward the C-terminus. Mutations known in the art that eliminate or substantially reduce the activity of a protein are preferably avoided. In some embodiments, the variant lacks the N- and / or C-terminal portions of the full-length polypeptide. For example, up to 10, 20, or 50 amino acids are missing from either end. In some embodiments, the polypeptide has the sequence of a mature (full-length) polypeptide. The mature polypeptide refers to a polypeptide that has one or more portions, such as a signal peptide, that are removed during normal intracellular proteolytic processing (e.g., during co-translational or post-translational processing). In some embodiments, if the protein is produced by other than purifying it from cells that naturally express it, the protein is a chimeric polypeptide. The chimeric polypeptide refers to the inclusion of portions from two or more different species. In some embodiments, if the protein is produced by other than purifying it from cells that naturally express it, the protein is a derivative. The derivative refers to the inclusion of additional sequences not related to the protein, so long as those sequences do not substantially reduce the biological activity of the protein.

[0129] As used herein, the term "functional fragment" refers to a polypeptide that is smaller in size than the polypeptide from which it is a fragment, but has an amino acid sequence that is substantially identical to the polypeptide from which it is a fragment, wherein the polypeptide sequence of the functional fragment has at least about 50%, 60%, 70%, 80%, 90%, or 100%, or 100% or more, e.g., 1.5-fold, 2-fold, 3-fold, 4-fold, or 4-fold more, effective biological activity of the polypeptide from which it is a fragment. A functional fragment polypeptide 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).

[0130] 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 capable of autonomous replication and / or expression of nucleic acids linked thereto. Vectors capable of directing the expression of genes operably linked thereto are referred to herein as "expression vectors." Thus, an "expression vector" is a specialized vector that contains the necessary regulatory regions required for the expression of a desired gene in a host cell. In some embodiments, the desired gene is operably linked to another sequence in the vector. Vectors can be viral or non-viral. Viral vectors will be used. Preferably, the viral vector is replication-deficient. This can be achieved, for example, by removing all viral nucleic acid encoding for replication. Replication-deficient viral vectors also retain their infectious properties and will enter cells in a manner similar to replicating adenoviral vectors. However, once inside the cell, replication-deficient viral vectors do not reproduce or propagate. Vectors also encompass liposomes, nanoparticles, and other means of delivering DNA molecules into cells.

[0131] The term "operably linked" means that regulatory sequences necessary for the expression of a coding sequence are positioned in a DNA molecule in the appropriate position relative to the coding sequence to achieve expression of the coding sequence. This same definition may also be applied to the positioning of a coding sequence and transcription control elements (e.g., promoters, enhancers, and termination elements) in an expression vector. The term "operably linked" also includes having an appropriate initiation signal (e.g., ATG) in front of the polynucleotide sequence to be expressed, maintaining the correct reading frame, and allowing expression of the polynucleotide sequence under the control of the expression control sequences and production of the desired polypeptide encoded by the polynucleotide sequence.

[0132] The term "viral vector" refers to the use of a viral vector or virus-associated vector as a carrier of a nucleic acid construct into a cell. The construct can be incorporated into and packaged in, for example, retroviral and lentiviral vectors for infection or transduction of cells, such as non-replication-deficient viral genomes like adenovirus, adeno-associated virus (AAV), or herpes simplex virus (HSV). The vector may or may not integrate into the cellular genome. The construct may optionally include viral sequences for transfection. Alternatively, the construct may be incorporated into an episomal replication-competent vector, such as EPV and EBV vectors.

[0133] The terms "regulatory sequence" and "promoter" are used interchangeably herein to refer to nucleic acid sequences, e.g., initiation signals, enhancers, and promoters, that induce or control the transcription of a protein-coding sequence operably linked to them. In some instances, transcription of a recombinant gene is under the control of a promoter sequence (or other transcriptional regulatory sequence) that controls expression of the recombinant gene in a cell type in which expression is intended. The recombinant gene may be under the control of transcriptional regulatory sequences that are the same as or different from those that control transcription of the naturally occurring form of the protein. In some instances, the promoter sequence is recognized by or introduced into the synthetic machinery of the cell and is required to initiate transcription of a particular gene.

[0134] As used herein, the term "transcription factor" refers to a protein that uses a DNA-binding domain to bind to specific sites on DNA and is part of a system that controls the transfer (or transcription) of genetic information from DNA to RNA. As used herein, "growing" and "proliferation" refer to an increase in cell number (growth) in a population through cell division. Cell proliferation is generally understood to result from the coordinated activation of multiple signal transduction pathways in response to the environment, e.g., growth factors and other mitogens. Cell proliferation can also be promoted by relief from the action of intracellular or extracellular signals and mechanisms that inhibit or negatively affect cell proliferation.

[0135] The term "selectable marker" refers to a gene, RNA, or protein that, when expressed, confers on a cell a selectable phenotype, such as resistance to cytotoxic or cytostatic agents (e.g., antibiotic resistance), nutrient prototrophy, or expression of a specific protein that can be used as a basis for distinguishing between cells expressing the specific protein and cells that do not. Proteins whose expression can be easily detected, such as fluorescent or luminescent proteins or enzymes that react with a substrate to produce a chromogenic, fluorescent, or luminescent substance ("detectable markers"), constitute a subset of selectable markers. The presence of a selectable marker linked to expression control elements native to a gene that is normally expressed selectively or exclusively in pluripotent cells makes it possible to identify and select somatic cells that have been reprogrammed to the pluripotent state. Various selectable marker genes can be used, such as the neomycin resistance gene (neo), puromycin resistance gene (puro), guanine phosphoribosyltransferase (gpt), dihydrofolate reductase (DHFR), adenosine diaminase (ada), puromycin-N-acetyltransferase (PAC), hygromycin resistance gene (hyg), multidrug resistance gene (mdr), thymidine kinase (TK), hypoxanthine-guanine phosphoribosyltransferase (HPRT), and hisD gene. Detectable markers include green fluorescent protein (GFP), blue, sapphire, yellow, red, orange, and cyan fluorescent proteins, and mutants of any of these. Luminescent proteins, such as luciferase (e.g., firefly or Renilla luciferase), are also useful. As will be evident to those skilled in the art, the term "selectable marker" as used herein can refer to a gene or the expression product of the gene, e.g., the encoded protein.

[0136] In some embodiments, the selectable marker confers a growth and / or survival advantage to cells expressing it over cells that do not express it or express it at significantly lower levels. Such a growth 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 not expressing the marker do not grow and / or survive and are removed from the population or are reduced in number to only a very small percentage of the population. The method of selecting cells expressing a marker that confers a growth and / or survival advantage by maintaining a cell population under selective conditions to largely or completely eliminate cells that do not express the marker is referred to herein as "positive selection." The marker is said to be "useful for positive selection." Negative selection and markers useful for negative selection are also subject to certain methods described herein. Expression of such a marker confers a growth and / or survival disadvantage on cells expressing the marker relative to cells that do not express the marker or express it at significantly lower levels (or, alternatively, cells that do not express the marker have a growth and / or survival advantage over cells that express the marker). Thus, cells expressing the marker are largely or completely eliminated from the cell population if maintained under selective conditions for a sufficient period of time.

[0137] As used herein, the term "reporter gene" encompasses any gene that is genetically introduced into a cell and adds to the stem cell phenotype. Reporter genes disclosed in this invention include fluorescent, luminescent, enzymatic, and resistance genes, and are intended to encompass other genes that can be easily detected by one skilled in the art. In some embodiments of the present invention, reporter genes are used as markers for identifying specific stem cells, cardiovascular stem cells, and their differentiated progeny. Reporter genes are genetically operably linked to sequences that regulate their expression in a manner that responds to one or more conditions monitored by measuring reporter gene expression. In some cases, reporter gene expression may be measured in living cells. When live cell reporter gene assays are used, reporter gene expression may be monitored at multiple time points, e.g., 2, 3, 4, 5, 6, 8, or 10 or more time points. In some cases, when a reporter assay in live cells is used, expression of the reporter gene is monitored at a frequency of at least about 10 minutes to about 24 hours, e.g., 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 another frequency from any integer between about 10 minutes and about 24 hours.

[0138] The terms "subject" and "individual" are used interchangeably herein to refer to an animal, e.g., a human, from which cells may be obtained and / or to which treatment, e.g., prophylactic treatment, with the cells described herein may be provided. For treatment of infections, symptoms, or disease states specific to a particular animal, e.g., a human subject, the term subject refers to that particular animal. The terms "non-human animal" and "non-human mammal," used interchangeably herein, include mammals, e.g., rats, mice, rabbits, sheep, cats, dogs, cows, pigs, and non-human primates. The term "subject" also encompasses any vertebrate, e.g., but is not limited to, mammals, reptiles, amphibians, and fish. Preferably, however, the subject is a mammal, e.g., a human or other mammal, e.g., a domestic mammal, e.g., a dog, cat, horse, or a production mammal, e.g., a cow, sheep, pig, or the like.

[0139] The terms "diabetes" and "diabetes mellitus" are used interchangeably herein. The World Health Organization defines diabetes as a fasting plasma glucose concentration of 7.0 mmol / L (126 mg / dL) or higher (whole blood 6.1 mmol / L or 110 mg / dL) or a 2-hour glucose level of 11.1 mmol / L or higher (200 mg / dL or higher). Other values ​​suggestive of diabetes or indicating a high risk of diabetes include elevated arterial blood pressure of 140 / 90 mmHg or higher; elevated plasma triglycerides (1.7 mmol / L; 150 mg / dL) and / or low HDL-cholesterol (0.9 mmol / L, less than 35 mg / dL for men; 1.0 mmol / L, less than 39 mg / dL for women); central obesity (men: waist-to-hip ratio > 0.90; women: waist-to-hip ratio > 0.85) and / or a body mass index of 30 kg / m 2 Ultra- or microalbuminuria (in which the urinary albumin excretion rate is 20 μg / min or more, or the albumin:creatinine ratio is 30 mg / g or more) is included. The term diabetes includes all types of diabetes, for example, type I, type II, and type 1.5.

[0140] When applied to isolated cells, the terms "treat," "treating," "treatment," and the like include subjecting the cells to any kind of process or condition, or performing any kind of treatment or procedure on the cells. When applied to a subject, the terms refer to providing medical or surgical treatment, care, or management to an individual who is typically ill or injured, or at increased risk of developing a disease relative to the average member of the population, and who is in need of such treatment, care, or management.

[0141] As used herein, the terms "treating" and "treatment" refer to administering an effective amount of a composition to a subject so that the subject experiences a reduction in at least one symptom of a disease or an improvement in the disease, e.g., a beneficial or desired clinical result. For purposes of this invention, a beneficial or desired clinical result includes, but is not limited to, an improvement in one or more symptoms, whether detectable or not, a reduction in the extent of the disease, a stable (i.e., not worsening) state of the disease, a delay or slowing of disease progression, an improvement or palliation, and abatement (whether partial or total) of the disease state. Treating can also mean prolonging survival compared to expected survival in the absence of treatment. Thus, one of skill in the art will understand that treatment may improve the disease symptoms, but may not completely cure the disease. As used herein, the term "treatment" includes prophylaxis. Alternatively, treatment is "effective" if the progression of the disease is reduced or halted. "Treatment" can also mean prolonging survival compared to expected survival in the absence of treatment. Those in need of treatment include those already diagnosed with a cardiac condition, as well as those who may be at risk of developing a cardiac condition due to genetic susceptibility or other factors, such as weight, diet, and health.

[0142] As used herein, the terms "administering," "introducing," and "implanting" are used interchangeably to refer to the placement of cells of the present invention (e.g., SC-β cells) into a subject by a method or route that results in at least partial localization of the introduced cells at a desired site. The cells, e.g., SC-β cells (e.g., pancreatic β cells or pancreatic β-like cells), can be directly implanted into the pancreas or administered by any suitable route that results in delivery to a desired location in the subject. In this case, at least some of the implanted cells or components of the cells remain viable. The survival period of the cells after administration to a subject can be as short as a few hours, e.g., 24 hours, to several days or years. In some cases, the cells can be administered to a non-pancreatic site, e.g., the liver, or can be administered subcutaneously, e.g., in a capsule (e.g., microcapsule) that maintains the implanted cells in place and prevents migration of the implanted cells.

[0143] As used herein, the terms "parenteral administration" and "parenterally administered" refer to modes of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intrathecal, intraspinal, and intrasternal injection and infusion. As used herein, the terms "systemic administration," "administered systemically," "peripheral administration," and "peripherally administered" refer to administration of cardiovascular stem cells and / or their progeny and / or compounds and / or other materials other than directly into the central nervous system, and thus subject to processes such as metabolism as they enter the animal's organs, e.g., subcutaneous administration.

[0144] The term "tissue" refers to a group or layer of specialized cells that together perform a particular specialized function. The term "tissue-specific" refers to cells sourced from a particular tissue.

[0145] The terms "reduce," "decreased," "reduction," "decrease," or "inhibit" are all used generally herein to mean a decrease by a statistically significant amount. For the avoidance of doubt, however, "decreased," "reduction," "reduce," or "inhibit" means a decrease of at least 10% compared to a reference level, for example, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least 60%, or at least about 70%, or at least about 80%, or at least about 90%; or a decrease of up to and including 100% (i.e., an absent level compared to a reference sample), or any decrease between 10-100% compared to a reference level.

[0146] The terms "increased," "increase," "enhance," or "activate" are all used generally herein to mean an increase by a statistically significant amount. For the avoidance of doubt, the terms "increased," "increase," "enhance," or "activate" mean an increase of at least 10% compared to a reference level, 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 an increase up to and including 100%, or any increase between 10-100% compared to a reference level, or an increase of at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold, or any increase between 2-fold and 10-fold compared to a reference level.

[0147] The term "statistically significant" or "significantly" refers to statistical significance, and generally refers to two standard deviations (2SD) of the marker concentration below normal or lower. The term refers to statistical evidence that a difference exists. The term is defined as the probability of making a decision to reject the null hypothesis when the null hypothesis is actually true. The decision is often made using p-value.

[0148] As used herein, the terms "comprising" or "comprises" are used in reference to compositions, methods and each component thereof that are essential to the invention, but are open to the inclusion of unspecified components, whether essential or not.

[0149] As used herein, the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristics of that embodiment of the invention.

[0150] The term "consisting of" refers to the compositions, methods, and components thereof described herein, exclusively with respect to any element not recited in that description of an embodiment.

[0151] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "the method" includes one or more methods and / or steps of the type described herein, which would be apparent to one of ordinary skill in the art based on a reading of this disclosure.

[0152] stem cells

[0153] Stem cells are cells that retain the ability to renew themselves through cell division and can differentiate into a range of specialized cell types. Two broad types of mammalian stem cells are embryonic stem (ES) cells found in blastocysts and adult stem cells found in adult tissues. In the developing embryo, stem cells can differentiate into all specialized embryonic tissues. In adult organisms, stem and progenitor cells serve as the body's repair system, replenishing specialized cells and maintaining the normal turnover of regenerating organs, such as blood, skin, or gastrointestinal tissues. Pluripotent stem cells can differentiate into cells derived from any of the three germ layers.

[0154] 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 their precursors, germline cells may be used in place of or in conjunction with the stem cells to provide at least one SC-β cell using protocols similar to the exemplary protocols described herein. Suitable germline cells can be prepared, for example, from primordial germline cells present in human fetal material harvested approximately 8-11 weeks after the last menstrual period. Exemplary methods for preparing germline cells are described, for example, in Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998 and U.S. Pat. No. 6,090,622.

[0155] ES cells, such as human embryonic stem cells (hESCs) or mouse embryonic stem cells (mESCs), which have virtually limitless replicative potential and the potential to differentiate into most cell types, in principle provide an unlimited starting material for generating differentiated cells for clinical therapy (http: / / stemcells.nih.gov / info / scireport / 2006report.htm, 2006). One potential use of ES cells is to generate new pancreatic β cells for cell replacement therapy for type 1 diabetes, for example, by first producing endoderm, e.g., definitive endoderm, from hESCs, then further differentiating the definitive endoderm into at least one insulin-positive endocrine cell or precursor thereof, and then further differentiating the at least one insulin-positive endocrine cell or precursor thereof into SC-β cells.

[0156] hESCs are described, for example, in Cowan et al. (N Engl. J. Med. 350:1353, 2004) and Thomson et al. (Science 282:1145, 1998), and embryonic stem cells from other primates, such as rhesus monkey 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 line (hEG) cells (Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998), can also be used in the methods described herein. mESCs are described, for example, in Tremml et al. (Curr Protoc Stem Cell Biol. Chapter 1:Unit 1C.4, 2008). The stem cells can be, for example, unipotent, totipotent, multipotent, or pluripotent. In some examples, any cell of primate origin capable of producing progeny from at least one germ layer or all three germ layers can be used in the methods disclosed herein.

[0157] In certain instances, ES cells can be isolated as described, for example, in Cowan et al. (N Engl. J. Med. 350:1353, 2004) and U.S. Pat. No. 5,843,780, and Thomson et al., Proc. Natl. Acad. Sci. USA 92:7844, 1995. For example, hESC cells can be prepared from human blastocyst cells using the techniques described in Thomson et al. (U.S. Pat. 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. Equivalent cell types to hESCs include their pluripotent derivatives, such as primitive ectoderm-like (EPL) cells, as reviewed in WO 01 / 51610 (Bresagen). hESCs can also be derived from human preimplantation embryos. Alternatively, in vitro fertilized (IVF) embryos can be used, or single-cell human embryos 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 media (Gardner et al., Fertil. Steril. 69:84, 1998). The zona pellucida is removed from developed blastocysts by brief exposure to pronase (Sigma). Inner cell populations can be isolated by immunosurgery. In this immunosurgery, blastocysts are exposed to a 1:50 dilution of rabbit anti-human spleen cell antiserum for 30 minutes, then washed three times for 5 minutes in DMEM, and exposed to a 1:5 dilution of guinea pig complement (Gibco) for 3 minutes (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 gentle pipetting. The ICM is then plated onto mEF feeder layers.After 9 to 15 days, outgrowths from the inner cell population can be dissociated into clumps 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, and then replated onto mEFs in fresh medium. Outgrowth colonies with undifferentiated morphology can be individually selected with a micropipette, mechanically dissociated into clumps, and replated. ES-like morphology is characterized by small colonies with a clearly high nucleus / cytoplasm ratio and prominent nuclei. The resulting hESCs are then routinely split every 1–2 weeks, for example, by simple trypsinization, exposure to Dulbecco's PBS (containing 2 mM EDTA), exposure to type IV collagenase (approximately 200 U / mL; Gibco), or by selecting individual colonies with a micropipette. In some cases, a clump size of approximately 50 to 100 cells is optimal. mESC cells can be prepared, for example, using the techniques described in Conner et al. (Curr. Prot. in Mol. Biol. Unit 23.4, 2003).

[0158] Embryonic stem cells can be isolated from blastocysts of members of primate species (U.S. Pat. No. 5,843,780; Thomson et al., Proc. Natl. Acad. Sci. USA 92:7844, 1995). Human embryonic stem (hES) cells can be prepared from human blastocyst cells using techniques described by Thomson et al. (U.S. Pat. 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. Equivalent cell types to hES cells include their pluripotent derivatives, such as primitive ectoderm-like (EPL) cells, as reviewed in WO 01 / 51610 (Bresagen).

[0159] Alternatively, in some embodiments, hES cells can be obtained from human preimplantation embryos. Alternatively, in vitro fertilized (IVF) embryos can be used, or single-cell human embryos 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 media (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 population is isolated by immunosurgery. In this immunosurgery, blastocysts are exposed to a 1:50 dilution of rabbit anti-human spleen cell antiserum for 30 minutes, then washed three times for 5 minutes in DMEM, and exposed to a 1:5 dilution of guinea pig complement (Gibco) for 3 minutes (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 gentle pipetting. The ICM is then plated onto mEF feeder layers.

[0160] After 9 to 15 days, outgrowths from the inner cell population are dissociated into clumps 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, and then replated onto mEFs in fresh medium. Outgrowth colonies with undifferentiated morphology are individually selected with a micropipette, mechanically dissociated into clumps, and replated. ES-like morphology is characterized by small colonies with a clearly high nucleus / cell ratio and prominent nuclei. The resulting ES cells can then be routinely split every 1–2 weeks, for example, by simple trypsinization, exposure to Dulbecco's PBS (containing 2 mM EDTA), exposure to type IV collagenase (approximately 200 U / mL; Gibco), or by selecting individual colonies with a micropipette. A clump size of approximately 50 to 100 cells is optimal.

[0161] In some embodiments, human embryonic germline (hEG) cells are pluripotent stem cells that can be used in the methods disclosed herein to differentiate into primitive endoderm cells. hEG cells can be prepared from primordial germline cells present in human fetal material harvested approximately 8-11 weeks after the last menstrual period. Suitable preparation methods are described in Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998, and U.S. Patent No. 6,090,622, which are hereby incorporated by reference in their entireties.

[0162] Briefly, genital ridges were processed to form disaggregated cells. EG growth medium was DMEM, containing 4500 mg / L D-glucose, 2200 mg / L mM NaHCO3, 15% ES-grade fetal bovine 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). A 96-well tissue culture plate was prepared containing a partially confluent layer of feeder cells (e.g., STO cells, ATCC No. CRL1503) cultured for 3 days in modified EG growth medium without LIF, bFGF, or forskolin and inactivated by 5000 rads of γ-irradiation. Approximately 0.2 mL of primordial germline cell (PGC) suspension is added to each well. The first passage is performed after 7-10 days in EG growth medium, and each well is transferred to one well of a 24-well culture dish previously primed with irradiated STO mouse fibroblasts. The cells are cultured with daily medium changes until a cell morphology consistent with EG cells is observed, typically 7-30 days or 1-4 passages.

[0163] In certain instances, the stem cells may be undifferentiated (e.g., cells not committed to a particular lineage) before exposure to at least one beta cell maturation factor according to the methods described herein. In other instances, it may be desirable to differentiate the stem cells into one or more intermediate cell types before exposure to at least one beta cell maturation factor 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 instances, undifferentiated cells may be evident in a two-dimensional microscopic view in colonies of cells with a high nucleus-to-cytoplasm ratio and prominent nuclei. The stem cells may be used by themselves (e.g., substantially free of any undifferentiated cells) or in the presence of differentiated cells. In certain instances, the stem cells may be cultured in the presence of appropriate nutrients and, optionally, other cells, to allow the stem cells to proliferate and, optionally, differentiate. For example, embryonic fibroblasts or fibroblast-like cells may be present in the culture to support the proliferation of the stem cells. The fibroblasts may be present during one stage of stem cell expansion, but not all stages, for example, the fibroblasts may be added to a stem cell culture at an early culture stage and not be added to the stem cells at one or more subsequent culture stages.

[0164] Stem cells used in all aspects of the present invention can be any cells obtained from any type of tissue (e.g., embryonic tissue, e.g., fetal or prefetal tissue, or adult tissue). In this case, the stem cells have the characteristic of being capable, under appropriate conditions, of producing progeny of various cell types, e.g., derivatives of at least one of all three germ layers (endoderm, mesoderm, and ectoderm). These cell types can be provided in the form of established cell lines, or they can be obtained directly from primary embryonic tissue and used immediately for differentiation. Cells listed in the NIH Human Embryonic Stem Cell Registry include, for example, hESBGN-01, hESBGN-02, hESBGN-03, and hESBGN-04 (BresaGen, Inc.); HES-1, HES-2, HES-3, HES-4, HES-5, and HES-6 (ES Cell International); Miz-hES1 (MizMedi Hospital-Seoul National University); HSF-1 and HSF-6 (University of California at San Francisco); and H1, H7, H9, H13, and H14 (Wisconsin Alumni Research Foundation (WiCell Research Institute)). In some embodiments, the source of human stem cells or pluripotent stem cells used for chemically induced differentiation into mature insulin-positive cells did not involve the destruction of human embryos.

[0165] In another embodiment, the stem cells can be isolated from tissue, for example, solid tissue. In some embodiments, the tissue is skin, adipose tissue (e.g., adipose 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.

[0166] Desired stem cells also include various types of embryonic cells, exemplified by human embryonic stem (hES) cells, as described in Thomson et al. (1998) Science 282:1145; embryonic stem cells from other primates, such as rhesus monkey 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 line (hEG) cells (Shambloft et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998). Lineage-committed stem cells, such as mesodermal stem cells and other early cardiogenic cells, are also of interest (see, e.g., Reyes et al. (2001) Blood 98:2615-2625; Eisenberg & Bader (1996) Circ Res. 78(2):205-16). The stem cells may be obtained from any mammalian species, e.g., human, horse, cow, pig, dog, cat, rodent, e.g., mouse, rat, hamster, primate, etc. In some embodiments, human embryos were not destroyed for the source of pluripotent cells used in the methods and compositions disclosed herein.

[0167] ES cells are considered undifferentiated once they have not committed to a specific lineage of differentiation. Such cells display 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 are typically evident in two-dimensional microscopic views of cell colonies with distinctly high nuclear / cytoplasmic ratios and prominent nuclei. Undifferentiated ES cells express genes that can be used as markers to detect the presence of undifferentiated cells, the polypeptide products of which can be used as markers for negative selection. See, e.g., U.S. Patent Application Publication No. 2003 / 0224411; 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 characteristic of undifferentiated non-human primate ES cells and human EC cells, including stage-specific embryonic antigen (SSEA)-3, SSEA-4, TRA-1-60, TRA-1-81, and alkaline phosphatase. The globo-series glycolipid GL7 contains the SSEA-4 epitope and is formed by adding sialic acid to the globo-series glycolipid GbS. The globo-series glycolipid GbS contains the SSEA-3 epitope. Therefore, GL7 reacts with antibodies against both SSEA-3 and SSEA-4. The undifferentiated human ES cell lines do not stain with SSEA-1, whereas differentiated cells stain strongly with SSEA-1. Methods for propagating hES cells in an undifferentiated state are described in WO99 / 20741, WO01 / 51616, and WO03 / 020920.

[0168] A mixture of cells from a suitable source of endothelial, muscle, and / or neural stem cells can be collected from a mammalian donor by methods known in the art. A suitable source is the hematopoietic microenvironment. For example, preferably, mobilized (i.e., mobilized) circulating peripheral blood is removed from the subject. Alternatively, bone marrow may be obtained from a mammal, e.g., a human patient, undergoing an autologous transplant. In some embodiments, stem cells can be obtained from a subject's adipose tissue using, for example, the CELUTION™ SYSTEM from Cytori, as disclosed in U.S. Pat. Nos. 7,390,484 and 7,429,488, which are incorporated herein by reference in their entireties.

[0169] In some embodiments, human umbilical cord blood cells (HUCBCs) are useful in the methods disclosed herein. Human Umbilical Cord Blood Cells (HUCBCs) are 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 blood and placental blood were considered waste products that were typically discarded 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 reconstituting 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). Another advantage of HUCBCs is the immature immune properties of these cells, which are very similar to fetal cells. The immature immunity significantly reduces the risk of rejection by the host (Taylor & Bryson, 1985 J. Immunol. 134:1493-1497).Human umbilical cord blood contains mesodermal and hematopoietic progenitor cells and endothelial cell precursors 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. Haematology 98:775-777; Erices et al., 2000 Br. J. Haematology 109:235-242). The total number of hematopoietic progenitor cells in umbilical cord blood is equal to or exceeds that of bone marrow. Furthermore, highly proliferative hematopoietic cells are eight times more abundant in HUCBC than in bone marrow and express 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).

[0170] In another embodiment, the pluripotent cells are cells in the hematopoietic microenvironment, for example, circulating peripheral blood, preferably from the mononuclear fraction of mammalian peripheral blood, umbilical cord blood, bone marrow, fetal liver or yolk sac. The stem cells, in particular neural stem cells, can also be obtained from the central nervous system, for example, from the meninges.

[0171] In another embodiment, the pluripotent cells are present in embryoid bodies. The embryoid bodies are formed by harvesting ES cells by brief protease digestion and allowing small clumps of undifferentiated human ESCs to grow in suspension culture. Differentiation is induced by withdrawal of conditioned medium. The resulting embryoid bodies are plated onto a semi-solid substrate. The formation of differentiated cells may be observed after about 7 days to about 4 weeks. Viable, differentiating cells from in vitro culture of stem cells are selected by partially dissociating embryoid bodies or similar structures that provide cell aggregation. Aggregates containing desired cells are selected for phenotypic characteristics using methods that substantially maintain cells in cell-cell contact in the aggregates.

[0172] In another embodiment, the stem cells can be reprogrammed stem cells, e.g., stem cells obtained from somatic cells or differentiated cells. In such embodiments, the dedifferentiated stem cells can be, for example, but not limited to, neoplastic cells, tumor cells, and cancer cells, or induced reprogrammed cells, e.g., induced pluripotent stem cells or iPS cells.

[0173] Cloning and cell culture

[0174] Exemplary methods for molecular genetics and genetic engineering that can be used in the techniques described herein can be found, for example, in the latest 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, for example, in 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 kits for genetic manipulation are commercially available from, for example, BioRad, Stratagene, Invitrogen, ClonTech, and Sigma-Aldrich Co.

[0175] Suitable cell culture methods can be found, for example, in the latest editions 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 University Press); and "Embryonic Stem Cells: Methods and Protocols" (K. Turksen ed., Humana Press). Suitable tissue culture supplies and reagents are commercially available from, for example, Gibco / BRL, Nalgene-Nunc International, Sigma Chemical Co., and ICN Biomedicals.

[0176] Pluripotent stem cells can be continuously propagated in culture by those skilled in the art using culture conditions that promote proliferation without promoting differentiation. A typical serum-containing ES medium consists of 80% DMEM (e.g., Knock-Out DMEM, Gibco), 20% defined fetal bovine serum (FBS, Hyclone) or serum replacement (WO98 / 30679), 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 (WO99 / 20741, Geron Corp.). Traditionally, ES cells are cultured on a layer of feeder cells, typically fibroblasts obtained from embryonic or fetal tissue.

[0177] Geron scientists have discovered that pluripotent SCs can be maintained in an undifferentiated state without feeder cells. The environment for feeder-free culture includes a suitable cell substrate, particularly an extracellular matrix such as Matrigel® or laminin. Typically, cells are arrested before complete dispersion by enzymatic digestion (e.g., collagenase IV for about 5 minutes). Then, clumps of about 10 to 2,000 cells are seeded directly onto the substrate without further dispersion.

[0178] Feeder-free cultures are supported by a nutrient medium containing factors that support cell growth without differentiation. Such factors may be introduced into the medium by culturing the medium with cells that secrete such factors, such as irradiated (approximately 4,000 rads) primary mouse embryonic fibroblasts, telomerized mouse fibroblasts, or fibroblast-like cells obtained from pPS cells. The medium is cultured in serum-free medium, such as KO DMEM supplemented with 20% serum replacement and 4 ng / mL bFGF, at a concentration of approximately 5-6 x 10 cells. 4 pieces / cm -2 The medium can be conditioned by seeding feeders at a density of 1000 x 1000 cells / well. The medium conditioned for 1-2 days is further supplemented with bFGF and used to support pluripotent SC cultures for 1-2 days. The characteristics of feeder-free culture methods are further discussed in WO 01 / 51616; and Xu et al., Nat. Biotechnol. 19:971, 2001.

[0179] Under microscopic observation, ES cells appear as small colonies with a high nuclear / cytoplasmic ratio, prominent nuclei, and barely discernible intercellular junctions. Primate ES cells express markers detectable using antibodies designated stage-specific embryonic antigens (SSEA) 3 and 4, as well as Tra-1-60 and Tra-1-81 (Thomson et al., Science 282:1145, 1998). Mouse ES cells can be used as a positive control for SSEA-1 and as a negative control for SSEA-4, Tra-1-60, and Tra-1-81. SSEA-4 is consistently present in human embryonic carcinoma (hEC) cells. In vitro differentiation of pluripotent SCs results in the loss of expression of SSEA-4, Tra-1-60, and Tra-1-81 and increased expression of SSEA-1. Increased expression of SSEA-1 is also found in undifferentiated hEC cells.

[0180] Stem cell line β cell (SC-β)

[0181] In some aspects, the present disclosure provides stem cell-based β cells (SC-β). The SC-β cells disclosed herein share many distinguishing characteristics of native β cells but differ in certain aspects (e.g., gene expression profiles). In some embodiments, the SC-β cells are non-naturally occurring. As used herein, "non-naturally occurring" means that the SC-β cells, in certain aspects, are significantly different from naturally occurring β cells, i.e., native β cells. However, it should be understood that these significant differences typically relate to structural features that may result in the SC-β cells exhibiting specific functional differences; for example, the gene expression patterns of SC-β cells differ from those of native β cells. The SC-β cells behave in a manner similar to native β cells, but may have certain altered (e.g., improved) functions compared to native β cells. For example, as shown in Figure 2E, SC-β cells respond to 20 mM glucose at a higher frequency compared to the frequency of native β cells. The differences between SC-β cells and native β cells will be apparent to those skilled in the art based on the data disclosed herein.

[0182] The SC-β cells of the present disclosure share many of the characteristic properties of β cells that are important for normal β cell function. In some embodiments, the SC-β cells exhibit a glucose-stimulated insulin secretion (GSIS) response in vitro. In some embodiments, the SC-β cells exhibit a GSIS response in vivo. In some embodiments, the SC-β cells exhibit a GSIS response in vitro and in vivo. In some embodiments, the GSIS response resembles the GSIS response of endogenous mature pancreatic β cells. In some embodiments, the SC-β cells exhibit a GSIS response to at least one glucose challenge. In some embodiments, the SC-β cells exhibit a GSIS response to at least two consecutive glucose challenges. In some embodiments, the SC-β cells exhibit a GSIS response to at least three consecutive glucose challenges. In some embodiments, the GSIS response resembles the GSIS response of endogenous human pancreatic islets to multiple glucose challenges. In some embodiments, the GSIS response is observed immediately after transplantation of the cells into a human or animal. In some embodiments, the GSIS response is observed within about 24 hours after transplantation of the cells into a human or animal. In some embodiments, the GSIS response is observed within about one week after transplantation of the cells into a human or animal. In some embodiments, the GSIS response is observed within about two weeks after transplantation of the cells into a human or animal. In some embodiments, the stimulation index of the cells, characterized by the ratio of insulin secreted in response to a high glucose concentration compared to a low glucose concentration, is similar to that of endogenous mature pancreatic beta cells. In some embodiments, the SC-beta cells exhibit a stimulation index greater than 1. In some embodiments, the SC-beta cells exhibit a stimulation index of 1 or greater. In some embodiments, the SC-beta cells exhibit a stimulation index greater than 1.1. In some embodiments, the SC-beta cells exhibit a stimulation index of 1.1 or greater. In some embodiments, the SC-beta cells exhibit a stimulation index greater than 2. In some embodiments, the SC-beta cells exhibit a stimulation index of 2 or greater.In some embodiments, the SC-beta cells exhibit 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 greater.

[0183] In some embodiments, the SC-β cells exhibit cytokine-induced apoptosis in response to a cytokine, ie, the SC-β cells exhibit cytokine-induced apoptosis in response to a cytokine selected from the group consisting of interleukin-1β (IL-β), interferon-γ (INF-γ), tumor necrosis factor-α (TNF-α), and combinations thereof.

[0184] In some embodiments, insulin secretion from the SC-β cells is enhanced in response to a known antidiabetic agent (e.g., an antidiabetic agent acting on β cells ex vivo or in vitro and / or an antidiabetic agent in vivo generally). The present disclosure contemplates any known antidiabetic agent. In some embodiments, insulin secretion from the SC-β cells 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.

[0185] In some embodiments, the SC-β cells are monohormonal. In some embodiments, the SC-β cells exhibit a morphology similar to that of endogenous mature pancreatic β cells. In some embodiments, the SC-β cells encapsulate crystalline insulin granules. In some embodiments, the SC-β cells exhibit encapsulated crystalline insulin granules under electron microscopy similar to insulin granules in endogenous mature pancreatic β cells. In some embodiments, the SC-β cells exhibit a slow rate of replication. In some embodiments, the SC-β cells exhibit a slow rate of replication. In some embodiments, the SC-β cells exhibit a slow, but improved rate of replication in response to treatment with prolactin, as measured by staining with C-peptide and Ki67.

[0186] In some embodiments, the SC-β cells increase intracellular Ca in response to glucose. 2+ In some embodiments, the SC-β cells increase glucose-stimulated Ca2+ expression similar to the GSCF of endogenous mature pancreatic β cells. 2+ In some embodiments, the SC-β cells exhibit a GSCF response to at least three consecutive glucose challenges in a manner similar to the GSCF response of endogenous mature pancreatic β cells to multiple glucose challenges.

[0187] In some embodiments, the SC-beta cells express at least one marker characteristic of endogenous mature pancreatic beta cells selected from the group consisting of insulin, C-peptide, PDX1, MAFA, NKX6-1, PAX6, neuroD1, glucokinase (GCK), SLC2A1, PCSK1, KCNJ11, ABCC8, SLC30A8, SNAP25, RAB3A, GAD2, and PTPRN.

[0188] In some embodiments, the SC-beta cells do not express at least one marker (e.g., a marker that is not expressed by endogenous mature pancreatic beta cells) selected from the group consisting of: a) a hormone selected from the group consisting of i) glucagon (GCG) and ii) somatostatin (SST); b) a glandular cell marker selected from the group consisting of i) amylase and ii) carboxypeptidase A (CPA1); c) an alpha cell marker selected from the group consisting of i) GCG, Arx, Irx1, and Irx2; d) a ductal cell marker selected from the group consisting of i) CFTR and ii) Sox9.

[0189] The SC-β cells can be differentiated in vitro from any starting cell, as the present invention is not intended to be limited by the starting cell from which the SC-β cells are derived. Exemplary starting cells include, but are not limited to, insulin-positive endocrine cells or any precursors thereof, such as Nkx6-1-positive pancreatic progenitor cells, Pdx1-positive pancreatic progenitor cells, and pluripotent stem cells, embryonic stem cells, and induced pluripotent stem cells. In some embodiments, the SC-β cells can be differentiated in vitro from reprogrammed cells, partially reprogrammed cells (i.e., partially reprogrammed somatic cells, e.g., fibroblasts, that exist in an intermediate state between induced pluripotent cells and the somatic cells from which they are derived), or transdifferentiated cells. In some embodiments, the SC-β cells disclosed herein can be differentiated in vitro from insulin-positive endocrine cells or precursors thereof. In some embodiments, the SC-β cells can be differentiated in vitro from precursors selected from the group consisting of Nkx6-1-positive pancreatic progenitor cells, Pdx1-positive pancreatic progenitor cells, and pluripotent stem cells. In some embodiments, the pluripotent stem cells are selected from the group consisting of embryonic stem cells and induced pluripotent stem cells. In some embodiments, the SC-β cells or the pluripotent stem cells from which the SC-β cells are derived are human. In some embodiments, the SC-β cells are human.

[0190] In some embodiments, the SC-β cells are not genetically modified. In some embodiments, the SC-β cells acquire characteristics shared in common with native β cells in the absence of genetic modification of the cells. In some embodiments, the SC-β cells are genetically modified.

[0191] In some embodiments, the insulin produced per SC-β cell is at least 0.5 μIU per 1000 cells upon 30 minutes of incubation (eg, ex vivo) in high glucose concentrations.

[0192] In some embodiments, the insulin produced per SC-beta 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 after 30 minutes of incubation at a high glucose concentration. In some embodiments, the insulin produced per SC-beta cell is between 0.5 μIU and 10 μIU per 1000 cells after 30 minutes of incubation at a high glucose concentration. In some embodiments, the insulin produced per SC-beta cell is about 2.5 μIU per 1000 cells after 30 minutes of incubation at a high glucose concentration.

[0193] In some aspects, the present disclosure provides cell lines comprising the SC-β cells described herein. In some embodiments, the SC-β cells stably express insulin. In some embodiments, the SC-β cells can be frozen, thawed, and expanded for at least 30 passages without obvious morphological changes, with a doubling time of 24 to 44 hours.

[0194] Generation of SC-β cells

[0195] Aspects of the present disclosure relate to generating SC-β cells (e.g., pancreatic β cells). Generally, at least one SC-β cell or precursor thereof, e.g., a pancreatic progenitor produced according to the methods disclosed herein, can include a mixture or combination of various cells, such as Pdx1-positive pancreatic progenitors, pancreatic progenitors co-expressing Pdx1 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.

[0196] The at least one SC-β cell or precursor thereof can be produced based on any suitable culture protocol that allows stem cells or pluripotent cells to differentiate 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 differentiating the at least one pluripotent cell into the at least one SC-β cell or precursor thereof.

[0197] 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, the population of SC-β cells or precursors thereof comprises 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 devoid of embryonic stem cells or pluripotent cells or iPS cells.

[0198] In some embodiments, somatic cells, e.g., fibroblasts, can be isolated from a subject, e.g., from a tissue biopsy, e.g., a skin biopsy, and reprogrammed into induced pluripotent stem cells for further differentiation to produce at least one SC-β cell or precursor thereof for use in the compositions and methods described herein. In some embodiments, somatic cells, e.g., fibroblasts, can be maintained in culture by methods known to those of skill in the art, and in some embodiments, expanded before being converted into SC-β cells by methods disclosed herein.

[0199] In some embodiments, the at least one SC-β cell or precursor thereof is maintained in culture by methods known to those of skill in the art, and in some embodiments, expanded before being converted to an SC-β cell by the methods disclosed herein.

[0200] Furthermore, the at least one SC-β cell or its precursor, e.g., pancreatic precursor, can be derived from any mammalian species. Non-limiting examples include murine, bovine, simian, porcine, equine, bovine, or human cells. For clarity and simplicity, the methods described herein refer to at least one mammalian SC-β cell or its precursor, but all methods described herein can be readily applied to at least one SC-β cell or its precursor of other cell types. In some embodiments, the at least one SC-β cell or its precursor is obtained from a human individual.

[0201] Induction of differentiation of pluripotent stem cells into definitive endoderm cells

[0202] Embodiments of the present disclosure include definitive endoderm cells. The definitive endoderm cells useful herein can be obtained from any source or generated according to any suitable protocol. In some embodiments, pluripotent stem cells, such as iPSCs or hESCs, are differentiated into endoderm cells. In some embodiments, the endoderm cells are further differentiated into primitive gut cells, Pdx1-positive pancreatic progenitor cells, NKX6-1-positive pancreatic progenitor cells, Ngn3-positive endocrine progenitor cells, or insulin-positive endocrine cells, and subsequently induced or matured into SC-β cells.

[0203] In some embodiments, the stem cells may be seeded onto a new substrate, or the medium may be changed to remove extracellular matrix or soluble factors that inhibit differentiation. This may be referred to as the "direct differentiation method," and is described in general terms in International Publication WO 01 / 51616 and U.S. Patent Application Publication No. 2002 / 0019046, both of which are incorporated herein by reference in their entireties. To avoid potential complications in the differentiation process caused by residual feeder cells, direct differentiation methods that begin with feeder-free culture of stem cells are generally preferred. Another approach is to place undifferentiated stem cells in suspension culture, which will often cause them to form differentiated and undifferentiated cell aggregates. For example, stem cells can be collected by simple collagenase digestion, dissociated into clusters, and passaged in non-adherent cell culture plates. The aggregates can be fed every few days and then harvested after an appropriate period, typically 4-8 days. Depending on the conditions, aggregation generally begins by forming a heterogeneous collection of cell types, including a substantial frequency of endodermal cells. The aggregates can then be dispersed and replated onto a substrate such as laminin or fibronectin for the next step in the differentiation process, or passaged in suspension culture, for example, using non-adherent plates and appropriate media.

[0204] Direct differentiation or differentiation in aggregates can be monitored for the presence of endodermal cells using appropriate markers, such as those listed in U.S. Patent No. 7,326,572. In some preferred embodiments, differentiation can be monitored for the presence of endodermal cells using markers such as Sox17. Once a sufficient proportion of endoderm is obtained, the cells can be replated or otherwise manipulated to initiate another stage of differentiation. In certain situations, cell differentiation or maintenance can be improved if the cells are maintained in micropopulation clusters (e.g., 50 to 5,000 cells). Further stages of differentiation contemplated in this disclosure are shown in FIG. 1.

[0205] In some embodiments, definitive endoderm cells are produced by contacting (e.g., culturing) pluripotent stem cells with a compound of Formula (I) described in U.S. Pat. No. 8,507,274 (the "'274 patent"), which is incorporated herein by reference. The compounds of Formula (I) described in the '274 patent are cell-permeable small molecules that can regulate cellular processes by modulating signaling pathways, gene expression, or metabolism, and have been effectively used in stem cell differentiation protocols. Small molecules can be synthesized with high quality and purity and can be conveniently delivered or removed, offering great potential for therapeutic applications. High-throughput screens have been conducted to identify novel small molecules that can 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), and induction of specific cell types, particularly neural and muscle cells (reviewed in Ding and Schultz, 2004). It is expected that compounds of formula (I) from the '274 patent can be used to differentiate pluripotent stem cells into definitive endoderm cells.

[0206] In some embodiments, the compound of formula (I) from the '274 patent is

[0207] [ka]

[0208] Contains formula (I).

[0209] During the ceremony,

[0210] R 1 and R 2are independently H, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cyclyl, or cyclyl, each of which can be optionally substituted and / or interrupted in the backbone by one or more O, N, S, S(O), and C(O);

[0211] R 3 and R 4 are 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 taken together with the carbon to which they are attached form a heterocyclyl optionally substituted cyclyl; and

[0212] L is C1-C 10 Alkylenyl, C2- 10 Alkenylenyl or C2- 10 alkynylenyl, each of which can be optionally substituted and / or interrupted in the backbone by one or more O, N, S, S(O) and C(O).

[0213] In some embodiments, the compound of Formula (I) from the '274 patent includes IDE1:

[0214] [ka]

[0215] In some embodiments, the compound of formula (I) from the '274 patent includes IDE2:

[0216] [ka]

[0217] The '274 patent then describes methods for confirming the identity of the resulting definitive endoderm cells, as well as methods for isolating, sorting, expanding, and further differentiating the definitive endoderm, all of which may be used in the compositions and methods described herein, as will be understood by those skilled in the art.

[0218] In some embodiments, definitive endoderm cells can be obtained by differentiating at least some of the pluripotent cells in the population into definitive endoderm cells, e.g., by contacting the 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.

[0219] The present disclosure contemplates the use of any growth factor from the TGF-β superfamily (e.g., alone or in combination with a WNT signaling pathway activator) to induce the differentiation of the pluripotent stem cells into definitive endoderm cells. 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).

[0220] The present disclosure contemplates the use of any WNT signaling pathway activator (e.g., alone or in combination with a growth factor from the TGF-β superfamily) to induce the differentiation of the pluripotent stem cells into definitive endoderm cells. In some embodiments, the WNT signaling pathway activator comprises CHIR99021. In some embodiments, the WNT signaling pathway activator comprises Wnt3a recombinant protein.

[0221] Those skilled in the art will appreciate that the concentration of the agent (e.g., growth factor) used 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 between 10 ng / mL and 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 110 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 101ng / mL, 102ng / mL, 103ng / mL, 104ng / mL, 105ng / mL, 106ng / mL, 107ng / mL, 108ng / mL or 109ng / mL.

[0222] In some embodiments, the pluripotent cells are contacted with the WNT signaling pathway activator at a concentration between 1.4 μg / mL and 140 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 μg / mL, 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.

[0223] Generally, the pluripotent cells are maintained in a suitable culture medium (e.g., suspension culture) for a period of time sufficient to induce differentiation of at least some of the pluripotent cells into definitive endoderm cells. Exemplary suitable culture media are shown in Table 1 below.

[0224] [Table 1]

[0225] In some embodiments, the culture medium suitable for differentiating pluripotent cells into definitive endoderm cells comprises S1 medium.

[0226] In some embodiments, contacting the pluripotent cells is accomplished 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 a WNT signaling pathway activator are added to the suspension culture on day 1. In some embodiments, the at least one growth factor from the TGF-β superfamily is replenished during the suspension culture on day 2. In some embodiments, the WNT signaling pathway activator is not replenished during the suspension culture on day 2. In some embodiments, the WNT signaling pathway activator is removed from the suspension culture on day 2. In some embodiments, the at least one growth factor from the TGF-β superfamily is replenished during the suspension culture on day 2, and the WNT signaling pathway activator is removed from the suspension culture on day 2 or is not replenished during the suspension culture. In some embodiments, neither the at least one growth factor from the TGF-β superfamily nor the WNT signaling pathway activator is replenished in the suspension culture on day 3. In some embodiments, the at least one growth factor from the TGF-β superfamily and the WNT signaling pathway activator are both removed from the suspension culture on day 3.

[0227] The method 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 the methods described herein. In some embodiments, the pluripotent cell comprises an induced pluripotent stem cell. In some embodiments, the pluripotent cell comprises an embryonic stem cell. In some embodiments, the pluripotent cell comprises a human cell.

[0228] In some embodiments, differentiating at least some of the pluripotent cells in the collection into definitive endoderm cells is accomplished by contacting the collection of pluripotent cells with i) activin A and ii) CHIR99021 to induce differentiation of at least some of the pluripotent cells in the collection into definitive endoderm cells, wherein the definitive endoderm cells express at least one marker characteristic of definitive endoderm.

[0229] Other methods for producing definitive endoderm cells are known in the art, such as those described in U.S. Patent Application Publication Nos. 2006 / 0003446 (G. Keller, et al.), 2006 / 0003313 (K. D'Amour, et al.), 2005 / 0158853 (K. D'Amour, et al.), and 2005 / 0260749 (Jon Odorico, et al.), the relevant portions of which are incorporated herein by reference.

[0230] In some embodiments, definitive endoderm cells produced by the methods disclosed herein express at least one marker selected from the group consisting of Nodal, Tmprss2, Tmem30b, St14, Spink3, Sh3gl2, Ripk4, Rab15, Npnt, Clic6, Cldn8, Cacna1b, Bnipl, Anxa4, Emb, FoxA1, Sox17, and Rbm35a, wherein expression of the at least one marker is upregulated at a statistically significant amount in the definitive endoderm cells relative to the pluripotent cells from which they are derived. In some embodiments, definitive endoderm cells produced by the methods disclosed herein do not express at least one marker selected from the group consisting of Gata4, SPARC, AFP, and Dab2 at a statistically significant amount relative to the pluripotent cells from which they are derived. In some embodiments, definitive endoderm cells produced by the methods disclosed herein do not express at least one marker selected from the group consisting of Zicl, Pax6, Flk1, and CD31 at a statistically significant amount relative to the pluripotent cells from which they are derived.

[0231] In some embodiments, definitive endoderm cells produced by the methods disclosed herein have a statistically significantly higher level of Smad2 phosphorylation relative to the pluripotent cells from which they are derived. In some embodiments, definitive endoderm cells produced by the methods disclosed herein have the ability to form a digestive tract in vivo. In some embodiments, definitive endoderm cells produced by the methods disclosed herein can be differentiated into cells with enterocyte-specific morphology, where the enterocyte-specific morphology expresses FoxA2 and / or claudin-6. In some embodiments, definitive endoderm cells produced by the methods disclosed herein can be further differentiated into cells of endodermal origin.

[0232] In some embodiments, the population of pluripotent cells is cultured in the presence of at least one beta cell maturation factor before any differentiation or during the first stage of differentiation. Any pluripotent stem cells 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, the beta cell maturation factor described herein may be present in the culture medium of the population of pluripotent stem cells or may be added in a bolus or periodically during the growth (e.g., replication or proliferation) of the population of pluripotent stem cells. In certain examples, the population of pluripotent stem cells may be exposed to at least one beta cell maturation factor before any differentiation. In other examples, the population of pluripotent stem cells may be exposed to at least one beta cell maturation factor during the first stage of differentiation.

[0233] Induction of differentiation of adult endoderm cells into primitive gut cells

[0234] Embodiments of the present disclosure include primitive gut cells. The primitive gut cells useful herein can be obtained from any source or generated according to any suitable protocol. In some embodiments, definitive endoderm cells are differentiated into primitive gut cells. In some embodiments, the primitive gut cells are further differentiated into, for example, Pdx1-positive pancreatic progenitor cells, NKX6-1-positive pancreatic progenitor cells, Ngn3-positive endocrine progenitor cells, or insulin-positive endocrine cells, and subsequently induced or matured into SC-β cells.

[0235] In some embodiments, primitive gut cells can be obtained by differentiating at least some of the definitive endoderm cells in the collection into primitive gut cells, e.g., by 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 primitive gut cells, wherein the primitive gut cells express at least one marker specific for primitive gut cells.

[0236] The present disclosure contemplates the use of any growth factor from the FGF family (e.g., alone or in combination with other factors) to induce definitive endoderm cells to differentiate into primitive gut cells. 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.

[0237] Those skilled in the art will appreciate that the concentration of the growth factor used may vary. In some embodiments, the definitive endoderm cells are contacted with the at least one growth factor from the FGF family at a concentration between 5 ng / mL and 500 ng / mL. In some embodiments, the definitive 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 definitive 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 definitive 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 definitive 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 definitive endoderm cells are contacted with the at least one growth factor from the FGF family at a concentration of 50 ng / mL.

[0238] In some embodiments, the definitive endoderm cells are cultured in a suitable culture medium.

[0239] Generally, the definitive endoderm cells are maintained in a suitable culture medium (e.g., suspension culture) for a period of time sufficient to induce differentiation of at least some of the definitive endoderm cells into primitive gut cells. Exemplary suitable culture media are shown in Table 2 below.

[0240] [Table 2]

[0241] In some embodiments, a culture medium suitable for differentiating definitive endoderm cells into primitive gut cells comprises S2 medium.

[0242] In some embodiments, the contacting of the definitive endoderm cells is accomplished in suspension culture. In some embodiments, the suspension culture is maintained in a spinner flask. In some embodiments, the period is between 2 and 5 days. In some embodiments, the period is 3 days. In some embodiments, the suspension culture is replenished every other day.

[0243] In some embodiments, definitive endoderm cells can be obtained by differentiating at least some of the definitive endoderm cells in the collection into gastrula cells, e.g., by contacting the definitive endoderm cells with KGF to induce differentiation of at least some of the definitive endoderm cells into gastrula cells, wherein the gastrula cells express at least one marker characteristic of definitive endoderm.

[0244] Induction of differentiation of primitive gut cells into Pdx1-positive pancreatic progenitor cells

[0245] Embodiments of the present disclosure include Pdx1-positive pancreatic progenitor cells. Pdx1-positive pancreatic progenitor cells useful herein can be obtained from any source or generated according to any suitable protocol. In some embodiments, primitive gut cells are differentiated into Pdx1-positive pancreatic progenitor cells. In some embodiments, the Pdx1-positive pancreatic progenitor cells are further differentiated into, for example, NKX6-1-positive pancreatic progenitor cells, Ngn3-positive endocrine progenitor cells, or insulin-positive endocrine cells, and subsequently induced or matured into SC-β cells.

[0246] In some embodiments, Pdx1-positive pancreatic progenitor cells can be obtained by differentiating at least a portion of the primitive gut cells in the collection into Pdx1-positive pancreatic progenitor cells, e.g., by contacting the primitive gut cells with: i) at least one bone morphogenetic protein (BMP) signaling 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) signaling pathway activator; and v) at least one protein kinase C activator to induce differentiation of at least a portion of the primitive gut cells into Pdx1-positive pancreatic progenitor cells, wherein the Pdx1-positive pancreatic progenitor cells express Pdx1.

[0247] The present disclosure contemplates the use of any BMP signaling pathway inhibitor (e.g., alone or in any combination with at least one growth factor from the FGF family, at least one SHH pathway inhibitor, at least one retinoic acid signaling pathway activator, and at least one protein kinase C activator) to induce primitive gut cells to differentiate into Pdx1-positive pancreatic progenitor cells. In some embodiments, the BMP signaling pathway inhibitor comprises LDN193189.

[0248] The present disclosure contemplates the use of any growth factor from the FGF family (e.g., alone or in any combination with at least one BMP signaling pathway inhibitor, at least one SHH pathway inhibitor, at least one retinoic acid signaling pathway activator, and at least one protein kinase C activator) to induce primitive gut cells to differentiate into Pdx1-positive pancreatic progenitor cells. 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, and FGF21.

[0249] The present disclosure contemplates the use of any SHH pathway inhibitor (e.g., alone or in any combination with at least one BMP signaling pathway inhibitor, at least one growth factor from the FGF family, at least one retinoic acid signaling pathway activator, and at least one protein kinase C activator) to induce primitive gut cells to differentiate into Pdx1-positive pancreatic progenitor cells. In some embodiments, the SHH pathway inhibitor comprises Sant1.

[0250] The present disclosure contemplates the use of any RA signaling pathway activator (e.g., alone or in any combination with at least one BMP signaling pathway inhibitor, at least one growth factor from the FGF family, at least one SHH pathway inhibitor, and at least one protein kinase C activator) to induce primitive gut cells to differentiate into Pdx1-positive pancreatic progenitor cells. In some embodiments, the RA signaling pathway activator comprises retinoic acid.

[0251] The present disclosure contemplates the use of any PKC activator (e.g., alone or in any combination with at least one BMP signaling pathway inhibitor, at least one growth factor from the FGF family, at least one SHH pathway inhibitor, and at least one RA signaling pathway activator) to induce primitive gut cells to differentiate into Pdx1-positive pancreatic progenitor cells. In some embodiments, the PKC activator comprises PdbU. In some embodiments, the PKC activator comprises TPB.

[0252] Those skilled in the art will understand that the concentration of the agent (e.g., growth factor) used can vary. In some embodiments, the gastrula cells are contacted with the BMP signaling pathway inhibitor at a concentration between 20 nM and 2000 nM. In some embodiments, the gastrula cells are contacted with the BMP signaling pathway inhibitor at a concentration of 3040 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180 nM, or 190 nM. In some embodiments, the gastrula cells are contacted with the BMP signaling 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 gastrula cells are contacted with the BMP signaling pathway inhibitor at a concentration of 300 nM, 400 nM, 500 nM, 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, or 1900 nM. In some embodiments, the gastrula cells are contacted with the BMP signaling 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 gastrula cells are contacted with the BMP signaling pathway inhibitor at a concentration of 200 nM.

[0253] In some embodiments, the primitive gut 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 primitive gut 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 primitive gut 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 primitive gut 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 primitive gut 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 primitive gut cells are contacted with the at least one growth factor from the FGF family at a concentration of 50 ng / mL.

[0254] In some embodiments, the primitive gut cells are contacted with the at least one SHH pathway inhibitor at a concentration between 0.1 μM and 0.5 μM, hi some embodiments, the primitive gut cells are contacted with the at least one SHH 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 primitive gut cells are contacted with the at least one SHH 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, or 0.49 μM. In some embodiments, the primitive gut cells are contacted with the at least one SHH pathway inhibitor at a concentration of 0.25 μM.

[0255] In some embodiments, the primitive gut cells are contacted with the RA signaling pathway activator at a concentration between 0.01 μM and 1.0 μM. In some embodiments, the primitive gut 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 primitive gut 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 primitive gut cells are contacted with the RA signaling pathway activator at a concentration of 0.1 μM.

[0256] In some embodiments, the gastrula cells are contacted with the PKC activator at a concentration between 50 nM and 5000 nM. In some embodiments, the gastrula 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 gastrula 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 gastrula 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 gastrula 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. In some embodiments, the gastrula cells are contacted with the PKC activator at a concentration of 500 nM.

[0257] Generally, the primitive gut cells are maintained in a suitable culture medium (e.g., suspension culture) for a period of time sufficient to induce differentiation of at least some of the primitive gut cells into Pdx1-positive pancreatic progenitor cells. Exemplary suitable culture media are shown in Table 3 below.

[0258] [Table 3]

[0259] In some embodiments, S3 medium may be used as a suitable culture medium for differentiating primitive gut cells into pancreatic progenitor cells.

[0260] In some embodiments, the contacting of the primitive gut cells is accomplished 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.

[0261] In some embodiments, primitive gut cells can be obtained by inducing differentiation of at least some of the primitive gut cells in the population into Pdx1-positive pancreatic progenitor cells, e.g., by contacting the primitive gut cells with i) LDN193189, ii) KGF, iii) Sant1; iv) RA, and iv) PdbU to induce differentiation of at least some of the primitive gut cells into Pdx1-positive pancreatic progenitor cells, wherein the Pdx1-positive pancreatic progenitor cells express Pdx1.

[0262] Induction of differentiation of Pdx1-positive pancreatic progenitor cells into NKX6-1+ pancreatic progenitor cells

[0263] Embodiments of the present disclosure include NKX6-1-positive pancreatic progenitor cells. NKX6-1-positive pancreatic progenitor cells useful herein can be obtained from any source or generated according to any suitable protocol. In some embodiments, Pdx1-positive pancreatic progenitor cells are differentiated into NKX6-1-positive pancreatic progenitor cells. In some embodiments, the NKX6-1-positive pancreatic progenitor cells are further differentiated into, for example, Ngn3-positive endocrine progenitor cells or insulin-positive endocrine cells, and subsequently induced or matured into SC-β cells.

[0264] In some aspects, a method for producing NKX6-1-positive pancreatic progenitor cells from Pdx1-positive pancreatic progenitor cells includes contacting a cell population comprising Pdx1-positive pancreatic progenitor cells (e.g., under conditions that promote cell cluster formation) with a) at least one growth factor from the fibroblast growth factor (FGF) family, b) at least two beta cell maturation factors including a sonic hedgehog pathway inhibitor, and optionally c) a low concentration of a retinoic acid (RA) signaling pathway activator for a period of at least 5 days to induce differentiation of at least one Pdx1-positive pancreatic progenitor cell in the population into an NKX6-1-positive pancreatic progenitor cell, wherein the NKX6-1-positive pancreatic progenitor cell expresses NKX6-1.

[0265] In some embodiments, the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells can be obtained by contacting the Pdx1-positive pancreatic progenitor cells with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally iii) a low concentration of an RA signaling pathway activator for a period of 5 days under conditions that promote cell cluster formation, to induce differentiation of at least some of the Pdx1-positive pancreatic progenitor cells into Pdx1-positive, NKX6-1-positive pancreatic progenitor cells, wherein the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells express Pdx1 and NKX6-1.

[0266] In some embodiments, the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells can be obtained by contacting the Pdx1-positive pancreatic progenitor cells with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and iii) an RA signaling pathway activator to induce differentiation of at least some of the Pdx1-positive pancreatic progenitor cells into Pdx1-positive, NKX6-1-positive pancreatic progenitor cells, wherein the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells express Pdx1 and NKX6-1.

[0267] In some embodiments, the Pdx1-positive pancreatic progenitor cells are generated from a population of pluripotent cells. In some embodiments, the Pdx1-positive pancreatic progenitor cells are generated from a population of iPS cells. In some embodiments, the Pdx1-positive pancreatic progenitor cells are generated from a population of ESC cells. In some embodiments, the Pdx1-positive pancreatic progenitor cells are generated from a population of definitive endoderm cells. In some embodiments, the Pdx1-positive pancreatic progenitor cells are generated from a population of primitive gut cells.

[0268] The present disclosure contemplates the use of any growth factor from the FGF family (e.g., alone or in any combination with at least one SHH pathway inhibitor, or optionally at least one retinoic acid signaling pathway activator) to induce differentiation of Pdx1-positive pancreatic progenitor cells into NKX6-1-positive pancreatic progenitor cells. 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, and FGF21.

[0269] The present disclosure contemplates the use of any SHH pathway inhibitor (e.g., alone or in any combination with at least one growth factor from the FGF family or at least one retinoic acid signaling pathway activator) to induce Pdx1-positive pancreatic progenitor cells to differentiate into NKX6-1-positive pancreatic progenitor cells. In some embodiments, the SHH pathway inhibitor comprises Sant1.

[0270] The present disclosure contemplates the use of any RA signaling pathway activator (e.g., alone or in any combination with at least one growth factor from the FGF family and at least one SHH pathway inhibitor) to induce Pdx1-positive pancreatic progenitor cells to differentiate into NKX6-1-positive pancreatic progenitor cells. In some embodiments, the RA signaling pathway activator comprises retinoic acid.

[0271] In some embodiments, the method comprises contacting the cell population (e.g., Pdx1-positive pancreatic progenitor cells) with at least one additional beta cell maturation factor. In some embodiments, the at least one additional beta cell maturation factor comprises at least one growth factor from the EGF family. In some embodiments, the method comprises contacting the Pdx1-positive pancreatic progenitor cells with at least one growth factor from the EGF family. The present disclosure contemplates the use of any growth factor from the EGF family to promote differentiation of Pdx1-positive pancreatic progenitor cells into NKX6-1-positive pancreatic progenitor cells (e.g., with any combination of at least one growth factor from the FGF family, at least one SHH 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.

[0272] Those skilled in the art will appreciate that the concentration of the agent (e.g., growth factor) used may vary. In some embodiments, the Pdx1-positive pancreatic progenitor cells are contacted with the at least one growth factor from the FGF family at a concentration between 1 ng / mL and 100 ng / mL. In some embodiments, the Pdx1-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 Pdx1-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 Pdx1-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 Pdx1-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 Pdx1-positive pancreatic progenitor cells are contacted with the at least one growth factor from the FGF family at a concentration of 50 ng / mL.

[0273] In some embodiments, the Pdx1-positive pancreatic progenitor cells are contacted with the at least one SHH pathway inhibitor at a concentration between 0.1 μM and 0.5 μM. In some embodiments, the Pdx1-positive pancreatic progenitor cells are contacted with the at least one SHH 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 Pdx1 positive pancreatic progenitor cells are contacted with the at least one SHH 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, or 0.49 μM. In some embodiments, the Pdx1 positive pancreatic progenitor cells are contacted with the at least one SHH pathway inhibitor at a concentration of 0.25 μM.

[0274] In some embodiments, the Pdx1 positive pancreatic progenitor cells are contacted with the RA signaling pathway activator at a concentration between 0.01 μM-1.0 μM. In some embodiments, the Pdx1 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 Pdx1 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 Pdx1-positive pancreatic progenitor cells are contacted with the RA signaling pathway activator at a concentration of 0.1 μM.

[0275] In some embodiments, the Pdx1-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 and 200 ng / mL. In some embodiments, the Pdx1-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 Pdx1-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 Pdx1-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 Pdx1-positive pancreatic progenitor cells are contacted with the at least one growth factor from the EGF family at a concentration of 20 ng / mL.

[0276] Generally, the Pdx1-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 Pdx1-positive pancreatic progenitor cells in the population into the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells. Exemplary suitable culture media are set forth in Table 3 above. In some embodiments, the conditions promoting cell cluster formation include 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 replenished every other day. In some embodiments, the beta cell maturation factor is replenished every other day.

[0277] 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 day 5. 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, the BMP signaling pathway inhibitor is removed from the suspension culture prior to day 5. In some embodiments, the BMP signaling pathway inhibitor comprises LDN193189.

[0278] In some embodiments, at least 10% of the Pdx1-positive pancreatic progenitor cells in the population are induced to differentiate into Pdx1-positive, NKX6-1-positive pancreatic progenitor cells, hi some embodiments, at least 95% of the Pdx1-positive pancreatic progenitor cells are induced to differentiate into Pdx1-positive, NKX6-1-positive pancreatic progenitor cells.

[0279] Generally, any Pdx1-positive pancreatic progenitor cell can differentiate into a Pdx1-positive, NKX6-1-positive pancreatic progenitor cell. In some embodiments, the NKX6-1-positive pancreatic progenitor cell expresses Pdx1, NKX6-1, and / or FoxA2.

[0280] In some embodiments, the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells can be obtained by contacting the Pdx1-positive pancreatic progenitor cells with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally iii) a low concentration of an RA signaling pathway activator for a period of 5 days under conditions that promote cell cluster formation, to induce differentiation of at least some of the Pdx1-positive pancreatic progenitor cells into Pdx1-positive, NKX6-1-positive pancreatic progenitor cells, wherein the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells express Pdx1 and NKX6-1.

[0281] In some embodiments, NKX6-1-positive pancreatic progenitor cells can be obtained by differentiating at least some of the Pdx1-positive pancreatic progenitor cells into Pdx1-positive, NKX6-1-positive pancreatic progenitor cells by contacting the Pdx1-positive pancreatic progenitor cells with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally iii) an RA signaling pathway activator once every two days for a period of five days under conditions that promote cell cluster formation, thereby inducing differentiation of at least some of the Pdx1-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.

[0282] In some embodiments, NKX6-1-positive pancreatic progenitor cells can be obtained by differentiating at least a portion of the Pdx1-positive pancreatic progenitor cells in the population into Pdx1-positive, NKX6-1-positive pancreatic progenitor cells, e.g., by contacting the Pdx1-positive pancreatic progenitor cells with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally iii) an RA signaling pathway activator, to induce differentiation of at least a portion of the Pdx1-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.

[0283] Induction of differentiation of NKX6-1+ pancreatic progenitor cells into insulin+ endocrine cells

[0284] Embodiments of the present disclosure include insulin-positive endocrine cells. Insulin-positive endocrine cells useful herein can be obtained from any source or produced according to any suitable protocol. In some embodiments, NKX6-1-positive pancreatic progenitor cells are differentiated into insulin-positive endocrine cells. In some embodiments, the insulin-positive endocrine cells are further differentiated, for example, by induction or maturation into SC-β cells.

[0285] In some aspects, a method for producing insulin-positive endocrine cells from NKX6-1-positive pancreatic progenitor cells includes contacting a cell population comprising NKX6-1-positive pancreatic progenitor cells with at least two beta cell maturation factors, including a) a TGF-β signaling inhibitor and b) a thyroid hormone signaling pathway activator (e.g., under conditions that promote cell cluster formation), 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.

[0286] The present disclosure contemplates the use of any TGF-β signaling pathway inhibitor (e.g., alone or in combination with other β cell maturation factors, such as thyroid hormone signaling pathway activators) to induce differentiation of the NKX6-1-positive pancreatic progenitor cells into insulin-positive endocrine cells. In some embodiments, the TGF-β signaling pathway comprises TGF-β receptor type I kinase signaling. In some embodiments, the TGF-β signaling pathway inhibitor comprises Alk5 inhibitor II.

[0287] The present disclosure contemplates the use of any thyroid hormone signaling pathway activator (e.g., alone or in combination with other beta cell maturation factors, such as TGF-β signaling pathway inhibitors) to induce differentiation of the NKX6-1-positive pancreatic progenitor cells into insulin-positive endocrine cells. In some embodiments, the thyroid hormone signaling pathway activator comprises triiodothyronine (T3).

[0288] In some embodiments, the method comprises contacting the cell population (e.g., NKX6-1-positive pancreatic progenitor cells) with at least one additional beta cell maturation factor. In some embodiments, the method comprises contacting the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells with at least one of: i) an SHH 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.

[0289] In some embodiments, the at least one additional beta cell maturation factor comprises a gamma-secretase inhibitor. The present disclosure contemplates the use of any gamma-secretase inhibitor (e.g., alone or in combination with either a TGF-β signaling pathway inhibitor and / or a thyroid hormone signaling pathway activator) capable of inducing differentiation of collecting NKX6-1-positive pancreatic progenitor cells into insulin-positive endocrine cells. In some embodiments, the gamma-secretase inhibitor comprises XXI. In some embodiments, the gamma-secretase inhibitor comprises DAPT.

[0290] In some embodiments, the at least one additional beta cell maturation factor comprises at least one growth factor from the EGF family. The present disclosure contemplates the use of any growth factor from the EGF family (e.g., alone or in combination with either a TGF-β signaling pathway inhibitor and / or a thyroid hormone signaling pathway activator) capable of inducing differentiation of collecting NKX6-1-positive pancreatic progenitor cells into insulin-positive endocrine cells. 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.

[0291] In some embodiments, the at least one additional beta cell maturation factor comprises a low concentration of a retinoic acid (RA) signaling pathway activator. The present disclosure contemplates the use of any RA signaling pathway activator (e.g., alone or in combination with either a TGF-β signaling pathway inhibitor and / or a thyroid hormone signaling pathway activator) to induce differentiation of NKX6-1-positive pancreatic progenitor cells into insulin-positive endocrine cells. In some embodiments, the RA signaling pathway activator comprises RA.

[0292] In some embodiments, the at least one additional beta cell maturation factor comprises a sonic hedgehog (SHH) pathway inhibitor. The present disclosure contemplates the use of any SHH pathway inhibitor (e.g., alone or in combination with either a TGF-β signaling pathway inhibitor and / or a thyroid hormone signaling pathway activator) to induce differentiation of NKX6-1-positive pancreatic progenitor cells into insulin-positive endocrine cells. In some embodiments, the SHH pathway inhibitor comprises Sant1.

[0293] In some embodiments, the cell population (eg, NKX6-1 positive pancreatic progenitor cells) is exposed to glucose.

[0294] 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 present disclosure contemplates the use of any protein kinase inhibitor (e.g., alone or in combination with either a TGF-β signaling pathway inhibitor and / or a thyroid hormone signaling pathway activator) capable of inducing differentiation of the NKX6-1-positive pancreatic progenitor cells in the population into insulin-positive endocrine cells. In some embodiments, the protein kinase inhibitor comprises staurosporine.

[0295] In some embodiments, insulin-positive endocrine cells can be obtained by contacting Pdx1-positive, NKX6-1-positive pancreatic progenitor cells with i) at least one SHH pathway inhibitor, ii) RA signaling pathway activator, iii) γ-secretase inhibitor, iv) TGF-β signaling pathway inhibitor, v) TH signaling pathway activator, and vi) at least one growth factor from the epidermal growth factor (EGF) family to induce 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.

[0296] Those skilled in the art will appreciate that the concentration of agents (eg, growth factors) used may vary.

[0297] 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 between 100nM-100μM. 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 10μM. 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 100nM, 200nM, 300nM, 400nM, 500nM, 600nM, 700nM, 800nM or 900nM. 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 2μM, 3μM, 4μM, 5μM, 6μM, 7μM, 8μM or 9μM. 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 NKX6-1 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.

[0298] In some embodiments, the NKX6-1-positive pancreatic progenitor cells are contacted with the thyroid hormone signaling pathway activator at a concentration between 0.1 μM and 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 gamma thyroid hormone signaling 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.

[0299] In some embodiments, the NKX6-1 positive pancreatic progenitor cells are contacted with the gamma-secretase inhibitor at a concentration between 0.1 μM and 10 μM. In some embodiments, the NKX6-1 positive pancreatic progenitor cells are contacted with the gamma-secretase inhibitor at a concentration of 1 μM. In some embodiments, the NKX6-1 positive pancreatic progenitor cells are contacted with the gamma-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 gamma-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 gamma secretase inhibitor at a concentration of 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM or 9 μM.

[0300] 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 between 2 ng / mL and 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 30ng / mL, 35ng / mL, 40ng / mL, 45ng / mL, 50ng / mL, 55ng / mL, 60ng / mL, 65ng / mL, 70ng / mL, 75ng / mL, 80ng / mL, 85ng / mL, 90ng / mL, 95ng / mL, or 100ng / 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 21ng / mL, 22ng / mL, 23ng / mL, 24ng / mL, 25ng / mL, 26ng / mL, 27ng / mL, 28ng / mL, or 29ng / 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.

[0301] In some embodiments, the NKX6-1 positive pancreatic progenitor cells are contacted with the RA signaling pathway activator at a concentration between 0.01 μM-1.0 μM. In some embodiments, the NKX6-1 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.

[0302] In some embodiments, the NKX6-1 positive pancreatic progenitor cells are contacted with the RA signaling pathway activator at a low concentration, at a concentration between 0.01 μM-1.0 μM. In some embodiments, the NKX6-1 positive pancreatic progenitor cells are contacted with the RA signaling pathway activator at a low concentration, 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 low concentration, 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 a low concentration of the RA signaling pathway activator, at a concentration of 0.1 μM.

[0303] In some embodiments, the NKX6-1 positive pancreatic progenitor cells are contacted with the at least one SHH pathway inhibitor at a concentration 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 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 contacted with the at least one SHH 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 NKX6-1 positive pancreatic progenitor cells are contacted with the at least one SHH pathway inhibitor at a concentration of 0.25 μM.

[0304] 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 NKX6-1 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 NKX6-1 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.

[0305] In some embodiments, the NKX6-1 positive pancreatic progenitor cells are contacted with glucose at a concentration between 1 mM and 50 mM. In some embodiments, the NKX6-1 positive pancreatic progenitor cells are contacted with glucose at a concentration between 25 mM.

[0306] In some embodiments, the insulin-positive endocrine cells can be obtained by differentiating at least a portion of the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells into Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells by contacting the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells with i) a TGF-β signaling pathway inhibitor, b) a thyroid hormone signaling pathway activator, and optionally c) at least one SHH 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 once every two days for a period of between 5 and 7 days under conditions that promote cell cluster formation, thereby inducing differentiation of at least a portion of the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells into Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells. In this case, 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.

[0307] In some embodiments, the insulin-positive endocrine cells can be obtained by differentiating at least some of the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells in the population into Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells, for example, by contacting the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells with i) at least one SHH pathway inhibitor, ii) an RA signaling pathway activator, iii) a γ-secretase inhibitor, vi) a TGF-β signaling pathway inhibitor, v) a TH signaling pathway activator, and at least one growth factor from the epidermal growth factor (EGF) family to induce 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. In this case, the Pdx1-positive, NKX6-1-, and insulin-positive endocrine cells express Pdx1, NKX6-1, NKX2-2, Mafb, glis3, Sur1, Kir6.2, Znt8, SLC2A1, SLC2A3, and / or insulin.

[0308] Generally, the cell population is maintained in an appropriate culture medium for a period of time sufficient to induce differentiation of at least one of the NKX6-1-positive pancreatic progenitor cells in the population into an insulin-positive endocrine cell. Exemplary culture media are shown in Table 4.

[0309] [Table 4]

[0310] In some embodiments, BE5 medium can be used as a suitable culture medium 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.

[0311] In some embodiments, the conditions that promote cell cluster formation include suspension culture. In some embodiments, the period includes a period sufficient to maximize the number of cells co-expressing C-peptide and Nkx6-1. In some embodiments, the period is at least 5 days. In some embodiments, the period is between 5 and 7 days. In some embodiments, the period is at least 7 days. In some embodiments, the suspension culture is replenished daily (e.g., with beta cell maturation factors). In some embodiments, a period of between 5 and 7 days maximizes the number of cells co-expressing C-peptide and Nkx6-1.

[0312] In some embodiments, at least 15% of said NKX6-1-positive pancreatic progenitor cells in said population are induced to differentiate into insulin-positive endocrine cells.

[0313] In some embodiments, at least 99% of said NKX6-1-positive pancreatic progenitor cells in said population are induced to differentiate into insulin-positive endocrine cells.

[0314] Induction of insulin+ endocrine cells into SC-β cells

[0315] Embodiments of the present disclosure include SC-β cells. SC-β cells useful herein can be obtained from any source or generated according to any suitable protocol. In some embodiments, insulin-positive endocrine cells are induced to mature into SC-β cells.

[0316] In some aspects, the present disclosure provides a method for generating mature glucose-responsive beta cells from insulin-positive endocrine cells, comprising contacting a cell population comprising insulin-positive endocrine cells (e.g., under conditions that promote cell cluster formation) with at least two beta cell maturation factors, including a) a transforming growth factor beta (TGF-β) signaling inhibitor and b) a thyroid hormone (TH) signaling pathway activator, to induce in vitro maturation of at least one insulin-positive endocrine cell in the population into an SC-β cell.

[0317] Aspects of the present disclosure include generating SC-β cells that are similar in shape and function to endogenous mature β cells, yet are distinct from natural 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., consecutive) glucose challenges similar to the response of endogenous human islets to multiple glucose challenges. In some embodiments, the SC-β cells are capable of releasing or secreting insulin in response to two consecutive glucose challenges. In some embodiments, the SC-β cells are capable of releasing or secreting insulin in response to three consecutive glucose challenges. In some embodiments, the SC-β cells are capable of releasing or secreting insulin in response to four consecutive glucose challenges. In some embodiments, the SC-β cells are capable of releasing or secreting insulin in response to five consecutive glucose challenges. In some embodiments, the SC-β cells release or secrete insulin in response to continuous consecutive glucose challenges. In some embodiments, the cells are capable of releasing or secreting insulin in response to continuous ... 2+ These can be assayed to determine whether they respond to successive glucose challenges by determining whether they repeatedly increase β.

[0318] In some embodiments, the morphology of the SC-β cells is similar to that of endogenous β cells. In some embodiments, the SC-β cells exhibit a glucose-stimulated insulin secretion (GSIS) response in vitro. In some embodiments, the SC-β cells exhibit a GSIS response in vivo. In some embodiments, the SC-β cells exhibit a GSIS response in vitro and / or in vivo. In some embodiments, the in vitro and / or in vitro GSIS response is similar to the GSIS response of endogenous mature β cells. In some embodiments, the SC-β cells exhibit an in vitro (GSIS) response similar to the GSIS response of endogenous β cells. In some embodiments, the SC-β cells exhibit an in vivo GSIS response similar to the GSIS response of endogenous β cells. The GSIS response may be observed immediately after transplantation into a human or animal subject. In some embodiments, the GSIS response is observed within two weeks of transplantation of the SC-β cells into a human or animal subject. In some embodiments, the GSIS response is observed within 2 weeks after transplantation of the SC-β cells into a human or animal subject, or by 3 weeks, 4 weeks, 5 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 by 1 year or more after transplantation of the SC-β cells into a human or animal subject.

[0319] In some embodiments, the SC-β cells display at least one marker of mature endogenous pancreatic β cells. Exemplary markers include, but are not limited to, Pdx1, HNF6, Ptf1a, Sox9, FoxA2, ​​Nkx2.2, Ngn3, and NKX6-1. In some embodiments, expression of the marker selected from the group consisting of HNF6, Ptf1a, Sox9, FoxA2, ​​Nkx2.2, Ngn3, and NKX6-1 is statistically significantly upregulated in the SC-β cells relative to the pluripotent stem cells (e.g., embryonic stem cells or induced pluripotent cells) from which the SC-β cells are derived.

[0320] The present disclosure contemplates the use of any TGF-β signaling pathway inhibitor (e.g., alone or in any combination with at least one thyroid hormone (TH) signaling pathway activator, or optionally, a protein kinase inhibitor) to induce insulin-positive endocrine cells to differentiate and / or mature into SC-β cells. In some embodiments, the TGF-β signaling pathway comprises TGF-β receptor type I kinase signaling. In some embodiments, the TGF-β signaling pathway inhibitor comprises Alk5 inhibitor II.

[0321] The present disclosure contemplates the use of any thyroid hormone signaling pathway activator (e.g., alone or in any combination with at least one TGF-β signaling pathway inhibitor, or optionally, a protein kinase inhibitor) to induce insulin-positive endocrine cells to differentiate and / or mature into SC-β cells. In some embodiments, the thyroid hormone signaling pathway activator comprises T3.

[0322] In some embodiments, the Pdx1-positive, NKX6-1-positive, insulin-positive cells are optionally contacted with a protein kinase inhibitor. 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 Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with the protein kinase inhibitor. The present disclosure contemplates the use of any protein kinase inhibitor (e.g., alone or in any combination with at least one TGF-β signaling pathway inhibitor and / or thyroid hormone signaling pathway activator) to induce insulin-positive endocrine cells to differentiate and / or mature into SC-β cells. In some embodiments, the protein kinase inhibitor comprises staurosporine.

[0323] In some embodiments, the method includes contacting the cell population (eg, insulin-positive endocrine cells) with at least one additional beta cell maturation factor.

[0324] In some embodiments, the at least one additional beta cell maturation factor comprises a cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor. In some embodiments, the method comprises contacting the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells with a CFTR inhibitor. The present disclosure contemplates the use of any CFTR inhibitor (e.g., alone or in any combination with at least one TGF-β signaling pathway inhibitor and / or thyroid hormone signaling pathway activator, and optionally a protein kinase inhibitor) to induce insulin-positive endocrine cells to differentiate and / or mature into SC-β cells. In some embodiments, the CFTR inhibitor comprises Gly-H101.

[0325] 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 Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells with an O-GlcNAcase inhibitor. The present disclosure contemplates the use of any O-GlcNAcase inhibitor (e.g., alone or in any combination with at least one TGF-β signaling pathway inhibitor and / or thyroid hormone signaling pathway activator, and optionally, a protein kinase inhibitor) to induce insulin-positive endocrine cells to differentiate and / or mature into SC-β cells. In some embodiments, the O-GlcNAcase inhibitor comprises Thiamet G.

[0326] Those skilled in the art will understand that the concentration of the agent (e.g., growth factor) used 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 between 100 nM and 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.

[0327] In some embodiments, the insulin-positive endocrine cells are contacted with the thyroid hormone signaling pathway activator at a concentration between 0.1 μM and 10 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 endocrine cells are contacted with the thyroid hormone signaling 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 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.

[0328] In some embodiments, the insulin-positive endocrine cells are contacted with the protein kinase inhibitor at a concentration between 10 nM-1 μM. In some embodiments, the insulin-positive endocrine cells are contacted with the protein kinase inhibitor at a concentration between 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.

[0329] In some embodiments, the Pdx1-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 Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with the CFTR inhibitor at a concentration of between 10 nM and 10 μM.

[0330] In some embodiments, the Pdx1-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, hi some embodiments, the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with the O-GlcNAcase inhibitor at a concentration of between 10 nM and 10 μM.

[0331] In some embodiments, SC-β cells can be obtained by differentiating at least some of the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells into SC-β cells by contacting the Pdx1-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 once every two days for a period of between 7 and 14 days under conditions that promote cell cluster formation, thereby inducing in vitro maturation of at least some of the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells into SC-β cells. In this case, the SC-β cells exhibit a GSIS response both in vitro and / or in vivo. In some embodiments, the GSIS response is similar to that of endogenous β cells.

[0332] In some embodiments, SC-β cells can be obtained by differentiating at least some of the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells in the population into SC-β cells, e.g., by contacting the Pdx1-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 Pdx1-positive, NKX6-1-positive, insulin-producing endocrine cells into SC-β cells, where the SC-β cells exhibit a GSIS response both in vitro and / or in vivo similar to that of endogenous β cells.

[0333] In some aspects, the present disclosure provides a method for generating SC-β cells, comprising contacting Pdx1-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 under conditions that promote cell cluster formation, thereby inducing in vitro maturation of at least some of the Pdx1-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 endogenous β cells.

[0334] In some aspects, the disclosure provides a method for differentiation of at least some of the Pdx1-positive pancreatic progenitor cells in the collection, comprising: a) differentiating pluripotent stem cells in a collection into Pdx1-positive pancreatic progenitor cells; b) differentiating at least some of the Pdx1-positive pancreatic progenitor cells into Pdx1-positive, NKX6-1-positive pancreatic progenitor cells by contacting the Pdx1-positive pancreatic progenitor cells with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally iii) an RA signaling pathway activator, once every two days for a period of five days under conditions that promote cell cluster formation, to induce differentiation of at least some of the Pdx1-positive pancreatic progenitor cells in the collection into NKX6-1-positive pancreatic progenitor cells, wherein the NKX6-1-positive pancreatic progenitor cells express Pdx1 and NKX6-1; c) differentiating the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells in the collection under conditions that promote cell cluster formation. and inducing differentiation of at least a portion of the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells into Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells by contacting the cells with i) a TGF-β signaling pathway inhibitor, b) a TH signaling pathway activator, and optionally c) at least one SHH 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 once every two days for a period of between five and seven days, wherein the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells are differentiated into Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells, wherein the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are induced by the following: Pdx1, NKX6-1, NKX2-2, Mafb, glis3, Sur1, Kir6.and d) differentiating at least some of the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells into SC-β cells by contacting the Pdx1-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 once every two days for a period of between 7 and 14 days under conditions that promote cell cluster formation, thereby inducing in vitro maturation of at least some of the Pdx1-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 is similar to that of endogenous β cells.

[0335] In some aspects, the disclosure provides a method for producing a method of differentiation comprising: a) differentiating at least a portion of the pluripotent cells in a population into Pdx1-positive pancreatic progenitor cells; b) differentiating at least a portion of the Pdx1-positive pancreatic progenitor cells into Pdx1-positive, NKX6-1-positive pancreatic progenitor cells by contacting the Pdx1-positive pancreatic progenitor cells with i) KGF, ii) Sant1, and optionally iii) a low concentration of RA, once every two days for a period of five days under conditions that promote cell cluster formation, to induce differentiation of at least one of the Pdx1-positive pancreatic progenitor cells in the population into an NKX6-1-positive pancreatic progenitor cell, wherein the NKX6-1-positive pancreatic progenitor cells express Pdx1 and NKX6-1; and c) differentiating at least a portion of the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells into Pdx1-positive, NKX6-1-positive pancreatic progenitor cells by contacting the Pdx1-positive pancreatic progenitor cells with i) KGF, ii) Sant1, and optionally iii) a low concentration of RA, once every two days for a period of five days under conditions that promote cell cluster formation, to induce differentiation of at least one of the Pdx1-positive pancreatic progenitor cells in the population into an NKX6-1-positive pancreatic progenitor cell, wherein the NKX6-1-positive pancreatic progenitor cells express Pdx1 and NKX6-1; and (v) XXI and vi) betacellulin, once every two days for a period of between five and seven days, to induce differentiation of at least a portion 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 are differentiated into Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells by a method comprising contacting Pdx1-positive, NKX6-1-positive pancreatic progenitor cells with i) Alk5 inhibitor II, ii) T3, and optionally iii) Sant1, iv) RA, v) XXI and vi) betacellulin once every two days for a period of between five and seven days.and d) differentiating at least some of the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells into SC-β cells by contacting the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells with i) Alk5 inhibitor II, ii) T3, and optionally iii) staurosporine once every two days for a period of between 7 and 14 days under conditions that promote cell cluster formation, thereby inducing in vitro maturation of at least some of the Pdx1-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 similar to that of endogenous β cells.

[0336] Generally, the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are maintained in a suitable culture medium for a period of time sufficient to induce in vitro maturation of at least some of the Pdx1-positive, NKX6-1-positive, insulin-positive SC-β cells. Exemplary suitable culture media are listed in Table 4 above and Table 5 below.

[0337] [Table 5]

[0338] In some embodiments, the suitable culture medium comprises Connought Medical Research Laboratories 1066 supplemented with pancreatic islet growth medium (CMRLS). In some embodiments, the suitable culture medium comprises a component of CMRLS (supplemented zinc). In some embodiments, the suitable culture medium is set forth 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, the culture medium suitable for differentiating insulin-positive endocrine cells into SC-β cells comprises S3 medium. In some embodiments, the conditions promoting cell cluster formation comprise suspension culture. In some embodiments, the period comprises at least 7 days. In some embodiments, the period comprises between 7 and 21 days. In some embodiments, the period comprises between 7 and 14 days. In some embodiments, the period comprises between 10 and 14 days. In some embodiments, the period comprises 14 days. In some embodiments, the suspension culture is replenished (e.g., with the beta cell maturation factors) every two days.

[0339] 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%, or at least 50% of the Pdx1-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%, or at least 99% of the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are induced to mature into SC-β cells. In some embodiments, at least 30% of the generated cells comprise SC-β cells. In some embodiments, the SC-β cells express C-peptide, insulin, NKX6-1, and Pdx1, and co-express NKX6-1 and C-peptide.

[0340] In some embodiments, the SC-β cells comprise human cells. In some embodiments, the in vitro generation of the SC-β cells is scalable.

[0341] Isolated collection of cells

[0342] Aspects of the present disclosure relate to isolated populations of cells produced according to the methods described herein. In some embodiments, the population of SC-β cells is produced by contacting at least one insulin-positive endocrine cell, or its precursor, with at least one β cell maturation factor described herein. In some embodiments, the population of SC-β cells is produced by contacting at least one insulin-positive endocrine cell, or its precursor, with at least two β cell maturation factors described herein. In some embodiments, the population of SC-β cells is produced by contacting at least one insulin-positive endocrine cell, or its precursor, with at least three β cell maturation factors described herein. In some embodiments, the population of SC-β cells is produced by contacting at least one insulin-positive endocrine cell, or its precursor, with at least four β cell maturation factors described herein. In some embodiments, the population of SC-β cells is produced by contacting at least one insulin-positive endocrine cell, or its precursor, with at least five β cell maturation factors described herein. In some embodiments, the population of SC-β cells is produced 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, or at least ten of the β cell maturation factors described herein.

[0343] In some aspects, the present disclosure provides an isolated population of definitive endoderm cells. For example, the isolated population of definitive endoderm cells can be obtained by differentiating at least some of the pluripotent cells in the population into definitive endoderm cells by contacting the population 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.

[0344] In some aspects, the present disclosure provides an isolated population of primitive gut cells. For example, the isolated population of primitive gut cells can be obtained by differentiating at least some of the definitive endoderm cells in the population into primitive gut cells by contacting the definitive endoderm cells with at least one growth factor selected from fibroblast growth factors (FGFs) to induce differentiation of at least some of the definitive endoderm cells into primitive gut cells. In this case, the primitive gut cells express at least one marker specific to definitive endoderm.

[0345] In some aspects, the present disclosure provides an isolated population of Pdx1-positive pancreatic progenitor cells. For example, the isolated population of Pdx1-positive pancreatic progenitor cells can be obtained by differentiating at least some of the primitive gut cells in the population into Pdx1-positive pancreatic progenitor cells by contacting the primitive gut cells with: i) at least one bone morphogenetic protein (BMP) signaling 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) signaling pathway activator; and v) at least one protein kinase C activator to induce differentiation of at least some of the primitive gut cells into Pdx1-positive pancreatic progenitor cells. In this case, the Pdx1-positive pancreatic progenitor cells express Pdx1.

[0346] In some aspects, the present disclosure provides an isolated population of NKX6-1-positive pancreatic progenitor cells. For example, the isolated population of NKX6-1-positive pancreatic progenitor cells can be obtained by differentiating at least some of the Pdx1-positive pancreatic progenitor cells in the population into Pdx1-positive, NKX6-1-positive pancreatic progenitor cells by contacting the Pdx1-positive pancreatic progenitor cells with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally iii) an RA signaling pathway activator to induce differentiation of at least one Pdx1-positive pancreatic progenitor cell in the population into an NKX6-1-positive pancreatic progenitor cell. In this case, the NKX6-1-positive pancreatic progenitor cells express Pdx1 and NKX6-1.

[0347] In some embodiments, the present disclosure provides an isolated population of insulin-positive endocrine cells. For example, the isolated population of insulin-positive endocrine cells can be obtained by differentiating at least some of the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells in the population into Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells by contacting the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells with: i) a TGF-β signaling 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 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, thereby inducing 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. In this case, the Pdx1-positive, NKX6-1-, and insulin-positive endocrine cells express Pdx1, NKX6-1, NKX2-2, Mafb, glis3, Sur1, Kir6.2, Znt8, SLC2A1, SLC2A3, and / or insulin.

[0348] In some aspects, the present disclosure provides an isolated population of SC-β cells. For example, the isolated population of SC-β cells can be obtained by differentiating at least some of the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells in the population into SC-β cells by contacting the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells with i) a transforming growth factor β (TGF-β) signaling 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 Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells into SC-β cells. In this case, the SC-β cells exhibit a GSIS response in vitro and / or in vivo. In some embodiments, the GSIS response is similar to that of endogenous β cells.

[0349] Aspects of the present disclosure include microcapsules comprising an isolated population of cells (e.g., SC-β cells) described herein. Microcapsules are well known in the art. Suitable examples of microcapsules can be found 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 technology: Towards clinical application," Journal of Controlled Release 2008; 132:76-83; and Zanin et al. al., "The development of encapsulated cell technologies as therapies for neurological and sensory diseases," Journal of Controlled Release 2012;160:3-13). Microcapsules can be formulated in a variety of ways. A typical microcapsule contains an alginate core surrounded by a polycation layer, which is covered by an outer alginate membrane. The polycation membrane forms a semipermeable membrane.The semipermeable membrane confers stability and biocompatibility. Examples of polycations include, but are not limited to, poly-L-lysine, poly-L-ornithine, chitosan, lactose-modified chitosan, and photopolymerizable biomaterials. In some embodiments, the alginate core is modified to produce a scaffold comprising, for example, an alginate core covalently bonded to an oligopeptide having an RGD sequence (arginine, glycine, aspartic acid). In some embodiments, the alginate core is modified to produce a covalently reinforced microcapsule comprising, for example, chemoenzymatically modified alginate for improved stability. In some embodiments, the alginate core is modified to produce a membrane-like film constructed by, for example, in situ polymerization of acrylate-functional phospholipids. In some embodiments, the microcapsules are composed of alginate enzymatically modified using an epimerase. In some embodiments, the microcapsules comprise covalent bonds between adjacent layers of the microcapsule membrane. In some embodiments, the microcapsules comprise subsieve-sized capsules comprising alginate conjugated with a phenolic moiety. In some embodiments, the microcapsules comprise a scaffold comprising alginate-agarose. In some embodiments, the SC-β cells are modified with PEG before being encapsulated in alginate. In some embodiments, cells, e.g., isolated populations of SC-β cells, are encapsulated in photoreactive liposomes and alginate. 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, RGD-containing dextran, EHD and PEGDA, PMBV and PVA, PGSAS, agarose, gelatin-containing agarose, PLGA, and multilayer embodiments thereof.

[0350] In some embodiments, compositions containing cell populations produced according to the methods described herein can also be used as functional components in mechanical devices designed to produce one or more endocrine polypeptides from pancreatic islet cells. In its simplest form, the device contains a population of pancreatic beta cells (e.g., produced from a population of insulin-positive endocrine cells or their precursors) behind a semipermeable membrane that prevents the passage of the cell population, retaining them within the device but allowing the passage of insulin, glucagon, or somatostatin secreted by the cell population. This typically involves a population of microencapsulated pancreatic beta cells in the form of cell clusters that allow cell interactions that inhibit differentiation. For example, U.S. Patent No. 4,391,909 describes pancreatic islet cells encapsulated in an ellipsoidal, semipermeable membrane composed of polysaccharide polymers greater than 3,000 mol wt, cross-linked to allow permeability to insulin-sized proteins but not to molecules greater than 100,000 mol wt. U.S. Patent No. 6,023,009 describes pancreatic islet cells encapsulated in a semipermeable membrane formed from agarose and agaropectin. Microcapsules of this nature are adapted for intracorporeal administration to diabetic patients and are believed to have particular advantages in reducing tissue compatibility issues or susceptibility to bacteria.

[0351] More sophisticated devices containing a population of pancreatic beta cells generated from insulin-positive endocrine cells or their precursors based on the methods described herein are also contemplated, for either implantation in diabetic patients or extracorporeal treatment. U.S. Patent No. 4,378,016 describes an artificial endocrine gland comprising an extracorporeal segment, a subcutaneous segment, and a replaceable envelope containing hormone-producing cells. U.S. Patent No. 5,674,289 describes a bioartificial pancreas having a pancreatic islet chamber separated by a semipermeable membrane from one or more vascularized chambers open to the surrounding tissue. Useful devices typically have a chamber adapted to contain pancreatic islet cells and a chamber separated from the islet cells by a semipermeable membrane that collects secreted proteins from the islet cells and, in some cases, allows for signaling of circulating glucose levels back to the islet cells.

[0352] Aspects of the present disclosure include assays comprising isolated populations of cells (e.g., SC-β cells) described herein. In some embodiments, the assays may 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, β-cell death, β-cell function, β-cell susceptibility to immune attack, or β-cell susceptibility to dedifferentiation or differentiation. In some embodiments, the assays may be used to identify one or more candidate agents that promote the differentiation of at least one insulin-positive endocrine cell or its precursor into an SC-β cell. In some embodiments, the assays may be used to identify one or more candidate agents that stimulate β-cells to produce insulin or increase insulin production or secretion.

[0353] Based on the methods described herein, the present disclosure contemplates methods in which SC-β cells are generated from iPS cells obtained from cells extracted or isolated from an individual suffering from a disease (e.g., diabetes, obesity, or a β-cell-related disorder), and the SC-β cells are compared to normal β cells from a healthy individual without the disease to identify differences between the SC-β cells and normal β cells that may be useful as markers for the disease (e.g., acquired and / or genetic). In some embodiments, β cells are obtained from a diabetic individual and compared to normal β cells, and the β cells are then reprogrammed into iPS cells. The iPS cells are analyzed for genetic and / or acquired markers present in β cells obtained from the diabetic individual but absent from the normal β cells to identify (e.g., prediabetic) markers. In some embodiments,...

Claims

1. 1. A composition comprising a population of cells, the population comprising a plurality of non-native pancreatic beta cells, the non-native pancreatic beta cells comprising: (a) expressing INS, PDX1, NKX6.1, and one or more of the following genes: PC2, MNX1, or ABCC8; (b) exhibiting an in vitro glucose-stimulated insulin secretory response when subjected to a first glucose challenge; (c) comprises one or more crystalline insulin granules; (d) does not express one or both of somatostatin and glucagon; The composition, wherein the collection further comprises a plurality of cells that express glucagon.

2. 2. The composition of claim 1, wherein the plurality of non-native pancreatic beta cells exhibits an in vitro glucose-stimulated insulin secretion response when subjected to a first glucose challenge, a second glucose challenge, and a third glucose challenge, where the first glucose challenge, the second glucose challenge, and the third glucose challenge are applied sequentially.

3. 10. The composition of claim 1, wherein the plurality of non-native pancreatic beta cells is generated in vitro from human stem cells.

4. The composition of claim 3, wherein the stem cells are embryonic stem cells.

5. The composition of claim 3 , wherein the stem cells are induced pluripotent stem cells.

6. The composition of claim 1 , wherein the plurality of non-native pancreatic beta cells expresses PC2.

7. 2. The composition of claim 1, wherein the plurality of non-native pancreatic beta cells expresses MNX1.

8. The composition of claim 1 , wherein the plurality of non-native pancreatic beta cells expresses ABCC8.

9. 2. The composition of claim 1, wherein the plurality of non-native pancreatic beta cells expresses chromogranin A.

10. 10. The composition of claim 1, wherein the plurality of non-native pancreatic beta cells expresses C-peptide.

11. 10. The composition of claim 1, wherein the plurality of non-native pancreatic beta cells expresses MAFB.

12. 2. The composition of claim 1, wherein the plurality of non-naturally occurring pancreatic beta cells exhibits an in vitro glucose-stimulated insulin secretion response when subjected to a first glucose challenge at a first glucose concentration and a second glucose challenge at a second glucose concentration, where the first glucose challenge and second glucose challenge are applied sequentially, wherein the first glucose concentration is greater than the second glucose concentration, and the plurality of non-naturally occurring pancreatic beta cells exhibits a stimulation index of at least 1.1, wherein the stimulation index is equal to the ratio of insulin secreted in response to the first glucose concentration to the second glucose concentration.

13. 10. The composition of claim 1, wherein said plurality of non-native pancreatic beta cells is monohormonal.

14. 13. The composition of claim 12, wherein the insulin secreted by said plurality of non-naturally occurring pancreatic beta cells is at least 0.5 μIU per 1000 cells per 30 minute incubation when said plurality of non-naturally occurring pancreatic beta cells is exposed to at least 20 mM glucose.

15. The composition of claim 1 , wherein the plurality of non-native pancreatic beta cells is genetically modified.

16. 2. The composition of claim 1, wherein at least 10% of the cells in the population of cells are a plurality of non-native pancreatic beta cells.

17. The composition of claim 1 , wherein the population of cells is a cluster of cells, with the proviso that the non-naturally occurring pancreatic beta cells do not include ABCC8-expressing non-naturally occurring pancreatic beta cells.

18. 18. The composition of claim 17, wherein the composition comprises a plurality of clusters of cells.

19. The population of cells comprises: a) C-peptide negative / glucagon positive cells; b) C-peptide negative / somatostatin positive cells; c) glucagon-positive / somatostatin-negative cells; d) glucagon-negative / somatostatin-positive cells; or e) any combination of these The composition of claim 1 comprising one of:

20. 2. The composition of claim 1, wherein at least 3% of the cell population are C-peptide negative / glucagon positive cells or glucagon positive / somatostatin negative cells.

21. the plurality of non-native pancreatic beta cells comprising: a) expresses MNX1; b) generated in vitro from human stem cells; c) the composition of claim 1, having a gene expression profile that differs from that of native beta cells.

22. 22. The composition of claim 21, wherein the plurality of non-native pancreatic beta cells expresses MAFB.

23. 22. The composition of claim 21, wherein the plurality of non-native pancreatic beta cells expresses chromogranin A and C-peptide.

24. 22. The composition of claim 21, wherein the plurality of non-native pancreatic beta cells exhibits an in vitro glucose-stimulated insulin secretion response when subjected to a first glucose challenge, a second glucose challenge, and a third glucose challenge, where the first glucose challenge, the second glucose challenge, and the third glucose challenge are applied sequentially.

25. 22. The composition of claim 21, wherein the population of cells is a cluster of cells.

26. 22. The composition of claim 21, wherein the plurality of non-native pancreatic beta cells are genetically modified.

27. 22. The composition of claim 21, wherein the plurality of non-native pancreatic beta cells expresses PC2 and ABCC8.

28. the plurality of non-native pancreatic beta cells comprising: a) expressing PC2, ABCC8, MAFB, chromogranin A and C peptide; b) exhibiting an in vitro glucose-stimulated insulin secretion response when subjected 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.

29. 30. The composition of claim 28, wherein the plurality of non-native pancreatic beta cells are genetically modified.

30. 29. The composition of claim 28, wherein the population of cells is a cluster of cells.

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