RU2813705C1 - SC-β CELLS AND COMPOSITIONS AND METHODS FOR THEIR CREATION - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: device for insulin secretion containing a population of non-native pancreatic β-cells, and non-native pancreatic β-cells demonstrate an in vitro response consisting of glucose-stimulated insulin secretion to glucose stimulation. Moreover, the device contains a semi-permeable membrane, which is designed to retain non-native pancreatic β-cells in the device and ensure the passage of insulin secreted by non-native pancreatic β-cells.

EFFECT: invention can be effectively used in cell therapy.

18 cl, 17 dwg, 5 tbl

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[1] This application claims the benefit of U.S. Provisional Patent Application No. 61/833898, filed June 11, 2013, and U.S. Provisional Patent Application No. 61/972212, filed March 28, 2014, the entire contents of which are incorporated herein by links.

BACKGROUND OF THE ART

[2] Research to date has only produced abnormally functioning insulin-expressing cells that do not secrete adequate amounts of insulin in response to successive changes in glucose levels, or pancreatic progenitor cells that can mature into functioning insulin-expressing cells in only 3 years. 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; Nostra et al., 2011; Rezania et al., 2012; Schulz et al., 2012; Xie et al., 2013). Unlike normal islet cells or dispersed adult β-cells, which release high levels of insulin in response to high glucose levels in the glucose-stimulated insulin secretion (GSIS) assay and can do so repeatedly, insulin-expressing cells obtained from existing methods from hPSCs are not able to properly secrete insulin in response to the addition of different concentrations of glucose. Accordingly, there is a need for a method of obtaining cells from hPSCs that exhibit a normal islet cell or adult β-cell phenotype.

SUMMARY OF THE INVENTION

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

[4] In some embodiments, the cell is mature. In some embodiments, the cell exhibits a glucose-stimulated insulin secretion (GSIS) response in vitro. In some embodiments, the cell exhibits an iv vivo GSIS response. In some embodiments, the cell exhibits in vitro and in vivo responses consisting of glucose-stimulated insulin secretion (GSIS). In some embodiments, the cell exhibits a GSIS response to at least one glucose stimulation. In some embodiments, the cell exhibits a GSIS response to at least two sequential glucose stimulations. In some embodiments, the cell exhibits a GSIS response to at least three consecutive glucose stimulations. In some embodiments, the GSIS response is observed immediately after cell transplantation into a human or animal. In some embodiments, the GSIS response is observed within approximately 24 hours after the cell is transplanted into a human or animal body. In some embodiments, the GSIS response is observed within approximately two weeks after the cell is transplanted into a human or animal body. In some embodiments, the stimulation index, characterized by the ratio of insulin secreted in response to high glucose concentrations and low glucose concentrations, is similar to the stimulation index of an endogenous mature pancreatic β-cell. In some embodiments, the stimulation index is greater than or equal to 1, or greater than or equal to 1.1, or greater than or equal to 1.3, or greater than or equal to 2, or greater than or equal to 2.3, or greater than or equal to 2.6. In some embodiments, the cell exhibits cytokine-induced apoptosis in response to the 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 cell is increased in response to the 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 cell is monohormonal. In some embodiments, the cell exhibits a morphology that is similar to that of an endogenous mature pancreatic β cell. In some embodiments, encapsulated crystalline insulin granules that resemble endogenous mature pancreatic β-cell insulin granules are observed in the cell under an electron microscope. In some embodiments, the cell exhibits a low replication coefficient. In some embodiments, the cell exhibits glucose-stimulated Ca ²⁺ flux (GSCF), which is similar to the GSCF of an endogenous mature pancreatic β-cell. In some embodiments, the cell exhibits a GSCF response to at least one glucose stimulation. In some embodiments, the cell exhibits a GSCF response to at least two glucose stimulations. In some embodiments, the cell exhibits a GSCF response to at least three glucose stimulations. In some embodiments, the cell exhibits increased calcium flux. In some embodiments, the increased calcium flux includes an increased influx or an increased ratio between influxes at low and high glucose concentrations. In some embodiments, the cell expresses at least one endogenous mature pancreatic β-cell marker 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, Pax4. In some embodiments, the cell does not express at least one marker selected from the group consisting of a) a hormone selected from the group consisting of i) glucagon (GCG) and ii) somatostatin (SST); or b) an acinar cell marker selected from the group consisting of i) amylase and ii) carboxypeptidase A (CPA1); c) an α-cell marker selected from the group consisting of i) GCG, ii) Arx, iii) Irx1 and Irx2; and d) a ductal cell marker selected from the group consisting of i) CFTR and ii) Sox9. In some embodiments, the cell is differentiated in vitro from an insulin-positive endocrine cell or a precursor thereof selected from the group consisting of an Nkx6-1-positive pancreatic progenitor cell, a Pdx1-positive pancreatic progenitor cell, and a pluripotent stem cell. In some embodiments, the pluripotent stem cell is selected from the group consisting of an embryonic stem cell and an induced pluripotent stem cell. In some embodiments, the cell is human. In some embodiments, the cell is not genetically modified. In some embodiments, the cell is genetically modified. In some embodiments, the amount of insulin produced per cell is between 0.5 and 10 μIU per 1000 cells per 30 minutes of incubation at a high glucose concentration. In some embodiments, the amount of insulin produced per cell is approximately 2.5 μIU per 1000 cells per 30 minutes of incubation at high glucose concentration. In some embodiments, the incubation is carried out ex vivo.

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

[6] In some aspects, the invention provides a method of generating an SC-β cell from insulin-positive endocrine cells, comprising bringing a population of cells comprising insulin-positive endocrine cells into contact, under conditions that promote cell clustering, with at least two factors β-cell maturation agents comprising a) an inhibitor of the transforming growth factor β (TGF-β) signaling pathway and b) an activator of the thyroid hormone signaling pathway to induce in vitro maturation of at least one insulin-positive endocrine cell in the population into an SC-β cell .

[7] In some embodiments, the SC-β cell exhibits a response to at least one glucose stimulation. In some embodiments, the SC-β cell exhibits a response to at least two sequential glucose stimulations. In some embodiments, the SC-β cell exhibits a response to at least three sequential glucose stimulations. In some embodiments, the morphology of the SC-β cell is similar to the morphology of an endogenous mature β cell. In some embodiments, the SC-β cell exhibits in vitro and/or in vivo responses consisting of glucose-stimulated insulin secretion (GSIS). In some embodiments, the GSIS response is observed immediately after transplantation of an SC-β cell into a subject. In some embodiments, the GSIS response is observed for approximately 24 hours after transplantation of the SC-β cell into the subject. In some embodiments, the GSIS response is observed for approximately two weeks after transplantation of the SC-β cell into the subject. In some embodiments, a population of cells is contacted with a TGF-β signaling pathway inhibitor at a concentration of 100 nM to 100 μM. In some embodiments, a population of cells is contacted with a TGF-β signaling pathway inhibitor at a concentration of 10 μM. In some embodiments, the TGF-β signaling pathway includes TGF-β receptor type I kinase signaling. In some embodiments, the TGF-β signaling pathway inhibitor includes an Alk5 inhibitor II. In some embodiments, the TGF-β signaling pathway inhibitor includes an analogue or derivative of Alk5 inhibitor II. In some embodiments, a population of cells is contacted with a thyroid hormone signaling pathway activator at a concentration of 0.1 μM to 10 μM. In some embodiments, a population of cells is contacted with a thyroid hormone signaling pathway activator at a concentration of 1 μM. In some embodiments, the thyroid hormone signaling pathway activator includes triiodothyronine (T3). In some embodiments, the population of cells is optionally contacted with a protein kinase inhibitor. In some embodiments, the population of cells is not brought into contact with the protein kinase inhibitor. In some embodiments, a population of cells is contacted with a protein kinase inhibitor. In some embodiments, a population of cells is contacted with a protein kinase inhibitor at a concentration of 10 nM to 1 μM. In some embodiments, a population of cells is contacted with a protein kinase inhibitor at a concentration of 100 nM. In some embodiments, the protein kinase inhibitor includes staurosporine. In some embodiments, the method includes contacting a population of cells with at least one additional β-cell maturation factor. In some embodiments, the at least one additional β-cell maturation factor comprises a cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor. In some embodiments, a population of cells is contacted with a CFTR inhibitor at a concentration of 100 nM - 100 μM. In some embodiments, a population of cells is contacted with a CFTR inhibitor at a concentration of 10 nM - 10 μM. In some embodiments, the CFTR inhibitor includes Gly-H101. In some embodiments, the at least one additional β-cell maturation factor comprises an O-GlcNAase inhibitor. In some embodiments, a population of cells is contacted with an O-GlcNAase inhibitor at a concentration of 100 nM to 100 μM. In some embodiments, a population of cells is contacted with an O-GlcNAase inhibitor at a concentration of 10 nM to 10 μM. In some embodiments, the O-GlcNAase inhibitor includes Thiamet-G. In some embodiments, the population of cells is cultured in a suitable culture medium. In some embodiments, a suitable culture medium includes Connought Medical Research Laboratories Supplemented Islet Medium 1066 (CMRLS) or a CMRLS component. In some embodiments, the CMRLS is supplemented with serum. In some embodiments, the CMRLS is supplemented with 10% fetal bovine serum. In some embodiments, conditions that promote cell clustering include suspension culture. In some embodiments, the cell population is maintained in suspension culture for a period of time sufficient to induce in vitro maturation of at least one of the insulin-positive endocrine cells in the cell population into at least one SC-β cell. In some embodiments of the invention, the time period is at least 7 days. In some embodiments of the invention, the time period is from 7 days to 21 days. In some embodiments of the invention, the time period is from 7 to 14 days. In some embodiments of the invention, the time period is from 10 to 14 days. In some embodiments of the invention, the time period is 14 days. In some embodiments, β-cell maturation factors are replenished every other day. In some embodiments, at least 1% of the insulin-positive endocrine cells in the cell population are induced to mature into SC-β cells. In some embodiments, at least 99% of the insulin-positive endocrine cells in the population are induced to mature into SC-β cells. In some embodiments, at least 30% of the resulting cells in the population include SC-β cells. In some embodiments, SC-β cells express C-peptide, insulin, NKX6-1, Pdx1, and 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 derived from a population of pluripotent stem cells selected from the group consisting of embryonic stem cells and induced pluripotent stem cells. In some embodiments, SC-β cells include human cells. In some embodiments, in vitro generation of SC-β cells is scalable.

[8] In some aspects, the invention provides an isolated population of SC-β cells obtained in accordance with the methods described herein.

[9] In some aspects, the invention provides a microcapsule containing an isolated population of SC-β cells encapsulated therein.

[10] In some aspects, the invention provides a composition comprising a population of SC-β cells obtained in accordance with the method described herein.

[11] In some aspects, the invention provides an assay method comprising an isolated population of SC-β cells obtained in accordance with the method described herein.

[12] In some embodiments, the assay is designed to identify one or more candidate agents that promote or inhibit β-cell fate selected from the group consisting of β-cell proliferation, β-cell replication, β-cell death, function β cells, β cell susceptibility to immune attack, or β cell susceptibility to dedifferentiation or differentiation. In some embodiments, the assay is designed to identify one or more candidate agents that stimulate the differentiation of at least one insulin-positive endocrine cell or precursor thereof into at least one SC-β cell.

[13] In some aspects, the invention provides a method of treating a subject in need thereof, comprising administering to the subject a composition comprising an isolated population of SC-β cells obtained in accordance with a method described herein. In some embodiments, the SC-β cells are encapsulated in a microcapsule. In some embodiments, the SC-β cells are derived from a population of pluripotent stem cells obtained from the subject to whom the SC-β cells are administered. In some embodiments, the SC-β cells are derived from a population of iPS (induced pluripotent stem cells) cells, wherein the iPS cells are derived from a cell obtained from the subject to whom 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 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.

[14] In some aspects, the invention relates to the use of an isolated population of SC-β cells obtained by the methods of any one of claims. 41-102, for administration to a subject in need.

[15] In some embodiments, the subject is administered an isolated population of SC-β cells encapsulated in microcapsules. 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 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.

[16] In some aspects, the invention provides a culture medium containing a) an Alk5 inhibitor, b) optionally triiodothyronine (T3), c) staurosporine, and optionally d) CMRLS or a CMRLS component.

[17] In some aspects, the invention relates to the use of a culture medium to induce in vitro maturation of insulin-positive endocrine cells into SC-β cells, wherein the SC-β cells exhibit an in vitro and/or in vivo GSIS response.

[18] In some aspects, the invention provides a method of obtaining an NKX6-1-positive pancreatic progenitor cell from a Pdx1-positive pancreatic progenitor cell, comprising bringing a population of cells containing Pdx1-positive pancreatic progenitor cells into contact under conditions that stimulate cell clustering , with at least two β-cell maturation factors including a) at least one growth factor from the fibroblast growth factor (FGF) family, b) a sonic hedgehog pathway inhibitor, and optionally c) a low concentration of a retinoic acid signaling pathway activator ( RA), over a period of at least five days, to induce the differentiation of at least one Pdx1-positive pancreatic progenitor cell in the population into NKX6-1-positive pancreatic progenitor cells, wherein NKX6-1-positive pancreatic cells -progenitors express NKX6-1.

[19] In some embodiments, a population of cells is contacted with at least one growth factor from the FGF family at a concentration of 1 ng/ml to 100 ng/ml. In some embodiments, a population of cells is contacted with at least one growth factor from the FGF family at a concentration of 50 ng/ml. In some embodiments, at least one growth factor from the FGF family includes keratinocyte growth factor (KGF). In some embodiments, 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 population of cells is not brought into contact with the RA signaling pathway activator. In some embodiments, a population of cells is contacted with an RA signaling pathway activator at a concentration of 0.01 μM to 1.0 μM. In some embodiments, a population of cells is contacted with a RA signaling pathway activator at a concentration of 0.1 μM. In some embodiments, the RA signaling pathway activator includes RA. In some embodiments, a population of cells is contacted with an SHH pathway inhibitor at a concentration of 0.1 μM to 0.5 μM. In some embodiments, a population of cells is contacted with an SHH pathway inhibitor at a concentration of 0.25 μM. In some embodiments, the SHH pathway inhibitor includes Sant1. In some embodiments, the method comprises treating a population of cells with at least one additional β-cell maturation factor. In some embodiments, the at least one additional β-cell maturation factor includes at least one growth factor from the EGF family. In some embodiments, the cell population is treated with at least one growth factor from the EGF family at a concentration of 2 ng/ml to 200 ng/ml. In some embodiments, the cell population is treated with at least one growth factor from the EGF family at a concentration of 20 ng/ml. In some embodiments, at least one growth factor from the EGF family is selected from the group consisting of betacellulin and EGF. In some embodiments, the population of cells is cultured in a suitable culture medium. In some embodiments, conditions that promote cell clustering include suspension culture. In some embodiments, β-cell maturation factors are replenished every other day. In some embodiments, no protein kinase C activator is added to the suspension culture for 5 days. In some embodiments, the protein kinase C activator is removed from the suspension culture before the 5 day period has elapsed. In some embodiments, the protein kinase C activator includes PdbU. In some embodiments, no BMP (bone morphogenetic protein) 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 over a period of less than 5 days. In some embodiments, the BMP signaling pathway inhibitor includes LDN193189. In some embodiments, at least 10% of 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, NKX6-1-positive pancreatic progenitor cells express Pdx1, NKX6-1, and FoxA2. In some embodiments, Pdx1-positive pancreatic progenitor cells are derived from a population of pluripotent stem cells selected from the group consisting of embryonic stem cells and induced pluripotent stem cells.

[20] In some aspects, the invention provides an isolated population of NKX6-1 positive pancreatic progenitor cells obtained in accordance with the methods described herein.

[21] In some aspects, the invention provides a microcapsule containing a selected population of NKX6-1 positive pancreatic progenitor cells encapsulated therein.

[22] In some aspects, the invention provides a composition comprising a population of NKX6-1 positive pancreatic progenitor cells obtained in accordance with the method described herein.

[23] In some aspects, the invention provides an assay method comprising an isolated population of NKX6-1 positive pancreatic progenitor cells obtained in accordance with the method described herein.

[24] In some embodiments, the assay is designed to identify one or more candidate agents that stimulate the differentiation of at least one Pdx1-positive pancreatic progenitor cell or a precursor thereof into NKX6-1-positive pancreatic progenitor cells.

[25] In some aspects, the invention 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 obtained in accordance with a method described herein.

[26] In some embodiments, the NKX6-1-positive pancreatic progenitor cells are derived from a population of pluripotent stem cells obtained from the subject to whom the NKX6-1-positive pancreatic progenitor cells are administered. In some embodiments, the NKX6-1-positive pancreatic progenitor cells are encapsulated in a microcapsule. 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 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.

[27] In some aspects, the invention relates to the use of an isolated population of NKX6-1-positive pancreatic progenitor cells obtained by the methods of any one of claims. 113-142, for differentiation into SC-β cells.

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

[29] In some embodiments, the subject is administered an isolated population of NKX6-1 positive pancreatic progenitor cells encapsulated in microcapsules. 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 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.

[30] In some aspects, the invention provides a culture medium comprising KGF, b) SANT1) and optionally c) RA, wherein the culture medium is substantially free of PdbU and LDN 193189. In some embodiments, the invention relates to the use of a culture medium for p. 160 to induce in vitro differentiation of Pdx1-positive pancreatic progenitor cells into NKX6-1-positive pancreatic progenitor cells.

[31] In some aspects, the invention provides a method of producing an insulin-positive endocrine cell from an NKX6-1-positive pancreatic progenitor cell, comprising bringing a population of cells containing NKX6-1-positive pancreatic progenitor cells into contact under conditions that promote clustering cells, with at least two β-cell maturation factors, including a) an inhibitor of the TGF-β signaling pathway and b) an activator of the thyroid hormone signaling pathway, to induce the differentiation of at least one NKX6-1-positive pancreatic progenitor cell in the population according to at least one insulin-positive endocrine cell, wherein the at least one insulin-positive pancreatic progenitor cell expresses insulin. In some embodiments, a population of cells is contacted with a TGF-β signaling pathway inhibitor at a concentration of 100 nM to 100 μM. In some embodiments, a population of cells is contacted with a TGF-β signaling pathway inhibitor at a concentration of 10 μM. In some embodiments, the TGF-β signaling pathway includes TGF-β receptor type I kinase signaling. In some embodiments, the TGF-β signaling pathway inhibitor includes an Alk5 inhibitor II. In some embodiments, a population of cells is contacted with a thyroid hormone signaling pathway activator at a concentration of 0.1 μM to 10 μM. In some embodiments, a population of cells is contacted with a thyroid hormone signaling pathway activator at a concentration of 1 μM. In some embodiments, the thyroid hormone signaling pathway activator includes triiodothyronine (T3). In some embodiments, the method includes contacting a population of cells with at least one additional β-cell maturation factor. In some embodiments, the at least one additional β-cell maturation factor includes a γ-secretase inhibitor. In some embodiments, a population of cells is contacted with a γ-secretase inhibitor at a concentration of 0.1 μM to 10 μM. In some embodiments, a population of cells is contacted with a 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 includes DAPT. In some embodiments, the at least one additional β-cell maturation factor includes at least one growth factor from the EGF family. In some embodiments, a population of cells is contacted with at least one growth factor from the EGF family at a concentration of 2 ng/ml to 200 ng/ml. In some embodiments, a population of cells is contacted with at least one growth factor from the EGF family at a concentration of 20 ng/ml. In some embodiments, at least one growth factor from the EGF family includes betacellulin. In some embodiments, at least one growth factor from the EGF family includes EGF. In some embodiments, the at least one additional β-cell maturation factor comprises a low concentration of a retinoic acid (RA) signaling pathway activator. In some embodiments, a population of cells is contacted with an RA signaling pathway activator at a concentration of 0.01 μM to 1.0 μM. In some embodiments, a population of cells is contacted with a RA signaling pathway activator at a concentration of 0.1 μM. In some embodiments, the RA signaling pathway activator includes RA. In some embodiments, the at least one additional β-cell maturation factor comprises a sonic hedgehog (SHH) pathway inhibitor. In some embodiments, a population of cells is contacted with an SHH pathway inhibitor at a concentration of 0.1 μM to 0.5 μM. In some embodiments, a population of cells is contacted with an SHH pathway inhibitor at a concentration of 0.25 μM. In some embodiments, the SHH pathway inhibitor includes Sant1. In some embodiments, the population of cells is optionally contacted with a protein kinase inhibitor. In some embodiments, the population of cells is not brought into contact with the protein kinase inhibitor. In some embodiments, a population of cells is contacted with a protein kinase inhibitor. In some embodiments, a population of cells is contacted with a protein kinase inhibitor at a concentration of 10 nM to 1 μM. In some embodiments, a population of cells is contacted with a protein kinase inhibitor at a concentration of 100 nM. In some embodiments, the protein kinase inhibitor includes staurosporine. In some embodiments, the method includes treating a population of cells with glucose. In some embodiments, the cell population is treated with glucose at a concentration of 1 mM to 50 mM. In some embodiments, the cell population is treated with glucose at a concentration of 25 mM. In some embodiments, conditions that promote cell clustering include suspension culture. In some embodiments, the population of cells 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 of the invention, the specified period of time is at least 7 days. In some embodiments, β-cell maturation factors are replenished in the suspension culture every other day. In some embodiments, at least 15% of the NKX6-1-positive pancreatic progenitor cells in the population are induced to differentiate into insulin-positive endocrine cells. In some embodiments, at least 99% of the NKX6-1-positive pancreatic progenitor cells in the population are induced to differentiate into insulin-positive endocrine cells. In some embodiments, the insulin-positive endocrine cells express 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 derived from a population of pluripotent stem cells selected from the group consisting of embryonic stem cells and induced pluripotent stem cells.

[32] In some aspects, the invention provides an isolated population of insulin-positive endocrine cells obtained in accordance with the method described herein.

[33] In some aspects, the invention provides a microcapsule containing a selected population of insulin-positive endocrine cells encapsulated therein. In some embodiments, the invention provides a composition comprising a population of insulin-positive endocrine cells obtained in accordance with the method described herein.

[34] In some aspects, the invention provides a method of treating a subject in need thereof, comprising administering to the subject a composition comprising an isolated population of insulin-positive endocrine cells obtained in accordance with a method described herein.

[35] In some embodiments, the insulin-positive endocrine cells are derived from a population of pluripotent stem cells obtained from the subject to whom the insulin-positive endocrine cells are administered. In some embodiments, the insulin-positive endocrine cells are encapsulated in a microcapsule. 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 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.

[36] In some aspects, the invention relates to the use of an isolated population of insulin-positive endocrine cells obtained by the method described herein for differentiation into SC-β cells.

[37] In some aspects, the invention relates to the use of an isolated population of insulin-positive endocrine cells obtained by a method described herein for administration to a subject in need thereof.

[38] In some embodiments, the subject is administered a selected population of insulin-positive endocrine cells encapsulated in microcapsules. 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 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.

[39] In some aspects, the invention provides a culture medium containing a) an inhibitor of the TGF-β signaling pathway, b) an activator of the TH (thyroid hormone) pathway, and at least one additional β-cell maturation factor selected from the group consisting of i ) XXI, ii) betacellulin, iii) low concentration of RA signaling pathway activator and iv) SHH pathway inhibitor.

[40] In some aspects, the invention relates to the use of the culture medium of claim 221 to induce in vitro differentiation of NKX6-1-positive pancreatic progenitor cells into insulin-positive endocrine cells.

[41] In some aspects, the invention provides a method of producing SC-β cells, comprising: bringing Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells into contact under conditions that stimulate cell clustering with i) an inhibitor of the transforming signaling pathway growth factor β (TGF-β), ii) an activator of the thyroid hormone signaling pathway and, optionally, iii) a protein kinase inhibitor to induce in vitro maturation of at least one of Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells into SC-β cells, with SC-β cells demonstrating a GSIS response in vitro and/or in vivo.

[42] In some embodiments, the GSIS response is observed (i) immediately after transplantation of an SC-β cell into a subject; (ii) within approximately 24 hours after transplantation of the cell into the subject; or (iii) within approximately two weeks after the cell is transplanted into the subject. In some embodiments, SC-β cells exhibit a response to (i) at least one glucose stimulation; (ii) at least two consecutive glucose stimulations; or (iii) at least three consecutive glucose stimulations. In some embodiments, the morphology of SC-β cells resembles the morphology of endogenous β cells. In some embodiments, Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with a TGF-β signaling pathway inhibitor at a concentration of 100 nM to 100 μM. In some embodiments, Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with a TGF-β signaling pathway inhibitor at a concentration of 10 μM. In some embodiments, the TGF-β signaling pathway includes TGF-β receptor type I kinase signaling. In some embodiments, the TGF-β signaling pathway inhibitor includes an Alk5 inhibitor II. In some embodiments, Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with a thyroid hormone signaling pathway activator at a concentration of 0.1 μM to 10 μM. In some embodiments, Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with a thyroid hormone signaling pathway activator at a concentration of 1 μM. In some embodiments, the thyroid hormone signaling pathway activator includes triiodothyronine (T3). In some embodiments, the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are not brought into contact with the protein kinase inhibitor. In some embodiments, Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with a protein kinase inhibitor. In some embodiments, Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with a protein kinase inhibitor at a concentration of 10 nM to 1 μM. In some embodiments, Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with a protein kinase inhibitor at a concentration of 100 nM. In some embodiments, the protein kinase inhibitor includes staurosporine. In some embodiments, the method comprises contacting Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells with a cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor. In some embodiments, Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with a CFTR inhibitor at a concentration of 100 nM - 100 μM. In some embodiments, Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with a CFTR inhibitor at a concentration of 10 nM to 10 μM. In some embodiments, the CFTR inhibitor includes Gly-H101. In some embodiments, the method comprises contacting Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells with an O-GlcNAase inhibitor. In some embodiments, Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with an O-GlcNAase inhibitor at a concentration of 100 nM to 100 μM. In some embodiments, Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with an O-GlcNAase inhibitor at a concentration of 10 nM to 10 μM. In some embodiments, the O-GlcNAase inhibitor includes Thiamet-G. In some embodiments, Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are cultured in a suitable culture medium. In some embodiments, a suitable culture medium includes Connought Medical Research Laboratories Supplemented Islet Medium 1066 (CMRLS) or a CMRLS component. In some embodiments, the CMRLS is supplemented with serum. In some embodiments, the CMRLS is supplemented with 10% fetal bovine serum. In some embodiments, conditions that promote cell clustering include 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 of the invention, the time period is at least 7 days. In some embodiments of the invention, the time period is from 7 days to 21 days. In some embodiments of the invention, the time period is from 7 to 14 days. In some embodiments of the invention, the time period is 14 days. In some embodiments, the suspension culture is replenished every other day. In some embodiments, at least 30% of the resulting cells contain SC-β cells. In some embodiments, SC-β cells express C-peptide, insulin, NKX6-1, Pdx1, and co-express NKX6-1 and C-peptide. In some embodiments, SC-β cells include human cells. In some embodiments, in vitro generation of SC-β cells is scalable.

[43] In some embodiments, insulin-positive endocrine cells are produced by contacting Pdx1-positive, NKX6-1-positive pancreatic progenitor cells under conditions that promote cell clustering with i) an inhibitor of the TGF-β signaling pathway, and ii ) an activator of the thyroid hormone signaling pathway to induce the differentiation of at least some Pdx1-positive, NKX6-1-positive pancreatic progenitor cells into Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells, wherein 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.

[44] In some embodiments, Pdx1-positive, NKX6-1-positive pancreatic progenitor cells are contacted with a TGF-β signaling pathway inhibitor at a concentration of 100 nM to 100 μM. In some embodiments, Pdx1-positive, NKX6-1-positive pancreatic progenitor cells are contacted with a TGF-β signaling pathway inhibitor at a concentration of 10 μM. In some embodiments, the TGF-β signaling pathway includes TGF-β receptor type I kinase signaling. In some embodiments, the TGF-β signaling pathway inhibitor includes an Alk5 inhibitor II. In some embodiments, Pdx1-positive, NKX6-1-positive pancreatic progenitor cells are contacted with a thyroid hormone signaling pathway activator at a concentration of 0.1 μM to 10 μM. In some embodiments, Pdx1-positive, NKX6-1-positive pancreatic progenitor cells are contacted with a thyroid hormone signaling pathway activator at a concentration of 1 μM. In some embodiments, the thyroid hormone signaling pathway activator includes triiodothyronine (T3). In some embodiments, the method comprises contacting Pdx1-positive, NKX6-1-positive pancreatic progenitor cells with at least one component 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, Pdx1-positive, NKX6-1-positive pancreatic progenitor cells are contacted with an SHH pathway inhibitor at a concentration of 0.1 μM to 0.5 μM. In some embodiments, Pdx1-positive, NKX6-1-positive pancreatic progenitor cells are contacted with an SHH pathway inhibitor at a concentration of 0.25 μM. In some embodiments, the SHH pathway inhibitor includes Sant1. In some embodiments, Pdx1-positive, NKX6-1-positive pancreatic progenitor cells are contacted with a RA signaling pathway activator at a concentration of 0.01 μM to 1.0 μM. In some embodiments, Pdx1-positive, NKX6-1-positive pancreatic progenitor cells are contacted with an RA signaling pathway activator at a concentration of 0.1 μM. In some embodiments, the RA signaling pathway activator includes RA. In some embodiments, Pdx1-positive, NKX6-1-positive pancreatic progenitor cells are contacted with a γ-secretase inhibitor at a concentration of 0.1 μM to 10 μM. In some embodiments, Pdx1-positive, NKX6-1-positive pancreatic progenitor cells are contacted with a 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 includes DAPT. In some embodiments, Pdx1-positive, NKX6-1-positive pancreatic progenitor cells are contacted with at least one growth factor from the EGF family at a concentration of 2 ng/ml to 200 ng/ml. In some embodiments, Pdx1-positive, NKX6-1-positive pancreatic progenitor cells are contacted with at least one growth factor from the EGF family at a concentration of 20 ng/ml. In some embodiments, at least one growth factor from the EGF family includes betacellulin. In some embodiments, at least one growth factor from the EGF family includes EGF. In some embodiments, Pdx1-positive, NKX6-1-positive pancreatic progenitor cells are not brought into contact with the protein kinase inhibitor. In some embodiments, Pdx1-positive, NKX6-1-positive pancreatic progenitor cells are contacted with a protein kinase inhibitor. In some embodiments, Pdx1-positive, NKX6-1-positive pancreatic progenitor cells are contacted with a protein kinase inhibitor at a concentration of 10 nM to 1 μM. In some embodiments, Pdx1-positive, NKX6-1-positive pancreatic progenitor cells are contacted with a protein kinase inhibitor at a concentration of 100 nM. In some embodiments, the protein kinase inhibitor includes staurosporine. In some embodiments, the method includes treating a population of cells with glucose. In some embodiments, the cell population is treated with glucose at a concentration of 1 mM to 50 mM. In some embodiments, the cell population is treated with glucose at a concentration of 25 mM. In some embodiments, conditions that promote cell clustering include 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 some of the Pdx1-positive, NKX6-1-positive pancreatic progenitor cells c Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells. In some embodiments of the invention, the specified period of time is at least 7 days. In some embodiments, the suspension culture is replenished every other day. In some embodiments, at least 15% of 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.

[45] In some embodiments, Pdx1-positive, NKX6-1-positive pancreatic progenitor cells are produced by contacting Pdx1-positive pancreatic progenitor cells, under conditions that promote cell clustering, with i) at least one growth factor of the FGF family, ii) at least one SHH pathway inhibitor and, optionally, iii) low concentrations of an RA signaling pathway activator, over a period of five days, to induce the differentiation of at least some Pdx1-positive pancreatic progenitor cells into Pdx1-positive, NKX6-1-positive pancreatic progenitor cells, where Pdx1-positive, NKX6-1-positive pancreatic progenitor cells express Pdx1 and NKX6-1.

[46] In some embodiments, Pdx1-positive pancreatic progenitor cells are contacted with at least one growth factor from the FGF family at a concentration of 1 ng/ml to 100 ng/ml. In some embodiments, Pdx1-positive pancreatic progenitor cells are contacted with at least one growth factor from the FGF family at a concentration of 50 ng/ml. In some embodiments, at least one growth factor from the FGF family includes keratinocyte growth factor (KGF). In some embodiments, at least one growth factor from the FGF family is selected from the group consisting of FGF2, FGF8B, FGF10 and FGF21. In some embodiments, Pdx1-positive pancreatic progenitor cells are contacted with at least one SHH pathway inhibitor at a concentration of 0.1 μM to 0.5 μM. In some embodiments, Pdx1-positive pancreatic progenitor cells are contacted with at least one SHH pathway inhibitor at a concentration of 0.25 μM. In some embodiments, the at least one SHH pathway inhibitor includes Sant1. In some embodiments, Pdx1-positive pancreatic progenitor cells are contacted with RA signaling pathway activator at a concentration of 0.01 μM to 1.0 μM. In some embodiments, Pdx1-positive pancreatic progenitor cells are contacted with a RA signaling pathway activator at a concentration of 0.1 μM. In some embodiments, the RA signaling pathway activator includes RA. In some embodiments, the method comprises contacting Pdx1-positive pancreatic progenitor cells with at least one growth factor from the EGF family. In some embodiments, Pdx1-positive pancreatic progenitor cells are contacted with at least one growth factor from the EGF family at a concentration of 2 ng/ml to 200 ng/ml. In some embodiments, Pdx1-positive pancreatic progenitor cells are contacted with at least one growth factor from the EGF family at a concentration of 20 ng/ml. In some embodiments, at least one growth factor from the EGF family includes betacellulin. In some embodiments, at least one growth factor from the EGF family includes EGF. In some embodiments, Pdx1-positive pancreatic progenitor cells are cultured in a suitable culture medium. In some embodiments, conditions that promote cell clustering include suspension culture. In some embodiments, the suspension culture is replenished every other day. In some embodiments, no protein kinase C activator is added to the suspension culture for 5 days. In some embodiments, the protein kinase C activator is removed from the suspension culture before the 5 day period has elapsed. In some embodiments, the protein kinase C activator includes 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 over a period of less than 5 days. In some embodiments, the BMP signaling pathway inhibitor includes LDN193189. In some embodiments, at least 10% of 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 Pdx1-positive pancreatic progenitor cells are induced to differentiate into Pdx1-positive, NKX6-1-positive pancreatic progenitor cells.

[47] In some aspects, the invention provides a method for generating SC-β cells from pluripotent cells, comprising: a) differentiating a population of pluripotent stem cells 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 bringing the Pdx1-positive pancreatic progenitor cells, under conditions that stimulate cell clustering, into contact 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, every other day for a period of five days to induce differentiation of at least some Pdx1-positive pancreatic progenitor cells in a population of NKX6-1-positive pancreatic progenitor cells, wherein the NKX6-1-positive pancreatic progenitor cells express Pdx1 and NKX6-1; c) differentiating at least some Pdx1-positive, NKX6-1-positive pancreatic progenitor cells into Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells by bringing Pdx1-positive, NKX6-1-positive pancreatic cells- progenitors under conditions that promote cell clustering, in contact with i) an inhibitor of the TGF-β signaling pathway, b) an activator of the TH signaling pathway and, optionally, c) at least one inhibitor of the SHH pathway, ii) an activator of the RA signaling pathway, iii) a γ-secretase inhibitor and vi) at least one growth factor from the epidermal growth factor (EGF) family, every other day for a period of five to seven days to induce differentiation of at least some Pdx1-positive, NKX6-1-positive pancreatic progenitor cells into Pdx1-positive, NKX6-1, insulin-positive endocrine cells, while Pdx1-positive, NKX6-1, insulin-positive endocrine cells express Pdx1, NKX6-1, NKX2-2, Mafb, glis3, Sur1 , Kir6.2, Znt8, SLC2A1, SLC2A3 and/or insulin; and d) differentiating at least some of the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells into SC-β cells by exposing the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells to conditions that stimulate cell clustering, in contact with i) a transforming growth factor β (TGF-β) signaling pathway inhibitor, ii) a thyroid hormone signaling pathway activator, and optionally iii) a protein kinase inhibitor, every other day for a period of seven to 14 days, to induce in vitro maturation of at least some 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.

[48] In some embodiments, the invention provides a method of producing SC-β cells from pluripotent cells, comprising: a) differentiating at least some of the pluripotent cells in the population 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 bringing the Pdx1-positive pancreatic progenitor cells, under conditions that stimulate cell clustering, into contact with i) KGF, ii) Sant1 and, optionally, iii) low concentrations of RA, every other day for a period of five days, to induce the differentiation of at least some of the Pdx1-positive pancreatic progenitor cells in the population into NKX6-1-positive pancreatic cells- progenitors, wherein NKX6-1-positive pancreatic progenitor cells express Pdx1 and NKX6-1; c) differentiating at least some Pdx1-positive, NKX6-1-positive pancreatic progenitor cells into Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells by bringing Pdx1-positive, NKX6-1-positive pancreatic cells- progenitors into contact with i) Alk5 inhibitor II, ii) T3 and optionally iii) Sant1, iv) RA, v) XXI and vi) betacellulin, every other day for a period of five to seven days to induce differentiation at least some Pdx1-positive, NKX6-1-positive pancreatic progenitor cells into Pdx1-positive, NKX6-1, insulin-positive endocrine cells, while Pdx1-positive, NKX6-1, insulin-positive endocrine cells express Pdx1, NKX6-1 , NKX2-2, Mafb, glis3, Sur1, Kir6.2, Znt8, SLC2A1, SLC2A3 and/or insulin; and d) differentiating at least some of the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells into SC-β cells by exposing the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells to conditions that stimulate cell clustering, in contact with i) Alk5 inhibitor II, ii) T3 and optionally iii) staurosporine, every other day for a period of seven to 14 days to induce in vitro maturation of at least some Pdx1-positive, NKX6 -1-positive, insulin-positive endocrine cells into SC-β cells, with SC-β cells demonstrating a GSIS response in vitro and/or in vivo.

[49] In some aspects, the invention provides an artificial islet containing SC-β cells differentiated in vitro from pluripotent stem cells.

[50] In some aspects, the invention provides an artificial pancreas containing SC-β cells differentiated in vitro from pluripotent stem cells.

[51] The practice of the present invention generally employs, unless otherwise noted, conventional methods of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant nucleic acid (e.g., DNA) technology, immunology, and RNA interference (RNAi), which are consistent with the art.

Non-limiting descriptions of specific methods 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, John Wiley. & Sons, N.Y., 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, R.I., "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, V.A.: Mendelian Inheritance in Man. A Catalog of Human Genes and Genetic Disorders. Baltimore: Johns Hopkins University Press, 1998 (12th edition) or more recent 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), accessed 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 characteristics of different animal species (other than humans and mice), at http://omia. angis.org.au/contact.shtml. All patents, patent publications, and other publications (such as scientific articles, books, web pages, and databases) referenced herein are incorporated by reference in their entirety. In the event of any conflict between the description of the invention and the included references, the description of the invention (including any modifications thereto that may be based on the included reference) shall control. Unless otherwise indicated, the standard meanings of terms used in this document are used in this document. This document uses standard abbreviations for various terms.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

[52] The patent or application file contains at least one graphic image in color. Copies of this patent or patent application with color graphics will be made available by the patent office upon request and upon payment of the required fee.

[53] In FIG. 1A and 1B show a comparison of a previously published control differentiation method and a new controlled differentiation method. In FIG. 1A is a schematic comparison of an exemplary guided differentiation method of the invention for producing INS+ cells from hPSCs and a previously published control differentiation method. FIG. 1B illustrates histological sections of undifferentiated HUES8 (top), DE differentiated (middle) and PP1 differentiated (bottom) and stained with OCT4, SOX17 and PDX1, respectively, using a previously published control differentiation method. Scale bar = 100 µm.

[54] FIG. 2A, 2B, and 2C demonstrate that stem cell-derived β-cells (SC-β) generated in vitro secrete insulin in response to repeated sequential stimulation with high glucose concentrations, like primary human β-cells. FIG. 2A, 2B, and 2C are graphs illustrating met ELISA measurements of human insulin secreted from SC-β (FIG. 2A), primary β cells (FIG. 2B), and PH cells (FIG. 2C) that were sequentially stimulated with 2 , 20, 2, 20, 2 and 20 mM glucose. After sequential stimulation with low/high glucose concentrations, cells were depolarized with 30 mM KCl.

[55] FIG. 3A, 3B, and 3C demonstrate additional biological replicas of in vitro-derived SC-β cells that secrete insulin in response to repeated sequential stimulation with high glucose concentrations, like primary β-cells. The left panels are the same as in FIG. 2. SC-β cells (SC-β; FIG. 3A), primary β-cells (1° β; FIG. 3B), and PH cells (FIG. 3C) were sequentially stimulated with 2, 20, 2, 20, 2, and 20 mM glucose and 30 mM KCl, and human insulin levels were measured using ELISA.

[56] FIG. 4A, 4B, 4C, 4D, and 4E demonstrate the presence of a cytosolic Ca ²⁺ flux in SC-β cells in response to repeated sequential stimulation with high glucose concentrations, as in the case of primary β-cells. FIG. 4A is a diagram representing the detection of cytosolic Ca ²⁺ at the population level and single cell level using Fluo-4 AM staining. Population-level measurements were made on individual intact clusters (indicated in the diagram by a large red circle), and individual cells within intact clusters (indicated by small red circles) were analyzed by single-cell analysis. FIG. 4B is a graph illustrating population measurements of dynamic normalized fluorescence intensity of Fluo-4 for SC-β cells, primary β cells, and PH cells sequentially stimulated with 2, 20, 2, 20, 2, and 20 mM glucose and 30 mM KCl. In FIG. 4C shows fluorescence images obtained from Fluo-4 AM staining used for single cell analysis. In FIG. 4D shows representative images indicating the locations of single cells that exhibited responses to 3 (yellow), 2 (orange), 1 (blue), and 0 (red) glucose stimulation. In FIG. 4E shows a graphical quantitation of the frequency of SC-β cells (n=156), primary β-cells (n=114) and PH cells (n=138) that showed a response to 20 mM glucose. Scale bar = 100 µm.

[57] FIG. 5A, 5B, 5C, 5D, 5E and 5F demonstrate that SC-β express human β-cell markers at the protein and gene expression level. In FIG. Figure 5A shows immunohistochemical images of cells stained for C-peptide (green), NKX6-1 (red), and somatostatin (gray). In FIG. Figure 5B shows immunohistochemical images of cells stained for C-peptide (green) and PDX1 (red). In FIG. Figure 5C shows immunohistochemical images of cells stained for C-peptide (green) and glucagon (red) with the corresponding DAPI dye (blue). In FIG. 5D shows typical dot plots from flow cytometry data and the percentage of cells in the population stained for C-peptide and NKX6-1. In FIG. 5E shows hierarchical clustering analysis based on all genes measured by microarray analysis of undifferentiated HUES8, PH cells, fetal β cells, and primary adult β cells sorted based on INS (data from Hrvatin et al. ., 2014)), and SC-β cells (SC-β), sorted based on INS and NKX6-1. In FIG. Figure 5F shows a heat map of the 100 genes with the highest variation among all samples. CP = C-peptide, SST = somatostatin, GCG = glucagon. Scale bar = 100 µm.

[58] FIG. 6 illustrates the histology of a cluster of SC-β cells stained for DAPI (blue), insulin (green), C-peptide (red). Scale bar = 100 µm.

[59] FIG. 7A, 7B and 7C show additional histological staining of SC-β cells. FIG. 7A illustrates staining for C-peptide (green) and ISL1 (red). FIG. 7B illustrates staining for C-peptide (green) and MAFA (red). FIG. 7C illustrates staining for C-peptide (green) and MAFB (red). Scale bar = 100 µm.

[60] In FIG. 8A, 8B, and 8C show typical flow cytometry dot plots and the percentage of SC-β cells and PH cells in the population stained for C-peptide and SST (FIG. 8A), C-peptide and GCG (FIG. 8B), and SST and GCG (FIG. 8C).

[61] FIG. 9A, 9B and 9C demonstrate that SC-β cell granules are structurally similar to those of primary human β cells. In FIG. 9A shows electron microscope images of granules highlighting typical crystallized insulin granules (red), early insulin granules (yellow), and mixed endocrine granules (blue). Scale bar = 500 nm. In FIG. 9B shows enlarged images of the granules highlighted in (FIG. 9A). Scale bar = 500 nm. In FIG. 9C shows electron microscope images of cells labeled by gold immunostaining, illustrating granules that contain insulin (smaller 5 nm black dots) and/or glucagon (larger 15 nm black dots). Representative gold particles are indicated by red arrows (insulin) and blue arrows (glucagon). Scale bar = 100 nm.

[62] FIG. 10A and 10B demonstrate that stem cell-derived β-cells (SC-β) derived from hiPSCs in vitro secrete insulin in response to repeated sequential stimulation with high glucose concentrations, like primary human β-cells. FIG. 10A and 10B are graphs illustrating met ELISA measurements of human insulin secreted from SC-β derived from non-diabetic cells (FIG. 10A) and type 1 diabetic cells (FIG. 10B) sequentially stimulated with 2, 20, 2. 20, 2 and 20 mM glucose.

[63] In FIG. 11A, 11B, 11C, 11D, 11E and 11F show representative flow cytometry dot plots and the percentage of cells stained for C-peptide and NKX6-1 from multiple hiPSC lines. In FIG. 11A, 11B and 11C show representative flow cytometry dot plots and the percentage of cells stained for C-peptide and NKX6-1 from non-diabetic hiPSC lines. In FIG. 11D, 11E and 11F show representative flow cytometry dot plots and the percentage of cells stained for C-peptide and NKX6-1 from type 1 diabetic hiPSC lines.

[64] FIG. 12A, 12B, 12C and 12D demonstrate that transplanted SC-β cells rapidly become functional in vivo. FIG. 12A is a graph illustrating met ELISA measurements of human insulin from the serum of individual mice transplanted with SC-β cells (cultured for 1 week at the final in vitro stage), primary human β-cells (1° β) or PH cells . Measurements were taken before (white bars) and 30 minutes after (black bars) glucose injection into mice two weeks after transplantation. In FIG. 12B shows immunohistochemical images of transplanted cells from (FIG. 12A) stained for C-peptide (green) and PDX1 (red) to confirm the presence of a graft. FIG. 12C is a graph illustrating met ELISA measurements of human insulin from the serum of individual mice transplanted with pancreatic progenitor cells. Measurements were taken before (white bars) and 30 minutes after (black bars) glucose injection into mice two weeks after transplantation. FIG. 12D is a graph illustrating met ELISA measurements of human insulin from the serum of individual mice transplanted with SC-β cells cultured for 2 weeks in the final in vitro step. Measurements were taken 30 minutes after (black bars) glucose injection into mice two weeks after transplantation, n/a = not determined. Scale bar = 100 µm.

[65] FIG. 13A and 13B illustrate additional histological sections of SC-β cells and PH cells transplanted into mice over 2 weeks. In FIG. 13A shows low magnification images of grafts stained for DAPI (blue), C-peptide (green), and GCG (red). Scale bar = 200 µM. In FIG. 13B shows higher magnification images of grafts stained for C-peptide (green) and GCG (red). Scale bar = 100 µM.

[66] FIG. 14A, 14B and 14C demonstrate the use of media at the last stage of differentiation to enable SC-β cells to secrete more insulin in vivo. In FIG. 14A is a diagram illustrating the use of various media at various stages of the differentiation process. FIG. 14B illustrates that the addition of additional factors such as Sant1, XXI and SSP to CMRL medium at the last stage of differentiation leads to a better SC-β cell response of glucose stimulated insulin secretion (GSIS), as determined by the stimulation index at high and high stimulation. low glucose concentrations. FIG. 14C illustrates that the addition of additional factors such as Sant1, XXI and SSP to the CMRL medium at the last stage of differentiation results in a better glucose-stimulated insulin secretion (GSIS) response of SC-β cells, as measured by the amount of insulin released.

[67] FIG. 15A, 15B, 15C, 15D, 15E, 15F, 15G, 15H, and 15I demonstrate protocol modifications that can improve the survival and quality of the resulting SC-β cells. FIG. 15A is a schematic illustration of the protocol. FIG. 15B shows how much more pure NKX6.1+ endocrine cell clusters can be obtained (FIG. 15B) using the modified protocol. FIG. 15C demonstrates how application of Rock inhibitor in steps 3-5 can improve cell survival. FIG. 15D demonstrates how the use of activin A along with nicotinamide can lead to down-regulation of SOX2 and improve cell survival. FIG. 15E shows that SOX2 and NKX6-1 are mutually exclusive. FIG. 15F demonstrates how the use of staurosporine in step 6 generates a substantially pure population of endocrine cells, and FIG. 15G demonstrates how the use of staurosporine in step 6 produces a higher percentage of NKX6-1/C-peptide+ cells. FIG. 15I demonstrates how the use of XXI in combination with Alk5i and T3 at stages 5-6 increases the NeuroD+ population compared to the use of Alk5i and T3 alone (FIG. 15H).

[68] FIG. 16A, 16B, 16C, 16D, 16E, 16F, 16G, 16H and 16I demonstrate the clinical application of SC-β cells as a diabetes therapy or a platform for the development of new drugs. FIG. 16A is a schematic illustration of the use of SC-β cells for treating diabetes or screening drugs to improve function or replication. FIG. 16B is a table showing the investigational diabetes medications and their overall therapeutic category. FIG. 16C is a graph illustrating met ELISA measurements of human insulin from seeded SC-β cells treated with the indicated drugs in 2 and 20 mM glucose. The reported p-values compare the value of insulin levels in 20 mM glucose between drug and control. FIG. 16D is an immunofluorescence image of dispersed and seeded SC-β cells stained with DAPI (blue), C-peptide (green), and Ki67 (red) without treatment. FIG. 16E is an immunofluorescence image of dispersed and seeded SC-β cells stained with DAPI (blue), C-peptide (green), and Ki67 (red) treated with prolactin for 48 hours. FIG. 16F shows a graphical quantitation of the proportion of cells that coexpress C-peptide and Ki67. *p<0.05. FIG. 16G is a graph illustrating fasting blood glucose measurements for Akita mice transplanted with SC-β cells (n=6) or PH cells (n=6). *p<0.05 when comparing two groups of cells on the same day. FIG. 16H is a graph illustrating blood glucose measurements in 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 into mice 2 weeks after transplantation. Saturation for glucose measurements was observed at 550 mg/dL. *p<0.05 when comparing two groups of cells at the same time after glucose injection. FIG. 16I is a graph illustrating met ELISA measurements of human insulin from the serum of Akita mice 20 minutes after glucose injection. Mice were stimulated with glucose 2 weeks after transplantation. *p<0.05 when comparing two groups of cells. Scale bar = 50 µm.

[69] FIG. 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 when comparing two groups of cells at the time point corresponding to 18 and 28 days.

DETAILED DESCRIPTION OF THE INVENTION

[70] Aspects of the invention relate to compositions, methods, kits and agents for generating stem cell-derived β-cells (SC-β) (eg, mature pancreatic β-cells) from at least one insulin-positive endocrine cell or a precursor thereof (e.g. iPS cells, hESCs, definitive endoderm cells, subembryonic cavity 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 using these compositions, methods, kits and agents, for use in cell therapies, assays (eg, drug screening) and various treatments.

[71] In addition, aspects of the invention relate to methods for identifying SC-β cells that are identified based on morphological criteria without the need for a selectable marker, as well as functional characteristics such as the ability to express insulin, secrete insulin in response to one or more stimulations glucose, exhibit a mature GSIS response and form islets in pancreatic tissue in vivo, and typically contain small spindle cells with a diameter of about 9-15 µm.

[72] In addition, aspects of the invention relate to methods for identifying β-cell maturation factors. It is obvious or not difficult for one skilled in the art to verify, using assays known in the art, whether a particular β-cell maturation factor is functional. For example, the ability of 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 assay methods described herein. Other convenient assays include determining the ability to activate transcription of a reporter construct containing a β-cell marker binding site operably linked to a nucleotide sequence encoding a detectable marker, such as luciferase. One assay involves determining whether a candidate β-cell maturation factor induces the conversion of at least one insulin-positive endocrine cell to an SC-β cell, or the expression of β-cell markers, or the expression of functional characteristics of a mature β-cell, as described in this document. Determination of the expression of β-cell markers can be carried out using any suitable method, for example immunoblotting. Such assays can be readily adapted to detect or confirm the presence of activity of agents that directly convert at least one insulin-positive endocrine cell or its precursor into an SC-β cell.

[73] In vitro-matured SC-β cells (i.e., pancreatic β-cells) obtained in accordance with the methods of the invention described herein demonstrate a number of advantages, for example, they perform glucose-stimulated insulin secretion in vitro, similar in gene expression and ultrastructure with human islet β-cells, secrete human insulin and alleviate hyperglycemia when transplanted into mice, represent a new platform for cell therapy (for example, transplantation of additional and/or functional β-cells into the body of a patient in need), drug screening drugs (eg, for insulin production/secretion, survival, differentiation, etc.), research (eg, determining the difference between the functioning of normal and diabetic β-cells) and tissue engineering (eg, using SC-β cells as the first type cells during islet reconstruction).

[74] DEFINITIONS

[75] For convenience, individual terms used in this document are collected below - in the description of the invention, examples and the attached claims. Unless otherwise specified, all technical and scientific terms used herein have the meanings commonly understood by one skilled in the art to which this invention relates.

[76] The term “differentiated cell”, as defined herein, means any primary cell that is not pluripotent in its native form. According to another definition, the term "differentiated cell" refers to a cell of a more specialized type derived from a cell of a less specialized type (eg, a stem cell, such as an induced pluripotent stem cell) through the process of cell differentiation. Without being limited by theory, it can be said that during normal ontogeny, a pluripotent stem cell may differentiate first into an endodermal cell, which is capable of forming pancreatic cells and other types of endoderm cells. Further differentiation of endodermal cells results in initiation of the pancreatic pathway, with ^~ 98% of cells becoming exocrine, ductular or matrix cells and ^~ 2% becoming endocrine cells. Early endocrine cells are the precursors of islets, which can then differentiate into insulin-producing cells (eg, functional endocrine cells) that secrete insulin, glucagon, somatostatin, or pancreatic polypeptide. Endodermal cells can also differentiate into other cells of endodermal origin, for example, cells of the lungs, liver, intestines, thymus, etc.

[77] As used herein, the term “somatic cell” refers to any cell that forms the body of an organism, as opposed to germ line cells. In the case of mammals, the germline cells (also known as "gametes") are sperm and eggs that fuse during fertilization to form a cell called a zygote from which the entire mammalian embryo develops. Every other type of cell in a mammal except sperm and eggs, the cells from which they are formed (gametocytes), and undifferentiated stem cells are somatic cells: internal organs, skin, bones, blood, and connective tissue are composed of somatic cells. In some embodiments, the somatic cell is a “non-embryonic somatic cell,” by which is meant a somatic cell that is not present in or derived from an embryo and is not the result of proliferation of such a cell in vitro. In some embodiments, the somatic cell is an “adult somatic cell,” which means a somatic cell that is present in or derived from an organism other than an embryo or fetus, or that results from the proliferation of such a cell in vitro. Unless otherwise indicated, methods for converting at least one insulin-positive endocrine cell or precursor thereof into an insulin-producing, glucose-sensitive cell can be carried out either in vivo or in vitro (in vivo methods are practiced when at least one the insulin-positive endocrine cell or its precursor is present in the body of the subject, and in vitro methods are practiced using at least one isolated insulin-positive endocrine cell or its precursor contained in culture).

[78] As used herein, the term “adult cell” refers to a cell that can be found in the body after embryonic development.

[79] As used herein, the term “endodermal cell” refers to a cell from one of the three primary germ cell layers at a very early embryonic stage (the other two germ cell layers are mesoderm and ectoderm). The endoderm is the deepest layer of the three. The endoderm may differentiate first into the primary gut tube of the embryo, and then into the lining of the respiratory and digestive tracts (eg, intestines), liver and pancreas.

[80] As used herein, the term “cell of endodermal origin” refers to any cell that has developed or differentiated from an endodermal cell. For example, a cell of endodermal origin includes cells of the liver, lung, pancreas, thymus, intestine, stomach, and thyroid. Without being limited by theory, liver and pancreatic progenitor cells (also called pancreatic progenitor cells) develop from endoderm cells in the embryonic foregut. Shortly after specification, liver and pancreatic progenitor cells rapidly acquire significantly different cellular functions and regenerative capabilities. These changes are driven by inductive signals and genetic regulatory factors that are highly conserved among vertebrates. Interest in organ development and regeneration has been fueled by the urgent need for hepatocytes and pancreatic β-cells in the therapeutic treatment of liver failure and type I diabetes. Studies in a variety of model organisms and humans have identified evolutionarily conserved inductive signals and transcription factor systems that drive the differentiation of liver and pancreatic cells and provide guidance for promoting the differentiation of hepatocytes and β-cells from various types of stem and pancreatic cells. predecessors.

[81] As used herein, the term “definitive endoderm” refers to a cell differentiated from an endodermal cell that can differentiate into an SC-β cell (eg, a pancreatic β cell). The definitive endoderm cell expresses the Sox17 marker. Other markers characteristic of definitive endoderm cells include, but are not limited to, MIXL2, GATA4, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CXCR4, cerberus, OTX2, goosecoid, C-Kit, CD99, CMKOR1, and CRIP1. In particular, the definitive endoderm cells described herein express Sox17 and, in some embodiments, Sox17 and HNF3B, and do not express GATA4, SPARC, APF or DAB to significant levels. Definitive endoderm cells are not positive for the Pdx1 marker (eg, they are Pdx1 negative). Definitive endoderm cells have the ability to differentiate into cells including liver, lung, pancreatic, thymus, intestinal, stomach, and thyroid cells. The expression of Sox17 and other definitive endoderm markers can be assessed by any method known to one skilled in the art, such as immunochemistry, for example using an anti-Sox17 antibody, or quantitative RT-PCR.

[82] The term "pancreatic endoderm" refers to a cell of endodermal origin that is capable of differentiating into multiple pancreatic lineages, including pancreatic β-cells, but cannot differentiate into non-pancreatic lineages.

[83] As used herein, the term “primordial tube cell” or “gut cell” refers to a cell differentiated from an endodermal cell that can differentiate into an SC-β cell (eg, a pancreatic β cell). The primary gut tube cell expresses at least one of the following markers: HNF1-β, HNF3-β, or HNF4-α. Cells of the primary intestinal tube have the ability to differentiate into cells including lung, liver, pancreatic, stomach and intestinal cells. The expression of HNF1-β and other markers of the primary intestinal tube can be assessed by any method known to one skilled in the art, such as immunochemistry, for example using an anti-HNF1-β antibody.

[84] The terms "pancreatic progenitor cell", "pancreatic endocrine progenitor cell", "pancreatic progenitor" or "pancreatic endocrine progenitor" are used interchangeably herein and refer to a stem cell that is capable of becoming a pancreatic hormone-expressing cell capable of forming pancreatic endocrine cells, pancreatic exocrine cells or pancreatic ductal cells. These cells are designed to differentiate into at least one type of pancreatic cell, such as 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 express at least one of the following markers: NGN3, NKX2.2, NeuroD, ISL-1, Pax4, Pax6, or ARX.

[85] As used herein, the term “pdx1-positive pancreatic progenitor cell” refers to a cell that is a pancreatic endoderm (PE) cell that can differentiate into SC-β cells, such as pancreatic β cells. Pdx1-positive pancreatic progenitor cells express the Pdx1 marker. Other markers include, but are not limited to, Cdcp1 or Ptf1a, or HNF6, or NRx2.2. Pdx1 expression can be assessed by any method known to one skilled in the art, such as immunochemistry using an anti-Pdx1 antibody or quantitative RT-PCR.

[86] As used herein, the term “pdx1-positive, NKX6-1-positive pancreatic progenitor cell” refers to a cell that is a pancreatic endoderm (PE) cell that can differentiate into insulin-producing cells such as pancreatic β-cells. cells. pdx1-positive, NKX6-1-positive pancreatic progenitor cells express Pdx1 NKX6-1 markers. Other markers include, but are not limited to, Cdcp1 or Ptf1a, or HNF6, or NRx2.2. Expression of NKX6-1 can be assessed by any method known to one skilled in the art, such as immunochemistry using an anti-NKX6-1 antibody or quantitative RT-PCR.

[87] As used herein, the term “Ngn3-positive endocrine progenitor cell” refers to pancreatic endocrine cell progenitors expressing the transcription factor neurogenin-3 (Ngn3). Progenitor cells are more differentiated than multipotent stem cells and can only differentiate into a small number of cell types. In particular, Ngn3-positive endocrine progenitor cells have the ability to differentiate into five types of pancreatic endocrine cells (α, β, δ, ε and PP). Ngn3 expression can be assessed by any method known to one skilled in the art, such as immunochemistry using an anti-Ngn3 antibody or quantitative RT-PCR.

[88] The terms "NeuroD" and "NeuroD1" are used interchangeably herein and refer to a protein expressed in pancreatic endocrine progenitor cells and the gene encoding it.

[89] The terms "insulin-positive β-like cell" and "insulin-positive endocrine cell" refer to a cell (eg, pancreatic endocrine cell) that exhibits at least one marker characteristic of a pancreatic β-cell and also expresses insulin does not exhibit the characteristic endogenous β-cell GSIS response.

[90] The term “precursor thereof” when used with respect to an insulin-positive endocrine cell refers to any cell that is capable of differentiating into an insulin-positive endocrine cell, including, for example, a pluripotent stem cell, a definitive endoderm cell, a primitive gut tube cell, a pancreatic progenitor cell or an endocrine progenitor cell, when cultured under conditions suitable for differentiation of the progenitor cell into an insulin-positive endocrine cell.

[91] The terms “stem cell-derived β-cell,” “SC-β cell,” “functional β-cell,” “functional pancreatic β-cell,” and “mature SC-β cell” refer to cells (eg, pancreatic β cells) that exhibit at least one marker characteristic of a pancreatic β cell (eg, PDX-1 or NKX6-1), express insulin and exhibit a GSIS response characteristic of an endogenous mature β cell. In some embodiments, an “SC-β cell” includes mature pancreatic β cells. It should be understood that SC-β cells do not necessarily have to be derived (e.g., directly) from stem cells, since the methods of the invention make it possible to obtain SC-β cells from any insulin-positive endocrine cell or its precursor using as starting material any cell (for example, embryonic stem cells, induced pluripotent stem cells, progenitor cells, partially reprogrammed somatic cells can be used (for example, a somatic cell that has been reprogrammed to an intermediate state between the induced pluripotent stem cell and the somatic cell from which it was derived ), multipotent cells, totipotent cells, transdifferentiated versions of any of the above cells, etc., since the invention is not limited in this sense). In some embodiments, SC-β cells exhibit a response to multiple glucose stimulations (eg, at least one, at least two, or at least three or more sequential glucose stimulations). In some embodiments, the response is similar to the response of endogenous islets (eg, human islets) to repeated glucose stimulation. In some embodiments, the morphology of the SC-β cell is similar to the morphology of an endogenous β cell. In some embodiments, an SC-β cell exhibits an in vitro GSIS response that is similar to the GSIS response of an endogenous β cell. In some embodiments, the SC-β cell exhibits an in vivo GSIS response that is similar to the GSIS response of an endogenous β cell. In some embodiments, the SC-β cell exhibits both in vitro and in vivo GSIS responses that are similar to the GSIS response of an endogenous β cell. The GSIS response of an SC-β cell can be observed within two weeks of transplantation of an SC-β cell into a host (eg, human or animal). In some embodiments, insulin in SC-β cells is packaged into secretory granules. In some embodiments, encapsulated crystalline insulin granules are observed in SC-β cells. In some embodiments, SC-β cells have a stimulation index greater than 1. In some embodiments, SC-β cells have a stimulation index greater than 1.1. In some embodiments, SC-β cells have a stimulation index greater than 2. In some embodiments, SC-β cells exhibit cytokine-induced apoptosis in response to cytokines. In some embodiments, insulin secretion from SC-β cells is increased in response to known antidiabetic drugs (eg, secretagogues). In some embodiments, SC-β cells are monohormonal. In some embodiments, SC-β cells do not exhibit abnormal coexpression of other hormones, such as glucagon, somatostatin, or pancreatic polypeptide. In some embodiments, SC-β cells exhibit a low replication coefficient. In some embodiments, SC-β cells increase intracellular Ca ²⁺ in response to glucose. As used herein, the term “exocrine cell” refers to a cell of an exocrine gland, i.e. a gland whose secretion occurs through a duct. In specific embodiments, exocrine cells refer to a pancreatic exocrine cell, which is a pancreatic cell that produces enzymes that are secreted into the small intestine. These enzymes help break down food as it passes through the gastrointestinal tract. Pancreatic exocrine cells are also known as islets of Langerhans, which secrete two hormones - insulin and glucagon. A pancreatic exocrine cell can be one of several types: alpha-2 cells (which produce the hormone glucagon); or β-cells (which produce the hormone insulin); and alpha-1 cells (which produce the regulatory agent somatostatin). As used herein, non-insulin-producing exocrine cells are referred to as alpha-2 cells or alpha-1 cells. It should be noted that the term "pancreatic exocrine cells" includes "pancreatic endocrine cells", which refers to the pancreatic cell that produces hormones (eg, insulin (produced by β-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.

[92] As used herein, the term “insulin-producing cell” refers to a cell differentiated from a pancreatic progenitor cell, or a precursor thereof, that secretes insulin. An insulin-producing cell, as used herein, includes pancreatic β-cells as well as pancreatic β-like cells (e.g., insulin-positive endocrine cells) that synthesize (i.e., transcribe the insulin gene, translate proinsulin mRNA and modify proinsulin mRNA into insulin protein), express (ie, exhibit the phenotypic trait carried by the insulin gene) or secrete (release insulin into the extracellular space) insulin in a persistent or inducible manner. The population of insulin-producing cells, for example, obtained by differentiating insulin-positive endocrine cells or their precursors into SC-β cells in accordance with the methods of the present invention, can be pancreatic β-cells or β-like cells (for example, cells that have at least one or at least two characteristics of an endogenous β-cell and exhibit a GSIS response similar to that of an endogenous adult β-cell). The novelty of the presented compositions and methods is not offset by the presence in the population of cells that produce insulin naturally (for example, β-cells). It is also contemplated that the population of insulin-producing cells, for example, obtained by methods disclosed herein, may comprise mature pancreatic β-cells or SC-β cells, and may also contain non-insulin-producing cells (i.e., cells with a phenotype similar to β cells except that they do not produce or secrete insulin).

[93] As used herein, the terms “endogenous β-cell,” “endogenous mature pancreatic β-cell,” or “endogenous pancreatic β-cell” refer to an insulin-producing cell of the pancreas or a cell having a pancreatic β-cell phenotype (β -cells). The pancreatic β-cell phenotype is well known to those skilled in the art and includes, for example, insulin secretion in response to elevated glucose levels, expression of markers such as c-peptide, Pdx1 and Glut 2 polypeptide, and distinct morphological characteristics such as formation islets in the pancreas in vivo, and typically includes small spindle-like cells with a diameter of about 9-15 μm.

[94] As used herein, the terms “SC-β cell,” “pancreatic β-like cell,” and “mature pancreatic β-like cell” refer to cells produced by the methods disclosed herein that express at least 15% of the amount insulin expressed by an endogenous pancreatic β cell, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100%, or more than 100%, for example at least about 1.5 times, 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 5-fold multiples of the amount of insulin secreted by an endogenous pancreatic β-cell, or, alternatively, exhibit at least one or at least two characteristics of an endogenous pancreatic β-cell, such as, but not limited to, insulin secretion in response to glucose and expression of β-cell markers cells, such as, for example, c-peptide, Pdx1 and glut-2. In one embodiment, the SC-β cell is not an immortalized cell (eg, proliferates in culture indefinitely). In one embodiment, the SC-β cell is a non-transformed cell, for example, a cell that exhibits transformation properties such as growth in soft agar or lack of contact inhibition.

[95] The term “β-cell marker” refers, without limitation, 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. Typical β-cell markers include, but are not limited to, pancreatic and duodenal homeobox polypeptide 1 (Pdx1), 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 θ MafA, β Zhang et al., Diabetes. 50 (10): 2231-6 (2001). IN β-cell is a nuclear marker of β-cells. In some embodiments, the β-cell marker is Pdx1 or PH3.

[96] The term "pancreatic endocrine marker" refers, without limitation, 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. Typical pancreatic endocrine cell markers include, but are not limited to, Ngn-3, NeuroD and Islet-1.

[97] As used herein, the term “non-insulin-producing cell” means any cell of endodermal origin that does not naturally synthesize, express or secrete insulin either chronically or upon induction. Thus, as used herein, the term “non-insulin-producing cells” does not include pancreatic β-cells. Examples of non-insulin-producing cells that can be used in the methods of the present invention include pancreatic cells other than β-cells, such as amylase-producing cells, acinar cells, cells from ductal adenocarcinoma cell lines (eg, CD18, CD11, and Capan-cells). I (see Busik et al. 1997; Schaffert et al. 1997) Non-pancreatic cells of endodermal origin may also be used, such as non-pancreatic stem cells from other endocrine or exocrine organs, including, for example, liver cells, thymus cells, thyroid cells , intestinal cells, lung cells, and pituitary cells. In some embodiments, the non-insulin-producing endodermal cells may be mammalian cells, or more specifically, human cells. Examples of implementation of the proposed method using mammalian pancreatic non-islet cells, pancreatic amylase-producing cells, are provided herein. mammalian cells, mammalian pancreatic acinar cells.

[98] The term "phenotype" refers to one or a number of general biological characteristics that are characteristic of a cell or organism under the influence of certain external conditions and factors, regardless of the actual genotype.

[99] As used herein, the term “pluripotent” refers to a cell that has the ability under different conditions to differentiate into more than one differentiated cell type and preferentially differentiate into cell types characteristic of all three germ layers of cells. Pluripotent cells are characterized primarily by their ability to differentiate into more than one cell type, preferably into cells from all three germ layers, for example in the teratoma formation assay in nude mice. Pluripotency is also indicated by the expression of embryonic stem (ES) cell markers, although the preferred hallmark of pluripotency is the demonstration of the ability to differentiate into cells of each of the three germ layers. It should be noted that culturing such cells in itself is not enough to determine their pluripotency. Reprogrammed pluripotent cells (eg, as defined herein, iPS cells) also have the characteristic ability to undergo extended passage without loss of growth capacity compared to the original parent cells, which generally have the ability to undergo a limited number of divisions in culture.

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

[101] The terms "progenitor cell" or "precursor" are used interchangeably herein and refer to cells having a cellular phenotype that is more primitive (i.e., at an earlier stage of development or progression than a fully differentiated cell) compared to the cells that can arise from them during the process of differentiation. Often progenitor cells have significant or very high proliferative potential. Progenitor cells can develop into a large number of different types of differentiated cells or a single type of differentiated cell, depending on the developmental path and the environment in which the cells develop and differentiate.

[102] As used herein, the term “stem cell” refers to an undifferentiated cell capable of proliferating and developing into new progenitor cells that have the ability to generate a large number of mother cells, which in turn can develop into differentiated or differentiable daughter cells. The daughter cells themselves can be induced to proliferate and give rise to progeny, which subsequently differentiate into one or more mature cell types, retaining one or more cells with the developmental capacity of the parent cells. The term "stem cell" refers to a subset of progenitor cells that have the ability or ability under certain circumstances to differentiate into a more specialized or differentiated phenotype and which under certain circumstances retain the ability to proliferate without any significant differentiation. In one embodiment, the term stem cell generally refers to a mother cell of natural origin, whose descendants (progeny) specialize, often in different directions, by differentiation, for example, acquiring completely individual characteristics, as occurs during the progressive diversification of embryonic cells and fabrics. Cellular differentiation is a complex process, usually involving a large number of cell division stages. A differentiated cell can be derived from a multipotent cell, which in turn is derived from a multipotent cell, and so on. Although any of these multipotent cells can be considered stem cells, the range of cell types that can develop from each of them can vary significantly. Some differentiated cells also have the ability to develop into cells with greater developmental capacity. This ability may be natural or may be induced artificially by treatment with various factors. In many biological systems, stem cells are also "multipotent" in that they can give rise to offspring belonging to more than one cell type, although this is not a necessary condition for being a "stem cell". Self-renewal represents another classical part of the definition of stem cells and is important in the context of this paper. According to the theory, self-renewal can occur through either of two mechanisms. Stem cells can divide asymmetrically, with one daughter cell remaining a stem cell and the other daughter cell expressing some distinct specific function and phenotype. Alternatively, some of the stem cells in the population may divide symmetrically in two stages, such that a number of stem cells are retained in the population as a whole, while other cells in the population produce exclusively differentiated progeny. Formally, it is possible for cells that were originally stem cells to develop to a differentiated phenotype, but then “reverse” and again express the stem cell phenotype; this phenomenon is often referred to by those skilled in the art as "dedifferentiation" or "reprogramming" or "retrodifferentiation". As used herein, the term “pluripotent stem cell” includes embryonic stem cells, induced pluripotent stem cells, placental stem cells, etc.

[103] In the context of cellular ontogeny, the adjective "differentiated" or "differentiating" is a relative concept denoting a "differentiated cell" - a cell that has made more developmental progress than the cell to which it is compared. Thus, stem cells can differentiate into lineage-specific progenitor cells (such as mesodermal stem cells), which in turn can, during development, differentiate into other types of progenitor cells (such as cardiomyocyte progenitors) and then into the final differentiated cell , which plays a characteristic role in a particular tissue type and may or may not retain the ability to further proliferate.

[104] The term "embryonic stem cell" is used to refer to the pluripotent stem cells of the inner cell mass of the embryonic blastocyst (see US Pat. Nos. 5,843,780, 6,200,806). Such cells can be obtained from the inner cell mass of blastocysts obtained by somatic cell nuclear transfer (see, for example, US patents No. 5945577, 5994619, 6235970). The distinctive characteristics of an embryonic stem cell determine the phenotype of the embryonic stem cell. Accordingly, a cell has an embryonic stem cell phenotype if it has one or more unique characteristics of an embryonic stem cell such that the cell can be distinguished from other cells. Exemplary distinguishing characteristics of an embryonic stem cell include, but are not limited to, gene expression profile, proliferative capacity, differentiation capacity, karyotype, responsiveness to particular culture conditions, and the like.

[105] The term “adult stem cell” or “ASC” (“adult stem cell”) is used to refer to any multipotent stem cell derived from non-embryonic tissue, including fetal, juvenile and adult tissue. Stem cells have been isolated from a wide variety of adult tissues, including blood, bone marrow, brain, olfactory epithelium, skin, thyroid, skeletal muscle, and cardiac muscle. Any of these stem cells can be characterized based on gene expression, responsiveness to factors, and morphology in culture. Typical adult stem cells include neural stem cells, neural crest stem cells, mesenchymal stem cells, hematopoietic stem cells, and pancreatic stem cells. As stated above, stem cells have been found in almost all tissues. Accordingly, the present invention contemplates that stem cell populations can be isolated from virtually any animal tissue.

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

[107] As used herein, the term “reprogramming” refers to a process that changes or reverses the differentiation state of a somatic cell. Before reprogramming, the cell may be partially or completely differentiated. Reprogramming involves a complete reversal of the differentiation state of a somatic cell into a pluripotent cell. This complete reversal of differentiation results in an induced pluripotent (iPS) cell. As used herein, reprogramming also includes partially reversing a cell's differentiated state, for example, to a multipotent state or to a somatic cell that is neither pluripotent nor multipotent, but is a cell that has lost one or more specific characteristics of the differentiated cell from which it was obtained, for example, in the case of direct reprogramming of a differentiated cell into a different type of somatic cell. In general, reprogramming involves changing, e.g., reversing, at least some of the heritable features of nucleotide modification (e.g., methylation), chromatin condensation, epigenetic changes, genomic imprinting, etc. that occur during cell differentiation as the zygote develops into adult organism.

[108] As used herein, the term “agent” refers to any compound or substance, such as, without limitation, a small molecule, nucleic acid, polypeptide, peptide, drug, ion, etc. "Agent" can be any chemical substance, compound or component, including, without limitation, synthetic and natural protein and non-protein compounds. In some embodiments, the agent is a nucleic acid, nucleic acid analogs, proteins, antibodies, peptides, aptamers, nucleic acid oligomers, amino acids, or carbohydrates, including, but not limited to, proteins, oligonucleotides, ribozymes, DNA enzymes, glycoproteins, shRNA, lipoproteins, aptamers, as well as their modifications and combinations, etc. In certain embodiments of the invention, the agents are small molecules containing a chemical moiety. For example, chemical components include unsubstituted or substituted alkyl, aromatic or heterocyclic compounds, including macrolides, leptomycins and related natural products and analogs. The compounds may be known to have the desired activity and/or properties, or they may be selected from a library of different compounds.

[109] As used herein, the term “contacting” (i.e., bringing at least one insulin-positive endocrine cell or its precursor into contact with a β-cell maturation factor or a combination of β-cell maturation factors) includes in vitro co-incubation of β-cell maturation factor and cells (for example, adding β-cell maturation factors to cells in culture). In some embodiments, the term “contacting” does not include in vivo exposure of cells to compounds as described herein that may occur naturally in a subject's body (i.e., exposure that may occur as a result of natural physiological process). The step of contacting at least one insulin-positive endocrine cell or its precursor with a β-cell maturation factor, as described in embodiments of the invention associated with the production of SC-β cells, can be carried out by any suitable method. For example, the cells can be treated in an adherent culture or in a suspension culture. In some embodiments, the cells are treated under conditions that promote cell clustering. The invention contemplates any conditions that stimulate cell clustering. Examples of conditions that promote cell clustering include, but are not limited to, suspension culture in low adhesion tissue culture plates, roller bottles, AggreWell plates. In some embodiments, the inventors observed that the clusters remained stable in media containing 10% serum. In some embodiments, conditions that promote cell clustering include a low serum environment.

[110] It should be understood that cells that are contacted with β-cell maturation factor can also be simultaneously or sequentially contacted with another agent, such as a growth factor or other differentiation agent or media to stabilize the cells or to further differentiate the cells .

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

[112] As used herein, the term “cell culture medium” (also referred to herein as “culture medium” or “medium”) is a cell culture medium containing nutrients that support cell viability and promote proliferation. The cell culture medium may contain any of the following components in a suitable combination: salt(s), buffer(s), amino acids, glucose or other sugar(s), antibiotics, serum or serum substitute, and other components such as peptide growth factors, etc. Cell culture media traditionally used for specific cell types are known to those skilled in the art.

[113] The term "cell line" refers to a population of largely or substantially identical cells that are typically derived from a single parent cell or from a defined and/or substantially identical population of parent cells. The cell line may be or may be maintained in culture for an extended period of time (eg, months, years, or indefinitely). It could undergo a spontaneous or induced transformation process, which resulted in an unlimited life span of cells in culture. Cell lines include all cell lines known in the art. It should be understood that over time, cells accumulate mutations and possible epigenetic changes, so that at least some properties of individual cells in a cell line may differ from each other. In some embodiments, the cell line comprises an SC-β cell described herein.

[114] The term "exogenous" refers to a substance present in a cell or organism other than its native source. For example, the terms "exogenous nucleic acid" or "exogenous protein" refer to a nucleic acid or protein that has been introduced through a human process into a biological system, such as a cell or organism, in which it is not normally present or in which it is present in smaller quantities. quantities. A substance is considered exogenous if it is introduced into a cell or a precursor cell that inherits the substance. The term "endogenous", in contrast, refers to a substance that is native to a biological system.

[115] The term "expression" refers to the cellular processes associated with the production of RNA and proteins and, as appropriate, the secretion of proteins, including but not limited to, as appropriate, transcription, translation, folding, modification and processing." Expression products include RNA transcribed from a gene and polypeptides produced by translation of mRNA transcribed from the gene.

[116] As used herein, the terms “genetically modified” or “engineered” cell refer to a cell into which an exogenous nucleic acid has been introduced through a human process (or to the progeny of such a cell which has inherited at least a portion of that nucleic acid). A nucleic acid may, for example, contain a sequence that is exogenous to a cell; it may contain native sequences (i.e., sequences that can be found in cells in their natural state) but in an order that is not natural (for example, when the coding region is linked to the promoter of another gene), or modified versions of native sequences, etc. The process of transferring a nucleic acid into a cell can be accomplished by any suitable method. Suitable methods include calcium phosphate or lipid mediated transfection, electroporation and transduction or infection with a viral vector. In some embodiments, a polynucleotide or a portion thereof is integrated into the genome of a cell. The nucleic acid may subsequently be removed or excluded from the genome, provided that such removal or exclusion results in a detectable change in the cell compared to an unmodified but otherwise equivalent cell. It should be understood that the term genetically modified includes the introduction of modified RNA directly into a cell (eg, synthetic modified RNA). Such synthetic modified RNAs include modifications that prevent rapid destruction by endo- and exonucleases and avoid or reduce the cellular natural immune or interferon response to the RNA. Modifications include, but are not limited to, (a) terminal modifications, e.g., 5' end modifications (phosphorylation, dephosphorylation, conjugation, inverted bonds, etc.), 3' end modifications (conjugation, DNA nucleotides, inverted bonds, etc.), (b) modifications of bases, e.g. substitution with modified bases, stabilizing bases, destabilizing bases or bases that are capable of pairing with an expanded set of partners, or conjugate bases, (c) modifications of sugars (e.g. position 2' or at position 4') or sugar substitution, and (d) modification of internucleoside bonds, including modification or substitution of phosphodiester bonds. Where such modifications interfere with translation (i.e., result in a reduction in translation of 50% or more if no modification were present, such as in vitro translation assays in rabbit reticulocytes), such modification is not suitable for use in the applications described herein. document methods and compositions. In some embodiments, the SC-β cell is genetically modified to express neurogenin 3. In some embodiments, the genetic modification of the SC-β cell includes the introduction of a synthetically modified mRNA encoding neurogenin 3. The genetic modification of the SC-β cells is said to be synthetic modified mRNA encoding neurogenin 3 increases insulin production from cells. This genetic modification of any insulin-producing cell is expected to increase insulin production in that cell.

[117] In some aspects, the invention provides an SC-β cell genetically modified to contain a detectable marker at the insulin locus. In some embodiments, the SC-β cell is modified such that both alleles at the insulin locus are replaced by a detectable marker. In some embodiments, the SC-β cell is genetically modified such that a detectable marker is inserted into the insulin locus and is expressed along with insulin in the SC-β cell in response to glucose stimulation. In some embodiments, the SC-β cell is genetically modified such that a detectable marker is inserted into the insulin locus in place of insulin and is expressed in place of insulin in the SC-β cell in response to glucose stimulation. It is contemplated that any detectable marker may be inserted into the insulin locus, including, for example, a nucleic acid encoding a fluorescent protein (eg, ZFP). Those skilled in the art will appreciate that such genetically modified SC-β cells can be used in various screening methods, for example, to identify agents that stimulate insulin expression and/or secretion from β cells by analyzing a detectable marker in response to the agent. For example, an SC-β cell genetically modified so that the insulin gene is replaced (eg, ZPB) in both alleles can be exposed to a test agent, and those agents that cause SC-β cells to fluoresce due to expression of ZPB can be considered candidate agents that are capable of activating insulin gene expression in β-cells. In other words, the detectable marker can be used as a surrogate marker for insulin expression in such genetically modified SC-β cells.

[118] As used herein, the term “identity” refers to the degree of sequence similarity of two or more nucleic acids or polypeptides. The percentage identity between a sequence of interest and a second sequence in a scoring window, e.g., over the sequence of interest, can be estimated by aligning the sequences, determining the number of residues (nucleotides or amino acids) within the comparison window that are opposite an identical residue, while allowing for inclusion gaps to maximize identity, dividing by the total number of residues in the sequence of interest or the second sequence (whichever is longer) falling within the comparison window, and multiplying by 100. When calculating the number of identical residues needed to achieve specific percentage of identity, fractional numbers should be rounded to the nearest whole number. The percent identity can be calculated using a variety of computer programs known in the art. For example, computer programs such as BLAST2, BLASTN, BLASTP, Gapped BLAST, etc. create alignments and calculate the percentage of identity between sequences of interest. The Karlin and Altschul algorithm (Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:22264-2268, 1990), modified from Karlin and Altschul, Proc. Natl. Acad. ScL USA 90:5873-5877, 1993, part of the NBLAST and XBLAST programs developed by Altschul et al. (Altschul, et al., J. MoT Biol. 215:403-410, 1990). To obtain alignments containing gaps for comparison purposes, use Gapped BLAST as described by Altschul et al. (Altschul, et al. Nucleic Acids Res. 25: 3389-3402, 1997). When using the BLAST and Gapped BLAST programs, you can use the default settings of the respective programs. You can use the RAM250 or BLOSUM62 matrix. BLAST analysis software is publicly available from the National Center for Biotechnology Information (NCBI). To view these programs, see the website URL: “ncbi.nlm nih.gov.” In a specific embodiment of the invention, percent identity is calculated using BLAST2 with default parameters provided by NCBI.

[119] As used herein, the term “isolated” or “partially purified” in the case of a nucleic acid or polypeptide refers to a nucleic acid or polypeptide that is separated from at least one of the other components (e.g., nucleic acid or polypeptide) that are present together with a nucleic acid or polypeptide in a natural source and/or that would be present with the nucleic acid or polypeptide if expressed by a cell or secreted in the case of secreted polypeptides. A nucleic acid or polypeptide that is chemically synthesized or synthesized by in vitro transcription/translation is considered to be “isolated.”

[120] As used herein, the term “isolated cell” refers to a cell that has been removed from the organism in which it originally resided, or the progeny of that cell. Optionally, the cell has been cultured in vitro, for example, in the presence of other cells. Optionally, the cell is later introduced into a second organism or reintroduced into the organism from which it (or the cell of which it is a progeny) was isolated.

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

[122] The term "substantially pure" when used with respect to a particular cell population refers to a population of cells that is at least about 75% pure, preferably at least about 85% pure, more preferably at least about 90% pure. % and most preferably at least about 95% pure compared to the cells constituting the general cell population. The terms “substantially pure” or “substantially pure” as used with respect to a population of SC-β cells then refer to a population of cells that contains less than about 20%, more preferably less than about 15%, 10%, 8%, 7%, most preferably less than about 5%, 4%, 3%, 2%, 1%, or less than 1% of cells that are not SC-β cells as those terms are defined herein. In some embodiments, the present invention includes 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.

[123] Likewise, the terms “substantially pure” or “substantially purified” when used with respect to a population of insulin-positive endocrine cells refer to a population of cells that contains less than about 20%, more preferably less than about 15%, 10 %, 8%, 7%, most preferably less than about 5%, 4%, 3%, 2%, 1%, or less than 1% of cells that are not insulin-positive endocrine cells as those terms are defined herein. In some embodiments, the present invention includes methods for expanding a population of insulin-positive endocrine cells, wherein the expanded population of insulin-positive endocrine cells is a substantially pure population of insulin-positive endocrine cells.

[124] Likewise, the terms “substantially pure” or “substantially pure” as used with respect to a population of Ngn3-positive endocrine progenitor cells refer to a population of cells that contains less than about 20%, more preferably less than about 15% 10%, 8%, 7%, most preferably less than about 5%, 4%, 3%, 2%, 1%, or less than 1% of cells that are not Ngn3-positive endocrine progenitor cells or their progeny, according to the interpretation of these terms in this document. In some embodiments, the present invention includes methods for expanding a population of Ngn3-positive endocrine progenitor cells, wherein the expanded population of Ngn3-positive endocrine progenitor cells is a substantially pure population of Ngn3-positive endocrine progenitor cells.

[125] Likewise, the terms "substantially pure" or "substantially pure" as used in reference to a population of Pdx1-positive, NKX6-1-positive pancreatic progenitor cells refer to a population of cells that contains less than about 20%, more preferably less than about 15%, 10%, 8%, 7%, most preferably less than about 5%, 4%, 3%, 2%, 1% or less than 1% of cells that are not Pdx1-positive, NKX6- 1-positive pancreatic progenitor cells or their progeny, as those terms are defined herein. In some embodiments, the present invention includes methods for expanding a population of Pdx1-positive, NKX6-1-positive pancreatic progenitor cells, wherein the expanded population of Pdx1-positive, NKX6-1-positive pancreatic progenitor cells is a substantially pure Pdx1- positive, NKX6-1-positive pancreatic progenitor cells.

[126] Likewise, the terms “substantially pure” or “substantially pure” as used with respect to a population of Pdx1-positive pancreatic progenitor cells refer to a population of cells that contains less than about 20%, more preferably less than about 15% 10%, 8%, 7%, most preferably less than about 5%, 4%, 3%, 2%, 1%, or less than 1% of cells that are not Pdx1-positive pancreatic progenitor cells or their progeny, according to the interpretation of these terms in this document. In some embodiments, the present invention includes methods for expanding a population of Pdx1-positive pancreatic progenitor cells, wherein the expanded population of Pdx1-positive pancreatic progenitor cells is a substantially pure population of Pdx1-positive pancreatic progenitor cells.

[127] Likewise, the terms “substantially pure” or “substantially pure” when used with respect to a population of cells of the primary intestinal tube refer to a population of cells that contains less than about 20%, more preferably less than about 15%, 10% , 8%, 7%, most preferably less than about 5%, 4%, 3%, 2%, 1%, or less than 1% of cells that are not primary gut tube cells or progeny thereof, as those terms are defined herein . In some embodiments, the present invention includes methods for expanding a population of primary intestinal tube cells, wherein the expanded population of primary intestinal tube cells is a substantially pure population of primary intestinal tube cells.

[128] Likewise, the terms "substantially pure" or "substantially pure" as used with respect to a population of definitive endoderm cells refer to a population of cells that contains less than about 20%, more preferably less than about 15%, 10%, 8%, 7%, most preferably less than about 5%, 4%, 3%, 2%, 1%, or less than 1% of cells that are not definitive endoderm cells or progeny thereof, as those terms are defined herein. In some embodiments, the present invention includes methods for expanding a population of definitive endoderm cells, wherein the expanded population of definitive endoderm cells is a substantially pure population of definitive endoderm cells.

[129] Likewise, the terms "substantially pure" or "substantially pure" when used with respect to a population of pluripotent cells refer to a population of cells that contains less than about 20%, more preferably less than about 15%, 10%, 8 %, 7%, most preferably less than about 5%, 4%, 3%, 2%, 1%, or less than 1% of cells that are not pluripotent cells or their progeny, as those terms are defined herein. In some embodiments, the present invention includes methods for expanding a population of pluripotent cells, wherein the expanded population of pluripotent cells is a substantially pure population of pluripotent cells.

[130] The terms "enrichment" or "enriched" are used interchangeably herein and mean that the yield of (a portion of) cells of one type is increased by at least 10% compared to the portion of cells of that type in the starter culture or preparation.

[131] The terms "renewal" or "self-renewal" or "proliferation" are used interchangeably herein and refer to the ability of stem cells to renew themselves by dividing into the same unspecialized cell types over a long period of time and/or many months to years. In some cases, proliferation refers to the increase in cell number by repeated divisions of single cells into two identical daughter cells.

[132] As used herein, the term “lineages” describes cells of common origin or cells with a common developmental path. For example, in the context of a cell that is of endodermal origin or belongs to an "endodermal lineage", this means that the cell was derived from an endoderm cell and can differentiate according to a pathway limited by the endodermal lineage, e.g., according to one or more lineage developmental pathways, which give rise to definitive endoderm cells, which in turn can differentiate into cells of the liver, thymus, pancreas, lungs and intestines.

[133] As used herein, the term “xenogeneic” refers to cells obtained from different species.

[134] 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 that have characteristics of interest. The markers are cell specific. Markers are characteristics, both morphological, functional or biochemical (enzymatic) characteristics of a particular cell type, and the molecules that that cell type expresses. Preferably, such markers are proteins and more preferably contain epitopes for antibodies or other binding molecules known in the art. The marker may contain any molecule that can be found in a cell, including, but not limited to, proteins (peptides and polypeptides), lipids, polysaccharides, nucleic acids and steroids. Examples of morphological characteristics or features include, but are not limited to, shape, size, and nuclear to cytoplasmic ratio. Examples of functional characteristics or traits include, but are not limited to, the ability to bind to specific substrates, the ability to incorporate or exclude specific dyes, the ability to migrate under specific conditions, and the ability to differentiate along specific lineages. Markers can be detected by any means known to one skilled in the art. Markers may also be the absence of any morphological characteristics or the absence of proteins, lipids, etc. Markers may be a panel of unique characteristics corresponding to the presence or absence of polypeptides and other morphological characteristics.

[135] The term "modulate" is used in accordance with its meaning accepted in the art, i.e. means to cause or promote a qualitative or quantitative change, transformation, or modification in a process, pathway, or phenomenon of interest. Without limitation, such a change may be an increase, decrease, or change in the relative strength or activity of different components or directions of a process, pathway, or phenomenon. A "modulator" is an agent that causes or promotes a qualitative or quantitative change, transformation, or modification in a process, pathway, or phenomenon of interest.

[136] As used herein, the term “DNA” means deoxyribonucleic acid.

[137] The term "polynucleotide" is used interchangeably herein with the term "nucleic acid" and refers to a polymer of nucleosides. Typically, the polynucleotide of the present invention consists of nucleosides that can be found in DNA or RNA in their natural state (eg, adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine and deoxycytidine) linked by phosphodiester bonds. However, the term also includes molecules containing nucleosides or nucleoside analogs containing chemically or biologically modified bases, modified skeletons, etc., regardless of whether they can be found in naturally occurring nucleic acids, and such molecules may be preferred in certain applications. Where a polynucleotide is mentioned herein, it is understood that both DNA and RNA are meant, and, in each case, both single- and double-stranded forms (as well as complementary molecules of single-stranded molecules). As used herein, "polynucleotide sequence" may refer to the polynucleotide material itself and/or to sequence information (ie, the order of letters used as base abbreviations) that biochemically characterizes a particular nucleic acid. The polynucleotide sequence presented herein is presented in the 5' to 3' direction unless otherwise indicated.

[138] As used herein, the term “polypeptide” refers to a polymer of amino acids. The terms “protein” and “polypeptide” are used interchangeably herein. A peptide is a relatively short polypeptide, typically ranging from 2 to 60 amino acids in length. Polypeptides as used herein typically contain amino acids, such as the 20 L-amino acids, which are most commonly found in proteins. In this case, other amino acids and/or amino acid analogues known in the art can be used. One or more amino acids in a polypeptide may be modified, for example, by adding a chemical component such as a carbohydrate group, phosphate group, fatty acid group or linker for conjugation, functionalization, etc. A polypeptide that contains a non-polypeptide component covalently or non-covalently linked to it is considered a "polypeptide". Typical modifications include glycosylation and palmitoylation. Polypeptides can be isolated from natural sources, produced using recombinant DNA technology, synthesized chemically such as traditional solid-phase peptide synthesis, etc. As used herein, the term “polypeptide sequence” or “amino acid sequence” may refer to the polypeptide material itself and/or to sequence information (i.e., the order of letters or three-letter codes used as abbreviations for amino acid names) that biochemically characterizes the polypeptide . The polypeptide sequence presented herein is presented in the direction from N-terminus to C-terminus unless otherwise indicated.

[139] The term “variant” when used with respect to a polypeptide can be, for example, a polypeptide that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the full-length polypeptide. The variant may be a fragment of a full-length polypeptide. The variant may be a naturally occurring splice variant. The variant may be a polypeptide that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to a polypeptide fragment, wherein the length of the fragment is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% of the total length of a wild-type polypeptide, or a domain thereof, having an activity of interest, such as the ability to detect the presence of an SC-β cell or an insulin-positive endocrine cell or precursor thereof from which SC-β cell was obtained. 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. It is preferable to avoid variations known in the art to eliminate or significantly reduce the activity of the protein. In some embodiments, the variant is missing the N- and/or C-terminal portion of the full-length polypeptide, for example, up to 10, 20, or 50 amino acids from either end are missing. In some embodiments, the polypeptide has a mature (full-length) polypeptide sequence, which is a polypeptide in which one or more portions, such as a signal peptide, have been removed during normal intracellular proteolytic processing (eg, co-translational or post-translational processing). In some embodiments, in which the protein is produced by a process other than isolating it from cells that naturally express it, the protein is a chimeric polypeptide, meaning that it contains portions obtained from two or more different species. In some embodiments of the invention, in which the protein is produced by a process other than isolating it from cells that naturally express it, the protein is a derivative, meaning that the protein contains additional sequences unrelated to the protein, provided that those sequences are substantially do not reduce the biological activity of the protein.

[140] As used herein, the term “functional fragment” means a polypeptide having an amino acid sequence that is smaller in size but substantially homologous to the polypeptide of which it is a fragment, and wherein the polypeptide sequence of the functional fragment is at least about 50% or 60%, or 70%, or 80%, or 90%, or 100% or more than 100%, for example, 1.5 times, 2 times, 3 times, 4 times or more than 4 times effective biological effect compared to the polypeptide of which it is a fragment. Functional polypeptide fragments may have additional functions, which may include reduced antigenicity, increased ability to bind DNA (as in the case of transcription factors), or altered ability to bind RNA (as in the case of regulating RNA stability or degradation).

[141] The term "vector" refers to a DNA molecule carrying a DNA sequence that can be inserted for introduction into a host cell. Preferred vectors are those that are capable of autonomous replication and/or expression of the nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are operably linked are referred to herein as “expression vectors”. Thus, an “expression vector” is a specialized vector that contains the necessary regulatory regions that are needed for the expression of a gene of interest in a host cell. In some embodiments, the gene of interest is operably linked to another sequence in the vector. The vectors can be viral vectors and non-viral vectors. When viral vectors are used, it is preferred that the viral vectors be replication defective, which can be achieved, for example, by removing all viral nucleic acids that encode replication. A replication-defective viral vector retains its infectious properties and is introduced into the cell in the same way as a replicating adenoviral vector, and after entering the cell, the replication-defective viral vector is not able to reproduce or multiply. Vectors also include liposomes and nanoparticles, as well as other means of delivering DNA molecules into the cell.

[142] The term "operably linked" means that the regulatory sequences necessary for the expression of the coding sequence are placed in the DNA molecule at appropriate positions relative to the coding sequence so as to influence the expression of the coding sequence. The same definition is sometimes used for the location of coding sequences and transcriptional regulatory elements (eg, promoters, enhancers, and termination elements) in an expression vector. The term "operably linked" includes the presence of an appropriate start signal (eg, ATG) at the beginning of the polynucleotide sequence to be expressed and maintenance of the correct reading frame to allow expression of the polynucleotide sequence under the control of an expression regulatory sequence and production of the desired polypeptide encoded by the polynucleotide sequence.

[143] The term “viral vectors” refers to the use of viruses or virus-associated vectors as carriers of nucleotide constructs for introduction into a cell. The constructs can be integrated and packaged into non-replicating defective viral genomes such as adenovirus, adeno-associated virus (AAV) or herpes simplex virus (HSV) or others, including retroviral and lentiviral vectors, for infection or transduction of cells. The vector may or may not be integrated into the cellular genome. If necessary, the constructs may contain viral sequences for transfection. Alternatively, the construct may be incorporated into vectors capable of episomal replication, such as EPV and EBV vectors.

[144] The terms “regulatory sequence” and “promoter” are used interchangeably herein and refer to nucleotide sequences, such as initiation signals, enhancers and promoters, that induce or regulate transcription of protein-coding sequences to which they are operably linked. In some examples, transcription of the recombinant gene is under the control of a promoter sequence (or other transcriptional control sequence) that directs expression of the recombinant gene in the cell type in which expression is to occur. It should also be understood that the recombinant gene may be under the control of transcriptional control sequences that are the same as or that are different from those sequences that control transcription of the native form of the protein. In some cases, a promoter sequence is recognized by the cellular synthesis machinery or contributed synthesis machinery necessary to initiate transcription of a particular gene.

[145] As used herein, the term “transcription factor” refers to a protein that binds to specific parts of DNA through DNA-binding domains and is part of the system that controls the transfer (or transcription) of genetic information from DNA to RNA. As used herein, "proliferative" or "proliferation" refers to the increase in the number of cells in a population (growth) through cell division. Cell proliferation is generally understood to result from the coordinated activation of multiple signal transduction pathways in response to environmental factors, including growth factors and other mitogens. Cell proliferation can also be stimulated by the release of intra- and extracellular signals and mechanisms that block or negatively affect cell proliferation.

[146] The term "selectable marker" refers to a gene, RNA, or protein that, when expressed, confers a selectable phenotype on a cell, such as resistance to cytotoxic or cytostatic agents (e.g., antibiotic resistance), nutrient prototrophy, or expression of a particular protein. which can be used as a basis to distinguish between cells that express the protein and cells that do not. Proteins whose expression can be readily detected, such as a fluorescent or luminescent protein or an enzyme whose action on a substrate results in the production of a colored, fluorescent or luminescent substance (“detectable markers”), constitute a subset of selectable markers. The presence of a selectable marker associated with expression control elements native to a gene that is usually selectively or exclusively expressed in pluripotent cells makes it possible to identify and select somatic cells that have been reprogrammed into a pluripotent state. A large number of selectable marker genes can be used, such as neomycin resistance gene (neo), puromycin resistance gene (puro), guanine phosphoribosyltransferase gene (gpt), dihydrofolate reductase (DHFR), adenosine deaminase (ada), puromycin-N-acetyltransferase (PAC) , hygromycin resistance gene (hyg), multidrug resistance gene (mdr), thymidine kinase (TK) gene, hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene and hisD gene. Detectable markers include green fluorescent protein (GFP), blue, sapphire, yellow, red, orange and cyan fluorescent proteins, as well as variants of any of these. Luminescent proteins such as luciferase (eg firefly luciferase or Renilla luciferase) can also be used. It will be apparent to one skilled in the art that the term “selectable marker” as used herein can refer to a gene or a gene expression product, such as an encoded protein.

[147] In some embodiments, the selectable marker provides cells that express it with a proliferation and/or viability advantage over cells that do not express it or that express it at substantially lower levels. This proliferation and/or viability advantage typically occurs when cells are maintained under certain conditions, e.g. "selective conditions". To ensure that selection is effective, a population of cells can be maintained under conditions and for a sufficient period of time such that cells that do not express the marker do not proliferate and/or do not survive and are excluded from the population or constitute only a very small portion of the population. The process of selecting cells that express a marker that provides a proliferation and/or viability advantage by maintaining a population of cells under selective conditions so as to largely or completely exclude cells that do not express the marker is referred to herein as “positive selection.” ", and the marker is considered "applicable for positive selection". Negative selection and markers useful for negative selection are also of interest in the context of some of the methods described herein. Expression of such markers provides cells that express the marker with a proliferation and/or viability disadvantage compared to cells that do not express the marker or that express it at substantially lower levels (or, in other words, cells that do not express the marker have a proliferation and/or viability advantage over cells that express the marker). Consequently, cells that express the marker can be largely or completely eliminated from the cell population when maintained under selective conditions for a sufficient period of time.

[148] As used herein, the term “reporter gene” includes any gene genetically introduced into a cell that complements the stem cell phenotype. Reporter genes disclosed herein include fluorescent, luminescent, enzymatic, and resistance-conferring genes, as well as other genes that can be readily identified by those skilled in the art. In some embodiments, reporter genes are used as markers to identify specific stem cells, cardiovascular stem cells, and their differentiated progeny. In general, a reporter gene is operably linked to sequences that direct its expression in a manner that depends on one or more conditions that are monitored by determining expression of the reporter gene. In some cases, reporter gene expression can be determined in living cells. In cases where reporter gene assays are used in living cells, reporter gene expression can be monitored over a number of time points, for example, 2, 3, 4, 5, 6, 8 or 10 or more time points. In some cases, when reporter gene assays are used in living cells, reporter gene expression is monitored at a frequency of at least about 10 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 other frequency equal to any integer value from about 10 minutes to about 24 hours.

[149] The terms “subject” and “individual” are used interchangeably herein and refer to an animal, such as a human, from which cells can be obtained and/or treated, including prophylactic cell therapy, as described herein. document. In the case of treating those infections, pathological or disease states that are specific to a particular animal, such as a human, the term subject refers to that particular animal. The expressions “non-human animals” and “non-human mammals” are used interchangeably herein and include mammals such as rats, mice, rabbits, sheep, cats, dogs, cows, pigs and monkeys. The term "subject" also includes any vertebrates, including, but not limited to, mammals, reptiles, amphibians and fish. The subject is preferably a mammal such as a human or other mammals such as domestic mammals such as dogs, cats, horses and the like, or farm mammals such as cows, pigs and the like.

[150] The terms “diabetes” and “diabetes mellitus” are used interchangeably throughout this document. According to the World Health Organization definition, in the case of diabetes mellitus, the diagnostic value of fasting plasma glucose is 7.0 mmol/L (126 mg/dL) or higher (in whole blood - 6.1 mmol/L or 110 mg/dL) or level glucose 2 hours after eating is 11.1 mmol/L or higher (200 mg/dL or higher). Other indicators that suggest or indicate a high risk of diabetes include elevated blood pressure of 140/90 mmHg. or higher; elevated plasma triglycerides (1.7 mmol/L, 150 mg/dL) and low high-density lipoprotein cholesterol (less than 0.9 mmol/L, 35 mg/dL for men; less than 1.0 mmol/L, 39 mg /dl for women); central obesity (in men: the ratio between the waist and hips exceeds 0.90; in women: the ratio between the waist and hips exceeds 0.85) and/or a body mass index exceeding 30 kg/ ^m2 ; microalbuminuria, in which the urinary albumin excretion rate is 20 mcg/min or higher, or the albumin-to-reatinin ratio is 30 mg/g or higher). The term diabetes includes all forms of diabetes, such as type I, type II and type 1.5.

[151] The terms “treat” (“treat”), “treatment” (“treatment”), etc., when used in relation to an isolated cell, include subjecting the cell to a process or condition of any type or performing any type of manipulation or procedure with cell. When used in relation to a subject, these terms refer to the provision of medical or surgical care, monitoring or treatment to an individual. Typically, an individual has a disease or injury or is at increased risk of developing a disease compared to the average member of the population and requires such care, observation or treatment.

[152] As used herein, the term “treating” refers to administering to a subject an effective amount of a composition so as to cause the subject to experience relief of at least one symptom of a disease or improvement of a disease, eg, favorable or desired clinical results. In the context of this invention, beneficial or desired clinical results include, but are not limited to, relief of one or more symptoms, reduction in the severity of a disease, stabilization (ie, non-worsening) of a disease state, arrest or slowing of disease progression, improvement or alleviation of a disease state, and remission (both partial and complete), both detected and not detected. Treatment may refer to prolongation of survival time compared to life expectancy without treatment. Thus, it will be apparent to one skilled in the art that treatment may improve the disease state without resulting in a complete cure of the disease. As used herein, the term “treatment” includes prophylaxis. Alternatively, treatment is “effective” if disease progression is slowed or stopped. “Treatment” can also mean prolonging the lifespan compared to the expected lifespan without treatment. Those in need of treatment include those who have already been diagnosed with a heart condition, as well as those who are at high risk of developing a heart condition due to genetic susceptibility or other factors such as weight, diet and health status.

[153] As used herein, the terms "administration", "deposition" and "transplantation" are used interchangeably in the context of introducing cells, e.g., SC-β cells according to the invention, into the body of a subject in a manner or manner that results in at least partial placement introduced cells in the required area. Cells, for example, SC-β cells (eg, pancreatic β-cells or pancreatic β-like cells), can be implanted directly into the pancreas or, alternatively, they can be administered by any suitable route that results in delivery to the desired site in in the subject's body, wherein at least a portion of the implanted cells or cell components remain viable. The period of cell viability after introduction into the body of a subject can range from several hours, for example twenty-four hours, to several days and up to several years. In some cases, the cells can also be administered not into the pancreas, but, for example, into the liver or subcutaneously, for example, in a capsule (eg, microcapsule), in order to maintain the implanted cells at the implantation site and to avoid migration of the implanted cells.

[154] As used herein, the expressions “parenteral administration” and “parenterally administered” mean modes of administration other than enteral and topical administration, usually administered by injection and including, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraocular, intracardial, intradermal, intraperitoneal, transtracheal, subcutaneous, intradermal, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal and intrathoracic injection and infusion. As used herein, the expressions “systemically administered,” “systemically administered,” “peripherally administered,” and “peripherally administered” mean the administration of cardiovascular stem cells and/or their progeny and/or compound and/or other material other than direct administration to the central nervous system so that it enters the animal's body and undergoes metabolism and other similar processes, for example, as in the case of subcutaneous administration.

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

[156] The terms “reduced,” “reduced,” “reduced,” “reduced,” or “inhibited” are generally used to denote a reduction by a statically significant amount. However, for the avoidance of doubt, “reduced,” “reduced,” or “reduced,” or “inhibited,” means a reduction of at least 10% relative to a control level, e.g., a reduction of at least about 20% or at least by about 30%, or by at least about 40%, or by at least about 50%, or by at least about 60%, or by at least about 70%, or by at least about 80%, or at least about 90%, or up to and including 100% reduction (ie, no levels compared to the control sample), or any reduction corresponding to a value in the range of 10-100% compared to the control level.

[157] The terms "increased", "raise" or "increase", or "activate" are generally used to denote an increase of a statically significant amount; For the avoidance of doubt, the terms “increased,” “increase,” or “increase,” or “activate” mean an increase of at least 10% above the reference level, e.g., an increase of at least about 20% or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including 100% increase, or any increase corresponding to a value in the range of 10-100% compared to the control level, or at least about 2-fold, or at least about 3-fold a fold, or at least about a 4-fold, or at least about a 5-fold, or at least about a 10-fold increase, or any increase corresponding to a range of 2 to 10 times the control level.

[158] The term “statistically significant” or “significantly” refers to statistical significance and generally means twice the standard deviation (2SD) of the marker concentration at or below normal. This term refers to statistical evidence of a difference. It is defined as the probability of deciding to reject the null hypothesis when the null hypothesis is actually true. This decision is often made using the p-value.

[159] As used herein, the terms “comprising” or “comprises” are used to refer to compositions, methods and their respective component(s) that are relevant to the present invention, but also refer to the inclusion of optional elements, both essential and and no.

[160] As used herein, the term “consists primarily of” refers to those elements that are necessary for a given embodiment of the invention. The term allows for the presence of additional elements that do not significantly affect the basic or functional characteristic(s) of this embodiment of the invention.

[161] The term “consisting of” refers to the compositions, methods and their respective components described herein, which do not include elements not set forth in this embodiment description.

[162] In the specification and the accompanying claims, the use of the singular form includes a reference to the plural unless the context clearly requires otherwise. Thus, for example, a reference to “method” refers to one or more methods and/or steps of a method described herein as will become apparent to those skilled in the art upon reading the specification, etc.

[163] Stem cells

[164] Stem cells are cells that retain the ability to self-renew through mitotic cell division and can differentiate into a wide range of specialized cell types. The two main types of mammalian stem cells are: embryonic stem (ES) cells, which are found in blastocysts, and adult stem cells, which are found in adult tissues. In the developing embryo, stem cells can differentiate into all the specialized embryonic tissues. In adult organisms, stem and progenitor cells serve as the body's repair system, replenishing the population of specialized cells, and also support the normal renewal of regenerative organs such as blood, skin or intestinal tissue. Pluripotent stem cells can differentiate into cells derived from any of the three germ layers.

[165] Although certain embodiments of the invention are described below in connection with the use of stem cells to produce SC-β cells (eg, pancreatic β-cells or β-like cells) or their precursors, germ cells can be used instead of or in conjunction with stem cells to obtaining at least one SC-β cell using protocols similar to the exemplary protocols described herein. Suitable germ cells can be obtained, for example, from primordial cells that are present in human fetal material taken approximately 8-11 weeks after the last menstrual cycle. Exemplary methods for obtaining germ cells are described, for example, in Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998 and US Patent No. 6090622.

[166] ES cells, such as human embryonic stem cells (hESC) or mouse embryonic stem cells (mESC), with the ability to replicate virtually endlessly and differentiate into most cell types, represent a potentially unlimited starting material for the generation of differentiated cells for clinical therapy. (http://stemcells.nih.gov/info/scireport/2006report.htm, 2006). One possible application of ES cells is to generate new pancreatic β-cells for cell replacement therapy for type I diabetes by obtaining endoderm, e.g., definitive endoderm, from, e.g., hESCs, and further differentiating the definitive endoderm into at least one insulin-positive endocrine cell or a precursor thereof, and then differentiating at least one insulin-positive endocrine cell or precursor thereof into an SC-β cell.

[167] hESCs are described, for example, in Cowan et al. (N Engl. J. Med. 350:1353, 2004) and Thomson et al. (Science 282:1145, 1998); embryonic stem cells from other primates, rhesus 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 (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). Stem cells can be, for example, unipotent, totipotent, multipotent or pluripotent. In some examples, the methods disclosed herein can use any primate-derived progeny-producing cells that are derived from at least one germ layer or all three germ layers.

[168] In some examples, ES cells can be isolated, for example, as described in Cowan et al. (N Engl. J. Med. 350:1353, 2004) and US patent No. 5843780, as well as Thomson et al., Proc. Natl. Acad. Sci. USA 92:7844, 1995. For example, hESC can be obtained from human blastocyst cells using the methods 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. hESC-equivalent cell types include their pluripotent derivatives, such as primary ectoderm-like (EPL) cells, as set forth, for example, in WO 01/51610 (Bresagen). hESC can also be obtained from human preimplantation embryos. Alternatively, in vitro fertilized (IVF) embryos can be used or single-cell human embryos can be grown to the blastocyst stage (Bongso et al., Hum Reprod 4: 706, 1989). Embryos are cultured to blastocyst stage in G1.2 and G2.2 media (Gardner et al., Fertil. Steril. 69:84, 1998). The zona pellucida is removed from the formed blastocysts by briefly treating with pronase (Sigma). The inner cell mass can be isolated using immunosurgery, during which blastocysts are exposed to a 1:50 solution of rabbit anti-human antiserum to spleen cells for 30 minutes, then washed three times for 5 minutes in DMEM and exposed to a 1:5 solution for 3 minutes. guinea pig complement (Gibco) (Solter et al., Proc. Natl. Acad. Sci. USA 72:5099, 1975). After two additional washes in DMEM, lysed trophectoderm cells were removed from the intact inner cell mass (ICM) by gentle pipetting, and the ICM was seeded onto mEF feeder layers. After 9-15 days, the inner cell mass proliferation can be separated into groups by treatment with calcium-magnesium-free phosphate-buffered saline (PBS) with 1 mM EDTA, by treatment with dispase or trypsin, or by mechanical separation using a micropipette; and then transfer to mEF in fresh medium. Growing colonies with undifferentiated morphology can be individually collected using a micropipette, mechanically separated into groups and subcultured. ES-like morphology has the characteristics of compact colonies with an apparently high nuclear-to-cytoplasmic ratio and prominent nucleoli. The resulting hESCs can then be separated routinely every 1 to 2 weeks, for example by brief trypsin treatment, treatment with Dulbecco's PBS (containing 2 mM EDTA), treatment with type IV collagenase (about 200 U/ml; Gibco), or by picking individual colonies using a micropipette. In some examples, optimal group sizes are around 50-100 cells. mESC can be obtained using methods described, for example, in Conner et al. (Curr. Prot. in Mol. Biol. Unit 23.4, 2003).

[169] Embryonic stem cells can be isolated from blastocysts of primates (US Pat. No. 5,843,780; Thomson et al., Proc. Natl. Acad. Sci. USA 92:7844, 1995). Human embryonic stem (hES) can be obtained from human blastocyst cells using the methods described in Thomson et al. (US Patent No. 6200806; Science 282:1145, 1998; Curr. Top. Dev. Biol. 38:133 ff., 1998) and Reubinoff et al, Nature Biotech. 18:399, 2000. HES cell-equivalent cell types include their pluripotent derivatives, such as primary ectoderm-like (EPL) cells, as set forth, for example, in WO 01/51610 (Bresagen).

[170] In some alternative embodiments, hES cells can be obtained from human preimplantation embryos. Alternatively, in vitro fertilized (TVF) embryos can be used or single-cell human embryos can be grown to the blastocyst stage (Bongso et al., Hum Reprod 4: 706, 1989). Embryos are cultured to blastocyst stage in G1.2 and G2.2 media (Gardner et al., Fertil. Steril. 69:84, 1998). The zona pellucida is removed from the formed blastocysts by briefly treating with pronase (Sigma). The inner cell mass is isolated using immunosurgery, during which blastocysts are exposed to a 1:50 solution of rabbit anti-human antiserum to spleen cells for 30 minutes, then washed three times for 5 minutes in DMEM and exposed to a 1:5 solution of complement for 3 minutes. guinea pig (Gibco) (Solter et al., Proc. Natl. Acad. Sci. USA 72:5099, 1975). After two additional washes in DMEM, lysed trophectoderm cells were removed from the intact inner cell mass (ICM) by gentle pipetting, and the ICM was seeded onto mEF feeder layers.

[171] After 9-15 days, the inner cell mass proliferation is separated into groups by treatment with calcium- and magnesium-free phosphate-buffered saline (PBS) with 1 mM EDTA, by treatment with dispase or trypsin, or by mechanical separation using a micropipette; and then transferred to mEF in fresh medium. Growing colonies exhibiting an undifferentiated morphology are individually collected using a micropipette, mechanically separated into groups and subcultured. ES-like morphology has the characteristics of compact colonies with an apparently high nuclear-to-cytoplasmic ratio and prominent nucleoli. The resulting ES cells are then routinely separated every 1–2 weeks by brief trypsin treatment, treatment with Dulbecco's PBS (containing 2 mM EDTA), treatment with collagenase type IV ( ^~ 200 U/ml; Gibco), or by collecting individual colonies using a micropipette . Optimal group sizes are about 50-100 cells.

[172] In some embodiments, human embryonic germ cells (hEG) are pluripotent stem cells that can be used in the methods described herein to differentiate into primary endoderm cells. hEG cells can be obtained from primordial cells that are present in human fetal material taken approximately 8-11 weeks after the last menstrual cycle. Suitable preparation methods are described in Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998 and US Patent No. 6,090,622, which are incorporated herein by reference in their entirety.

[173] Briefly, sex cords are processed to form disaggregated cells. The EG growth medium consists of DMEM, 4500 mg/L D-glucose, 2200 mg/L mM NaHCO ₃ ; 15% ES-matched 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). Ninety-six-well tissue culture plates are coated with a subconfluent layer of feeder cells (e.g., STO cells, ATCC No. CRL 1503), cultured for 3 days in modified EG growth medium containing no LIF, bFGF, or forskolin, with ^~ 0 added to each well. 2 ml of a suspension of primordial germ cells (PGC) inactivated by γ-irradiation at 5000 rad. The first passage is completed after 7-10 days in EG growth medium by transferring the contents of each well to one of the wells of a 24-well culture plate pretreated with irradiated STO mouse fibroblasts. Cells are cultured with daily replacement of the medium until cellular morphology consistent with EG cells is observed, usually after 7-60 days or 1-4 passages.

[174] In certain examples, before treatment with at least one β-cell maturation factor in accordance with the methods disclosed herein, the stem cells may be undifferentiated (e.g., a cell not belonging to a particular lineage), while in other examples it may be desirable to differentiate stem cells into one or more intermediate cell types before treatment with at least one β-cell maturation factor described herein. For example, stem cells may exhibit morphological, biological or physical characteristics of undifferentiated cells that can distinguish them from differentiated cells of an embryo or mature organism. In some examples, undifferentiated cells may appear in two dimensions when microscopically observing colonies of cells with a high nuclear/cytoplasmic ratio and prominent nucleoli. Stem cells can be present on their own (for example, in the virtual absence of any undifferentiated cells) or they can be used in the presence of differentiated cells. In certain examples, stem cells can be cultured in the presence of suitable nutrients and, optionally, other cells, so that the stem cells can grow and, optionally, differentiate. For example, embryonic fibroblasts or fibroblast-like cells may be present in culture to promote the growth of stem cells. Fibroblasts may be present at one stage of stem cell growth, but not necessarily at all stages. For example, fibroblasts can be added to stem cell cultures in the first culture step and not added to the stem cell cultures in one or more subsequent culture steps.

[175] Stem cells used in all aspects of the present invention can be any cells derived from any type of tissue (for example, fetal tissue, such as fetal or prefetal tissue, or adult tissue), and the stem cells are characterized by the ability to under appropriate conditions, produce offspring belonging to different cell types, for example, derivatives of at least one of the 3 germ layers (endoderm, mesoderm and ectoderm). These cell types may be in the form of a stable cell line, or they may be directly obtained from primary embryonic tissue and used directly for differentiation. Cells included are those listed in the NIH Human Embryonic Stem Cell Registry, e.g., hESBGN-01, hESBGN-02, hESBGN-03, hESBGN-04 (BresaGen, Inc.); HES-1, HES-2, HES-3, HES-4, HES-5, HES-6 (ES Cell International); Miz-hES1 (MizMedi Hospital-Seoul National University); HSF-1, HSF-6 (University of California at San Francisco); and H1, H7, H9, H13, 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 does not involve destruction of a human embryo.

[176] In another embodiment, stem cells can be isolated from tissue, including solid tissue. In some embodiments, the tissue is skin, adipose tissue (eg, adipose tissue), muscle tissue, heart, or cardiac tissue. In other embodiments, the tissue is, without limitation, umbilical cord blood, placenta, bone marrow, or cartilage.

[177] Stem cells of interest also include various types of embryonic cells, exemplified by human embryonic stem (hES) cells 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 (hEG) cells (Shambloft et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998). Also of interest are lineage-specific stem cells, such as mesodermal stem cells and other early cardiogenic cells (see Reyes et al. (2001) Blood 98:2615-2625; Eisenberg & Bader (1996) Circ Res. 78(2) :205-16; etc.). These stem cells can be obtained from any mammalian species such as humans, horses, ruminants, pigs, dogs, cats, rodents such as mice, rats, hamsters, primates, etc. In some embodiments, a human embryo is not destroyed to provide a source of pluripotent cells used in the methods and compositions disclosed herein.

[178] ES cells are considered undifferentiated if they do not belong to a specific lineage. Such cells exhibit morphological characteristics that distinguish them from differentiated embryonic or adult cells. Those skilled in the art will readily identify undifferentiated ES cells, which typically appear in two dimensions when observing microscopic colonies of cells with a high nuclear/cytoplasmic ratio and prominent nucleoli. Undifferentiated ES cells express genes that can be used as markers to determine the presence of undifferentiated cells, and whose polypeptide products can be used as markers for negative selection. For example, see US Patent Application No. 2003/0224411 A1; Bhattacharya (2004) Blood 103(8):2956-64; and Thomson (1998), supra, each of which is incorporated herein by reference. Human ES cell lines express cell surface markers that are characteristic of undifferentiated monkey ES cells and human ES cells, including stage-specific embryonic antigen (SSEA)-3, SSEA-4, TRA-1-60, TRA-1-81, and alkaline phosphatase. The globo-series glycolipid GL7, which carries the SSEA-4 epitope, is formed by adding sialic acid to the globo-series glycolipid GbS, which carries the SSEA-3 epitope. Thus, GL7 reacts with antibodies to both SSEA-3 and SSEA-4. Undifferentiated human ES cell lines do not stain with SSEA-1, but differentiated cells stain intensely with SSEA-I. Methods for the proliferation of hES cells in an undifferentiated form are described in WO 99/20741, WO 01/51616 and WO 03/020920.

[179] A mixture of cells from a suitable source of endothelial, muscle and/or neural stem cells can be obtained from a donor mammal by methods known in the art. A suitable source is the hematopoietic microenvironment. For example, circulating peripheral blood, preferably mobilized (ie, stimulated), can be collected from a patient. Alternatively, bone marrow may be obtained from a mammal, such as a human autologous transplant patient. In some embodiments, stem cells can be obtained from adipose tissue of subjects, for example, using the CELUTION™ SYSTEM from Cytori, as disclosed in US Pat. Nos. 7,390,484 and 7,429,488, which are incorporated herein by reference in their entirety.

[180] In some embodiments, the methods disclosed herein utilize human umbilical cord blood cells (HUCBC). Human UBC cells are considered a rich source of hematopoietic and mesenchymal progenitor cells (Broxmeyer et al., 1992 Proc. Natl. Acad. Sci. USA 89:4109-4113). Previously, umbilical cord and placental blood were considered byproducts that usually accompanied the birth of a child. Cord blood cells are used as a source of transplantable stem and progenitor cells and as a source of repopulating bone marrow cells for the treatment of malignant diseases (i.e. acute lymphoid 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). An important advantage of HUCBC is the immature immunity of these cells, very similar to fetal cells, which significantly reduces the risk of host rejection (Taylor & Bryson, 1985 J. Immunol. 134:1493-1497). Human cord blood contains mesenchymal and hematopoietic progenitor cells, as well as endothelial progenitor cells, which can be expanded in tissue culture (Broxmeyer et al., 1992 Proc. Natl. Acad. Sci. USA 89:4109-4113; Kohli-Kumar et al., 1992 Proc. Natl. Acad. Sci. USA 89:4109-4113; 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., 1996 Crit. Rev. Oncol. Hematol. 22:61-78 al., 1995 Cell Transplantation 4:493-503; Taylor & Bryson, 1985 J. Immunol. 134:1493-1497 Broxmeyer, 1995 Transfusion 35:694-702; Chenetal., 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 content of hematopoietic progenitor cells in cord blood is equal to or greater than that in bone marrow and, in addition, the number of highly proliferative hematopoietic cells is eight times higher in HUCBC than in bone marrow and they 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).

[181] In another embodiment, pluripotent cells are cells in the hematopoietic microenvironment, such as circulating peripheral blood, preferably from the mononuclear fraction of peripheral blood, umbilical cord blood, bone marrow, fetal liver or mammalian yolk sac. Stem cells, especially neural stem cells, can also be obtained from the central nervous system, including the meninges.

[182] In another embodiment, pluripotent cells present in embryoid bodies are obtained by harvesting ES cells with brief protease treatment and allowing small groups of undifferentiated human ESCs to grow in suspension culture. Differentiation is induced by removing the conditioned medium. The resulting embryoid bodies are plated on semi-solid substrates. The formation of differentiated cells can be observed after a time ranging from about 7 days to about 4 weeks. The selection of viable differentiating cells from in vitro stem cell cultures is carried out by partial dissociation of embryoid bodies or similar structures to obtain cellular aggregates. The selection of aggregates containing cells of interest is carried out on the basis of phenotypic traits, using methods that largely preserve contacts between cells in the aggregate.

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

[184] Cloning and cell culture

[185] Illustrative molecular genetics and genetic engineering techniques that can be used in the methods described herein can be found, for example, in current editions of Molecular Cloning: A Laboratory Manual, (Sambrook et al., Cold Spring Harbor); Gene Transfer Vectors for Mammalian Cells (Miller & Calos eds.); and Current Protocols in Molecular Biology (F.M. Ausubel et al. eds., Wiley & Sons). Materials concerning cell biology, protein chemistry, and antibody-related methods 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 genetic manipulation kits are available commercially from, for example, BioRad, Stratagene, Invitrogen, Clon Tech, and Sigma-Aldrich Co.

[186] A general description of suitable cell culture methods can be found, for example, in the modern publication Culture of Animal Cells: A Manual of Basic Technique (R.I. Freshney ed., Wiley &Sons); General Techniques of Cell Culture (M.A. Harrison & I.F. Rae, Cambridge Univ. Press) and Embryonic Stem Cells: Methods and Protocols (K. Turksen ed., Humana Press). Suitable nutrients and reagents for tissue culture are available, for example, from Gibco/BRL, Nalgene-Nunc Herternational, Sigma Chemical Co. and ICN Biomedicals.

[187] One skilled in the art can grow pluripotent stem cells in culture over a long period of time using culture conditions that stimulate proliferation but do not stimulate differentiation. A typical serum-containing medium for ES cells consists of 80% DMEM (e.g., Knock-Out DMEM, Gibco), 20% chemically defined fetal bovine serum (FB S, Hyclone) serum substituent (WO 98/30679), 1% nonessential amino acids, 1 mM L-glutamine and 0.1 mM β-mercaptoethanol. Immediately before use, human bFGF is added to 4 ng/ml (WO 99/20741, Geron Corp.). Traditionally, ES cells are cultured on a layer of feeder cells, typically fibroblasts derived from embryonic or fetal tissue.

[188] Geron scientists discovered that pluripotent SCs can be maintained in an undifferentiated state even in the absence of nurse cells. Media for nutrient-free cultures includes a suitable culture substrate, namely an extracellular matrix such as Matrigel® or laminin. Typically, enzymatic digestion is stopped until the cells are completely dispersed (eg, this corresponds to about 5 min of collagenase IV treatment). Groups containing from ^~ 10 to 2000 cells are then seeded directly onto the substrate without additional scattering.

[189] Nutrient-free cultures are maintained using a growth medium containing factors that stimulate cell proliferation without differentiation. Such factors can be introduced into the medium by culturing the medium with cells secreting such factors, such as irradiated ( ^~ 4000 rad) primary mouse embryonic fibroblasts, telomerized mouse fibroblasts, or fibroblast-like cells derived from pPS cells. The medium can be conditioned by plating feeders at a density of ^~ 5-6×10 ⁴ cm ^-2 in a serum-free medium such as KO DMEM supplemented with 20% serum substituent and 4 ng/ml bFGF. The medium, conditioned for 1-2 days, is supplemented with additional bFGF and used to maintain the pluripotent SC culture for 1-2 days. Features of the nutrient-free culture method are further discussed in international patent publication WO 01/51616; and in Xu et al., Nat. Biotechnol. 19:971, 2001.

[190] When observed microscopically, ES cells are characterized by a high nuclear-to-cytoplasmic ratio and pronounced nucleoli, as well as the formation of compact colonies with weak intercellular contacts. Primate ES cells express stage-specific embryonic antigens (SSEA) 3 and 4, as well as markers detected by antibodies called 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 obligatorily present in human embryonal carcinoma (hEC) cells. Differentiation of pluripotent SCs in vitro results in a loss of expression of SSEA-4, Tra-1-60, and Tra-1-81 and an increase in the expression of SSEA-1, which can also be detected in undifferentiated hEG cells.

[191] Stem cell-derived β-cell (SC-β)

[192] In some aspects, the invention provides a stem cell-derived β-cell (SC-β). The SC-β cells disclosed herein have many of the hallmarks of β cells but differ from them in certain aspects (eg, gene expression profiles). In some embodiments, the SC-β cell is not native. In the context of this document, “non-native” means that SC-β cells are significantly different in some respects from naturally occurring β cells, i.e. native β cells. It should be understood that these significant differences typically relate to structural features that can result in SC-β cells exhibiting certain functional differences, such as SC-β cells having different gene expression levels from native β-cells. cells, SC-β cells behave in a manner similar to native β-cells, but some functions may be altered (eg, enhanced) compared to native β-cells. For example, as shown in FIG. 2E, the response of SC-β cells to 20 mM glucose occurs at a higher frequency compared to the frequency exhibited by native β-cells. Other differences between SC-β cells and native β cells will become apparent to one skilled in the art based on the data disclosed herein.

[193] The SC-β cells of the invention exhibit many of the characteristic features of β cells that are important for normal β cell function. In some embodiments, an SC-β cell exhibits a glucose-stimulated insulin secretion (GSIS) response in vitro. In some embodiments, the SC-β cell exhibits a GSIS response in vivo. In some embodiments, the SC-β cell exhibits in vitro and in vivo GSIS responses. In some embodiments, the GSIS response is similar to the GSIS response of an endogenous mature pancreatic β cell. In some embodiments, the SC-β cell exhibits a GSIS response to at least one glucose stimulation. In some embodiments, the SC-β cell exhibits a GSIS response to at least two sequential glucose stimulations. In some embodiments, the SC-β cell exhibits a GSIS response to at least three consecutive glucose stimulations. In some embodiments, the GSIS response is similar to the GSIS response of endogenous human islets to repeated glucose stimulation. In some embodiments, the GSIS response is observed immediately after the cell is transplanted into a human or animal body. In some embodiments, the GSIS response is observed within approximately 24 hours after the cell is transplanted into a human or animal body. In some embodiments, the GSIS response is observed within approximately one week after the cell is transplanted into a human or animal body. In some embodiments, the GSIS response is observed within approximately two weeks after the cell is transplanted into a human or animal body. In some embodiments, the stimulation index, characterized by the ratio of insulin secreted in response to high glucose concentrations and low glucose concentrations, is similar to the stimulation index of an endogenous mature pancreatic β-cell. In some embodiments, the SC-β cell exhibits a stimulation index greater than 1. In some embodiments, the SC-β cell exhibits a stimulation index greater than or equal to 1. In some embodiments, the SC-β cell exhibits a stimulation index greater than 1. 1. In some embodiments, the SC-β cell exhibits a stimulation index greater than or equal to 1.1. In some embodiments, the SC-β cell exhibits a stimulation index greater than 2. In some embodiments, the SC-β cell exhibits a stimulation index greater than or equal to 2. In some embodiments, the SC-β cell exhibits a stimulation index of at least at least 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3 ,3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5 , 4.6, 4.7, 4.8, 4.9 or 5.0 or more.

[194] In some embodiments, an SC-β cell exhibits cytokine-induced apoptosis in response to cytokines. In some embodiments, an SC-β cell exhibits 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-γ), α) and their combinations.

[195] In some embodiments, insulin secretion from a SC-β cell is enhanced in response to known antidiabetic drugs (e.g., antidiabetic drugs that target β cells ex vivo or in vitro, and/or antidiabetic drugs in general case in vivo). The present invention provides for any antidiabetic drug. In some embodiments, insulin secretion from an SC-β cell 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.

[196] In some embodiments, the SC-β cell is monohormonal. In some embodiments, the SC-β cell exhibits a morphology that is similar to the morphology of an endogenous mature pancreatic β cell. In some embodiments, crystalline insulin granules are encapsulated within the SC-β cell. In some embodiments, encapsulated crystalline insulin granules are observed in the SC-β cell under an electron microscope, which resemble the insulin granules of an endogenous mature pancreatic β-cell. In some embodiments, the SC-β cell exhibits a low replication coefficient. In some embodiments, the SC-β cell exhibits a low replication coefficient. In some embodiments, the SC-β cell exhibits a low but increased replication rate as measured by C-peptide and Ki67 staining in response to prolactin treatment.

[197] In some embodiments, the SC-β cell increases intracellular Ca ²⁺ in response to glucose. In some embodiments, the SC-β cell exhibits glucose-stimulated Ca ²⁺ flux (GSCF), which is similar to the GSCF of an endogenous mature pancreatic β cell. In some embodiments, the SC-β cell exhibits a GSCF response to at least three consecutive glucose stimulations that is similar to the GSCF response of an endogenous mature pancreatic β cell to multiple glucose stimulations.

[198] In some embodiments, the SC-β cell expresses at least one endogenous mature pancreatic β-cell marker 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.

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

[200] SC-β cells are differentiated in vitro from any starter cell, since the invention is not limited to the type of starter cell from which the SC-β cells are derived. Exemplary starter cells include, but are not limited to, insulin-positive endocrine cells or any of their progenitors, such as Nkx6-1-positive pancreatic progenitor cell, Pdx1-positive pancreatic progenitor cell and pluripotent stem cell, embryonic stem cell and induced pluripotent stem cell . In some embodiments, SC-β cells are differentiated in vitro from a reprogrammed cell, a partially reprogrammed cell (i.e., a somatic cell, e.g., a fibroblast, that has been partially reprogrammed to exist in an intermediate state between an induced pluripotent cell and a somatic cell, from which it was obtained), a transdifferentiated cell. In some embodiments, SC-β cells disclosed herein can be differentiated in vitro from an insulin-positive endocrine cell or a precursor thereof. In some embodiments, the SC-β cell is differentiated in vitro from a progenitor cell selected from the group consisting of an Nkx6-1-positive pancreatic progenitor cell, a Pdx1-positive pancreatic progenitor cell, and a pluripotent stem cell. In some embodiments, the pluripotent stem cell is selected from the group consisting of an embryonic stem cell and an induced pluripotent stem cell. In some embodiments, the SC-β cell or pluripotent stem cell from which the SC-β cell is derived is human. In some embodiments, the SC-β cell is human.

[201] In some embodiments, the SC-β cell is not genetically modified. In some embodiments, in the absence of genetic modification of the cells, the SC-β cell shares characteristics with native β cells. In some embodiments, the SC-b cell is genetically modified.

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

[203] In some embodiments, the amount of insulin produced per SC-β cell is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8 or at least 9 µIU per 1000 cells per 30 minutes of incubation at high glucose concentration. In some embodiments, the amount of insulin produced per SC-β cell is 0.5-10 μIU per 1000 cells per 30 minutes of incubation at high glucose concentration. In some embodiments, the amount of insulin produced per SC-β cell is approximately 2.5 μIU per 1000 cells per 30 minutes of incubation at high glucose concentration.

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

[205] Generation of SC-β cells

[206] Aspects of the invention relate to the generation of SC-β cells (eg, pancreatic β cells). In general, at least one SC-β cell or a progenitor thereof, e.g., pancreatic progenitor cells obtained in accordance with the methods disclosed herein, may comprise a mixture or combination of different cells, e.g., a mixture of cells such as Pdx1- positive pancreatic progenitor cells, pancreatic progenitor cells coexpressing Pdx1 and NKX6-1, Ngn3-positive endocrine progenitor cell, insulin-positive endocrine cell (eg, β-like cell) and insulin-positive endocrine cell and/or other pluripotent or stem cells.

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

[208] In some embodiments, the at least one SC-β cell or precursor thereof belongs to a substantially purified population of SC-β cells or precursors thereof. In some embodiments, the population of SC-β cells or progenitors thereof comprises a mixture of pluripotent cells or differentiated cells. In some embodiments, the population of SC-β cells or their progenitors contains substantially no or no embryonic stem cells or pluripotent cells or iPS cells.

[209] In some embodiments, a somatic cell, such as a fibroblast, can be isolated from a subject, such as a tissue biopsy, such as a skin biopsy, and reprogrammed into an induced pluripotent stem cell 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, a somatic cell, such as a fibroblast, is maintained in culture by methods known to one skilled in the art, and in some embodiments, expanded before being converted into SC-β cells by methods disclosed herein.

[210] In some embodiments, at least one SC-β cell or progenitor thereof is maintained in culture by methods known to one skilled in the art, and in some embodiments, expanded before conversion to SC-β cells by methods disclosed herein.

[211] In addition, the at least one SC-β cell or a precursor thereof, such as a pancreatic progenitor cell, may be from any mammalian species, and non-limiting examples of such cells include murine, bovine, simian, porcine, equine, ovine, or human cell. For convenience and simplicity, the description of the methods described herein relates to at least one SC-β cell or progenitor thereof from a mammal, but it should be understood that all methods described herein can be readily applied to other cell types to which belongs to at least one SC-β cell or its predecessor. In some embodiments, the at least one SC-β cell or precursor thereof is derived from a human.

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

[213] Included in aspects of the invention are definitive endoderm cells. The definitive endoderm cells used herein can be obtained from any source or generated according to any suitable protocol. In some aspects, pluripotent stem cells, such as iPSCs or hESCs, are differentiated into endoderm cells. In some aspects, endoderm cells are further differentiated, for example, into subembryonic cavity cells, Pdx1-positive pancreatic progenitor cells, NKX6-1-positive pancreatic progenitor cells, Ngn3-positive endocrine progenitor cells, or insulin-positive endocrine cells, followed by induction or maturation into SC-β cells.

[214] In some embodiments, the stem cells can be seeded onto a new substrate or the medium can be changed to remove extracellular matrix or soluble factors that inhibit differentiation. This is sometimes referred to as the "direct differentiation method", which is generally described in International Patent Publication WO 01/51616 and US Patent Publication 2002/0019046, both of which are incorporated herein by reference in their entirety. In direct differentiation, it is usually preferable to start with a feeder-free stem cell culture to avoid potential complications in the differentiation process that may be caused by residual feeder cells. Another approach is to place undifferentiated stem cells in a suspension culture, which will result in the continuous formation of aggregates of differentiated and undifferentiated cells. For example, stem cells can be collected by briefly treating with collagenase, separated into clusters, and passaged in culture plates coated with nonadherent cells. The units can be fed every few days and then harvested after an appropriate period of time, usually 4-8 days. Depending on the conditions, aggregates generally begin to form a heterogeneous population of different cell types, including the fairly common endoderm cells. The aggregates can then be dispersed and subcultured onto substrates such as laminin or fibronectin for the next step in the differentiation process; or passage in suspension culture using, for example, non-adhesive plates and suitable media.

[215] During direct differentiation or differentiation in aggregates, the presence of endoderm cells can be monitored using suitable markers, such as those described in US Pat. No. 7,326,572. In some preferred embodiments, the presence of endoderm cells can be monitored during differentiation using markers such as Sox17. Once a sufficient proportion of endoderm cells have been obtained, the cells can be subseeded or otherwise manipulated to begin another stage of differentiation. Under certain conditions, cell differentiation or maintenance can be enhanced if the cells are in microclusters (eg, 50 to 5000 cells). Additional differentiation steps contemplated by the invention are shown in FIG. 1.

[216] In some embodiments, definitive endoderm cells are produced by contacting (eg, culturing) a pluripotent stem cell with a compound of formula (I), as described in US Pat. No. 8,507,274 (the "274th patent"), included in this document by reference. The compound having formula (I) as described in the '274 patent is a small molecule capable of penetrating cells and regulating cellular processes by modulating signal transduction pathways, gene expression or metabolism, and is effectively used in stem cell differentiation protocols. Small molecules can be synthesized in large quantities and with high purity, and can be conveniently delivered or removed, providing them with great potential in therapeutic applications. A tremendous amount of research has been conducted to identify new small molecules that can support self-renewal of ES cells (Chen et al., 2006; Desbordes et al., 2008), cardiogenic-specific murine ES cells (Wu et al., 2004) or neural cells -progenitors (Diamandis et al., 2007), and also induce certain cell types, particularly neuronal and muscle cells (reviewed by Ding and Schultz, 2004). It is expected that the compounds of formula (I) of the '274 patent can be used to differentiate a pluripotent stem cell into a definitive endoderm cell.

[217] In some embodiments, the compound of formula (I) of the '274 patent contains:

[218]

[219]

[220] where:

[221] R ¹ and R ² independently represent H, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cyclyl or cyclyl, each of which may be optionally substituted and/or one or more elements of O may be inserted into its skeleton. N, S, S(O) and C(O);

[222] R ³ and R ⁴ independently represent H, halogen, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, cyclyl or cyclyl, each of which may be optionally substituted, or R ³ and R ⁴ together with a carbon atom, to which they are attached form an optionally substituted cyclyl or heterocyclyl; And

[223] L is C ₁ -C ₁₀ alkylenyl, C ₂ -C ₁₀ alkenylenyl or C ₂ -C ₁₀ alkynylenyl, each of which may be optionally substituted and/or have one or more elements of O inserted into its skeleton N, S, S(O) and C(O).

[224] In some embodiments, the compound of formula (I) of the '274 patent contains the following IDE1:

[225]

[226] In some embodiments, the compound of formula (I) of the '274 patent contains the following IDE2:

[227]

[228] The '274 patent describes methods for confirming the identity of the definitive endoderm cell thus obtained, as well as methods for isolating, storing, propagating, and further differentiating the definitive endoderm, which, as one skilled in the art will understand, can be used in those described herein compositions and methods.

[2-29] 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, for example, by contacting the population of pluripotent cells with i) at least one growth factor from the TGF superfamily -β and ii) an activator of the WNT signaling pathway 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 the definitive endoderm.

[230] The invention provides for the use of any growth factor from the TGF-β superfamily that induces the differentiation of pluripotent stem cells into definitive endoderm cells (eg, alone or in combination with an activator of the WNT signaling pathway). In some embodiments, at least one growth factor from the TGF-β superfamily includes activin A. In some embodiments, at least one growth factor from the TGF-β superfamily includes growth and differentiation factor 8 (GDF8).

[231] The invention contemplates the use of any activator of the WNT signaling pathway that induces differentiation of pluripotent stem cells into definitive endoderm cells (eg, alone or in combination with a growth factor from the TGF-β superfamily). In some embodiments, the WNT signaling pathway activator includes CHIR99021. In some embodiments, the WNT signaling pathway activator comprises recombinant Wnt3a protein.

[232] One skilled in the art will appreciate that the concentrations of agents (eg, growth factors) used may vary. In some embodiments, pluripotent cells are contacted with at least one growth factor from the TGF-β superfamily at a concentration of 10 ng/ml to 1000 ng/ml. In some embodiments, the pluripotent cells are contacted with 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 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 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 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 at least one growth factor from the TGF-β superfamily at a concentration of 101 ng/ml, 102 ng/ml, 103 ng/ml, 104 ng/ml, 105 ng/ml, 106 ng/ml, 107 ng/ml, 108 ng/ml or 109 ng/ml.

[233] In some embodiments, pluripotent cells are contacted with a WNT signaling pathway activator at a concentration of 1.4 μg/ml to 140 μg/ml. In some embodiments, the pluripotent cells are contacted with a WNT signaling pathway activator at a concentration of 14 μg/ml. In some embodiments, the pluripotent cells are contacted with a 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 a 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 a 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, pluripotent cells are contacted with a 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.

[234] In general, the pluripotent cells are maintained in a suitable culture medium (eg, suspension culture) for a period of time sufficient to induce differentiation of at least some of the pluripotent cells into definitive endoderm cells. A typical suitable culture medium is given below in Table 1.

[235]

[236] In some embodiments, a suitable culture medium for differentiating pluripotent cells into definitive endoderm cells includes S1 medium.

[237] In some embodiments, the contacting of pluripotent cells is carried out in a suspension culture. In some embodiments, the suspension culture is maintained in a roller bottle. In some embodiments of the invention, the time period is 3 days. In some embodiments, at least one growth factor from the TGF-β superfamily and a WNT signaling pathway activator are added to the suspension culture on the first day. In some embodiments, on the second day, the suspension culture is replenished with at least one growth factor from the TGF-β superfamily. In some embodiments, the suspension culture is not replenished with WNT signaling pathway activator on the second day. In some embodiments, the WNT signaling pathway activator is removed from the suspension culture on the second day. In some embodiments, at least one growth factor from the TGF-β superfamily is replenished in the suspension culture on the second day, and the WNT signaling pathway activator is removed or not replenished in the suspension culture on the second day. In some embodiments, on the third day, neither at least one growth factor from the TGF-β superfamily nor the WNT signaling pathway activator is replenished in the suspension culture. In some embodiments, on the third day, both at least one growth factor from the TGF-β superfamily and an activator of the WNT signaling pathway are removed from the suspension culture.

[238] Using these methods, at least one pluripotent cell in a population of cells can be induced to differentiate into a definitive endoderm cell. In general, using the method described herein, it is possible to differentiate any pluripotent cell into a definitive endoderm cell. In some embodiments, the pluripotent cells include induced pluripotent cells. In some embodiments, the pluripotent cells include embryonic stem cells. In some embodiments, the pluripotent cells include human cells.

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

[240] Other methods for obtaining definitive endoderm cells are known in the art, including, for example, those described in United States Patent Application US 2006/0003446 to G. Keller, et al.; US 2006/0003313 by K. D'Amour, et al., US 2005/0158853 by K. D'Amour, et al. and US 2005/0260749 by Jon Odorico, et al., the relevant sections of which are incorporated herein by reference.

[241] In some embodiments, a definitive endoderm cell produced by the methods described herein expresses 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, with the definitive endoderm cell having increased expression of at least one marker by a statistically significant amount compared to the pluripotent stem cell from which it was derived. In some embodiments, a definitive endoderm cell produced by the methods described herein does not express in a statistically significant amount at least one marker selected from the group consisting of: Gata4, SPARC, AFP, and Dab2, compared to a pluripotent stem cell. from which it was derived. In some embodiments, a definitive endoderm cell produced by the methods described herein does not express in a statistically significant amount at least one marker selected from the group consisting of: Zicl, Pax6, Flk2, and CD31, compared to a pluripotent stem cell. from which it was derived.

[242] In some embodiments, a definitive endoderm cell produced by methods described herein has a statistically significantly higher level of Smad2 phosphorylation compared to the pluripotent stem cell from which it was derived. In some embodiments, a definitive endoderm cell produced by the methods described herein has the ability to form a gut tube in vivo. In some embodiments, a definitive endoderm cell produced by the methods described herein can differentiate into a cell with the morphology of an intestinal cell, and wherein the cell with the morphology of an intestinal cell expresses FoxA2 and/or Claudin6. In some embodiments, a definitive endoderm cell produced by the methods described herein may further differentiate into a cell of endodermal origin.

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

[244] Induction of differentiation of definitive endoderm cells into cells of the primary intestinal tube

[245] Aspects of the invention include cells of the primary intestinal tube. The primary gut tube cells used herein can be obtained from any source or generated according to any suitable protocol. In some aspects, the cells of the definitive endoderm differentiate into cells of the primary intestinal tube. In some aspects, primary gut tube cells further differentiate, for example, into Pdx1-positive pancreatic progenitor cells, NKX6-1-positive pancreatic progenitor cells, Ngn3-positive endocrine progenitor cells, insulin-positive endocrine cells, followed by induction or maturation into SC-β cells.

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

[247] The invention provides for the use of any growth factor from the FGF family that induces the differentiation of definitive endoderm cells into primary gut tube cells (eg, alone or in combination with other factors). In some embodiments, at least one growth factor from the FGF family includes keratinocyte growth factor (KGF). In some embodiments, at least one growth factor from the FGF family includes FGF2. In some embodiments, at least one growth factor from the FGF family includes FGF8B. In some embodiments, at least one growth factor from the FGF family includes FGF10. In some embodiments, at least one growth factor from the FGF family includes FGF21.

[248] One skilled in the art will appreciate that the concentrations of growth factors used may vary. In some embodiments, definitive endoderm cells are contacted with at least one growth factor from the FGF family at a concentration of 5 ng/ml to 500 ng/ml. In some embodiments, the definitive endoderm cells are contacted with 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 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 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 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, definitive endoderm cells are contacted with at least one growth factor from the FGF family at a concentration of 50 ng/ml.

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

[250] Generally, the definitive endoderm cells are maintained in a suitable culture medium (eg, suspension culture) for a period of time sufficient to induce differentiation of at least some of the definitive endoderm cells into primary gut tube cells. A typical suitable culture medium is given below in Table 2.

[251]

[252] In some embodiments, a suitable culture medium for differentiating definitive endoderm cells into primary gut tube cells includes S2 medium.

[253] In some embodiments of the invention, contacting definitive endoderm cells is carried out in suspension culture. In some embodiments, the suspension culture is maintained in a roller bottle. In some embodiments of the invention, the time period is from 2 days to 5 days. In some embodiments of the invention, the time period is 3 days. In some embodiments, the suspension culture is replenished every other day.

[254] In some embodiments, definitive endoderm cells can be produced by differentiating at least some of the definitive endoderm cells in the population into primary gut tube cells, for example, by contacting the definitive endoderm cells with KGF to induce differentiation of at least some of the cells definitive endoderm into cells of the primary intestinal tube, wherein the cells of the primary intestinal tube express at least one marker characteristic of definitive endoderm.

[255] Induction of differentiation of primary intestinal tube cells into Pdx1-positive pancreatic progenitor cells

[256] Aspects of the invention include Pdx1-positive pancreatic progenitor cells. The Pdx1-positive pancreatic progenitor cells used herein can be obtained from any source or generated according to any suitable protocol. In some aspects, primary gut tube cells differentiate into Pdx1-positive pancreatic progenitor cells. In some aspects, Pdx1-positive pancreatic progenitor cells further differentiate, for example, into NKX6-1-positive pancreatic progenitor cells, Ngn3-positive endocrine progenitor cells, insulin-positive endocrine cells, followed by induction or maturation into SC-β cells.

[257] In some aspects, Pdx1-positive pancreatic progenitor cells can be obtained by differentiating at least some of the cells of the primary intestine in the population into Pdx1-positive pancreatic progenitor cells, for example, by bringing the cells of the primary intestine into contact with i) to 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 cells of the primary gut tube into Pdx1-positive pancreatic progenitor cells, wherein the Pdx1-positive pancreatic progenitor cells express Pdx1.

[258] The invention provides for the use of any inhibitor of the BMP signaling pathway that induces the differentiation of primary gut tube cells into Pdx1-positive pancreatic progenitor cells (for example, alone or in combination with at least one growth factor from the FGF family, at least one an SHH pathway inhibitor, at least one retinoic acid signaling pathway activator, and at least one protein kinase C activator). In some embodiments, the BMP signaling pathway inhibitor includes LDN193189.

[259] The invention provides for the use of any growth factor from the FGF family that induces the differentiation of primary gut tube cells into Pdx1-positive pancreatic progenitor cells (e.g., alone or in combination with at least one BMP signaling pathway inhibitor, at least one an SHH pathway inhibitor, at least one retinoic acid signaling pathway activator, and at least one protein kinase C activator). In some embodiments, at least one growth factor from the FGF family includes keratinocyte growth factor (KGF). In some embodiments, at least one growth factor from the FGF family is selected from the group consisting of FGF2, FGF8B, FGF10 and FGF21.

[260] The invention provides for the use of any SHH pathway inhibitor that induces differentiation of primary gut tube cells into Pdx1-positive pancreatic progenitor cells (e.g., alone or in combination with at least one BMP signaling pathway inhibitor, at least one growth factor from the FGF family, at least one activator of the retinoic acid signaling pathway and at least one activator of protein kinase C). In some embodiments, the SHH pathway inhibitor includes Sant1.

[261] The invention provides for the use of any RA signaling pathway activator that induces the differentiation of primary gut tube cells into Pdx1-positive pancreatic progenitor cells (e.g., alone or in combination with at least one BMP signaling pathway inhibitor, at least one factor growth member of the FGF family, at least one SHH pathway inhibitor and at least one protein kinase C activator). In some embodiments, the RA signaling pathway activator includes retinoic acid.

[262] The invention provides for the use of any protein kinase C (PKC) activator that induces the differentiation of primary gut tube cells into Pdx1-positive pancreatic progenitor cells (e.g., alone or in 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 activator of the retinoic acid signaling pathway). In some embodiments, the PKC activator includes PdbU. In some embodiments, the PKC activator includes TPB.

[263] One skilled in the art will appreciate that the concentrations of agents (eg, growth factors) used may vary. In some embodiments, primary gut tube cells are contacted with a BMP signaling pathway inhibitor at a concentration of 20 nM to 2000 nM. In some embodiments, primary gut tube cells are contacted with a BMP signaling pathway inhibitor at a concentration of 30 nM, 40 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, primary gut tube cells are contacted with a 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, primary gut tube cells are contacted with a 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, primary gut tube cells are contacted with a 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, primary gut cells are contacted with a BMP signaling pathway inhibitor at a concentration of 200 nM.

[264] In some embodiments, primary gut cells are contacted with at least one growth factor from the FGF family at a concentration of 5 ng/ml to 500 ng/ml. In some embodiments, the cells of the primary intestinal tube are contacted with 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 cells of the primary intestinal tube are contacted with 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, primary gut tube cells are contacted with 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, primary gut tube cells are contacted with 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, primary gut cells are contacted with at least one growth factor from the FGF family at a concentration of 50 ng/ml.

[265] In some embodiments, primary gut tube cells are contacted with at least one SHH signaling pathway inhibitor at a concentration of 0.1 μM to 0.5 μM. In some embodiments, primary gut tube cells are contacted with at least one SHH signaling pathway inhibitor at a concentration of 0.11 μM, 0.12 μM, 0.13 μM, 0.14 μM, 0.15 μM, 0. 16 µM, 0.17 µM, 0.18 µM, 0.19 µM, 0.2 µM, 0.21 µM, 0.22 µM, 0.23 µM or 0.24 µM. In some embodiments, primary gut tube cells are contacted with at least one SHH signaling pathway inhibitor at a concentration of 0.26 μM, 0.27 μM, 0.28 μM, 0.29 μM, 0.30 μM, 0. 31 µM, 0.32 µM, 0.33 µM, 0.34 µM, 0.35 µM, 0.36 µM, 0.37 µM, 0.38 µM, 0.39 µM, 0.40 µM, 0, 41 µM, 0.42 µM, 0.43 µM, 0.44 µM, 0.45 µM, 0.46 µM, 0.47 µM, 0.48 µM, 0.49 µM. In some embodiments, primary gut tube cells are contacted with at least one SHH signaling pathway inhibitor at a concentration of 0.25 μM.

[266] In some embodiments, primary gut tube cells are contacted with an RA signaling pathway activator at a concentration of 0.01 μM to 1.0 μM. In some embodiments, primary gut tube cells are contacted with 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, primary gut tube cells are contacted with 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, primary gut tube cells are contacted with a RA signaling pathway activator at a concentration of 0.1 μM.

[267] In some embodiments, primary gut tube cells are contacted with a PKC activator at a concentration of 50 nM to 5000 nM. In some embodiments, primary gut tube cells are contacted with a 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, primary gut tube cells are contacted with a 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, primary gut tube cells are contacted with a 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, primary gut tube cells are contacted with a 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, primary gut tube cells are contacted with a PKC activator at a concentration of 500 nM.

[268] In general, the primary gut tube cells are maintained in a suitable culture medium (eg, suspension culture) for a period of time sufficient to induce the differentiation of at least some of the primary gut tube cells into Pdx1-positive pancreatic progenitor cells. A typical suitable culture medium is given below in Table 3.

[269]

[270] In some embodiments, S3 medium can be used as a suitable culture medium for the differentiation of primary intestinal tube cells into pancreatic progenitor cells.

[271] In some embodiments of the invention, contacting the cells of the primary intestinal tube is carried out in suspension culture. In some embodiments, the suspension culture is maintained in a roller bottle. In some embodiments of the invention, the time period is at least 2 days. In some embodiments of the invention, the suspension culture is replenished every day.

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

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

[274] Aspects of the invention include NKX6-1-positive pancreatic progenitor cells. The NKX6-1 positive pancreatic progenitor cells used herein can be obtained from any source or generated according to any suitable protocol. In some aspects, Pdx1-positive pancreatic progenitor cells differentiate into NKX6-1-positive pancreatic progenitor cells. In some aspects, NKX6-1-positive pancreatic progenitor cells are further differentiated, for example, into Ngn3-positive endocrine progenitor cells or insulin-positive endocrine cells, followed by induction or maturation into SC-β cells.

[275] In some aspects, a method of producing an NKX6-1-positive pancreatic progenitor cell from a Pdx1-positive pancreatic progenitor cell includes bringing a population of cells (eg, under conditions that promote cell clustering) containing Pdx1-positive pancreatic progenitor cells into contact with at least two β-cell maturation factors including a) at least one growth factor from the fibroblast growth factor (FGF) family, b) a sonic hedgehog pathway inhibitor, and optionally c) a low concentration of a retinoic signaling pathway activator acid (RA) over a period of at least five 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 NKX6-1- positive pancreatic progenitor cells express NKX6-1.

[276] In some embodiments, Pdx1-positive, NKX6-1-positive pancreatic progenitor cells are produced by contacting Pdx1-positive pancreatic progenitor cells, under conditions that promote cell clustering, with i) at least one growth factor of the FGF family, ii) at least one SHH pathway inhibitor and, optionally, iii) low concentrations of an RA signaling pathway activator, over a period of five days, to induce the differentiation of at least some Pdx1-positive pancreatic progenitor cells into Pdx1-positive, NKX6-1-positive pancreatic progenitor cells, where Pdx1-positive, NKX6-1-positive pancreatic progenitor cells express Pdx1 and NKX6-1.

[277] In some embodiments, Pdx1-positive, NKX6-1-positive pancreatic progenitor cells are generated by contacting Pdx1-positive pancreatic progenitor cells with i) at least one FGF family growth factor, ii) at least at least one SHH pathway inhibitor; and iii) an RA signaling pathway activator to induce differentiation of at least some 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.

[278] In some embodiments, Pdx1-positive pancreatic progenitor cells are derived from a population of pluripotent cells. In some embodiments, Pdx1-positive pancreatic progenitor cells are derived from the iPS cell population. In some embodiments, Pdx1-positive pancreatic progenitor cells are derived from a population of ESC cells. In some embodiments, the Pdx1-positive pancreatic progenitor cells are derived from a population of definitive endoderm cells. In some embodiments, Pdx1-positive pancreatic progenitor cells are derived from a population of primary gut tube cells.

[279] The invention provides for the use of any growth factor from the FGF family that induces the differentiation of Pdx1-positive pancreatic progenitor cells into NKX6-1-positive pancreatic progenitor cells (for example, alone or in combination with at least one SHH pathway inhibitor or , optionally at least one activator of the retinoic acid signaling pathway). In some embodiments, at least one growth factor from the FGF family includes keratinocyte growth factor (KGF). In some embodiments, at least one growth factor from the FGF family is selected from the group consisting of FGF2, FGF8B, FGF10 and FGF21.

[280] The invention provides for the use of any SHH pathway inhibitor that induces the differentiation of Pdx1-positive pancreatic progenitor cells into NKX6-1 positive pancreatic progenitor cells (for example, alone or in combination with at least one growth factor from the FGF family or at least one activator of the retinoic acid signaling pathway). In some embodiments, the SHH pathway inhibitor includes Sant1.

[281] The invention provides for the use of any RA signaling pathway activator that induces the differentiation of Pdx1-positive pancreatic progenitor cells into NKX6-1 positive pancreatic progenitor cells (for example, alone or in combination with at least one growth factor from the FGF family or at least one SHH pathway inhibitor). In some embodiments, the RA signaling pathway activator includes retinoic acid.

[282] In some embodiments, the method includes contacting a population of cells (eg, Pdx1-positive pancreatic progenitor cells) with at least one additional β-cell maturation factor. In some embodiments, the at least one additional β-cell maturation factor includes at least one growth factor from the EGF family. In some embodiments, the method comprises contacting Pdx1-positive pancreatic progenitor cells with at least one growth factor from the EGF family. The invention provides for the use of any growth factor from the EGF family that facilitates the differentiation of Pdx1-positive pancreatic progenitor cells into NKX6-1-positive pancreatic progenitor cells (for example, together with any combination of at least one growth factor from the FGF family, according to at least one SHH pathway inhibitor and, optionally, at least one RA signaling pathway activator). In some embodiments, at least one growth factor from the EGF family includes betacellulin. In some embodiments, at least one growth factor from the EGF family includes EGF.

[283] One skilled in the art will appreciate that the concentrations of agents (eg, growth factors) used may vary. In some embodiments, Pdx1-positive pancreatic progenitor cells are contacted with at least one growth factor from the FGF family at a concentration of 1 ng/ml to 100 ng/ml. In some embodiments, Pdx1-positive pancreatic progenitor cells are contacted with 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 ml, 30 ng/ml, 35 ng/ml or 40 ng/ml. In some embodiments, Pdx1-positive pancreatic progenitor cells are contacted with 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 ml, 85 ng/ml, 90 ng/ml, 95 ng/ml or 100 ng/ml. In some embodiments, Pdx1-positive pancreatic progenitor cells are contacted with 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 ml, 46 ng/ml, 47 ng/ml, 48 ng/ml or 49 ng/ml. In some embodiments, Pdx1-positive pancreatic progenitor cells are contacted with 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 ml, 56 ng/ml, 57 ng/ml, 58 ng/ml or 59 ng/ml. In some embodiments, Pdx1-positive pancreatic progenitor cells are contacted with at least one growth factor from the FGF family at a concentration of 50 ng/ml.

[284] In some embodiments, Pdx1-positive pancreatic progenitor cells are contacted with at least one SHH pathway inhibitor at a concentration of 0.1 μM to 0.5 μM. In some embodiments, Pdx1-positive pancreatic progenitor cells are contacted with 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, Pdx1-positive pancreatic progenitor cells are contacted with 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, Pdx1-positive pancreatic progenitor cells are contacted with at least one SHH pathway inhibitor at a concentration of 0.25 μM.

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

[286] In some embodiments, Pdx1-positive pancreatic progenitor cells are contacted with at least one growth factor from the EGF family at a concentration of 2 ng/ml to 200 ng/ml. In some embodiments, Pdx1-positive pancreatic progenitor cells are contacted with 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, Pdx1-positive pancreatic progenitor cells are contacted with 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 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 ml. In some embodiments, Pdx1-positive pancreatic progenitor cells are contacted with 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 ml, 26 ng/ml, 27 ng/ml, 28 ng/ml or 29 ng/ml. In some embodiments, Pdx1-positive pancreatic progenitor cells are contacted with at least one growth factor from the EGF family at a concentration of 20 ng/ml.

[287] In general, 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 Pdx1-positive, NKX6-1- positive pancreatic progenitor cells. An exemplary suitable culture medium is listed above in Table 3. In some embodiments, conditions that promote cell clustering include suspension culture. In some embodiments, the suspension culture is maintained in a roller bottle. In some embodiments of the invention, the time period is at least 5 days. In some embodiments, the suspension culture is replenished every other day. In some embodiments, β-cell maturation factors are replenished every other day.

[288] 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 before the 5 day period has elapsed. In some embodiments, the protein kinase C activator includes 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 over a period of less than 5 days. In some embodiments, the BMP signaling pathway inhibitor includes LDN193189.

[289] In some embodiments, at least 10% of 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 Pdx1-positive pancreatic progenitor cells are induced to differentiate into Pdx1-positive, NKX6-1-positive pancreatic progenitor cells.

[290] In general, any Pdx1-positive pancreatic progenitor cell can differentiate into a Pdx1-positive, NKX6-1-positive pancreatic progenitor cell. In some embodiments, NKX6-1-positive pancreatic progenitor cells express Pdx1, NKX6-1, and/or FoxA2.

[291] In some embodiments, Pdx1-positive, NKX6-1-positive pancreatic progenitor cells are generated by contacting Pdx1-positive pancreatic progenitor cells, under conditions that promote cell clustering, with i) at least one growth factor of the FGF family, ii) at least one SHH pathway inhibitor and, optionally, iii) low concentrations of an RA signaling pathway activator, over a period of five days, to induce the differentiation of at least some Pdx1-positive pancreatic progenitor cells into Pdx1-positive, NKX6-1-positive pancreatic progenitor cells, where Pdx1-positive, NKX6-1-positive pancreatic progenitor cells express Pdx1 and NKX6-1.

[292] In some embodiments, NKX6-1-positive pancreatic progenitor cells can be obtained by differentiating at least some Pdx1-positive pancreatic progenitor cells into Pdx1-positive, NKX6-1-positive pancreatic progenitor cells by bringing Pdx1- positive pancreatic progenitor cells under conditions that stimulate cell clustering, in contact 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 every other day for over a period of five days to induce 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.

[293] In some embodiments, 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, for example , 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) an RA signaling pathway activator to induce differentiation at least at least some Pdx1-positive pancreatic progenitor cells in the population are NKX6-1-positive pancreatic progenitor cells, with NKX6-1-positive pancreatic progenitor cells expressing Pdx1 and NKX6-1.

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

[295] Aspects of the invention include insulin-positive endocrine cells. The insulin-positive endocrine cells used herein can be obtained from any source or generated according to any suitable protocol. In some aspects, NKX6-1-positive pancreatic progenitor cells differentiate into insulin-positive endocrine cells. In some aspects, insulin-positive endocrine cells are further differentiated, for example, by induction or maturation into SC-β cells.

[296] In some aspects, a method of producing insulin-positive endocrine cells from NKX6-1-positive pancreatic progenitor cells includes bringing a population of cells (for example, under conditions that promote cell clustering) containing NKX6-1-positive pancreatic progenitor cells, into contact with at least two β-cell maturation factors, including a) an inhibitor of the TGF-β signaling pathway and b) an activator of the thyroid hormone signaling pathway, to induce the differentiation of at least one NKX6-1-positive pancreatic progenitor cell in the population in an insulin-positive endocrine cell, wherein the insulin-positive pancreatic progenitor cell expresses insulin.

[297] The invention provides for the use of any inhibitor of the TGF-β signaling pathway that induces the differentiation of NKX6-1-positive pancreatic progenitor cells into insulin-positive endocrine cells (e.g., alone or in combination with other β-cell maturation factors, e.g. activator of the thyroid hormone signaling pathway). In some embodiments, the TGF-β signaling pathway includes TGF-β receptor type I kinase signaling. In some embodiments, the TGF-β signaling pathway inhibitor includes an Alk5 inhibitor II.

[298] The invention provides for the use of any activator of the thyroid hormone signaling pathway that induces the differentiation of NKX6-1-positive pancreatic progenitor cells into insulin-positive endocrine cells (for example, alone or in combination with other β-cell maturation factors, for example, an inhibitor TGF-β signaling pathway). In some embodiments, the thyroid hormone signaling pathway activator includes triiodothyronine (T3).

[299] In some embodiments, the method includes contacting a population of cells (eg, NKX6-1 positive pancreatic progenitor cells) with at least one additional β-cell maturation factor. In some embodiments, the method comprises contacting 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.

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

[301] In some embodiments, the at least one additional β-cell maturation factor includes at least one growth factor from the EGF family. The invention provides for the use of any growth factor from the EGF family that is capable of inducing the differentiation of NKX6-1-positive pancreatic progenitor cells in the population into insulin-positive endocrine cells (for example, alone or in combination with any component of a TGF-β signaling pathway inhibitor and /or activator of the thyroid hormone signaling pathway). In some embodiments, at least one growth factor from the EGF family includes betacellulin. In some embodiments, at least one growth factor from the EGF family includes EGF.

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

[303] In some embodiments, the at least one additional β-cell maturation factor comprises a sonic hedgehog (SHH) pathway inhibitor. The invention provides for the use of any SHH pathway inhibitor that induces the differentiation of NKX6-1-positive pancreatic progenitor cells into insulin-positive endocrine cells (for example, alone or in combination with any component of a TGF-β signaling pathway inhibitor and/or a signaling pathway activator thyroid hormone). In some embodiments, the SHH pathway inhibitor includes Sant1.

[304] In some embodiments, a population of cells (eg, NKX6-1 positive pancreatic progenitor cells) is treated with glucose.

[305] In some embodiments, the population of cells is optionally contacted with a protein kinase inhibitor. In some embodiments, the population of cells is not brought into contact with the protein kinase inhibitor. In some embodiments, a population of cells is contacted with a protein kinase inhibitor. The invention provides for the use of any protein kinase inhibitor that is capable of inducing the differentiation of NKX6-1-positive pancreatic progenitor cells in the population into insulin-positive endocrine cells (for example, alone or in combination with any component of a TGF-β signaling pathway inhibitor and/or activator thyroid hormone signaling pathway). In some embodiments, the protein kinase inhibitor includes staurosporine.

[306] In some embodiments, insulin-positive endocrine cells are produced by contacting 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, iv) an inhibitor of the TGF-β signaling pathway, v) an activator of the TH signaling pathway, and vi) at least one growth factor from the epidermal growth factor (EGF) family to induce the differentiation of at least some Pdx1-positive, NKX6-1-positive pancreatic progenitor cells into Pdx1-positive, NKX6-1, insulin-positive endocrine cells, while Pdx1-positive, NKX6-1, insulin-positive endocrine cells express Pdx1, NKX6-1, NKX2-2, Mafb, glis3, Sur1, Kir6.2, Znt8, SLC2A1, SLC2A3 and/or insulin.

[307] One skilled in the art will appreciate that the concentrations of agents (eg, growth factors) used may vary.

[308] In some embodiments, NKX6-1-positive pancreatic progenitor cells are contacted with at least one TGF-β signaling pathway inhibitor at a concentration of 100 nM to 100 μM. In some embodiments, NKX6-1-positive pancreatic progenitor cells are contacted with at least one TGF-β signaling pathway inhibitor at a concentration of 10 μM. In some embodiments, NKX6-1-positive pancreatic progenitor cells are contacted with at least one TGF-β signaling pathway inhibitor at a concentration of 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM , 800 nM or 900 nM. In some embodiments, NKX6-1-positive pancreatic progenitor cells are contacted with 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, NKX6-1-positive pancreatic progenitor cells are contacted with 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, NKX6-1-positive pancreatic progenitor cells are contacted with 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, NKX6-1-positive pancreatic progenitor cells are contacted with 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.

[309] In some embodiments, NKX6-1-positive pancreatic progenitor cells are contacted with a thyroid hormone signaling pathway activator at a concentration of 0.1 μM to 10 μM. In some embodiments, NKX6-1-positive pancreatic progenitor cells are contacted with a thyroid hormone signaling pathway activator at a concentration of 1 μM. In some embodiments, NKX6-1-positive pancreatic progenitor cells are contacted with a 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, NKX6-1-positive pancreatic progenitor cells are contacted with a 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, NKX6-1-positive pancreatic progenitor cells are contacted with a 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.

[310] In some embodiments, NKX6-1-positive pancreatic progenitor cells are contacted with a γ-secretase inhibitor at a concentration of 0.1 μM to 10 μM. In some embodiments, NKX6-1-positive pancreatic progenitor cells are contacted with a γ-secretase inhibitor at a concentration of 1 μM. In some embodiments, NKX6-1-positive pancreatic progenitor cells are contacted with a γ-secretase inhibitor at a concentration of 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0 .7 µM, 0.8 µM or 0.9 µM. In some embodiments, NKX6-1-positive pancreatic progenitor cells are contacted with a γ-secretase inhibitor at a concentration of 1.1 μM, 1.2 μM, 1.3 μM, 1.4 μM, 1.5 μM, 1 .6 µM, 1.7 µM, 1.8 µM or 1.9 µM. In some embodiments, NKX6-1-positive pancreatic progenitor cells are contacted with a γ-secretase inhibitor at a concentration of 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, or 9 μM.

[311] In some embodiments, NKX6-1-positive pancreatic progenitor cells are contacted with at least one growth factor from the EGF family at a concentration of 2 ng/ml to 200 ng/ml. In some embodiments, NKX6-1-positive pancreatic progenitor cells are contacted with 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, NKX6-1-positive pancreatic progenitor cells are contacted with 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, NKX6-1-positive pancreatic progenitor cells are contacted with 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, NKX6-1-positive pancreatic progenitor cells are contacted with at least one growth factor from the EGF family at a concentration of 20 ng/ml.

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

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

[314] In some embodiments, NKX6-1-positive pancreatic progenitor cells are contacted with at least one SHH pathway inhibitor at a concentration of 0.1 μM to 0.5 μM. In some embodiments, NKX6-1-positive pancreatic progenitor cells are contacted with 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, NKX6-1-positive pancreatic progenitor cells are contacted with 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, NKX6-1-positive pancreatic progenitor cells are contacted with at least one SHH pathway inhibitor at a concentration of 0.5 μM.

[315] In some embodiments, NKX6-1-positive pancreatic progenitor cells are contacted with a protein kinase inhibitor at a concentration of 10 nM to 1 μM. In some embodiments, NKX6-1-positive pancreatic progenitor cells are contacted with a protein kinase inhibitor at a concentration of 100 nM. In some embodiments, NKX6-1-positive pancreatic progenitor cells are contacted with a 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, NKX6-1-positive pancreatic progenitor cells are contacted with a 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, NKX6-1-positive pancreatic progenitor cells are contacted with a protein kinase inhibitor at a concentration of 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, or 900 nM.

[316] In some embodiments, NKX6-1-positive pancreatic progenitor cells are contacted with glucose at a concentration of 1 mM to 50 mM. In some embodiments, NKX6-1-positive pancreatic progenitor cells are contacted with glucose at a concentration of 25 mM.

[317] In some embodiments, insulin-positive endocrine cells can be obtained by differentiating at least some Pdx1-positive, NKX6-1-positive pancreatic progenitor cells into Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells by bringing Pdx1-positive, NKX6-1-positive pancreatic progenitor cells, under conditions that promote cell clustering, into contact with i) an inhibitor of the TGF-β signaling pathway, b) an activator of the TH signaling pathway, 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, every other day for a period of five to seven days, to induce the differentiation of at least some Pdx1-positive, NKX6-1-positive pancreatic progenitor cells into Pdx1-positive, NKX6-1, insulin-positive endocrine cells, wherein the Pdx1-positive, NKX6-1, insulin-positive endocrine cells express Pdx1, NKX6-1, NKX2-2, Mafb, glis3, Sur1, Kir6.2, Znt8, SLC2A1, SLC2A3 and/or insulin.

[318] In some embodiments, insulin-positive endocrine cells can be obtained by differentiating at least some 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 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) inhibitor TGF-β signaling pathway, v) an activator of the TH signaling pathway, and vi) at least one growth factor from the epidermal growth factor (EGF) family to induce the differentiation of at least some Pdx1-positive, NKX6-1-positive pancreatic progenitor cells c Pdx1-positive, NKX6-1, insulin-positive endocrine cells, while Pdx1-positive, NKX6-1, insulin-positive endocrine cells express Pdx1, NKX6-1, NKX2-2, Mafb, glis3, Sur1, Kir6.2 , Znt8, SLC2A1, SLC2A3 and/or insulin.

[319] Generally, a population of cells is maintained in a suitable culture medium for a period of time sufficient to induce differentiation of at least one NKX6-1 positive pancreatic progenitor cell in the population into an insulin-positive endocrine cell. A typical suitable culture medium is given in Table 4.

[320]

[321] 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 listed in Table 5.

[322] In some embodiments, conditions that promote cell clustering include suspension culture. In some embodiments, the time period corresponds to a period of time sufficient to maximize the number of cells coexpressing C-peptide and Nkx6-1. In some embodiments of the invention, the time period is at least 5 days. In some embodiments of the invention, the time period is from 5 days to 7 days. In some embodiments of the invention, the specified period of time is at least 7 days. In some embodiments, the suspension culture is replenished daily (e.g., with maturation factors (3-cells). In some embodiments, a period of 5 days to 7 days maximizes the number of cells coexpressing C-peptide and Nkx6-1 .

[323] 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.

[324] 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.

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

[326] Aspects of the invention include SC-β cells. The SC-β cells used herein can be obtained from any source or generated according to any suitable protocol. In some aspects, insulin-positive endocrine cells are induced to mature into SC-β cells.

[327] In some aspects, the invention provides a method for generating mature glucose-dependent β-cells from insulin-positive endocrine cells, comprising bringing a population of cells (eg, under conditions that promote cell clustering) containing insulin-positive endocrine cells into contact with at least with at least two β-cell maturation factors, including a) an inhibitor of the transforming growth factor β (TGF-β) signaling pathway, b) an activator of the thyroid hormone (TH) signaling pathway, to induce in vitro maturation of at least one insulin-positive endocrine cell in the SC-β cell population.

[328] Aspects of the invention include the generation of SC-β cells that are similar in form and functionality to endogenous mature β cells, but differ from native β cells. SC-β cells may demonstrate a response to at least one glucose stimulation. In some embodiments, SC-β cells exhibit a response to at least two sequential glucose stimulations. In some embodiments, SC-β cells exhibit a response to at least three sequential glucose stimulations. In some embodiments, SC-β cells exhibit a response to repeated (eg, sequential) glucose stimulation that is similar to the response of endogenous human islets to repeated glucose stimulation. In some embodiments, SC-β cells are capable of releasing or secreting insulin in response to two sequential glucose stimulations. In some embodiments, SC-β cells are capable of releasing or secreting insulin in response to three consecutive glucose stimulations. In some embodiments, SC-β cells are capable of releasing or secreting insulin in response to four consecutive glucose stimulations. In some embodiments, SC-β cells are capable of releasing or secreting insulin in response to five consecutive glucose stimulations. In some embodiments, SC-β cells release or secrete insulin in response to chronic sequential glucose stimulation. In some embodiments, cells can be analyzed to determine whether they respond to sequential glucose stimulation by determining whether there is a systematic increase in intracellular Ca ²⁺ , as described in the Examples provided herein.

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

[330] In some embodiments, SC-β cells exhibit at least one marker of mature endogenous pancreatic β cells. Exemplary markers include, but are not limited to, Pdx1, HNF6, Ptfla, Sox9, FoxA2, Nkx2.2, Ngn3, and NKX6-1. In some embodiments, SC-β cells exhibit up-regulation of expression of a marker selected from the group consisting of HNF6, Ptfla, Sox9, FoxA2, Nkx2.2, Ngn3, and NKX6-1 by a statistically significant amount compared to pluripotent stem cells (eg, embryonic stem cells or induced pluripotent cells) from which SC-β cells were derived.

[331] The invention provides for the use of any inhibitor of the TGF-β signaling pathway that induces the differentiation and/or maturation of insulin-positive endocrine cells into SC-β cells (for example, alone or in any combination with at least one activator of the thyroid hormone signaling pathway (TH) or, optionally, a protein kinase inhibitor). In some embodiments, the TGF-β signaling pathway includes TGF-β receptor type I kinase signaling. In some embodiments, the TGF-β signaling pathway inhibitor includes an Alk5 inhibitor II.

[332] The invention provides for the use of any thyroid hormone signaling pathway activator that induces differentiation and/or maturation of insulin-positive endocrine cells into SC-β cells (e.g., alone or in any combination with at least one TGF-β signaling pathway inhibitor or optionally a protein kinase inhibitor). In some embodiments, the thyroid hormone signaling pathway activator includes T3.

[333] 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 brought into contact with the protein kinase inhibitor. In some embodiments, Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with a protein kinase inhibitor. The invention provides for the use of any protein kinase inhibitor that induces the differentiation and/or maturation of insulin-positive endocrine cells into SC-β cells (for example, alone or in any combination with at least one TGF-β signaling pathway inhibitor and/or a signaling pathway activator thyroid hormone). In some embodiments, the protein kinase inhibitor includes staurosporine.

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

[335] In some embodiments, the at least one additional β-cell maturation factor comprises a cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor. In some embodiments, the method comprises contacting Pdx1-positive, NKX6-1-positive, insulin-positive cells with a CFTR inhibitor. The invention provides for the use of any CFTR inhibitor that induces the differentiation and/or maturation of insulin-positive endocrine cells into SC-β cells (for example, alone or in any combination with at least one TGF-β signaling pathway inhibitor and/or a signaling pathway activator thyroid hormone, and optionally a protein kinase inhibitor). In some embodiments, the CFTR inhibitor includes Gly-H101.

[336] In some embodiments, the at least one additional β-cell maturation factor comprises an O-GlcNAase inhibitor. In some embodiments, the method comprises contacting Pdx1-positive, NKX6-1-positive, insulin-positive cells with an O-GlcNAase inhibitor. The invention provides for the use of any O-GlcNAase inhibitor that induces differentiation and/or maturation of insulin-positive endocrine cells into SC-β cells (for example, alone or in any combination with at least one TGF-β signaling pathway inhibitor and/or activator thyroid hormone signaling pathway, and optionally a protein kinase inhibitor). In some embodiments, the O-GlcNAase inhibitor includes Thiamet-G.

[337] One skilled in the art will appreciate that the concentrations of agents (eg, growth factors) used may vary. In some embodiments, insulin-positive endocrine cells are contacted with at least one TGF-β signaling pathway inhibitor at a concentration of 100 nM to 100 μM. In some embodiments, insulin-positive endocrine cells are contacted with at least one TGF-β signaling pathway inhibitor at a concentration of 10 μM. In some embodiments, insulin-positive endocrine cells are contacted with 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, insulin-positive endocrine cells are contacted with at least one TGF-β signaling 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, insulin-positive endocrine cells are contacted with 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, insulin-positive endocrine cells are contacted with 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, insulin-positive endocrine cells are contacted with 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.

[338] In some embodiments, insulin-positive endocrine cells are contacted with a thyroid hormone signaling pathway activator at a concentration of 0.1 μM to 10 μM. In some embodiments, insulin-positive endocrine cells are contacted with a thyroid hormone signaling pathway activator at a concentration of 1 μM. In some embodiments, insulin-positive endocrine cells are contacted with a 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, insulin-positive endocrine cells are contacted with a 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, insulin-positive endocrine cells are contacted with a 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.

[339] In some embodiments, insulin-positive endocrine cells are contacted with a protein kinase inhibitor at a concentration of 10 nM to 1 μM. In some embodiments, insulin-positive endocrine cells are contacted with a protein kinase inhibitor at a concentration of 100 nM. In some embodiments, insulin-positive endocrine cells are contacted with a 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, insulin-positive endocrine cells are contacted with a 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, insulin-positive endocrine cells are contacted with a protein kinase inhibitor at a concentration of 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, or 900 nM.

[340] In some embodiments, Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with a CFTR inhibitor at a concentration of 100 nM - 100 μM. In some embodiments, Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with a CFTR inhibitor at a concentration of 10 nM to 10 μM.

[341] In some embodiments, Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with an O-GlcNAase inhibitor at a concentration of 100 nM to 100 μM. In some embodiments, Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells are contacted with an O-GlcNAase inhibitor at a concentration of 10 nM to 10 μM.

[342] In some embodiments, an SC-β cell can be obtained by differentiating at least some Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells into SC-β cells by bringing Pdx1-positive, NKX6-1- positive, insulin-positive endocrine cells under conditions that stimulate cell clustering, in contact with i) an inhibitor of the transforming growth factor-β (TGF-β) signaling pathway, ii) an activator of the thyroid hormone signaling pathway and, optionally, iii) a protein kinase inhibitor, every other day for a period of seven to 14 days to induce in vitro maturation of at least some Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells into SC-β cells, wherein the SC-β cells exhibit GSIS response in vitro and/or in vivo. In some embodiments, the GSIS response is similar to the GSIS response of an endogenous β cell.

[343] In some embodiments, an SC-β cell 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, for example, by making Pdx1-positive , NKX6-1-positive, insulin-positive endocrine cells into contact with i) an inhibitor of the transforming growth factor-β (TGF-β) signaling pathway, ii) an activator of the thyroid hormone signaling pathway and, optionally, iii) a protein kinase inhibitor to induce in in vitro maturation of at least some Pdx1-positive, NKX6-1-positive, insulin-producing endocrine cells into SC-β cells, wherein the SC-β cells exhibit an in vitro and/or in vivo GSIS response that is similar to the GSIS response endogenous β-cell.

[344] In some aspects, the invention provides a method of producing SC-β cells, comprising: bringing Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells into contact under conditions that stimulate cell clustering with i) an inhibitor of the transforming signaling pathway growth factor β (TGF-β), ii) an activator of the thyroid hormone signaling pathway and, optionally, iii) a protein kinase inhibitor to induce in vitro maturation of at least one of Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells into SC-β cells, with SC-β cells demonstrating a GSIS response in vitro and/or in vivo. In some embodiments, the GSIS response is similar to the GSIS response of an endogenous β cell.

[345] In some aspects, the invention provides a method for generating SC-β cells from pluripotent cells, comprising: a) differentiating the pluripotent cells in a population 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 bringing the Pdx1-positive pancreatic progenitor cells, under conditions that stimulate cell clustering, into contact 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, every other day for a period of five days to induce differentiation of at least some Pdx1- positive pancreatic progenitor cells in a population of NKX6-1-positive pancreatic progenitor cells, wherein the NKX6-1-positive pancreatic progenitor cells express Pdx1 and NKX6-1; c) differentiating at least some Pdx1-positive, NKX6-1-positive pancreatic progenitor cells into Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells by bringing Pdx1-positive, NKX6-1-positive pancreatic cells- progenitors under conditions that stimulate cell clustering, in contact with i) an inhibitor of the TGF-β signaling pathway, b) an activator of the TH signaling pathway, and optionally c) at least one inhibitor of the SHH pathway, ii) an activator of the RA signaling pathway, iii) an inhibitor γ-secretase and vi) at least one growth factor from the epidermal growth factor (EGF) family, every other day for a period of five to seven days to induce differentiation of at least some Pdx1-positive, NKX6-1-positive pancreatic progenitor cells into Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells, while 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; and d) differentiating at least some of the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells into SC-β cells by exposing the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells to conditions that stimulate cell clustering, in contact with i) a transforming growth factor-β (TGF-β) signaling pathway inhibitor, ii) a thyroid hormone signaling pathway activator, and optionally iii) a protein kinase inhibitor, every other day for a period of seven to 14 days to induce in vitro maturation of at least some 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 the GSIS response of endogenous mature β cells.

[346] In some aspects, the invention provides a method for generating SC-β cells from pluripotent cells, comprising: a) differentiating at least some of the pluripotent cells in the population 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 bringing the Pdx1-positive pancreatic progenitor cells, under conditions that stimulate cell clustering, into contact with i) KGF, ii) Sant1 and, optionally, iii) low concentrations of RA, every other day for a period of time of five days, to induce the differentiation of at least one Pdx1-positive pancreatic progenitor cell in the population into NKX6-1-positive pancreatic cells -progenitors, wherein NKX6-1-positive pancreatic progenitor cells express Pdx1 and NKX6-1; c) differentiating at least some Pdx1-positive, NKX6-1-positive pancreatic progenitor cells into Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells by bringing Pdx1-positive, NKX6-1-positive pancreatic cells- progenitors under conditions that stimulate cell clustering, in contact with i) Alk5 inhibitor II, ii) T3 and, optionally, iii) Sant1, iv) RA, v) XXI and vi) betacellulin, every other day for a period of time ranging from five to seven days to induce the differentiation of at least some Pdx1-positive, NKX6-1-positive pancreatic progenitor cells into Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells, wherein 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; and d) differentiating at least some of the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells into SC-β cells by exposing the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells to conditions that stimulate clustering of cells, in contact with i) Alk5 inhibitor II, ii) T3 and, optionally, iii) staurosporine, every other day for a period of seven to 14 days to induce in vitro maturation of at least some Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells into SC-β cells, with SC-β cells exhibiting in vitro and in vivo GSIS responses that bear similarities to the endogenous β-cell GSIS response.

[347] In general, 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 endocrine cells into SC-β cells. A typical suitable culture medium is given above in Table 4 and below in Table 5.

[348]

[349] In some embodiments, a suitable culture medium includes Supplemented Islet Medium 1066 from Connought Medical Research Laboratories (CMRLS). In some embodiments, a suitable culture medium includes a CMRLS component (eg, zinc support). In some embodiments, a suitable culture medium is listed in Table 3. In some embodiments, the CMRLS is supplemented with serum (eg, human). In some embodiments, the CMRLS is supplemented with serum substituents (eg, 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, a suitable culture medium for differentiating insulin-positive endocrine cells into SC-β cells includes S3 medium. In some embodiments, conditions that promote cell clustering include suspension culture. In some embodiments of the invention, the time period is at least 7 days. In some embodiments of the invention, the time period is from 7 days to 21 days. In some embodiments of the invention, the time period is from 7 to 14 days. In some embodiments of the invention, the time period is from 10 to 14 days. In some embodiments of the invention, the time period is 14 days. In some embodiments, the suspension culture is replenished every other day (eg, with β-cell maturation factors).

[350] In some embodiments, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50% Pdx1-positive, NKX6-1-positive , insulin-positive endocrine cells are induced to mature into SC-β cells. In some embodiments, at least at least 60%, at least 70%, at least 80%, at least 90%, at least 99% 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 resulting cells contain SC-β cells. In some embodiments, SC-β cells express C-peptide, insulin, NKX6-1, Pdx1, and co-express NKX6-1 and C-peptide.

[351] In some embodiments, SC-β cells include human cells. In some embodiments, in vitro generation of SC-β cells is scalable.

[352] Isolated cell populations

[353] Aspects of the invention relate to isolated populations of cells obtained in accordance with the methods described herein. In some embodiments, a population of SC-β cells is generated by contacting at least one insulin-positive endocrine cell or precursor thereof with at least one β-cell maturation factor described herein. In some embodiments, a population of SC-β cells is generated 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, a population of SC-β cells is generated 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, a population of SC-β cells is generated 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, a population of SC-β cells is generated 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, a population of SC-β cells is generated by bringing at least one insulin-positive endocrine cell or precursor cell into contact with at least seven, at least eight, at least nine, or at least ten β-cell maturation factors described herein.

[354] In some aspects, the invention provides a selected population of definitive endoderm cells. An 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, for example, by contacting the population of pluripotent cells with i) at least one growth factor from the TGF-β superfamily and ii) a signaling activator Wnt pathway 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 the definitive endoderm.

[355] In some aspects, the invention provides a selected population of cells of the primary intestinal tube. A selected population of primary gut tube cells can be obtained by differentiating at least some of the definitive endoderm cells in the population into primary gut tube cells, for example, 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 primary gut tube cells, wherein the primary gut tube cells express at least one marker characteristic of the definitive endoderm.

[356] In some aspects, the invention provides a selected population of Pdx1-positive pancreatic progenitor cells. A selected population of Pdx1-positive pancreatic progenitor cells can be obtained by differentiating at least some of the primordial cells in the population into Pdx1-positive pancreatic progenitor cells, for example, by contacting the primordial cells with i) at least one inhibitor bone morphogenetic protein (BMP) signaling pathway, 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 at least one protein kinase C activator to induce differentiation of at least some cells of the primary gut tube into Pdx1-positive pancreatic progenitor cells, wherein the Pdx1-positive pancreatic progenitor cells express Pdx1.

[357] In some aspects, the invention provides a selected population of NKX6-1 positive pancreatic progenitor cells. A selected 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, e.g. pancreatic progenitor cells in contact with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor and, optionally, iii) an activator of the RA signaling pathway to induce differentiation of at least one Pdx1-positive pancreatic progenitor cells in a population of NKX6-1-positive pancreatic progenitor cells, wherein NKX6-1-positive pancreatic progenitor cells express Pdx1 and NKX6-1.

[358] In some aspects, the invention provides a selected population of insulin-positive endocrine cells. A selected 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, e.g. contacting Pdx1-positive, NKX6-1-positive pancreatic progenitor cells with i) an inhibitor of the TGF-β signaling pathway, ii) an activator of the TH signaling pathway, 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 to induce differentiation at least some Pdx1-positive, NKX6-1-positive pancreatic progenitor cells into Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells, wherein Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells cells express Pdx1, NKX6-1, NKX2-2, Mafb, glis3, Sur1, Kir6.2, Znt8, SLC2A1, SLC2A3 and/or insulin.

[359] In some aspects, the invention provides an isolated population of SC-β cells. An isolated population of SC-β cells can be obtained by differentiating at least some of the Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells in the population into SC-β cells, for example, by converting Pdx1-positive, NKX6-1-positive , insulin-positive endocrine cells into contact with i) an inhibitor of the transforming growth factor-β (TGF-β) signaling pathway, ii) an activator of the thyroid hormone signaling pathway, and optionally iii) a protein kinase inhibitor to induce in vitro maturation of at least some Pdx1-positive, NKX6-1-positive, insulin-positive endocrine cells into SC-β cells, with SC-β cells demonstrating a GSIS response in vitro and/or in vivo. In some embodiments, the GSIS response is similar to the GSIS response of an endogenous β cell.

[360] Aspects of the invention include microcapsules containing isolated populations of cells described herein (eg, SC-β cells). Microcapsules are well known in the art. Suitable examples of microcapsules are described in the literature (eg, 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., "The development of encapsulated cell technologies as therapies for neurological and sensory diseases", Journal of Controlled Release 2012; 160: 3-13). Microcapsules can be prepared in a variety of ways. Typical microcapsules contain an alginate core surrounded by a polycationic layer covered by an outer alginate membrane. The polycationic membrane forms a semi-permeable membrane that ensures stability and biocompatibility. Examples of polycations include, but are not limited to, poly-L-lysine, poly-L-ornithine, chitosan, lactose-modified chitosan, and photopolymerized biomaterials. In some embodiments, the alginate core is modified, for example, to provide a core comprising an alginate core containing covalently conjugated oligopeptides with an RGD (arginine, glycine, aspartic acid) sequence. In some embodiments, the alginate core is modified, for example, to provide a covalently reinforced microcapsule containing a chemoenzymatically engineered alginate with increased stability. In some embodiments, the alginate core is modified, for example, to provide membrane-mimetic films formed by in-situ polymerization of acrylate-functionalized phospholipids. In some embodiments, the microcapsules consist of alginates enzymatically modified with epimerases. In some embodiments, the microcapsules contain covalent bonds between adjacent layers of the microcapsule membrane. In one embodiment of the invention, the microcapsule comprises a below-screen size capsule containing alginate conjugated with phenolic components. In some embodiments, the microcapsule contains a core containing alginate-agarose. In some embodiments, the SC-β cell is modified with PEG prior to encapsulation in alginate. In some embodiments, isolated cell populations, such as SC-β cells, are encapsulated in photoreactive liposomes and alginate. It should be understood that the alginate used in the microcapsules can be replaced by other suitable biomaterials, including, without limitation, PEG, chitosan, PES hollow fibers, collagen, hyaluronic acid, dextran with RGD, EHD and PEGDA, PMBV and PVA, PGSAS, agarose, agarose with gelatin, PLGA and multilayer embodiments thereof.

[361] In some embodiments, compositions containing populations of cells obtained in accordance with the methods described herein can also be used as a functional component in a mechanical device designed to produce one or more endocrine islet cell polypeptides. The simplest form of this device contains a population of pancreatic beta cells (for example, derived from a population of insulin-positive endocrine cells or their precursors) bounded by a semi-permeable membrane that prevents the passage of the population of cells, retaining them within the device, but does not interfere with the passage of insulin, glucagon or somatostatin secreted by a population of cells. The above involves a microencapsulated population of beta cells, typically in the form of cell clusters, to allow interactions that inhibit differentiation. For example, US patent No. 4391909 describes islet cells encapsulated in a spherical semi-permeable membrane consisting of polysaccharide polymers with a molecular weight. weighing >3000, which are cross-linked in such a way that the membrane is permeable to proteins the size of insulin, but impermeable to molecules of mol. weighing more than 100,000. US Patent No. 6,023,009 describes islet cells encapsulated in a semipermeable membrane consisting of agarose and agaropectin. Microcapsules of this origin are intended for administration into the abdominal cavity of a diabetic patient and are believed to have certain advantages related to the reduction of histocompatibility problems or susceptibility to bacteria.

[362] Other devices have also been developed containing a population of pancreatic beta cells derived from insulin-positive endocrine cells or their precursors in accordance with the methods described herein, intended for both implantation in patients with diabetes and for extracorporeal therapy. US Pat. No. 4,378,016 describes an artificial endocrine gland comprising an extracorporeal segment, a subcutaneous segment, and a replaceable capsule containing hormone-producing cells. US Pat. No. 5,674,289 describes a biosynthetic pancreas containing an islet cavity separated by a semipermeable membrane from one or more vascular cavities with access to surrounding tissue. The devices used typically comprise a cavity adapted to contain islet cells and a cavity separated from the islet cells by a semi-permeable membrane that collects proteins secreted from the islet cells and which can also transmit a feedback signal to the islet cells, for example regarding circulating glucose levels.

[363] Aspects of the invention include assay methods associated with isolated populations of cells described herein (eg, SC-β cells). In some embodiments, these assays can be used to identify one or more candidate agents that promote or inhibit β cell fate selected from the group consisting of β cell proliferation, β cell replication, and β cell death, β function -cells, β-cell susceptibility to immune system attack, or β-cell susceptibility to dedifferentiation or differentiation. In some embodiments, these assays can be used to identify one or more candidate agents that stimulate the differentiation of at least one insulin-positive endocrine cell or its precursor into SC-β cells. In some embodiments, these assays can be used to identify one or more candidate agents that stimulate β-cell insulin production or increase insulin production or secretion.

[364] The invention provides methods in which SC-β cells are obtained in accordance with the methods described herein from iPS cells obtained from cells extracted or isolated from the body of individuals suffering from a disease (for example, diabetes, obesity or disorder, β-cell-associated) and these SC-β cells are compared with normal β-cells from healthy individuals who do not have disease to identify differences between SC-β cells and normal β-cells that may serve as markers of disease (eg , epigenetic and/or genetic). In some embodiments, β cells are obtained from an individual with diabetes and compared to normal β cells, and the β cells are then reprogrammed into iPS cells and the iPS cells are analyzed for epigenetic and/or genetic markers that are present in the β cells , obtained from an individual with diabetes, but are not present in normal β-cells to detect these markers (eg, prediabetic). In some embodiments, iPS and/or SC-β cells obtained from diabetic patients are used to screen for agents (eg, agents that are capable of modulating genes responsible for the diabetic phenotype).

[365] Methods for differentiating insulin-positive endocrine cells into SC-β cells

[366] Generation of SC-β cells by conversion of at least one insulin-positive endocrine cell or its precursor using the methods of the invention has many advantages. First, the methods of the invention make it possible to generate autologous SC-β cells that are specific and genetically matched to an individual. In general, autologous cells are less likely than non-autologous cells to be subject to immunological rejection. The cells are derived from at least one insulin-positive endocrine cell or a precursor thereof, e.g., a pancreatic progenitor cell, obtained by reprogramming a somatic cell (e.g., fibroblast) from an individual into an induced pluripotent state and then culturing the pluripotent cells to differentiate at least some pluripotent cells into at least one insulin-positive endocrine cell or precursor thereof, followed by transplantation of the at least one insulin-positive endocrine cell or precursor thereof into the individual so that the at least one insulin-positive endocrine cell or precursor thereof matures in vivo into an SC-β cell, or by inducing in vitro maturation of at least one insulin-positive endocrine cell or its precursor into an SC-β cell.

[367] In some embodiments, the subject from whose body the at least one insulin-positive endocrine cell or precursor thereof is obtained is a mammalian subject, such as a human subject. In some embodiments, the subject suffers from a β-cell disorder. In some embodiments, the subject suffers from diabetes. In some embodiments, the subject suffers from prediabetes. In such embodiments, at least one insulin-positive endocrine cell or precursor thereof can be differentiated into an SC-β cell ex vivo using the methods described herein and can then be administered to the subject from whom the cells were collected to effect treating a subject for a β-cell disorder (eg, diabetes).

[368] In some embodiments, at least one insulin-positive endocrine cell or precursor thereof is present in the subject's body (in vivo) and is converted to become an SC-β cell using in vivo methods described herein. In some embodiments, conversion of at least one insulin-positive endocrine cell or precursor thereof to an SC-β cell in vivo can be achieved by administering to a subject a composition comprising at least one, at least two, at least three, at least four, at least five, or at least six β-cell maturation factors as described herein. In some embodiments, conversion of at least one insulin-positive endocrine cell or precursor thereof to an SC-β cell in vivo can be achieved by administering to a subject a composition comprising at least one, at least two, at least three, at least four, at least five, or at least six β-cell maturation factors as described herein.

[369] In some embodiments, contacting may be accomplished by maintaining at least one insulin-positive endocrine cell or precursor thereof in a culture medium containing one or more β-cell maturation factors. In some embodiments, the at least one insulin-positive endocrine cell or precursor thereof may be genetically engineered. In some embodiments, the at least one insulin-positive endocrine cell or precursor thereof may be genetically engineered to express one or more β-cell maturation factors as described herein, for example, to express at least one polypeptide selected from pancreatic and duodenal homeobox polypeptide 1 (PDX-1), insulin, c-peptide, amylin, E-cadherin, Hnf3β, PCI/3, B2, Nkx2.2, NKX6-1, GLUT2, PC2, ZnT-8 or significantly the degree of amino acid sequences homologous to them, or their functional fragments or functional variants.

[370] When at least one insulin-positive endocrine cell or its precursor is maintained in vitro, conventional tissue culture conditions and methods known to those skilled in the art may be used. Methods for isolating and culturing various cells are known to those skilled in the art.

[371] In general, in the methods of the invention, at least one insulin-positive endocrine cell or its precursor can be cultured under standard temperature, pH and other environmental conditions, for example, as adherent cells on tissue culture plates at 37°C C and in an atmosphere containing 5-10% CO2. Cells and/or culture medium are appropriately modified to achieve SC-β cell conversion as described herein. In certain embodiments, at least one insulin-positive endocrine cell or progenitor thereof, for example, a pancreatic progenitor cell, can be cultured on or in the presence of a material that mimics one or more characteristics of the extracellular matrix or contains one or more components of the extracellular matrix or basal matrix. membranes. In some embodiments, Matrigel™ is used. Other materials include proteins or mixtures thereof such as gelatin, collagen, fibronectin, etc. In certain embodiments of the invention, at least one insulin-positive endocrine cell or its precursor can be cultured in the presence of a layer of feeder cells. Such cells can be, for example, of mouse or human origin. They may also be irradiated, chemically inactivated by treatment with a chemical inactivator such as mitomycin C, or otherwise treated to inhibit their proliferation if desired. In other embodiments, at least one insulin-positive endocrine cell or precursor thereof is cultured without feeder cells. In some embodiments, insulin-positive endocrine cells or their precursors are cultured under conditions that promote cell clustering. As used herein, “conditions that promote cell clustering” refers to any conditions that promote cell clustering during differentiation into SC-β cells. In some embodiments, conditions that promote cell clustering include suspension culture. Boretti and Gooch (Tissue Eng. 2006 Apr; 12(4): 939-48) reported that culture under less adhesive conditions (low serum medium, low adhesiveness substrate) stimulated cell clustering during transdifferentiation of adult pancreatic ductal epithelial cells organism into beta cells in vitro. Accordingly, without being limited by theory, in some embodiments, conditions that promote cell clustering include minimally adhesive conditions, eg, low serum media, a low adhesive substrate.

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

[373] Accordingly, cells and compositions prepared by the methods described herein are included herein. The exact amount and type of β-cell maturation factor may vary depending on the number of insulin-positive endocrine cells or their precursors, the stage of differentiation required, and the number of stages of differentiation that have occurred.

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

[375] In further examples, β-cell maturation factors may be present in the culture medium of at least one insulin-positive endocrine cell or precursor thereof, or, alternatively, β-cell maturation factors may be added to at least one insulin-positive endocrine cell. cell or its predecessor at some stages of growth.

[376] Confirmation and identification of SC-β cells

[377] Any means known to one skilled in the art can be used to confirm the presence of an SC-β cell, for example, a mature pancreatic β-cell obtained by inducing differentiation of at least one insulin-positive endocrine cell or its precursor by treating it with at least one β-cell maturation factor as described herein.

[378] In some embodiments, the presence of β cell markers, such as chemically induced SC-β cells, can be confirmed by determining the presence or absence of one or more endogenous β cell markers. In some embodiments, the method may include determining the positive expression (eg, presence) of a mature β-cell marker. In some embodiments, the marker can be detected using a reagent, such as an NKX6-1 and C-peptide detection reagent. In particular, SC-β cells herein express NKX6-1 and C-peptide and do not express at significant levels other markers that would be characteristic of immature β cells (eg, MafB). The reagent for the marker can be, for example, an antibody to this marker or primers for an RT-PCR or PCR reaction, for example, a semi-quantitative or quantitative RT-PCR or PCR reaction. Such markers can be used to determine whether a SC-β cell is obtained. The antibody or other detection reagent may be coupled to a label, such as a radioactive, fluorescent (eg, ZPB) or colorimetric label for use in detection. If the detection reagent is a primer, it may be provided as a dry preparation, such as lyophilized, or in solution.

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

[380] As described in connection with tracking the derivation of SC-β cells (eg, mature pancreatic β-cells) from insulin-positive endocrine cells, qualitative or semi-quantitative methods, such as immunoblotting and immunocytochemistry techniques known in the art, can be used to determine marker expression in this field of technology. Alternatively, marker expression can be accurately quantified using techniques such as quantitative PCR, methods well known in the art. In addition, it should be understood that at the polypeptide level, many markers of pancreatic islet hormone-expressing cells are secreted proteins. Therefore, methods for determining the extracellular content of markers, such as ELISA, can be used.

[381] SC-β cells may also be characterized by down-regulation of markers characteristic of the pluripotent stem cells from which SC-β cells were induced. For example, SC-β cells derived from pluripotent stem cells may, in their mature state, be characterized by statistically significant down-regulation of pluripotent stem cell markers - alkaline phosphatase (AP), NANOG, OCT-4, SOX-2, SSEA4, TRA-1-60 or TRA-1-81 - compared to the pluripotent stem cells from which they were derived. Other markers expressed by pluripotent stem cells include, but are not limited to, alkaline phosphatase (AP); ABCG2; stage-specific embryonic antigen-1 (SSEA-1); SSEA-3; SSEA-4; TRA-1-60; TRA-1-81; Tga-2-49/6E; ERas/ECATS, E-cadherin; βIII-tubulin; α-smooth muscle actin (α-SMA); fibroblast growth factor 4 (Fgf4), Cripto, Dax1; zinc finger protein 296 (Zfp296); N-acetyltransferase-1 (Nat1); (ES cell-associated transcript 1 (ECAT1); ESG1/DPPAS/ECAT2; ECAT3; ECAT6; ECAT7; ECAT8; ECAT9; ECAT10; ECAT15-1; ECAT15-2; Fth117; Sa114; undifferentiated embryonic cell transcription factor (Utf1); Rex1; p53; G3PDH; telomerase, including TERT; silent genes of the X chromosome; Dnmt3a; Dnmt3b; TRIM28; F-box containing protein 15 (Fbx15); Nanog/ECAT4; Oct3/4; Sox2; Klf4; c-Myc; Esrrb; TDGF1; GABRB3; Zfp42, FoxD3; GDF3; CYP25A1; pluripotency factor 2 (DPPA2); T-cell lymphoma associated breakpoint 1 (Tcl1); DPPA3/Stella; DPPA4; Dnmt3L; Soxl5; Stat3; Grb2; SV40 large T antigen; HPV16 E6; HPV16 E7, β-catenin and Bmi1 and other general markers of pluripotency, with at least one or more of them down-regulated by a statistically significant amount in adulthood compared with pluripotent stem cells from which they were obtained.

[382] It is understood that the present invention is not limited to the markers described herein as markers of mature β-cells, but the present invention also includes markers such as cell surface markers, antigens and other gene products, including ESTs, RNA (including microRNA and antisense RNA), DNA (including genes and cDNA), as well as their fragments.

[383] Enrichment, isolation and purification of SC-β cells

[384] Another aspect of the present invention relates to isolating a population of SC-β cells from a heterogeneous population of cells, wherein the mixed population of cells comprises SC-β cells and insulin-positive endocrine cells or precursors thereof from which the SC-β cells were derived. A population of SC-β cells obtained by any of the methods described above can be enriched, isolated and/or purified using any cell surface marker present on SC-β cells that is not present on insulin-positive endocrine cells or their progenitors from which they were derived received. Such cell surface markers are also called SC-β cell-specific affinity tags. Examples of SC-β cell-specific affinity tags are antibodies, ligands, or other binding agents specific to a marker molecule, such as a polypeptide present on the cell surface of SC-β cells that is essentially not present on other cell types (eg, insulin -positive endocrine cells or their precursors). In some methods, an antibody that binds to a cell surface antigen on an SC-β cell (e.g., a human SC-β cell) is used as an affinity tag to enrich, isolate, or purify chemically induced (e.g., by contacting at least one β-cell maturation factor, as described herein) of SC-β cells obtained by the methods described herein. Such antibodies are known and commercially available.

[385] Methods for using antibodies to enrich, isolate and/or purify SC-β cells are known to one of ordinary skill in the art. For example, in some embodiments, a reagent, such as an antibody, is incubated with a population of cells containing SC-β cells, wherein the population of cells is treated to reduce intracellular and substrate adhesion. The cell population is then washed, centrifuged and redissolved. In some embodiments, if the antibody is not labeled, the cell suspension is then incubated with a secondary antibody, such as a FITC-conjugated antibody, that is capable of binding to the primary antibody. SC-β cells are then washed, centrifuged, and redissolved in buffer. The SC-β cell suspension is then analyzed and sorted using a fluorescence-activated cell sorter (FACS). Antibody-bound fluorescent reprogrammed cells are collected separately from unbound non-fluorescent cells (eg, immature insulin-producing cells), thereby separating SC-β cells from other cells present in the cell suspension, such as insulin-positive endocrine cells or their precursors or immature insulin-producing cells (eg, other differentiated cell types).

[386] In another embodiment of the methods described herein, the isolated cell composition containing SC-β cells can be further purified using an alternative affinity-based method or through additional sorting steps using the same or different SC cell-specific markers -β. For example, in some embodiments, FACS sorting is used to first separate SC-β cells that express NKX6-1, either alone or in conjunction with C-peptide expression or, alternatively, a β- marker disclosed herein. cells, from cells that do not express one of these markers (eg, negative cells) in the cell population. A second FAC sort, such as positive cell sorting using FACS to isolate cells that are positive for a marker different from that used in the first sort, allows the cell population to be enriched with reprogrammed cells.

[387] In an alternative embodiment, FACS sorting is used to separate cells using negative sorting for a marker that is present on most insulin-positive endocrine cells or their precursors, but is not present on SC-β cells.

[388] In some embodiments of the methods described herein, SC-β cells are fluorescently labeled without the use of an antibody and then separated from unlabeled cells using a fluorescence-activated cell sorter (FACS). In such embodiments, a nucleic acid encoding a GFB, YFP, or other nucleic acid encoding an expressible fluorescent marker gene, such as a gene encoding luciferase, is used to label reprogrammed cells using the methods described above. For example, in some embodiments of the invention, at least one copy of a nucleic acid encoding a ZPB or a biologically active fragment thereof is introduced into at least one insulin-positive endocrine cell, which is first chemically induced to an SC-β cell, downstream of a promoter expressed in SC-β cell, such as an insulin promoter, so that the expression of the ZPB gene product or a biologically active fragment thereof is under the control of the insulin promoter.

[389] In addition to the above procedures, chemically induced SC-β cells can also be isolated using other cell isolation methods. In addition, SC-β cells can also be enriched or isolated by serial subculture methods under growth conditions that promote selective survival or selective expansion of SC-β cells. Such methods are known to those skilled in the art and may include the use of agents such as insulin, members of the TGF-beta family including activin A, TGF-beta 1, 2 and 3, bone morphogenetic proteins (BMP-2, -3 , -4, -5, -6, -7, -11, -12 and -13), fibroblast growth factors 1 and 2, platelet-derived growth factor AA and BB, platelet-rich plasma, insulin-like growth factors (IGF- I, II), growth and differentiation factor (GDF-5, -6, -7, -8, -10, -11, -15), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), pleotrophin, endothelin, epidermal growth factor (EGF), beta-cellulin. Other pharmaceutical compounds may include, for example, nicotinamide, glucagon-like peptide-I (GLP-1) and II, GLP-1 and 2 mimebody, exendin-4, retinoic acid, parathyroid hormone.

[390] Using the methods described herein, it is possible to obtain in vitro enriched, isolated and/or purified populations of SC-β cells from insulin-positive endocrine cells or their precursors (that have been differentiated from pluripotent stem cells by the methods described herein). In some embodiments, preferred enrichment, isolation, and/or purification methods relate to the in vitro production of human SC-β cells from human insulin-positive endocrine cells or precursors thereof that have been differentiated from human pluripotent stem cells or from human induced pluripotent stem cells ( iPS). In an embodiment of the invention in which SC-β cells are differentiated from insulin-positive endocrine cells that were previously derived from definitive endoderm cells that were previously derived from iPS cells, the SC-β cells may be autologous to the subject from which they were derived. cells for generating iPS cells were obtained.

[391] Using the methods described herein, isolated cell populations of SC-β cells are enriched in SC-β cell content by at least about 2 to about 1000 times the cell population prior to chemical induction of the insulin-positive endocrine cell population or their predecessors. In some embodiments, SC-β cells may be enriched by at least about 5 to about 500 times the population prior to chemical induction of the population of insulin-positive endocrine cells or their precursors. In other embodiments, SC-β cells may be enriched at least about 10-fold to about 200-fold over the population prior to chemical induction of the population of insulin-positive endocrine cells or their precursors. In other embodiments, SC-β cells may be enriched from about 20 to about 100 times the population prior to chemical induction of the population of insulin-positive endocrine cells or their precursors. In other embodiments, SC-β cells may be enriched by at least about 40 to about 80 times the population prior to chemical induction of the population of insulin-positive endocrine cells or their precursors. In certain embodiments, SC-β cells may be enriched by at least about 2 to about 20 times the population prior to chemical induction of the population of insulin-positive endocrine cells or their precursors.

[392] Compositions containing SC-β cells

[393] Some embodiments of the present invention relate to cell compositions, such as cell cultures or cell populations, containing SC-β cells, wherein the SC-β cells were derived from at least one insulin-positive endocrine cell or precursor thereof. In some embodiments, the cell compositions comprise insulin-positive endocrine cells. In some embodiments, the cell compositions comprise NKX6-1 pancreatic progenitor cells. In some embodiments, the cell compositions comprise Pdx1 pancreatic progenitor cells. In some embodiments, the cell compositions comprise cells of the primary intestinal tube. In some embodiments, the cell compositions comprise definitive endoderm cells.

[394] In certain embodiments, the chemically induced SC-β cells are mammalian cells, and in a preferred embodiment, such SC-β cells are human SC-β cells. In some embodiments, the insulin-positive endocrine cells are derived from definitive endoderm cells, for example, from human definitive endoderm stem cells. In certain embodiments, the chemically induced Pdx1-positive pancreatic progenitor cells are mammalian cells, and in a preferred embodiment, the Pdx1-positive pancreatic progenitor cells are human Pdx1-positive pancreatic progenitor cells.

[395] Other embodiments of the present invention relate to compositions, such as an isolated cell population or cell culture, containing SC-β cells obtained by the methods disclosed herein. In some embodiments, the present invention provides compositions, such as isolated cell populations or cell cultures, containing chemically induced SC-β cells produced by methods disclosed herein. In such embodiments, SC-β cells comprise less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60 %, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 12%, less than about 10%, less than about 8%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2% or less than about 1% of the total number of cells in the SC-β cell population. In some embodiments, the composition comprises a population of SC-β cells that constitutes more than about 90% of the total number of cells in the cell population, such as at least about 95%, or at least 96%, or at least 97%, or at least 98%, or at least about 99%, or at least about 100% of the total number of cells in the cell population are SC-β cells.

[396] Certain other embodiments of the present invention relate to compositions, such as isolated cell populations or cell cultures, containing a combination of SC-β cells and insulin-positive endocrine cells or precursors thereof from which the SC-β cells were derived. In some embodiments, the insulin-positive endocrine cells from which the SC-β cells are derived comprise less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5% less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the total number of cells in the isolated cell population or culture.

[397] Additional embodiments of the present invention relate to compositions, such as isolated cell populations or cell cultures, obtained by methods described herein, which contain chemically induced SC-β cells as the main cell type. In some embodiments, the methods and processes described herein enable the production of isolated cell cultures and/or cell populations containing at least about 99%, at least about 98%, at least about 97%, at least about 96%, at least about 95%, at least about 94%, at least about 93%, at least about 92%, at least about 91%, at least about 90%, at least about 89 %, at least about 88%, at least about 87%, at least about 86%, at least about 85%, at least about 84%, at least about 83%, at least about 82% , at least about 81%, at least about 80%, at least about 79%, at least about 78%, at least about 77%, at least about 76%, at least about 75%, at least about 74%, at least about 73%, at least about 72%, at least about 71%, at least about 70%, at least about 69%, at least about 68%, according to at least about 67%, at least about 66%, at least about 65%, at least about 64%, at least about 63%, at least about 62%, at least about 61%, at least at least about 60%, at least about 59%, at least about 58%, at least about 57%, at least about 56%, at least about 55%, at least about 54%, at least about 53%, at least about 52%, at least about 51%, or at least about 50% SC-β cells.

[398] In another embodiment, the isolated cell populations or cell compositions (or cell cultures) comprise human SC-β cells. In other embodiments, the methods and processes described herein make it possible to obtain isolated cell populations containing at least about 50%, at least about 45%, at least about 40%, at least about 35%, at least at least about 30%, at least about 25%, at least about 24%, at least about 23%, at least about 22%, at least about 21%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5 %, at least about 4%, at least about 3%, at least about 2%, or at least about 1% SC-β cells. In preferred embodiments of the invention, the isolated cell populations may comprise human SC-β cells. In some embodiments, the percentage of SC-β cells in cell cultures or populations is calculated without taking into account the feeder cells remaining in the culture.

[399] Other embodiments of the present invention relate to compositions, such as isolated cell populations or cell cultures, containing a mixture of SC-β cells and insulin-positive endocrine cells or their precursors from which they have been differentiated. For example, it is possible to obtain cell cultures or cell populations containing at least about 5 SC-β cells for every 95 insulin-positive endocrine cells or their precursors. In other embodiments, it is possible to obtain cell cultures or cell populations containing at least about 95 SC-β cells for every 5 insulin-positive endocrine cells or precursors thereof. In addition, cell cultures or cell populations containing other ratios of SC-β cells and insulin-positive endocrine cells or their precursors are provided. For example, compositions may be prepared containing at least about 1 SC-β cell for every 1,000,000, or at least 100,000 cells, or at least 10,000 cells, or at least 1,000 cells, or 500, or at least 250, or at least 100, or at least 10 insulin-positive endocrine cells or their precursors.

[400] Additional embodiments of the present invention relate to compositions, such as cell cultures or cell populations, containing human cells, including a human SC-β cell, that have at least one characteristic of an endogenous β cell.

[401] In preferred embodiments of the present invention, cell cultures and/or cell populations of SC-β cells comprise human SC-β cells that are non-recombinant cells. In such embodiments, the cell cultures and/or cell populations contain little or no recombinant human SC-β cells.

[402] β-cell maturation factors

[403] Aspects of the invention include bringing insulin-positive endocrine cells or their precursors into contact with β-cell maturation factors, for example, to induce the maturation of insulin-positive endocrine cells or the differentiation of their precursors into SC-β cells (eg, mature pancreatic β-cells). cells). The term “β-cell maturation factor” refers to an agent that stimulates or promotes the conversion of at least one insulin-positive endocrine cell or its precursor into an SC-β cell. In some embodiments, the β-cell maturation factor induces differentiation of pluripotent cells (eg, iPSCs or hESCs) into definitive endoderm cells, for example, in accordance with the method described herein. In some embodiments, the β-cell maturation factor induces the differentiation of definitive endoderm cells into primary gut tube cells, for example, in accordance with the method described herein. In some embodiments, the β-cell maturation factor induces the differentiation of primary intestinal tube cells into Pdx1-positive pancreatic progenitor cells, for example, in accordance with the method described herein. In some embodiments, the β-cell maturation factor induces the differentiation of Pdx1-positive pancreatic progenitor cells into NKX6-1-positive pancreatic progenitor cells, for example, in accordance with the method described herein. In some embodiments, the β-cell maturation factor induces the differentiation of NKX6-1-positive pancreatic progenitor cells into insulin-positive endocrine cells, for example, in accordance with the method described herein. In some embodiments, the β-cell maturation factor induces the maturation of insulin-positive endocrine cells into SC-β cells, for example, in accordance with the method described herein.

[404] In general, at least one β-cell maturation factor described herein can be used alone or in combination with other β-cell maturation factors to generate SC-β cells in accordance with the methods disclosed herein. In some embodiments, at least one, 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 β-cell maturation factors described herein are used in methods for generating SC-β cells.

[405] Transforming growth factor β (TGF-β) superfamily

[406] Aspects of the invention relate to the use of growth factors from the transforming growth factor β (TGF-β) superfamily as β-cell maturation factors. The “TGF-β” superfamily refers to proteins that have the structural and functional characteristics of known members of the TGFβ family. The characteristics of the TGFβ family proteins are well known, both in terms of structural and functional aspects. It includes the TGFβ protein groups inhibins (including inhibin A and inhibin B), activins (including activin A, activin B and activin AB), MIS (Muller inhibitory substance), BMP (bone morphogenetic proteins), dpp (decapentaplegic), Vg-1 , MNSF (monoclonal nonspecific suppression factor) and others. The activity of this family of proteins is based on specific binding to specific receptors on different cell types. Members of this family contain regions of sequence identity, particularly at the C-terminus, that are consistent with their function. The TGFβ family includes more than one hundred different proteins, all of which contain at least one region of amino acid sequence identity. Members of this family include, but are not limited to, the following proteins, identified by their GenBank accession numbers: P07995, P18331, P08476, Q04998, P03970, P43032, P55102, P27092, P42917, P09529, P27093, P04088, Q04999, P174 91, P55104 , Q9WUK5, Р55103, O88959, O08717, Р58166, O61643, Р35621, Р09534, Р48970, Q9NR23, Р25703, Р30884, Р12643, Р49001, Р21274, O46564, O19006, Р220 04, P20722, Q04906, Q07104, P30886, P18075, P23359, P22003 , Р34821, Р49003, Q90751, Р21275, Q06826, Р30885, Р34820, Q29607, Р12644, Q90752, O46576, Р27539, Р48969, Q26974, Р07713, Р91706, Р91699, Р27 091, O42222, Q24735, P20863, O18828, P55106, Q9PTQ2, O14793 , O08689, O42221, O18830, O18831, O18836, O35312, O42220, Р43026, Р43027, Р43029, O95390, Q9R229, O93449, Q9Z1W4, Q9BDW8, Р43028, Q7Z4P5, Р504 14, P17246, P54831, P04202, P01137, P09533, P18341, O19011 , Q9Z1Y6, P07200, Q9Z217, O95393, P55105, P30371, Q9MZE2, Q07258, Q96S42, P97737, AAA97415.1, NP-776788.1, NP-058824.1, EAL24001.1, 1S4Y, NP-001009856.1, NP-032406.1, NP-999193.1 , XP-519063.1, AAG17260.1, CAA40806.1, NP-001009458.1, AAQ55808.1, AAK40341.1, AAP33019.1, AAK21265.1, AAC59738.1, CAI46003.1, B40905, AAQ55 811.1,AAK40342.1 , XP-540364.1, P55102, AAQ55810.1, NP-990727.1, CAA51163.1, AAD50448.1, JC4862, PN0504, BAB17600.1, AAH56742.1, BAB17596.1, CAG06183.1, CAG0533 9.1, BAB17601.1 , CAB43091.1, A36192, AAA49162.1, AAT42200.1, NP-789822.1, AAA59451.1, AAA59169.1, XP-541000.1, NP-990537.1, NP-002184.1, AAC14187.1, AAP83319 .1,AAA59170.1 , BAB16973.1, AAM66766.1, WFPGBB, 1201278C, AAH30029.1, CAA49326.1, XP-344131.1, AAN48845.1, XP-148966.3, I48235, B41398, AAH77857.1, AAB26863. 1, 1706327A, VAA83804.1 , NP-571143.1, CAG00858.1, VAV17599.1, VAV17602.1, AAV61468.1, PN0505, PN0506, SAV43092.1, VAV17598.1, VAA22570.1, VAV16972.1, VAS81672.1 , ВАА12694.1, ВАА08494 .1, В36192, С36192, ВАВ16971.1, NP-034695.1, AAA49160.1, CAA62347.1, AAA49161.1, AAD30132.1, CAA58290.1, NP-005529.1, XP-522443.1, AAM27448.1 , XP-538247.1 , AAD30133.1, AAC36741.1, AAH10404.1, NP-032408.1, AAN03682.1, XP-509161.1, AAC32311.1, NP-651942.2, AAL51005.1, AAC39083.1, AAH85547.1, NP-571 023.1, CAF94113 .1, EAL29247.1, AAW30007.1, AAH90232.1, A29619, NP-001007905.1, AAH73508.1, AAD02201.1, NP-999793.1, NP-990542.1, AAF19841.1, AAC97488.1, AAC60 038.1, NP 989197.1, NP-571434.1, EAL41229.1, AAT07302.1, CAI19472.1, NP-031582.1, AAA40548.1, XP-535880.1, NP-037239.1, AAT72007.1, XP-418956.1, CAA41 634.1, BAC30864.1, CAA38850.1, CAB81657.2, CAA45018.1, CAA45019.1, BAC28247.1, NP-031581.1, NP-990479.1, NP-999820.1, AAB27335.1, S45355, CAB82007.1, XP-534351.1 ,NP-058874.1, NP-031579.1, 1REW, AAB96785.1, AAB46367.1, CAA05033.1, BAA89012.1, 1ES7, AAP20870.1, BAC24087.1, AAG09784.1, BAC06352.1, AAQ89234.1, AAM27000.1, A AH30959. 1, CAG01491.1, NP-571435.1, 1REU, AAC60286.1, BAA24406.1, A36193, AAH55959.1, AAH54647.1, AAH90689.1, CAG09422.1, BAD16743.1, NP-032134.1, XP-53 2179.1, AAB24876.1, AAH57702.1, AAA82616.1, CAA40222.1, CAB90273.2, XP-342592.1, XP-534896.1, XP-534462.1, 1LXI, XP-417496.1, AAF34179.1, AAL73188.1, CAF96266.1, AAB34226.1, AAB33846.1, AAT12415.1, AA033819.1, AAT72008.1, AAD38402.1, BAB68396.1, CAA45021.1, AAB27337.1, AAP69917.1, AAT12416.1, NP-571396.1, C AA53513. 1, AAO33820.1, AAA48568.1, BAC02605.1, BAC02604.1, BAC02603.1, BAC02602.1, BAC02601.1, BAC02599.1, BAC02598.1, BAC02597.1, BAC02595.1, BAC02593.1, BAC02592.1, BAC02590.1, AAD28039.1, AAP74560.1, AAB94786.1, NP-001483.2, XP-528195.1, NP-571417.1, NP-001001557.1, AAH43222.1, AAM33143.1, CAG1 0381.1, BAA31132. 1, EAL39680.1, EAA12482.2, P34820, AAP88972.1, AAP74559.1, CAI16418.1, AAD30538.1, XP-345502.1, NP-038554.1, CAG04089.1, CAD60936.2, NP-031584 .1, B55452, AAC60285.1, BAA06410.1, AAH52846.1, NP-031580.1, NP-036959.1, CAA45836.1, CAA45020.1, Q29607, AAB27336.1, XP-547817.1, AAT12414.1, AAM54049.1, A AH78901.1, AA025745.1, NP-570912.1, XP-392194.1, AAD20829.1, AAC97113.1, AAC61694.1, AAH60340.1, AAR97906.1, BAA32227.1, BAB68395.1, BAC02895.1, AAW51451.1 , AAF82188. 1, XP-544189.1, NP-990568.1, BAC80211.1, AAW82620.1, AAF99597.1, NP-571062.1, CAC44179.1, AAB97467.1, AAT99303.1, AAD28038.1, AAH52168.1, NP-00 1004122.1, CAA72733.1, NP-032133.2, XP-394252.1, XP-224733.2, JH0801, AAP97721.1, NP-989669.1, S43296, P43029, A55452, AAH32495.1, XP-542974.1, NP-032 135.1, AAK30842.1, AAK27794. 1, BAC30847.1, EAA12064.2, AAP97720.1, XP-525704.1, AAT07301.1, BAD07014.1, CAF94356.1, AAR27581.1, AAG13400.1, AAC60127.1, CAF92055.1, XP-540103 .1, AAO20895.1, CAF97447.1, AAS01764.1, BAD08319.1, CAA10268.1, NP-998140.1, AAR03824.1, AAS48405.1, AAS48403.1, AAK53545.1, AAK84666.1, XP-395420. 1, AAK56941. 1, AAC47555.1, AAR88255.1, EAL33036.1, AAW47740.1, AAW29442.1, NP-722813.1, AAR08901.1, AAO15420.2, CAC59700.1, AAL26886.1, AAK71708.1, AAK71707 .1, CAC51427.2, AAK67984.1, AAK67983.1, AAK28706.1, P07713, P91706, P91699, CAG02450.1, AAC47552.1, NP-005802.1, XP-343149.1, AW34055.1, XP-538 221.1, AAR27580.1, XP-125935.3, AAF21633.1, AAF21630.1, AAD05267.1, Q9Z1W4, NP-031585.2, NP-571094.1, CAD43439.1, CAF99217.1, CAB63584.1, NP-722840.1, CAE46407.1 , XP-417667.1, BAC53989.1, BAB19659.1, AAM46922.1, AAA81169.1, AAK28707.1, AAL05943.1, AAB17573.1, CAH25443.1, CAG10269.1, BAD16731.1, EAA00276.2, AAT07320.1, AAT0 7300. 1, AAN15037.1, CAH25442.1, AAK08152.2, 2009388A, AAR12161.1, CAG01961.1, CAB63656.1, CAD67714.1, CAF94162.1, NP-477340.1, EAL24792.1, NP-00100 9428.1, AAB86686. 1, AAT40572.1, AAT40571.1, AAT40569.1, NP-033886.1, AAB49985.1, AAG39266.1, Q26974, AAC77461.1, AAC47262.1, BAC05509.1, NP-055297.1, XP-546146. 1, XP- 525772.1, NP-060525.2, AAH33585.1, AAH69080.1, CAG12751.1, AAH74757.2, NP-034964.1, NP-038639.1, O42221, AAF02773.1, NP-062024.1, AAR18244.1 , AAR14343.1, XP- 228285.2, AAT40573.1, AAT94456.1, AAL35278.1, AAL35277.1, AAL17640.1, AAC08035.1, AAB86692.1, CAB40844.1, BAC38637.1, BAB16046.1, AAN63522.1, NP-5 71041.1, AAB04986.2, AAC26791.1, AAB95254.1, BAA11835.1, AAR18246.1, XP-538528.1, BAA31853.1, AAK18000.1, XP-420540.1, AAL35276.1, AAQ98602.1, CAE71944. 1, AAW50585. 1, AAV63982.1, AAW29941.1, AAN87890.1, AAT40568.1, CAD57730.1, AAB81508.1, AAS00534.1, AAC59736.1, BAB79498.1, AAA97392.1, AAP85526.1, NP-999600.2 , NP-878293.1, BAC82629.1, CAC60268.1, CAG04919.1, AAN10123.1, CAA07707.1 AAK20912.1, AAR88254.1, CAC34629.1, AAL35275.1, AAD46997.1, AAN03842.1, NP -571951.2 , CAC50881.1, AAL99367.1, AAL49502.1, AAB71839.1, AAB65415.1, NP-624359.1, NP-990153.1, AAF78069.1, AAK49790.1, NP-919367.2, NP-001192.1, XP- 544948.1, AAQ18013 .1, AAV38739.1, NP-851298.1, CAA67685.1, AAT67171.1, AAT37502.1, AAD27804.1, AAN76665.1, BAC11909.1, XP-421648.1, CAB63704.1, NP-037306.1, A557 06, AAF02780 .1, CAG09623.1, NP-067589.1, NP-035707.1, AAV30547.1, AAP49817.1, BAC77407.1, AAL87199.1, CAG07172.1, B36193, CAA33024.1, NP-001009400.1, AAP36 538.1, XP -512687.1, XP-510080.1, AAH05513.1, 1KTZ, AAH14690.1, AAA31526.1.

[407] Any growth factor of the TGF-β superfamily is contemplated that is capable, alone or in combination with one or more other β-cell maturation factors, of inducing the differentiation of at least one insulin-producing endocrine cell or its precursor into an SC cell -β can be used in the methods, compositions and kits described herein.

[408] Growth factor from TGF-β can be obtained naturally or recombinantly. In some embodiments, the growth factor of the TGF-β superfamily includes activin A. The term “activin A” includes fragments and derivatives of activin A. The sequence of the exemplary activin A is disclosed as SEQ ID No: 1 in US Patent Publication No. 2009/0155218 (“ publication 218"). Other non-limiting examples of activin A are provided in SEQ ID Nos: 2-16 of publication 218, and non-limiting examples of nucleic acids encoding activin A are provided in SEQ ID nos: 33-34 of publication 218. In some embodiments, a growth factor from the TGF-β superfamily β contains a polypeptide having an amino acid sequence of at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% or more identical to SEQ ID No: 1 of publication 218.

[409] In some embodiments, a growth factor from the TGF-β superfamily includes growth and differentiation factor 8 (GDF8). The term "GDF8" includes fragments and derivatives of GDF8. GDF8 polypeptide sequences are available to those skilled in the art. In some embodiments, a growth factor from the TGF-β superfamily comprises a polypeptide having an amino acid sequence of at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% or more identical to the human GDF8 polypeptide sequence (GenBank accession number EAX10880).

[410] In some embodiments, a growth factor from the TGF-β superfamily includes a growth factor closely related to GDF8, such as growth and differentiation factor 11 (GDF11). GDF11 polypeptide sequences are available to those skilled in the art. In some embodiments, a growth factor from the TGF-β superfamily comprises a polypeptide having an amino acid sequence of at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% or more identical to the human GDF11 polypeptide sequence (GenBank accession number AAF21630).

[411] In certain embodiments, the methods, compositions, and kits disclosed herein do not include at least one growth factor from the TGF-β superfamily.

[412] In some embodiments, the at least one growth factor from the TGF-β superfamily may be replaced by an agent that mimics the at least one growth factor from the TGF-β superfamily. Exemplary agents that mimic at least one growth factor from the TGF-β superfamily include, but are not limited to, IDE1 and IDE2.

[413] TGF-β signaling pathway inhibitors

[414] Aspects of the invention relate to the use of inhibitors of the TGF-β signaling pathway as β-cell maturation factors. Any inhibitor of the TGF-β signaling pathway that is capable, alone or in combination with one or more other β-cell maturation factors, of inducing the differentiation of at least one insulin-producing endocrine cell or its precursor into an SC-β cell is contemplated. used in the methods, compositions and kits described herein. In certain embodiments, the methods, compositions, and kits disclosed herein do not include a TGF-β signaling pathway inhibitor.

[415] In some embodiments, the TGF-β signaling pathway inhibitor involves TGF-β receptor type I (TGF-β RI) kinase signaling. In some embodiments, the TGF-β signaling pathway inhibitor includes ALK5 inhibitor II (CAS 446859-33-2, ATP-competitive TGF-B RI kinase inhibitor, also known as RepSox, IUPAC name: 2-[5-(6- methylpyridin-2-yl)-1H-pyrazol-4-yl]-1,5-naphthyridine In some embodiments, the TGF-β signaling pathway inhibitor is an analogue or derivative of ALK5 inhibitor II.

[416] In some embodiments, the ALK5 inhibitor II analog or derivative is a compound of Formula I, as described in US Patent Publication No. 2012/0021519, incorporated herein by reference in its entirety.

[417] In some embodiments, the TGF-β signaling pathway inhibitor is a TGF-β receptor inhibitor described in US Patent Publication No. 2010/0267731. In some embodiments, the TGF-β signaling pathway inhibitor includes an ALK5 inhibitor described in US Patent Publications No. 2009/0186076 and 2007/0142376.

[418] In some embodiments, the TGF-β signaling pathway inhibitor is A 83-01. In some embodiments, the TGF-β signaling pathway inhibitor is not A 83-01. In some embodiments, the compositions and methods described herein do not include A 83-01.

[419] In some embodiments, the TGF-β signaling pathway inhibitor is SB 431542. In some embodiments, the TGF-β signaling pathway inhibitor is not SB 431542. In some embodiments, the compositions and methods described herein are not included SB 431542.

[420] In some embodiments, the TGF-β signaling pathway inhibitor is D 4476. In some embodiments, the TGF-β signaling pathway inhibitor is not D 4476. In some embodiments, the compositions and methods described herein are not includes D 4476.

[421] In some embodiments, the TGF-β signaling pathway inhibitor is GW 788388. In some embodiments, the TGF-β signaling pathway inhibitor is not GW 788388. In some embodiments, the compositions and methods described herein are not included GW 788388.

[422] In some embodiments, the TGF-β signaling pathway inhibitor is LY 364947. In some embodiments, the TGF-β signaling pathway inhibitor is not LY 364947. In some embodiments, the compositions and methods described herein are not includes LY 364947.

[423] In some embodiments, the TGF-β signaling pathway inhibitor is LY 580276. In some embodiments, the TGF-β signaling pathway inhibitor is not LY 580276. In some embodiments, the compositions and methods described herein are not included LY 580276.

[424] In some embodiments, the TGF-β signaling pathway inhibitor is SB 525334. In some embodiments, the TGF-β signaling pathway inhibitor is not SB 525334. In some embodiments, the compositions and methods described herein are not included SB 525334.

[425] In some embodiments, the TGF-β signaling pathway inhibitor is SB 505124. In some embodiments, the TGF-β signaling pathway inhibitor is not SB 505124. In some embodiments, the compositions and methods described herein are not includes SB 505124.

[426] In some embodiments, the TGF-β signaling pathway inhibitor is SD 208. In some embodiments, the TGF-β signaling pathway inhibitor is not SD 208. In some embodiments, the compositions and methods described herein are not SD 208 included.

[427] In some embodiments, the TGF-β signaling pathway inhibitor is GW 6604. In some embodiments, the TGF-β signaling pathway inhibitor is not GW 6604. In some embodiments, the compositions and methods described herein are not GW 6604 included.

[428] In some embodiments, the TGF-β signaling pathway inhibitor is GW 788388. In some embodiments, the TGF-β signaling pathway inhibitor is not GW 788388. In some embodiments, the compositions and methods described herein are not included GW 788388.

[429] The following compounds from the set of compounds described above can be obtained from various sources: LY-364947, SB-525334, SD-208 and SB-505124 are available from Sigma, P.O. Box 14508, St. Louis, Mo., 63178-9916; 616452 and 616453 are available from Calbiochem (EMD Chemicals, Inc.), 480 S. Democrat Road, Gibbstown, N.J., 08027; GW788388 and GW6604 are available from GlaxoSmithKline, 980 Great West Road, Brentford, Middlesex, TW8 9GS, United Kingdom; LY580276 available from Lilly Research, Indianapolis, Ind. 46285; and SM16 available from Biogen Idec, P.O. Box 14627, 5000 Davis Drive, Research Triangle Park, N.C., 27709-4627.

[430] WNT Signal Path

[431] Aspects of the invention relate to the use of activators of the WNT signaling pathway as β-cell maturation factors. It is contemplated that any activator of the WNT signaling pathway that is capable, alone or in combination with one or more other β-cell maturation factors, of inducing the differentiation of at least one insulin-producing endocrine cell or its precursor into an SC-β cell can be used in the described methods, compositions, and kits described herein.

[432] In some embodiments, the WNT signaling pathway activator includes CHIR99021. In some embodiments, the WNT signaling pathway activator comprises a derivative of CHTR99021, such as a salt of CHIR99021, such as the trichlorohydrate salt of CHIR99021. In some embodiments, the WNT signaling pathway activator comprises recombinant Wnt3a protein. In some embodiments, the WNT signaling pathway activator includes a glycogen synthase kinase 3 (GSK3) inhibitor. Typical GSK3 inhibitors include, but are not limited to, 3F8, A 1070722, AR-A 014418, BIO, BIO-acetoxime, FRAT-ide, 10Z-hymenialdisine, indirubin-3-oxime, kenpaullone, L803, L803-mts, lithium carbonate, NSC 693868, SB 216763, SB 415286, TC-G 24, TCS 2002, TCS 21311, TWS 119, as well as analogs and derivatives of any of these compounds. In certain embodiments, the methods, compositions, and kits disclosed herein do not include a WNT signaling pathway activator.

[433] Fibroblast growth factor (FGF) family

[434] Aspects of the invention relate to the use of growth factors from the FGF family as β-cell maturation factors. It is contemplated that any growth factor from the FGF family that is capable, alone or in combination with one or more other β-cell maturation factors, of inducing the differentiation of at least one insulin-producing endocrine cell or its precursor into an SC-β cell can be used in the methods, compositions and kits described herein. In certain embodiments, the methods, compositions, and kits disclosed herein do not include a growth factor from the FGF family.

[435] In some embodiments, at least one growth factor from the FGF family includes keratinocyte growth factor (KGF). KGF polypeptide sequences are available to those skilled in the art. In some embodiments, a growth factor from the FGF family comprises a polypeptide having an amino acid sequence of at least 30%, at least 40%, at least 50%, at least 60%, at least 70 %, at least 80%, at least 90%, at least 95%, or at least 99% or more identical to the human KGF polypeptide sequence (GenBank accession number AAB21431).

[436] In some embodiments, at least one growth factor from the FGF family includes FGF2. FGF2 polypeptide sequences are available to those skilled in the art. In some embodiments, a growth factor from the FGF family comprises a polypeptide having an amino acid sequence of at least 30%, at least 40%, at least 50%, at least 60%, at least 70 %, at least 80%, at least 90%, at least 95%, or at least 99% or more identical to the human FGF2 polypeptide sequence (GenBank accession number NP_001997).

[437] In some embodiments, at least one growth factor from the FGF family includes FGF8B. FGF8B polypeptide sequences are available to those skilled in the art. In some embodiments, a growth factor from the FGF family comprises a polypeptide having an amino acid sequence of at least 30%, at least 40%, at least 50%, at least 60%, at least 70 %, at least 80%, at least 90%, at least 95%, or at least 99% or more identical to the human FGF8B polypeptide sequence (GenBank accession number AAB40954).

[438] In some embodiments, at least one growth factor from the FGF family includes FGF10. FGF10 polypeptide sequences are available to those skilled in the art. In some embodiments, a growth factor from the FGF family comprises a polypeptide having an amino acid sequence of at least 30%, at least 40%, at least 50%, at least 60%, at least 70 %, at least 80%, at least 90%, at least 95%, or at least 99% or more identical to the human FGF10 polypeptide sequence (GenBank accession number CAG46489).

[439] In some embodiments, at least one growth factor from the FGF family includes FGF21. FGF21 polypeptide sequences are available to those skilled in the art. In some embodiments, a growth factor from the FGF family comprises a polypeptide having an amino acid sequence of at least 30%, at least 40%, at least 50%, at least 60%, at least 70 %, at least 80%, at least 90%, at least 95%, or at least 99% or more identical to the human FGF21 polypeptide sequence (GenBank accession number AAQ89444.1).

[440] Bone morphogenetic protein (BMP) signaling pathway inhibitors

[441] Aspects of the invention relate to the use of BMP signaling pathway inhibitors as β-cell maturation factors. The BMP signaling family is a diverse subgroup of the TGF-β superfamily (Sebald et al. Biol. Chem. 385: 697-710, 2004). More than twenty known BMP ligands belong to three different type II receptors (BMPRII, ActRIIa and ActRIIb) and at least three type I receptors (ALK2, ALK3 and ALK6). Dimeric ligands facilitate the assembly of receptor heteromers by enabling constitutively active type II receptor serine-threonine kinases to phosphorylate type I receptor serine-threonine kinases. Activated type I receptors phosphorylate BMP-sensitive (BR) SMAD effectors (SMADs 1, 5, and 8) to facilitate nuclear translocation in complex with SMAD4, a co-SMAD that also facilitates TGF signaling. In addition, BMP signals can activate intracellular effectors such as p38 MAPK in a SMAD-independent manner (Nohe et al. Cell Signal 16: 291-299, 2004). Soluble BMP antagonists, such as noggin, chordin, gremlin, and follistatin, limit BMP signaling by sequestering ligands.

[442] Any inhibitor of the BMP signaling pathway that is capable, alone or in combination with one or more other β-cell maturation factors, is contemplated to induce the differentiation of at least one insulin-producing endocrine cell or its precursor into an SC-β cell, can be used in the methods, compositions and kits described herein. In certain embodiments, the methods, compositions, and kits disclosed herein do not include a BMP signaling pathway inhibitor.

[443] In some embodiments, the BMP signaling pathway inhibitor includes LDN 193189 (also known as LDN193189, 1062368-24-4, LDN-193189, DM 3189, DM-3189, IUPAC name: 4-[6-(4- piperazin-1-ylphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinolone).

[444] In some embodiments, the BMP signaling pathway inhibitor includes an analogue or derivative of LDN 193189, such as a salt, hydrate, solvent, ester, or prodrug of LDN 193189. In some embodiments, the derivative (e.g., salt) of LDN 193189 includes LDN193189 hydrochloride.

[445] In some embodiments, the BMP signaling pathway inhibitor includes a compound of Formula I from US Patent Publication No. 2011/0053930.

[446] Sound Hedgehog (SHH) signaling pathway

[447] Aspects of the invention relate to the use of SHH signaling pathway inhibitors as β-cell maturation factors. It is contemplated that any inhibitor of the SHH signaling pathway that is capable, alone or in combination with one or more other β-cell maturation factors, of inducing the differentiation of at least one insulin-producing endocrine cell or its precursor into an SC-β cell can be used in the methods, compositions and kits described herein.

[448] In some embodiments, the SHH signaling pathway inhibitor includes Sant1. In some embodiments, the SHH signaling pathway inhibitor includes SANT2. In some embodiments, the SHH signaling pathway inhibitor includes SANT3. In some embodiments, the SHH signaling pathway inhibitor includes SANT4. In some embodiments, the SHH signaling pathway inhibitor includes Cur61414. In some embodiments, the SHH signaling pathway inhibitor includes forskolin. In some embodiments, the SHH signaling pathway inhibitor includes tomatidine. In some embodiments, the SHH signaling pathway inhibitor includes AY9944. In some embodiments, the SHH signaling pathway inhibitor includes triparanol. In some embodiments, the SHH signaling pathway inhibitor includes Compound A or Compound B (as disclosed in US Pat. No. 2004/0060568). In some embodiments, the SHH signaling pathway inhibitor includes a steroidal alkaloid that antagonizes hedgehog signaling (eg, cyclopamine or a derivative thereof), as disclosed in US Pat. publ. US No. 2006/0276391. In certain embodiments, the methods, compositions, and kits disclosed herein do not include an SHH signaling pathway inhibitor.

[449] Retinoic acid signaling pathway

[450] Aspects of the invention relate to the use of retinoic acid signaling modulators as β-cell maturation factors. It is contemplated that any modulator of retinoic acid signaling that is capable, alone or in combination with one or more other β-cell maturation factors, of inducing the differentiation of at least one insulin-producing endocrine cell or its precursor into an SC-β cell can be used in the methods, compositions and kits described herein.

[451] In some embodiments, the retinoic acid signaling modulator includes an activator of retinoic acid signaling. In some embodiments, the RA signaling pathway activator includes retinoic acid. In some embodiments, the RA signaling pathway activator includes a retinoic acid receptor agonist. Exemplary retinoic acid receptor agonists include, but are not limited to, CD 1530, AM 580, TTNPB, CD 437, Ch 55, BMS 961, AC 261066, AC 55649, AM 80, BMS 753, tazarogen, adapalene, and CD 2314.

[452] In some embodiments, the retinoic acid signaling modulator includes an inhibitor of retinoic acid signaling. In some embodiments, the retinoic acid signaling pathway inhibitor includes DEAB (IUPAC name: 2-[2-(diethylamino)ethoxy]-3-prop-2-enylbenzaldehyde). In some embodiments, the retinoic acid signaling pathway inhibitor includes a DEAB analogue or derivative.

[453] In some embodiments, the retinoic acid signaling pathway inhibitor includes a retinoic acid receptor antagonist. In some embodiments, the retinoic acid receptor antagonist comprises (E)-4-[2-(5,6-dihydro-5,5-dimethyl-8-phenyl-2-naphthalenyl)ethenyl]benzoic acid, (E)-4 -[[(5,6-dihydro-5,5-dimethyl-8-phenylethynyl)-2-naphthalenyl]ethenyl]benzoic acid, (E)-4-[2-[5,6-dihydro-5,5- dimethyl-8-(2-naphthalenyl)-2-naphthalenyl]ethenyl]benzoic acid and (E)-4-[2-[5,6-dihydro-5,5-dimethyl-8-(4-methoxyphenyl)-2 -naphthalenyl]ethenyl]benzoic acid. In some embodiments, the retinoic acid receptor antagonist includes BMS 195614 (CAS No. 253310-42-8), ER 50891 (CAS No. 187400-85-7), BMS 493 (CAS No. 170355-78-9), CD 2665 (CAS No. 170355-78-9), LE 135 (CAS No. 155877-83-1), BMS 453 (CAS No. 166977-43-1) or MM 11253 (CAS No. 345952-44-5).

[454] In certain embodiments, the methods, compositions, and kits disclosed herein do not include a retinoic acid signaling modulator. In certain embodiments, the methods, compositions, and kits disclosed herein do not include a retinoic acid signaling pathway activator. In certain embodiments, the methods, compositions, and kits disclosed herein do not include a retinoic acid signaling pathway inhibitor.

[455] Protein kinase C

[456] Aspects of the invention relate to the use of protein kinase C activators as β-cell maturation factors. Protein kinase C belongs to one of the largest families of protein kinase enzymes and has many isoforms. Traditional isoforms include a, βI, βII, γ; new isoforms include δ, ε, η, Θ; and atypical isoforms include ξ and ι/λ. PKC enzymes are predominantly cytosolic, but upon activation they translocate to the membrane. In the cytoplasm, PKC is phosphorylated by other kinases or autophosphorylated. Activation of some PKC isoforms (eg, PKC-ε) requires a molecule to bind to the diacylglycerol (“DAG”) binding site or phosphatadylserine (“PS”) binding site. Others are able to activate without the involvement of any secondary messengers. PKC activators that bind to the DAG site include, but are not limited to, bryostatin, picologs, phorbol esters, aplysiatoxin, and gnidimacrine. PKC activators that bind to the PS site include, but are not limited to, polyunsaturated fatty acids and their derivatives. It is contemplated that any protein kinase C activator that is capable, alone or in combination with one or more other β-cell maturation factors, of inducing the differentiation of at least one insulin-producing endocrine cell or its precursor into an SC-β cell can be used in the described methods, compositions, and kits described herein.

[457] In some embodiments, the PKC activator includes PdbU. In some embodiments, the PKC activator includes TPB. In some embodiments, the PKC activator includes cyclopropanated polyunsaturated fatty acids, cyclopropanated monounsaturated fatty acids, cyclopropanated polyunsaturated fatty alcohols, cyclopropanated monounsaturated fatty alcohols, cyclopropanated polyunsaturated fatty acid esters, cyclopropanated monounsaturated fatty acid esters, cyclopropanated polyunsaturated fat sulfates acids, cyclopropanated monounsaturated fatty sulfates acids, cyclopropanated polyunsaturated fatty acid phosphates, cyclopropanated monounsaturated fatty acid phosphates, macrocyclic lactones, DAG derivatives, isoprenoids, octilindolactam V, gnidimacrine, iripallidal, ingenol, naphthalene sulfonamides, diacylglycerol kinase inhibitors, fibroblast growth factor 18 (FGF-18), insulin growth factor , hormones and growth factor activators as described in WIPO publication No. WO/2013/071282. In some embodiments, bryostatin includes bryostatin-1, bryostatin-2, bryostatin-3, bryostatin-4, bryostatin-5, bryostatin-6, bryostatin-7, bryostatin-8, bryostatin-9, bryostatin-10, bryostatin-11 , bryostatin-12, bryostatin-13, bryostatin-14, bryostatin-15, bryostatin-16, bryostatin-17 or bryostatin-18. In certain embodiments, the methods, compositions, and kits disclosed herein do not include a protein kinase C activator.

[458] γ-secretase inhibitors

[459] Aspects of the invention relate to the use of γ-secretase inhibitors as β-cell maturation factors. It is contemplated that any γ-secretase inhibitor that is capable, alone or in combination with one or more other β-cell maturation factors, of inducing the differentiation of at least one insulin-producing endocrine cell or its precursor into an SC-β cell can be used in the described methods, compositions, and kits described herein. There are many known γ-secretase inhibitors. In some embodiments, the γ-secretase inhibitor includes XXI. In some embodiments, the γ-secretase inhibitor includes DAPT. Additional exemplary γ-secretase inhibitors include, but are not limited to, the γ-secretase inhibitors described in US Pat. Nos. 7,049,296, 8,481,499, 8,501,813 and WIPO Publication No. WO/2013/052700. In certain embodiments, the methods, compositions, and kits disclosed herein do not include a γ-secretase inhibitor.

[460] Activators of the thyroid hormone signaling pathway

[461] Aspects of the invention relate to the use of thyroid hormone signaling pathway activators as β-cell maturation factors. It is contemplated that any activator of the thyroid hormone signaling pathway that is capable, alone or in combination with one or more other β-cell maturation factors, of inducing the differentiation of at least one insulin-producing endocrine cell or its precursor into an SC-β cell can be used in the methods, compositions and kits described herein. In certain embodiments of any aspect described herein, the methods, compositions, and kits disclosed herein do not include a thyroid hormone signaling pathway activator. In certain embodiments of any aspect described herein, the methods, compositions and kits disclosed herein do not include T3 or a T3 analogue described herein.

[462] In some embodiments, the thyroid hormone signaling pathway activator includes triiodothyronine (T3). In some embodiments, the thyroid hormone signaling pathway activator includes a T3 analogue or derivative. Exemplary T3 analogs include, but are not limited to, selective and non-selective thyromimetics, selective GC-1 TRβ agonist, GC-24,4-hydroxy-RSB 106, MB07811, MB07344,3,5-diiodothyropropionic acid (DITPA); selective GC-1 TR-β agonist; 3-iodothyronamine (T(1)AM) and 3,3',5-triiodothyroacetic acid (Triac) (bioactive metabolites of the hormone thyroxine (T(4)); KB-2115 and KB-141; thyronamines; SKF L-94901; DIBIT; 3'-AC-T2; tetraiodothyroacetic acid (Tetrac) and triiodothyroacetic acid (Triac) (by oxidative deamination and decarboxylation of the alanine chain of thyroxine [T4] and triiodothyronine [T3]), 3,3',5'-triiodothyronine (rT3 ) (by deiodination of T4 and T3), 3,3'-diiodothyronine (3,3'-T2) and 3,5-diiodothyronine (T2) (by deiodination of T4, T3 and rT3) and 3-iodothyronamine (T1AM) and thyronamine (T0AM) (by deiodination and amino acid decarboxylation of T4 and T3), as well as structural analogues of TH, such as 3,5,3'-triiodotyropropionic acid (Triprop), 3,5-dibromo-3-pyridazinone-1-thyronine (L -940901), N-[3,5-dimethyl-4-(4'-hydroxy-3'-isopropylphenoxy)-phenyl]-oxamic acid (CGS 23425), 3,5-dimethyl-4-[(4'- hydroxy-3'-isopropylbenzyl)-phenoxy]acetic acid (GC-1), 3,5-dichloro-4-[(4-hydroxy-3-isopropylphenoxy)-phenyl]acetic acid (KB-141) and 3,5 -diiodotyropropionic acid (DITPA).

[463] In some embodiments, the thyroid hormone signaling pathway activator includes a T3 prodrug or prohormone, such as T4 thyroid hormone (eg, thyroxine or L-3,5,3',5'-tetraiodothyronine).

[464] In some embodiments, the thyroid hormone signaling pathway activator is an iodothyronine composition described in US Pat. No. 7,163,918.

[465] Epidermal growth factor (EGF) family

[466] Aspects of the invention relate to the use of growth factors from the EGF family as β-cell maturation factors. It is contemplated that any growth factor from the EGF family that is capable, alone or in combination with one or more other β-cell maturation factors, of inducing the differentiation of at least one insulin-producing endocrine cell or its precursor into an SC-β cell can be used in the methods, compositions and kits described herein. In some embodiments, at least one growth factor from the EGF family includes betacellulin. In some embodiments, at least one growth factor from the EGF family includes EGF. Epidermal growth factor (EGF) is a 53-amino acid cytokine that is proteolytically cleaved from a large integral membrane protein precursor. In some embodiments, a growth factor from the EGF family includes a variant EGF polypeptide, such as an isolated epidermal growth factor polypeptide, having at least 90% amino acid identity to the wild-type human EGF polypeptide sequence as disclosed in US Pat. No. 7,084,246. In some embodiments, a growth factor from the EGF family includes an engineered mutant of EGF that binds to and agonizes the EGF receptor, as disclosed in US Pat. No. 8,247,531. In some embodiments, at least one growth factor from the EGF family is replaced with an agent that activates the EGF family signaling pathway. In some embodiments, a growth factor from the EGF family includes a compound that mimics EGF. In certain embodiments, the methods, compositions, and kits disclosed herein do not include a growth factor from the EGF family.

[467] Protein kinase inhibitors

[468] Aspects of the invention relate to the use of protein kinase inhibitors as β-cell maturation factors. It is contemplated that any protein kinase inhibitor that is capable, alone or in combination with other β-cell maturation factors, of inducing the differentiation of at least one insulin-producing endocrine cell or its precursor into an SC-β cell can be used in the methods described herein , compositions and sets.

[469] In some embodiments, the protein kinase inhibitor includes staurosporine. In some embodiments, the protein kinase inhibitor includes a staurosporine analogue. Exemplary staurosporine analogues include, but are not limited to, Ro-31-8220, bisindolylmaleimide (Bis) compound, 10'-{5''-[(methoxycarbonyl)amino]-2''-methyl}-phenylaminocarbonylstaurosporine, staralog (see, for example, Lopez et al., “Staurosporine-derived inhibitors broaden the scope of analog-sensitive kinase technology,” J. Am. Chem. Soc. Soc. 2013; 135(48):18153-18159) and cgp41251.

[470] In some embodiments, the protein kinase inhibitor is a PKCβ inhibitor. In some embodiments, the protein kinase inhibitor is a PKCβ inhibitor with the structure below, or a derivative, analog, or variant of the compound below:

[471] In some embodiments, the PKCβ inhibitor is a GSK-2 compound with the structure below, or a derivative, analog, or variant of the compound below:

[472] In some embodiments, the PKC inhibitor is bisindolylmaleimide. Exemplary bisindolylmaleimides include, without limitation, bisindolylmaleimide I, bisindolylmaleimide II, bisindolylmaleimide III, hydrochloride or derivative, analog or variant. In some embodiments, the derivative, analog, or variant of the compound is selected from the derivative, analog, or variant of the compound selected from the compounds:

[473] In some embodiments, the PKC inhibitor is pseudohypericin or a derivative, analog, or variant of the following:

[474] In some embodiments, the PKC inhibitor is indorublin-3-monoxime, 5-iodo, or a derivative, analog, or variant of the compound below:

[475] In certain embodiments, the methods, compositions, and kits disclosed herein do not include protein kinase inhibitors.

[476] Cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor

[477] Aspects of the invention relate to the use of CFTR inhibitors as β-cell maturation factors. It is contemplated that any CFTR inhibitor that is capable, alone or in combination with one or more other β-cell maturation factors, of inducing the differentiation of at least one insulin-producing endocrine cell or its precursor into an SC-β cell may be used as described in methods, compositions and sets in this document.

[478] Numerous CFTR inhibitors used herein are available to those skilled in the art. Exemplary CFTR inhibitors include, but are not limited to, the LPA2 receptor agonist CFTR inhibitors disclosed in US Pat. publ. US No. 2007/0078111, hydrazine-containing CFTR inhibitors disclosed in US Patent No. 7888332, and CFTR inhibitors disclosed in WIPO publication No. WO/2008/121877, CFTR inhibitor compound disclosed in US Pat. publ. US No. 2008/0269206. In some embodiments, the CFTR inhibitor includes a pore glycine-hydrazine scavenging CFTR inhibitor. In some embodiments, the CFTR inhibitor includes Gly-H101. In some embodiments, the CFTR inhibitor includes a derivative or analogue of Gly-H101 (see, for example, Muanprasat et al., "Discovery of Glyciine Hydrazide Pore-occluding CFTR Inhibitors", J. Gen. PHysiol 2004; 124(2):125- 137). In certain embodiments, the methods, compositions, and kits disclosed herein do not include a CFTR inhibitor.

[479] O-GlcNAase inhibitor

[480] Aspects of the invention relate to the use of O-GlcNAse inhibitors as β-cell maturation factors. It is contemplated that any O-GlcNAase inhibitor that is capable, alone or in combination with one or more other β-cell maturation factors, of inducing the differentiation of at least one insulin-producing endocrine cell or its precursor into an SC-β cell can be used in the methods, compositions and kits described herein. Numerous O-GlcNAase inhibitors used herein are available to those skilled in the art. Exemplary O-GlcNAase inhibitors include, but are not limited to, permeable glycosidase inhibitors (see, for example, WIPO Publication No. WO/2013/169576 and WO/2013/166654), and selective glycosidase inhibitors (see, for example, WIPO Publication No. WO/2013/ 000084 and WO/2013/000085). In some embodiments, the O-GlcNAase inhibitor includes Thiamet-G. In certain embodiments, the methods, compositions, and kits disclosed herein do not include an O-GlcNAase inhibitor.

[481] Mixed compositions

[482] Another aspect of the present invention relates to mixing insulin-positive endocrine cells or their precursors and at least one β-cell maturation factor, for example, to induce the differentiation of at least one insulin-positive endocrine cell or its precursor into SC cells. β.

[483] In another aspect, the present invention provides a composition, such as a reaction mixture, comprising at least one insulin-positive endocrine cell or precursor thereof (e.g., a population of insulin-positive endocrine cells or precursors thereof for differentiation into SC-β cells) and at least one β-cell maturation factor. In an alternative embodiment, the present invention provides a reaction mixture comprising (i) a population of SC-β cells obtained by chemically inducing differentiation of a population of insulin-positive endocrine cells or their precursors to differentiate into an SC-β cell, and (ii) at least one β-cell maturation factor.

[484] In some embodiments, the concentration of at least one β-cell maturation factor added to the reaction mixture is a sufficient dose to induce differentiation of at least one insulin-positive endocrine cell or precursor thereof into an SC-β cell, as described in this document.

[485] In some embodiments, the composition contains a concentration of at least one β-cell maturation factor of from about 25 nM to 10 μM, or from about 25 nM to 50 nM, or from about 50 nM to 100 nM, or from about 100 nM to 200 nM, or from about 200 nM to about 500 nM, or from about 500 nM to about 1 μM, or from about 1 μM to 2 μM, or from about 2 μM to 5 μM, or from about 5 μM to 10 µM.

[486] In some embodiments, the composition or mixture contains a concentration of at least one β-cell maturation factor of at least about 5 nM, at least about 7 nM, at least about 10 nM, at least about 12 nM, at least about 15 nM, at least about 17 nM, at least about 20 nM, at least about 25 nM, at least about 30 nM, at least about 35 nM, at least about 40 nM , at least about 45 nM, at least about 50 nM, at least about 100 nM, or at least about 200 nM, or at least about 300 nM, or at least about 400 nM, or at least about 500 nM, or more than 500 nM, or any integer in the range of 10-500 nM, or any integer in the range of 5-50 nM, or any integer in the range of 50-100 nM, or any integer in the range of 100 nM - 200 nM, or any integer in the range 200 nM - 500 nM. In some embodiments, the composition or mixture contains a concentration of at least one β-cell maturation factor of at least about 0.1 μM, or at least about 0.2 μM, or at least about 0.3 μM, or at least about 0.4 μM, or at least about 0.5 μM, or at least about 1 μM, at least about 1.5 μM, at least about 2 μM, at least about 2.5 µM, at least about 3 µM, at least about 3.5 µM, at least about 4 µM, at least about 4.5 µM, at least about 5 µM, at least about 6 µM, at least at least about 7 μM, at least about 8 μM, at least about 9 μM, or at least about 10 μM, or more than 10 μM, or any integer in the range of 0.1-0.5 μM, or any an integer in the range of 0.5-10 μM, or any integer in the range 0.1-10 μM, or any integer in the range 0.5-5 μM, or any integer in the range 5 μM-10 μM.

[487] Compositions and sets

[488] Described herein are compositions that contain a cell described herein (eg, an SC-β cell or a mature pancreatic β cell). In some embodiments, the composition also contains a β-cell maturation factor and/or cell culture media described herein. Also described herein are compositions containing compounds described herein (eg, cell culture media containing one or more compounds described herein). This document describes the kits.

[489] Another aspect of the present invention relates to kits for practicing the methods disclosed herein and for producing SC-β cells or mature pancreatic β cells disclosed herein. In one aspect, the kit contains at least one insulin-positive endocrine cell or precursor thereof and at least one β-cell maturation factor, as described herein, and, optionally, the kit may further contain instructions for converting at least one insulin -positive endocrine cell or its precursor into the SC-β cell population using the methods described herein. In some embodiments, the kit contains at least two β-cell maturation factors. In some embodiments, the kit contains at least three β-cell maturation factors. In some embodiments, the kit contains at least four β-cell maturation factors. In some embodiments, the kit contains at least five β-cell maturation factors. In some embodiments, the kit contains at least six β-cell maturation factors. In some embodiments, the kit contains at least seven β-cell maturation factors. In some embodiments, the kit contains at least eight β-cell maturation factors. In some embodiments, the kit contains at least nine β-cell maturation factors. In some embodiments, the kit contains at least ten β-cell maturation factors. In some embodiments, the kit contains β-cell maturation factors for the differentiation of pluripotent stem cells into definitive endoderm cells. In some embodiments, the kit contains β-cell maturation factors for the differentiation of definitive endoderm cells into primary gut tube cells. In some embodiments, the kit contains β-cell maturation factors for differentiating primary gut tube cells into Pdx1-positive pancreatic progenitor cells. In some embodiments, the kit contains β-cell maturation factors for differentiating NKX6-1-positive pancreatic progenitor cells into insulin-positive endocrine cells. In some embodiments, the kit contains β-cell maturation factors for differentiating insulin-positive endocrine cells into SC-β cells.

[490] In some embodiments, the kit contains any combination of β-cell maturation factors, for example, for differentiating pluripotent stem cells into definitive endoderm cells, differentiating definitive endoderm cells into primary gut tube cells, differentiating primary gut tube cells into Pdx1-positive pancreatic cells -progenitors, differentiation of NKX6-1-positive pancreatic progenitor cells into insulin-positive endocrine cells and differentiation of insulin-positive endocrine cells into SC-β cells.

[491] In one embodiment, the kit may contain a pluripotent stem cell for use as a positive control, for example, to evaluate or monitor the effectiveness or ability of a compound of formula (I) to chemically induce the differentiation of a pluripotent stem cell into at least one insulin-positive endocrine cell or its precursor and subsequently into the SC-β cell. Accordingly, the kit may contain a sufficient amount of at least one β-cell maturation factor to induce differentiation of a control population of pluripotent stem cells (positive control), as well as induce differentiation of a population of pluripotent stem cells of interest (e.g., a user's preferred pluripotent stem cell, e.g. , iPS cells) into at least one insulin-positive endocrine cell or precursor thereof or an SC-β cell.

[492] In some embodiment, the compound in the kit may be provided in a waterproof or gas-tight container, which in some embodiments contains substantially no other components of the kit. The compound may be supplied in more than one container, for example it may be supplied in a container containing sufficient reagents for a predetermined number of reactions, for example 1, 2, 3 or more separate reactions to induce pluripotent stem cells into definitive endoderm cells and subsequently into insulin-positive endocrine cells, and subsequently into SC-β cells. The β-cell maturation factor may be provided in any form, such as liquid, dry or lyophilized form. It is preferred that the compound(s) described herein (eg, β-cell maturation factors) be substantially purified and/or sterile. When the compound(s) described herein are provided as a liquid solution, the liquid solution is preferably an aqueous solution, with a sterile aqueous solution being preferred. If the compound(s) described herein are provided in dry form, recovery is generally accomplished by adding a suitable solvent. A solvent, such as sterile water or buffer, may optionally be provided in the kit.

[493] In some embodiments of the invention, the kit optionally further contains information material. The informational material may be descriptive, instructional, marketing or other material that relates to the methods described herein and/or the use of the compound(s) described herein in the methods described herein.

[494] The information material in the kits is not limited in its instructional or informational content. In one embodiment of the invention, the information material may include information about the production of the compound, molecular weight of the compound, concentration, expiration date, lot number and place of manufacture information, and so on. In one embodiment of the invention, the information material relates to methods of administering the compound. In addition, the information material from the sets is not limited in its form. In many cases, information material, such as instructions, is provided in printed form, such as printed text, an image and/or a photograph, such as a label or printed sheet. However, the information material may also be provided in other formats, such as braille, machine-readable material, video or audio. In another embodiment of the invention, the information material from the kits contains contact information, for example, a physical address, email address, website or telephone number, where the user of the kit can obtain comprehensive information about the compound described herein and/or its use in the described in methods in this document. Of course, information material can also be provided in any combined format.

[495] In one embodiment, the information material may contain instructions for administering the compound(s) described herein (e.g., β-cell maturation factor) in a manner suitable for carrying out the methods described herein, e.g., in a suitable dosage, dosage form or by a suitable route of administration (eg, in the dosage, dosage form, or route of administration described herein) (eg, into an in vitro cell or an in vivo cell). In another embodiment, the information material may contain instructions for administering the compound(s) described herein to a suitable subject, for example, a human, for example, a person at risk of having a disorder described herein, or into a cell in vitro.

[496] In addition to the compound(s) described herein, the kit composition may contain other ingredients, such as a solvent or buffer, a stabilizer, a preservative, a flavoring agent (e.g., a bittering agent or sweetener), a fragrance or other cosmetic ingredient, and/or an additional agent, for example, for the induction of pluripotent stem cells (eg, in vitro) or for the treatment of a pathological condition or disorder described herein. Alternatively, other ingredients may be included in the kit, but in different compositions or in different containers than the compound described herein. In such embodiments, the kit may contain instructions for mixing the compound(s) described herein and other ingredients or for using the compound(s) described herein together with other ingredients, for example, instructions for mixing the two agents before administration.

[497] The β-cell maturation factor described herein may be provided in any form, such as liquid, dry or lyophilized form. Preferably, the compound(s) described herein are substantially purified and/or sterile. When the compound(s) described herein are provided as a liquid solution, the liquid solution is preferably an aqueous solution, with a sterile aqueous solution being preferred. If the compound(s) described herein are provided in dry form, recovery is generally accomplished by adding a suitable solvent. A solvent, such as sterile water or buffer, may optionally be provided in the kit.

[498] The kit may contain one or more containers for a composition containing at least one β-cell maturation factor described herein. In some embodiments, the kit contains separate containers (eg, two separate containers for two agents), partitions or compartments for the composition(s) and information material. For example, the composition may be in a bottle, vial or syringe, and the information material may be in a plastic file or bag. In other embodiments of the invention, the individual elements of the set are contained in a single, non-divided container. For example, the composition is in a bottle, vial or syringe with attached information material in the form of a label. In some embodiments, the kit contains a plurality (eg, a package) of individual containers, each of which contains one or more unit dosage forms (eg, a dosage form described herein) of a compound described herein. For example, a kit contains a plurality of syringes, ampoules, foil pouches or blister packs, each containing a unit dose of a compound described herein. Kit containers may be airtight, waterproof (for example, resistant to changes in humidity or evaporation) and/or lightproof.

[499] The kit optionally contains a device suitable for administering the composition, such as a syringe, inhaler, pipette, forceps, measuring spoon, dropper (eg, eye dropper), swab (eg, cotton swab or wood swab), or any similar device For delivery. In a preferred embodiment of the invention, the device is a medical implant device, for example, packaged for surgical use.

[500] The kit may also contain a component for detecting an SC-β cell marker, such as a marker described herein, such as a positive SC-β cell detection reagent. Or, in some embodiments, the kit may also contain reagents for detecting negative SC-β cell markers for negative selection of SC-β cells or for identifying cells that do not express negative markers (eg, SC-β cells). The reagents may be, for example, an antibody to a marker or primers for an RT-PCR or PCR reaction, for example, a semi-quantitative or quantitative RT-PCR or PCR reaction. Such markers can be used to determine whether an iPS cell has been obtained. If the detection reagent is an antibody, it may be provided as a dry preparation, such as lyophilized, or in solution. The antibody or other detection reagent may be coupled to a label, such as a radioactive, fluorescent (eg, ZPB) or colorimetric label for use in detection. If the detection reagent is a primer, it may be provided as a dry preparation, such as lyophilized, or in solution.

[501] There may be a need to perform karyotype analysis of SC-β cells. Accordingly, the kit may contain a karyotyping component, such as a probe, dye, substrate, enzyme, antibody, or other reagents used to obtain a karyotype of a cell.

[502] The kit may contain SC-β cells, for example, mature pancreatic β-cells, derived from insulin-positive endocrine cells or their precursors of the same type, for example, for use as a positive control cell type.

[503] The kit may also contain informational materials, such as instructions, for the use of two or more components included in the kit.

[504] The informational material may be descriptive, instructional, marketing or other material that relates to the methods described herein and/or the use of the compound(s) described herein for differentiating a pluripotent stem cell in accordance with the methods described herein. In one embodiment of the invention, the information material may include information about the production of the compound, molecular weight of the compound, concentration, expiration date, lot number and place of manufacture information, and so on. In one embodiment, the information material relates to methods of culturing a population of insulin-positive endocrine cells in the presence of at least one β-cell maturation factor described herein.

[505] Methods of introducing cells

[506] In one embodiment, the cells described herein, eg, a population of SC-β cells, are transplantable, eg, a population of SC-β cells can be administered to a subject. In some embodiment, the subject to whom the SC-β cell population is administered is the same subject from which the pluripotent stem cell was obtained for differentiation into an SC-β cell (eg, for autologous cell therapy). In some embodiments, the subject is another subject. In some embodiments, the subject suffers from diabetes, such as type I diabetes, or is a normal subject. For example, cells for transplantation (eg, a composition containing a population of SC-β cells) may be in a form suitable for transplantation, eg, organ transplantation.

[507] The method may further comprise administering the cells to a subject in need thereof, such as a mammalian subject, such as a human subject. The source of the cells may be a mammal, preferably a human. The source or recipient of the cells may also be a non-human subject, such as an animal model. The term "mammal" includes organisms that include mice, rats, cows, sheep, pigs, rabbits, goats, horses, monkeys, dogs, cats and, preferably, humans. Likewise, the transplanted cells can be obtained from any of these organisms, including a non-human transgenic organism. In one embodiment of the invention, the transplanted cells are genetically engineered, eg, the cells contain an exogenous gene or have been genetically engineered to inactivate or alter an endogenous gene.

[508] A composition containing a population of SC-β cells can be administered to a subject using an implantable device. Implantable devices and related technologies are known in the art and are useful as delivery systems when sustained or time-limited release delivery of the compounds or compositions described herein is desired. In addition, an implantable device delivery system is useful for targeting specific sites for delivery of a compound or composition (eg, specific sites, organs). Negrin et al., Biomaterials, 22(6):563 (2001). Time-limited release technology that utilizes alternative delivery methods can also be used in this invention. For example, time-limited release formulations based on polymer technologies, sustained release technologies, and encapsulation technologies (eg, polymer, liposomal) can also be used to deliver the compounds or compositions described herein.

[509] Pharmaceutical compositions containing a population of insulin-producing glucose-sensitive cells

[510] To administer to a subject a population of cells obtained by methods disclosed herein, for example, a population of SC-β cells (obtained by contacting at least one insulin-positive endocrine cell with at least one β-cell maturation factor (eg , one, two, three, four, five or more β-cell maturation factors described herein)) can be administered to a subject, for example, in pharmaceutically acceptable compositions. These pharmaceutically acceptable compositions contain a therapeutically effective amount of the SC-β cell population as described above, admixed together with one or more pharmaceutically acceptable carriers (additives) and/or diluents.

[511] As described in more detail below, the pharmaceutical compositions of the present invention can be specifically formulated for administration in solid or liquid form, including those suitable for: (1) oral administration, for example, liquid dosage forms (aqueous or non-aqueous solutions or suspensions), lozenges, dragees, capsules, pills, tablets (for example, intended for buccal, sublingual or systemic absorption), boluses, powders, granules, tongue pastes; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection such as a sterile solution or suspension or sustained release preparation; (3) topical administration, such as a cream, ointment, or controlled-release skin patch or spray; (4) intravaginal or rectal, for example, in the form of a pessary, cream or foam; (5) sublingual; (6) ophthalmic; (7) transdermal; (8) transmucosal; or (9) nasal administration. In addition, the compounds can be implanted or injected into the patient's body using a drug delivery system. See, for example, Urquhart, et al., Ann. Rev. Pharmacol. Toxicol. 24: 199-236 (1984); Lewis, ed. "Controlled Release of Pesticides and Pharmaceuticals" (Plenum Press, New York, 1981); US Patent No. 3,773,919; and US Patent No. 35 3270960.

[512] As used herein, the term “pharmaceutically acceptable” refers to those compounds, materials, compositions and/or dosage forms that are medically suitable for use in contact with tissues of humans and animals without causing undue toxicity, irritation, or allergic reactions. or other problems or complications and are characterized by a rational benefit/risk ratio.

[513] As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid excipient, diluent, excipient, manufacturing aid (e.g., lubricant, talc, calcium or zinc stearate or steric acid) or solvent-encapsulating material involved in the transfer of a specific compound from one organ or part of the body to another organ or part of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the drug and not harmful to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars such as glucose, lactose and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients such as coconut oil and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols such as propylene glycol; (11) polyols such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline solution; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents such as polypeptides and amino acids (23) serum components such as serum albumin, HDL and LDL; (22) C ₂ -C ₁₂ alcohols such as ethanol; and (23) other non-toxic compatible substances used in pharmaceutical preparations. The preparation may also contain wetting agents, dyes, release agents, coating agents, sweeteners, flavoring agents, flavoring agents, preservatives and antioxidants. Terms such as “excipient”, “carrier”, “pharmaceutically acceptable carrier” and the like are used interchangeably herein.

[514] The expression "therapeutically effective amount" as used herein in relation to a population of cells means that the number of corresponding cells in a population of cells, for example, SC-β cells or mature pancreatic β cells, or a composition containing SC-β cells according to the present invention, is effective in the sense of providing the desired therapeutic effect in at least a subpopulation of cells in the animal's body with a rational benefit/risk ratio applicable to any type of treatment. For example, an amount of a population of SC-β cells administered to a subject that is sufficient to result in a statistically significant, detectable change in at least one symptom of type 1, type 1.5 or type 2 diabetes, such as glycosylated hemoglobin level, glucose level in the blood on an empty stomach, hypoinsulinemia, etc. Methods for determining a therapeutically effective amount are well known to those skilled in the art. In general, the therapeutically effective amount may vary depending on the subject's medical history, age, condition, sex, as well as the severity and type of condition of the subject and the administration of other pharmaceutically active agents.

[515] As used herein, the term “administer” refers to introducing the composition into the body of a subject in a manner or manner that results in at least partial localization of the composition at the desired location in the body so that the desired effect is produced. A compound or composition described herein can be administered by any suitable route known in the art, including, but not limited to, oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, inhalation (aerosol), inhalation, nasal, rectal and local (including buccal and sublingual) administration.

[516] Typical modes of administration include, but are not limited to, injection, infusion, infusion, inhalation, or ingestion. “Injection” includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraocular, intracardial, intradermal, intraperitoneal, transtracheal, subcutaneous, intradermal, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrathoracic injection and infusion. ju. In preferred embodiments of the invention, the compositions are administered by intravenous infusion or injection.

[517] By “treating,” “preventing,” or “alleviating” a disease or disorder, we mean delaying or preventing the onset of such disease or disorder, reversing, mitigating, alleviating, suppressing, slowing, or stopping the progression, aggravation, or worsening of the progression or severity of a pathological condition, associated with such a disease or disorder. In one embodiment of the invention, the symptoms of the disease or disorder are alleviated by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%.

[518] Treatment of diabetes is determined by standard medical methods. The goal of diabetes treatment is to reduce blood sugar levels to as close to normal as safely possible. Typically goals are 80-120 milligrams per deciliter (mg/dL) on an empty stomach and 100-140 mg/dL at bedtime. A given healthcare provider may have different goals for a patient depending on other factors, such as how often the patient experiences hypoglycemic reactions. Medical tests used include tests of the patient's blood and urine to determine blood sugar levels, studies of glycosylated hemoglobin (HbA1c; a measurement of the average blood glucose level over the past 2-3 months, the normal range of which is 4-6%), studies of cholesterol levels and fat, as well as testing the level of protein in the urine. Such studies are standard studies known to those skilled in the art (see, for example, American Diabetes Association, 1998). A successful treatment program can also be determined by reducing the number of patients in the program who have diabetes-related complications, such as eye disease, kidney disease, or nerve disease.

[519] Delaying the onset of diabetes in a subject refers to delaying the onset of at least one symptom of diabetes, for example, hyperglycemia, hypoinsulinemia, diabetic retinopathy, diabetic nephropathy, blindness, memory loss, renal failure, cardiovascular disease (including coronary artery disease, peripheral artery disease, cerebrovascular disease, atherosclerosis and high blood pressure), neuropathy, autonomic dysfunction, hyperglycemic hyperosmolar coma or combinations thereof, for at least 1 week, at least 2 weeks, at least 1 month, at least for 2 months, at least 6 months, at least 1 year, at least 2 years, at least 5 years, at least 10 years, at least 20 years, at least 30 years, at least 40 years or more, and may include the entire lifespan of the subject.

[520] In certain embodiments of the invention, the subject is a mammal, such as a primate, such as a human. The terms “patient” and “subject” are used interchangeably throughout this document. Preferably the subject is a mammal. The mammal may be, but is not limited to, a human, monkey, mouse, rat, dog, cat, horse or cow. Mammals other than humans may be advantageously used as subjects that represent animal models of type 1 diabetes, type 2 diabetes, diabetes mellitus or pre-diabetic conditions. In addition, the methods described herein can be used to treat domesticated animals and/or companion animals. The subject can be male or female. The subject may be someone who has previously been diagnosed or diagnosed with diabetes (eg, type 1 or type 2), one or more complications associated with diabetes, or a prediabetic condition, and, optionally, who has previously been treated for diabetes, one or more complications diabetes-related, or pre-diabetic conditions. The subject may also be someone who does not suffer from diabetes or a pre-diabetic condition. A subject may also be someone who has been diagnosed or diagnosed with diabetes, one or more diabetes-related complications, or a prediabetic condition, but who has demonstrated improvement in known diabetes risk factors as a result of receiving one or more diabetes treatments, one or more complications associated with diabetes or prediabetic conditions. Alternatively, the subject may also be one who has not previously been diagnosed with diabetes, one or more complications associated with diabetes, or a pre-diabetic condition. For example, a subject may be one who demonstrates the presence of one or more risk factors for diabetes, complications associated with diabetes, or a prediabetic condition, or a subject who does not demonstrate risk factors for diabetes, or a subject who is asymptomatic with respect to diabetes, one or more complications, associated with diabetes, or prediabetic condition. The subject may also be someone who has or is at risk of diabetes or a pre-diabetic condition. A subject may also be one who has been diagnosed or identified with one or more complications associated with diabetes or a prediabetic condition as defined herein, or alternatively, a subject may be one who has not previously been diagnosed or identified with one or more more complications associated with diabetes, or a prediabetic condition.

[521] As used herein, the term “subject requiring SC-β cells” refers to a subject who has been diagnosed, identified, or is at risk for developing diabetes (eg, type 1, type 1.5, or type 2), one or more diabetes-related complications or a prediabetic condition.

[522] A subject requiring a population of SC-β cells can be identified using any of the methods used to diagnose diabetes. For example, type 1 diabetes can be diagnosed using a glycosylated hemoglobin (A1C) test, a random blood glucose test, and/or a fasting blood glucose test. Parameters for diagnosing diabetes are known in the art and are readily available to those skilled in the art.

[523] In some embodiments, the methods of the invention further comprise selecting a subject that is in need of additional SC-β cells. The subject in need of the SC-β cell population may be selected based on presenting symptoms, such as symptoms of type 1, type 1.5 or type 2 diabetes. Typical symptoms of diabetes include, but are not limited to, increased thirst (polydipsia), frequent urination (polyuria), increased hunger (polyphagia), increased fatigue, weight loss, hyperglycemia, low insulin levels, high blood sugar (eg, sugars greater than 250 mg, greater than 300 mg), ketones in the urine, fatigue, dry and/or irritated skin, blurred vision, slow-healing cuts and wounds, more infections than usual, numbness and tingling in the feet, diabetic retinopathy, diabetic nephropathy, blindness, memory loss, renal failure, cardiovascular disease (including coronary artery disease, peripheral artery disease, cerebrovascular disease, atherosclerosis and high blood pressure), neuropathy, autonomic dysfunction, hyperglycemic hyperosmolar coma, or combinations thereof.

[524] In some embodiments, a composition comprising a population of SC-β cells for administration to a subject may further comprise a pharmaceutically active agent, such as agents known in the art for the treatment of diabetes or agents having hypoglycemic activity, such as inhibitors dipeptidyl peptidases 4 (DPP-4) (eg, alogliptin, linagliptin, saxagliptin, sitagliptin, vildagliptin and berberine), biguanides (eg, metformin, buformin and phenformin), peroxisome proliferator-activated receptor (PPAR) modulators such as thiazolidinediones (TZDs) (eg, pioglitazone, rivoglitazone, rosiglitazone, and troglitazone), dual PPAR agonists (eg, aleglitazar, muraglitazar, and tezaglitazar), sulfonylureas (eg, acetohexamide, carbutamide, chlorpropamide, gliclazide, tolbutamide, tolazamide, glibenclamide (glyburide), glipizide, glyquidone , glyclopyramide and glimepiride), meglitinides (“glinides”) (eg, nateglinide, repaglinide and mitiglinide), glucagon-like peptide-1 (GLP-1) and its analogues (eg, exendin-4, exenatide, liraglutide, albiglutide), insulin and insulin analogues (eg, insulin lispro, insulin aspart, insulin glulisine, insulin glargine, insulin detemir, Exubera and NPH insulin), alpha-glucosidase inhibitors (eg, acarbose, miglitol and voglibose), amylin analogues (eg, pramlintide), inhibitors sodium-dependent glucose cotransporter T2 (SGLT T2) (eg, dapagliflozin, remogliflozin and sergliflozin) and others (eg, benfluorex and tolrestat).

[525] In type 1 diabetes, unwanted destruction of β-cells occurs due to a continuous autoimmune response. Therefore, this autoimmune response can be weakened by the use of compounds that suppress or block such an autoimmune response. In some embodiments, a composition comprising a population of SC-P cells for administration to a subject may further comprise a pharmaceutically active agent that is a modulator of the immune response. As used herein, the term “immune response modulator” refers to a compound (eg, a small molecule, antibody, peptide, nucleic acid, or gene therapy reagent) that suppresses an autoimmune response in a subject. Without being limited by theory, an immune response modulator suppresses the autoimmune response by inhibiting the activity, activation, or expression of inflammatory cytokines (eg, IL-12, IL-23, or IL-27) or STAT-4. Exemplary immune response modulators include, but are not limited to, members of the group consisting of lysophylline (LSF) and LSF analogs and derivatives described in US Pat. No. 6,774,130, the contents of which are incorporated herein by reference in their entirety.

[526] The composition containing SC-β cells can be administered to the subject at the same time or at different times with the administration of the pharmaceutically active agent or composition containing it. When administered at different times, compositions containing a population of SC-β cells and/or a pharmaceutically active agent for administration to a subject may be administered over 5 minutes, 10 minutes, 20 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 8 hours, 12 hours, 24 hours after the introduction of another. If the compositions containing the SC-β cell population and the composition containing the pharmaceutically active agent are administered in different pharmaceutical compositions, the route of administration may be different. In some embodiments, a composition comprising SC-β cells is administered to a subject. In other embodiments, the subject is administered a composition containing a pharmaceutically active agent. In another embodiment, the subject is administered compositions containing a population of SC-β cells admixed with a pharmaceutically active agent. In another embodiment, the subject is administered a composition comprising a population of SC-β cells and a composition containing a pharmaceutically active agent, the administration occurring at substantially the same time or sequentially.

[527] The toxicity and therapeutic efficacy of administration of compositions containing a population of SC-β cells can be determined using standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose , therapeutically effective for 50% of the population). Preferred are compositions containing a population of SC-β cells that exhibit high therapeutic indices.

[528] The amount of a composition containing a population of SC-β cells can be tested in several well-known animal models.

[529] The non-obese diabetic (NOD) mouse carries a genetic defect that results in insulitis manifesting within a few weeks of age (Yoshida et al., Rev. Immunogenet. 2:140, 2000). 60-90% of females develop clinical diabetes mellitus after 20-30 weeks. The immune pathology is similar to that observed in type I diabetes in humans. Other models of type I diabetes are mice carrying transgenic mutations and gene knockout mutations (Wong et al., Immunol. Rev. 169:93, 1999). Recently, Lenzen et al. (Diabetologia 44:1189, 2001) reported a rat model of spontaneous type I diabetes. Hyperglycemia (>500 mg glucose/dL) can also be induced in mice by a single intraperitoneal injection of streptozotocin (Soria et al., Diabetes 49:157, 2000) or by sequential application of low doses of streptozotocin (Ito et al., Environ. Toxicol. Pharmacol 9:71, 2001). To examine the effectiveness of implanted islet cells, mice are monitored for the return of glucose to normal levels (<200 mg/dL).

[530] Larger animals provide a good model for studying the complications of chronic hyperglycemia. Dogs can be rendered insulin dependent by removal of the pancreas (J. Endocrinol. 158:49, 2001) or by feeding galactose (Kador et al., Arch. Opthalmol. 113:352, 1995). There is also a hereditary model of type I diabetes in Keeshond dogs (Am. J. Pathol. 105:194, 1981). One of the first works with a canine model (Banting et al., Can. Med. Assoc. J. 22:141, 1922) led to two Canadians making a long ocean voyage to Stockholm in February 1925.

[531] By way of illustration, a pilot study can be performed by implanting SC-β cells into the following animals: a) non-diabetic nude (T-cell deficient) mice; b) nude mice with streptozotocin-induced diabetes; and c) nude mice during islet regeneration after partial pancreatectomy. The number of transplanted cells is equivalent to ^~ 1000-2000 normal human β-cells implanted under the renal capsule, liver or pancreas. In nondiabetic mice, endpoints may include graft survival (by histology) and insulin production by biochemical assays, RIA, ELISA, and immunohistochemistry. Survival, metabolic regulation (blood glucose), and weight gain can also be assessed in streptozotocin-treated and partially pancreatectomized animals.

[532] In some embodiments, data obtained from cell culture assays and animal studies can be used to determine dosage ranges for use in humans. The dosage of such compounds preferably corresponds to a circulating concentration range that includes the ED50 with little or no toxicity. Dosage may vary within this range depending on the dosage form used and route of administration.

[533] The therapeutically effective dose of a composition containing a population of SC-β cells can also be initially assessed using cell culture assays. Dosing can be determined in in vivo animal models to achieve insulin secretion at an appropriate concentration in response to circulating plasma glucose levels. Alternatively, the effect of any particular dosage can be monitored using a suitable bioassay method.

[534] To make decisions regarding the duration and frequency of treatment, clinicians typically monitor subjects to determine whether the treatment is having a beneficial therapeutic effect and to determine whether the dosage should be reduced or increased, or the frequency of administration reduced or increased. interrupt treatment, continue treatment, or make other changes to the treatment regimen. The dosage regimen may vary from once weekly to daily depending on a number of clinical factors such as the subject's sensitivity to the polypeptides. The required amount can be administered at one time or divided into divided doses, for example, 2-4 divided doses, and administered over a period of time, for example, at appropriate intervals throughout the day or according to another schedule. Such fractional doses may be administered in unit dosage forms. In some embodiments, administration is continuous, such as one or more doses daily over a period of weeks or months. Examples of dosage regimens include daily administration, twice daily, three times daily, or four or more times daily for a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months. , 5 months or 6 months or more.

[535] In another aspect of the invention, the methods provide the use of a population of SC-β cells as disclosed herein. In one embodiment of the invention, an isolated population of SC-β cells disclosed herein can be used to produce a pharmaceutical composition for transplantation into subjects in need of treatment, for example, a subject who has or is at risk of developing diabetes, for example, without limitation, subjects with hereditary or acquired diabetes. In one embodiment of the invention, the isolated SC-β cell population may be genetically modified. In another aspect, the subject may be ill or at risk for diabetes and/or a metabolic disorder. In some embodiments, the isolated SC-β cell population disclosed herein may be autologous and/or allogeneic. In some embodiments, the subject is a mammal, and in other embodiments, the mammal is a human.

[536] The use of an isolated SC-β cell population disclosed herein has advantages over existing methods because the SC-β cell population can be differentiated from insulin-positive endocrine cells or stem cell-derived precursors thereof, e.g. iPS obtained or collected from a subject injected with an isolated population of SC-β cells. This is a huge advantage as it provides a renewable source of SC-β cells that can be differentiated from stem cells into insulin-positive endocrine cells by methods known to one skilled in the art, and then further differentiated by the methods described herein into pancreatic β-like cells. cells or cells having the characteristics of pancreatic β-like cells for transplantation into a subject, particularly a largely pure population of mature pancreatic β-like cells that do not carry the risks and limitations of cells derived from other systems.

[537] In another embodiment, an isolated population of SC-β cells (eg, mature pancreatic β-cells or β-like cells) can be used as models to study the differentiation properties of insulin-producing cells, such as pancreatic β-cells or pancreatic β-like cells, or pathways for cells of endodermal origin to develop into pancreatic β-cells.

[538] In some embodiments, insulin-positive endocrine cells or SC-β cells can be genetically engineered to contain markers operably linked to promoters that are expressed when the marker is expressed or secreted, for example, the marker can be operably linked to insulin promoter and thus marker expression occurs when insulin-positive endocrine cells or their precursors differentiate into SC-β cells that express and secrete insulin. In some embodiments, the SC-β cell population can be used as a model to study the differentiation pathway of cells that differentiate into islet β cells or pancreatic β-like cells.

[539] In other embodiments, insulin-producing glucose-sensitive cells can be used as models to study the role of islet β cells in the pancreas and in the development of diabetes and metabolic disorders. In some embodiments, the SC-β cells may come from a normal subject or a subject that carries a mutation and/or polymorphism (for example, in the Pdx1 gene, which leads to early-onset non-insulin-dependent diabetes mellitus (NIDDM) as well as adult-onset diabetes mellitus in young type 4 (MODY4), which can be used to identify small molecules and other therapeutic agents that can be used to treat diabetic subjects carrying a mutation or polymorphism in Pdx1. In some embodiments, SC-β cells can be genetically engineered to to correct a polymorphism in the Pdx1 gene prior to administration to a subject during therapeutic treatment of a subject with diabetes.In some embodiments, SC-β cells may be genetically engineered to carry the mutation and/or polymorphism.

[540] In one embodiment, the invention provides a method of treating diabetes or a metabolic disorder in a subject, comprising administering an effective amount of a composition comprising a population of SC-β cells disclosed herein to a subject with diabetes and/or a metabolic disorder. In a further embodiment, the invention provides a method of treating diabetes, comprising administering a composition comprising a population of SC-β cells disclosed herein to a subject who has or is at increased risk of developing diabetes in an effective amount sufficient to produce insulin in response to the elevated levels of blood glucose.

[541] In one embodiment of the above methods, the subject is a human and the SC-β cell population disclosed herein is human cells. In some embodiments, the invention provides that the SC-β cell population disclosed herein is administered directly to the pancreas of a subject or administered in a systemic manner. In some embodiments, the SC-β cell population disclosed herein can be administered at any suitable location in the body of a subject, such as in a capsule in a blood vessel or liver, or at any suitable site where the administered SC-β cell population can secrete insulin in response to elevated blood glucose levels.

[542] The present invention also provides a method for treating a subject with diabetes or a metabolic disorder that occurs due to a genetic defect, physical trauma, environmental or air pollution, poor health, obesity, and other risk factors for diabetes known to one skilled in the art. technology. The effectiveness of treatment of a subject administered a composition containing a population of SC-β cells can be monitored by clinically accepted criteria and tests, which include, for example, (i) a glycated hemoglobin (A1C) test, which provides information on the average blood sugar level of the subject over the past two to three months, based on determining the percentage of blood sugar attached to hemoglobin, the oxygen-carrying protein in red blood cells. The higher the blood sugar level, the more sugar is attached to hemoglobin. An A1C level of 6.5 percent or higher on two separate samples indicates the subject has diabetes. A value of 6-6.5% suggests that the subject has prediabetes. (ii) Random blood sugar levels. A blood sample is drawn from the subject at a randomly determined time, and a random blood sugar level of 200 milligrams per deciliter (mg/dL) - 11.1 millimoles per liter (mmol/L) or higher indicates that the subject has diabetes, ( iii) Fasting blood sugar test. A blood sample is taken from the subject after an overnight fast. A fasting blood sugar level of 70–99 mg/dL (3.9 and 5.5 mmol/L) is normal. If a subject's fasting blood sugar level is 126 mg/dL (7 mmol/L) or higher on two separate samples, the subject has diabetes. A fasting blood sugar level of 100 to 125 mg/dL (5.6 to 6.9 mmol/L) indicates that the subject has prediabetes. (iv) Oral glucose tolerance test. A blood sample is taken from the subject after the subject has fasted for at least eight hours or overnight and then drunk a sugar solution, and the blood sugar level is determined two hours later. A blood sugar level of less than 140 mg/dL (7.8 mmol/L) is normal. A blood sugar level between 140 and 199 mg/dL (7.8 and 11 mmol/L) is considered prediabetic. This is sometimes called impaired glucose tolerance (IGT). A blood sugar level of 200 mg/dL (11.1 mmol/L) or higher indicates that the subject has diabetes.

[543] In some embodiments, the effect of administering a population of SC-β cells disclosed herein to a subject in need thereof is associated with improved exercise capacity or other quality of life measures, as well as reduced mortality. The effect of cell therapy with a population of SC-β cells can occur within days to weeks after the procedure. However, beneficial effects can appear within a few hours after the procedure and can last for several years. In some embodiments, the effect of cell therapy with a population of SC-β cells is evident within two weeks after the procedure.

[544] In some embodiments, the SC-β cell population disclosed herein can be used to repair or regenerate tissue of a human patient or other subject in need of such treatment. In some embodiments, compositions containing populations of SC-β cells can be administered in a manner that allows engraftment or migration to the desired tissue site and repair or regenerate the functionally deficient area. Special devices are available that are adapted to administer cells capable of restoring the β-cell population in the pancreas or other desired location. Accordingly, SC-β cells can be introduced into the recipient's pancreas by injection or administered by intramuscular injection.

[545] In some embodiments, compositions containing the SC-β cell population disclosed herein have a variety of clinical, research, development, and commercial uses. For example, for therapeutic purposes, the SC-β cell population disclosed herein can be administered to enhance insulin production in response to elevated blood glucose levels in any apparent need, such as an inborn error of metabolic function resulting from a disease condition (eg, diabetes) or the result of a characteristic injury (ie, pancreatic injury or islet β-cell loss or damage). In some embodiments, a population of SC-β cells disclosed herein is administered to a subject not only to help restore the function of damaged or otherwise unhealthy tissues, but also to facilitate the remodeling of damaged tissues.

[546] To determine whether cell compositions are suitable for therapeutic administration, the SC-β cell population can first be tested in a suitable animal model. At one level, cells are assessed for their ability to survive and maintain their phenotype in vivo. Cellular compositions containing SC-β cells can be administered to immunodeficient animals (such as nude mice, or animals with chemically or radiation-induced immunodeficiency). Tissues are collected during regrowth and the presence of injected cells or their progeny is assessed.

[547] This can be accomplished by introducing cells that express a detectable label (such as green fluorescent protein or β-galactosidase); that have been pre-labeled (eg with BrdU or [3H]thymidine), or by subsequent detection of a constitutive cellular marker (eg using a human-specific antibody). The presence and phenotype of the administered SC-β cell population can be assessed by immunohistochemistry or ELISA using a human-specific antibody or by RT-PCR analysis using primers and hybridization conditions that result in amplification specific for human polynucleotides in accordance with published data on conditions.

[548] A large number of animal models for diabetes research are available and known in the art, for example, as disclosed in US Pat. No. 6,187,991, which is incorporated herein by reference, as well as rodent-based models; NOD (non-obese) mice, BB_DB mice, KDP rats and TCR mice and other animal models of diabetes as described in Rees et al, Diabet Med. April 2005; 22(4):359-70; Srinivasan K, et al., Indian J Med. Res. March 2007; 125(3):451-7; Chatzigeorgiou A, et al., In Vivo. 2009 March-April; 23(2):245-58, which are incorporated herein by reference.

[549] In some embodiments, the SC-β cell population disclosed herein can be administered in any physiologically acceptable excipient and the SC-β cells can find an appropriate site for replication, proliferation, and/or engraftment. In some embodiments, the SC-β cell population disclosed herein can be administered by injection, catheter, or the like. In some embodiments, the SC-β cell population disclosed herein can be frozen at liquid nitrogen temperature and stored for an extended period of time for use after thawing. If frozen, the SC-β cell population is typically maintained in a medium containing 10% DMSO, 50% FBS, 40% RPMI 1640. Once thawed, the cells can be expanded by application of growth factors and/or cell feeders relevant to SC-β cell culture , as described in this document.

[550] In some embodiments, the SC-β cell population disclosed herein may be provided in the form of a pharmaceutical composition containing an isotonic excipient prepared under sufficiently sterile conditions for use in humans. For general principles of drug preparation, see Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy, by G. Morstyn & W. Sheridan eds, Cambridge University Press, 1996; and Hematopoietic Stem Cell Therapy, E. D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000. The selection of cellular excipient and any accompanying elements for a composition containing the SC-β cell population disclosed herein is tailored according to the route and device used for administration. In some embodiments, a composition comprising a population of SC-β cells may also contain or be supplemented with one or more other ingredients that facilitate the engraftment or functional mobilization of SC-β cells. Suitable ingredients include matrix proteins that promote or stimulate adhesion of SC-β cells or complementary cell types, in particular epithelial cells. In another embodiment of the invention, the composition may contain absorbable or biodegradable matrix scaffolds.

[551] In some embodiments, a population of SC-β cells disclosed herein can be genetically modified to introduce genes beneficial to insulin-producing cells, such as pancreatic β cells, for example, to correct a genetic defect in an individual, introducing a selectable marker, etc., or genes useful for selection against non-insulin producing cells differentiated from at least one insulin-positive endocrine cell or its precursor, or for selective suicide of implanted SC-β cells. In some embodiments, the SC-β cell population can also be genetically modified to increase survival, regulate proliferation, and the like. In some embodiments, a population of SC-β cells disclosed herein can be genetically altered by transfection or transduction with a suitable vector, homologous recombination, or other appropriate method such that the cells express a gene of interest. In one embodiment, a population of SC-β cells is transfected with genes encoding the catalytic component of telomerase (TERT), typically under the control of a heterologous promoter, which increases telomerase expression above that observed with the endogenous promoter (see WO 98 /14592, which is incorporated herein by reference). In other embodiments, a selectable marker is added to provide greater purification of the SC-β cell population. In some embodiments, the SC-β cell population can be genetically altered using vector-containing supernatants for a period of 8-16 hours and then exchanged for growth media for 1-2 days. Selection of genetically altered SC-β cells can be performed using a drug sensitivity selection agent such as puromycin, G418 or blasticidin and then repeating the culture.

[552] Gene therapy can be used to modify a cell to replace a gene product, to facilitate tissue regeneration, to treat a disease, or to improve the survival of cells after implantation in a subject (ie, prevent rejection).

[553] In an alternative embodiment, the population of SC-β cells disclosed herein can also be genetically modified to enhance their ability to participate in tissue regeneration or to deliver a therapeutic gene to the site of administration. The vector is constructed using a known coding sequence of the desired gene operably linked to a promoter that is polyspecific or specifically active in a differentiated cell type. In particular, cells of interest are those that have been genetically modified to express one or more different types of growth factors, such as somatostatin, glucagon, and other factors.

[554] Many vectors are available to transfer exogenous genes into the SC-β target cells disclosed herein. These vectors may be episomal, such as plasmids, vectors derived from viruses such as cytomegalovirus, adenovirus, etc., or may be integrated into the genome of the target cell by homologous recombination or random integration, such as vectors derived from retroviruses , such as MMLV, HIV-1, ALV, etc. In some embodiments, combinations of retroviruses and appropriate packaging cell lines may also be used, wherein the capsid proteins remain functional to infect SC-β cells disclosed herein. Typically, SC-β cells and virus are incubated for at least 24 hours in culture medium. In some embodiments, SC-β cells are allowed to grow in culture medium for short intervals, in some applications 24-73 hours, or for at least two weeks, and may be allowed to grow for five weeks or more before analysis. Typically used retroviral vectors are “defective”, i.e. unable to produce viral proteins necessary for productive infection. Replication of vectors requires growth in a packaging cell line.

[555] The host cell specificity of the retrovirus is determined by the envelope protein env (p120). The envelope protein is provided by the packaging cell line. Envelope proteins are divided into at least three types - ecotropic, amphotropic and xenotropic. Retroviruses packaged with ecotropic envelope proteins, such as MMLV, are capable of infecting most murine and rat cell types. Ecotropic packaging cell lines include BOSC23 (Pear et al. (1993) P.N.A.S. 90:8392-8396). Retroviruses carrying amphotropic envelope proteins, such as 4070A (Danos et al, supra.), are capable of infecting most mammalian cell types, including humans, dogs and mice. Amphotropic packaging cell lines include PAL2 (Miller et al. (1985) Mol. Cell. Biol. 5:431-437); PA317 (Miller et al. (1986) Mol. Cell. Biol. 6:2895-2902); GRIP (Danos et al. (1988) PNAS 85:6460-6464). Retroviruses packaged with xenotropic envelope proteins, such as AKR env, are capable of infecting most types of mammalian cells with the exception of mouse cells. In some embodiments, the vectors may contain genes that subsequently need to be removed, for example, using a recombinase system such as Cre/Lox, or destroy cells that express them, for example, by including genes that enable selective toxicity, such as herpes virus TK genes, Bc1-Xs, etc.

[556] Suitable inducible promoters are activated in the desired target cell type, both in the transfected cells and in their progeny. By transcriptional activation is meant that transcription in target cells is increased above basal levels by at least about 100-fold, more often by at least about 1000-fold. Various promoters are known that are induced in different cell types.

[557] In one aspect of the present invention, the SC-β cell population disclosed herein is suitable for systemic administration or for targeting to an anatomical site. The population of SC-β cells can, for example, be engrafted in or near the pancreas of a subject, or can be administered systemically, for example, without limitation, by intra-arterial or intravenous administration. In alternative embodiments, the SC-β cell population of the present invention can be administered by various routes suitable for implantation into the pancreatic or secretory system, including, but not limited to, parenteral, including intravenous and intra-arterial, intrathecal, intraventricular, intraparenchymal, intracranial, intracisternal, intrastriatal and intranigral administration. Optionally, the SC-β cell population is administered along with an immunosuppressive agent.

[558] In some embodiments, the SC-β cell population may be administered and dosed in accordance with good medical practice, taking into account the clinical condition of the individual patient, site and route of administration, mode of administration, age, sex, and body weight of the patient, and other factors known to medical practitioners. Thus, as used herein, a pharmaceutically “effective amount” is determined based on considerations known in the art. The amount must be effective to achieve improvement, including, but not limited to, increased survival or faster recovery, or improvement or resolution of symptoms and other indicators that are selected as appropriate by those skilled in the art. The SC-β cell population can be administered to a subject in the following settings: clinic, clinical practice, emergency department, hospital ward, intensive care unit, operating room, catheterization unit, and radiology department.

[559] In other embodiments, the SC-β cell population is retained for further implantation/infusion. The SC-β cell population can be divided into more than one aliquot or unit so that part of the SC-β cell population is retained for later use and part is immediately administered to the subject. Medium or long-term storage of all or a portion of cells in a cell bank is also within the scope of this invention, as disclosed in US Patent Application No. 20030054331 and Patent Application No. WO 03024215, which are incorporated herein by reference in their entirety. At the end of the treatment, the concentrated cells can be loaded into a delivery device, such as a syringe, for introduction into the recipient's body by any means known to one skilled in the art.

[560] In some embodiments, the SC-β cell population can be used alone or in combination with other cells, tissues,

Claims (23)

1. An insulin secretion device comprising a population of non-native pancreatic β-cells, wherein the non-native pancreatic β-cells are: demonstrate an in vitro response consisting of glucose-stimulated insulin secretion to glucose stimulation, express INS, PDX1, NKX6.1 and one or more of the following genes: PC2, MNX1 or ABCC8, and do not express one or both of somatostatin and glucagon, and wherein the device comprises a semi-permeable membrane, wherein the semi-permeable membrane is configured to retain non-native pancreatic β-cells in the device and allow the passage of insulin secreted by the non-native pancreatic β-cells, and wherein the device is configured to enable non-native pancreatic β-cells to produce and release insulin when implanted in a subject. 2. The device according to claim 1, containing non-native pancreatic β-cells encapsulated with a biomaterial that contains alginate, polyethylene glycol (PEG), chitosan, polyester hollow fibers (PES), collagen, hyaluronic acid, dextran with arginine, glycine, aspartic acid acid (RGD), 5-ethyl-5-(hydroxymethyl)-β,β-dimethyl-1,3-dioxane-2-ethanol diacrylate (EHD), polyethylene glycol diacrylate (PEGDA), copolymer of 2-methacryloyloxyethylphosphorylcholine, n-butyl methacrylate and p -vinylphenylboronic acid (PMBV), polyvinyl alcohol (PVA), polyglycerol sialosides (PGSAS), agarose, gelatin agarose, copolymer of lactic-glycolic acid (PLGA), or any combination thereof. 3. The device according to claim 1, characterized in that at least one of the non-native pancreatic β-cells is modified with PEG. 4. The device according to claim 1, containing microcarriers or microcapsules. 5. The device according to claim 2, characterized in that the non-native pancreatic β-cells are separated by a semi-permeable membrane in the islet chamber from one or more vascular chambers of the device. 6. The device according to claim 2, characterized in that the semi-permeable membrane is made of a polysaccharide or polycation. 7. The device according to claim 1, additionally containing an immunomodulator. 8. The device according to claim 1, additionally containing an extracorporeal segment. 9. The device according to claim 1, additionally containing a frame made of an absorbable or biodegradable matrix. 10. The device according to claim 1, characterized in that non-native pancreatic β-cells demonstrate an in vitro response consisting of glucose-stimulated insulin secretion to a first glucose stimulation, a second glucose stimulation and a third glucose stimulation, wherein the first glucose stimulation, the second stimulation glucose and the third stimulation with glucose is carried out sequentially. 11. The device according to claim 1, characterized in that the secretion of insulin by a non-native pancreatic β-cell in response to stimulation with glucose is proportional to the concentration of glucose upon stimulation with glucose. 12. The device according to claim 1, characterized in that the non-native pancreatic β-cells contain one or more crystalline insulin granules. 13. The device of claim 1, wherein the non-native pancreatic β-cells exhibit a cytosolic Ca ²⁺ flux in response to sequential first glucose stimulation and second glucose stimulation. 14. The device according to claim 1, characterized in that non-native pancreatic β-cells are monohormonal. 15. The device according to claim 1, characterized in that non-native pancreatic β-cells, when incubated in the presence of at least 20 mM glucose in vitro, secrete at least 0.5 μIU insulin per 1000 cells per 30 minutes of incubation. 16. The device according to claim 1, characterized in that the non-native pancreatic β-cells are characterized by a gene expression profile that differs from the gene expression profile of the native β-cell. 17. The device according to claim 1, characterized in that the population of non-native pancreatic β-cells contains 340-748 million non-native pancreatic β-cells. 18. The device according to claim 1, characterized in that non-native pancreatic β-cells are genetically modified.