Stem cell-derived pancreatic islet differentiation

Abstract

Disclosed herein are compositions and methods for the differentiation of stem cells into pancreatic islet cells.In some aspects, the methods provided herein relate to the in vitro generation of pancreatic beta cells, alpha cells, delta cells and EC cells.In some aspects, the disclosure provides pharmaceutical compositions comprising the cells produced according to the methods disclosed herein, as well as the methods of treatment using them.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 274,402, filed November 1, 2021, entitled "STEM CELL DERIVED PANCREATIC ISLET DIFFERENTIATION," and U.S. Provisional Patent Application No. 63 / 274,391, filed November 1, 2021, entitled "ENHANCED DIFFERENTIATION OF PANCREATIC ISLET CELLS," the entire contents of each of which are incorporated herein by reference.

[0002] Reference to Electronic Sequence Listing The contents of the electronic sequence listing (V013870082WO00-SEQ-ZJG.xml; size: 40,337 bytes; and creation date: October 27, 2022) are incorporated herein by reference in their entirety. [Background technology]

[0003] Pancreas or pancreatic islet transplantation has been used to treat diabetes, including type 1 diabetes. Pancreatic islet transplantation does not require major surgery, and islet graft function can be maintained in the recipient for years. However, a shortage of islet donors has prevented this treatment from being effectively implemented. Artificial pancreases or islets offer an alternative source of transplantable islets. Therefore, there is a need for an in vitro method to restore pancreatic islets with functions and properties similar to those of endogenous islets.

[0004] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Unless otherwise indicated, all publications, patents, and patent applications mentioned in this specification are herein incorporated by reference in their entirety. Summary of the Invention

[0005] In some aspects, compositions and methods for generating pancreatic islet cells (e.g., stem cell-derived pancreatic islet cells) are provided herein. In some embodiments, the methods described herein include contacting pancreatic progenitor cells (e.g., PDX1-positive, NKX6.1-negative pancreatic progenitor cells) with a medium containing a forkhead box O1 (FoxO1) inhibitor and a Notch signaling pathway inhibitor, and optionally a Notch signaling pathway inhibitor. In some embodiments, the methods described herein include contacting pancreatic progenitor cells (e.g., PDX1-positive, NKX6.1-positive pancreatic progenitor cells) with a medium containing a Wnt signaling pathway inhibitor and a PKC activator. In some embodiments, the methods described herein include contacting pancreatic progenitor cells (e.g., PDX1-positive, NKX6.1-negative pancreatic progenitor cells) with a medium containing a forkhead box O1 (FoxO1) inhibitor and a Notch signaling pathway inhibitor, and contacting the resulting cells with a medium containing a Wnt signaling pathway inhibitor and a PKC activator. In some aspects, populations of in vitro differentiated cells (e.g., stem cell-derived pancreatic islet cells) generated using the compositions and methods described herein are also provided. In some embodiments, the population of in vitro differentiated cells (e.g., stem cell-derived pancreatic islet cells) comprises NKX6.1-positive, ISL1-positive cells and NKX6.1-negative, ISL1-positive cells. In some embodiments, the population comprises more NKX6.1-positive, ISL1-positive cells than NKX6.1-negative, ISL1-positive cells. In some embodiments, at least 15% of the cells in the population are NKX6.1-negative, ISL1-positive cells, and less than 12% of the cells in the population are NKX6.1-negative, ISL1-negative cells. In some aspects, methods are provided for using the population of in vitro differentiated cells (e.g., stem cell-derived pancreatic islet cells) described herein to treat diseases (e.g., diabetes).

[0006] Some embodiments of the present disclosure provide an in vitro composition comprising a pancreatic progenitor cell population and a culture medium comprising a forkhead box O1 (FoxO1) inhibitor and a Notch signaling pathway inhibitor, wherein the pancreatic progenitor cell population comprises cells that are PDX1-positive, NKX6.1-negative and cells that are PDX1-positive, NKX6.1-positive.

[0007] In some embodiments, the medium further comprises a PKC activator. In some embodiments, the Notch signaling pathway inhibitor is a gamma-secretase inhibitor. In some embodiments, the gamma-secretase inhibitor is XXI, DAPT or a derivative thereof. In some embodiments, the gamma-secretase inhibitor is XXI. In some embodiments, the FOXO1 inhibitor is represented by formula (IB):

[0008] [ka]

[0009] or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, cocrystal, tautomer, stereoisomer, isotope-labeled derivative, prodrug, composition, or mixture thereof. In some embodiments, the FoxO1 inhibitor is present in the medium at a concentration of 0.1 to 10 μM. In some embodiments, the FoxO1 inhibitor is present in the medium at a concentration of 1 μM. In some embodiments, the PKC activator is phorbol 12,13-dibutyrate (PdBU), TPB, phorbol 12-myristate 13-acetate, bryostatin 1, or a derivative thereof. In some embodiments, the PKC activator is PdBU. In some embodiments, the medium further comprises one or more agents selected from a fibroblast growth factor, a sonic hedgehog (SHH) signaling pathway inhibitor, a retinoic acid, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, and a TGF-β ligand; optionally, the medium further comprises a fibroblast growth factor, a sonic hedgehog (SHH) signaling pathway inhibitor, a retinoic acid, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, and a TGF-β ligand. In some embodiments, the medium further comprises keratinocyte growth factor (KGF), SANT-1, RA, triazovidine, and activin A. In some embodiments, the medium further comprises a water-soluble synthetic polymer. In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol, poloxamer, polyvinylpyrrolidone, polyethylene glycol (PEG), PEG copolymer, poly(N-isopropylacrylamide), or polyacrylamide. In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol (PVA). In some embodiments, the water-soluble synthetic polymer has a concentration of 0.005% to 0.5% (w / v), 0.01% to 0.2% (w / v), 0.02% to 0.1% (w / v), or 0.03% to 0.08% (w / v) of the medium. In some embodiments, the PVA is at most 85% hydrolyzed. In some embodiments, the PVA is about 80% hydrolyzed.

[0010] In some embodiments, at least 50% of the pancreatic progenitor cell population is PDX1 positive and NKX6.1 negative, and / or 50% or less of the pancreatic progenitor cell population is PDX1 positive and NKX6.1 positive. In some embodiments, 50% or less of the pancreatic progenitor cell population is PDX1 positive and NKX6.1 negative, and / or at least 50% of the pancreatic progenitor cell population is PDX1 positive and NKX6.1 positive. In some embodiments, the culture medium does not contain a Wnt inhibitor.

[0011] Another aspect of the present disclosure provides an in vitro composition comprising a pancreatic progenitor cell population comprising PDX1-positive, NKX6.1-positive, insulin-negative cells; and a medium comprising a protein kinase C (PKC) activator and a Wnt signaling pathway inhibitor, wherein the medium does not contain a FOXO1 inhibitor and the pancreatic progenitor cell population has previously been cultured in a medium comprising a FOXO1 inhibitor. In some embodiments, the PKC activator is phorbol 12,13-dibutyrate (PdBU), phorbol 12-myristate 13-acetate, bryostatin 1, or a derivative thereof. In some embodiments, the PKC activator is PdBU. In some embodiments, the Wnt signaling pathway inhibitor is a tankyrase inhibitor. In some embodiments, the tankyrase inhibitor is NVP-TNKS656. In some embodiments, the medium further comprises one or more agents selected from a sonic hedgehog (SHH) signaling pathway inhibitor, an epidermal growth factor, a notch signaling pathway inhibitor, a TGFβ-R1 kinase inhibitor, a thyroid hormone, a bone morphogenetic (BMP) signaling pathway inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a protein kinase inhibitor, a histone methyltransferase EZH2 inhibitor, and retinoic acid. In some embodiments, the medium further comprises a sonic hedgehog (SHH) signaling pathway inhibitor, an epidermal growth factor, a notch signaling pathway inhibitor, a TGFβ-R1 kinase inhibitor, a thyroid hormone, a bone morphogenetic (BMP) signaling pathway inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a protein kinase inhibitor, a histone methyltransferase EZH2 inhibitor, and retinoic acid. In some embodiments, the medium further comprises SANT-1, betacellulin, XXI, Alk5i, GC-1, LDN-193189, thiazovivin, staurosporine, DZNEP, and retinoic acid. In some embodiments, the medium further comprises one or more agents selected from acetyl-CoA-related metabolites, HDAC inhibitors, redox homeostasis regulators, and one-carbon metabolic pathway intermediates.In some embodiments, the medium further comprises an acetyl-CoA-related metabolite, an HDAC inhibitor, a redox homeostasis regulator, and a one-carbon metabolic pathway intermediate. In some embodiments, the medium further comprises acetate, β-hydroxybutyrate, taurine, and formate. In some embodiments, the medium further comprises a vitamin. In some embodiments, the vitamin is biotin. In some embodiments, the medium further comprises glutamine. In some embodiments, the medium comprises PDBU, NVP-TNKS656, SANT-1, betacellulin, XXI, Alk5i, GC-1, LDN-193189, thiazovivin, staurosporine, DZNEP, retinoic acid, acetate, β-hydroxybutyrate, taurine, formate, biotin, and glutamine. In some embodiments, the medium further comprises a water-soluble synthetic polymer. In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol, poloxamer, polyvinylpyrrolidone, polyethylene glycol (PEG), PEG copolymer, poly(N-isopropylacrylamide), or polyacrylamide. In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol (PVA). In some embodiments, the water-soluble synthetic polymer has a concentration of 0.005% to 0.5% (w / v), 0.01% to 0.2% (w / v), 0.02% to 0.1% (w / v), or 0.03% to 0.08% (w / v) of the medium. In some embodiments, the PVA is at most 90% hydrolyzed. In some embodiments, the PVA is about 87% to 89% hydrolyzed.

[0012] In some embodiments, at least 50% of the pancreatic progenitor cell population is PDX1-positive, NKX6.1-positive, and insulin-negative. In some embodiments, the in vitro composition further comprises pancreatic endocrine cells that are PDX1-positive, NKX6.1-positive, and insulin-positive.

[0013] In some embodiments, the Wnt signaling pathway inhibitor is present in the medium at a concentration of 0.2 μM to 2 μM, hi some embodiments, the Wnt signaling pathway inhibitor is present in the medium at a concentration of about 2 μM.

[0014] Further provided herein is a method comprising culturing a first cell population in a first culture medium, wherein the first cell population comprises pancreatic progenitor cells that are PDX1-positive, NKX6.1-negative and pancreatic progenitor cells that are PDX1-positive, NKX6.1-positive; and the first culture medium comprises a Forkhead Box O1 (FoxO1) inhibitor and a Notch signaling pathway inhibitor.

[0015] In some embodiments, the first culture medium further comprises a PKC activator. In some embodiments, the Notch signaling pathway inhibitor is a gamma-secretase inhibitor. In some embodiments, the gamma-secretase inhibitor is XXI, DAPT or a derivative thereof. In some embodiments, the gamma-secretase inhibitor is XXI. In some embodiments, the FoxO1 inhibitor is represented by formula (IB):

[0016] [ka]

[0017] or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, cocrystal, tautomer, stereoisomer, isotope-labeled derivative, prodrug, composition, or mixture thereof. In some embodiments, the FoxO1 inhibitor is present in the first culture medium at a concentration of 0.1 to 10 μM. In some embodiments, the FoxO1 inhibitor is present in the first culture medium at a concentration of 1 μM. In some embodiments, the PKC activator is phorbol 12,13-dibutyrate (PdBU), TPB, phorbol 12-myristate 13-acetate, bryostatin 1, or a derivative thereof. In some embodiments, the PKC activator is PdBU. In some embodiments, the first culture medium further comprises one or more agents selected from a fibroblast growth factor, a sonic hedgehog (SHH) signaling pathway inhibitor, retinoic acid, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, and a TGF-β ligand. In some embodiments, the first culture medium further comprises a fibroblast growth factor, a sonic hedgehog (SHH) signaling pathway inhibitor, retinoic acid, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, and a TGF-β ligand. In some embodiments, the first culture medium further comprises keratinocyte growth factor (KGF), SANT-1, RA, triazovidine, and activin A. In some embodiments, the first culture medium does not comprise a Wnt signaling pathway inhibitor. In some embodiments, the first culture medium further comprises a water-soluble synthetic polymer. In some embodiments, the water-soluble synthetic polymer is PVA, and optionally, the PVA is at most 85% hydrolyzed (e.g., 80% hydrolyzed). In some embodiments, the first cell population is cultured in the first culture medium for about 20 to 80 hours, 20 to 60 hours, 20 to 50 hours, 30 to 50 hours, 30 to 55 hours, or 24 to 72 hours to produce a second cell population. In some embodiments, the first cell population is cultured in the first medium for 24 to 48 hours to become the second cell population.

[0018] In some embodiments, the method further comprises culturing the second cell population in a second culture medium comprising a Wnt signaling pathway inhibitor and a PKC activator. In some embodiments, the Wnt signaling pathway inhibitor is a tankyrase inhibitor. In some embodiments, the tankyrase inhibitor is NVP-TNKS656. In some embodiments, the PKC activator is phorbol 12,13-dibutyrate (PdBU), TPB, phorbol 12-myristate 13-acetate, bryostatin 1, or a derivative thereof. In some embodiments, the PKC activator is PdBU. In some embodiments, the second culture medium further comprises one or more agents selected from epidermal growth factor, thyroid hormone, TGFβ-R1 kinase inhibitor, Notch signaling pathway inhibitor, Sonic hedgehog (SHH) signaling pathway inhibitor, retinoic acid, Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, protein kinase inhibitor, bone morphogenetic (BMP) signaling pathway inhibitor, and histone methyltransferase EZH2 inhibitor. In some embodiments, the second culture medium further comprises epidermal growth factor, thyroid hormone, TGFβ-R1 kinase inhibitor, Notch signaling pathway inhibitor, Sonic hedgehog (SHH) signaling pathway inhibitor, retinoic acid, Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, protein kinase inhibitor, bone morphogenetic (BMP) signaling pathway inhibitor, and histone methyltransferase EZH2 inhibitor. In some embodiments, the second culture medium further comprises SANT-1, betacellulin, XXI, Alk5i, GC-1, LDN-193189, thiazovivin, staurosporine, DZNEP, and retinoic acid. In some embodiments, the second culture medium further comprises one or more agents selected from acetyl-CoA-related metabolites, HDAC inhibitors, redox homeostasis regulators, and one-carbon metabolic pathway intermediates. In some embodiments, the second culture medium further comprises acetyl-CoA-related metabolites, HDAC inhibitors, redox homeostasis regulators, and one-carbon metabolic pathway intermediates. In some embodiments, the second medium further comprises acetate, beta-hydroxybutyrate, taurine, and formate.In some embodiments, the second culture medium further comprises a vitamin. In some embodiments, the vitamin is biotin. In some embodiments, the second culture medium further comprises glutamine. In some embodiments, the second culture medium comprises PDBU, NVP-TNKS656, SANT-1, betacellulin, XXI, Alk5i, GC-1, LDN-193189, thiazovivin, staurosporine, DZNEP, retinoic acid, acetate, β-hydroxybutyrate, taurine, formate, biotin, and glutamine. In some embodiments, the second culture medium does not comprise a FOXO1 inhibitor. In some embodiments, the second culture medium further comprises a water-soluble synthetic polymer. In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol, poloxamer, polyvinylpyrrolidone, polyethylene glycol (PEG), PEG copolymer, poly(N-isopropylacrylamide), or polyacrylamide. In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol (PVA). In some embodiments, the water-soluble synthetic polymer has a concentration of 0.005%-0.5% (w / v), 0.01%-0.2% (w / v), 0.02%-0.1% (w / v), or 0.03%-0.08% (w / v) of the medium. In some embodiments, the PVA is at most 90% hydrolyzed, and optionally, the PVA is about 87%-89% hydrolyzed. In some embodiments, the Wnt signaling pathway inhibitor is present in the second medium at a concentration of 0.2 μM-2 μM. In some embodiments, the Wnt signaling pathway inhibitor is present in the second medium at a concentration of about 2 μM. In some embodiments, the second cell population is cultured in the second culture medium for about 20-80, 20-60, 20-50, 30-50, 30-55, 40-120, 40-100, 60-110, 70-110, 80-110, 80-100, 90-100, or 24-72 hours to become a third cell population. In some embodiments, the second cell population is cultured in the second culture medium for about 24-72 hours to become a third cell population. In some embodiments, the second cell population is cultured in the second culture medium for about 72-120 hours to become a third cell population.

[0019] In some embodiments, the method further comprises culturing the third cell population in a third medium comprising thyroid hormone, a TGFβ-R1 kinase inhibitor, a Notch signaling pathway inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a protein kinase inhibitor, a bone morphogenetic (BMP) signaling pathway inhibitor, a histone methyltransferase EZH2 inhibitor, an acetyl-CoA-related metabolite, an HDAC inhibitor, a redox homeostasis regulator, a one-carbon metabolic pathway intermediate, a vitamin, glutamine, and a water-soluble synthetic polymer. In some embodiments, the third medium comprises GC-1, ALK5i II, XXI, triazovidine, staurosporine (SSP), LDN193189, DZNep, acetate, β-hydroxybutyrate, taurine, formate, biotin, glutamine, and PVA, optionally, the PVA being at most 90% hydrolyzed. In some embodiments, the third cell population is cultured in the third medium for about 20-80, 20-60, 20-50, 30-50, 30-55, 40-120, 40-100, 60-110, 70-110, 80-110, 90-110, 70-120, 80-100, 90-100, or 24-72 hours to become a fourth cell population. In some embodiments, the third cell population is cultured in the third medium for about 48-96 hours to become a fourth cell population.

[0020] In some embodiments, the method further comprises culturing the fourth cell population in a fourth medium comprising a TGFβ-R1 kinase inhibitor, thyroid hormone, a bone morphogenetic (BMP) signaling pathway inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a protein kinase inhibitor, a histone methyltransferase EZH2 inhibitor, an acetyl-CoA-related metabolite, an HDAC inhibitor, a redox homeostasis regulator, a one-carbon metabolic pathway intermediate, a vitamin, glutamine, glutamate, carnitine, albumin, and ZnSO. In some embodiments, the fourth medium comprises ALK5i, GC-1, LDN-193189, thiazovivin, staurosporine (SSP), DZBEP, acetate, β-hydroxybutyrate, taurine, formate, biotin, glutamine, glutamate, carnitine, HSA, and ZnSO. In some embodiments, the fourth cell population is cultured in the fourth medium for about 20-80, 20-60, 20-50, 30-50, 30-55, 40-120, 40-100, 60-110, 70-110, 80-110, 90-110, 70-120, 80-100, 90-100, or 24-72 hours to form a fifth cell population. In some embodiments, the fourth cell population is cultured in the fourth medium for about 48-96 hours to form a fifth cell population.

[0021] In some embodiments, the method further comprises culturing the fifth cell population in a fifth culture medium comprising glutamine, HSA, and ZnSO4, and optionally the fifth culture medium further comprises acetate, β-hydroxybutyrate, taurine, formate, biotin, glutamate, and carnitine. In some embodiments, the fifth cell population is cultured in the fifth culture medium for at least 72 hours to become a sixth cell population. In some embodiments, at least 15% of the cells of the sixth cell population are NKX6.1-negative and ISL1-positive; and less than 12% of the fifth cell population are NKX6.1-negative and ISL1-negative.

[0022] moreover, (i) culturing a first cell population in a first culture medium to obtain a second cell population, wherein the first cell population comprises PDX1-positive, NKX6.1-negative pancreatic progenitor cells and PDX1-positive, NKX6.1-positive pancreatic progenitor cells, and the first culture medium comprises a FoxO1 inhibitor, a Notch signaling pathway inhibitor, a PKC activator, a fibroblast growth factor, a sonic hedgehog (SHH) signaling pathway inhibitor, retinoic acid, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a TGF-β ligand, and a water-soluble synthetic polymer; (ii) culturing the second cell population obtained in (i) in a second culture medium to obtain a third cell population, wherein the second culture medium comprises a Wnt signaling pathway inhibitor, a PKC activator, an epidermal growth factor, a thyroid hormone, a TGFβ-R1 kinase inhibitor, a Notch signaling pathway inhibitor, a Sonic hedgehog (SHH) signaling pathway inhibitor, retinoic acid, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a protein kinase inhibitor, a bone morphogenetic (BMP) signaling pathway inhibitor, a histone methyltransferase EZH2 inhibitor, an acetyl-CoA-related metabolite, an HDAC inhibitor, a redox homeostasis regulator, a one-carbon metabolic pathway intermediate, a vitamin, glutamine, and a water-soluble synthetic polymer (e.g., PVA), and the second culture medium does not comprise a FOXO1 inhibitor; (iii) culturing the third cell population obtained in (ii) in a third culture medium to obtain a fourth cell population, wherein the third culture medium comprises a Notch signaling pathway inhibitor, a TGFβ-R1 kinase inhibitor, a thyroid hormone, a bone morphogenetic (BMP) signaling pathway, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a protein kinase inhibitor, and a histone methyltransferase EZH2 inhibitor, an acetyl-CoA-related metabolite, an HDAC inhibitor, a redox homeostasis regulator, a one-carbon metabolic pathway intermediate, a vitamin, glutamine, and a water-soluble synthetic polymer, and the third culture medium is free of a Wnt signaling pathway inhibitor and a PKC activator; (iv) culturing the fourth cell population obtained in (iii) in a fourth culture medium to obtain a fifth cell population, wherein the fourth culture medium comprises a TGFβ-R1 kinase inhibitor, a thyroid hormone, a bone morphogenetic (BMP) signaling pathway inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a protein kinase inhibitor, a histone methyltransferase EZH2 inhibitor, an acetyl-CoA-related metabolite, an HDAC inhibitor, a redox homeostasis regulator, a one-carbon metabolic pathway intermediate, a vitamin, glutamine, glutamate, carnitine, albumin, and ZnSO4, and the fourth culture medium does not comprise a Wnt signaling pathway inhibitor or a PKC activator; (v) culturing the fifth cell population obtained in (iv) in a fifth culture medium to obtain a sixth cell population, wherein the fifth culture medium contains albumin and ZnSO4; Provided herein is a method comprising:

[0023] In some embodiments, the first cell population is obtained by differentiating human embryonic stem cells. Another aspect of the present disclosure provides an in vitro differentiated cell population comprising NKX6.1-positive, ISL1-positive cells and NKX6.1-negative, ISL1-positive cells; wherein at least 15% of the cells in the population are NKX6.1-negative, ISL1-positive cells; and less than 12% of the cells in the population are NKX6.1-negative, ISL1-negative cells. In some embodiments, the in vitro differentiated cell population comprises at least 60%, at least 70%, at least 75%, at least 80%, about 50-90%, or about 70-85% ISL1-positive cells. In some embodiments, at least 20% of the ISL1-positive cells are NKX6.1-negative. In some embodiments, at least 25%, at least 30%, at least 40%, about 30-50%, or about 40-50% of the ISL1-positive cells are NKX6.1-negative. In some embodiments, the in vitro differentiated cell population comprises up to 20%, up to 30%, up to 40%, or up to 50% NK6.1-positive, ISL1-positive cells. In some embodiments, the in vitro differentiated cell population comprises less than 25% or less than 20% NKX6.1-positive, ISL1-negative cells. In some embodiments, the in vitro differentiated cell population comprises less than 10% or less than 5% NKX6.1-negative, ISL1-negative cells.

[0024] In some embodiments, the C-peptide content per 1,000 in vitro differentiated cells is at least 300 pM, and optionally, the C-peptide content per 1,000 in vitro differentiated cells is at least 400 pM. In some embodiments, the glucagon content per 1,000 in vitro differentiated cells is at least 100 pM. In some embodiments, the glucagon content per 1,000 in vitro differentiated cells is at least 200 pM, at least 300 pM, at least 500 pM, at least 600 pM, at least 700 pM, or at least 800 pM. In some embodiments, at least 10% of the in vitro differentiated cells are stem cell-derived alpha cells. In some embodiments, at least 20%, at least 25%, at least 30%, at least 35%, about 20-40%, or about 30-40% of the in vitro differentiated cells are stem cell-derived alpha cells. In some embodiments, at least 35% of the in vitro differentiated cells are stem cell-derived beta cells. In some embodiments, at least 40%, at least 45%, or about 35-50% of the in vitro differentiated cells are stem cell-derived beta cells. In some embodiments, at least 5% of the in vitro differentiated cells are stem cell-derived delta cells. In some embodiments, about 5-10% of the in vitro differentiated cells are stem cell-derived delta cells. In some embodiments, at least 5% of the in vitro differentiated cells are glucagon-positive and somatostatin-negative. In some embodiments, at least 5% of the in vitro differentiated cells are glucagon-negative and somatostatin-positive. In some embodiments, less than 10% of the in vitro differentiated cells are both ISL1-1-negative and NKX6.1-negative. In some embodiments, about 5-10% of the in vitro differentiated cells are both ISL1-1-negative and NKX6.1-negative. In some embodiments, less than 20% of the in vitro differentiated cells are both ISL1-negative and NKX6.1-positive. In some embodiments, about 10-20% of the in vitro differentiated cells are both ISL1-negative and NKX6.1-positive.

[0025] Further provided herein is an in vitro differentiated cell population comprising NKX6.1-positive, ISL1-positive cells and NKX6.1-negative, ISL1-positive cells, wherein at least 15% of the cells in the population are NKX6.1-positive, ISL1-positive cells, at least 10% of the cells in the population are NKX6.1-negative, ISL1-positive cells, and less than 12% of the cells in the population are NKX6.1-negative, ISL1-negative cells.

[0026] Further provided herein is an in vitro differentiated cell population comprising NKX6.1-positive, ISL1-positive cells and NKX6.1-negative, ISL1-positive cells, wherein at least 15% of the cells in the population are NKX6.1-positive, ISL1-positive cells, at least 10% of the cells in the population are NKX6.1-negative, ISL1-positive cells, and 5-25% of the cells in the population are NKX6.1-positive, ISL1-negative cells.

[0027] In some embodiments, at least 30% of the cells in the population are NKX6.1-positive, ISL1-positive cells. In some embodiments, 30-60% of the cells in the population are NKX6.1-positive, ISL1-positive cells. In some embodiments, 35-50% of the cells in the population are NKX6.1-positive, ISL1-positive cells.

[0028] In some embodiments, at least 25% of the cells in the population are NKX6.1-negative, ISL1-positive cells. In some embodiments, 25-50% of the cells in the population are NKX6.1-negative, ISL1-positive cells. In some embodiments, 30-45% of the cells in the population are NKX6.1-negative, ISL1-positive cells.

[0029] In some embodiments, 1-12% of the cells in the population are NKX6.1-negative, ISL1-negative cells, hi some embodiments, 2-12% of the cells in the population are NKX6.1-negative, ISL1-negative cells.

[0030] In some embodiments, 5-25% of the cells in the population are NKX6.1-positive, ISL1-negative cells, hi some embodiments, 9-25% of the cells in the population are NKX6.1-positive, ISL1-negative cells.

[0031] In some embodiments, there are more NKX6.1-positive, ISL1-positive cells than NKX6.1-negative, ISL1-positive cells in the population. In some embodiments, the in vitro differentiated cell population further comprises a culture medium. In some embodiments, the culture medium comprises a sugar. In some embodiments, the sugar is sucrose or glucose. In some embodiments, the culture medium comprises a sugar at a concentration of about 0.05% to about 1.5%. In some embodiments, the culture medium is CMRL medium, or the culture medium is HypoThermosol® FRS storage medium.

[0032] In some embodiments, the cell population is in cell clusters, hi some embodiments, the cell clusters are 125-225 microns, 130-160, 170-225, 140-200, 140-170, 160-220, 170-215, and 170-200 microns in diameter.

[0033] In some embodiments, the population comprises one or more NKX6.1-positive, ISL1-positive cells that express CHGA, MAFB, and / or ESRRG at a level higher (e.g., at least 10%, 30%, 50%, 70%, 100%, 125%, 150%, or 200% higher) than NKX6.1-positive, ISL1-positive cells derived from the pancreas of a healthy adult control subject. In some embodiments, at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the NKX6.1-positive, ISL1-positive cells in the cell population express CHGA, MAFB, and / or ESRRG at a level higher than at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the NKX6.1-positive, ISL1-positive cells derived from the pancreas of a healthy adult control subject. In some embodiments, the population comprises one or more NKX6.1-positive, ISL1-positive cells that express SIX3, MAFA, CHGB, RBP4 and / or FXYD2 at a lower level (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% lower) than NKX6.1-positive, ISL1-positive cells from the pancreas of a healthy control adult subject. In some embodiments, at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the NKX6.1-positive, ISL1-positive cells in the cell population express SIX3, MAFA, CHGB, RBP4, and / or FXYD2 at levels lower than at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the NKX6.1-positive, ISL1-positive cells from the pancreas of a healthy adult control subject. In some embodiments, the population comprises NKX6.1-positive, ISL1-positive cells that express MAFA at a lower level than the NKX6.1-positive, ISL1-positive cells from the pancreas of a healthy adult control subject. In some embodiments, the population comprises NKX6.1-positive, ISL1-positive cells that express SIX3 at a lower level than the NKX6.1-positive, ISL1-positive cells from the pancreas of a healthy adult control subject. In some embodiments, the population comprises NKX6.1-positive, ISL1-positive cells that express CHGB at a lower level than NKX6.1-positive, ISL1-positive cells from the pancreas of a healthy control adult subject.In some embodiments, the population comprises NKX6.1-positive, ISL1-positive cells that express RBP4 at a lower level than NKX6.1-positive, ISL1-positive cells from the pancreas of a healthy adult control subject. In some embodiments, the population comprises NKX6.1-positive, ISL1-positive cells that express FXYD2 at a lower level than NKX6.1-positive, ISL1-positive cells from the pancreas of a healthy adult control subject. In some embodiments, the population comprises NKX6.1-positive, ISL1-positive cells that express MAFB at a higher level than NKX6.1-positive, ISL1-positive cells from the pancreas of a healthy adult control subject. In some embodiments, the population comprises NKX6.1-positive, ISL1-positive cells that express SIX2, HOPX, IAPP, and / or UCN3 at a higher level than NKX6.1-positive, ISL1-positive cells from the pancreas of a healthy adult control subject. In some embodiments, the population comprises NKX6.1-positive, ISL1-positive cells that do not express MAFA. In some embodiments, the population comprises NKX6.1-positive, ISL1-positive cells that express MAFB.

[0034] In some embodiments, as an alternative to comparing gene expression with cells of a healthy adult pancreas, the comparison could be to gene expression in one or more cadaveric islets (e.g., cadaveric islets suitable for transplantation in diabetic patients - see, e.g., Hering et al., 2016, Diabetes Care, 39(7):1230-1240) and / or primary human pancreatic islet cells (e.g., primary adult human pancreatic islet cells such as those available from the Nordic Network for Islet Transplantation (Uppsala University) and the University of Alberta IsletCore (Canada) - see, e.g., Balboa et al., 2022, Nature Biotechnology, 40:1042-1055).

[0035] In some embodiments, the population comprises one or more NKX6.1-positive, ISL1-positive cells that express CHGA, MAFB, and / or ESRRG at a higher level (e.g., at least 10%, 30%, 50%, 70%, 100%, 125%, 150%, or 200% higher) than the NKX6.1-positive, ISL1-positive cells of the cadaveric islets or primary human islet cells. In some embodiments, at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the NKX6.1-positive, ISL1-positive cells in the cell population express CHGA, MAFB, and / or ESRRG at a higher level than at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the NKX6.1-positive, ISL1-positive cells of the cadaveric islets or primary human islet cells. In some embodiments, the population comprises one or more NKX6.1-positive, ISL1-positive cells that express SIX3, MAFA, CHGB, RBP4 and / or FXYD2 at a lower level (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% lower) than the NKX6.1-positive, ISL1-positive cells in cadaveric islets or primary human islet cells. In some embodiments, at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the NKX6.1-positive, ISL1-positive cells in the cell population express SIX3, MAFA, CHGB, RBP4, and / or FXYD2 at levels lower than at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the NKX6.1-positive, ISL1-positive cells in the cadaveric islets or primary human islet cells. In some embodiments, the population comprises NKX6.1-positive, ISL1-positive cells that express lower levels of MAFA than the NKX6.1-positive, ISL1-positive cells in the cadaveric islets or primary human islet cells. In some embodiments, the population comprises NKX6.1-positive, ISL1-positive cells that express lower levels of SIX3 than NKX6.1-positive, ISL1-positive cells of cadaveric islets or primary human islet cells. In some embodiments, the population comprises NKX6.1-positive, ISL1-positive cells that express lower levels of CHGB than NKX6.1-positive, ISL1-positive cells of cadaveric islets or primary human islet cells.In some embodiments, the population comprises NKX6.1-positive, ISL1-positive cells that express lower levels of RBP4 than NKX6.1-positive, ISL1-positive cells of cadaveric islets or primary human islet cells. In some embodiments, the population comprises NKX6.1-positive, ISL1-positive cells that express lower levels of FXYD2 than NKX6.1-positive, ISL1-positive cells of cadaveric islets or primary human islet cells. In some embodiments, the population comprises NKX6.1-positive, ISL1-positive cells that express higher levels of MAFB than NKX6.1-positive, ISL1-positive cells of cadaveric islets or primary human islet cells. In some embodiments, the population comprises NKX6.1-positive, ISL1-positive cells that express higher levels of SIX2, HOPX, IAPP, and / or UCN3 than NKX6.1-positive, ISL1-positive cells of cadaveric islets or primary human islet cells.

[0036] In some embodiments, any of the NKX6.1-positive, ISL1-positive cells disclosed herein also express any one or more of the following genes: PC2, MNX1, or ABCC8.

[0037] In some embodiments, any of the compositions or cell populations described herein comprises less than 40% VMAT1 positive cells.In some embodiments, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, or less than 10% of the cells in any of the compositions or cell populations disclosed herein are VMAT1 positive cells. In some embodiments, the composition or cell population comprises 1-40%, 1-35%, 1-30%, 1-25%, 1-20%, 1-15%, 1-10%, 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, 5-10%, 10-40%, 10-35%, 10-30%, 10-25%, 15-25%, 10-20%, 10-15%, 20-40%, 20-35%, 20-30%, 20-25%, or 30-40% VMAT1-positive cells.

[0038] In some embodiments, the cell population is derived from in vitro stem cells. In some embodiments, the stem cells are genetically modified. In some embodiments, the stem cells have reduced expression of one or more of beta-2 microglobulin, CXCL10, linalase, CIITA, HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and / or HLADR compared to stem cells that have not been genetically modified. In some embodiments, the stem cells have increased expression of one or more of CD47, PDL1, HLA-G, CD46, CD55, CD59, and / or CTLA compared to stem cells that have not been genetically modified.

[0039] In some embodiments, the in vitro differentiated cell population is contained within a device for implantation into a subject. Further provided herein is an in vitro composition comprising the in vitro differentiated cell population described herein in a culture medium. In some embodiments, the culture medium comprises human serum albumin. In some embodiments, the culture medium comprises glutamine. In some embodiments, the culture medium comprises any one or more of an inorganic compound, an Alk5 inhibitor, a thyroid hormone receptor beta-specific agonist, a BMPI-type receptor inhibitor, a RHO / ROCK pathway inhibitor, a protein kinase inhibitor, or an S-adenosylhomocysteine ​​hydrolase inhibitor. In some embodiments, the culture medium comprises any one or more of ZnSO4, Alk5i, GC-1, LDN-193189, thiazovivin, staurosporine, or DZNEP. In some embodiments, the culture medium comprises any one or more of L-glutamate, L-carnitine, taurine, acetate, β-hydroxybutarate, biotin, or formate.

[0040] Further provided herein is an implantable encapsulation device comprising the in vitro differentiated cell population described herein. In some embodiments, the device comprises a first membrane having a first surface comprising a plurality of channels and a plurality of second surfaces opposite the first surface; and a second membrane attached opposite the plurality of second surfaces of the first membrane. In some embodiments, the first membrane and the second membrane comprise PVDF, PTFE, ePTFE, PCL, PE / PES, PP, PS, PMMA, PLGA, PLLA, or any combination thereof.

[0041] Also provided herein is an implantable encapsulation device comprising an internal volume containing a composition described herein disposed therein. In some embodiments, the implantable encapsulation device comprises at least one membrane at least partially defining the internal volume. In some embodiments, the at least one membrane comprises a first membrane and a second membrane, the first membrane and the second membrane being bonded together to form a seal extending at least partially around the periphery of the internal volume disposed between the first membrane and the second membrane. In some embodiments, the at least one membrane comprises at least one selected from PVDF, PTFE, ePTFE, PCL, PE / PES, PP, PS, PMMA, PLGA, and PLLA. In some embodiments, the at least one membrane comprises ePTFE.

[0042] In some embodiments, the device is implanted in a subject with diabetes. In some embodiments, the subject has type 1 diabetes. Methods of treating a subject are provided, the methods comprising administering to the subject a composition comprising an in vitro differentiated cell population described herein. In some embodiments, the methods comprise administering to the subject a composition comprising an in vitro differentiated cell population comprising NKX6.1-positive, ISL1-positive cells and NKX6.1-negative, ISL1-positive cells, wherein the population comprises more NKX6.1-positive, ISL1-positive cells than NKX6.1-negative, ISL1-positive cells, wherein at least 15% of the cells in the population are NKX6.1-negative, ISL1-positive cells, and wherein less than 12% of the cells in the population are NKX6.1-negative, ISL1-negative cells. In some embodiments, the method includes implanting into the subject an implantable encapsulation device containing an in vitro differentiated cell population comprising NKX6.1-positive, ISL1-positive cells and NKX6.1-negative, ISL1-positive cells, wherein the population comprises more NKX6.1-positive, ISL1-positive cells than NKX6.1-negative, ISL1-positive cells, wherein at least 15% of the cells in the population are NKX6.1-negative, ISL1-positive cells, and wherein less than 12% of the cells in the population are NKX6.1-negative, ISL1-negative cells.

[0043] The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the invention will be apparent from the following drawings and detailed description of certain embodiments, as well as from the appended claims.

[0044] The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component shown in various figures is represented by a like numeral. For clarity, not every component is shown in every figure. [Brief explanation of the drawings]

[0045] [Figure 1]Figure 1 is a graph showing blood glucose levels in mice transplanted with SC-islet cells differentiated using Protocol 1 or Protocol 2. SC-islet cells differentiated using Protocol 2 reduced blood glucose levels in diabetic mice to levels achieved with twice the dose of SC-islet cells differentiated using Protocol 1. [Figure 2A] Figures 2A-2B are graphs showing the results of an oral glucose tolerance test (OGTT) in mice transplanted with SC-islet cells differentiated using Protocol 1 or Protocol 2. Figure 2A shows glucose levels up to 180 minutes after glucose administration. [Figure 2B] Figures 2A-2B are graphs showing the results of an oral glucose tolerance test (OGTT) of mice transplanted with SC-islet cells differentiated using Protocol 1 or Protocol 2. Figure 2B shows the area under the curve (AUC) derived from the oral glucose tolerance test (OGTT). [Figure 3A] Figures 3A-3C are graphs showing the percentage of C-peptide-positive cells (β cells), glucagon-positive cells (α cells), and somatostatin-positive cells (δ cells) in kidney grafts from mice transplanted with SC-islet cells differentiated using Protocol 1 (Groups 3 and 4) or Protocol 2 (Group 5). Groups 3 and 5 were transplanted with 4 million SC-islet cells, and Group 4 was transplanted with 8 million SC-islet cells. [Figure 3B] Figures 3A-3C are graphs showing the percentage of C-peptide-positive cells (β cells), glucagon-positive cells (α cells), and somatostatin-positive cells (δ cells) in kidney grafts from mice transplanted with SC-islet cells differentiated using Protocol 1 (Groups 3 and 4) or Protocol 2 (Group 5). Groups 3 and 5 were transplanted with 4 million SC-islet cells, and Group 4 was transplanted with 8 million SC-islet cells. [Figure 3C]Figures 3A-3C are graphs showing the percentage of C-peptide-positive cells (β cells), glucagon-positive cells (α cells), and somatostatin-positive cells (δ cells) in kidney grafts from mice transplanted with SC-islet cells differentiated using Protocol 1 (Groups 3 and 4) or Protocol 2 (Group 5). Groups 3 and 5 were transplanted with 4 million SC-islet cells, and Group 4 was transplanted with 8 million SC-islet cells. [Figure 4A] Figures 4A-4B are graphs showing the percentage of serotonin-positive cells (Figure 4A) and the percentage of proliferating EC cells (Figure 4B) in kidney grafts from mice transplanted with SC-islet cells differentiated using Protocol 1 (Groups 3 and 4) or Protocol 2 (Group 5). Groups 3 and 5 were transplanted with 4 million SC-islet cells, and Group 4 was transplanted with 8 million SC-islet cells. [Figure 4B] Figures 4A-4B are graphs showing the percentage of serotonin-positive cells (Figure 4A) and the percentage of proliferating EC cells (Figure 4B) in kidney grafts from mice transplanted with SC-islet cells differentiated using Protocol 1 (Groups 3 and 4) or Protocol 2 (Group 5). Groups 3 and 5 were transplanted with 4 million SC-islet cells, and Group 4 was transplanted with 8 million SC-islet cells. [Figure 5] Figure 5 is a graph showing the percentage of Ki67-positive cells in kidney grafts from mice transplanted with SC-islet cells differentiated using Protocol 1 (Groups 3 and 4) or Protocol 2 (Group 5). Groups 3 and 5 received 4 million SC-islet cells, and Group 4 received 8 million SC-islet cells. [Figure 6A]Figures 6A-6C are graphs showing the percentages of SOX9-positive cells (Figure 6A), Ki67-positive cells (Figure 6B), and SOX9- and Ki67-positive cells (Figure 6C) in kidney grafts from mice transplanted with SC-islet cells differentiated using Protocol 1 (Groups 3 and 4) or Protocol 2 (Group 5). Groups 3 and 5 were transplanted with 4 million SC-islet cells, and Group 4 was transplanted with 8 million SC-islet cells. [Figure 6B] Figures 6A-6C are graphs showing the percentages of SOX9-positive cells (Figure 6A), Ki67-positive cells (Figure 6B), and SOX9- and Ki67-positive cells (Figure 6C) in kidney grafts from mice transplanted with SC-islet cells differentiated using Protocol 1 (Groups 3 and 4) or Protocol 2 (Group 5). Groups 3 and 5 were transplanted with 4 million SC-islet cells, and Group 4 was transplanted with 8 million SC-islet cells. [Figure 6C] Figures 6A-6C are graphs showing the percentages of SOX9-positive cells (Figure 6A), Ki67-positive cells (Figure 6B), and SOX9- and Ki67-positive cells (Figure 6C) in kidney grafts from mice transplanted with SC-islet cells differentiated using Protocol 1 (Groups 3 and 4) or Protocol 2 (Group 5). Groups 3 and 5 were transplanted with 4 million SC-islet cells, and Group 4 was transplanted with 8 million SC-islet cells. [Figure 7] Figure 7 is a graph showing quantification of angiogenesis in renal grafts from mice transplanted with SC-islet cells differentiated using Protocol 1 (Groups 3 and 4) or Protocol 2 (Group 5). Angiogenesis was indicated by CD31 expression levels. Groups 3 and 5 were transplanted with 4 million SC-islet cells, and Group 4 was transplanted with 8 million SC-islet cells. [Figure 8A]Figures 8A-8B are flow cytometry graphs showing the percentage of ISL1+ cells in S6 SC-islets. SC-islets differentiated using Protocol 2 (Figure 8B) showed an increase in ISL1+ cells compared to SC-islets differentiated using Protocol 1 (Figure 8A). [Figure 8B] Figures 8A-8B are flow cytometry graphs showing the percentage of ISL1+ cells in S6 SC-islets. SC-islets differentiated using Protocol 2 (Figure 8B) showed an increase in ISL1+ cells compared to SC-islets differentiated using Protocol 1 (Figure 8A). [Figure 9] Figures 9A-9B are flow cytometry graphs showing ISL1 / NKX6.1 expression in pancreatic progenitor cells differentiated using protocol 1 (Figure 9A) or in pancreatic progenitor cells differentiated using protocol 1 with the addition of FoxO1 inhibitor to S4D5 (Figure 9B). [Figure 10] Figures 10A-10B are flow cytometry graphs showing ISL1 / Nkx6.1 expression in pancreatic progenitor cells differentiated using protocol 1 (Figure 10A) or protocol 1 with the addition of Wnt inhibitor (XAV) on S5 days 1-4 (Figure 10B). [Figure 11] Figures 11A-11B are flow cytometry graphs showing ISL1 / NKX6.1 expression in pancreatic progenitor cells differentiated using protocol 1 with the addition of a FoxO1 inhibitor at S4D5 (Figure 11A) or with the addition of a FoxO1 inhibitor at S4D5 and a Wnt inhibitor (XAV) at S5 days 1-4 (Figure 11B). [Figure 12] Figures 12A-12B are flow cytometry graphs showing ISL1 / NKX6.1 expression in pancreatic progenitor cells differentiated using protocol 1 (Figure 12A) or using protocol 1 with the addition of PKC activator (TPB) on S4D5 and S5 days 1 and 2 (Figure 12B). [Figure 13]Figures 13A-13B are flow cytometry graphs showing ISL1 / NKX6.1 expression in pancreatic progenitor cells differentiated using protocol 1 (Figure 13A) or using protocol 1 with the addition of a PKC activator (TPB) and a Notch inhibitor (XXI) at S4D5, followed by the addition of PKC activator (TPB) alone on S5 days 1-4 (Figure 13B). [Figure 14] Figures 14A-14B are flow cytometry graphs showing ISL1 / NKX6.1 expression in pancreatic progenitor cells differentiated using protocol 1, which involves the addition of a FoxO1 inhibitor, a PKC activator (TPB), and a Notch inhibitor (XXI) on S4D5 (Figure 14A), or using protocol 1, which involves the addition of a FoxO1 inhibitor, a PKC activator (TPB), and a Notch inhibitor (XXI) on S4D5, followed by the addition of a PKC activator (TPB) and a Wnt inhibitor (XAV) on days 1-4 of S5 (Figure 14B). [Figure 15A] Figures 15A-15C are flow cytometry graphs showing ISL1 / NKX6.1 expression in pancreatic progenitor cells differentiated using Protocol 1 with the addition of a PKC activator (PDBU) at S4D5 (Figure 15A), Protocol 1 with the addition of a FoxO1 inhibitor, a PKC activator (PDBU), and a Notch inhibitor (XXI) at S4D5 (Figure 15B), and Protocol 1 with the addition of a FoxO1 inhibitor, a PKC activator (PDBU), and a Notch inhibitor (XXI) at S4D5 followed by the addition of a Wnt inhibitor (XAV) on days 1-4 of S5 (Figure 15C). [Figure 15B] Figures 15A-15C are flow cytometry graphs showing ISL1 / NKX6.1 expression in pancreatic progenitor cells differentiated using Protocol 1 with the addition of a PKC activator (PDBU) at S4D5 (Figure 15A), Protocol 1 with the addition of a FoxO1 inhibitor, a PKC activator (PDBU), and a Notch inhibitor (XXI) at S4D5 (Figure 15B), and Protocol 1 with the addition of a FoxO1 inhibitor, a PKC activator (PDBU), and a Notch inhibitor (XXI) at S4D5 followed by the addition of a Wnt inhibitor (XAV) on days 1-4 of S5 (Figure 15C). [Figure 15C] Figures 15A-15C are flow cytometry graphs showing ISL1 / NKX6.1 expression in pancreatic progenitor cells differentiated using Protocol 1 with the addition of a PKC activator (PDBU) at S4D5 (Figure 15A), Protocol 1 with the addition of a FoxO1 inhibitor, a PKC activator (PDBU), and a Notch inhibitor (XXI) at S4D5 (Figure 15B), and Protocol 1 with the addition of a FoxO1 inhibitor, a PKC activator (PDBU), and a Notch inhibitor (XXI) at S4D5 followed by the addition of a Wnt inhibitor (XAV) on days 1-4 of S5 (Figure 15C). [Figure 16A-B] 16A-16H are flow cytometry graphs showing ISL1 / NKX6.1 expression of S6D4 in SC islet cells differentiated using the methods described in Table 6. [Figure 16C-D] 16A-16H are flow cytometry graphs showing ISL1 / NKX6.1 expression of S6D4 in SC islet cells differentiated using the methods described in Table 6. [Figure 16E-F] 16A-16H are flow cytometry graphs showing ISL1 / NKX6.1 expression of S6D4 in SC islet cells differentiated using the methods described in Table 6. [Figure 16G-H] 16A-16H are flow cytometry graphs showing ISL1 / NKX6.1 expression of S6D4 in SC islet cells differentiated using the methods described in Table 6. [Figure 17A-B] 17A-17H are flow cytometry graphs showing chromogranin A (CHGA) / C-peptide expression of S6D4 in SC-islet cells differentiated using the methods described in Table 6. [Figure 17C-D] 17A-17H are flow cytometry graphs showing chromogranin A (CHGA) / C-peptide expression of S6D4 in SC-islet cells differentiated using the methods described in Table 6. [Figure 17E-F]17A-17H are flow cytometry graphs showing chromogranin A (CHGA) / C-peptide expression of S6D4 in SC-islet cells differentiated using the methods described in Table 6. [Figure 17G-H] 17A-17H are flow cytometry graphs showing chromogranin A (CHGA) / C-peptide expression of S6D4 in SC-islet cells differentiated using the methods described in Table 6. [Figure 18A-B] 18A-18H are flow cytometry graphs showing SOX9 / Ki67 expression of S6D4 in SC-islet cells differentiated using the methods described in Table 6. [Figure 18C-D] 18A-18H are flow cytometry graphs showing SOX9 / Ki67 expression of S6D4 in SC-islet cells differentiated using the methods described in Table 6. [Figure 18E-F] 18A-18H are flow cytometry graphs showing SOX9 / Ki67 expression of S6D4 in SC-islet cells differentiated using the methods described in Table 6. [Figure 18G-H] 18A-18H are flow cytometry graphs showing SOX9 / Ki67 expression of S6D4 in SC-islet cells differentiated using the methods described in Table 6. [Figure 19A-B] 19A-19H are flow cytometry graphs showing ISL1 / NKX6.1 expression of S6D7 in SC-islet cells differentiated using the methods described in Table 6. [Figure 19C-D] 19A-19H are flow cytometry graphs showing ISL1 / NKX6.1 expression of S6D7 in SC-islet cells differentiated using the methods described in Table 6. [Figure 19E-F] 19A-19H are flow cytometry graphs showing ISL1 / NKX6.1 expression of S6D7 in SC-islet cells differentiated using the methods described in Table 6. [Figure 19G-H]19A-19H are flow cytometry graphs showing ISL1 / NKX6.1 expression of S6D7 in SC-islet cells differentiated using the methods described in Table 6. [Figure 20A-B] 20A-20H are flow cytometry graphs showing chromogranin A (CHGA) / C-peptide expression of S6D7 in SC-islet cells differentiated using the methods described in Table 6. [Figure 20C-D] 20A-20H are flow cytometry graphs showing chromogranin A (CHGA) / C-peptide expression of S6D7 in SC-islet cells differentiated using the methods described in Table 6. [Figure 20E-F] 20A-20H are flow cytometry graphs showing chromogranin A (CHGA) / C-peptide expression of S6D7 in SC-islet cells differentiated using the methods described in Table 6. [Figure 20G-H] 20A-20H are flow cytometry graphs showing chromogranin A (CHGA) / C-peptide expression of S6D7 in SC-islet cells differentiated using the methods described in Table 6. [Figure 21A-B] 21A-21H are flow cytometry graphs showing glucagon (GCG) / somatostatin (SST) expression of S6D7 in SC-islet cells differentiated using the methods described in Table 6. [Figure 21C-D] 21A-21H are flow cytometry graphs showing glucagon (GCG) / somatostatin (SST) expression of S6D7 in SC-islet cells differentiated using the methods described in Table 6. [Figure 21E-F] 21A-21H are flow cytometry graphs showing glucagon (GCG) / somatostatin (SST) expression of S6D7 in SC-islet cells differentiated using the methods described in Table 6. [Figure 21G-H] 21A-21H are flow cytometry graphs showing glucagon (GCG) / somatostatin (SST) expression of S6D7 in SC-islet cells differentiated using the methods described in Table 6. [Figure 22A-B] 22A-22H are flow cytometry graphs showing ISL1 / NKX6.1 expression of S6D12 in SC-islet cells differentiated using the methods described in Table 6. [Figure 22C-D] 22A-22H are flow cytometry graphs showing ISL1 / NKX6.1 expression of S6D12 in SC-islet cells differentiated using the methods described in Table 6. [Figure 22E-F] 22A-22H are flow cytometry graphs showing ISL1 / NKX6.1 expression of S6D12 in SC-islet cells differentiated using the methods described in Table 6. [Figure 22G-H] 22A-22H are flow cytometry graphs showing ISL1 / NKX6.1 expression of S6D12 in SC-islet cells differentiated using the methods described in Table 6. [Figure 23A-B] 23A-23H are flow cytometry graphs showing chromogranin A (CHGA) / C-peptide expression of S6D12 in SC-islet cells differentiated using the methods described in Table 6. [Figure 23C-D] 23A-23H are flow cytometry graphs showing chromogranin A (CHGA) / C-peptide expression of S6D12 in SC-islet cells differentiated using the methods described in Table 6. [Figure 23E-F] 23A-23H are flow cytometry graphs showing chromogranin A (CHGA) / C-peptide expression of S6D12 in SC-islet cells differentiated using the methods described in Table 6. [Figure 23G-H] 23A-23H are flow cytometry graphs showing chromogranin A (CHGA) / C-peptide expression of S6D12 in SC-islet cells differentiated using the methods described in Table 6. [Figure 24A-B] 24A-24H are flow cytometry graphs showing glucagon (GCG) / somatostatin (SST) expression in S6D12 SC-islet cells differentiated using the method described in Table 6. [Figure 24C-D]24A-24H are flow cytometry graphs showing glucagon (GCG) / somatostatin (SST) expression in S6D12 SC-islet cells differentiated using the method described in Table 6. [Figure 24E-F] 24A-24H are flow cytometry graphs showing glucagon (GCG) / somatostatin (SST) expression in S6D12 SC-islet cells differentiated using the method described in Table 6. [Figure 24G-H] 24A-24H are flow cytometry graphs showing glucagon (GCG) / somatostatin (SST) expression in S6D12 SC-islet cells differentiated using the method described in Table 6. [Figure 25] Figures 25A-25B are graphs showing the percentage of ISL1+ cells (Figure 25A) and the percentage of SC-EC cells (Figure 25B) in SC-islets differentiated using Protocol 1, Protocol 2, or Protocol 3. SC-islets differentiated using Protocol 2 or Protocol 3 show an increase in total ISL1+ cells and a decrease in ISL1- cells (SC-EC cells). [Figure 26] Figures 26A-26C are flow cytometry graphs showing ISL1 / NKX6.1 expression of S6D4 in SC-islet cells differentiated using Protocol 1 (Figure 26A), Protocol 2 (Figure 26B), or Protocol 3 (Figure 26C). [Figure 27] Figure 27 is a graph of cell density throughout differentiation using Protocol 1, Protocol 2, or Protocol 3. "Spinner" indicates that the cells were cultured in spinner flasks, and "3-MAG" indicates that the cells were cultured in a 3-liter reactor. [Figure 28] Figures 28A-28B are graphs showing the C-peptide (Figure 28A) and glucagon (Figure 28B) content of SC-islet cells differentiated using Protocol 1 (Table 2), Protocol 2 (Table 2), or Protocol 3 (Table 9). [Figure 29A]Figures 29A-29D are flow cytometry graphs showing ISL1 / Nkx6.1 expression in S6D3 SC-islet cells differentiated using Protocol 1 (Figure 29A), Protocol 2 (Figure 29B), Protocol 3 (Figure 29C), or Protocol 3 with the addition of FGF2 (100 ng / ml) (Figure 29D) in Stage 6 medium (Medium A shown in Table 7A). [Figure 29B] Figures 29A-29D are flow cytometry graphs showing ISL1 / Nkx6.1 expression in S6D3 SC-islet cells differentiated using Protocol 1 (Figure 29A), Protocol 2 (Figure 29B), Protocol 3 (Figure 29C), or Protocol 3 with the addition of FGF2 (100 ng / ml) (Figure 29D) in Stage 6 medium (Medium A shown in Table 7A). [Figure 29C] Figures 29A-29D are flow cytometry graphs showing ISL1 / Nkx6.1 expression in S6D3 SC-islet cells differentiated using Protocol 1 (Figure 29A), Protocol 2 (Figure 29B), Protocol 3 (Figure 29C), or Protocol 3 with the addition of FGF2 (100 ng / ml) (Figure 29D) in Stage 6 medium (Medium A shown in Table 7A). [Figure 29D] Figures 29A-29D are flow cytometry graphs showing ISL1 / Nkx6.1 expression in S6D3 SC-islet cells differentiated using Protocol 1 (Figure 29A), Protocol 2 (Figure 29B), Protocol 3 (Figure 29C), or Protocol 3 with the addition of FGF2 (100 ng / ml) (Figure 29D) in Stage 6 medium (Medium A shown in Table 7A). [Figure 30A] Figures 30A-30D are flow cytometry graphs showing aristas-related homeobox (ARX) / glucagon (GCG) expression of S6D3 in SC-islet cells differentiated using Protocol 1 (Figure 30A), Protocol 2 (Figure 30B), Protocol 3 (Figure 30C), or Protocol 3 with the addition of FGF2 (100 ng / ml) (Figure 30D) in Stage 6 medium (Medium A shown in Table 7A). [Figure 30B]Figures 30A-30D are flow cytometry graphs showing aristas-related homeobox (ARX) / glucagon (GCG) expression of S6D3 in SC-islet cells differentiated using Protocol 1 (Figure 30A), Protocol 2 (Figure 30B), Protocol 3 (Figure 30C), or Protocol 3 with the addition of FGF2 (100 ng / ml) (Figure 30D) in Stage 6 medium (Medium A shown in Table 7A). [Figure 30C] Figures 30A-30D are flow cytometry graphs showing aristas-related homeobox (ARX) / glucagon (GCG) expression of S6D3 in SC-islet cells differentiated using Protocol 1 (Figure 30A), Protocol 2 (Figure 30B), Protocol 3 (Figure 30C), or Protocol 3 with the addition of FGF2 (100 ng / ml) (Figure 30D) in Stage 6 medium (Medium A shown in Table 7A). [Figure 30D] Figures 30A-30D are flow cytometry graphs showing aristas-related homeobox (ARX) / glucagon (GCG) expression of S6D3 in SC-islet cells differentiated using Protocol 1 (Figure 30A), Protocol 2 (Figure 30B), Protocol 3 (Figure 30C), or Protocol 3 with the addition of FGF2 (100 ng / ml) (Figure 30D) in Stage 6 medium (Medium A shown in Table 7A). [Figure 31A] Figures 31A-31D are flow cytometry graphs showing chromogranin A (CHGA) / Nkx6.1 expression in S6D3 SC-islet cells differentiated using Protocol 1 (Figure 31A), Protocol 2 (Figure 31B), Protocol 3 (Figure 31C), or Protocol 3 with the addition of FGF2 (100 ng / ml) (Figure 31D) in Stage 6 medium (Medium A shown in Table 7A). [Figure 31B]Figures 31A-31D are flow cytometry graphs showing chromogranin A (CHGA) / Nkx6.1 expression in S6D3 SC-islet cells differentiated using Protocol 1 (Figure 31A), Protocol 2 (Figure 31B), Protocol 3 (Figure 31C), or Protocol 3 with the addition of FGF2 (100 ng / ml) (Figure 31D) in Stage 6 medium (Medium A shown in Table 7A). [Figure 31C] Figures 31A-31D are flow cytometry graphs showing chromogranin A (CHGA) / Nkx6.1 expression in S6D3 SC-islet cells differentiated using Protocol 1 (Figure 31A), Protocol 2 (Figure 31B), Protocol 3 (Figure 31C), or Protocol 3 with the addition of FGF2 (100 ng / ml) (Figure 31D) in Stage 6 medium (Medium A shown in Table 7A). [Figure 31D] Figures 31A-31D are flow cytometry graphs showing chromogranin A (CHGA) / Nkx6.1 expression in S6D3 SC-islet cells differentiated using Protocol 1 (Figure 31A), Protocol 2 (Figure 31B), Protocol 3 (Figure 31C), or Protocol 3 with the addition of FGF2 (100 ng / ml) (Figure 31D) in Stage 6 medium (Medium A shown in Table 7A). [Figure 32A] Figures 32A-32D are flow cytometry graphs showing SOX9 / Ki67 expression in S6D3 SC-islet cells differentiated using Protocol 1 (Figure 32A), Protocol 2 (Figure 32B), Protocol 3 (Figure 32C), or Protocol 3 with the addition of FGF2 (at 100 ng / ml) (Figure 32D) in Stage 6 medium (Medium A shown in Table 7A). [Figure 32B] Figures 32A-32D are flow cytometry graphs showing SOX9 / Ki67 expression in S6D3 SC-islet cells differentiated using Protocol 1 (Figure 32A), Protocol 2 (Figure 32B), Protocol 3 (Figure 32C), or Protocol 3 with the addition of FGF2 (at 100 ng / ml) (Figure 32D) in Stage 6 medium (Medium A shown in Table 7A). [Figure 32C]Figures 32A-32D are flow cytometry graphs showing SOX9 / Ki67 expression in S6D3 SC-islet cells differentiated using Protocol 1 (Figure 32A), Protocol 2 (Figure 32B), Protocol 3 (Figure 32C), or Protocol 3 with the addition of FGF2 (at 100 ng / ml) (Figure 32D) in Stage 6 medium (Medium A shown in Table 7A). [Figure 32D] Figures 32A-32D are flow cytometry graphs showing SOX9 / Ki67 expression in S6D3 SC-islet cells differentiated using Protocol 1 (Figure 32A), Protocol 2 (Figure 32B), Protocol 3 (Figure 32C), or Protocol 3 with the addition of FGF2 (at 100 ng / ml) (Figure 32D) in Stage 6 medium (Medium A shown in Table 7A). [Figure 33A] Figures 33A-33D are flow cytometry graphs showing ISL1 / Nkx6.1 expression in S6D7 SC-islet cells differentiated using Protocol 1 (Figure 33A), Protocol 2 (Figure 33B), Protocol 3 (Figure 33C), or Protocol 3 with the addition of FGF2 (at 100 ng / ml) (Figure 33D) in Stage 6 medium (Medium A shown in Table 7A). [Figure 33B] Figures 33A-33D are flow cytometry graphs showing ISL1 / Nkx6.1 expression in S6D7 SC-islet cells differentiated using Protocol 1 (Figure 33A), Protocol 2 (Figure 33B), Protocol 3 (Figure 33C), or Protocol 3 with the addition of FGF2 (at 100 ng / ml) (Figure 33D) in Stage 6 medium (Medium A shown in Table 7A). [Figure 33C] Figures 33A-33D are flow cytometry graphs showing ISL1 / Nkx6.1 expression in S6D7 SC-islet cells differentiated using Protocol 1 (Figure 33A), Protocol 2 (Figure 33B), Protocol 3 (Figure 33C), or Protocol 3 with the addition of FGF2 (at 100 ng / ml) (Figure 33D) in Stage 6 medium (Medium A shown in Table 7A). [Figure 33D]Figures 33A-33D are flow cytometry graphs showing ISL1 / Nkx6.1 expression in S6D7 SC-islet cells differentiated using Protocol 1 (Figure 33A), Protocol 2 (Figure 33B), Protocol 3 (Figure 33C), or Protocol 3 with the addition of FGF2 (at 100 ng / ml) (Figure 33D) in Stage 6 medium (Medium A shown in Table 7A). [Figure 34A] Figures 34A-34D are flow cytometry graphs showing ISL1 / Nkx6.1 expression in S6D11 SC-islet cells differentiated using Protocol 1 (Figure 34A), Protocol 2 (Figure 34B), Protocol 3 (Figure 34C), or Protocol 3 with the addition of FGF2 (at 100 ng / ml) (Figure 34D) in Stage 6 medium (Medium A shown in Table 7A). [Figure 34B] Figures 34A-34D are flow cytometry graphs showing ISL1 / Nkx6.1 expression in S6D11 SC-islet cells differentiated using Protocol 1 (Figure 34A), Protocol 2 (Figure 34B), Protocol 3 (Figure 34C), or Protocol 3 with the addition of FGF2 (at 100 ng / ml) (Figure 34D) in Stage 6 medium (Medium A shown in Table 7A). [Figure 34C] Figures 34A-34D are flow cytometry graphs showing ISL1 / Nkx6.1 expression in S6D11 SC-islet cells differentiated using Protocol 1 (Figure 34A), Protocol 2 (Figure 34B), Protocol 3 (Figure 34C), or Protocol 3 with the addition of FGF2 (at 100 ng / ml) (Figure 34D) in Stage 6 medium (Medium A shown in Table 7A). [Figure 34D] Figures 34A-34D are flow cytometry graphs showing ISL1 / Nkx6.1 expression in S6D11 SC-islet cells differentiated using Protocol 1 (Figure 34A), Protocol 2 (Figure 34B), Protocol 3 (Figure 34C), or Protocol 3 with the addition of FGF2 (at 100 ng / ml) (Figure 34D) in Stage 6 medium (Medium A shown in Table 7A). [Figure 35A]Figures 35A-35B are graphs showing human C-peptide levels (Figure 35A) and human glucagon levels (Figure 35B) in SC-islet cells differentiated using Protocol 1, Protocol 2, Protocol 3, or Protocol 3 with the addition of FGF2 (at 100 ng / ml) in Stage 6 medium (Medium A shown in Table 7A). [Figure 35B] Figures 35A-35B are graphs showing human C-peptide levels (Figure 35A) and human glucagon levels (Figure 35B) in SC-islet cells differentiated using Protocol 1, Protocol 2, Protocol 3, or Protocol 3 with the addition of FGF2 (at 100 ng / ml) in Stage 6 medium (Medium A shown in Table 7A). DETAILED DESCRIPTION OF THE INVENTION

[0046] The following description and examples will explain the embodiments of the present disclosure in detail. It should be understood that the present disclosure is not limited to the specific embodiments described herein and may therefore vary. Those skilled in the art will recognize that there are many variations and modifications to the present disclosure, which are encompassed within the scope of the present disclosure.

[0047] All terms are intended to be understood as understood by one of ordinary skill in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0048] The headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. Although various features of the present disclosure may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination. Conversely, although for clarity the present disclosure may be described herein in the context of separate embodiments, the present disclosure may also be implemented in a single embodiment.

[0049] The following definitions supplement the definitions in the art and are directed to this application, and are not attributable to any related or unrelated examples, such as any commonly owned patents or patent applications. Although any methods and materials similar or equivalent to those described herein can be used in the practice of testing the present disclosure, preferred materials and methods are described herein. Therefore, the terms used herein are only for the purpose of describing specific embodiments and are not intended to be limiting.

[0050] In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that as used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0051] The use of "or" in this application means "and / or" unless stated otherwise. As used herein, the terms "and / or" and "any combinations thereof," and their grammatical equivalents, can be used interchangeably. These terms can convey that any combination is specifically intended. For illustrative purposes only, the following phrases, "A, B, and / or C" or "A, B, C, or any combinations thereof," can mean "A individually, B individually, C individually, A and B, B and C, A and C, and A, B, and C." The term "or" can be used conjunctively or disjunctively unless the context specifically implies a disjunctive use.

[0052] Furthermore, use of the term "including" and other forms such as "include," "includes," and "included" is not limiting. References in the specification to "some embodiments," "an embodiment," "one embodiment," or "other embodiments" mean that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least some embodiments of the present disclosure, but not necessarily in all embodiments.

[0053] As used in the specification and claims, the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is intended that any embodiment discussed herein can be implemented with respect to any method or composition of the disclosure, and vice versa. Furthermore, the compositions of the disclosure can be used to achieve the methods of the disclosure.

[0054] The terms "about" or "approximately" refer to within an acceptable error range for a particular value, as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean within or more than one standard deviation, as practiced in the art. Alternatively, "about" can mean a range of up to 20%, 10%, 5%, or 1% of a given value. In another example, the amount "about 10" includes 10 and any amount from 9 to 11. In yet another example, the term "about" in connection with a reference numerical value can also include ranges of plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of that value. Alternatively, particularly with respect to biological systems or processes, the term "about" can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a value. When specific values ​​are described in applications and claims, unless otherwise stated, the term "about" should be assumed to mean within an acceptable error range for the particular value.

[0055] The term "diabetes" and its grammatical equivalents used herein can refer to a disease characterized by high blood sugar levels over a long period of time.For example, the term "diabetes" and its grammatical equivalents used herein can refer to all or any type of diabetes, including but not limited to type 1 diabetes, type 2 diabetes, cystic fibrosis-related diabetes, surgical diabetes, gestational diabetes, and mitochondrial diabetes.In some embodiments, diabetes can be a form of hereditary diabetes.In some embodiments, diabetes can be autoimmune type diabetes.

[0056] The term "endocrine cells," unless otherwise specified, may refer to hormone-producing cells present in the pancreas of a living organism, such as "pancreatic islets," "pancreatic islet cells," "pancreatic islet equivalents," "pancreatic islet-like cells," "pancreatic islets," and grammatical equivalents thereof. In one embodiment, endocrine cells can be differentiated from pancreatic progenitor cells or precursors. Pancreatic islet cells may include various types of cells, including, but not limited to, pancreatic alpha cells, pancreatic beta cells, pancreatic delta cells, pancreatic F cells, and / or pancreatic epsilon cells. Pancreatic islet cells may also refer to groups of cells, cell clusters, etc.

[0057] The terms "progenitor cell" and "precursor" cell are used interchangeably herein to refer to cells that have a more primitive cellular phenotype (e.g., at an earlier step in the developmental pathway or development than a fully differentiated cell) compared to cells that can result from differentiation. Progenitor cells can also often have significant or extremely high proliferative potential. Progenitor cells can give rise to many different differentiated cell types or a single differentiated cell type, depending on the developmental pathway and the environment in which the cells develop and differentiate.

[0058] The term "precursor thereof" in reference to an insulin-positive endocrine cell can mean any cell that can differentiate into an insulin-positive endocrine cell, including, for example, a pluripotent stem cell, a definitive endoderm cell, a primitive gut cell, a pancreatic progenitor cell, or an endocrine precursor cell, and when cultured under appropriate conditions, the precursor cell can be differentiated into an insulin-positive endocrine cell.

[0059] The terms "stem cell-derived β cells," "SC-β cells," "functional β cells," "functional pancreatic β cells," "mature SC-β cells," "β-like cells," and their grammatical equivalents can refer to cells (e.g., non-native pancreatic β cells) that display at least one marker indicative of pancreatic β cells (e.g., PDX-1 or NKX6.1), express insulin, and exhibit a glucose-stimulated insulin secretion (GSIS) response equivalent to or greater than that of endogenous mature β cells (e.g., mature β cells from a healthy, functional pancreas from a healthy, adult, non-diabetic patient). For simplicity, SC-β cells may be referred to simply as "β cells" in this disclosure. In some embodiments, the terms "SC-β cells" and "non-native β cells" are interchangeable as used herein. In some embodiments, "SC-β cells" express lower levels of MAFA than pancreatic β cells from a healthy adult human patient. In some embodiments, "SC-β cells" express higher levels of MAFB than pancreatic β cells from a healthy adult human patient. In some embodiments, "SC-β cells" express higher levels of SIX2, HOPX, IAPP, and / or UCN3 than pancreatic β cells from healthy adult human patients. In some embodiments, "SC-β cells" include mature pancreatic cells. It should be understood that SC-β cells need not be derived (e.g., directly) from stem cells, as the methods of the present disclosure can derive SC-β cells from any insulin-positive endocrine cell or its precursor using any cell as a starting point (e.g., progenitor cells such as embryonic stem cells, induced pluripotent stem cells, definitive endoderm cells, partially reprogrammed somatic cells (somatic cells partially reprogrammed to an intermediate state between induced pluripotent stem cells and the somatic cells from which they are derived), pluripotent cells, totipotent cells, transdifferentiated versions of any of the foregoing cells, etc., can be used, and the invention is not intended to be limited in this manner). In some embodiments, SC-β cells exhibit a response to multiple glucose challenges (e.g., a series of at least one, at least two, or at least three or more glucose challenges). In some embodiments, the response resembles the response of endogenous pancreatic islets (eg, human pancreatic islets) to multiple glucose challenges.In some embodiments, the morphology of SC-β cells is similar to that of endogenous β cells. In some embodiments, SC-β cells exhibit an in vitro GSIS response similar to that of endogenous β cells. In some embodiments, SC-β cells exhibit an in vivo GSIS response similar to that of endogenous β cells. In some embodiments, SC-β cells exhibit both in vitro and in vivo GSIS responses similar to that of endogenous β cells. In some embodiments, the GSIS response of SC-β cells can be observed within two weeks after transplantation of the SC-β cells into a host (e.g., a human or animal). In some embodiments, the GSIS response of SC-β cells can be observed within three weeks after transplantation of the SC-β cells into a host (e.g., a human or animal). In some embodiments, the GSIS response of SC-β cells can be observed within four weeks after transplantation of the SC-β cells into a host (e.g., a human or animal). In some embodiments, the GSIS response of SC-β cells can be observed between one and three months after transplantation of the SC-β cells into a host (e.g., a human or animal). In some embodiments, SC-beta cells package insulin into secretory granules. In some embodiments, SC-beta cells exhibit encapsulated crystalline insulin granules when viewed by electron microscopy. In some embodiments, SC-beta cells exhibit a stimulation index greater than 1. In some embodiments, SC-beta cells exhibit a stimulation index greater than 1.1. In some embodiments, SC-beta cells exhibit a stimulation index greater than 2. In some embodiments, the stimulation index of a cell is characterized by the ratio of insulin secreted in response to a high glucose concentration (e.g., 15 mM) compared to a low glucose concentration (e.g., 2.5 mM).

[0060] In some embodiments, SC-β cells exhibit cytokine-induced apoptosis in response to cytokines. In some embodiments, insulin secretion from SC-β cells is enhanced in response to known antidiabetic drugs (e.g., secretagogues). In some embodiments, SC-β cells are monohormonal. In some embodiments, SC-β cells do not abnormally co-express other hormones, such as glucagon, somatostatin, or pancreatic polypeptide. In some embodiments, SC-β cells exhibit a low replication rate. In some embodiments, SC-β cells increase intracellular Ca2+ in response to glucose.

[0061] The terms "stem cell-derived α cells," "SC-α cells," "functional α cells," "functional pancreatic α cells," "mature SC-α cells," "α-like cells," and their grammatical equivalents can refer to cells (e.g., non-native pancreatic α cells) that display at least one marker indicative of pancreatic α cells (e.g., express glucagon, ISL1, but not NKX6.1), express glucagon, and are capable of secreting functional glucagon in response to stimuli that induce endogenous pancreatic α cells to secrete functional glucagon. In some embodiments, "SC-α cells" do not express somatostatin. In some embodiments, "SC-α cells" do not express insulin. In some embodiments, the terms "SC-α cells" and "non-native α cells" as used herein are interchangeable. In some embodiments, "SC-α cells" include mature pancreatic cells. For brevity, these cells may be referred to simply as "α cells" in the present disclosure.

[0062] The terms "stem cell-derived δ cells," "SC-δ cells," "functional δ cells," "functional pancreatic δ cells," "mature SC-δ cells," "δ-like cells," and their grammatical equivalents can refer to cells (e.g., non-native pancreatic δ cells) that display and express at least one marker indicative of pancreatic δ cells (e.g., somatostatin) and are capable of secreting somatostatin in response to stimuli that induce endogenous pancreatic δ cells to secrete functional glucagon. For simplicity, SC-δ cells may be referred to simply as "δ cells" in the present disclosure. In some embodiments, "SC-δ cells" do not express glucagon. In some embodiments, "SC-δ cells" do not express insulin. In some embodiments, the terms "SC-δ cells" and "non-native δ cells" used herein are interchangeable. In some embodiments, "SC-δ cells" include mature pancreatic cells.

[0063] The terms "stem cell-derived enterochromaffin (EC) cells," "SC-EC cells," and their grammatical equivalents can refer to cells (e.g., non-native pancreatic EC cells) that exhibit at least one marker indicative of pancreatic EC cells (e.g., expressing VMAT1 (vesicular monoamine transporter 1), NKX6.1, but not ISL1). In some embodiments, the terms "SC-EC cells" and "non-native EC cells" as used herein are interchangeable.

[0064] It should be understood that, like SC-β cells, SC-α, SC-δ, and SC-EC cells need not be derived (e.g., directly) from stem cells, as the disclosed methods can derive SC-α cells from other progenitor cells produced during in vitro differentiation of SC-β cells as a starting point (e.g., embryonic stem cells, induced pluripotent stem cells, progenitor cells, partially reprogrammed somatic cells (e.g., somatic cells partially reprogrammed to an intermediate state between an induced pluripotent stem cell and the somatic cell from which it was derived), pluripotent cells, totipotent cells, transdifferentiated versions of any of the foregoing cells, etc., can be used, as the invention is not intended to be so limited).

[0065] As used herein, the term "insulin-producing cells" and its grammatical equivalents refer to cells that differentiate from pancreatic progenitor cells or their precursors and secrete insulin. Insulin-producing cells, as that term is used herein, can include pancreatic β cells and pancreatic β-like cells (e.g., insulin-positive endocrine cells) that synthesize (e.g., transcribe the insulin gene, translate proinsulin mRNA, and modify proinsulin mRNA to form insulin protein), express (e.g., realize the phenotypic trait conveyed by the insulin gene), or secrete (release insulin into the extracellular space) insulin in a constitutive or inducible manner. For example, a population of insulin-producing cells generated by differentiating insulin-positive endocrine cells or their precursors into SC-β cells according to the methods of the present disclosure can be pancreatic β cells or β-like cells (e.g., cells that have at least one or at least two characteristics of endogenous β cells and exhibit a glucose-stimulated insulin secretion (GSIS) response similar to that of endogenous mature β cells). For example, a population of insulin-producing cells produced by the methods disclosed herein may include mature pancreatic β cells or SC-β cells, and may also include non-insulin-producing cells (e.g., cells that do not produce or secrete insulin but have a cell-like phenotype).

[0066] The terms "insulin-positive β-like cells," "insulin-positive endocrine cells," and their grammatical equivalents can refer to cells (e.g., pancreatic endocrine cells) that exhibit at least one marker indicative of pancreatic β cells and also express insulin, but, unless otherwise specified, lack the glucose-stimulated insulin secretion (GSIS) response characteristic of endogenous β cells. Exemplary markers of "insulin-positive endocrine cells" include, but are not limited to, NKX6.1 (NK6 homeobox 1), ISL1 (Islet1), and insulin.

[0067] 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 expressed or present in pancreatic β cells. Exemplary β cell markers include, but are not limited to, pancreatic and duodenal homeobox 1 (PDX1) polypeptide, insulin, c-peptide, amylin, E-cadherin, Hnf3β, PCI / 3, B2, Nkx2.2, GLUT2, PC2, ZnT-8, ISL1, Pax6, Pax4, NeuroD, 1 Inf1b, Hnf-6, Hnf-3beta, VMAT2, NKX6.1, and MafA, as well as those described by Zhang et al., Diabetes. 50(10):2231-6 (2001). In some embodiments, the β cell marker is a nuclear β-cell marker. In some embodiments, the beta cell marker is PDX1 or PH3.

[0068] The term "pancreatic endocrine marker" can refer, without limitation, to proteins, peptides, nucleic acids, protein and nucleic acid polymorphisms, splice variants, protein or nucleic acid fragments, elements, and other analytes expressed or present in pancreatic endocrine cells. Exemplary pancreatic endocrine cell markers include, but are not limited to, Ngn-3, NeuroD, and Islet-1.

[0069] The terms "pancreatic progenitor cells," "pancreatic endocrine precursor cells," "pancreatic precursors," "pancreatic endocrine precursors," and their grammatical equivalents are used interchangeably herein and can refer to stem cells that can become pancreatic hormone-expressing cells that can form pancreatic endocrine cells, pancreatic exocrine cells, or pancreatic duct cells. These cells are committed to differentiation toward at least one type of pancreatic cell, such as insulin-producing beta cells, glucagon-producing alpha cells, somatostatin-producing delta cells (or D cells), and / or pancreatic polypeptide-producing F cells. Such cells can express at least one of the following markers: NGN3, NKX2.2, NeuroD, ISL-1, Pax4, Pax6, or ARX.

[0070] As used herein, the term "PDX1-positive pancreatic progenitor cells" may refer to cells that are pancreatic endoderm (PE) cells capable of differentiating into SC-β cells, such as pancreatic β cells. PDX1-positive pancreatic progenitor cells express the marker PDX1. Other markers include, but are not limited to, Cdcp1, Ptf1a, HNF6, or NRx2.2. PDX1 expression can be assessed by any method known to those skilled in the art, such as immunochemistry using an anti-PDX1 antibody or quantitative RT-PCR. In some embodiments, PDX1-positive pancreatic progenitor cells lack NKX6.1 expression. In some embodiments, PDX1-positive pancreatic progenitor cells can also be referred to as PDX1-positive, NKX6.1-negative pancreatic progenitor cells because they lack NKX6.1 expression. In some embodiments, PDX1-positive pancreatic progenitor cells can also be referred to as "pancreatic foregut endoderm cells."

[0071] The terms "PDX1-positive, NKX6.1-positive pancreatic progenitor cells" and "NKX6.1-positive pancreatic precursors" are used interchangeably herein and can refer to cells that are pancreatic endoderm (PE) cells capable of differentiating into insulin-producing cells, such as pancreatic β cells. PDX1-positive, NKX6-1-positive pancreatic progenitor cells express the markers PDX1 and NKX6-1. Other markers may include, but are not limited to, Cdcp1, Ptf1a, HNF6, or NRx2.2. NKX6-1 expression can be assessed by any method known to those skilled in the art, such as immunochemistry or quantitative RT-PCR using an anti-NKX6-1 antibody. As used herein, the terms "NKX6.1" and "NKX6-1" are equivalent and interchangeable. In some embodiments, PDX1-positive, NKX6-1-positive pancreatic progenitor cells can also be referred to as "pancreatic foregut precursor cells."

[0072] The terms "NeuroD" and "NeuroD1" are used interchangeably to identify proteins and their encoding genes expressed in pancreatic endocrine precursor cells. The term "differentiated cell" or its grammatical equivalents, as defined herein, refers to any primary cell that is not inherently pluripotent. Alternatively, the term "differentiated cell" can refer to a cell of a more specialized cell type derived from a cell of a less specialized cell type (e.g., a stem cell, such as an induced pluripotent stem cell) during the cell differentiation process. Without wishing to be limited by theory, pluripotent stem cells during normal ontogeny can initially differentiate into endodermal cells, which can form pancreatic cells and other endodermal cell types. Further differentiation of endodermal cells leads to the pancreatic pathway, in which approximately 98% of cells become exocrine, ductal, or matrix cells, and approximately 2% become endocrine cells. Early endocrine cells are pancreatic islet precursor cells, which can then further differentiate into insulin-producing cells (e.g., functional endocrine cells) that secrete insulin, glucagon, somatostatin, or pancreatic polypeptide. Endodermal cells can also differentiate into other cells of endodermal origin, such as lung, liver, intestine, and thymus.

[0073] As used herein, the term "somatic cell" can refer to any cell that forms an organism, as opposed to a germline cell. In mammals, germline cells (also known as "gametes") are sperm and eggs that fuse during fertilization to produce a cell called a zygote, from which the entire mammalian embryo develops. Aside from sperm and eggs (gametocytes) and undifferentiated stem cells, from which somatic cells are made, all other cell types in the mammalian body are somatic cells. Internal organs, skin, bone, blood, and connective tissue are all made from somatic cells. In some embodiments, the somatic cell is a "non-embryonic somatic cell," which refers to a somatic cell that is not present in or obtained from an embryo and that does not result from in vitro propagation of such a cell. In some embodiments, the somatic cell is an "adult somatic cell," which refers to a cell that is present in or obtained from an organism other than an embryo or fetus, or that results from in vitro propagation of such a cell. Unless otherwise indicated, the method for converting at least one insulin-positive endocrine cell or precursor thereof into an insulin-producing, glucose-responsive cell can be performed both in vivo and in vitro (in vivo performed when at least one insulin-positive endocrine cell or precursor thereof is present in the subject, and in vitro performed using at least one insulin-positive endocrine cell or precursor thereof that has been isolated and maintained in culture).

[0074] As used herein, the term "adult cell" may refer to cells found throughout the body after embryonic development. As used herein, the term "endodermal cell" can refer to a cell that is from one of the three primary germ cell layers in a very early embryo (the other two being mesoderm and ectoderm). The endoderm is the innermost of the three layers. Endodermal cells differentiate to give rise first to the fetal gut, then to the lining of the respiratory and digestive tract (including the intestine), liver, and pancreas.

[0075] As used herein, the term "cells of endodermal origin" refers to any cell that develops or differentiates from endodermal cells. For example, cells of endodermal origin include cells of the liver, lung, pancreas, thymus, intestine, stomach, and thyroid. Without wishing to be bound by theory, liver and pancreatic progenitor cells (also referred to as pancreatic progenitor cells) arise from endodermal cells of the embryonic foregut. Shortly after their identification, liver and pancreatic progenitors rapidly acquire distinct 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 strong need for hepatocytes and pancreatic β cells in the therapeutic treatment of liver failure and type 1 diabetes. Studies in various model organisms and humans have revealed a network of evolutionarily conserved inductive signals and transcription factors that trigger hepatocyte and pancreatic cell differentiation and provide guidance on how to promote hepatocyte and β cell differentiation from diverse stem and progenitor cell types.

[0076] As used herein, the term "definitive endoderm" may refer to cells that can differentiate from endoderm cells and differentiate into SC-β cells (e.g., pancreatic β cells). Definitive endoderm cells express the marker Sox17. Other markers characteristic of definitive endoderm cells may 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, definitive endoderm cells herein express Sox17 and in some embodiments, Sox17 and HNF3B, but do not express significant levels of GATA4, SPARC, APF, or DAB. Definitive endoderm cells are not positive for the marker PDX1 (e.g., they are PDX1 negative). Definitive endoderm cells have the potential to differentiate into cells including liver, lung, pancreatic, thymus, intestine, stomach, and thyroid cells. Expression of Sox17 and other definitive endoderm markers can be assessed by any method known to those skilled in the art, such as immunochemistry using anti-Sox17 antibodies or quantitative RT-PCR.

[0077] The term "pancreatic endoderm" can refer to cells of endodermal origin that can differentiate into multiple pancreatic lineages, including pancreatic beta cells, but no longer have the capacity to differentiate into non-pancreatic lineages. The term "pancreatic islet cells" refers to a cell population comprising different types of pancreatic endocrine cells (β cells, α cells, δ cells, ε cells) and enterochromaffin (EC) cells, as described, for example, in Xavier et al., (J Clin Med. 2018 Mar; 7(3): 54), which is incorporated herein by reference.

[0078] As used herein, the term "primitive gut cells" or "gut cells" can refer to cells that differentiate from endoderm cells and can differentiate into SC-β cells (e.g., pancreatic β cells). Primitive gut cells express at least one of the following markers: HNF1-β, HNF3-β, or HNF4-α. In some embodiments, primitive gut cells are FOXA2-positive and SOX2-positive, i.e., express both FOXA2 (also known as HNF3-β) and SOX2. In some embodiments, primitive gut cells are FOXA2-positive and PDX1-negative, i.e., express FOXA2 but not PDX1. Primitive gut cells have the potential to differentiate into cells including lung, liver, pancreatic, stomach, and intestinal cells. Expression of HNF1-β and other primitive gut markers can be assessed by any method known to those skilled in the art, such as immunochemistry using an anti-HNF1-β antibody.

[0079] The term "phenotype" can refer to all of the one or several biological characteristics that define a cell or organism under a particular set of environmental conditions and factors, regardless of the actual genotype. The terms "patient," "subject," and "individual" are used interchangeably and can refer to either a human or a non-human animal. As used interchangeably herein, "non-human animal" and "non-human mammal" include mammals such as rats, mice, rabbits, sheep, cats, dogs, cows, pigs, and non-human primates. The term "subject" also encompasses any vertebrate, including, but not limited to, mammals, reptiles, amphibians, and fish. Advantageously, however, the subject is a mammal, such as a human, or other mammal, such as, for example, a dog, cat, horse, or other livestock, or a cow, sheep, pig, or other production mammal. A "patient in need thereof" or "subject in need thereof," as used herein, refers to a patient diagnosed with or suspected of having a disease or disorder, for example, but not limited to, diabetes.

[0080] As used herein, "administering" can mean providing one or more compositions described herein to a patient or subject. By way of example and not limitation, administration, e.g., injection, of a composition can be performed via intravenous (iv), subcutaneous (sc), intradermal (id), intraperitoneal (ip), or intramuscular (im) injection. One or more such routes can be employed. Parenteral administration can be, for example, by bolus injection or gradual perfusion over time. Alternatively, or concurrently, administration can be via the oral route. Additionally, administration can involve surgical deposition of a bolus or pellet of cells or positioning of a medical device. In one embodiment, a composition of the present disclosure can comprise engineered or host cells expressing a nucleic acid sequence described herein or a vector comprising at least one nucleic acid sequence described herein in an amount effective to treat or prevent a proliferative disorder. A pharmaceutical composition can comprise a cell population described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include a buffer such as neutral buffered saline or phosphate buffered saline, a carbohydrate such as glucose, mannose, sucrose, or dextran, mannitol, a protein, polypeptide, or an amino acid such as glycine, an antioxidant, a chelating agent such as EDTA or glutathione, an adjuvant (e.g., aluminum hydroxide), and a preservative.

[0081] The generally disclosed ranges may be referred to as, for example, "X is administered on or about days 1-2, or on or about days 2-3 [or any range of values]," and the ranges include the values ​​themselves (e.g., the endpoints of the ranges) and each individual value within the range.

[0082] All of these different combinations are contemplated by the ranges disclosed throughout. Unless otherwise specifically indicated to the contrary, all disclosed ranges, whether in terms of administration of therapeutic agents, days, months, years, weights, dosages, etc., should be interpreted in this manner.

[0083] Differentiation stage Pancreatic differentiation as disclosed herein can be carried out in a stepwise manner. In exemplary embodiments of the stepwise progression, "Stage 1" or "S1" or "St1" refers to the first stage of the differentiation process, where pluripotent stem cells are differentiated into cells expressing markers characteristic of definitive endoderm cells ("DE," "Stage 1 cells," "St1 cells," or "S1 cells"). "Stage 2" or "S2" or "St2" refers to the second stage, where cells expressing markers characteristic of definitive endoderm cells are differentiated into cells expressing markers characteristic of intestinal cells ("GT," "Stage 2 cells," "St2 cells," or "S2 cells"). "Stage 3" or "S3" or "St3" refers to the third stage, where cells expressing markers characteristic of intestinal cells are differentiated into cells expressing markers characteristic of pancreatic progenitor 1 cells ("PP1," "Stage 3 cells," "St3 cells," or "S3 cells"). "Stage 4" or "S4" or "St4" refers to the fourth stage, differentiation of cells expressing markers characteristic of pancreatic progenitor 1 cells to cells expressing markers characteristic of pancreatic progenitor 2 cells ("PP2," "Stage 4 cells" or "St4 cells" or "S4 cells"). "Stage 5" or "S5" or "St5" refers to the fifth stage, differentiation of cells expressing markers characteristic of pancreatic progenitor 2 cells (e.g., PDX.1+, NKX6.1+) to cells expressing markers characteristic of pancreatic endoderm cells and / or pancreatic endocrine progenitor cells (e.g., insulin+) ("EN," "Stage 5 cells" or "St5 cells" or "S5 cells"). "Stage 6" or "S6" or "St6" refers to the differentiation of cells expressing markers characteristic of pancreatic endocrine precursor cells (e.g., insulin) into cells expressing markers characteristic of pancreatic endocrine beta cells ("SC-beta cells") or pancreatic endocrine alpha cells ("SC-alpha cells"). However, it should be understood that not all cells within a particular population progress through these stages at the same rate, i.e., some cells may progress less or more along the differentiation pathway than the majority of cells present in that population.For example, in some embodiments, SC-β cells can be identified during stage 5, at the end of stage 5, at the beginning of stage 6, etc. Examples of methods for generating cells at any one of stages 1-6 are provided in, for example, U.S. Patent No. 10,030,229, U.S. Patent No. 10,443,042, U.S. Patent Application Publication No. 20200332262, U.S. Patent Application Publication No. 20210198632, U.S. Patent Application Publication No. 20220090020, and International Application Publication No. WO2022147056, each of which is incorporated by reference in its entirety.

[0084] Compositions and methods for generating pancreatic islet cells In some embodiments, the present disclosure provides compositions and methods for differentiating pancreatic islet cells (e.g., from stem cells, such as human embryonic stem cells or human pluripotent stem cells). The compositions and methods provided herein can, in some embodiments, provide pancreatic SC-islet cells, cell populations, or cell clusters comprising pancreatic SC-β cells and pancreatic SC-α cells. In some embodiments, such pancreatic SC-islet cells, cell populations, or cell clusters exhibit high insulin content, an excellent glucose-dependent insulin secretion response, and a high percentage of pancreatic SC-α, SC-β, SC-δ, and enterochromaffin (EC) cells, making them structurally and functionally similar to native pancreatic islets. In some embodiments, populations of pancreatic islet cells (e.g., stem cell-derived islet cells) generated using the compositions and methods described herein comprise approximately 30%-45% pancreatic SC-β cells, 40%-50% pancreatic α cells, 3-10% pancreatic SC-δ cells, and / or less than 20% SC-EC cells. In some embodiments, populations of pancreatic islet cells (e.g., stem cell-derived islet cells) produced using the compositions and methods described herein have improved glucose-stimulated insulin secretion (GSIS) responses compared to cell compositions produced according to conventional methods. In some embodiments, populations of pancreatic islet cells (e.g., stem cell-derived islet cells) produced using the compositions and methods described herein have dynamic GSIS responses similar to those of native pancreatic islets (e.g., pancreatic islets from a healthy, functioning pancreas from a healthy, adult, non-diabetic subject).

[0085] In some embodiments, the methods for generating pancreatic islet cells (e.g., SC-beta cells, SC-alpha cells, SC-delta cells) described herein include contacting pancreatic progenitor cells (e.g., PDX1-positive, NKX6.1-negative pancreatic progenitor cells) with a medium containing a forkhead box O1 (FoxO1) inhibitor and / or a Notch signaling inhibitor (e.g., a gamma-secretase inhibitor). In some embodiments, the methods described herein include contacting pancreatic progenitor cells in culture with a FOXO1 inhibitor and / or a Notch signaling pathway inhibitor (e.g., a gamma-secretase inhibitor), wherein the culture comprises PDX1-positive, NKX6.1-negative pancreatic progenitor cells and PDX1-positive, NKX6.1-positive pancreatic progenitor cells. In some embodiments, the methods described herein include contacting pancreatic progenitor cells (e.g., PDX1-positive, NKX6.1-positive pancreatic progenitor cells) with a medium containing a Wnt signaling pathway inhibitor and / or a PKC activator. In some embodiments, the methods described herein include contacting pancreatic progenitor cells (e.g., PDX1-positive, NKX6.1-negative pancreatic progenitor cells) with a medium containing a Forkhead Box O1 (FoxO1) inhibitor and / or a Notch signaling pathway inhibitor (e.g., a γ-secretase inhibitor), and contacting the resulting cells with a medium containing a Wnt signaling pathway inhibitor and / or a PKC activator. In some embodiments, the methods do not include simultaneously contacting the cells with a Wnt signaling pathway inhibitor and a FoxO1 inhibitor.

[0086] Compositions Comprising a FoxO1 Inhibitor and Optionally a Notch Signaling Inhibitor In some aspects, the present disclosure provides an in vitro composition comprising a pancreatic progenitor cell population and a medium comprising a forkhead box O1 (FoxO1) inhibitor. In some embodiments, the medium further comprises a Notch signaling pathway inhibitor (e.g., a gamma-secretase inhibitor). In some embodiments, the medium further comprises a PKC activator. In some embodiments, the medium further comprises one or more (e.g., 1, 2, 3, 4, 5) agents selected from a fibroblast growth factor, a sonic hedgehog (SHH) signaling pathway inhibitor, a retinoic acid, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, and a TGF-β ligand. In some embodiments, the medium further comprises a fibroblast growth factor, a sonic hedgehog (SHH) signaling pathway inhibitor, a retinoic acid, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, and a TGF-β ligand.

[0087] In some embodiments, the medium comprises a FoxO1 inhibitor, a Notch signaling pathway inhibitor (e.g., a gamma-secretase inhibitor), a PKC activator, a fibroblast growth factor, a sonic hedgehog (SHH) signaling pathway inhibitor, retinoic acid, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, and a TGF-β ligand. In some embodiments, the medium comprises a FoxO1 inhibitor AS1842856 (e.g., Sigma-Aldrich catalog number 344355), XXI, PdBu, keratinocyte growth factor (KGF), SANT-1, RA, triazovidine, and activin A. In some embodiments, the medium does not comprise a Wnt signaling pathway inhibitor.

[0088] In some embodiments, the FoxO1 inhibitor (e.g., AS1842856) is present in the medium at a concentration of 0.1 μM to 10 μM. In some embodiments, the FoxO1 inhibitor (AS1842856) is present at a concentration of 0.1 μM to 10 μM, 0.1 μM to 9 μM, 0.1 μM to 8 μM, 0.1 μM to 7 μM, 0.1 μM to 6 μM, 0.1 μM to 5 μM, 0.1 μM to 4 μM, 0.1 μM to 3 μM, 0.1 μM to 2 μM, 0.1 μM to 1 μM, 0.1 μM to 0.5 μM, 0.5 μM to 10 μM, 0. 5μM~9μM, 0.5μM~8μM, 0.5μM~7μM, 0.5μM~6μM, 0.5μM~5μM, 0.5μM~4μM, 0.5μM~3μM, 0.5μM~2μM, 0.5μM~1μM, 1μM~10μM, 1μM~9μM, 1μM~8μM, 1μM~7μM, 1μM~6μM, 1μM~5μM, 1μM~4μM, 1μM~3μM, 1μM~ 2μM, 2μM~10μM, 2μM~9μM, 2μM~8μM, 2μM~7μM, 2μM~6μM, 2μM~5μM, 2μM~4μM, 2μM~3μM, 3μM~10μM, 3μM~9μM, 3μM~8μM, 3μM~7μM, 3μM~6μM, 3μM~5μM, 3μM~4μM, 4μM~10μM, 4μM~9μM, 4μM~8μM, 4μM~7μ In some embodiments, the FoxO1 inhibitor (e.g., AS1842856) is present in the medium at a concentration of 0.5 μM to 5 μM (e.g., 0.5 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, or 5 μM). In some embodiments, the FoxO1 inhibitor (e.g., AS1842856) is present in the medium at a concentration of 0.7-1.3 μM, 0.8-1.2 μM, or 0.9-1.1 μM. In some embodiments, the FoxO1 inhibitor (e.g., AS1842856) is present in the medium at a concentration of 1 μM.

[0089] In some embodiments, the Notch signaling pathway inhibitor (e.g., a γ-secretase inhibitor such as XXI) is present in the medium at a concentration of 0.1 μM to 10 μM. In some embodiments, the Notch signaling pathway inhibitor (e.g., a γ-secretase inhibitor such as XXI) is present in the medium at a concentration of 0.1 μM to 10 μM, 0.1 μM to 9 μM, 0.1 μM to 8 μM, 0.1 μM to 7 μM, 0.1 μM to 6 μM, 0.1 μM to 5 μM, 0.1 μM to 4 μM, 0.1 μM to 3 μM, 0.1 μM to 2 μM, 0.1 μM to 1 μM, or 0.1 μM to 0.5 μM. , 0.5μM~10μM, 0.5μM~9μM, 0.5μM~8μM, 0.5μM~7μM, 0.5μM~6μM, 0.5μM~5μM, 0.5μM~4μM, 0.5μM~3μM , 0.5μM~2μM, 0.5μM~1μM, 1μM~10μM, 1μM~9μM, 1μM~8μM, 1μM~7μM, 1μM~6μM, 1μM~5μM, 1μM~4μM, 1μM ~3μM, 1μM~2μM, 2μM~10μM, 2μM~9μM, 2μM~8μM, 2μM~7μM, 2μM~6μM, 2μM~5μM, 2μM~4μM, 2μM~3μM, 3μM ~10μM, 3μM~9μM, 3μM~8μM, 3μM~7μM, 3μM~6μM, 3μM~5μM, 3μM~4μM, 4μM~10μM, 4μM~9μM, 4μM~8μM, 4μM Present in the medium at a concentration of 4 μM to 7 μM, 4 μM to 6 μM, 4 μM to 5 μM, 5 μM to 10 μM, 5 μM to 9 μM, 5 μM to 8 μM, 5 μM to 7 μM, 5 μM to 6 μM, 6 μM to 10 μM, 6 μM to 9 μM, 6 μM to 8 μM, 6 μM to 7 μM, 7 μM to 10 μM, 7 μM to 9 μM, 7 μM to 8 μM, 8 μM to 10 μM, 8 μM to 9 μM, or 9 μM to 10 μM. In some embodiments, the Notch signaling pathway inhibitor (e.g., a γ-secretase inhibitor such as XXI) is present in the medium at a concentration of 0.5 μM to 5 μM (e.g., 0.5 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, or 5 μM). In some embodiments, the Notch signaling pathway inhibitor (e.g., a γ-secretase inhibitor such as XXI) is present in the medium at a concentration of 1.5 μM to 2.5 μM or 1.8 μM to 2.2 μM. In some embodiments, the Notch signaling pathway inhibitor (e.g., a γ-secretase inhibitor such as XXI) is present in the medium at a concentration of 2 μM.

[0090] In some embodiments, the PKC activator (e.g., PdBu) is present in the medium at a concentration of 0.1 μM to 10 μM. In some embodiments, the PKC activator (e.g., PdBu) is present in the medium at a concentration of 0.1 μM to 10 μM, 0.1 μM to 9 μM, 0.1 μM to 8 μM, 0.1 μM to 7 μM, 0.1 μM to 6 μM, 0.1 μM to 5 μM, 0.1 μM to 4 μM, 0.1 μM to 3 μM, 0.1 μM to 2 μM, 0.1 μM to 1 μM, 0.1 μM to 0.5 μM, 0.5 μM to 10 μM, 0.5 μM to 1 ... μM~9μM, 0.5μM~8μM, 0.5μM~7μM, 0.5μM~6μM, 0.5μM~5μM, 0.5μM~4μM, 0.5μM~3μM, 0.5μM~2μM, 0 .5μM~1μM, 1μM~10μM, 1μM~9μM, 1μM~8μM, 1μM~7μM, 1μM~6μM, 1μM~5μM, 1μM~4μM, 1μM~3μM, 1μM~2 μM, 2μM~10μM, 2μM~9μM, 2μM~8μM, 2μM~7μM, 2μM~6μM, 2μM~5μM, 2μM~4μM, 2μM~3μM, 3μM~10μM, 3 μM~9μM, 3μM~8μM, 3μM~7μM, 3μM~6μM, 3μM~5μM, 3μM~4μM, 4μM~10μM, 4μM~9μM, 4μM~8μM, 4μM~7μM In some embodiments, the PKC activator (e.g., PdBu) is present in the medium at a concentration of 0.2 μM to 1 μM (e.g., 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, or 1 μM). In some embodiments, the PKC activator (e.g., PdBu) is present in the medium at a concentration of 0.3 μM to 0.7 μM or 0.4 μM to 0.6 μM, hi some embodiments, the PKC activator (e.g., PdBu) is present in the medium at a concentration of 0.5 μM.

[0091] In some embodiments, the sonic hedgehog (SHH) signaling pathway inhibitor (e.g., SANT-1) is present in the medium at a concentration of 0.1 μM to 10 μM. In some embodiments, the sonic hedgehog (SHH) signaling pathway inhibitor (e.g., SANT-1) is present in the medium at a concentration of 0.1 μM to 10 μM, 0.1 μM to 9 μM, 0.1 μM to 8 μM, 0.1 μM to 7 μM, 0.1 μM to 6 μM, 0.1 μM to 5 μM, 0.1 μM to 4 μM, 0.1 μM to 3 μM, 0.1 μM to 2 μM, 0.1 μM to 1 μM, or 0.1 μM to 0.5 μM. , 0.5μM~10μM, 0.5μM~9μM, 0.5μM~8μM, 0.5μM~7μM, 0.5μM~6μM, 0.5μM~5μM, 0.5μM~4μM, 0.5μM~3μM , 0.5μM~2μM, 0.5μM~1μM, 1μM~10μM, 1μM~9μM, 1μM~8μM, 1μM~7μM, 1μM~6μM, 1μM~5μM, 1μM~4μM, 1μM ~3μM, 1μM~2μM, 2μM~10μM, 2μM~9μM, 2μM~8μM, 2μM~7μM, 2μM~6μM, 2μM~5μM, 2μM~4μM, 2μM~3μM, 3μM ~10μM, 3μM~9μM, 3μM~8μM, 3μM~7μM, 3μM~6μM, 3μM~5μM, 3μM~4μM, 4μM~10μM, 4μM~9μM, 4μM~8μM, 4μM Present in the medium at a concentration of 4 μM to 7 μM, 4 μM to 6 μM, 4 μM to 5 μM, 5 μM to 10 μM, 5 μM to 9 μM, 5 μM to 8 μM, 5 μM to 7 μM, 5 μM to 6 μM, 6 μM to 10 μM, 6 μM to 9 μM, 6 μM to 8 μM, 6 μM to 7 μM, 7 μM to 10 μM, 7 μM to 9 μM, 7 μM to 8 μM, 8 μM to 10 μM, 8 μM to 9 μM, or 9 μM to 10 μM. In some embodiments, the sonic hedgehog (SHH) signaling pathway inhibitor (e.g., SANT-1) is present in the medium at a concentration of 0.1 μM to 0.5 μM (e.g., 0.1 μM, 0.15 μM, 0.2 μM, 0.25 μM, 0.3 μM, 0.35 μM, 0.4 μM, 0.45 μM, or 0.5 μM). In some embodiments, the sonic hedgehog (SHH) signaling pathway inhibitor (e.g., SANT-1) is present in the medium at a concentration of 0.25 μM.

[0092] In some embodiments, the Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor (e.g., thiazovivin) is present in the medium at a concentration of 1 μM to 10 μM. In some embodiments, the Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor (e.g., thiazovivin) is present in the medium at a concentration of 1 μM to 10 μM, 1 μM to 9 μM, 1 μM to 8 μM, 1 μM to 7 μM, 1 μM to 6 μM, 1 μM to 5 μM, 1 μM to 4 μM, 1 μM to 3 μM, 1 μM to 2 μM, 2 μM to 10 μM, 2 μM to 9 μM, 2 μM to 8 μM, 2 μM to 7 μM, 2 μM to 6 μM, 2 μM to 5 μM, 2 μM to 4 μM, 2 μM to 3 μM, 3 μM to 10 μM, 3 μM to 9 μM, 3 μM to 8 μM, 3 μM to 8 μM, 3 μM to 9 μM, 3 μM to 8 μM, 3 μM to 5 μM, 3 μM to 4 μM, 3 μM to 3 μM, 3 μM to 10 μM, 3 μM to 9 μM, 3 μM to 8 μM, 3 μM to 5 ... μM~7μM, 3μM~6μM, 3μM~5μM, 3μM~4μM, 4μM~10μM, 4μM~9μM, 4μM~8μM, 4μM~7μM, 4μM~6μM, 4μM~5μM, 5μM~10μM, 5μM~9μM, 5μM~8μM, 5μM~7 present in the culture medium at a concentration of μM, 5 μM to 6 μM, 6 μM to 10 μM, 6 μM to 9 μM, 6 μM to 8 μM, 6 μM to 7 μM, 7 μM to 10 μM, 7 μM to 9 μM, 7 μM to 8 μM, 8 μM to 10 μM, 8 μM to 9 μM, or 9 μM to 10 μM. In some embodiments, the Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor (e.g., thiazovivin) is present in the medium at a concentration of 1 μM to 5 μM (e.g., 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, or 5 μM). In some embodiments, the Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor (e.g., thiazovivin) is present in the medium at a concentration of 2.5 μM.

[0093] In some embodiments, retinoic acid is present in the medium at a concentration of 0.05 μM to 0.5 μM. In some embodiments, retinoic acid is present in the medium at a concentration of 0.05 μM to 0.5 μM, 0.1 μM to 0.5 μM, 0.15 μM to 0.5 μM, 0.2 μM to 0.5 μM, 0.25 μM to 0.5 μM, 0.3 μM to 0.5 μM, 0.35 μM to 0.5 μM, 0.4 μM to 0.5 μM, 0.45 μM to 0.5 μM, 0.05 μM to 0.4 μM, 0.1 μM to 0.4 μM, 0.15 μM to 0.4 μM, 0.2 μM In some embodiments, retinoic acid is present in the medium at a concentration of 0.05 μM to 0.2 μM (e.g., 0.05 μM, 0.1 μM, 0.15 μM, or 0.2 μM). In some embodiments, retinoic acid is present in the medium at a concentration of 0.1 μM.

[0094] In some embodiments, the TGF-β ligand (eg, activin A) is present in the medium at a concentration of 1 ng / ml to 10 ng / ml. In some embodiments, the TGF-β ligand (e.g., activin A) is 1ng / ml to 10ng / ml, 1ng / ml to 9ng / ml, 1ng / ml to 8ng / ml, 1ng / ml to 7n g / ml, 1ng / ml~6ng / ml, 1ng / ml~5ng / ml, 1ng / ml~4ng / ml, 1ng / ml~3ng / ml, 1ng / ml~2ng / ml, 2ng / ml~10ng / ml, 2ng / ml~9ng / ml, 2ng / ml~8ng / ml, 2ng / ml~7ng / ml, 2ng / ml~6ng / ml, 2ng / ml~5ng / ml, 2ng / ml~4ng / m l, 2ng / ml~3ng / ml, 3ng / ml~10ng / ml, 3ng / ml~9ng / ml, 3ng / ml~8ng / ml, 3ng / ml~7ng / ml, 3ng / ml~6ng / ml , 3ng / ml~5ng / ml, 3ng / ml~4ng / ml, 4ng / ml~10ng / ml, 4ng / ml~9ng / ml, 4ng / ml~8ng / ml, 4ng / ml~7ng / ml, 4ng / ml~6ng / ml, 4ng / ml~5ng / ml, 5ng / ml~10ng / ml, 5ng / ml~9ng / ml, 5ng / ml~8ng / ml, 5ng / ml~7ng / ml, 5 ng / ml~6ng / ml, 6ng / ml~10ng / ml, 6ng / ml~9ng / ml, 6ng / ml~8ng / ml, 6ng / ml~7ng / ml, 7ng / ml~10ng / ml, 7 Present in the medium at a concentration of ng / ml to 9ng / ml, 7ng / ml to 8ng / ml, 8ng / ml to 10ng / ml, 8ng / ml to 9ng / ml, or 9ng / ml to 10ng / ml. In some embodiments, the TGF-β ligand (e.g., activin A) is present in the medium at a concentration of 2 ng / ml to 8 ng / ml (e.g., 2 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml). In some embodiments, the TGF-β ligand (e.g., activin A) is present in the medium at a concentration of 5 ng / ml.

[0095] In some embodiments, the fibroblast growth factor (e.g., keratinocyte growth factor (KGF)) is present in the medium at a concentration of 10 ng / ml to 100 ng / ml. In some embodiments, the fibroblast growth factor (e.g., keratinocyte growth factor (KGF)) is present in the medium at a concentration of 10 ng / ml to 100 ng / ml, 10 ng / ml to 90 ng / ml, 10 ng / ml to 80 ng / ml, 10 ng / ml to 70 ng / ml, 10 ng / ml to 60 ng / ml, 10 ng / ml to 50 ng / ml, 10 ng / ml to 40 ng / ml, 10 ng / ml to 30 ng / ml, 10 ng / ml to 20 ng / ml, 20 ng / ml to 100ng / ml, 20ng / ml~90ng / ml, 20ng / ml~80ng / ml, 20ng / ml~70ng / ml, 20ng / ml~60ng / ml, 20ng / ml~50ng / ml, 20ng / ml~4 0ng / ml, 20ng / ml~30ng / ml, 30ng / ml~100ng / ml, 30ng / ml~90ng / ml, 30ng / ml~80ng / ml, 30ng / ml~70ng / ml, 30ng / ml~60n g / ml, 30ng / ml~50ng / ml, 30ng / ml~40ng / ml, 40ng / ml~100ng / ml, 40ng / ml~90ng / ml, 40ng / ml~80ng / ml, 40ng / ml~70ng / ml, 40ng / ml~60ng / ml, 40ng / ml~50ng / ml, 50ng / ml~100ng / ml, 50ng / ml~90ng / ml, 50ng / ml~80ng / ml, 50ng / ml~70ng / m In some embodiments, a fibroblast growth factor (e.g., keratinocyte growth factor (KGF)) is present in the medium at a concentration of 20 ng / ml to 80 ng / ml (e.g., 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml to 60 ng / ml, 60 ng / ml to 100 ng / ml, 60 ng / ml to 90 ng / ml, 60 ng / ml to 80 ng / ml, 60 ng / ml to 70 ng / ml, 70 ng / ml to 100 ng / ml, 70 ng / ml to 90 ng / ml, 70 ng / ml to 80 ng / ml, 80 ng / ml to 100 ng / ml, or 90 ng / ml to 100 ng / ml).In some embodiments, fibroblast growth factor (eg, keratinocyte growth factor (KGF)) is present in the medium at a concentration of 50 ng / ml.

[0096] In some embodiments, the medium contains a FoxO1 inhibitor (e.g., AS1842856) at a concentration of 0.5 μM to 5 μM, a Notch signaling pathway inhibitor (e.g., a γ-secretase inhibitor such as XXI) at a concentration of 0.5 μM to 5 μM, a PKC activator (e.g., PdBu) at a concentration of 0.2 μM to 1 μM, a fibroblast growth factor (e.g., KGF) at a concentration of 20 ng / ml to 80 ng / ml, a Sonic hedgehog (SHH) signaling pathway inhibitor (e.g., SANT-1) at a concentration of 0.1 μg / ml to 0.5 μg / ml, retinoic acid at a concentration of 0.05 μM to 0.2 μM, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor (e.g., thiazovivin) at a concentration of 1 μM to 5 μM, and a TGF-β ligand (e.g., activin A) at a concentration of 2 ng / ml to 8 ng / ml.

[0097] In some embodiments, the medium comprises a FoxO1 inhibitor (e.g., AS1842856) at a concentration of 1 μM, a Notch signaling pathway inhibitor (e.g., a γ-secretase inhibitor such as XXI) at a concentration of 2 μM, a PKC activator (e.g., PdBu) at a concentration of 0.5 μM, a fibroblast growth factor (e.g., KGF) at a concentration of 50 ng / ml, a sonic hedgehog (SHH) signaling pathway inhibitor (e.g., SANT-1) at a concentration of 0.25 μM, retinoic acid at a concentration of 0.1 μM, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor (e.g., thiazovivin) at a concentration of 0.5 μM, and a TGF-β ligand (e.g., activin A) at a concentration of 5 ng / ml.

[0098] In some embodiments, the in vitro compositions described herein further comprise a water-soluble synthetic polymer. In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol (PVA), poloxamer, polyvinylpyrrolidone, polyethylene glycol (PEG), a PEG copolymer, poly(N-isopropylacrylamide), or polyacrylamide, and optionally, the water-soluble synthetic polymer is polyvinyl alcohol. In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol (PVA). In some embodiments, the water-soluble synthetic polymer has a concentration of 0.005% to 0.5% (w / v), 0.01% to 0.2% (w / v), 0.02% to 0.1% (w / v), or 0.03% to 0.08% (w / v) of the medium. In some embodiments, the water-soluble synthetic polymer has a concentration of 0.005% (w / v), 0.01% (w / v), 0.05% (w / v), 0.1% (w / v), 0.15% (w / v), 0.2% (w / v), 0.25% (w / v), 0.3% (w / v), 0.35% (w / v), 0.4% (w / v), 0.45% (w / v), or 0.5% (w / v) of the medium. In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol (PVA), and the PVA is at most 85% (e.g., 75%-80%) hydrolyzed.

[0099] In some embodiments, the in vitro composition comprises a pancreatic progenitor cell population comprising PDX1-positive, NKX6.1-negative cells. In some embodiments, the in vitro composition described herein comprises a pancreatic progenitor cell population comprising PDX1-positive, NKX6.1-positive cells. In some embodiments, the in vitro composition described herein comprises a pancreatic progenitor cell population comprising PDX1-positive, NKX6.1-negative cells and PDX1-positive, NKX6.1-positive cells. In some embodiments, the PDX1-positive, NKX6.1-positive cells are insulin-negative.

[0100] In some embodiments, the pancreatic progenitor cell population comprises more PDX1-positive, NKX6.1-negative cells than PDX1-positive, NKX6.1-positive cells. In some embodiments, at least 50% (e.g., at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%) of the pancreatic progenitor cell population are PDX1-positive, NKX6.1-negative. In some embodiments, 50% or less (e.g., 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less) of the pancreatic progenitor cell population are PDX1-positive, NKX6.1-positive pancreatic progenitor cells. In some embodiments, 50% to 90% (e.g., 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 90%, 60% to 80%, 60% to 70%, 30% to 80%, 70% to 90%, 70% to 80%, or 80% to 90%) of the pancreatic progenitor cell population are PDX1-positive and NKX6.1-negative. In some embodiments, 10% to 50% (e.g., 10% to 50%, 20% to 50%, 30% to 50%, 40% to 50%, 10% to 40%, 20% to 40%, 30% to 40%, 10% to 30%, 20% to 30%, or 10% to 20%) of the pancreatic progenitor cell population are PDX1-positive and NKX6.1-positive pancreatic progenitor cells. In some embodiments, 50% to 90% (e.g., 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 90%, 60% to 80%, 60% to 70%, 30% to 80%, 70% to 90%, 70% to 80%, or 80% to 90%) of the pancreatic progenitor cell population are PDX1-positive and NKX6.1-negative, and 10% to 50% (e.g., 10% to 50%, 20% to 50%, 30% to 50%, 40% to 50%, 10% to 40%, 20% to 40%, 30% to 40%, 10% to 30%, 20% to 30%, or 10% to 20%) of the pancreatic progenitor cell population are PDX1-positive and NKX6.1-positive pancreatic progenitor cells. In some embodiments, at least 50% of the pancreatic progenitor cell population are PDX1-positive, NKX6.1-negative, and no more than 50% of the pancreatic progenitor cell population are PDX1-positive, NKX6.1-positive pancreatic progenitor cells.

[0101] In some embodiments, the pancreatic progenitor cell population comprises more PDX1-positive, NKX6.1-positive cells than PDX1-positive, NKX6.1-negative cells. In some embodiments, at least 50% (e.g., at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%) of the pancreatic progenitor cell population are PDX1-positive, NKX6.1-positive. In some embodiments, 50% or less (e.g., 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less) of the pancreatic progenitor cell population are PDX1-positive, NKX6.1-negative pancreatic progenitor cells. In some embodiments, 50% to 90% (e.g., 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 90%, 60% to 80%, 60% to 70%, 30% to 80%, 70% to 90%, 70% to 80%, or 80% to 90%) of the pancreatic progenitor cell population are PDX1-positive and NKX6.1-positive. In some embodiments, 10% to 50% (e.g., 10% to 50%, 20% to 50%, 30% to 50%, 40% to 50%, 10% to 40%, 20% to 40%, 30% to 40%, 10% to 30%, 20% to 30%, or 10% to 20%) of the pancreatic progenitor cell population are PDX1-positive and NKX6.1-negative pancreatic progenitor cells. In some embodiments, 50% to 90% (e.g., 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 90%, 60% to 80%, 60% to 70%, 30% to 80%, 70% to 90%, 70% to 80%, or 80% to 90%) of the pancreatic progenitor cell population are PDX1-positive and NKX6.1-positive, and 10% to 50% (e.g., 10% to 50%, 20% to 50%, 30% to 50%, 40% to 50%, 10% to 40%, 20% to 40%, 30% to 40%, 10% to 30%, 20% to 30%, or 10% to 20%) are PDX1-positive, NKX6.1-negative pancreatic progenitor cells. In some embodiments, at least 50% of the pancreatic progenitor cell population are PDX1-positive, NKX6.1-positive, and 50% or less of the pancreatic progenitor cell population are PDX1-positive, NKX6.1-negative pancreatic progenitor cells.

[0102] Compositions Comprising a Wnt Inhibitor and Optionally a PKC Activator In another aspect, the present disclosure provides an in vitro composition comprising a pancreatic progenitor cell population comprising PDX1-positive, NKX6.1-positive, insulin-negative cells and a medium containing a Wnt signaling pathway inhibitor (e.g., a tankyrase inhibitor). In some embodiments, the medium further comprises a protein kinase C (PKC) activator. In some embodiments, the medium does not contain a FOXO1 inhibitor. In some embodiments, the pancreatic progenitor cell population has previously been cultured in a medium containing a FOXO1 inhibitor. In some embodiments, the medium further comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) agents selected from a sonic hedgehog (SHH) signaling pathway inhibitor, an epidermal growth factor receptor (EGFR), a Notch signaling pathway inhibitor (e.g., a γ-secretase inhibitor), a TGFβ-R1 kinase inhibitor, a thyroid hormone, a bone morphogenetic (BMP) signaling pathway inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a protein kinase inhibitor, a histone methyltransferase EZH2 inhibitor, and retinoic acid. In some embodiments, the medium further comprises a sonic hedgehog (SHH) signaling pathway inhibitor, an epidermal growth factor, a notch signaling pathway inhibitor (e.g., a gamma-secretase inhibitor), a TGFβ-R1 kinase inhibitor, a thyroid hormone, a bone morphogenetic (BMP) signaling pathway inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a protein kinase inhibitor, a histone methyltransferase EZH2 inhibitor, and retinoic acid.

[0103] In some embodiments, the medium further comprises one or more (e.g., 1, 2, 3, or 4) agents selected from acetyl-CoA-related metabolites, HDAC inhibitors, redox homeostasis regulators, and one-carbon metabolic pathway intermediates. In some embodiments, the medium further comprises acetyl-CoA-related metabolites, HDAC inhibitors, redox homeostasis regulators, and one-carbon metabolic pathway intermediates.

[0104] In some embodiments, the medium further comprises vitamins, hi some embodiments, the medium further comprises glutamine. In some embodiments, the medium comprises a PKC activator, a Wnt signaling pathway inhibitor (e.g., a tankyrase inhibitor), a sonic hedgehog (SHH) signaling pathway inhibitor, an epidermal growth factor, a notch signaling pathway inhibitor (e.g., a gamma-secretase inhibitor), a TGFβ-R1 kinase inhibitor, a thyroid hormone, a bone morphogenetic (BMP) signaling pathway inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a protein kinase inhibitor, a histone methyltransferase EZH2 inhibitor, retinoic acid, an acetyl-CoA-related metabolite, an HDAC inhibitor, a redox homeostasis regulator, a one-carbon metabolic pathway intermediate, a vitamin, and glutamine. In some embodiments, the medium comprises PdBu, NVP-TNKS656, SANT-1, betacellulin, XXI, Alk5i, GC-1, LDN-193189, thiazovivin, staurosporine, DZNEP, retinoic acid, acetate, beta-hydroxybutyrate, taurine, formate, biotin, and glutamine.

[0105] In some embodiments, the PKC activator (e.g., PdBu) is present in the medium at a concentration of 0.1 μM to 10 μM. In some embodiments, the PKC activator (e.g., PdBu) is present in the medium at a concentration of 0.1 μM to 10 μM, 0.1 μM to 9 μM, 0.1 μM to 8 μM, 0.1 μM to 7 μM, 0.1 μM to 6 μM, 0.1 μM to 5 μM, 0.1 μM to 4 μM, 0.1 μM to 3 μM, 0.1 μM to 2 μM, 0.1 μM to 1 μM, 0.1 μM to 0.5 μM, 0.5 μM to 10 μM, 0.5 μM to 1 ... μM~9μM, 0.5μM~8μM, 0.5μM~7μM, 0.5μM~6μM, 0.5μM~5μM, 0.5μM~4μM, 0.5μM~3μM, 0.5μM~2μM, 0 .5μM~1μM, 1μM~10μM, 1μM~9μM, 1μM~8μM, 1μM~7μM, 1μM~6μM, 1μM~5μM, 1μM~4μM, 1μM~3μM, 1μM~2 μM, 2μM~10μM, 2μM~9μM, 2μM~8μM, 2μM~7μM, 2μM~6μM, 2μM~5μM, 2μM~4μM, 2μM~3μM, 3μM~10μM, 3 μM~9μM, 3μM~8μM, 3μM~7μM, 3μM~6μM, 3μM~5μM, 3μM~4μM, 4μM~10μM, 4μM~9μM, 4μM~8μM, 4μM~7μM In some embodiments, the PKC activator (e.g., PdBu) is present in the medium at a concentration of 0.2 μM to 1 μM (e.g., 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, or 1 μM). In some embodiments, the PKC activator (e.g., PdBu) is present in the medium at a concentration of 0.3 μM to 0.7 μM or 0.4 μM to 0.6 μM, hi some embodiments, the PKC activator (e.g., PdBu) is present in the medium at a concentration of 0.5 μM.

[0106] In some embodiments, the Wnt signaling pathway inhibitor (e.g., a tankyrase inhibitor such as NVP-TNKS656) is present in the medium at a concentration of 0.1 μM to 10 μM. In some embodiments, the Wnt signaling pathway inhibitor (e.g., a tankyrase inhibitor such as NVP-TNKS656) is present in the medium at a concentration of 0.1 μM to 10 μM, 0.1 μM to 9 μM, 0.1 μM to 8 μM, 0.1 μM to 7 μM, 0.1 μM to 6 μM, 0.1 μM to 5 μM, 0.1 μM to 4 μM, 0.1 μM to 3 μM, 0.1 μM to 2 μM, 0.1 μM to 1 μM, 0.1 μM to 0.5 μM, 0.1 μM to 0.2 μM, 0.2 μM to 10 μM, 0.2 μM to 1 ... μM~9μM, 0.2μM~8μM, 0.2μM~7μM, 0.2μM~6μM, 0.2μM~5μM, 0.2μM~4μM, 0.2μM~3μM, 0.2μM~2μM, 0.2μM~1μM, 0.2μM~0.5μ M, 0.5μM~10μM, 0.5μM~9μM, 0.5μM~8μM, 0.5μM~7μM, 0.5μM~6μM, 0.5μM~5μM, 0.5μM~4μM, 0.5μM~3μM, 0.5μM~2μM, 0.5μM ~1 μM, 1 μM ~ 10 μM, 1 μM ~ 9 μM, 1 μM ~ 8 μM, 1 μM ~ 7 μM, 1 μM ~ 6 μM, 1 μM ~ 5 μM, 1 μM ~ 4 μM, 1 μM ~ 3 μM, 1 μM ~ 2 μM, 2 μM ~ 10 μM, 2 μM ~ 9 μM, 2 μM M~8μM, 2μM~7μM, 2μM~6μM, 2μM~5μM, 2μM~4μM, 2μM~3μM, 3μM~10μM, 3μM~9μM, 3μM~8μM, 3μM~7μM, 3μM~6μM, 3μM~5μM, 3μM Present in the medium at a concentration of 4 μM, 4 μM to 10 μM, 4 μM to 9 μM, 4 μM to 8 μM, 4 μM to 7 μM, 4 μM to 6 μM, 4 μM to 5 μM, 5 μM to 10 μM, 5 μM to 9 μM, 5 μM to 8 μM, 5 μM to 7 μM, 5 μM to 6 μM, 6 μM to 10 μM, 6 μM to 9 μM, 6 μM to 8 μM, 6 μM to 7 μM, 7 μM to 10 μM, 7 μM to 9 μM, 7 μM to 8 μM, 8 μM to 10 μM, 8 μM to 9 μM, or 9 μM to 10 μM. In some embodiments, the Wnt signaling pathway inhibitor (e.g., a tankyrase inhibitor such as NVP-TNKS656) is present in the medium at a concentration of 0.5 μM to 5 μM (e.g., 0.5 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, or 5 μM).In some embodiments, the Wnt signaling pathway inhibitor (e.g., a tankyrase inhibitor such as NVP-TNKS656) is present in the medium at a concentration of 1.5 μM to 2.5 μM or 1.8 μM to 2.2 μM. In some embodiments, the Wnt signaling pathway inhibitor (e.g., a tankyrase inhibitor such as NVP-TNKS656) is present in the medium at a concentration of 2 μM.

[0107] In some embodiments, the sonic hedgehog (SHH) signaling pathway inhibitor (e.g., SANT-1) is present in the medium at a concentration of 0.1 μM to 10 μM. In some embodiments, the sonic hedgehog (SHH) signaling pathway inhibitor (e.g., SANT-1) is present in the medium at a concentration of 0.1 μM to 10 μM, 0.1 μM to 9 μM, 0.1 μM to 8 μM, 0.1 μM to 7 μM, 0.1 μM to 6 μM, 0.1 μM to 5 μM, 0.1 μM to 4 μM, 0.1 μM to 3 μM, 0.1 μM to 2 μM, 0.1 μM to 1 μM, or 0.1 μM to 0.5 μM. , 0.5μM~10μM, 0.5μM~9μM, 0.5μM~8μM, 0.5μM~7μM, 0.5μM~6μM, 0.5μM~5μM, 0.5μM~4μM, 0.5μM~3μM , 0.5μM~2μM, 0.5μM~1μM, 1μM~10μM, 1μM~9μM, 1μM~8μM, 1μM~7μM, 1μM~6μM, 1μM~5μM, 1μM~4μM, 1μM ~3μM, 1μM~2μM, 2μM~10μM, 2μM~9μM, 2μM~8μM, 2μM~7μM, 2μM~6μM, 2μM~5μM, 2μM~4μM, 2μM~3μM, 3μM ~10μM, 3μM~9μM, 3μM~8μM, 3μM~7μM, 3μM~6μM, 3μM~5μM, 3μM~4μM, 4μM~10μM, 4μM~9μM, 4μM~8μM, 4μM Present in the medium at a concentration of 4 μM to 7 μM, 4 μM to 6 μM, 4 μM to 5 μM, 5 μM to 10 μM, 5 μM to 9 μM, 5 μM to 8 μM, 5 μM to 7 μM, 5 μM to 6 μM, 6 μM to 10 μM, 6 μM to 9 μM, 6 μM to 8 μM, 6 μM to 7 μM, 7 μM to 10 μM, 7 μM to 9 μM, 7 μM to 8 μM, 8 μM to 10 μM, 8 μM to 9 μM, or 9 μM to 10 μM. In some embodiments, the sonic hedgehog (SHH) signaling pathway inhibitor (e.g., SANT-1) is present in the medium at a concentration of 0.1 μM to 0.5 μM (e.g., 0.1 μM, 0.15 μM, 0.2 μM, 0.25 μM, 0.3 μM, 0.35 μM, 0.4 μM, 0.45 μM, or 0.5 μM). In some embodiments, the sonic hedgehog (SHH) signaling pathway inhibitor (e.g., SANT-1) is present in the medium at a concentration of 0.25 μM.

[0108] In some embodiments, the epidermal growth factor (e.g., betacellulin) is present in the medium at a concentration of 10 ng / ml to 50 ng / ml. In some embodiments, the epidermal growth factor (e.g., betacellulin) is present in the medium at a concentration of 10 ng / ml to 50 ng / ml, 10 ng / ml to 40 ng / ml, 10 ng / ml to 30 ng / ml, 10 ng / ml to 20 ng / ml, 20 ng / ml to 50 ng / ml, 20 ng / ml to 40 ng / ml, 20 ng / ml to 30 ng / ml, 30 ng / ml to 50 ng / ml, 30 ng / ml to 40 ng / ml, or 40 ng / ml to 50 ng / ml. In some embodiments, the epidermal growth factor (e.g., betacellulin) is present in the medium at a concentration of 10 ng / ml to 30 ng / ml (e.g., 10 ng / ml, 20 ng / ml, 20 ng / ml). In some embodiments, epidermal growth factor (eg, betacellulin) is present in the medium at a concentration of 20 ng / ml.

[0109] In some embodiments, the Notch signaling pathway inhibitor (e.g., a γ-secretase inhibitor such as XXI) is present in the medium at a concentration of 0.1 μM to 10 μM. In some embodiments, the Notch signaling pathway inhibitor (e.g., a γ-secretase inhibitor such as XXI) is present in the medium at a concentration of 0.1 μM to 10 μM, 0.1 μM to 9 μM, 0.1 μM to 8 μM, 0.1 μM to 7 μM, 0.1 μM to 6 μM, 0.1 μM to 5 μM, 0.1 μM to 4 μM, 0.1 μM to 3 μM, 0.1 μM to 2 μM, 0.1 μM to 1 μM, or 0.1 μM to 0.5 μM. , 0.5μM~10μM, 0.5μM~9μM, 0.5μM~8μM, 0.5μM~7μM, 0.5μM~6μM, 0.5μM~5μM, 0.5μM~4μM, 0.5μM~3μM , 0.5μM~2μM, 0.5μM~1μM, 1μM~10μM, 1μM~9μM, 1μM~8μM, 1μM~7μM, 1μM~6μM, 1μM~5μM, 1μM~4μM, 1μM ~3μM, 1μM~2μM, 2μM~10μM, 2μM~9μM, 2μM~8μM, 2μM~7μM, 2μM~6μM, 2μM~5μM, 2μM~4μM, 2μM~3μM, 3μM ~10μM, 3μM~9μM, 3μM~8μM, 3μM~7μM, 3μM~6μM, 3μM~5μM, 3μM~4μM, 4μM~10μM, 4μM~9μM, 4μM~8μM, 4μM Present in the medium at a concentration of 4 μM to 7 μM, 4 μM to 6 μM, 4 μM to 5 μM, 5 μM to 10 μM, 5 μM to 9 μM, 5 μM to 8 μM, 5 μM to 7 μM, 5 μM to 6 μM, 6 μM to 10 μM, 6 μM to 9 μM, 6 μM to 8 μM, 6 μM to 7 μM, 7 μM to 10 μM, 7 μM to 9 μM, 7 μM to 8 μM, 8 μM to 10 μM, 8 μM to 9 μM, or 9 μM to 10 μM. In some embodiments, the Notch signaling pathway inhibitor (e.g., a γ-secretase inhibitor such as XXI) is present in the medium at a concentration of 0.5 μM to 5 μM (e.g., 0.5 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, or 5 μM). In some embodiments, the Notch signaling pathway inhibitor (e.g., a γ-secretase inhibitor such as XXI) is present in the medium at a concentration of 2 μM.

[0110] In some embodiments, the TGFβ-R1 kinase inhibitor (e.g., ALK5i) is present in the medium at a concentration of 1 μM to 50 μM. In some embodiments, the TGFβ-R1 kinase inhibitor (e.g., ALK5i) is present in the medium at a concentration of 1 μM to 50 μM, 1 μM to 40 μM, 1 μM to 30 μM, 1 μM to 20 μM, 1 μM to 10 μM, 10 μM to 50 μM, 10 μM to 40 μM, 10 μM to 30 μM, 10 μM to 20 μM, 20 μM to 50 μM, 20 μM to 40 μM, 20 μM to 30 μM, 30 μM to 50 μM, 30 μM to 40 μM, or 40 μM to 50 μM. In some embodiments, the TGFβ-R1 kinase inhibitor (e.g., ALK5i) is present in the medium at a concentration of 5 μM to 20 μM (e.g., 5 μM, 10 μM, 15 μM, or 20 μM). In some embodiments, the TGFβ-R1 kinase inhibitor (e.g., ALK5i) is present in the medium at a concentration of 10 μM.

[0111] In some embodiments, the thyroid hormone (e.g., GC-1) is present in the medium at a concentration of 0.1 μM to 10 μM. In some embodiments, the thyroid hormone (e.g., GC-1) is present in the medium at a concentration of 0.1 μM to 10 μM, 0.1 μM to 9 μM, 0.1 μM to 8 μM, 0.1 μM to 7 μM, 0.1 μM to 6 μM, 0.1 μM to 5 μM, 0.1 μM to 4 μM, 0.1 μM to 3 μM, 0.1 μM to 2 μM, 0.1 μM to 1 μM, 0.1 μM to 0.5 μM, 0.5 μM to 10 μM, 0.5 μM to 1 ... μM~9μM, 0.5μM~8μM, 0.5μM~7μM, 0.5μM~6μM, 0.5μM~5μM, 0.5μM~4μM, 0.5μM~3μM, 0.5μM~2μM, 0 .5μM~1μM, 1μM~10μM, 1μM~9μM, 1μM~8μM, 1μM~7μM, 1μM~6μM, 1μM~5μM, 1μM~4μM, 1μM~3μM, 1μM~2 μM, 2μM~10μM, 2μM~9μM, 2μM~8μM, 2μM~7μM, 2μM~6μM, 2μM~5μM, 2μM~4μM, 2μM~3μM, 3μM~10μM, 3 μM~9μM, 3μM~8μM, 3μM~7μM, 3μM~6μM, 3μM~5μM, 3μM~4μM, 4μM~10μM, 4μM~9μM, 4μM~8μM, 4μM~7μM In some embodiments, thyroid hormone (e.g., GC-1) is present in the medium at a concentration of 0.5 μM to 5 μM (e.g., 0.5 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, or 5 μM). In some embodiments, thyroid hormone (eg, GC-1) is present in the medium at a concentration of 1 μM.

[0112] In some embodiments, the osteogenic (BMP) signaling pathway inhibitor (e.g., LDN-193189) is present in the medium at a concentration of 0.05 μM to 0.5 μM. In some embodiments, the osteogenic (BMP) signaling pathway inhibitor (e.g., LDN-193189) is present in the medium at a concentration of 0.05 μM to 0.5 μM, 0.1 μM to 0.5 μM, 0.15 μM to 0.5 μM, 0.2 μM to 0.5 μM, 0.25 μM to 0.5 μM, 0.3 μM to 0.5 μM, 0.35 μM to 0.5 μM, 0.4 μM to 0.5 μM, 0.45 μM to 0.5 μM, 0.05 μM to 0.4 μM, 0.1 μM to 0.4 μM, 0.1 μM to 0.1 μM. Present in the medium at a concentration of 5 µM to 0.4 µM, 0.2 µM to 0.4 µM, 0.25 µM to 0.4 µM, 0.3 µM to 0.4 µM, 0.35 µM to 0.4 µM, 0.05 µM to 0.3 µM, 0.1 µM to 0.3 µM, 0.15 µM to 0.3 µM, 0.2 µM to 0.3 µM, 0.25 µM to 0.3 µM, 0.05 µM to 0.2 µM, 0.1 µM to 0.2 µM, 0.15 µM to 0.2 µM, or 0.05 µM to 0.1 µM. In some embodiments, the bone morphogenetic (BMP) signaling pathway inhibitor (e.g., LDN-193189) is present in the medium at a concentration of 0.05 μM to 0.2 μM (e.g., 0.05 μM, 0.1 μM, 0.15 μM, or 0.2 μM). In some embodiments, the bone morphogenetic (BMP) signaling pathway inhibitor (e.g., LDN-193189) is present in the medium at a concentration of 0.1 μM.

[0113] In some embodiments, the Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor (e.g., thiazovivin) is present in the medium at a concentration of 1 μM to 10 μM. In some embodiments, the Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor (e.g., thiazovivin) is present in the medium at a concentration of 1 μM to 10 μM, 1 μM to 9 μM, 1 μM to 8 μM, 1 μM to 7 μM, 1 μM to 6 μM, 1 μM to 5 μM, 1 μM to 4 μM, 1 μM to 3 μM, 1 μM to 2 μM, 2 μM to 10 μM, 2 μM to 9 μM, 2 μM to 8 μM, 2 μM to 7 μM, 2 μM to 6 μM, 2 μM to 5 μM, 2 μM to 4 μM, 2 μM to 3 μM, 3 μM to 10 μM, 3 μM to 9 μM, 3 μM to 8 μM, 3 μM to 8 μM, 3 μM to 9 μM, 3 μM to 8 μM, 3 μM to 5 μM, 3 μM to 4 μM, 3 μM to 3 μM, 3 μM to 10 μM, 3 μM to 9 μM, 3 μM to 8 μM, 3 μM to 5 ... μM~7μM, 3μM~6μM, 3μM~5μM, 3μM~4μM, 4μM~10μM, 4μM~9μM, 4μM~8μM, 4μM~7μM, 4μM~6μM, 4μM~5μM, 5μM~10μM, 5μM~9μM, 5μM~8μM, 5μM~7 present in the culture medium at a concentration of μM, 5 μM to 6 μM, 6 μM to 10 μM, 6 μM to 9 μM, 6 μM to 8 μM, 6 μM to 7 μM, 7 μM to 10 μM, 7 μM to 9 μM, 7 μM to 8 μM, 8 μM to 10 μM, 8 μM to 9 μM, or 9 μM to 10 μM. In some embodiments, the Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor (e.g., thiazovivin) is present in the medium at a concentration of 1 μM to 5 μM (e.g., 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, or 5 μM). In some embodiments, the Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor (e.g., thiazovivin) is present in the medium at a concentration of 2.5 μM.

[0114] In some embodiments, the protein kinase inhibitor (eg, staurosporine) is present in the medium at a concentration of 0.5 nM to 10 nM. In some embodiments, the protein kinase inhibitor (e.g., staurosporine) is administered at a concentration of 0.5 nM to 10 nM, 0.5 nM to 9 nM, 0.5 nM to 8 nM, 0.5 nM to 7 nM, 0.5 nM to 6 nM, 0.5 nM to 5 nM, 0.5 nM to 4 nM, 0.5 nM to 3 nM, 0.5 nM to 2 nM, 0.5 nM to 1 nM, 1 nM to 10 nM, 1 nM to 9 nM, 1 nM to 8 nM, 1 nM to 7 nM, 1 nM to 6 nM, 1 nM to 5 nM, 1 nM to 4 nM, 1 nM to 3 nM, 1 nM to 2 nM, 2 nM to 10 nM, 2 nM to 9 nM, 2 nM to 8 nM, 2 nM to 7 nM, 2 nM to 6 nM, 2 nM to 5 nM, 2nM~4nM, 2nM~3nM, 3nM~10nM, 3nM~9nM, 3nM~8nM, 3nM~7nM, 3nM~6nM, 3nM~5nM, 3nM~4nM, 4nM~10nM, 4nM~9nM, 4nM~8nM, 4nM~7nM, 4nM~6nM, 4nM~5nM, 5nM~10nM, In some embodiments, the protein kinase inhibitor (e.g., staurosporine) is present in the medium at a concentration of 1 nM to 5 nM (e.g., 1 nM, 2 nM, 3 nM, 4 nM, or 5 nM). In some embodiments, the protein kinase inhibitor (e.g., staurosporine) is present in the medium at a concentration of 3 nM.

[0115] In some embodiments, the histone methyltransferase EZH2 inhibitor (e.g., DZNEP) is present in the medium at a concentration of 0.05 μM to 0.5 μM. In some embodiments, the histone methyltransferase EZH2 inhibitor (e.g., DZNEP) is present in the medium at a concentration of 0.05 μM to 0.5 μM, 0.1 μM to 0.5 μM, 0.15 μM to 0.5 μM, 0.2 μM to 0.5 μM, 0.25 μM to 0.5 μM, 0.3 μM to 0.5 μM, 0.35 μM to 0.5 μM, 0.4 μM to 0.5 μM, 0.45 μM to 0.5 μM, 0.05 μM to 0.4 μM, 0.1 μM to 0.4 μM, 0.15 μM to 0.4 μM, 0.15 μM to 0.5 μM, 0.2 μM to 0.5 μM, 0.25 μM to 0.5 μM, 0.3 μM to 0.5 μM, 0.35 μM to 0.5 μM, 0.4 μM to 0.5 μM, 0.45 μM to 0.5 μM, 0.05 μM to 0.4 μM, 0.1 μM to 0.4 μM, 0.15 μM to 0.5 μM, 0.4 ... Present in the medium at concentrations of 0.4 μM, 0.2 μM to 0.4 μM, 0.25 μM to 0.4 μM, 0.3 μM to 0.4 μM, 0.35 μM to 0.4 μM, 0.05 μM to 0.3 μM, 0.1 μM to 0.3 μM, 0.15 μM to 0.3 μM, 0.2 μM to 0.3 μM, 0.25 μM to 0.3 μM, 0.05 μM to 0.2 μM, 0.1 μM to 0.2 μM, 0.15 μM to 0.2 μM, or 0.05 μM to 0.1 μM. In some embodiments, the histone methyltransferase EZH2 inhibitor (e.g., DZNEP) is present in the medium at a concentration of 0.05 μM to 0.2 μM (e.g., 0.05 μM, 0.1 μM, 0.15 μM, or 0.2 μM). In some embodiments, the histone methyltransferase EZH2 inhibitor (e.g., DZNEP) is present in the medium at a concentration of 0.1 μM.

[0116] In some embodiments, retinoic acid is present in the medium at a concentration of 0.02 μM to 0.5 μM. In some embodiments, retinoic acid is present in the medium at a concentration of 0.02 μM to 0.5 μM, 0.05 μM to 0.5 μM, 0.1 μM to 0.5 μM, 0.15 μM to 0.5 μM, 0.2 μM to 0.5 μM, 0.25 μM to 0.5 μM, 0.3 μM to 0.5 μM, 0.35 μM to 0.5 μM, 0.4 μM to 0.5 μM, 0.45 μM to 0.5 μM, 0.02 μM to 0.4 μM, 0.05 μM to 0.4 μM, 0.1 μM to 0.4 μM, 0.15 μM to 0.4 μM, 0.2 μM to 0.4 μM, 0.25 μM Present in the medium at a concentration of 0.4 µM, 0.3 µM to 0.4 µM, 0.35 µM to 0.4 µM, 0.02 µM to 0.3 µM, 0.05 µM to 0.3 µM, 0.1 µM to 0.3 µM, 0.15 µM to 0.3 µM, 0.2 µM to 0.3 µM, 0.25 µM to 0.3 µM, 0.02 µM to 0.2 µM, 0.05 µM to 0.2 µM, 0.1 µM to 0.2 µM, 0.15 µM to 0.2 µM, 0.02 µM to 0.1 µM, 0.05 µM to 0.1 µM, or 0.02 µM to 0.05 µM. In some embodiments, retinoic acid is present in the medium at a concentration of 0.02 μM to 0.2 μM (e.g., 0.02 μM, 0.05 μM, 0.1 μM, 0.15 μM, or 0.2 μM). In some embodiments, retinoic acid is present in the medium at a concentration of 0.05 μM.

[0117] In some embodiments, the acetyl-CoA-related metabolite (e.g., acetate) is present in the medium at a concentration of 0.1 mM to 10 mM. In some embodiments, the acetyl-CoA-related metabolite (e.g., acetate) is present in the medium at a concentration of 0.1 mM to 10 mM, 0.1 mM to 9 mM, 0.1 mM to 8 mM, 0.1 mM to 7 mM, 0.1 mM to 6 mM, 0.1 mM to 5 mM, 0.1 mM to 4 mM, 0.1 mM to 3 mM, 0.1 mM to 2 mM, 0.1 mM to 1 mM, 0.1 mM to 0.5 mM, 0.5 mM to 10 mM, 0.5mM~9mM, 0.5mM~8mM, 0.5mM~7mM, 0.5mM~6mM, 0.5mM~5mM, 0.5mM~4mM, 0.5mM~3mM, 0.5mM~2mM , 0.5mM~1mM, 1mM~10mM, 1mM~9mM, 1mM~8mM, 1mM~7mM, 1mM~6mM, 1mM~5mM, 1mM~4mM, 1mM~3mM, 1mM ~2mM, 2mM~10mM, 2mM~9mM, 2mM~8mM, 2mM~7mM, 2mM~6mM, 2mM~5mM, 2mM~4mM, 2mM~3mM, 3mM~10mM, 3mM~9mM, 3mM~8mM, 3mM~7mM, 3mM~6mM, 3mM~5mM, 3mM~4mM, 4mM~10mM, 4mM~9mM, 4mM~8mM, 4mM~7m In some embodiments, the acetyl-CoA-related metabolite (e.g., acetate) is present in the medium at a concentration of 0.5 mM to 5 mM (e.g., 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, or 5 mM). In some embodiments, the acetyl-CoA-related metabolite (eg, acetate) is present in the medium at a concentration of 1 mM.

[0118] In some embodiments, the HDAC inhibitor (e.g., β-hydroxybutyrate) is present in the medium at a concentration of 0.05 μM to 0.5 μM. In some embodiments, the HDAC inhibitor (e.g., β-hydroxybutyrate) is present in the medium at a concentration of 0.05 μM to 0.5 μM, 0.1 μM to 0.5 μM, 0.15 μM to 0.5 μM, 0.2 μM to 0.5 μM, 0.25 μM to 0.5 μM, 0.3 μM to 0.5 μM, 0.35 μM to 0.5 μM, 0.4 μM to 0.5 μM, 0.45 μM to 0.5 μM, 0.05 μM to 0.4 μM, 0.1 μM to 0.4 μM, 0.15 μM to 0.5 μM, In some embodiments, the HDAC inhibitor (e.g., β-hydroxybutyrate) is present in the medium at a concentration of 0.1 μM to 0.5 μM (e.g., 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.2 μM to 0.4 μM, 0.25 μM to 0.4 μM, 0.3 μM to 0.4 μM, 0.35 μM to 0.4 μM, 0.05 μM to 0.3 μM, 0.1 μM to 0.3 μM, 0.15 μM to 0.3 μM, 0.2 μM to 0.3 μM, 0.25 μM to 0.3 μM, 0.05 μM to 0.2 μM, 0.1 μM to 0.2 μM, 0.15 μM to 0.2 μM, or 0.05 μM to 0.1 μM). In some embodiments, the HDAC inhibitor is present in the medium at a concentration of 0.2 μM.

[0119] In some embodiments, the redox homeostasis regulator (e.g., taurine) is present in the medium at a concentration of 20 μM to 100 μM. In some embodiments, the redox homeostasis regulator (e.g., taurine) is present in the medium at a concentration of 20 μM to 100 μM, 20 μM to 90 μM, 20 μM to 80 μM, 20 μM to 70 μM, 20 μM to 60 μM, 20 μM to 50 μM, 20 μM to 40 μM, 20 μM to 30 μM, 30 μM to 100 μM, 30 μM to 90 μM, 30 μM to 80 μM, 30 μM to 70 μM, 30 μM to 60 μM, 30 μM to 50 μM, 30 μM to 40 μM, 40 μM to 100 μM, 40 μM to 90 μM, Present in the medium at a concentration of 40 μM to 80 μM, 40 μM to 70 μM, 40 μM to 60 μM, 40 μM to 50 μM, 50 μM to 100 μM, 50 μM to 90 μM, 50 μM to 80 μM, 50 μM to 70 μM, 50 μM to 60 μM, 60 μM to 100 μM, 60 μM to 90 μM, 60 μM to 80 μM, 60 μM to 70 μM, 70 μM to 100 μM, 70 μM to 90 μM, 70 μM to 80 μM, 80 μM to 100 μM, 80 μM to 90 μM, or 90 μM to 100 μM. In some embodiments, the redox homeostasis regulator (e.g., taurine) is present in the medium at a concentration of 50 μM to 100 μM (e.g., 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, or 100 μM). In some embodiments, the redox homeostasis regulator (e.g., taurine) is present in the medium at a concentration of 90 μM.

[0120] In some embodiments, the one-carbon metabolic pathway intermediate (e.g., formate) is present in the medium at a concentration of 20 μM to 100 μM. In some embodiments, the one-carbon metabolic pathway intermediate (e.g., formate) is present in the medium at a concentration of 20 μM to 100 μM, 20 μM to 90 μM, 20 μM to 80 μM, 20 μM to 70 μM, 20 μM to 60 μM, 20 μM to 50 μM, 20 μM to 40 μM, 20 μM to 30 μM, 30 μM to 100 μM, 30 μM to 90 μM, 30 μM to 80 μM, 30 μM to 70 μM, 30 μM to 60 μM, 30 μM to 50 μM, 30 μM to 40 μM, 40 μM to 100 μM, 40 μM to 90 μM, 40 μM to 40 μM. Present in the medium at a concentration of 40 μM to 80 μM, 40 μM to 70 μM, 40 μM to 60 μM, 40 μM to 50 μM, 50 μM to 100 μM, 50 μM to 90 μM, 50 μM to 80 μM, 50 μM to 70 μM, 50 μM to 60 μM, 60 μM to 100 μM, 60 μM to 90 μM, 60 μM to 80 μM, 60 μM to 70 μM, 70 μM to 100 μM, 70 μM to 90 μM, 70 μM to 80 μM, 80 μM to 100 μM, 80 μM to 90 μM, or 90 μM to 100 μM. In some embodiments, the one-carbon metabolic pathway intermediate (e.g., formate) is present in the medium at a concentration of 20 μM to 80 μM (e.g., 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, or 80 μM). In some embodiments, the one-carbon metabolic pathway intermediate (e.g., formate) is present in the medium at a concentration of 50 μM.

[0121] In some embodiments, the vitamin (e.g., biotin) is present in the medium at a concentration of 0.1 μM to 5 μM. In some embodiments, the vitamin (e.g., biotin) is present in the medium at a concentration of 0.1 μM to 5 μM, 0.1 μM to 4 μM, 0.1 μM to 3 μM, 0.1 μM to 2 μM, 0.1 μM to 1 μM, 0.1 μM to 0.5 μM, 0.5 μM to 5 μM, 0.5 μM to 4 μM, 0.5 μM to 3 μM, 0.5 μM to 2 μM, 0.5 μM to 1 μM, 1 μM to 5 μM, 1 μM to 4 μM, 1 μM to 3 μM, 1 μM to 2 μM, 2 μM to 5 μM, 2 μM to 4 μM, 2 μM to 3 μM, 3 μM to 5 μM, 3 μM to 4 μM, or 4 μM to 5 μM. In some embodiments, the vitamin (e.g., biotin) is present in the medium at a concentration of 0.5 μM to 2 μM (e.g., 0.5 μM, 0.8 μM, 1 μM, 1.5 μM, 2 μM). In some embodiments, the vitamin (e.g., biotin) is present in the medium at a concentration of 0.8 μM.

[0122] In some embodiments, glutamine (e.g., L-glutamine) is present in the medium at a concentration of 1 mM to 10 mM. In some embodiments, glutamine (e.g., L-glutamine) is present at a concentration of 1 mM to 10 mM, 1 mM to 9 mM, 1 mM to 8 mM, 1 mM to 7 mM, 1 mM to 6 mM, 1 mM to 5 mM, 1 mM to 4 mM, 1 mM to 3 mM, 1 mM to 2 mM, 2 mM to 10 mM, 2 mM to 9 mM, 2 mM to 8 mM, 2 mM to 7 mM, 2 mM to 6 mM, 2 mM to 5 mM, 2 mM to 4 mM, 2 mM to 3 mM, 3 mM to 10 mM, 3 mM to 9 mM, 3 mM to 8 mM, 3 mM to 7 mM, 3 mM to 6 mM. It is present in the medium at a concentration of 0.05 mM, 3 mM to 5 mM, 3 mM to 4 mM, 4 mM to 10 mM, 4 mM to 9 mM, 4 mM to 8 mM, 4 mM to 7 mM, 4 mM to 6 mM, 4 mM to 5 mM, 5 mM to 10 mM, 5 mM to 9 mM, 5 mM to 8 mM, 5 mM to 7 mM, 5 mM to 6 mM, 6 mM to 10 mM, 6 mM to 9 mM, 6 mM to 8 mM, 6 mM to 7 mM, 7 mM to 10 mM, 7 mM to 9 mM, 7 mM to 8 mM, 8 mM to 10 mM, 8 mM to 9 mM, or 9 mM to 10 mM. In some embodiments, glutamine (e.g., L-glutamine) is present in the medium at a concentration of 2 mM to 6 mM (e.g., 2 mM, 3 mM, 4 mM, 5 mM, or 6 mM). In some embodiments, glutamine (e.g., L-glutamine) is present in the medium at a concentration of 4 mM.

[0123] In some embodiments, the medium contains an inhibitor of the Wnt signaling pathway (e.g., a tankyrase inhibitor such as NVP-TNKS656) at a concentration of 0.5 μM to 5 μM, an inhibitor of the Sonic Hedgehog (SHH) signaling pathway (e.g., SANT-1) at a concentration of 0.1 μM to 0.5 μM, an epidermal growth factor (EGF) (e.g., EGFR-1) at a concentration of 10 ng / ml to 30 ng / ml, or an inhibitor of the Wnt signaling pathway (e.g., a tankyrase inhibitor such as NVP-TNKS656) at a concentration of 0.5 μM to 5 μM. betacellulin), a Notch signaling pathway inhibitor (e.g., a γ-secretase inhibitor such as XXI) at a concentration of 0.1 μM to 0.5 μM, a TGFβ-R1 kinase inhibitor (e.g., ALK5i) at a concentration of 5 μM to 20 μM, a thyroid hormone (e.g., GC-1) at a concentration of 0.5 μM to 5 μM, a bone morphogenetic (BMP) signaling pathway inhibitor (e.g., LDN-193189) at a concentration of 0.05 μM to 0.2 μM, and a 1 μM Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitors (e.g., thiazovivin) at concentrations of ~5 μM, protein kinase inhibitors (e.g., staurosporine) at concentrations of 1 nM to 5 nM, histone methyltransferase EZH2 inhibitors (e.g., DZNEP) at concentrations of 0.05 μM to 0.2 μM, retinoic acid at concentrations of 0.02 μM to 0.2 μM, and acetyl-CoA-related metabolites at concentrations of 0.5 mM to 5 mM. The solution contains a metabolic product (e.g., acetate) at a concentration of 0.1 μM to 0.5 μM, an HDAC inhibitor (e.g., β-hydroxybutyrate) at a concentration of 0.1 μM to 0.5 μM, a redox homeostasis regulator (e.g., taurine) at a concentration of 50 μM to 100 μM, a one-carbon metabolic pathway intermediate (e.g., formate) at a concentration of 20 μM to 80 μM, a vitamin (e.g., biotin) at a concentration of 0.5 μM to 2 μM, and glutamine (e.g., L-glutamine) at a concentration of 2 mM to 6 mM.

[0124] In some embodiments, the medium contains a PKC activator (e.g., PdBu) at a concentration of 0.5 μM, a Wnt signaling pathway inhibitor (e.g., a tankyrase inhibitor such as NVP-TNKS656) at a concentration of 2 μM, a Sonic Hedgehog (SHH) signaling pathway inhibitor (e.g., SANT-1) at a concentration of 0.25 μM, an epidermal growth factor (e.g., betacellulin) at a concentration of 20 ng / ml, a Notch signaling pathway inhibitor (e.g., a γ-secretase inhibitor such as XXI) at a concentration of 2 μM, a TGFβ-R1 kinase inhibitor (e.g., ALK5i) at a concentration of 10 μM, a thyroid hormone (e.g., GC-1) at a concentration of 1 μM, a bone morphogenetic (BMP) signaling pathway inhibitor (e.g., LDN-193189) at a concentration of 0.1 μM, and a 2.5 μM ATP-dependent ... The medium contains a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor (e.g., thiazovivin) at a concentration of 1 μM, a protein kinase inhibitor (e.g., staurosporine) at a concentration of 3 μM, a histone methyltransferase EZH2 inhibitor (e.g., DZNEP) at a concentration of 0.1 μM, retinoic acid at a concentration of 0.05 μM, an acetyl-CoA-related metabolite (e.g., acetate) at a concentration of 4 mM, an HDAC inhibitor (e.g., β-hydroxybutyrate) at a concentration of 50 μM, a redox homeostasis regulator (e.g., taurine) at a concentration of 90 μM, a one-carbon metabolic pathway intermediate (e.g., formate) at a concentration of 50 μM, a vitamin (e.g., biotin) at a concentration of 0.8 μM, and glutamine (e.g., L-glutamine) at a concentration of 4 mM.

[0125] In some embodiments, the in vitro compositions described herein further comprise a water-soluble synthetic polymer. In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol (PVA), poloxamer, polyvinylpyrrolidone, polyethylene glycol (PEG), a PEG copolymer, poly(N-isopropylacrylamide), or polyacrylamide, and optionally, the water-soluble synthetic polymer is polyvinyl alcohol. In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol (PVA). In some embodiments, the water-soluble synthetic polymer has a concentration of 0.005% to 0.5% (w / v), 0.01% to 0.2% (w / v), 0.02% to 0.1% (w / v), or 0.03% to 0.08% (w / v) of the medium. In some embodiments, the water-soluble synthetic polymer has a concentration of 0.005% (w / v), 0.01% (w / v), 0.05% (w / v), 0.1% (w / v), 0.15% (w / v), 0.2% (w / v), 0.25% (w / v), 0.3% (w / v), 0.35% (w / v), 0.4% (w / v), 0.45% (w / v), or 0.5% (w / v) of the medium. Polyvinyl alcohol as described herein can refer to a water-soluble synthetic polymer having the idealized formula [CHCH(OH)] that can be partially or fully hydrolyzed. In some examples, polyvinyl alcohol is produced by either partial or complete hydrolysis of polyvinyl acetate to remove the acetate groups. In some examples, the polyvinyl alcohol is at most 85% hydrolyzed, for example, 80% hydrolyzed. The percentage of hydrolysis measures the approximate percentage (e.g., average percentage) of acetate residues that are hydrolyzed in the polyvinyl acetate precursor polymer. In some examples, the polyvinyl alcohol is at least 85% hydrolyzed, e.g., 87-89% hydrolyzed, 87-90% hydrolyzed, or 99% hydrolyzed. In some embodiments, the polyvinyl alcohol is 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% hydrolyzed.In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol (PVA), and the PVA is at most 90% hydrolyzed (e.g., 87%-89%). In some embodiments, the PVA is 80% hydrolyzed (e.g., in stages 1-4). In some embodiments, the PVA is 89% hydrolyzed (e.g., in stage 5).

[0126] In some embodiments, the in vitro compositions described herein comprise a pancreatic progenitor cell population comprising PDX1-positive, NKX6.1-positive, insulin-negative cells. In some embodiments, the in vitro compositions described herein comprise a pancreatic progenitor cell population comprising PDX1-positive, NKX6.1-positive, insulin-positive cells. In some embodiments, the in vitro compositions described herein comprise a pancreatic progenitor cell population comprising PDX1-positive, NKX6.1-positive, insulin-negative cells and PDX1-positive, NKX6.1-positive, insulin-positive cells.

[0127] In some embodiments, the pancreatic progenitor cell population comprises more PDX1-positive, NKX6.1-positive, insulin-negative cells than PDX1-positive, NKX6.1-positive, insulin-positive cells. In some embodiments, at least 50% (e.g., at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%) of the pancreatic progenitor cell population are PDX1-positive, NKX6.1-positive, insulin-negative. In some embodiments, 50% or less (e.g., 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less) of the pancreatic progenitor cell population are PDX1-positive, NKX6.1-positive, insulin-positive pancreatic progenitor cells. In some embodiments, 50% to 90% (e.g., 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 90%, 60% to 80%, 60% to 70%, 30% to 80%, 70% to 90%, 70% to 80%, or 80% to 90%) of the pancreatic progenitor cell population are PDX1 positive, NKX6.1 positive, and insulin negative. In some embodiments, 10% to 50% (e.g., 10% to 50%, 20% to 50%, 30% to 50%, 40% to 50%, 10% to 40%, 20% to 40%, 30% to 40%, 10% to 30%, 20% to 30%, or 10% to 20%) of the pancreatic progenitor cell population are pancreatic progenitor cells that are PDX1-positive, NKX6.1-positive, and insulin-positive. In some embodiments, 50% to 90% (e.g., 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 90%, 60% to 80%, 60% to 70%, 30% to 80%, 70% to 90%, 70% to 80%, or 80% to 90%) of the pancreatic progenitor cell population are PDX1-positive, NKX6.1-positive, and insulin-negative, and 10% to 50% (e.g., 10% to 50%, 20% to 50%, 30% to 50%, 40% to 50%, 10% to 40%, 20% to 40%, 30% to 40%, 10% to 30%, 20% to 30%, or 10% to 20%) are PDX1-positive, NKX6.1-positive, and insulin-positive pancreatic progenitor cells. In some embodiments, at least 50% of the pancreatic progenitor cell population are PDX1-positive, NKX6.1-positive, insulin-negative, and no more than 50% of the pancreatic progenitor cell population are PDX1-positive, NKX6.1-positive, insulin-positive pancreatic progenitor cells.

[0128] In some embodiments, the pancreatic progenitor cell population comprises more PDX1-positive, NKX6.1-positive, insulin-positive cells than PDX1-positive, NKX6.1-positive, insulin-negative cells. In some embodiments, at least 50% (e.g., at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%) of the pancreatic progenitor cell population are PDX1-positive, NKX6.1-positive, insulin-positive. In some embodiments, 50% or less (e.g., 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less) of the pancreatic progenitor cell population are PDX1-positive, NKX6.1-positive, insulin-negative pancreatic progenitor cells. In some embodiments, 50% to 90% (e.g., 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 90%, 60% to 80%, 60% to 70%, 30% to 80%, 70% to 90%, 70% to 80%, or 80% to 90%) of the pancreatic progenitor cell population are PDX1-positive, NKX6.1-positive, and insulin-positive. In some embodiments, 10% to 50% (e.g., 10% to 50%, 20% to 50%, 30% to 50%, 40% to 50%, 10% to 40%, 20% to 40%, 30% to 40%, 10% to 30%, 20% to 30%, or 10% to 20%) of the pancreatic progenitor cell population are pancreatic progenitor cells that are PDX1-positive, NKX6.1-positive, and insulin-negative. In some embodiments, 50% to 90% (e.g., 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 90%, 60% to 80%, 60% to 70%, 30% to 80%, 70% to 90%, 70% to 80%, or 80% to 90%) of the pancreatic progenitor cell population are PDX1-positive, NKX6.1-positive, and insulin-positive, and 10% to 50% (e.g., 10% to 50%, 20% to 50%, 30% to 50%, 40% to 50%, 10% to 40%, 20% to 40%, 30% to 40%, 10% to 30%, 20% to 30%, or 10% to 20%) of the pancreatic progenitor cell population are PDX1-positive, NKX6.1-positive, and insulin-negative pancreatic progenitor cells. In some embodiments, at least 50% of the pancreatic progenitor cell population are PDX1-positive, NKX6.1-positive, and insulin-positive, and 50% or less of the pancreatic progenitor cell population are PDX1-positive, NKX6.1-positive, and insulin-negative pancreatic progenitor cells.

[0129] Methods for generating pancreatic islet cells In aspects, the present disclosure relates to compositions and methods for producing endocrine cells from pancreatic progenitor cells or precursors. Certain exemplary detailed protocols for producing endocrine cells to provide at least one SC-β cell are described in U.S. Patent Application Publication Nos. 20150240212, 20150218522, 20200332262, 20210198632, 20220090020, 20210238553, U.S. Patent No. 10,030,229; U.S. Patent No. 10,443,042; and International Patent Publication No. WO2022147056, each of which is incorporated herein by reference in its entirety.

[0130] In some embodiments, the methods for producing endocrine cell populations result in an increased proportion of pancreatic α cells and / or pancreatic δ cells and a decreased proportion of pancreatic EC cells when producing pancreatic β cells. In some embodiments, the methods described herein can be used to obtain an enriched population of α cells. In some embodiments, the methods described herein can be used to obtain an enriched population of β cells. In some embodiments, the methods described herein can be used to obtain an enriched population of α cells and β cells. In some embodiments, the methods described herein can be used to increase the yield of pancreatic endocrine cells.

[0131] hPSCs can be differentiated into hormone-expressing pancreatic endocrine cells by transitioning them through key stages of embryonic development: differentiation into mesendoderm and definitive endoderm, establishment of primitive gut endoderm, patterning of the posterior foregut, and specification and maturation of pancreatic endoderm and endocrine precursors. Through these stages, hPSCs can acquire a pancreatic endocrine phenotype and the ability to secrete insulin in response to glucose in vitro.

[0132] Generally, at least one pancreatic SC-α, SC-β and / or SC-δ cell or precursor thereof, e.g., pancreatic progenitor cell, produced according to the methods disclosed herein can comprise a mixture or combination of different cells, such as, for example, PDX1-positive pancreatic progenitor cells, pancreatic progenitor cells co-expressing PDX1 and NKX6-1, Ngn3-positive endocrine progenitor cells, insulin-positive endocrine cells (e.g., NKX6.1-positive cells, ISL1-positive cells, or β-like cells), and / or other pluripotent or stem cells.

[0133] Any suitable culture protocol can be followed to generate at least one pancreatic alpha, beta, and / or delta cell, or a precursor thereof, for differentiating stem cells or pluripotent cells to a desired stage of differentiation. In some embodiments, at least one pancreatic alpha, beta, and / or delta cell, or a precursor thereof, is generated by culturing at least one pluripotent cell for a period of time and under conditions suitable for the at least one pluripotent cell to differentiate into at least one pancreatic alpha, beta, and / or delta cell, or a precursor thereof.

[0134] In some embodiments, the at least one pancreatic alpha, beta, and / or delta cell or its precursor is a substantially pure population of pancreatic alpha, beta, and / or delta cells or their precursors. In some embodiments, the population of pancreatic alpha, beta, and / or delta cells or their precursors comprises a mixture of pluripotent cells or differentiated cells. In some embodiments, the population of pancreatic alpha, beta, and / or delta cells or their precursors is substantially free of or devoid of embryonic stem cells or pluripotent cells or iPS cells. In some embodiments, the methods described herein produce a cell population comprising pancreatic alpha, beta, and / or delta cells in ratios similar to those of natural pancreatic islets.

[0135] In some embodiments, the methods described herein include (i) culturing a first cell population containing pancreatic progenitor cells (e.g., PDX1-positive, NKX6.1-negative cells; or a mixture of PDX1-positive, NKX6.1-negative cells and PDX1-positive, NKX6.1-positive cells) in a first medium containing a forkhead box O1 (FoxO1) inhibitor and a Notch signaling pathway inhibitor for a period of time to obtain a second cell population (e.g., a cell population containing more PDX1-positive, NKX6.1-positive cells than the first population); and (ii) culturing the second cell population in a second medium containing a PKC activator and a Wnt signaling pathway inhibitor. In some embodiments, the method produces a cell population containing PDX1-positive, NKX6.1-positive, insulin-positive cells.

[0136] In some embodiments, the method described herein comprises culturing a first cell population in a first culture medium, wherein the first cell population comprises PDX1-positive, NKX6.1-negative pancreatic progenitor cells and PDX1-positive, NKX6.1-positive pancreatic progenitor cells; the first culture medium comprises a Forkhead Box O1 (FoxO1) inhibitor (e.g., AS1842856 or a derivative thereof). In some embodiments, the first culture medium further comprises a Notch signaling pathway inhibitor. In some embodiments, the Notch signaling pathway inhibitor is a γ-secretase inhibitor (e.g., XXI, DAPT or a derivative thereof). In some embodiments, the γ-secretase inhibitor is XXI. In some embodiments, the first culture medium does not comprise a Wnt signaling pathway inhibitor.

[0137] In some embodiments, the first cell population comprises pancreatic progenitor cells that are PDX1-positive and NKX6.1-positive. In some embodiments, the first cell population comprises more PDX1-positive and NKX6.1-negative pancreatic progenitor cells than PDX1-positive and NKX6.1-positive pancreatic progenitor cells. In some embodiments, the first cell population comprises more PDX1-positive and NKX6.1-positive pancreatic progenitor cells than PDX1-positive and NKX6.1-negative pancreatic progenitor cells.

[0138] In some embodiments, the first culture medium further comprises a PKC activator (e.g., PdBU, TPB, phorbol 12-myristate 13-acetate, bryostatin 1, or a derivative thereof). In some embodiments, the PKC activator is PdBU. In some embodiments, the first culture medium further comprises one or more (e.g., 1, 2, 3, 4, or 5) agents selected from a fibroblast growth factor (e.g., KGF), a sonic hedgehog (SHH) signaling pathway inhibitor (e.g., SANT-1), retinoic acid, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor (e.g., triazovidine), and a TGF-kinase inhibitor (e.g., activin A). In some embodiments, the first culture medium further comprises a water-soluble synthetic polymer (e.g., PVA, such as PVA 80%). In some embodiments, the first culture medium comprises a FoxO1 inhibitor (e.g., AS1842856 or a derivative thereof), a Notch signaling pathway inhibitor (e.g., a γ-secretase inhibitor such as XXI), a PKC activator (e.g., PdBU), a fibroblast growth factor (e.g., KGF), a sonic hedgehog (SHH) signaling pathway inhibitor (e.g., SANT-1), retinoic acid, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor (e.g., triazovidine), and a TGF-β ligand (e.g., activin A), as well as a water-soluble synthetic polymer (e.g., PVA such as PVA80%).

[0139] In some embodiments, the first cell population is cultured for about 12 to 72 hours (e.g., about 12 to 72 hours, 12 to 66 hours, 12 to 60 hours, 12 to 54 hours, 12 to 48 hours, 12 to 42 hours, 12 to 36 hours, 12 to 30 hours, 12 to 24 hours, 12 to 18 hours, 18 to 72 hours, 18 to 66 hours, 18 to 60 hours, 18 to 54 hours, 18 to 48 hours, 18 to 42 hours, 18 to 36 hours, 18 to 30 hours, 18 to 24 hours, 24 to 72 hours, 24 to 66 hours, 24 to 60 hours, 24 to 54 hours, 24 to 48 hours, 24 to 42 hours, 24 to 36 ... 0 hours, 30-72 hours, 30-66 hours, 30-60 hours, 30-54 hours, 30-48 hours, 30-42 hours, 30-36 hours, 36-72 hours, 36-66 hours, 36-60 hours, 36-54 hours, 36-48 hours, 36-42 hours, 42-72 hours, 42-66 hours, 42-60 hours, 42-54 hours, 42-48 hours, 48-72 hours, 48-66 hours, 48-60 hours, 48-54 hours, 54-72 hours, 54-66 hours, 54-60 hours, 60-72 hours, 60-66 hours, or 66-72 hours), and cultured in a first medium. In some embodiments, the first cell population is cultured in the first medium for about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72 hours. In some embodiments, the first cell population is cultured in the first medium for about 24 hours. In some embodiments, the first cell population is cultured in the first medium for about 48 hours.

[0140] In some embodiments, culturing the first cell population in the first culture medium for the contact period described herein (e.g., 24 hours or 48 hours) results in a second cell population. In some embodiments, the second cell population comprises PDX1-positive, NKX6.1-positive pancreatic progenitor cells and PDX1-positive, NKX6.1-negative pancreatic progenitor cells. In some embodiments, the second cell population comprises more PDX1-positive, NKX6.1-positive pancreatic progenitor cells than the first cell population. In some embodiments, the second cell population comprises more PDX1-positive, NKX6.1-negative pancreatic progenitor cells than PDX1-positive, NKX6.1-positive pancreatic progenitor cells. In some embodiments, the second cell population comprises a trace amount of PDX1-positive, NKX6.1-negative pancreatic progenitor cells (e.g., less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the second cell population).

[0141] In some embodiments, the methods described herein further include culturing the second cell population in a second culture medium comprising a Wnt signaling pathway inhibitor (e.g., a tankyrase inhibitor such as NVP-TNKS656). In some embodiments, the second culture medium comprises a PKC activator (e.g., PdBu). In some embodiments, the second culture medium further comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) agents selected from epidermal growth factor (e.g., betacellulin), thyroid hormone (e.g., GC-1), TGFβ-R1 kinase inhibitor (e.g., ALK5i), Notch signaling pathway inhibitor (e.g., a γ-secretase inhibitor such as XXI), sonic hedgehog (SHH) signaling pathway inhibitor (e.g., SANT-1), retinoic acid, Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor (e.g., triazovidine), protein kinase inhibitor (e.g., staurosporine), bone morphogenetic (BMP) signaling pathway inhibitor (e.g., LDN193189), and histone methyltransferase EZH2 inhibitor (e.g., DZNep). In some embodiments, the second medium further comprises one or more (e.g., 1, 2, 3, 4) agents selected from an acetyl-CoA-related metabolite (e.g., acetate), an HDAC inhibitor (e.g., β-hydroxybutyrate), a redox homeostasis regulator (e.g., taurine), and a one-carbon metabolic pathway intermediate (e.g., formate). In some embodiments, the second medium further comprises a vitamin (e.g., biotin). In some embodiments, the second medium further comprises glutamine. In some embodiments, the second medium further comprises a water-soluble synthetic polymer (e.g., PVA, such as 87-89% PVA). In some embodiments, the second medium does not comprise a FOXO1 inhibitor.In some embodiments, the second culture medium contains an inhibitor such as a Wnt signaling pathway inhibitor (e.g., a tankyrase inhibitor such as NVP-TNKS656), a PKC activator (e.g., PdBu), an epidermal growth factor (e.g., betacellulin), a thyroid hormone (e.g., GC-1), a TGFβ-R1 kinase inhibitor (e.g., ALK5i), a Notch signaling pathway inhibitor (e.g., a γ-secretase inhibitor such as XXI), a sonic hedgehog (SHH) signaling pathway inhibitor (e.g., SANT-1), retinoic acid, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor (e.g., trimethoprim-1), a phosphodiesterase inhibitor (e.g., phosphodiesterase inhibitor ... azovivin), protein kinase inhibitors (e.g., staurosporine), bone formation (BMP) signaling pathway inhibitors (e.g., LDN193189), histone methyltransferase EZH2 inhibitors (e.g., DZNep), acetyl-CoA-related metabolites (e.g., acetate), HDAC inhibitors (e.g., β-hydroxybutyrate), redox homeostasis regulators (e.g., taurine), one-carbon metabolic pathway intermediates (e.g., formate), vitamins (e.g., biotin), glutamine, and water-soluble synthetic polymers (e.g., PVA such as PVA87-89%), but do not contain FOXO1 inhibitors.

[0142] In some embodiments, the second cell population is cultured for about 12 to 72 hours (e.g., about 12 to 72 hours, 12 to 66 hours, 12 to 60 hours, 12 to 54 hours, 12 to 48 hours, 12 to 42 hours, 12 to 36 hours, 12 to 30 hours, 12 to 24 hours, 12 to 18 hours, 18 to 72 hours, 18 to 66 hours, 18 to 60 hours, 18 to 54 hours, 18 to 48 hours, 18 to 42 hours, 18 to 36 hours, 18 to 30 hours, 18 to 24 hours, 24 to 72 hours, 24 to 66 hours, 24 to 60 hours, 24 to 54 hours, 24 to 48 hours, 24 to 42 hours, 24 to 36 ... 0 hours, 30-72 hours, 30-66 hours, 30-60 hours, 30-54 hours, 30-48 hours, 30-42 hours, 30-36 hours, 36-72 hours, 36-66 hours, 36-60 hours, 36-54 hours, 36-48 hours, 36-42 hours, 42-72 hours, 42-66 hours, 42-60 hours, 42-54 hours, 42-48 hours, 48-72 hours, 48-66 hours, 48-60 hours, 48-54 hours, 54-72 hours, 54-66 hours, 54-60 hours, 60-72 hours, 60-66 hours, or 66-72 hours), and cultured in a second medium. In some embodiments, the second cell population is cultured in the second medium for about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72 hours. In some embodiments, the second cell population is cultured in the second medium for about 48 hours.

[0143] In some embodiments, culturing the second cell population in the second medium for the contact time described herein (e.g., 48 hours) results in a third cell population. In some embodiments, the third cell population comprises pancreatic progenitor cells that are PDX1-positive and NKX6.1-positive. In some embodiments, the third cell population comprises ISL1-positive cells. In some embodiments, the third cell population comprises ISL1-negative cells. In some embodiments, the third cell population comprises ISL1-positive cells. In some embodiments, the third cell population comprises more ISL1-positive cells than the first and second cell populations. In some embodiments, the third cell population comprises more ISL1-negative cells than ISL1-positive cells. In some embodiments, the third cell population comprises insulin-negative cells. In some embodiments, the third cell population comprises insulin-positive cells. In some embodiments, the third cell population comprises more insulin-negative cells than insulin-positive cells. In some embodiments, the third cell population comprises more cells that are insulin positive than the first and second cell populations.

[0144] In some embodiments, the method further comprises culturing the third cell population in a third medium comprising one or more agents selected from a Notch signaling pathway inhibitor (e.g., a gamma-secretase inhibitor such as XXI), a TGFβ-R1 kinase inhibitor (e.g., ALK5i), a thyroid hormone (e.g., GC-1), a bone morphogenetic (BMP) signaling pathway inhibitor (e.g., LDN193189), a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor (e.g., triazovidine), a protein kinase inhibitor (e.g., staurosporine), and a histone methyltransferase EZH2 inhibitor (e.g., DZNep). In some embodiments, the third medium further comprises one or more agents selected from an acetyl-CoA-related metabolite (e.g., acetate), an HDAC inhibitor (e.g., β-hydroxybutyrate), a redox homeostasis regulator (e.g., taurine), and a one-carbon metabolic pathway intermediate (e.g., formate). In some embodiments, the third medium further comprises a vitamin (e.g., biotin). In some embodiments, the third medium further comprises glutamine. In some embodiments, the third medium further comprises a water-soluble synthetic polymer (e.g., PVA, such as 87-89% PVA).

[0145] In some embodiments, the third culture medium does not contain a Wnt signaling pathway inhibitor or a PKC activator. In some embodiments, the third culture medium contains a Notch signaling pathway inhibitor (e.g., a γ-secretase inhibitor such as XXI), a TGFβ-R1 kinase inhibitor (e.g., ALK5i), a thyroid hormone (e.g., GC-1), a bone morphogenetic (BMP) signaling pathway inhibitor (e.g., LDN193189), a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor (e.g., triazovidine), a protein kinase inhibitor (e.g., staurosporine), a histone methyltransferase EZH2 inhibitor (e.g., DZNep), an acetyl-CoA-related metabolite (e.g., acetate), an HDAC inhibitor (e.g., β-hydroxybutyrate), a redox homeostasis regulator (e.g., taurine), a one-carbon metabolic pathway intermediate (e.g., formate), a vitamin (e.g., biotin), glutamine, and a water-soluble synthetic polymer (e.g., PVA such as PVA 87-89%), but does not contain a Wnt signaling pathway inhibitor and a PKC activator. In some embodiments, the third cell population is cultured in a third medium (e.g., a third medium that does not contain a Wnt signaling pathway inhibitor or a PKC activator) for about 24 to 96 hours (e.g., about 24 to 96 hours, about 24 to 84 hours, about 24 to 72 hours, about 24 to 60 hours, 24 to 48 hours, 24 to 36 hours, 36 to 96 hours, 36 to 84 hours, 36 to 72 hours, 36 to 60 hours, 36 to 48 hours, 48 ​​to 96 hours, 48 ​​to 84 hours, 48 ​​to 72 hours, 48 ​​to 60 hours, 60 to 96 hours, 60 to 84 hours, 60 to 72 hours, 72 to 96 hours, 72 to 84 hours, or 84 to 96 hours). In some embodiments, the third cell population is cultured in the third medium for about 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96 hours.In some embodiments, the third cell population is cultured in the third medium for about 96 hours.

[0146] In some embodiments, the third culture medium further comprises a Wnt signaling pathway inhibitor but does not comprise a PKC activator. In some embodiments, the third culture medium further comprises a Wnt signaling pathway inhibitor (e.g., a tankyrase inhibitor such as NVP-TNKS656), a Notch signaling pathway inhibitor (e.g., a γ-secretase inhibitor such as XXI), a TGFβ-R1 kinase inhibitor (e.g., ALK5i), a thyroid hormone (e.g., GC-1), a bone morphogenetic (BMP) signaling pathway inhibitor (e.g., LDN193189), a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor (e.g., triazovidine), a protease inhibitor (e.g., ribozyme), a phospholipase C (POI) ... These include protein kinase inhibitors (e.g., staurosporine), histone methyltransferase EZH2 inhibitors (e.g., DZNep), acetyl-CoA-related metabolites (e.g., acetate), HDAC inhibitors (e.g., β-hydroxybutyrate), redox homeostasis regulators (e.g., taurine), one-carbon metabolic pathway intermediates (e.g., formate), vitamins (e.g., biotin), glutamine, and water-soluble synthetic polymers (e.g., PVA such as PVA 87-89%), but do not include PKC activators. In some embodiments, the third cell population is cultured in a third medium (e.g., a third medium containing a Wnt signaling pathway inhibitor but not a PKC activator) for about 24-48 hours (e.g., about 24-48 hours, 24-36 hours, or 36-48 hours), after which the Wnt signaling pathway inhibitor is removed from the third medium and the cells are further cultured for about 24-48 hours (e.g., about 24-48 hours, 24-36 hours, or 36-48 hours). In some embodiments, the third cell population is cultured in a third medium (e.g., a third medium containing a Wnt signaling pathway inhibitor but not a PKC activator) for about 48 hours, after which the Wnt signaling pathway inhibitor is removed from the third medium and the cells are further cultured for about 48 hours.

[0147] In some embodiments, culturing the third cell population in the third medium for a contact period described herein (e.g., 96 hours) results in a fourth cell population. In some embodiments, the fourth cell population comprises cells that are PDX1-positive and NKX6.1-positive. In some embodiments, the fourth cell population comprises cells that are insulin-positive. In some embodiments, the fourth cell population comprises cells that are PDX1-positive, NKX6.1-positive, and insulin-positive. In some embodiments, the fourth cell population comprises cells that are ISL1-positive. In some embodiments, the fourth cell population comprises cells that are ISL-1-negative. In some embodiments, at least 30% (e.g., at least 30%, at least 40%, at least 50%, or at least 60%) of the fourth cell population is insulin-positive. In some embodiments, 30%-50%, 30%-40%, or 40%-50% of the fourth cell population is insulin-positive.

[0148] In some embodiments, the method further comprises culturing the fourth cell population in a fourth medium comprising one or more agents selected from a TGFβ-R1 kinase inhibitor (e.g., ALK5i), a thyroid hormone (e.g., GC-1), an osteogenic (BMP) signaling pathway inhibitor (e.g., LDN193189), a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor (e.g., triazovidine), a protein kinase inhibitor (e.g., staurosporine), and a histone methyltransferase EZH2 inhibitor (e.g., DZNep). In some embodiments, the fourth medium further comprises one or more agents selected from an acetyl-CoA-related metabolite (e.g., acetate), an HDAC inhibitor (e.g., β-hydroxybutyrate), a redox homeostasis regulator (e.g., taurine), and a one-carbon metabolic pathway intermediate (e.g., formate). In some embodiments, the fourth medium further comprises a vitamin (e.g., biotin). In some embodiments, the fourth culture medium further comprises one or more of glutamine (e.g., L-glutamine), glutamate (e.g., L-glutamate), and carnitine (e.g., L-carnitine). In some embodiments, the fourth culture medium further comprises albumin (e.g., human serum albumin or HSA). In some embodiments, the fourth culture medium further comprises ZnSO. In some embodiments, the fourth culture medium does not comprise a Wnt signaling pathway inhibitor or a PKC activator.In some embodiments, the fourth culture medium comprises a TGFβ-R1 kinase inhibitor (e.g., ALK5i), a thyroid hormone (e.g., GC-1), an osteogenic (BMP) signaling pathway inhibitor (e.g., LDN193189), a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor (e.g., triazovidine), a protein kinase inhibitor (e.g., staurosporine), a histone methyltransferase EZH2 inhibitor (e.g., DZNep), an acetyl-CoA-related metabolite (e.g., acetate), an HDAC inhibitor (e.g., β-hydroxybutyrate), a redox homeostasis regulator (e.g., taurine), a one-carbon metabolic pathway intermediate (e.g., formate), a vitamin (e.g., biotin), glutamine, glutamate, carnitine, albumin (e.g., human serum albumin or HSA), and ZnSO, but does not comprise a Wnt signaling pathway inhibitor or a PKC activator.

[0149] In some embodiments, the fourth cell population is cultured in the fourth medium for about 24 to 96 hours (e.g., about 24 to 96 hours, 24 to 84 hours, 24 to 72 hours, 24 to 60 hours, 24 to 48 hours, 24 to 36 hours, 36 to 96 hours, 36 to 84 hours, 36 to 72 hours, 36 to 60 hours, 36 to 48 hours, 48 ​​to 96 hours, 48 ​​to 84 hours, 48 ​​to 72 hours, 48 ​​to 60 hours, 60 to 96 hours, 60 to 84 hours, 60 to 72 hours, 72 to 96 hours, 72 to 84 hours, or 84 to 96 hours). In some embodiments, the fourth cell population is cultured in the fourth medium for about 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96 hours. In some embodiments, the fourth cell population is cultured in the fourth medium for about 72 hours.

[0150] In some embodiments, culturing the fourth cell population in the fourth culture medium for the contact period described herein (e.g., 96 hours) results in a fifth cell population. In some embodiments, the methods described herein further include culturing the fifth cell population in a fifth culture medium containing glutamine, albumin (e.g., human serum albumin or HSA), and ZnSO. In some embodiments, the fifth culture medium contains glutamine, albumin (e.g., human serum albumin or HSA), and ZnSO, and does not contain any one agent selected from a TGFβ-R1 kinase inhibitor (e.g., ALK5i), a thyroid hormone (e.g., GC-1), an osteogenic (BMP) signaling pathway inhibitor (e.g., LDN193189), a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor (e.g., triazovidine), a protein kinase inhibitor (e.g., staurosporine), and a histone methyltransferase EZH2 inhibitor (e.g., DZNep). In some embodiments, the fifth medium further comprises a histone methyltransferase EZH2 inhibitor (e.g., DZNep), an acetyl-CoA-related metabolite (e.g., acetate), an HDAC inhibitor (e.g., β-hydroxybutyrate), a redox homeostasis regulator (e.g., taurine), a one-carbon metabolic pathway intermediate (e.g., formate), a vitamin (e.g., biotin), glutamine, glutamate, and carnitine.In some embodiments, the fifth culture medium comprises a histone methyltransferase EZH2 inhibitor (e.g., DZNep), an acetyl-CoA-related metabolite (e.g., acetate), an HDAC inhibitor (e.g., β-hydroxybutyrate), a redox homeostasis regulator (e.g., taurine), a one-carbon metabolic pathway intermediate (e.g., formate), a vitamin (e.g., biotin), glutamate, glutamine, carnitine, albumin (e.g., human serum albumin or HSA), and ZnSO4, and does not contain any one agent selected from a TGFβ-R1 kinase inhibitor (e.g., ALK5i), a thyroid hormone (e.g., GC-1), a bone morphogenetic (BMP) signaling pathway inhibitor (e.g., LDN193189), a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor (e.g., thiazovivin), a protein kinase inhibitor (e.g., staurosporine), and a histone methyltransferase EZH2 inhibitor (e.g., DZNep). In some embodiments, the fifth culture medium comprises albumin (e.g., human serum albumin or HSA) and ZnSO4, and does not contain any one agent selected from a TGFβ-R1 kinase inhibitor (e.g., ALK5i), a thyroid hormone (e.g., GC-1), an osteogenic (BMP) signaling pathway inhibitor (e.g., LDN193189), a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor (e.g., triazovidine), a protein kinase inhibitor (e.g., staurosporine), a histone methyltransferase EZH2 inhibitor (e.g., DZNep), an acetyl-CoA-related metabolite (e.g., acetate), an HDAC inhibitor (e.g., β-hydroxybutyrate), a redox homeostasis regulator (e.g., taurine), a one-carbon metabolic pathway intermediate (e.g., formate), a vitamin (e.g., biotin), carnitine, glutamate, and glutamine.

[0151] In some embodiments, the fifth cell population is cultured in the fifth medium for about 96 to 240 hours (e.g., about 96 to 240 hours, 96 to 216 hours, 96 to 192 hours, 96 to 168 hours, 96 to 144 hours, 96 to 120 hours; 120 to 240 hours, 120 to 216 hours, 120 to 192 hours, 120 to 168 hours, 120 to 144 hours, 144 to 240 hours, 144 to 216 hours, 144 to 192 hours, 144 to 168 hours, 168 to 240 hours, 168 to 216 hours, 168 to 192 hours, 192 to 240 hours, 192 to 216 hours, or 192 to 240 hours). In some embodiments, the fifth cell population is cultured in the fifth medium for about 24, 48, 72, 96, 120, 144, 168, 192, 216, or 240 hours. In some embodiments, the fifth cell population is cultured in the fifth medium for about 192 hours.

[0152] In some embodiments, culturing the fifth cell population in the fifth culture medium for the contact time described herein (e.g., 192 hours) results in a sixth cell population. In some embodiments, at least 15% (e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40% or more) of the sixth cell population is NKX6.1-negative, ISL-positive; and less than 12% (e.g., less than 12%, less than 10%, less than 8%, less than 6%, less than 4%, less than 2% or less) of the sixth cell population is NKX6.1-negative, ISL-negative.

[0153] In some embodiments, the methods described herein include: (i) culturing a first cell population in a first culture medium to obtain a second cell population, wherein the first cell population comprises PDX1-positive, NKX6.1-negative pancreatic progenitor cells and PDX1-positive, NKX6.1-positive pancreatic progenitor cells, and the first culture medium comprises a FoxO1 inhibitor, a Notch signaling pathway inhibitor, a PKC activator, a fibroblast growth factor, a sonic hedgehog (SHH) signaling pathway inhibitor, retinoic acid, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a TGF-β ligand, and a water-soluble synthetic polymer; (ii) culturing the second cell population obtained in (i) in a second culture medium to obtain a third cell population, wherein the second culture medium comprises a Wnt signaling pathway inhibitor, a PKC activator, an epidermal growth factor, a thyroid hormone, a TGFβ-R1 kinase inhibitor, a Notch signaling pathway inhibitor, a Sonic hedgehog (SHH) signaling pathway inhibitor, retinoic acid, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a protein kinase inhibitor, a bone morphogenetic (BMP) signaling pathway inhibitor, a histone methyltransferase EZH2 inhibitor, an acetyl-CoA-related metabolite, an HDAC inhibitor, a redox homeostasis regulator, a one-carbon metabolic pathway intermediate, a vitamin, glutamine, and a water-soluble synthetic polymer (e.g., PVA), and the second culture medium does not comprise a FOXO1 inhibitor; (iii) culturing the third cell population obtained in (ii) in a third culture medium to obtain a fourth cell population, wherein the third culture medium comprises one or more of a Notch signaling pathway inhibitor, a TGFβ-R1 kinase inhibitor, a thyroid hormone, a bone morphogenetic (BMP) signaling pathway inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a protein kinase inhibitor, a histone methyltransferase EZH2 inhibitor, an acetyl-CoA-related metabolite, an HDAC inhibitor, a redox homeostasis regulator, a one-carbon metabolic pathway intermediate, a vitamin, glutamine, and a water-soluble synthetic polymer, and the third culture medium is free of a Wnt signaling pathway inhibitor and a PKC activator; (iv) culturing the fourth cell population obtained in (iii) in a fourth culture medium to obtain a fifth cell population, wherein the fourth culture medium comprises a Notch signaling pathway inhibitor, a TGFβ-R1 kinase inhibitor, a thyroid hormone, a bone morphogenetic (BMP) signaling pathway inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a protein kinase inhibitor, a histone methyltransferase EZH2 inhibitor, an acetyl-CoA-related metabolite, an HDAC inhibitor, a redox homeostasis regulator, an acetyl-CoA-related metabolite, an HDAC inhibitor, a redox homeostasis regulator, a one-carbon metabolic pathway intermediate, a vitamin, glutamine, glutamate, carnitine, albumin, and ZnSO4, and the fourth culture medium does not comprise a Wnt signaling pathway inhibitor or a PKC activator; (v) culturing the fifth cell population obtained in (iv) in a fifth culture medium to obtain a sixth cell population, wherein the fifth culture medium comprises albumin (e.g., human serum albumin or HSA) and ZnSO; Includes:

[0154] In some embodiments, the methods described herein further comprise producing a first cell population comprising PDX1-positive, NKX6.1-negative pancreatic progenitor cells and PDX1-positive, NKX6.1-positive pancreatic progenitor cells. In some embodiments, the first cell population is differentiated from stem cells (e.g., embryonic stem cells or pluripotent stem cells). In some embodiments, the stem cells (e.g., embryonic stem cells) are produced from the inner cell mass corresponding to a blastocyst-stage embryo. Stem cells can be maintained in culture, self-renewing, and can proliferate indefinitely as undifferentiated ES cells, and can differentiate into any cell type in the body as cells or tissues of the ectodermal, mesodermal, and endodermal lineages.

[0155] Cell types during pancreatic differentiation Aspects of the present disclosure provide cell types of pancreatic lineage obtained upon differentiation of stem cells to produce pancreatic islet cells, including any cell that can differentiate into a pancreatic islet cell, such as, for example, pluripotent stem cells, definitive endoderm cells, primitive gut cells, pancreatic progenitor cells, and endocrine progenitor cells, when cultured under conditions suitable for differentiating the progenitor cells into pancreatic islet cells.

[0156] stem cells "Stem cell" refers to a cell (e.g., plant stem cell, vertebrate stem cell) that has both the capacity for self-renewal and the ability to generate differentiated cell types (Morrison et al., (1997) Cell 88:287-298). In the context of cellular ontogeny, the adjectives "differentiated" and "differentiating" are relative terms. A "differentiated cell" is a cell that has progressed further along the developmental pathway than the cell to which it is being compared. Thus, pluripotent stem cells can differentiate into lineage-restricted progenitor cells (e.g., mesodermal stem cells), which can differentiate into further lineage-restricted cells (e.g., neuronal progenitor cells), which can differentiate into terminal cells that play characteristic roles in a tissue type (i.e., terminally differentiated cells, e.g., neurons, cardiomyocytes, etc.), and may or may not retain the ability to proliferate further. Stem cells can be characterized by the presence of certain markers (e.g., proteins, RNA, etc.) and the absence of certain markers. Stem cells can also be identified by functional assays, both in vitro and in vivo, particularly assays related to the ability of stem cells to give rise to multiple differentiated progeny. In one embodiment, the host cells are adult stem cells, somatic stem cells, non-embryonic stem cells, embryonic stem cells, hematopoietic stem cells, including pluripotent stem cells, and trophoblast stem cells.

[0157] Stem cells of interest include pluripotent stem cells (PSCs). The terms "pluripotent stem cells" or "PSCs" are used herein to refer to stem cells that can generate all cell types in an organism. Thus, PSCs can give rise to cells of all germ cell layers of an organism (e.g., endoderm, mesoderm, and ectoderm in vertebrates). Pluripotent cells can form teratomas and contribute to the ectodermal, mesodermal, and endodermal tissues of an organism. Plant pluripotent stem cells can give rise to all cell types of the plant (e.g., roots, stems, leaves, and other cells).

[0158] Animal PSCs can be derived in several different ways. For example, embryonic stem cells (ESCs) are derived from the inner cell mass of an embryo (Thomson et al., Science. 1998 Nov. 6; 282(5391):1145-7), while induced pluripotent stem cells (iPSCs) are derived from somatic cells (Takahashi et al., Cell. 2007 Nov. 30; 13 l(5):861-72; Takahashi et al., Nat Protoc. 2007; 2(l2):3081-9; Yu et al., Science. 2007 Dec. 21; 318(5858):1917-20. Epub 2007 Nov. 20). The term PSC refers to pluripotent stem cells regardless of their origin, and therefore encompasses the terms ESC and iPSC, as well as the term fetal embryonic stem cells (EGSC), which is another example of a PSC. PSCs can be in the form of established cell lines, which can be obtained directly from primary embryonic tissue or can be derived from somatic cells.

[0159] "Embryonic stem cells (ESCs)" refers to PSCs isolated from an embryo, typically from the inner cell mass of a blastocyst. ESC lines include, for example, hESBGN-01, hESBGN-02, hESBGN-03, and hESBGN-04 (BresaGen, Inc.); HES-1, HES-2, HES-3, HES-4, HES-5, and HES-6 (ES Cell International); Miz-hES1 (MizMedi Hospital-Seoul National University); HSF-1 and HSF-6 (University of California at San Francisco); and H1, H7, H9, H13, and H14 (Wisconsin Alumni Research Foundation (WiCell Research Institute)). Stem cells of interest also include embryonic stem cells from other primates, such as rhesus monkey stem cells and marmoset stem cells. Stem cells can be obtained from any mammalian species, such as humans, horses, cows, pigs, dogs, cats, rodents, such as mice, rats, hamsters, and primates (Thomson et al., (1998) Science 282:1145; Thomson et al., (1995) Proc. Natl. Acad. Sci. USA 92:7844; Thomson et al., (1996) Biol. Reprod. 55:254; Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998). In culture, ESCs typically grow as flat colonies with a large nucleus-to-cytoplasm ratio, distinct borders, and prominent nuclei. Furthermore, ESCs express SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and alkaline phosphatase, but not SSEA-1. Examples of methods for producing and characterizing ESCs can be found, for example, in U.S. Patent Nos. 7,029,913, 5,843,780, and 6,200,806, each of which is incorporated herein in its entirety.Methods for expanding undifferentiated forms of hESCs are described in WO 99 / 20741, WO 01 / 51616, and WO 03 / 020920, each of which is incorporated herein in its entirety.

[0160] "Fetal embryonic stem cells" (EGSCs) or "fetal embryonic cells" or "EG cells" refer to PSCs derived from embryonic cells and / or embryonic cell precursors, such as primordial embryonic cells, i.e., embryonic cells capable of developing into sperm and eggs. Fetal embryonic cells (EG cells) are believed to have similar properties to the above-described fetal stem cells. Examples of methods for producing and characterizing EG cells can be found, for example, in U.S. Patent No. 7,153,684; Matsui, Y. et al., (1992) Cell 70:841; Shamblott, M. et al., (2001) Proc. Natl. Acad. Sci. USA 98:113; Shamblott, M. et al., (1998) Proc. Natl. Acad. Sci. USA, 95:13726; and Koshimizu, U. et al., (1996) Development, 122:1235, each of which is incorporated herein in its entirety. "Induced pluripotent stem cells" or "iPSCs" refer to PSCs derived from non-PSC cells (i.e., from cells differentiated into PSCs). iPSCs can be derived from a variety of different cell types, including terminally differentiated cells. iPSCs have an ES cell-like morphology and grow as flat colonies with a large nuclear-cytoplasmic ratio, distinct borders, and prominent nuclei. Furthermore, iPSCs express one or more key pluripotency markers known to those skilled in the art, including, but not limited to, alkaline phosphatase, SSEA3, SSEA4, Sox2, Oct3 / 4, Nanog, TRA160, TRA181, TDGF1, Dnmt3b, FoxD3, GDF3, Cyp26al, TERT, and zfp42. Examples of methods for generating and characterizing iPSCs can be found, for example, in U.S. Patent Publication Nos. 2009 / 0047263, 2009 / 0068742, 2009 / 0191159, 2009 / 0227032, 2009 / 0246875, and 2009 / 0304646, each of which is incorporated herein in its entirety. Generally, to generate iPSCs, somatic cells are provided with reprogramming factors known in the art (e.g., Oct4, SOX2, KLF4, MYC, Nanog, Lin28, etc.) that reprogram somatic cells to pluripotent stem cells.

[0161] "Somatic cells" refers to any cell of an organism that does not normally give rise to all cell types of the organism without experimental manipulation. In other words, somatic cells are cells that are fully differentiated and therefore do not naturally give rise to cells of all three germ layers of an organism, i.e., ectoderm, mesoderm, and endoderm. For example, somatic cells include neurons and neuronal progenitor cells, which can naturally give rise to all or some cell types of the central nervous system but cannot give rise to cells of the mesodermal or endodermal lineages.

[0162] In certain instances, stem cells may be undifferentiated (e.g., cells not committed to a particular lineage) before exposure to at least one cell maturation factor according to the methods disclosed herein, while in other instances, it may be desirable to differentiate stem cells into one or more intermediate cell types before exposure to at least one cell maturation factor(s) described herein. For example, stem cells may exhibit morphological, biological, or physical characteristics of undifferentiated cells, which can be used to distinguish them from differentiated cells of fetal or adult origin. In some instances, undifferentiated cells may appear microscopically in two dimensions in colonies of cells with a high nucleus / cytoplasm ratio and prominent nuclei. Stem cells may be used by themselves (e.g., substantially free of any undifferentiated cells) or in the presence of differentiated cells. In certain instances, stem cells may be cultured in the presence of suitable nutrients and, optionally, other cells, to grow and optionally differentiate. For example, fetal fibroblasts or fibroblast-like cells may be present in the culture to support stem cell growth. Fibroblasts may be present during one stage of stem cell development, but not necessarily at all stages. For example, fibroblasts may be added to a culture of stem cells at an initial culture stage and not be added to the culture of stem cells at one or more subsequent culture stages.

[0163] Stem cells used in all aspects of the invention can be any cells derived from any type of tissue (e.g., embryonic tissue, such as fetal or pre-fetal tissue, or adult tissue), and these stem cells are characterized by their ability, under appropriate conditions, to generate progeny consisting of various cell types, e.g., derivatives of all or at least one of the three germ layers (endoderm, mesoderm, and ectoderm). These cell types can be provided in the form of established cell lines, or can be obtained directly from primary embryonic tissue and immediately used for differentiation. Cells listed in the NIH Human Embryonic Stem Cell Registry include hESBGN-O1, hESBGN-02, hESBGN-03, and hESBGN-04 (BresaGen, Inc.); HES-1, HES-2, HES-3, HES-4, HES-5, and HES-6 (ES Cell International); Miz-hES1 (MizMedi Hospital-Seoul National University); HSF-1 and FISF-6 (University of California at San Francisco); and H1, H7, H9, H13, and H14 (Wisconsin Alumni Research Foundation (WiCell Research Institute)). In some embodiments, the source of human stem cells or pluripotent stem cells used for chemically induced differentiation into mature insulin-positive cells did not involve the destruction of a human embryo.

[0164] In another embodiment, stem cells can be isolated from tissues, including solid tissues.In some embodiments, the tissue is skin, adipose tissue (for example, adipose tissue), muscle tissue, heart or cardiac tissue.In other embodiments, the tissue is, for example, but not limited to, umbilical cord blood, placenta, bone marrow, or cartilage.

[0165] Stem cells of interest include various types of embryonic cells, such as human embryonic stem cells (hES), as described in Thomson et al. (1998) Science 282:1145; embryonic stem cells from other primates, such as rhesus stem cells (Thomson et al. (1995) Proc. Natl. Acad. Sci. USA 92:7844); marmoset stem cells (Thomson et al. (1996) Biol. Reprod. 55:254); and human embryonic germ (hEG) cells (Shambloft et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998). Also applicable are stem cells committed to lineages such as mesodermal stem cells and other early cardiac developmental cells (see, e.g., Reyes et al. (2001) Blood 98:2615-2625; Eisenberg & Bader (1996) Circ Res. 78(2):205-16). Stem cells can be obtained from any mammalian species, e.g., human, bovine, porcine, canine, feline, rodent, e.g., mouse, rat, hamster, primate, etc. In some embodiments, human embryos were not destroyed for the source of pluripotent cells used in the methods and compositions disclosed herein.

[0166] A mixture of cells from a suitable source of endothelial, muscle, and / or neural stem cells can be collected from a mammalian donor by methods known in the art. A suitable source is the hematopoietic microenvironment. For example, preferably fixed (i.e., mobilized) circulating peripheral blood can be removed from a subject. In one embodiment, the stem cells can be reprogrammed stem cells, such as stem cells derived from somatic cells or differentiated cells. In such embodiments, the dedifferentiated stem cells can be, for example, but not limited to, neoplastic cells, tumor cells, and cancer cells, or induced reprogrammed cells, such as induced pluripotent stem cells or iPS cells.

[0167] Definitive endoderm cells Definitive endoderm is produced in vivo from the inner cell mass by the embryonic process of gastrulation, during which epiblast cells are directed to form three germ layers. Definitive endoderm can give rise to a variety of cells and tissues that contribute to vital organs, such as pancreatic beta cells, hepatocytes of the liver, alveolar cells of the lung, thyroid gland, thymus, and the epithelial lining of the nutrient and respiratory tracts. This differs from the primitive endoderm, which is an extraembryonic tissue that can give rise to visceral and parietal endoderm. Definitive endoderm derived from ES cells can theoretically become any endodermal derivative.

[0168] The precise patterning of the definitive endoderm along the anterior-posterior axis ultimately leads to the formation of the primitive gut. The primitive gut, derived from the definitive endoderm, gives rise to the pharynx, esophagus, stomach, duodenum, small intestine, and large intestine, as well as related organs such as the pancreas, lungs, thyroid gland, thymus, parathyroid gland, and liver along the anterior-posterior axis. The anterior portion of the foregut of the primitive gut gives rise to the lungs, thyroid gland, esophagus, and stomach. The pancreas, liver, and duodenum originate from the posterior portion of the foregut. The midgut and hindgut of the primitive gut give rise to the small intestine and large intestine. The anterior foregut expresses developmental markers NK2 homeobox (NKX2-1) and SRY (sex-determining region Y)-box 2 (SOX2); the posterior foregut expresses hematopoietically expressed homeobox (HHEX), pancreatic and duodenal homeobox 1 (PDX1), onecut homeobox 1 (ONECUT1, also known as HNF6), and hepatocyte nuclear factor 4 alpha (HNF4A); and the midgut / hindgut expresses caudal homeobox 1 (CDX1), caudal homeobox 2 (CDX2), and motor neuron and pancreatic homeobox 1 (MNX1) (3, 19, 20).

[0169] As described herein, the definitive endoderm cell used herein can be derived from any source or produced according to any suitable protocol.In some embodiments, pluripotent stem cells, such as iPSCs or hESCs, are differentiated into endoderm cells.In some embodiments, endoderm cells (stage 1) are further differentiated into, for example, primitive gut cells (stage 2), PDX1-positive pancreatic progenitor cells (stage 3), NKX6.1-positive pancreatic progenitor cells (stage 4), or Ngn3-positive endocrine progenitor cells or insulin-positive endocrine cells (stage 5), and then are induced into SC-β cells or matured (stage 6).

[0170] In some embodiments, definitive endoderm cells can be obtained by differentiating at least some pluripotent cells in a population into definitive endoderm cells, for example, by contacting a population of pluripotent cells with i) at least one growth factor from the TGF-β superfamily and ii) a WNT signaling pathway activator to induce differentiation of at least some of the pluripotent cells into definitive endoderm cells, wherein the definitive endoderm cells express at least one marker characteristic of definitive endoderm.

[0171] Any growth factor from the TGF-β superfamily that can induce pluripotent stem cells (e.g., alone or in combination with a WNT signaling pathway activator) to differentiate into definitive endoderm cells can be used in the methods provided herein. In some embodiments, a growth factor from the TGF-β superfamily comprises activin A. In some embodiments, a growth factor from the TGF-β superfamily comprises growth differentiation factor 8 (GDF8). Any WNT signaling pathway activator that can induce pluripotent stem cells (e.g., alone or in combination with a growth factor from the TGF-β superfamily) to differentiate into definitive endoderm cells can be used in the methods provided herein. In some embodiments, the WNT signaling pathway activator comprises CHIR99021. In some embodiments, the WNT signaling pathway activator comprises Wnt3a recombinant protein.

[0172] In some embodiments, differentiating at least some of the pluripotent cells in the population into definitive endoderm cells is accomplished by a process of contacting the population of pluripotent cells with i) activin A, and ii) CHIR99021 for a suitable period of time, for example, about 2 days, about 3 days, about 4 days, or about 5 days, 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. In some embodiments, this process comprises contacting the pluripotent cell population with activin A and CHIR99021 for 1 day, followed by contacting with activin A (in the absence of CHIR99021) for an additional 1 or 2 days.

[0173] In some examples, the methods include differentiating the pluripotent cells into definitive endoderm cells by contacting the population of pluripotent cells with a suitable concentration, e.g., about 10 ng / mL, about 20 ng / mL, about 50 ng / mL, about 75 ng / mL, about 80 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL, about 110 ng / mL, about 120 ng / mL, about 130 ng / mL, about 140 ng / mL, about 150 ng / mL, about 175 ng / mL, about 180 ng / mL, about 200 ng / mL, about 250 ng / mL, or about 300 ng / mL, of a growth factor from the TGF-β superfamily (e.g., activin A). In some embodiments, the methods involve using about 70-130 ng / ml, 80-120 ng / ml, or 90-110 ng / ml of activin A to differentiate pluripotent cells into definitive endoderm cells. In some embodiments, the methods involve using about 100 ng / mL of activin A to differentiate pluripotent cells into definitive endoderm cells. In some embodiments, the methods involve using about 200 ng / mL of activin A to differentiate pluripotent cells into definitive endoderm cells.

[0174] In some examples, the methods include differentiating the pluripotent cells into definitive endoderm cells by contacting the population of pluripotent cells with a suitable concentration of a WNT signaling pathway activator (e.g., CHIR99021), e.g., about 0.01 μM, about 0.05 μM, about 0.1 μM, about 0.2 μM, about 0.5 μM, about 0.8 μM, about 1 μM, about 1.5 μM, about 2 μM, about 2.5 μM, about 3 μM, about 3.5 μM, about 4 μM, about 5 μM, about 8 μM, about 10 μM, about 12 μM, about 15 μM, about 20 μM, about 30 μM, about 50 μM, about 100 μM, or about 200 μM. In some embodiments, the methods include using about 1-5 μM or 2-4 μM CHIR99021 for the differentiation of the pluripotent cells into definitive endoderm cells. In some embodiments, the method comprises using about 2 μM CHIR99021 to differentiate pluripotent cells into definitive endoderm cells. In some embodiments, the method comprises using about 3 μM CHIR99021 to differentiate pluripotent cells into definitive endoderm cells. In some embodiments, the method comprises using about 5 μM CHIR99021 to differentiate pluripotent cells into definitive endoderm cells.

[0175] In some embodiments, the cells are further contacted with a water-soluble synthetic polymer. In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol. In some examples, the polyvinyl alcohol is at least 78% hydrolyzed, e.g., 79-81% hydrolyzed, 87-89% hydrolyzed, 87-90% hydrolyzed, or 99% hydrolyzed. In some embodiments, the polyvinyl alcohol is 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% hydrolyzed. In some embodiments, the PVA is 80% hydrolyzed.

[0176] In some embodiments, definitive endoderm cells generated by the methods disclosed herein express at least one marker selected from the group consisting of Nodal, Tmprss2, Tmem30b, St14, Spink3, Sh3gl2, Ripk4, Rab1S, Npnt, Clic6, Cldn5, Cacna1b, Bnip1, Anxa4, Emb, FoxA1, Sox17, and Rbm35a, and the expression of the at least one marker is upregulated at a statistically significant amount in the definitive endoderm cells relative to the pluripotent stem cells from which they were derived. In some embodiments, definitive endoderm cells generated by the methods disclosed herein do not express at least one marker selected from the group consisting of Gata4, SPARC, AFP, and Dab2 at a statistically significant amount relative to the pluripotent stem cells from which they were derived. In some embodiments, the definitive endoderm cells generated by the methods disclosed herein do not express a statistically significant amount of at least one marker selected from the group consisting of Zic1, Pax6, Flk1, and CD31 compared to the pluripotent stem cells from which they were derived. In some embodiments, the definitive endoderm cells generated by the methods disclosed herein have a statistically significant higher level of Smad2 phosphorylation relative to the pluripotent stem cells from which they were derived. In some embodiments, the definitive endoderm cells generated by the methods disclosed herein have the ability to form a gut tube in vivo. In some embodiments, the definitive endoderm cells generated by the methods disclosed herein can be differentiated into cells having morphology characteristic of intestinal cells, and the cells having morphology characteristic of intestinal cells express FoxA2 and / or Claudin6. In some embodiments, the definitive endoderm cells generated by the methods disclosed herein can be further differentiated into cells of endodermal origin.

[0177] In some embodiments, the population of pluripotent stem cells is cultured in the presence of at least one beta cell differentiation factor before any differentiation or during the first stage of differentiation. Any pluripotent stem cells can be used, such as human pluripotent stem cells or human iPS cells, or any pluripotent stem cells discussed herein or other suitable pluripotent stem cells. In some embodiments, the beta cell differentiation factors described herein can be present in the culture medium of the population of pluripotent stem cells, or can be added once or periodically during the growth (e.g., replication or expansion) of the population of pluripotent stem cells. In certain examples, the population of pluripotent stem cells can be exposed to at least one beta cell differentiation factor before any differentiation. In other examples, the population of pluripotent stem cells can be exposed to at least one beta cell differentiation factor during the first stage of differentiation.

[0178] gastrula cells The embodiments of the present disclosure include primitive gut cells.The primitive gut cells used herein can be derived from any source or produced according to any suitable protocol.In some embodiments, definitive endoderm cells are differentiated into primitive gut cells.In some embodiments, primitive gut cells are further differentiated into, for example, PDX1 positive pancreatic progenitor cells, NKX6.1 positive pancreatic progenitor cells, Ngn3 positive endocrine progenitor cells, insulin positive endocrine cells, and then induce or mature into SC-β cells.

[0179] In some embodiments, primitive gut cells can be obtained by differentiating at least some of the definitive endoderm cells in the population into primitive gut cells, e.g., by contacting the definitive endoderm cells with at least one growth factor from the fibroblast growth factor (FGF) family to induce differentiation of at least some of the definitive endoderm cells into primitive gut cells, wherein the primitive gut cells express at least one marker characteristic of primitive gut cells.

[0180] Any growth factor from the FGF family that can induce definitive endoderm cells to differentiate into primitive gut cells (e.g., alone or in combination with other factors) can be used in the methods provided herein. In some embodiments, at least one growth factor from the FGF family comprises keratinocyte growth factor (KGF). In some embodiments, at least one growth factor from the FGF family comprises FGF2. In some embodiments, at least one growth factor from the FGF family comprises FGF8B. In some embodiments, at least one growth factor from the FGF family comprises FGF10. In some embodiments, at least one growth factor from the FGF family comprises FGF21.

[0181] In some embodiments, primitive gut tube cells can be obtained by differentiating at least some of the definitive endoderm cells in the population into primitive gut tube cells, for example, by contacting the definitive endoderm cells with KGF for a period of time, for example, about 1 day, about 2 days, about 3 days, or about 4 days, to induce differentiation of at least some of the definitive endoderm cells into primitive gut tube cells.

[0182] In some embodiments, the methods include differentiating definitive endoderm cells into primitive gut cells by contacting the cells with a growth factor from the FGF family (e.g., KGF) at a suitable concentration, e.g., about 10 ng / mL, about 20 ng / mL, about 50 ng / mL, about 75 ng / mL, about 80 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL, about 110 ng / mL, about 120 ng / mL, about 130 ng / mL, about 140 ng / mL, about 150 ng / mL, about 175 ng / mL, about 180 ng / mL, about 200 ng / mL, about 250 ng / mL, or about 300 ng / mL. In some embodiments, the methods include using about 20-80 ng / mL, 30-70 ng / mL, or 40-60 ng / mL of KGF to differentiate the definitive endoderm cells into primitive gut cells. In some embodiments, the methods comprise using about 50 ng / mL of KGF to differentiate the definitive endoderm cells into primitive gut cells, hi some embodiments, the methods comprise using about 100 ng / mL of KGF to differentiate the definitive endoderm cells into primitive gut cells.

[0183] In some embodiments, the cells are further contacted with a water-soluble synthetic polymer. In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol. In some examples, the polyvinyl alcohol is at least 78% hydrolyzed, e.g., 79-81% hydrolyzed, 87-89% hydrolyzed, 87-90% hydrolyzed, or 99% hydrolyzed. In some embodiments, the polyvinyl alcohol (PVA) is 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% hydrolyzed. In some embodiments, the PVA is 80% hydrolyzed.

[0184] PDX1-positive pancreatic progenitor cells The embodiments of the present disclosure include PDX1 positive pancreatic progenitor cells.The PDX1 positive pancreatic progenitor cells used herein can be derived from any source or produced according to any suitable protocol.In some embodiments, primitive gut cells are differentiated into PDX1 positive pancreatic progenitor cells.In some embodiments, PDX1 positive pancreatic progenitor cells are NKX6.1 negative, and can be further differentiated into, for example, NKX6.1 positive pancreatic progenitor cells, Ngn3 positive endocrine progenitor cells, insulin positive endocrine cells, and then can be induced or matured into SC-β cells.

[0185] In some embodiments, PDX1-positive pancreatic progenitor cells can be obtained by differentiating at least a portion of the primitive gut cells in the population into PDX1-positive pancreatic progenitor cells, for example, by contacting the primitive gut cells with one or more of: i) at least one BMP signaling pathway inhibitor, ii) a growth factor from the TGF-β superfamily, iii) at least one growth factor from the FGF family, iv) at least one SHH pathway inhibitor, v) at least one retinoic acid (RA) signaling pathway activator, i) at least one protein kinase C activator, and vii) a ROCK inhibitor, to induce differentiation of at least a portion of the primitive gut cells into PDX1-positive pancreatic progenitor cells, wherein the PDX1-positive pancreatic progenitor cells express PDX1.

[0186] In some embodiments, PDX1-positive pancreatic progenitor cells can be obtained by differentiating at least a portion of the primitive gut cells in the population into PDX1-positive pancreatic progenitor cells, for example, by contacting the primitive gut cells with one or more of: i) at least one BMP signaling pathway inhibitor, ii) a growth factor from the TGF-β superfamily, iii) at least one growth factor from the FGF family, iv) at least one SHH pathway inhibitor, v) at least one retinoic acid (RA) signaling pathway activator, and vi) at least one protein kinase C activator, and inducing differentiation of at least a portion of the primitive gut cells into PDX1-positive pancreatic progenitor cells, wherein the PDX1-positive pancreatic progenitor cells express PDX1.

[0187] In some embodiments, PDX1-positive pancreatic progenitor cells can be obtained by differentiating at least a portion of the primitive gut cells in the population into PDX1-positive pancreatic progenitor cells, for example, by contacting the primitive gut cells with one or more of: i) at least one BMP signaling pathway inhibitor, ii) at least one growth factor from the FGF family, iii) at least one SHH pathway inhibitor, iv) at least one retinoic acid (RA) signaling pathway activator, and v) at least one protein kinase C activator, to induce differentiation of at least a portion of the primitive gut cells into PDX1-positive pancreatic progenitor cells, wherein the PDX1-positive pancreatic progenitor cells express PDX1.

[0188] In some embodiments, PDX1-positive pancreatic progenitor cells can be obtained by differentiating at least a portion of the primitive gut cells in the population into PDX1-positive pancreatic progenitor cells, for example, by contacting the primitive gut cells with: i) at least one SHH pathway inhibitor; ii) at least one retinoic acid (RA) signaling pathway activator; and iii) at least one protein kinase C activator, wherein the PDX1-positive pancreatic progenitor cells express PDX1.

[0189] In some embodiments, PDX1-positive pancreatic progenitor cells can be obtained by differentiating at least some of the primitive gut cells in the population into PDX1-positive pancreatic progenitor cells, for example, by contacting the primitive gut cells with i) at least one growth factor from the FGF family and ii) at least one activator of the retinoic acid (RA) signaling pathway to induce differentiation of at least some of the primitive gut cells into PDX1-positive pancreatic progenitor cells, wherein the PDX1-positive pancreatic progenitor cells express PDX1.

[0190] Any BMP signaling pathway inhibitor that can induce primitive gut cells to differentiate into PDX1-positive pancreatic progenitor cells (e.g., alone or in combination with a growth factor from the TGF-β superfamily, at least one growth factor from the FGF family, at least one SHH pathway inhibitor, at least one retinoic acid signaling pathway activator, at least one protein kinase C activator, and a ROCK inhibitor) can be used in the methods provided herein. In some embodiments, the BMP signaling pathway inhibitor comprises LDN193189 or DMH-1. In some examples, the method includes contacting the primitive gut cells with a BMP signaling pathway inhibitor (e.g., LDN193189) at a concentration of, for example, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, about 100 nM, about 110 nM, about 120 nM, about 130 nM, about 140 nM, about 150 nM, about 160 nM, about 170 nM, about 180 nM, about 190 nM, about 200 nM, about 210 nM, about 220 nM, about 230 nM, about 240 nM, about 250 nM, about 280 nM, about 300 nM, about 400 nM, about 500 nM, or about 1 μM. In some examples, the method includes contacting the primitive gut cells with a BMP signaling pathway inhibitor (e.g., DMH-1) at a concentration of, for example, about 0.01 μM, about 0.02 μM, about 0.05 μM, about 0.1 μM, about 0.2 μM, about 0.5 μM, about 0.8 μM, about 1 μM, about 1.2 μM, about 1.5 μM, about 1.75 μM, about 2 μM, about 2.2 μM, about 2.5 μM, about 2.75 μM, about 3 μM, about 3.25 μM, about 3.5 μM, about 3.75 μM, about 4 μM, about 4.5 μM, about 5 μM, about 8 μM, about 10 μM, about 15 μM, about 20 μM, about 30 μM, about 40 μM, about 50 μM, or about 100 μM. In some examples, the method includes contacting the gastrula cells with a BMP signaling pathway inhibitor (e.g., DMH-1) at a concentration of about 220-280 nM, about 230-270 nM, about 240-260 nM, or about 245-255 nM, etc. In some examples, the method includes contacting the gastrula cells with a BMP signaling pathway inhibitor (e.g., DMH-1) at a concentration of about 250 nM.

[0191] Any growth factor from the TGF-β superfamily that can induce primitive gut cells to differentiate into PDX1-positive pancreatic progenitor cells (e.g., alone or in combination with at least one BMP signaling pathway inhibitor, a growth factor from the FGF family, at least one SHH pathway inhibitor, at least one retinoic acid signaling pathway activator, at least one protein kinase C activator, and a ROCK inhibitor) can be used. In some embodiments, the growth factor from the TGF-β family includes activin A. In some embodiments, the growth factor from the TGF-β family includes GDF8. In some examples, the method includes contacting the primitive gut cells with a growth factor from the TGF-β superfamily (e.g., activin A) at a concentration of about 5 ng / mL, about 7.5 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL, about 11 ng / mL, about 12 ng / mL, about 13 ng / mL, about 14 ng / mL, about 15 ng / mL, about 16 ng / mL, about 17 ng / mL, about 18 ng / mL, about 19 ng / mL, about 20 ng / mL, about 21 ng / mL, about 22 ng / mL, about 23 ng / mL, about 24 ng / mL, about 25 ng / mL, about 26 ng / mL, about 27 ng / mL, about 28 ng / mL, about 29 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 50 ng / mL, or about 100 ng / mL. In some examples, the method includes contacting the gastrula cells with a growth factor from the TGF-β superfamily (e.g., activin A) at a concentration of about 17-23 ng / ml, about 18-22 ng / ml, or about 19-21 ng / ml, etc. In some examples, the method includes contacting the gastrula cells with a growth factor from the TGF-β superfamily (e.g., activin A) at a concentration of about 20 ng / ml.

[0192] Any growth factor from the FGF family that can induce primitive gut cells to differentiate into PDX1-positive pancreatic progenitor cells (e.g., alone or in combination with at least one BMP signaling pathway inhibitor, a growth factor from the TGF-β superfamily, at least one SHH pathway inhibitor, at least one retinoic acid signaling pathway activator, at least one protein kinase C activator, and a ROCK inhibitor) can be used. In some embodiments, the at least one growth factor from the FGF family comprises keratinocyte growth factor (KGF). In some embodiments, the at least one growth factor from the FGF family is selected from the group consisting of FGF2, FGF8B, FGF10, and FGF21. In some examples, the method includes contacting the primary gut cells with a growth factor from the FGF family (e.g., KGF) at a concentration of, for example, about 10 ng / mL, about 20 ng / mL, about 50 ng / mL, about 75 ng / mL, about 80 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL, about 110 ng / mL, about 120 ng / mL, about 130 ng / mL, about 140 ng / mL, about 150 ng / mL, about 175 ng / mL, about 180 ng / mL, about 200 ng / mL, about 250 ng / mL, or about 300 ng / mL. In some examples, the method includes contacting the primary gut cells with a growth factor from the FGF family (e.g., KGF) at a concentration of, for example, about 20-80 ng / mL, about 30-70 ng / mL, about 40-60 ng / mL, or about 45-55 ng / mL. In some examples, the method includes contacting the primitive gut cells with a growth factor from the FGF family (eg, KGF) at a concentration of about 50 ng / ml.

[0193] Any SHH pathway inhibitor that can induce primitive gut cells to differentiate 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, a growth factor from the TGF-β superfamily, at least one retinoic acid signaling pathway activator, at least one protein kinase C activator, and a ROCK inhibitor) can be used. In some embodiments, the SHH pathway inhibitor comprises Sant1. In some examples, the method comprises administering to the primitive gut cells at a concentration of, for example, about 0.001 μM, about 0.002 μM, about 0.005 μM, about 0.01 μM, about 0.02 μM, about 0.03 μM, about 0.05 μM, about 0.08 μM, about 0.1 μM, about 0.12 μM, about 0.13 μM, about 0.14 μM, about 0.15 μM, about 0.16 μM, about 0.17 μM, about 0.18 μM, about 0.19 μM, about 0.2 μM, about 0.21 μM, about 0.22 μM, about 0.23 μM, about 0.24 μM, about 0.25 μM, about 0.26 μM, about 0.27 μM, about 0.28 μM, about 0.29 μM, about 0.30 μM, about 0.31 μM, about 0.32 μM, about 0.33 μM, about 0.34 μM, about 0.35 μM, about 0.36 μM, about 0.37 μM, about 0.38 μM, about 0.39 μM, about 10 ... The method includes contacting the primary gut cells with an SHH pathway inhibitor (e.g., Sant1) at a concentration of about 220-280 nM, about 230-270 nM, about 240-260 nM, or about 245-255 nM, or about 255 nM, or about 260 nM, or about 270 nM, or about 280 nM, or about 290 nM, or about 300 nM, or about 310 nM, or about 320 nM, or about 330 nM, or about 340 nM, or about 355 nM, or about 360 nM, or about 370 nM, or about 380 nM, or about 390 nM, or about 400 nM, or about 410 nM, or about 420 nM, or about 430 nM, or about 440 nM, or about 455 nM, or about 50 ... In some examples, the method includes contacting the primitive gut cells with an SHH pathway inhibitor (e.g., Sant1) at a concentration of about 250 nM.

[0194] Any RA signaling pathway activator that can induce primitive gut cells to differentiate 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, at least one protein kinase C activator, and a ROCK inhibitor) can be used. In some embodiments, the RA signaling pathway activator comprises retinoic acid. In some examples, the method comprises administering to the primitive gut cells at a concentration of, for example, about 0.02 μM, about 0.1 μM, about 0.2 μM, about 0.25 μM, about 0.3 μM, about 0.4 μM, about 0.45 μM, about 0.5 μM, about 0.55 μM, about 0.6 μM, about 0.65 μM, about 0.7 μM, about 0.75 μM, about 0.8 μM, about 0.85 μM, about 0.9 μM, about 1 μM, about 1.1 μM, about 1.2 μM, about 1.3 μM, about 1.4 μM, about 1.5 μM, about 1.6 μM, about 1.7 μM, about 1.8 μM, about 1.9 μM, about 2 μM, about 2.1 μM, about 2.2 μM, about 2.3 μM, about 2.4 μM, or about 2.5 μM. , about 2.6 μM, about 2.7 μM, about 2.8 μM, about 3 μM, about 3.2 μM, about 3.4 μM, about 3.6 μM, about 3.8 μM, about 4 μM, about 4.2 μM, about 4.4 μM, about 4.6 μM, about 4.8 μM, about 5 μM, about 5.5 μM, about 6 μM, about 6.5 μM, about 7 μM, about 7.5 μM, about 8 μM, about 8.5 μM, about 9 μM, about 9.5 μM, about 10 μM, about 12 μM, about 14 μM, about 15 μM, about 16 μM, about 18 μM, about 20 μM, about 50 μM, or about 100 μM of a RA signaling pathway activator (e.g., retinoic acid). In some examples, the method includes contacting the primitive gut cells with an RA signaling pathway activator (e.g., retinoic acid) at a concentration of about 1.7-2.3 μM, about 1.8-2.2 μM, or about 1.9-2.1 μM, etc. In some examples, the method includes contacting the primitive gut cells with an RA signaling pathway activator (e.g., retinoic acid) at a concentration of about 2 μM.

[0195] Any PKC activator that can induce primitive gut cells to differentiate into PDX1-positive pancreatic progenitor cells (e.g., alone or in any combination with at least one BMP signaling pathway inhibitor, at least one growth factor from the FGF family, at least one SHH pathway inhibitor, at least one RA signaling pathway activator, and a ROCK inhibitor) can be used. In some embodiments, the PKC activator comprises PdBU. In some embodiments, the PKC activator comprises TPPB. In some examples, the method comprises injecting primitive gut cells with at least one of the following: about 10 nM, 50 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM, 850 nM, 900 nM, 950 nM, 1 μM, 10 μM, about 20 μM, about 50 μM, about 75 μM, about 80 μM, about 100 μM, about 120 μM, about 140 μM, about 150 μM, about 175 μM, about 180 μM, about 200 μM, about 210 μM, about 220 μM, about 240 μM, about 250 μM, about 260 μM The method includes contacting the cells with a PKC activator (e.g., PdBU or TPPB) at a concentration of about 1 mM, about 280 μM, about 300 μM, about 320 μM, about 340 μM, about 360 μM, about 380 μM, about 400 μM, about 420 μM, about 440 μM, about 460 μM, about 480 μM, about 500 μM, about 520 μM, about 540 μM, about 560 μM, about 580 μM, about 600 μM, about 620 μM, about 640 μM, about 660 μM, about 680 μM, about 700 μM, about 750 μM, about 800 μM, about 850 μM, about 900 μM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, or about 5 mM. In some embodiments, the method includes contacting the gastrula cells with a PKC activator (e.g., PdBU or TPPB) at a concentration of, for example, 10 nM to 1 mM, 10 nM to 500 μM, 10 nM to 1 μM, 10 to 800 nM, 100 to 900 nM, 300 to 800 nM, 300 to 600 nM, 400 to 600 nM, 450 to 550 nM, or about 500 nM.In some examples, the method includes contacting the primary gut cells with a PKC activator (e.g., PdBU or TPPB) at a concentration of about 450-550 mM, about 475-525 nM, about 490-510 nM, or about 495-505 nM, etc. In some examples, the method includes contacting the primary gut cells with a PKC activator (e.g., PdBU or TPPB) at a concentration of about 500 nM. In some embodiments, the primary gut cells are not treated with a PKC activator (e.g., PDBU).

[0196] Any ROCK inhibitor that can induce primitive gut cells to differentiate 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, a PKC activator, and at least one RA signaling pathway activator) can be used. In some embodiments, the ROCK inhibitor comprises thiazovivin, Y-27632, Fasudil / HA1077, or H-1152. In some embodiments, the ROCK inhibitor comprises Y-27632. In some embodiments, the ROCK inhibitor comprises thiazovivin. In some examples, the method further comprises administering to the primitive gut cells a therapeutically effective amount of HCl at a concentration of, for example, about 0.2 μM, about 0.5 μM, about 0.75 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 7.5 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, about 18 μM, The method includes contacting the primary intestinal cells with a ROCK inhibitor (e.g., Y-27632 or Thiazovivin) at a concentration of about 19 μM, about 20 μM, about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM, about 26 μM, about 27 μM, about 28 μM, about 29 μM, about 30 μM, about 35 μM, about 40 μM, about 50 μM, or about 100 μM. In some examples, the method includes contacting the primary intestinal cells with a ROCK inhibitor (e.g., Y-27632 or Thiazovivin) at a concentration of about 2.2-2.8 μM, about 2.3-2.7 μM, or about 2.4-2.6 μM. In some examples, the method includes contacting the primary intestinal cells with a ROCK inhibitor (e.g., Y-27632 or Thiazovivin) at a concentration of about 2.5 μM.

[0197] In some embodiments, the cells are further contacted with a water-soluble synthetic polymer. In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol. In some examples, the polyvinyl alcohol is at least 78% hydrolyzed, e.g., 79-81% hydrolyzed, 87-89% hydrolyzed, 87-90% hydrolyzed, or 99% hydrolyzed. In some embodiments, the polyvinyl alcohol (PVA) is 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% hydrolyzed. In some embodiments, the PVA is 80% hydrolyzed.

[0198] In some embodiments, PDX1-positive pancreatic progenitor cells can be obtained by differentiating at least a portion of the primitive gut cells in the population into PDX1-positive pancreatic progenitor cells, e.g., by contacting the primitive gut cells with retinoic acid, KGF, Sant1, DMH-1, PdBU, thiazovivin, and activin A for an appropriate period of time, e.g., about 1 day, about 2 days, about 3 days, or about 4 days. In some embodiments, PDX1-positive pancreatic progenitor cells can be obtained by differentiating at least a portion of the primitive gut cells in the population into PDX1-positive pancreatic progenitor cells, e.g., by contacting the primitive gut cells with retinoic acid, KGF, Sant1, DMH-1, PdBU, thiazovivin, and activin A for about 2 days. In some embodiments, PDX1-positive pancreatic progenitor cells can be obtained by differentiating at least some of the primitive gut cells in the population into PDX1-positive pancreatic progenitor cells, for example, by contacting the primitive gut cells with retinoic acid, KGF, Sant1, DMH-1, PdBU, thiazovivin, and activin A for one day, followed by contacting them with retinoic acid, KGF, Sant1, PdBU, thiazovivin, and activin A for one day (in the absence of DMH-1).

[0199] NKX6.1-positive pancreatic progenitor cells The embodiments of the present disclosure include NKX6.1 positive pancreatic progenitor cells.The NKX6.1 positive pancreatic progenitor cells used herein can be derived from any source or produced according to any suitable protocol.In some embodiments, PDX1 positive, NKX6.1 negative pancreatic progenitor cells are differentiated into PDX1 positive, NKX6.1 positive pancreatic progenitor cells.In some embodiments, NKX6.1 positive pancreatic progenitor cells are further differentiated into, for example, Ngn3 positive endocrine progenitor cells or insulin positive endocrine cells, and then induce or mature into SC-β cells.

[0200] In some embodiments, a method for generating NKX6.1-positive pancreatic progenitor cells from PDX1-positive pancreatic progenitor cells includes contacting a cell population comprising PDX1-positive pancreatic progenitor cells (e.g., under conditions that promote cell clustering and / or conditions that promote cell survival) with at least two beta cell differentiation factors comprising: 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 (RA) signaling pathway activator, to induce differentiation of at least one PDX1-positive pancreatic progenitor cell in the population into an NKX6.1-positive pancreatic progenitor cell, wherein the NKX6.1-positive pancreatic progenitor cell expresses NKX6.1.

[0201] In some embodiments, the PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting PDX1-positive pancreatic progenitor cells with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally iii) an RA signaling pathway activator to induce differentiation of at least a portion of the PDX1-positive pancreatic progenitor cells into PDX1-positive, NKX6.1-positive pancreatic progenitor cells, wherein the PDX1-positive, NKX6.1-positive pancreatic progenitor cells express PDX1 and NKX6.1.

[0202] In some embodiments, the PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting PDX1-positive pancreatic progenitor cells with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally iii) an RA signaling pathway activator, iv) a ROCK inhibitor, and v) at least one growth factor from the TGF-β superfamily, to induce differentiation of at least a portion of the PDX1-positive pancreatic progenitor cells into PDX1-positive, NKX6.1-positive pancreatic progenitor cells. In some embodiments, after 3, 4 or 5 days of contact, PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting PDX1-positive pancreatic progenitor cells with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally iii) RA signaling pathway activator, iv) ROCK inhibitor, and v) at least one growth factor from the TGF-β superfamily; Then, cells are contacted with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally iii) RA signaling pathway activator, iv) ROCK inhibitor, and v) at least one growth factor from the TGF-β superfamily, and vi) PKC activator, and optionally vii) gamma-secretase inhibitor. In some examples, PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting PDX1-positive pancreatic progenitor cells with at least one growth factor from the FGF family under conditions that promote cell clustering. In some embodiments, the growth factor of the FGF family is KGF.

[0203] In some embodiments, the present disclosure provides a method of culturing a first cell population comprising PDX1-positive, NKX6.1-negative cells in a medium comprising any one or combination of: i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, iii) an RA signaling pathway activator, iv) a ROCK inhibitor, and v) a growth factor from the TGF-β superfamily for about 1, 2, 3, 4, or 5 days (e.g., 2-4 days, 3-4 days, or 4-5 days), thereby producing a second cell population. In some embodiments, the second cell population is then incubated for about 1, 2, or 3 days (e.g., 1-2 days, 1-3 days, or 2-3 days) in a composition comprising any one or combination of: i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, iii) an RA signaling pathway activator, iv) a ROCK inhibitor, v) a growth factor from the TGF-β superfamily, vi) a PKC activator, vii) a FoxO1 inhibitor, and optionally viii) a Notch signaling inhibitor.

[0204] In some embodiments, in the medium for culturing the first cell population, the growth factor from the FGF family is present at a concentration of about 45-55 ng / ml, about 46-54 ng / ml, about 47-53 ng / ml, about 48-52 ng / ml, or about 49-51 ng / ml, the SHH pathway inhibitor is present at a concentration of about 200-300 nM, about 220-280 nM, or about 240-260 nM, and the RA The signal transduction pathway inhibitor is present at a concentration of about 1.7-2.3 μM, about 1.8-2.2 μM, or about 1.9-2.1 μM, the ROCK inhibitor is present at a concentration of about 2-3 μM, about 2.2-2.8 μM, or about 2.4-2.6 μM, and / or the growth factor from the TGF-β superfamily is present at a concentration of about 2-8 ng / ml, about 3-7 ng / ml, or about 4-6 ng / ml.

[0205] In some embodiments, in the medium for culturing the second cell population, the growth factor from the FGF family is present at a concentration of about 45-55 ng / ml, about 46-54 ng / ml, about 47-53 ng / ml, about 48-52 ng / ml, or about 49-51 ng / ml, the SHH pathway inhibitor is present at a concentration of about 200-300 nM, about 220-280 nM, or about 240-260 nM, the RA signaling pathway inhibitor is present at a concentration of about 1.7-2.3 μM, about 1.8-2.2 μM, or about 1.9-2.1 μM, and the ROCK inhibitor is present at a concentration of about 2.2-2.3 μM. the growth factor from the TGF-β superfamily is present at a concentration of about 2-8 ng / ml, about 3-7 ng / ml, or about 4-6 ng / ml; the PKC activator is present at a concentration of about 0.2-0.8 μM, about 0.3-0.7 μM, or about 0.4-0.6 μM; the FoxO1 inhibitor is present at a concentration of about 0.7-1.3 μM, about 0.8-1.2 μM, or about 0.9-1.1 μM; and optionally the Notch signaling inhibitor is present at a concentration of about 1.7-2.3 μM, about 1.8-2.2 μM, or about 1.9-2.1 μM.

[0206] In some embodiments, the PDX1-positive pancreatic progenitor cells are generated from a pluripotent cell population. In some embodiments, the PDX1-positive pancreatic progenitor cells are generated from an iPS cell population. In some embodiments, the PDX1-positive pancreatic progenitor cells are generated from an ESC cell population. In some embodiments, the PDX1-positive pancreatic progenitor cells are generated from a definitive endoderm cell population. In some embodiments, the PDX1-positive pancreatic progenitor cells are generated from an primitive gut cell population.

[0207] Any growth factor from the FGF family that can induce PDX1-positive pancreatic progenitor cells to differentiate into NKX6.1-positive pancreatic progenitor cells (e.g., alone or in combination with at least one SHH pathway inhibitor, a ROCK inhibitor, a growth factor from the TGF-β superfamily, and at least one retinoic acid signaling pathway activator) can be used in the methods provided herein. In some embodiments, the at least one growth factor from the FGF family comprises keratinocyte growth factor (KGF). In some embodiments, the at least one growth factor from the FGF family is selected from the group consisting of FGF8B, FGF10, and FGF21. In some examples, the method includes contacting PDX1-positive pancreatic progenitor cells with a growth factor from the FGF family (e.g., KGF) at a concentration of, for example, about 10 ng / mL, about 20 ng / mL, about 50 ng / mL, about 75 ng / mL, about 80 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL, about 110 ng / mL, about 120 ng / mL, about 130 ng / mL, about 140 ng / mL, about 150 ng / mL, about 175 ng / mL, about 180 ng / mL, about 200 ng / mL, about 250 ng / mL, or about 300 ng / mL. In some examples, the method includes contacting the PDX1-positive pancreatic progenitor cells with a growth factor from the FGF family (e.g., KGF) at a concentration of about 20-80 ng / ml, about 30-70 ng / ml, about 40-60 ng / ml, or about 45-55 ng / ml, etc. In some examples, the method includes contacting the PDX1-positive pancreatic progenitor cells with about 50 ng / ml of a growth factor from the FGF family (e.g., KGF).

[0208] Any SHH pathway inhibitor that can induce PDX1-positive pancreatic progenitor cells to differentiate into NKX6.1-positive pancreatic progenitor cells (e.g., alone or in combination with at least one growth factor from the FGF family, a retinoic acid signaling pathway activator, a ROCK inhibitor, and at least one growth factor from the TGF-β superfamily) can be used in the methods provided herein. In some embodiments, the SHH pathway inhibitor comprises Sant1. In some examples, the method comprises administering to the patient a dose of PDX1-positive pancreatic progenitor cells at a concentration of, for example, about 0.001 μM, about 0.002 μM, about 0.005 μM, about 0.01 μM, about 0.02 μM, about 0.03 μM, about 0.05 μM, about 0.08 μM, about 0.1 μM, about 0.12 μM, about 0.13 μM, about 0.14 μM, about 0.15 μM, about 0.16 μM, about 0.17 μM, about 0.18 μM, about 0.19 μM, about 0.2 μM, about 0.21 μM, about 0.22 μM, about The method includes contacting the cells with an SHH pathway inhibitor (e.g., Sant1) at a concentration of 0.23 μM, about 0.24 μM, about 0.25 μM, about 0.26 μM, about 0.27 μM, about 0.28 μM, about 0.29 μM, about 0.3 μM, about 0.31 μM, about 0.32 μM, about 0.33 μM, about 0.34 μM, about 0.35 μM, about 0.4 μM, about 0.45 μM, about 0.5 μM, about 0.6 μM, about 0.8 μM, about 1 μM, about 2 μM, or about 5 μM. In some examples, the method includes contacting PDX1-positive pancreatic progenitor cells with an SHH pathway inhibitor (e.g., Sant1) at a concentration of about 220-280 nM, about 230-270 nM, about 240-260 nM, or about 245-255 nM, etc. In some examples, the method includes contacting PDX1-positive pancreatic progenitor cells with an SHH pathway inhibitor (e.g., Sant1) at a concentration of about 250 nM.

[0209] Any RA signaling pathway activator that can induce PDX1-positive pancreatic progenitor cells to differentiate into NKX6.1-positive pancreatic progenitor cells (e.g., alone or in combination with at least one growth factor from the FGF family, at least one SHH pathway inhibitor, a ROCK inhibitor, and at least one growth factor from the TGF-β superfamily) can be used. In some embodiments, the RA signaling pathway activator comprises retinoic acid. In some examples, the methods involve administering PDX1-positive pancreatic progenitor cells at a concentration of, for example, about 0.02 μM, about 0.1 μM, about 0.2 μM, about 0.25 μM, about 0.3 μM, about 0.4 μM, about 0.45 μM, about 0.5 μM, about 0.55 μM, about 0.6 μM, about 0.65 μM, about 0.7 μM, about 0.75 μM, about 0.8 μM, about 0.85 μM, about 0.9 μM, about 1 μM, about 1.1 μM, about 1.2 μM, about 1.3 μM, about 1.4 μM, about 1.5 μM, about 1.6 μM, about 1.7 μM, about 1.8 μM, about 1.9 μM, about 2 μM, about 2.1 μM, about 2.2 μM, about 2.3 μM, about 2.4 μM, or about 2.5 μM. The method includes contacting the RA signaling pathway activator (e.g., retinoic acid) at a concentration of 5 μM, about 2.6 μM, about 2.7 μM, about 2.8 μM, about 3 μM, about 3.2 μM, about 3.4 μM, about 3.6 μM, about 3.8 μM, about 4 μM, about 4.2 μM, about 4.4 μM, about 4.6 μM, about 4.8 μM, about 5 μM, about 5.5 μM, about 6 μM, about 6.5 μM, about 7 μM, about 7.5 μM, about 8 μM, about 8.5 μM, about 9 μM, about 9.5 μM, about 10 μM, about 12 μM, about 14 μM, about 15 μM, about 16 μM, about 18 μM, about 20 μM, about 50 μM, or about 100 μM. In some examples, the method includes contacting PDX1-positive pancreatic progenitor cells with an RA signaling pathway activator (e.g., retinoic acid) at a concentration of about 70-130 nM, about 80-120 nM, about 90-110 nM, or about 95-105 nM, etc. In some examples, the method includes contacting PDX1-positive pancreatic progenitor cells with an RA signaling pathway activator (e.g., retinoic acid) at a concentration of about 100 nM.

[0210] Any ROCK inhibitor that can induce PDX1-positive pancreatic progenitor cells to differentiate into NKX6.1-positive pancreatic progenitor cells (for example, alone or in combination with at least one growth factor from the FGF family, at least one SHH pathway inhibitor, an RA signaling pathway activator, and at least one growth factor from the TGF-β superfamily) can be used. In some embodiments, the ROCK inhibitor includes Thiazovivin, Y-27632, Fasudil / HA1077, or 14-1152. In some examples, the method comprises administering to the patient a dose of PDX1-positive pancreatic progenitor cells at a concentration of, for example, about 0.2 μM, about 0.5 μM, about 0.75 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 7.5 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, about 18 μM, about 20 μM, about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM, about 26 μM, about 27 μM, about 28 μM, about 29 μM, about 30 μM, about 31 μM, about 32 μM, about 33 μM, about 34 μM, about 35 μM, about 36 μM, about 37 μM, about 38 μM, about 39 μM, about 40 μM, about 41 μM, about 42 μM, about 43 μM, about 44 μM, about 45 μM, about 46 μM, about 47 μM, about 48 μM, about 49 μM, about 50 μM, about 51 μM, about 52 μM, about 53 μM, about 54 μM, about 55 μM, about 56 μM, about 57 μM, about 58 μM, about 59 μM, about 60 μM, about 61 μM, about 62 μM, about 63 μM, about 64 μM, about 65 μM, about 6 The method includes contacting the PDX1-positive pancreatic progenitor cells with a ROCK inhibitor (e.g., Y-27632 or Thiazovivin) at a concentration of about 2.2-2.8 μM, about 2.3-2.7 μM, or about 2.4-2.6 μM. In some examples, the method includes contacting PDX1-positive pancreatic progenitor cells with a ROCK inhibitor (e.g., Y-27632 or thiazovivin) at a concentration of about 2.5 μM.

[0211] Any activator from the TGF-β superfamily can be used to induce PDX1 positive pancreatic progenitor cells to differentiate into NKX6.1 positive pancreatic progenitor cells (for example, alone or in combination with at least one growth factor from the FGF family, at least one SHH pathway inhibitor, RA signaling pathway activator, and ROCK inhibitor).In some embodiments, the activator from the TGF-β superfamily comprises activin A or GDF8. In some examples, the methods involve treating PDX1-positive pancreatic progenitor cells with a concentration of a growth factor from the TGF-β superfamily (e.g., activin A), for example, about 0.1 ng / mL, about 0.2 ng / mL, about 0.3 ng / mL, about 0.4 ng / mL, about 0.5 ng / mL, about 0.6 ng / mL, about 0.7 ng / mL, about 0.8 ng / mL, about 1 ng / mL, about 1.2 ng / mL, about 1.4 ng / mL, about 1.6 ng / mL, about 1.8 ng / mL, about 2 ng / mL, about 2.2 ng / mL, about 2.4 ng / mL, about 2.6 ng / mL, about 2.8 ng / mL, about 3 ng / mL, about 3.2 ng / mL, about 3.2 ng / mL, about 3.4 ng / mL, about 3.6 ng / mL, about 3.8 ng / mL, about 3.9 ... The method includes contacting the antibody with about 3.4 ng / mL, about 3.6 ng / mL, about 3.8 ng / mL, about 4 ng / mL, about 4.2 ng / mL, about 4.4 ng / mL, about 4.6 ng / mL, about 4.8 ng / mL, about 5 ng / mL, about 5.2 ng / mL, about 5.4 ng / mL, about 5.6 ng / mL, about 5.8 ng / mL, about 6 ng / mL, about 6.2 ng / mL, about 6.4 ng / mL, about 6.6 ng / mL, about 6.8 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL, about 20 ng / mL, about 30 ng / mL, or about 50 ng / mL. In some examples, the method includes contacting the PDX1-positive pancreatic progenitor cells with a growth factor from the TGF-β superfamily (e.g., activin A) at a concentration of about 2-8 ng / ml, about 3-7 ng / ml, about 4-6 ng / ml, or about 4.5-5.5 ng / ml, etc. In some examples, the method includes contacting the PDX1-positive pancreatic progenitor cells with a growth factor from the TGF-β superfamily (e.g., activin A) at a concentration of about 5 ng / mL.

[0212] Any FoxO1 inhibitor that can induce PDX1-positive pancreatic progenitor cells to differentiate into NKX6.1-positive pancreatic progenitor cells (e.g., alone or in any combination with at least one growth factor from the FGF family, at least one retinoic acid signaling pathway activator, a ROCK inhibitor, at least one growth factor from the TGF-β superfamily, a PKC activator, and a Notch signaling inhibitor) can be used in the methods provided herein. In some embodiments, the FoxO1 inhibitor is AS1842856. In some examples, the methods include administering PDX1-positive pancreatic progenitor cells at about 0.1 μM, about 0.12 μM, about 0.13 μM, about 0.14 μM, about 0.15 μM, about 0.16 μM, about 0.17 μM, about 0.18 μM, about 0.19 μM, about 0.2 μM, about 0.21 μM, about 0.22 μM, about 0.23 μM, about 0.24 μM, about 0.25 μM, about 0.26 μM, about 0.27 μM, about 0.28 μM, about 0.29 μM, about 0.30 μM, about 0.31 μM, about 0.32 μM, about 0.33 μM, about 0.34 μM, about 0.35 μM, about 0.36 μM, about 0.37 μM, about 0.38 μM, about 0.39 μM, about 10 μM, about 10 μM, about 11 μM, about 11 μM, about 12 μM, about 12 μM, about 13 μM, about 13 μM, about 14 μM, about 14 μM, about 15 μM, about 15 μM, about 16 μM, about 16 μM, about 17 μM, about 17 μM, about 18 μM, about 18 μM, about 19 μM, about 20 μM, about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM, about 26 μM, about 27 μM, about 28 μM, about 29 μM, about 30 μM, about 31 μM, about 32 μM, about 33 μ The method includes contacting the PDX1-positive pancreatic progenitor cells with a FoxO1 inhibitor (e.g., AS1842856) at a concentration of about 0.7-1.3 μM, about 0.8-1.2 μM, about 0.9-1.1 μM, or the like. In some examples, the method includes contacting PDX1-positive pancreatic progenitor cells with a FoxO1 inhibitor (such as AS1842856) at a concentration such as about 1 μM.

[0213] Any PKC activator that can induce PDX1-positive pancreatic progenitor cells to differentiate into NKX6.1-positive pancreatic progenitor cells (e.g., alone or in any combination with at least one growth factor from the FGF family, at least one retinoic acid signaling pathway activator, a ROCK inhibitor, at least one growth factor from the TGF-β super pathway, a FoxO1 inhibitor, and a Notch signaling inhibitor) can be used in the methods provided herein. In some embodiments, the PKC activator is PDBU. In some examples, the method includes injecting PDX1-positive pancreatic progenitor cells into a pancreatic progenitor cell suspension at a concentration of about 0.1 μM, about 0.12 μM, about 0.13 μM, about 0.14 μM, about 0.15 μM, about 0.16 μM, about 0.17 μM, about 0.18 μM, about 0.19 μM, about 0.2 μM, about 0.21 μM, about 0.22 μM, about 0.23 μM, about 0.24 μM, about 0.25 μM, about 0.26 μM, or about 0. The method includes contacting PDX1-positive pancreatic progenitor cells with a PKC activator (e.g., PDBU) at a concentration of about 27 μM, about 0.28 μM, about 0.29 μM, about 0.3 μM, about 0.31 μM, about 0.32 μM, about 0.33 μM, about 0.34 μM, about 0.35 μM, about 0.4 μM, about 0.45 μM, about 0.5 μM, about 0.6 μM, about 0.8 μM, about 1 μM, about 2 μM, or about 5 μM. In some examples, the method includes contacting PDX1-positive pancreatic progenitor cells with a PKC activator (e.g., PDBU) at a concentration of about 0.2-0.8 μM, about 0.3-0.7 μM, about 0.4-0.6 μM, or the like. In some examples, the method includes contacting PDX1-positive pancreatic progenitor cells with a PKC activator (e.g., PDBU) at a concentration of about 0.5 μM, or the like.

[0214] Any Notch signaling inhibitor that can induce PDX1-positive pancreatic progenitor cells to differentiate into NKX6.1-positive pancreatic progenitor cells (e.g., alone or in any combination with at least one growth factor from the FGF family, at least one retinoic acid signaling pathway activator, a ROCK inhibitor, at least one growth factor from the TGF-β superfamily, a FoxO1 inhibitor, and a PKC activator) can be used in the methods provided herein. In some embodiments, the Notch signaling inhibitor is XXI. In some examples, the method includes administering to the patient a PDX1-positive pancreatic progenitor cell at a concentration of about 0.1 μM, about 0.12 μM, about 0.13 μM, about 0.14 μM, about 0.15 μM, about 0.16 μM, about 0.17 μM, about 0.18 μM, about 0.19 μM, about 0.2 μM, about 0.21 μM, about 0.22 μM, about 0.23 μM, about 0.24 μM, about 0.25 μM, about 0.26 μM, about 0.27 μM, about 0.28 μM, about 0.29 μM, about 0.30 μM, about 0.31 μM, about 0.32 μM, about 0.33 μM, about 0.34 μM, about 0.35 μM, about 0.36 μM, about 0.37 μM, about 0.38 μM, about 0.39 μM, about 10 μM, about 10 μM, about 11 μM, about 11 μM, about 12 μM, about 12 μM, about 13 μM, about 13 μM, about 14 μM, about 14 μM, about 15 μM, about 15 μM, about 16 μM, about 16 μM, about 17 μM, about 17 μM, about 18 μM, about 18 μM, about 19 μM, about 20 μM, about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM, about 26 μM, about 27 μM, about 28 μM, about 29 μM, about 30 μM, about 31 μM, about 32 μ The method includes contacting PDX1-positive pancreatic progenitor cells with a Notch signaling inhibitor (e.g., XXI) at a concentration of about 7 μM, about 0.28 μM, about 0.29 μM, about 0.3 μM, about 0.31 μM, about 0.32 μM, about 0.33 μM, about 0.34 μM, about 0.35 μM, about 0.4 μM, about 0.45 μM, about 0.5 μM, about 0.6 μM, about 0.8 μM, about 1 μM, about 2 μM, or about 5 μM. In some examples, the method includes contacting PDX1-positive pancreatic progenitor cells with a Notch signaling inhibitor (e.g., XXI) at a concentration of about 1.7-2.3 μM, about 1.8-2.2 μM, or about 1.9-2.1 μM. In some examples, the method includes contacting PDX1-positive pancreatic progenitor cells with a Notch signaling inhibitor (e.g., XXI) at a concentration of about 2 μM.

[0215] In some embodiments, the cells are further contacted with a water-soluble synthetic polymer. In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol. In some examples, the polyvinyl alcohol is at least 78% hydrolyzed, e.g., 79-81% hydrolyzed, 87-89% hydrolyzed, 87-90% hydrolyzed, or 99% hydrolyzed. In some embodiments, the polyvinyl alcohol (PVA) is 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% hydrolyzed. In some embodiments, the PVA is 80% hydrolyzed.

[0216] In some embodiments, PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting PDX1-positive pancreatic progenitor cells with KGF, Sant1, and RA for 5 or 6 days under conditions that promote cell clustering. In some embodiments, PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting PDX1-positive pancreatic progenitor cells with KGF, Sant1, RA, thiazovivin, and activin A for 5 or 6 days under conditions that promote cell clustering. In some embodiments, PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting PDX1-positive pancreatic progenitor cells with KGF for 5 days under conditions that promote cell clustering. In some embodiments, PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting PDX1-positive pancreatic progenitor cells with KGF for 6 days under conditions that promote cell clustering. In some embodiments, PDX1-positive, NKX6.1-positive pancreatic progenitor cells are obtained by: a) contacting PDX1-positive pancreatic progenitor cells with KGF, Sant1, RA, thiazovivin, and activin A for 3, 4, or 5 days (e.g., 4 days), followed by b) contacting the cells of a) with PDBU, XXI, KGF, Sant1, RA, thiazovivin, activin A, and optionally AS1842856 for 1, 2, or 3 days (e.g., 2 days).

[0217] Insulin-positive endocrine cells Embodiments of the present disclosure involve insulin-positive endocrine cells (e.g., NKX6.1-positive, ISL1-positive, or β-like cells), and additional methods for producing insulin-positive endocrine cells.The insulin-positive endocrine cells used herein can be derived from any source or produced according to any suitable protocol.In some embodiments, NKX6.1-positive pancreatic progenitor cells are differentiated into insulin-positive endocrine cells (e.g., NKX6.1-positive, ISL1-positive, or β-like cells).In some embodiments, insulin-positive endocrine cells are further differentiated, for example, by induction or maturation into SC-β cells.

[0218] In some aspects, a method for generating insulin-positive endocrine cells from NKX6.1-positive pancreatic progenitor cells includes contacting a cell population containing NKX6-1-positive pancreatic progenitor cells (e.g., under conditions that promote cell clustering) with: a) a TGF-β signaling pathway inhibitor; b) a thyroid hormone signaling pathway activator; c) a BMP pathway inhibitor; and / or d) a protein kinase inhibitor to induce differentiation of at least one NKX6.1-positive pancreatic progenitor cell in the population into an insulin-positive endocrine cell, wherein the insulin-positive endocrine cell expresses insulin. In some embodiments, the insulin-positive endocrine cell expresses PDX1, NKX6.1, ISL1, NKX2.2, Mafb, glis3, Sur1, Kir6.2, Znt8, SLC2A1, SLC2A3, and / or insulin.

[0219] Any TGF-β signaling pathway inhibitor that can induce differentiation of NKX6.1-positive pancreatic progenitor cells into insulin-positive endocrine cells (e.g., alone or in combination with other β cell differentiation factors, such as thyroid hormone signaling pathway activators) can be used. In some embodiments, the TGF-β signaling pathway comprises TGF-β type I receptor kinase signaling. In some embodiments, the TGF-β signaling pathway inhibitor comprises Alk5 inhibitor II. In some examples, the method includes administering NKX6.1 positive pancreatic progenitor cells at about 0.1 μM, about 0.5 μM, about 1 μM, about 1.5 μM, about 2 μM, about 2.5 μM, about 3 μM, about 3.5 μM, about 4 μM, about 4.5 μM, about 5 μM, about 5.5 μM, about 6 μM, about 6.5 μM, about 7 μM, about 7.5 μM, about 8 μM, about 8.5 μM, about 9 μM, about 9.5 μM, about 10 μM, about 10.5 μM, about 11 μM, about 11.5 μM, about 12 μM, about 12.5 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, about 18 μM, about 19 μM, about 20 μM, about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM, about 26 μM, about 27 μM, about 28 μM, about 29 μM, about 30 μM, about 31 μM, about 32 μM, about 33 μM, about 34 μM, about 35 μM, about 36 μM, about 37 μM, about 38 μM, about 39 μM, about 40 μM, about 41 μM, about 42 μM, about 43 μM, about 44 μM, about 45 μM, about 46 μM, about 47 μM, about 48 μM, about 49 μM, about 50 μM, about 51 μM, about 52 μM, about 53 μM, about 54 μM, about In some examples, the method includes contacting the NKX6.1-positive pancreatic progenitor cells with a TGF-β signaling pathway inhibitor (e.g., an Alk5 inhibitor such as Alk5 inhibitor II) at a concentration of about 7-13 μM, about 8-12 μM, or about 9-11 μM, such as about 13.5 μM, about 14 μM, about 14.5 μM, about 15 μM, about 15.5 μM, about 16 μM, about 16.5 μM, about 17 μM, about 17.5 μM, about 18 μM, about 18.5 μM, about 19 μM, about 19.5 μM, about 20 μM, about 25 μM, about 30 μM, about 35 μM, about 40 μM, about 45 μM, or about 50 μM. In some examples, the method includes contacting the NKX6.1-positive pancreatic progenitor cells with a TGF-β signaling pathway inhibitor (e.g., an Alk5 inhibitor such as Alk5 inhibitor II) at a concentration of about 7-13 μM, about 8-12 μM, or about 9-11 μM. In some examples, the method includes contacting the NKX6.1-positive pancreatic progenitor cells with a TGF-β signaling pathway inhibitor (e.g., an Alk5 inhibitor such as Alk5 inhibitor II) at a concentration of about 10 μM, or the like.

[0220] Any thyroid hormone signaling pathway activator that can induce differentiation of NKX6.1-positive pancreatic progenitor cells into insulin-positive endocrine cells (e.g., alone or in combination with other beta cell differentiation factors, such as TGF-β signaling pathway inhibitors) can be used. In some embodiments, the thyroid hormone signaling pathway activator comprises triiodothyronine (T3). In some embodiments, the thyroid hormone signaling pathway activator comprises GC-1. In some examples, the method comprises inducing NKX6.1-positive pancreatic progenitor cells with a thyroid hormone signaling pathway activator at about 0.1 μM, about 0.12 μM, about 0.13 μM, about 0.14 μM, about 0.15 μM, about 0.16 μM, about 0.17 μM, about 0.18 μM, about 0.19 μM, about 0.2 μM, about 0.21 μM, about 0.22 μM, about 0.23 μM, about 0.24 μM, about 0.25 μM, about 0.26 μM, about 0.27 μM, or about 0.30 μM. The method includes contacting the NKX6.1-positive pancreatic progenitor cells with a thyroid hormone signaling pathway activator (e.g., GC-1) at a concentration of about 0.28 μM, about 0.29 μM, about 0.3 μM, about 0.31 μM, about 0.32 μM, about 0.33 μM, about 0.34 μM, about 0.35 μM, about 0.4 μM, about 0.45 μM, about 0.5 μM, about 0.6 μM, about 0.8 μM, about 1 μM, about 2 μM, or about 5 μM, etc. In some examples, the method includes contacting the NKX6.1-positive pancreatic progenitor cells with a thyroid hormone signaling pathway activator (e.g., GC-1) at a concentration of about 0.7-1.3 μM, about 0.8-1.2 μM, or about 0.9-1.1 μM, etc. In some examples, the methods include contacting the NKX6.1-positive pancreatic progenitor cells with a thyroid hormone signaling pathway activator (eg, GC-1) at a concentration such as about 1 μM.

[0221] In some embodiments, the method comprises contacting a cell population (e.g., NKX6.1-positive pancreatic progenitor cells) with at least one additional factor. In some embodiments, the method comprises contacting PDX1-positive, NKX6.1-positive pancreatic progenitor cells with at least one of the following: i) an SHH pathway inhibitor, ii) a gamma-secretase inhibitor, iii) at least one growth factor from the epidermal growth factor (EGF) family, iv) a TGF-β signaling pathway inhibitor, or vii) a thyroid hormone signaling pathway activator. In some embodiments, the method comprises contacting a cell population (e.g., NKX6.1-positive pancreatic progenitor cells) with at least one additional factor. In some embodiments, the method comprises contacting PDX1-positive, NKX6.1-positive pancreatic progenitor cells with one of: i) an SHH pathway inhibitor, ii) an RA signaling pathway activator, iii) a gamma-secretase inhibitor, iv) at least one growth factor from the epidermal growth factor (EGF) family, v) a protein kinase inhibitor, vi) a TGF-β signaling pathway inhibitor, vii) a thyroid hormone signaling pathway activator, viiii) a wnt signaling pathway inhibitor, or ix) a PKC activator.

[0222] In some embodiments, the method includes 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 gamma-secretase inhibitor, iv) at least one growth factor from the epidermal growth factor (EGF) family, v) at least one bone morphogenetic protein (BMP) signaling pathway inhibitor, vi) a TGF-β signaling pathway inhibitor, vii) a thyroid hormone signaling pathway activator, viii) a protein kinase inhibitor, or ix) a ROCK inhibitor.

[0223] In some embodiments, the method includes 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 gamma-secretase inhibitor, iv) at least one growth factor from the epidermal growth factor (EGF) family, v) at least one bone morphogenetic protein (BMP) signaling pathway inhibitor, vi) a TGF-β signaling pathway inhibitor, vii) a thyroid hormone signaling pathway activator, viii) an epigenetic modifying compound, ix) a protein kinase inhibitor, or x) a ROCK inhibitor. In some embodiments, the method includes contacting PDX1-positive, NKX6.1-positive pancreatic progenitor cells in culture with 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, v) at least one bone morphogenetic protein (BMP) signaling pathway inhibitor, vi) a TGF-β signaling pathway inhibitor, vii) a thyroid hormone signaling pathway inhibitor, viii) an epigenetic modifying compound, ix) a protein kinase inhibitor, x) a ROCK inhibitor, xi) a PKC activator, and xii) a Wnt signaling pathway inhibitor for 1 day, 2 days, or 3 days (e.g., 1-2 days, 1-3 days, or 2-3 days), and then contacting the cells in culture with i) a γ-secretase inhibitor, v) at least one bone morphogenetic protein (BMP) signaling pathway inhibitor, vi) a TGF-β signaling pathway inhibitor, vii) a thyroid hormone signaling pathway inhibitor, viii) an epigenetic modifying compound, ix) a protein kinase inhibitor, x) a ROCK inhibitor, xi) a PKC activator, and xii) a Wnt signaling pathway inhibitor for 1 day, 2 days, or 3 days (e.g., 1-2 days, 1-3 days, or 2-3 days). the method comprises contacting the cells with a secretase inhibitor, ii) at least one growth factor from the epidermal growth factor (EGF) family, iii) at least one bone morphogenetic protein (BMP) signaling pathway inhibitor, iv) a TGF-β signaling pathway inhibitor, v) a thyroid hormone signaling pathway activator, vi) an epigenetic modifying compound, vii) a protein kinase inhibitor, and viii) a ROCK inhibitor for 1, 2, 3, 4, 5, 6, or 7 days (e.g., 1 to 7 days, 1 to 5 days, 1 to 3 days, 3 to 7 days, 3 to 5 days, 5 to 7 days, or 4 to 6 days) in the absence of an SHH pathway inhibitor, an RA signaling pathway activator, a Wnt signaling pathway inhibitor, a PKC activator, and / or an epidermal growth factor (EGF) family growth factor.

[0224] In some embodiments, in the method of generating insulin-positive endocrine cells from PDX1-positive, NKX6.1-positive pancreatic progenitor cells, some of the differentiation factors are present only for the first 1, 2, 3, 4, or 5 days during the differentiation step. In some embodiments, some of the differentiation factors, such as an SHH pathway inhibitor, an RA signaling pathway activator, a PKC activator, and at least one growth factor from the EGF family, are removed from the medium after the first 1, 2, or 3 days of culture.

[0225] Any gamma-secretase inhibitor that can induce the differentiation of NKX6.1-positive pancreatic progenitor cells in the population into insulin-positive endocrine cells (e.g., alone or in combination with a TGF-β signaling pathway inhibitor and / or a thyroid hormone signaling pathway activator) can be used. In some embodiments, the gamma-secretase inhibitor comprises XXI. In some embodiments, the gamma-secretase inhibitor comprises DAPT. In some examples, the method comprises administering to the patient a dose of NKX6.1-positive pancreatic progenitor cells at a concentration of, for example, about 0.01 μM, about 0.02 μM, about 0.05 μM, about 0.075 μM, about 0.1 μM, about 0.2 μM, about 0.3 μM, about 0.4 μM, about 0.5 μM, about 0.6 μM, about 0.7 μM, about 0.8 μM, about 0.9 μM, about 1 μM, about 1.1 μM, about 1.2 μM, about 1.3 μM, about 1.4 μM, about 1.5 μM, about 1.6 μM, about 1.7 μM, about 1.8 μM, about 1.9 μM, about 2 μM, about 2.1 μM, about 2.2 μM, about 2.3 μM, about 2.4 μM, about 2.5 μM, about 2 ... The method includes contacting the antibody with a gamma secretase inhibitor (e.g., XXI) at a concentration of about 0.6 μM, about 2.7 μM, about 2.8 μM, about 2.9 μM, about 3 μM, about 3.2 μM, about 3.4 μM, about 3.6 μM, about 3.8 μM, about 4 μM, about 4.2 μM, about 4.4 μM, about 4.6 μM, about 4.8 μM, about 5 μM, about 5.2 μM, about 5.4 μM, about 5.6 μM, about 5.8 μM, about 6 μM, about 6.2 μM, about 6.4 μM, about 6.6 μM, about 6.8 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 20 μM, about 30 μM, or about 50 μM. In some examples, the method includes contacting the NKX6.1-positive pancreatic progenitor cells with a gamma-secretase inhibitor (e.g., XXI) at a concentration of about 1.7-2.3 μM, about 1.8-2.2 μM, or about 1.9-2.1 μM, etc. In some examples, the method includes contacting the NKX6.1-positive pancreatic progenitor cells with a gamma-secretase inhibitor (e.g., XXI) at a concentration of about 2 μM, etc.

[0226] Any growth factor from the EGF family that can induce differentiation of NKX6.1-positive pancreatic progenitor cells in the population into insulin-positive endocrine cells (e.g., alone or in combination with any TGF-β signaling pathway inhibitor and / or thyroid hormone signaling pathway activator) can be used. In some embodiments, at least one growth factor from the EGF family comprises betacellulin. In some embodiments, at least one growth factor from the EGF family comprises EGF. In some examples, the method includes contacting the NKX6.1-positive pancreatic progenitor cells with a growth factor from the EGF family (e.g., betacellulin) at a concentration of, for example, about 1 ng / mL, about 2 ng / mL, about 4 ng / mL, about 6 ng / mL, about 8 ng / mL, about 10 ng / mL, about 12 ng / mL, about 14 ng / mL, about 16 ng / mL, about 18 ng / mL, about 20 ng / mL, about 22 ng / mL, about 24 ng / mL, about 26 ng / mL, about 28 ng / mL, about 30 ng / mL, about 40 ng / mL, about 50 ng / mL, about 75 ng / mL, about 80 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL, about 150 ng / mL, about 200 ng / mL, about 250 ng / mL, or about 300 ng / mL. In some examples, the method includes contacting the NKX6.1-positive pancreatic progenitor cells with an EGF family growth factor (e.g., betacellulin) at a concentration of about 17-23 ng / ml, about 18-22 ng / ml, or about 19-21 ng / ml, etc. In some examples, the method includes contacting the NKX6.1-positive pancreatic progenitor cells with an EGF family growth factor (e.g., betacellulin) at a concentration of about 20 ng / ml, etc.

[0227] Any RA signaling pathway activator that can induce the differentiation of NKX6.1-positive pancreatic progenitor cells into insulin-positive endocrine cells (e.g., alone or in combination with any TGF-β signaling pathway inhibitor and / or thyroid hormone signaling pathway activator) can be used. In some embodiments, the RA signaling pathway activator comprises RA. In some examples, the methods include administering NKX6.1 positive pancreatic progenitor cells to a patient at a concentration of, for example, about 0.02 μM, about 0.05 μM, about 0.1 μM, about 0.2 μM, about 0.25 μM, about 0.3 μM, about 0.4 μM, about 0.45 μM, about 0.5 μM, about 0.55 μM, about 0.6 μM, about 0.65 μM, about 0.7 μM, about 0.75 μM, about 0.8 μM, about 0.85 μM, about 0.9 μM, about 1 μM, about 1.1 μM, about 1.2 μM, about 1.3 μM, about 1.4 μM, about 1.5 μM, about 1.6 μM, about 1.7 μM, about 1.8 μM, about 1.9 μM, about 2 μM, about 2.1 μM, about 2.2 μM, about 2.3 μM, about 2.4 μM, The method includes contacting the subject with a RA signaling pathway activator (e.g., retinoic acid) at a concentration of about 1 μM, about 2.5 μM, about 2.6 μM, about 2.7 μM, about 2.8 μM, about 3 μM, about 3.2 μM, about 3.4 μM, about 3.6 μM, about 3.8 μM, about 4 μM, about 4.2 μM, about 4.4 μM, about 4.6 μM, about 4.8 μM, about 5 μM, about 5.5 μM, about 6 μM, about 6.5 μM, about 7 μM, about 7.5 μM, about 8 μM, about 8.5 μM, about 9 μM, about 9.5 μM, about 10 μM, about 12 μM, about 14 μM, about 15 μM, about 16 μM, about 18 μM, about 20 μM, about 50 μM, or about 100 μM. In some examples, the method includes contacting the NKX6.1-positive pancreatic progenitor cells with an RA signaling pathway activator (e.g., retinoic acid) at a concentration of about 20-80 nM, about 30-70 nM, or about 40-60 nM, etc. In some examples, the method includes contacting the NKX6.1-positive pancreatic progenitor cells with an RA signaling pathway activator (e.g., retinoic acid) at a concentration of about 50 nM, etc.

[0228] Any SHH pathway inhibitor that can induce differentiation of NKX6.1-positive pancreatic progenitor cells into insulin-positive endocrine cells (e.g., alone or in combination with any TGF-β signaling pathway inhibitor and / or thyroid hormone signaling pathway activator) can be used in the methods provided herein. In some embodiments, the SHH pathway inhibitor comprises Sant1. In some examples, the method comprises administering to the patient NKX6.1-positive pancreatic progenitor cells a concentration of, for example, about 0.001 μM, about 0.002 μM, about 0.005 μM, about 0.01 μM, about 0.02 μM, about 0.03 μM, about 0.05 μM, about 0.08 μM, about 0.1 μM, about 0.12 μM, about 0.13 μM, about 0.14 μM, about 0.15 μM, about 0.16 μM, about 0.17 μM, about 0.18 μM, about 0.19 μM, about 0.2 μM, about 0.21 μM, about 0.22 μM, The method includes contacting the cells with an SHH pathway inhibitor (e.g., Sant1) at a concentration of about 0.23 μM, about 0.24 μM, about 0.25 μM, about 0.26 μM, about 0.27 μM, about 0.28 μM, about 0.29 μM, about 0.3 μM, about 0.31 μM, about 0.32 μM, about 0.33 μM, about 0.34 μM, about 0.35 μM, about 0.4 μM, about 0.45 μM, about 0.5 μM, about 0.6 μM, about 0.8 μM, about 1 μM, about 2 μM, or about 5 μM. In some examples, the method includes contacting NKX6.1-positive pancreatic progenitor cells with an SHH pathway inhibitor (e.g., Sant1) at a concentration of about 220-280 nM, about 230-270 nM, about 240-260 nM, or about 245-255 nM, etc. In some examples, the method includes contacting NKX6.1-positive pancreatic progenitor cells with an SHH pathway inhibitor (e.g., Sant1) at a concentration of about 250 nM, etc.

[0229] Any BMP signaling pathway inhibitor that can induce the differentiation of NKX6.1-positive pancreatic progenitor cells into insulin-positive endocrine cells (e.g., alone or in combination with any TGF-β signaling pathway inhibitor and / or thyroid hormone signaling pathway activator) can be used. In some embodiments, the BMP signaling pathway inhibitor comprises LDN193189 or DMH-1. In some examples, the method includes contacting NKX6.1-positive pancreatic progenitor cells with a BMP signaling pathway inhibitor (e.g., LDN1931189) at a concentration of, for example, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, about 100 nM, about 110 nM, about 120 nM, about 130 nM, about 140 nM, about 150 nM, about 160 nM, about 170 nM, about 180 nM, about 190 nM, about 200 nM, about 210 nM, about 220 nM, about 230 nM, about 240 nM, about 250 nM, about 280 nM, about 300 nM, about 400 nM, about 500 nM, or about 1 μM. In some examples, the method includes contacting the NKX6.1-positive pancreatic progenitor cells with a BMP signaling pathway inhibitor (e.g., LDN1931189) at a concentration of about 70-130 nM, about 80-120 nM, about 90-110 nM, etc. In some examples, the method includes contacting the NKX6.1-positive pancreatic progenitor cells with a BMP signaling pathway inhibitor (e.g., LDN1931189) at a concentration of about 100 nM, etc.

[0230] Any ROCK inhibitor that can induce the differentiation of NKX6.1-positive pancreatic progenitor cells in the population into insulin-positive endocrine cells (e.g., alone or in combination with any TGF-β signaling pathway inhibitor and / or thyroid hormone signaling pathway activator) can be used. In some embodiments, the ROCK inhibitor comprises thiazovivin, Y-27632, Fasudil / HA1077, or H-1152. In some embodiments, the ROCK inhibitor comprises Y-27632. In some embodiments, the ROCK inhibitor comprises thiazovivin. In some examples, the method comprises administering to the patient a dose of PDX1-positive, NKX6.1-positive pancreatic progenitor cells at a concentration of, for example, about 0.2 μM, about 0.5 μM, about 0.75 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 7.5 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, or about 18 μM. The method includes contacting the PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a ROCK inhibitor (e.g., Y-27632 or thiazovivin) at a concentration of about 2.2-2.8 μM, about 2.3-2.7 μM, or about 2.4-2.6 μM. In some embodiments, the ROCK inhibitor comprises thiazovivin. In some examples, the method includes contacting the PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a ROCK inhibitor (e.g., Y-27632 or thiazovivin) at a concentration of about 2.2-2.8 μM, about 2.3-2.7 μM, or about 2.4-2.6 μM. In some embodiments, the ROCK inhibitor comprises thiazovivin. In some examples, the method includes contacting PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a ROCK inhibitor (e.g., Y-27632 or thiazovivin) at a concentration such as about 2.5 μM.

[0231] Any epigenetic modification compound that can induce the differentiation of NKX6.1-positive pancreatic progenitor cells in the population into insulin-positive endocrine cells (e.g., alone or in combination with any TGF-β signaling pathway inhibitor and / or thyroid hormone signaling pathway activator) can be used. In some embodiments, the epigenetic modification compound comprises a histone methyltransferase inhibitor or an HDAC inhibitor. In some embodiments, the epigenetic modification compound comprises a histone methyltransferase inhibitor, such as DZNep. In some embodiments, the epigenetic modification compound comprises an HDAC inhibitor, such as KD5170. In some examples, the method includes contacting PDX1-positive, NKX6.1-positive pancreatic progenitor cells with an epigenetic modifying compound (e.g., DZNep or KD5170) at a concentration of, for example, about 0.01 μM, about 0.025 μM, about 0.05 μM, about 0.075 μM, about 0.1 μM, about 0.15 μM, about 0.2 μM, about 0.5 μM, about 0.75 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 7.5 μM, about 8 μM, about 9 μM, about 10 μM, about 15 μM, about 20 μM, about 25 μM, about 30 μM, about 35 μM, about 40 μM, about 50 μM, or about 100 μM. In some examples, the method includes contacting PDX1-positive, NKX6.1-positive pancreatic progenitor cells with an epigenetic modifying compound (e.g., DZNep or KD5170) at a concentration of about 70-130 nM, about 80-120 nM, or about 90-110 nM, etc. In some examples, the method includes contacting PDX1-positive, NKX6.1-positive pancreatic progenitor cells with an epigenetic modifying compound (e.g., DZNep or KD5170) at a concentration of about 100 nM, etc.

[0232] Any Wnt signaling pathway inhibitor that can induce differentiation of NKX6.1-positive pancreatic progenitor cells in the population into insulin-positive endocrine cells (e.g., alone or in combination with a TGF-β signaling pathway inhibitor and / or a thyroid hormone signaling pathway activator) can be used. In some embodiments, the Wnt signaling pathway inhibitor comprises a tankyrase inhibitor. In some embodiments, the tankyrase inhibitor is NVP-TNKS656. In some examples, the method includes contacting PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a Wnt signaling pathway inhibitor (e.g., a tankyrase inhibitor such as NVP-TNKS656) at a concentration of about 0.1 μM, about 0.15 μM, about 0.2 μM, about 0.25 μM, about 0.3 μM, about 0.35 μM, about 0.4 μM, about 0.45 μM, about 0.5 μM, about 0.55 μM, about 0.6 μM, about 0.65 μM, about 0.7 μM, about 0.75 μM, about 0.8 μM, about 0.85 μM, about 0.9 μM, about 0.95 μM, about 1 μM, about 1.5 μM, about 2 μM, about 2.5 μM, about 3 μM, about 3.5 μM, about 4 μM, about 4.5 μM, or about 5 μM. In some examples, the method includes contacting PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a Wnt signaling pathway inhibitor (e.g., a tankyrase inhibitor such as NVP-TNKS656) at a concentration of about 1.7-2.3 μM, about 1.8-2.2 μM, or about 1.9-2.1 μM, etc. In some examples, the method includes contacting PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a Wnt signaling pathway inhibitor (e.g., a tankyrase inhibitor such as NVP-TNKS656) at a concentration of about 2 μM, etc.

[0233] Any PKC activator that can induce differentiation of NKX6.1-positive pancreatic progenitor cells in the population into insulin-positive endocrine cells (e.g., alone or in combination with either a TGF-β signaling pathway inhibitor and / or a thyroid hormone signaling pathway activator) can be used. In some embodiments, the PKC activator is TPB or PDBU. In some examples, the method involves inducing differentiation of PDX1-positive, NKX6.1-positive pancreatic progenitor cells at about 0.01 μM, about 0.025 μM, about 0.05 μM, about 0.075 μM, about 0.1 μM, about 0.15 μM, about 0.2 μM, about 0.25 μM, about 0.3 μM, about 0.35 μM, about 0.4 μM, about 0.45 μM, about 0.5 μM, about 0.55 μM, about 0.6 μM, about 0.65 μM, about 0.75 μM, about 0.8 μM, about 0.95 ... The method includes contacting PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a PKC activator (TPB or PDBU) at a concentration of about 450-550 nM, about 475-525 nM, about 490-510 nM, or about 495-505 nM, for example. In some examples, the methods include contacting PDX1-positive, NKX6.1-positive pancreatic progenitor cells with a PKC activator (TPB or PDBU) at a concentration such as about 500 nM.

[0234] In some embodiments, the cell population is optionally contacted with a protein kinase inhibitor. In some embodiments, the cell population is not contacted with a protein kinase inhibitor. In some embodiments, the cell population is contacted with a protein kinase inhibitor. Any protein kinase inhibitor that can induce the differentiation of NKX6.1-positive pancreatic progenitor cells in the population into insulin-positive endocrine cells (e.g., alone or in combination with any TGF-β signaling pathway inhibitor and / or thyroid hormone signaling pathway activator) can be used. In some embodiments, the protein kinase inhibitor comprises staurosporine. In some examples, the method comprises administering to the patient NKX6.1 positive pancreatic progenitor cells at about 0.1 nM, about 0.2 nM, about 0.3 nM, about 0.4 nM, about 0.5 nM, about 0.6 nM, about 0.7 nM, about 0.8 nM, about 0.9 nM, about 1 nM, about 1.1 nM, about 1.2 nM, about 1.3 nM, about 1.4 nM, about 1.5 nM, about 1.6 nM, about 1.7 nM, about 1.8 nM, about 1.9 nM, about 2.0 nM, about 2.1 nM, about 2.2 nM, about 2.3 nM, about 2.4 nM, about 2.5 nM, about 2.6 nM, about The method comprises contacting the antibody with a protein kinase inhibitor (e.g., staurosporine) at a concentration of about 2.7 nM, about 2.8 μM, about 2.9 nM, about 3 nM, about 3.1 nM, about 3.2 nM, about 3.3 nM, about 3.4 nM, about 3.5 nM, about 3.6 nM, about 3.7 nM, about 3.8 nM, about 3.9 nM, about 4.0 nM, about 4.1 nM, about 4.2 nM, about 4.3 nM, about 4.4 nM, about 4.5 nM, about 4.6 nM, about 4.7 nM, about 4.8 μM, about 4.9 nM, or about 5 nM. In some examples, the method includes contacting the NKX6.1-positive pancreatic progenitor cells with a protein kinase inhibitor (e.g., staurosporine) at a concentration of about 1-5 nM, about 2-4 nM, or about 2.5-3.5 nM, etc. In some examples, the method includes contacting the NKX6.1-positive pancreatic progenitor cells with a protein kinase inhibitor (e.g., staurosporine) at a concentration of about 3 nM, etc.

[0235] In some embodiments, the cells are further contacted with a water-soluble synthetic polymer. In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol. In some instances, the polyvinyl alcohol is at least 78% hydrolyzed, e.g., 79-81% hydrolyzed, 87-89% hydrolyzed, 87-90% hydrolyzed, or 99% hydrolyzed. In some embodiments, the polyvinyl alcohol (PVA) is 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% hydrolyzed. In some embodiments, the PVA is 89% hydrolyzed.

[0236] In some embodiments, the method includes contacting a population of cells (e.g., NKX6.1-positive pancreatic progenitor cells) with XXI, Alk5i, T3 or GC-1, RA, Sant1, and betacellulin, PDBU, and NVP-TNKS656 for 7 days to induce differentiation of at least one NKX6.1-positive pancreatic progenitor cell in the population into an insulin-positive endocrine cell, wherein the insulin-positive endocrine cell expresses insulin. In some embodiments, the method includes contacting a population of cells (e.g., NKX6.1-positive pancreatic progenitor cells) with XXI, Alk5i, T3 or GC-1, RA, Sant1, betacellulin, and LDN193189 for 7 days to induce differentiation of at least one NKX6.1-positive pancreatic progenitor cell in the population into an insulin-positive endocrine cell, wherein the insulin-positive endocrine cell expresses insulin. In some embodiments, one or more differentiation factors are added as part of Stage 5, e.g., only for the first 1, 2, 3, 4, 5, or 6 days of Stage 5, or only for the last 1, 2, 3, 4, 5, or 6 days of Stage 5. In one example, cells are contacted with an SHH signaling pathway inhibitor, a PKC activator, retinoic acid, and / or a wnt signaling pathway inhibitor for only the first 2, 3, 4, or 5 days of Stage 5, after which the SHH signaling pathway inhibitor, PKC activator, retinoic acid, and / or wnt signaling pathway inhibitor is not included in the culture medium or is removed from the medium. In another example, cells are contacted with a BMP signaling pathway inhibitor for only the first 1, 2, or 3 days of Stage 5, after which the BMP signaling pathway inhibitor is removed from the medium.

[0237] In some embodiments, the method includes contacting a cell population (e.g., NKX6.1-positive pancreatic progenitor cells) with one or more metabolites. In some embodiments, the method includes contacting a cell population (e.g., NKX6.1-positive pancreatic progenitor cells) with one or more of acetyl-CoA-related metabolites, vitamins, histone deacetylase inhibitors (HDACi), redox homeostasis regulators, intermediates of one-carbon metabolic pathways, and / or glutamine. Examples of metabolites include glutamine, taurine, acetate, beta-hydroxybutyrate, biotin, and formate.

[0238] In some embodiments, the composition (e.g., medium) of the present disclosure comprises an acetyl-CoA-related metabolite. Exemplary acetyl-CoA-related metabolites include, but are not limited to, acetate, pyruvate, ketonic amino acids, valine, leucine, isoleucine, phenylalanine, tyrosine, lysine, tryptophan, fatty acids, CoA, isovaleryl-CoA, and β-hydroxybutyrate. In some embodiments, the acetyl-CoA-related metabolite is acetate. In some embodiments, an acetyl-CoA-related metabolite is present in or added to a composition of the present disclosure at a concentration of about 10 nM, about 50 nM, about 80 nM, about 100 nM, about 120 nM, about 140 nM, about 150 nM, about 200 nM, about 300 nM, about 500 nM, about 800 nM, about 1 μM, about 10 μM, about 100 μM, about 500 μM, about 800 μM, about 900 μM, about 1 mM, about 2 mM, about 3 mM, about 5 mM, or about 10 mM. In some embodiments, the acetyl-CoA-related metabolite is present in or added to a composition of the present disclosure at a concentration of about 0.01-50 mM, 0.1-50 mM, 0.5-50 mM, 0.01-20 mM, 0.1-20 mM, 0.5-20 mM, 0.01-10 mM, 0.1-10 mM, 0.5-10 mM, 0.8-25 mM, 0.8-10 mM, 0.8-5 mM, 0.8-2 mM, 0.8-1.5 mM, 0.8-1.2 mM, 0.9-1.1 mM, or 0.95-1.05 mM. In some embodiments, the acetyl-CoA-related metabolite is acetate present at a concentration of about 1 mM. In some embodiments, the acetyl-CoA-related metabolite is acetate present at a concentration of about 50-1000 nM, 50-800 nM, 50-500 nM, 50-300 nM, 50-250 nM, 100-200 nM, or 125-175 nM, hi some embodiments, the acetyl-CoA-related metabolite is acetate present at a concentration of about 160 nM.

[0239] In some embodiments, the compositions (e.g., media) of the present disclosure comprise one or more vitamins. Exemplary vitamins include, but are not limited to, biotin, vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B6 (pyridoxine), and vitamin B12 (cyanocobalamin). In some embodiments, the vitamin regulates fatty acid synthesis. In some embodiments, the vitamin regulates branched-chain amino acid metabolism. In some embodiments, the vitamin regulates or participates as a cofactor in the TCA cycle, for example, as a cofactor for pyruvate carboxylase. In some embodiments, the vitamin is biotin. In some embodiments, the vitamin is present in or added to a composition of the present disclosure at a concentration of about 100 nM, about 300 nM, about 500 nM, about 600 nM, about 700 nM, about 800 nM, about 900 nM, about 1 μM, about 1.5 μM, about 3 μM, about 5 μM, about 10 μM, or about 100 μM. In some embodiments, the vitamin is biotin present at a concentration of about 800 nM. In some embodiments, the vitamin is from about 1 nM to about 500 μM, from about 1 nM to about 100 μM, from 1 nM to 10 μM, from 1 nM to 1 μM, from 1 nM to 800 nM, from 1 nM to 600 nM, from 1 nM to 400 nM, from 1 nM to 300 nM, from 1 nM to 200 nM, from 25 nM to 500 μM, from 25 nM to 100 μM, from 25 nM to 1 μM, from 25 nM to 800 nM, from 25 nM to 600 nM, from 25 nM to 400 nM, from 25 nM to 300 nM, from 25 nM to 200 nM, from 50 nM to 500 μM, from 50 nM to 10

[0033] The hydroxybenzoates may be present in or added to compositions of the present disclosure at a concentration of 0 μM, 50 nM to 10 μM, 50 nM to 1 μM, 50 nM to 800 nM, 50 nM to 600 nM, 50 nM to 400 nM, 50 nM to 300 nM, 50 nM to 200 nM, 100 nM to 500 μM, 100 nM to 100 μM, 100 nM to 10 μM, 100 nM to 1 μM, 100 nM to 800 nM, 100 nM to 600 nM, 100 nM to 400 nM, about 100 nM to 300 nM, or 100 nM to 200 nM.

[0240] In some embodiments, the compositions (e.g., media) of the present disclosure comprise a histone deacetylase inhibitor (HDACi). Exemplary histone deacetylase inhibitors (HDACi) include, but are not limited to, beta-hydroxybutyrate, butyrate, Class I HDACi, Class IIA HDACi, Class IIB HDACi, Class III HDACi, Class IV ... HDACi, HDAC-1, HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC-6, HDAC-7, HDAC-8, HDAC-9, HDAC-10, HDAC-11, sirtuin, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, vorinostat (suberoylanilide hydroxamic acid, SAHA, MK0683), entinostat (MS-275, SNDX-275), panobinostat (LBH589, NVP-LBH589), trichostatin A (TSA), mocetinostat (MGCD0103, MG0103), GSK3117391 (GSK3117391A, HDAC-IN-3), BRD3308, tubastatin A TFA (Tubastatin A trifluoroacetate), Tubastatin A, SIS17, NKL22, BML-210 (CAY10433), TC-H 106, SR-4370, Belinostat (PXD101, NSC726630, PX-105684), Romidepsin (FK228, Depsipeptide, FR901228, NSC630176), MC1568, Givinostat (ITF2357), Dacinostat (LAQ824, NVP-LAQ824), CUDC-101, Xynostat (JNJ-26481585), Prasinostat (SB939), PCI-34051, Droxinostat (NS41080), Abexinostat (PCI-24781), abexinostat (PCI-24781, CRA-024781), RGFP966, AR-42 (HDAC-42), ricolinostat (ACY-1215, rosirinostat), valproic acid sodium salt (sodium valproate), tacedinaline (CI994, PD-123654, GOE-5549, acetyldinaline), fimepinostat (CUDC-907), sodium butyrate (NaB), curcumin, diferuloylmethane, M344, tubacin,RG2833 (RGFP109), resminostat (RAS2410), divalproex sodium, scriptaid (GCK1026), sodium phenylbutyrate, sinapininc acid (sinapic acid), TMP269, santacruzamate A (CAY10683), TMP195 (TFMO2), valproic acid (VPA), UF010, tasquinimod (ABR-215050), SKLB-23bb, isoguanosine, ruforaphane, BRD73954, citalinostat (ACY-241, HDAC-IN-2), suberohydroxamic acid, prithomycin, HPOB, LMK-235, biphenyl-4-sulfonylmethane In some embodiments, the HDACi is β-hydroxybutyrate. In some embodiments, the HDACi is present in or added to the composition of the present disclosure at a concentration of about 100nM, about 300nM, about 500nM, about 600nM, about 700nM, about 800nM, about 900nM, about 1μM, about 1.5μM, about 3μM, about 5μM, about 10μM or about 100μM.In some embodiments, the HDACi is β-hydroxybutyrate and is present at a concentration of about 200nM. In some embodiments, the HDACi is 1 nM to 500 μM, 1 nM to 100 μM, 1 nM to 10 μM, 1 nM to 1 μM, 1 nM to 800 nM, 1 nM to 600 nM, 1 nM to 400 nM, 1 nM to 300 nM, 1 nM to 200 nM, 25 nM to 500 μM, 25 nM to 100 μM, 25 nM to 10 μM, 2 5nM~1μM, 25nM~800nM, 25nM~600nM, 25nM~400nM, 25nM~300nM, 25nM~200nM, 50nM~ 500μM, 50nM~100μM, 50nM~10μM, 50nM~1μM, 50nM~800nM, 50nM~600nM, 50nM~400nM,It is present in or added to the composition at a concentration of 50 nM to 300 nM, 50 nM to 200 nM, 100 nM to 500 μM, 100 nM to 100 μM, 100 nM to 10 μM, 100 nM to 1 μM, 100 nM to 800 nM, 100 nM to 600 nM, 100 nM to 400 nM, 100 nM to 300 nM, or 100 nM to 200 nM.

[0241] In some embodiments, the composition (e.g., medium) of the present disclosure comprises a redox homeostasis regulator. Exemplary redox homeostasis regulators include taurine, respiratory chain regulators, free radical scavengers, mitochondrial protein synthesis regulators, allium sulfur compounds, anthocyanins, beta-carotene, catechins, copper, cryptoxanthin, flavonoids, indoles, isoflavonoids, lignans, lutein, lycopene, alpha lipoic acid, ellagic acid, manganese, polyphenols, selenium, glutathione, vitamin A, vitamin C, vitamin E, zinc, superoxide dismutase, GSHPx, Prx-I, catalase, and coenzyme Q10. In some embodiments, the redox homeostasis regulator is taurine. In some embodiments, the redox homeostasis regulator is present in or added to the composition of the present disclosure at a concentration of about 100 nM, about 500 nM, 1 μM, about 10 μM, about 20 μM, about 30 μM, about 40 μM, about 50 μM, about 60 μM, about 70 μM, about 80 μM, about 90 μM, about 100 μM, about 110 μM, about 150 μM, or about 200 μM. In some embodiments, the redox homeostasis regulator is taurine. In some embodiments, the redox homeostasis regulator is taurine present at a concentration of about 90 μM. In some embodiments, the redox homeostasis regulator intermediate is between about 100 nM and 1 mM, 500 nM and 1 mM, 1 μM and 1 mM, 10 μM and 1 mM, 20 μM and 1 mM, 30 μM and 1 mM, 30 μM and 1 mM, 40 μM and 1 mM, 50 μM and 1 mM, 60 μM and 1 mM, 70 μM and 1 mM, 80 μM and 1 mM, 100 nM and 250 μM, 500 nM and 250 μM, 1 μM and 250 μM, 10 μM and 250 μM, 20 μM and 250 μM, 30 μM and 25 The compound may be present or added at a concentration of 0 μM, 30 μM to 250 μM, 40 μM to 250 μM, 50 μM to 250 μM, 60 μM to 250 μM, 70 μM to 250 μM, 100 nM to 100 μM, 500 nM to 100 μM, 1 μM to 100 μM, 10 μM to 100 μM, 20 μM...

Claims

1. 1. An in vitro composition comprising a plurality of non-native cells prepared in vitro, comprising: a) at least 30% of the cells in the composition are NKX6.1-positive, ISL1-positive cells; b) at least 25% of the cells in the composition are NKX6.1-negative, ISL1-positive cells; c) there are more NKX6.1-positive, ISL1-positive cells in the composition than NKX6.1-negative, ISL1-positive cells; and d) i) less than 6% of the cells in the composition are NKX6.1-negative, ISL1-negative cells, and / or ii) 9-25% of the cells in the composition are NKX6.1-positive, ISL1-negative cells; In vitro composition.

2. The in vitro composition of claim 1, wherein 30 to 60% of the cells among the plurality of cells are NKX6.1-positive, ISL1-positive cells, and 20 to 50% of the cells among the plurality of cells are NKX6.1-negative, ISL1-positive cells.

3. The in vitro composition of claim 1, wherein 35 to 55% of the cells among the plurality of cells are NKX6.1-positive, ISL1-positive cells, and 25 to 45% of the cells among the plurality of cells are NKX6.1-negative, ISL1-positive cells.

4. The in vitro composition according to any one of claims 1 to 3, wherein 1 to 6% of the cells in the plurality of cells are NKX6.1-negative, ISL1-negative cells.

5. The in vitro composition of claim 1, wherein 2 to 25% of the cells among the plurality of cells are NKX6.1-positive, ISL1-negative cells, and 1 to 6% of the cells among the plurality of cells are NKX6.1-negative, ISL1-negative cells.

6. The in vitro composition of claim 1 or 5, wherein 9 to 25% of the cells in the plurality of cells are NKX6.1-positive, ISL1-negative cells.

7. The in vitro composition of claim 1, wherein 35 to 55% of the cells among the plurality of cells are NKX6.1-positive, ISL1-positive cells, 25 to 45% of the cells among the plurality of cells are NKX6.1-negative, ISL1-positive cells, and 1 to 6% of the cells among the plurality of cells are NKX6.1-negative, ISL1-negative cells.

8. The in vitro composition of claim 1 , wherein the plurality of cells is in a cell cluster.

9. 4. The in vitro composition of claim 1, wherein the plurality of cells is in a plurality of cell clusters.

10. 9. The in vitro composition of claim 8, wherein the cell cluster comprises 500 to 2,500 cells.

11. 10. The in vitro composition of claim 9, wherein each of the plurality of cell clusters comprises 500 to 2,500 cells.

12. 9. The in vitro composition of claim 8, wherein the cell cluster comprises 500 to 2,500 NKX6.1-positive, ISL1-positive cells.

13. 10. The in vitro composition of claim 9, wherein each of the plurality of cell clusters comprises 500 to 2,500 NKX6.1-positive, ISL1-positive cells.

14. 9. The in vitro composition of claim 8, wherein the cell clusters are 80 to 270 microns in diameter.

15. 10. The in vitro composition of claim 9, wherein each of the plurality of cell clusters is between 80 and 270 microns in diameter.

16. 4. The in vitro composition of any one of claims 1 to 3, wherein the composition comprises cells genetically modified to reduce expression of any one or more of the following genes: B2M, CIITA, CXCL10, renalase, HLA-A, HLA-B, HLA-C, NLRC5, ABO, RHD, FUT1, KDM5D, PDGFRa, OLIG2, and / or GFAP compared to cells that are not genetically modified.

17. 4. The in vitro composition of any one of claims 1 to 3, wherein the composition comprises cells that have been genetically modified to increase expression of any one or more of CD47, PDL1, PDL2, HLA-E, HLA-G, CD46, CD55, CD59, and CTLA, compared to cells that are not genetically modified.

18. 4. The in vitro composition according to claim 1, further comprising a medium containing sugar at a concentration of about 0.05% to about 1.5%.

19. 4. The in vitro composition of claim 1, further comprising CMRL medium or HypoThermosol® FRS storage medium.

20. 4. The in vitro composition of claim 1, wherein the composition is in a device, the device comprising a semipermeable membrane configured to retain the cells within the device and to allow insulin secreted by the cells to pass out of the device.

21. 21. The in vitro composition of claim 20, wherein the semipermeable membrane can be made from poly(lactide) (PLA), poly(glycolic acid) (PGA), poly(lactide-co-glycolide) (PLGA), and other polyhydroxy acids, poly(caprolactone), polycarbonate, polyamide, polyanhydride, polyphosphazene, polyamino acids, polyorthoesters, polyacetal, polycyanoacrylate, biodegradable polyurethanes, albumin, collagen, fibrin, polyamino acids, prolamines, alginate, agarose, agarose including gelatin, dextran, polyacrylate, ethylene-vinyl acetate polymers and other acyl-substituted cellulose acetates and their derivatives, polyurethane, polystyrene, polyvinyl chloride, polyvinyl fluoride, poly(vinylimidazole), chlorosulfonated polyolefins, polyethylene oxide, or any combination thereof.

22. The in vitro composition according to any one of claims 1 to 3, wherein 65 to 85% of the cells express ISL1.

23. 4. The in vitro composition of claim 1, wherein the plurality of cells comprises 0.1 to 5% ghrelin-positive cells.

24. 4. The in vitro composition of claim 1, wherein the plurality of cells comprises 3% to 10% somatostatin-positive, glucagon-negative cells.

25. 4. The in vitro composition of claim 1, wherein the composition comprises NKX6.1-positive, ISL1-positive cells that exhibit a glucose-stimulated insulin secretion (GSIS) response equal to or greater than that of endogenous mature beta cells when tested in vitro.

26. 4. The in vitro composition of claim 1, comprising NKX6.1-positive, ISL1-positive cells that also express PC2, MNX1, or ABCC8.

27. 4. An in vitro composition according to any one of claims 1 to 3, wherein the C-peptide content per 1,000 in vitro differentiated cells is at least 300 pM, and the C-peptide content per 1,000 NKX6.1-positive, ISL1-positive cells may be at least 400 pM.

28. 4. An in vitro composition according to any one of claims 1 to 3, used to treat a subject with type 1 diabetes.

29. 30. A device comprising the in vitro composition of claim 28, the device comprising a semipermeable membrane configured to retain cells within the device and allow insulin secreted by the cells to pass out of the device.

30. 29. The in vitro composition of claim 28, prepared for application via the portal vein.

Images