Enhanced differentiation of beta cells

Abstract

To provide techniques usable in the large-scale manufacture of SC-islets for human therapeutic use.SOLUTION: Provided herein are methods of manufacturing β cells in vitro. Also provided herein are methods of treating a disease in a subject, comprising administering the β cells manufactured in vitro to the subject. Also provided herein are methods of differentiating stem cells into β cells.SELECTED DRAWING: Figure 1

Description

Related Applications

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 866,100, filed June 25, 2019, which is hereby incorporated by reference in its entirety into this specification. [Background technology]

[0002]

[0002] Pancreas or pancreatic islet transplantation has been used to treat diabetes, such as type 1 diabetes. Pancreatic islet transplantation does not require major surgery, and the function of the islet graft can be maintained in the recipient for a long period of time. However, a shortage of islet donors has prevented this therapy from being effectively implemented. Artificial pancreases or pancreatic islets offer an alternative source of transplantable pancreatic islets. Summary of the Invention

[0003] In some embodiments, the present disclosure provides a composition comprising dissociated cells. In some embodiments, the composition of the present disclosure does not include cell clusters. In some embodiments, the composition of the present disclosure does not include insulin-positive cell clusters. In some embodiments, the composition of the present disclosure does not include cell clusters comprising more than 5, 10, 20, 30, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 cells. In some embodiments, the composition of the present disclosure does not include cell clusters comprising more than 50 cells. In some embodiments, the composition of the present disclosure does not include cell clusters comprising more than 100 cells. In some embodiments, the composition of the present disclosure does not include cell clusters comprising more than 500 cells. In some embodiments, the present disclosure provides a composition comprising a plurality of dissociated insulin-positive endocrine precursor cells. In some embodiments, the dissociated cells are Ngn3-positive. In some embodiments, the dissociated cells are PDX.1-positive. In some embodiments, the dissociated cells are NKX6.1-positive. In some embodiments, the present disclosure provides a composition comprising dissociated cells (e.g., dissociated insulin-positive endocrine precursor cells) and a BMP signaling pathway inhibitor. In some embodiments, the BMP signaling pathway inhibitor is LDN193189 or a derivative thereof. In some embodiments, the present disclosure provides a composition comprising a plurality of dissociated cells (e.g., dissociated insulin-positive endocrine precursor cells) and a ROCK inhibitor. In some embodiments, the ROCK inhibitor is thiazovivin, Y-27632, fasudil / HA1077, or 14-1152, or a derivative thereof. In some embodiments, the present disclosure provides a composition comprising a plurality of dissociated cells (e.g., dissociated insulin-positive endocrine precursor cells) and a histone methyltransferase inhibitor. In some embodiments, the histone methyltransferase inhibitor is 3-deazaneplanocin A hydrochloride or a derivative thereof. In some embodiments, the present disclosure provides a composition comprising a plurality of dissociated cells (e.g., dissociated insulin-positive endocrine precursor cells) and zinc. In some embodiments, the zinc is in the form of ZnSO4.In some embodiments, the present disclosure provides a composition comprising a plurality of dissociated cells (e.g., dissociated insulin-positive endocrine precursor cells) and a monoglyceride lipase (MGLL) inhibitor. In some embodiments, the MGLL inhibitor is JJKK048, KML29, NF1819, JW642, JZL184, JZL195, JZP361, pristimerin, or URB602, or a derivative of any of the foregoing. In some embodiments, the present disclosure provides a composition comprising a plurality of dissociated cells (e.g., dissociated insulin-positive endocrine precursor cells) and a lipid. In some embodiments, the lipid is a saturated fatty acid. In some embodiments, the saturated fatty acid is palmitate. In some embodiments, the lipid is an unsaturated fatty acid. In some embodiments, the unsaturated fatty acid is oleic acid, linoleic acid, or palmitoleic acid ... The present disclosure provides a composition comprising a plurality of dissociated cells (e.g., dissociated insulin-positive endocrine precursor cells) and glutamate. In some embodiments, the present disclosure provides a composition comprising a plurality of dissociated cells (e.g., dissociated insulin-positive endocrine precursor cells) and acetate. In some embodiments, the present disclosure provides a composition comprising a plurality of dissociated cells (e.g., dissociated insulin-positive endocrine precursor cells) and β-hydroxybutarate. In some embodiments, the present disclosure provides a composition comprising a plurality of dissociated cells (e.g., dissociated insulin-positive endocrine precursor cells) and L-carnitine. In some embodiments, the present disclosure provides a composition comprising a plurality of dissociated cells (e.g., dissociated insulin-positive endocrine precursor cells) and taurine. In some embodiments, the present disclosure provides a composition comprising a plurality of dissociated cells (e.g., dissociated insulin-positive endocrine precursor cells) and formate. In some embodiments, the present disclosure provides a composition comprising a plurality of dissociated cells (e.g., dissociated insulin-positive endocrine precursor cells) and biotin. In some embodiments, the compositions of the present disclosure further comprise serum albumin protein. In some embodiments, the serum albumin protein is human serum albumin protein. In some embodiments, the compositions of the present disclosure further comprise a serum albumin concentration of 0.01% to 1%, 0.03 to 1%, 0.03 to 0.9%, 0.03 to 0.08%, 0.03 to 0.06%, 0.03 to 0.05%, 0.04 to 0.8%, 0.04 to 0.7%, 0.04 to 0.6%, 0.04 to 0.5%, 0.04 to 0.4%, 0.04 to 0.3%, 0.04 to 0.2%, 0.04 to 0.1%, 0.04 to 0.09%, 0.04 to 0.8%, 0.04 to 0. Contains 0.07%, 0.04-0.06%, 0.04-0.05%, 0.05-1%, 0.05-0.9%, 0.05-0.8%, 0.05-0.7%, 0.05-0.6%, 0.05-0.5%, 0.05-0.4%, 0.05-0.3%, 0.05-0.2%, 0.05-0.1%, 0.05-0.09%, 0.05-0.8%, 0.05-0.07%, or 0.05-0.06% serum albumin protein.In some embodiments, less than 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 1% of the cells in the composition are organized into cell clusters. In some embodiments, the compositions of the present disclosure include a TGF-β pathway inhibitor. In some embodiments, the TGF-β pathway inhibitor is Alk5i (SB505124) or a derivative thereof. In some embodiments, the compositions of the present disclosure include a thyroid hormone signaling pathway activator. In some embodiments, the thyroid hormone signaling pathway activator is GC-1 or T3, or a derivative thereof. In some embodiments, the compositions of the present disclosure include a protein kinase inhibitor. In some embodiments, the protein kinase inhibitor is staurosporine. In some embodiments, the compositions of the present disclosure include vitamin C. In some embodiments, the composition of the present disclosure comprises insulin. In certain embodiments, the composition of the present disclosure is in vitro. In some embodiments, the composition of the present disclosure does not comprise a gamma-secretase inhibitor (e.g., XXI). In some embodiments, the dissociated insulin-positive endocrine precursor cells have been previously frozen.

[0004] In some embodiments, the present disclosure provides a method comprising contacting a plurality of dissociated insulin-positive endocrine precursor cells with a BMP signaling pathway inhibitor. In some embodiments, the BMP signaling pathway inhibitor is LDN193189 or a derivative thereof. In some embodiments, the present disclosure provides a method comprising contacting a plurality of dissociated insulin-positive endocrine precursor cells with a ROCK inhibitor. In some embodiments, the ROCK inhibitor is thiazovivin, Y-27632, fasudil / HA1077, or 14-1152, or a derivative thereof. In some embodiments, the present disclosure provides a method comprising contacting a plurality of dissociated insulin-positive endocrine precursor cells with a histone methyltransferase inhibitor. In some embodiments, the histone methyltransferase inhibitor is 3-deazaneplanocin A hydrochloride or a derivative thereof. In some embodiments, the present disclosure provides a method comprising contacting a plurality of dissociated insulin-positive endocrine precursor cells with zinc and In some embodiments, the zinc is in the form of ZnSO. In some embodiments, the present disclosure provides a method comprising contacting a plurality of dissociated insulin-positive endocrine precursor cells with a monoglyceride lipase (MGLL) inhibitor. In some embodiments, the MGLL inhibitor is JJKK048, KML29, NF1819, JW642, JZL184, JZL195, JZP361, pristimerin, or URB602, or a derivative of any of the foregoing. In some embodiments, the present disclosure provides a method comprising contacting a plurality of dissociated insulin-positive endocrine precursor cells with a lipid. In some embodiments, the lipid is a saturated fatty acid. In some embodiments, the saturated fatty acid is palmitate. In some embodiments, the lipid is an unsaturated fatty acid. In some embodiments, the unsaturated fatty acid is oleic acid, linoleic acid, or palmitoleic acid. In some embodiments, the present disclosure provides a method comprising contacting a plurality of dissociated insulin-positive endocrine precursor cells with glutamate. In some embodiments, the present disclosure provides a method comprising contacting a plurality of dissociated insulin-positive endocrine precursor cells with acetate. In some embodiments, the present disclosure provides a method comprising contacting a plurality of dissociated insulin-positive endocrine precursor cells with β-hydroxybutarate. In some embodiments, the present disclosure provides a method comprising contacting a plurality of dissociated insulin-positive endocrine precursor cells with L-carnitine. In some embodiments, the present disclosure provides a method comprising contacting a plurality of dissociated insulin-positive endocrine precursor cells with taurine. In some embodiments, the present disclosure provides a method comprising contacting a plurality of dissociated insulin-positive endocrine precursor cells with formate. In some embodiments, the present disclosure provides a method comprising contacting a plurality of dissociated insulin-positive endocrine precursor cells with biotin. In some embodiments, the methods of the present disclosure comprise contacting the plurality of dissociated insulin-positive endocrine precursor cells with a serum albumin protein, hi some embodiments, the serum albumin protein is human serum albumin protein.In some embodiments, the compositions of the present disclosure may be administered in the presence of 0.01% to 1%, 0.03 to 1%, 0.03 to 0.9%, 0.03 to 0.08%, 0.03 to 0.06%, 0.03 to 0.05%, 0.04 to 0.8%, 0.04 to 0.7%, 0.04 to 0.6%, 0.04 to 0.5%, 0.04 to 0.4%, 0.04 to 0.3%, 0.04 to 0.2%, 0.04 to 0.1%, 0.04 to 0.09%, 0.04 to 0.8%, 0.04 to 0. Contains 0.07%, 0.04-0.06%, 0.04-0.05%, 0.05-1%, 0.05-0.9%, 0.05-0.8%, 0.05-0.7%, 0.05-0.6%, 0.05-0.5%, 0.05-0.4%, 0.05-0.3%, 0.05-0.2%, 0.05-0.1%, 0.05-0.09%, 0.05-0.8%, 0.05-0.07%, or 0.05-0.06% serum albumin protein. In some embodiments, less than 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 1% of the cells in the composition are organized into cell clusters. In some embodiments, the disclosed method includes contacting a plurality of dissociated insulin-positive endocrine precursor cells with a TGF-β pathway inhibitor. In some embodiments, the TGF-β pathway inhibitor is Alk5i (SB505124) or a derivative thereof. In some embodiments, the disclosed method includes contacting a plurality of dissociated insulin-positive endocrine precursor cells with a thyroid hormone signaling pathway activator. In some embodiments, the thyroid hormone signaling pathway activator is GC-1 or T3, or a derivative thereof. In some embodiments, the method of the present disclosure comprises contacting a plurality of dissociated insulin-positive endocrine precursor cells with a protein kinase inhibitor. In some embodiments, the protein kinase inhibitor is staurosporine. In some embodiments, the method of the present disclosure comprises contacting a plurality of dissociated insulin-positive endocrine precursor cells with vitamin C. In some embodiments, the method of the present disclosure comprises contacting a plurality of dissociated insulin-positive endocrine precursor cells with insulin.In some embodiments, the methods of the present disclosure include multiple. The method does not include contacting the dissociated insulin-positive endocrine precursor cells with a gamma-secretase inhibitor (e.g., XXI). In some embodiments, the dissociated insulin-positive endocrine precursor cells have been previously frozen. In some embodiments, the method of the present disclosure is carried out for 1-10 days, 1-9 days, 1-8 days, 1-7 days, 1-6 days, 1-5 days, 1-4 days, 1-3 days, 1-2 days, 2-10 days, 2-9 days, 2-8 days, 2-7 days, 2-6 days, 2-5 days, 2-4 days, 2-3 days, 3-10 days, 3-9 days, 3-8 days, 3-7 days, 3-6 days, 3-5 days, 3-4 days, 4-10 days, 4-9 days, 4-8 days, 4-7 days, 4-6 days, or 4-5 days. In some embodiments, the method of the present disclosure reaggregates the dissociated cells into multiple cell clusters. In some embodiments, at least about 40%, 50%, 60%, 70%, 80%, or 90% of the plurality of cell clusters have a diameter of about 50 μm to about 250 μm, about 75 μm to about 250 μm, or about 100 μm to about 200 μm. In some embodiments, at least about 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, or 99% of the cells of the plurality of cell clusters of the second cell population are viable. In some embodiments, the methods of the present disclosure reaggregate the dissociated cells into at least 2, 3, 4, 5, 10, 50, 100, 1,000, 10,000, 100,000, or 1,000,000 cell clusters.

[0005] In some embodiments, the present disclosure provides a composition comprising a plurality of cell clusters. In some embodiments, the present disclosure provides a composition comprising a plurality of cell clusters, the cell clusters comprising insulin-positive cells, and wherein at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, or at least 65% of the cells in the composition are viable after 11 days of in vitro culture. In some embodiments, the present disclosure provides a composition comprising a plurality of cell clusters, the cell clusters comprising insulin-positive cells, and wherein at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the cell clusters in the composition are 90-140 μm, 90-130 μm, 90-120 μm, 90-110 μm, 100-140 μm, 100-130 μm, 100-120 μm, or 100-110 μm in diameter. In some embodiments, the disclosure provides a composition comprising a plurality of cell clusters, wherein the cell clusters comprise insulin-positive cells, and wherein at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the cell clusters in the composition have an insulin-positive cell population of 1.5 to 4.5, 1. The compositions exhibit a glucose-stimulated insulin secretion (GSIS) stimulation index of 5 to 4.0, 1.5 to 3.5, 1.5 to 3.0, 1.5 to 2.5, 1.5 to 2.5, 1.5 to 2.0, 2.0 to 4.5, 2.0 to 4.0, 2.0 to 3.5, 2.0 to 3.0, 2.0 to 2.5, 2.5 to 4.5, 2.5 to 4.0, 2.5 to 3.5, 2.5 to 3.0, 3.0 to 4.5, 3.0 to 4.0, 3.0 to 3.5, 3.5 to 4.5, 3.5 to 4.0, or 4.0 to 4.5. In some embodiments, the cell clusters comprise C-peptide-positive cells. In some embodiments, the cell clusters comprise somatostatin-positive cells.In some embodiments, the cell clusters comprise glucagon-positive cells. In some embodiments, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, or at least 65% of the cells in the composition are glucagon-positive cells within 11 days. In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 100%, at least 15 ... In some embodiments, at least 95% of the cell clusters in the composition have a diameter of 90-140 μm, 90-130 μm, 90-120 μm, 90-110 μm, 100-140 μm, 100-130 μm, 100-120 μm, or 100-110 μm. In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the cell clusters in the composition have a diameter of 1.5-4.5, 1.5-4.0, 1.5-3.5, 1.5-3 The compositions exhibit a glucose-stimulated insulin secretion (GSIS) stimulation index of 0.0, 1.5-2.5, 1.5-2.5, 1.5-2.0, 2.0-4.5, 2.0-4.0, 2.0-3.5, 2.0-3.0, 2.0-2.5, 2.5-4.5, 2.5-4.0, 2.5-3.5, 2.5-3.0, 3.0-4.5, 3.0-4.0, 3.0-3.5, 3.5-4.5, 3.5-4.0, or 4.0-4.5. In some embodiments, there are at least 2, 3, 4, 5, 10, 50, 100, 1,000, 10,000, 100,000, or 1,000,000 cell clusters. In some embodiments, the compositions of the present disclosure are prepared according to any of the methods disclosed herein. In some embodiments, the present disclosure provides a device comprising any of the cell compositions disclosed herein. In some embodiments, the present disclosure provides a method of treating a subject having a disease characterized by prolonged high blood glucose levels (e.g., diabetes), the method comprising administering to the subject any of the compositions disclosed herein or any of the devices disclosed herein.

[0006]

[0006] In some embodiments, the present disclosure provides a composition comprising a plurality of cell clusters, wherein the cell clusters comprise insulin-positive cells, and wherein at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, or at least 65% of the cells in the composition are viable after 11 days of in vitro culture. In some embodiments, the present disclosure provides a composition comprising a plurality of cell clusters, the cell clusters comprising insulin-positive cells, and wherein at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the cell clusters in the composition are 90-140 μm, 90-130 μm, 90-120 μm, 90-110 μm, 100-140 μm, 100-130 μm, 100-120 μm, or 100-110 μm in diameter. In some embodiments, the disclosure provides a composition comprising a plurality of cell clusters, wherein the cell clusters comprise insulin-positive cells, and wherein at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the cell clusters in the composition have an insulin-positive cell population of 1.5 to 4.5, 1. The compositions exhibit a glucose-stimulated insulin secretion (GSIS) stimulation index of 5 to 4.0, 1.5 to 3.5, 1.5 to 3.0, 1.5 to 2.5, 1.5 to 2.5, 1.5 to 2.0, 2.0 to 4.5, 2.0 to 4.0, 2.0 to 3.5, 2.0 to 3.0, 2.0 to 2.5, 2.5 to 4.5, 2.5 to 4.0, 2.5 to 3.5, 2.5 to 3.0, 3.0 to 4.5, 3.0 to 4.0, 3.0 to 3.5, 3.5 to 4.5, 3.5 to 4.0, or 4.0 to 4.5. In some embodiments, the cell clusters comprise C-peptide-positive cells. In some embodiments, the cell clusters comprise somatostatin-positive cells. In some embodiments, the cell clusters comprise glucagon-positive cells.In some embodiments, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, or at least 65% of the cells in the composition are viable after 11 days of in vitro culture. In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 10% of the cell clusters in the composition are viable. At least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the cell clusters in the composition are 90-140 μm, 90-130 μm, 90-120 μm, 90-110 μm, 100-140 μm, 100-130 μm, 100-120 μm, or 100-110 μm in diameter. In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the cell clusters in the composition are 1.5-4.5, 1.5-4.0, 1.5-3.5, 1.5-3. The compositions exhibit a glucose-stimulated insulin secretion (GSIS) stimulation index of 0.0, 1.5-2.5, 1.5-2.5, 1.5-2.0, 2.0-4.5, 2.0-4.0, 2.0-3.5, 2.0-3.0, 2.0-2.5, 2.5-4.5, 2.5-4.0, 2.5-3.5, 2.5-3.0, 3.0-4.5, 3.0-4.0, 3.0-3.5, 3.5-4.5, 3.5-4.0, or 4.0-4.5. In some embodiments, there are at least 2, 3, 4, 5, 10, 50, 100, 1,000, 10,000, 100,000, or 1,000,000 cell clusters. In some embodiments, the compositions of the present disclosure are prepared according to any of the methods disclosed herein. In some embodiments, the present disclosure provides a device comprising any of the cell compositions disclosed herein. In some embodiments, the present disclosure provides a method of treating a subject having a disease characterized by prolonged high blood glucose levels (e.g., diabetes), the method comprising administering to the subject any of the compositions disclosed herein or any of the devices disclosed herein.

[0007]

[0007] The present specification provides a method for treating insulin-positive cells, comprising: (a) obtaining a first cell population comprising a plurality of cell clusters, each of which comprises insulin-positive cells; (b) dissociating in vitro at least a portion of the plurality of cell clusters in the first cell population; (c) contacting in vitro the first cell population comprising at least a portion of the dissociated cell clusters with a first composition to obtain a second cell population comprising a plurality of cell clusters, each of which comprises a plurality of insulin-positive cells, wherein the first composition comprises at least one agent selected from the group consisting of a monoglyceride lipase (MGLL) inhibitor, a bone morphogenetic protein (BMP) type 1 receptor inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a histone methyltransferase inhibitor, and a protein kinase inhibitor; and (d) contacting the second insulin-positive cell population with the second composition in vitro. and contacting in vitro at least a portion of the second insulin-positive cell population with a third cell population comprising a plurality of beta cells, wherein the second composition is different from the first composition, and the third cell population comprises a higher percentage of viable beta cells compared to a corresponding beta cell population comprising beta cells derived from the first cell population not contacted with the first composition.

[0008]

[0008] The present specification provides a method for treating endocrine cancer, comprising: (a) obtaining a first cell population comprising a plurality of cell clusters that include insulin-positive cells; (b) dissociating in vitro at least a portion of the plurality of cell clusters in the first cell population; (c) contacting in vitro the first cell population comprising at least a portion of the dissociated cell clusters with a first composition to obtain a second cell population comprising a plurality of cell clusters that include a plurality of insulin-positive endocrine cells, wherein the first composition comprises a transforming growth factor beta (TGF-β) signaling pathway inhibitor, a thyroid hormone signaling pathway activator, or both, and at least one agent selected from the group consisting of a monoglyceride lipase (MGLL) inhibitor, a bone morphogenetic protein (BMP) type 1 receptor inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a histone methyltransferase inhibitor, and a protein kinase inhibitor; and (d) contacting the second insulin-positive cell population with the second composition in vitro. contacting the second insulin-positive cell population in vitro, thereby causing at least a portion of the second insulin-positive cell population to differentiate into a third cell population comprising a plurality of β cells. wherein the second composition is different from the first composition and the third cell population comprises a higher percentage of viable beta cells compared to a corresponding beta cell population comprising beta cells derived from the first cell population not contacted with the first composition.

[0009]

[0009] The present specification provides a method for producing a cell population comprising: (a) obtaining a first cell population comprising a plurality of cell clusters that include insulin-positive cells; (b) dissociating in vitro at least a portion of the plurality of cell clusters in the first cell population; (c) contacting in vitro the first cell population comprising at least a portion of the dissociated cell clusters with a first composition to obtain a second cell population comprising a plurality of cell clusters that include a plurality of insulin-positive endocrine cells, wherein the first composition comprises a monoglyceride lipase (MGLL) inhibitor; and (d) contacting the second insulin-positive cell population with the second composition in vitro. and contacting in vitro at least a portion of the second insulin-positive cell population with a third cell population comprising a plurality of beta cells, wherein the second composition is different from the first composition, and the third cell population comprises a higher percentage of viable beta cells compared to a corresponding beta cell population comprising beta cells derived from the first cell population not contacted with the first composition.

[0010]

[0010] In some embodiments, the method of the present disclosure further includes the steps of freezing at least a portion of a first cell population comprising at least a portion of the dissociated cell clusters, thawing at least a portion of the frozen first cell population, and contacting at least a portion of the thawed first cell population with the first composition in vitro.

[0011] In some embodiments, at least a portion of the plurality of cell clusters of the second cell population have a diameter of about 50 μm to about 250 μm, about 75 μm to about 250 μm, or about 100 μm to about 200 μm. In some embodiments, at least about 40%, 50%, 60%, 70%, 80%, or 90% of the plurality of cell clusters of the second cell population have a diameter of about 50 μm to about 250 μm, about 75 μm to about 250 μm, or about 100 μm to about 200 μm. In some embodiments, at least about 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, or 99% of the cells of the second cell population are viable. In some embodiments, at least about 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, or 99% of the cells in the plurality of cell clusters of the second cell population are viable.

[0012]

[0012] In some embodiments, the second cell population comprises at least 2, 3, 4, 5, 10, 50, 100, 1000, 10000, 100,000, or 1,000,000 cell clusters.

[0013] In some embodiments, the second cell population comprises a higher percentage of insulin-positive endocrine cells compared to a corresponding cell population comprising insulin-positive endocrine cells not contacted with the first composition. In some embodiments, the second cell population comprises at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% more viable insulin-positive endocrine cells compared to a corresponding cell population comprising insulin-positive endocrine cells not contacted with the first composition. In some embodiments, the second cell population comprises at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% more viable insulin-positive endocrine cells compared to a corresponding cell population comprising insulin-positive endocrine cells not contacted with the first composition after about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days of contact between the first cell population and the first composition. In some embodiments, the second cell population is incubated with the first cell population and the first composition for about 1-10 days, 1-9 days, 1-8 days, 1-7 days, 1-6 days, 1-5 days, 1-4 days, or 2 days. After contact for 1-3 days, 1-2 days, 2-10 days, 2-9 days, 2-8 days, 2-7 days, 2-6 days, 2-5 days, 2-4 days, 2-3 days, 3-10 days, 3-9 days, 3-8 days, 3-7 days, 3-6 days, 3-5 days, 3-4 days, 4-10 days, 4-9 days, 4-8 days, 4-7 days, 4-6 days, or 4-5 days, the population contains at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% more viable insulin-positive endocrine cells compared to a corresponding cell population containing insulin-positive endocrine cells not contacted with the first composition.

[0014] In some embodiments, at least a portion of the plurality of beta cells form a plurality of cell clusters. In some embodiments, at least a portion of the plurality of cell clusters of the third cell population have a diameter of about 50 μm to about 250 μm, about 50 μm to about 150 μm, about 50 μm to about 100 μm, about 75 μm to about 250 μm, about 75 μm to about 150 μm, about 75 μm to about 125 μm, about 75 μm to about 100 μm, or about 100 μm to about 200 μm.

[0015] In some embodiments, at least about 40%, 50%, 60%, 70%, 80%, or 90% of the plurality of cell clusters of the third cell population have, without a selection step, a diameter of about 50 μm to about 250 μm, about 50 μm to about 150 μm, about 50 μm to about 100 μm, about 75 μm to about 250 μm, about 75 μm to about 150 μm, about 75 μm to about 125 μm, about 75 μm to about 100 μm, or about 100 μm to about 200 μm. In some embodiments, at least about 40%, 50%, 60%, 70%, 75%, 80%, 90%, or 95% of the cell clusters have, without a selection step, a diameter of about 50-150 μm, 75-12 μm, 80-120 μm, or 90-110 μm. In some embodiments, at least about 50%, 60%, 70%, 75%, 80%, 90%, or 95% of the cell clusters have a diameter of about 100 microns without a selection step. In some embodiments, at least about 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, or 99% of the cells of the third cell population are viable without a selection step. In some embodiments, at least about 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, or 99% of the cells of the plurality of cell clusters of the third cell population are viable without a selection step.

[0016] In some embodiments, the third cell population comprises at least 2, 3, 4, 5, 10, 50, 100, 1,000, 10,000, 100,000, or 1,000,000 cell clusters. In some embodiments, the third cell population comprises at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% more viable beta cells compared to a corresponding cell population comprising beta cells derived from the first cell population not contacted with the first composition. In some embodiments, the third cell population comprises at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% more viable beta cells after about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days of contact between the first cell population and the first composition, compared to a corresponding cell population comprising beta cells derived from the first cell population not contacted with the first composition. In some embodiments, the second cell population comprises at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% more viable beta cells after about 1-10 days, 1-9 days, 1-8 days, 1-7 days, 1-6 days, 1-5 days, 1-4 days, 1-3 days, 1-2 days, 2-10 days, 2-9 days, 2-8 days, 2-7 days, 2-6 days, 2-5 days, 2-4 days, 2-3 days, 3-10 days, 3-9 days, 3-8 days, 3-7 days, 3-6 days, 3-5 days, 3-4 days, 4-10 days, 4-9 days, 4-8 days, 4-7 days, 4-6 days, or 4-5 days of contact between the first cell population and the first composition, compared to a corresponding cell population comprising beta cells derived from the first cell population not contacted with the first composition.

[0017] In some embodiments, at least a portion of the plurality of beta cells of the third cell population are in The beta cells exhibit glucose-stimulated insulin secretion (GSIS) in response to a glucose load in vitro. In some embodiments, at least a portion of the plurality of beta cells of the third cell population express insulin.

[0018] In some embodiments, the first composition impairs two, three, four, or five of the drugs. In some embodiments, the first composition impairs three, four, five, six, or seven of the drugs. In some embodiments, the contacting step with the first composition comprises contacting the first cell population with the first composition for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days or more. In some embodiments, the contacting step with the first composition comprises contacting the first cell population with the first composition for about 4 days. In some embodiments, the contacting step with the first composition comprises contacting the first cell population with the first composition for about 6 hours, 10 hours, 12 hours, 24 hours, 30 hours, 36 hours, 40 hours, 48 ​​hours, 56 hours, 72 hours, or more. In some embodiments, the step of contacting the first cell population with the first composition comprises contacting the first cell population with the first composition for about 1-10 days, 1-9 days, 1-8 days, 1-7 days, 1-6 days, 1-5 days, 1-4 days, 1-3 days, 1-2 days, 2-10 days, 2-9 days, 2-8 days, 2-7 days, 2-6 days, 2-5 days, 2-4 days, 2-3 days, 3-10 days, 3-9 days, 3-8 days, 3- The method includes contacting the cells for 7 days, 3 to 6 days, 3 to 5 days, 3 to 4 days, 4 to 10 days, 4 to 9 days, 4 to 8 days, 4 to 7 days, 4 to 6 days, 4 to 5 days, 5 to 10 days, 5 to 9 days, 5 to 8 days, 5 to 7 days, 5 to 6 days, 6 to 10 days, 6 to 9 days, 6 to 8 days, 6 to 7 days, 7 to 10 days, 7 to 8 days, 8 to 10 days, 8 to 9 days, or 9 to 10 days. In some embodiments, contacting the first composition comprises contacting the first cell population with the first composition for about 6-96 hours, 6-72 hours, 6-48 hours, 6-24 hours, 6-12 hours, 12-96 hours, 12-72 hours, 12-48 hours, 12-24 hours, 24-96 hours, 24-72 hours, 24-45 hours, 48-96 hours, or 48-72 hours. In some embodiments, contacting the first composition comprises contacting the first cell population with the first composition for about 72 hours.

[0019] In some embodiments, the first composition further comprises a transforming growth factor β (TGF-β) signaling pathway inhibitor, a thyroid hormone signaling pathway activator, or both. In some embodiments, the first composition comprises an MGLL inhibitor. In some embodiments, the first composition comprises a TGF-β signaling pathway inhibitor. In some embodiments, the first composition comprises a thyroid hormone signaling pathway activator. In some embodiments, the first composition comprises a bone morphogenetic protein (BMP) type 1 receptor inhibitor. In some embodiments, the first composition comprises a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor. In some embodiments, the first composition comprises a histone methyltransferase inhibitor. In some embodiments, the first composition comprises a protein kinase inhibitor.

[0020]

[0020] In some embodiments, the first composition comprises a TGF-β signaling pathway inhibitor, a thyroid hormone signaling pathway activator, a bone morphogenetic protein (BMP) type 1 receptor inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a histone methyltransferase inhibitor, and a protein kinase inhibitor.

[0021]

[0021] In some embodiments, the first composition comprises an MGLL inhibitor, a TGF-β signaling pathway inhibitor, a thyroid hormone signaling pathway activator, a bone morphogenetic protein (BMP) type 1 receptor inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a histone methyltransferase inhibitor, and a protein kinase inhibitor.

[0022]

[0022] In some embodiments, the TGF-β signaling pathway inhibitor is Alk5i (SB505124).

[0023] In some embodiments, the thyroid hormone signaling pathway activator is T3 or an analog or derivative thereof.

[0023]

[0024] In some embodiments, the thyroid hormone signaling pathway activator is the TRβ selective agonist GC-1.

[0025] In some embodiments, the thyroid hormone signaling pathway activator is 3,5-dimethyl-4-[(4'-hydroxy-3'-isopropylbenzyl)-phenoxy]acetic acid.

[0024]

[0026] In some embodiments, the bone morphogenetic protein (BMP) type 1 receptor inhibitor is LDN193189 or a derivative thereof.

[0027] In some embodiments, the Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor is thiazovivin.

[0025]

[0028] In some embodiments, the histone methyltransferase inhibitor is 3-deazaneplanocin A.

[0029] In some embodiments, the protein kinase inhibitor is staurosporine (SSP).

[0026]

[0030] In some embodiments, the first composition does not include a gamma secretase inhibitor (eg, XXI), zinc sulfate, or both.

[0031] In some embodiments, the first composition further comprises a lipid. In some embodiments, the lipid is a saturated fatty acid. In some embodiments, the saturated fatty acid is palmitate. In some embodiments, the lipid is an unsaturated fatty acid. In some embodiments, the unsaturated fatty acid is oleic acid, linoleic acid, or palmitoleic acid.

[0027]

[0032] In some embodiments, the first composition comprises human serum albumin (HSA). In some embodiments, the first composition comprises about 0.01-5%, 0.01-4%, 0.01-3%, 0.01-2%, 0.01-1%, 0.01-0.5%, 0.01-0.06%, or 0.01-0.05% HSA. In some embodiments, the first composition comprises about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or greater than 0.1% HSA. In some embodiments, the first composition comprises less than about 5%, 4%, 3%, 2%, 1%, 0.5%, 0.06%, or 0.05% HSA. In some embodiments, the first composition comprises about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.9%, 1%, 2%, 3%, 4%, or 5% HSA, hi some embodiments, the first composition comprises about 0.05% HSA.

[0028]

[0033] In some embodiments, the first composition comprises MCDB 131. In some embodiments, the first composition comprises DMEM / F12. In some embodiments, the first composition comprises zinc. In some embodiments, the first composition comprises ZnSO4.

[0029]

[0034] In some embodiments, the first composition comprises at least one metabolite. In some embodiments, the at least one metabolite is glutamate, acetate, β-hydroxybutyrate, L-carnitine, taurine, formate, or biotin. In some embodiments, the first composition comprises at least one, two, or three of glutamate, acetate, β-hydroxybutyrate, L-carnitine, taurine, formate, or biotin. , including four, five, six, or seven.

[0030]

[0035] In some embodiments, the second composition comprises at least one amino acid.In some embodiments, at least one amino acid is alanine, glutamate, glycine, proline, threonine or tryptophan.In some embodiments, at least one amino acid is arginine, histidine, lysine, aspartic acid, glutamic acid, serine, asparagine, glutamine, cysteine, selenocysteine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, glutamate, glycine, proline, threonine or tryptophan.

[0031]

[0036] In some embodiments, the second composition comprises at least one vitamin, hi some embodiments, the at least one vitamin is biotin or riboflavin.

[0037] In some embodiments, contacting with the second composition comprises contacting the second cell population with the second composition for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days or more, hi some embodiments, contacting with the second composition comprises contacting the second cell population with the second composition for about 6 hours, 10 hours, 12 hours, 24 hours, 30 hours, 36 hours, 40 hours, 48 ​​hours, 56 hours, 72 hours, or more. In some embodiments, contacting the second cell population with the second composition comprises contacting the second cell population with the second composition for about 1-10 days, 1-9 days, 1-8 days, 1-7 days, 1-6 days, 1-5 days, 1-4 days, 1-3 days, 1-2 days, 2-10 days, 2-9 days, 2-8 days, 2-7 days, 2-6 days, 2-5 days, 2-4 days, 2-3 days, 3-10 days, 3-9 days, 3-8 days, 3- The method includes contacting the cells for 7 days, 3 to 6 days, 3 to 5 days, 3 to 4 days, 4 to 10 days, 4 to 9 days, 4 to 8 days, 4 to 7 days, 4 to 6 days, 4 to 5 days, 5 to 10 days, 5 to 9 days, 5 to 8 days, 5 to 7 days, 5 to 6 days, 6 to 10 days, 6 to 9 days, 6 to 8 days, 6 to 7 days, 7 to 10 days, 7 to 8 days, 8 to 10 days, 8 to 9 days, or 9 to 10 days. In some embodiments, contacting the second composition comprises contacting the second cell population with the second composition for about 6-96 hours, 6-72 hours, 6-48 hours, 6-24 hours, 6-12 hours, 12-96 hours, 12-72 hours, 12-48 hours, 12-24 hours, 24-96 hours, 24-72 hours, 24-45 hours, 48-96 hours, or 48-72 hours. In some embodiments, contacting the second composition comprises contacting the second cell population with the second composition for about 7 days.

[0032]

[0038] In some embodiments, the second composition does not include one or more of an MGLL inhibitor, a TGF-β signaling pathway inhibitor, a thyroid hormone signaling pathway activator, a bone morphogenetic protein (BMP) type 1 receptor inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a histone methyltransferase inhibitor, or a protein kinase inhibitor.

[0033]

[0039] In some embodiments, the second composition does not include an MGLL inhibitor.

[0040] In some embodiments, the second composition does not include a TGF-β signaling pathway inhibitor.

[0034]

[0041] In some embodiments, the second composition does not include a thyroid hormone signaling pathway activator.

[0042] In some embodiments, the second composition does not include a bone morphogenetic protein (BMP) type 1 receptor inhibitor.

[0035]

[0043] In some embodiments, the second composition does not include a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor.

[0044] In some embodiments, the second composition does not include a histone methyltransferase inhibitor.

[0036]

[0045] In some embodiments, the second composition does not include a protein kinase inhibitor.

[0046] In some embodiments, the second composition does not include a TGF-β signaling pathway inhibitor, a thyroid hormone signaling pathway activator, a bone morphogenetic protein (BMP) type 1 receptor inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a histone methyltransferase inhibitor, and a protein kinase inhibitor.

[0037]

[0047] In some embodiments, the second composition does not include an MGLL inhibitor, a TGF-β signaling pathway inhibitor, a thyroid hormone signaling pathway activator, a bone morphogenetic protein (BMP) type 1 receptor inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a histone methyltransferase inhibitor, and a protein kinase inhibitor.

[0038]

[0048] In some embodiments, the second composition comprises a lipid. In some embodiments, the lipid is a saturated fatty acid. In some embodiments, the saturated fatty acid is palmitate. In some embodiments, the lipid is an unsaturated fatty acid. In some embodiments, the unsaturated fatty acid is oleic acid, linoleic acid, or palmitoleic acid.

[0039]

[0049] In some embodiments, the second composition comprises an MGLL inhibitor.

[0050] In some embodiments, the second composition does not contain human serum albumin (HSA). In some embodiments, the second composition contains human serum albumin (HSA). In some embodiments, the second composition contains about 0.1-5%, 0.1-4%, 0.1-3%, 0.1-2%, 0.1-1%, or 0.1-0.5% HSA. In some embodiments, the second composition contains less than about 5%, 4%, 3%, 2%, 1%, 0.6%, or 0.5% HSA. In some embodiments, the second composition contains about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, or 5% HSA. In some embodiments, the second composition contains about 1% HSA.

[0040]

[0051] In some embodiments, the second composition comprises MCDB 131. In some embodiments, the second composition comprises DMEM / F12.

[0052] In some embodiments, the second composition comprises zinc. In some embodiments, the second composition comprises ZnSO4.

[0041]

[0053] In some embodiments, the second composition comprises at least one metabolite. In some embodiments, the at least one metabolite is glutamate, acetate, β-hydroxybutyrate, L-carnitine, taurine, formate, or biotin. In some embodiments, the second composition comprises one, two, three, four, five, six, or seven of glutamate, acetate, β-hydroxybutyrate, L-carnitine, taurine, formate, or biotin.

[0042]

[0054] In some embodiments, the second composition comprises at least one amino acid, hi some embodiments, the at least one amino acid is alanine, glutamate, glycine, proline, threonine, or tryptophan.

[0043]

[0055] In some embodiments, the second composition comprises at least one vitamin, hi some embodiments, the at least one vitamin is biotin or riboflavin.

[0056] In some embodiments, the dissociating step does not include subjecting the cell population to flow cytometry.

[0044]

[0057] (b) dissociating in vitro at least a portion of the plurality of cell clusters in the first cell population; (c) freezing at least a portion of the first cell population, the first cell population comprising at least a portion of the dissociated cell clusters; (d) thawing at least a portion of the frozen first cell population; (e) contacting in vitro at least a portion of the thawed first cell population with a first composition to obtain a second cell population, the second cell population comprising a plurality of insulin-positive endocrine cells comprising the plurality of cell clusters, the first composition comprising one, two, three, four, or five of the following agents: a monoglyceride lipase (MGLL) inhibitor, a bone morphogenetic protein (BMP) type 1 receptor inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a histone methyltransferase inhibitor, or a protein kinase inhibitor; and (f) contacting in vitro the second insulin-positive endocrine cell population with the second composition to obtain a second insulin-positive endocrine cell population. contacting in vitro, thereby differentiating at least a portion of the second insulin-positive endocrine cell population into a third cell population comprising a plurality of beta cells comprising a plurality of cell clusters, wherein the second composition is different from the first composition, and the third cell population comprises a higher percentage of viable beta cells compared to a corresponding beta cell population comprising beta cells derived from the first cell population not contacted with the first composition.

[0045]

[0058] In some embodiments, the first composition comprises a bone morphogenetic protein (BMP) type 1 receptor inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a histone methyltransferase inhibitor, and a protein kinase inhibitor.

[0046]

[0059] In some embodiments, the first composition further comprises a transforming growth factor beta (TGF-β) signaling pathway inhibitor.

[0060] In some embodiments, the first composition further comprises a thyroid hormone signaling pathway activator.

[0047]

[0061] In some embodiments, the first composition further comprises a monoglyceride lipase (MGLL) inhibitor.

[0062] The present invention provides a method for producing insulin-positive endocrine cells, comprising: (a) obtaining a first cell population comprising a plurality of cell clusters comprising insulin-positive endocrine cells; (b) dissociating in vitro at least a portion of the plurality of cell clusters in the first cell population; (c) freezing at least a portion of the first cell population comprising at least a portion of the dissociated cell clusters; (d) thawing at least a portion of the frozen first cell population; and (e) combining at least a portion of the thawed first cell population with a first composition in vitro. (f) contacting in vitro the first composition with a transforming growth factor beta (TGF-β) signaling pathway inhibitor, a thyroid hormone signaling pathway activator, or both, and one, two, three, four, or five of a monoglyceride lipase (MGLL) inhibitor, a bone morphogenetic protein (BMP) type 1 receptor inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a histone methyltransferase inhibitor, or a protein kinase inhibitor to obtain a second cell population comprising a plurality of insulin-positive endocrine cells comprising a plurality of cell clusters; contacting in vitro, thereby differentiating at least a portion of the second insulin-positive endocrine cell population into a third cell population comprising a plurality of beta cells comprising a plurality of cell clusters, wherein the second composition is different from the first composition, and the third cell population comprises a higher percentage of viable beta cells compared to a corresponding beta cell population comprising beta cells derived from the first cell population not contacted with the first composition.

[0048]

[0063] In some embodiments, the first composition comprises a monoglyceride lipase (MGLL) inhibitor, a bone morphogenetic protein (BMP) type 1 receptor inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a histone methyltransferase inhibitor, and a protein kinase inhibitor.

[0049]

[0064] Provided herein are methods including: (a) obtaining a first cell population comprising a plurality of cell clusters comprising insulin-positive endocrine cells; (b) dissociating in vitro at least a portion of the plurality of cell clusters in the first cell population; (c) contacting in vitro the first cell population comprising at least a portion of the dissociated cell clusters with a first composition to obtain a second cell population comprising a plurality of cell clusters, the plurality of cell clusters comprising a plurality of insulin-positive endocrine cells; and (d) contacting in vitro the second insulin-positive endocrine cell population with a second composition, thereby differentiating at least a portion of the second insulin-positive endocrine cell population into a third cell population comprising a plurality of beta cells, wherein the second composition is different from the first composition, and the second composition comprises at least one metabolite, and the third cell population comprises a higher percentage of viable cells compared to a corresponding cell population comprising beta cells derived from the first cell population not contacted with the second composition.

[0050]

[0065] Provided herein is a method comprising: (a) obtaining a first cell population comprising a plurality of cell clusters comprising insulin-positive endocrine cells; (b) dissociating in vitro at least a portion of the plurality of cell clusters in the first cell population; (c) contacting in vitro the first cell population comprising at least a portion of the dissociated cell clusters with a first composition to obtain a second cell population comprising a plurality of cell clusters, the plurality of insulin-positive endocrine cells; and (d) contacting in vitro the second insulin-positive endocrine cell population with a second composition, thereby differentiating at least a portion of the second insulin-positive endocrine cell population into a third cell population comprising a plurality of beta cells, wherein the second composition is different from the first composition, and the second composition comprises at least one metabolite, and the plurality of beta cells exhibits enhanced glucose-stimulated insulin secretion relative to a corresponding cell population comprising beta cells derived from the first cell population not contacted with the second composition.

[0051]

[0066] In some embodiments, the at least one metabolite is glutamate, acetate, beta-hydroxybutarate, L-carnitine, taurine, formate, or biotin.

[0052]

[0067] In some embodiments, the second composition comprises at least two, three, four, five, six, or seven metabolites of glutamate, acetate, beta-hydroxybutarate, L-carnitine, taurine, formate, or biotin.

[0053]

[0068] In some embodiments, the second composition comprises DMEM / F12.

[0069] In some embodiments, the second composition comprises about 0.05-2% HSA.

[0070] In some embodiments, the second composition comprises about 1% HSA.

[0054]

[0071] In some embodiments, the second composition comprises zinc.

[0072] In some embodiments, the second composition comprises ZnSO4.

[0073] In some embodiments, the second composition comprises at least one amino acid.

[0055]

[0074] In some embodiments, at least one amino acid is alanine, glutamate, glycine, proline, threonine, or tryptophan.

[0075] In some embodiments, the second composition comprises at least one vitamin.

[0056]

[0076] In some embodiments, the at least one vitamin is biotin or riboflavin.

[0077] In some embodiments, the plurality of beta cells exhibits enhanced glucose-stimulated insulin secretion relative to a corresponding cell population comprising beta cells derived from the first cell population that are not contacted with the second composition.

[0057]

[0078] In some embodiments, the third cell population comprises a higher percentage of viable cells compared to a corresponding cell population comprising beta cells derived from the first cell population that has not been contacted with the second composition.

[0058]

[0079] In some embodiments, the third cell population comprises a plurality of cell clusters, each having a diameter of about 50-150 microns. In some embodiments, the third cell population comprises a plurality of cell clusters, each having a diameter of about 100 microns. In some embodiments, the third cell population comprises a plurality of cell clusters, wherein at least 50%, 60%, 70%, 75%, 80%, 90%, or 95% of the cell clusters have a diameter of about 100 microns without a selection step. In some embodiments, the third cell population comprises a plurality of cell clusters, wherein at least 50%, 60%, 70%, 75%, 80%, 90%, or 95% of the cell clusters have a diameter of about 50-150 microns, 75-125 microns, 80-120 microns, or 90-110 microns without a selection step.

[0059]

[0080] In some embodiments, the first composition comprises at least one agent selected from the group consisting of a monoglyceride lipase (MGLL) inhibitor, a bone morphogenetic protein (BMP) type 1 receptor inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a histone methyltransferase inhibitor, and a protein kinase inhibitor.

[0060]

[0081] In some embodiments, the methods of the present disclosure further include (a) freezing at least a portion of a first cell population comprising at least a portion of the dissociated cell clusters; (b) thawing at least a portion of the frozen first cell population; and (c) contacting at least a portion of the thawed first cell population with the first composition in vitro.

[0061]

[0082] Provided herein are compositions comprising at least a portion of a second cell population comprising insulin-positive endocrine cells described herein or produced by a method described herein.

[0062]

[0083] Provided herein are compositions comprising at least a portion of a third cell population of beta cells described herein or produced by a method described herein.

[0084] Provided herein are compositions comprising at least a portion of a third cell population of beta cells described herein or produced by a method described herein, and at least a portion of a second cell population comprising insulin-positive endocrine cells described herein or produced by a method described herein.

[0063]

[0085] Provided herein are devices comprising compositions of beta cells described herein or made by the methods described herein.

[0086] Provided herein are devices comprising compositions of insulin-positive endocrine cells described herein or produced by the methods described herein.

[0064]

[0087] As used herein, the term "antibody" refers to a substance that is prepared by any method described herein or by any method described herein. The present invention provides a device comprising a composition of insulin-positive endocrine cells and beta cells produced by the method of the present invention.

[0065]

[0088] Provided herein are methods of treating a subject having a disease characterized by prolonged periods of high blood glucose levels (e.g., diabetes), the methods comprising administering to the subject a composition of cells described herein or produced by a method described herein.

[0066]

[0089] Provided herein are methods of treating a subject having a disease characterized by prolonged periods of high blood glucose levels (e.g., diabetes), the methods comprising administering to the subject a composition of cells described herein or produced by a method described herein.

[0067]

[0090] Provided herein are methods of treating a subject having a disease characterized by prolonged periods of high blood glucose levels (e.g., diabetes), the methods comprising administering to the subject a composition of cells described herein or produced by a method described herein.

[0068]

[0091] Provided herein is a method of treating a subject having a disease characterized by high blood glucose levels over an extended period of time (e.g., diabetes), the method comprising implanting a device described herein into the subject.

[0069]

[0092] Provided herein is a method of treating a subject having a disease characterized by high blood glucose levels over an extended period of time (e.g., diabetes), the method comprising implanting a device described herein into the subject.

[0070]

[0093] Provided herein is a method of treating a subject having a disease characterized by high blood glucose levels over an extended period of time (e.g., diabetes), the method comprising implanting a device described herein into the subject.

[0071]

[0094] In some embodiments, the disease is diabetes. In some embodiments, the disease is type I diabetes. In some embodiments, the disease is type II diabetes.

[0095] Provided herein are compositions comprising isolated insulin-positive endocrine cells that have been contacted in vitro with an agent that inhibits monoglyceride lipase (MGLL) expression or function and that exhibit a decreased rate of monoglyceride conversion to free fatty acids compared to a corresponding isolated insulin-positive endocrine cell population that has not been contacted in vitro with an agent that inhibits monoglyceride lipase (MGLL) expression or function.

[0072]

[0096] Provided herein are compositions comprising isolated insulin-positive endocrine cells that have been contacted in vitro with an agent that inhibits monoglyceride lipase (MGLL) expression or function and that exhibit an increased ratio of monoglycerides to free fatty acids compared to a corresponding isolated insulin-positive endocrine cell population that has not been contacted in vitro with an agent that inhibits monoglyceride lipase (MGLL) expression or function.

[0073]

[0097] Provided herein are compositions comprising isolated insulin-positive endocrine cells that have been contacted in vitro with an agent that inhibits monoglyceride lipase (MGLL) expression or function and that exhibit a decreased ratio of free fatty acids to monoglycerides compared to a corresponding isolated insulin-positive endocrine cell population that has not been contacted in vitro with an agent that inhibits monoglyceride lipase (MGLL) expression or function.

[0074]

[0098] Provided herein are compositions comprising isolated insulin-positive endocrine cells that have been contacted in vitro with an agent that inhibits monoglyceride lipase (MGLL) expression or function, and that exhibit reduced levels of free fatty acids compared to a corresponding isolated insulin-positive endocrine cell population that has not been contacted in vitro with an agent that inhibits monoglyceride lipase (MGLL) expression or function.

[0075]

[0099] Provided herein are compositions comprising isolated insulin-positive endocrine cells that have been contacted in vitro with an agent that inhibits monoglyceride lipase (MGLL) expression or function, and that exhibit elevated levels of monoglycerides compared to a corresponding isolated insulin-positive endocrine cell population that has not been contacted in vitro with an agent that inhibits monoglyceride lipase (MGLL) expression or function.

[0076]

[0100] Provided herein are compositions comprising a population of insulin-positive endocrine cells and an agent that inhibits the conversion of monoglycerides to free fatty acids.

[0101] In some embodiments, the agent inhibits the expression or function of monoglyceride lipase (MGLL).

[0077]

[0102] Provided herein are compositions comprising an insulin-positive cell population and an agent that inhibits monoglyceride lipase (MGLL) expression or function. In some embodiments, the agent that inhibits monoglyceride lipase (MGLL) expression or function is JJKK048, KML29, NF1819, JW642, JZL184, JZL195, JZP361, pristimerin, or URB602.

[0078]

[0103] Provided herein are compositions comprising a beta cell population that has been contacted in vitro with at least one agent selected from the group consisting of glutamate, acetate, beta-hydroxybutarate, L-carnitine, taurine, formate, or biotin, wherein the beta cell population exhibits increased glucose-stimulated insulin secretion compared to a corresponding beta cell population that has not been contacted with the at least one agent. In some embodiments, the cell population has been contacted with at least two, three, four, five, six, or seven of the agents selected from the group consisting of glutamate, acetate, beta-hydroxybutarate, L-carnitine, taurine, formate, or biotin.

[0079]

[0104] Provided herein are compositions comprising a beta cell population and at least one, two, three, four, five, six, or seven of the following agents selected from the group consisting of glutamate, acetate, beta-hydroxybutarate, L-carnitine, taurine, formate, or biotin.

[0080] Incorporation by Reference

[0105] All publications, patents, and patent applications mentioned in this specification are incorporated herein 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. To the extent that the publications and patents or patent applications incorporated by reference conflict with the present disclosure contained herein, it is intended that the present specification supersede and / or take precedence over any such conflicting material. This specification includes the following disclosure of the invention: [Item 1] A composition comprising a plurality of dissociated insulin-positive endocrine precursor cells and a BMP signaling pathway inhibitor. [Item 2] A composition comprising a plurality of dissociated insulin-positive endocrine precursor cells and a ROCK inhibitor. [Item 3] A composition comprising a plurality of dissociated insulin-positive endocrine precursor cells and a histone methyltransferase inhibitor. [Item 4] A composition comprising a plurality of dissociated insulin-positive endocrine precursor cells and zinc. [Item 5] A composition comprising a plurality of dissociated insulin-positive endocrine precursor cells and a monoglyceride lipase (MGLL) inhibitor. [Item 6] A composition comprising a plurality of dissociated insulin-positive endocrine precursor cells and lipids. [Item 7] A composition comprising a plurality of dissociated insulin-positive endocrine precursor cells and one or more of glutamate, acetate, beta-hydroxybutyrate, L-carnitine, taurine, formate, or biotin. [Item 8] A composition described in any one of Items 2 to 7, comprising a BMP signaling pathway inhibitor. [Item 9] The composition described in Item 1 or 8, wherein the BMP signaling pathway inhibitor is LDN193189 or a derivative thereof. [Item 10] The composition according to any one of Items 1 or 3 to 9, comprising a ROCK inhibitor. [Item 11] The composition according to Item 2 or 10, wherein the ROCK inhibitor is thiazovivin, Y-27632, fasudil / HA1077, or 14-1152, or a derivative thereof. [Item 12] The composition of any one of items 1, 2, or 4 to 11, comprising a histone methyltransferase inhibitor. [Item 13] The composition of Items 3 or 12, wherein the histone methyltransferase inhibitor is 3-deazaneplanocin A hydrochloride or a derivative thereof. [Item 14] The composition according to any one of items 1 to 3 or 5 to 13, which contains zinc. [Item 15] The composition according to Item 4 or 14, wherein the zinc is in the form of ZnSO4. [Item 16] The composition according to any one of items 1 to 4 or 6 to 15, comprising a monoglyceride lipase (MGLL) inhibitor. [Item 17] The composition of Items 5 or 16, wherein the MGLL inhibitor is JJKK048, KML29, NF1819, JW642, JZL184, JZL195, JZP361, pristimerin, or URB602, or a derivative of any of the foregoing. [Item 18] The composition according to any one of items 1 to 5 or 7 to 17, comprising a lipid. [Item 19] The composition according to Item 6 or 18, wherein the lipid is a saturated fatty acid. [Item 20] The composition according to Item 19, wherein the saturated fatty acid is palmitate. [Item 21] The composition according to Item 6 or 18, wherein the lipid is an unsaturated fatty acid. [Item 22] The composition according to Item 21, wherein the unsaturated fatty acid is oleic acid, linoleic acid, or palmitoleic acid. [Item 23] The composition described in any one of Items 1 to 22, further comprising a serum albumin protein. [Item 24] The composition described in Item 23, wherein the serum albumin protein is human serum albumin protein. [Item 25] 0.01%~1%, 0.03~1%, 0.03~0.9%, 0.03~0.08%, 0.03~0.06%, 0.03~0.05%, 0.04~0.8%, 0.04~0.7%, 0.04~0.6 %, 0.04~0.5%, 0.04~0.4%, 0.04~0.3%, 0.04~0.2%, 0.04~0.1%, 0.04~0.09%, 0.04~0.8%, 0.04~0.07%, 0.04~0.06% 25. The composition according to item 23 or 24, comprising 0.04 to 0.05%, 0.05 to 1%, 0.05 to 0.9%, 0.05 to 0.8%, 0.05 to 0.7%, 0.05 to 0.6%, 0.05 to 0.5%, 0.05 to 0.4%, 0.05 to 0.3%, 0.05 to 0.2%, 0.05 to 0.1%, 0.05 to 0.09%, 0.05 to 0.8%, 0.05 to 0.07%, or 0.05 to 0.06% serum albumin protein. [Item 26] The composition of any one of Items 1 to 25, wherein less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, or less than 1% of the cells in the composition are in cell clusters. [Item 27] ​​The composition described in any one of Items 1 to 26, comprising a TGF-β pathway inhibitor. [Item 28] The TGF-β pathway inhibitor is Alk5i (SB505124) or its 28. The composition according to item 27, which is a derivative of [Item 29] The composition described in any one of Items 1 to 28, comprising a thyroid hormone signaling pathway activator. [Item 30] The composition described in Item 29, wherein the thyroid hormone signaling pathway activator is GC-1 or T3, or a derivative thereof. [Item 31] The composition according to any one of Items 1 to 30, comprising a protein kinase inhibitor. [Item 32] The composition of Item 31, wherein the protein kinase inhibitor is staurosporine. [Item 33] The composition according to any one of Items 1 to 32, comprising glutamate. [Item 34] The composition according to any one of Items 1 to 33, comprising acetate. [Item 35] The composition according to any one of Items 1 to 34, comprising β-hydroxybutarate. [Item 36] The composition according to any one of Items 1 to 35, comprising L-carnitine. [Item 37] The composition according to any one of Items 1 to 36, comprising taurine. [Item 38] The composition according to any one of Items 1 to 37, comprising a formate. [Item 39] The composition according to any one of Items 1 to 38, which contains biotin. [Item 40] The composition according to any one of Items 1 to 39, containing vitamin C. [Item 41] The composition according to any one of Items 1 to 40, comprising insulin. [Item 42] The composition of any one of Items 1 to 41, wherein the dissociated insulin-positive endocrine precursor cells have been previously frozen. [Item 43] A method comprising the step of contacting a plurality of dissociated insulin-positive endocrine precursor cells with a BMP signaling pathway inhibitor. [Item 44] A method comprising the step of contacting a plurality of dissociated insulin-positive endocrine precursor cells with a ROCK inhibitor. [Item 45] A method comprising the step of contacting a plurality of dissociated insulin-positive endocrine precursor cells with a histone methyltransferase inhibitor. [Item 46] A method comprising the step of contacting a plurality of dissociated insulin-positive endocrine precursor cells with zinc. [Item 47] A method comprising the step of contacting a plurality of dissociated insulin-positive endocrine precursor cells with a monoglyceride lipase (MGLL) inhibitor. [Item 48] A method comprising the step of contacting a plurality of dissociated insulin-positive endocrine precursor cells with a lipid. [Item 49] A method comprising contacting a plurality of dissociated insulin-positive endocrine precursor cells with one or more of glutamate, acetate, β-hydroxybutarate, L-carnitine, taurine, formate, or biotin. [Item 50] The method described in any one of Items 44 to 49, comprising contacting the plurality of dissociated insulin-positive endocrine precursor cells with a BMP signaling pathway inhibitor. [Item 51] The method described in Item 43 or 50, wherein the BMP signaling pathway inhibitor is LDN193189 or a derivative thereof. [Item 52] The method of any one of Items 43 or 45 to 51, comprising contacting the plurality of dissociated insulin-positive endocrine precursor cells with a ROCK inhibitor. [Item 53] The composition according to Item 44 or 52, wherein the ROCK inhibitor is thiazovivin, Y-27632, fasudil / HA1077, or 14-1152, or a derivative thereof. [Item 54] The method of any one of Items 43, 44, or 46 to 53, comprising contacting the plurality of dissociated insulin-positive endocrine precursor cells with a histone methyltransferase inhibitor. [Item 55] The histone methyltransferase inhibitor is 3-deazaneplanocytidine. 55. The method according to item 45 or 54, wherein the compound is benzophenone A hydrochloride or a derivative thereof. [Item 56] The method of any one of Items 43 to 45 or 47 to 55, comprising contacting the plurality of dissociated insulin-positive endocrine precursor cells with zinc. [Item 57] The method according to Item 46 or 56, wherein the zinc is in the form of ZnSO4. [Item 58] The method described in any one of Items 43 to 46 or 48 to 57, comprising contacting the plurality of dissociated insulin-positive endocrine precursor cells with a monoglyceride lipase (MGLL) inhibitor. [Item 59] The method described in Item 47 or 58, wherein the MGLL inhibitor is JJKK048, KML29, NF1819, JW642, JZL184, JZL195, JZP361, pristimerin, or URB602, or a derivative of any of the foregoing. [Item 60] The method of any one of Items 43 to 47 or 49 to 59, comprising contacting the plurality of dissociated insulin-positive endocrine precursor cells with a lipid. [Item 61] The method described in Item 48 or 60, wherein the lipid is a saturated fatty acid. [Item 62] The method according to Item 61, wherein the saturated fatty acid is palmitate. [Item 63] The method described in Item 48 or 60, wherein the lipid is an unsaturated fatty acid. [Item 64] The method according to Item 63, wherein the unsaturated fatty acid is oleic acid, linoleic acid, or palmitoleic acid. [Item 65] The method of any one of Items 43 to 64, comprising contacting the plurality of dissociated insulin-positive endocrine precursor cells with serum albumin protein. [Item 66] The method described in Item 65, wherein the serum albumin protein is human serum albumin protein. [Item 67] The composition contains 0.01% to 1%, 0.03 to 1%, 0.03 to 0.9%, 0.03 to 0.08%, 0.03 to 0.06%, 0.03 to 0.05%, 0.04 to 0.8%, 0.04 to 0.7%, 0.04 to 0.6%, 0.04 to 0.5%, 0.04 to 0.4%, 0.04 to 0.3%, 0.04 to 0.2%, 0.04 to 0.1%, 0.04 to 0.09%, 0.04 to 0.8%, 0.04 to 0.07%, 0.04 to 0. 67. The method of claim 65 or 66, comprising 0.06%, 0.04-0.05%, 0.05-1%, 0.05-0.9%, 0.05-0.8%, 0.05-0.7%, 0.05-0.6%, 0.05-0.5%, 0.05-0.4%, 0.05-0.3%, 0.05-0.2%, 0.05-0.1%, 0.05-0.09%, 0.05-0.8%, 0.05-0.07%, or 0.05-0.06% serum albumin protein. [Item 68] The method of any one of Items 43 to 67, wherein less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, or less than 1% of the cells in the composition are in cell clusters. [Item 69] The method described in any one of Items 43 to 68, comprising contacting the plurality of dissociated insulin-positive endocrine precursor cells with a TGF-β pathway inhibitor. [Item 70] The method described in Item 69, wherein the TGF-β pathway inhibitor is Alk5i (SB505124) or a derivative thereof. [Item 71] The method of any one of Items 43 to 70, comprising contacting the plurality of dissociated insulin-positive endocrine precursor cells with a thyroid hormone signaling pathway activator. [Item 72] The method described in Item 71, wherein the thyroid hormone signaling pathway activator is GC-1 or T3, or a derivative thereof. [Item 73] The method of any one of Items 43 to 72, comprising contacting the plurality of dissociated insulin-positive endocrine precursor cells with a protein kinase inhibitor. [Item 74] The protein kinase inhibitor is staurosporine. The method described. [Item 75] The method of any one of Items 43 to 74, comprising contacting the plurality of dissociated insulin-positive endocrine precursor cells with glutamate. [Item 76] The method of any one of Items 43 to 75, comprising contacting the plurality of dissociated insulin-positive endocrine precursor cells with acetate. [Item 77] The method of any one of Items 43 to 76, comprising contacting the plurality of dissociated insulin-positive endocrine precursor cells with β-hydroxybutarate. [Item 78] The method of any one of Items 43 to 77, comprising contacting the plurality of dissociated insulin-positive endocrine precursor cells with L-carnitine. [Item 79] The method of any one of Items 43 to 78, comprising contacting the plurality of dissociated insulin-positive endocrine precursor cells with taurine. [Item 80] The method of any one of Items 43 to 79, comprising contacting the plurality of dissociated insulin-positive endocrine precursor cells with formate. [Item 81] The method of any one of Items 43 to 80, comprising contacting the plurality of dissociated insulin-positive endocrine precursor cells with biotin. [Item 82] The method of any one of Items 43 to 81, comprising contacting the plurality of dissociated insulin-positive endocrine precursor cells with vitamin C. [Item 83] The method of any one of Items 43 to 82, comprising contacting the plurality of dissociated insulin-positive endocrine precursor cells with insulin. [Item 84] The method according to any one of Items 43 to 83, wherein the dissociated insulin-positive endocrine precursor cells have been previously frozen. [Item 85] The method according to any one of Items 43 to 84, which is carried out over 1 to 10 days, 1 to 9 days, 1 to 8 days, 1 to 7 days, 1 to 6 days, 1 to 5 days, 1 to 4 days, 1 to 3 days, 1 to 2 days, 2 to 10 days, 2 to 9 days, 2 to 8 days, 2 to 7 days, 2 to 6 days, 2 to 5 days, 2 to 4 days, 2 to 3 days, 3 to 10 days, 3 to 9 days, 3 to 8 days, 3 to 7 days, 3 to 6 days, 3 to 5 days, 3 to 4 days, 4 to 10 days, 4 to 9 days, 4 to 8 days, 4 to 7 days, 4 to 6 days, or 4 to 5 days. [Item 86] The method described in any one of Items 43 to 85, wherein the dissociated cells are reaggregated into multiple cell clusters. [Item 87] The method of Item 86, wherein at least about 40%, 50%, 60%, 70%, 80%, or 90% of the plurality of cell clusters have a diameter of about 50 μm to about 250 μm, about 75 μm to about 250 μm, or about 100 μm to about 200 μm. [Item 88] The method of Items 86 or 87, wherein at least about 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, or 99% of the cells of the plurality of cell clusters of the second cell population are viable. [Item 89] The method of any one of Items 86 to 88, wherein the dissociated cells are reaggregated into at least 2, 3, 4, 5, 10, 50, 100, 1,000, 10,000, 100,000, or 1,000,000 cell clusters. [Item 90] A composition comprising a plurality of cell clusters, the cell clusters comprising insulin-positive cells, the composition comprising: a) at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, or at least 65% of the cells in the composition are viable after 11 days of in vitro culture; b) at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% of the cell clusters in the composition are 90-140 μm, 90-130 μm, 90-120 μm, 90-110 μm, 100-140 μm, 100-130 μm, 100-120 μm, 100-110 μm in diameter; and / or c) at least 10%, at least 20%, at least At least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% are 1.5-4.5, 1.5-4.0, 1.5-3.5, 1.5-3.0, 1.5-2.5, 1.5-2.5, 1.5- A glucose-stimulated insulin secretion (GSIS) stimulation index of 2.0, 2.0-4.5, 2.0-4.0, 2.0-3.5, 2.0-3.0, 2.0-2.5, 2.5-4.5, 2.5-4.0, 2.5-3.5, 2.5-3.0, 3.0-4.5, 3.0-4.0, 3.0-3.5, 3.5-4.5, 3.5-4.0, or 4.0-4.5 A composition comprising at least one of: [Item 91] The composition described in Item 90, wherein the cell clusters comprise C-peptide positive cells. [Item 92] The composition described in Item 90 or 91, wherein the cell clusters comprise somatostatin-positive cells. [Item 93] The composition described in Item 90 or 91, wherein the cell clusters comprise glucagon-positive cells. [Item 94] The composition of any one of Items 90 to 93, wherein at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, or at least 65% of the cells in the composition are viable after 11 days of in vitro culture. [Item 95] The composition according to any one of Items 90 to 94, wherein at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the cell clusters in the composition have a diameter of 90 to 140 μm, 90 to 130 μm, 90 to 120 μm, 90 to 110 μm, 100 to 140 μm, 100 to 130 μm, 100 to 120 μm, or 100 to 110 μm. [Item 96] At least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the cell clusters in the composition are 1.5 to 4.5, 1.5 to 4.0, 1.5 to 3.5, 1.5 to 3.0, 1.5 to 2.5, 1 96. The composition of any one of items 90 to 95, exhibiting a glucose-stimulated insulin secretion (GSIS) stimulation index of 0.5 to 2.5, 1.5 to 2.0, 2.0 to 4.5, 2.0 to 4.0, 2.0 to 3.5, 2.0 to 3.0, 2.0 to 2.5, 2.5 to 4.5, 2.5 to 4.0, 2.5 to 3.5, 2.5 to 3.0, 3.0 to 4.5, 3.0 to 4.0, 3.0 to 3.5, 3.5 to 4.5, 3.5 to 4.0, or 4.0 to 4.5. [Item 97] The composition of any one of items 90 to 96, comprising at least 2, 3, 4, 5, 10, 50, 100, 1,000, 10,000, 100,000, or 1,000,000 cell clusters. [Item 98] The composition according to any one of Items 90 to 97, prepared according to the method according to any one of Items 43 to 89. [Item 99] A device comprising the composition described in any one of Items 90 to 98. [Item 100] A method for treating a subject having a disease characterized by prolonged high blood glucose levels (e.g., diabetes), comprising administering to the subject a composition described in any one of Items 90 to 98 or a device described in Item 99. [Item 101] (a) obtaining a first cell population including a plurality of cell clusters including insulin-positive cells; (b) dissociating in vitro at least a portion of the plurality of cell clusters in the first cell population; (c) separating the first cell population containing at least a portion of the dissociated cell clusters into a second cell population; obtaining a second cell population comprising a plurality of cell clusters, each of which comprises a plurality of insulin-positive cells, by contacting the first cell population in vitro with the first composition, wherein the first composition comprises at least one agent selected from the group consisting of a monoglyceride lipase (MGLL) inhibitor, a bone morphogenetic protein (BMP) type 1 receptor inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a histone methyltransferase inhibitor, and a protein kinase inhibitor; (d) contacting the second insulin-positive cell population in vitro with a second composition, thereby differentiating at least a portion of the second insulin-positive cell population into a third cell population comprising a plurality of beta cells, wherein the second composition is different from the first composition, and the third cell population comprises a higher percentage of viable beta cells compared to a corresponding beta cell population comprising beta cells derived from the first cell population not contacted with the first composition; A method comprising: [Item 102] (a) obtaining a first cell population including a plurality of cell clusters including insulin-positive cells; (b) dissociating in vitro at least a portion of the plurality of cell clusters in the first cell population; (c) contacting the first cell population comprising at least a portion of the dissociated cell clusters with a first composition in vitro to obtain a second cell population comprising a plurality of cell clusters, the second cell population comprising a plurality of insulin-positive endocrine cells, wherein the first composition comprises a transforming growth factor beta (TGF-β) signaling pathway inhibitor, a thyroid hormone signaling pathway activator, or both, and at least one agent selected from the group consisting of a monoglyceride lipase (MGLL) inhibitor, a bone morphogenetic protein (BMP) type 1 receptor inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a histone methyltransferase inhibitor, and a protein kinase inhibitor; (d) contacting the second insulin-positive cell population in vitro with a second composition, thereby differentiating at least a portion of the second insulin-positive cell population into a third cell population comprising a plurality of beta cells, wherein the second composition is different from the first composition, and the third cell population comprises a higher percentage of viable beta cells compared to a corresponding beta cell population comprising beta cells derived from the first cell population not contacted with the first composition; A method comprising: [Item 103] (a) obtaining a first cell population including a plurality of cell clusters including insulin-positive cells; (b) dissociating in vitro at least a portion of the plurality of cell clusters in the first cell population; (c) contacting the first cell population comprising at least a portion of the dissociated cell clusters in vitro with a first composition to obtain a second cell population comprising a plurality of cell clusters, the second cell population comprising a plurality of insulin-positive endocrine cells, wherein the first composition comprises a monoglyceride lipase (MGLL) inhibitor; (d) contacting the second insulin-positive cell population in vitro with a second composition, thereby differentiating at least a portion of the second insulin-positive cell population into a third cell population comprising a plurality of beta cells, wherein the second composition is different from the first composition, and the third cell population comprises a higher percentage of viable beta cells compared to a corresponding beta cell population comprising beta cells derived from the first cell population not contacted with the first composition; A method comprising: [Item 104] (a) freezing at least a portion of the first cell population, including at least a portion of the dissociated cell clusters; (b) thawing at least a portion of the frozen first cell population; (c) contacting at least a portion of the thawed first cell population in vitro with the first composition; 104. The method of claim 101, 102, or 103, further comprising: [Item 105] The method according to any one of Items 101 to 104, wherein at least a portion of the plurality of cell clusters of the second cell population have a diameter of about 50 μm to about 250 μm, about 75 μm to about 250 μm, or about 100 μm to about 200 μm. [Item 106] The method of any one of Items 101 to 105, wherein at least about 40%, 50%, 60%, 70%, 80%, or 90% of the plurality of cell clusters of the second cell population have a diameter of about 50 μm to about 250 μm, about 75 μm to about 250 μm, or about 100 μm to about 200 μm. [Item 107] The method of any one of Items 101 to 106, wherein at least about 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, or 99% of the cells in the second cell population are viable. [Item 108] The method of any one of Items 101 to 107, wherein at least about 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, or 99% of the cells in the plurality of cell clusters of the second cell population are viable. [Item 109] The method of any one of Items 101 to 108, wherein the second cell population comprises at least 2, 3, 4, 5, 10, 50, 100, 1000, 10,000, 100,000, or 1,000,000 cell clusters. [Item 110] The method of any one of Items 101 to 109, wherein the second cell population comprises a higher percentage of insulin-positive endocrine cells compared to a corresponding cell population comprising insulin-positive endocrine cells not contacted with the first composition. [Item 111] The method of any one of Items 101 to 110, wherein the second cell population comprises at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% more viable insulin-positive endocrine cells compared to a corresponding cell population comprising insulin-positive endocrine cells not contacted with the first composition. [Item 112] The method of any one of Items 101 to 111, wherein the second cell population contains at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% more viable insulin-positive endocrine cells after about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days of contact between the first cell population and the first composition, compared to a corresponding cell population containing insulin-positive endocrine cells not contacted with the first composition. [Item 113] The second cell population is incubated with the first cell population and the first composition for about 1 to 10 days, 1 to 9 days, 1 to 8 days, 1 to 7 days, 1 to 6 days, 1 to 5 days, 1 to 4 days, 1 to 3 days, 1 to 2 days, 2 to 10 days, 2 to 9 days, 2 to 8 days, 2 to 7 days, 2 to 6 days, 2 to 5 days, 2 to 4 days, 2 to 3 days, 3 to 10 days, 3 to 9 days, 3 to 8 days, 3 to 7 days, 3 to 6 days, 3 to 5 days, 3 to 4 days 112. The method of any one of paragraphs 101 to 111, wherein after 4 to 10 days, 4 to 9 days, 4 to 8 days, 4 to 7 days, 4 to 6 days, or 4 to 5 days of contact, the cell population comprises at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% more viable insulin-positive endocrine cells compared to a corresponding cell population comprising insulin-positive endocrine cells not contacted with the first composition. [Item 114] The method described in any one of Items 101 to 113, wherein at least a portion of the plurality of beta cells form a plurality of cell clusters. [Item 115] The method according to Item 114, wherein at least a portion of the plurality of cell clusters of the third cell population have a diameter of about 50 μm to about 250 μm, about 50 μm to about 150 μm, about 50 μm to about 100 μm, about 75 μm to about 250 μm, about 75 μm to about 150 μm, about 75 μm to about 125 μm, about 75 μm to about 100 μm, or about 100 μm to about 200 μm. [Item 116] The method of Item 114 or 115, wherein at least about 40%, 50%, 60%, 70%, 80%, or 90% of the plurality of cell clusters of the third cell population have a diameter of about 50 μm to about 250 μm, about 50 μm to about 150 μm, about 50 μm to about 100 μm, about 75 μm to about 250 μm, about 75 μm to about 150 μm, about 75 μm to about 125 μm, about 75 μm to about 100 μm, or about 100 μm to about 200 μm without a selection step. [Item 117] The method of Item 114, wherein at least about 40%, 50%, 60%, 70%, 75%, 80%, 90%, or 95% of the cell clusters have a diameter of about 50 to 150 μm, 75 to 12 μm, 80 to 120 μm, or 90 to 110 μm without a selection step. [Item 118] The method of Item 114, wherein at least about 50%, 60%, 70%, 75%, 80%, 90%, or 95% of the cell clusters have a diameter of about 100 microns without a selection step. [Item 119] The method of any one of Items 114 to 118, wherein at least about 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, or 99% of the cells in the third cell population are viable without a selection step. [Item 120] The method of any one of Items 114 to 119, wherein at least about 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, or 99% of the cells of the plurality of cell clusters of the third cell population are viable without a selection step. [Item 121] The method of any one of Items 114 to 120, wherein the third cell population comprises at least 2, 3, 4, 5, 10, 50, 100, 1000, 10,000, 100,000, or 1,000,000 cell clusters. [Item 122] The method of any one of Items 114 to 121, wherein the third cell population comprises at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% more viable beta cells compared to a corresponding cell population comprising beta cells derived from the first cell population that have not been contacted with the first composition. [Item 123] The method of any one of Items 114 to 122, wherein the third cell population comprises at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% more viable beta cells after about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days of contact between the first cell population and the first composition, compared to a corresponding cell population comprising beta cells derived from the first cell population not contacted with the first composition. [Item 124] The second cell population is incubated with the first cell population and the first composition for about 1 to 10 days, 1 to 9 days, 1 to 8 days, 1 to 7 days, 1 to 6 days, 1 to 5 days, 1 to 4 days, 1 to 3 days, 1 to 2 days, 2 to 10 days, 2 to 9 days, 2 to 8 days, 2 to 7 days, 2 to 6 days, 2 to 5 days, 2 to 4 days, 2 to 3 days, 3 to 10 days, 3 to 9 days, 3 to 8 days, 3 to 7 days, 3 to 6 days, 3 to 5 days, 3 to 124. The method of any one of paragraphs 114 to 123, wherein after 4 days, 4 to 10 days, 4 to 9 days, 4 to 8 days, 4 to 7 days, 4 to 6 days, or 4 to 5 days of contact, the first cell population comprises at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% more viable beta cells compared to a corresponding cell population comprising beta cells derived from the first cell population not contacted with the first composition. [Item 125] The method described in any one of Items 114 to 124, wherein at least a portion of the plurality of beta cells of the third cell population exhibit glucose-stimulated insulin secretion (GSIS) in response to a glucose load in vitro. [Item 126] The method described in any one of Items 114 to 125, wherein at least a portion of the plurality of beta cells of the third cell population express insulin. [Item 127] The method of any one of Items 101 or 104 to 126, wherein the first composition impairs two, three, four, or five of the drugs. [Item 128] The method of any one of Items 102 or 104 to 126, wherein the first composition impairs three, four, five, six, or seven of the drugs. [Item 129] The method of any one of Items 101 to 128, wherein the step of contacting the first cell population with the first composition comprises contacting the first cell population with the first composition for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days or more. [Item 130] The method of Item 129, wherein the step of contacting with the first composition comprises contacting the first cell population with the first composition for about 4 days. [Item 131] The method of any one of Items 101 to 128, wherein the step of contacting the first cell population with the first composition comprises contacting the first cell population with the first composition for about 6 hours, 10 hours, 12 hours, 24 hours, 30 hours, 36 hours, 40 hours, 48 ​​hours, 56 hours, 72 hours, or longer. [Item 132] The step of contacting the first cell population with the first composition may include exposing the first cell population to the first composition for about 1 to 10 days, 1 to 9 days, 1 to 8 days, 1 to 7 days, 1 to 6 days, 1 to 5 days, 1 to 4 days, 1 to 3 days, 1 to 2 days, 2 to 10 days, 2 to 9 days, 2 to 8 days, 2 to 7 days, 2 to 6 days, 2 to 5 days, 2 to 4 days, 2 to 3 days, 3 to 10 days, 3 to 9 days, 3 to 8 days, 3 to 7 days, 3 to 6 days. 129. The method of any one of items 101 to 128, comprising contacting the cells for 3 to 5 days, 3 to 4 days, 4 to 10 days, 4 to 9 days, 4 to 8 days, 4 to 7 days, 4 to 6 days, 4 to 5 days, 5 to 10 days, 5 to 9 days, 5 to 8 days, 5 to 7 days, 5 to 6 days, 6 to 10 days, 6 to 9 days, 6 to 8 days, 6 to 7 days, 7 to 10 days, 7 to 8 days, 8 to 10 days, 8 to 9 days, or 9 to 10 days. [Item 133] The method of any one of Items 101 to 128, wherein the step of contacting the first cell population with the first composition comprises contacting the first cell population with the first composition for about 6 to 96 hours, 6 to 72 hours, 6 to 48 hours, 6 to 24 hours, 6 to 12 hours, 12 to 96 hours, 12 to 72 hours, 12 to 48 hours, 12 to 24 hours, 24 to 96 hours, 24 to 72 hours, 24 to 45 hours, 48 ​​to 96 hours, or 48 to 72 hours. [Item 134] The method of any one of Items 101 to 128, wherein the step of contacting with the first composition comprises contacting the first cell population with the first composition for about 72 hours. [Item 135] The method of any one of Items 101 or 103 to 134, wherein the first composition further comprises a transforming growth factor beta (TGF-β) signaling pathway inhibitor, a thyroid hormone signaling pathway activator, or both. [Item 136] The method according to any one of Items 101 to 135, wherein the first composition comprises an MGLL inhibitor. [Item 137] The method described in any one of Items 101 to 136, wherein the first composition comprises a TGF-β signaling pathway inhibitor. [Item 138] The method described in any one of Items 101 to 137, wherein the first composition comprises a thyroid hormone signaling pathway activator. [Item 139] The method described in any one of items 101 to 138, wherein the first composition comprises a bone morphogenetic protein (BMP) type 1 receptor inhibitor. [Item 140] The method described in any one of items 101 to 139, wherein the first composition comprises a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor. [Item 141] The method described in any one of Items 101 to 140, wherein the first composition comprises a histone methyltransferase inhibitor. [Item 142] The method of any one of Items 101 to 141, wherein the first composition comprises a protein kinase inhibitor. [Item 143] The first composition contains a TGF-β signaling pathway inhibitor, a thyroid hormone 143. The method of any one of items 101 to 142, comprising a Mon signaling pathway activator, a bone morphogenetic protein (BMP) type 1 receptor inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a histone methyltransferase inhibitor, and a protein kinase inhibitor. [Item 144] The method of any one of items 101 to 143, wherein the first composition comprises an MGLL inhibitor, a TGF-β signaling pathway inhibitor, a thyroid hormone signaling pathway activator, a bone morphogenetic protein (BMP) type 1 receptor inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a histone methyltransferase inhibitor, and a protein kinase inhibitor. [Item 145] The method described in any one of Items 101 to 144, wherein the TGF-β signaling pathway inhibitor is Alk5i (SB505124). [Item 146] The method of any one of Items 101 to 145, wherein the thyroid hormone signaling pathway activator is T3 or an analog or derivative thereof. [Item 147] The method described in any one of Items 101 to 146, wherein the thyroid hormone signaling pathway activator is the TRβ selective agonist GC-1. [Item 148] The method according to any one of Items 101 to 147, wherein the thyroid hormone signaling pathway activator is 3,5-dimethyl-4-[(4'-hydroxy-3'-isopropylbenzyl)-phenoxy]acetic acid. [Item 149] The method described in any one of items 101 to 148, wherein the bone morphogenetic protein (BMP) type 1 receptor inhibitor is LDN193189 or a derivative thereof. [Item 150] The method of any one of items 101 to 149, wherein the Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor is thiazovivin. [Item 151] The method of any one of Items 101 to 150, wherein the histone methyltransferase inhibitor is 3-deazaneplanocin A. [Item 152] The method of any one of items 101 to 151, wherein the protein kinase inhibitor is staurosporine (SSP). [Item 153] The method according to any one of Items 101 to 152, wherein the first composition does not contain zinc sulfate. [Item 154] The method of any one of Items 101 to 153, wherein the first composition further comprises a lipid. [Item 155] The method according to Item 154, wherein the lipid is a saturated fatty acid. [Item 156] The method according to Item 155, wherein the saturated fatty acid is palmitate. [Item 157] The method described in Item 154, wherein the lipid is an unsaturated fatty acid. [Item 158] The method according to Item 157, wherein the unsaturated fatty acid is oleic acid, linoleic acid, or palmitoleic acid. [Item 159] The method described in any one of Items 101 to 158, wherein the first composition comprises human serum albumin (HSA). [Item 160] The method according to Item 159, wherein the first composition contains about 0.01 to 5%, 0.01 to 4%, 0.01 to 3%, 0.01 to 2%, 0.01 to 1%, 0.01 to 0.5%, 0.01 to 0.06%, or 0.01 to 0.05% HSA. [Item 161] The method described in Item 159, wherein the first composition contains more than about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1% HSA. [Item 162] The method described in Item 159, wherein the first composition contains less than about 5%, 4%, 3%, 2%, 1%, 0.5%, 0.06%, or 0.05% HSA. [Item 163] The method described in Item 159, wherein the first composition contains about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.9%, 1%, 2%, 3%, 4%, or 5% HSA. [Item 164] The method described in Item 159, wherein the first composition contains about 0.05% HSA. [Item 165] The method of any one of Items 101 to 164, wherein the first composition comprises MCDB 131. [Item 166] The method of any one of Items 101 to 165, wherein the first composition comprises DMEM / F12. [Item 167] The method according to any one of Items 101 to 166, wherein the first composition contains zinc. [Item 168] The method according to any one of Items 101 to 167, wherein the first composition comprises ZnSO4. [Item 169] The method of any one of Items 101 to 168, wherein the first composition comprises at least one metabolite. [Item 170] The method of any one of Items 101 to 169, wherein the at least one metabolite is glutamate, acetate, β-hydroxybutarate, L-carnitine, taurine, formate, or biotin. [Item 171] The method of any one of items 101 to 168, wherein the first composition comprises one, two, three, four, five, six, or seven of glutamate, acetate, beta-hydroxybutarate, L-carnitine, taurine, formate, or biotin. [Item 172] The method of any one of Items 101 to 171, wherein the second composition contains at least one amino acid. [Item 173] The method of Item 172, wherein the at least one amino acid is alanine, glutamate, glycine, proline, threonine, or tryptophan. [Item 174] The method according to any one of Items 101 to 173, wherein the second composition contains at least one vitamin. [Item 175] The method according to Item 174, wherein the at least one vitamin is biotin or riboflavin. [Item 176] The method of any one of Items 101 to 175, wherein the step of contacting the second cell population with the second composition comprises contacting the second cell population with the second composition for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days or more. [Item 177] The method of any one of Items 101 to 175, wherein the step of contacting the second cell population with the second composition comprises contacting the second cell population with the second composition for about 6 hours, 10 hours, 12 hours, 24 hours, 30 hours, 36 hours, 40 hours, 48 ​​hours, 56 hours, 72 hours, or longer. [Item 178] The step of contacting the second cell population with the second composition may include contacting the second cell population with the second composition for about 1 to 10 days, 1 to 9 days, 1 to 8 days, 1 to 7 days, 1 to 6 days, 1 to 5 days, 1 to 4 days, 1 to 3 days, 1 to 2 days, 2 to 10 days, 2 to 9 days, 2 to 8 days, 2 to 7 days, 2 to 6 days, 2 to 5 days, 2 to 4 days, 2 to 3 days, 3 to 10 days, 3 to 9 days, 3 to 8 days, 3 to 7 days, 3 to 6 days. 176. The method of any one of items 101 to 175, comprising contacting the cells for 3 to 5 days, 3 to 4 days, 4 to 10 days, 4 to 9 days, 4 to 8 days, 4 to 7 days, 4 to 6 days, 4 to 5 days, 5 to 10 days, 5 to 9 days, 5 to 8 days, 5 to 7 days, 5 to 6 days, 6 to 10 days, 6 to 9 days, 6 to 8 days, 6 to 7 days, 7 to 10 days, 7 to 8 days, 8 to 10 days, 8 to 9 days, or 9 to 10 days. [Item 179] The method of any one of Items 101 to 175, wherein the step of contacting the second cell population with the second composition comprises contacting the second cell population with the second composition for about 6 to 96 hours, 6 to 72 hours, 6 to 48 hours, 6 to 24 hours, 6 to 12 hours, 12 to 96 hours, 12 to 72 hours, 12 to 48 hours, 12 to 24 hours, 24 to 96 hours, 24 to 72 hours, 24 to 45 hours, 48 ​​to 96 hours, or 48 to 72 hours. [Item 180] The step of contacting the second cell population with the second composition 176. The method of any one of items 101 to 175, comprising contacting with the second composition for about 7 days. [Item 181] The method of any one of items 101 to 180, wherein the second composition does not include one or more of an MGLL inhibitor, a TGF-β signaling pathway inhibitor, a thyroid hormone signaling pathway activator, a bone morphogenetic protein (BMP) type 1 receptor inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a histone methyltransferase inhibitor, or a protein kinase inhibitor. [Item 182] The method according to any one of Items 101 to 181, wherein the second composition does not contain an MGLL inhibitor. [Item 183] The method described in any one of Items 101 to 182, wherein the second composition does not contain a TGF-β signaling pathway inhibitor. [Item 184] The method of any one of Items 101 to 183, wherein the second composition does not contain a thyroid hormone signaling pathway activator. [Item 185] The method described in any one of Items 101 to 184, wherein the second composition does not contain a bone morphogenetic protein (BMP) type 1 receptor inhibitor. [Item 186] The method described in any one of Items 101 to 185, wherein the second composition does not contain a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor. [Item 187] The method described in any one of Items 101 to 186, wherein the second composition does not contain a histone methyltransferase inhibitor. [Item 188] The method of any one of Items 101 to 187, wherein the second composition does not contain a protein kinase inhibitor. [Item 189] The method described in any one of items 101 to 188, wherein the second composition does not contain a TGF-β signaling pathway inhibitor, a thyroid hormone signaling pathway activator, a bone morphogenetic protein (BMP) type 1 receptor inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a histone methyltransferase inhibitor, and a protein kinase inhibitor. [Item 190] The method of any one of items 101 to 188, wherein the second composition does not include an MGLL inhibitor, a TGF-β signaling pathway inhibitor, a thyroid hormone signaling pathway activator, a bone morphogenetic protein (BMP) type 1 receptor inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a histone methyltransferase inhibitor, and a protein kinase inhibitor. [Item 191] The method of any one of Items 101 to 190, wherein the second composition comprises a lipid. [Item 192] The method described in Item 191, wherein the lipid is a saturated fatty acid. [Item 193] The method according to Item 192, wherein the saturated fatty acid is palmitate. [Item 194] The method described in Item 191, wherein the lipid is an unsaturated fatty acid. [Item 195] The method according to Item 194, wherein the unsaturated fatty acid is oleic acid, linoleic acid, or palmitoleic acid. [Item 196] The method of any one of Items 101 to 181, 183 to 189, or 191 to 195, wherein the second composition comprises an MGLL inhibitor. [Item 197] The method described in any one of Items 101 to 196, wherein the second composition does not contain human serum albumin (HSA). [Item 198] The method described in any one of Items 101 to 196, wherein the second composition comprises human serum albumin (HSA). [Item 199] The method according to Item 198, wherein the second composition contains about 0.1 to 5%, 0.1 to 4%, 0.1 to 3%, 0.1 to 2%, 0.1 to 1%, or 0.1 to 0.5% HSA. [Item 200] The method described in Item 199, wherein the second composition contains less than about 5%, 4%, 3%, 2%, 1%, 0.6%, or 0.5% HSA. [Item 201] The second composition may comprise about 0.1%, 0.2%, 0.3%, 0.4%, 0 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, or 5% HSA. [Item 202] The method described in Item 199, wherein the second composition contains about 1% HSA. [Item 203] The method of any one of Items 101 to 202, wherein the second composition comprises MCDB 131. [Item 204] The method of any one of Items 101 to 202, wherein the second composition comprises DMEM / F12. [Item 205] The method according to any one of Items 101 to 204, wherein the second composition contains zinc. [Item 206] The method according to any one of Items 101 to 205, wherein the second composition comprises ZnSO4. [Item 207] The method of any one of Items 101 to 206, wherein the second composition comprises at least one metabolite. [Item 208] The method of Item 207, wherein the at least one metabolite is glutamate, acetate, β-hydroxybutarate, L-carnitine, taurine, formate, or biotin. [Item 209] The method of any one of Items 101 to 206, wherein the second composition comprises one, two, three, four, five, six, or seven of glutamate, acetate, beta-hydroxybutarate, L-carnitine, taurine, formate, or biotin. [Item 210] The method of any one of Items 101 to 209, wherein the second composition contains at least one amino acid. [Item 211] The method of Item 210, wherein the at least one amino acid is alanine, glutamate, glycine, proline, threonine, or tryptophan. [Item 212] The method according to any one of Items 101 to 211, wherein the second composition contains at least one vitamin. [Item 213] The method according to Item 212, wherein the at least one vitamin is biotin or riboflavin. [Item 214] The method of any one of Items 101 to 213, wherein the dissociating step does not include a step of subjecting the cell population to flow cytometry. [Item 215] (a) obtaining a first cell population including a plurality of cell clusters including insulin-positive endocrine cells; (b) dissociating in vitro at least a portion of the plurality of cell clusters in the first cell population; (c) freezing at least a portion of the first cell population, including at least a portion of the dissociated cell clusters; (d) thawing at least a portion of the frozen first cell population; (e) contacting in vitro the at least a portion of the thawed first cell population with a first composition to obtain a second cell population comprising a plurality of insulin-positive endocrine cells comprising a plurality of cell clusters, wherein the first composition comprises one, two, three, four, or five of the following agents: a monoglyceride lipase (MGLL) inhibitor, a bone morphogenetic protein (BMP) type 1 receptor inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a histone methyltransferase inhibitor, or a protein kinase inhibitor; (f) contacting the second insulin-positive endocrine cell population in vitro with a second composition, thereby differentiating at least a portion of the second insulin-positive endocrine cell population into a third cell population comprising a plurality of beta cells comprising a plurality of cell clusters, wherein the second composition is different from the first composition, and the third cell population comprises corresponding beta cells derived from the first cell population that were not contacted with the first composition. a step containing a higher percentage of viable beta cells compared to the beta cell population; A method comprising: [Item 216] The method described in Item 215, wherein the first composition comprises a bone morphogenetic protein (BMP) type 1 receptor inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a histone methyltransferase inhibitor, and a protein kinase inhibitor. [Item 217] The method described in Item 215 or 216, wherein the first composition further contains a transforming growth factor β (TGF-β) signaling pathway inhibitor. [Item 218] The method described in any one of Items 215 to 217, wherein the first composition further comprises a thyroid hormone signaling pathway activator. [Item 219] The method described in any one of Items 215 to 218, wherein the first composition further contains a monoglyceride lipase (MGLL) inhibitor. [Item 220] (a) obtaining a first cell population including a plurality of cell clusters including insulin-positive endocrine cells; (b) dissociating in vitro at least a portion of the plurality of cell clusters in the first cell population; (c) freezing at least a portion of the first cell population, including at least a portion of the dissociated cell clusters; (d) thawing at least a portion of the frozen first cell population; (e) contacting the at least a portion of the thawed first cell population in vitro with a first composition to obtain a second cell population comprising a plurality of insulin-positive endocrine cells comprising a plurality of cell clusters, wherein the first composition comprises a transforming growth factor beta (TGF-β) signaling pathway inhibitor, a thyroid hormone signaling pathway activator, or both, and one, two, three, four, or five of the following agents: a monoglyceride lipase (MGLL) inhibitor, a bone morphogenetic protein (BMP) type 1 receptor inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a histone methyltransferase inhibitor, or a protein kinase inhibitor; (f) contacting the second insulin-positive endocrine cell population in vitro with a second composition, thereby differentiating at least a portion of the second insulin-positive endocrine cell population into a third cell population comprising a plurality of beta cells comprising a plurality of cell clusters, wherein the second composition is different from the first composition, and the third cell population comprises a higher percentage of viable beta cells compared to a corresponding beta cell population comprising beta cells derived from the first cell population not contacted with the first composition; A method comprising: [Item 221] The method described in Item 220, wherein the first composition comprises a monoglyceride lipase (MGLL) inhibitor, a bone morphogenetic protein (BMP) type 1 receptor inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a histone methyltransferase inhibitor, and a protein kinase inhibitor. [Item 222] (a) obtaining a first cell population including a plurality of cell clusters including insulin-positive endocrine cells; (b) dissociating in vitro at least a portion of the plurality of cell clusters in the first cell population; (c) contacting the first cell population comprising at least a portion of the dissociated cell clusters with a first composition in vitro to obtain a second cell population comprising a plurality of cell clusters, the second cell population comprising a plurality of insulin-positive endocrine cells; (d) contacting the second insulin-positive endocrine cell population in vitro with a second composition, thereby differentiating at least a portion of the second insulin-positive endocrine cell population into a third cell population comprising a plurality of beta cells, wherein the second composition is different from the first composition, and the second composition comprises at least one metabolite. wherein the third cell population comprises a higher percentage of viable cells compared to a corresponding cell population comprising beta cells derived from the first cell population that has not been contacted with the second composition. A method comprising: [Item 223] (a) obtaining a first cell population including a plurality of cell clusters including insulin-positive endocrine cells; (b) dissociating in vitro at least a portion of the plurality of cell clusters in the first cell population; (c) contacting the first cell population comprising at least a portion of the dissociated cell clusters with a first composition in vitro to obtain a second cell population comprising a plurality of cell clusters, the second cell population comprising a plurality of insulin-positive endocrine cells; (d) contacting the second insulin-positive endocrine cell population in vitro with a second composition, thereby differentiating at least a portion of the second insulin-positive endocrine cell population into a third cell population comprising a plurality of beta cells, wherein the second composition is different from the first composition, the second composition comprising at least one metabolite, and the plurality of beta cells exhibits enhanced glucose-stimulated insulin secretion relative to a corresponding cell population comprising beta cells derived from the first cell population not contacted with the second composition; A method comprising: [Item 224] The method described in Item 222 or 223, wherein the metabolite is an amino acid, a vitamin, a polyol, an organic substance, an acid, an antioxidant, a nucleotide, or an alcohol. [Item 225] The method according to Item 222 or 223, wherein the at least one metabolite is glutamate, acetate, β-hydroxybutarate, L-carnitine, taurine, formate, or biotin. [Item 226] The method of Item 222 or 223, wherein the second composition comprises at least two, three, four, five, six, or seven metabolites of glutamate, acetate, beta-hydroxybutarate, L-carnitine, taurine, formate, or biotin. [Item 227] The method of any one of Items 222 to 226, wherein the second composition comprises DMEM / F12. [Item 228] The method according to any one of Items 222 to 227, wherein the second composition contains about 0.05 to 2% HSA. [Item 229] The method described in Item 228, wherein the second composition contains about 1% HSA. [Item 230] The method according to any one of Items 222 to 229, wherein the second composition contains zinc. [Item 231] The method according to any one of Items 222 to 230, wherein the second composition comprises ZnSO4. [Item 232] The method of any one of Items 222 to 231, wherein the second composition contains at least one amino acid. [Item 233] The method of Item 231, wherein the at least one amino acid is alanine, glutamate, glycine, proline, threonine, or tryptophan. [Item 234] The method according to any one of Items 222 to 233, wherein the second composition contains at least one vitamin. [Item 235] The method according to Item 234, wherein the at least one vitamin is biotin or riboflavin. [Item 236] The method described in any one of Items 222 to 235, wherein the plurality of beta cells exhibits enhanced glucose-stimulated insulin secretion compared to a corresponding cell population comprising beta cells derived from the first cell population that have not been contacted with the second composition. [Item 237] The third cell population is the first cell population that has not been contacted with the second composition. 237. The method of any one of paragraphs 222 to 236, wherein the cell population comprises a higher percentage of viable cells compared to a corresponding cell population comprising beta cells derived therefrom. [Item 238] The method of any one of Items 222 to 237, wherein the third cell population comprises a plurality of cell clusters, each having a diameter of approximately 50 to 150 microns. [Item 239] The method of any one of Items 222 to 238, wherein the third cell population comprises a plurality of cell clusters, each having a diameter of about 100 microns. [Item 240] The method of any one of Items 222 to 239, wherein the third cell population comprises a plurality of cell clusters, and at least 50%, 60%, 70%, 75%, 80%, 90%, or 95% of the cell clusters have a diameter of about 100 microns without a selection step. [Item 241] The method of any one of Items 222 to 240, wherein the third cell population comprises a plurality of cell clusters, and at least 50%, 60%, 70%, 75%, 80%, 90%, or 95% of the cell clusters have a diameter of approximately 50 to 150 microns, 75 to 125 microns, 80 to 120 microns, or 90 to 110 microns without a selection step. [Item 242] The method of any one of items 222 to 241, wherein the first composition comprises at least one agent selected from the group consisting of a monoglyceride lipase (MGLL) inhibitor, a bone morphogenetic protein (BMP) type 1 receptor inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a histone methyltransferase inhibitor, and a protein kinase inhibitor. [Item 243] (a) freezing at least a portion of the first cell population, including at least a portion of the dissociated cell clusters; (b) thawing at least a portion of the frozen first cell population; (c) contacting at least a portion of the thawed first cell population in vitro with the first composition; 239. The method of any one of items 219 to 239, further comprising: [Item 244] A composition comprising at least a portion of the second cell population comprising insulin-positive endocrine cells described in any one of Items 101 to 243. [Item 245] A composition comprising at least a portion of the third cell population of beta cells described in any one of Items 101 to 243. [Item 246] A composition comprising at least a portion of the third cell population of beta cells described in any one of Items 101 to 243 and at least a portion of the second cell population comprising insulin-positive endocrine cells described in any one of Items 101 to 243. [Item 247] A device comprising the beta cell composition described in Item 244. [Item 248] A device comprising the insulin-positive endocrine cell composition described in Item 245. [Item 249] A device comprising the composition of insulin-positive endocrine cells and beta cells described in Item 246. [Item 250] A method for treating a subject having a disease characterized by prolonged high blood glucose levels (e.g., diabetes), comprising administering to the subject a composition of cells described in Item 244. [Item 251] A method for treating a subject having a disease characterized by prolonged high blood glucose levels (e.g., diabetes), comprising administering to the subject a composition of cells described in Item 245. [Item 252] A method for treating a subject having a disease characterized by prolonged high blood sugar levels (e.g., diabetes), comprising administering to the subject a composition of cells described in Item 246. [Item 253] A method for treating a subject having a disease characterized by prolonged high blood glucose levels (e.g., diabetes), comprising implanting the device described in Item 247 into the subject. [Item 254] A method for treating a subject having a disease characterized by prolonged high blood glucose levels (e.g., diabetes), comprising implanting the device described in Item 248 into the subject. [Item 255] A method for treating a subject having a disease characterized by prolonged high blood glucose levels (e.g., diabetes), comprising implanting the device described in Item 249 into the subject. [Item 256] The method according to any one of items 250 to 255, wherein the disease is diabetes. [Item 257] A composition comprising isolated insulin-positive endocrine cells that have been contacted in vitro with an agent that inhibits the expression or function of monoglyceride lipase (MGLL) and that exhibit a reduced rate of conversion of monoglycerides to free fatty acids compared to a corresponding isolated insulin-positive endocrine cell population that has not been contacted in vitro with the agent that inhibits the expression or function of monoglyceride lipase (MGLL). [Item 258] A composition comprising isolated insulin-positive endocrine cells that have been contacted in vitro with an agent that inhibits the expression or function of monoglyceride lipase (MGLL) and that exhibit an increased ratio of monoglycerides to free fatty acids compared to a corresponding isolated insulin-positive endocrine cell population that has not been contacted in vitro with an agent that inhibits the expression or function of monoglyceride lipase (MGLL). [Item 259] A composition comprising isolated insulin-positive endocrine cells that have been contacted in vitro with an agent that inhibits the expression or function of monoglyceride lipase (MGLL) and that exhibit a decreased ratio of free fatty acids to monoglycerides compared to a corresponding isolated insulin-positive endocrine cell population that has not been contacted in vitro with an agent that inhibits the expression or function of monoglyceride lipase (MGLL). [Item 260] A composition comprising isolated insulin-positive endocrine cells that have been contacted in vitro with an agent that inhibits the expression or function of monoglyceride lipase (MGLL) and that exhibit reduced levels of free fatty acids compared to a corresponding isolated insulin-positive endocrine cell population that has not been contacted in vitro with an agent that inhibits the expression or function of monoglyceride lipase (MGLL). [Item 261] A composition comprising isolated insulin-positive endocrine cells that have been contacted in vitro with an agent that inhibits the expression or function of monoglyceride lipase (MGLL) and that exhibit elevated levels of monoglycerides compared to a corresponding isolated insulin-positive endocrine cell population that has not been contacted in vitro with an agent that inhibits the expression or function of monoglyceride lipase (MGLL). [Item 262] A composition comprising an insulin-positive endocrine cell population and an agent that inhibits the conversion of monoglycerides to free fatty acids. [Item 263] The composition described in Item 262, wherein the agent inhibits the expression or function of monoglyceride lipase (MGLL). [Item 264] A composition comprising an insulin-positive cell population and an agent that inhibits the expression or function of monoglyceride lipase (MGLL). [Item 265] The composition of any one of Items 257 to 264, wherein the agent that inhibits the expression or function of monoglyceride lipase (MGLL) is JJKK048, KML29, NF1819, JW642, JZL184, JZL195, JZP361, pristimerin, or URB602. [Item 266] A composition comprising a beta cell population that has been contacted in vitro with at least one agent selected from the group consisting of glutamate, acetate, beta-hydroxybutarate, L-carnitine, taurine, formate, or biotin, wherein the beta cell population exhibits increased glucose-stimulated insulin secretion compared to a corresponding beta cell population that has not been contacted with the at least one agent. [Item 267] The cell population is selected from the group consisting of glutamate, acetate, β-hydroxybutarate, L-carnitine, taurine, formate, or biotin. 267. The composition of claim 266, which has been in contact with at least two, three, four, five, six, or seven of the agents. [Item 268] A composition comprising a beta cell population and at least one, two, three, four, five, six, or seven of the following agents selected from the group consisting of glutamate, acetate, beta-hydroxybutarate, L-carnitine, taurine, formate, or biotin.

[0081] BRIEF DESCRIPTION OF THE DRAWINGS

[0106] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained from the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, as well as the accompanying drawings (which are also incorporated herein by reference as "Figures and Drawings"). and "FIG." [Brief explanation of the drawings]

[0082] [Figure 1]

[0107] FIG. 1 is a graph showing the percentage of viable stage 6 cells recovered after thawing of cryopreserved stage 5 cells using a thawing medium containing DMEM F12 and 1% human serum albumin (HSA). [Figure 2]

[0108] FIG. 2 is a diagram outlining one of the experimental protocols described in Example 1. [Figure 3]

[0109] 3 is a bar graph showing percent aggregation of cells recovered from seeded cells on day 4 of stage 6 (S6d4). Cells were cultured in the indicated stage 6 (S6) medium, modified stage 5 (S5) medium containing XXI, or modified stage 5 (S5) medium without XXI with the indicated supplements, i.e., glucose (Glc) and pyruvate (Pyr). [Figure 4]

[0110] Figure 4 is a bar graph showing the percentage of CHGA-positive cells recovered at stage 6 day 4 (S6d4). Cells were cultured in the indicated stage 6 (S6) medium, modified stage 5 (S5) medium containing XXI, or modified stage 5 (S5) medium without XXI with the indicated supplements, i.e., glucose (Glc) and pyruvate (Pyr). [Figure 5A]

[0111] Figure 5A is a FACS plot showing the percentage of stem cell-derived β cells (Nkx6.1 / Isl1 double-positive cells) at stage 6 day 4 (S6d4). Cells were cultured in the indicated stage 6 (S6) medium, modified stage 5 (S5) medium containing XXI, or modified stage 5 (S5) medium without XXI. [Figure 5B] Figure 5B is a table showing the percentage of stem cell-derived β cells (Nkx6.1 / Isl1 double-positive cells) at stage 6 day 4 (S6d4). Cells were cultured in the indicated stage 6 (S6) medium, modified stage 5 (S5) medium containing XXI, or modified stage 5 (S5) medium without XXI with the indicated supplements, i.e., glucose (Glc) and pyruvate (Pyr). [Figure 6]

[0112] Figure 6 is a bar graph showing the fold improvement in recovery of stem cell-derived beta cells at stage 6 day 4 (S6d4). Cells were cultured in the indicated Stage 6 (S6) medium, modified Stage 5 (S5) medium containing XXI, or modified Stage 5 (S5) medium without XXI with the indicated supplements, namely glucose (Glc) and pyruvate (Pyr). [Figure 7]

[0113] Figure 7 is a bar graph showing glucose-stimulated insulin secretion (GSIS) of cells at stage 6 day 11 (S6d11). GSIS is measured by the level of human C-peptide (pM) per 1000 cells. Cells were cultured in either stage 6 (S6) medium or modified stage 5 (S5) medium, as indicated, for days 1-4 of stage 6 (approximately 72 hours), and in stage 6 (S6) medium for days 4-7 or 4-11, as indicated. [Figure 8]

[0114] Figure 8 shows microscopic images of cultured cells at day 7 (S6d7) of stage 6. Cells were cultured in stage 6 (S6) medium, modified stage 5 (S5) medium containing XXI, or modified stage 5 (S5) medium without XXI, as indicated, for days 1–4 of stage 6 (approximately 72 hours). Cells were then cultured in S6 (S6) medium for days 4–7 of stage 6. As indicated, modified stage 5 basal medium was MCDB 131 containing 0.05% HSA. Stage 6 basal medium was DMEM F12 containing 1% HSA. [Figure 9]

[0115] Figure 9 shows microscopic images of stage 6 cell clusters arising from cells cultured in stage 6 (S6) medium containing 1.0% HSA for the duration of stage 6 (left) or in modified stage 5 (S5) medium containing 0.05% HSA for days 1-4 of stage 6 (approximately 72 hours) (right). [Figure 10]

[0116] Figure 10A is a bar graph showing the levels of human C-peptide (pM) per 1000 cells at stage 6 day 11 (S6d11). Cells were cultured in either stage 6 (S6) medium or modified stage 5 (S5) medium, as indicated, for days 1-4 of stage 6 (approximately 72 hours), followed by culture in stage 6 (S6) medium for days 4-11 of stage 6. Figure 10B is a FACS plot showing the percentage of stem cell-derived beta cells (Nkx6.1 / Isl1 double-positive cells) at stage 6 day 4 (S6d4). Cells were cultured in either stage 6 (S6) medium or modified stage 5 (S5) medium, as indicated, for days 1-4 of stage 6 (approximately 72 hours), followed by culture in stage 6 (S6) medium for days 4-11 of stage 6. [Figure 11]

[0117] Figure 11 is a graph showing the percentage of cells recovered from seeding when cells were cultured in Stage 6 medium (left) or modified Stage 5 medium (without XXI) (right) between days 1 and 4 of Stage 6 (approximately 72 hours). [Figure 12]

[0118] FIG. 12 is a table summarizing the results of Example 1, showing the effect of culturing cells in Stage 6 medium or Stage 5 day 6 medium (S5d6 medium) on days 1-4 of Stage 6 (approximately 72 hours) on total cell recovery, cell yield, percentage (composition) of beta cells, insulin content of recovered beta cells, and GSIS of the cells. [Figure 13]

[0119] FIG. 13 is a diagram outlining one of the experimental protocols described in Example 2. [Figure 14]

[0120] 14 is a bar graph showing the percentage of total cells and the percentage of stem cell-derived β cells recovered at stage 6 day 4 (S6d4). Cells were cultured in medium containing 1% HSA or 0.05% HSA as indicated, along with the indicated modified stage 5 medium factors. [Figure 15]

[0121] 15 is a bar graph showing the number of stem cell-derived beta cells at stage 6 day 4 (S6d4). Cells were cultured in medium containing 1% HSA or 0.05% HSA as indicated, along with the modified stage 5 medium factors as indicated. [Figure 16]

[0122] 16 is a bar graph showing the number of stem cell-derived beta cells at stage 6 day 12 (S6d12). Cells were cultured in medium containing 1% HSA or 0.05% HSA as indicated, along with the modified stage 5 medium factors as indicated. [Figure 17]

[0123] 17 is a bar graph showing human C-peptide levels (pM) per 1000 cells at stage 6 day 12 (S6d12). Cells were cultured in medium containing 1% HSA or 0.05% HSA as indicated, along with the modified stage 5 media factors indicated. [Figure 18]

[0124] Figure 18A is a bar graph showing glucose-stimulated insulin secretion (GSIS) of cells at stage 6 day 12 (S6d12). GSIS was measured as the level of human C-peptide (pM) per 1000 cells after glucose stimulation. Cells were cultured in stage 6 (S6) medium + 1% HSA from days 1 to 12 of stage 6. Figure 18B is a bar graph showing glucose-stimulated insulin secretion (GSIS) of cells at stage 6 day 12 (S6d12). GSIS was measured as the level of human C-peptide (pM) per 1000 cells after glucose stimulation. Cells were cultured in modified stage 5 (S5) medium + 0.05% HSA from days 1 to 4 of stage 6 (approximately 72 hours) and in basal stage 5 (S5) medium + 0.05% HSA from days 4 to 12 of stage 6. [Figure 19]

[0125] FIG. 19 is a table illustrating the results of Example 2, showing the effect of Stage 6 medium or Stage 5 day 6 medium (S5d6 medium) for culturing cells on days 1-4 of Stage 6 on total cell recovery, cell yield, composition of recovered cells, insulin content of cells, and GSIS of cells. [Figure 20]

[0126] FIG. 20 is a diagram outlining one of the experimental protocols described in Example 3. [Figure 21]

[0127] Figure 21 is a bar graph showing the percentage of recovered SC-β cells (Nkx6.1 / Isl1 double positive cells) when stage 5 cells were cultured in the indicated media for days 1-4 of stage 6 (approximately 72 hours). [Figure 22]

[0128] Figure 22 is a bar graph showing the number of SC-β cells at stage 6 day 10 (S6d10) when stage 5 cells were cultured in the indicated media for stage 6 days 1-4 (approximately 72 hours). [Figure 23]

[0129] Figure 23 shows microscopic images of stage 6 day 4 (S6d4) cell clusters arising from stage 5 cells cultured in modified stage 5 (S5) medium containing 0.5% HSA (left), modified stage 5 (S5) medium containing 0.5% HSA and palmitate (center), or modified stage 5 (S5) medium containing 0.5% HSA and linoleic acid (right) for stage 6 days 1-4 (approximately 72 hours). [Figure 24]

[0130] Figure 24A is a bar graph showing the number of cells at stage 6 day 10 (S6d10) when stage 5 cells were cultured in the indicated medium for days 1-4 of stage 6 (approximately 72 hours) and in stage 6 (S6) medium for days 4-10 of stage 6. Figure 24B is a bar graph showing the number of stem cell-derived β cells (SC-β) at stage 6 day 10 (S6d10) when stage 5 cells were cultured in the indicated medium for days 1-4 of stage 6 (approximately 72 hours) and in stage 6 (S6) medium for days 4-10 of stage 6. [Figure 25]

[0131] FIG. 25 is a bar graph showing C-peptide content in 16 different samples of stage 6 day 14 (S6d14) cells cultured in medium containing MGLL inhibitors (1 μM JJKK048, 10 μM KML-29, 10 μM NF1819). [Figure 26]

[0132] Figure 26A is a bar graph showing glucose-stimulated insulin secretion (GSIS) of Stage 6 day 10 (S6d10) cells cultured in Stage 6 (S6) medium from days 1 to 10 of Stage 6. Figure 26B is a bar graph showing glucose-stimulated insulin secretion (GSIS) of Stage 6 day 10 (S6d10) cells cultured in modified Stage 5 (S5) medium containing 0.05% HSA from days 1 to 4 of Stage 6 and in Stage 6 (S6) medium from days 4 to 10 of Stage 6. [Figure 27]

[0133] Figure 27A is a bar graph showing glucose-stimulated insulin secretion (GSIS) of stage 6 day 10 (S6d10) cells cultured in modified stage 5 (S5) medium containing 0.5% HSA for days 1-4 of stage 6 (approximately 72 hours). Figure 27B is a bar graph showing glucose-stimulated insulin secretion (GSIS) of stage 6 day 10 (S6d10) cells cultured in modified stage 5 (S5) medium containing 0.5% HSA and palmitate for days 1-4 of stage 6. [Figure 28]

[0134] Figure 28 is a series of FACS plots showing the percentage of stem cell-derived β (SC-β) cells (Nkx6.1 / Isl1 double positive cells) recovered at stage 6 when cells were cultured in modified S5d6 medium and MGLL inhibitor (days 1-10 of stage 6) (right two plots) compared to cells cultured in S6 control medium (left) or modified S5 medium containing factors (center). [Figure 29]

[0135] FIG. 29 is a table summarizing the results of Examples 3 and 4, showing the effect of 1% HSA, fatty acid, and MGLL inhibitor supplementation (to Stage 5 medium MCBD containing factors and 0.05% HSA) in culture of cells at Stage 6 days 1-4 (approximately 72 hours) on the yield of stem cell-derived beta cells, the percentage of stem cell-derived beta cells (composition), the insulin content of harvested stem cell-derived beta cells (content), and glucose-stimulated insulin secretion (GSIS) of stem cell-derived beta cells. [Figure 30]

[0136] Figure 30 is a diagram depicting Stage 6, where Day 1 (D1) begins with thawing of cryopreserved Stage 5 cells, Day 4 (D4) is a process intermediate, Day 7 (D7) is the drug substance, and Day 11 (D11) is cells with GSIS activity and insulin content. DS2 medium is an optional Stage 6 medium containing DMDM / F12 and 1% HSA used over Days 1-11. DS3 medium is a second optional Stage 6 medium. During days 1-4 of stage 6, DS3 medium contains MCDB 131 supplemented with S5d6 factors (Alk5i (10 µM), GC-1 (1 µM), LDN-193189 (100 nM), thiazovinin (2.5 µM), SSP (3 nM), DZNEP (100 nM)), 0.05% HSA, ITS-X, glutamax, VitC, and, if necessary, additional drugs such as lipids and MGLL inhibitors. During days 5-11 of stage 6, DS3 medium contains MCDB 131 supplemented with 0.05% HSA. [Figure 31]

[0137] FIG. 31 is a table listing the compositions of Stage 6 Medium DS2 and Stage 6 Medium DS3. [Figure 32]

[0138] 32 is a table illustrating the recovery and functional properties of DS2 Stage 6 medium compared to DS3 Stage 6 medium. As shown, DS3 medium improved cell recovery at Stage 6 day 4 (S6d4) and Stage 6 day 7 (S6d7) compared to DS2 Stage 6 medium. DS3 Stage 6 medium did not improve cell recovery at Stage 6 day 11 (S6d11), nor did it improve the insulin content of cells or the percentage of SC-β cells compared to DS2 Stage 6 medium. DS3 Stage 6 medium reduced glucose-stimulated insulin secretion (GSIS) function compared to DS2 Stage 6 medium. [Figure 33]

[0139] 33 is a bar graph showing the percent of cells recovered from viable seeded cells at S6d4, S6d7, or S6d11 from either DS2 cultured stage 6 cells or DS3 cultured stage 6 cells. The results show, on average, improved percent cell recovery over stage 6 when using DS3 medium. [Figure 34]

[0140] Figure 34A is a bar graph showing the percentage of SC-β cells (Nkx6.1 / Isl1 cells) at S6d4 or S6d11 derived from either DS2 or DS3 stage 6 cultured cells. The results show that DS3 stage 6 medium maintains a similar percentage of SC-β cells as DS2 stage 6 medium. Figure 34B shows the percentage of endocrine cells (chga+ cells) at S6d4 or S6d11 derived from either DS2 or DS3 stage 6 cultured cells. The results show that DS3 stage 6 medium maintains a similar percentage of endocrine cells as DS2 stage 6 medium. [Figure 35] 35 is a bar graph showing the total levels of insulin content of SC islets at S6d4 or S6d11 derived from either DS2-cultured stage 6 cells or DS3-cultured stage 6 cells. The results show that cells cultured in DS3 stage 6 medium have similar insulin content to cells cultured in DS2 stage 6 medium. [Figure 36] 36 is a bar graph showing glucose-stimulated insulin secretion of cells cultured in DS2 stage 6 medium or DS3 stage 6 medium and either high glucose stimulation (HG), low glucose stimulation (LG), or KLC treatment (positive control). The results show that DS3 medium produces SC islets with lower GSIS function compared to DS2 stage 6 medium. [Figure 37] 37 is a line graph showing blood glucose levels (mg / dL) in mice implanted with devices containing SC islets cultured in DS3 stage 6 medium or DS2 stage 6 medium. The results demonstrate that DS2 and DS3 SC islets can function in vivo. [Figure 38]

[0145] Figure 38 is a line graph showing blood glucose levels (mg / dL) in mice implanted with devices containing SC islets cultured in DS3 stage 6 medium, demonstrating in vivo blood glucose regulation. [Figure 39]

[0146] Figure 39 is a diagram illustrating a third Stage 6 medium ("DS6") and its components in comparison with DS2 Stage 6 medium and DS3 Stage 6 medium. [Figure 40]

[0147] Figure 40 is a scatter plot showing the ratios of amino acids in human plasma-like medium (HPLM) to those in MCDB 131 (circles), HPLM to DMEM / F12 (squares), and HPLM to CRML (triangles). The results show that MCDB 131 medium contains lower levels of certain amino acids, including, for example, alanine, glutamate, and glycine. [Figure 41]

[0148] Figure 41 is a scatter plot showing the ratios of vitamins in human plasma-like medium (HPLM) to those in MCDB 131 (circles), HPLM to DMEM / F12 (squares), and HPLM to CRML (triangles). The results show that MCDB 131 medium contains lower levels of certain vitamins, including, for example, biotin and riboflavin. [Figure 42] 42 is a table showing the total number of viable cells at S6d4, S6d7, and S6d11 cultured in DS2 Stage 6 medium, DS3 Stage 6 medium, or DS3 Stage 6 medium containing ZnSO. The results show that including ZnSO in DS3 medium greatly improves the number of viable S6d4 cells, but cell loss still exists after S6d4. [Figure 43] 43 is a bar graph showing the percent of cells recovered on S6d4, S6d7, and S6d11 cultured in DS2, DS3, or DS3 containing ZnSO. The results show that including ZnSO in DS3 medium significantly improves S6d4 cell recovery, but cell loss still exists after S6d4. [Figure 44]

[0151] Figure 44 is a FACS plot showing the number of β cells (chga+ / Nkx6.1+) recovered on S6d4 using DS3 stage 6 medium supplemented with ZnSO4. [Figure 45] Figure 45A is a table showing the total number of viable S6d11 cells cultured in DS2 Stage 6 medium, DS3 Stage 6 medium, DS3 Stage 6 medium supplemented with metabolites, or MCDB 131 medium without vitamins but supplemented with amino acids and metabolites. The results show that including metabolites and vitamins in DS3 medium significantly improves the number of viable S6d11 cells. Figure 45B is a bar graph showing the percentage of cells recovered in S6d4 or S6d11 cultured in DS2, DS3, or DS3 supplemented with metabolites. The results show that including metabolites in DS3 medium significantly improves S6d11 cell recovery. [Figure 46] 46 is a bar graph showing the percentage of SC-β cells (Nkx6.1 / Isl1+ cells) at S6d11 cultured in DS2 medium, DS3 medium containing the indicated supplements, and MCDB 131 medium containing the indicated supplements. The data show that additional medium supplements (e.g., vitamins, amino acids, metabolites, and lipids) can improve the percentage of SC-β cells over S6d11. [Figure 47] 47 is a bar graph showing glucose-stimulated insulin secretion (GSIS) of cells cultured in MCDB 131 medium containing DS2, DS3, or the indicated supplements. The results show that the supplements are insufficient to improve high-glucose GSIS function in MCDB 131-cultured cells to the level of GSIS in DS2-cultured cells, but do improve some aspects, such as the magnitude of KCL-induced insulin secretion. [Figure 48]Figure 48A is a bar graph showing the number of viable cells recovered at S6d7 and S6d11 from DS2, DS3, DS6 (metabolite-free), or DS6 (metabolite-containing) cultures. The data show that DS6 medium using DMEM / F12 further improves viable cell numbers throughout Stage 6. Figure 48B is a bar graph showing the percentage of cells recovered at S6d7 and S6d11 from DS2, DS3, DS6 (metabolite-free), or DS6 (metabolite-containing) cultures. The data show that DS6 medium using DMEM / F12 further improves cell recovery throughout Stage 6. [Figure 49]

[0158] Figure 49 shows microscopic images of SC islet clusters at S6d7 and S6d11 from DS2, DS3, or DS6 (metabolite-containing) cultures. The images show that DS6 clusters exhibit greater uniformity throughout stage 6. [Figure 50]

[0159] Figure 50 is a series of area graphs showing the frequency of cell clusters and cluster size at S6d4, S6d7, and S6d11 from DS2, DS3, and DS6 cultures. The results show that DS6 clusters are smaller and exhibit greater uniformity at S6d11. [Figure 51]

[0160] Figure 51 is a bar graph showing GSIS function of S6d11 cells derived from DS2 culture. [Figure 52]

[0161] Figure 52 is a bar graph showing GSIS function of S6d11 cells from DS3 culture. The results show that SC islets in DS3 medium do not exhibit as much GSIS function as those in DS2 (compare Figure 51). [Figure 53]

[0162] Figure 53 is a bar graph showing GSIS function of S6d11 cells from DS6 (metabolite-free) culture. The results show that SC islets in DS6 (metabolite-free) medium exhibit improved GSIS function (compare Figure 51). [Figure 54]

[0163] Figure 54 is a bar graph showing GSIS function of S6d11 cells from DS6 (metabolite-containing) culture. The results show that SC islets in DS6 medium exhibit identical GSIS function to DS2 culture (compare Figure 51). [Figure 55]

[0164] Figure 55 is a table showing the effect of DS5 Stage 6 medium. DS6 Stage 6 medium shows improved S6d11 cell recovery compared to DS3 medium and improved GSIS function compared to DS2 medium. [Figure 56]

[0165] Figure 56 is an area graph showing cluster size of SC islets cultured in Stage 5 medium with and without lipid supplementation. The results show that cluster size is increased by lipid supplementation. [Figure 57]

[0166] Figure 57 is a bar graph showing the percent of blood glucose control in mice implanted with intracellularly cultured SC islets. The results show that the modified formulation not only improves cluster reaggregation after cryopreservation, but also that the cells exhibit in vivo efficacy. DETAILED DESCRIPTION OF THE INVENTION

[0083]

[0167] Provided herein, inter alia, are methods for increasing reaggregation efficiency and optimizing cluster size and in vitro SC-β cell function. The present specification discloses the identification of novel signaling requirements that not only enhance CDGA-positive endocrine cell reaggregation efficiency but also improve SC islet composition and cluster size, as well as SC-β cell function in vitro. The novel methods can be used for large-scale production of SC islets for human therapeutic use.

[0084]

[0168] While various embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, modifications, and substitutions will occur to those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be employed.

[0085] definition

[0169] 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.

[0086]

[0170] In this application, the use of "or" means "and / or" unless stated otherwise. The terms "and / or" and "any combination thereof," as well as their grammatical equivalents, can be used interchangeably when used herein. These terms can convey that any combination is specifically contemplated. For illustrative purposes only, the following phrases, "A, B, and / or C" or "A, B, C, or any combination thereof," can be used interchangeably with "A only, B only, C only, A and B, B and C." , A and C, and A and B and C." The term "or" can be used conjunctively or disjunctively, unless the context clearly indicates a reference to the disjunctive use.

[0087]

[0171] Furthermore, use of the term "including" as well as other word forms such as "include," "includes," and "included" is not limiting.

[0088]

[0172] References herein to "some embodiments," "an embodiment," "one embodiment," or "other embodiments" mean that the particular feature, structure, or characteristic described in connection with the embodiment is included in at least some embodiments of the present disclosure, but not necessarily in all embodiments.

[0089]

[0173] As used in the specification and claims, the words "comprising" (and any form of comprising, e.g., "comprise" and "comprises"), "having" (and any form of having, e.g., "have" and "has"), "including" (and any form of including, e.g., "includes" and "include"), or "containing" (and any form of containing, e.g., "contains" and "contain") are non-exclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated 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.

[0090]

[0174] The term "about" and its grammatical equivalents, as used herein, in reference to a reference numerical value, can include both the numerical value itself and values ​​within a range of plus or minus 10% from the numerical value.

[0091]

[0175] The term "about" or "approximately" means within an acceptable error range of a particular value, as determined by one of ordinary skill in the art, and depends, in part, on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean within 1 or more standard deviations, as is customary in the art. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. In another example, the amount "about 10" includes any amount between 10 and 9-11. In yet another example, the term "about" in reference to a numerical reference can also include values ​​within a range 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 10-fold, preferably within 5-fold, and more preferably within 2-fold of a value. When particular values ​​are described in this application and claims, unless otherwise stated, the term "about" meaning within an acceptable error range of the particular value should be assumed.

[0092]

[0176] The term "diabetes" and its grammatical equivalents, as used herein, can refer to the disease characterized by long-term high blood sugar levels.For example, the term "diabetes" and its grammatical equivalents, as used herein, can refer to all or any type of diabetes, for example, but not limited to, type 1, type 2, cystic fibrosis-related, surgical, gestational diabetes, and mitochondrial diabetes.In some cases, diabetes can be a form of hereditary diabetes.

[0093]

[0177] The term "endocrine cells," unless otherwise specified, can refer to hormone-producing cells present in the pancreas of an organism, e.g., "islets," "islet cells," "islet equivalents," "islet-like cells," "pancreatic islets," and grammatical equivalents thereof. In certain embodiments, endocrine cells may be differentiated from pancreatic progenitor or precursor cells. Islet cells can 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. Islet cells can also refer to groups of cells, cell clusters, and the like.

[0094]

[0178] The terms "progenitor" and "precursor" cells are used interchangeably herein and refer to cells that have a cellular phenotype that is more primitive than cells that can result from differentiation (e.g., that are at an earlier step in a developmental pathway or progression than fully differentiated cells). In many cases, progenitor cells can also have significant or very high proliferative potential. Progenitor cells can give rise to multiple distinct differentiated cell types or a single differentiated cell type, depending on the developmental pathway and the environment in which the cells develop and differentiate.

[0095]

[0179] The term "progenitor thereof" in relation to an insulin-positive endocrine cell can refer to any 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, that can be differentiated into an insulin-positive endocrine cell when cultured under conditions suitable for differentiating the progenitor cell into an insulin-positive endocrine cell.

[0096]

[0180] The term "exocrine cells," as used herein, can refer to cells of an exocrine gland, i.e., a gland that excretes its secretions through a duct. In certain embodiments, exocrine cells can refer to exocrine pancreatic cells, which are pancreatic cells capable of producing enzymes secreted into the small intestine. These enzymes can help digest food passing through the gastrointestinal tract. Exocrine pancreatic cells are also known as islets of Langerhans, which can secrete two hormones: insulin and glucagon. Exocrine pancreatic cells can be one of several cell types, such as alpha-2 cells (which can produce the hormone glucagon), or beta cells (which can produce the hormone insulin), and alpha-1 cells (which can produce the regulatory substance somatostatin). Non-insulin-producing exocrine cells, as used herein, can refer to alpha-2 cells or alpha-1 cells. The term pancreatic exocrine cells encompasses "pancreatic endocrine cells," which can refer to pancreatic cells that produce hormones secreted into the bloodstream, such as insulin (produced by beta cells), glucagon (produced by alpha-2 cells), somatostatin (produced by delta cells), and pancreatic polypeptide (produced by F cells).

[0097]

[0181] The terms "stem cell-derived beta cells," "SC-beta cells," "functional beta cells," "functional pancreatic beta cells," "mature SC-beta cells," and their grammatical equivalents can refer to cells (e.g., non-native pancreatic beta cells) that exhibit at least one marker indicative of a pancreatic beta cell (e.g., PDX-1 or NKX6.1), express insulin, and exhibit a glucose-stimulated insulin secretion (GSIS) response characteristic of endogenous mature beta cells. In some embodiments, the terms "SC-beta cells" and "non-native beta cells" are interchangeable as used herein. In some embodiments, "SC-beta 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 disclosed methods can be used to derive SC-β cells from any insulin-positive endocrine cell or its precursor using any cell as a starting point (e.g., although the invention is not intended to be limited in this manner, embryonic stem cells, induced pluripotent stem cells, progenitor cells, partially reprogrammed somatic cells (e.g., somatic cells partially reprogrammed to an intermediate state between induced pluripotent stem cells and the somatic cell from which they were derived), multipotent cells, totipotent cells, transdifferentiated forms of any of the foregoing cells, etc.). In some embodiments, SC-β cells exhibit a response to multiple glucose loads (e.g., at least one, at least two, or at least three or more sequential glucose loads). In some embodiments, the response is similar to the response of endogenous islets (e.g., human islets) to multiple glucose loads. 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 an in vitro GSIS response and an in vivo GSIS response similar to that of endogenous β cells. The GSIS response of SC-β cells can be observed within two weeks of transplantation of SC-β cells into a host (e.g., a human or animal). In some embodiments, SC-β cells encapsulate insulin in secretory granules.In some embodiments, the SC-β cells exhibit encapsulated crystalline insulin granules. In some embodiments, the SC-β cells exhibit a stimulation index greater than 1. In some embodiments, the SC-β cells exhibit a stimulation index greater than 1.1. In some embodiments, the SC-β cells exhibit a stimulation index greater than 2. In some embodiments, the SC-β cells exhibit cytokine-induced apoptosis in response to a cytokine. In some embodiments, insulin secretion from SC-β cells is enhanced in response to known antidiabetic agents (e.g., secretagogues). In some embodiments, the SC-β cells are monohormonal. In some embodiments, the SC-β cells do not abnormally co-express other hormones, e.g., glucagon, somatostatin, or pancreatic polypeptide. In some embodiments, the SC-β cells exhibit a low replication rate. In some embodiments, the SC-β cells increase intracellular Ca2+ in response to glucose.

[0098]

[0182] As used herein, the term "insulin-producing cells" and its grammatical equivalents refer to insulin-secreting cells differentiated from pancreatic precursors or their precursors. Insulin-producing cells can include pancreatic β cells, as that term is described herein, and pancreatic β-like cells (e.g., insulin-positive endocrine cells) that constitutively or inducibly synthesize (e.g., transcribe the insulin gene, translate proinsulin mRNA, and modify proinsulin mRNA to form insulin protein), express (e.g., express the phenotype carried by the insulin gene), or secrete (release insulin into the extracellular space) insulin. For example, insulin-producing cell populations 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 adult β cells. For example, insulin-producing cell populations generated by the methods disclosed herein can include mature pancreatic β cells or SC-β cells, and can also contain non-insulin-producing cells (e.g., cells that do not produce or secrete insulin but have a cell-like phenotype).

[0099]

[0183] The terms "insulin-positive beta-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 beta cells and further express insulin, but lack the glucose-stimulated insulin secretion (GSIS) response characteristic of endogenous beta cells.

[0100]

[0184] The term "β-cell marker" refers to, but is not limited to, proteins, peptides, nucleic acids, protein and nucleic acid polymorphisms, splice variants, protein or nucleic acid fragments, elements, and other analytes that are specifically expressed or present in pancreatic β-cells. Exemplary β-cell markers include, but are not limited to, pancreatic and duodenal homeobox 1 (Pdx1) polypeptide, insulin, C-peptide, amylin, E-cadherin, Hnf3β, PCI / 3, B2, Nkx2.2, GLUT2, PC2, ZnT-8, Examples include ISL1, Pax6, Pax4, NeuroD, 1 Inf1b, Hnf-6, Hnf-3 beta, and MafA, as well as those described in Zhang et al., Diabetes. 50(10):2231-6 (2001). In some embodiments, the beta cell marker is a nuclear 3-cell marker. In some embodiments, the beta cell marker is Pdx1 or PH3.

[0101]

[0185] The term "pancreatic endocrine marker" can refer to, but is not limited to, proteins, peptides, nucleic acids, protein and nucleic acid polymorphisms, splice variants, protein or nucleic acid fragments, elements, and other analytes that are specifically expressed or present in pancreatic endocrine cells. Exemplary pancreatic endocrine cell markers include, but are not limited to, Ngn-3, NeuroD, and Islet-1.

[0102]

[0186] The terms "pancreatic progenitor," "pancreatic endocrine precursor," "pancreatic precursor," "pancreatic endocrine precursor," 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 differentiate into 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 marker of NGN3, NKX2.2, NeuroD, ISL-1, Pax4, Pax6, or ARX.

[0103]

[0187] The term "Pdx1-positive pancreatic progenitor" as used herein can refer to cells that are pancreatic endoderm (PE) cells capable of differentiating into SC-β cells, e.g., pancreatic β cells. Pdx1-positive pancreatic progenitors 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 cases, Pdx1-positive pancreatic progenitor cells lack NKX6.1 expression. In some cases, Pdx1-positive pancreatic progenitor cells can also be referred to as Pdx1-positive NKX6.1-negative pancreatic progenitor cells due to the lack of NKX6.1 expression. In some cases, Pdx1-positive pancreatic progenitor cells can also be referred to as "pancreatic foregut endoderm cells."

[0104]

[0188] The term "Pdx1-positive, NKX6-1-positive pancreatic progenitors" as used herein 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 progenitors express the markers Pdx1 and NKX6-1. Other markers include, but are not limited to, Cdcp1, Ptf1a, HNF6, or NRx2.2. NKX6-1 expression can be assessed by any method known to those skilled in the art, such as immunochemistry or quantitative RT-PCR using anti-NKX6-1 antibodies. As used herein, the terms "NKX6.1" and "NKX6-1" are synonymous and interchangeable. In some cases, Pdx1-positive, NKX6-1-positive pancreatic progenitor cells may also be referred to as "pancreatic foregut precursor cells."

[0105]

[0189] The term "Ngn3-positive endocrine precursors," as used herein, can refer to precursors of pancreatic endocrine cells that express the transcription factor Neurogenin-3 (Ngn3). Precursor cells are more differentiated than pluripotent stem cells and can differentiate into only a few cell types. In particular, Ngn3-positive endocrine precursor cells have the ability to differentiate into five pancreatic endocrine cell types (α, β, δ, ε, and PP). Ngn3 expression can be detected by any method known to those skilled in the art, such as immunochemistry using anti-Ngn3 antibodies or quantitative RT-PCR. Therefore, it can be evaluated.

[0106]

[0190] The terms "NeuroD" and "NeuroD1" are used interchangeably to identify proteins and their encoding genes expressed in pancreatic endocrine precursor cells.

[0191] The term "selection marker" refers to a gene, RNA, or protein that, when expressed, confers a selectable phenotype on a cell, such as resistance to cytotoxic or cytostatic agents (e.g., antibiotic resistance), auxotrophy, or expression of a specific protein that can be used to distinguish between cells that express the protein and cells that do not. As used herein, the term "selection marker" can refer to a gene or the expression product of a gene, e.g., the encoded protein. In some embodiments, a selection marker confers a growth and / or survival advantage on cells that express it relative to cells that do not express it or express it at significantly lower levels. Such a growth and / or survival advantage typically occurs when cells are maintained under certain conditions, i.e., "selection conditions." To ensure effective selection, a cell population can be maintained under conditions long enough that cells that do not express the marker do not grow and / or survive and are removed from the population or their number is reduced to only a very small percentage of the population. Methods of selecting cells expressing markers that confer a growth and / or survival advantage by maintaining a cell population under selective conditions that largely or completely eliminate cells that do not express the marker are referred to herein as "positive selection," and markers are described as being "useful for positive selection." Negative selection and markers useful for negative selection are also the subject of some of the methods described herein. Expression of such markers confers a growth and / or survival disadvantage on cells that express the marker relative to cells that do not express the marker or express it at significantly lower levels (or, considered another way, cells that do not express the marker have a growth and / or survival advantage relative to cells that express the marker). Thus, cells that express this marker can be largely or completely eliminated from a cell population if maintained under selective conditions for a sufficient period of time.

[0107]

[0192] The term "epigenetics" refers to heritable changes in gene function that do not involve changes in DNA sequence. While epigenetics most often refers to chromosomal changes that affect gene activity and expression, it can also be used to describe any heritable phenotypic change that does not result from modifications to the genome. Such effects on cellular and physiological traits can result from external or environmental factors or be part of a normal developmental program. Epigenetics can also refer to functionally relevant changes in the genome that do not involve changes in the nucleotide sequence. Examples of mechanisms that produce such changes are DNA methylation and histone modifications, each of which alters how genes are expressed without altering the underlying DNA sequence. Gene expression can be controlled through the action of repressor proteins that bind to silencer regions of DNA. These epigenetic changes can persist throughout the lifespan of a cell through cell division and can even continue for multiple generations despite not involving changes in the underlying organism's DNA sequence. One example of epigenetic changes in eukaryotic biology is the process of cellular differentiation. During morphogenesis, totipotent stem cells can give rise to a variety of pluripotent cells, and pluripotent cells can give rise to fully differentiated cells.

[0108]

[0193] The term "epigenetic modifying compound" refers to chemical compounds that can cause epigenetic changes to genes, i.e., alter gene expression without changing the DNA sequence. Epigenetic changes can help determine whether genes are turned on or off and can affect the production of proteins in certain cells, e.g., beta cells. Epigenetic modifications, such as DNA methylation and histone modifications, can affect DNA accessibility and chromatin structure. Epigenetic modification modifies the expression of pluripotent genes, thereby regulating the pattern of gene expression. These processes are crucial for the normal development and differentiation of distinct cell lineages in adult organisms. These processes can be altered by external influences, and thus can contribute to or be the result of environmentally induced phenotypic or pathophenotypic changes. Importantly, epigenetic modification plays a crucial role in regulating pluripotent genes that are inactivated during differentiation. Non-limiting exemplary epigenetic modification compounds include DNA methylation inhibitors, histone acetyltransferase inhibitors, histone deacetylase inhibitors, histone methyltransferase inhibitors, bromodomain inhibitors, or any combination thereof.

[0109]

[0194] The term "differentiated cell" or its grammatical equivalents refers to any primary cell that is not pluripotent as defined herein in its natural form. In another sense, the term "differentiated cell" can refer to a cell of a more specialized cell type obtained from a cell of a less specialized cell type (e.g., a stem cell, e.g., 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 that can form pancreatic cells and other endodermal cell types. Further differentiation of endodermal cells leads to the pancreatic pathway, where approximately 98% of cells become exocrine, ductular, or stromal cells, and approximately 2% become endocrine cells. Early endocrine cells are islet precursors, 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, thymus, etc.

[0110]

[0195] As used herein, the term "somatic cell" can refer to any cell that forms the body of 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. All other cell types in a mammal's body—except sperm and eggs, the cells from which they are produced (gametocytes), and undifferentiated stem cells—are somatic cells, and internal organs, skin, bone, blood, and connective tissue are all composed of somatic cells. In some embodiments, the somatic cell is a "non-embryonic somatic cell," which refers to a somatic cell that is not present in or obtained from an embryo or 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 methods for converting at least one insulin-positive endocrine cell or precursor thereof to an insulin-producing glucose-responsive cell can be performed both in vivo and in vitro (wherein in vivo is performed when at least one insulin-positive endocrine cell or precursor thereof is present in the subject's body, and in vitro is performed using isolated at least one insulin-positive endocrine cell or precursor thereof maintained in culture).

[0111]

[0196] As used herein, the term "adult cell" can refer to cells found throughout the body after embryonic development.

[0197] The term "endodermal cells," as used herein, can refer to cells derived from one of the three primary germ cell layers in a very early embryo (the other two are mesoderm and ectoderm). The endoderm is the innermost of the three layers. Endodermal cells first give rise to the embryonic gut, and then differentiate to give rise to the lining of the respiratory and digestive tracts (e.g., intestine), the liver, and the pancreas.

[0112]

[0198] The term "cells of endodermal origin" as used herein refers to cells derived from endodermal cells. Endodermal cell development can refer to any cell that develops or differentiates from an organ. 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 progenitors (also called pancreatic precursors) develop from endodermal cells of the embryonic foregut. Shortly after specification, liver and pancreatic progenitors rapidly acquire significantly different cellular functions and regenerative potential. These changes are induced by inductive signals and gene 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 evolutionarily conserved inductive signal and transcription factor networks that induce hepatic and pancreatic cell differentiation, providing guidance for methods to promote hepatocyte and β cell differentiation from diverse stem and progenitor cell types.

[0113]

[0199] The term "definitive endoderm" as used herein can 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 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 ability to differentiate into cells including liver, lung, pancreas, thymus, intestine, stomach, and thyroid cells. Expression of Sox17 and other markers of definitive endoderm can be assessed by any method known to those skilled in the art, such as immunochemistry using anti-Sox17 antibodies or quantitative RT-PCR.

[0114]

[0200] 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.

[0115]

[0201] The term "primitive gut cells" or "gut cells," as used herein, can refer to cells that can differentiate from endoderm cells and into SC-β cells (e.g., pancreatic β cells). Primitive gut cells express at least one marker, HNF1-β, HNF3-β, or HNF4-α. Primitive gut cells have the ability to differentiate into cells including lung, liver, pancreatic, stomach, and intestinal cells. The expression of HNF1-β and other markers of the primitive gut can be assessed by any method known to those skilled in the art, such as immunochemistry using an anti-HNF1-β antibody.

[0116]

[0202] The term "stem cell," as used herein, can refer to an undifferentiated cell that can proliferate and give rise to more progenitor cells that have the potential to generate a large number of mother cells that can give rise to differentiated or differentiable daughter cells. The daughter cells themselves retain one or more cells with the developmental potential of their parent, while also being able to be induced to proliferate and produce progeny that subsequently differentiate into one or more mature cell types. The term "stem cell" can refer to a subset of precursors that, under certain circumstances, have the ability or potential to differentiate into a more specialized or differentiated phenotype, and, under certain circumstances, retain the ability to proliferate without substantial differentiation. In one embodiment, the term stem cell, taken as a whole, refers to naturally occurring mother cells whose progeny (progeny) often specialize in different directions by differentiation, for example, by acquiring entirely individual characteristics that occur in the gradual diversification of germ cells and tissues. Cells Differentiation is a complex process that typically occurs through many cell divisions. Differentiated cells can be derived from pluripotent cells, which themselves are derived from multipotent cells, etc. Each of these pluripotent cells can be considered a stem cell, but the range of cell types each can give rise to can vary greatly. Some differentiated cells also have the ability to give rise to cells of higher developmental potential. Such ability can occur naturally or be artificially induced based on treatment with various factors. In many biological cases, stem cells are also "pluripotent" because they can produce progeny of two or more distinct cell types, but this is not required for "stemness." Self-renewal is another classic part of the definition of stem cells and, as used in this document, is essential. Theoretically, self-renewal can occur through either of two main mechanisms. Stem cells can divide asymmetrically, such that one daughter retains the stem cell state while the other daughter expresses some other distinct, specific function and phenotype. Alternatively, some of the stem cells in the population can divide symmetrically into two stem cells, thus maintaining some of the stem cells in the population as a whole, while other cells in the population can only give rise to differentiated progeny.Formally, cells that start as stem cells can progress to differentiated phenotypes, but can then "reverse" and re-express stem cell phenotypes, which is often referred to by those skilled in the art as "dedifferentiation" or "reprogramming" or "reverse differentiation".As used herein, the term "pluripotent stem cells" includes embryonic stem cells, induced pluripotent stem cells, placental stem cells, etc.

[0117]

[0203] The term "pluripotent," as used herein, can refer to cells that have the ability to differentiate into two or more differentiated cell types under various conditions, preferably into cell types characteristic of all three germ cell layers. Pluripotent cells are primarily characterized by their ability to differentiate into two or more cell types, preferably into all three germ layers, using, for example, a nude mouse teratoma formation assay. While pluripotency can also be demonstrated by the expression of embryonic stem (ES) cell markers, the preferred test for pluripotency is the demonstration of the ability to differentiate into cells of each of the three germ layers. It should be noted that simply culturing such cells alone does not render them pluripotent. Reprogrammed pluripotent cells (e.g., iPS cells, as defined herein) are also characterized by the ability to be passaged for extended periods without loss of growth potential, compared to their primary cell parents, which generally have the ability to divide only a limited number of times in culture.

[0118]

[0204] As used herein, the terms "iPS cells" and "induced pluripotent stem cells" are used interchangeably and can refer to pluripotent stem cells that are artificially obtained (e.g., by induced or complete reversion) from non-pluripotent cells, typically adult somatic cells, for example, by inducing forced expression of one or more genes.

[0119]

[0205] The term "phenotype" can refer to one or several overall biological characteristics that define a cell or organism under a particular set of environmental conditions and factors, regardless of the actual genotype.

[0120]

[0206] The terms "subject," "patient," or "individual" are used interchangeably herein and can refer to an animal, e.g., a human, from which cells may be obtained and / or to which treatment, including prophylactic treatment, using the cells described herein is provided. In the case of treatment of an infection, condition, or disease state specific to a particular animal, such as a human subject, the term subject can refer to that particular animal. "Non-human animal" and "non-human mammal," used interchangeably herein, include mammals, e.g., rats, mice, rabbits, sheep, cats, dogs, cows, pigs, and non-human primates. The term "subject" also encompasses any vertebrate, including, but not limited to, mammals, reptiles, amphibians, and fish. Advantageously, however, the subject is a mammal, such as a human, or other mammal, e.g., a domestic mammal, e.g., a dog, cat, horse, etc., or a production mammal, e.g., a cow, sheep, pig, etc. A "patient in need thereof" or "subject in need thereof," as used herein, refers to a human diagnosed with a disease or disorder, for example, but not limited to, diabetes. "A" refers to a patient diagnosed with or suspected of having a pulmonary embolism.

[0121]

[0207] As used herein, "administering" can refer to providing one or more compositions described herein to a patient or subject. By way of example, and not limitation, composition administration, e.g., injection, can be performed via intravenous (iv), subcutaneous (sc), intradermal (id), intraperitoneal (ip), or intramuscular (im) injection. One or more such routes can be used. Parenteral administration can be performed, for example, via bolus injection or gradual perfusion over time. Alternatively, or concurrently, administration can be performed via the oral route. In addition, administration can also be performed via surgical deposition of a bolus or pellet of cells or placement of a medical device. In certain embodiments, a composition of the present disclosure can comprise an amount of engineered or host cells expressing a nucleic acid sequence described herein or a vector comprising at least one nucleic acid sequence described herein effective to treat or prevent a proliferative disorder. A pharmaceutical composition can include a cell population described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffers, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose, or dextran, mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0122]

[0208] The terms "treat," "treating," "treatment," and their grammatical equivalents, when applied to isolated cells, include subjecting the cells to any kind of process or condition, or performing any kind of operation or procedure on the cells. When applied to a subject, these terms refer to providing medical or surgical treatment, care, or management to an individual. An individual is typically ill or in impaired health, or at increased risk of developing a disease compared to the average member of the population, and is in need of such treatment, care, or management.

[0123]

[0209] As used herein, the terms "treating" and "treatment" can refer to administering an effective amount of a composition to a subject, resulting in the subject experiencing a reduction in at least one symptom of the disease or an improvement in the disease, e.g., a beneficial or desired clinical result. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, whether detectable or undetectable, a decrease in the extent of the disease, a stable (e.g., not worsening) state of the disease, a delay or slowing of disease progression, an improvement or palliation of the disease state, and remission (whether partial or complete). Treating can refer to extending survival compared to the expected survival in the absence of treatment. Thus, those skilled in the art will recognize that treatment may improve the disease state but may not be a complete cure of the disease. As used herein, the term "treatment" includes prophylaxis. Alternatively, treatment is "effective" if the progression of the disease is reduced or halted. "Treatment" can also mean extending survival compared to the expected survival in the absence of treatment. Those in need of treatment include those already diagnosed with heart disease, as well as those who are more likely to develop heart disease due to genetic susceptibility or other factors, such as weight, diet, and health.

[0124]

[0210] The terms "therapeutically effective amount," "therapeutic amount," or grammatical equivalents thereof, can refer to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount can vary depending on factors such as the disease state, the age, sex, and weight of the individual, and the ability of the compositions described herein to elicit a desired response in one or more subjects. The exact amount of the composition of the present disclosure to be administered will depend on the patient's (subject's) age, weight, tumor size, and other factors. The treatment can be determined by a physician taking into account the extent of infection or metastasis and individual differences in condition.

[0125]

[0211] Alternatively, the pharmacological and / or physiological effect of administering one or more compositions described herein to a patient or subject may be "prophylactic," e.g., the effect completely or partially prevents a disease or symptom in the patient or subject. A "prophylactically effective amount" can refer to an amount effective, at dosages and for periods of time necessary, to achieve a desired prophylactic result (e.g., prevention of disease onset).

[0126]

[0212] Some numerical values ​​disclosed throughout are referred to as, for example, "X is at least or at least about 100, or 200 [or any number]." This includes the number itself as well as aX is at least 100, bX is at least 200, cX is at least about 100, and dX is at least about 200 All of the above are included.

[0127]

[0213] All of these different combinations are contemplated by the numerical values ​​disclosed throughout. All disclosed numerical values, whether referring to administration of a therapeutic agent or to days, months, years, body weight, dosage, etc., should be construed in this manner unless otherwise specifically indicated to the contrary.

[0128]

[0214] Ranges disclosed throughout may be referred to, for example, as "X is administered on or about days 1-2, or on or about days 2-3 [or any range of values]." The ranges may include the numbers themselves (e.g., the endpoints of the ranges) as well as the ranges themselves. i) X is administered between days 1 and 2; ii) X is administered between days 2 and 3; iii) X is administered between about days 1 and 2; iv) X is administered between about days 2 and 3; v) X is administered between day 1 and about day 2; vi) X is administered between day 2 and about day 3; vii) X is administered between about day 1 and about day 2, and viii) X is administered between about day 2 and about day 3 All of the above are included.

[0129]

[0215] All of these different combinations are contemplated by the ranges disclosed throughout. All disclosed ranges, whether referring to administration of therapeutic agent or to number of days, months, years, body weight, dosage, etc., should be construed in this manner unless otherwise specifically indicated to the contrary.

[0130] Stages of Differentiation

[0216] In some embodiments, the pancreatic differentiation disclosed herein is carried out in a stepwise manner. In the stepwise progression, "Stage 1" or "S1" refers to the first step of the differentiation process, i.e., the differentiation of pluripotent stem cells into cells expressing markers characteristic of definitive endoderm cells ("DE") ("Stage 1 cells" or "S1 cells"). "Stage 2" refers to the second step, i.e., the differentiation of cells expressing markers characteristic of definitive endoderm cells into cells expressing markers characteristic of gut cells ("GT") ("Stage 2 cells" or "S2 cells"). "Stage 3" refers to the third step, i.e., the differentiation of cells expressing markers characteristic of gut cells into cells expressing markers characteristic of gut cells ("GT"). "Stage 4" refers to the fourth step, i.e., the differentiation of cells expressing markers characteristic of pancreatic progenitor 1 cells ("PP1") into cells expressing markers characteristic of pancreatic progenitor 2 cells ("PP2") ("Stage 4 cells" or "S4 cells"). "Stage 5" refers to the fifth step, i.e., the differentiation of cells expressing markers characteristic of pancreatic progenitor 2 cells into cells expressing markers characteristic of pancreatic endoderm cells and / or pancreatic endocrine progenitor cells ("EN") ("Stage 5 cells" or "S5 cells"). "Stage 6" refers to the differentiation of cells expressing markers characteristic of pancreatic endocrine progenitor cells into cells expressing markers characteristic of pancreatic endocrine beta cells ("SC-β cells") or pancreatic endocrine alpha cells ("SC-α cells"). However, it should be understood that not all cells in a particular population progress through these stages at the same rate, i.e., some cells may be less or more advanced along the differentiation pathway than the majority of cells present in the population.

[0131] Culture media and drugs TGF-β signaling pathway inhibitors

[0217] Exemplary TGF-β signaling pathway inhibitors include ALK5 inhibitor II (CAS 446859-33-2, an ATP-competitive inhibitor of TGF-B Ri kinase, also known as RepSox, IIJPAC name: 2-[5-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl]-1,5-naphthyridine), analogs or derivatives of ALK5 inhibitor II, such as those described in U.S. Patent Publication No. 2012 / 0021519, and those described in U.S. Patent Publication No. 2010 / 0267731. These include, but are not limited to, TGF-β receptor inhibitors, ALK5 inhibitors described in U.S. Patent Publication Nos. 2009 / 0186076 and 2007 / 0142376 (e.g., including A83-01, 431542, D4476, GW788388, LY364947, LY580276, SB525334, SB505124, SD208, GW6604, or GW788388).

[0132]

[0218] In some embodiments, the TGF-β signaling pathway inhibitor may have the following structure:

[0219]

[0133] [ka]

[0134]

[0220] In some embodiments, the concentration of the TGF-β signaling pathway inhibitor can be about 0.1-110 μM, 0.1-50 μM, 0.1-25 μM, or 0.1-10 μM. In some embodiments, the concentration of the TGF-β signaling pathway inhibitor can be about 10 μM. In some embodiments, the TGF-β signaling pathway inhibitor is Alk5 inhibitor II, and the concentration of the inhibitor is about 10 μM.

[0135] Thyroid hormone signaling pathway activators

[0221] Exemplary thyroid hormone signaling pathway activators include triiodothyronine (T3), analogs or derivatives of T3, e.g., selective and non-selective thyromimetics, TRJ selective agonists GC-1, GC-2, 4,4-hydroxy-PCB 106, MB0781 1, MB07344, 3,5-diiodo thyropropionic acid (DITPA); the selective TR-β agonist GC-1; 3-iodothyronamine (T(l)AM) and 3,3',5-triiodothyroacetic acid (Triac) (bioactive metabolites of the hormone thyroxine (T(4))); KB-21 15 and KB-141; thyronamine; SKF L-94901; DIBIT; 3'-AC-T2; tetraiodothyroacetic acid (Tetrac) and triiodothyroacetic acid (Triac) (by oxidative deamination and decarboxylation of the alanine chain of thyroxine [T4] and triiodothyronine [T3]), 3,3',5'-triiodothyronine (rT3) (by deiodination of T4 and T3), 3,3'-diiodothyronine (3,3'-T2) and 3,5-diiodothyronine (T2) by deiodination of T4, T3, and rT3), and 3-iodothyronamine (T1AM) and thyronamine (T0AM) (by deiodination of T4 and T3 and decarboxylation of amino acids), as well as TH structural analogs such as 3,5,3'-triiodothyropropionic acid (Triprop), 3,5-dibromo-3-pyridazinone-l-thyronine (L-940901), N-[3,5-dimethyl-4-(4'-hydroxy-3 f

[0033] Examples of suitable iodine-modifying agents include, but are not limited to, 3,5-dimethyl-4-[(4'-hydroxy-3'-isopropylbenzyl)-phenoxy]acetic acid (CGS 23425), 3,5-dimethyl-4-[(4'-hydroxy-3'-isopropylbenzyl)-phenoxy]acetic acid (GC-1), 3,5-dichloro-4-[(4-hydroxy-3-isopropylphenoxy)phenyl]acetic acid (KB-141), and 3,5-diiodothyropropionic acid (DITPA).

[0136]

[0222] In some embodiments, the thyroid hormone signaling pathway activator can include a prodrug or prohormone of T3, such as a T4 thyroid hormone (e.g., thyroxine or L-3,5,3',5'-tetraiodothyronine). In some embodiments, the thyroid hormone signaling pathway activator can be an iodothyronine composition described in U.S. Pat. No. 7,163,918. In some embodiments, the thyroid hormone signaling pathway activator can be 2-[4-[[4-hydroxy-3-(1-methylethyl)phenyl]methyl]-3,5-dimethylphenoxy]acetic acid (GC-1). GC-1 is a thyromimetic and a high-affinity agonist of the thyroid hormone receptor (TR) β and TR oc receptors (K D (The values ​​are 67p and 440p, respectively.) GC-1 is 5-fold and 100-fold more potent in vitro than the endogenous agonist T3 at TRoti and TRi receptors, respectively.

[0137]

[0223] In some embodiments, the thyroid hormone signaling pathway activator may have the structure:

[0224]

[0138] [ka]

[0139]

[0225] In some embodiments, the concentration of the thyroid hormone signaling pathway activator can be about 0.1-110 μM, 0.1-50 μM, 0.1-25 μM, or 0.1-10 μM. In some embodiments, the concentration of the thyroid hormone signaling pathway activator can be about 1 μM. In some embodiments, the thyroid hormone signaling pathway activator is GC-1, and the concentration of the activator is about 1 μM.

[0140] Protein kinase inhibitors

[0226] Exemplary protein kinase inhibitors include staurosporine, analogs of staurosporine, e.g., Ro-31-8220, bisindolylmaleimide (Bis) compounds, and the like. In some embodiments, the protein kinase inhibitor may be staurosporine.

[0141]

[0227] In some embodiments, the concentration of the protein kinase inhibitor can be about 0.1-110 nM, 0.1-50 nM, 0.1-25 nM, 0.1-10 nM, or 0.1-5 nM. In some embodiments, the concentration of the protein kinase inhibitor can be about 3 nM. In some embodiments, the protein kinase inhibitor is staurosporine (SSP) and the concentration of the activator is about 3 nM.

[0142]

[0228] In some embodiments, the protein kinase inhibitor may have the structure:

[0229]

[0143] [ka]

[0144] Bone morphogenetic protein (BMP) signaling pathway inhibitors

[0230] Exemplary BMP signaling pathway inhibitors include, but are not limited to, 4-[6-(4-piperazin-1-ylphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinolone (LDN 193 189; also known as LDN1931 89, 1062368-24-4, LDN-193189, DM 3189, DM-3189, and referred to herein as LDN), an analog or derivative of LDN193189, such as a salt (e.g., LDN193189 hydrochloride), hydrate, solvent, ester, or prodrug of LDN193189, or a compound of Formula I of U.S. Patent Application Publication No. 2011 / 0053930. In embodiments of the present invention, the BMP signaling pathway inhibitor comprises LDN193189.

[0145]

[0231] In some embodiments, the BMP signaling pathway inhibitor has the following structure:

[0232]

[0146] [ka]

[0147] can have:

[0233] In some embodiments, the concentration of the BMP signaling pathway inhibitor can be about 0.1-110 nM, 0.1-100 nM, or 0.1-50 nM. In some embodiments, the concentration of the BMP signaling pathway inhibitor can be about 100 nM. In some embodiments, the protein BMP signaling pathway inhibitor is LDN193189, and the concentration of the activator is about 100 nM.

[0148] Rho-associated protein kinase (ROCK) inhibitors

[0234] Exemplary ROCK inhibitors include, but are not limited to, N-[(1S)-2-hydroxy-1-phenylethyl]-N'-[4-(4-pyridinyl)phenyl]-urea (AS 1892802), fasudil hydrochloride (also known as HA 1077), N-[3-[[2-(4-amino-1,2,5-oxadiazol-3-yl)-1-ethyl-1H-imidazo[4,5-c]pyridin-6-yl]oxy]phenyl]-4-[2-(4-morpholinyl)ethoxy]benzamide (GS 269962), 4-[4-(trifluoromethyl)phenyl]-N-(6-fluoro-1H-indazol-5-yl)-2-methyl-6-oxo-1,4,5,6-tetrahydro-3-pyridinecarboxamide (GS 429286), (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine dihydrochloride (H 1 152 dihydrochloride), (S)-(+)-4-glycyl-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine dihydrochloride (glycyl-M 1 152 dihydrochloride), N-[(3-hydroxyphenyl)methyl]-N'-[4-(4-pyridinyl)-2-thiazolyl]urea dihydrochloride (RKI 1447 dihydrochloride), (3S)-1-[[2-(4-amino-1,2,5-oxadiazol-3-yl)-1-ethyl-1H-imidazo[4,5-c]pyridin-7-yl]carbonyl]-3-pyrrolidinamine dihydrochloride (SB772077B dihydrochloride), N-[2-[2-(dimethylamino)ethoxy]-4-(1H-pyrazol-4-yl)phenyl-2,3-dihydro-1,4-benzodioxine-2-carboxamide dihydrochloride (SR 3677 dihydrochloride), and trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide dihydrochloride (Y-27632 dihydrochloride), N-benzyl-[2-(pyrimidin-4-yl)amino]thiazole-4-carboxamide (thiazovivin), ROCK inhibitors, isoquinoline sulfonamide compounds (Rho kinase inhibitors), N-(4-pyridyl)-N'-(2,4,6-trichlorophenyl)urea (Rho kinase inhibitor II), 3-(4-pyridyl)-1H-indole (Rho a small organic molecule ROCK inhibitor selected from the group consisting of Rock-1(B1), Rock-1(C-19), Rock-1(H-11), Rock-1(G-6), Rock-1(H-85), Rock-1(K-18), Rock-2(C-20), Rock-2(D-2), Rock-2(D-11), Rock-2(N-19), Rock-2(H-85), Rock-2(30-J); Rock antibody commercially available from Biosciences; ROCK selected from the group consisting of Rock-1 CRISPR / Cas9 KO Plasmid (h), Rock-2 CRISPR / Cas9 KO Plasmid (h), Rock-1 CRISPR / Cas9 KO Plasmid (m), and Rock-2 CRISPR / Cas9 KO Plasmid (m). CRISPR / Cas9 knockout plasmid; Rock-1 siRNA(h):sc-29473, Rock-1 siRNA(m):sc-36432, Rock-1 and ROCK siRNA, shRNA plasmid, and / or shRNA lentiviral particle gene silencers selected from the group consisting of siRNA(r):sc-72179, Rock-2 siRNA(h):sc-29474, Rock-2 siRNA(m):sc-36433, Rock-2 siRNA(r):sc-108088.

[0149]

[0235] In some embodiments, the ROCK inhibitor comprises Y-27632. In some embodiments, the ROCK inhibitor is thiazovivin.

[0236] In some embodiments, the ROCK inhibitor has the following structure:

[0237]

[0150] [ka]

[0151] can have:

[0238] In some embodiments, the concentration of the ROCK inhibitor can be about 0.1-110 μM, 0.1-50 μM, 0.1-25 μM, or 0.1-10 μM. In some embodiments, the concentration of the ROCK inhibitor can be about 2.5 μM. In some embodiments, the ROCK inhibitor is thiazovivin, and the concentration of the inhibitor is about 2.5 μM.

[0152]

[0239] In some embodiments, the concentration of the ROCK inhibitor (e.g., Y-27632 or thiazovivin) is 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 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 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 66 μM It can be 3 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 35 μM, about 40 μM, about 50 μM, or about 100 μM.

[0153] Histone methyltransferase inhibitors

[0240] In some embodiments, histone methyltransferase inhibitors can be used as epigenetic modifiers. Exemplary histone methyltransferase inhibitors include, but are not limited to, 3-deazaneplanocin A hydrochloride (DZNep-(1S,2R,5R)-5-(4-amino-1H-imidazo[4,5-c]pyridin-1-yl)-3-(hydroxymethyl)cyclopent-3-ene-1,2-diol); Bix-01294, UNC0638, BRDD4770, EPZ004777, AZ505, PDB4e47, alproic acid, vorinostat, romidepsin, entinostat, abexinostat, gibinostat, and mocetinostat, butyrate, a member of the serine protease inhibitor (serpin) family. In some embodiments, histone methyltransferase inhibitors can be used as epigenetic modifiers. In some embodiments, the histone methyltransferase inhibitor has the following structure:

[0241]

[0154] [ka]

[0155] can have:

[0242] In some embodiments, the concentration of the histone methyltransferase inhibitor can be about 0.1-110 nM, 0.1-100 nM, or 0.1-50 nM. In some embodiments, the concentration of the histone methyltransferase inhibitor can be about 100 nM. In some embodiments, the histone methyltransferase inhibitor is DZNep, and the concentration of the inhibitor is about 100 nM.

[0156]

[0243] In some embodiments, the concentration of the histone methyltransferase inhibitor can be 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.

[0157] MGLL inhibitors

[0244] Exemplary MGLL (monoglyceride lipase) inhibitors include, but are not limited to, for example, JJKK048, KML29, NF1819, JW642, JZL184, JZL195, JZP361, pristimerin, or URB602.

[0158]

[0245] In some embodiments, the MGLL inhibitor can be JJKK048. In some embodiments, the MGLL inhibitor can be KML29. In some embodiments, the MGLL inhibitor can be NF1819.

[0159]

[0246] In some embodiments, the MGLL inhibitor may have the structure:

[0247]

[0160] [ka]

[0161]

[0248] In some embodiments, the MGLL inhibitor may have the structure:

[0249]

[0162] [ka]

[0163]

[0250] In some embodiments, the MGLL inhibitor may have the structure:

[0251]

[0164] [ka]

[0165]

[0252] In some embodiments, the concentration of the MGLL inhibitor is about 0.1 μM to 100 μM, or about 0.1 μM, 1 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, or 100 μM.

[0166]

[0253] In some embodiments, the MGLL inhibitor is JJKK048 and is present at a concentration of about 0.1 μM to 100 μM. In some embodiments, the MGLL inhibitor is KML29 and is present at a concentration of about 0.1 μM to 100 μM. In some embodiments, the MGLL inhibitor is NF1819 and is present at a concentration of about 0.1 μM to 100 μM.

[0167]

[0254] In some embodiments, the MGLL inhibitor is JJKK048 and has a concentration of 1 μM. In some embodiments, the MGLL inhibitor is KML29 and has a concentration of 10 μM. In some embodiments, the MGLL inhibitor is NF1819 and has a concentration of 10 μM.

[0168] lipids

[0255] Exemplary lipids include, but are not limited to, fatty acids, such as saturated or unsaturated fatty acids. In some embodiments, the lipid is a saturated fatty acid. In some embodiments, the lipid is an unsaturated fatty acid. Exemplary saturated fatty acids include, for example, palmitate, palmitic acid, butyric acid, valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, undecylic acid, lauric acid, tridecylic acid, myristic acid, pentadecylic acid, margaric acid, stearic acid, nonadecylic acid, arachidic acid, heneicosylic acid, behenic acid, tricosylic acid, lignoceric acid, pentacosylic acid, cerotic acid, heptacosylic acid, montanic acid, nonacosylic acid, melissic acid, hentriacontylic acid, russellic acid, silicic acid, gedic acid, ceroplastic acid, hexatriacontylic acid, and heptatriacontylic acid. Examples of suitable carboxylic acids include, but are not limited to, octatriacontanoic acid, nonatriacontanoic acid, or tetracontanoic acid, or salts or esters thereof.

[0169]

[0256] In some embodiments, the saturated fatty acid is palmitic acid or a salt or ester thereof. In some embodiments, the saturated fatty acid is palmitate.

[0257] Exemplary unsaturated fatty acids include, but are not limited to, for example, oleic acid, linoleic acid, palmitoleic acid, stearidonic acid, eicosapentaenoic acid, docosahexaenoic acid, linoledic acid, gamma-linolenic acid, dihomo-gamma-linolenic acid, arachidonic acid, docosatetraenoic acid, vaccenic acid, paulic acid, elaidic acid, gondoic acid, erucic acid, nervonic acid, or mead acid, or a salt or ester thereof.

[0170]

[0258] In some embodiments, the unsaturated fatty acid is oleic acid. In some embodiments, the unsaturated fatty acid is linoleic acid. In some embodiments, the unsaturated fatty acid is palmitoleic acid.

[0171] Initialization

[0259] The term "reprogramming" as used herein refers to the reprogramming of somatic cells. Reprogramming refers to the process of changing or reversing the differentiation state of a cell. Cells may be reprogrammed after partial differentiation or after terminal differentiation. Reprogramming encompasses the complete reversal of the differentiation state of a somatic cell to a pluripotent cell. Such a complete reversal of differentiation results in induced pluripotent (iPS) cells. As used herein, reprogramming also encompasses the partial reversal of a cell's differentiation state, e.g., to a pluripotent state, or to a somatic cell that is neither pluripotent nor multipotent but has lost one or more characteristics specific to the differentiated cell from which it arose, e.g., the direct reprogramming of a differentiated cell to a different somatic cell type. Reprogramming, as a whole, involves the alteration, e.g., reversal, of at least some of the inheritance patterns, such as nucleic acid modifications (e.g., methylation), chromatin condensation, epigenetic changes, and genomic imprinting, that occur during the differentiation of cells as a zygote develops into an adult.

[0172]

[0260] As used herein, the term "reprogramming factor" is intended to refer to a molecule associated with cell "reprogramming," i.e., differentiation and / or dedifferentiation and / or transdifferentiation, such that cells transform into a different cell type or phenotype. Reprogramming factors as a whole affect the expression of genes associated with cell differentiation, dedifferentiation, and / or transdifferentiation. Transcription factors are examples of reprogramming factors.

[0173]

[0261] The term "differentiation" and their grammatical synonyms, as used herein, refer to the process by which a less specialized cell (i.e., a more naive cell with greater differentiation potential) becomes a more specialized cell type (i.e., a less naive cell with less differentiation potential); the term "dedifferentiation" refers to the process by which a more specialized cell becomes a less specialized cell type (i.e., a more naive cell with greater differentiation potential); and the term "transdifferentiation" refers to the process by which cells of a particular cell type are converted into another cell type without significantly changing their level of "differentiation potential" or "naivety." Without wishing to be bound by theory, cells are considered to "transdifferentiate" if they are converted from one lineage-committed or terminally differentiated cell type to another lineage-committed or terminally differentiated cell type without significantly changing their level of "differentiation potential" or "naivety."

[0174]

[0262] As used herein, the term "differentiation potential" should be understood to refer to the ability of a cell to differentiate into cells of different lineages. For example, pluripotent cells (e.g., stem cells) have the potential to differentiate into cells of any of the three germ layers, i.e., endoderm (the lining of the stomach, gastrointestinal tract, lungs), mesoderm (muscle, bone, blood, urogenital tract), or ectoderm (epidermal tissue and nervous system), and thus have high differentiation potential, and are called multipotent cells (e.g., stem cells or Stem cells (certain types of induced stem cells, such as pluripotent stem cells) have the ability to give rise to cells from multiple but limited lineages (e.g., hematopoietic stem cells, cardiac stem cells, or neural stem cells) and have relatively lower differentiation potential than pluripotent cells. Lineage-committed or terminally differentiated cells may have even lower differentiation potential. Specific examples of transdifferentiation known in the art include, for example, the conversion of fibroblasts to beta cells or exocrine pancreatic cells to beta cells.

[0175]

[0263] Thus, cells can be differentiated into more naive cells (e.g., terminally differentiated cells can be differentiated to become multipotent or pluripotent), or cells can be dedifferentiated into less naive cells (e.g., multipotent or pluripotent cells can be differentiated into lineage-committed or terminally differentiated cells). However, in certain embodiments, cells can be converted or transdifferentiated from one cell type (or phenotype) to another cell type (or phenotype), e.g., having a similar level of differentiation potential. Thus, in certain embodiments of the present disclosure, the induction step of the present disclosure can prime the cells of the present disclosure to differentiate, dedifferentiate, and / or transdifferentiate. In certain embodiments of the present disclosure, the induction step of the present disclosure can prime the cells to transdifferentiate.

[0176]

[0264] For example, methods for reprogramming or inducing a specific type of cell into another type of cell by differentiation, dedifferentiation, and / or transdifferentiation using one or more exogenous polynucleotide or polypeptide reprogramming factors are known to those skilled in the art. Such methods may rely on the introduction of genetic material encoding one or more transcription factors or other polypeptides associated with cellular reprogramming. For example, Pdx1, Ngn3, and MafA, or functional fragments thereof, are all known to encode peptides capable of inducing cellular differentiation, dedifferentiation, and / or transdifferentiation of the cells of the present disclosure. In some methods known to those skilled in the art, an exogenous polypeptide (e.g., a recombinant polypeptide) encoded by a reprogramming gene (e.g., a gene described above) is contacted with a cell to induce, for example, the cells of the present disclosure. Those skilled in the art will understand that other genes may be associated with cellular reprogramming, and that exogenous molecules encoding such genes (or functional fragments thereof) and the encoded polypeptides are also considered polynucleotide or polypeptide reprogramming factors (e.g., polynucleotides or polypeptides that affect the expression level of another gene associated with cellular reprogramming). For example, it has been shown that the introduction of exogenous polynucleotides or polypeptide epigenetic gene silencers that reduce p53 inactivation increases the efficiency of inducing induced pluripotent stem cells (iPSCs).Therefore, exogenous polynucleotides or polypeptides that encode epigenetic silencers and other genes or proteins that can be directly or indirectly involved in increasing cell reprogramming or cell programming efficiency can be considered to constitute exogenous polynucleotides or polypeptide reprogramming factors.Those skilled in the art will understand that there are other ways to affect cell reprogramming, such as introducing RNAi molecules (or genetic material encoding RNAi molecules) that can knock down the expression of genes involved in inhibiting cell reprogramming.Therefore, any exogenous polynucleotide or polypeptide molecule that is associated with or enhances cell reprogramming should be understood to be an exogenous polynucleotide or polypeptide reprogramming factor as described herein.

[0177]

[0265] In some embodiments of the present disclosure, the methods exclude the use of reprogramming factors that are not small molecules. However, the methods do not include "traditional" tissue culture components, such as culture media, serum, serum replacement substances, supplements, antibiotics, etc., such as RPMI, renal epithelial basal medium (REBM), Dulbecco's modified Eagle's medium (DMEM), MCDB 131 medium, CMRL 1066 medium, F12, fetal calf serum (FCS), fetal bovine serum (FBS), bovine serum albumin (BSA), D-glucose, L-glutamine, GlutaMAX™-1 (a dipeptide of L-alanine-L-glutamine), B27, heparin, progesterone, putrescine, laminin, nicotinamide, insulin, transferrin, sodium selenite, selenite, thiamin mononitrate ... It will be understood that typical tissue culture components, such as benzodiazepines, ethanolamine, human epidermal growth factor (hEGF), basic fibroblast growth factor (bFGF), hydrocortisone, epinephrine, normacin, penicillin, streptomycin, gentamicin, and amphotericin, may also be utilized. It should be understood that these typical tissue culture components (and other similar tissue culture components routinely used in tissue culture) are not small molecule reprogramming molecules for the purposes of the present disclosure. Indeed, these components are not small molecules as defined herein and / or are not reprogramming factors as defined herein.

[0178]

[0266] Thus, in certain embodiments, the present disclosure does not involve culturing cells with one or more exogenous polynucleotide or polypeptide reprogramming factors. Thus, in certain embodiments, the methods of the present disclosure do not involve introducing one or more exogenous polynucleotide or polypeptide reprogramming factors, for example, by introducing a transposon, a viral transgenic vector (e.g., a retroviral vector), a plasmid, mRNA, miRNA, a peptide, or a fragment of any of these molecules, involved in producing artificial beta cells or otherwise inducing the cells of the present disclosure to differentiate, dedifferentiate, and / or transdifferentiate.

[0179]

[0267] That is, in an embodiment, the method is carried out without the presence of one or more exogenous polynucleotides or polypeptide reprogramming factors.Therefore, it should be understood that in an embodiment, the method of the present disclosure utilizes small molecules that reprogram cells without adding polypeptide transcription factors; other polypeptide factors that are specifically associated with inducing differentiation, dedifferentiation, and / or transdifferentiation; polynucleotide sequences that code for polypeptide transcription factors, polynucleotide sequences that code for other polypeptide factors that are specifically associated with inducing differentiation, dedifferentiation, and / or transdifferentiation; mRNA; interfering RNA; microRNA, and fragments thereof.

[0180] stem cells

[0268] The term "stem cell" is used herein to refer to cells (e.g., plant stem cells, vertebrate stem cells) that have the capacity to both self-renew and generate differentiated cell types (Morrison et al. (1997) Cell 88:287-298). In the context of cellular ontogeny, the adjectives "differentiated" or "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 restricted cells (e.g., neuronal precursors), which can differentiate into end-stage cells (i.e., terminally differentiated cells, e.g., neurons, cardiomyocytes, etc.) that play characteristic roles in a particular tissue type and may or may not retain the ability to proliferate further. Stem cells can be characterized by both the presence and absence of specific markers (e.g., proteins, RNA, etc.). Stem cells can also be identified both in vitro and in vivo by functional assays, particularly assays relating to the ability of stem cells to give rise to multiple differentiated progeny. In certain embodiments, the host cells are adult stem cells, somatic stem cells, non-embryonic stem cells, embryonic stem cells, hematopoietic stem cells, induced pluripotent stem cells, and trophoblast stem cells.

[0181]

[0269] Stem cells of interest include pluripotent stem cells (PSCs). The term "pluripotent stem cells" or "PSCs" is used herein to mean stem cells that can produce all cell types of an organism. Thus, PSCs can give rise to cells of all germ layers of an organism (e.g., endoderm, mesoderm, and ectoderm of vertebrates). Pluripotent cells can form teratomas and contribute to ectodermal, mesodermal, or endodermal tissues of living organisms. Plant pluripotent stem cells can give rise to all cell types of a plant (e.g., root, stem, It can produce cells such as leaves.

[0182]

[0270] Animal PSCs can be derived in several different ways. For example, embryonic stem cells (ESCs) are derived from the inner cell mass of embryos (Thomson et al., Science. 1998 Nov. 6;282(5391):1145-7), and induced pluripotent stem cells (iPSCs) are derived from somatic cells (Takahashi et al., Cell. 2007 Nov. 30;131(5):861-72; Takahashi et al., Nat. Protoc. 2007 Nov. 2(12):3081-9; Yu et al., Science. 2007 Dec. 21;318(5858):1917-20. Epub. Nov. 20, 2007). Because the term PSC refers to pluripotent stem cells regardless of their origin, the term PSC encompasses the terms ESC and iPSC, as well as embryonic germ stem cells (EGSCs), another example of PSCs. PSCs may be in the form of established cell lines, obtained directly from primary embryonic tissue, or derived from somatic cells.

[0183]

[0271] "Embryonic stem cells" (ESCs) refer to PSCs isolated from embryos, typically from the inner cell mass of blastocysts. ESC lines, such as hESBGN-01, hESBGN-02, hESBGN-03, hESBGN-04 (BresaGen, Inc.); HES-1, HES-2, HES-3, HES-4, HES-5, HES-6 (ES Cell International); Miz-hES1 (MizMedi Hospital-Seoul National University); HSF-1, HSF-6 (University of California, San Francisco); and H1, H7, H9, H13, H14 (Wisconsin Alumni Research Foundation (WiCell Research Institute)) are listed in the NIH Human Embryonic Stem Cell Registry. 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, equines, bovines, porcines, canines, felines, 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 large nucleocytoplasmic ratios, distinct borders, and prominent nucleoli. In addition, ESCs express SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and alkaline phosphatase, but do not express SSEA-1. Examples of methods for generating 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 propagating undifferentiated hESCs are described in WO99 / 20741, WO01 / 51616, and WO03 / 020920, each of which is incorporated herein in its entirety.

[0184]

[0272] "Embryonic germ stem cells" (EGSCs) or "embryonic germ cells" or "EG cells" refer to PSCs derived from germ cells and / or germ cell precursors, such as primordial germ cells, i.e., cells that can give rise to sperm and eggs. Embryonic germ cells (EG cells) are believed to have properties similar to the embryonic stem cells described above. Examples of methods for generating and characterizing EG cells are 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. and the like, each of which is incorporated herein in its entirety.

[0185]

[0273] "Induced pluripotent stem cells" or "iPSCs" refer to PSCs obtained from cells that are not PSCs (i.e., from cells that have differentiated relative to PSCs). iPSCs can be obtained from several different cell types, including terminally differentiated cells. iPSCs have an ES cell-like morphology, growing as flat colonies with a large nuclear-cytoplasmic ratio, distinct borders, and prominent nuclei. In addition, iPSCs express one or more key pluripotency markers known by those skilled in the art, including, but not limited to, alkaline phosphatase, SSEA3, SSEA4, Sox2, Oct3 / 4, Nanog, TRA160, TRA181, TDGF1, Dnmt3b, FoxD3, GDF3, Cyp26a1, TERT, and zfp42. Examples of methods for generating and characterizing iPSCs can be found, for example, in U.S. Patent Application Publication Nos. 20090047263, 20090068742, 20090191159, 20090227032, 20090246875, and 20090304646, each of which is incorporated herein in its entirety. Generally, to generate iPSCs, somatic cells are provided with reprogramming factors known in the art to reprogram somatic cells into pluripotent stem cells (e.g., Oct4, SOX2, KLF4, MYC, Nanog, Lin28, etc.).

[0186]

[0274] "Somatic cells" refers to any cell of an organism that does not normally give rise to all types of cells in the organism in the absence of experimental manipulation. In other words, somatic cells are fully differentiated cells that do not naturally generate all cells of the body's three germ layers, i.e., ectoderm, mesoderm, and endoderm. For example, somatic cells can include both neurons and neural precursors, the latter of which can naturally give rise to all or some cell types of the central nervous system, but cannot give rise to cells of either the mesodermal or endodermal lineages.

[0187]

[0275] In certain instances, stem cells can be undifferentiated (e.g., cells not committed to a particular lineage) before exposure to at least one beta cell maturation factor according to the methods 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 described herein. For example, stem cells can exhibit morphological, biological, or physical characteristics of undifferentiated cells that can be used to distinguish stem cells from differentiated cells of embryonic or adult origin. In some instances, undifferentiated cells can appear in two-dimensional microscopic images as colonies of cells with a high nuclear / cytoplasmic ratio and prominent nucleoli. Stem cells can be used by themselves (e.g., substantially free of any undifferentiated cells) or in the presence of differentiated cells. In certain instances, stem cells can be cultured with appropriate nutrients and, if necessary, in the presence of other cells that allow the stem cells to grow and differentiate as needed. For example, embryonic fibroblasts or fibroblast-like cells can be present in the culture to support stem cell growth. Fibroblasts can be present during one stage of stem cell development, but not necessarily at all stages. For example, fibroblasts may be added to the stem cell culture in a first culture step and not be added to the stem cell culture in one or more subsequent culture steps.

[0188]

[0276] Stem cells used in all aspects of the present invention can be any cell obtained from any type of tissue (e.g., embryonic tissue, e.g., fetal or prefetal tissue, or adult tissue) and are characterized by their ability, under appropriate conditions, to produce a variety of cell types, e.g., progeny of all derivatives of 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, for example, hESBGN-01, hESBGN-02, hESBGN-03, hESBGN-04 (BresaGen, Inc.); HES-1, HES-2, HES-3, HES-4, HES-5, and HES-6. , HES-6 (ES Cell International); Miz-hES1 (MizMedi Hospital-Seoul National University); HSF-1, HSF-6 (University of California, San Francisco); and H1, H7, H9, H13, H14 (Wisconsin Alumni Research Foundation (WiCell Research Institute)). In some embodiments, the source of human stem cells or pluripotent stem cells used for chemically induced differentiation into mature insulin-positive cells did not involve the destruction of human embryos.

[0189]

[0277] In another embodiment, stem cells can be isolated from tissues, including solid tissues.In some embodiments, the tissue is skin, adipose tissue (e.g., adipose tissue), muscle tissue, heart or cardiac tissue.In other embodiments, the tissue is, for example, but not limited to, umbilical cord blood, placenta, bone marrow, or cartilage.

[0190]

[0278] Stem cells of interest also include various types of embryonic cells exemplified by human embryonic stem (hES) cells, as described by Thomson et al. (1998) Science 282:1145; embryonic stem cells from other primates, such as rhesus stem cells (Thomson et al., (1995) Proc. Natl. Acad. Sci. USA 92:7844); marmoset stem cells (Thomson et al., (1996) Biol. Reprod. 55:254); and human embryonic germ (hEG) cells (Shambloft et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998). Lineage-committed stem cells, such as mesodermal stem cells and other early cardiac progenitor cells, are also of interest (see, e.g., Reyes et al. (2001) Blood 98:2615-2625; Eisenberg and Bader (1996) Circ Res. 78(2):205-16). Stem cells can be obtained from any mammalian species, e.g., human, equine, bovine, porcine, canine, feline, rodent, e.g., mouse, rat, hamster, primate, etc. In some embodiments, human embryos are not destroyed for the source of pluripotent cells used in the methods and compositions disclosed herein.

[0191]

[0279] A mixture of cells from a suitable source of endothelial stem cells, muscle stem cells, 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 collected (i.e., mobilized) circulating peripheral blood can be extracted from a subject.In some embodiments, stem cells can be reprogrammed stem cells, such as stem cells obtained from somatic cells or differentiated cells.In such embodiments, dedifferentiated stem cells can be, for example, but not limited to, neoplastic cells, tumor cells, and cancer cells, or alternatively, artificially reprogrammed cells, such as induced pluripotent stem cells or iPS cells.

[0192]

[0280] In some embodiments, SC-β cells are derived from hair follicles, keratinocytes, gonadotrophs, corticotrophs, thyrotrophs, growth hormone-producing cells, prolactin-producing cells, chromaffin cells, parafollicular cells, glomus cells, melanocytes, nevus cells, Merkel cells, odontoblasts, cementoblasts, keratocytes, retinal Müller cells, retinal pigment epithelial cells, neurons, glial cells (e.g., oligodendrocytes), stellate cells), ependymal cells, pineal cells, lung cells (e.g., type I pneumocytes and type II pneumocytes), Clara cells, goblet cells, G cells, D cells, ECL cells, gastric chief cells, gastric parietal cells, pit cells, K cells, D cells, I cells, goblet cells, Paneth cells, enterocytes, lesser fold cells, hepatocytes, hepatic stellate cells (e.g., mesodermally derived Kupffer cells), gallbladder cells, acinar center cells, pancreatic stellate cells, pancreatic alpha cells, pancreatic beta cells, pancreatic delta cells, pancreatic F cells (e.g., PP cells), pancreatic epsilon cells, thyroid gland (e.g., follicular cells), parathyroid gland (e.g., parathyroid chief cells), eosinophilic cells, urothelial cells, osteoblasts, osteocytes, chondroblasts, chondrocytes, fibroblasts, fibrocytes, myoblasts, muscle cells, muscle satellite cells, tendon cells, The endothelial cells may be derived from one or more of: cardiomyocytes, lipoblasts, adipocytes, interstitial cells of Cajal, hemangioblasts, endothelial cells, mesangial cells (e.g., intraglomerular mesangial cells and extraglomerular mesangial cells), juxtaglomerular cells, macula densa cells, interstitial cells, interstitial cells, telocytes, simple epithelial cells, podocytes, renal proximal tubule brush border cells, Sertoli cells, Leydig cells, granulosa cells, plug cells, germ cells, sperm, eggs, lymphocytes, myeloid cells, endothelial progenitor cells, endothelial stem cells, hemangioblasts, mesoangioblasts, pericytes, mural cells, spleen cells (e.g., T lymphocytes, B lymphocytes, dendritic cells, microphages, leukocytes), trophoblast stem cells, or any combination thereof.

[0193] Pancreatic progenitor cells or precursors

[0281] In some aspects, the present disclosure provides a method for generating NKX6.1-positive pancreatic progenitor cells from Pdx1-positive pancreatic progenitor cells, comprising contacting a cell population comprising Pdx1-positive pancreatic progenitor cells or precursors with at least two beta cell maturation 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, under conditions that promote cell cluster formation, for a period of at least 5 days to induce differentiation of at least one Pdx1-positive pancreatic progenitor cell in the population into an NKX6.1-positive pancreatic progenitor cell, wherein the NKX6.1-positive pancreatic progenitor cell expresses NKX6.1.

[0194]

[0282] In some embodiments, at least 10% of the Pdx1-positive pancreatic progenitor cells in the population are induced to differentiate into NKX6-1-positive pancreatic progenitor cells. In some embodiments, at least 95% of the Pdx1-positive pancreatic progenitor cells in the population are induced to differentiate into NKX6.1-positive pancreatic progenitor cells. In some embodiments, the NKX6.1-positive pancreatic progenitor cells express Pdx1, NKX6.1, and FoxA2. In some embodiments, the Pdx1-positive pancreatic progenitor cells are generated from a pluripotent stem cell population selected from the group consisting of embryonic stem cells and induced pluripotent stem cells.

[0195] Stem cell-derived beta cells

[0283] Provided herein, in some embodiments, are methods for generating SC-beta cells (e.g., mature pancreatic beta cells or beta-like cells) or their precursors using stem cells. In certain embodiments, germ cells can be used in place of or in conjunction with stem cells to provide at least one SC-beta cell using similar protocols described in U.S. Patent Application Publication Nos. 20150240212 and 20150218522, each of which is incorporated herein by reference in its entirety. Suitable germ cells can be prepared, for example, from primordial germ cells present in human fetal material obtained approximately 8-11 weeks after the last menstrual period. Exemplary germ cell preparation methods are described, for example, in Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998, and U.S. Patent No. 6,090,622.

[0196]

[0284] In some embodiments, compositions and methods for generating SC-β cells (e.g., pancreatic β cells) are provided herein. Overall, at least one SC-β cell or its precursor, such as a pancreatic progenitor generated according to the methods disclosed herein, can include a mixture or combination of different cells, such as Pdx1-positive pancreatic progenitors, pancreatic progenitors co-expressing Pdx1 and NKX6.1, Ngn3-positive endocrine precursor cells, insulin-positive endocrine cells (e.g., β-like cells), and insulin-positive endocrine cells, and / or other pluripotent or stem cell-like cells.

[0197]

[0285] At least one SC-β cell or progenitor thereof is generated according to any culture protocol suitable for differentiating stem cells or pluripotent cells to a desired stage of differentiation. In some embodiments, the at least one SC-β cell or 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 SC-β cell or precursor thereof.

[0198]

[0286] In some embodiments, at least one SC-β cell or precursor thereof is a substantially pure population of SC-β cells or precursors thereof. In some embodiments, the population of SC-β cells or precursors thereof comprises a mixture of pluripotent cells or differentiated cells. In some embodiments, the population of SC-β cells or precursors thereof is substantially free of or devoid of embryonic stem cells, pluripotent cells, or iPS cells.

[0199]

[0287] In some embodiments, somatic cells, e.g., fibroblasts, can be isolated from a subject, e.g., as a tissue biopsy, e.g., a skin biopsy, and reprogrammed into induced pluripotent stem cells for further differentiation to generate at least one SC-β cell or progenitor thereof for use in the compositions and methods described herein. In some embodiments, somatic cells, e.g., fibroblasts, are maintained in culture by methods known to those of skill in the art, and in some embodiments, expanded and then converted into SC-β cells by the methods disclosed herein.

[0200]

[0288] In some embodiments, at least one SC-β cell or progenitor thereof is maintained in culture by methods known to those skilled in the art, and in some embodiments, is expanded and then converted to an SC-β cell by the methods disclosed herein.

[0201]

[0289] Furthermore, the at least one SC-β cell or its precursor, e.g., pancreatic progenitor, can be derived from any mammalian species, including, but not limited to, murine, bovine, ape, porcine, equine, ovine, or human cells. For clarity and simplicity of exposition, the description of the methods herein refers to at least one mammalian SC-β cell or its precursor, but it should be understood that all methods described herein can be readily applied to at least one SC-β cell or its precursor of other cell types. In some embodiments, the at least one SC-β cell or its precursor is derived from a human individual.

[0202] Cell clusters of stem cell-derived beta cells

[0290] In some embodiments, provided herein is a cell cluster that is similar to the function and characteristics of endogenous pancreatic islets.Such cell clusters can mimic the function of endogenous pancreatic islets, such as regulating the metabolism of a subject, for example, glucose metabolism.Therefore, cell clusters can be transplanted into a subject to treat diseases caused by insufficient pancreatic islet function, such as diabetes.The terms "cluster" and "aggregate" can be used interchangeably and refer to a group of cells that have close cell-to-cell contact, and in some cases, the cells in the cluster can adhere to each other.

[0203]

[0291] A cell cluster comprises a plurality of cells, hi some embodiments, a cell cluster comprises at least 10, at least 50, at least 200, at least 500, at least 750, at least 1000, at least 1500, at least 2000, at least 2500, at least 3000, at least 3500, at least 4000, at least 4500, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 20,000, at least 30,000, or at least 50,000 cells. In some embodiments, cell clusters are between 10 and 10,000 cells, between 50 and 10,000, between 100 and 10,000, between 100 and 10,000, between 1,000 and 10,000, between 500 and 10,000, between 500 and 5,000, between 500 and 2,500 In some embodiments, the cell clusters comprise between 500 and 2,000 cells, between 1,000 and 100,000 cells, between 1,000 and 50,000 cells, between 1,000 and 40,000 cells, between 1,000 and 20,000 cells, between 1,000 and 10,000 cells, between 1,000 and 5,000 cells, and between 1,000 and 3,000 cells. In some embodiments, the cell clusters comprise at least 500 cells. In some embodiments, the cell clusters comprise at least 1,000 cells. In some embodiments, the cell clusters comprise at least 2,000 cells. In some embodiments, the cell clusters comprise at least 5,000 cells. In some embodiments, the cell clusters comprise no more than 100,000, no more than 90,000, no more than 80,000, no more than 70,000, no more than 60,000, no more than 50,000, no more than 40,000, no more than 30,000, no more than 20,000, no more than 10,000, no more than 7,000, no more than 5,000, no more than 3,000, no more than 2,000 cells, or no more than 1,000 cells.

[0204]

[0292] The cell clusters herein can comprise at least one non-native cell, e.g., a non-native pancreatic beta cell. Non-native cells (e.g., non-native pancreatic beta cells) can share characteristics of endogenous cells (e.g., endogenous mature pancreatic beta cells) but differ in certain aspects (e.g., gene expression profiles). Non-native cells can be genetically modified cells. Non-native cells can be cells differentiated from progenitor cells, e.g., stem cells. Stem cells can be embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). In some cases, non-native cells can be cells differentiated in vitro from progenitor cells. In some cases, non-native cells can be cells differentiated in vivo from progenitor cells. For example, a cell cluster can comprise at least one non-native pancreatic beta cell. Non-native pancreatic beta cells can be those described in U.S. Patent Application Nos. 14 / 684,129 and 14 / 684,101, the entire contents of which are incorporated herein. A cell cluster can comprise multiple non-native pancreatic beta cells. In some cases, at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% of the cells in the cell cluster are non-native pancreatic beta cells. The cell cluster can include one or more native cells. For example, the cell cluster can include one or more primary cells, such as primary cells derived from endogenous pancreatic islets.

[0205]

[0293] A cell cluster can include one or more cells expressing at least one marker of endogenous cells, such as endogenous mature pancreatic beta cells. The term "marker" can refer to a molecule that can be observed or detected. For example, a marker can include, but is not limited to, a nucleic acid, such as a transcript of a specific gene, a polypeptide product of a gene, a non-gene product polypeptide, a glycoprotein, a carbohydrate, a glycolipid, a lipid, a lipoprotein, or a small molecule. In many cases, a marker can refer to a molecule that can be characteristic of a specific cell type, and thus can be referred to as a cell-type marker. For example, the insulin gene can be referred to as a marker of beta cells. In some cases, a marker is a gene. Non-limiting examples of markers of endogenous mature pancreatic beta cells include insulin, C-peptide, PDX1, NKX6.1, CHGA, MAFA, ZNT8, PAX6, NEUROD1, glucokinase (GCK), SLC2A, PCSK1, KCNJ11, ABCC8, SLC30A8, SNAP25, RAB3A, GAD2, and PTPRN.

[0206]

[0294] The cell cluster can comprise one or more cells that express one or more markers of endogenous cells, for example, endogenous mature pancreatic beta cells. For example, the cell cluster can comprise one or more cells that co-express at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 markers of endogenous cells, for example, endogenous mature pancreatic beta cells. In some cases, the cell cluster comprises cells that express NKX6.1 and C-peptide, both of which can be markers of beta cells.

[0207]

[0295] A cell cluster can include a plurality of cells expressing at least one marker of an endogenous cell. For example, at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the cells in the cell cluster can express at least one marker of an endogenous cell. In some cases, all cells in the cell cluster can express a marker of an endogenous cell. In some cases, the endogenous cell can be a pancreatic cell, such as a pancreatic beta cell, a pancreatic alpha cell, a pancreatic beta cell, a pancreatic delta cell, or a pancreatic gamma cell. The cell clusters provided herein can include a heterogeneous cell population, e.g., a variety of cell types. For example, a cell cluster can include cells expressing insulin / C-peptide, which can be a marker of pancreatic beta cells, cells expressing glucagon, which can be a marker of pancreatic alpha cells, cells expressing somatostatin, which can be a marker of pancreatic delta cells, cells expressing pancreatic polypeptide, or any combination thereof.

[0208]

[0296] For example, the cell clusters herein can comprise a plurality of cells that express one or more markers of endogenous mature pancreatic beta cells, e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the cells in the cell cluster can express one or more markers of endogenous mature pancreatic beta cells.

[0209]

[0297] The cell cluster can comprise a plurality of cells that express CHGA. In some cases, at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the cells in the cell cluster express CHGA. In some cases, at least about 85% of the cells in the cell cluster can express CHGA. In some cases, the cell cluster can comprise about 90% cells that express CHGA. In some cases, the cell cluster can comprise about 95% cells that express CHGA. In certain cases, all of the cells in the cell cluster can express CHGA.

[0210]

[0298] A cell cluster can comprise a plurality of cells that express NKX6.1. For example, at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% of the cells in the cell cluster can express NKX6.1. In some cases, at least about 50% of the cells in the cell cluster can express NKX6.1. In some cases, all of the cells in the cell cluster can express NKX6.1.

[0211]

[0299] The cell cluster can include a plurality of cells that express C-peptide. For example, at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the cells in the cell cluster can express C-peptide. In some cases, at least about 60% of the cells in the cell cluster can express C-peptide. In some cases, all of the cells in the cell cluster can express C-peptide.

[0212]

[0300] A cell cluster can contain a plurality of cells that express both NKX6.1 and C-peptide. For example, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the cells in a cell cluster can express C-peptide. In some cases, at least about 35% of the cells in a cell cluster can express NKX6.1 and C-peptide. In some cases, at least about 40% of the cells in a cell cluster can express NKX6.1 and C-peptide. In some cases, at least about 35% of the cells in a cell cluster can express NKX6.1 and C-peptide. In some cases, a cell cluster can contain about 60% cells that express NKX6.1 and C-peptide. In some cases, a cell cluster can contain about 75% cells that express NKX6.1 and C-peptide. In some cases, all cells in a cell cluster can express NKX6.1 and C-peptide.

[0213]

[0301] Cell clusters may contain little or no stem cells or progenitor cells, such as pancreatic progenitor cells.For example, the cell clusters provided herein may contain up to about 5%, up to about 5%, up to about 5%, up to about 5%, up to about 5%, up to about 2%, up to about 1%, up to about 0.5%, up to about 0.1%, up to about 0.05%, up to about 0.01%, or no cells that express LIN28.In some examples, the cell clusters provided herein may contain up to about 5%, up to about 5%, up to about 5%, up to about 5%, up to about 5%, up to about 2%, up to about 1%, up to about 0.5%, up to about 0.1%, up to about 0.05%, up to about 0.01%, or no cells that express Ki67.

[0214]

[0302] In some cases, the cell clusters may contain up to 3%, up to about 2%, up to about 1%, up to about 0.5%, up to about 0.1%, up to about 0.05%, up to about 0.01%, or no cells expressing SOX2. In some cases, the cell clusters may contain about 1% cells expressing SOX2. In some cases, the cell clusters may contain about 0.6% cells expressing SOX2. In some cases, the cell clusters may contain about 0.3% cells expressing SOX2. In some cases, the cell clusters may contain about 0.1% cells expressing SOX2.

[0215]

[0303] In some examples, the cell clusters may contain up to 10%, up to about 8%, up to about 6%, up to about 5%, up to about 2%, up to about 1%, up to about 0.5%, up to about 0.1%, up to about 0.05%, up to about 0.01%, or no cells expressing SOX9. In some cases, the cell clusters may contain about 2% cells expressing SOX9. In some cases, the cell clusters may contain about 6% cells expressing SOX9. In some cases, the cell clusters may contain about 1.2% cells expressing SOX9.

[0216]

[0304] The cell clusters herein can exhibit one or more glucose-stimulated insulin secretion (GSIS) responses in vitro when exposed to glucose load(s). This GSIS response may be similar to the GSIS response of endogenous pancreatic islets. In some cases, the cell clusters exhibit an in vitro GSIS response to a glucose load. In some cases, the cell clusters exhibit an in vitro GSIS response to multiple glucose loads, for example, sequential glucose loads. For example, the cell clusters can exhibit an in vitro GSIS response to at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 sequential glucose loads.

[0217]

[0305] The cell clusters provided herein can include at least one cell that exhibits in vitro GSIS. For example, at least one cell in a cell cluster can be referred to as a mature pancreatic beta cell. In some cases, at least one cell is a non-native pancreatic beta cell. In some cases, at least one cell is a pancreatic beta cell similar to a natural / endogenous beta cell. In some cases, the cells exhibit an in vitro glucose-stimulated insulin secretion (GSIS) response. In some cases, at least one cell exhibits a GSIS response to at least one glucose load. In some cases, the cells exhibit a GSIS response to at least two sequential glucose loads. In some cases, the cells exhibit a GSIS response to at least three sequential glucose loads.

[0218]

[0306] As provided herein, cell clusters can exhibit a GSIS stimulation index similar to that of endogenous pancreatic islets. The stimulation index of a cell cluster or cell is characterized by the ratio of insulin secreted in response to high glucose concentrations compared to low glucose concentrations. For example, the stimulation index of a cell cluster or cell provided herein can be calculated as the ratio of insulin secreted in response to 20 mM glucose stimulation to insulin secreted in response to 2.8 mM glucose stimulation. In some examples, the stimulation index of a cell cluster or cell provided herein is 1 or greater, or 1.1 or greater, or 1.3 or greater, or 2 or greater, or 2.3 or greater, or 2.6 or greater. In some cases, the cell cluster or cell exhibits cytokine-induced apoptosis in response to a cytokine. In some cases, the cytokine comprises interleukin-β (IL-β), interferon-γ (INF-γ), tumor necrosis factor-α (TNF-α), or any combination thereof. In some cases, insulin secretion from the cell cluster or cell is enhanced in response to an antidiabetic agent. In some cases, the antidiabetic agent comprises a secretagogue selected from the group consisting of an incretin mimetic, a sulfonylurea, a meglitinide, and a combination thereof. In some cases, the cell cluster or cell is monohormonal. In some cases, the cell clusters or cells exhibit a morphology similar to that of endogenous mature pancreatic beta cells. In some cases, the cell clusters or cells exhibit encapsulated crystalline insulin granules similar to those of endogenous mature pancreatic beta cells under an electron microscope. In some cases, the cell clusters or cells exhibit a low replication rate. In some cases, the cell clusters or cells exhibit a glucose-stimulated Ca2+ response similar to that of endogenous mature pancreatic beta cells. 2+ In some cases, the cell clusters or cells exhibit a GSCF response similar to GSCF (GSCF) flux. In some cases, the cell clusters or cells exhibit a GSCF response to at least one glucose load. In some cases, the cell clusters or cells exhibit a GSCF response to at least two glucose loads. In some cases, the cell clusters or cells exhibit a GSCF response to at least three glucose loads. In some cases, the cell clusters or cells exhibit increased calcium flux. In some cases, the increased calcium flux includes an increased amount of influx or a ratio of influx at low glucose concentrations to high glucose concentrations.

[0219]

[0307] The cell clusters provided herein can exhibit biphasic insulin secretion in response to high glucose stimulation, similar to endogenous pancreatic islets, e.g., human pancreatic islets. Biphasic insulin secretion can be a characteristic phenomenon of endogenous pancreatic islets, e.g., human islets. In some embodiments, the response of the cell clusters provided herein, e.g., reaggregated pancreatic cell clusters, to a high glucose concentration load, e.g., 10 mM, 15 mM, 20 mM, or 30 mM, can exhibit a transient increase in insulin secretion to a peak value, followed by a rapid decline to a relatively elevated insulin secretion level, e.g., a level higher than the insulin secretion level in response to a lower glucose concentration, e.g., 2.8 mM glucose. Such a transient increase and decline process may be referred to as the first phase of a biphasic insulin secretion pattern. Thus, with persistent high glucose loading, the first phase can be followed by a second phase in which insulin secretion by the cell clusters can be maintained at a relatively elevated level. The second phase can continue for a long period of time, e.g., as long as the high glucose concentration load continues, or for a longer period than the first phase. Such a biphasic insulin secretion pattern may result from intrinsic cell signaling changes characteristic of mature native pancreatic β-cells.

[0220]

[0308] When transplanted into a subject, the cell clusters can exhibit one or more in vivo GSIS responses when exposed to glucose load(s). The cell clusters herein may be able to exhibit in vivo GSIS responses within a short period of time after transplantation into a subject. For example, the cell clusters can exhibit in vivo GSIS within about 6, 12, or 24 hours after transplantation. In some cases, the cell clusters exhibit in vivo GSIS within about 2, 4, 6, 8, 10, 12, 14, 21, 28, 35, or 42 days after transplantation. The amount of insulin secreted by the cell clusters may be similar to or higher than that of endogenous pancreatic islets. The term "about" when used throughout this application in reference to a numerical value can include values ​​within a range of plus or minus 10% from that value. For example, "about 1 The amount "0" includes amounts from 9 to 11. For example, the term "about" in reference to a reference numerical value can also include values ​​within a range of plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value.

[0221]

[0309] The cell clusters can maintain the ability to exhibit an in vivo GSIS response for a period of time after transplantation into a subject. For example, the in vivo GSIS response of the cell clusters can be observed for at least 2 weeks, 3 weeks, 4 weeks, 5 weeks, 10 weeks, 15 weeks, 20 weeks, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 10 years, 20 years, 30 years, 40 years, 60 years, 80 years, or up to 100 years after transplantation of the cell clusters into a subject (e.g., a human).

[0222]

[0310] The GSIS of cell clusters can be measured by a stimulation index. The stimulation index of a cell cluster can be equal to the ratio of insulin secreted in response to high glucose concentrations compared to insulin secreted in response to low glucose concentrations. The cell cluster can have a stimulation index similar to that of endogenous pancreatic islets. In some cases, the cell cluster has a stimulation index of at least 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0.

[0223]

[0311] The amount of insulin secreted by a cell cluster in response to a glucose load (e.g., a high concentration, e.g., 20 mM glucose) is approximately 0.1 μIU / 10 3 Cells ~ approx. 5μIU / 10 3 cells, approximately 0.2μIU / 10 3 Cells ~ approx. 4μIU / 10 3 cells, approximately 0.2μIU / 10 3 Cells ~ approx. 3μIU / 10 3 cells, or approximately 0.23 μIU / 10 3 Cells ~ approx. 2.7μIU / 10 3 In some cases, the amount of insulin secreted by the cell cluster in response to a glucose load (e.g., a high concentration, e.g., 20 mM glucose) can be at least 0.05, 0.1, 0.15, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 μIU / 10 3 It is a cell.

[0224]

[0312] The cell clusters can secrete both proinsulin and insulin. For example, the cell clusters can secrete proinsulin and insulin at a proinsulin to insulin ratio substantially the same as the ratio of proinsulin to insulin secreted by endogenous pancreatic islets. In some cases, the cell clusters secrete proinsulin and insulin at a proinsulin to insulin ratio of about 0.01 to about 0.05, about 0.02 to about 0.04, about 0.02 to about 0.03, or 0.029 to about 0.031. In some cases, the cell clusters secrete proinsulin and insulin at a proinsulin to insulin ratio of about 0.02, 0.021, 0.022, 0.023, 0.024, 0.025, 0.026, 0.027, 0.028, 0.029, 0.03, 0.031, 0.032, 0.033, 0.034, 0.035, 0.036, 0.037, 0.038, 0.039, or 0.04.

[0225]

[0313] The cell cluster can be similar in size to endogenous pancreatic islets. For example, the cell cluster can have a diameter similar to endogenous pancreatic islets. The diameter of the cell cluster can refer to the maximum linear distance between two points on the surface of the cell cluster. In some cases, the diameter of the cell cluster is up to 300 μm, 200 μm, 150 μm, 100 μm, 90 μm, 80 μm, 70 μm, 60 μm, 50 μm, or 40 μm. The diameter of the cell cluster can be The diameter of the cell clusters can be about 75 μm to about 250 μm.

[0226]

[0314] The cell clusters may contain few or no dead cells. The cell clusters may be of a size that allows for the effective diffusion of molecules (e.g., nutrients and gases) from the surrounding environment to the center of the cell cluster. The diffused molecules may be important for the survival and function of the cells in the center. In some cases, the cell clusters may have less than about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% dead cells, for example, dead cells in the center. In some cases, the cell clusters may have no dead cells at all. The dead cells may be apoptotic cells, narcotic cells, or any combination thereof.

[0227]

[0315] The cell clusters can include one or more types of cells. In some cases, the cell clusters include one or more types of pancreatic cells. For example, the cell clusters can include one or more pancreatic beta cells, pancreatic alpha cells, pancreatic delta cells, pancreatic gamma cells, and any combination thereof. In some cases, the pancreatic cells can be non-native pancreatic cells, for example, cells derived from stem cells such as ESCs and / or iPSCs. In some cases, the cell clusters can also include one or more progenitor cells of mature pancreatic cells, including iPSCs, ESCs, definitive endoderm cells, primitive gut cells, Pdx1-positive pancreatic progenitor cells, Pdx1-positive / NKX6.1-positive pancreatic progenitor cells, Ngn3-positive endocrine progenitor cells, and any combination thereof.

[0228]

[0316] The cell clusters can undergo cytokine-induced apoptosis in response to cytokines, such as interleukin-1β (IL-β), interferon-γ (INF-γ), tumor necrosis factor-α (TNF-α), and combinations thereof.

[0229]

[0317] Insulin secretion from the cell clusters herein is associated with the administration of antidiabetic drugs (e.g., ex The secretion of insulin from the cell clusters may be enhanced by a secretagogue (an antidiabetic drug acting on pancreatic beta cells in vivo, in vitro, and / or in vivo). The present disclosure contemplates any known antidiabetic drug. In some cases, insulin secretion from the cell clusters may be enhanced by a secretagogue. The secretagogue may be an incretin mimetic, a sulfonylurea, a meglitinide, or a combination thereof.

[0230]

[0318] The cell clusters can comprise a single hormone. For example, the cell clusters can comprise pancreatic cells that are single hormones (e.g., pancreatic beta cells, pancreatic alpha cells, pancreatic beta cells, pancreatic delta cells, or pancreatic gamma cells). In some cases, the cell clusters comprise insulin-secreting non-native pancreatic cells that are single hormones. The cell clusters can comprise multiple hormones. In some cases, the cell clusters include single hormone cells and multiple hormone cells.

[0231]

[0319] A cell cluster can include cells (e.g., non-native pancreatic cells) having a morphology similar to that of endogenous mature pancreatic beta cells. In some cases, a cell cluster can include cells that encapsulate crystalline insulin granules similar to those of endogenous mature pancreatic beta cells, as detected, for example, by electron microscopy. A cell cluster can include a plurality of cells having a morphology similar to that of endogenous mature pancreatic beta cells. For example, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the cells in a cell cluster can encapsulate crystalline insulin granules similar to those of endogenous mature pancreatic beta cells. In some cases, 100% of the cells in a cell cluster encapsulate crystalline insulin granules similar to those of endogenous mature pancreatic beta cells.

[0232]

[0320] Cell clusters respond to glucose-stimulated calcium (Ca) responses in response to one or multiple glucose loads. 2+ In some cases, cell clusters can exhibit endogenous islet glucose-stimulated Ca flux. 2+In some cases, the cell clusters exhibit a GSCF response to at least 1, 2, 3, 4, 5, 6, 8, or 10 sequential glucose loads in a manner similar to the GSCF response of endogenous pancreatic islets to multiple glucose loads. The cell clusters can exhibit an in vitro and / or in vivo GSCF response when exposed to a glucose load.

[0233]

[0321] The cell clusters can include cells of any species of origin. For example, the cell clusters can include cells from mammalian species, including, but not limited to, mouse, bovine, ape, porcine, equine, ovine, or human cells. In some cases, at least one cell in the cell cluster is a human cell.

[0234]

[0322] Also provided herein are compositions comprising the cell clusters disclosed throughout this application. In addition to the cell clusters, the compositions can further comprise a scaffold or matrix that can be used to transplant the cell clusters into a subject. The scaffold can provide a structure to which the cell clusters adhere. The cell clusters can be transplanted into a subject along with the scaffold. The scaffold can be biodegradable. In some cases, the scaffold comprises a biodegradable polymer. The biodegradable polymer can be a synthetic polymer, such as poly(lactide) (PLA), poly(glycolic acid) (PGA), poly(lactide-co-glycolide) (PLGA), and other polyhydroxy acids, poly(caprolactone), polycarbonate, polyamide, polyanhydride, polyphosphazene, polyamino acid, polyorthoester, polyacetal, polycyanoacrylate, and biodegradable polyurethane. Biodegradable polymers can also be natural polymers, such as albumin, collagen, fibrin, polyamino acids, prolamines, and polysaccharides (e.g., alginate, heparin, and other naturally occurring biodegradable polymers of sugar units). Alternatively, the scaffold can be non-biodegradable. For example, the scaffold can include non-biodegradable polymers such as polyacrylates, ethylene-vinyl acetate polymers, and other acyl-substituted cellulose acetates and their derivatives, polyurethanes, polystyrenes, polyvinyl chlorides, polyvinyl fluorides, poly(vinylimidazole), chlorosulfonated polyolefins, and polyethylene oxides.

[0235] Dissociated cell compositions and methods for producing cell clusters of stem cell-derived beta cells

[0323] Further disclosed herein is a method for producing a cell cluster that resembles the functions and characteristics of endogenous tissue or cell clusters, such as endogenous pancreatic islets. The disclosed method may include dissociating a first cell cluster and reaggregating the dissociated cells into a second cell cluster, wherein the second cell cluster more closely resembles the functions and characteristics of endogenous tissue or cell clusters, such as endogenous pancreatic islets, compared to the first cell cluster. The term "reaggregation" and its grammatical equivalents, as used herein, may refer to the dissociation of a cluster into smaller clusters or single cells, and the dissociated cells then forming new intercellular junctions to form a new cluster. The disclosed method may be used to generate a cell cluster in vitro by a) dissociating a plurality of cells from the first cell cluster, and b) culturing the plurality of cells of a) in a medium, thereby allowing the plurality of cells to form a second cell cluster. Optionally, the second cell cluster is an in vitro cell cluster. The first cell clusters can be in vitro cell clusters, e.g., clusters formed by a suspension of single cells in vitro in a culture medium. Optionally, the first cell clusters can be ex vivo cell clusters, e.g., cell clusters formed within a living organism and isolated from said organism. For example, the first cell cluster to which the methods provided herein are applicable may be human pancreatic islets. Optionally, the first cell cluster may be cadaveric pancreatic islets.

[0236]

[0324] The methods provided herein can enrich pancreatic cells, such as pancreatic beta cells, endocrine cells, or endocrine progenitor cells, in cell clusters.In some examples, the methods of the present disclosure can reduce or eliminate stem cells or pancreatic progenitor cells from cell clusters.Optionally, the second cell cluster contains a higher percentage of cells expressing chromogranin A compared to the first cell cluster.Optionally, the second cell cluster contains a higher percentage of cells expressing NKX6.1 and C-peptide compared to the first cell cluster.Optionally, the second cell cluster contains a lower percentage of cells expressing SOX2 compared to the first cell cluster.Optionally, the second in vitro cell cluster contains a lower percentage of cells expressing SOX9 compared to the first cell cluster.

[0237]

[0325] Optionally, the medium contains a thyroid hormone signaling pathway activator and a transforming growth factor β (TGF-β) signaling pathway inhibitor. Optionally, the medium contains a) serum, and b) one or both of a thyroid hormone signaling pathway activator and a TGF-β signaling pathway inhibitor. Optionally, the medium for reaggregation (reaggregation medium) as provided herein may not contain a small molecule compound. For example, the reaggregation medium may not contain a thyroid hormone signaling pathway activator. Optionally, the reaggregation medium does not contain triiodothyronine (T3) or contains only trace amounts of T3. The reaggregation medium may not contain a TGFβ signaling pathway inhibitor. Optionally, the reaggregation medium does not contain an Alk5 inhibitor (Alk5i) or contains only trace amounts of Alk5i.

[0238]

[0326] Dissociation of the first cell cluster can be carried out using methods known in the art. Non-limiting exemplary methods for dissociating cell clusters include physical force (e.g., mechanical dissociation, such as using a cell scraper, trituration with a narrow-bore pipette, fine needle aspiration, vortex disaggregation, and forced filtration through a fine nylon or stainless steel mesh), enzymatic dissociation using enzymes such as trypsin, collagenase, TrypLE™, etc., or a combination thereof. After dissociation, the cells of the first cell cluster can be in a cell suspension, for example, a single cell suspension. The term "suspension" as used herein can refer to a cell culture condition in which cells are not attached to a solid support. Cells grown in suspension can be agitated during growth using devices well known to those skilled in the art.

[0239]

[0327] In some embodiments, the present disclosure provides a composition comprising dissociated cells. In some embodiments, the composition of the present disclosure does not comprise cell clusters. In some embodiments, the composition of the present disclosure does not comprise insulin-positive cell clusters. In some embodiments, the composition of the present disclosure does not comprise cell clusters comprising more than 5, 10, 20, 30, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 cells. In some embodiments, the composition of the present disclosure does not comprise cell clusters comprising more than 50 cells. In some embodiments, the composition of the present disclosure does not comprise cell clusters comprising more than 100 cells. In some embodiments, the composition of the present disclosure does not comprise cell clusters comprising more than 500 cells. In some embodiments, the present disclosure provides a composition comprising a plurality of dissociated insulin-positive endocrine precursor cells. In some embodiments, the dissociated cells are Ngn3-positive. In some embodiments, the dissociated cells are PDX.1-positive. In some embodiments, the dissociated cells are NKX6.1-positive. In some embodiments, the present disclosure provides a composition comprising dissociated cells (e.g., dissociated insulin-positive endocrine precursor cells) and a BMP signaling pathway inhibitor. The BMP signaling pathway inhibitor is LDN193189 or a derivative thereof. In some embodiments, the present disclosure provides a composition comprising a plurality of dissociated cells (e.g., dissociated insulin-positive endocrine precursor cells) and a ROCK inhibitor. In some embodiments, the ROCK inhibitor is thiazovivin, Y-27632, fasudil / HA1077, or 14-1152, or a derivative thereof. In some embodiments, the present disclosure provides a composition comprising a plurality of dissociated cells (e.g., dissociated insulin-positive endocrine precursor cells) and a histone methyltransferase inhibitor. In some embodiments, the histone methyltransferase inhibitor is 3-deazaneplanocin A hydrochloride or a derivative thereof. In some embodiments, the present disclosure provides a composition comprising a plurality of dissociated cells (e.g., dissociated insulin-positive endocrine precursor cells) and zinc. In some embodiments, the zinc is in the form of ZnSO4. In some embodiments, the present disclosure provides a composition comprising a plurality of dissociated cells (e.g., dissociated insulin-positive endocrine precursor cells) and a monoglyceride lipase (MGLL) inhibitor. In some embodiments, the MGLL inhibitor is JJKK048, KML29, NF1819, JW642, JZL184, JZL195, JZP361, pristimerin, or URB602, or a derivative of any of the foregoing. In some embodiments, the present disclosure provides a composition comprising a plurality of dissociated cells (e.g., dissociated insulin-positive endocrine precursor cells) and a lipid. In some embodiments, the lipid is a saturated fatty acid. In some embodiments, the saturated fatty acid is palmitate. In some embodiments, the lipid is an unsaturated fatty acid. In some embodiments, the unsaturated fatty acid is oleic acid, linoleic acid, or palmitoleic acid. In some embodiments, the present disclosure provides a composition comprising a plurality of dissociated cells (e.g., dissociated insulin-positive endocrine precursor cells) and glutamate. In some embodiments, the present disclosure provides a composition comprising a plurality of dissociated cells (e.g., dissociated insulin-positive endocrine precursor cells) and acetate. In some embodiments, the present disclosure provides a composition comprising a plurality of dissociated cells (e.g., dissociated insulin-positive endocrine precursor cells) and β-hydroxybutarate.In some embodiments, the present disclosure provides a composition comprising a plurality of dissociated cells (e.g., dissociated insulin-positive endocrine precursor cells) and L-carnitine. In some embodiments, the present disclosure provides a composition comprising a plurality of dissociated cells (e.g., dissociated insulin-positive endocrine precursor cells) and taurine. In some embodiments, the present disclosure provides a composition comprising a plurality of dissociated cells (e.g., dissociated insulin-positive endocrine precursor cells) and formate. In some embodiments, the present disclosure provides a composition comprising a plurality of dissociated cells (e.g., dissociated insulin-positive endocrine precursor cells) and biotin. In some embodiments, the composition of the present disclosure further comprises a serum albumin protein. In some embodiments, the serum albumin protein is human serum albumin protein. In some embodiments, the compositions of the present disclosure may be administered in the presence of 0.01% to 1%, 0.03 to 1%, 0.03 to 0.9%, 0.03 to 0.08%, 0.03 to 0.06%, 0.03 to 0.05%, 0.04 to 0.8%, 0.04 to 0.7%, 0.04 to 0.6%, 0.04 to 0.5%, 0.04 to 0.4%, 0.04 to 0.3%, 0.04 to 0.2%, 0.04 to 0.1%, 0.04 to 0.09%, 0.04 to 0.8%, 0.04 to 0. Contains 0.07%, 0.04-0.06%, 0.04-0.05%, 0.05-1%, 0.05-0.9%, 0.05-0.8%, 0.05-0.7%, 0.05-0.6%, 0.05-0.5%, 0.05-0.4%, 0.05-0.3%, 0.05-0.2%, 0.05-0.1%, 0.05-0.09%, 0.05-0.8%, 0.05-0.07%, or 0.05-0.06% serum albumin protein. In some embodiments, less than 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 1% of the cells in the composition are in cell clusters. In some embodiments, the compositions of the present disclosure comprise a TGF-β pathway inhibitor. In some embodiments, the TGF-β pathway inhibitor is Alk5i (SB505124) or a derivative thereof. In some embodiments, the compositions of the present disclosure inhibit thyroid hormone signaling pathways. In some embodiments, the thyroid hormone signaling pathway activator is GC-1 or T3, or a derivative thereof. In some embodiments, the composition of the present disclosure comprises a protein kinase inhibitor. In some embodiments, the protein kinase inhibitor is staurosporine. In some embodiments, the composition of the present disclosure comprises vitamin C. In certain embodiments, the composition of the present disclosure is in vitro. In some embodiments, the composition of the present disclosure comprises insulin. In some embodiments, the composition of the present disclosure does not comprise a gamma-secretase inhibitor (e.g., XXI). In some embodiments, the dissociated insulin-positive endocrine precursor cells have been previously frozen.

[0240]

[0328] In some embodiments, the present disclosure provides a composition comprising a plurality of cell clusters. In some embodiments, the present disclosure provides a composition comprising a plurality of cell clusters, wherein the cell clusters comprise insulin-positive cells, and wherein at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, or at least 65% of the cells in the composition are viable after 11 days of in vitro culture. In some embodiments, the present disclosure provides a composition comprising a plurality of cell clusters, the cell clusters comprising insulin-positive cells, and wherein at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the cell clusters in the composition are 90-140 μm, 90-130 μm, 90-120 μm, 90-110 μm, 100-140 μm, 100-130 μm, 100-120 μm, or 100-110 μm in diameter. In some embodiments, the disclosure provides a composition comprising a plurality of cell clusters, wherein the cell clusters comprise insulin-positive cells, and wherein at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the cell clusters in the composition have an insulin-positive cell population of 1.5 to 4.5, 1. The compositions exhibit a glucose-stimulated insulin secretion (GSIS) stimulation index of 5 to 4.0, 1.5 to 3.5, 1.5 to 3.0, 1.5 to 2.5, 1.5 to 2.5, 1.5 to 2.0, 2.0 to 4.5, 2.0 to 4.0, 2.0 to 3.5, 2.0 to 3.0, 2.0 to 2.5, 2.5 to 4.5, 2.5 to 4.0, 2.5 to 3.5, 2.5 to 3.0, 3.0 to 4.5, 3.0 to 4.0, 3.0 to 3.5, 3.5 to 4.5, 3.5 to 4.0, or 4.0 to 4.5. In some embodiments, the cell clusters comprise C-peptide-positive cells. In some embodiments, the cell clusters comprise somatostatin-positive cells.In some embodiments, the cell clusters comprise glucagon-positive cells. In some embodiments, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, or at least 65% of the cells in the composition are viable after 11 days of in vitro culture. In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the cell clusters in the composition are 90-140 μm, 90-130 μm, 90-120 μm, 90-110 μm, 100-140 μm, 100-130 μm, 100-120 μm, or 100-110 μm in diameter. In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the cell clusters in the composition are 1.5-4.5, 1.5-4.0, 1.5-3.5, 1.5-3.0, 1.5-2.5, 1.5-2.5, 1.5-2.0, 2.0-4.5, 2.0-4.0, 2.0-3.5, 2.0-3.0, 2.0-2.5, 2.5-4.5, 2.5-4.0, or 2.5-3.5. The cells exhibit a glucose-stimulated insulin secretion (GSIS) stimulation index of 2.5 to 3.0, 3.0 to 4.5, 3.0 to 4.0, 3.0 to 3.5, 3.5 to 4.5, 3.5 to 4.0, or 4.0 to 4.5. In some embodiments, there are at least 2, 3, 4, 5, 10, 50, 100, 1,000, 10,000, 100,000, or 1,000,000 cell clusters. In some embodiments, the compositions of the present disclosure are prepared according to any method disclosed herein. In some embodiments, the present disclosure provides a device comprising any of the cell compositions disclosed herein. In some embodiments, the present disclosure provides a method of treating a subject with a disease characterized by prolonged elevated blood glucose levels (e.g., diabetes), comprising administering to the subject any of the compositions disclosed herein or any of the devices disclosed herein.

[0241]

[0329] In some embodiments, the present disclosure provides a method comprising contacting a plurality of dissociated insulin-positive endocrine precursor cells with a BMP signaling pathway inhibitor. In some embodiments, the BMP signaling pathway inhibitor is LDN193189 or a derivative thereof. In some embodiments, the present disclosure provides a method comprising contacting a plurality of dissociated insulin-positive endocrine precursor cells with a ROCK inhibitor. In some embodiments, the ROCK inhibitor is thiazovivin, Y-27632, fasudil / HA1077, or 14-1152, or a derivative thereof. In some embodiments, the present disclosure provides a method comprising contacting a plurality of dissociated insulin-positive endocrine precursor cells with a histone methyltransferase inhibitor. In some embodiments, the histone methyltransferase inhibitor is 3-deazaneplanocin A hydrochloride or a derivative thereof. In some embodiments, the present disclosure provides a method comprising contacting a plurality of dissociated insulin-positive endocrine precursor cells with zinc. In some embodiments, the zinc is in the form of ZnSO. In some embodiments, the present disclosure provides a method comprising contacting a plurality of dissociated insulin-positive endocrine precursor cells with a monoglyceride lipase (MGLL) inhibitor. In some embodiments, the MGLL inhibitor is JJKK048, KML29, NF1819, JW642, JZL184, JZL195, JZP361, pristimerin, or URB602, or a derivative of any of the foregoing. In some embodiments, the present disclosure provides a method comprising contacting a plurality of dissociated insulin-positive endocrine precursor cells with a lipid. In some embodiments, the lipid is a saturated fatty acid. In some embodiments, the saturated fatty acid is palmitate. In some embodiments, the lipid is an unsaturated fatty acid. In some embodiments, the unsaturated fatty acid is oleic acid, linoleic acid, or palmitoleic acid. In some embodiments, the present disclosure provides a method comprising contacting a plurality of dissociated insulin-positive endocrine precursor cells with glutamate. In some embodiments, the present disclosure provides a method comprising contacting a plurality of dissociated insulin-positive endocrine precursor cells with acetate.In some embodiments, the present disclosure provides a method comprising contacting a plurality of dissociated insulin-positive endocrine precursor cells with β-hydroxybutarate. In some embodiments, the present disclosure provides a method comprising contacting a plurality of dissociated insulin-positive endocrine precursor cells with L-carnitine. In some embodiments, the present disclosure provides a method comprising contacting a plurality of dissociated insulin-positive endocrine precursor cells with taurine. In some embodiments, the present disclosure provides a method comprising contacting a plurality of dissociated insulin-positive endocrine precursor cells with formate. In some embodiments, the present disclosure provides a method comprising contacting a plurality of dissociated insulin-positive endocrine precursor cells with biotin. In some embodiments, the method of the present disclosure comprises contacting a plurality of dissociated insulin-positive endocrine precursor cells with serum albumin protein. In some embodiments, the serum albumin protein is human serum albumin protein. In some embodiments, the compositions of the present disclosure may be administered in the form of 0.01% to 1%, 0.03 to 1%, 0.03 to 0.9%, 0.03 to 0.08%, 0.03 to 0.06%, 0.03 to 0.05%, 0.04 to 0.8%, 0.04 to 0.7%, 0.04 to 0.6%, 0.04 to 0.5%, 0.04 to 0.4%, 0.04 to 0.3%, 0.04 to 0.5%. Contains 0.04-0.2%, 0.04-0.1%, 0.04-0.09%, 0.04-0.8%, 0.04-0.07%, 0.04-0.06%, 0.04-0.05%, 0.05-1%, 0.05-0.9%, 0.05-0.8%, 0.05-0.7%, 0.05-0.6%, 0.05-0.5%, 0.05-0.4%, 0.05-0.3%, 0.05-0.2%, 0.05-0.1%, 0.05-0.09%, 0.05-0.8%, 0.05-0.07%, or 0.05-0.06% serum albumin protein. In some embodiments, less than 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 1% of the cells in the composition are organized into cell clusters. In some embodiments, the disclosed method includes contacting a plurality of dissociated insulin-positive endocrine precursor cells with a TGF-β pathway inhibitor. In some embodiments, the TGF-β pathway inhibitor is Alk5i (SB505124) or a derivative thereof. In some embodiments, the disclosed method includes contacting a plurality of dissociated insulin-positive endocrine precursor cells with a thyroid hormone signaling pathway activator. In some embodiments, the thyroid hormone signaling pathway activator is GC-1 or T3, or a derivative thereof. In some embodiments, the disclosed method includes contacting the plurality of dissociated insulin-positive endocrine precursor cells with a protein kinase inhibitor. In some embodiments, the protein kinase inhibitor is staurosporine. In some embodiments, the disclosed method includes contacting the plurality of dissociated insulin-positive endocrine precursor cells with vitamin C. In some embodiments, the disclosed method includes contacting the plurality of dissociated insulin-positive endocrine precursor cells with insulin. In some embodiments, the disclosed method does not include contacting the plurality of dissociated insulin-positive endocrine precursor cells with a gamma-secretase inhibitor (e.g., XXI). In some embodiments, the dissociated insulin-positive endocrine precursor cells have been previously frozen.In some embodiments, the methods of the disclosure are carried out over a period of 1 to 10 days, 1 to 9 days, 1 to 8 days, 1 to 7 days, 1 to 6 days, 1 to 5 days, 1 to 4 days, 1 to 3 days, 1 to 2 days, 2 to 10 days, 2 to 9 days, 2 to 8 days, 2 to 7 days, 2 to 6 days, 2 to 5 days, 2 to 4 days, 2 to 3 days, 3 to 10 days, 3 to 9 days, 3 to 8 days, 3 to 7 days, 3 to 6 days, 3 to 5 days, 3 to 4 days, 4 to 10 days, 4 to 9 days, 4 to 8 days, 4 to 7 days, 4 to 6 days, or 4 to 5 days. In some embodiments, the methods of the disclosure reaggregate the dissociated cells into multiple cell clusters. In some embodiments, at least about 40%, 50%, 60%, 70%, 80%, or 90% of the plurality of cell clusters have a diameter of about 50 μm to about 250 μm, about 75 μm to about 250 μm, or about 100 μm to about 200 μm. In some embodiments, at least about 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, or 99% of the cells of the plurality of cell clusters of the second cell population are viable. In some embodiments, the methods of the present disclosure reaggregate the dissociated cells into at least 2, 3, 4, 5, 10, 50, 100, 1,000, 10,000, 100,000, or 1,000,000 cell clusters.

[0242]

[0330] In some cases, the method provided herein does not include an active cell sorting process, such as flow cytometry. In some cases, the cell clusters described herein can be unsorted cell clusters. In some cases, the method provided herein does not rely on active cell sorting to enrich or eliminate specific cell types in a first cell cluster. In some cases, a method only requires the steps of dissociating a first cell cluster and culturing a plurality of cells dissociated from the first cell cluster in a medium, thereby allowing the formation of a second cell cluster.

[0243]

[0331] Optionally, the methods provided herein can be applied to dissociate and re-aggregate cell clusters more than once. For example, according to the methods provided herein, The cell cluster can be dissociated and re-aggregated to form a second cell cluster, the second cell cluster can be further dissociated and re-aggregated to form a third cell cluster, etc. Re-aggregation as provided herein can be performed on the cell cluster at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive times.

[0244]

[0332] As described herein, cell sorting can refer to the process of isolating a group of cells from a plurality of cells by relying on differences in cell size, shape (morphology), surface protein expression, endogenous signal protein expression, or any combination thereof. In some cases, cell sorting involves subjecting the cells to flow cytometry. Flow cytometry can be a biophysical technique based on lasers or impedance. In flow cytometry, cells can be suspended in a fluid stream and passed through an electronic detection device. Fluorescence-activated cell sorting (FACS), a type of flow cytometry, can be used to physically separate and purify cells of interest based on one or more parameters of the cells' optical properties (e.g., emission wavelength upon laser excitation). As described herein, unsorted cell clusters can be cell clusters formed by a plurality of cells that have not been subjected to an active cell sorting process, e.g., flow cytometry. Unsorted cell clusters, sometimes referred to as "reaggregated cell clusters," may be formed by multiple cells dissociated from a pre-existing cell cluster, and there may be no active cell sorting process, e.g., flow cytometry or other methods, to isolate one or more specific cell types for reaggregation as provided herein before they reaggregate into a new cell cluster. Optionally, the flow cytometry described herein may be based on one or more signal peptides expressed in the cells. For example, the cell clusters may include cells expressing a signal peptide (e.g., a fluorescent protein, e.g., green fluorescent protein (GFP) or tdTomato). Optionally, the signal peptide is expressed to indicate insulin expression in the cells. For example, the cell clusters may include cells having an exogenous nucleic acid sequence encoding GFP under the control of an insulin promoter. The insulin promoter may be an endogenous or exogenous promoter. Optionally, the expression of GFP in these cells may indicate insulin expression in the cells. Thus, the GFP signal may be a marker for pancreatic beta cells.In some cases, the cell sorting described herein may involve magnetically activated flow cytometry, in which magnetic antibodies or other ligands are used to label different types of cells, and differences in magnetic properties can be used to sort the cells.

[0245]

[0333] The cells dissociated from the first cell cluster can be cultured in a medium for reaggregation into a second cell cluster. The medium can include Connought Medical Research Laboratories 1066 supplemented pancreatic islet medium (CMRLS). Optionally, a suitable culture medium includes a component of CMRLS (e.g., supplemental zinc). CMRLS can be supplemented with, for example, serum (e.g., human serum, human platelet lysate, fetal bovine serum, or serum substitute such as Knockout Serum Replacement).

[0246]

[0334] The medium may contain one or more compounds that regulate a particular signal transduction pathway in cells. For example, the medium may contain a thyroid hormone signal transduction pathway activator, a transforming growth factor β (TGF-β) signal transduction pathway inhibitor, or both.

[0247]

[0335] The thyroid hormone signaling pathway activator in the medium used herein may be triiodothyronine (T3). Optionally, the thyroid hormone signaling pathway activator may be an analog or derivative of T3. Non-limiting exemplary analogs of T3 include: These include selective and non-selective thyromimetics, such as the TRβ-selective agonists GC-1, GC-24, 4-hydroxy-PCB 106, MB07811, MB07344, 3,5-diiodothyropropionic acid (DITPA); the selective TRβ agonist GC-1; 3-iodothyronamine (T(1)AM) and 3,3',5-triiodothyroacetic acid (Triac) (bioactive metabolites of the hormone thyroxine (T(4))); KB-2115 and KB-141; thyronamine; and SKF L-94901; DIBIT; 3'-AC-T2; tetraiodothyroacetic acid (Tetrac) and triiodothyroacetic acid (Triac) (by oxidative deamination and decarboxylation of thyroxine (T4) and triiodothyronine (T3) alanine chains), 3,3',5'-triiodothyronine (rT3) (by deiodination of T4 and T3), 3,3'-diiodothyronine (3,3'-T2) and 3,5-diiodothyronine (T2) (by deiodination of T4, T3, and rT3) thyronamine (TAM) (by deiodination of T4 and T3 and decarboxylation of amino acids), and 3-iodothyronamine (TAM) and thyronamine (T0AM) (by deiodination of T4 and T3 and decarboxylation of amino acids), as well as TH structural analogs such as 3,5,3'-triiodothyropropionic acid (Triprop), 3,5-dibromo-3-pyridazinone-l-thyronine (L-940901), N-[3,5-dimethyl-4-(4'-hydroxy-3'-isopropylphenoxy)-phenyl]-oxamic acid (CGS 23425), 3,5-dimethyl-4-[(4'-hydroxy-3'-isopropylbenzyl)-phenoxy]acetic acid (GC-1), 3,5-dichloro-4-[(4-hydroxy-3-isopropylphenoxy)phenyl]acetic acid (KB-141), and 3,5-diiodothyropropionic acid (DITPA). In some cases, the thyroid hormone signaling pathway activator is a prodrug or prohormone of T3, such as a T4 thyroid hormone (e.g., thyroxine or L-3,5,3',5'-tetraiodothyronine). The thyroid hormone signaling pathway activator may be an iodothyronine composition described in U.S. Pat. No. 7,163,918, the entire contents of which are incorporated herein by reference.

[0248]

[0336] The concentration of the thyroid hormone signaling pathway activator in the medium can be within a range suitable for cell aggregation. In some cases, the concentration of the thyroid hormone signaling pathway activator in the medium is about 0.1 μM to about 10 μM, for example, about 0.5 μM to about 2 μM, about 0.8 μM to about 1.5 μM, about 0.9 μM to about 1.5 μM, about 0.9 μM to about 1.2 μM, or about 0.9 μM to about 1.2 μM. In some cases, the concentration of the thyroid hormone signaling pathway activator in the medium is at least about 0.1 μM, 0.2 μM, 0.4 μM, 0.8 μM, 0.9 μM, 1 μM, 1.1 μM, 1.2 μM, 1.3 μM, 1.4 μM, 1.5 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, or 10 μM. Optionally, the concentration of the thyroid hormone signaling pathway activator (e.g., T3) in the medium is about 1 μM.

[0249]

[0337] The TGF-β signaling pathway inhibitor used in the medium herein can be a TGF-β receptor type I kinase (TGF-β RI) signaling inhibitor.The TGF-β signaling pathway inhibitor can be an activin receptor-like kinase-5 (Alk5) inhibitor, for example, ALK5 inhibitor II (CAS 446859-33-2, an ATP-competitive inhibitor of TGF-β RI kinase, also known as RepSox, IUPAC name: 2-[5-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl]-1,5-naphthyridine).Optionally, the TGF-β signaling pathway inhibitor is an analog or derivative of ALK5 inhibitor II, including those described in U.S. Patent Publication Nos. 2012 / 0021519, 2010 / 0267731, 2009 / 0186076 and 2007 / 0142376, all of which are incorporated herein by reference. Examples of TGF-β signaling pathway inhibitors that may optionally be used in the medium herein include D 4476, SB431542, A-83-01, also known as 3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbohydrate. amide; 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine, Wnt3a / BIO, BMP4, GW788388 (-(4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]pyridin(pyridm)-2-yl}-N-(tetrahydro-2H-pyran-4-yl)benzamide), SMI 6, ΓΝ-1130 (3-((5-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imidazol-2-yl)methyl)benzamide, GW6604 (2-phenyl-4-(3-pyridin-2-yl-1H-pyrazol-4-yl)pyridine), SB-505124 (2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine hydrochloride), SU5416, lerdelimumab (CAT-152), methimumab (CAT-192), GC-1008, ID1 1, AP-12009, AP-1 1014, LY550410, LY580276, LY364947, LY2109761, SD-208, SM16, NPC-30345, KI26894, SB-203580, SD-093, ALX-270-448, EW-7195, SB-525334, ΓΝ-1233, SKI2162, Gleevec, 3,5,7,2',4'-pentahydroxyflavone (Morin), Activin Also included are M108A, P144, soluble TBR2-Fc, pyrimidine derivatives and indolinones.Inhibition of TGF-β / activin pathway can have similar effects.Therefore, any inhibitor of TGF-β / activin pathway (for example, upstream or downstream) can be used in combination with or instead of the TGF-β / ALK5 inhibitor described herein.Exemplary TGF-β / activin pathway inhibitors include, but are not limited to, TGF-β receptor inhibitors, SMAD2 / 3 phosphorylation inhibitors, SMAD2 / 3 and SMAD4 interaction inhibitors, and SMAD6 and SMAD7 activators / agonists.Furthermore, the categorizations described herein are for organizational purposes only, and those skilled in the art will recognize that a compound may function in more than one of the defined categories because it may act at one or more points in the pathway. TGF-β receptor inhibitors may include any general TGF signaling inhibitors or inhibitors specific to TGF-β receptors (e.g., ALK5) inhibitors, which may include antibodies against TGF-β receptors, dominant-negative variants of TGF-β receptors, and siRNAs and antisense nucleic acids that suppress the expression of TGF-β receptors.

[0250]

[0338] The concentration of the TGF-β signaling pathway inhibitor in the medium can be within a range suitable for cell aggregation. In some cases, the concentration of the TGF-β signaling pathway inhibitor in the medium is about 1 μM to about 50 μM, for example, about 5 μM to about 15 μM, about 8 μM to about 12 μM, or about 9 μM to about 11 μM. In some cases, the concentration of the TGF-β signaling pathway inhibitor in the medium is at least about 1 μM, 5 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, or 50 μM. In some cases, the concentration of the TGF-β signaling pathway inhibitor (e.g., Alk5 inhibitor II) in the medium is about 10 μM.

[0251]

[0339] The medium used to culture the cells dissociated from the first cell cluster may be xeno-free. Xeno-free medium for culturing cells and / or cell clusters of animal origin may not contain any products derived from other animals. Optionally, xeno-free medium for culturing human cells and / or cell clusters may not contain any products derived from non-human animals. For example, xeno-free medium for culturing human cells and / or cell clusters may contain human platelet lysate (PLT) instead of fetal bovine serum (FBS). For example, the medium may contain about 1% to about 20%, about 5% to about 15%, about 8% to about 12%, or about 9 to about 11% serum. Optionally, the medium may contain about 10% serum. Optionally, the medium may be free of small molecules and / or FBS. For example, the medium may contain about 10% serum. The medium may comprise MCDB131 basal medium supplemented with 2% BSA. Optionally, the medium is serum-free. In some examples, the medium may contain exogenous small molecules or signaling pathway agonists or antagonists, such as growth factors from the fibroblast growth factor family (FGFs, e.g., FGF2, FGF8B, FGF10, or FGF21), sonic hedgehog antagonists (e.g., Sant1, Sant2, Sant3), or erythrocyte proliferation inhibitors (e.g., erythrocyte stimulatory ... 4, Sant4, Cur61414, forskolin, tomatidine, AY9944, triparanol, cyclopamine, or their derivatives), retinoic acid signaling agonists (e.g., retinoic acid, CD1530, AM580, TTHPB, CD437, Ch55, BMS961, AC261066, AC55649, AM80, BMS753, tazarotene, adapalene, or CD2314), inhibitors of Rho-associated coiled-coil-containing protein kinase (ROCK) (e.g., thiazovivin, Y-27632, fasudil / HA1077, or 14-1152), activators of protein kinase C (PKC) (e.g., phorbol 12,13-dibutyrate (PDBU), TPB, phorbol 12-myristate 13-acetate, bryostatin 1, or derivatives thereof), antagonists of the TGF beta superfamily (e.g., Alk5 inhibitor II (CAS 446859-33-2), A83-01, SB431542, D4476, GW788388, LY364947, LY580276, SB505124, GW6604, SB-525334, SD-208, SB-505124, or derivatives thereof), inhibitors of bone morphogenetic protein (BMP) type 1 receptor (e.g., LDN193189 or derivatives thereof), thyroid hormone signaling pathway activators (e.g., T3 or derivatives thereof), gamma-secretase inhibitors (e.g., XXI, DAPT, or derivatives thereof), activators of the TGF-β signaling pathway (e.g., WNT3a or activin A) growth factors from the epidermal growth factor (EGF) family (e.g., betacellulin or EGF), broad-spectrum kinases (e.g., staurosporine or derivatives thereof), non-essential amino acids, vitamins or antioxidants (e.g., cyclopamine, vitamin D, vitamin C, vitamin A, or derivatives thereof), or other additives such as N-acetylcysteine, zinc sulfate, or heparin. Optionally, the reaggregation medium may be free of exogenous extracellular matrix molecules. Optionally, the reaggregation medium does not include Matrigel™. Optionally, the reaggregation medium does not include other extracellular matrix molecules or substances, such as collagen, gelatin, poly-L-lysine, poly-D-lysine, vitronectin, laminin, fibronectin, PLO laminin, fibrin, thrombin, and RetroNectin, and mixtures thereof, or lysed cell membrane preparations.

[0252]

[0340] Those skilled in the art will understand that the concentration of BSA supplemented to the medium can vary. For example, a medium (e.g., MCDB131) can contain about 0.01%, 0.05%, 0.1%, 1%, about 2%, about 3%, about 4%, about 5%, about 10%, or about 15% BSA. The medium (e.g., MCDB131 medium) used can contain components not found in conventional basal media, such as trace elements, putrescine, adenine, thymidine, and higher levels of several amino acids and vitamins. These additions can allow the medium to be supplemented with very low levels of serum or defined components. The medium can be protein- and / or growth factor-free and can be supplemented with EGF, hydrocortisone, and / or glutamine.

[0253]

[0341] The medium may include one or more extracellular matrix molecules (e.g., extracellular proteins). Non-limiting exemplary extracellular matrix molecules used in the medium may include collagen, placental matrix, fibronectin, laminin, merosin, tenascin, heparin, heparin sulfate, chondroitin sulfate, dermatan sulfate, aggrecan, biglycan, thrombospondin, vitronectin, and decorin. Optionally, the medium includes a laminin such as LN-332. Optionally, the medium includes heparin.

[0254]

[0342] For example, the medium can be periodically changed during culture to provide the optimal environment for the cells in the medium.When culturing the cells dissociated from the first cell cluster for reaggregation, the medium can be changed at least or about every 4 hours, 12 hours, 24 hours, 48 ​​hours, 3 days or 4 days.For example, the medium can be changed about every 48 hours.

[0255]

[0343] Cells dissociated from a first cell cluster can be seeded into a container for reaggregation. The seeding density can be correlated with the size of the second cell cluster to be reaggregated. The seeding density can be controlled so that the size of the second cell cluster can be similar to that of endogenous pancreatic islets. In some cases, the seeding density is controlled so that the size of the second cell cluster can be about 75 μm to about 250 μm. Cells dissociated from a first cell cluster can be seeded at a density of about 100,000 cells per mL to about 10 million cells per mL, for example, about 500,000 cells per mL to about 1.5 million cells per mL, about 800,000 cells per mL to about 1.2 million cell...

Claims

1. A composition comprising a plurality of dissociated insulin-positive endocrine precursor cells and a BMP signaling pathway inhibitor.

2. A composition comprising a plurality of dissociated insulin-positive endocrine precursor cells and a ROCK inhibitor.

3. A composition comprising a plurality of dissociated insulin-positive endocrine precursor cells and a histone methyltransferase inhibitor.

4. A composition comprising a plurality of dissociated insulin-positive endocrine precursor cells and zinc.

5. A composition comprising a plurality of dissociated insulin-positive endocrine precursor cells and a monoglyceride lipase (MGLL) inhibitor.

6. A composition comprising a plurality of dissociated insulin-positive endocrine precursor cells and a lipid.

7. A composition comprising a plurality of dissociated insulin-positive endocrine precursor cells and one or more of glutamate, acetate, β-hydroxybutarate, L-carnitine, taurine, formate, or biotin.

8. The composition of any one of claims 2 to 7, comprising a BMP signaling pathway inhibitor.

9. The composition of claim 1 or 8, wherein the BMP signaling pathway inhibitor is LDN193189 or a derivative thereof.

10. 10. The composition of any one of claims 1 or 3 to 9, comprising a ROCK inhibitor.

11. The composition of claim 2 or 10, wherein the ROCK inhibitor is thiazovivin, Y-27632, fasudil / HA1077, or 14-1152, or a derivative thereof.

12. 12. The composition of any one of claims 1, 2, or 4 to 11, comprising a histone methyltransferase inhibitor.

13. The composition of claim 3 or 12, wherein the histone methyltransferase inhibitor is 3-deazaneplanocin A hydrochloride or a derivative thereof.

14. 14. The composition of any one of claims 1 to 3 or 5 to 13, comprising zinc.

15. The zinc is ZnSO ４ 15. The composition of claim 4 or 14 in the form of:

16. 16. The composition of any one of claims 1 to 4 or 6 to 15, comprising a monoglyceride lipase (MGLL) inhibitor.

17. 17. The composition of claim 5 or 16, wherein the MGLL inhibitor is JJKK048, KML29, NF1819, JW642, JZL184, JZL195, JZP361, pristimerin, or URB602, or a derivative of any of the foregoing.

18. 18. The composition of any one of claims 1 to 5 or 7 to 17, comprising a lipid.

19. 20. The composition of claim 6 or 18, wherein the lipid is a saturated fatty acid.

20. 20. The composition of claim 19, wherein the saturated fatty acid is palmitate.

21. 20. The composition of claim 6 or 18, wherein the lipid is an unsaturated fatty acid.

22. 22. The composition of claim 21, wherein the unsaturated fatty acid is oleic acid, linoleic acid, or palmitoleic acid.

23. 23. The composition of any one of claims 1 to 22, further comprising a serum albumin protein.

24. 24. The composition of claim 23, wherein the serum albumin protein is human serum albumin protein.

25. 0.01% to 1%, 0.03 to 1%, 0.03 to 0.9%, 0.03 to 0.08%, 0.03 to 0.06%, 0.03 to 0.05%, 0.04 to 0.8%, 0.04 to 0.7%, 0.04 to 0.6%, 0.04 to 0.5%, 0.04 to 0.4%, 0.04 to 0.3%, 0.04 to 0.2%, 0.04 to 0.1%, 0.04 to 0.09%, 0.04 to 0.8%, 0.04 to 0.07%, 0.04 to 0.06%, 0.0 25. The composition of claim 23 or 24, comprising 4-0.05%, 0.05-1%, 0.05-0.9%, 0.05-0.8%, 0.05-0.7%, 0.05-0.6%, 0.05-0.5%, 0.05-0.4%, 0.05-0.3%, 0.05-0.2%, 0.05-0.1%, 0.05-0.09%, 0.05-0.8%, 0.05-0.07%, or 0.05-0.06% serum albumin protein.

26. 26. The composition of any one of claims 1 to 25, wherein less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, or less than 1% of the cells in the composition are in cell clusters.

27. 27. The composition of any one of claims 1 to 26, comprising a TGF-β pathway inhibitor.

28. 28. The composition of claim 27, wherein the TGF-β pathway inhibitor is Alk5i (SB505124) or a derivative thereof.

29. 29. The composition of any one of claims 1 to 28, comprising a thyroid hormone signaling pathway activator.

30. 30. The composition of claim 29, wherein the thyroid hormone signaling pathway activator is GC-1 or T3, or a derivative thereof.

31. 31. The composition of any one of claims 1 to 30, comprising a protein kinase inhibitor.

32. 32. The composition of claim 31, wherein the protein kinase inhibitor is staurosporine.

33. 33. The composition of any one of claims 1 to 32, comprising glutamate.

34. 34. The composition of any one of claims 1 to 33, comprising acetate.

35. 35. The composition of any one of claims 1 to 34, comprising β-hydroxybutarate.

36. 36. The composition of any one of claims 1 to 35, comprising L-carnitine.

37. 37. The composition of any one of claims 1 to 36, comprising taurine.

38. 38. The composition of any one of claims 1 to 37, comprising a formate.

39. 39. The composition of any one of claims 1 to 38, comprising biotin.

40. 40. The composition of any one of claims 1 to 39, comprising vitamin C.

41. 41. The composition of any one of claims 1 to 40, comprising insulin.

42. 42. The composition of any one of claims 1 to 41, wherein the dissociated insulin-positive endocrine precursor cells have been previously frozen.

43. A method comprising contacting a plurality of dissociated insulin-positive endocrine precursor cells with a BMP signaling pathway inhibitor.

44. A method comprising contacting a plurality of dissociated insulin-positive endocrine precursor cells with a ROCK inhibitor.

45. A method comprising contacting a plurality of dissociated insulin-positive endocrine precursor cells with a histone methyltransferase inhibitor.

46. A method comprising contacting a plurality of dissociated insulin-positive endocrine precursor cells with zinc.

47. A method comprising contacting a plurality of dissociated insulin-positive endocrine precursor cells with a monoglyceride lipase (MGLL) inhibitor.

48. A method comprising contacting a plurality of dissociated insulin-positive endocrine precursor cells with a lipid.

49. A method comprising contacting a plurality of dissociated insulin-positive endocrine precursor cells with one or more of glutamate, acetate, β-hydroxybutarate, L-carnitine, taurine, formate, or biotin.

50. 50. The method of any one of claims 44 to 49, comprising contacting the plurality of dissociated insulin-positive endocrine precursor cells with a BMP signaling pathway inhibitor.

51. 51. The method of claim 43 or 50, wherein the BMP signaling pathway inhibitor is LDN193189 or a derivative thereof.

52. The plurality of dissociated insulin-positive endocrine precursor cells are contacted with a ROCK inhibitor.

52. The method of any one of claims 43 or 45 to 51, comprising the steps of:

53. The composition of claim 44 or 52, wherein the ROCK inhibitor is thiazovivin, Y-27632, fasudil / HA1077, or 14-1152, or a derivative thereof.

54. 54. The method of any one of claims 43, 44, or 46-53, comprising contacting the plurality of dissociated insulin-positive endocrine precursor cells with a histone methyltransferase inhibitor.

55. 55. The method of claim 45 or 54, wherein the histone methyltransferase inhibitor is 3-deazaneplanocin A hydrochloride or a derivative thereof.

56. 56. The method of any one of claims 43-45 or 47-55, comprising contacting the plurality of dissociated insulin-positive endocrine precursor cells with zinc.

57. The zinc is ZnSO ４ 57. The method of claim 46 or 56, in the form of

58. 58. The method of any one of claims 43-46 or 48-57, comprising contacting the plurality of dissociated insulin-positive endocrine precursor cells with a monoglyceride lipase (MGLL) inhibitor.

59. 59. The method of claim 47 or 58, wherein the MGLL inhibitor is JJKK048, KML29, NF1819, JW642, JZL184, JZL195, JZP361, pristimerin, or URB602, or a derivative of any of the foregoing.

60. 60. The method of any one of claims 43-47 or 49-59, comprising contacting the plurality of dissociated insulin-positive endocrine precursor cells with a lipid.

61. 61. The method of claim 48 or 60, wherein the lipid is a saturated fatty acid.

62. 62. The method of claim 61, wherein the saturated fatty acid is palmitate.

63. 61. The method of claim 48 or 60, wherein the lipid is an unsaturated fatty acid.

64. 64. The method of claim 63, wherein the unsaturated fatty acid is oleic acid, linoleic acid, or palmitoleic acid.

65. 65. The method of any one of claims 43 to 64, comprising contacting the plurality of dissociated insulin-positive endocrine precursor cells with serum albumin protein.

66. 66. The method of claim 65, wherein the serum albumin protein is human serum albumin protein.

67. The composition may contain 0.01% to 1%, 0.03 to 1%, 0.03 to 0.9%, 0.03 to 0.08%, 0.03 to 0.06%, 0.03 to 0.05%, 0.04 to 0.8%, 0.04 to 0.7%, 0.04 to 0.6%, 0.04 to 0.5%, 0.04 to 0.4%, 0.04 to 0.3%, 0.04 to 0.2%, 0.04 to 0.1%, 0.04 to 0.09%, 0.04 to 0.8%, 0.04 to 0.07%, 0.04 to 0.06%, 0.04 to 0.05%, 0.05 to 1%, 0.05 to 0.9%, 0.05 to 0.8%, 0.05 to 0.7%, 67. The method of claim 65 or 66, comprising 0.05-0.6%, 0.05-0.5%, 0.05-0.4%, 0.05-0.3%, 0.05-0.2%, 0.05-0.1%, 0.05-0.09%, 0.05-0.8%, 0.05-0.07%, or 0.05-0.06% serum albumin protein.

68. 68. The method of any one of claims 43 to 67, wherein less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, or less than 1% of the cells in the composition are in cell clusters.

69. 69. The method of any one of claims 43 to 68, comprising contacting the plurality of dissociated insulin-positive endocrine precursor cells with a TGF-β pathway inhibitor.

70. 70. The method of claim 69, wherein the TGF-β pathway inhibitor is Alk5i (SB505124) or a derivative thereof.

71. 71. The method of any one of claims 43 to 70, comprising contacting the plurality of dissociated insulin-positive endocrine precursor cells with a thyroid hormone signaling pathway activator.

72. 72. The method of claim 71, wherein the thyroid hormone signaling pathway activator is GC-1 or T3, or a derivative thereof.

73. 73. The method of any one of claims 43 to 72, comprising contacting the plurality of dissociated insulin-positive endocrine precursor cells with a protein kinase inhibitor.

74. 74. The method of claim 73, wherein the protein kinase inhibitor is staurosporine.

75. 75. The method of any one of claims 43 to 74, comprising contacting the plurality of dissociated insulin-positive endocrine precursor cells with glutamate.

76. 76. The method of any one of claims 43 to 75, comprising contacting the plurality of dissociated insulin-positive endocrine precursor cells with acetate.

77. 77. The method of any one of claims 43 to 76, comprising contacting the plurality of dissociated insulin-positive endocrine precursor cells with β-hydroxybutarate.

78. 78. The method of any one of claims 43 to 77, comprising contacting the plurality of dissociated insulin-positive endocrine precursor cells with L-carnitine.

79. 79. The method of any one of claims 43 to 78, comprising contacting the plurality of dissociated insulin-positive endocrine precursor cells with taurine.

80. 80. The method of any one of claims 43 to 79, comprising contacting the plurality of dissociated insulin-positive endocrine precursor cells with formate.

81. 81. The method of any one of claims 43 to 80, comprising contacting the plurality of dissociated insulin-positive endocrine precursor cells with biotin.

82. 82. The method of any one of claims 43 to 81, comprising contacting the plurality of dissociated insulin-positive endocrine precursor cells with vitamin C.

83. 83. The method of any one of claims 43 to 82, comprising contacting the plurality of dissociated insulin-positive endocrine precursor cells with insulin.

84. 84. The method of any one of claims 43 to 83, wherein the dissociated insulin-positive endocrine precursor cells have been previously frozen.

85. 85. The method of any one of claims 43 to 84, wherein the method is carried out over a period of 1 to 10 days, 1 to 9 days, 1 to 8 days, 1 to 7 days, 1 to 6 days, 1 to 5 days, 1 to 4 days, 1 to 3 days, 1 to 2 days, 2 to 10 days, 2 to 9 days, 2 to 8 days, 2 to 7 days, 2 to 6 days, 2 to 5 days, 2 to 4 days, 2 to 3 days, 3 to 10 days, 3 to 9 days, 3 to 8 days, 3 to 7 days, 3 to 6 days, 3 to 5 days, 3 to 4 days, 4 to 10 days, 4 to 9 days, 4 to 8 days, 4 to 7 days, 4 to 6 days, or 4 to 5 days.

86. 86. The method of any one of claims 43 to 85, wherein the dissociated cells are reaggregated into multiple cell clusters.

87. 87. The method of claim 86, wherein at least about 40%, 50%, 60%, 70%, 80%, or 90% of the plurality of cell clusters have a diameter of about 50 μm to about 250 μm, about 75 μm to about 250 μm, or about 100 μm to about 200 μm.

88. 88. The method of claim 86 or 87, wherein at least about 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, or 99% of the cells of the plurality of cell clusters of the second cell population are viable.

89. 89. The method of any one of claims 86 to 88, wherein the dissociated cells are reaggregated into clusters of at least 2, 3, 4, 5, 10, 50, 100, 1000, 10,000, 100,000, or 1,000,000 cells.

90. 1. A composition comprising a plurality of cell clusters, said cell clusters comprising insulin positive cells, said composition comprising: a) at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, or at least 65% of the cells in the composition are viable after 11 days of in vitro culture; b) at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% of the cell clusters in the composition are 90-140 μm, 90-130 μm, 90-120 μm, 90-110 μm, 100-140 μm, 100-130 μm, 100-120 μm, 100-110 μm in diameter; and / or c) at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% of the cell clusters in the composition are 1.5-4.5, 1.5-4.0, 1.5-3.5, 1.5-3.0, 1.5-2.5, 1.5-2.5, 1.5-2.0, 2.0-4.5, 2.0-4.0, 2.0-3.5, 2.0-3.0, 2.0-2.5, 2.5-4.5, 2.5-4.0, 2.5-3.5, 2.5-3.0, 3.0-4 exhibiting a glucose-stimulated insulin secretion (GSIS) stimulation index of 3.5, 3.0-4.0, 3.0-3.5, 3.5-4.5, 3.5-4.0, or 4.0-4.5; A composition comprising at least one of:

91. 91. The composition of claim 90, wherein the cell clusters comprise C-peptide positive cells.

92. 92. The composition of claim 90 or 91, wherein the cell clusters comprise somatostatin-positive cells.

93. 92. The composition of claim 90 or 91, wherein the cell clusters comprise glucagon-positive cells.

94. 94. The composition of any one of claims 90 to 93, wherein at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, or at least 65% of the cells in the composition are viable after 11 days of in vitro culture.

95. 95. The composition of any one of claims 90 to 94, wherein at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the cell clusters in the composition are 90-140 μm, 90-130 μm, 90-120 μm, 90-110 μm, 100-140 μm, 100-130 μm, 100-120 μm, or 100-110 μm in diameter.

96. At least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% of the cell clusters in the composition are 1.5 to 4.5, 1.5 to 4.0, 1.5 to 3.5, 1.5 to 3.0, 1.5 to 2.5, 1.5 to 2.

96. The composition of any one of claims 90-95, wherein the composition exhibits a glucose-stimulated insulin secretion (GSIS) stimulation index of 0.5, 1.5-2.0, 2.0-4.5, 2.0-4.0, 2.0-3.5, 2.0-3.0, 2.0-2.5, 2.5-4.5, 2.5-4.0, 2.5-3.5, 2.5-3.0, 3.0-4.5, 3.0-4.0, 3.0-3.5, 3.5-4.5, 3.5-4.0, or 4.0-4.

5.

97. 97. The composition of any one of claims 90 to 96, comprising at least 2, 3, 4, 5, 10, 50, 100, 1000, 10,000, 100,000, or 1,000,000 cell clusters.

98. 98. The composition of any one of claims 90 to 97, prepared according to the method of any one of claims 43 to 89.

99. 99. A device comprising the composition of any one of claims 90 to 98.

100. A method of treating a subject having a disease characterized by high blood glucose levels over a prolonged period of time (e.g., diabetes), comprising administering to the subject a composition described in any one of claims 90 to 98 or a device described in claim 99.

101. (a) obtaining a first cell population comprising a plurality of cell clusters containing insulin-positive cells; and (b) dissociating in vitro at least a portion of the plurality of cell clusters in the first cell population; (c) contacting the first cell population comprising at least a portion of the dissociated cell clusters with a first composition in vitro to obtain a second cell population comprising a plurality of cell clusters, the second cell population comprising a plurality of insulin-positive cells, wherein the first composition comprises at least one agent selected from the group consisting of a monoglyceride lipase (MGLL) inhibitor, a bone morphogenetic protein (BMP) type 1 receptor inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a histone methyltransferase inhibitor, and a protein kinase inhibitor; (d) contacting the second insulin-positive cell population in vitro with a second composition, thereby differentiating at least a portion of the second insulin-positive cell population into a third cell population comprising a plurality of beta cells, wherein the second composition is different from the first composition, and the third cell population comprises a higher percentage of viable beta cells compared to a corresponding beta cell population comprising beta cells derived from the first cell population not contacted with the first composition; A method comprising:

102. (a) obtaining a first cell population comprising a plurality of cell clusters comprising insulin-positive cells; (b) dissociating in vitro at least a portion of the plurality of cell clusters in the first cell population; (c) contacting the first cell population comprising at least a portion of the dissociated cell clusters with a first composition in vitro to obtain a second cell population comprising a plurality of cell clusters, the second cell population comprising a plurality of insulin-positive endocrine cells, wherein the first composition comprises a transforming growth factor beta (TGF-β) signaling pathway inhibitor, a thyroid hormone signaling pathway activator, or both, and at least one agent selected from the group consisting of a monoglyceride lipase (MGLL) inhibitor, a bone morphogenetic protein (BMP) type 1 receptor inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a histone methyltransferase inhibitor, and a protein kinase inhibitor; (d) contacting the second insulin-positive cell population in vitro with a second composition, thereby differentiating at least a portion of the second insulin-positive cell population into a third cell population comprising a plurality of beta cells, wherein the second composition is different from the first composition, and the third cell population comprises a higher percentage of viable beta cells compared to a corresponding beta cell population comprising beta cells derived from the first cell population not contacted with the first composition; A method comprising:

103. (a) obtaining a first cell population comprising a plurality of cell clusters comprising insulin-positive cells; (b) dissociating in vitro at least a portion of the plurality of cell clusters in the first cell population; (c) contacting the first cell population comprising at least a portion of the dissociated cell clusters in vitro with a first composition to obtain a second cell population comprising a plurality of cell clusters, the second cell population comprising a plurality of insulin-positive endocrine cells, wherein the first composition comprises a monoglyceride lipase (MGLL) inhibitor; (d) contacting the second insulin-positive cell population in vitro with a second composition, thereby differentiating at least a portion of the second insulin-positive cell population into a third cell population comprising a plurality of beta cells, wherein the second composition is in vitro contacted with the first insulin-positive cell population. wherein the third cell population is different from said composition and comprises a higher percentage of viable beta cells compared to a corresponding beta cell population comprising beta cells derived from said first cell population that have not been contacted with said first composition. A method comprising:

104. (a) freezing at least a portion of the first cell population, the first cell population comprising at least a portion of the dissociated cell clusters; (b) thawing at least a portion of the frozen first cell population; (c) contacting at least a portion of the thawed first cell population in vitro with the first composition; 104. The method of claim 101, 102, or 103, further comprising:

105. 105. The method of any one of claims 101 to 104, wherein at least some of the plurality of cell clusters of the second cell population have a diameter of about 50 μm to about 250 μm, about 75 μm to about 250 μm, or about 100 μm to about 200 μm.

106. 106. The method of any one of claims 101-105, wherein at least about 40%, 50%, 60%, 70%, 80%, or 90% of the plurality of cell clusters of the second cell population have a diameter of about 50 μm to about 250 μm, about 75 μm to about 250 μm, or about 100 μm to about 200 μm.

107. 107. The method of any one of claims 101-106, wherein at least about 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, or 99% of the cells of the second cell population are viable.

108. 108. The method of any one of claims 101-107, wherein at least about 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, or 99% of the cells of the plurality of cell clusters of the second cell population are viable.

109. 109. The method of any one of claims 101 to 108, wherein the second cell population comprises at least 2, 3, 4, 5, 10, 50, 100, 1000, 10,000, 100,000, or 1,000,000 cell clusters.

110. 110. The method of any one of claims 101 to 109, wherein the second cell population comprises a higher percentage of insulin-positive endocrine cells compared to a corresponding cell population comprising insulin-positive endocrine cells not contacted with the first composition.

111. 111. The method of any one of claims 101 to 110, wherein the second cell population comprises at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% more viable insulin-positive endocrine cells compared to a corresponding cell population comprising insulin-positive endocrine cells not contacted with the first composition.

112. 112. The method of any one of claims 101 to 111, wherein the second cell population comprises at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% more viable insulin-positive endocrine cells after about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days of contact between the first cell population and the first composition, compared to a corresponding cell population comprising insulin-positive endocrine cells not contacted with the first composition.

113. The second cell population is incubated with the first cell population and the first composition for about 1 to 10 days.

112. The method of any one of claims 101-111, wherein after contact for up to 9 days, 1-8 days, 1-7 days, 1-6 days, 1-5 days, 1-4 days, 1-3 days, 1-2 days, 2-10 days, 2-9 days, 2-8 days, 2-7 days, 2-6 days, 2-5 days, 2-4 days, 2-3 days, 3-10 days, 3-9 days, 3-8 days, 3-7 days, 3-6 days, 3-5 days, 3-4 days, 4-10 days, 4-9 days, 4-8 days, 4-7 days, 4-6 days, or 4-5 days, the population comprises at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% more viable insulin-positive endocrine cells compared to a corresponding cell population comprising insulin-positive endocrine cells not contacted with the first composition.

114. 114. The method of any one of claims 101 to 113, wherein at least a portion of the plurality of beta cells form a plurality of cell clusters.

115. 115. The method of claim 114, wherein at least some of the plurality of cell clusters of the third cell population have a diameter of about 50 μm to about 250 μm, about 50 μm to about 150 μm, about 50 μm to about 100 μm, about 75 μm to about 250 μm, about 75 μm to about 150 μm, about 75 μm to about 125 μm, about 75 μm to about 100 μm, or about 100 μm to about 200 μm.

116. 116. The method of claim 114 or 115, wherein at least about 40%, 50%, 60%, 70%, 80%, or 90% of the plurality of cell clusters of the third cell population have a diameter of about 50 μm to about 250 μm, about 50 μm to about 150 μm, about 50 μm to about 100 μm, about 75 μm to about 250 μm, about 75 μm to about 150 μm, about 75 μm to about 125 μm, about 75 μm to about 100 μm, or about 100 μm to about 200 μm, without a selection step.

117. 115. The method of claim 114, wherein at least about 40%, 50%, 60%, 70%, 75%, 80%, 90%, or 95% of the cell clusters have a diameter of about 50-150 μm, 75-12 μm, 80-120 μm, or 90-110 μm without a selection step.

118. 115. The method of claim 114, wherein at least about 50%, 60%, 70%, 75%, 80%, 90%, or 95% of the cell clusters have a diameter of about 100 microns without a selection step.

119. 119. The method of any one of claims 114 to 118, wherein at least about 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, or 99% of the cells of the third cell population are viable without a selection step.

120. 120. The method of any one of claims 114-119, wherein at least about 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, or 99% of the cells of the plurality of cell clusters of the third cell population are viable without a selection step.

121. 121. The method of any one of claims 114 to 120, wherein the third cell population comprises at least 2, 3, 4, 5, 10, 50, 100, 1000, 10,000, 100,000, or 1,000,000 cell clusters.

122. The third cell population exhibits at least about 10%, 20%, 30%, or 40% reduction in beta cells from the first cell population that has not been contacted with the first composition compared to a corresponding cell population comprising beta cells derived from the first cell population that has not been contacted with the first composition.

122. The method of any one of claims 114 to 121, comprising 40%, 50%, 60%, 70%, 80%, or 90% more viable beta cells.

123. 123. The method of any one of claims 114-122, wherein the third cell population comprises at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% more viable beta cells after about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days of contact between the first cell population and the first composition, compared to a corresponding cell population comprising beta cells derived from the first cell population not contacted with the first composition.

124. The second cell population may be incubated with the first cell population and the first composition for about 1-10 days, 1-9 days, 1-8 days, 1-7 days, 1-6 days, 1-5 days, 1-4 days, 1-3 days, 1-2 days, 2-10 days, 2-9 days, 2-8 days, 2-7 days, 2-6 days, 2-5 days, 2-4 days, 2-3 days, 3-10 days, 3-9 days, 3-8 days, 3-7 days, 3-6 days, 3-5 days, 3-4 days, 124. The method of any one of claims 114-123, wherein after 4 to 10 days, 4 to 9 days, 4 to 8 days, 4 to 7 days, 4 to 6 days, or 4 to 5 days of contact, the first cell population comprises at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% more viable beta cells compared to a corresponding cell population comprising beta cells derived from the first cell population not contacted with the first composition.

125. 125. The method of any one of claims 114 to 124, wherein at least a portion of the plurality of beta cells of the third cell population exhibit glucose-stimulated insulin secretion (GSIS) in response to a glucose load in vitro.

126. 126. The method of any one of claims 114 to 125, wherein at least a portion of the plurality of beta cells of the third cell population express insulin.

127. 127. The method of any one of claims 101 or 104-126, wherein the first composition impairs two, three, four, or five of the agents.

128. 127. The method of any one of claims 102 or 104-126, wherein the first composition impairs three, four, five, six, or seven of the agents.

129. 129. The method of any one of claims 101-128, wherein the step of contacting with the first composition comprises contacting the first cell population with the first composition for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days or more.

130. 130. The method of claim 129, wherein said contacting with said first composition comprises contacting said first cell population with said first composition for about 4 days.

131. 129. The method of any one of claims 101-128, wherein the step of contacting with the first composition comprises contacting the first cell population with the first composition for about 6 hours, 10 hours, 12 hours, 24 hours, 30 hours, 36 hours, 40 hours, 48 ​​hours, 56 hours, 72 hours, or more.

132. The step of contacting the first cell population with the first composition may include contacting the first cell population with the first composition for about 1-10 days, 1-9 days, 1-8 days, 1-7 days, 1-6 days, 1-5 days, 1-4 days, 1-3 days, 1-2 days, 2-10 days, 2-9 days, 2-8 days, 2-7 days, 2-6 days, 2-5 days, 2-4 days, 2-3 days, 3-10 days, 3-9 days, 3-8 days, 3-7 days, 3-6 days, 3-5 days, 3-4 days, 4-10 days, 4-9 days, 129. The method of any one of claims 101 to 128, comprising contacting for 4-8 days, 4-7 days, 4-6 days, 4-5 days, 5-10 days, 5-9 days, 5-8 days, 5-7 days, 5-6 days, 6-10 days, 6-9 days, 6-8 days, 6-7 days, 7-10 days, 7-8 days, 8-10 days, 8-9 days, or 9-10 days.

133. 129. The method of any one of claims 101-128, wherein the step of contacting with the first composition comprises contacting the first cell population with the first composition for about 6-96 hours, 6-72 hours, 6-48 hours, 6-24 hours, 6-12 hours, 12-96 hours, 12-72 hours, 12-48 hours, 12-24 hours, 24-96 hours, 24-72 hours, 24-45 hours, 48-96 hours, or 48-72 hours.

134. 129. The method of any one of claims 101 to 128, wherein the step of contacting with the first composition comprises contacting the first cell population with the first composition for about 72 hours.

135. 135. The method of any one of claims 101 or 103-134, wherein the first composition further comprises a transforming growth factor beta (TGF-β) signaling pathway inhibitor, a thyroid hormone signaling pathway activator, or both.

136. 136. The method of any one of claims 101 to 135, wherein the first composition comprises an MGLL inhibitor.

137. 137. The method of any one of claims 101 to 136, wherein the first composition comprises a TGF-β signaling pathway inhibitor.

138. 138. The method of any one of claims 101 to 137, wherein the first composition comprises a thyroid hormone signaling pathway activator.

139. 139. The method of any one of claims 101 to 138, wherein the first composition comprises a bone morphogenetic protein (BMP) type 1 receptor inhibitor.

140. 140. The method of any one of claims 101 to 139, wherein the first composition comprises a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor.

141. 141. The method of any one of claims 101 to 140, wherein the first composition comprises a histone methyltransferase inhibitor.

142. 142. The method of any one of claims 101 to 141, wherein the first composition comprises a protein kinase inhibitor.

143. 143. The method of any one of claims 101 to 142, wherein the first composition comprises a TGF-β signaling pathway inhibitor, a thyroid hormone signaling pathway activator, a bone morphogenetic protein (BMP) type 1 receptor inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a histone methyltransferase inhibitor, and a protein kinase inhibitor.

144. The first composition may comprise an MGLL inhibitor, a TGF-β signaling pathway inhibitor, a thyroid hormone signaling pathway activator, a bone morphogenetic protein (BMP) type 1 receptor inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a histone methyltransferase inhibitor, a thyroid hormone receptor activator ...

144. The method of any one of claims 101 to 143, comprising a phosphodiesterase inhibitor, a phosphodiesterase inhibitor, and a protein kinase inhibitor.

145. 145. The method of any one of claims 101 to 144, wherein the TGF-β signaling pathway inhibitor is Alk5i (SB505124).

146. 146. The method of any one of claims 101 to 145, wherein the thyroid hormone signaling pathway activator is T3 or an analog or derivative thereof.

147. 147. The method of any one of claims 101 to 146, wherein the thyroid hormone signaling pathway activator is the TRβ selective agonist GC-1.

148. 148. The method of any one of claims 101 to 147, wherein the thyroid hormone signaling pathway activator is 3,5-dimethyl-4-[(4'-hydroxy-3'-isopropylbenzyl)-phenoxy]acetic acid.

149. 149. The method of any one of claims 101 to 148, wherein the bone morphogenetic protein (BMP) type 1 receptor inhibitor is LDN193189 or a derivative thereof.

150. 150. The method of any one of claims 101 to 149, wherein the Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor is thiazovivin.

151. 151. The method of any one of claims 101 to 150, wherein the histone methyltransferase inhibitor is 3-deazaneplanocin A.

152. 152. The method of any one of claims 101 to 151, wherein the protein kinase inhibitor is staurosporine (SSP).

153. 153. The method of any one of claims 101 to 152, wherein the first composition does not contain zinc sulfate.

154. 154. The method of any one of claims 101 to 153, wherein the first composition further comprises a lipid.

155. 155. The method of claim 154, wherein the lipid is a saturated fatty acid.

156. 156. The method of claim 155, wherein the saturated fatty acid is palmitate.

157. 155. The method of claim 154, wherein the lipid is an unsaturated fatty acid.

158. 158. The method of claim 157, wherein the unsaturated fatty acid is oleic acid, linoleic acid, or palmitoleic acid.

159. 159. The method of any one of claims 101 to 158, wherein the first composition comprises human serum albumin (HSA).

160. 160. The method of claim 159, wherein the first composition comprises about 0.01-5%, 0.01-4%, 0.01-3%, 0.01-2%, 0.01-1%, 0.01-0.5%, 0.01-0.06%, or 0.01-0.05% HSA.

161. 160. The method of claim 159, wherein the first composition comprises more than about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1% HSA.

162. 160. The method of claim 159, wherein the first composition comprises less than about 5%, 4%, 3%, 2%, 1%, 0.5%, 0.06%, or 0.05% HSA.

163. 160. The method of claim 159, wherein the first composition comprises about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.9%, 1%, 2%, 3%, 4%, or 5% HSA.

164. 160. The method of claim 159, wherein the first composition comprises about 0.05% HSA.

165. 165. The method of any one of claims 101 to 164, wherein the first composition comprises MCDB 131.

166. 166. The method of any one of claims 101 to 165, wherein the first composition comprises DMEM / F12.

167. 167. The method of any one of claims 101 to 166, wherein the first composition comprises zinc.

168. 168. The method of any one of claims 101 to 167, wherein the first composition comprises ZnSO4.

169. 169. The method of any one of claims 101 to 168, wherein the first composition comprises at least one metabolite.

170. 170. The method of any one of claims 101 to 169, wherein the at least one metabolite is glutamate, acetate, β-hydroxybutarate, L-carnitine, taurine, formate, or biotin.

171. 169. The method of any one of claims 101 to 168, wherein the first composition comprises one, two, three, four, five, six, or seven of glutamate, acetate, beta-hydroxybutarate, L-carnitine, taurine, formate, or biotin.

172. 172. The method of any one of claims 101 to 171, wherein the second composition comprises at least one amino acid.

173. 173. The method of claim 172, wherein the at least one amino acid is alanine, glutamate, glycine, proline, threonine, or tryptophan.

174. 174. The method of any one of claims 101 to 173, wherein the second composition comprises at least one vitamin.

175. 175. The method of claim 174, wherein the at least one vitamin is biotin or riboflavin.

176. The step of contacting the second cell population with the second composition may include contacting the second cell population with the second composition for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days or more.

176. The method of any one of claims 101 to 175, comprising contacting for a period of time.

177. 176. The method of any one of claims 101-175, wherein the step of contacting with the second composition comprises contacting the second cell population with the second composition for about 6 hours, 10 hours, 12 hours, 24 hours, 30 hours, 36 hours, 40 hours, 48 ​​hours, 56 hours, 72 hours, or more.

178. The step of contacting the second cell population with the second composition may include contacting the second cell population with the second composition for about 1-10 days, 1-9 days, 1-8 days, 1-7 days, 1-6 days, 1-5 days, 1-4 days, 1-3 days, 1-2 days, 2-10 days, 2-9 days, 2-8 days, 2-7 days, 2-6 days, 2-5 days, 2-4 days, 2-3 days, 3-10 days, 3-9 days, 3-8 days, 3-7 days, 3-6 days, 3-5 days, 176. The method of any one of claims 101 to 175, comprising contacting for days, 3-4 days, 4-10 days, 4-9 days, 4-8 days, 4-7 days, 4-6 days, 4-5 days, 5-10 days, 5-9 days, 5-8 days, 5-7 days, 5-6 days, 6-10 days, 6-9 days, 6-8 days, 6-7 days, 7-10 days, 7-8 days, 8-10 days, 8-9 days, or 9-10 days.

179. 176. The method of any one of claims 101-175, wherein the step of contacting with the second composition comprises contacting the second cell population with the second composition for about 6-96 hours, 6-72 hours, 6-48 hours, 6-24 hours, 6-12 hours, 12-96 hours, 12-72 hours, 12-48 hours, 12-24 hours, 24-96 hours, 24-72 hours, 24-45 hours, 48-96 hours, or 48-72 hours.

180. 176. The method of any one of claims 101 to 175, wherein the step of contacting with the second composition comprises contacting the second cell population with the second composition for about 7 days.

181. 181. The method of any one of claims 101 to 180, wherein the second composition does not include one or more of an MGLL inhibitor, a TGF-β signaling pathway inhibitor, a thyroid hormone signaling pathway activator, a bone morphogenetic protein (BMP) type 1 receptor inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a histone methyltransferase inhibitor, or a protein kinase inhibitor.

182. 182. The method of any one of claims 101 to 181, wherein the second composition does not comprise an MGLL inhibitor.

183. 183. The method of any one of claims 101 to 182, wherein the second composition does not comprise a TGF-β signaling pathway inhibitor.

184. 184. The method of any one of claims 101 to 183, wherein the second composition does not contain a thyroid hormone signaling pathway activator.

185. 185. The method of any one of claims 101 to 184, wherein the second composition does not include a bone morphogenetic protein (BMP) type 1 receptor inhibitor.

186. 186. The method of any one of claims 101 to 185, wherein the second composition does not include a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor.

187. 187. The method of any one of claims 101 to 186, wherein the second composition does not include a histone methyltransferase inhibitor.

188. 188. The method of any one of claims 101 to 187, wherein the second composition does not include a protein kinase inhibitor.

189. The method of any one of claims 101 to 188, wherein the second composition does not comprise a TGF-β signaling pathway inhibitor, a thyroid hormone signaling pathway activator, a bone morphogenetic protein (BMP) type 1 receptor inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a histone methyltransferase inhibitor, and a protein kinase inhibitor.

190. The method of any one of claims 101 to 188, wherein the second composition does not comprise an MGLL inhibitor, a TGF-β signaling pathway inhibitor, a thyroid hormone signaling pathway activator, a bone morphogenetic protein (BMP) type 1 receptor inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a histone methyltransferase inhibitor, and a protein kinase inhibitor.

191. 191. The method of any one of claims 101 to 190, wherein the second composition comprises a lipid.

192. 192. The method of claim 191, wherein the lipid is a saturated fatty acid.

193. 193. The method of claim 192, wherein the saturated fatty acid is palmitate.

194. 192. The method of claim 191, wherein the lipid is an unsaturated fatty acid.

195. 195. The method of claim 194, wherein the unsaturated fatty acid is oleic acid, linoleic acid, or palmitoleic acid.

196. 196. The method of any one of claims 101-181, 183-189, or 191-195, wherein the second composition comprises an MGLL inhibitor.

197. 197. The method of any one of claims 101 to 196, wherein the second composition does not contain human serum albumin (HSA).

198. 197. The method of any one of claims 101 to 196, wherein the second composition comprises human serum albumin (HSA).

199. 200. The method of claim 198, wherein the second composition comprises about 0.1-5%, 0.1-4%, 0.1-3%, 0.1-2%, 0.1-1%, or 0.1-0.5% HSA.

200. 200. The method of claim 199, wherein the second composition comprises less than about 5%, 4%, 3%, 2%, 1%, 0.6%, or 0.5% HSA.

201. 200. The method of claim 199, wherein the second composition comprises about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, or 5% HSA.

202. 200. The method of claim 199, wherein the second composition comprises about 1% HSA.

203. 203. The method of any one of claims 101 to 202, wherein the second composition comprises MCDB 131.

204. 203. The method of any one of claims 101 to 202, wherein the second composition comprises DMEM / F12.

205. 205. The method of any one of claims 101 to 204, wherein the second composition comprises zinc.

206. 206. The method of any one of claims 101 to 205, wherein the second composition comprises ZnSO4.

207. 207. The method of any one of claims 101 to 206, wherein the second composition comprises at least one metabolite.

208. 208. The method of claim 207, wherein said at least one metabolite is glutamate, acetate, beta-hydroxybutarate, L-carnitine, taurine, formate, or biotin.

209. 207. The method of any one of claims 101-206, wherein the second composition comprises one, two, three, four, five, six, or seven of glutamate, acetate, beta-hydroxybutarate, L-carnitine, taurine, formate, or biotin.

210. 210. The method of any one of claims 101 to 209, wherein the second composition comprises at least one amino acid.

211. 211. The method of claim 210, wherein the at least one amino acid is alanine, glutamate, glycine, proline, threonine, or tryptophan.

212. 212. The method of any one of claims 101 to 211, wherein the second composition comprises at least one vitamin.

213. 213. The method of claim 212, wherein the at least one vitamin is biotin or riboflavin.

214. 214. The method of any one of claims 101 to 213, wherein the dissociating step does not include subjecting the cell population to flow cytometry.

215. (a) obtaining a first cell population comprising a plurality of cell clusters comprising insulin-positive endocrine cells; (b) dissociating in vitro at least a portion of the plurality of cell clusters in the first cell population; (c) freezing at least a portion of the first cell population, including at least a portion of the dissociated cell clusters; (d) thawing at least a portion of the frozen first cell population; (e) contacting the at least a portion of the thawed first cell population in vitro with a first composition to obtain a second cell population comprising a plurality of insulin-positive endocrine cells comprising a plurality of cell clusters, wherein the first composition contains a monoglyceride lipase (MGLL) inhibitor, a bone morphogenetic protein (BMP) type 1 receptor inhibitor, a Rho-associated protein (RIP) inhibitor, a phospholipase C (PGC ... and one, two, three, four, or five of the following agents: a coiled-coil-containing protein kinase (ROCK) inhibitor, a histone methyltransferase inhibitor, or a protein kinase inhibitor; (f) contacting the second insulin-positive endocrine cell population in vitro with a second composition, thereby differentiating at least a portion of the second insulin-positive endocrine cell population into a third cell population comprising a plurality of beta cells comprising a plurality of cell clusters, wherein the second composition is different from the first composition, and the third cell population comprises a higher percentage of viable beta cells compared to a corresponding beta cell population comprising beta cells derived from the first cell population not contacted with the first composition; A method comprising:

216. The method of claim 215, wherein the first composition comprises a bone morphogenetic protein (BMP) type 1 receptor inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a histone methyltransferase inhibitor, and a protein kinase inhibitor.

217. 217. The method of claim 215 or 216, wherein the first composition further comprises a transforming growth factor beta (TGF-β) signaling pathway inhibitor.

218. 218. The method of any one of claims 215 to 217, wherein the first composition further comprises a thyroid hormone signaling pathway activator.

219. 219. The method of any one of claims 215 to 218, wherein the first composition further comprises a monoglyceride lipase (MGLL) inhibitor.

220. (a) obtaining a first cell population comprising a plurality of cell clusters comprising insulin-positive endocrine cells; (b) dissociating in vitro at least a portion of the plurality of cell clusters in the first cell population; (c) freezing at least a portion of the first cell population, including at least a portion of the dissociated cell clusters; (d) thawing at least a portion of the frozen first cell population; (e) contacting the at least a portion of the thawed first cell population in vitro with a first composition to obtain a second cell population comprising a plurality of insulin-positive endocrine cells comprising a plurality of cell clusters, wherein the first composition comprises a transforming growth factor beta (TGF-β) signaling pathway inhibitor, a thyroid hormone signaling pathway activator, or both, and one, two, three, four, or five of the following agents: a monoglyceride lipase (MGLL) inhibitor, a bone morphogenetic protein (BMP) type 1 receptor inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a histone methyltransferase inhibitor, or a protein kinase inhibitor; (f) contacting the second insulin-positive endocrine cell population in vitro with a second composition, thereby differentiating at least a portion of the second insulin-positive endocrine cell population into a third cell population comprising a plurality of beta cells comprising a plurality of cell clusters, wherein the second composition is different from the first composition, and the third cell population comprises a higher percentage of viable beta cells compared to a corresponding beta cell population comprising beta cells derived from the first cell population not contacted with the first composition; A method comprising:

221. The first composition comprises a monoglyceride lipase (MGLL) inhibitor, a bone morphogenetic protein (BMP) type 1 receptor inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROK) inhibitor, and a steroid hormone receptor inhibitor.

221. The method of claim 220, comprising a phosphodiesterase (CK) inhibitor, a histone methyltransferase inhibitor, and a protein kinase inhibitor.

222. (a) obtaining a first cell population comprising a plurality of cell clusters comprising insulin-positive endocrine cells; (b) dissociating in vitro at least a portion of the plurality of cell clusters in the first cell population; (c) contacting the first cell population comprising at least a portion of the dissociated cell clusters with a first composition in vitro to obtain a second cell population comprising a plurality of cell clusters, the second cell population comprising a plurality of insulin-positive endocrine cells; (d) contacting the second insulin-positive endocrine cell population in vitro with a second composition, thereby differentiating at least a portion of the second insulin-positive endocrine cell population into a third cell population comprising a plurality of beta cells, wherein the second composition is different from the first composition, the second composition comprises at least one metabolite, and the third cell population comprises a higher percentage of viable cells compared to a corresponding cell population comprising beta cells derived from the first cell population not contacted with the second composition; A method comprising:

223. (a) obtaining a first cell population comprising a plurality of cell clusters comprising insulin-positive endocrine cells; (b) dissociating in vitro at least a portion of the plurality of cell clusters in the first cell population; (c) contacting the first cell population comprising at least a portion of the dissociated cell clusters with a first composition in vitro to obtain a second cell population comprising a plurality of cell clusters, the second cell population comprising a plurality of insulin-positive endocrine cells; (d) contacting the second insulin-positive endocrine cell population in vitro with a second composition, thereby differentiating at least a portion of the second insulin-positive endocrine cell population into a third cell population comprising a plurality of beta cells, wherein the second composition is different from the first composition, the second composition comprising at least one metabolite, and the plurality of beta cells exhibits enhanced glucose-stimulated insulin secretion relative to a corresponding cell population comprising beta cells derived from the first cell population not contacted with the second composition; A method comprising:

224. 224. The method of claim 222 or 223, wherein the metabolite is an amino acid, a vitamin, a polyol, an organic substance, an acid, an antioxidant, a nucleotide, or an alcohol.

225. 224. The method of claim 222 or 223, wherein the at least one metabolite is glutamate, acetate, beta-hydroxybutarate, L-carnitine, taurine, formate, or biotin.

226. 224. The method of claim 222 or 223, wherein the second composition comprises at least two, three, four, five, six, or seven metabolites of glutamate, acetate, beta-hydroxybutarate, L-carnitine, taurine, formate, or biotin.

227. 227. The method of any one of claims 222 to 226, wherein the second composition comprises DMEM / F12.

228. 228. The method of claim 222, wherein the second composition comprises about 0.05 to 2% HSA. The method according to any one of claims 1 to 4.

229. 229. The method of claim 228, wherein the second composition comprises about 1% HSA.

230. 230. The method of any one of claims 222 to 229, wherein the second composition comprises zinc.

231. 231. The method of any one of claims 222 to 230, wherein the second composition comprises ZnSO4.

232. 232. The method of any one of claims 222 to 231, wherein the second composition comprises at least one amino acid.

233. 232. The method of claim 231, wherein the at least one amino acid is alanine, glutamate, glycine, proline, threonine, or tryptophan.

234. 234. The method of any one of claims 222 to 233, wherein the second composition comprises at least one vitamin.

235. 235. The method of claim 234, wherein the at least one vitamin is biotin or riboflavin.

236. 236. The method of any one of claims 222 to 235, wherein the plurality of beta cells exhibits enhanced glucose-stimulated insulin secretion compared to a corresponding cell population comprising beta cells derived from the first cell population that have not been contacted with the second composition.

237. 237. The method of any one of claims 222-236, wherein the third cell population comprises a higher percentage of viable cells compared to a corresponding cell population comprising beta cells derived from the first cell population that have not been contacted with the second composition.

238. 238. The method of any one of claims 222 to 237, wherein the third cell population comprises a plurality of cell clusters, each having a diameter of about 50 to 150 microns.

239. 239. The method of any one of claims 222 to 238, wherein the third cell population comprises a plurality of cell clusters, each having a diameter of about 100 microns.

240. 240. The method of any one of claims 222-239, wherein the third cell population comprises a plurality of cell clusters, and at least 50%, 60%, 70%, 75%, 80%, 90%, or 95% of the cell clusters have a diameter of about 100 microns without a selection step.

241. 241. The method of any one of claims 222-240, wherein the third cell population comprises a plurality of cell clusters, and at least 50%, 60%, 70%, 75%, 80%, 90%, or 95% of the cell clusters have a diameter of about 50-150 microns, 75-125 microns, 80-120 microns, or 90-110 microns without a selection step.

242. The first composition may comprise a monoglyceride lipase (MGLL) inhibitor, a bone morphogenetic protein (BMP) type 1 receptor inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a histone methyltransferase inhibitor, or a protein kinase inhibitor.

242. The method of any one of claims 222 to 241, comprising at least one agent selected from the group consisting of a toxic agent.

243. (a) freezing at least a portion of the first cell population, the first cell population comprising at least a portion of the dissociated cell clusters; (b) thawing at least a portion of the frozen first cell population; (c) contacting at least a portion of the thawed first cell population in vitro with the first composition; 240. The method of any one of claims 219 to 239, further comprising:

244. 244. A composition comprising at least a portion of the second cell population comprising insulin-positive endocrine cells according to any one of claims 101 to 243.

245. 244. A composition comprising at least a portion of the third cell population of beta cells described in any one of claims 101 to 243.

246. 244. A composition comprising at least a portion of the third cell population of beta cells described in any one of claims 101 to 243 and at least a portion of the second cell population comprising insulin-positive endocrine cells described in any one of claims 101 to 243.

247. A device comprising the beta cell composition of claim 244.

248. A device comprising the insulin-positive endocrine cell composition of claim 245.

249. A device comprising the insulin-positive endocrine cell and beta cell composition of claim 246.

250. A method for treating a subject having a disease characterized by prolonged high blood sugar levels (e.g., diabetes), comprising administering to the subject a composition of cells described in claim 244.

251. A method for treating a subject having a disease characterized by prolonged high blood sugar levels (e.g., diabetes), comprising administering to the subject a composition of cells described in claim 245.

252. A method for treating a subject having a disease characterized by prolonged high blood sugar levels (e.g., diabetes), comprising administering to the subject a composition of cells described in claim 246.

253. A method for treating a subject having a disease characterized by high blood glucose levels over a prolonged period of time (e.g., diabetes), comprising implanting a device described in claim 247 into the subject.

254. A method for treating a subject having a disease (e.g., diabetes) characterized by high blood glucose levels over a prolonged period of time, comprising implanting a device described in claim 248 into the subject.

255. A method of treating a subject having a disease characterized by high blood glucose levels over a prolonged period of time (e.g., diabetes), comprising implanting a device described in claim 249 into the subject.

256. 256. The method of any one of claims 250 to 255, wherein the disease is diabetes.

257. A composition comprising isolated insulin-positive endocrine cells that have been contacted in vitro with an agent that inhibits the expression or function of monoglyceride lipase (MGLL) and that exhibit a reduced rate of conversion of monoglycerides to free fatty acids compared to a corresponding isolated insulin-positive endocrine cell population that has not been contacted in vitro with the agent that inhibits the expression or function of monoglyceride lipase (MGLL).

258. A composition comprising isolated insulin-positive endocrine cells that have been contacted in vitro with an agent that inhibits the expression or function of monoglyceride lipase (MGLL) and that exhibit an increased ratio of monoglycerides to free fatty acids compared to a corresponding isolated insulin-positive endocrine cell population that has not been contacted in vitro with an agent that inhibits the expression or function of monoglyceride lipase (MGLL).

259. A composition comprising isolated insulin-positive endocrine cells that have been contacted in vitro with an agent that inhibits the expression or function of monoglyceride lipase (MGLL) and that exhibit a decreased ratio of free fatty acids to monoglycerides compared to a corresponding isolated insulin-positive endocrine cell population that has not been contacted in vitro with an agent that inhibits the expression or function of monoglyceride lipase (MGLL).

260. A composition comprising isolated insulin-positive endocrine cells that have been contacted in vitro with an agent that inhibits monoglyceride lipase (MGLL) expression or function and that exhibit reduced levels of free fatty acids compared to a corresponding isolated insulin-positive endocrine cell population that has not been contacted in vitro with an agent that inhibits monoglyceride lipase (MGLL) expression or function.

261. A composition comprising isolated insulin-positive endocrine cells that have been contacted in vitro with an agent that inhibits monoglyceride lipase (MGLL) expression or function and that exhibit elevated levels of monoglycerides compared to a corresponding isolated insulin-positive endocrine cell population that has not been contacted in vitro with an agent that inhibits monoglyceride lipase (MGLL) expression or function.

262. A composition comprising a population of insulin-positive endocrine cells and an agent that inhibits the conversion of monoglycerides to free fatty acids.

263. The composition of claim 262, wherein the agent inhibits the expression or function of monoglyceride lipase (MGLL).

264. A composition comprising an insulin-positive cell population and an agent that inhibits the expression or function of monoglyceride lipase (MGLL).

265. 265. The composition of any one of claims 257 to 264, wherein the agent that inhibits the expression or function of monoglyceride lipase (MGLL) is JJKK048, KML29, NF1819, JW642, JZL184, JZL195, JZP361, pristimerin, or URB602.

266. 1. A composition comprising a population of beta cells that has been contacted in vitro with at least one agent selected from the group consisting of glutamate, acetate, beta-hydroxybutarate, L-carnitine, taurine, formate, or biotin, wherein the beta cell population has been previously A composition that exhibits increased glucose-stimulated insulin secretion compared to a corresponding beta cell population that has not been contacted with the at least one agent.

267. 267. The composition of claim 266, wherein the cell population has been contacted with at least two, three, four, five, six, or seven of the agents selected from the group consisting of glutamate, acetate, beta-hydroxybutarate, L-carnitine, taurine, formate, or biotin.

268. A composition comprising a beta cell population and at least one, two, three, four, five, six, or seven of the following agents selected from the group consisting of glutamate, acetate, beta-hydroxybutarate, L-carnitine, taurine, formate, or biotin.

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