Enhanced beta cell differentiation

Abstract

Provided herein are compositions and methods for improved generation of SC-β cells in vitro, among others. Provided are novel preparations and differentiation methods that result in SC islet clusters with advantageous characteristics such as higher cell yield and recovery rate, increased number and relative percentage of SC-β cells, enhanced stability and shelf life of SC-β cells, reduced size and increased uniformity, improved function of SC-β cells in vitro, and improved survival rate, function, and reduced immunogenicity after transplantation. The compositions and methods of the present disclosure can be used in the production of SC islets for human therapeutic applications.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 131,471, filed December 29, 2020, which is hereby incorporated by reference in its entirety into this specification. [Background technology]

[0002]

[0002] Pancreas or islet transplantation has been used to treat diabetes, such as type I diabetes. 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 islets provide an alternative source of transplantable islets. Summary of the Invention

[0003]

[0003] Pancreatic islet transplantation is a promising therapy that can achieve significant clinical benefits for diabetic subjects, e.g., subjects with type I diabetes. Because the supply of pancreatic islet sources from donor pancreatic tissue is limited, improved techniques are needed to generate transplantable islets from alternative sources, such as stem cells. Improved methods of generating islet components (e.g., SC-β cells) may result in more effective therapeutic products (e.g., SC-β cells with improved functionality), improved methods of producing SC islets for human therapeutic use (e.g., higher cell yields), or a combination thereof.

[0004]

[0004] Provided herein, inter alia, are compositions and methods for improved generation of SC-β cells in vitro. Novel formulations and differentiation methods are provided that result in SC islet clusters with advantageous characteristics such as higher cell yields and recovery rates, increased numbers and relative percentages of SC-β cells, enhanced stability and shelf life of SC-β cells, reduced size and increased uniformity, improved function of SC-β cells in vitro, and improved survival, function, and reduced immunogenicity after transplantation. The compositions and methods of the present disclosure can be used in the large-scale production of SC islets for human therapeutic applications.

[0005]

[0005] In some aspects, disclosed herein is an in vitro composition comprising a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells and one or more of an acetyl-CoA-related metabolite, an HDAC inhibitor, a redox homeostasis regulator, or a one-carbon metabolic pathway intermediate.

[0006]

[0006] In some aspects, disclosed herein is an in vitro composition comprising a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells, and acetyl-CoA-related metabolites.

[0007]

[0007] In some aspects, disclosed herein is an in vitro composition comprising a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells and one or more of acetate, β-hydroxybutyrate, taurine, or formate.

[0008]

[0008] In some aspects, disclosed herein is an in vitro composition comprising a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells, and acetate.

[0009] In some cases, the compositions of the present disclosure include acetate at 0.01-50 mM, 0.1-50 mM, 0.5-50 mM, 0.01-20 mM, 0.1-20 mM, 0.5-20 mM, 0.01-10 mM, 0.1-10 mM, 0.5-10 mM, 0.8-25 mM, 0.8-10 mM, 0.8-5 mM, 0.8-2 mM, 0.8-1.5 mM, 0.8-1.2 mM, 0.9-1.1 mM, or 0.95-1.05 mM. In some cases, the compositions of the present disclosure further include glutamine. In some cases, compositions of the present disclosure include between 0.5 and 20 mM, between 0.5 and 10 mM, between 0.5 and 5 mM, between 1 and 5 mM, between 2 and 5 mM, or between 1 mM and 10 mM glutamine.

[0010]

[0010] In some aspects, disclosed herein is an in vitro composition comprising a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells and 3-10, 3-7, 3-8, 3-6, 3-5, 3-4, 3.5-4.5, 3.8-4.2, or 3.9-4.1 mM glutamine.

[0011] In some cases, glutamine is at a concentration of 3.8-4.2 mM. In some cases, at least 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, or 5 mM of glutamine is not in the dipeptide form. In some cases, at least 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, or 5 mM of glutamine is not in the alanine-glutamine dipeptide form. In some cases, at least 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 5.5 mM, 6 mM, 6.5 mM, 7 mM, 7.5 mM, 8 mM, 8.5 mM, 9 mM, 9.5 mM, or 10 mM of the glutamine is in the form of free glutamine. In some cases, the compositions of the present disclosure further comprise a plurality of insulin-positive endocrine precursor cells.

[0012]

[0012] In some aspects, disclosed herein is an in vitro composition comprising a plurality of insulin-positive endocrine precursor cells and 0.01-50 mM, 0.1-50 mM, 0.5-50 mM, 0.01-20 mM, 0.1-20 mM, 0.5-20 mM, 0.01-10 mM, 0.1-10 mM, 0.5-10 mM, 0.8-25 mM, 0.8-10 mM, 0.8-5 mM, 0.8-2 mM, 0.8-1.5 mM, 0.8-1.2 mM, 0.9-1.1 mM, or 0.95-1.05 mM of an acetyl-CoA-related metabolite.

[0013]

[0013] In some aspects, disclosed herein is an in vitro composition comprising a plurality of insulin-positive endocrine precursor cells and 0.01-50 mM, 0.1-50 mM, 0.5-50 mM, 0.01-20 mM, 0.1-20 mM, 0.5-20 mM, 0.01-10 mM, 0.1-10 mM, 0.5-10 mM, 0.8-25 mM, 0.8-10 mM, 0.8-5 mM, 0.8-2 mM, 0.8-1.5 mM, 0.8-1.2 mM, 0.9-1.1 mM, or 0.95-1.05 mM acetate.

[0014]

[0014] In some cases, the compositions of the present disclosure contain about 1 mM acetate.

[0015] In some embodiments, disclosed herein is an in vitro composition comprising a plurality of insulin-positive endocrine precursor cells and 3-10, 3-7, 3-8, 3-6, 3-5, 3-4, 3.5-4.5, 3.8-4.2, or 3.9-4.1 mM glutamine. In some cases, the disclosed composition comprises about 4 mM glutamine. In some cases, the disclosed composition comprises glutamine and acetate.

[0015]

[0016] In some aspects, disclosed herein is an in vitro composition comprising a plurality of insulin-positive endocrine precursor cells and one or more of an acetyl-CoA-related metabolite, an HDAC inhibitor, a redox homeostasis regulator, a one-carbon metabolic pathway intermediate, glutamate, or L-carnitine.

[0016]

[0017] In some aspects, disclosed herein is an in vitro composition comprising a plurality of insulin-positive endocrine precursor cells and one or more of glutamate, acetate, beta-hydroxybutyrate, taurine, L-carnitine, or formate.

[0017]

[0018] In some cases, the compositions of the present disclosure include 50-1000 nM, 50-800 nM, 50-500 nM, 50-300 nM, 50-250 nM, 100-200 nM, or 125-175 nM acetate. In some cases, the compositions of the present disclosure include β-hydroxybutyrate. In some cases, the compositions of the present disclosure include taurine. In some cases, the compositions of the present disclosure include formate. In some cases, the compositions of the present disclosure include a vitamin. In some cases, the compositions of the present disclosure include biotin. In some cases, the compositions of the present disclosure include glutamine, acetate, β-hydroxybutyrate, taurine, formate, and biotin. In some cases, the compositions of the present disclosure further include L-carnitine. In some cases, the compositions of the present disclosure include L-carnitine. In some cases, the compositions of the present disclosure further include a BMP signaling pathway inhibitor. In some cases, the BMP signaling pathway inhibitor is LDN193189 or DMH-1 or their derivatives.In some cases, the composition of the present disclosure further comprises a ROCK inhibitor.In some cases, the ROCK inhibitor is thiazovivin, Y-27632, fasudil / HA1077 or 14-1152 or their derivatives.In some cases, the composition of the present disclosure further comprises a histone methyltransferase inhibitor. In some cases, the histone methyltransferase inhibitor is 3-deazaneplanocin A hydrochloride, Bix-01294, UNC0638, BRDD4770, EPZ004777, AZ505, PDB4e47, alproic acid, vorinostat, romidepsin, entinostat abexinostat, gibinostat, and mocetinostat, butyrate, a serine protease inhibitor, a serpin, or a derivative thereof. In some cases, the compositions of the present disclosure further comprise a TGF-β pathway inhibitor. In some cases, the TGF-β pathway inhibitor is ALK5 inhibitor II, A83-01, 431542, D4476, GW788388, LY364947, LY580276, SB525334, SB505124, SD208, GW6604, or GW788388, or a derivative thereof.In some cases, the composition of the present disclosure further comprises a thyroid hormone signaling pathway activator. In some cases, the thyroid hormone signaling pathway activator is GC-1, T3, T1AM, T0AM, Triprop, L-940901, CGS 23425, KB-141, or DITPA, or a derivative thereof. In some cases, the composition of the present disclosure further comprises a protein kinase inhibitor. In some cases, the protein kinase inhibitor is staurosporine or Ro-31-8220, or a derivative thereof. In some cases, the composition of the present disclosure further comprises a sonic hedgehog pathway inhibitor. In some cases, the sonic hedgehog pathway inhibitor comprises Sant1, Sant2, Sant 4, Sant4, Cur61414, forskolin, tomatidine, AY9944, triparanol, cyclopamine, or a derivative thereof. In some cases, the composition of the present disclosure further comprises a growth factor from the epidermal growth factor (EGF) family. In some cases, the growth factor from EGF family comprises betacellulin or EGF. In some cases, the composition of the present disclosure further comprises a gamma secretase inhibitor. In some cases, the gamma secretase inhibitor comprises XXI, DAPT, or a derivative thereof. In some cases, the composition of the present disclosure further comprises zinc. In some cases, the zinc is in the form of ZnSO4. In some cases, the composition of the present disclosure further comprises serum albumin protein. In some cases, the serum albumin protein is human serum albumin protein.In some cases, the compositions of the present disclosure may be 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. In some cases, the composition of the present disclosure further comprises DMEM. In some cases, the DMEM is DMEM / F12. In some cases, the composition of the present disclosure does not comprise vitamin C.

[0018]

[0019] In some cases, the plurality of insulin-positive endocrine precursor cells are dissociated. In some cases, at least 50%, 60%, 65%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the plurality of insulin-positive endocrine precursor cells are not in cell clusters. In some cases, 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. In some cases, the insulin-positive endocrine precursor cells have been previously frozen.

[0019]

[0020] In some cases, at least 30%, 40%, 50%, 60%, 65%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the plurality of insulin-positive endocrine precursor cells are arranged in cell clusters. In some cases, the plurality of insulin-positive endocrine precursor cells are arranged in cell clusters. In some cases, at least about 40%, 50%, 60%, 70%, 80%, or 90% of the 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 cases, at least about 40%, 50%, 60%, 70%, 80%, or 90% of the cell clusters have a diameter of about 80-150, about 100-150, about 120-150, about 140-150, about 80-130, about 100-130, about 120-130, about 80-120, about 90-120, or about 100-120 μm. In some cases, the cell clusters have a mean or median diameter of up to 120, up to 130, up to 140, up to 150, up to 160, or up to 170 μm. In some cases, the cell clusters have an average or median diameter of about 80-150, about 100-150, about 120-150, about 140-150, about 80-130, about 100-130, about 120-130, about 80-120, about 90-120, or about 100-120 μm. In some cases, at least about 50%, at least about 60%, at least about 70%, or at least about 80% of the cell clusters have a diameter less than 150 μm. In some cases, at least about 50%, at least about 60%, or at least about 70% of the cell clusters have a diameter less than 130 μm. In some cases, the compositions of the present disclosure comprise at least 35%, at least 38%, at least 40%, at least 42%, at least 44%, or at least 46% ISL1 positive, NKX6.1 positive cells. In some cases, compositions of the disclosure comprise up to 2%, up to 4%, up to 6%, up to 8%, up to 10%, up to 12%, up to 14%, up to 16%, up to 18%, up to 20%, up to 22%, or up to 22% ISL1 negative, NKX6.1 negative cells.

[0020]

[0021] In some cases, the compositions of the present disclosure include cells that are engineered to include a genomic disruption in at least one gene sequence, the disruption reducing or eliminating expression of a protein encoded by the gene sequence. In some cases, the at least one gene sequence is any one or more of beta-2-microglobulin (B2M), HLA-A, HLA-B, or CIITA. In some cases, the compositions of the present disclosure include cells that are engineered to increase expression of PD-L1 and / or CD47. In some cases, the cells are engineered using a CRISPR / Cas system.

[0021]

[0022] In some aspects, a method is disclosed herein that includes contacting a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells in vitro with a first composition comprising one or more of an acetyl-CoA-related metabolite, an HDAC inhibitor, a redox homeostasis regulator, or a one-carbon metabolic pathway intermediate.

[0022]

[0023] In some aspects, a method is disclosed herein that includes contacting a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells in vitro with a first composition comprising an acetyl-CoA related metabolite.

[0023]

[0024] In some aspects, a method is disclosed herein that includes contacting a plurality of PDX1 positive, NKX6.1 positive, insulin negative cells in vitro with a first composition comprising one or more of acetate, beta-hydroxybutyrate, taurine, or formate.

[0024]

[0025] In some aspects, disclosed herein is a method comprising contacting a plurality of PDX1 positive, NKX6.1 positive, insulin negative cells in vitro with a first composition comprising acetate.

[0025]

[0026] In some cases, the disclosed methods include contacting the cells with 0.01-50 mM, 0.1-50 mM, 0.5-50 mM, 0.01-20 mM, 0.1-20 mM, 0.5-20 mM, 0.01-10 mM, 0.1-10 mM, 0.5-10 mM, 0.8-25 mM, 0.8-10 mM, 0.8-5 mM, 0.8-2 mM, 0.8-1.5 mM, 0.8-1.2 mM, 0.9-1.1 mM, or 0.95-1.05 mM acetate. In some cases, the first composition further comprises glutamine. In some cases, the first composition further comprises between 0.5 and 20 mM, between 0.5 and 10 mM, between 0.5 and 5 mM, between 1 and 5 mM, between 2 and 5 mM, or between 1 mM and 10 mM glutamine.

[0026]

[0027] In some aspects, disclosed herein are methods that include contacting a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells with a first composition comprising 3-10, 3-7, 3-8, 3-6, 3-5, 3-4, 3.5-4.5, 3.8-4.2, or 3.9-4.1 mM glutamine.

[0027]

[0028] In some cases, the glutamine is at a concentration of 3.8-4.2 mM. In some cases, at least 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, or 5 mM of the glutamine is not in the dipeptide form. In some cases, at least 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, or 5 mM of the glutamine is not in the alanine-glutamine dipeptide form. In some cases, at least 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 5.5 mM, 6 mM, 6.5 mM, 7 mM, 7.5 mM, 8 mM, 8.5 mM, 9 mM, 9.5 mM, or 10 mM of the glutamine is in the form of free glutamine. In some cases, the first composition comprises about 4 mM glutamine. In some cases, the first composition comprises glutamine and acetate. In some cases, the first composition comprises β-hydroxybutyrate. In some cases, the first composition comprises taurine. In some cases, the first composition comprises formate. In some cases, the first composition comprises a vitamin. In some cases, the first composition further comprises biotin. In some cases, the first composition comprises glutamine, acetate, β-hydroxybutyrate, taurine, formate, and biotin. In some cases, the first composition further comprises a BMP signaling pathway inhibitor. In some cases, the BMP signaling pathway inhibitor is LDN193189 or DMH-1 or a derivative thereof. In some cases, the first composition further comprises a ROCK inhibitor. In some cases, the ROCK inhibitor is thiazovivin, Y-27632, fasudil / HA1077, or 14-1152, or a derivative thereof. In some cases, the first composition further comprises a histone methyltransferase inhibitor.In some cases, the histone methyltransferase inhibitor is 3-deazaneplanocin A hydrochloride, Bix-01294, UNC0638, BRDD4770, EPZ004777, AZ505, PDB4e47, valproic acid, vorinostat, romidepsin, entinostat abexinostat, gibinostat, and mocetinostat, butyrate, a serine protease inhibitor, a serpin, or a derivative thereof. In some cases, the first composition further comprises zinc. In some cases, the zinc is in the form of ZnSO4. In some cases, the first composition further comprises serum albumin protein. In some cases, the serum albumin protein is human serum albumin protein. In some cases, the first composition may be 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%, 0.04-0.0 In some cases, the first composition further comprises a TGF-β pathway inhibitor. In some cases, the TGF-β pathway inhibitor is Alk5 inhibitor II, A83-01, 431542, D4476, GW788388, LY364947, LY580276, SB525334, SB505124, SD208, GW6604, or GW788388, or a derivative thereof. In some cases, the first composition further comprises a thyroid hormone signaling pathway activator.In some cases, the thyroid hormone signaling pathway activator is GC-1 or T3, T1AM, T0AM, Triprop, L-940901, CGS 23425, KB-141, or DITPA, or a derivative thereof. In some cases, the first composition further comprises a protein kinase inhibitor. In some cases, the protein kinase inhibitor is staurosporine or Ro-31-8220, or a derivative thereof. In some cases, the first composition further comprises a gamma-secretase inhibitor. In some cases, the gamma-secretase inhibitor is XXI, DAPT, or a derivative thereof.

[0028]

[0029] In some cases, the first composition further comprises L-carnitine. In some cases, the method of the present disclosure comprises contacting a plurality of PDX1 positive, NKX6.1 positive cells with a first composition for 1-3 days, and then contacting with a second composition comprising one or more of acetate, beta-hydroxybutyrate, taurine, or formate for 3-7 days.

[0029]

[0030] In some cases, the disclosed method includes contacting a plurality of PDX1 positive, NKX6.1 positive cells with a first composition for 1-3 days, followed by contacting with a second composition comprising one or more of a TGF-β pathway inhibitor, a thyroid hormone signaling pathway activator, a BMP signaling pathway inhibitor, a ROCK inhibitor, a histone methyltransferase inhibitor, or a protein kinase inhibitor for 2-7 days. In some cases, the disclosed method includes contacting a plurality of PDX1 positive, NKX6.1 positive cells with a first composition for 1-3 days, followed by contacting with a second composition comprising one or more of Alk5i, GC-1, LDN193189, thiazovivin, SSP, or DZNEP for 2-7 days. In some cases, the first composition further comprises a sonic hedgehog pathway inhibitor. In some cases, the sonic hedgehog pathway inhibitor comprises Sant1, Sant2, Sant 4, Sant4, Cur61414, forskolin, tomatidine, AY9944, triparanol, cyclopamine, or derivatives thereof. In some cases, the first composition further comprises a growth factor from the epidermal growth factor (EGF) family. In some cases, the growth factor from the EGF family comprises betacellulin or EGF. In some cases, the second composition lacks the sonic hedgehog pathway inhibitor and the growth factor from the epidermal growth factor (EGF) family, and is otherwise the same as the first composition.

[0030]

[0031] In some cases, the contacting step differentiates at least a portion of the plurality of PDX1 positive, NKX6.1 positive, insulin negative cells into insulin positive endocrine precursor cells. In some cases, the insulin positive endocrine precursor cells include ISL1 positive, NKX6.1 positive cells. In some cases, the contacting step results in the generation of a cell population having a higher percentage of ISL1 positive, NKX6.1 positive cells compared to the step of contacting the plurality of PDX1 positive, NKX6.1 positive, insulin negative cells with a composition that is the same as the first composition except that it lacks one or more of acetate, β-hydroxybutyrate, taurine, or formate. In some cases, the contacting step results in the generation of a cell population having at least 35%, at least 38%, at least 40%, at least 42%, at least 44%, or at least 46% ISL1 positive, NKX6.1 positive cells. In some cases, the contacting step results in the generation of a cell population having about 35%, about 38%, about 40%, about 41%, about 42%, or about 47% ISL1-positive, NKX6.1-positive cells. In some cases, the contacting step results in the generation of a cell population having at least 35%, at least 38%, or at least 40% ISL1-positive, NKX6.1-positive cells after 4 days, 7 days, or 10 days of culture. In some cases, the contacting step results in the generation of a cell population having a lower percentage of ISL1-negative, NKX6.1-negative cells compared to contacting a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells with a composition that is the same as the first composition except that it lacks one or more of acetate, beta-hydroxybutyrate, taurine, or formate. In some cases, the contacting step results in the production of a cell population having up to 22%, up to 20%, or up to 18%, up to 16%, up to 14%, up to 12%, up to 10%, up to 8%, up to 6%, or up to 4% ISL1-negative, NKX6.1-negative cells. In some cases, the contacting step results in the production of a cell population having about 2%, 11%, 22%, 20%, or 18% ISL1-negative, NKX6.1-negative cells.

[0031]

[0032] In some aspects, disclosed herein is a method comprising contacting a plurality of insulin-positive endocrine precursor cells in vitro with a first composition comprising one or more of an acetyl-CoA related metabolite, an HDAC inhibitor, a redox homeostasis regulator, a vitamin, a one-carbon metabolic pathway intermediate, L-carnitine, or glutamate.

[0032]

[0033] In some aspects, disclosed herein are methods that include contacting a plurality of insulin-positive endocrine precursor cells in vitro with a first composition comprising one or more of glutamate, acetate, beta-hydroxybutyrate, taurine, biotin, L-carnitine, or formate.

[0033]

[0034] In some cases, the first composition includes glutamate. In some cases, the first composition includes acetate. In some cases, the first composition includes 50-1000 nM, 50-800 nM, 50-500 nM, 50-300 nM, 50-250 nM, 100-200 nM, or 125-175 nM acetate. In some cases, the first composition includes beta-hydroxybutyrate. In some cases, the first composition includes taurine. In some cases, the first composition includes formate. In some cases, the first composition further includes biotin. In some cases, the first composition includes L-carnitine. In some cases, the first composition includes glutamate, acetate, beta-hydroxybutyrate, taurine, formate, L-carnitine, and biotin. In some cases, the first composition further comprises a BMP signaling pathway inhibitor. In some cases, the BMP signaling pathway inhibitor is LDN193189 or DMH-1 or their derivatives. In some cases, the first composition further comprises a ROCK inhibitor. In some cases, the ROCK inhibitor is thiazovivin, Y-27632, fasudil / HA1077 or 14-1152 or their derivatives. In some cases, the first composition further comprises a histone methyltransferase inhibitor. In some cases, the histone methyltransferase inhibitor is 3-deazaneplanocin A hydrochloride, Bix-01294, UNC0638, BRDD4770, EPZ004777, AZ505, PDB4e47, valproic acid, vorinostat, romidepsin, entinostat abexinostat, gibinostat, and mocetinostat, butyrate, a serine protease inhibitor, a serpin, or a derivative thereof. In some cases, the first composition further comprises a TGF-β pathway inhibitor. In some cases, the TGF-β pathway inhibitor is ALK5 inhibitor II, A83-01, 431542, D4476, GW788388, LY364947, LY580276, SB525334, SB505124, SD208, GW6604, or GW788388, or a derivative thereof. In some cases, the first composition further comprises a thyroid hormone signaling pathway activator.In some cases, the thyroid hormone signaling pathway activator is GC-1, T3, T1AM, T0AM, Triprop, L-940901, CGS 23425, KB-141, or DITPA, or a derivative thereof. In some cases, the first composition further comprises a protein kinase inhibitor. In some cases, the protein kinase inhibitor is staurosporine or Ro-31-8220, or a derivative thereof. In some cases, the first composition further comprises vitamin C. In some cases, the first composition does not comprise vitamin C. In some cases, the first composition further comprises zinc. In some cases, the zinc is in the form of ZnSO4. In some cases, the first composition further comprises serum albumin protein. In some cases, the serum albumin protein is human serum albumin protein. In some cases, the first composition may be 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%, 0.04-0.0 In some cases, the composition of the present disclosure comprises DMEM. In some cases, the DMEM is DMEM / F12.

[0034]

[0035] In some cases, the plurality of insulin-positive endocrine precursor cells are dissociated. In some cases, the dissociated insulin-positive endocrine precursor cells have been previously frozen. In some cases, the method of the present disclosure reaggregates the dissociated cells into a plurality of cell clusters. In some cases, 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 cases, at least about 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, or 99% of the cells of the plurality of cell clusters are viable. In some cases, the disclosed methods reaggregate dissociated cells into at least 2, 3, 4, 5, 10, 50, 100, 1000, 10000, 100000, or 1,000,000 cell clusters. In some cases, at least about 40%, 50%, 60%, 70%, 80%, or 90% of the cell clusters have a diameter of about 80-150, about 100-150, about 120-150, about 140-150, about 80-130, about 100-130, about 120-130, about 80-120, about 90-120, or about 100-120 μm. In some cases, the cell clusters have a mean or median diameter of up to 120, up to 130, up to 140, up to 150, up to 160, or up to 170 μm. In some cases, the cell clusters have an average or median diameter of about 80-150, about 100-150, about 120-150, about 140-150, about 80-130, about 100-130, about 120-130, about 80-120, about 90-120, or about 100-120 μm. In some cases, at least about 50%, at least about 60%, at least about 70%, or at least about 80% of the cell clusters have a diameter less than 150 microns. In some cases, at least about 50%, at least about 60%, or at least about 70% of the cell clusters have a diameter less than 130 μm.In some cases, the methods of the disclosure are 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 cases, the methods of the present disclosure are carried out for 1-8 days, 1-7 days, 1-6 days, 1-5 days, 1-4 days, 1-3 days, 1-2 days, 2-8 days, 2-7 days, 2-6 days, 2-5 days, 2-4 days, 2-3 days, 3-8 days, 3-7 days, 3-6 days, 3-5 days, 3-4 days, 4-8 days, 4-7 days, 4-6 days, or 4-5 days.

[0035]

[0036] In some cases, the plurality of insulin-positive endocrine precursor cells are contacted with the first composition for about 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days, thereby generating a second cell population. In some cases, the disclosed method further comprises contacting the second cell population with a second composition different from the first 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. In some cases, the second cell population is contacted with the second composition for 1-8 days, 1-7 days, 1-6 days, 1-5 days, 1-4 days, 1-3 days, 1-2 days, 2-8 days, 2-7 days, 2-6 days, 2-5 days, 2-4 days, 2-3 days, 3-8 days, 3-7 days, 3-6 days, 3-5 days, 3-4 days, 4-8 days, 4-7 days, 4-6 days, or 4-5 days. In some cases, the second cell population is contacted with the second composition for about 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days.In some cases, the second composition does not include a BMP signaling pathway inhibitor, a ROCK inhibitor, a histone methyltransferase inhibitor, a TGF-β pathway inhibitor, a thyroid hormone signaling pathway inhibitor, or a protein kinase inhibitor.In some cases, the second composition does not include glutamate, acetate, β-hydroxybutyrate, taurine, biotin, L-carnitine, or formate.

[0036]

[0037] In some aspects, disclosed herein is a method comprising: (a) contacting a plurality of PDX1 positive, NKX6.1 positive, insulin negative cells in vitro with a first composition, thereby generating a first cell population comprising a plurality of cell clusters comprising insulin positive endocrine precursor cells, the first composition comprising one or more of acetate, glutamine, and beta-hydroxybutyrate, taurine, formate, or biotin; (b) dissociating at least a portion of the plurality of cell clusters in the first cell population in vitro; and (c) contacting the first cell population comprising at least a portion of the dissociated cell clusters in vitro with a second composition, thereby generating a second cell population comprising a plurality of cell clusters comprising a plurality of insulin positive cells, the second composition comprising one or more of glutamate, acetate, beta-hydroxybutyrate, taurine, biotin, L-carnitine, or formate. In some cases, the methods of the present disclosure further include contacting in vitro a second insulin-positive cell population with a third 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 third composition is different from the second composition.

[0037]

[0038] In some cases, the third composition does not include glutamate, acetate, beta-hydroxybutyrate, taurine, biotin, L-carnitine, or formate. In some cases, the first composition includes 3-10, 3-7, 3-8, 3-6, 3-5, 3-4, 3.5-4.5, 3.8-4.2, or 3.9-4.1 mM glutamine. In some cases, the first composition includes about 4 mM glutamine. In some cases, the first composition comprises 0.01-50 mM, 0.1-50 mM, 0.5-50 mM, 0.01-20 mM, 0.1-20 mM, 0.5-20 mM, 0.01-10 mM, 0.1-10 mM, 0.5-10 mM, 0.8-25 mM, 0.8-10 mM, 0.8-5 mM, 0.8-2 mM, 0.8-1.5 mM, 0.8-1.2 mM, 0.9-1.1 mM, or 0.95-1.05 mM acetate. In some cases, the first composition comprises about 1 mM acetate. In some cases, the second composition comprises 50-1000 nM, 50-800 nM, 50-500 nM, 50-300 nM, 50-250 nM, 100-200 nM, or 125-175 nM acetate. In some cases, the second composition comprises glutamate, acetate, beta-hydroxybutyrate, taurine, formate, L-carnitine, and biotin.

[0038]

[0039] In some cases, the first composition further comprises at least one agent selected from the group consisting of a TGF-β signaling pathway inhibitor, a growth factor from the EGF family, a retinoic acid signaling pathway activator, a sonic hedgehog pathway inhibitor, a thyroid hormone signaling pathway activator, a protein kinase inhibitor, a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, a bone morphogenetic protein (BMP) type 1 receptor inhibitor, a gamma-secretase inhibitor, or a histone methyltransferase inhibitor.

[0039]

[0040] In some cases, the second composition further comprises at least one agent selected from the group consisting of a TGF-β signaling pathway inhibitor, a retinoic acid signaling pathway activator, a sonic hedgehog pathway inhibitor, a thyroid hormone signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and a histone methyltransferase inhibitor. In some cases, (A) the sonic hedgehog pathway inhibitor comprises SANT1, (B) the retinoic acid signaling pathway activator comprises retinoic acid, (C) the gamma-secretase inhibitor comprises XXI, (D) the growth factor from the EGF family comprises betacellulin or EGF, (E) the BMP signaling pathway inhibitor comprises LDN193189 or a derivative thereof, and (F) the TGF-β signaling pathway inhibitor comprises an ALK5 inhibitor II or or derivatives thereof; (G) the thyroid hormone signaling pathway activator comprises GC-1 or T3, or derivatives thereof; (H) the protein kinase inhibitor comprises staurosporine; (I) the ROCK inhibitor comprises thiazovivin, Y-27632, fasudil / HA1077, or 14-1152, or derivatives thereof; or (J) the histone methyltransferase inhibitor comprises 3-deazaneplanocin A hydrochloride or a derivative thereof.

[0040]

[0041] In some aspects, disclosed herein are methods comprising contacting a NKX6.1 positive, ISL1 positive, insulin positive cell population with one or more of a serum albumin protein, a TGF-β signaling pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, an epigenetic modifying compound, an acetyl-CoA related metabolite, a vitamin, a histone deacetylase inhibitor (HDACi), a redox homeostasis regulator, a one-carbon metabolic pathway intermediate, glutamate, and / or carnitine for a first period of 1, 2, 3, 4, 5, 6, or 7 days (e.g., 4 days). In some cases, the methods of the disclosure further include, after the first period of time, contacting the cell population with one or more of a serum albumin protein, an acetyl-CoA related metabolite, a vitamin, a histone deacetylase inhibitor (HDACi), a redox homeostasis regulator, a one-carbon metabolic pathway intermediate, glutamate, and / or carnitine for a second period of 1, 2, 3, 4, 5, 6, or 7 days (e.g., 3 days) or more in the absence of a TGF-β signaling pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and / or an epigenetic modifying compound.

[0041]

[0042] In some aspects, disclosed herein are methods that include contacting a NKX6.1 positive, ISL1 positive, insulin positive cell population with one or more of HSA, Alk5 inhibitor II, GC-1, staurosporine, thiazovivin, LDN193189, DZNEP, taurine, acetate, beta hydroxybutyrate, biotin, carnitine, glutamate, and formate for a first period of 1, 2, 3, 4, 5, 6, or 7 days (e.g., 4 days). In some cases, the methods of the disclosure further include, after the first period of time, contacting the cell population with one or more of HSA, taurine, acetate, beta-hydroxybutyrate, biotin, carnitine, glutamate, and formate for a second period of 1, 2, 3, 4, 5, 6, or 7 days (e.g., 3 days) or more in the absence of Alk5 inhibitor II, GC-1, staurosporine, thiazovivin, LDN193189, and DZNEP.

[0042]

[0043] In some cases, the disclosed method further comprises contacting the cell population with ZnSO4 during the first and / or second time period. In some cases, the disclosed method further comprises contacting the cell population with DMEM during the first and / or second time period. In some cases, the DMEM is DMEM / F12. In some cases, the cells are contacted during the second time period with the same concentration of serum albumin (e.g., about 0.05% HSA) compared to the first time period. In some cases, the cells are contacted during the second time period with a higher concentration of HSA (e.g., about 1.0%) compared to the first time period (e.g., about 0.05%). In some cases, the cells are engineered to include a genomic disruption in at least one gene sequence, the disruption reducing or eliminating expression of a protein encoded by the gene sequence. In some cases, the at least one gene sequence is any one or more of beta-2-microglobulin (B2M), HLA-A, HLA-B, or CIITA. In some cases, the compositions of the present disclosure include cells that have been engineered to increase expression of PD-L1 and / or CD47. In some cases, the cells have been engineered using a CRISPR / Cas system.

[0043]

[0044] In some aspects, disclosed herein are compositions comprising the insulin-positive endocrine precursor cells disclosed herein.

[0044]

[0045] In some aspects, disclosed herein are compositions comprising at least a portion of the third cell population of beta cells disclosed herein.

[0045]

[0046] In some aspects, disclosed herein is a device that comprises the cells disclosed herein.In some cases, the device is configured to produce and release insulin when implanted in a subject.In some cases, the cells are encapsulated.In some cases, the device further comprises a semipermeable membrane, which is configured to retain the cells in the device and allow insulin to pass through.

[0046]

[0047] In some aspects, disclosed herein are methods of treating a subject having a disease characterized by high blood glucose levels over an extended period of time (e.g., diabetes), comprising administering a cell disclosed herein or implanting a device disclosed herein. In some cases, the disease is diabetes.

[0047]

[0048] In some aspects, disclosed herein is a composition comprising a plurality of insulin positive cells and a cryopreservation agent, and one or more of an acetyl-CoA related metabolite, an HDAC inhibitor, a redox homeostasis regulator, a one-carbon metabolic pathway intermediate, a vitamin, or L-glutamine.

[0048]

[0049] In some cases, the compositions of the present disclosure are cryopreserved. In some cases, the compositions of the present disclosure include an acetyl-CoA related metabolite. In some cases, the acetyl-CoA related metabolite is acetate. In some cases, the compositions of the present disclosure include 0.01-50 mM, 0.1-50 mM, 0.5-50 mM, 0.01-20 mM, 0.1-20 mM, 0.5-20 mM, 0.01-10 mM, 0.1-10 mM, 0.5-10 mM, 0.8-25 mM, 0.8-10 mM, 0.8-5 mM, 0.8-2 mM, 0.8-1.5 mM, 0.8-1.2 mM, 0.9-1.1 mM, or 0.95-1.05 mM acetate. In some cases, the compositions of the present disclosure include glutamine. In some cases, the compositions of the present disclosure include 0.5-20 mM, 0.5-10 mM, 0.5-5 mM, 1-5 mM, 2-5 mM, 3.8-4.2, or 1 mM-10 mM glutamine. In some cases, at least 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, or 5 mM of the glutamine is not in the alanine-glutamine dipeptide form. In some cases, at least 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 5.5 mM, 6 mM, 6.5 mM, 7 mM, 7.5 mM, 8 mM, 8.5 mM, 9 mM, 9.5 mM, or 10 mM of the glutamine is in the form of free glutamine. In some cases, the composition of the present disclosure includes a redox homeostasis regulator. In some cases, the redox homeostasis regulator is taurine. In some cases, the composition of the present disclosure includes a one-carbon metabolic pathway intermediate. In some cases, the one-carbon metabolic pathway intermediate is formate. In some cases, the composition of the present disclosure includes a vitamin. In some cases, the vitamin is biotin.

[0049] Incorporation by Reference

[0050] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. 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 specification supersede and / or take precedence over any such conflicting material.

[0050] BRIEF DESCRIPTION OF THE DRAWINGS

[0051] 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 by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings (also referred to herein as "Figure" and "FIG."). [Brief description of the drawings]

[0051] [Figure 1A]

[0052] 1A and 1B provide the results of a Seahorse assay performed on human embryonic stem cells incubated for 48 hours with or without the MQ medium supplement disclosed herein. MQ refers to a medium containing formate, acetate, biotin, β-hydroxybutyrate, taurine, and glutamine. FIG. 1A shows the oxygen consumption rate (OCR) over multiple stages of the assay. FIG. 1B shows the ATP-associated oxygen consumption calculated based on the assay. CTL indicates control cells incubated in the absence of MQ. +MQ or Met* indicates cells incubated in the presence of MQ. hESCs cultured in the presence of MQ exhibited increased basal metabolic rage (see FIG. 1A, e.g., the left side of the graph) and increased ATP-associated oxygen consumption (FIG. 1B), indicating that MQ can alter intracellular metabolism. [Figure 1B]Figure 1B shows the ATP-associated oxygen consumption calculated based on the assay. CTL indicates control cells incubated in the absence of MQ. +MQ or Met* indicates cells incubated in the presence of MQ. hESCs cultured in the presence of MQ exhibited increased basal metabolic rage (see Figure 1A, e.g., the left side of the graph) and increased ATP-associated oxygen consumption (Figure 1B), indicating that MQ can alter intracellular metabolism. [Figure 1C]

[0053] FIG. 1C is a bright field microscopy image of a culture of human embryonic stem cells incubated for 48 hours with (MQ) or without (CTL) the MQ medium supplement disclosed herein, or incubated with MQ supplements excluding glutamine and acetate. Cultures incubated with MQ exhibited increased cell density, suggesting enhanced growth. When glutamine and acetate were removed from the MQ cocktail, cell density decreased compared to the complete set of supplements, suggesting that glutamine and acetate contribute to the observed effects of MQ on cells. [Diagram 2]

[0054] FIG. 2A is an exemplary scatter plot from samples assessed at the end of stage 5 of cultures incubated with MQ (+ metabolites) or in the absence of MQ (control). SC-β cells are shown in the upper right quadrant (see Nkx6.1+ / Isl1+). Addition of MQ to the medium throughout stage 5 substantially increased the percentage of SC-β cells from 25.9% in control cultures to 38.1% in cultures with MQ. FIG. 2B is a graph quantifying the percentage of Nkx6.1+ / Isl1+ SC-β cells obtained at the end of stage 5 from repetitions performed without MQ, showing that the SC-β% achieved in the absence of MQ is reproducibly lower than that observed with MQ in FIG. 2A. These data suggest that addition of MQ during stage 5 can enhance SC-β cell differentiation. [Diagram 3]3 provides exemplary flow cytometry results of experiments in which MQ was absent throughout stage 5 (control), present throughout stage 5 (+MQ), or MQ supplemented without acetate and glutamine (+MQ*-Acetate-Q). The percentage of Nkx6.1+ / Isl1+ SC-β cells was 29.8% in control conditions, 41.6% in the presence of MQ, and 34.3% with MQ supplemented without acetate and glutamine. [Figure 4] FIG. 4 is a graph providing summary data from two experiments comparing the percentage of SC-β cells present at the end of stage 5 following incubation in the presence or absence of MQ throughout stage 5. [Diagram 5]

[0058] Figure 5 provides exemplary flow cytometry data from day 4 of stage 6, showing that in cultures in which MQ was present at stage 5 (+ metabolites), 47.3% of cells were Nkx6.1+ / Isl1+ compared to 37% in control cultures. [Figure 6]

[0059] Figure 6A is a graph demonstrating the percent recovery of all cells at days 4 and 10 of stage 6 relative to the number of viable cells seeded at the beginning of stage 6. The total cell yield was higher in cultures where MQ was present at stage 5. Figure 6B is a graph quantifying the number of SC-β cells at day 4 of stage 6, calculated from the product of the total cell yield and the percent of cells that were SC-β cells. The yield of SC-β cells was higher in cultures where MQ was present at stage 5. [Figure 7]

[0061] Figure 7A shows an exemplary flow plot of cells cultured with or without MQ during stage 5 and analyzed at day 7 of stage 6. It was found that 45.1% of cells were Nkx6.1+ / Isl1+ in cultures where MQ was present at stage 5, compared to 34.3% in cells not incubated with MQ. Figure 7B shows a graph of the percentage of Nkx6.1+ / Isl1+ cells at days 4, 7, and 10 of stage 6 for cells cultured with or without MQ during stage 5, demonstrating that the high percentage of SC-β cells observed with MQ treatment at stage 5 is maintained over time (MQ St5). [Figure 8] 8 is a graph illustrating the percent recovery (yield) of all cells at days 4, 7, and 10 of stage 6 versus the number of viable cells seeded at the beginning of stage 6. Cells were cultured in the presence (MQ) or absence (Control) of MQ and thawed into Medium A or Control Medium 1 at stage 6. Higher yields were observed in cells incubated with MQ at stage 5 (MQ St5). [Figure 9]

[0064] Figure 9A is a bright field microscopy image of cells incubated with or without MQ (control) during stage 5 and then thawed into medium A or control medium 1 at stage 6. Images were taken on day 4 of stage 6. Cells incubated with MQ during stage 5 and then thawed into medium A formed smaller clusters. Figure 9B provides bright field microscopy images of cells incubated with or without MQ (control) during stage 5 and then thawed into medium A or control medium 1 at stage 6. Images were taken on day 7 of stage 6. Cells incubated with MQ during stage 5 and then thawed into medium A formed smaller clusters. [Figure 10]10 is a graph quantifying cluster size frequency at day 7 of stage 6, showing that cluster size is smaller in cells incubated with MQ at stage 5 and then thawed into medium A at stage 6. Cells incubated with MQ during stage 5 and then thawed into medium A formed smaller clusters. [Figure 11]

[0067] Figure 11 illustrates Stage 6 medium ("Medium A") and its components compared to Control Medium 1 and Control Medium 2. During days 1-4 of Stage 6, S5d6 factors include Alk5i (10 μM), GC-1 (1 μM), LDN-193189 (100 nM), thiazovinin (2.5 μM), SSP (3 nM), and DZNEP (100 nM). [Figure 12] FIG. 12A is a bar graph showing the number of viable cells recovered at S6d7 and S6d11 from control medium 1, control medium 2, medium A (no metabolites), or medium A (containing metabolites) cultures. The data shows that medium A cultures using DMEM / F12 further improve the number of viable cells throughout stage 6. FIG. 12B is a bar graph showing the percentage of cells recovered at S6d7 and S6d11 from control medium 1, control medium 2, medium A (no metabolites), or medium A (containing metabolites) cultures. The data shows that medium A cultures using DMEM / F12 further improve the cell recovery throughout stage 6. [Figure 13]

[0070] Figure 13 shows microscopy images of SC islet clusters at S6d7 and S6d11 from control medium 1, control medium 2, or medium A (containing metabolites) cultures. The images show that medium A clusters exhibit greater uniformity throughout stage 6. [Figure 14]14 is a series of area graphs showing the frequency and cluster size of cell clusters in S6d4, S6d7, and S6d11 from control medium 1, control medium 2, and medium A cultures. The results show that medium A clusters are smaller and exhibit greater uniformity in S6d11. [Figure 15] FIG. 15 is a bar graph showing GSIS function of S6d11 cells from control medium 1 culture. [Figure 16]

[0073] Figure 16 is a bar graph showing GSIS function of S6d11 cells cultured from control medium 2. The results show that SC islets in control medium 2 do not exhibit as much GSIS function as those in control medium 1 (compare Figure 15). [Figure 17]

[0074] Figure 17 is a bar graph showing the GSIS function of S6d11 cells from medium A (no metabolites) culture. The results show that SC islets from medium A (no metabolites) cultures exhibit improved GSIS function (compare with Figure 15). [Figure 18]

[0075] Figure 18 is a bar graph showing GSIS function of S6d11 cells from medium A (containing metabolites) culture. The results show that SC islets from medium A cultures exhibit similar GSIS function to control medium 1 cultures (compare Figure 15). [Figure 19] 19 is a table showing the effect of medium A. Medium A shows improved S6d11 cell recovery rate compared to control medium 2, and improved GSIS function compared to control medium 1. [Figure 20]

[0077] Figure 20A is a graph showing the viability of SC islet cells 6 weeks after transplantation into nude rats, comparing cells generated by regimen 1 at stage 6 of differentiation with cells generated by regimen 2 at stage 6. Cells generated using regimen 1 at stage 6 exhibited higher viability compared to cells generated using regimen 2 when assessed 6 weeks after implantation. Figure 20B is a graph showing the viability of SC islet cells at stage 6 of differentiation generated by regimen 2 at stage 6 6 and 13 weeks after transplantation into nude rats. [Figure 21]

[0078] Figure 21 is a graph quantifying the number of multinucleated giant cells (MNG) detected in histopathological evaluation of devices 6 weeks after implantation in nude rats. SC islands generated by regimen 1 at stage 6 of differentiation are compared to SC islands generated by regimen 2 at stage 6. Fewer multinucleated giant cells (MNG) were observed in cells generated using regimen 1 6 weeks after implantation. [Figure 22] 22 is an exemplary histopathology image of H&E stained sections from nude rats 6 weeks after implantation of devices of the present disclosure containing SC islets generated by methods including regimen 2 or regimen 1 at stage 6 of differentiation. Six weeks after implantation, reduced immune infiltrates and fewer multinucleated giant cells (MNGs) were observed in cells generated using regimen 1. [Figure 23] 23 is a graph illustrating non-fasting blood glucose levels in diabetic NOD Scid Gamma (NSG) mice receiving one (1×) or two (2×) devices loaded with SC islets generated using Regimen 2 or Regimen 1 during stage 6 of differentiation. Animals receiving two devices (2×) maintained normoglycemia until approximately 8 weeks after implantation. [Figure 24]FIG. 24A is a graph illustrating non-fasting human C-peptide levels over time in diabetic NOD Scid gamma (NSG) mice administered one (1×) or two (2×) devices loaded with SC islets generated using regimen 2 or regimen 1 during stage 6 of differentiation. In contrast to naive and diabetic control mice, in which C-peptide levels were undetectable, an increase in C-peptide was observed in all experimental groups after implantation, with sustained levels observed in animals administered two devices. CAD refers to cell-containing device. FIG. 24B is a graph comparing fasting human C-peptide levels measured 153 days after implantation with non-fasting C-peptide levels measured 112 days after implantation in diabetic NOD Scid gamma (NSG) mice administered one (1×) or two (2×) devices loaded with SC islets generated using regimen 2 or regimen 1 during stage 6 of differentiation. The data show that SC islets produced by methods created using the compositions and methods of the present disclosure, including Regimen 2 and Regimen 1, can exhibit efficacy in increasing insulin production after transplantation into diabetic subjects. [Diagram 25]

[0083] Figure 25 is a graph providing survival data for SC islets assessed 5 months (one device) or 6 months (two devices) after implantation in diabetic NSG mice. SC islets were generated using either regimen 2 or regimen 1 during stage 6 of differentiation. High survival is observed in devices with cells generated by either regimen 2 or regimen 1 at stage 6 of differentiation. [Figure 26]

[0084] Figure 26 shows exemplary histology images of sections stained for C-peptide and glucagon (left panel) or H&E stained (right panel) 6 months after implantation of SC islets into diabetic NSG mice. These results show that SC islets generated using the disclosed compositions and methods, including Regimen 2 and Regimen 1, can remain viable after implantation and produce insulin and glucagon. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052]

[0085] Transplantation of pancreatic islets is a promising therapy that can achieve significant clinical benefits for diabetic subjects, such as subjects with type I diabetes. Because the supply of pancreatic islet sources from donor pancreatic tissue is limited, there is a need for improved techniques to generate implantable islets from alternative sources, such as stem cells. Improved methods for generating islet components (e.g., SC-β cells) may result in more effective therapeutic products (e.g., SC-β cells with improved functionality), improved methods for producing SC islets (e.g., higher cell yields) for human therapeutic applications, or a combination thereof.

[0053]

[0086] Provided herein, inter alia, are compositions and methods for improved in vitro generation of SC-β cells. Certain compositions and combinations of agents disclosed herein improve the fitness and metabolic flexibility of differentiated cells and the resulting SC-β cells by targeting specific aspects of the cell's metabolism. For example, the present disclosure provides novel formulations and differentiation methods that result in SC islet clusters with advantageous characteristics such as higher cell yields and recovery rates, increased numbers and relative percentages of SC-β cells, enhanced stability and shelf life of SC-β cells, reduced size and increased uniformity, improved function of SC-β cells in vitro, and improved viability, function, and reduced immunogenicity after transplantation. The disclosed compositions and methods can be used in the large-scale production of SC islets for human therapeutic applications.

[0054]

[0087] The metabolic pathways that can be targeted by the compositions disclosed herein include, for example, one-carbon metabolism, acetyl-CoA synthesis for lipid production and protein acetylation, promotion of mitochondrial oxidative phosphorylation and TCA cycle, and the production of intermediates that maintain redox homeostasis.The compositions disclosed herein that are useful for improving the production of SC-β cells can include, for example, one-carbon metabolic pathway intermediates (e.g., formate), acetyl-CoA-related metabolites (e.g., acetate), vitamins (e.g., biotin), HDAC inhibitors (e.g., β-hydroxybutyrate), redox homeostasis regulators (e.g., taurine), glutamine, glutamate, carnitine, or any combination thereof.

[0055]

[0088] 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 used.

[0056] definition

[0089] In this application, the use of the singular includes the plural unless specifically stated otherwise. It should be noted that as used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0057]

[0090] In this application, the use of "or" means "and / or" unless otherwise stated. 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 mean "A only, B only, C only, A and B, B and C, A and C, and A, B and C." The term "or" can be used conjunctively or disjunctively, unless the context clearly indicates a reference to disjunctive use.

[0058]

[0091] Furthermore, use of the term "including" as well as other forms such as "include," "includes," and "included" is not limiting.

[0059]

[0092] References herein to "some embodiments," "an embodiment," "one embodiment," or "other embodiments" mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least some embodiments of the present disclosure, but not necessarily in all embodiments.

[0060]

[0093] 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.

[0061]

[0094] The term "about" and its grammatical equivalents as used herein in reference to a numerical value can include both the numerical value itself and a range of plus or minus 10% from the numerical value.

[0062]

[0095] 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, depending 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 ​​in a range of plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from 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 a particular value is described in the present application and claims, the term "about" should be assumed to mean within an acceptable error range of the particular value, unless otherwise stated.

[0063]

[0096] The term "diabetes" and its grammatical equivalents, as used herein, can refer to a disease characterized by long-term high blood glucose 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.

[0064]

[0097] 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.

[0065]

[0098] The terms "progenitor" and "precursor" cells are used interchangeably herein and refer to cells that have a cell phenotype that is more primitive than cells that can be generated by differentiation (e.g., at an earlier step in a developmental pathway or progression than a fully differentiated cell). In many cases, progenitor cells can also have significant or very high proliferation 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.

[0066]

[0099] 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 intestinal 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.

[0067]

[0100] 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 pancreatic exocrine cells, which are pancreatic cells that can produce enzymes that are secreted into the small intestine. These enzymes can help digest food that passes through the gastrointestinal tract. Pancreatic exocrine cells are also known as islets of Langerhans, which can secrete two hormones, insulin and glucagon. Pancreatic exocrine cells can be one of several cell types, for example, alpha-2 cells (which can produce the hormone glucagon), or beta cells (which can generate the hormone insulin), and alpha-1 cells (which can produce the regulator somatostatin). Non-insulin-producing exocrine cells, as the term is 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).

[0068]

[0101] 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 the SC-β cells do not have to be derived (e.g., directly) from stem cells, since the disclosed method can be derived 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 cells from which they are derived), multipotent cells, totipotent cells, transdifferentiated forms of any of the foregoing cells, etc.). In some embodiments, the 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 the SC-β cells is similar to that of endogenous β cells. In some embodiments, the SC-β cells exhibit an in vitro GSIS response similar to that of endogenous β cells. In some embodiments, SC-β cells exhibit in vivo GSIS response similar to that of endogenous β cells. In some embodiments, SC-β cells exhibit both in vitro GSIS response and in vivo GSIS response similar to that of endogenous β cells. GSIS response of SC-β cells can be observed within 2 weeks after transplantation of SC-β cells into a host (e.g., 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 cytokines. In some embodiments, insulin secretion from SC-β cells is enhanced in response to known antidiabetic drugs (e.g., secretagogues). In some embodiments, the SC-β cells are monohormonal. In some embodiments, the SC-β cells do not abnormally co-express other hormones, 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.

[0069]

[0102] As used herein, the term "insulin-producing cell" and its grammatical equivalents refer to cells that secrete insulin and are 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 insulin gene, translate proinsulin mRNA, and modify proinsulin mRNA to insulin protein), express (e.g., express the phenotype carried by insulin gene), or secrete (release insulin into extracellular space) insulin. For example, an insulin-producing cell population generated by differentiating insulin-positive endocrine cells or their precursors into SC-β cells according to the methods of the present disclosure can be a pancreatic β cell or a β-like cell (e.g., a cell having at least one or at least two characteristics of an endogenous β cell) and exhibits a glucose-stimulated insulin secretion (GSIS) response similar to an endogenous adult β cell. For example, an insulin-producing cell population 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 an otherwise cell-like phenotype).

[0070]

[0103] The terms "insulin-positive β-like cell," "insulin-positive endocrine cell," and their grammatical synonyms, can refer to cells (e.g., pancreatic endocrine cells) that exhibit at least one marker indicative of a pancreatic β cell and further express insulin, but lack the glucose-stimulated insulin secretion (GSIS) response characteristic of endogenous β cells.

[0071]

[0104] 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, ISL1, Pax6, Pax4, NeuroD, 1 Inf1b, Hnf-6, Hnf-3 beta, and MafA, and those described in Zhang et al., Diabetes. 50(10):2231-6 (2001). In some embodiments, the β-cell marker is a nuclear 3-cell marker. In some embodiments, the β-cell marker is Pdx1 or PH3.

[0072]

[0105] 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.

[0073]

[0106] 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.

[0074]

[0107] The term "Pdx1 positive pancreatic progenitor" as used herein can refer to a cell that is a pancreatic endoderm (PE) cell that has the ability to differentiate into SC-β cells, e.g., pancreatic β cells. Pdx1 positive pancreatic progenitor expresses the marker Pdx1. Other markers include, but are not limited to, Cdcp1, or Ptf1a, or HNF6, or NRx2.2. The expression of Pdx1 can be evaluated by any method known by those skilled in the art, such as immunochemistry using anti-Pdx1 antibodies or quantitative RT-PCR. In some cases, Pdx1 positive pancreatic progenitor cells lack the expression of NKX6.1. 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 expression of NKX6.1. In some cases, Pdx1 positive pancreatic progenitor cells can also be referred to as "pancreatic foregut endoderm cells".

[0075]

[0108] The term "Pdx1 positive NKX6-1 positive pancreatic progenitor" as used herein can refer to cells that are pancreatic endoderm (PE) cells that have the ability to differentiate into insulin-producing cells, such as pancreatic β cells. Pdx1 positive NKX6-1 positive pancreatic progenitor expresses markers Pdx1 and NKX6-1. Other markers can include, but are not limited to, Cdcp1, or Ptf1a, or HNF6, or NRx2.2. NKX6-1 expression can be evaluated by any method known by 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 can also be referred to as "pancreatic foregut precursor cells".

[0076]

[0109] 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). Progenitor 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). The expression of Ngn3 can be assessed by any method known by those skilled in the art, such as immunochemistry using anti-Ngn3 antibodies or quantitative RT-PCR.

[0077]

[0110] The terms "NeuroD" and "NeuroD1" are used interchangeably to identify a protein and its encoding gene that are expressed in pancreatic endocrine precursor cells.

[0078]

[0111] 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), nutrient prototrophy, or expression of a specific protein that can be used as a criterion to distinguish cells that express the protein from cells that do not. The term "selection marker" as used herein can refer to a gene or the expression product of a gene, such as the encoded protein. In some embodiments, a selection marker confers a growth and / or survival advantage on cells that express it compared to cells that do not express it or express it at a significantly lower level. 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 for a sufficient period of time under conditions such that cells that do not express the marker do not grow and / or survive, and are removed from the population or the number of such cells is reduced to only a very small percentage of the population. The method of selecting cells expressing a marker that confers a growth and / or survival advantage by maintaining a cell population under selective conditions that largely or completely remove cells that do not express the marker is referred to herein as "positive selection" and the marker is described as "useful for positive selection". Negative selection and markers useful for negative selection are also the subject of some methods described herein. Expression of such a marker creates a growth and / or survival disadvantage for cells expressing the marker compared to cells that do not express the marker or express it at a significantly lower level (or, considered differently, cells that do not express the marker have a growth and / or survival advantage compared to cells that express the marker). Thus, cells expressing this marker can be largely or completely removed from a cell population if maintained under selective conditions for a sufficient period of time.

[0079]

[0112] The term "epigenetics" refers to heritable changes in gene function that do not involve changes in the DNA sequence. Epigenetics most often refers to chromosomal changes that affect gene activity and expression, but 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 phenotypes can be due to external or environmental factors or can be part of a normal developmental program. Epigenetics can also refer to functionally related 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 changing 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 continue throughout the lifespan of a cell through cell division and can also continue for multiple generations, despite not involving changes in the DNA sequence of the underlying organism. 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.

[0080]

[0113] The term "epigenetic modifying compound" refers to a chemical compound that can cause epigenetic changes to genes, i.e., can change gene expression without changing the DNA sequence. Epigenetic changes can help determine whether a gene is 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, alter DNA accessibility and chromatin structure, thereby regulating patterns 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 alterations. Importantly, epigenetic modifications have a crucial role in regulating pluripotency genes that become inactivated during differentiation. Non-limiting exemplary epigenetic modifying compounds include DNA methylation inhibitors, histone acetyltransferase inhibitors, histone deacetylase inhibitors, histone methyltransferase inhibitors, bromodomain inhibitors, or any combination thereof.

[0081]

[0114] 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 aspect, the term "differentiated cell" can refer to a more specialized cell type obtained from a less specialized cell type (e.g., stem cell, e.g., induced pluripotent stem cell) during the process of cell differentiation. 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 about 98% of cells become exocrine, ductular, or stromal cells, and about 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.

[0082]

[0115] As used herein, the term "somatic cell" can refer to any cell that forms the body of an organism, unlike 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 for sperm and eggs, the cells from which they are produced (gametocytes), and undifferentiated stem cells - are somatic cells, and the internal organs, skin, bone, blood, and connective tissues are all composed of somatic cells. In some embodiments, the somatic cell is a "non-embryonic somatic cell," which means a somatic cell that is not present in or obtained from an embryo, or that does not result from the in vitro propagation of such a cell. In some embodiments, the somatic cell is an "adult somatic cell," which means a cell that is present in or obtained from an organism other than an embryo or fetus, or that results from the 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 an isolated at least one insulin-positive endocrine cell or precursor thereof maintained in culture).

[0083]

[0116] As used herein, the term "adult cell" can refer to cells found throughout the body after embryonic development.

[0084]

[0117] The term "endodermal cell", 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 germ cell layers are mesoderm and ectoderm). Endoderm is the innermost of the three layers. Endoderm cells first give rise to the embryonic gut, then differentiate to give rise to the inner lining of the respiratory and digestive tracts (e.g., gut), liver, and pancreas.

[0085]

[0118] The term "cells of endodermal origin", as used herein, can refer to any cell that develops or differentiates from endoderm cells. For example, cells of endodermal origin include cells of the liver, lung, pancreas, thymus, intestine, stomach, and thyroid. Without wishing to be bound by theory, liver and pancreatic precursors (also called pancreatic precursors) develop from endoderm cells of the embryonic foregut. Shortly after specification, liver and pancreatic precursors rapidly acquire strikingly distinct cellular functions and regenerative potential. These changes are triggered by inductive signals and gene regulators that are highly conserved among vertebrates. Interest in organ development and regeneration is driven by the strong need for hepatocytes and pancreatic β cells in the therapeutic treatment of liver failure and type I diabetes. Studies in various model organisms and humans have revealed evolutionarily conserved inductive signals and transcription factor networks that induce differentiation of liver and pancreatic cells, providing guidance on how to promote hepatocyte and β cell differentiation from diverse stem and progenitor cell types.

[0086]

[0119] 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, and 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, for example, immunochemistry using anti-Sox17 antibodies or quantitative RT-PCR.

[0087]

[0120] The term "pancreatic endoderm" can refer to cells of endodermal origin that can differentiate into multiple pancreatic lineages, including pancreatic β cells, but no longer have the capacity to differentiate into non-pancreatic lineages.

[0088]

[0121] The term "primitive gut cell" or "gut cell" as used herein can refer to a cell that can differentiate from an endoderm cell and differentiate into a SC-β cell (e.g., a pancreatic β cell). Primitive gut cells express at least one marker of HNP1-β, HNF3-β, or HNF4-α. Primitive gut cells have the ability to differentiate into cells including lung, liver, pancreas, stomach, and intestinal cells. The expression of HNF1-β and other markers of primitive gut can be evaluated by any method known by those skilled in the art, such as immunochemistry using anti-HNF1-β antibodies.

[0089]

[0122] The term "stem cell" as used herein can refer to an undifferentiated cell that can proliferate and give rise to many more progenitor cells that have the capacity to generate a large number of mother cells that can give rise to differentiated or differentiable daughter cells. The daughter cells themselves can be induced to proliferate and produce progeny that subsequently differentiate into one or more mature cell types while retaining one or more cells with parental developmental potential. The term "stem cell" can refer to a subset of progenitors that, under certain circumstances, have the capacity or potential to differentiate into a more specialized or more differentiated phenotype and, under certain circumstances, retain the capacity to proliferate without substantial differentiation. In one embodiment, the term stem cell refers, as a whole, to naturally occurring mother cells whose progeny (progeny) often specialize in different directions by differentiation, e.g., by acquiring entirely individual traits that arise in the gradual diversification of germ cells and tissues. Differentiation of cells is a complex process that typically occurs through many cell divisions. Differentiated cells can be obtained from pluripotent cells, which themselves are obtained from pluripotent cells, etc. Each of these pluripotent cells can be considered stem cells, although the range of cell types each can give rise to can vary widely. Some differentiated cells also have the ability to give rise to cells of higher developmental potential. Such ability may be natural or 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 necessary for "stemness". Self-renewal is another classical part of the definition of stem cells, and as used in this document, is essential. In theory, self-renewal can be achieved by either of two main mechanisms. Stem cells can divide asymmetrically, such that one daughter retains the stem cell state and the other 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 begin as stem cells may progress to a differentiated phenotype, but then "reverse" and re-express the stem cell phenotype, a term 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, and the like.

[0090]

[0123] 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 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, nude mouse teratoma formation assays. Pluripotency can also be demonstrated by the expression of embryonic stem (ES) cell markers, but 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 does not render them pluripotent by itself. Reprogrammed pluripotent cells (e.g., iPS cells, as the term is defined herein) are also characterized by the ability to be passaged for long periods of time without loss of growth potential, compared to primary cell parents that generally have the ability to divide only a limited number of times in culture.

[0091]

[0124] 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 reversal) from non-pluripotent cells, typically adult somatic cells, for example by inducing forced expression of one or more genes.

[0092]

[0125] 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.

[0093]

[0126] 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, with 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 animals" and "non-human mammals", as 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. However, advantageously, 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. "Patient in need thereof" or "subject in need thereof" as used herein means a patient diagnosed with or suspected of having a disease or disorder, e.g., but not limited to, diabetes.

[0094]

[0127] 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 by intravenous (iv), subcutaneous (sc), intradermal (id), intraperitoneal (ip), or intramuscular (im) injection. One or more such routes can be used. Parenteral administration can be by, for example, bolus injection or gradual perfusion over time. Alternatively, or simultaneously, administration can be by oral route. In addition, administration can also be by surgical deposition of a bolus or pellet of cells, or placement of a medical device. In an embodiment, the composition of the present disclosure can include 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 proliferation disorder. A pharmaceutical composition can include a cell population described herein in combination with one or more pharma- ceutical or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffers, such as neutral buffered saline, phosphate buffered saline, and the like; 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.

[0095]

[0128] The terms "treat," "treating," "treatment," and their grammatical equivalents, when applied to isolated cells, include subjecting the cells to any type of process or condition, or performing any type 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 diseased or in poor 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.

[0096]

[0129] As used herein, the terms "treating" and "treatment" can refer to administering an effective amount of a composition to a subject, such that the subject experiences a reduction in at least one symptom of the disease or an improvement in the disease, e.g., a beneficial or desired clinical outcome. For purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, alleviation of one or more symptoms, whether detectable or undetectable, a reduction 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 alleviation of the disease state, and remission (whether partial or complete). Treating can refer to extending survival time compared to the expected survival time in the absence of treatment. Thus, one of skill 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 prevention. Alternatively, treatment is "effective" if the progression of the disease is reduced or stopped. "Treatment" can also mean extending survival time compared to the expected survival time in the absence of treatment. Those in need of treatment include those already diagnosed with heart disease, as well as those who are likely to develop heart disease due to genetic susceptibility or other factors, such as weight, diet, and health status.

[0097]

[0130] The term "therapeutically effective amount", "therapeutic amount", or its grammatical equivalents, can refer to an amount effective at the dosage and duration required to achieve the desired therapeutic result. The therapeutically effective amount can vary depending on factors such as disease state, individual age, sex, and weight, and the ability of the compositions described herein to induce a desired response in one or more individual subjects. The exact amount of the composition of the present disclosure to be administered can be determined by a physician who takes into account the individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and condition.

[0098]

[0131] Alternatively, the pharmacological and / or physiological effect of administration of 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 condition 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).

[0099]

[0132] 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 numerical value includes both the number itself and 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.

[0100]

[0133] All of these different combinations are contemplated by the numerical values ​​disclosed throughout. All numerical values ​​disclosed, whether referring to administration of therapeutic agent or to days, months, years, body weight, dosage, etc., should be construed in this manner unless otherwise specifically indicated to the contrary.

[0101]

[0134] 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 numeric range]." The ranges may include any number of terms, including the numbers themselves (e.g., the endpoints of the ranges) as well as any other terms that may be used. 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.

[0102]

[0135] All of these different combinations are contemplated by the ranges disclosed throughout. All disclosed ranges, whether referring to administration of therapeutic agent or to days, months, years, body weight, dosage, etc., should be construed in this manner unless otherwise specifically indicated to the contrary.

[0103] Differentiation stages

[0136] 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., 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., 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., differentiation of cells expressing markers characteristic of gut cells into cells expressing markers characteristic of pancreatic progenitor 1 cells ("PP1") ("Stage 3 cells" or "S3 cells"). "Stage 4" refers to the fourth step, i.e., differentiation of cells expressing markers characteristic of pancreatic progenitor 1 cells to cells expressing markers characteristic of pancreatic progenitor 2 cells ("PP2") ("Stage 4 cells" or "S4 cells"). "Stage 5" refers to the fifth step, i.e., differentiation of cells expressing markers characteristic of pancreatic progenitor 2 cells (e.g., PDX.1 + , NKX6.1 + ) of cells expressing markers characteristic of pancreatic endoderm cells and / or pancreatic endocrine precursor cells (e.g., insulin +"Stage 5 cells" or "S5 cells") expressing markers characteristic of endocrine precursor cells ("EN"). "Stage 6" refers to the differentiation of cells expressing markers characteristic of pancreatic endocrine progenitor cells (e.g., insulin) 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 in the differentiation pathway than the majority of cells present in the population. For example, in some embodiments, SC-β cells can be identified during stage 5, at the end of stage 5, at the beginning of stage 6, etc. Examples of methods for generating any one of stages 1-6 cells are provided, for example, in U.S. Pat. No. 10,030,229, U.S. Pat. No. 10,443,042, U.S. Published Application No. 20200332262, and Pending U.S. Patent Application No. 17 / 010,346, each of which is incorporated by reference herein in its entirety.

[0104] Culture media and drugs Acetyl-CoA related metabolites

[0137] In some embodiments, the compositions (e.g., media) of the present disclosure include acetyl-CoA-related metabolites. Metabolism of acetyl-CoA can provide multiple metabolic functions, including energy production, lipid synthesis, and protein acetylation.

[0105]

[0138] In some embodiments, contacting cells with an acetyl-Coenzyme A-related metabolite (e.g., acetate) can result in improved in vitro generation of SC-β cells, e.g., higher cell yield and recovery, increased numbers and relative percentages of SC-β cells, enhanced stability and shelf life of SC-β cells, SC islet clusters with advantageous characteristics such as reduced size and increased uniformity, improved function of SC-β cells in vitro, improved cell survival after transplantation, improved cell function, reduced immunogenicity, or a combination thereof, e.g., compared to a composition lacking or containing a lower concentration of an acetyl-Coenzyme A-related metabolite.

[0106]

[0139] Exemplary acetyl-CoA-related metabolites include, but are not limited to, acetate, pyruvate, ketogenic amino acids, valine, leucine, isoleucine, phenylalanine, tyrosine, lysine, tryptophan, fatty acids, CoA, isovaleryl-CoA, and β-hydroxybutyrate.In some embodiments, the acetyl-CoA-related metabolite is acetate.In some embodiments, the composition of the present disclosure comprises two or more different acetyl-CoA-related metabolites, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different acetyl-CoA-related metabolites.In some embodiments, the acetyl-CoA-related metabolite is acetate.

[0107]

[0140] In some embodiments, an acetyl-CoA related metabolite is present in or added to a composition of the disclosure at a concentration of at least 0.1 nM, at least 1 nM, at least 10 nM, at least 50 nM, at least 80 nM, at least 100 nM, at least 120 nM, at least 140 nM, at least 150 nM, at least 200 nM, at least 300 nM, at least 500 nM, at least 800 nM, at least 1 μM, at least 10 μM, at least 100 μM, at least 500 μM, at least 800 μM, at least 900 μM, at least 1 mM, at least 2 mM, at least 3 mM, at least 5 mM, or at least 10 mM.

[0108]

[0141] In some embodiments, acetyl-CoA related metabolites are present in or added to a composition of the disclosure at a concentration of up to 200 nM, up to 300 nM, up to 500 nM, up to 800 nM, up to 1 μM, up to 10 μM, up to 100 μM, up to 500 μM, up to 800 μM, up to 900 μM, up to 1 mM, up to 2 mM, up to 3 mM, up to 5 mM, or up to 10 mM.

[0109]

[0142] In some embodiments, an acetyl-CoA related metabolite is present in or added to a composition of the disclosure at a concentration of about 10 nM, about 50 nM, about 80 nM, about 100 nM, about 120 nM, about 140 nM, about 150 nM, about 200 nM, about 300 nM, about 500 nM, about 800 nM, about 1 μM, about 10 μM, about 100 μM, about 500 μM, about 800 μM, about 900 μM, about 1 mM, about 2 mM, about 3 mM, about 5 mM, or about 10 mM.

[0110]

[0143] In some embodiments, the acetyl-CoA related metabolite is present or added to the compositions of the present disclosure at a concentration of about 0.01-50 mM, 0.1-50 mM, 0.5-50 mM, 0.01-20 mM, 0.1-20 mM, 0.5-20 mM, 0.01-10 mM, 0.1-10 mM, 0.5-10 mM, 0.8-25 mM, 0.8-10 mM, 0.8-5 mM, 0.8-2 mM, 0.8-1.5 mM, 0.8-1.2 mM, 0.9-1.1 mM, or 0.95-1.05 mM. In some embodiments, the acetyl-CoA related metabolite is acetate, which is present at a concentration of about 1 mM.

[0111]

[0144] In some embodiments, the acetyl-CoA related metabolite is acetate present at a concentration of about 50-1000 nM, 50-800 nM, 50-500 nM, 50-300 nM, 50-250 nM, 100-200 nM, or 125-175 nM. In some embodiments, the acetyl-CoA related metabolite is acetate present at a concentration of about 160 nM.

[0112]

[0145] In some embodiments, the cells are contacted with an acetyl-CoA-related metabolite (e.g., acetate) in the methods of the present disclosure. The cells can be PDX1 positive, NKX6.1 positive, insulin negative cells. The cells can be insulin positive endocrine precursor cells. The cells can be SC-β cells. In some embodiments, the cells are frozen. In some embodiments, the cells have been previously frozen. In some embodiments, the cells are in cell clusters. In some embodiments, the cells are dissociated.

[0113]

[0146] In some embodiments, cells (e.g., PDX1 positive, NKX6.1 positive, insulin negative cells, or insulin positive endocrine precursor cells) are contacted with an acetyl-CoA related metabolite (e.g., acetate) for about 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 84 hours, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days.

[0114]

[0147] In some embodiments, cells (e.g., PDX1 positive, NKX6.1 positive, insulin negative cells, or insulin positive endocrine precursor cells) are contacted with an acetyl-CoA related metabolite (e.g., acetate) for at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, at least 72 hours, at least 84 hours, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days.

[0115]

[0148] In some embodiments, cells (e.g., PDX1 positive, NKX6.1 positive, insulin negative cells, or insulin positive endocrine precursor cells) are contacted with an acetyl-CoA related metabolite (e.g., acetate) for up to 12 hours, up to 24 hours, up to 36 hours, up to 48 hours, up to 60 hours, up to 72 hours, up to 84 hours, up to 4 days, up to 5 days, up to 6 days, up to 7 days, up to 8 days, up to 9 days, up to 10 days, up to 11 days, up to 12 days, up to 13 days, up to, or up to 14 days.

[0116]

[0149] In some embodiments, cells (e.g., PDX1 positive, NKX6.1 positive, insulin negative cells, or insulin positive endocrine precursor cells) are contacted with an acetyl-CoA related metabolite (e.g., acetate) 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, 4-8 days, 4-7 days, 4-6 days, or 4-5 days.

[0117]

[0150] In some embodiments, a composition or method of the disclosure does not include acetyl-CoA related metabolites (eg, does not include acetate).

[0118] vitamin

[0151] In some embodiments, the compositions (e.g., media) of the present disclosure include one or more vitamins. In some embodiments, contacting cells with a vitamin (e.g., biotin) can result in improved in vitro generation of SC-β cells, e.g., higher cell yield and recovery, increased number and relative percentage of SC-β cells, enhanced stability and shelf life of SC-β cells, SC islet clusters with advantageous characteristics such as reduced size and increased uniformity, improved function of SC-β cells in vitro, improved cell survival after transplantation, improved cell function, reduced immunogenicity, or a combination thereof, e.g., compared to a composition that does not contain the vitamin or contains it at a lower concentration.

[0119]

[0152] Exemplary vitamins include, but are not limited to, biotin, vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B6 (pyridoxine), and vitamin B12 (cyanocobalamin). In some embodiments, the vitamin regulates the synthesis of fatty acids. In some embodiments, the vitamin regulates the metabolism of branched chain amino acids. In some embodiments, the vitamin regulates or participates as a cofactor in the TCA cycle, e.g., as a cofactor for pyruvate carboxylase. In some embodiments, the vitamin is biotin. In some embodiments, the compositions of the present disclosure include two or more different vitamins, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different vitamins.

[0120]

[0153] In some embodiments, vitamins are present in or added to compositions of the present disclosure at a concentration of at least 0.1 nM, at least 1 nM, at least 10 nM, at least 50 nM, at least 80 nM, at least 100 nM, at least 120 nM, at least 140 nM, at least 150 nM, at least 200 nM, at least 300 nM, at least 500 nM, at least 600 nM, at least 700 nM, at least 800 nM, at least 900 nM, at least 1 μM, at least 10 μM, at least 100 μM, at least 500 μM, at least 800 μM, at least 900 μM, at least 1 mM, at least 2 mM, at least 3 mM, at least 5 mM, or at least 10 mM.

[0121]

[0154] In some embodiments, vitamins are present in or added to a composition of the disclosure at a concentration of up to 200 nM, up to 300 nM, up to 500 nM, up to 600 nM, up to 700 nM, up to 800 nM, up to 900 nM, up to 1 μM, up to 1.5 μM, up to 3 μM, up to 5 μM, up to 10 μM, up to 100 μM, up to 500 μM, up to 800 μM, up to 1 mM, or up to 10 mM.

[0122]

[0155] In some embodiments, the vitamin is present or added to the composition of the present disclosure at a concentration of about 100 nM, about 300 nM, about 500 nM, about 600 nM, about 700 nM, about 800 nM, about 900 nM, about 1 μM, about 1.5 μM, about 3 μM, about 5 μM, about 10 μM, or about 100 μM. In some embodiments, the vitamin is biotin, which is present at a concentration of about 800 nM.

[0123]

[0156] In some embodiments, the vitamin is at about 1 nM to 500 μM, 1 nM to 100 μM, 1 nM to 10 μM, 1 nM to 1 μM, 1 nM to 800 nM, 1 nM to 600 nM, 1 nM to 400 nM, 1 nM to 300 nM, 1 nM to 200 nM, 25 nM to 500 μM, 25 nM to 100 μM, 25 nM to 10 μM, 25 nM to 1 μM, 25 nM to 800 nM, 25 nM to 600 nM, 25 nM to 400 nM, 25 nM to 300 nM, 25 nM to 200 nM, 50 nM to 500 μM, 50 nM

[0043] The riboflavin may be present in or added to a composition of the present disclosure at a concentration of from 100 μM, 50 nM to 10 μM, 50 nM to 1 μM, 50 nM to 800 nM, 50 nM to 600 nM, 50 nM to 400 nM, 50 nM to 300 nM, 50 nM to 200 nM, 100 nM to 500 μM, 100 nM to 100 μM, 100 nM to 10 μM, 100 nM to 1 μM, 100 nM to 800 nM, 100 nM to 600 nM, 100 nM to 400 nM, 100 nM to 300 nM, or 100 nM to 200 nM.

[0124]

[0157] In some embodiments, the cells are contacted with a vitamin (e.g., biotin) in the methods of the present disclosure. The cells can be PDX1 positive, NKX6.1 positive, insulin negative cells. The cells can be insulin positive endocrine precursor cells. The cells can be SC-β cells. In some embodiments, the cells are frozen. In some embodiments, the cells have been previously frozen. In some embodiments, the cells are in cell clusters. In some embodiments, the cells are dissociated.

[0125]

[0158] In some embodiments, cells (e.g., PDX1 positive, NKX6.1 positive, insulin negative cells, or insulin positive endocrine precursor cells) are contacted with a vitamin (e.g., biotin) for about 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 84 hours, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days.

[0126]

[0159] In some embodiments, cells (e.g., PDX1 positive, NKX6.1 positive, insulin negative cells, or insulin positive endocrine precursor cells) are contacted with a vitamin (e.g., biotin) for at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, at least 72 hours, at least 84 hours, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least or at least 14 days.

[0127]

[0160] In some embodiments, cells (e.g., PDX1 positive, NKX6.1 positive, insulin negative cells, or insulin positive endocrine precursor cells) are contacted with a vitamin (e.g., biotin) for up to 12 hours, up to 24 hours, up to 36 hours, up to 48 hours, up to 60 hours, up to 72 hours, up to 84 hours, up to 4 days, up to 5 days, up to 6 days, up to 7 days, up to 8 days, up to 9 days, up to 10 days, up to 11 days, up to 12 days, up to 13 days, up to 14 days.

[0128]

[0161] In some embodiments, cells (e.g., PDX1 positive, NKX6.1 positive, insulin negative cells, or insulin positive endocrine precursor cells) are contacted with a vitamin (e.g., biotin) 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, 4-8 days, 4-7 days, 4-6 days, or 4-5 days.

[0129]

[0162] In some embodiments, a composition or method of the present disclosure does not include a vitamin (eg, does not include biotin).

[0130] Histone deacetylase inhibitors (HDACi)

[0163] In some embodiments, the compositions (e.g., media) of the present disclosure comprise a histone deacetylase inhibitor (HDACi), a class of compounds that increase the acetylation of lysine residues in histone proteins and other non-histone proteins by inhibiting the activity of HDAC enzymes.

[0131]

[0164] In some embodiments, contacting cells with an HDACi can result in improved in vitro generation of SC-β cells, e.g., higher cell yield and recovery, increased numbers and relative percentages of SC-β cells, enhanced stability and shelf life of SC-β cells, SC islet clusters with advantageous characteristics such as reduced size and increased uniformity, improved function of SC-β cells in vitro, improved cell survival after transplantation, improved cell function, reduced immunogenicity, or a combination thereof, e.g., compared to compositions lacking or containing a lower concentration of HDACi.

[0132]

[0165] Exemplary histone deacetylase inhibitors (HDACi) include β-hydroxybutyrate, butyrate, class I HDACi, class IIA HDACi, class IIB HDACi, class III HDACi, class IV HDACi, HDACi, HDAC-1, HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC-6, HDAC-7, HDAC-8, HDAC-9, HDAC-10, HDAC-11, Sirtuin, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, Vorinostat (Suberoylanilide hydroxamic acid, SAHA, MK0683), Entinostat (MS-275, SNDX-275), Panobinostat (LBH589, NVP-LBH589), Trichostatin A (TSA), Mocetinostat (MGCD0103, MG0103), GSK3117391 (GSK3117391A, HDAC-IN-3), BRD3308, BRD3308, Tubastatin A TFA (Tubastatin A trifluoroacetate), Tubastatin A, SIS17, NKL 22, BML-210 (CAY10433), TC-H 106, SR-4370, Belinostat (PXD101, NSC726630, PX-105684), Romidepsin (FK228, Depsipeptide, FR 901228, NSC 630176), MC1568, Gibinostat (ITF2357), Dacinostat (LAQ824, NVP-LAQ824), CUDC-101, Xinostat (JNJ-26481585), Prasinostat (SB939), PCI-34051, Droxinostat (NS41080), abexinostat (PCI-24781), abexinostat (PCI-24781, CRA-024781), RGFP966, AR-42 (HDAC-42), ricolinostat (ACY-1215, rosilinostat), valproic acid sodium salt (sodium valproate), tacedinaline (CI994, PD-123654, GOE-5549, acetyldinaline), fimepinostat (CUDC-907), sodium butyrate (NaB), curcumin, diferuloylmethane, M344, tubacin, RG2833 (RGFP109), RG2833 (RGFP109), resminostat (RAS2410), divalproex sodium, scriptaid (GCK 1026), sodium phenylbutyrate, sinapic acid (sinapic acid), TMP269, santacruzamate A (CAY10683), TMP195 (TFMO 2), valproic acid (VPA), UF010, tasquinimod (ABR-215050), SKLB-23bb, isoguanosine, sulforaphane, BRD73954, citalinostat (ACY-241, HDAC-IN-2), suberohydroxamic acid, splitomycin, HPOB, LMK-235, biphenyl-4-sulfonyl chloride (p-phenylbenzenesulfonyl, 4-phenylbenzene sulfonyl, p-biphenylsulfonyl), nextulastat A, TH34, tushidinostat (chidamide, HBI-8000, CS-055), (-)-parthenolide, WT161, CAY10603, CAY10603, ACY-738, radeanin A, tinostamustin (EDO-S101), domatinostat (4SC-202), and BG45.

[0133]

[0166] In some embodiments, the HDACi is β-hydroxybutyrate. β-hydroxybutyrate is a ketone body that, together with butyrate, is an agonist of hydroxycarboxylic acid receptor 2 (HCA2), a Gi / o-coupled GPCR. In some embodiments, the HDACi inhibitor is an agonist of hydroxycarboxylic acid receptor 2.

[0134]

[0167] In some embodiments, a composition of the disclosure comprises two or more different HDACi's, for example 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different HDACi's.

[0135]

[0168] In some embodiments, the HDACi is present in or added to a composition of the disclosure at a concentration of at least 0.1 nM, at least 1 nM, at least 10 nM, at least 50 nM, at least 80 nM, at least 100 nM, at least 120 nM, at least 140 nM, at least 150 nM, at least 200 nM, at least 300 nM, at least 500 nM, at least 600 nM, at least 700 nM, at least 800 nM, at least 900 nM, at least 1 μM, at least 1.2 μM, at least 1.5 μM, at least 1.8 μM, at least 2 μM, at least 3 μM, at least 5 μM, at least 10 μM, at least 100 μM, at least 500 μM, at least 800 μM, at least 900 μM, at least 1 mM, at least 2 mM, at least 3 mM, at least 5 mM, or at least 10 mM.

[0136]

[0169] In some embodiments, HDACi's are present in or added to compositions of the disclosure at a concentration of up to 150 nM, up to 200 nM, up to 300 nM, up to 500 nM, up to 600 nM, up to 700 nM, up to 800 nM, up to 900 nM, up to 1 μM, up to 1.2 μM, up to 1.5 μM, up to 1.8 μM, up to 2 μM, up to 3 μM, up to 5 μM, up to 10 μM, up to 100 μM, up to 500 μM, up to 800 μM, up to 1 mM, or up to 10 mM.

[0137]

[0170] In some embodiments, HDACi is present or added in the composition of the present disclosure at a concentration of about 100nM, about 300nM, about 500nM, about 600nM, about 700nM, about 800nM, about 900nM, about 1μM, about 1.5μM, about 3μM, about 5μM, about 10μM or about 100μM.In some embodiments, HDACi is beta-hydroxybutyrate present at a concentration of about 200nM.

[0138]

[0171] In some embodiments, the HDACi is from about 1 nM to 500 μM, 1 nM to 100 μM, 1 nM to 10 μM, 1 nM to 1 μM, 1 nM to 800 nM, 1 nM to 600 nM, 1 nM to 400 nM, 1 nM to 300 nM, 1 nM to 200 nM, 25 nM to 500 μM, 25 nM to 100 μM, 25 nM to 10 μM, 25 nM to 1 μM, 25 nM to 800 nM, 25 nM to 600 nM, 25 nM to 400 nM, 25 nM to 300 nM, 25 nM to 200 nM, 50 nM to 500 μM, 50 nM to 50 nM In some embodiments, the agonist is present in or added to a composition of the present disclosure at a concentration of from 100 nM to 10 μM, 50 nM to 10 μM, 50 nM to 1 μM, 50 nM to 800 nM, 50 nM to 600 nM, 50 nM to 400 nM, 50 nM to 300 nM, 50 nM to 200 nM, 100 nM to 500 μM, 100 nM to 100 μM, 100 nM to 10 μM, 100 nM to 1 μM, 100 nM to 800 nM, 100 nM to 600 nM, 100 nM to 400 nM, 100 nM to 300 nM, or 100 nM to 200 nM.

[0139]

[0172] In some embodiments, the cells are contacted with HDACi (e.g., β-hydroxybutyrate) in the methods of the present disclosure. The cells can be PDX1 positive, NKX6.1 positive, insulin negative cells. The cells can be insulin positive endocrine precursor cells. The cells can be SC-β cells. In some embodiments, the cells are frozen. In some embodiments, the cells have been previously frozen. In some embodiments, the cells are in cell clusters. In some embodiments, the cells are dissociated.

[0140]

[0173] In some embodiments, cells (e.g., PDX1 positive, NKX6.1 positive, insulin negative cells, or insulin positive endocrine precursor cells) are contacted with an HDACi (e.g., β-hydroxybutyrate) for about 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 84 hours, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days.

[0141]

[0174] In some embodiments, cells (e.g., PDX1 positive, NKX6.1 positive, insulin negative cells, or insulin positive endocrine precursor cells) are contacted with an HDACi (e.g., β-hydroxybutyrate) for at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, at least 72 hours, at least 84 hours, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least or at least 14 days.

[0142]

[0175] In some embodiments, cells (e.g., PDX1 positive, NKX6.1 positive, insulin negative cells, or insulin positive endocrine precursor cells) are contacted with an HDACi (e.g., β-hydroxybutyrate) for up to 12 hours, up to 24 hours, up to 36 hours, up to 48 hours, up to 60 hours, up to 72 hours, up to 84 hours, up to 4 days, up to 5 days, up to 6 days, up to 7 days, up to 8 days, up to 9 days, up to 10 days, up to 11 days, up to 12 days, up to 13 days, up to 14 days.

[0143]

[0176] In some embodiments, cells (e.g., PDX1 positive, NKX6.1 positive, insulin negative cells, or insulin positive endocrine precursor cells) are contacted with an HDACi (e.g., β-hydroxybutyrate) 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, 4-8 days, 4-7 days, 4-6 days, or 4-5 days.

[0144]

[0177] In some embodiments, a composition or method of the disclosure does not include an HDACi (eg, does not include β-hydroxybutyrate).

[0145] Redox homeostasis regulators

[0178] In some embodiments, the compositions (e.g., media) of the present disclosure include redox homeostasis regulators. Redox homeostasis inhibitors can, for example, improve viability and cell function by minimizing oxidative damage to cells, or promote proper oxidation of substrates as part of a biological process.

[0146]

[0179] In some embodiments, contacting the cells with a redox homeostasis regulator (e.g., taurine) can result in improved in vitro generation of SC-β cells, e.g., higher cell yield and recovery, increased number and relative percentage of SC-β cells, enhanced stability and shelf life of SC-β cells, SC islet clusters with advantageous characteristics such as reduced size and increased uniformity, improved function of SC-β cells in vitro, improved cell survival after transplantation, improved cell function, reduced immunogenicity, or a combination thereof, e.g., compared to a composition lacking or comprising a lower concentration of the redox homeostasis regulator.

[0147]

[0180] Exemplary redox homeostasis regulators include, but are not limited to, taurine, respiratory chain regulators, free radical scavengers, regulators of mitochondrial protein synthesis, allium sulfur compounds, anthocyanins, beta-carotene, catechins, copper, cryptoxanthin, flavonoids, indoles, isoflavonoids, lignans, lutein, lycopene, alpha lipoic acid, ellagic acid, manganese, polyphenols, selenium, glutathione, vitamin A, vitamin C, vitamin E, zinc, superoxide dismutase, GSHPx, Prx-I, catalase, and coenzyme Q10.

[0148]

[0181] In some embodiments, the redox homeostasis regulator is taurine.

[0182] Taurine is a non-proteinogenic β-aminosulfonic acid that can be derived from the metabolism of methionine and cysteine. In some embodiments, taurine can inhibit ROS generation in the respiratory chain.

[0149]

[0183] In some embodiments, compositions of the present disclosure comprise two or more different redox homeostasis regulators, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different redox homeostasis regulators.

[0150]

[0184] In some embodiments, the redox homeostasis regulator is present in or added to a composition of the present disclosure at a concentration of at least 100 nM, at least 500 nM, 1 μM, at least 10 μM, at least 20 μM, at least 30 μM, at least 40 μM, at least 50 μM, at least 60 μM, at least 70 μM, at least 80 μM, at least 90 μM, at least 100 μM, at least 110 μM, at least 110 μM, at least 150 μM, at least 200 μM, or at least 500 μM.

[0151]

[0185] In some embodiments, the redox homeostasis regulator is present in or added to a composition of the present disclosure at a concentration of up to 10 μM, up to 20 μM, up to 30 μM, up to 40 μM, up to 50 μM, up to 60 μM, up to 70 μM, up to 80 μM, up to 90 μM, up to 100 μM, up to 110 μM, up to 110 μM, up to 150 μM, up to 200 μM, or up to 500 μM.

[0152]

[0186] In some embodiments, the redox homeostasis regulator is present or added to the composition of the present disclosure at a concentration of about 100nM, about 500nM, 1μM, about 10μM, about 20μM, about 30μM, about 40μM, about 50μM, about 60μM, about 70μM, about 80μM, about 90μM, about 100μM, about 110μM, about 110μM, about 150μM or about 200μM.In some embodiments, the redox homeostasis regulator is taurine.In some embodiments, the redox homeostasis regulator is taurine present at a concentration of about 90μM.

[0153]

[0187] In some embodiments, the redox homeostasis regulator intermediate is from about 100 nM to 1 mM, 500 nM to 1 mM, 1 μM to 1 mM, 10 μM to 1 mM, 20 μM to 1 mM, 30 μM to 1 mM, 30 μM to 1 mM, 40 μM to 1 mM, 50 μM to 1 mM, 60 μM to 1 mM, 70 μM to 1 mM, 80 μM to 1 mM, 100 nM to 250 μM, 500 nM to 250 μM, 1 μM to 250 μM, 10 μM to 250 μM, 20 μM to 250 μM, 30 μM to 250 μM, The compound may be present or added at a concentration of 100 nM to 100 μM, 30 μM to 250 μM, 40 μM to 250 μM, 50 μM to 250 μM, 60 μM to 250 μM, 70 μM to 250 μM, 100 nM to 100 μM, 500 nM to 100 μM, 1 μM to 100 μM, 10 μM to 100 μM, 20 μM to 100 μM, 30 μM to 100 μM, 40 μM to 100 μM, 50 μM to 100 μM, 60 μM to 100 μM, 70 μM to 100 μM, or 80 μM to 100 μM.

[0154]

[0188] In some embodiments, the cells are contacted with a redox homeostasis regulator (e.g., taurine) in the methods of the present disclosure. The cells can be PDX1 positive, NKX6.1 positive, insulin negative cells. The cells can be insulin positive endocrine precursor cells. The cells can be SC-β cells. In some embodiments, the cells are frozen. In some embodiments, the cells have been previously frozen. In some embodiments, the cells are in cell clusters. In some embodiments, the cells are dissociated.

[0155]

[0189] In some embodiments, cells (e.g., PDX1 positive, NKX6.1 positive, insulin negative cells, or insulin positive endocrine precursor cells) are contacted with a redox homeostasis regulator (e.g., taurine) for about 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 84 hours, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days.

[0156]

[0190] In some embodiments, cells (e.g., PDX1 positive, NKX6.1 positive, insulin negative cells, or insulin positive endocrine precursor cells) are contacted with a redox homeostasis regulator (e.g., taurine) for at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, at least 72 hours, at least 84 hours, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least or at least 14 days.

[0157]

[0191] In some embodiments, cells (e.g., PDX1 positive, NKX6.1 positive, insulin negative cells, or insulin positive endocrine precursor cells) are contacted with a redox homeostasis regulator (e.g., taurine) for up to 12 hours, up to 24 hours, up to 36 hours, up to 48 hours, up to 60 hours, up to 72 hours, up to 84 hours, up to 4 days, up to 5 days, up to 6 days, up to 7 days, up to 8 days, up to 9 days, up to 10 days, up to 11 days, up to 12 days, up to 13 days, up to, or up to 14 days.

[0158]

[0192] In some embodiments, cells (e.g., PDX1 positive, NKX6.1 positive, insulin negative cells, or insulin positive endocrine precursor cells) are contacted with a redox homeostasis regulator (e.g., taurine) 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, 4-8 days, 4-7 days, 4-6 days, or 4-5 days.

[0159]

[0193] In some embodiments, a composition or method of the present disclosure does not include a redox homeostasis regulator (eg, does not include taurine).

[0160] One-carbon metabolic pathway intermediates

[0194] In some embodiments, the compositions (e.g., media) of the present disclosure include one-carbon metabolic pathway intermediates. One-carbon metabolism mediated by folate cofactors supports multiple physiological processes, including amino acid homeostasis (methionine, glycine, and serine), nucleotide (purine, thymidine) biosynthesis, epigenetic maintenance, and redox protection.

[0161]

[0195] In some embodiments, contacting cells with a one-carbon metabolic pathway intermediate can result in improved in vitro production of SC-β cells, e.g., higher cell yield and recovery, increased numbers and relative percentages of SC-β cells, enhanced stability and shelf life of SC-β cells, SC islet clusters with advantageous characteristics such as reduced size and increased uniformity, improved function of SC-β cells in vitro, improved cell survival after transplantation, improved cell function, reduced immunogenicity, or a combination thereof, e.g., compared to compositions lacking or containing a lower concentration of the one-carbon metabolic pathway intermediate.

[0162]

[0196] Exemplary one-carbon metabolic pathway intermediates include, but are not limited to, formate, tetrahydrofolate (THF), 10-formylTHF, 5,10-meTHF, 5,10-meTHF, and 10-formylTHF.

[0163]

[0197] In some embodiments, a composition of the disclosure comprises two or more different one-carbon metabolic pathway intermediates, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different one-carbon metabolic pathway intermediates.

[0164]

[0198] In some embodiments, a one-carbon metabolic pathway intermediate is present in or added to a composition of the disclosure at a concentration of at least 100 nM, at least 500 nM, at least 1 μM, at least 10 μM, at least 20 μM, at least 30 μM, at least 40 μM, at least 50 μM, at least 75 μM, or at least 100 μM.

[0165]

[0199] In some embodiments, one-carbon metabolic pathway intermediates are present in or added to compositions of the disclosure at a concentration of up to 10 μM, up to 20 μM, up to 30 μM, up to 40 μM, up to 50 μM, up to 75 μM, up to 100 μM, up to 200 μM, up to 300 μM, up to 400 μM, up to 500 μM, up to 1 mM, or up to 10 mM.

[0166]

[0200] In some embodiments, a one-carbon metabolic pathway intermediate is present in or added to a composition of the disclosure at a concentration of about 100 nM, about 500 nM, 1 μM, about 10 μM, about 20 μM, about 30 μM, about 40 μM, about 50 μM, about 75 μM, or about 100 μM.

[0167]

[0201] In some embodiments, the one-carbon metabolic pathway intermediate is formate present at a concentration of about 50 μM.

[0168]

[0202] In some embodiments, the one-carbon metabolic pathway intermediate is at about 100 nM to 1 mM, 500 nM to 1 mM, 1 μM to 1 mM, 10 μM to 1 mM, 20 μM to 1 mM, 30 μM to 1 mM, 100 nM to 250 μM, 500 nM to 250 μM, 1 μM to 250 μM, 10 μM to 250 μM, 20 μM to 250 μM, 30 μM to 250 μM, 100 nM to 100 μM, The compound may be present or added at a concentration of 500 nM to 100 μM, 1 μM to 100 μM, 10 μM to 100 μM, 20 μM to 100 μM, 30 μM to 100 μM, 100 nM to 60 μM, 500 nM to 60 μM, 1 μM to 60 μM, 10 μM to 60 μM, 20 μM to 60 μM, 30 μM to 60 μM, 40 μM to 60 μM, or 45 μM to 55 μM.

[0169]

[0203] In some embodiments, the cells are contacted with a one-carbon metabolic pathway intermediate (e.g., formate) in the methods of the disclosure. The cells can be PDX1 positive, NKX6.1 positive, insulin negative cells. The cells can be insulin positive endocrine precursor cells. The cells can be SC-β cells. In some embodiments, the cells are frozen. In some embodiments, the cells have been previously frozen. In some embodiments, the cells are in cell clusters. In some embodiments, the cells are dissociated.

[0170]

[0204] In some embodiments, cells (e.g., PDX1 positive, NKX6.1 positive, insulin negative cells, or insulin positive endocrine precursor cells) are contacted with a one-carbon metabolic pathway intermediate (e.g., formate) for about 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 84 hours, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days.

[0171]

[0205] In some embodiments, cells (e.g., PDX1 positive, NKX6.1 positive, insulin negative cells, or insulin positive endocrine precursor cells) are contacted with a one-carbon metabolic pathway intermediate (e.g., formate) for at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, at least 72 hours, at least 84 hours, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least or at least 14 days.

[0172]

[0206] In some embodiments, cells (e.g., PDX1 positive, NKX6.1 positive, insulin negative cells, or insulin positive endocrine precursor cells) are contacted with a one-carbon metabolic pathway intermediate (e.g., formate) for up to 12 hours, up to 24 hours, up to 36 hours, up to 48 hours, up to 60 hours, up to 72 hours, up to 84 hours, up to 4 days, up to 5 days, up to 6 days, up to 7 days, up to 8 days, up to 9 days, up to 10 days, up to 11 days, up to 12 days, up to 13 days, up to, or up to 14 days.

[0173]

[0207] In some embodiments, cells (e.g., PDX1 positive, NKX6.1 positive, insulin negative cells, or insulin positive endocrine precursor cells) are contacted with a one-carbon metabolic pathway intermediate (e.g., formate) 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, 4-8 days, 4-7 days, 4-6 days, or 4-5 days.

[0174]

[0208] In some embodiments, a composition or method of the disclosure does not include a one-carbon metabolic pathway intermediate (e.g., does not include formate).

[0175] glutamine

[0209] In some embodiments, a composition (eg, medium) of the present disclosure comprises glutamine.

[0176]

[0210] In some embodiments, contacting cells with glutamine as disclosed herein can result in improved in vitro generation of SC-β cells, e.g., higher cell yield and recovery, increased numbers and relative percentages of SC-β cells, enhanced stability and shelf-life of SC-β cells, SC islet clusters with advantageous characteristics such as reduced size and increased uniformity, improved function of SC-β cells in vitro, improved cell survival after transplantation, improved cell function, reduced immunogenicity, or a combination thereof, e.g., compared to compositions that lack glutamine, contain less glutamine, or contain a different (e.g., less bioavailable) form of glutamine.

[0177]

[0211] Glutamine (Gln or Q) is an alpha-amino acid. Glutamine may be an essential amino acid in in vitro cell cultures. Glutamine supports the growth of cells, including those with high energy demands and that synthesize large amounts of protein and nucleic acid. Glutamine is an alternative energy source for rapidly dividing cells and cells that use glucose inefficiently.

[0178]

[0212] In some embodiments, the compositions and methods of the present disclosure utilize forms of glutamine with increased bioavailability. Because L-glutamine is chemically unstable and important for cell growth and function, it is important to tailor its delivery to each unique cell culture process. Free glutamine (e.g., L-glutamine) is unstable in liquid media at physiological pH and can degrade into ammonium and pyroglutamate at rates that are problematic in many cell culture and biomanufacturing applications. Therefore, many cell culture media contain stabilized forms of glutamine, including dipeptide forms such as alanyl-l-glutamine and glycyl-l-glutamine. However, these more stable forms of L-glutamine may have limited bioavailability due to the requirement for processing by enzymes such as cell surface peptidases. Thus, in some embodiments, the compositions and methods of the present disclosure utilize glutamine in a form that has increased bioavailability, such as free glutamine, such as non-dipeptide, non-alanine-glutamine dipeptide (e.g., non-alanyl-l-glutamine), non-glycine-glutamine dipeptide (e.g., non-glycyl-l-glutamine), where glutamine is not conjugated to another amino acid or stabilizing moiety, monomeric, free, or combinations thereof. In some embodiments, glutamine is provided as a protein hydrolysate.

[0179]

[0213] In some embodiments, the basal medium contains glutamine. In some embodiments, glutamine in a form as disclosed herein is added to a medium that already contains glutamine. In some embodiments, glutamine in a form as disclosed herein is added to a basal medium that does not contain glutamine or that contains only low levels of glutamine to increase the bioavailability of glutamine.

[0180]

[0214] Glutamine can contribute to multiple cellular metabolic processes important to the embodiments of the present disclosure, such as redox regulation, NAD recycling, TCA cycle replenishing reactions, and nitrogen metabolism. Glutamine contains one nitrogen atom as an amide and another as an amine, and transports and delivers nitrogen to cells in toxic amounts as free ammonium. Glutamine amide nitrogen can be used for the synthesis of NAD and NADP, purine nucleotides, CTP from UTP, and asparagine. Nitrogen initially stored as glutamine can also be used to generate carbamyl phosphate for the synthesis of pyrimidines. Glutamine is the precursor of glutamate, the major amino acid used in the transamination of alpha keto acids to form other alpha amino acids. When glucose levels are low and energy demands are high, cells can metabolize amino acids for energy. Glutamine is one of the most readily available amino acids used as an energy source and is the primary energy source for many rapidly dividing cell types in vitro.

[0181]

[0215] Cells require nitrogen atoms to build molecules such as nucleotides, amino acids, amino-sugars, and vitamins. Ammonium is an inorganic nitrogen source that exists primarily as the positively charged cation NH4+ at physiological pH. The ammonium nitrogen used by cells is initially incorporated into organic nitrogen as the amine of glutamate or the amide of glutamine. These two amino acids provide the primary reservoir of nitrogen for the synthesis of proteins, nucleic acids, and other nitrogenous compounds. The reactions that fix nitrogen into glutamate and glutamine consume equal amounts of energy. Glutamate can be synthesized from ammonium and alpha-ketoglutarate, a tricarboxylic acid (TCA) cycle intermediate. Its synthesis may require the oxidation of either NADH or NADPH. Glutamine is formed from ammonium and glutamate, and its synthesis consumes ATP. The enzymes involved in glutamate synthesis, glutamate dehydrogenase (EC 1.4.1.4) and glutamate synthase (EC 1.4.1.13), are reversible. Glutamine synthetase (EC 6.3.1.2), the enzyme responsible for glutamine synthesis, is highly regulated to limit glutamine production to cellular requirements. Catabolism of glutamine to glutamate and ammonium is mediated by a mitochondrial enzyme called glutaminase (EC 3.5.1.2). Ammonium produced in vivo can be metabolized to urea. In vitro, ammonium is not metabolized to urea. Under some in vitro conditions, ammonia accumulates in the extracellular medium as ammonium ions.

[0182]

[0216] Cellular glutamine demand can be increased by stress conditions. In some embodiments, glutamine is a conditionally essential amino acid, e.g., must be provided at sufficient levels in stress conditions. Like other amino acids, glutamine is biochemically important as a building block of proteins. Glutamine can also be crucial in nitrogen metabolism. Ammonia (formed by nitrogen fixation) can be assimilated into organic compounds by converting glutamic acid to glutamine. The enzyme that does this is called glutamine synthetase. Glutamine can be used as a nitrogen donor in the biosynthesis of many compounds, including other amino acids, purines, and pyrimidines. L-glutamine can improve nicotinamide adenine dinucleotide (NAD) redox potential.

[0183]

[0217] In some embodiments, glutamine may be present in a composition of the present disclosure at a concentration of at least 100 μM, at least 250 μM, at least 500 μM, at least 750 μM, at least 1 mM, at least 1.5 mM, at least 2 mM, at least 2.5 mM, at least 2.6 mM, at least 2.7 mM, at least 2.8 mM, at least 2.9 mM, at least 3 mM, at least 3.1 mM, at least 3.2 mM, at least 3.3 mM, at least 3.4 mM, at least 3.5 mM, at least 3.6 mM, at least 3.7 mM, at least 3.8 mM, at least 3.9 mM, at least 4 mM, or at least 5 mM.

[0184]

[0218] In some embodiments, glutamine may be present in a composition of the present disclosure at a concentration of up to 2 mM, up to 3 mM, up to 3.1 mM, up to 3.2 mM, up to 3.3 mM, up to 3.4 mM, up to 3.5 mM, up to 3.6 mM, up to 3.7 mM, up to 3.8 mM, up to 3.9 mM, up to 4 mM, up to 4.1 mM, up to 4.2 mM, up to 4.2 mM, up to 4.4 mM, up to 4.5 mM, up to 4.75 mM, up to 5 mM, up to 6 mM, up to 7 mM, up to 8 mM, up to 9 mM, up to 10 mM, up to 15 mM, up to 20 mM, up to 30 mM, up to 40 mM, or up to 50 mM.

[0185]

[0219] In some embodiments, glutamine may be present in a composition of the present disclosure at a concentration of about 100 μM, about 250 μM, about 500 μM, about 750 μM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 2.6 mM, about 2.7 mM, about 2.8 mM, about 2.9 mM, about 3 mM, about 3.1 mM, about 3.2 mM, about 3.3 mM, about 3.4 mM, about 3.5 mM, about 3.6 mM, about 3.7 mM, about 3.8 mM, about 3.9 mM, about 4 mM, or about 5 mM.

[0186]

[0220] In some embodiments, glutamine is present in or added to the disclosed compositions at a concentration of 0.5-20 mM, 0.5-10 mM, 0.5-5 mM, 1-5 mM, 2-5 mM, or 1 mM-10 mM. In some embodiments, glutamine is present in or added to the disclosed compositions at a concentration of 3.8-4.2 mM. In some embodiments, glutamine is present in or added to the disclosed compositions at a concentration of 1-10, 1-7, 1-8, 1-6, 1-5, 1-4, 2-10, 2-7, 2-8, 2-6, 2-5, 2-4, 3-10, 3-7, 3-8, 3-6, 3-5, 3-4, 3.5-4.5, 3.8-4.2, or 3.9-4.1 mM. In some embodiments, glutamine is present in or added to a composition of the present disclosure at a concentration of about 4 mM.

[0187]

[0221] In some embodiments, at least 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, or 5 mM of the glutamine is not in the dipeptide form.

[0188]

[0222] In some embodiments, at least 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, or 5 mM of the glutamine is not in the alanine-glutamine dipeptide form.

[0189]

[0223] In some embodiments, at least 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, or 5 mM of the glutamine is not in the glycine-glutamine dipeptide form.

[0190]

[0224] In some embodiments, at least 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, or 5 mM of the glutamine is not in multimeric form.

[0191]

[0225] In some embodiments, at least 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, or 5 mM of the glutamine is not conjugated to another amino acid or stabilizing moiety.

[0192]

[0226] In some embodiments, at least 500 μM, at least 750 μM, at least 1 mM, at least 1.5 mM, at least 2 mM, at least 2.5 mM, at least 2.6 mM, at least 2.7 mM, at least 2.8 mM, at least 2.9 mM, at least 3 mM, at least 3.1 mM, at least 3.2 mM, at least 3.3 mM, at least 3.4 mM, at least 3.5 mM, at least 3.6 mM, at least 3.7 mM, at least 3.8 mM, at least 3.9 mM, at least 4 mM, or at least 5 mM of the glutamine is in monomeric form.

[0193]

[0227] In some embodiments, at least 500 μM, at least 750 μM, at least 1 mM, at least 1.5 mM, at least 2 mM, at least 2.5 mM, at least 2.6 mM, at least 2.7 mM, at least 2.8 mM, at least 2.9 mM, at least 3 mM, at least 3.1 mM, at least 3.2 mM, at least 3.3 mM, at least 3.4 mM, at least 3.5 mM, at least 3.6 mM, at least 3.7 mM, at least 3.8 mM, at least 3.9 mM, at least 4 mM, at least 5 mM, at least 5.5 mM, at least 6 mM, at least 6.5 mM, at least 7 mM, at least 7.5 mM, at least 8 mM, at least 8.5 mM, at least 9 mM, at least 9.5 mM, or at least 10 mM of the glutamine is free.

[0194]

[0228] In some embodiments, the method of the present disclosure contacts the cells with glutamine. The cells can be PDX1 positive, NKX6.1 positive, insulin negative cells. The cells can be insulin positive endocrine precursor cells. The cells can be SC-β cells. In some embodiments, the cells are frozen. In some embodiments, the cells have been previously frozen. In some embodiments, the cells are in cell clusters. In some embodiments, the cells are dissociated.

[0195]

[0229] In some embodiments, cells (e.g., PDX1 positive, NKX6.1 positive, insulin negative cells, or insulin positive endocrine precursor cells) are contacted with glutamine for about 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 84 hours, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days.

[0196]

[0230] In some embodiments, cells (e.g., PDX1 positive, NKX6.1 positive, insulin negative cells, or insulin positive endocrine precursor cells) are contacted with glutamine for at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, at least 72 hours, at least 84 hours, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least or at least 14 days.

[0197]

[0231] In some embodiments, cells (e.g., PDX1 positive, NKX6.1 positive, insulin negative cells, or insulin positive endocrine precursor cells) are contacted with glutamine for up to 12 hours, up to 24 hours, up to 36 hours, up to 48 hours, up to 60 hours, up to 72 hours, up to 84 hours, up to 4 days, up to 5 days, up to 6 days, up to 7 days, up to 8 days, up to 9 days, up to 10 days, up to 11 days, up to 12 days, up to 13 days, up to 14 days, or up to 14 days.

[0198]

[0232] In some embodiments, cells (e.g., PDX1 positive, NKX6.1 positive, insulin negative cells, or insulin positive endocrine precursor cells) are contacted with glutamine 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, 4-8 days, 4-7 days, 4-6 days, or 4-5 days.

[0199]

[0233] In some embodiments, a composition or method of the present disclosure does not include glutamine. Glutamate

[0234] In some embodiments, the composition (e.g., medium) of the present disclosure comprises glutamate (e.g., L-glutamate). Glutamate can be converted to, for example, g-aminobutyric acid (GABA), ornithine, 2-oxoglutarate, glucose, or glutathione. Glutamate and the metabolites generated therefrom can contribute to, for example, redox homeostasis, cell signaling, nitrogen assimilation, amine catabolism, amino acid biosynthesis, nucleoside biosynthesis, and cofactor production.

[0200]

[0235] In some embodiments, contacting the cells with glutamate can result in improved in vitro generation of SC-β cells, e.g., higher cell yield and recovery, increased numbers and relative percentages of SC-β cells, enhanced stability and shelf life of SC-β cells, SC islet clusters with advantageous characteristics such as reduced size and increased uniformity, improved function of SC-β cells in vitro, improved cell survival after transplantation, improved cell function, reduced immunogenicity, or a combination thereof, e.g., compared to compositions lacking glutamate or comprising a lower concentration of glutamate.

[0201]

[0236] In some embodiments, glutamate may be present in a composition of the present disclosure at a concentration of at least 100 μM, at least 200 μM, at least 300 μM, at least 400 μM, at least 450 μM, at least 500 μM, at least 600 μM, at least 700 μM, at least 800 μM, at least 900 μM, at least 1 mM, at least 1.5 mM, at least 2 mM, at least 2.5 mM, at least 3 mM, at least 4 mM, or at least 5 mM.

[0202]

[0237] In some embodiments, glutamate may be present in a composition of the present disclosure at a concentration of up to 500 μM, up to 600 μM, up to 700 μM, up to 800 μM, up to 900 μM, up to 1 mM, up to 2 mM, up to 3 mM, up to 4 mM, up to 5 mM, up to 6 mM, up to 7 mM, up to 8 mM, up to 9 mM, up to 10 mM, up to 15 mM, up to 20 mM, up to 30 mM, up to 40 mM, or up to 50 mM.

[0203]

[0238] In some embodiments, glutamate may be present in the disclosed compositions at a concentration of about 100 μM, about 200 μM, about 300 μM, about 400 μM, about 450 μM, about 500 μM, about 550 μM, about 600 μM, about 700 μM, about 800 μM, about 900 μM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 4 mM, or about 5 mM. In some embodiments, glutamate is present or added to the disclosed compositions at a concentration of about 500 μM.

[0204]

[0239] In some embodiments, glutamate is between about 100 μM and 5 mM, 200 μM and 5 mM, 300 μM and 5 mM, 400 μM and 5 mM, 100 μM and 3 mM, 200 μM and 3 mM, 300 μM and 3 mM, 400 μM and 3 mM, 100 μM and 2 mM, 200 μM and 2 mM, 300 μM and 2 mM, 400 μM and 2 mM, 100 μM and 1 mM, 200 μM and 1 mM, 300 μM and 1 mM, 400 μM and 1 mM, 100 μM and 700 μM, 200 μM and 700 μM, 300 μM and 700 μM,

[0240] It is present in or added to the compositions of the present disclosure at a concentration of 400 μM to 700 μM, 100 μM to 600 μM, 200 μM to 600 μM, 300 μM to 600 μM, or 400 μM to 600 μM.

[0205]

[0241] In some embodiments, the method of the present disclosure contacts the cells with glutamate. The cells can be PDX1 positive, NKX6.1 positive, insulin negative cells. The cells can be insulin positive endocrine precursor cells. The cells can be SC-β cells. In some embodiments, the cells are frozen. In some embodiments, the cells have been previously frozen. In some embodiments, the cells are in cell clusters. In some embodiments, the cells are dissociated.

[0206]

[0242] In some embodiments, cells (e.g., PDX1 positive, NKX6.1 positive, insulin negative cells, or insulin positive endocrine precursor cells) are contacted with glutamate for about 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 84 hours, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days.

[0207]

[0243] In some embodiments, cells (e.g., PDX1 positive, NKX6.1 positive, insulin negative cells, or insulin positive endocrine precursor cells) are contacted with glutamate for at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, at least 72 hours, at least 84 hours, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least or at least 14 days.

[0208]

[0244] In some embodiments, cells (e.g., PDX1 positive, NKX6.1 positive, insulin negative cells, or insulin positive endocrine precursor cells) are contacted with glutamate for up to 12 hours, up to 24 hours, up to 36 hours, up to 48 hours, up to 60 hours, up to 72 hours, up to 84 hours, up to 4 days, up to 5 days, up to 6 days, up to 7 days, up to 8 days, up to 9 days, up to 10 days, up to 11 days, up to 12 days, up to 13 days, up to 14 days.

[0209]

[0245] In some embodiments, cells (e.g., PDX1 positive, NKX6.1 positive, insulin negative cells, or insulin positive endocrine precursor cells) are contacted with glutamate 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, 4-8 days, 4-7 days, 4-6 days, or 4-5 days.

[0210]

[0246] In some embodiments, a composition or method of the present disclosure does not include glutamate.

[0211] Carnitine

[0247] In some embodiments, the compositions (e.g., media) of the present disclosure include carnitine, which can transport long-chain fatty acids into mitochondria for oxidation for energy production and support cellular fitness.

[0212]

[0248] In some embodiments, contacting the cells with carnitine can result in improved in vitro generation of SC-β cells, e.g., higher cell yield and recovery, increased number and relative percentage of SC-β cells, enhanced stability and shelf life of SC-β cells, SC islet clusters with advantageous characteristics such as reduced size and increased uniformity, improved function of SC-β cells in vitro, improved cell survival after transplantation, improved cell function, reduced immunogenicity, or a combination thereof, e.g., compared to compositions lacking carnitine or comprising a lower concentration of carnitine.

[0213]

[0249] In some embodiments, carnitine is present in or added to a composition of the present disclosure at a concentration of at least 100 nM, at least 500 nM, at least 1 μM, at least 10 μM, at least 15 μM, at least 20 μM, at least 25 μM, at least 30 μM, at least 35 μM, at least 40 μM, at least 50 μM, at least 75 μM, or at least 100 μM.

[0214]

[0250] In some embodiments, carnitine is present in or added to a composition of the disclosure at a concentration of up to 20 μM, up to 30 μM, up to 40 μM, up to 50 μM, up to 60 μM, 70 μM, up to 80 μM, up to 90 μM, up to 100 μM, up to 200 μM, up to 300 μM, up to 400 μM, up to 500 μM, or up to 1 mM.

[0215]

[0251] In some embodiments, carnitine is present or added to the composition of the present disclosure at a concentration of about 100nM, about 500nM, about 1μM, about 10μM, about 15μM, about 20μM, about 25μM, about 30μM, about 35μM, about 40μM, about 45μM, about 50μM, about 55μM, about 60μM, about 75μM, or about 100μM.In some embodiments, carnitine is present or added at a concentration of about 40μM.

[0216]

[0252] In some embodiments, carnitine is at about 100 nM to 1 mM, 500 nM to 1 mM, 1 μM to 1 mM, 10 μM to 1 mM, 20 μM to 1 mM, 30 μM to 1 mM, 100 nM to 250 μM, 500 nM to 250 μM, 1 μM to 250 μM, 10 μM to 250 μM, 20 μM to 250 μM, 30 μM to 250 μM, 100 nM to 100 μM, 500 nM to The agonist may be present in or added to a composition of the present disclosure at a concentration of 100 μM, 1 μM-100 μM, 10 μM-100 μM, 20 μM-100 μM, 30 μM-100 μM, 100 nM-60 μM, 500 nM-60 μM, 1 μM-60 μM, 10 μM-60 μM, 20 μM-60 μM, 30 μM-60 μM, 35 μM-60 μM, or 30 μM-50 μM.

[0217]

[0253] In some embodiments, the cell is contacted with carnitine in the method of the present disclosure. The cell can be a PDX1 positive, NKX6.1 positive, insulin negative cell. The cell can be an insulin positive endocrine precursor cell. The cell can be an SC-β cell. In some embodiments, the cell is frozen. In some embodiments, the cell has been previously frozen. In some embodiments, the cell is in cell clusters. In some embodiments, the cell is dissociated.

[0218]

[0254] In some embodiments, cells (e.g., PDX1 positive, NKX6.1 positive, insulin negative cells, or insulin positive endocrine precursor cells) are contacted with carnitine for about 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 84 hours, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days.

[0219]

[0255] In some embodiments, cells (e.g., PDX1 positive, NKX6.1 positive, insulin negative cells, or insulin positive endocrine precursor cells) are contacted with carnitine for at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, at least 72 hours, at least 84 hours, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least or at least 14 days.

[0220]

[0256] In some embodiments, cells (e.g., PDX1 positive, NKX6.1 positive, insulin negative cells, or insulin positive endocrine precursor cells) are contacted with carnitine for up to 12 hours, up to 24 hours, up to 36 hours, up to 48 hours, up to 60 hours, up to 72 hours, up to 84 hours, up to 4 days, up to 5 days, up to 6 days, up to 7 days, up to 8 days, up to 9 days, up to 10 days, up to 11 days, up to 12 days, up to 13 days, up to 14 days, or up to 14 days.

[0221]

[0257] In some embodiments, cells (e.g., PDX1-positive, NKX6.1-positive, insulin-negative cells, or insulin-positive endocrine precursor cells) are contacted with carnitine 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, 4-8 days, 4-7 days, 4-6 days, or 4-5 days.

[0222]

[0258] In some embodiments, the compositions or methods of the present disclosure do not include carnitine.

[0223] TGF-β signaling pathway inhibitors

[0259] Exemplary TGF-β signaling pathway inhibitors include ALK5 Inhibitor II (CAS 446859-33-2, 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, analogs or derivatives of ALK5 Inhibitor II, such as 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 (including, for example, A83-01, 431542, D4476, GW788388, LY364947, LY580276, SB525334, SB505124, SD208, GW6604, or GW788388).

[0224]

[0260] In some embodiments, the TGF-β signaling pathway inhibitor may have the structure:

[0225] [ka]

[0226]

[0261] 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.

[0227] Thyroid hormone signaling pathway activators

[0262] Exemplary thyroid hormone signaling pathway activators include triiodothyronine (T3), analogs or derivatives of T3, such as selective and nonselective thyromimetics, TRJ selective agonists GC-1, GC-24, 4-hydroxy-PCB 106, MB0781 1, MB07344, 3,5-diiodothyropropionic acid (DITPA); 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 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) ( Thyronamine (TAM) (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-1-thyronine (L-940901), N-[3,5-dimethyl-4-(4'-hydroxy-3 f Examples of suitable iodine-modifying agents include, but are not limited to, 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).

[0228]

[0263] In some embodiments, the thyroid hormone signaling pathway activator can include a prodrug or prohormone of T3, such as 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) beta and TRoc 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 the TRoti and TRi receptors, respectively.

[0229]

[0264] In some embodiments, a thyroid hormone signaling pathway activator may have the structure:

[0230] [ka]

[0231]

[0265] 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.

[0232] Protein Kinase Inhibitors

[0266] Exemplary protein kinase inhibitors include, but are not limited to, staurosporine, analogs of staurosporine such as Ro-31-8220, bisindolylmaleimide (Bis) compounds, 10'-{5"-[(methoxycarbonyl)amino]-2"-methyl}-phenylaminocarbonyl staurosporine, stalalogues (see, e.g., Lopez et al., "Staurosporine-derived inhibitors broaden the scope of analog-sensitive kinase technology," J. Am. Chem. Soc. 2013;i 35(48):1 8153-18159), and cgp4125 1. In some embodiments, the protein kinase inhibitor may be staurosporine.

[0233]

[0267] 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.

[0234]

[0268] In some embodiments, the protein kinase inhibitor may have the structure:

[0235] [ka]

[0236] Bone morphogenetic protein (BMP) signaling pathway inhibitors

[0269] 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), analogs or derivatives of LDN193189, such as salts of LDN193189 (e.g., LDN193189 hydrochloride), hydrates, solvents, esters, or prodrugs, or compounds of formula I of US Patent Application Publication No. 2011 / 0053930. In aspects of the invention, the BMP signaling pathway inhibitor comprises LDN193189. In some embodiments, the BMP signaling pathway inhibitor comprises DMH-1 or an analog or derivative thereof.

[0237]

[0270] In some embodiments, the BMP signaling pathway inhibitor has the following structure:

[0238] [ka]

[0239] may have the following structure:

[0271] 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.

[0240] Rho-associated protein kinase (ROCK) inhibitors

[0272] 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 (Η 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 kinase inhibitor III, Rockout), and 4-pyrazoleboronic acid pinacol ester; Rock antibodies commercially available from Santa Cruz Biotechnology, 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-1 CRISPR / Cas9 KO plasmid (h), Rock-2 CRISPR / Cas9 ROCK CRISPR / Cas9 knockout plasmids selected from the group consisting of Rock-1 CR1SPR / Cas9 KO Plasmid (h), Rock-1 CRISPR / Cas9 KO Plasmid (m), and Rock-2 CRISPR / Cas9 KO Plasmid (m); ROCK siRNAs, shRNA plasmids, and / or shRNA lentiviral particle gene silencers selected from the group consisting of Rock-1 siRNA (h): sc-29473, Rock-1 siRNA (m): sc-36432, Rock-1 siRNA (r): sc-72179, Rock-2 siRNA (h): sc-29474, Rock-2 siRNA (m): sc-36433, and Rock-2 siRNA (r): sc-108088.

[0241]

[0273] In some embodiments, the ROCK inhibitor comprises Y-27632. In some embodiments, the ROCK inhibitor is thiazovivin.

[0242]

[0274] In some embodiments, the ROCK inhibitor has the following structure:

[0243] [ka]

[0244] may have the following structure:

[0275] In some embodiments, the concentration of the ROCK inhibitor may 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 may be about 2.5 μM. In some embodiments, the ROCK inhibitor is thiazovivin and the concentration of the inhibitor is about 2.5 μM.

[0245]

[0276] 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 16 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, about 67 μM, about 68 μM It can be about 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.

[0246] Histone methyltransferase inhibitors

[0277] In some embodiments, histone methyltransferase inhibitors can be used as epigenetic modifiers. Exemplary histone methyltransferase inhibitors can 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 serine protease inhibitor (serpin) family member. In some embodiments, the histone methyltransferase inhibitor can be DZNep. In some embodiments, the histone methyltransferase inhibitor has the structure:

[0247] [ka]

[0248] may have the following structure:

[0278] 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.

[0249]

[0279] 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.

[0250] MGLL inhibitors

[0280] Exemplary MGLL (monoglyceride lipase) inhibitors include, but are not limited to, for example, JJKK048, KML29, NF1819, JW642, JZL184, JZL195, JZP361, pristimerin, or URB602.

[0251]

[0281] 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.

[0252]

[0282] In some embodiments, the MGLL inhibitor may have the structure:

[0253] [ka]

[0254]

[0283] In some embodiments, the MGLL inhibitor may have the structure:

[0255] [ka]

[0256]

[0284] In some embodiments, the MGLL inhibitor may have the structure:

[0257] [ka]

[0258]

[0285] In some embodiments, the concentration of the MGLL inhibitor is about 0.1 μM to 100 μM. In some embodiments, the concentration of the MGLL inhibitor is about 0.1 μM, 1 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM.

[0259]

[0286] In some embodiments, the MGLL inhibitor is JJKK048 and is at a concentration of about 0.1 μM to 100 μM. In some embodiments, the MGLL inhibitor is KML29 and is at a concentration of about 0.1 μM to 100 μM. In some embodiments, the MGLL inhibitor is NF1819 and is at a concentration of about 0.1 μM to 100 μM.

[0260]

[0287] In some embodiments, the MGLL inhibitor is JJKK048 and the concentration is 1 μM. In some embodiments, the MGLL inhibitor is KML29 and the concentration is 10 μM. In some embodiments, the MGLL inhibitor is NF1819 and the concentration is 10 μM.

[0261] Lipids

[0288] 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, but are not limited to, 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, hentriacontyl acid, russellic acid, silylic acid, gedic acid, celloplastic acid, hexatriacontyl acid, heptatriacontanoic acid, octatriacontanoic acid, nonatriacontanoic acid, or tetracontanoic acid, or a salt or ester thereof.

[0262]

[0289] 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.

[0263]

[0290] Exemplary unsaturated fatty acids include, but are not limited to, for example, oleic acid, linoleic acid, palmitoleic acid, stearidonic acid, eicosapentaenoic acid, docosahexaenoic acid, linolelide 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.

[0264]

[0291] 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.

[0265] Additional Reagents

[0292] In some embodiments, any of the compositions disclosed herein comprises DMEM / F12. In some embodiments, any of the compositions disclosed herein may contain any of the following: glycine (e.g., at a concentration of 0.25 mM), L-alanine (e.g., at a concentration of 0.049999997 mM), L-arginine hydrochloride (e.g., at a concentration of 0.69905216 mM), L-asparagine-H2O (0.05 mM), L-aspartic acid (e.g., at a concentration of 0.05 mM), L-cysteine ​​hydrochloride-H2O (e.g., at a concentration of 0.09977272 mM), L-cystine 2HCl (e.g., at a concentration of 0.09996805 mM), L-glutamic acid (e.g., at a concentration of 0.05 mM), L-glutamine (e.g., at a concentration of 2.5 mM), L-histidine hydrochloride-H2O (e.g., at a concentration of 0.14990476 mM), L-isoleucine (e.g., For example, L-leucine (e.g., at a concentration of 0.41580153 mM), L-lysine hydrochloride (e.g., at a concentration of 0.45076334 mM), L-lysine hydrochloride (e.g., at a concentration of 0.4986339 mM), L-methionine (e.g., at a concentration of 0.11570469 mM), L-phenylalanine (e.g., at a concentration of 0.2150303 mM), L-proline (e.g., at a concentration of 0.15 mM), L -contains any one or more of the following amino acids: serine (e.g., at a concentration of 0.25 mM), L-threonine (e.g., at a concentration of 0.44915968 mM), L-tryptophan (e.g., at a concentration of 0.04421569 mM), L-tyrosine (e.g., at a concentration of 0.21375479 mM), or L-valine (e.g., at a concentration of 0.4517094 mM).In some embodiments, any of the compositions disclosed herein may contain additional ingredients such as biotin (e.g., at a concentration of 1.4344263E-5 mM), choline chloride (e.g., at a concentration of 0.06414285 mM), calcium D-pantothenate (e.g., at a concentration of 0.0046960167 mM), folic acid (e.g., at a concentration of 0.0060090707 mM), niacinamide (e.g., at a concentration of 0.016557377 In some embodiments, the vitamins include any of the following: vitamin B12 (e.g., at a concentration of 5.0184503E-4 mM), vitamin C (e.g., at a concentration of 0.07 mM), vitamin D3 (e.g., at a concentration of 0.07 mM), vitamin B4 (e.g., at a concentration of 0.07 mM), vitamin D5 (e.g., at a concentration of 0.07 mM), vitamin D6 (e.g., at a concentration of 0.07 mM), vitamin D7 (e.g., at a concentration of 0.07 mM), vitamin D8 (e.g., at a concentration of 0.07 mM), vitamin D9 (e.g., at a concentration of 0.07 mM), vitamin D10 (e.g., at a concentration of 0.07 mM), vitamin D11 (e.g., at a concentration of 0.07 mM), vitamin D12 (e.g., at a concentration of 0.07 mM), or vitamin D12 (e.g., at a concentration of 0.07 mM). In some embodiments, any of the compositions disclosed herein include any of the following components: dextrose (17.505556 mM), hypoxanthine Na (0.015031448 mM), linoleic acid (1.4999999E-4 mM), lipoic acid (5.097087E-4 mM), phenol red (0.021519661 mM), putrescine 2HCl (5.031056E-4 mM), sodium pyruvate (0.5 mM), or thymidine (0.0015082645 mM).

[0266]

[0293] In some embodiments, any of the compositions disclosed herein comprises about 0.01%, about 0.05%, about 0.1%, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, or about 15% human serum albumin (HSA). In some embodiments, the compositions of the present disclosure comprise 0.01-1%, 0.01-0.08%, 0.02-0.07%, 0.03-0.06%, 0.04-0.06%, 0.045-0.055%, 0.8-1.2%, or 0.9-1.1% HSA.

[0267]

[0294] In some embodiments, any of the compositions disclosed herein comprises zinc (ZnSO 4 In some embodiments, the compositions of the present disclosure include 1-100 μM, 1-50 μM, 1-20 μM, 1-12 μM, 5-15 μM, 8-12 μM, or 9-11 μM ZnSO.4 In some embodiments, the compositions of the present disclosure comprise about 10 μM ZnSO 4 Includes.

[0268]

[0295] In some embodiments, any of the compositions disclosed herein does not include ascorbic acid.

[0269] Exemplary Drug Combinations

[0296] In some embodiments, the composition of the present disclosure comprises two or more drugs disclosed herein in combination.Two or more drugs can be present in cell culture medium, optionally in the medium containing cells undergoing the differentiation or reprogramming method of the present disclosure.The combination of drugs disclosed herein exhibits surprising and unexpected benefits for the generation of SC-β cells in vitro, such as higher cell yield and recovery rate, increased number and relative percentage of SC-β cells (e.g., NKX6.1 positive and / or ISL1 positive cells), enhanced stability and shelf life of SC-β cells, SC islet clusters with advantageous characteristics such as reduced size and increased uniformity, improved function of SC-β cells in vitro, and improved survival rate, function, and reduced immunogenicity after transplantation.

[0270]

[0297] In some embodiments, the drug combinations improve the fitness and metabolic flexibility of differentiated cells and the resulting SC-β cells by targeting specific aspects of the cell's metabolism. Non-limiting pathways and aspects of metabolism that can be modulated by the drug combinations include one-carbon metabolism, acetyl-CoA synthesis for lipid production and protein acetylation, promotion of mitochondrial oxidative phosphorylation and the TCA cycle, which generate intermediates that maintain redox homeostasis.

[0271]

[0298] In some embodiments, contacting the cell population with the drug combination increases the growth of the cell population. In some embodiments, contacting the cell population with the drug combination alters or increases the metabolism of the cell population. For example, in some cases, contacting the cell population with the drug combination increases the oxygen consumption rate (OCR) of the cell population by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 50%, as determined, for example, by Seahorse assay. In some cases, contacting the cell population with the drug combination increases the ATP-related oxygen consumption rate of the cell population by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 50%.

[0272]

[0299] In some embodiments, the composition of the present disclosure comprises a one-carbon metabolic pathway intermediate (e.g., formate), an acetyl-CoA-related metabolite (e.g., acetate), a vitamin (e.g., biotin), an HDAC inhibitor (e.g., β-hydroxybutyrate), and a redox homeostasis regulator (e.g., taurine), and glutamine. The combination of agents comprising acetate, β-hydroxybutyrate, taurine, formate, biotin, and glutamine may be collectively referred to herein as "MQ". The composition may comprise a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells, for example, in a cell culture medium as disclosed herein. The composition may comprise a plurality of insulin-positive endocrine precursor cells, for example, in a cell culture medium as disclosed herein. In some embodiments, the composition of the present disclosure comprises cell clusters. In some embodiments, the cells of the culture of the present disclosure are dissociated cells or are mostly dissociated cells. In some embodiments, the cells are frozen. In some embodiments, the cells of the compositions of the present disclosure have been previously frozen.

[0273]

[0300] In some embodiments, the composition of the present disclosure comprises at least one of a one-carbon metabolic pathway intermediate (e.g., formate), an acetyl-CoA-related metabolite (e.g., acetate), a vitamin (e.g., biotin), an HDAC inhibitor (e.g., β-hydroxybutyrate), and a redox homeostasis regulator (e.g., taurine), and glutamine. The composition may comprise a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells, for example, in a cell culture medium as disclosed herein. The composition may comprise a plurality of insulin-positive endocrine precursor cells, for example, in a cell culture medium as disclosed herein. In some embodiments, the composition of the present disclosure comprises cell clusters. In some embodiments, the cells of the culture of the present disclosure are dissociated cells or are mostly dissociated cells. In some embodiments, the cells are frozen. In some embodiments, the cells of the composition of the present disclosure have been previously frozen.

[0274]

[0301] In some embodiments, the composition of the present disclosure comprises two of a one-carbon metabolic pathway intermediate (e.g., formate), an acetyl-CoA-related metabolite (e.g., acetate), a vitamin (e.g., biotin), an HDAC inhibitor (e.g., β-hydroxybutyrate), and a redox homeostasis regulator (e.g., taurine), and glutamine. The composition may comprise a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells, for example, in a cell culture medium as disclosed herein. The composition may comprise a plurality of insulin-positive endocrine precursor cells, for example, in a cell culture medium as disclosed herein. In some embodiments, the composition of the present disclosure comprises cell clusters. In some embodiments, the cells of the culture of the present disclosure are dissociated cells or are mostly dissociated cells. In some embodiments, the cells are frozen. In some embodiments, the cells of the composition of the present disclosure have been previously frozen.

[0275]

[0302] In some embodiments, the composition of the present disclosure comprises three of a one-carbon metabolic pathway intermediate (e.g., formate), an acetyl-CoA-related metabolite (e.g., acetate), a vitamin (e.g., biotin), an HDAC inhibitor (e.g., β-hydroxybutyrate), and a redox homeostasis regulator (e.g., taurine), and glutamine. The composition may comprise a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells, for example, in a cell culture medium as disclosed herein. The composition may comprise a plurality of insulin-positive endocrine precursor cells, for example, in a cell culture medium as disclosed herein. In some embodiments, the composition of the present disclosure comprises cell clusters. In some embodiments, the cells of the culture of the present disclosure are dissociated cells or are mostly dissociated cells. In some embodiments, the cells are frozen. In some embodiments, the cells of the composition of the present disclosure have been previously frozen.

[0276]

[0303] In some embodiments, the composition of the present disclosure comprises four of a one-carbon metabolic pathway intermediate (e.g., formate), an acetyl-CoA-related metabolite (e.g., acetate), a vitamin (e.g., biotin), an HDAC inhibitor (e.g., β-hydroxybutyrate), and a redox homeostasis regulator (e.g., taurine), and glutamine. The composition may comprise a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells, for example, in a cell culture medium as disclosed herein. The composition may comprise a plurality of insulin-positive endocrine precursor cells, for example, in a cell culture medium as disclosed herein. In some embodiments, the composition of the present disclosure comprises cell clusters. In some embodiments, the cells of the culture of the present disclosure are dissociated cells or are mostly dissociated cells. In some embodiments, the cells are frozen. In some embodiments, the cells of the composition of the present disclosure have been previously frozen.

[0277]

[0304] In some embodiments, the composition of the present disclosure comprises five of a one-carbon metabolic pathway intermediate (e.g., formate), an acetyl-CoA-related metabolite (e.g., acetate), a vitamin (e.g., biotin), an HDAC inhibitor (e.g., β-hydroxybutyrate), and a redox homeostasis regulator (e.g., taurine), and glutamine. The composition may comprise a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells, for example, in a cell culture medium as disclosed herein. The composition may comprise a plurality of insulin-positive endocrine precursor cells, for example, in a cell culture medium as disclosed herein. In some embodiments, the composition of the present disclosure comprises cell clusters. In some embodiments, the cells of the culture of the present disclosure are dissociated cells or are mostly dissociated cells. In some embodiments, the cells are frozen. In some embodiments, the cells of the composition of the present disclosure have been previously frozen.

[0278]

[0305] In some embodiments, the compositions of the present disclosure include one-carbon metabolic pathway intermediates (e.g., formate), acetyl-CoA-related metabolites (e.g., acetate), vitamins (e.g., biotin), HDAC inhibitors (e.g., β-hydroxybutyrate), and redox homeostasis regulators (e.g., taurine). In some embodiments, the compositions of the present disclosure include one-carbon metabolic pathway intermediates (e.g., formate), acetyl-CoA-related metabolites (e.g., acetate), vitamins (e.g., biotin), HDAC inhibitors (e.g., β-hydroxybutyrate), and glutamine. In some embodiments, the compositions of the present disclosure include one-carbon metabolic pathway intermediates (e.g., formate), acetyl-CoA-related metabolites (e.g., acetate), vitamins (e.g., biotin), redox homeostasis regulators (e.g., taurine), and glutamine. In some embodiments, the composition of the present disclosure comprises a one-carbon metabolic pathway intermediate (e.g., formate), an acetyl-CoA-related metabolite (e.g., acetate), an HDAC inhibitor (e.g., β-hydroxybutyrate), a redox homeostasis regulator (e.g., taurine), and glutamine. In some embodiments, the composition of the present disclosure comprises a one-carbon metabolic pathway intermediate (e.g., formate), a vitamin (e.g., biotin), an HDAC inhibitor (e.g., β-hydroxybutyrate), a redox homeostasis regulator (e.g., taurine), and glutamine. In some embodiments, the composition of the present disclosure comprises an acetyl-CoA-related metabolite (e.g., acetate), a vitamin (e.g., biotin), an HDAC inhibitor (e.g., β-hydroxybutyrate), a redox homeostasis regulator (e.g., taurine), and glutamine. The composition may comprise a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells, for example, in a cell culture medium as disclosed herein. The composition may comprise a plurality of insulin-positive endocrine precursor cells, for example in a cell culture medium as disclosed herein. In some embodiments, the composition of the present disclosure comprises cell clusters. In some embodiments, the cells of the culture of the present disclosure are dissociated cells or are mostly dissociated cells. In some embodiments, the cells are frozen. In some embodiments, the cells of the composition of the present disclosure have been previously frozen.

[0279]

[0306] In some embodiments, the compositions of the present disclosure include one-carbon metabolic pathway intermediates (e.g., formate), acetyl CoA-related metabolites (e.g., acetate), vitamins (e.g., biotin), and HDAC inhibitors (e.g., β-hydroxybutyrate). In some embodiments, the compositions of the present disclosure include one-carbon metabolic pathway intermediates (e.g., formate), acetyl CoA-related metabolites (e.g., acetate), vitamins (e.g., biotin), and redox homeostasis regulators (e.g., taurine). In some embodiments, the compositions of the present disclosure include one-carbon metabolic pathway intermediates (e.g., formate), acetyl CoA-related metabolites (e.g., acetate), vitamins (e.g., biotin), and glutamine. In some embodiments, the compositions of the present disclosure include one-carbon metabolic pathway intermediates (e.g., formate), acetyl CoA-related metabolites (e.g., acetate), HDAC inhibitors (e.g., β-hydroxybutyrate), and redox homeostasis regulators (e.g., taurine). In some embodiments, the compositions of the present disclosure include one-carbon metabolic pathway intermediates (e.g., formate), acetyl CoA-related metabolites (e.g., acetate), HDAC inhibitors (e.g., β-hydroxybutyrate), and glutamine. In some embodiments, the compositions of the present disclosure include one-carbon metabolic pathway intermediates (e.g., formate), acetyl CoA-related metabolites (e.g., acetate), redox homeostasis regulators (e.g., taurine), and glutamine. In some embodiments, the compositions of the present disclosure include one-carbon metabolic pathway intermediates (e.g., formate), vitamins (e.g., biotin), HDAC inhibitors (e.g., β-hydroxybutyrate), and redox homeostasis regulators (e.g., taurine). In some embodiments, the compositions of the present disclosure include one-carbon metabolic pathway intermediates (e.g., formate), vitamins (e.g., biotin), HDAC inhibitors (e.g., β-hydroxybutyrate), and glutamine. In some embodiments, compositions of the present disclosure include a one-carbon metabolic pathway intermediate (e.g., formate), a vitamin (e.g., biotin), a redox homeostasis regulator (e.g., taurine), and glutamine.In some embodiments, the compositions of the present disclosure include one-carbon metabolic pathway intermediates (e.g., formate), HDAC inhibitors (e.g., β-hydroxybutyrate), redox homeostasis regulators (e.g., taurine), and glutamine. In some embodiments, the compositions of the present disclosure include acetyl-CoA-related metabolites (e.g., acetate), vitamins (e.g., biotin), HDAC inhibitors (e.g., β-hydroxybutyrate), and redox homeostasis regulators (e.g., taurine). In some embodiments, the compositions of the present disclosure include acetyl-CoA-related metabolites (e.g., acetate), vitamins (e.g., biotin), HDAC inhibitors (e.g., β-hydroxybutyrate), and glutamine. In some embodiments, the compositions of the present disclosure include acetyl-CoA-related metabolites (e.g., acetate), vitamins (e.g., biotin), redox homeostasis regulators (e.g., taurine), and glutamine. In some embodiments, the composition of the present disclosure comprises an acetyl-CoA-related metabolite (e.g., acetate), an HDAC inhibitor (e.g., β-hydroxybutyrate), a redox homeostasis regulator (e.g., taurine), and glutamine. In some embodiments, the composition of the present disclosure comprises a vitamin (e.g., biotin), an HDAC inhibitor (e.g., β-hydroxybutyrate), a redox homeostasis regulator (e.g., taurine), and glutamine. The composition may comprise a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells, for example, in a cell culture medium as disclosed herein. The composition may comprise a plurality of insulin-positive endocrine precursor cells, for example, in a cell culture medium as disclosed herein. In some embodiments, the composition of the present disclosure comprises cell clusters. In some embodiments, the cells of the culture of the present disclosure are dissociated cells or are mostly dissociated cells. In some embodiments, the cells are frozen. In some embodiments, the cells of the composition of the present disclosure have been previously frozen.

[0280]

[0307] In some embodiments, the compositions of the present disclosure include one-carbon metabolic pathway intermediates (e.g., formate), acetyl-CoA-related metabolites (e.g., acetate), vitamins (e.g., biotin), HDAC inhibitors (e.g., β-hydroxybutyrate), redox homeostasis regulators (e.g., taurine), glutamate, and carnitine. In some embodiments, the compositions of the present disclosure include DMEM / F12. In some embodiments, the compositions of the present disclosure include zinc (e.g., ZnSO 4 ). In some embodiments, the composition of the present disclosure comprises human serum albumin. The composition may comprise a plurality of PDX1 positive, NKX6.1 positive, insulin negative cells, for example in a cell culture medium as disclosed herein. The composition may comprise a plurality of insulin positive endocrine precursor cells, for example in a cell culture medium as disclosed herein. In some embodiments, the composition of the present disclosure comprises cell clusters. In some embodiments, the cells of the culture of the present disclosure are dissociated cells or are mostly dissociated cells. In some embodiments, the cells are frozen. In some embodiments, the cells of the composition of the present disclosure have been previously frozen.

[0281]

[0308] In some embodiments, the composition of the present disclosure comprises at least one of a one-carbon metabolic pathway intermediate (e.g., formate), an acetyl-CoA-related metabolite (e.g., acetate), a vitamin (e.g., biotin), an HDAC inhibitor (e.g., β-hydroxybutyrate), a redox homeostasis regulator (e.g., taurine), glutamate, and carnitine. The composition may comprise a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells, for example, in a cell culture medium as disclosed herein. The composition may comprise a plurality of insulin-positive endocrine precursor cells, for example, in a cell culture medium as disclosed herein. In some embodiments, the composition of the present disclosure comprises cell clusters. In some embodiments, the cells of the culture of the present disclosure are dissociated cells or are mostly dissociated cells. In some embodiments, the cells are frozen. In some embodiments, the cells of the composition of the present disclosure have been previously frozen.

[0282]

[0309] In some embodiments, the compositions of the present disclosure include two of a one-carbon metabolic pathway intermediate (e.g., formate), an acetyl-CoA-related metabolite (e.g., acetate), a vitamin (e.g., biotin), an HDAC inhibitor (e.g., β-hydroxybutyrate), a redox homeostasis regulator (e.g., taurine), glutamate, and carnitine. In some embodiments, the compositions of the present disclosure include DMEM / F12. In some embodiments, the compositions of the present disclosure include zinc (e.g., ZnSO 4 ). In some embodiments, the composition of the present disclosure comprises human serum albumin. The composition may comprise a plurality of PDX1 positive, NKX6.1 positive, insulin negative cells, for example in a cell culture medium as disclosed herein. The composition may comprise a plurality of insulin positive endocrine precursor cells, for example in a cell culture medium as disclosed herein. In some embodiments, the composition of the present disclosure comprises cell clusters. In some embodiments, the cells of the culture of the present disclosure are dissociated cells or are mostly dissociated cells. In some embodiments, the cells are frozen. In some embodiments, the cells of the composition of the present disclosure have been previously frozen.

[0283]

[0310] In some embodiments, the compositions of the present disclosure include three of a one-carbon metabolic pathway intermediate (e.g., formate), an acetyl-CoA-related metabolite (e.g., acetate), a vitamin (e.g., biotin), an HDAC inhibitor (e.g., β-hydroxybutyrate), a redox homeostasis regulator (e.g., taurine), glutamate, and carnitine. In some embodiments, the compositions of the present disclosure include DMEM / F12. In some embodiments, the compositions of the present disclosure include zinc (e.g., ZnSO 4). In some embodiments, the composition of the present disclosure comprises human serum albumin. The composition may comprise a plurality of PDX1 positive, NKX6.1 positive, insulin negative cells, for example in a cell culture medium as disclosed herein. The composition may comprise a plurality of insulin positive endocrine precursor cells, for example in a cell culture medium as disclosed herein. In some embodiments, the composition of the present disclosure comprises cell clusters. In some embodiments, the cells of the culture of the present disclosure are dissociated cells or are mostly dissociated cells. In some embodiments, the cells are frozen. In some embodiments, the cells of the composition of the present disclosure have been previously frozen.

[0284]

[0311] In some embodiments, the compositions of the present disclosure include four of a one-carbon metabolic pathway intermediate (e.g., formate), an acetyl-CoA-related metabolite (e.g., acetate), a vitamin (e.g., biotin), an HDAC inhibitor (e.g., β-hydroxybutyrate), a redox homeostasis regulator (e.g., taurine), glutamate, and carnitine. In some embodiments, the compositions of the present disclosure include DMEM / F12. In some embodiments, the compositions of the present disclosure include zinc (e.g., ZnSO 4 ). In some embodiments, the composition of the present disclosure comprises human serum albumin. The composition may comprise a plurality of PDX1 positive, NKX6.1 positive, insulin negative cells, for example in a cell culture medium as disclosed herein. The composition may comprise a plurality of insulin positive endocrine precursor cells, for example in a cell culture medium as disclosed herein. In some embodiments, the composition of the present disclosure comprises cell clusters. In some embodiments, the cells of the culture of the present disclosure are dissociated cells or are mostly dissociated cells. In some embodiments, the cells are frozen. In some embodiments, the cells of the composition of the present disclosure have been previously frozen.

[0285]

[0312] In some embodiments, the compositions of the present disclosure include five of a one-carbon metabolic pathway intermediate (e.g., formate), an acetyl-CoA-related metabolite (e.g., acetate), a vitamin (e.g., biotin), an HDAC inhibitor (e.g., β-hydroxybutyrate), a redox homeostasis regulator (e.g., taurine), glutamate, and carnitine. In some embodiments, the compositions of the present disclosure include DMEM / F12. In some embodiments, the compositions of the present disclosure include zinc (e.g., ZnSO 4 ). In some embodiments, the composition of the present disclosure comprises human serum albumin. The composition may comprise a plurality of PDX1 positive, NKX6.1 positive, insulin negative cells, for example in a cell culture medium as disclosed herein. The composition may comprise a plurality of insulin positive endocrine precursor cells, for example in a cell culture medium as disclosed herein. In some embodiments, the composition of the present disclosure comprises cell clusters. In some embodiments, the cells of the culture of the present disclosure are dissociated cells or are mostly dissociated cells. In some embodiments, the cells are frozen. In some embodiments, the cells of the composition of the present disclosure have been previously frozen.

[0286]

[0313] In some embodiments, the compositions of the present disclosure include six of a one-carbon metabolic pathway intermediate (e.g., formate), an acetyl-CoA-related metabolite (e.g., acetate), a vitamin (e.g., biotin), an HDAC inhibitor (e.g., β-hydroxybutyrate), a redox homeostasis regulator (e.g., taurine), glutamate, and carnitine. In some embodiments, the compositions of the present disclosure include DMEM / F12. In some embodiments, the compositions of the present disclosure include zinc (e.g., ZnSO 4). In some embodiments, the composition of the present disclosure comprises human serum albumin. The composition may comprise a plurality of PDX1 positive, NKX6.1 positive, insulin negative cells, for example in a cell culture medium as disclosed herein. The composition may comprise a plurality of insulin positive endocrine precursor cells, for example in a cell culture medium as disclosed herein. In some embodiments, the composition of the present disclosure comprises cell clusters. In some embodiments, the cells of the culture of the present disclosure are dissociated cells or are mostly dissociated cells. In some embodiments, the cells are frozen. In some embodiments, the cells of the composition of the present disclosure have been previously frozen.

[0287]

[0314] In some embodiments, the compositions of the present disclosure include one-carbon metabolic pathway intermediates (e.g., formate), acetyl CoA-related metabolites (e.g., acetate), vitamins (e.g., biotin), HDAC inhibitors (e.g., β-hydroxybutyrate), redox homeostasis regulators (e.g., taurine), and glutamate. In some embodiments, the compositions of the present disclosure include one-carbon metabolic pathway intermediates (e.g., formate), acetyl CoA-related metabolites (e.g., acetate), vitamins (e.g., biotin), HDAC inhibitors (e.g., β-hydroxybutyrate), redox homeostasis regulators (e.g., taurine), and carnitine. In some embodiments, the compositions of the present disclosure include one-carbon metabolic pathway intermediates (e.g., formate), acetyl CoA-related metabolites (e.g., acetate), vitamins (e.g., biotin), HDAC inhibitors (e.g., β-hydroxybutyrate), glutamate, and carnitine. In some embodiments, the compositions of the present disclosure include one-carbon metabolic pathway intermediates (e.g., formate), acetyl-CoA-related metabolites (e.g., acetate), vitamins (e.g., biotin), redox homeostasis regulators (e.g., taurine), glutamate, and carnitine. In some embodiments, the compositions of the present disclosure include one-carbon metabolic pathway intermediates (e.g., formate), acetyl-CoA-related metabolites (e.g., acetate), HDAC inhibitors (e.g., β-hydroxybutyrate), redox homeostasis regulators (e.g., taurine), glutamate, and carnitine. In some embodiments, the compositions of the present disclosure include one-carbon metabolic pathway intermediates (e.g., formate), vitamins (e.g., biotin), HDAC inhibitors (e.g., β-hydroxybutyrate), redox homeostasis regulators (e.g., taurine), glutamate, and carnitine. In some embodiments, the compositions of the present disclosure include acetyl-CoA-related metabolites (e.g., acetate), vitamins (e.g., biotin), HDAC inhibitors (e.g., β-hydroxybutyrate), redox homeostasis regulators (e.g., taurine), glutamate, and carnitine. In some embodiments, the compositions of the present disclosure include DMEM / F12. In some embodiments, the compositions of the present disclosure include zinc (e.g., ZnSO 4). In some embodiments, the composition of the present disclosure comprises human serum albumin. The composition may comprise a plurality of PDX1 positive, NKX6.1 positive, insulin negative cells, for example in a cell culture medium as disclosed herein. The composition may comprise a plurality of insulin positive endocrine precursor cells, for example in a cell culture medium as disclosed herein. In some embodiments, the composition of the present disclosure comprises cell clusters. In some embodiments, the cells of the culture of the present disclosure are dissociated cells or are mostly dissociated cells. In some embodiments, the cells are frozen. In some embodiments, the cells of the composition of the present disclosure have been previously frozen.

[0288]

[0315] In some embodiments, the compositions of the present disclosure include one-carbon metabolic pathway intermediates (e.g., formate), acetyl-CoA-related metabolites (e.g., acetate), vitamins (e.g., biotin), HDAC inhibitors (e.g., β-hydroxybutyrate), and redox homeostasis regulators (e.g., taurine). In some embodiments, the compositions of the present disclosure include one-carbon metabolic pathway intermediates (e.g., formate), acetyl-CoA-related metabolites (e.g., acetate), vitamins (e.g., biotin), HDAC inhibitors (e.g., β-hydroxybutyrate), and glutamate. In some embodiments, the compositions of the present disclosure include one-carbon metabolic pathway intermediates (e.g., formate), acetyl-CoA-related metabolites (e.g., acetate), vitamins (e.g., biotin), HDAC inhibitors (e.g., β-hydroxybutyrate), and carnitine. In some embodiments, the compositions of the present disclosure include one-carbon metabolic pathway intermediates (e.g., formate), acetyl-CoA-related metabolites (e.g., acetate), vitamins (e.g., biotin), redox homeostasis regulators (e.g., taurine), and glutamate. In some embodiments, the compositions of the present disclosure include one-carbon metabolic pathway intermediates (e.g., formate), acetyl-CoA-related metabolites (e.g., acetate), vitamins (e.g., biotin), redox homeostasis regulators (e.g., taurine), and carnitine. In some embodiments, the compositions of the present disclosure include one-carbon metabolic pathway intermediates (e.g., formate), acetyl-CoA-related metabolites (e.g., acetate), vitamins (e.g., biotin), glutamate, and carnitine. In some embodiments, the compositions of the present disclosure include one-carbon metabolic pathway intermediates (e.g., formate), acetyl-CoA-related metabolites (e.g., acetate), HDAC inhibitors (e.g., β-hydroxybutyrate), redox homeostasis regulators (e.g., taurine), and glutamate. In some embodiments, the compositions of the present disclosure include one-carbon metabolic pathway intermediates (e.g., formate), acetyl-CoA-related metabolites (e.g., acetate), HDAC inhibitors (e.g., β-hydroxybutyrate), redox homeostasis regulators (e.g., taurine), and carnitine.In some embodiments, the compositions of the present disclosure include one-carbon metabolic pathway intermediates (e.g., formate), acetyl CoA-related metabolites (e.g., acetate), HDAC inhibitors (e.g., β-hydroxybutyrate), glutamate, and carnitine. In some embodiments, the compositions of the present disclosure include one-carbon metabolic pathway intermediates (e.g., formate), acetyl CoA-related metabolites (e.g., acetate), redox homeostasis regulators (e.g., taurine), glutamate, and carnitine. In some embodiments, the compositions of the present disclosure include one-carbon metabolic pathway intermediates (e.g., formate), vitamins (e.g., biotin), HDAC inhibitors (e.g., β-hydroxybutyrate), redox homeostasis regulators (e.g., taurine), and glutamate. In some embodiments, the compositions of the present disclosure include one-carbon metabolic pathway intermediates (e.g., formate), vitamins (e.g., biotin), HDAC inhibitors (e.g., β-hydroxybutyrate), redox homeostasis regulators (e.g., taurine), and carnitine. In some embodiments, the compositions of the present disclosure include one-carbon metabolic pathway intermediates (e.g., formate), vitamins (e.g., biotin), HDAC inhibitors (e.g., β-hydroxybutyrate), glutamate, and carnitine. In some embodiments, the compositions of the present disclosure include one-carbon metabolic pathway intermediates (e.g., formate), vitamins (e.g., biotin), redox homeostasis regulators (e.g., taurine), glutamate, and carnitine. In some embodiments, the compositions of the present disclosure include one-carbon metabolic pathway intermediates (e.g., formate), HDAC inhibitors (e.g., β-hydroxybutyrate), redox homeostasis regulators (e.g., taurine), glutamate, and carnitine. In some embodiments, the compositions of the present disclosure include an acetyl-CoA-related metabolite (e.g., acetate), a vitamin (e.g., biotin), an HDAC inhibitor (e.g., β-hydroxybutyrate), a redox homeostasis regulator (e.g., taurine), and glutamate. In some embodiments, the compositions of the present disclosure include an acetyl-CoA-related metabolite (e.g., acetate), a vitamin (e.g., biotin), an HDAC inhibitor (e.g., β-hydroxybutyrate), a redox homeostasis regulator (e.g., taurine), and carnitine.In some embodiments, the compositions of the present disclosure include acetyl-CoA-related metabolites (e.g., acetate), vitamins (e.g., biotin), HDAC inhibitors (e.g., β-hydroxybutyrate), glutamate, and carnitine. In some embodiments, the compositions of the present disclosure include acetyl-CoA-related metabolites (e.g., acetate), vitamins (e.g., biotin), redox homeostasis regulators (e.g., taurine), glutamate, and carnitine. In some embodiments, the compositions of the present disclosure include acetyl-CoA-related metabolites (e.g., acetate), HDAC inhibitors (e.g., β-hydroxybutyrate), redox homeostasis regulators (e.g., taurine), glutamate, and carnitine. In some embodiments, the compositions of the present disclosure include vitamins (e.g., biotin), HDAC inhibitors (e.g., β-hydroxybutyrate), redox homeostasis regulators (e.g., taurine), glutamate, and carnitine. In some embodiments, the compositions of the present disclosure include DMEM / F12. In some embodiments, the compositions of the present disclosure include zinc (e.g., ZnSO. 4 ). In some embodiments, the composition of the present disclosure comprises human serum albumin. The composition may comprise a plurality of PDX1 positive, NKX6.1 positive, insulin negative cells, for example in a cell culture medium as disclosed herein. The composition may comprise a plurality of insulin positive endocrine precursor cells, for example in a cell culture medium as disclosed herein. In some embodiments, the composition of the present disclosure comprises cell clusters. In some embodiments, the cells of the culture of the present disclosure are dissociated cells or are mostly dissociated cells. In some embodiments, the cells are frozen. In some embodiments, the cells of the composition of the present disclosure have been previously frozen.

[0289]

[0316] The compositions comprising the drug combinations may further comprise any one or more of the additional factors disclosed herein, such as BMP signaling pathway inhibitors, LDN193189, ROCK inhibitors, thiazovivin, Y-27632, fasudil / HA1077, or 14-1152, histone methyltransferase inhibitors, 3-deazaneplanocin A hydrochloride, TGF-β pathway inhibitors, ALK5 inhibitor II, thyroid hormone signaling pathway activators, GC-1, T3, protein kinase inhibitors, staurosporine, sonic hedgehog pathway inhibitors, Sant1, Sant2, Sant 4, Sant4, Cur61414, forskolin, tomatidine, AY9944, triparanol, cyclopamine, growth factors from the epidermal growth factor (EGF) family, gamma secretase inhibitors, XXI, DAPT, zinc, ZnSO4, serum albumin protein, or derivatives thereof.

[0290]

[0317] In some embodiments, the present disclosure provides a composition comprising any combination of a cryopreservative (e.g., DMSO), any of the cells disclosed herein, and any of the media components disclosed herein (e.g., acetate, beta-hydroxybutyrate, taurine, formate, biotin, L-glutamine, L-carnitine, or L-glutamate). In some embodiments, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the cells in the composition are dissociated (not clustered). In some embodiments, the composition of the present disclosure is frozen. In some embodiments, the composition of the present disclosure has been previously frozen.

[0291] Initialization

[0318] The term "reprogramming" as used herein refers to the process of changing or reversing the differentiation state of somatic cells. Cells may be reprogrammed after partial differentiation or after terminal differentiation. Reprogramming encompasses the complete reversal of the differentiation state of somatic cells to pluripotent cells. Such a complete reversal of differentiation results in induced pluripotent (iPS) cells. Reprogramming as used herein also encompasses the partial reversal of the differentiation state of cells, 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 originated, e.g., direct reprogramming of differentiated cells to different somatic cell types. Reprogramming generally 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, genomic imprinting, etc., that occur during the differentiation of cells as the zygote develops into an adult.

[0292]

[0319] 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 different cell types or phenotypes. 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.

[0293]

[0320] 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 a higher differentiation potential) becomes a more specialized cell type (i.e., a less naive cell with a lower 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 a higher differentiation potential), and the term "transdifferentiation" refers to the process by which a cell of a particular cell type is converted to another cell type without significantly changing its level of "differentiation potential" or "naivety." Without wishing to be bound by theory, a cell is considered to "transdifferentiate" if it is converted from one lineage committed or terminally differentiated cell type to another lineage committed or terminally differentiated cell type without significantly changing its level of "differentiation potential" or "naivety."

[0294]

[0321] 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 any of the three germ layers, i.e., endoderm (the inner lining of the stomach, gastrointestinal tract, lungs), mesoderm (muscle, bone, blood, urogenital tract), or ectoderm (epidermal tissue and nervous system), and therefore have high differentiation potential, whereas multipotent cells (e.g., stem cells or certain types of artificial stem cells) have the ability to generate cells from multiple but limited lineages (e.g., hematopoietic stem cells, cardiac stem cells, or neural stem cells, etc.), and relatively have lower differentiation potential than pluripotent cells. Cells that are committed to a specific lineage or terminally differentiated may have even lower differentiation potential. Specific examples of transdifferentiation known in the art include, for example, the conversion of fibroblasts into beta cells, or exocrine pancreatic cells into beta cells, etc.

[0295]

[0322] 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.

[0296]

[0323] For example, methods are known to those skilled in the art for reprogramming or inducing a particular type of cell into another type of cell by differentiation, dedifferentiation, and / or transdifferentiation using one or more exogenous polynucleotide or polypeptide reprogramming factors. Such methods may rely on the introduction of genetic material that encodes one or more transcription factors or other polypeptides associated with cell reprogramming. For example, Pdx1, Ngn3, and MafA, or functional fragments thereof, are all known to encode peptides that can induce cell differentiation, dedifferentiation, and / or transdifferentiation of 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) contacts a cell to induce, for example, a cell of the present disclosure. Those skilled in the art will understand that other genes may be associated with cell reprogramming, and exogenous molecules encoding such genes (or functional fragments thereof) and the encoded polypeptides are also considered polynucleotide or polypeptide reprogramming factors (e.g., a polynucleotide or polypeptide that affects the expression level of another gene associated with cell reprogramming). For example, it has been shown that the introduction of exogenous polynucleotide or polypeptide epigenetic gene silencers that reduce p53 inactivation enhances the efficiency of inducing induced pluripotent stem cells (iPSCs). Therefore, exogenous polynucleotides or polypeptides that code for epigenetic silencers and other genes or proteins that can be directly or indirectly involved in enhancing cell reprogramming or cell programming efficiency can be considered to constitute exogenous polynucleotide 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 that codes for 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.

[0297]

[0324] In some embodiments of the present disclosure, the method excludes the use of reprogramming factors that are not small molecules. However, the method does not include the use of tissue culture components, such as culture media, serum, serum replacements, supplements, antibiotics, etc., such as RPMI, renal epithelial basal medium (REBM), Dulbecco's modified Eagle medium (DMEM), MCDB 131 medium, CMRL, etc. It will be understood that 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, selenium, ethanolamine, human epidermal growth factor (hEGF), basic fibroblast growth factor (bFGF), hydrocortisone, epinephrine, normacin, penicillin, streptomycin, gentamicin, and amphotericin may be utilized. It should be understood that these 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. In fact, these components are not small molecules as defined herein and / or are not reprogramming factors as defined herein.However, the cell culture components and metabolites disclosed herein can be used to enhance the cell reprogramming and differentiation methods disclosed herein.For example, the combination of the cell culture components / additives and metabolites disclosed herein can improve the efficiency of SC-β cell generation and their function.

[0298]

[0325] Thus, in an embodiment, the present disclosure does not involve a step of culturing cells with one or more exogenous polynucleotides or polypeptide reprogramming factors. Thus, in an embodiment, the method of the present disclosure does not involve the introduction of one or more exogenous polynucleotides or polypeptide reprogramming factors, for example, by introducing a transposon, a viral transgenic vector (e.g., a retroviral vector), a plasmid, an mRNA, an miRNA, a peptide, or a fragment of any of these molecules involved in producing an artificial beta cell or otherwise inducing a cell of the present disclosure to differentiate, dedifferentiate, and / or transdifferentiate.

[0299]

[0326] That is, in an embodiment, the method is carried out in the absence 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 specifically associated with inducing differentiation, dedifferentiation, and / or transdifferentiation; polynucleotide sequences that code polypeptide transcription factors, other polypeptide factors specifically associated with inducing differentiation, dedifferentiation, and / or transdifferentiation; mRNA; interference RNA; microRNA, and fragments thereof.

[0300] stem cells

[0327] The term "stem cell" is used herein to refer to a cell (e.g., plant stem cell, vertebrate stem cell) that has the capacity to both self-renew and generate differentiated cell types (Morrison et al. (1997) Cell 88:287-298). In the context of cell ontogeny, the adjectives "differentiated" or "differentiating" are relative terms. A "differentiated cell" is a cell that has progressed further down 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., beta cell precursors), which can differentiate into end-stage cells (i.e., terminally differentiated cells, e.g., beta cells, 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 of specific markers (e.g., proteins, RNA, etc.) and the absence of specific markers. 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 some 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.

[0301]

[0328] 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 ectoderm, mesoderm, or endoderm tissues of a living organism. Plant pluripotent stem cells can give rise to all cell types of a plant (e.g., cells of roots, stems, leaves, etc.).

[0302]

[0329] PSCs in animals can be obtained in several different ways. For example, embryonic stem cells (ESCs) are obtained 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 obtained from somatic cells (Takahashi et al., Cell. 2007 Nov. 30;131(5):861-72; Takahashi et al., Nat Protoc. 2007;2(12):3081-9; Yu et al., Science. 2007 Dec. 21;318(5858):1917-20. Epub 20 Nov. 20). The term PSC refers to pluripotent stem cells regardless of their origin, and therefore the term PSC encompasses the terms ESC and iPSC, as well as the term embryonic germ stem cell (EGSC), which is another example of a PSC. PSCs may be in the form of established cell lines, obtained directly from primary embryonic tissue, or derived from somatic cells.

[0303]

[0330] "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, primates, etc. (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 flattened colonies with large nuclear-cytoplasmic ratios, well-defined 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 expanding hESCs in an undifferentiated form are described in WO99 / 20741, WO01 / 51616, and WO03 / 020920, each of which is incorporated herein in its entirety.

[0304]

[0331] "Embryonic germ stem cells" (EGSCs) or "embryonic germ cells" or "EG cells" refer to germ cells and / or germ cell precursors, such as PSCs derived from primordial germ cells, i.e., cells that can become sperm and eggs. Embryonic germ cells (EG cells) are believed to have similar properties to the embryonic stem cells described above. Examples of methods for generating and characterizing EG cells can be found, for example, in U.S. Patent No. 7,153,684; Matsui, Y. et al. (1992) Cell 70:841; Shamblott, M. et al. (2001) Proc. Natl. Acad. Sci. USA 98:113; Shamblott, M. et al. (1998) Proc. Natl. Acad. Sci. USA, 95:13726; and Koshimizu, U. et al. (1996) Development, 122:1235, each of which is incorporated herein in its entirety.

[0305]

[0332] "Induced pluripotent stem cells" or "iPSCs" refer to PSCs obtained from cells that are not PSCs (i.e., from cells that are differentiated relative to PSCs). iPSCs can be obtained from multiple different cell types, including terminally differentiated cells. iPSCs have an ES cell-like morphology, growing as flat colonies with large nuclear-cytoplasmic ratios, clear borders, and prominent nuclei. In addition, iPSCs express one or more important 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. In general, 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.).

[0306]

[0333] "Somatic cell" refers to any cell of an organism that does not normally give rise to all types of cells of 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 three germ layers of the body, namely 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 mesodermal or endodermal lineages.

[0307]

[0334] In certain instances, the stem cells may be undifferentiated (e.g., cells not committed to a particular lineage) prior to exposure to at least one β-cell maturation factor according to the methods disclosed herein, while in other instances, it may be desirable to differentiate the stem cells into one or more intermediate cell types prior to exposure to at least one cell maturation factor described herein. For example, the stem cells may exhibit morphological, biological, or physical characteristics of undifferentiated cells that can be used to distinguish the stem cells from differentiated cells of embryonic or adult origin. In some instances, the undifferentiated cells may appear in colonies of cells with high nuclear / cytoplasmic ratios and prominent nucleoli in two-dimensional microscopic images. The stem cells may be used by themselves (e.g., substantially free of any undifferentiated cells) or in the presence of differentiated cells. In certain instances, the stem cells may be cultured with suitable nutrients and, if necessary, in the presence of other cells such that the stem cells can grow and differentiate as necessary. For example, embryonic fibroblasts or fibroblast-like cells may be present in the culture to support the growth of the stem cells. Fibroblasts may be present during one stage of stem cell growth, but not necessarily at all stages. For example, fibroblasts may be added to the stem cell culture in a first culture step and not added to the stem cell culture in one or more subsequent culture steps.

[0308]

[0335] The stem cells used in all aspects of the invention may be any cell obtained from any kind of tissue (e.g., embryonic tissue, such as fetal or pre-fetal tissue, or adult tissue) and are characterized by their ability, under appropriate conditions, to produce a variety of cell types, including progeny of all derivatives of at least one of the three germ layers (endoderm, mesoderm, and ectoderm). These cell types may be provided in the form of established cell lines or may 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, 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.

[0309]

[0336] In another embodiment, stem cells can be isolated from tissues including solid tissues.In some embodiments, tissue is skin, adipose tissue (e.g., adipose tissue), muscle tissue, heart or cardiac tissue.In other embodiments, tissue is, for example, but not limited to, umbilical cord blood, placenta, bone marrow, or cartilage.

[0310]

[0337] 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 progenitors, 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 may 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.

[0311]

[0338] 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 a hematopoietic microenvironment. For example, circulating peripheral blood, preferably collected (i.e., mobilized), can be taken from a subject. In some embodiments, the stem cells can be reprogrammed stem cells, such as stem cells obtained from somatic cells or differentiated cells. In such embodiments, the dedifferentiated stem cells can be, for example, but not limited to, neoplastic cells, tumor cells, and cancer cells, or alternatively, artificially reprogrammed cells, such as induced pluripotent stem cells or iPS cells.

[0312]

[0339] In some embodiments, the SC-β cells are hair follicles, keratinocytes, gonadotropes, adrenocorticotropes, thyrotropes, growth hormone cells, prolactinocytes, 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, foveolar cells, K cells, D cells, I cells, goblet cells, Paneth cells, intestinal cells, fold cells, hepatocytes, hepatic stellate cells (e.g., mesodermally derived Kupffer cells), gallbladder cells, central acinar cells, pancreatic stellate cells, pancreatic α cells, pancreatic β cells, pancreatic δ cells, pancreatic F cells (e.g., PP cells), pancreatic ε cells, thyroid (e.g., follicular cells), parathyroid gland (e.g., parathyroid chief cells), eosinophilic cells, urothelial cells, osteoblasts, osteocytes, chondroblasts, chondrocytes, fibroblasts, fibrocytes, myoblasts, myocytes, muscle satellite cells, tendon cells, cardiac myocytes, 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 The cells may be derived from one or more of: 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.

[0313] Pancreatic progenitor cells or precursors

[0340] In some aspects, the 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, for a period of at least 5 days under conditions promoting cell cluster formation to induce differentiation of at least one Pdx1-positive pancreatic progenitor cell in the population into an NKX6.1-positive pancreatic progenitor cell, wherein the NKX6.1-positive pancreatic progenitor cells express NKX6.1.

[0314]

[0341] 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.

[0315] Stem Cell-Derived Beta Cells

[0342] Provided herein in some embodiments are methods for generating SC-beta cells (e.g., mature pancreatic beta cells or beta-like cells) or precursors thereof using stem cells. In certain embodiments, germ cells can be used in place of or together 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 about 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.

[0316]

[0343] In some embodiments, compositions and methods are provided herein for generating SC-β cells (e.g., pancreatic β cells).In general, at least one SC-β cell or its precursor, for example, the pancreatic precursor generated according to the method disclosed herein, can comprise a mixture or combination of different cells, for example, Pdx1 positive pancreatic precursor, Pdx1 and NKX6.1 co-expressing pancreatic precursor, Ngn3 positive endocrine precursor, insulin positive endocrine cell (e.g., β-like cell), and insulin positive endocrine cell, and / or other multipotent or stem cell, etc.

[0317]

[0344] At least one SC-β cell or its precursor can be generated according to any culture protocol suitable for differentiating stem cells or pluripotent cells to a desired stage of differentiation. In some embodiments, at least one SC-β cell or its precursor is generated by culturing at least one pluripotent cell for a period of time and under conditions suitable for the differentiation of at least one pluripotent cell into at least one SC-β cell or its precursor. In some embodiments, the generation of SC-β cell or its precursor is enhanced by contacting the cell with one or more agents disclosed herein (e.g., one or more of glutamine, glutamate, carnitine, taurine, β-hydroxybutyrate, biotin, acetate, and formate).

[0318]

[0345] In some embodiments, at least one SC-β cell or its precursor is a substantially pure population of SC-β cells or its precursor. In some embodiments, the population of SC-β cells or its precursor comprises a mixture of pluripotent cells or differentiated cells. In some embodiments, the population of SC-β cells or its precursor is substantially free of or devoid of embryonic stem cells, pluripotent cells, or iPS cells.

[0319]

[0346] 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 can be reprogrammed into induced pluripotent stem cells for further differentiation to generate at least one SC-β cell or a precursor 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 to SC-β cells by the methods disclosed herein.

[0320]

[0347] In some embodiments, at least one SC-β cell or a precursor thereof is maintained in culture by methods known to those of skill in the art, and in some embodiments, is expanded and then converted to an SC-β cell by the methods disclosed herein.

[0321]

[0348] Furthermore, at least one SC-β cell or its precursor, e.g., pancreatic progenitor, can be from any mammalian species, including, but not limited to, mouse, bovine, ape, porcine, equine, ovine, or human cells. For clarity and simplicity of description, the method description herein refers to at least one SC-β cell or its precursor from a mammal, but it should be understood that all methods described herein can be easily applied to at least one SC-β cell or its precursor from other cell types. In some embodiments, at least one SC-β cell or its precursor is from a human individual.

[0322]

[0349] In some embodiments, the cell population of the present disclosure comprises a plurality of cells that express both NKX6.1 and ISL1. For example, at least about 10%, 20%, 30%, 35%, 38%, 40%, 42%, 44%, 46%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the cells can express ISL1. In some cases, at least about 35% of the cells express NKX6.1 and ISL1. In some cases, at least about 38% of the cells express NKX6.1 and ISL1. In some cases, at least about 40% of the cells express NKX6.1 and ISL1. In some cases, at least about 42% of the cells express NKX6.1 and ISL1. In some cases, at least about 44% of the cells express NKX6.1 and ISL1. In some cases, at least about 46% of cells express NKX6.1 and ISL1.In some cases, at least about 50% of cells express NKX6.1 and ISL1.The compositions and methods of the present disclosure can be useful for increasing the percentage of cells that express both NKX6.1 and ISL1.For example, after contacting cells that undergo differentiation into SC-β cells according to the methods of the present disclosure, the percentage of cells that express NKX6.1 and ISL1 can increase compared to the method that lacks one or more agents or contains different (e.g., lower) concentrations of agents.

[0323]

[0350] In some cases, the cell population of the present disclosure contains a limited percentage of cells that are negative for both NKX6.1 and ISL1. For example, the cell population may contain up to 2%, up to 4%, up to 6%, up to 8%, up to 10%, up to 12%, up to 14%, up to 16%, up to 18%, up to 20%, up to 22%, up to 22%, up to 25%, or up to 30% ISL1-negative, NKX6.1-negative cells.

[0324] Cell clusters of stem cell-derived beta cells

[0351] In some aspects, cell clusters are provided herein that are similar to the functions and characteristics of endogenous pancreatic islets.Such cell clusters can mimic the functions of endogenous pancreatic islets, such as regulating the metabolism of a subject, such as 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 with close cell-cell contact, and in some cases, the cells in the cluster can adhere to each other.

[0325]

[0352] 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, the cell clusters comprise between 10 and 10,000 cells, between 50 and 10,000 cells, between 100 and 10,000 cells, between 100 and 10,000 cells, between 1,000 and 10,000 cells, between 500 and 10,000 cells, between 500 and 5,000 cells, between 500 and 2,500 cells, 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 cells, no more than 90,000 cells, no more than 80,000 cells, no more than 70,000 cells, no more than 60,000 cells, no more than 50,000 cells, no more than 40,000 cells, no more than 30,000 cells, no more than 20,000 cells, no more than 10,000 cells, no more than 7,000 cells, no more than 5,000 cells, no more than 3,000 cells, no more than 2,000 cells, or no more than 1,000 cells.

[0326]

[0353] The cell clusters herein can include at least one non-native cell, e.g., a non-native pancreatic beta cell. A non-native cell (e.g., a non-native pancreatic beta cell) can share characteristics of an endogenous cell (e.g., an endogenous mature pancreatic beta cell), but differ in certain aspects (e.g., gene expression profile). A non-native cell can be a genetically modified cell. A non-native cell can be a cell differentiated from a progenitor cell, e.g., a stem cell. The stem cell can be an embryonic stem cell (ESC) or an induced pluripotent stem cell (iPSC). In some cases, a non-native cell can be a cell differentiated in vitro from a progenitor cell. In some cases, a non-native cell can be a cell differentiated in vivo from a progenitor cell. For example, a cell cluster can include at least one non-native pancreatic beta cell. A non-native pancreatic beta cell can be one described in U.S. Patent Application Nos. 14 / 684,129 and 14 / 684,101, which are incorporated herein in their entirety. A cell cluster can include a plurality of non-native pancreatic beta cells. In some cases, at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 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.

[0327]

[0354] A cell cluster can include one or more cells that express at least one marker of an endogenous cell, such as an endogenous mature pancreatic beta cell. 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 particular 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 particular cell type, and thus the marker can be referred to as a marker of a cell type. For example, the insulin gene can be referred to as a marker of beta cells. In some cases, the 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.

[0328]

[0355] The cell cluster can include one or more cells that express one or more markers of endogenous cells, such as endogenous mature pancreatic β cells.For example, the cell cluster can include 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, such as endogenous mature pancreatic β cells.In some cases, the cell cluster includes cells that express NKX6.1 and C-peptide, both of which can be markers of β cells.

[0329]

[0356] 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%, 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 population of cells, such as cells of various types. For example, the 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.

[0330]

[0357] For example, the cell clusters herein can comprise a plurality of cells expressing 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 a cell cluster can express one or more markers of endogenous mature pancreatic beta cells.

[0331]

[0358] The cell cluster can include 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 include about 90% cells that express CHGA. In some cases, the cell cluster can include about 95% cells that express CHGA. In certain cases, all of the cells in the cell cluster can express CHGA.

[0332]

[0359] A cell cluster can include 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 the cells in the cell cluster can express NKX6.1.

[0333]

[0360] The cell cluster can include a plurality of cells expressing ISL1. 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 ISL1. In some cases, all the cells in the cell cluster can express ISL1.

[0334]

[0361] 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.

[0335]

[0362] A cell cluster can include 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 the cell cluster can express C-peptide. In some cases, at least about 35% of the cells in the cell cluster can express NKX6.1 and C-peptide. In some cases, at least about 40% of the cells in the cell cluster can express NKX6.1 and C-peptide. In some cases, at least about 35% of the cells in the cell cluster can express NKX6.1 and C-peptide. In some cases, a cell cluster can include about 60% of cells that express NKX6.1 and C-peptide. In some cases, a cell cluster can include about 75% of cells that express NKX6.1 and C-peptide. In some cases, all of the cells in the cell cluster can express NKX6.1 and C-peptide.

[0336]

[0363] The cell cluster can include a plurality of cells expressing both NKX6.1 and ISL1. For example, at least about 10%, 20%, 30%, 35%, 38%, 40%, 42%, 44%, 46%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the cells in the cell cluster can express NKX6.1 and ISL1. In some cases, at least about 35% of the cells in the cell cluster can express NKX6.1 and ISL1. In some cases, at least about 40% of the cells in the cell cluster can express NKX6.1 and ISL1. In some cases, at least about 35% of the cells in the cell cluster can express NKX6.1 and ISL1. In some cases, the cell cluster can include about 60% of the cells expressing NKX6.1 and ISL1. In some cases, the cell cluster can include about 75% of the cells expressing NKX6.1 and ISL1. In some cases, all cells in a cell cluster can express NKX6.1 and ISL1.

[0337]

[0364] The cell clusters can contain a limited percentage of cells that are negative for both NKX6.1 and ISL1. For example, the cell clusters can contain up to 2%, up to 4%, up to 6%, up to 8%, up to 10%, up to 12%, up to 14%, up to 16%, up to 18%, up to 20%, up to 22%, up to 22%, up to 25%, or up to 30% ISL1-negative, NKX6.1-negative cells.

[0338]

[0365] Cell clusters may contain few or no stem cells or progenitor cells, such as pancreatic progenitor cells.For example, 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 expressing LIN28.In some examples, 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 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 Ki67.

[0339]

[0366] 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.

[0340]

[0367] In some cases, the cell cluster 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 cluster may contain about 2% cells expressing SOX9. In some cases, the cell cluster may contain about 6% cells expressing SOX9. In some cases, the cell cluster may contain about 1.2% cells expressing SOX9.

[0341]

[0368] 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 in vitro GSIS responses to glucose load. In some cases, the cell clusters exhibit in vitro GSIS responses to multiple glucose loads, for example, sequential glucose loads. For example, the cell clusters can exhibit in vitro GSIS responses to at least 2, 3, 4, 5, 6, 7, 8, 9, 10 sequential glucose loads.

[0342]

[0369] The cell clusters provided herein can include at least one cell that exhibits in vitro GSIS. For example, at least one cell in the cell cluster may be referred to as a mature pancreatic β cell. In some cases, at least one cell is a non-native pancreatic β cell. In some cases, at least one cell is a pancreatic β cell that resembles a native / endogenous β cell. In some cases, the cell exhibits in vitro glucose-stimulated insulin secretion (GSIS) response. In some cases, at least one cell exhibits GSIS response to at least one glucose load. In some cases, the cell exhibits GSIS response to at least two successive glucose loads. In some cases, the cell exhibits GSIS response to at least three successive glucose loads.

[0343]

[0370] As provided herein, the cell clusters can exhibit a GSIS stimulation index similar to that of endogenous pancreatic islets. The stimulation index of the cell clusters or cells can be characterized by the ratio of insulin secreted in response to high glucose concentrations compared to low glucose concentrations. For example, the stimulation index of the cell clusters or cells 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 the cell clusters or cells provided herein is 1 or more, or 1.1 or more, or 1.3 or more, or 2 or more, or 2.3 or more, or 2.6 or more. In some cases, the cell clusters or cells exhibit cytokine-induced apoptosis in response to cytokines. In some cases, the cytokines include 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 clusters or cells is enhanced in response to antidiabetic agents. In some cases, the antidiabetic agent comprises a secretagogue selected from the group consisting of incretin mimetics, sulfonylureas, meglitinides, and combinations thereof. In some cases, the cell clusters or cells are 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 insulin granules of endogenous mature pancreatic beta cells under electron microscopy. In some cases, the cell clusters or cells exhibit a low replication rate. In some cases, the cell clusters or cells exhibit glucose-stimulated Ca2+ stimulatory activity of endogenous mature pancreatic beta cells. 2+In some cases, the cell cluster or cell exhibits a GSCF response similar to glucose load (GSCF). In some cases, the cell cluster or cell exhibits a GSCF response to at least one glucose load. In some cases, the cell cluster or cell exhibits a GSCF response to at least two glucose loads. In some cases, the cell cluster or cell exhibits an increased calcium flux. In some cases, the increased calcium flux comprises an increased amount of influx or a ratio of influx of low glucose concentration to high glucose concentration.

[0344]

[0371] The cell clusters provided herein can exhibit biphasic insulin secretion in response to high glucose concentration stimulation, similar to endogenous pancreatic islets, e.g., human pancreatic islets. Biphasic insulin secretion can be a phenomenon characteristic 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 high glucose concentration loading, 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., 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, during 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 loading continues, or relatively longer than the first phase. Such a biphasic insulin secretion pattern may result from intrinsic cell signaling changes characteristic of mature native pancreatic β-cells.

[0345]

[0372] When transplanted into a subject, the cell clusters can exhibit one or more in vivo GSIS responses when exposed to a glucose load(s). The cell clusters herein may be capable of exhibiting an in vivo GSIS response 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 endogenous pancreatic islets. The term "about" as used throughout this application in reference to a numerical value may include values ​​in a range of plus or minus 10% from that value. For example, an amount of "about 10" includes an amount of 9 to 11. For example, the term "about" in reference to a 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.

[0346]

[0373] 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).

[0347]

[0374] The GSIS of cell clusters can be measured by stimulation index. The stimulation index of cell clusters can be equal to the ratio of insulin secreted in response to high glucose concentration compared to insulin secreted in response to low glucose concentration. The cell clusters can have a stimulation index similar to that of endogenous pancreatic islets. In some cases, the cell clusters have 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.

[0348]

[0375] 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 0.23μIU / 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 challenge (e.g., a high concentration, e.g., 20 mM glucose) can be in the range of 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.

[0349]

[0376] 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 about 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.

[0350]

[0377] The cell cluster may be of a similar size to an endogenous pancreatic islet. For example, the cell cluster may have a similar diameter to an endogenous pancreatic islet. The diameter of the cell cluster may 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 may be about 75 μm to about 250 μm. The diameter of the cell cluster may be up to 100 μm.

[0351]

[0378] In some embodiments, at least about 40%, 50%, 60%, 70%, 80%, or 90% of the 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.

[0352]

[0379] In some embodiments, at least about 40%, 50%, 60%, 70%, 80%, or 90% of the cell clusters have a diameter of about 80-150, about 100-150, about 120-150, about 140-150, about 80-130, about 100-130, about 120-130, about 80-120, about 90-120, or about 100-120 μm.

[0353]

[0380] In some embodiments, the cell clusters have a mean or median diameter of at most 120, at most 130, at most 140, at most 150, at most 160, or at most 170 μm.

[0354]

[0381] In some embodiments, the cell clusters have a mean or median diameter of about 80-150, about 100-150, about 120-150, about 140-150, about 80-130, about 100-130, about 120-130, about 80-120, about 90-120, or about 100-120 μm.

[0355]

[0382] In some embodiments, at least about 50%, at least about 60%, at least about 70%, or at least about 80% of the cell clusters have a diameter of less than 150 μm.

[0356]

[0383] In some embodiments, at least about 50%, at least about 60%, or at least about 70% of the cell clusters have a diameter of less than 140 μm.

[0357]

[0384] In some embodiments, at least about 50%, at least about 60%, or at least about 70% of the cell clusters have a diameter of less than 130 μm.

[0358]

[0385] The cell cluster may contain few or no dead cells. The cell cluster may be of a size that allows for 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 cluster may have less than about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% dead cells, e.g., dead cells in the center. In some cases, the cell cluster may have no dead cells at all. The dead cells may be apoptotic cells, narcotic cells, or any combination thereof.

[0359]

[0386] The cell cluster can include one or more types of cells. In some cases, the cell cluster can include one or more types of pancreatic cells. For example, the cell cluster can include one or more pancreatic β cells, pancreatic α cells, pancreatic Δ cells, pancreatic γ cells, and any combination thereof. In some cases, the pancreatic cells can be non-native pancreatic cells, such as cells derived from stem cells, such as ESCs and / or iPSCs. In some cases, the cell cluster can also include one or more progenitors 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.

[0360]

[0387] 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.

[0361]

[0388] Insulin secretion from the cell clusters herein can be enhanced by antidiabetic drugs (e.g., antidiabetic drugs acting on pancreatic β cells ex vivo, in vitro, and / or in vivo). The present disclosure can contemplate any known antidiabetic drug. In some cases, insulin secretion from the cell clusters can be enhanced by secretagogues. Secretagogues can be incretin mimetics, sulfonylureas, meglitinides, and combinations thereof.

[0362]

[0389] The cell cluster can include a single hormone. For example, the cell cluster can include pancreatic cells that are single hormone (e.g., pancreatic β cells, pancreatic α cells, pancreatic β cells, pancreatic Δ cells, or pancreatic γ cells). In some cases, the cell cluster includes insulin-secreting non-native pancreatic cells that are single hormone. The cell cluster can include multiple hormones. In some cases, the cell cluster includes single hormone and multiple hormone cells.

[0363]

[0390] The 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, the cell cluster can include cells that encapsulate crystalline insulin granules similar to those of endogenous mature pancreatic beta cells, e.g., as detected by electron microscopy. The 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%, 90% of the cells in the cell cluster can encapsulate crystalline insulin granules similar to those of endogenous mature pancreatic beta cells. In some cases, 100% of the cells in the cell cluster encapsulate crystalline insulin granules similar to those of endogenous mature pancreatic beta cells.

[0364]

[0391] Cell clusters respond to glucose-stimulated calcium (Ca) responses 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 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.

[0365]

[0392] The cell cluster can include cells originating from any species. For example, the cell cluster can include cells from mammalian species, including, but not limited to, mouse, cow, ape, pig, horse, sheep, or human cells. In some cases, at least one cell in the cell cluster is a human cell.

[0366]

[0393] Provided herein also includes compositions comprising cell clusters as disclosed throughout this application. In addition to the cell clusters, the compositions can further include 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 includes 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. The biodegradable polymer can also be a natural polymer, 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(vinyl imidazoles), chlorosulfonated polyolefins, and polyethylene oxides.

[0367] Dissociated cell compositions and methods for producing cell clusters of stem cell derived beta cells - Patents.com

[0394] Further disclosed herein are methods for generating endogenous tissues or cell clusters, such as cell clusters that resemble the functions and characteristics of endogenous pancreatic islets.

[0368]

[0395] The method of the present disclosure may include dissociating a first cell cluster and reaggregating the dissociated cells into a second cell cluster, where the second cell cluster more closely resembles the function and characteristics of an endogenous tissue or cell cluster, e.g., an endogenous pancreatic islet, compared to the first cell cluster. The term "reaggregation" and its grammatical equivalents, as used herein, may mean that the cluster dissociates into smaller clusters or single cells, and the dissociated cells then form new intercellular junctions to form new clusters. The method of the present disclosure may be used to generate cell clusters 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 cluster may be an in vitro cell cluster, e.g., a cluster formed by a suspension of a single cell in a culture medium in vitro. Optionally, the first cell cluster can be an ex vivo cell cluster, for example, a cell cluster formed in a living organism and isolated from said organism.For example, the first cell cluster to which the method provided herein is applicable can be a human pancreatic islet.Optionally, the first cell cluster can be a cadaveric pancreatic islet.In some embodiments, the dissociated cells are previously frozen.

[0369]

[0396] The method provided herein can enrich pancreatic cells, such as pancreatic β cells, endocrine cells, or endocrine precursor cells, in cell clusters. In some examples, the method of the present disclosure can reduce or eliminate stem cells or pancreatic precursor cells from cell clusters. Optionally, the second cell cluster comprises a higher percentage of cells expressing chromogranin A compared to the first cell cluster. Optionally, the second cell cluster comprises a higher percentage of cells expressing NKX6.1 and C-peptide compared to the first cell cluster. Optionally, the second cell cluster comprises a higher percentage of cells expressing NKX6.1 and ISL1 compared to the first cell cluster. Optionally, the second cell cluster comprises a lower percentage of cells negative for NKX6.1 and C-peptide compared to the first cell cluster. Optionally, the second cell cluster comprises a lower percentage of cells expressing SOX2 compared to the first cell cluster. Optionally, the second in vitro cell cluster comprises a lower percentage of cells that express SOX9 compared to the first cell cluster.

[0370]

[0397] Optionally, the medium comprises a thyroid hormone signaling pathway activator and a transforming growth factor beta (TGF-β) signaling pathway inhibitor. Optionally, the medium comprises 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 as provided herein (reaggregation medium) may not comprise small molecule compounds. For example, the reaggregation medium may not comprise a thyroid hormone signaling pathway activator. Optionally, the reaggregation medium does not comprise triiodothyronine (T3) or only comprises trace amounts of T3. The reaggregation medium may not comprise a TGFβ signaling pathway inhibitor. Optionally, the reaggregation medium does not comprise Alk5 inhibitor (Alk5i) or only comprises trace amounts of Alk5i.

[0371]

[0398] In some cases, the reaggregation medium includes one or more of a one-carbon metabolic pathway intermediate (e.g., formate), an acetyl-CoA related metabolite (e.g., acetate), a vitamin (e.g., biotin), an HDAC inhibitor (e.g., β-hydroxybutyrate), a redox homeostasis regulator (e.g., taurine), glutamate, and carnitine.

[0372]

[0399] In some cases, the reaggregation medium contains two, three, four, five, or six of a one-carbon metabolic pathway intermediate (e.g., formate), an acetyl-CoA related metabolite (e.g., acetate), a vitamin (e.g., biotin), an HDAC inhibitor (e.g., β-hydroxybutyrate), a redox homeostasis regulator (e.g., taurine), glutamate, and carnitine.

[0373]

[0400] Optionally, the reaggregation medium contains one-carbon metabolic pathway intermediates (e.g., formate), acetyl-CoA related metabolites (e.g., acetate), vitamins (e.g., biotin), HDAC inhibitors (e.g., β-hydroxybutyrate), redox homeostasis regulators (e.g., taurine), glutamate, and carnitine.

[0374]

[0401] In some embodiments, the methods of the disclosure include contacting a cell population (e.g., NKX6.1 positive, ISL1 positive, insulin positive cells) with one or more of serum albumin protein, TGF-β signaling pathway inhibitors, TH signaling pathway activators, protein kinase inhibitors, ROCK inhibitors, BMP signaling pathway inhibitors, epigenetic modifying compounds, acetyl-CoA related metabolites, vitamins, histone deacetylase inhibitors (HDACi), redox homeostasis regulators, one-carbon metabolic pathway intermediates, glutamate, and / or carnitine for a first period of 1, 2, 3, 4, 5, 6, or 7 days (e.g., 4 days). In some embodiments, the method of the present disclosure further comprises contacting the cell population after the first period with one or more of serum albumin protein, acetyl-CoA-related metabolites, vitamins, histone deacetylase inhibitors (HDACi), redox homeostasis regulators, one-carbon metabolic pathway intermediates, glutamate, and / or carnitine for a second period of 1, 2, 3, 4, 5, 6, or 7 days (e.g., 3 days) or more in the absence of TGF-β signaling pathway inhibitors, TH signaling pathway activators, protein kinase inhibitors, ROCK inhibitors, BMP signaling pathway inhibitors, and / or epigenetic modification compounds. In some embodiments, the cells are contacted with the same concentration of serum albumin (e.g., 0.05% HSA) during the second period compared to the first period. In some embodiments, the cells are contacted with a higher concentration of serum albumin during the second period compared to the first period. In some embodiments, the composition of the present disclosure is ZnSO 4 In some embodiments, the compositions of the present disclosure further comprise DMEM / F12.

[0375]

[0402] In some embodiments, the methods of the disclosure include contacting a cell population (e.g., NKX6.1-positive, ISL1-positive, insulin-positive cells) with one or more of HSA, Alk5 inhibitor II, GC-1, staurosporine, thiazovivin, LDN193189, DZNEP, taurine, acetate, beta-hydroxybutyrate, biotin, carnitine, glutamate, and formate for a first period of 1, 2, 3, 4, 5, 6, or 7 days (e.g., 4 days). In some embodiments, the disclosed method further comprises contacting the cell population after the first period with one or more of HSA, taurine, acetate, beta-hydroxybutyrate, biotin, carnitine, glutamate, and formate for a second period of 1, 2, 3, 4, 5, 6, or 7 days (e.g., 3 days) or more in the absence of Alk5 inhibitor II, GC-1, staurosporine, thiazovivin, LDN193189, and DZNEP. In some embodiments, the disclosed composition comprises ZnSO 4 In some embodiments, the composition of the present disclosure further comprises DMEM / F12. In some embodiments, the cells are contacted with the same concentration of serum albumin (e.g., 0.05% HSA) during the second period compared to the first period. In some embodiments, the cells are contacted with a higher concentration of HSA (e.g., about 1.0%) during the second period compared to the first period (e.g., about 0.05%).

[0376]

[0403] In some embodiments, the compositions and methods disclosed herein improve cell recovery or yield after cells are dissociated and then reaggregated. The increased cell recovery may include an increase in the number or percentage of viable cells. The increased cell recovery may include an increase in the number or percentage of a population of interest (e.g., ISL1-positive and NKX6.1-positive cells, or another population disclosed herein). In some embodiments, the increased cell recovery is achieved after contacting the cells with a combination of agents disclosed herein. For example, in some embodiments, contacting the cells with any one or more of the one-carbon metabolic pathway intermediates (e.g., formate), acetyl-CoA-related metabolites (e.g., acetate), vitamins (e.g., biotin), HDAC inhibitors (e.g., β-hydroxybutyrate), redox homeostasis regulators (e.g., taurine), glutamine, glutamate, and / or carnitine disclosed herein increases cell recovery. In some embodiments, the cell recovery increases when the agent is contacted with the cells before dissociation. In some embodiments, cell recovery is increased when the agent is contacted with the cells after dissociation.

[0377]

[0404] In some embodiments, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, or at least 60% of the total viable cells present prior to dissociation are recovered as viable cells when assessed following reaggregation, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after dissociation, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after thawing of dissociated and cryopreserved cells.

[0378]

[0405] In some embodiments, the yield of total viable cells obtained by the methods of the present disclosure is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 4-fold, or at least 5-fold higher compared to a method that does not utilize an agent or combination of agents disclosed herein.

[0379]

[0406] In some embodiments, the yield of total viable cells obtained by the methods of the disclosure is, for example, about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days after dissociation, or 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days after thawing dissociated and cryopreserved cells. is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 4-fold, or at least 5-fold higher after reaggregation when assessed after 14 days, compared to a method not utilizing an agent or combination of agents disclosed herein. In some embodiments, the yield of total viable cells is increased when the agent or combination of agents disclosed herein is contacted with the cells prior to earlier dissociation of the clusters. In some embodiments, the yield of total viable cells is increased when the agent or combination of agents disclosed herein is contacted with the cells after earlier dissociation of the clusters.

[0380]

[0407] In some embodiments, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, or at least 60% of the cells of the population of the present disclosure present before dissociation (e.g., NKX6.1 opositive and ISL1 positive cells, or another population disclosed herein) are recovered as viable cells when assessed after reaggregation, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after dissociation, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after thawing of dissociated and cryopreserved cells.

[0381]

[0408] In some embodiments, the yield of cells of the population of the disclosure (e.g., NKX6.1 positive (poositive) and ISL1 positive cells, or another population disclosed herein) obtained by the method of the disclosure is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 4-fold, or at least 5-fold higher than a method that does not utilize an agent or combination of agents disclosed herein.

[0382]

[0409] In some embodiments, the yield of cells of the population of the disclosure (e.g., NKX6.1 positive and ISL1 positive cells, or another population disclosed herein) obtained by the methods of the disclosure is, for example, about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days after dissociation, or 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days after thawing dissociated and cryopreserved cells. When assessed after 10, 11, 12, 13, or 14 days, the yield is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 4-fold, or at least 5-fold higher after reaggregation compared to a method that does not utilize an agent or combination of agents disclosed herein. In some embodiments, the yield is increased when the agent or combination of agents is contacted with the cells prior to earlier dissociation of the clusters. In some embodiments, the yield is increased when the agent or combination of agents is contacted with the cells after earlier dissociation of the clusters.

[0383]

[0410] In some embodiments, the compositions and methods disclosed herein increase the percentage of cells in a cluster that express both NKX6.1 and ISL1 after dissociation and reaggregation.For example, at least about 10%, 20%, 30%, 35%, 38%, 40%, 42%, 44%, 46%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of cells in a cell cluster can express NKX6.1 and ISL1 after dissociation and reaggregation as disclosed herein.In some cases, at least about 35% of cells in a cell cluster can express NKX6.1 and ISL1.In some cases, at least about 38% of cells in a cell cluster can express NKX6.1 and ISL1.In some cases, at least about 40% of cells in a cell cluster can express NKX6.1 and ISL1. In some cases, at least about 45% of cells in cell clusters can express NKX6.1 and ISL1. In some cases, at least about 50% of cells in cell clusters can express NKX6.1 and ISL1. In some embodiments, the percentage of cells in reaggregated clusters that express both NKX6.1 and ISL1 increases when cells are contacted with the agent or combination of agents disclosed herein before dissociation of earlier clusters. In some embodiments, the percentage of cells in reaggregated clusters increases when cells are contacted with the agent or combination of agents disclosed herein after dissociation of earlier clusters.

[0384]

[0411] Dissociation of the first cell cluster can be performed using methods known in the art. Non-limiting exemplary methods for dissociating cell clusters include mechanical dissociation such as mechanical dissociation 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™, and the like, or combinations thereof. After dissociation, the cells of the first cell cluster can be in a cell suspension, e.g., a single cell suspension. The term "suspension" as used herein can refer to a cell culture condition in which the cells are not attached to a solid support. Cells growing in suspension can be agitated during growth using equipment well known to those skilled in the art.

[0385]

[0412] In some embodiments, the present disclosure provides a composition comprising dissociated 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 ZnSO 4In 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.

[0386]

[0413] 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 monoglyceride to free fatty acid ratio 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.

[0387]

[0414] 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.

[0388]

[0415] 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.

[0389]

[0416] 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.

[0390]

[0417] Provided herein are compositions comprising an insulin positive endocrine cell population and an agent that inhibits the conversion of monoglycerides to free fatty acids.

[0391]

[0418] 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 an acetyl-CoA-related metabolite (e.g., 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 a histone deacetylase inhibitor (HDACi; e.g., β-hydroxybutyrate). 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 glutamine (e.g., L-glutamine). 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 redox homeostasis regulator (e.g., 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 a one-carbon metabolic pathway intermediate (e.g., 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 a vitamin (e.g., biotin). In some embodiments, the present disclosure provides a composition comprising a plurality of dissociated cells (e.g., dissociated insulin-positive endocrine precursor cells) and DMEM / F12. 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 (e.g., ZnSO 4In some embodiments, the compositions of the present disclosure further comprise 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 comprise a 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. 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%, 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. In some embodiments, the composition of the present disclosure comprises a TGF-β pathway inhibitor. In some embodiments, the TGF-β pathway inhibitor is Alk5i (SB505124) or a derivative thereof. In some embodiments, the composition of the present disclosure comprises 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 composition of the present disclosure comprises a ROCK inhibitor. In some embodiments, the ROCK inhibitor is thiazovivin. In some embodiments, the composition of the present disclosure comprises a histone methyltransferase inhibitor. In some embodiments, the histone methyltransferase inhibitor is 3-deazaneplanocin A hydrochloride.In some embodiments, the compositions of the present disclosure comprise 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 does not comprise a gamma secretase inhibitor (e.g., XXI). In some embodiments, the dissociated insulin-positive endocrine precursor cells have been previously frozen.

[0392]

[0419] 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.

[0393]

[0420] In some embodiments, the 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%, 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.

[0394]

[0421] In some embodiments, the disclosure provides a composition comprising a plurality of cell clusters, the cell clusters comprising insulin positive cells, and at least about 40%, 50%, 60%, 70%, 80%, or 90% of the cell clusters have a diameter of about 80-150, about 100-150, about 120-150, about 140-150, about 80-130, about 100-130, about 120-130, about 80-120, about 90-120, or about 100-120 μm.

[0395]

[0422] In some embodiments, the disclosure provides a composition comprising a plurality of cell clusters, wherein the cell clusters comprise insulin positive cells, and wherein the cell clusters have a mean or median diameter of up to 120, up to 130, up to 140, up to 150, up to 160, or up to 170 μm.

[0396]

[0423] In some embodiments, the disclosure provides a composition comprising a plurality of cell clusters, the cell clusters comprising insulin positive cells, and the cell clusters having a mean or median diameter of about 80-150, about 100-150, about 120-150, about 140-150, about 80-130, about 100-130, about 120-130, about 80-120, about 90-120, or about 100-120 μm.

[0397]

[0424] In some embodiments, the disclosure provides a composition comprising a plurality of cell clusters, wherein the cell clusters comprise insulin positive cells, and at least about 50%, at least about 60%, at least about 70%, or at least about 80% of the cell clusters have a diameter of less than 150 μm.

[0398]

[0425] In some embodiments, the disclosure provides a composition comprising a plurality of cell clusters, wherein the cell clusters comprise insulin positive cells, and at least about 50%, at least about 60%, at least about 70%, or at least about 80% of the cell clusters have a diameter of less than 140 μm.

[0399]

[0426] In some embodiments, the disclosure provides a composition comprising a plurality of cell clusters, wherein the cell clusters comprise insulin positive cells, and at least about 50%, at least about 60%, at least about 70%, or at least about 80% of the cell clusters have a diameter of less than 150 μm.

[0400]

[0427] In some embodiments, the disclosure provides a composition comprising a plurality of cell clusters, the cell clusters comprising insulin positive cells, and 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 have an insulin posit...

Claims

1. An in vitro composition comprising a plurality of insulin-positive endocrine precursor cells and one or more of an acetyl-CoA-related metabolite, an HDAC inhibitor, a redox homeostasis regulator, a one-carbon metabolic pathway intermediate, glutamate, or L-carnitine.

2. The composition of claim 1 comprising β-hydroxybutyrate.

3. 3. The composition of claim 1 or 2, comprising taurine.

4. The composition according to any one of claims 1 to 3, comprising a formate.

5. The composition according to any one of claims 1 to 4, comprising a vitamin.

6. The composition according to any one of claims 1 to 5, comprising biotin.

7. Glutamine, acetate, beta-hydroxybutyrate, taurine, formate, and The composition according to any one of claims 1 to 6, comprising biotin.

8. The composition according to any one of claims 2 to 7, further comprising L-carnitine.

9. The composition according to any one of claims 1 to 7, comprising L-carnitine.

10. The composition of any one of claims 1 to 9, further comprising a BMP signaling pathway inhibitor.

11. The composition of claim 10, wherein the BMP signaling pathway inhibitor is LDN193189 or DMH-1 or a derivative thereof.

12. The composition according to any one of claims 1 to 11, further comprising a ROCK inhibitor.

13. The composition of claim 12, wherein the ROCK inhibitor is thiazovivin, Y-27632, fasudil / HA1077, or 14-1152, or a derivative thereof.

14. The composition of any one of claims 1 to 13, further comprising a histone methyltransferase inhibitor.

15. 15. The composition of claim 14, wherein the histone methyltransferase inhibitor is 3-deazaneplanocin A hydrochloride, Bix-01294, UNC0638, BRDD4770, EPZ004777, AZ505, PDB4e47, valproic acid, vorinostat, romidepsin, entinostat abexinostat, gibinostat, mocetinostat, butyrate, a serine protease inhibitor, a serpin, or a derivative thereof.

16. The composition of any one of claims 1 to 15, further comprising a TGF-β pathway inhibitor.

17. 17. The composition of claim 16, wherein the TGF-β pathway inhibitor is ALK5 inhibitor II, A83-01, 431542, D4476, GW788388, LY364947, LY580276, SB525334, SB505124, SD208, GW6604, or GW788388, or a derivative thereof.

18. The composition of any one of claims 1 to 17, further comprising a thyroid hormone signaling pathway activator.

19. 19. The composition of claim 18, wherein the thyroid hormone signaling pathway activator is GC-1, T3, T1AM, T0AM, Triprop, L-940901, CGS 23425, KB-141, or DITPA, or a derivative thereof.

20. The composition according to any one of claims 1 to 19, wherein the plurality of insulin-positive endocrine precursor cells are in cell clusters.

21. 21. The composition of claim 20, wherein at least about 40%, 50%, 60%, 70%, 80%, or 90% of the 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.

22. 22. The composition of any one of claims 20 or 21, wherein the cell clusters have a mean or median diameter of about 80-150, about 100-150, about 120-150, about 140-150, about 80-130, about 100-130, about 120-130, about 80-120, about 90-120, or about 100-120 μm.

23. 23. The composition of any of claims 1 to 22 or claim 1, comprising at least 35%, at least 38%, at least 40%, at least 42%, at least 44%, or at least 46% ISL1 positive, NKX6.1 positive cells.

24. 24. The composition of any one of claims 1 to 23, comprising a cell that has been engineered to contain a genomic disruption in at least one genetic sequence, said disruption reducing or eliminating expression of a protein encoded by said genetic sequence.

25. 25. The composition of claim 24, wherein the at least one gene sequence is any one or more of beta-2-microglobulin (B2M), HLA-A, HLA-B, or CIITA.