Improvement of beta cell differentiation

Abstract

This invention provides compositions and methods for the improved generation of SC-β cells in vitro. [Solution] Disclosed are novel formulations and differentiation methods that result in SC island clusters having advantageous features 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 in vitro function of SC-β cells, and improved post-transplant survival, improved function, and reduced immunogenicity. The compositions and methods of this disclosure can be used in the production of SC islands for human therapeutic use. In one embodiment, an in vitro composition is provided comprising a plurality of PDX1-positive, NKX6.1-positive, insulin-negative endocrine progenitor cells, glutamine, and acetate. The composition preferably further comprises β-hydroxybutyrate, taurine, formate, and biotin.

Description

[Technical Field]

[0001] Cross-reference of related applications

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

[0002]

[0002] Pancreatic or islet transplantation has been used to treat diabetes, such as type 1 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 hindered the effective implementation of this therapy. Artificial pancreases or islets can serve as an alternative source of transplantable islets. [Overview of the initiative]

[0003] This specification includes the disclosure of the following inventions. [Item 1] An in vitro composition comprising multiple PDX1-positive, NKX6.1-positive, insulin-negative cells, and one or more of acetyl-CoA-related metabolites, HDAC inhibitors, redox homeostasis regulators, or monocarbonate pathway intermediates. [Item 2] An in vitro composition comprising multiple PDX1-positive, NKX6.1-positive, insulin-negative cells and acetyl-CoA-related metabolites. [Item 3] An in vitro composition comprising multiple PDX1-positive, NKX6.1-positive, insulin-negative cells, and one or more of acetate, β-hydroxybutyrate, taurine, or formate. [Item 4] An in vitro composition comprising multiple PDX1-positive, NKX6.1-positive, insulin-negative cells and acetate. [Item 5] A composition according to item 3 or 4, comprising acetate in an amount of 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. [Item 6] A composition according to any one of items 1 to 5, further comprising glutamine. [Item 7] The composition described in Item 6, comprising 0.5–20 mM, 0.5–10 mM, 0.5–5 mM, 1–5 mM, 2–5 mM, or 1 mM–10 mM of glutamine. [Item 8] An in vitro composition containing multiple PDX1-positive, NKX6.1-positive, insulin-negative cells, and glutamine in concentrations of 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. [Item 9] The composition according to Item 8, wherein the glutamine is concentrated at a concentration of 3.8 to 4.2 mM. [Item 10] The composition according to any one of items 6 to 9, wherein 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 dipeptide form. [Item 11] The composition according to Item 10, wherein 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. [Item 12] The composition according to any one of items 6 to 11, wherein 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 free glutamine form. [Item 13] Any one of items 1 through 12, further comprising multiple insulin-positive endocrine progenitor cells. The composition described above. [Item 14] An in vitro composition comprising multiple insulin-positive endocrine progenitor cells and acetyl-CoA-related metabolites in concentrations of 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. [Item 15] An in vitro composition comprising multiple insulin-positive endocrine progenitor cells and acetate in concentrations of 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. [Item 16] A composition according to any one of items 1 to 15, comprising approximately 1 mM acetate. [Item 17] An in vitro composition containing multiple insulin-positive endocrine progenitor cells and glutamine in concentrations of 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. [Item 18] A composition according to any one of items 1 to 17, comprising approximately 4 mM glutamine. [Item 19] A composition according to any one of items 1 to 18, comprising glutamine and acetate. [Item 20] An in vitro composition comprising multiple insulin-positive endocrine progenitor cells, and one or more of the following: acetyl-CoA-related metabolites, HDAC inhibitors, redox homeostasis regulators, monocarbonate pathway intermediates, glutamates, or L-carnitine. [Item 21] An in vitro composition comprising a plurality of insulin-positive endocrine progenitor cells and one or more of glutamate, acetate, β-hydroxybutyrate, taurine, L-carnitine, or formate. [Item 22] The composition according to Item 21, comprising acetate with a concentration of 50-1000 nM, 50-800 nM, 50-500 nM, 50-300 nM, 50-250 nM, 100-200 nM, or 125-175 nM. [Item 23] A composition according to any one of items 1 to 22, comprising β-hydroxybutyrate. [Item 24] A composition according to any one of items 1 to 23, comprising taurine. [Item 25] A composition according to any one of items 1 to 24, comprising a formate. [Item 26] A composition containing a vitamin, as described in any one of items 1 to 25. [Item 27] ​​A composition according to any one of items 1 to 26, comprising biotin. [Item 28] A composition according to any one of items 1 to 27, comprising glutamine, acetate, β-hydroxybutyrate, taurine, formate, and biotin. [Item 29] A composition according to any one of items 1 to 19 or 22 to 28, further comprising L-carnitine. [Item 30] A composition according to any one of items 20 to 28, comprising L-carnitine. [Item 31] The composition according to any one of items 1 to 30, further comprising a BMP signaling pathway inhibitor. [Item 32] The composition according to Item 31, wherein the BMP signaling pathway inhibitor is LDN193189 or DMH-1 or a derivative thereof. [Item 33] The composition according to any one of items 1 to 32, further comprising a ROCK inhibitor. [Item 34] The composition according to Item 33, wherein the ROCK inhibitor is thiazovibin, Y-27632, fasudil / HA1077, or 14-1152, or a derivative thereof. [Item 35] The composition according to any one of Items 1 to 34, further comprising a histone methyltransferase inhibitor. [Item 36] The composition according to Item 35, wherein the histone methyltransferase inhibitor is 3-deazaneplanocin A hydrochloride, Bix-01294, UNC0638, BRDD4770, EPZ004777, AZ505, PDB4e47, valproic acid, vorinostat, romidepsin, entinostat abexinostat, givinostat, and mocetinostat, butyrate, a serine protease inhibitor, serpin, or a derivative thereof. [Item 37] The composition according to any one of Items 1 to 36, further comprising a TGF-β pathway inhibitor. [Item 38] The composition according to Item 37, 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. [Item 39] The composition according to any one of Items 1 to 38, further comprising a thyroid hormone signaling pathway activator. [Item 40] The composition according to Item 39, 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. [Item 41] The composition according to any one of Items 1 to 40, further comprising a protein kinase inhibitor. [Item 42] The composition according to Item 41, wherein the protein kinase inhibitor is staurosporine, Ro-31-8220, or a derivative thereof. [Item 43] The composition according to any one of Items 1 to 42, further comprising a Sonic Hedgehog pathway inhibitor. [Item 44] The composition according to Item 43, wherein the Sonic Hedgehog pathway inhibitor comprises Sant1, Sant2, Sant 4, Sant4, Cur61414, forskolin, tomatidine, AY9944, triparanol, cyclopamine, or derivatives thereof. [Item 45] The composition according to any one of Items 1 to 44, further comprising a growth factor from the epidermal growth factor (EGF) family. [Item 46] The composition according to Item 45, wherein the growth factor from the EGF family comprises betacellulin or EGF. [Item 47] The composition according to any one of Items 1 to 20 or Items 22 to 46, further comprising a gamma-secretase inhibitor. [Item 48] The composition according to Item 47, wherein the gamma-secretase inhibitor comprises XXI, DAPT, or derivatives thereof. [Item 49] The composition according to any one of Items 1 to 48, further comprising zinc. [Item 50] The composition according to Item 49, wherein the zinc is in the form of ZnSO₄. [Item 51] The composition according to any one of Items 1 to 50, further comprising serum albumin protein. [Item 52] The composition according to Item 51, wherein the serum albumin protein is human serum albumin protein. [Item 53] 0.01%~1%, 0.03~1%, 0.03~0.9%, 0.03~0.08%, 0.03~0.06%, 0.03~0.05%, 0.04~0.8%, 0.04~0.7%, 0.04~0.6 %, 0.04~0.5%, 0.04~0.4%, 0.04~0.3%, 0.04~0.2%, 0.04~0.1%, 0.04~0.09%, 0.04~0.8%, 0.04~0.07%, 0.04~0.06% The composition according to item 51 or 52, comprising serum albumin protein in amounts of 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%. [Item 54] The composition according to any one of items 1 to 53, further comprising DMEM. [Item 55] The composition according to Item 54, wherein the DMEM is DMEM / F12. [Item 56] A composition according to any one of items 1 to 54, which does not contain vitamin C. [Item 57] The composition according to any one of items 14 to 56, wherein the plurality of insulin-positive endocrine progenitor cells are dissociated. [Item 58] The composition according to any one of items 14 to 57, wherein 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 progenitor cells are not in cell clusters. [Item 59] The composition according to any one of items 1 to 58, wherein less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, or less than 1% of the cells in the composition are in the form of cell clusters. [Item 60] The composition according to any one of items 57 to 59, wherein the insulin-positive endocrine progenitor cells have been previously frozen. [Item 61] The composition according to any one of items 14 to 56, wherein 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 progenitor cells are in cell clusters. [Item 62] The composition according to any one of items 14 to 56, wherein the plurality of insulin-positive endocrine progenitor cells are arranged in a cell cluster. [Item 63] The composition according to Item 62, 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. [Item 64] The composition according to Item 62, wherein 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. [Item 65] The composition according to any one of items 62 to 64, wherein the cell cluster has an average or median diameter of up to 120, up to 130, up to 140, up to 150, up to 160, or up to 170 μm. [Item 66] The composition according to any one of items 62 to 64, wherein the cell clusters have an average diameter 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. [Item 67] The composition according to any one of items 62 to 66, wherein 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. [Item 68] The composition according to any one of items 62 to 66, wherein 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. [Item 69] A composition according to any one of items 1 to 68, 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. [Item 70] A composition according to any one of items 1 to 69, comprising 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. [Item 71] A composition according to any one of items 1 to 70, comprising a cell which has been manipulated to include a genomic disruption in at least one gene sequence, wherein the disruption reduces or eliminates the expression of a protein encoded by the gene sequence. [Item 72] The composition according to Item 71, wherein the at least one gene sequence is one or more of beta-2-microglobulin (B2M), HLA-A, HLA-B, or CIITA. [Item 73] A composition according to any one of items 1 to 72, comprising cells that have been manipulated to increase the expression of PD-L1 and / or CD47. [Item 74] The composition according to any one of items 71 to 73, wherein the cells are manipulated using a CRISPR / Cas system. [Item 75] A method comprising the step of contacting a first composition comprising one or more of acetyl-CoA-related metabolites, HDAC inhibitors, redox homeostasis regulators, or monocarbonate pathway intermediates with a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells in vitro. [Item 76] A method comprising the step of contacting a first composition containing an acetyl-CoA-related metabolite with a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells in vitro. [Item 77] A method comprising the step of contacting a first composition comprising one or more of acetate, β-hydroxybutyrate, taurine, or formate with a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells in vitro. [Item 78] A method comprising the step of contacting a first composition comprising acetate with a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells in vitro. [Item 79] The method according to any one of items 75 to 78, comprising the step of contacting the cells with an acetate of 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. [Item 80] The method according to any one of items 75 to 78, wherein the first composition further comprises glutamine. [Item 81] The method according to any one of items 75 to 78, wherein the first composition further comprises 0.5–20 mM, 0.5–10 mM, 0.5–5 mM, 1–5 mM, 2–5 mM, or 1 mM–10 mM of glutamine. [Item 82] A method comprising the step of contacting a first composition containing glutamine in an amount of 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 with a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells. [Item 83] The glutamine is present in the manner described in Item 82, with a concentration of 3.8 to 4.2 mM. [Item 84] The method according to any one of items 80 to 83, wherein 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 dipeptide form. [Item 85] The method according to Item 84, wherein 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. [Item 86] The method according to any one of items 80 to 85, wherein 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 free glutamine form. [Item 87] The first composition according to any one of items 75 to 86, comprising about 4 mM glutamine. [Item 88] The method according to any one of items 75 to 87, wherein the first composition comprises glutamine and acetate. [Item 89] The first composition is the method according to any one of items 75 to 88, comprising β-hydroxybutyrate. [Item 90] The method according to any one of items 75 to 89, wherein the first composition comprises taurine. [Item 91] The first composition is a method according to any one of items 75 to 90, comprising a formate. [Item 92] The method according to any one of Items 75 to 91, wherein the first composition comprises a vitamin. [Item 93] The method according to any one of items 75 to 92, wherein the first composition further comprises biotin. [Item 94] The method according to any one of items 75 to 93, wherein the first composition comprises glutamine, acetate, β-hydroxybutyrate, taurine, formate, and biotin. [Item 95] The method according to any one of items 75 to 94, wherein the first composition further comprises a BMP signaling pathway inhibitor. [Item 96] The method according to Item 95, wherein the BMP signaling pathway inhibitor is LDN193189 or DMH-1 or a derivative thereof. [Item 97] The method according to any one of items 75 to 96, wherein the first composition further comprises a ROCK inhibitor. [Item 98] The composition according to Item 97, wherein the ROCK inhibitor is thiazovibin, Y-27632, fasudil / HA1077, or 14-1152, or a derivative thereof. [Item 99] The method according to any one of items 75 to 98, wherein the first composition further comprises a histone methyltransferase inhibitor. [Item 100] The method according to Item 99, wherein the histone methyltransferase inhibitor is 3-deazanepranosin A hydrochloride, Bix-01294, UNC0638, BRDD4770, EPZ004777, AZ505, PDB4e47, valproic acid, vorinostat, romidepsin, entinostat, avexinostat, gibinostat, and mosetinostat, butyrate, serine protease inhibitors, serpine, or derivatives thereof. [Item 101] The method according to any one of Items 75 to 100, wherein the first composition further comprises zinc. [Item 102] The method according to Item 101, wherein the zinc is in the form of ZnSO4. [Item 103] The method according to any one of Items 75 to 102, wherein the first composition further comprises serum albumin protein. [Item 104] The method according to Item 103, wherein the serum albumin protein is human serum albumin protein. [Item 105] The composition of XL1 is 0.01%~1%, 0.03~1%, 0.03~0.9%, 0.03~0.08%, 0.03~0.06%, 0.03~0.05%, 0.04~0.8%, 0.04~0.7%, 0.04~0.6%, 0.04~0.5%, 0.04~0.4%, 0.04~0.3%, 0.04~0.2%, 0.04~0.1%, 0.04~0.09%, 0.04~0.8%, 0.04~0.07%, 0.04~ The method according to item 103 or 104, comprising serum albumin protein in the following proportions: 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%. [Item 106] The method according to any one of items 75 to 105, wherein the first composition further comprises a TGF-β pathway inhibitor. [Item 107] The method according to Item 106, 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. [Item 108] The method according to any one of items 75 to 107, wherein the first composition further comprises a thyroid hormone signaling pathway activator. [Item 109] The thyroid hormone signaling pathway activators mentioned above include GC-1 or T3, T1AM, T0AM, Triprop, L-940901, CGS 23425, KB-141, and The method described in item 108, which is DITPA or a derivative thereof. [Item 110] The method according to any one of items 75 to 109, wherein the first composition further comprises a protein kinase inhibitor. [Item 111] The method according to Item 110, wherein the protein kinase inhibitor is staurosporine or Ro-31-8220 or a derivative thereof. [Item 112] The method according to any one of items 75 to 111, wherein the first composition further comprises a gamma-secretase inhibitor. [Item 113] The method according to Item 112, wherein the gamma-secretase inhibitor is XXI, DAPT, or a derivative thereof. [Item 114] The method according to any one of items 75 to 113, wherein the first composition further comprises L-carnitine. [Item 115] The method according to any one of items 75 to 114, comprising the steps of contacting the plurality of PDX1-positive, NKX6.1-positive cells with the first composition for 1 to 3 days, and then contacting them with a second composition comprising one or more of acetate, β-hydroxybutyrate, taurine, or formate for 3 to 7 days. [Item 116] The method according to any one of items 75 to 114, comprising the steps of contacting the plurality of PDX1-positive, NKX6.1-positive cells with the first composition for 1 to 3 days, and then contacting them 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 to 7 days. [Item 117] The method according to any one of items 75 to 114, comprising the step of contacting the plurality of PDX1-positive, NKX6.1-positive cells with the first composition for 1 to 3 days, and then contacting them with a second composition comprising one or more of Alk5i, GC-1, LDN193189, thiazovibin, SSP, or DZNEP for 2 to 7 days. [Item 118] The method according to any one of Items 115 to 117, wherein the first composition further comprises a sonic hedgehog pathway inhibitor. [Item 119] The sonic hedgehog pathway inhibitor is the method described in Item 118, comprising Sant1, Sant2, Sant4, Sant4, Cur61414, forskolin, tomatidine, AY9944, tripanol, cyclopamine, or derivatives thereof. [Item 120] The method according to any one of items 115 to 119, wherein the first composition further comprises a growth factor from the epidermal growth factor (EGF) family. [Item 121] The method according to Item 120, wherein the growth factor from the EGF family comprises beta-cellulline or EGF. [Item 122] The method according to any one of items 115 to 121, wherein the second composition is the same as the first composition, except that it lacks a sonic hedgehog pathway inhibitor and a growth factor from the epidermal growth factor (EGF) family. [Item 123] The method according to any one of Items 115 to 122, wherein the contact step differentiates at least a portion of the plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells into insulin-positive endocrine progenitor cells. [Item 124] The insulin-positive endocrine progenitor cells include ISL1-positive, NKX6.1-positive cells, as described in Item 123. [Item 125] The method according to any one of Items 75 to 124, wherein the contact step results in the generation of a cell population having a higher proportion 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 the composition which is the same as the first composition except that it lacks one or more of acetate, β-hydroxybutyrate, taurine, or formate. [Item 126] The method according to any one of Items 75 to 124, wherein the contact 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. [Item 127] The method according to any one of Items 75 to 124, wherein the contact 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. [Item 128] The method according to any one of items 75 to 127, wherein the contact step yields a cell population having at least 35%, at least 38%, or at least 40% ISL1-positive, NKX6.1-positive cells after culturing for 4, 7, or 10 days. [Item 129] The method according to any one of Items 75 to 128, wherein the contact step results in the generation of a cell population having a lower proportion of ISL1-negative, NKX6.1-negative cells compared to the step of contacting the plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells with the composition which is the same as the first composition except that it lacks one or more of acetate, β-hydroxybutyrate, taurine, or formate. [Item 130] The method according to any one of Items 75 to 127, wherein the contact step results in the generation of a cell population having up to 22%, up to 20%, 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. [Item 131] The method according to any one of items 75 to 127, wherein the contact step results in the generation of a cell population having about 2%, 11%, 22%, 20%, or 18% ISL1-negative, NKX6.1-negative cells. [Item 132] A method comprising the step of contacting a plurality of insulin-positive endocrine progenitor cells in vitro with a first composition comprising one or more of acetyl-CoA-related metabolites, HDAC inhibitors, redox homeostasis regulators, vitamins, monocarbon metabolic pathway intermediates, L-carnitine, or glutamates. [Item 133] A method comprising the step of contacting a plurality of insulin-positive endocrine progenitor cells in vitro with a first composition comprising one or more of glutamate, acetate, β-hydroxybutyrate, taurine, biotin, L-carnitine, or formate. [Item 134] The first composition is the method according to item 132 or 133, comprising glutamate. [Item 135] The first composition is the method according to any one of items 132 to 134, comprising acetate. [Item 136] The method according to any one of Items 132 to 135, wherein the first composition comprises an acetate of 50-1000 nM, 50-800 nM, 50-500 nM, 50-300 nM, 50-250 nM, 100-200 nM, or 125-175 nM. [Item 137] The first composition is the method according to any one of items 132 to 136, comprising β-hydroxybutyrate. [Item 138] The first composition is the method according to any one of items 132 to 137, comprising taurine. [Item 139] The first composition is the method according to any one of items 132 to 138, comprising a formate. [Item 140] The method according to any one of items 132 to 139, wherein the first composition further comprises biotin. [Item 141] The first composition is the method according to any one of items 132 to 140, comprising L-carnitine. [Item 142] The method according to any one of Items 132 to 141, wherein the first composition comprises glutamate, acetate, β-hydroxybutyrate, taurine, formate, L-carnitine, and biotin. [Item 143] The method according to any one of Items 132 to 142, wherein the first composition further comprises a BMP signaling pathway inhibitor. [Item 144] The method according to Item 143, wherein the BMP signaling pathway inhibitor is LDN193189 or DMH-1 or a derivative thereof. [Item 145] The method according to any one of Items 132 to 144, wherein the first composition further comprises a ROCK inhibitor. [Item 146] The composition according to Item 145, wherein the ROCK inhibitor is thiazovibin, Y-27632, fasudil / HA1077, or 14-1152, or a derivative thereof. [Item 147] The method according to any one of Items 132 to 146, wherein the first composition further comprises a histone methyltransferase inhibitor. [Item 148] The method according to Item 147, wherein the histone methyltransferase inhibitor is 3-deazanepranosin A hydrochloride, Bix-01294, UNC0638, BRDD4770, EPZ004777, AZ505, PDB4e47, valproic acid, vorinostat, romidepsin, entinostat, avexinostat, gibinostat, and mosetinostat, butyrate, serine protease inhibitors, serpine, or derivatives thereof. [Item 149] The method according to any one of items 132 to 148, wherein the first composition further comprises a TGF-β pathway inhibitor. [Item 150] The method according to Item 149, 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. [Item 151] The method according to any one of Items 132 to 150, wherein the first composition further comprises a thyroid hormone signaling pathway activator. [Item 152] The method according to Item 151, 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. [Item 153] The method according to any one of Items 132 to 152, wherein the first composition further comprises a protein kinase inhibitor. [Item 154] The method according to Item 153, wherein the protein kinase inhibitor is staurosporine or Ro-31-8220 or a derivative thereof. [Item 155] The method according to any one of Items 132 to 154, wherein the first composition further comprises vitamin C. [Item 156] The first composition is the method according to any one of Items 132 to 154, wherein the first composition is free of vitamin C. [Item 157] The method according to any one of Items 132 to 156, wherein the first composition further comprises zinc. [Item 158] The method according to Item 157, wherein the zinc is in the form of ZnSO4. [Item 159] The method according to any one of items 132 to 158, wherein the first composition further comprises serum albumin protein. [Item 160] The method according to Item 159, wherein the serum albumin protein is human serum albumin protein. [Item 161] The composition of XL1 is 0.01%~1%, 0.03~1%, 0.03~0.9%, 0.03~0.08%, 0.03~0.06%, 0.03~0.05%, 0.04~0.8%, 0.04~0.7%, 0.04~0.6%, 0.04~0.5%, 0.04~0.4%, 0.04~0.3%, 0.04~0.2%, 0.04~0.1%, 0.04~0.09%, 0.04~0.8%, 0.04~0.07%, 0.04~ The method according to item 159 or 160, containing serum albumin protein in the following proportions: 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%. [Item 162] The composition comprising DMEM, according to any one of Items 132 to 161. [Item 163] The method according to Item 162, wherein the DMEM is DMEM / F12. [Item 164] The method according to any one of items 132 to 163, wherein the plurality of insulin-positive endocrine progenitor cells are dissociated. [Item 165] The method according to Item 164, wherein the dissociated insulin-positive endocrine progenitor cells are previously frozen. [Item 166] The method of Item 164 or 165, wherein the dissociated cells are re-aggregated into multiple cell clusters. [Item 167] The method according to Item 166, wherein at least about 40%, 50%, 60%, 70%, 80%, or 90% of the plurality of cell clusters have a diameter of about 50 μm to about 250 μm, about 75 μm to about 250 μm, or about 100 μm to about 200 μm. [Item 168] The method according to Item 166 or 167, wherein at least about 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, or 99% of the cells in the plurality of cell clusters are viable. [Item 169] The method according to any one of Items 164 to 168, wherein the dissociated cells are re-aggregated into at least 2, 3, 4, 5, 10, 50, 100, 1,000, 10,000, 1,000,000, or 1,000,000 cell clusters. [Item 170] The method according to any one of Items 164 to 169, wherein 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. [Item 171] The method according to any one of Items 164 to 170, wherein the cell cluster has an average or median diameter of up to 120, up to 130, up to 140, up to 150, up to 160, or up to 170 μm. [Item 172] The cell cluster having 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, according to any one of items 164 to 171. [Item 173] The method according to any one of items 164 to 172, wherein at least about 50%, at least about 60%, at least about 70%, or at least about 80% of the cell cluster has a diameter of less than 150 μm. [Item 174] The method according to any one of items 164 to 173, wherein at least about 50%, at least about 60%, or at least about 70% of the cell cluster has a diameter of less than 130 μm. [Item 175] 1-10 days, 1-9 days, 1-8 days, 1-7 days, 1-6 days, 1-5 days, 1- The method described in any one of items 132 to 174, carried out over a period of 4 days, 1 to 3 days, 1 to 2 days, 2 to 10 days, 2 to 9 days, 2 to 8 days, 2 to 7 days, 2 to 6 days, 2 to 5 days, 2 to 4 days, 2 to 3 days, 3 to 10 days, 3 to 9 days, 3 to 8 days, 3 to 7 days, 3 to 6 days, 3 to 5 days, 3 to 4 days, 4 to 10 days, 4 to 9 days, 4 to 8 days, 4 to 7 days, 4 to 6 days, or 4 to 5 days. [Item 176] A method described in any one of Items 132 to 175, carried out over a period of 1 to 8 days, 1 to 7 days, 1 to 6 days, 1 to 5 days, 1 to 4 days, 1 to 3 days, 1 to 2 days, 2 to 8 days, 2 to 7 days, 2 to 6 days, 2 to 5 days, 2 to 4 days, 2 to 3 days, 3 to 8 days, 3 to 7 days, 3 to 6 days, 3 to 5 days, 3 to 4 days, 4 to 8 days, 4 to 7 days, 4 to 6 days, or 4 to 5 days. [Item 177] The method according to any one of items 132 to 176, wherein the plurality of insulin-positive endocrine progenitor cells are brought into contact with the first composition for about 3, 4, 5, 6, 7, or 8 days, thereby generating a second cell population. [Item 178] The method according to Item 177, further comprising the step of 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 β cells. [Item 179] The method according to Item 178, wherein the second cell population is brought into contact with the second composition for 1 to 8 days, 1 to 7 days, 1 to 6 days, 1 to 5 days, 1 to 4 days, 1 to 3 days, 1 to 2 days, 2 to 8 days, 2 to 7 days, 2 to 6 days, 2 to 5 days, 2 to 4 days, 2 to 3 days, 3 to 8 days, 3 to 7 days, 3 to 6 days, 3 to 5 days, 3 to 4 days, 4 to 8 days, 4 to 7 days, 4 to 6 days, or 4 to 5 days. [Item 180] The method according to Item 179, wherein the second cell population is brought into contact with the second composition for about 3, 4, 5, 6, 7, or 8 days. [Item 181] The method according to any one of Items 178 to 180, wherein the second composition is not 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. [Item 182] The method according to any one of Items 178 to 181, wherein the second composition is free from glutamate, acetate, β-hydroxybutyrate, taurine, biotin, L-carnitine, or formate. [Item 183] (a) A step of contacting a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells in vitro with a first composition to generate a first cell population comprising a plurality of cell clusters including insulin-positive endocrine progenitor cells, wherein the first composition comprises acetate, glutamine, and one or more of β-hydroxybutyrate, taurine, formate, or biotin, (b) The step of dissociating at least a portion of the plurality of cell clusters in the first cell population in vitro, (c) A step of contacting the first cell population, which includes at least a portion of the dissociated cell clusters, with a second composition in vitro to generate a second cell population, which includes a plurality of cell clusters, each comprising a plurality of insulin-positive cells, wherein the second composition comprises one or more of glutamate, acetate, β-hydroxybutyrate, taurine, biotin, L-carnitine, or formate. A method that includes this. [Item 184] A step of contacting the second insulin-positive cell population with a third composition in vitro, thereby differentiating at least a portion of the second insulin-positive cell population into a third cell population comprising a plurality of β cells, wherein the third composition is different from the second composition. The method described in item 183, which further includes the method described in item 183. [Item 185] The method according to Item 184, wherein the third composition is free from glutamate, acetate, β-hydroxybutyrate, taurine, biotin, L-carnitine, or formate. [Item 186] The method according to any one of items 183 to 185, wherein the first composition comprises 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 of glutamine. [Item 187] The first composition is the method according to any one of items 183 to 185, comprising about 4 mM glutamine. [Item 188] The first composition according to any one of items 183 to 187, comprising 0.01 to 50 mM, 0.1 to 50 mM, 0.5 to 50 mM, 0.01 to 20 mM, 0.1 to 20 mM, 0.5 to 20 mM, 0.01 to 10 mM, 0.1 to 10 mM, 0.5 to 10 mM, 0.8 to 25 mM, 0.8 to 10 mM, 0.8 to 5 mM, 0.8 to 2 mM, 0.8 to 1.5 mM, 0.8 to 1.2 mM, 0.9 to 1.1 mM, or 0.95 to 1.05 mM. [Item 189] The first composition according to any one of items 183 to 187, comprising about 1 mM acetate. [Item 190] The method according to any one of items 183 to 189, wherein the second composition comprises an acetate of 50-1000 nM, 50-800 nM, 50-500 nM, 50-300 nM, 50-250 nM, 100-200 nM, or 125-175 nM. [Item 191] The method according to any one of items 183 to 190, wherein the second composition comprises glutamate, acetate, β-hydroxybutyrate, taurine, formate, L-carnitine, and biotin. [Item 192] The method according to any one of Items 183 to 191, further comprising at least one agent from among 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-related coiled-coil-containing protein kinase (ROCK) inhibitor, a bone morphogenetic protein (BMP) type 1 receptor inhibitor, a γ-secretase inhibitor, or a histone methyltransferase inhibitor. [Item 193] The method according to any one of Items 183 to 192, wherein the second composition further comprises at least one agent selected from 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, or a histone methyltransferase inhibitor. [Item 194] (A) The sonic hedgehog pathway inhibitor contains SANT1 or (B) The retinoic acid signaling pathway activator contains retinoic acid, (C) The γ-secretase inhibitor contains XXI or (D) The growth factors from the EGF family include beta-cell phosphate or EGF, (E) The BMP signaling pathway inhibitor comprises LDN193189 or a derivative thereof, (F) The TGF-β signaling pathway inhibitor includes ALK5 inhibitor II or a derivative thereof, (G) The thyroid hormone signaling pathway activator comprises GC-1 or T3, or derivatives thereof. (H) The protein kinase inhibitor contains staurosporine or (I) The ROCK inhibitor comprises thiazovibin, Y-27632, fasudil / HA1077, or 14-1152, or derivatives thereof, or (J) The histone methyltransferase inhibitor comprises 3-deazanepranosin A hydrochloride or a derivative thereof. The method described in item 192 or 193. [Item 195] A method comprising the step of exposing an NKX6.1-positive, ISL1-positive, insulin-positive cell population to one or more of the following: serum albumin protein, TGF-β signaling pathway inhibitor, TH signaling pathway activator, protein kinase inhibitor, ROCK inhibitor, BMP signaling pathway inhibitor, epigenetic modification compound, acetyl-CoA-related metabolite, vitamin, histone deacetylase inhibitor (HDACi), redox homeostasis regulator, monocarbon metabolic pathway intermediate, glutamate, and / or carnitine, over a first period of 1, 2, 3, 4, 5, 6, or 7 days (e.g., 4 days). [Item 196] The method according to Item 195, further comprising the step of 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, monocarbon metabolic pathway intermediates, glutamates, 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 modifying compounds. [Item 197] A method comprising the step of exposing an NKX6.1-positive, ISL1-positive, insulin-positive cell population to one or more of HSA, Alk5 inhibitor II, GC-1, staurosporine, thiazovibin, LDN193189, DZNEP, taurine, acetate, beta-hydroxybutyrate, biotin, carnitine, glutamate, and formate over a first period of 1, 2, 3, 4, 5, 6, or 7 days (e.g., 4 days). [Item 198] The method according to Item 197, further comprising the step of 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 longer, in the absence of Alk5 inhibitor II, GC-1, staurosporine, thiazovibin, LDN193189, and DZNEP, after the first period. [Item 199] During the first and / or second period, the cell population is brought into contact with ZnSO4. The method described in any one of items 195 to 198, including further steps. [Item 200] The method according to any one of items 195 to 199, further comprising the step of bringing the cell population into contact with DMEM during the first and / or second period. [Item 201] The method according to Item 200, wherein the DMEM is DMEM / F12. [Item 202] The method according to any one of items 196 or 198 to 200, wherein, during the second period, the cells are brought into contact with the same concentration of serum albumin (e.g., about 0.05% HSA) as in the first period. [Item 203] The method according to any one of items 196 or 198 to 200, wherein the cells are brought into contact with the HSA at a higher concentration (e.g., about 1.0%) compared to the first period (e.g., about 0.05%) during the second period. [Item 204] The method according to any one of items 68 to 203, wherein the cells are manipulated to include a genomic disruption in at least one gene sequence, the disruption reducing or eliminating the expression of the protein encoded by the gene sequence. [Item 205] The method according to Item 204, wherein the at least one gene sequence is one or more of beta-2-microglobulin (B2M), HLA-A, HLA-B, or CIITA. [Item 206] The method according to any one of Items 68 to 203, wherein the composition comprises cells that have been manipulated to increase the expression of PD-L1 and / or CD47. [Item 207] The method according to any one of Items 204 to 206, wherein the cells are manipulated using a CRISPR / Cas system. [Item 208] A composition comprising insulin-positive endocrine progenitor cells as described in any one of items 14 to 74 or 123 to 182. [Item 209] A composition comprising at least a portion of the third cell population described in any one of items 184 to 194. [Item 210] A device containing cells as described in any one of items 1 through 74, 208, 209, or 193 through 207. [Item 211] The device described in Item 210, which is configured to produce and release insulin when implanted in a subject. [Item 212] The device according to item 210 or 211, in which the cells are enclosed. [Item 213] The device according to any one of items 210 to 212, further comprising a semipermeable membrane, the semipermeable membrane being configured to retain the cells in the device and to allow the passage of insulin. [Item 214] A method for treating a subject having a disease characterized by long-term high blood glucose levels (e.g., diabetes mellitus), comprising the step of administering to the subject cells described in any one of items 1 to 74, 208, 209, or 193 to 207, or item 1 A method comprising the step of embedding a device as described in any one of paragraphs 87 to 190. [Item 215] The disease is diabetes mellitus, as described in Item 214. [Item 216] A composition comprising multiple insulin-positive cells and cryopreserving agents, and one or more of the following: acetyl-CoA-related metabolites, HDAC inhibitors, redox homeostasis regulators, monocarbon metabolic pathway intermediates, vitamins, or L-glutamine. [Item 217] The composition described in Item 216, which is stored frozen. [Item 218] The composition according to item 216 or 217, comprising an acetyl-CoA-related metabolite. [Item 219] The composition according to Item 218, wherein the acetyl-CoA related metabolite is acetate. [Item 220] A composition according to Item 219, comprising acetate in the following concentrations: 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. [Item 221] A composition comprising glutamine, as described in any one of items 216 to 220. [Item 222] The composition described in Item 221, comprising 0.5–20 mM, 0.5–10 mM, 0.5–5 mM, 1–5 mM, 2–5 mM, 3.8–4.2 mM, or 1 mM–10 mM of glutamine. [Item 223] The composition according to Item 221 or 222, wherein 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. [Item 224] The composition according to any one of items 221 to 223, wherein 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 free glutamine form. [Item 225] A composition according to any one of items 216 to 224, comprising a redox homeostasis regulator. [Item 226] The composition according to Item 225, wherein the redox homeostasis regulator is taurine. [Item 227] A composition according to any one of items 216 to 226, comprising a monocarbon metabolic pathway intermediate. [Item 228] The composition according to Item 227, wherein the monocarbon metabolic pathway intermediate is a formate. [Item 229] A composition containing a vitamin, as described in any one of items 216 to 228. [Item 230] The composition according to Item 229, wherein the vitamin is biotin.

[0003] Islet transplantation is a promising therapy that can achieve significant clinical benefits for patients with diabetes, for example, those with type 1 diabetes. Because the supply of 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 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 for human therapeutic use (e.g., higher cell yield), or a combination of these.

[0004]

[0004] This specification provides, in particular, compositions and methods for the improved production of SC-β cells in vitro. For example, novel formulations and differentiation methods are provided that result in SC island clusters having advantageous features 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 post-transplant survival, improved function and reduced immunogenicity. The compositions and methods of this disclosure can be used in the large-scale production of SC islands for human therapeutic applications.

[0005]

[0005] In some embodiments, in vitro compositions comprising a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells, and one or more of acetyl-CoA-related metabolites, HDAC inhibitors, redox homeostasis regulators, or monocarbonate pathway intermediates are disclosed herein.

[0006]

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

[0007]

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

[0008]

[0008] In some embodiments, in vitro compositions comprising a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells and acetates are disclosed herein.

[0009]

[0009] In some cases, the compositions of the present disclosure include acetate in concentrations of 0.01 to 50 mM, 0.1 to 50 mM, 0.5 to 50 mM, 0.01 to 20 mM, 0.1 to 20 mM, 0.5 to 20 mM, 0.01 to 10 mM, 0.1 to 10 mM, 0.5 to 10 mM, 0.8 to 25 mM, 0.8 to 10 mM, 0.8 to 5 mM, 0.8 to 2 mM, 0.8 to 1.5 mM, 0.8 to 1.2 mM, 0.9 to 1.1 mM, or 0.95 to 1.05 mM. In some cases, the compositions of the present disclosure further include glutamine. In some cases, the compositions of this disclosure contain 0.5–20 mM, 0.5–10 mM, 0.5–5 mM, 1–5 mM, 2–5 mM, or 1 mM–10 mM of glutamine.

[0010]

[0010] In some embodiments, in vitro compositions 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 are disclosed herein.

[0011]

[0011] In some cases, the glutamine concentration is 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 glutamine is in the free glutamine form. In some cases, the compositions of this disclosure further comprise a plurality of insulin-positive endocrine progenitor cells.

[0012]

[0012] In some embodiments, in vitro compositions comprising a plurality of insulin-positive endocrine progenitor cells and acetyl-CoA-related metabolites in amounts of 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 are disclosed herein.

[0013]

[0013] In some embodiments, in vitro compositions comprising a plurality of insulin-positive endocrine progenitor 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 acetate are disclosed herein.

[0014]

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

[0015] In some embodiments, in vitro compositions comprising a plurality of insulin-positive endocrine progenitor 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 are disclosed herein. In some cases, the compositions of the disclosure contain about 4 mM glutamine. In some cases, the compositions of the disclosure contain glutamine and acetate.

[0015]

[0016] In some embodiments, in vitro compositions comprising a plurality of insulin-positive endocrine progenitor cells and one or more of acetyl-CoA-related metabolites, HDAC inhibitors, redox homeostasis regulators, monocarbonate pathway intermediates, glutamates, or L-carnitine are disclosed herein.

[0016]

[0017] In some embodiments, in vitro compositions comprising a plurality of insulin-positive endocrine progenitor cells and one or more of glutamate, acetate, β-hydroxybutyrate, taurine, L-carnitine, or formate are disclosed herein.

[0017]

[0018] In some cases, the compositions of the Disclosure include acetates in concentrations of 50-1000 nM, 50-800 nM, 50-500 nM, 50-300 nM, 50-250 nM, 100-200 nM, or 125-175 nM. In some cases, the compositions of the Disclosure include β-hydroxybutyrate. In some cases, the compositions of the Disclosure include taurine. In some cases, the compositions of the Disclosure include formate. In some cases, the compositions of the Disclosure include vitamins. In some cases, the compositions of the Disclosure include biotin. In some cases, the compositions of the Disclosure include glutamine, acetate, β-hydroxybutyrate, taurine, formate, and biotin. In some cases, the compositions of the Disclosure further include L-carnitine. In some cases, the compositions of the Disclosure include L-carnitine. In some cases, the compositions of the Disclosure further include 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 composition of the Disclosure further comprises a ROCK inhibitor. In some cases, the ROCK inhibitor is thiazovibin, Y-27632, fasudil / HA1077, or 14-1152 or a derivative thereof. In some cases, the composition of the Disclosure further comprises a histone methyltransferase inhibitor. In some cases, histone methyltransferase inhibitors include 3-deazanepranosin A hydrochloride, Bix-01294, UNC0638, BRDD4770, EPZ004777, AZ505, PDB4e47, alproic acid, vorinostat, romidepsin, entinostat, avexinostat, gibinostat, and mosetinostat, butyrate, serine protease inhibitors, serpine, or derivatives thereof. In some cases, the compositions of this disclosure further include TGF-β pathway inhibitors. In some cases, TGF-β pathway inhibitors are ALK5 inhibitors II, A83-01, 431542, D4476, GW788388, LY364947, LY580276, SB525334, SB505124, SD208, GW6604, or GW788388, or their derivatives.In some cases, the compositions of the present disclosure further comprise 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 derivatives thereof. In some cases, the compositions of the present disclosure further comprise a protein kinase inhibitor. In some cases, the protein kinase inhibitor is staurosporine or Ro-31-8220, or derivatives thereof. In some cases, the compositions of the present disclosure further comprise a sonic hedgehog pathway inhibitor. In some cases, the sonic hedgehog pathway inhibitor comprises Sant1, Sant2, Sant4, Sant4, Cur61414, forskolin, tomatidine, AY9944, tripanol, cyclopamine, or derivatives thereof. In some cases, the compositions of the present disclosure further comprise a growth factor from the epidermal growth factor (EGF) family. In some cases, the growth factor from the EGF family includes beta-cell phosphate or EGF. In some cases, the composition of the disclosure further includes a gamma-secretase inhibitor. In some cases, the gamma-secretase inhibitor includes XXI, DAPT, or derivatives thereof. In some cases, the composition of the disclosure further includes zinc. In some cases, the zinc is in the form of ZnSO4. In some cases, the composition of the disclosure further includes serum albumin protein. In some cases, the serum albumin protein is human serum albumin protein.In some cases, the compositions of this disclosure are found in concentrations of 0.01% to 1%, 0.03 to 1%, 0.03 to 0.9%, 0.03 to 0.08%, 0.03 to 0.06%, 0.03 to 0.05%, 0.04 to 0.8%, 0.04 to 0.7%, 0.04 to 0.6%, 0.04 to 0.5%, 0.04 to 0.4%, 0.04 to 0.3%, 0.04 to 0.2%, 0.04 to 0.1%, 0.04 to 0.09%, 0.04 to 0.8%, and 0.04 to 0. The compositions contain serum albumin protein in the following proportions: 0.7%, 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%. In some cases, the compositions of this disclosure further include DMEM. In some cases, the DMEM is DMEM / F12. In some cases, the compositions of this disclosure do not contain vitamin C.

[0018]

[0019] In some cases, multiple insulin-positive endocrine progenitor 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 multiple insulin-positive endocrine progenitor 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 progenitor cells are 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 multiple insulin-positive endocrine progenitor cells are clustered together. In some cases, multiple insulin-positive endocrine progenitor cells are clustered together. 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 cell clusters have a diameter of about 80–150, 100–150, 120–150, 140–150, 80–130, 100–130, 120–130, 80–120, 90–120, or 100–120 μm. In some cases, cell clusters have an average or median diameter of up to 120, 130, 140, 150, 160, or 170 μm. In some cases, cell clusters have an average or median diameter of approximately 80–150, 100–150, 120–150, 140–150, 80–130, 100–130, 120–130, 80–120, 90–120, or 100–120 μm. In some cases, at least approximately 50%, at least approximately 60%, at least approximately 70%, or at least approximately 80% of cell clusters have a diameter of less than 150 μm. In some cases, at least approximately 50%, at least approximately 60%, or at least approximately 70% of cell clusters have a diameter of less than 130 μm. In some cases, the compositions of the present disclosure contain 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 compositions of the present disclosure 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%, or up to 22% ISL1-negative, NKX6.1-negative cells.

[0020]

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

[0021]

[0022] In some embodiments, a method is disclosed herein that includes contacting a first composition comprising one or more of acetyl-CoA-related metabolites, HDAC inhibitors, redox homeostasis regulators, or monocarbonate pathway intermediates with a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells in vitro.

[0022]

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

[0023]

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

[0024]

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

[0025]

[0026] In some cases, the method of the present disclosure includes the step of contacting cells with an acetate in a concentration of 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 first composition further comprises glutamine. In some cases, the first composition further comprises 0.5–20 mM, 0.5–10 mM, 0.5–5 mM, 1–5 mM, 2–5 mM, or 1 mM–10 mM of glutamine.

[0026]

[0027] In some embodiments, a method is disclosed herein that includes contacting a first composition containing 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 with a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells.

[0027]

[0028] In some cases, glutamine is present at concentrations 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 glutamine is in the free glutamine form. In some cases, the first composition contains about 4 mM of glutamine. In some cases, the first composition contains glutamine and acetate. In some cases, the first composition contains β-hydroxybutyrate. In some cases, the first composition contains taurine. In some cases, the first composition contains formate. In some cases, the first composition contains vitamins. 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 thiazovibin, 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-deazanepranosin A hydrochloride, Bix-01294, UNC0638, BRDD4770, EPZ004777, AZ505, PDB4e47, valproic acid, vorinostat, romidepsin, entinostat, avexinostat, gibinostat, and mosetinostat, butyrate, serine protease inhibitors, serpine, or derivatives 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 is 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 The first composition contains serum albumin protein in amounts of 7%, 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%. 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 derivatives 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 derivatives 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 derivatives thereof. In some cases, the first composition further comprises a gamma-secretase inhibitor. In some cases, the gamma-secretase inhibitor is XXI, DAPT, or derivatives thereof.

[0028]

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

[0029]

[0030] In some cases, the method of the present disclosure includes the step of contacting a plurality of PDX1-positive, NKX6.1-positive cells with a first composition for 1 to 3 days, and then contacting them with a second composition comprising one or more of the following for 2 to 7 days: 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. In some cases, the method of the present disclosure includes the step of contacting a plurality of PDX1-positive, NKX6.1-positive cells with a first composition for 1 to 3 days, and then contacting them with a second composition comprising one or more of the following for 2 to 7 days: Alk5i, GC-1, LDN193189, thiazovibin, SSP, or DZNEP. In some cases, the first composition further comprises a sonic hedgehog pathway inhibitor. In some cases, the sonic hedgehog pathway inhibitor comprises Sant1, Sant2, Sant4, Sant4, Cur61414, forskolin, tomatidine, AY9944, tripanol, 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 beta-cellulline or EGF. In some cases, the second composition is the same as the first composition, except that it lacks the sonic hedgehog pathway inhibitor and the growth factor from the epidermal growth factor (EGF) family.

[0030]

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

[0031]

[0032] In some embodiments, a method is disclosed herein that includes contacting a plurality of insulin-positive endocrine progenitor cells in vitro with a first composition comprising one or more of acetyl-CoA-related metabolites, HDAC inhibitors, redox homeostasis regulators, vitamins, monocarbonate pathway intermediates, L-carnitine, or glutamates.

[0032]

[0033] In some embodiments, a method is disclosed herein that includes the step of contacting a plurality of insulin-positive endocrine progenitor cells in vitro with a first composition comprising one or more of glutamate, acetate, β-hydroxybutyrate, taurine, biotin, L-carnitine, or formate.

[0033]

[0034] In some cases, the first composition contains glutamate. In some cases, the first composition contains acetate. In some cases, the first composition contains acetate of 50-1000 nM, 50-800 nM, 50-500 nM, 50-300 nM, 50-250 nM, 100-200 nM, or 125-175 nM. In some cases, the first composition contains β-hydroxybutyrate. In some cases, the first composition contains taurine. In some cases, the first composition contains formate. In some cases, the first composition further contains biotin. In some cases, the first composition contains L-carnitine. In some cases, the first composition contains glutamate, acetate, β-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 a derivative thereof. In some cases, the first composition further comprises a ROCK inhibitor. In some cases, the ROCK inhibitor is thiazovibin, 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 inhibitors are 3-deazanepranosin A hydrochloride, Bix-01294, UNC0638, BRDD4770, EPZ004777, AZ505, PDB4e47, valproic acid, vorinostat, romidepsin, entinostat, avexinostat, gibinostat, and mosetinostat, butyrate, serine protease inhibitors, serpine, or derivatives 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 contain 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 is 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 The compositions contain serum albumin protein in amounts of 7%, 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%. In some cases, the compositions of this disclosure contain DMEM. In some cases, the DMEM is DMEM / F12.

[0034]

[0035] In some cases, multiple insulin-positive endocrine progenitor cells are dissociated. In some cases, the dissociated insulin-positive endocrine progenitor cells are previously frozen. In some cases, the method of this disclosure re-aggregates the dissociated cells into multiple cell clusters. In some cases, at least about 40%, 50%, 60%, 70%, 80%, or 90% of the multiple 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 in the multiple cell clusters are viable. In some cases, the method of the present disclosure re-aggregates dissociated cells into clusters of at least 2, 3, 4, 5, 10, 50, 100, 1000, 10000, 100000, or 1,000,000 cells. 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 an average 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, cell clusters have an average or median diameter of approximately 80–150, 100–150, 120–150, 140–150, 80–130, 100–130, 120–130, 80–120, 90–120, or 100–120 μm. In some cases, at least approximately 50%, at least approximately 60%, at least approximately 70%, or at least approximately 80% of cell clusters have a diameter of less than 150 microns. In some cases, at least approximately 50%, at least approximately 60%, or at least approximately 70% of cell clusters have a diameter of less than 130 μm.In some cases, the method of disclosure may be carried out over a period of 1 to 10 days, 1 to 9 days, 1 to 8 days, 1 to 7 days, 1 to 6 days, 1 to 5 days, 1 to 4 days, 1 to 3 days, 1 to 2 days, 2 to 10 days, 2 to 9 days, 2 to 8 days, 2 to 7 days, 2 to 6 days, 2 to 5 days, 2 to 4 days, 2 to 3 days, 3 to 10 days, 3 to 9 days, 3 to 8 days, 3 to 7 days, 3 to 6 days, 3 to 5 days, 3 to 4 days, 4 to 10 days, 4 to 9 days, 4 to 8 days, 4 to 7 days, 4 to 6 days, or 4 to 5 days. In some cases, the method of disclosure may be carried out over a period of 1 to 8 days, 1 to 7 days, 1 to 6 days, 1 to 5 days, 1 to 4 days, 1 to 3 days, 1 to 2 days, 2 to 8 days, 2 to 7 days, 2 to 6 days, 2 to 5 days, 2 to 4 days, 2 to 3 days, 3 to 8 days, 3 to 7 days, 3 to 6 days, 3 to 5 days, 3 to 4 days, 4 to 8 days, 4 to 7 days, 4 to 6 days, or 4 to 5 days.

[0035]

[0036] In some cases, a plurality of insulin-positive endocrine progenitor cells are brought into contact with a first composition for approximately 3, 4, 5, 6, 7, or 8 days to generate a second cell population. In some cases, the method of the present disclosure further includes the step of bringing the second cell population into contact with a second composition different from the first composition to differentiate at least a portion of the second insulin-positive cell population into a third cell population comprising a plurality of β-cells. In some cases, the second cell population is brought into contact with the second composition for 1 to 8 days, 1 to 7 days, 1 to 6 days, 1 to 5 days, 1 to 4 days, 1 to 3 days, 1 to 2 days, 2 to 8 days, 2 to 7 days, 2 to 6 days, 2 to 5 days, 2 to 4 days, 2 to 3 days, 3 to 8 days, 3 to 7 days, 3 to 6 days, 3 to 5 days, 3 to 4 days, 4 to 8 days, 4 to 7 days, 4 to 6 days, or 4 to 5 days. In some cases, the second cell population is exposed to the second composition for approximately 3, 4, 5, 6, 7, or 8 days. In some cases, the second composition does not contain BMP signaling pathway inhibitors, ROCK inhibitors, histone methyltransferase inhibitors, TGF-β pathway inhibitors, thyroid hormone signaling pathway inhibitors, or protein kinase inhibitors. In some cases, the second composition does not contain glutamate, acetate, β-hydroxybutyrate, taurine, biotin, L-carnitine, or formate.

[0036]

[0037] In some embodiments, a method is disclosed herein that includes the steps of (a) contacting a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells in vitro with a first composition to generate a first cell population comprising a plurality of cell clusters comprising insulin-positive endocrine progenitor cells, wherein the first composition comprises acetate, glutamine, and one or more of β-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, with a second composition in vitro to generate a second cell population comprising a plurality of cell clusters comprising a plurality of insulin-positive cells, wherein the second composition comprises one or more of glutamate, acetate, β-hydroxybutyrate, taurine, biotin, L-carnitine, or formate. In some cases, the method of the present disclosure includes the step of contacting a second insulin-positive cell population with a third composition in vitro, thereby differentiating at least a portion of the second insulin-positive cell population into a third cell population comprising a plurality of β-cells, wherein the third composition further includes steps different from those of the second composition.

[0037]

[0038] In some cases, the third composition does not contain glutamate, acetate, β-hydroxybutyrate, taurine, biotin, L-carnitine, or formate. In some cases, the first composition contains 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 contains approximately 4 mM glutamine. In some cases, the first composition contains 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 acetate. In some cases, the first composition contains approximately 1 mM of acetate. In some cases, the second composition contains acetate in concentrations of 50-1000 nM, 50-800 nM, 50-500 nM, 50-300 nM, 50-250 nM, 100-200 nM, or 125-175 nM. In some cases, the second composition contains glutamate, acetate, β-hydroxybutyrate, taurine, formate, L-carnitine, and biotin.

[0038]

[0039] In some cases, the first composition further comprises at least one agent selected from among TGF-β signaling pathway inhibitors, growth factors from the EGF family, retinoic acid signaling pathway activators, sonic hedgehog pathway inhibitors, thyroid hormone signaling pathway activators, protein kinase inhibitors, Rho-related coiled-coil-containing protein kinase (ROCK) inhibitors, bone morphogenetic protein (BMP) type 1 receptor inhibitors, γ-secretase inhibitors, or histone methyltransferase inhibitors.

[0039]

[0040] In some cases, the second composition further comprises at least one agent from among 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, or 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 γ-secretase inhibitor comprises XXI, (D) the growth factor from the EGF family comprises beta-cell phosphate or EGF, (E) the BMP signaling pathway inhibitor comprises LDN193189 or a derivative thereof, or (F) the TGF-β signaling pathway inhibitor comprises ALK5 inhibitor II or (G) The thyroid hormone signaling pathway activator comprises GC-1 or T3, or a derivative thereof; (H) The protein kinase inhibitor comprises staurosporine; (I) The ROCK inhibitor comprises thiazovibin, Y-27632, fasudil / HA1077, or 14-1152, or a derivative thereof; or (J) The histone methyltransferase inhibitor comprises 3-deazanepranosin A hydrochloride or a derivative thereof.

[0040]

[0041] In some embodiments, a method is disclosed herein that includes the step of exposing an NKX6.1-positive, ISL1-positive, insulin-positive cell population to one or more of the following: serum albumin protein, TGF-β signaling pathway inhibitors, TH signaling pathway activators, protein kinase inhibitors, ROCK inhibitors, BMP signaling pathway inhibitors, epigenetic modification compounds, acetyl-CoA-related metabolites, vitamins, histone deacetylase inhibitors (HDACi), redox homeostasis regulators, monocarbon metabolic pathway intermediates, glutamates, and / or carnitine, over a first period of 1, 2, 3, 4, 5, 6, or 7 days (e.g., 4 days). In some cases, the method of the present disclosure further includes, after a first period, contacting a cell population with one or more of the following: serum albumin protein, acetyl-CoA-related metabolites, vitamins, histone deacetylase inhibitors (HDACi), redox homeostasis regulators, monocarbonate pathway intermediates, glutamates, and / or carnitine, over 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 modifying compounds.

[0041]

[0042] In some embodiments, a method is disclosed herein that includes the step of contacting an NKX6.1-positive, ISL1-positive, insulin-positive cell population with one or more of the following: HSA, Alk5 inhibitor II, GC-1, staurosporine, thiazovibin, LDN193189, DZNEP, taurine, acetate, beta-hydroxybutyrate, biotin, carnitine, glutamate, and formate, over a first period of 1, 2, 3, 4, 5, 6, or 7 days (e.g., 4 days). In some cases, the method of the present disclosure further includes, after a first period, contacting a cell population with one or more of HSA, taurine, acetate, beta-hydroxybutyrate, biotin, carnitine, glutamate, and formate over 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, thiazovibin, LDN193189, and DZNEP.

[0042]

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

[0043]

[0044] In some embodiments, compositions comprising insulin-positive endocrine progenitor cells as disclosed herein are disclosed herein.

[0044]

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

[0045]

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

[0046]

[0047] In some embodiments, methods for treating subjects having a disease characterized by long-term high blood glucose levels (e.g., diabetes mellitus) are disclosed herein, comprising the steps of administering cells disclosed herein or implanting a device disclosed herein. In some cases, the disease is diabetes mellitus.

[0047]

[0048] In some embodiments, compositions comprising a plurality of insulin-positive cells and a cryopreservation agent, and one or more of acetyl-CoA-related metabolites, HDAC inhibitors, redox homeostasis regulators, monocarbon metabolic pathway intermediates, vitamins, or L-glutamine are disclosed herein.

[0048]

[0049] In some cases, the compositions of this disclosure are stored cryopreserved. In some cases, the compositions of this disclosure contain acetyl-CoA-related metabolites. In some cases, the acetyl-CoA-related metabolites are acetates. In some cases, the compositions of this disclosure contain acetates in concentrations of 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 this disclosure contain glutamine. In some cases, the compositions of this disclosure contain 0.5–20 mM, 0.5–10 mM, 0.5–5 mM, 1–5 mM, 2–5 mM, 3.8–4.2 mM, or 1–10 mM of 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 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 glutamine is in the free glutamine form. In some cases, the compositions of the disclosure contain a redox homeostasis regulator. In some cases, the redox homeostasis regulator is taurine. In some cases, the compositions of the disclosure contain a monocarbonate pathway intermediate. In some cases, the monocarbonate pathway intermediate is a formate. In some cases, the compositions of the disclosure contain a vitamin. In some cases, the vitamin is biotin.

[0049] Embedding by reference

[0050] All publications, patents, and patent applications referenced herein are incorporated by reference to the same extent as each individual publication, patent, or patent application is specifically and individually indicated to be incorporated by reference. To the extent that any publications and patents or patent applications incorporated by reference conflict with the disclosures contained herein, this specification is intended to supersede and / or take precedence over any such conflicting material.

[0050] Brief explanation of the drawing

[0051] Novel features of this disclosure are described in detail in the appended claims. A better understanding of the features and advantages of this disclosure will be obtained by referring to the following detailed description of exemplary embodiments in which the principles of this disclosure are utilized, as well as to the appended drawings (furthermore, "Figure" and "FIG." as described herein). [Brief explanation of the drawing]

[0051] [Figure 1A]

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

[0053] Figure 1C shows bright-field microscopy images of human embryonic stem cell cultures incubated for 48 hours with the MQ medium additives disclosed herein (MQ) or without them (CTL), or incubated with MQ additives 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 additives, suggesting that glutamine and acetate contribute to the observed effects of MQ on cells. [Figure 2]

[0054] Figure 2A is an exemplary dispersion plot from samples evaluated 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 culture medium throughout Stage 5 substantially increased the percentage of SC-β cells from 25.9% in the control culture to 38.1% in the culture with MQ. Figure 2B is a graph quantifying the percentage of Nkx6.1+ / Isl1+ SC-β cells obtained at the end of Stage 5 from repeats performed without MQ, showing that the SC-β% achieved in the absence of MQ is consistently lower than that observed with MQ in Figure 2A. These data suggest that the addition of MQ during Stage 5 may enhance SC-β cell differentiation. [Figure 3]

[0056] Figure 3 shows exemplary flow cytometry results for experiments in which MQ was absent throughout Stage 5 (control), present throughout Stage 5 (+MQ), or with MQ additives excluding acetate and glutamine (+MQ*-acetate-Q). The percentage of Nkx6.1+ / Isl1+ SC-β cells was 29.8% under control conditions, 41.6% in the presence of MQ, and 34.3% with MQ additives excluding acetate and glutamine. [Figure 4]

[0057] Figure 4 is a graph that provides summary data for two experiments comparing the percentage of SC-β cells present at the end of Stage 5 after incubation in or without MQ throughout Stage 5. [Figure 5]

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

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

[0061] Figure 7A is a diagram showing an exemplary flow plot of cells cultured in or without MQ during Stage 5 and analyzed on day 7 of Stage 6. Compared to 34.3% in cells not incubated with MQ, 45.1% of cells were found to be Nkx6.1+ / Isl1+ in cultures with MQ in Stage 5. Figure 7B is a graph showing the percentage of Nkx6.1+ / Isl1+ cells on days 4, 7, and 10 of Stage 6 for cells cultured in or without MQ during Stage 5, demonstrating that the high percentage of SC-β cells observed in the case of MQ treatment in Stage 5 is maintained over time (MQ St5). [Figure 8]

[0063] Figure 8 is a graph illustrating the percentage (yield) of all cells recovered at days 4, 7, and 10 of Stage 6, relative to the number of viable cells seeded at the start of Stage 6. Cells were cultured in the presence or absence of MQ (control) and thawed in 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 in the presence or absence (control) of MQ during Stage 5, and then thawed in medium A or control medium 1 in Stage 6. The image was taken on day 4 of Stage 6. Cells incubated with MQ during Stage 5 and then thawed in medium A formed smaller clusters. Figure 9B is a figure providing a bright-field microscopy image of cells incubated in the presence or absence (control) of MQ during Stage 5, and then thawed in medium A or control medium 1 in Stage 6. The image was taken on day 7 of Stage 6. Cells incubated with MQ during Stage 5 and then thawed in medium A formed smaller clusters. [Figure 10]

[0066] Figure 10 is a graph quantifying the cluster size frequency on day 7 of stage 6, showing that the cluster size is smaller in cells incubated with MQ in stage 5 and then thawed in medium A in stage 6. Cells incubated with MQ during stage 5 and then thawed in medium A formed smaller clusters. [Figure 11]

[0067] Figure 11 shows the stage 6 medium ("Medium A") and its components in comparison with control medium 1 and control medium 2. Between days 1 and 4 of stage 6, the S5d6 factor contains Alk5i (10 μM), GC-1 (1 μM), LDN-193189 (100 nM), thiazovinin (2.5 μM), SSP (3 nM), and DZNEP (100 nM). [Figure 12]

[0068] Figure 12A is a bar graph showing the number of viable cells recovered to S6d7 and S6d11 from cultures in control medium 1, control medium 2, medium A (without metabolites), or medium A (with metabolites). The data shows that medium A using DMEM / F12 further improves the number of viable cells throughout stage 6. Figure 12B is a bar graph showing the percentage of cells recovered to S6d7 and S6d11 from cultures in control medium 1, control medium 2, medium A (without metabolites), or medium A (with metabolites). The data shows that medium A using DMEM / F12 further improves the cell recovery rate throughout stage 6. [Figure 13]

[0070] Figure 13 shows microscopic images of SC island clusters in S6d7 and S6d11 from cultures in control medium 1, control medium 2, or medium A (containing metabolites). The images show that medium A clusters exhibit higher homogeneity throughout stage 6. [Figure 14]

[0071] Figure 14 is a series of area graphs showing the frequency and cluster size of cell clusters in control medium 1, control medium 2, and S6d4, S6d7, and S6d11 derived from cultures in medium A. The results show that the clusters in medium A are smaller and exhibit greater uniformity in S6d11. [Figure 15]

[0072] Figure 15 is a bar graph showing the GSIS function of S6d11 cells derived from control medium 1 culture. [Figure 16]

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

[0074] Figure 17 is a bar graph showing the GSIS function of S6d11 cells cultured in medium A (without metabolites). The results show that the SC islands in medium A (without metabolites) exhibit improved GSIS function (compare with Figure 15). [Figure 18]

[0075] Figure 18 is a bar graph showing the GSIS function of S6d11 cells derived from culture in medium A (containing metabolites). The results show that the SC islands in medium A exhibit similar GSIS function to those in control medium 1 culture (compare with Figure 15). [Figure 19]

[0076] Figure 19 is a table showing the effects 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 survival rate 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 showed a higher survival rate compared to cells generated using regimen 2 when evaluated 6 weeks after implantation. Figure 20B is a graph showing the survival rate of stage 6 SC islet cells generated by regimen 2 at stage 6 of differentiation, 6 and 13 weeks after transplantation into nude rats. [Figure 21]

[0078] Figure 21 is a graph quantifying the number of multinucleated giant cells (MNGs) detected in the histopathological evaluation of the device 6 weeks after transplantation into nude rats (rates). SC islands generated by regimen 1 at differentiation stage 6 are compared with SC islands generated by regimen 2 at stage 6. Six weeks after implantation, fewer multinucleated giant cells (MNGs) were observed in cells generated using regimen 1. [Figure 22]

[0079] Figure 22 is an exemplary histopathological image of H&E stained sections from nude rats 6 weeks after implantation of the device of this disclosure containing SC islands generated by a method comprising regimen 2 or regimen 1 at differentiation stage 6. Six weeks after implantation, reduced immunoinfiltration and fewer multinucleated giant cells (MNGs) were observed in cells generated using regimen 1. [Figure 23]

[0080] Figure 23 is a graph illustrating non-fasting blood glucose levels in diabetic NOD Scid gamma (NSG) mice administered one (1×) or two (2×) devices filled with SC islands generated using regimen 2 or regimen 1 during stage 6 of differentiation. Animals administered with two devices (2×) maintained normal blood glucose levels until approximately 8 weeks after implantation. [Figure 24]

[0081] Figure 24A is a graph illustrating the time course of non-fasting human C-peptide levels in diabetic NOD Scid gamma (NSG) mice administered with one (1×) or two (2×) devices filled with SC islands generated using regimen 2 or regimen 1 during differentiation stage 6. In contrast to naive control and diabetic control mice, where C-peptide levels were undetectable, an increase in C-peptide was observed in all experimental groups after implantation, and sustained levels were observed in animals administered with two devices. CAD refers to the cell-containing device. Figure 24B is a graph comparing the fasting level of human C-peptide measured at 153 days post-transplant with the non-fasting level of C-peptide measured at 112 days post-transplant in diabetic NOD Scid gamma (NSG) mice administered with one (1×) or two (2×) devices filled with SC islands generated using regimen 2 or regimen 1 during differentiation stage 6. The data demonstrate that SC islands produced by methods using the compositions and methods of this disclosure, including Regimen 2 and Regimen 1, may be effective in increasing insulin production after transplantation in diabetic subjects. [Figure 25]

[0083] Figure 25 is a graph showing SC island survival data evaluated 5 months (1 device) or 6 months (2 devices) after implantation in diabetic NSG mice. SC islands were generated using regimen 2 or regimen 1 during differentiation stage 6. High survival rates were observed in devices with cells generated by either regimen 2 or regimen 1 during differentiation stage 6. [Figure 26]

[0084] Figure 26 shows exemplary histological images of sections stained for C peptide and glucagon (left panel) or H&E stained (right panel) six months after implantation of SC islands in diabetic NSG mice. These results demonstrate that SC islands produced using the compositions and methods of this disclosure, including Regimen 2 and Regimen 1, can remain viable after implantation and produce insulin and glucagon. [Modes for carrying out the invention]

[0052]

[0085] Islet transplantation is a promising therapy that can achieve significant clinical benefits in patients with diabetes, such as those with type 1 diabetes. Because the supply of 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. Improvements in methods for generating islet components (e.g., SC-β cells) could lead to more effective therapeutic products (e.g., SC-β cells with improved functionality), improved methods for producing SC islets for human therapeutic use (e.g., higher cell yield), or a combination of these.

[0053]

[0086] This specification provides, in particular, compositions and methods for improving the generation of SC-β cells in vitro. Certain compositions and combinations of the agents disclosed herein improve the fitness and metabolic flexibility of differentiated cells and the resulting SC-β cells by targeting specific aspects of cellular metabolism. For example, this disclosure provides novel formulations and differentiation methods that result in SC island clusters with advantageous features such as higher cell yield and recovery, increased number and relative percentage of SC-β cells, enhanced stability and shelf life of SC-β cells, reduced size and increased uniformity, improved in vitro function of SC-β cells, and improved viability, function, and reduced immunogenicity after transplantation. The disclosed compositions and methods can be used in the large-scale production of SC islands for human therapeutic applications.

[0054]

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

[0055]

[0088] While various embodiments of the Disclosure are shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided only as examples. Numerous variations, modifications, and substitutions will be conceivable to those skilled in the art without departing from the Disclosure. It should be understood that various alternative forms of the embodiments of the Disclosure described herein may be used.

[0056] definition

[0089] In this application, the use of the singular form includes the plural form unless otherwise specified. It should be noted that, as used herein, the singular forms “a,” “an,” and “the” refer to multiple objects unless the context clearly indicates 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 synonyms, can be used interchangeably as used herein. These terms can convey that any combination is specifically intended. For illustrative purposes only, the following phrases, “A, B, and / or C” or “A, B, C, or any 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 makes disjunctive use clear.

[0058]

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

[0059]

[0092] References to “some embodiments,” “a certain embodiment,” “one embodiment,” or “other embodiments” in this specification mean that certain features, structures, or characteristics described in conjunction with the embodiments are included in at least some embodiments of this disclosure, but not necessarily in all embodiments.

[0060]

[0093] As used herein and in the 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 non-limiting and do not preclude any additional undescribed elements or method steps. 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 "approximately" and its grammatical synonyms in relation to a reference number may, as used herein, include the number itself and values ​​within a range of plus or minus 10% of that number.

[0062]

[0095] The terms “about” or “approximately” mean that a particular value is within the tolerance range determined by those skilled in the art, and in part depends on the method by which that value is measured or determined, for example, on the limitations of the measurement system. For example, “about” may mean a standard deviation of 1 or more than 1, as is customary in the art. Alternatively, “about” may 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 quantity “about 10” includes 10 and any quantities from 9 to 11. In yet another example, the term “about” in relation to a reference number may also include values ​​within a range of plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of that value. Alternatively, particularly in relation to biological systems or processes, the term “about” may mean within 10 times a certain value, preferably within 5 times, and more preferably within 2 times. Where a particular value is described in this application and claims, unless otherwise stated, the term “about” should be assumed to mean that the particular value is within the tolerance range.

[0063]

[0096] The term “diabetes” and its grammatical synonyms, as used herein, can refer to a disease characterized by long-term high blood glucose levels. For example, the term “diabetes” and its grammatical synonyms, 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 may be a form of hereditary diabetes.

[0064]

[0097] The term “endocrine cells” can refer, unless otherwise specified, to hormone-producing cells present in the pancreas of an organism, such as “islands,” “island cells,” “island equivalents,” “island-like cells,” “pancreatic islets,” and their grammatical synonyms. In some embodiments, endocrine cells may be differentiated from pancreatic progenitor cells or precursors. Island cells can include, but are not limited to, pancreatic α-cells, pancreatic β-cells, pancreatic δ-cells, pancreatic F-cells, and / or pancreatic ε-cells, and may also refer to groups of cells, cell clusters, etc.

[0065]

[0098] The terms “progenitor” and “early-stage” cells are used interchangeably herein and refer to cells that have a cellular phenotype that is more primitive than cells that may arise through differentiation (e.g., at an earlier step in the developmental pathway or progression than fully differentiated cells). Often, progenitor cells may also have significant or very high proliferative capacity. Depending on the developmental pathway and the environment in which the cells develop and differentiate, progenitor cells may give rise to multiple distinct differentiated cell types or a single differentiated cell type.

[0066]

[0099] In relation to insulin-positive endocrine cells, the term "precursor" can refer to any cell that, when cultured under conditions favorable for differentiation into insulin-positive endocrine cells, can be differentiated into insulin-positive endocrine cells, including, for example, pluripotent stem cells, endoderm cells, primitive intestinal cells, pancreatic progenitor cells, or endocrine progenitor cells.

[0067]

[0100] The term “exocrine cell,” as used herein, may refer to the cells of an exocrine gland, i.e., a gland that discharges its secretions through a duct. In certain embodiments, exocrine cells may refer to pancreatic exocrine cells, which are pancreatic cells that can produce enzymes secreted into the small intestine. These enzymes can help digest food as it passes through the gastrointestinal tract. Pancreatic exocrine cells are also known as the islets of Langerhans, which can secrete two hormones, namely insulin and glucagon. Pancreatic exocrine cells can be one of several cell types, e.g., α-2 cells (which can produce the hormone glucagon), or β-cells (which can produce the hormone insulin), and α-1 cells (which can produce the regulatory substance somatostatin). Non-insulin-producing exocrine cells, as used herein, may refer to α-2 cells or α-1 cells. The term "exocrine pancreatic cells" can encompass "endocrine pancreatic cells," which are pancreatic cells that produce hormones secreted into the bloodstream, such as insulin (produced by β cells), glucagon (produced by alpha-2 cells), somatostatin (produced by delta cells), and pancreatic polypeptides (produced by F cells).

[0068]

[0101] The terms “stem cell-derived β-cells,” “SC-β-cells,” “functional β-cells,” “functional pancreatic β-cells,” and “mature SC-β-cells,” and their grammatical synonyms, may refer to cells (e.g., non-native pancreatic β-cells) that exhibit at least one marker indicating pancreatic β-cells (e.g., PDX-1 or NKX6.1), express insulin, and exhibit a glucose-stimulated insulin secretion (GSIS) response characteristic of endogenous mature β-cells. In some embodiments, the terms “SC-β-cells” and “non-native β-cells” are interchangeable as used herein. In some embodiments, “SC-β-cells” include mature pancreatic cells. It should be understood that the SC-β cells do not need to be derived from stem cells (e.g., directly) since the method of this disclosure can be used to obtain SC-β cells from any insulin-positive endocrine cell or its precursor, using any cell as a starting point (for example, embryonic stem cells, induced pluripotent stem cells, progenitor cells, partially reprogrammed somatic cells (e.g., somatic cells partially reprogrammed to an intermediate state between induced pluripotent stem cells and the somatic cells from which they originate), multipotent cells, totipotent cells, or any of the aforementioned cell differentiation forms, etc., although the present invention is not intended to be limited to this form). In some embodiments, SC-β cells exhibit a response to multiple glucose loadings (e.g., at least one, at least two, or at least three or more sequential glucose loadings). In some embodiments, the response is similar to the response of endogenous islets (e.g., human islets) to multiple glucose loadings. In some embodiments, the morphology of SC-β cells is similar to that of endogenous β cells. In some embodiments, SC-β cells exhibit an in vitro GSIS response similar to that of endogenous β cells. In some embodiments, SC-β cells exhibit an in vivo GSIS response similar to that of endogenous β cells. In some embodiments, SC-β cells exhibit both an in vitro GSIS response similar to that of endogenous β cells and an in vivo GSIS response. The GSIS response of SC-β cells can be observed within two weeks of 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, SC-β cells exhibit encapsulated crystalline insulin granules. In some embodiments, SC-β cells exhibit a stimulation index greater than 1. In some embodiments, SC-β cells exhibit a stimulation index greater than 1.1. In some embodiments, SC-β cells exhibit a stimulation index greater than 2. In some embodiments, SC-β cells exhibit cytokine-induced apoptosis in response to cytokines. In some embodiments, insulin secretion from SC-β cells is enhanced in response to known antidiabetic drugs (e.g., secretagogues). In some embodiments, SC-β cells are monohormone. In some embodiments, SC-β cells do not abnormally co-express other hormones, such as glucagon, somatostatin, or pancreatic polypeptides. In some embodiments, SC-β cells exhibit a low replication rate. In some embodiments, SC-β cells increase intracellular Ca2+ in response to glucose.

[0069]

[0102] As used herein, the term “insulin-producing cell” and its grammatical synonyms refer to insulin-secreting cells differentiated from the pancreatic precursor or its progenitor. Examples of insulin-producing cells include pancreatic β-cells as the term is used herein, and pancreatic β-like cells (e.g., insulin-positive endocrine cells) that constitutively or inductively synthesize insulin (e.g., transcribe the insulin gene, translate proinsulin mRNA, modify proinsulin mRNA to produce insulin protein), express (e.g., express the phenotype held by the insulin gene), or secrete (release insulin into the extracellular space). 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 this disclosure may be pancreatic β cells or β-like cells (e.g., cells having at least one or at least two endogenous β-cell characteristics) and exhibit a glucose-stimulated insulin secretion (GSIS) response similar to that of endogenous adult β cells. For example, an insulin-producing cell population generated by the methods disclosed herein may include mature pancreatic β cells or SC-β cells and may also include non-insulin-producing cells (e.g., cells with a cell-like phenotype except that they neither produce nor secrete insulin).

[0070]

[0103] The terms "insulin-positive β-like cells," "insulin-positive endocrine cells," and their grammatical synonyms can refer to cells (e.g., pancreatic endocrine cells) that exhibit at least one marker indicating pancreatic β-cells 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, 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) polypeptides, 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, as well as those described in Zhang et al., Diabetes. 50(10):2231~6 (2001). In some embodiments, the β-cell marker is a nuclear 3-cell marker. In some embodiments, the β-cell marker is Pdx1 or PH3.

[0072]

[0105] The term “pancreatic endocrine markers” can refer, but are 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 precursor,” “pancreatic endocrine precursor,” “pancreatic progenitor,” and “pancreatic endocrine progenitor,” and their grammatical synonyms, are used interchangeably herein and may refer to stem cells that can become pancreatic hormone-expressing cells capable of forming pancreatic endocrine cells, pancreatic exocrine cells, or pancreatic duct cells. These cells are determined to differentiate into at least one type of pancreatic cell, e.g., insulin-producing β cells, glucagon-producing α cells, somatostatin-producing δ cells (or D cells), and / or pancreatic polypeptide-producing F cells. Such cells may express at least one marker from among NGN3, NKX2.2, NeuroD, ISL-1, Pax4, Pax6, or ARX.

[0074]

[0107] The term “Pdx1-positive pancreatic precursor,” as used herein, may refer to cells that are pancreatic endoderm (PE) cells capable of differentiating into SC-β cells, such as pancreatic β cells. Pdx1-positive pancreatic precursors express the marker Pdx1. Other markers include, but are not limited to, Cdcp1, or Ptf1a, or HNF6, or NRx2.2. Pdx1 expression can be assessed by any method known to those skilled in the art, such as immunochemistry or quantitative RT-PCR using an anti-Pdx1 antibody. In some cases, Pdx1-positive pancreatic progenitor cells lack NKX6.1 expression. In some cases, Pdx1-positive pancreatic progenitor cells may also be referred to as Pdx1-positive NKX6.1-negative pancreatic progenitor cells due to the lack of NKX6.1 expression. In some cases, Pdx1-positive pancreatic progenitor cells may also be referred to as “foregut endoderm cells.”

[0075]

[0108] The term “Pdx1-positive NKX6-1-positive pancreatic precursor,” as used herein, may refer to cells that are pancreatic endodermal (PE) cells capable of differentiating into insulin-producing cells, such as pancreatic β-cells. Pdx1-positive NKX6-1-positive pancreatic precursors express the markers Pdx1 and NKX6-1. Other markers, but not limited to these, may include Cdcp1, or Ptf1a, or HNF6, or NRx2.2. NKX6-1 expression can be assessed by any method known to those skilled in the art, such as immunochemistry or quantitative RT-PCR using an anti-NKX6-1 antibody. As used herein, the terms “NKX6.1” and “NKX6-1” are synonymous and interchangeable. In some cases, Pdx1-positive NKX6-1-positive pancreatic progenitor cells may also be referred to as “pancreatic foregut progenitor cells.”

[0076]

[0109] The term “Ngn3-positive endocrine precursor,” as used herein, may refer to a pancreatic endocrine cell precursor expressing 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). Ngn3 expression can be assessed by any method known to those skilled in the art, such as immunochemistry using an anti-Ngn3 antibody or quantitative RT-PCR.

[0077]

[0110] The terms "NeuroD" and "NeuroD1" are used interchangeably to identify proteins expressed in pancreatic endocrine progenitor cells and the genes that encode them.

[0078]

[0111] The term “selection marker” refers to a gene, RNA, or protein that, when expressed, confers to a cell the expression of a specific protein, which can be used as a criterion for distinguishing between cells that express the protein and those that do not, resulting in a selectable phenotype, such as resistance to cytotoxic agents or cell division inhibitors (e.g., antibiotic resistance), trophotrophy, or the expression of a particular protein. As used herein, the term “selection marker” may also refer to a gene or the expression product of a gene, such as the protein it encodes. In some embodiments, a selection marker confers a proliferation and / or survival advantage to cells that express it compared to cells that do not express it or express it at significantly low levels. Such a proliferation and / or survival advantage typically arises when the cells are maintained under certain conditions, i.e., “selection conditions.” To ensure effective selection, the cell population may be maintained for a sufficient period of time under conditions such that cells that do not express the marker do not proliferate and / or survive and are removed from the population or the number of such cells is reduced to only a very small proportion of the population. A method of selecting cells that express a marker in which a proliferation and / or survival advantage is conferred by maintaining a cell population under selective conditions in which cells that do not express the marker are largely or completely removed 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 causes proliferation and / or survival disadvantages to cells that express the marker compared to cells that do not express the marker or express it at significantly low levels (or, conversely, cells that do not express the marker have a proliferation and / or survival advantage compared to cells that express the marker). Therefore, cells that express this marker can be largely or completely removed from a cell population if they are maintained under selective conditions for a sufficient period of time.

[0079]

[0112] The term "epigenetics" refers to genetic changes in gene function that do not involve alterations in the DNA sequence. While epigenetics most often refers to chromosomal changes that affect gene activity and expression, it can also be used to describe any genetic phenotypic changes that do not originate from genome modifications. Such effects on a cell's phenotypic and physiological phenotypic traits may be due to external or environmental factors, or they may be part of a normal developmental program. Epigenetics can also refer to functionally relevant changes in the genome that do not involve alterations in the nucleotide sequence. Examples of mechanisms that produce such changes include DNA methylation and histone modification, each altering how genes are expressed without changing the underlying DNA sequence. Gene expression can be regulated through the action of repressor proteins that bind to the silencer region of DNA. These epigenetic changes can persist through cell division until the cell reaches the end of its lifespan and may even persist across multiple generations despite the absence of changes in the underlying organism's DNA sequence. An example of epigenetic change in eukaryotic biology is the process of cell differentiation. During morphogenesis, totipotent stem cells can differentiate into various pluripotent cells, and these pluripotent cells can then differentiate into fully differentiated cells.

[0080]

[0113] The term "epigenetic modifier" refers to chemical compounds that can induce epigenetic changes in genes, i.e., alter gene expression without altering the DNA sequence. Epigenetic changes can help determine whether a gene is "on" or "off" and can affect protein production in certain cells, such as beta cells. Epigenetic modifications, such as DNA methylation and histone modifications, alter DNA accessibility and chromatin structure, thereby regulating gene expression patterns. 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 can therefore contribute to or result from changes in phenotypic or pathological phenotypes due to the environment. Importantly, epigenetic modifications play a critical role in regulating pluripotency genes that are inactivated during differentiation. Non-limiting and exemplary epigenetic modifiers 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 synonyms refers to any primary cell that is not pluripotent as defined herein in its natural form. Alternatively, the term “differentiated cell” can refer to a more specialized cell type obtained from a less specialized cell type (e.g., stem cells, e.g., induced pluripotent stem cells) in the process of cell differentiation. While not wishing to be limited to theory, pluripotent stem cells in the course of normal ontogeny can initially differentiate into endodermal cells capable of forming pancreatic cells and other endodermal cell types. Further differentiation of endodermal cells leads to the pancreatic pathway, where approximately 98% of cells become exocrine, tubule, or matrix cells, and approximately 2% become endocrine cells. Early endocrine cells are island precursors, which can then further differentiate into insulin-producing cells (e.g., functional endocrine cells) that secrete insulin, glucagon, somatostatin, or pancreatic polypeptides. Endoderm cells can also differentiate into other cells of endodermal origin, such as the lungs, liver, intestines, thymus, etc.

[0082]

[0115] As used herein, the term “somatic cell” can refer to any cell that makes up the body of an organism, distinct from germline cells. In mammals, germline cells (also known as “gametes”) are sperm and eggs, which fuse during fertilization to produce a cell called a zygote, from which the entire mammalian embryo develops. All other cell types in the mammalian body—with the exception of sperm and eggs, the cells from which they are produced (germ cells), and undifferentiated stem cells—are somatic cells, and viscera, skin, bone, blood, and connective tissue are all composed of somatic cells. In some embodiments, somatic cells are “non-embryonic cells,” meaning somatic cells that are neither present in an embryo nor obtained from an embryo, and that do not result from the in vitro proliferation of such cells. In some embodiments, somatic cells are “adult somatic cells,” meaning cells that are present in or obtained from an organism other than an embryo or fetus, or that result from the in vitro proliferation of such cells. Unless otherwise specified, methods for converting at least one insulin-positive endocrine cell or its precursor into an insulin-producing glucose-responsive cell can be performed both in vivo and in vitro (where in vivo is performed when at least one insulin-positive endocrine cell or its precursor is present in the subject's body, and in vitro is performed using at least one isolated insulin-positive endocrine cell or its precursor maintained in culture).

[0083]

[0116] As used herein, the term “adult cells” may refer to cells found throughout the body after embryonic development.

[0084]

[0117] As used herein, the term “endodermal cell” can refer to a cell derived from one of the three primary germ cell layers in a very early embryo (the other two germ cell layers are the mesoderm and ectoderm). The endoderm is the innermost of the three layers. Endoderm cells differentiate to first give rise to the embryonic intestine, and then to the inner surfaces of the airways and digestive tract (e.g., the intestines), the liver, and the pancreas.

[0085]

[0118] The term “endodermal-derived cells,” as used herein, can refer to any cells that develop or differentiate from endodermal cells. Examples of endodermal-derived cells include those of the liver, lungs, pancreas, thymus, intestines, stomach, and thyroid. While not wishing to be constrained by theory, liver and pancreatic precursors (also referred to as pancreatic precursors) arise from endodermal cells of the pre-embryonic gut. Immediately after specification, liver and pancreatic precursors rapidly acquire remarkably different cellular functions and regenerative capabilities. These changes are induced by highly conserved inductive signals and genetic regulators across vertebrates. Interest in organ development and regeneration is heightened by the strong need for hepatocytes and pancreatic β-cells in the treatment of hepatic failure and type 1 diabetes. Studies in diverse model organisms and humans have revealed evolutionarily conserved inductive signaling and transcription factor networks that induce hepatocyte and pancreatic cell differentiation, providing guidance on methods to promote hepatocyte and β-cell differentiation from diverse stem and progenitor cell types.

[0086]

[0119] The term “embryonic endoderm,” as used herein, can refer to cells that differentiate from endodermal cells and can differentiate into SC-β cells (e.g., pancreatic β cells). Embryonic endoderm cells express the marker Sox17. Other markers characteristic of embryonic endoderm cells, but not limited to these, include MIXL2, GATA4, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CXCR4, Cerberus, OTX2, goosecoid, C-Kit, CD99, CMKOR1, and CRIP1. In particular, embryonic endoderm cells expressed herein express Sox17, and in some embodiments express Sox17 and HNF3B, but do not express significant levels of GATA4, SPARC, APF, or DAB. Embryonic endoderm cells are not positive for the marker Pdx1 (e.g., they are Pdx1 negative). Embryonic endoderm cells have the ability to differentiate into cells including those of the liver, lungs, pancreas, thymus, intestines, stomach, and thyroid. The expression of Sox17 and other endoderm markers can be evaluated by any method known to those skilled in the art, such as immunochemistry or quantitative RT-PCR using anti-Sox17 antibodies.

[0087]

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

[0088]

[0121] The terms “primitive intestinal cells” or “intestinal cells,” as used herein, may refer to cells that differentiate from endodermal cells and can differentiate into SC-β cells (e.g., pancreatic β cells). Primitive intestinal cells express at least one marker from among HNP1-β, HNF3-β, or HNF4-α. Primitive intestinal 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 the primitive intestine can be evaluated by any method known to those skilled in the art, such as immunochemistry using an anti-HNF1-β antibody.

[0089]

[0122] As used herein, the term “stem cell” can refer to an undifferentiated cell capable of producing many progenitor cells that are proliferative and capable of producing a large number of parent cells that can give rise to differentiated or differentiateable daughter cells. The daughter cells themselves can retain one or more cells that have the developmental potential to be parents, while at the same time being induced to proliferate and produce progeny that will later differentiate into one or more mature cell types. The term “stem cell” can refer to a subset of precursors that, under certain circumstances, have the ability or potential to differentiate into a more specialized or more differentiated phenotype, and under certain circumstances, retain the ability to proliferate substantially without differentiation. In one embodiment, the term stem cell as a whole refers to a naturally occurring parent cell whose offspring (progeny) often specialize in different directions by acquiring entirely distinct characteristics through differentiation, for example, in the stepwise diversification of germ cells and tissues. Cell differentiation is a complex process that typically occurs through many cell divisions. Differentiated cells can be obtained from pluripotent cells, and pluripotent cells themselves can be obtained from pluripotent cells, etc. Each of these pluripotent cells can be considered a stem cell, but the range of cell types that each can give rise to can vary considerably. Some differentiated cells also possess the ability to produce cells with higher developmental potential. Such ability may be naturally occurring or artificially induced based on treatment with various factors. In many biological cases, stem cells are also "pluripotent," meaning they can produce offspring of two or more distinct cell types, but this is not necessary for "stem cell-ness." Self-renewal is another classic part of the definition of stem cells and is essential when used in this document. Theoretically, self-renewal can occur by one of two main mechanisms. Stem cells may divide asymmetrically, such that one daughter retains the stem cell state while the other daughter expresses some other distinct specific function and phenotype. Alternatively, some stem cells in a population may divide symmetrically into two stem cells, thus maintaining some stem cells in the population as a whole while other cells in the population can only produce differentiated offspring.Formally, cells that begin as stem cells may progress to a differentiated phenotype, but then "reverse" to re-express the stem cell phenotype. This term is often referred to by those skilled in the art as "dedifferentiation," "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] As used herein, the term "pluripotency" can refer to cells that have the ability to differentiate into two or more differentiated cell types, preferably into cell types characteristic of all three germ layers, under diverse conditions. Pluripotent cells are primarily characterized by their ability to differentiate into two or more cell types, preferably into all three germ layers, for example, using a nude mouse teratoma formation assay. While pluripotency can also be demonstrated by the expression of embryonic stem (ES) cell markers, a preferred test for pluripotency is the demonstration of the ability to differentiate into cells of each of the three germ layers. It should be noted that simply culturing such cells alone does not make them pluripotent. Reprogrammed pluripotent cells (e.g., iPS cells as defined herein) also have the characteristic of being able to be passed through for long periods without loss of growth capacity, compared to primary cell parents, which 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 interchangeable and may refer to pluripotent stem cells artificially obtained (e.g., by induction or complete inversion) from non-pluripotent cells, typically adult somatic cells, by inducing the forced expression of one or more genes.

[0092]

[0125] The term "phenotype" can refer to one or more overall biological characteristics that define a cell or organism under a specific set of environmental conditions and factors, regardless of its actual genotype.

[0093]

[0126] The terms “subject,” “patient,” or “individual” are used interchangeably herein and may refer to an animal, such as a human, from which cells can be obtained and / or to which treatment is provided, including prophylactic treatment using the cells described herein. In the case of treatment for an infectious disease, condition, or disease state specific to a particular animal, such as a human subject, the term “subject” may refer to that particular animal. “Non-human animals” and “non-human mammals” are used interchangeably herein and include mammals, such as rats, mice, rabbits, sheep, cats, dogs, cattle, pigs, and non-human primates. The term “subject” also encompasses all vertebrates, including but not limited to mammals, reptiles, amphibians, and fish. However, advantageously, a subject is a mammal such as a human, or another mammal, such as a domestic mammal, such as a dog, cat, horse, etc., or a production mammal, such as a cattle, sheep, pig, etc. “Patient requiring it” or “subject requiring it” means herein a patient who has been diagnosed with or is suspected of having a disease or disorder, such as, for example, diabetes, but not limited to, this.

[0094]

[0127] As used herein, “administering” may mean providing one or more compositions described herein to a patient or subject. For example, but not limited to, administration of compositions, e.g., by injection, may be carried out by intravenous (iv), subcutaneous (sc), intradermal (id), intraperitoneal (ip), or intramuscular (im) injection. One or more such routes may be used. Parenteral administration may be carried out, e.g., by bolus injection or gradual perfusion over time. Alternatively or concurrently, administration may be carried out by an oral route. In addition, administration may also be carried out by surgical deposition of a bolus or pellet of cells, or by implantation of a medical device. In some embodiments, the compositions of the Disclosure may include engineered cells or host cells expressing a nucleic acid sequence described herein, or a vector containing at least one nucleic acid sequence described herein, in an amount effective to treat or prevent a proliferation disorder. Pharmaceutical compositions may include a cell population described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffers, such as neutral buffered saline or phosphate-buffered saline; carbohydrates, such as glucose, mannose, sucrose, or dextran, or mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (such as aluminum hydroxide); and preservatives.

[0095]

[0128] The terms “to treat,” “to treat,” and “treatment,” and their grammatical synonyms, when applied to isolated cells, include subjecting cells to any kind of process or condition, or performing any kind of operation or procedure on cells. When applied to an object, these terms refer to providing medical or surgical treatment, nursing, or management to an individual. The individual is typically diseased or unhealthy, or at increased risk of disease compared to the average member of the population, and therefore requires such treatment, nursing, or management.

[0096]

[0129] As used herein, the terms “to treat” and “treatment” may mean administering an effective amount of a composition to a subject, resulting in the subject obtaining a reduction in at least one symptom of the disease or improvement of the disease, e.g., a beneficial or desired clinical outcome. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, the reduction of one or more symptoms, whether detectable or undetectable; a reduction in the severity of the disease; a stable (e.g., non-exacerbating) state of the disease; a delay or slowing of disease progression; improvement or mitigation of the disease state; and remission (whether partial or complete). To treat may mean extending survival compared to the survival expected without treatment. Those skilled in the art will therefore recognize that treatment may improve the state of the disease but not necessarily result in a complete cure. As used herein, the term “treatment” includes prevention. Alternatively, treatment is “effective” if the progression of the disease is reduced or stopped. “Treatment” may also mean extending survival compared to the survival expected without treatment. Those requiring treatment include individuals already diagnosed with heart disease, as well as those at high risk of developing heart disease due to genetic susceptibility or other factors, such as weight, diet, and health status.

[0097]

[0130] The terms “therapeutic dose,” “therapeutic dose,” or their grammatical synonyms can refer to the effective amount of medication and duration required to achieve the desired therapeutic outcome. The therapeutic dose may vary depending on factors such as disease status, age, sex, and weight of the individual, and the ability of the composition described herein to induce a desired response in one or more subjects. The exact amount of the composition of this disclosure administered can be determined by a physician, taking into account individual differences in the patient's (subject's) age, weight, tumor size, extent of infection or metastasis, and condition.

[0098]

[0131] Alternatively, the pharmacological and / or physiological effects of administering one or more compositions described herein to a patient or subject may be “preventive,” for example, the effect completely or partially prevents the disease or symptoms of the patient or subject. “Preventive effective dose” can mean an effective dose in terms of dosage and duration required to achieve the desired preventive outcome (e.g., prevention of disease onset).

[0099]

[0132] Some of the numbers disclosed throughout are referred to as, for example, "X is at least or at least about 100 or 200 [or any number]." This number includes the number itself as well as aX is at least 100. bX is at least 200. cX is at least about 100, and dX is at least approximately 200. It includes all of the above.

[0100]

[0133] All of these different combinations are intended by the figures disclosed throughout. All disclosed figures, whether referring to the administration of therapeutic agents, days, months, years, weight, dosage, etc., should be interpreted in this way unless otherwise specifically indicated.

[0101]

[0134] The range disclosed throughout may be referred to, for example, as "X is administered on day 1-2 or approximately day 1-2, or on day 2-3 [or any numerical range] or approximately day 2-3 [or any numerical range]." This range includes the number itself (e.g., the endpoints of the range) as well as i) X is administered between day 1 and day 2. ii) X is administered between day 2 and day 3. iii) X is administered between approximately day 1 and day 2. iv) X is administered between approximately the second and third day. v) X is administered between day 1 and approximately day 2. vi) X is administered between day 2 and approximately day 3. vii)X is administered between approximately day 1 and day 2, and viii)X is administered between approximately the second and third day. It includes all of the above.

[0102]

[0135] All of these different combinations are intended by the scope disclosed throughout. The entire scope disclosed, whether referring to the administration of therapeutic agents, or to days, months, years, body weight, dosage, etc., should be interpreted in this way unless otherwise specifically indicated.

[0103] Stages of differentiation

[0136] In some embodiments, the pancreatic differentiation disclosed herein is performed stepwise. In stepwise progression, “Stage 1” or “S1” refers to the first step of the differentiation process, namely the differentiation of pluripotent stem cells into cells expressing markers characteristic of endoderm cells (“DE”) (“Stage 1 cells” or “S1 cells”). “Stage 2” refers to the second step, namely the differentiation of cells expressing markers characteristic of endoderm cells into cells expressing markers characteristic of intestinal cells (“GT”) (“Stage 2 cells” or “S2 cells”). “Stage 3” refers to the third step, namely the differentiation of cells expressing markers characteristic of intestinal 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, namely the differentiation of cells expressing a marker characteristic of pancreatic progenitor 1 cells into cells expressing a marker characteristic of pancreatic progenitor 2 cells ("PP2") ("stage 4 cells" or "S4 cells"). "Stage 5" refers to the fifth step, namely the differentiation of pancreatic progenitor 2 cells (e.g., PDX.1). + NKX6.1 + ) Pancreatic endodermal cells and / or pancreatic endocrine progenitor cells (e.g., insulin cells) that express markers characteristic of ) +Cells that express a characteristic marker of ("EN") ("stage 5 cells", or "S Stage 5 refers to differentiation into cells. 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 β-cells ("SC-β-cells") or pancreatic endocrine α-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; that is, some cells may progress through the differentiation pathway less or more than the majority of cells 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 producing any one of the Stage 1-6 cells are provided, for example, in U.S. Patent No. 10,030,229, U.S. Patent No. 10,443,042, U.S. Publication No. 20200332262, and U.S. Patent Pending Application No. 17 / 010,346, each of which is incorporated herein by reference in its entirety.

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

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

[0105]

[0138] In some embodiments, the step of contacting cells with an acetyl coenzyme A-related metabolite (e.g., acetate) can improve the generation of SC-β cells in vitro compared to compositions lacking or containing the acetyl coenzyme A-related metabolite at lower concentrations, resulting in SC island clusters with advantageous characteristics such as higher cell yield and recovery, 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, improved cell viability after transplantation, improved cell function, reduced immunogenicity, or a combination thereof.

[0106]

[0139] Examples of acetyl-CoA-related metabolites include, but are not limited to, acetate, pirubate, 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 compositions of the present disclosure comprise two or more different acetyl-CoA-related metabolites, e.g., 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, acetyl-CoA-related metabolites are present in or added to the composition of the Disclosure at concentrations 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 the composition of the Disclosure at concentrations 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, acetyl-CoA-related metabolites are present in or added to the compositions of the Disclosure at concentrations of approximately 10 nM, 50 nM, 80 nM, 100 nM, 120 nM, 140 nM, 150 nM, 200 nM, 300 nM, 500 nM, 800 nM, 1 μM, 10 μM, 100 μM, 500 μM, 800 μM, 900 μM, 1 mM, 2 mM, 3 mM, 5 mM, or 10 mM.

[0110]

[0143] In some embodiments, the acetyl-CoA-related metabolite is present in or added to the composition of the Disclosure at concentrations of approximately 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 an acetate present at a concentration of approximately 1 mM.

[0111]

[0144] In some embodiments, the acetyl-CoA-related metabolite is an acetate present at concentrations of approximately 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 an acetate present at a concentration of approximately 160 nM.

[0112]

[0145] In some embodiments, the method of this disclosure involves contacting cells with an acetyl-CoA-related metabolite (e.g., acetate). The cells may be PDX1-positive, NKX6.1-positive, insulin-negative cells. The cells may be insulin-positive endocrine progenitor cells. The cells may be SC-β cells. In some embodiments, the cells are frozen. In some embodiments, the cells are 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 progenitor cells) are exposed to acetyl-CoA-related metabolites (e.g., acetate) for approximately 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, or insulin-positive endocrine progenitor cells) are brought into contact 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 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 progenitor cells) are exposed to acetyl-CoA-related metabolites (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, or up to 14 days.

[0116]

[0149] In some embodiments, cells (e.g., PDX1-positive, NKX6.1-positive, insulin-negative, or insulin-positive endocrine progenitor cells) are exposed to acetyl-CoA-related metabolites (e.g., acetate) for approximately 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, the compositions or methods of the present disclosure do not contain acetyl-CoA-related metabolites (e.g., acetate-free).

[0118] vitamin

[0151] In some embodiments, the compositions of the present disclosure (e.g., culture media) include one or more vitamins. In some embodiments, the step of contacting cells with a vitamin (e.g., biotin) can improve the generation of SC-β cells in vitro compared to compositions that do not contain the vitamin or contain it at a lower concentration, resulting in SC island clusters with advantageous characteristics such as higher cell yield and recovery, 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, improved cell viability after transplantation, improved cell function, reduced immunogenicity, or a combination thereof.

[0119]

[0152] Examples of 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 vitamins regulate fatty acid synthesis. In some embodiments, the vitamins regulate the metabolism of branched-chain amino acids. In some embodiments, the vitamins regulate or participate as cofactors in the TCA cycle, for example, as cofactors in pyruvate carboxylase. In some embodiments, the vitamin is biotin. In some embodiments, the compositions of the present disclosure include two or more different vitamins, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different vitamins.

[0120]

[0153] In some embodiments, the vitamin is present in or added to the 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 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, the vitamins are present in or added to the composition of the Disclosure at concentrations 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 in or added to the composition of the Disclosure at concentrations of approximately 100 nM, approximately 300 nM, approximately 500 nM, approximately 600 nM, approximately 700 nM, approximately 800 nM, approximately 900 nM, approximately 1 μM, approximately 1.5 μM, approximately 3 μM, approximately 5 μM, approximately 10 μM, or approximately 100 μM. In some embodiments, the vitamin is biotin present at a concentration of approximately 800 nM.

[0123]

[0156] In some embodiments, the vitamins are approximately 1nM to 500μM, 1nM to 100μM, 1nM to 10μM, 1nM to 1μM, 1nM to 800nM, 1nM to 600nM, 1nM to 400nM, 1nM to 300nM, 1nM to 200nM, 25nM to 500μM, 25nM to 100μM, 25nM to 1μM, 25nM to 800nM, 25nM to 600nM, 25nM to 400nM, 25nM to 300nM, 25nM to 200nM, 50nM to 500μM, and 50nM These substances are present in or added to the compositions of this disclosure at concentrations of ~100 μM, 50 nM~10 μM, 50 nM~1 μM, 50 nM~800 nM, 50 nM~600 nM, 50 nM~400 nM, 50 nM~300 nM, 50 nM~200 nM, 100 nM~500 μM, 100 nM~100 μM, 100 nM~10 μM, 100 nM~1 μM, 100 nM~800 nM, 100 nM~600 nM, 100 nM~400 nM, 100 nM~300 nM, or 100 nM~200 nM.

[0124]

[0157] In some embodiments, the method of this disclosure involves contacting cells with a vitamin (e.g., biotin). The cells may be PDX1-positive, NKX6.1-positive, insulin-negative cells. The cells may be insulin-positive endocrine progenitor cells. The cells may be SC-β cells. In some embodiments, the cells are frozen. In some embodiments, the cells are 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 progenitor cells) are exposed to a vitamin (e.g., biotin) for approximately 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 progenitor cells) are brought into contact 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, or 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 progenitor cells) are exposed to vitamins (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, or up to 14 days.

[0128]

[0161] In some embodiments, cells (e.g., PDX1-positive, NKX6.1-positive, insulin-negative cells, or insulin-positive endocrine progenitor cells) are exposed to a vitamin (e.g., biotin) for approximately 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, the compositions or methods of the present disclosure are vitamin-free (e.g., biotin-free).

[0130] Histone deacetylase inhibitors (HDACi)

[0163] In some embodiments, the compositions of the present disclosure (e.g., culture media) include a histone deacetylase inhibitor (HDACi). A histone deacetylase inhibitor (HDACi) is 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, the step of contacting cells with HDACi can improve the generation of SC-β cells in vitro compared to compositions lacking HDACi or containing it at lower concentrations, resulting in SC island 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, improved cell viability after transplantation, improved cell function, reduced immunogenicity, or a combination thereof.

[0132]

[0165] Exemplary histone deacetylase inhibitors (HDACi) include β-hydroxybutyrate, butyrate, class I HDACi, class IIA HDACi, and class IIB. HDACi, Class III HDACi, Class IV HDACi, HDAC-1, HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC-6, HDAC-7, HDAC-8, HDAC-9, HDAC-10, HDAC-11, Sirtuin, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, Vorinostat (Suberoylanilide Hydroxamic Acid, SAHA, MK0683), Entinostat (MS-275, SNDX-275), Panobinostat (LBH589, NVP-LBH589), Trichostatin A (TSA), Mosetinostat (MGCD0103, MG0103), GSK3117391 (GSK3117391A, HDAC-IN-3), BRD3308, BRD3308, Tuvastatin A TFA (Tuvastatin A trifluoroacetate), Tuvastatin A, SIS17, NKL 22, BML-210 (CAY10433), TC-H 106, SR-4370, Bellinostat (PXD101, NSC726630, PX-105684), Romidepsin (FK228, Depsipeptide, FR 901228, NSC 630176), MC1568, Gibinostat (ITF2357), Dasinostat (LAQ824, NVP-LAQ824), CUDC-101, Xinostat (JNJ-26481585), Prasinostat (SB939), PCI-34051, Droxinostat (NS 41080), avexinostat (PCI-24781), avexinostat (PCI-24781, CRA-024781), RGFP966, AR-42 (HDAC-42), licorinostat (ACY-1215, rosirinostat), sodium valproate (sodium valproate), tasedinarin (CI994, PD-123654, GOE-5549, acetyldinarin), fimepinostat (CUDC-907), sodium butyrate (NaB), curcumin, diferuloylmethane, M344, tubacin, RG2833 (RGFP109), RG2833 (RGFP109), resminostat (RAS2410), divalproex sodium, scriptide (GCK1026), sodium phenylbutyrate, synapic acid (synapic acid), TMP269, Santa Cruzamate A (CAY10683), TMP195 (TFMO2), valproic acid (VPA), UF010, Tascinimod (ABR-215050), SKLB-23bb, isoguanosine, sulforaphane, BRD73954, sitalinostat (ACY-241, HDAC-IN-2), suberohydroxamic acid, spritomycin, HPOB, LMK-235, biphenyl-4-sulfonyl chloride (p-phenylbenzenesulfonyl, 4-phenylbenzene Examples include, but are not limited to, sulfonyl (p-biphenylsulfonyl), nextulastat A, TH34, tusidinostat (thidamide, HBI-8000, CS-055), (-)-parthenolide, WT161, CAY10603, CAY10603, ACY-738, radianine A, tinostamstine (EDO-S101), domatinostat (4SC-202), and BG45.

[0133]

[0166] In some embodiments, HDACi is β-hydroxybutyrate. β-hydroxybutyrate, along with butyrate, is a ketone body that 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, the compositions of the present disclosure include two or more different HDACi, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different HDACi.

[0135]

[0168] In some embodiments, HDACi is present in or added to the composition of the Disclosure at concentrations 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 may be present in or added to the composition of the Disclosure at concentrations 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 in or added to the composition of the Disclosure at concentrations of approximately 100 nM, approximately 300 nM, approximately 500 nM, approximately 600 nM, approximately 700 nM, approximately 800 nM, approximately 900 nM, approximately 1 μM, approximately 1.5 μM, approximately 3 μM, approximately 5 μM, approximately 10 μM, or approximately 100 μM. In some embodiments, HDACi is a β-hydroxybutyrate present at a concentration of approximately 200 nM.

[0138]

[0171] In some embodiments, HDACi has a range of approximately 1nM to 500μM, 1nM to 100μM, 1nM to 10μM, 1nM to 1μM, 1nM to 800nM, 1nM to 600nM, 1nM to 400nM, 1nM to 300nM, 1nM to 200nM, 25nM to 500μM, 25nM to 100μM, 25nM to 1μM, 25nM to 800nM, 25nM to 600nM, 25nM to 400nM, 25nM to 300nM, 25nM to 200nM, 50nM to 500μM, and 50n These substances are present in or added to the compositions of this disclosure at concentrations of M~100 μM, 50 nM~10 μM, 50 nM~1 μM, 50 nM~800 nM, 50 nM~600 nM, 50 nM~400 nM, 50 nM~300 nM, 50 nM~200 nM, 100 nM~500 μM, 100 nM~100 μM, 100 nM~10 μM, 100 nM~1 μM, 100 nM~800 nM, 100 nM~600 nM, 100 nM~400 nM, 100 nM~300 nM, or 100 nM~200 nM.

[0139]

[0172] In some embodiments, the method of this disclosure involves contacting cells with HDACi (e.g., β-hydroxybutyrate). The cells may be PDX1-positive, NKX6.1-positive, insulin-negative cells. The cells may be insulin-positive endocrine progenitor cells. The cells may be SC-β cells. In some embodiments, the cells are frozen. In some embodiments, the cells are 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 progenitor cells) are exposed to HDACi (e.g., β-hydroxybutyrate) for approximately 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, or insulin-positive endocrine progenitor cells) are brought into contact with 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, or 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 progenitor cells) are exposed to 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, or up to 14 days.

[0143]

[0176] In some embodiments, cells (e.g., PDX1-positive, NKX6.1-positive, insulin-negative, or insulin-positive endocrine progenitor cells) are exposed to HDACi (e.g., β-hydroxybutyrate) for approximately 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, the compositions or methods of the present disclosure are HDACi-free (e.g., β-hydroxybutyrate-free).

[0145] Redox homeostasis regulator

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

[0146]

[0179] In some embodiments, the step of contacting cells with a redox homeostasis regulator (e.g., taurine) can improve the generation of SC-β cells in vitro compared to compositions lacking or containing the redox homeostasis regulator at lower concentrations, resulting in SC island clusters with advantageous characteristics such as higher cell yield and recovery, 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, improved cell viability after transplantation, improved cell function, reduced immunogenicity, or a combination thereof.

[0147]

[0180] Exemplary redox homeostasis regulators include, but are not limited to, taurine, respiratory chain regulators, free radical scavengers, mitochondrial protein synthesis regulators, Allium sulfur compounds, anthocyanins, beta-carotenes, catechins, copper, cryptoxanthin, flavonoids, indole, isoflavonoids, lignans, lutein, lycopene, alpha-lipoic acid, ellagic acid, manganese, polyphenols, selenium, glutathione, vitamin A, vitamin C, vitamin E, zinc, superoxide distase, 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 production in the respiratory chain.

[0149]

[0183] In some embodiments, the compositions of the present disclosure include two or more different redox homeostasis regulators, e.g., 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 the composition of the 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 the composition of the Disclosure at concentrations 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 in or added to the composition of the Disclosure at concentrations of approximately 100 nM, 500 nM, 1 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 110 μM, 110 μM, 150 μM, or 200 μM. In some embodiments, the redox homeostasis regulator is taurine. In some embodiments, the redox homeostasis regulator is taurine present at a concentration of approximately 90 μM.

[0153]

[0187] In some embodiments, the redox homeostasis regulator intermediate is approximately 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 It is present or added at concentrations of μM, 30μM-250μM, 40μM-250μM, 50μM-250μM, 60μM-250μM, 70μM-250μM, 100nM-100μM, 500nM-100μM, 1μM-100μM, 1μM-100μM, 10μM-100μM, 20μM-100μM, 30μM-100μM, 40μM-100μM, 50μM-100μM, 60μM-100μM, 70μM-100μM, or 80μM-100μM.

[0154]

[0188] In some embodiments, the method of this disclosure involves contacting cells with a redox homeostasis regulator (e.g., taurine). The cells may be PDX1-positive, NKX6.1-positive, insulin-negative cells. The cells may be insulin-positive endocrine progenitor cells. The cells may be SC-β cells. In some embodiments, the cells are frozen. In some embodiments, the cells are 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 progenitor cells) are exposed to a redox homeostasis regulator (e.g., taurine) for approximately 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, or insulin-positive endocrine progenitor cells) are brought into contact 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, or at least or at least 14 days.

[0157]

[0191] In some embodiments, cells (e.g., PDX1-positive, NKX6.1-positive, insulin-negative, or insulin-positive endocrine progenitor cells) are exposed to 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, 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 progenitor cells) are exposed to a redox homeostasis regulator (e.g., taurine) for approximately 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, the compositions or methods of the present disclosure do not contain redox homeostasis regulators (e.g., do not contain taurine).

[0160] One-carbon metabolic pathway intermediate

[0194] In some embodiments, a composition of the present disclosure (e.g., a medium) comprises a one-carbon metabolic pathway intermediate. Folate cofactor-mediated one-carbon metabolism supports multiple physiological processes including amino acid homeostasis (methionine, glycine, and serine), nucleotide (purine, thymidine) biosynthesis, epigenetic maintenance, and redox defense.

[0161]

[0195] In some embodiments, contacting cells with a one-carbon metabolic pathway intermediate improves the production of SC-β cells in vitro compared with, for example, a composition lacking the one-carbon metabolic pathway intermediate or comprising the intermediate at a lower concentration, and can result in, for example, higher cell yield and recovery rate, increased number and relative percentage of SC-β cells, enhanced stability and shelf life of SC-β cells, SC-islet clusters with advantageous features such as reduced size and increased homogeneity, improved function of SC-β cells in vitro, improved cell survival after transplantation, improved cell function, reduced immunogenicity, or combinations thereof.

[0162]

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

[0163]

[0197] In some embodiments, the composition of the present 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, the one-carbon metabolic pathway intermediate is present in or added to the 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 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, the one-carbon metabolic pathway intermediate is present in or added to the 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 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, the one-carbon metabolic pathway intermediate is present in or added to the composition of the present disclosure at a concentration of about 100 nM, about 500 nM, 1 μM, about 10 μM, about 20 μM, about 30 μM, about 40 μM, about 50 μM, about 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 present in or added at a concentration of 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 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 method of this disclosure involves contacting cells with a monocarbon metabolic pathway intermediate (e.g., phormate). The cells may be PDX1-positive, NKX6.1-positive, insulin-negative cells. The cells may be insulin-positive endocrine progenitor cells. The cells may be SC-β cells. In some embodiments, the cells are frozen. In some embodiments, the cells are 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 progenitor cells) are exposed to a monocarbon metabolic pathway intermediate (e.g., phormate) for approximately 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, or insulin-positive endocrine progenitor cells) are brought into contact with a monocarbon metabolic pathway intermediate (e.g., phormate) 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 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 progenitor cells) are brought into contact with a monocarbon metabolic pathway intermediate (e.g., phormate) 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, 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 progenitor cells) are brought into contact with a monocarbon metabolic pathway intermediate (e.g., phormate) for approximately 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, the compositions or methods of the present disclosure do not contain a single-carbon metabolic pathway intermediate (e.g., they do not contain a formate).

[0175] glutamine

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

[0176]

[0210] In some embodiments, the step of contacting cells with glutamine as disclosed herein is, for example, in a composition lacking glutamine, containing less glutamine, or containing a different (e.g., less bioavailable) form of glutamine. This can improve the generation of in vitro SC-β cells, for example, by resulting in SC island clusters with favorable 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 in vitro SC-β cell function, improved cell viability after transplantation, enhanced cell function, reduced immunogenicity, or a combination thereof.

[0177]

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

[0178]

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

[0179]

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

[0180]

[0214] Glutamine can contribute to several cellular metabolic processes important to the embodiments of this disclosure, such as redox regulation, NAD recycling, TCA cycle replenishment, and nitrogen metabolism. Glutamine, containing one nitrogen atom as an amide and another as an amine, transports and delivers nitrogen to cells in toxic amounts as free ammonium. The glutamine amide nitrogen can be used in the synthesis of NAD and NADP, purine nucleotides, CTP from UTP, and asparagine. Nitrogen initially stored as glutamine can also be used to produce carbamyl phosphate for pyrimidine synthesis. Glutamine is a precursor of glutamates, 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 in vitro for many rapidly dividing cell types.

[0181]

[0215] Cells require nitrogen atoms to construct molecules such as nucleotides, amino acids, amino sugars, and vitamins. Ammonium is an inorganic nitrogen source that primarily exists as a positively charged cation NH4+ at physiological pH. The ammonium nitrogen used by cells is first incorporated into organic nitrogen as the amine of glutamate or the amide of glutamine. These two amino acids provide a primary storage of nitrogen for the synthesis of proteins, nucleic acids, and other nitrogenous compounds. The reactions that fix nitrogen to glutamate and glutamine consume energy equivalents. 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. The enzyme responsible for glutamine synthesis, glutamine synthetase (EC 6.3.1.2), is highly regulated to limit glutamine production to cellular requirements. Catabolism from glutamine to glutamate and ammonium is mediated by a mitochondrial enzyme called glutaminase (EC 3.5.1.2). In vivo, ammonium produced 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 requirements can increase under stressful conditions. In some embodiments, glutamine is a conditionally essential amino acid and must be supplied in sufficient levels, for example, under stressful conditions. Like other amino acids, glutamine is biochemically important as a building block of proteins. Glutamine can also be extremely important 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 the nicotinamide adenine dinucleotide (NAD) redox potential.

[0183]

[0217] In some embodiments, glutamine may be present in the compositions of the Disclosure at concentrations 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 the compositions of the Disclosure at concentrations 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 the compositions 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 compositions of the present disclosure at a concentration of 0.5 to 20 mM, 0.5 to 10 mM, 0.5 to 5 mM, 1 to 5 mM, 2 to 5 mM, or 1 mM to 10 mM. In some embodiments, glutamine is present in or added to the compositions of the present disclosure at a concentration of 3.8 to 4.2 mM. In some embodiments, glutamine is present in or added to the compositions of the present disclosure at a concentration of 1 to 10, 1 to 7, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 2 to 10, 2 to 7, 2 to 8, 2 to 6, 2 to 5, 2 to 4, 3 to 10, 3 to 7, 3 to 8, 3 to 6, 3 to 5, 3 to 4, 3.5 to 4.5, 3.8 to 4.2, or 3.9 to 4.1 mM. In some embodiments, glutamine is present in or added to the compositions 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 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 form of alanine-glutamine dipeptide.

[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 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 glutamine is not in the multimer 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 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 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 glutamine is in the free form.

[0194]

[0228] In some embodiments, the method of this disclosure involves contacting glutamine with cells. The cells may be PDX1-positive, NKX6.1-positive, insulin-negative cells. The cells may be insulin-positive endocrine progenitor cells. The cells may be SC-β cells. In some embodiments, the cells are frozen. In some embodiments, the cells are 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, or insulin-positive endocrine progenitor cells) are exposed to glutamine for approximately 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, or insulin-positive endocrine progenitor cells) are brought into contact 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, or 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 progenitor cells) are exposed to 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, 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 progenitor cells) are exposed to glutamine for approximately 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, the compositions or methods disclosed herein do not contain glutamine. Glutamate

[0234] In some embodiments, the compositions of the present disclosure (e.g., culture media) include glutamates (e.g., L-glutamates). Glutamates can be converted to, for example, g-aminobutyric acid (GABA), ornithine, 2-oxoglutaric acid, glucose, or glutathione. Glutamates and their metabolites can contribute, for example, to redox homeostasis, cell signaling, nitrogen assimilation, amine catabolism, amino acid biosynthesis, nucleoside biosynthesis, and cofactor production.

[0200]

[0235] In some embodiments, the step of contacting cells with glutamate can improve the generation of SC-β cells in vitro compared to compositions lacking glutamate or containing glutamate at lower concentrations, resulting in SC island clusters with advantageous characteristics such as higher cell yield and recovery, 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, improved cell viability after transplantation, improved cell function, reduced immunogenicity, or a combination thereof.

[0201]

[0236] In some embodiments, glutamate may be present in the compositions of the Disclosure at concentrations 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 the compositions of the Disclosure at concentrations 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 compositions of the Disclosure at concentrations 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 may be present in or added to the compositions of the Disclosure at a concentration of about 500 μM.

[0204]

[0239] In some embodiments, glutamate is present in concentrations of approximately 100 μM to 5 mM, 200 μM to 5 mM, 300 μM to 5 mM, 400 μM to 5 mM, 100 μM to 3 mM, 200 μM to 3 mM, 300 μM to 3 mM, 400 μM to 3 mM, 100 μM to 2 mM, 200 μM to 2 mM, 300 μM to 2 mM, 400 μM to 2 mM, 100 μM to 1 mM, 200 μM to 1 mM, 300 μM to 1 mM, 400 μM to 1 mM, 100 μM to 700 μM, 200 μM to 700 μM, 300 μM to 700 μM.

[0240] It may be present in or added to the compositions of this disclosure at concentrations 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, glutamate is brought into contact with cells in the method of this disclosure. The cells may be PDX1-positive, NKX6.1-positive, insulin-negative cells. The cells may be insulin-positive endocrine progenitor cells. The cells may be SC-β cells. In some embodiments, the cells are frozen. In some embodiments, the cells are 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 progenitor cells) are exposed to glutamate for approximately 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, or insulin-positive endocrine progenitor cells) are brought into contact 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, or 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 progenitor cells) are exposed to 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, or up to 14 days.

[0209]

[0245] In some embodiments, cells (e.g., PDX1-positive, NKX6.1-positive, insulin-negative cells, or insulin-positive endocrine progenitor cells) are exposed to glutamate for approximately 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, the compositions or methods of the present disclosure do not contain glutamate.

[0211] Carnitine

[0247] In some embodiments, the composition of the present disclosure (e.g., a culture medium) comprises carnitine. Carnitine can transport long-chain fatty acids into mitochondria for oxidation for energy production and support cytocompatibility.

[0212]

[0248] In some embodiments, the step of contacting cells with carnitine can improve the generation of in vitro SC-β cells compared to compositions lacking carnitine or containing lower concentrations of carnitine, for example, resulting in SC island 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 in vitro SC-β cells, improved cell viability after transplantation, improved cell function, reduced immunogenicity, or a combination thereof.

[0213]

[0249] In some embodiments, carnitine is present in or added to the composition of the Disclosure at concentrations 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 may be present in or added to the composition of the Disclosure at concentrations 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 in or added to the composition of the Disclosure at concentrations of approximately 100 nM, approximately 500 nM, approximately 1 μM, approximately 10 μM, approximately 15 μM, approximately 20 μM, approximately 25 μM, approximately 30 μM, approximately 35 μM, approximately 40 μM, approximately 45 μM, approximately 50 μM, approximately 55 μM, approximately 60 μM, approximately 75 μM, or approximately 100 μM. In some embodiments, carnitine is present in or added at a concentration of approximately 40 μM.

[0216]

[0252] In some embodiments, carnitine is expressed in concentrations of approximately 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, and 500 nM. It may be present in or added to the compositions of this disclosure at concentrations of 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, 35 μM to 60 μM, or 30 μM to 50 μM.

[0217]

[0253] In some embodiments, carnitine is brought into contact with cells in the method of this disclosure. The cells may be PDX1-positive, NKX6.1-positive, insulin-negative cells. The cells may be insulin-positive endocrine progenitor cells. The cells may be SC-β cells. In some embodiments, the cells are frozen. In some embodiments, the cells are previously frozen. In some embodiments, the cells are in cell clusters. In some embodiments, the cells are dissociated.

[0218]

[0254] In some embodiments, cells (e.g., PDX1-positive, NKX6.1-positive, insulin-negative cells, or insulin-positive endocrine progenitor cells) are exposed to carnitine for approximately 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, or insulin-positive endocrine progenitor cells) are brought into contact 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, or 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 progenitor cells) are exposed to 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, 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 progenitor cells) are brought into contact with carnitine for approximately 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 disclosed herein do not contain 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-β Ri kinase, also known as RepSox, IIJPAC name: 2-[5-(6-methylpyridine-2-yl)-1H-pyrazole-4-yl]-1,5-naphthiridine, analogs or derivatives of ALK5 inhibitor II, for example, analogs or derivatives of ALK5 inhibitor II described in U.S. Patent Publication 2012 / 0021519, and those described in U.S. Patent Publication 2010 / 0267731). Examples of TGF-β receptor inhibitors include, but are not limited to, ALK5 inhibitors described in U.S. Patent Publications 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 following structure.

[0225] [ka]

[0226]

[0261] In some embodiments, the concentration of the TGF-β signaling pathway inhibitor may be approximately 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 may be approximately 10 μM. In some embodiments, the TGF-β signaling pathway inhibitor is Alk5 inhibitor II, and the concentration of the inhibitor is approximately 10 μM.

[0227] Thyroid hormone signaling pathway activators

[0262] Exemplary thyroid hormone signaling pathway activators include triiodothyronine (T3), analogs or derivatives of T3, e.g., selective and non-selective thyromimetics, TRJ selective agonists GC-1, GC-24,4-hydroxy-PCB 106, MB0781 1, MB07344,3,5-diiodotyropropionic acid (DITPA); selective TR-β agonist GC-1; 3-iodothyronamine (T(1)AM) and 3,3',5-triiodotyroacetate (Triac) (biologically active metabolites of the hormone thyroxine (T(4)); KB-21 15 and KB-141; thyronamine; SKF L-94901; DIBIT; 3'-AC-T2; Tetraiodotyroacetate (Tetrac) and triiodotyroacetate (Triac) (by oxidative deamination and decarboxylation of thyroxine [T4] and triiodothyronine [T3] alanine chains), 3,3',5'-triiodothyronine (rT3) (by deiodination of T4 and T3), 3,3'-diiodothyronine (3,3'-T2) and 3,5-diiodothyronine (T2) ( (by deiodination of T4, T3, and rT3), as well as 3-iodothyronamine (T1AM) and thyronamine (T0AM) (by deiodination of T4 and T3 and decarboxylation of amino acids), and TH structural analogs, e.g., 3,5,3'-triiodotyropropionic acid (Triprop), 3,5-dibromo-3-pyridazinone-1-thyronine (L-940901), N-[3,5-dimethyl-4-(4'-hydroxy-3 f Examples include, but are not limited to, 3,5-isopropylphenoxy)phenyl]oxamic acid (CGS 23425), 3,5-dimethyl-4-[(4'-hydroxy-3'-isopropylbenzyl)-phenoxy]acetic acid (GC-1), 3,5-dichloro-4-[(4-hydroxy-3-isopropylphenoxy)phenyl]acetic acid (KB-141), and 3,5-diiodotyropropionic acid (DITPA).

[0228]

[0263] In some embodiments, the thyroid hormone signaling pathway activator may include a T3 prodrug or prohormone such as a T4 thyroid hormone (e.g., thyroxine or L-3,5,3',5'-tetraiodothyronine). In some embodiments, the thyroid hormone signaling pathway activator may be an iodothyronine composition described in U.S. Patent No. 7,163,918. In some embodiments, the thyroid hormone signaling pathway activator may be 2-[4-[[4-hydroxy-3-(1-methylethyl)phenyl]methyl]-3,5-dimethylphenoxy]acetic acid (GC-1). GC-1 is thyromimetic and a high-affinity agonist of the thyroid hormone receptor (TR)β and TRoc receptor (K D The values ​​are 67p and 440p, respectively. GC-1 shows 5-fold and 100-fold higher potency in vitro than the TRoti receptor and the endogenous TRi receptor agonist T3, respectively.

[0229]

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

[0230] [ka]

[0231]

[0265] In some embodiments, the concentration of the thyroid hormone signaling pathway activator 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 thyroid hormone signaling pathway activator may 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] Examples of protein kinase inhibitors include, but are not limited to, staurosporines, staurosporine analogs such as Ro-31-8220, bisindylmaleimide (Bis) compounds, 10'-{5”-[(methoxycarbonyl)amino]-2”-methyl}phenylaminocarbonylstaurosporine, staurologs (see, for example, 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 a staurosporine.

[0233]

[0267] In some embodiments, the concentration of the protein kinase inhibitor may 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 may 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 following structure.

[0235] [ka]

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

[0269] Examples of BMP signaling pathway inhibitors include, but are not limited to, 4-[6-(4-piperazine-1-ylphenyl)pyrazolo[1,5-a]pyrimidine-3-yl]quinolone (also known as LDN 193 189; LDN1931 89, 1062368-24-4, LDN-193189, DM 3189, DM-3189, and referred to herein as LDN), analogs or derivatives of LDN193189, e.g., salts of LDN193189 (e.g., LDN193189 hydrochloride), hydrates, solvents, esters, or prodrugs, or compounds of formula I of U.S. Patent Application Publication No. 2011 / 0053930. In several embodiments of the present invention, the BMP signaling pathway inhibitor comprises LDN193189. In some embodiments, the BMP signaling pathway inhibitor comprises DMH-1 or analogs or derivatives thereof.

[0237]

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

[0238] [ka]

[0239] It can have.

[0271] In some embodiments, the concentration of the BMP signaling pathway inhibitor may 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 may 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-related protein kinase (ROCK) inhibitors

[0272] Examples of 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-oxadiazole-3-yl)-1-ethyl-1H-imidazo[4,5-c]pyridine-6-yl]oxy]phenyl]-4-[2-(4-morpholinyl)ethoxy]benzamide (GS 269962), and 4-[4-(trifluoromethyl)phenyl]-N-(6-fluoro-1H-indazole-5-yl)-2-methyl-6-oxo-1,4,5,6-tetrahydro-3-pyridinecarboxamide (GSK 429286), (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine dihydrochloride (H1152 dihydrochloride), (S)-(+)-4-glycyl-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine dihydrochloride (glycyl-M1152 dihydrochloride), N-[(3-hydroxyphenyl)methyl]-N'-[4-(4-pyridinyl)-2-thiazolyl]urea dihydrochloride (RKI 1447 dihydrochloride), (3S)-1-[[2-(4-amino-1,2,5-oxadiazole-3-yl)-1-ethyl-1H-imidazo[4,5-c]pyridine-7-yl]carbonyl]-3-pyrrolidineamine dihydrochloride (SB772077B dihydrochloride), N-[2-[2-(dimethylamino)ethoxy]-4-(1H-pyrazole-4-yl)phenyl-2,3-dihydro-1,4-benzodioxin-2-carboxamide dihydrochloride (SR 3677 dihydrochloride), and trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide dihydrochloride (Y-27632 dihydrochloride), N-benzyl-[2-(pyrimidine-4-yl)amino]thiazole-4-carboxamide (thiazovibin), ROCK inhibitor, isoquinoline sulfonamide compound (Rho kinase inhibitor), N-(4-pyridyl)-N'-(2,4,Organic small molecule ROCK inhibitors selected from the group consisting of 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), and Rock-2(30-J); Rock-1 CRISPR / Cas9 KO plasmid (h), Rock-2 CRISPR / Cas9 Examples include ROCK CRISPR / Cas9 knockout plasmids selected from the group consisting of KO plasmid (h), Rock-1 CR1SPR / Cas9 KO plasmid (m), and Rock-2 CRISPR / Cas9 KO plasmid (m); ROCK siRNAs 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; shRNA plasmids; and / or shRNA lentiviral particle gene silencers.

[0241]

[0273] In some embodiments, the ROCK inhibitor includes Y-27632. In some embodiments, the ROCK inhibitor is thiazovibin.

[0242]

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

[0243] [ka]

[0244] It can have.

[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 thiazovibin, and the concentration of the inhibitor is about 2.5 μM.

[0245]

[0276] In some embodiments, the concentrations of the ROCK inhibitor (e.g., Y-27632 or thiazovibin) are approximately 0.2 μM, 0.5 μM, 0.75 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 7.5 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, and 1 It may be 3 μM, approximately 14 μM, approximately 15 μM, approximately 16 μM, approximately 17 μM, approximately 18 μM, approximately 19 μM, approximately 20 μM, approximately 21 μM, approximately 22 μM, approximately 23 μM, approximately 24 μM, approximately 25 μM, approximately 26 μM, approximately 27 μM, approximately 28 μM, approximately 29 μM, approximately 30 μM, approximately 35 μM, approximately 40 μM, approximately 50 μM, or approximately 100 μM.

[0246] Histone methyltransferase inhibitors

[0277] In some embodiments, histone methyltransferase inhibitors can be used as epigenetic modifiers. Examples of histone methyltransferase inhibitors, but not limited to these, include 3-deazanepranosin A hydrochloride (DZNep-(1S,2R,5R)-5-(4-amino-1H-imidazo[4,5-c]pyridine-1-yl)-3-(hydroxymethyl)cyclopenta-3-en-1,2-diol); Bix-01294, UNC0638, BRDD4770, EPZ004777, AZ505, PDB4e47, alproic acid, vorinostat, romidepsin, entinostat, avexinostat, gibinostat, and mosetinostat, butyrate, and members of the serine protease inhibitor (serpine) family. In some embodiments, the histone methyltransferase inhibitor may be DZNep. In some embodiments, the histone methyltransferase inhibitor has the following structure:

[0247] [ka]

[0248] It can have.

[0278] In some embodiments, the concentration of the histone methyltransferase inhibitor may 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 may 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 may be approximately 0.01 μM, approximately 0.025 μM, approximately 0.05 μM, approximately 0.075 μM, approximately 0.1 μM, approximately 0.15 μM, approximately 0.2 μM, approximately 0.5 μM, approximately 0.75 μM, approximately 1 μM, approximately 2 μM, approximately 3 μM, approximately 4 μM, approximately 5 μM, approximately 6 μM, approximately 7 μM, approximately 7.5 μM, approximately 8 μM, approximately 9 μM, approximately 10 μM, approximately 15 μM, approximately 20 μM, approximately 25 μM, approximately 30 μM, approximately 35 μM, approximately 40 μM, approximately 50 μM, or approximately 100 μM.

[0250] MGLL inhibitors

[0280] Examples of MGLL (monoglyceride lipase) inhibitors include, but are not limited to, JJKK048, KML29, NF1819, JW642, JZL184, JZL195, JZP361, pristimerine, or URB602.

[0251]

[0281] In some embodiments, the MGLL inhibitor may be JJKK048. In some embodiments, the MGLL inhibitor may be KML29. In some embodiments, the MGLL inhibitor may be NF1819.

[0252]

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

[0253] [ka]

[0254]

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

[0255] [ka]

[0256]

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

[0257] [ka]

[0258]

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

[0259]

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

[0260]

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

[0261] Lipids

[0288] Examples of 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. Examples of saturated fatty acids include, but are not limited to, 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, henicosyl acid, behenic acid, tricosylic acid, lignoceric acid, pentacosylic acid, cerotic acid, heptacosylic acid, montanic acid, nonacosyl acid, melissic acid, hentriaconcylic acid, russellic acid, silyl acid, gesic acid, ceroplastic acid, hexatriacontylic acid, heptatriacontanoic acid, octatriacontanoic acid, nonatriacontanoic acid, or tetracontanoic acid, or their salts or esters.

[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] Examples of unsaturated fatty acids include, but are not limited to, oleic acid, linoleic acid, palmitoleic acid, stearidonic acid, eicosapentaenoic acid, docosahexaenoic acid, linolelideic acid, gamma-linolenic acid, dihomo-gamma-linolenic acid, arachidonic acid, docosatetraenoic acid, vaccenic acid, pauric acid, elaidic acid, gondouic acid, erucic acid, nervonic acid, or meadic acid, or their salts or esters.

[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 includes DMEM / F12. In some embodiments, any of the compositions disclosed herein may be 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 (at a concentration of 0.41580153 mM), L-lysine hydrochloride (at a concentration of 0.4986339 mM), L-methionine (at a concentration of 0.11570469 mM), L-phenylalanine (at a concentration of 0.2150303 mM), L-proline (at a concentration of 0.15 mM), L - Contains one or more amino acids, e.g., serine (at a concentration of 0.25 mM), L-threonine (at a concentration of 0.44915968 mM), L-tryptophan (at a concentration of 0.04421569 mM), L-tyrosine (at a concentration of 0.21375479 mM), or L-valine (at a concentration of 0.4517094 mM).In some embodiments, any of the compositions disclosed herein may contain 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., 0.016557377 mM). It contains one of the following vitamins: (at a concentration of mM), pyridoxine hydrochloride (e.g., at a concentration of 0.009771844 mM), riboflavin (e.g., at a concentration of 5.824468E-4 mM), thiamine hydrochloride (e.g., at a concentration of 0.0064391694 mM), vitamin B12 (e.g., at a concentration of 5.0184503E-4 mM), or i-inositol (e.g., at a concentration of 0.07 mM). In some embodiments, any of the compositions disclosed herein contains one of the following components: dextrose (17.505556 mM), sodium hypoxanthine (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 contains 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 disclosed herein contain 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 contains zinc (ZnSO4). In some embodiments, the compositions disclosed herein contain 1 to 100 μM, 1 to 50 μM, 1 to 20 μM, 1 to 12 μM, 5 to 15 μM, 8 to 12 μM, or 9 to 11 μM of ZnSO4. In some embodiments, the compositions disclosed herein contain about 10 μM of ZnSO4.

[0268]

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

[0269] Example drug combinations

[0296] In some embodiments, the compositions of the Disclosure comprise a combination of two or more agents disclosed herein. The two or more agents may be present in cell culture media, and optionally in media containing cells undergoing the differentiation or reprogramming methods of the Disclosure. The combinations of agents disclosed herein exhibit remarkable and unexpected benefits in in vitro generation of SC-β cells, resulting, for example, higher cell yield and recovery, 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 island clusters with advantageous characteristics such as reduced size and increased uniformity, improved in vitro function of SC-β cells, and improved post-transplant survival, improved function, and reduced immunogenicity.

[0270]

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

[0271]

[0298] In some embodiments, the step of contacting a cell population with a drug combination increases the growth of the cell population. In some embodiments, the step of contacting a cell population with a drug combination alters or increases the metabolism of the cell population. For example, in some cases, the step of contacting a cell population with a 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 by, for example, the Seahorse assay. In some cases, the step of contacting a cell population with a 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 compositions of the Disclosure include a monocarbon 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. A combination of agents including acetate, β-hydroxybutyrate, taurine, formate, biotin, and glutamine may be collectively referred to herein as "MQ". The compositions may include a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells in, for example, a cell culture medium disclosed herein. The compositions may include a plurality of insulin-positive endocrine progenitor cells in, for example, a cell culture medium disclosed herein. In some embodiments, the compositions of the Disclosure include cell clusters. In some embodiments, the cells of the culture of the 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 this disclosure are previously frozen.

[0273]

[0300] In some embodiments, the compositions of the Disclosure comprise at least one of the following: a monocarbon 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), and glutamine. The compositions may comprise a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells in, for example, a cell culture medium disclosed herein. The compositions may comprise a plurality of insulin-positive endocrine progenitor cells in, for example, a cell culture medium disclosed herein. In some embodiments, the compositions of the Disclosure comprise cell clusters. In some embodiments, the cells of the culture of the 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 Disclosure are previously frozen.

[0274]

[0301] In some embodiments, the compositions of the Disclosure include two of the following: a monocarbon 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 compositions may include a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells in, for example, a cell culture medium disclosed herein. The compositions may include a plurality of insulin-positive endocrine progenitor cells in, for example, a cell culture medium disclosed herein. In some embodiments, the compositions of the Disclosure include cell clusters. In some embodiments, the cells of the culture of the 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 Disclosure are previously frozen.

[0275]

[0302] In some embodiments, the compositions of the Disclosure comprise three of the following: a monocarbon 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), and glutamine. The compositions may comprise a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells in, for example, a cell culture medium disclosed herein. The compositions may comprise a plurality of insulin-positive endocrine progenitor cells in, for example, a cell culture medium disclosed herein. In some embodiments, the compositions of the Disclosure comprise cell clusters. In some embodiments, the cells of the culture of the 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 Disclosure are previously frozen.

[0276]

[0303] In some embodiments, the compositions of the Disclosure comprise four of the following: a monocarbon 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), and glutamine. The compositions may comprise a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells in, for example, a cell culture medium disclosed herein. The compositions may comprise a plurality of insulin-positive endocrine progenitor cells in, for example, a cell culture medium disclosed herein. In some embodiments, the compositions of the Disclosure comprise cell clusters. In some embodiments, the cells of the culture of the 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 Disclosure are previously frozen.

[0277]

[0304] In some embodiments, the compositions of the Disclosure comprise five of the following: a monocarbon 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), and glutamine. The compositions may comprise a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells in, for example, a cell culture medium disclosed herein. The compositions may comprise a plurality of insulin-positive endocrine progenitor cells in, for example, a cell culture medium disclosed herein. In some embodiments, the compositions of the Disclosure comprise cell clusters. In some embodiments, the cells of the culture of the 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 Disclosure are previously frozen.

[0278]

[0305] In some embodiments, the compositions of the present disclosure include a monocarbon 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). In some embodiments, the compositions of the present disclosure include a monocarbon 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 glutamine. In some embodiments, the compositions of the present disclosure include a monocarbon pathway intermediate (e.g., formate), an acetyl-CoA related metabolite (e.g., acetate), a vitamin (e.g., biotin), a redox homeostasis regulator (e.g., taurine), and glutamine. In some embodiments, the compositions of the disclosure include a monocarbon 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 compositions of the disclosure include a monocarbon 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 compositions of the 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 glutamine. The compositions may contain a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells in, for example, a cell culture medium disclosed herein. The composition may contain a plurality of insulin-positive endocrine progenitor cells in, for example, a cell culture medium disclosed herein. In some embodiments, the composition of the disclosure includes a cell cluster. In some embodiments, the cells of the culture of the 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 disclosure are previously frozen.

[0279]

[0306] In some embodiments, the compositions of the disclosure include a monocarbon pathway intermediate (e.g., formate), an acetyl-CoA related metabolite (e.g., acetate), a vitamin (e.g., biotin), and an HDAC inhibitor (e.g., β-hydroxybutyrate). In some embodiments, the compositions of the disclosure include a monocarbon pathway intermediate (e.g., formate), an acetyl-CoA related metabolite (e.g., acetate), a vitamin (e.g., biotin), and a redox homeostasis regulator (e.g., taurine). In some embodiments, the compositions of the disclosure include a monocarbon pathway intermediate (e.g., formate), an acetyl-CoA related metabolite (e.g., acetate), a vitamin (e.g., biotin), and glutamine. In some embodiments, the compositions of the disclosure include a monocarbon pathway intermediate (e.g., formate), an acetyl-CoA related metabolite (e.g., acetate), an HDAC inhibitor (e.g., β-hydroxybutyrate), and a redox homeostasis regulator (e.g., taurine). In some embodiments, the compositions of the disclosure include a monocarbon pathway intermediate (e.g., formate), an acetyl-CoA-related metabolite (e.g., acetate), an HDAC inhibitor (e.g., β-hydroxybutyrate), and glutamine. In some embodiments, the compositions of the disclosure include a monocarbon pathway intermediate (e.g., formate), an acetyl-CoA-related metabolite (e.g., acetate), a redox homeostasis regulator (e.g., taurine), and glutamine. In some embodiments, the compositions of the disclosure include a monocarbon pathway intermediate (e.g., formate), a vitamin (e.g., biotin), an HDAC inhibitor (e.g., β-hydroxybutyrate), and a redox homeostasis regulator (e.g., taurine). In some embodiments, the compositions of the disclosure include a monocarbon pathway intermediate (e.g., formate), a vitamin (e.g., biotin), an HDAC inhibitor (e.g., β-hydroxybutyrate), and glutamine. In some embodiments, the compositions of the present disclosure include a monocarbon 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 disclosure include a monocarbon metabolic pathway intermediate (e.g., formate), an HDAC inhibitor (e.g., β-hydroxybutyrate), a redox homeostasis regulator (e.g., taurine), and glutamine. In some embodiments, the compositions of the disclosure include 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). In some embodiments, the compositions of the disclosure include an acetyl-CoA related metabolite (e.g., acetate), a vitamin (e.g., biotin), an HDAC inhibitor (e.g., β-hydroxybutyrate), and glutamine. In some embodiments, the compositions of the disclosure include an acetyl-CoA related metabolite (e.g., acetate), a vitamin (e.g., biotin), a redox homeostasis regulator (e.g., taurine), and glutamine. In some embodiments, the compositions of the Disclosure include 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 compositions of the Disclosure include a vitamin (e.g., biotin), an HDAC inhibitor (e.g., β-hydroxybutyrate), a redox homeostasis regulator (e.g., taurine), and glutamine. The compositions may include a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells in, for example, a cell culture medium disclosed herein. The compositions may include a plurality of insulin-positive endocrine progenitor cells in, for example, a cell culture medium disclosed herein. In some embodiments, the compositions of the Disclosure include a cell cluster. In some embodiments, the cells of the culture of the 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 Disclosure are previously frozen.

[0280]

[0307] In some embodiments, the compositions of the Disclosure include monocarbonate pathway intermediates (e.g., formates), acetyl-CoA related metabolites (e.g., acetates), vitamins (e.g., biotin), HDAC inhibitors (e.g., β-hydroxybutyrates), redox homeostasis regulators (e.g., taurine), glutamates, and carnitine. In some embodiments, the compositions of the Disclosure include DMEM / F12. In some embodiments, the compositions of the Disclosure include zinc (e.g., ZnSO4). In some embodiments, the compositions of the Disclosure include human serum albumin. The compositions may include a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells in, for example, a cell culture medium disclosed herein. The compositions may include a plurality of insulin-positive endocrine progenitor cells in, for example, a cell culture medium disclosed herein. In some embodiments, the compositions of the Disclosure include cell clusters. In some embodiments, the cells of the culture of the 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 this disclosure are previously frozen.

[0281]

[0308] In some embodiments, the compositions of the Disclosure comprise at least one of the following: a monocarbon 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 compositions may comprise a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells in, for example, a cell culture medium disclosed herein. The compositions may comprise a plurality of insulin-positive endocrine progenitor cells in, for example, a cell culture medium disclosed herein. In some embodiments, the compositions of the Disclosure comprise a cell cluster. In some embodiments, the cells of the culture of the 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 Disclosure are previously frozen.

[0282]

[0309] In some embodiments, the compositions of the Disclosure include two of the following: monocarbon metabolic pathway intermediates (e.g., formates), acetyl-CoA related metabolites (e.g., acetates), vitamins (e.g., biotin), HDAC inhibitors (e.g., β-hydroxybutyrates), redox homeostasis regulators (e.g., taurine), glutamates, and carnitine. In some embodiments, the compositions of the Disclosure include DMEM / F12. In some embodiments, the compositions of the Disclosure include zinc (e.g., ZnSO4). In some embodiments, the compositions of the Disclosure include human serum albumin. The compositions may include a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells in, for example, a cell culture medium disclosed herein. The compositions may include a plurality of insulin-positive endocrine progenitor cells in, for example, a cell culture medium disclosed herein. In some embodiments, the compositions of the Disclosure include cell clusters. In some embodiments, the cells of the culture of the 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 this disclosure are previously frozen.

[0283]

[0310] In some embodiments, the compositions of the Disclosure include three of the following: a monocarbon 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 Disclosure include DMEM / F12. In some embodiments, the compositions of the Disclosure include zinc (e.g., ZnSO4). In some embodiments, the compositions of the Disclosure include human serum albumin. The compositions may include a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells in, for example, a cell culture medium disclosed herein. The compositions may include a plurality of insulin-positive endocrine progenitor cells in, for example, a cell culture medium disclosed herein. In some embodiments, the compositions of the Disclosure include cell clusters. In some embodiments, the cells of the culture of the 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 this disclosure are previously frozen.

[0284]

[0311] In some embodiments, the compositions of the Disclosure include four of the following: a monocarbon 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 Disclosure include DMEM / F12. In some embodiments, the compositions of the Disclosure include zinc (e.g., ZnSO4). In some embodiments, the compositions of the Disclosure include human serum albumin. The compositions may include a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells in, for example, a cell culture medium disclosed herein. The compositions may include a plurality of insulin-positive endocrine progenitor cells in, for example, a cell culture medium disclosed herein. In some embodiments, the compositions of the Disclosure include cell clusters. In some embodiments, the cells of the culture of the 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 this disclosure are previously frozen.

[0285]

[0312] In some embodiments, the compositions of the Disclosure include five of the following: a monocarbon 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 Disclosure include DMEM / F12. In some embodiments, the compositions of the Disclosure include zinc (e.g., ZnSO4). In some embodiments, the compositions of the Disclosure include human serum albumin. The compositions may include a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells in, for example, a cell culture medium disclosed herein. The compositions may include a plurality of insulin-positive endocrine progenitor cells in, for example, a cell culture medium disclosed herein. In some embodiments, the compositions of the Disclosure include cell clusters. In some embodiments, the cells of the culture of the 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 this disclosure are previously frozen.

[0286]

[0313] In some embodiments, the compositions of the Disclosure include six of the following: monocarbonate pathway intermediates (e.g., formates), acetyl-CoA related metabolites (e.g., acetates), vitamins (e.g., biotin), HDAC inhibitors (e.g., β-hydroxybutyrates), redox homeostasis regulators (e.g., taurine), glutamates, and carnitine. In some embodiments, the compositions of the Disclosure include DMEM / F12. In some embodiments, the compositions of the Disclosure include zinc (e.g., ZnSO4). In some embodiments, the compositions of the Disclosure include human serum albumin. The compositions may include a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells in, for example, a cell culture medium disclosed herein. The compositions may include a plurality of insulin-positive endocrine progenitor cells in, for example, a cell culture medium disclosed herein. In some embodiments, the compositions of the Disclosure include cell clusters. In some embodiments, the cells of the culture of the 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 this disclosure are previously frozen.

[0287]

[0314] In some embodiments, the compositions of the disclosure include a monocarbon 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), and glutamate. In some embodiments, the compositions of the disclosure include a monocarbon 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), and carnitine. In some embodiments, the compositions of the disclosure include a monocarbon 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), glutamate, and carnitine. In some embodiments, the compositions of the present disclosure include a monocarbon metabolic pathway intermediate (e.g., formate), an acetyl-CoA related metabolite (e.g., acetate), a vitamin (e.g., biotin), a redox homeostasis regulator (e.g., taurine), glutamate, and carnitine. In some embodiments, the compositions of the present disclosure include a monocarbon 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), glutamate, and carnitine. In some embodiments, the compositions of the present disclosure include a monocarbon 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), glutamate, and carnitine. In some embodiments, the compositions of the 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 Disclosure include DMEM / F12. In some embodiments, the compositions of the Disclosure include zinc (e.g., ZnSO4).In some embodiments, the compositions of the disclosure include human serum albumin. The compositions may include a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells in, for example, a cell culture medium disclosed herein. The compositions may include a plurality of insulin-positive endocrine progenitor cells in, for example, a cell culture medium disclosed herein. In some embodiments, the compositions of the disclosure include cell clusters. In some embodiments, the cells of the culture of the 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 disclosure are previously frozen.

[0288]

[0315] In some embodiments, the compositions of the present disclosure include a monocarbon 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). In some embodiments, the compositions of the present disclosure include a monocarbon 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 glutamate. In some embodiments, the compositions of the present disclosure include a monocarbon 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 carnitine. In some embodiments, the compositions of the present disclosure include a monocarbon pathway intermediate (e.g., formate), an acetyl-CoA related metabolite (e.g., acetate), a vitamin (e.g., biotin), a redox homeostasis regulator (e.g., taurine), and glutamate. In some embodiments, the compositions of the present disclosure include a monocarbon pathway intermediate (e.g., formate), an acetyl-CoA related metabolite (e.g., acetate), a vitamin (e.g., biotin), a redox homeostasis regulator (e.g., taurine), and carnitine. In some embodiments, the compositions of the present disclosure include a monocarbon pathway intermediate (e.g., formate), an acetyl-CoA related metabolite (e.g., acetate), a vitamin (e.g., biotin), glutamate, and carnitine. In some embodiments, the compositions of the present disclosure include a monocarbon 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 glutamate. In some embodiments, the compositions of the present disclosure include a monocarbon 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 carnitine.In some embodiments, the compositions of the present disclosure include a monocarbon pathway intermediate (e.g., formate), an acetyl-CoA related metabolite (e.g., acetate), an HDAC inhibitor (e.g., β-hydroxybutyrate), glutamate, and carnitine. In some embodiments, the compositions of the present disclosure include a monocarbon pathway intermediate (e.g., formate), an acetyl-CoA related metabolite (e.g., acetate), a redox homeostasis regulator (e.g., taurine), glutamate, and carnitine. In some embodiments, the compositions of the present disclosure include a monocarbon 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 glutamate. In some embodiments, the compositions of the present disclosure include a monocarbon 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 carnitine. In some embodiments, the compositions of the present disclosure include a monocarbon metabolic pathway intermediate (e.g., formate), a vitamin (e.g., biotin), an HDAC inhibitor (e.g., β-hydroxybutyrate), glutamate, and carnitine. In some embodiments, the compositions of the present disclosure include a monocarbon metabolic pathway intermediate (e.g., formate), a vitamin (e.g., biotin), a redox homeostasis regulator (e.g., taurine), glutamate, and carnitine. In some embodiments, the compositions of the present disclosure include a monocarbon metabolic pathway intermediate (e.g., formate), 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 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 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 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 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 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 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 Disclosure include DMEM / F12. In some embodiments, the compositions of the Disclosure include zinc (e.g., ZnSO4). In some embodiments, the compositions of the Disclosure include human serum albumin. The compositions may include a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells in, for example, a cell culture medium disclosed herein. The compositions may include a plurality of insulin-positive endocrine progenitor cells in, for example, a cell culture medium disclosed herein. In some embodiments, the compositions of the Disclosure include cell clusters. In some embodiments, the cells of the culture of the 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 Disclosure are previously frozen.

[0289]

[0316] Compositions comprising a combination of drugs may further include one or more of the additional factors disclosed herein, such as BMP signaling pathway inhibitors, LDN193189, ROCK inhibitors, thiazovibin, Y-27632, fasudil / HA1077, or 14-1152, histone methyltransferase inhibitors, 3-deazanepranosin 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, Sant4, Sant4, Cur61414, forskolin, tomatidine, AY9944, tripanol, 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 Disclosure provides compositions comprising any combination of a cryopreservative (e.g., DMSO), any of the cells disclosed herein, and any of the culture medium components disclosed herein (e.g., acetate, β-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 compositions of the Disclosure are frozen. In some embodiments, the compositions of the Disclosure are previously frozen.

[0291] Initialization

[0318] The term “reprogramming,” as used herein, refers to the process of changing or reversing the differentiation state of a somatic cell. Cells may be reprogrammed after partial differentiation or after terminal differentiation. Reprogramming encompasses the complete reversal of the differentiation state of a somatic cell to a pluripotent cell. Such a complete reversal of differentiation results in induced pluripotent (iPS) cells. Reprogramming also, as used herein, encompasses the partial reversal of a cell's differentiation state to, for example, a pluripotent state, or to a somatic cell that is neither pluripotent nor pluripotent but has lost one or more characteristics specific to the differentiated cell from which it is derived, such as a direct reprogramming of a differentiated cell to a different somatic cell type. Reprogramming as a whole involves at least some alteration, e.g., reversal, of genetic patterns such as nucleic acid modifications (e.g., methylation), chromatin condensation, epigenetic changes, and genomic imprinting that occur during cell differentiation as a zygote develops into an adult.

[0292]

[0319] As used herein, the term “reprogramming factor” is intended to refer to molecules associated with cellular “reprogramming,” i.e., differentiation and / or dedifferentiation and / or transdifferentiation, which result in a cell being transformed into a different cell type or phenotype. Reprogramming factors, as a whole, influence the expression of genes associated with cellular differentiation, dedifferentiation, and / or transdifferentiation. Transcription factors are an example of reprogramming factors.

[0293]

[0320] As used herein, the terms “differentiation” and their grammatical synonyms refer to the process by which less specialized cells (i.e., more naive cells with higher differentiation potential) become more specialized cell types (i.e., less naive cells with lower differentiation potential); the term “dedifferentiation” refers to the process by which more specialized cells become less specialized cell types (i.e., more naive cells with higher differentiation potential); and the term “transdifferentiation” refers to the process by which cells of a particular cell type are converted to another cell type without a significant change in their “differentiation potential” or “naiveness” levels. While we do not wish to be bound by theory, cells are considered to “transdifferentiate” when they are converted from one phylogenetic or terminally differentiated cell type to another phylogenetic or terminally differentiated cell type without a significant change in their “differentiation potential” or “naiveness” levels.

[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 cells of any of the three germ layers: endoderm (inner surface of the stomach, gastrointestinal tract, lungs), mesoderm (muscle, bone, blood, genitourinary tract), or ectoderm (epidermal tissue and nervous system), and therefore have high differentiation potential. Multipotent cells (e.g., stem cells or certain types of induced stem cells) have the ability to produce cells derived from multiple but limited number of lineages (e.g., hematopoietic stem cells, cardiac stem cells, or neural stem cells), and have relatively lower differentiation potential than pluripotent cells. Cells that are determined to a particular lineage or that differentiate into terminal cells may have even lower differentiation potential. Specific examples of transdifferentiation known in the art include, for example, the conversion of fibroblasts to beta cells, or the conversion of pancreatic exocrine cells to beta cells.

[0295]

[0322] Therefore, cells can be differentiated into more naive cells (for example, terminally differentiated cells can be differentiated into multipotent or pluripotent cells), or cells can be dedifferentiated into less naive cells (for example, multipotent or pluripotent cells can be differentiated into lineage-determining cells or terminally differentiated cells). However, in some embodiments, cells can be converted or transdifferentiated from one cell type (or phenotype) to another cell type (or phenotype) having, for example, a similar level of differentiation potential. Therefore, in some embodiments of the present disclosure, the induction step of the present disclosure can be used to reprogram the cells of the present disclosure to differentiate, dedifferentiate, and / or transdifferentiate.

[0296]

[0323] For example, methods for reprogramming or inducing cells into a particular type of cell by differentiation, dedifferentiation, and / or transdifferentiation using one or more exogenous polynucleotides or polypeptide reprogramming factors are known to those skilled in the art. Such methods may depend on the introduction of genetic material encoding one or more transcription factors or other polypeptides associated with cell reprogramming. For example, Pdx1, Ngn3, and MafA, or their functional fragments, are all known to encode peptides that can induce cell differentiation, dedifferentiation, and / or transdifferentiation of the cells of this 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., the genes described above) comes into contact with the cell to induce, for example, the cells of this disclosure. Those skilled in the art will understand that other genes may also be associated with cell reprogramming, and that the exogenous molecules encoding such genes (or their functional fragments) and the encoded polypeptides can also be considered polynucleotides or polypeptide reprogramming factors (e.g., polynucleotides or polypeptides that affect the expression level of another gene associated with cell reprogramming). For example, the introduction of exogenous polynucleotide or polypeptide epigenetic gene silencers that reduce p53 inactivation has been shown to increase the efficiency of inducing induced pluripotent stem cells (iPSCs). Therefore, exogenous polynucleotides or polypeptides encoding epigenetic silencers and other genes or proteins that may be directly or indirectly involved in enhancing cell reprogramming or cell reprogramming 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 influence cell reprogramming, such as introducing RNAi molecules (or genetic material encoding RNAi molecules) that can knock down the expression of genes involved in inhibiting cell reprogramming. Therefore, any exogenous polynucleotide or polypeptide molecule that is associated with or enhances cell reprogramming should be understood to be an exogenous polynucleotide or polypeptide reprogramming factor as described herein.

[0297]

[0324] In some embodiments of this disclosure, the method excludes the use of reprogramming factors that are not small molecules. However, the method does not use tissue culture components, such as culture media, serum, serum substitutes, supplements, antibiotics, etc., such as RPMI, renal epithelial basal medium (REBM), Dulbecco's modified Eagle medium (DMEM), MCDB 131 medium, CMRL It will be understood that 1066 medium, F12, fetal bovine serum (FCS), fetal bovine serum (FBS), bovine serum albumin (BSA), D-glucose, L-glutamine, GlutaMAX™-1 (L-alanine-L-glutamine dipeptide), 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 used. It should be understood that these tissue culture components (and other similar tissue culture components conventionally used in tissue culture) are not small molecule reprogramming molecules for the purposes of this disclosure. In fact, these components are not small molecules as defined herein, and / or 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, combinations of the cell culture components / additives and metabolites disclosed herein can improve the efficiency of SC-β cell generation and their function.

[0298]

[0325] Accordingly, in some embodiments, the Disclosure does not involve a cell culture step using one or more exogenous polynucleotides or polypeptide reprogramming factors. Accordingly, in some embodiments, the method of the Disclosure does not involve the introduction of one or more exogenous polynucleotides or polypeptide reprogramming factors by introducing, for example, transposons, viral transgenic vectors (e.g., retroviral vectors), plasmids, mRNA, miRNA, peptides, or fragments of any of these molecules that are involved in producing artificial beta cells or inducing the cells of the Disclosure to otherwise differentiate, dedifferentiate, and / or transdifferentiate.

[0299]

[0326] In other words, in some embodiments, the method is carried out in the absence of one or more exogenous polynucleotides or polypeptide reprogramming factors. Therefore, in some embodiments, it should be understood that the method of the present disclosure utilizes small molecules that reprogram cells without the addition of polypeptide transcription factors; other polypeptide factors specifically associated with inducing differentiation, dedifferentiation, and / or transdifferentiation; polynucleotide sequences encoding polypeptide transcription factors, polynucleotide sequences encoding other polypeptide factors specifically associated with inducing differentiation, dedifferentiation, and / or transdifferentiation; mRNA; interfering RNA; microRNA; and fragments thereof.

[0300] stem cells

[0327] The term “stem cell” is used herein to refer to cells that have the ability to both self-replicate and generate differentiated cell types (e.g., plant stem cells, vertebrate stem cells) (Morrison et al. (1997) Cell 88:287-298). In the context of cell-on-onset, the adjectives “differentiated” or “to differentiate” are relative terms. A “differentiated cell” is a cell that has progressed further in the developmental pathway than the cell being compared. Thus, pluripotent stem cells can differentiate into lineage-restricted progenitor cells (e.g., mesodermal stem cells), lineage-restricted progenitor cells can differentiate into further restricted cells (e.g., beta cell precursors), which can then differentiate into final-stage cells (i.e., terminally differentiated cells, e.g., beta cells, etc.) that may or may not retain the ability to proliferate, playing a characteristic role in a particular tissue type. Stem cells can be characterized by both the presence and absence of specific markers (e.g., proteins, RNA, etc.). Stem cells can also be identified both in vitro and in vivo by functional assays, particularly those concerning the ability of stem cells to produce offspring with multiple differentiations. In some embodiments, host cells include 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] The target stem cells include pluripotent stem cells (PSCs). The terms “pluripotent stem cells” or “PSCs” are used herein to mean stem cells capable of producing 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 in vertebrates). Pluripotent cells can form teratomas and contribute to the ectoderm, mesoderm, or endodermal tissue of a living organism. Plant pluripotent stem cells can give rise to all cell types of a plant (e.g., root, stem, leaf, etc.).

[0302]

[0329] Animal PSCs can be obtained in several different ways. For example, embryonic stem cells (ESCs) are obtained from the inner cell mass of an embryo (Thomson et al., Science. November 6, 1998; 282(5391): 1145-7), and induced pluripotent stem cells (iPSCs) are obtained from somatic cells (Takahashi et al., Cell. November 30, 2007; 131(5): 861-72; Takahashi et al., Nat Protoc. 2007; 2(12): 3081-9; Yu et al., Science. December 21, 2007; 318(5858): 1917-20. Published online November 20, 2007). Since the term PSC refers to pluripotent stem cells regardless of its origin, the term PSC encompasses the terms ESC and iPSC, as well as embryonic germ stem cells (EGSCs), another example of PSCs. PSCs may be in the morphology of an established cell line, 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 the blastocyst. Examples of ESC lines include 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. Other target stem cells include embryonic stem cells derived 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, equids, bovines, pigs, canines, felines, rodents, such as mice, rats, hamsters, and primates (Thomson et al., (1998) Science 282:1145; Thomson et al., (1995) Proc. Natl. Acad. Sci USA 92:7844; Thomson et al., (1996) Biol. Reprod. 55:254; Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998). In culture, ESCs typically grow as flattened colonies with a large nucleocytoplasmic ratio, well-defined boundaries, 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. Patents 7,029,913, 5,843,780, and 6,200,806, each of which is incorporated herein in whole. Methods for growing undifferentiated hESCs are described in WO99 / 20741, WO01 / 51616, and WO03 / 020920, each of which is incorporated herein in whole.

[0304]

[0331] “Embryo germ stem cells” (EGSCs), or “embryo germ cells” or “EG cells” means germ cells and / or germ cell precursors, such as primordial germ cells, i.e., PSCs obtained from cells that can become sperm and eggs. Embryogery germ cells (EG cells) are thought to have similar properties to embryonic stem cells as 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 by reference in whole.

[0305]

[0332] "Induced pluripotent stem cells" or "iPSCs" refers to PSCs obtained from cells that are not PSCs (i.e., from cells differentiated compared to PSCs). iPSCs can be obtained from several different cell types, including terminally differentiated cells. iPSCs have an ES cell-like morphology, growing as flattened colonies with a large nucleocytoplasmic ratio, well-defined boundaries, and prominent nuclei. In addition, iPSCs express one or more important pluripotency markers known to those skilled in the art, including but not limited to alkaline phosphatase, SSEA3, SSEA4, Sox2, Oct3 / 4, Nanog, TRA160, TRA181, TDGF1, Dnmt3b, FoxD3, GDF3, Cyp26a1, TERT, and zfp42. Examples of methods for generating and characterizing iPSCs can be found, for example, in U.S. Patent Application Publications 20090047263, 20090068742, 20090191159, 20090227032, 20090246875, and 20090304646, each of which is incorporated herein in whole. Overall, to generate iPSCs, somatic cells are provided, along 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 cells refer to all cells of an organism that do not normally produce all types of cells in the absence of experimental manipulation. In other words, somatic cells are fully differentiated cells that do not naturally produce all cells of the body's three germ layers: the ectoderm, mesoderm, and endoderm. For example, somatic cells can include both neurons and neural precursors, the latter of which can naturally produce all or some cell types of the central nervous system, but not cells of the mesodermal or endodermal lineages.

[0307]

[0334] In certain cases, stem cells may be undifferentiated (e.g., cells not determined to a particular lineage) before exposure to at least one β-cell maturation factor according to the methods disclosed herein, while in other cases, it may be desirable to differentiate stem cells into one or more intermediate cell types before exposure to at least one cell maturation factor described herein. For example, stem cells may exhibit morphological, biological, or physical characteristics of undifferentiated cells that can be used to distinguish stem cells from differentiated cells of embryonic or adult origin. In some cases, undifferentiated cells may appear in two-dimensional microscopic images as colonies of cells with a high nucleus / cytoplasmic ratio and prominent nucleoli. Stem cells may be used on their own (e.g., substantially no undifferentiated cells present) or in the presence of differentiated cells. In certain cases, stem cells may be cultured in suitable nutrients and, if necessary, in the presence of other cells that allow the stem cells to grow and differentiate as needed. For example, embryonic fibroblasts or fibroblast-like cells may be present in the culture to assist in the growth of 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 the first culture stage, but may not be added to the stem cell culture in one or more subsequent culture stages.

[0308]

[0335] The stem cells used in all aspects of the present invention may be any cells obtained from any type of tissue (e.g., embryonic tissue, e.g., fetal or pre-fetal tissue, or adult tissue) and are characterized by being able to produce progeny of all derivatives of at least one of the three germinal layers (endoderm, mesoderm, and ectoderm) under appropriate conditions. 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 chemoinducible 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 containing solid tissue. In some embodiments, the tissue is skin, adipose tissue (e.g., fatty tissue), muscle tissue, heart, or cardiac tissue. In other embodiments, the tissue is, for example, but not limited to, umbilical cord blood, placenta, bone marrow, or cartilage.

[0310]

[0337] Target stem cells include human embryonic stem (hES) cells, as described by Thomson et al. (1998) Science 282:1145; embryonic stem cells from other primates, such as rhesus monkey stem cells (Thomson et al., (1995) Proc. Natl. Acad. Sci. USA 92:7844); marmoset stem cells (Thomson et al., (1996) Biol. Reprod. 55:254); and various types of embryonic cells exemplified by human embryonic germ (hEG) cells (Shambloft et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998). Furthermore, lineage-determining stem cells, such as mesodermal stem cells and other early cardiogenic cells, are also target stem cells (see Reyes et al., (2001) Blood 98:2615~2625; Eisenberg and Bader (1996) Circ Res. 78(2):205~16, etc.). Stem cells can be obtained from any mammalian species, such as humans, equids, bovines, pigs, canids, felines, rodents, such as mice, rats, hamsters, and primates. In some embodiments, human embryos are not destroyed for use as a source of pluripotent cells in the methods and compositions disclosed herein.

[0311]

[0338] A mixture of cells from suitable sources of endothelial stem cells, muscle stem cells, and / or neural stem cells can be collected from a mammalian donor by methods known in the art. A suitable source is the hematopoietic microenvironment. For example, preferably aggregated (i.e., mobilized) circulating peripheral blood can be taken from the subject. In some embodiments, the stem cells may be reprogrammed stem cells, such as stem cells obtained from somatic cells or differentiated cells. In such embodiments, the dedifferentiated stem cells may be, for example, 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, SC-β cells include hair follicles, keratinocytes, gonadotropin-producing cells, adrenocorticotropin-producing cells, thyroid-stimulating hormone-producing cells, growth hormone-producing cells, prolactin-producing cells, chromaffin cells, parafollicular cells, glomus cells, melanocytes, nevus cells, Merkel cells, odontoblasts, cementoblasts, corneal stromal cells, retinal Müller cells, retinal pigment epithelial cells, neurons, and glial cells (e.g., oligodendrocytes). Star cells), ependymal cells, pineal cells, lung cells (e.g., type I and type II lung cells), Clara cells, goblet cells, G cells, D cells, ECL cells, gastric chief cells, parietal cells, pit cells, K cells, D cells, I cells, goblet cells, Paneth cells, intestinal cells, fold cells, hepatocytes, hepatic stellate cells (e.g., mesoderm-derived Kupffer cells), gallbladder cells, acinar central 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., For example, follicular cells, parathyroid cells (e.g., chief parathyroid cells), eosinophilic cells, urothelial cells, osteoblasts, osteocytes, chondrocytes, chondrocytes, fibroblasts, fibrocytes, myoblasts, myocytes, muscle satellite cells, tendinocytes, cardiomyocytes, lipoblasts, adipocytes, interstitial cells of Cajal, angioblasts, endothelial cells, mesangial cells (e.g., intraglomerular mesangial cells and extraglomerular mesangial cells), juxtaglomerular cells, macula densa cells, stromal cells, interstitial cells, telocytes They can be obtained from one or more of the following: simple epithelial cells, podocytes, renal proximal tubular brush border cells, Sertoli cells, Leydig cells, granulosa cells, stent cells, germ cells, sperm, oocytes, lymphocytes, myeloid cells, endothelial progenitor cells, endothelial stem cells, angioblasts, mesoangioblasts, pericytes, parietal cells, splenic 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 embodiments, the Disclosure provides a method for generating NKX6.1-positive pancreatic progenitor cells from Pdx1-positive pancreatic progenitor cells, comprising the steps of: inducing differentiation of at least one Pdx1-positive pancreatic progenitor cell in the population into an NKX6.1-positive pancreatic progenitor cell by exposing the NKX6.1-positive pancreatic progenitor cell to NKX6.1-positive pancreatic progenitor cell, wherein the NKX6.1-positive pancreatic progenitor cell expresses NKX6.1.

[0314]

[0341] In some embodiments, at least 10% of Pdx1-positive pancreatic progenitor cells in a population are induced to differentiate into NKX6-1-positive pancreatic progenitor cells. In some embodiments, at least 95% of Pdx1-positive pancreatic progenitor cells in a population are induced to differentiate into NKX6.1-positive pancreatic progenitor cells. In some embodiments, NKX6.1-positive pancreatic progenitor cells express Pdx1, NKX6.1, and FoxA2. In some embodiments, Pdx1-positive pancreatic progenitor cells are 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] In this specification, in some embodiments, methods are provided for generating SC-beta cells (e.g., mature pancreatic β-cells or β-like cells) or their precursors using stem cells. In some embodiments, germ cells may be used instead of or in conjunction with stem cells to provide at least one SC-β cell using similar protocols described in U.S. Patent Application Publications 20150240212 and 20150218522, each of which is incorporated herein by whole reference. Suitable germ cells can be prepared, for example, from primordial germ cells present in human fetal material obtained about 8 to 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 this specification, in some embodiments, compositions and methods for generating SC-β cells (e.g., pancreatic β cells) are provided. Overall, at least one SC-β cell or its precursor, for example, a pancreatic precursor generated according to the methods disclosed herein, may include mixtures or combinations of different cells, such as Pdx1-positive pancreatic precursors, pancreatic precursors co-expressing Pdx1 and NKX6.1, Ngn3-positive endocrine progenitor cells, insulin-positive endocrine cells (e.g., β-like cells), and insulin-positive endocrine cells, as well as / or mixtures of other pluripotent or stem cell cells.

[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 under conditions and for a period of time 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 cells or its precursor is enhanced by contacting the cells with one or more of the 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 their precursors. In some embodiments, the population of SC-β cells or its precursors includes a mixture of pluripotent cells or differentiated cells. In some embodiments, the population of SC-β cells or its precursors substantially does not include or lacks embryonic stem cells, pluripotent cells, or iPS cells.

[0319]

[0346] In some embodiments, somatic cells, such as fibroblasts, can be isolated from a subject, for example, by a tissue biopsy, such as a skin biopsy, and can be reprogrammed into induced pluripotent stem cells for further differentiation to produce at least one SC-β cell or its precursor for use in the compositions and methods described herein. In some embodiments, somatic cells, such as fibroblasts, are maintained in culture by methods known to those skilled in the art, and in some embodiments, after amplification, are converted to SC-β cells by methods disclosed herein.

[0320]

[0347] In some embodiments, at least one SC-β cell or its precursor is maintained in culture by a method known to those skilled in the art, and in some embodiments, after amplification, is converted to SC-β cells by a method disclosed herein.

[0321]

[0348] Furthermore, at least one SC-β cell or its precursor, for example, a pancreatic precursor, can be derived from any mammalian species, non-limiting examples including mouse, cattle, ape, pig, horse, sheep, or human cells. For clarity and simplicity of explanation, the methods described herein refer to at least one SC-β cell or its precursor from a mammal, but it should be understood that all methods described herein are readily applicable 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 derived from a human individual.

[0322]

[0349] In some embodiments, the cell population of this disclosure comprises 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 cells may express ISL1. In some cases, at least about 35% of cells express NKX6.1 and ISL1. In some cases, at least about 38% of cells express NKX6.1 and ISL1. In some cases, at least about 40% of cells express NKX6.1 and ISL1. In some cases, at least about 42% of cells express NKX6.1 and ISL1. In some cases, at least about 44% of cells express NKX6.1 and ISL1. In some cases, at least about 46% of cells express NKX6.1 and ISL1. In other cases, at least about 50% of cells express NKX6.1 and ISL1. The compositions and methods of this disclosure may be useful in increasing the percentage of cells expressing both NKX6.1 and ISL1. For example, after contacting cells differentiated into SC-β cells according to the methods of this disclosure, the percentage of cells expressing NKX6.1 and ISL1 may be increased compared to methods lacking one or more agents or containing different (e.g., lower) concentrations of agents.

[0323]

[0350] In some cases, the cell populations of this disclosure contain a limited proportion of cells that are negative for both NKX6.1 and ISL1. For example, the cell populations 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 cluster of stem cell-derived beta cells

[0351] In this specification, in some embodiments, cell clusters similar in function and characteristics to endogenous pancreatic islets are provided. Such cell clusters can mimic the function of endogenous pancreatic islets in regulating the metabolism of a subject, for example, glucose metabolism. Thus, cell clusters can be transplanted into a subject to treat diseases resulting from insufficient pancreatic islet function, such as diabetes. The terms “cluster” and “aggregate” can be used interchangeably to refer to groups of cells having close intercellular contact, and in some cases, cells in a cluster that can adhere to one another.

[0325]

[0352] A cell cluster contains multiple cells. In some embodiments, a cell cluster contains at least 10, at least 50, at least 200, at least 500, at least 750, at least 1000, at least 1500, at least 2000, at least 2500, at least 3000, at least 3500, at least 4000, at least 4500, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 20,000, at least 30,000, or at least 50,000 cells. In some embodiments, cell clusters include cells in the following ranges: 10 to 10,000, 50 to 10,000, 100 to 10,000, 1,000 to 10,000, 500 to 10,000, 500 to 5,000, 500 to 2,500, 500 to 2,000, 1,000 to 100,000, 1,000 to 50,000, 1,000 to 40,000, 1,000 to 20,000, 1,000 to 10,000, 1,000 to 5,000, and 1,000 to 3,000. In some embodiments, cell clusters include at least 500 cells. In some embodiments, the cell cluster contains at least 1,000 cells. In some embodiments, the cell cluster contains at least 2,000 cells. In some embodiments, the cell cluster contains at least 5,000 cells. In some embodiments, the cell cluster contains 100,000 or fewer cells, 90,000 or fewer cells, 80,000 or fewer cells, 70,000 or fewer cells, 60,000 or fewer cells, 50,000 or fewer cells, 40,000 or fewer cells, 30,000 or fewer cells, 20,000 or fewer cells, 10,000 or fewer cells, 7,000 or fewer cells, 5,000 or fewer cells, 3,000 or fewer cells, 2,000 or fewer cells, or 1,000 or fewer cells.

[0326]

[0353] The cell clusters described herein may include at least one non-natural cell, e.g., a non-natural pancreatic β-cell. Non-natural cells (e.g., non-natural pancreatic β-cells) may share characteristics with endogenous cells (e.g., endogenous mature pancreatic β-cells), but differ in certain aspects (e.g., gene expression profiles). Non-natural cells may be genetically modified cells. Non-natural cells may be cells differentiated from progenitor cells, e.g., stem cells. Stem cells may be embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). In some cases, non-natural cells may be cells differentiated in vitro from progenitor cells. In some cases, non-natural cells may be cells differentiated in vivo from progenitor cells. For example, a cell cluster may include at least one non-natural pancreatic β-cell. Non-natural pancreatic β-cells may be those described in U.S. Patent Applications 14 / 684,129 and 14 / 684,101, which are incorporated herein by reference. A cell cluster may include multiple non-natural pancreatic β-cells. In some cases, at least approximately 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 99% of the cells in a cell cluster are non-native pancreatic β-cells. A cell cluster can contain one or more native cells. For example, a cell cluster can contain one or more primary cells, such as primary cells derived from endogenous pancreatic islets.

[0327]

[0354] A cell cluster may include one or more cells expressing at least one marker for endogenous cells, such as endogenous mature pancreatic β-cells. The term "marker" can refer to a molecule that can be observed or detected. For example, markers can include, but are not limited to, nucleic acids, such as transcripts of specific genes, polypeptide products of genes, non-gene product polypeptides, glycoproteins, carbohydrates, glycolipids, lipids, lipoproteins, or small molecules. Often, a marker can refer to a molecule that may be characteristic of a particular cell type, and therefore, a marker can be called a cell type marker. For example, the insulin gene can be called a marker for β-cells. In some cases, the marker is a gene. Non-exclusive markers for endogenous mature pancreatic β-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] A cell cluster may include one or more cells expressing one or more markers of endogenous cells, such as endogenous mature pancreatic β-cells. For example, a cell cluster may include one or more cells co-expressing 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 may include cells expressing NKX6.1 and C peptide, both of which may be β-cell markers.

[0329]

[0356] A cell cluster can include multiple cells expressing at least one marker of endogenous cells. For example, at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, and 99% of the cells in a cell cluster can express at least one marker of endogenous cells. In some cases, all cells in a cell cluster can express the marker of endogenous cells. In some cases, the endogenous cells can be pancreatic cells, e.g., pancreatic β-cells, pancreatic α-cells, pancreatic β-cells, pancreatic Δ-cells, or pancreatic γ-cells. The cell clusters provided herein can include heterogeneous cell populations, e.g., diverse types of cells. For example, a cell cluster can include cells expressing insulin / C-peptide, which may be a marker of pancreatic β-cells; cells expressing glucagon, which may be a marker of pancreatic α-cells; cells expressing somatostatin, which may be a marker of pancreatic Δ-cells; cells expressing pancreatic polypeptides; or any combination thereof.

[0330]

[0357] For example, the cell clusters described herein may include a plurality of cells expressing one or more markers of endogenous mature pancreatic β-cells. For example, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the cells in the cell cluster may express one or more markers of endogenous mature pancreatic β-cells.

[0331]

[0358] A cell cluster can contain multiple cells expressing CHGA. In some cases, at least approximately 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the cells in the cell cluster express CHGA. In some cases, at least approximately 85% of the cells in the cell cluster can express CHGA. In some cases, a cell cluster can contain approximately 90% of cells expressing CHGA. In some cases, a cell cluster can contain approximately 95% of cells expressing CHGA. In certain specific cases, all cells in the cell cluster can express CHGA.

[0332]

[0359] A cell cluster can contain multiple cells expressing NKX6.1. For example, at least approximately 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, and 99% of the cells in a cell cluster can express NKX6.1. In some cases, at least approximately 50% of the cells in a cell cluster can express NKX6.1. In some cases, all cells in a cell cluster can express NKX6.1.

[0333]

[0360] A cell cluster can contain multiple cells expressing ISL1. For example, at least approximately 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, and 99% of the cells in a cell cluster can express NKX6.1. In some cases, at least approximately 50% of the cells in a cell cluster can express ISL1. In some cases, all cells in a cell cluster can express ISL1.

[0334]

[0361] A cell cluster can contain multiple cells expressing C-peptide. For example, at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the cells in a cell cluster can express C-peptide. In some cases, at least about 60% of the cells in a cell cluster can express C-peptide. In some cases, all cells in a cell cluster can express C-peptide.

[0335]

[0362] A cell cluster can contain multiple cells expressing both NKX6.1 and C-peptide. For example, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the cells in a cell cluster can express C-peptide. In some cases, at least about 35% of the cells in a cell cluster can express both NKX6.1 and C-peptide. In some cases, at least about 40% of the cells in a cell cluster can express both NKX6.1 and C-peptide. In some cases, at least about 35% of the cells in a cell cluster can express both NKX6.1 and C-peptide. In some cases, a cell cluster may contain about 60% of cells expressing both NKX6.1 and C-peptide. In some cases, a cell cluster may contain about 75% of cells expressing both NKX6.1 and C-peptide. In some cases, all cells in a cell cluster can express both NKX6.1 and C-peptide.

[0336]

[0363] A cell cluster can contain multiple 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 cells in a cell cluster can express NKX6.1 and ISL1. 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 40% of cells in a cell cluster can express NKX6.1 and ISL1. In some cases, at least about 35% of cells in a cell cluster can express NKX6.1 and ISL1. In some cases, a cell cluster can contain about 60% of cells expressing NKX6.1 and ISL1. In some cases, a cell cluster can contain about 75% of cells expressing NKX6.1 and ISL1. In some cases, all cells in a cell cluster can express NKX6.1 and ISL1.

[0337]

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

[0338]

[0365] Cell clusters may contain little to no stem cells or progenitor cells, such as pancreatic progenitor cells. For example, the cell clusters provided herein may contain or contain up to approximately 5%, up to approximately 5%, up to approximately 5%, up to approximately 5%, up to approximately 5%, up to approximately 2%, up to approximately 1%, up to approximately 0.5%, up to approximately 0.1%, up to approximately 0.05%, up to approximately 0.01% or none of cells expressing LIN28. In some examples, the cell clusters provided herein may contain or contain up to approximately 5%, up to approximately 5%, up to approximately 5%, up to approximately 5%, up to approximately 5%, up to approximately 2%, up to approximately 1%, up to approximately 0.5%, up to approximately 0.1%, up to approximately 0.05%, up to approximately 0.01% or none of cells expressing Ki67.

[0339]

[0366] In some cases, a cell cluster may contain up to 3%, up to approximately 2%, up to approximately 1%, up to approximately 0.5%, up to approximately 0.1%, up to approximately 0.05%, up to approximately 0.01%, or contain none at all. In some cases, a cell cluster may contain approximately 1% of cells expressing SOX2. In some cases, a cell cluster may contain approximately 0.6% of cells expressing SOX2. In some cases, a cell cluster may contain approximately 0.3% of cells expressing SOX2. In some cases, a cell cluster may contain approximately 0.1% of cells expressing S...

Claims

1. An in vitro composition comprising multiple PDX1-positive, NKX6.1-positive, insulin-negative cells, and one or more of acetyl-CoA-related metabolites, HDAC inhibitors, redox homeostasis regulators, or monocarbonate pathway intermediates.

2. An in vitro composition comprising multiple PDX1-positive, NKX6.1-positive, insulin-negative cells, and acetyl-CoA-related metabolites.

3. An in vitro composition comprising multiple PDX1-positive, NKX6.1-positive, insulin-negative cells, and one or more of acetate, β-hydroxybutyrate, taurine, or formate.

4. An in vitro composition comprising multiple PDX1-positive, NKX6.1-positive, insulin-negative cells, and acetate.

5. The composition according to claim 3 or 4, comprising 0.01 to 50 mM, 0.1 to 50 mM, 0.5 to 50 mM, 0.01 to 20 mM, 0.1 to 20 mM, 0.5 to 20 mM, 0.01 to 10 mM, 0.1 to 10 mM, 0.5 to 10 mM, 0.8 to 25 mM, 0.8 to 10 mM, 0.8 to 5 mM, 0.8 to 2 mM, 0.8 to 1.5 mM, 0.8 to 1.2 mM, 0.9 to 1.1 mM, or 0.95 to 1.05 mM of acetate.

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

7. The composition according to claim 6, comprising 0.5 to 20 mM, 0.5 to 10 mM, 0.5 to 5 mM, 1 to 5 mM, 2 to 5 mM, or 1 mM to 10 mM of glutamine.

8. An in vitro composition comprising multiple PDX1-positive, NKX6.1-positive, insulin-negative cells, and glutamine in concentrations of 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.

9. The composition according to claim 8, wherein the glutamine is concentrated at a concentration of 3.8 to 4.2 mM.

10. The composition according to any one of claims 6 to 9, wherein 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 dipeptide form.

11. The composition according to claim 10, wherein 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.

12. The composition according to any one of claims 6 to 11, wherein 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 free glutamine form.

13. The composition according to any one of claims 1 to 12, further comprising a plurality of insulin-positive endocrine progenitor cells.

14. An in vitro composition comprising multiple insulin-positive endocrine progenitor cells and acetyl-CoA-related metabolites in concentrations of 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.

15. An in vitro composition comprising multiple insulin-positive endocrine progenitor cells and acetate in concentrations of 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.

16. A composition according to any one of claims 1 to 15, comprising approximately 1 mM acetate.

17. An in vitro composition comprising multiple insulin-positive endocrine progenitor cells and glutamine in concentrations of 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.

18. A composition according to any one of claims 1 to 17, comprising approximately 4 mM glutamine.

19. A composition according to any one of claims 1 to 18, comprising glutamine and acetate.

20. An in vitro composition comprising multiple insulin-positive endocrine progenitor cells, and one or more of the following: acetyl-CoA-related metabolites, HDAC inhibitors, redox homeostasis regulators, monocarbonate pathway intermediates, glutamates, or L-carnitine.

21. An in vitro composition comprising multiple insulin-positive endocrine progenitor cells and one or more of glutamate, acetate, β-hydroxybutyrate, taurine, L-carnitine, or formate.

22. The composition according to claim 21, comprising acetate in a concentration of 50-1000 nM, 50-800 nM, 50-500 nM, 50-300 nM, 50-250 nM, 100-200 nM, or 125-175 nM.

23. A composition according to any one of claims 1 to 22, comprising β-hydroxybutyrate.

24. A composition according to any one of claims 1 to 23, comprising taurine.

25. A composition according to any one of claims 1 to 24, comprising a formate.

26. A composition according to any one of claims 1 to 25, comprising a vitamin.

27. A composition according to any one of claims 1 to 26, comprising biotin.

28. A composition according to any one of claims 1 to 27, comprising glutamine, acetate, β-hydroxybutyrate, taurine, formate, and biotin.

29. The composition according to any one of claims 1 to 19 or 22 to 28, further comprising L-carnitine.

30. A composition according to any one of claims 20 to 28, comprising L-carnitine.

31. The composition according to any one of claims 1 to 30, further comprising a BMP signaling pathway inhibitor.

32. The composition according to claim 31, wherein the BMP signaling pathway inhibitor is LDN193189 or DMH-1 or a derivative thereof.

33. The composition according to any one of claims 1 to 32, further comprising a ROCK inhibitor.

34. The composition according to claim 33, wherein the ROCK inhibitor is thiazovibin, Y-27632, fasudil / HA1077, or 14-1152, or a derivative thereof.

35. The composition according to any one of claims 1 to 34, further comprising a histone methyltransferase inhibitor.

36. The composition according to claim 35, wherein the histone methyltransferase inhibitor is 3-deazanepranosin A hydrochloride, Bix-01294, UNC0638, BRDD4770, EPZ004777, AZ505, PDB4e47, valproic acid, vorinostat, romidepsin, entinostat, avexinostat, gibinostat, and mosetinostat, butyrate, serine protease inhibitors, serpine, or derivatives thereof.

37. The composition according to any one of claims 1 to 36, further comprising a TGF-β pathway inhibitor.

38. The composition according to claim 37, 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.

39. The composition according to any one of claims 1 to 38, further comprising a thyroid hormone signaling pathway activator.

40. The composition according to claim 39, 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.

41. The composition according to any one of claims 1 to 40, further comprising a protein kinase inhibitor.

42. The composition according to claim 41, wherein the protein kinase inhibitor is staurosporine, Ro-31-8220, or a derivative thereof.

43. The composition according to any one of claims 1 to 42, further comprising a sonic hedgehog pathway inhibitor.

44. The composition according to claim 43, wherein the sonic hedgehog pathway inhibitor comprises Sant1, Sant2, Sant4, Sant4, Cur61414, forskolin, tomatidine, AY9944, tripanol, cyclopamine, or derivatives thereof.

45. The composition according to any one of claims 1 to 44, further comprising a growth factor from the epidermal growth factor (EGF) family.

46. The composition according to claim 45, wherein the growth factor from the EGF family comprises beta-cellulin or EGF.

47. The composition according to any one of claims 1 to 20 or 22 to 46, further comprising a gamma secretase inhibitor.

48. The composition according to claim 47, wherein the gamma-secretase inhibitor comprises XXI, DAPT, or derivatives thereof.

49. The composition according to any one of claims 1 to 48, further comprising zinc.

50. The aforementioned zinc is ZnSO ４ The composition according to claim 49, in the form of...

51. The composition according to any one of claims 1 to 50, further comprising serum albumin protein.

52. The composition according to claim 51, wherein the serum albumin protein is human serum albumin protein.

53. 0.01%–1%, 0.03–1%, 0.03–0.9%, 0.03–0.08%, 0.03–0.06%, 0.03–0.05%, 0.04–0.8%, 0.04–0.7%, 0.04–0.6%, 0.04–0.5%, 0.04–0.4%, 0.04–0.3%, 0.04–0.2%, 0.04–0.1%, 0.04–0.09%, 0.04–0.8%, 0.04–0.07%, 0.04–0.06%, 0.0 The composition according to claim 51 or 52, comprising 4-0.05%, 0.05-1%, 0.05-0.9%, 0.05-0.8%, 0.05-0.7%, 0.05-0.6%, 0.05-0.5%, 0.05-0.4%, 0.05-0.3%, 0.05-0.2%, 0.05-0.1%, 0.05-0.09%, 0.05-0.8%, 0.05-0.07%, or 0.05-0.06% of serum albumin protein.

54. The composition according to any one of claims 1 to 53, further comprising DMEM.

55. The composition according to claim 54, wherein the DMEM is DMEM / F12.

56. A composition according to any one of claims 1 to 54, which does not contain vitamin C.

57. The composition according to any one of claims 14 to 56, wherein the plurality of insulin-positive endocrine progenitor cells are dissociated.

58. The composition according to any one of claims 14 to 57, wherein 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 progenitor cells are not in cell clusters.

59. The composition according to any one of claims 1 to 58, wherein less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, or less than 1% of the cells in the composition are in the form of cell clusters.

60. The composition according to any one of claims 57 to 59, wherein the insulin-positive endocrine progenitor cells are previously frozen.

61. The composition according to any one of claims 14 to 56, wherein 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 progenitor cells are in cell clusters.

62. The composition according to any one of claims 14 to 56, wherein the plurality of insulin-positive endocrine progenitor cells are arranged in cell clusters.

63. The composition according to claim 62, 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.

64. The composition according to claim 62, wherein 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.

65. The composition according to any one of claims 62 to 64, wherein the cell cluster has an average diameter or median diameter of up to 120, up to 130, up to 140, up to 150, up to 160, or up to 170 μm.

66. The composition according to any one of claims 62 to 64, wherein the cell clusters have an average diameter or median diameter of about 80 to 150, about 100 to 150, about 120 to 150, about 140 to 150, about 80 to 130, about 100 to 130, about 120 to 130, about 80 to 120, about 90 to 120, or about 100 to 120 μm.

67. The composition according to any one of claims 62 to 66, wherein 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.

68. The composition according to any one of claims 62 to 66, wherein 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.

69. The composition according to any one of claims 1 to 68, 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.

70. The composition according to any one of claims 1 to 69, comprising 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.

71. The composition according to any one of claims 1 to 70, comprising a cell manipulated to include a genomic disruption in at least one gene sequence, wherein the disruption reduces or eliminates the expression of a protein encoded by the gene sequence.

72. The composition according to claim 71, wherein the at least one gene sequence is one or more of beta-2-microglobulin (B2M), HLA-A, HLA-B, or CIITA.

73. The composition according to any one of claims 1 to 72, comprising cells that have been manipulated to increase the expression of PD-L1 and / or CD47.

74. The composition according to any one of claims 71 to 73, wherein the cells are manipulated using a CRISPR / Cas system.

75. A method comprising the step of contacting a first composition comprising one or more of acetyl-CoA-related metabolites, HDAC inhibitors, redox homeostasis regulators, or monocarbonate pathway intermediates with a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells in vitro.

76. A method comprising the step of contacting a first composition containing an acetyl-CoA-related metabolite with a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells in vitro.

77. A method comprising the step of contacting a first composition comprising one or more of acetate, β-hydroxybutyrate, taurine, or formate with a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells in vitro.

78. A method comprising the step of contacting a first composition containing acetate with a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells in vitro.

79. The method according to any one of claims 75 to 78, comprising the step of bringing the cells into contact with an acetate in a concentration of 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.

80. The method according to any one of claims 75 to 78, wherein the first composition further comprises glutamine.

81. The method according to any one of claims 75 to 78, wherein the first composition further comprises 0.5 to 20 mM, 0.5 to 10 mM, 0.5 to 5 mM, 1 to 5 mM, 2 to 5 mM, or 1 mM to 10 mM of glutamine.

82. A method comprising the step of contacting a first composition containing glutamine in an amount of 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 with a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells.

83. The method according to claim 82, wherein the glutamine is at a concentration of 3.8 to 4.2 mM.

84. The method according to any one of claims 80 to 83, wherein 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 dipeptide form.

85. The method according to claim 84, wherein 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.

86. The method according to any one of claims 80 to 85, wherein 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 free glutamine form.

87. The method according to any one of claims 75 to 86, wherein the first composition comprises about 4 mM glutamine.

88. The method according to any one of claims 75 to 87, wherein the first composition comprises glutamine and acetate.

89. The method according to any one of claims 75 to 88, wherein the first composition comprises β-hydroxybutyrate.

90. The method according to any one of claims 75 to 89, wherein the first composition comprises taurine.

91. The method according to any one of claims 75 to 90, wherein the first composition comprises a formate.

92. The method according to any one of claims 75 to 91, wherein the first composition comprises a vitamin.

93. The method according to any one of claims 75 to 92, wherein the first composition further comprises biotin.

94. The method according to any one of claims 75 to 93, wherein the first composition comprises glutamine, acetate, β-hydroxybutyrate, taurine, formate, and biotin.

95. The method according to any one of claims 75 to 94, wherein the first composition further comprises a BMP signaling pathway inhibitor.

96. The method according to claim 95, wherein the BMP signaling pathway inhibitor is LDN193189 or DMH-1 or a derivative thereof.

97. The method according to any one of claims 75 to 96, wherein the first composition further comprises a ROCK inhibitor.

98. The composition according to claim 97, wherein the ROCK inhibitor is thiazovibin, Y-27632, fasudil / HA1077, or 14-1152, or a derivative thereof.

99. The method according to any one of claims 75 to 98, wherein the first composition further comprises a histone methyltransferase inhibitor.

100. The method according to claim 99, wherein the histone methyltransferase inhibitor is 3-deazanepranosin A hydrochloride, Bix-01294, UNC0638, BRDD4770, EPZ004777, AZ505, PDB4e47, valproic acid, vorinostat, romidepsin, entinostat, avexinostat, gibinostat, and mosetinostat, butyrate, serine protease inhibitors, serpine, or derivatives thereof.

101. The method according to any one of claims 75 to 100, wherein the first composition further comprises zinc.

102. The aforementioned zinc is ZnSO ４ The method according to claim 101, in the form described above.

103. The method according to any one of claims 75 to 102, wherein the first composition further comprises serum albumin protein.

104. The method according to claim 103, wherein the serum albumin protein is human serum albumin protein.

105. The first composition, 0.01% to 1%, 0.03 to 1%, 0.03 to 0.9%, 0.03 to 0.08%, 0.03 to 0.06%, 0.03 to 0.05%, 0.04 to 0.8%, 0.04 to 0.7%, 0.04 to 0.6%, 0.04 to 0.5%, 0.04 to 0.4%, 0.04 to 0.3%, 0.04 to 0.2%, 0.04 to 0.1%, 0.04 to 0.09%, 0.04 to 0.8%, 0.04 to 0.07%, 0.04 to 0.06 The method according to claim 103 or 104, comprising serum albumin protein in the following proportions: 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%.

106. The method according to any one of claims 75 to 105, wherein the first composition further comprises a TGF-β pathway inhibitor.

107. The method according to claim 106, 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.

108. The method according to any one of claims 75 to 107, wherein the first composition further comprises a thyroid hormone signaling pathway activator.

109. The method according to claim 108, wherein 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.

110. The method according to any one of claims 75 to 109, wherein the first composition further comprises a protein kinase inhibitor.

111. The method according to claim 110, wherein the protein kinase inhibitor is staurosporine, Ro-31-8220, or a derivative thereof.

112. The method according to any one of claims 75 to 111, wherein the first composition further comprises a gamma-secretase inhibitor.

113. The method according to claim 112, wherein the gamma-secretase inhibitor is XXI, DAPT, or a derivative thereof.

114. The method according to any one of claims 75 to 113, wherein the first composition further comprises L-carnitine.

115. The method according to any one of claims 75 to 114, comprising the steps of contacting the plurality of PDX1-positive, NKX6.1-positive cells with the first composition for 1 to 3 days, and then contacting them with a second composition comprising one or more of acetate, β-hydroxybutyrate, taurine, or formate for 3 to 7 days.

116. The method according to any one of claims 75 to 114, comprising the steps of contacting the plurality of PDX1-positive, NKX6.1-positive cells with the first composition for 1 to 3 days, and then contacting them 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 to 7 days.

117. The method according to any one of claims 75 to 114, comprising the steps of contacting the plurality of PDX1-positive, NKX6.1-positive cells with the first composition for 1 to 3 days, and then contacting them with a second composition comprising one or more of Alk5i, GC-1, LDN193189, thiazovibin, SSP, or DZNEP for 2 to 7 days.

118. The method according to any one of claims 115 to 117, wherein the first composition further comprises a sonic hedgehog pathway inhibitor.

119. The method according to claim 118, wherein the sonic hedgehog pathway inhibitor comprises Sant1, Sant2, Sant4, Sant4, Cur61414, forskolin, tomatidine, AY9944, tripanol, cyclopamine, or derivatives thereof.

120. The method according to any one of claims 115 to 119, wherein the first composition further comprises a growth factor from the epidermal growth factor (EGF) family.

121. The method according to claim 120, wherein the growth factor from the EGF family comprises beta-cellulin or EGF.

122. The method according to any one of claims 115 to 121, wherein the second composition is the same as the first composition, except that it lacks a sonic hedgehog pathway inhibitor and a growth factor from the epidermal growth factor (EGF) family.

123. The method according to any one of claims 115 to 122, wherein the contact step differentiates at least a portion of the plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells into insulin-positive endocrine progenitor cells.

124. The method according to claim 123, wherein the insulin-positive endocrine progenitor cells include ISL1-positive and NKX6.1-positive cells.

125. The method according to any one of claims 75 to 124, wherein the contact step results in the generation of a cell population having a higher proportion of ISL1-positive, NKX6.1-positive cells compared to the step of contacting a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells with the composition which is the same as the first composition except that it lacks one or more of acetate, β-hydroxybutyrate, taurine, or formate.

126. The method according to any one of claims 75 to 124, wherein the contact 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.

127. The method according to any one of claims 75 to 124, wherein the contact 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.

128. The method according to any one of claims 75 to 127, wherein the contact step yields a cell population having at least 35%, at least 38%, or at least 40% ISL1-positive, NKX6.1-positive cells after culturing for 4, 7, or 10 days.

129. The method according to any one of claims 75 to 128, wherein the contact step results in the generation of a cell population having a lower proportion of ISL1-negative, NKX6.1-negative cells compared to the step of contacting the plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells with the composition which is the same as the first composition except that it lacks one or more of acetate, β-hydroxybutyrate, taurine, or formate.

130. The method according to any one of claims 75 to 127, wherein the contact step results in the generation of a cell population having up to 22%, up to 20%, 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.

131. The method according to any one of claims 75 to 127, wherein the contact step results in the generation of a cell population having about 2%, 11%, 22%, 20%, or 18% ISL1-negative, NKX6.1-negative cells.

132. A method comprising the step of contacting a plurality of insulin-positive endocrine progenitor cells in vitro with a first composition comprising one or more of acetyl-CoA-related metabolites, HDAC inhibitors, redox homeostasis regulators, vitamins, monocarbon metabolic pathway intermediates, L-carnitine, or glutamates.

133. A method comprising the step of contacting a plurality of insulin-positive endocrine progenitor cells in vitro with a first composition comprising one or more of glutamate, acetate, β-hydroxybutyrate, taurine, biotin, L-carnitine, or formate.

134. The method according to claim 132 or 133, wherein the first composition comprises glutamate.

135. The method according to any one of claims 132 to 134, wherein the first composition comprises acetate.

136. The method according to any one of claims 132 to 135, wherein the first composition comprises an acetate with a molecular weight of 50 to 1000 nM, 50 to 800 nM, 50 to 500 nM, 50 to 300 nM, 50 to 250 nM, 100 to 200 nM, or 125 to 175 nM.

137. The method according to any one of claims 132 to 136, wherein the first composition comprises β-hydroxybutyrate.

138. The method according to any one of claims 132 to 137, wherein the first composition comprises taurine.

139. The method according to any one of claims 132 to 138, wherein the first composition comprises a formate.

140. The method according to any one of claims 132 to 139, wherein the first composition further comprises biotin.

141. The method according to any one of claims 132 to 140, wherein the first composition comprises L-carnitine.

142. The method according to any one of claims 132 to 141, wherein the first composition comprises glutamate, acetate, β-hydroxybutyrate, taurine, formate, L-carnitine, and biotin.

143. The method according to any one of claims 132 to 142, wherein the first composition further comprises a BMP signaling pathway inhibitor.

144. The method according to claim 143, wherein the BMP signaling pathway inhibitor is LDN193189 or DMH-1 or a derivative thereof.

145. The method according to any one of claims 132 to 144, wherein the first composition further comprises a ROCK inhibitor.

146. The composition according to claim 145, wherein the ROCK inhibitor is thiazovibin, Y-27632, fasudil / HA1077, or 14-1152, or a derivative thereof.

147. The method according to any one of claims 132 to 146, wherein the first composition further comprises a histone methyltransferase inhibitor.

148. The method according to claim 147, wherein the histone methyltransferase inhibitor is 3-deazanepranosin A hydrochloride, Bix-01294, UNC0638, BRDD4770, EPZ004777, AZ505, PDB4e47, valproic acid, vorinostat, romidepsin, entinostat, avexinostat, gibinostat, and mosetinostat, butyrate, serine protease inhibitors, serpine, or derivatives thereof.

149. The method according to any one of claims 132 to 148, wherein the first composition further comprises a TGF-β pathway inhibitor.

150. The method according to claim 149, 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.

151. The method according to any one of claims 132 to 150, wherein the first composition further comprises a thyroid hormone signaling pathway activator.

152. The method according to claim 151, 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.

153. The method according to any one of claims 132 to 152, wherein the first composition further comprises a protein kinase inhibitor.

154. The method according to claim 153, wherein the protein kinase inhibitor is staurosporine, Ro-31-8220, or a derivative thereof.

155. The method according to any one of claims 132 to 154, wherein the first composition further comprises vitamin C.

156. The method according to any one of claims 132 to 154, wherein the first composition does not contain vitamin C.

157. The method according to any one of claims 132 to 156, wherein the first composition further comprises zinc.

158. The aforementioned zinc is ZnSO ４ The method according to claim 157, in the form of...

159. The method according to any one of claims 132 to 158, wherein the first composition further comprises serum albumin protein.

160. The method according to claim 159, wherein the serum albumin protein is human serum albumin protein.

161. The first composition, 0.01% to 1%, 0.03 to 1%, 0.03 to 0.9%, 0.03 to 0.08%, 0.03 to 0.06%, 0.03 to 0.05%, 0.04 to 0.8%, 0.04 to 0.7%, 0.04 to 0.6%, 0.04 to 0.5%, 0.04 to 0.4%, 0.04 to 0.3%, 0.04 to 0.2%, 0.04 to 0.1%, 0.04 to 0.09%, 0.04 to 0.8%, 0.04 to 0.07%, 0.04 to 0.06 The method according to claim 159 or 160, comprising serum albumin protein in the following proportions: %, 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%.

162. The method according to any one of claims 132 to 161, wherein the composition comprises DMEM.

163. The method according to claim 162, wherein the DMEM is DMEM / F12.

164. The method according to any one of claims 132 to 163, wherein the plurality of insulin-positive endocrine progenitor cells are dissociated.

165. The method according to claim 164, wherein the dissociated insulin-positive endocrine progenitor cells were previously frozen.

166. The method according to claim 164 or 165, wherein the dissociated cells are re-aggregated into a plurality of cell clusters.

167. The method according to claim 166, wherein at least about 40%, 50%, 60%, 70%, 80%, or 90% of the plurality of cell clusters have a diameter of about 50 μm to about 250 μm, about 75 μm to about 250 μm, or about 100 μm to about 200 μm.

168. The method according to claim 166 or 167, wherein at least about 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, or 99% of the cells in the plurality of cell clusters are viable.

169. The method according to any one of claims 164 to 168, wherein the dissociated cells are re-aggregated into at least 2, 3, 4, 5, 10, 50, 100, 1,000, 10,000, 1,000,000, or 1,000,000 cell clusters.

170. The method according to any one of claims 164 to 169, wherein 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.

171. The method according to any one of claims 164 to 170, wherein the cell cluster has an average diameter or median diameter of up to 120, up to 130, up to 140, up to 150, up to 160, or up to 170 μm.

172. The method according to any one of claims 164 to 171, wherein the cell cluster has an average diameter or median diameter of about 80 to 150, about 100 to 150, about 120 to 150, about 140 to 150, about 80 to 130, about 100 to 130, about 120 to 130, about 80 to 120, about 90 to 120, or about 100 to 120 μm.

173. The method according to any one of claims 164 to 172, wherein at least about 50%, at least about 60%, at least about 70%, or at least about 80% of the cell cluster has a diameter of less than 150 μm.

174. The method according to any one of claims 164 to 173, wherein at least about 50%, at least about 60%, or at least about 70% of the cell cluster has a diameter of less than 130 μm.

175. The method according to any one of claims 132 to 174, carried out over a period of 1 to 10 days, 1 to 9 days, 1 to 8 days, 1 to 7 days, 1 to 6 days, 1 to 5 days, 1 to 4 days, 1 to 3 days, 1 to 2 days, 2 to 10 days, 2 to 9 days, 2 to 8 days, 2 to 7 days, 2 to 6 days, 2 to 5 days, 2 to 4 days, 2 to 3 days, 3 to 10 days, 3 to 9 days, 3 to 8 days, 3 to 7 days, 3 to 6 days, 3 to 5 days, 3 to 4 days, 4 to 10 days, 4 to 9 days, 4 to 8 days, 4 to 7 days, 4 to 6 days, or 4 to 5 days.

176. The method according to any one of claims 132 to 175, carried out over a period of 1 to 8 days, 1 to 7 days, 1 to 6 days, 1 to 5 days, 1 to 4 days, 1 to 3 days, 1 to 2 days, 2 to 8 days, 2 to 7 days, 2 to 6 days, 2 to 5 days, 2 to 4 days, 2 to 3 days, 3 to 8 days, 3 to 7 days, 3 to 6 days, 3 to 5 days, 3 to 4 days, 4 to 8 days, 4 to 7 days, 4 to 6 days, or 4 to 5 days.

177. The method according to any one of claims 132 to 176, wherein the plurality of insulin-positive endocrine progenitor cells are brought into contact with the first composition for about three, four, five, six, seven, or eight days, thereby generating a second cell population.

178. The method according to claim 177, further comprising the step of 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 β cells.

179. The method according to claim 178, wherein the second cell population is brought into contact with the second composition for 1 to 8 days, 1 to 7 days, 1 to 6 days, 1 to 5 days, 1 to 4 days, 1 to 3 days, 1 to 2 days, 2 to 8 days, 2 to 7 days, 2 to 6 days, 2 to 5 days, 2 to 4 days, 2 to 3 days, 3 to 8 days, 3 to 7 days, 3 to 6 days, 3 to 5 days, 3 to 4 days, 4 to 8 days, 4 to 7 days, 4 to 6 days, or 4 to 5 days.

180. The method according to claim 179, wherein the second cell population is brought into contact with the second composition for about three, four, five, six, seven, or eight days.

181. The method according to any one of claims 178 to 180, wherein 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.

182. The method according to any one of claims 178 to 181, wherein the second composition does not contain glutamate, acetate, β-hydroxybutyrate, taurine, biotin, L-carnitine, or formate.

183. (a) A step of contacting a plurality of PDX1-positive, NKX6.1-positive, insulin-negative cells in vitro with a first composition to generate a first cell population comprising a plurality of cell clusters including insulin-positive endocrine progenitor cells, wherein the first composition comprises acetate, glutamine, and one or more of β-hydroxybutyrate, taurine, formate, or biotin. (b) The step of dissociating at least a portion of the plurality of cell clusters in the first cell population in vitro, (c) The step of bringing the first cell population, which includes at least a portion of the dissociated cell clusters, into contact in vitro with a second composition to generate a second cell population, which includes a plurality of cell clusters, each comprising a plurality of insulin-positive cells, wherein the second composition comprises one or more of glutamate, acetate, β-hydroxybutyrate, taurine, biotin, L-carnitine, or formate. A method that includes this.

184. The steps include bringing the second insulin-positive cell population into contact with a third composition in vitro, thereby differentiating at least a portion of the second insulin-positive cell population into a third cell population containing a plurality of β-cells, wherein the third composition is different from the second composition. The method according to claim 183, further comprising:

185. The method according to claim 184, wherein the third composition does not contain glutamate, acetate, β-hydroxybutyrate, taurine, biotin, L-carnitine, or formate.

186. The method according to any one of claims 183 to 185, wherein the first composition comprises 3 to 10, 3 to 7, 3 to 8, 3 to 6, 3 to 5, 3 to 4, 3.5 to 4.5, 3.8 to 4.2, or 3.9 to 4.1 mM of glutamine.

187. The method according to any one of claims 183 to 185, wherein the first composition comprises about 4 mM glutamine.

188. The method according to any one of claims 183 to 187, wherein the first composition comprises 0.01 to 50 mM, 0.1 to 50 mM, 0.5 to 50 mM, 0.01 to 20 mM, 0.1 to 20 mM, 0.5 to 20 mM, 0.01 to 10 mM, 0.1 to 10 mM, 0.5 to 10 mM, 0.8 to 25 mM, 0.8 to 10 mM, 0.8 to 5 mM, 0.8 to 2 mM, 0.8 to 1.5 mM, 0.8 to 1.2 mM, 0.9 to 1.1 mM, or 0.95 to 1.05 mM of acetate.

189. The method according to any one of claims 183 to 187, wherein the first composition comprises about 1 mM acetate.

190. The method according to any one of claims 183 to 189, wherein the second composition comprises an acetate with a molecular weight of 50 to 1000 nM, 50 to 800 nM, 50 to 500 nM, 50 to 300 nM, 50 to 250 nM, 100 to 200 nM, or 125 to 175 nM.

191. The method according to any one of claims 183 to 190, wherein the second composition comprises glutamate, acetate, β-hydroxybutyrate, taurine, formate, L-carnitine, and biotin.

192. The method according to any one of claims 183 to 191, wherein the first composition further comprises at least one agent selected from 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-related coiled-coil protein kinase (ROCK) inhibitor, a bone morphogenetic protein (BMP) type 1 receptor inhibitor, a γ-secretase inhibitor, or a histone methyltransferase inhibitor.

193. The method according to any one of claims 183 to 192, wherein the second composition further comprises at least one agent selected from 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, or a histone methyltransferase inhibitor.

194. (A) The sonic hedgehog pathway inhibitor contains SANT1 or (B) The retinoic acid signaling pathway activator contains retinoic acid or (C) The γ-secretase inhibitor includes XXI, (D) The growth factors from the EGF family include beta-cell phosphate or EGF, (E) The BMP signaling pathway inhibitor includes LDN193189 or a derivative thereof, (F) The TGF-β signaling pathway inhibitor includes ALK5 inhibitor II or a derivative thereof, (G) The thyroid hormone signaling pathway activator comprises GC-1 or T3, or derivatives thereof. (H) The protein kinase inhibitor contains staurosporine or (i) The ROCK inhibitor comprises thiazovibin, Y-27632, fasudil / HA1077, or 14-1152, or derivatives thereof, (J) The histone methyltransferase inhibitor comprises 3-deazanepranosin A hydrochloride or a derivative thereof. The method according to claim 192 or 193.

195. A method comprising the step of exposing an NKX6.1-positive, ISL1-positive, insulin-positive cell population to one or more of the following: serum albumin protein, TGF-β signaling pathway inhibitor, TH signaling pathway activator, protein kinase inhibitor, ROCK inhibitor, BMP signaling pathway inhibitor, epigenetic modification compound, acetyl-CoA-related metabolite, vitamin, histone deacetylase inhibitor (HDACi), redox homeostasis regulator, monocarbon metabolic pathway intermediate, glutamate, and / or carnitine, over a first period of 1, 2, 3, 4, 5, 6, or 7 days (e.g., 4 days).

196. The method according to claim 195, further comprising the step of contacting the cell population with one or more of serum albumin protein, acetyl-CoA related metabolites, vitamins, histone deacetylase inhibitors (HDACi), redox homeostasis regulators, monocarbon metabolic pathway intermediates, glutamates, 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 modifying compounds.

197. A method comprising the step of exposing an NKX6.1-positive, ISL1-positive, insulin-positive cell population to one or more of HSA, Alk5 inhibitor II, GC-1, staurosporine, thiazovibin, LDN193189, DZNEP, taurine, acetate, beta-hydroxybutyrate, biotin, carnitine, glutamate, and formate over a first period of 1, 2, 3, 4, 5, 6, or 7 days (e.g., 4 days).

198. The method according to claim 197, further comprising the step of 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, thiazovibin, LDN193189, and DZNEP, after the first period.

199. The method according to any one of claims 195 to 198, further comprising the step of bringing the cell population into contact with ZnSO4 during the first and / or second period.

200. The method according to any one of claims 195 to 199, further comprising the step of bringing the cell population into contact with DMEM during the first and / or second period.

201. The method according to claim 200, wherein the DMEM is DMEM / F12.

202. The method according to any one of claims 196 or 198 to 200, wherein, during the second period, the cells are brought into contact with the serum albumin (e.g., about 0.05% HSA) at the same concentration as during the first period.

203. The method according to any one of claims 196 or 198 to 200, wherein, during the second period, the cells are brought into contact with a higher concentration of the HSA (e.g., about 1.0%) compared to the first period (e.g., about 0.05%).

204. The method according to any one of claims 68 to 203, wherein the cells are manipulated to include a genomic disruption in at least one gene sequence, the disruption reducing or eliminating the expression of the protein encoded by the gene sequence.

205. The method according to claim 204, wherein the at least one gene sequence is one or more of beta-2-microglobulin (B2M), HLA-A, HLA-B, or CIITA.

206. The method according to any one of claims 68 to 203, wherein the composition comprises cells manipulated to increase the expression of PD-L1 and / or CD47.

207. The method according to any one of claims 204 to 206, wherein the cells are manipulated using a CRISPR / Cas system.

208. A composition comprising insulin-positive endocrine progenitor cells according to any one of claims 14 to 74 or 123 to 182.

209. A composition comprising at least a portion of the third cell population described in any one of claims 184 to 194.

210. A device comprising cells according to any one of claims 1 to 74, 208, 209, or 193 to 207.

211. The device according to claim 210, configured to produce and release insulin when implanted in a target.

212. The device according to claim 210 or 211, wherein the aforementioned cells are enclosed.

213. The device according to any one of claims 210 to 212, further comprising a semipermeable membrane, the semipermeable membrane being configured to retain the cells in the device and to allow the passage of insulin.

214. A method for treating a subject having a disease characterized by long-term high blood glucose levels (e.g., diabetes mellitus), comprising the steps of administering to the subject cells according to any one of claims 1 to 74, 208, 209, or 193 to 207, or implanting a device according to any one of claims 187 to 190.

215. The method according to claim 214, wherein the disease is diabetes.

216. A composition comprising multiple insulin-positive cells and a cryopreservation agent, and one or more of the following: acetyl-CoA-related metabolites, HDAC inhibitors, redox homeostasis regulators, monocarbon metabolic pathway intermediates, vitamins, or L-glutamine.

217. The composition according to claim 216, which is stored by freezing.

218. The composition according to claim 216 or 217, comprising an acetyl-CoA-related metabolite.

219. The composition according to claim 218, wherein the acetyl-CoA-related metabolite is acetate.

220. The composition according to claim 219, comprising 0.01 to 50 mM, 0.1 to 50 mM, 0.5 to 50 mM, 0.01 to 20 mM, 0.1 to 20 mM, 0.5 to 20 mM, 0.01 to 10 mM, 0.1 to 10 mM, 0.5 to 10 mM, 0.8 to 25 mM, 0.8 to 10 mM, 0.8 to 5 mM, 0.8 to 2 mM, 0.8 to 1.5 mM, 0.8 to 1.2 mM, 0.9 to 1.1 mM, or 0.95 to 1.05 mM of acetate.

221. A composition according to any one of claims 216 to 220, comprising glutamine.

222. The composition according to claim 221, comprising 0.5 to 20 mM, 0.5 to 10 mM, 0.5 to 5 mM, 1 to 5 mM, 2 to 5 mM, 3.8 to 4.2 mM, or 1 mM to 10 mM of glutamine.

223. The composition according to claim 221 or 222, wherein 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.

224. The composition according to any one of claims 221 to 223, wherein 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 free glutamine form.

225. A composition according to any one of claims 216 to 224, comprising a redox homeostasis regulator.

226. The composition according to claim 225, wherein the redox homeostasis regulator is taurine.

227. A composition according to any one of claims 216 to 226, comprising a monocarbon metabolic pathway intermediate.

228. The composition according to claim 227, wherein the aforementioned one-carbon metabolic pathway intermediate is a formate.

229. A composition according to any one of claims 216 to 228, comprising a vitamin.

230. The composition according to claim 229, wherein the vitamin is biotin.

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