Methods of making stem cell-derived islet-like cells, as well as populations and compositions including the same

Abstract

Methods are disclosed for making a stem cell-derived islet-like cell (SC-IC) population, where such methods include culturing precursor cells such as pancreatic progenitor cells, pancreatic endocrine precursor cells or immature SC-ICs one or more differentiation mediums that promote the differentiation of the precursor cells to more differentiated cells to obtain an SC-IC population that includes mature pancreatic-beta-like cells. Also disclosed are cell-differentiating compositions useful in performing the methods and SC-IC populations obtained by performing the methods. Also disclosed are compositions and implantable devices comprising the SC-IC populations, which are useful thereof for treatment of metabolic disorders such as diabetes mellitus (diabetes).

Description

METHODS OF MAKING STEM CELL-DERIVED ISLET-LIKE CELLS, AS WELL AS POPULATIONS AND COMPOSITIONS INCLUDING THE SAME REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0001] The disclosure is being filed along with a Sequence Listing in ST.26 XML format. The Sequence Listing is provided as a file titled “30174_WO” created 8 November 2024 and is 6.6 kilobytes (kb) in size. The Sequence Listing information in the ST.26 XML format is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0002] The disclosure relates generally to biology and medicine, and more particularly it relates to methods of making stem cell-derived islet-like cells (SC-ICs), populations thereof and compositions including the same, as well as relates to uses thereof in treating metabolic disorders such as diabetes mellitus (diabetes). BACKGROUND

[0003] Diabetes is a major, global healthcare problem and is a group of metabolic disorders characterized by abnormal glucose homeostasis / metabolism. A hallmark of diabetes is an elevated blood glucose concentration. One form of diabetes is Type I diabetes (T1D), which results from autoimmune destruction of beta cells in pancreatic islets and which leads to a lack of insulin (INS). Another form of diabetes is Type II diabetes (T2D), which results from peripheral tissue INS resistance and beta (β) cell dysfunction.

[0004] Individuals having diabetes manage it by keeping their blood glucose concentration close to normal (i.e., between 70-120 mg / dL or 3.9-6.7 mmol / L). Diabetes management includes diet, exercise, weight loss, use of therapeutic agents (e.g., exogenous INS and / or anti- diabetics) or a combination thereof.

[0005] A more recent treatment for diabetes, especially T1D, is islet transplantation with donor islets. See, e.g., Shapiro et al. (2000) N. Engl. J. Med.343:230-238. There are, however, a number of disadvantages to islet transplantation including a scarcity and lack of quality of donor islets, as well as a need for an immunosuppressive cocktail.

[0006] An alternative approach to islet transplantation with donor islets is to derive a population of islet-like cells from stem cells. Several in vitro methods are known for differentiating stem cells into islet-like cells. See, e.g., Ameri et al. (2017) Cell Rep.19:36-49;D’Amour et al. (2006) Nat. Biotechnol. 24:1392-1401; Millman et al. (2015) Nat. Commun. 7:11463; Mfopou et al. (2010) Gastroenterol. 138:2233-2245; Pagliuca et al. (2014) Cell 159:428-439; Rezania et al. (2014) Nat. Biotechnol.32:1121-1133; and Sui et al. (2018) Curr. Protoc. Hum. Gene.99:e68; Veres et al. (2019) Nature.569: 368-373, including Methods and Extended Data; Nair et al. (2020) Nature Reviews Endocrinology 16: 508-518. See also, Intl. Patent Application Publication Nos. WO 2014 / 160413, WO 2016 / 170067, WO 2017 / 222879, WO 2019 / 018818, WO 2019 / 099725, WO 2019 / 169351, WO 2019 / 227198, WO 2020264072, WO 2020033879, WO 2022204377, WO 2023076554, US Patent 9388386, US Patent 10975355, US Patent 11299711, US Patent 11466256, US Patent 11525120, and US Patent 11332716.

[0007] Despite the existence of these methods, there is a need for additional in vitro methods of differentiating stem cells, including induced pluripotent stem cells (iPSCs) into functional islet-like cells that can effectively produce and secrete INS, particularly in response to glucose challenge. These functional islet-like cells are referred to herein as SC-ICs. In particular, there is a need for in vitro methods that produce cell populations comprising a large number and / or proportion of differentiated functional islet-like cells. BRIEF SUMMARY

[0008] To address this need, the disclosure provides methods of deriving a SC-IC population from a precursor cell population comprising pancreatic progenitor (PP) cells (e.g., a PP cell population defined herein), pancreatic endocrine precursor (PEP) cells (e.g., a PEP cell population defined herein) or SC-ICs (e.g., a precursor SC-IC population defined herein) to derive a SC-IC population. In some instances, these methods produce a SC-IC population having one or more altered characteristics as compared to a control SC-IC population. In some instances, the methods are adapted and scalable for bioreactors such as, for example, large scale bioreactors, suitable to produce differentiated cell populations comprising a large number of differentiated functional islet-like cells.

[0009] In one aspect, a SC-IC population can be derived by a method that includes or begins with culturing a PP cell population, a PEP cell population or a precursor SC-IC population in one or more differentiating mediums (e.g., as defined herein), at least one of which is a low- glucose defined medium comprising < about 2.5 mM of glucose, or < about 2 mM of glucose, for example, without limitation zero-glucose. In some instances, the SC-IC population can haveone or more altered characteristics as compared to a control SC-IC population obtained by performing the same method in the same differentiating medium(s) except that each control differentiating medium has ≥ 2 mM glucose or ≥ 2.5 mM glucose. In some instances, the SC- IC population can include one or more of the following: increased INS content, increased INS secretion, increased glucose-stimulated INS secretion (GSIS), a lower percentage of non- pancreatic endocrine cells (NPECs), a lower percentage of proliferating cells, a higher percentage of pancreatic endocrine cells (PECs) and a higher percentage of pancreatic beta- like cells (PBLCs).

[0010] In another aspect, a SC-IC population can be derived by a method that includes or begins with culturing a PP cell population or a PEP cell population in one or more differentiating mediums, at least one of which is a defined medium comprising an epigenetic modifier. In some instances, the SC-IC population can have one or more altered characteristics as compared to a control SC-IC population obtained by performing the same method in the same differentiating medium(s) except that each control differentiating medium lacks an epigenetic modifier. In some instances, the altered characteristics can include one or more of the following: a higher percentage of PECs, a higher percentage of PBLCs and a lower percentage of polyhormonal cells. In some instances, the epigenetic modifier can be an S- adenosylhomocysteine hydrolase (ADOHCYASE) inhibitor, a (HMT) inhibitor, a DNA methyltransferase (DNMT) inhibitor, a histone deacetylase (HDAC) inhibitor, a sirtuin 1 (SIRT1) activator or a sirtuin 6 (SIRT6) activator. In other instances, the epigenetic modifier can be an inhibitor of euchromatic histone-lysine N-methyltransferase 2 (EHMT2), also known as G9a.

[0011] In another aspect, a SC-IC population can be derived by a method that includes or begins with culturing a PP cell population or a PEP cell population in one or more differentiating mediums, wherein culturing is at pH in the range of: pH7.2 to pH7.8, pH7.3 to pH7.8, pH7.4 to pH7.6, pH7.4 to pH7.8, pH 7.2+ / - 0.2 (pH7.0 to pH7.4), pH 7.4+ / - 0.2 (pH7.2 to pH7.6), pH7.6+ / - 0.2 (pH7.4 to pH7.8) or pH7.8+ / - 0.2 (pH7.6 to pH8.0). In some instances, the SC-IC population can have one or more altered characteristics as compared to a control SC- IC population obtained by performing the same method in the same differentiating medium(s) except that the culturing is at a pH below pH7.4.

[0012] In another aspect, a SC-IC population can be derived by a method that includes or begins with culturing a PP cell population in one or more PP-differentiating mediums toproduce a PEP cell population, then culturing the PEP cell population in one or more PEP- differentiating mediums to produce a precursor SC-IC population, and then culturing the precursor SC-IC population in a SC-IC-differentiating medium to obtain the SC-IC population, where each of the PP-, PEP- and SC-IC-differentiating mediums can be a defined medium comprising a G9a inhibitor. In some instances, the SC-IC population has one or more altered characteristics as compared to a control SC-IC population and can be obtained by performing the same method in the same differentiating mediums except that each control differentiating medium lacks a G9a inhibitor. In some instances, the altered characteristics in the SC-IC population can include one or more of the following: a higher percentage of PECs, a higher percentage of PBLCs, a lower percentage of polyhormonal cells, increased INS content, increased INS secretion and increased GSIS. In some instances, the G9a inhibitor can be CM- 272, UNC0321, UNC0638 or a combination thereof.

[0013] In another aspect, a SC-IC population can be derived by a method that includes or begins with culturing a PEP cell population or a precursor SC-IC population in one or more differentiating mediums, where at least one of the differentiating mediums is a defined medium comprising ≤ about 1 mM pyruvate. In some instances, the SC-IC population has one or more altered characteristics as compared to a control SC-IC population obtained by performing the same method in the same differentiating mediums except that each control differentiating medium comprises > about 1 mM pyruvate. In some instances, the altered characteristics in the derived SC-IC population can include one or more of the following: increased INS secretion and increased GSIS. In some instances, the defined medium can include about 0.05 mM pyruvate. In some instances, the defined medium can include a human plasma-like medium (HPLM). In some instances, the altered characteristics in the SC-IC population can be one or more of the following: a higher percentage of PECs, a higher percentage of PBLCs, a lower percentage of polyhormonal cells, increased INS content, increased INS secretion and increased GSIS as compared to a control SC-IC population obtained by performing the same method in differentiating medium(s) that lack HPLM.

[0014] In another aspect, differentiation methods and processes for generating a population of INS producing cells, called SC-ICs, with improved characteristics. In one embodiment, the methods described herein are based upon a finding that culturing cells in a differentiation medium with a concentration of glucose below 2.5mM, e.g. without limitation about 0mM glucose, and optionally adding galactose (or another alternative nutrient) while differentiatingPP cells to PEPs, or PEPs to immature SC-ICs decreases (i.e., reduces or eliminates) EC-like cells (i.e., SLC18A1+cells). In one embodiment, the methods comprise at least one step of culturing cells, e.g. without limitation in a bioreactor culturing PP cells to PEPs, or PEPs to immature SC-ICs, at a pH in the range of: pH7.2 to pH7.8, pH7.3 to pH7.8, pH7.4 to pH7.6, pH7.4 to pH7.8, pH 7.2+ / - 0.2 (pH7.0 to pH7.4), pH 7.4+ / - 0.2 (pH7.2 to pH7.6), pH7.6+ / - 0.2 (pH7.4 to pH7.8) or pH7.8+ / - 0.2 (pH7.6 to pH8.0). In one embodiment, the methods comprise at least one step of culturing cells, e.g. without limitation in a bioreactor culturing PP cells to PEPs, or PEPs to immature SC-ICs, in a differentiation medium with a concentration of glucose below 2.5mM, e.g. without limitation about 0mM glucose, and at a pH in the range of: pH7.2 to pH7.8, pH7.3 to pH7.8, pH7.4 to pH7.6, pH7.4 to pH7.8, pH 7.2+ / - 0.2 (pH7.0 to pH7.4), pH 7.4+ / - 0.2 (pH7.2 to pH7.6), pH7.6+ / - 0.2 (pH7.4 to pH7.8) or pH7.8+ / - 0.2 (pH7.6 to pH8.0). Without being bound by theory, the pH and glucose concentration effectively selects against and thereby decreases (i.e., reduces or eliminates) EC-like cells (i.e., SLC18A1+cells) in the differentiated population. In one embodiment, the methods described herein are based on the finding that differentiating PPs is optimal in the presence of two tankyrase 1 / 2 inhibitors, at least one of which is Wiki4. In some embodiments, at least about 67% of the cells in population are CPEP+ / GCG- cells, e.g.67% to 80% of the cells in population are CPEP+ / GCG- , and at least about 99% of the cells in the population are CHGA+, e.g. 99% to 99.99% of the cells in the population are CHGA+, the population of insulin producing cells can be used for cell therapy to treat, for example, diabetes mellitus (e.g., T1D).

[0015] Second, the disclosure describes methods that incorporate one or more of the above differentiation methods to derive SC-ICs from pluripotent stem cells such as iPSCs or from partially differentiated cells.

[0016] In some instances, the method can include or can begin with a step of differentiating pluripotent stem cells (PSCs) to mesendoderm (ME) cells by culturing a population of cells including PSCs (a PSC population) in a PSC-differentiating medium for about 0.5 day to about 2 days, especially for about 1 day, to obtain a population of cells including ME cells (an ME cell population). In some instances, the PSC population can be iPSCs (an iPSC population) and the PSC-differentiating medium can be a defined medium supplemented with one or more differentiation factors in amount(s) effective to promote differentiation of at least a portion of the iPSC population to ME cells.

[0017] In some instances, the PSC-differentiating medium can include glucose and / or fructose and can be supplemented with one or more of a GSK-3α and GSK-3β inhibitor / Wnt pathway signaling activator (e.g., CHIR99021), a Rho kinase (ROCK) inhibitor (e.g., Y- 27632), a growth factor from the transforming growth factor β (TGF-β) superfamily (e.g., Activin A) and a Wnt / β-catenin pathway activator (e.g., a Wnt3a protein).

[0018] In some instances, the PSC-differentiating medium also can include one or more of a buffer (e.g., sodium bicarbonate (NaHCO3)), albumin, glutamine (e.g., a glutamine dipeptide), glutamate and pyruvate (e.g., sodium pyruvate).

[0019] In some instances, the PSC-differentiating medium also can include a serum replacement supplement that includes one or more of the following: INS, transferrin, a selenium (e.g., sodium selenite) and ethanolamine (e.g., an ITS-G or ITS-X supplement described herein).

[0020] In some instances, the PSC population can include one or more cell aggregates, with each cell aggregate having an initial aggregate diameter of about 150 μm to about 170 μm.

[0021] In some instances, the PSC population can be a human iPSC (hiPSC) population and optionally about 95% of the cells in the PSC population can be OCT4+ / NANOG+, and the ME cell population can be characterized as including at least about 50% to about 70% TBXT+ / MIXL1+cells.

[0022] The method also can include expanding and / or aggregating steps for the PSCs prior to initiating the differentiating step. In some instances, the method can include washing the PSCs in a defined medium prior to initiating the differentiating step.

[0023] In addition, the method can include or can begin with a step of differentiating ME cells to definitive endoderm (DE) cells by culturing a ME cell population (e.g., as defined herein) in a ME-differentiating medium for about 0.5 day to about 2 days, especially for about 1 day, to obtain a population of cells including DE cells (i.e., a DE cell population). The ME- differentiating medium can be a defined medium supplemented with one or more differentiation factors in amount(s) effective to promote differentiation of at least a portion of the ME cell population to DE cells.

[0024] In some instances, the ME-differentiating medium can include glucose and / or fructose and can be supplemented with one or more of a bone morphogenetic protein (BMP) inhibitor (e.g., LDN-193189) and a TGF-β superfamily growth factor (e.g., Activin A).

[0025] In some instances, the ME-differentiating medium also can include one or more of a buffer (e.g., NaHCO3), albumin, glutamine (e.g., a glutamine dipeptide), glutamate and pyruvate (e.g., sodium pyruvate).

[0026] In some instances, the ME-differentiating medium also can include a serum replacement supplement that includes one or more of INS, transferrin, a selenium (e.g., sodium selenite) and ethanolamine (e.g., an ITS-G or ITS-X supplement).

[0027] In some instances, the ME cell population can be derived from a hiPSC population, and the DE cell population can be characterized as including > about 90% GATA6+ / SOX17+cells and at least any of about 40% to about 80% FOXA2+ / SOX17+cells.

[0028] In some instances, the method can include washing the ME cell population in a defined medium prior to initiating the differentiating step.

[0029] In addition, the method can include or can begin with a step of differentiating DE cells to primitive gut tube (PGT) cells by culturing a DE cell population (e.g., as defined herein) in a DE-differentiating medium for about 2 days to about 4 days, especially for about 3 days, to obtain a population of cells including PGT cells (e.g., a PGT cell population defined herein). The DE-differentiating medium can be a defined medium supplemented with one or more differentiation factors in amount(s) effective to promote differentiation of at least a portion of the DE cell population to PGT cells.

[0030] In some instances, the DE-differentiating medium can include glucose and / or fructose and can be supplemented with one or more of a Vitamin C compound (e.g., ascorbic acid) and a growth factor from the fibroblast growth factor (FGF) family (e.g., keratinocyte growth factor (KGF)).

[0031] In some instances, the DE-differentiating medium also can include one or more of the following: a buffer (e.g., NaHCO3), albumin, glutamine (e.g., a glutamine dipeptide), glutamate and pyruvate (e.g., sodium pyruvate), and glutamine (e.g., a glutamine dipeptide), glutamate and pyruvate (e.g., sodium pyruvate).

[0032] In some instances, the DE-differentiating medium also can include a serum replacement supplement that includes one or more of the following: INS, transferrin, a selenium (e.g., sodium selenite) and ethanolamine (e.g., an ITS-G or ITS-X supplement described herein).

[0033] In some instances, the DE cell population can be human cells, and the PGT cell population can be characterized as including at least about 50% to about 70% FOXA2+cells.

[0034] In addition, the method can include or can begin with a step of differentiating PGT cells to foregut endoderm (FE) cells by culturing a PGT cell population in a first PGT- differentiating medium for about 0.5 day to about 2 days, especially for about 1 day, and then in a second PGT-differentiating medium for about 12 hours (hr) to about 48 hr, especially for about 1 day, to obtain a population of cells including FE cells (e.g., a FE cell population). Each PGT-differentiating medium can be a defined medium supplemented with one or more differentiation factors in amount(s) effective to promote differentiation of at least a portion of the PGT cell population to FE cells.

[0035] In some instances, the first PGT-differentiating medium can include glucose and / or fructose and can be supplemented with one or more of the following: a Vitamin C compound (e.g., ascorbic acid), a small molecule BMP inhibitor (e.g., DMH-1), a FGF family growth factor (e.g., a KGF protein), a protein kinase C (PKC) activator (e.g., 2S,5S-E,E-8-5-4- trifluoromethyl phenyl-2,4-pentadienoylamino benzolactam (TPPB)), a retinoid (e.g., all-trans retinoic acid (ATRA)), a ROCK inhibitor (e.g., Y-27632), a cell-permeable Sonic Hedgehog (SHH) signaling inhibitor (e.g., SANT-1), a TGF-β superfamily growth factor (e.g., Activin A) and at least one tankyrase 1 / 2 inhibitor as defined herein (e.g., IWR-1 and / or WIKI4). In some instances, the first PGT-differentiating medium can include only one tankyrase 1 / 2 inhibitor (e.g., IWR-1 or WIKI4).

[0036] In some instances, the second PGT-differentiating medium lacks a BMP inhibitor but can otherwise be identical to the first PGT-differentiating medium. In some instances, the second PGT-differentiating medium includes two tankyrase 1 / 2 inhibitors (e.g., IWR-1 and WIKI4).

[0037] In some instances, one or both PGT-differentiating mediums also can include one or more of a buffer (e.g., NaHCO3), albumin, glutamine (e.g., a glutamine dipeptide), glutamate and pyruvate (e.g., sodium pyruvate).

[0038] In some instances, one or both PGT-differentiating mediums also can include a serum replacement supplement that includes one or more of the following: INS, transferrin, a selenium (e.g., sodium selenite) ethanolamine, biotin, alpha tocopherol, Vitamin A, albumin, catalase, superoxide dismutase, corticosterone, galactose, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine and triiodo-L-thyronine (e.g., a B27 supplement described herein).

[0039] In some instances, the PGT cell population can be human cells, and the FE cell population can be characterized as including PDX1+cells.

[0040] In addition, the method can include or can begin with a step of differentiating FE cells to PP cells by culturing a FE cell population in a FE-differentiating medium for about 2 days to about 6 days, especially for about 3 days, to obtain a population of cells including PP cells (e.g., a PP cell population). The FE-differentiating medium can be a defined medium supplemented with one or more differentiation factors in amount(s) effective to promote differentiation of at least a portion of the FE cell population to PP cells.

[0041] In some instances, the FE-differentiating medium can include glucose and / or fructose and can be supplemented with one or more of the following: a Vitamin C compound (e.g., ascorbic acid), a growth factor from the epidermal growth factor (EGF) family (e.g., an EGF protein), a FGF family growth factor (e.g., a KGF protein), a Vitamin B3 compound (e.g., nicotinamide (NAM)), a PKC activator (e.g., TPPB), a retinoid (e.g., ATRA), a ROCK inhibitor (e.g., Y-27632), a cell-permeable SHH signaling inhibitor (e.g., SANT-1) and at least one tankyrase 1 / 2 inhibitor as defined herein (e.g., IWR-1 and / or WIKI4).

[0042] In some instances, the FE-differentiating medium also can include an epigenetic modifier (e.g., a G9a inhibitor such as UNC321).

[0043] In some instances, the FE-differentiating medium also can include one or more of a buffer (e.g., NaHCO3), albumin, glutamine (e.g., a glutamine dipeptide), glutamate and pyruvate (e.g., sodium pyruvate).

[0044] In some instances, the FE-differentiating medium also can include a serum replacement supplement that includes one or more of the following: INS, transferrin, a selenium (e.g., sodium selenite) ethanolamine, biotin, alpha tocopherol, Vitamin A, albumin, catalase, superoxide dismutase, corticosterone, galactose, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine and triiodo-L-thyronine (e.g., a B27 supplement described herein).

[0045] In some instances, the FE cell population includes of human cells, and the PP cell population can be characterized as including at least about 70% PDX1+cells, at least about 30% PDX1+ / NKX6.1+cells and less than about 40% CHGA+cells.

[0046] In addition, the method can include or can begin with a step of differentiating PP cells to PEP cells by culturing a PP cell population (e.g., as defined herein) in a first PP- differentiating medium for about 3 days to about 6 days, especially for about 4 days, then in asecond PP-differentiating medium for about 1 day to about 3 days, especially for about 2 days, to obtain a population of cells including PEP cells (e.g., a PEP cell population defined herein). Each PP-differentiating medium can be a defined medium supplemented with one or more differentiation factors in amount(s) effective to promote differentiation of at least a portion of the PP cell population to PEP cells.

[0047] PP and PP / PEP cell populations cultured in a bioreactor are typically cultured at a range of pH between pH7.0 and pH7.2. The present methods unexpectedly demonstrate that culturing certain cell populations such as, for example, populations comprising PP cells, at a pH higher than pH7.0 to pH7.2, selectively improves the proportion of mature SC-ICs in the in vitro differentiated cell population. In some instances, the culturing comprises monitoring and adjusting the pH in the bioreactor so as to maintain a pH that is not higher than pH7.8. In some instances, the culturing comprises monitoring and adjusting the pH in the bioreactor so as to maintain a pH that is a pH in the range of: pH7.2 to pH7.8, pH7.3 to pH7.8, pH7.4 to pH7.8, pH 7.2+ / - 0.2 (pH7.0 to pH7.4), pH 7.4+ / - 0.2 (pH7.2 to pH7.6), pH7.6+ / - 0.2 (pH7.4 to pH7.8) or pH7.8+ / - 0.2 (pH7.6 to pH8.0). In certain embodiments, the culturing is performed under conditions where dissolved oxygen is >50mmHg, about 100mmHg to about 110mmHg, optionally about 102mmHg to about 107mmHg.

[0048] In some instances, the first PP-differentiating medium can include about 2 mM to ≤ about 50 mM glucose (e.g., about 25 mM) while the concentration of glucose in the second PP- differentiating medium can be < about 2 mM (e.g., ≤ about 1 mM or ≤ about 0.5 mM) or is glucose-free (i.e., < about 0.01 mM, < about 0.001 mM or 0 mM).

[0049] In some instances, the first PP-differentiating medium can include about 5 mM to about 40 mM glucose and can be supplemented with one or more of a small molecule BMP inhibitor (e.g., LDN-193189), a zinc compound (e.g., zinc sulfate (ZnSO4)), a thyroid hormone signaling pathway activator (e.g., triiodothyronine (T3)), an ATP-competitive inhibitor of TGF-β RI kinase (e.g., ALK5 inhibitor II (ALK5iII)), a cell-permeable SHH signaling inhibitor (e.g., SANT-1), a ROCK inhibitor (e.g., Y-27632), a Vitamin C compound (e.g., ascorbic acid), a γ-secretase inhibitor (GSI; e.g., GSI-XX), an epigenetic modifier (e.g. a G9a inhibitor) and at least one tankyrase 1 / 2 inhibitor (e.g., IWR-1 and / or WIKI4).

[0050] In some instances, the second PP-differentiating medium can include glucose at ≤ about 0.05 mM (or is glucose-free; i.e., < about 0.01 mM, < about 0.001 mM or 0 mM), an alternative nutrient (e.g., galactose) and can be supplemented with one or more of thefollowing: an epigenetic modifier (e.g. a G9a inhibitor), a small molecule BMP inhibitor (e.g., LDN-193189), a zinc compound (e.g., ZnSO4), a thyroid hormone signaling pathway activator (e.g., T3), an ATP-competitive inhibitor of TGF-β RI kinase (e.g. ALK5iII), a cell-permeable SHH signaling inhibitor (e.g., SANT-1), a ROCK inhibitor (e.g., Y-27632), a Vitamin C compound (e.g., ascorbic acid), a GSI (e.g., GSI-XX) and at least one tankyrase 1 / 2 inhibitor as defined herein (e.g., IWR-1 and / or WIKI4).

[0051] In some instances, one or both PP-differentiating mediums also can include one or more of a buffer (e.g., NaHCO3), albumin, glutamine (e.g., a glutamine dipeptide), glutamate and pyruvate (e.g., sodium pyruvate).

[0052] In some instances, one or both PP-differentiating mediums also can include a serum replacement supplement that includes a mixture of two or more of INS, transferrin, a selenium (e.g., sodium selenite) ethanolamine, biotin, alpha tocopherol, Vitamin A, albumin, catalase, superoxide dismutase, corticosterone, galactose, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine and T3 (e.g., a B27 supplement described herein).

[0053] In some instances, one or both PP-differentiating mediums also can include a heparin (e.g., an unfractionated heparin (UFH)).

[0054] In some instances, one or both PP-differentiating mediums also can include a non- essential amino acid (NEAA) supplement that includes two or more non-essential amino acids.

[0055] In some instances, the PP cell population includes human cells, and the PEP cell population can be characterized as including at least about 70% CHGA+cells, at least about 40% CHGA+ / PDX1+cells and less than about 30% Ki67+cells.

[0056] In some instances, the method also includes a step of dissociating cell aggregates in the PEP cell population into single cells to obtain a dissociated PEP cell population before performing any subsequent step. In some instances, the method also can include a step of washing the dissociated PEP cell population before performing any subsequent step.

[0057] In addition, the method can include or can begin with a step of differentiating PEP cells to SC-ICs by culturing a dissociated PEP cell population (e.g., as defined herein) in a first PEP-differentiating medium for about 1 day to about 3 days, especially for about 2 days, to obtain a reaggregated cell population, then culturing the reaggregated cell population in a second PEP-differentiating medium for about 1 day to about 3 days, especially for about 2 days, to obtain a precursor SC-IC population. Each of the PEP-differentiating mediums is a defined medium that includes a pyruvate concentration of < about 1 mM (e.g., about 0.01 mMto about 0.5 mM) supplemented with one or more differentiation factors in amount(s) effective to promote differentiation of at least a portion of the PEP cell population to immature pancreatic endocrine cells (e.g., immature PBLCs and immature pancreatic alpha-like cells (PALCs)).

[0058] In some instances, the defined medium in each of the PEP-differentiating mediums can include glucose and / or fructose, < about 0.5 mM pyruvate and can be supplemented with one of more of the following: an epigenetic modifier (e.g., a G9a inhibitor), a thiol-based antioxidant (e.g., N-acetyl cysteine (NAC)), a Vitamin C compound (e.g., ascorbic acid), an ATP-competitive inhibitor of TGF-β RI kinase (e.g., ALKViII)), a small molecule BMP inhibitor (e.g., LDN-193189), a heparin (e.g., an UFH), a thiol-based antioxidant (e.g., N-acetyl cysteine (NAC)), a cell-permeable SHH signaling inhibitor (e.g., SANT-1), a thyroid hormone signaling pathway activator (e.g.,T3) and a zinc compound (e.g., ZnSO4).

[0059] In some instances, the first PEP-differentiating medium also can include a deoxyribonuclease (e.g., a recombinant mammalian DNAse I).

[0060] In some instances, the defined medium in one or both PP-differentiating mediums also can include one or more of a buffer (e.g., NaHCO3), albumin, galactose, glutamine (e.g., a glutamine dipeptide), glutamate and pyruvate (e.g., sodium pyruvate).

[0061] In some instances, the defined medium in one or both PP-differentiating mediums can be HPLM, which can include about 5 mM glucose, about 0.04 mM fructose, about 0.06 mM galactose, about 0.55 mM glutamine and about 0.05 mM pyruvate.

[0062] In some instances, one or both PEP-differentiating mediums also can include a serum replacement supplement that includes a mixture of at least two the following components: INS, transferrin, a selenium (e.g., sodium selenite) ethanolamine, biotin, alpha tocopherol, Vitamin A, an albumin, catalase, superoxide dismutase, corticosterone, galactose, glutathione, L- carnitine, linoleic acid, linolenic acid, progesterone, putrescine and triiodo-L-thyronine (e.g., a B27 supplement).

[0063] In some instances, one or both PEP-differentiating mediums also can include a heparin (e.g., a UFH).

[0064] In some instances, the dissociated PEP cell population includes human cells, and the precursor SC-IC cell population can be characterized as including one or more of: (i) about 50% to about 90% INS+ / SLC18A1- cells, (ii) about 0% to about 20% INS- / SLC18A1+cells, (iii) about 45% to about 75% CPEP+ / GCG- cells, (iv) about 5% to about 45% CPEP+ / GCG+cells and / or (v) about90% to about 100% CHGA+ / Ki67- cells.

[0065] In addition, the method can include or can begin with a step of differentiating immature SC-ICs to mature SC-ICs by culturing a precursor SC-IC population in a SC-IC- differentiating medium for about 8 days to about 15 days or for about 8 days to about 10 days, especially for about 9 days, to obtain a population of cells including mature SC-ICs (e.g., a mature SC-IC population defined herein). The SC-IC-differentiating medium can be a defined medium having a pyruvate concentration of < about 1 mM (e.g., about 0.01 mM to about 0.5 mM) and can be supplemented with one or more differentiation factors in amount(s) effective to promote differentiation of at least a portion of the precursor SC-IC population to mature PBLCs.

[0066] In some instances, the defined medium in the SC-IC-differentiating medium can include glucose and / or fructose, < about 0.5 mM pyruvate (e.g., sodium pyruvate) and can be supplemented with one of more of the following: a Vitamin C compound (e.g., ascorbic acid), a small molecule BMP inhibitor (e.g., LDN-193189), a carnitine compound (e.g., acetyl-L- carnitine), a thiol-based antioxidant (e.g., NAC), a thyroid hormone signaling pathway activator (e.g.,T3), a cell-permeable Vitamin E analog / antioxidant (e.g., Trolox), a zinc compound (e.g., ZnSO4) and an epigenetic modifier (e.g., a G9a inhibitor).

[0067] In some instances, the defined medium in the SC-IC-differentiating medium also can include one or more of a buffer (e.g., NaHCO3), albumin, galactose, glutamine (e.g., a glutamine dipeptide) and glutamate.

[0068] In some instances, the defined medium in the SC-IC-differentiating medium can be a HPLM (e.g., as defined herein), which can include about 5 mM glucose, about 0.04 mM fructose, about 0.06 mM galactose, about 0.55 mM glutamine and about 0.05 mM pyruvate.

[0069] In some instances, the SC-IC-differentiating medium also can include a serum replacement medium that includes a mixture of at least two of the following components: glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L- hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid 2-phosphate, transferrin, INS, a selenium (e.g., sodium selenite), a lipid-rich albumin and salts containing the trace element moieties Ag+, Al3+, Ba2+, Cd2+, Co2+, Cr3+, Ge4+, Se4+, Br-, I-, F-, Mn2+, Si4+, V5+, Mo6+, Ni2+, Rb+, Sn2+and Zr4+(e.g., a KnockOut serum replacement (KOSR) medium).

[0070] In some instances, the SC-IC-differentiating medium also can include a trace elements A supplement that includes one or more of the following: cupric sulfate, ferric citrate, a selenium (e.g., sodium selenite) and zinc sulfate (e.g., a trace elements A supplement).

[0071] In some instances, the SC-IC-differentiating medium also can include a trace elements B supplement that includes one or more of the following: ammonium molybdate, ammonium vanadate, manganese sulfate, nickel sulfate, sodium silicate, stannous chloride and hydrochloric acid (e.g., a trace elements B supplement).

[0072] In some instances, the SC-IC-differentiating medium can also include a chemically defined lipid mixture (CDLM) that includes two or more of the following: arachidonic acid, cholesterol, DL-alpha-tocopherol acetate, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, palmitoleic acid and stearic acid (e.g., a CDLM).

[0073] In some instances, the SC-IC-differentiating medium also can include a heparin (e.g., a UFH).

[0074] In some instances, the SC-IC population includes human cells, and the mature SC-IC population can be characterized as including one or more of: (i) about 50% to about 90% INS+ / SLC18A1- cells, (ii) about 0% to about 20% INS- / SLC18A1+cells (iii) about 45% to about 75% CPEP+ / GCG- cells, (iv) about 5% to about 45% CPEP+ / GCG+cells and (v) about 90% to about 100% CHGA+ / Ki67- cells.

[0075] Alternatively, the methods of deriving SC-ICs from PSCs can include differentiating PSCs (e.g., iPSCs, especially hiPSCs) into cells with expression markers characteristic of the ME and DE (i.e., Stage 1 cells) as described herein, differentiating the Stage 1 cells into cells expressing markers characteristic of the PGT (i.e., Stage 2 cells) as described herein, differentiating the Stage 2 cells into cells expressing markers characteristic of the FE (i.e., Stage 3 cells) as described herein, differentiating the Stage 3 cells into cells expressing markers of PPs (i.e., Stage 4 cells) as described herein, differentiating the Stage 4 cells into cells expressing markers of PEPs (i.e., Stage 5 cells) as described herein, differentiating the Stage 5 cells into cells expressing markers of immature SC-ICs (i.e., Stage 6 cells) as described herein, and differentiating the Stage 6 cells into cells expressing markers of mature SC-ICs (i.e., Stage 7 cells) as described herein.

[0076] Alternatively, the methods of deriving SC-ICs from partially differentiated cells can include differentiating Stage 1 cells into cells expressing markers characteristic of the PGT (i.e., Stage 2 cells) as described herein, differentiating the Stage 2 cells into cells expressingmarkers characteristic of the FE (i.e., Stage 3 cells) as described herein, differentiating the Stage 3 cells into cells expressing markers of PPs (i.e., Stage 4 cells) as described herein, differentiating the Stage 4 cells into cells expressing markers of PEPs (i.e., Stage 5 cells) as described herein, differentiating the Stage 5 cells into cells expressing markers of immature SC-ICs (i.e., Stage 6 cells) as described herein, and differentiating the Stage 6 cells into cells expressing markers of mature SC-ICs (i.e., Stage 7 cells) as described herein.

[0077] Alternatively, the methods of deriving SC-ICs from partially differentiated cells can include differentiating Stage 2 cells into cells expressing markers characteristic of the FE (i.e., Stage 3 cells) as described herein, differentiating the Stage 3 cells into cells expressing markers of PPs (i.e., Stage 4 cells) as described herein, differentiating the Stage 4 cells into cells expressing markers of PEPs (i.e., Stage 5 cells) as described herein, differentiating the Stage 5 cells into cells expressing markers of immature SC-ICs (i.e., Stage 6 cells) as described herein, and differentiating the Stage 6 cells into cells expressing markers of mature SC-ICs (i.e., Stage 7 cells) as described herein.

[0078] Alternatively, the methods of deriving SC-ICs from partially differentiated cells can include differentiating Stage 3 cells into cells expressing markers of PPs (i.e., Stage 4 cells) as described herein, differentiating the Stage 4 cells into cells expressing markers of PEPs (i.e., Stage 5 cells) as described herein, differentiating the Stage 5 cells into cells expressing markers of immature SC-ICs (i.e., Stage 6 cells) as described herein, and differentiating the Stage 6 cells into cells expressing markers of mature SC-ICs (i.e., Stage 7 cells) as described herein.

[0079] Alternatively, the methods of deriving SC-ICs from partially differentiated cells can include differentiating Stage 4 cells into cells expressing markers of PEPs (i.e., Stage 5 cells) as described herein, differentiating the Stage 5 cells into cells expressing markers of immature SC-ICs (i.e., Stage 6 cells) as described herein, and differentiating the Stage 6 cells into cells expressing markers of mature SC-ICs (i.e., Stage 7 cells) as described herein.

[0080] Alternatively, the methods of deriving SC-ICs from partially differentiated cells can include differentiating Stage 5 cells into cells expressing markers of immature SC-ICs (i.e., Stage 6 cells) as described herein, and differentiating the Stage 6 cells into cells expressing markers of mature SC-ICs (i.e., Stage 7 cells) as described herein.

[0081] Alternatively, the methods of deriving SC-ICs from partially differentiated cells can include differentiating Stage 6 cells into cells expressing markers of mature SC-ICs (i.e., Stage 7 cells) as described herein.

[0082] In any of the above, the methods can include the steps of dissociating and reaggregating any of the cell populations before initiating differentiation of that cell population, for example, dissociating and reaggregating an FE cell population (e.g., Stage 3 cells) before culturing in any PP-differentiating medium and / or dissociating and reaggregating a precursor SC-IC population (e.g., Stage 6 cells) before culturing in any SC-IC differentiating medium.

[0083] In any of the above, the methods can begin with human PSCs (e.g., hiPSCs) or with more differentiated cells derived from human PSCs (e.g., hiPSCs).

[0084] In any of the above, the methods also can include a step of isolating or purifying a cell type of interest to obtain an essentially pure population of the cell type of interest.

[0085] In any of the above, the methods also can include a step of reaggregating at least two isolated or purified populations of cells into pseudoislets. In some instances, the isolated or purified populations of cells are alpha (α)-like cells and beta-like cells (e.g., PALCs and PBLCs) obtained from mature SC-ICs.

[0086] Third, the disclosure describes methods of differentiating certain less differentiated cells into more differentiated cells, and in particular a method of differentiating PP cells into PEP cells and a method of differentiating PEP cells into a SC-IC population.

[0087] Fourth, the disclosure describes compositions that include a SC-IC population obtained by performing a differentiation method described herein as well as compositions useful for performing certain differentiation steps in the methods: a composition that includes PP cells and a G9a inhibitor and optionally at least one tankyrase 1 / 2 inhibitor, a composition that includes PEP cells and a G9a inhibitor, and a composition that includes immature PBLCs and a G9a inhibitor.

[0088] Fifth, the disclosure describes compositions that include a human SC-IC population having characteristics that are desirable for use as an implantable cell therapy to treat diabetes, and pharmaceutical compositions including the same. In some instances, the SC-IC population includes greater than 60% PBLCs, about 25% PALCs and about 15% EC-like cells.

[0089] Sixth, the disclosure describes compositions and implantable devices that encapsulate an SC-IC population described herein. In some instances, the implantable device can be a hydrogel capsule including an afibrotic compound (e.g., on an outer layer of the hydrogel capsule) and a population of SC-IC described herein.

[0090] Seventh, the disclosure describes methods of treating metabolic disorders such as diabetes (e.g., Type 1 diabetes) by administering to the individual an effective amount of a SC-IC population described herein. The SC-IC population can be administered as a composition, as a device including encapsulated SC-ICs or as a composition including unencapsulated SC- ICs.

[0091] Eighth, the disclosure describes uses for SC-IC populations, compositions and implantable devices described herein in treating metabolic disorders such as diabetes (e.g., T1D). Likewise, the disclosure describes uses for the SC-IC populations and compositions herein in manufacturing a medicament or implantable device for treating metabolic disorders such as diabetes (e.g., T1D).

[0092] Ninth, the disclosure describes in vitro methods that are adapted and scalable for bioreactors, e.g. large scale bioreactors, suitable to produce large number of differentiated stem cells. In a non-limiting embodiment, the bioreactor is stirred-tanked bioreactor.

[0093] An advantage of the SC-IC derivation methods herein is that differentiating certain precursor cell populations (i.e., PP, PEP and / or precursor SC-IC populations) following the steps and differentiating mediums described herein results in mature SC-IC populations with desirable characteristics as compared to SC-IC populations derived using standard methods known in the art. These desirable characteristics include: lower percentages of off-target cells such as EC-like cells (i.e., SLC18A1+cells) and proliferating Ki67+cells, a higher percentages of PECs (i.e., CPEP+cells) and mature PBLCs (i.e., CPEP+ / GCG- cells), as well as increased INS content and GSIS.

[0094] An advantage of the methods herein is that the resulting SC-IC populations have appropriate INS content, INS secretion and GSIS response (i.e., are functional) for use in islet cell therapy of an individual having or suspected of having diabetes (e.g., T1D).

[0095] Another advantage of the methods herein is that this functionality of the resulting SC- IC populations is durable in vivo when encapsulated in a device that protects the SC-ICs from an individual’s immune system.

[0096] In one aspect, the disclosure provides a method of deriving a cell population comprising mature stem cell-derived islet-like cells (SC-ICs), the method comprising a step of: (a) culturing a first precursor cell population in one or more differentiating mediums to obtain the mature SC-IC population, wherein at least one of the differentiating mediums is a defined medium comprising glucose at less than about 2.5 mM or at less than about 2 mM, and wherein the first precursor cell population is selected from the group consisting of a foregut endoderm (FE) population, a pancreatic progenitor (PP) cellpopulation, a pancreatic endocrine precursor (PEP) cell population and a precursor SC-IC cell population.

[0097] In some embodiments, the first precursor PP cell population comprises PDX1+ cells, the method comprising: (a) culturing the first precursor PP cell population comprising PDX1+cells, optionally PDX1+ / NKX6.1+cells and CHGA- cells (optionally PDX1+ / CHGA- cells), wherein the at least one of the differentiating mediums is a defined medium comprising glucose at a concentration of about 0 mM to less than about 2.5 mM and a G9a inhibitor, thereby obtaining a second cell population comprising PDX1+ / CHGA+cells (PDX1+ / NKX6.1+ / CHGA+cells).

[0098] In some embodiments, the first precursor PP cell population comprises PDX1+ / NKX6.1+cells and CHGA- cells , optionally PDX1+ / CHGA- cells.

[0099] In some embodiments, the at least one of the cell differentiating mediums is a defined medium comprising glucose at a concentration of about 0 to about 0.02 mM, about 0 to about 0.04 mM, about 0 to about 0.06 mM, about 0 to about 0.08 mM, about 0 to about 0.1 mM, about 0 to about 0.2 mM, about 0 to about 0.3 mM, about 0 to about 0.4 mM, about 0 to about 0.5 mM, about 0 to about 0.6 mM, about 0 to about 0.7 mM, about 0 to about 0.8 mM, about 0 to about 0.9 mM, about 0 to about 1.0 mM, about 0 to about 1.1 mM, about 0 to about 1.2 mM, about 0 to about 1.3 mM, about 0 to about 1.4 mM, about 0 to about 1.5 mM, about 0 to about 1.6 mM, about 0 to about 1.7 mM, about 0 to about 1.8 mM, about 0 to about 1.9 mM, about 0 to about 2.0 mM, about 0 to about 2.1 mM, about 0 to about 2.2 mM, about 0 to about 2.3 mM, about 0 to about 2.4 mM, about 0 to about 2.5 mM, about 0 to about 2.6 mM, about 0 to about 2.7 mM, about 0 to less than about 2.5 mM, about 0 to less than about 2.6mM, about 0 to less than about 2.7mM, or about 0 to less than about 2.8mM.

[0100] In some embodiments, the at least one of the cell differentiating mediums is a defined medium comprising glucose at a concentration of about 0mM, about 0.1mM, about 0.2mM, about 0.3mM, about 0.4mM, about 0.5mM, about 0.6mM, about 0.7mM, about 0.8mM, about 0.9mM, about 1.0mM, about 1.1mM, about 1.2mM, about 1.3mM, about 1.4mM, about 1.5mM, about 1.6mM, about 1.7mM, about 1.8mM, about 1.9mM, about 2.0mM, about 2.1mM, about 2.2mM, about 2.3mM, about 2.4mM, about 2.5mM, about 2.6mM, or less than 2.8mM.

[0101] In some embodiments, the at least one of the cell differentiating mediums is a defined medium comprising glucose at a concentration of 0mM, 0.1mM, 0.2mM, 0.3mM, 0.4mM,0.5mM, 0.6mM, 0.7mM, 0.8mM, 0.9mM, 1.0mM, 1.1mM, 1.2mM, 1.3mM, 1.4mM, 1.5mM, 1.6mM, 1.7mM, 1.8mM, 1.9mM, 1.0mM, 2.1mM, 2.2mM, 2.3mM, 2.4mM, 2.5mM, 2.6mM, 2,7 or less than 2.8mM.

[0102] In some embodiments, the culturing in step (a) further comprises monitoring the pH. In some embodiments, the culturing in step (a) is at a pH in the range of: pH7.2 to pH7.8, pH7.4 to pH7.8, pH 7.2+ / - 0.2 (pH7.0 to pH7.4), pH 7.4+ / - 0.2 (pH7.2 to pH7.6), pH7.6+ / - 0.2 (pH7.4 to pH7.8) or pH7.8+ / - 0.2 (pH7.6 to pH8.0). In some embodiments, the culturing in step (a) is in a bioreactor and comprises monitoring and maintaining a pH which is not higher than pH7.8, a pH in the range of: pH7.2 to pH7.8, pH7.4 to pH7.8, pH 7.2+ / - 0.2 (pH7.0 to pH7.4), pH 7.4+ / - 0.2 (pH7.2 to pH7.6), pH7.6+ / - 0.2 (pH7.4 to pH7.8) or pH7.8+ / - 0.2 (pH7.6 to pH8.0).

[0103] In some embodiments, at least one of the one or more differentiating mediums of steps (a) comprises a tankyrase 1 / 2 inhibitor, wherein the tankyrase ½ inhibitor is Wiki4.

[0104] In some embodiments, the defined medium is glucose-free.

[0105] In some embodiments, the defined medium comprises galactose at a concentration of about 4 mM to about 7 mM, about 5 mM to about 6.0 mM, about 5.5mM optionally and wherein the defined medium is pyruvate-free.

[0106] The method of any one of claims 1-11, wherein at least one of the differentiating mediums of step (a) comprises: 5.5mM galactose, glutamine, and at least two differentiation factors selected from the group consisting of: a small molecule BMP inhibitor, a zinc compound, a thyroid hormone signaling pathway activator, a heparin, an ATP-competitive inhibitor of TGF-β RI kinase, a cell permeable Sonic Hedgehog (SHH) signaling inhibitor, a non-essential amino acids (NEAA) supplement, a Rho kinase (ROCK) inhibitor, a Vitamin C compound, and a γ-secretase inhibitor (GSI), and optionally further comprises one or more of an albumin, a buffer and a serum replacement supplement.

[0107] In some embodiments, the methods further comprise: (b) culturing the second cell population comprising CHGA+ / PDX1+cells in a differentiating medium in the presence of an enzymatic aggregate-dissociating solution, to obtain a dissociated cell population comprising CHGA+ / PDX1+cells single cells, optionally wherein the enzymatic aggregate dissociating solution comprises trypsin activity, chymotrypsin / elastase activity, and collagenase type 1 activity, optionally including a chelating agent such as EDTA; and (c) culturing the dissociated cell population comprising CHGA+ / PDX1+cells single cells in a differentiating medium comprising DNAase I and G9a inhibitor for an additional time period sufficient to obtain a reaggregated population, wherein the reaggregated population comprises NKX6.1+ / CPEP+ cells, CPEP+ / GCG- cells, INS+ / SLC18A1- cells, CHGA+ / Ki67- cells, or a combination thereof, which cells are comprised in cell aggregates, optionally wherein the cell aggregates have an average size of about 40 µm to about 100 µm, or about 70 microns.

[0108] In some embodiments, the differentiating mediums of step (b) and step (c) each comprises glucose at a concentration of about 1 mM to ≤ about 25 mM, glutamine, and at least two differentiation factors selected from the group consisting of: a thiol-based antioxidant, a small molecule BMP inhibitor, a zinc compound, a thyroid hormone signaling pathway activator, an ATP-competitive inhibitor of TGF-β RI kinase, a cell permeable SHH signaling inhibitor, a Vitamin C compound, a heparin, and optionally wherein each differentiating mediums further comprises one or more of an albumin, a buffer and a serum replacement supplement.

[0109] In some embodiments, the enzymatic aggregate dissociating solution comprises chymotrypsin / elastase activity and EDTA at a concentration of 0.5mM.

[0110] In some embodiments, filtering of the dissociated cell population is through a 40micron filter and at least about 80% of the cells in the population are single cells.

[0111] In some embodiments, the methods further comprise:(d) culturing the reaggregated population, wherein the population comprises NKX6.1+ / CPEP+ cells, CPEP+ / GCG- cells, INS+ / SLC18A1- cells, CHGA+ / Ki67- cells, or a combination thereof, in a differentiating medium for a time period sufficient to obtain the mature (SC-ICs) cell population comprising NKX6.1+ / CPEP+ cells and CPEP+ / GCG- cells, wherein the differentiating medium comprises glucose at a concentration of about 1 mM to ≤ about 25 mM, glutamine and at least two differentiation factors selected from the group consisting of: a cell-permeable vitamin E analog / antioxidant, a carnitine compound, a chemically defined lipid mixture (CDLM), a thiol-based antioxidant, a small molecule bone morphogenetic protein (BMP) inhibitor; a zinc compound, a thyroid hormone signaling pathway activator, a Vitamin C compound, a heparin, a G9A inhibitor, and optionally wherein the differentiating medium further comprises one or more of an albumin, a buffer and a serum replacement supplement.

[0112] In some embodiments, the G9a inhibitor is CM-272, UNC0321 or UNC0638. In some embodiments, the G9A inhibitor is UNC0321.

[0113] In some embodiments, the differentiating medium comprises the human plasma-like medium (HPLM).

[0114] In some embodiments, the methods further comprise before step (a), a step(s) of culturing a foregut endoderm (FE) cell population comprising a combination of PDX1+, FOXA2+, NKX6.1-, and CHGA- cells in one or more cell differentiating mediums comprising two tankyrase inhibitors, wherein one of the tankyrase inhibitors is Wiki4, thereby obtaining the (PP) precursor cell population comprising PDX1+, optionally comprising PDX1+ / NKX6.1+cells and CHGA-cells.

[0115] In some embodiments, the methods do not include a step of sorting or isolating individual cells or cell populations comprising a cell marker or a combination of cell markers, optionally a cell surface marker or a combination of cell surface cell markers. In someembodiments, the methods do not include sorting or isolating individual cells or cell populations by means of fluorescent activated cell sorting, or magnetic beads sorting. In some embodiments, the sorting or isolating uses a selection marker such as, for example, a positive selection marker, to enrich for CPEP+ / GCG- cells, CPEP+ / NKX6.1+ cells, or CPEP+ cells, wherein the selection marker is a cell marker, e.g. without limitation cell markers such as TSQ, CD49A, ST8SIA1, GLUT2, ZNT8, CD9. In some embodiments, the sorting or isolating uses a selection marker, e.g. a negative selection marker, to deplete cell populations other than CPEP+ / GCG-, or CPEP+ / NKX6.1+ or CPEP+, wherein the selection marker a is a cell marker such as, for example and without limitation, CD26, SLC18A, or a combination thereof.

[0116] In some embodiments, the mature SC-ICs cell population comprises at least about 54% to about 60% NKX6.1+ / CPEP+cells and at least about 60% to about 80% CPEP+ / GCG- cells.

[0117] In some embodiments, the mature SC-ICs cell population further comprises about 99.8% CHGA+cells.

[0118] In some embodiments, the culturing in (a), (b), (c) and (d) is in a bioreactor and produces at least 1.5E5cells / ml, optionally 1.5E5 cells / ml to 5E5 cells / ml, or about 3E5 cells / ml. In some embodiments, the bioreactor is a 1L, 2L , 3L , 5L, 10L, 15L, 20L, 25L, 30L, 35L, 40L, 45L, or 50L bioreactor. In some embodiments, the bioreactor is a large bioreactor.

[0119] The disclosure also provides a method of deriving a cell population comprising mature stem cell-derived islet-like cells (SC-ICs) from a cell population comprising pancreatic progenitor (PP) cells comprising PDX1+cells, wherein the method comprises: (i) culturing the PP cell population in a first PP-differentiating medium for a time period sufficient to obtain an intermediate PP / PEP cell population, wherein the PP cell population comprises PDX1+, optionally PDX1+ / NKX6.1+cells and CHGA- cells (PDX1+ / CHGA- cells) and the PP / PEP cell population comprises PDX1+ / CHGA+, wherein the first PP-differentiating medium is a defined medium comprising about 5 mM to about 50 mM glucose and a set of differentiation factors in amounts sufficient to promote differentiation of a least a portion of the PP cell population to PEP cells, wherein the set of differentiation factors comprises a G9a inhibitor and at least one factor selected from the group consisting of: a small molecule bone morphogenetic protein (BMP) inhibitor, a zinc compound, a thyroid hormone signaling pathway activator,an ATP-competitive inhibitor of TGF-β RI kinase, a cell-permeable Sonic Hedgehog (SHH) signaling inhibitor, a Rho kinase (ROCK) inhibitor, a Vitamin C compound, a γ-secretase inhibitor (GSI), a heparin, and optionally at least one tankyrase 1 / 2 inhibitor; optionally two tankyrase 1 / 2 inhibitors; (ii) washing the intermediate PP / PEP cell population in a defined wash medium comprising < about 1 mM glucose; (iii) culturing the washed intermediate PP / PEP cell population in a second PP- differentiating medium for a time period sufficient to obtain a PEP cell population comprising cell aggregates, wherein the second PP-differentiating medium is a defined medium comprising < about 1 mM glucose and a set of differentiation factors in amounts sufficient to promote differentiation of at least a portion of the intermediate PP / PEP cell population to PEP population comprising PDX1+ / CHGA+(PDX1+ / NKX6.1+ / CHGA+cells) cells, wherein the set of differentiation factors comprises a G9a inhibitor and at least one factor selected from the group consisting of: a small molecule BMP inhibitor, a zinc compound, a thyroid hormone signaling pathway activator, an ATP-competitive inhibitor of TGF-β RI kinase, a cell-permeable SHH signaling inhibitor, a ROCK inhibitor, a Vitamin C compound, a GSI, a heparin, and at least one tankyrase 1 / 2 inhibitor; optionally two tankyrase 1 / 2 inhibitors; (iv) dissociating at least about 80% of the cell aggregates in the aggregated PEP cell population into single cells to obtain a dissociated PEP cell population;(v) culturing the dissociated PEP cell population in a first PEP-differentiating medium comprising a deoxyribonuclease (DNAse) for a time period sufficient to obtain a reaggregated, intermediate PEP / SC-IC population, wherein the first PEP-differentiating medium is a defined medium comprising the DNAse, ≤ about 0.5 mM pyruvate, about 1 mM to ≤ about 25 mM glucose and a set of differentiation factors in amounts sufficient to promote differentiation of at least a portion of the PEP cell population to immature pancreatic endocrine cells (PECs), wherein the set of differentiation factors comprises a G9a inhibitor and at least one factor selected from the group consisting of: a thiol-based antioxidant, a small molecule BMP inhibitor; a zinc compound, a thyroid hormone signaling pathway activator, an ATP-competitive inhibitor of TGF-β RI kinase, a cell-permeable SHH signaling inhibitor, a Vitamin C compound, and a heparin; (vi) washing the reaggregated, intermediate PEP / SC-IC population in a defined wash medium comprising ≤ about 0.5 mM pyruvate and about 1 mM to ≤ about 25 mM glucose; (vii) culturing the washed intermediate PEP / SC-IC population in a second PEP- differentiating medium for a time period sufficient to obtain a precursor SC-IC population, wherein the second PEP-differentiating medium is a defined medium comprising ≤ about 0.5 mM pyruvate, about 1 mM to ≤ about 25 mM glucose and a set of differentiation factors in amounts sufficient to promote differentiation of at least a portion of the intermediate PEP / SC- IC population to a precursor SC-IC population comprising CHGA+ / PDX1+cells, wherein the set of differentiation factors comprises a G9a inhibitor and at least one factor selected from the group consisting of: a thiol-based antioxidant, a small molecule BMP inhibitor, a zinc compound, a thyroid hormone signaling pathway activator, an ATP-competitive inhibitor of TGF-β RI kinase, a cell-permeable SHH signaling inhibitor,a Vitamin C compound, and a heparin; and (viii) culturing the precursor SC-IC population in an SC-IC-differentiating medium for a time period sufficient to obtain the mature SC-IC population comprising a combination of NKX6.1+ / CPEP+cells, CPEP+ / GCG- cells, INS+ / SLC18A1- cells, or CHGA+ / Ki67- cells, wherein the SC-IC-differentiating medium comprises ≤ about 0.5 mM pyruvate, about 1 mM to ≤ about 25 mM glucose and a set of differentiation factors in amounts sufficient to promote differentiation of at least a portion of the precursor SC-IC population to mature PBLCs, wherein the set of differentiation factors comprises a G9a inhibitor and at least one factor selected from the group consisting of: a cell-permeable Vitamin E analog / antioxidant, a carnitine compound, a chemically defined lipid mixture (CDLM), a thiol-based antioxidant, a small molecule BMP inhibitor, a zinc compound, a thyroid hormone signaling pathway activator, a Vitamin C compound, and a heparin; and wherein in all steps (i)-(viii) at least one of the tankyrase 1 / 2 inhibitor is Wiki4 and the G9a inhibitor is UNC0321.

[0120] In some embodiments, the methods further comprise obtaining the PP cell population used in step (i) by culturing a foregut endoderm (FE) population of cells comprising PDX1+cells in an FE-differentiating medium for a time period sufficient to obtain the PP cell population, wherein the FE-differentiating medium is a defined medium comprising about 5 mM to about 50 mM glucose and a set of differentiation factors in amounts sufficient to promote differentiation of at least a portion of the FE cell population to PP cells, wherein the set of differentiation factors comprises at least one factor selected from the group consisting of: an EGF family growth factor, a Vitamin B3 compound, a Vitamin C compound, a FGF family growth factor,a PKC activator, a retinoid, a ROCK inhibitor, a cell-permeable SHH signaling inhibitor, at least one tankyrase 1 / 2 inhibitor, optionally two tankyrase 1 / 2 inhibitors, wherein at least one of the trankyrase ½ inhibitor is Wiki4.

[0121] In some embodiments, the methods further comprise the step of: obtaining the FE cell population by: (i) culturing a primitive gut tube (PGT) population of cells comprising FOXA2+cells or a PGT cell population in a first PGT-differentiating medium for a first time period of about 12 hours to about 48 hours to obtain an intermediate PGT / FE cell population; and (ii) culturing the intermediate PGT / FE cell population in a second PGT medium for a second time period of about 12 hours to about 48 hours to obtain the FE cell population, wherein the first PGT-differentiating medium is a defined medium comprising about 5 mM to about 50 mM glucose and a set of differentiation factors in amounts sufficient to promote differentiation of at least a portion of the PGT cell population to FE cells, wherein the factors are selected from the group consisting of a small molecule BMP inhibitor and at least one of an FGF family growth factor, a PKC activator, a retinoid, a ROCK inhibitor, a cell- permeable SHH signaling inhibitor and at least one tankyrase 1 / 2 inhibitor; and wherein the second PGT-differentiating medium is a defined medium that lacks a BMP inhibitor and comprises about 5 mM to about 50 mM glucose, about 0.5 mM to about 1.5 mM pyruvate and a set of differentiation factors in amounts sufficient to promote differentiation of at least a portion of the intermediate PGT / FE cell population to FE cells, wherein the factors comprise at least one factor selected from the group consisting of: a Vitamin C compound, an FGF family growth factor, a PKC activator, a retinoid, a ROCK inhibitor, a cell-permeable SHH signaling inhibitor, andat least one tankyrase 1 / 2 inhibitor, optionally two tankyrase 1 / 2 inhibitors, wherein at least one of the trankyrase ½ inhibitor is Wiki4.

[0122] In some embodiments, the methods further comprise obtaining the PGT cell population by culturing a definitive endoderm (DE) population of cells comprising PDX1- cells, and FOXA2+ / SOX17+ cells or GATA6+ / SOX17+ cells, in a DE-differentiating medium for a time period sufficient to obtain the PGT cell population, wherein the DE-differentiating medium is a defined medium comprising about 5 mM to about 20 mM glucose and a set of differentiation factors in amounts sufficient to promote differentiation of at least a portion of the DE cell population to PGT cells, wherein the factors comprise at least one factor selected from the group consisting of a Vitamin C compound and an FGF family growth factor.

[0123] In some aspects, the disclosure provides a composition comprising a cell population such as, for example, an in vitro cell population, and optionally a carrier, wherein: (i) < about 2% of the cells in the cell population or population cells are non- endocrine cells (CHGA-), or at least about 98% of the population cells express chromogranin A (CHGA+); (ii) at least about 50% of the population cells are CPEP+ / GCG- cells or at least about 50% of the population cells produce C-peptide (CPEP+) and do not express glucagon (GCG-) (CPEP+ / GCG-); (iii) < about 40% of the population cells express glucagon (GCG+); (iv) at least about 45%of the population cells are pancreatic endocrine cells (PDX+ / CHGA+), or at least about 45% of the population cells produce CPEP (CPEP+) and express NK6 homeobox 1 (NKX6.l+) (CPEP+ / NKX6.1+); (v) at least about 60% of the population cells produce insulin (INS+) and do not express solute carrier family 18 member 1 (SLC18A1-) (INS+ / SLC18A1-); (vi) an insulin content of at least about 150 nU / cell; (vii) < about 16% of the population cells are INS- / SLC+ cells (CPEP- / SLC18A1+) that do not produce insulin, or < about 16% of the population cells do not produce insulin and express solute carrier family 18 member 1 (SLC18A1+) (INS- / SLC18A1+); (viii) < about 5% of the population cells are proliferating cells (Ki67+), or less than about 5% of the population cells express (Ki67+);(ix) at least about 99.5% of the population cells are CHGA+, at least about 60% of the population cells are CPEP+ / GCG-, at least about 50% of the population cells are CPEP+ / NKX6.1+and at least about 70% of the population cells are INS+ / SLC18A1-; (x) < about 12% of the population cells are INS- / SLC18A1+and < about 4% of the population cells are Ki67-; (xi) the cell population does not produce lactate; (xii) < about 0.5% or about 0.2% of the population cells are non-endocrine cells, at least about 60% of the population cells are CPEP+ / GCG- cells, at least about 50% or about 60% of the population cells are PBLCs expressing NKX6.1 (NKX6.1+), at least about 70% or about 75% of the population cells are insulin-producing cells that are not ECLCs (INS- / SLC+); < about 11% or about 7% of the population are INS- / SLC+ cells that do not produce insulin and < about 4% or about 2% of the population cells are proliferating cells; the cell population does not produce lactate and optionally the cell population has an insulin content of at least about 325 nU / cell, or 150nU / cell to at least about 200nU / cell; and (xiii) at least about 98% or at least about 99.5% of the population cells are CHGA+, at least about 60% or about 65% of the population cells are CPEP+ / GCG-, at least about 50% or about 60% of the population cells are CPEP+ / NKX6.1+, at least about 70% or about 75% of the population cells are INS+ / SLC18A1-; < about 11% or about 7% of the population are INS- / SLC18A1+and < about 4% or about 2% of the population cells are Ki67-; the cell population does not produce lactate; and optionally the cell population has an insulin content of at least about 325 nU / cell, or at least 150nU / cell to about 200nU / cell.

[0124] In some aspects, the disclosure provides an in vitro cell population such as, for example, an in vitro differentiated cell population, comprising cells wherein: (i) about 40% to about 60% or about 45% to about 55% of the cells in the population are PDX1+ / NKX6.1+cells; optionally wherein about 60% of the cells in the population are PDX1+ / NKX6.1+; (ii) about 60% to about 90% or about 65% to about 75% of the cells in the population are PDX1+ / CHGA- cells; (iii) about 50% to about 65% or about 50% to about 60% of the cells in the population are NKX6.1+cells; (iv) about 65% to about 97% or about 80% to about 85% of the cells in the population are PDX1+cells; or(v) < about 5% to about 15% or < about 9% to about 13% of the cells in the population are CHGA+cells.

[0125] In some aspects, the disclosure provides an in vitro cell population such as, for example, an in vitro differentiated cell population, comprising cells wherein: (i) at least about 60% of the cells in the population are CPEP+ / GCG- cells; optionally about 60%, about 60% to about 68% of the cells in the population are CPEP+ / GCG- cells; (ii) no more than about 23% of the cells in the population are GCG+cells; optionally about 10% to about 20%, about 10% to about 23% of the cells in the population are GCG+cells; (iii) at least about 54% of the cells in the population are NKX6.1+ / CPEP+cells; optionally about 54% to about 65%, about 54% of the cells in the population are NKX6.1+ / CPEP+cells; (iv) at least about 68% of the cells in the population are INS+ / SLC- cells; optionally about 68%, about 68% to about 77% of the cells in the population are INS+ / SLC- cells; (v) less than about 11% of the cells in the population are INS- / SLC+cells; optionally about 11%, about 7% to about 11% of the cells in the population are INS- / SLC+cells; (vi) at least about 99.5% of the cells in the cell population are CHGA+cells; about 99.5% of the cells in the cell population are CHGA+cells; (vii) less than about 4% of the cells in the cell population are Ki67+cells; optionally about 3.5%, about 1% to 4% of the cells in the population are Ki67+cells; (viii) an insulin content of at least about 150nU / cell to about 200nU / cell, or (ix) lactate production by the cell population in 48hrs of less than 0.5mM, about 0 to about 0.02 mM, about 0 to about 0.04 mM, about 0 to about 0.06 mM, about 0 to about 0.08 mM about 0 to about 0.1 mM 0, about 0 to about 0.2 mM, about 0 to about 0.3 mM, about 0 to about 0.4 mM, about 0 to about 0.5 mM, wherein the population comprises at least two of the above characteristics.

[0126] In some embodiments of the in vitro cell population, at least about 60% to about 68% of the cells in the population are CPEP+ / GCG- and at least about 99.5% of the cells in the population are CHGA+. In some embodiments of the in vitro cell population at least about 54% to about 65% of the cells in the population are NKX6.1+ / CPEP+and less than about 11% of the cells in the population are INS- / SLC+. In some embodiments of the in vitro cell population atleast about 60% to about 68% of the cells in the population are CPEP+ / GCG- and at least about 68% of the cells in the population are INS+ / SLC-. In some embodiments of the in vitro cell population at least about 54% of the cells in the population are NKX6.1+ / CPEP+and at least about 68% to about 77% of the cells in the population are INS+ / SLC-. In some embodiments of the in vitro cell population no more than about 23% of the cells in the population are GCG+. In some embodiments of the in vitro cell population at least about 99.5% of the cells in the population are CHGA+.

[0127] In some aspect, the disclosure provides an in vitro cell population such as, for example, an in vitro differentiated cell population, comprising cells wherein: (i) at least about 67% of the cells in population are CPEP+ / GCG-; optionally about 67% to about 80%, about 67% about 70%, about 70% to about 80%, about 70% to about 85% of the cells in the population are CPEP+ / GCG- cells; (ii) no more than about 22% of the cells in the cell population are GCG+cells; optionally about 10% to about 20%, about 10% to about 22% of the cells in the population are GCG+cells; (iii) at least about 60% of the cells in the cell population are NKX6.1+ / CPEP+cells; optionally about 60% to about 70%, about 65% to about 75%, about 60% of the cells in the population are NKX6.1+ / CPEP+cells; (iv) about 68% of the cells in the cell population are INS+ / SLC- cells; optionally about 77%, about 70% to about 80%, about 70% to about 85% of the cells in the population are INS+ / SLC- cells; (v) less than about 7% of the cells in the cell population are INS- / SLC+cells; optionally about 7%, about 3-7% of the cells in the population are INS- / SLC+cells; (vi) at least about 99.8% of the cells in the cell population are CHGA+cells; about 99.8% of the cells in the cell population are CHGA+cells; (vii) less than about 2% of the cells in the cell population are Ki67+cells; optionally 0.3-2%, about 2% of the cells in the population are Ki67+cells; (viii) an insulin content of at least 150nu / cell to about 200nU / cell, or (ix) lactate production by the cell population in 48hrs of less than 0.5mM, about 0 to about 0.02 mM, about 0 to about 0.04 mM, about 0 to about 0.06 mM, about 0 to about 0.08 mM about 0 to about 0.1 mM 0, about 0 to about 0.2 mM, about 0 to about 0.3 mM, about 0 toabout 0.4 mM, about 0 to about 0.5 mM, wherein the population comprises at least two of the above characteristics.

[0128] In some embodiments of the in vitro cell population, about 67% of the cells in population are CPEP+ / GCG- and about 99.8% of the cells in the population are CHGA+cells. In some embodiments of the in vitro cell population at least about 54% of the cells in the population are NKX6.1+ / CPEP+and less than about 11% of the cells in the population are INS- / SLC+. In some embodiments of the in vitro cell population about 67% of the cells in the population are CPEP+ / GCG- and about 68% of the cells in the population are INS+ / SLC-. In some embodiments of the in vitro cell population about 60% of the cells in the population are NKX6.1+ / CPEP+and about 68% of the cells in the population are INS+ / SLC-. In some embodiments of the in vitro cell population no more than about 22% of the cells in the population are GCG+. In some embodiments of the in vitro cell population at least about 99.8% of the cells in the population are CHGA+.

[0129] In some embodiments of the in vitro cell population, the population has an insulin content of at least 150nU / cell to about 200nU / cell. In some embodiments of the in vitro cell population lactate production by the cell population of less than 0.5 mM in 48hrs.

[0130] In one aspect provided is a liquid cell differentiating composition comprising: (a) a serum-free basal culture media; and (b) a set of differentiation factors, wherein the set of differentiation factors is: (i) a set of factors promoting differentiation of foregut endoderm (FE) cells to pancreatic progenitor (PP) cells (a FE factor set); (ii) a set of factors promoting differentiation of pancreatic progenitor (PP) comprising PDX1+ cells to pancreatic endocrine precursor (PEP) population comprising PDX1+ / CHGA+cells (a PP factor set); (iii) a set of factors promoting differentiation of PEP cells to immature SC- IC (a PEP factor set); or (iv) a set of factors capable of promoting differentiation of immature SC-IC to mature SC-IC (a SC-IC factor set).

[0131] In some embodiments, the serum-free basal culture media comprises 0 mM to less than 2.5 mM glucose and the PP factor set comprises a G9a inhibitor, optionally UNC0321. In some embodiments, the liquid cell differentiating composition further comprising at least one tankyrase ½ inhibitor, optionally wherein the tankyrase ½ inhibitor is Wiki4. In someembodiments, the liquid cell differentiating composition is used for differentiating progenitor populations comprising PDX1+ cells, optionally a PP population comprising PDX1+ cells to a PEP population comprising PDX1+ / CHGA+cells.

[0132] In some aspects, the disclosure provides a pharmaceutical composition comprising the compositions comprising differentiated SC-IC cells as described or in vitro cell populations as described, and a carrier, for example a suitable pharmaceutical carrier.

[0133] In some aspects, the disclosure provides methods of treating an individual having diabetes mellitus, one or more complications related to diabetes mellitus or a pre-diabetic condition, the method comprising: (a) administering to the individual an effective amount of a composition comprising differentiated SC-IC cells as described herein, any one of the in vitro cell populations or a pharmaceutical composition comprising the same; (b) administering to the individual an effective amount of a pharmaceutical composition as described or any one of the in vitro cell populations, each encapsulated in a device that provides immune protection of the encapsulated compositions and / or cell populations; or (c) administering to the individual a device comprising a composition comprising differentiated SC-IC cells as described, any one of the in vitro cell populations or the pharmaceutical composition comprising these. In some embodiments, the methods further comprising administering to the individual an immunosuppressant before and / or after the administering of step (a), (b) or (c).

[0134] In some embodiments of the methods, the device comprises alginate chemically modified with an afibrotic-effective amount of a compound of Formula I.

[0135] In some embodiments, diabetes mellitus is T1D.

[0136] In some aspects, the disclosure provides compositions of comprising differentiated SC-IC cells as described, any one of the in vitro cell populations, pharmaceutical compositions comprising these or devices comprising these for use in the treatment of diabetes mellitus, one or more complications related to diabetes or a pre-diabetic condition. In some embodiments, the use is in the treatment of Type 1 Diabetes.

[0137] In some aspects, the disclosure provides use of compositions of comprising differentiated SC-IC cells as described, any one of the in vitro cell populations, pharmaceutical compositions comprising these or devices comprising these in the manufacture of amedicament for treating diabetes, one or more complications related to diabetes mellitus or a pre-diabetic condition. In some embodiments, the medicament is for treating of Type 1 Diabetes.

[0138] In some aspects, the disclosure provides methods of culturing stem cells to obtain a population comprising differentiated cells, the method comprising at least one step of culturing a population cells at a pH that is not higher than pH7.8, a pH in the range of: pH7.2 to pH7.8, pH7.3 to pH 7.8, pH7.4 to pH7.8, pH 7.2+ / - 0.2 (pH7.0 to pH7.4), pH 7.4+ / - 0.2 (pH7.2 to pH7.6), pH7.6+ / - 0.2 (pH7.4 to pH7.8) or pH7.8+ / - 0.2 (pH7.6 to pH8.0), wherein the at least one step is conducted in a bioreactor. In some embodiments, the at least one step of culturing a population cells at a pH, which pH is not higher than pH7.8, a pH in the range of: pH7.2 to pH7.8, pH7.3 to pH 7.8, pH7.4 to pH7.8, pH 7.2+ / - 0.2 (pH7.0 to pH7.4), pH 7.4+ / - 0.2 (pH7.2 to pH7.6), pH7.6+ / - 0.2 (pH7.4 to pH7.8) or pH7.8+ / - 0.2 (pH7.6 to pH8.0), is conducted in at least one differentiation medium comprising a defined medium comprising 0mM to less than 2.5mM glucose, 0mM to less than 2mM glucose, 0mM to 25mM glucose, 0mM to 50mM glucose, or any glucose concentration in these ranges.

[0139] In some aspects, provided are methods of deriving a population of differentiated cells derived from stem cells, the method comprising at least one step of culturing a population cells at a pH that is not higher than pH7.8, a pH in the range of: pH7.2 to pH7.8, pH7.3 to pH 7.8, pH7.4 to pH7.8, pH 7.2+ / - 0.2 (pH7.0 to pH7.4), pH 7.4+ / - 0.2 (pH7.2 to pH7.6), pH7.6+ / - 0.2 (pH7.4 to pH7.8) or pH7.8+ / - 0.2 (pH7.6 to pH8.0) in at least one differentiation medium. In some embodiments, the differentiation medium comprises a defined medium comprising 0 mM to less than 2.5mM glucose, 0 mM to less than 2mM glucose, 0mM to 25mM glucose, 0mM to 50mM glucose, or any glucose concentration in these ranges, and wherein the method is conducted in a bioreactor.

[0140] In some embodiments, the culturing step is in a differentiation protocol that differentiates stem cells such as, for example and without limitation, iPSCs to mature SC-ICs. In some embodiments, the culturing is of a population of cells, e.g. without limitation PDX1+ cells. In some embodiments, the population of differentiated cells is mature SC-ICs comprising CPEP+ / GCG- cells and GCG+cells.

[0141] In one aspect, the disclosure provides methods of deriving a cell population comprising mature SC-ICs, the method comprising a step of:(a) culturing a first precursor cell population comprising PDX1+ cells in one or more differentiating mediums to obtain the mature SC-IC population, wherein the first precursor cell population is selected from the group consisting of a FE population, a PP cell population, a PEP cell population and a precursor SC-IC cell population, wherein at least one of the differentiating mediums is a defined medium comprising G9a inhibitor.

[0142] In some embodiments, the G9a inhibitor is UNC0321 and optionally wherein at least one of the differentiating mediums is a defined medium comprising glucose at less than about 2.5 mM or at less than about 2 mM.

[0143] In some embodiments, the culturing of the first precursor cell population comprising PDX1+ cells is at pH in the range of: pH7.2 to pH7.8, pH7.3 to pH7.8, pH7.4 to pH7.6, pH7.4 to pH7.8, pH 7.2+ / - 0.2 (pH7.0 to pH7.4), pH 7.4+ / - 0.2 (pH7.2 to pH7.6), pH7.6+ / - 0.2 (pH7.4 to pH7.8) or pH7.8+ / - 0.2 (pH7.6 to pH8.0).

[0144] In some embodiments, the methods comprise additional culturing steps in one or more differentiating mediums as described herein, so as to derive the population of differentiated cells is mature SC-ICs comprising CPEP+ / GCG- cells and GCG+cells. In certain embodiments, the culturing steps of the methods described herein are conducted in a bioreactor. In certain embodiments, the bioreactor is a large-scale bioreactor such as, for example and without limitation, having a volume over 500mL. Alternatively, the bioreactor is a large-scale bioreactor such as, for example and without limitation, having a volume over 1L. Alternatively, the bioreactor is a large-scale bioreactor such as, for example and without limitation, having a volume over 3L. Alternatively, the bioreactor is a large scale bioreactor such as, for example and without limitation, having a volume over 5L. Alternatively, the bioreactor is a large-scale bioreactor such as, for example and without limitation, having a volume over 10L. Alternatively, the bioreactor is a large-scale bioreactor such as, for example and without limitation, having a volume over 25L. Alternatively, the bioreactor is a large-scale bioreactor such as, for example and without limitation, having a volume over 50L.

[0145] In some aspects, the disclosure provides an in vitro cell population such as, for example and without limitation, comprising mature SC-ICs, wherein the population is produced by any one of the methods as described.

[0146] In some aspects, provided is an in vitro cell population comprising cells wherein: (i) at least about 67% of the cells in population are CPEP+ / GCG-; (ii) no more than about 22% of the cells in the cell population are GCG+cells;(iii) at least about 60% of the cells in the cell population are NKX6.1+ / CPEP+cells; (iv) about 68% of the cells in the cell population are INS+ / SLC- cells; (v) less than about 7% of the cells in the cell population are INS- / SLC+cells; (vi) at least about 99.8% of the cells in the cell population are CHGA+cells; (vii) less than about 2% of the cells in the cell population are Ki67+cells; (viii) an insulin content of 150nu / cell to at least about 200nU / cell; or (ix) lactate production by the cell population of less than 0.5 mM in 48hrs, wherein the population comprises at least two of the above characteristics, and wherein the cell population is produced by any one of the methods as described. BRIEF DESCRIPTION OF THE DRAWINGS

[0147] The advantages, effects, features, and objects other than those set forth above will become more readily apparent when consideration is given to the detailed description below. Such detailed description refers to the following drawing(s), where:

[0148] FIG.1A shows an overview of an exemplary iPSC to SC-IC differentiation protocol, and FIG.1B shows a more detailed schematic of the exemplary protocol shown in FIG.1A.

[0149] FIGS. 2A-2D show graphs illustrating the effects of reaggregation and glucose deprivation on SC-IC composition and potency relative to other contemporaneous treatments.

[0150] FIGS. 3A-3C shows graphs illustrating correlations (or lack thereof) between INS secretion and SC-IC composition or viability, and effects of reaggregation or glucose deprivation on these parameters relative to other contemporaneous treatments.

[0151] FIG. 4 shows the effects on INS content (FIG. 4A) and INS secretion (FIG. 4B) of glucose deprivation and / or reaggregation at various times in stages 5 and 6, with synergistic benefits observed when glucose deprivation and reaggregation were performed sequentially at the end of Stage 5 and beginning of Stage 6 (D14-D15, Zero Glucose, D16 Reagg).

[0152] FIG. 5A shows the effects of the D14-D15, Zero Glucose, D16 Reagg treatment on LDHA expression in the mature SC-IC composition, and FIG.5B shows the effects of the same on lactate production by the differentiating cells during Stages 6 and 7.

[0153] FIG.6 shows the effects on INS content of including UNC0321 during all of Stage 5 (D10-16) and including MDL-800 or butyrate starting near the end of Stage 5 and continuing through Stage 6 (D14-20) relative to matched controls.

[0154] FIG.7 shows the effects of varying pyruvate concentrations during Stages 6 and 7 on INS secretion by the resulting SC-IC composition.

[0155] FIGS. 8A-8D shows the effects of HPLM vs MCDB basal media during Stage 7 on SC-IC composition and potency.

[0156] FIG. 9 shows in vivo glucose lowering effects of encapsulated SC-ICs derived from the methods described herein. DETAILED DESCRIPTION

[0157] Overview

[0158] Diabetes is a family of disorders characterized by chronic hyperglycemia and the development of long-term complications. This family of disorders includes T1D, T2D, gestational diabetes, and other types of diabetes. Persons with diabetes, especially those diagnosed with T1D, could potentially be cured through transplantation of an exogenous supply of beta cells. This approach, however, is limited because of the scarcity and quality of donor islets. As such, the generation from stem cells of an unlimited supply of functional islet-like cells that can produce and secrete INS can make this therapeutic approach available to a greater number of individuals.

[0159] Estimates put the number of functional cells required for treating diabetes in the order of 109per individual. As such, differentiation strategies are needed for generating sufficient beta-like cells for treating diabetes as an alternative to islet transplantation with donor islets.

[0160] The description below thus provides embodiments of differentiation methods and processes for generating a population of insulin producing cells, called SC-ICs, at least about 67% of the cells in population are CPEP+ / GCG-, e.g.67% to 80% of the cells in population are CPEP+ / GCG-, and at least about 99% of the cells in the population are CHGA+, e.g. 99% to 99.99% of the cells in the population are CHGA+, that can be used for cell therapy to treat, for example, diabetes mellitus (e.g., T1D). In one embodiment, the methods described herein are based on the finding that differentiating PPs is optimal in the presence of two tankyrase 1 / 2 inhibitors, at least one of which is Wiki4. In one embodiment, the methods described herein are based upon a finding that culturing cells in a differentiation medium with a concentration of glucose below 2.5mM, e.g. without limitation about 0mM glucose, and optionally adding galactose (or another alternative nutrient) while differentiating PP cells to PEPs, or PEPs to immature SC-ICs decreases (i.e., reduces or eliminates) EC-like cells (i.e., SLC18A1+cells).In another embodiment, the methods comprise at least one step of culturing cells, e.g. without limitation in a bioreactor culturing PP cells to PEPs, or PEPs to immature SC-ICs, at a pH in the range of: pH7.2 to pH7.8, pH7.3 to pH7.8, pH7.4 to pH7.6, pH7.4 to pH7.8, pH 7.2+ / - 0.2 (pH7.0 to pH7.4), pH 7.4+ / - 0.2 (pH7.2 to pH7.6), pH7.6+ / - 0.2 (pH7.4 to pH7.8) or pH7.8+ / - 0.2 (pH7.6 to pH8.0). Without being bound by theory, the pH and glucose concentration effectively selects against and thereby decreases (i.e., reduces or eliminates) EC-like cells (i.e., SLC18A1+cells) in the differentiated population. In another embodiment, the methods comprise at least one step of culturing cells, e.g. without limitation in a bioreactor culturing PP cells to PEPs, or PEPs to immature SC-ICs, in a differentiation medium with a concentration of glucose below 2.5mM and at a pH in the range of: pH7.2 to pH7.8, pH7.3 to pH7.8, pH7.4 to pH7.6, pH7.4 to pH7.8, pH 7.2+ / - 0.2 (pH7.0 to pH7.4), pH 7.4+ / - 0.2 (pH7.2 to pH7.6), pH7.6+ / - 0.2 (pH7.4 to pH7.8) or pH7.8+ / - 0.2 (pH7.6 to pH8.0), which combination effectively selects against and thereby decreases (i.e., reduces or eliminates) EC-like cells (i.e., SLC18A1+cells) in the differentiated population. Additionally, the methods described herein are based upon a finding that dissociating a population comprising immature SC-ICs into single cells, wherein a substantial portion of the population is dissociated into singe cells, separating the dissociated immature SC-ICs single cells from the undissociated cells, and then reaggregating the dissociated immature SC-ICs single cells prior to differentiating the cells to mature SC-ICs produces an enriched population that exhibits a dynamic INS secretion response. Moreover, the methods described herein are based upon a finding that adding an epigenetic modifier, e.g. a G9a inhibitor including without limitation a G9a inhibitor such as UNC0321, when obtaining PEPs and / or immature SC-ICs improves cell composition and potency of mature SC-ICs.

[0161] Abbreviations and Definitions

[0162] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art to which the disclosure pertains. Although any methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the SC-ICs, pharmaceutical compositions including the same and methods of making and using SC-ICs, the preferred methods and materials are described herein.

[0163] Additionally, reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one element is present, unless the context clearly requires that there be one and only one element. The indefinite article “a” or “an” thus usually means “at least one.”

[0164] Moreover, use of “including,” as well as other forms, such as “include,” “includes” and “included” is not limiting. Similarly, use of “comprising,” as well as other forms, such as “comprise”, “comprises” and “comprised” is not limiting. The presence of including or comprising (or any of their other forms) in the description or claims herein does not exclude additional, unrecited elements or method steps.

[0165] The terms “and / or” and “any combination thereof’ and their grammatical equivalents can be used interchangeably herein to convey that any combination is specifically contemplated. Solely for illustrative purposes, the following phrases “A, B and / or C” or “A, B, C or any combination thereof” can mean “A individually; B individually; C individually; A and B; B and C; A and C; and A, B and C.”

[0166] Certain abbreviations used herein are as follows:

[0167] “ADOHCYASE” refers to S-adenosylhomocysteine hydrolase; “ADRA2A” refers to adrenoceptor alpha 2A; “ALC” refers to O-acetyl-L-carnitine hydrochloride; “ALK5iII” refers to ALK5 inhibitor II; “amu” refers to atomic mass unit(s); “APP” refers to amyloid precursor protein; “ARX” refers to aristaless-related homeobox; “ATRA” refers to all-trans retinoic acid; “ATP” refers to adenosine triphosphate; “AZA” refers to azacytidine; “BMAL1” and “ARNTL” refer to aryl hydrocarbon receptor nuclear translocator-like protein 1; “BMP” refers to bone morphogenetic protein; “BR” refers to bioreactor; “BSA” refers to bovine serum albumin; “CDLC” refers to chemically defined lipid concentrate; “CDLM” refers to chemically defined lipid mixture, “CHGA” refers to chromogranin A; “CPEP” refers to C-peptide; “CXCL14” refers to C-X-C motif chemokine ligand 14; “CXCR4” refers to C-X-C chemokine receptor type 4; “D” refers to day; “DACDM” refers to N,N′-diacetyl-L-cystine dimethylester; “DE” refers to definitive endoderm or definitive endodermal cell(s); “DiNAC” refers to N,N′- diacetyl-L-cystine; “dL” refers to decilitier(s); “DMEM” refers to Dulbecco’s Modified Eagle Medium; “DNMT” refers to DNA methyltransferase; “DZNep” refers to 3-deazaneplanocin A; “E8 medium” refers to Essential 8 medium; “EC” refers to ectoderm or ectodermal cell; “ECC” refers to enterochromaffin cell; “ECLC” refers to enterochromaffin-like cell; “EGF” refers to epidermal growth factor; “EGSC” refers to embryonic germ stem cell; “EHMT1” refers toeuchromatic histone lysine methyltransferase 1; “EHMT2” refers to euchromatic histone lysine methyltransferase 1; “EN” refers to endoderm or endodermal cell; “EOMES” refers to eomesodermin; “FAF-BSA” refers to fatty acid-free bovine serum albumin; “FAF-HSA” refers to fatty acid-free human serum albumin; “FE” refers to foregut endoderm; “FEV” refers to FEV transcription factor; “FGF” refers to fibroblast growth factor; “FOXA2” refers to forkhead box A2; “G6PC2” refers to glucose-6-phosphatase catalytic subunit 2; “Gal” refers to galactose; “GATA4” refers to GATA Binding Protein 4; “GATA6” refers to GATA Binding Protein 6; “GCG” refers to glucagon; “GDF8” refers to growth differentiating factor 8; “GLP- 1” refers to glucagon like peptide 1; “Glu” refers to glucose; “GRL” refers to ghrelin; “GSC” refers to goosecoid; “GSI” refers to gamma (γ) secretase inhibitor; “GSIS” refers to glucose- stimulated insulin secretion; “GSK” refers to glycogen synthase kinase-3; “HbA1c” refers to hemoglobin A1c; “HB9” refers to homeobox 9; “HDAC” refers to histone deacetylase; “HHEX” refers to hematopoietically expressed homeobox; “hiPSC” refers to human induced pluripotent stem cell; “HMT” refers to histone methyltransferase; “HNFlβ” refers to hepatocyte nuclear factor 1-beta; “HNF3-β” refers to hepatocyte nuclear factor 3-beta; “HNF4α” refers to hepatocyte nuclear factor 4 alpha; “HNF6” refers to hepatocyte nuclear factor 6; “hr” refers to hour(s); “HPLM” refers to human plasma-like medium; “HSA” refers to human serum albumin; “ILC” refers to isovaleryl L-carnitine; “INS” refers to insulin; “iPSC” refers to induced pluripotent stem cell; “ISL1” refers to islet-1; “ITS-X” refers to insulin-transferrin- selenium-ethanolamine supplement; “ILV” refers to indolactam; “K+” refers to potassium ion; “KGF” refers to keratinocyte growth factor; “KOSR” refers to KnockOut serum replacement; “KRB” refers to Kreb’s Ringer Buffer; “L” refers to liter(s); “LDHA” refers to lactate dehydrogenase A; “LDHB” refers to lactate dehydrogenase B; “LMX1A” LIM homeobox transcription factor 1 alpha; “MAFA” refers to MAF bZIP transcription factor A; “MAFB” refers to MAF bZIP transcription factor B; “ME” refers to mesendoderm or mesendodermal cell; “MEM” refers to minimum essential medium; “mg” refers to milligram(s); “min” refers to minute or minutes; “MIXL1” refers to mix1 homeobox-like protein 1; “mmol” refers to millimole(s); “Mpy” refers to methylpyruvate; “MSC” refers to multipotent stem cell; “NAC” refers to N-acetyl cysteine; “NAM” refers to nicotinamide; “NaPyr” refers to sodium pyruvate; “NEAA” refers to nonessential amino acids; “NeruroD1” refers to neurogenic differentiation 1; “NGN3” refers to neurogenin-3; “NKX2.2” refers to NK2 homeobox 2; “NKX6.l” refers to NK6 homeobox 1; “nM” refers to nanomolar; “NODAL” refers to nodal growth differentiationfactor; “NPEC” refers to non-pancreatic endocrine cell(s); “NPTX2” refers to neuronal pentraxin 2; “NR1D1” refers to nuclear receptor subfamily 1 group D member 1; “OCT4” refers to Octamer-binding transcription factor 4; “OTX2” refers to orthodenticle homeobox 2; “PALC” refers to pancreatic alpha-like cell(s); “PAX4” refers to paired box gene 4; “PAX6” refers to paired box gene 6; “PARP-1” refers to poly(ADP-ribose)polymerase 1; “PBLC” refers to pancreatic beta-like cell(s); “PC1 / 3” refers to prohormone convertase; “PDX1” refers to pancreatic and duodenal homeobox 1; “PEC” refers to pancreatic endocrine cell(s); “PEP” refers to pancreatic endocrine precursor(s); “PGT” refers to primitive gut tube; “PKC” refers to protein kinase c; “PLC” refers to propionyl-L-carnitine; “PP” refers to pancreatic progenitor(s) (e.g., PP1, PP2); “PPP” refers to pancreatic polypeptide; “PROX1” refers to prospero homeobox protein 1; “PSC” refers to pluripotent stem cell; “PTF1α” refers to pancreas transcription factor 1-alpha; “ROCK” refers to Rho kinase; “SAM” refers to sterile alpha motif; “SC-IC” refers to stem-cell derived islet-like cell; “SHH” refers to Sonic Hedgehog; “SIRT1” refers to sirtuin 1; “SIRT6” refers to sirtuin 6; “SIX2” refers to SIX homeobox 2; “SIX3” refers to SIX homeobox 3; “SLC18A1” refers to solute carrier family 18 member 1; “SOX2” refers to sex determining region Y (SRY)-Box Transcription Factor 2; “SOX9” refers to SRY-Box Transcription Factor 9; “SOX17” refers to SRY-Box Transcription Factor 17; “SRT” refers to serotonin; “SST” refers to somatostatin; “T1D” refers to Type 1 diabetes; “T2D” refers to Type 2 diabetes; “T3” refers to triiodothyronine; “TAC1” refers to tachykinin precursor 1; “TBXT” refers to T-box transcription factor; “TGF-β” refers to transforming growth factor beta; “TGF-β RI” refers to transforming growth factor beta receptor type I; “TNKS1” refers to tankyrase 1; “TNKS2” refers to tankyrase 2; “TSC” refers to totipotent stem cell; “U” refers to unit(s); “µM” refers to micromoles(s) or micromolar; “µm” refers to micron(s) or micrometer(s); “UCN3” refers to urocortin-3; “UFH” refers to unfractionated heparin; “VTN-N” refers to vitronectin-N; “Wnt3a” refers to wingless-type MMTV integration site family, member 3A; “XF” refers to xeno-free; “ZnSO4” refers to zinc sulfate; and “ZnT8” refers to zinc transporter 8.

[0168] Certain definitions used herein are defined as follows:

[0169] As used herein, “about” means within a statistically meaningful range of a value or values such as, for example, a stated concentration, length, molecular weight, pH, pressure, sequence similarity, time frame, temperature, volume, etc. Such a value or range can be within 20%, within 15%, within 10%, or more typically within 5% of a given value or range.Alternatively, and with respect to biological systems or processes, the term “about” can mean within an order of magnitude such as, for example, within five-fold or more typically within two-fold of a given value. The allowable variation encompassed by “about” will depend upon the system under study, and can be readily appreciated by one of skill in the art. In some instances of when the term “about X%” refers to the percent of a specified cell in a cell population, the term “about X%” means within a range of 15% below X to 15% above X, or 10% below X to 10% above X or 5% below X to 5% above X.

[0170] As used herein, “Activin A” means a homodimer of two beta A chains from the Activin family, which are nonglycosylated homodimers or heterodimers of various beta subunits (beta A, beta B, beta C and beta E in mammals). The 14 kDa mature human beta A chain shares 100% amino acid sequence identity with bovine, feline, mouse, porcine, and rat beta A. Unless otherwise specified, the Activin A protein in a differentiation medium described herein is recombinant human Activin A, which is commercially available from, for example, R&D Systems (Minneapolis, MN USA).

[0171] As used herein, “albumin” means a mammalian albumin protein, which has been either isolated from the serum of the mammalian species or has been recombinantly produced. In some instances, the albumin used in the methods described herein is bovine serum albumin or human serum albumin. In some instances, the albumin is a fatty-acid free (FAF) albumin, which means that all or substantially all of the fatty acids that bind to the albumin when present in serum have been removed. FAF-BSA and FAF-HSA are commercially available from, for example, Millipore Sigma (Burlington, MA USA). Other albumins suitable for use in the differentiation methods described herein include a recombinant mammalian (e.g., bovine or human) albumin, which can be part of an albumin composition that includes (a) one or more phospholipids (e.g., sphingoine-1-phosphate and / or lysophsphatidic acid) or (b) a mixture of fatty acids (e.g., as described in US Patent No. 11,767,504; or a deAlbumin™ composition commercially available from Albcura Corp (New Taipai City, Taiwan)). Another albumin suitable for use herein is a lipid-rich albumin, which can be a recombinant albumin in a composition that includes the same lipid composition present in the commercially available AlbuMax™, which are shown in Table 2 of Garcia-Gonzalo & Belmonte (2008) PLoS One 3:e1384.

[0172] As used herein, ALK5 inhibitor II is the small molecule compound, 2-(3-(6- methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine (C17H13N5, CAS No. 446859-33-2)).ALK inhibitor II, which is also known as RepSox, E-616452 and SJN 2511 and is a selective and ATP-competitive inhibitor of TGF-β type I receptor ALK5 with an IC50of 4 nM. ALK5 inhibitor II is commercially available from, for example, ReproCell (Beltsville, MD USA).

[0173] As used herein, “ALK5 inhibitor compound” and “ALK5i compound” mean a compound (e.g., a small molecule compound) that is an ATP competitive inhibitor of the TGF- β type I receptor ALK5. In some instances, the ALK5i compound is 3-(pyridineyridin-2-yl)-4- (4-quinonyl)]-1H-pyrazole; (C17H12N4, CAS No. 396129-53-6), which is also known as LY364947 and is an ATP competitive inhibitor of with an IC50of 59 nM in a cell-free assay and exhibits 7-fold selectivity over TGFβR-II. LY364947 is commercially available from, for example, Selleck Chemicals (Houston, TX USA). In some instances, the ALK5i compound is ALK inhibitor II as defined herein.

[0174] As used herein, “alpha-like cell,” “α-like cell,” “pancreatic alpha-like cell,” “pancreatic α-like cell” and “PALC” may be used interchangeably to mean a pancreatic endocrine cell that at least expresses and secretes glucagon (GCG; i.e., GCG+) or otherwise has characteristics such that the cell is functionally equivalent to a native, human alpha cell. A PALC is sometimes referred to herein as being immature or mature based on, for example, its functional characteristics. In some instances, immature PALCs (or pre-alpha cells) proliferate, express prohormone convertase 1 / 3 (PC1 / 3), produce GLP-1 and express the GLP-1 receptor. In some instances, immature PALCs, or pre-alpha cells, are polyhormonal (i.e., co-express INS and GCG). In some instances, a PALC expresses both CPEP and GCG (i.e., CPEP+ / GCG+cell). In some instances, a mature PALC is a monohormonal GCG-expressing cell. In some instances, a mature PALC expresses both CPEP and GCG but does not secrete INS (i.e., CPEP+ / GCG+ / INS- cell).

[0175] As used herein, “alternative nutrient” means a non-glucose energy source such as an amino acid, lipid and / or non-glucose carbohydrate that allows the cell to produce ATP through mitochondrial respiration but does not allow the cell to rely solely on anaerobic glycolysis for survival. Examples of alternative nutrients include, but are not limited to, galactose, methyl pyruvate, methyl succinate and pyruvate.

[0176] As used herein “all-trans retinoic acid” and “ATRA” means an oxidized form of Vitamin A that acts by binding to heterodimers of the retinoic acid receptor (RAR) and the retinoid × receptor (RXR), which then bind to retinoic acid response elements (RAREs) in the regulatory regions activating gene transcription (Marshall et al. (1996) FASEB J 10:969-978).ATRA, which has the chemical name and structure shown in Table 15 below, is commercially available from, for example, ReproCell USA, Inc. (Beltsville, MD USA).

[0177] As used herein, “betacellulin” means a member of the EGF family and signals through the EGF receptor (EGFR) and the receptor tyrosine-protein kinase, ERBB4. A recombinant human betacullulin protein is commercially available from, for example, Stemcell Technologies (Vancouver, BC Canada) and has an amino acid sequence of SEQ ID NO:1.

[0178] As used herein, “B27 supplement” means a defined mixture of antioxidant enzymes, proteins, Vitamins and fatty acids that are combined in optimized ratios to support neuronal survival in culture and is based on the serum-free neuronal culture supplement formula developed by Dr. Gregory Brewer and colleagues. See, e.g., Brewer & Cotman (1989) Brain Res.494:65-74; and Brewer et al. (1993) J. Neuroscience Res.35:567-576. In some instances, the B27 supplement is a 50x concentrated solution. The composition of an exemplary B27 (50x) supplement is shown in Table 28 in the Examples below. Another exemplary B27 (50x) supplement is the commercially available serum-free B-27TMSupplement (50X) from, for example, ThermoFisher Scientific (Waltham, MA USA).

[0179] As used herein, “beta-like cell,” “β-like cell,” “pancreatic beta-like cell” and PBLC may be used interchangeably and mean a pancreatic endocrine cell that at least makes and secretes INS (i.e., INS+), but does not express GCG (i.e., GCG-), somatostatin, ghrelin or pancreatic polypeptide. A PBLC is sometimes referred to herein as being immature or mature based on, for example, whether it exhibits functional characteristics that are substantially similar to functional characteristics exhibited by human endogenous immature or to functional characteristics exhibited by human endogenous mature beta cells. The differences in the functional characteristics of immature and mature beta cells are well-known in the art (see, e.g., Barsby & Otonkoski (2022) Diabetologia 65:917-930; Sun et al. (2021) World J. Stem Cells 13:193-207; Intl. Patent Application Publication No. WO 2020 / 247954 and US Patent Application Publication No.2014 / 0287944). Both immature and mature PBLCs express CPEP but do not express GCG (i.e., CPEP+ / GCG-). In some instances, a PBLC expresses CPEP and NKX6.1 (i.e., CPEP+ / NKX6.1+). In some instances, a PBLC is INS+ / CPEP+ / GCG- and / or INS+ / CPEP+ / NKX6.1+.

[0180] As used herein, “immature PBLC” means a pancreatic endocrine cell that produces INS, but lacks a GSIS response that is characteristic of an endogenous human beta cell (e.g., a biphasic GSIS). In some instances, immature PBLCs expressing markers characteristic ofhuman beta cells can be characterized by their expression of INS and at least one of the following transcription factors: PDX1, NKX2.2, NKX6.1, NeuroD1, GLIS3, ISL1, HNF3β, HB9, MAFA, MAFB, NEUROG3, RFX3 and PAX6. In some instances, an immature PBLC expresses INS and NKX6.1 and does not substantially express NGN3. In some instances, an immature PBLC expresses INS but does not express UCN3 protein, or another mature beta cell marker as described below.

[0181] As used herein, “intermediate [x / y] cell population” means a heterogenous cell population that exists at a referenced time point during a method of differentiating cells of a less specialized x cell type (e.g., PGT cells, PP cells, PEP cells, immature PBLCs) to cells of a more specialized y cell type (e.g., FE cells, PEP cells, SC-ICs, mature PBLCs, respectively). An intermediate [x / y] cell population is generally more phenotypically heterogenous than the starting x cell population or the ending y cell population and can include cells that exhibit only x cell markers, cells that exhibit only y cell markers, and cells that exhibit both x cell markers and y cell markers. The methods described herein can generate various intermediate [x / y] cell populations, including an intermediate PGT / FE cell population, an intermediate PP / PEP cell population, an intermediate PEP / SC-IC population, and an intermediate precursor / mature SC- IC population.

[0182] As used herein, “intermediate PGT / FE cell population” means a population of cells that are at various points of differentiation between PGT cells and FE cells, and may include, for example, various percentages of: (i) PGT cells (e.g., exhibiting only PGT markers, (ii) FE cells (e.g., exhibiting only FE cell markers, and (iii) cells with intermediate phenotypes, which can be phenotypically more similar to PGT cells than to FE cells (e.g., exhibit more PGT cell markers than FE cell markers) and / or phenotypically more similar to FE cells than PGT cells (e.g., exhibit more FE cell markers than PGT cell markers).

[0183] As used herein, “intermediate PP / PEP cell population” means a population of cells that exists at a referenced time point during a method of differentiating a PP cell population to a PEP cell population, and can include various percentages of PP cells (e.g., express only PP cell markers), PEP cells (e.g., exhibit only PEP cell markers) and cells with intermediate phenotypes (e.g., exhibit both PP cell markers and PEP cell markers).

[0184] As used herein, “intermediate PEP / SC-IC population” means a population of cells that exists at a referenced time point during a method of differentiating a PEP cell population to a precursor SC-IC population and may include various percentages of: PEP cells (e.g.,exhibit only PEP cell markers), cells that exhibit one or more markers of different SC-IC types that have varying degrees of maturity (e.g., cells that exhibit marker(s) for immature / mature PALCs and / or immature / mature PBLCs), and cells with intermediate phenotypes (e.g., exhibit both PEP cell marker(s) and markers for PLACs and / or PBLCs).

[0185] As used herein, “intermediate precursor / mature SC-IC population” means a population of cells that exists at a referenced time point during a method of differentiating a precursor SC-IC population (e.g., includes immature and / or maturing beta-like cells and alpha- like cells) to a mature SC-IC population (e.g., includes higher percentages of mature SC-ICs such as mature beta-like cells and mature alpha-like cells than in the precursor SC-IC population).

[0186] As used herein, a “mature PBLC” means a pancreatic endocrine cell that produces INS, expresses at least one marker indicative of an endogenous mature beta cell (e.g., UCN3 and / or MAFA) and displays a GSIS response to a glucose challenge that is substantially similar to the biphasic GSIS response exhibited by an endogenous mature pancreatic beta cell, or otherwise has characteristics such that the PBLC is functionally equivalent to an endogenous mature human beta cell. In some instances, mature PBLCs exhibit at least one of the following characteristics of biphasic GSIS: (i) coupling of mitochondrial respiration / activity with INS secretion; (ii) rapid INS secretion response to heightened demand (here defined as high glucose concentration); (iii) ability to rapidly turn off INS secretion after demand has subsided; (iv) ability for multiple rounds of “on-off” switching of INS secretion; (v) ability to secrete the correct amount of INS as dictated by demand; and (vi) ability to respond to multiple INS secretagogues (for example, Exendin-4, or amino acids L-glutamine and L-arginine). In some instances, a mature PBLC may be identified as having one or more of the following markers: single hormonal INS, NKX6.1, UCN3, GLUT2, SLC2A1, SIX2, SIX3, BMAL and PDX1, and MAFA expression at a higher level than a less mature pancreatic endocrine cell, in particular an immature PBLC. In some instances, a mature PBLC may be identified as having one or more of these markers in addition to the absence of one or more of MCT1 (SLC16A1), LDHA, and HK1 expression.

[0187] As used herein, “BMP inhibitor” means a compound such as, for example, a small molecule compound, that inhibits the BMP type-I receptor activin receptor-like kinase 2, also known as the ALK2 receptor. Exemplary BMP inhibitors include, but are not limited to, DMH- 1, LDN-193189 and KO2288 (CAS No.1431985-92-0).

[0188] As used herein, “carnitine compound” means L-carnitine and derivatives thereof, such as O-acetyl-L-carnitine hydrochloride (ALC), propionyl-L-carnitine (PLC), and isovaleryl L-carnitine (ILC). L-carnitine (also known as Vitamin BT) is involved in metabolism and transports long-chain fatty acids from the cytosol into mitochondria to be oxidized for free energy production, and also participates in removing products of metabolism from cells. The chemical names and structures for L-Carnitine and ALC are shown in Table 15 below, and each compound is commercially available from, for example, Millipore Sigma.

[0189] As used herein, "cell marker" means a marker (i.e., a peptide, a protein) expressed in, or produced by, a cell that is specific to a particular cell type or particular class of cells present in a population of cells (e.g., CPEP+is a marker for pancreatic endocrine cells and Ki-67+is a marker for proliferating cells). Various cell types described herein may be characterized as being positive or negative for one or more cell markers.

[0190] As used herein, “chemically defined lipid mixture” and “CDLM” mean a liquid composition that includes two or more of arachidonic acid, cholesterol, DL-alpha-tocopherol acetate, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, palmitoleic acid and stearic acid in defined concentrations. In some instances, a CDLM includes three, four, five, six, seven, eight, nine or all ten of these lipids. In some instances, a CDLM can include one of the lipid combinations set forth in Table 6 of US Patent Application Publication No. 2013 / 0273010. In some instances, a CDLM can include one or more emulsifiers such as, for example, Pluronic F68® and Tween 80®. An exemplary CDLM composition is shown in Table 27 herein below. In some instances, a CDLM used in the differentiation methods described herein includes components 1 to 10 of Table 27 herein below. In some instances, the CDLM does not include palmitoleic acid. In some instances, the CDLM includes components 1 to 12 or components 1 to 13 of Table 27. CDLM compositions are commercially available, e.g., Sigma-Aldrich Lipid Mixture 1 and GibcoTMChemically Defined Lipid Concentrate.

[0191] As used herein “CHIR98014” means a small molecule compound having the chemical name and structure shown in Table 15 herein below. CHIR98014, which inhibits GSK-3α and GSK-3β with IC50 values of 0.65 nM and 0.58 nM, respectively, is commercially available from, for example, APExBIO (Houston, TX USA).

[0192] As used herein, “CHIR99021” means a small molecule compound known as laduviglusib (chemical name and structure shown in Table 15) and pharmaceutically acceptable salts thereof. CHIR99021, which acts as an inhibitor of GSK-3 (GSK3β, IC507nM) and as aWnt / β-catenin activator, is commercially available from, for example, Selleck Chemicals (Houston, TX USA).

[0193] As used herein, “Chroman 1” means a small molecule compound having the chemical name and structure shown in Table 15 and pharmaceutically acceptable salts thereof. Chroman 1, which is a ROCK inhibitor that is more potent against ROCK2 (IC50 = 1 pM) than ROCK1 (IC50= 52 pM), is commercially available from, for example, Selleck Chemicals.

[0194] As used herein, “consists essentially of”, and variations such as “consist essentially of” or “consisting essentially of” as used throughout the specification and claims, indicate the inclusion of any recited elements or group of elements, and the optional inclusion of other elements, of similar or different nature than the recited elements, that do not materially change the basic or novel properties of the specified molecule, cell population, composition, device, or method. As a non-limiting example, a cell population which consists essentially of a specified cell type (e.g., CPEP+cells) may include a minor amount of other cell types (e.g., CPEP- cells), but the presence of such unrecited cells do not materially affect the relevant biological activity of the cell population. In another non-limiting example, a differentiating medium that consists essentially of a recited list of components may have other components that do not materially change the cell culturing or differentiating properties of the medium.

[0195] As used herein, “culturing” means an in vitro process unless otherwise specified.

[0196] As used herein, “defined medium” means an aqueous cell growth medium in which the amounts or quantities of the chemical components or ingredients (i.e., formulation) are known. A defined medium minimally includes at least one nutrient and at least one electrolyte and can also include one or more other components typically present in cell culture mediums, (e.g., a buffer, albumin or albumin substitute, galactose, INS, transferrin or transferrin substitute, one or more amino acids, one or more antioxidants, one or more lipids, one or more Vitamins, one or more trace elements such as selenium, and the like). All references to glucose and galactose in a defined medium herein refer to the D form (e.g., D-glucose and D-galactose) unless otherwise specified. In addition, the use of glucose as a nutrient in any defined medium described herein may be partially or completely replaced with fructose (e.g., the medium may include glucose, fructose or glucose and fructose). All references to glutamine in a defined medium herein refer to the L form (L-glutamine) unless otherwise specified. Examples of defined media include, but are not limited to, DMEM (commercially available from Gibco; the components of DMEM are described in Dulbecco & Freeman (1959) Virol.8:396-397), MCDB131 (no glutamine; commercially available from ThermoFisher Scientific; the components for MCDB 131 are described in Knedler & Ham (1987) In Vitro Cell. Dev. Biol. Anim. 23:481- 491) and HPLM (commercially available from ThermoFisher Scientific; the components for HPLM are described in Cantor et al. (2017) Cell 169:258-272; see also, Intl. Patent Application Publication No. WO 2018 / 089928).

[0197] As used herein, “definitive endoderm cell population” or “DE cell population” means a cell population obtained by: (i) differentiating pluripotent stem cells (e.g., iPSCs) by any method known in the art or as described herein or (ii) differentiating a precursor cell population derived from PSCs (e.g., a ME cell population). Greater than 50% of the cells in a DE cell population are DE cells (i.e., do not express PDX1 (PDX1-) and express at least one of the following DE cell markers: brachyury, cerberus, C-Kit, CD99, CXCR4, FOXA2, GATA4, GATA6, GSC, MIXL1, SOX17, and OTX2, especially GATA6, SOX17 and / or FOXA2. In some instances, a DE cell population can be > about 80% GATA6+ / SOX17+and > about 90% FOXA2+ / SOX17+. In some instances, a DE cell population includes less differentiated and / or more differentiated cells (e.g., ME and / or PGT cells) and / or cells of other cell types. In some instances, a DE cell population is derived from human PSCs (e.g., hiPSCs) or from a ME cell population that was derived from human PSCs (e.g., hiPSCs).

[0198] As used herein, “delta-like cell” and “δ-like cell” mean an SC-IC that at least makes and secretes somatostatin (SST) (e.g., SST+cell) or otherwise has characteristics such that the cell is functionally equivalent to a native, human delta cell. In some instances, a delta-like cell is SST+ / HHEX+.

[0199] As used herein, “diabetes” means a disease characterized by high blood glucose levels over a prolonged period. That is, “diabetes” can refer to all or any type of diabetes, including, but not limited to, T1D, T2D, cystic fibrosis-related, surgical, gestational diabetes and mitochondrial diabetes.

[0200] As used herein, “differentiate,” “differentiated,” and “differentiating” are relative terms that mean a process by which a less specialized cell (e.g., a more naive cell with a higher cell potency) becomes a more specialized cell type (e.g., a less naive cell with a lower cell potency). Stated differently, a differentiated call can be a cell that has progressed further down a developmental pathway than the cell it is being compared with (e.g., from an immature state to a less immature state; e.g., from a partially differentiated cell to a more differentiated cell) or from an immature state to a mature state (e.g., from a partially differentiated cell to a fullydifferentiated cell). Thus, pluripotent cells can differentiate into lineage-restricted progenitor cells (e.g., ectoderm, endoderm, and mesoderm), which in turn can differentiate into cells that are further restricted (e.g., PEP), which can differentiate into end-stage cells (e.g., terminally differentiated cells; e.g., cardiomyocytes, neurons, beta cells, etc.), and which play a characteristic role in a certain tissue type and which can or cannot retain the capacity to proliferate further.

[0201] As used herein, “DMH-1” means a small molecule compound having the chemical name and structure shown in Table 15. DMH-1, which is a selective inhibitor of BMP type-I receptor activin receptor-like kinase 2 (ALK2) receptor, exhibits 6- and 19-fold selectivity for ALK-2 over ALK-1 and ALK-3, respectively, and no significant inhibition of AMPK, ALK5, KDR (VEGFR-2) or PDGFR receptors, is commercially available from, for example, Bio- Techne Corporation (Minneapolis, MN USA).

[0202] As used herein, “Deoxyribonuclease I” and “DNAse I” mean a mammalian DNA- specific endonuclease that hydrolyzes double-stranded or single-stranded DNA to a mixture of oligonucleotides and mononucleotides. DNase I is often included in tissue dissociation protocols to digest DNA that has leaked into the dissociation medium because of cell damage. In some instances, the DNase I is a recombinant DNAse I, which has the same amino acid sequence as bovine DNAse I and is recombinantly expressed (e.g., in Pichia pastoris) without using any animal cells or other materials derived from animals. In some instances, the recombinant bovine DNAse I is a glycoprotein with a molecular weight of approximately 39 kDa. A recombinant bovine DNAse I is commercially available from, for example, Millipore Sigma.

[0203] As used herein, “ectoderm cell, “ectodermal cell” and the like means a cell or cells from the ectoderm (EC), which is one of the three primary germ cells layers in the very early embryo. These cells can differentiate to form epithelial and neural tissues.

[0204] As used herein, “effective amount” means an amount, concentration or dose of, for example, an SC-IC or an SC-IC population described herein or a composition including the same that upon single or multiple dose administration to an individual in need thereof, provides a desired effect in such an individual under diagnosis or treatment (i.e., may produce a clinically measurable difference in a condition of the individual such as, for example, a reduction in blood glucose, a reduction in HbA1c, and / or a reduction in weight or body fat). An effective amount can be readily determined by one of skill in the art by using known techniques and by observingresults obtained under analogous circumstances. In determining the effective amount for an individual, a number of factors are considered, including, but not limited to, the species of the individual (e.g., a mammalian species; e.g., a human), its size, age and general health, the specific disease or disorder involved, the degree of or involvement or the severity of the disease or disorder, the response of the individual, the particular form in which the SC-ICs are administered, the mode of administration, the bioavailability characteristics of the preparation administered, the dose regimen selected, the use of concomitant medication, and other relevant circumstances.

[0205] As used herein, “endocrine cell” means a cell that expresses CHGA (i.e., CHGA+).

[0206] As used herein, “endoderm cell,” “endodermal cell”, and “EN cell” mean a cell from the endoderm (EN), which is one of the three primary germ cell layers in the very early embryo. EN cells can first differentiate to the embryonic gut and then to the linings of the respiratory and digestive tracts, the liver, and the pancreas. EN cells express at least one of SOX17 and FOXA2 (i.e., SOX17+ / FOXA2+cells).

[0207] As used herein, “enterochromaffin-like cell” or “EC-like cell” or “ECLC” means a cell that at least makes and secretes serotonin (SRT; i.e., SRT+) or otherwise has characteristics such that the cell is functionally equivalent to a native, human enterochromaffin cell (ECC) (e.g., ECC cell markers such as ADRΑ2A, CXCL14, FEV, LMX1A, SLC18A1 and TAC1). In some instances, ECLCs are CHGA+ / NKX6.1+but lack PBLC markers such as PDX1, ISL1, G6PC2 and NPTX2. In some instances, ECLCs may be called non-pancreatic cells.

[0208] As used herein, “epidermal growth factor family” and “EGF family” mean the family of EGF proteins that include EGF, heparin-binding EGF-like growth factor (HB-EGF), transforming growth factor-alpha (TGF-α), amphiregulin (AR), epiregulin, epigen, betacellulin, neuregulin-1, neuregulin-2, neuregulin-3, and neuregulin-4. In some instances, the EGF family member protein used in the differentiation methods described herein is a recombinant human EGF protein or a recombinant human betacellulin protein.

[0209] As used herein, “EGF protein”, means the mammalian epidermal growth factor protein which is the founding member of the EGF family and signals through the class I tyrosine kinase receptor c-erbB. In some instances, the mammalian EGF protein used in the differentiation methods herein is recombinant human EGF protein, which is commercially available from R&D Systems (Minneapolis, MN USA) and has an amino acid sequence of SEQ ID NO:2.

[0210] As used herein, “epigenetic modifier” means a chemical agent used to modulate gene activity and / or expression in a cell or cell population by exerting changes in DNA methylation, histone modification and chromatin organization but not by changing a DNA sequence itself. Of interest herein are epigenetic modifiers that influence a cell or cell population to more likely differentiate into pancreatic beta-like cells (e.g., immature PBLCs and / or mature PBLCs in an SC-IC population). Examples of epigenetic modifiers include, but are not limited to, bromodomain inhibitors, DNA methylation inhibitors, histone acetyltransferase inhibitors, histone deacetylase inhibitors and histone methyltransferase (e.g., G9a) inhibitors, as well as combinations thereof. Exemplary epigenetic modifiers include, but are not limited to, azacytidine, butyrate, DZNep, EPZ004777, MDL-800, CM-272, UNC0321 and UNC0638.

[0211] As used herein, “epsilon-like cell” and “ε-like cell” mean a cell that at least makes and secretes ghrelin (GRL; i.e., GRL+) or otherwise has characteristics such that the cell is functionally equivalent to a native, human epsilon cell.

[0212] As used herein, “express” and the like, with regard to a marker, means to have an observable and / or a measurable amount or presence thereof (i.e., capable of qualitative or quantitative characterization).

[0213] As used herein, “foregut endoderm cell”, “FE cell” and the like mean a cell derived from PGT cells and expresses at least one of the following markers: CDX2, FOXA2, HNF4α, PDX1 and SOX2, especially PDX1.

[0214] As used herein, “FE cell population” means a cell population obtained by (i) differentiating pluripotent stem cells (e.g., iPSCs) by any method known in the art or as described herein or (ii) differentiating a precursor cell population derived from PSCs (e.g., a PGT cell population defined herein). The majority of the cells in an FE cell population are FE cells (e.g., express one or more FE cell markers listed above), but the population may also include less and / or more differentiated cells (e.g., PGT and / or PP cells) and / or other cell types (e.g., off-target cells). An FE cell population typically has a higher amount of PDX1 expression than the PGT cell population from which it was derived. In some instances, an FE cell population comprises greater than about any of 50%, 70%, 80%, 90% or 95% FE cells. In some instances, at least 60%, 70%, 80%, 90% or 95% of the cells in an FE cell population are PDX1+cells. In some instances, an FE cell population is derived from human PSCs (e.g., hiPSCs) or from a PGT cell population that was derived from human PSCs (e.g., hiPSCs).

[0215] As used herein, “functionally equivalent,” in describing a differentiating or differentiated cell type herein, means the cell performs the same function and / or provides the same utility as a referenced, native human cell type even though not identical thereto. For example, a beta-like cell can be functionally equivalent to a native, human beta cell if it displays at least one marker indicative of a native, human beta cell, has an INS content (i.e., has observable INS granules), and / or secretes INS in response to appropriate stimuli such as, for example, glucose (i.e., has a regulated GSIS). Alternatively, other characteristics of a beta-like cell include, but are not limited to, (i) coupling of mitochondrial respiration / activity with INS secretion; (ii) rapid INS secretion response to heightened demand (here defined as high glucose concentration); (iii) ability to rapidly turn off INS secretion after demand subsides; (iv) ability for multiple rounds of INS secretion; (v) ability to secrete an amount of INS as dictated by demand; and (vi) ability to respond to multiple INS secretagogues (e.g., Exendin-4 or amino acids such as L-glutamine and L-arginine).

[0216] As used herein, “G9a inhibitor” means a compound such as, for example, a small molecule compound, that inhibits the activity of one or both of (i) the histone methyltransferase G9a (also known as euchromatic histone lysine methyltransferase 2 (EHMT2)) and (ii) the histone methyltransferase G9a-like protein (also known as euchromatic histone lysine methyltransferase 1 (EHMT1)). In some instances, a G9a inhibitor is more selective for G9a than G9a-like. In other instances, a G9a inhibitor is more selective for G9a-like protein than G9a. Exemplary inhibitors include CM-272, UNC0321 and UNC0638, whose chemical names and structures are shown in Table 15 herein below.

[0217] As used herein, “gamma-like cell” and “γ-like cell” mean a cell that at least makes and secretes pancreatic polypeptide (PPP; i.e., a PPP+cell) or otherwise has characteristics such that the cell is functionally equivalent to a native, human gamma cell.

[0218] As used herein, “γ-Secretase Inhibitor” and “GSI” mean a compound such as, for example, a small molecule compound, that inhibits γ-secretase, a multimeric membrane protein complex. In some instances, the GSI is γ-Secretase Inhibitor XX (GSI-XX), which is a cell- permeable dibenzazepine compound that acts as a potent inhibitor of γ-secretase. GSI-XX is commercially available from, for example, MilliporeSigma. In some instances, the GSI is DAPT (N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester), DAPT, also known as GSI-IX is commercially available from, for example, MedChemExpress (Monmouth Junction, NJ USA).

[0219] As used herein, “glucose-free medium” means a defined medium that contains no glucose (i.e., < about 0.01 mM, < about 0.001 mM or 0 mM).

[0220] As used herein, “glucose-stimulated insulin secretion” or “GSIS” means an ability of a native, human beta cell or a beta-like cell in an in vitro or in vivo environment to sense glucose and to secrete INS via potassium ion (K+) channel-dependent and / or K+channel-independent mechanisms.

[0221] As used herein, “glutamine dipeptide” means a dipeptide of L-glutamine and another amino acid. Exemplary glutamine dipeptides are L-alanyl-L-glutamine (also referred to herein as L-alanine-L-glutamine) and glycyl-L-glutamine.

[0222] As used herein, the terms “grafting,” “administering,” “introducing,” “implanting” and “transplanting,” as well as grammatical variations thereof, are used interchangeably and mean in the context of the placement of cells (e.g., SC-IC cells herein) or a population of cells (e.g., SC-IC populations herein) into an individual by a method or route that results in at least partial localization of the introduced cells at a desired site. The SC-IC cells can be implanted directly to the desired site, or alternatively can be administered by any appropriate route that results in delivery to a desired location in the individual where at least a portion of the implanted cells or components of the cells remain viable. The period of viability of the cells after administration to a subject can be as short as a few hours (e.g., 24 hr), to a few days or even to as long as several years.

[0223] As used herein, “GSK-3α and GSK-3β inhibitor / Wnt pathway signaling activator” means a compound such as, for example, a small molecule compound, that acts as an inhibitor of GSK-3 and as a Wnt / β-catenin activator. Exemplary GSK-3α and GSK-3β inhibitor / Wnt pathway signaling activators include the GSK-3 β inhibitor compounds described in Intl. Patent Application Publication No. WO 2013 / 192005. In some instances, a GSK-3α and GSK-3β inhibitor / Wnt pathway signaling activator suitable for use in the differentiation methods described herein is CHIR98014, CHIR99021 or GSK inhibitor IX.

[0224] As used herein, “GSK-3 inhibitor IX" means a small molecule compound known as 6-bromo-3-[3-(hydroxyamino)indol-2-ylidene]-1H-indol-2-one (C16H10BrN3O2, CAS No. 667463-62-9). GSK-3 inhibitor IX, also known as 6-bromoindirubin-3'-oxime, is a selective, cell-permeable, ATP-competitive and reversible inhibitor of GSK-3α and GSK-3β (IC50 = 5nM for GSK-3β). GSK-3 inhibitor IX is commercially available from, for example, APExBIO.

[0225] As used herein, “heparin” and “heparin sulfate” means a linear, unbranched and highly sulfated polysaccharide with anticoagulant activity that belongs to the family of glycosaminoglycans (GAGs). The repeating disaccharide units of heparin consist of uronic acid and D-glucosamine connected by α-glycosidic linkage. At least three forms of heparin are commercially available: UFH (average molecular weight of 19 kDa), low molecular weight heparin (LMWH) and ultralow molecular weight heparin (ULMWH), which have the characteristics described in Plamberger et al. (2021) Int. J. Mol. Sci.22:12041. Commercially available UFH is isolated from porcine intestinal mucosa (UFH-PIM) or lung and intestine from cattle (UFH-C). In some instances, the differentiation methods described herein use UFH- PIM, which is a heparin sulfate commercially available from, for example, Sigma Aldrich. In other instances, the heparin form is a LMWH or a ULMWH. In some instances, the differentiation methods described herein use a synthetic ULMWH (e.g., fondaparinux; CAS No.114870-03-0, commercially available from Dr.Reddy’s).

[0226] As used herein, “human plasma like medium” or “HPLM” mean a basal cell culture medium that (a) includes the components and concentration ranges of any of the basal culture mediums described in Intl. Patent Application No. WO 2018 / 089927 or (b) includes some or all components in Table 18 or Table 19 herein below that would correspond to the following exemplary HPLMs. One exemplary HPLM includes: (a) at least 9 of the following proteinogenic amino acids: glycine, L- alanine, L-arginine, L-asparagine, L-aspartate, L- cysteine, L-glutamate, L-glutamine, L- histidine, L-isoleucine, L-leucine, L-lysine, L- methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L- valine and L-cystine; (b) at least 8, 9, 10 or 11 of the following Vitamins: D-biotin, choline, folic acid, myo-inositol, niacinamide, p-aminobenzoic acid, D-pantothenic acid, Vitamin B6, riboflavin, thiamine and Vitamin B12; (c) 6, 7, 8 or 9 inorganic salts selected from: CaCl2, KCl, MgCl2, MgS04, NaCl, NaHCO3, Na2HPO4, Ca(NO3)24H2O and NH4Cl; (d) glucose; and (e) at least 10 small organic compounds selected from: 4-hydroxyproline, acetylglycine, alpha- aminobutyrate, betaine, carnitine, citrulline, ornithine, taurine, 2-hydroxybutyrate, 3- hydroxybutyrate, acetate, citrate, formate, lactate, malonate, pyruvate, succinate, acetone, creatine, creatinine, glutathione, glycerol, urea, galactose, fructose, hypoxanthine and uric acid. One exemplary HPLM includes each of the components and mg / mL concentrations shown in Table 18 herein below. Another exemplary HPLM includes, or consists essentially of, each of the components and mg / mL concentrations shown in Table 19 herein below.

[0227] As used herein, “LDN-193189” means a small molecule compound having the chemical name and structure shown in Table 15 herein below and pharmaceutically acceptable salts thereof (e.g., a hydrochloride salt). LDN-193189, which is a cell- permeable selective inhibitor of BMP type I receptors ALK2 and ALK3 with IC50values of 5 nM and 30 nM, respectively, is commercially available from, for example, Reprocell (Beltsville, MD USA).

[0228] As used herein, “low-glucose medium” means a defined medium that contains less than about 2.5 mM glucose or less than about 2 mM glucose. In some instances, the glucose concentration in a low-glucose medium ≤ about 1 mM, ≤ about 0.5 mM, ≤ about 0.1 mM or ≤ about 0.05 mM. In some instances, a low-glucose medium is a glucose-free medium (i.e., < about 0.01 mM, < about 0.001 mM or 0 mM).

[0229] As used herein, “individual” means any mammal including, but not limited to, cats, dogs, mice, rats and primates, especially humans. Moreover, “subject, “participant” or “patient” may be used interchangeably with “individual.”

[0230] As used herein, “induced pluripotent stem cell,” “iPSC” and the like mean a pluripotent stem cell artificially derived (e.g., induced or by complete reversal) from a non- pluripotent cell, typically an adult somatic cell by, for example, inducing a forced expression of one or more reprogramming factors (e.g., Klf4, Lin28, Myc, Oct3 / 4, Sox2 and / or Nanog). See, e.g., Takahashi et al. (2007) Cell 131:861-872; and Yu et al. (2007) Science 318:1917- 1920. iPSCs have an ESC-like morphology, growing as flat colonies with large nucleo- cytoplasmic ratios, defined borders, and prominent nuclei. Like ESCs, iPSCs express one or more pluripotency markers including, but not limited to, alkaline phosphatase, SSEA3, SSEA4, Sox2, Oct3 / 4, Nanog, TRA160, TRA181, TDGF 1, Dnmt3b, FoxD3, GDF3, Cyp26al, TERT and zfp42, which may be detected via RT-PCR, Northern blots, in situ hybridization (see, e.g., “Current Protocols in Molecular Biology,” (Ausubel et al., eds., John Wiley & Sons, Inc. (1988)), as well as immunoassays such as immunohistochemical analysis of sectioned material, Western blotting and for markers that are accessible in intact cells, flow cytometry analysis (FACS) (see, e.g., Harlow & Lane, “Using Antibodies: A Laboratory Manual,” (Cold Spring Harbor Laboratory Press (l998)). See also, “Current Protocols in Cell Biology,” (Bonifacino et al., Wiley & Sons (2016)); “Current Protocols in Immunology,” (Colligan et al., eds., Wiley & Sons (1991)); “Current Protocols in Protein Science,” (Colligan et al., eds., Wiley & Sons(1995)); and “Gene Transfer Vectors for Mammalian Cells,” (Miller & Calos, eds., Cold Spring Harbor Laboratory Press (1987)).

[0231] As used herein, “human induced pluripotent stem cell” or “hiPSC” means an iPSC derived from a human somatic cell.

[0232] As used herein, “induced pluripotent stem cell population” or “iPSC population” means a cell population in which a majority of cells are iPSCs (e.g., as defined above). In some instances, > about 80%, > about 90% or more of the cells in an iPSC population express one or more of the pluripotency markers listed above. In some instances, > about 80%, > about 90%, > about 95%, > about 99% or more of the cells in an iPSC population co-express Oct4 and Nanog. In some instances, all of the iPSCs in an iPSC population are hiPSCs, and the iPSC population is referred to as an hiPSC population. In some instances, > about 95% of the cells in an hiPSC population are OCT4+ / NANOG+cells.

[0233] As used herein, “insulin-producing cell(s)” or “INS-producing cell(s)” means cell(s) that produce and store or secrete a detectable amount of INS.

[0234] As used herein, “ITS-G supplement” and “ITSG supplement” may be used interchangeably and mean a serum replacement supplement comprising INS, transferrin and a selenium (e.g., sodium selenite). In some instances, the ITS-G supplement is a 100x concentrated solution. The composition of an exemplary ITS-G (100x) supplement is a solution comprising INS, transferrin, sodium selenite and ethanol at the concentrations shown in Table 23 in the Examples below. Another exemplary ITS-G (100x) supplement is the Gibco™ Insulin-Transferrin-Selenium-Ethanolamine (ITS-G) (100X) solution, which is commercially available from, for example, ThermoFisher Scientific (Waltham, MA USA).

[0235] As used herein, “ITS-X supplement” and “ITSX supplement” may be used interchangeably and mean a cell culture medium supplement comprising INS, transferrin, a selenium (e.g., sodium selenite) and ethanolamine. In some instances, the ITS-X supplement is a 100x concentrated solution. The composition of an exemplary ITS-X (100x) supplement is a solution having the composition shown in Table 23 in the Examples below. Another exemplary ITS-X (100x) supplement is the Gibco™ Insulin-Transferrin-Selenium- Ethanolamine (ITS-X) (100X) solution, which is commercially available from, for example, ThermoFisher Scientific.

[0236] As used herein, “IWR-1-endo” and “IWR-1” may be used interchangeably and mean a small molecule compound having the chemical name and structure shown in Table 15. IWR- 1 is an inhibitor of Tankyrase 1 and 2.

[0237] As used herein, “keratinocyte growth factor protein,” “KGF,” “KGF protein,” “FGF- 7” and “FGF-7 protein” may be used interchangeably and mean a mammalian protein that is a member of the fibroblast growth factor (FGF) family and signals through FGF Receptor 2b. In some instances, the mammalian KGF protein used herein is a recombinant human KGF. Recombinant human KGF (SEQ ID NO:3) is commercially available from, for example, Peprotech, which is part of ThermoFisher Scientific.

[0238] As used herein, “KnockOut serum replacement medium” or “KOSR medium” mean a serum-free medium including small organic molecules (e.g., amino acids, Vitamins and antioxidants), trace elements, INS, transferrin, selenite and albumin (e.g., a lipid-rich albumin as described herein). Exemplary KOSR mediums include, but are not limited to, any of the serum-free culture medium supplements described in Intl. Patent Application Publication No. WO 1998 / 030679. The components of an exemplary KOSR medium are shown in Tables 29 and 30 below. In some instances, the KOSR medium is the KOSR composition shown in Table 30 or the commercially available Gibco KOSR or xeno-free (XF) version thereof from ThermoFisher Scientific.

[0239] As used herein, “lactate dehydrogenase A” or “LDHA” means an enzyme that preferentially catalyzes the conversion of pyruvate to lactate. LDHA is a monomer of lactate dehydrogenase, which exists as a tetramer that includes lactate dehydrogenase B (LDHB) as the other main subunit. LDHA is highly expressed in many tissues and in adult alpha cells within pancreatic islets; however, normal adult beta cells within pancreatic islets do not express LDHA.

[0240] As used herein, “marker,” “cell marker” and the like mean any molecule that can be observed or detected. Examples of a marker include, but are not limited to, a nucleic acid, such as a transcript of a specific gene; a polypeptide, such as a membrane protein or a glycoprotein; a carbohydrate; a lipid, such as a glycolipid or a lipoprotein; or a small molecule (e.g., molecules having a molecular weight of less than 10,000 amu). A marker is differentially expressed by or in a cell of interest. In this context, differential expression of a positive marker means an increased level for that marker as compared to an undifferentiated cell or a cell at another stage of differentiation. Similarly, differential expression of a negative marker meansa decreased level of that marker as compared to an undifferentiated cell or a cell at another stage of differentiation. The detectable level of the marker is sufficiently higher or lower in the cell of interest compared to another cell, such that the cell of interest can be identified and distinguished from the other using any of a variety of detection methods known in the art.

[0241] As used herein, “mesoderm cell,” and “mesodermal cell” mean a cell that is from the mesoderm, which is one of the three primary germ cell layers in the very early embryo. Mesoderm cells can differentiate to form mesenchyme, mesothelium, non-epithelial blood cells and coelomocytes and express at least one of the following markers: eomesodermin (EOMES) and nodal growth differentiation factor (NODAL).

[0242] As used herein, “mesendoderm cell,” “mesendodermal cell” and “ME cell” mean a cell that is from the mesendoderm, an embryonic tissue layer that can differentiate into ME and EN (i.e., is bipotent). ME cells express at least one of the following markers: TBXT (also known as brachyury) and MIXL1.

[0243] As used herein, “ME cell population” means a cell population obtained by differentiating pluripotent stem cells (e.g., iPSCs) by any method known in the art or as described herein. The majority of the cells in a ME cell population are ME cells (e.g., express one or more ME cell markers listed above), but the cell population may also include less and / or more differentiated cells (e.g., iPSCs and / or DE cells) and / or other cell types (e.g., off-target cells). In some instances, the ME cell population comprises greater than about any of about 70%, 80%, 90% or 95% ME cells. In some instances, at least about 70%, 80%, 90% or 95% of the cells in the ME cell population are TBXT+ / MIXL1+cells. In some instances, the ME cell population is derived from human PSCs (e.g., an hiPSC population).

[0244] As used herein, “multipotent stem cell” or “MSC” means a cell that is committed to one or more embryonic cell fate(s) / lineage(s) and retains a capacity to self-renew but, in contrast to a pluripotent cell, cannot give rise to each of the three germ cell layers. Examples of multipotent stem cells include, for example, hematopoietic stem cells, mesenchymal stem cells and neural stem cells.

[0245] As used herein, “N-acetyl-L-cysteine,” N-acetyl-cysteine,” “N-acetylcysteine” and “NAC” can be used interchangeably and mean a compound having the chemical name and structure shown in Table 15. NAC, which is a cell-permeable antioxidant and a precursor of reduced glutathione (GSH), is commercially available from, for example, Millipore Sigma.

[0246] As used herein, “nicotinamide” and “NAM” can be used interchangeably and mean a small molecule compound having the chemical name and structure shown in Table . NAM, which is the amide form of Vitamin B3, is an inhibitor of multiple enzymes (including poly(ADP-ribose)polymerase 1 (PARP-1), ROCK and casein kinase 1) and is commercially available from, for example, Millipore Sigma.

[0247] As used herein, “NEAA supplement” means a defined mixture of two, three, four, five or more of the following non-essential amino acids: alanine, arginine, asparagine, aspartic acid (or aspartate), cysteine, glutamic acid (or glutamate), glycine, proline, serine, tyrosine and selenocysteine. An exemplary NEAA supplement includes the non-essential amino acids listed in Table 24 herein below, which are the same non-essential amino acids present in the standard minimum essential medium (MEM) well-known in the art. In some instances, a NEAA supplement is a 100x concentrated solution. The composition of an exemplary NEAA (100x) supplement is shown in Table 24. Another exemplary NEAA (100x) supplement is Gibco™ MEM Non-Essential Amino Acids Solution that is commercially available from, for example, ThermoFisher Scientific.

[0248] As used herein, “non-pancreatic cell” means a cell from a developmental lineage outside the pancreas (e.g., an ECLC that expresses SLC18A1; i.e., SLC18A1+ECLC).

[0249] As used herein, “non-proliferative cell” or “non-proliferating cell” means a cell that has exited the cell cycle and no longer undergoes division.

[0250] As used herein, “pancreatic endocrine cell” or “PEC” means a cell that expresses chromogranin A (CHGA) and at least one pancreatic islet hormone (e.g., INS, GCG, SST, GRL and / or PPP). Other markers characteristic of PECs include one or more of HB9, ISL1, NeuroD1, NKX2.2, NKX6.1, PAX4, PAX6 and PDX1. In some instances, a PEC is a CHGA+ / INS+cell or a CHGA+ / GCG+cell. In some instances, a PEC is a PALC (e.g., CHGA+ / GCG+) or a PBALC (e.g., CHGA+ / INS+).

[0251] As used herein, “pancreatic endocrine precursor cell,” “PEP cell” and the like mean a cell derived from PP cells, where PEP cells express at least one of the following markers: ARX, CHGA, ISL1, NeuroDl, NGN3, NKX2.2, PAX4, PAX6 and PDX1, especially CHGA, NGN3 and PDX1. In some instances, a PEP cell is NKX2.2+ / NeuroD+. In some instances, a PEP cell is CHGA+ / NGN3+, CHGA+ / PDX1+or NGN3+ / PDX1+.

[0252] As used herein, “PEP cell population” means a cell population obtained by (i) differentiating pluripotent stem cells (e.g., iPSCs) by any method known in the art or asdescribed herein or (ii) differentiating a precursor cell population derived from PSCs (e.g., a PP cell population as defined herein). The majority of the cells in a PEP cell population are PEP cells (e.g., express one or more PEP cell markers listed above), but the population may also include less and / or more differentiated cells (e.g., PP cells and / or PECs) and / or other cell types (e.g., off-target cells such as SOX9+cells or ECLCs). In some instances, a PEP cell population comprises greater than about 70%, 80%, 90% or 95% PEP cells. In some instances, at least about 70%, 80%, 90% or 95% of the cells in a PEP cell population are CHGA+ cells and at least about 40% of the cells are CHGA+ / PDX1+cells. In some instances, less than about 10% of the cells in a PEP cell population are off-target cells. In some instances, a PEP cell population is derived from human PSCs (e.g., hiPSCs) or from a PP cell population that was derived from human PSCs (e.g., hiPSCs).

[0253] As used herein, “pancreatic progenitor,” “PP” and the like mean a cell derived from FE cells, where PP cells express at least one of the following markers: gastrin, HB9, HNFlβ, HNF4α, HNF6, NGN3, NKX6.l, PDX1, PTF1α, PROX1 and SOX9, especially NKX6.1 and PDX1.

[0254] As used herein, “PKC activator” is a compound such as, for example, a small molecule compound that activates PKC. Exemplary PKC activators, include, but are not limited to, TPPB, phorbol 12,13-dibutyrate (PdBU) (CAS No.37558-16-0), phorbol-12-myristate-13- acetate (PMA) (CAS No. 16561-29-8), (-)-indolactam V (ILV) (CAS No. 90365-57-4), bryostatin 1 (CAS No.83314-01-6) and derivatives of these compounds.

[0255] As used herein, “PP cell population” means a cell population obtained by (i) differentiating pluripotent stem cells (e.g., iPSCs) by any method known in the art or as described herein or (ii) differentiating a precursor cell population derived from PSCs (e.g., an FE cell population as defined herein). The majority of the cells in a PP cell population are PP cells (e.g., express one or more PP cell markers listed above), but the population may also include less and / or more differentiated cells (e.g., FE and / or PEP cells) and / or other cell types (e.g., off-target cells such as SOX2+and / or CDX2+cells). In some instances, about 50% of the cells in a PP cell population are proliferative cells (e.g., Ki67+cells) and about 20% to about 80% of the cells are PDX1+ / NKX6.1+but CHGA-. In some instances, a PP cell population comprises greater than about 70%, 80%, 90% or 95% PP cells. In some instances, at least about 70%, 80%, 90% or 95% of the cells in a PP cell population are PDX1+cells, at least about 30% of the cells are PDX1+ / NKX6.1+cells, and less than about 30%, 20% or 10% of the cells areCHGA+. In some instances, a PP cell population is derived from human PSCs (e.g., hiPSCs) or from an FE cell population that was derived from human PSCs (e.g., hiPSCs).

[0256] As used herein, “pluripotent stem cell” or “PSC” means a cell having a capacity, under defined conditions, to differentiate to more than one differentiated cell type, and preferably to differentiate to a cell type characteristic of all three germ cell layers. Pluripotent cells are characterized primarily by, for example, a nude mouse teratoma formation assay. Pluripotency is also evidenced by the expression of embryonic stem cell (ESC) markers, although the preferred test for pluripotency is the demonstration of the capacity to differentiate into cells of each of the three germ layers. It should be noted that simply culturing such cells does not, on its own, render them pluripotent. Reprogrammed pluripotent cells (e.g., iPSCs) also have the characteristic of the capacity of extended passaging without loss of growth potential, relative to primary cell parents, which generally have capacity for only a limited number of divisions in culture. as used herein can refer to a stem cell capable of producing all cell types of the organism. Therefore, a pluripotent stem cell can give rise to cells of all germ layers (e.g., the endoderm, mesoderm and ectoderm). Pluripotent cells can be capable of forming teratomas and of contributing to ectoderm, mesoderm, or endoderm tissues in a living organism. Moreover, pluripotent stem cell can refer to pluripotent stem cells regardless of their derivation. That is, the term pluripotent stem cell can encompass the terms embryonic stem cell and iPSC, as well as the term embryonic germ stem cell (EGSC). PSCs can be in the form of an established cell line, can be obtained directly from primary embryonic tissue or can be derived from a somatic cell.

[0257] As used herein, “polyhormonal cell” or “PHC” means a cell that at least expresses (i.e., makes and / or secretes) GCG and INS, and may be detected by flow cytometry analysis for expression of GCG and INS (GCG+ / INS+) or for expression of GCG and CPEP (GCG+ / CPEP+). Thus, the terms GCG+ / INS+cells and GCG+ / CPEP+cells are used herein interchangeably to refer to the same type of polyhormonal cell unless otherwise specified.

[0258] As used herein, “potency,” with regard to SC-ICs, means cellular INS content, INS release / secretion, GSIS, and / or marker expression akin to that of a native, human beta cell.

[0259] As used herein, “primitive gut tube” or “PGT” means a cell or cells derived from DE, where PGT cells express at least one of the following markers: FOXA2, GATA4, HNF1β and hepatocyte nuclear factor 4 alpha (HNF4α), especially FOXA2.

[0260] As used herein, “PGT cell population” means a cell population obtained by (i) differentiating pluripotent stem cells (e.g., iPSCs) by any method known in the art or as described herein or (ii) differentiating a precursor cell population derived from PSCs (e.g., a DE cell population defined herein). The majority of the cells in a PGT cell population are PGT cells (e.g., express one or more PGT cell markers listed above), but the population may also include less and / or differentiated cells (e.g., DE and / or FE cells) and / or other cell types (e.g., off-target cells). In some instances, a PGT cell population comprises greater than about 70%, 80%, 90% or 95% PGT cells. In some instances, at least about 70%, 80%, 90% or 95% of the cells in a PGT cell population are FOXA2+cells (i.e., FOXA2+ / PDX1- cells). In some instances, a PGT cell population is derived from human PSCs (e.g., hiPSCs) or from a DE cell population that was derived from human PSCs (e.g., hiPSCs).

[0261] As used herein, “progenitor cell” and “precursor cell” are used interchangeably herein and mean a cell that has a cellular phenotype that is more primitive (e.g., is at an earlier step along a developmental pathway or progression than is a fully differentiated cell) relative to a cell that it can give rise to by differentiation. Often, progenitor cells can have significant or very high proliferative potential. Progenitor cells can give rise to multiple distinct differentiated cell types or to a single differentiated cell type, depending on the developmental pathway and on the environment in which the cells develop and differentiate.

[0262] As used herein, “proliferative cell” or “proliferating cell” means a cell that undergoes active cell division to produce two daughter cells. Proliferating cells may be identified by any means known in the art, for example, by expressing Ki67 (i.e., Ki67+cells).

[0263] As used herein, “reprogramming factor(s)” means one or more molecules that are associated with cell “reprogramming,” that is, differentiation, and / or de-differentiation, and / or trans-differentiation, such that a cell converts to a different cell type or phenotype. Reprogramming factors generally affect expression of genes associated with cell differentiation, dedifferentiation and / or transdifferentiation. Transcription factors are examples of reprogramming factors such as Klf4, Lin28, Myc, Oct3 / 4, Sox2 and / or Nanog.

[0264] As used herein, “ROCK inhibitor” means a compound such as, for example, a small molecule compound that inhibits one or both of Rho kinase family members ROCK1 and ROCK2. Exemplary ROCK inhibitors useful in the differentiation methods described herein include, but are not limited to, Chroman 1, Y-27632, thiazovivin (CAS No. 1226056-71-8),fasudil hydrochloride, also known as HA1077 HCl (CAS No. 105628-07-7) and H-1152 dihydrochloride (CAS No.871543-07-6).

[0265] As used herein, “SANT-1” means a small molecule compound known as N-[(3,5- dimethyl-1-phenyl-1H-pyrazol-4-yl)methylene]-4-(phenylmethyl)-1-piperazinamine (C23H27N5, CAS No. 304909-07-7). SANT-1 is a cell-permeable antagonist of the SHH signaling pathway by binding to Smoothened, a distant relative of G protein-coupled receptors.

[0266] As used herein, “Sonic Hedgehog signaling pathway inhibitor” or “SHH signaling pathway inhibitor” means a compound such as, for example, a small molecule compound capable of inhibiting the SHH signaling pathway. Exemplary SHH signaling pathway inhibitors suitable for use in the differentiation methods described herein include cyclopamine, glasdegib, saridegib, sonedegib and vismodegib.

[0267] As used herein, “stem cell” or “SC” means a cell having an ability to self-renew and differentiate to another cell having a more differentiated state. Stem cells can be characterized by both the presence of specific markers (e.g., RNAs, proteins, etc.) and the absence of specific markers. Stem cells can also be identified by functional assays both in vitro and in vivo, particularly assays relating to the ability of stem cells to give rise to multiple differentiated progenies. Examples of stem cells include, but are not limited to, totipotent stem cells, pluripotent stem cells and multipotent stem cells.

[0268] As used herein, “stem cell-derived islet-like cell,” “SC-IC” and the like mean a cell derived from, for example, a stem cell such as an ESC or an iPSC that possess characteristics akin to one of the different endocrine cell types (e.g., α, β, δ, ε and / or γ cells) present in native islet cells (e.g., express markers characteristic of the different endocrine cell types). Examples of SC-ICs include PALC, PBLC, delta-like cell (PDLC), epsilon-like cell (EDLC) and gamma- like cell (PGLC). In some instances, an SC-IC can be an immature or precursor SC-IC (i.e., has a less mature phenotype than the corresponding native endocrine cell type).

[0269] As used herein, “mature stem cell-derived islet-like cell,” “mature SC-IC” and the like means an SC-IC cell type that has phenotypic and functional characteristics that are closer to the phenotype and function of the corresponding cell type in native, human islets relative to immature SC-ICs (e.g., SC-ICs obtained by culturing PEP cells in a PEP differentiating medium for about 4 or 5 days). In some instances, a mature SC-IC is a mature PBLC (e.g., displays at least one marker indicative of a pancreatic beta cell (e.g., PDX1 or NKX6.1), expresses INS and displays a GSIS response to a glucose challenge characteristic of anendogenous mature pancreatic beta cell. In some instances, a mature SC-IC is a mature PALC (e.g., a monohormonal GCG+cell).

[0270] As used herein, “mature SC-IC population” means a cell population obtained by (i) differentiating pluripotent stem cells (e.g., iPSCs) by any method known in the art or as described herein or (ii) differentiating a precursor cell population derived from PSCs (e.g., a precursor SC-IC population as defined herein). In some instances, the majority of the cells in a mature SC-IC cell population are mature SC-ICs (i.e., express one or more of the markers of native, mature, human islet cells listed below) and include mature PBLCs and mature PALCs. In some instances, a mature SC-IC population includes less differentiated cells (e.g., immature PBLCs and immature PALCs) and can include other cell types (e.g., off-target cells such as proliferating cells and ECLCs). In some instances, the mature SC-IC population includes a greater percentage of mature PBLCs than immature PBLCs and / or a greater percentage of mature PALCs than immature PALCs. In some instances, a mature SC-IC population is characterized as comprising at least about 99.5% CHGA+cells, at least about 60% CPEP+ / GCG- cells, at least about 50% CPEP+ / NKX6.1+cells, at least about 70% INS+ / SLC18A1- cells; less than about 12% INS- / SLC18A1+cells and less than about 4% Ki67- cells and optionally an INS content of at least about 325 nU / cell. In some instances, a mature SC-IC population is derived from human PSCs (e.g., hiPSCs) or from a precursor SC-IC population that was derived from human PSCs (e.g., hiPSCs).

[0271] Exemplary markers of native, mature, human pancreatic alpha (α) cells include, but are not limited to, GCG and ARX, but not PDX1, NKX6.1 or INS. Other examples of markers of native, mature, human pancreatic alpha cells include, but are not limited to, expressing GCG, secreting GCG, and / or displaying a response to a stimulus akin to that of a native, mature, human pancreatic alpha cell; and expressing or secreting GCG, GLP-1, GRL, INS, PPP and / or SST akin to that of a native, mature, human pancreatic α cell. Other markers of native, mature, human pancreatic alpha cells include expressing ARX and MAFB but not PDX1 and NKX6.1. Another marker of native, mature human pancreatic alpha cells is expression of the prohormone convertase PC2.

[0272] Examples of markers of native, mature, human pancreatic beta (β) cells include, but are not limited to, amylin (IAPP), B2, CPEP, E-cadherin (ECAD), glucagon-like peptide 1 receptor (GLIP1R), glucose transporter 1 (GLUT1), glucose transporter 2 (GLUT2), HNF3β, HNF6, INS, MAFA, NeuroD1, NKX2.2, Pax4, Pax6, prohormone convertase enzyme 2 (PC2),PC1 / 3, PDXl, urocortin 3 (UCN3), and zinc transporter 8 (ZnT8), especially INS, NKX6.1 and / or CPEP without GCG. Other examples of markers of native, mature, human pancreatic beta cells include expressing INS, secreting INS, and / or displaying a GSIS response akin to that of a native, mature, human pancreatic beta cell; and expressing or secreting GCG, GRL, INS, PPP and / or SST akin to that of a native, mature, human pancreatic beta cell.

[0273] Exemplary markers of native, mature, human pancreatic delta (δ) cells include, but are not limited to, expressing SST, secreting SST and / or displaying a response to a stimulus akin to that of a native, mature, human pancreatic delta cell; and expressing or secreting GCG, GRL, INS, PPP and / or SST akin to that of a native, mature, human pancreatic delta cell.

[0274] Exemplary markers of native, mature, human pancreatic epsilon (ε) cells include, but are not limited to expressing GRL, secreting GRL, and / or displaying a response to a stimulus akin to that of a native, mature, human pancreatic epsilon cell; and expressing or secreting GCG, GRL, INS, PPP and / or SST akin to that of a native, mature, human pancreatic epsilon cell.

[0275] Exemplary markers of native, mature human pancreatic gamma (γ) cells include, but are not limited to expressing PPP, secreting PPP, and / or displaying a response to a stimulus akin to that of a native, mature, human pancreatic gamma cell; and expressing or secreting GCG, GRL, INS, PPP and / or SST akin to that of a native, mature, human pancreatic gamma cell.

[0276] As used herein, “precursor SC-IC” or “immature SC-IC” means a cell derived from PEP cells and is functionally less mature than a mature SC-IC of the same cell type. In some instances, a precursor (immature) SC-IC can express at least one of the following markers: CHGA, CPEP, GCG, GHRL, INS, PPP, SST, ARX, HB9, ISLl, NeuroD1, NKX2.2, NKX6.l, PAX4, PAX6 and PDX1, especially GCG, CPEP and / or INS. In some instances, a precursor (immature) SC-IC is an immature PBLC or an immature PALC, each as defined above.

[0277] As used herein, “precursor SC-IC population” and “immature SC-IC population” may be used interchangeably to mean a cell population obtained by (i) differentiating pluripotent stem cells (e.g., iPSCs) by any method known in the art or as described herein or (ii) differentiating a precursor cell population derived from PSCs (e.g., a PEP cell population as defined herein). In some instances, many of the cells in a precursor SC-IC cell population are immature SC-ICs (i.e., express one or more of the precursor SC-IC markers listed above) and include immature PBLCs and immature PALCs. In some instances, a precursor SC-ICpopulation includes less and / or more differentiated cells (e.g., PEP cells and / or mature PBLCs and mature PALCs) and can include other cell types (e.g., off-target cells such as proliferating cells and ECLCs). In some instances, the precursor SC-IC population includes a greater percentage of immature PBLCs than mature PBLCs and / or a greater percentage of immature PALCs than mature PALCs. In some instances, a precursor SC-IC population is about 40% to about 90% INS+ / SLC18A1- (or alternatively, less than about 25% INS- / SLC18A1+), about 35% to about 80% CPEP+ / GCG- (or alternatively, less than about 10% to about 40% CPEP+ / GCG+) and / or about 90% to about 100% CHGA+ / Ki67-. In some instances, the precursor SC-IC population obtained by culturing a PEP cell population as described herein can be characterized by flow cytometry as including: about 50% to about 85% INS+ / SLC18A- cells; ≤ about 25% INS- / SLC+cells, about 40% to about 75% CPEP+ / GCG- cells; CPEP+ / GCG+cells; and about 95% to about 100% CHGA+ / Ki67- cells. In some instances, a precursor SC-IC population is derived from human PSCs (e.g., hiPSCs) or from a PEP cell population that was derived from human PSCs (e.g., hiPSCs).

[0278] As used herein, the term “supplemented with” means that a supplement has been added to a starting material to arrive at an ending material. Unless specifically indicated, the supplement or supplements need not be added at a specific time or in a specific order. The term “supplemented with” does not preclude the starting material from being additionally supplemented with other supplements, at any point in time, before or after being supplemented with the present supplement. Unless specifically indicated, supplements are added to a culture media or differentiation medium in a “substantially pure” form. The term “substantially pure” indicates that a supplement is substantially free of components with which it naturally occurs in nature. For example, a substantially pure albumin could be a purified albumin or an albumin that is recombinantly produced.

[0279] As used herein, “tankyrase 1 / 2 inhibitor” means a compound such as, for example, a small molecule compound that binds to tankyrase 1 and / or 2 and antagonizes the Wnt signal transduction pathway by stabilizing axin and promoting β-catenin degradation. Tankyrase 1 (TNKS1 / ARTD5 / PARP5a) and Tankyrase 2 (TNKS2 / ARTD6 / PARP5b) form a distinct subgroup of the polymer forming ARTDs. TNKS1 and TNKS2 share 82% sequence identity and are distinguished from the rest of the family by a unique domain structure containing several ankyrin repeats and a sterile alpha motif (SAM). In some instances, a tankyrase 1 / 2 inhibitor inhibits binding of a substrate to a nicotinamide subsite or an adenosine subsite, orboth, of tankyrase 1 and / or tankyrase 2. Exemplary tankyrase 1 / 2 inhibitors include, but are not limited to, AZ 6102, JW55, MN64, IWR-l-endo, TC-E5001, WIKI4, TNKS 22, TNKS 49, 2X- 121 (E7449), XAV-939, G007-LK and NVP-TNKS656. In some instances, the tankyrase 1 / 2 inhibitor binds to the nicotinamide pocket of tankyrases 1 and 2 and can be XAV939. In some instances, the tankyrase 1 / 2 inhibitor does not bind to the nicotinamide pocket but instead binds to the adenosine subsite of the catalytic domains in tankyrases 1 and 2. This class of adenosine subsite specific binding tankyrase 1 / 2 inhibitors includes the various adenosine binding site compounds listed in Table 1 of Mariotti et al. (2017) Brit. J. Pharmacol. 174:461-4636, the ADE subsite binding compounds shown in Fig. 5 of Haikarainen et al. (2014) Curr. Pharm. Des.20:6472-6488 and the inhibitors based on the 1,2,4 triazole scaffold disclosed in Table 1 of Leenders et al. (2021) J. Med. Chem.64:17936-17040. Exemplary adenosine subsite binding compounds described in these references include G007-LK, IWR-1, JW55, CMP4, CMP24 and CMP40 in Fig.5 of Haikarainen et al., supra; the JW74-based Compound No.15 in Table 1 of Mariotti et al., supra; K-756; OM-153; OM-1700; oxazolidinone and the oxazoldinone- based compound 20 in Table 1 of Mariotti et al., supra and WIKI4. In some instances, the tankyrase 1 / 2 inhibitor binds to the adenosine subsite of tankyrases 1 and 2, but also interacts with the G-loop (see, Hakiarainen et al. (2013) PLoS One 8:e65404). This adenosine subsite / G- loop interacting class of tankyrase 1and 2 inhibitors includes WIKI4.

[0280] As used herein, “totipotent stem cell” or “TSC” means a cell having an ability to self- renew and differentiate to another cell having a more differentiated state.

[0281] As used herein, “TPPB” means a small molecule compound known as 2S,5S-E,E-8- 5-4-trifluoromethyl phenyl-2,4-pentadienoylamino benzolactam (CAS No. 497259-23-1). TPPB, which is also known as PKC Activator V, is a cell-permeable benzolactam derived PKC activator (Ki = 11.9 nM for PKCα) that efficiently enhances non-amyloidogenic α-processing of amyloid precursor protein (APP). TPPB is commercially available from, for example, Millipore Sigma.

[0282] As used herein, “trace elements A supplement” means a liquid composition that includes one, two, three or all four of cupric sulfate, ferric citrate, a selenium (e.g., sodium selenite) and zinc sulfate. Likewise, “trace elements B supplement” means a liquid composition that includes one, two, three, four, five, six or all seven of ammonium molybdate, ammonium vanadate, manganese sulfate, nickel sulfate, sodium silicate, stannous chloride and hydrochloric acid. In some instances, each of the trace elements A and B supplements is a 100xconcentrated solution. The compositions of exemplary 100x and 1x trace elements A and trace elements B supplements are shown below in Tables 25 and 26, respectively. Concentrated trace elements A (100x) and trace elements B (100x) supplements are commercially available from, for example, Fisher Scientific.

[0283] As used herein, “treat” or “treating” means an act of providing care to an individual in need thereof, for example, by administering a therapeutic agent (e.g., an SC-IC or composition including the same) to the individual for purposes of improving the health and / or well-being of the individual with respect to an existing condition (e.g., a disease, disorder) or to prevent or decrease the likelihood of the occurrence of a condition. Treating also can involve decreasing the frequency or severity of at least one sign, symptom or contributing factor of a condition (e.g., disease or disorder) experienced by the individual.

[0284] As used herein, “triiodo-L-thyronine,” triiodothyronine” and “T3” may be used interchangeably to mean the thyroid hormone having a chemical name and structure shown in Table 15. T3, also known as liothyronine, is commercially available from, for example, Millipore Sigma.

[0285] As used herein, “trolox” means a small molecule compound known as 6-hydroxy- 2,5,7,8-tetramethylchroman-2-carboxylic acid (C14H18O4; CAS No. 53188-07-1). Trolox is a cell-permeable, water-soluble derivative of Vitamin E with potent antioxidant properties. Trolox is commercially available from, for example, Millipore Sigma.

[0286] As used herein, “retinoid” means Vitamin A compounds and vitamers of Vitamin A. Retinoids include, but are not limited to, retinol, retinal, retinoic acid, beta carotene, isotrentinoin, tretinoin (also known as ATRA), alitrentinoin, etretinate and its metabolite acitretin, and retinoidal benzoic acid derivatives such as adapalene, bexarotene and tazarotene.

[0287] As used herein, “thiol-based antioxidant” means a compound that contains a sulfhydryl (SH) side chain group or a disulfide bond and acts as an antioxidant. Exemplary thiol-based antioxidants for use herein include, but are not limited to, cysteine, NAC, cystine and cystine analogues such as N,N′-diacetyl-l-cystine (DiNAC) (CAS No. 5545-17-5) and N,N′-diacetyl-l-cystine dimethylester (DACDM) (CAS No.32381-28-5).

[0288] As used herein, “TPPB” means a small molecule compound having the chemical name and structure shown in Table 15. Also known as PKC Activator V, TPPB is a cell- permeable benzolactam derived PKC activator (Ki = 11.9 nM for PKCα) that inducesdifferentiation of PSCs into Pdx-1 expressing pancreatic progenitor cells. TPPB is commercially available from, for example, Millipore Sigma.

[0289] As used herein, “urocortin 3” and “UCN3” mean a peptide hormone that is expressed in mature endogenous pancreatic islet beta cells. UCN3, which is a member of the corticotropin-releasing factor (CRF) family that selectively binds the G-protein coupled receptor CRFR2, is co-released with INS under high glucose conditions and stimulates somatostatin secretion from delta cells, which are the primary cells within the islet that express CRHR2.

[0290] As used herein, “Vitamin B3 compound” means (a) niacin, nicotinamide, nicotinic acid and / or nicotinamide riboside; (b) nicotinamide derivatives and analogues with activity in the differentiation methods described herein that is substantially similar to nicotinamide; and (c) pharmaceutically acceptable salts of any of the compounds listed in (a) and (b).

[0291] As used herein, “Vitamin C compound” means (a) ascorbic acid; (b) analogues or derivatives and analogues of ascorbic acid having activity substantially similar to ascorbic acid in the differentiation methods described herein; and (c) pharmaceutically acceptable salts of the compounds in (a) and (b). In some instances, a Vitamin C compound is any of dehydroascorbic acid and pharmaceutically acceptable salts thereof, ascorbyl phosphate and pharmaceutically acceptable salts thereof, sodium ascorbate, calcium ascorbate, zinc ascorbate, niacinamide ascorbate. Dehydroascorbic acid (chemical name and structure shown in Table 15) is made from the oxidation of ascorbic acid. Ascorbyl phosphate is a synthetic form of Vitamin C and is found in different salt forms such as magnesium ascorbyl phosphate and sodium ascorbyl phosphate (chemical name and structure are shown in Table 15).

[0292] As used herein, “Wnt-3a” and Wnt3a” means a mammalian wingless-type MMTV integration site family, member 3A protein. In some instances, Wnt3a is a recombinant, mammalian protein (e.g., recombinant mouse Wnt3a or recombinant human Wnt3a). Recombinant mouse and human Wnt3a are commercially available from, for example, R&D Systems (Minneapolis, MN).

[0293] As used herein, “WNT / β-catenin signaling pathway activator” means a compound such as, for example, a small molecule compound or a protein, capable of activating this signaling pathway at a similar level as achieved by a Wnt3 ligand (e.g., Wnt3a. Wnt1, Wnt3a and spondin).

[0294] As used herein, “Wnt Inhibitor Kinase Inhibitor 4” and “WIKI4” mean a small molecule compound having the chemical name and structure shown in Table 15. WIKI4, also known as Tankyrase 1 / 2 Inhibitor V, is a selective inhibitor of TNKS1 and TNKS2 (IC50 = 26 and 15 nM, respectively). Through its effects on TNK2, WIKI4 prevents AXIN ubiquitinylation and degradation and inhibits signaling through the Wnt / beta-catenin pathway. WIKI4 is commercially available from, for example, Cayman Chemical (Ann Arbor, MI USA).

[0295] As used herein, “Y-27632” means a small molecule compound having the chemical name and structure shown in Table 15 and pharmaceutically acceptable salts thereof. Y-27632 is a specific inhibitor of the ROCK family with Ki values of 0.22 μM and 0.30 μM for ROCK1 and ROCK2, respectively. In some instances, the methods described herein use the dihydrochloride salt of Y-27632 (Y-276322HCl), which is commercially available from, for example, Selleck Chemicals.

[0296] As used herein, “zinc compound” means a small molecule compound containing zinc2+(Zn2+). In some instances, zinc compounds useful in the differentiation methods described herein is a salt of Zn2+(e.g., zinc sulfate (ZnSO4), zinc acetate (Zn(O2CCH3)2), zinc nitrate (Zn(NO3)2), zinc chlorate (Zn(ClO3)2) and zinc phosphate Zn(PO4)2. In some instances, the zinc compound is a pharmaceutically acceptable salt of zinc.

[0297] Methods

[0298] Methods of Differentiating Stem Cells to SC-ICs

[0299] Pluripotent Cell Lines: The differentiation methods can begin with stem cells, especially pluripotent stem cells such as, for example, iPSCs. iPSCs can be derived from multiple different cell types, including terminally differentiated cells (i.e., somatic cells). In some instances, the iPSCs are derived from a human cell type.

[0300] One can generate iPSCs via any of the reprogramming methods for somatic cells that are well-known in the art. See, e.g., US Patent Application Publication Nos. 2009 / 0047263, 2009 / 0068742, 2009 / 0191159, 2009 / 0227032, 2009 / 0246875 and 2009 / 0304646. Generally, to generate iPSCs, somatic cells are provided with reprogramming factors (e.g., Oct4, SOX2, KLF4, MYC, Nanog, Lin28, etc.; see, e.g., Takahashi et al. (2007), supra, and Yu et al. (2007), supra. Alternatively, iPSCs can be obtained from commercial suppliers including, but not limited to, Cell and Gene Therapy Catapult (London, United Kingdom), FujiFilm CellularDynamics, Inc. (Madison, WI, USA), Healios K.K. (Tokyo, Japan) and Lonza Group Ltd. (Basel, Switzerland).

[0301] In other instances, the differentiation methods can begin with ESCs or EGSCs (e.g., human ESCs or human EGSCs).

[0302] PSC Expansion: Although presumably immortal in their renewal capacity, the stress of in vitro culture on PSCs is known to cause genetic alterations that eventually may impair a cell line’s capacity to “perform” as desired. For that reason, characterizing, expanding and banking of PSCs may be necessary. Here, a PSC line, such as an iPSC line (e.g., hiPSC line), can be expanded by culturing a cell population of > about 98% Oct4+ / Nanog+cells for about 3 days to about 5 days in a two-dimensional (2D) culture. In some instances, the cells can be at a density of about 150,000 cells / cm2to about 500,000 cells / cm2. In some instances, the cells can be at a density of about 175,000 cells / cm2to about 475,000 cells / cm2, about 200,000 cells / cm2to about 450,000 cells / cm2, about 225,000 cells / cm2to about 425,000 cells / cm2, about 250,000 cells / cm2to about 400,000 cells / cm2, about 275,000 cells / cm2to about 375,000 cells / cm2, about 300,000 cells / cm2to about 350,000 cells / cm2, or about 325,000 cells / cm2. In some instances, the cells can be at a density of about any of 250,000 cells / cm2, 275,000 cells / cm2, 300,000 cells / cm2, 325,000 cells / cm2, 350,000 cells / cm2, 375,000 cells / cm2, 400,000 cells / cm2, 425,000 cells / cm2, and 450,000 cells / cm2, especially about 350,000 cells / cm2. The cells can be initially cultured in a medium such as Essential 8 (E8) including vitronectin-N (VTN-N). A final passage prior to using such cells can be in a medium such as mTESR™ including VTN-N. In some embodiments, the medium is E8 flex + pluronic.

[0303] Alternatively, one can expand iPSCs via any of the methods that are well-known in the art. See, e.g., Intl. Patent Application Publication No. WO 2017 / 222879, as well as Kwok et al. (2022) Reprod. Toxicol. 112:22-35, Manstein et al. (2021) Stem Cells Trans. Med. 10- 7:1063-1080, Marotta et al. (2022) Methods Mol. Biol.2454:1-15, Mesquita et al. (2021) Stem Cell Biol.5:209-229 and Van der Wal et al. (2018) Stem Cell Rep.10:1975-1990.

[0304] iPSC Aggregation: The methods also can include an iPSC aggregation step to increase the surface area for cell growth per media volume. Here, about 0.25 x 106cells / mL to about 2 x 106cells / mL, especially about 1 x 106cells / mL, of the expanded iPSCs can be aggregated for about 1 day in spinner flasks, a conventional stirred-tank bioreactor, or a vertical wheel bioreactor (such as a bioreactor in the PBS vertical wheel family (PBS Biotech; Camarillo, CA USA) in a defined medium such as mTESR™, Essential 8™ , Essential 8™Flex including a ROCK inhibitor and Poloxamer 188, or Essentual 8™ flex + pluronic. In some instances, the culture pH can range from about 6.6 to about 7.4 and dissolved oxygen can be at a concentration from about 20 mmHg to about 100 mmHg. In some instances, agitation rates in a vertical wheel bioreactor can be from about any of about 20 rpm, 30 rpm, 40 rpm and 50 rpm but up to about 60 rpm. Aggregate morphology at the end can be from about 50 μm to about 170 μm, depending on the choice of aggregation medium used. In some instances, the culture can be controlled at a temperature of about 36°C to about 38°C, especially about 37°C. In some instances, air, carbon dioxide and oxygen are supplied to the bioreactor via passive diffusion, active overlay or sparging.

[0305] In some instances, the ROCK inhibitor is Y-27632 (e.g., Y-276322HCl), which can be at a concentration from about 1 μM to about 20 μM. In some instances, Y-276322HCl can be at a concentration from about 2 μM to about 19 μM, about 3 μM to about 18 μM, about 4 μM to about 17 μM, about 5 μM to about 16 μM, about 6 μM to about 15 μM, about 7 μM to about 14 μM, about 8 μM to about 13 μM, about 9 μM to about 12 μM, or about 10 μM to about 11 μM. In yet other instances, the ROCK inhibitor can be at a concentration of about 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, or 15 μM especially about 10 μM. Other suitable ROCK inhibitors include, but are not limited to, Chroman 1, thiazovivin, fasudil / HA1077 and H-1152.

[0306] Alternatively, one can aggregate iPSCs via any of the methods that are well-known in the art. See, e.g., Intl. Patent Application Publication No. WO 2017 / 222879, as well as Kwok et al. (2022) Reprod. Toxicol. 112:22-35, Manstein et al. (2021) Stem Cells Trans. Med. 10- 7:1063-1080, Marotta et al. (2022) Methods Mol. Biol.2454:1-15, Mesquita et al. (2021) Stem Cell Biol.5:209-229 and Van der Wal et al. (2018) Stem Cell Rep.10:1975-1990.

[0307] Differentiation: After cells from the pluripotent stem cell line, such as an iPSC line, are aggregated, they are subjected to one or more differentiation steps which typically include culturing in vitro a population of cells in one or more differentiating mediums which include a basal cell culture medium (e.g. a serum-free medium) and one or more molecules (referred to herein as “differentiation factors”) to promote the differentiation of the cells from one cell type to a more differentiated cell type. The differentiation steps can be performed in the order below or can begin at any particular stage and commence from there. That is, in some instances, the differentiation methods can begin with pluripotent stem cells such as iPSCs; however, in other instances the methods can begin with a more differentiated cell type, such as PP cells or PEPcells, and proceed from there. In some instances, there can be seven differentiation stages. In other instances, there can be less than seven differentiation stages such as, for example, six differentiation stages, five differentiation stages, four differentiation stages, three differentiation stages, two differentiation stages or even one differentiation stage. In some instances, any differentiation stage can include two or more sub-stages. In some instances, the cell population generated in the first sub-stage of a differentiation stage has one or more different characteristics than the cell population generated in the next sub-stage (e.g., the cell population generated in a second sub-stage is more differentiated (i.e., more mature)) than the first sub-stage cell population. In some instances, the cell population generated in the first sub- stage is washed in a defined medium before carrying out the second sub-stage.

[0308] In some instances, one or more of the individual differentiation stages in the methods described herein (i.e., one or more of Stages 1a, 1b, 2, 3 and 4) can be carried out by a method well-known the art and / or may include alternative differentiation factors and culturing techniques well-known in the art. Exemplary methods, differentiation factors and culturing techniques used in various stages of PSC to SC-IC differentiation protocols are described in the following published patent applications: Intl. Patent Application Publication Nos. WO 2003 / 050249, WO 2013 / 192005, WO 2014 / 105543, WO 2015 / 028614, WO 2016 / 100930, WO 2017 / 222879, WO 2019 / 048690, WO 2019 / 099725, WO 2019 / 169351, WO 2019 / 227198, WO 2020 / 033879, WO 2022 / 026932, WO 2023 / 077140 and WO 2023 / 133568.

[0309] Any differentiating medium described herein can optionally include an antibiotic to minimize the risk of bacterial contamination of the cell culture(s). When used, the antibiotic can be a penicillin-streptomycin solution at a concentration from about 0.5% to about 1.5%, about 0.7% to about 1.3%, about 0.9% to about 1.1%, or about 1.0%. In some instances, the concentration of penicillin-streptomycin solution in a differentiating medium can be at a concentration of about any of about 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4% or 1.5%.

[0310] Stage 1a Cells and Cell Populations (iPSCs to ME cells):

[0311] In some instances, the differentiation methods can begin with or can include differentiating PSCs (e.g., iPSCs) into ME cells by culturing a population of PSCs (e.g., a PSC population that includes Oct4+ / Nanog+iPSCs) in a PSC-differentiating medium for a time period of about 1 day (e.g., Day 0 in FIG. 1B) to obtain a cell population including ME cells(e.g., the ME cell population includes TBXT+ cells and / or MIXL1+ cells. In some instances, the PSC population consists essentially of hiPSCs.

[0312] In some instances, the culturing of the PSC population is performed in a bioreactor and includes cell transfer densities from about 3 x 105cells / mL to about 2 x 106cells / mL. In some instances, the culture pH can range from about 6.6 to about 7.4. In yet other instances, pH can be about 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3 or 7.4. In some instances, the dissolved oxygen can be controlled to a concentration from about 20 mmHg to about 60 mmHg. In some instances, agitation rates can be from about 20 rpm to about 60 rpm depending on the bioreactor size. In some instances, the culture is controlled at a temperature of about 36°C to about 38°C, especially about 37°C. In some instances, air, carbon dioxide, and oxygen are supplied to the bioreactor via passive diffusion, active overlay or sparging.

[0313] PSC-differentiating medium: The PSC-differentiating medium can include a defined medium including glucose and / or fructose and one or more of glutamine (e.g., a glutamine dipeptide), albumin (e.g., BSA or HSA), a buffer (e.g., NaHCO3), a serum replacement supplement (e.g., an ITS-G or ITS-X supplement described herein), a TGF-β superfamily growth factor (e.g., Activin A), a Wnt / β-catenin pathway signaling activator (a Wnt3a protein), a GSK-3α and GSK-3β inhibitor / Wnt pathway signaling activator (e.g., CHIR99021) and a ROCK inhibitor (e.g., Y-27632). In some instances, the PSC-differentiating medium includes the MCDB media described in Table 16 herein below.

[0314] In some instances, the PSC-differentiating medium includes glucose at a concentration from about 5 mM to about 20 mM. In some instances, the glucose concentration can be about 6 mM to about 19 mM, about 8 mM to about 17 mM, about 10 mM to about 15 mM, or about 12 mM to about 13 mM. In some instances, the glucose concentration in the PSC-differentiating medium can be about 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM or 17 mM. In some instances, the PSC-differentiating medium includes about 12 mM glucose.

[0315] In some instances, the PSC-differentiating medium includes glutamine, which can be in the form of a glutamine-containing dipeptide compound (e.g., L-alanine-L-glutamine or glycyl-L-glutamine). In some instances, the PSC-differentiating medium includes L-alanine- L-glutamine at a concentration of about 1 mM to about 4 mM. In some instances, the L-alanine- L-glutamine concentration is about 1.25 mM to about 3.5 mM, about 1.5 mM to about 3 mMor about 1.75 mM to about 2.25 mM. In some instances, each the PSC-differentiating medium includes about 2 mM L-alanine-L-glutamine.

[0316] In some instances, the PSC-differentiating medium includes an albumin (e.g., a serum albumin or recombinant albumin described herein) at a concentration of about 0.05% to about 2%. In some instances, the albumin can be a recombinant human albumin, which can be provided in a composition comprising a mixture of fatty acids and / or lipids. In some instances, the PSC-differentiating medium includes FAF-BSA or FAF-HSA at a concentration from about 0.05% to about 1%. In some instances, the concentration of FAF-BSA or FAF-HSA can be about 0.05% to about 0.5%, about 0.07% to about 0.25%, about 0.09% to about 0.23%, about 0.11% to about 0.21%, about 0.13% to about 0.19%, or about 0.15% to about 0.17%. In some instances, the FAF-BSA or FAF-HSA concentration in the PSC-differentiating medium can be about 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.20%, 0.21%, 0.22%, 0.23%, 0.24% or 0.25%. In some instances, the PSC-differentiating medium includes about 0.2% FAF-BSA or about 0.2% FAF-HSA.

[0317] In some instances, the PSC-differentiating medium includes a buffer, which can be NaHCO3 at a concentration from about 25 mM to about 60 mM. In some instances, the concentration of NaHCO3in the PSC-differentiating medium is about 30 mM to about 55 mM, about 35 mM to about 50 mM, about 40 mM to about 50 mM, or about 42 mM to about 48 mM. In some instances, the NaHCO3concentration can be about 42 mM, 43 mM, 44 mM, 45 mM, 46 mM, 47 mM or 48 mM. In some instances, the PSC-differentiating medium includes about 45.2 mM NaHCO3(3.8 g / L NaHCO3).

[0318] In some instances, the PSC-differentiating medium includes a serum replacement supplement, which can include one, two, three or all four of INS, transferrin, a selenium (e.g., sodium selenite) and ethanolamine. In some instances, the serum replacement supplement includes INS, transferrin and sodium selenite, which can be provided in a concentrated ITS-G supplement (e.g., as defined herein). In some instances, the serum replacement supplement includes all four of these components, which can be provided in a concentrated ITS-X supplement (e.g., as defined herein). In some instances, the serum replacement supplement in the PSC-differentiating medium is an ITS-X (100x) solution, which has the composition shown in Table 23. In some instances, the concentration (v:v) of the ITS-X (100x) solution in the PSC- differentiating medium can be about 1:1000 to about 1:8000. In some instances, the ITS-X (100x) solution can be present at a concentration (v:v) of about 1:1500 to about 1:7500, about1:2000 to about 1:7000, about 1:2500 to about 1:6500, about 1:3000 to about 1:6000, about 1:3500 to about 1:5500, or about 1:4000 to about 1:5000. In some instances, the ITS-X (100x) supplement concentration (v:v) is about any of 1:3000, 1:3500, 1:4000, 1:4500, 1:5000, 1:5500, 1:6000, 1:6500, 1:7000, 1:7500 and 1:8000. In some instances, the PSC-differentiating medium includes the ITS-X (100x) solution shown in Table 23 at a concentration (v:v) of about 1:5500.

[0319] In some instances, the PSC-differentiating medium includes a TGF-β growth factor, which can be Activin A at a concentration from about 50 ng / mL to about 300 ng / mL. In some instances, the concentration of Activin A in the PSC-differentiating medium can be about 60 ng / mL to about 290 ng / mL, about 80 ng / mL to about 270 ng / mL, about 100 ng / mL to about 250 ng / mL, about 120 ng / mL to about 230 ng / mL, about 140 ng / mL to about 210 ng / mL, about 160 ng / mL to about 190 ng / mL, or about 180 ng / mL. In some instances, the Activin A concentration can be about 150 ng / mL, 160 ng / mL, 170 ng / mL, 180 ng / mL, 190 ng / mL, 200 ng / mL, 210, ng / mL, 220 ng / mL, 230 ng / mL, 240 ng / mL or 250 ng / mL. In some instances, the PSC-differentiating medium includes about 200 ng / mL Activin A. Other suitable TGF-β growth factors include, but are not limited to, growth differentiating factor 8 (GDF8).

[0320] In some instances, the PSC-differentiating medium includes a Wnt pathway signaling activator, which can be a Wnt3a protein, as defined herein, at a concentration from about 5 ng / mL to about 20 ng / mL. In some instances, the Wnt3a protein is recombinant human Wnt3a. In some instances, the Wnt3a protein is recombinant mouse Wnt3a. In some instances, the concentration of the Wnt3a protein in the PSC-differentiating medium can be about 6 ng / mL to about 19 ng / mL, about 8 ng / mL to about 18 ng / mL, about 10 ng / mL to about 16 ng / mL or about 12 ng / mL to about 14 ng / mL. In some instances, the Wnt3a protein concentration in the PSC-differentiating medium can be about 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL or 15 ng / mL. In some instances, the PSC-differentiating medium includes about 12.5 ng / mL recombinant mouse Wnt3a protein or recombinant human Wnt3a protein.

[0321] In some instances, the PSC-differentiating medium includes a GSK-3α and GSK-3β inhibitor / Wnt pathway signaling activator, which can be CHIR99021 at a concentration from about 1 μM to about 5 μM. In some instances, the CHIR99021 concentration in the PSC- differentiating medium can be about 1 μM, 2 μM, 3 μM, 4 μM or 5 μM. In some instances, the PSC-differentiating medium includes about 3 μM CHIR99021. Other suitable GSK-3α and GSK-3β inhibitors / Wnt pathway signaling activators include, but are not limited to, 6- bromoindirubin-3′-oxime (BIO).

[0322] In some instances, the PSC-differentiating medium includes a ROCK inhibitor, which can be Y-27632 (e.g., Y-276322HCl) at a concentration from about 5 μM to about 15 μM. In some instances, the Y-27632 concentration in the PSC-differentiating medium can be about 6 μM to about 14 μM, about 8 μM to about 12 μM, or about 10 μM. In some instances, the Y- 27632 concentration in the PSC-differentiating medium can be about 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM or 15 μM. In some instances, the PSC- differentiating medium includes about 10 μM Y-27532 (e.g., Y-276322HCl). Other suitable ROCK inhibitors include, but are not limited to, Chroman 1, thiazovivin, fasudil / HA1077 and H-1152.

[0323] In some instances, the PSC-differentiating medium includes (i) about 11 mM to about 13 mM glucose; (ii) about 1.75 mM to about 2.25 mM L-alanine-L-glutamine; (iii) about 0.15% to about 0.25% of FAF-BSA or FAF-HSA; (iv) about 42 mM to about 48 mMNaHCO3; (v) an ITS-X (100x) solution (e.g., the composition shown in Table 23) at a ratio of about 1:4000 to about 1:6000; (vi) about 190 ng / mL to about 210 ng / mL Activin A; (vii) about 12 ng / mL to about 13 ng / mL recombinant mouse Wnt3a protein or recombinant human Wnt3a; (viii) about 2.5 μM to about 3.5 μM CHIR99021: and (ix) about 9 μM to about 11 μM Y-27632 (e.g., Y- 27632 2HCl). In some instances, the PSC-differentiating medium also includes the MCDB media shown in Table 16 herein below.

[0324] In some instances, the PSC-differentiating medium includes the MCDB media shown in Table 16, about 12 mM glucose, about 0.25% of FAF-BSA or FAF-HSA, about 45.2 mM NaHCO3, the ITS-X (100x) solution shown in Table 23 at a ratio of about 1:5000, about 200 ng / mL Activin A, about 12.5 ng / mL recombinant mouse Wnt3a or the corresponding concentration of recombinant human Wnt3a, about 3.0 μM CHIR99021 and about 10 μM Y- 27632 (e.g., Y-276322HCl).

[0325] Alternatively, one can differentiate PSCs into ME cells via any of the methods that are well-known in the art.

[0326] Stage 1b Cells and Cell Populations (ME to DE):

[0327] The differentiation methods can begin with or can include differentiating ME cells into DE cells by culturing an ME cell population in an ME-differentiating medium for about 1 day (i.e., Day 1 in FIG. 1B) to obtain a DE cell population including Sox17+cells. In some instances, the method uses an ME cell population obtained by: (a) performing the Stage 1a differentiating method or (b) differentiating PSCs into ME cells by any method known in theart. In some instances, the method includes washing the ME cell population in a wash media before culturing in the ME-differentiating medium.

[0328] In some instances, the culturing of the ME cell population is performed in a bioreactor and includes cell transfer densities from about 5 x 105cells / mL to about 3 x 106cells / mL. In some instances, the culture pH can range from about 6.6 to about 7.4. In yet other instances, pH can be about 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3 or 7.4. In some instances, the dissolved oxygen can be controlled to a concentration from about 20 mmHg to about 100 mmHg. In some instances, agitation rates can be from about 20 rpm to about 60 rpm depending on the bioreactor size. In some instances, the culture is controlled at a temperature of about 36°C to about 38°C, especially about 37°C. In some instances, air, carbon dioxide and oxygen are supplied to the bioreactor via passive diffusion, active overlay or sparging.

[0329] The wash media can include a defined medium comprising glucose (e.g., about 4 mM to about 7 mM) and NaHCO3 (e.g., about 10 mM to about 20 mM). In some instances, the wash media can include about 5.6 mM glucose, about 14 mM NaHCO3 and the MCDB media described in Table 16 herein below.

[0330] ME-differentiating medium: The ME-differentiating medium can include a defined medium comprising glucose and / or fructose and one or more of glutamine (e.g., L-alanine-L- glutamine or glycyl-L-glutamine), albumin (e.g., BSA or HSA), a buffer (e.g., NaHCO3), a serum replacement supplement (e.g., an ITS-G or an ITS-X supplement), a TGF-β superfamily growth factor (e.g., Activin A) and a BMP inhibitor (e.g., LDN-193189). In some instances, the ME-differentiating medium includes the MCDB media described in Table 16 herein below.

[0331] In some instances, the ME-differentiating medium can include glucose at a concentration from about 5 mM to about 20 mM. In some instances, the ME-differentiating medium includes glucose at a concentration of any of the glucose concentration ranges and concentrations described above for the PSC-differentiating medium. In some instances, the glucose concentration in the ME-differentiating medium can be about 11 mM to about 13 mM glucose. In some instances, the ME-differentiating medium can include about 12 mM glucose.

[0332] In some instances, the ME-differentiating medium includes glutamine, which can be in the form of a glutamine-containing dipeptide compound (e.g., L-alanine-L-glutamine or glycyl-L-glutamine). In some instances, the ME-differentiating medium includes L-alanine-L- glutamine at a concentration of about 1 mM to about 4 mM. In some instances, the L-alanine- L-glutamine concentration in the ME-differentiating medium can be any of the L-alanine-L-glutamine concentration ranges and concentrations described above for the PSC-differentiating medium. In some instances, the L-alanine-L-glutamine concentration in the ME-differentiating medium can be about 1.75 mM to about 2.5 mM. In some instances, the ME-differentiating medium includes about 2 mM L-alanine-L-glutamine.

[0333] In some instances, the ME-differentiating medium includes an albumin (e.g., a FAF- albumin or recombinant albumin described herein), which can be at a concentration of about 0.05% to about 2%. In some instances, the albumin in each medium can be a recombinant albumin (e.g., recombinant human albumin), which can be provided in a composition comprising fatty acids and / or lipids. In some instances, the albumin can be FAF-BSA or FAF- HSA, which can be at a concentration from about 0.05% to about 1%. In some instances, the FAF-albumin (e.g., FAF-BSA or FAF-HSA) concentration in the ME-differentiating medium can be selected from any of the FAF-BSA / FAF-HSA concentration ranges and concentrations described above for the PSC-differentiating medium. In some instances, the FAF-BSA or FAF- HSA albumin concentration in the ME-differentiating medium can be about 0.19% to about 0.21%. In some instances, the ME-differentiating medium includes about 0.20% FAF-BSA or about 0.20% FAF-HSA.

[0334] In some instances, the ME-differentiating medium includes a buffer, which can be NaHCO3 at a concentration from about 24 mM to about 60 mM. In some instances, the concentration of NaHCO3in the ME-differentiating medium can be selected from any of the NaHCO3 concentration ranges and concentrations described above for the PSC-differentiating medium. In some instances, the NaHCO3concentration in the ME-differentiating medium can be about 43 mM to about 47 mM. In some instances, the ME-differentiating medium includes about 45.2 mM NaHCO3.

[0335] In some instances, the ME-differentiating medium includes a serum replacement supplement, which can include one, two, three or all four of INS, transferrin, a selenium (e.g., sodium selenite) and ethanolamine. In some instances, the serum replacement supplement includes INS, transferrin and sodium selenite (e.g., the serum replacement supplement can be a concentrated ITS-G supplement). In some instances, the serum replacement supplement includes all four of these components (e.g., the serum replacement supplement can be a concentrated ITS-X supplement). In some instances, the serum replacement supplement in the ME-differentiating medium is an ITS-X (100x) solution, which has the composition shown in Table 23. In some instances, the concentration of the ITS-X (100x) solution in the ME-differentiating medium can be selected from any of the ITS-X (100x) solution concentration ranges and concentrations described above for the PSC-differentiating medium. In some instances, the concentration of the ITS-X (100x) solution in the ME-differentiating medium can be about 1:4500 to about 1:5500. In some instances, the ME-differentiating medium includes the ITS-X (100x) solution shown in Table 23 at a concentration of about 1:5500.

[0336] In some instances, the ME-differentiating medium includes a TGF-β growth factor, which can be Activin A at a concentration from about 50 ng / mL to about 300 ng / mL. In some instances, the concentration of Activin A in the ME-differentiating medium can be selected from any of the Activin A concentration ranges and concentrations described above for the PSC-differentiating medium. In some instances, the Activin A concentration in the ME- differentiating medium can be about 180 ng / mL, 190 ng / mL, 200 ng / mL, 210, ng / mL or 220 ng / mL. In some instances, ME-differentiating medium includes about 200 ng / mL Activin A. Other suitable TGF-β growth factors include, but are not limited to, GDF8.

[0337] In some instances, the ME-differentiating medium includes a BMP inhibitor, which can be LDN-193189 at a concentration from about 5 nM to about 20 nM. In some instances, the concentration of LDN-193189 in the ME-differentiating medium can be about 6 nM to about 18 nM, about 7 nM to about 16 nM, about 8 nM to about 14 nM, or about 9 nM to about 12 nM. In some instances, the LDN-193189 concentration in the ME-differentiating medium can be about 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 11 nM, 12 nM, 13 nM, 14 nM or 15 nM. In some instances, ME-differentiating medium includes about 10 nM LDN-193189. Other suitable BMP inhibitors include, but are not limited to, DMH-1 (e.g., at a concentration of about 150 nM).

[0338] In some instances, the ME-differentiating medium includes (i) about 11 mM to about 13 mM glucose, (ii) about 1.75 mM to about 2.25 mM L-alanine-L-glutamine, (iii) about 0.15% to about 0.25% FAF-BSA or FAF-HSA; (iv) about 42 mM to about 48 mM NaHCO3, (v) about 190 ng / mL to about 210 ng / mL Activin A, and (vi) about 9 nM to about 11 nM LDN-193189. In some instances, the ME-differentiating medium also includes an ITS-X (100x) supplement solution (e.g., the composition shown in Table 23) at a ratio of about 1:4000 to about 1:6:000. In some instances, the ME-differentiating medium also includes the MCDB media shown in Table 16 herein below.

[0339] In some instances, the ME-differentiating medium includes the MCDB media shown in Table 16, about 12 mM glucose, about 0.2% FAF-BSA or about 0.2% FAF-HSA, about 42.5mM NaHCO3, about 200 ng / mL Activin A and about 10 nM LDN-193189. In some instances, ME-differentiating medium also includes the ITSX (100x) solution shown in Table 23 at a ratio of about 1:5000.

[0340] Alternatively, one can differentiate ME cells into DE cells via any of the methods that are well-known in the art.

[0341] Stage 2 Cells and Cell Populations (DE to PGT):

[0342] The differentiation methods can begin with or can include differentiating DE cells into PGT cells by culturing a DE cell population in a DE-differentiating medium for a time period of about 3 days (i.e., Days 2-4 in FIG. 1B) to obtain a PGT cell population including FOXA2+cells (e.g., FOXA2+, PDX1- cells). In some instances, the method uses a DE cell population obtained by: (a) performing the Stage 1b differentiating method, and optionally also performing the Stage 1a differentiating method, or (b) differentiating PSCs or ME cells into DE cells by any method known in the art. In some instances, the method includes replacing the DE-differentiating medium in the culture with fresh DE-differentiating medium one or two times during the time period (i.e., at about 24 hr and / or about 48 hr after initiating the culturing step).

[0343] In some instances, the culturing of the DE cell population is performed in a bioreactor and includes cell transfer densities from about 5 x 105cells / mL to about 3 x 106cells / mL. In some instances, the culture pH can range from about 6.6 to about 7.4. In yet other instances, pH can be about 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3 or 7.4. In some instances, the dissolved oxygen can be controlled to a concentration from about 20 mmHg to about 100 mmHg. In some instances, agitation rates can be from about 20 rpm to about 60 rpm depending on the bioreactor size. In some instances, the culture is controlled at a temperature of about 36°C to about 38°C, especially about 37°C. In some instances, air, carbon dioxide and oxygen are supplied to the bioreactor via passive diffusion, active overlay or sparging.

[0344] DE-differentiating medium. The DE-differentiating medium can include a defined medium comprising glucose and / or fructose and one or more of glutamine (e.g., a glutamine dipeptide), albumin (e.g., FAF-BSA or FAF-HSA), a buffer (e.g., NaHCO3), a Vitamin C compound (e.g., ascorbic acid), a serum replacement supplement (e.g., an ITS-G or ITS-X supplement) and a growth factor from the FGF family (e.g., KGF). In some instances, the DE- differentiating medium includes the Basal A Media described in Table 3 herein below.

[0345] In some instances, DE-differentiating medium includes each of glucose, L-alanine-L- glutamine, FAF-BSA (or FAF-HSA) and NaHCO3, which can be present at concentrations selected from the respective glucose, L-alanine-L-glutamine and FAF-BSA (or FAF-HSA) and NaHCO3concentration ranges and concentrations described above for the PSC-differentiating and ME-differentiating mediums. In some instances, the concentrations of glucose, L-alanine- L-glutamine, FAF-BSA (or FAF-HSA) and NaHCO3in the DE-differentiating medium can be about 10 to about 15 mM, about 1 to about 3 mM, about 0.19% to about 0.21% or about 35 mM to 54 mM, respectively. In some instances, the DE-differentiating medium includes about 12 mM glucose, about 2 mM L-alanine-L-glutamine, about 0.2% FAF-BSA (or FAF-HSA) and about 45.2 mM (3.8 g / L) NaHCO3.

[0346] In some instances, the DE-differentiating medium includes a Vitamin C compound, which can be ascorbic acid at a concentration of about 0.05 mM to about 0.50 mM. In some instances, the ascorbic acid concentration in the DE-differentiating medium can be about 0.10 mM to about 0.45 mM, about 0.15 mM to about 0.40 mM, about 0.20 mM to about 0.35 mM, or about 0.30 mM. In some instances, the ascorbic acid concentration can be about 0.05 mM, 0.10 mM, 0.15 mM, 0.20 mM, 0.25 mM, 0.30 mM, 0.35 mM, 0.40 mM, 0.45 mM or 0.50 mM. In some instances, the DE-differentiating medium includes about 0.25 mM ascorbic acid. In some instances, the Vitamin C compound is dehydroascorbic acid.

[0347] In some instances, the DE-differentiating medium includes a serum replacement supplement, which can include a mixture of two, three or all four of INS, transferrin, a selenium (e.g., sodium selenite) and ethanolamine. In some instances, the serum replacement supplement includes INS, transferrin and sodium selenite (e.g., the serum replacement can be a concentrated ITS-G supplement). In some instances, the serum replacement supplement includes all four of these components (e.g., the serum replacement can be a concentrated ITS- X supplement). In some instances, the serum replacement supplement in the DE-differentiating medium can be an ITS-X (100x) solution, which has the composition shown in Table 23. In some instances, the concentration (v:v) of the ITS-X (100x) solution in the DE-differentiating medium can be about 1:50 to about 1:400. In some instances, the ITS-X (100x) solution can be present at a concentration (v:v) of about 1:75 to about 1:350, about 1:100 to about 1:300, about 1:125 to about 1:250, about 1:150 to about 1:225 or about 1:175 to about 1:200. In some instances, the ITS-X (100x) supplement concentration (v:v) can be about 1:100, 1:150, 1:200,1:250 or 1:300. In some instances, the PSC-differentiating medium includes the ITS-X (100x) solution shown in Table 23 at a concentration (v:v) of about 1:200.

[0348] In some instances, the DE-differentiating medium includes a FGF family growth factor, which can be a KGF protein at a concentration from about 10 ng / mL to about 200 ng / mL. In some instances, the KGF protein is a recombinant human KGF protein can be at a concentration from about 15 ng / mL to about 200 ng / mL, about 20 ng / mL to about 150 ng / mL, about 25 ng / mL to about 100 ng / mL, about 30 ng / mL to about 75 ng / mL or about 35 ng / mL to about 50 ng / mL. In some instances, the concentration of recombinant human KGF protein in the DE-differentiating medium can be about 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, 55 ng / mL, 60 ng / mL, 65 ng / mL, 70 ng / mL or about 75 ng / mL. In some instances, the DE-differentiating medium includes about 50 ng / mL of recombinant human KGF protein. Other suitable FGF family growth factors include FGF2, FGF8B, FGF10 and FGF21. In some instances, the DE-differentiating medium also includes an ITS-X (100x) solution (e.g., the composition shown in Table 23) at a ratio of about 1:175 to about 1:225.

[0349] In some instances, the DE-differentiating medium includes (i) about 11 mM to about 13 mM glucose, (ii) about 1.75 mM to about 2.25 mM L-alanine-L-glutamine, (iii) about 0.15% to about 0.25% FAF-BSA or 0.25% FAF-HSA, (iv) about 40 mM to about 50 mM NaHCO3, (v) about 0.20 mM to about 0.3 mM ascorbic acid and (vi) about 40 ng / ml to about 60 ng / mL recombinant human KGF protein. In some instances, the DE-differentiating medium also includes an ITS-X (100x) solution (e.g., the composition shown in Table 23) at a ratio of about 1:175 to about 1:225. In some instances, the PSC-differentiating medium also includes the MCDB A / B media shown in Table 16 herein below.

[0350] In some instances, the DE-differentiating medium includes the MCDB media shown in Table 16, about 12 mM glucose, about 0.2% FAF-BSA (or FAF-HSA), about 45.2 mM NaHCO3, about 0.25 mM ascorbic acid and about 50 ng / mL recombinant human KGF protein. In some instances, ME-differentiating medium also includes the ITSX (100x) solution shown in Table 23 at a ratio of about 1:200.

[0351] Alternatively, one can differentiate DE cells into PGT cells via any of the methods that are well-known in the art.

[0352] Stage 3 Cells and Cell Populations (PGT to FE):

[0353] The methods can include differentiating PGT cells to FE cells by culturing a PGT cell population in a first PGT-differentiating medium for a first time period of about 1 day (i.e., Day 5 in FIG. 1B) to obtain an intermediate PGT / FE cell population (e.g., as defined herein) and then culturing the intermediate PGT / FE cell population in a second PGT-differentiating medium for a second time period of about 1 day (i.e., Day 6 in FIG. 1B) to obtain a FE cell population including PDX1+cells. In some instances, the method uses a PGT cell population obtained by: (a) performing the Stage 2 differentiation method, and optionally also performing the Stage 1a and Stage 1b differentiation methods, or (b) differentiating PSCs into FE cells by any method known in the art.

[0354] In some instances, the culturing of the PGT and PGT / FE cell populations can be performed in a bioreactor and includes cell transfer densities from about 5 x 105cells / mL to about 3.5 x 106cells / mL. In some instances, the culture pH can range from about 6.6 to 7.4. In yet other instances, pH can be about 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3 or 7.4. In some instances, the dissolved oxygen can be controlled to a concentration from about 20 mmHg to about 100 mmHg. In some instances, agitation rates can be from about 20 rpm to about 60 rpm depending on the bioreactor size. In some instances, the culture is controlled at a temperature of about 36°C to about 38°C, especially about 37°C. In some instances, air, carbon dioxide and oxygen are supplied to the bioreactor via passive diffusion, active overlay or sparging.

[0355] PGT-differentiating mediums. Each of the first and second PGT-differentiating mediums can include a defined medium comprising glucose and / or fructose and one or more of glutamine (e.g., a glutamine dipeptide), albumin (e.g., BSA or HSA), a buffer (e.g., NaHCO3), a Vitamin C compound (e.g., ascorbic acid), a serum replacement supplement (e.g., a B27 supplement), a FGF family growth factor (e.g., KGF), a PKC activator (e.g., TPPB), a retinoid (e.g., ATRA), a ROCK inhibitor (e.g., Y-27632), a cell-permeable SHH signaling inhibitor (e.g., SANT-1), a TGF-β superfamily growth factor (e.g., Activin A) and at least one tankyrase 1 / 2 inhibitor (e.g., IWR-1 and / or WIKI4). In some instances, the first PGT- differentiating medium also includes a small molecule BMP inhibitor (e.g., DMH-1). In some instances, each PGT-differentiating medium includes the MCDB Media described in Table 16 herein below.

[0356] In some instances, the glucose and / or fructose concentrations in each PGT- differentiating medium can be the same or different. In some instances, each PGT- differentiating medium includes glucose at a concentration from about 5 mM to about 50 mM.In some instances, the glucose concentration can be about 10 mM to about 45 mM, about 15 mM to about 40 mM, about 20 mM to about 35 mM or about 25 mM to about 30 mM. In some instances, the glucose concentration can be about 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 45 mM or 50 mM. In some instances, the glucose concentration in each of the first and second PGT-differentiating mediums can be about 25 mM.

[0357] In some instances, one or both PGT-differentiating mediums includes glutamine, which can be provided in the form of L-alanine-L-glutamine at a concentration that can be the same or different in each medium.

[0358] In some instances, one or both PGT-differentiating mediums includes a buffer, which can be NaHCO3at a concentration that is the same or different in each medium.

[0359] In some instances, each PGT-differentiating medium includes L-alanine-L-glutamine and NaHCO3 at concentrations that can be selected from the respective L-alanine-L-glutamine and NaHCO3 concentration ranges and concentrations described above for the DE- differentiating medium. In some instances, each of the PGT-differentiating mediums includes about 1.75 mM to about 2.25 mM L-alanine-L-glutamine and about 42 mM to about 48 mM NaHCO3. In some instances, each of the PGT-differentiating mediums includes about 2 mM L-alanine-L-glutamine and about 42.5 mM NaHCO3.

[0360] In some instances, one or both PGT-differentiating mediums can include an albumin, which can be at the same or different concentration in each medium. In some instances, the albumin concentration in each PGT-differentiating medium can be about 0.5% to about 5%. In some instances, the albumin in each medium can be a recombinant human albumin, which can be provided in a composition comprising fatty acids and / or lipids. In some instances, the albumin can be FAF-BSA or FAF-HSA, which can be at a concentration from about 1% to about 3%. In some instances, the FAF-BSA or FAF-HSA concentration in each of the PGT- differentiating mediums can be about 0.5%, 1%, 1.5%, 2%, 2.5%, 3% or 3.5%. In some instances, each of the PGT-differentiating mediums includes about 2% FAF-BSA (or FAF- HSA).

[0361] In some instances, each PGT-differentiating medium includes a Vitamin C compound, which can be ascorbic acid at the same or different concentrations in each medium.

[0362] In some instances, each PGT-differentiating medium includes a FGF family growth factor, which can be KGF at the same or different concentrations in each medium.

[0363] In some instances, each PGT-differentiating medium includes ascorbic acid and KGF, which can be present at concentrations selected from the respective ascorbic acid and KGF concentration ranges and concentrations described above for the DE-differentiating medium. In some instances, each of the PGT-differentiating mediums includes about 0.20 mM to about 0.30 mM ascorbic acid, and about 45 ng / mL to about 55 ng / mL recombinant human KGF. In some instances, each of the PGT-differentiating mediums includes about 0.25 mM ascorbic acid and about 50 ng / ml KGF.

[0364] In some instances, each PGT-differentiating medium includes a serum replacement supplement, which can include two, three, four, five or more of the components of a B27 supplement as defined herein. In some instances, the serum replacement supplement can be a concentrated B27 supplement (e.g., B27 (50x)) that can be added to the defined medium at a volume that is selected to achieve a desired final concentration, which can be the same or different in each PGT-differentiating medium. In some instances, the B27 (50x) supplement can be present in each PGT-differentiating medium at a concentration of about 0.1x to about 1.0x. In some instances, the serum replacement supplement is the B27 (50x) supplement shown in Table 28 herein below, or the commercially available B-27TMSupplement (50x), either of which can be present in each PGT-differentiating medium at a concentration of about 0.2x to about 0.9x, about 0.3x to about 0.8x, about 0.4x to about 0.7x or about 0.5x to about 0.6x. In some instances, the B27 (50x) supplement concentration in each PGT-differentiating medium can be about 0.1x, 0.2x, 0.3x, 0.4x, 0.5x, 0.6x, 0.7x ,0.8x, 0.9x or 1.0x. In some instances, each of the first and second PGT-differentiating mediums includes the B27 (50x) supplement at about 0.5x concentration.

[0365] In some instances, each PGT-differentiating medium includes a PKC activator, which may be the same or different in each medium. In some instances, the PKC activator in each PGT-differentiating medium is TPPB, which can be at the same or different concentration in each medium. In some instances, the TPPB concentration in each PGT-differentiating medium can be from about 5 nM to about 100 nM. In some instances, the concentration of TPPB in each PGT-differentiating medium can be about 10 nM to about 90 nM, about 20 nM to about 80 nM, about 30 nM to about 70 nM, about 40 nM to about 60 nM or about 45 nM to about 55 nM. In some instances, each PGT-differentiating medium includes TPPB at a concentration of about 20 nM, 25 nM, 30 nM, 35 nM, 40 nM, 45 nM, 50 nM, 55 nM, 60 nM, 65 nM, 70 nM, 75 or 80 nM. In some instances, each PGT-differentiating medium includes about 50 nM TPPB.

[0366] In some instances, each PGT-differentiating medium includes a retinoid, which may be the same or different in each medium. In some instances, the retinoid in each medium is ATRA, which can be at the same or different concentration in each medium. In some instances, the ATRA concentration in each PGT-differentiating medium is from about 0.25 μM to about 10 μM. In some instances, the concentration of ATRA in each PGT-differentiating medium can be about 0.50 μM to about 8 μM, about 1 μM to about 6 μM, about 1.5 μM to about 4 μM or about 2 μM to about 5 μM. In some instances, the ATRA concentration can be about 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM or 5 μM. In some instances, each PGT- differentiating medium includes about 3 μM ATRA.

[0367] In some instances, each PGT-differentiating medium includes a Rock inhibitor, which may be the same or different in each medium. In some instances, the ROCK inhibitor in each PGT-differentiating medium is Y-27632 (e.g., Y-276322HCl), which can be at the same or different concentration in each medium. In some instances, the Y-27632 concentration in each PGT-differentiating medium can be about 1 μM to about 20 μM. In some instances, the Y- 27632 concentration in each PGT-differentiating medium can be selected from the Y-27632 2HCl concentration ranges and concentrations described above for the PSC-differentiating medium. In some instances, the Y-27632 (e.g., Y-276322HCl) concentration in each PGT- medium can be about 9 μM to about 11 μM. In some instances, each PGT-differentiating medium includes about 10 μM Y-27632 (Y-27632 2HCl). Other suitable ROCK inhibitors include, but are not limited to, Chroman 1, thiazovivin, fasudil / HA1077 and H-1152.

[0368] In some instances, each PGT-differentiating medium includes a cell-permeable SHH signaling inhibitor, which may be the same or different in each medium. In some instances, the SHH signaling inhibitor in each PGT-differentiating medium is SANT-1, which can be at the same or different concentration in each medium. In some instances, the SANT-1 concentration in each PGT-differentiating medium can be about 0.1 μM to about 0.5 μM. In some instances, the concentration of SANT-1 in each PTG-differentiating medium can be about 0.15 μM to about 0.4 μM or about 0.2 μM to about 0.3 μM. In some instances, the SANT-1 concentration in each PGT-differentiating medium can be about 0.15 μM, 0.2 μM, 0.25 μM, 0.3 μM, 0.35 μM or 0.4 μM. In some instances, each PGT-differentiating medium includes about 0.25 μM SANT-1.

[0369] In some instances, each PGT-differentiating medium includes a TGF-β superfamily growth factor, which may be the same or different in each medium. In some instances, theTGF-β growth factor in each PGT-differentiating is Activin A, which can be at the same or different concentration in each medium. In some instances, the Activin A concentration in each PGT-differentiating medium can be from about 5 ng / mL to about 40 ng / mL. In some instances, the concentration of Activin A can be about 5 ng / mL to about 35 ng / mL, about 10ng / mL to about 30 ng / mL or about 15 ng / mL to about 20 ng / mL. In some instances, the concentration of Activin A in each PGT-differentiating medium can be about 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 21, ng / mL, 22 ng / mL, 23 ng / mL, 24 ng / mL, 25 ng / mL, about 26 ng / mL, 27 ng / mL, 28 ng / mL, 29 ng / mL or 30 ng / mL. In some instances, each PGT-differentiating medium includes about 20 ng / mL Activin A.

[0370] In some instances, each PGT-differentiating medium includes a tankyrase 1 / 2 inhibitor (e.g., an adenosine subsite binding inhibitor or an adenosine subsite binding / G loop interacting inhibitor), which can be the same or different in each medium, or at different concentrations in each medium. In some instances, the adenosine subsite binding inhibitor is IWR-1, JW55 or JW74. In some instances, the adenosine subsite binding / G loop interacting inhibitor is WIKI4.

[0371] In some instances, the tankyrase 1 / 2 inhibitor in one or both PGT-differentiating mediums is an adenosine subsite binding inhibitor, which can be IWR-1 at the same or different concentration in each medium. In some instances, the IWR-1 concentration in each PGT- differentiating medium can be about 50 nM to about 400 nM. In some instances, the concentration of IWR-1 can be about 75 nM to about 375 nM, about 100 nM to about 350 nM, about 125 to about 325 nM, about 150 nM to about 300 nM or about 175 nM to about 275 nM. In some instances, the IWR-1 concentration in each PGT-differentiating medium can be about 100 nM, 125 nM, 150 nM, 175 nM, 200 nM, 225 nM, 250 nM, 275 nM or 300 nM. In some instances, each PGT-differentiating medium includes about 200 nM IWR-1. In some instances, each PGT-differentiating medium lacks any other tankyrase inhibitor.

[0372] In some instances, the tankyrase 1 / 2 inhibitor in one or both PGT-differentiating mediums is an adenosine subsite binding / G loop interacting inhibitor, which can be WIKI4 at the same or different concentration in each medium. In some instances, the WIKI4 concentration in each PGT-differentiating medium can be from about 1 µM to about 30 µM. In some instances, the concentration of WIKI4 can be about 3 µM to about 20 µM, about 4 µM to about 15 µM, about 6 µM to about 12 µM or about 8 µM to about 10 µM. In some instances,the WIKI4 concentration in each PGT-differentiating medium can be about 3 µM, 6 µM, 9 µM, 12 µM or 15 µM. In some instances, each PGT-differentiating medium includes about 9 µM WIKI4. In some instances, each PGT-differentiating medium lacks any other tankyrase inhibitor.

[0373] In some instances, one or both PGT-differentiating mediums includes two tankyrase 1 / 2 inhibitors: one is an adenosine subsite specific binding inhibitor (e.g., IWR-1. JW55, or JW74) and the other is an adenosine subsite / G-loop interacting inhibitor (e.g., WIKI4). In some instances, only the second PGT-differentiating medium includes two tankyrase 1 / 2 inhibitors.

[0374] In some instances, each PGT-differentiating medium includes both IWR-1 and WIKI4, which can be present at the same or different concentrations in each medium. In some instances, the IWR-1 and WIKI 4 concentrations in any PGT-differentiating medium that includes both compounds can be selected from any of the IWR-1 and WIKI4 concentrations described above. In some instances, the IWR-1 and WIKI4 concentrations in each PGT- differentiating medium, or in just the second PGT-differentiating medium, can be about 180 nM to about 220 nM and about 8 µM to about 10 µM, respectively. In some instances, each PGT-differentiating medium, or just the second PGT-differentiating medium, includes about 200 nM IWR-1 and about 9 µM WIKI4.

[0375] In some instances, the first PGT-differentiating medium includes a small molecule BMP inhibitor, which can be DMH-1 at a concentration from about 50 nM to about 250 nM. In some instances, the concentration of DMH-1 in the first PGT-differentiating medium can be about 75 nM to about 225 nM, about 100 nM to about 200 nM, about 125 nM to about 175 nM or about 140 nM to about 160 mM. In some instances, the DMH-1 concentration can be about 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180 nM, 190 nM or 200 nM. In some instances, the first PGT-differentiating medium includes about 150 nM DMH- 1. Other suitable BMP inhibitors include, but are not limited to, LDN-193189.

[0376] In some instances, the first PGT-differentiating medium includes about 125 nM to about 175 nM DMH-1 and each of the first and second PGT-differentiating mediums includes about 20 mM to about 30 mM glucose, about 1.75 mM to about 2.25 mM L-alanine-L- glutamine, about 1.5% to about 2.5% BSA or HSA, about 42 mM3.6 g / L to about 48 mM NaHCO3, about 0.20 mM to about 0.30 mM ascorbic acid, about 45 ng / mL to about 55 ng / mL recombinant human KGF, 45 nM to about 55 nM TPPB, about 2.5 μM to 3.5 μM ATRA, about 9 μM to about 11 μM Y-27632 (e.g., Y-276322HCl), about 0.2 μM to about 0.3 μM SANT-1,about 15 ng / mL to about 25 ng / mL Activin A, and optionally one or both of IWR-1 and WIKI4 at concentrations of about 180 nM to about 220 nM and about 8 µM to about 10 µM, respectively. In some instances, the first PGT-differentiating medium includes IWR-1 or WIKI4 but does not include both compounds. In some instances, each PGT-differentiating medium also includes a B27 (50x) supplement at about 0.25x to about 0.75x. In some instances, each PGT-differentiating medium also includes the Table 1 MCDB media.

[0377] In some instances, the first PGT-differentiating medium includes about 150 nM DMH-1 and each of the first and second PGT-differentiating mediums includes the Table 16 MCDB media, about 25 mM glucose, about 2 mM L-alanine-L-glutamine, about 2% BSA (or HSA), about 45.2 mM NaHCO3, about 0.25 mM ascorbic acid, about 50 ng / mL recombinant human KGF, about 50 nM TPPB, about 3 μM ATRA, about 10 μM Y-27632 (e.g., Y-27632 2HCl), about 0.25 μM SANT-1, about 20 ng / mL Activin A and about 200 nM IWR-1-Endo. In some instances, each PGT-differentiating medium includes about 9 µM WIKI4 and does not include IWR-1-Endo. In some instances, each PGT-differentiating medium also includes a B27 (50x) supplement at about 0.5x.

[0378] Alternatively, one can differentiate PGT cells into FE cells via any of the methods that are well-known in the art.

[0379] Stage 4 Cells and Cell Populations (FE to PP):

[0380] The methods can begin with or can include differentiating FE cells to PP cells by culturing an FE cell population in a FE-differentiating medium for a time period of about 3 days (e.g., Days 7-9 in FIG. 1B) to obtain a PP cell population including PDX1+ / NKX6.1+cells. In some instances, the method uses an FE cell population obtained by: (a) performing the Stage 3 differentiation method, and optionally also performing the Stage 1a, Stage 1b and Stage 2 differentiation methods, or (b) differentiating PSCs into FE cells by any method known in the art. In some instances, the method includes replacing the FE-differentiating medium in the culture with fresh FE-differentiating medium one or two times during the time period (e.g., at about 24 hr and / or about 48 hr after initiating the culturing step).

[0381] In some instances, the culturing of the FE cell population can be performed in a bioreactor and includes cell transfer densities from about 1 x 106cells / mL to about 5 x 106cells / mL. In some instances, the culture pH can range from about 6.6 to about 8.0. In instances, pH can be about 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4. 7.5, 7.6, 7.8, 7.9 or 8.0. In some instances, the dissolved oxygen can be controlled to a concentration from about 20 mmHg toabout 150 mmHg. In some instances, agitation rates can be from about 20 rpm to about 60 rpm depending on the bioreactor size. In some instances, the culture can be controlled at a temperature of about 36°C to about 38°C, especially about 37°C. In some instances, air, carbon dioxide and oxygen can be supplied to the bioreactor via passive diffusion, active overlay or sparging.

[0382] FE-differentiating Medium. The FE-differentiating medium can include a defined medium comprising glucose and / or fructose and one or more of glutamine (e.g., a glutamine dipeptide), an albumin (e.g., BSA or HSA), a buffer (e.g., NaHCO3) and one or more of an EGF family growth factor (e.g., recombinant human EGF protein), a Vitamin B3 compound (e.g., NAM), a Vitamin C compound (e.g., ascorbic acid), a serum replacement supplement (e.g., a B27 supplement defined herein), a FGF family growth factor (e.g., a KGF protein), a PKC activator (e.g., TPPB), a retinoid (e.g., ATRA), a ROCK inhibitor (e.g., Y-27632), a cell- permeable SHH signaling inhibitor (e.g., SANT-1), at least one tankyrase 1 / 2 inhibitor (e.g., IWR-1-Endo and / or WIKI4) and a G9a inhibitor (e.g., UNC0321). In some instances, the FE- differentiating medium includes the MCDB media described in Table 16 herein below.

[0383] In some instances, the FE-differentiating medium includes each of glucose, L- alanine-L-glutamine, albumin and NaHCO3, which can be present at concentrations selected from the respective glucose, L-alanine-L-glutamine, albumin and NaHCO3 concentration ranges and concentrations described above for the PGT-differentiating mediums. In some instances, the glucose concentration in the FE-differentiating medium can be about 20 mM to about 30 mM. In some instances, the L-alanine-L-glutamine concentration in the FE- differentiating medium can be about 1.8 mM to about 2.2 mM L-alanine-L-glutamine. In some instances, the albumin is FAF-BSA or FAF-HSA, and the FE-differentiating medium includes FAF-BSA or FAF-HSA at about 1%, 1.5%, 2%, 2.5% or 3%. In some instances, the NaHCO3 concentration is about 42 mM to about 48 mM NaHCO3. In some instances, the FE- differentiating medium includes about 25 mM glucose, about 2 mM L-alanine-L-glutamine, about 2% FAF-BSA or FAF-HSA and about 45.2 mM NaHCO3.

[0384] In some instances, the FE-differentiating medium includes an EGF family growth factor, which can be a recombinant EGF protein at a concentration from about 50 ng / mL to about 350 ng / mL. In some instances, the EGF protein is recombinant human EGF, which can be at a concentration from about 100 ng / mL to about 300 ng / mL, about 125 ng / mL to about 275 ng / mL, about 150 ng / mL to about 250 ng / mL or about 175 ng / mL to about 225 ng / mL. Insome instances, the concentration of recombinant human EGF in the FE-differentiating medium can be about 100 ng / mL, 125 ng / mL, 150 ng / mL, 175 ng / mL, 200 ng / mL, 225 ng / mL, 250 ng / mL, 275 ng / mL or 300 ng / mL. In some instances, the FE-differentiating medium includes about 200 ng / mL recombinant human EGF.

[0385] In some instances, the FE-differentiating medium includes a Vitamin B3 compound, which can NAM at a concentration of about 1 µM to about 20 µM, about 2.5 µM to about 17.5 µM, about 5 µM to about 15 µM or about 7.5 µM to about 12.5 µM. In some instances, the NAM concentration can be about 2 µM, 4 µM, 6 µM, 8 µM, 10 µM, 12 µM, 14 µM, 16 µM or 18 µM. In some instances, the FE-differentiating medium includes 10 µM NAM. In some instances, the FE-differentiating medium does not include NAM. In some instances, the FE- differentiating medium does not include a Vitamin B3 compound in addition to any amount of a Vitamin B3 compound that can be present in the basal medium or in any multi-component supplement present in or added to the FE-differentiating medium.

[0386] In some instances, the FE-differentiating medium includes a Vitamin C compound, which can be ascorbic acid at a concentration of about 0.05 mM to about 0.50 mM. In some instances, the ascorbic acid concentration in the FE-differentiating medium can be selected from the ascorbic acid concentration ranges and concentrations described above for the DE- differentiating mediums. In some instances, the ascorbic acid concentration in the FE- differentiating medium can be about 0.20 mM to about 0.30 mM. In some instances, the FE- differentiating medium includes about 0.25 mM ascorbic acid. In some instances, the Vitamin C compound is dehydroascorbic acid.

[0387] In some instances, FE-differentiating medium includes a serum replacement supplement, which can include the components of a B27 supplement as defined herein. In some instances, the serum replacement supplement can be a concentrated B27 supplement (e.g., B27 (50x)) which can be added to the defined medium at a volume that is selected to achieve a desired final concentration. In some instances, the B27 supplement is a B27 (50x) supplement as described above, which can be present at a concentration from about 0.2x to about 2.0x. In some instances, the B27 supplement is the B27 (50x) supplement shown in Table 28 herein below, or the commercially available B-27TMSupplement (50x), either of which can be present from about 0.3x to about 1.9x, about 0.5x to about 1.7x, about 0.7x to about 1.5x, about 0.9x to about 1.3x or about 1.0x to about 1.1x. In some instances, the FE-differentiating medium includes a B27 (50x) supplement at about 0.6x, 0.7x, 0.8x, 0.9x, 1.0x, 1.1x, 1.2x, 1.3x or1.4x.In some instances, the FE-differentiating medium includes the B27 (50x) supplement at about 1x concentration.

[0388] In some instances, the FE-differentiating medium includes a FGF family growth factor, which can be KGF (e.g., recombinant human KGF) at a concentration selected from the KGF concentration ranges and concentrations described above for the PGT-differentiating mediums. In some instances, the FE-differentiating medium includes recombinant human KGF at a concentration of about 45 ng / mL to about 55 ng / mL. In some instances, the FE- differentiating medium includes about 50 ng / ml recombinant human KGF.

[0389] In some instances, the FE-differentiating medium includes a PKC activator, which can be TPPB at a concentration from about 20 nM to about 200 nM. In some instances, the concentration of TPPB in the FE-differentiating medium can be about 40 nM to about 180 nM, about 60 nM to about 160 nM, about 80 nM to about 140 nM, about 90 nM to about 120 nM or about 100 nM to about 110 nM. In some instances, the FE-differentiating medium includes TPPB at a concentration of about 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM or 150 nM. In some instances, the FE-differentiating medium includes about 100 nM TPPB.

[0390] In some instances, the FE-differentiating medium includes a retinoid, which can be ATRA at a concentration from about 50 nM to about 200 nM. In some instances, the concentration of ATRA in the FE-differentiating medium can be about 60 nM to about 180 μM, about 70 nM to about 160 nM, about 80 nM to about 150 nM or about 90 nM to about 130 nM. In some instances, the ATRA concentration can be about 80 nM, 90 nM, 100 nM, 110 nM or 120 nM. In some instances, the FE-differentiating medium includes about 100 nM ATRA.

[0391] In some instances, the FE-differentiating medium includes one or both of a ROCK inhibitor and a cell-permeable SHH signaling inhibitor. In some instances, the concentrations for the ROCK inhibitor and cell-permeable SHH signaling inhibitor in the FE-differentiating medium can be selected from their respective concentration ranges and concentrations described above for the PGT-differentiating mediums. In some instances, the ROCK inhibitor can be Y-27632 (e.g., Y-276322HCl), which can be at a concentration of about 9 μM to about 11 μM. In some instances, the cell-permeable SHH signaling inhibitor is SANT-1, which can be present at a concentration of about 0.2 μM to about 0.3 μM. In some instances, the FE- differentiating medium includes about 10 μM Y-27632 (e.g., Y-276322HCl) and about 0.25 μM SANT-1.

[0392] In some instances, the FE-differentiating medium includes a tankyrase 1 / 2 inhibitor (e.g., an adenosine subsite binding inhibitor or an adenosine subsite binding / G loop interacting inhibitor), which may be the same or different compound, or may be present at the same or different concentration, than any tankyrase 1 / 2 inhibitor present in the PGT-differentiating mediums. In some instances, the adenosine subsite binding inhibitor can be IWR-1, JW55 or JW74. In some instances, the adenosine subsite binding / G loop interacting inhibitor can be WIKI4.

[0393] In some instances, the tankyrase 1 / 2 inhibitor in the FE-differentiating medium can be an adenosine subsite binding inhibitor, which can be IWR-1 at a concentration from about 50 nM to about 400 nM. In some instances, the concentration of IWR-1 can be about 75 nM to about 375 nM, about 100 nM to about 350 nM, about 125 to about 325 nM, about 150 nM to about 300 nM or about 175 nM to about 275 nM. In some instances, the IWR-1 concentration in the FE-differentiating medium can be about 100 nM, 125 nM, 150 nM, 175 nM, 200 nM, 225 nM, 250 nM, 275 nM or 300 nM. In some instances, the FE-differentiating medium includes about 200 nM IWR-1. Other exemplary tankyrase 1 / 2 inhibitors for use in the FE- differentiating medium instead of IWR-1 include G007-LK (e.g., at a concentration of about 5 µM), JW55 (e.g., at a concentration of about 5 µM) and JW74 (e.g., at a concentration of about 105 µM). In some instances, FE-differentiating medium lacks any other tankyrase inhibitor.

[0394] In some instances, the tankyrase 1 / 2 inhibitor in the FE-differentiating medium can be an adenosine subsite binding / G loop interacting inhibitor, which can be WIKI4 at a concentration from about 1 µM to about 30 µM. In some instances, the concentration of WIKI4 can be about 3 µM to about 20 µM, about 4 µM to about 15 µM, about 6 µM to about 12 µM or about 8 µM to about 10 µM. In some instances, the WIKI4 concentration in the FE- differentiating medium can be about 3 µM, 6 µM, 9 µM, 12 µM or 15 µM. In some instances, the FE-differentiating medium includes about 9 µM WIKI4. In some instances, the FE- differentiating medium lacks any other tankyrase inhibitor.

[0395] In some instances, the FE-differentiating medium includes two tankyrase 1 / 2 inhibitors, where one is an adenosine subsite specific binding inhibitor (e.g., G007-LK, IWR- 1, JW55, or JW74) and the other is an adenosine subsite / G-loop interacting inhibitor (e.g., WIKI4). In some instances, FE-differentiating medium includes both IWR-1 and WIKI4, which can be present at concentrations selected from any of the IWR-1 and WIKI4 concentrations described above. In some instances, the FE-differentiating medium includesabout 180 nM to about 220 nM IWR-1 and about 8 µM to about 10 µM WIKI4. In some instances, the FE-differentiating medium includes about 200 nM IWR-1 and about 9 µM WIKI4.

[0396] In some instances, the FE-differentiating medium includes a G9a inhibitor, which can be UNC0321, UNC0638 or CM-272.

[0397] In some instances, the G9a inhibitor is UNC0321, which can be present in the FE- differentiating medium at a concentration of about 1 μM to about 10 μM. In some instances, the UNC0321 concentration can be about 2 μM to about 9 μM, about 3 μM to about 8 μM, about 4 μM to about 7 μM or about 4.5 μM to about 5.5 μM. In some instances, the UNC0321 concentration can be about 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM or 10 μM. In some instances, the FE-differentiating medium includes about 5 μM UNC0321.

[0398] In some instances, the G9a inhibitor is UNC0638, which can be present in the FE- differentiating medium at a concentration of about 0.1 μM to about 1.0 μM. In some instances, the UNC0638 concentration can be about 0.2 μM to about 0.9 μM, about 0.3 μM to about 0.8 μM, about 0 / 4 μM to about 0.7 μM or about 0.45 μM to about 0.55 μM. In some instances, the UNC0638 concentration can be about 0.2 μM, 0.4 μM, 0.6 μM or 0.8 μM. In some instances, the FE-differentiating medium includes about 0.5 μM UNC0638.

[0399] In some instances, the FE-differentiating medium includes about 20 mM to about 30 mM glucose, about 1.8 mM to about 2.2 mM L-alanine-L-glutamine, about 1.5% to about 2.5% BSA or HSA, about 42 mM to about 48 mM NaHCO3, about 175 ng / mL to about 225 ng / mL recombinant human EGF, about 9 µM to about 11 µM NAM, 0.20 mM to about 0.30 mM ascorbic acid, about 45 ng / mL to about 55 ng / mL recombinant human KGF, about 90 nM to about 110 nM TPPB, 90 nM to about 110 nM ATRA, 9 μM to about 11 μM Y-27632 (e.g., Y- 276322HCl), about 0.2 μM to about 0.3 μM SANT-1, about 180 nM to about 220 nM IWR-1- Endo and / or about 8 uM to about 10 uM WIKI4, and optionally about 4.0 μM to about 6.0 μM UNC0321 (or about 0.4 μM to about 0.6 μM UNC0638). In some instances, the FE- differentiating medium also includes a B27 (50x) supplement at about 0.5x to about 1.5x. In some instances, FE-differentiating medium also includes the Table 16 MCDB media.

[0400] In some instances, the FE-differentiating medium includes the MCDB A / B media shown in Table 16, about 25 mM glucose, about 2 mM L-alanine-L-glutamine, about 2% BSA (or HSA), about 45.2 mM NaHCO3, about 20 ng / mL recombinant human EGF, about 10 µM NAM, about 0.25 mM ascorbic acid, about 50 ng / mL recombinant human KGF, about 100 nMTPPB, about 100 nM ATRA, about 10 μM Y-27632 (e.g., Y-276322HCl), about 0.25 μM SANT-1, one or both of about 200 nM IWR-1-Endo and about 9 µM WIKI4, and optionally about 5 μM UNC0321 (or about 0.5 μM UNC0638). In some instances, the FE-differentiating medium also includes a B27 (50x) supplement at about 1x.

[0401] Alternatively, one can differentiate FE cells to PP cells via any of the methods that are well-known in the art.

[0402] Stage 5 Cells and Cell Populations (PP to PEP):

[0403] The differentiation methods can begin with or can include differentiating PP cells to PEP cells by culturing a PP cell population in a first PP-differentiating medium for a first time period of about 4 days (i.e., Days 10-13 in FIG. 1B) to obtain an intermediate PP / PEP cell population (e.g., as defined herein), washing the intermediate PP / PEP cell population in a PP / PEP wash media comprising less than about 2.5, less than about 2 mM glucose (e.g., < about 1 mM glucose or glucose-free (i.e., 0 mM)), and then culturing the washed, intermediate PP / PEP cell population in a second PP-differentiating medium comprising < about 2 mM glucose (e.g., ≤ about 1 mM glucose or glucose-free) for a second time period of about 2 days (i.e., Days 14-15 in FIG. 1B) to obtain a PEP cell population including CHGA+ / PDX1+cells. In some instances, the first and / or second PP-differentiating medium comprises glucose at concentration of about 0 to about 0.02 mM, about 0 to about 0.04 mM, about 0 to about 0.06 mM, about 0 to about 0.08 mM about 0 to about 0.1 mM 0, about 0 to about 0.2 mM, about 0 to about 0.3 mM, about 0 to about 0.4 mM, about 0 to about 0.5 mM, about 0 to about 0.6 mM, about 0 to about 0.7 mM, about 0 to about 0.8 mM, about 0 to about 0.9 mM, about 0 to about 1.0 mM, about 0 to about 1.1 mM, about 0 to about 1.2 mM, about 0 to about 1.3 mM, about 0 to about 1.4 mM, about 0 to about 1.5 mM, about 0 to about 1.6 mM, about 0 to about 1.7 mM, about 0 to about 1.8 mM, about 0 to about 1.9 mM, about 0 to about 2.0 mM, about 0 to about 2.1 mM, about 0 to about 2.2 mM, about 0 to about 2.3 mM, about 0 to about 2.4 mM, or about 0 to about 2.5 mM. In some instances the PP-differentiating medium comprises glucose at concentration of about 0mM, about 0.1mM, about 0.2mM, about 0.3mM, about 0.4mM, about 0.5mM, about 0.6mM, about 0.7mM, about 0.8mM, about 0.9mM, about 1.0mM, about 1.1mM, about 1.2mM, about 1.3mM, about 1.4mM, about 1.5mM, about 1.6mM, about 1.7mM, about 1.8mM, about 1.9mM, about 2.0mM, about 2.1mM, about 2.2mM, about 2.3mM, about 2.4mM, or about 2.5mM. In some instances, the method uses a PP cell population obtained by: (a) performing the Stage 4 differentiation method described herein,and optionally also performing the Stage 1a, Stage 1b, Stage 2 and Stage 3 differentiation methods, or (b) differentiating PSCs into PP cells by any method known in the art.

[0404] In some instances, the PP to PEP differentiating method includes replacing the first PP-differentiating medium in the culture with fresh first PP-differentiating medium one or more times during the first time period (e.g., at one or more of about 24 hr, 48 hr or 72 hr after initiating the culturing in the first PP-differentiating medium), and replacing the second PP- differentiating medium in the culture with fresh second PP-differentiating medium one time during the second time period (e.g., at about 24 hr after starting culturing in the second PP- differentiating medium).

[0405] In some instances, the PEP cell population includes cell aggregates, and the method can include dissociating the cell aggregates into single cells. The dissociating step can be chemically, enzymatically or mechanically mediated via methods that are well-known in the art. See, e.g., Veres et al. (2019) Nature.569:368-373; Ali et al. (2023) Biol Open 12(3); and Velazco-Cruz et al. (2019) Stem Cell Rep 12(2): 351-365. In some instances, the dissociating step includes collecting the aggregates from the PEP cell population and contacting the collected aggregates with an enzymatic solution comprising trypsin activity, chymotrypsin / elastase activity and collagenase type 1 activity for a time period sufficient to obtain a dissociated PEP cell population including CHGA+ / PDX1+cells. In some instances, the time period can be from about 3 minutes to about 12 minutes, about 4 minutes to about 11 minutes, about 5 minutes to about 10 minutes or about 6 minutes to about 8 minutes.

[0406] In some instances, the culturing of the PP and PP / PEP cell populations can be performed in a bioreactor and includes cell transfer densities from about 1 x 106cells / mL to about 5 x 106cells / mL. In some instances, the culture pH can range from about 6.6 to about 8.0. In yet other instances, pH is about: 6.6, 6.8, 7.0, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0. PP and PP / PEP cell populations cultured at this stage of differentiation in a bioreactor are typically cultured at a range of pH between pH7.0 and pH7.2. The present methods unexpectedly demonstrate that culturing certain cell populations such as, for example, populations comprising PP cells, at a pH higher than pH7.0 to pH7.2, selectively improves the proportion of mature SC-ICs in the in vitro differentiated cell population. In some instances, culturing comprises monitoring and adjusting the pH in the bioreactor so as to maintain a pH that is not higher than pH7.8. In some instances culturing comprises monitoring and adjusting the pH in the bioreactor so as to maintain a pH that is a pH in the range of: pH7.2 to pH7.8,pH7.4 to pH7.8, pH 7.2+ / - 0.2 (pH7.0 to pH7.4), pH 7.4+ / - 0.2 (pH7.2 to pH7.6), pH7.6+ / - 0.2 (pH7.4 to pH7.8) or pH7.8+ / - 0.2 (pH7.6 to pH8.0). Adjusting the pH is achieved by any suitable means such as, for example, supplying a suitable buffer to maintain the desired pH range. In some instances, the dissolved oxygen can be controlled to a concentration from about 20 mmHg to about 150 mmHg. In some instances, agitation rates can be from about 20 rpm to 60 rpm depending on the bioreactor size. In some instances, the culture is controlled at a temperature of about 36°C to about 38°C, especially about 37°C. In some instances, air, carbon dioxide and oxygen are supplied to the bioreactor via passive diffusion, active overlay or sparging.

[0407] First PP-differentiating Medium. The first PP-differentiating medium can include a defined medium having glucose and / or fructose and one or more of glutamine (e.g., a glutamine dipeptide), pyruvate, albumin (e.g., BSA or HSA), a buffer (e.g., NaHCO3), a small molecule BMP inhibitor (e.g., LDN-193189), a zinc compound (e.g., ZnSO4), a thyroid hormone signaling pathway activator (e.g., T3), a heparin (e.g., a UFH), an ATP-competitive inhibitor of TGF-β RI kinase (e.g., ALK5iII), a serum replacement supplement (e.g., a B27 supplement described herein), a cell-permeable SHH signaling inhibitor (e.g.,SANT-1), a NEAA supplement, a ROCK inhibitor (e.g., Y-27632), a Vitamin C compound (e.g., ascorbic acid), a γ-secretase inhibitor (e.g., GSI-XX), an epigenetic modifier (e.g., a G9a inhibitor such as UNC0321) and at least one tankyrase 1 / 2 inhibitor (e.g., IWR-1 and / or WIKI4). In some instances, the first PP-differentiating medium includes the MCDB media described in Table 16 herein below.

[0408] In some instances, the first PP-differentiating medium includes each of glucose or is glucose-free, L-alanine-L-glutamine, an albumin and NaHCO3, which can be at concentrations selected from the respective glucose, L-alanine-L-glutamine, albumin, and NaHCO3 concentration ranges and concentrations described above for the PGT-differentiating mediums. In some instances, the glucose concentration in the first PP-differentiating medium can be about 20 mM to about 30 mM. In some instances, the L-alanine-L-glutamine concentration in the first PP-differentiating medium can be about 1.8 mM to about 2.2 mM. In some instances, the albumin can be FAF-BSA or FAF-HSA, and the first PP-differentiating medium includes FAF-BSA or FAF-HSA at about 1.5% to about 2.5%. In some instances, the NaHCO3 concentration can be about 42 mM to about 48 mM. In some instances, the first PP-differentiating medium includes about 25 mM glucose, about 2 mM L-alanine-L-glutamine, about 2% FAF-BSA and about 45.2 mM NaHCO3.

[0409] In some instances, the first PP-differentiating medium optionally includes pyruvate, which can be at a concentration of about 0.25 mM to 2.0 mM, about 0.5 mM to about 1.50 mM or about 0.75 mM to about 1.25 mM. In some instances, the pyruvate concentration in the first PP-differentiating medium can be about 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM or 1.5 mM. In some instances, the first PP-differentiating medium includes about 1.0 mM pyruvate. In some instances, the first PP-differentiating medium is substantially pyruvate free.

[0410] In some instances, the first PP-differentiating medium includes a small molecule BMP inhibitor (e.g., LDN-193189 or DMH-1). In some instances, the BMP inhibitor is LDN- 193189, which can be present at a concentration of about 50 nM to about 200 nM. In some instances, the concentration of LDN-193189 in the first PP-differentiating medium can be about 60 nM to about 190 nM, about 70 nM to about 180 nM, about 80 nM to about 170 nM, about 90 nM to about 160 nM, about 100 nM to about 150 nM, about 110 nM to about 140 nM or about 120 nM to about 130 nM. In some instances, the LDN-193189 concentration in the first PP-differentiating medium can be about 70 nM, 80 nM, 90 nM, 100 nM, 110 nM, 120 nM or 130 nM. In some instances, the first PP-differentiating medium includes about 100 nM LDN- 193189.

[0411] In some instances, the first PP-differentiating medium includes a zinc compound, which can be ZnSO4, at a concentration of about 1 μM to about 5 μM, about 2 μM to about 4 μM or about 3 μM. In some instances, the ZnSO4 concentration in the first PP-differentiating medium can be about 1 μM, 2 μM, 3 μM or 4 μM. In some instances, the first PP-differentiating medium includes about 2 μM ZnSO4. In some instances, the first PP-differentiating medium does not include a zinc compound in addition to any amount of a zinc compound that can be present in the basal medium or in any multi-component supplement present in or added to the first PP-differentiating medium.

[0412] In some instances, the first PP-differentiating medium includes a thyroid hormone signaling pathway activator, which can be T3 at a concentration of about 1 μM to about 5 μM, about 2 μM to about 4 μM or about 2.5 μM to about 3.5 μM. In some instances, the T3 concentration can be about 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM or 4 μM. In some instances, the first PP-differentiating medium includes about 3 μM T3. Other suitable thyroid hormonesignaling pathway activators include, but are not limited to, other thyroid hormones (e.g., GC- 1) and the T3 analogs and derivatives described in Intl. Patent Application Publication No. WO 2019 / 018818. In some instances, the first PP-differentiating medium does not contain a thyroid hormone signaling pathway activator in addition to any amount of a thyroid hormone signaling pathway activator that can be present in the basal medium or in any multi-component supplement present in or added to the first PP-differentiating medium.

[0413] In some instances, the first PP-differentiating medium includes a heparin (e.g., a UFH) at a concentration of about 1 μg / mL to about 20 μg / mL, about 3 μg / mL to about 18 μg / mL, about 5 μg / mL to about 16 μg / mL, about 7 μg / mL to about 14 μg / mL or about 9 μg / mL to about 12 μg / mL. In some instances, the heparin concentration can be about 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL, 10 μg / mL, 11 μg / mL, 12 μg / mL, 13 μg / mL or 14 μg / mL. In some instances, the first PP-differentiating medium includes about 10 μg / mL UFH-PIM. Other suitable heparins include, but are not limited to, fondaparinux.

[0414] In some instances, the first PP-differentiating medium includes an ATP-competitive inhibitor of TGF-β RI kinase, which can be ALK5iII at a concentration of about 1 μM to about 10 μM. In some instances, ALK5iII concentration can be about 2 μM to about 9 μM, about 3 μM to about 8 μM, about 4 μM to about 7 μM or about 5 μM to about 6 μM. In some instances, the ALK5iII concentration can be about 3 μM, 4 μM, 5 μM, 6 μM or 7 μM. In some instances, the first PP-differentiating medium includes about 5 μM ALK5iII.

[0415] In some instances, the first PP-differentiating medium includes the components of a B27 supplement (e.g., as defined herein). In some instances, the B27 supplement can be a B27 (50x) supplement as described above, which can be present at a concentration selected from the B27 (50x) supplement concentration ranges and concentrations described above for the FE- differentiating medium. In some instances, the concentration of the B27 (50x) supplement in the first PP-differentiating medium can be about 0.9x to about 1.1x concentration. In some instances, the first PP-differentiating medium includes the B27 (50x) supplement at about 1x concentration.

[0416] In some instances, the first PP-differentiating medium includes a cell-permeable SHH signaling inhibitor, which can be SANT-1 at a concentration of about 0.1 μM to about 0.5 μM or about 0.2 μM to about 0.3 μM. In some instances, the SANT-1 concentration can be selected from the SANT-1 concentration ranges and concentrations described above for the PGT-differentiating mediums. In some instances, the first PP-differentiating medium includes about 0.25 μM SANT-1.

[0417] In some instances, the first PP-differentiating medium includes two or more of the components of an NEAA supplement described herein. In some instances, the NEAA supplement components can be provided as an about 100x concentrate solution (e.g., NEAA (100x)) at a volume selected to achieve a desired final concentration in the differentiating medium. In some instances, the NEAA supplement can be the NEAA (100x) supplement shown in Table 24, which can be present in the first PP-differentiating medium at a concentration of about 0.5x to about 1.5x, about 0.7x to about 1.3x or about 0.9x to about 1.1x. In some instances, the concentration of the NEAA (100x) supplement can be about 0.7x, 0.8x, 0.9x, 1.0x, 1.1x ,1.2x or 1.3x. In some instances, the first PP-differentiating medium includes the NEAA (100x) supplement at about 1x concentration. In some instances, the first PP- differentiating medium does not contain a NEAA supplement.

[0418] In some instances, the first PP-differentiating medium includes a ROCK inhibitor, which can be Y-27632 (e.g., Y-276322HCl) at a concentration from about 1 μM to about 20 μM or about 9 μM to about 11 μM. In some instances, the concentration of Y-276322HCl can be selected from the Y-276322HCl concentration ranges and concentrations described above for the PGT-differentiating mediums. In some instances, the first PP-differentiating medium includes about 10 μM Y-27632 2HCl. Other suitable ROCK inhibitors include, but are not limited to, Chroman 1, thiazovivin, fasudil / HA1077 and H-1152.

[0419] In some instances, the first PP-differentiating medium includes a Vitamin C compound, which can be ascorbic acid at a concentration of about 0.05 mM to about 0.50 mM, about 0.10 mM to about 0.45 mM, about 0.15 mM to about 0.40 mM or about 0.20 mM to about 0.35 mM. In some instances, the ascorbic acid concentration can be about 0.15 mM, 0.20 mM, 0.25 mM, 0.30 mM or 0.35 mM. In some instances, the first PP-differentiating medium includes about 0.25 nM ascorbic acid. In some instances, the Vitamin C compound is dehydroascorbic acid.

[0420] In some instances, the first PP-differentiating medium includes a γ-secretase inhibitor (GSI), which can be GSI-XX at a concentration from about 50 nM to about 200 nM. In other instances, GSI-XX can be at a concentration from about 60 nM to about 180 nM, about 70 nM to about 160 nM, about 80 nM to about 140 nM or about 90 nM to about 120 nM. In some instances, the GSI-XX concentration can be about 70 nM, 80 nM, 90 nM, 100 nM, 110 nM,120 nM or 130 nM. In some instances, the first PP-differentiating medium includes about 100 nM GSI-XX. Other suitable GSIs include, but are not limited to, DAPT.

[0421] In some instances, the first PP-differentiating medium includes a G9a inhibitor, which can be UNC0321, UNC0638 or CM-272.

[0422] In some instances, the first PP-differentiating medium includes UNC0321 at a concentration of about 1 μM to about 10 μM. In some instances, the UNC0321 concentration can be about 2 μM to about 9 μM, about 3 μM to about 8 μM, about 4 μM to about 7 μM or about 4.5 μM to about 5.5 μM. In some instances, the UNC0321 concentration can be about 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM or 10 μM. In some instances, the first PP-differentiating medium includes about 5 μM UNC0321.

[0423] In some instances, the first PP-differentiating medium includes UNC0638 at a concentration of about 0.1 μM to about 1.0 μM. In some instances, the UNC0638 concentration can be about 0.2 μM to about 0.9 μM, about 0.3 μM to about 0.8 μM, about 0 / 4 μM to about 0.7 μM or about 0.45 μM to about 0.55 μM. In some instances, the UNC0638 concentration can be about 0.2 μM, 0.4 μM, 0.6 μM or 0.8 μM. In some instances, the first PP-differentiating medium includes about 0.5 μM UNC0638.

[0424] In some instances, the first PP-differentiating medium includes a tankyrase 1 / 2 inhibitor (e.g., an adenosine subsite binding inhibitor or an adenosine subsite binding / G loop interacting inhibitor), which may be the same or different compound, or may be present at the same or different concentration, than any tankyrase 1 / 2 inhibitor present in any of the PGT- differentiating and FE-differentiating mediums. In some instances, the adenosine subsite binding inhibitor can be IWR-1, JW55 or JW74. In some instances, the adenosine subsite binding / G loop interacting inhibitor can be WIKI4.

[0425] In some instances, the tankyrase 1 / 2 inhibitor in the first PP-differentiating medium is an adenosine subsite binding inhibitor, which can be IWR-1 at a concentration from about 50 nM to about 400 nM. In some instances, the concentration of IWR-1 can be about 75 nM to about 375 nM, about 100 nM to about 350 nM, about 125 to about 325 nM, about 150 nM to about 300 nM or about 175 nM to about 275 nM. In some instances, the IWR-1 concentration in the first PP-differentiating medium can be about 100 nM, 125 nM, 150 nM, 175 nM, 200 nM, 225 nM, 250 nM, 275 nM or 300 nM. In some instances, the first PP-differentiating medium includes about 200 nM IWR-1. Other exemplary tankyrase 1 / 2 inhibitors for use in the first PP-differentiating medium instead of IWR-1 include G007-LK (e.g., at a concentrationof about 5 µM), JW55 (e.g., at a concentration of about 5 uM) and JW74 (e.g., at a concentration of about 10 µM). In some instances, the first PP-differentiating medium lacks any other tankyrase inhibitor.

[0426] In some instances, the tankyrase 1 / 2 inhibitor in the first PP-differentiating medium can be an adenosine subsite binding / G loop interacting inhibitor, which can be WIKI4 at a concentration from about 1 µM to about 30 µM. In some instances, the concentration of WIKI4 can be about 3 µM to about 20 µM, about 4 µM to about 15 µM, about 6 µM to about 12 µM or about 8 µM to about 10 µM. In some instances, the WIKI4 concentration in the first PP- differentiating medium can be about 3 µM, 6 µM, 9 µM, 12 µM or 15 µM. In some instances, the first PP-differentiating medium includes about 9 µM WIKI4. In some instances, the first PP-differentiating medium lacks any other tankyrase inhibitor.

[0427] In some instances, the first PP-differentiating medium includes two tankyrase 1 / 2 inhibitors: one can be an adenosine subsite specific binding inhibitor (e.g., G007-LK, IWR-1. JW55 or JW74), and the other can be an adenosine subsite / G-loop interacting inhibitor (e.g., WIKI4). In some instances, the first PP-differentiating medium includes both IWR-1 and WIKI4, which can be present at concentrations selected from any of the IWR-1 and WIKI4 concentrations described above. In some instances, the first PP-differentiating medium includes about 180 nM to about 220 nM IWR-1 and about 8 µM to about 10 µM WIKI4. In some instances, the first PP-differentiating medium includes about 200 nM IWR-1 and about 9 µM WIKI4.

[0428] In some instances, the first PP-differentiating medium includes about 20 mM to about 30 mM glucose, about 1.8 mM to about 2.2 mM L-alanine-L-glutamine, about 0.75 mM to about 1.25 mM pyruvate, about 1.5% to about 2.5% FAF-BSA (or FAF-HSA), about 43 mM to about 48 mM NaHCO3, about 90 nM to about 110 nM LDN-193189, about 1.5 μM to about 2.5 μM ZnSO4, about 2.5 μM to about 3.5 μM T3, about 9 μg / mL to about 11 μg / mL UFH- PIM, about 4 μM to about 6 μM ALK5i II, about 0.9x to about 1.1x of a B27 (50x) supplement, 0.20 μM to about 0.30 μM SANT-1, about 0.75x to about 1.25x of a MEM NEAA (100x) supplement, about 9 μM to about 11 μM Y-276322HCl, about 0.20 mM to about 0.30 mM ascorbic acid, about 90 nM to about 110 nM GSI-XX and optionally about 4.5 μM to about 5.5 μM UNC0321 (or about 0.4 μM to about 0.6 μM UNC0638). In some instances, the first PP- differentiating medium also includes the MCDB media shown in Table 16.

[0429] In some instances, the first PP-differentiating medium includes the MCDB media shown in Table 16, about 25 mM glucose, about 2 mM L-alanine-L-glutamine, about 1 mM pyruvate, about 2% FAF-BSA (or FAF-HSA), about 42.5 mM NaHCO3, about 100 nM LDN- 193189, about 2 μM ZnSO4, about 3 μM T3, about 10 μg / mL UFH-PIM, about 5 μM ALK5i II, about 1x of the B27 (50x) supplement shown in Table 28, about 0.25 μM SANT-1, about 1.0x of the MEM NEAA (100x) supplement shown in Table 23, about 10 μM Y-276322HCl, about 0.25 mM ascorbic acid, about 100 nM GSI-XX and about 5 μM UNC0321 (or about 0.5 μM UNC0638).

[0430] PP / PEP Wash Media. The PP / PEP wash media can include a defined medium comprising ≤ about 1 mM, ≤ about 0.5 mM, ≤ about 0.1 mM, ≤ about 0.05 mM or ≤ about 0.01 mM glucose and optionally albumin (e.g., BSA or HSA). In some instances, the PP / PEP wash medium can be glucose-free, pyruvate-free, HEPES-free and includes about 0.02% to about 2% FAF-BSA (or FAF-HSA). In some instances, the PP / PEP wash medium includes the no glucose, DMEM composition shown in Table 20 herein below, which can optionally be supplemented with about 0.2% FAF-BSA (or FAF-HSA).

[0431] Second PP-differentiating Medium. The second PP-differentiating medium can include a defined medium comprising < about 2 mM glucose and one or more of an alternative nutrient (e.g., galactose), albumin (e.g., BSA or HSA), a buffer (e.g., NaHCO3), glutamine (e.g., a glutamine dipeptide), a small molecule BMP inhibitor (e.g., LDN-193189), a zinc compound (e.g., ZnSO4), a thyroid hormone signaling pathway activator (e.g., T3), a heparin (e.g., UFH), an ATP-competitive inhibitor of TGF-β RI kinase (e.g., ALK5iII), a serum replacement supplement (e.g., a B27 supplement), a cell-permeable SHH signaling inhibitor (e.g., SANT-1), a NEAA supplement, a ROCK inhibitor (e.g., Y-27632), a Vitamin C compound (e.g., ascorbic acid), a γ-secretase inhibitor (e.g., GSI-XX), an epigenetic modifier (e.g., a G9a inhibitor such as UNC0321) and at least one tankyrase 1 / 2 inhibitor (e.g., IWR-1 and / or WIKI4).

[0432] In some instances, the concentration of glucose in the second PP-differentiating medium can be ≤ about 1.5 mM, ≤ about 1 mM, ≤ about 0.5 mM, ≤ about 0.25 mM, ≤ about 0.1 mM, ≤ about 0.05 mM or ≤ about 0.01 mM. In some instances, the glucose concentration in the second PP-differentiating medium can be ≤ about 0.1 mM, ≤ about 0.05 mM or ≤ about 0.01 mM glucose. In some instances, the second PP-differentiating medium can be glucose- free (e.g., 0 mM).

[0433] In some instances, the second PP-differentiating medium optionally includes an alternative nutrient, which can be galactose, methyl pyruvate, methyl succinate or pyruvate. In some instances, the alternative nutrient can be galactose, which can be present at a concentration of about 1 mM to about 40 mM, about 2 mM to about 30 mM, about 3 mM to about 20 mM, about 4 mM to about 10 mM, about 5 mM to about 9 mM or about 4 mM to about 6 mM. In some instances, the galactose concentration can be about 4.0 mM, 4.5 mM, 5.0 mM, 5.5 mM or 6.0 mM. In some instances, the second PP-differentiating medium includes galactose at a concentration of about 5.6 mM.

[0434] In some instances, the second PP-differentiating medium includes each of a glutamine dipeptide, albumin and NaHCO3, which can be present at concentrations selected from the corresponding glutamine dipeptide, albumin and NaHCO3 concentration ranges and concentrations described above for the PGT-differentiating mediums. In some instances, the glutamine dipeptide can be L-alanine-L-glutamine at a concentration of about 1.8 mM to about 2.2 mM. In some instances, the albumin is FAF-BSA (or FAF-HSA), which can be present at about 1%, 1.5%, 2%, 2.5% or 3%. In some instances, the NaHCO3concentration in the second PP-differentiating medium can be about 20 mM to about 60 mM, about 20 mM to about 50 mM, about 25 mM to about 45 mM or about 42 mM to about 48 mM. In some instances, the second PP-differentiating medium includes about 2.0 mM L-alanine-L-glutamine, about 2% FAF-BSA (or FAF-HSA) and about 25 mM or about 42.5 mM NaHCO3.

[0435] In some instances, the second PP-differentiating medium includes a small molecule BMP inhibitor (e.g., LDN-193189 or DMH-1). In some instances, the BMP inhibitor can be LDN-193189, which can be present in the second PP-differentiating medium at a concentration of about 50 nM to about 200 nM or about 90 nM to about 110 nM. In some instances, the LDN- 193189 concentration can be selected from the LDN-193189 concentration ranges and concentrations described above for the first PP-differentiating medium. In some instances, the second PP-differentiating medium includes about 100 nM LDN-193189.

[0436] In some instances, the second PP-differentiating medium includes a zinc compound, which can be ZnSO4 at a concentration of about 1 μM to about 5 μM, about 2 μM to about 4 μM or about 3 μM. In some instances, the ZnSO4concentration in the second PP-differentiating medium can be about 1 μM, 2 μM, 3 μM or 4 μM. In some instances, the second PP- differentiating medium includes about 2 μM ZnSO4. In some instances, the second PP- differentiating medium does not include a zinc compound in addition to any amount of a zinccompound that can be present in the basal medium or in any multi-component supplement present in or added to the second PP-differentiating medium.

[0437] In some instances, the second PP-differentiating medium includes a thyroid hormone signaling pathway activator, which can be T3 at a concentration of about 1 μM to about 5 μM, about 2 μM to about 4 μM or about 2.5 μM to about 3.5 μM. In some instances, the T3 concentration can be about 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM or 4 μM. In some instances, the second PP-differentiating medium includes about 3 μM T3. In some instances, T3 is not present in the second PP-differentiating medium in addition to any amount present in the basal medium or multi-component supplement added to the differentiating medium.

[0438] In some instances, the second PP-differentiating medium includes a heparin, which can be an UFH at a concentration of about 1 μg / mL to about 20 μg / mL or about 9 μg / mL to about 11 μg / mL. In some instances, the heparin concentration can be selected from the heparin concentration ranges and concentrations described above for the first PP-differentiating medium. In some instances, the second PP-differentiating medium includes about 10 μg / mL UFH-PIM.

[0439] In some instances, the second PP-differentiating medium includes an ATP- competitive inhibitor of TGF-β RI kinase, which can be ALK5iII at a concentration of about 1 μM to about 10 μM or about 4 μM to about 6 μM. In some instances, the ALK5iII concentration can be selected from the ALK5iII concentration ranges and concentrations described above for the first PP-differentiating medium. In some instances, the second PP-differentiating medium includes about 5 μM ALK5iII.

[0440] In some instances, the second PP-differentiating medium includes the components of a B27 supplement (e.g., as defined herein). In some instances, the B27 supplement can be a B27 (50x) supplement as described above, which can be present at a concentration selected from the B-27 concentration ranges and concentrations described above for the FE- differentiating medium. In some instances, the concentration of the B27 (50x) supplement in the second PP-differentiating medium can be about 0.9x to about 1.1x. In some instances, the second PP-differentiating medium includes the B27 (50x) supplement shown in Table 28 at about 1x concentration.

[0441] In some instances, the second PP-differentiating medium includes a cell-permeable SHH signaling inhibitor, which can be SANT-1 at a concentration of about 0.1 μM to about 0.5 μM or about 0.2 μM to about 0.3 μM. In some instances, the SANT-1 concentration can beselected from the SANT-1 concentration ranges and concentrations described above for the PGT-differentiating mediums. In some instances, the second PP-differentiating medium includes about 0.25 μM SANT-1.

[0442] In some instances, the second PP-differentiating medium includes two or more of the components of a NEAA supplement described herein. In some instances, the NEAA supplement can be a NEAA (100x) supplement as described above, which can be present at a concentration selected from the NEAA concentration ranges and concentrations described above for the first PP-differentiating medium. In some instances, the concentration of the NEAA (100x) supplement is about 0.9x to about 1.1x. In some instances, the second PP- differentiating medium includes the NEAA (100x) supplement shown in Table 24 at about 1x concentration. In some instances, the first PP-differentiating medium does not contain a NEAA supplement.

[0443] In some instances, the second PP-differentiating medium includes a ROCK inhibitor, which can be Y-27632 (e.g., Y-276322HCl) at a concentration from about 1 μM to about 20 μM or about 9 μM to about 11 μM. In some instances, the concentration of Y-276322HCl can be selected from the Y-276322HCl concentration ranges and concentrations described above for the PGT-differentiating mediums. In some instances, the second PP-differentiating medium includes about 10 μM Y-276322HCl.

[0444] In some instances, the second PP-differentiating medium includes a Vitamin C compound, which can be ascorbic acid at a concentration of about 0.05 mM to about 0.50 mM or about 0.20 mM to about 0.3 mM. In some instances, the ascorbic acid concentration can be selected from the ascorbic acid concentration ranges and concentrations described above for the first PP-differentiating medium. In some instances, the second PP-differentiating medium includes about 0.25 nM ascorbic acid. In some instances, the Vitamin C compound can be dehydroascorbic acid.

[0445] In some instances, the second PP-differentiating medium includes a γ-secretase inhibitor (GSI), which can be GSI-XX at a concentration from about 50 nM to about 200 nM or about 90 nM to about 110 nM. In some instances, the GSI-XX concentration can be selected from the GSI-XX concentration ranges and concentrations described above for the first PP- differentiating medium. In some instances, the second PP-differentiating medium includes about 100 nM GSI-XX. Other suitable GSIs include, but are not limited to, DAPT.

[0446] In some instances, the second PP-differentiating medium includes a G9a inhibitor, which may be the same or different than any G9a inhibitor present in the first PP-differentiating medium. In some instances, the G9a inhibitor can be UNC0321, UNC0638 or CM-272.

[0447] In some instances, the G9a inhibitor in the second PP-differentiating medium can be UNC0321, which can be present at a concentration of about 1 μM to about 10 μM. In some instances, the UNC0321 concentration can be selected from the UNC0321 concentration ranges and concentrations described above for the first PP-differentiating medium. In some instances, the UNC0321 concentration in the second PP-differentiating medium is about 4.5 μM to about 5.5 μM. In some instances, the second PP-differentiating medium includes about 5 μM UNC0321.

[0448] In some instances, the second PP-differentiating medium includes UNC0638 at a concentration of about 0.1 μM to about 1.0 μM. In some instances, the UNC0638 concentration can be about 0.2 μM to about 0.9 μM, about 0.3 μM to about 0.8 μM, about 0 / 4 μM to about 0.7 μM or about 0.45 μM to about 0.55 μM. In some instances, the UNC0638 concentration can be about 0.2 μM, 0.4 μM, 0.6 μM or 0.8 μM. In some instances, the first PP-differentiating medium includes about 0.5 μM UNC0638.

[0449] In some instances, the second PP-differentiating medium includes a tankyrase 1 / 2 inhibitor (e.g., an adenosine subsite binding inhibitor or an adenosine subsite binding / G loop interacting inhibitor), which may be the same or different compound, or may be present at the same or different concentration, than any tankyrase 1 / 2 inhibitor present in any of the PGT, FE and first PP-differentiating mediums. In some instances, the adenosine subsite binding inhibitor can be IWR-1, JW55 or JW74. In some instances, the adenosine subsite binding / G loop interacting inhibitor can be WIKI4.

[0450] In some instances, the tankyrase 1 / 2 inhibitor in the second PP-differentiating medium can be an adenosine subsite binding inhibitor, which can be IWR-1 at a concentration from about 50 nM to about 400 nM. In some instances, the concentration of IWR-1 can be about 75 nM to about 375 nM, about 100 nM to about 350 nM, about 125 to about 325 nM, about 150 nM to about 300 nM or about 175 nM to about 275 nM. In some instances, the IWR- 1 concentration in the FE-differentiating medium can be about 100 nM, 125 nM, 150 nM, 175 nM, 200 nM, 225 nM, 250 nM, 275 nM or 300 nM. In some instances, the first PP- differentiating medium includes about 200 nM IWR-1. Other exemplary tankyrase 1 / 2 inhibitors for use in the second PP-differentiating medium instead of IWR-1 include G007-LK(e.g., at a concentration of about 5 µM), JW55 (e.g., at a concentration of about 5 µM) and JW74 (e.g., at a concentration of about 10 µM). In some instances, the first PP-differentiating medium lacks any other tankyrase inhibitor.

[0451] In some instances, the tankyrase 1 / 2 inhibitor in the second PP-differentiating medium can be an adenosine subsite binding / G loop interacting inhibitor, which can be WIKI4 at a concentration from about 1 µM to about 30 µM. In some instances, the concentration of WIKI4 can be about 3 µM to about 20 µM, about 4 µM to about 15 µM, about 6 µM to about 12 µM or about 8 µM to about 10 µM. In some instances, the WIKI4 concentration in the first PP-differentiating medium can be about 3 µM, 6 µM, 9 µM, 12 µM or 15µM. In some instances, the first PP-differentiating medium includes about 9 µM WIKI4. In some instances, the second PP-differentiating medium lacks any other tankyrase inhibitor.

[0452] In some instances, the second PP-differentiating medium includes two tankyrase 1 / 2 inhibitors: one can be an adenosine subsite specific binding inhibitor (e.g., IWR-1, JW55 or JW74), and the other can be an adenosine subsite / G-loop interacting inhibitor (e.g., WIKI4). In some instances, the second PP-differentiating medium includes both IWR-1 and WIKI4, which can be present at concentrations selected from any of the IWR-1 and WIKI4 concentrations described above. In some instances, the second PP-differentiating medium includes about 180 nM to about 220 nM IWR-1 and about 8 µM to about 10 µM WIKI4. In some instances, the second PP-differentiating medium includes about 200 nM IWR-1 and about 9 µM WIKI4.

[0453] In some instances, the second PP-differentiating medium includes glucose at ≤ about 0.05 mM or is glucose-free (i.e., 0 mM), about 5 mM to 6 mM galactose, about 1.8 mM to about 2.2 mM L-alanine-L-glutamine, about 1% to about 3% FAF-BSA (or FAF-HSA), about 22 mM to about 26 mM NaHCO3 (or about 42 mM to about 48 mM NaHCO3), about 90 nM to about 110 nM LDN193189, about 1 μM to about 3 μM ZnSO4, about 2.5 μM to about 3.5 μM T3, about 9 μg / mL to about 11 μg / mL UFH-PIM, about 4 μM to about 6 μM ALK5iII, a B27 (50x) supplement (e.g., the composition in Table 28) at about 09.x to about 1.1x, about 0.2 μM to about 0.3 μM SANT-1, a NEAA (100x) supplement at about 0.9x to about 1.1x, about 9 μM to about 11 μM Y-27632 (e.g., Y-276322HCl), 0.20 mM to about 0.3 mM ascorbic acid, 90 nM to about 110 nM GSI-XX, about 4.5 μM to about 5.5 μM UNC0321 and optionally one or both of IWR-1 (at about 180 nM to about 220 nM) and WIKI4 (at about 8 µM to about 10 µM).In some instances, the second PP-differentiating medium also includes the MCDB media shown in Table 16.

[0454] In some instances, the second PP-differentiating medium includes the MCDB media shown in Table 16, about 5.5 mM galactose, about 2.0 mM L-alanine-L-glutamine, about 2% FAF-BSA (FAF-HSA), about 24 mM or about 42 mM NaHCO3, about 100 nM LDN193189, about 2 μM ZnSO4, about 3 μM T3, about 10 μg / mL UFH-PIM, about 5 μM ALK5iII, the B27 (50x) supplement shown in Table 28 at about 1x, about 0.25 μM SANT-1, the NEAA (100x) supplement shown in Table 24 at about 1x concentration, about 10 μM Y-276322HCl, about 0.25 mM ascorbic acid, about 100 nM GSI-XX and about 5 μM UNC0321. In some instances, the second PP-differentiating medium also includes about 200 nM IWR-1-Endo and / or about 9 µM WIKI4.

[0455] Stage 6 Cells and Cell Populations (PEP to Precursor SC-IC):

[0456] The differentiation methods can begin with or can include differentiating PEP cells to SC-ICs by (i) culturing a dissociated PEP cell population in a first PEP-differentiating medium including ≤ about 1 mM pyruvate (e.g., ≤ about 0.1 mM pyruvate) and DNAse I for a first time period of about 2 days (i.e., Days 16-17 in FIG. 1B) to obtain a reaggregated cell population having PEP cells and / or SC-ICs, (ii) washing the reaggregated PEP / SC-IC population in a wash medium, and (iii) culturing the washed PEP / SC-IC population in a second PEP-differentiating medium for a second time period of about 2 days (i.e., Days 18-19 in FIG. 1B) to obtain a precursor SC-IC population including CPEP+cells and GCG+cells. In some instances, one or both PEP-differentiating mediums includes an epigenetic modifier. In some instances, each PEP-differentiating medium includes an HPLM composition (e.g., one of the HPLM compositions shown in Table 18 and Table 19 herein below).

[0457] In some instances, the method includes replacing the second PEP-differentiating medium with fresh second PEP-differentiating medium one time during the second time period (i.e., at about 24 hr after initiating the culturing step (iii)).

[0458] In some instances, the concentration of the dissociated PEP cell population present in the culturing step (i) is about 1 X 106per mL.

[0459] In some instances, the method uses a dissociated PEP cell population obtained by: (a) performing the Stage 5 differentiation method, and optionally also performing the Stage 1a, Stage 1b, Stage 2, Stage 3 and Stage 4 differentiation methods, or (b) differentiating PSCs into PEP cells by any method known in the art.

[0460] In some instances, the reaggregated PEP / SC-IC population includes cell aggregates having an average size of about 40 µm to about 100 µm, about 50 µm to about 90 µm, about 60 µm to about 80 µm or about 70 µm.

[0461] In some instances, the Stage 6 differentiation method can include analyzing the precursor SC-IC population via flow cytometry to determine the concentration of one or more of INS+ / SLC- cells, INS- / SLC+cells, CPEP+ / GCG- cells, CPEP+ / GCG+cells and / or CHGA+ / Ki67- cells.

[0462] In some instances, the culturing of the PEP cell and PEP / SC-IC populations can be performed in a bioreactor and includes cell transfer densities from about 1 x 105cells / mL to about 1 x 106cells / mL. In yet other instances, the cell transfer density can be about 1 x 106cells / mL, 1.5 x 106cells / mL, 2 x 106cells / mL, 2.5 x 106cells / mL, 3.5 x 106cells / mL or up to about 4 x 106cells / mL. In some instances, the culture pH can range from about 6.8 to about 7.6. In yet other instances, pH can be about 6.9, 7.0, 7.1, 7.2, 7.3, 7.4 or 7.5. In some instances, the dissolved oxygen can be controlled to a concentration from about 60 mmHg to about 150 mmHg. In some instances, agitation rates can be from about 20 rpm to about 60 rpm depending on the bioreactor size. In some instances, the culture is controlled at a temperature of about 36°C to about 38°C, especially about 37°C. In some instances, air, carbon dioxide and oxygen can be supplied to the bioreactor via passive diffusion, active overlay or sparging.

[0463] PEP-differentiating and Wash Mediums. Each of the first and second PEP- differentiating mediums can include a defined medium comprising about 1 mM to about 10 mM glucose, about 0.01 mM to about 0.1 mM fructose, about 0.01 mM to about 0.1 mM galactose, about 0.01 mM to about 0.10 mM pyruvate, about 0.1 mM to about 1 mM glutamine, about 20 mM to about 30 mM NaHCO3, one or more of an albumin (e.g., BSA or HSA), an ATP-competitive inhibitor of TGF-β RI kinase (e.g., ALK5iII), a small molecule BMP inhibitor (e.g., LDN-193189), a zinc compound (e.g., ZnSO4), a heparin (e.g., a UFH), a thyroid hormone signaling pathway activator (e.g., T3), a Vitamin C compound (e.g., ascorbic acid), a thiol-based antioxidant (e.g., NAC), a B27 supplement, a G9a inhibitor (e.g., UNC0321) and a cell-permeable SHH signaling inhibitor (e.g., SANT-1). In some instances, the first PEP- differentiating medium also includes a DNAse I (e.g., a recombinant DNAse I). In some instances, each PEP-differentiating medium includes the HPLM shown in Table 18 herein below.

[0464] In some instances, one or both PEP-differentiating mediums do not include one or more factors selected from (i) a small molecule BMP inhibitor, (ii) zinc, (iii) a heparin, (iv) a thyroid hormone signaling pathway activator, (v) a Vitamin C compound, (vi) a thiol-based antioxidant, (vii) a B-27 supplement, (viii) a SHH signaling inhibitor, and (ix) an albumin in addition to any amount of the selected factor(s) present in the basal medium or in any multi- component supplement present in or added to the PEP-differentiating medium(s).

[0465] In some instances, the wash medium used between the first and second culturing steps includes the same defined medium as described above for the first and second PEP- differentiating mediums but is supplemented only with albumin (e.g., as defined herein). In some instances, the wash medium includes an HPLM composition (e.g., the HPLM composition shown in Table 19 herein below).

[0466] In some instances, the first PEP-differentiating medium includes DNAse I, which can be a recombinant bovine DNAse I (e.g., as defined herein) at a concentration of about 1 U / mL to about 100 Us / mL, about 2 U / mL to about 50 U / mL, about 5 U / mL to about 20 U / mL or about 8 U / mL to about 12 U / mL. In some instances, the first PEP-differentiating medium includes about 10 U / mL recombinant bovine DNAse I.

[0467] In some instances, each PEP-differentiating medium includes glucose, which can be at the same or different concentration in each medium. In some instances, the glucose concentration in each PEP-differentiating medium can be about 2 mM to about 9 mM, about 3 mM to about 8 mM, about 3 mM to about 7 mM, about 4 mM to about 6 mM or about 4.5 mM to about 5.5 mM. In some instances, the glucose concentration in each PEP-differentiating medium can be about 3 mM, 4 mM, 5 mM, 6 mM, 7 mM or 8 mM. In some instances, each of the first and second PEP-differentiating mediums includes about 5 mM glucose. In some instances, each of the first and second PEP-differentiating mediums includes less than 5 mM glucose. In some instances, each of the first and second PEP-differentiating mediums includes less than 2.5 mM glucose. In some instances, each of the first and second PEP-differentiating mediums includes less than 2mM glucose.

[0468] In some instances, each PEP-differentiating medium includes fructose, which can be at the same or different concentration in each medium. In some instances, the fructose concentration in each PEP-differentiating medium can be about 0.02 mM to about 0.08 mM, about 0.03 mM to about 0.06 mM or about 0.04 mM to about 0.05 mM. In some instances, the fructose concentration in each PEP-differentiating medium can be about 0.02 mM, 0.03 mM,0.04 mM, 0.05 mM or 0.06 mM. In some instances, each PEP-differentiating medium includes about 0.04 mM fructose.

[0469] In some instances, each PEP-differentiating medium includes galactose, which can be at the same or different concentration in each medium. In some instances, the galactose concentration in each PEP-differentiating medium can be about 0.02 mM to about 0.09 mM, about 0.03 mM to about 0.08 mM, about 0.04 mM to about 0.07 mM, or about 0.05 mM to about 0.06 mM. In some instances, the galactose concentration in each PEP-differentiating medium can be about 0.04 mM, 0.05 mM, 0.06 mM, 0.07 mM or 0.08 mM. In some instances, each PEP-differentiating medium includes 0.06 mM galactose.

[0470] In some instances, each PEP-differentiating medium includes pyruvate, which can be at the same or different concentration in each medium. In some instances, the pyruvate concentration in each PEP-differentiating medium can be about 0.02 mM to about 0.09 mM, about 0.03 mM to about 0.08 mM, about 0.04 mM to about 0.07 mM or about 0.05 mM to about 0.06 mM. In some instances, the pyruvate concentration in each PEP-differentiating medium can be about 0.03 mM, 0.04 mM, 0.05 mM, 0.06 mM or 0.07 mM. In some instances, each PEP-differentiating medium includes about 0.05 mM pyruvate.

[0471] In some instances, each PEP-differentiating medium includes glutamine, which can be at the same or different concentration in each medium. In some instances, the glutamine concentration in each PEP-differentiating medium can be about 0.2 mM to about 0.9 mM, about 0.3 mM to about 0.8 mM, about 0.4 mM to about 0.7 mM or about 0.5 mM to about 0.6 mM. In some instances, the glutamine concentration in each PEP-differentiating medium can be about 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM or 0.7 mM. In some instances, each PEP- differentiating medium includes about 0.55 mM glutamine.

[0472] In some instances, each PEP-differentiating medium includes NaHCO3, which can be at the same or different concentration in each medium. In some instances, the NaHCO3concentration in each PEP-differentiating medium can be about 20 mM to about 29 mM, about 21 mM to about 28 mM, about 22 mM to about 27 mM, about 23 mM to about 26 mM or about 24 mM to about 25 mM. In some instances, the NaHCO3, concentration in each PEP- differentiating medium can be about 22 mM, 23 mM, 24 mM, 25 mM or 26 mM. In some instances, each PEP-differentiating medium includes about 24 mM NaHCO3.

[0473] In some instances, each PEP-differentiating medium includes an albumin, which can be at the same or different concentration in each medium. In some instances, the albuminconcentration in each PEP-differentiating medium can be about 0.5% to about 5%. In some instances, the albumin can be FAF-BSA or FAF-HSA, which can be at a concentration in each medium from about 1% to about 3%. In some instances, the FAF-BSA or FAF-HSA concentration in each PEP-differentiating medium can be about 0.5%, 1%, 1.5%, 2%, 2.5%, 3% or 3.5%. In some instances, each PEP-differentiating medium includes about 2% FAF-BSA (or FAF-HSA).

[0474] In some instances, each PEP-differentiating medium includes an ATP-competitive inhibitor of TGF-β RI kinase, which may be the same or different in each medium. In some instances, the ATP-competitive inhibitor of TGF-β RI kinase in each PEP-differentiating medium can be ALK5iII, which can be present in each medium at the same or different concentration. In some instances, the ALK5iII concentration in each PEP-differentiating medium can be about 0.5 μM to about 5 μM. In some instances, the ALK5iII concentration in each PEP-differentiating medium can be about 1.0 μM to about 4 μM, about 1.5 μM to about 3.5 μM or about 2.0 μM to about 3 μM. In some instances, each PEP-differentiating medium includes about 2.5 μM ALK5iII.

[0475] In some instances, each PEP-differentiating medium includes a small molecule BMP inhibitor, which can be the same or different in each medium. In some instances, the BMP inhibitor in each PEP-differentiating medium can be DMH-1 or LDN-193189, either of which can be present at the same or different concentration in each medium.

[0476] In some instances, each PEP-differentiating medium includes LDN-193189, which can be present at the same or different concentration in each medium. In some instances, the LDN-193189 concentration in each medium can be about 50 nM to about 200 nM. In some instances, the LDN-193189 concentration in each PEP-differentiating medium can be selected from the LDN-193189 concentration ranges and concentrations described above for the first PP-differentiating medium. In some instances, the LDN-193189 concentration in each PEP- differentiating medium can be about 90 nM to about 110 nM. In some instances, each PEP- differentiating medium includes about 100 nM LDN-193189.

[0477] In some instances, each PEP-differentiating medium includes a zinc compound, which can be ZnSO4at the same or different concentration in each medium. In some instances, the ZnSO4 concentration in each PEP-differentiating medium can be about 1 μM to about 5 μM, about 2 μM to about 4 μM or about 3 μM. In some instances, the ZnSO4 concentration ineach PEP-differentiating medium can be about 1 μM, 2 μM, 3 μM or 4 μM. In some instances, each PEP-differentiating medium includes about 2 μM ZnSO4.

[0478] In some instances, each PEP-differentiating medium includes a heparin, which can be UFH at the same or different concentration in each medium. In some instances, the heparin can be UFH, which can be at a concentration in each medium from about 1 μg / mL to about 20 μg / mL. In some instances, the heparin concentration in each PEP-differentiating medium can be selected from the heparin concentration ranges and concentrations described above for the first PP-differentiating medium. In some instances, the UFH concentration in each PEP- differentiating medium can be about 9 μg / mL to about 11 μg / mL. In some instances, each PEP- differentiating medium includes about 10 μg / mL UFH-PIM.

[0479] In some instances, each PEP-differentiating medium includes a thyroid hormone signaling pathway activator, which can be at the same or different concentration in each medium. In some instances, the thyroid hormone signaling pathway activator in each PEP- differentiating medium can be T3 at a concentration from about 1 μM to about 5 μM, about 2 μM to about 4 μM or about 2.5 μM to about 3.5 μM. In some instances, the T3 concentration in each PEP-differentiating medium can be about 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM or 4 μM. In some instances, each PEP-differentiating medium includes about 3 μM T3.

[0480] In some instances, each PEP-differentiating medium includes a Vitamin C compound, which can be at the same or different concentration in each medium. In some instances, the Vitamin C compound can be ascorbic acid at a concentration in each PEP-differentiating medium from about 0.05 mM to about 0.50 mM, about 0.10 mM to about 0.45 mM, about 0.15 mM to about 0.40 mM or about 0.20 mM to about 0.35 mM. In some instances, the ascorbic acid concentration in each PEP-differentiating medium can be about 0.15 mM, 0.20 mM, 0.25 mM, 0.30 mM or 0.35 mM. In some instances, each PEP-differentiating medium includes about 0.25 nM ascorbic acid. In some instances, the Vitamin C compound is dehydroascorbic acid.

[0481] In some instances, each PEP-differentiating medium includes NAC, which can be at the same or different concentration in each medium. In some instances, the NAC concentration in each PEP-differentiating medium can be about 0.5 mM to about 1.5 mM, about 0.6 mM to about 1.4 mM, about 0.7 mM to about 1.3 mM, about 0.8 mM to about 1.2 mM or about 0.9 mM to about 1.1 mM. In some instances, the NAC concentration in each PEP-differentiating medium can be at a concentration of about 0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM or 1.2 mM. In some instances, each PEP-differentiating medium includes about 1.0 mM NAC.

[0482] In some instances, each PEP-differentiating medium includes the components of a B27 supplement, which can be at the same or different concentration in each medium. In some instances, the B27 supplement can be a B27 (50x) supplement as described above, which can be present in each PEP-differentiating medium at a concentration selected from the B-27 concentration ranges and concentrations described above for the FE-differentiating medium. In some instances, the concentration of the B27 (50x) supplement (e.g., the B27 (50x) supplement shown in Table 28) in each PEP-differentiating medium can be about 0.9x to about 1.1x. In some instances, each PEP-differentiating medium includes the B27 (50x) supplement at about 1x concentration.

[0483] In some instances, each PEP-differentiating medium includes a G9a inhibitor, which can be the same or different in each PEP-differentiating medium. In some instances, the G9a inhibitor in each PEP-differentiating medium is at the same or different concentration in each medium. In some instances, the G9a inhibitor in each PEP-differentiating medium can be UNC0321, UNC0631 or CM-272, which can be present at the same or different concentration in each medium.

[0484] In some instances, each PEP-differentiating medium includes UNC0321, which can be at a concentration from about 1 μM to about 10 μM. In some instances, the UNC0321 concentrat...

Claims

CLAIMS What is claimed is:

1. A method of deriving a cell population comprising mature stem cell-derived islet-like cells (SC-ICs), the method comprising a step of: (a) culturing a first precursor cell population in one or more differentiating mediums to obtain the mature SC-IC population, wherein at least one of the differentiating mediums is a defined medium comprising glucose at less than about 2.5 mM or at less than about 2 mM, and wherein the first precursor cell population is selected from the group consisting of a foregut endoderm (FE) population, a pancreatic progenitor (PP) cell population, a pancreatic endocrine precursor (PEP) cell population and a precursor SC-IC cell population.

2. The method of claim 1, wherein the first precursor PP cell population comprises PDX1+cells, the method comprising: (a) culturing the first precursor PP cell population comprising PDX1+cells, optionally PDX1+ / NKX6.1+cells and CHGA- cells, wherein the at least one of the differentiating mediums is a defined medium comprising glucose at a concentration of about 0 mM to less than about 2.5 mM and a G9a inhibitor, thereby obtaining a second cell population comprising PDX1+ / CHGA+cells.

3. The method of claim 2, wherein the first precursor PP cell population comprises PDX1+ / NKX6.1+cells and CHGA- cells.

4. The method of any one of claims 1-3, wherein the at least one of the cell differentiating mediums is a defined medium comprising glucose at a concentration of about 0 to about 0.02 mM, about 0 to about 0.04 mM, about 0 to about 0.06 mM, about 0 to about 0.08 mM, about 0 to about 0.1 mM, 0, about 0 to about 0.2 mM, about 0 to about 0.3 mM, about 0 to about 0.4 mM, about 0 to about 0.5 mM, about 0 to about 0.6 mM, about 0 to about 0.7 mM, about 0 to about 0.8 mM, about 0 to about 0.9 mM, about 0 to about 1.0 mM, about 0 to about 1.1 mM, about 0 to about 1.2 mM, about 0 to about 1.3 mM, about 0 to about 1.4 mM, about 0 to about 1.5 mM, about 0 to about 1.6 mM, about 0 to about 1.7 mM, about 0 to about 1.8 mM, about 0 to about 1.9 mM, about 0 to about 2.0 mM, about 0 to about 2.1 mM, about 0 to about 2.2 mM, about 0 to about 2.3 mM, about 0 to about 2.4 mM, or about 0 to less than about 2.5 mM.

5. The method of any one of claims 1-4, wherein the at least one of the cell differentiating mediums is a defined medium comprising glucose at a concentration of about 0mM, about 0.1mM, about 0.2mM, about 0.3mM, about 0.4mM, about 0.5mM, about 0.6mM, about 0.7mM, about 0.8mM, about 0.9mM, about 1.0mM, about 1.1mM, about 1.2mM, about 1.3mM, about 1.4mM, about 1.5mM, about 1.6mM, about 1.7mM, about 1.8mM, about 1.9mM, about 2.0mM, about 2.1mM, about 2.2mM, about 2.3mM, about 2.4mM, or less than 2.5mM.

6. The method of any one of claims 1-5, wherein the at least one of the cell differentiating mediums is a defined medium comprising glucose at a concentration of 0mM, 0.1mM, 0.2mM, 0.3mM, 0.4mM, 0.5mM, 0.6mM, 0.7mM, 0.8mM, 0.9mM, 1.0mM, 1.1mM, 1.2mM, 1.3mM, 1.4mM, 1.5mM, 1.6mM, 1.7mM, 1.8mM, 1.9mM, 1.0mM, 2.1mM, 2.2mM, 2.3mM, 2.4mM, or less than 2.5mM.

7. The method of any one of claims 1-6, wherein the culturing in step (a) further comprises monitoring the pH.

8. The method of any one of claims 1-7, wherein the culturing in step (a) is at a pH in the range of: pH7.2 to pH7.8, pH7.4 to pH7.8, pH 7.2+ / - 0.2 (pH7.0 to pH7.4), pH 7.4+ / - 0.2 (pH7.2 to pH7.6), pH7.6+ / - 0.2 (pH7.4 to pH7.8) or pH7.8+ / - 0.2 (pH7.6 to pH8.0).

9. The method of any one of claims 1-8, wherein at least one of the one or more differentiating mediums of steps (a) comprises a tankyrase 1 / 2 inhibitor, wherein the tankyrase ½ inhibitor is Wiki4.

10. The method of any one of claims 1-9, wherein the defined medium is glucose-free.

11. The method of any one of claims 1-10, wherein the defined medium comprises galactose at a concentration of about 4 mM to about 7 mM, about 5 mM to about 6.0 mM, about 5.5mM and optionally wherein the defined medium is pyruvate-free.

12. The method of any one of claims 1-11, wherein at least one of the differentiating mediums of step (a) comprises: 5.5mM galactose, glutamine, and at least two differentiation factors selected from the group consisting of: a small molecule BMP inhibitor, a zinc compound, a thyroid hormone signaling pathway activator, a heparin,an ATP-competitive inhibitor of TGF-β RI kinase, a cell permeable Sonic Hedgehog (SHH) signaling inhibitor, a non-essential amino acids (NEAA) supplement, a Rho kinase (ROCK) inhibitor, a Vitamin C compound, and a γ-secretase inhibitor (GSI), and optionally further comprises one or more of an albumin, a buffer and a serum replacement supplement.

13. The method of any one of claims 1-2, further comprising: (b) culturing the second cell population comprising CHGA+ / PDX1+cells in a differentiating medium in the presence of an enzymatic aggregate-dissociating solution, to obtain a dissociated cell population comprising CHGA+ / PDX1+cells single cells, optionally wherein the enzymatic aggregate dissociating solution comprises trypsin activity, chymotrypsin / elastase activity, and collagenase type 1 activity, optionally including a chelating agent such as EDTA; and (c) culturing the dissociated cell population comprising CHGA+ / PDX1+cells single cells in a differentiating medium comprising DNAase I and G9a inhibitor for an additional time period sufficient to obtain a reaggregated population of cells, which cells are comprised in cell aggregates, optionally wherein the cell aggregates have an average size of about 40 µm to about 100 µm, or about 70 microns.

14. The method of claim 13, wherein the differentiating mediums of step (b) and step (c) each comprises glucose at a concentration of about 1 mM to ≤ about 25 mM, glutamine, and at least two differentiation factors selected from the group consisting of: a thiol-based antioxidant, a small molecule BMP inhibitor, a zinc compound, a thyroid hormone signaling pathway activator, an ATP-competitive inhibitor of TGF-β RI kinase, a cell permeable SHH signaling inhibitor, a Vitamin C compound, a heparin, andoptionally wherein each differentiating mediums further comprises one or more of an albumin, a buffer and a serum replacement supplement.

15. The method of claim 13 or 14, wherein the enzymatic aggregate dissociating solution comprises chymotrypsin / elastase activity and EDTA at a concentration of 0.5mM.

16. The method of claim 15, wherein filtering of the dissociated cell population is through a 40micron filter and at least about 80% of the cells in the population are single cells.

17. The method of any one of claims 13 to 16, further comprising : (d) culturing the reaggregated population of cells in a differentiating medium for a time period sufficient to obtain the mature (SC-ICs) cell population comprising NKX6.1+ / CPEP+ cells and CPEP+ / GCG- cells, wherein the differentiating medium comprises glucose at a concentration of about 1 mM to ≤ about 25 mM, glutamine and at least two differentiation factors selected from the group consisting of: a cell-permeable vitamin E analog / antioxidant, a carnitine compound, a chemically defined lipid mixture (CDLM), a thiol-based antioxidant, a small molecule bone morphogenetic protein (BMP) inhibitor; a zinc compound, a thyroid hormone signaling pathway activator, a Vitamin C compound, a heparin, a G9A inhibitor, and optionally wherein the differentiating medium further comprises one or more of an albumin, a buffer and a serum replacement supplement.

18. The method of any one of claims 2 to 17, wherein the G9a inhibitor is CM-272, UNC0321 or UNC0638.

19. The method of claim 18, wherein the G9A inhibitor is UNC0321.

20. The method of any one of claims 13-19, wherein the differentiating medium comprises the human plasma-like medium (HPLM).

21. The method of any one of claims 1- 20 further comprising before step (a), culturing a foregut endoderm (FE) cell population comprising a combination of PDX1+, FOXA2+, NKX6.1-, and CHGA- cells in one or more cell differentiating mediums comprising twotankyrase inhibitors, wherein one of the tankyrase inhibitors is Wiki4, thereby obtaining the (PP) precursor cell population comprising PDX1+, optionally comprising PDX1+ / NKX6.1+cells and CHGA-cells.

22. The method of any one of claims 1-21 wherein the method does not include a step of sorting or isolating individual cells or cell populations comprising a cell marker or a combination of cell markers.

23. The method of claim 22, wherein the sorting or isolating uses a selection marker to enrich for CPEP+ / GCG- cells, CPEP+ / NKX6.1+ cells, or CPEP+ cells, wherein the selection marker is any one of a cell marker TSQ, CD49A, ST8SIA1, GLUT2, ZNT8, CD9, or a combination thereof.

24. The method of claim 22 , wherein the sorting or isolating uses a selection marker to deplete cell populations other than CPEP+ / GCG-, or CPEP+ / NKX6.1+ or CPEP+, wherein the selection marker is any one of a cell marker CD26, SLC18A, or a combination thereof.

25. The method of any one of claims 1-24, wherein the mature SC-ICs cell population comprises at least about 54% to about 60% NKX6.1+ / CPEP+cells and at least about 60% to about 80% CPEP+ / GCG- cells.

26. The method of any one of claims 1-25, wherein the mature SC-ICs cell population further comprises about 99.8% CHGA+cells.

27. The method of any one of claims 1-26, wherein the culturing in (a), (b), (c) and (d) is in a bioreactor and produces at least 1.5E5cells / ml, optionally 1.5E5 cells / ml to 5E5 cells / ml, or about 3E5 cells / ml.

28. A method of deriving a cell population comprising mature stem cell-derived islet-like cells (SC-ICs) from a cell population comprising pancreatic progenitor (PP) cells comprising PDX1+cells, wherein the method comprises: (i) culturing the PP cell population in a first PP-differentiating medium for a time period sufficient to obtain an intermediate PP / PEP cell population, wherein the PP cell population comprises PDX1+, optionally PDX1+ / NKX6.1+cells and CHGA- cells (PDX1+ / CHGA- cells) and the PP / PEP cell population comprises PDX1+ / CHGA+, wherein the first PP-differentiating medium is a defined medium comprising about 5 mM to about 50 mM glucose and a set of differentiation factors in amounts sufficient to promote differentiation of a least a portion of the PP cell population to PEP cells, wherein the set of differentiation factors comprises a G9a inhibitor and at least one factor selected from the group consisting of:a small molecule bone morphogenetic protein (BMP) inhibitor, a zinc compound, a thyroid hormone signaling pathway activator, an ATP-competitive inhibitor of TGF-β RI kinase, a cell-permeable Sonic Hedgehog (SHH) signaling inhibitor, a Rho kinase (ROCK) inhibitor, a Vitamin C compound, a γ-secretase inhibitor (GSI), a heparin, and optionally at least one tankyrase 1 / 2 inhibitor; optionally two tankyrase 1 / 2 inhibitors; (ii) washing the intermediate PP / PEP cell population in a defined wash medium comprising < about 1 mM glucose; (iii) culturing the washed intermediate PP / PEP cell population in a second PP- differentiating medium for a time period sufficient to obtain a PEP cell population comprising cell aggregates, wherein the second PP-differentiating medium is a defined medium comprising < about 1 mM glucose and a set of differentiation factors in amounts sufficient to promote differentiation of at least a portion of the intermediate PP / PEP cell population to PEP population comprising PDX1+ / CHGA+(PDX1+ / NKX6.1+ / CHGA+cells) cells, wherein the set of differentiation factors comprises a G9a inhibitor and at least one factor selected from the group consisting of: a small molecule BMP inhibitor, a zinc compound, a thyroid hormone signaling pathway activator, an ATP-competitive inhibitor of TGF-β RI kinase, a cell-permeable SHH signaling inhibitor, a ROCK inhibitor, a Vitamin C compound, a GSI, a heparin, and at least one tankyrase 1 / 2 inhibitor; optionally two tankyrase 1 / 2 inhibitors;(iv) dissociating at least about 80% of the cell aggregates in the aggregated PEP cell population into single cells to obtain a dissociated PEP cell population; (v) culturing the dissociated PEP cell population in a first PEP-differentiating medium comprising a deoxyribonuclease (DNAse) for a time period sufficient to obtain a reaggregated, intermediate PEP / SC-IC population, wherein the first PEP-differentiating medium is a defined medium comprising the DNAse, ≤ about 0.5 mM pyruvate, about 1 mM to ≤ about 25 mM glucose and a set of differentiation factors in amounts sufficient to promote differentiation of at least a portion of the PEP cell population to immature pancreatic endocrine cells (PECs), wherein the set of differentiation factors comprises a G9a inhibitor and at least one factor selected from the group consisting of: a thiol-based antioxidant, a small molecule BMP inhibitor; a zinc compound, a thyroid hormone signaling pathway activator, an ATP-competitive inhibitor of TGF-β RI kinase, a cell-permeable SHH signaling inhibitor, a Vitamin C compound, and a heparin; (vi) washing the reaggregated, intermediate PEP / SC-IC population in a defined wash medium comprising ≤ about 0.5 mM pyruvate and about 1 mM to ≤ about 25 mM glucose; (vii) culturing the washed intermediate PEP / SC-IC population in a second PEP- differentiating medium for a time period sufficient to obtain a precursor SC-IC population, wherein the second PEP-differentiating medium is a defined medium comprising ≤ about 0.5 mM pyruvate, about 1 mM to ≤ about 25 mM glucose and a set of differentiation factors in amounts sufficient to promote differentiation of at least a portion of the intermediate PEP / SC- IC population to a precursor SC-IC population comprising CHGA+ / PDX1+cells, wherein the set of differentiation factors comprises a G9a inhibitor and at least one factor selected from the group consisting of: a thiol-based antioxidant, a small molecule BMP inhibitor, a zinc compound, a thyroid hormone signaling pathway activator,an ATP-competitive inhibitor of TGF-β RI kinase, a cell-permeable SHH signaling inhibitor, a Vitamin C compound, and a heparin; and (viii) culturing the precursor SC-IC population in an SC-IC-differentiating medium for a time period sufficient to obtain the mature SC-IC population comprising a combination of NKX6.1+ / CPEP+cells, CPEP+ / GCG- cells, INS+ / SLC18A1- cells, or CHGA+ / Ki67- cells, wherein the SC-IC-differentiating medium comprises ≤ about 0.5 mM pyruvate, about 1 mM to ≤ about 25 mM glucose and a set of differentiation factors in amounts sufficient to promote differentiation of at least a portion of the precursor SC-IC population to mature PBLCs, wherein the set of differentiation factors comprises a G9a inhibitor and at least one factor selected from the group consisting of: a cell-permeable Vitamin E analog / antioxidant, a carnitine compound, a chemically defined lipid mixture (CDLM), a thiol-based antioxidant, a small molecule BMP inhibitor, a zinc compound, a thyroid hormone signaling pathway activator, a Vitamin C compound, and a heparin; and wherein in all steps (i)-(viii) at least one of the tankyrase 1 / 2 inhibitor is Wiki4 and the G9a inhibitor is UNC0321.

29. The method of Claim 28 further comprising obtaining the PP cell population used in step (i) by culturing a foregut endoderm (FE) population of cells comprising PDX1+cells in an FE-differentiating medium for a time period sufficient to obtain the PP cell population, wherein the FE-differentiating medium is a defined medium comprising about 5 mM to about 50 mM glucose and a set of differentiation factors in amounts sufficient to promote differentiation of at least a portion of the FE cell population to PP cells, wherein the set of differentiation factors comprises at least one factor selected from the group consisting of: an EGF family growth factor, a Vitamin B3 compound, a Vitamin C compound,a FGF family growth factor, a PKC activator, a retinoid, a ROCK inhibitor, a cell-permeable SHH signaling inhibitor, at least one tankyrase 1 / 2 inhibitor, optionally two tankyrase 1 / 2 inhibitors, wherein at least one of the trankyrase ½ inhibitor is Wiki4.

30. The method of Claim 28 or 29, further comprising the step of: obtaining the FE cell population by: (i) culturing a primitive gut tube (PGT) population of cells comprising FOXA2+cells or a PGT cell population in a first PGT-differentiating medium for a first time period of about 12 hours to about 48 hours to obtain an intermediate PGT / FE cell population; and (ii) culturing the intermediate PGT / FE cell population in a second PGT medium for a second time period of about 12 hours to about 48 hours to obtain the FE cell population, wherein the first PGT-differentiating medium is a defined medium comprising about 5 mM to about 50 mM glucose and a set of differentiation factors in amounts sufficient to promote differentiation of at least a portion of the PGT cell population to FE cells, wherein the factors are selected from the group consisting of a small molecule BMP inhibitor and at least one of an FGF family growth factor, a PKC activator, a retinoid, a ROCK inhibitor, a cell- permeable SHH signaling inhibitor and at least one tankyrase 1 / 2 inhibitor; and wherein the second PGT-differentiating medium is a defined medium that lacks a BMP inhibitor and comprises about 5 mM to about 50 mM glucose, about 0.5 mM to about 1.5 mM pyruvate and a set of differentiation factors in amounts sufficient to promote differentiation of at least a portion of the intermediate PGT / FE cell population to FE cells, wherein the factors comprise at least one factor selected from the group consisting of: a Vitamin C compound, an FGF family growth factor, a PKC activator, a retinoid, a ROCK inhibitor, a cell-permeable SHH signaling inhibitor, andat least one tankyrase 1 / 2 inhibitor, optionally two tankyrase 1 / 2 inhibitors, wherein at least one of the trankyrase ½ inhibitor is Wiki4.

31. The method of any one of Claims 28 to 30, further comprising obtaining the PGT cell population by culturing a definitive endoderm (DE) population of cells comprising PDX1- cells, and FOXA2+ / SOX17+ cells or GATA6+ / SOX17+ cells, in a DE-differentiating medium for a time period sufficient to obtain the PGT cell population, wherein the DE- differentiating medium is a defined medium comprising about 5 mM to about 20 mM glucose and a set of differentiation factors in amounts sufficient to promote differentiation of at least a portion of the DE cell population to PGT cells, wherein the factors comprise at least one factor selected from the group consisting of a Vitamin C compound and an FGF family growth factor.

32. A composition comprising a cell population, wherein: (i) < about 2% of the cells in the cell population or population cells are non- endocrine cells (CHGA-), or at least about 98% of the population cells express chromogranin A (CHGA+); (ii) at least about 50% of the population cells are CPEP+ / GCG- cells or at least about 50% of the population cells produce C-peptide (CPEP+) and do not express glucagon (GCG-) (CPEP+ / GCG-); (iii)< about 40% of the population cells express glucagon (GCG+); (iv) at least about 45%of the population cells are pancreatic endocrine cells (PDX+ / CHGA+), or at least about 45% of the population cells produce CPEP (CPEP+) and express NK6 homeobox 1 (NKX6.l+) (CPEP+ / NKX6.1+); (v) at least about 60% of the population cells produce insulin (INS+) and do not express solute carrier family 18 member 1 (SLC18A1-) (INS+ / SLC18A1-); (vi) an insulin content of at least about 150 nU / cell; (vii) < about 16% of the population cells are INS- / SLC+ cells that do not produce insulin, or < about 16% of the population cells do not produce insulin and express solute carrier family 18 member 1 (SLC18A1+) (INS- / SLC18A1+); (viii) < about 5% of the population cells are proliferating cells (Ki67+), or less than about 5% of the population cells express (Ki67+); (ix) at least about 99.5% of the population cells are CHGA+, at least about 60% of the population cells are CPEP+ / GCG-, at least about 50% of the population cells areCPEP+ / NKX6.1+and at least about 70% of the population cells are INS+ / SLC18A1- ; (x) < about 12% of the population cells are INS- / SLC18A1+and < about 4% of the population cells are Ki67-; (xi) the cell population does not produce lactate; (xii) < about 0.5% or about 0.2% of the population cells are non-endocrine cells, at least about 60% of the population cells are CPEP+ / GCG- cells, at least about 50% or about 60% of the population cells are PBLCs expressing NKX6.1 (NKX6.1+), at least about 70% or about 75% of the population cells are insulin-producing cells that are not ECLCs (INS- / SLC+); < about 11% or about 7% of the population are INS- / SLC+ cells that do not produce insulin and < about 4% or about 2% of the population cells are proliferating cells; the cell population does not produce lactate and optionally the cell population has an insulin content of at least about 325 nU / cell, or 150nU / cell to at least about 200nU / cell; and (xiii) at least about 98% or at least about 99.5% of the population cells are CHGA+, at least about 60% or about 65% of the population cells are CPEP+ / GCG-, at least about 50% or about 60% of the population cells are CPEP+ / NKX6.1+, at least about 70% or about 75% of the population cells are INS+ / SLC18A1-; < about 11% or about 7% of the population are INS- / SLC18A1+and < about 4% or about 2% of the population cells are Ki67-; the cell population does not produce lactate; and optionally the cell population has an insulin content of at least about 325 nU / cell, or at least 150nU / cell to about 200nU / cell.

33. An in vitro cell population comprising cells wherein: (i) about 40% to about 60% or about 45% to about 55% of the cells in the population are PDX1+ / NKX6.1+cells; optionally wherein about 60% of the cells in the population are PDX1+ / NKX6.1+; (ii) about 60% to about 90% or about 65% to about 75% of the cells in the population are PDX1+ / CHGA- cells; (iii) about 50% to about 65% or about 50% to about 60% of the cells in the population are NKX6.1+cells; (iv) about 65% to about 97% or about 80% to about 85% of the cells in the population are PDX1+cells; or(v) < about 5% to about 15% or < about 9% to about 13% of the cells in the population are CHGA+cells.

34. An in vitro cell population comprising cells wherein: (i) at least about 60% of the cells in the population are CPEP+ / GCG- cells; optionally about 60%, about 60% to about 68% of the cells in the population are CPEP+ / GCG- cells; (ii) no more than about 23% of the cells in the population are GCG+cells; optionally about 10% to about 20%, about 10% to about 23% of the cells in the population are GCG+cells; (iii) at least about 54% of the cells in the population are NKX6.1+ / CPEP+cells; optionally about 54% to about 65%, about 54% of the cells in the population are NKX6.1+ / CPEP+cells, (iv) at least about 68% of the cells in the population are INS+ / SLC- cells; optionally about 68%, about 68% to about 77% of the cells in the population are INS+ / SLC- cells; (v) less than about 11% of the cells in the population are INS- / SLC+cells; optionally about 11%, about 7% to about 11% of the cells in the population are INS- / SLC+cells; (vi) at least about 99.5% of the cells in the cell population are CHGA+cells; about 99.5% of the cells in the cell population are CHGA+cells; (vii) less than about 4% of the cells in the cell population are Ki67+cells; optionally about 3.5%, about 1% to 4% of the cells in the population are Ki67+cells; (viii) an insulin content of at least about 150nU / cell to about 200nU / cell, or (ix) lactate production by the cell population in 48hrs of less than 0.5mM, about 0 to about 0.02 mM, about 0 to about 0.04 mM, about 0 to about 0.06 mM, about 0 to about 0.08 mM about 0 to about 0.1 mM 0, about 0 to about 0.2 mM, about 0 to about 0.3 mM, about 0 to about 0.4 mM, about 0 to about 0.5 mM, wherein the population comprises at least two of the above characteristics.

35. The in vitro cell population of claim 34, wherein at least about 60% to about 68% of the cells in the population are CPEP+ / GCG- and at least about 99.5% of the cells in the population are CHGA+.

36. The in vitro cell population of claim 34, wherein at least about 54% to about 65% of the cells in the population are NKX6.1+ / CPEP+and less than about 11% of the cells in the population are INS- / SLC+.

37. The in vitro cell population of claim 34, wherein at least about 60% to about 68% of the cells in the population are CPEP+ / GCG- and at least about 68% of the cells in the population are INS+ / SLC-.

38. The in vitro cell population of claim 34, wherein at least about 54% of the cells in the population are NKX6.1+ / CPEP+and at least about 68% to about 77% of the cells in the population are INS+ / SLC-.

39. The in vitro cell population of claim 34-38, wherein no more than about 23% of the cells in the population are GCG+.

40. The in vitro cell population of claim 34-39, wherein at least about 99.5% of the cells in the population are CHGA+.

41. An in vitro cell population comprising cells wherein: (i) at least about 67% of the cells in population are CPEP+ / GCG-; optionally about 67% to about 80%, about 67% about 70%, about 70% to about 80%, about 70% to about 85% of the cells in the population are CPEP+ / GCG- cells; (ii) no more than about 22% of the cells in the cell population are GCG+cells; optionally about 10% to about 20%, about 10% to about 22% of the cells in the population are GCG+cells; (iii) at least about 60% of the cells in the cell population are NKX6.1+ / CPEP+cells; optionally about 60% to about 70%, about 65% to about 75%, about 60% of the cells in the population are NKX6.1+ / CPEP+cells; (iv) about 68% of the cells in the cell population are INS+ / SLC- cells; optionally about 77%, about 70% to about 80%, about 70% to about 85% of the cells in the population are INS+ / SLC- cells; (v) less than about 7% of the cells in the cell population are INS- / SLC+cells; optionally about 7%, about 3-7% of the cells in the population are INS- / SLC+cells; (vi) at least about 99.8% of the cells in the cell population are CHGA+cells; about 99.8% of the cells in the cell population are CHGA+cells; (vii) less than about 2% of the cells in the cell population are Ki67+cells; optionally 0.3- 2%, about 2% of the cells in the population are Ki67+cells;(viii) an insulin content of at least 150nu / cell to about 200nU / cell, or (ix) lactate production by the cell population in 48hrs of less than 0.5mM, about 0 to about 0.02 mM, about 0 to about 0.04 mM, about 0 to about 0.06 mM, about 0 to about 0.08 mM about 0 to about 0.1 mM 0, about 0 to about 0.2 mM, about 0 to about 0.3 mM, about 0 to about 0.4 mM, about 0 to about 0.5 mM, wherein the population comprises at least two of the above characteristics.

42. The in vitro cell population of claim 41, wherein about 67% of the cells in population are CPEP+ / GCG- and about 99.8% of the cells in the population are CHGA+cells.

43. The in vitro cell population of claim 41, wherein at least about 54% of the cells in the population are NKX6.1+ / CPEP+and less than about 11% of the cells in the population are INS- / SLC+.

44. The in vitro cell population of claim 41, wherein about 67% of the cells in the population are CPEP+ / GCG- and about 68% of the cells in the population are INS+ / SLC-.

45. The in vitro cell population of claim 41, wherein about 60% of the cells in the population are NKX6.1+ / CPEP+and about 68% of the cells in the population are INS+ / SLC-.

46. The in vitro cell population of claim 41-45, wherein no more than about 22% of the cells in the population are GCG+.

47. The in vitro cell population of claim 41-45, wherein at least about 99.8% of the cells in the population are CHGA+.

48. The in vitro cell population of any one of claims 35-40, 42-47, wherein the population has an insulin content of at least 150nU / cell to about 200nU / cell.

49. The in vitro cell population of any one of claims 35-40, 42-47, wherein lactate production by the cell population of less than 0.5 mM in 48hrs.

50. A liquid cell differentiating composition comprising: (a) a serum-free basal culture media; and (b) a set of differentiation factors, wherein the set of differentiation factors is: (i) a set of factors promoting differentiation of foregut endoderm (FE) cells to pancreatic progenitor (PP) cells (a FE factor set); (ii) a set of factors promoting differentiation of pancreatic progenitor (PP) comprising PDX1+ cells to pancreatic endocrine precursor (PEP) population comprising PDX1+ / CHGA+cells (a PP factor set);(iii) a set of factors promoting differentiation of PEP cells to immature SC- IC (a PEP factor set); or (iv) a set of factors capable of promoting differentiation of immature SC-IC to mature SC-IC (a SC-IC factor set).

51. The liquid cell differentiating composition of claim 50, wherein the serum-free basal culture media comprises 0 mM to less than 2.5 mM glucose and the PP factor set comprises a G9a inhibitor, optionally UNC0321.

52. The liquid cell differentiating composition of claim 51 further comprising at least one tankyrase ½ inhibitor, optionally wherein the tankyrase ½ inhibitor is Wiki4.

53. Use of the liquid cell differentiating composition of claim 51 or 52 for differentiating progenitor populations comprising PDX1+ cells, optionally a PP population comprising PDX1+ cells to a PEP population comprising PDX1+ / CHGA+cells.

54. A pharmaceutical composition comprising the composition of claim 32 or the in vitro cell populations of any one of claims 33-49, and a carrier.

55. A method of treating an individual having diabetes mellitus, one or more complications related to diabetes mellitus or a pre-diabetic condition, the method comprising: (a) administering to the individual an effective amount of the composition of claim 33, the in vitro cell population of any one of claims 33-49 or the pharmaceutical composition of claim 54; (b) administering to the individual an effective amount of a pharmaceutical composition comprising the composition of claim 33 or the in vitro cell population of any one of claims 33-49 encapsulated in a device that provides immune protection of the encapsulated cells; or (c) administering to the individual a device comprising the composition of claim 33, the in vitro cell population of any one of claims 33-49 or the pharmaceutical composition of claim 54.

56. The method of claim 55, the method further comprising administering to the individual an immunosuppressant before and / or after the administering of step (a), (b) or (c).

57. The method of claim 55 or 56 wherein the device comprises alginate chemically modified with an afibrotic-effective amount of a compound of Formula I.

58. The method of any one of claims claim 57-59, wherein diabetes mellitus is Type 1 Diabetes.

59. The composition of claim 33, the in vitro cell population of any one of claims 33-49 or the pharmaceutical composition of claim 54 for use in the treatment of diabetes mellitus, one or more complications related to diabetes or a pre-diabetic condition.

60. Use of the composition of claim 33, the in vitro cell population of any one of claims 33-49 or the pharmaceutical composition of claim 54 in the manufacture of a medicament for treating diabetes, one or more complications related to diabetes mellitus or a pre-diabetic condition.

61. A method of culturing stem cells to obtain a population of differentiated cells, the method comprising at least one step of culturing a population of cells at a pH that is not higher than pH7.8, a pH in the range of: pH7.2 to pH7.8, pH7.3 to pH 7.8, pH7.4 to pH7.8, pH 7.2+ / - 0.2 (pH7.0 to pH7.4), pH 7.4+ / - 0.2 (pH7.2 to pH7.6), pH7.6+ / - 0.2 (pH7.4 to pH7.8) or pH7.8+ / - 0.2 (pH7.6 to pH8.0), wherein the at least one step is conducted in a bioreactor.

62. The method of claim 61 wherein the at least one step of culturing a population of cells at a pH, which pH is not higher than pH7.8, a pH in the range of: pH7.2 to pH7.8, pH7.3 to pH 7.8, pH7.4 to pH7.8, pH 7.2+ / - 0.2 (pH7.0 to pH7.4), pH 7.4+ / - 0.2 (pH7.2 to pH7.6), pH7.6+ / - 0.2 (pH7.4 to pH7.8) or pH7.8+ / - 0.2 (pH7.6 to pH8.0), is conducted in at least one differentiation medium comprising a defined medium comprising 0mM to less than 2.5mM glucose or 0mM to less than 2mM glucose.

63. A method of deriving a population of differentiated cells derived from stem cells, the method comprising at least one step of culturing a population cells at a pH that is not higher than pH7.8, a pH in the range of: pH7.2 to pH7.8, pH7.3 to pH 7.8, pH7.4 to pH7.8, pH 7.2+ / - 0.2 (pH7.0 to pH7.4), pH 7.4+ / - 0.2 (pH7.2 to pH7.6), pH7.6+ / - 0.2 (pH7.4 to pH7.8) or pH7.8+ / - 0.2 (pH7.6 to pH8.0) in at least one differentiation medium.

64. The method of claim 63, wherein the differentiation medium comprises a defined medium comprising 0 mM to less than 2.5mM glucose or 0 mM to less than 2mM glucose, and wherein the method is conducted in a bioreactor.

65. The method of any one of claims 61- 64, wherein the population of cells comprises PDX1+cells.

66. The method of any one of claims 61- 65, wherein the population of differentiated cells is mature stem cell-derived islet-like cells (SC-ICs) comprising CPEP+ / GCG- cells and GCG+cells.

67. A method of deriving a cell population comprising mature stem cell-derived islet-like cells (SC-ICs), the method comprising a step of: (a) culturing a first precursor cell population comprising PDX1+ cells in one or more differentiating mediums to obtain the mature SC-IC population, wherein the first precursor cell population is selected from the group consisting of a foregut endoderm (FE) population, a pancreatic progenitor (PP) cell population, a pancreatic endocrine precursor (PEP) cell population and a precursor SC-IC cell population, wherein at least one of the differentiating mediums is a defined medium comprising G9a inhibitor.

68. The method of claim 67, wherein the G9a inhibitor is UNC0321 and optionally wherein at least one of the differentiating mediums is a defined medium comprising glucose at less than about 2.5 mM or at less than about 2 mM.

69. The method of claim 68, wherein the culturing of the first precursor cell population comprising PDX1+ cells is at pH in the range of: pH7.2 to pH7.8, pH7.3 to pH7.8, pH7.4 to pH7.6, pH7.4 to pH7.8, pH 7.2+ / - 0.2 (pH7.0 to pH7.4), pH 7.4+ / - 0.2 (pH7.2 to pH7.6), pH7.6+ / - 0.2 (pH7.4 to pH7.8) or pH7.8+ / - 0.2 (pH7.6 to pH8.0).

70. An in vitro cell population comprising mature SC-ICs wherein the population is produced by the method of any one of claims 1-31 or 61-69.

71. An in vitro cell population comprising cells wherein: (i) at least about 67% of the cells in population are CPEP+ / GCG-; (ii) no more than about 22% of the cells in the cell population are GCG+cells; (iii) at least about 60% of the cells in the cell population are NKX6.1+ / CPEP+cells; (iv) about 68% of the cells in the cell population are INS+ / SLC- cells; (v) less than about 7% of the cells in the cell population are INS- / SLC+cells; (vi) at least about 99.8% of the cells in the cell population are CHGA+cells; (vii) less than about 2% of the cells in the cell population are Ki67+cells; (viii) an insulin content of 150nu / cell to at least about 200nU / cell, or (ix) lactate production by the cell population of less than 0.5 mM in 48hrs, wherein the population comprises at least two of the above characteristics, and wherein the cell population is produced by the method of any one of claims 1-31 or 61-69.