Methods for preparing stem cell-derived islet-like cells, populations thereof, and compositions comprising them.

Abstract

Disclosed are methods for preparing a population of stem cell-derived islet-like cells (SC-ICs), wherein such methods comprise culturing a precursor cell, e.g., a pancreatic progenitor cell, a pancreatic endocrine precursor cell, or an immature SC-IC, in one or more differentiation media that promote differentiation of the precursor cell to a more highly differentiated cell to obtain a population of SC-ICs comprising mature pancreatic beta-like cells. Also disclosed are cell differentiation compositions useful for performing the methods and populations of SC-ICs obtained by performing the methods. Also disclosed are compositions and implantable devices comprising the population of SC-ICs, which are useful for treating metabolic disorders, e.g., diabetes mellitus (diabetes).

Description

[0001] Reference to the electronically submitted sequence list This disclosure is submitted together with a sequence list in ST.26 XML format. This sequence list is provided as a file named “30174_WO”, created on November 8, 2024, and is 6.6 kilobytes (kb) in size. The sequence list information in ST.26 XML format is incorporated herein by reference in its entirety. Technical Field

[0002] This disclosure relates generally to biology and medicine, and more specifically, to methods for preparing stem cell-derived islet-like cells (SC-IC), populations thereof, and compositions comprising thereof, and to their use in the treatment of metabolic disorders such as diabetes mellitus.

[0003] background Diabetes is a major global healthcare problem and a group of metabolic disorders characterized by abnormal glucose homeostasis / metabolism. A hallmark of diabetes is elevated blood glucose levels. One form of diabetes is type 1 diabetes (T1D), which results from the autoimmune destruction of beta cells in the pancreas, leading to insulin (INS) deficiency. Another form of diabetes is type 2 diabetes (T2D), which results from peripheral tissue INS resistance and beta (β) cell dysfunction.

[0004] Individuals with diabetes manage their condition by keeping their blood glucose levels close to normal (i.e., 70–120 mg / dL or 3.9–6.7 mmol / L). Diabetes management includes diet, exercise, weight loss, the use of therapeutic agents (e.g., exogenous insulin and / or antidiabetic medications) or a combination thereof.

[0005] A newer treatment for diabetes, particularly type 1 diabetes (T1D), islet transplantation using donor islets. See, for example, Shapiro et al. (2000). N. Engl. J. Med. 343:230-238. However, islet transplantation has many drawbacks, including a shortage and poor quality of donor islets, and the need for immunosuppressive cocktail therapy.

[0006] An alternative to islet transplantation using donor islets is to derive islet-like cell populations from stem cells. Several in vitro methods for differentiating stem cells into islet-like cells are known. See, for example, 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 International 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, WO2020033879, WO 2022204377, WO 2023076554, U.S. Patent Nos. 9388386, 10975355, 11299711, 11466256, 11525120 and 11332716.

[0007] Despite the existence of these methods, there is still a need for additional in vitro methods to differentiate stem cells, including induced pluripotent stem cells (iPSCs), into functional islet-like cells, which are particularly capable of efficiently producing and secreting insulin in response to glucose challenge. These functional islet-like cells are referred to herein as SC-IC. In particular, there is a need for in vitro methods to generate cell populations containing large numbers and / or high proportions of differentiated functional islet-like cells.

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

[0009] In one aspect, SC-IC populations can be derived by methods including or beginning with culturing PP cell populations, PEP cell populations, or precursor SC-IC populations in one or more differentiation media (e.g., as defined herein), at least one of which is a low-glucose defined-component medium containing <about 2.5 mM glucose or <about 2 mM glucose, such as, but not limited to, zero glucose. In some cases, SC-IC populations may have one or more altered characteristics compared to control SC-IC populations obtained by performing the same methods in the same differentiation media (except that each control differentiation medium has ≥2 mM glucose or ≥2.5 mM glucose). In some cases, SC-IC populations may 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 β-like cells (PBLCs).

[0010] In another aspect, SC-IC populations can be derived by methods including or beginning with culturing PP or PEP cell populations in one or more differentiation media, at least one of which is a defined component containing an epigenetic modifier. In some cases, SC-IC populations may have one or more altered characteristics compared to control SC-IC populations obtained by performing the same methods in the same differentiation media (except that each control differentiation media lacks an epigenetic modifier). In some cases, altered characteristics may include one or more of the following: a higher percentage of PECs, a higher percentage of PBLCs, and a lower percentage of multi-hormone cells. In some cases, the epigenetic modifier may be an S-adenosylhomocysteine ​​hydrolase (ADOHCYASE) inhibitor, a (HMT) inhibitor, a DNA methyltransferase (DNMT) inhibitor, a histone deacetylase (HDAC) inhibitor, a silencing information regulator 1 (SIRT1) activator, or a silencing information regulator 6 (SIRT6) activator. In other cases, epigenetic modifiers can be inhibitors of euchromatin histone-lysine N-methyltransferase 2 (EHMT2) (also known as G9a).

[0011] In another aspect, SC-IC populations can be derived by methods including or beginning with culturing PP or PEP cell populations in one or more differentiation media, wherein the culture is conducted at pH ranges including: pH 7.2 to pH 7.8, pH 7.3 to pH 7.8, pH 7.4 to pH 7.6, pH 7.4 to pH 7.8, pH 7.2 + / - 0.2 (pH 7.0 to pH 7.4), pH 7.4 + / - 0.2 (pH 7.2 to pH 7.6), pH 7.6 + / - 0.2 (pH 7.4 to pH 7.8), or pH 7.8 + / - 0.2 (pH 7.6 to pH 8.0). In some cases, SC-IC populations may exhibit one or more altered characteristics compared to control SC-IC populations obtained by performing the same methods in the same differentiation media (except for culturing at pH values ​​below 7.4).

[0012] In another aspect, SC-IC populations can be derived by methods including or beginning with: culturing PP cell populations in one or more PP differentiation media to generate PEP cell populations, then culturing PEP cell populations in one or more PEP differentiation media to generate precursor SC-IC populations, and then culturing the precursor SC-IC populations in SC-IC differentiation media to obtain SC-IC populations, wherein the PP differentiation media, PEP differentiation media, and SC-IC differentiation media can each be a defined component medium containing a G9a inhibitor. In some cases, SC-IC populations have one or more altered characteristics compared to control SC-IC populations and can be obtained by performing the same methods in the same differentiation media (except that each control differentiation medium lacks a G9a inhibitor). In some cases, altered characteristics in SC-IC populations may include one or more of the following: a higher percentage of PECs, a higher percentage of PBLCs, a lower percentage of multi-hormone cells, increased INS content, increased INS secretion, and increased GSIS. In some cases, the G9a inhibitor may be CM-272, UNC0321, UNC0638, or a combination thereof.

[0013] In another aspect, SC-IC populations can be derived by methods including or beginning with culturing PEP cell populations or precursor SC-IC populations in one or more differentiation media, wherein at least one of the differentiation media is a defined component medium containing ≤ about 1 mM pyruvate. In some cases, SC-IC populations have one or more altered characteristics compared to control SC-IC populations obtained by performing the same method in the same differentiation media (except that each control differentiation media contains > about 1 mM pyruvate). In some cases, altered characteristics in derived SC-IC populations may include one or more of the following: increased INS secretion and increased GSIS. In some cases, the defined component medium may include about 0.05 mM pyruvate. In some cases, the defined component medium may include human plasma-like medium (HPLM). In some cases, altered characteristics in SC-IC populations compared to control SC-IC populations obtained by performing the same method in a differentiation medium lacking HPLM may be one or more of the following: a higher percentage of PECs, a higher percentage of PBLCs, a lower percentage of multi-hormone cells, increased INS content, increased INS secretion, and increased GSIS.

[0014] In another aspect, methods and processes for generating a population of INS-producing cells (referred to as SC-IC) with improved properties are described herein. In one embodiment, the method described herein is based on the finding that cells are cultured in a differentiation medium having a glucose concentration of less than 2.5 mM (e.g., but not limited to 0 mM glucose), and that, while differentiating PP cells into PEP cells or PEP cells into immature SC-IC cells, galactose (or another alternative nutrient) is optionally added to reduce (i.e., decrease or eliminate) EC-like cells (i.e., SLC18A1). + (Cells). In one embodiment, the method includes at least one step of culturing cells, such as, but not limited to, culturing PP cells into PEP cells or culturing PEP cells into immature SC-IC cells in a bioreactor at pH ranges including: pH 7.2 to pH 7.8, pH 7.3 to pH 7.8, pH 7.4 to pH 7.6, pH 7.4 to pH 7.8, pH 7.2 + / - 0.2 (pH 7.0 to pH 7.4), pH 7.4 + / - 0.2 (pH 7.2 to pH 7.6), pH 7.6 + / - 0.2 (pH 7.4 to pH 7.8), or pH 7.8 + / - 0.2 (pH 7.6 to pH 8.0). In one embodiment, the method includes at least one cell culture step, such as, but not limited to, culturing PP cells into PEP cells, or culturing PEP cells into immature SC-IC cells, in a bioreactor in a differentiation medium having a glucose concentration of less than 2.5 mM (e.g., but not limited to 0 mM glucose) and at pH ranges including: pH 7.2 to pH 7.8, pH 7.3 to pH 7.8, pH 7.4 to pH 7.6, pH 7.4 to pH 7.8, pH 7.2 + / - 0.2 (pH 7.0 to pH 7.4), pH 7.4 + / - 0.2 (pH 7.2 to pH 7.6), pH 7.6 + / - 0.2 (pH 7.4 to pH 7.8), or pH 7.8 + / - 0.2 (pH 7.6 to pH 8.0). Unbound by theory, pH and glucose concentrations are effectively targeted at EC-like cells (i.e., SLC18A1) in the differentiation population. + The method selects cells and thereby reduces (i.e., decreases or eliminates) such cells. In one embodiment, the method described herein is based on the finding that differentiation of PPs is optimal in the presence of two end-anchored polymerase 1 / 2 inhibitors (at least one of which is Wiki4). In some embodiments, at least about 67% of the cells in the population are CPEPs. + / GCG - Cells, for example, 67% to 80% of the cells in a population are CPEP. + / GCG - Furthermore, at least approximately 99% of the cells in the population are CHGA. +For example, 99% to 99.99% of the cells in the population are CHGA + Insulin-producing cell populations can be used in cell therapy to treat conditions such as diabetes (e.g., T1D).

[0015] Secondly, this disclosure describes methods for deriving SC-ICs from pluripotent stem cells such as iPSCs or partially differentiated cells by incorporating one or more of the above differentiation methods.

[0016] In some cases, the method may include or may begin with the steps of differentiating pluripotent stem cells (PSCs) into mesoendothelial (ME) cells by culturing a cell population comprising PSCs (PSC population) in a PSC differentiation medium for about 0.5 days to about 2 days, particularly about 1 day, to obtain a cell population comprising ME cells (ME cell population). In some cases, the PSC population may be iPSCs (iPSC population), and the PSC differentiation medium may be a defined component medium supplemented with one or more differentiation factors, the amount of which effectively promotes the differentiation of at least a portion of the iPSC population into ME cells.

[0017] In some cases, the PSC differentiation medium may include glucose and / or fructose and may be supplemented with one or more of the following: GSK-3α and GSK-3β inhibitors / Wnt pathway signaling activators (e.g., CHIR99021), Rho kinase (ROCK) inhibitors (e.g., Y-27632), growth factors from the transforming growth factor β (TGF-β) superfamily (e.g., activin A), and Wnt / β-catenin pathway activators (e.g., Wnt3a protein).

[0018] In some cases, the PSC differentiation medium may also include one or more of the following: buffers (e.g., sodium bicarbonate (NaHCO3)), albumin, glutamine (e.g., glutamine dipeptide), glutamate, and pyruvate (e.g., sodium pyruvate).

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

[0020] In some cases, a PSC population may include one or more cell aggregates, each of which has an initial aggregate diameter of about 150 μm to about 170 μm.

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

[0022] The method may also include amplification and / or aggregation steps for PSCs prior to the differentiation initiation step. In some cases, the method may include washing PSCs in a defined composition medium prior to the differentiation initiation step.

[0023] Alternatively, the method may include or may begin with the steps of differentiating ME cells into defined endoderm (DE) cells by culturing a population of ME cells in an ME differentiation medium (e.g., as defined herein) for about 0.5 days to about 2 days, particularly about 1 day, to obtain a cell population comprising DE cells (i.e., a DE cell population). The ME differentiation medium may be a defined component medium supplemented with one or more differentiation factors, the amount of which effectively promotes the differentiation of at least a portion of the ME cell population into DE cells.

[0024] In some cases, ME differentiation medium may include glucose and / or fructose and may be supplemented with one or more of the following: bone morphogenetic protein (BMP) inhibitors (e.g., LDN-193189) and TGF-β superfamily growth factors (e.g., activin A).

[0025] In some cases, ME differentiation medium may also include one or more of the following: buffer (e.g., NaHCO3), albumin, glutamine (e.g., glutamine dipeptide), glutamate, and pyruvate (e.g., sodium pyruvate).

[0026] In some cases, ME differentiation medium may also include serum alternative supplements, which include one or more of the following: INS, transferrin, selenium (e.g., sodium selenite) and ethanolamine (e.g., ITS-G or ITS-X supplements).

[0027] In some cases, ME cell populations can be derived from hiPSC populations, and DE cell populations can be characterized as including >approximately 90% GATA6. + / SOX17 + Cells and at least one of about 40% to about 80% FOXA2 + / SOX17 + cell.

[0028] In some cases, the method may include washing the ME cell population in a defined culture medium before initiating the differentiation step.

[0029] Alternatively, the method may include or may begin by differentiating DE cells into primitive intestinal (PGT) cells by culturing a population of DE cells in a DE differentiation medium (e.g., as defined herein) for approximately 2 to approximately 4 days, particularly approximately 3 days, to obtain a cell population comprising PGT cells (e.g., a PGT cell population as defined herein). The DE differentiation medium may be a defined component medium supplemented with one or more differentiation factors, the amount of which effectively promotes the differentiation of at least a portion of the DE cell population into PGT cells.

[0030] In some cases, the DE differentiation medium may include glucose and / or fructose and may be supplemented with one or more of the following: vitamin C compounds (e.g., ascorbic acid) and growth factors from the fibroblast growth factor (FGF) family (e.g., keratinocyte growth factor (KGF)).

[0031] In some cases, the DE differentiation medium may also include one or more of the following: buffer (e.g., NaHCO3), albumin, glutamine (e.g., glutamine dipeptide), glutamate and pyruvate (e.g., sodium pyruvate), and glutamine (e.g., glutamine dipeptide), glutamate and pyruvate (e.g., sodium pyruvate).

[0032] In some cases, the DE differentiation medium may also include serum replacement supplements, including one or more of the following: INS, transferrin, selenium (e.g., sodium selenite) and ethanolamine (e.g., the ITS-G or ITS-X supplements described herein).

[0033] In some cases, 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. + cell.

[0034] Alternatively, the method may include or may begin with the steps of differentiating PGT cells into foregut endoderm (FE) cells by culturing a population of PGT cells in a first PGT differentiation medium for about 0.5 days to about 2 days, particularly about 1 day, and then culturing them in a second PGT differentiation medium for about 12 hours (hr) to about 48 hours, particularly about 1 day, to obtain a cell population comprising FE cells (e.g., an FE cell population). Each PGT differentiation medium may be a defined component medium supplemented with one or more differentiation factors, the amount of which effectively promotes the differentiation of at least a portion of the PGT cell population into FE cells.

[0035] In some cases, the first PGT differentiation medium may include glucose and / or fructose and may be supplemented with one or more of the following: vitamin C compounds (e.g., ascorbic acid), small molecule BMP inhibitors (e.g., DMH-1), FGF family growth factors (e.g., KGF protein), protein kinase C (PKC) activators (e.g., 2S,5S-E,E-8-5-4-trifluoromethylphenyl-2,4-pentadienoylaminobenzolactam (TPPB)), retinoids (e.g., all-trans retinoic acid (ATRA)), ROCK inhibitors (e.g., Y-27632), cell-permeable sound hedgehog (SHH) signaling inhibitors (e.g., SANT-1), TGF-β superfamily growth factors (e.g., activin A), and at least one end-anchored polymerase 1 / 2 inhibitor as defined herein (e.g., IWR-1 and / or WIKI4). In some cases, the first PGT differentiation medium may include only one end-anchored polymerase 1 / 2 inhibitor (e.g., IWR-1 or WIKI4).

[0036] In some cases, the second PGT differentiation medium lacks BMP inhibitors, but in other respects it may be the same as the first PGT differentiation medium. In some cases, the second PGT differentiation medium includes two end-anchored polymerase 1 / 2 inhibitors (e.g., IWR-1 and WIKI4).

[0037] In some cases, one or two PGT differentiation media may also include one or more of the following: buffer (e.g., NaHCO3), albumin, glutamine (e.g., glutamine dipeptide), glutamate and pyruvate (e.g., sodium pyruvate).

[0038] In some cases, one or both PGT differentiation media may also include serum replacement supplements comprising one or more of the following: INS, transferrin, selenium (e.g., sodium selenite), ethanolamine, biotin, α-tocopherol, vitamin A, albumin, catalase, superoxide dismutase, corticosterone, galactose, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, and triiodo-L-thyroxine (e.g., the B27 supplement described herein).

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

[0040] Alternatively, the method may include or may begin with the step of differentiating FE cells into PP cells by culturing a population of FE cells in an FE differentiation medium for approximately 2 to approximately 6 days, particularly approximately 3 days, to obtain a cell population comprising PP cells (e.g., a PP cell population). The FE differentiation medium may be a defined component medium supplemented with one or more differentiation factors, the amount of which effectively promotes the differentiation of at least a portion of the FE cell population into PP cells.

[0041] In some cases, the FE differentiation medium may include glucose and / or fructose and may be supplemented with one or more of the following: vitamin C compounds (e.g., ascorbic acid), growth factors from the epidermal growth factor (EGF) family (e.g., EGF protein), FGF family growth factors (e.g., KGF protein), vitamin B3 compounds (e.g., nicotinamide (NAM)), PKC activators (e.g., TPPB), retinoids (e.g., ATRA), ROCK inhibitors (e.g., Y-27632), cell-permeable SHH signaling inhibitors (e.g., SANT-1), and at least one end-anchored polymerase 1 / 2 inhibitor as defined herein (e.g., IWR-1 and / or WIKI4).

[0042] In some cases, FE differentiation media may also include epigenetic modifiers (e.g., G9a inhibitors such as UNC321).

[0043] In some cases, FE differentiation medium may also include one or more of the following: buffers (e.g., NaHCO3), albumin, glutamine (e.g., glutamine dipeptide), glutamate, and pyruvate (e.g., sodium pyruvate).

[0044] In some cases, FE differentiation media may also include serum replacement supplements, which include one or more of the following: INS, transferrin, selenium (e.g., sodium selenite), ethanolamine, biotin, α-tocopherol, vitamin A, albumin, catalase, superoxide dismutase, corticosterone, galactose, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, and triiodo-L-thyroxine (e.g., the B27 supplement described herein).

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

[0046] Alternatively, the method may include or may begin by differentiating PP cells into PEP cells by culturing a PP cell population in a first PP differentiation medium (e.g., as defined herein) for about 3 to about 6 days, particularly about 4 days, and then culturing it in a second PP differentiation medium for about 1 to about 3 days, particularly about 2 days, to obtain a cell population comprising PEP cells (e.g., a PEP cell population as defined herein). Each PP differentiation medium may be a defined component medium supplemented with one or more differentiation factors, the amount of which effectively promotes the differentiation of at least a portion of the PP cell population into PEP cells.

[0047] PP and PP / PEP cell populations cultured in bioreactors are typically cultured within a pH range of 7.0 to 7.2. The methods described in this paper unexpectedly demonstrate that culturing certain cell populations, such as those containing PP cells, at pH levels above 7.0 to 7.2 selectively improves the proportion of mature SC-IC cells in in vitro differentiated cell populations. In some cases, the culture involves monitoring and adjusting the pH in the bioreactor to maintain a pH not exceeding 7.8. In some cases, cultivation involves monitoring and adjusting the pH in the bioreactor to maintain it within the following pH ranges: pH 7.2 to pH 7.8, pH 7.3 to pH 7.8, pH 7.4 to pH 7.8, pH 7.2 + / - 0.2 (pH 7.0 to pH 7.4), pH 7.4 + / - 0.2 (pH 7.2 to pH 7.6), pH 7.6 + / - 0.2 (pH 7.4 to pH 7.8), or pH 7.8 + / - 0.2 (pH 7.6 to pH 8.0). In some embodiments, cultivation is carried out under conditions where dissolved oxygen is >50 mmHg, about 100 mmHg to about 110 mmHg, and optionally about 102 mmHg to about 107 mmHg.

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

[0049] In some cases, the first PP differentiation medium may include about 5 mM to about 40 mM of glucose and may be supplemented with one or more of the following: small molecule BMP inhibitors (e.g., LDN-193189), zinc compounds (e.g., zinc sulfate (ZnSO4)), thyroid hormone signaling pathway activators (e.g., triiodothyronine (T3)), ATP-competitive inhibitors of TGF-β RI kinase (e.g., ALK5 inhibitor II (ALK5iII)), cell-permeable SHH signaling inhibitors (e.g., SANT-1), ROCK inhibitors (e.g., Y-27632), vitamin C compounds (e.g., ascorbic acid), γ-secretase inhibitors (GSI; such as GSI-XX), epigenetic modifiers (e.g., G9a inhibitors), and at least one end-anchored polymerase 1 / 2 inhibitor (e.g., IWR-1 and / or WIKI4).

[0050] In some cases, the second PP differentiation medium may include glucose at ≤ about 0.05 mM (or glucose-free; i.e., < about 0.01 mM, < about 0.001 mM, or 0 mM), alternative nutrients (e.g., galactose), and may be supplemented with one or more of the following: epigenetic modifiers (e.g., G9a inhibitors), small molecule BMP inhibitors (e.g., LDN-193189), zinc compounds (e.g., ZnSO4), thyroid hormone signaling pathway activators (e.g., T3), ATP-competitive inhibitors of TGF-β RI kinase (e.g., ALK5iII), cell permeability SHH signaling inhibitors (e.g., SANT-1), ROCK inhibitors (e.g., Y-27632), vitamin C compounds (e.g., ascorbic acid), GSI (e.g., GSI-XX), and at least one end-anchored polymerase 1 / 2 inhibitor as defined herein (e.g., IWR-1 and / or WIKI4).

[0051] In some cases, one or two PP differentiation media may also include one or more of the following: buffer (e.g., NaHCO3), albumin, glutamine (e.g., glutamine dipeptide), glutamate and pyruvate (e.g., sodium pyruvate).

[0052] In some cases, one or both PP differentiation media may also include a serum replacement supplement comprising a mixture of two or more of the following: INS, transferrin, selenium (e.g., sodium selenite), ethanolamine, biotin, α-tocopherol, vitamin A, albumin, catalase, superoxide dismutase, corticosterone, galactose, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, and T3 (e.g., the B27 supplement described herein).

[0053] In some cases, one or both PP differentiation media may also include heparin (e.g., unfractionated heparin (UFH)).

[0054] In some cases, one or two PP differentiation media may also include non-essential amino acid (NEAA) supplements, which include two or more non-essential amino acids.

[0055] In some cases, PP cell populations include human cells, and PEP cell populations can be characterized as including at least about 70% CHGA. + Cells, at least approximately 40% CHGA + / PDX1 + Cells and less than about 30% Ki67 + cell.

[0056] In some cases, the method further includes the step of dissociating cell aggregates in the PEP cell population into individual cells before performing any subsequent steps, to obtain a dissociated PEP cell population. In some cases, the method may also include the step of washing the dissociated PEP cell population before performing any subsequent steps.

[0057] Alternatively, the method may include or may begin by differentiating PEP cells into SC-IC cells by culturing a dissociated population of PEP cells (e.g., as defined herein) in a first PEP differentiation medium for about 1 to about 3 days, particularly about 2 days, to obtain a reaggregated cell population, and then culturing the reaggregated cell population in a second PEP differentiation medium for about 1 to about 3 days, particularly about 2 days, to obtain a precursor SC-IC population. Each PEP differentiation medium is a defined component medium supplemented with one or more differentiation factors, comprising a pyruvate concentration of <1 mM (e.g., about 0.01 mM to about 0.5 mM), the amount of which effectively promotes the differentiation of at least a portion of the PEP cell population into immature pancreatic endocrine cells (e.g., immature PBLCs and immature pancreatic α-like cells (PALCs)).

[0058] In some cases, the specific components of the PEP differentiation medium may include glucose and / or fructose, <about 0.5 mM pyruvate, and may be supplemented with one or more of the following: epigenetic modifiers (e.g., G9a inhibitors), thiol-based antioxidants (e.g., N-acetylcysteine ​​(NAC)), vitamin C compounds (e.g., ascorbic acid), ATP-competitive inhibitors of TGF-β RI kinase (e.g., ALKViII)), small molecule BMP inhibitors (e.g., LDN-193189), heparin (e.g., UFH), thiol-based antioxidants (e.g., N-acetylcysteine ​​(NAC)), cell-permeable SHH signaling inhibitors (e.g., SANT-1), thyroid hormone signaling pathway activators (e.g., T3), and zinc compounds (e.g., ZnSO4).

[0059] In some cases, the first PEP differentiation medium may also include deoxyribonuclease (e.g., recombinant mammalian DNAase I).

[0060] In some cases, the defined components of one or two PP differentiation media may also include one or more of the following: buffers (e.g., NaHCO3), albumin, galactose, glutamine (e.g., glutamine dipeptide), glutamate, and pyruvate (e.g., sodium pyruvate).

[0061] In some cases, the defined components of one or two PP differentiation media may be HPLM, which may 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 cases, one or both PEP differentiation media may also include a serum replacement supplement comprising a mixture of at least two of the following components: INS, transferrin, selenium (e.g., sodium selenite), ethanolamine, biotin, α-tocopherol, vitamin A, albumin, catalase, superoxide dismutase, corticosterone, galactose, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, and triiodo-L-thyroxine (e.g., B27 supplement).

[0063] In some cases, one or both PEP differentiation media may also include heparin (e.g., UFH).

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

[0065] Alternatively, the method may include or may begin by differentiating immature SC-ICs into mature SC-ICs by culturing a precursor SC-IC population in an SC-IC differentiation medium for approximately 8 to approximately 15 days, or approximately 8 to approximately 10 days, particularly approximately 9 days, to obtain a cell population comprising mature SC-ICs (e.g., a mature SC-IC population as defined herein). The SC-IC differentiation medium may be a defined-component medium having a pyruvate concentration of <1 mM (e.g., approximately 0.01 mM to approximately 0.5 mM) and may be supplemented with one or more differentiation factors in amounts that effectively promote the differentiation of at least a portion of the precursor SC-IC population into mature PBLCs.

[0066] In some cases, the defined components of the SC-IC differentiation medium may include glucose and / or fructose, <0.5 mM pyruvate (e.g., sodium pyruvate), and may be supplemented with one or more of the following: vitamin C compounds (e.g., ascorbic acid), small molecule BMP inhibitors (e.g., LDN-193189), carnitine compounds (e.g., acetyl-L-carnitine), thiol-based antioxidants (e.g., NAC), thyroid hormone signaling pathway activators (e.g., T3), cell-permeable vitamin E analogs / antioxidants (e.g., Trolox), zinc compounds (e.g., ZnSO4), and epigenetic modifiers (e.g., G9a inhibitors).

[0067] In some cases, the defined components of the SC-IC differentiation medium may also include one or more of the following: buffers (e.g., NaHCO3), albumin, galactose, glutamine (e.g., glutamine dipeptide), and glutamate.

[0068] In some cases, the defined components of the SC-IC differentiation medium may be HPLM (e.g., as defined herein), which may 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 cases, SC-IC differentiation medium may also include a serum-alternative medium comprising 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, selenium (e.g., sodium selenite), lipid-rich albumin, and a trace element fraction (Ag). + Al3 + Ba 2+ Cd 2+ Co 2+ Cr 3+ 、Ge 4+ Se 4+ ,Br - I - F - Mn 2+ Si 4+ V 5+ Mo 6+ Ni 2+ 、Rb + Sn 2+ and Zr 4+ ) salts (e.g., KnockOut serum replacement (KOSR) medium).

[0070] In some cases, SC-IC differentiation media may also include a trace element A supplement, which includes one or more of the following: copper sulfate, ferric citrate, selenium (e.g., sodium selenite) and zinc sulfate (e.g., a trace element A supplement).

[0071] In some cases, SC-IC differentiation media may also include a trace element B supplement, which includes one or more of the following: ammonium molybdate, ammonium vanadate, manganese sulfate, nickel sulfate, sodium silicate, stannous chloride, and hydrochloric acid (e.g., trace element B supplement).

[0072] In some cases, SC-IC differentiation media may also include a chemically defined lipid mixture (CDLM) comprising two or more of the following: arachidonic acid, cholesterol, DL-α-tocopherol acetate, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, palmitoleic acid, and stearic acid (e.g., CDLM).

[0073] In some cases, SC-IC differentiation medium may also include heparin (e.g., UFH).

[0074] In some cases, SC-IC populations include human cells, and mature SC-IC populations can be characterized by including one or more of the following: (i) approximately 50% to approximately 90% INS + / SLC18A1 - Cells, (ii) approximately 0% to approximately 20% INS - / SLC18A1 + Cells, (iii) approximately 45% to approximately 75% CPEP + / GCG - Cells, (iv) approximately 5% to approximately 45% CPEP + / GCG + Cells, and (v) approximately 90% to approximately 100% CHGA + / Ki67 - cell.

[0075] Alternatively, methods for deriving SC-IC from PSC may include: differentiating PSC (e.g., iPSC, especially hiPSC) into cells expressing ME and DE characteristic markers as described herein (i.e., stage 1 cells); differentiating stage 1 cells into cells expressing PGT characteristic markers as described herein (i.e., stage 2 cells); differentiating stage 2 cells into cells expressing FE characteristic markers as described herein (i.e., stage 3 cells); differentiating stage 3 cells into cells expressing PP markers as described herein (i.e., stage 4 cells); differentiating stage 4 cells into cells expressing PEP markers as described herein (i.e., stage 5 cells); differentiating stage 5 cells into cells expressing immature SC-IC markers as described herein (i.e., stage 6 cells); and differentiating stage 6 cells into cells expressing mature SC-IC markers as described herein (i.e., stage 7 cells).

[0076] Alternatively, methods for deriving SC-IC from partially differentiated cells may include: differentiating stage 1 cells into cells expressing the PGT characteristic marker as described herein (i.e., stage 2 cells), differentiating stage 2 cells into cells expressing the FE characteristic marker as described herein (i.e., stage 3 cells), differentiating stage 3 cells into cells expressing the PP marker as described herein (i.e., stage 4 cells), differentiating stage 4 cells into cells expressing the PEP marker as described herein (i.e., stage 5 cells), differentiating stage 5 cells into cells expressing immature SC-IC markers as described herein (i.e., stage 6 cells), and differentiating stage 6 cells into cells expressing mature SC-IC markers as described herein (i.e., stage 7 cells).

[0077] Alternatively, methods for deriving SC-IC from partially differentiated cells may include: differentiating stage 2 cells into cells expressing FE characteristic markers as described herein (i.e., stage 3 cells), differentiating stage 3 cells into cells expressing PP markers as described herein (i.e., stage 4 cells), differentiating stage 4 cells into cells expressing PEP markers as described herein (i.e., stage 5 cells), differentiating stage 5 cells into cells expressing immature SC-IC markers as described herein (i.e., stage 6 cells), and differentiating stage 6 cells into cells expressing mature SC-IC markers as described herein (i.e., stage 7 cells).

[0078] Alternatively, methods for deriving SC-IC from partially differentiated cells may include: differentiating stage 3 cells into cells expressing PP markers as described herein (i.e., stage 4 cells), differentiating stage 4 cells into cells expressing PEP markers as described herein (i.e., stage 5 cells), differentiating stage 5 cells into cells expressing immature SC-IC markers as described herein (i.e., stage 6 cells), and differentiating stage 6 cells into cells expressing mature SC-IC markers as described herein (i.e., stage 7 cells).

[0079] Alternatively, methods for deriving SC-IC from partially differentiated cells may include: differentiating stage 4 cells into cells expressing PEP markers as described herein (i.e., stage 5 cells), differentiating stage 5 cells into cells expressing immature SC-IC markers as described herein (i.e., stage 6 cells), and differentiating stage 6 cells into cells expressing mature SC-IC markers as described herein (i.e., stage 7 cells).

[0080] Alternatively, methods for deriving SC-IC from partially differentiated cells may include differentiating stage 5 cells into cells expressing immature SC-IC markers as described herein (i.e., stage 6 cells), and differentiating stage 6 cells into cells expressing mature SC-IC markers as described herein (i.e., stage 7 cells).

[0081] Alternatively, methods for deriving SC-IC from partially differentiated cells may include differentiating stage 6 cells into cells expressing mature SC-IC markers as described herein (i.e., stage 7 cells).

[0082] In any of the above cases, the method may include the steps of dissociating and re-aggregating any cell population before initiating differentiation of the cell population, for example, dissociating and re-aggregating an FE cell population (e.g., stage 3 cells) before culturing in any PP differentiation medium, and / or dissociating and re-aggregating a precursor SC-IC population (e.g., stage 6 cells) before culturing in any SC-IC differentiation medium.

[0083] In either of the above cases, the method may 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 cases, the method may further include the step of isolating or purifying the target cell type to obtain a substantially pure population of the target cell type.

[0085] In any of the above cases, the method may further include the step of reassembling at least two isolated or purified cell populations into pseudoislets. In some cases, the isolated or purified cell populations are alpha (α)-like cells and β-like cells (e.g., PALCs and PBLCs) obtained from mature SC-ICs.

[0086] Third, this disclosure describes methods for differentiating certain less differentiated cells into more differentiated cells, and in particular methods for differentiating PP cells into PEP cells and PEP cells into SC-IC populations.

[0087] Fourth, this disclosure describes compositions comprising SC-IC populations obtained by performing the differentiation methods described herein, and compositions that can be used to perform certain differentiation steps in the methods: compositions comprising PP cells and G9a inhibitors and optionally at least one end-anchored polymerase 1 / 2 inhibitor, compositions comprising PEP cells and G9a inhibitors, and compositions comprising immature PBLCs and G9a inhibitors.

[0088] Fifth, this disclosure describes compositions comprising human SC-IC populations, and pharmaceutical compositions comprising them, wherein the human SC-IC populations possess desirable properties for use as implantable cell therapies for the treatment of diabetes. In some cases, the SC-IC populations comprise greater than 60% PBLCs, about 25% PALCs, and about 15% EC-like cells.

[0089] Sixth, this disclosure describes compositions and implantable devices encapsulating the SC-IC group described herein. In some cases, the implantable device may be a hydrogel capsule comprising an antifibrotic compound (e.g., on the outer layer of the hydrogel capsule) and the SC-IC group described herein.

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

[0091] Eighth, this disclosure describes the use of the SC-IC groups, compositions, and implantable devices described herein in the treatment of metabolic disorders such as diabetes (e.g., T1D). Similarly, this disclosure describes the use of the SC-IC groups and compositions described herein in the manufacture of pharmaceuticals or implantable devices for the treatment of metabolic disorders such as diabetes (e.g., T1D).

[0092] Ninth, this disclosure describes in vitro methods suitable and scalable for bioreactors (e.g., large-scale bioreactors) adapted to produce large quantities of differentiated stem cells. In one non-limiting embodiment, the bioreactor is a stirred tank bioreactor.

[0093] One advantage of the SC-IC derivatization method described herein is that, by following the steps and differentiation media described herein, certain precursor cell populations (i.e., PP, PEP, and / or precursor SC-IC populations) can be differentiated to obtain mature SC-IC populations with desired characteristics compared to SC-IC populations derived using standard methods known in the art. These desired characteristics include: a lower percentage of off-target cells, such as EC-like cells (i.e., SLC18A1). + (cells) and proliferating Ki67 + Cells, a higher percentage of PEC (i.e., CPEP) + Cellular cells) and mature PBLCs (i.e., CPEP) + / GCG - (cells), as well as increased INS content and GSIS.

[0094] One advantage of the method described in this paper is that the resulting SC-IC population has appropriate INS content, INS secretion, and GSIS response (i.e., is functional) for islet cell therapy in individuals with or suspected of having diabetes (e.g., T1D).

[0095] Another advantage of the method described in this paper is that the functionality of the resulting SC-IC population is durable in vivo when encapsulated in a device that protects SC-ICs from an individual's immune system.

[0096] In one aspect, this disclosure provides a method for deriving a cell population comprising mature stem cell-derived islet-like cells (SC-IC), the method comprising the following steps: (a) A first precursor cell population is cultured in one or more differentiation media to obtain a mature SC-IC population, wherein at least one of the differentiation media is a defined component medium containing glucose at less than about 2.5 mM or less than about 2 mM, and wherein the first precursor cell population is selected from foregut endoderm (FE) populations, pancreatic progenitor (PP) cell populations, pancreatic endocrine precursor (PEP) cell populations, and precursor SC-IC cell populations.

[0097] In some implementations, the first precursor PP cell population comprises PDX1+ cells, and the method includes: (a) Cultivating PDX1 + Cells, optionally PDX1 + / NKX6.1 + Cells and CHGA - Cells (optionally PDX1) + / CHGA - The first precursor PP cell population of PDX1 cells, wherein at least one of the differentiation media is a defined component medium containing glucose and a G9a inhibitor at a concentration of about 0 mM to less than about 2.5 mM, thereby obtaining PDX1 cells. + / CHGA + Cells (PDX1) + / NKX6.1 + / CHGA + The second cell population (cells).

[0098] In some implementations, the first precursor PP cell population contains PDX1 + / NKX6.1 + Cells and CHGA - Cells, optionally PDX1 + / CHGA - cell.

[0099] In some embodiments, at least one of the cell differentiation culture media is a defined component culture medium containing glucose at concentrations 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. 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 2.6 mM, about 0 to less than about 2.7 mM, or about 0 to less than about 2.8 mM.

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

[0101] In some embodiments, at least one of the cell differentiation culture media is a defined component culture medium containing glucose at concentrations of: 0 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 1.0 mM, 2.1 mM, 2.2 mM, 2.3 mM, 2.4 mM, 2.5 mM, 2.6 mM, 2.7 mM, or less than 2.8 mM.

[0102] In some embodiments, the culture in step (a) further includes pH monitoring. In some embodiments, the culture in step (a) is conducted at pH values ​​within the following ranges: pH 7.2 to pH 7.8, pH 7.4 to pH 7.8, pH 7.2 + / - 0.2 (pH 7.0 to pH 7.4), pH 7.4 + / - 0.2 (pH 7.2 to pH 7.6), pH 7.6 + / - 0.2 (pH 7.4 to pH 7.8), or pH 7.8 + / - 0.2 (pH 7.6 to pH 8.0). In some embodiments, the cultivation in step (a) takes place in a bioreactor and includes monitoring and maintaining a pH not higher than pH 7.8, within the following pH ranges: pH 7.2 to pH 7.8, pH 7.4 to pH 7.8, pH 7.2 + / - 0.2 (pH 7.0 to pH 7.4), pH 7.4 + / - 0.2 (pH 7.2 to pH 7.6), pH 7.6 + / - 0.2 (pH 7.4 to pH 7.8), or pH 7.8 + / - 0.2 (pH 7.6 to pH 8.0).

[0103] In some embodiments, at least one of the one or more differentiation media in step (a) contains an end-anchor polymerase 1 / 2 inhibitor, wherein the end-anchor polymerase 1 / 2 inhibitor is Wiki4.

[0104] In some implementations, the component culture medium is determined to be glucose-free.

[0105] In some embodiments, the defined component culture medium contains galactose at concentrations of about 4 mM to about 7 mM, about 5 mM to about 6.0 mM, or about 5.5 mM, optionally and wherein the defined component culture medium is free of pyruvate.

[0106] The method according to any one of claims 1-11, wherein at least one of the differentiation culture media in step (a) comprises: 5.5 mM galactose, glutamine, and at least two differentiation factors selected from the following: Small molecule BMP inhibitors, Zinc compounds, Activators of thyroid hormone signaling pathways heparin, TGF-β RI kinase competitive inhibitor, Inhibitors of cell-permeable sound hedgehog (SHH) signaling, Non-essential amino acid (NEAA) supplements Rho kinase (ROCK) inhibitors, Vitamin C compounds, and Gamma-secretase inhibitors (GSIs) It may also optionally further include one or more of albumin, buffers, and serum replacement supplements.

[0107] In some implementations, the method further includes: (b) In the presence of an enzyme-catalyzed aggregate dissociation solution, CHGA-containing proteins were cultured in differentiation medium. + / PDX1 + A second cell population of cells was obtained to contain CHGA. + / PDX1 + The dissociation of single cells into a cell population, optionally wherein the enzymatic aggregate dissociation solution contains trypsin activity, chymotrypsin / elastase activity, and type I collagenase activity, optionally including a chelating agent such as EDTA; and (c) Culturing CHGA in differentiation medium containing DNase I and G9a inhibitor + / PDX1 + The dissociation of individual cells into a cell population is sustained for an additional period of time sufficient to obtain a re-aggregated population, wherein the re-aggregated population comprises NKX6.1+ / CPEP+ cells, CPEP+ / GCG- cells, INS+ / SLC18A1- cells, CHGA+ / Ki67- cells, or combinations thereof, the cells being contained in cell aggregates, optionally wherein the cell aggregates have an average size of about 40 µm to about 100 µm, or about 70 micrometers.

[0108] In some embodiments, the differentiation medium in steps (b) and (c) each contains glucose, glutamine, and at least two differentiation factors selected from the following at a concentration of about 1 mM to ≤ about 25 mM: Thiol-based antioxidants, Small molecule BMP inhibitors, Zinc compounds, Activators of thyroid hormone signaling pathways TGF-β RI kinase competitive inhibitor, Cellular permeability SHH signaling inhibitors Vitamin C compounds, Heparin, and Optionally, each differentiation medium may further contain one or more of albumin, buffers, and serum replacement supplements.

[0109] In some embodiments, the enzymatic aggregate dissociation solution contains chymotrypsin / elastase activity and 0.5 mM EDTA.

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

[0111] In some implementations, the method further includes: (d) Culture a reaggregated population in a differentiation medium, wherein the population comprises NKX6.1+ / CPEP+ cells, CPEP+ / GCG- cells, INS+ / SLC18A1- cells, CHGA+ / Ki67- cells, or combinations thereof, for a period sufficient to obtain a mature (SC-IC) cell population comprising NKX6.1+ / CPEP+ cells and CPEP+ / GCG- cells, wherein the differentiation medium comprises glucose, glutamine, and at least two differentiation factors selected from the group consisting of, at a concentration of about 1 mM to ≤ about 25 mM: Cell-permeable vitamin E analogs / antioxidants Carnitine compounds, Chemically defined lipid mixtures (CDLM) Thiol-based antioxidants, Small molecule bone morphogenetic protein (BMP) inhibitors; Zinc compounds, Activators of thyroid hormone signaling pathways Vitamin C compounds, heparin, G9A inhibitors, and Optionally, the differentiation medium further comprises one or more of albumin, buffer, and serum replacement supplement.

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

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

[0114] In some embodiments, the method further includes, prior to step (a), culturing cells containing PDX1 in one or more cell differentiation media containing two end-anchor polymerase inhibitors. + FOXA2 + NKX6.1 - and CHGA - The step of assembling a population of foregut endoderm (FE) cells, wherein one of the end-anchor polymerase inhibitors is Wiki4, thereby obtaining a population containing PDX1. + Optionally includes PDX1 + / NKX6.1+ Cells and CHGA - (PP) precursor cell population of cells.

[0115] In some embodiments, the method does not include the step of sorting or separating individual cells or cell populations containing cell markers or combinations of cell markers (optionally cell surface markers or combinations of cell surface markers). In some embodiments, the method does not include sorting or separating individual cells or cell populations by means of fluorescence-activated cell sorting or magnetic bead sorting. In some embodiments, sorting or separation uses selective markers, such as positive selective markers, to enrich CPEP+ / GCG- cells, CPEP+ / NKX6.1+ cells, or CPEP+ cells, wherein said selective markers are cell markers, such as, but not limited to, cell markers such as TSQ, CD49A, ST8SIA1, GLUT2, ZNT8, CD9. In some embodiments, sorting or separation uses selective markers, such as negative selective markers, to deplete cell populations other than CPEP+ / GCG-, or CPEP+ / NKX6.1+, or CPEP+, wherein said selective markers are cell markers, such as, but not limited to, CD26, SLC18A, or combinations thereof.

[0116] In some implementations, the mature SC-IC cell population contains at least about 54% to about 60% NKX6.1 + / CPEP + Cells and at least about 60% to about 80% CPEP + / GCG - cell.

[0117] In some implementations, the mature SC-IC cell population further comprises approximately 99.8% CHGA. + cell.

[0118] In some embodiments, the cultures in (a), (b), (c), and (d) are conducted in a bioreactor and produce at least 1.5E5 cells / ml, optionally from 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-scale bioreactor.

[0119] This disclosure also provides information from sources including PDX1. + A method for deriving a cell population comprising mature stem cell-derived islet-like cells (SC-IC) from a population of pancreatic progenitor (PP) cells, wherein the method includes: (i) A PP cell population is cultured in a first PP differentiation medium for a period sufficient to obtain an intermediate PP / PEP cell population, wherein the PP cell population contains PDX1+, optionally PDX1 + / NKX6.1 + Cells and CHGA - Cells (PDX1) + / CHGA - The PP / PEP cell population comprises PDX1+ / CHGA+ cells, wherein the first PP differentiation medium is a defined component medium containing approximately 5 mM to approximately 50 mM glucose and a set of differentiation factors sufficient to promote differentiation of at least a portion of the PP cell population into PEP cells, wherein the set of differentiation factors comprises a G9a inhibitor and at least one factor selected from the following: Small molecule bone morphogenetic protein (BMP) inhibitors, Zinc compounds, Activators of thyroid hormone signaling pathways TGF-β RI kinase competitive inhibitor, Inhibitors of cell-permeable sound hedgehog (SHH) signaling, Rho kinase (ROCK) inhibitors, Vitamin C compounds, Gamma-secretase inhibitors (GSIs) Heparin, and Optionally at least one end-anchored polymerase 1 / 2 inhibitor; or optionally two end-anchored polymerase 1 / 2 inhibitors; (ii) Wash the intermediate PP / PEP cell population in a wash medium containing a defined component of <1 mM glucose; (iii) The washed intermediate PP / PEP cell population is cultured in a second PP differentiation medium for a period sufficient to obtain a PEP cell population containing cell aggregates, wherein the second PP differentiation medium is a defined component medium containing <1 mM glucose and a set of differentiation factors, the amount of which is sufficient to promote the differentiation of at least a portion of the intermediate PP / PEP cell population into cells containing PDX1. + / CHGA + (PDX1) + / NKX6.1 + / CHGA + A population of PEP cells, wherein the set of differentiation factors includes a G9a inhibitor and at least one factor selected from the following: Small molecule BMP inhibitors, Zinc compounds, Activators of thyroid hormone signaling pathways TGF-β RI kinase competitive inhibitor, Cellular permeability SHH signaling inhibitors ROCK inhibitors Vitamin C compounds, GSI, Heparin, and At least one end-anchored polymerase 1 / 2 inhibitor; optionally two end-anchored polymerase 1 / 2 inhibitors; (iv) Dissociate 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) A dissociated population of PEP cells is cultured in a first PEP differentiation medium containing deoxyribonuclease (DNase) for a duration sufficient to yield a reaggregated intermediate PEP / SC-IC population, wherein the first PEP differentiation medium is a defined component medium containing DNase, ≤ about 0.5 mM pyruvate, about 1 mM to ≤ about 25 mM glucose, and a set of differentiation factors in an amount sufficient to promote differentiation of at least a portion of the PEP cell population into immature pancreatic endocrine cells (PECs), wherein the set of differentiation factors includes a G9a inhibitor and at least one factor selected from the following: Thiol-based antioxidants, Small molecule BMP inhibitors; Zinc compounds, Activators of thyroid hormone signaling pathways TGF-β RI kinase competitive inhibitor, Cellular permeability SHH signaling inhibitors Vitamin C compounds, and heparin; (vi) Wash the reaggregated intermediate PEP / SC-IC population in a wash medium containing ≤ about 0.5 mM pyruvate and about 1 mM to ≤ about 25 mM glucose. (vii) The washed intermediate PEP / SC-IC population is cultured in a second PEP differentiation medium for a period sufficient to obtain a precursor SC-IC population, wherein the second PEP differentiation medium is a defined component medium containing ≤ about 0.5 mM pyruvate, about 1 mM to ≤ about 25 mM glucose, and a set of differentiation factors sufficient to promote the differentiation of at least a portion of the intermediate PEP / SC-IC population into CHGA-containing cells. + / PDX1 +A precursor SC-IC population of cells, wherein the set of differentiation factors includes a G9a inhibitor and at least one factor selected from the following: Thiol-based antioxidants, Small molecule BMP inhibitors, Zinc compounds, Activators of thyroid hormone signaling pathways TGF-β RI kinase competitive inhibitor, Cellular permeability SHH signaling inhibitors Vitamin C compounds, and Heparin; and (viii) Culture the precursor SC-IC population in SC-IC differentiation medium for a period sufficient to obtain a mature SC-IC population containing NKX6.1. + / CPEP + Cells, CPEP + / GCG - Cells, INS + / SLC18A1 - Cells, or CHGA + / Ki67 - The cell combination, wherein the SC-IC differentiation medium comprises ≤0.5 mM pyruvate, about 1 mM to ≤25 mM glucose, and a set of differentiation factors, the amount of which is sufficient to promote at least a portion of the precursor SC-IC population to differentiate into mature PBLCs, wherein the set of differentiation factors comprises a G9a inhibitor and at least one factor selected from the following: Cell-permeable vitamin E analogs / antioxidants Carnitine compounds, Chemically defined lipid mixtures (CDLM) Thiol-based antioxidants, Small molecule BMP inhibitors, Zinc compounds, Activators of thyroid hormone signaling pathways Vitamin C compounds, and Heparin; and wherein in all steps (i)-(viii), at least one end-anchored polymerase 1 / 2 inhibitor is Wiki4 and the G9a inhibitor is UNC0321.

[0120] In some embodiments, the method further includes obtaining the PP cell population used in step (i) by culturing cells containing PDX1 in FE differentiation medium. +A foregut endoderm (FE) cell population sustained for a period sufficient to yield a PP cell population, wherein the FE differentiation medium is a defined component medium containing approximately 5 mM to approximately 50 mM glucose and a set of differentiation factors sufficient to promote differentiation of at least a portion of the FE cell population into PP cells, wherein the set of differentiation factors comprises at least one factor selected from the following: EGF family growth factors, Vitamin B3 compounds, Vitamin C compounds, FGF family growth factors, PKC activator, retinoids ROCK inhibitors Cellular permeability SHH signaling inhibitors At least one end-anchored polymerase 1 / 2 inhibitor, optionally two end-anchored polymerase 1 / 2 inhibitors, wherein at least one of the end-anchored polymerase 1 / 2 inhibitors is Wiki4.

[0121] In some implementations, the method further includes the following steps: The FE cell population was obtained through the following methods: (i) Cultured in first PGT differentiation medium containing FOXA2 + The primitive gut tract (PGT) cell population, or PGT cell population, was sustained for approximately 12 to 48 hours in a first time period to obtain an intermediate PGT / FE cell population; and (ii) An intermediate PGT / FE cell population was cultured in a second PGT medium for a second time period of approximately 12 to approximately 48 hours to obtain an FE cell population. The first PGT differentiation medium is a defined culture medium containing approximately 5 mM to approximately 50 mM glucose and a set of differentiation factors, wherein the amount of differentiation factors is sufficient to promote the differentiation of at least a portion of the PGT cell population into FE cells, wherein the factors are selected from at least one of small molecule BMP inhibitors and FGF family growth factors, PKC activators, retinoids, ROCK inhibitors, cell permeability SHH signaling inhibitors, and at least one end-anchored polymerase 1 / 2 inhibitor; and The second PGT differentiation medium is a defined-component medium that is deficient in BMP inhibitors and contains approximately 5 mM to approximately 50 mM glucose, approximately 0.5 mM to approximately 1.5 mM pyruvate, and a set of differentiation factors sufficient to promote the differentiation of at least a portion of the intermediate PGT / FE cell population into FE cells, wherein the factors comprise at least one selected from the following: Vitamin C compounds, FGF family growth factors, PKC activator retinoids, ROCK inhibitors Inhibitors of cell-permeable SHH signaling, and At least one end-anchored polymerase 1 / 2 inhibitor, optionally two end-anchored polymerase 1 / 2 inhibitors, wherein at least one of the end-anchored polymerase 1 / 2 inhibitors is Wiki4.

[0122] In some embodiments, the method further includes obtaining a PGT cell population by culturing a defined endoderm (DE) cell population containing PDX1- cells and FOXA2+ / SOX17+ cells or GATA6+ / SOX17+ cells in a DE differentiation medium for a period of time sufficient to obtain a PGT cell population, wherein the DE differentiation medium is a defined component medium containing about 5 mM to about 20 mM of glucose and a set of differentiation factors, the amount of which is sufficient to promote at least a portion of the DE cell population to differentiate into PGT cells, wherein the factors include at least one factor selected from vitamin C compounds and FGF family growth factors.

[0123] In some aspects, this disclosure provides compositions comprising cell populations, such as in vitro cell populations, and optionally a carrier, wherein: (i) Cell population or <2% of cells in the population are non-endocrine cells (CHGA) - ), or at least about 98% of the population cells express chromogranin A (CHGA). + ); (ii) At least approximately 50% of the cell population are CPEP+ / GCG- cells, or at least approximately 50% of the cell population produce C-peptide (CPEP). + It does not express glucagon (GCG) - ) (CPEP + / GCG - ); (iii) Approximately 40% of the cell population express glucagon (GCG). + ); (iv) At least approximately 45% of the cell population are pancreatic endocrine cells (PDX+ / CHGA+), or at least approximately 45% of the cell population produce CPEP (CPEP...). + And it expresses the NK6 homologous frame 1 (NKX6.l) + ) (CPEP + / NKX6.1 + ); (v) At least approximately 60% of the cell population produces insulin (INS). +It does not express solute carrier family 18 member 1 (SLC18A1) - ) (INS + / SLC18A1 - ); (vi) Insulin content of at least approximately 150 nU / cell; (vii) < Approximately 16% of the cell population consists of insulin-free INS- / SLC+ cells (CPEP- / SLC18A1+), or < Approximately 16% of the cell population consists of insulin-free cells that express solute carrier family 18 member 1 (SLC18A1). + ) (INS - / SLC18A1 + ); (viii) < Approximately 5% of the cell population are proliferating cells (Ki67+), or less than approximately 5% of the cell population express (Ki67+). + ); (ix) At least approximately 99.5% of the cell population is CHGA + At least 60% of the cell population is CPEP. + / GCG - At least 50% of the cell population is CPEP. + / NKX6.1 + And at least about 70% of the population cells are INS. + / SLC18A1 - ; (x) < Approximately 12% of the cell population is INS - / SLC18A1 + And approximately 4% of the cell population is Ki67. - ; (xi) The cell population does not produce lactic acid; (xii) < Approximately 0.5% or 0.2% of the cell population are non-endocrine cells, at least approximately 60% of the cell population are CPEP+ / GCG- cells, and at least approximately 50% or 60% of the cell population are PBLCs expressing NKX6.1 (NKX6.1). + At least 70% or about 75% of the cell population are insulin-producing cells (INS) that are not ECLC. - / SLC + The cell population comprises approximately 11% or 7% non-insulin-producing INS- / SLC+ cells, and approximately 4% or 2% of the cells are proliferating cells; the cell population does not produce lactate, and optionally the cell population has an insulin content of at least approximately 325 nU / cell, or from 150 nU / cell to at least approximately 200 nU / cell; and (xiii) At least approximately 98% or at least approximately 99.5% of the cell population is CHGA + At least 60% or about 65% of the cell population is CPEP. + / GCG - At least 50% or 60% of the cell population is CPEP. + / NKX6.1 + At least 70% or about 75% of the cell population is INS. + / SLC18A1 - Approximately 11% or 7% of the cells are INS. - / SLC18A1 + And <4% or about 2% of the population cells are Ki67 - The cell population does not produce lactic acid; and optionally the cell population has an insulin content of at least about 325 nU / cell, or at least 150 nU / cell to about 200 nU / cell.

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

[0125] In some respects, this disclosure provides in vitro cell populations comprising cells, such as in vitro differentiated cell populations, wherein: (i) At least about 60% of the cells in the population are CPEP + / GCG - Cells; optionally, approximately 60% to approximately 68% of the cells in the population are CPEP.+ / GCG - cell; (ii) No more than about 23% of the cells in the population are GCG. + Cells; optionally, approximately 10% to 20% and approximately 10% to 23% of the cells in the population are GCG. + cell; (iii) At least approximately 54% of the cells in the population are NKX6.1. + / CPEP + Cells; optionally, approximately 54% to 65% of the cells in the population were NKX6.1. + / CPEP + cell; (iv) At least approximately 68% of the cells in the population are INS. + / SLC - Cells; optionally, approximately 68% to approximately 77% of the cells in the population are INS. + / SLC - cell; (v) Less than 11% of the cells in the population are INS + / SLC - Cells; optionally, approximately 11% to 11% of the cells in the population are INS. + / SLC - cell; (vi) At least approximately 99.5% of the cells in the cell population are CHGA + Cells; approximately 99.5% of the cells in the cell population are CHGA. + cell; (vii) Less than 4% of the cells in the cell population are Ki67. + Cells; approximately 3.5% to 1% to 4% of the cells in the optional population are Ki67. + cell; (viii) Insulin levels of at least about 150 nU / cell to about 200 nU / cell, or (ix) Lactic acid is produced within 48 hours by cell populations of less than 0.5 mM, 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, or about 0 to about 0.5 mM, wherein said populations contain at least two of the above characteristics.

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

[0127] In some respects, this disclosure provides in vitro cell populations comprising cells, such as in vitro differentiated cell populations, wherein: (i) At least approximately 67% of the cells in the population are CPEP + / GCG - In the selected population, approximately 67% to 80%, 67% to 70%, 70% to 80%, and 70% to 85% of the cells were CPEP. + / GCG - cell; (ii) No more than about 22% of the cells in the cell population are GCG. + Cells; optionally, approximately 10% to 20% and approximately 10% to 22% of the cells in the population are GCG. + cell; (iii) At least approximately 60% of the cells in the cell population are NKX6.1. + / CPEP + Cells; optionally, approximately 60% to 70%, approximately 65% ​​to 75%, and approximately 60% of the cells in the population are NKX6.1 + / CPEP + cell; (iv) Approximately 68% of the cells in the cell population are INS.+ / SLC - Cells; optionally, approximately 77%, approximately 70% to approximately 80%, and approximately 70% to approximately 85% of the cells in the population are INS. + / SLC - cell; (v) Less than 7% of the cells in the cell population are INS + / SLC - Cells; approximately 7% to 3-7% of the cells in the population are INS. + / SLC - cell; (vi) At least approximately 99.8% of the cells in the cell population are CHGA + Cells; approximately 99.8% of the cells in the cell population are CHGA. + cell; (vii) Less than 2% of the cells in the cell population are Ki67. + Cells; optionally, 0.3-2%, approximately 2%, of the cells in the population are Ki67. + cell; (viii) Insulin levels of at least 150 nU / cell to approximately 200 nU / cell, or (ix) Lactic acid is produced within 48 hours by cell populations of less than 0.5 mM, 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, or about 0 to about 0.5 mM, wherein said populations contain at least two of the above characteristics.

[0128] In some implementations of in vitro cell populations, approximately 67% of the cells in the population are CPEP. + / GCG - Furthermore, approximately 99.8% of the cells in the population are CHGA. + Cells. In some implementations of in vitro cell populations, at least approximately 54% of the cells in the population are NKX6.1. + / CPEP + Furthermore, less than 11% of the cells in the population are INS. - / SLC + In some implementations of in vitro cell populations, approximately 67% of the cells in the population are CPEP. + / GCG - Furthermore, approximately 68% of the cells in the population are INS. + / SLC - In some implementations of in vitro cell populations, approximately 60% of the cells in the population are NKX6.1.+ / CPEP + Furthermore, approximately 68% of the cells in the population are INS. + / SLC - In some implementations of in vitro cell populations, no more than about 22% of the cells in the population are GCG. + In some implementations of in vitro cell populations, at least approximately 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 150 nU / cell to about 200 nU / cell. In some embodiments of the in vitro cell population, lactate is produced over 48 hours via a concentration of less than 0.5 mM in the cell population.

[0130] In one aspect, a liquid cell differentiation composition is provided, comprising: (a) Serum-free basal medium; and (b) A set of differentiation factors, wherein the set of differentiation factors is: (i) A set of factors that promote the differentiation of foregut endoderm (FE) cells into pancreatic progenitor (PP) cells (FE factor set); (ii) Promote the differentiation of pancreatic progenitor cells (PP) containing PDX1+ cells into cells containing PDX1+ cells. + / CHGA + The set of factors (PP factor set) of the pancreatic endocrine precursor (PEP) population of cells. (iii) A set of factors that promote the differentiation of PEP cells into immature SC-IC cells (PEP factor set); or (iv) The set of factors that can promote the differentiation of immature SC-IC into mature SC-IC (SC-IC factor set).

[0131] In some embodiments, the serum-free basal medium contains 0 mM to less than 2.5 mM glucose, and the PP factor set contains a G9a inhibitor, optionally UNC0321. In some embodiments, the liquid cell differentiation composition further contains at least one end-anchored polymerase 1 / 2 inhibitor, optionally said end-anchored polymerase 1 / 2 inhibitor being Wiki4. ​​In some embodiments, the liquid cell differentiation composition is used to differentiate a progenitor cell population containing PDX1+ cells, optionally a PP population containing PDX1+ cells, into a cell line containing PDX1+ cells. + / CHGA + PEP population of cells.

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

[0133] In some aspects, this disclosure provides methods for treating individuals with diabetes, one or more diabetes-related complications, or prediabetes, said methods comprising: (a) Administering an effective amount of the composition to an individual, said composition comprising any of the differentiated SC-IC cells, in vitro cell populations, or pharmaceutical compositions comprising them as described herein; (b) Administering to an individual an effective amount of either the pharmaceutical composition described herein or an in vitro cell population, each encapsulated in a device providing immunoprotection for the encapsulated composition and / or cell population; or (c) Administering to an individual a device comprising a composition containing differentiated SC-IC cells as described, any of an in vitro cell population, or a pharmaceutical composition comprising such compositions. In some embodiments, the method further comprises administering an immunosuppressant to the individual before and / or after the administration in steps (a), (b), or (c).

[0134] In some embodiments of the method, the apparatus comprises alginate chemically modified with an effective amount of a compound of formula I for anti-fibrosis.

[0135] In some implementations, diabetes is T1D.

[0136] In some aspects, this disclosure provides compositions comprising differentiated SC-IC cells as described, any of in vitro cell populations, pharmaceutical compositions comprising these, or devices comprising these for the treatment of diabetes, one or more diabetes-related complications, or prediabetes. In some embodiments, the use is for the treatment of type 1 diabetes.

[0137] In some aspects, this disclosure provides the use of compositions comprising differentiated SC-IC cells as described, any of in vitro cell populations, pharmaceutical compositions comprising these, or devices comprising these in the manufacture of an agent for treating diabetes, one or more diabetes-related complications, or prediabetes. In some embodiments, the agent is used to treat type 1 diabetes.

[0138] In some aspects, this disclosure provides a method for culturing stem cells to obtain a population comprising differentiated cells, the method comprising at least one step of culturing the population of cells at a pH not higher than 7.8, within the following pH ranges: pH 7.2 to pH 7.8, pH 7.3 to pH 7.8, pH 7.4 to pH 7.8, pH 7.2 + / - 0.2 (pH 7.0 to pH 7.4), pH 7.4 + / - 0.2 (pH 7.2 to pH 7.6), pH 7.6 + / - 0.2 (pH 7.4 to pH 7.8), or pH 7.8 + / - 0.2 (pH 7.6 to pH 8.0), wherein the at least one step is carried out in a bioreactor. In some embodiments, at least one step of culturing the population of cells at a pH not higher than 7.8, within the following pH ranges: pH 7.2 to pH 7.8, pH 7.3 to pH 7.8, pH 7.4 to pH 7.8, pH 7.2 + / - 0.2 (pH 7.0 to pH 7.4), pH 7.4 + / - 0.2 (pH 7.2 to pH 7.6), pH 7.6 + / - 0.2 (pH 7.4 to pH 7.8), or pH 7.8 + / - 0.2 (pH 7.6 to pH 8.0), is carried out in at least one differentiation medium comprising a defined component medium containing 0 mM to less than 2.5 mM glucose, 0 mM to less than 2 mM glucose, 0 mM to 25 mM glucose, 0 mM to 50 mM glucose, or any glucose concentration within these ranges.

[0139] In some aspects, methods are provided for deriving a population of differentiated cells derived from stem cells, the methods comprising at least one step of culturing the population of cells in at least one differentiation medium at a pH not higher than pH 7.8, within the following pH ranges: pH 7.2 to pH 7.8, pH 7.3 to pH 7.8, pH 7.4 to pH 7.8, pH 7.2 + / - 0.2 (pH 7.0 to pH 7.4), pH 7.4 + / - 0.2 (pH 7.2 to pH 7.6), pH 7.6 + / - 0.2 (pH 7.4 to pH 7.8), or pH 7.8 + / - 0.2 (pH 7.6 to pH 8.0). In some embodiments, the differentiation medium comprises a defined component medium containing 0 mM to less than 2.5 mM glucose, 0 mM to less than 2 mM glucose, 0 mM to 25 mM glucose, 0 mM to 50 mM glucose, or any glucose concentration within these ranges, and said method is carried out in a bioreactor.

[0140] In some embodiments, the culture step is within a differentiation protocol that differentiates stem cells, such as but not limited to iPSCs, into mature SC-ICs. In some embodiments, a cell population is cultured, such as but not limited to PDX1+ cells. In some embodiments, the population of differentiated cells contains CPEP. + / GCG - Cells and GCG + Cell maturation SC-IC.

[0141] In one aspect, this disclosure provides a method for deriving a cell population comprising mature SC-IC, the method comprising the following steps: (a) A first precursor cell population containing PDX1+ cells is cultured in one or more differentiation media to obtain a mature SC-IC population, wherein the first precursor cell population is selected from FE population, PP cell population, PEP cell population and precursor SC-IC cell population, and wherein at least one of the differentiation media is a defined component medium containing a G9a inhibitor.

[0142] In some embodiments, the G9a inhibitor is UNC0321, and optionally, at least one of the differentiation media is a defined component medium containing glucose at less than about 2.5 mM or less than about 2 mM.

[0143] In some implementations, the first precursor cell population containing PDX1+ cells is cultured at pH ranges of: pH 7.2 to pH 7.8, pH 7.3 to pH 7.8, pH 7.4 to pH 7.6, pH 7.4 to pH 7.8, pH 7.2 + / - 0.2 (pH 7.0 to pH 7.4), pH 7.4 + / - 0.2 (pH 7.2 to pH 7.6), pH 7.6 + / - 0.2 (pH 7.4 to pH 7.8), or pH 7.8 + / - 0.2 (pH 7.6 to pH 8.0).

[0144] In some embodiments, the method includes an additional culture step in one or more differentiation media as described herein, in order to derive it as containing CPEP. + / GCG - Cells and GCG +The mature SC-IC differentiated cell population. In some embodiments, the culture steps of the methods described herein are performed in a bioreactor. In some embodiments, the bioreactor is a large-scale bioreactor, such as, but not limited to, a bioreactor with a volume exceeding 500 mL. Alternatively, the bioreactor is a large-scale bioreactor, such as, but not limited to, a bioreactor with a volume exceeding 1 L. Alternatively, the bioreactor is a large-scale bioreactor, such as, but not limited to, a bioreactor with a volume exceeding 3 L. Alternatively, the bioreactor is a large-scale bioreactor, such as, but not limited to, a bioreactor with a volume exceeding 5 L. Alternatively, the bioreactor is a large-scale bioreactor, such as, but not limited to, a bioreactor with a volume exceeding 10 L. Alternatively, the bioreactor is a large-scale bioreactor, such as, but not limited to, a bioreactor with a volume exceeding 25 L. Alternatively, the bioreactor is a large-scale bioreactor, such as, but not limited to, a bioreactor with a volume exceeding 50 L.

[0145] In some aspects, this disclosure provides, for example, but not limited to, in vitro cell populations comprising mature SC-ICs, said populations being generated by any of the methods described.

[0146] In some respects, in vitro cell populations comprising cells are provided, wherein: (i) At least approximately 67% of the cells in the population are CPEP + / GCG - ; (ii) No more than about 22% of the cells in the cell population are GCG. + cell; (iii) At least approximately 60% of the cells in the cell population are NKX6.1. + / CPEP + cell; (iv) Approximately 68% of the cells in the cell population are INS. + / SLC - cell; (v) Less than 7% of the cells in the cell population are INS - / SLC + cell; (vi) At least approximately 99.8% of the cells in the cell population are CHGA + cell; (vii) Less than 2% of the cells in the cell population are Ki67. + cell; (viii) Insulin levels ranging from 150 nU / cell to at least approximately 200 nU / cell; or (ix) is produced within 48 hours by a cell population of less than 0.5 mM lactate, wherein the cell population contains at least two of the above-described properties, and wherein the cell population is produced by any of the methods described above. Brief description of the attached diagram The advantages, effects, features, and purposes beyond those described above will become more apparent when considered in the detailed description below. Such detailed description is illustrated in the accompanying drawings, in which: Figure 1A An overview of an exemplary iPSC to SC-IC differentiation scheme is shown, and Figure 1B Showing Figure 1A A more detailed schematic diagram of the exemplary scheme shown is provided.

[0148] Figure 2A-2D The effects of reaggregation and glucose deprivation on the SC-IC composition and potency relative to other concurrent treatments were demonstrated.

[0149] Figures 3A-3C The graph shows the correlation (or lack thereof) between INS secretion and SC-IC composition or activity, and the effect of reaggregation or glucose deprivation on these parameters relative to other concurrent treatments.

[0150] Figure 4 shows the effect of glucose deprivation and / or reaggregation on INS levels at different times in stages 5 and 6. Figure 4A ) and INS secretion ( Figure 4B The effect of glucose deprivation and reaggregation was observed when they were sequentially performed at the end of stage 5 and the beginning of stage 6 (D14-D15, zero glucose, D16 reaggregation), with synergistic benefits observed.

[0151] Figure 5A The effects of D14-D15, zero glucose, and D16 reaggregation treatments on LDHA expression in mature SC-IC compositions were demonstrated, and Figure 5B It showed its role in lactate production through cell differentiation during stages 6 and 7.

[0152] Figure 6 The effects of UNC0321 on INS levels were shown relative to the matched control throughout Phase 5 (D10-16), and from the near end of Phase 5 until Phase 6 (D14-20), including MDL-800 or butyrate.

[0153] Figure 7 The effects of various pyruvate concentrations on INS secretion via the obtained SC-IC composition were shown during stages 6 and 7.

[0154] Figures 8A-8DThis study demonstrates the effect of HPLM relative to MCDB basal medium on the SC-IC composition and efficacy during Phase 7.

[0155] Figure 9 The in vivo hypoglycemic effect of the encapsulated SC-IC derived from the methods described herein is demonstrated.

[0156] Detailed description Overview Diabetes is a group of diseases characterized by chronic hyperglycemia and the development of long-term complications. This group includes type 1 diabetes (T1D), type 2 diabetes (T2D), gestational diabetes, and other types of diabetes. Individuals with diabetes, especially those diagnosed with T1D, can potentially be cured through transplantation of exogenous beta-cell donors. However, this approach is limited by the scarcity and quality of donor islets. Therefore, generating an unlimited supply of functional islet-like cells from stem cells that can produce and secrete insulin (INS) could make this treatment available to a larger number of individuals.

[0157] It is estimated that 10 functional cells are needed to treat diabetes. 9 Approximately one per individual. Therefore, differentiation strategies are needed to generate sufficient β-like cells for the treatment of diabetes as an alternative to islet transplantation using donor islets.

[0158] The following describes an implementation scheme for a differentiation method and process to generate a population of insulin-producing cells (referred to as SC-IC), in which at least approximately 67% of the cells are CPEP. + / GCG - For example, 67% to 80% of the cells in the population are CPEP. + / GCG - Furthermore, at least approximately 99% of the cells in the population are CHGA. + For example, a population in which 99% to 99.99% of the cells are CHGA+ can be used for cell therapy to treat, for example, diabetes (e.g., T1D). In one embodiment, the method described herein is based on the finding that differentiation of PP is optimal in the presence of two end-anchor polymerase 1 / 2 inhibitors (at least one of which is Wiki4). In one embodiment, the method described herein is based on the finding that culturing cells in a differentiation medium with a glucose concentration of less than 2.5 mM (e.g., but not limited to 0 mM glucose), and optionally adding galactose (or another alternative nutrient) to reduce (i.e., decrease or eliminate) EC-like cells (i.e., SLC18A1) while differentiating PP cells into PEP or PEP into immature SC-IC, is also effective. +(Cells). In another embodiment, the method includes at least one step of culturing cells, such as, but not limited to, culturing PP cells as PEP, or culturing PEP as immature SC-IC in a bioreactor at pH ranges including: pH 7.2 to pH 7.8, pH 7.3 to pH 7.8, pH 7.4 to pH 7.6, pH 7.4 to pH 7.8, pH 7.2 + / - 0.2 (pH 7.0 to pH 7.4), pH 7.4 + / - 0.2 (pH 7.2 to pH 7.6), pH 7.6 + / - 0.2 (pH 7.4 to pH 7.8), or pH 7.8 + / - 0.2 (pH 7.6 to pH 8.0). Unbound by theory, pH and glucose concentrations are effectively targeted at EC-like cells (i.e., SLC18A1) in the differentiation population. + The method selects cells and thereby reduces (i.e., lowers or eliminates) such cells. In another embodiment, the method includes at least one step of culturing cells, such as, but not limited to, culturing PP cells as PEP, or culturing PEP as immature SC-IC in a bioreactor in a differentiation medium having a glucose concentration of less than 2.5 mM and at pH ranges including: pH 7.2 to pH 7.8, pH 7.3 to pH 7.8, pH 7.4 to pH 7.6, pH 7.4 to pH 7.8, pH 7.2 + / - 0.2 (pH 7.0 to pH 7.4), pH 7.4 + / - 0.2 (pH 7.2 to pH 7.6), pH 7.6 + / - 0.2 (pH 7.4 to pH 7.8), or pH 7.8 + / - 0.2 (pH 7.6 to pH 8.0), the combination of which effectively targets EC-like cells (i.e., SLC18A1) in the differentiation population. + The method involves selecting cells and thereby reducing (i.e., decreasing or eliminating) such cells. Additionally, the method described herein is based on the finding that dissociating a population containing immature SC-ICs into single cells, with the majority of the population dissociated into single cells, separating the dissociated immature SC-IC single cells from the undissociated cells, and then re-aggregating the dissociated immature SC-IC single cells before differentiating them into mature SC-ICs, yields rich clusters exhibiting dynamic INS secretion responses. Furthermore, the method described herein is based on the finding that adding epigenetic modifiers, such as G9a inhibitors (including but not limited to G9a inhibitors such as UNC0321), improves the cellular composition and potency of mature SC-ICs when obtaining PEPs and / or immature SC-ICs.

[0159] Abbreviations and Definitions Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may be used to practice or test SC-ICs, pharmaceutical compositions including those thereof, and methods of preparing and using SC-ICs.

[0160] Furthermore, mentioning an element by the indefinite article "a" or "a kind" does not preclude the possibility of more than one / a kind of element, unless the context explicitly requires the presence of one / a kind and only one / a kind of element. Therefore, the indefinite article "a" or "a kind" generally means "at least one / a kind".

[0161] Furthermore, the use of "including" and other forms such as "include," "includes," and "included" is non-limiting. Similarly, the use of "comprising" and other forms such as "comprise," "comprises," and "comprised" is non-limiting. In the specification or claims herein, the presence of "including" or "comprising" (or any other form thereof) does not exclude additional unlisted elements or method steps.

[0162] The terms “and / or” and “any combination thereof” and their grammatical equivalents are used interchangeably in this document to indicate that any combination is specifically considered. For illustrative purposes only, the phrases “A, B and / or C” or “A, B, C or any combination thereof” can mean “A alone; B alone; C alone; A and B; B and C; A and C; and A, B and C”.

[0163] Some of the abbreviations used in this article are as follows: "ADOHCYASE" refers to S-adenosylhomocysteine ​​hydrolase; "ADRA2A" refers to adrenaline receptor α2A; "ALC" refers to O-acetyl-L-carnitine hydrochloride; "ALK5iII" refers to ALK5 inhibitor II; "amu" refers to atomic mass unit; "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 azacitidine; "BMAL1" and "ARNTL" refer to aryl hydrocarbon receptor nuclear transporter-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... "CXC motif chemokine ligand 14"; "CXCR4" refers to CXC chemokine receptor type 4; "D" refers to tantalum; "DACDM" refers to N,N′-diacetyl-L-cysteine ​​dimethyl ester; "DE" refers to typed endoderm or typed endoderm cells; "DiNAC" refers to N,N′-diacetyl-L-cysteine; "dL" refers to deciliter; "DMEM" refers to Dalbeco modified Eagle medium; "DNMT" refers to DNA methyltransferase; "DZNep" refers to 3-deadenine A; "E8 medium" refers to Essential medium. 8. Culture medium; “EC” refers to ectoderm or ectoderm cells; “ECC” refers to enterochromaffin cells; “ECLC” refers to enterochromaffin-like cells; “EGF” refers to epidermal growth factor; “EGSC” refers to embryonic germline stem cells; “EHMT1” refers to euchromatin histone lysine methyltransferase 1; “EHMT2” refers to euchromatin histone lysine methyltransferase 1; “EN” refers to endoderm or endoderm cells; “EOMES” refers to demesoderm protein; “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 FEV1 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 differentiation 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 hematopoietic expression homeobox; "hiPSC" refers to human induced pluripotent stem cells; "HMT" refers to histone methyltransferase; "HNF1β" refers to hepatocyte nuclear factor 1-β; "HNF3-β" refers to hepatocyte nuclear factor 3-β; "HNF4α" refers to hepatocyte nuclear factor 4α; "HNF6" refers to hepatocyte nuclear factor 6; "hr" refers to hours; "HPLM" refers to human plasma-like culture medium; "HSA" refers to human serum albumin; "ILC" refers to isovaleryl L-carnitine; "INS" refers to insulin; "iPSC" refers to induced pluripotent stem cells; "ISL1" refers to islet-1; "ITS-X" refers to insulin-transferrin-selenoethanolamine supplement; "ILV" refers to indolelactam; "K" +"KGF" refers to keratinocyte growth factor; "KOSR" refers to KnockOut serum substitute; "KRB" refers to Kreb's Ringer Buffer; "L" refers to liter; "LDHA" refers to lactate dehydrogenase A; "LDHB" refers to lactate dehydrogenase B; "LMX1A" refers to LIM homeobox transcription factor 1α; "MAFA" refers to MAF bZIP transcription factor A; "MAFB" refers to MAF... bZIP transcription factor B; “ME” refers to mesoderm or mesoderm cells; “MEM” refers to minimum essential culture medium; “mg” refers to milligrams; “min” refers to minutes; “MIXL1” refers to mix1 homeobox-like protein 1; “mmol” refers to millimoles; “Mpy” refers to methyl pyruvate; “MSC” refers to pluripotent stem cells; “NAC” refers to N-acetylcysteine; “NAM” refers to nicotinamide; “NaPyr” refers to sodium pyruvate; “NEAA” refers to non-essential amino acids; “NeruroD1” refers to neurogenic differentiation factor 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 and differentiation factor; "NPEC" refers to non-pancreatic endocrine cells; "NPTX2" refers to neuronal pentamin 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. 2); “PALC” refers to pancreatic α-like cells; “PAX4” refers to PAX4; “PAX6” refers to PAX6; “PARP-1” refers to poly(ADP-ribose) polymerase 1; “PBLC” refers to pancreatic β-like cells; “PC1 / 3” refers to prohormone convertase; “PDX1” refers to pancreatic and duodenal homology box 1; “PEC” refers to pancreatic endocrine cells; “PEP” refers to pancreatic endocrine precursors; “PGT” refers to primitive intestine; “PKC” refers to protein kinase c; “PLC” refers to propionyl-L-carnitine; “PP” refers to pancreatic progenitor cells (e.g., PP1, PP2); “PPP” refers to pancreatic progenitor cells. Polypeptide; "PROX1" refers to prospero homeobox 1; "PSC" refers to pluripotent stem cells; "PTF1α" refers to pancreatic transcription factor 1-α; "ROCK" refers to Rho kinase; "SAM" refers to sterile α motif; "SC-IC" refers to stem cell-derived islet-like cells; "SHH" refers to sound hedgehog factor; "SIRT1" refers to silencing information regulator 1; "SIRT6" refers to silencing information regulator 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 the 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 β; "TGF-β" refers to... "RI" refers to transforming growth factor β receptor type I; "TNKS1" refers to end-anchor polymerase 1; "TNKS2" refers to end-anchor polymerase 2; "TSC" refers to pluripotent stem cells; "U" refers to units; "µM" refers to micromoles or micromolars; "µm" refers to micrometers or micrometers; "UCN3" refers to urocortin-3; "UFH" refers to unfractionated heparin; "VTN-N" refers to vitrin-N; "Wnt3a" refers to wingless MMTV integration site family member 3A; "XF" indicates no xenogeneic component; "ZnSO4" refers to zinc sulfate; and "ZnT8" refers to zinc transporter 8.

[0164] Some of the definitions used in this article are as follows: As used herein, “about” means within a statistically significant range of one or more values, such as concentration, length, molecular weight, pH, pressure, sequence similarity, time frame, temperature, volume, etc. Such values ​​or ranges may be within 20%, 15%, 10%, or more typically 5% of a given value or range. Alternatively, and in relation to a biological system or process, the term “about” may mean within an order of magnitude of a given value, such as within five times or more typically within two times. The permissible variation covered by “about” will depend on the system under study and may be readily understood by those skilled in the art. In some cases, when the term “about X%” refers to the percentage of a specified number of cells in a cell population, the term “about X%” means within 15% below X and above 15%, or within 10% below X and above 10%, or within 5% below X and above 5%.

[0165] As used herein, “activin A” refers to a homodimer of two βA chains from the activin family, which is a non-glycosylated homodimer or heterodimer of various β subunits (βA, βB, βC, and βE in mammals). The 14 kDa mature human βA chain shares 100% amino acid sequence identity with βA from bovine, cat, mouse, pig, and rat animals. Unless otherwise stated, the activin A protein in the differentiation medium described herein is recombinant human activin A, commercially available from, for example, R&D Systems (Minneapolis, MN USA).

[0166] As used herein, “albumin” means mammalian albumin, which has either been isolated from the serum of a mammalian species or has been recombinantly produced. In some cases, the albumin used in the methods described herein is bovine serum albumin or human serum albumin. In some cases, the albumin is fatty acid-free (FAF) albumin, which means that all or substantially all fatty acids bound to 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 the differentiation methods described herein include recombinant mammalian (e.g., bovine or human) albumin, which may be part of an albumin composition comprising (a) one or more phospholipids (e.g., sphingosine-1-phosphate and / or lysophosphatidic acid) or (b) a mixture of fatty acids (e.g., as described in U.S. Patent No. 11,767,504; or deAlbumin™ compositions commercially available from Albcura Corp (New Taipai City, Taiwan, China). Another albumin suitable for this article is a lipid-rich albumin, which may be a recombinant albumin in a composition comprising the same lipid composition present in commercially available AlbuMax™, as shown in Garcia-Gonzalo & Belmonte (2008). PLoS One Table 2 of 3:e1384.

[0167] As used in this article, ALK5 inhibitor II is a small molecule compound, 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthidine (C 17 H 13 N5 (CAS No. 446859-33-2). An ALK inhibitor II, also known as RepSox, E-616452, and SJN 2511, is a selective and ATP-competitive inhibitor of the TGF-β type I receptor ALK5, with an IC50... 50 The value is 4 nM. ALK5 inhibitor II is commercially available, for example, from ReproCell (Beltsville, MD USA).

[0168] As used herein, “ALK5 inhibitor compound” and “ALK5i compound” refer to a compound (e.g., a small molecule compound) that is an ATP-competitive inhibitor of the TGF-β type I receptor ALK5. In some cases, ALK5i compounds are 3-(pyridineyridin-2-yl)-4-(4-quinoyl)-1H-pyrazole; (C 17 H 12N4 (CAS No. 396129-53-6), also known as LY364947, is an IC50 assay used in cell-free assays. 50 It is a 59 nM ATP-competitive inhibitor and exhibits 7-fold selectivity for TGFβR-II. LY364947 is commercially available from, for example, Selleck Chemicals (Houston, TXUSA). In some cases, ALK5i compounds are ALK inhibitors II as defined herein.

[0169] As used herein, “alpha-like cell,” “pancreatic alpha-like cell,” “pancreatic alpha-like cell,” and “PALC” are used interchangeably to refer to pancreatic endocrine cells that at least express and secrete glucagon (GCG; i.e., GCG+) or otherwise possess such characteristics that the cell is functionally equivalent to a natural human alpha cell. PALCs are sometimes referred to herein as immature or mature based on, for example, their functional characteristics. In some cases, immature PALCs (or pre-alpha cells) proliferate, express prohormone-converting enzyme 1 / 3 (PC1 / 3), produce GLP-1, and express the GLP-1 receptor. In some cases, immature PALCs or pre-alpha cells are multi-hormonal (i.e., co-expressing INS and GCG). In some cases, PALCs express both CPEP and GCG (i.e., CPEP...). + / GCG + (Cells). In some cases, mature PALCs are cells that express the single hormone GCG. In other cases, mature PALCs express both CPEP and GCG, but do not secrete INS (i.e., CPEP). + / GCG + / INS - cell).

[0170] As used herein, “alternative nutrient” means a non-glucose energy source such as amino acids, lipids, and / or non-glucose carbohydrates that allows cells to produce ATP through mitochondrial respiration but does not allow cells 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.

[0171] As used herein, “all-trans retinoic acid” and “ATRA” refer to the oxidized form of vitamin A, which functions by binding to a heterodimer of the retinoic acid receptor (RAR) and the retinoid × receptor (RXR), which then binds to retinoic acid response elements (RAREs) in the regulatory region, activating gene transcription (Marshall et al. (1996)). FASEB J 10:969-978). ATRAs with the chemical names and structures shown in Table 15 below are commercially available from, for example, ReproCell USA, Inc. (Beltsville, MD USA).

[0172] As used herein, “β-cytokinin” refers to a member of the EGF family and is involved in signal transduction via the EGF receptor (EGFR) and the receptor tyrosine protein kinase ERBB4. Recombinant human β-cytokinin protein is commercially available from, for example, Stemcell Technologies (Vancouver, BC, Canada) and has the amino acid sequence of SEQ ID NO:1.

[0173] As used herein, “B27 supplement” refers to a defined blend of antioxidant enzymes, proteins, vitamins, and fatty acids in optimized proportions to support neuronal survival in culture, and is based on a serum-free neuronal culture supplement formulation developed by Dr. Gregory Brewer and colleagues. See, for example, Brewer & Cotman (1989). Brain Res. 494:65-74; and Brewer et al. (1993) J. Neuroscience Res. 35:567-576. In some cases, B27 supplements are 50x concentrated solutions. The composition of exemplary B27 (50x) supplements is shown in Table 28 of the examples below. Another exemplary B27 (50x) supplement is serum-free B-27 commercially available, for example, from ThermoFisher Scientific (Waltham, MA USA). TM Supplement (50X).

[0174] As used in this article, “beta-like cell,” “pancreatic beta-like cell,” and PBLC are used interchangeably and refer to cells that at least produce and secrete INS (i.e., INS). + However, it does not express glucagon (i.e., GCG). -Pancreatic endocrine cells (PBLCs) are those that produce somatostatin, ghrelin, or pancreatic polypeptides. Based on whether they exhibit functional characteristics substantially similar to those exhibited by human endogenous immature β cells or by human endogenous mature β cells, PBLCs are sometimes referred to herein as immature or mature. The differences in functional characteristics between immature and mature β cells are well known in the art (see, for example, Barsby & Otonkoski (2022)). Diabetologia 65:917-930; Sun et al. (2021) World J. Stem Cells 13:193-207; International Patent Application Publication No. WO 2020 / 247954 and U.S. Patent Application Publication No. 2014 / 0287944). Both immature and mature PBLCs express CPEP but not GCG (i.e., CPEP). + / GCG - In some cases, PBLC expresses CPEP and NKX6.1 (i.e., CPEP). + / NKX6.1 + In some cases, PBLC is INS. + / CPEP + / GCG - and / or INS + / CPEP + / NKX6.1 + .

[0175] As used herein, “immature PBLC” refers to pancreatic endocrine cells that produce INS but lack the GSIS response characteristic of endogenous human β cells (e.g., biphasic GSIS). In some cases, immature PBLCs expressing characteristic human β cell markers can be characterized by their INS expression 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 cases, immature PBLCs express INS and NKX6.1 and substantially do not express NGN3. In some cases, immature PBLCs express INS but do not express UCN3 protein, or another mature β cell marker as described below.

[0176] As used herein, “intermediate [x / y] cell population” refers to a heterogeneous cell population present at a reference time point during the process of differentiating poorly specialized x-cell types (e.g., PGT cells, PP cells, PEP cells, immature PBLCs) into highly specialized y-cell types (e.g., FE cells, PEP cells, SC-IC, mature PBLCs, respectively). Intermediate [x / y] cell populations are generally more phenotypically heterogeneous than the initial x-cell population or the final y-cell population, and may include cells exhibiting only x-cell markers, cells exhibiting only y-cell markers, and cells exhibiting both x-cell and y-cell markers. The methods described herein can generate various intermediate [x / y] cell populations, including intermediate PGT / FE cell populations, intermediate PP / PEP cell populations, intermediate PEP / SC-IC populations, and intermediate precursor / mature SC-IC populations.

[0177] As used herein, “intermediate PGT / FE cell population” means a cell population at various differentiation points between PGT cells and FE cells, and may include, for example, various percentages of the following: (i) PGT cells (e.g., exhibiting only PGT markers), (ii) FE cells (e.g., exhibiting only FE cell markers), and (iii) cells with an intermediate phenotype that may be phenotypically more similar to PGT cells than FE cells (e.g., exhibiting more PGT cell markers than FE cell markers), and / or phenotypically more similar to FE cells than PGT cells (e.g., exhibiting more FE cell markers than PGT cell markers).

[0178] As used herein, “intermediate PP / PEP cell population” means the cell population present at a reference time point during the process of differentiating a PP cell population into a PEP cell population, and may include various percentages of PP cells (e.g., expressing only PP cell markers), PEP cells (e.g., expressing only PEP cell markers), and cells with an intermediate phenotype (e.g., expressing both PP cell markers and PEP cell markers).

[0179] As used herein, “intermediate PEP / SC-IC population” means the cell population present at a reference time point during the process of differentiating a PEP cell population into a precursor SC-IC population, and may include various percentages of the following: PEP cells (e.g., exhibiting only PEP cell markers), cells exhibiting one or more markers of different SC-IC types with various maturities (e.g., cells exhibiting markers of immature / mature PALCs and / or immature / mature PBLCs), and cells with an intermediate phenotype (e.g., exhibiting both PEP cell markers and markers relating to PLACs and / or PBLCs).

[0180] As used herein, “intermediate precursor / mature SC-IC population” means the cell population present at a reference time point during the process of differentiating a precursor SC-IC population (e.g., including immature and / or mature β-like and α-like cells) into a mature SC-IC population (e.g., including a higher percentage of mature SC-ICs, such as mature β-like and mature α-like cells, compared to the precursor SC-IC population).

[0181] As used herein, “mature PBLC” means pancreatic endocrine cells that produce INS, express at least one marker indicating endogenous mature β cells (e.g., UCN3 and / or MAFA), and exhibit a GSIS response to glucose attack substantially similar to that exhibited by endogenous mature pancreatic β cells, or possess other properties that make PBLCs functionally equivalent to endogenous mature human β cells. In some cases, mature PBLCs exhibit at least one of the following properties of biphasic GSIS: (i) coupling of mitochondrial respiration / activity with INS secretion; (ii) a rapid INS secretion response to enhanced demand (defined herein as high glucose concentration); (iii) the ability to rapidly shut down INS secretion after the demand has subsided; (iv) the ability to perform multiple rounds of “on-off” switching with respect to INS secretion; (v) the ability to secrete the correct amount of INS as indicated by demand; and (vi) the ability to respond to multiple INS secretagogues (e.g., exendin-4, or the amino acids L-glutamine and L-arginine). In some cases, mature pancreatic PBLCs can be identified by the presence of one or more of the following markers: single hormones INS, NKX6.1, UCN3, GLUT2, SLC2A1, SIX2, SIX3, BMAL, and PDX1, as well as MAFA expression (at higher levels than in less mature pancreatic endocrine cells, particularly immature PBLCs). In other cases, mature PBLCs can be identified by the presence of one or more of these markers in addition to the absence of MCT1 (SLC16A1), LDHA, and HK1 expression.

[0182] As used herein, “BMP inhibitor” means a compound, such as a small molecule compound, that inhibits BMPI-type 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).

[0183] As used herein, “carnitine compound” refers to L-carnitine and its derivatives, 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 the mitochondria for oxidation to generate free energy, and also participates in the removal of metabolites from the cell. The chemical names and structures of L-carnitine and ALC are shown in Table 15 below, and each compound is commercially available from, for example, Millipore Sigma.

[0184] As used herein, “cell marker” means a marker (i.e., peptide, protein) expressed or produced by a cell that is specific to a particular cell type or cell category present in a cell population (e.g., CPEP). + It is a marker of pancreatic endocrine cells, and Ki-67 + These are markers for proliferating cells. The various cell types described in this article can be characterized as being positive or negative for one or more cell markers.

[0185] As used herein, “chemically defined lipid mixture” and “CDLM” mean a liquid composition comprising two or more of arachidonic acid, cholesterol, DL-α-tocopheryl acetate, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, palmitoleic acid, and stearic acid in defined concentrations. In some cases, a CDLM comprises three, four, five, six, seven, eight, nine, or all ten of these lipids. In some cases, a CDLM may comprise one of the lipid combinations shown in Table 6 of U.S. Patent Application Publication No. 2013 / 0273010. In some cases, a CDLM may comprise one or more emulsifiers such as Pluronic F68® and Tween 80®. Exemplary CDLM compositions are shown in Table 27 below. In some cases, the CDLM used in the differentiation methods described herein comprises components 1 through 10 of Table 27 below. In some cases, a CDLM does not include palmitoleic acid. In some cases, a CDLM comprises components 1 through 12 or components 1 through 13 of Table 27. CDLM compositions are commercially available, such as Sigma-Aldrich Lipid Mixture 1 and Gibco. TM ChemicallyDefined Lipid Concentrate.

[0186] As used herein, “CHIR98014” refers to a small molecule compound having the chemical name and structure shown in Table 15 below. Inhibition of GSK-3α and GSK-3β (IC50) 50 CHIR98014 (with values ​​of 0.65 nM and 0.58 nM respectively) is commercially available from, for example, APExBIO (Houston, TX USA).

[0187] As used herein, “CHIR99021” refers to the small molecule compound called laduviglusib (chemical name and structure shown in Table 15) and its pharmaceutically acceptable salt. It acts as a GSK-3 inhibitor (GSK3β, IC50). 50 CHIR99021, a 7nM agonist and Wnt / β-catenin activator, is commercially available from companies such as Selleck Chemicals (Houston, TX USA).

[0188] As used herein, "Seman 1" refers to small molecule compounds having the chemical names and structures shown in Table 15 and their pharmaceutically acceptable salts. Seman 1 is a target for ROCK2 (IC). 50 = 1 pM) compared to ROCK1 (IC 50= 52 pM) more effective ROCK inhibitors, which are commercially available from, for example, Selleck Chemicals.

[0189] 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 of the listed elements or groups of elements, as well as the optional inclusion of other elements that are similar or different in nature from the listed elements, which do not substantially alter the fundamental or novel properties of the specified molecules, cell populations, compositions, devices, or methods. As a non-limiting example, “consists essentially of” refers to the inclusion of a specified cell type (e.g., CPEP). + A cell population consisting of cells may include a small number of other cell types (e.g., CPEP cells). - (cells), but the presence of such unlisted cells does not substantially affect the relevant biological activity of the cell population. In another non-limiting example, a differentiation medium consisting essentially of the listed components may have other components that do not substantially alter the cell culture or differentiation properties of the medium.

[0190] Unless otherwise stated, as used herein, “culture” means an in vitro process.

[0191] As used herein, “defined-component medium” means an aqueous cell growth medium in which the amount or quantity of chemical components or ingredients (i.e., formulations) is known. A defined-component medium includes at least one nutrient and at least one electrolyte, and may also include one or more other components commonly found in cell culture media (e.g., buffers, albumin or albumin substitutes, galactose, INS, transferrin or transferrin substitutes, 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, etc.). Unless otherwise stated, all references to glucose and galactose in defined-component mediums herein refer to the D-form (e.g., D-glucose and D-galactose). Additionally, glucose used as a nutrient in any defined-component medium described herein may be partially or completely replaced with fructose (e.g., the medium may include glucose, fructose, or glucose and fructose). Unless otherwise stated, all references to glutamine in defined-component mediums herein refer to the L-form (L-glutamine). Examples of culture media with defined components include, but are not limited to, DMEM (commercially available from Gibco; the components of DMEM are described in Dulbecco & Freeman (1959)). Virol.(Described in 8:396-397), MCDB 131 (glutamine-free; commercially available from Thermo Fisher Scientific; composition of MCDB 131 is described in Knedler & Ham (1987)). In Vitro Cell. Dev. Biol. Anim. (described in 23:481-491) and HPLM (commercially available from ThermoFisher Scientific; composition of HPLM is described in Cantor et al. (2017)). Cell As described in 169:258-272; see also International Patent Application Publication No. WO 2018 / 089928).

[0192] As used herein, “defined endoderm cell population” or “DE cell population” means a cell population obtained by: (i) differentiation of pluripotent stem cells (e.g., iPSCs) using any method known in the art or as described herein, or (ii) differentiation of a precursor cell population derived from PSCs (e.g., ME cell population). More than 50% of the cells in the DE cell population are DE cells (i.e., cells that do not express PDX1). - The cells 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 cases, the DE cell population may be >approximately 80% GATA6. + / Sox17 + and > 90% FoxA2 + / Sox17 + In some cases, the DE cell population includes cells with lower and / or higher differentiation (e.g., ME and / or PGT cells) and / or other cell types. In some cases, the DE cell population is derived from human PSCs (e.g., hiPSCs) or from ME cell populations derived from human PSCs (e.g., hiPSCs).

[0193] As used herein, “delta-like cell” and “δ-like cell” refer to SC-ICs that at least produce and secrete somatostatin (SST) (e.g., SST). + These cells (or other properties) are functionally equivalent to natural human delta cells. In some cases, delta-like cells are SSTs. + / HHEX + .

[0194] As used in this article, “diabetes” refers to a disease characterized by elevated blood glucose levels over a prolonged period. That is, “diabetes” can refer to all or any type of diabetes, including but not limited to type 1 diabetes, type 2 diabetes, cystic fibrosis-associated diabetes, surgical diabetes, gestational diabetes, and mitochondrial diabetes.

[0195] As used herein, “differentiate,” “differentiated,” and “differentiating” are relative terms that refer to the process by which a less specialized cell (e.g., a more primitive cell with higher cellular potential) becomes a more specialized cell type (e.g., a less primitive cell with lower cellular potential). In other words, a differentiated cell can be a cell that has progressed further down the developmental pathway than the cell to which it is compared (e.g., from an immature state to a less mature state; e.g., from a partially differentiated cell to a more differentiated cell), or a cell that has progressed from an immature state to a mature state (e.g., from a partially differentiated cell to a fully differentiated cell). Thus, pluripotent cells can differentiate into lineage-restricted progenitor cells (e.g., ectoderm, endoderm, and mesoderm), which in turn can differentiate into further restricted cells (e.g., PEP), which can differentiate into terminal stage cells (e.g., terminally differentiated cells; e.g., cardiomyocytes, neurons, β cells, etc.), and play characteristic roles in certain tissue types, and may or may not retain the ability to proliferate further.

[0196] As used herein, “DMH-1” refers to a small molecule compound having the chemical name and structure shown in Table 15. DMH-1 is a selective inhibitor of the BMP type I receptor activin receptor-like kinase 2 (ALK2) receptor, exhibiting 6-fold and 19-fold selectivity for ALK-2 relative to ALK-1 and ALK-3, respectively, and no significant inhibition of AMPK, ALK5, KDR (VEGFR-2) or PDGFR receptors. It is commercially available, for example, from Bio-Techne Corporation (Minneapolis, MN USA).

[0197] As used herein, “deoxyribonuclease I” and “DNase I” refer to mammalian DNA-specific endonucleases that hydrolyze double-stranded or single-stranded DNA into a mixture of oligonucleotides and mononucleotides. DNase I is frequently included in tissue dissociation protocols to digest DNA that has leaked into the dissociation medium due to cell damage. In some cases, DNase I is recombinant DNase I, which has the same amino acid sequence as bovine DNase I and does not require recombinant expression using any animal cells or other animal-derived materials (e.g., in Pichia pastoris). Pichia pastoris (In some cases, recombinant bovine DNase I is a glycoprotein with a molecular weight of approximately 39 kDa. Recombinant bovine DNase I is commercially available from, for example, MilliporeSigma.)

[0198] As used herein, "ectoderm cell" or "ectodermal cell" refers to one or more cells derived from the ectoderm (EC), one of the three major germ layers in the very early embryo. These cells can differentiate to form epithelial and neural tissues.

[0199] As used herein, “effective amount” means, for example, the amount, concentration, or dose of an SC-IC or SC-IC group or composition comprising thereof, which, upon administration of a single or multiple doses to an individual in need, provides the desired effect in such an individual undergoing diagnosis or treatment (i.e., produces a clinically measurable difference in the individual’s condition, such as a decrease in blood glucose, a decrease in HbA1c, and / or a decrease in weight or body fat). Effective amounts can be readily determined by those skilled in the art using known techniques and observations obtained in similar circumstances. In determining the effective amount for an individual, a variety of factors are taken into account, including but not limited to the individual’s species (e.g., mammalian species; e.g., human), their size, age, and general health, the specific disease or condition involved, the extent or severity of the disease or condition, the individual’s response, the specific form in which the SC-IC is administered, the mode of administration, the bioavailability characteristics of the administered formulation, the selected dosing regimen, the use of concomitant medications, and other relevant factors.

[0200] As used in this article, "endocrine cells" refers to cells expressing CHGA (i.e., CHGA). + ) cells.

[0201] As used herein, “endoderm cell,” “endodermal cell,” and “EN cell” refer to cells derived from the endoderm (EN), one of the three major germ cell layers in the very early embryo. EN cells can first differentiate into the embryonic intestine, and then into the lining 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 + cell).

[0202] As used herein, “enterochromaffin-like cells” or “EC-like cells” or “ECLC” refers to cells that at least produce and secrete serotonin (SRT); that is, SRT + (or otherwise possesses properties that make the cells functionally equivalent to native human enterochromaffin cells (ECCs) (e.g., ECC cell markers such as ADRA2A, CXCL14, FEV, LMX1A, SLC18A1, and TAC1). In some cases, ECLCs are CHGAs. + / NKX6.1 + However, PBLC markers such as PDX1, ISL1, G6PC2, and NPTX2 are lacking. In some cases, ECLC can be referred to as non-pancreatic cells.

[0203] As used herein, "epidermal growth factor family" and "EGF family" refer to the EGF protein family, which includes EGF, heparin-bound EGF-like growth factor (HB-EGF), transforming growth factor-α (TGF-α), amphotericin (AR), epidermal regulatory protein, epigen, β-cytokinin, neuroregulatory protein-1, neuroregulatory protein-2, neuroregulatory protein-3, and neuroregulatory protein-4. In some cases, the EGF family members used in the differentiation methods described herein are recombinant human EGF proteins or recombinant human β-cytokinin proteins.

[0204] As used herein, “EGF protein” refers to mammalian epidermal growth factor protein, a founding member of the EGF family that signals via the class I tyrosine kinase receptor c-erbB. In some cases, the mammalian EGF protein used in the differentiation methods described herein is recombinant human EGF protein, commercially available from R&D Systems (Minneapolis, MN USA), and has the amino acid sequence SEQ ID NO:2.

[0205] As used herein, “epigenetic modifier” means a chemical agent used to regulate gene activity and / or expression in a cell or cell population by applying changes in DNA methylation, histone modification, and chromatin fabrication, rather than altering the DNA sequence itself. This article is of interest to epigenetic modifiers that affect cells or cell populations to a greater degree of likelihood of differentiating into pancreatic β-like cells (e.g., immature 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, and combinations thereof. Exemplary epigenetic modifiers include, but are not limited to, azacitidine, butyrate, DZNep, EPZ004777, MDL-800, CM-272, UNC0321, and UNC0638.

[0206] As used herein, "epsilon-like cell" and "ε-like cell" refer to cells that at least produce and secrete ghrelin (GRL); that is, GRL + It may possess such characteristics in other ways that the cell is functionally equivalent to a natural human ε cell.

[0207] As used herein, the term “expression” of a marker implies an observable and / or measurable quantity or its presence (i.e., capable of being qualitatively or quantitatively characterized).

[0208] As used herein, “foregut endoderm cells”, “FE cells”, etc., refer to cells derived from PGT cells that express at least one of the following markers: CDX2, FOXA2, HNF4α, PDX1, and SOX2, especially PDX1.

[0209] As used herein, “FE cell population” means a cell population obtained by: (i) differentiation of pluripotent stem cells (e.g., iPSCs) by any method known in the art or as described herein, or (ii) differentiation of a precursor cell population derived from PSCs (e.g., the PGT cell population as defined herein). The majority of cells in an FE cell population are FE cells (e.g., expressing one or more FE cell markers listed above), but the population may also include cells with lower and / or higher differentiation levels (e.g., PGT and / or PP cells) and / or other cell types (e.g., off-target cells). FE cell populations typically have higher PDX1 expression levels than the PGT cell population from which they are derived. In some cases, an FE cell population contains more than about 50%, 70%, 80%, 90%, or 95% of any of the FE cells. In some cases, at least 60%, 70%, 80%, 90%, or 95% of the cells in an FE cell population are PDX1. + Cells. In some cases, FE cell populations are derived from human PSCs (e.g., hiPSCs) or from PGT cell populations derived from human PSCs (e.g., hiPSCs).

[0210] As used herein, in describing differentiated or differentiated cell types, "functionally equivalent" means that the cell performs the same function and / or provides the same utility as the mentioned natural human cell type, even if it is not exactly the same. For example, a β-like cell may be functionally equivalent to a natural human β cell if it exhibits at least one marker indicating a natural human β cell, has INS content (i.e., has observable INS granules), and / or secretes INS in response to appropriate stimuli such as glucose (i.e., has regulated GSIS). Alternatively, other characteristics of β-like cells include, but are not limited to: (i) coupling of mitochondrial respiration / activity with INS secretion; (ii) a rapid INS secretion response to increased demand (defined herein as high glucose concentration); (iii) the ability to rapidly shut down INS secretion after the demand subsides; (iv) the ability to secrete multiple rounds of INS; (v) the ability to secrete INS amounts as indicated by demand; and (vi) the ability to respond to multiple INS secretagogues (e.g., exenatide or amino acids such as L-glutamine and L-arginine).

[0211] As used herein, “G9a inhibitor” means a compound, such as a small molecule compound, that inhibits the activity of one or both of the following: (i) histone methyltransferase G9a (also known as euchromatin histone lysine methyltransferase 2 (EHMT2)) and (ii) histone methyltransferase G9a-like protein (also known as euchromatin histone lysine methyltransferase 1 (EHMT1)). In some cases, G9a inhibitors are more selective for G9a than for G9a-like proteins. In other cases, G9a inhibitors are more selective for G9a-like proteins than for G9a. Exemplary inhibitors include CM-272, UNC0321, and UNC0638, whose chemical names and structures are shown in Table 15 below.

[0212] As used herein, “gamma-like cell” and “γ-like cell” refer to cells that at least produce and secrete pancreatic polypeptide (PPP); that is, PPP + (The cell) or otherwise possesses such characteristics that it is functionally equivalent to a natural human γ cell.

[0213] As used herein, "γ-secretase inhibitor" and "GSI" refer to compounds such as small molecule compounds that inhibit γ-secretase, a multimeric membrane protein complex. In some cases, GSI is γ-secretase inhibitor XX (GSI-XX), which is a cell-permeable dibenzodiazepine compound that acts as a potent inhibitor of γ-secretase. GSI-XX is commercially available from, for example, Millipore Sigma. In some cases, GSI is DAPT (N-[N-(3,5-difluorophenylacetyl)- L [-alanyl]-S-phenylglycine tert-butyl ester), DAPT, also known as GSI-IX, is commercially available from, for example, MedChemExpress (Monmouth Junction, NJ USA).

[0214] As used in this article, “glucose-free medium” means a defined medium that does not contain glucose (i.e., <0.01 mM, <0.001 mM or 0 mM).

[0215] As used in this article, "glucose-stimulated insulin secretion" or "GSIS" refers to the sensing of glucose by natural human β-cells or β-like cells in vitro or in vivo, and the response via potassium ions (K+) to insulin secretion. + Channel dependency mechanism and / or K + The ability of INS to secrete via a channel-independent mechanism.

[0216] As used herein, “glutamine dipeptide” refers to 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.

[0217] As used herein, the terms “grafting,” “application,” “introduction,” “implantation,” and “transplanting,” as well as their grammatical variations, are used interchangeably and refer to placing cells (e.g., SC-IC cells, as used herein) or cell populations (e.g., SC-IC populations, as used herein) within an individual by means or pathways that result in the introduced cells being at least partially localized at the desired site. SC-IC cells may be directly implanted at the desired site, or alternatively, may be applied via any suitable pathway that results in delivery to the desired location in the individual, wherein at least a portion of the implanted cells or components of the cells remain viable. The viability period of cells after application to a subject can range from a few hours (e.g., 24 hours) to several days or even years.

[0218] As used herein, “GSK-3α and GSK-3β inhibitors / Wnt pathway signaling activators” means compounds such as small molecule compounds that act as inhibitors of GSK-3 and activators of Wnt / β-catenin. Exemplary GSK-3α and GSK-3β inhibitors / Wnt pathway signaling activators include the GSK-3β inhibitor compounds described in International Patent Application Publication No. WO 2013 / 192005. In some cases, the GSK-3α and GSK-3β inhibitors / Wnt pathway signaling activators suitable for the differentiation methods described herein are CHIR98014, CHIR99021, or GSK inhibitor IX.

[0219] As used in this article, “GSK-3 inhibitor IX” refers to 6-bromo-3-[3-(hydroxyamino)indole-2-yl]-1H-indole-2-one (C 16 H 10 BrN3O2 (CAS No. 667463-62-9) is a small molecule compound. GSK-3 inhibitor IX, also known as 6-bromoindorubin-3'-oxime, is a selective, cell-permeable, ATP-competitive, and reversible inhibitor of both GSK-3α and GSK-3β (for GSK-3β, IC50 is 1000 mg / L). 50 = 5nM). GSK-3 inhibitor IX is commercially available from companies such as APExBIO.

[0220] As used herein, “heparin” and “heparin sulfate” refer to linear, unbranched, and highly sulfated polysaccharides with anticoagulant activity, belonging to the family of glycosaminoglycans (GAGs). The repeating disaccharide unit of heparin consists of a uronic acid and a D-glucosamine linked by an α-glycosidic bond. At least three forms of heparin are commercially available: UFH (average molecular weight 19 kDa), low molecular weight heparin (LMWH), and ultra-low molecular weight heparin (ULMWH), which have been described by Palmberger et al. (2021). Int. J. Mol. Sci. The properties described in 22:12041. Commercially available UFH is isolated from porcine intestinal mucosa (UFH-PIM) or from bovine lungs and intestines (UFH-C). In some cases, the differentiation method described herein uses UFH-PIM, which is heparin sulfate commercially available from, for example, Sigma Aldrich. In other cases, the heparin form is LMWH or ULMWH. In some cases, the differentiation method described herein uses synthetic ULMWH (e.g., fondaparinux sodium; CAS number 114870-03-0, commercially available from Dr. Reddy's).

[0221] As used herein, “human plasma-like culture medium” or “HPLM” means a basal cell culture medium that (a) includes the components and concentration ranges of any basal culture medium described in International Patent Application No. WO 2018 / 089927, or (b) includes some or all of the components in Tables 18 or 19 below, which correspond to the exemplary HPLMs described below. An exemplary HPLM comprises: (a) at least nine of the following protein amino acids: glycine, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, 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-cysteine; (b) at least eight, nine, ten, or eleven of the following vitamins: D-biotin, choline, folic acid, inositol, nicotinamide, para-aminobenzoic acid, D-pantothenic acid, vitamin B6, riboflavin, thiamine, and vitamin B12; and (c) six, seven, eight, or nine inorganic salts selected from: CaCl2, KCl, MgCl2, MgSO4, NaCl, NaHCO3, Na2HPO4, Ca(NO3)2. 4H₂O and NH₄Cl; (d) glucose; and (e) at least 10 small organic compounds selected from the following: 4-hydroxyproline, acetylglycine, α-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. An exemplary HPLM comprises each of the components and mg / mL concentrations shown in Table 18 below. Another exemplary HPLM comprises or is substantially composed of the following: each of the components and mg / mL concentrations shown in Table 19 below.

[0222] As used herein, “LDN-193189” refers to a small molecule compound having the chemical name and structure shown in Table 15 below, and its pharmaceutically acceptable salt (e.g., hydrochloride). It is a cell-permeability-selective inhibitor of BMP type I receptors ALK2 and ALK3 (ICV). 50 LDN-193189 (with values ​​of 5 nM and 30 nM) is commercially available from Reprocell (Beltsville, MD USA).

[0223] As used herein, “low-glucose medium” means a defined medium containing less than about 2.5 mM or less than about 2 mM of glucose. In some cases, the glucose concentration in a low-glucose medium is ≤ about 1 mM, ≤ about 0.5 mM, ≤ about 0.1 mM, or ≤ about 0.05 mM. In some cases, a low-glucose medium is a glucose-free medium (i.e., < about 0.01 mM, < about 0.001 mM, or 0 mM).

[0224] As used herein, “individual” means any mammal, including but not limited to cats, dogs, mice, rats, and primates, especially humans. Furthermore, “subject,” “participant,” or “patient” may be used interchangeably with “individual.”

[0225] As used herein, "induced pluripotent stem cells," "iPSCs," etc., refer to pluripotent stem cells artificially derived (e.g., induced or by complete reversal) from non-pluripotent cells (typically adult somatic cells) through, for example, the forced expression of one or more reprogramming factors (e.g., Klf4, Lin28, Myc, Oct3 / 4, Sox2, and / or Nanog). See, for example, Takahashi et al. (2007). Cell 131:861-872; and Yu et al. (2007) Science318:1917-1920. iPSCs have an ESC-like morphology, grow as flat colonies, and have a high nucleus-to-mass ratio, clear boundaries, 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, TDGF1, Dnmt3b, FoxD3, GDF3, Cyp26al, TERT, and zfp42, which can be detected by: RT-PCR, RNA blotting, in situ hybridization (see, e.g., “Current Protocols in Molecular Biology”, (Ausubel et al., editors, John Wiley & Sons, Inc. (1988)), and immunoassays such as immunohistochemical analysis of tissue sections, Western blotting, and for markers accessible in intact cells, flow cytometry (FACS) (see, e.g., Harlow & Lane, “Using Antibodies: A Laboratory Manual”, (ColdSpring Harbor Laboratory Press (1998)). Also see, “Current Protocols in Cell Biology” (Bonifacino et al., Wiley & Sons). (2016)); “Current Protocols in Immunology” (Colligan et al., editors, Wiley & Sons (1991)); “Current Protocols in Protein Science” (Colligan et al., editors, Wiley & Sons (1995)); and “Gene Transfer Vectors for Mammalian Cells” (Miller & Calos, editors, Cold Spring Harbor Laboratory Press (1987)).

[0226] As used in this article, "human induced pluripotent stem cells" or "hiPSC" refers to iPSCs derived from human cells.

[0227] As used herein, “induced pluripotent stem cell population” or “iPSC population” means a cell population in which the majority of cells are iPSCs (e.g., as defined above). In some cases, >approximately 80%, >approximately 90% or more of the cells in an iPSC population express one or more of the pluripotency markers listed above. In some cases, >approximately 80%, >approximately 90%, >approximately 95%, >approximately 99% or more of the cells in an iPSC population co-express Oct4 and Nanog. In some cases, all iPSCs in an iPSC population are hiPSCs, and the iPSC population is referred to as a hiPSC population. In some cases, >approximately 95% of the cells in a hiPSC population are OCT4. + / NANOG + cell.

[0228] As used in this article, “insulin-producing cells” or “INS-producing cells” refers to cells that produce, store, or secrete detectable amounts of INS.

[0229] As used herein, “ITS-G supplement” and “ITSG supplement” are used interchangeably and refer to serum replacement supplements containing INS, transferrin, and selenium (e.g., sodium selenite). In some cases, ITS-G supplements are 100x concentrated solutions. An exemplary ITS-G (100x) supplement consists of a solution containing INS, transferrin, sodium selenite, and ethanol at concentrations shown in Table 23 of the examples below. Another exemplary ITS-G (100x) supplement is a Gibco™ Insulin-Transferrin-Selenium-Ethanolamine (ITS-G) (100X) solution, which is commercially available, for example, from ThermoFisher Scientific (Waltham, MA USA).

[0230] As used herein, “ITS-X supplement” and “ITSX supplement” are used interchangeably and refer to a cell culture medium supplement containing INS, transferrin, selenium (e.g., sodium selenite), and ethanolamine. In some cases, the ITS-X supplement is a 100x concentrated solution. An exemplary ITS-X (100x) supplement is a solution having the composition shown in Table 23 of the examples below. Another exemplary ITS-X (100x) supplement is a Gibco™ insulin-transferrin-selenoethanolamine (ITS-X) (100X) solution, which is commercially available, for example, from ThermoFisher Scientific.

[0231] As used herein, “IWR-1-endo” and “IWR-1” are used interchangeably and refer to small molecule compounds having the chemical names and structures shown in Table 15. IWR-1 is an inhibitor of end-anchored polymerases 1 and 2.

[0232] As used herein, “keratinocyte growth factor protein,” “KGF,” “KGF protein,” “FGF-7,” and “FGF-7 protein” are used interchangeably and refer to mammalian proteins that are members of the fibroblast growth factor (FGF) family and signal transduction via the FGF receptor 2b. In some cases, the mammalian KGF protein used herein is recombinant human KGF. Recombinant human KGF (SEQ ID NO:3) is commercially available, for example, from Peprotech, a subsidiary of ThermoFisher Scientific.

[0233] As used herein, “KnockOut serum alternative medium” or “KOSR medium” means serum-free medium that includes small organic molecules (e.g., amino acids, vitamins, and antioxidants), trace elements, INS, transferrin, selenite, and albumin (e.g., lipid-rich albumin as described herein). Exemplary KOSR mediums include, but are not limited to, any serum-free medium supplements described in International Patent Application Publication No. WO 1998 / 030679. The components of exemplary KOSR mediums are shown in Tables 29 and 30 below. In some cases, KOSR medium is the KOSR composition shown in Table 30, or Gibco KOSR or its xenogeneic (XF) form, commercially available from ThermoFisher Scientific.

[0234] As used herein, “lactate dehydrogenase A” or “LDHA” refers to the enzyme that preferentially catalyzes the conversion of pyruvate to lactate. LDHA is a monomer of lactate dehydrogenase, which exists as a tetramer, including lactate dehydrogenase B (LDHB) as another major subunit. LDHA is highly expressed in many tissues and in adult α cells within the pancreatic islets; however, normal adult β cells within the pancreatic islets do not express LDHA.

[0235] As used herein, "marker," "cell marker," etc., means any molecule that can be observed or detected. Examples of markers include, but are not limited to, nucleic acids, such as transcripts of specific genes; polypeptides, such as membrane proteins or glycoproteins; carbohydrates; lipids, such as glycolipids or lipoproteins; or small molecules (e.g., molecules with a molecular weight less than 10,000 amu). Markers are expressed by or differentially within target cells. In this context, differential expression of a positive marker means an increased level of that marker compared to undifferentiated cells or cells at another stage of differentiation. Similarly, differential expression of a negative marker means a decreased level of that marker compared to undifferentiated cells or cells at another stage of differentiation. The detectable level of a marker in the target cell is sufficiently higher or lower than that in another cell type, such that the target cell can be identified and distinguished from others using any of the various detection methods known in the art.

[0236] As used herein, “mesoderm cell” and “mesodermal cell” refer to cells derived from the mesoderm, one of the three major germ cell layers in the very early embryo. Mesodermal cells can differentiate to form mesenchyme, mesothelium, non-epithelial hemocytes, and coelomic cells, and express at least one of the following markers: demesoderm protein (EOMES) and nodal growth differentiation factor (NODAL).

[0237] As used herein, “mesendoderm cell”, “mesendodermal cell”, and “ME cell” refer to cells derived from the mesendodermis, an embryonic tissue layer capable of differentiating into ME and EN (i.e., having dual potential). ME cells express at least one of the following markers: TBXT (also known as brachyury) and MIXL1.

[0238] As used herein, “ME cell population” means a population of cells obtained by differentiating pluripotent stem cells (e.g., iPSCs) using methods known in the art or as described herein. The majority of cells in a ME cell population are ME cells (e.g., expressing one or more ME cell markers listed above), but the cell population may also include cells with lower and / or higher differentiation (e.g., iPSCs and / or DE cells) and / or other cell types (e.g., off-target cells). In some cases, an ME cell population contains more than about 70%, 80%, 90%, or 95% of any of the ME cells. In some cases, at least about 70%, 80%, 90%, or 95% of the cells in an ME cell population are TBXT. + / MIXL1 +Cells. In some cases, ME cell populations are derived from human PSCs (e.g., hiPSC populations).

[0239] As used herein, "pluripotent stem cells" or "MSCs" refers to cells that are classified into one or more embryonic cell fates / lineages and retain the ability to self-renew, but in contrast to pluripotent cells, they are unable to generate each of the three embryonic cell layers. Examples of pluripotent stem cells include, for example, hematopoietic stem cells, mesenchymal stem cells, and neural stem cells.

[0240] As used herein, “N-acetyl-L-cysteine,” “N-acetyl-cysteine,” “N-acetylcysteine,” and “NAC” are used interchangeably and refer to compounds having the chemical names and structures shown in Table 15. NAC, a precursor to cell-penetrating antioxidants and reduced glutathione (GSH), is commercially available from, for example, Millipore Sigma.

[0241] As used herein, “nicotinamide” and “NAM” are used interchangeably and refer to small molecule compounds having the chemical names and structures shown in the table. NAM, 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.

[0242] 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 aspartic acid), cysteine, glutamic acid (or glutamic acid), glycine, proline, serine, tyrosine, and selenocysteine. Exemplary NEAA supplements include the non-essential amino acids listed in Table 24 below, which are the same non-essential amino acids present in standard minimum essential medium (MEM) well known in the art. In some cases, the 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, which is commercially available, for example, from ThermoFisher Scientific.

[0243] As used herein, “non-pancreatic cells” refers to cells from developmental lineages outside the pancreas (e.g., ECLCs expressing SLC18A1; i.e., SLC18A1). + ECLC).

[0244] As used in this article, "non-proliferative cell" or "non-proliferating cell" refers to a cell that has exited the cell cycle and is no longer undergoing division.

[0245] As used herein, “pancreatic endocrine cells” or “PEC” refers to cells expressing chromogranin A (CHGA) and at least one insulin hormone (e.g., INS, GCG, SST, GRL, and / or PPP). Other markers characteristic of PEC include one or more of HB9, ISL1, NeuroD1, NKX2.2, NKX6.1, PAX4, PAX6, and PDX1. In some cases, PEC is CHGA. + / INS + Cells or CHGA + / GCG + Cells. In some cases, PEC is PALC (e.g., CHGA). + / GCG + ) or PBALC (e.g., CHGA) + / INS + ).

[0246] As used herein, "pancreatic endocrine progenitor cells" and "PEP cells" refer to cells derived from PP cells, wherein PEP cells express at least one of the following markers: ARX, CHGA, ISL1, NeuroD1, NGN3, NKX2.2, PAX4, PAX6, and PDX1, especially CHGA, NGN3, and PDX1. In some cases, PEP cells express NKX2.2. + / NeuroD + In some cases, PEP cells are CHGA. + / NGN3 + CHGA + / PDX1 + or NGN3 + / PDX1 + .

[0247] As used herein, “PEP cell population” means a cell population obtained by: (i) differentiation of pluripotent stem cells (e.g., iPSCs) using any method known in the art or as described herein, or (ii) differentiation of a precursor cell population derived from PSCs (e.g., a PP cell population as defined herein). The majority of cells in a PEP cell population are PEP cells (e.g., expressing one or more of the PEP cell markers listed above), but the population may also include cells with lower and / or higher differentiation levels (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 cases, a PEP cell population contains more than approximately 70%, 80%, 90%, or 95% PEP cells. In some cases, at least approximately 70%, 80%, 90%, or 95% of the cells in a PEP cell population are CHGA+ cells, and at least approximately 40% of the cells are CHGA. + / PDX1 + Cells. In some cases, less than about 10% of the cells in a PEP cell population are off-target cells. In some cases, the PEP cell population is derived from human PSCs (e.g., hiPSCs) or from PP cell populations derived from human PSCs (e.g., hiPSCs).

[0248] As used in this article, "pancreatic progenitor cells" and "PP" refer to cells derived from FE cells, wherein PP cells express at least one of the following markers: gastrin, HB9, HNF1β, HNF4α, HNF6, NGN3, NKX6.1, PDX1, PTF1α, PROX1, and SOX9, especially NKX6.1 and PDX1.

[0249] As used herein, “PKC activator” is a compound that activates PKC, such as a small molecule compound. Exemplary PKC activators include, but are not limited to, TPPB, phorbol 12,13-dibutyrate (PdBU) (CAS No. 37558-16-0), phorbol-12-tetradecanoate-13-acetate (PMA) (CAS No. 16561-29-8), (-)-indolinamide V (ILV) (CAS No. 90365-57-4), lichenin 1 (CAS No. 83314-01-6), and derivatives of these compounds.

[0250] As used herein, “PP cell population” means a cell population obtained by: (i) differentiation of pluripotent stem cells (e.g., iPSCs) using any method known in the art or as described herein, or (ii) differentiation of a precursor cell population derived from PSCs (e.g., FE cell population as defined herein). The majority of cells in a PP cell population are PP cells (e.g., expressing one or more PP cell markers listed above), but the population may also include cells with lower and / or higher differentiation levels (e.g., FE and / or PEP cells) and / or other cell types (e.g., off-target cells such as SOX2). + Cells and / or CDX2 + (Cells). In some cases, approximately 50% of the cells in a PP cell population are proliferating cells (e.g., Ki67). + (cells), and approximately 20% to approximately 80% of the cells are PDX1. + / NKX6.1 + But CHGA - In some cases, the PP cell population contains more than approximately 70%, 80%, 90%, or 95% PP cells. In other cases, at least approximately 70%, 80%, 90%, or 95% of the cells in the PP cell population are PDX1. + The cells, at least about 30% of which are PDX1. + / NKX6.1 + Cells, and less than approximately 30%, 20%, or 10% of the cells are CHGA + In some cases, PP cell populations are derived from human PSCs (e.g., hiPSCs) or FE cell populations derived from human PSCs (e.g., hiPSCs).

[0251] As used herein, "pluripotent stem cell" or "PSC" means a cell, under specific conditions, capable of differentiating into more than one cell type, and preferably into a cell type characteristic of all three germ cell layers. Pluripotent cells are primarily characterized by, for example, nude mouse teratoma formation assays. Pluripotency is also demonstrated by the expression of embryonic stem cell (ESC) markers, although the preferred test for pluripotency is confirming the ability to differentiate into cells of each of the three germ layers. It should be noted that simply culturing such cells does not make them pluripotent. Reprogrammed pluripotent cells (e.g., iPSCs) also possess the ability to prolong passage without loss of growth potential, relative to primary cell parents that generally have only a limited number of divisions in culture. As used herein, "stem cell" can refer to stem cells capable of generating all cell types of an organism. Thus, pluripotent stem cells can generate cells of all germ layers (e.g., endoderm, mesoderm, and ectoderm). Pluripotent cells may be able to form teratomas and can promote ectodermal, mesodermal, or endoderm tissues in a living organism. Furthermore, pluripotent stem cells can refer to any pluripotent stem cell, regardless of its derivative. That is, the term pluripotent stem cells can encompass the terms embryonic stem cells and iPSCs, as well as the term embryonic germline stem cells (EGSCs). PSCs can be in the form of established cell lines, which can be obtained directly from primary embryonic tissues or can be derived from autologous cells.

[0252] As used herein, “multi-hormone cells” or “PHC” refers to cells that express (i.e., produce and / or secrete) at least GCG and INS, and whose GCG and INS expression can be analyzed by flow cytometry. + / INS + ) or the expression of GCG and CPEP (GCG) + / CPEP + Therefore, unless otherwise stated, the term GCG is used for detection. + / INS + The terms "cells" and "GCG+ / CPEP+ cells" are used interchangeably in this article to refer to the same type of multi-hormone cells.

[0253] As used in this article, “potency” in SC-IC refers to cellular INS content, INS release / secretion, GSIS and / or expression of markers similar to those in natural human β cells.

[0254] As used herein, “primitive gut” or “PGT” means one or more cells derived from DE, wherein PGT cells express at least one of the following markers: FOXA2, GATA4, HNF1β and hepatocyte nuclear factor 4α (HNF4α), especially FOXA2.

[0255] As used herein, “PGT cell population” means a cell population obtained by: (i) differentiation of pluripotent stem cells (e.g., iPSCs) using any method known in the art or as described herein, or (ii) differentiation of a precursor cell population derived from PSCs (e.g., a DE cell population as defined herein). The majority of cells in a PGT cell population are PGT cells (e.g., expressing one or more PGT cell markers listed above), but the population may also include lower and / or differentiated cells (e.g., DE and / or FE cells) and / or other cell types (e.g., off-target cells). In some cases, a PGT cell population contains more than about 70%, 80%, 90%, or 95% PGT cells. In some cases, 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 cases, the PGT cell population is derived from human PSCs (e.g., hiPSCs) or from DE cell populations derived from human PSCs (e.g., hiPSCs).

[0256] As used herein, "progenitor cell" and "precursor cell" are used interchangeably and refer to cells with a more primitive cellular phenotype (e.g., at an earlier stage in the developmental pathway or process than fully differentiated cells) relative to the cells they can differentiate into. Typically, progenitor cells can have significant or very high proliferative potential. Depending on the developmental pathway and the environment in which the cell develops and differentiates, progenitor cells can produce multiple distinct differentiated cell types or a single differentiated cell type.

[0257] As used herein, "proliferative cell" or "proliferating cell" means a cell that undergoes active cell division to produce two daughter cells. Proliferative cells can be produced by any means known in the art, for example, by expressing Ki67 (i.e., Ki67). + Identification was performed using cells.

[0258] As used herein, “reprogramming factor” refers to one or more molecules associated with cellular “reprogramming” (i.e., differentiation, and / or dedifferentiation and / or transdifferentiation), resulting in the transformation of cells into different cell types or phenotypes. Reprogramming factors generally affect gene expression 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.

[0259] As used herein, “ROCK inhibitor” means a compound, such as a small molecule compound, that inhibits one or both of the Rho kinase family members ROCK1 and ROCK2. Exemplary ROCK inhibitors that may be used in the differentiation methods described herein include, but are not limited to, sema1, 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).

[0260] As used in this article, "SANT-1" refers to a small molecule compound called N-[(3,5-dimethyl-1-phenyl-1H-pyrazol-4-yl)methylene]-4-(phenylmethyl)-1-piperazinamine (C 23 H 27 N5 (CAS No. 304909-07-7). SANT-1 is a cell-permeable SHH signaling pathway antagonist that binds to Smoothened, a distant relative of the G protein-coupled receptor.

[0261] As used herein, “sound hedgehog factor signaling pathway inhibitor” or “SHH signaling pathway inhibitor” means a compound that can inhibit the SHH signaling pathway, such as a small molecule compound. Exemplary SHH signaling pathway inhibitors applicable to the differentiation methods described herein include cyclopamine, glasdegib, saridegib, sonedegib, and vismodegib.

[0262] As used herein, "stem cell" or "SC" refers to a cell capable of self-renewal and differentiation into another cell with a higher degree of differentiation. Stem cells can be characterized by both the presence and absence of specific markers (e.g., RNA, proteins, etc.). Stem cells can also be identified by both in vitro and in vivo functional assays, particularly assays related to the ability of stem cells to produce multiple differentiated progeny. Examples of stem cells include, but are not limited to, totipotent stem cells, pluripotent stem cells, and multipotent stem cells.

[0263] As used herein, "stem cell-derived islet-like cells" and "SC-IC" refer to cells derived from, for example, stem cells such as ESCs or iPSCs, that possess characteristics similar to one of the different endocrine cell types present in natural islet cells (e.g., expressing markers characteristic of the different endocrine cell types). Examples of SC-ICs include PALCs, PBLCs, delta-like cells (PDLCs), ε-like cells (EDLCs), and γ-like cells (PGLCs). In some cases, SC-ICs can be immature or precursor SC-ICs (i.e., having a less mature phenotype than the corresponding natural endocrine cell type).

[0264] As used herein, "mature stem cell-derived islet-like cells" and "mature SC-IC" refer to SC-IC cell types that possess phenotypic and functional characteristics more closely resembling those of the corresponding cell types in natural human islets compared to immature SC-ICs (e.g., SC-ICs obtained by culturing PEP cells in PEP differentiation medium for approximately 4 or 5 days). In some cases, mature SC-ICs are mature PBLCs (e.g., those exhibiting at least one marker indicating pancreatic β-cells (e.g., PDX1 or NKX6.1), expressing INS, and demonstrating the GSIS response to glucose challenge characteristic of endogenous mature pancreatic β-cells). In other cases, mature SC-ICs are mature PALCs (e.g., those exhibiting a single hormone GCG). + cell).

[0265] As used herein, “mature SC-IC population” means a cell population obtained by: (i) differentiation of pluripotent stem cells (e.g., iPSCs) using any method known in the art or as described herein, or (ii) differentiation of a precursor cell population derived from PSCs (e.g., a precursor SC-IC population as defined herein). In some cases, the majority of cells in a mature SC-IC cell population are mature SC-ICs (i.e., expressing one or more of the markers of native, mature human islet cells listed below) and include mature PBLCs and mature PALCs. In some cases, a mature SC-IC population includes cells with lower degrees of differentiation (e.g., immature PBLCs and immature PALCs) and may include other cell types (e.g., off-target cells such as proliferating cells and ECLCs). In some cases, a 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 cases, a mature SC-IC population is characterized as containing at least about 99.5% CHGA. + Cells, at least approximately 60% CPEP + / GCG -Cells, at least approximately 50% CPEP + / NKX6.1 + Cells, at least about 70% of INS + / SLC18A1 - Cells; less than approximately 12% INS - / SLC18A1 + Cells and less than about 4% Ki67 - Cells, and optionally at least about 325 nU / cell INS content. In some cases, the mature SC-IC population is derived from human PSCs (e.g., hiPSCs) or from precursor SC-IC populations derived from human PSCs (e.g., hiPSCs).

[0266] Exemplary markers of native, mature human pancreatic alpha (α) cells include, but are not limited to, GCG and ARX, but exclude PDX1, NKX6.1, or INS. Other examples of markers of native, mature human pancreatic α cells include, but are not limited to, expression of GCG, secretion of GCG, and / or display of stimulus-response similar to that of native, mature human pancreatic α cells; and expression or secretion of GCG, GLP-1, GRL, INS, PPP, and / or SST similar to those of native, mature human pancreatic α cells. Other markers of native, mature human pancreatic α cells include expression of ARX and MAFB, but not PDX1 and NKX6.1. Another marker of native, mature human pancreatic α cells is the expression of prohormone convertase PC2.

[0267] Examples of markers for 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-converting enzyme 2 (PC2), PC1 / 3, PDX1, urocortin 3 (UCN3), and zinc transporter 8 (ZnT8), especially INS, NKX6.1, and / or CPEP, without GCG. Other examples of markers for native, mature human pancreatic β cells include expression of INS, secretion of INS, and / or display of a GSIS response similar to that of native, mature human pancreatic β cells; and expression or secretion of GCG, GRL, INS, PPP, and / or SST similar to those of native, mature human pancreatic β cells.

[0268] Exemplary markers of natural, mature human pancreatic delta (δ) cells include, but are not limited to, expressing SST, secreting SST, and / or exhibiting a response to stimuli similar to that of natural, mature human pancreatic δ cells; and expressing or secreting GCG, GRL, INS, PPP, and / or SST similar to those of natural, mature human pancreatic δ cells.

[0269] Exemplary markers of native, mature human pancreatic epsilon (ε) cells include, but are not limited to, expression of GRL, secretion of GRL and / or display of a response to a stimulus similar to that of native, mature human pancreatic ε cells; and expression or secretion of GCG, GRL, INS, PPP and / or SST similar to those of native, mature human pancreatic ε cells.

[0270] Exemplary markers of natural, mature human pancreatic gamma (γ) cells include, but are not limited to, expressing PPP, secreting PPP, and / or exhibiting a response to stimuli similar to that of natural, mature human pancreatic γ cells; and expressing or secreting GCG, GRL, INS, PPP, and / or SST similar to those of natural, mature human pancreatic γ cells.

[0271] As used herein, “precursor SC-IC” or “immature SC-IC” refers to cells derived from PEP cells and functionally less mature than mature SC-ICs of the same cell type. In some cases, precursor (immature) SC-ICs may express at least one of the following markers: CHGA, CPEP, GCG, GHRL, INS, PPP, SST, ARX, HB9, ISL1, NeuroD1, NKX2.2, NKX6.1, PAX4, PAX6, and PDX1, especially GCG, CPEP, and / or INS. In some cases, precursor (immature) SC-ICs are immature PBLCs or immature PALCs, each as defined above.

[0272] As used herein, the terms “precursor SC-IC population” and “immature SC-IC population” are used interchangeably to refer to a cell population obtained by: (i) differentiation of pluripotent stem cells (e.g., iPSCs) by any method known in the art or as described herein, or (ii) differentiation of a precursor cell population derived from PSCs (e.g., a PEP cell population as defined herein). In some cases, many cells in a precursor SC-IC population are immature SC-ICs (i.e., expressing one or more of the precursor SC-IC markers listed above) and include immature PBLCs and immature PALCs. In some cases, a precursor SC-IC population includes cells with lower and / or higher differentiation levels (e.g., PEP cells and / or mature PBLCs and mature PALCs) and may include other cell types (e.g., off-target cells such as proliferating cells and ECLCs). In some cases, a precursor SC-IC population includes a larger percentage of immature PBLCs than mature PBLCs, and / or a larger percentage of immature PALCs than mature PALCs. In some cases, the precursor SC-IC population accounts for approximately 40% to approximately 90% of INS. + / SLC18A1 - (Or alternatively, less than about 25% INS) - / SLC18A1 + ), approximately 35% to approximately 80% of CPEP + / GCG - (Or alternatively, less than about 10% to about 40% of CPEP) + / GCG + ), and / or approximately 90% to approximately 100% CHGA + / Ki67 - In some cases, the precursor SC-IC population obtained by culturing PEP cell populations as described herein can be characterized by flow cytometry as comprising approximately 50% to approximately 85% INS. + / SLC18A - Cells; ≤25% INS - / SLC + Cells, approximately 40% to approximately 75% CPEP + / GCG - Cells; CPEP + / GCG + Cells; and approximately 95% to 100% CHGA + / Ki67 - Cells. In some cases, the precursor SC-IC population is derived from human PSCs (e.g., hiPSCs) or from PEP cell populations derived from human PSCs (e.g., hiPSCs).

[0273] As used herein, the term "supplemented" means that a supplement has been added to the raw materials to obtain the final material. Unless otherwise expressly stated, it is not necessary to add one or more supplements at a specific time or in a specific order. The term "supplemented" does not preclude the addition of other supplements to the raw materials at any point in time before or after the supplementation described herein. Unless otherwise expressly stated, supplements are added to the culture medium or differentiation medium in a "substantially pure" form. The term "substantially pure" indicates that the supplement is substantially free of its components that are naturally occurring in nature. For example, substantially pure albumin can be purified albumin or recombinant albumin.

[0274] As used herein, "anchor polymerase 1 / 2 inhibitors" refers to compounds such as small molecules that bind to anchor polymerases 1 and / or 2 and antagonize the Wnt signaling pathway by stabilizing axonal proteins and promoting β-catenin degradation. Anchor polymerase 1 (TNKS1 / ARTD5 / PARP5a) and anchor polymerase 2 (TNKS2 / ARTD6 / PARP5b) form a distinct subgroup of polymers that constitute ARTDs. TNKS1 and TNKS2 share 82% sequence identity and are distinguished from the remaining members of the family by a unique domain structure containing several anchorin repeats and a sterile α motif (SAM). In some cases, anchor polymerase 1 / 2 inhibitors inhibit the binding of substrates to the nicotinamide or adenosine subsites, or both, of anchor polymerase 1 and / or anchor polymerase 2. Exemplary end-anchored polymerase 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 cases, end-anchored polymerase 1 / 2 inhibitors bind to the nicotinamide pocket of end-anchored polymerases 1 and 2, and this can be XAV939. In other cases, end-anchored polymerase 1 / 2 inhibitors do not bind to the nicotinamide pocket, but rather to the adenosine subsite of the catalytic domain in end-anchored polymerases 1 and 2. Such adenosine subsite-specific binding end-anchored polymerase 1 / 2 inhibitors include those by Mariotti et al. (2017). Brit. J. Pharmacol. The various adenosine-binding site compounds listed in Table 1 of 174:461-4636, Haikarainen et al. (2014) Curr. Pharm. Des. The ADE subsite binding compounds shown in Figure 5 for 20:6472-6488, and Leenders et al. (2021). J. Med. Chem.Inhibitors based on the 1,2,4-triazole scaffold disclosed in Table 1 of 64:17936-17040. Exemplary adenosine subsite-binding compounds described in these references include Haikarainen et al., G007-LK, IWR-1, JW55, CMP4, CMP24, and CMP40 in Figure 5 above; Mariotti et al., compound number 15 based on JW74 in Table 1 above; K-756; OM-153; OM-1700; Mariotti et al., oxazolidinones and oxazolidinone-based compound 20 in Table 1 above; and WIKI4. In some cases, end-anchored polymerase 1 / 2 inhibitors bind to the adenosine subsites of end-anchored polymerases 1 and 2, and also interact with the G ring (see, Haikarainen et al. (2013)). PLoS One 8:e65404). Inhibitors of these adenosine subsite / G-ring interaction-mediated end-anchored polymerase 1 and 2 include WIKI4.

[0275] As used in this article, "totipotent stem cell" or "TSC" refers to a cell that has the ability to self-renew and differentiate into another type of cell with a higher degree of differentiation.

[0276] As used herein, "TPPB" refers to the small molecule compound known as 2S,5S-E,E-8-5-4-trifluoromethylphenyl-2,4-pentadienylaminobenzolactam (CAS No. 497259-23-1). Also known as PKC activator V, TPPB is a cell-permeable benzolactam-derived PKC activator (regarding PKCα,K...). i = 11.9 nM), which effectively enhances the non-amyloid α-processing of amyloid precursor protein (APP). TPPB is commercially available from, for example, Millipore Sigma.

[0277] As used herein, “trace element A supplement” refers to a liquid composition comprising one, two, three, or all four of the following: copper sulfate, ferric citrate, selenium (e.g., sodium selenite), and zinc sulfate. Similarly, “trace element B supplement” refers to a liquid composition comprising one, two, three, four, five, six, or all seven of the following: ammonium molybdate, ammonium vanadate, manganese sulfate, nickel sulfate, sodium silicate, stannous chloride, and hydrochloric acid. In some cases, trace element A and B supplements are each a 100x concentrated solution. The compositions of exemplary 100x and 1x trace element A and B supplements are shown in Tables 25 and 26 below, respectively. Concentrated trace element A (100x) and trace element B (100x) supplements are available, for example, from Fisher Scientific.

[0278] As used herein, “treat” or “treating” means, for example, the act of providing care to an individual in need by administering a therapeutic agent (e.g., SC-IC or a composition comprising thereto) for the purpose of improving the individual’s health and / or well-being with respect to an existing condition (e.g., disease, disorder) or preventing or reducing the likelihood of the condition occurring. Treatment may also involve reducing the frequency or severity of at least one sign, symptom, or contributing factor of a condition (e.g., disease or symptom) experienced by the individual.

[0279] As used herein, “triiodo-L-thyroxine,” “triiodothyroxine,” and “T3” are used interchangeably to refer to thyroid hormones having the chemical names and structures shown in Table 15. T3, also known as iodoxylone, is commercially available from, for example, Millipore Sigma.

[0280] As used herein, "trolox" refers to 6-hydroxy-2,5,7,8-tetramethyl-2-carboxylic acid (C6H2O) 14 H 18 Trolox is a small molecule compound (O4; CAS No. 53188-07-1). It is a cell-permeable, water-soluble derivative of vitamin E with potent antioxidant properties. Trolox is commercially available from sources such as Millipore Sigma.

[0281] As used herein, "retinoids" refers to vitamin A compounds and vitamin A synergists. Retinoids include, but are not limited to, retinol, retinal, retinoic acid, beta-carotene, isotretinoin, retinoic acid (also known as ATRA), retinoic acid, acitretin ester and its metabolite acitretin, as well as retinoid benzoic acid derivatives such as adapalene, bexarotine and tazarotene.

[0282] As used herein, “thiol-based antioxidants” means compounds containing a thiohydrogen (SH) side chain group or a disulfide bond and acting as antioxidants. Exemplary thiol-based antioxidants used herein include, but are not limited to, cysteine, NAC, cystine, and cystine analogues such as N,N′-diacetyl-L-cysteine ​​(DiNAC) (CAS No. 5545-17-5) and N,N′-diacetyl-L-cysteine ​​dimethyl ester (DACDM) (CAS No. 32381-28-5).

[0283] As used herein, “TPPB” refers to 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 (for PKCα, K...). i=11.9 nM), which induces PSCs to differentiate into pancreatic progenitor cells expressing Pdx-1. TPPB is commercially available from, for example, Millipore Sigma.

[0284] As used herein, “urinary corticosteroid 3” and “UCN3” refer to peptide hormones expressed in mature endogenous pancreatic β cells. UCN3, a member of the corticotropin-releasing factor (CRF) family (which selectively binds to the G protein-coupled receptor CRFR2), is co-released with INS under hyperglycemic conditions and stimulates somatostatin secretion from delta cells, the main cells in the pancreas that express CRFR2.

[0285] As used herein, “vitamin B3 compound” means (a) nicotinic acid, nicotinamide, nicotinic acid and / or nicotinamide nucleoside; (b) nicotinamide derivatives and analogs having substantially similar activity to nicotinamide in the differentiation methods described herein; and (c) pharmaceutically acceptable salts of any of the compounds listed in (a) and (b).

[0286] As used herein, “vitamin C compound” means (a) ascorbic acid; (b) ascorbic acid analogs or derivatives and analogues having substantially similar activity to ascorbic acid in the differentiation methods described herein; and (c) pharmaceutically acceptable salts of the compounds in (a) and (b). In some cases, vitamin C compound is any one of dehydroascorbic acid and its pharmaceutically acceptable salts, ascorbate phosphate and its pharmaceutically acceptable salts, sodium ascorbate, calcium ascorbate, zinc ascorbate, and nicotinamide ascorbate. Dehydroascorbic acid (chemical names and structures are shown in Table 15) is prepared by oxidation of ascorbic acid. Ascorbate phosphate is the synthetic form of vitamin C and is found in various salt forms such as magnesium ascorbate phosphate and sodium ascorbate phosphate (chemical names and structures are shown in Table 15).

[0287] As used herein, “Wnt-3a” and “Wnt3a” refer to protein 3A, a member of the mammalian wingless MMTV integration site family. In some cases, 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).

[0288] As used herein, “WNT / β-catenin signaling pathway activator” means a compound, such as a small molecule or protein, that can activate the signaling pathway at a similar level as that achieved by Wnt3 ligands (e.g., Wnt3a, Wnt1, Wnt3a, and cytokinin).

[0289] As used herein, “Wnt inhibitor kinase inhibitor 4” and “WIKI4” refer to small molecule compounds having the chemical names and structures shown in Table 15. WIKI4, also known as end-anchored polymerase 1 / 2 inhibitor V, is a selective inhibitor of TNKS1 and TNKS2 (IC50, IC50, and IC50, respectively). 50 = 26 and 15 nM). Through its action on TNK2, WIKI4 prevents AXIN ubiquitination and degradation, and inhibits signal transduction via the Wnt / β-catenin pathway. WIKI4 is commercially available from, for example, Cayman Chemical (AnnArbor, MI USA).

[0290] As used herein, “Y-27632” refers to a small molecule compound having the chemical names and structures shown in Table 15 and its pharmaceutically acceptable salts. 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 cases, the methods described herein use the dihydrochloride of Y-27632 (Y-27632 2HCl), which is commercially available from, for example, Selleck Chemicals.

[0291] As used in this article, "zinc compound" means containing zinc. 2+ (Zn 2+ Small molecule compounds. In some cases, zinc compounds that can be used in the differentiation methods described herein are Zn. 2+ Zinc salts (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 cases, the zinc compounds are pharmaceutically acceptable zinc salts.

[0292] method Methods for differentiating stem cells into SC-IC Pluripotent cell lines: Differentiation can begin with stem cells, especially pluripotent stem cells such as iPSCs. iPSCs can be derived from many different cell types, including terminally differentiated cells (i.e., somatic cells). In some cases, iPSCs are derived from human cell types.

[0293] iPSCs can be generated via any reprogramming method known in the art for somatic cells. See, for example, U.S. Patent Application Publication Nos. 2009 / 0047263, 2009 / 0068742, 2009 / 0191159, 2009 / 0227032, 2009 / 0246875, and 2009 / 0304646. Generally, to generate iPSCs, somatic cells are supplied with reprogramming factors (e.g., Oct4, SOX2, KLF4, MYC, Nanog, Lin28, etc.; see, for example, Takahashi et al. (2007), ibid., and Yu et al. (2007), ibid.). Alternatively, iPSCs may be obtained from commercial suppliers, including but not limited to Cell and GeneTherapy Catapult (London, UK), FujiFilm Cellular Dynamics, Inc. (Madison, WI, USA), Healios KK (Tokyo, Japan), and Lonza Group Ltd. (Basel, Switzerland).

[0294] In other cases, the differentiation method may begin with ESC or EGSC (e.g., human ESC or human EGSC).

[0295] PSC Amplification: Although their renewal capacity is presumed to be immortal, it is known that stress on PSCs during in vitro culture leads to genetic alterations that can ultimately impair the cell line's ability to "perform" as needed. Therefore, the characterization, amplification, and banking of PSCs can be necessary. Here, PSC lines, such as iPSC lines (e.g., hiPSC lines), can be cultured in two-dimensional (2D) cultures with >approximately 98% Oct4. + / Nanog + The cell population expands in approximately 3 to 5 days. In some cases, the cell count can reach approximately 150,000 cells / cm³. 2 Approximately 500,000 cells / cm² 2 The density. In some cases, the cell density can be as follows: approximately 175,000 cells / cm³. 2 Approximately 475,000 cells / cm² 2 Approximately 200,000 cells / cm³ 2 Approximately 450,000 cells / cm² 2 Approximately 225,000 cells / cm³ 2 Approximately 425,000 cells / cm² 2 Approximately 250,000 cells / cm³ 2 Approximately 400,000 cells / cm² 2Approximately 275,000 cells / cm³ 2 Approximately 375,000 cells / cm² 2 Approximately 300,000 cells / cm³ 2 Approximately 350,000 cells / cm² 2 or approximately 325,000 cells / cm³ 2 In some cases, cells can be at a density of approximately 250,000 cells / cm³. 2 275,000 cells / cm 2 300,000 cells / cm 2 325,000 cells / cm 2 350,000 cells / cm 2 375,000 cells / cm³ 2 400,000 cells / cm 2 425,000 cells / cm 2 and 450,000 cells / cm 2 Especially approximately 350,000 cells / cm 2 Cells can initially be cultured in a medium containing vilinkin-N (VTN-N), such as Essential 8 (E8). The final passage before using these cells can be in a medium containing VTN-N, such as mTESR™. In some embodiments, the medium is E8 flex + Pranick.

[0296] Alternatively, the iPSC can be amplified by any method well known in the art. See, for example, International Patent Application Publication No. WO 2017 / 222879, and 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 Vander Wal et al. (2018) Stem Cell Rep. 10:1975-1990.

[0297] iPSC aggregation: This method may also include an iPSC aggregation step to increase the surface area available for cell growth per volume of culture medium. Here, approximately 0.25 x 10⁻⁶ 6 1 cell / mL to approximately 2 x 10⁻⁶ cells / mL 6 cells / mL, especially about 1 x 10⁻⁶ cells / mL. 6Expanded iPSCs at 100 cells / mL can aggregate for approximately 1 day in a spin flask, a conventional stirred tank bioreactor, or a vertical wheel bioreactor (e.g., the bioreactors in the PBS Vertical Wheel series (PBS Biotech; Camarillo, CA USA)) in a defined medium such as mTESR™, Essential 8™, Essential 8™ Flex including the ROCK inhibitor and Poloxamer 188, or Essential 8™ Flex + Pluronic. In some cases, the culture pH can range from approximately 6.6 to approximately 7.4, and the dissolved oxygen concentration can be from approximately 20 mmHg to approximately 100 mmHg. In some cases, the stirring rate in the vertical wheel bioreactor can be any of approximately 20 rpm, 30 rpm, 40 rpm, and 50 rpm, but at most approximately 60 rpm. Depending on the choice of aggregation medium used, the final aggregate morphology can range from approximately 50 μm to approximately 170 μm. In some cases, the culture can be controlled at a temperature of about 36°C to about 38°C, especially about 37°C. In some cases, air, carbon dioxide, and oxygen are supplied to the bioreactor via passive diffusion, active covering, or injection.

[0298] In some cases, the ROCK inhibitor is Y-27632 (e.g., Y-27632 2HCl), which can be at concentrations from about 1 μM to about 20 μM. In some cases, Y-27632 2HCl can be at concentrations of: 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 still other cases, the ROCK inhibitor can be at concentrations 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, particularly about 10 μM. Other suitable ROCK inhibitors include, but are not limited to, Seman 1, Tiazovivan, Fasudil / HA1077, and H-1152.

[0299] Alternatively, iPSCs can be collected via any method well known in the art. See, for example, International Patent Application Publication No. WO 2017 / 222879, and Kwok et al. (2022). Reprod. Toxicol. 112:22-35, Manstein et al. (2021) Stem Cells Trans. Med10-7:1063-1080, Marotta et al. (2022) Methods Mol. Biol. 2454:1-15, Mesquita et al. (2021) Stem Cell Biol. 5:209-229 and Vander Wal et al. (2018) Stem Cell Rep. 10:1975-1990.

[0300] Differentiation: After cells from pluripotent stem cell lines such as iPSCs aggregate, they are subjected to one or more differentiation steps, which typically involve culturing the cell population in vitro in one or more differentiation media, including a basal cell culture medium (e.g., serum-free medium) and one or more molecules (referred to herein as “differentiation factors”) to promote differentiation of cells from one cell type to a more differentiated cell type. Differentiation steps can be performed in the order described below, or can begin and proceed from any particular stage. That is, in some cases, the differentiation approach can begin with pluripotent stem cells such as iPSCs; however, in other cases, the approach can begin with a more differentiated cell type such as PP cells or PEP cells, and proceed from there. In some cases, there may be seven differentiation stages. In other cases, there may be fewer than seven differentiation stages, such as six, five, four, three, two, or even one differentiation stage. In some cases, any differentiation stage may include two or more sub-stages. In some cases, the cell population generated in the first sub-stage of the differentiation phase may have one or more different characteristics compared to the cell population generated in the next sub-stage (e.g., the cell population generated in the second sub-stage may be more differentiated (i.e., more mature) than the cell population generated in the first sub-stage). In some cases, the cell population generated in the first sub-stage is washed in a defined-component culture medium before proceeding to the second sub-stage.

[0301] In some cases, one or more of the differentiation stages described herein (i.e., one or more of stages 1a, 1b, 2, 3 and 4) may be performed by methods well known in the art, and / or may include alternative differentiation factors and culture techniques well known in the art. Exemplary methods, differentiation factors, and culture techniques used in each stage of the PSC to SC-IC differentiation scheme are described in the following published patent applications: International Patent Application Publications Nos. WO2003 / 050249, WO 2013 / 192005, WO 2014 / 105543, WO 2015 / 028614, WO 2016 / 100930, WO2017 / 222879, WO 2019 / 048690, WO 2019 / 099725, WO 2019 / 169351, WO 2019 / 227198, WO2020 / 033879, WO 2022 / 026932, WO 2023 / 077140, and WO 2023 / 133568.

[0302] Any differentiation medium described herein may optionally include antibiotics to minimize the risk of bacterial contamination of the cell cultures. When used, the antibiotic may be a penicillin-streptomycin solution at a concentration of 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 cases, the concentration of the penicillin-streptomycin solution in the differentiation medium may be any one 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%.

[0303] Phase 1a cells and cell populations (iPSCs to ME cells): In some cases, the differentiation approach may begin with or may include differentiating PSCs (e.g., iPSCs) into ME cells by: culturing a PSC population (e.g., including Oct4 cells) in a PSC differentiation medium. + / Nanog + The iPSC PSC group), lasting approximately 1 day (e.g., Figure 1B The time period (day 0) is used to obtain a cell population that includes ME cells (e.g., an ME cell population includes TBXT+ cells and / or MIXL1+ cells). In some cases, the PSC population consists primarily of hiPSCs.

[0304] In some cases, the cultivation of PSC populations is performed in bioreactors and includes approximately 3 x 10⁻⁶ cells / years. 5 1 cell / mL to approximately 2 x 10⁻⁶ cells / mL 6Cell transfer density of cells / mL. In some cases, the culture pH range can be from about 6.6 to about 7.4. In other cases, the 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 cases, dissolved oxygen can be controlled at a concentration of about 20 mmHg to about 60 mmHg. In some cases, depending on the size of the bioreactor, the stirring rate can be from about 20 rpm to about 60 rpm. In some cases, the culture is controlled at a temperature of about 36°C to about 38°C, especially about 37°C. In some cases, air, carbon dioxide, and oxygen are supplied to the bioreactor via passive diffusion, active covering, or jetting.

[0305] PSC differentiation medium: PSC differentiation medium may include a defined component medium comprising glucose and / or fructose and one or more of the following: glutamine (e.g., glutamine dipeptide), albumin (e.g., BSA or HSA), buffer (e.g., NaHCO3), serum replacement supplements (e.g., ITS-G or ITS-X supplements described herein), TGF-β superfamily growth factors (e.g., activin A), Wnt / β-catenin pathway signaling activator (Wnt3a protein), GSK-3α and GSK-3β inhibitors / Wnt pathway signaling activators (e.g., CHIR99021), and ROCK inhibitors (e.g., Y-27632). In some cases, PSC differentiation medium includes MCDB medium as described in Table 16 below.

[0306] In some cases, the PSC differentiation medium contains glucose at concentrations ranging from approximately 5 mM to 20 mM. In some cases, the glucose concentration may be approximately 6 mM to approximately 19 mM, approximately 8 mM to approximately 17 mM, approximately 10 mM to approximately 15 mM, or approximately 12 mM to approximately 13 mM. In some cases, the glucose concentration in the PSC differentiation medium may be approximately 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 cases, the PSC differentiation medium contains approximately 12 mM glucose.

[0307] In some cases, the PSC differentiation medium includes glutamine, which may be in the form of a dipeptide compound containing glutamine (e.g., L-alanine-L-glutamine or glycyl-L-glutamine). In some cases, the PSC differentiation medium includes L-alanine-L-glutamine at a concentration of about 1 mM to about 4 mM. In some cases, the concentration of L-alanine-L-glutamine is about 1.25 mM to about 3.5 mM, about 1.5 mM to about 3 mM, or about 1.75 mM to about 2.25 mM. In some cases, each PSC differentiation medium includes about 2 mM L-alanine-L-glutamine.

[0308] In some cases, the PSC differentiation medium comprises albumin (e.g., serum albumin or recombinant albumin as described herein) at a concentration of about 0.05% to about 2%. In some cases, the albumin may be recombinant human albumin, which may be provided in a composition comprising a mixture of fatty acids and / or lipids. In some cases, the PSC differentiation medium comprises FAF-BSA or FAF-HSA at a concentration of about 0.05% to about 1%. In some cases, the concentration of FAF-BSA or FAF-HSA may 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 cases, the concentration of FAF-BSA or FAF-HSA in the PSC differentiation medium may be approximately 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 cases, the PSC differentiation medium includes approximately 0.2% FAF-BSA or approximately 0.2% FAF-HSA.

[0309] In some cases, the PSC differentiation medium includes a buffer, which may be NaHCO3 at a concentration of about 25 mM to about 60 mM. In some cases, the NaHCO3 concentration in the PSC differentiation 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 cases, the NaHCO3 concentration may be about 42 mM, 43 mM, 44 mM, 45 mM, 46 mM, 47 mM, or 48 mM. In some cases, the PSC differentiation medium includes about 45.2 mM NaHCO3 (3.8 g / L NaHCO3).

[0310] In some cases, the PSC differentiation medium includes a serum replacement supplement, which may include one, two, three, or all four of INS, transferrin, selenium (e.g., sodium selenite), and ethanolamine. In some cases, the serum replacement supplement comprises INS, transferrin, and sodium selenite, which may be provided in a concentrated ITS-G supplement (e.g., as defined herein). In some cases, the serum replacement supplement comprises all four components, which may be provided in a concentrated ITS-X supplement (e.g., as defined herein). In some cases, the serum replacement supplement in the PSC differentiation medium is an ITS-X (100x) solution having the composition shown in Table 23. In some cases, the concentration (v:v) of the ITS-X (100x) solution in the PSC differentiation medium may be from about 1:1000 to about 1:8000. In some cases, ITS-X (100x) solutions may be present at concentrations (v:v) of approximately 1:1500 to approximately 1:7500, approximately 1:2000 to approximately 1:7000, approximately 1:2500 to approximately 1:6500, approximately 1:3000 to approximately 1:6000, approximately 1:3500 to approximately 1:5500, or approximately 1:4000 to approximately 1:5000. In some cases, the concentration (v:v) of the ITS-X (100x) supplement is any one of approximately 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 cases, the PSC differentiation medium comprises the ITS-X (100x) solution shown in Table 23 at a concentration of about 1:5500 (v:v).

[0311] In some cases, the PSC differentiation medium includes TGF-β growth factor, which may be activator A at a concentration of about 50 ng / mL to about 300 ng / mL. In other cases, the concentration of activator A in the PSC differentiation medium may 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 cases, the concentration of activin A can be approximately 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 cases, the PSC differentiation medium includes approximately 200 ng / mL of activin A. Other suitable TGF-β growth factors include, but are not limited to, growth differentiation factor 8 (GDF8).

[0312] In some cases, the PSC differentiation medium includes a Wnt pathway signaling activator, which may be Wnt3a protein as defined herein, at a concentration of about 5 ng / mL to about 20 ng / mL. In some cases, the Wnt3a protein is recombinant human Wnt3a. In some cases, the Wnt3a protein is recombinant mouse Wnt3a. In some cases, the concentration of Wnt3a protein in the PSC differentiation medium may 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 cases, the concentration of Wnt3a protein in the PSC differentiation medium may 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 cases, the PSC differentiation medium contains approximately 12.5 ng / mL of recombinant mouse Wnt3a protein or recombinant human Wnt3a protein.

[0313] In some cases, the PSC differentiation medium includes GSK-3α and GSK-3β inhibitors / Wnt pathway signaling activators, which may be CHIR99021 at concentrations of approximately 1 μM to 5 μM. In some cases, the concentration of CHIR99021 in the PSC differentiation medium may be approximately 1 μM, 2 μM, 3 μM, 4 μM, or 5 μM. In some cases, the PSC differentiation medium includes approximately 3 μM CHIR99021. Other suitable GSK-3α and GSK-3β inhibitors / Wnt pathway signaling activators include, but are not limited to, 6-bromoindorubin-3′-oxime (BIO).

[0314] In some cases, the PSC differentiation medium includes a ROCK inhibitor, which may be Y-27632 at a concentration of about 5 μM to about 15 μM (e.g., Y-27632 2HCl). In some cases, the concentration of Y-27632 in the PSC differentiation medium may be about 6 μM to about 14 μM, about 8 μM to about 12 μM, or about 10 μM. In some cases, the concentration of Y-27632 in the PSC differentiation medium may 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 cases, the PSC differentiation medium includes about 10 μM Y-27532 (e.g., Y-27632 2HCl). Other suitable ROCK inhibitors include, but are not limited to, Seman 1, Tiazovivan, Fasudil / HA1077, and H-1152.

[0315] In some cases, the PSC differentiation medium comprises: (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) ITS-X (100x) solution at a ratio of about 1:4000 to about 1:6000 (e.g., the composition shown in Table 23); (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 cases, the PSC differentiation medium also includes MCDB medium as shown in Table 16 below.

[0316] In some cases, the PSC differentiation medium includes MCDB medium as shown in Table 16, approximately 12 mM glucose, approximately 0.25% FAF-BSA or FAF-HSA, approximately 45.2 mM NaHCO3, ITS-X (100x) solution as shown in Table 23 at a ratio of approximately 1:5000, approximately 200 ng / mL activin A, approximately 12.5 ng / mL recombinant mouse Wnt3a or a corresponding concentration of recombinant human Wnt3a, approximately 3.0 μM CHIR99021, and approximately 10 μM Y-27632 (e.g., Y-27632 2HCl).

[0317] Alternatively, PSCs can be differentiated into ME cells by any method known in the art.

[0318] Stage 1b cells and cell populations (ME to DE): Differentiation methods may begin with or include differentiating ME cells into DE cells by culturing a population of ME cells in ME differentiation medium for approximately 1 day (i.e., Figure 1B Day 1 of the program), to obtain Sox17 + DE cell population. In some cases, the method uses a ME cell population obtained by: (a) performing the Phase 1a differentiation method, or (b) differentiating PSCs into ME cells by any method known in the art. In some cases, the method includes washing the ME cell population in washing medium before culturing in ME differentiation medium.

[0319] In some cases, the culture of ME cell populations is performed in a bioreactor and includes approximately 5 x 10⁻⁶ cells. 5 1 cell / mL to approximately 3 x 10 6 Cell transfer density of cells / mL. In some cases, the culture pH can range from about 6.6 to about 7.4. In other cases, the 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 cases, dissolved oxygen can be controlled at a concentration of about 20 mmHg to about 100 mmHg. In some cases, depending on the size of the bioreactor, the stirring rate can be from about 20 rpm to about 60 rpm. In some cases, the culture is controlled at a temperature of about 36°C to about 38°C, especially about 37°C. In some cases, air, carbon dioxide, and oxygen are supplied to the bioreactor via passive diffusion, active covering, or jetting.

[0320] Washing media may include a defined composition medium containing glucose (e.g., about 4 mM to about 7 mM) and NaHCO3 (e.g., about 10 mM to about 20 mM). In some cases, washing media may include about 5.6 mM glucose, about 14 mM NaHCO3, and MCDB medium as described in Table 16 below.

[0321] ME differentiation medium: ME differentiation medium may include a defined component medium containing glucose and / or fructose, and one or more of the following: glutamine (e.g., L-alanine-L-glutamine or glycyl-L-glutamine), albumin (e.g., BSA or HSA), buffer (e.g., NaHCO3), serum replacement supplement (e.g., ITS-G or ITS-X supplement), TGF-β superfamily growth factor (e.g., activin A), and BMP inhibitor (e.g., LDN-193189). In some cases, ME differentiation medium includes the MCDB medium described in Table 16 below.

[0322] In some cases, ME differentiation medium may include glucose at a concentration of about 5 mM to about 20 mM. In some cases, ME differentiation medium includes glucose at any of the glucose concentration ranges and concentrations described above for PSC differentiation medium. In some cases, the glucose concentration in ME differentiation medium may be about 11 mM to about 13 mM. In some cases, ME differentiation medium may include about 12 mM glucose.

[0323] In some cases, the ME differentiation medium includes glutamine, which may be in the form of a dipeptide compound containing glutamine (e.g., L-alanine-L-glutamine or glycyl-L-glutamine). In some cases, the ME differentiation medium includes L-alanine-L-glutamine at a concentration of about 1 mM to about 4 mM. In some cases, the concentration of L-alanine-L-glutamine in the ME differentiation medium can be any of the L-alanine-L-glutamine concentration ranges and concentrations described above for the PSC differentiation medium. In some cases, the concentration of L-alanine-L-glutamine in the ME differentiation medium can be about 1.75 mM to about 2.5 mM. In some cases, the ME differentiation medium includes about 2 mM L-alanine-L-glutamine.

[0324] In some cases, the ME differentiation medium includes albumin (e.g., FAF-albumin or recombinant albumin as described herein), which may be at a concentration of about 0.05% to about 2%. In some cases, the albumin in each medium may be recombinant albumin (e.g., recombinant human albumin), which may be provided in a composition containing fatty acids and / or lipids. In some cases, the albumin may be FAF-BSA or FAF-HSA, which may be at a concentration of about 0.05% to about 1%. In some cases, the concentration of FAF-albumin (e.g., FAF-BSA or FAF-HSA) in the ME differentiation medium may be selected from any of the FAF-BSA / FAF-HSA concentration ranges and concentrations described above for the PSC differentiation medium. In some cases, the concentration of FAF-BSA or FAF-HSA albumin in the ME differentiation medium may be about 0.19% to about 0.21%. In some cases, the ME differentiation medium includes about 0.20% FAF-BSA or about 0.20% FAF-HSA.

[0325] In some cases, the ME differentiation medium includes a buffer, which may be NaHCO3 at a concentration of about 24 mM to about 60 mM. In some cases, the concentration of NaHCO3 in the ME differentiation medium may be selected from any of the NaHCO3 concentration ranges and concentrations described above for PSC differentiation medium. In some cases, the NaHCO3 concentration in the ME differentiation medium may be about 43 mM to about 47 mM. In some cases, the ME differentiation medium includes about 45.2 mM NaHCO3.

[0326] In some cases, the ME differentiation medium includes a serum replacement supplement, which may include one, two, three, or all four of INS, transferrin, selenium (e.g., sodium selenite), and ethanolamine. In some cases, the serum replacement supplement includes INS, transferrin, and sodium selenite (e.g., the serum replacement supplement may be a concentrated ITS-G supplement). In some cases, the serum replacement supplement includes all four components (e.g., the serum replacement supplement may be a concentrated ITS-X supplement). In some cases, the serum replacement supplement in the ME differentiation medium is an ITS-X (100x) solution having the composition shown in Table 23. In some cases, the concentration of the ITS-X (100x) solution in the ME differentiation medium may be selected from any of the ITS-X (100x) solution concentration ranges and concentrations described above for the PSC differentiation medium. In some cases, the concentration of the ITS-X (100x) solution in the ME differentiation medium may be from about 1:4500 to about 1:5500. In some cases, the ME differentiation medium comprises an ITS-X (100x) solution as shown in Table 23 at a concentration of approximately 1:5500.

[0327] In some cases, the ME differentiation medium includes TGF-β growth factor, which may be activator A at a concentration of about 50 ng / mL to about 300 ng / mL. In some cases, the concentration of activator A in the ME differentiation medium may be selected from any of the activator A concentration ranges and concentrations described above for PSC differentiation medium. In some cases, the concentration of activator A in the ME differentiation medium may be about 180 ng / mL, 190 ng / mL, 200 ng / mL, 210 ng / mL, or 220 ng / mL. In some cases, the ME differentiation medium includes about 200 ng / mL of activator A. Other suitable TGF-β growth factors include, but are not limited to, GDF8.

[0328] In some cases, the ME differentiation medium includes a BMP inhibitor, which may be LDN-193189 at a concentration of about 5 nM to about 20 nM. In some cases, the concentration of LDN-193189 in the ME differentiation medium may 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 cases, the concentration of LDN-193189 in the ME differentiation medium may 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 cases, the ME differentiation 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).

[0329] In some cases, the ME differentiation medium comprises: (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 activator A, and (vi) about 9 nM to about 11 nM LDN-193189. In some cases, the ME differentiation medium also includes an ITS-X (100x) supplement solution at a ratio of about 1:4000 to about 1:6000 (e.g., the composition shown in Table 23). In some cases, the ME differentiation medium also includes the MCDB medium shown in Table 16 below.

[0330] In some cases, the ME differentiation medium includes MCDB medium as shown in Table 16, approximately 12 mM glucose, approximately 0.2% FAF-BSA or approximately 0.2% FAF-HSA, approximately 42.5 mM NaHCO3, approximately 200 ng / mL activator A, and approximately 10 nM DN-193189. In some cases, the ME differentiation medium also includes an ITSX (100x) solution as shown in Table 23 at a ratio of approximately 1:5000.

[0331] Alternatively, ME cells can be differentiated into DE cells by any method known in the art.

[0332] Phase 2 cells and cell populations (DE to PGT): Differentiation methods may begin with or include differentiating DE cells into PGT cells by culturing a population of DE cells in a DE differentiation medium for approximately 3 days (i.e., Figure 1B The time period (days 2-4 in the original text) is used to obtain information including FOXA2. + Cells (e.g., FOXA2) + PDX1 - The method uses a population of PGT cells (cells). In some cases, the method uses a population of DE cells obtained by: (a) performing a phase 1b differentiation method, and optionally also performing a phase 1a differentiation method, or (b) differentiating PSC or ME cells into DE cells by any method known in the art. In some cases, the method includes replacing the DE differentiation medium in the culture with fresh DE differentiation medium once or twice during this time period (i.e., about 24 hours and / or about 48 hours after the initiation of the culture step).

[0333] In some cases, the culture of DE cell populations is performed in a bioreactor and includes approximately 5 x 10⁻⁶ cells. 5 1 cell / mL to approximately 3 x 10 6 Cell transfer density of cells / mL. In some cases, the culture pH can range from about 6.6 to about 7.4. In other cases, the 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 cases, dissolved oxygen can be controlled at a concentration of about 20 mmHg to about 100 mmHg. In some cases, depending on the size of the bioreactor, the stirring rate can be from about 20 rpm to about 60 rpm. In some cases, the culture is controlled at a temperature of about 36°C to about 38°C, especially about 37°C. In some cases, air, carbon dioxide, and oxygen are supplied to the bioreactor via passive diffusion, active covering, or jetting.

[0334] DE differentiation medium. DE differentiation medium may include a defined component medium containing glucose and / or fructose, and one or more of the following: glutamine (e.g., glutamine dipeptide), albumin (e.g., FAF-BSA or FAF-HSA), buffer (e.g., NaHCO3), vitamin C compound (e.g., ascorbic acid), serum replacement supplement (e.g., ITS-G or ITS-X supplement), and growth factors from the FGF family (e.g., KGF). In some cases, DE differentiation medium includes basal A medium as described in Table 3 below.

[0335] In some cases, the DE differentiation medium includes each of glucose, L-alanine-L-glutamine, FAF-BSA (or FAF-HSA), and NaHCO3, which may be present at concentrations selected from the corresponding glucose, L-alanine-L-glutamine, FAF-BSA (or FAF-HSA), and NaHCO3 concentration ranges and concentrations described above for PSC and ME differentiation media. In some cases, the concentrations of glucose, L-alanine-L-glutamine, FAF-BSA (or FAF-HSA), and NaHCO3 in the DE differentiation medium may 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 cases, the DE differentiation medium consists of approximately 12 mM glucose, approximately 2 mM L-alanine-L-glutamine, approximately 0.2% FAF-BSA (or FAF-HSA), and approximately 45.2 mM (3.8 g / L) NaHCO3.

[0336] In some cases, the DE differentiation medium includes a vitamin C compound, which may be ascorbic acid at a concentration of about 0.05 mM to about 0.50 mM. In some cases, the concentration of ascorbic acid in the DE differentiation medium may 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 cases, the ascorbic acid concentration may 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 cases, the DE differentiation medium includes about 0.25 mM ascorbic acid. In some cases, the vitamin C compound is dehydroascorbic acid.

[0337] In some cases, the DE differentiation medium includes a serum replacement supplement, which may comprise a mixture of two, three, or all four of the following: INS, transferrin, selenium (e.g., sodium selenite), and ethanolamine. In some cases, the serum replacement supplement includes INS, transferrin, and sodium selenite (e.g., the serum substitute may be a concentrated ITS-G supplement). In some cases, the serum replacement supplement includes all four components (e.g., the serum substitute may be a concentrated ITS-X supplement). In some cases, the serum replacement supplement in the DE differentiation medium may be an ITS-X (100x) solution with the composition shown in Table 23. In some cases, the concentration (v:v) of the ITS-X (100x) solution in the DE differentiation medium may be from about 1:50 to about 1:400. In some cases, ITS-X (100x) solutions may be present at concentrations (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 cases, ITS-X (100x) supplement concentrations (v:v) may be about 1:100, 1:150, 1:200, 1:250, or 1:300. In some cases, PSC differentiation media comprise ITS-X (100x) solutions as shown in Table 23 at a concentration of about 1:200 (v:v).

[0338] In some cases, the DE differentiation medium includes FGF family growth factors, which may be KGF protein at concentrations of about 10 ng / mL to about 200 ng / mL. In some cases, the KGF protein is recombinant human KGF protein, which may be at concentrations of 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 cases, the concentration of recombinant human KGF protein in the DE differentiation medium may be approximately 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 approximately 75 ng / mL. In some cases, the DE differentiation medium includes approximately 50 ng / mL of recombinant human KGF protein. Other suitable FGF family growth factors include FGF2, FGF8B, FGF10, and FGF21. In some cases, the DE differentiation medium also includes an ITS-X (100x) solution at a ratio of approximately 1:175 to approximately 1:225 (e.g., the composition shown in Table 23).

[0339] In some cases, the DE differentiation medium comprises: (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 cases, the DE differentiation medium also includes an ITS-X (100x) solution at a ratio of about 1:175 to about 1:225 (e.g., the composition shown in Table 23). In some cases, the PSC differentiation medium also includes the MCDB A / B medium shown in Table 16 below.

[0340] In some cases, the DE differentiation medium includes MCDB medium as shown in Table 16, approximately 12 mM glucose, approximately 0.2% FAF-BSA (or FAF-HSA), approximately 45.2 mM NaHCO3, approximately 0.25 mM ascorbic acid, and approximately 50 ng / mL recombinant human KGF protein. In some cases, the ME differentiation medium also includes an ITSX (100x) solution as shown in Table 23 at a ratio of approximately 1:200.

[0341] Alternatively, DE cells can be differentiated into PGT cells by any method known in the art.

[0342] Phase 3 cells and cell populations (PGT to FE): This method may include differentiating PGT cells into FE cells by culturing a population of PGT cells in a first PGT differentiation medium for approximately 1 day (i.e., Figure 1B The intermediate PGT / FE cell population (e.g., as defined herein) is obtained during the first time period (day 5 of the study), and then cultured in a second PGT differentiation medium for approximately 1 day (i.e., day 5 of the study). Figure 1B The second time period (day 6) is used to obtain PDX1. + FE cell population. In some cases, the method uses a PGT cell population obtained by: (a) performing a stage 2 differentiation method, and optionally also performing stage 1a and stage 1b differentiation methods, or (b) differentiating PSCs into FE cells by any method known in the art.

[0343] In some cases, the culture of PGT and PGT / FE cell populations can be performed in a bioreactor and includes approximately 5 x 10⁻⁶ cells. 5 Cells / mL to approximately 3.5 x 10⁻⁶ 6 Cell transfer density of cells / mL. In some cases, the culture pH range can be from about 6.6 to 7.4. In other cases, the 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 cases, dissolved oxygen concentration can be controlled from about 20 mmHg to about 100 mmHg. In some cases, depending on the size of the bioreactor, the stirring rate can be from about 20 rpm to about 60 rpm. In some cases, the culture is controlled at a temperature of about 36°C to about 38°C, especially about 37°C. In some cases, air, carbon dioxide, and oxygen are supplied to the bioreactor via passive diffusion, active covering, or jetting.

[0344] PGT differentiation media. The first and second PGT differentiation media may each comprise a defined component medium containing glucose and / or fructose, and one or more of the following: glutamine (e.g., glutamine dipeptide), albumin (e.g., BSA or HSA), buffer (e.g., NaHCO3), vitamin C compound (e.g., ascorbic acid), serum replacement supplement (e.g., B27 supplement), FGF family growth factors (e.g., KGF), PKC activators (e.g., TPPB), retinoids (e.g., ATRA), ROCK inhibitors (e.g., Y-27632), cell-permeable SHH signaling inhibitors (e.g., SANT-1), TGF-β superfamily growth factors (e.g., activin A), and at least one end-anchored polymerase 1 / 2 inhibitor (e.g., IWR-1 and / or WIKI4). In some cases, the first PGT differentiation medium also includes a small molecule BMP inhibitor (e.g., DMH-1). In some cases, each PGT differentiation medium comprises the MCDB medium described in Table 16 below.

[0345] In some cases, the glucose and / or fructose concentrations in each PGT differentiation medium may be the same or different. In some cases, each PGT differentiation medium includes glucose at a concentration of about 5 mM to about 50 mM. In some cases, the glucose concentration may 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 cases, the glucose concentration may be about 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 45 mM, or 50 mM. In some cases, the glucose concentration in each of the first and second PGT differentiation media may be about 25 mM.

[0346] In some cases, one or both PGT differentiation media include glutamine, which may be provided in the form of L-alanine-L-glutamine, and the concentration may be the same or different in each medium.

[0347] In some cases, one or both PGT differentiation media include a buffer, which may be NaHCO3 at a concentration that is the same or different in each medium.

[0348] In some cases, each PGT differentiation medium comprises L-alanine-L-glutamine and NaHCO3, the concentrations of which may be selected from the respective L-alanine-L-glutamine and NaHCO3 concentration ranges and concentrations described above for the DE differentiation medium. In some cases, each PGT differentiation medium comprises approximately 1.75 mM to approximately 2.25 mM L-alanine-L-glutamine and approximately 42 mM to approximately 48 mM NaHCO3. In some cases, each PGT differentiation medium comprises approximately 2 mM L-alanine-L-glutamine and approximately 42.5 mM NaHCO3.

[0349] In some cases, one or both PGT differentiation media may include albumin, which may be at the same or different concentrations in each medium. In some cases, the albumin concentration in each PGT differentiation medium may be from about 0.5% to about 5%. In some cases, the albumin in each medium may be recombinant human albumin, which may be provided in a composition containing fatty acids and / or lipids. In some cases, the albumin may be FAF-BSA or FAF-HSA, which may be at a concentration of from about 1% to about 3%. In some cases, the concentration of FAF-BSA or FAF-HSA in each PGT differentiation medium may be about 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, or 3.5%. In some cases, each PGT differentiation medium includes about 2% FAF-BSA (or FAF-HSA).

[0350] In some cases, each PGT differentiation medium includes a vitamin C compound, which may be ascorbic acid in the same or different concentrations in each medium.

[0351] In some cases, each PGT differentiation medium includes FGF family growth factors, which may be KGF at the same or different concentrations in each medium.

[0352] In some cases, each PGT differentiation medium includes ascorbic acid and KGF, which may be present at concentrations selected from the respective ascorbic acid and KGF concentration ranges and concentrations described above for the DE differentiation medium. In some cases, each PGT differentiation medium comprises 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 cases, each PGT differentiation medium comprises about 0.25 mM ascorbic acid and about 50 ng / mL KGF.

[0353] In some cases, each PGT differentiation medium includes a serum replacement supplement, which may include two, three, four, five, or more components of B27 supplement as defined herein. In some cases, the serum replacement supplement may be a concentrated B27 supplement (e.g., B27 (50x)) that may be added to a specific component medium in a volume selected to achieve a desired final concentration, which may be the same or different in each PGT differentiation medium. In some cases, the B27 (50x) supplement may be present in each PGT differentiation medium at a concentration of about 0.1x to about 1.0x. In some cases, the serum replacement supplement is the B27 (50x) supplement shown in Table 28 below, or commercially available B-27. TM Supplement (50x), any of which may be present in each PGT differentiation 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 cases, the concentration of B27 (50x) supplement in each PGT differentiation medium may 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 cases, the first PGT differentiation medium and the second PGT differentiation medium each include a B27 (50x) supplement at a concentration of about 0.5x.

[0354] In some cases, each PGT differentiation medium includes a PKC activator, which may be the same or different in each medium. In some cases, the PKC activator in each PGT differentiation medium is TPPB, which may be the same or different concentrations in each medium. In some cases, the TPPB concentration in each PGT differentiation medium may be from about 5 nM to about 100 nM. In some cases, the TPPB concentration in each PGT differentiation medium may be from about 10 nM to about 90 nM, from about 20 nM to about 80 nM, from about 30 nM to about 70 nM, from about 40 nM to about 60 nM, or from about 45 nM to about 55 nM. In some cases, each PGT differentiation medium includes TPPB at concentrations 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 cases, each PGT differentiation medium contains approximately 50 nMTPPB.

[0355] In some cases, each PGT differentiation medium includes retinoids, which may be the same or different in each medium. In some cases, the retinoid in each medium is ATRA, which may be the same or different in each medium. In some cases, the ATRA concentration in each PGT differentiation medium is from about 0.25 μM to about 10 μM. In some cases, the ATRA concentration in each PGT differentiation medium may be from about 0.50 μM to about 8 μM, from about 1 μM to about 6 μM, from about 1.5 μM to about 4 μM, or from about 2 μM to about 5 μM. In some cases, the ATRA concentration may be from 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 cases, each PGT differentiation medium includes about 3 μM ATRA.

[0356] In some cases, each PGT differentiation medium includes a rock inhibitor, which may be the same or different in each medium. In some cases, the rock inhibitor in each PGT differentiation medium is Y-27632 (e.g., Y-27632 2HCl), which may be the same or different concentrations in each medium. In some cases, the concentration of Y-27632 in each PGT differentiation medium may be from about 1 μM to about 20 μM. In some cases, the concentration of Y-27632 in each PGT differentiation medium may be selected from the Y-27632 2HCl concentration range and concentration described above for PSC differentiation media. In some cases, the concentration of Y-27632 (e.g., Y-27632 2HCl) in each PGT medium may be from about 9 μM to about 11 μM. In some cases, each PGT differentiation medium includes about 10 μM Y-27632 (Y-27632 2HCl). Other suitable ROCK inhibitors include, but are not limited to, Seman 1, Tiazovivan, Fasudil / HA1077, and H-1152.

[0357] In some cases, each PGT differentiation medium includes a cell-permeable SHH signaling inhibitor, which may be the same or different in each medium. In some cases, the SHH signaling inhibitor in each PGT differentiation medium is SANT-1, which may be the same or different in each medium. In some cases, the SANT-1 concentration in each PGT differentiation medium may be from about 0.1 μM to about 0.5 μM. In some cases, the SANT-1 concentration in each PGT differentiation medium may be from about 0.15 μM to about 0.4 μM or from about 0.2 μM to about 0.3 μM. In some cases, the SANT-1 concentration in each PGT differentiation medium may be about 0.15 μM, 0.2 μM, 0.25 μM, 0.3 μM, 0.35 μM, or 0.4 μM. In some cases, each PGT differentiation medium includes about 0.25 μM SANT-1.

[0358] In some cases, each PGT differentiation medium includes TGF-β superfamily growth factors, which may be the same or different in each medium. In some cases, the TGF-β growth factor in each PGT differentiation is activin A, which may be the same or different in each medium. In some cases, the concentration of activin A in each PGT differentiation medium may be from about 5 ng / mL to about 40 ng / mL. In some cases, the concentration of activin A may be from about 5 ng / mL to about 35 ng / mL, from about 10 ng / mL to about 30 ng / mL, or from about 15 ng / mL to about 20 ng / mL. In some cases, the concentration of activin A in each PGT differentiation medium can be approximately 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, approximately 26 ng / mL, 27 ng / mL, 28 ng / mL, 29 ng / mL, or 30 ng / mL. In some cases, each PGT differentiation medium includes approximately 20 ng / mL of activin A.

[0359] In some cases, each PGT differentiation medium includes an end-anchored polymerase 1 / 2 inhibitor (e.g., an adenosine subsite binding inhibitor or an adenosine subsite binding / G-ring interaction inhibitor), which may be the same or different in each medium, or may be at different concentrations in each medium. In some cases, the adenosine subsite binding inhibitor is IWR-1, JW55, or JW74. In some cases, the adenosine subsite binding / G-ring interaction inhibitor is WIKI4.

[0360] In some cases, the terminal anchored polymerase 1 / 2 inhibitor in one or two PGT differentiation media is an adenosine subsite binding inhibitor, which may be IWR-1 at the same or different concentrations in each medium. In some cases, the IWR-1 concentration in each PGT differentiation medium may be from about 50 nM to about 400 nM. In some cases, the IWR-1 concentration may be from about 75 nM to about 375 nM, from about 100 nM to about 350 nM, from about 125 nM to about 325 nM, from about 150 nM to about 300 nM, or from about 175 nM to about 275 nM. In some cases, the IWR-1 concentration in each PGT differentiation medium may be from about 100 nM, 125 nM, 150 nM, 175 nM, 200 nM, 225 nM, 250 nM, 275 nM, or 300 nM. In some cases, each PGT differentiation medium includes about 200 nM of IWR-1. In some cases, each PGT differentiation medium lacks any other end-anchor polymerase inhibitors.

[0361] In some cases, the end-anchored polymerase 1 / 2 inhibitor in one or both PGT differentiation media is an adenosine subsite binding / G-ring interaction inhibitor, which may be WIKI4 at the same or different concentrations in each medium. In some cases, the WIKI4 concentration in each PGT differentiation medium may be from about 1 µM to about 30 µM. In some cases, the WIKI4 concentration may be from about 3 µM to about 20 µM, from about 4 µM to about 15 µM, from about 6 µM to about 12 µM, or from about 8 µM to about 10 µM. In some cases, the WIKI4 concentration in each PGT differentiation medium may be from about 3 µM, 6 µM, 9 µM, 12 µM, or 15 µM. In some cases, each PGT differentiation medium includes about 9 µM of WIKI4. In some cases, each PGT differentiation medium lacks any other end-anchored polymerase inhibitor.

[0362] In some cases, one or both PGT differentiation media include two end-anchored polymerase 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-ring interaction inhibitor (e.g., WIKI4). In some cases, only the second PGT differentiation medium includes both end-anchored polymerase 1 / 2 inhibitors.

[0363] In some cases, each PGT differentiation medium includes both IWR-1 and WIKI4, which may be present in each medium at the same or different concentrations. In some cases, the concentrations of IWR-1 and WIKI4 in any PGT differentiation medium including both compounds may be selected from any of the IWR-1 and WIKI4 concentrations described above. In some cases, the concentrations of IWR-1 and WIKI4 may be approximately 180 nM to approximately 220 nM and approximately 8 µM to approximately 10 µM, respectively, in each PGT differentiation medium or only in the second PGT differentiation medium. In some cases, each PGT differentiation medium or only the second PGT differentiation medium includes approximately 200 nM IWR-1 and approximately 9 µM WIKI4.

[0364] In some cases, the first PGT differentiation medium includes a small molecule BMP inhibitor, which may be DMH-1 at a concentration of about 50 nM to about 250 nM. In some cases, the concentration of DMH-1 in the first PGT differentiation medium may 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 cases, the DMH-1 concentration may 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 cases, the first PGT differentiation medium includes about 150 nM DMH-1. Other suitable BMP inhibitors include, but are not limited to, LDN-193189.

[0365] In some cases, the first PGT differentiation medium comprises approximately 125 nM to approximately 175 nM DMH-1, and the first and second PGT differentiation media each comprise approximately 20 mM to approximately 30 mM glucose, approximately 1.75 mM to approximately 2.25 mM L-alanine-L-glutamine, approximately 1.5% to approximately 2.5% BSA or HSA, approximately 42 mM 3.6 g / L to approximately 48 mM NaHCO3, approximately 0.20 mM to approximately 0.30 mM ascorbic acid, approximately 45 ng / mL to approximately 55 ng / mL recombinant human KGF, 45 nM to approximately 55 nM TPPB, approximately 2.5 μM to 3.5 μM ATRA, approximately 9 μM to approximately 11 μM Y-27632 (e.g., Y-27632 2HCl), approximately 0.2 μM to approximately 0.3 μM SANT-1, and approximately 15 ng / mL to approximately 25 nM DMH-1. The medium contains ng / mL activator 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 cases, the first PGT differentiation medium includes either IWR-1 or WIKI4, but not both compounds. In some cases, each PGT differentiation medium also includes a B27 (50x) supplement at about 0.25x to about 0.75x. In some cases, each PGT differentiation medium also includes the MCDB medium listed in Table 1.

[0366] In some cases, the first PGT differentiation medium comprises approximately 150 nM DMH-1, and the first and second PGT differentiation media each comprise the MCDB medium listed in Table 16, approximately 25 mM glucose, approximately 2 mM L-alanine-L-glutamine, approximately 2% BSA (or HSA), approximately 45.2 mM NaHCO3, approximately 0.25 mM ascorbic acid, approximately 50 ng / mL recombinant human KGF, approximately 50 nM TPPB, approximately 3 μM ATRA, approximately 10 μM Y-27632 (e.g., Y-27632 2HCl), approximately 0.25 μM MSANT-1, approximately 20 ng / mL activin A, and approximately 200 nM IWR-1-Endo. In some cases, each PGT differentiation medium comprises approximately 9 µM WIKI4 and does not include IWR-1-Endo. In some cases, each PGT differentiation medium also includes a supplement of approximately 0.5x B27 (50x).

[0367] Alternatively, PGT cells can be differentiated into FE cells by any method well known in the art.

[0368] Phase 4 cells and cell populations (FE to PP): This method may begin with or may include differentiating FE cells into PP cells by: culturing a population of FE cells in FE differentiation medium for approximately 3 days (e.g., Figure 1B The time period (days 7-9 in the original text) is used to obtain PDX1. + / NKX6.1 + PP cell population. In some cases, the method uses an FE cell population obtained by: (a) performing a stage 3 differentiation method, and optionally also performing 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 cases, the method includes replacing the FE differentiation medium in the culture once or twice with fresh FE differentiation medium during this time period (e.g., about 24 hours and / or about 48 hours after the initiation of the culture step).

[0369] In some cases, the culture of FE cell populations can be performed in a bioreactor and includes approximately 1 x 10⁻⁶ cells. 6 1 cell / mL to approximately 5 x 10⁻⁶ cells / mL 6 Cell transfer density of cells / mL. In some cases, the culture pH range can be from about 6.6 to about 8.0. In some cases, the 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 cases, dissolved oxygen concentration can be controlled from about 20 mmHg to about 150 mmHg. In some cases, depending on the size of the bioreactor, the stirring rate can be from about 20 rpm to about 60 rpm. In some cases, the culture can be controlled at a temperature of about 36°C to about 38°C, especially about 37°C. In some cases, air, carbon dioxide, and oxygen can be supplied to the bioreactor via passive diffusion, active covering, or jetting.

[0370] FE differentiation medium. FE differentiation medium may include a defined component medium containing glucose and / or fructose, and one or more of the following: glutamine (e.g., glutamine dipeptide), albumin (e.g., BSA or HSA), buffer (e.g., NaHCO3), and one or more of the EGF family growth factors (e.g., recombinant human EGF protein), vitamin B3 compounds (e.g., NAM), vitamin C compounds (e.g., ascorbic acid), serum replacement supplements (e.g., B27 supplements as defined herein), FGF family growth factors (e.g., KGF protein), PKC activators (e.g., TPPB), retinoids (e.g., ATRA), ROCK inhibitors (e.g., Y-27632), cell-permeable SHH signaling inhibitors (e.g., SANT-1), at least one end-anchored polymerase 1 / 2 inhibitor (e.g., IWR-1-Endo and / or WIKI4), and G9a inhibitors (e.g., UNC0321). In some cases, FE differentiation medium includes the MCDB medium described in Table 16 below.

[0371] In some cases, the FE differentiation medium comprises each of glucose, L-alanine-L-glutamine, albumin, and NaHCO3, which may be present at concentrations selected from the respective concentration ranges and concentrations of glucose, L-alanine-L-glutamine, albumin, and NaHCO3 described above for the PGT differentiation medium. In some cases, the glucose concentration in the FE differentiation medium may be from about 20 mM to about 30 mM. In some cases, the L-alanine-L-glutamine concentration in the FE differentiation medium may be from about 1.8 mM to about 2.2 mM L-alanine-L-glutamine. In some cases, the albumin is FAF-BSA or FAF-HSA, and the FE differentiation medium includes FAF-BSA or FAF-HSA at about 1%, 1.5%, 2%, 2.5%, or 3%. In some cases, the NaHCO3 concentration is from about 42 mM to about 48 mM NaHCO3. In some cases, the FE differentiation medium includes approximately 25 mM glucose, approximately 2 mM L-alanine-L-glutamine, approximately 2% FAF-BSA or FAF-HSA, and approximately 45.2 mM NaHCO3.

[0372] In some cases, the FE differentiation medium includes EGF family growth factors, which can be recombinant EGF protein at concentrations of about 50 ng / mL to about 350 ng / mL. In some cases, the EGF protein is recombinant human EGF, which can be at concentrations of 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. In some cases, the concentration of recombinant human EGF in the FE differentiation 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 cases, the FE differentiation medium includes about 200 ng / mL of recombinant human EGF.

[0373] In some cases, the FE differentiation medium includes a vitamin B3 compound, which may be NAM at concentrations 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 cases, the NAM concentration may be about 2 µM, 4 µM, 6 µM, 8 µM, 10 µM, 12 µM, 14 µM, 16 µM, or 18 µM. In some cases, the FE differentiation medium includes 10 µM NAM. In some cases, the FE differentiation medium does not include NAM. In some cases, the FE differentiation medium does not include any amount of vitamin B3 compound other than that present in or added to the basal medium or any multi-component supplement.

[0374] In some cases, the FE differentiation medium includes a vitamin C compound, which may be ascorbic acid at a concentration of about 0.05 mM to about 0.50 mM. In some cases, the ascorbic acid concentration in the FE differentiation medium may be selected from the ascorbic acid concentration range and concentration described above for the DE differentiation medium. In some cases, the ascorbic acid concentration in the FE differentiation medium may be about 0.20 mM to about 0.30 mM. In some cases, the FE differentiation medium includes about 0.25 mM ascorbic acid. In some cases, the vitamin C compound is dehydroascorbic acid.

[0375] In some cases, the FE differentiation medium includes a serum replacement supplement, which may include components of a B27 supplement as defined herein. In some cases, the serum replacement supplement may be a concentrated B27 supplement (e.g., B27(50x)) that may be added to a defined component medium in a volume selected to achieve the desired final concentration. In some cases, the B27 supplement is the B27(50x) supplement as described above, which may be present at a concentration of about 0.2x to about 2.0x. In some cases, the B27 supplement is the B27(50x) supplement shown in Table 28 below, or commercially available B-27. TM Supplement (50x), any of which may be present at 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 cases, the FE differentiation medium includes a B27 (50x) supplement at about 0.6x, 0.7x, 0.8x, 0.9x, 1.0x, 1.1x, 1.2x, 1.3x, or 1.4x. In some cases, the FE differentiation medium includes a B27 (50x) supplement at a concentration of about 1x.

[0376] In some cases, the FE differentiation medium includes FGF family growth factors, which may be KGF (e.g., recombinant human KGF), at a concentration selected from the KGF concentration range and concentration described above for PGT differentiation medium. In some cases, the FE differentiation medium includes recombinant human KGF at a concentration of about 45 ng / mL to about 55 ng / mL. In some cases, the FE differentiation medium includes about 50 ng / mL of recombinant human KGF.

[0377] In some cases, the FE differentiation medium includes a PKC activator, which may be TPPB at a concentration of about 20 nM to about 200 nM. In some cases, the concentration of TPPB in the FE differentiation medium may 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 cases, the FE differentiation medium includes TPPB at concentrations 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 cases, the FE differentiation medium includes about 100 nM TPPB.

[0378] In some cases, the FE differentiation medium includes retinoids, which can be ATRA at a concentration of about 50 nM to about 200 nM. In some cases, the concentration of ATRA in the FE differentiation 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 cases, the ATRA concentration can be about 80 nM, 90 nM, 100 nM, 110 nM, or 120 nM. In some cases, the FE differentiation medium includes about 100 nM ATRA.

[0379] In some cases, the FE differentiation medium includes one or both of a ROCK inhibitor and a cell-permeable SHH signaling inhibitor. In some cases, the concentrations of the ROCK inhibitor and the cell-permeable SHH signaling inhibitor in the FE differentiation medium may be selected from the respective concentration ranges and concentrations described above for the PGT differentiation medium. In some cases, the ROCK inhibitor may be Y-27632 (e.g., Y-27632 2HCl), which may be present at a concentration of about 9 μM to about 11 μM. In some cases, the cell-permeable SHH signaling inhibitor is SANT-1, which may be present at a concentration of about 0.2 μM to about 0.3 μM. In some cases, the FE differentiation medium includes about 10 μM Y-27632 (e.g., Y-27632 2HCl) and about 0.25 μM SANT-1.

[0380] In some cases, the FE differentiation medium includes an anchored polymerase 1 / 2 inhibitor (e.g., an adenosine subsite binding inhibitor or an adenosine subsite binding / G-ring interaction inhibitor), which may be the same or different compound as any anchored polymerase 1 / 2 inhibitor present in the PGT differentiation medium, or may be present at the same or different concentrations. In some cases, the adenosine subsite binding inhibitor may be IWR-1, JW55, or JW74. In some cases, the adenosine subsite binding / G-ring interaction inhibitor may be WIKI4.

[0381] In some cases, the end-anchored polymerase 1 / 2 inhibitor in the FE differentiation medium can be an adenosine subsite binding inhibitor, which can be IWR-1 at a concentration of about 50 nM to about 400 nM. In some cases, the concentration of IWR-1 can be about 75 nM to about 375 nM, about 100 nM to about 350 nM, about 125 nM to about 325 nM, about 150 nM to about 300 nM, or about 175 nM to about 275 nM. In some cases, the concentration of IWR-1 in the FE differentiation 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 cases, the FE differentiation medium includes about 200 nM of IWR-1. Other exemplary end-anchored polymerase 1 / 2 inhibitors that can be used in place of IWR-1 in FE differentiation media 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 5 µM). In some cases, FE differentiation media lack any other end-anchored polymerase inhibitors.

[0382] In some cases, the end-anchored polymerase 1 / 2 inhibitor in the FE differentiation medium can be an adenosine subsite binding / G-ring interaction inhibitor, which can be WIKI4 at a concentration of about 1 µM to 30 µM. In some cases, 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 cases, the concentration of WIKI4 in the FE differentiation medium can be about 3 µM, 6 µM, 9 µM, 12 µM, or 15 µM. In some cases, the FE differentiation medium includes about 9 µM of WIKI4. In some cases, the FE differentiation medium lacks any other end-anchored polymerase inhibitors.

[0383] In some cases, the FE differentiation medium includes two end-anchored polymerase 1 / 2 inhibitors, one of which is an adenosine subsite-specific binding inhibitor (e.g., G007-LK, IWR-1, JW55, or JW74), and the other is an adenosine subsite / G-ring interaction inhibitor (e.g., WIKI4). In some cases, the FE differentiation medium includes both IWR-1 and WIKI4, which may be present at concentrations selected from any of the IWR-1 and WIKI4 concentrations described above. In some cases, the FE differentiation medium includes approximately 180 nM to approximately 220 nM of IWR-1 and approximately 8 µM to approximately 10 µM of WIKI4. In some cases, the FE differentiation medium includes approximately 200 nM of IWR-1 and approximately 9 µM of WIKI4.

[0384] In some cases, the FE differentiation medium includes a G9a inhibitor, which may be UNC0321, UNC0638, or CM-272.

[0385] In some cases, the G9a inhibitor is UNC0321, which can be present in FE differentiation medium at concentrations of about 1 μM to about 10 μM. In some cases, the concentration of UNC0321 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 cases, the concentration of UNC0321 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 cases, the FE differentiation medium contains about 5 μM UNC0321.

[0386] In some cases, the G9a inhibitor is UNC0638, which may be present in FE differentiation medium at concentrations of about 0.1 μM to about 1.0 μM. In some cases, the UNC0638 concentration may 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 cases, the UNC0638 concentration may be about 0.2 μM, 0.4 μM, 0.6 μM, or 0.8 μM. In some cases, the FE differentiation medium includes about 0.5 μM UNC0638.

[0387] In some cases, the FE differentiation medium includes approximately 20 mM to approximately 30 mM glucose, approximately 1.8 mM to approximately 2.2 mM L-alanine-L-glutamine, approximately 1.5% to approximately 2.5% BSA or HSA, approximately 42 mM to approximately 48 mM NaHCO3, approximately 175 ng / mL to approximately 225 ng / mL recombinant human EGF, approximately 9 µM to approximately 11 µM NAM, 0.20 mM to approximately 0.30 mM ascorbic acid, approximately 45 ng / mL to approximately 55 ng / mL recombinant human KGF, approximately 90 nM to approximately 110 nM TPPB, 90 nM to approximately 110 nM ATRA, 9 μM to approximately 11 μM Y-27632 (e.g., Y-27632 2HCl), approximately 0.2 μM to approximately 0.3 μM SANT-1, approximately 180 nM to approximately 220 nM IWR-1-Endo, and / or approximately 8 μM to approximately 10 μM μM 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 cases, the FE differentiation medium also includes a B27 (50x) supplement at about 0.5x to about 1.5x. In some cases, the FE differentiation medium also includes the MCDB medium listed in Table 16.

[0388] In some cases, the FE differentiation medium includes the MCDB A / B medium shown in Table 16, approximately 25 mM glucose, approximately 2 mM L-alanine-L-glutamine, approximately 2% BSA (or HSA), approximately 45.2 mM NaHCO3, approximately 20 ng / mL recombinant human EGF, approximately 10 µM NAM, approximately 0.25 mM ascorbic acid, approximately 50 ng / mL recombinant human KGF, approximately 100 nM TPPB, approximately 100 nM ATRA, approximately 10 μM Y-27632 (e.g., Y-27632 2HCl), approximately 0.25 μM SANT-1, approximately 200 nM IWR-1-Endo, and approximately 9 µM WIKI4, or both, and optionally approximately 5 μM UNC0321 (or approximately 0.5 μM UNC0638). In some cases, the FE differentiation medium also includes a supplement of approximately 1x B27 (50x).

[0389] Alternatively, FE cells can be differentiated into PP cells by any method known in the art.

[0390] Phase 5 cells and cell populations (PP to PEP): Differentiation methods may begin with or include differentiating PP cells into PEP cells by culturing a PP cell population in a first PP differentiation medium for approximately 4 days (i.e., Figure 1B During the first time period (days 10-13), an intermediate PP / PEP cell population (e.g., as defined herein) was obtained. This intermediate PP / PEP cell population was washed in PP / PEP wash medium containing less than about 2.5 mM glucose (e.g., < about 1 mM glucose or no glucose (i.e., 0 mM)). The washed intermediate PP / PEP cell population was then cultured in a second PP differentiation medium containing < about 2 mM glucose (e.g., ≤ about 1 mM glucose or no glucose) for approximately 2 days (i.e., days 10-13). Figure 1B The second time period (days 14-15) is used to obtain CHGA. + / PDX1 + PEP cell population of cells. In some cases, the first PP differentiation medium and / or the second PP differentiation medium contain glucose at concentrations 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, or about 0 to about 2.0 mM. 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 cases, PP differentiation medium contains glucose at the following concentrations: about 0 mM, about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 1.0 mM, about 1.1 mM, about 1.2 mM, about 1.3 mM, about 1.4 mM, about 1.5 mM, about 1.6 mM, about 1.7 mM, about 1.8 mM, about 1.9 mM, about 2.0 mM, about 2.1 mM, about 2.2 mM, about 2.3 mM, about 2.4 mM, or about 2.5 mM. In some cases, the method uses a population of PP cells obtained by: (a) performing the Phase 4 differentiation method described herein, and optionally also performing Phase 1a, Phase 1b, Phase 2 and Phase 3 differentiation methods, or (b) differentiating PSCs into PP cells by any method known in the art.

[0391] In some cases, the PP to PEP differentiation method includes replacing the first PP differentiation medium in the culture once or more with fresh first PP differentiation medium during a first time period (e.g., about 24 hours, 48 ​​hours or 72 hours after initiation of culture in the first PP differentiation medium), and replacing the second PP differentiation medium in the culture once with fresh second PP differentiation medium during a second time period (e.g., about 24 hours after initiation of culture in the second PP differentiation medium).

[0392] In some cases, the PEP cell population comprises cell aggregates, and the method may include dissociating the cell aggregates into individual cells. The dissociation step may be chemically, enzymatically, or mechanically mediated by methods well known in the art. See, for example, 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 cases, the dissociation step involves collecting aggregates from the PEP cell population and contacting the collected aggregates with an enzymatic solution containing trypsin activity, chymotrypsin / elastase activity, and type I collagenase activity for a duration sufficient to obtain CHGA. + / PDX1 + The time period during which cells dissociate from the PEP cell population. In some cases, the time period can be approximately 3 minutes to approximately 12 minutes, approximately 4 minutes to approximately 11 minutes, approximately 5 minutes to approximately 10 minutes, or approximately 6 minutes to approximately 8 minutes.

[0393] In some cases, the culture of PP and PP / PEP cell populations can be performed in a bioreactor and includes approximately 1 x 10⁻⁶ cells. 6 1 cell / mL to approximately 5 x 10 6Cell transfer density of cells / mL. In some cases, the culture pH range can be from about 6.6 to about 8.0. In other cases, the 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. In bioreactors, PP and PP / PEP cell populations cultured at this differentiation stage are typically cultured in a pH range of 7.0 to 7.2. The methods described in this paper unexpectedly demonstrated that culturing certain cell populations, such as those containing PP cells, at pH levels above 7.0 to 7.2 selectively improves the proportion of mature SC-IC in in vitro differentiated cell populations. In some cases, the culture involves monitoring and adjusting the pH in the bioreactor to maintain a pH not exceeding 7.8. In some cases, cultivation involves monitoring and adjusting the pH in the bioreactor to maintain it within the following pH ranges: pH 7.2 to pH 7.8, pH 7.4 to pH 7.8, pH 7.2 + / - 0.2 (pH 7.0 to pH 7.4), pH 7.4 + / - 0.2 (pH 7.2 to pH 7.6), pH 7.6 + / - 0.2 (pH 7.4 to pH 7.8), or pH 7.8 + / - 0.2 (pH 7.6 to pH 8.0). pH adjustment is achieved by any suitable means, such as supplying a suitable buffer solution to maintain the desired pH range. In some cases, dissolved oxygen concentrations can be controlled at approximately 20 mmHg to approximately 150 mmHg. In some cases, depending on the size of the bioreactor, the stirring rate can be approximately 20 rpm to 60 rpm. In some cases, cultivation is controlled at temperatures from approximately 36°C to approximately 38°C, particularly approximately 37°C. In some cases, air, carbon dioxide, and oxygen are supplied to the bioreactor via passive diffusion, active covering, or injection.

[0394] First PP differentiation medium. The first PP differentiation medium may include a defined component medium containing glucose and / or fructose, and one or more of the following: glutamine (e.g., glutamine dipeptide), pyruvate, albumin (e.g., BSA or HSA), buffer (e.g., NaHCO3), small molecule BMP inhibitor (e.g., LDN-193189), zinc compound (e.g., ZnSO4), thyroid hormone signaling pathway activator (e.g., T3), heparin (e.g., UFH), TGF-β. ATP-competitive inhibitors of RI kinase (e.g., ALK5iII), serum replacement supplements (e.g., the B27 supplement described herein), cell-permeable SHH signaling inhibitors (e.g., SANT-1), NEAA supplements, ROCK inhibitors (e.g., Y-27632), vitamin C compounds (e.g., ascorbic acid), γ-secretase inhibitors (e.g., GSI-XX), epigenetic modifiers (e.g., G9a inhibitors such as UNC0321), and at least one end-anchored polymerase 1 / 2 inhibitor (e.g., IWR-1 and / or WIKI4). In some cases, the first PP differentiation medium includes the MCDB medium described in Table 16 below.

[0395] In some cases, the first PP differentiation medium comprises glucose or none of glucose, L-alanine-L-glutamine, albumin, and NaHCO3, which may be selected from the respective concentration ranges and concentrations of glucose, L-alanine-L-glutamine, albumin, and NaHCO3 described above for the PGT differentiation medium. In some cases, the glucose concentration in the first PP differentiation medium may be from about 20 mM to about 30 mM. In some cases, the L-alanine-L-glutamine concentration in the first PP differentiation medium may be from about 1.8 mM to about 2.2 mM. In some cases, the albumin may be FAF-BSA or FAF-HSA, and the first PP differentiation medium includes FAF-BSA or FAF-HSA at about 1.5% to about 2.5%. In some cases, the NaHCO3 concentration may be from about 42 mM to about 48 mM. In some cases, the first PP differentiation medium consists of approximately 25 mM glucose, approximately 2 mM L-alanine-L-glutamine, approximately 2% FAF-BSA, and approximately 45.2 mM NaHCO3.

[0396] In some cases, the first PP differentiation medium optionally includes pyruvate, which may 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 cases, the pyruvate concentration in the first PP differentiation medium may 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 cases, the first PP differentiation medium includes about 1.0 mM pyruvate. In some cases, the first PP differentiation medium is substantially free of pyruvate.

[0397] In some cases, the first PP differentiation medium includes a small molecule BMP inhibitor (e.g., LDN-193189 or DMH-1). In some cases, the BMP inhibitor is LDN-193189, which may be present at a concentration of about 50 nM to about 200 nM. In some cases, the concentration of LDN-193189 in the first PP differentiation medium may 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 cases, the concentration of LDN-193189 in the first PP differentiation medium may be about 70 nM, 80 nM, 90 nM, 100 nM, 110 nM, 120 nM, or 130 nM. In some cases, the first PP differentiation medium consists of approximately 100 nM LDN-193189.

[0398] In some cases, the first PP differentiation medium includes a zinc compound, which may 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 cases, the ZnSO4 concentration in the first PP differentiation medium may be about 1 μM, 2 μM, 3 μM, or 4 μM. In some cases, the first PP differentiation medium includes about 2 μM ZnSO4. In some cases, the first PP differentiation medium does not contain any zinc compound other than any amount of zinc compound that may be present in the basal medium or any multi-component supplement present in or added to the first PP differentiation medium.

[0399] In some cases, the first PP differentiation medium includes a thyroid hormone signaling pathway activator, which may 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 cases, the T3 concentration may be about 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, or 4 μM. In some cases, the first PP differentiation medium includes about 3 μM T3. Other suitable thyroid hormone signaling pathway activators include, but are not limited to, other thyroid hormones (e.g., GC-1) and T3 analogs and derivatives as described in International Patent Application Publication No. WO 2019 / 018818. In some cases, the first PP differentiation medium does not contain any amount of thyroid hormone signaling pathway activator other than that present in or added to the first PP differentiation medium in the basal medium or any multi-component supplement.

[0400] In some cases, the first PP differentiation medium comprises heparin (e.g., UFH) at concentrations 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 cases, the heparin concentration may 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 cases, the first PP differentiation medium comprises about 10 μg / mL UFH-PIM. Other suitable heparin includes, but is not limited to, fondaparinux sodium.

[0401] In some cases, the first PP differentiation medium includes an ATP-competitive inhibitor of TGF-β RI kinase, which may be ALK5iII at a concentration of about 1 μM to about 10 μM. In some cases, the ALK5iII concentration may 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 cases, the ALK5iII concentration may be about 3 μM, 4 μM, 5 μM, 6 μM, or 7 μM. In some cases, the first PP differentiation medium includes about 5 μM ALK5iII.

[0402] In some cases, the first PP differentiation medium includes a component of B27 supplement (e.g., as defined herein). In some cases, the B27 supplement may be a B27 (50x) supplement as described above, which may be present at a concentration selected from the concentration range and concentration of the B27 (50x) supplement described above for the FE differentiation medium. In some cases, the concentration of the B27 (50x) supplement in the first PP differentiation medium may be from about 0.9x to about 1.1x. In some cases, the first PP differentiation medium includes a B27 (50x) supplement at a concentration of about 1x.

[0403] In some cases, the first PP differentiation medium includes a cell-permeable SHH signaling inhibitor, which may 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 cases, the SANT-1 concentration may be selected from the SANT-1 concentration range and concentration described above for PGT differentiation medium. In some cases, the first PP differentiation medium includes about 0.25 μM SANT-1.

[0404] In some cases, the first PP differentiation medium includes two or more components of the NEAA supplement described herein. In some cases, the NEAA supplement component may be provided as a concentrated solution of about 100x (e.g., NEAA (100x)), the volume of which is selected to achieve the desired final concentration in the differentiation medium. In some cases, the NEAA supplement may be the NEAA (100x) supplement shown in Table 24, which may be present in the first PP differentiation medium at concentrations 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 cases, the concentration of the NEAA (100x) supplement may be about 0.7x, 0.8x, 0.9x, 1.0x, 1.1x, 1.2x, or 1.3x. In some cases, the first PP differentiation medium includes the NEAA (100x) supplement at a concentration of about 1x. In some cases, the first PP differentiation medium does not contain the NEAA supplement.

[0405] In some cases, the first PP differentiation medium includes a ROCK inhibitor, which may be Y-27632 (e.g., Y-27632 2HCl) at a concentration of about 1 μM to about 20 μM or about 9 μM to about 11 μM. In some cases, the concentration of Y-27632 2HCl may be selected from the Y-27632 2HCl concentration range and concentration described above for PGT differentiation medium. In some cases, the first PP differentiation medium includes about 10 μM Y-27632 2HCl. Other suitable ROCK inhibitors include, but are not limited to, Seman 1, Tiazoveven, Fasudil / HA1077, and H-1152.

[0406] In some cases, the first PP differentiation medium includes a vitamin C compound, which may be ascorbic acid at concentrations 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 cases, the ascorbic acid concentration may be about 0.15 mM, 0.20 mM, 0.25 mM, 0.30 mM, or 0.35 mM. In some cases, the first PP differentiation medium includes about 0.25 nM ascorbic acid. In some cases, the vitamin C compound is dehydroascorbic acid.

[0407] In some cases, the first PP differentiation medium includes a γ-secretase inhibitor (GSI), which can be a GSI-XX at a concentration of about 50 nM to about 200 nM. In other cases, the GSI-XX can be at a concentration of 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 cases, the GSI-XX concentration can be about 70 nM, 80 nM, 90 nM, 100 nM, 110 nM, 120 nM, or 130 nM. In some cases, the first PP differentiation medium includes about 100 nM GSI-XX. Other suitable GSIs include, but are not limited to, DAPT.

[0408] In some cases, the first PP differentiation medium includes a G9a inhibitor, which may be UNC0321, UNC0638, or CM-272.

[0409] In some cases, the first PP differentiation medium comprises UNC0321 at a concentration of about 1 μM to about 10 μM. In some cases, the concentration of UNC0321 may 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 cases, the concentration of UNC0321 may 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 cases, the first PP differentiation medium comprises about 5 μM UNC0321.

[0410] In some cases, the first PP differentiation medium comprises UNC0638 at a concentration of about 0.1 μM to about 1.0 μM. In some cases, the UNC0638 concentration may 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 cases, the UNC0638 concentration may be about 0.2 μM, 0.4 μM, 0.6 μM, or 0.8 μM. In some cases, the first PP differentiation medium comprises about 0.5 μM UNC0638.

[0411] In some cases, the first PP differentiation medium includes an anchored polymerase 1 / 2 inhibitor (e.g., an adenosine subsite binding inhibitor or an adenosine subsite binding / G-ring interaction inhibitor), which can be the same as or different from any anchored polymerase 1 / 2 inhibitor present in either the PGT or FE differentiation medium, or can be present at the same or different concentrations. In some cases, the adenosine subsite binding inhibitor can be IWR-1, JW55, or JW74. In some cases, the adenosine subsite binding / G-ring interaction inhibitor can be WIKI4.

[0412] In some cases, the end-anchored polymerase 1 / 2 inhibitor in the first PP differentiation medium is an adenosine subsite binding inhibitor, which can be IWR-1 at a concentration of about 50 nM to about 400 nM. In some cases, the concentration of IWR-1 can be about 75 nM to about 375 nM, about 100 nM to about 350 nM, about 125 nM to about 325 nM, about 150 nM to about 300 nM, or about 175 nM to about 275 nM. In some cases, the concentration of IWR-1 in the first PP differentiation 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 cases, the first PP differentiation medium includes about 200 nM of IWR-1. Other exemplary end-anchored polymerase 1 / 2 inhibitors that can be used in place of IWR-1 in the first PP differentiation medium 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 cases, the first PP differentiation medium lacks any other end-anchored polymerase inhibitors.

[0413] In some cases, the end-anchored polymerase 1 / 2 inhibitor in the first PP differentiation medium can be an adenosine subsite binding / G-ring interaction inhibitor, which can be WIKI4 at a concentration of about 1 µM to 30 µM. In some cases, 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 cases, the concentration of WIKI4 in the first PP differentiation medium can be about 3 µM, 6 µM, 9 µM, 12 µM, or 15 µM. In some cases, the first PP differentiation medium includes about 9 µM WIKI4. In some cases, the first PP differentiation medium lacks any other end-anchored polymerase inhibitors.

[0414] In some cases, the first PP differentiation medium includes two end-anchored polymerase 1 / 2 inhibitors: one may be an adenosine subsite-specific binding inhibitor (e.g., G007-LK, IWR-1, JW55, or JW74), and the other may be an adenosine subsite / G-ring interaction inhibitor (e.g., WIKI4). In some cases, the first PP differentiation medium includes both IWR-1 and WIKI4, which may be present at concentrations selected from any of the IWR-1 and WIKI4 concentrations described above. In some cases, the first PP differentiation medium includes approximately 180 nM to approximately 220 nM of IWR-1 and approximately 8 µM to approximately 10 µM of WIKI4. In some cases, the first PP differentiation medium includes approximately 200 nM of IWR-1 and approximately 9 µM of WIKI4.

[0415] In some cases, the first PP differentiation medium includes approximately 20 mM to approximately 30 mM glucose, approximately 1.8 mM to approximately 2.2 mM L-alanine-L-glutamine, approximately 0.75 mM to approximately 1.25 mM pyruvate, approximately 1.5% to approximately 2.5% FAF-BSA (or FAF-HSA), approximately 43 mM to approximately 48 mM NaHCO3, approximately 90 nM to approximately 110 nM LDN-193189, approximately 1.5 μM to approximately 2.5 μM ZnSO4, approximately 2.5 μM to approximately 3.5 μM T3, approximately 9 μg / mL to approximately 11 μg / mL UFH-PIM, approximately 4 μM to approximately 6 μM ALK5i II, approximately 0.9x to approximately 1.1x B27 (50x) supplement, 0.20 μM to approximately 0.30 μM SANT-1, and approximately 0.75x to approximately 1.25x MEM NEAA. (100x) supplement, about 9 μM to about 11 μM Y-27632 2HCl, 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 MUNC0321 (or about 0.4 μM to about 0.6 μM UNC0638). In some cases, the first PP differentiation medium also includes MCDB medium as shown in Table 16.

[0416] In some cases, the first PP differentiation medium includes MCDB medium as shown in Table 16, approximately 25 mM glucose, approximately 2 mM L-alanine-L-glutamine, approximately 1 mM pyruvate, approximately 2% FAF-BSA (or FAF-HSA), approximately 42.5 mM NaHCO3, approximately 100 nM LDN-193189, approximately 2 μM ZnSO4, approximately 3 μM T3, approximately 10 μg / mL UFH-PIM, approximately 5 μM ALK5i II, approximately 1x of B27 (50x) supplement as shown in Table 28, approximately 0.25 μM SANT-1, approximately 1.0x of MEM NEAA (100x) supplement as shown in Table 23, approximately 10 μM Y-27632 2HCl, approximately 0.25 mM ascorbic acid, approximately 100 nM MGS1-XX, and approximately 5 μM UNC0321 (or approximately 0.5 μM... UNC0638).

[0417] PP / PEP wash medium. PP / PEP wash medium may comprise a defined composition medium containing ≤1 mM, ≤0.5 mM, ≤0.1 mM, ≤0.05 mM, or ≤0.01 mM glucose, and optionally albumin (e.g., BSA or HSA). In some cases, PP / PEP wash medium may be glucose-free, pyruvate-free, HEPES-free, and contain about 0.02% to about 2% FAF-BSA (or FAF-HSA). In some cases, PP / PEP wash medium comprises the glucose-free DMEM composition shown in Table 20 below, which may optionally be supplemented with about 0.2% FAF-BSA (or FAF-HSA).

[0418] Secondary PP differentiation medium. The secondary PP differentiation medium may include a defined component medium containing <about 2 mM glucose and one or more of the following: alternative nutrients (e.g., galactose), albumin (e.g., BSA or HSA), buffer (e.g., NaHCO3), glutamine (e.g., glutamine dipeptide), small molecule BMP inhibitors (e.g., LDN-193189), zinc compounds (e.g., ZnSO4), thyroid hormone signaling pathway activators (e.g., T3), heparin (e.g., UFH), TGF-β. ATP-competitive inhibitors of RI kinases (e.g., ALK5iII), serum replacement supplements (e.g., B27 supplements), cell-permeable SHH signaling inhibitors (e.g., SANT-1), NEAA supplements, ROCK inhibitors (e.g., Y-27632), vitamin C compounds (e.g., ascorbic acid), γ-secretase inhibitors (e.g., GSI-XX), epigenetic modifiers (e.g., G9a inhibitors such as UNC0321), and at least one end-anchored polymerase 1 / 2 inhibitor (e.g., IWR-1 and / or WIKI4).

[0419] In some cases, the glucose concentration in the second PP differentiation medium may 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 cases, the glucose concentration in the second PP differentiation medium may be ≤ about 0.1 mM, ≤ about 0.05 mM, or ≤ about 0.01 mM glucose. In some cases, the second PP differentiation medium may be glucose-free (e.g., 0 mM).

[0420] In some cases, the second PP differentiation medium optionally includes alternative nutrients, which may be galactose, methyl pyruvate, methyl succinate, or pyruvate. In some cases, the alternative nutrient may be galactose, which may be present at concentrations 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 cases, the galactose concentration may be about 4.0 mM, 4.5 mM, 5.0 mM, 5.5 mM, or 6.0 mM. In some cases, the second PP differentiation medium includes galactose at a concentration of about 5.6 mM.

[0421] In some cases, the second PP differentiation medium comprises each of glutamine dipeptide, albumin, and NaHCO3, which may be present at concentrations selected from the corresponding concentration ranges and concentrations of glutamine dipeptide, albumin, and NaHCO3 described above for the PGT differentiation medium. In some cases, the glutamine dipeptide may be L-alanine-L-glutamine at a concentration of about 1.8 mM to about 2.2 mM. In some cases, the albumin is FAF-BSA (or FAF-HSA), which may be present at about 1%, 1.5%, 2%, 2.5%, or 3%. In some cases, the NaHCO3 concentration in the second PP differentiation medium may 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 cases, the second PP differentiation medium includes approximately 2.0 mM L-alanine-L-glutamine, approximately 2% FAF-BSA (or FAF-HSA), and approximately 25 mM or approximately 42.5 mM NaHCO3.

[0422] In some cases, the second PP differentiation medium includes a small molecule BMP inhibitor (e.g., LDN-193189 or DMH-1). In some cases, the BMP inhibitor may be LDN-193189, which may be present in the second PP differentiation medium at a concentration of about 50 nM to about 200 nM or about 90 nM to about 110 nM. In some cases, the LDN-193189 concentration may be selected from the LDN-193189 concentration range and concentration described above for the first PP differentiation medium. In some cases, the second PP differentiation medium includes about 100 nM LDN-193189.

[0423] In some cases, the second PP differentiation medium includes a zinc compound, which may 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 cases, the ZnSO4 concentration in the second PP differentiation medium may be about 1 μM, 2 μM, 3 μM, or 4 μM. In some cases, the second PP differentiation medium includes about 2 μM ZnSO4. In some cases, the second PP differentiation medium does not contain any zinc compound other than any amount of zinc compound that may be present in the basal medium or any multi-component supplement present in or added to the second PP differentiation medium.

[0424] In some cases, the second PP differentiation medium includes a thyroid hormone signaling pathway activator, which can be T3 at concentrations 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 cases, the T3 concentration can be about 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, or 4 μM. In some cases, the second PP differentiation medium includes about 3 μM T3. In some cases, T3 is not present in the second PP differentiation medium except in any amount present in the basal medium or multicomponent supplement added to the differentiation medium.

[0425] In some cases, the second PP differentiation medium includes heparin, which may be 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 cases, the heparin concentration may be selected from the heparin concentration range and concentration described above for the first PP differentiation medium. In some cases, the second PP differentiation medium includes about 10 μg / mL UFH-PIM.

[0426] In some cases, the second PP differentiation medium includes an ATP-competitive inhibitor of TGF-β RI kinase, which may be ALK5iII at a concentration of about 1 μM to about 10 μM or about 4 μM to about 6 μM. In some cases, the ALK5iII concentration may be selected from the ALK5iII concentration range and concentration described above for the first PP differentiation medium. In some cases, the second PP differentiation medium includes about 5 μM ALK5iII.

[0427] In some cases, the second PP differentiation medium includes a component of a B27 supplement (e.g., as defined herein). In some cases, the B27 supplement may be a B27 (50x) supplement as described above, which may be present at a concentration selected from the B-27 concentration range and concentration described above for the FE differentiation medium. In some cases, the concentration of the B27 (50x) supplement in the second PP differentiation medium may be from about 0.9x to about 1.1x. In some cases, the second PP differentiation medium includes a B27 (50x) supplement as shown in Table 28 at a concentration of about 1x.

[0428] In some cases, the second PP differentiation medium includes a cell-permeable SHH signaling inhibitor, which may 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 cases, the SANT-1 concentration may be selected from the SANT-1 concentration range and concentration described above for PGT differentiation medium. In some cases, the second PP differentiation medium includes about 0.25 μM SANT-1.

[0429] In some cases, the second PP differentiation medium includes two or more components of the NEAA supplement described herein. In some cases, the NEAA supplement may be the NEAA (100x) supplement as described above, which may be present at a concentration selected from the NEAA concentration range and concentration described above for the first PP differentiation medium. In some cases, the concentration of the NEAA (100x) supplement is from about 0.9x to about 1.1x. In some cases, the second PP differentiation medium includes the NEAA (100x) supplement shown in Table 24 at a concentration of about 1x. In some cases, the first PP differentiation medium does not contain a NEAA supplement.

[0430] In some cases, the second PP differentiation medium includes a ROCK inhibitor, which may be Y-27632 (e.g., Y-27632 2HCl) at a concentration of about 1 μM to about 20 μM or about 9 μM to about 11 μM. In some cases, the concentration of Y-27632 2HCl may be selected from the Y-27632 2HCl concentration range and concentration described above for PGT differentiation medium. In some cases, the second PP differentiation medium includes about 10 μM Y-27632 2HCl.

[0431] In some cases, the second PP differentiation medium includes a vitamin C compound, which may 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 cases, the ascorbic acid concentration may be selected from the ascorbic acid concentration range and concentration described above for the first PP differentiation medium. In some cases, the second PP differentiation medium includes about 0.25 nM ascorbic acid. In some cases, the vitamin C compound may be dehydroascorbic acid.

[0432] In some cases, the second PP differentiation medium includes a gamma-secretase inhibitor (GSI), which may be a GSI-XX at a concentration of about 50 nM to about 200 nM or about 90 nM to about 110 nM. In some cases, the GSI-XX concentration may be selected from the GSI-XX concentration range and concentration described above for the first PP differentiation medium. In some cases, the second PP differentiation medium includes about 100 nM GSI-XX. Other suitable GSIs include, but are not limited to, DAPT.

[0433] In some cases, the second PP differentiation medium includes a G9a inhibitor, which may be the same as or different from any G9a inhibitor present in the first PP differentiation medium. In some cases, the G9a inhibitor may be UNC0321, UNC0638, or CM-272.

[0434] In some cases, the G9a inhibitor in the second PP differentiation medium may be UNC0321, which may be present at a concentration of about 1 μM to about 10 μM. In some cases, the UNC0321 concentration may be selected from the UNC0321 concentration range and concentration described above for the first PP differentiation medium. In some cases, the UNC0321 concentration in the second PP differentiation medium is about 4.5 μM to about 5.5 μM. In some cases, the second PP differentiation medium includes about 5 μM UNC0321.

[0435] In some cases, the second PP differentiation medium comprises UNC0638 at a concentration of about 0.1 μM to about 1.0 μM. In some cases, the UNC0638 concentration may 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 cases, the UNC0638 concentration may be about 0.2 μM, 0.4 μM, 0.6 μM, or 0.8 μM. In some cases, the first PP differentiation medium comprises about 0.5 μM UNC0638.

[0436] In some cases, the second PP differentiation medium includes an anchored polymerase 1 / 2 inhibitor (e.g., an adenosine subsite binding inhibitor or an adenosine subsite binding / G-ring interaction inhibitor), which can be the same as or different from any anchored polymerase 1 / 2 inhibitor present in any of the PGT differentiation medium, FE differentiation medium, and the first PP differentiation medium, or can be present at the same or different concentrations. In some cases, the adenosine subsite binding inhibitor can be IWR-1, JW55, or JW74. In some cases, the adenosine subsite binding / G-ring interaction inhibitor can be WIKI4.

[0437] In some cases, the end-anchored polymerase 1 / 2 inhibitor in the second PP differentiation medium can be an adenosine subsite binding inhibitor, which can be IWR-1 at a concentration of about 50 nM to about 400 nM. In some cases, the concentration of IWR-1 can be about 75 nM to about 375 nM, about 100 nM to about 350 nM, about 125 nM to about 325 nM, about 150 nM to about 300 nM, or about 175 nM to about 275 nM. In some cases, the concentration of IWR-1 in the FE differentiation 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 cases, the first PP differentiation medium includes about 200 nM of IWR-1. Other exemplary end-anchored polymerase 1 / 2 inhibitors that can replace IWR-1 in the second PP differentiation medium 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 cases, the first PP differentiation medium lacks any other end-anchored polymerase inhibitors.

[0438] In some cases, the end-anchored polymerase 1 / 2 inhibitor in the second PP differentiation medium can be an adenosine subsite binding / G-ring interaction inhibitor, which can be WIKI4 at a concentration of about 1 µM to 30 µM. In some cases, 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 cases, the concentration of WIKI4 in the first PP differentiation medium can be about 3 µM, 6 µM, 9 µM, 12 µM, or 15 µM. In some cases, the first PP differentiation medium includes about 9 µM WIKI4. In some cases, the second PP differentiation medium lacks any other end-anchored polymerase inhibitors.

[0439] In some cases, the second PP differentiation medium includes two end-anchored polymerase 1 / 2 inhibitors: one may be an adenosine subsite-specific binding inhibitor (e.g., IWR-1, JW55, or JW74), and the other may be an adenosine subsite / G-ring interaction inhibitor (e.g., WIKI4). In some cases, the second PP differentiation medium includes both IWR-1 and WIKI4, which may be present at concentrations selected from any of the IWR-1 and WIKI4 concentrations described above. In some cases, the second PP differentiation medium includes approximately 180 nM to approximately 220 nM of IWR-1 and approximately 8 µM to approximately 10 µM of WIKI4. In some cases, the second PP differentiation medium includes approximately 200 nM of IWR-1 and approximately 9 µM of WIKI4.

[0440] In some cases, the second PP differentiation medium comprises glucose ≤ about 0.05 mM or 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 at about 0.9x to about 1.1x (e.g., the compositions in Table 28), and about 0.2 μM to about 0.3 μM SANT-1, NEAA (100x) supplement at about 0.9x to about 1.1x, Y-27632 (e.g., Y-27632 2HCl) at about 9 μM to about 11 μM, ascorbic acid at about 0.20 mM to about 0.3 mM, GSI-XX at about 90 nM to about 110 nM, UNC0321 at about 4.5 μM to about 5.5 μM, 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 cases, the second PP differentiation medium also includes MCDB medium as shown in Table 16.

[0441] In some cases, the second PP differentiation medium includes MCDB medium as shown in Table 16, approximately 5.5 mM galactose, approximately 2.0 mM L-alanine-L-glutamine, approximately 2% FAF-BSA (FAF-HSA), approximately 24 mM or approximately 42 mM NaHCO3, approximately 100 nM LDN193189, approximately 2 μM ZnSO4, approximately 3 μM T3, approximately 10 μg / mL UFH-PIM, approximately 5 μM ALK5iII, B27 (50x) supplement as shown in Table 28 at approximately 1x, approximately 0.25 μM SANT-1, NEAA (100x) supplement as shown in Table 24 at approximately 1x concentration, approximately 10 μM Y-27632 2HCl, approximately 0.25 mM ascorbic acid, approximately 100 nM MGS1-XX, and approximately 5 μM UNC0321. In some cases, the second PP differentiation medium also includes approximately 200 nM IWR-1-Endo and / or approximately 9 µM WIKI4.

[0442] Stage 6 cells and cell populations (PEP to precursor SC-IC): Differentiation methods may begin with or may include differentiating PEP cells into SC-IC cells by: (i) culturing a dissociated population of PEP cells in a first PEP differentiation medium comprising ≤ about 1 mM pyruvate (e.g., ≤ about 0.1 mM pyruvate) and DNase I for about 2 days (i.e., Figure 1B The process involves: (i) the first time period (days 16-17) to obtain a reaggregated cell population with PEP cells and / or SC-IC cells; (ii) washing the reaggregated PEP / SC-IC population in washing medium; and (iii) culturing the washed PEP / SC-IC population in a second PEP differentiation medium for approximately 2 days (i.e., day 16-17). Figure 1B The second time period (days 18-19) is used to obtain CPEP. + Cells and GCG + Precursor SC-IC populations of cells. In some cases, one or both PEP differentiation media contain epigenetic modifiers. In some cases, each PEP differentiation medium includes an HPLM composition (e.g., one of the HPLM compositions shown in Tables 18 and 19 below).

[0443] In some cases, the method includes replacing the second PEP differentiation medium once with fresh second PEP differentiation medium during the second time period (i.e., approximately 24 hours after initiation of culture step (iii)).

[0444] In some cases, the concentration of the dissociated PEP cell population present in culture step (i) is approximately 1 x 10⁻⁶. 6 per mL.

[0445] In some cases, the method uses a dissociated PEP cell population obtained by: (a) performing a stage 5 differentiation method, and optionally also performing 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.

[0446] In some cases, reaggregated PEP / SC-IC populations comprise cell aggregates with 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.

[0447] In some cases, stage 6 differentiation methods may include analyzing the precursor SC-IC population via flow cytometry to determine INS levels. + / SLC - Cells, INS - / SLC +Cells, CPEP + / GCG - Cells, CPEP + / GCG + Cells and / or CHGA + / Ki67 - One or more concentrations in cells.

[0448] In some cases, the culture of PEP cells and PEP / SC-IC populations can be performed in a bioreactor and includes approximately 1 x 10⁻⁶ cells. 5 cells / mL to approximately 1 x 10⁻⁶ cells / mL 6 Cell transfer density of cells / mL. In other cases, the cell transfer density can be approximately 1 x 10⁻⁶ cells / mL. 6 cells / mL, 1.5 x 10 6 cells / mL, 2 x 10 6 cells / mL, 2.5 x 10 6 cells / mL, 3.5 x 10 6 1 cell / mL or up to about 4 x 10 6 Cells / mL. In some cases, the culture pH can range from about 6.8 to about 7.6. In other cases, the pH can be about 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5. In some cases, dissolved oxygen can be controlled at a concentration of about 60 mmHg to about 150 mmHg. In some cases, depending on the size of the bioreactor, the stirring rate can be from about 20 rpm to about 60 rpm. In some cases, the culture is controlled at a temperature of about 36°C to about 38°C, especially about 37°C. In some cases, air, carbon dioxide, and oxygen can be supplied to the bioreactor via passive diffusion, active covering, or jetting.

[0449] PEP differentiation medium and washing medium. The first and second PEP differentiation media may each comprise a defined component medium containing approximately 1 mM to approximately 10 mM glucose, approximately 0.01 mM to approximately 0.1 mM fructose, approximately 0.01 mM to approximately 0.1 mM galactose, approximately 0.01 mM to approximately 0.10 mM pyruvate, approximately 0.1 mM to approximately 1 mM glutamine, approximately 20 mM to approximately 30 mM NaHCO3, and one or more of the following: albumin (e.g., BSA or HSA), TGF-β. ATP-competitive inhibitors of RI kinases (e.g., ALK5iII), small molecule BMP inhibitors (e.g., LDN-193189), zinc compounds (e.g., ZnSO4), heparin (e.g., UFH), thyroid hormone signaling pathway activators (e.g., T3), vitamin C compounds (e.g., ascorbic acid), thiol-based antioxidants (e.g., NAC), B27 supplements, G9a inhibitors (e.g., UNC0321), and cell-permeable SHH signaling inhibitors (e.g., SANT-1). In some cases, the first PEP differentiation medium also includes DNase I (e.g., recombinant DNase I). In some cases, each PEP differentiation medium includes the HPLMs shown in Table 18 below.

[0450] In some cases, one or both PEP differentiation media do not include any of the following factors selected in any amount present in the basal medium or any multi-component supplement, other than those present in the PEP differentiation medium or added to the basal medium: (i) small molecule BMP inhibitors, (ii) zinc, (iii) heparin, (iv) thyroid hormone signaling pathway activators, (v) vitamin C compounds, (vi) thiol-based antioxidants, (vii) B-27 supplements, (viii) SHH signaling inhibitors, and (ix) albumin.

[0451] In some cases, the wash medium used between the first and second culture steps comprises the same defined composition medium as described above for the first and second PEP differentiation media, but supplemented only with albumin (e.g., as defined herein). In some cases, the wash medium comprises an HPLM composition (e.g., the HPLM compositions shown in Table 19 below herein).

[0452] In some cases, the first PEP differentiation medium includes DNase I, which may be recombinant bovine DNase I at a concentration of about 1 U / mL to about 100 U / 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 (e.g., as defined herein). In some cases, the first PEP differentiation medium includes about 10 U / mL of recombinant bovine DNase I.

[0453] In some cases, each PEP differentiation medium includes glucose, which may be at the same or different concentrations in each medium. In some cases, the glucose concentration in each PEP differentiation medium may 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 cases, the glucose concentration in each PEP differentiation medium may be about 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, or 8 mM. In some cases, the first and second PEP differentiation media each contain about 5 mM of glucose. In some cases, the first and second PEP differentiation media each contain less than 5 mM of glucose. In some cases, the first and second PEP differentiation media each contain less than 2.5 mM of glucose. In some cases, the first and second PEP differentiation media each contain less than 2 mM of glucose.

[0454] In some cases, each PEP differentiation medium includes fructose, which may be at the same or different concentrations in each medium. In some cases, the fructose concentration in each PEP differentiation medium may 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 cases, the fructose concentration in each PEP differentiation medium may be about 0.02 mM, 0.03 mM, 0.04 mM, 0.05 mM, or 0.06 mM. In some cases, each PEP differentiation medium includes about 0.04 mM fructose.

[0455] In some cases, each PEP differentiation medium includes galactose, which may be at the same or different concentrations in each medium. In some cases, the galactose concentration in each PEP differentiation medium may 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 cases, the galactose concentration in each PEP differentiation medium may be about 0.04 mM, 0.05 mM, 0.06 mM, 0.07 mM, or 0.08 mM. In some cases, each PEP differentiation medium includes 0.06 mM galactose.

[0456] In some cases, each PEP differentiation medium includes pyruvate, which may be at the same or different concentrations in each medium. In some cases, the pyruvate concentration in each PEP differentiation medium may 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 cases, the pyruvate concentration in each PEP differentiation medium may be about 0.03 mM, 0.04 mM, 0.05 mM, 0.06 mM, or 0.07 mM. In some cases, each PEP differentiation medium includes about 0.05 mM pyruvate.

[0457] In some cases, each PEP differentiation medium includes glutamine, which may be at the same or different concentrations in each medium. In some cases, the glutamine concentration in each PEP differentiation medium may 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 cases, the glutamine concentration in each PEP differentiation medium may be about 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, or 0.7 mM. In some cases, each PEP differentiation medium includes about 0.55 mM glutamine.

[0458] In some cases, each PEP differentiation medium includes NaHCO3, which may be the same or different concentrations in each medium. In some cases, the NaHCO3 concentration in each PEP differentiation medium may 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 cases, the NaHCO3 concentration in each PEP differentiation medium may be about 22 mM, 23 mM, 24 mM, 25 mM, or 26 mM. In some cases, each PEP differentiation medium includes about 24 mM NaHCO3.

[0459] In some cases, each PEP differentiation medium includes albumin, which may be at the same or different concentrations in each medium. In some cases, the albumin concentration in each PEP differentiation medium may be from about 0.5% to about 5%. In some cases, the albumin may be FAF-BSA or FAF-HSA, which may be at a concentration from about 1% to about 3% in each medium. In some cases, the FAF-BSA or FAF-HSA concentration in each PEP differentiation medium may be about 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, or 3.5%. In some cases, each PEP differentiation medium includes about 2% FAF-BSA (or FAF-HSA).

[0460] In some cases, each PEP differentiation medium includes an ATP-competitive inhibitor of TGF-β RI kinase, which may be the same or different in each medium. In some cases, the ATP-competitive inhibitor of TGF-β RI kinase in each PEP differentiation medium may be ALK5iII, which may be present in each medium at the same or different concentrations. In some cases, the ALK5iII concentration in each PEP differentiation medium may be from about 0.5 μM to about 5 μM. In some cases, the ALK5iII concentration in each PEP differentiation medium may be from about 1.0 μM to about 4 μM, from about 1.5 μM to about 3.5 μM, or from about 2.0 μM to about 3 μM. In some cases, each PEP differentiation medium includes about 2.5 μM ALK5iII.

[0461] In some cases, each PEP differentiation medium includes a small molecule BMP inhibitor, which may be the same or different in each medium. In some cases, the BMP inhibitor in each PEP different...

Claims

1. A method for generating a cell population comprising mature stem cell-derived islet-like cells (SC-IC), the method comprising the following steps: (a) A first precursor cell population is cultured in one or more differentiation media to obtain a mature SC-IC population, wherein at least one of the differentiation media is a defined component medium containing glucose at less than about 2.5 mM or less than about 2 mM, and wherein the first precursor cell population is selected from foregut endoderm (FE) populations, pancreatic progenitor (PP) cell populations, pancreatic endocrine precursor (PEP) cell populations, and precursor SC-IC cell populations.

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

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

4. The method according to any one of claims 1-3, wherein at least one of the cell differentiation culture media is a defined component culture medium containing glucose at concentrations 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. 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 according to any one of claims 1-4, wherein at least one of the cell differentiation culture media is a defined component culture medium containing glucose at concentrations of: about 0 mM, about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 1.0 mM, about 1.1 mM, about 1.2 mM, about 1.3 mM, about 1.4 mM, about 1.5 mM, about 1.6 mM, about 1.7 mM, about 1.8 mM, about 1.9 mM, about 2.0 mM, about 2.1 mM, about 2.2 mM, about 2.3 mM, about 2.4 mM, or less than 2.5 mM.

6. The method according to any one of claims 1-5, wherein at least one of the cell differentiation culture media is a defined component culture medium containing glucose at concentrations of: 0 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 1.0 mM, 2.1 mM, 2.2 mM, 2.3 mM, 2.4 mM, or less than 2.5 mM.

7. The method according to any one of claims 1-6, wherein the culture in step (a) further includes monitoring pH.

8. The method according to any one of claims 1-7, wherein the culture in step (a) is carried out at pH values ​​in the following ranges: pH 7.2 to pH 7.8, pH 7.4 to pH 7.8, pH 7.2 + / - 0.2 (pH 7.0 to pH 7.4), pH 7.4 + / - 0.2 (pH 7.2 to pH 7.6), pH 7.6 + / - 0.2 (pH 7.4 to pH 7.8), or pH 7.8 + / - 0.2 (pH 7.6 to pH 8.0).

9. The method according to any one of claims 1-8, wherein at least one of the one or more differentiation media in step (a) comprises an end-anchor polymerase 1 / 2 inhibitor, wherein the end-anchor polymerase 1 / 2 inhibitor is Wiki4.

10. The method according to any one of claims 1-9, wherein the determined component culture medium is glucose-free.

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

12. The method according to any one of claims 1-11, wherein at least one of the differentiation culture media in step (a) comprises: 5.5 mM galactose, glutamine, and at least two differentiation factors selected from the following: Small molecule BMP inhibitors, Zinc compounds, Activators of thyroid hormone signaling pathways heparin, TGF-β RI kinase competitive inhibitor, Inhibitors of cell-permeable sound hedgehog (SHH) signaling, Non-essential amino acid (NEAA) supplements Rho kinase (ROCK) inhibitors, Vitamin C compounds, and Gamma-secretase inhibitors (GSI) It may also optionally further include one or more of albumin, buffers, and serum replacement supplements.

13. The method according to any one of claims 1-2, further comprising: (b) In the presence of an enzyme-catalyzed aggregate dissociation solution, CHGA-containing proteins were cultured in differentiation medium. + / PDX1 + A second cell population of cells was obtained to contain CHGA. + / PDX1 + The dissociation of single cells into a cell population, optionally wherein the enzymatic aggregate dissociation solution contains trypsin activity, chymotrypsin / elastase activity, and type I collagenase activity, optionally including a chelating agent such as EDTA; and (c) Culturing CHGA in differentiation medium containing DNase I and G9a inhibitor + / PDX1 + The dissociation of individual cells into a cell population, sustained for an additional period of time sufficient to obtain a re-aggregated cell population, the cells being contained in cell aggregates, optionally wherein the cell aggregates have an average size of about 40 µm to about 100 µm, or about 70 micrometers.

14. The method of claim 13, wherein the differentiation medium in steps (b) and (c) each comprises glucose, glutamine, and at least two differentiation factors selected from the group consisting of glucose at a concentration of about 1 mM to ≤ about 25 mM: Thiol-based antioxidants, Small molecule BMP inhibitors, Zinc compounds, Activators of thyroid hormone signaling pathways TGF-β RI kinase competitive inhibitor, Cellular permeability SHH signaling inhibitors Vitamin C compounds, Heparin, and Optionally, each differentiation medium may further contain one or more of albumin, buffers, and serum replacement supplements.

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

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

17. The method according to any one of claims 13 to 16, further comprising: (d) Culture the reaggregated cell population in a differentiation medium for a period of time sufficient to obtain a mature (SC-IC) cell population comprising NKX6.1+ / CPEP+ cells and CPEP+ / GCG- cells, wherein the differentiation medium contains glucose, glutamine, and at least two differentiation factors selected from the group consisting of glucose at a concentration of about 1 mM to ≤ about 25 mM: Cell-permeable vitamin E analogs / antioxidants Carnitine compounds, Chemically defined lipid mixtures (CDLM) Thiol-based antioxidants, Small molecule bone morphogenetic protein (BMP) inhibitors; Zinc compounds, Activators of thyroid hormone signaling pathways Vitamin C compounds, heparin, G9A inhibitors, and Optionally, the differentiation medium further comprises one or more of albumin, buffer, and serum replacement supplement.

18. The method according to 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 according to any one of claims 13-19, wherein the differentiation medium comprises human plasma-like medium (HPLM).

21. The method according to any one of claims 1-20, further comprising, prior to step (a), culturing cells containing PDX1 in one or more cell differentiation media containing two end-anchor polymerase inhibitors. + FOXA2 + NKX6.1 - and CHGA - A population of foregut endoderm (FE) cells, comprising a combination of cells, wherein one of the end-anchor polymerase inhibitors is Wiki4, thereby obtaining a cell containing PDX1. + Optionally includes PDX1 + / NKX6.1 + Cells and CHGA - (PP) precursor cell population of cells.

22. The method according to any one of claims 1-21, wherein the method does not include the step of sorting or separating individual cells or cell populations containing cell markers or combinations of cell markers.

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

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

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

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

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

28. From including PDX1 + A method for deriving a cell population comprising mature stem cell-derived islet-like cells (SC-IC) from a population of pancreatic progenitor (PP) cells, wherein the method includes: (i) A PP cell population is cultured in a first PP differentiation medium for a period sufficient to obtain an intermediate PP / PEP cell population, wherein the PP cell population contains PDX1+, optionally PDX1 + / NKX6.1 + Cells and CHGA - Cells (PDX1) + / CHGA - The first PP differentiation medium is a defined component medium containing about 5 mM to about 50 mM glucose and a set of differentiation factors sufficient to promote at least a portion of the PP cell population to differentiate into PEP cells. The set of differentiation factors includes a G9a inhibitor and at least one factor selected from the group consisting of: Small molecule bone morphogenetic protein (BMP) inhibitors, Zinc compounds, Activators of thyroid hormone signaling pathways TGF-β RI kinase competitive inhibitor, Inhibitors of cell-permeable sound hedgehog (SHH) signaling, Rho kinase (ROCK) inhibitors, Vitamin C compounds, Gamma-secretase inhibitors (GSI) Heparin, and Optionally at least one end-anchored polymerase 1 / 2 inhibitor; or optionally two end-anchored polymerase 1 / 2 inhibitors; (ii) Wash the intermediate PP / PEP cell population in a wash medium containing a defined component of <1 mM glucose; (iii) The washed intermediate PP / PEP cell population is cultured in a second PP differentiation medium for a period sufficient to obtain a PEP cell population containing cell aggregates, wherein the second PP differentiation medium is a defined component medium containing <1 mM glucose and a set of differentiation factors, the amount of which is sufficient to promote the differentiation of at least a portion of the intermediate PP / PEP cell population into cells containing PDX1. + / CHGA + (PDX1) + / NKX6.1 + / CHGA + A population of PEP cells, wherein the set of differentiation factors includes a G9a inhibitor and at least one factor selected from the following: Small molecule BMP inhibitors, Zinc compounds, Activators of thyroid hormone signaling pathways TGF-β RI kinase competitive inhibitor, Cellular permeability SHH signaling inhibitors ROCK inhibitors Vitamin C compounds, GSI, Heparin, and At least one end-anchored polymerase 1 / 2 inhibitor; optionally two end-anchored polymerase 1 / 2 inhibitors; (iv) Dissociate 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) A dissociated population of PEP cells is cultured in a first PEP differentiation medium containing deoxyribonuclease (DNase) for a duration sufficient to yield a reaggregated intermediate PEP / SC-IC population, wherein the first PEP differentiation medium is a defined component medium containing DNase, ≤ about 0.5 mM pyruvate, about 1 mM to ≤ about 25 mM glucose, and a set of differentiation factors in an amount sufficient to promote differentiation of at least a portion of the PEP cell population into immature pancreatic endocrine cells (PECs), wherein the set of differentiation factors includes a G9a inhibitor and at least one factor selected from the following: Thiol-based antioxidants, Small molecule BMP inhibitors; Zinc compounds, Activators of thyroid hormone signaling pathways TGF-β RI kinase competitive inhibitor, Cellular permeability SHH signaling inhibitors Vitamin C compounds, and heparin; (vi) Wash the reaggregated intermediate PEP / SC-IC population in a wash medium containing ≤ about 0.5 mM pyruvate and about 1 mM to ≤ about 25 mM glucose. (vii) The washed intermediate PEP / SC-IC population is cultured in a second PEP differentiation medium for a period sufficient to obtain a precursor SC-IC population, wherein the second PEP differentiation medium is a defined component medium containing ≤ about 0.5 mM pyruvate, about 1 mM to ≤ about 25 mM glucose, and a set of differentiation factors sufficient to promote the differentiation of at least a portion of the intermediate PEP / SC-IC population into CHGA-containing cells. + / PDX1 + A precursor SC-IC population of cells, wherein the set of differentiation factors includes a G9a inhibitor and at least one factor selected from the following: Thiol-based antioxidants, Small molecule BMP inhibitors, Zinc compounds, Activators of thyroid hormone signaling pathways TGF-β RI kinase competitive inhibitor, Cellular permeability SHH signaling inhibitors Vitamin C compounds, and Heparin; and (viii) Culture the precursor SC-IC population in SC-IC differentiation medium for a period sufficient to obtain a mature SC-IC population containing NKX6.

1. + / CPEP + Cells, CPEP + / GCG - Cells, INS + / SLC18A1 - Cells, or CHGA + / Ki67 - The cell combination, wherein the SC-IC differentiation medium comprises ≤0.5 mM pyruvate, about 1 mM to ≤25 mM glucose, and a set of differentiation factors, the amount of which is sufficient to promote at least a portion of the precursor SC-IC population to differentiate into mature PBLCs, wherein the set of differentiation factors comprises a G9a inhibitor and at least one factor selected from the following: Cell-permeable vitamin E analogs / antioxidants Carnitine compounds, Chemically defined lipid mixtures (CDLM) Thiol-based antioxidants, Small molecule BMP inhibitors, Zinc compounds, Activators of thyroid hormone signaling pathways Vitamin C compounds, and Heparin; and wherein in all steps (i)-(viii), at least one end-anchored polymerase 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 cells containing PDX1 in FE differentiation medium. + A foregut endoderm (FE) cell population sustained for a period sufficient to yield a PP cell population, wherein the FE differentiation medium is a defined component medium containing approximately 5 mM to approximately 50 mM glucose and a set of differentiation factors sufficient to promote differentiation of at least a portion of the FE cell population into PP cells, wherein the set of differentiation factors comprises at least one factor selected from the following: EGF family growth factors, Vitamin B3 compounds, Vitamin C compounds, FGF family growth factors, PKC activator, retinoids ROCK inhibitors Cellular permeability SHH signaling inhibitors At least one end-anchored polymerase 1 / 2 inhibitor, optionally two end-anchored polymerase 1 / 2 inhibitors, wherein at least one of the end-anchored polymerase 1 / 2 inhibitors is Wiki4.

30. The method according to claim 28 or 29, further comprising the following steps: The FE cell population was obtained through the following methods: (i) Cultured in first PGT differentiation medium containing FOXA2 + The primitive gut tract (PGT) cell population or PGT cell population was used for a first time period of approximately 12 to 48 hours to obtain an intermediate PGT / FE cell population; and (ii) An intermediate PGT / FE cell population was cultured in a second PGT medium for a second time period of approximately 12 to approximately 48 hours to obtain an FE cell population. The first PGT differentiation medium is a defined culture medium containing approximately 5 mM to approximately 50 mM glucose and a set of differentiation factors, wherein the amount of differentiation factors is sufficient to promote the differentiation of at least a portion of the PGT cell population into FE cells, wherein the factors are selected from at least one of small molecule BMP inhibitors, FGF family growth factors, PKC activators, retinoids, ROCK inhibitors, cell permeability SHH signaling inhibitors, and at least one end-anchored polymerase 1 / 2 inhibitor; and The second PGT differentiation medium is a defined-component medium that is deficient in BMP inhibitors and contains approximately 5 mM to approximately 50 mM glucose, approximately 0.5 mM to approximately 1.5 mM pyruvate, and a set of differentiation factors sufficient to promote the differentiation of at least a portion of the intermediate PGT / FE cell population into FE cells, wherein the factors comprise at least one selected from the following: Vitamin C compounds, FGF family growth factors, PKC activator retinoids, ROCK inhibitors Inhibitors of cell-permeable SHH signaling, and At least one end-anchored polymerase 1 / 2 inhibitor, optionally two end-anchored polymerase 1 / 2 inhibitors, wherein at least one of the end-anchored polymerase 1 / 2 inhibitors is Wiki4.

31. The method according to any one of claims 28 to 30, further comprising obtaining a PGT cell population by culturing a defined endoderm (DE) cell population comprising PDX1- cells and FOXA2+ / SOX17+ cells or GATA6+ / SOX17+ cells in a DE differentiation medium for a period of time sufficient to obtain a PGT cell population, wherein the DE differentiation medium is a defined component medium comprising about 5 mM to about 20 mM glucose and a set of differentiation factors, wherein the amount of differentiation factors is sufficient to promote at least a portion of the DE cell population to differentiate into PGT cells, wherein the factors comprise at least one factor selected from vitamin C compounds and FGF family growth factors.

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

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

34. An in vitro cell population containing cells, wherein: (i) At least approximately 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 - cell; (ii) No more than about 23% of the cells in the population are GCG. + Cells; optionally, about 10% to about 20% and about 10% to about 23% of the cells in the population are GCG. + cell; (iii) At least approximately 54% of the cells in the population are NKX6.

1. + / CPEP + Cells; optionally, approximately 54% to approximately 65% ​​of the cells in the population are NKX6.

1. + / CPEP + cell; (iv) At least approximately 68% of the cells in the population are INS. + / SLC - Cells; optionally, approximately 68%, approximately 68% to approximately 77% of the cells in the population are INS. + / SLC - cell; (v) Less than 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 + cell; (vi) At least approximately 99.5% of the cells in the cell population are CHGA. + Cells; approximately 99.5% of the cells in the cell population are CHGA. + cell; (vii) Less than 4% of the cells in the cell population are Ki67. + Cells; optionally, about 3.5% to about 1% to 4% of the cells in the population are Ki67. + cell; (viii) Insulin levels of at least about 150 nU / cell to about 200 nU / cell, or (ix) Lactic acid is produced within 48 hours by cell populations of less than 0.5 mM, 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, or about 0 to about 0.5 mM, wherein said populations contain 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 - Furthermore, at least approximately 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 + Furthermore, less than approximately 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 - Furthermore, at least approximately 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 + Furthermore, at least approximately 68% to approximately 77% of the cells in the population are INS. + / SLC - .

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

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

41. An in vitro cell population containing cells, wherein: (i) At least approximately 67% of the cells in the population are CPEP. + / GCG - Optionally, approximately 67% to 80%, approximately 67% to 70%, approximately 70% to 80%, and approximately 70% to 85% of the cells in the population are CPEP. + / GCG - cell; (ii) No more than about 22% of the cells in the cell population are GCG. + Cells; optionally, about 10% to about 20% and about 10% to about 22% of the cells in the population are GCG. + cell; (iii) At least approximately 60% of the cells in the cell population are NKX6.

1. + / CPEP + Cells; optionally, about 60% to about 70%, about 65% to about 75%, and about 60% of the cells in the population are NKX6.1 + / CPEP + cell; (iv) Approximately 68% of the cells in the cell population are INS. + / SLC - Cells; optionally, about 77%, about 70% to about 80%, and about 70% to about 85% of the cells in the population are INS. + / SLC - cell; (v) Less than 7% of the cells in the cell population are INS. + / SLC + Cells; optionally, about 7% to about 3-7% of the cells in the population are INS. + / SLC + cell; (vi) At least approximately 99.8% of the cells in the cell population are CHGA. + Cells; approximately 99.8% of the cells in the cell population are CHGA. + cell; (vii) Less than 2% of the cells in the cell population are Ki67. + Cells; optionally, 0.3-2%, or about 2%, of the cells in the population are Ki67. + cell; (viii) Insulin levels of at least 150 nU / cell to approximately 200 nU / cell, or (ix) Lactic acid is produced within 48 hours by cell populations of less than 0.5 mM, 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, or about 0 to about 0.5 mM, wherein said populations contain at least two of the above characteristics.

42. The in vitro cell population according to claim 41, wherein approximately 67% of the cells in the population are CPEP. + / GCG - Furthermore, approximately 99.8% of the cells in this population are CHGA. + cell.

43. The in vitro cell population of claim 41, wherein at least about 54% of the cells in the population are NKX6.

1. + / CPEP + Furthermore, less than approximately 11% of the cells in the population are INS. - / SLC + .

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

45. The in vitro cell population of claim 41, wherein approximately 60% of the cells in the population are NKX6.

1. + / CPEP + Furthermore, approximately 68% of the cells in this population are INS. + / SLC - .

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

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

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

49. The in vitro cell population according to any one of claims 35-40, 42-47, wherein lactate is produced within 48 hours by the cell population at a concentration of less than 0.5 mM.

50. A liquid cell differentiation composition comprising: (a) Serum-free basal medium; and (b) A set of differentiation factors, wherein the set of differentiation factors is: (i) A set of factors that promote the differentiation of foregut endoderm (FE) cells into pancreatic progenitor (PP) cells (FE factor set); (ii) Promote the differentiation of pancreatic progenitor cells (PP) containing PDX1+ cells into cells containing PDX1+ cells. + / CHGA + The set of factors (PP factor set) of the pancreatic endocrine precursor (PEP) population of cells. (iii) A set of factors that promote the differentiation of PEP cells into immature SC-IC cells (PEP factor set); or (iv) The set of factors that can promote the differentiation of immature SC-IC into mature SC-IC (SC-IC factor set).

51. The liquid cell differentiation composition of claim 50, wherein the serum-free basal medium 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 differentiation composition according to claim 51, further comprising at least one end-anchored polymerase 1 / 2 inhibitor, optionally wherein the end-anchored polymerase 1 / 2 inhibitor is Wiki4.

53. The liquid cell differentiation composition according to claim 51 or 52 is used to differentiate a progenitor cell population containing PDX1+ cells, optionally a PP population containing PDX1+ cells, into a cell line containing PDX1+ cells. + / CHGA + Applications of PEP populations in cells.

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

55. A method for treating an individual with diabetes, one or more diabetes-related complications, or prediabetes, said method comprising: (a) Administering to an individual an effective amount of the composition according to claim 33, an in vitro cell population according to any one of claims 33-49, or a pharmaceutical composition according to claim 54; (b) Administering to an individual an effective amount of a pharmaceutical composition encapsulated in a device providing immune protection to the encapsulated cells, comprising the composition according to claim 33 or an in vitro cell population according to any one of claims 33-49; or (c) An apparatus for administering to an individual a composition comprising the composition according to claim 33, an in vitro cell population according to any one of claims 33-49, or a pharmaceutical composition according to claim 54.

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

57. The method according to claim 55 or 56, wherein the apparatus comprises alginate chemically modified with an effective amount of an anti-fibrotic compound of formula I.

58. The method according to any one of claims 57-59, wherein the diabetes is type 1 diabetes.

59. The composition according to claim 33, the in vitro cell population according to any one of claims 33-49, or the pharmaceutical composition according to claim 54, for the treatment of diabetes, one or more diabetes-related complications, or prediabetes.

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 diabetes-related complications, or prediabetes.

61. A method for culturing stem cells to obtain a population of differentiated cells, the method comprising at least one step of culturing the cell population at a pH not higher than 7.8, within the following pH ranges: pH 7.2 to pH 7.8, pH 7.3 to pH 7.8, pH 7.4 to pH 7.8, pH 7.2 + / - 0.2 (pH 7.0 to pH 7.4), pH 7.4 + / - 0.2 (pH 7.2 to pH 7.6), pH 7.6 + / - 0.2 (pH 7.4 to pH 7.8), or pH 7.8 + / - 0.2 (pH 7.6 to pH 8.0), wherein the at least one step is performed in a bioreactor.

62. The method of claim 61, wherein at least one step of culturing the cell population at a pH not higher than 7.8, or at a pH in the following ranges: pH 7.2 to pH 7.8, pH 7.3 to pH 7.8, pH 7.4 to pH 7.8, pH 7.2 + / - 0.2 (pH 7.0 to pH 7.4), pH 7.4 + / - 0.2 (pH 7.2 to pH 7.6), pH 7.6 + / - 0.2 (pH 7.4 to pH 7.8), or pH 7.8 + / - 0.2 (pH 7.6 to pH 8.0), is carried out in at least one differentiation medium comprising a defined component medium containing 0 mM to less than 2.5 mM glucose or 0 mM to less than 2 mM glucose.

63. A method for deriving a population of differentiated cells derived from stem cells, the method comprising at least one step of culturing the population of cells in at least one differentiation medium at a pH not higher than 7.8, within the following pH ranges: pH 7.2 to pH 7.8, pH 7.3 to pH 7.8, pH 7.4 to pH 7.8, pH 7.2 + / - 0.2 (pH 7.0 to pH 7.4), pH 7.4 + / - 0.2 (pH 7.2 to pH 7.6), pH 7.6 + / - 0.2 (pH 7.4 to pH 7.8), or pH 7.8 + / - 0.2 (pH 7.6 to pH 8.0).

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

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

66. The method according to any one of claims 61-65, wherein the population of said differentiated cells comprises CPEP. + / GCG - Cells and GCG + Mature stem cell-derived islet-like cells (SC-IC).

67. A method for deriving a cell population comprising mature stem cell-derived islet-like cells (SC-IC), the method comprising the steps of: (a) A first precursor cell population containing PDX1+ cells is cultured in one or more differentiation media to obtain a mature SC-IC population, wherein the first precursor cell population is selected from 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 differentiation media is a defined component medium containing a G9a inhibitor.

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

69. The method of claim 68, wherein the culture of the first precursor cell population comprising PDX1+ cells is carried out at pH values ​​in the following ranges: pH 7.2 to pH 7.8, pH 7.3 to pH 7.8, pH 7.4 to pH 7.6, pH 7.4 to pH 7.8, pH 7.2 + / - 0.2 (pH 7.0 to pH 7.4), pH 7.4 + / - 0.2 (pH 7.2 to pH 7.6), pH 7.6 + / - 0.2 (pH 7.4 to pH 7.8), or pH 7.8 + / - 0.2 (pH 7.6 to pH 8.0).

70. An in vitro cell population comprising mature SC-IC, wherein the population is generated by the method according to any one of claims 1-31 or 61-69.

71. An in vitro cell population containing cells, wherein: (i) At least approximately 67% of the cells in the population are CPEP. + / GCG - ; (ii) No more than about 22% of the cells in the cell population are GCG. + cell; (iii) At least approximately 60% of the cells in the cell population are NKX6.

1. + / CPEP + cell; (iv) Approximately 68% of the cells in the cell population are INS. + / SLC - cell; (v) Less than 7% of the cells in the cell population are INS. - / SLC + cell; (vi) At least approximately 99.8% of the cells in the cell population are CHGA. + cell; (vii) Less than 2% of the cells in the cell population are Ki67. + cell; (viii) Insulin levels ranging from 150 nU / cell to at least approximately 200 nU / cell; or (ix) is produced within 48 hours by a cell population of less than 0.5 mM lactate, wherein the population contains at least two of the above-described properties, and wherein the cell population is produced by the method according to any one of claims 1-31 or 61-69.

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