Engineered stem cell derived pancreatic beta cell compositions and methods of use thereof

Abstract

The present disclosure provides compositions of stem cell-derived pancreatic islet cells engineered for improved engraftment and methods of use thereof for treating a subject having an insulin deficiency.

Description

Attorney Docket: MINU-012 / 01WO 339336.2063ENGINEERED STEM CELL DERIVED PANCREATIC BETA CELL COMPOSITIONS AND METHODS OF USE THEREOF CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U. S. Provisional Patent Application No. 63 / 738,410, filed December 23, 2024, all of which are incorporated herein by reference in their entirety.STATEMENT CONCERNING GOVERNMENT SUPPORT

[0002] This invention was made with government support under contracts 1R43DK138729-01A1 and R01DK129523 awarded by the National Institutes of Health (NIH). The government has certain rights to the invention.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0003] The contents of the electronic sequence listing (MINU M2 HWO_SeqList_ST26.xml; Size: 2,868 bytes; and Date of Creation: December 22, 2025) are herein incorporated by reference in their entirety.BACKGROUND

[0004] Diabetes occurs when the mass or function of insulin producing beta cells is insufficient to meet metabolic demands. In Type 1 diabetes, the beta cells are killed by an autoimmune attack. As a result, patients with this form of diabetes are rendered dependent on exogenous insulin, administered by injection, for survival. In patients with Type 2 diabetes, insulin producing beta cells are present, but they do not secrete sufficient quantities of insulin for normal metabolic control.

[0005] Restoration of lost or insufficient beta cell mass has been performed by transplanting either whole pancreas organs or islets of Langerhans isolated from donated pancreas organs.However, access to transplantation is limited by the number of deceased donors and the need for life-long immunosuppressive therapy since they are recognized as foreign to the hosts immune system. Stem cells are being explored as a source of islets. However, there remains a need for329282749 1Attorney Docket: MINU-012 / 01WO 339336,2063approaches that prevent immune rejection of transplanted islets in order to achieve therapeutic efficacy. The methods and compositions of the present disclosure address this need.SUMMARY

[0006] In some aspects, the present disclosure provides a method for stem cell therapy in a subject having an insulin deficiency, comprising administering to the subject a composition comprising a population of pancreatic islet cells, wherein the population of pancreatic islet cells is obtained by in vitro differentiation from a pluripotent stem cell prior to the administering, wherein the pluripotent stem cell comprises a loss of function mutation in a let methylcytosine dioxygenase 2 (TET2) gene, wherein the pluripotent stem cell is allogeneic with respect to the subject, wherein the population of pancreatic islet cells or a portion thereof undergoes engraftment following the administering, thereby treating the insulin deficiency in the subject.

[0007] In some embodiments of the foregoing or related aspects, the engraftment is characterized by (i) increased plasma levels of C-peptide when measured in a fasting state as part of a mixed meal tolerance test, (ii) reduction in HbAlc levels, and / or (iii) increased Time in Range (TIR). In some embodiments, the engraftment occurs without acute rejection of the population. In some embodiments, the engraftment is improved as compared to administering a control composition comprising a population of pancreatic islet cells, wherein the composition is obtained by in vitro differentiation from a pluripotent stem cell comprising a wild-type TET2 gene. In some embodiments, the engraftment is characterized by a reduced frequency of severe hypoglycemia, e.g,, as compared to prior to the administering. In some embodiments, the engraftment is maintained for a duration of time following the administering. In some embodiments, the duration of time is at least about 6 months, about 7 months, about 8 months, or about 9 months. In some embodiments, the engraftment is maintained in the absence of the subject receiving an immunosuppressant.

[0008] In some aspects, the disclosure provides a method for stem cell therapy in a subject having an insulin deficiency, comprising administering to the subject a composition comprising a population of pancreatic islet cells, wherein the population of pancreatic islet cells is obtained by in vitro differentiation from a pluripotent stem cell prior to the administering, wherein the pluripotent stem cell comprises a loss of function mutation in a TET2 gene, wherein the pluripotent stem cell is allogeneic with respect to the subject, and wherein the administering is329282749 2Attorney Docket: MINU-012 / 01WO 339336,2063associated with a reduced alloimmune response compared to administering a population of unmodified pancreatic islet ceils.

[0009] In some embodiments of the foregoing or related aspects, the reduced alloimmune response is characterized by increased plasma levels of C-peptide when measured in a fasting state as part of a mixed meal tolerance test, (ii) reduction in HbAlc levels, and / or (iii) increased TIR. In some embodiments, the unmodified population comprises of pancreatic islet cells obtained from a pluripotent stem cell comprising a wild-type TET2 gene. In some embodiments, the reduced alloimmune response is characterized by a reduced frequency of severe hypoglycemia, e.g., as compared to prior to the administering,

[0010] In some embodiments of the foregoing or related aspects, a plurality of the pancreatic islet cells in the population are insulin producing cells. In some embodiments, at least about 30% to about 80% of the population of pancreatic islet cells are insul in producing cells. In some embodiments, at least about 30% to about 60% of the population of pancreatic islet cells are insulin producing cells. In some embodiments, the insulin producing cells comprises beta cells. In some embodiments, the population further comprises alpha cells, delta cells, or a combination thereof. In some embodiments, less than about 10% of cells of the population are epsilon cells, pancreatic progenitor cells, acinar and ductal cells, enterochromaffin cells, and / or proliferative cells, optionally wherein less than 1% are proliferative cells. In some embodiments, a plurality of pancreatic islet cells in the population are present in cell clusters. In some embodiments, the cell clusters comprise a longest diameter of about 50 pm to 350 pm or about 100 pm to about 200 pm. In some embodiments, the cell clusters comprise about 500, about 600, about 700, about 800, about 900, about 1000, about 1100, about 1200, about 1300, about 1400, or about 1500 cells.

[0011] In some embodiments of the foregoing or related aspects, the composition further comprises a support factors, optionally wherein the support factor is selected from the group consisting of gamma-aminobutyric acid (GABA), parathyroid hormone (PTH), PTH-related peptide (PTHrP), vascular endothelial growth factor (VEGF), platelet- derived growth factor (PDGF), angiopoietin, and a combination thereof. In some embodiments, the support factor is a soluble factor. In some embodiments, the support factor is operably linked to a surface of a plurality of pancreatic islet cells in the population. In some embodiments, the factors are introduced to the composition following the in vitro differentiation and prior to the329282749 3Attorney Docket: MINU-012 / 01WO 339336,2063administering. In some embodiments, the composition comprises the population of pancreatic islet cells in a liquid suspension. In some embodiments, the composition comprises the population of pancreatic islet ceils in an encapsulation device or a degradable material.

[0012] In some embodiments of the foregoing or related aspects, the pluripotent stem cell is a human cell. In some embodiments, the pluripotent stem cell comprises an embryonic stem cell or an induced pluripotent stem cell. In some embodiments, the pluripotent stem cell is an embryonic pluripotent stem ceil. In some embodiments, the pluripotent stem cell is an induced pluripotent stem cell. In some embodiments, the in vitro differentiation comprises a step-wise differentiation of (i) the pluripotent stem cell to a definitive endoderm cell, (ii) the definitive endoderm cell to a posterior foregut cell, (iii) the posterior foregut cell to a pancreatic progenitor cell, and (iv) the pancreatic progenitor cell to the pancreatic islet cell. In some embodiments, differentiation of the pluripotent stem cell to the definitive endoderm cell comprises contacting the pluripotent stem cell with a differentiation factor selected from a ROCK inhibitor, a TGFP superfamily growth factor, an epidermal growth factor family polypeptide, a WNT activator, and a combination thereof. In some embodiments, the definitive endoderm cell is characterized by expression of CXCR4, SOX17, and / or FOXA2. In some embodiments, the definitive endoderm cell does not substantially express PDX1. In some embodiments, differentiation of the definitive endoderm cell to the posterior foregut cell comprises contacting the definitive endoderm cell with a differentiation factor selected from a growth factor of the fibroblast growth factor ( FGF) family, an activator of the retinoic acid signaling pathway, an activator of protein kmase C, an inhibitor of bone morphogenetic protein (BMP) signaling, an inhibitor of sonic hedgehog (SHH) signaling, and a combination thereof. In some embodiments, the posterior foregut cell is characterized by expression of PDX1, HNF6, FOXA2, HNF4A, HNF1B, and / or FOXA1. In some embodiments, differentiation of the posterior foregut cell to the pancreatic progenitor cell comprises contacting the posterior foregut cell with a differentiation factor selected from an epidermal growth factor (EGF), a retinoic pathway activator, a growth factor of the FGF family, and a combination thereof. In some embodiments, the pancreatic progenitor cell is characterized by expression of PDX1 and NKX6-1. In some embodiments, differentiation of the pancreatic progenitor cell to the pancreatic islet cell comprises contacting the pancreatic progenitor cell with a differentiation factor selected from an inhibitor of TGFP type I receptor, a thyroid hormone, a BMP signaling inhibitor, an inhibitor of gamma secretase, and a combination thereof.329282749 4Attorney Docket: MINU-012 / 01WO 339336,2063In some embodiments, the pancreatic islet cell is characterized by expression of PDX1, NKX6-1, and C-peptide. In some embodiments, the pancreatic islet cell is further characterized by expression of INS, PDX1, CHGA, SIX2, MAFA, NKX2.2, NEUROD, ISL1, UCN3, ENTPD3, and / or MAFB.

[0013] In some embodiments of the foregoing or related aspects, the population of pancreatic islet cells express an amount of PDX1, wherein the amount is at least about 70% to about 130% of the amount of PDX1 expressed by a control population of pancreatic islet cells comprising a wild-type TET2 gene. In some embodiments, the population of pancreatic islet cells express an amount of NKX6-1, wherein the amount is at least about 70% to about 130% of the amount of NKX6-1 expressed by a control population of pancreatic islet cells comprising a wild-type TET2 gene. In some embodiments, the population of pancreatic islet cells express an amount of ('-peptide, wherein the amount is at least about 40% to about 100% of the amount of C-peptide expressed by a control population of pancreatic islet cells comprising a wild-type TET2 gene.

[0014] In some embodiments of the foregoing or related aspects, the pluripotent stem cell comprises an alloantigen with respect to the subject. In some embodiments, the alloantigen comprises a major histocompatibility complex (MHC) class I molecule, an MHCII molecule, a minor histocompatibility antigen, a blood type antigen or a combination thereof. In some embodiments, the pluripotent stem cell is MHC -mismatched with respect to the subject. In some embodiments, the population of pancreatic islet cells is resistant to killing by an effector CD8+ T cell reactive to the alloantigen. In some embodiments, upon contacting the population of pancreatic islet cells with an effector CD 8+ T cell reactive to the alloantigen, the population is characterized by about 10% to about 90% higher viability as compared to a control population of pancreatic islet cells comprising a wild-type TET2 gene, optionally wherein the contacting comprises an effector cell to target cell ratio of about 1: 1 to about 10:1.

[0015] In some embodiments of the foregoing or related aspects, the population of pancreatic islet cells is characterized by expression of at least one diabetogenic antigen in an amount. In some embodiments, the at least one diabetogenic antigen is selected from the group consisting of: proinsulin, insulinoma-associated protein 2 (IA-2), zinc transporter 8 (ZnT8), islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), insulin, glutamic acid decarboxylase 65 (GAD65), chromogranin A (ChgA), islet amyloid polypeptide (IAPP), and a combination thereof. In some embodiments, the amount of the at least one diabetogenic antigen329282749 5Attorney Docket: MINU-012 / 01WO 339336,2063is at least about 70% to about 130% of the amount expressed by a control population of pancreatic islet cells comprising a wild-type TET2 gene. In some embodiments, the at least one diabetogenic antigen comprises proinsulin, IA-2, ZnT8, insulin, GAD65, ChgA, and IAPP. In some embodiments, the amount of the at least one diabetogenic antigen is less than about 70% of the amount expressed by a control population of pancreatic islet cells comprising a wild-type TET2 gene. In some embodiments, the at least one diabetogenic antigen comprises IGRP.

[0016] In some embodiments of the foregoing or related aspects, upon contacting the population of pancreatic islet cells with an inflammatory cytokine, the population is characterized by a survival property as compared to a control population of pancreatic islet cells comprising a wildtype TET2 gene, wherein the survival property is selected from the group consisting of: (i) decreased endosomal stress response; (ii) decreased inflammatory response; (ui) decreased activation of cellular death pathways; (iv) decreased cell apoptosis; and (v) a combination of (i)-(iv). In some embodiments, the inflammatory cytokine is selected from the group consisting of: TNFa, IL-1 P, IFNy, IL-6, and a combination thereof. In some embodiments, the endosomal stress response is characterized by expression of a gene selected from the group consisting of: ATF4, BiP, CHOP, ATF3, ATF6, XBP1, and a combination thereof. In some embodiments, the inflammatory stress response is characterized by expression of a gene selected from the group consisting of: CXCL10, STAT1, CXCL16, CD47, MX1, CCL2, TNF, and a combination thereof. In some embodiments, the cellular death pathway is characterized by expression of a gene selected from the group consisting of: FAS, PDL1, IDO, BACH1, BACH2, CASP3, and a combination thereof.

[0017] In some embodiments of the foregoing or related aspects, upon contacting the population of pancreatic islet cells with ER stress inducer, the population is characterized by increased viability as compared to a control population of pancreatic islet cells comprising a wild-type TET2 gene.

[0018] In some embodiments of the foregoing or related aspects, the loss of function mutation is introduced to the pluripotent stem cell prior to the in vitro differentiation. In some embodiments, the loss of function mutation is introduced using a gene editing system. In some embodiments, the gene editing system comprises a CRISPR / Cas system. In some embodiments, the gene editing system introduces a deletion to the TET2 gene. In some embodiments, the gene editing system introduces a frameshift mutation to the TET2 gene. In some embodiments, the pluripotent329282749 6Attorney Docket: MINU-012 / 01WO 339336.2063stem cell comprises a loss of function mutation in one or both alleles comprising the TET2 gene. In some embodiments, the pluripotent stem cell comprises the loss of function mutation in one allele comprising the TET2 gene. In some embodiments, the pluripotent stem cell comprises the loss of function mutation in both alleles comprising the TET2 gene. In some embodiments, the deletion spans a region of the TET2 gene, wherein the region comprises all or a portion of exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, or exon 11. In some embodiments, the region comprises a portion of exon 3. In some embodiments, the region comprises a 3' portion of exon 8, exon 9, and a 5' portion of exon 10.

[0019] In some embodiments of the foregoing or related aspects, the composition is implanted at an extrahepatic site. In some embodiments, the composition is implanted at a subcutaneous or intramuscular site. In some embodiments, the pancreatic islet cells comprise a function of a human donor pancreatic islet selected from the group consisting of (i) glucose-responsive secretion of C-peptide; (ii) glucose-responsive secretion of insulin; (lii) insulin granule biogenesis, trafficking, and / or exocytosis; and (iv) a combination of (i)-(iii).

[0020] In some embodiments of the foregoing or related aspects, the pancreatic islet cells secrete an increased level of insulin in response to an increased level of glucose. In some embodiments, the pancreatic islet cells are characterized by a glucose stimulated insulin secretion (GSIS) response. In some embodiments, the GSIS response is in vitro and / or in vivo. In some embodiments, the pancreatic islet cells secrete an increased level of C-peptide in response to an increased level of glucose.

[0021] In some embodiments of the foregoing or related aspects, prior to the administration of the composition, the subject received a daily infusion of insulin. In some embodiments, the subject has a medical history of severe hypoglycemic events. In some embodiments, the subject is characterized by no residual endogenous islet cell function. In some embodiments, prior to the administration of the composition, the subject has undetectable blood C-peptide level when measured using a mixed meal tolerance test. In some embodiments, the insulin deficiency is characterized by high blood sugar levels for a prolonged period of time. In some embodiments, the insulin deficiency is Type 1 diabetes. In some embodiments, the insulin deficiency is Type 2 diabetes. In some embodiments, the subject is human.BRIEF DESCRIPTION OF THE FIGURES329282749 7Attorney Docket: MINU-012 / 01WO 339336,2063

[0022] FIG. 1A is a schematic showing an exemplary CRISPR / Cas approach described herein to generate a TET2 knockout (KO). The approach involves dual guide RNAs to introduce a deletion of a genomic region spanning exon 9 of TET2.

[0023] FIG. IB is a schematic showing the portion of the TET2 protein deleted as a result of CRISPR / Cas gene editing, which begins within the first DSBH (doubles stranded P-helix) of the catalytic domain of TET2.

[0024] FIG. 1C is a gel electrophoresis image showing presence of gene fragments expected for the TET2 gene truncation represented in FIG. 1 A in embryonic stem cells (ESCs) having the TET2 KO, Control cells were wild-type (WT) ESCs.

[0025] FIG.2A is a graph showing expression of TET2 is increased over the course of differentiation from WT ESCs to pancreatic islets organoids.

[0026] FIG.2B is a graph showing no expression of TET2 is observed in pancreatic islet organoids differentiated from a clonal population of TET2 KO ESCs (clone 15 or 59). Control pancreatic islet organoids were differentiated from WT ESCs.

[0027] FIG.2C is a graph showing TET2 activity is reduced in pancreatic islet organoids differentiated from a clonal population of TET2 KO ESCs (clone 15 or 59) as compared to control pancreatic islet organoids differentiated from WT ESCs.

[0028] FIGs.3A-3B are graphs showing expression of pancreatic beta cell markers PDX1 (FIG.3A) and NKX6.1 (FIG. 3B) is comparable between pancreatic islet organoids differentiated from a clonal population of TET2 KO ESCs (clone 15 or 59) and from WT ESCs.

[0029] FIG.4A are brightfield (top) and fluorescence (bottom) images of islet organoids differentiated from a clonal population of TET2 KO ESCs (clone 15 or 59) or from WT ESCs, showing comparable morphology and expression of C-peptide.

[0030] FIG. 4B are flow cytometry scatter plots showing cells positive for NKX6.1 and / or C-peptide expression in a population of islet organoids differentiated from a clonal population of TET2 KO ESCs. Control islet organoids were differentiated from WT ESCs.

[0031] FIGs.4C-4D are graphs showing glucose-responsive C-peptide secretion is comparable between islet organoids differentiated from a clonal population of TET2 KO ESCs (clone 15) and from WT ESCs.

[0032] FIG. 5 is a graph showing elevated human insulin levels in serum (micro units per niL) under glucose-stimulated (T30) vs fasting conditions (TO) for mice that received a transplant of329282749 8Attorney Docket: MINU-012 / 01WO 339336.2063islet organoids differentiated from a clonal population of TET2 KO ESCs (clone 15 or 59) or WT ESCs.

[0033] FIG. 6A is a graph showing pancreatic islet cells differentiated from a clonal population of TET2 KO ESCs (clone 15 or 59) are more resistant to killing by effector CD8+ T cells selected for reactivity to diabetic autoantigens than pancreatic islet cells differentiated from WT ESCs. Effector and target cells were combined in the killing assay at the indicated (E: T) ratio.

[0034] FIG. 6B is a graph showing expression of HLA-A2.1 is comparable for pancreatic islet cells differentiated from a clonal population of TET2 KO ESCs (clone 15 or 59) or WT ESCs following culture without IFNy (left) or with IFNy (right).

[0035] FIG.6C is a graph showing expression of diabetic autoantigens by pancreatic islet cells differentiated from a clonal population of TET2 KO ESCs (clone 15 or 59) or from WT ESCs with or without cytokine stimulation (“cytoplus”).

[0036] FIG. 7 is a graph showing pancreatic islet cells differentiated from a clonal population of TET2 KO ESCs (clone 15 or 59) and pulsed with diabetic autoantigens are more resistant to killing by effector CD8 + T cells reactive to the diabetic autoantigens relative to pancreatic islet cells differentiated from WT ESCs.

[0037] FIG. 7B is a graph showing pancreatic islet cells differentiated from a clonal population of TET2 KO ESCs (clone 15 or 59) and pulsed with EBV are more resistant to killing by effector CD8+ T cells reactive to EBV relative to pancreatic islet cells differentiated from WT ESCs.

[0038] FIG. 8A is a graph showing reduced expression of genes associated with ER stress response by pancreatic islet cells differentiated from a clonal population of TET2 KO ESCs (clone 15 or 59) relative to WT ESCs following cytokine stimulation (“cytoplus”).

[0039] FIG. 8B is a graph showing reduced expression of genes associated with inflammation response by pancreatic islet cells differentiated from a clonal population of TET2 KO ESCs (clone 15 or 59) relative to WT ESCs following exposure to cytokine stimulation (“cytoplus”).

[0040] FIG. 8C is a graph showing reduced expression of STAT1 and CXCL10 by pancreatic islet cells differentiated from a clonal population of TET2 KO ESCs (clone 15 or 59) relative to WT ESCs following exposure to elevated levels of cytokine stimulation.

[0041] FIG. 8D is a graph showing reduced expression of cell death pathway genes by pancreatic islet cells differentiated from a clonal population of TET2 KO ESCs (clone 15 or 59) relative to WT ESCs following exposure to cytokine stimulation.329282749 9Attorney Docket: MINU-012 / 01WO 339336.2063

[0042] FIG.9 is a graph showing a higher proportion of living cells following exposure to cytokine stimulation relative to unstimulated for pancreatic islet cells differentiated from a clonal population of TET2 KO ESCs (clone 15 or 59) relative to pancreatic islet cells differentiated from WT ESCs.

[0043] FIG. 10 is a graph showing an increased percentage of live cells following exposure to ER stress inducers for pancreatic islet cells differentiated from a clonal population of TET2 KO ESCs (clone 15 or 59) relative to WT ESCs.

[0044] FIG. 11 is a graph showing pancreatic islet cells differentiated from a clonal population of TET2 KO iPSCs (produced from a HL A-A2.1 + donor) are more resistant to killing by effector CD8+ T cells selected for HLA-A2.1+ reactivity relative to pancreatic islet cells differentiated from WT iPSCs.DETAILED DESCRIPTION

[0045] The present disclosure provides compositions of stem cell-derived (SCD) pancreatic islets, and methods of use thereof for beta cell replacement, e.g., in a subject having an insulin deficiency.

[0046] Pluripotent stem cells offer an unlimited cell source for producing cellular replacement therapies. Islets derived from pluripotent stem cells offer a promising cell therapy for restoring pancreatic function, e.g,, in subject having diabetes. One source of pluripotent stem cells is induced pluripotent stem cells (iPSCs) that are generated from the subject’s own somatic cells (see, e.g,, Cell (2024) 187:6152), For example, somatic cells (e.g., adipose cells) are harvested from the subject, reprogrammed to iPSCs, and differentiated to islets that are transplanted in the subject. A benefit of this approach is the subject is less likely to experience a host-versus-graft response, at least because the transplanted cells lack non-self antigens that have the potential to trigger an alloimmune response in the subject. However, this approach is typically labor intensive and expensive as it requires harvesting and manipulating the subject’s cells.

[0047] To make SCD pancreatic islet therapies more cost-effective and accessible, it is desirable to develop an allogeneic transplantation strategy, m which SCD pancreatic islets are derived from an allogeneic stem cell source. Allogeneic stem cells are preferred for generating islets, as they provide an accessible, readily administered product that may be selected for desirable clinical attributes (e.g., insulin production). However, allogeneic cell therapies are at risk for329282749 10Attorney Docket: MINU-012 / 01WO 339336,2063immune rejection. Immune rejection of allogeneic cells results, at least in part, from the recipient immune’s system recognizing the implanted donor cells as foreign or “non-self.” Rejection of transplanted cells can occur over a timescale of minutes / hours (hyperacute), days / months (acute), and months / years (chronic). This rejection results from action of the innate and adaptive immune system (see, e.g., Murphy et al (2011) Immunol Rev 241:39-48; Issa et al (2010) Expert Rev Clin Immunol 6:155-69). Current approaches to prevent rejection of an allograft includes treatment with immunosuppressant drugs. However, these drugs must be taken for the remainder of the subject’s life, and do not always work. Moreover, they are systemic-acting and leave the subject immunocompromised with an increased risk of cancer and / or infection.

[0048] Allogeneic SCD pancreatic islets are also at risk of autoimmune rejection in subjects having 1D. Autoimmune rejection of beta cells is the primary driver for T1D, and typically results, at least in part, from destruction of beta cells by islet-reactive effector CD8+ T cells (see, e.g., Burrack et al (2017) Front Endocrinol Vol 8), as well as by pro-inflammatory cytokines (e.g., IFNy and TNFa) secreted by, e.g., autoreactive CD4+ and CD8+ T cells and innate immune cells. As a result, for transplantation of SCD pancreatic islet cells in recipients having T1D (allogeneic or autologous), the recipient’s autoimmune response against beta cells can result in killing of the transplanted cells. Factors such as inflammation, impaired insulin processing, endoplasmic reticulum stress, and oxidative stress can also damage the transplanted cells, making them more susceptible to killing by activated immune cells. Immunosuppressive drugs used to prevent allograft rejection don’t fully block the underlying autoimmune response, resulting in graft loss over time. Thus, SCD pancreatic islet cells engineered for immune protection against both alloimmune and autoimmune responses are needed for an effective stem cell therapy for treatment of T1D.

[0049] As described herein, it was surprisingly discovered that SCD pancreatic islet cells comprising a loss of function mutation in the epigenetic modulator TET2 are protected from autoimmune responses associated with cell killing in T1D, as well as immune responses directed to alloantigens. As shown herein, modified SCD pancreatic islet cells comprising a loss of function mutation in TET2 are more resistant to cell killing by effector CD8+ T cells reactive to islet autoantigens, as well as to effector CD8+ T cells reactive to alloantigens, as compared to control SCD pancreatic islet cells comprising wild-type TET2. Moreover, it is shown that the modified SCD pancreatic islet cells are resistant to expression of genes associated with ER stress329282749 11Attorney Docket: MINU-012 / 01WO 339336,2063response, inflammation, and cell death when contacted with pro-inflammatory cytokines, as compared to the control SCD pancreatic islet cells. Without being bound by theory, SCD pancreatic islet cells comprising a TET2 loss of function mutation (e.g., in both alleles encoding TET2) are resistant to islet- and alloantigen-specific immune responses, as a result of reduced expression of genes associated with cell death in the presence of pro-inflammatory cytokines and / or reduced susceptibility to killing by reactive effector CD8+ T cells.

[0050] It was further determined that pluripotent stem cells comprising a loss of function mutation in TET2 are effectively differentiated into functional pancreatic islets. This was surprising, given that TET2 function is associated with numerous processes that occur during differentiation of pluripotent stem cells to functional cells (e.g., pancreatic islet cells) (see, e.g., Yang, et al (2020) Development 147: dev 183129), and it was appreciated that one or more of these processes could be hampered by eliminating TET2 function in pluripotent stems cells when induced to differentiate. Although, other TET proteins (including TET1 and TET3) are expressed, the function, DNA targets, and expression patterns of the TET family member protein do not fully overlap, such that loss of one TET protein can detrimentally affect normal cellular processes. Despite this, it was shown that pluripotent stem cells (including embryonic stem cells or induced pluripotent stem cells) are differentiated to pancreatic islet cells comprising functionality substantially comparable to control SCD pancreatic islet cells derived from pluripotent stem cells comprising wild-type TET2. Such functionality includes secretion of insulin and C-peptide in response to glucose stimulation (in vitro or in vivo), and comparable levels of expression of markers associated with β cells (e.g., PDX1 and NKX6.1) to control SCD pancreatic islet cells.

[0051] Accordingly, the present disclosure provides compositions of allogeneic SCD pancreatic islet cells engineered for immune suppression, e.g., when administered to a subject. In some embodiments, the SCD pancreatic islet cells comprise a gene disruption that reduces immune rejection following administration to a subject. In some embodiments, the gene disruption reduces an alloimmune response and / or an autoimmune response. In some embodiments, the gene disruption comprises a loss of function mutation in a gene encoding an epigenetic modulator. In some embodiments, the gene disruption comprises a loss of function mutation in a gene encoding a ten-eleven-translocation methylcytosine dioxygenase (TET) protein. In some embodiments, the gene encodes TET2 (“TET2 gene”). Sequence information for the TET2 gene329282749 12Attorney Docket: MINU-012 / 01WO 339336,2063is provided in the NCBI database under gene ID number 54790. Sequence information for the protein encoded by the TET2 gene is provided in the Uniprot database under accession number Q6N021. In some embodiments, the gene disruption comprises a loss of function mutation in a TET2 gene, wherein the loss of function mutation results in a reduced expression and / or reduced activity of a transcriptional or translational product of the TET2 gene.Definitions

[0052] In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0053] As used herein, the term “stem cell-derived cell” or “SCD cell” are interchangeably used to refer to a cell differentiated from a stem cell (such as a pluripotent stem cell described herein (e.g., embryonic stem cells, induced pluripotent stem cells (iPSC)) a multipotent cell, or an unipotent cell), a precursor cell differentiated from a stem cell, or a partially reprogrammed somatic cell (i.e., a somatic cell which has been partially reprogrammed to an intermediate state between an iPSC and the somatic cell from which it was derived). The term is intended to encompass cells differentiated from the indicated source cell using human or machine intervention.

[0054] As used herein, the term “stem cell” refers to cells of the body characterized by the potential to differentiate into a cell having a more specialized or differentiated phenotype and the potential to proliferate without substantially differentiating. For example, in some embodiments, a stem cell refers to an undifferentiated mother cell whose descendants (progeny) specialize by differentiation, e.g., by acquiring one or more functional properties. Stem cells are classified, at least in part, based upon their development potential. A stem cell that is “totipotent” refers to one having the capacity to differentiate into all embryonic and extraembryonic cell types. A stem cell that is “pluripotent” refers to one having the capacity to differentiate to each of the three germ cell layers. A stem cell that is “multipotent” refers to one having the capacity to differentiate into all cell types within one particular lineage (e.g., hematopoietic stem cells are multipotent stem cells having capacity to differentiate into all blood cell types). A stem cell that is “unipotent” refers to one having the capacity to differentiate into a single cell lineage (e.g., spermatogenic stem cells).329282749 13Attorney Docket: MINU-012 / 01WO 339336,2063

[0055] As used herein, the term “differentiate” or “differentiation” refers to the process in which a stem cell progresses from a less specialized state to a more specialized state.

[0056] As used herein, the term “pluripotent cell” refers to a cell characterized by the capacity to self-renew and proliferate while remaining in an undifferentiated state and that can, under proper conditions, be induced to differentiate into a more specialized cell type.

[0057] As used herein, the term “pluripotent stem cell” refers to a pluripotent cell having the potential to differentiate into any of the three germ layers: endoderm (e.g., the stomach lining, gastrointestinal tract, lungs, etc,), mesoderm (e.g., blood, bone, muscle, urogenital tissue, etc), or ectoderm (e.g., epidermal tissue and nervous system tissue).

[0058] As used herein, the terms “progenitor” and “precursor” cell are used interchangeably herein and refer to cells that have a cellular phenotype that is more primitive (e.g., is at an earlier step along a developmental pathway or progression than is a fully differentiated cell) relative to a cell which it can give rise to by differentiation. In some embodiments, a progenitor cell gives rise to multiple distinct differentiated cell types or to a single differentiated cell type by exposure to an environment and set of conditions that promote differentiation along a certain developmental pathway.

[0059] As used herein, the term “endoderm” refers to a cell of one of the three primary germ cell layers in the early embryo (the other two germ cell layers are mesoderm and ectoderm). An endoderm cell differentiates to give rise to the embryonic gut and then to linings of the respiratory tract, the digestive tract, the liver, and the pancreas. The term encompasses endogenous endoderm cells in a subject and SCD endoderm cells (e.g., endoderm cells differentiated from a pluripotent stem cell, such as by the methods described herein).

[0060] A “cell of endoderm origin” refers to one having differentiated from an endoderm cell. Exemplary cells of endoderm origin include, but are not limited to cells of the liver, lung, pancreas, thymus, intestine, stomach, and thyroid. In some embodiments, the cell of endoderm origin is a definitive endoderm cell.

[0061] As used herein, the term “definitive endoderm” refers to a cell differentiated from an endoderm cell and which retains capability to be differentiated into cells of the liver, lung, pancreas, thymus, intestine, stomach and / or thyroid. In some embodiments, the definitive endoderm cell is characterized by expression of one or more cell markers. In some embodiments, the one or more cell markers comprises SOX17. In some embodiments, the one or more cell329282749 14Attorney Docket: MINU-012 / 01WO 339336,2063markers comprises SOX17, CXCR4, FOXA2, and CD177. In some embodiments, the definitive endoderm cells do not substantially express PDX1 (i.e., PDX1 negative). In some embodiments, the definitive endoderm cells do not substantially express pluripotency markers such as OCT3 / 4, SOX2, TRA-1-60, and NANOG (i.e., negative for these proteins.) The expression of SOX17 and other cell markers of definitive endoderm may be assessed by any method known by the skilled person such as immunochemistry or flow cytometry, e.g., using marker-specific antibody, or quantitative RT-PCR.

[0062] As used herein, the term “posterior foregut” refers to a cell differentiated from a definitive endoderm cell and which retains capability to be differentiated into cells of the lung, liver, pancreas, stomach, and intestine. In some embodiments, the posterior foregut cell is characterized by expression of one or more cell markers. In some embodiments, the one or more cell markers comprises CDX2, FOXA2, HNF6, SOX17, HNF4A, HNF1B, FOXA1, and c-KIT In some embodiments, the posterior foregut cell is FOXA-2 positive. In some embodiments, the posterior foregut cell expresses SOX2.

[0063] A cell of the “pancreatic endoderm” refers to a cell differentiated from definitive endoderm that retains the capability to differentiate into multiple pancreatic lineages, but no longer has the capacity to differentiate into non-pancreatic lineages accessible to definitive endoderm cells. The term encompasses endogenous pancreatic endoderm cells in a subject and pancreatic endoderm cells differentiated from a stem cell (e.g., a pluripotent stem cell), such as by the methods described herein.

[0064] As used herein, the term “pancreatic progenitor,” “pancreatic endocrine progenitor,” “pancreatic precursor,” pancreatic endocrine precursor” are interchangeably used to refer to a cell of endoderm origin that has the capacity to differentiate into a pancreatic endocrine cell, pancreatic exocrine cell, or a pancreatic duct cell. Such cells are committed to differentiating to at least one type of pancreatic cell, e.g., a pancreatic α cell, β cell, δ cell, PP cell, or ε cell, acinar cell, duct cell etc. The term encompasses endogenous pancreatic progenitor cells in a subject and pancreatic progenitor cells differentiated from a stem cell (e.g., a pluripotent stem cell), such as by the methods described herein. Pancreatic progenitor cells are characterized by expression of at least one of the following cell markers: NGN3, NKX2.2, NKX6.1, NEUROD, ISL-1, PAX4, PAX6, SOX9, HNF6, FOXA2, PDX1, and ARX. The expression of cell markers of pancreatic progenitor cell may be assessed by any method known by the skilled person such as329282749 15Attorney Docket: MINU-012 / 01WO 339336,2063immunochemistry or flow cytometry, e.g., using marker-specific antibody, or quantitative RT-PCR.

[0065] As used herein, the term “PDX- 1 -positive pancreatic progenitor” refers to a pancreatic endoderm cell having capacity to differentiate into a pancreatic cell or P-like cell. A PDX-1 positive cell is characterized by expression of pancreatic and duodenal homeobox 1 (PDX-1). In some embodiments, the PDX-1 positive cell is further characterized by expression of at least one of the following cell markers: PTF1 A, HNF6, NKX2.2, HNF4A, HNF1B, FOXA1, FOXA2, SOX9, NGN3, and NKX6.1. In some embodiments, the PDX-1 positive cell is further characterized by expression of NKX6.1 (referred to herein as a “PDX-1, NKX6.1 -positive pancreatic progenitor”).

[0066] As used herein, the term “insulin-producing cell” refers to any cell that produces, stores, and / or secretes detectable amounts of insulin. In some embodiments, an insulin producing cell has the capacity to produce and secrete insulin similar to that produced and secreted by a beta cell of the islets of Langerhans. The term encompasses pancreatic β cells (e.g., human donor pancreatic β cells), pancreatic-like β cells, and SCD β cells that synthesize insulin (i.e., transcribe the insulin gene and translate the mRNA product thereof to generate insulin), express insulin (e.g., package and store insulin into secretory granules), and secrete insulin (e.g., release insulin into the extracellular space) in a constitutive or inducible manner. In some embodiments, a population of insulin producing cells is characterized by a glucose stimulated insulin secretion (GSIS) response that resembles that exhibited by a pancreatic β cell. In some embodiments, the population of insulin producing cells comprises non-insulin producing cells.

[0067] As used herein, the term “glucose stimulated insulin secretion response” or “GSIS response,” used interchangeably herein, refers to secretion of insulin by an insulin producing cell upon contact with glucose. According to the canonical model, the GSIS response results from the uptake of glucose (through a glucose transporter) by the insulin producing cell, which in turn increases production of adenosine triphosphate (ATP). An increase in the ATP / ADP ratio results in closing of potassium channels of the cell, resulting in depolarization of the cell and opening of calcium channels. This results in insulin secretion. In some embodiments, the GSIS response is measured in vitro by contacting the insulin producing cell with glucose at a concentration of about 1 mM to about 30 mM and measuring extracellular secretion of insulin (or c-peptide as an insulin surrogate) using, e.g., an ELISA. Functional assessment of purified human pancreatic329282749 16Attorney Docket: MINU-012 / 01WO 339336,2063islets: glucose stimulated insulin release by ELISA - A Standard Operating Procedure of the NIH Clinical Islet Transplantation Consortium CellR42014; 2(2):e900 for methods to measure GSIS in vitro. In some embodiments, an increase in extracellular secretion of insulin as compared to an insulin producing cell not contacted with glucose indicates a GSIS response. In some embodiments, the extracellular secretion of insulin is at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50% of the total insulin stored by the insulin producing cell following contacting with glucose (e.g., at a concentration of about 1 mM to about 30 mM). In some embodiments, the extracellular secretion of insulin is about 1% to about 20%, of the total insulin stored by the insulin producing cell following contacting with glucose (e.g., at a concentration of about 1 mM to about 30 mM). In some embodiments, the GSIS response is measured in vivo by administering a glucose solution to a subject following a period of fasting and measuring blood insulin levels. In some embodiments, an increase in blood insulin levels following the administering as compared to prior to the administering indicates a GSIS response.

[0068] As used herein, the term “SCD P cell” or “stem cell-derived cell” are interchangeably used to refer to an SCD cell that displays at least one pancreatic P cell marker, expresses insulin, and displays a glucose stimulated insulin secretion (GSIS) response mimicking an endogenous mature pancreatic cell.

[0069] As used herein, the term “pancreatic P cell marker” refers to polypeptides, nucleic acids, metabolites, or analytes expressed by pancreatic P cells. In some embodiments, the pancreatic β cell marker is selected from: NKX6.1, PDX1, insulin, c-peptide, amylin, E-cadherin, Hnf3β, PC1 / 3, GLUT2, PC2, ZnT-8, ISL1, Pax6, Pax4, NeuroD, Hnf1b, Hnf-6, MAFA, MAFB, NKX2.2, PHB, UCN3, ENTPD3, and a combination thereof. In some embodiments, the pancreatic P cell markers comprise NKX6.1 and c-peptide. In some embodiments, the pancreatic P cell markers comprise NKX6.1 and insulin. In some embodiments, the pancreatic P cell markers comprise C-peptide, PDX1, NKX6.1, MAFA, MAFB, UCN3, and ENTPD3.

[0070] As used herein, the term “SCD a cell” or “stem cell-derived a cell” are interchangeably used to refer to an SCD cell that displays at least one marker of a pancreatic a cell (e.g., GCG, PDX1, IRX, PC2), does not express a pancreatic β cell marker (e.g., PCI, NKX6-1), a pancreatic 8 cell marker (e.g., somatostatin), a pancreatic y cell marker (e.g., pancreatic polypeptide), or a pancreatic s cell marker (e.g., ghrelin), and express and secrete glucagon. In some embodiments,329282749 17Attorney Docket: MINU-012 / 01WO 339336,2063a SCD a does not express a pancreatic hormone other than glucagon. In some embodiments, a SCD a cell does not express insulin and / or somatostatin.

[0071] As used herein, the term “SCD 5 cell” or “stem cell-derived 6 cell” are interchangeably used to refer to an SCD cell that displays at least one marker of a pancreatic 6 cell and express and secrete somatostatin. In some embodiments, a SCD δ cell does not express a pancreatic hormone other than somatostatin. In some embodiments, a SCD δ cell does not express insulin, glucagon, pancreatic polypeptide, ghrelin, or a combination thereof.

[0072] As used herein, the term “SCD y cell” or “stem cell-derived y cell” are interchangeably used to refer to an SCD cell that displays at least one marker of a pancreatic y cell and express and secrete pancreatic polypeptide. In some embodiments, a SCD y cell does not express a pancreatic hormone other than pancreatic polypeptide. In some embodiments, a SCD y cell does not express insulin, glucagon, somatostatin, Ghrelin, or a combination thereof.

[0073] As used herein, the term “SCD s cell” or “stem cell-derived s cell” are interchangeably used to refer to an SCD cell that displays at least one marker of a pancreatic s cell and express and secrete ghrelin. In some embodiments, a SCD ε cell does not express a pancreatic hormone other than ghrelin. In some embodiments, a SCD ε cell does not express insulin, glucagon, somatostatin, pancreatic polypeptide, or a combination thereof.

[0074] As used herein, the term “cell cluster,” “cluster,” “cell aggregate,” or “aggregate” are interchangeably used to refer to a group of cells having direct cell-to-cell contact. For example, in some embodiments, a plurality of cells in a cell cluster are in directed contact with a least one additional cell present m the cluster.

[0075] As used herein, the term “longest diameter” in reference to a cell cluster described herein refers to the distance between the two farthest points present on the surface of the cell cluster.

[0076] As used herein, the term “allogeneic” refers to a cell, a cell population, or a biological sample comprising the cells that are obtained from a donor that is different than the subject that receives the cell, cell population, or biological sample, or a downstream product thereof. For example, the term encompasses stem cells or progenitor cells that are derived from a donor and differentiated into SCD cells that are administered to a subject that is different than the donor. A transplant comprising the allogeneic cells is referred to as an “allogeneic transplant.” A subject’s immune response against an allogeneic transplant is referred to as an “allogeneic immune response.”329282749 18Attorney Docket: MINU-012 / 01WO 339336,2063

[0077] As used herein, the term “autologous” refers to a cell, a cell population, or a biological sample comprising the cells that are obtained from an individual that also receives the cell, cell population, or biological sample, or a downstream product thereof. For example, the term encompasses adult somatic cells derived from an individual that are reprogrammed (e.g. reprogrammed to iPSCs) ex vivo, differentiated to SCD cells, and administered to the same individual. A transplant comprising the autologous cells is referred to as an “autologous transplant.”

[0078] As used herein, the term “Major histocompatibility complex class I” or “MHC-I” generally refer to a class of biomolecules that are found on the cell surface of all nucleated cells in vertebrates, including mammals, e.g., humans; and function to display peptides of non-self or foreign antigens, e.g, proteins, from within the cell (i.e. cytosolic) to cytotoxic T cells, e.g, CD8+ T cells, in order to stimulate an immune response. In some embodiments, a MHC-I biomolecule is HLA-A (NCBI Gene ID No: 3105), HLA-B (NCBI Gene ID No: 3106), HLA-C (NCBI Gene ID No: 3107), or B2M (NCBI Gene ID No: 567).

[0079] As used herein, the term “Major histocompatibility complex class II” or “MIIC-II” generally refer to a class of biomolecules that are typically found on the cell surface of antigen-presenting cells in vertebrates, including mammals, e.g., humans; and function to display peptides of non-self or foreign antigens, e.g., proteins, from outside of the cell (extracellular) to cytotoxic T cells, e.g., CD8+ T cells, m order to stimulate an immune response. In some embodiments, a MHC-II biomolecule is HLA-DPA(NCBI Gene ID No: 3113), HLA-DPB (NCBI Gene ID No: 3115), HLA-DMA (NCBI Gene ID No: 3108), HLA-DMB (NCBI Gene ID No: 3109), HLA-DOA (NCBI Gene ID No: 3111), HLA- DOB (NCBI Gene ID No: 3112), HLA-DQA (NCBI Gene ID No: 3117), HLA-DQB (NCBI Gene ID No: 3119), HLA-DRA (NCBI Gene ID No: 3122), or HLA-DRB (NCBI Gene ID No: 3123).

[0080] As used herein, the term “unmodified” in reference to a population of cells (e.g., a population of pancreatic islet cells) refers to a population of cells that has not been subjected to a gene disruption of the disclosure (e.g., a loss of function mutation in the TET2 gene).

[0081] As used herein, the term “support factor” refers to any component that when combined with a SCD pancreatic islet composition of the disclosure, promotes a desired function of the cell composition compared to a cell composition lacking the support factor. The term encompasses, but is not limited to, components that are a molecule (e.g., a nucleic acid, protein, hormone,329282749 19Attorney Docket: MINU-012 / 01WO 339336,2063carbohydrate, lipid, steroid, a pharmacological agent) or a cell (e g., a CD34+ cell or a cell derived from PTG tissue). The desired functions encompass, but are not limited to, improved survival (e.g., in vitro or in vivo), insulin production (for an insulin producing cell), and / or tolerance to hypoxic and / or nutrient deficient conditions.

[0082] As used herein, the term “diabetes” or “diabetic disorder” or “diabetes mellitus,” are used interchangeably to refer to a disease which is marked by elevated levels of sugar (glucose) in the blood. Diabetes can be caused by too little insulin (a protein produced by the pancreas to regulate blood sugar), resistance to insulin, or both. Diabetes mellitus includes, without limitation, type 1 diabetes, type 2 diabetes, or surgical diabetes,

[0083] As used herein, the term “type 1 diabetes,” as used herein, refers to a chronic disease that occurs when the pancreas produces too little insulin to regulate blood sugar levels appropriately. Type 1 diabetes is also interchangeably referred to as “insulin-dependent diabetes mellitus,” “IDDM,” “juvenile onset diabetes,” “autoimmune diabetes,” and “diabetes — type 1.” Type 1 diabetes is the result of a progressive autoimmune destruction of pancreatic p cells with subsequent insulin deficiency.

[0084] As used herein, the term "type 2 diabetes" (also referred to as “non-insulin-dependent diabetes mellitus” or “adult-onset diabetes”) refers to a metabolic disorder in individuals who exhibit insulin resistance and who usually exhibit relative, rather than absolute, insulin deficiency. Illustrative, but non limiting criteria for determining whether an individual has type 2 diabetes, include one or more of the following: (1) a confirmed fasting plasma glucose value of greater than or equal to 126 milligrams / deciliter (mg / dL), (2) in the presence of symptoms of diabetes, a confirmed non-fasting plasma glucose value of greater than or equal to 200 mg / dL, and (3) with an oral glucose tolerance test (by administering 75 grams of anhydrous glucose dissolved in water, in accordance with World Health Organization standards, and then measuring the plasma glucose concentration 2 hours later), a confirmed glucose value of greater than or equal to 200 mg / dL.

[0085] As used herein, the term “surgically induced diabetes” or “surgical diabetes” refers to diabetes cause by some surgical procedure, such as when surgery on the pancreas impacts its ability to produce insulin either permanently or temporarily.329282749 20Attorney Docket: MINU-012 / 01WO 339336,2063

[0086] As used herein, the term “transplanting” refers to placement of cells or cell clusters of the cell composition into a subject by a method or route of administration that results in at least partial localization of the administered cell composition at the transplant site.

[0087] As used herein, the term “treating” and “treatment” refer to administering to the subject an effective amount of the cell composition such that the subject experiences a reduction in at least one symptom of the disease and / or an improvement in a clinical outcome associated with the disease. In some embodiments, the clinical outcome comprises alleviation of one or more symptoms, stabilized (e.g., not worsened) state of disease, delayed or slowed progression of the disease, ameliorated or palliation of the disease state, and remission (e.g., partial or total) of the disease. Treating can refer to prolonging survival as compared to expected survival if not receiving treatment. Treatment can further refer to an improvement in a clinical outcome without further therapeutic intervention,

[0088] As used herein, the terms “subject,” “patient,” or “individual” are used interchangeably herein, and refer to an animal, for example, a human from whom cells can be obtained and / or to whom treatment, including prophylactic treatment, with the cell compositions as described herein, is provided. The terms “non-human animals” and “non-human mammals” as used interchangeably herein, includes mammals such as rats, mice, rabbits, sheep, cats, dogs, cows, pigs, and non-human primates. The term “subject” also encompasses any vertebrate including but not limited to mammals, reptiles, amphibians and fish. “Patient m need thereof’ or “subject in need thereof’ is referred to herein as a patient diagnosed with or suspected of having a disease or disorder (e.g., diabetes).

[0089] As used herein, the term “parathyroid gland factor” or “PTG factor” are interchangeably used to refer to soluble factors present in parathyroid gland (PTG) tissue. Methods to identify soluble factors present in PTG tissue are known in the art (see, e.g., US Pat No 11,951,136). It is to be understood the term need not be limited to soluble factors harvested from PTG tissue and encompasses the soluble factors when obtained through other means (e.g., chemical synthesis, recombinant protein expression). Further, the term encompasses derivatives or variants of soluble present in PTG tissue. In some embodiments, the PTG factor comprises a proangiogenic factor. In some embodiments, the PTG factor comprises a hormone.

[0090] As used herein, the term “pro-angiogenic factor” refers to a molecule (e g., a polypeptide, small molecule, nucleic acid, etc) that triggers angiogenic signaling pathways to329282749Attorney Docket: MINU-012 / 01WO 339336.2063induce formation of new blood vessels. Exemplary pro-angiogenic factors of the disclosure include, but are not limited to, fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR), NRP-1, angiopoietin (Ang) 1, Ang2, platelet derived growth factor (PDGF), PDGFR receptor (PDGFR), TGFP, endoglin, TGFP receptor, CCL2, VE-cadherin, CD31, ephrin, plasminogen activators, AC 133, 1D1, semaphorins, and Nogo-A.

[0091] As used herein, the term “loss of function mutation” refers to a mutation within or proximal to a gene (e.g., within or proximal to the coding sequence, a transcriptional control element, and / or a splicing sequence of the gene), wherein the presence of the mutation results in a reduced expression level and / or activity of the transcriptional and / or translational product of the gene (e.g., as compared to the transcriptional and / or translational product of the gene in the absence of the mutation).

[0092] A “diabetogenic antigen” is a molecule (e.g,, antigenic peptide) present in pancreatic P cells that triggers an autoimmune attack in subjects having T1D.Methods of Treatment

[0093] Provided herein are methods for treating a subject having an insulin deficiency, comprising administering to the subject a SCD pancreatic islet composition described herein, or a pharmaceutical composition thereof, wherein the SCD pancreatic islet composition is generated from stem cells comprising a gene disruption of the disclosure (e.g., a gene disruption of the TET2 gene), wherein the stem cells are allogeneic with respect to the subject, and wherein the gene disruption improves survival, persistence, and / or a pancreatic function of the SCD pancreatic islet composition following administration to the subject.

[0094] In some embodiments, the SCD pancreatic islet composition comprises insulin producing cells (e.g., SCD p cells), wherein the insulin producing cells comprise the gene disruption (e.g., the gene disruption of the TET2 gene), and wherein the gene disruption improves survival, persistence, and or a pancreatic function of the insulin producing cells following administration to the subject.

[0095] In some embodiments, the gene disruption (e.g., the gene disruption of the TET2 gene) reduces an alloimmune response to cells (e.g., insulin producing cells) of the SCD pancreatic islet composition following transplantation. In some embodiments, the gene disruption reduces an autoimmune response to cells (e.g., insulin producing cells) of the SCD pancreatic islet329282749 22Attorney Docket: MINU-012 / 01WO 339336,2063composition following transplantation. In some embodiments, cells (e.g., insulin producing cells) of the SCD pancreatic islet composition are less immunogenic as compared to cells of the same type lacking the gene disruption.

[0096] SCD pancreatic islet cells differentiated from stem cells comprising a TET2 gene disruption are referred to herein as “TET2 engineered SCD pancreatic islet cells.”Engineered SCD Pancreatic Islets

[0097] The present disclosure provides SCD pancreatic islet compositions for use in the methods described herein. In some embodiments, the SCD pancreatic islet composition is generated from stem cells using a method described herein. In some embodiments, the SCD pancreatic islet composition is obtained by providing a stem cell (e.g., ESC or iPSC) comprising a gene disruption described herein (e.g., a TET2 gene disruption), and differentiating the stem cell to SCD pancreatic islet cells according to a method known in the art or described herein.

[0098] In some embodiments, the stem cells comprising the gene disruption, wherein the gene disruption results in a reduced expression of and / or a reduced activity of an epigenetic modulator, e.g., as compared to stem cells without the gene disruption. In some embodiments, the epigenetic modulator is a TET protein. In some embodiments, the TET protein is TET2. In some embodiments, the stem cells are differentiated to SCD pancreatic islet cells according to a method described herein, wherein the SCD pancreatic islet cells comprise the gene disruption. It follows that SCD cells (e.g., SCD pancreatic islet cells) differentiated from stem cells comprising the gene disruption likewise comprise the gene disruption.

[0099] In some embodiments, the stem cells comprise a gene disruption in the TET2 gene. In some embodiments, the gene disruption is present in one allele comprising the TET2 gene. In some embodiments, the gene disruption is present in both alleles comprising the TET2 gene. In some embodiments, the gene disruption results in a reduced expression of TET2. In some embodiments, the gene disruption results in a reduced activity of TET2.

[0100] In some embodiments, the SCD pancreatic islet cells differentiated from stem cells comprising a gene disruption in the TET2 gene are characterized by reduced TET2 expression (e.g., reduced expression of a TET2 transcriptional and / or translation product) as compared to control SCD pancreatic islet cells (e.g., SCD pancreatic islet cells differentiated from stem cells comprising a wild- type TET2 gene). In some embodiments, the reduced TET2 expression is at329282749 23Attorney Docket: MINU-012 / 01WO 339336.2063least 2-fold, 5-fold, 10-fold, 100-fold, or 1000-fold lower. In some embodiments, the reduced TET2 expression is substantially no expression of TET2 (e.g., a TET2 transcriptional and / or translation product) above a level of detection for an assay used to measure TET2 expression. In some embodiments, the assay is any known in the art for quantifying RNA transcripts (e.g., quantitative PCR) or protein (e g., immunoblot).

[0101] In some embodiments, the gene disruption comprises a deletion, an insertion, a translocation, an inversion, or a substitution. In some embodiments, the stem cells comprise a deletion in the TET2 gene. In some embodiments, the deletion is present in one allele comprising the TET2 gene. In some embodiments, the deletion is present in both alleles comprising the TET2 gene. In some embodiments, the deletion is homozygous (i.e,, the alleles comprising the TET2 gene comprise the same deletion). In some embodiments, the deletion is heterozygous (i.e., the alleles comprising the TET2 gene comprise a different deletion),

[0102] In some embodiments, the gene disruption comprises a frameshift mutation in the TET2 gene. In some embodiments, the frameshift mutation is present in one allele comprising the TET2 gene. In some embodiments, the frameshift mutation is present in both alleles comprising the TET2 gene.

[0103] In some embodiments, the stem cells further comprise an insertion of an immune suppression sequence, e.g., in a safe harbor locus. In some embodiments, the immune suppression sequence encodes CD47, CTLA-4, PDL1, PDL2, HLA-C, HLA-E, HLA-G, CI-inhibitor, IL-35, DUX4, IDO1, IL10, CCL21, CCL22, CD16, CD52, H2-M3, CD200, FASLG, MFGE8, TGFB, IL6, and / or SERPINB9.

[0104] In some embodiments, the stem cells further comprise a reduced expression of MHC-I and / or MHC-II. In some embodiments, the stem cells comprise a gene disruption in an allele encoding P-2 microglobulin (B2M), thereby resulting in reduced expression of MHC-I. In some embodiments, the stem cells comprise a gene disruption in an allele encoding class II major histocompatibility complex transactivator (CIITA), thereby resulting in reduced expression of MHC-II.

[0105] In some embodiments, the stem cells are pluripotent stem cells. In some embodiments, the pluripotent stem cells are human pluripotent stem cells. In some embodiments, the pluripotent stem cells are embryonic stem cells. In some embodiments, the pluripotent stem cells are iPSCs.329282749 24Attorney Docket: MINU-012 / 01WO 339336,2063Functional Characteristics of SCD Pancreatic Islet Compositions

[0106] The disclosure provides TET2 engineered SCD pancreatic islet cells that are characterized by an indicia of pancreatic function and / or reduced susceptibility to immune response with respect to control SCD pancreatic islet cells, e.g., as measured using an assay described herein. In some embodiments, the indicia of pancreatic function is improved with respect to control SCD pancreatic islet cells. In some embodiments, the indicia of pancreatic function is substantially similar with respect to control SCD pancreatic islet cells. In some embodiments, the indicia of pancreatic function is any described herein. In some embodiments, the control SCD pancreatic islet cells are differentiated from stem cells comprising a wild-type TET2 gene and / or lacking the TET2 gene disruption.(i) Indicia of Pancreatic Function

[0107] TET2 engineered SCD pancreatic islet cells of the disclosure are characterized by an indicia of pancreatic function. In some embodiments, the TET2 engineered SCD pancreatic islet cells are characterized by at least one (e.g., 1, 2, 3, 4, 5 or more) indicia of pancreatic function described herein.

[0108] In some embodiments, the indicia of pancreatic function is a quantity of insulin producing cells as a proportion of total TET2 engineered SCD pancreatic islet cells, wherein the proportion is at least about 20%. In some embodiments, at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% of total TET2 engineered SCD pancreatic islet cells are insulin producing cells. In some embodiments, at least about 20% to about 70% of total TET2 engineered SCD pancreatic islet cells are insulin producing cells. In some embodiments, at least about 30% to about 60% of total TET2 engineered SCD pancreatic islet cells are insulin producing cells. In some embodiments, at least about 40% to about 60% of total TET2 engineered SCD pancreatic islet cells are insulin producing cells. In some embodiments, the TET2 engineered SCD pancreatic islet cells further comprise SCD a cells, SCD 0 cells, or a combination thereof. In some embodiments, less than about 10%. 8%, 5%, 3%, or 1% of the TET2 engineered SCD pancreatic islet cells are epsilon cells, pancreatic progenitor cells, acinar and ductal cells, enterochromaffin cells, and / or proliferative cells. In some embodiments, less329282749 25Attorney Docket: MINU-012 / 01WO 339336,2063than about 5%, 4%, 3%, 2%, or 1% of the TET2 engineered SCD pancreatic islet cells are proliferative cells.

[0109] In some embodiments, the indicia of pancreatic function comprises formation of the TET2 engineered SCD pancreatic islet cells into cell clusters. In some embodiments, the cell clusters are spherical or substantially spherical. In some embodiments, the cell clusters are characterized by a longest diameter described herein (e.g., at least about 50 pm and up to about 1000 pm).

[0110] In some embodiments, the indicia of pancreatic function comprises expression of a pancreatic β cell marker described herein by the TET2 engineered SCD pancreatic islet cells. In some embodiments, the pancreatic cell marker comprises C-peptide, PDX1 and / or NKX6-1. In some embodiments, expression of the pancreatic P cell marker is measured according to a technique known in the art and / or described herein for measuring expression of cell markers (e.g., flow cytometry, PCR, western blot). In some embodiments, expression of the pancreatic P cell marker by the SCD pancreatic islet cells is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, or 130% of the expression of the pancreatic cell marker by control SCD pancreatic islet cells. In some embodiments, the pancreatic P cell marker is C-peptide, and expression thereof by the SCD pancreatic islet cells is about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the expression of the pancreatic P cell marker by control SCD pancreatic islet cells. In some embodiments, the pancreatic P cell marker is PDX1, and expression thereof by the SCD pancreatic islet cells is about 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, or 130% of the expression of the pancreatic P cell marker by control SCD pancreatic islet cells. In some embodiments, the pancreatic P cell marker is NKX6-1, and expression thereof by the SCD pancreatic islet cells is about 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, or 130% of the expression of the pancreatic P cell marker by control SCD pancreatic islet cells.

[0111] In some embodiments, the indicia of pancreatic function comprises an insulin response by the TET2 engineered SCD pancreatic islet cells. In some embodiments, the insulin response is characterized by the level of insulin and / or C-peptide secreted in response to a glucose challenge. In some embodiments, the level of insulin and / or C-peptide secreted is substantially similar for SCD pancreatic islet cells as compared to control SCD pancreatic islet cells. In some329282749 26Attorney Docket: MINU-012 / 01WO 339336,2063embodiments, the level of insulin and / or C-peptide secreted is at least about 70% to 130% of the level of insulin and / or C-peptide secreted by control SCD pancreatic islet cells in response to glucose challenge. In some embodiments, the insulin response is characterized by the total level of insulin and / or C-peptide produced. In some embodiments, the total level of insulin and / or C-peptide produced by the SCD pancreatic islet cells is substantially similar to the total level of insulin and / or C-peptide produced by control SCD pancreatic islet cells. In some embodiments, the total level of insulin and / or C-peptide produced is at least about 70% to 130% of the total level of insulin and / or C-peptide produced by control SCD pancreatic islet cells.

[0112] In some embodiments, the insulin response is measured in vitro. In some embodiments, the insulin response is measured in vivo.(ii) Reduced Susceptibility to Immune Responses

[0113] The TET2 engineered SCD pancreatic islet cells are characterized by reduced susceptibility to an immune response, e.g., as compared to control SCD pancreatic islet cells.

[0114] In some embodiments, the TET2 engineered SCD pancreatic islet cells express an alloantigen, wherein at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the TET2 engineered SCD pancreatic islet cells remain viable following exposure to effector CD8+ T cells reactive to the alloantigen, e.g., as measured in an in vitro killing assay described herein. In some embodiments, the contacting is performed at a target cell to effector cell ratio of at least about 1: 1 and up to about 10: 1, wherein the target cells are the TET2 engineered SCD pancreatic islet cells and the effector cells are the effector CD8+ T cells. In some embodiments, the alloantigen is an MHC class I molecule, an MHC class II molecule, a minor histocompatibility antigen, and / or a blood type antigen.

[0115] In some embodiments, the TET2 engineered SCD pancreatic islet cells express a diabetogenic antigen, wherein at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the TET2 engineered SCD pancreatic islet cells remain viable following exposure to effector CD8+ T cells reactive to the diabetogenic antigen, e.g., as measured in an in vitro killing assay described herein. In some embodiments, the contacting is performed at a target cell to effector cell ratio of at least about 1: 1 and up to about 10:1, wherein the target cells are the TET2 engineered SCD pancreatic islet329282749 27Attorney Docket: MINU-012 / 01WO 339336,2063cells and the effector cells are the effector CD8+ T cells. In some embodiments, the diabetogenic antigen is any described herein. In some embodiments, the diabetogenic antigen comprises proinsulin, insulinoma-associated protein 2 (IA-2), zinc transporter 8 (ZnT8), islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), insulin, glutamic acid decarboxylase 65 (GAD65), chromogranin A (ChgA), islet amyloid polypeptide (IAPP), or a fragment thereof.

[0116] In some embodiments, the TET2 engineered SCD pancreatic islet cells are characterized by a decreased endosomal stress response upon exposure to cytokine stimulation and / or an ER stress inducer as compared to control SCD pancreatic islet cells. In some embodiments, the endosomal stress response is characterized by expression of a gene selected from the group consisting of: ATF4, BiP, CHOP, ATF3, ATF6, XBP1, and a combination thereof.

[0117] In some embodiments, the TET2 engineered SCD pancreatic islet cells are characterized by a decreased inflammatory response upon exposure to cytokine stimulation and / or an ER stress inducer as compared to control SCD pancreatic islet cells. In some embodiments, the inflammatory response is characterized by expression of a gene selected from the group consisting of CXCL10, STAT1, CXCL16, CD47, MX1, CCL2, TNF, and a combination thereof.

[0118] In some embodiments, the TET2 engineered SCD pancreatic islet cells are characterized by a decreased activation of cell death pathways upon exposure to cytokine stimulation and / or an ER stress inducer as compared to control SCD pancreatic islet cells. In some embodiments, the cell death pathway is characterized by expression of a gene selected from the group consisting of: FAS, PDL1, IDO, BACH1, BACH2, CASP3, and a combination thereof.

[0119] In some embodiments, the TET2 engineered SCD pancreatic islet cells are characterized by increased viability upon exposure to cytokine stimulation and / or an ER stress inducer as compared to control SCD pancreatic islet cells.

[0120] In some embodiments, the TET2 engineered SCD pancreatic islet cells are characterized by increased viability following administration to a subject as compared to control SCD pancreatic islet cells. In some embodiments, the TET2 engineered SCD pancreatic islet cells are characterized by increased engraftment following administration to a subject as compared to control SCD pancreatic islet cells.Treatment Parameters329282749 28Attorney Docket: MINU-012 / 01WO 339336,2063

[0121] The present disclosure provides methods of treating, or delaying onset of, a subject having diabetes, comprising administering to the subject a composition of SCD pancreatic islet cells described herein (e.g., a composition of TET2 engineered SCD pancreatic islet cells described herein). In some embodiments, the SCD pancreatic islet composition is administered to the subject to provide a pancreatic function that is otherwise impaired in the subject due to diabetes. In some embodiments, the SCD pancreatic islet composition is contained in a delivery device described herein. In some embodiments, the SCD pancreatic islet composition is not contained in a delivery device. In some embodiments, the administration comprises a subcutaneous infusion of the SCD pancreatic islet composition. In some embodiments, the administration comprises an intramuscular infusion of the SCD pancreatic islet composition.

[0122] In some embodiments, the disclosure provides methods for treating or delaying onset of diabetes mellitus (such as, but not limited to, type 1, type 2, or surgical diabetes mellitus) in a subject, comprising administering to the subject an effective amount of a SCD pancreatic islet composition described herein, or a pharmaceutical composition thereof, wherein the SCD pancreatic islet composition is generated from stem cells comprising a gene disruption, wherein the gene disruption comprises a loss of function mutation in the TET2 gene, and wherein the stem cells are allogeneic with respect to the subject.

[0123] In some embodiments, the TET2 engineered SCD pancreatic islet composition comprises an alloantigen described herein (e.g., an MHC class I molecule, an MHC class II molecule, a minor histocompatibility antigen, or a blood type antigen) with respect to the subject. In some embodiments, the TET2 engineered SCD pancreatic islet composition is MHC mismatched with respect to the subject. It is appreciated that allogeneic pancreatic islet cells administered to a subject are typically susceptible to an alloimmune response upon administration to the subject, e.g., unless the subject is immunocompromised. It is further appreciated that allogeneic pancreatic islet cells administered to a subject having T1D are typically susceptible to an isletspecific autoimmune response upon administration to the subject. An alloimmune and / or autoimmune response may be avoided or reduced by administering an immunosuppressant to the subject prior to, concurrently with, or soon after administration of the allogeneic cells. However, administering immunosuppressants to the subject is undesirable due to toxicity effects and / or introducing a risk of infection disease and / or cancer.329282749Attorney Docket: MINU-012 / 01WO 339336,2063

[0124] In some embodiments, the TET2 engineered SCD pancreatic islet cells are characterized by reduced susceptibility to an alloimmune response upon administration to the subject, e.g., as compared to control SCD pancreatic islet cells. In some embodiments, the SCD pancreatic islet composition are characterized by reduced susceptibility to an autoimmune response upon administration to the subject, e.g., as compared to control SCD pancreatic islet cells. In some embodiments, the SCD pancreatic islet composition are characterized by reduced susceptibility to an autoimmune response directed to a diabetogenic antigen upon administration to the subject, e.g., as compared to control SCD pancreatic islet cells.

[0125] In some embodiments, the loss of function mutation in the TET2 gene increases resistance of the SCD pancreatic islet composition to cytokine stress following the administering. In some embodiments, the loss of function mutation in the TET2 gene increases engraftment of the SCD pancreatic islet composition following the administration.

[0126] In some embodiments, the subject does not receive systemic immunosuppression prior to, concurrently with, or after administering the SCD pancreatic islet composition. Exemplary agents for systemic immunosuppression are known in the art, and include, tacrolimus, sirolimus, everolimus, cyclosporine, alemtuzumab, basiliximab, daclizumab, tegoprubart, MMF, anti-TNFa, belatacept, anti-CD40 ligand, and anti-thymocyte globulin (ATG).

[0127] In some embodiments, the SCD pancreatic islet composition is administered to an extrahepatic site, e.g., subcutaneously (SQ) or intramuscularly (IM).

[0128] In some embodiments, the SCD pancreatic islet composition further comprises a support factor described herein. In some embodiments, the support factor comprises a proangiogenic factor, a PTG hormone, a cytokine, or a combination thereof.

[0129] In some embodiments, the methods comprise transplanting the SCD pancreatic islet composition. In some embodiments, the SCD pancreatic islet composition is transplanted directly in the pancreas. In some embodiments, the SCD pancreatic islet composition is transplanted in the liver. In some embodiments, the SCD pancreatic islet composition is transplanted at an extrahepatic site. In some embodiments, the SCD pancreatic islet composition is transplanted at a subcutaneous site. In some embodiments, the SCD pancreatic islet composition is transplanted at an intramuscular site. In some embodiments, the SCD pancreatic islet composition is transplanted in a capsule, e.g., to maintain a plurality of cells of the SCD pancreatic islet composition at the transplant site.329282749 30Attorney Docket: MINU-012 / 01WO 339336,2063

[0130] In some embodiments, the administration results in treatment of diabetes without the subject receiving systemic immunosuppression prior to, concurrently with, or after administering the SCD pancreatic islet composition.Treatment Outcome

[0131] The present disclosure provides desirable clinical outcomes for treatment of a subject having diabetes according to the methods described herein. Criteria for treatment of diabetes that results in an improvement to a clinical outcome are known in the art.

[0132] In some embodiments, the clinical outcome is a reduction of an HbAlc level (e.g., an HbAlc level measured in a blood sample). In some embodiments, the reduced level of HbAlc is less than about 6%, about 5.9%, about 5.8%, about 5.7%, about 5.6%, or about 5.5%.

[0133] In some embodiments, the clinical outcome is a reduction in blood sugar levels to normal levels, e.g., blood sugar levels in a healthy subject. In some embodiments, the clinical outcome is a blood sugar level of about 80-120 mg / dL before meals and 100-140 mg / dL at bedtime. The particular target blood sugar level may vary for subjects, depending on other factors, and as determined by a clinician skilled in the art. In some embodiments, measurement of the clinical outcome comprises determining blood sugar level, glycosylated hemoglobin level, cholesterol and fat levels, and / or urine protein levels. In some embodiments, the clinical outcome is a reduced risk of complications associated with diabetes, e.g., disease of the eye, kidney disease, and / or nerve disease. In some embodiments, the clinical outcome is a reduced occurrence of severe hypoglycemia.

[0134] In some embodiments, the administration delays onset of diabetes without the subject receiving systemic immunosuppression prior to, concurrently with, or after administering the SCD pancreatic islet composition.

[0135] In some embodiments, delaying onset of diabetes comprises delaying onset of a symptom of diabetes (e.g., hyperglycemia, hypoinsulinemia, diabetic retinopathy, diabetic nephropathy, blindness, memory loss, renal failure, cardiovascular disease, neuropathy, autonomic dysfunction, hyperglycemic hyperosmolar coma) for at least about 2 weeks, about 4 weeks, about 2 months, about 6 months, about 12 months, about 24 months, about 36 months, about 48 months, about 72 months, about 8 years, about 9 years, about 10 years, about 15 years, about 20 years, about 30 years, or longer and up to the entire lifespan of the subject.329282749 31Attorney Docket: MINU-012 / 01WO 339336,2063

[0136] In some embodiments, the method results in engraftment of the SCD pancreatic islet composition following administration, without the subject receiving systemic immunosuppression prior to, concurrently with, or after administering the SCD pancreatic islet composition.

[0137] In some embodiments, the subject experiences increased engraftment as compared to administrating a SCD pancreatic islet composition without the gene disruption (e.g., the TET2 gene disruption).

[0138] In some embodiments, the increased engraftment is characterized by insulin production within three days (such as any of 1, 2, or 3 days or any of about 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, or 72 hours, inclusive of times falling in between these values) of administration. In some embodiments, the increased engraftment is characterized by insulin production that is sufficient to reduce blood glucose levels in the subject. In some embodiments, the increased engraftment is characterized by an amount of glucose that is lower than the threshold associated with pathology of diabetes.

[0139] In some embodiments, the increased engraftment is characterized by increased glucose-or mixed meal-stimulated C-peptide expression for a duration following the administration. In some embodiments, the increased engraftment is characterized by increased levels of C-peptide when measured in a fasting state as part of a mixed meal tolerance test. C-peptide (PubChem CID: 16132309) is a short 31-amino-acid polypeptide that connects insulin's A-chain to its B-chain m the proinsulin molecule and is a byproduct of insulin production. In diabetes and other diseases, a measurement of stimulated C-peptide blood serum levels can be used to distinguish between certain diseases with similar clinical features. A mixed meal tolerance test measures the amount of insulin secreted by the pancreas after ingesting a mixed meal, e.g., by monitoring C-peptide levels in the blood. The procedure is performed by having a subject fast overnight, followed by consuming a standardized “mixed meal” beverage. Blood samples are obtained prior to and following the consumption, and C-peptide levels in the blood samples are measured. It is within the knowledge of the person of ordinary skill in the art to determine changes in the level of C-peptide in the blood following the consumption that are indicative of insulin production.

[0140] In some embodiments, the increased engraftment is characterized by a normalized level of hemoglobin Ale (HbAlc) following the administration. In some embodiments, the level of329282749 32Attorney Docket: MINU-012 / 01WO 339336,2063HbAlc is measured in a blood sample. In some embodiments, the normalized level of HbAlc is less than about 6%, about 5.9%, about 5.8%, about 5.7%, about 5.6%, or about 5.5%. In some embodiments, the normalized level of HbAlc is maintained for at least about 2 weeks, about 4 weeks, about 6 weeks, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 8 months, about 10 months, about 1 year, or about 2 years following the administration (e.g., without the subject receiving systemic immune suppression).

[0141] In some embodiments, the increased engraftment is characterized by an increased Time in Range (TIR). “Time in Range” is a measure of the percentage of time a subject has blood glucose levels in a target range (e.g., 70-180 mg / dl). In some embodiments, the increased TIR is greater than about 80%, about 85%, or about 90%. In some embodiments, the normalized TIR is maintained for at least about 2 weeks, about 4 weeks, about 6 weeks, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 8 months, about 10 months, about 1 year, or about 2 years following the administration (e.g., without the subject receiving systemic immune suppression).

[0142] In some embodiments, the increased engraftment is characterized by improved survival of the SCD pancreatic islet composition for a duration following the administration. In some embodiments, survival of the SCD pancreatic islet composition is maintained for a duration of at least about 2 weeks, about 4 weeks, about 2 months, about 6 months, about 12 months, about 24 months, about 36 months, about 48 months, or about 72 months following the administration. In some embodiments, the survival of insulin producing cells contained in the SCD pancreatic islet composition is maintained for a duration of at least about 2 weeks, about 4 weeks, about 2 months, about 6 months, about 12 months, about 24 months, about 36 months, about 48 months, or about 72 months following the administration.

[0143] In some embodiments, the increased engraftment is characterized by a reduced frequency of severe hypoglycemia. In some embodiments, the frequency of severe hypoglycemia is reduced as compared to prior to the administering.

[0144] In some embodiments, the subject experiences a reduced immune response as compared to administrating a SCD pancreatic islet composition without the gene disruption (e.g., the TET2 gene disruption). In some embodiments, the immune response is an alloimmune response. In some embodiments, the alloimmune response is characterized by (i) stimulating alloreactive T cell expansion, activation, and / or effector function, (ii) stimulating alloreactive natural killer329282749 33Attorney Docket: MINU-012 / 01WO 339336,2063(NK) cells expansion, activation, and / or effector function, (iii) stimulating alloantibody production, (iv) stimulating activation of alloantigen-presenting dendritic cells, (v) stimulating complement-mediated cytotoxicity, or (vi) a combination of (i)-(v).

[0145] In some embodiments, the SCD pancreatic islet composition exhibits reduced susceptibility to a beta cell targeted autoimmune response as compared to administering a SCD pancreatic islet composition without the gene disruption (e.g., the TET2 gene disruption).Methods to determine whether a cell composition described herein evades an immune response include, but are not limited to, IFNy Elispot assays, microglia killing assays, cell engraftment animal models, cytokine release assays, mixed- lymphocyte reactions, and immunofluorescent analysis. Exemplary assays are described in WO2023133568, WO2016183041, and WO2018132783, each of which are hereby incorporated by reference,

[0146] In some embodiments, the SCD pancreatic islet composition exhibits survival without substantially inducing a host immune response for a duration of at least about 1 week, about 2 weeks, about 4 weeks about 2 months, about 6 months, about 12 months, about 24 months, about 36 months, about 48 months, or about 72 months following the administration.

[0147] In some embodiments, the method comprises administering the SCD pancreatic islet composition and a support factor, wherein survival of cells (e.g., insulin producing cells) contained in the SCD pancreatic islet composition is increased as compared to administering a SCD pancreatic islet composition lacking the support factors. In some embodiments, the survival is increased by at least about 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.5-fold, about 1.7-fold, about 1.9-fold, about 5-fold, about 10-fold, about 50-fold, about 100-fold, about 500-fold, or about 1000-fold compared to administering a SCD pancreatic islet composition lacking the support factors. In some embodiments, the survival is increased for a duration following the administration of at least about 2 weeks, about 4 weeks, about 2 months, about 6 months, about 12 months, about 24 months, about 36 months, about 48 months, or about 72 months. In some embodiments, administration of the SCD pancreatic islet composition results in angiogenesis at the transplant site that is increased as compared to administering a SCD pancreatic islet composition lacking the support factors. In some embodiments, the angiogenesis is increased for a duration following the administration of at least about 2 weeks, about 4 weeks, about 2 months, about 6 months, about 12 months, about 24 months, about 36 months, about 48 months, or about 72 months.329282749 34Attorney Docket: MINU-012 / 01WO 339336,2063Subject

[0148] In some embodiments, the subject is a mammal. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is human. In some embodiments, the non-human mammal is used as an animal model for type 1 diabetes, type 2 diabetes, or pre¬ diabetic conditions. In some embodiments, the subject is male or female. In some embodiments, the subject has been previously diagnosed with diabetes prior to receiving a SCD pancreatic islet composition according to the methods described herein. In some embodiments, the subject has experienced a complication associated with diabetes or a pre-diabetic condition. In some embodiments, the subject has received a therapeutic intervention for treatment of diabetes prior to receiving a SCD pancreatic islet composition according to the methods described herein. In some embodiments, the subject has received a dose (e.g., a daily dose) of exogenous insulin prior to receiving a cell composition according to the methods described herein. In some embodiments, the subject no longer requires exogenous insulin upon receiving the cell composition according to the methods described herein for a duration of at least about 2 weeks, about 4 weeks, about 2 months, about 6 months, about 12 months, about 24 months, about 36 months, about 48 months, about 72 months, about 8 years, about 9 years, about 10 years, about 15 years, about 20 years, about 30 years, or longer and up to an entire lifespan. In some embodiments, the subject has not experienced a complication associated with diabetes or a pre-diabetic condition. In some embodiments, the subject is asymptomatic for diabetes. In some embodiments, the subject exhibits a diabetes risk factor.Administration

[0149] In some embodiments, the SCD pancreatic islet compositions, the pharmaceutical compositions thereof, or the delivery devices comprising the SCD pancreatic islet compositions or pharmaceutical compositions are administered to a subject according to the method described herein. In some embodiments, the administering is performed according to any means known in the art for delivering a cell therapy to a subject.

[0150] In some embodiments, the administration is performed by injection, infusion, instillation, or ingestion. In some embodiments, the administration is performed by an intravenous, intramuscular, intradermal, intraperitoneal, or subcutaneous injection or infusion.329282749 35Attorney Docket: MINU-012 / 01WO 339336.2063

[0151] In some embodiments, the method comprises transplanting via the intraperitoneal space, renal subcapsule, renal capsule, omentum, subcutaneous space, or via pancreatic bed infusion. For example, transplanting can be subcapsular transplanting, intramuscular transplanting, or intraportal transplanting, e.g., intraportal infusion.

[0152] In some embodiments, the SCD pancreatic islet composition is administered in a delivery device described herein to provide immune protection.Producing SCD Pancreatic Islet Compositions

[0153] The present disclosure provides SCD pancreatic islet compositions for use in the methods described herein, and methods for producing the compositions.

[0154] In some embodiments, the SCD pancreatic islet composition comprises a population of cells, wherein a plurality' of the cells are SCD pancreatic islet cells described herein. In some embodiments, the SCD pancreatic islet composition comprises SCD beta cells or SCD beta-like cells.

[0155] In some embodiments, the cells or the plurality of cells are formed into an organoid. In some embodiments, the cells or the plurality of cells are formed into clusters. In some embodiments, the cells or the plurality of cells are formed into spherical or substantially spherical clusters. In some embodiments, the clusters comprise a longest diameter of about 50 pm to about 1000 pm. In some embodiments, the clusters comprise a longest diameter of about 100 pm to about 350 pm. In some embodiments, the clusters comprise a longest diameter of about 100 pm to about 200 pm. In some embodiments, the population of cells further comprises at least one additional cell type of endoderm origin.

[0156] In some embodiments, the cells or a plurality of cells of the population comprise a gene disruption described herein. In some embodiments, the gene disruption reduces an autoimmune and an allogeneic immune response to the cells or the plurality of cells following in vivo administration. In some embodiments, the gene disruption reduces an allogeneic immune response to the cells or the plurality of cells following in vivo administration. In some embodiments, the gene disruption reduces an autoimmune immune response to the cells or the plurality of cells following in vivo administration.

[0157] In some embodiments, the SCD pancreatic islet composition further comprises a support factor described herein. In some embodiments, the cells or a plurality of cells of the population329282749 36Attorney Docket: MINU-012 / 01WO 339336,2063are characterized by an improved property' in the presence of the support factor. In some embodiments, the improved property comprises increased survival, e.g., under hypoxic, inflamed, and / or nutrient deprived conditions, and / or reduced sensitivity to immune killing following administration to a subject.(I) Stem Cell Source

[0158] In some embodiments, the SCD pancreatic islets of the disclosure are generated from pluripotent stem cells (e.g., human pluripotent stem cells), wherein the pluripotent stem cells comprise a gene disruption described herein (e.g., a TET2 gene disruption).

[0159] In some embodiments, the pluripotent stem cells are obtained from a source that is not the subject intended to receive a transplant comprising the SCD pancreatic islets. In some embodiments, the pluripotent stem cells comprise an alloantigen with respect to the subject intended to receive a transplant comprising the SCD pancreatic islets. In some embodiments, the alloantigen comprises an MHC class I molecule. In some embodiments, the alloantigen comprises an MHC class II molecule. In some embodiments, the alloantigen comprises a minor histocompatibility antigen. In some embodiments, the alloantigen comprises a blood type antigen.

[0160] Pluripotent stem cells have the ability to differentiate into cells of all three germ layers (i.e., ectoderm, mesoderm, and endoderm) and have the characteristics of self-renewability. As understood by the skilled artisan, sources of pluripotent stem include embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). The generation of pluripotent stem cells (e.g., human pluripotent stem cells) is generally known in the art,

[0161] In some embodiments, the SCD pancreatic islets are differentiated from ESCs, wherein the ESCs comprise a gene disruption described herein (e.g., a TET2 gene disruption). In some embodiments, the ESCs are human ESCs. ESCs are pluripotent cells isolated from the inner cell mass of the early mammalian embryo that implants into the uterus that have capacity for selfrenewal and can be differentiated to multiple types of adult cells in vitro (Thomson, et al (1998) Science 282: 1145-1147). Methods for maintaining ESCs in culture are known in the art.Exemplary methods are described in US10 / 486,408; US11 / 021,618; USll / 165,305;USll / 573,662; US12 / 729,084; US12 / 093,590; USll / 993,399; US11588,693; USll / 681,687; USll / 807,223; USll / 773,944; US12 / 099759; US12 / 107020; US12 / 618649; US12 / 765714;329282749 37Attorney Docket: MINU-012 / 01WO 339336,2063US11 / 838054; US12 / 264760; US14 / 201630; US14 / 106330; and PCT / US2016 / 061442, each of which are hereby incorporated by reference.

[0162] In some embodiments, the SCD pancreatic islets are differentiated from an ESC cell line. In some embodiments, the ESC cell line is established from cells of blastocytes / unfertilized embryos. In some embodiments, the ESC cell line is established from cells that received a somatic cell nuclear transfer (e.g., ooplasm or nuclear transfer). In some embodiments, the ESC cell line is established from germ cell lineage cells (e.g., oocyte or testicular cells).

[0163] Exemplary ESC cell lines are known in the art and include, but are not limited to the H9, CyT49, CyT25, CyT203, CyT212, MEL-1 ESC cell lines. In some embodiments, the ESC cell line is one made available through the NIH Embryonic Stem Cell Registry or the HHMI HUES collection. In some embodiments, the ESC cell line is the MEL-1 ESC cell line (NIH registration number: 0139) (Micallef et al (2011) Diabetologia 55:694). In some embodiments, the ESC cell line is one made available through the California Institute for Regenerative Medicine (CIRM) (word wide web: cirm.ca.gov / cirm-information-applicants-clinically-compatible-hpsc-lines / ).

[0164] In some embodiments, the SCD pancreatic islets are differentiated from an ESC cell line established under current Good Manufacturing Practices (cGMP). Clinical ESC cell lines refer to ESC cell lines cultured in the absence of animal-derived components and under cGMP conditions. Exemplary ESC cell lines established under cGMP include, but are not limited to, those made commercially available by WiCell (see world wide web: wicell.org / home / stem-cells / catalog-of-stem-cell-lines / collections / cgmp-cell-banks.cmsx).

[0165] In some embodiments, the SCD pancreatic islets are differentiated from an iPSC. In some embodiments, the SCD pancreatic islets are differentiated from a human iPSC. As used herein, the terms “iPS cell” and “induced pluripotent stem cell” are used interchangeably and refer to a pluripotent stem cell artificially derived (e.g., induced or by complete reversal) from a non- pluripotent cell, typically an adult somatic cell, for example, by inducing a forced expression of one or more genes. iPSCs are reprogrammed from somatic cells into the embryonic-like pluripotent state by overexpression of key reprogramming genes and have a similar capacity for self-renewal and differentiation (Takahashi, et al (2007) Cell 131:861-72; Yu, et al (2007) & / er?ce318:1917 -20; Seki, etal (2015) World J. Stem CellsTAIM, Lakshmipathy & Vermuri Ed. Methods in Molecular Biology: Pluripotent Stem. Cells, Methods and Protocols Springer 2013; each of which are herein incorporated by reference). Generally, iPSCs are generated from329282749 38Attorney Docket: MINU-012 / 01WO 339336,2063somatic cells (e.g., blood cells, fibroblasts) by transient expression of a reprogramming factor using, e.g., an episomal vector. Once the cells are reprogrammed due to expression of the reprogramming factors, they become pluripotent and express the factors from endogenous genes. Exemplary reprogramming factors include, but are not limited to, OCT4, KLF4, SOX2, c-Myc, SOKMNLT, NANOG, LIN28, and SV40L T antigen.

[0166] In some embodiments, the SCD pancreatic islets are differentiated from an iPSC generated from a somatic cell (e.g., a blood cell, a fibroblast) induced to express a reprogramming factor selected from OCT4, KLF4, SOX2, c-Myc, SOKMNLT, NANOG, LIN28, SV40L T antigen, and a combination thereof. In some embodiments, the reprogramming factor comprises OCT4. In some embodiments, the reprogramming factor comprises OCT4 and SOX2, In some embodiments, the reprogramming factor comprises OCT4, SOX2, and KLF4. In some embodiments, the reprogramming factor comprises OCT4, SOX2, KLF4, and c-Myc, In some embodiments, the reprogramming factor further comprises one or more of SOKMNLT, NANOG, LIN28, and SV40L T antigen. In some embodiments, the iPSC is generated from a somatic cell using chemical reprogramming, e.g., as described in Guan, et al (2022) Nature 605:325. In some embodiments, the somatic cell is obtained from a donor other than the subject intended to receive a transplant comprising the SCD pancreatic islets.

[0167] Methods for establishing or determining pluripotency of a pluripotent stem cell described herein are known in the art. In some embodiments, the method comprises assaying for expression of a pluripotency-specific factor using, e.g., flow cytometry, immunohistochemistry, Western blot, and / or ELISA. In some embodiments, the pluripotency-specific factor comprises NANOG, OCT4, SOX2, ESRRB, TRA-1-60, TRA-1-81, SSEA4, or a combination thereof. In some embodiments, the method comprises differentiating the pluripotent stem cell, wherein successful differentiation is an indication of pluripotency.(II) Methods of Generating SCD Pancreatic Islets

[0168] Provided herein are methods for generating SCD pancreatic islet compositions, e.g., SCD pancreatic islet compositions comprising SCD p cells. In some embodiments, the SCD pancreatic islet composition is generated from pluripotent stem cells described herein. In some embodiments, the SCD pancreatic islet composition comprises SCD P cells and at least one additional endocrine cell type. In some embodiments, the at least one additional endocrine cell329282749 39Attorney Docket: MINU-012 / 01WO 339336,2063type is selected from an SCD a cell, a SCD 8 cell, a SCD y cell, and a SCD E cell. In some embodiments, the SCD pancreatic islet composition comprises not more than about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% pancreatic progenitor cells.

[0169] Differentiation of stem cells into specialized cell types often involve providing signal modulators at specific time points in a manner that mimics signals and development pathways from the embryo (Irion, et al (2008) Cold Spring Harb Symp Quant Biol 73: 101-110). For example, strategies developed for generating insulin producing cells from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) are based upon approaches that mimic embryonic pancreatic development. Without wishing to be limited to theory, a pluripotent stem cell in the course of normal ontogeny can differentiate first to an endoderm cell that is capable of forming pancreas cells and other endoderm cell types. Further differentiation of an endoderm cell leads to the pancreatic pathway, where, in some embodiments, -98% of the cells become exocrine, ductular, or matrix cells, and -2% become endocrine cells. Endoderm cells can also be differentiated into other cells of endodermal origin, e.g. lung, liver, intestine, thymus etc. Early endocrine cells are islet progenitors, which can then differentiate further into functional endocrine cells which secrete insulin, glucagon, somatostatin, or pancreatic polypeptide. / Xs appreciated by the skilled artisan, directed differentiation is performed by application of cytokines (e.g., epidermal growth factor, bFGF) and signaling modulators (e.g., bone morphogenetic proteins, y-secretase inhibitors) at each stage of development to activate or inhibit specific signaling pathways involved in the generation of adult pancreatic P cells (D’ Amour, et al (2006) Nat Biotech 24:1392-1401; Rezania, et al (2014) Nat. Biotechnol 32:1121-33; Zhang, etal (2009) Cell Res 19:429-38). Exemplary methods for differentiating a pluripotent stem cell to generate a SCD P cell of the disclosure are further described in PC17US2016 / 028963, PCT / US2021 / 044080, and PCT / US2021 / 013735, each of which is hereby incorporated by reference. In some embodiments, the method is described in Parent, et al (2022) Stem Cell Reports, 17:979; Nair, etal (2019) Pro toe Exchange doi.org / 10.1038 / protex.2018.140; and Nair et al (2019) Nat Cell Biol 21:263, each of which are herein incorporated by reference.

[0170] In some embodiments, the SCD pancreatic islet compositions of the disclosure are generated by differentiating pluripotent stem cells (e.g., ESCs or iPSCs) into a committed pancreatic cell lineage. In some embodiments, the method comprises stepwise differentiation of329282749 40Attorney Docket: MINU-012 / 01WO 339336,2063pluripotent stem cells (e.g., an ESC or an iPSC) to definitive endoderm cells, cells of the pancreatic lineage, and then to pancreatic P ceils. In some embodiments, the pluripotent stem cells (e.g., human ESCs) are characterized by expression of OCT4, NANOG, and SOX2. In some embodiments, the pluripotent stem cells are human ESCs. In some embodiments, the pluripotent stem ceils are a human ESC cell line described herein or known in the art. In some embodiments, the pluripotent stem cells are a cGMP human ESC ceil line described herein or known in the art.

[0171] In vivo, the definitive endoderm is generated by the process of gastrulation of embryogenesis, in which epibiast cells differentiate to form the three germ iayers. Definitive endoderm ceils then give rise to cells of various tissues, including pancreatic, liver, lung, thyroid, thymus, and epithelial lining ceils (“endoderm lineage” cells). The definitive endoderm forms the primitive gut tube, which becomes the pharynx, esophagus, stomach, duodenum, small and iarge intestine, and associated organs (e.g., pancreas, lung, thyroid, thymus, parathyroid, and liver). The pancreas, liver, and duodenum differentiate from cells of the posterior gut tube, which are characterized by expression of haeinatopoietically expressed homeobox (III IEX), pancreatic and duodenal homeobox 1 (PDX1), one cut homeobox 1 (ONECUT1 or FINF6), and hepatocyte nuclear factor 4 alpha (FINF4A).

[0172] In some embodiments, the directed differentiation of pluripotent stem cells to a population comprising definitive endoderm cells is the first stage of generating a SCD pancreatic islet composition of the disclosure. In some embodiments, the first stage is performed by contacting the pluripotent stem cells with culture conditions described herein. In some embodiments, the contacting results in a population comprising definitive endoderm cells following a duration of about 1 day, about 2 days, about 3 days, or about 4 days. In some embodiments, the contacting results in a population comprising definitive endoderm cells following a duration of about 3 days or about 4 days.

[0173] In some embodiments, the directed differentiation of a population comprising definitive endoderm cells to a population comprising posterior foregut is the second stage of generating a SCD pancreatic islet composition of the disclosure. In some embodiments, the second stage is performed by contacting the population comprising definitive endoderm cells with a culture condition described herein. In some embodiments, the contacting results in a population comprising posterior foregut following a duration of about 1 day, about 2 days, about 3 days,329282749 41Attorney Docket: MINU-012 / 01WO 339336,2063about 4 days, about 5 days, or about 6 days. In some embodiments, the contacting results in a population comprising primitive gut tube following a duration of about 2 days, about 3 days, or about 4 days. In some embodiments, the contacting results in a population comprising posterior foregut following a duration of about 2 days, about 3 days, or about 4 days.

[0174] In some embodiments, the directed differentiation of a population comprising posterior gut tube to a population comprising pancreatic progenitor cells is the third stage of generating a SCD pancreatic islet composition of the disclosure. In some embodiments, the third stage is performed by contacting the population comprising posterior foregut with a culture condition described herein. In some embodiments, the contacting results in a population comprising pancreatic progenitor cells following a duration of about 1 day, about 2 days, about 3 days, or about 4 days.

[0175] In some embodiments, the directed differentiation of a population comprising pancreatic progenitor cells to a population comprising insulin producing pancreatic cells is the fourth stage of generating a SCD pancreatic islet composition of the disclosure. In some embodiments, the fourth stage is performed by contacting the population comprising pancreatic progenitor cells with a culture condition described herein. In some embodiments, the contacting results in a SCD pancreatic islet composition following a duration of about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, or longer. In some embodiments, the contacting results in a population comprising endocrine progenitor cells following a duration of about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, or about 8 days. In some embodiments, the contacting results in a population comprising immature islets following a duration of about 8 days, about 9 days, about 10 days, or about 11 days. In some embodiments, the contacting results in a population comprising mature islets following a duration of about 15 days, about 16 days, about 17 days, about 18 days, or longer.

[0176] In some embodiments, contacting a cell population described herein with a culture medium is performed according to any cell culture modality known in the art. For example, in some embodiments, the contacting is performed using 2D culture (see, e.g., Marek-Trzonkowska, et al (2012) Diabetes Care 35:1817; Schulz, et al (2015) Stem Cell Transl Med 4:927, each of which are hereby incorporated by reference). In some embodiments, the contacting is performed in multi-well plates under orbital stirring (see, e.g., Nair, et al (2019) Nat329282749 42Attorney Docket: MINU-012 / 01WO 339336,2063Cell Biol 21:263; Schulz, et al (2012) PLoS One 7:e37004, each of which are hereby incorporated by reference). In some embodiments, the contacting is performed using roller bottles (see, e.g., Schulz, et al (2015) Stem Cell Transl Med 4:927). In some embodiments, the contacting is performed using a bioreactor, such as a vertical- wheel bioreactor (e.g., a PBS Vertical-Wheel bioreactor), or wave-agitation bioreactor (see, e.g., Somerville, et al (2012) J. Transl Med 10:69; Fraser, et al (2018) Mol Ther Methods Clin Dev 8:198, each of w’hich are hereby incorporated by reference). In some embodiments, the contacting is performed using a stirred suspension bioreactor (see, e.g., Krawetz, et al (2010) Tissue Eng Part C Methods 16:573; Kempf, et al (2014) Stem Cell Rep 3:1132, each of which are hereby incorporated by reference). In some embodiments, the contacting is performed in the presence of a microcarrier (e.g., polystyrene, glass, alginate, or dextran beads) (see, e.g., Lock, etal (2009) Tissue Eng Part A 15:2051; Bardy, etal (2013) Tissue Eng Part C Methods 19:166, each of which are hereby incorporated by reference). In some embodiments, the culturing described herein is performed under stirring or shaking. In some embodiments, the population comprising cells are suspended in a container, wherein the suspension is stirred or shaken.

[0177] In some embodiments, the culture medium is xeno-free. In some embodiments, the culture medium does not comprise animal products derived from a non-human.(i) Directed Differentiation to Definitive Endoderm Cells

[0178] In some embodiments, the method comprises a first stage of differentiation, wherein the first stage of differentiation comprises differentiating the pluripotent stem cells (e.g., human ESC or iPSC cells) to a second population comprising definitive endoderm cells. In some embodiments, the differentiating comprises contacting the pluripotent stem cells with (i) a first culture medium comprising a ROCK inhibitor, a TGFP superfamily growth factor described herein (e.g., activin A), and an epidermal growth factor family polypeptide (e.g., Heregulin P-1), (li) a second culture medium comprising a TGFp superfamily growth factor described herein (e.g., activin A) and a WNT activator described herein (e.g., CHIR99021 ), and (iii) a third culture medium comprising a TGF superfamily growth factor described herein (e.g., activin A).

[0179] In some embodiments, the first stage of differentiation comprises contacting the pluripotent stem cells with a first culture medium comprising (i) a ROCK inhibitor; (ii) Heregulin P-1; and (iii) activin A. In some embodiments, the contacting is performed for a329282749 43Attorney Docket: MINU-012 / 01WO 339336,2063duration of at least 1 day. In some embodiments, the contacting is performed for about 1 to about 5 days. In some embodiments, the contacting is performed for about 1 day. In some embodiments, the first culture medium comprises a concentration of ROCK inhibitor, wherein the concentration is at least about 1 pM, about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 6 pM, about 7 pM, about 8 pM, about 9 pM, or about 10 pM. In some embodiments, the concentration is about 5 pM to about 50 pM. In some embodiments, the concentration of ROCK inhibitor is about 5 pM to about 20 pM. In some embodiments, the concentration of ROCK inhibitor is about 10 pM. In some embodiments, the first culture medium comprises a concentration of Heregulin P-1, wherein the concentration is at least about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, or about 10 ng / ml. In some embodiments, the concentration of Heregulin p-1 is about 5 ng / ml to about 50 ng / ml. In some embodiments, the concentration of Heregulin P-1 is about 5 ng / ml to about 20 ng / ml. In some embodiments, the concentration of Heregulin P-1 is about 10 ng / ml. In some embodiments, the first culture medium comprises a concentration of activin A, wherein the concentration is at least about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, or about 10 ng / ml. In some embodiments, the concentration is about 5 ng / ml to about 50 ng / ml. In some embodiments, the concentration of activin A is about 5 ng / ml to about 20 ng / ml. In some embodiments, the concentration of activin A is about 10 ng / ml.10180] In some embodiments, the first stage of differentiation further comprises contacting the pluripotent stem cells with a second culture medium, wherein the second culture medium comprises a TGFP superfamily growth factor described herein (e.g., activin A), and a WNT activator described herein (e.g., CHIR99021). In some embodiments, the pluripotent stem cells contacted with the first culture medium, or a portion thereof, are transferred to the second culture medium.10181] In some embodiments, the second culture medium comprises (i) a concentration of activin A, w’herein the concentration is at least about 10 ng / mL, about 20 ng / mL, about 30 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, or about 100 ng / mL; and (ii) a concentration of the CHIR99021, wherein the concentration is at least about 0.1 pM, 0.5 pM, 1.0 pM, 1.5 pM, 2.0 pM, 2.5 pM, or 3 pM. In329282749 44Attorney Docket: MINU-012 / 01WO 339336,2063some embodiments, the concentration of activin A is about 10 ng / mL to about 200 ng / mL. In some embodiments, the concentration of the CHIR99021 is about 0.1 pMto about 10 pM.

[0182] In some embodiments, the second culture medium further comprises a concentration of vitamin C, wherein the concentration is at least about 0.05 mM, about 0.1 mM, about 0.15 mM, about 0.2 mM, or about 0.25 mM. In some embodiments, the concentration of vitamin C is about 0.05 mM to about 2 mM. In some embodiments, the concentration of vitamin C is about 0.05 mM to about 0.25 mM.

[0183] In some embodiments, the second culture medium further comprises an ITS supplement (e.g., an Gibco ITS supplement). In some embodiments, the ITS supplement comprises insulin, transferrin, and selenium.

[0184] In some embodiments, the second culture medium further comprises fetal bovine serum (FBS). In some embodiments, the second culture medium further comprises a concentration of FBS, wherein the concentration of FBS is at least about 0.05%, 0.1%, 0.15%, or 0,2%. In some embodiments, the concentration of FBS is about 0.05% to about 10%. In some embodiments, the concentration of FBS is about 0.2%.

[0185] In some embodiments, contacting with the second culture medium is performed for a duration of at least about I day. In some embodiments, the duration is about I day to about 10 days. In some embodiments, the duration is about 1 day.

[0186] In some embodiments, the first stage of differentiation further comprises contacting the pluripotent stem cells with a third culture medium, wherein the third culture medium a TGFP superfamily growth factor described herein (e.g., activin A). In some embodiments, the pluripotent stem cells contacted with the second culture medium, or a portion thereof, are transferred to the third culture medium.

[0187] In some embodiments, the third culture medium comprises a concentration of activin A, wherein the concentration is at least about 10 ng / mL, about 20 ng / mL, about 30 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, or about 100 ng / mL. In some embodiments, the concentration of activin A is about 10 ng / mL to about 200 ng / mL.

[0188] In some embodiments, the third culture medium further comprises a concentration of vitamin C, wherein the concentration is at least about 0.05 mM, about 0.1 mM, about 0.15 mM, about 0.2 mM, or about 0.25 mM. In some embodiments, the concentration of vitamin C is about329282749 45Attorney Docket: MINU-012 / 01WO 339336,20630.05 mM to about 2 mM. In some embodiments, the concentration of vitamin C is about 0.05 mM to about 0.25 mM.

[0189] In some embodiments, the third culture medium comprises an ITS supplement (e.g., Gibco ITS supplement). In some embodiments, the ITS supplement comprises insulin, transferrin, and selenium.

[0190] In some embodiments, the third culture medium further comprises fetal bovine serum (FBS). In some embodiments, the third culture medium further comprises a concentration of FBS, wherein the concentration of FBS is at least about 0.05%, 0.1%, 0,15%, or 0.2%. In some embodiments, the concentration of FBS is about 0.05% to about 10%. In some embodiments, the concentration of FBS is about 0.2%,

[0191] In some embodiments, contacting with the third culture medium is performed for a duration of at least about 1 day. In some embodiments, the duration is about 1 day to about 10 days. In some embodiments, the duration is about 1 day.

[0192] In some embodiments, the first culture medium, the second culture medium, and / or the third culture medium each comprise a base media, wherein the base media is any defined media for human pluripotent stem cell culture known in the art. In some embodiments, the defined media is free of ancillary materials that are animal-derived (e.g., non-human animal or human- derived). In some embodiments, the defined media is StemFit Basisc03 complete cell medium.

[0193] In some embodiments, a plurality of cells of the second population are characterized by¬ expression of a cell marker. In some embodiments, expression of the cell marker is increased in a plurality of cells of the second population as compared to the population comprising pluripotent stem cells from which it was derived. In some embodiments, a plurality of cells of the second population is characterized by expression of CXCR4, SOX17, and / or FOXA2, but do not substantially express PDXl. In some embodiments, at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% of cells of the second population are characterized by expression of CXCR4, SOX17, and FOXA2, but do not substantially express PDXl. In some embodiments, a plurality of cells of the second population are definitive endoderm cells. In some embodiments, at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%,329282749 46Attorney Docket: MINU-012 / 01WO 339336,2063about 90%, about 95%, about 98%, or about 99% of cells of the second population are definitive endoderm cells.(ii) Directed Differentiation to Posterior Foregut Cells

[0194] In some embodiments, the methods described herein for generating a SCD pancreatic islet composition further comprise a second stage of differentiation, wherein the second stage of differentiation comprises differentiating the second population comprising definitive endoderm cells to a third population comprising posterior foregut cells. In some embodiments, a plurality of cells of the second population are definitive endoderm cells, wherein the second stage of differentiation comprises differentiating the definitive endoderm cells to posterior foregut cells. In some embodiments, the differentiating comprises contacting the second population with a first culture medium comprising a growth factor of the fibroblast growth factor (FGF) family described herein (e.g., KGF). In some embodiments, the differentiating further comprising contacting the second population with a second culture medium comprising an activator of the retinoic acid signaling pathway described herein (e.g., TTNPB), an activator of protein kinase C (PKC) described herein (e.g., PMA), an inhibitor of bone morphogenetic protein (BMP) signaling described herein (e.g., LDN193189), and / or an inhibitor of sonic hedgehog (SHH) signaling described herein (e.g., SANT1). In some embodiments, the population comprising definitive endoderm contacted with the first culture medium, or a portion thereof, are then contacted with the second culture medium.

[0195] In some embodiments, the second stage of differentiation comprises contacting the second population comprising definitive endoderm cells with a first culture medium, wherein the first culture medium comprises the fibroblast growth factor (FGF) family (e.g., KGF).

[0196] In some embodiments, the first culture medium comprises a concentration of KGF, wherein the concentration is at least about 5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, or about 50 ng / mL. In some embodiments, the concentration of KGF is about 5 ng / mL to about 200 ng / mL. In some embodiments, the concentration of KGF is about 50 ng / mL.

[0197] In some embodiments, the first culture medium further comprises vitamin C. In some embodiments, the first culture medium further comprises a concentration of vitamin C, wherein329282749 47Attorney Docket: MINU-012 / 01WO 339336,2063the concentration is about at least about 0.1 mM, about 0.15 mM, about 0.2 mM, or about 0.25 mM. In some embodiments, the concentration of vitamin C is about 0.1 mM to about 3 mM.

[0198] In some embodiments, the first culture medium comprises an ITS supplement (e.g., Gibco ITS supplement). In some embodiments, the ITS supplement comprises insulin, transferrin, and selenium.

[0199] In some embodiments, the first culture medium further comprises FBS. In some embodiments, the first culture medium further comprises a concentration of FBS, wherein the concentration of FBS is at least about 0.5%, about 1%, about 1.5%, or about 2%. In some embodiments, the concentration of FBS is about 0.5% to about 10%. In some embodiments, the concentration of FBS is about 2%.

[0200] In some embodiments, the first culture medium comprises a base media, wherein the base media is any defined media for human pluripotent stem cell culture known in the art. In some embodiments, the defined media is free of ancillary materials that are animal-derived (e.g., nonhuman animal or human-derived). In some embodiments, the defined media is StemFit Basisc03 complete cell medium.

[0201] In some embodiments, contacting with the first culture medium is performed for a duration of at least about 1 day, about 2 days, or about 3 days. In some embodiments, the contacting is performed for a duration of about I day to about 5 days. In some embodiments, the contacting is performed for a duration of about 1 day, about 2 days, or about 3 days.

[0202] In some embodiments, the second culture medium comprises a retinoic pathway activator described herein (e.g., TTNPB), a SHH signaling inhibitor described herein (e.g., Sant-1), a PKC activator described herein (e.g., PMA), and / or a BMP signaling inhibitor described herein (e.g., LDN193189). In some embodiments, the second culture medium comprises the retinoic pathway activator, the SHH signaling inhibitor, the PKC activator, and the BMP signaling inhibitor. In some embodiments, the second culture medium comprises TTNPB, Sant-1, PMA, and LDN193189.

[0203] In some embodiments the second culture medium comprises (i) a concentration of TTNPB, wherein the concentration is at least about 0.5 nM, about 1 nM, about 1.5 nM, about 2 nM, about 2.5 n, or about 3 nM; (ii) a concentration of Sant-1, wherein the concentration is at least about 50 nM, about 100 M, about 150 nM, about 200 nM, or about 250 M; (iii) a concentration of PMA, wherein the concentration is at least about 5 n, about 10 nM, about 15329282749 48Attorney Docket: MINU-012 / 01WO 339336,2063nM, about 20 nM, about 25 nM, or about 30 nM; and (iv) a concentration of LDN193189, wherein the concentration is at least about 50 nM, about 100 nM, about 150 nM, about 200 nM, or about 250 nM. In some embodiments the concentration of TTNPB is about 0.5 to about 10 n. In some embodiments the concentration of TTNPB is about 3 nM. In some embodiments, the concentration of Sant-1 is about 50 nM to about 500 n. In some embodiments, the concentration of Sant-1 is about 250 nM. In some embodiments, the concentration of PMA is about 5 nM to about 100. In some embodiments, the concentration of PMA is about 30 nM. In some embodiments, the concentration of LDN193189 is about 50 nM to about 500 nM. In some embodiments, the concentration of LDN193189 is about 250 nM.

[0204] In some embodiments, the second culture medium further comprises non-essential amino acids, sodium pyruvate, and neurocult.

[0205] In some embodiments, the second culture medium further comprises a concentration of vitamin C, wherein the concentration is about at least about 0.1 mM, about 0, 15 mM, about 0.2 mM, or about 0,25 mM, In some embodiments, the concentration of vitamin C is about 0.1 mM to about 3 mM.

[0206] In some embodiments, contacting with the second culture medium is performed for a duration of at least about 1 day, about 2 days, or about 3 days. In some embodiments, the contacting is performed for a duration of about I day to about 5 days. In some embodiments, the contacting is performed for a duration of about 1 day, about 2 days, or about 3 days.

[0207] In some embodiments, a plurality of cells of the third population are characterized by expression of a cell marker. In some embodiments, expression of the cell marker is increased in a plurality of cells of the third population as compared to the population comprising definitive endoderm cells from which it was derived. In some embodiments, at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% of cells of the third population are characterized by expression of PDX1, HNF6, FOXA2, HNF4A, HNF1B, and / or FOXA1. In some embodiments, a plurality of cells of the third population are posterior foregut cells. In some embodiments, at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% of cells of the third population are posterior foregut cells.329282749 49Attorney Docket: MINU-012 / 01WO 339336.2063(iii) Directed Differentiation to PDX1, NKX6.1 -Positive Pancreatic Progenitor Cells

[0208] In some embodiments, the methods described herein for generating a SCD pancreatic islet composition further comprise a third stage of differentiation, wherein the third stage of differentiation comprises differentiating a third population comprising posterior foregut cells to a fourth population comprising pancreatic progenitor cells (e.g., PDX1, NKX6.1 -positive pancreatic progenitor cells). In some embodiments, the differentiating comprises contacting the population comprising posterior foregut cells with (i) a first culture medium culture medium comprising epidermal growth factor (EGF) and / or a retinoic pathway activator described herein (e.g., TTNPB); and (ii) a second culture medium comprising EGF and / or a growth factor of the fibroblast growth factor (FGF) family described herein (e.g., KGF). In some embodiments, the population comprising posterior foregut cells contacted with the first culture medium, or a portion thereof, are then contacted with the second culture medium. In some embodiments, the population contacted with the second culture medium is then contacted with the third culture medium.

[0209] In some embodiments, the third stage of differentiation comprises contacting the population comprising posterior foregut cells with a first culture medium, wherein the first culture medium comprises EGE In some embodiments, the first culture medium comprises a concentration of EGF, wherein the concentration is at least about 10 ng / mL, about 20 ng / mL, about 30 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, or about 100 ng / mL. In some embodiments, the concentration of EGF is about 10 ng / mL to about 1,000 ng / mL. In some embodiments, the concentration of EGF is about 100 ng / mL.

[0210] In some embodiments, the first culture medium further comprises a retinoic pathway activator described herein (e.g., TTNPB). In some embodiments, the first culture medium further comprises a concentration of TTNPB, wherein the concentration is at least about 0.5 nM, about 1 nM, about 1.5 nM, about 2 nM, about 2.5 nM, or about 3 n. In some embodiments the concentration of TTNPB is about 0.5 nM to about 10 nM. In some embodiments the concentration of TTNPB is about 3 nM.

[0211] In some embodiments, the first culture medium further comprises non-essential amino acids, sodium pyruvate, and neurocult.329282749 50Attorney Docket: MINU-012 / 01WO 339336,2063

[0212] In some embodiments, the first culture medium further comprises a concentration of vitamin C, wherein the concentration is about at least about 0.1 mM, about 0.15 mM, about 0.2 mM, or about 0.25 mM. In some embodiments, the concentration of vitamin C is about 0.1 mM to about 3 mM.

[0213] In some embodiments, contacting with the first culture medium is performed for a duration of at least about 1 day, about 2 days, or about 3 days. In some embodiments, the contacting is performed for a duration of about 1 day to about 5 days. In some embodiments, the contacting is performed for a duration of about 1 day, about 2 days, or about 3 days.

[0214] In some embodiments, the population of cells is further contacted with a second culture medium, wherein the second culture medium comprises EGF. In some embodiments, the second culture medium comprises a concentration of EGF, wherein the concentration is at least about 10 ng / mL, about 20 ng / mL, about 30 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, or about 100 ng / mL. In some embodiments, the concentration of EGF is about 10 ng / mL to about 1,000 ng / mL. In some embodiments, the concentration of EGF is about 100 ng / mL.

[0215] In some embodiments, the second culture medium further comprises a concentration of KGF, wherein the concentration is at least about 5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, or about 50 ng / mL. In some embodiments, the concentration of KGF is about 5 ng / mL to about 200 ng / mL. In some embodiments, the concentration of KGF is about 50 ng / mL.

[0216] In some embodiments, the second culture medium further comprises non-essential amino acids, sodium pyruvate, and neurocult.

[0217] In some embodiments, the second culture medium further comprises a concentration of vitamin C, wherein the concentration is about at least about 0.1 mM, about 0.15 mM, about 0.2 mM, or about 0.25 mM. In some embodiments, the concentration of vitamin C is about 0.1 mM to about 3 mM.

[0218] In some embodiments, the first culture medium and the second culture medium each comprises a base media, wherein the base media is any defined media for human pluripotent stem cell culture known in the art. In some embodiments, the defined media is free of ancillary materials that are animal- derived (e g., non-human animal or human-derived). In some embodiments, the defined media is StemFit Basisc03 complete cell medium.329282749 51Attorney Docket: MINU-012 / 01WO 339336,2063

[0219] In some embodiments, a plurality of cells of the fourth population are characterized by expression of PDX1 (e.g., as measured by immunohistochemistry, ELISA, or flow cytometry). In some embodiments, the plurality is further characterized by expression of HNF4A, HNF1B, FOXA1, FOXA2, SOX9, NKX6-1, NGN3, and / or PTF1 A (e.g., as measured by immunohistochemistry, qPCR, ELISA, or flow cytometry). In some embodiments, a plurality of cells of the fourth population are characterized by expression of PDX1 and NKX6-1 (e.g., as measured by immunohistochemistry, qPCR, ELISA, or flow cytometry). In some embodiments, a plurality of cells of the fourth population are characterized by expression of PDX1, HNF4A, HNF1B, FOXA1, FOXA2, SOX9, NKX6-1, NGN3, and PTF1 A (e.g., as measured by immunohistochemistry, qPCR, ELISA, or flow cytometry).

[0220] In some embodiments, at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% of cells of the fourth population are characterized by expression of PDX1 (e.g,, as measured by immunohistochemistry or flow cytometry). In some embodiments, at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% of cells of the fourth population are characterized by expression of PDX1 and NKX6-1 (e.g., as measured by immunohistochemistry or flow cytometry). In some embodiments, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70% of cells of the fourth population are characterized by expression of PDX1 and NKX6-1 (e.g., as measured by immunohistochemistry or flow cytometry).

[0221] In some embodiments, at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% of cells of the fourth population are pancreatic progenitor cells (e.g., PDX1+, NKX6-1+ pancreatic progenitor cells). In some embodiments, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70% of cells of the fourth population are pancreatic progenitor cells (e.g., PDX1+, NKX6-1+ pancreatic progenitor cells).(iv) Insulin Producing Endocrine Cells329282749 52Attorney Docket: MINU-012 / 01WO 339336.2063

[0222] In some embodiments, the method further comprises a fourth stage of differentiation, wherein the fourth stage of differentiation comprises differentiating the fourth population comprising pancreatic progenitor cells (e.g., PDX1, NKX6.1 -positive pancreatic progenitor cells) to a fifth population comprising insulin producing endocrine cells (e g., SCD P cells). In some embodiments, a plurality of cells of the fourth population are PDX1+, NKX6-1+ pancreatic progenitor cells, wherein the fourth stage of differentiation comprises differentiating the PDX1+, NKX6-1+ pancreatic progenitor cells to insulin producing endocrine cells (e.g., SCD P cells). In some embodiments, the differentiation comprises contacting the population comprising PDX1 +, NKX6-1 + pancreatic progenitor cells with (i) a first culture medium comprising an inhibitor of TGFp type I receptor (Alk5) described herein (e.g., Alk5i II), a thyroid hormone described herein (e.g., triiodothyronine), a BMP signaling inhibitor described herein (e.g., LDN193189), and / or an inhibitor of gamma secretase (Notch signaling) described herein (e.g., XXi); and (ii) a second culture medium comprising an inhibitor of Alk5 described herein (e.g., Alk5i II), a thyroid hormone described herein (e.g., triiodothyronine), a BMP signaling inhibitor described herein (e.g., LDN193189), and / or an inhibitor of gamma secretase (Notch signaling) described herein (e.g., XXi). In some embodiments, the population comprising PDX1+, NKX6-1+ pancreatic progenitor cells is first contacted with the first culture medium, whereupon the population or a portion thereof, is then contacted with the second culture medium.

[0223] In some embodiments, the first culture medium comprises an inhibitor of TGFp type I receptor (Alk5) described herein (e.g., Alk5i II), a thyroid hormone described herein (e.g., triiodothyronine), a BMP signaling inhibitor described herein (e.g., LDN193189), and / or an inhibitor of gamma secretase (Notch signaling) described herein (e.g., XXi). In some embodiments, the first culture medium comprises the inhibitor of TGFP type I receptor (Alk5), the thyroid hormone, the BMP signaling inhibitor, and the inhibitor of gamma secretase (Notch signaling).

[0224] In some embodiments, the first culture medium comprises (i) a concentration of Alk5i 11, wherein the concentration is at least about 1 pM, about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 6 pM, about 7 pM, about 8 pM, about 9 pM, or about 10 pM; (ii) a concentration of triiodothyronine, wherein the concentration is at least about 0.1 pM, about 0.2 pM, about 0.3 pM, about 0.4 pM, about 0.5 pM, about 0.6 pM, about 0.7 pM, about 0.8 pM, about 0.9 pM, or about 1 pM; (iii) a concentration of LDN193189, wherein the concentration is at least about 0.01329282749 53Attorney Docket: MINU-012 / 01WO 339336,2063pM, about 0.05 pM, about 0.1 pM, about 0.2 pM, about 0.3 pM, about 0.4 pM, or about 0.5 pM; and (iv) a concentration of XXi, wherein the concentration is at least about 0.1 pM, about 0.2 pM, about 0.3 pM, about 0.4 pM, about 0.5 pM, about 0.6 pM, about 0.7 pM, about 0.8 pM, about 0.9 pM, or about 1 pM.

[0225] In some embodiments, the first culture medium comprises a concentration of Alk5i II of about 1 pM to about 20 pM. In some embodiments, the first culture medium comprises a concentration of Alk5i II of about 10 pM.

[0226] In some embodiments, the first culture medium comprises a concentration of triiodothyronine of about 0.1 pM to about 10 pM. In some embodiments, the first culture medium comprises a concentration of triiodothyronine of 1 pM,

[0227] In some embodiments, the first culture medium comprises a concentration of LDN193189 of about 0.01 pM to about 5 pM. In some embodiments, the first culture medium comprises a concentration of LDN193189 of about 0.5 pM.

[0228] In some embodiments, the first culture medium comprises a concentration of XXi of about 0.1 pM to about 10 pM. In some embodiments, the first culture medium comprises a concentration of XXi of about 1 pM.

[0229] In some embodiments, the first culture medium further comprises non-essential amino acids, sodium pyruvate, insulin, selenium, transferrin, zinc, cysteine, vitamin C, and bovine serum albumin (BSA).

[0230] In some embodiments, the first culture medium comprises a concentration of zinc, wherein the concentration is at least about 1 pM, about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 6 pM, about 7 pM, about 8 pM, about 9 pM, or about 10 pM. In some embodiments, the concentration of zinc is about 10 pM.

[0231] In some embodiments, the first culture medium comprises a concentration of vitamin C, wherein the concentration is at least about 10 pM, about 50 pM, about 100 pM, or about 150 pM. In some embodiments, the concentration of vitamin C is about 155 pM.

[0232] In some embodiments, the first culture medium comprises a concentration of BSA, wherein the concentration is at least about 0.1%, about 0.5%, about 1%, about 1.5%, or about 2%. In some embodiments, the concentration of BSA is about 2%.

[0233] In some embodiments, contacting with the first culture medium is performed for a duration of at least about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6329282749 54Attorney Docket: MINU-012 / 01WO 339336,2063days, or about 7 days. In some embodiments, the contacting is performed for a duration of about 3 days to about 10 days. In some embodiments, the contacting is performed for a duration of about 6 days, about 7 days, or about 8 days.

[0234] In some embodiments, the second culture medium comprises an inhibitor of Alk5 described herein (e.g., Alk5i II), a thyroid hormone described herein (e.g., triiodothyronine), a BMP signaling inhibitor described herein (e.g., LDN193189), and / or an inhibitor of gamma secretase (Notch signaling) described herein (e.g., XXi). In some embodiments, the second culture medium comprises the inhibitor of TGFp type I receptor (Alk5), the thyroid hormone, the BMP signal ing inhibitor, and the inhibitor of gamma secretase (Notch signaling).

[0235] In some embodiments, the second culture medium comprises (i) a concentration of Alk5i II, wherein the concentration is at least about 1 pM, about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 6 pM, about 7 pM, about 8 pM, about 9 pM, or about 10 pM; (ii) a concentration of triiodothyronine, wherein the concentration is at least about 0.1 pM, about 0.2 pM, about 0.3 pM, about 0.4 pM, about 0.5 pM, about 0.6 pM, about 0.7 pM, about 0.8 pM, about 0.9 pM, or about 1 pM; (iii) a concentration of LDN193189, wherein the concentration is at least about 0.01 pM, about 0.05 pM, about 0.1 pM, about 0.2 pM, about 0.3 pM, about 0.4 pM, or about 0.5 pM; and (iv) a concentration of XXi, wherein the concentration is at least about 0.1 pM, about 0.2 pM, about 0.3 pM, about 0.4 pM, about 0.5 pM, about 0.6 pM, about 0.7 pM, about 0.8 pM, about 0.9 pM, or about 1 pM.

[0236] In some embodiments, the second culture medium comprises a concentration of Alk5i II of about 1 pM to about 20 pM. In some embodiments, the second culture medium comprises a concentration of Alk5i II of about 10 pM.

[0237] In some embodiments, the second culture medium comprises a concentration of triiodothyronine of about 0.1 pM to about 10 pM. In some embodiments, the second culture medium comprises a concentration of triiodothyronine of 1 pM.

[0238] In some embodiments, the second culture medium comprises a concentration of LDN193189 of about 0.01 pM to about 5 pM. In some embodiments, the second culture medium comprises a concentration of LDN193189 of about 0.5 pM.

[0239] In some embodiments, the second culture medium comprises a concentration of XXi of about 0.1 pM to about 10 pM. In some embodiments, the second culture medium comprises a concentration of XXi of about 1 pM.329282749 55Attorney Docket: MINU-012 / 01WO 339336,2063

[0240] In some embodiments, the second culture medium further comprises non-essential amino acids, sodium pyruvate, zinc, cysteine, vitamin C, heparin, glutamax, and BSA.

[0241] In some embodiments, the first second medium comprises a concentration of zinc, wherein the concentration is at least about 1 pM, about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 6 pM, about 7 pM, about 8 pM, about 9 pM, or about 10 pM. In some embodiments, the concentration of zinc is about 10 pM.

[0242] In some embodiments, the second culture medium further comprises a concentration of vitamin C, wherein the concentration is at least about 10 pM, about 50 pM, about 100 pM, or about 150 pM, In some embodiments, the concentration of vitamin C is about 155 pM.

[0243] In some embodiments, the second culture medium comprises a concentration of BSA, wherein the concentration is at least about 0.1%, about 0.5%, about 1%, about 1.5%, or about 2%. In some embodiments, the concentration of BSA is about 2%. In some embodiments, the second culture medium comprises a concentration of BSA, wherein the concentration is at least about 1 pg / mL, about 5 pg / mL, or about 10 pg / mL. In some embodiments, the concentration of BSA is about 10 pg / mL.

[0244] In some embodiments, contacting with the second culture medium is performed for a duration of at least about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days. In some embodiments, the contacting is performed for a duration of about 3 days to about 10 days. In some embodiments, the contacting is performed for a duration of about 6 days, about 7 days, or about 8 days.

[0245] In some embodiments, a plurality of cells of the fifth population are characterized by expression of a marker (e.g., as measured by immunohistochemistry, ELISA, or flow cytometry). In some embodiments, a plurality of cells of the fifth population are characterized by expression of NKX6-1 and C-peptide (e.g., as measured by immunohistochemistry, ELISA, or flow cytometry). In some embodiments, a plurality of cells of the fifth population are characterized by expression of NKX6-1 and INS (e.g., as measured by immunohistochemistry, ELISA, qPCR, or flow cytometry). In some embodiments, the plurality' of cells of the fifth population are further characterized by expression of PDX1, CHGA, SIX2, MAFA, NKX2.2, NEUROD, ISL1, UCN3, ENTPD3, and / or MAFB (e.g., as measured by immunohistochemistry, ELISA, qPCR, or flow cytometry). In some embodiments, at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about329282749 56Attorney Docket: MINU-012 / 01WO 339336,206365%, about 70%, about 75%, or about 80% of the fifth population are characterized by expression of NKX6-1 and c-peptide (e.g., as measured by immunohistochemistry or flow cytometry). In some embodiments, about 5% to about 50% of the fifth population are characterized by expression of NKX6-1 and c-peptide (e.g., as measured by immunohistochemistry or flow cytometry). In some embodiments, at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% of cells of the fifth population are pancreatic P cells. In some embodiments, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% of cells of the fifth population are SCD cells. In some embodiments, about 5% to about 50% of cells of the fifth population are SCD P cells,

[0246] In some embodiments, the fifth population comprises clusters of cells, wherein the clusters of cells comprise the SCD p cells. In some embodiments, the cluster of cells further comprises SCD a cells and / or SCD 8 cells. In some embodiments, the cluster of cells comprises at least about 10, about 50, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 600, about 700, about 800, about 900, about IxlO3, about 1.5xl03, about 2xl03, about 3xl03, about 4xl03, about 5x103, or about IxlO4. In some embodiments, the cell cluster comprises not more than about IxlO5, 50xl04, 40xl04, 30x104, 20xl04, 10xl04, 5xl04, IxlO4, 50xl03, 40xl03, 30x103, 20xl03, 10xl03, or 5xlO3cells.

[0247] In some embodiments, the cell cluster is substantially spherical. In some embodiments, the cell cluster is not spherical. In some embodiments, the cell cluster comprises a longest diameter of about 40 pm, about 50 pm, about 60 pm, about 70 pm, about 80 pm, about 90 pm, about 100 pm, about 110 pm, about 120 pm, about 130 pm, about 140 pm, about 150 pm, about 160 pm, about 170 pm, about 180 pm, about 190 pm, about 200 pm, about 210 pm, about 220 pm, about 230 pm, about 240 pm, about 250 pm, about 260 pm, about 270 pm, about 280 pm, about 290 pm, about 300 pm, about 310 pm, about 320 pm, about 330 pm, about 340 pm, about 350 pm, about 360 pm, about 370 pm, about 380 pm, about 390 pm, about 400 pm, about 410 pm, about 420 pm, about 430 pm, about 440 pm, about 450 pm, about 460 pm, about 470 pm, about 480 pm, about 490 pm, or about 500 pm. In some embodiments, the cell cluster comprises a longest diameter of about 100 pm to about 300 pm. In some embodiments, the cell cluster comprises a longest diameter of about 100 pm to about 200 pm.329282749 57Attorney Docket: MINU-012 / 01WO 339336,2063

[0248] In some embodiments, the SCD pancreatic islet composition described herein (e.g., the a SCD pancreatic islet compositions comprising insulin-producing cells) are cultured under conditions and supplemented with nutrients to improve survival rates. It has been shown in vitro that nutrient deprivation and hypoxia can independently kill mature insulin producing cells such as human islets. Combination of nutrient deprivation and hypoxia that occurs during ischemia act additively to kill insulin producing cells. SCD pancreatic precursor cells and immature insulin producing cells are more resistant to nutrient deprivation and hypoxia alone, but these two factors act synergistically to kill pancreatic precursor cells and immature insulin producing cells in vitro. However, generating pancreatic precursor cells and immature insulin producing cells under physiological oxygen tension of 5% can confer hypoxia resistance without affecting differentiation or function. As such, in some embodiments, insulin-producing cells are cultured prior to transplantation in an atmosphere having any of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% oxygen concentration. In another embodiment, the transplanted insulin producing cells are supplemented during and / or after transplantation with one or more amino acids. In some nonlimiting embodiments, the amino acids are alanine and glutamine. Further information regarding pre-culturing insulin-producing cells under low oxygen conditions prior to transplant and nutritional supplementation of these cells can be found in Faleo, et al., Stem Cell Reports, 2017, 9(3): 807- 19, the disclosure of which is incorporated by reference herein in its entirety.(Ill) Differentiation Factors

[0249] The present disclosure provides factors for differentiating pluripotent stem cells described herein (e.g., a population comprising ESCs or a population comprising iPSCs) to a population comprising insulin producing endocrine cells (e.g., insulin producing SCD P cells). In some embodiments, the factors are used in the methods described herein to differentiate the population comprising pluripotent stem cells to a population comprising definitive endoderm cells. In some embodiments, the factors are used in the methods described herein to differentiate a population comprising definitive endoderm cells to a population comprising posterior foregut cells. In some embodiments, the factors are used in the methods described herein to differentiate a population comprising posterior foregut cells to a population comprising PDX1, NKX6.1 -positive pancreatic progenitor cells to a population comprising insulin producing endocrine cells (e.g., insulin producing SCD P cells).329282749 58Attorney Docket: MINU-012 / 01WO 339336.2063(i) Rho Kinase (ROCK) Signaling Pathway

[0250] As described herein, the methods of the disclosure for generating populations comprising SCD insulin producing pancreatic cells (e.g., SCD cells) use inhibitors of the ROCK signaling pathway. ROCK is a serine / threonine kinase downstream of Ras homolog gene family, member A (RhoA), a small GTPase protein in the Rho family. RhoA is converted between an inactive and active state by binding to GTP. Once activated, RhoA associates with ROCK to mediate downstream signaling.

[0251] In some embodiments, the ROCK inhibitor comprises a compound set forth in Table 1.Table 1: Exemplary ROCK inhibitors of the DisclosureCompound Name Structure SourceY-27632 Selleckchem Cat No SI 049H N. —, HN~ / X- A. / 02mThiazovivin Selleckchem Cat No SI 459Fasudil Selleckchem Cat No S1573NH HCI GSK429286A H(R. NS / Selleckchem Cat No ThSI 474k,bk,,7 H 7 7 \O " ^'1XF329282749 59Attorney Docket: MINU-012 / 01WO 339336,2063ZINC00881524 Selleckchem Cat No S8448- Oo-A / > / Y / — dRKI-1447 Selleckchem Cat No S719500(ii) Transforming Growth Factor P (TGFP) Superfamily

[0252] As described herein, the methods of the disclosure for generating populations comprising SCD insulin producing pancreatic cells (e.g., SCD P cells) use growth factors of the TGFP superfamily. As used herein, the term “TGFP superfamily” refers to polypeptides that interact with TGFP receptors. In some embodiments, the TFGP superfamily comprises polypeptides of the activin subfamily (e.g., activin A, activin B, and activin AB), polypeptides of the inhibin subfamily (e.g., inhibin A and inhibin B), bone morphogenetic proteins, polypeptides of the TGF subfamily (e.g., TGFpi, TGFp2, TGF 3), polypeptides of the growth and differentiation factor (GDF) family (e.g., GDF1, GDF2, GDF3, GDF5, GDF6, GDF8, GDF9, GDF10, GDF11, and GDF15), and polypeptides of the left-right determination factor family (e.g., lefty-1).

[0253] In some embodiments, the growth factor of the TGF superfamily is activin A. In some embodiments, the growth factor of the TGFβ superfamily is human activin A. The amino acid sequence for human activin A is publicly available through the Uniprot database using reference number A0A1B0GXA9.(iii) WNT Signaling Pathway Activators

[0254] As described herein, the methods of the disclosure for generating populations comprising SCD insulin producing pancreatic cells (e.g., SCD P cells) use activators of the WNT signaling pathway. Wnt proteins are secreted morphogens that play roles in stem cell proliferation and selfrenewal (see, e.g,, Bonnet et al (2021) RSC Chem Biol 2:1144). In some embodiments, the activator is a small molecule that activates the Wnt signaling pathway. In some embodiments, the329282749 60Attorney Docket: MINU-012 / 01WO 339336,2063activator is a small molecule inhibitor of GSK-3p. Exemplary' activator include, but are not limited to, TWS119 (Selleckchem Cat No S1590), IQ-1 (StemCell Technologies Cat No 72774), SB216763 (Tocris Cat No 1616), SB415286 (Tocris Cat No S2729), CHIR9902 (Selleckchem Cat No S1263), and L807mts (see Licht-Murava, et al (2016) Sci Signal 9:rall0). In some embodiments, the activator is CHIR99021. In some embodiments, the activator is a salt of CHIR99021.(iv) Fibroblast Growth Factor (FGF) Family

[0255] As described herein, the methods of the disclosure for generating populations comprising SCD insulin producing pancreatic cells (e.g., SCD cells) use a polypeptide of the FGF family. In some embodiments, the polypeptide of the FGF family comprises keratinocyte growth factor (KGF). In some embodiments, the polypeptide of the FGF family is human KGF. Human KGF is also referred to as fibroblast growth factor 7. Sequence information for KGF is known in the art (see, e.g., UniProt Ref No P21781).

[0256] In some embodiments, the polypeptide of the FGF family comprises FGF2. Sequence information for FGF2 is known in the ait (see, e.g., UniProt Ref No P09038).

[0257] In some embodiments, the polypeptide of the FGF family comprises FGF8. Sequence information for FGF8 is known in the art (see, e.g,, UniProt Ref No P55075),

[0258] In some embodiments, the polypeptide of the FGF family comprises FGF10. Sequence information for FGF10 is known m the art (see, e.g., UniProt Ref No 015520),

[0259] In some embodiments, the polypeptide of the FGF family comprises FGF21, Sequence information for FGF21 is known m the art (see, e.g., UniProt Ref No Q9NSA1),(v) Retinoic Acid Signaling Pathway Modulation

[0260] As described herein, the methods of the discl osure for generating populations comprising SCD insulin producing pancreatic cells (e.g., SCD P cells) use a modulator of retinoic acid signaling. Retinoic acid (RA) is a metabolite of retinol (vitamin A) that functions as a ligand of nuclear RA receptors (RARs), which include RARa, RARP, and RARy. RARs bind to DNA and recruit nuclear receptor coactivators or nuclear receptor corepressors to activate or repress transcription.329282749 61Attorney Docket: MINU-012 / 01 WO 339336.2063

[0261] In some embodiments, the modulator is an activator (agonist) of RA signaling, such as any activator of RA signaling known in the art. In some embodiments, the modulator is an agonist of RARa, RARP, and / or RARy. In some embodiments, the modulator is an agonist of RARa, RARP, and RARy. In some embodiments, the modulator is a selective RARP agonist. In some embodiments, the modulator is a selective RARa agonist. In some embodiments, the modulator is a selective RARy agonist. In some embodiments, the activator comprises retinoic acid. In some embodiments, the activator comprises an analog of retinoic acid. In some embodiments, the activator is a compound set forth in Table 2. In some embodiments, the activator is TTNPB.Table 2: Exemplary RA Signaling Pathway Activators of the DisclosureCompound Name Structure SourceTTNPB StemCelln Technologies Cat No 1, 72892\ ' '' ' IfF AC 261066 Tocris Cat No 4046v / jA---\ KAvz' radapalene Tocris Cat No 2852tazarotene Selleckchem Cat No SI 569 OCkA6AM80 Tocris Cat No 3507A CO?H329282749 62Attorney Docket: MINU-012 / 01WO 339336,2063AM580, X;XHM e M s 0 X Tocns Cat No 0760 X A A A CJJs'M R M R BMS753 \ / Tocris Cat No 3505 v y H | i y^o / / V\ A X oF BMS961 Q Tocris Cat No 3410 >o-- i5ZI ''C02l-lCD 1530 X- 1 „ X J Tocris Cat No 2554~ X I s- x '^-0HCD2314 Tocris Cat No 3824 _ / — CCVH000^CD437 XX^C°2HTocris Cat No 3824MeCh55 M e M e Tocris Cat No 2020.......v ADC2 M 0a ~r,71 0 Tocris Cat No 6873A 'H-(vi) Protein Kinase C (PKC) Activators10262] As described herein, the methods of the disclosure for generating populations comprising SCD insulin producing pancreatic cells (e.g., SCD P cells) use an activator of one or more PKCs. PKCs are serine / threonine kinases that function in multiple cellular signaling pathways. There are multiple PKC isoforms, including conventional PKCs (a, pi), alternatively spliced PCKs (PIT329282749 63Attorney Docket: MINU-012 / 01WO 339336.2063and y), novel PCKs (5, 0, s, rj), and atypical PCKs (2, and 1 / ). Canonical PKC signaling is activated by phospholipase C (PLC)-mediated hydrolysis of phosphatidylinositol 4,5-biphosphate (PIP2) to diacylglycerol (DAG), which in turn activates PKC and inositol triphosphate (IPa) to mobilize intracellular calcium. Many PKCs are activated by phorbol esters, e.g., phorbol 12-myristate 13-acetate (PMA), that anchor PCKs in their active conformation to membranes. In some embodiments, the activator is bryostatin-1. In some embodiments, the activator is diacylglycerol. In some embodiments, the activator is PMA. In some embodiments, the activator is prostatin (13-O-acetyl-12-deoxyphorbol), In some embodiments, the activator is Phorbol 12,13-dibutyrate. See Kawano, et al (2021) Pharmaceutics 13:1748 (herein incorporated by reference) for exemplary PKC activators for use in the methods of the disclosure.(vii) Bone Morphogenetic Protein (BMP) Signaling Pathway Inhibitors

[0263] As described herein, the methods of the disclosure for generating populations comprising SCD insulin producing pancreatic cells (e.g., SCD ]3 cells) use inhibitors of the BMP signaling pathway. The BMP signaling family is a subset of the TGFP superfamily. In humans, the family contains at least 20 family members (see Bragdon, et al (2011) Cell Signal 23:609). For example, the family includes multiple TGFp / GDF / activin type I receptors (ALK2, ALK3, ALK1, and ALK6) and type II receptors (BMPRII, ActRIIa, and ActRIIb). Endogenously, dimeric ligands facilitate assembly of receptor heterodimers, such that the constitutively active type II receptor phosphorylates a type I receptor. Activated type I receptors phosphorylate SMAD effectors (SMADs 1, 5, and 8) to facilitate nuclear translocation in complex with SMAD4.

[0264] In some embodiments, the BMP signaling pathway inhibitor comprises a compound set forth in Table 3. In some embodiments, the BMP signaling pathway inhibitor is a compound described in Dinter, et al (2019) Methods Mol Biol 1891:221, herein incorporated by reference.

[0265] In some embodiments, the BMP signaling pathway inhibitor comprises a compound described in Hao, et al (2010) ACS Chem Biol 5:245, herein incorporated by reference.

[0266] In some embodiments, the BMP signaling pathway inhibitor comprises LDN-193189. LDN-193189 is a selective inhibitor of ALK1, ALK2, ALK3, and ALK6 (IC50 = 0.8, 0.8, 5.3, and 16.7 nM respectively; see Sanvitale, etal (2013) PLOS One 8:e62721). In some embodiments, the BMP signaling pathway inhibitor comprises any salt of LDN- 193189. In some329282749 64Attorney Docket: MINU-012 / 01WO 339336.2063embodiments, the BMP signaling pathway inhibitor comprises the dihydrochloride salt of LDN-193189.Table 3: Exemplary BMP Signaling Pathway Inhibitors of the DisclosureCompound Name Structure Source Dorsomorphin Tocris Bioscience Cat dihydrochloride I'T No 3093-. r i?r / \ / —\ \ / / \ / X _>K' \.. \ y\ X\ / oLDN-193189 HN~-\ StemCell dihydrochloride Technologies ky2HCi [XHjO]SB-431452 n SelleckchemXi i T 4^Ay „J..(zXV / YV- / .0Ox / OK02288 Selleckchem329282749 65Attorney Docket: MINU-012 / 01WO 339336,2063Compound Name Structure SourceLDN-212854 Tocris Bioscience HNAO DMH1 SelleckchemO-,■ L J. >f K- / LDN-214117 HN '•'J 0 Selleckchem Q.( i:■ t N 'x ' °(viii) Sonic Hedgehog (SHH) Signaling Pathway Inhibitors

[0267] As described herein, the methods of the disclosure for generating populations comprising SCD insulin producing pancreatic cells (e g., SCD p cells) uses an inhibitor of the SHH pathway. The hedgehog signaling pathway is initiated by SHH, Indian hedgehog, and Desert hdedgehog, with SHH being the most widely expressed and potent of the three ligands. The receptor for SHH is Patched! (PITCH!), Binding of SHH to PITCH! relieves repressed of Smoothed (SMO) by PITCH!, which activates SMO signaling activity. SMO signaling activates GU transcription factors, resulting in changes in gene expression. See Carpenter (2019) Drug Saf 42:263.

[0268] In some embodiments, the SHH signaling pathway inhibitor is a compound set forth in Table 4. In some embodiments, the SHH signaling pathway inhibitor is SANT1. In some embodiments, the SHH signaling pathway inhibitor is a salt of SANT2,Table 4: Exemplary SHH signaling pathway inhibitors of the disclosure329282749 66Attorney Docket: MINU-012 / 01WO 339336,2063Compound Name Structure SourceCur-61414 MedChemExpress Cat No HY-113965O / "~Q J b QO 7"! \. z / / / \1SANT1 StemCell / z \5 < Technologies Cat \#100-0539X " xX") '■ •XSANT2 MedChemExpress Cat No HY-107408CiVF oUO„ ’ » 0.20,^ / 'AY9944 zx.. Cl x-x Abeam Cat No A Y CX.2..( F- 1 2 9944,2HCi(ix) Epidermal Growth Factor (EGF) Family

[0269] As described herein, the methods of the discl osure for generating populations comprising SCD insulin producing pancreatic cells (e.g., SCD P cells) use polypeptides of the EGF family. In some embodiments, the polypeptide of the EGF family is human wild-type EGF polypeptide. Sequence information for human wild-type EGF polypeptide is accessible via UniProt database reference Q6QBS2 In some embodiments, the polypeptide is a variant of human wild-type EGF329282749 67Attorney Docket: MINU-012 / 01WO 339336.2063polypeptide. In some embodiments, the variant comprises at least 80%, 85%, 90%, or 95% identity to human wild-type EGF polypeptide. In some embodiments, the polypeptide is a truncation of human wild-type EGF polypeptide, wherein the truncation retains binding to the EGF receptor.(x) TGF-P Signaling Pathwa Xy O X Inhibitors\ / /

[0270] As described herein, the methods of the disclosure for generating populations comprising SCD insulin producing pancreatic c Mells (e.g., SCD P cells) use inhibitors of the TGF'P signaling pathway. In some embodiments, the inhibitor of the TGF'P signaling pathway comprises an inhibitor of TGF'P receptor (also referred to o as “ALK5”).o• /

[0271] In some embodiments, the inhibitor is a compound identified in Table 5.

[0272] In some embodiments, the TGFP signaling pathway comprises a ALK5 inhibitor described m US Pat Pub 20100267731, US20090186076, US20070142376, and US20230092449, each of which are herein incorporated by reference.Table 5: Exemplary TGFP Signaling Pathway Inhibitors of the DisclosureCompound Name Structure SourceLY 3200882R-268712 Selleckchem Cat No S7710HNZ / / f - ~\\ U / V-O8329282749 68Attorney Docket: MINU-012 / 01WO 339336,2063Compound Name Structure Source TP0427736 Selleckchem Cat No S8700S / ZHciNRepSox (E-616452) HN Tocris Cat No 3742 ' " I / ~ _ - / [f T T / . / \SB525334 / \J \ _ Sellechchem Cat No SI 476GS788388 Selleckchem Cat No S2750H. N'A \\ / O' \ KZ^\ / \v / - -N 1 z""vT A ZA,N-Z \:JBIBF-0775 Selleckchem Cat No -O S2234"" \-- NH\„, N i. f if^ > oil H0329282749 69Attorney Docket: MINU-012 / 01 WO 339336.2063Compound Name Structure SourceSD-208 Selleckchem Cat No X J S7624HN 'xftXY N FlL.,1 x J,.N N jj' 'YClGalunisertib ^^Y Z, Selleckchem Cat No (LY2157299) \ S2230< 7i \.\ - 1 < yX(xi) Thyroid Hormone Signaling Pathway Activators

[0273] As described herein, the methods of the disclosure for generating populations comprising SCD insulin producing pancreatic cells (e g., SCD cells) use activators of the thyroid hormone signaling pathway. In some embodiments, the activator comprises a compound set forth in Table 6. In some embodiments, the activator comprises triiodothyronine (T3).Table 6: Exemplary Thyroid Hormone Signaling Pathway Activators of the DisclosureCompound Name Structure Source Triiodothyronine SteniCell1 Technologies Cat No 100-0549-o r xX x}'O y -0H0• Na* [XH2O]!Liothyronine sodium a SelleckChem Cat NO S4217NH2329282749 70Attorney Docket: MINU-012 / 01WO 339336.2063(xii) y-Secretase Inhibitors

[0274] As described herein, the methods of the disclosure for generating populations comprising SCD insulin producing pancreatic cells (e.g., SCD P cells) use inhibitors of y-secretase.

[0275] In some embodiments, the inhibitor of y-secretase comprises a compound set forth in Table 7. In some embodiments, the inhibitor comprises XXI. In some embodiments, the inhibitor comprises DAPT.Table 7: Exemplary Inhibitors of y-Secretase of the DisclosureCompound Name Structure SourceXXI >>:5Sigma Alrich 565790 T I O r 7 CH, re 1,.<f / "'7•DAPT phH H I Tocns Cat No. 2634AN JL XT 7 T Y TrFunctional Characteristics of Insulin Producing SCD Pancreatic Islets

[0276] In some embodiments, provided herein are SCD pancreatic islet compositions comprising insulin producing cells, wherein the insulin producing cells comprise a gene disruption described herein (e.g., a gene disruption comprising a loss of function mutation in a TET2 gene). In some embodiments, the insulin producing cells comprise SCD p cells. In some embodiments, the cell composition further comprises SCD a cells, SCD S cells, SCD y, or SCD s cells. In some embodiments, endocrine progenitor cells account for not more than about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1% of total cells in the cell composition.

[0277] In some embodiments, the insulin producing cells comprise one or more functional properties comparable to primary human beta cells. Suitable methods for measuring the functionality of insulin producing cells are known in the art, see e.g., Nair, et al (2019) Nat Cell Biol 21:263-274; Velazco-Cruz et al, (2019) Stem Cell Reports 12:351-65; Hogrebe et al., (2020),329282749 71Attorney Docket: MINU-012 / 01WO 339336,2063Nat Biotech, 38:460-70; Rezania et al., (2014) Nature Biotech 32: 1121-33; Russ et al., (2015), EMBO Journal 34:1759-72; and Pagliuca et al., (2014), Cell, 159:428-39.

[0278] In some embodiments, the insulin producing cells (e.g., SCD P cells) are characterized by the capacity to regulate insulin secretion in a manner similar to in situ pancreatic beta cells. For example, in some embodiments, the insulin producing cells secrete insulin in response to an increase in glucose in the extracellular environment.

[0279] In some embodiments, the insulin producing cells (e g., SCD P cells) are characterized by a response to a glucose challenge. In some embodiments, the response is substantially comparable to the response of endogenous islets to a glucose challenge.

[0280] In some embodiments, the insulin producing cells (e.g,, SCD cells) are characterized by a total insulin content that is substantially comparable to primary human islet beta cells.

[0281] In some embodiments, the insulin producing cells (e.g,, SCD cells) are characterized by an expression of one or more cell-specific markers (e.g., C-peptide, NKX6.1, and PDX1) that is substantially comparable to that of primary human islet beta cells, e.g,, as measured by flow cytometry.

[0282] In some embodiments, the insulin producing cells (e.g., SCD P cells) are characterized by an expression of mRNAs encoding prohormone convertase enzymes that are substantially comparable to that of primary human islet beta cells, e.g., as measured by quantifying the levels of PCI and PC2 mRNAs. In some embodiments, PC I and PC2 mRNA levels are measured by qPCR, RNA-seq, or any other method of measuring gene expression levels that is known in the art.

[0283] In some embodiments, the insulin producing cells (e.g., SCD P cells) are characterized by a metabolic state that is substantially comparable to primary human islet beta cells, e.g., as measured by a Seahorse analyzer.

[0284] In some embodiments, the insulin producing cells (e.g., SCD P cells) are characterized by increased calcium signaling (e.g., as measured by microscopy or flow cytometry) in response to a secretagogue. In some embodiments, the insulin producing cells (e.g., SCD P cells) is characterized by increased calcium signaling (e.g., as measured by microscopy or flow cytometry) m response to glucose. In some embodiments, the insulin producing cells (e.g., SCD P cells) are characterized by insulin secretion that is enhanced in response to a secretagogue.329282749 72Attorney Docket: MINU-012 / 01WO 339336,2063

[0285] In some embodiments, the insulin producing cells (e.g., SCD P cells) packages insulin into secretory granules (e.g., as measured by electron microscopy). In some embodiments, the insulin producing cells (e.g., SCD P cells) are characterized by a quantity of insulin secretory’ granules that is substantially comparable to that present in primary’ human islet beta cells (e.g., as measured by electron microscopy). In some embodiments, the insulin producing cells (e.g., SCD P cells) is characterized by the presence of crystalline insulin granules (e.g., as measured by electron microscopy).

[0286] In some embodiments, the insulin producing cells (e.g,, SCD cells) are characterized by a level of mitochondrial energization that is substantially comparable to that of primary human islet beta cells.

[0287] In some embodiments, the insulin producing cells (e.g., SCD p cells) secrete a level of c-peptide following administration in vivo that is substantially comparable to that produced following administration of primary human islet beta cells.

[0288] In some embodiments, the insulin producing cells (e.g,, SCD cells) are characterized by a morphology substantially similar to the morphology of endogenous pancreatic islet p cells.

[0289] In some embodiments, the insulin producing cells (e.g., SCD P cells) are characterized by an m vitro GSIS response that is substantially similar to the GSIS response of endogenous pancreatic islet P cells. In some embodiments, the insulin producing cells (e.g., SCD P cells) exhibit a response to multiple glucose challenges (e.g, at least one, at least two, or at least three or more sequential glucose challenges), e.g., in vitro. In some embodiments, the response resembles the response of endogenous pancreatic islet P cells to multiple glucose challenges.

[0290] In some embodiments, the insulin producing cells (e.g., SCD P cells) are characterized by an in vivo GSIS response that is substantially similar to the GSIS response of endogenous pancreatic islet P cells. In some embodiments, the insulin producing cells (e.g., SCD P cells) are characterized by an in vitro and an in vivo GSIS response that is substantially similar to the GSIS response of endogenous pancreatic islet P cells. In some embodiments, the insulin producing cells (e.g., SCD P cells) exhibit a GSIS response within about 2 weeks of in vivo transplantation. In some embodiments, the insulin producing cells (e.g., SCD P cells) exhibit a GSIS response within about 3 weeks of in vivo transplantation. In some embodiments, the insulin producing cells (e.g., SCD P cells) exhibit a GSIS response within about 3 weeks of in vivo transplantation. In some embodiments, the insulin producing cells (e.g., SCD P cells) exhibit a GSIS response329282749 73Attorney Docket: MINU-012 / 01WO 339336.2063within about 4 weeks of in vivo transplantation. In some embodiments, the insulin producing cells (e.g., SCD P cells) exhibit a GSIS response within about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, or about 12 weeks of in vivo transplantation. In some embodiments, the insulin producing cells (e.g., SCD P cells) exhibit a GSIS response within about 12 weeks of in vivo transplantation.

[0291] In some embodiments, the insulin producing cells (e.g., SCD P cells) are characterized by a stimulation index (i.e., ratio of insulin produced at high to low glucose concentration) of greater than 1. In some embodiments, the insulin producing cells (e.g., SCD [3 cells) are characterized by a stimulation index of greater than 1,1. In some embodiments, the insulin producing cells (e.g,, SCD P cells) are characterized by a stimulation index of greater than 2, See Functional assessment of purified human pancreatic islets: glucose stimulated insulin release by ELISA- A Standard Operating Procedure of the NIH Clinical Islet Transplantation Consortium CellR42014; 2(2):e900 for methods to determine the stimulation index,

[0292] In some embodiments, the insulin producing cells (e.g,, SCD cells) are characterized as monohormonal. In some embodiments, the insulin producing cells (e.g., SCD p cells) express insulin, but does not substantially express another hormone (e.g., glucagon, somatostatin, PP).

[0293] In some embodiments, the insulin producing cells (e.g., SCD P cells) are characterized by apoptosis in response to cytokines.

[0294] In some embodiments, the insulin producing cells (e.g., SCD P cells) are characterized by an ability to reverse diabetes in an animal model of diabetes. Animal models of diabetes are known in the art, see, e.g., Kottaisamy et al (2021) Laboratory animal research, 37(1), 23; and Singh et al (2024) Front. Endocrinol. 15, 1359685. In some embodiments, the animal model of diabetes uses NOD-SCID Gamma mice, NOD mice, B6 mice, or Ball / c mice. In some embodiments, the animal model of diabetes uses immunodeficient mice. In some embodiments, diabetes is induced in the mice by injecting with streptozotocin. In some embodiments, the insulin producing cells (e.g., SCD P cells) are characterized by lowering blood glucose levels in an animal model of diabetes to a greater extent than animals that are not treated with insulin producing cells. In some embodiments, the insulin producing cells (e.g., SCD P cells) are characterized by an ability' to lower blood glucose levels in the animals to blood glucose levels of less than about 250 mg / dL, about 200 mg / dL, or about 150 mg / dL.Gene Disruption329282749 74Attorney Docket: MINU-012 / 01WO 339336,2063

[0295] In some embodiments, the SCD pancreatic islet composition described herein comprise a plurality' of cells (e.g., SCD cells) comprising a gene disruption to reduce an immune response (e.g., an alloimmune response) following administration to a subject.

[0296] In some embodiments, the SCD pancreatic islet composition is characterized by reduced susceptibility to an allogeneic immune response following administration to a subject as compared to a SCD pancreatic islet composition lacking the gene disruption. For example, in some embodiments, the SCD pancreatic islet composition comprises SCD pancreatic islets differentiated from pluripotent stem cells not obtained from the subject, wherein the SCD pancreatic islets comprise the gene disruption, and wherein the SCD pancreatic islet composition is characterized by a reduced allogenic immune response as compared to the SCD pancreatic islet composition lacking the gene disruption. In some embodiments, the genetic modification renders the SCD composition less susceptible to host-versus-graft disease following administration to the subject.

[0297] In some embodiments, the SCD pancreatic islet composition is characterized by reduced susceptibility to an autoimmune response following administration to a subject as compared to a cell composition lacking the genetic modification. For example, in some embodiments, the SCD pancreatic islet composition comprises a plurality of cells (e.g., SCD P cells) comprising the gene disruption, wherein the SCD pancreatic islet composition is administered to a subject having type 1 diabetes, and wherein the SCD pancreatic islet composition is characterized by reduced susceptibility to the subject’s autoimmune response as compared to a SCD pancreatic islet composition lacking the genetic modification. In some embodiments, the subject’s autoimmune response is characterized by a T cell mediated immune response against pancreatic beta cells.

[0298] In some embodiments, the gene disruption reduces or eliminates expression of a gene product encoded by the at least one gene. In some embodiments, the gene disruption results in expression of an inactive gene product.

[0299] In some embodiments, the gene encodes an epigenetic modulator. In some embodiments, the gene encodes tet methylcytosine dioxygenase 2 (TET2).

[0300] In some embodiments, the SCD pancreatic islet composition comprises a plurality of cells (e g., SCD P cells) comprising a TET2 knockout or a TET2 knockdown.329282749 75Attorney Docket: MINU-012 / 01WO 339336.2063

[0301] In some embodiments, the TET2 knockout is introduced using a method of gene editing described herein.

[0302] In some embodiments, the mutation is a loss of function mutation. In some embodiments, the mutation is a frameshift mutation. In some embodiments, the mutation comprises a deletion.(I) Methods of Gene Editing

[0303] In some embodiments, the disclosure provides a method of introducing a gene disruption to a stem cell described herein using a gene editing system. In some embodiments, upon introducing the gene editing system to the cell, the system introduces a double-stranded break ( DSB) in a gene described herein (e.g., a TET2 gene), wherein repair of the DSB reduces and expression level and / or activity of a transcriptional and / or translation product of the gene (e.g., a TET2 protein). In some embodiments, the gene-editing system comprises a CRISPR system, a TALEN, or a zinc finger nuclease.

[0304] In some embodiments, the stem cell is a pluripotent stem cell described herein. In some embodiments, the gene disruption comprises a TET2 knockout.

[0305] In some embodiments, the gene editing system comprises a CRISPR system. In some embodiments, the CRISPR system comprises a CRISPR nuclease, or a polynucleotide encoding the CRISPR nuclease, and an RNA molecule comprising a guide sequence complementary to a target sequence in a target gene described herein (e.g., a TET2 gene). The “target sequence” is a sequence of about 15-50 nucleotides present in the target gene. In some embodiments, the target sequence is 15-50 contiguous nucleotides (i.e., covalently linked and immediately adjacent to each other). In some embodiments, the target sequence is 19-30 contiguous nucleotides.

[0306] In some embodiments, the CRISPR nuclease comprises a Cas protein. In some embodiments, the Cas protein is any Cas type (e.g., Cas I, Cas I A, Cas IB, Cas IC, Cas ID, Cas IE, Cas IF, Cas IU, Cas III, Cas IIIA, Cas IIIB, Cas IIIC, Cas HID, Cas IV, Cas IVA, Cas IVB, Cas II, Cas HA, Cas IIB, Cas IIC, Cas V, Cas VI). In some embodiments, the CRISPR nuclease comprises a Cas9 protein. In some embodiments, the CRI SPR nuclease comprises a fusion of the Cas protein and an additional polypeptide, e.g., an enzyme (e.g., ligase, transcriptase, transposase, nuclease, endonuclease, reverse transcriptase, polymerase, helicase).

[0307] In some embodiments, the CRISPR system is introduced to the cell in a lipid nanoparticle. In some embodiments, the CRISPR system is introduced to the cell as a329282749 76Attorney Docket: MINU-012 / 01WO 339336.2063ribonucleoprotein complex. In some embodiments, the CRISPR system is introduced to the cell in a viral vector.

[0308] In some embodiments, the method comprises contacting the stem cells with the CRISPR system, wherein the CRISPR system introduces a mutation in the TET2 gene. In some embodiments, the CRISPR system comprises a CRISPR nuclease, or a polynucleotide encoding the CRISPR nuclease, and an RNA molecule comprising a guide sequence complementary to a target sequence in the TET2 gene. In some embodiments, the target sequence is present in an exon of the TET2 gene. In some embodiments, the target sequence is present in an intron of the TET2 gene. In some embodiments, the target sequence is present in a regulatory region of the TET2 gene. In some embodiments, the target sequence is present in any one of exons 3-11 of the TET2 gene.

[0309] In some embodiments, the method comprises contacting the stem cells with a CRISPR system comprising a CRISPR nuclease, or a polynucleotide encoding the CRISPR nuclease, an RNA molecule comprising a guide sequence complementary to a target sequence in the TET2 gene, wherein the CRISPR nuclease combines with the RNA molecule to induce cleavage proximal to the target sequence to introduce an insertion and / or deletion in the TET2 gene, thereby resulting in a reduction or elimination of TET2 expression in the stem cells.

[0310] In some embodiments, the method comprises contacting the stem cells with a CRISPR system comprising a CRISPR nuclease, or a polynucleotide encoding the CRISPR nuclease, a first RNA molecule comprising a guide sequence complementary to a first target sequence in the TET2 gene, and a second RN A molecule comprising a guide sequence complementary to a second target sequence in the TET2 gene, wherein the CRISPR nuclease combines with the first RN A molecule and the second RNA molecule to induce cleavage proximal to the first and second target sequences to introduce an approximately 2-10 kb deletion in the TET2 gene, thereby resulting in a reduction or elimination of TET2 expression in the stem cells. In some embodiments, the method introduces a 5-15 kb deletion in the TET2 gene. In some embodiments, the deletion removes all or a portion of an exon of the TET2 gene. In some embodiments, the deletion removes all or a portion of exon 3, 4, 5, 6, 7, 8, 9, 10 or 11 of the TET2 gene. In some embodiments, the deletion removes all or a portion of exon 3 the TET2 gene. In some embodiments, the deletion removes all or a portion of exon 9 the TET2 gene. In329282749 77Attorney Docket: MINU-012 / 01WO 339336,2063some embodiments, the deletion removes a 3' portion of exon 8, exon 9, and a 5' portion of exon 10.

[0311] Methods for selecting a gRNA directed to a target gene are known in the art. For example, in some embodiments, the spacer sequence is designed in silico by locating a target sequence (e.g., a 19-30 bp sequence) adjacent a PAM sequence in the target gene (e.g., a TET2 gene), wherein the PAM sequence is recognized by a Cas nuclease described herein. Non¬ limiting exemplary PAM sequences are described in Cong et al., (2013) Science 339:819-823; Kleinstiver et al,, (2015) Nat Biotechnol 33:1293-1298; Kleinstiver et al., (2015) Nature 523:481-485; Kleinstiver et al., (2016) Nature 529:490- 495; Tsai et al., (2014) Nat Biotechnol 32:569-576; Slaymaker et al,, (2016) Science 351:84-88; Anders et al., (2016) Mol Cell 61:895-902; Kim et al., (2017) Nat Comm 8: 14500; Fonfara et al., (2013) Nucleic Acids Res 42:2577-2590; Garneau et al., (2010) Nature 468:67-71; Magadan et al., (2012) PLoS ONE 7:e40913; Esvelt et al., (2013) Nat Methods 10(11):! 116-1121.

[0312] In some embodiments, the method of in silico screening is used to predict cleavage efficiency of a gRNA spacer sequence at both on-target and off-target sites, thereby allowing selection of a gRNA with high cleavage efficiency at a target sequence in the genome comprising a target gene described herein, with low or minimal cutting efficiency at off-target sites in the genome (i.e., low or minimal frequency of DSBs occurring at sites other than the selected target sequence).

[0313] In some embodiments, the nucleotide sequence of the spacer is designed or chosen using an algorithm or method known in the art. In some embodiments, the algorithm uses variables to screen for suitable gRNA spacer sequences and corresponding target sequences. Non-limiting examples of such variables include predicted melting temperature of the gRNA sequence, secondary structure formation of the gRNA sequence, predicted annealing temperature of the gRN A sequence, sequence identity, genomic context of the target sequence, chromatin accessibility of the target sequence, % GC, frequency of genomic occurrence of the target sequence (e.g., of sequences that are identical or are similar but vary in one or more spots as a result of mismatch, insertion or deletion), methylation status of the target sequence, and / or presence of SNPs within the target sequence.

[0314] In some embodiments, one or more bioinformatics tools known in the art are used to predict the off-target activity of a gRNA spacer sequence and / or identify the most likely sites of329282749 78Attorney Docket: MINU-012 / 01WO 339336,2063off-target activity. Non-limiting examples of bioinformatics tools for use in the present disclosure include CCTop, CRISPOR, and COSMID.

[0315] In some embodiments, identification of gRNA target sequences is best achieved through a combination of in silico selection and experimental evaluation. Experimental methods to evaluate, for example, gRNA on-target and off-target cleavage efficiency are known in the art and further described herein.

[0316] In some embodiments, cleavage efficiency is measured as frequency of INDELs proximal the target sequence targeted by the guide sequence. Methods to measure frequency of INDELs at a particular target sequence in a genome are known in the art. An exemplary method to measure frequency of INDELs at a predicted cut site in a given target sequence comprises, (i) isolation of genomic DNA from the edited cell population and / or tissue, (li) amplification of the DNA region comprising the target sequence (e.g., by PCR), (iii) sequencing of the amplified DNA region (e.g., by Sanger sequencing), and (iv) determining frequency of INDELs at the predicted cut site by Tracking of In dels decomposition (TIDE) assay, for example, as described by Brinkman, et al (2014) NUCLEIC ACIDS RESEARCH 42: el 68. A further exemplary method comprises sequencing of the amplified DNA region by next-generation sequencing (NGS) and analysis of INDEL frequency at the predicted cut site in the target sequence, for example, as described by Bell et al (2014) BMC Genomics 15:1002.Support Factors

[0317] In some embodiments, the SCD pancreatic islet compositions of the disclosure comprise cells described herein (e.g., SCD cells and / or insulin producing cells) and a support factor. In some embodiments, the cells and the support factor are combined in a suspension. In some embodiments, the support factor is operably linked to the surface of a plurality of the cells. In some embodiments, the cells and the support factor are encapsulated in a delivery device described herein. In some embodiments, the support factor is a gene product described herein, wherein a plurality of cells of the composition are engineered to express the gene product. In some embodiments, the cells of the composition are transfected with the support factor, such that the support factor is internalized by a plurality of the cells.

[0318] In some embodiments, the cell composition comprising the support factor exhibits improved survival compared to a cell composition lacking the support factor. In some329282749 79Attorney Docket: MINU-012 / 01WO 339336,2063embodiments, the survival is improved in vitro. In some embodiments, the survival is improved following administration in vivo. For example, in some embodiments, the cell composition comprising the support factor exhibits improved survival for duration of at least about 2 weeks, about 4 weeks, about 8 weeks, about 12 weeks, about 24 weeks, or longer compared to a cell composition lacking the support factors. In some embodiments, the cell composition comprising the support factor exhibits improved angiogenesis compared to a cell composition lacking the support factor. In some embodiments, the angiogenesis is improved following administration in vivo, e.g., within about 2 weeks, about 4 weeks, about 8 weeks, or about 12 weeks following administration in vivo.

[0319] In some embodiments, the SCD pancreatic islet composition comprises an insulin producing cell described herein (e.g., a SCD p cell) and a support factor, wherein the SCD pancreatic islet composition exhibits an improved GSIS response compared to a cell composition lacking the support factor. In some embodiments, the improved GSIS response is an in vitro or in vivo GSIS response. In some embodiments, the SCD pancreatic islet composition exhibits increased insulin expression compared to a SCD pancreatic islet composition lacking the support factor. In some embodiments, the SCD pancreatic islet composition exhibits increased c-peptide expression compared to a SCD pancreatic islet composition lacking the support factor

[0320] In some embodiments, the SCD pancreatic islet composition comprises an insulin producing cell describe herein and a support factor, wherein the support factor comprises a cell capable of secreting a proangiogemc factor, a PTG factor, or both. In some embodiments, the SCD pancreatic islet composition comprises a first cell capable of secreting a PTG hormone and a second cell capable of secreting a proangiogemc factor. In some embodiments, the SCD pancreatic islet composition comprises a cell capable of secreting both a proangiogemc factor and P TG hormone. In some embodiments, the cell is one derived from a PTG tissue. In some embodiments, the cell is a CD34+ cell. In some embodiments, the cell is a hematopoietic progenitor cell. Methods for co-transplanting an insulin producing cell and a cell derived from a PTG tissue or a CD34+ cell are further described in US Pat No. 11,951,136 (herein incorporated by reference).

[0321] In some embodiments, the SCD pancreatic islet composition comprises an insulin producing cell describe herein and a support factor, wherein the support factor comprises a PTG factor (e.g., a PTG hormone), a proangiogemc factor, a cytokine, an anti-inflammatory factor, or329282749 80Attorney Docket: MINU-012 / 01WO 339336.2063a combination thereof. In some embodiments, the support factor is selected from gamma- aminobutyric acid (GABA), parathyroid hormone (PTH), PTH-related peptide (PTHrP), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), angiopoietin, and a combination thereof.

[0322] In some embodiments, the SCD pancreatic islet composition comprises an insulin producing cell describe herein and 1, 2, 3, 4, 5, 6, or 7 support factors selected from GABA, PTH, HGF, PTHrP, VEGF, angiopoietin, PDGF, and a combination thereof.Pharmaceutical Compositions

[0323] The present disclosure provides pharmaceutical compositions comprising SCD pancreatic islet compositions described herein. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the SCD pancreatic islet cells comprise SCD P cells, SCD a cells, and / or SCD 5 cells. In some embodiments, the SCD pancreatic islet cells are present in one or more clusters. In some embodiments, the SCD pancreatic islet cells are present in a cell suspension.

[0324] In some embodiments, the pharmaceutical compositions are formulated for administration to a subject to treat, prevent, or mitigate one or more symptoms of a disease or disorder (e.g., diabetes).

[0325] In some embodiments, the SCD pancreatic islet composition is sorted or enriched, e g., to increase the proportion of the population that are SCD P cells and / or to remove not fully differentiated progenitor cells or pluripotent stem cells. In some embodiments, the SCD pancreatic islet composition is sorted or enriched using FACS. In some embodiments, the SCD pancreatic islet composition is sorted or enriched using a microfluidic cell sorter.

[0326] In some embodiments, the pharmaceutical composition is formulated as a liquid dosage form comprising the population of cells, and a pharmaceutically acceptable carrier. In some embodiments, the liquid dosage form is a sterile solution or suspension.

[0327] In some embodiments, the pharmaceutical composition is formulated as a solid dosage form comprising the SCD pancreatic islet composition, and a pharmaceutically acceptable carrier. In some embodiments, the solid dosage form is for oral administration. In some embodiments, the solid dosage form is a drench, lozenge, dragees, capsule, pill, tablet, bolus, powder, granule, or paste.329282749 81Attorney Docket: MINU-012 / 01WO 339336,2063

[0328] As used herein, the term “pharmaceutically acceptable carrier” refers to reagents, cells, compounds, materials, compositions, and / or dosage forms that are compatible with the cell compositions described herein for administration to a subject. Exemplary pharmaceutically acceptable carriers include, but are not limited to, water, a salt solution, alcohols, oils, gelatins, carbohydrates (e.g., lactose, amylose, starch), glycolipids, cellulose derivatives, and polymers (e.g., PEG, polyvinyl pyrrolidine). In some embodiments, the pharmaceutically acceptable carrier is sterilized. In some embodiments, pharmaceutically acceptable carrier further comprises a lubricant, preservative, stabilizer, wetting agent, emulsifier, salts (e.g., to modulate osmotic pressure), buffers, and coloring.

[0329] As used herein, the term “pharmaceutically acceptable” refers to compounds, materials, compositions, and / or dosage forms that are suitable for administering to a subject without substantial risk of toxicity, allergic response, and or other complications, as ascertained by an individual skilled in the art. The term implies that each carrier present m the pharmaceutical composition is compatible with other components of the composition, and when combined to form the composition can be administered to a subject without substantial risk.

[0330] In some embodiments, the pharmaceutical composition comprises an effective amount of the population of cells. / Xs used herein, the term “effective amount” used in respect to a population of cells described herein refers to an amount of cells in the population that is sufficient to produce an effect following administration to a subject that is characterized by one or more therapeutic and / or prophylactic benefits. For example, in some embodiments, the pharmaceutical composition comprises an effective amount of SCD pancreatic islets (e.g., SCD pancreatic islets comprising SCD P cells), wherein the amount is sufficient to produce a measurable change in at least one symptom of diabetes (e.g., glycosylated hemoglobin level, fasting blood glucose level, hypoinsulinemia) following administration to a subject having diabetes. Determination of an effective amount is within the capability of the skilled clinician and is adapted based upon the subject’s medical history, age, condition, sex, severity of the medical condition, and administration of other pharmaceutically active agents.

[0331] In some embodiments, the data obtained from cell culture assays and animal studies, such as any described herein, is used in formulating a range of dosages for use in humans. The dosage of a pharmaceutical composition described herein can be estimated initially from preclinical data comparing the relative potency of the new’ formulation with the standard formulation in animal329282749 82Attorney Docket: MINU-012 / 01WO 339336,2063studies. A minimal cell dose to achieve diabetes remission in animal studies can be compared between old and new formulations. In these dose-finding studies, C-peptide (a non-metabolized byproduct of insulin production) concentration at baseline and after administration of glucose can also be measured and compared between the old and new formulation. Such information can be used to more accurately determine useful doses in humans. Levels of C-peptide in plasma may be measured, for example, using enzyme-linked immunosorbent assay.

[0332] The dosages administered will vary from individual to individual; a therapeutically effective dose in humans can be estimated, for example but not limited to, by the level of enhancement of function (e.g,, C-peptide concentration or minimal cell dose required for diabetes remission). Monitoring levels of co-transpl anted cell introduction, the level of expression of certain genes affected by such transfer, and / or the presence or level s of the encoded product will also enable one skilled in the art to select and adjust the dosages administered. Generally, a composition including co-transplanted cells will be administered in a single dose m the range of 105- 106cells per kg body weight, preferably in the range of 106- 107cells per kg body weight. This dosage may be repeated as considered appropriate by the treating physician

[0333] In some embodiments, the pharmaceutical composition is formulated for administration via a parental route. Examples of parenteral routes of administration include, for example, intramuscular, intravenous, intradermal, subcutaneous, transdennal (topical), transinucosal, intra¬ peritoneal and intraomental administration. In some embodiments, the pharmaceutical composition is formulated for administration to an extrahepatic site of the subject. In some embodiments, the extrahepatic site is subcutaneous. In some embodiments, the extrahepatic site is intramuscular. Methods to formulate a pharmaceutical composition based upon the route of administration are known in the art. See, e.g., Remington: The Science and Practice of Pharmacy, 19thEd (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E. Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton Pennsylvania, 1975; Liberman, H. A. and Lachman, L, Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N. Y. 1980; and Pharmaceutical Dosage Forms and Drug Delivery’ Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999).

[0334] Solutions or suspensions used for parenteral application can include the following components: a sterile diluent such as water for injection, saline solution, tissue preservation329282749 83Attorney Docket: MINU-012 / 01WO 339336,2063solution, heparin containing isotonic fluid (Plasma-LyteA, normal saline), CMRL 1066, +50 mb 25% human serum albumin containing heparin, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as mono- and / or di -basic sodium phosphate, hydrochloric acid or sodium hydroxide (e.g., to a pH of about 7.2-7.8, e.g., 7.5). Agents that increases viscosity, such as sodium carboxymethyl cellulose, sorbitol, dextran, hydrogel, or fibrin, may be included to facilitate cell aggregation. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0335] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. Suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N. J.), or phosphate buffered saline (PBS). In all cases, the composition should be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of preparation and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosai, and the like. In many cases, isotonic agents can be included, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition.

[0336] Pharmaceutical formulations for parenteral administration include aqueous solutions of the cell compositions described herein. For injection, the pharmaceutical compositions of the present disclosure may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer' solution, Wisconsin Solution, Plasma-LyteA, or physiologically buffered saline. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, dextran, hydrogel, or fibrin. Human serum albumin may be included to support cell viability.329282749 84Attorney Docket: MINU-012 / 01WO 339336,2063Additionally, suspensions of the active solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the cell compositions described herein to allow for the preparation of highly concentrated solutions.

[0337] In some embodiments, the pharmaceutical compositions are prepared with carriers that protect the cell compositions against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery’ systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid, and poly-lactic-co-gly colic acid (PLGA). Methods for preparation of such formulations will be apparent to those skilled in the art. In some embodiments, the pharmaceutical compositions of the disclosure are prepared with poly-lactic-co-glycolic acid (PLGA).

[0338] In some embodiments, the pharmaceutical composition is manufactured according to known techniques, e.g., by means of conventions mixing, dissolving, granulating, levigating, emulsifying, encapsulating, entrapping, or compression processes.

[0339] After the pharmaceutical compositions disclosed herein have been prepared and are formulated in a pharmaceutically acceptable carrier, they can be placed in an appropriate container and labeled for treatment of an indicated condition with information including amount, frequency and method of administration. In some embodiments, any of the pharmaceutical compositions disclosed herein are loaded into a syringe (such as a pre-tilled syringe), a catheter, or a cannula prior to administration to a subject.Delivery Devices

[0340] The present disclosure provides SCD pancreatic islet compositions or pharmaceutical compositions thereof that are transplanted using a delivery device, such as, for example, a retrievable net-like delivery device, a 3D scaffold-based delivery device, or by encapsulation (with limited permeability for cells and macromolecules). Use of delivery devices in the methods and compositions disclosed herein facilitates easy monitoring and removal of insulin-producing grafts transplanted into the individual either subcutaneously, intramuscularly, or by any other method known in the art. Delivery devices for transplantation of an insulin producing cell graft are further described in US Pat No 11,951,136, herein incorporated by reference.329282749 85Attorney Docket: MINU-012 / 01WO 339336,2063

[0341] Preferred devices may have certain characteristics which are desirable but are not limited to one or a combination of the following: (i) comprised of a biologically derived or synthetic biocompatible material that functions under physiologic conditions, including pH and temperature; (ii) releases no toxic compounds harming the transplanted ceil composition delivered with the device; (hi) promotes secretion or release of a biologically active agent or macromolecule present in the cell composition (e.g., a support factor described herein) across the device; (iv) promotes long-term stability of the delivered cell composition; (v) promotes vascularization; (vi) comprised of membranes or housing structure that is chemically inert; (vii) provides stable mechanical properties; (viii) maintains structure / housing integrity (e.g., prevents unintended leakage of toxic or harmful agents and / or cells (such as neoplastic cells); (ix) is refillable and / or flushable; (x) is mechanically expandable; (xi) provides a means for retrieving and / or monitoring the transplanted cell composition from the host tissue; (xi) is easy to fabricate and manufacture; and (xii) can be sterilized,

[0342] In some embodiments, the delivery device is configured to allow for the delayed or controlled release of the cell compositions and / or pharmaceutical compositions.

[0343] In some embodiments, the delivery device can be made of biologically derived or synthetic polymers and adopts a mesh or lattice-like configuration in which the transplanted cell composition is placed. This configuration permits newly formed blood vessels to reach the transplanted cell composition, thereby improving the likelihood of survival in nutrient and oxygen poor transplantation sites (such as the subcutaneous or intramuscular space).Additionally, the mesh or lattice configuration of the delivery device permits removal or monitoring of the transplanted cell composition following administration.

[0344] Some exemplary, non-limiting examples of biologic derived materials that can be used in conjunction with the disclosures provided for herein include platelet poor plasma (PPP), platelet rich plasma (PRP), starch, chitosan, alginate, fibrin, thrombin, polysaccharide, cellulose, collagen, bovine collagen, bovine pericardium, gelatin-resorcin- formalm adhesive, oxidized cellulose, mussel-based adhesive, poly (amino acid), agarose, polyetheretherketones, amylose, hyaluronan, hyaluronic acid, whey protein, cellulose gum, starch, gelatin, silk, or other material suitable to be mixed with biological material and introduced into a transplantation site, including combinations of materials, or any material apparent to those skilled in the art in view of the disclosures provided for herein.329282749 86Attorney Docket: MINU-012 / 01WO 339336,2063

[0345] Biologic materials can be derived from a number of sources, including from the subject in which the biologic material is to be implanted, a donor that is not the subject in which the biologic material is to be implanted, or other animals. Synthetic materials can be any synthetic material that can be useful in an implantable surgical device, such as a biocompatible polymeric material or a biocompatible non-polymeric synthetic material. Non-limiting examples of useful synthetic polymeric materials include thermoplastic polymeric materials such as polyolefins (e.g., polypropylenes), polyurethanes, acetel materials, Teflon® materials, and the like; thermoset materials such as silicones; and materials that are otherwise curable, e.g,, that can be cured by ultraviolet radiation or chemical reactions, including curable materials such as curable urethanes, epoxies, acrylates, cyanoacrylates, and the like. Any of these materials may be homopolymers, copolymers, or a blend or other combination of homopolymers, copolymers, or both. Other suitable synthetic materials include, without limitation, metals (e.g, silver filigree, tantalum gauze mesh, and stainless steel mesh).

[0346] In some embodiments, the scaffold is gel-like in nature. Examples of gel-like scaffolds for use m the methods and compositions disclosed herein include, without limitation, fibrin, collagen, alginate, matrigel, and gelatin. In other embodiments, the scaffold is fibrous in nature and each fiber of the scaffold comprises a biocompatible material. Optionally, the biocompatible material comprises a material selected from the group including, but not limited to, an absorbable material, a non-absorbable material and combinations thereof. Further, the three-dimensional matrices can be formed of a biodegradable, non-degradable, or combination of biodegradable and non-degradable materials which have been configured to produce high cell densities by allowing adequate diffusion of nutrients, hormones, growth factors, insulin, and waste as well as gas exchange, while in vitro or in vivo.

[0347] Absorbable material for use in the fiber scaffold can be selected from the group including, but not limited to, polygly colic acid (PGA), polylactic acid (PLA), polyglycolide-lactide, polycaprolactone, polydioxanone, polyoxalate, a polyanhydride, a poly(phosphoester), catgut suture, collagen, silk, chitin, chitosan, hydroxyapatite, bioabsorbable calcium phosphate, hyaluronic acid, elastin, and combinations thereof. Non-absorbable material for use in 3-D fiber scaffolds can be selected from the group including, but not limited to, polypropylene, polyester, pol ytetrafl uoroethyl en e (PTFE), expanded PTFE (ePTFE), polyethylene, polyurethane, polyamide, nylon, polyetheretherketone (PEEK), polysulfone, a cellulosic, fiberglass, an acrylic,329282749 87Attorney Docket: MINU-012 / 01WO 339336,2063tantalum, polyvinyl alcohol, carbon, ceramic, a metal, and combinations thereof. The fiber scaffold can be made from biocompatible fibers, including textured fibers that provide a much lower bulk density filling than non-texturized fiber. The low bulk density of textured fibers can provide for implantation of a significant numbers of cells.

[0348] In yet another embodiment, the transplanted cell composition is encapsulated prior to transplantation into the individual. For example, such devices can house therapeutically effective quantities of cells within a semi-permeable membrane having a pore size such that oxygen and other molecules important to cell survival and function can move through the semi-permeable membrane but the cells of the immune system cannot permeate or traverse through the pores. Similarly, such devices can contain therapeutically effective quantities of a biologically active agent, e.g., insulin, an angiogenic factor, a growth factor, a hormone and the like. Ideally, the transplanted cell composition is wholly encapsulated or enclosed in at least one internal space or are encapsulation chambers, which are bounded by at least one or more semi -permeable membranes. Such a semi-permeable membrane should allow the encapsulated biologically active substance of interest to pass (e.g, insulin, glucagon, pancreatic polypeptide and the like), making the active substance available to the target cells outside the device and in the patient's body. In some embodiments, the semi-permeable membrane allows nutrients naturally present in the subject to pass through the membrane to provide essential nutrients to the encapsulated cells. At the same time, such a semi-permeable membrane prohibits or prevents the patient's cells, more particularly the immune system cells, from passing through and into the device and harming the encapsulated cells in the device as well as prevents any potentially neoplastic cell originating in the co transplanted cells or tissue from passing into the patient’s body. For example, in the case of diabetes, this approach can allow glucose and oxygen to stimulate insulin-producing cells to release insulin as required by the body in real time while preventing immune system cells from recognizing and destroying the implanted cells. In some embodiments, the semi-permeable membrane prohibits the implanted cells from escaping encapsulation.

[0349] Cell permeable and impermeable membranes comprising of have been described in the art including those patents previously described above by Baxter or otherwise previously referred to as TheraCyte cell encapsulation devices including, U. S. Pat. Nos. 6,773,458; 6,520,997; 6, 156,305; 6,060,640; 5,964,804; 5,964,261; 5,882,354; 5,807,406; 5,800,529; 5,782,912;5,741,330; 5,733,336; 5,713,888; 5,653,756; 5,593,440; 5,569,462; 5,549,675; 5,545,223;329282749 88Attorney Docket: MINU-012 / 01WO 339336.20635,453,278; 5,421,923; 5,344,454; 5,314,471; 5,324,518; 5,219,361; 5,100,392; and 5,011,494, which are herein incorporated by reference in their entireties.

[0350] In some embodiments, the encapsulating devices are comprised of a biocompatible material including, but are not limited to, anisotropic materials, polysulfone (PSF), nano-fiber mats, polyimide, tetrafluoroethylene / polytetrafluoroetliylene (PTFE; also known as Teflon®), ePTFE (expanded polytetrafluoroethylene), polyacrylonitrile, polyethersulfone, acrylic resin, cellulose acetate, cellulose nitrate, polyamide, as well as hydroxylpropyl methyl cellulose (HPMC) membranes. These and substantially similar membrane types and components are manufactured by at least Gore®, Phillips Scientific®, Zeus®, Pall® and Dewal® to name a few. Further information regarding encapsulation of insulin-producing cells can be found in Nyitray et al. (ACS Nano., 2015 May 14;9(6): 5675-82) and Chang et al. (ACS Nano. 2017 Aug 7; 1 l(8):7747-57) the disclosures of which are incorporated by reference hereinKits

[0351] The present disclosure provides kits comprising SCD pancreatic islet compositions described herein, or pharmaceutical compositions thereof, and instructions for use.

[0352] In some embodiments, the kit comprises a SCD pancreatic islet composition described herein, a pharmaceutical composition comprising SCD pancreatic islet composition, or a delivery device comprising the SCD pancreatic islet composition or the pharmaceutical composition, and instructions for administering the SCD pancreatic islet composition, the pharmaceutical composition, or the delivery device to a subject for treatment of a disease or disorder in a subject. In some embodiments, the disease or disorder is associated with an insulin deficiency. In some embodiments, the disease or disorder is type I or type II diabetes.EXEMPLARY EMBODIMENTS

[0353] The disclosure further provides the following exemplary embodiments.

[0354] Embodiment 1-1. A method for stem cell therapy in a subject having an insulin deficiency, comprising administering to the subject a composition comprising a population of pancreatic islet cells, wherein the population of pancreatic islet cells is obtained by in vitro differentiation from a pluripotent stem cell prior to the administering, wherein the pluripotent stem cell comprises a loss of function mutation in a Tet methylcytosine dioxygenase 2 (TET2)329282749 89Attorney Docket: MINU-012 / 01WO 339336,2063gene, wherein the pluripotent stem cell is allogeneic with respect to the subject, wherein the population of pancreatic islet cells or a portion thereof undergoes engraftment following the administering, thereby treating the insulin deficiency in the subject.

[0355] Embodiment 1-2. The method of Embodiment I- 1, wherein the engraftment is characterized by (i) increased plasma levels of C-peptide when measured in a fasting state as part of a mixed meal tolerance test, (ii) reduction in HbAlc levels, and / or (iii) increased Time in Range (TIR).

[0356] Embodiment 1-3. The method of Embodiment 1-1 or Embodiment 1-2, wherein the engraftment occurs without acute rejection of the population.

[0357] Embodiment 1-4. The method of any of Embodiment 1-1 to Embodiment 1-3, wherein the engraftment is improved as compared to administering a control composition comprising a population of pancreatic islet cells, wherein the composition is obtained by in vitro differentiation from a pluripotent stem cell comprising a wild-type TET2 gene.

[0358] Embodiment 1-5. The method of any of Embodiment 1-1 to Embodiment 1-4, wherein the engraftment is maintained for a duration of time following the administering.

[0359] Embodiment 1-6. The method of Embodiment 1-5, w'herein the duration of time is at least about 6 months, about 7 months, about 8 months, or about 9 months.

[0360] Embodiment 1-7. The method of Embodiment 1-5 or Embodiment 1-6, w'herein the engraftment is maintained in the absence of the subject receiving an immunosuppressant.

[0361] Embodiment 1-8. A method for stem cell therapy in a subject having an insulin deficiency, comprising administering to the subject a composition comprising a population of pancreatic islet cells, w'herein the population of pancreatic islet cells is obtained by in vitro differentiation from a pluripotent stem cell prior to the administering, wherein the pluripotent stem cell comprises a loss of function mutation in a TET2 gene, wherein the pluripotent stem cell is allogeneic with respect to the subject, and wherein the administering is associated with a reduced alloimmune response compared to administering a population of unmodified pancreatic islet cells.

[0362] Embodiment 1-9. The method of Embodiment 1-8, w'herein the reduced alloimmune response is characterized by increased plasma levels of C-peptide when measured in a fasting state as part of a mixed meal tolerance test, (ii) reduction in HbAlc levels, and / or (iii) increased TIR.329282749 90Attorney Docket: MINU-012 / 01WO 339336,2063

[0363] Embodiment I- 10. The method of Embodiment 1-8 or Embodiment 1-9, wherein the unmodified population comprises of pancreatic islet cells obtained from a pluripotent stem cell comprising a wild-type TET2 gene.

[0364] Embodiment I- 11. The method of any of Embodiment I- 1 to Embodiment I- 10, wherein a plurality of the pancreatic islet cells m the population are insulin producing cells.

[0365] Embodiment 1-12. The method of any of Embodiment I- 1 to Embodiment I- 11, wherein at least about 30% to about 60% of the population of pancreatic islet cells are insulin producing cells.

[0366] Embodiment 1-13, The method of Embodiment 1-11 or Embodiment 1-12, wherein the insulin producing cells comprises beta cells.

[0367] Embodiment 1-14, The method of any of Embodiment 1-11 to Embodiment 1-13, wherein the population further comprises alpha cells, delta cells, or a combination thereof.

[0368] Embodiment 1-15, The method of Embodiment 1-14, wherein less than about 10% of cells of the population are epsilon cells, pancreatic progenitor cells, acinar and ductal cells, enterochromaffin cells, and / or proliferative cells

[0369] Embodiment 1-16. The method of Embodiment 1-15, wherein less than about 1% of cells of the population are proliferative cells.

[0370] Embodiment 1-17. The method of any of Embodiment 1-1 to Embodiment 1-16, wherein a plurality of pancreatic islet cells in the population are present in cell clusters.

[0371] Embodiment 1-18. The method of Embodiment 1-17, wherein the cell clusters comprise a longest diameter of about 50 pm to 350 um or about 100 pm to about 200 pm.

[0372] Embodiment 1-19. The method of Embodiment 1-17 or Embodiment 1-18, wherein the cell clusters comprise about 500, about 600, about 700, about 800, about 900, about 1000, about 1100, about 1200, about 1300, about 1400, or about 1500 cells.

[0373] Embodiment 1-20. The method of any of Embodiment 1-1 to Embodiment 1-19, wherein the composition further comprises a support factors, optionally wherein the support factor is selected from the group consisting of gamma-aminobutyric acid (GABA), parathyroid hormone (PTH), PTH-related peptide (PTHrP), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), angiopoietin, and a combination thereof.

[0374] Embodiment 1-21. The method of Embodiment 1-20, wherein the support factor is a soluble factor.329282749 91Attorney Docket: MINU-012 / 01WO 339336,2063

[0375] Embodiment 1-22. The method of Embodiment 1-20, wherein the support factor is operably linked to a surface of a plurality of pancreatic islet ceils in the population.

[0376] Embodiment 1-23. The method of any of Embodiment 1-20 to Embodiment 1-22, wherein the factors are introduced to the composition following the in vitro differentiation and prior to the administering.

[0377] Embodiment 1-24. The method of any of Embodiment 1-1 to Embodiment 1-23, wherein the composition comprises the population of pancreatic islet cells in a liquid suspension.

[0378] Embodiment 1-25, The method of any of Embodiment I- 1 to Embodiment 1-23, wherein the composition comprises the population of pancreatic islet cells in an encapsulation device or a degradable material.

[0379] Embodiment 1-26. The method of any of Embodiment 1-1 to Embodiment 1-25, wherein the pluripotent stem cell is a human cell.

[0380] Embodiment 1-27. The method of any of Embodiment 1-1 to Embodiment 1-26, wherein the pluripotent stem cell comprises an embryonic stem cell or an induced pluripotent stem cell.

[0381] Embodiment 1-28. The method of any of Embodiment 1-1 to Embodiment 1-27, wherein the in vitro differentiation comprises a step-wise differentiation of (i) the pluripotent stem cell to a definitive endoderm cell, (ii) the definitive endoderm cell to a posterior foregut cell, (iii) the posterior foregut cell to a pancreatic progenitor cell, and (iv) the pancreatic progenitor cell to the pancreatic islet cell.

[0382] Embodiment 1-29. The method of Embodiment 1-28, wherein differentiation of the pluripotent stem cell to the definitive endoderm cell comprises contacting the pluripotent stem cell with a differentiation factor selected from a ROCK inhibitor, a TGFβ superfamily growth factor, an epidermal growth factor family polypeptide, a WNT activator, and a combination thereof.

[0383] Embodiment 1-30. The method of Embodiment I- 28 or Embodiment 1-29, wherein the definitive endoderm cell is characterized by expression of CXCR4, SOX17, and / or FOXA2.

[0384] Embodiment 1-31. The method of Embodiment 1-30, wherein the definitive endoderm cell does not substantially express PDX1329282749 92Attorney Docket: MINU-012 / 01WO 339336,2063

[0385] Embodiment 1-32. The method of any of Embodiment 1-28 to Embodiment 1-31, wherein differentiation of the definitive endoderm cell to the posterior foregut cell comprises contacting the definitive endoderm cell with a differentiation factor selected from a growth factor of the fibroblast growth factor (FGF) family, an activator of the retinoic acid signaling pathway, an activator of protein kinase C, an inhibitor of bone morphogenetic protein (BMP) signaling, an inhibitor of sonic hedgehog (SHH) signaling, and a combination thereof

[0386] Embodiment 1-33. The method of any of Embodiment 1-28 to Embodiment 1-32, wherein the posterior foregut cell is characterized by expression of PDX1, HNF6, FOXA2, HNF4A, HNF1B, and / or FOXA1,

[0387] Embodiment 1-34. The method of any of Embodiment 1-28 to Embodiment 1-33, wherein differentiation of the posterior foregut cell to the pancreatic progenitor cell comprises contacting the posterior foregut cell with a differentiation factor selected from an epidermal growth factor (EGF), a retinoic pathway activator, a growth factor of the FGF family, and a combination thereof.

[0388] Embodiment 1-35. The method of any of Embodiment 1-28 to Embodiment 1-34, wherein the pancreatic progenitor cell is characterized by expression of PDX1 and NKX6-1.

[0389] Embodiment 1-36. The method of any of Embodiment 1-28 to Embodiment 1-35, wherein differentiation of the pancreatic progenitor cell to the pancreatic islet cell comprises contacting the pancreatic progenitor cell with a differentiation factor selected from an inhibitor of TGFβ type I receptor, a thyroid hormone, a BMP signaling inhibitor, an inhibitor of gamma secretase, and a combination thereof.

[0390] Embodiment 1-37. The method of any of Embodiment 1-28 to Embodiment 1-36, wherein the pancreatic islet cell is characterized by expression of PDX1, NKX6-1, and C-peptide.

[0391] Embodiment 1-38. The method of Embodiment 1-37, wherein the pancreatic islet cell is further characterized by expression of INS, PDX1, CHGA, S1X2, MAFA, NKX2.2, NEUROD, ISL1, UCN3, ENTPD3, and / or MAFB.

[0392] Embodiment 1-39. The method of any of Embodiment 1-1 to Embodiment 1-38, wherein the pluripotent stem cell comprises an alloantigen with respect to the subject.329282749 93Attorney Docket: MINU-012 / 01WO 339336.2063

[0393] Embodiment 1-40. The method of Embodiment 1-39, wherein the alloantigen comprises a major histocompatibility complex (MHC) class I molecule, an MHCII molecule, a minor histocompatibility antigen, a blood type antigen or a combination thereof.

[0394] Embodiment 1-41. The method of any of Embodiment 1-1 to Embodiment 1-40, wherein the loss of function mutation is introduced to the pluripotent stem cell prior to the in vitro differentiation.

[0395] Embodiment 1-42. The method of Embodiment 1-41, wherein the loss of function mutation is introduced using a gene editing system.

[0396] Embodiment 1-43, The method of Embodiment 1-42, wherein the gene editing system comprises a CRISPR / Cas system.

[0397] Embodiment 1-44, The method of Embodiment 1-42 or Embodiment 1-43, wherein the gene editing system introduces a deletion to the TET2 gene.

[0398] Embodiment 1-45, The method of Embodiment I- 42 or Embodiment 1-43, wherein the gene editing system introduce a frameshift mutation to the TET2 gene.

[0399] Embodiment 1-46. The method of any of Embodiment 1-1 to Embodiment 1-45, wherein the pluripotent stem cell comprises a loss of function mutation in one or both alleles comprising the TET2 gene.

[0400] Embodiment 1-47. The method of any of Embodiment 1-1 to Embodiment 1-46, wherein the composition is implanted at an extrahepatic site.

[0401] Embodiment 1-48. The method of any of Embodiment 1-1 to Embodiment 1-47, wherein the composition is implanted at a subcutaneous or intramuscular site.

[0402] Embodiment 1-49. The method of any of Embodiment 1-1 to Embodiment 1-48, wherein the pancreatic islet cells comprise a function of a human donor pancreatic islet selected from the group consisting of: (i) glucose-responsive secretion of C-peptide; (ii) glucoseresponsive secretion of insulin; (iii) insulin granule biogenesis, trafficking, and / or exocytosis; and (iv) a combination of (i)-(iii).

[0403] Embodiment 1-50. The method of Embodiment 1-49, wherein the pancreatic islet cells secrete an increased level of insulin in response to an increased level of glucose.

[0404] Embodiment 1-51. The method of Embodiment I- 49 or Embodiment 1-50, wherein the pancreatic islet cells are characterized by a glucose stimulated insulin secretion (GSIS) response329282749 94Attorney Docket: MINU-012 / 01WO 339336.2063

[0405] Embodiment 1-52. The method of Embodiment 1- 51, wherein the GSIS response is in vitro and / or in vivo.

[0406] Embodiment 1-53. The method of any of Embodiment 1-49 to Embodiment 1-52, wherein the pancreatic islet cells secrete an increased level of C-peptide in response to an increased level of glucose.

[0407] Embodiment 1-54. The method of any of Embodiment 1-1 to Embodiment 1-53, wherein prior to the administration, the subject received a daily infusion of insulin.

[0408] Embodiment 1-55. The method of any of Embodiment 1-1 to Embodiment 1-54, wherein the subject has a medical history of severe hypoglycemic events.

[0409] Embodiment 1-56. The method of any of Embodiment 1-1 to Embodiment 1-55, wherein the subject is characterized by no residual endogenous islet cell function.

[0410] Embodiment 1-57. The method of any of Embodiment 1-1 to Embodiment 1-56, wherein prior to the administration, the subject has undetectable blood C-peptide level when measured using a mixed meal tolerance test,

[0411] Embodiment 1-58. The method of any one of Embodiment 1-1 to Embodiment 1-57, wherein the insulin deficiency is characterized by high blood sugar levels for a prolonged period of time.

[0412] Embodiment 1-59. The method of any one of Embodiment I- 1 to Embodiment 1-58, wherein the insulin deficiency is Type I diabetes.

[0413] Embodiment 1-60. The method of any one of Embodiment 1-1 to Embodiment 1-58, wherein the insulin deficiency is Type 2 diabetes.

[0414] Embodiment 1-61. The method of any one of Embodiment I- 1 to Embodiment 1-60, wherein the subject is human.EXAMPLESExample I: Generation of Islet Organoids from TET2 Knockout (KO) Embryonic Stem Cells (ESCs)

[0415] This Example describes generation of islet organoids from ESCs having a TET2 KO.329282749 95Attorney Docket: MINU-012 / 01WO 339336.2063

[0416] Experiments were performed using the NIH-approved human embryonic stem cell (hESC) line MEL-1 (NIH registration number: 0139) in which a GFP reporter was inserted into an allele of the endogenous insulin locus (INSGFP / W hESCs; see Micallef et al., 2012 Diabetologia 55:694). INSGFP / W hESCs were cultured on irradiated mouse embryonic fibroblasts (Thermo Fisher) m hESC maintenance media composed of Dulbecco's Modified Eagle Medium (DMEM) / F12, 20% (vol / vol) KnockOut serum replacement (Thermo Fisher Scientific), nonessential amino acids (Thermo Fisher Scientific), GlutaMAX (Thermo Fisher Scientific), and beta-mercaptoethanol (Millipore). The maintenance media was supplemented with 10 ng / mL recombinant human FGF-2 (R& D Systems). Confluent hESCs were dissociated into single-cell suspension with TrypLE Select (Gibco) and passaged every 3 4 days,

[0417] A dual guide CRISPR / Cas9 strategy was used to delete exon 9 and parts of exons 8 and 10 in the TET2 gene in wild-type MEL-1 INS-GFP cells. See FIGs. 1A-1B. The sequences for the gRNA targeting exon 8 and exon 10 are SEQ ID NOs: 1-2 respectively. Lyophilized crRNAs and tracrRNAs (Dharmacon) were resuspended in RNase-free water at a concentration of 160 mM, and stored in aliquots at 80°C. crRNA and tracrRNA aliquots were thawed, mixed at a ratio of 1:1, and annealed by incubation at 37°C for 30 min to form an 80 mM gRNA solution.Recombinant Cas9 (QB3 Macrolab) was then mixed 1:1 by volume with the 80 mM gRNA (2:1 gRNA to Cas9 molar ratio) at 37°C for 15 min to form an RNP at 20 mM. During the incubation, WT INSGFP / W hPSCs were dissociated into single cells using TrypLE Select (Thermo Fisher Scientific) and 400,000 cells were resuspended in P3 primary cell solution (Lonza). Cas9 RNP and the cell solution were added to a Nucleocuvette (Lonza) and nucleofection was carried out using the setting CAI 37 on the Amaxa 4D-Nucleofector. Cells were replated on MEFs in hESC maintenance media supplemented with ROCK inhibitor.

[0418] FIG. 1C shows gel electrophoresis of PCR-amplified fragments from WT ESCs and TET2 KO ESCs. Only TET2 KO ESCs exhibited PCR fragments corresponding to the deletion of exon 9.

[0419] Two clones were generated as shown in Table 8. The deletions observed resulted in frameshift mutations.Table 8: Exemplary TET2 Gene Knockout Clones in MEL-1 INS-GFP Cells329282749 96Attorney Docket: MINU-012 / 01WO 339336.2063Clone Gene mutation (Allele 1) Gene mutation (Allele 2) 15 (heterozygous) deletion of 36 bp at 3 'end of deletion of 34 bp at 3 'end of exon8, all of exon 9, and exon8, all of exon9, and 347bp at 5 'end of exonlO; 343bp at 5'end of exonlO; mRNA product has a 521 bp mRNA product has a 515bp deletion deletion59 (homozygous) deletion of 36 bp at 3 'end of exon8, all of exon9, 347bp at the5'end of exonlO; mRNA product has a 521bp deletion

[0420] To initiate differentiation, confluent cultures were dissociated into single-cell suspensions using TrypLE Select, counted, and seeded them in six-well suspension plates at a density of 5.5 x 106 cells per 5.5 mL of hESC maintenance media supplemented with 10 ng / mL activin A (R& D Systems) and 10 ng / mL heregulin B (PeproTech). The plates were incubated at 37C with 5% CO2 on an orbital shaker set at 100 rpm to induce 3D sphere formation. After 24 hours, the spheres were collected in 1.5 mL microcentrifuge tubes, one well per tube, and allowed to settle by gravity, washed once with RPMI medium (Gibco), and resuspended in day 1 differentiation media. The resuspended spheres were distributed into fresh six- well suspension plates for a final volume of 5.5 mL of day 1 media per well. Until day 3, spheres were fed daily by removing about 5 mL of the media and replenishing with 5.5 ml. From day 4 to day 20, media was removed daily, and 5 mL of fresh media was added. Media compositions for differentiation of INSGFP / W hESCs are as follows: day 1, RPMI (Gibco) containing 0.2% fetal bovine serum (FBS, Corning), 1: 5000 insulin transferrin selenium G (ITS-G, Gibco), 100 ng / mL activin A, and 1uM CHIR99021 (STEMCELL Technologies); day 2, RPMI containing 0.2% FBS, 1:2000 ITS, and 100 ng / mL activin A; day 3, RPMI containing 0.2% FBS, 1: 1000 ITS, 2.5 pM TGFbi IV (Calbio chem), and 25 ng / mL keratinocyte growth factor (KGF; R& D Systems); day 4-5, RPMI containing 0.2% FBS, 1: 1000 I TS, and 25 ng / mL KGF; day 6-7, DMEM (Gibco) with 25 mM glucose containing 1:100 B27 (Gibco) and 3 nM TTNPB (Sigma); day 8, DMEM with 25 mM glucose containing 1:100 B27, 3 nM TTNPB, and 50 ng / mL epidermal growth factor (EGF; R& D Systems); day 9-11, DMEM with 25 mM glucose containing 1: 100 B27, 50 ng / mL EGF, and 50 ng / mL KGF; day 12+, DMEM with 25 mM glucose containing 1: 100 B27, 1: 100 Gluta-MAX (Gibco), 1:100 NEAA (Gibco), 10 μM ALKiII (Axxora), 500 nMLDN-193189 (Stemgent), 1 μM Xxi (Millipore), 1 μM T3 (Sigma-Aldrich), 0.5 mM vitamin C, 1 mMN- acetylcysteine (Sigma- Aldrich), 10 pMzinc sulfate (Sigma-Aldrich), and 10 pg / mL heparin sulfate. At day 19-20 of differentiation, the cells were digested into single cells with Accumax329282749 97Attorney Docket: MINU-012 / 01WO 339336.2063(STEMCELL Technologies) prior to reaggregation in Connaught Medical Research Laboratories (CMRL) medium containing 10% FBS, 1:100 Glutamax (Gibco), 1:100 NEAA(Gibco), 10 μM ALKi II (Axxora), 0.5 mM vitamin C, 1 μM T3 (Sigma- Aldrich), 1 mM N-acetyl Cysteine (Sigma- Aldrich), 10 μM zinc sulfate (Sigma- Aldrich), and 10 μg / ml of heparin sulfate supplemented with 10 μM of the ROCK inhibitor Y-27632 (Tocris) and Penicillin Streptomycin (Corning). The cells were then cultured until day 30 in the above media without ROCK inhibitor Y-27632.

[0421] TET2 expression was measured using quantitative real-time PCR, PCR Samples were harvested at indicated stages of differentiation and homogenized in RLT buffer (QIAGEN), RNA was extracted and purified using RNeasy Mmi / Micro kits (QIAGEN), cDNA was synthesized with iScript (Biorad). qPCR was performed using SYBR green on Biorad CFX Connect Real-Time PCR Detection System. Assay was performed using diluted cDNA samples and primers against genes of interest and TBP as internal control,

[0422] FIG.2A shows TET2 expression depending on the stage of differentiation from wildtype MEL-1 INS-GFP cells. MEL-1 INS-GFP had substantially no expression of TET2, while cells at stages of differentiation progressing to islet cells had increasing expression of TET2.

[0423] FIG.2B shows TET2 expression in islet cells differentiated from clone 15 or 59 TET2 KO MEL-1 INS-GFP cells or from wild-type MEL-1 INS-GFP cells.

[0424] FIG.2C shows TET2 activity in islet cells differentiated from clone 15 or 59 TET2 KO MEL-1 INS-GFP cells or from wild-type MEL-1 INS-GFP cells.Example 2: In Vitro Characterization ofTET2 KO Pancreatic Islet Organoid Function

[0425] Islet organoids differentiated from TET2 deficient ESCs were characterized for beta cell function. Islet organoids were differentiated from clone 15 and 59 TET2 KO MEL-1 INS-GFP cells as described in Example I (“TET2 KO islet organoids”). Control pancreatic islet organoids were differentiated from wild-type MEL-1 INS-GFP cells.

[0426] Islet organoids at day 23 of the differentiation protocol were characterized for expression of beta cell markers PDX1 and NKX6.1. Expression was measured by qRT-PCR as described in Example 1. As shown in FIGs.3A-3B, TET2 KO pancreatic islet organoids had comparable levels ofPDXl and NKX6.1 expression to control cells.329282749 98Attorney Docket: MINU-012 / 01WO 339336.2063

[0427] Pancreatic islet organoids at day 23 of the differentiation protocol were characterized for expression of C-peptide. GFP expression in MEL-1 INS-GFP cells was used to quantify the level of C-peptide expression. GFP expression was measured by microscopy.

[0428] FIG.4A shows uniform clusters of C-peptide were observed for TET2 KO pancreatic islet organoids, which had similar morphology compared to control cells.

[0429] Additionally, flow cytometry was used to evaluate marker expression. Differentiated clusters were dissociated into single cells using Accumax (Thermo Fisher Scientific), fixed using 4% paraformaldehyde, permeabilized, and stained for intracellular markers. Anti-human C-peptide was conjugated using a Molecular Probes Antibody Labeling Kits according to manufacturer’s instructions. Data were analyzed using FlowJo software.

[0430] FIG. 4B shows the proportion of TET2 KO pancreatic islet organoids having NKX6.1 and C-peptide expression. FIG. 4C shows TET2 KO pancreatic islet organoids have lower expression of C-peptide compared to control organoids as measured by median fluorescent intensity.

[0431] Dynamic glucose-stimulated insulin secretion was measured using a perifusion system (Biorep technologies). For each sample, one hundred sorted clusters were handpicked and placed on filters in plastic chambers that were maintained under temperature and CO2 controlled conditions. After an initial about one hour preincubation period in 2.8 mM glucose in KRB buffer, chambers were sequentially perfused at a flow rate of 100 μl / min with 2.8 mM glucose for 20 min and 20 mM glucose for 30 mm. Flow- through was collected over the course of the experiment, and insulin concentrations in the supernatant were determined using an ultrasensitive insulin ELISA kit (STELLUX Chemi Human C-peptide ELISA from Alpco).

[0432] FIG.4D shows TET2 KO pancreatic islet organoids have a robust C-peptide response as demonstrated by increased C-peptide in response to high glucose stimulation (20 mM) vs low glucose stimulation (2.8 mM). These data demonstrate that TET2 KO pancreatic islet organoids have hormone secretion capability in response to glucose stimulation that is comparable to control organoids.Example 3: In Vivo Characterization of TET2 KO Pancreatic Islet Organoid Function

[0433] Pancreatic islet organoids differentiated from TET2 KO ESCs were characterized for beta cell function in vivo. TET2 KO pancreatic islet organoids (islet organoids differentiated from329282749 99Attorney Docket: MINU-012 / 01WO 339336.2063clone 15 and 59 TET2 deficient MEL-1 INS-GFP cells as described in Example 1) were evaluated for in vivo glucose stimulation response. Control pancreatic islet organoids were differentiated from wild-type MEL-1 INS-GFP cells.

[0434] Studies were performed in NOD / SCID Gamma mice. Recipient mice were anesthetized, shaved and cleaned. A small incision was made on the left flank of the mouse and the kidney was exposed. Approximately 450 islet organoids were pipetted under the kidney capsule. The kidney was placed back into the cavity and the peritoneum and skin were sutured and stapled, micro¬ units per mL (uU / mL) insulin in NOD / SCID mice were used as a measure of pancreatic function, with low or undetectable insulin expected in diabetic states and increased ranges upon engrafting functional pancreatic islets.

[0435] FIG. 5 shows insulin production in vivo was similar for TET2 deficient islet organoids as compared to control organoids. Unstimulated levels of circulating human insulin (“TO”) were substantially similar for TET2 deficient islet organoids as compared to control organoids.Stimulated level was significantly lower for TET2 deficient islet organoids derived from clone 59 (p=0.01). The fold change between the islet donors was similar (2.12+0.28 pU / ml in KOI 5, 2.23+0.34 in KO59 vs 2.46+0.31 in WT).Example 4: TET2 KO Promotes Resistance of Pancreatic Islet Organoids to CD8+ T Cell Killing

[0436] Pancreatic islet organoids differentiated from TET2 KO ESCs were characterized in vitro for killing by CD8+ effector T cells enriched for responsiveness to diabetogenic autoantigens. TET2 KO pancreatic islet organoids (islet organoids differentiated from clone 15 and 59 TET2 deficient MEL-1 INS-GFP cells as described in Example 1) were evaluated for in vivo glucose stimulation response. Control pancreatic islet organoids were differentiated from wild-type MEL-1 INS-GFP cells.

[0437] CD8+ effector cells were enriched for diabetogenic antigens (i.e. a pool of peptides of proinsulin, IA-2, ZnT8, IGRP, insulin, and GAD65) or EBV BMLF1. A2+EBV+ PBMC to start with, 2,500 cells / well(96wp), PHA grow for 10 days, stimulate with Diab or EBV antigens for 2-3days and screen IFNr+ / - wells via ELISA- pool the IFNr+ wells or IFNr- wells to use as effectors cells.329282749 100Attorney Docket: MINU-012 / 01WO 339336.2063

[0438] A 24 hour CTL assay was performed. Pancreatic islet organoids were dissociated into single cells and cocultured with T cells at an E / T ratio of 0, 1, 2, or 5. The T cells were seeded in a 96 flat well plat prior to addition of target cells. Coculture was performed for about 24hr.Readout of killing was performed by flow cytometry to determine ratio of live to dead cells.

[0439] FIG. 6A shows killing of approximately 50% of the control organoid cells at an E: T ratio of 5:1, whereas the TET2 KO pancreatic islet organoid cells showed significantly reduced killing (pcO. OOOl two way ANOVA).

[0440] Expression of Class I MHC was evaluated to determine if reduced expression thereof contributed to resistance to killing. To evaluate the expression of Class I MHC, Day 30 islet organoids clusters were treated with 50ng / mLIFNy for 20 hours. They were dissociated with Accutase (six minutes at 37 degrees Celsius) and stained HLA-A2 APC (1: 100) and HLA-ABC-Pecy5 (1: 100) in FACS buffer at 4 degrees Celsius for one hour. DAPI was added for live / dead staining.

[0441] FIG. 6B shows the expression of HLA-A2.1 was not reduced on the TET2 KO pancreatic islet organoids (measured by flow cytometry in the presence or absence of IFNy treatment). Thus, it was determined that the resistance to killing did not result from a lower expression of Class 1 MHC allele.

[0442] Expression of autoantigens was measured by RT-PCR. FIG. 6C shows expression of IGRP was reduced for TET2 KO pancreatic islet organoids compared to control islet organoids, but not for the other the other autoantigens evaluated. This was with or without culture with a panel of four cytokines (TNFa, IL-ip, IFNy, and IL-6).

[0443] To further understand the resistance to killing, islet organoids were pulsed with autoantigen or EBV peptide and the CTL assay was performed.

[0444] FIG. 7A-7B show that under conditions in which antigen was loaded onto the cells, there was reduced sensitivity to killing of the TET2 deficient islet organoids using the autoantigen- reactive or EB V peptide-reactive effector cells.Example 5: TET2 KO Promotes Resistance of Pancreatic Islet Organoids to Inflammatory Cytokines

[0445] The expression of genes associated with ER stress, cell death, and inflammatory responses were evaluated in TET2 KO pancreatic islet organoids (islet organoids differentiated329282749 101Attorney Docket: MINU-012 / 01WO 339336.2063from clone 15 and 59 TET2 deficient MEL-1 INS-GFP cells as described in Example 1). Control islet organoids were differentiated from wild-type MEL-1 INS-GFP cells.

[0446] Cells obtained from TET2 KO or control pancreatic islet organoids were cultured with a pool of 4 cytokines implicated in immune killing of P cells in Type 1 diabetes: IFNa, IFNy, TNFa, and IL-ip. Expression of genes associated with ER stress (ATF4, BiP, CHOP, ATF3, ATF6, and XBP1), inflammatory response (CXCL10, STAT1, CXCL16, CD47, MX1, CCL2, and TNF), and cell death pathway (FAS, PDL1, IDO, BACH1, BACH2, and CASP3) were measured by RT-PCR,

[0447] FIG. 8A shows TET2 KO pancreatic islet organoids had reduced expression of several ER stress response genes (ATF4, BiP, CHOP, XBP1, ATF6) following cytokine stimulation.

[0448] FIG. 8B shows TET2 KO pancreatic islet organoids had reduced expression of inflammatory response genes (STAT1, CXCL10, and IDO) following cytokine stimulation. At higher doses of IFNa (10,000 U / ml) the expression of CXCL10 and STAT1 was reduced to a greater extent (FIG. 8C),

[0449] FIG. 8D shows TET2 KO pancreatic islet organoids had reduced expression of several cell death pathway genes following cytokine stimulation.

[0450] Cell death was measured by flow cytometry for TET2 KO pancreatic organoids cultured with inflammatory cytokines or with ER stress inducers brefeldin A or MG132.

[0451] FIG. 9 show the frequency of live p cells was increased for TET2 KO pancreatic islet organoids following culture with cytokines.

[0452] FIG. 10 shows reduced cell death in response to ER stress inducers in TET2 KO pancreatic islet organoids as compared to pancreatic islet organoids differentiated from WT ESCs.Example 6: Generation of Beta Cells from TET2 Deficient iPSCs

[0453] iPSC derived from a HLA-A2.1+ donor were produced as described in Pauli, et al (2015) Nat Methods 12:885.

[0454] A dual guide CRISPR / Cas9 strategy was used to delete exon 3 in the TET2 gene in iPSC cells. Lyophilized crRNAs and tracrRNAs (Dharmacon) were resuspended in RNase-free water at a concentration of 160 mM, and stored in aliquots at 80C. crRNA and tracrRNA aliquots were thawed, mixed at a ratio of 1: 1, and annealed by incubation at 37 C for 30 min to form an 80329282749 102Attorney Docket: MINU-012 / 01WO 339336.2063gRNA solution. Recombinant Cas9 (QB3 Macrolab) was then mixed 1:1 by volume with the 80 mM gRNA (2: 1 gRNA to Cas9 molar ratio) at 37C for 15 min to form an RNP at 20 mM. During the incubation, iPSCs were dissociated into single cells using TrypLE Select (Thermo Fisher Scientific) and 400,000 cells were resuspended in P3 primary cell solution (Lonza). Cas9 RNP and the cell solution were added to a Nucleocuvette (Lonza) and nucleofection was carried out using the setting CA137 on the Amaxa 4D-Nucleofector. Cells were replated on MEFs in hESC maintenance media supplemented with ROCK inhibitor.

[0455] TET2-KO iPSCs were differentiated to islet cells as described in Example 1.

[0456] CD8+ T cells from an HLA-2.1 negative donor were selected for reactivity to HLA-A2.1 and then expanded.

[0457] A CTL assay was performed as described in Example 4.

[0458] FIG. 11 shows pancreatic islet cells having the TET2-KO were resistant to killing by alloantigen-specific effector T cells as compared to control islets.329282749 103

Claims

Attorney Docket: MINU-012 / 01WO 339336,2063CLAIMS1. A method for stem cell therapy m a subject having an insulin deficiency, comprising administering to the subject a composition comprising a population of pancreatic islet cells, wherein the population of pancreatic islet cells is obtained by in vitro differentiation from a pluripotent stem cell prior to the administering, wherein the pluripotent stem cell comprises a loss of function mutation in a Tet methylcytosine dioxygenase 2 (TET2) gene, wherein the pluripotent stem cell is allogeneic with respect to the subject, wherein the population of pancreatic islet cells or a portion thereof undergoes engraftment following the administering, thereby treating the insulin deficiency in the subject.

2. The method of claim 1, w’herein the engraftment is characterized by (i) increased plasma levels of C-peptide when measured in a fasting state as part of a mixed meal tolerance test, (ii) reduction in HbAlc levels, and / or (iii) increased Time in Range (TIR),3. The method of claim 1 or 2, wherein the engraftment occurs without acute rejection of the population.

4. The method of any of claims 1-3, wherein the engraftment is improved as compared to administering a control composition comprising a population of pancreatic islet cells, wherein the composition is obtained by in vitro differentiation from a pluripotent stem cell comprising a wild-type TET2 gene.

5. The method of any of claims 1-4, wherein the engraftment is maintained for a duration of time following the administering.

6. The method of claim 5, wherein the duration of time is at least about 6 months, about 7 months, about 8 months, or about 9 months.

7. The method of claim 5 or 6, wherein the engraftment is maintained in the absence of the subject receiving an immunosuppressant.329282749 104Attorney Docket: MINU-012 / 01WO 339336,20638. A method for stem cell therapy in a subject having an insulin deficiency, comprising administering to the subject a composition comprising a population of pancreatic islet cells, wherein the population of pancreatic islet cells is obtained by in vitro differentiation from a pluripotent stem cell prior to the administering, wherein the pluripotent stem cell comprises a loss of function mutation in a TET2 gene, wherein the pluripotent stem cell is allogeneic with respect to the subject, and wherein the administering is associated with a reduced alloimmune response compared to administering a population of unmodified pancreatic islet cells.

9. The method of claim 8, wherein the reduced alloimmune response is characterized by increased plasma levels of C-peptide when measured in a fasting state as part of a mixed meal tolerance test, (ii) reduction in HbAlc levels, and / or (iii) increased TIR.

10. The method of claim 8 or 9, wherein the unmodified population of pancreatic islet cells is obtained from a pluripotent stem cell comprising a wild-type TET2 gene.

11. The method of any of claims 1-10, wherein a plurality of the pancreatic islet cells in the population are insulin producing cells.

12. The method of any of claims 1-11, wherein at least about 30% to about 60% of the population of pancreatic islet cells are insulin producing cells.

13. The method of claim 11 or 12, wherein the insulin producing cells comprises beta cells.

14. The method of any of claims 11-13, wherein the population further comprises alpha cells, delta cells, or a combination thereof.

15. The method of claim 14, wherein less than about 10% of cells of the population are epsilon cells, pancreatic progenitor cells, acinar and ductal cells, enterochromaffin cells, and / or proliferative cells.329282749 105Attorney Docket: MINU-012 / 01WO 339336,206316. The method of claim 15, wherein less than about 1% of cells of the population are proliferative cells.

17. The method of any of claims 1-16, wherein a plurality of pancreatic islet cells in the population are present in cell clusters.

18. The method of claim 17, w’herein the cell clusters comprise a longest diameter of about 50 pm to 350 pm or about 100 pm to about 200 pm,19. The method of claim 17 or 18, wherein the cell clusters comprise about 500, about 600, about 700, about 800, about 900, about 1000, about 1100, about 1200, about 1300, about 1400, or about 1500 cells.

20. The method of any of claims 1-19, wherein the composition further comprises a support factor, optionally wherein the support factor is selected from the group consisting of gamma-aminobutyric acid (GABA), parathyroid hormone (PTH), PTH-related peptide (PTHrP), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), angiopoietm, and a combination thereof.

21. The method of claim 20, wherein the support factor is a soluble factor.

22. The method of claim 20, wherein the support factor is operably linked to a surface of a plurality of pancreatic islet cells in the population.

23. The method of any of claim 20-22, wherein the factors are introduced to the composition following the in vitro differentiation and prior to the administering.

24. The method of any of claims 1-23, wherein the composition comprises the population of pancreatic islet cells in a liquid suspension.329282749 106Attorney Docket: MINU-012 / 01WO 339336,206325. The method of any of claims 1-23, wherein the composition comprises the population of pancreatic islet cells in an encapsulation device or a degradable material.

26. The method of any of claims 1-25, wherein the pluripotent stem cell is a human cell.

27. The method of any of claims 1 -26, wherein the pluripotent stem cell comprises an embryonic stem cell.

28. The method of any of claims 1 -26, wherein the pluripotent stem cell comprises an induced pluripotent stem cell.

29. The method of any of claims 1-28, wherein the in vitro differentiation comprises a step-wise differentiation of (i) the pluripotent stem cell to a definitive endoderm cell, (ii) the definitive endoderm cell to a posterior foregut cell, (iii) the posterior foregut cell to a pancreatic progenitor cell, and (iv) the pancreatic progenitor cell to the pancreatic islet cell.

30. The method of claim 29, wherein differentiation of the pluripotent stem cell to the definitive endoderm cell comprises contacting the pluripotent stem cell with a differentiation factor selected from a ROCK inhibitor, a TGFP superfamily growth factor, an epidermal growth factor family polypeptide, a WNT activator, and a combination thereof.

31. The method of claim 29 or 30, wherein the definitive endoderm cell is characterized by expression of CXCR4, SOX17, and / or FOXA2.

32. The method of claim 31, wherein the definitive endoderm cell does not substantially express PDX1.

33. The method of any of claims 29-32, wherein differentiation of the definitive endoderm cell to the posterior foregut cell comprises contacting the definitive endoderm cell with a differentiation factor selected from a growth factor of the fibroblast growth factor (FGF) family, an activator of the retinoic acid signaling pathway, an activator of protein kinase C, an inhibitor329282749 107Attorney Docket: MINU-012 / 01WO 339336,2063of bone morphogenetic protein (BMP) signaling, an inhibitor of sonic hedgehog (SHH) signaling, and a combination thereof.

34. The method of any of claims 29-33, wherein the posterior foregut cell is characterized by expression of PDX1, HNF6, FOXA2, HNF4A, HNF1B, and / or F0XA1.

35. The method of any of claims 29-34, wherein differentiation of the posterior foregut cell to the pancreatic progenitor cell comprises contacting the posterior foregut cell with a differentiation factor selected from an epidermal growth factor (EGF), a retinoic pathway activator, a growth factor of the FGF family, and a combination thereof,36. The method of any of claims 29-35, wherein the pancreatic progenitor cell is characterized by expression of PDX1 and NKX6-1.

37. The method of any of claims 29-36, wherein differentiation of the pancreatic progenitor cell to the pancreatic islet cell comprises contacting the pancreatic progenitor cell with a differentiation factor selected from an inhibitor of TGFp type I receptor, a thyroid hormone, a BMP signaling inhibitor, an inhibitor of gamma secretase, and a combination thereof.

38. The method of any of claims 29-37, wherein the pancreatic islet cell is characterized by expression of PDX1, NKX6-1, and C-peptide.

39. The method of claim 38, wherein the pancreatic islet cell is further characterized by expression of INS, CHGA, SIX2, MAFA, NKX2.2, NEUROD, ISL1, UCN3, ENTPD3, and / or MAFB.

40. The method of any of claims 1-39, wherein the population of pancreatic islet cells express an amount of PDX1, wherein the amount is at least about 70% to about 130% of the amount of PDX1 expressed by a control population of pancreatic islet cells comprising a wild¬ type TET2 gene.329282749 108Attorney Docket: MINU-012 / 01WO 339336,206341. The method of any of claims 1-40, wherein the population of pancreatic islet cells express an amount of NKX6-1, wherein the amount is at least about 70% to about 130% of the amount of NKX6-1 expressed by a control population of pancreatic islet cells comprising a wild¬ type TET2 gene.

42. The method of any of claims 1-41, wherein the population of pancreatic islet cells express an amount of C-peptide, wherein the amount is at least about 40% to about 100% of the amount of C-peptide expressed by a control population of pancreatic islet cells comprising a wild-type TET2 gene.

43. The method of any of claims 1 -42, wherein the pluripotent stem cell is MHC mismatched with respect to the subject.

44. The method of any of claims 1-43, wherein the pluripotent stem cell comprises an alloantigen with respect to the subject.

45. The method of claim 44, wherein the alloantigen comprises a major histocompatibility complex (MHC) class I molecule, an MHCII molecule, a minor histocompatibility antigen, a blood type antigen or a combination thereof.

46. The method of claim 44 or 45, wherein the population of pancreatic islet cells is resistant to killing by an effector CD8+ T cell reactive to the alloantigen.

47. The method of claim 44 or 45, wherein upon contacting the population of pancreatic islet cells with an effector CD8+ T cell reactive to the alloantigen, the population is characterized by about 10% to about 90% higher viability as compared to a control population of pancreatic islet cells comprising a wild-type TET2 gene, optionally wherein the contacting comprises an effector cell to target cell ratio of about 1: 1 to about 10:1.

48. The method of any of claims 1-47, wherein the population of pancreatic islet cells is characterized by expression of at least one diabetogenic antigen in an amount, optionally the at329282749 109Attorney Docket: MINU-012 / 01WO 339336,2063least one diabetogenic antigen is selected from the group consisting of: proinsulin, insulinoma-associated protein 2 (IA-2), zinc transporter 8 (ZnT8), islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), insulin, glutamic acid decarboxylase 65 (GAD65), chromogranin A (ChgA), islet amyloid polypeptide (IAPP), and a combination thereof.

49. The method of claim 48, wherein the amount is at least about 70% to about 130% of the amount expressed by a control population of pancreatic islet cells comprising a wild-type TET2 gene.

50. The method of claim 49, wherein the at least one diabetogenic antigen comprises proinsulin, IA-2, ZnT8, insulin, GAD65, ChgA, and IAPP.

51. The method of claim 48, wherein the amount is less than about 70% of the amount expressed by a control population of pancreatic islet cells comprising a wild-type TET2 gene.

52. The method of claim 51, wherein the at least one diabetogenic antigen comprises IGRP.

53. The method of any of claims 1-50, wherein upon contacting the population of pancreatic islet cells with an inflammatory cytokine, the population is characterized by a survival property as compared to a control population of pancreatic islet cells comprising a wild-type TET2 gene, wherein the survival property is selected from the group consisting of:(i) decreased endosomal stress response;(ii) decreased inflammatory response;(iii) decreased activation of cellular death pathways;(iv) decreased cell apoptosis; and(v) a combination of (i)-(iv).

54. The method of claim 53, wherein the inflammatory cytokine is selected from the group consisting of: TNFa, IL-ip, IFNy, IL-6, and a combination thereof.329282749 110Attorney Docket: MINU-012 / 01WO 339336,206355. The method of claim 53 or 54, wherein the endosomal stress response is characterized by expression of a gene selected from the group consisting of ATF4, BiP, CHOP, ATF3, ATF6, XBP1, and a combination thereof.

56. The method of any of claim 53-55, wherein the inflammatory stress response is characterized by expression of a gene selected from the group consisting of: CXCL10, STAT1, CXCL16, CD47, MX1, CCL2, TNF, and a combination thereof.

57. The method of any of claim 53-56, wherein the cellular death pathway is characterized by expression of a gene selected from the group consisting of: FAS, PDL1, IDO, BACH1, BACH2, CASP3, and a combination thereof.

58. The method of any of claims 1 -57, wherein upon contacting the population of pancreatic islet cells with ER stress inducer, the population is characterized by increased viability as compared to a control population of pancreatic islet cells comprising a wild-type TET2 gene.

59. The method of claim 58, wherein the loss of function mutation is introduced using a gene editing system.

60. The method of claim 59, wherein the gene editing system comprises a CRISPR / Cas system.

61. The method of claim 59 or 60, wherein the gene editing system introduces an insertion and / or deletion to the TET2 gene.

62. The method of claim 61, wherein the insertion and / or deletion results in a frameshift mutation to the TET2 gene.

63. The method of claim 61 or 62, wherein the gene editing system introduces the deletion to the TET2 gene.329282749 111Attorney Docket: MINU-012 / 01WO 339336,206364. The method of claim 63, wherein the deletion spans a region of the TET2 gene, w’herein the region comprises all or a portion of exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, or exon 11.

65. The method of claim 64, w’herein the region comprises a portion of exon 3.

66. The method of claim 64, w’herein the region comprises a 3' portion of exon 8, exon 9, and a 5' portion of exon 10.

67. The method of any of claims 1-66, wherein the pluripotent stem cell comprises the loss of function mutation in one allele comprising the TET2 gene.

68. The method of any of claims 1 -66, wherein the pluripotent stem cell comprises the loss of function mutation in both alleles comprising the TET2 gene.

69. The method of any of claims 1-68, wherein the composition is implanted at an extrahepatic site.

70. The method of any of claims 1 -69, wherein the composition is implanted at a subcutaneous or intramuscular site.

71. The method of any of claims 1-70, wherein the population of pancreatic islet cells comprise a function of a human donor pancreatic islet selected from the group consisting of:(i) glucose-responsive secretion of C-peptide;(li) glucose-responsive secretion of insulin;(in) insulin granule biogenesis, trafficking, and / or exocytosis; and(iv) a combination of (i)-(iii).

72. The method of claim 71, wherein the population of pancreatic islet cells secrete an increased level of insulin in response to an increased level of glucose.329282749 112Attorney Docket: MINU-012 / 01WO 339336,206373. The method of claim 71 or 72, wherein the population of pancreatic islet ceils are characterized by a glucose stimulated insulin secretion (GSIS) response.

74. The method of claim 73, wherein the GSIS response is in vitro.

75. The method of claim 73, wherein the GSIS response is in vivo.

76. The method of any of claims 71-75, wherein the population of pancreatic islet cells secrete an increased level of C-peptide in response to an increased level of glucose.

77. The method of any of claims 1 -76, wherein the subject received a daily infusion of insulin prior to administration of the composition,78. The method of any of claims 1-77, wherein the subject has a medical history of severe hypoglycemic events.

79. The method of any of claims 1 -78, wherein the subject is characterized by no residual endogenous islet cell function.

80. The method of any of claims 1-79, wherein the subject has undetectable blood C-peptide level when measured using a mixed meal tolerance test prior to administration of the composition.

81. The method of any one of claims 1-80, wherein the insulin deficiency is characterized by high blood sugar levels for a prolonged period of time.

82. The method of any one of claims 1-81, wherein the insulin deficiency is Type 1 diabetes.

83. The method of any one of claims 1-81, wherein the insulin deficiency is Type 2 diabetes.

84. The method of any one of claims 1-83, wherein the subject is human.329282749 113

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