Hypoimmunogenic β cells and method for producing same

Abstract

Disclosed herein are hypoimmunogenic stem cell-derived beta cells for use in a variety of applications, including cell therapy.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 311,037, filed February 16, 2022, and U.S. Provisional Patent Application No. 63 / 332,302, filed April 19, 2022, the teachings of which are incorporated herein by reference in their entireties. [Background technology]

[0002] One hundred years ago, the first diabetic patient with type 1 diabetes (T1D) was treated with “pancreatic extract” in Toronto as part of a series of experiments that would later lead to the discovery of insulin (Banting et al., 1922). Since then, the scientific basis of T1D has been explained as an autoimmune attack of insulin-producing β cells in the pancreas. However, for a long time, exogenous insulin administration was the only option to regulate blood glucose levels, despite technological advances such as insulin pumps and continuous glucose monitoring devices (Kovatchev, 2019). However, in the past decades, β cell replacement strategies have emerged as a new hope for patients, starting with cadaveric islet transplantation that demonstrated insulin independence (Shapiro et al., 2000), followed by human induced pluripotent cells (hPSCs) being investigated as an inexhaustible source of β cell differentiation and replacement.

[0003] Despite these advances, a major challenge that remains is to protect SC islets from autoimmune alloresponses and recurrent β-cell destruction. The use of immunosuppressive drugs can cause complications and may lead to graft failure in the long term (Lehmann et al., 2008). Encapsulation methods and cell capture devices are still under development (Alagpulinsa et al., 2019). Modulation of the immune system by regulatory T cells has been proposed (Raffin et al., 2020), and recently, significant efforts have been expended on genetically modifying hPSCs to create donor universal lines for cell replacement therapy that can be transplanted “naked” without the use of immunosuppressive drugs. However, these naked universal lines are still under development. Summary of the Invention [Means for solving the problem]

[0004] We performed a comprehensive analysis to define the forces that induce immune attack in endocrine tissues in various modeling systems. For the first time in human SC islets, we explored new genes that could serve as potential targets for gene editing to help cells escape allogeneic attack. The focus of this evaluation was to determine the key transcriptional events that occur in the inflammatory environment caused by introducing SC islets into an allogeneic immune system, and the ultimate strategies to reverse / prevent them with just one perturbation. By using single-cell RNA sequencing (scRNA-seq) and whole-genome CRISPR screening, we found that the JAK / STAT type II interferon (IFN) pathway is a key modulator of early and late inflammatory response events in vivo and in vitro. Although manipulation of upstream and central mediators of this pathway allows some reduction in the immunogenicity of SC islets, one unique approach is to target downstream elements by depleting C-X-C motif chemokine ligand 10 (CXCL10).

[0005] Disclosed herein are engineered beta cells, e.g., engineered stem cell-derived beta cells, which can contain one or more perturbations in the JAK / STAT type II interferon (IFN) pathway.

[0006] In some embodiments, the one or more perturbations occur in the IFNγ signaling mediator or downstream elements of the IFN pathway, e.g., downstream inflammatory elements, e.g., by a gene editing system such as CRISPR. In some embodiments, the one or more perturbations comprise reduced or eliminated expression of one or more downstream elements of the IFN pathway. In some embodiments, the one or more perturbations comprise reduced or eliminated expression of one or more genes listed in Table 1. In some embodiments, the one or more perturbations comprise reduced expression of one or more genes selected from the group consisting of CXCL10, STAT1, and TAP1.

[0007] Also disclosed herein are modified β cells, e.g., stem cell-derived β cells, that do not express CXCL10 (i.e., modified to lack expression of CXCL10).Also disclosed herein are modified β cells, e.g., stem cell-derived β cells, that do not express STAT1 (i.e., modified to lack expression of CXCL10).

[0008] In some embodiments, the engineered beta cells further exhibit increased expression or activation of one or more immune modulators. In some embodiments, the one or more immune modulators are selected from Table 2. In one embodiment, the one or more immune modulators are selected from the group consisting of PD-L1, CD47, SOCS1, and HLA-E.

[0009] In some embodiments, the engineered beta cells are hypoimmunogenic beta cells, e.g., hypoimmunogenic stem cell-derived beta cells. In some embodiments, the engineered beta cells exhibit one or more of protection against self and / or allogeneic rejection, increased cell survival under immune rejection, and / or reduced T cell activation and / or NK cell activation upon transplantation. In some embodiments, the beta cells are human or non-human cells, e.g., mouse cells.

[0010] Disclosed herein is a method for generating hypoimmunogenic beta cells, e.g., hypoimmunogenic stem cell-derived beta cells, comprising attenuating or silencing the IFN pathway in the beta cells.

[0011] In some embodiments, the method comprises reducing or eliminating expression of one or more genes selected from Table 1. In some embodiments, the method comprises reducing or eliminating expression of one or more genes selected from the group consisting of CXCL10, STAT1, and TAP1, e.g., eliminating expression of CXCL10 or STAT1.

[0012] Also disclosed herein is a method for generating hypoimmunogenic beta cells, e.g., hypoimmunogenic stem cell-derived beta cells, comprising perturbing one or more downstream elements of the IFN pathway in beta cells.

[0013] In some embodiments, perturbation comprises reducing or eliminating expression of one or more downstream elements of the IFN pathway, e.g., using a gene editing system such as CRISPR. In some embodiments, the one or more downstream elements are selected from Table 1. In one embodiment, the one or more downstream elements are selected from the group consisting of CXCL10, STAT1, and TAP1, and one embodiment comprises eliminating expression of CXCL10 or STAT1.

[0014] In some embodiments, hypoimmunogenic beta cells exhibit one or more of protection against auto- and / or allo-rejection, increased cell survival under immune rejection, and / or reduced T cell activation and / or NK cell activation upon transplantation.

[0015] In some embodiments, the method further comprises increasing or activating expression of one or more immune modulators. In some embodiments, the one or more immune modulators are selected from Table 2. In one embodiment, the one or more immune modulators are selected from the group consisting of PD-L1, CD47, SOCS1, and HLA-E.

[0016] In some embodiments, the hypoimmunogenic beta cells are human cells or non-human cells, such as mouse cells.

[0017] The patent or application file contains at least one color drawing. Copies of this patent or patent application publication containing color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0018] [Figure 1A] Single cell transcriptional profile of SC islet graft rejection in a humanized model. SC islets were transplanted under the kidney capsule of MHC null NSG mice. After 2-4 weeks, graft function was assessed by human insulin detection in mouse serum 30 min after glucose injection. Half of the mice were then injected with human PBMCs and human insulin was continuously monitored until graft engraftment failure was observed. Transplanted cells were then extracted and analyzed for gene expression by 10x scRNA-seq. [Figure 1B] Figure 1 shows single cell transcriptional profile of SC islet graft rejection in a humanized model. Representative images of transplanted kidneys after 10 weeks with or without PBMC injection are shown. Bar = 2 mm. [Figure 1C]Figure 1 shows single cell transcriptional profile of SC islet graft rejection in a humanized model.IVIS imaging of control and humanized mice transplanted with GAPDH-luciferase SC islets at 1 and 8 weeks after PBMC injection. [Figure 1D] Single cell transcriptional profile of SC islet graft rejection in a humanized model. Rejection of SC islet grafts over time as assessed by IVIS imaging quantification of radioluminescence following PBMC injection 30 min after glucose injection in overnight fasted mice (n=15 mice) or blood human insulin detection by ELISA (lower panel). [Figure 1E] Single cell transcriptional profile of SC islet graft rejection in a humanized model. Immunofluorescence staining of kidney SC islet graft sections from either humanized or control mice 6 weeks after PBMC injection. Each bar is 10 μm in the main panel and 3 μm in the enlarged panel. The kidney (K) and graft (G) margins are shown. CHGA = chromogranin A. [Figure 1F] Single cell transcriptional profile of SC islet graft rejection in a humanized model. Flow cytometry of SC-α (glucagon+C-peptide-) and SC-β (glucagon-C-peptide+) in grafts extracted 10 weeks after PBMC. HK) 10x scRNA-seq analysis. [Figure 1G] Figure 1 shows single cell transcriptional profile of SC islet graft rejection in a humanized model. Flow cytometry of human T lymphocyte infiltration in blood, spleen, and grafts of humanized mice 10 weeks after PBMC injection. Percentage of human CD45+ is gated from mouse CD45 negative population. Percentage of CD4+ and CD8+ is gated from hCD45+ / hCD3+. [Figure 1H]Figure 1 shows single cell transcriptional profile of SC islet graft rejection in a humanized model. UMAP plots of human transplanted cells extracted from mice after 10 weeks with or without PBMC injection. n=6 mice per group included. SC-endocrine cell clusters are indicated. [Figure 1I] Figure 1 shows single cell transcriptional profiles of SC islet graft rejection in a humanized model. Volcano plots of differentially expressed genes in SC-β and SC-α in rejected (+PBMC) grafts compared to non-rejected (no PBMC) grafts. [Figure 1J] Figure 1 shows single cell transcriptional profile of SC islet graft rejection in a humanized model. Violin plots of selected genes expressed in SC endocrine cells associated with IFNγ response are provided. [Figure 2A] Initial response of immune-challenged SC islets profiled by single-cell transcriptional analysis after co-culture with human allogeneic PBMCs. Enriched (CD49A magnetically sorted) human hESC-derived SC islets pre-treated with thapsigargin (5 μM for 5 h) and co-cultured with human allogeneic PBMCs (n=2 donors) for 0, 24, and 48 h were shown and subjected to 10x scRNA-seq. [Figure 2B] Figure 1 shows the initial response of immune-challenged SC islets profiled by single-cell transcriptional analysis after co-culture with human allogeneic PBMCs.Dot plots depicting the expression of activating / inhibitory genes in specific T and NK cell populations in response to 0, 24, and 48 h of stimulation with SC islets. [Figure 2C] Figure 1 shows the initial response of immune-challenged SC islets profiled by single-cell transcriptional analysis after co-culture with human allogeneic PBMCs. Volcano plot of differentially expressed genes in SC-α after 24 h co-culture with PBMCs compared to control (t=0, no co-culture). [Figure 2D]Figure 1 shows the initial response of immune-challenged SC islets profiled by single-cell transcriptional analysis after co-culture with human allogeneic PBMCs. Volcano plot of differentially expressed genes in SC-β after 24 h co-culture with PBMCs compared to control (t=0, no co-culture). [Figure 2E] Figure 1 shows the initial response of immune-challenged SC islets profiled by single-cell transcriptional analysis after co-culture with human allogeneic PBMCs. Top pathway and gene set enrichment analysis (GSEA) results of upregulated genes in co-cultured SC-β after 48 hours are shown. [Figure 2F] Figure 1 shows the initial response of immune-challenged SC islets profiled by single-cell transcriptional analysis after co-culture with human allogeneic PBMCs. Expression of selected inflammatory genes in SC-α and SC-β in response to 0, 24, and 48 h of co-culture with PBMCs. [Figure 2G] Figure 1 shows the initial response of immune-challenged SC islets profiled by single-cell transcriptional analysis after co-culture with human allogeneic PBMCs. Violin plots of SC-β timed expression of selected genes are shown. [Figure 2H] Figure 2 shows the initial response of immune-challenged SC islets profiled by single cell transcriptional analysis after co-culture with human allogeneic PBMCs. ELISA of human CXCL10 from supernatants of 24 / 48 hour co-cultures of SC islets and PBMCs. n=2 donors, Figures 2I-2J). [Figure 2I] Figure 1 shows the initial response of immune-challenged SC islets profiled by single-cell transcriptional analysis after co-culture with human allogeneic PBMCs. Immunofluorescence staining of CXCL10 in SC islet clusters 48 hours after treatment with 20 ng / ml rhIFNγ. C-peptide staining of SC-β. Each bar is 10 μm in the main panel and 2 μm in the enlarged panel. [Figure 2J]Figure 1 shows the initial response of immune-challenged SC islets profiled by single-cell transcriptional analysis after co-culture with human allogeneic PBMCs. Immunofluorescence staining of phosphorylated STAT1 in SC islet clusters 48 hours after treatment with 20 ng / ml rhIFNγ. C-peptide staining of SC-β. Each bar is 10 μm in the main panel and 2 μm in the enlarged panel. [Figure 3A] We demonstrate that genome-wide CRISPR screening identifies tolerizing perturbations in SC islet grafts under allogeneic rejection. We show that SC islets were transduced with a pooled lentiviral CRISPR library (Brunello) expressing Cas9 and 77,441 gRNAs in pLentiCRISPRv2. SC islets were then transplanted into a humanized mouse line as previously described (see Figure 2). Grafts were harvested at the time of failure / rejection and genomic DNA was extracted and sequenced. [Figure 3B] We demonstrate that genome-wide CRISPR screening identifies tolerizing perturbations in SC islet grafts under allo-rejection. SC islet graft rejection as measured by circulating human insulin and C-peptide detection by ELISA over time after PBMC injection 30 min after glucose injection in overnight fasted mice. n=6 per group. [Figure 3C] 14 demonstrates that genome-wide CRISPR screening identifies tolerizing perturbations in SC islet grafts under allo-rejection. Analysis of enriched and depleted gene knockouts by mean fold change in gRNA counts is shown. Ranking of gene knockouts is shown as gene vs. environment interaction, with the y-axis being the interaction between resistance and knockout on PBMCs and the x-axis being the KO effect across environments. [Figure 3D]We demonstrate that genome-wide CRISPR screening identifies tolerizing perturbations in SC islet grafts under allo-rejection. Violin plots showing normalized counts of individual gRNAs from mouse replicates (n=6 per condition) of IFN-related genes positively and negatively enriched in the screen are shown. Comparisons were made between each condition (±PBMC) and control gRNA (n=4 random control gRNA). Red lines in each violin represent median values. Dashed lines represent the mean log2norm counts of intergenic gRNAs. Error bars or shaded regions are mean ±SD. *p<0.05, **p<0.01, unpaired two-tailed t-test. [Figure 4A] Perturbation of individual components of the IFNγ pathway in the search for potential gene targets to reduce PBMC activation and enhance co-cultured SC-β survival is shown. Venn diagram featuring significantly upregulated genes (log2 fold change >1 and regulation p-value <0.05) from in vivo (blue) and in vitro (red) co-culture scRNA-seq data common to each hit of the CRISPR screen (positive enrichment in humanized mice, log2 fold change >1) (green). On the right are genes from the SC-α population and on the right are genes from the SC-β population. [Figure 4B] Perturbation of individual components of the IFNγ pathway in search of potential gene targets to reduce PBMC activation and increase survival of co-cultured SC-β. Human hESC-derived SD islets pre-transduced with lentivirus carrying Cas9+gRNA (KO in pLentiCRISPRv2) or specific ORF inserts (OE in pLX_317), pretreated with thapsigargin (5 μM for 5 h) and co-cultured with human allogeneic PBMCs are shown. [Figure 4C]Perturbation of individual components of the IFNγ pathway in searching for potential gene targets to reduce PBMC activation and increase survival of co-cultured SC-β. Flow cytometry analysis of the percentage (%) of TUNEL+ (apoptotic) SC islet cells and SC-β (C-peptide+) after 48 h of PBMC co-culture. Percent apoptosis (%) was calculated as a percentage from baseline (no PBMC addition). SC islets transduced with gRNA lentivirus were compared to non-targeting (NT) gRNA and SC islets transduced with ORF overexpression (OE) were compared to eGFP OE. n=2-3 PBMC donors. [Figure 4D] Perturbation of individual components of the IFNγ pathway in the search for potential gene targets to reduce PBMC activation and increase survival of co-cultured SC-β. Transduced SC islets were co-cultured with Cell Trace Violet (CTV)-labeled PBMC for 48 hours. PBMC were then isolated and expanded in culture for an additional 7 days before CD3 staining for flow cytometry. CD3+ were gated on the CTV-negative percentage of divided cells. PBMC treated with anti-CD3 / CD28 activation beads served as positive control. n=2–7 donors. [Figure 4E] Perturbation of individual components of the IFNγ pathway in the search for potential gene targets to reduce PBMC activation and increase survival of co-cultured SC-β is shown. Blocking antibody experiments in which PBMCs were pretreated with neutralizing anti-CXCR3, or SC islets were pretreated with anti-TLR4, or anti-CXCL10 was added to the co-culture medium are shown. [Figure 4F] Perturbation of individual components of the IFNγ pathway in searching for potential gene targets to reduce PBMC activation and increase viability of co-cultured SC-β. Flow cytometry analysis of the percentage of TUNEL+SC-β (C-peptide+) after 48 h of PBMC co-culture. n=2–6 donors. [Figure 4G]Perturbation of individual components of the IFNγ pathway in search of potential gene targets to reduce PBMC activation and increase survival of co-cultured SC-β. Percentage of expanded CD3 T cells measured by CTV-negative T cells after co-culture is shown. n=3 donors. Error bars are mean ± SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 unpaired two-tailed t-test. [Figure 5A] 11 shows the generation and performance of CXCL10 KO and STAT1 KO hESC lines. A schematic diagram of targeting of the CXCL10 locus by nucleofection of hESCs with a targeting vector containing Cas9 / sgRNA ribonucleoprotein complex (RNP) and site-specific homology arms (HA) as indicated. Red and blue arrows indicate the location of PCR primers used for genotyping of clones as shown in FIG. 11. [Figure 5B] 11 shows the generation and performance of CXCL10 KO and STAT1 KO hESC lines. Schematic diagram of targeting of the STAT1 locus by nucleofection of hESCs with Cas9 / sgRNA ribonucleoprotein complexes (RNPs) and site-specific homology arm (HA) containing targeting vectors as indicated. Red and blue arrows indicate the position of PCR primers used for genotyping of clones as shown in FIG. 11. [Figure 5C] Generation and performance of CXCL10 KO and STAT1 KO hESC lines. Percentage of SC-β in multiple batches of C10G and ST1L differentiations compared to control wild type (WT) and GAPDH-luc (GL) lines. n=1-3 differentiations. [Figure 5D] Generation and performance of CXCL10 KO and STAT1 KO hESC lines are shown. SC islet GSIS functional assay of the different lines after 12-15 weeks of transplantation into NSG mice is shown. Results are shown as stimulation ratios (ELISA) of human insulin in blood before and after glucose injection (2 g / kg). [Figure 5E]Generation and performance of CXCL10 KO and STAT1 KO hESC lines. Flow cytometry analysis of intracellular CXCL10 protein in WT / C10G SC ​​islets and SC-β (C-peptide+) with or without 48 h treatment with rhIFNγ and 5 h monensin treatment to prevent protein secretion. n=1-3. [Figure 5F] Generation and performance of CXCL10 KO and STAT1 KO hESC lines. CXCL10 ELISA assay on supernatants from WT / C10G / ST1L SC islets with or without treatment with rhIFNγ. Dashed line indicates lower limit of detection. n=2-3 differentiations. [Figure 5G] Generation and performance of CXCL10 KO and STAT1 KO hESC lines. Flow cytometry analysis of protein expression in rhIFNγ GL / ST1L SC islets as indicated. n=3-4. [Figure 5H] Generation and performance of CXCL10 KO and STAT1 KO hESC lines. Genetically modified (GM;C10G / ST1L) and control (WT / GL) lines were differentiated into SC islets, pretreated with thapsigargin (5 μM for 5 h) and co-cultured with human PBMCs or purified T cells / NKs. [Figure 5I] Generation and performance of CXCL10 KO and STAT1 KO hESC lines. Results are shown as assessed by flow cytometry using the percentage of TUNEL+C-peptide+WT or C10G SC-β cells (n=3-5 donors). [Figure 5J] Generation and performance of CXCL10 KO and STAT1 KO hESC lines. Results are shown of the percentage of TUNEL+C-peptide+GL or ST1L SC-β cells assessed using flow cytometry (n=2-3 donors). [Figure 5K]Generation and performance of CXCL10 KO and STAT1 KO hESC lines using flow cytometry to assess the percentage of proliferating CD3 T cells as measured by CTV-negative T cells after the indicated co-cultures (n=2-4 donors). [Figure 6A] We show that CXCL10 KO SC islet grafts avoid allo-rejection in humanized mice. We show that WT or C10G SC ​​islets were transplanted under the kidney capsule of MHC null NSG mice (n=10 from each strain). After 4 weeks, graft function was assessed by human insulin detection in mouse serum 30 min after glucose injection. Then, 6-7 mice from each group were injected with human PBMCs (n=2 human donors), and the rest served as non-humanized controls. Human insulin was continuously monitored until graft engraftment failure was observed. [Figure 6B] Figure 1 shows that CXCL10 KO SC islet grafts avoid allo-rejection in humanized mice. Rejection of SC islet grafts measured over time by detection of human insulin in the blood by ELISA after PBMC injection 30 min after glucose injection in overnight fasted mice. Error bars are mean ± SD. *p<0.05, **p<0.01, unpaired two-tailed t-test. [Figure 7A] Single cell transcriptional profile of SC islet graft rejection in a humanized model. Immunofluorescence staining of kidney SC islet graft sections from either humanized or control mice 6 weeks after PBMC injection. Each bar is 10 μm in the main panel and 3 μm in the enlarged panel. The kidney (K) and graft (G) margins are shown. CHGA = chromogranin A. [Figure 7B] Single-cell transcriptional profiles of SC islet graft rejection in a humanized model. Cell counts of cell populations obtained from endocrine scRNAseq analysis in Figures 1H-1I are shown. [Figure 7C] Figure 1 shows single cell transcriptional profiles of SC islet graft rejection in a humanized model. Cell markers for endocrine cells are shown. [Figure 7D]Figure 1 shows single cell transcriptional profiles of SC islet graft rejection in a humanized model, showing differential expression of selected genes in various cell populations (SC-β, SC-α, and SC-EC cells). [Figure 7E] Figure 1 shows single cell transcriptional profiles of SC islet graft rejection in a humanized model. Pathway analysis of upregulated genes in different cell populations (SC-β, SC-α, and SC-EC cells). [Figure 7F] Figure 1 shows single cell transcriptional profile of SC islet graft rejection in a humanized model.Violin plot of CXCL10 expressed in SC endocrine cells. [Figure 8A] Figure 2 shows the initial response of immune-challenged SC islets profiled by single-cell transcriptional analysis after co-culture with human allogeneic PBMCs. UMAP of in vitro PBMC and SC islet co-cultures over time (see Figure 2). [Figure 8B] Figure 2 shows the initial response of immune-challenged SC islets profiled by single-cell transcriptional analysis after co-culture with human allogeneic PBMCs. UMAPs of in vitro PBMC and SC islet co-cultures are shown by cell type (see Figure 2). [Figure 8C] Figure 1 shows the initial response of immune-challenged SC islets profiled by single-cell transcriptional analysis after co-culture with human allogeneic PBMCs.Dot blots representing marker genes used to define cell types in the analysis are shown. [Figure 8D] Figure 1 shows the initial response of immune-challenged SC islets profiled by single-cell transcriptional analysis after co-culture with human allogeneic PBMCs. Pathway analysis of upregulated genes in different cell populations is shown. [Figure 8E] Figure 1 shows the initial response of immune-challenged SC islets profiled by single-cell transcriptional analysis after co-culture with human allogeneic PBMCs.Figure 2 shows GSEA analysis of differentially expressed genes in SC β-cells. [Figure 8F]Figure 1 shows the initial response of immune-challenged SC islets profiled by single-cell transcriptional analysis after co-culture with human allogeneic PBMCs. Expression of inflammatory genes in different cell populations in response to 0, 24, and 48 h of co-culture with PBMCs. [Figure 9A] We demonstrate that genome-wide CRISPR screening identifies tolerizing perturbations in SC islet grafts under allogeneic rejection. We show the gating strategy used for flow cytometry of humanized mouse blood to detect human T cells. [Figure 9B] We demonstrate that genome-wide CRISPR screening identifies tolerizing perturbations in SC islet grafts under allogeneic rejection. Detection of human T cells in humanized mouse blood over the course of the experiment described in Figure 3 is shown. [Figure 9C] We demonstrate that genome-wide CRISPR screening identifies tolerizing perturbations in SC islet grafts under allogeneic rejection. Human insulin and C-peptide detected by ELISA at the end of the experiment in non-fasted mice are shown (see Figure 3B). [Figure 10A] Perturbation of individual components of the IFNγ pathway in the search for potential gene targets that reduce PBMC activation and increase the viability of co-cultured SC-β cells. Expression of target proteins after lentiviral transduction with non-targeting (NT) or targeted (as indicated) gRNAs is shown. [Figure 10B] Perturbation of individual components of the IFNγ pathway in the search for potential gene targets to reduce PBMC activation and increase the viability of co-cultured SC-β is shown. T cell activation marker expression (CD25) after 48 h of co-culture with pre-transduced SC islets is shown. [Figure 10C] Perturbation of individual components of the IFNγ pathway in the search for potential gene targets to reduce PBMC activation and increase the viability of co-cultured SC-β is shown. T cell activation marker expression (CD69) after 48 h of co-culture with pre-transduced SC islets is shown. [Figure 10D]Perturbation of individual components of the IFNγ pathway in the search for potential gene targets to reduce PBMC activation and increase the viability of co-cultured SC-β. Detection of CXCL10 in cell supernatants after 48 h of co-culture. [Figure 10E] Perturbation of individual components of the IFNγ pathway in the search for potential gene targets to reduce PBMC activation and increase the viability of co-cultured SC-β. Expression of T cell activation markers (CD25 or CD69) after 48 hours of co-culture with blocking antibodies is shown. [Figure 11A] Generation and performance of CXCL10 KO and STAT1 KO hESC lines. Clonal genotyping of endogenous or targeted alleles is shown. Endogenous amplified PCR bands, indicated by dashed blue boxes, were isolated and sequenced for detection of indels. [Figure 11B] Generation and performance of CXCL10 KO and STAT1 KO hESC lines. Karyotype analysis of G10G and ST1L. [Figure 11C] 1 shows the generation and performance of CXCL10 KO and STAT1 KO hESC lines. Pluripotency marker expression in all four lines: WT, C10G, GL, and ST1L. [Figure 11D] Generation and performance of CXCL10 KO and STAT1 KO hESC lines. Flow cytometry analysis to assess % SC-β (% C-peptide+ / NKX6.1+ or % C-peptide+ / glucagon-) in C10G and ST1L hESCs at stage 6 of the Melton β cell differentiation protocol. [Figure 11E] Generation and performance of CXCL10 KO and STAT1 KO hESC lines. Relative light units (RLU) detected by a luminescence plate reader of ST1L hESCs at different stages of β-cell differentiation (S0 = before ES stage) with increasing numbers of cells per well. [Figure 11F]Generation and performance of CXCL10 KO and STAT1 KO hESC lines. Relative light units (RLU) detected by a luminescence plate reader of ST1L hESCs at different stages of β-cell differentiation (S0 = before ES stage) with increasing numbers of cells per well. [Figure 11G] Generation and performance of CXCL10 KO and STAT1 KO hESC lines using flow cytometry to assess the percentage of TUNEL+C-peptide+WT or C10G SC ​​islets (n=3-5 donors) and the percentage of GL or ST1L SC islets (n=2-3 donors). [Figure 11H] Generation and performance of CXCL10 KO and STAT1 KO hESC lines. T cell activation marker expression (CD25 or CD69) after 48 hours of co-culture. [Figure 11I] Generation and performance of CXCL10 KO and STAT1 KO hESC lines. T cell activation marker expression (CD25 or CD69) after 48 hours of co-culture. [Figure 11J] Generation and performance of CXCL10 KO and STAT1 KO hESC lines. Expression of NK cell activation marker (CD107a) after 48 hours of co-culture. [Figure 12] As shown in FIG. 6B, the absolute values ​​of insulin detected by ELISA are shown. [Figure 13] FIG. 1 shows a flow chart of the differentiation stages for obtaining stem cell-derived β cells. [Figure 14] Single cell profiling of allogeneic immune responses in vitro. The diagram illustrates the in vitro co-culture of ESC-derived SC islets and the process for performing scRNA-seq to reveal potential targetable genes (IFN pathway "alarm" genes) that enhance cell survival under immune rejection. UMAP projections are shown for various cell types and over different time periods. [Figure 15]Alarm gene activation plots for +PBMC-2 and +PBMC-1 at t=0, 24, and 48 hours with different agents are shown, demonstrating that interferon pathway alarm genes are upregulated in immune-challenged allogeneic SC-β. [Figure 16-1] Figure 1 illustrates the process of single cell profiling of in vitro autoimmune responses from type I diabetes patients. UMAP projections are shown for different cell types and comparing co-cultured iPSC-β cells with control iPSC-β cells. [Figure 16-2] Same as above. [Figure 17] We show that immune-challenged autologous iPSC-β cells upregulate alarm genes in the interferon pathway. In vitro co-culture of T1D-derived iPSC-islets and scRNA-seq reveal potential targetable genes (IFN pathway "alarm" genes) to enhance cell survival under immune rejection. UMAP projections compare "alarmed" iPSC-β to iPSC-β cells, which vary based on target genes. [Figure 18] Figure 1 shows the identification of potential targets affecting the immunogenicity of SC islets, including CXCL9, CXCL10, CXCL11 depletion as downstream effectors of JAK / STAT; IFNGR1, STAT1, IRF1, JAK1 / 2 depletion as upstream regulators of JAK / STAT; SOCS1 expression; and TNNFSF10 and TNFRSF12A as part of the TNF apoptosis pathway. Depletion of B2M and expression of PD-L1 / CD274 are known to regulate perturbation and affect SC islets. [Figure 19] A target list of genes that can be depleted or expressed to generate hypoimmunogenic SC islets is shown, with each target identified using scRNA sequencing of SC islet co-cultures with allogeneic PBMCs. [Figure 20]A diagram of the in vivo genome-wide pooled CRSPR screening model is shown. Genome-wide CRISPR survival screening in humanized mice revealed potential targetable genes (IFN pathway "alarm" genes) that enhance cell survival under immune rejection. [Figure 21-1] FIG. 1 shows post-glucose mouse SC islet allograft survival screening comparing SC islet Tx with and without PMBC measuring human insulin and human C-peptide over time as well as the percentage (%) of human CD45 and CD4 and CD8 expression over time after SC islet Tx. [Figure 21-2] Same as above. [Figure 22] Results of SC islet allograft survival screening with gRNA enrichment. Mean gRNA number in mice without PBMC (control) vs with PBMC based on agent used. Genome-wide CRISPR survival screening in humanized mice reveals potential targetable genes to enhance cell survival under immune rejection, including IFN pathway "alarm" genes. [Figure 23] Figure 1 shows a targeted list of genes that can be depleted or expressed to generate hypoimmunogenic SC islets. Targets are identified using CRISPR survival screening in H. mice. [Figure 24-1] Figure 1 shows that CXCL10 and STAT1 inhibition reduces the immunogenicity of SC islets. CXCL10 and STAT1 inhibition using CRISPR KO shows reduced activation / proliferation of T cells co-cultured with modified SC islets and improved viability of modified SC islets. Each plot shows inhibition of CXCL10 and STAT1 using CRISPR KO by measuring the percentage of CD3+ T cells expanded and the percentage of viable SC-β. Co-culture diagram shows lentiviral transduction with gRNA and CRISPR / Cas9 with SC islets and allogeneic PMBCs, and measurements of CXCL10 and STAT1 on IFNγ expression when non-targeting gRNA is compared to CXCL10 / STAT1 gRNA. [Figure 24-2] Same as above. [Diagram 25] Expression of immune regulators in SC islets is shown. Overexpression of SOCS1 has been shown to increase viability and reduce chemokine secretion (CXCL10) of engineered SC islets, an effect comparable to that of overexpression of PD-L1. Plots shown show the percentage of apoptotic SC-β cells for overexpression of eGFP, PD-L1, and SOCS1, as well as empty vector and overexpression of SOCS1. [Figure 26] The design of CXCL10 (left) and STAT1 (right) knockout hESC lines is shown. [Figure 27] Figure 1 shows improved viability of CXCL10 KO SC islets by co-cultured immune cells. Percentage of apoptotic cells in PBMCs, T cells, and NK cells from WT and C10G2 co-cultures are shown. [Figure 28] CXCL10 expression and secretion are shown for WT, CXCL10 KO, and STAT1 KO SC islets and SC islets+IFNγ. [Figure 29] The effect of the STAT1 KO luciferase line (ST1L) is shown. Expression of STAT1 and HLA is shown for Hues8 cells + IFNγ. Luciferase activity in vitro and in vivo is also shown. [Diagram 30] 1 shows an analysis of a CXCL10 knockout cell line (C10G). Fluorescence plots of GM SC islets are shown for the CXCL10 knockout line (C10G) along with graphs showing % CXCL10.0 expression of SC islets and SC islets + IFNγ for wild type Hues8 and CXCL10 knockout Hues8. [Diagram 31] Analysis of STAT1 knockout luciferase cell line (ST1L) is shown. C-peptide / NKX6.1 plots of STL15.1 and STL15.2 are shown. Graphs showing the percentage of cells expressing wild type and ST1L15 for STAT1, phos-STAT1, and HLA-ABC cells are also shown. Additionally, luciferase activity of ST1L cell line is shown in vitro and in vivo. [Diagram 32]Immune tacked iPSC-β cells upregulate alarm genes. A shows a UMAP projection comparing alarmed iPSC-β to iPSC-β cells. Activation was indicated by increased B2M, HLA-A, and TAP1, while inhibition was indicated by increased CD274. B shows the log p-values ​​of different enriched pathways in activated endocrine cells. [Figure 33A] Figure 2 shows that T cell activation was suppressed by genetically modified iPSC-β cells. Figure 3 shows the effect of β2M depletion on T cell activation. [Figure 33B] Figure 2 shows that T cell activation was suppressed by genetically modified iPSC-β cells. Figure 3 shows the effect of PD-L1 overexpression on T cell activation. [Diagram 34] Figure 2 shows that immune-challenged iPSC-β cells express and secrete CXCL10. UMAP projections show the expression of CXCL10 in alerted iPSC-β cells and iPSC β cells. [Figure 35A] Figure 1 shows that CXCL10 inhibition reduces the immunogenicity of SC islets. Figure 2 shows that inhibition of CXCL10 increases the percentage of live SC-β cells and decreases the percentage of apoptotic SC-β cells. [Figure 35B] Figure 1 shows that CXCL10 inhibition reduces the immunogenicity of SC islets. Figure 2 shows the generation of co-cultures combining transduction with allogeneic PBMC to form SC islets and the percentage of CD3+ T cells expanded using non-targeting gRNA and CDCL10 gRNA. [Figure 35C] Figure 1 shows that CXCL10 inhibition reduces the immunogenicity of SC islets.Plots comparing intracellular CXCL10 using NT gRNA vs. CXCL10 gRNA are shown. [Diagram 36] Single cell profiling of T cells displaying activation markers / immune checkpoint molecules, apoptosis induction, and nuclear factors from CD4 and CD8 PBMC and SC co-cultures and enrichment pathway comparison of co-cultured versus unstimulated CD4 and CD8 T cells. [Figure 37] UMAP projections are shown for various cell types and different time periods. [Figure 38] 1 shows plots of treated versus control expression for various alarm genes over 24 and 48 hours, showing that co-cultured SC-β cells upregulate alarm genes (IFN response). [Figure 39A] Figure 1 shows alarm gene expression over time in co-cultures. Figure 1 shows CXCL10 secretion in co-cultures with SC islets + PBMCs for 0, 24 and 48 hours. [Figure 39B] Figure 2 shows the time course expression of alarm genes in co-cultures. UMAP projections over different time periods are shown. [Figure 39C] Figure 1 shows the time course of alarm gene expression in co-culture. Violin plots of the time course of expression of CXCL10, B2M, and STAT1 in SC-β cells. [Figure 40A] Figure 1 shows that SC-β cells secrete CXCL10 and stimulate immune cells via CXCR3.Figure 2 shows a diagram of SC islets secreting CXCL10, CXCL11, and CXCL9 when targeted by T cells. [Figure 40B] Figure 1 shows that SC-β cells secrete CXCL10 and stimulate immune cells via CXCR3.Figure 2 shows the co-culture of modified SC islets and allogeneic PBMCs with anti-CXCR3 Ab. [Figure 40C] Figure 1 shows that SC-β cells secrete CXCL10 and stimulate immune cells via CXCR3. Figure 1 shows plots measuring the percentage of proliferated CD3+ T cells, CD3+CD69+ T cells, and the percentage of apoptotic SC-β cells. [Diagram 41] FIG. 1 shows the effect of CXCL10 KO cell lines on T cell activation, NK cell activation, and SC islet viability. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Human embryonic stem cells (hESCs) offer new possibilities in cell replacement therapy for T1D. Therapeutic amounts of human stem cell-derived islet cells (SC islets) can be obtained in vitro following a stepwise differentiation protocol. However, preventing allorejection of transplanted cells and recurrent autoimmunity without lifelong immunosuppressive drugs remains a major challenge. One option being actively pursued is physical encapsulation to prevent direct immune cell-cell contact. Another option being pursued here is transplanting “naked” SC islet cells genetically modified to evade the immune system. To determine the underlying factors inducing immunogenicity of SC islets in an inflammatory environment, we performed single-cell RNA sequencing (scRNA-seq) of SC islets in co-culture with allogeneic peripheral blood mononuclear cells (PBMCs) or under immune challenge with PBMCs in humanized mouse transplants. Data analysis identifies an "alarmed" population of SC islet cells and upregulated genes in the interferon (IFN) pathway that contribute to inflammatory stimulation of co-cultured / infiltrating T cells. In vivo genome-wide CRISPR screening confirms that targeting IFNγ signaling mediators and downstream inflammatory components has a favorable effect on SC islet cell survival under immune attack. Manipulating the IFN response by knocking out CXCL10 in SC islet grafts improves survival against allo-rejection in humanized mice compared to wild-type grafts. Overall, these results provide insight into the nature of immune destruction of SC islets in the allo-response and provide potential targets for gene editing.

[0020] definition For convenience, certain terms used in the specification, examples, and appended claims are collected here. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0021] The term "differentiated cell" refers to any primary cell that is not pluripotent in its native form, as defined herein. In other words, the term "differentiated cell" refers to a more specialized cell type derived from a less specialized cell type (e.g., stem cell, such as induced pluripotent stem cell) during the cell differentiation process. Without wishing to be limited by theory, pluripotent stem cells can differentiate during normal development first into endodermal cells that can form pancreatic cells and other endodermal cell types. Further differentiation of endodermal cells leads to the pancreatic pathway, where about 98% of cells become exocrine, ductal, or matrix cells, and about 2% become endocrine cells.

[0022] As used herein, the term "somatic cell" refers to any cell that forms the body of an organism, as opposed to a germ cell. In mammals, germ cells (also called "gametes") are sperm and eggs that fuse during fertilization to produce a cell called a zygote, from which the entire mammalian embryo develops. Apart from sperm and eggs, the cells from which they arise (gamete cells), and undifferentiated stem cells, all other cell types in a mammal's body are somatic cells: internal organs, skin, bones, blood, and connective tissues are all made up of somatic cells. In some embodiments, the somatic cell is a "non-embryonic somatic cell," which refers to a somatic cell that is not present in or obtained from an embryo, and not by propagation of such a cell in vitro. In some embodiments, the somatic cell is an "adult somatic cell," which refers to a cell that is present in or obtained from an organism other than an embryo or fetus, or by propagation of such a cell in vitro. Unless otherwise indicated, the methods described herein can be performed in vivo or in vitro.

[0023] As used herein, the term "adult cell" refers to cells found throughout the body after embryonic development.

[0024] As used herein, the term "endodermal cells" refers to cells derived from one of the three major germ cell layers in the very early embryo (the other two are mesoderm and ectoderm). The endoderm is the innermost of the three layers. Endoderm cells differentiate to give rise first to the embryonic gut, then to the lining of the respiratory and digestive tracts (such as the intestine), the liver, and the pancreas.

[0025] As used herein, the term "endodermal derived cells" refers to any cell that develops or differentiates from an endoderm cell. For example, endoderm derived cells include liver, lung, pancreas, thymus, intestine, stomach and thyroid cells. Without wishing to be limited by theory, liver and pancreatic progenitor cells (also called pancreatic progenitor cells) develop from endoderm cells in the embryonic foregut. Shortly after their differentiation, liver and pancreatic progenitor cells rapidly acquire strikingly different cellular functions and regenerative capabilities. These changes are driven by inductive signals and genetic regulators that are highly conserved among vertebrates.

[0026] The term "pancreatic progenitor cell" or "pancreatic precursor" is used interchangeably herein and refers to a stem cell that can form either pancreatic endocrine cell, pancreatic exocrine cell, or pancreatic duct cell. As used herein, the term "Pdx1 positive pancreatic progenitor cell" or "Pdx1+ pancreatic progenitor cell" refers to a cell that is a pancreatic endoderm (PE) cell. Pdx1 positive pancreatic progenitor cell expresses the marker Pdx1. Other markers include, but are not limited to, Cdcp1, or Ptf1a, or HNF6, or NRx2.2. The expression of Pdx1 may be evaluated by any method known to those skilled in the art, such as immunochemistry or quantitative RT-PCR using anti-Pdx1 antibody. As used herein, the term "Pdx1 positive, NKX6-1 positive pancreatic progenitor cell" or "Pdx1+, NKX6-1+ pancreatic progenitor cell" refers to a cell that is a pancreatic endoderm (PE) cell. Pdx1 positive, NKX6-1 positive pancreatic progenitor cells express markers Pdx1 and NKX6-1.Other markers include, but are not limited to, Cdcp1, or Ptf1a, or HNF6, or NRx2.2.NKX6-1 expression may be evaluated by methods known to those skilled in the art, such as immunochemistry or quantitative RT-PCR using anti-NKX6-1 antibody.

[0027] The terms "stem cell-derived beta cells", "SC-beta cells", and "mature SC-beta cells" refer to cells (e.g., pancreatic beta cells) that exhibit at least one marker indicative of a pancreatic beta cell, express insulin, and exhibit a GSIS response characteristic of endogenous mature beta cells. In some embodiments, "SC-beta cells" include mature pancreatic beta cells. It will be understood that the methods of the present disclosure can use any cell as a starting point to derive SC-beta cells from any insulin-positive endocrine cell or its precursor, and thus the SC-beta cells need not be derived (e.g., directly) from stem cells (e.g., embryonic stem cells, induced pluripotent stem cells, progenitor cells, partially reprogrammed somatic cells (e.g., somatic cells partially reprogrammed to an intermediate state between induced pluripotent stem cells and the somatic cells from which they are derived), pluripotent cells, totipotent cells, retrodifferentiated versions of any of the aforementioned cells, etc. can be used, as the invention is not intended to be so limited). Furthermore, it should be understood that the SC-β cells of the present invention are non-native, i.e., non-naturally occurring, non-endogenous cells, and have at least one characteristic that distinguishes them from natural / naturally occurring / endogenous cells. Examples of SC-β cells, and methods for obtaining such SC-β cells, are described in WO2015 / 002724 and WO2014 / 201167, both of which are incorporated herein by reference in their entirety. In some embodiments, the "SC-β cells" are hypoimmunogenic stem cell-derived β cells, e.g., that generate limited or no immune response.

[0028] The terms "stem cell-derived α-cells," "SC-α-cells," and "mature SC-α-cells" refer to cells (e.g., pancreatic α-cells) that exhibit at least one marker indicative of pancreatic α-cells, express and secrete glucagon, and exhibit an ultrastructure similar to cadaveric α-cells. In some embodiments, "SC-α-cells" include mature pancreatic α-cells. It will be understood that the methods of the present disclosure can use any cell as a starting point to derive SC-α-cells from any insulin-positive endocrine cell or precursor thereof, and thus the SC-α-cells need not be derived (e.g., directly) from stem cells (e.g., embryonic stem cells, induced pluripotent stem cells, progenitor cells, partially reprogrammed somatic cells (e.g., somatic cells partially reprogrammed to an intermediate state between induced pluripotent stem cells and the somatic cells from which they are derived), pluripotent cells, totipotent cells, retrodifferentiated versions of any of the foregoing cells, and the like can be used, as the invention is not intended to be so limited). Furthermore, it should be understood that the SC-α cells of the present invention are non-native, i.e., non-naturally occurring, non-endogenous cells, and have at least one characteristic that distinguishes them from natural / naturally occurring / endogenous cells. Examples of SC-α cells, and methods for obtaining such SC-α cells, are described in WO2019 / 217487, which is incorporated herein by reference in its entirety.

[0029] The terms "stem cell-derived enterochromaffin cells", "SC-EC cells", and "mature SC-EC cells" refer to cells (e.g., enterochromaffin cells) that exhibit at least one marker indicative of enterochromaffin cells, express SLC18A1, and are capable of producing and releasing serotonin (5-HT). In some embodiments, "SC-EC cells" include mature enterochromaffin cells. It will be understood that the methods of the present disclosure can use any cell as a starting point to derive SC-EC cells from any progenitor cell, and thus the SC-EC cells need not be derived (e.g., directly) from stem cells (e.g., embryonic stem cells, induced pluripotent stem cells, progenitor cells, partially reprogrammed somatic cells (e.g., somatic cells partially reprogrammed to an intermediate state between induced pluripotent stem cells and the somatic cells from which they are derived), pluripotent cells, totipotent cells, retrodifferentiated versions of any of the aforementioned cells, and the like can be used, as the invention is not intended to be so limited). Furthermore, it should be understood that the SC-EC cells of the present invention are non-native, i.e., non-naturally occurring, non-endogenous cells, and have at least one characteristic that differs from natural / naturally occurring / endogenous cells. Examples of SC-EC cells, and methods for obtaining such SC-EC cells, are described in WO2019 / 217493, which is incorporated herein by reference in its entirety.

[0030] As used herein, the term "exocrine cells" refers to cells of exocrine glands, i.e., glands that excrete secretions through ducts. In certain embodiments, exocrine cells refer to exocrine pancreatic cells, which are pancreatic cells that produce enzymes that are secreted into the small intestine. These enzymes aid in the digestion of food as it passes through the digestive tract. Exocrine pancreatic cells, also called the islets of Langerhans, secrete two hormones: insulin and glucagon.

[0031] As used herein, the term "pluripotent" refers to cells that have the ability to differentiate under different conditions into one or more differentiated cell types, preferably into cell types characteristic of all three germ cell layers. Pluripotent cells are primarily characterized by their ability to differentiate into one or more cell types, preferably into all three germ cell layers, using, for example, nude mouse teratoma formation assays. Pluripotency can also be demonstrated by expression of embryonic stem (ES) cell markers, but the preferred test of pluripotency is the demonstration of the ability to differentiate into cells of each of the three germ layers. It should be noted that merely culturing such cells does not, in and of itself, make them pluripotent. Reprogrammed pluripotent cells (e.g., iPS cells as the term is defined herein) are also characterized by the ability to be passaged for extended periods without loss of growth potential, compared to their primary parent cells, which generally only undergo a limited number of divisions in culture.

[0032] As used herein, the terms "iPS cells" and "induced pluripotent stem cells" are used interchangeably and refer to pluripotent stem cells that have been artificially derived (e.g., by induction or complete reversal) from non-pluripotent cells, typically adult somatic cells, for example, by inducing forced expression of one or more genes.

[0033] The terms "progenitor cell" or "precursor" cell are used interchangeably herein and refer to a cell that has a more primitive cellular phenotype (i.e., earlier in a developmental pathway or progression than a fully differentiated cell) compared to the cell it may give rise to by differentiation. In many cases, progenitor cells also have significant, or even very high, proliferative potential. Progenitor cells can give rise to multiple different differentiated cell types, or a single differentiated cell type, depending on the developmental pathway and environment in which the cell develops and differentiates.

[0034] As used herein, the term "stem cell" refers to an undifferentiated cell that is capable of proliferation and of giving rise to more progenitor cells, which in turn have the capacity to give rise to a number of mother cells, which in turn can give rise to differentiated or differentiable daughter cells. The daughter cells themselves can be induced to proliferate and give rise to progeny that subsequently differentiate into one or more mature cells, while at the same time retaining one or more cells of the parent's developmental potential. The term "stem cell" refers to a subset of progenitor cells that, under certain conditions, have the capacity or potential to differentiate into a more specialized or differentiated phenotype, and also retain the capacity to proliferate without substantial differentiation, under certain conditions. In one embodiment, the term stem cell refers to a naturally occurring mother cell whose progeny (progeny cells) often specialize in different directions by differentiation, e.g., by acquiring entirely separate characteristics, as they occur in the progressive diversification of embryonic cells and tissues. Cell differentiation is a complex process, and typically occurs through many cell divisions. Differentiated cells may be derived from pluripotent cells, which themselves are derived from pluripotent cells, and so on. Although each of these pluripotent cells may be considered stem cells, the range of cell types each can give rise to may vary considerably. Some differentiated cells also have the ability to give rise to more developmentally potent cells. This ability may be natural or may be induced artificially by treatment with various factors. In many biological examples, stem cells are "pluripotent" because they can give rise to more than one different type of cell progeny, but this does not require "stemness". Self-renewal is another classical part of the definition of a stem cell, and is mandatory as used in this document. In theory, self-renewal can occur primarily by one of two main mechanisms. Stem cells may divide asymmetrically, with one daughter maintaining the stem cell state and the other daughter expressing some other specific function and phenotype. Alternatively, some of the stem cells in a population can divide symmetrically into two stem cells, thus maintaining some stem cells in the population as a whole, while other cells in the population give rise only to differentiated progeny.Formally, cells that begin as stem cells progress toward a differentiated phenotype, but may then "reverse" and re-express the stem cell phenotype, a term often referred to by those skilled in the art as "dedifferentiation" or "reprogramming," or "redifferentiation." As used herein, the term "pluripotent stem cells" includes embryonic stem cells, induced pluripotent stem cells, placental stem cells, and the like.

[0035] The term "embryonic stem cells" is used to refer to pluripotent stem cells of the inner cell mass of blastocysts (see U.S. Patent Nos. 5,843,780 and 6,200,806). Such cells can also be obtained from the inner cell mass of blastocysts derived from somatic cell nuclear transfer (see, e.g., U.S. Patent Nos. 5,945,577, 5,994,619, and 6,235,970). The distinguishing characteristics of embryonic stem cells define the phenotype of the embryonic stem cells. Thus, a cell has an embryonic stem cell phenotype if it possesses one or more of the distinctive characteristics of embryonic stem cells that distinguish the cell from other cells. Exemplary distinguishing characteristics of embryonic stem cells include, but are not limited to, gene expression profile, proliferation potential, differentiation potential, karyotype, responsiveness to specific culture conditions, and the like.

[0036] The term "adult stem cell" or "ASC" refers to pluripotent stem cells derived from non-embryonic tissues, including fetal, juvenile and adult tissues. Stem cells have been isolated from a variety of adult tissues, including blood, bone marrow, brain, olfactory epithelium, skin, pancreas, skeletal and muscle. Each of these stem cells has been characterized based on gene expression, factor responsiveness, and morphology in culture. Exemplary adult stem cells include neural stem cells, neural crest stem cells, mesenchymal stem cells, hematopoietic stem cells and pancreatic stem cells. As discussed above, stem cells have been found to be present in virtually every tissue. Thus, the present invention recognizes that stem cell populations can be isolated from virtually every animal tissue.

[0037] As used herein, the term "reprogramming" refers to the process of changing or reversing the differentiation state of a somatic cell. Prior to reprogramming, the cell may be partially or terminally differentiated. Reprogramming includes the complete reversion of the differentiation state of a somatic cell to a pluripotent cell. Such a complete reversal of differentiation produces an induced pluripotent (iPS) cell. As used herein, reprogramming also includes the partial reversal of the differentiation state of a cell, e.g., to a pluripotent state, or to a somatic cell that is neither pluripotent nor multipotent, but has lost one or more specific characteristics of the differentiated cell from which it was derived, e.g., characteristics that directly reprogram a differentiated cell to a different somatic cell type. Reprogramming generally involves changing, e.g., reversing, at least a portion of the genetic patterns, such as nucleic acid modifications (e.g., methylation), chromatin condensation, epigenetic changes, genomic imprinting, etc., that occur during cell differentiation as a zygote develops into an adult.

[0038] As used herein, the term "agent" refers to any compound or substance, such as, but not limited to, small molecules, nucleic acids, polypeptides, peptides, drugs, ions, and the like. An "agent" can be any chemical, entity, or moiety, including, but not limited to, synthetic and naturally occurring proteinaceous and non-proteinaceous entities. In some embodiments, an agent is a nucleic acid, a nucleic acid analog, a protein, an antibody, a peptide, an aptamer, an oligomer of nucleic acid, an amino acid, or a carbohydrate, including, but not limited to, a protein, an oligonucleotide, a ribozyme, a DNAzyme, a glycoprotein, an siRNA, a lipoprotein, an aptamer, and modifications and combinations thereof. In certain embodiments, an agent is a small molecule having a chemical moiety. For example, the chemical moiety includes unsubstituted or substituted alkyl, aromatic, or heterocyclic moieties, including macrolides, leptomycin, and related natural products or analogs thereof. The compound is known to have the desired activity and / or characteristics, or can be selected from a diverse library of compounds.

[0039] As used herein, the term "contacting" (i.e., contacting at least one endocrine cell or precursor thereof with a maturation factor or combination of maturation factors) is intended to include culturing the cells together with the maturation factor in vitro (e.g., adding the maturation factor to the cells in culture). In some embodiments, the term "contacting" is not intended to include in vivo exposure of the cells to a compound as disclosed herein that may occur naturally in a subject (i.e., exposure that may occur as a result of a natural physiological process). As in the embodiments described herein, the step of contacting at least one endocrine cell or precursor thereof with a maturation factor can be performed in any suitable manner. For example, the cells can be treated in adherent or suspension culture. In some embodiments, the cells can be treated under conditions that promote cell clustering. The present disclosure contemplates any condition that promotes cell clustering. Examples of conditions that promote cell clustering include, but are not limited to, suspension culture in low-attachment tissue culture plates, spinner flasks, or aggrewell plates. In some embodiments, the inventors have observed that the clusters are stably maintained in medium containing 10% serum. In some embodiments, the conditions that promote cluster formation include low serum medium.

[0040] It is understood that cells contacted with a maturation factor may be simultaneously or subsequently contacted with another agent, such as a growth factor or other differentiation agent or environment, to stabilize the cells or to further differentiate the cells.

[0041] As used herein, a "cell culture medium" (also referred to herein as "culture medium" or "culture medium") is a medium for culturing cells that contains nutrients that maintain cell viability and support growth. Cell culture media may contain any of the following in appropriate combinations: salt(s), buffer(s), amino acids, glucose or other sugar(s), antibiotics, serum or serum substitutes, and other components such as peptide growth factors. Cell culture media commonly used for particular cell types are known to those of skill in the art.

[0042] The term "cell line" refers to a population of largely or substantially identical cells, typically derived from a single ancestral cell or from a population of distinct and / or substantially identical ancestral cells. A cell line may have been or may be maintained in culture for extended periods (e.g., months, years, indefinitely). A cell line may have undergone a spontaneous or induced process of transformation that confers unlimited culture life to the cells. A cell line includes all cell lines recognized in the art as such. It is recognized that at least some characteristics of individual cells of a cell line may differ from one another, as cells acquire mutations over time and, in some cases, undergo epigenetic changes. In some embodiments, a cell line comprises cells derived from stem cells as described herein.

[0043] The term "exogenous" refers to a substance present in a cell or organism other than its native source. For example, the term "exogenous nucleic acid" or "exogenous protein" refers to a nucleic acid or protein that is introduced by a human-mediated process into a biological system, such as a cell or organism, in which it is not normally present or is present in low abundance. A substance is considered exogenous if it is introduced into a cell or an ancestor of a cell that inherits the substance. In contrast, the term "endogenous" refers to a substance that is native to a biological system.

[0044] The term "expression" refers to the cellular processes involved in producing RNA and proteins and, if appropriate, secreting proteins, including, but not limited to, transcription, translation, folding, modification, and processing, as applicable. "Expression product" includes RNA transcribed from a gene and polypeptides resulting from translation of mRNA transcribed from a gene.

[0045] As used herein, the term "genetically modified" or "engineered" cell refers to a cell into which an exogenous nucleic acid has been introduced by a process mediated by the hand of man (or the progeny of such a cell that inherits at least a portion of the nucleic acid). The nucleic acid may, for example, contain a sequence exogenous to the cell, or may contain a native sequence (i.e., a sequence naturally found in the cell), but may also contain a sequence that does not occur in nature (e.g., a coding region linked to a promoter from a different gene), or a modified version of a native sequence, etc. Introducing the nucleic acid into the cell can be accomplished by any suitable technique. Suitable techniques include calcium phosphate or lipid-mediated transfection, electroporation, and introduction or infection with a viral vector. In some embodiments, the polynucleotide or a portion thereof is integrated into the genome of the cell. The nucleic acid may then be removed or excised from the genome, provided that such removal or excision results in a detectable change in the cell compared to an unmodified but otherwise equivalent cell. It should be recognized that the term genetic modification is intended to include the direct introduction of modified RNA into the cell (e.g., synthetic modified RNA). Such synthetic modified RNAs include modifications to prevent rapid degradation by endonucleases and exonucleases and to avoid or reduce cellular innate immune or interferon responses to RNA. Modifications include, for example, (a) terminal modifications, such as 5'-end modifications (phosphorylation, dephosphorylation, splicing, reverse linkage, etc.), 3'-end modifications (splicing, DNA nucleotides, reverse linkage, etc.), (b) base modifications, such as modified bases, stabilizing bases, destabilizing bases, or bases that base-pair with an expanded repertoire of partners, or substitutions with conjugate bases, (c) sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions, and (d) internucleoside linkage modifications, including modifications or substitutions of phosphodiester linkages. If such modifications prevent translation (i.e., translation is reduced by 50% or more relative to the absence of the modification), then the modifications are not suitable for the methods and compositions described herein.

[0046] As used herein, the term "identity" refers to the degree to which two or more nucleic acid or polypeptide sequences are the same. The percent identity between a sequence of interest and a second sequence over an evaluation range, such as the length of a sequence of interest, can be calculated by aligning the sequences, determining the number of residues (nucleotides or amino acids) in the evaluation range opposite the identical residues, allowing for the introduction of gaps to maximize identity, dividing by the total number of residues in the sequence of interest or the second sequence (whichever is larger) that fall within the range, and multiplying by 100. When calculating the number of identical residues required to achieve a particular percent identity, fractions are rounded to the nearest whole number. Percent identity can be calculated using various computer programs known in the art. For example, computer programs such as BLAST2, BLASTN, BLASTP, Gapped BLAST generate alignments and provide percent identity between sequences of interest. The algorithm of Karlin and Altschul (Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:22264-2268, 1990), modified as in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993, has been incorporated into the NBLAST and XBLAST programs of Altschul et al. (Altschul, et al., J. MoI. Biol. 215:403-410, 1990). To obtain gapped alignments for comparison, Gapped BLAST is utilized as described by Altschul et al. (Altschul, et al. Nucleic Acids Res. 25:3389-3402, 1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs can be used. PAM250 or BLOSUM62 matrices may be used. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI).For these programs, see the website with the URL World Wide Web address "ncbi.nlmnih.gov." In a specific embodiment, the percent identity is calculated using BLAST2 with the default parameters as provided by NCBI.

[0047] As used herein, the terms "isolated" or "partially purified" refer to a nucleic acid or polypeptide that has been separated from at least one other component (e.g., nucleic acid or polypeptide) that is present with the nucleic acid or polypeptide in a natural source and / or that would be present with the nucleic acid or polypeptide when expressed by a cell or that would be secreted, in the case of a secreted polypeptide. Chemically synthesized nucleic acids or polypeptides or those synthesized using in vitro transcription / translation are considered "isolated."

[0048] As used herein, the term "isolated cell" refers to a cell that has been removed from the organism in which it was originally found, or the progeny of such a cell. Optionally, the cell has been cultured in vitro, for example in the presence of other cells. Optionally, the cell is subsequently introduced into a second organism, or the cell (or the progeny of the cell) is reintroduced into the organism from which it was isolated.

[0049] As used herein, the term "isolated population" with respect to an isolated population of cells refers to a cell population that is removed and separated from a mixed or heterogeneous cell population. In some embodiments, an isolated population is a substantially pure cell population compared to the heterogeneous population from which the cells are isolated or enriched.

[0050] The term "substantially pure" with respect to a particular cell population refers to a cell population that is at least about 75%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% pure with respect to the cells that make up the total cell population.

[0051] The terms "enrich" or "enriched" are used interchangeably herein and mean that the yield (proportion) of a type of cell is increased by at least 10% over the proportion of that type of cell in the starting culture or preparation.

[0052] As used herein, the terms "renewal" or "self-renewal" or "proliferation" are used interchangeably and refer to the ability of a stem cell to renew itself by dividing into the same non-specific cell type over an extended period of time and / or over a period of months to years. In some cases, proliferation refers to the expansion of a cell by repeatedly dividing into two identical daughter cells.

[0053] As used herein, the term "lineage" refers to cells that share a common ancestor or a common developmental fate. For example, in the context of a cell that is of endoderm origin or of "endodermal lineage," it means that the cell is derived from an endoderm cell and can differentiate along one or more developmental lineage pathways that are restricted to the endodermal lineage, such as giving rise to a committed endoderm cell, which can then differentiate into liver cells, thymus, pancreas, lung, and intestine.

[0054] As used herein, a "marker" is used to describe the characteristics and / or phenotype of a cell. Markers can be used to select cells containing a characteristic of interest. Markers vary depending on the particular cell. A marker is a characteristic, such as a morphological, functional, or biochemical (enzymatic) characteristic of a particular cell type of cell or a molecule expressed by a cell type. Preferably, such a marker is a protein, more preferably having an epitope for an antibody or other binding molecule available in the art. However, a marker may be composed of any molecule found within a cell, including, but not limited to, proteins (peptides and polypeptides), lipids, polysaccharides, nucleic acids, and steroids. Examples of morphological characteristics or traits include, but are not limited to, shape, size, and nucleus to cytoplasm ratio. Examples of functional characteristics or traits include, but are not limited to, the ability to adhere to a particular substrate, the ability to take up or exclude a particular dye, the ability to migrate under particular conditions, and the ability to differentiate along a particular lineage. Markers can be detected by any method available to one of skill in the art. A marker can also be the absence of a morphological feature, or the absence of a protein, lipid, etc. A marker can be a combination of a panel of unique features related to the presence and absence of polypeptides, as well as other morphological features.

[0055] The term "modulate" is used as it is in the art, i.e., to cause or promote a qualitative or quantitative change, alteration, or modification in a process, pathway, or phenomenon of interest. Without being limited thereto, such a change can be an increase, decrease, or change in the relative strength or activity of different components or branches of the process, pathway, or phenomenon. A "modulator" is an agent that causes or promotes a qualitative or quantitative change, alteration, or modification in a process, pathway, or phenomenon of interest.

[0056] The term "polynucleotide" is used interchangeably with "nucleic acid" herein to refer to a polymer of nucleosides. Typically, the polynucleotides of the present invention are nucleosides found naturally in DNA or RNA (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) linked by phosphodiester bonds. However, the term encompasses molecules containing nucleosides, whether or not found in naturally occurring nucleic acids, or nucleoside analogs, including chemically or biologically modified bases, modified backbones, and the like, which may be preferred for certain applications. When the present application refers to polynucleotides, it is understood that DNA, RNA, and in each case both single-stranded and double-stranded forms (and the complement of each single-stranded molecule) are provided. As used herein, "polynucleotide sequence" can refer to the polynucleotide material itself and / or the sequence information (i.e., the sequence of letters used as abbreviations for bases) that biochemically characterize a particular nucleic acid. Polynucleotide sequences presented herein are presented in the 5' to 3' orientation unless otherwise indicated.

[0057] As used herein, the term "polypeptide" refers to a polymer of amino acids. The terms "protein" and "polypeptide" are used interchangeably herein. A peptide is a relatively short polypeptide, typically about 2-60 amino acids in length. A polypeptide as used herein typically contains amino acids, such as the 20 L-amino acids most commonly found in proteins. However, other amino acids and / or amino acid analogs known in the art can be used. One or more amino acids in a polypeptide can be modified, for example, by the addition of a chemical entity, such as a carbohydrate group, a phosphate group, a fatty acid group, a linker for conjugation, functionalization, etc. Polypeptides to which non-polypeptide moieties are covalently or non-covalently attached are also considered to be "polypeptides". Exemplary modifications include glycosylation and palmitoylation. Polypeptides can be purified from natural sources, produced using recombinant DNA technology, synthesized by chemical means such as conventional solid-phase peptide synthesis, etc. As used herein, the terms "polypeptide sequence" or "amino acid sequence" can refer to the polypeptide material itself and / or to the sequence information that biochemically characterizes the polypeptide (i.e., the sequence of letters or three-letter codes used as an abbreviation for the names of amino acids). The sequences of the polypeptides presented herein are presented in the N-terminal to C-terminal direction unless otherwise indicated.

[0058] The term "variant" when referring to a polypeptide may be, for example, a polypeptide that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the full-length polypeptide. A variant may be a fragment of a full-length polypeptide. A variant may be a naturally occurring splice variant. A variant may be a polypeptide that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to a fragment of a polypeptide, where the fragment is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% in length of the full-length wild-type polypeptide or a domain thereof that has the desired activity. In some embodiments, the domain is at least 100, 200, 300, or 400 amino acids in length, beginning at any amino acid position in the sequence and extending toward the C-terminus. Variants known in the art that eliminate or substantially reduce the activity of a protein are preferably avoided. In some embodiments, the variant lacks the N-terminal and / or C-terminal portions of the full-length polypeptide, e.g., up to 10, 20, or 50 amino acids from either end. In some embodiments, the polypeptide has the sequence of a mature (full-length) polypeptide, meaning a polypeptide from which one or more portions, such as a signal peptide, have been removed during normal intracellular proteolytic processing (e.g., during or after co-translational processing). In some embodiments, where the protein is produced by other than purification from a cell that naturally expresses it, the protein is a chimeric polypeptide, meaning that it contains portions from two or more different species. In some embodiments, where the protein is produced by other than purification from a cell that naturally expresses it, the protein is a derivative, meaning that it contains additional sequences not associated with the protein, so long as those sequences do not substantially reduce the biological activity of the protein.

[0059] As used herein, the term "functional fragment" refers to a polypeptide having a substantially homologous amino acid sequence but smaller in size than the polypeptide from which the fragment is derived, where the functional fragment polypeptide sequence has at least 50%, or 60%, or 70%, or 80%, or 90%, or 100% or more, e.g., 1.5-fold, 2-fold, 3-fold, 4-fold, or more than 4-fold, effective biological activity of the polypeptide from which the fragment is derived. Functional fragment polypeptides can have additional functions including reduced antigenicity, increased DNA binding (such as a transcription factor), or altered RNA binding (such as regulating RNA stability or degradation).

[0060] The term "vector" refers to a carrier DNA molecule into which a DNA sequence can be inserted for introduction into a host cell. Preferred vectors are capable of autonomous replication and / or expression of the nucleic acid to which it is linked. A vector capable of directing the expression of a gene to which it is operably linked is referred to herein as an "expression vector." Thus, an "expression vector" is a specialized vector that contains the necessary regulatory regions for the expression of a gene of interest in a host cell. In some embodiments, the gene of interest is operably linked to another sequence in the vector. The vector may be a viral vector or a non-viral vector. When a viral vector is used, it is preferred that the viral vector is replication-deficient, which can be achieved, for example, by removing all viral nucleic acid that codes for replication. Replication-deficient viral vectors still retain infectious properties and enter cells in a manner similar to replicating adenoviral vectors, but once inside the cell, replication-deficient viral vectors do not replicate or grow. Vectors also include means of delivering DNA molecules to cells, such as liposomes and nanoparticles.

[0061] The term "operably linked" means that the control sequences necessary for expression of a coding sequence are positioned in a DNA molecule in the appropriate position relative to the coding sequence to effect expression of the coding sequence. This same definition may also be applied to the positioning of coding sequences and transcription control elements (e.g., promoters, enhancers and termination elements) in an expression vector. The term "operably linked" includes having an appropriate initiation signal (e.g., ATG) in front of the polynucleotide sequence to be expressed, and maintaining the correct reading frame to allow expression of the polynucleotide sequence under the control of the expression control sequences and production of the desired polypeptide encoded by the polynucleotide sequence.

[0062] The term "viral vector" refers to the use of a virus or virus-associated vector as a carrier of a nucleic acid construct into a cell. The construct may be incorporated and packaged into an adenovirus, adeno-associated virus (AAV), or herpes simplex virus (HSV), or other non-replicating defective viral genome, including retroviral and lentiviral vectors, to infect or introduce into a cell. The vector may or may not be integrated into the genome of the cell. The construct may optionally include viral sequences for transfection. Alternatively, the construct may be incorporated into a vector capable of episomal replication, such as EPV and EBV vectors.

[0063] The terms "control sequence" and "promoter" are used interchangeably herein and refer to nucleic acid sequences, such as initiation signals, enhancers, and promoters, that induce or control the transcription of a protein coding sequence to which they are operably linked. In some examples, transcription of a recombinant gene is under the control of a promoter sequence (or other transcription control sequence) that controls expression of the recombinant gene in a cell type in which expression is intended. It is also understood that a recombinant gene can be under the control of a transcription control sequence that is the same or different from the sequence that controls transcription of the naturally occurring protein. In some cases, the promoter sequence is recognized by the synthetic machinery of the cell, or introduced synthetic machinery, necessary to initiate transcription of a particular gene.

[0064] As used herein, the term "transcription factor" refers to a protein that binds to a specific portion of DNA using a DNA-binding domain and is part of a system that controls the transfer (or transcription) of genetic information from DNA to RNA. As used herein, "proliferate" and "proliferation" refer to an increase in the number of cells in a population (growth) by cell division. Cell proliferation is generally understood to result from the coordinated activation of multiple signaling pathways in response to an environment that includes growth factors and other mitogens. Cell proliferation can also be promoted by relief from the action of intracellular or extracellular signals and mechanisms that inhibit or adversely affect cell proliferation.

[0065] The term "selectable marker" refers to a gene, RNA, or protein that, when expressed, confers on a cell a selectable phenotype, such as resistance to cytotoxic or cytostatic agents (e.g., antibiotic resistance), trophogenicity, or expression of a specific protein that can be used as a basis for distinguishing cells that express the protein from those that do not. Proteins whose expression can be easily detected ("detectable markers"), such as fluorescent or luminescent proteins, or enzymes that act on a substrate to produce a colored, fluorescent, or luminescent substance, constitute a subset of selectable markers. The presence of a selectable marker linked to expression control elements native to genes that are normally selectively or exclusively expressed in pluripotent cells allows for the identification and selection of somatic cells that have been reprogrammed to the pluripotent state. Various selectable marker genes can be used, such as the neomycin resistance gene (neo), puromycin resistance gene (puro), guanine phosphoribosyltransferase (gpt), dihydrofolate reductase (DHFR), adenosine deaminase (ada), puromycin-N-acetyltransferase (PAC), hygromycin resistance gene (hyg), multidrug resistance gene (mdr), thymidine kinase (TK), hypoxanthine-guanine phosphoribosyltransferase (HPRT), and hisD gene. Detectable markers include green fluorescent protein (GFP), blue, sapphire, yellow, red, orange, and cyan fluorescent proteins, and mutants of any of these. Luminescent proteins such as luciferase (e.g., firefly or Renilla luciferase) are also useful. As will be appreciated by those skilled in the art, the term "selectable marker" as used herein can refer to a gene or the expression product of a gene, such as an encoded protein.

[0066] In some embodiments, a selectable marker confers a growth and / or survival advantage to cells expressing it compared to cells that do not express it or express it at a significantly lower level. Such a growth and / or survival advantage usually occurs when cells are maintained under specific conditions, i.e., "selective conditions". To ensure effective selection, a population of cells can be maintained for a sufficient period of time under conditions such that cells that do not express the marker do not grow and / or survive and are eliminated from the population or are reduced in number to a small fraction of the population. The process of selecting cells expressing a marker that confers a growth and / or survival advantage by maintaining a population of cells under selective conditions that largely or completely eliminate cells that do not express the marker is referred to herein as "positive selection" and a marker is said to be "useful for positive selection". Negative selection and markers useful for negative selection are also of interest in certain methods described herein. Expression of such a marker confers a growth and / or survival disadvantage on cells expressing the marker relative to cells that do not express the marker or express the marker at significantly lower levels (or, viewed alternatively, cells that do not express the marker have a growth and / or survival advantage relative to cells that express the marker.) Thus, cells expressing the marker can be largely or completely eliminated from a cell population when maintained under selective conditions for a sufficient period of time.

[0067] As used herein, "reporter gene" includes any gene genetically introduced into a cell that adds to the stem cell phenotype. The reporter genes disclosed in the present invention are intended to include fluorescent genes, luminescent genes, enzyme genes, and resistance genes, but also include other genes that can be easily detected by a person skilled in the art. In some embodiments of the present invention, reporter genes are used as markers to identify specific stem cells, cardiovascular stem cells, and their differentiated progeny. Reporter genes are generally operably linked to sequences that control their expression in a manner that depends on one or more conditions monitored by measuring the expression of the reporter gene. In some cases, the expression of the reporter gene is determined in living cells. When a live cell reporter gene assay is used, the expression of the reporter gene can be monitored at multiple time points, for example, 2, 3, 4, 5, 6, 8, or 10 or more time points. In some cases, when a live cell reporter assay is used, reporter gene expression is monitored at a frequency of at least about 10 minutes to about 24 hours, e.g., 20 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 18 hours, or another frequency from any integer between about 10 minutes and about 24 hours.

[0068] The terms "subject" and "individual" are used interchangeably herein and refer to an animal, e.g., a human, from which cells can be obtained and / or to which cell therapy (including prophylactic therapy) as described herein is provided. For the treatment of an infection, condition, or disease state specific to a particular animal, such as a human subject, the term subject refers to that particular animal. As used interchangeably herein, "non-human animals" and "non-human mammals" include 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. However, advantageously, the subject is a mammal, such as a human, or other mammal, such as a domesticated mammal, e.g., dog, cat, horse, or a production mammal, e.g., cow, sheep, pig, etc.

[0069] When applied to isolated cells, the terms "treat," "treating," "treatment," and the like include subjecting the cells to any process or condition, or performing any operation or procedure on the cells. When applied to a subject, these terms refer to the medical or surgical treatment, care, or management of an individual, who is usually ill or injured, or at increased risk of developing a disease compared to the average member of the population, and who is in need of such attention, care, or management.

[0070] As used herein, the terms "treat" and "treatment" refer to administering an effective amount of a composition to a subject so that the subject achieves at least one symptom of the disease or an improvement in the disease, e.g., a beneficial or desired clinical outcome. For purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, the reduction of one or more symptoms, the reduction in the extent of the disease, the stabilization of the disease state (i.e., not worsening), the delay or slowing of the progression of the disease, the improvement or mitigation of the disease state, and remission (whether partial or complete), whether detectable or undetectable. "Treatment" may also refer to the prolongation of survival compared to the expected survival in the absence of treatment. Thus, one of ordinary skill in the art will recognize that treatment may improve the disease state, but may not result in a complete cure of the disease. As used herein, the term "treatment" includes prevention. Alternatively, treatment is "effective" if the progression of the disease is inhibited or stopped. "Treatment" may also mean the prolongation of survival compared to the expected survival in the absence of treatment.

[0071] As used herein, the terms "administer", "introduce" and "implant" are used interchangeably in the context of introducing the cells of the present invention into a subject in a manner or route such that the introduced cells are at least partially localized at a desired site. The cells can be directly implanted into the pancreas or digestive tract, or can be administered by a suitable route such that at least some of the implanted cells or components of the cells remain viable and are delivered to the desired site in the subject. The viability of the cells after administration to the subject can be as short as a few hours, e.g., 24 hours, to as long as several days or years. In some cases, the cells can be administered subcutaneously, e.g., in a capsule (e.g., microcapsule) to maintain the implanted cells at the implanted site and avoid migration of the implanted cells.

[0072] As used herein, the phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. As used herein, the phrases "systemic administration", "administered systemically", "peripheral administration", "administered peripherally" refer to administration of stem cell-derived cells and / or their progeny and / or compounds and / or other substances other than directly into the central nervous system, such that it enters the animal's body and thus undergoes metabolic and other similar processes, e.g., subcutaneous administration.

[0073] The term "tissue" refers to a group or layer of specialized cells that together perform a particular specialized function. The term "tissue-specific" refers to the source of cells that are derived from a particular tissue.

[0074] As used herein, the terms "reduce", "reduced", "reduction", "reduction" or "inhibit" are all commonly used to mean a statistically significant amount of reduction. However, for the avoidance of doubt, "reduced", "reduction" or "reduce" or "inhibit" means a reduction of at least 10% compared to the reference level, for example, a reduction of at least about 20%, or a reduction of at least about 30%, or a reduction of at least about 40%, or a reduction of at least about 50%, or a reduction of at least about 60%, or a reduction of at least about 70%, or a reduction of at least about 80%, or a reduction of at least about 90%, or a reduction of up to 100% (i.e., absent levels compared to the reference sample), or any reduction between 10-100% compared to the reference level.

[0075] The terms "increased", "increase", "enhance" or "activate" are all used herein to generally mean an increase of a statistically significant amount. For the avoidance of any doubt, the terms "increase", "increase", "enhance" or "activate" mean an increase of at least 10% compared to a reference level, for example, an increase of at least about 20%, or an increase of at least about 30%, or an increase of at least about 40%, or an increase of at least about 50%, or an increase of at least about 60%, or an increase of at least about 70%, or an increase of at least about 80%, or an increase of at least about 90%, or an increase of up to 100%, or any increase between 10-100% compared to a reference level, or an increase of at least about 2-fold compared to a reference level, or an increase of at least about 3-fold, or an increase of at least about 4-fold, or an increase of at least about 5-fold, or an increase of at least about 10-fold, or any increase between 2-fold and 10-fold or more compared to a reference level.

[0076] The term "statistically significant" or "significantly" refers to statistical significance, which generally means that the concentration of the marker is two standard deviations (2SD) or lower than the normal value. The term refers to statistical evidence that there is a difference. It is defined as the probability of making a decision to reject the null hypothesis when the null hypothesis is actually true. It is often determined using a p-value.

[0077] As used herein, the terms "comprising" or "comprises" are used in reference to compositions, methods, and their respective component(s) that are essential to the invention, but are open to the inclusion of unspecified elements, whether essential or not.

[0078] As used herein, the term "consisting essentially of" refers to elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[0079] The term "comprising" refers to compositions, methods, and components thereof described herein, which do not include any element not recited in that description of an embodiment.

[0080] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "the method" includes one or more methods, and / or steps of the type described herein and / or that will become apparent to those skilled in the art upon reading this disclosure, etc.

[0081] stem cells Stem cells are cells that retain the ability to renew themselves by mitotic cell division and can differentiate into a variety of specialized cell types. There are two types of mammalian stem cells: embryonic stem (ES) cells, found in blastocysts, and adult stem cells, found in adult tissues. In the developing embryo, stem cells can differentiate into all specialized embryonic tissues. In adult organisms, stem and progenitor cells act as the body's repair system, not only replenishing specialized cells but also maintaining the normal turnover of regenerative organs such as blood, skin, and intestinal tissues. Pluripotent stem cells can differentiate into cells derived from any of the three germ cell layers.

[0082] Below, certain embodiments are described with respect to the use of stem cells, however, germ cells can be used in place of or together with stem cells to provide at least one differentiated cell using protocols similar to the exemplary protocols described herein. Suitable germ cells can be prepared, for example, from primordial germ cells present in human fetal material harvested approximately 8-11 weeks after the last menstrual period. Exemplary methods for preparing germ cells are described, for example, in Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998 and U.S. Patent No. 6,090,622.

[0083] ES cells, such as human embryonic stem cells (hESCs) or mouse embryonic stem cells (mESCs), have a virtually unlimited replicative capacity and the potential to differentiate into most cell types, in principle offering an unlimited starting material for generating differentiated cells for clinical therapy (stemcells.nih.gov / info / scireport / 2006report.htm, 2006).

[0084] hESC cells have been described, for example, in Cowan et al. (N Engl. J. Med. 350:1353, 2004) and Thomson et al. (Science 282:1145, 1998), and embryonic stem cells from other primates, such as rhesus monkey stem cells (Thomson et al., Proc. Natl. Acad. Sci. USA 92:7844, 1995), marmoset stem cells (Thomson et al., Biol. Reprod. 55:254, 1996), and human embryonic germ (hEG) cells (Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998), may also be used in the methods disclosed herein. mESCs are described, for example, in Tremml et al. (Curr Protoc Stem Cell Biol. Chapter 1:Unit 1C.4, 2008). Stem cells can be, for example, unipotent, totipotent, pluripotent, or multipotent. In some examples, any cell from a primate that can produce progeny that are derivatives of at least one germ cell layer, or all three germ cell layers, can be used in the methods disclosed herein.

[0085] In certain instances, ES cells can be isolated as described, for example, in Cowan et al. (N Engl. J. Med. 350:1353, 2004) and U.S. Patent No. 5,843,780, and Thomson et al., Proc. Natl. Acad. Sci. USA 92:7844, 1995. For example, hESC cells have been described by Thomson et al. (U.S. Patent No. 6,200,806, Science 282:1145, 1998, Curr. Top. Dev. Equivalent cell types to hESCs include their pluripotent derivatives, such as primitive ectoderm-like (EPL) cells, as reviewed, for example, in WO 01 / 51610 (Bresagen). hESCs can also be obtained from human preimplantation embryos. Alternatively, in vitro fertilized (IVF) embryos can be used, or human one-cell embryos can be expanded to the blastocyst stage (Bongso et al., Hum Reprod 4:706, 1989). Embryos are cultured to the blastocyst stage in G1.2 and G2.2 media (Gardner et al. al., Fertil. Steril. 69:84, 1998). The zona pellucida is removed from the developing blastocysts by brief exposure to pronase (Sigma). The inner cell mass may be isolated by immunosurgery, in which the blastocysts are exposed to a 1:50 dilution of rabbit anti-human spleen cell antiserum for 30 min, washed three times for 5 min in DMEM, and exposed to a 1:5 dilution of guinea pig complement (Gibco) for 3 min (Solter et al., Proc. Natl. Acad. Sci. USA 72:5099, 1975). After two further washes with DMEM, the lysed trophectoderm cells are removed from the intact inner cell mass (ICM) by gentle pipetting, and the ICM is plated onto a layer of mEF feeders. After 9–15 days, the inner cell mass-derived outgrowths are cultured in 1 mM calcium- and magnesium-free medium. They can be dissociated into clumps by exposure to phosphate-buffered saline (PBS) with EDTA, by exposure to dispase or trypsin, or by mechanical dissociation using a micropipette, and then replated onto mEFs in fresh medium.Growing colonies with undifferentiated morphology can be individually selected with a micropipette, mechanically dissociated into clumps, and replated. ES-like morphology is characterized as small colonies with a clearly high nuclear to cytoplasmic ratio and prominent nucleoli. The resulting hESCs can be split periodically every 1-2 weeks, for example, by brief trypsinization, exposure to Dulbecco's PBS (containing 2 mM EDTA), exposure to collagenase type IV (approximately 200 U / mL; Gibco), or by selection of individual colonies with a micropipette. In some examples, clump sizes of approximately 50-100 cells are optimal. mESC cells can be prepared, for example, using techniques described in Conner et al. (Curr.Prot.in Mol.Biol.Unit 23.4, 2003).

[0086] Embryonic stem cells can be isolated from blastocysts of members of the primate species (U.S. Pat. No. 5,843,780; Thomson et al., Proc. Natl. Acad. Sci. USA 92:7844, 1995). Human embryonic stem (hES) cells have been described by Thomson et al. (U.S. Pat. No. 6,200,806; Science 282:1145, 1998; Curr. Top. Dev. Equivalent cell types to hES cells include their pluripotent derivatives, such as primitive ectoderm-like (EPL) cells, as reviewed in WO01 / 51610 (Bresagen).

[0087] Alternatively, in some embodiments, hESCs can be obtained from human preimplantation embryos. Alternatively, in vitro fertilized (IVF) embryos can be used, or human one-cell embryos can be expanded to the blastocyst stage (Bongso et al., Hum Reprod 4:706, 1989). Embryos are cultured to the blastocyst stage in G1.2 and G2.2 media (Gardner et al., Fertil. Steril. 69:84, 1998). The zona pellucida is removed from the developed blastocysts by brief exposure to pronase (Sigma). The inner cell mass is isolated by immunosurgery by exposing blastocysts to a 1:50 dilution of rabbit anti-human spleen cell antiserum for 30 min, followed by three 5-min washes in DMEM, and a 3-min exposure to a 1:5 dilution of guinea pig complement (Gibco) (Solter et al., Proc. Natl. Acad. Sci. USA 72:5099, 1975). After two additional washes in DMEM, lysed trophectoderm cells are removed from the intact inner cell mass (ICM) by gentle pipetting, and the ICM is plated onto mEF feeder layers.

[0088] After 9–15 days, the inner cell mass-derived outgrowths are dissociated into clumps by exposure to phosphate-buffered saline (PBS) containing 1 mM EDTA without calcium and magnesium, by exposure to dispase or trypsin, or by mechanical dissociation with a micropipette, and then replated on mEFs in fresh medium. Growing colonies with undifferentiated morphology are individually selected with a micropipette, mechanically dissociated into clumps, and replated. ES-like morphology is characterized as small colonies with a clearly high nuclear to cytoplasmic ratio and prominent nucleoli. The resulting ES cells are routinely split every 1–2 weeks by brief trypsinization, exposure to Dulbecco's PBS (containing 2 mM EDTA), exposure to collagenase type IV (approximately 200 U / mL; Gibco), or by selection of individual colonies with a micropipette. A clump size of approximately 50–100 cells is optimal.

[0089] In some embodiments, human embryonic germ (hEG) cells are pluripotent stem cells that can be used to differentiate into primitive endoderm cells in the manner disclosed herein. hEG cells can be prepared from primordial germ cells present in human fetal material harvested approximately 8-11 weeks after the last menstrual period. Suitable preparation methods are described in Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998, and U.S. Patent No. 6,090,622, which are incorporated herein by reference in their entireties.

[0090] Briefly, genital ridges are treated to form disaggregated cells. EG growth medium is DMEM, 4500 mg / L D-glucose, 2200 mg / L mM NaHCO3, 15% ES-qualified fetal bovine serum (BRL), 2 mM glutamine (BRL), 1 mM sodium pyruvate (BRL), 1000-2000 U / mL human recombinant leukemia inhibitory factor (LIF, Genzyme), 1-2 ng / mL human recombinant bFGF (Genzyme), and 10 µM forskolin (10% in DMSO). 96-well tissue culture plates are prepared with a subconfluent layer of feeder cells (e.g., STO cells, ATCC number CRL1503) cultured for 3 days in modified EG growth medium without LIF, bFGF, or forskolin inactivated by 5000 rad gamma irradiation, and approximately 0.2 mL of primary germ cell (PGC) suspension is added to each well. The first passage is performed after 7-10 days in EG growth medium, transferring each well into one well of a 24-well culture dish previously prepared with irradiated STO mouse fibroblasts. Culture the cells with daily changes of medium until cell morphology consistent with EG cells is observed, typically after 7-30 days or 1-4 passages.

[0091] In some examples, the stem cells may be undifferentiated cells (e.g., cells that are not committed to a particular lineage) prior to exposure to at least one maturation factor according to the methods as disclosed herein, while in other examples, it may be desirable to differentiate the stem cells into one or more intermediate cell types prior to exposure to at least one maturation factor(s) as described herein. For example, the stem cells may exhibit morphological, biological, or physical characteristics of undifferentiated cells that can be used to distinguish them from differentiated cells of embryonic or adult origin. In some examples, undifferentiated cells may be seen in colonies of cells viewed in two dimensions under a microscope, with high nuclear / cytoplasmic ratios and prominent nucleoli. The stem cells may be used by themselves (e.g., in the substantial absence of undifferentiated cells) or in the presence of differentiated cells. In some examples, the stem cells may be cultured in the presence of appropriate nutrients and, optionally, other cells, to allow the stem cells to proliferate and, optionally, differentiate. For example, embryonic fibroblasts or fibroblast-like cells may be present in the culture to support the growth of the stem cells. Fibroblasts may be present at one stage of stem cell proliferation, but not necessarily at all stages, for example, fibroblasts may be added to the stem cell culture medium during an initial culture stage, but not during one or more subsequent culture stages.

[0092] The stem cells used in all aspects of the invention can be any cell derived from any type of tissue (e.g., embryonic tissue, such as fetal or pre-fetal tissue, or adult tissue), characterized in that under appropriate conditions they can produce progeny of different cell types, such as all derivatives of at least one of the three germ layers (endoderm, mesoderm, and ectoderm). These cell types can be provided in the form of established cell lines, or can be obtained directly from primary embryonic tissue and used immediately for differentiation. Cells listed in the NIH Human Embryonic Stem Cell Registry include hESBGN-01, hESBGN-02, hESBGN-03, hESBGN-04 (BresaGen, Inc.); HES-1, HES-2, HES-3, HES-4, HES-5, HES-6 (ES Cell International); Miz-hES1 (MizMedi Hospital-Seoul National University); HSF-1, HSF-6 (University of California at San Francisco); and H1, H7, H9, H13, H14 (Wisconsin Alumni Research Foundation (WiCell Research Institute)). In some embodiments, the source of human stem cells or pluripotent stem cells used for chemically induced differentiation into stem cell-derived cells did not involve the destruction of human embryos.

[0093] In another embodiment, stem cells can be isolated from tissues, including solid tissues. In some embodiments, the tissue is skin, fat tissue (e.g., adipose tissue), muscle tissue, heart or cardiac tissue. In other embodiments, the tissue is, for example, but not limited to, umbilical cord blood, placenta, bone marrow, or cartilage.

[0094] Stem cells of interest also include various types of embryonic cells, such as human embryonic stem (hES) cells, as described by Thomson et al. (1998) Science 282:1145; embryonic stem cells from other primates, such as rhesus monkey stem cells (Thomson et al. (1995) Proc. Natl. Acad. Sci. USA 92:7844); marmoset stem cells (Thomson et al. (1996) Biol. Reprod. 55:254); and human embryonic germ (hEG) cells (Shambloft et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998). Also of interest are lineage-committed stem cells such as mesodermal stem cells and other early cardiac progenitors (see, e.g., Reyes et al. (2001) Blood 98:2615-2625; Eisenberg & Bader (1996) Circ Res. 78(2):205-16). Stem cells can be obtained from any mammalian species, e.g., human, horse, cow, pig, dog, cat, rodent, e.g., mouse, rat, hamster, primate, etc. In some embodiments, human embryos were not destroyed for the source of pluripotent cells used in the methods and compositions as disclosed herein.

[0095] ES cells are considered undifferentiated if they are not committed to a particular lineage of differentiation. Such cells display morphological characteristics that distinguish them from differentiated cells of embryonic or adult origin. Undifferentiated ES cells are easily recognized by those skilled in the art and are usually seen in colonies of cells with high nuclear / cytoplasmic ratios and prominent nucleoli when viewed two-dimensionally under a microscope. Undifferentiated ES cells express genes that can be used as markers to detect the presence of undifferentiated cells, and the polypeptide products of which can be used as markers for negative selection. See, for example, U.S. Patent Application No. 2003 / 0224411 A1, Bhattacharya (2004) Blood 103(8):2956-64, and Thomson (1998), supra, each of which is incorporated herein by reference. Human ES cell lines express cell surface markers that characterize undifferentiated non-human primate ES cells and human EC cells, including stage-specific embryonic antigen (SSEA)-3, SSEA-4, TRA-1-60, TRA-1-81, and alkaline phosphatase. The globo-series glycolipid GL7, which carries the SSEA-4 epitope, is formed by the addition of sialic acid to the globo-series glycolipid GbS, which carries the SSEA-3 epitope. Thus, GL7 reacts with antibodies against both SSEA-3 and SSEA-4. Undifferentiated human ES cell lines did not stain with SSEA-1, whereas differentiated cells stained strongly with SSEA-I. Methods for expanding undifferentiated hES cells are described in WO99 / 20741, WO01 / 51616, and WO03 / 020920.

[0096] A mixture of cells from a suitable source of endothelial, muscle, and / or neural stem cells can be harvested from a mammalian donor by methods known in the art. A suitable source is the hematopoietic microenvironment. For example, circulating peripheral blood, preferably mobilized (i.e., recruited) circulating peripheral blood, may be removed from a subject. Alternatively, bone marrow may be harvested from a mammal, such as a human patient undergoing an autologous transplant. In some embodiments, stem cells can be obtained from adipose tissue of a subject, for example, using Cytori's CELUTION™ SYSTEM, as disclosed in U.S. Pat. Nos. 7,390,484 and 7,429,488, which are incorporated herein by reference in their entirety.

[0097] In some embodiments, human umbilical cord blood cells (HUCBCs) are useful in the methods disclosed herein. Human UBC cells are recognized as a rich source of hematopoietic and mesenchymal progenitor cells (Broxmeyer et al., 1992 Proc. Natl. Acad. Sci. USA 89:4109-4113). Until now, umbilical cord and placental blood have been considered waste products that are usually discarded at the birth of an infant. Umbilical cord blood cells have been used as a source of transplantable stem and progenitor cells and as a source of bone marrow repopulating cells for the treatment of malignant diseases (i.e., acute lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndromes, and neuroblastoma) and non-malignant diseases such as Fanconi anemia and aplastic anemia (Kohli-Kumar et al., 1993 Br. J. Haematol. 85:419-422; Wagner et al., 1992 Blood 79; 1874-1881; Lu et al., 1996 Crit. A distinct advantage of HUCBC is that these cells have immature immune properties very similar to fetal cells, which significantly reduces the risk of rejection by the host (Taylor & Bryson, 1985 J. Immunol. 134: 1493-1497).Human umbilical cord blood contains mesenchymal and hematopoietic progenitor cells, as well as endothelial cell precursor cells that can be expanded in tissue culture (Broxmeyer et al., 1992 Proc. Natl. Acad. Sci. USA 89:4109-4113; Kohli-Kumar et al., 1993 Br. J. Haematol. 85:419-422; Wagner et al., 1992 Blood 79;1874-1881; Lu et al., 1996 Crit. Rev. Oncol. Hematol 22:61-78; Lu et al., 1995 Cell Transplantation 4:493-503; Taylor & Bryson, 1985J. Immunol. 134:1493-1497; Broxmeyer, 1995 Transfusion 35:694-702;Chen et al., 2001 Stroke 32:2682-2688;Nieda et al., 1997 Br. J. Haematology 98:775-777, Erices et al., 2000 Br. J. Haematology 109:235-242). The total content of hematopoietic progenitor cells in umbilical cord blood is equal to or exceeds that of bone marrow, and moreover, highly proliferative hematopoietic cells express hematopoietic markers such as CD14, CD34, and CD45 at eight-fold higher levels in HUCBC than in bone marrow (Sanchez-Ramos et al., 2001 Exp. Neur. 171:109-115; Bicknese et al., 2002 Cell Transplantation 11:261-264; Lu et al., 1993 J. Exp Med. 178:2089-2096).

[0098] In another embodiment, the pluripotent cells are cells in a hematopoietic microenvironment, such as circulating peripheral blood, preferably the peripheral blood mononuclear fraction of a mammal, umbilical cord blood, bone marrow, fetal liver, or yolk sac. Stem cells, particularly neural stem cells, may be derived from the central nervous system, including the meninges.

[0099] In another embodiment, pluripotent cells present in embryoid bodies are formed by harvesting ES cells with brief protease digestion and growing small clumps of undifferentiated human ESCs in suspension culture. Differentiation is induced by removal of conditioned medium. The resulting embryoid bodies are plated on a semi-solid substrate. Formation of differentiated cells is observed after about 7 days to about 4 weeks. To select viable differentiated cells from in vitro cultures of stem cells, embryoid bodies or similar structures are partially dissociated to obtain cell aggregates. Aggregates containing cells of interest are selected for phenotypic characteristics using methods that substantially maintain contact between cells in the aggregates.

[0100] In another embodiment, the stem cells can be reprogrammed stem cells, such as stem cells derived from somatic cells or differentiated cells. In such embodiments, the dedifferentiated stem cells can be, for example, but not limited to, neoplastic cells, tumor cells, and cancer cells, or alternatively derived reprogrammed cells, such as induced pluripotent stem cells, or iPS cells.

[0101] Cloning and cell culture Exemplary methods of molecular genetics and genetic engineering that can be used in the techniques described herein can be found, for example, in Molecular Cloning: A Laboratory Manual (Sambrook et al., Cold Spring Harbor), Gene Transfer Vectors for Mammalian Cells (Miller & Calos eds.), and current editions of Current Protocols in Molecular Biology (FMA Usubel et al. eds., Wiley & Sons). Cell biology, protein chemistry, and antibody techniques can be found, for example, in Current Protocols in Protein Science (JE Colligan et al. eds., Wiley & Sons), Current Protocols in Cell Biology (JS Bonifacino et al., Wiley & Sons), and Current Protocols in Immunology (JE Colligan et al. eds., Wiley & Sons). Exemplary reagents, cloning vectors, and kits for genetic manipulation can be obtained commercially from, for example, BioRad, Stratagene, Invitrogen, ClonTech, and Sigma-Aldrich Co.

[0102] Suitable cell culture techniques can be found, for example, in the most recent editions of Culture of Animal Cells: A Manual of Basic Technique (RI Freshney ed., Wiley & Sons), General Techniques of Cell Culture (MA Harrison & IF Rae, Cambridge Univ. Press), and Embryonic Stem Cells: Methods and Protocols (K. Turksen ed., Humana Press). Suitable tissue culture supplies and reagents are commercially available, for example, from Gibco / BRL, Nalgene-Nunc International, Sigma Chemical Co., and ICN Biomedicals.

[0103] Pluripotent stem cells can be continuously expanded in culture by those skilled in the art using culture conditions that promote proliferation without promoting differentiation. An exemplary serum-containing ES medium is made with 80% DMEM (e.g., Knock-Out DMEM, Gibco), 20% either defined fetal bovine serum (FBS, Hyclone) or serum replacement (WO98 / 30679), 1% non-essential amino acids, 1 mM L-glutamine, and 0.1 mM β-mercaptoethanol. Human bFGF is added to 4 ng / mL immediately before use (WO99 / 20741, Geron Corp.). Traditionally, ES cells are cultured on a layer of feeder cells, typically fibroblasts derived from embryonic or fetal tissue.

[0104] Multipotent SCs can be maintained in an undifferentiated state even in the absence of feeder cells. The environment for feeder-free culture includes a suitable culture substrate, especially an extracellular matrix such as Matrigel® or laminin. Usually, the enzymatic digestion is stopped before the cells are completely dispersed (e.g., about 5 min with collagenase IV). Then, clumps of about 10 to 2,000 cells are plated directly on the substrate without further dispersion.

[0105] Feeder-free culture is supported by a nutrient medium containing factors that support the growth of the cells without causing differentiation. Such factors can be introduced into the medium by culturing the medium with cells that secrete such factors, such as irradiated (approximately 4,000 rad) primary mouse embryonic fibroblasts, telomerized mouse fibroblasts, or fibroblast-like cells derived from pPS cells. The medium is cultured with approximately 5-6 × 10 cells in a serum-free medium such as KO DMEM supplemented with 20% serum replacement and 4 ng / mL bFGF. 4 cm -2 The medium can be conditioned by plating the feeders at a density of 100-200 nm. The medium conditioned for 1-2 days is further supplemented with bFGF and used to support pluripotent SC culture for 1-2 days. The characteristics of feeder-free culture methods are further discussed in International Patent Publication No. WO 01 / 51616 and Xu et al., Nat. Biotechnol. 19:971, 2001.

[0106] Under the microscope, ES cells appear with a high nuclear / cytoplasmic ratio, prominent nucleoli, and small colony formation with poorly discernible cell junctions. Primate ES cells express markers detectable with antibodies designated stage-specific embryonic antigens (SSEA) 3 and 4, as well as Tra-1-60 and Tra-1-81 (Thomson et al., Science 282:1145, 1998). Mouse ES cells can be used as a positive control for SSEA-1 and as a negative control for SSEA-4, Tra-1-60, and Tra-1-81. SSEA-4 is consistently present in human embryonic carcinoma (hEC) cells. Differentiation of pluripotent stem cells in vitro results in loss of expression of SSEA-4, Tra-1-60, and Tra-1-81, and increased expression of SSEA-1, which is also seen in undifferentiated hEG cells.

[0107] Method for producing hypoimmunogenic stem cell-derived beta cells Aspects of the present disclosure relate to generating hypoimmunogenic stem cell-derived β cells. Generally, hypoimmunogenic stem cell-derived β cells or their precursors, e.g., pancreatic progenitor cells, can include a mixture or combination of different cells, e.g., a mixture of cells such as Pdx1+ pancreatic progenitor cells, pancreatic progenitor cells co-expressing Pdx1 and NKX6-1, Ngn3-positive endocrine precursor cells, endocrine cells (e.g., β-like cells), and / or other pluripotent or stem cells.

[0108] In some embodiments, somatic cells, e.g., fibroblasts, can be isolated from a subject, e.g., a tissue biopsy, such as a skin biopsy, and reprogrammed into induced pluripotent stem cells and further differentiated to generate stem cell-derived beta cells or their precursors. In some embodiments, somatic cells, e.g., fibroblasts, are maintained in culture by methods known to those of skill in the art, and in some embodiments, expanded and then converted into stem cell-derived beta cells.

[0109] In some embodiments, the beta cells or progenitor cells thereof can be produced according to any suitable culture protocol for differentiating stem cells or pluripotent cells to a desired differentiation stage. In some embodiments, the stem cell-derived beta cells or progenitor cells thereof are produced by culturing at least one pluripotent cell for a time and under conditions suitable for the pluripotent cell to differentiate into a stem cell-derived beta cell or progenitor cell. In some embodiments, the non-native beta cells or stem cell-derived beta cells (e.g., pancreatic stem cell-derived beta cells) may be produced using methods known to those of skill in the art. In certain embodiments, the stem cell-derived beta cells may be produced using methods disclosed in WO2015 / 002724, WO2014 / 201167, WO2019 / 217493, and / or WO2020 / 247954, all of which are incorporated herein by reference.

[0110] In some embodiments, stem cells are modified to generate hypoimmunogenic stem cells that are differentiated into beta cells. In some embodiments, pluripotent or iPS cells are modified before differentiating the cells into hypoimmunogenic beta cells. In some embodiments, stem cell-derived beta cells obtained by the skilled artisan are further modified to produce hypoimmunogenic stem cell-derived beta cells. Stem cell-derived beta cells can be modified to reduce the immunogenicity of stem cell-derived beta cells, and thus may exhibit improved survival upon, for example, transplantation. In some embodiments, stem cell-derived beta cells are modified to reduce or prevent auto- and / or allogeneic rejection of transplanted SC islets. Modifications to stem cell-derived beta cells can reduce T cell activation, reduce NK cell activation, and increase survival of SC islets upon transplantation.

[0111] In some embodiments, stem cells, e.g., pluripotent stem cells, iPSCs, pancreatic progenitor cells, or non-native beta cells, are modified to overexpress or activate protective genes, e.g., to protect SC islets or SC beta cells from immune rejection. In some embodiments, stem cells, e.g., iPSCs, pancreatic progenitor cells, or non-native beta cells, are modified to reduce or eliminate expression of genes that contribute to inflammatory responses, e.g., that are directly or indirectly detrimental to SC islets or SC-beta cells. In some embodiments, non-native beta cells or precursor cells thereof are modified to perturb the JAK / STAT type II interferon (IFN) pathway. In some embodiments, stem cells are modified to perturb the IFN pathway prior to differentiating the stem cells into non-native beta cells, e.g., stem cell-derived beta cells. In some embodiments, IFNγ signaling mediators and downstream inflammatory elements of the IFN pathway are targeted. In some embodiments, stem cell-derived beta cells or precursor cells thereof are modified to reduce expression of one or more genes in the interferon (IFN) pathway. In some embodiments, the stem cell-derived beta cells or precursor cells thereof are modified to reduce or eliminate expression of one or more genes listed in Table 1. In certain embodiments, the stem cell-derived beta cells or precursor cells thereof are modified to reduce or eliminate expression of one or more genes selected from the group consisting of CXCL10 and STAT1. In one embodiment, the stem cell-derived beta cells or precursor cells thereof are modified to reduce or eliminate expression of CXCL10. In one embodiment, the stem cell-derived beta cells or precursor cells thereof are modified to reduce or eliminate expression of STAT1. In some embodiments, the stem cell-derived beta cells or precursor cells thereof are further modified to reduce or eliminate expression of a gene selected from the group consisting of B2M, HLA-A, HLA-B, and TAP1.

[0112] [Table 1]

[0113] In some embodiments, the stem cell-derived beta cells or progenitor cells thereof are modified to increase expression of one or more immune modulators or immune checkpoint inhibitors. In some embodiments, the stem cell-derived beta cells or progenitor cells thereof are modified to increase expression of one or more genes listed in Table 2. In certain embodiments, the stem cell-derived beta cells or progenitor cells thereof are modified to increase expression of one or more genes selected from the group consisting of CD47, HLA-E, SOCS1, and PD-L1. In one embodiment, the stem cell-derived beta cells or progenitor cells thereof are modified to increase expression of CD47. In one embodiment, the stem cell-derived beta cells or progenitor cells thereof are modified to increase expression of PD-L1. In one embodiment, the stem cell-derived beta cells or progenitor cells thereof are modified to increase expression of SOCS1.

[0114] [Table 2]

[0115] In some embodiments, the expression of one or more genes of stem cell-derived beta cells is modified using any gene editing tool known to those skilled in the art (e.g., TALENS, CRISPR, etc.). In some embodiments, the gene editing tool is delivered to stem cells using a retrovirus (e.g., lentivirus). In some embodiments, gene editing (e.g., CRISPR) can be used to target one or more genes to regulate the expression of one or more genes.

[0116] In some embodiments, the hypoimmunogenic stem cell-derived beta cells or their precursor cells are maintained in culture by methods known to those of skill in the art, and in some embodiments, expanded and then converted to hypoimmunogenic stem cell-derived beta cells by the methods disclosed herein.

[0117] Furthermore, at least one hypoimmunogenic stem cell-derived β cell or progenitor cell thereof, e.g., pancreatic progenitor cell, can be from any mammalian species, including, but not limited to, murine, bovine, simian, porcine, equine, ovine, or human cells. Although the description of the methods herein refers to mammalian hypoimmunogenic stem cell-derived β cells or progenitor cells thereof for clarity and simplicity, it should be understood that all methods described herein can be readily applied to hypoimmunogenic stem cell-derived β cells or progenitor cells thereof of other cell types. In some embodiments, the hypoimmunogenic stem cell-derived β cell or progenitor cell thereof is from a human individual.

[0118] In some embodiments, the hypoimmunogenic stem cell-derived beta cells or progenitor cells thereof is a substantially pure population of hypoimmunogenic stem cell-derived beta cells or progenitor cells. In some embodiments, the hypoimmunogenic stem cell-derived beta cells or progenitor cell population comprises a mixture of pluripotent or differentiated cells (e.g., a mixture of SC-beta cells, SC-alpha cells, SC-EC cells, and / or other differentiated cell types). In some embodiments, the SC islets (e.g., hypoimmunogenic SC islets) comprise a mixture of pluripotent or differentiated cells (e.g., a mixture of SC-beta cells, SC-alpha cells, SC-EC cells, and / or other differentiated cell types). In some embodiments, the hypoimmunogenic SC-beta cells or progenitor cell population is substantially free of or devoid of embryonic stem cells or pluripotent cells or iPS cells.

[0119] Low immunogenicity of stem cell-derived beta cells In some aspects of the present disclosure, modified stem cell-derived β cells (e.g., hypoimmunogenic stem cell-derived β cells) are provided. The hypoimmunogenic stem cell-derived β cells disclosed herein share many distinguishing features of native pancreatic cells, but differ in certain aspects. In some embodiments, the stem cell-derived β cells are non-native, i.e., non-endogenous cells that do not occur in nature. As used herein, "non-native" means that the modified stem cell-derived β cells are significantly different in some aspects from cells that occur in nature, i.e., native cells. However, it should be recognized that, although such significant differences may result in modified stem cell-derived β cells exhibiting certain differences, modified stem cell-derived β cells may nevertheless behave similarly to native cells, with certain functions altered (e.g., improved) compared to native cells.

[0120] The present invention is not intended to be limited to the starting cells from which the stem cell-derived beta cells are derived, and thus the stem cell-derived beta cells may be differentiated in vitro from any starting cell. Exemplary starting cells include, but are not limited to, NKX6-1+ pancreatic progenitor cells, Pdx1+ pancreatic progenitor cells, and endocrine cells or precursors thereof, such as pluripotent stem cells, embryonic stem cells, and induced pluripotent stem cells. In some embodiments, the stem cell-derived cells are differentiated in vitro from reprogrammed cells, partially reprogrammed cells (i.e., somatic cells, e.g., fibroblasts that have been partially reprogrammed to exist in an intermediate state between an induced pluripotent cell and the somatic cell from which it is derived), transdifferentiated cells. In some embodiments, the stem cell-derived beta cells disclosed herein may be differentiated in vitro from endocrine cells or precursors thereof. In some embodiments, the stem cell-derived beta cells are differentiated in vitro from precursor cells selected from the group including NKX6-1+ pancreatic progenitor cells, Pdx1+ pancreatic progenitor cells, and pluripotent stem cells. In some embodiments, the pluripotent stem cells are selected from the group comprising embryonic stem cells and induced pluripotent stem cells. In some embodiments, the stem cell-derived beta cells or the pluripotent stem cells from which the stem cell-derived beta cells are derived are human. In some embodiments, the stem cell-derived beta cells are human.

[0121] In some embodiments, the stem cell-derived beta cells are modified to overexpress or activate protective genes. In some embodiments, the stem cell-derived beta cells are modified to eliminate genes that contribute to inflammatory responses. In certain embodiments, the stem cell-derived beta cells contain perturbations in the IFN pathway, e.g., modifications to IFNγ signaling mediators and / or downstream inflammatory elements. Stem cell-derived beta cells containing perturbations in the IFN pathway can be hypoimmunogenic stem cell-derived beta cells.

[0122] Hypoimmunogenic stem cell-derived β cells may exhibit a reduced risk of auto- and / or allogeneic rejection upon transplantation. Additionally, hypoimmunogenic stem cell-derived β cells may exhibit, for example, reduced T cell activation, reduced NK cell activation, and / or increased survival upon transplantation. In some embodiments, hypoimmunogenic stem cell-derived β cells may evade the immune system upon transplantation. In some embodiments, hypoimmunogenic stem cell-derived β cells exhibit improved cell survival under immune rejection.

[0123] In some embodiments, the hypoimmunogenic stem cell-derived beta cells comprise reduced or absent expression of one or more genes selected from Table 1. In certain embodiments, the hypoimmunogenic stem cell-derived beta cells comprise reduced expression of one or more genes selected from the group consisting of CXCL10 and STAT1. In one embodiment, the hypoimmunogenic stem cell-derived beta cells do not express CXCL10. In one embodiment, the hypoimmunogenic stem cell-derived beta cells do not express STAT1. In some embodiments, the hypoimmunogenic stem cell-derived beta cells further comprise reduced expression of one or more genes selected from the group consisting of B2M, HLA-A, HLA-B, and TAP1. In some embodiments, the hypoimmunogenic stem cell-derived beta cells comprise increased or activated expression of one or more genes selected from Table 2. In certain embodiments, the hypoimmunogenic stem cell-derived beta cells comprise increased or activated expression of one or more genes selected from the group consisting of CD47, PD-L1, SOCS1, and HLA-E. In some embodiments, the hypoimmunogenic stem cell-derived beta cells comprise increased expression of CD47. In some embodiments, the hypoimmunogenic stem cell-derived beta cells comprise increased expression of PD-L1, hi some embodiments, the hypoimmunogenic stem cell-derived beta cells comprise increased expression of SOCS1.

[0124] In some embodiments, the hypoimmunogenic stem cell-derived beta cells are not genetically modified. In some embodiments, the hypoimmunogenic stem cell-derived beta cells acquire characteristics in common with native cells without genetic modification of the cells. In some embodiments, the hypoimmunogenic stem cell-derived beta cells are genetically modified.

[0125] In some aspects, the disclosure provides cell lines comprising the hypoimmunogenic stem cell-derived β cells described herein. In some aspects, the disclosure provides SC islets comprising the hypoimmunogenic stem cell-derived β cells described herein in combination with SC-α cells, SC-δ cells, and / or SC-EC cells.

[0126] In some embodiments, the cells described herein, e.g., a population of hypoimmunogenic stem cell-derived beta cells, can be transplanted, e.g., a population of hypoimmunogenic stem cell-derived beta cells can be administered to a subject. In some embodiments, SC islets comprising hypoimmunogenic stem cell-derived beta cells can be transplanted, e.g., SC islets can be administered to a subject. In some embodiments, the subject receiving the population of hypoimmunogenic stem cell-derived beta cells is the same subject from which the pluripotent stem cells used for differentiation into hypoimmunogenic stem cell-derived beta cells were obtained (e.g., for autologous cell therapy). In some embodiments, the subject is a different subject. In some embodiments, the subject suffers from an intestinal disease, such as enteritis, or is a normal subject. For example, the cells for transplantation (e.g., a composition comprising a population of hypoimmunogenic stem cell-derived beta cells, or a SC islet comprising a population of hypoimmunogenic stem cell-derived beta cells) can be in a form suitable for transplantation, e.g., organ transplantation.

[0127] The method may further comprise administering the cells to a subject in need thereof, e.g., a mammalian subject, e.g., a human subject. The source of the cells may be a mammal, preferably a human. The source or recipient of the cells may also be a non-human subject, e.g., an animal model. The term "mammal" includes organisms including mice, rats, cows, sheep, pigs, rabbits, goats, horses, monkeys, dogs, cats, and preferably humans. Similarly, transplantable cells may be obtained from any of these organisms, including non-human transgenic organisms. In one embodiment, the transplantable cells are genetically engineered, e.g., the cells are genetically engineered to contain an exogenous gene or to inactivate or modify an endogenous gene.

[0128] A composition comprising a population of hypoimmunogenic stem cell-derived cells (e.g., hypoimmunogenic pancreatic stem cell-derived cells such as SC-β cells and / or SC-α cells) can be administered to a subject using an implantable device. Implantable devices and related technologies are well known in the art and are useful as delivery systems when continuous or sustained release of the compounds or compositions described herein is desired. In addition, implantable device delivery systems are useful for targeting a specific point of delivery of the compound or composition (e.g., localized site, organ) (Negrin et al., Biomaterials, 22(6):563 (2001). Timed release technologies involving alternative delivery methods can also be used in the present invention. For example, timed release formulations based on polymeric technologies, sustained release technologies, and encapsulation technologies (e.g., polymers, liposomes) can also be used for delivery of the compounds and compositions detailed herein.

[0129] For administration to a subject, a cell population produced by a method as disclosed herein, e.g., hypoimmunogenic stem cell-derived cells (e.g., hypoimmunogenic stem cell-derived β cells) or SC islets comprising hypoimmunogenic stem cell-derived cells, can be administered to a subject, for example, in a pharma- ceutically acceptable composition. These pharma-ceutically acceptable compositions comprise a therapeutically effective amount of such a hypoimmunogenic stem cell-derived cell population formulated with one or more pharma-ceutically acceptable carriers (additives) and / or diluents.

[0130] As described in detail below, the pharmaceutical compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for (1) oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), lozenges, dragees, capsules, pills, tablets (e.g., buccal, sublingual, and those targeted for systemic absorption), boluses, powders, granules, pastes for application to the tongue, (2) parenteral administration, e.g., as a sterile solution or suspension, or as a sustained release formulation, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection, (3) topical application, e.g., as a cream, ointment, or controlled release patch or spray applied to the skin, (4) vaginal or rectal administration, e.g., as a pessary, cream, or foam, (5) sublingual administration, (6) ophthalmic administration, (7) transdermal administration, (8) transmucosal administration, or (9) nasal administration. Additionally, the compounds may be implanted into the patient or injected using a drug delivery system. See, e.g., Urquhart, et al., Ann. Rev. Pharmacol. Toxicol. 24: 199-236 (1984); Lewis, ed. "Controlled Release of Pesticides and Pharmaceuticals" (Plenum Press, New York, 1981); U.S. Pat. No. 3,773,919; and U.S. Pat. No. 353,270,960.

[0131] As used herein, the term "pharmacologically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio.

[0132] As used herein, the term "pharmaceutically acceptable carrier" refers to a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricants, talc, magnesium, calcium, or zinc stearate, or steric acid), or solvent encapsulating material involved in carrying or transporting a compound of interest from one organ or part of the body to another. Each carrier should be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient. Examples of substances which may function as pharma- ceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants, such as magnesium stearate, sodium lauryl sulfate, and talc; (8) lubricants, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil. (10) oils, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffer solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids; (23) serum components, such as serum albumin, HDL, and LDL; (24) C2-C 12Alcohols, e.g., ethanol; and (25) other non-toxic, compatible substances used in pharmaceutical formulations. Wetting agents, coloring agents, releasing agents, coating agents, sweetening agents, flavoring agents, perfumes, preservatives and antioxidants may also be present in the formulation. The terms "excipient", "carrier", "pharmaceutical acceptable carrier", and the like, are used interchangeably herein.

[0133] The phrase "therapeutically effective amount" as used herein with respect to a cell population refers to an amount of relevant cells in a cell population, such as hypoimmunogenic stem cell-derived cells, or a composition comprising hypoimmunogenic stem cell-derived cells of the present invention, that is effective to produce some desired therapeutic effect on at least a subpopulation of cells in an animal, with a reasonable benefit / risk ratio applicable to any medical treatment. For example, the administration of a population of hypoimmunogenic stem cell-derived cells to a subject is sufficient to produce a statistically significant and measurable change in at least one symptom of type 1, type 1.5 or type 2 diabetes, such as glycosylated hemoglobin levels, fasting blood glucose levels, hypoinsulinemia, etc. The determination of a therapeutically effective amount is well within the capabilities of one of ordinary skill in the art. In general, a therapeutically effective amount may vary depending on the subject's medical history, age, condition, sex, as well as the severity and type of the condition in the subject, and the administration of other pharmacologic active agents.

[0134] As used herein, the term "administering" refers to placing a composition in a subject by a method or route that results in at least partial localization of the composition at a desired site so that a desired effect occurs. The compounds or compositions described herein may be administered by any suitable route known in the art, including, but not limited to, oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, nasal, rectal, and topical (including buccal and sublingual) administration.

[0135] Exemplary modes of administration include, but are not limited to, injection, infusion, instillation, inhalation, or ingestion. "Injection" includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. In a preferred embodiment, the composition is administered by intravenous infusion or injection.

[0136] "Treating," "preventing," or "ameliorating" a disease or disorder means delaying or preventing the onset of such disease or disorder, reversing, alleviating, ameliorating, inhibiting, slowing or halting the progression or severity of the conditions associated with such disease or disorder. In one embodiment, symptoms of the disease or disorder are alleviated by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%.

[0137] Treatment of diabetes is determined by standard medical methods. The goal of diabetes treatment is to lower sugar levels as close to normal as safely possible. Commonly set goals are 80-120 milligrams per deciliter (mg / dl) before meals and 100-140 mg / dl at bedtime. A particular physician may set different goals for a patient depending on other factors such as how often the patient has hypoglycemic reactions. Useful medical tests include testing the patient's blood and urine to measure blood glucose levels, testing for glycosylated hemoglobin levels (HbA1c; an indicator of average blood glucose levels over the past 2-3 months, normal range is 4-6%), testing for cholesterol and fat levels, and testing for urinary protein levels. Such tests are standard tests known to those skilled in the art (see, for example, American Diabetes Association, 1998). A successful treatment program can also be determined by the patient's low incidence of diabetes-related complications, such as eye disease, kidney disease, or nerve disease.

[0138] Delaying the onset of diabetes in a subject refers to delaying the onset of at least one symptom of diabetes, e.g., hyperglycemia, hypoinsulinemia, diabetic retinopathy, diabetic nephropathy, blindness, memory loss, renal failure, cardiovascular disease (including coronary artery disease, peripheral artery disease, cerebrovascular disease, atherosclerosis, and hypertension), neuropathy, autonomic dysfunction, hyperglycemic hyperosmolar coma, or a combination thereof, for at least 1 week, at least 2 weeks, at least 1 month, at least 2 months, at least 6 months, at least 1 year, at least 2 years, at least 5 years, at least 10 years, at least 20 years, at least 30 years, at least 40 years or more, and can include the entire lifespan of the subject.

[0139] In certain embodiments, the subject is a mammal, such as a primate, such as a human. The terms "patient" and "subject" are used interchangeably herein. Preferably, the subject is a mammal. The mammal may be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow. A non-human mammal may be advantageously used as a subject representing an animal model of type 1 diabetes, type 2 diabetes, or a pre-diabetic condition. Furthermore, the methods described herein may be used to treat livestock animals and / or pets. The subject may be male or female. The subject may be one who has previously been diagnosed or identified as suffering from or having diabetes (e.g., type 1 or type 2), one or more complications associated with diabetes, or a pre-diabetic condition, and optionally, need not have already been treated for diabetes, one or more complications associated with diabetes, or a pre-diabetic condition. The subject may also be one who does not suffer from diabetes or a pre-diabetic condition. The subject may also be a person who has been diagnosed with or identified as suffering from diabetes, one or more complications associated with diabetes, or a pre-diabetic condition, but who has received one or more treatments for diabetes, one or more complications associated with diabetes, or a pre-diabetic condition, and as a result shows an improvement in known diabetes risk factors. Alternatively, the subject may not have been previously diagnosed with diabetes, one or more complications associated with diabetes, or a pre-diabetic condition. For example, the subject may be a subject who shows one or more risk factors for diabetes, one or more complications associated with diabetes, or a pre-diabetic condition, or a subject who does not show risk factors for diabetes, or a subject who is asymptomatic for diabetes, one or more complications associated with diabetes, or a pre-diabetic condition. The subject may also be a person who has or is at risk of developing diabetes or a pre-diabetic condition. The subject may also be a person who has been diagnosed with or identified as having one or more complications associated with diabetes or a pre-diabetic condition, as defined herein, or the subject may not have been previously diagnosed with or identified as having one or more complications associated with diabetes or a pre-diabetic condition.

[0140] As used herein, a "subject in need of pancreatic hypoimmunogenic stem cell-derived cells" refers to a subject who has been diagnosed or identified as suffering from, having, or at risk of developing diabetes (e.g., type 1, type 1.5, or type 2), one or more complications associated with diabetes, or a pre-diabetic condition.

[0141] The subject who needs the population of low immunogenicity pancreatic stem cell-derived cells can be identified by any method used for diagnosing diabetes.For example, type 1 diabetes can be diagnosed by glycosylated hemoglobin (A1C) test, random blood glucose test, and / or fasting blood glucose test.The parameters for diagnosing diabetes are known in the art and can be obtained by those skilled in the art without much effort.

[0142] In some embodiments, the method of the present invention further comprises selecting the subject identified as needing additional pancreatic hypoimmunogenic stem cell derived cells.The subject needing a population of pancreatic hypoimmunogenic stem cell derived cells can be selected based on presented symptoms, such as type 1, type 1.5 or type 2 diabetes symptoms. Exemplary symptoms of diabetes include, but are not limited to, excessive thirst (polydipsia), frequent urination (polyuria), extreme hunger (polyphagia), extreme fatigue, weight loss, hyperglycemia, low insulin levels, hyperglycemia (e.g., blood glucose levels above 250 mg, above 300 mg), presence of ketones in the urine, fatigue, dry and / or itchy skin, blurred vision, slow healing of cuts or sores, more infections than usual, numbness and tingling in the feet, diabetic retinopathy, diabetic nephropathy, blindness, memory loss, kidney failure, cardiovascular disease (including coronary artery disease, peripheral artery disease, cerebrovascular disease, atherosclerosis, high blood pressure), neuropathy, autonomic dysfunction, hyperglycemic hyperosmolar coma, and combinations thereof.

[0143] In some embodiments, compositions comprising a population of hypoimmunogenic stem cell-derived cells for administration to a subject may further comprise a pharma- ceutically active agent, such as an agent known in the art as a treatment for diabetes and / or having antihyperglycemic activity, for example, dipeptidyl peptidase 4 (DPP-4) inhibitors (e.g., alogliptin, linagliptin, saxagliptin, sitagliptin, vildagliptin, and berberine), biguanides (e.g., metformin, buformin, and phenformin), peroxisome proliferator-activated receptor (PPAR) modulators such as thiazolidinediones (TZDs) (e.g., pioglitazone, rivoglitazone, rosiglitazone, and troglitazone), dual PPAR agonists (e.g., aleglitazar, muraglitazar, and tesaglitazar), sulfonylureas (e.g., acetonitrile, cefolia, and cefolia), and the like. hexamide, carbutamide, chlorpropamide, gliclazide, tolbutamide, tolazamide, glibenclamide (glyburide), glipizide, gliquidone, glyclopyramide, and glimepiride), meglitinides ("glinides") (e.g., nateglinide, repaglinide, and mitiglinide), glucagon-like peptide-1 (GLP-1) and its analogs (e.g., exendin-4, exenatide, liraglutide, albiglutide), insulin and insulin analogs (e.g., insulin lispro, insulin aspart, insulin glargine, insulin detemir, Exubera, and NPH insulin), α-glucosidase inhibitors (e.g., acarbose, miglitol, and voglibose), amylin analogs (e.g., pramlintide), sodium-dependent glucose cotransporter T2 (SGLT T2) inhibitors (e.g., dapriflozin, remogliflozin, and sergliflozin), and others (e.g., benfluorex, and trestat).

[0144] The composition comprising hypoimmunogenic stem cell-derived cells can be administered to a subject at the same time as or at a different time from the administration of a pharmacologic agent or a composition comprising the same. When administered at different times, the composition comprising hypoimmunogenic stem cell-derived cells and / or the pharmacologic agent for administration to a subject can be administered within 5 minutes, 10 minutes, 20 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 8 hours, 12 hours, 24 hours of the other. When the composition comprising hypoimmunogenic stem cell-derived cells and the composition comprising the pharmacologic agent are administered as different pharmaceutical compositions, the routes of administration can be different. In some embodiments, the subject is administered a composition comprising hypoimmunogenic stem cell-derived cells. In other embodiments, the subject is administered a composition comprising a pharmacologic agent. In another embodiment, the subject is administered a composition comprising hypoimmunogenic stem cell-derived cells mixed with a pharmacologic agent. In another embodiment, a subject is administered a composition comprising a population of hypoimmunogenic stem cell-derived cells and a composition comprising a pharma- ceutically active agent, the administrations being substantially simultaneous or subsequent to each other.

[0145] Toxicity and therapeutic efficacy of administration of compositions comprising populations of stem cell-derived cells can be determined by standard pharmaceutical procedures using cell cultures or experimental animals, for example to determine LD50 (the dose lethal to 50% of the population) and ED50 (the dose therapeutically effective in 50% of the population). Compositions comprising populations of low immunogenic stem cell-derived cells that exhibit large therapeutic indices are preferred.

[0146] The amount of a composition comprising a population of stem cell-derived cells with low immunogenicity can be tested using a number of established animal models.

[0147] Nonobese diabetic (NOD) mice have a genetic defect that causes insulitis at a few weeks of age (Yoshida et al., Rev. Immunogenet. 2:140, 2000). 60-90% of female mice develop overt diabetes by 20-30 weeks of age. The immune-related pathology appears to be similar to that of human type 1 diabetes. Other models of type 1 diabetes include transgenic and knockout mutant mice (Wong et al., Immunol. Rev. 169:93, 1999). Recently, Lenzen et al. reported a rat model of spontaneous type 1 diabetes (Diabetologia 44:1189, 2001). Hyperglycemia can also be induced in mice (glucose >500 mg / dL) by a single intraperitoneal injection of streptozotocin (Soria et al., Diabetes 49:157, 2000) or by continuous administration of low doses of streptozotocin (Ito et al., Environ. Toxicol. Pharmacol. 9:). To examine the efficacy of the transplanted islet cells, mice are monitored for the return of glucose to normal levels (<200 mg / dL).

[0148] Large animals provide a good model for tracking the sequelae of chronic hyperglycemia. Dogs can be made insulin-dependent by removing the pancreas (J. Endocrinol.158:49,2001) or feeding galactose (Kador et al.,Arch.Opthalmol.113:352,1995). The Keeshond is a genetic model for type 1 diabetes (Am.J.Pathol.105:194,1981). Early studies using the dog model (Banting et al.,Can.Med.Assoc.J.22:141,1922) led to the discovery in February 1925 of a group of two or three Canadians who made a long voyage to Stockholm.

[0149] In some embodiments, data obtained from cell culture assays and animal studies can be used in formulating a range of dosages for use in humans. The dosage of such compounds preferably falls within a range of circulating concentrations that include the ED50 with little or no toxicity. Dosages can vary within this range depending on the dosage form employed and the route of administration utilized.

[0150] The therapeutically effective dose of a composition comprising a population of hypoimmunogenic stem cell-derived cells can be estimated initially from cell culture assays, or the effect of any particular dosage can be monitored by a suitable bioassay.

[0151] With regard to duration and frequency of treatment, a skilled clinician will typically monitor the subject to determine when a therapeutic benefit is being achieved and decide whether to increase or decrease the dosage, increase or decrease the frequency of administration, discontinue treatment, resume treatment, or make other changes to the treatment regimen. The administration schedule can vary from once a week to daily, depending on various clinical factors, such as the subject's sensitivity to the stem cell-derived cells. The desired dosage can be administered all at once, or in smaller doses, e.g., 2-4 doses, administered over a period of time, e.g., at suitable intervals throughout the day, or on any other suitable schedule. Such divided doses can be administered as unit dosage forms. In some embodiments, administration is chronic, e.g., one or more doses per day over a period of several weeks or months. Exemplary administration schedules are daily, twice a day, three times a day, or four or more doses per day over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months or more.

[0152] In another aspect of the invention, a method provides for the use of an isolated population of hypoimmunogenic stem cell-derived cells as disclosed herein. In one embodiment of the invention, an isolated population of hypoimmunogenic stem cell-derived cells as disclosed herein can be used for the manufacture of a pharmaceutical composition for use in transplantation into a subject in need of treatment, such as, for example, a subject having diabetes or at risk of developing diabetes, including, but not limited to, a subject having congenital and acquired diabetes. In one embodiment, the isolated population of hypoimmunogenic stem cell-derived cells can be genetically modified. In another aspect, the subject has or is at risk of having diabetes and / or a metabolic disorder. In some embodiments, the isolated population of hypoimmunogenic stem cell-derived cells disclosed herein can be autologous and / or allogeneic. In some embodiments, the subject is a mammal, and in other embodiments, the mammal is a human.

[0153] The use of an isolated population of hypoimmunogenic stem cell-derived cells as disclosed herein provides an advantage over existing methods because the hypoimmunogenic stem cell-derived cell population can be differentiated from stem cell-derived endocrine precursor cells or their precursors, such as iPS cells, obtained or harvested from a subject administered the hypoimmunogenic stem cell-derived cell isolated population. This is highly advantageous in that it provides a renewable source of hypoimmunogenic stem cell-derived cells that can be differentiated into endocrine precursor cells from stem cells by methods commonly known to those skilled in the art, and further differentiated into pancreatic β-like cells, particularly SC islets comprising hypoimmunogenic pancreatic β-like cells, by the methods described herein, for transplantation into a subject, without the risks and limitations of cells derived from other systems.

[0154] One embodiment of the invention relates to a method of treating diabetes or a metabolic disorder in a subject comprising administering to a subject having diabetes and / or a metabolic disorder an effective amount of a composition comprising a population of hypoimmunogenic stem cell-derived cells (e.g., hypoimmunogenic pancreatic stem cell-derived cells) as disclosed herein. In a further embodiment, the invention provides a method of treating diabetes comprising administering to a subject having or at increased risk of developing diabetes a composition comprising a population of hypoimmunogenic stem cell-derived cells as disclosed herein.

[0155] In one embodiment of the above method, the subject is a human and the population of hypoimmunogenic stem cell-derived cells as disclosed herein are human cells. In some embodiments, the present invention contemplates that the population of hypoimmunogenic stem cell-derived cells as disclosed herein are administered directly to the pancreas of the subject or administered systemically. In some embodiments, the population of hypoimmunogenic stem cell-derived cells as disclosed herein may be administered to any suitable location within the subject, for example, within the capsule of a blood vessel or liver.

[0156] The present invention is also directed to a method of treating a subject having diabetes or metabolic disorders resulting from genetic defects, physical injury, external stimuli or environmental conditioning, poor health, obesity, and other diabetes risk factors commonly known to those skilled in the art. The effectiveness of treatment of a subject administered a composition comprising a population of hypoimmunogenic stem cell-derived cells (e.g., hypoimmunogenic pancreatic stem cell-derived cells) can be monitored by clinically accepted standards and tests, including, for example: (i) Glycosylated hemoglobin (A1C) test, which indicates the subject's average blood glucose level over the past 2-3 months by measuring the percentage of blood glucose attached to hemoglobin, the oxygen-carrying protein in red blood cells. The higher the blood glucose level, the higher the percentage of hemoglobin with sugar attached. An A1C value of 6.5% or higher on two tests indicates diabetes. A test value of 6-6.5% suggests that the subject has prediabetes. (ii) Random blood glucose tests. A blood sample is drawn from the subject at random times, and a random blood glucose value of 200 milligrams per deciliter (mg / dL), or 11.1 millimoles per liter (mmol / L), or greater, indicates that the subject has diabetes. (iii) Fasting Blood Glucose Test. The subject's blood is drawn after fasting overnight. A fasting blood glucose value between 70 and 99 mg / dL (3.9 and 5.5 mmol / L) is normal. A fasting blood glucose value of 126 mg / dL (7 mmol / L) or greater over two tests indicates diabetes. A blood glucose value between 100 and 125 mg / dL (5.6 and 6.9 mmol / L) indicates prediabetes. (iv) Oral Glucose Tolerance Test. Blood is drawn after the subject has fasted for at least 8 hours or overnight, then ingests a glucose solution, and blood glucose is measured two hours later. A blood glucose value less than 140 mg / dL (7.8 mmol / L) is normal. A person is considered to have prediabetes if their blood sugar level is between 140 and 199 mg / dL (7.8 and 11 mmol / L). This is sometimes called impaired glucose tolerance (IGT). A blood sugar level of 200 mg / dL or higher may indicate diabetes.

[0157] In some embodiments, the effect of administering a hypoimmunogenic stem cell-derived cell population (e.g., hypoimmunogenic pancreatic stem cell-derived cells) as disclosed herein to a subject in need thereof is associated with improved exercise tolerance or other quality of life measures, and reduced mortality. The effect of cell therapy with hypoimmunogenic stem cell-derived cell populations is evident within days to weeks after treatment. However, beneficial effects may be observed as early as hours after treatment and may persist for several years. In some embodiments, the effect of cell therapy with hypoimmunogenic stem cell-derived cell populations is apparent within two weeks after procedure.

[0158] In some embodiments, the hypoimmunogenic stem cell-derived cell population (e.g., hypoimmunogenic pancreatic stem cell-derived cells) as disclosed herein may be used for tissue reconstruction or regeneration in human patients or other subjects in need of such treatment. In some embodiments, the composition of the hypoimmunogenic stem cell-derived cell population may be administered in a manner that allows them to attach or migrate to the intended tissue site and reconstruct or regenerate the functionally deficient site. Specialized devices are available that are suitable for administering cells capable of reconstructing hypoimmunogenic pancreatic cell populations (e.g., beta cells) to the pancreas or another desired site. Thus, the hypoimmunogenic stem cell-derived cells may be administered by injection into the pancreas of the recipient subject or by intramuscular injection. EXAMPLES

[0159] One hundred years ago, the first diabetic patient with type 1 diabetes (T1D) was treated with “pancreatic extract” in Toronto as part of a series of experiments that would later lead to the discovery of insulin (Banting et al., 1922). Since then, the scientific basis of T1D has been explained as an autoimmune attack of insulin-producing β cells in the pancreas. However, despite technological advances such as insulin pumps and continuous glucose monitoring devices (Kovatchev, 2019), exogenous insulin administration remains the only option to regulate blood glucose levels. However, in the past decades, β cell replacement strategies have emerged as a new hope for patients, starting with cadaveric islet transplantation that demonstrated insulin independence (Shapiro et al., 2000), followed by human induced pluripotent cells (hPSCs) being investigated as an inexhaustible source of β cell differentiation and replacement.

[0160] Despite these advances, protecting SC islets from autoimmune alloresponses and recurrent β-cell destruction remains a major challenge. The use of immunosuppressive drugs can cause complications and may lead to graft failure in the long term (Lehmann et al., 2008). Encapsulation methods and cell capture devices are useful for immune protection and possible graft extraction, but there are problems with maintaining oxygen and nutrients within the graft, and insulin release from the graft, while avoiding issues such as fibrotic foreign body reactions and hypoxia (Kharbiker et al., 2021) (Alagpulinsa et al., 2019).

[0161] Although regulation of the immune system by regulatory T cells has been proposed (Raffin et al., 2020), recently, considerable efforts have been expended on genetically modifying hPSC4s to generate donor universal lines for cell replacement therapy that can be transplanted “naked” without the use of immunosuppressive drugs. Strategies include β-2-microglobulin (B2M) or HLA-I / II depletion to prevent donor cell presentation of allo- and autoantigens (Castro-Gutierrez et al., 2021; Deuse et al., 2019; Han et al., 2019; Parent et al., 2021; Wang et al., 2015), as well as expression of immune checkpoint inhibitors such as PD-L1 (Castro-Gutierrez et al., 2021; Yoshihara et al., 2020). Other approaches, such as expression of CD47 (Deuse et al., 2021; Deuse et al., 2019) and HLA-E (Gornalusse et al., 2017), face NK killing when HLA-A, -B, -C are directly or indirectly ablated (e.g., by B2M knockout), or by ablation of only HLA-A and HLA-B in iPSCs but retaining one HLA-C allele, meaning that only a small number of matched strains are needed to cover most of the recipient population worldwide (Xu et al., 2019). Although these strategies are extremely promising, they are all based on previous knowledge based on many studies in cancer, pathogens, or maternal-fetal interactions. There is limited understanding of newly discovered endocrine cell-associated targets for immune regulation against β-cell attack (Cai et al., 2020; Wei et al., 2018).

[0162] result Single-cell transcriptional analysis reveals "alarm" genes that induce immunogenicity in SC islets and lead to T cell activation and killing.

[0163] To investigate immune responses in the context of human allograft rejection, Hu-PBL-NSG-MHC ヌル The platform chosen (Brehm et al., 2019) was NOD-scid IL-2 receptor subunit gamma (IL2rg) lacking mouse MHC class I and II. ヌル (NSG) immunodeficient mice were transplanted (under the kidney capsule) with SC islets (HLA-A2 positive) followed by human PBMC injections from healthy mismatched donors (HLA-A2 negative) (n = 6 mice, referred to as "humanized"), while half of the cohort (n = 6 mice) were not injected as controls (Figure 1A). The lack of mouse MHC allowed long-term monitoring of functional grafts without the risk of xenograft-versus-host disease (GVHD). Graft dysfunction due to rejection was measured by bioluminescence from transplanted GAPDH-luciferase SC islets (Gerace et al., 2021) (Figure 1C-1D) or by human insulin detection from mouse blood (Figure 1D) 30 min after glucose injection. The reduction in graft size (Figure 1B-1C) and loss of function due to glucose sensitivity were mainly due to T cells retained in mouse tissues throughout the experiment (Figure 1G). Furthermore, CD8 cytotoxic T cells (CTLs) are clearly seen to infiltrate SC islet grafts in humanized mice at early time points (Figure 1E) (control mice show no T cells, only autofluorescence from red blood cells) and to be in contact with endocrine cells (chromogranin A positive) and SC-β cells (C-peptide positive). SC islets are composed of several pancreatic hormone-producing cell populations, including glucagon-expressing SC-α cells and insulin-expressing SC-β cells. Ten weeks after PBMC injection, the numbers of both SC-α and SC-β were observed to be decreased in humanized mouse grafts compared to controls (Figure 1F), which is typical of a nonselective alloreaction.

[0164] As graft destruction was not complete and endocrine cells remained in the humanized mouse grafts, graft extraction and scRNA-seq analysis were performed. Extracted grafts were dispersed into single cells and enriched for human cells using mouse cell depletion magnetic beads. These samples, in addition to pre-transplanted SC islets and pre-injected PBMCs (cryopreserved from the same batch / donor), were used for the preparation of 10x Genomics 3' Chromium expression libraries and Illumina NovaSeq sequencing. After filtration and clearing, mouse kidney cells were used for analysis. Datasets from multiple graft and cell samples were then merged using Seurat. As seen in the UMAP plots (Figure 1H and Figure 7C), the integrated engrafted endocrine cells (SC-Endo) from control and humanized mice appeared to maintain their cellular identity based on genetic markers of SC-α (INS-GCG+), SC-β (INS+GCG-) and the recently identified (Veres et al., 2019) SC-enterochromaffin cells (SC-EC; TPH1+). Humanized mouse grafts consisted of fewer endocrine cells (Figure 7B) compared to control grafts (a reduction of approximately 50%), consistent with what was observed in flow cytometry staining (Figure 1F). We next performed pseudobulk differential expression analysis of the different SC-Endo types to compare engrafted cells from humanized and control mice. SC-α, SC-β, and SC-EC showed a similar pattern of upregulated genes in immune-infiltrated (humanized) grafts, which differed only in expression levels compared to control grafts (Figure 1I and Figure 7D). This suggests that the response is not restricted to a specific cell population within the SC islet, but that all cells contribute to some degree to its progression. Among the most highly upregulated were prominent transcripts involved in antigen processing and presentation (B2M, HLA-A, -B, -C, -F, TAP1 / 2, CD74), inflammatory pathway mediators (STAT1, JAK1 / 2, IRF1 / 2), and proinflammatory cytokines such as IL32. When translated into proteins, these transcripts are known to induce T cell activation and contribute to the inflammatory environment.Furthermore, upregulated genes that are inhibitory to the immune system were identified, namely HLA-E, SOCS1, CD274 (PD-L1), WARS, and CD47. Expression of these genes is evidence of induction of interferon type I (IFNα / β) and type II (IFNγ) pathways via JAK / STAT signaling. Other genes play diverse roles in relation to these pathways (e.g., PSMB9, PLAAT4, ISG20) (Figure 1I and Figure 7D). Pathway analysis (Panther / Reactome) and gene ontology (GO) confirm that the SC islet response is IFN-induced and "alerts" the immune system through antigen presentation and cytokine secretion while inducing apoptosis of target cells (Figure 7D). Because IFN-inducible genes were upregulated, we selected several to focus on in the following experiments (Figs. 4–6), and in this analysis, we examined their expression in SC-Endo, including the IFNγ receptor gene IFNGR1, although no obvious changes were observed in humanized mouse grafts (Fig. 1J).

[0165] Correlating with the alert state of SC islet cells, human CD4 and CD8 T cells identified in explant scRNA-seq data from humanized mice displayed expression profiles of activation (e.g., CD69, CD40LG), cytokine and chemokine signaling (e.g., IFNG), and killing (CD8 CTLs via GZMB, a.k.a. Granzyme B) when compared to native PBMCs (pre-injected).

[0166] SC islets are involved in early immune cell activation via "alarm" genes as revealed by in vitro single-cell transcriptional analysis Although the use of the humanized mouse model used in this study proved to be powerful in recapitulating the alloimmune challenge resulting in reduction of SC islet mass and loss of function, it also has some major limitations to this study: 1) NK cells were not present in the long-term experiments, rather than total PBMC injected, and the model is limited to T cell responses, and 2) due to variability in HLA matching of human donors to Hues8 SC islets, the dynamics of graft rejection differed between the PBMCs of the donors used (Shultz et al., 2019). Thus, it proved difficult to pinpoint the exact timing of the appearance of genes that define the progression of graft rejection.

[0167] Therefore, we designed and performed an in vitro experiment in which SC islet clusters were enriched for SC-β (using CD49A magnetic sorting to deplete non-endocrine and undifferentiated cells) as previously reported (Veres et al., 2019), dissociated, and reaggregated in V-shaped 96-well plates to obtain uniform counts between each well. SC islets were then co-cultured with human allogeneic PBMCs for 24 and 48 h. As a control (t=0), SC islets were maintained in culture without the addition of PBMCs. These samples (t=0), besides PBMCs without co-culture, were used for 10× Genomics 3' Chromium expression library preparation and sequencing (Figure 2A). PBMCs were derived from the same donor used in vivo (Figure 1). Prior to co-culture, all SC islets were pre-treated with thapsigargin to induce ER stress in endocrine cells. As previously reported, ER stressed SC islets can induce and accelerate T cell activation (Leite et al., 2020). Focusing on cell populations of interest defined by genetic markers, we performed differential expression analysis of the combined data from all samples (Figures 2 and 8).

[0168] When compared to non-cocultured samples, CD4 and CD8 T cells and NK cells at 24 and 48 h of coculture displayed a gene expression profile of immune activation (Figures 2B and 8D). Genes transcribed for T cell costimulatory molecules (CD28, CD58 (LFA-3), CD40LG, TNFRSF9 (4-1BB), TNFRSF4 (OX40), etc.) and other activation markers (CD69, IL2RA (CD25), CD38) are upregulated in T cells, as are inhibitory and exhaustion markers (HAVCR2 (TIM-3), LAG3, PDCD1 (PD-1) (Figure 2B, left)). Co-proinflammatory cytokines (IFN G and TNF) and chemokines (XCL1 / 2) are expressed over time in NK and T cells, whereas anti-inflammatory cytokines (IL10 and TGFB1) are not detected or are downregulated. Sensitization of T cells and NK to proinflammatory chemokines is increased based on elevated levels of CXCR3, a chemokine receptor that binds CXCL9 / 10 / 11 (Fig. 2B, center). Other major transcripts are those involved in the killing function of CTL and NK (Fig. 2B, right: PRF1 (perforin), GZMB (granzyme B), FASLG), further highlighting their role in allo-attack in the co-culture system.

[0169] Due to CD49A enrichment performed prior to the co-culture experiments, we detected a large number of SC-α and SC-β, but only a very low number of SC-EC cells. Therefore, we focused on the differential expression of SC-α and SC-β cells in co-cultured cells compared to the control without PBMCs. Similar to what was observed in the in vivo analysis (Figure 1), in the co-culture experiments, the upregulated profile was not different between co-cultured SC-α and SC-β cells (Figures 2C-2D), and consisted of a clear IFN response via the JAK / STAT pathway, suggesting a link with T cell activation, apoptotic signaling, and allogeneic rejection (Figures 2E-2F, Figures 8D-8E, pathway analysis and GSEA). Unlike in vivo experiments, in the short 24-48 h co-culture time frame, chemokine signaling was most prominent with CXCL10 as the most upregulated gene in SC islets after co-culture, together with the same chemokine family members CXCL9 and CXCL11 (Figure 2C-2D and Figure 2F, center). Chemokine signaling plays a key role in attracting immune cell recruitment to inflamed tissues, which may be due to an early inflammatory response that was not seen in the timing of graft retrieval in the humanized mouse model (Figure 1). CXCL10 (IP-10) is also an IFN-inducible protein, and according to the data, it peaked at 24 h co-culture (Figure 2G), whereas other IFN-inducible genes maintained or increased their levels in the 48 h time frame in vivo (Figure 2G), and even into the 10 week window under immune challenge (Figure 1I-1J). After 10 weeks, CXCL10 was still upregulated in humanized mouse grafts, although few SC islets still expressed it (Figure 7D and 7F). Although CXCL10 accumulated in the coculture medium (Figure 2H) and was detected in endocrine cells within SC islet clusters under inflammatory signals (Figure 2I), we conclude that CXCL10 plays a central role in stimulating T and NK cells, a role that needs to be fully evaluated.

[0170] As the entire JAK / STAT pathway is largely upregulated in SC islets during coculture with PBMCs, we focused on genes mediating this pathway, starting with the IFNγ receptor gene IFNGR1, the intracellular regulator STAT1, the negative regulator SOCS1, and further downstream effectors B2M (activator of antigen presentation-mediated activator) and CD274 (PD-L1 co-inhibitor surface molecule) (Figure 2G). STAT1 is known to be a master regulator of the JAK / STAT pathway (Gurzov et al., 2016) and is enriched in the GSEA transcription factor motif analysis (Figure 8E). As further evidence of the importance of the pathway in the immunogenicity of SC islets, it has been shown that upon IFN stimulation, STAT1 is phosphorylated and translocates to the nucleus of SC islets (Figure 2J), where it induces the transcription of IFN response elements, including CXCL10 (Moore et al., 2011).

[0171] Genome-wide CRISPR screen identifies components of the IFN response as candidates for perturbation to enhance SC islet survival The transcriptional profile of SC islets under immune-induced inflammatory milieu presented here would give active genes that could be targeted to achieve immune surveillance or reduced immune recognition. However, expression of specific genes could favor either 1) pro-stimulatory, 2) anti-stimulatory immunological induction toward destroying islet cells, or 3) a balanced effect or no effect. A validation system was required to filter and evaluate what was learned from the single-cell RNA-seq data (Figures 1-2). Therefore, we applied the whole genome Brunello CRISPR lentiviral library (Doench et al., 2016) to screen for gene edits that could affect SC islet viability in allogeneic challenge / rejection grafts. The Brunello library consists of a pool of 76,441 human targeting guide RNAs (gRNAs) and 1000 control gRNAs (non-targeting or intergenic) in a lentiviral vector (lentiCRISPRv2) that also expresses Cas9. The pooled library targets 19,114 human genes, many of which are targeted by four gRNAs per gene.

[0172] To avoid multiple different gRNAs in cells and non-specific effects on screening results (Doench, 2018), dissociated SC islets were transduced with the Brunello library using low infectious titer lentivirus (multiplicity of infection < 1). The library-transduced cells (LT SC islets) were then subjected to optimal CRISPR editing and reaggregation of cell clusters for at least 10 days before transplantation into NSG-MHC null humanized mouse models (Figure 3A). Graft function and subsequent engraftment failure (by human PBMC injection) (Figure 1) were measured by detection of human insulin and C-peptide from (fasted) mouse blood 30 min after glucose administration (Figure 1B) and from non-fasted mice at the 10-week endpoint (Figure 9C). Humanized mice maintained circulating levels of T cells throughout the experiment (Figures 9A-9B). Ten weeks after PBMC injection, when graft failure was confirmed, the grafts were harvested and genomic DNA (gDNA) was extracted, amplified by PCR, and subjected to Illumina sequencing.

[0173] In the analysis, the impact of perturbation was evaluated based on the average number of gRNAs from immune-challenged LT SC islet grafts versus control grafts, while also considering the distance from the control intergenic gRNA number. A ranking method was applied based on the gene-environment interaction model (Caspi et al., 2003). As seen in Figure 3C, the axes are the z-scores of the relative fold change between the average number of four gRNAs targeting a particular gene, with respect to condition (y-axis: humanized vs. control mice) and / or with respect to the average number of intergenic gRNAs (y-axis and x-axis). Thus, within the top left quadrant, each dot is a gene that, when expressed, is favorable for graft survival under normal conditions, but detrimental upon immune challenge, based on its distance from 0, e.g., when perturbed, NCF1 is a strong hit with respect to graft survival against immune destruction, but is essential under normal conditions, and perturbing it may produce adverse effects upon transplantation. Within the same quadrant, perturbations of B2M and HLA-A favor cytoprotection under immune challenge but generally have little effect on graft viability. This is consistent with previous reports of the protective effect of HLA-I knockout, which prevents allorecognition by T cells (Castro-Gutierrez et al., 2021; Han et al., 2019; Parent et al., 2021; Wang et al., 2015). Consistent with this, hits in the upper right quadrant are deleterious genes both during regular transplantation and under PBMC injection and infiltration. Conspicuously, one of the top hits in this quadrant and generally on the positive y-axis is perturbation of CXCL10. This implies that CXCL10 expression in SC islets could be deleterious in the face of the alloimmune response modeled here using humanized mice. Even in the absence of PBMCs, cells that may express them have poor viability. Therefore, depletion of CXCL10 is strongly suggested to be beneficial for SC islet transplantation. In addition to B2M, HLA-A, and CXCL10, other canonical IFN pathway-related genes, STAT1, JAK1, and JAK2, were found to be associated with immune-protective effects.Looking at the enrichment per selected gRNA for each of these genes across mouse replicates (n=6 per group), the number of these perturbations is higher in the humanized mouse explants compared to control mice and compared to the control gRNA counts (Figure 3D). In the case of CXCL10, there is a notable change between CXCL10 gRNAs compared to the control gRNA in control mice (dashed line).

[0174] The lower quadrant of the gene vs. environment interaction plot (Figure 3C) has gene hits that favor graft survival under immune infiltration of PBMCs. gRNAs targeting these genes are the most depleted in humanized mouse grafts. Thus, artificially these genes may be protective against immune destruction. One such example is PTPRA, which belongs to the family of protein tyrosine phosphatases (PTPs) known to be negative regulators of JAK / STAT signaling (Gurzov et al., 2015; Stanley et al., 2015). Another member of PTPs, PTPN2, is a T1D risk gene (Barrett et al., 2009; Espino-Paisan et al., 2011) and ranked lower as a favorable gene for screening (dots not labeled in Figure 3C), but when perturbed, showed lower counts in humanized mice compared to controls (Figure 3D). Furthermore, suppressor of cytokine signaling 1 (SOCS1), also a negative regulator of JAK / STAT (Galic et al., 2014; Solomon et al., 2011), was upregulated in our scRNA-seq data (Figures 1-2) and showed potency as a tolerizing gene in the CRISPR screen (Figure 3D). Other previously reported protective / tolerizing molecules, namely HLA-E (Gornalusse et al., 2017) and PD-L1 (CD274) (Castro-Gutierrez et al., 2021; Yoshihara et al., 2020), showed protective effects (Figure 3D), but surprisingly, were less potent compared to the overall ranking.

[0175] Attenuation of the IFNγ pathway affects the immunogenicity of SC islets In vitro and in vivo scRNA-seq experiments have provided a wealth of data on the impact of immune challenge on and by internal signaling (JAK / STAT) in SC islets that are translated into external signals manifested by antigen presentation and chemokine secretion. Comparison of the data with an unbiased approach of whole genome screening provided further confirmation of IFN signaling as a key factor. All significant positive readouts from all assays, upregulated scRNA-seq genes, and gRNAs enriched in humanized mouse grafts were compared (Figure 4A). Within the CRISPR screening hits, there are four common genes that are also upregulated in immune-challenged engrafted or co-cultured SC-β and SC-α cells. These observed genes are strong evidence of the well-known importance of antigen processing (TAP1) and presentation (B2M, HLA-A) in MHC class I initiation of immune responses. STAT1 coordinates external signals of the IFNγ (and also IFNα and β) receptor with downstream effects including MHC-I stimulation as well as secreted substances such as CXCL10. CXCL10 was the most prominent gene in this study and turned out to be one of the strongest hits in the CRISPR survival screen (Figure 3). Although not many cells express CXCL10 during the late graft rejection phase (Figure 1), cells lacking CXCL10 have enhanced survival capacity under the same in vivo conditions (Figure 3), suggesting that CXCL10 has a role at the time point of early immune-graft interactions (Figure 3). Its widespread expression in short-term co-culture experiments supports this hypothesis (Figure 2).

[0176] To evaluate IFN signaling as a target for genetic manipulation, we performed co-culture experiments with human allogeneic PBMCs with SC islets pre-transduced with lentiviral vectors (Figure 4B). For gene knockout, we used vectors expressing Cas9 and gRNAs (in pLentiCRISRv2) targeting deleterious parts of the IFN pathway, namely the upstream IFNγ receptor α chain (IFNGR1), the central regulator STAT1, and the downstream effectors B2M and CXCL10. For overexpression vectors (pLX_317) expressing the open reading frame (ORF) of the intercellular negative regulator SOCS1, the downstream immune cell inhibitor PD-L1 (CD274), and CXCL10 (to evaluate the impact of its abundance), we chose them. All viral transductions had perturbative effects on target protein expression in SC islets under IFNγ stimulation that fell within the limits of transduction efficiency (Figure 10A). After 48 h of co-culture, SC islets were stained for apoptotic markers (C-peptide staining) to note SC-β viability by flow cytometry (Figure 4C). In parallel, PBMC fractions were either partially stained for T cell activation markers (Figures 10B-10C) or transferred after 7 days for new cultures to measure proliferation rates (Figure 4D).

[0177] B2M knockout did not reduce SC islet stimulation of T cell activation and proliferation when compared to non-targeting (NT) gRNA transduction, but it did reduce the rate of apoptotic SC-β and total SC islets. In contrast, depletion of CXCL10 and STAT1 improved SC-β survival under immune challenge with PBMCs, comparable to the known effects of knockout of B2M and overexpression of PD-L1 (Figure 4C). SC islets depleted of both CXCL10 and STAT1 reduced co-cultured T cell activation and proliferation compared to NT gRNA SC islets (Figure 4D and Figures 10B-10C). Decreased secretion of CXCL10 was observed during co-culture (Figure 10D).

[0178] Transduction of IFNGR1 gRNA reduced SC-β cell death but, surprisingly, increased T cell activation and T cell proliferation. Overexpression of SOCS1 in SC islets had a positive effect on SC islet viability and significantly reduced co-culture T cell proliferation compared to eGFP overexpression. Evidence of the detrimental effect of CXCL10 on SC-β under immune challenge can be seen by the significantly increased apoptosis in SC-β overexpressing CXCL10.

[0179] CXCR3 is a chemokine receptor expressed on T helper cells, CD8 T cells, NK cells, and monocytes, and reacts with the IFN-inducible chemokines CXCL4, CXCL9, CXCL10, and CXCL11. CXCR3 plays a traditional role in providing chemotactic and cell proliferation signals in IFNγ-induced immune responses (Loetscher et al., 1996), but it can also affect the polarization of effector T cells (Wildbaum et al., 2002). To evaluate the extent of the importance of the CXCL10-CXCR3 axis in the immunogenicity of SC islets, we performed co-culture experiments of SC islets + PBMCs with a blocking antibody to CXCR3 (Figure 4E). Anti-CXCR3 Ab treatment ("αCXCR3") prior to co-culture with SC islets reduced T cell activation and proliferation, as well as the subsequent apoptotic effect of SC-β, compared to non-specific IgG treatment (Figures 4F-4G and Figure 10E). Because CXCL10 is not the only chemokine that binds to CXCR3, we introduced anti-CXCL10 neutralizing antibodies during co-culture and observed a significant improvement in SC-β viability (Figure 4F). This indicates a large contribution of CXCL10 to SC-β cell death. Since it has been suggested that CXCL10 induces apoptosis in β cells by binding to TLR4 (Schulthes et al., 2009), we pretreated SC islets with TLR4 blocking antibodies before co-culture with PBMCs. SC islets treated with TLR4 antibodies showed a non-significant reduction in apoptotic events (Figure 4F). This suggests that CXCR3-induced immune cells are the main contributors to CXCL10-mediated SC islet killing, although it does not completely exclude the role of TLR4.

[0180] The generation of genetically modified hPSC lines comes with the challenges of risk of off-target effects and obtaining clones that have lost their preferred differentiation potential. These obstacles increase exponentially with the need to edit multiple genes with multiple rounds of transfection and clonal isolation. Therefore, the aim was to obtain low-immunogenic SC islets with a single perturbation. The data obtained in this study by scRNA-seq and CRISPR screening established the need to silence the IFN pathway to obtain low immunogenicity. Therefore, two Hues8 hESC CRISPR knockout lines were selected and generated: CXCL10 KO and STAT1 KO, based on the rationale of reducing overall IFN signaling either through the master regulator (STAT1) or by limiting it to its downstream effects (CXCL10).

[0181] Each line was generated by homology-directed repair (HDR) via nucleofection of Cas9 / sgRNA ribonucleoprotein complexes (RNPs) and a targeting vector. The targeting vectors were designed to facilitate in-frame integration of a GFP or luciferase cassette into exon 2 or exon 3 of the CXCL10 or STAT1 locus, respectively, along with puromycin resistance (Figures 5A-5B). Several heterozygous clones were obtained from each knockout, and CXCL10-GFP (C10G) and STAT1-luciferase (ST1L) clones were selected that carried the transgene integrated into one allele and a non-homologous end joining (NHEJ) mutation in the intact endogenous allele, as determined by PCR and Sanger sequencing (Figure 10A). Each clone displayed a normal karyotype (Figure 10B) and expression of pluripotency markers (Figure 10C). In the following experiments, these clones were compared with a wild-type Hues8 batch (WT) and a luciferase-expressing Hues8 line (GAPDH-luc, GL) (Gerace et al., 2021). C10G, ST1L, and control lines were successfully differentiated into SC-β using the Melton lab's β cell differentiation protocol (Pagliuca et al., 2014; Veres et al., 2019) (Figures 5C and 10D), and transplanted mice showed positive glucose-stimulated insulin secretion (Figure 5D).

[0182] As intended, C10G differentiated into SC islets showed negligible intracellular CXCL10 staining and barely detectable secretion of CXCL10 (ELISA) with or without IFNγ stimulation (Figure 5E-5F). Less than 1% of ST1L SC islets were STAT1 positive, and even less stained for activated phosphorylated STAT1 under IFNγ treatment (Figure 5G). As a regulator of the IFN pathway, the absence of STAT1 in ST1L also resulted in desensitization to IFNγ manifested by downregulation of downstream activating components such as HLA proteins and CXCL10, as well as inhibitory (HLA-E, PD-L1, and SOCS1) (Figure 5F-5G). ST1L also showed in-frame luciferase activity throughout the differentiation stages, which decreased over the course of differentiation (Figure 10E) and was lower than GL at stage 6 (Figure 10F).

[0183] In vitro coculture assays are powerful tools to predict the outcome of immune attacking cells upon transplantation. Therefore, genetically modified (GM) SC islets and control SC islets were cocultured with allogeneic PBMCs. To evaluate the contribution of specific immune populations to SC islet killing, GM SC islets were cocultured with blood purified T cells (C10G only) and NK cells (Figure 5H). Compared to WT, C10G cocultures showed remarkable protective performance against allogeneic PBMCs, T cells, and NK cells in terms of improved survival of SC-β (Figure 5I) and SC islets (Figure 10G), as well as reduced T cell activation and proliferation in cocultured PBMCs (Figure 5K and Figure 10H). In contrast, ST1L showed mild to low protection against cocultured PBMCs, while significantly more SC islets became apoptotic after NK cell coculture (Figure 5J and Figure 10G). T cells from PBMCs co-cultured with ST1L were less activated and proliferated than those co-cultured with control GL SC islets (Fig. 5K and Fig. 10I). NK cell activation was not significantly reduced after co-culture with either C10G or ST1L (Fig. 10J). The fact that ST1L does not show lower immunogenicity in co-cultures may be due to the lack of IFN-induced negative regulation by PD-L1 and SOCS1 (discussed above and in Fig. 5G).

[0184] CXCL10-depleted SC islets are less immunogenic in an in vivo allogeneic rejection model Given the equivocal results of ST1L in co-culture experiments, we focused on null C10G for in vivo studies. In a humanized mouse (MHC NSG) model similar to that used in Figures 1 and 3, C10G or WT SC islets were transplanted followed by injection of PBMCs from two human donors, with three mice per group serving as non-humanized controls. Graft function was continuously monitored by human insulin ELISA of plasma samples from overnight fasted mice (30 min after glucose administration). Graft engraftment failure was observed in humanized mice carrying WT SC islets starting at 11 weeks after PBMC injection and continued until week 13, whereas control grafts continued to function. Interestingly, insulin levels in C10G SC ​​islet grafts were stable and increased over time, with no obvious difference between humanized (+PBMC) and control mice (Figures 6A and 11A). Thus, SC islets with impaired ability to express and secrete CXCL10 can avoid immune attack and allograft rejection.

[0185] Consideration To protect insulin-producing SC-β cells from immune destruction, in this study we applied two main approaches to uncover the core players inducing SC islet immunogenicity: observing the events that occur upon immune challenge (using transcriptomics) and filtering the causes of those events to the most pragmatic mechanisms for obtaining immune protection (CRISPR screening). In other words, answering the questions: what happens and how can we reverse it?

[0186] The strongest effect seen in SC islets facing allogeneic immune cells is the upregulation of interferon-stimulated genes (ISGs). Data show that T cells are activated in their co-culture / graft environment and express IFNγ, among many other inflammatory genes. Secreted IFNγ binds to receptors on SC islets, leading to an inflammatory cascade leading to the upregulation of ISGs. A plausible explanation for T cell activation is antigen presentation via MHC class I molecules, evident by the differential expression of B2M, HLA genes, and antigen processing genes in SC islets in vivo (Figure 1) and in vitro (Figure 2). Cytokines secreted by primed T cells activate more immune cells, including NK cells. However, the most remarkable observation was the involvement of chemokines secreted by SC islets. CXCL10 is 8-fold more expressed in co-cultured SC-β cells (with PBMCs) compared to SC-β cells alone. Transplanted CXCL10-depleted SC islet cells under allogeneic immune challenge have a higher chance of survival compared to surrounding cells with other perturbations, as judged by the results of in vivo CRISPR screening (Figure 3). Furthermore, evidence from in vitro and in vivo immune challenge models indicates that CXCL10-deficient SC islets are immune evasive compared to wild-type cells (Figures 4-6).

[0187] CXCL10 is one of the most upregulated cytokines and chemokines in primary human islets (Eizirik et al., 2012) as well as hPSC-derived islets (Demine et al., 2020; Dettmer et al., 2022) under exposure to proinflammatory cytokines. CXCL10 is known as a chemoattractant for NK cells (Ali et al., 2021), monocytes and activated T cells (Taub et al., 1993) through binding to CXCR3 (Loetscher et al., 1996). Evidence from recent-onset T1D islets shows expression of CXCL10 in affected areas of insulitis with infiltrating lymphocytes expressing CXCR3 (Roep et al., 2010; Uno et al., 2010). These results indicate that CXCL10 induction is not restricted to SC-β cells but is also differentially expressed by other stem cell-derived endocrine cells, SC-α and SC-EC cells, which may correlate with a recent study showing evidence of pancreatic α cell contribution to CXCL10 expression in NOD mice and recent-onset T1D islets (Nigi et al., 2020).

[0188] In a previous study, CXCL10 was found to be highly secreted from iPSC islets in co-culture with matched T1D PBMCs using a T1D autologous in vitro model (Leite et al., 2020). In the present study, CXCL10 expression was found mainly in co-culture (Figure 2F-2H), but not in the later stages of graft rejection (Figure 7D and Figure 7F), proving that CXCL10 is a first-response "alarm" protein whose action is critical in initiating SC islet interaction with the aggressive immune system and determines the fate of SC islet survival (Figure 3, in vivo CRISPR screen). In T1D, islet CXCL10 expression occurs at an early stage (Roep et al., 2010, Uno et al., 2010), and the levels of CXCL10 in the serum of patients are mostly elevated in recent onset compared to established patients (autologous Ab+) (Shimada et al., 2001). Murine islet allografts expressed high levels of CXCL10 2 days after transplantation into diabetic C57BL / 6 mice, but to a lesser extent by day 100 (Bencer et al., 2017). Analysis of plasma samples from human islet transplant patients revealed that CXCL10 was one of the most abundantly released inflammatory mediators, peaking at 24 hours after transplantation (Yoshimatsu et al., 2017).

[0189] CRISPR screening in humanized mice reveals that CXCL10 is a deleterious gene for graft survival under standard transplant conditions, mostly under human T cell infiltration (Figure 3C). These stress conditions may induce inflammatory responses manifested by CXCL10. Paracrine or autocrine CXCL10 was suggested to mediate β-cell engraftment failure and apoptosis via CXCR3 (Jveed et al., 2021) or TLR4 binding (Schulthess et al., 2009) on β-cells. This could explain the enrichment of CXCL10 gRNAs compared to cells transduced with intergenic gRNAs in mouse grafts, independent of human T cell activity (Figure 3C). Co-culture systems using receptor-blocking antibodies only showed evidence of conventional PBMC-mediated cell death via the CXCL10-CXCR3 axis (Figures 4E-4G).

[0190] In T1D, pancreatic islets have been shown to respond to proinflammatory cytokines by regulating NF-κB and STAT1, which are part of the immune destruction mechanism of β-cells (Cnop et al., 2005; Eizirik et al., 2012). Although the conditions were homogenous, both transcription factors were upregulated in SC islets, although only STAT1 depletion showed as a hit in the CRISPR screen (Figure 3). Transduction of STAT1 gRNA with lentivirus rescued SC islets from immune destruction (Figure 4C-4D). However, this rescue was not reproduced when a pure line of STAT1 KO (ST1L) SC islets was used in the same co-culture conditions (Figure 5I-5K). The reason for this could be derived from the efficiency of lentiviral transduction. STAT1-deficient SC islets lose immune inductive elements such as HLA molecules and CXCL10, but also immune suppressive functions such as PD-L1 and SOCS1 (Figure 5G). As a constituent of the unperturbed cellular fraction (conditional on lentiviral efficiency), STAT1-deficient cells may reduce the overall inflammatory response together with unperturbed cells that retain endogenous inhibitory ligand function.

[0191] Downstream of STAT1 is the transcription factor IRF1, which has been reported to have some opposing effects on β-cells through induction of SOCS1 (Moore et al., 2011). SOCS1 and PTPN2 are negative regulators of cytokine signaling (Chong et al., 2002; Elvira et al., 2022; Moore et al., 2009), and both have been linked to T1D risk loci (Onengut-Gumuscu et al., 2015; Ram and Morahan, 2017). Previous reports have shown that overexpression of SOCS1 in NOD mouse islets prevents diabetes (Flodstrom-Tullberg et al., 2003) and delays rejection of allogeneic islet grafts in a mouse model (Solomon et al., 2011). These data indicate that under PBMC+SC islet inflammatory responses (humanized mouse grafts or co-culture), both IRF1 and SOCS1 are differentially upregulated (Figures 1-2). SOCS1 KO SC islets were depleted in CRISPR screens in humanized mouse grafts among other PTP family members, PTPN2 KO and PTPRA KO (Stanley et al., 2015) (Figure 5D). The effect of SOCS1 overexpression was also later verified and shown to be pro-survival in co-cultured SC islets (Figures 4C-4D).

[0192] Based on the unclear results of the STAT1 KO line (ST1L) (Figure 5), it was concluded that similar pan-JAK / STAT reduction strategies should be considered with caution. These approaches include overexpression of SOCS1 and depletion of IFNGR1. Although these proved to be potent in immune protection of SC islets in co-culture experiments (Figure 4C-4D), pure transgenic lines of SOCS1 OE or IFNGR1 KO may result in loss of inflammatory negative regulatory feedback of JAK / STAT signaling. Downregulation of PD-L1 under JAK / STAT silencing exposes SC islets to T cell attack, whereas downregulation of HLA results in NK cell killing. Such stem cell lines can be further edited with additional modification(s) that would address these concerns.

[0193] References Alagpulinsa, DA, Cao, JJL, Driscoll, RK, Sirbulescu, RF, Penson, MFE, Sremac, M., Engquist, EN, Brauns, TA, Markmann, JF, Melton, DA, et al. (2019). Alginate microencapsulation of human stem cell-derived beta cells with CXCL12 prolongs their survival and function in immunocompetent mice without systemic Immunosuppression. Am J Transplant 19, 1930-1940. Banting, FG, Best, CH, Collip, JB, Campbell, WR, and Fletcher, AA (1922). Pancreatic Extracts in the Treatment of Diabetes Mellitus. Can Med Assoc J 12, 141-146. Barrett, J.C., Clayton, D.G., Concannon, P., Akolkar, B., Cooper, J.D., Erlich, H.A., Julier, C., Morahan, G., Nerup, J., Nierras, C., et al. (2009). Genome-wide association study and meta-analysis find that over 40 loci affect risk of type 1 diabetes. Nat Genet 41, 703-707. Brehm, M.A., Kenney, L.L., Wiles, M.V., Low, B.E., Tisch, R.M., Burzenski, L., Mueller, C., Greiner, D.L., and Shultz, L.D. (2019). Lack of acute xenogeneic graft- versus-host disease, but retention of T-cell function following engraftment of human peripheral blood mononuclear cells in NSG mice deficient in MHC class I and II expression. FASEB J 33, 3137-3151. Cai, E.P., Ishikawa, Y., Zhang, W., Leite, N.C., Li, J., Hou, S., Kiaf, B., Hollister-Lock, J., Yilmaz, N.K., Schiffer, C.A., et al. (2020). Genome-scale in vivo CRISPR screen identifies RNLS as a target for beta cell protection in type 1 diabetes. Nat Metab 2, 934-945. Caspi, A., Sugden, K., Moffitt, T.E., Taylor, A., Craig, I.W., Harrington, H., McClay, J., Mill, J., Martin, J., Braithwaite, A., et al. (2003). Influence of life stress on depression: moderation by a polymorphism in the 5-HTT gene. Science 301, 386-389. Castro-Gutierrez, R., Alkanani, A., Mathews, C.E., Michels, A., and Russ, H.A. (2021). Protecting Stem Cell Derived Pancreatic Beta-Like Cells From Diabetogenic T Cell Recognition. Front Endocrinol (Lausanne) 12, 707881. Deuse, T., Hu, X., Agbor-Enoh, S., Jang, M.K., Alawi, M., Saygi, C., Gravina, A., Tediashvili, G., Nguyen, V.Q., Liu, Y., et al. (2021). The SIRPalpha-CD47 immune checkpoint in NK cells. J Exp Med 218. Deuse, T., Hu, X., Gravina, A., Wang, D., Tediashvili, G., De, C., Thayer, W.O., Wahl, A., Garcia, J.V., Reichenspurner, H., et al. (2019). Hypoimmunogenic derivatives of induced pluripotent stem cells evade immune rejection in fully immunocompetent allogeneic recipients. Nat Biotechnol 37, 252-258. Doench, J.G. (2018). Am I ready for CRISPR? A user’s guide to genetic screens. Nat Rev Genet 19, 67-80. Doench, J.G., Fusi, N., Sullender, M., Hegde, M., Vaimberg, E.W., Donovan, K.F., Smith, I., Tothova, Z., Wilen, C., Orchard, R., et al. (2016). Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol 34, 184-191. Espino-Paisan, L., de la Calle, H., Fernandez-Arquero, M., Figueredo, M.A., de la Concha, E.G., Urcelay, E., and Santiago, J.L. (2011). A polymorphism in PTPN2 gene is associated with an earlier onset of type 1 diabetes. Immunogenetics 63, 255-258. Galic, S., Sachithanandan, N., Kay, T.W., and Steinberg, G.R. (2014). Suppressor of cytokine signalling (SOCS) proteins as guardians of inflammatory responses critical for regulating insulin sensitivity. Biochem J 461, 177-188. Gerace, D., Boulanger, K.R., Hyoje-Ryu Kenty, J., and Melton, D.A. (2021). Generation of a heterozygous GAPDH-Luciferase human ESC line (HVRDe008-A-1) for in vivo monitoring of stem cells and their differentiated progeny. Stem Cell Res 53, 102371. Gornalusse, G.G., Hirata, R.K., Funk, S.E., Riolobos, L., Lopes, V.S., Manske, G., Prunkard, D., Colunga, A.G., Hanafi, L.A., Clegg, D.O., et al. (2017). HLA-E-expressing pluripotent stem cells escape allogeneic responses and lysis by NK cells. Nat Biotechnol 35, 765-772. Gurzov, E.N., Stanley, W.J., Brodnicki, T.C., and Thomas, H.E. (2015). Protein tyrosine phosphatases: molecular switches in metabolism and diabetes. Trends Endocrinol Metab 26, 30-39. Gurzov, E.N., Stanley, W.J., Pappas, E.G., Thomas, H.E., and Gough, D.J. (2016). The JAK / STAT pathway in obesity and diabetes. FEBS J 283, 3002-3015. Han, X., Wang, M., Duan, S., Franco, P.J., Kenty, J.H., Hedrick, P., Xia, Y., Allen, A., Ferreira, L.M.R., Strominger, J.L., et al. (2019). Generation of hypoimmunogenic human pluripotent stem cells. Proc Natl Acad Sci U S A 116, 10441-10446. Kharbikar, B.N., Chendke, G.S., and Desai, T.A. (2021). Modulating the foreign body response of implants for diabetes treatment. Adv Drug Deliv Rev 174, 87-113. Kovatchev, B. (2019). A Century of Diabetes Technology: Signals, Models, and Artificial Pancreas Control. Trends Endocrinol Metab 30, 432-444. Lehmann, R., Spinas, G.A., Moritz, W., and Weber, M. (2008). Has time come for new goals in human islet transplantation? Am J Transplant 8, 1096-1100. Leite, N.C., Sintov, E., Meissner, T.B., Brehm, M.A., Greiner, D.L., Harlan, D.M., and Melton, D.A. (2020). Modeling Type 1 Diabetes In Vitro Using Human Pluripotent Stem Cells. Cell Rep 32, 107894. Loetscher, M., Gerber, B., Loetscher, P., Jones, S.A., Piali, L., Clark-Lewis, I., Baggiolini, M., and Moser, B. (1996). Chemokine receptor specific for IP10 and mig: structure, function, and expression in activated T-lymphocytes. J Exp Med 184, 963-969. Moore, F., Naamane, N., Colli, M.L., Bouckenooghe, T., Ortis, F., Gurzov, E.N., Igoillo-Esteve, M., Mathieu, C., Bontempi, G., Thykjaer, T., et al. (2011). STAT1 is a master regulator of pancreatic {beta}-cell apoptosis and islet inflammation. J Biol Chem 286, 929-941. Nostro, M.C., Sarangi, F., Yang, C., Holland, A., Elefanty, A.G., Stanley, E.G., Greiner, D.L., and Keller, G. (2015). Efficient generation of NKX6-1+ pancreatic progenitors from multiple human pluripotent stem cell lines. Stem Cell Reports 4, 591-604. Pagliuca, F.W., Millman, J.R., Gurtler, M., Segel, M., Van Dervort, A., Ryu, J.H., Peterson, Q.P., Greiner, D., and Melton, D.A. (2014). Generation of functional human pancreatic beta cells in vitro. Cell 159, 428-439. Parent, A.V., Faleo, G., Chavez, J., Saxton, M., Berrios, D.I., Kerper, N.R., Tang, Q., and Hebrok, M. (2021). Selective deletion of human leukocyte antigens protects stem cell-derived islets from immune rejection. Cell Rep 36, 109538. Raffin, C., Vo, L.T., and Bluestone, J.A. (2020). Treg cell-based therapies: challenges and perspectives. Nat Rev Immunol 20, 158-172. Ramzy, A., Thompson, D.M., Ward-Hartstonge, K.A., Ivison, S., Cook, L., Garcia, R.V., Loyal, J., Kim, P.T.W., Warnock, G.L., Levings, M.K., et al. (2021). Implanted pluripotent stem-cell-derived pancreatic endoderm cells secrete glucose-responsive C-peptide in patients with type 1 diabetes. Cell Stem Cell 28, 2047-2061 e2045. Rezania, A., Bruin, J.E., Arora, P., Rubin, A., Batushansky, I., Asadi, A., O’Dwyer, S., Quiskamp, N., Mojibian, M., Albrecht, T., et al. (2014). Reversal of diabetes with insulin producing cells derived in vitro from human pluripotent stem cells. Nat Biotechnol 32, 1121-1133. Russ, H.A., Parent, A.V., Ringler, J.J., Hennings, T.G., Nair, G.G., Shveygert, M., Guo, T., Puri, S., Haataja, L., Cirulli, V., et al. (2015). Controlled induction of human pancreatic progenitors produces functional beta-like cells in vitro. EMBO J 34, 1759-1772. Schulthess, F.T., Paroni, F., Sauter, N.S., Shu, L., Ribaux, P., Haataja, L., Strieter, R.M., Oberholzer, J., King, C.C., and Maedler, K. (2009). CXCL10 impairs beta cell function and viability in diabetes through TLR4 signaling. Cell Metab 9, 125-139. Shapiro, A.M., Lakey, J.R., Ryan, E.A., Korbutt, G.S., Toth, E., Warnock, G.L., Kneteman, N.M., and Rajotte, R.V. (2000). Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 343, 230-238. Shultz, L.D., Keck, J., Burzenski, L., Jangalwe, S., Vaidya, S., Greiner, D.L., and Brehm, M.A. (2019). Humanized mouse models of immunological diseases and precision medicine. Mamm Genome 30, 123-142. Solomon, M., Flodstrom-Tullberg, M., and Sarvetnick, N. (2011). Beta-cell specific expression of suppressor of cytokine signaling-1 (SOCS-1) delays islet allograft rejection by down-regulating Interferon Regulatory Factor-1 (IRF-1) signaling. Transpl Immunol 24, 181-188. Stanley, W.J., Litwak, S.A., Quah, H.S., Tan, S.M., Kay, T.W., Tiganis, T., de Haan, J.B., Thomas, H.E., and Gurzov, E.N. (2015). Inactivation of Protein Tyrosine Phosphatases Enhances Interferon Signaling in Pancreatic Islets. Diabetes 64, 2489-2496. Veres, A., Faust, A.L., Bushnell, H.L., Engquist, E.N., Kenty, J.H., Harb, G., Poh, Y.C., Sintov, E., Gurtler, M., Pagliuca, F.W., et al. (2019). Charting cellular identity during human in vitro beta-cell differentiation. Nature 569, 368-373. Wang, D., Quan, Y., Yan, Q., Morales, J.E., and Wetsel, R.A. (2015). Targeted Disruption of the beta2-Microglobulin Gene Minimizes the Immunogenicity of Human Embryonic Stem Cells. Stem Cells Transl Med 4, 1234-1245. Wei, Z., Yoshihara, E., He, N., Hah, N., Fan, W., Pinto, A.F.M., Huddy, T., Wang, Y., Ross, B., Estepa, G., et al. (2018). Vitamin D Switches BAF Complexes to Protect beta Cells. Cell 173, 1135-1149 e1115. Wildbaum, G., Netzer, N., and Karin, N. (2002). Plasmid DNA encoding IFN-gamma inducible protein 10 redirects antigen-specific T cell polarization and suppresses experimental autoimmune encephalomyelitis. J Immunol 168, 5885-5892. Xu, H., Wang, B., Ono, M., Kagita, A., Fujii, K., Sasakawa, N., Ueda, T., Gee, P., Nishikawa, M., Nomura, M., et al. (2019). Targeted Disruption of HLA Genes via CRISPR-Cas9 Generates iPSCs with Enhanced Immune Compatibility. Cell Stem Cell 24, 566-578 e567. Yoshihara, E., O’Connor, C., Gasser, E., Wei, Z., Oh, T.G., Tseng, T.W., Wang, D., Cayabyab, F., Dai, Y., Yu, R.T., et al. (2020). Immune-evasive human islet-like organoids ameliorate diabetes. Nature 586, 606-611.

Claims

1. Modified stem cell-derived beta cells containing one or more perturbations in the JAK / STAT type II interferon (IFN) pathway.

2. 2. The modified stem cell-derived beta cell of claim 1, wherein the perturbation occurs in an IFNγ signaling mediator or downstream element of the IFN pathway.

3. 2. The modified stem cell-derived beta cell of claim 1, wherein the perturbation occurs in a downstream inflammatory element of the IFN pathway.

4. 2. The modified stem cell-derived beta cell of claim 1, wherein the perturbation comprises a decrease in expression of one or more downstream elements of the IFN pathway.

5. 2. The modified stem cell-derived beta cell of claim 1, wherein the perturbation comprises abolishing expression of one or more downstream elements of the IFN pathway.

6. 2. The modified stem cell-derived beta cell of claim 1, wherein the perturbation comprises a decrease in expression of one or more genes listed in Table 1.

7. 2. The modified stem cell-derived beta cell of claim 1, wherein the perturbation comprises abolishing expression of one or more genes listed in Table 1.

8. 2. The modified stem cell-derived beta cell of claim 1, wherein the perturbation comprises a decrease in expression of one or more genes selected from the group consisting of CXCL10, STAT1, and TAP1.

9. Modified stem cell-derived beta cells modified to abolish expression of CXCL10.

10. Modified stem cell-derived beta cells modified to abolish STAT1 expression.

11. The modified stem cell-derived beta cell of claim 1, which is a low-immunogenic stem cell-derived beta cell.

12. 2. The modified stem cell-derived beta cells of claim 1, which exhibit protection against autologous and / or allogeneic rejection.

13. 2. The modified stem cell-derived beta cell of claim 1, which exhibits increased cell survival under immune rejection conditions.

14. 2. The modified stem cell-derived beta cells of claim 1, which exhibit reduced T cell activation and / or NK cell activation upon transplantation.

15. 2. The modified stem cell-derived beta cell of claim 1, further comprising increased expression or activation of one or more immunomodulatory factors.

16. 16. The modified stem cell-derived beta cell of claim 15, wherein the one or more immunomodulatory factors are selected from Table 2.

17. 16. The modified stem cell-derived beta cell of claim 15, wherein the one or more immune modulators are selected from the group consisting of PD-L1, CD47, SOCS1, and HLA-E.

18. The modified stem cell-derived beta cell of any one of claims 1 to 17, wherein the beta cell is a human cell.

19. The modified stem cell-derived beta cell of any one of claims 1 to 17, wherein the beta cell is a non-human cell.

20. 20. The modified stem cell-derived beta cell of claim 19, wherein the non-human cell is a mouse cell.

Images