Compositions and methods for enhancing immune tolerance

Abstract

Disclosed herein are methods of generating low immune cells and methods of mediating local immune tolerance.

Description

Related Applications

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 340,453, filed May 10, 2022. The entire teachings of the above application are incorporated herein by reference. Background Art

[0002] Type 1 diabetes (T1D) is an autoimmune disease that results in the destruction of insulin-producing β cells in the pancreas. Whole cadaveric pancreas or islet transplantation is a successful treatment for T1D; however, these treatments are hampered by limited donor availability and the need for lifelong immunosuppression (Shapiro et al., 2006). To address the shortage of islet material, several protocols have been developed to direct the in vitro differentiation of human induced pluripotent stem cells (iPSCs) into functional SC islets containing glucose-responsive β cells (D'Amour et al., 2006; Millman et al., 2016; Nair et al., 2019; Pagliuca et al., 2014; Rezania et al., 2014; Russ et al., 2015; Veres et al., 2019).

[0003] Current strategies to protect allogeneic islet cells include encapsulation (Alagpulinsa et al., 2019; Bochenek et al., 2018), altering the patient's immune system by co-administering biologics such as low-dose IL-2 and anti-CD3 (Tepluzimab) (Hartemann et al., 2013; Herold et al., 2019), and / or genetic modification of SC islets. Since the main factor in immune recognition and rejection is human leukocyte antigen (HLA), HLA targeting has been performed in iPSCs to reduce or eliminate immune responses to foreign cells (Castro-Gutierrez et al., 2021; Deuse et al., 2019; Gornalusse et al., 2017; Han et al., 2019; Harding et al., 2019; Riolobos et al., 2013; Xu et al., 2019; Yoshihara et al., 2020). To date, engineering strategies that demonstrate some of the immunoprotective effects of SC islet cells have employed lentiviral overexpression of PD-L1 and selective retention of a single HLA-A2 allele in HLA-B / C-deficient cells (Parent et al., 2021; Yoshihara et al., 2020). However, lentiviral transgene overexpression is limited by transgene silencing (Herbst et al., 2012; Wen et al., 2021), and retention of a single HLA-A2 allele on SC-islet cells may lead to recurrent autoimmune rejection mediated by antigen-specific and tissue-retaining memory T cells (Abou-Daya et al., 2021; Monti et al., 2008; Stegall et al., 1996). Summary of the invention

[0004] In some embodiments, the present invention relates to a cell that is engineered (i.e., modified) to induce a tolerant local microenvironment. In some embodiments, the cell is an induced pluripotent stem cell; in some embodiments, the cell is a stem cell. In some embodiments, the cell is a progenitor or precursor cell, such as a β cell progenitor or a β cell precursor cell. The cell is engineered to express one or more tolerogenic genes (e.g., one or more transgenes) and / or one or more immunomodulators from at least one locus in the cell, which is not substantially silenced during the differentiation of the cell into the desired cell type. As used herein, a locus that is not substantially silenced is a locus that is measurably expressed after the cell is differentiated into the desired cell type. In some embodiments, the locus is measurably expressed both during and after the cell is differentiated into the desired cell type. The present invention relates to both engineered progenitor or precursor cells as described above, and to differentiated cells derived from engineered precursor or progenitor cells and comprising an engineered locus. The present invention further relates to methods for preparing cells and methods for using cells, for example, in cell therapy to treat subjects in need.

[0005] In some embodiments, the present invention relates to a method of producing a low immunogenic cell, comprising engineering stem cells or progenitor cells to express one or more tolerogenic transgenes from a locus in a stem cell or progenitor cell that will not be silenced during differentiation of the cell into a desired cell type, and exposing the stem cell or progenitor cell to conditions suitable for differentiation of the stem cell or progenitor cell into a desired cell type. The present invention also relates to cells produced by the method, including engineered stem cells, engineered progenitor cells, and engineered differentiated cells. In some embodiments, the cell is an induced pluripotent stem cell. In some embodiments, the progenitor cell is a β cell progenitor cell. In some embodiments, the desired cell type is a β cell.

[0006] In some aspects, the locus is a locus of a housekeeping gene. In some embodiments, the locus is constitutively expressed in all cells of an islet (e.g., a human islet). In some embodiments, the locus is constitutively expressed in a beta cell (e.g., a human beta cell). In some embodiments, the housekeeping gene is selected from the group consisting of actin, ubiquitin, and GAPDH.

[0007] In some aspects, one or more tolerogenic agents / transgenes are selected from the group consisting of: PDL1, HLA-E / G, CD47, SERPINB9, CCL21, FASL, CD200, MFGE8, CD55, CD46, HLS-G single chain fusion, soluble PDL1-Ig and CTLA4-Ig. In some aspects, the tolerogenic agent is an HLA-E single chain fusion. In some embodiments, the single chain fusion is loaded with a peptide capable of activating NK cells, for example, by interacting with inhibitory receptors NKG2A and / or NKG2C. In some embodiments, the peptide is selected from the group consisting of VMAPRTLLL (SEQ ID NO: 2), VMAPRTLL (SEQ ID NO: 3), VMAPRTLFL (SEQ ID NO: 4), VMAPRTLVL (SEQ ID NO: 5), VMAPRTLIL (SEQ ID NO: 6), IMAPRTLVL (SEQ ID NO: 7), VMPPRTLLL (SEQ ID NO: 8), VMAPRTVLL (SEQ ID NO: 9), VTAPRTLLL (SEQ ID NO: 10), VTAPRTVLL (SEQ ID NO: 11), VMAPRTLTL (SEQ ID NO: 12), and VMAPRALLL (SEQ ID NO: 13).

[0008] In other aspects, the present invention relates to a method of mediating local immune tolerance, which includes engineering stem cells or progenitor cells to express one or more immunomodulators that are not silenced during cell differentiation into a desired cell type from a locus in a stem cell or progenitor cell, and exposing the stem cell or progenitor cell to conditions suitable for differentiation of the stem cell or progenitor cell into a desired cell type, thereby producing a desired cell type that secretes one or more immunomodulators. The present invention also relates to cells produced by the method, including engineered stem cells, engineered progenitor cells, and engineered differentiated cells. In some embodiments, the cell is an induced pluripotent stem cell. In some embodiments, the progenitor cell is a β cell progenitor cell. In some embodiments, the desired cell type is a β cell. The present invention also relates to cells produced by the method, including engineered stem cells, engineered progenitor cells, and engineered differentiated cells.

[0009] In some aspects, the locus is a locus of a housekeeping gene. In some embodiments, the locus is constitutively expressed in all cells of islets (e.g., human islets). In some embodiments, the locus is constitutively expressed in β cells (e.g., human β cells). In some embodiments, the housekeeping gene is selected from the group consisting of actin, ubiquitin and GAPDH. In some aspects, one or more immunomodulators are cytokines. In some aspects, one or more immunomodulators are selected from the group consisting of IL-10, TGF-β and IL-2 and modified IL-2. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0011] Figures 1A to 1G : Generation of immune-evading SC islet cells. Figure 1A Schematic diagram of the generation of low immunogenic hESCs through genetic engineering strategy. Figure 1B Shown is FACS analysis of Nkx6.1 / C-peptide SC-β cells derived from low immunogenic hESC (S6d14). Figure 1C PDL1 and HLA-ABC expression on SC-β cells is shown. Data are presented as MFI (gated on C-pep+ / Nkx6.1+ SC-β cells) and normalized to mode. Figure 1D HLA-ABC and HLA-E expression on SC-β cells is shown. Data are presented as MFI (gated on C-pep+ / Nkx6.1+ SC-β cells) and normalized to mode. Figure 1E Immunofluorescence staining of HLA-E, Nkx6.1, and C-peptide in CD49a+ enriched SC-β cells (S6d14) is shown. Bar size = 100 µm. Figure 1F is a schematic diagram of the in vivo SC-islet cell xenorejection assay. Figure 1G Quantitative analysis of xenograft rejection is shown. Data are presented as mean ± SEM (n = 5 / group). Dashed line represents background luminescence.

[0012] FIG. 2A to FIG. 2I : In vitro co-culture of low immunogenic SC-islet cells with allogeneic human immune cells. Figure 2A A schematic diagram of the in vitro immune SC-islet cell co-culture assay is shown. Figure 2B Shown is the quantification of SC-islet cell viability when co-cultured with primary human PBMCs at a 1:1 ratio. Cell viability is expressed as mean ± SD (n = 5). Figure 2CShown is the quantification of SC-islet cell viability when co-cultured with primary human PBMCs at a ratio of 3: 1. Cell viability is expressed as mean ± SD (n = 5). Figure 2D IFN-γ-treated CD49a + Heat map of T cell ligand gene expression in SC-β cells. Ligand expression is expressed as fold change (log2). Figure 2E IFN-γ treated CD49a + Differential expression of T cell co-inhibitory ligands in SC-β cells. Ligand expression is expressed as fold change (log2). Figure 2F IFN-γ treated CD49a + Differential expression of T cell coactivating ligands in SC-β cells. Ligand expression is expressed as fold change (log2). Figure 2G showed that when compared with primary human CD56 + Quantification of SC-β cell survival in co-culture with NK cells. Cell viability is expressed as mean ± SD (n = 5). Figure 2H is a schematic diagram of the in vivo NK cell cytotoxicity assay. Fig.2I Quantitative analysis of in vivo NK cell assays is shown. Data are presented as mean ± SEM. The dashed line represents background luminescence.

[0013] Figures 3A to 3G : Characterization of the SC-islet cell-specific NK cell ligand profile. Figure 3A A schematic diagram of the SC-β and SC-endothelial cell bulk RNA sequencing workflow is shown. Figure 3B Figure 2 is a heat map of NK cell ligand expression in SC-β and SC-endothelial cells (-IFN-γ vs. +IFN-γ). Ligand expression is expressed as fold change (log2). Figure 3C The expression of NK cell activating ligands on SC-β and SC-endothelial cells is shown. Data are presented as MFI, normalized to mode, and are representative of three independent experiments. Figure 3D Expression of NK cell inhibitory ligands on SC-β and SC-endothelial cells is shown. Data are presented as MFI, normalized to mode, and are representative of three independent experiments. Figure 3E Schematic diagram of SC-islet cell transplantation in diabetic humanized NSG-DKO mice. Figure 3F Non-fasting blood glucose concentrations after SC-islet transplantation and PBMC injection are shown. Data are presented as mean ± SD (n = 12 / group). Figure 3G The in vivo GSIS before and after PBMC injection is shown. Plasma insulin concentrations were measured at t = 0 and 30 min after glucose injection. Data are presented as mean ± SD (n = 5 / group).

[0014] 4A to 4H :Immune tolerant SC-islet cells survive xenogeneic rejection. Figure 4A Schematic representation of the genetic engineering strategy for generating immunomodulatory hESCs and the GAP-2B10 HDR plasmid. Figure 4B is a FACS analysis of Nkx6.1 / C-peptide SC-β cells derived from immunoregulatory hESCs (S6d8). Figure 4C In vitro GSIS of 2B10 SC-islet cells is shown. Data are presented as mean ± SD (n = 4). Figure 4D Graph depicts quantification of cytokine secretion by 2B10 SC-islet cells. Data are presented as mean ± SD (n = 2). Figure 4E Shown is the quantification of SC-islet cell viability when co-cultured with primary human PBMCs at a 1:1 ratio. Cell viability is expressed as mean ± SD (n = 5). Figure 4F Shown are in vivo bioluminescence imaging of B6 / albino mice transplanted with 2B10SC-islet cells (n = 3 / group). Figure 4G Quantitative analysis of in vivo graft survival in B6 / albino mice is shown. Data are presented as mean ± SD (n = 3 / group). Figure 4H Immunostaining of 2B10 and WT SC-islet transplants is shown, demonstrating the presence of INS 5 weeks after transplantation + Cells and T reg Raised. Bar size = 100µm.

[0015] FIG. 5A to FIG. 5H : Figure 5A t-SNE projections of primary human pancreatic islet cells are shown. Figure 5B t-SNE projections of GAPDH expression in the indicated populations are shown. Cells are colored according to their assigned cluster. (Adapted from Segerstolpe et al., 2016). Figure 5C Schematic diagram of the homology-directed repair plasmid used to integrate Luc2 and PD-L1 at the GAPDH locus. Figure 5D Schematic diagram of Luc2 and peptide::B2M::HLA-E HDR plasmid targeting GAPDH. Figure 5E is a schematic diagram of the peptide::B2M::HLA-E long chain fusion. Fig. 5F PD1-Fc binding assay is shown. PD-L1 and WT SC-islet cells were dissociated and stained with a 2-fold dilution series of PE-conjugated human and mouse PD1-Fc. Data are presented as MFI, normalized to the mode. Figure 5G The FACS sorting strategy for HLA-ABC- / - hESCs is shown. Figure 5HShown is a method for quantifying Nkx6.1 after in vitro differentiation + / C-pep + FACS gating strategy for SC-β cells.

[0016] 6A to 6H : Fig. 6A FACS profiles of the HLA-A2 status of five PBMC donors are shown. Figure 6B CD4 in human apheresis-enriched PBMCs is shown + and CD8 + T cells and CD56 + FACS gating strategy for NK cells. Figure represents 5 donors. Figure 6C Shown is the quantification of SC-islet cell viability when co-cultured with purified human CD8+ T cells at a 1:1 ratio. Cell viability is expressed as mean ± SD (n = 5). Fig.6D Shown is the quantification of SC-islet cell viability when co-cultured with purified human CD8+ T cells at a ratio of 3: 1. Cell viability is expressed as mean ± SD (n = 5). Fig. 6E shows that when mixed at a 1:1 ratio with purified human CD4 + Quantification of SC-islet cell viability during T cell co-culture. Cell viability is expressed as mean ± SD (n = 5). Fig. 6F Shown is the quantification of SC-islet cell viability when co-cultured with purified human CD4+ T cells at a ratio of 3: 1. Cell viability is expressed as mean ± SD (n = 5). Figure 6G Shown is the FACS gating strategy for CD4+ T cells enriched from human apheresis. Graphs represent 5 donors. Figure 6H CD8 cells enriched from human leukapheresis were shown + FACS gating strategy for T cells. Figure represents 5 donors.

[0017] FIG. 7A to FIG. 7G : Fig. 7A Quantification of SC-β cell viability when co-cultured with NK92mi cells is shown. K562 and Raji cells were used as positive and negative controls, respectively. Cell viability is expressed as mean ± SD (n = 5). Figure 7B NKG2A / NKG2C expression on NK92mi cells is shown. Figure 7C CD56 enriched from human apheresis leukocytes + FACS gating strategy for NK cells. The gating of a specific population includes all CD56 + NK cells (red), CD56 高(green) and CD56 低(dim) (Pink). Graph represents 5 donors. Fig.7D Quantitative analysis of CD56 expression on enriched primary human NK cells is shown. Data are presented as %CD56 expression (n = 5). Fig. 7E In vivo NK cell assay is shown. Bioluminescence imaging was performed on days 1 and 5 after transplantation. Figure 7F is a schematic diagram of the SC-endothelial cell differentiation protocol. Figure 7G Shown is FACS analysis of the endothelial cell marker CD31 in SC-endothelial cells derived from WT and B2M- / - hESCs. DETAILED DESCRIPTION

[0018] Immunoprotection of transplanted stem cell-derived islet cells (sc-islets) has not been achieved without chronic immunosuppression or encapsulation. Existing genetic engineering approaches to generate low-immunogenic SC-islet cells have shown mixed results to date. Here we show that targeting human leukocyte antigen (HLA) and PD-L1 alone is not sufficient to protect SC-islet cells from xeno- or allogeneic rejection. Here we describe genetically engineered SC-islet cells that secrete the cytokines IL-10, TGF-β, and modified IL-2 to promote a tolerant local microenvironment by activating and expanding regulatory T cells (Tregs). These cytokine-secreting human SC-islet cells prevent xenogeneic rejection up to 9 weeks after transplantation in B6 / albino mice. Therefore, hESCs engineered to induce a tolerant local microenvironment may represent a source of alternative SC-islet cells that do not require encapsulation or immunosuppression for diabetic cell replacement therapy. The described cell modifications can be used alone or in conjunction with other approaches to generate low-immunogenic SC-islet cells.

[0019] To address the issue of transgene silencing during differentiation into SC-islet cells, we employed a transgene targeting strategy that exploits the constitutively expressed GAPDH gene in primary human islets and SC-islet cells (Gerace et al., 2021; Sintov et al., 2021). We generated low immunogenic SC-islet cells that overexpressed PD-L1 and HLA-E long-chain fusions in both HLA-normal and HLA-deficient settings. In these studies, PD-L1 overexpression did not protect SC-islet cells from xenogeneic rejection, and overexpression of HLA-E single-chain fusions was not required to suppress primary human NK cells. In addition to immune escape, we also addressed immune regulation and showed that constitutive secretion of IL-10, TGF-β, and IL-2 mutant proteins did not impair SC-islet cell function in vitro and indeed protected SC-islet cells from xenogeneic rejection up to 9 weeks after transplantation. Therefore, these immunoregulatory SC-islet cells represent a step forward in providing a source of islets for the treatment of T1D, with the long-term goal of eliminating the need for encapsulation or systemic immunosuppression.

[0020] The utility of immune escape / tolerance islet cell replacement therapy relies on the ability to maintain transgene expression during cell differentiation and after transplantation. Therefore, to analyze the effects of various gene editing strategies on the immune protection of SC-islets, SC-islets were engineered to constitutively express tolerance molecules, including PD-L1 from the GAPDH locus (Castro-Gutierrez et al., 2021; Yoshihara et al., 2020) and HLA-E long chain fusions (Gornalusse et al., 2017), both in HLA-competent and HLA-deficient backgrounds. We report that PD-L1 overexpression in SC-islets lacks a xenoprotective effect. This may be explained by species-specific differences in PD-L1 / PD-1 binding (Viricel et al., 2015) and the fact that human and mouse PD-1 share only 60% homology at the amino acid level (Finger et al., 1997). Indeed, we observed reduced binding of soluble mouse PD-1 to human PD-L1 expressed on SC-islet cells. Although these results suggest that overexpression of human PD-L1 is not sufficient to overcome xenogeneic rejection, PD-L1 overexpression may still be useful in the allogeneic setting.

[0021] Consistent with previous studies, HLA-deficient SC-islet cells are resistant to PBMC cytotoxicity in vitro (Han et al., 2019; Leite et al., 2022). We also found that SC-β cells modulate their T cell ligand profile in response to some inflammatory stimuli. Although we and others have attempted to exploit the PD-L1 / PD-1 T cell signaling axis to engineer immune-evading SC-islet cells (Yoshihara et al., 2020), our transcriptional analysis of T cell ligands in SC-β cells suggests that exploiting the LGALS9 / TIM-3 signaling axis may also be of interest. Indeed, like PD-L1, LGALS9 is frequently upregulated in cancer cells, where it promotes tumor progression by inhibiting T cell function (Heusschen et al., 2013; Yang et al., 2021). In addition, we identified the T cell activating ligands HVEM, CD40, and BTN3A1 as potential targets for knockout in SC-islet cells to further affect T cell function.

[0022] The work described here also shows that HLA-deficient SC-islet cells are resistant to cytotoxicity of preactivated NK cells both in vitro and in vivo, and that overexpression of HLA-E long-chain fusions does not provide any additional protective benefit. This is due to the SC-islet cell-specific lack of NK cell activating ligands, such as MIC and ULBP proteins, which are expressed in SC-endothelial cells and may explain why SC-endothelial cells are susceptible to cytotoxicity of preactivated NK cells, while SC-islet cells are resistant (Deuse et al., 2021). However, since many immunodeficient mouse models lack key components of NK cell survival and function, such as SIRPα and IL-15 (Herndler-Brandstetter et al., 2017), they do not support long-term engraftment of human NK cells, and we cannot rule out the possibility that HLA-deficient SC-islet cell destruction occurs in mouse models that better support NK cell engraftment. In addition, although transplantation of HLA-deficient SC-islet cells was able to normalize blood glucose levels in diabetic mice, these cells were ultimately rejected after injection of PBMCs, although the rejection kinetics were delayed. This could be explained by the presence of other immune cell subsets such as macrophages, monocytes, and dendritic cells that may play a role in indirect allograft rejection (Oberbarnscheidt et al., 2014; Wyburn et al., 2005; Zhuang et al., 2016). Ultimately, this highlights the limitations of using only in vitro immune cell co-culture assays to assess the effects of gene modification on protecting SC-islet cells from immune destruction (Castro-Gutierrez et al., 2021; Leite et al., 2022).

[0023] Finally, the work described here demonstrates for the first time that SC-islet cells engineered to secrete modified IL-2, TGF-β, and IL-10 are protected from xenogeneic rejection. This finding has several important implications. First, as β cells are professional secretory cells with large translational requirements, this demonstrates that SC-islet cells can be selected to secrete additional proteins while maintaining their designed function (Lim et al., 2020). Second, when determining the set of genetic modifications required to generate immune-evading cells, the intrinsic ligand profile of the desired cell type should be considered, as some cell types may not require extensive genetic manipulation. As the safety of immune-evading / immune-tolerant cell therapies is critical for their clinical translation, genetically engineered products should ideally be generated with minimal genetic perturbations to limit off-target events and chromosomal instability. Third, these results validate the use of immune-tolerance approaches (alone or in combination with immune-evasion approaches) as a way to protect SC-islet cells from the immune system. A variant of this approach could be to include some, but not all, cells secreting these tolerance molecules in the transplanted organ. In summary, this approach may eliminate the need for encapsulation or immunosuppression, a long-standing goal in the field of islet transplantation.

[0024] Although HLA-deficient SC-islet cells had improved survival in an in vitro PBMC co-culture assay ( Figure 2B and Figure 2C ), but these results have not been replicated in vivo. The selection of an appropriate humanized mouse model is critical for evaluating immune escape / immune tolerance genetic engineering strategies. Many PBMC humanized mouse models (such as the one used in this study) cannot fully reproduce human allograft rejection because they are biased towards CD3+ cell engraftment and lack other immune cell subsets. Therefore, although HLA-deficient SC-islets show delayed rejection in PBMC humanized mouse models ( Figure 3F ), but the possibility that these cells will be rejected earlier in CD34+ or BLT mice (humanized mouse models that better reproduce human immune components) cannot be ruled out. For these reasons, it is ideal to verify the protective effect of immune escape / tolerance genetic engineering strategies in humanized mouse models that more accurately reflect allogeneic transplant rejection. Although xenogeneic rejection does not simulate allogeneic rejection, the xenogeneic rejection model is used to evaluate this immune tolerance genetic engineering strategy because it is a stronger immune rejection model and eliminates the immune cell bias of the humanized mouse model.

[0025] By using this strong transplant rejection model, additional immune barriers are introduced, which require complex genetic engineering approaches to overcome. This means combining gene knockout and gene knockin to generate the desired immune escape / tolerant cell products. Knocking in multiple genes at specific locations in the genome requires large homology-directed repair templates, which are associated with poor integration efficiency and recovery of genetically unstable clones. Exploring the possibility of introducing tolerogenic molecules at multiple constitutively expressed loci may reduce the size of the HDR template and recover genetically stable homozygous knockin clones. Striking a balance between HDR length and the number of editing events may allow more foreign genetic material to be introduced into the genome without producing destabilizing effects.

[0026] Furthermore, since immune-tolerant SC-islets secrete cytokines from all cells within the islets, the concentration of constitutively secreted cytokines could result in chronic immunosuppression. Therefore, it is important to evaluate the immune status of mice receiving immune-tolerant SC-islet cells, as chronic immunosuppression should be avoided. However, if transplantation of immune-tolerant SC-islets does result in chronic immunosuppression, the ability to enrich for specific endocrine cell populations may allow us to tune the dose of cytokine secretion and thereby achieve local transplant tolerance by generating designer islets composed of both cytokine-secreting and non-cytokine-secreting endocrine cells.

[0027] definition

[0028] For convenience, certain terms employed herein 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 the invention belongs.

[0029] The term "differentiated cell" means any primary cell that is not pluripotent as defined herein in its native form. In other words, the term "differentiated cell" refers to a cell of a specialized more cell type derived from a cell of a specialized less cell type (e.g., a stem cell, such as an induced pluripotent stem cell) during cell differentiation. Without being limited by theory, pluripotent stem cells can first differentiate into endoderm cells and other endoderm cell types that can form pancreatic cells during normal individual development. Further differentiation of endoderm cells leads to a pancreatic pathway, in which about 98% of the cells become exocrine cells, ductal cells or stromal cells, and about 2% become endocrine cells.

[0030] As used herein, the term "somatic cell" refers to any cell that forms the body of an organism, relative to a germ cell. In mammals, germ cells (also referred to as "gametes") are sperm and eggs that fuse during fertilization to produce cells called zygotes, and the entire mammalian embryo is developed by the cells. Every other cell type except the cell sperm and egg (gametocyte) and undifferentiated stem cells that form them in mammals is a somatic cell: internal organs, skin, bones, blood, and connective tissues are all composed of somatic cells. In some embodiments, a somatic cell is a "non-embryonic somatic cell", which means a somatic cell that is not present in an embryo or is not obtained from an embryo and is not produced by the proliferation of such a cell in vitro. In some embodiments, a somatic cell is an "adult somatic cell", which means a cell that is present in an organism other than an embryo or a fetus or is obtained from an organism other than an embryo or a fetus or produced by the proliferation of such a cell in vitro. Unless otherwise indicated, methods described herein can be carried out in vivo and in vitro.

[0031] As used herein, the term "adult cell" refers to cells found throughout the body after embryonic development.

[0032] As used herein, the term "endodermal cell" refers to a cell from one of the three primary germ cell layers in the very early embryo (the other two germ cell layers are the mesoderm and ectoderm). The endoderm is the innermost of the three layers. Endoderm cells differentiate to form first the embryonic intestine, then the lining of the respiratory and digestive tracts (e.g., intestines), the liver, and the pancreas.

[0033] As used herein, the term "endoderm-derived cell" refers to any cell that has developed or differentiated from an endoderm cell. For example, endoderm-derived cells include cells of the liver, lungs, pancreas, thymus, intestines, stomach, and thyroid. Without being bound by theory, liver and pancreatic progenitor cells (also referred to as pancreatic progenitor cells) develop from endoderm cells in the embryonic foregut. Soon after their specialization, liver and pancreatic progenitor cells rapidly acquire significantly different cell functions and regenerative abilities. These changes are caused by highly conserved inductive signals and genetic regulators in vertebrates.

[0034] The terms "stem cell-derived β cells", "SC-β cells" and "mature SC-β cells" refer to cells (e.g., pancreatic β cells) that exhibit at least one marker indicative of pancreatic β cells, express insulin, and exhibit a characteristic GSIS response of endogenous mature β cells. In some embodiments, "SC-β cells" include mature pancreatic β cells. It should be understood that SC-β cells do not need to be derived (e.g., directly) from stem cells. In addition, 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 different from that of native / naturally occurring / endogenous cells. Examples of SC-β cells and methods for obtaining such SC-β cells are described in WO 2015 / 002724, WO 2014 / 201167, Millman et al., 2016 and Pagliuca et al., 2014, all of which are incorporated herein by reference in their entirety. In some embodiments, a "SC-β cell" is a low-immunogenic stem cell-derived β cell, eg, a β cell that produces limited or no immune response.

[0035] As used herein, the term "exocrine cell" refers to cells of an exocrine gland (i.e., a gland that discharges its secretions through a duct). In a specific embodiment, an exocrine cell refers to a pancreatic exocrine cell, which is a pancreatic cell that produces enzymes that are secreted into the small intestine. These enzymes help digest food as it passes through the gastrointestinal tract. Pancreatic exocrine cells are also called islets of Langerhans, which secrete two hormones, insulin and glucagon.

[0036] As used herein, term "pluripotency" refers to the cell having the ability of being differentiated into the cell type exceeding a differentiation and preferably being differentiated into the characteristic cell type of all three germ cell layers under different conditions.The feature of pluripotent cells is mainly that, using for example nude mouse teratoma formation assay to obtain, they are differentiated into the ability exceeding a cell type, preferably being differentiated into all three germ layers.The expression of embryonic stem (ES) cell markers has also proved pluripotency, although the preferred test for pluripotency is to prove the ability of the cell of each germ layer in three germ layers.It should be noted that only cultivating such cells themselves can not make them have pluripotency.Relative to primary cell parent (it usually only has the ability of limited number of divisions in culture), the pluripotent cells of reprogramming (for example, iPS cells as defined herein) also have the feature of extending passage and not losing the ability of growth potential.

[0037] As used herein, the terms "iPS cell" and "induced pluripotent stem cell" are used interchangeably and refer to pluripotent stem cells artificially derived (e.g., induced or by complete reversal) from non-pluripotent cells, typically adult somatic cells (e.g., by inducing forced expression of one or more genes).

[0038] The terms "progenitor cell" or "precursor" cell are used interchangeably herein and refer to cells that have a more primitive (i.e., at an earlier step along a developmental pathway or progression than a fully differentiated cell) cellular phenotype relative to cells that can be produced by differentiation. Typically, progenitor cells also have a significant or very high proliferation potential. Progenitor cells can give rise to a variety of different differentiated cell types or a single differentiated cell type, depending on the developmental pathway and the environment in which the cell develops and differentiates.

[0039] As used herein, the term "stem cell" refers to an undifferentiated cell that can proliferate and produce more progenitor cells, and the progenitor cell has the ability to generate a large number of mother cells, which in turn can produce differentiated or differentiable daughter cells. The daughter cell itself can be induced to proliferate and produce offspring, which then differentiate into one or more mature cell types, while also retaining one or more cells with parental developmental potential. The term "stem cell" refers to a subset of progenitor cells, which has the ability or potential to differentiate into a more specialized or differentiated phenotype under certain circumstances, and retains the ability to proliferate in the case of being substantially undifferentiated under certain circumstances. In one embodiment, the term stem cell generally refers to a naturally occurring mother cell, and its offspring (offspring) are generally differentiated in different directions, for example, by obtaining the characteristics of a complete individual and specialized, as occurs in the progressive diversification of embryonic cells and tissues. Cell differentiation is a complex process that generally occurs through many cell divisions. Differentiated cells can be derived from pluripotent cells, which themselves are derived from pluripotent cells, etc. Although each pluripotent cell in these pluripotent cells can be considered a stem cell, the range of cell types that can be produced can vary greatly. Some differentiated cells also have the ability to produce cells with greater developmental potential. This ability can be natural, or can be artificially induced after being treated with various factors. In many biological situations, stem cells can also be "multipotent" because they can produce more than one progeny of different cell types, but this is not necessary for "stem-ness". Self-renewal is another classical part of the definition of stem cells, and as used in this document, it is indispensable. In theory, self-renewal can occur by any of two main mechanisms. Stem cells can be asymmetrically divided, in which one of the daughters retains the stem cell state, and the other daughter expresses some different other specific functions and phenotypes. Alternatively, some stem cells in a colony can be symmetrically divided into two stem cells, thereby maintaining some stem cells in the colony as a whole, and other cells in the colony only produce differentiated progeny. Formally, cells starting with stem cells may develop toward a differentiated phenotype, but then "reverse" and re-express the stem cell phenotype, and the term is usually referred to as "dedifferentiation" or "reprogramming" or "reverse differentiation" by those of ordinary skill in the art. As used herein, the term "pluripotent stem cells" includes embryonic stem cells, induced pluripotent stem cells, placental stem cells, and the like.

[0040] The term "embryonic stem cell" is used to refer to the pluripotent stem cells of the inner cell mass of the embryonic blastocyst (see U.S. Patent Nos. 5,843,780, 6,200,806). Such cells can be similarly obtained from the inner cell mass of the blastocyst derived from somatic cell nuclear transfer (see, e.g., U.S. Patent Nos. 5,945,577, 5,994,619, 6,235,970). The distinctive features of embryonic stem cells define the embryonic stem cell phenotype. Therefore, if the cell has one or more unique features of embryonic stem cells so that the cell can be distinguished from other cells, the cell has the phenotype of embryonic stem cells. Exemplary distinctive embryonic stem cell features include but are not limited to gene expression profiles, proliferation capacity, differentiation capacity, karyotype, responsiveness to specific culture conditions, etc.

[0041] The term "adult stem cell" or "ASC" is used to refer to any pluripotent stem cell derived from non-embryonic tissues (including fetuses, young bodies and adult tissues). Stem cells have been isolated from various adult tissues, including blood, bone marrow, brain, olfactory epithelium, skin, pancreas, skeletal muscle and cardiac muscle. Each of these stem cells can be characterized according to the morphology in gene expression, factor responsiveness and cultivation. Exemplary adult stem cells include neural stem cells, neural crest stem cells, mesenchymal stem cells, hematopoietic stem cells and pancreatic stem cells. As indicated above, it has been found that stem cells are present in almost every tissue. Therefore, the present invention recognizes that stem cell colonies can be isolated from almost any animal tissue.

[0042] As used herein, the term "reprogramming" refers to the process of changing or reversing the state of differentiation of somatic cells. Before reprogramming, cells can be partially or terminally differentiated. Reprogramming encompasses the complete reversal of the state of differentiation of somatic cells to pluripotent cells. Such complete reversal of differentiation produces inducible pluripotent (iPS) cells. Reprogramming as used herein also encompasses the partial reversal of the state of cell differentiation, such as partial reversal to a pluripotent state or a somatic cell that is neither pluripotent nor pluripotent, but a cell that has lost one or more specific features of the differentiated cells that produce the cell, such as directly reprogramming differentiated cells to different somatic cell types. Reprogramming generally relates to changes in at least some heritable patterns of nucleic acid modifications (e.g., methylation), chromatin condensation, epigenetic changes, genomic imprinting, etc. that occur during cell differentiation when the zygote develops into an adult, such as reversal.

[0043] As used herein, the term "agent" means any compound or substance, such as but not limited to small molecules, nucleic acids, polypeptides, peptides, drugs, ions, etc. "Agent" can be any chemical entity or part, including but not limited to synthetic and naturally occurring protein entities and non-protein entities. In some embodiments, the agent is a nucleic acid, a nucleic acid analog, a protein, an antibody, a peptide, an oligomer, an amino acid or a carbohydrate of an aptamer, including but not limited to a protein, an oligonucleotide, a ribozyme, a DNA enzyme, a glycoprotein, siRNA, a lipoprotein, an aptamer and its modification and combination, etc. In certain embodiments, the agent is a small molecule with a chemical part. For example, the chemical part includes an unsubstituted or substituted alkyl, aromatic or heterocyclic moiety, including macrolide, leptomycin (leptomycin) and related natural products or their analogs. It can be known that the compound has the desired activity and / or characteristics, or the compound can be selected from a library of various compounds.

[0044] The term "cell culture medium" as referred to herein (also referred to herein as "culture medium" or "medium") is a culture medium for culturing cells, which contains nutrients to maintain cell viability and support proliferation. The cell culture medium may contain any of the following substances in appropriate combinations: one or more salts, one or more buffers, amino acids, glucose or one or more other sugars, antibiotics, serum or serum replacements, and other components such as peptide growth factors, etc. The cell culture medium commonly used for a particular cell type is known to those skilled in the art.

[0045] The term "cell line" refers to a colony of cells that are largely or substantially identical, and the cell colony is generally derived from a single ancestral cell or derived from a limited and / or substantially identical ancestral cell colony. A cell line may have been or may be able to be maintained in culture for an extended period of time (e.g., months, years, an unlimited period of time). It may have undergone a spontaneous or induced transformation process, thereby giving the cell an unlimited culture lifespan. Cell lines include all those cell lines that are recognized in the art as such. It should be understood that cells acquire mutations and possible epigenetic changes over time, so that at least some characteristics of the individual cells of the cell line may be different from each other. In some embodiments, a cell line comprises a stem cell-derived cell as described herein.

[0046] The term "exogenous" refers to a substance that is 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 has been introduced into a biological system (such as a cell or organism) by a process involving human intervention, where the nucleic acid or protein is not normally present or is present in lower amounts. A substance would be considered exogenous if it was introduced into a cell or an ancestor of a cell that inherited the substance. In contrast, the term "endogenous" refers to a substance that is native to a biological system.

[0047] The term "expression" refers to the cellular processes involved in producing RNA and protein and, where appropriate, secreting protein, including, but not limited to, transcription, translation, folding, modification and processing, where appropriate."Expression products" include RNA transcribed from a gene and polypeptides obtained by translating mRNA transcribed from a gene.

[0048] As used herein, the term "genetically modified" or "engineered" cell refers to a cell (or a descendant of such a cell that has inherited at least a portion of the nucleic acid) into which an exogenous nucleic acid is introduced by a process involving human action. The nucleic acid may, for example, contain a sequence that is exogenous to the cell, which may contain a native sequence (i.e., a sequence naturally found in the cell), but in a non-naturally occurring arrangement (e.g., a coding region connected to a promoter from a different gene), or contain an altered form of the native sequence, etc. The process of transferring the nucleic acid into the cell may be achieved by any suitable technique. Suitable techniques include calcium phosphate or lipid-mediated transfection, electroporation, and transduction or infection using a viral vector. In some embodiments, a 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 causes a detectable change in the cell relative to an unmodified but otherwise equivalent cell. It should be understood that the term genetically modified is intended to include the introduction of modified RNA directly into the cell (e.g., synthetic, modified RNA). Such synthetic modified RNA includes modifications for preventing rapid degradation by endonucleases and exonucleases and for avoiding or reducing the innate immunity or interferon response of cells to RNA. Modifications include, but are not limited to, for example (a) terminal modifications, such as 5' terminal modifications (phosphorylation, dephosphorylation, conjugation, reverse linkage, etc.), 3' terminal modifications (conjugation, DNA nucleotides, reverse linkage, etc.), (b) base modifications, such as bases paired with modified bases, stable bases, destabilized bases, or bases paired with amplified partner pools, or conjugated base substitutions, (c) sugar modifications (e.g., at the 2' position or the 4' position) or sugar substitutions, and (d) internucleoside linkage modifications, including modifications or substitutions of phosphodiester linkages. If such modifications interfere with translation (i.e., causing translation to be reduced by 50% or more relative to the lack of modification), the modifications are not applicable to the methods and compositions described herein.

[0049] In the case of nucleic acids or polypeptides, the terms "isolated" or "partially purified" as used herein refer to a nucleic acid or polypeptide that is separated from at least one other component (e.g., nucleic acid or polypeptide) that is present with the nucleic acid or polypeptide as found in its natural source and / or that is present with the nucleic acid or polypeptide when expressed by a cell or secreted in the case of a secreted polypeptide. Chemically synthesized nucleic acids or polypeptides or nucleic acids or polypeptides synthesized using in vitro transcription / translation are considered "isolated."

[0050] 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 a descendant 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 reintroduced into the organism from which the cell was isolated (or the cell from which the cell originated).

[0051] As used herein, the term "isolated population" regarding an isolated population of cells refers to a population of cells that has been removed and separated from a mixed or heterogeneous population of cells. In some embodiments, an isolated population is a substantially pure population of cells, as compared to the heterogeneous population from which the cells are isolated or enriched.

[0052] The term "substantially pure" with respect to a particular cell population refers to a population of cells 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 relative to the cells making up the total cell population.

[0053] The terms "enriching" or "enriched" are used interchangeably herein and mean that the yield (fraction) of one type of cells is increased by at least 10% relative to the fraction of cells of that type in the starting culture or preparation.

[0054] The terms "renewal" or "self-renewal" or "proliferation" are used interchangeably herein and are used to refer to the ability of stem cells to self-renew by dividing into the same non-specialized cell type over long periods of time and / or months to years. In some cases, proliferation refers to the expansion of a cell by repeated division of a single cell into two identical daughter cells.

[0055] As used herein, the term "lineage" describes cells with a common ancestor or cells with a common developmental fate. For example, in the context of cells of endoderm origin or "endodermal lineage", this means that the cells are derived from endoderm cells and can differentiate along endoderm lineage restricted pathways such as one or more developmental lineage pathways that produce definitive endoderm cells, which in turn can differentiate into hepatocytes, thymus, pancreas, lung and intestine.

[0056] As used herein, "marker" is used to describe the characteristics and / or phenotype of a cell. Markers can be used to select cells containing features of interest. Markers vary from cell to cell. Markers are features, whether morphological, functional or biochemical (enzyme) features of cells of a particular cell type, or molecules expressed by the cell type. Preferably, such markers are proteins, and more preferably, have epitopes of antibodies or other binding molecules available in the art. However, markers can be composed of any molecule found in cells (including but not limited to proteins (peptides and polypeptides), lipids, polysaccharides, nucleic acids and steroids). Examples of morphological features or traits include but are not limited to shape, size and nuclear-cytoplasmic ratio. Examples of functional features or traits include but are not limited to the ability to adhere to a specific substrate, the ability to incorporate or exclude specific dyes, the ability to migrate under specific conditions, and the ability to differentiate along a specific lineage. Markers can be detected by any method available to those skilled in the art. Markers can also be the absence of morphological features or the absence of proteins, lipids, etc. Markers can be a combination of a group of unique features of the presence and absence of polypeptides and other morphological features.

[0057] The term "modulate" is used consistently with its use in the art, i.e., to mean to cause or promote a qualitative or quantitative change, alteration, or modification in a process, pathway, or phenomenon of interest. Without limitation, such a change may be an increase, decrease, or change in the relative strength or activity of different components or branches of a 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.

[0058] The term "polynucleotide" is used interchangeably with "nucleic acid" herein to indicate a polymer of nucleoside. Typically, the polynucleotide of the present invention is composed of nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine and deoxycytidine) naturally present in DNA or RNA connected by phosphodiester bonds. However, the term encompasses molecules including nucleosides or nucleoside analogs containing chemically or biologically modified bases, modified backbones, etc., whether or not found in naturally occurring nucleic acids, and such molecules may be preferred for certain applications. When the application refers to polynucleotides, it should be understood that DNA, RNA, and single-stranded and double-stranded forms (and the complement of each single-stranded molecule) in each case are provided. "Polynucleotide sequence" as used herein may refer to polynucleotide material itself and / or sequence information (i.e., continuous letters used as base abbreviations) that characterizes specific nucleic acids in a biochemical manner. Unless otherwise indicated, the polynucleotide sequences presented herein are presented in 5' to 3' directions.

[0059] As used herein, the term "polypeptide" refers to a polymer of amino acids. The terms "protein" and "polypeptide" are used interchangeably herein. Peptides are relatively short polypeptides, typically between about 2 and 60 amino acids in length. The polypeptides used herein typically contain amino acids, such as the 20 most common L-amino acids in proteins. However, other amino acids and / or amino acid analogs known in the art may be used. One or more amino acids in a polypeptide may be modified, for example, by adding chemical entities such as carbohydrate groups, phosphate groups, fatty acid groups, joints for conjugation, functionalization, etc. A polypeptide having a non-polypeptide portion covalently or non-covalently associated therewith is still considered a "polypeptide". Exemplary modifications include glycosylation and palmitoylation. Polypeptides can be purified from natural sources, produced using recombinant DNA technology, and synthesized by chemical means such as conventional solid phase peptide synthesis. As used herein, the term "polypeptide sequence" or "amino acid sequence" may refer to the polypeptide material itself and / or sequence information (i.e., consecutive letters or three-letter codes used as abbreviations of amino acid names) that characterizes a polypeptide in a biochemical manner. Unless otherwise indicated, the polypeptide sequences presented herein are presented in the direction from the N-terminus to the C-terminus.

[0060] When referring to a polypeptide, the term "variant" can be, for example, a polypeptide having at least 80%, 85%, 90%, 95%, 98% or 99% identity with a full-length polypeptide. A variant can be a fragment of a full-length polypeptide. A variant can be a naturally occurring splice variant. A variant can be a polypeptide having at least 80%, 85%, 90%, 95%, 98% or 99% identity with a fragment of a polypeptide, wherein the length of the fragment is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98% or 99% of the full-length wild-type polypeptide or its active domain of interest. In some embodiments, the length of the domain is at least 100, 200, 300 or 400 amino acids, starting from any amino acid position in the sequence and extending to the C-terminus. It is preferred to avoid variants known in the art that eliminate or significantly reduce protein activity. In some embodiments, the variant lacks the N and / or C-terminal portion of the full-length polypeptide, for example lacking 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, which means a polypeptide from which one or more portions (such as a signal peptide) have been removed during normal intracellular proteolytic processing (e.g., during co-translational or post-translational processing). In some embodiments in which the protein is not produced by purification from cells that naturally express it, the protein is a chimeric polypeptide, which means that it contains portions from two or more different species. In some embodiments in which the protein is not produced by purification from cells that naturally express it, the protein is a derivative, which means that the protein contains additional sequences that are unrelated to the protein, as long as those sequences do not substantially reduce the biological activity of the protein.

[0061] As used herein, the term "functional fragment" is a polypeptide having an amino acid sequence that is smaller in size than the polypeptide from which the fragment is derived, but substantially homologous to the polypeptide, and wherein the effective biological effect of the functional fragment polypeptide sequence is about at least 50%, or 60% or 70% or 80% or 90% or 100% or greater than 100%, such as 1.5 times, 2 times, 3 times, 4 times or greater than 4 times that of the polypeptide from which the fragment is derived. Functional fragment polypeptides may have additional functions, which may include reducing antigenicity, increasing DNA binding (such as in transcription factors) or altering RNA binding (such as in regulating RNA stability or degradation).

[0062] 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 those capable of autonomous replication and / or expression of nucleic acids connected thereto. A vector capable of directing the expression of genes operably connected thereto is referred to herein as an "expression vector". Therefore, an "expression vector" is a specialized vector containing the necessary regulatory regions required for expressing a gene of interest in a host cell. In some embodiments, the gene of interest is operably connected to another sequence in the vector. The vector may be a viral vector or a non-viral vector. If a viral vector is used, it is preferred that the viral vector is replication-defective, which can be achieved, for example, by removing all viral nucleic acids encoded for replication. Replication-defective viral vectors will still retain their infection properties and enter cells in a manner similar to that of replicating adenoviral vectors, but once entering cells, replication-defective viral vectors will not replicate or propagate. The vector also encompasses liposomes and nanoparticles and other ways of delivering DNA molecules to cells.

[0063] The term "operably linked" means that the regulatory sequences necessary for the expression of the coding sequence are placed in the appropriate position of the DNA molecule relative to the coding sequence to achieve the expression of the coding sequence. This same definition is sometimes applied to the arrangement of the coding sequence and transcription control elements (e.g., promoters, enhancers and termination elements) in the expression vector. The term "operably linked" includes having an appropriate start signal (e.g., ATG) in front of the polynucleotide sequence to be expressed, and maintaining the correct reading frame to allow the expression of the polynucleotide sequence under the control of the expression control sequence, and producing the desired polypeptide encoded by the polynucleotide sequence.

[0064] The term "viral vector" refers to the use of a virus or a virus-associated vector as a carrier for the entry of a nucleic acid construct into a cell. The construct can be integrated and packaged into a non-replicating defective viral genome such as adenovirus, adeno-associated virus (AAV) or herpes simplex virus (HSV) or other (including retrovirus and lentiviral vectors) for infection or transduction into a cell. The vector may or may not be incorporated into the genome of the cell. If desired, the construct may include a viral sequence for transfection. Alternatively, the construct may be incorporated into a vector capable of additional replication, such as EPV and EBV vectors.

[0065] The terms "regulatory sequence" and "promoter" are used interchangeably herein and refer to nucleic acid sequences that induce or control the transcription of a protein coding sequence that is operably connected thereto, such as start signals, enhancers, and promoters. In some instances, the transcription of a recombinant gene is under the control of a promoter sequence (or other transcriptional regulatory sequences), which controls the expression of the recombinant gene in the cell type in which it is expected to be expressed. It should also be understood that the recombinant gene can be under the control of a transcriptional regulatory sequence that is identical or different from those sequences that control the transcription of a naturally occurring form of protein. In some cases, the promoter sequence is identified by the synthetic machinery of the cell required to initiate transcription of a specific gene or by the synthetic machinery introduced.

[0066] 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, "proliferation" and "proliferation" refer to increasing the number of cells (growth) in a population by cell division. Cell proliferation is generally understood to be caused by the coordinated activation of multiple signal transduction pathways in response to an environment including growth factors and other mitogens. Cell proliferation can also be promoted by releasing from the action of intracellular or extracellular signals and blocking or negatively affecting the mechanism of cell proliferation.

[0067] The term "selectable marker" refers to a gene, RNA or protein that confers a selectable phenotype to a cell when expressed, such as resistance to cytotoxic agents or cell growth inhibitors (e.g., antibiotic resistance), nutritional prototrophy, or expression of a specific protein that can be used as a basis for distinguishing cells expressing the protein from cells that do not express the protein. Proteins whose expression can be easily detected, such as fluorescent or luminescent proteins or enzymes that act on substrates to produce colored, fluorescent or luminescent substances ("detectable markers") constitute a subset of selectable markers. The presence of selectable markers connected to expression control elements that are native to genes that are typically selectively or exclusively expressed in pluripotent cells makes it possible to identify and select somatic cells that have been reprogrammed to a pluripotent state. A variety of selectable marker genes can be used, such as neomycin resistance gene (neo), puromycin resistance gene (puro), guanine phosphoribosyl transferase (gpt), dihydrofolate reductase (DHFR), adenosine deaminase (ada), puromycin-N-acetyl transferase (PAC), hygromycin resistance gene (hyg), multidrug resistance gene (mdr), thymidine kinase (TK), hypoxanthine-guanine phosphoribosyl transferase (HPRT) and hisD gene. Detectable markers include green fluorescent protein (GFP), blue fluorescent protein, sky blue fluorescent protein, yellow fluorescent protein, red fluorescent protein, orange fluorescent protein and cyan fluorescent protein and variants of any of these. Fluorescent proteins such as luciferase (e.g., firefly luciferase or Renilla luciferase) can also be used. As will be apparent to 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.

[0068] In some embodiments, relative to the cell that does not express the selectable marker or expresses the selectable marker at a significantly lower level, the selectable marker confers the cell proliferation and / or survival advantage of expressing it. Such proliferation and / or survival advantage usually occurs when the cell is maintained under certain conditions, i.e., "selective conditions". To ensure effective selection, the colony of the cell can be maintained under certain conditions and kept for a sufficiently long time so that the cell that does not express the marker does not proliferate and / or does not survive, and eliminate or reduce the number of the cell to only a small part of the colony from the colony. The process of selecting the cell that expresses the marker that gives proliferation and / or survival advantage by maintaining the colony of the cell under selective conditions to a great extent or completely eliminate the cell that does not express the marker is referred to as "positive selection" in this article, and the marker is referred to as "can be used for positive selection". In some methods described herein, negative selection and the marker that can be used for negative selection are also concerned. Relative to the cell that does not express the marker or expresses the marker at a significantly lower level, the expression of such markers confers the cell proliferation and / or survival disadvantage of expressing the marker (or, considering another way, relative to the cell that expresses the marker, the cell that does not express the marker has proliferation and / or survival advantage). Thus, when maintained under selective conditions for a sufficient period of time, cells expressing the marker can be largely or completely eliminated from the population of cells.

[0069] As used herein, "reporter gene" encompasses any gene introduced into a cell genetically, which increases the phenotype of a stem cell. Reporter genes such as disclosed in the present invention are intended to encompass fluorescence, luminescence, enzymes and resistance genes, but also encompass other genes that can be easily detected by a person of ordinary skill in the art. In some embodiments of the present invention, reporter genes are used as markers for identifying specific stem cells, cardiovascular stem cells and their differentiated offspring. Reporter genes are usually operably connected to a sequence that regulates their expression in a manner that depends on one or more conditions, and the conditions are monitored by measuring the expression of reporter genes. In some cases, the expression of reporter genes can be determined in living cells. When assayed using live cell reporter genes, reporter gene expression can be monitored at multiple time points, such as 2, 3, 4, 5, 6, 8 or 10 or more time points. In some cases, when assayed using live cell reporter genes, reporter gene expression is monitored at a frequency of at least about 10 minutes to about 24 hours, such as 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 about 10 minutes to about 24 hours. Another frequency of any integer between the monitoring reporter gene expression.

[0070] The terms "subject" and "individual" are used interchangeably herein and refer to an animal, such as a human, from which cells can be obtained and / or to which treatment (including prophylactic treatment) with cells as described herein can be provided. For treatment of those infections, illnesses or disease states that are specific to a particular animal, such as a human subject, the term subject refers to the particular animal. "Non-human animals" and "non-human mammals" as used interchangeably herein 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 mammals, such as domesticated mammals, such as dogs, cats, horses, etc., or production mammals, such as cows, sheep, pigs, etc.

[0071] The terms "treat," "treating," "treatment," and the like as applied to isolated cells include subjecting cells to any kind of process or condition or performing any kind of manipulation or surgery on cells. When applied to a subject, these terms refer to providing medical or surgical attention, care, or management to an individual. The individual is typically ill or injured, or is at increased risk of illness relative to the average member of the population, and is in need of such attention, care, or management.

[0072] As used herein, the terms "treating" and "treatment" refer to administering an effective amount of a composition to a subject so that at least one symptom of the subject's disease is alleviated or the disease is improved, such as a beneficial or desired clinical outcome. For purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, alleviation of one or more symptoms, reduction in disease extent, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial or total relief), whether detectable or undetectable. Treatment may refer to, for example, prolonged survival compared to the expected survival when not receiving treatment. Therefore, those skilled in the art recognize that treatment may improve the disease condition, but may not completely cure the disease. As used herein, the term "treatment" includes prevention. Alternatively, treatment is "effective" if the progression of the disease is reduced or stopped. "Treatment" may also mean prolonged survival compared to the expected survival when not receiving treatment.

[0073] As used herein, the terms "administering", "introducing" and "transplanting" are used interchangeably in the context of placing the cells of the present invention in a subject by a method or route that results in at least a portion of the introduced cells being located at a desired site. The cells can be implanted directly into the pancreas or gastrointestinal tract, or alternatively administered by any appropriate route that results in delivery to a desired location in a subject, where at least a portion of the implanted cells or cell components remain viable. The viability time of the cells after administration to the subject can be as short as a few hours, such as twenty-four hours, to a few days, to as long as several years. In some cases, the cells can also be administered subcutaneously, for example in the form of capsules (e.g., microcapsules), to maintain the implanted cells at the implantation site and to avoid migration of the implanted cells.

[0074] As used herein, the phrases "parenteral administration" and "administered parenterally" mean modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. As used herein, the phrases "systemic administration," "systemically administered," "peripherally administered," and "peripherally administered" mean administration of stem cell-derived cells and / or their progeny and / or compounds and / or other materials other than direct administration into the central nervous system so that they enter the animal's system and are thus subject to metabolism and other similar processes, such as subcutaneous administration.

[0075] The term "tissue" refers to a group or layer of specialized cells that work together to perform some specific function. The term "tissue specificity" refers to the origin of cells from a specific tissue.

[0076] The terms "decrease", "reduced", "reduction", "decrease" or "inhibit" are generally used herein to mean a reduction in a statistically significant amount. However, for the avoidance of doubt, "reduced", "reduction" or "reduction" or "inhibit" means a reduction of at least 10% compared to a reference level, such as a reduction of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% reduction (i.e., a level that is not present compared to a reference sample), or any reduction between 10%-100%.

[0077] The terms "increased", "increase", "enhance" or "activate" are all generally used herein to mean an increase by a statistically significant amount; for the avoidance of any doubt, the terms "increased", "increase", "enhance" or "activate" mean an increase of at least 10% compared to a reference level, such as an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase, or any increase between 10%-100%, or an increase of at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold, or any increase between 2-fold and 10-fold or greater compared to a reference level.

[0078] The term "statistically significant" or "significantly" refers to statistical significance and generally means two standard deviations (2SD) below normal or lower marker concentrations. 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. The decision is generally made using a p-value.

[0079] Stem Cells

[0080] Stem cells are cells that retain the ability to self-renew through mitotic cell division and can differentiate into a variety of specialized cell types. Two broad types of mammalian stem cells are: embryonic stem cells (ES) 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 cells and progenitor cells serve as the body's repair system, replenishing specialized cells, but also maintaining the normal turnover of regenerated organs (such as blood, skin or intestinal tissue). Pluripotent stem cells can differentiate into cells derived from any of the three germ layers.

[0081] Although certain embodiments are described below with reference to the use of stem cells, germ cells may be used in place of stem cells or in combination with stem cells to provide at least one differentiated cell, using protocols similar to the illustrative protocols described herein. Suitable germ cells may be prepared, for example, from primordial germ cells present in human fetal material collected about 8-11 weeks after the last menstrual period. For example, illustrative germ cell preparation methods are described, for example, in Shamblott et al. Proc. Natl. Acad. Sci. USA 95:13726, 1998 and U.S. Patent No. 6,090,622.

[0082] ES cells, such as human embryonic stem cells (hESC) or mouse embryonic stem cells (mESC), have almost unlimited replication capacity and the potential to differentiate into most cell types, providing in principle unlimited starting material to generate differentiated cells for clinical therapy (stemcells.nih.gov / info / scireport / 2006report.htm, 2006).

[0083] hESC cells are described, for example, by Cowan et al. (N Engl. J. Med. 350:1353, 2004) and Thomson et al. (Science 282:1145, 1998); embryonic stem cells from other primates, 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) can also be used in the methods disclosed herein. mESCs are described, for example, by Tremml et al. (Curr Protoc Stem Cell Biol. Chapter 1:Unit 1C.4, 2008). Stem cells can be, for example, unipotent, totipotent, multipotent, or pluripotent.In some embodiments, any cell of primate origin that is capable of producing progeny that are derivatives of at least one germinal layer or all three germinal layers can be used in the methods disclosed herein.

[0084] In certain examples, ES cells can be isolated, for example, as described in Cowan et al. (N Engl. J. Med. 350:1353, 2004) and U.S. Pat. No. 5,843,780 and Thomson et al. Proc. Natl. Acad. Sci. USA 92:7844, 1995. For example, hESC cells can be prepared from human blastocyst cells using techniques described by Thomson et al. (U.S. Pat. No. 6,200,806; Science 282:1145, 1998; Curr. Top. Dev. Biol. 38:133 ff., 1998) and Reubinoff et al. Nature Biotech. 18:399, 2000. Equivalent cell types of hESC include their pluripotent derivatives, such as primitive ectoderm-like (EPL) cells, as summarized in, for example, WO01 / 51610 (Bresagen). hESC can also be obtained from human preimplantation embryos. Alternatively, in vitro fertilization (IVF) embryos can be used, or single-cell human 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 culture media (Gardner et al., Fertil. Steril. 69: 84, 1998). The zona pellucida is removed from the developed blastocyst by short exposure to pronase (Sigma). The inner cell mass can be separated by immunosurgery, wherein the blastocyst is exposed to rabbit anti-human spleen cell antiserum 1: 50 dilution 30 minutes, then washed three times in DMEM, 5 minutes each time, and exposed to guinea pig complement (Gibco) 1: 5 dilution 3 minutes (Solter et al., Proc. Natl. Acad. Sci. USA 72: 5099, 1975). After washing twice more in DMEM, the lysed trophoblast cells are removed from the intact inner cell mass (ICM) by gentle pipetting, and the ICM is plated on the mEF feeder layer. After 9 to 15 days, the inner cell mass-derived outgrowths can be dissociated into clumps by exposure to phosphate buffered saline (PBS) containing 1 mM EDTA, exposure to dispase or trypsin, or by mechanical dissociation with a micropipette; and then re-plated on mEFs in fresh culture medium. Growing colonies with undifferentiated morphology can be selected individually by a micropipette, mechanically dissociated into clumps, and re-plated. ES-like morphology is characterized as dense colonies with a significantly high nuclear-cytoplasmic ratio and prominent nucleoli.The resulting hESCs can then be routinely isolated every 1-2 weeks, for example, by brief trypsinization, exposure to Dulbecco's PBS (containing 2 mM EDTA), exposure to type IV collagenase (about 200 U / mL; Gibco), or by selecting single colonies with a micropipette. In some examples, a clump size of about 50 to 100 cells is optimal. mESC cells can be prepared using techniques described, for example, by Conner et al. (Curr. Prot. in Mol. Biol. Unit 23.4, 2003).

[0085] Embryonic stem cells can be isolated from blastocysts of members of 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 can be prepared from human blastocyst cells using techniques described by Thomson et al. (U.S. Pat. No. 6,200,806; Science 282:1145, 1998; Curr. Top. Dev. Biol. 38:133 ff., 1998) and Reubinoff et al., Nature Biotech. 18:399, 2000. Cell types equivalent to hES cells include their pluripotent derivatives, such as primitive ectoderm-like (EPL) cells, as outlined in WO01 / 51610 (Bresagen).

[0086] Alternatively, in some embodiments, hES cells can be obtained from human preimplantation embryos. Alternatively, in vitro fertilization (IVF) embryos can be used, or single-cell human embryos can be expanded to the blastocyst stage (Bongso et al., Hum Reprod4: 706, 1989). Embryos are cultured to the blastocyst stage in G1.2 and G2.2 culture media (Gardner et al., Fertil.Steril. 69:84, 1998). Zona pellucida is removed from developed blastocysts by short exposure to pronase (Sigma). Inner cell mass is separated by immunosurgery, wherein blastocysts are exposed to rabbit anti-human spleen cell antiserum 1:50 diluents 30 minutes, then washed three times in DMEM, 5 minutes each time, and exposed to 1:5 diluents 3 minutes of guinea pig complement (Gibco) (Solter et al., Proc. Natl. Acad. Sci. USA 72:5099, 1975). After two more washes in DMEM, lysed trophoblast cells were removed from the intact inner cell mass (ICM) by gentle pipetting, and the ICM was plated on the mEF feeder layer.

[0087] After 9 to 15 days, the inner cell mass-derived outgrowths were dissociated into clumps by exposure to phosphate buffered saline (PBS) containing 1 mM EDTA, exposure to dispase or trypsin, or by mechanical dissociation with a micropipette; and then re-plated on mEFs in fresh culture medium. Growing colonies with undifferentiated morphology were selected individually by a micropipette, mechanically dissociated into clumps, and re-plated. ES-like morphology is characterized by dense colonies with a significantly high nuclear-cytoplasmic ratio and prominent nucleoli. The resulting ES cells were then routinely split every 1-2 weeks by brief trypsin digestion, exposure to Dulbecco's PBS (containing 2 mM EDTA), exposure to type IV collagenase (about 200 U / mL; Gibco), or by selecting single colonies via a micropipette. The clump size of about 50 to 100 cells is optimal.

[0088] In some embodiments, human embryonic germ (hEG) cells are pluripotent stem cells that can be used in methods as disclosed herein to differentiate into primitive endoderm cells. hEG cells can be prepared from primitive germ cells present in human fetal material collected about 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. Pat. No. 6,090,622, which are incorporated herein by reference in their entirety.

[0089] Briefly, genital ridges were processed to form disaggregated cells. EG growth medium was 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 (in 10% DMSO). Ninety-six-well tissue culture plates are prepared in which a subconfluent layer of feeder cells (e.g., STO cells, ATCC No. CRL1503) is cultured in modified EG growth medium without LIF, bFGF, or forskolin for 3 days, inactivated by 5000 rads of γ-irradiation, and about 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, and each well is transferred to one well of a previously prepared 24-well culture dish containing irradiated STO mouse fibroblasts. The cells are cultured with daily changes of medium until cell morphology consistent with EG cells is observed, usually after 7-30 days or 1-4 generations.

[0090] In some instances, according to methods as disclosed herein, before being exposed to at least one maturation factor, stem cells can be undifferentiated (e.g., unstereotyped as cells of a specific lineage), and in other instances, stem cells may need to be differentiated into one or more intermediate cell types before being exposed to at least one maturation factor as described herein. For example, stem cells can show the morphological, biological or physical properties of undifferentiated cells, which can be used to distinguish them from differentiated cells of embryonic or adult origin. In some instances, undifferentiated cells can appear in a two-dimensional microscope view of a cell colony with a high nuclear / cytoplasmic ratio and prominent nucleoli. Stem cells can be themselves (e.g., substantially absent any undifferentiated cells), or can be used in the presence of differentiated cells. In some instances, stem cells can be cultured in the presence of suitable nutrients and optional other cells so that stem cells can grow and optionally differentiate. For example, embryonic fibroblasts or fibroblast-like cells can be present in cultures to help stem cell growth. Fibroblasts can exist during a stage of stem cell growth, but not necessarily in all stages. For example, fibroblasts may be added to a stem cell culture in a first culture stage and not added to the stem cell culture in one or more subsequent culture stages.

[0091] The stem cells used in all aspects of the invention can be any cell derived from any kind of tissue (e.g., embryonic tissue, such as fetal or pre-fetal tissue, or adult tissue) that has the characteristic of being able to produce offspring of different cell types under appropriate conditions, 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 they can be obtained directly from the original embryonic tissue and immediately used for differentiation. Included are cells listed in the NIH human embryonic stem cell record, e.g., 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-Seou National University, Seoul); HSF-1, HSF-6 (University of California, San Francisco); and H1, H7, H9, H13, H14 (Wisconsin Alumni Research Foundation (WiCell Research Institute)). In some embodiments, the source of human stem cells or pluripotent stem cells for chemically induced differentiation into stem cell-derived cells does not involve destroying human embryos.

[0092] 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.

[0093] Stem cells of interest also include various types of embryonic cells, such as human embryonic stem (hES) cells 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 cardiogenic cells (see Reyes et al. (2001) Blood 98:2615-2625; Eisenberg and Bader (1996) Circ Res. 78(2):205-16; etc.). Stem cells can be obtained from any mammalian species, such as humans, horses, cattle, pigs, dogs, cats, rodents (e.g., mice, rats, hamsters), primates, etc. In some embodiments, for the source of pluripotent cells used in the methods and compositions disclosed herein, human embryos are not destroyed.

[0094] When ES cells are not stereotyped as a specific differentiation lineage, they are considered to be undifferentiated. Such cells exhibit morphological features that distinguish them from differentiated cells of embryonic or adult origin. Undifferentiated ES cells are easily identified by those skilled in the art, and typically appear in a two-dimensional microscopic view in a cell colony with a high nuclear / cytoplasmic ratio and prominent nucleoli. Undifferentiated ES cells express genes that can be used as markers to detect the presence of undifferentiated cells, and the polypeptide products of the genes can be used as markers for negative selection. For example, see U.S. Application Serial 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 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 adding 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 do not stain for SSEA-1, but differentiated cells stain strongly for SSEA-1. Methods for propagating hES cells in an undifferentiated form are described in WO 99 / 20741, WO 01 / 51616, and WO 03 / 020920.

[0095] Cell mixtures from suitable sources of endothelial, muscle and / or neural stem cells can be harvested from mammalian donors by methods known in the art. Suitable sources are hematopoietic microenvironments. For example, circulating peripheral blood can be taken out from a subject, preferably mobilized (i.e., raised). Alternatively, bone marrow can be obtained from a mammal undergoing autologous transplantation, such as a human patient. In some embodiments, stem cells can be obtained from adipose tissue of a subject, for example, using the CELUTION™ system from Cytori as disclosed in U.S. Patent Nos. 7,390,484 and 7,429,488, which are incorporated herein by reference in their entirety.

[0096] In some embodiments, human umbilical cord blood cells (HUCBC) can be used in the methods disclosed herein. Human UBC cells are considered to be a rich source of hematopoietic and mesenchymal progenitor cells (Broxmeyer et al., 1992 Proc. Natl. Acad. Sci. USA 89: 4109-4113). Previously, umbilical cord blood and placental blood were considered waste products that were usually discarded when a baby was born. Umbilical cord blood cells are 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 lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome, and neuroblastoma) and non-malignant diseases such as Fanconi's 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. Rev. Oncol. Hematol 22:61-78; Lu et al., 1995 Cell Transplantation 4:493-503). A distinct advantage of HUCBC is that the immature immunity of these cells is very similar to fetal cells, thus significantly reducing the risk of host rejection (Taylor and Bryson, 1985 J. Immunol. 134: 1493-1497).Human umbilical cord blood contains mesenchymal and hematopoietic progenitor cells and endothelial cell precursors 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 and Bryson, 1985 J. Immunol. 134:1493-1497 Broxmeyer, 1995 Transfusion 35:694-702; Chen et al., 1996 Blood 79: ... 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 in bone marrow, and in addition, highly proliferative hematopoietic cells are eight times higher in HUCBC than in bone marrow and express hematopoietic markers such as CD14, CD34 and CD45 (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).

[0097] In another embodiment, the pluripotent cells are cells in a hematopoietic microenvironment, such as circulating peripheral blood, preferably the mononuclear fraction of peripheral blood, umbilical cord blood, bone marrow, fetal liver or yolk sac from a mammal. Stem cells, especially neural stem cells, may also be derived from the central nervous system, including the meninges.

[0098] In another embodiment, the pluripotent cells present in the embryoid bodies are formed by harvesting ES cells with a transient protease digestion and allowing small clumps of undifferentiated human ESC to grow in suspension culture. Differentiation is induced by removing the conditioned medium. The resulting embryoid bodies are spread on a semisolid substrate. The formation of differentiated cells can be observed after about 7 days to about 4 weeks. By dissociating embryoid bodies or similar structures to provide cell aggregates, differentiated cells that can live are selected from the in vitro culture of stem cells. The method of maintaining cell-to-cell contact in aggregates is used to select aggregates containing cells of concern for phenotypic characteristics.

[0099] In alternative embodiments, stem cells can be reprogrammed stem cells, such as stem cells derived from somatic cells or differentiated cells. In such embodiments, dedifferentiated stem cells can be, for example, but not limited to, neoplastic cells, tumor cells, and cancer cells, or alternatively inducible reprogrammed cells, such as induced pluripotent stem cells or iPS cells.

[0100] Cloning and cell culture

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

[0102] Suitable cell culture methods can be found, for example, in Cell culture methods, generally described in the current edition of Culture of Animal Cells: A Manual of Basic Technique (RI Freshney, ed., Wiley and Sons); General Techniques of Cell Culture (MA Harrison and IFRae, 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 used by those of ordinary skill in the art to promote proliferation without promoting differentiation in culture conditions for continuous proliferation. Exemplary serum-containing ES culture medium is prepared with 80%DMEM (such as knocking out DMEM, Gibco), 20% definite fetal bovine serum (FBS, Hyclone) or serum substitute (WO98 / 30679), 1% non-essential amino acids, 1mM L-glutamine and 0.1mM β-mercaptoethanol. Before use, human bFGF is added to 4 ng / mL (WO 99 / 20741, GeronCorp.). Traditionally, ES cells are cultivated on a feeder cell layer, and the feeder cells are typically fibroblasts derived from embryonic or fetal tissue.

[0104] Even without feeder cells, pluripotent SCs can maintain an undifferentiated state. The environment for feeder-free culture includes a suitable culture substrate, particularly an extracellular matrix, such as MATRIGEL® or laminin. Typically, enzymatic digestion is stopped before the cells are fully dispersed (that is, using collagenase IV for about 5 min). Then a mass of about 10 to 2,000 cells is directly plated on a substrate without further dispersion.

[0105] Feeder-free culture is supported by a nutrient medium containing factors that support cell proliferation but not differentiation. Such factors can be introduced into the culture medium by culturing the medium with cells that secrete such factors, such as irradiated (about 4,000 rads) primary mouse embryonic fibroblasts, telomerized mouse fibroblasts, or fibroblast-like cells derived from pPS cells. Feeder-free culture can be achieved by culturing the medium with about 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 is conditioned by plating feeder cells at a density of 1:1.5 . The medium conditioned for 1-2 days is supplemented with additional 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 WO 01 / 51616 and Xu et al., Nat. Biotechnol. 19:971, 2001.

[0106] Under the microscope, ES cells show high nuclear / cytoplasmic ratio, prominent nucleoli and tight colony formation, and indistinguishable cell connections. Primate ES cells express stage-specific embryonic antigens (SSEA) 3 and 4, and markers detectable using antibodies called 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 can be used as a negative control for SSEA-4, Tra-1-60 and Tra-1-81. SSEA-4 is a human embryonic carcinoma (hEC) cell that is always present. In vitro differentiation of pluripotent SCs leads to the loss of SSEA-4, Tra-1-60 and Tra-1-81 expression and the increase of SSEA-1 expression, which can also be seen in undifferentiated hEG cells.

[0107] Methods for generating low immunogenic cells

[0108] Aspects of the present disclosure relate to generating low immunogenic cells. Low immunogenic cells may include pancreatic islets or β cells, such as stem cell-derived β cells. In some embodiments, stem cells or progenitor cells are engineered to express one or more tolerogenic transgenes from a locus in the cell, wherein the tolerogenic transgene is not silenced during cell differentiation into a desired cell type. Engineered stem cells or progenitor cells may be exposed to conditions suitable for cell differentiation into a desired cell type (e.g., SC-islets or SC-β cells).

[0109] In some embodiments, stem cells or progenitor cells are pluripotent stem cells (e.g., iPSC), pancreatic progenitor cells, non-native endocrine cells, or beta cell progenitor cells. In some embodiments, stem cells or progenitor cells may be engineered to express one or more tolerogenic transgenes from loci in stem cells or progenitor cells, wherein the tolerogenic transgenes are not silenced during differentiation. Non-limiting examples of tolerogenic transgenes include PDL1, HLA-E / G, CD47, SERPINB9, CCL21, FASL, CD200, MFGE8, CD55, CD46, HLS-G single-chain fusions, soluble PDL1-Ig, and CTLA4-Ig. In one embodiment, the tolerogenic agent is an HLA-E single-chain fusion. Single-chain fusions may be loaded with peptides capable of activating NK cells, for example, by interacting with inhibitor receptors NKG2A and / or NKG2C. In some embodiments, the peptide may be VMAPRTLL (SEQ ID NO: 3), VMAPRTLFL (SEQ ID NO: 4), VMAPRTLVL (SEQ ID NO: 5), VMAPRTLIL (SEQ ID NO: 6), IMAPRTLVL (SEQ ID NO: 7), VMPPRTLLL (SEQ ID NO: 8), VMAPRTVLL (SEQ ID NO: 9), VTAPRTLLL (SEQ ID NO: 10), VTAPRTVLL (SEQ ID NO: 11), VMAPRTLTL (SEQ ID NO: 12) and / or VMAPRALLL (SEQ ID NO: 13).

[0110] In some embodiments, stem cells or progenitor cells can be engineered to express one or more immunomodulators from a locus in a stem cell or progenitor cell, wherein the one or more immunomodulators are not silenced during cell differentiation. In some embodiments, the immunomodulator includes a cytokine. In certain embodiments, the immunomodulator includes IL-10, TGF-β and / or IL-2. In one embodiment, the immunomodulator is a cytokine. In one embodiment, the immunomodulator is IL-10. In one embodiment, the immunomodulator is TGF-β. In one embodiment, the immunomodulator is IL-2.

[0111] In some embodiments, any gene editing tool known to those skilled in the art (e.g., TALENS, CRISPR, etc.) is used to modify the expression of one or more genes in stem cells or progenitor cells. In some embodiments, a retrovirus (e.g., a lentivirus) is used to deliver the gene editing tool to stem cells. In some embodiments, gene editing (e.g., CRISPR) can be used to target one or more genes to regulate the expression of the one or more genes.

[0112] In some embodiments, engineered stem cells or progenitor cells (e.g., iPSC cells or β cell progenitor cells) differentiate into β cells (e.g., SC-β cells). In some embodiments, engineered stem cells or progenitor cells (e.g., iPSC cells or β cell progenitor cells) differentiate into pancreatic islets (e.g., SC-islets). In some embodiments, engineered stem cells or progenitor cells differentiate into desired cell types, such as β cells, which secrete one or more immunomodulators from the locus in the cell.

[0113] In some embodiments, stem cells or progenitor cells may be engineered to express one or more tolerogenic transgenes from a locus in a stem cell or progenitor cell. In some embodiments, stem cells or progenitor cells may be engineered to express one or more immunomodulators from a locus in a stem cell or progenitor cell. In some embodiments, the locus is constitutively expressed in all cells of an islet (e.g., a human islet). In some embodiments, the locus is constitutively expressed in a beta cell (e.g., in a human beta cell). The locus may be a locus for a housekeeping gene. In some embodiments, the housekeeping gene includes actin, ubiquitin, and / or GAPDH. In one embodiment, the housekeeping gene is GAPDH.

[0114] At least one low immunogenic cell or its precursor (e.g., stem cell or progenitor cell) may be from any mammalian species, non-limiting examples include mouse, cow, ape, pig, horse, sheep or human cells. For clarity and simplicity, the description of the methods herein refers to mammalian low immunogenic cells or their precursors, but it should be understood that all methods described herein can be easily applied to other cell types of low immunogenic cells or their precursors. In some embodiments, low immunogenic cells or their precursors are derived from human individuals.

[0115] In some embodiments, low immunogenic cells or their precursors are substantially pure low immunogenic cells or their precursors. In one embodiment, low immunogenic cells or their precursors are substantially pure low immunogenic β cells or their precursors. In some embodiments, the colony of low immunogenic cells or their precursors includes a mixture of pluripotent cells or differentiated cells (e.g., a mixture of SC-β cells and / or other differentiated cell types). In some embodiments, SC-islets (e.g., low immunogenic SC-islets) include a mixture of pluripotent cells or differentiated cells (e.g., a mixture of SC-β cells and / or other differentiated cell types). In some embodiments, the colony of low immunogenic SC-islet cells or their precursors is substantially free of or lacks embryonic stem cells or pluripotent cells or iPS cells.

[0116] Low immunogenic cells

[0117] In some aspects of the present disclosure, low immunogenic cells (e.g., low immunogenic β cells) are provided. The low immunogenic cells disclosed herein share many distinguishing features of native pancreatic cells, but differ in some aspects. In some embodiments, the low immunogenic cells are non-native, i.e., non-naturally occurring, non-endogenous cells. As used herein, "non-native" means that the modified low immunogenic cells are significantly different from cells existing in nature (i.e., native cells) in some aspects. However, it should be understood that these significant differences may cause the low immunogenic cells to exhibit certain differences, but the low immunogenic cells may still behave in a manner similar to native cells (e.g., with respect to insulin secretion by β cells), and certain functions have been changed (e.g., improved) compared to native cells.

[0118] Low immunogenic cells are differentiated in vitro from any starting cells, because the present invention is not intended to be limited to the starting cells from which low immunogenic cells are derived. Exemplary starting cells include, but are not limited to, endocrine cells or any precursors thereof, such as NKX6-1+ pancreatic progenitor cells, Pdx1+ pancreatic progenitor cells and pluripotent stem cells, embryonic stem cells and induced pluripotent stem cells. In some embodiments, low immunogenic cells are differentiated in vitro from reprogrammed cells, partially reprogrammed cells (i.e., somatic cells, such as fibroblasts that have been partially reprogrammed so that they exist in an intermediate state between induced pluripotent cells and somatic cells derived therefrom), and transdifferentiated cells. In some embodiments, low immunogenic cells disclosed herein may be differentiated in vitro from endocrine cells or their precursors. In some embodiments, low immunogenic cells are differentiated in vitro from stem cells or progenitor cells, which may include β cell progenitor cells or pluripotent stem cells. In some embodiments, pluripotent stem cells are selected from the group consisting of embryonic stem cells and induced pluripotent stem cells. In some embodiments, low immunogenic cells or pluripotent stem cells derived from low immunogenic cells are people. In some embodiments, the poorly immunogenic cells are human.

[0119] In some embodiments, low immunogenic cells (e.g., SC-β cells) are modified to express or overexpress one or more tolerogenic transgenes from loci in stem cells or progenitor cells. Non-limiting examples of tolerogenic transgenes include PDL1, HLA-E / G, CD47, SERPINB9, CCL21, FASL, CD200, MFGE8, CD55, CD46, HLS-G single-chain fusions, soluble PDL1-Ig and CTLA4-Ig. In one embodiment, the tolerogenic agent is an HLA-E single-chain fusion. Single-chain fusions can be loaded with peptides capable of activating NK cells, for example, by interacting with inhibitor receptors NKG2A and / or NKG2C. In some embodiments, the peptide may be VMAPRTLL (SEQ ID NO: 3), VMAPRTLFL (SEQ ID NO: 4), VMAPRTLVL (SEQ ID NO: 5), VMAPRTLIL (SEQ ID NO: 6), IMAPRTLVL (SEQ ID NO: 7), VMPPRTLLL (SEQ ID NO: 8), VMAPRTVLL (SEQ ID NO: 9), VTAPRTLLL (SEQ ID NO: 10), VTAPRTVLL (SEQ ID NO: 11), VMAPRTLTL (SEQ ID NO: 12) and / or VMAPRALLL (SEQ ID NO: 13).

[0120] In some embodiments, low immunogenic cells (e.g., SC-β cells) are modified to express or overexpress one or more immunomodulators from a locus in a stem cell or progenitor cell. In some embodiments, the immunomodulator includes a cytokine. In certain embodiments, the immunomodulator includes IL-10, TGF-β and / or IL-2. In one embodiment, the immunomodulator is a cytokine. In one embodiment, the immunomodulator is IL-10. In one embodiment, the immunomodulator is TGF-β. In one embodiment, the immunomodulator is IL-2.

[0121] In some embodiments, the locus is constitutively expressed in all cells of the islets (e.g., human islets). In some embodiments, the locus is constitutively expressed in β cells (e.g., in human β cells). The locus may be a locus of a housekeeping gene. In some embodiments, the housekeeping gene includes actin, ubiquitin and / or GAPDH. In one embodiment, the housekeeping gene is GAPDH.

[0122] Low immunogenic cells may show reduced risk of autologous and / or allogeneic rejection after transplantation. In some aspects, low immunogenic cells secrete one or more immunomodulators. For example, low immunogenic cells (e.g., low immunogenic β cells) secrete cytokines, such as IL-10, TGF-β and IL-2 or modified IL-2.

[0123] In some aspects, the disclosure provides a cell line comprising the low immunogenic cells described herein (e.g., low immunogenic β cells). In some aspects, the disclosure provides a SC-islet comprising low immunogenic stem cell-derived β cells.

[0124] In some embodiments, the cells described herein (e.g., a colony of low immunogenic cells) are transplantable, for example, a colony of low immunogenic cells can be applied to a subject. In some embodiments, SC-islets comprising low immunogenic cells (e.g., low immunogenic SC-β cells) are transplantable, for example, SC-islets can be applied to a subject. In some embodiments, the subject to which a colony of low immunogenic cells is administered is the same subject (e.g., for autologous cell therapy) as the subject from which the pluripotent stem cells for differentiation into low immunogenic cells are obtained. In some embodiments, the subject is a different subject. In some embodiments, the subject suffers from diabetes such as insulin-dependent diabetes, or is a normal subject. For example, cells for transplantation (e.g., a composition comprising a colony of low immunogenic cells or a SC-islet comprising a colony of low immunogenic cells) can be in a form suitable for transplantation (e.g., organ transplantation).

[0125] The method may further include administering the cells to a subject in need, such as a mammalian subject, such as a human subject. The source of the cell may be a mammal, preferably a human. The source or recipient of the cell may also be a non-human subject, such as 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 can be obtained from any organism in these organisms (including non-human transgenic organisms). In one embodiment, the transplantable cells are genetically engineered, for example, the cells contain exogenous genes or have been genetically engineered to inactivate or change endogenous genes.

[0126] An implantable device can be used to administer a composition comprising a population of low immunogenic cells (e.g., low immunogenic pancreatic stem cell-derived cells, such as SC-β cells) to a subject. Implantable devices and related technologies are known in the art and can be used as delivery systems that require continuous or timed release delivery of the compounds or compositions described herein. In addition, implantable device delivery systems can be used to target specific points (e.g., local sites, organs) for delivery of targeted compounds or compositions. 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 (e.g., polymers, liposomes) based on polymer technology, sustained release technology, and encapsulation technology can also be used to deliver the compounds and compositions described herein.

[0127] For administration to a subject, a cell population produced by a method as disclosed herein, for example, a population of low immunogenic cells (e.g., low immunogenic β cells) or SC-islets comprising low immunogenic cells can be administered to a subject, for example, in a pharmaceutically acceptable composition. These pharmaceutically acceptable compositions include a therapeutically effective amount of a population of low immunogenic cells as described above formulated with one or more pharmaceutically acceptable carriers (additives) and / or diluents.

[0128] As described in detail below, the pharmaceutical compositions of the present invention can be specifically formulated for administration in solid or liquid form, including those suitable for the following: (1) oral administration, such as drenches (aqueous or non-aqueous solutions or suspensions), lozenges, dragees, capsules, pills, tablets (such as those targeted for buccal, sublingual and systemic absorption), boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, such as by subcutaneous, intramuscular, intravenous or epidural injection in the form of, for example, a sterile solution or suspension, or a sustained release formulation; (3) topical application, such as in the form of a cream, ointment or controlled release patch or spray applied to the skin; (4) intravaginally or intrarectally, such as as a pessary, cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; (8) transmucosally; or (9) nasally. In addition, the compounds can be implanted in 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.

[0129] As used herein, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0130] As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or vehicle that participates in carrying or transporting the subject compound from one organ or part of the body to another organ or part of the body, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., a lubricant, talc, magnesium, calcium or zinc stearate, or stearic acid), or solvent encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can serve as pharmaceutically 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) excipients 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) glycols, such as propylene glycol; (11) polyols, such as glycerol, 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) ethanol; (20) pH buffered solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; (22) fillers, such as polypeptides and amino acids; (23) serum components, such as serum albumin, HDL, and LDL; (24) C2-C12 alcohols, such as ethanol; and (25) other nontoxic compatible substances used in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweeteners, flavoring agents, fragrances, preservatives and antioxidants may also be present in the formulation.Terms such as "excipient", "carrier", "pharmaceutically acceptable carrier" and the like are used interchangeably herein.

[0131] As used herein, the phrase "therapeutically effective amount" with respect to a population of cells means that the relevant cells (e.g., low immunogenic cells) in the population of cells, or the composition comprising the low immunogenic cells of the present invention, are effective in producing some desired therapeutic effect in at least one cell subpopulation in an animal at a reasonable benefit / risk ratio applicable to any medical treatment. For example, the amount of the population of low immunogenic cells administered to the subject is sufficient to produce a statistically significant, measurable change in at least one symptom of type 1, type 1.5, or type 2 diabetes (such as glycated hemoglobin levels, fasting blood glucose levels, hypoinsulinemia, etc.). The determination of a therapeutically effective amount is within the capabilities of those skilled in the art. Typically, the therapeutically effective amount varies with the subject's medical history, age, illness, sex, and the severity and type of the subject's medical illness, as well as the administration of other pharmaceutically active agents.

[0132] As used herein, the term "administering" refers to placing a composition in a subject by a method or approach that causes the composition to be at least partially positioned at a desired site so as to produce a desired effect. The compounds or compositions described herein can 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.

[0133] 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, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. In a preferred embodiment, the composition is administered by intravenous infusion or injection.

[0134] "Treatment," "prevention," or "improvement" of a disease or condition means delaying or preventing the onset of such a disease or condition, reversing, alleviating, ameliorating, inhibiting, slowing down, or stopping the progression of a condition associated with such a disease or condition, or the progression, aggravation, or worsening of the severity of the condition. In one embodiment, the symptoms of a disease or condition are alleviated by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%.

[0135] Treatment of diabetes is determined by standard medical methods. The goal of diabetes treatment is to reduce blood sugar levels to near normal levels as safely as possible. The goal usually set is 80-120 mg / dl (mg / dl) before meals and 100-140 mg / dl before bedtime. A specific physician may set different goals for patients based on other factors (such as the frequency of hypoglycemic reactions in patients). Useful medical tests include testing the patient's blood and urine to determine blood sugar levels, testing glycosylated hemoglobin levels (HbA1c; measuring the average blood sugar level in the past 2-3 months, the normal range is 4-6%), testing cholesterol and fat levels, and testing urine protein levels. Such tests are standard tests known to those skilled in the art (see, for example, American Diabetes Association, 1998). A successful treatment plan can be determined by the small number of patients with diabetes-related complications (such as eye diseases, kidney diseases, or neurological diseases) in the plan.

[0136] 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 arterial disease, cerebrovascular disease, atherosclerosis and hypertension), neuropathy, dysautonomia, hyperglycemic hyperosmolar coma, or a combination thereof) by 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 may include the entire lifetime of the subject.

[0137] 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 a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse or a cow, but is not limited to these examples. Mammals other than humans may be advantageously used as subjects representing animal models of type 1 diabetes, type 2 diabetes or prediabetes. In addition, the methods described herein may be used to treat domesticated animals and / or pets. The subject may be male or female. The subject may be a subject who has previously been diagnosed with or identified as suffering from or having diabetes (e.g., type 1 or type 2) or one or more complications associated with diabetes, or prediabetes, and optionally, but without having undergone treatment for diabetes, one or more complications associated with diabetes or prediabetes. The subject may also be a subject who does not suffer from diabetes or prediabetes. The subject may also be a subject who has been diagnosed or identified as suffering from diabetes, one or more complications associated with diabetes, or prediabetes, but who has shown known diabetes risk factors improved due to receiving one or more treatments for diabetes, one or more complications associated with diabetes, or prediabetes. Alternatively, the subject may also be a subject who has not been previously diagnosed as suffering from diabetes or one or more complications associated with diabetes or prediabetes. For example, the subject may be a subject who exhibits one or more diabetes risk factors, complications associated with diabetes, or prediabetes, or a subject who does not exhibit diabetes risk factors, or a subject who is asymptomatic for diabetes, one or more complications associated with diabetes, or prediabetes. The subject may also be a subject who suffers from diabetes or prediabetes or is at risk of developing diabetes or prediabetes. The subject may also be a subject who has been diagnosed with or identified as suffering from one or more complications associated with diabetes or prediabetes as defined herein, or alternatively, the subject may be a subject who has not previously been diagnosed with or identified as suffering from one or more complications associated with diabetes or prediabetes.

[0138] As used herein, the phrase "a subject in need of hypoimmunogenic β cells" refers to a subject diagnosed with 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 prediabetic condition.

[0139] Any method for diagnosing diabetes can be used to identify subjects in need of a population of low immunogenic β cells. For example, a glycosylated hemoglobin (A1C) test, a random blood glucose test, and / or a fasting blood glucose test can be used to diagnose type 1 diabetes. Parameters for diagnosing diabetes are known in the art and are available to those skilled in the art without further ado.

[0140] In some embodiments, the method of the present invention further includes selecting a subject identified as needing additional low immunogenic β cells. Subjects of a population of low immunogenic β cells may be selected based on the symptoms presented (such as symptoms of type 1, type 1.5 or type 2 diabetes). Exemplary symptoms of diabetes include, but are not limited to, extreme thirst (polydipsia), frequent urination (polyuria), extreme hunger (polyphagia), extreme fatigue, weight loss, high blood sugar, low insulin levels, high blood sugar (e.g., blood sugar levels exceeding 250 mg, exceeding 300 mg), ketones in 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, renal failure, cardiovascular disease (including coronary artery disease, peripheral artery disease, cerebrovascular disease, atherosclerosis and hypertension), neuropathy, autonomic dysfunction, hyperglycemic hyperosmolar coma and combinations thereof.

[0141] In some embodiments, the composition comprising a population of low immunogenic β cells for administration to a subject may further comprise a pharmaceutically active agent, such as those known in the art for treating diabetes and / or having anti-hyperglycemic 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) The sulfonylureas (e.g., acetohexamide, amoxicillin, chlorpropamide, gliclazide, tolbutamide, tolazamide, glyburide (Glyburide), glipizide, gliquidone, glypyramide, and glimepiride), the meglitinides (“glinides”) (e.g., nateglinide, repaglinide, and mitiglinide), glucagon-like peptide-1 (GLP-1) and analogs (e.g., exenatide-4 (Exendin-4), Exenatide, Liraglutide, Albiglutide), insulin and insulin analogs (e.g., Insulin lispro, Insulin aspart, Insulin glulisine, Insulin glargine, Insulin detemir, Exubera, and NPH insulin), alpha-glucosidase inhibitors (e.g., Acarbose, Miglitol, and Voglibose), amylin analogs (e.g., Pramlintide), sodium-dependent glucose co-transporter T2 (SGLT T2) inhibitors (e.g., Dapagliflozin, Remogliflozin, and Sergliflozin), and others (e.g., Benfluorex and Torestat).

[0142] In some aspects, the composition of the colony comprising low immunogenic β cells for use in a subject may further include a pharmaceutically active agent, such as those agents known in the art for containing the immune system, i.e., an immunosuppressant. Non-limiting examples of immunosuppressants include biologics (e.g., adalimumab and infliximab), calcineurin inhibitors (e.g., tacrolimus and cyclosporin), corticosteroids (e.g., prednisone), inosine monophosphate dehydrogenase (IMDH) inhibitors (e.g., mycophenolate mofetil), Janus kinase inhibitors (e.g., tofacitinib), rapamycin mechanism target (mTOR) inhibitors (e.g., sirolimus) and monoclonal antibodies (e.g., basiliximab).

[0143] Compositions comprising low immunogenic cells can be administered to subjects simultaneously or at different times with the administration of pharmaceutically active agents or compositions comprising them. When administered at different times, the composition of the colony of cells comprising low immunogenicity and / or pharmaceutically active agents administered to subjects 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 administering another. When the composition of the colony comprising low immunogenic cells and / or the composition comprising pharmaceutically active agents are administered with different pharmaceutical compositions, the route of administration can also be different. In some embodiments, compositions comprising low immunogenic cells are administered to subjects. In other embodiments, compositions comprising pharmaceutically active agents are administered to subjects. In another embodiment, compositions comprising colonies of low immunogenic cells mixed with pharmaceutically active agents are administered to subjects. In another embodiment, compositions comprising colonies of low immunogenic cells and compositions comprising pharmaceutically active agents are administered to subjects, wherein administration is substantially performed simultaneously, or is performed one after another.

[0144] Toxicity and therapeutic efficacy of administered compositions comprising a population of low immunogenic cells can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective to 50% of the population). Compositions comprising a population of low immunogenic cells that exhibit large therapeutic indices are preferred.

[0145] Several well-established animal models can be used to test the amount of a composition comprising a population of poorly immunogenic cells.

[0146] Non-obese diabetic (NOD) mice carry a genetic defect that causes them to show insulitis at a few weeks of age (Yoshida et al., Rev. Immunogenet. 2:140, 2000). 60%-90% of female mice develop overt diabetes at 20-30 weeks of age. The immune-related pathology appears to be similar to human type I diabetes. Other models of type I diabetes are mice with transgenic and knockout mutations (Wong et al., Immunol. Rev. 169:93, 1999). Lenzen et al. recently reported a rat model of spontaneous type I diabetes (Diabetologia 44:1189, 2001). Hyperglycemia (>500 mg glucose / dL) can also be induced in mice by a single intraperitoneal injection of streptozotocin (Soria et al., Diabetes 49:157, 2000) or by continuous low-dose streptozotocin (Ito et al., Environ. Toxicol. Pharmacol. 9:71, 2001). To test the efficacy of implanted islet cells, monitor whether the mouse blood glucose returns to normal levels (<200 mg / dL).

[0147] Larger animals provide a good model for following the sequelae of chronic hyperglycemia. Dogs can be made insulin dependent by removing the pancreas (J. Endocrinol. 158:49, 2001) or by feeding galactose (Kador et al., Arch. Opthalmol. 113:352, 1995). The keeshond dog also provides 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 a Canadian couple to embark on a long sea voyage to Stockholm in February 1925.

[0148] In some embodiments, the data obtained from cell culture assays and in animal studies can be used to formulate a dosage range for humans. The dosage of such compounds is preferably within a circulating concentration range including an ED50 with little or no toxicity. The dosage can vary within this range depending on the dosage form used and the route of administration utilized.

[0149] The therapeutically effective dose of a composition comprising a population of low immunogenic cells can also be estimated initially from cell culture assays. Alternatively, the effect of any particular dose can be monitored by a suitable bioassay.

[0150] About the duration and frequency of treatment, skilled clinicians usually monitor the subject to determine the time that treatment provides therapeutic benefit, and determine whether to increase or reduce dosage, increase or reduce the frequency of application, whether to stop treatment, resume treatment or make other changes to the treatment plan.Dosing schedule can change from once a week to once a day according to many clinical factors (such as the sensitivity of the subject to stem cell-derived cells).Required dosage can be disposable, or it can be divided into sub-doses (for example, 2-4 sub-doses) and for a period of time, for example, all day at appropriate intervals or with other appropriate schedules.Such sub-doses can be used as unit dosage forms.In some embodiments, it is long-term to use, for example, one or more doses are used every day in the period of several weeks or months.The example of dosing schedule is once a day, twice a day, three times a day or four times a day or more in the period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months or 6 months or longer.

[0151] In another aspect of the present invention, the method provides the use of a population of low immunogenic cells as disclosed herein. In one embodiment of the present invention, a population of low immunogenic cells as disclosed herein can be used to produce a pharmaceutical composition, for transplanting into a subject in need of treatment, such as a subject suffering from diabetes or a risk of diabetes, such as, but not limited to, a subject suffering from congenital and acquired diabetes. In one embodiment, the population of low immunogenic cells can be genetically modified. In another aspect, the subject can suffer from diabetes and / or metabolic disorders, or be at risk of diabetes and / or metabolic disorders. In some embodiments, the population of low immunogenic cells as disclosed herein can be autologous and / or allogeneic. In some embodiments, the subject is a mammal, and in other embodiments, the mammal is a person.

[0152] Use of isolated populations of low immunogenic cells as disclosed herein is advantageous over existing methods because the population of low immunogenic cells can be differentiated from endocrine progenitor cells derived from stem cells or their precursors, such as iPS cells obtained or harvested from a subject to whom the isolated population of low immunogenic cells is administered. This is very advantageous because it provides a renewable source of low immunogenic cells that can be differentiated from stem cells into endocrine progenitor cells by methods generally known to those of ordinary skill in the art, and then further differentiated into pancreatic β-like cells by the methods described herein for transplantation into a subject, particularly SC-islets containing low immunogenic pancreatic endocrine cells that do not have the risks and limitations of cells derived from other systems.

[0153] One embodiment of the present invention relates to a method for treating diabetes or metabolic disorders in a subject, comprising administering to a subject suffering from diabetes and / or metabolic disorders an effective amount of a composition comprising a population of low immunogenic cells (e.g., low immunogenic β cells) as disclosed herein. In another embodiment, the present invention provides a method for treating diabetes comprising administering to a subject suffering from diabetes or having an increased risk of diabetes a composition comprising a population of low immunogenic cells as disclosed herein.

[0154] In one embodiment of the above method, the subject is a human, and the population of low immunogenic cells as disclosed herein is a human cell. In some embodiments, the present invention contemplates administering the population of low immunogenic cells as disclosed herein directly to the pancreas of the subject or systemically. In some embodiments, the population of low immunogenic cells as disclosed herein can be administered to any suitable location in the subject, such as a blood vessel or a capsule in the liver.

[0155] The present invention also relates to a method for treating a patient with diabetes or a metabolic disorder that occurs due to genetic defects, physical injury, environmental damage or regulation, poor health, obesity, and other diabetes risk factors generally known to those of ordinary skill in the art. The efficacy of treatment of a subject administered with a composition comprising a population of low immunogenic cells (e.g., low immunogenic β cells) can be monitored by clinically acceptable standards and tests, including, for example, (i) a glycosylated hemoglobin (A1C) test, which indicates the average blood sugar level of a subject over the past two to three months by measuring the percentage of blood sugar attached to hemoglobin (the oxygen-carrying protein in red blood cells). The higher the blood sugar level, the more hemoglobin is attached to sugar. An A1C level of 6.5% or more in two independent tests indicates that the subject has diabetes. A test value of 6-6.5% indicates that the subject has prediabetes. (ii) a random blood sugar test. A blood sample is collected from the subject at a random time, and a random blood sugar level of 200 milligrams per deciliter (mg / dL)-11.1 millimoles per liter (mmol / L) or more indicates that the subject has diabetes. (iii) Fasting blood glucose test. A blood sample is collected from the subject after an overnight fast. A fasting blood glucose level between 70 and 99 mg / dL (3.9 and 5.5 mmol / L) is normal. If the subject's fasting blood glucose level is 126 mg / dL (7 mmol / L) or higher in two separate tests, the subject has diabetes. A blood glucose level of 100 to 125 mg / dL (5.6 to 6.9 mmol / L) indicates that the subject has prediabetes. (iv) Oral glucose tolerance test. A sample is collected after the subject has fasted for at least eight hours or overnight, then ingested a sugar solution, and the blood glucose level is measured two hours later. A blood glucose level below 140 mg / dL (7.8 mmol / L) is normal. A blood glucose level between 140 and 199 mg / dL (7.8 to 11 mmol / L) is considered prediabetes. This is sometimes called impaired glucose tolerance (IGT). A blood sugar level of 200 mg / dL (11.1 mmol / L) or higher may indicate diabetes.

[0156] In some embodiments, the effect of administering a population of low immunogenic cells (e.g., low immunogenic β cells) as disclosed herein to a subject in need thereof is associated with improved exercise tolerance or other quality of life indicators and reduced mortality. The effects of cell therapy using a population of low immunogenic cells may be apparent within a few days to a few weeks after surgery. However, beneficial effects may be observed as early as a few hours after surgery and may last for several years. In some embodiments, the effects of cell therapy using a population of low immunogenic cells appear within two weeks after surgery.

[0157] In some embodiments, a population of low immunogenic cells (e.g., low immunogenic β cells) as disclosed herein can be used for tissue reconstruction or regeneration of human patients or other subjects in need of such treatment. In some embodiments, the composition of a population of low immunogenic cells can be administered in a manner that allows it to be transplanted or migrated to the intended tissue site and to reconstruct or regenerate a functional defect area. Specific devices suitable for administering cells capable of reconstructing a population of low immunogenic pancreatic cells (e.g., β cells) in the pancreas or at another desired location are available. Therefore, low immunogenic cells can be administered by injection to the pancreas of a recipient subject or by intramuscular injection.

[0158] Example

[0159] Sustained transgene expression is essential to maintain the desired low immunogenic phenotype during differentiation and after transplantation. Previously, transgene silencing was observed when the expression of tolerogenic molecules was targeted to the AAVS1 safe harbor locus in hESCs followed by differentiation into SC-β cells. Since housekeeping genes such as GAPDH are constitutively expressed in all cells of human islets, it was hypothesized that targeting the expression of tolerogenic molecules at the GAPDH locus in hESCs would result in strong and sustained transgene expression after SC-islet cell differentiation and transplantation. Table 1 provides a non-exhaustive list of other housekeeping genes suitable for use in the present invention.

[0160] Generation of low immunogenic SC-islet cells

[0161] A targeted approach was adopted to engineer low immunogenic SC-islet cells using minimal editing steps. First, we interfered with the ability of stem cells and their differentiated progeny to present antigens by eliminating HLA class I expression by knocking out the B2M gene in hESC. By doing so, we reduced the ability of T cells to engage and destroy allogeneic SC-islet cells. We also targeted the expression of the immunomodulatory molecule PDL1 at the GAPDH locus to provide additional inhibitory signaling for T cells. Other candidate molecules for expression from the GAPDH locus include soluble PDL1-Ig and CTLA4-Ig, CD47 (macrophage and NK cell inhibition), HLA-G single-chain fusion (NK cell inhibition), SERPINB9 (granzyme inhibitor), CCL21 (macrophage cytokine inhibitor), FASL (T cell apoptosis), CD200 (myeloid cell inhibition), CD55 and CD46 (complement inactivation). In the case of multiple transgene expression, polycistronic RNA is transcribed from the GADPH locus encoding GAPDH and the tolerogenic molecule separated by a self-cleaving peptide. We chose not to interfere with HLA class II expression since its expression was not observed in our SC-islet cells.

[0162] Since HLA class I defective cells are susceptible to NK cell toxicity, we introduced HLA-E single-chain fusion genes at the GAPDH locus so that SC-islet cells would express single HLA molecules involved in NK cell inhibition. In addition, we also selectively loaded the peptide VMAPRTLLL, which has been shown to cause preferential interactions between HLA-E and the homologous inhibitory receptor NKG2A on NK cells 12. In contrast, other HLA-E single-chain fusions loaded with HLA-G leader sequence derived peptides (VMAPRTLFL) 3 have been shown to activate NK cells by interacting with the HLA-E homologous activation receptor NKG2C. Other potential candidate peptides for loading into HLA-E fusions but not activating NK cells by NKG2C include VMAPRTLVL, VMAPRTLIL, IMAPRTLVL, VMPPRTLLL, VMAPRTVLL, VTAPRTLLL, VTAPRTVLL, VMAPRTLTL and VMAPRALLL. Following the generation of hypoimmunogenic hESCs, these cells differentiate into SC-β cells that lack classical HLA expression and constitutively express tolerogenic molecules capable of suppressing T- and NK-cell responses in an allogeneic context.

[0163] SC-islet cells as mediators of local immune tolerance

[0164] We also sought to use our engineering strategy to endow SC-islet cells with the ability to modulate their local immune microenvironment by secreting immunomodulatory cytokines. By doing so, it was hypothesized that immunomodulatory cytokines secreted by islet cells could induce local immune tolerance, thereby protecting allogeneic transplants from immune destruction. We modified the GAPDH locus targeting construct to contain the tolerogenic cytokines IL-10, TGF-β, and modified IL-2, and introduced these cytokines into the GAPDH locus of hESCs. Thereafter, the cytokine-secreting hESCs were differentiated into SC-islet cells and transplanted into immune-competent mice.

[0165] method

[0166] Mouse

[0167] Male B6 / albino mice, Scid / beige mice, and NSG-MHC class I / II KO mice (6–8 weeks old) were purchased from Jackson Labs. Mice were housed in a specific pathogen-free environment at Harvard University. All animal studies were performed with approval from the Harvard University IACUC.

[0168] Cell culture

[0169] Maintenance and differentiation of human ES cells were performed as previously described (Millman et al., 2016; Pagliuca et al., 2014). SC-islet cell differentiation was initiated 72 h after the initial passage by aspiration of mTeSR1 and supplemented with stage- and day-specific media supplemented with appropriate small molecules or growth factors as previously described (Millman et al., 2016; Pagliuca et al., 2014; Veres et al., 2019). All cell lines were routinely tested for mycoplasma and were mycoplasma-free. All experiments involving human cells were approved by the Harvard University IRB and ESCRO committees.

[0170] Generation of immune-evading hESCs

[0171] The existing GAPluc (WT) hESC line serves as the starting material for generating HLA-deficient hESCs (Gerace et al., 2021). To generate HLA-deficient hESCs, 1x106 WT hESCs were nuclear transfected using 4D-Nucleofector (Lonza) according to the manufacturer's instructions, where RNPs were complexed with 120 pmol B2M gRNA (5'-GCTACTCTCTCTTTCTGGCC'3; SEQ ID NO: 1)(Mandal et al., 2014) (IDT) and 104 pmol Alt-R® Sp HiFi Cas9 nuclease V3 (IDT). The nuclear transfected cells were resuspended in mTesR1 (STEMCELL Technologies, 85850) + 10 µM Y27632 (DNSK International, DNSK-KI-15-02) and plated in matrigel-coated tissue culture plates. After 48 h, the cells were treated with 10 ng / ml IFN-γ (R&D, 285-IF-100) for 24 h and then stained with APC anti-human HLA-ABC (W6 / 32, 1:100) (Biolegend, 311409). HLA-ABC- / - cells were sorted on FACS Aria II (BD Biosciences) and seeded in a matrix gel-coated tissue culture plate containing mTesR1 + CloneR (STEMCELL Technologies, 05888), and single colonies were selected for amplification. The resulting HLA-deficient line was named B2M- / -.

[0172] To generate GAP-PD and GAP-BEC hESCs, human PD-L1 and peptide::B2M::HLA-E fusion sequences were synthesized as gBlocks (Genscript) and cloned into our existing GAPluc-targeted plasmid downstream of the Luc2 gene. The sequence of the covalently attached peptide (VMAPRTLLL (SEQ ID NO: 2)) in the peptide::B2M::HLA-E fusion was derived from the HLA-Cw7 molecule (Kaiser et al., 2005). hESCs were nucleofected with plasmids targeting GAP-PD or GAP-BEC and RNPs targeting GAPDH as previously described (Gerace et al., 2021). For GAP-B2P and GAP-BEC lines, polyclonal populations of puromycin-selected cells were subsequently nucleofected with RNPs targeting B2M and single colonies were picked for expansion after FACS sorting as described above. The gating strategy for sorting HLA-ABC- / - hESCs is described in Figure 5G middle.

[0173] Flow cytometry

[0174] Before staining, differentiated WT, B2M- / -, BEC, PD-L1 and B2P SC-islet cells were treated with 10ng / ml IFN-γ for 24 h and untreated. Cells were dissociated with Accutase (STEMCELL Technologies, 07920), washed twice with PBS +0.1% BSA (Gibco, A10008-01), and blocked on ice for 30 min with PBS + 5% donkey serum (Jackson Labs; 100181-234). The cells were then stained on ice in blocking buffer with PE or APC anti-human HLA-ABC (Biolegend, 311405), PE anti-human HLA-E (Biolegend, 342603) and PE anti-human PD-L1 (Biolegend, 393607) for 30 min. The cells were then washed three times and fixed at 4 ° C for 15 min in 4% PFA (EMS, 15710). Fixed cells were then incubated with rat anti-human C-peptide (DHSB; GN-ID4) and mouse anti-human Nkx6.1 (DHSB; F55A12) in blocking buffer (overnight at 4°C), washed three times with blocking buffer, incubated with goat anti-mouse 647 (Life Technologies, A-21247; 1:300) and goat anti-mouse 405 (Life Technologies, A-31553; 1:300) in blocking solution (1 h at room temperature), washed three times and resuspended in PBS + 0.1% BSA. Samples were captured on an LSR II (BD) flow cytometer and analyzed using FlowJo 10.7.1 (BD). All antibodies were used at a ratio of 1:100 unless otherwise stated. The gating strategy used to identify SC-β cells is described in. Figure 5H PE mouse IgG2a (Biolegend, 400213) and PE mouse IgG1 (Biolegend, 400111) served as isotype controls.

[0175] Magnetic enrichment and reaggregation using CD49a

[0176] WT and BEC SC-β cells were magnetically enriched from SC-islet clusters as previously described (Veres et al., 2019). The enriched cells were resuspended in S6 medium and plated at a density of 5x103 cells / well in low-attachment 96-well V-bottom tissue culture plates (Thermo Scientific, 277143), centrifuged at 300 g for 1 min and incubated at 37°C for 4-7 days. CD49a-enriched SC-β cell clusters were fed with fresh S6 medium every 2 days.

[0177] SC-β cell immunohistochemistry

[0178] CD49a-enriched SC-β cell clusters were fixed with 4% PFA for 1 h at room temperature, washed and frozen in OCT (Tissue-Tek, 4583), and then cut into 14 µm sections. Paraffin-embedded samples were treated with Histo-Clear (EMS, 64110-01) to remove paraffin before staining. For staining, slides were incubated in blocking buffer (PBS + 0.1% Triton-X + 5% donkey serum) for 1 h at room temperature, incubated in PBS + 5% donkey serum containing rat anti-human C-peptide (1:300), mouse anti-human Nkx6.1 (1:100), and rabbit anti-human HLA-E (Sigma-Aldrich, HPA031454, 1:100) for 1 h at room temperature, washed three times, incubated in goat anti-mouse 594 (Life Technologies, A-11032; 1:500), goat anti-rat 488 (Life Technologies, A-11006; 1:500), and goat anti-rabbit 647 (Life technologies, A-21244; 1:500) for 2 h at room temperature, washed, mounted in Vectashield with DAPI (Vector Laboratories; H-1200), covered with a coverslip and sealed with clear nail polish. Representative fields were imaged using a Zeiss.Z2 and Apotome microscopes.

[0179] Immune cell isolation

[0180] In accordance with IRB approval, PBMC, CD8 and CD4 T cells and NK cells were isolated from apheresis rings (n = 5 donors) obtained from Brigham and Women's Hospital. According to the manufacturer's instructions, PBMC were separated by density gradient centrifugation using lymphoprep (STEMCELL Technologies, 07801) SepMate™-50 (IVD) tubes (STEMCELL Technologies, 85450). CD4, CD8 and NK cells were isolated by density gradient centrifugation using Lymphoprep and SepMate™-50 (IVD) tubes, followed by addition of RosetteSep human CD4+ T cell enrichment mixture (STEMCELL Technologies, 15062), CD8+ T cell enrichment mixture (STEMCELL Technologies, 15063) and NK cell enrichment mixture (STEMCELL Technologies, 15065). The enriched immune cells were cryopreserved at a concentration of 10 M cells / vial in CryoStor CS10 (STEMCELL Technologies, 07930).

[0181] Flow cytometry analysis of enriched immune cell subsets was performed by staining cells in blocking buffer containing APC anti-human CD3 (Biolegend, 300311), PE anti-human CD4 (Biolegend, 357403), Pacific Blue anti-human CD8 (Biolegend, 344717), and Pacific Blue anti-human CD56 (Biolegend, 362519). Isotype controls were Pacific Blue mouse IgG1 (Biolegend, 400131), APC mouse IgG2a (Biolegend, 400221), and PE rat IgG2b (Biolegend, 400607). Unless otherwise stated, all antibodies were used at a ratio of 1:100. Representative gating strategies are shown in Figure 6G To Figure 6I.

[0182] In vitro T cell cytotoxicity assay

[0183] WT, B2M- / - and BEC SC-islet cells were seeded in S3 medium in a 96-well black flat-bottom plate (Corning, 3916) coated with matrigel at a density of 5 x 104 cells / well in the presence or absence of IFN-γ (10 ng / ml). After 24 h, the medium was switched to T cell medium (ImmunoCult™-XF T cell expansion medium (STEMCELL Technologies, 10981) + 100U / ml rhIL-2) for immune cell co-culture. Primary human PBMC, CD4 or CD8 cells (n = 5 donors) cultured in T cell medium were added to SC-islet cells at an effector: target ratio of 1:1 and 3:1 with or without the addition of ImmunoCult™ human CD3 / CD28 T cell activator (STEMCELL Technologies, 10991). All T cell cytotoxicity assays were co-cultured for 72 h, after which luminescence was measured on a CLARIOstar microplate reader (BMG LABTECH) after the addition of 150 µg / ml D-luciferin (Gold Biotechnology, LUCK-2G). SC-islet cell viability was calculated as a percentage of luminescence in the absence of T cells (test SC-islet cell luminescence / SC-islet cell luminescence without T cells x 100).

[0184] In vitro NK cell cytotoxicity assay

[0185] WT, B2M- / - and BEC SC-islet cells were seeded at a density of 2 x 104 cells / well in 96-well black round-bottom ultra-low attachment plates (Corning, 4591) in NK cell culture medium (NK MACS medium (Milteny Biotec, 130-114-429) + 5% human AB serum (Valley Biomed, HP1022HI) + 5% HyClone FBS (GE Healthcare, SH30070.03) + 0.5 ng / ml rhIL-2 (Peprotech, 200-02)). Primary NK cells (n = 5 donors) cultured in NK cell culture medium for 5 days were added to SC-islet cells at an effector: target ratio of 1:1 and 10:1. K562-Luc2 (Biocytogen, BCG-PS-015-luc) and Raji-GFP-Luc2 (Biocytogen, BCG-PS-087-luc) cells were used as positive and negative controls, respectively. All NK cell cytotoxicity assays were co-cultured for 5 h, after which luminescence and SC-islet cell viability were measured as described above.

[0186] In vivo NK cell cytotoxicity

[0187] 2.5 x 105 WT and B2M- / - SC-islet cell clusters were resuspended in phenol-free Matrigel (Corning, 356237) alone or together with 7.5 x 105 primary human NK cells (pretreated with 0.5 ng / ml rhIL-2 for 12 h) and then transplanted subcutaneously into Scid / beige mice (n = 5). On days 1 and 5 after cell transplantation, bioluminescence was measured on an IVIS Spectrum (PerkinElmer, 124262) 10 min after ip injection of 10 µL / g D-luciferin (15 mg / ml) as previously described (Gerace et al., 2021).

[0188] Endothelial cell differentiation

[0189] WT and B2M- / - hESCs were cultured at 7.5 x 10 4The concentration of cells / well was seeded in mTesR1 + 10 µM Y27632 in a 6-well tissue culture plate coated with matrigel (Corning, 3516). Afterwards, the cells were differentiated into endothelial cells using the STEMdiff™ Endothelial Differentiation Kit (STEMCELL Technologies, 08005) according to the manufacturer's instructions. After staining the endothelial cells with Pacific Blue anti-human CD31 (Biologend, 303113), the differentiation efficiency was quantified by FACS analysis. Pacific Blue Mouse IgG1 (Biolegend, 400131) was used as an isotype control. Unless otherwise stated, all antibodies were used at a ratio of 1:100.

[0190] Bulk RNA Sequencing

[0191] Magnetic enriched CD49a+ SC-β cells and SC-endothelial cells were cultured at 1 x 10 6 Cells / well (repeated three times) were inoculated in 6-well plates and treated with 10ng / ml IFN-γ for 24 h and without treatment. Cells were then harvested using Accutase and pellets were quickly frozen on dry ice. RNA extraction, library preparation and sequencing reactions were all performed at GENEWIZ, LLC (South Plainfield, NJ, USA). Total RNA was extracted from cell pellet samples using RNeasy Plus Mini Kit (Qiagen, Germantown, MD, USA) and quantified using Qubit Fluorometer (Life Technologies, Carlsbad, CA, USA). Full-length cDNA synthesis and amplification (Clontech, Mountain View, CA) were performed using SMART-Seq v4 ultra-low input sequencing kit, and sequencing library preparation was performed using Illumina Nextera XT library.

[0192] The multiplexed sequencing libraries were loaded onto the Illumina HiSeq instrument and sequenced using the 2x150 Paired End (PE) configuration. Image analysis and base calling were performed by the HiSeq Control Software (HCS). The raw sequence data (.bcl files) generated by the Illumina HiSeq were converted to FASTQ files and demultiplexed using Illumina's bcl2fastq 2.17 software. Index sequence identification allowed one mismatch.

[0193] After exploring the quality of the raw data, the sequence reads were trimmed using Trimmomatic v.0.36 to remove possible adapter sequences and poor quality nucleotides. The trimmed reads were mapped to the Homo sapiens reference genome available at ENSEMBL using STAR aligner v.2.5.2b. STAR aligner is a splice aligner that detects splice junctions and merges them to help align the entire read sequence. After this step was completed, a BAM file was generated. Unique gene hit counts were calculated using the Counts function from the Subread package v.1.5.2. Only unique reads located within exonic regions were counted.

[0194] After extracting the gene hit counts, the gene hit count table was used for downstream differential expression analysis. Gene expression between sample groups was compared using DESeq2. Wald test was used to generate p-values ​​and Log2 fold changes. For each comparison, genes with adjusted p-value < 0.05 and absolute log2 fold change > 1 were called differentially expressed genes. Gene ontology analysis was performed on statistically significant gene sets by implementing the software g:Profiler (Raudvere et al., 2019). The gene accession number of the unique RNAseq dataset generated in this study is GSE200021.

[0195] NK cell ligand analysis

[0196] WT and B2M- / - SC-islet cells and SC-endothelial cells were washed twice with washing buffer and blocked with blocking buffer on ice for 30 min. The cells were then incubated on ice in the presence of APC anti-human CD47 (Biolegend, 323123), APC anti-human CD324 (E-cadherin) (Biolegend, 324107), PE anti-human CD325 (N-cadherin) (Biolegend, 350806), PE anti-human CD112 (nectin-2) (Biolegend, 337409), APC anti-human CD155 (PVR) (Biolegend, 337617), PE anti-human CD111 (nectin-1) (Biolegend, 340404), APC anti-human MICA / MICB (Biolegend, 320907), AF647 anti-human PCNA (Biolegend, 307912), PE anti-human BAG6 (Abcam, ab210838), APC anti-human CD48 (Biolegend, 336713), PE anti-human CD70 (Biolegend, 355103), mouse anti-human CD113 (fibronectin-3) (Millipore-Sigma, MABT63), APC anti-human ULBP1 (R&D, FAB1380A), PE anti-human ULBP2 / 5 / 6 (R&D, FAB1298P), APC anti-human CLEC2D (R&D, FAB3480A) and PE anti-human CD72 (Biolegend, 316207) were stained in blocking buffer for 30 min. PE mouse IgG1 (Biolegend, 400111), APC mouse IgG1 (Biolegend, 400121), APC mouse IgG2a (Biolegend, 400221), PE mouse IgG2a (Biolegend, 400213), APC rat IgG1 (Biolegend, 401903), PE rabbit IgG (CellSignaling technology, 5742S) and goat anti-mouse 488 (Thermofisher, A-11001, 1:300) served as isotype controls. Unless otherwise stated, all antibodies were used at a ratio of 1:100.

[0197] Transplantation of HLA-deficient SC-islet cells

[0198] Diabetes was induced in NSG-MHC class I / II KO mice by multiple ip injections of low-dose (40 mg / kg) streptozotocin (STZ) as previously described (Furman, 2021). Once the blood glucose of the animals reached >500 mg / dL, 5x 10 6 WT or B2M- / - SC-islet cells were transplanted under the renal capsule (n = 10) as previously described (Millman et al., 2016; Pagliuca et al., 2014). Blood glucose and body weight were measured twice a week after transplantation.

[0199] Ten weeks after transplantation (before PBMC injection) and seven weeks after PBMC injection, the function of the transplanted cells was assessed by performing in vivo peritoneal glucose-stimulated insulin secretion (IPGTT) as previously described (Millman et al., 2016; Pagliuca et al., 2014). Insulin secretion was quantified using a human ultrasensitive insulin ELISA (ALPCO Diagnostics; 80-INSHUU-E01.1.).

[0200] Generation of immune-tolerant SC-islet cells

[0201] cDNAs for human IL-2, TGF-β, and IL-10 were synthesized as polycistronic gBlocks (Genscript) and cloned into an existing plasmid targeting GAPluc as previously described to generate the GAP-2B10 plasmid. The human IL-2 sequence was modified by replacing a single amino acid (N88D) as previously described (Peterson et al., 2018). GAP-2B10 hESCs were generated by co-nucleofection of a plasmid targeting GAP-2B10 and a RNP targeting GAPDH as previously described. GAP-2B10 SC-islet cells were differentiated as previously described (Millman et al., 2016; Pagliuca et al., 2014).

[0202] Glucose-stimulated insulin secretion in vitro

[0203] In vitro function was assessed by measuring glucose-stimulated insulin secretion (GSIS) as previously described (Millman et al., 2016; Pagliuca et al., 2014). SC-islet clusters were washed twice in Krebs buffer (KRB) and preincubated for 1 h at 37°C in KRB containing 2 mM glucose (low glucose). Three consecutive treatments were then performed alternating between low-high-low KRB containing glucose (high; 20 mM), followed by depolarization with low KRB containing 30 mM KCl. Each treatment lasted 30 min, after which 100 μl of supernatant was collected and human insulin was quantified using a human ultrasensitive insulin ELISA. Human insulin measurements were normalized by viable cell counts obtained by dispersing clusters using TrypLEExpress (Thermofisher, 12604013) and counting using ViCell (Beckman Coulter).

[0204] In vitro SC-islet cell cytokine secretion and PBMC cell cytotoxicity assays

[0205] GAP-2B10 SC-islet cell clusters were dispersed with TrypLE Express and cultured at linear concentrations (1, 2, 4, 6, 8 x10 4 and 1 x 10 5 Cells / well) were seeded in duplicate in 96-well matrigel-coated plates in S3 medium. After 24 h, the supernatant was collected, centrifuged at 3000 g for 5 min, and the cytokines IL-2, TGF-β, and IL-10 were quantified using Legend Max ELISA (IL-2, Biolegend, 431807; TGF-β, Biolegend, 436707; IL-10, Biolegend, 430607) according to the manufacturer's instructions. For cytotoxicity assays, WT and 2B10 SC-islet cells were co-cultured with human PBMCs as described above.

[0206] Xenotransplantation of GAP-2B10 SC-islet cells

[0207] WT and GAP-2B10 SC-islet cell clusters (5 x 10 6 SC islet grafts were transplanted under the renal capsule of B6 / albino mice (n = 3 / group) and graft survival was monitored weekly for nine weeks by bioluminescence after ip injection of D-luciferin as described above. Five weeks after transplantation, SC islet grafts were removed and immunohistochemical analysis of surviving INS+ cells and CD8+ T cells and FOXP3+ Treg cells was performed.

[0208] Immunohistochemistry of SC-islet transplants

[0209] Kidneys from B6 / albino mice transplanted with WT and 2B10 SC-islets were excised, fixed in 4% paraformaldehyde (PFA) overnight at room temperature, and embedded in paraffin. Sections were pre-cleared with Histo-Clear, rehydrated using an ethanol gradient, and antigens were fixed by incubation in boiling antigen retrieval reagent (10 mM sodium citrate, pH 6.0) for 50 min. Slides were then blocked in 5% donkey serum for 1 h and stained with guinea pig anti-human insulin (DAKO, A0564), rat anti-mouse CD8α (Biolegend, 100702), and mouse anti-mouse FOXP3 (Biolegend, 320002) at 4°C overnight. The slides were then washed three times, incubated in secondary antibodies goat anti-mouse 594 (Life Technologies, A-11032), goat anti-rat 488 (Life Technologies, A-11006), and goat anti-guinea pig 647 (Life technologies, A-21450) for 2 h at room temperature, washed, mounted in Vectashield with DAPI (Vector Laboratories; H-1200), covered with a coverslip, and sealed with clear nail polish. Representative areas were imaged using Zeiss.Z2 and Apotome microscopes. All primary and secondary antibodies were used at dilutions of 1:200 and 1:500, respectively.

[0210] statistics

[0211] All data are expressed as mean ± SD and analyzed by GraphPad Prism 9 (GraphPadSoftware). Statistically significant differences were determined by one-way ANOVA or two-way ANOVA with Tukey and Sidak post hoc test for multiple comparisons and two-tailed t-test for pairwise comparisons. P values ​​are indicated in the figures as *p < 0.05, **p < 0.01, ***p < 0.005 and ****p < 0.001.

[0212] result

[0213] Engineering low immunogenic SC-islet cells

[0214] Since GAPDH is constitutively expressed in all cells of human pancreatic islets ( FIG. 5A to FIG. 5B), we targeted the expression of PD-L1 and HLA-E long chain fusions to the GAPDH locus of hESCs and used luminescence as a reporter for cell viability. We chose to overexpress PD-L1 because it has previously been shown to protect SC-islet cells from xenogeneic rejection (Yoshihara et al., 2020), while HLA-E long chain fusions inhibit NK cells in an HLA-deficient background (Gornalusse et al., 2017; Mattapally et al., 2018; Riolobos et al., 2013; Wang et al., 2015). Plasmids targeting GAPDH were modified to include either PD-L1 or HLA-E ( FIG. 5C to FIG. 5E ) and used to create five hESC lines ( Figure 1A ), namely wild type (WT), HLA-deficient (B2M- / -), PD-L1-expressing (PD-L1), HLA-deficient / PD-L1-expressing (B2P), and HLA-deficient / HLA-E-expressing (BEC). All five genetically modified hESC lines were successfully differentiated into SC-β cells with similar efficiency as assessed by C-peptide and Nkx6.1 expression ( Figure 1B ). Knockout of the β2-microglobulin (B2M) gene effectively abolished HLA class I expression on B2M- / -, B2P, and BEC SC-islet cells. After IFN-γ stimulation, HLA class I and PD-L1 were upregulated in WT SC-islet cells (Castro-Gutierrez et al., 2021), while B2P SC-islet cells constitutively overexpressed PD-L1 and lacked HLA class I expression ( Figure 1C Similar HLA-E and HLA class I expression profiles were observed on WT and BEC SC-islet cells ( Figure 1D ) (Gornalusse et al., 2017). Immunohistochemistry of magnetically enriched SC-β cells showed that HLA-E long chain fusions were localized to the membrane of BEC SC-β cells ( Figure 1E ) (Veres et al., 2019).

[0215] To confirm that human PD-L1 expressed on the surface of SC-islet cells binds to PD-1, we evaluated the binding of fluorescently labeled soluble human and mouse PD-1-Fc on WT and B2P SC-islet cells ( Fig. 5F). Both soluble human and mouse PD-1 bind to membrane-bound PD-L1 on B2P SC-islet cells, whereas WT SC-islet cells do not endogenously express sufficient levels of PD-L1 to detectably bind either human or mouse PD-1. We also observed that soluble mouse PD-1 has reduced binding affinity to human PD-L1 (Freeman et al., 2000). When WT, PD-L1, and B2P SC-islet cells were transplanted under the renal capsule of B6 / albino mice ( Figure 1F ), all genetically modified SC-islet cells were rejected within 10 days after transplantation ( Figure 1G ). These results suggest that PD-L1 overexpression is not sufficient to protect HUES8-derived SC-islet cells from xenogeneic rejection and that ablation of HLA expression does not improve xenograft survival in our animal model of xenogeneic rejection.

[0216] HLA-deficient SC-islet cells are resistant to destruction by allogeneic immune cells in vitro

[0217] Next, we evaluated the in vitro survival of gene-modified SC-islet cells when co-cultured with allogeneic human immune cells ( Figure 2A ). Consistent with previous studies, NK cells as well as CD4+ and CD8+ T cells accounted for approximately 8%, 35%, and 10% of the enriched PBMCs, respectively ( FIG. 6A to FIG. 6B ) (Kleiveland, 2015). When SC-islet cells were pretreated with IFN-γ and co-cultured with human PBMCs at multiple effector:target ratios, B2M- / - and BEC SC-islet cells exhibited significantly improved survival compared to WT ( Figure 2B to Figure 2C ). HLA-E overexpression did not provide additional protective benefit. In addition, similar survival patterns were observed when SC-islet cells were co-cultured with purified CD8+ and CD4+ T cells ( FIG. 6C to FIG. 6F In all co-culture assays, there were no significant differences in the survival rates of all gene-modified SC-islet cells cultured with CD3 / CD28 activated cells.

[0218] Since the expression of T cell coactivating and co-inhibitory ligands determines T cell function and is regulated by various stimuli including IFN-γ stimulation (Chen and Flies, 2013), we performed bulk RNA sequencing of WT CD49a+ SC-β cells and assessed T cell ligand expression after IFN-γ stimulation ( Figure 2D As expected, IFN-γ stimulation upregulated the T cell co-inhibitory ligands PD-L1 and LGALS9 (galectin 9) in SC-β cells ( Figure 2E) (Garcia-Diaz et al., 2017; Imaizumi et al., 2002). In contrast, although we did not detect transcripts for many T cell coactivating ligands other than HLA genes, we found that IFN-γ stimulation upregulated members of the TNF receptor superfamily HVEM (TNF receptor superfamily member 14) and CD40 (tumor necrosis factor receptor superfamily member 5) in SC-β cells ( Figure 2F ) (Benci et al., 2016; Wagner et al., 2002). Interestingly, BNT3A1 (butyrophilin subfamily 3 member A1), an MHC-related gene that regulates T cell activation and proliferation, was also upregulated (Rigau et al., 2020). These results suggest that due to the loss of classical HLA-TCR signaling caused by HLA knockout, other co-stimulatory and co-inhibitory T cell ligands are expressed in SC-β cells, which may affect T cell function.

[0219] HLA-deficient SC-islet cells are resistant to allogeneic NK cell destruction in vitro and in vivo

[0220] Next, we evaluated the effects of HLA deficiency and HLA-E overexpression on NK cell antagonism of SC-islet function. When co-cultured with NK92mi cells, BEC SC-islet cells showed no significant difference in survival compared with WT SC-islet cells, whereas HLA-deficient SC-islet cells were susceptible to NK92mi cytotoxicity ( Fig. 7A ) (Gornalusse et al., 2017). The strong protective effect of HLA-E on NK92mi cells may be due to the higher percentage of NKG2A+ / NKG2C- cells (about 96.1%) ( Figure 7B ), which favors NK cell inhibition.

[0221] Although NK cell lines are useful tools for assessing NK cell cytotoxicity, they do not accurately recapitulate primary NK cell receptor expression and function. Previous studies have shown that human NK cells do not destroy HLA-deficient endothelial cells and platelets in the absence of IL-2 activation (Deuse et al., 2021; Suzuki et al., 2020). Therefore, we pre-activated human NK cells with IL-2 for 5 days as previously described before co-culturing with genetically modified SC-islet cells (Deuse et al., 2021). The enriched NK cells consisted of approximately 80% CD56 low and approximately 5% CD56 high NK cells ( FIG. 7C to FIG. 7D Surprisingly, despite IL-2 pre-activation, we did not observe significant differences in survival between genetically modified SC-islet cells at various effector:target ratios ( Figure 2G ). Since HLA-E overexpression did not provide any additional protective benefit to HLA-deficient SC-islet cells against NK cell cytotoxicity, we chose to continue studying WT and B2M- / - SC-islet cells. We evaluated the survival of WT and B2M- / - SC-islet cells in Scid / beige mice after transplantation with IL-2 pre-activated NK cells ( Figure 2H ). Similarly, there was no significant difference in the survival rate of WT and B2M- / - SC-islet cells in vivo ( Fig.2I , Fig. 7E ). Taken together, these results indicate that HLA-deficient SC-islet cells are intrinsically resistant to NK cell cytotoxicity and that their in vitro survival is recapitulated in vivo.

[0222] Exclusively, SC-β cells intrinsically possess and maintain NK cell escape ligand profiles following inflammatory stimulation

[0223] Since NK cell function is determined by the balance of inhibitory and activating signals, we compared the ligand profiles of CD49a+ SC-β cells with those of stem cell-derived endothelial (SC-endo) cells. We chose endothelial cells as a comparison cell type because HLA-deficient endothelial cells have previously been shown to be susceptible to preactivated NK cells (Deuse et al., 2021). Endothelial cell differentiation generated >95% CD31+ SC-Endo cells derived from WT and B2M- / - hESCs ( FIG. 7F to FIG. 7G Bulk RNA sequencing and transcriptional analysis of NK cell ligands in SC-β and SC-endo cells confirmed that SC-endo cells had an activating NK cell ligand profile characterized by the expression of MICA, MICB, ULBP1, ULBP2, and RAET1G, whereas transcripts of these genes were undetectable in SC-β cells ( FIG. 3A to FIG. 3B Importantly, we found that, with the exception of HLA molecules, the expression of most NK cell ligands was not regulated by IFN-γ. Thus, in an HLA-deficient background, the expression of non-HLA NK cell ligands was stable after inflammatory stimulation. We confirmed bulk RNA sequencing analysis of NK cell ligand expression by flow cytometry, which showed that MIC and ULBP proteins were absent or poorly expressed on SC-β cells ( FIG. 3C to FIG. 3D Taken together, these data suggest that SC-β cells intrinsically possess and maintain an attenuated NK cell-activating ligand profile following inflammatory stimulation and that this ligand phenotype may explain their resistance to cytotoxicity of IL-2-preactivated NK cells.

[0224] HLA-deficient SC-islet cells reverse diabetes and exhibit delayed graft rejection in humanized mice

[0225] Having demonstrated that HLA-deficient SC-islet cells are resistant to PBMC cytotoxicity in vitro, as well as NK cell cytotoxicity in vitro and in vivo, we evaluated the survival of HLA-deficient SC-islet cells in diabetic NSG-DKO mice before PBMC injection ( Figure 3E ). Ten weeks after transplantation, there was no significant difference in blood glucose levels between mice transplanted with WT and B2M- / - SC-islet cells ( Figure 3F ). After blood glucose levels were normalized by SC-islet transplantation, we injected HLA-A2- (mismatched) PBMCs and assessed allograft rejection by monitoring blood glucose levels. WT SC-islet cells were destroyed within 2 weeks, whereas rejection of B2M- / - SC-islet cells was delayed. At 7 weeks after PBMC injection, in vivo GSIS assays demonstrated that animals transplanted with WT SC-islet cells had lost in vivo graft function, whereas some graft function was observed in animals transplanted with B2M- / - SC-islet cells ( Figure 3G ). These results indicate that B2M- / - SC-islet cells reverse diabetes and exhibit delayed allograft rejection.

[0226] Cytokine-secreting SC-β cells induce a local tolerogenic microenvironment that prevents xenogeneic rejection

[0227] Since immune escape engineering has had varying success in protecting SC-islets, we took a complementary strategy by engineering SC-islets to secrete cytokines IL-2 muteins, TGF-β, and IL-10 as mediators of local immunity ( Figure 4A ). The IL-2 mutant protein (N88D) has reduced affinity for the IL-2Rβγ receptor, resulting in a Treg-selective molecule that preferentially expands Tregs while having minimal effects on CD4+ and CD8+ memory T cells (Khoryati et al., 2020; Peterson et al., 2018). Since the immunosuppressive phenotype of Tregs requires IL-10 and TGF-β, we included these two cytokines in our immune tolerance constructs (Horwitz et al., 2008).

[0228] Differentiation of the genetically modified cell line (termed 2B10) resulted in approximately 30% Nkx6.1+ / C-peptide+ SC-β cells ( Figure 4B ), and its physiological function was evaluated in vitro by GSIS ( Figure 4C We also demonstrated that 2B10 SC-islet cells secreted modified IL-2, TGF-β, and IL-10, and the amounts depended on the cell concentration ( Figure 4DWhen WT and 2B10 SC-islet cells were co-cultured with human PBMCs in vitro, 2B10 SC-islet cells showed significantly improved survival rate ( Figure 4E ). Notably, 2B10 SC-islets transplanted under the renal capsule of B6 / albino mice survived for up to 9 weeks after transplantation, whereas WT SC-islets were destroyed within 2 weeks ( Figures 4F to 4G ). In addition, we found that WT grafts contained almost no residual SC-islet cells, whereas 2B10 grafts contained viable insulin-producing cells and Tregs located within the graft ( Figure 4H Collectively, these data suggest that SC-islet cells can be engineered to co-secrete immunomodulatory cytokines and induce a tolerogenic local microenvironment characterized by Treg cell infiltration, thereby maintaining xenograft survival.

[0229] References

Claims

1. A low immunogenic cell engineered to express one or more tolerogenic transgenes from one or more loci in a stem cell or progenitor cell, wherein the one or more tolerogenic transgenes are not silenced during differentiation of the stem cell or progenitor cell into a desired differentiated cell type.

2. The low immunogenicity cell according to claim 1, wherein the cell is a stem cell.

3. The low immunogenic cell of claim 1, wherein the cell is an induced pluripotent cell.

4. The low immunogenicity cell of claim 1, wherein the cell is a progenitor cell.

5. The low immunogenic cell of claim 1, wherein the cell is a β cell progenitor cell. The low immunogenicity cell of claim 1 , wherein the cell is a differentiated cell.

7. The low immunogenic cell of claim 1, wherein the cell is a β cell.

8. The low immunogenicity cell of claim 1, wherein the one or more loci are loci of housekeeping genes.

9. The low immunogenic cell according to claim 8, wherein the housekeeping gene is selected from the group consisting of actin, ubiquitin and GAPDH.

10. The low immunogenic cell of claim 1, wherein the one or more tolerogenic transgenes are selected from the group consisting of PDL1, HLA-E / G, CD47, SERPINB9, CCL21, FASL, CD200, MFGE8, CD55, CD46, HLS-G single chain fusion, soluble PDL1-Ig and CTLA4-Ig.

11. The low immunogenic cell of claim 1, wherein the one or more tolerogenic transgenes are HLA-E single chain fusions.

12. The low immunogenicity cell according to claim 11, wherein the single-chain fusion is loaded with a peptide capable of activating NK cells.

13. The low immunogenicity cell according to claim 12, wherein the peptide activates NK cells by interacting with inhibitory receptors NKG2A and / or NKG2C.

14. The low immunogenic cell of claim 12, wherein the peptide is selected from the group consisting of VMAPRTLLL (SEQ ID NO: 2), VMAPRTLL (SEQ ID NO: 3), VMAPRTLFL (SEQ ID NO: 4), VMAPRTLVL (SEQ ID NO: 5), VMAPRTLIL (SEQ ID NO: 6), IMAPRTLVL (SEQ ID NO: 7), VMPPRTLLL (SEQ ID NO: 8), VMAPRTVLL (SEQ ID NO: 9), VTAPRTLLL (SEQ ID NO: 10), VTAPRTVLL (SEQ ID NO: 11), VMAPRTLTL (SEQ ID NO: 12) and VMAPRALLL (SEQ ID NO: 13).

15. The low immunogenic cell of claim 1, wherein the cell is HLA class I deficient. The low immunogenicity cell according to claim 15 , wherein the expression of the B2M gene is eliminated.

17. A method of producing a hypoimmunogenic cell comprising engineering a stem or progenitor cell to express one or more tolerogenic transgenes from a locus in the stem or progenitor cell that are not silenced during differentiation of the cell into a desired cell type, and exposing the stem or progenitor cell to conditions suitable for differentiation of the stem or progenitor cell into a desired cell type.

18. The method of claim 17, wherein the stem cells are induced pluripotent cells.

19. The method of claim 17, wherein the progenitor cells are β-cell progenitor cells.

20. The method of claim 17, wherein the desired cell type is a β cell.

21. The method of claim 17, wherein the locus is constitutively expressed in all cells of the islets.

22. The method of claim 17, wherein the locus is constitutively expressed in all cells of human pancreatic islets.

23. The method of claim 17, wherein the locus is constitutively expressed in β cells.

24. The method of claim 17, wherein the locus is constitutively expressed in human β cells.

25. The method of claim 17, wherein the locus is a locus of a housekeeping gene.

26. The method of claim 25, wherein the housekeeping gene is selected from the group consisting of actin, ubiquitin and GAPDH.

27. The method of claim 26, wherein the housekeeping gene is GAPDH.

28. The method of claim 17, wherein the one or more tolerogenic transgenes are selected from the group consisting of PDL1, HLA-E / G, CD47, SERPINB9, CCL21, FASL, CD200, MFGE8, CD55, CD46, HLS-G single chain fusion, soluble PDL1-Ig, and CTLA4-Ig.

29. The method of claim 17, wherein the tolerogenic transgene is an HLA-E single chain fusion.

30. The method of claim 29, wherein the single-chain fusion is loaded with a peptide capable of activating NK cells.

31. The method of claim 30, wherein the peptide activates NK cells by interacting with inhibitory receptors NKG2A and / or NKG2C.

32. The method of claim 30, wherein the peptide is selected from the group consisting of VMAPRTLLL (SEQ ID NO: 2), VMAPRTLL (SEQ ID NO: 3), VMAPRTLFL (SEQ ID NO: 4), VMAPRTLVL (SEQ ID NO: 5), VMAPRTLIL (SEQ ID NO: 6), IMAPRTLVL (SEQ ID NO: 7), VMPPRTLLL (SEQ ID NO: 8), VMAPRTVLL (SEQ ID NO: 9), VTAPRTLLL (SEQ ID NO: 10), VTAPRTVLL (SEQ ID NO: 11), VMAPRTLTL (SEQ ID NO: 12) and VMAPRALLL (SEQ ID NO: 13).

33. A method for mediating local immune tolerance, comprising engineering stem cells or progenitor cells to express one or more immunomodulators from a locus in the stem cells or progenitor cells that are not silenced during differentiation of the cells into a desired cell type, and exposing the stem cells or progenitor cells to conditions suitable for differentiation of the stem cells or progenitor cells into the desired cell type, thereby producing the desired cell type that secretes the one or more immunomodulators.

34. The method of claim 33, wherein the stem cells are induced pluripotent cells.

35. The method of claim 33, wherein the progenitor cells are β cell progenitor cells.

36. The method of claim 33, wherein the desired cell type is a β cell.

37. The method of claim 33, wherein the locus is constitutively expressed in all cells of the islets.

38. The method of claim 33, wherein the locus is constitutively expressed in all cells of human pancreatic islets.

39. The method of claim 33, wherein the locus is constitutively expressed in β cells.

40. The method of claim 33, wherein the locus is constitutively expressed in human β cells.

41. The method of claim 33, wherein the locus is a locus of a housekeeping gene.

42. The method of claim 41, wherein the housekeeping gene is selected from the group consisting of actin, ubiquitin and GAPDH.

43. The method of claim 42, wherein the housekeeping gene is GAPDH.

44. The method of claim 33, wherein the one or more immunomodulators are cytokines.

45. The method of claim 33, wherein the one or more immunomodulators are selected from the group consisting of IL-10, TGF-β, and IL-2 or modified IL-2.

46. ​​A cell engineered to express one or more immunomodulators from one or more loci in a stem or progenitor cell, said one or more immunomodulators being not silenced during differentiation of said stem or progenitor cell into a desired differentiated cell type.

47. The cell of claim 46, wherein the cell is a stem cell.

48. The cell of claim 46, wherein the cell is an induced pluripotent cell.

49. The cell of claim 46, wherein the cell is a progenitor cell.

50. The cell of claim 46, wherein the cell is a β cell progenitor cell.

51. The cell of claim 46, wherein the cell is a differentiated cell.

52. The cell of claim 46, wherein the cell is a β cell.

53. The cell of claim 46, wherein the cell is an alpha cell.

54. The cell of claim 46, wherein the cell is a pancreatic islet cell.

55. The cell of claim 46, wherein the one or more loci are loci of housekeeping genes.

56. The cell of claim 55, wherein the housekeeping gene is selected from the group consisting of actin, ubiquitin and GAPDH.

57. The cell of claim 46, wherein the one or more immunomodulators are cytokines.

58. The cell of claim 46, wherein the one or more immunomodulators are selected from the group consisting of IL-10, TGF-β, and IL-2 or modified IL-2.

59. A cell produced by the method of claim 17.

60. A cell produced by the method of claim 33.

61. A method of treating a subject in need thereof, comprising administering to the subject the cell of claim 1.

62. A method of treating a subject in need thereof, comprising administering to the subject the cell of claim 46.

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