IMMUNODESIGNED PLURIPOTENT CELLS.

Abstract

The invention provides pluripotent cells for therapeutic use in tissue regeneration that prevent rejection by recipient subjects. In particular, the invention provides hypoimmunogenic pluripotent cells that prevent immune rejection by the host. These cells lack important immune antigens that trigger immune responses and are designed to avoid phagocytic endocytosis. The invention further provides universally acceptable, commercially available pluripotent cells and their derivatives for generating or regenerating specific tissues and organs.

Description

IMMUNO-ENGINEERED PLURIPOTENT CELLS Cross-reference to related application This application claims the benefit of United States Provisional Application No. 62 / 445,969 filed on January 13, 2017. Field of invention Regenerative cell therapy is an important potential treatment for regenerating damaged organs and tissues. With the limited availability of organs for transplantation and the long waiting times associated with it, the possibility of regenerating tissue by transplanting readily available cell lines into patients is understandably attractive. Regenerative cell therapy has shown promising initial results for rehabilitating damaged tissue after transplantation in animal models (e.g., after a myocardial infarction). However, the propensity of the transplant recipient's immune system to reject allogeneic material greatly reduces the potential efficacy of these therapeutic agents and diminishes the potential positive effects surrounding such treatments. Background of the invention Regenerative cell therapy is an important potential treatment for regenerating damaged organs and tissues. With the limited availability of organs for transplantation and the long waiting times associated with it, the possibility of regenerating tissue by transplanting readily available cell lines into patients is understandably attractive. Regenerative cell therapy has shown promising initial results in rehabilitating damaged tissue after transplantation in animal models (e.g., after a myocardial infarction). However, the propensity of the transplant recipient's immune system to reject allogeneic material greatly reduces the potential efficacy of therapeutic agents and diminishes the possible positive effects surrounding such treatments. Autologous induced pluripotent stem cells (PSCs) theoretically represent an unlimited source of cells for patient-specific cell-based organ repair strategies. Their generation, however, presents technical and manufacturing challenges and is a lengthy process that conceptually precludes any acute treatment modality. Allogeneic PSC-based therapies are easier to manufacture and allow for the generation of well-selected, standardized, and high-quality cell products. However, due to their allogeneic origin, such cell products would be rejected. By reducing or eliminating cell antigenicity, universally acceptable cell products could be realized. Because pluripotent stem cells can differentiate into any cell type of the three germ layers, the potential application of stem cell therapy is broad.Differentiation can be performed ex vivo or in vivo by transplanting progenitor cells that continue to differentiate and mature in the organic environment of the implantation site. Ex vivo differentiation allows researchers or clinicians to closely monitor the procedure and ensure that the appropriate cell population is generated before transplantation. In most cases, however, undifferentiated pluripotent stem cells are avoided. in clinical transplant therapies due to their propensity to form teratomas. Rather, such therapies tend to use differentiated cells (for example, stem cell-derived cardiomyocytes transplanted into the myocardium of patients with heart failure). Clinical applications of such pluripotent cells or tissues would benefit from a "safety feature" that controls cell growth and survival after transplantation. The technique seeks stem cells capable of producing cells used to regenerate diseased or defective cells. Pluripotent stem cells (PSCs) can be used because they proliferate and differentiate rapidly into many possible cell types. The PSC family includes several members generated through different techniques and possessing distinct immunogenic characteristics. The patient's compatibility with the engineered cells or tissues derived from PSCs determines the risk of immune rejection and the need for immunosuppression. Embryonic stem cells (ESCs) isolated from the inner cell mass of beta-fast cytes exhibit histocompatibility antigens that do not match recipients. This immunological barrier cannot be overcome with human leukocyte antigen (HLA) ESC banks, as even HLA-matched PSC grafts suffer rejection due to mismatches in non-HLA molecules that function as minor antigens. To date, preclinical success of PSC-based approaches has only been achieved in immunocompromised or immunodeficient models, or when the cells are encapsulated and protected from the host immune system. However, the systemic immunosuppression used in allogeneic organ transplantation is not justifiable for regenerative approaches. Immunosuppressive drugs have serious side effects and significantly increase the risk of infections and malignancies. To avoid the problem of rejection, different techniques have been developed for generating patient-specific pluripotent stem cells. These include the transfer of a somatic cell nucleus into an enucleated oocyte (somatic cell nuclear transfer (SCNT) stem cells), the fusion of a somatic cell with an ESC (hybrid cell), and the reprogramming of somatic cells using certain transcription factors (induced pluripotent stem cells (IPSCs) or induced pluripotent stem cells (iPSCs)). However, both SCNT stem cells and iPSCs can have immune incompatibilities with the nucleus or donor cell, respectively, despite chromosomal identity. SCNT stem cells carry mitochondrial DNA (mtDNA) that passes through the oocyte. Proteins encoded by mtDNA can act as relevant minor antigens and trigger rejection.DNA and mtDNA mutations, along with the genetic instability associated with iPSC reprogramming and culture expansion, can also create minor antigens relevant to immune rejection. This previously unknown immune obstacle reduces the likelihood of successful large-scale engineering of patient-specific compatible tissues using SCNT stem cells or iPSCs. Brief description of the invention Hypoimmune pluripotent cells (HIPs) that evade rejection by the host immune system were generated. Syncytiotrophobiast cells from the placenta, which form the interface between maternal blood and fetal tissue, were used. The expression of MHC class I (HLA-I) and MHC class II (HLA-IL) was reduced. CD47 This pattern of impaired antigen-presenting capacity and protection against innate immune clearance evaded host immune rejection. This was demonstrated for HIP cells and the specific cells derived from ectoderm, mesoderm, and endoderm into which the HIP cells differentiated. Thus, the invention provides a method for generating a hypoimmunogenic pluripotent stem cell comprising: eliminating the activity of both alleles of a B2M gene in an induced pluripotent stem cell (iPSC); eliminating the activity of both alleles of a CUTA gene in the iPSC; and increasing the expression of CD47 in the iPSC. In a preferred embodiment of the method, the iPSC is human, the B2M gene is human, the CUTA gene is human, and the increased CD47 expression results from the introduction of at least one copy of a human CD47 gene under the control of a promoter into the iPSC. In another preferred embodiment of the method, the iPSC is marine, the 82m gene is marine, the Ciita gene is marine, and the increased CD47 expression results from the introduction of at least one copy of a murine CD47 gene under the control of a promoter into the iPSC. In a more preferred embodiment, the promoter is a constitutive promoter. In some forms of the methods described herein, the disruption of both alleles of the B2M gene results from a Clustered Regularly Interspaced Short Palindromic Repeats / Cas9 (CRISPR) reaction that disrupts both alleles of the B2M gene. In other forms of the method, the disruption of both alleles of the CUTA gene results from a CRISPR reaction that alters both alleles of the CUTA gene. The invention provides a human hypoimmunogenic pluripotent stem cell (hHIP) comprising: one or more alterations that inactivate both alleles of an endogenous B2M gene; one or more alterations that inactivate both alleles of an endogenous CUTA gene; and one or more alterations that cause increased expression of a CD47 gene in the hHIP stem cell; wherein the hHIP stem cell elicits a first natural killer (NK) cell response that is less than a second NK cell response elicited by an induced pluripotent stem cell (iPSC) comprising said B2M and CUTA alterations but not comprising said increased expression of the CD47 gene, and wherein the first and second NK cell responses are measured by determining the IFN-γ levels of NK cells incubated with either the hHIP or iPSC in vitro. The invention provides a human hypoimmunogenic pluripotent stem cell (hHIP) comprising: one or more alterations that inactivate both alleles of an endogenous B2M gene; one or more alterations that inactivate both alleles of an endogenous CUTA gene; and an alteration that causes an increase in the expression of a CD47 gene in the hHIP stem cell; wherein the hHIP stem cell elicits a first T cell response in a humanized mouse strain that is less than a second T cell response in the humanized mouse strain elicited by an iPSC, and wherein the first and second T cell responses are measured by determining the IFN-γ levels of the humanized mice in an ELISPOT assay. The invention provides a method comprising the transplantation of hHIP stem cells described herein into a human subject. The invention further provides for the use of the stem cells. hHIPs described herein for the preparation of a drug to treat conditions requiring cell transplantation. The invention provides a hypoimmunogenic pluripotent cell comprising reduced endogenous Major Histocompatibility Antigen Class I (HLA-I) function compared to a pluripotent stem cell; reduced endogenous Major Histocompatibility Antigen Class II (HLA-II) function compared to the pluripotent stem cell; and reduced susceptibility to NK cell killing compared to the pluripotent stem cell; wherein the hypoimmunogenic pluripotent cell is less susceptible to rejection when transplanted into a subject as a result of the reduced HLA-I function, the reduced HLA-II function, and the reduced susceptibility to NK cell destruction. In some embodiments, the hypoimmunogenic pluripotent cell is reduced by a reduction in the expression of the p-2 microglobulin protein. In a preferred embodiment, a gene encoding the B-2 microglobulin protein is deleted. In a more preferred embodiment, the B-2 microglobulin protein has at least 90% sequence identity with SEQ ID NO. 1. In a more preferred embodiment, the S-2 microglobulin protein has the sequence of SEQ ID NO. 1. In some modality, HLA-I function is reduced by a reduction in the expression of the HLA-A protein. In a preferred modality, a gene encoding the HLA-A protein is deleted. In some modality, HLA-I function is reduced by a reduction in the expression of the HLA-B protein. In a preferred modality, a gene encoding the HLA-B protein is deleted. In some modality, HLA-I function is reduced by a reduction in the expression of the HLA-C protein. In a preferred modality, a gene encoding the HLA-C protein is deleted. In another modality, hypoimmunogenic pluripotent cells do not comprise an HLA-I function. The invention provides a hypoimmunogenic pluripotent cell in which HLA-II function is reduced by a reduction in the expression of the CUTA protein. In a preferred embodiment, a gene encoding the CUTA protein is deleted. In a more preferred embodiment, the CUTA protein has at least 90% sequence identity with SEQ ID NO: 2. In some modality, HLA-II function is reduced by a reduction in HLA-DP protein expression. In a preferred modality, a gene encoding the HLA-DP protein is deleted. In some modality, HLA-II function is reduced by a reduction in HLA-DR protein expression. In a preferred modality, a gene encoding the HLA-DR protein is deleted. In some modality, HLA-H function is reduced by a reduction in HLA-DQ protein expression. In a preferred modality, a gene encoding the HLA-DQ protein is deleted. The invention provides hypoimmunogenic pluripotent cells that do not comprise an HLA-II function. The invention provides hypoimmunogenic pluripotent cells with reduced susceptibility to macrophage phagocytosis or NK cell destruction. The reduced susceptibility is caused by increased expression of a CD47 protein. In some embodiments, the increased CD47 expression results from a modification at a locus of the endogenous CD47 gene. In other embodiments, the increased CD47 expression results from a CD47 transgene. In a preferred embodiment, the CD47 protein has at least 90% sequence identity with SEQ ID NO: 3. In a more preferred embodiment, the CD47 protein has the sequence of SEQ ID NO: 3. The invention provides hypoimmunogenic pluripotent cells comprising a suicide gene that is activated by a trigger, causing the hypoimmunogenic pluripotent or differentiated progeny cell to die. In a preferred embodiment, the suicide gene is a herpes simplex virus thymidine kinase (HSV-tk) gene, and the trigger is ganciclovir. In a more preferred embodiment, the HSV-tk gene encodes a protein having at least 90% sequence identity with SEQ ID NO: 4. In another preferred embodiment, the suicide gene is an Escherichia coli cytosine deaminase gene (EC-CD) and the activator is 5-fluorocytosine (5-FC). In a more preferred embodiment, the EC-CD gene encodes a protein that has at least 90% sequence identity with SEQ ID NO: 5. In another preferred embodiment, the suicide gene encodes an inducible caspase protein, and the trigger is a chemical inducer of dimerization (CID). In a more preferred embodiment, the inducible gene encodes a caspase protein comprising at least 90% sequence identity with SEQ ID NO: 6. In a more preferred embodiment, the gene encodes a caspase protein comprising the sequence of SEQ ID NO: 6. In a more preferred embodiment, the CID is AP1903. The invention provides a method for producing a hypoimmunogenic pluripotent cell, comprising reducing an endogenous function of Major Histocompatibility Antigen Class I (HLA-I) in a pluripotent cell; reducing an endogenous function of Major Histocompatibility Antigen Class II (HLA-II) in a pluripotent cell; and increasing the expression of a protein that reduces the susceptibility of the pluripotent cell to macrophage phagocytosis or NK cell destruction In one embodiment of the method, HLA-I function is reduced by decreasing the expression of a microglobulin protein 6-2. In a preferred embodiment, the expression of the 6-2 microglobulin protein is reduced by deleting a gene encoding the 6-2 microglobulin protein. In a more preferred embodiment, the 6-2 microglobulin protein has at least 90% sequence identity with SEQ ID NO: 1. In a further preferred embodiment, the 6-2 microglobulin protein has the sequence of SEQ ID NO: 1. In another embodiment of the method, HLA-I function is reduced by reducing the expression of the HLA-A protein. In a preferred embodiment, HLA-A protein expression is reduced by deleting a gene encoding the HLA-A protein. In another embodiment of the method, HLA-I function is reduced by reducing the expression of the HLA-B protein. In a preferred embodiment, HLA-B protein expression is reduced by deleting a gene encoding the HLA-B protein. In another embodiment of the method, HLA-I function is reduced by reducing the expression of the HLA-C protein. In a preferred embodiment, HLA-C protein expression is reduced by deleting a gene encoding the HLA-C protein. In another modality of the method, the hypoimmunogenic pluripotent cell does not comprise a function HLA-I. In another embodiment of the method, HLA-II function is reduced by decreasing the expression of a CUTA protein. In a preferred embodiment, CUTA protein expression is reduced by deleting a gene encoding the CUTA protein. In a more preferred embodiment, the CUTA protein has at least 90% sequence identity with SEQ ID NO: 2. In a more preferred embodiment, the CUTA protein has the sequence of SEQ ID NO. 2. In another embodiment of the method, HLA-II function is reduced by reducing the expression of an HLA-DP protein. In a preferred embodiment, HLA-DP protein expression is reduced by deleting a gene that encodes the HLA-DP protein. In another embodiment of the method, HLA-II function is reduced by reducing the expression of an HLA-DR protein. In a preferred embodiment, HLA-DR protein expression is reduced by deleting a gene that encodes the HLA-DR protein. In some embodiments of the method, HLA-II function is reduced by reducing the expression of an HLA-DQ protein. In a preferred embodiment, HLA-DQ protein expression is reduced by deleting a gene that encodes the HLA-DQ protein. In another modality of the method, the hypoimmunogenic pluripotent cell does not comprise an HLA-II function. In another embodiment of the method, increased expression of a protein that reduces the susceptibility of pluripotent cells to macrophage phagocytosis results from a modification to an endogenous gene locus. In a preferred embodiment, the endogenous gene locus encodes a CD47 protein. In another embodiment, increased expression of the protein results from the expression of a transgene. In a preferred embodiment, the transgene encodes a CD47 protein. In a more preferred embodiment, the CD47 protein has at least 90% sequence identity with SEQ ID NO: 3. In a most preferred embodiment, the CD47 protein has the sequence of SEQ ID NQ: 3 Another embodiment of the method further comprises the expression of a suicide gene that is activated by a trigger, causing the hypoimmunogenic pluripotent or differentiated progeny cell to die. In a preferred embodiment, the suicide gene is a herpes simplex virus thymidine kinase (HSV-tk) gene, and the trigger is ganciclovir. In a more preferred embodiment, the HSV-tk gene encodes a protein that has at least 90% sequence identity with SEQ ID NO: 4. In another embodiment of the method, the suicide gene is an Escherichia coli cytosine deaminase (EC-CD) gene and the activator is 5-fluorocytosine (5-FC). In a preferred embodiment, the EC-CD gene encodes a protein that has at least 90% sequence identity with SEQ ID NO: 5. In a more preferred embodiment, the EC-CD gene encodes a protein that has the sequence of SEQ ID NQ: 5. In another iteration of the method, the suicide gene encodes an inducible caspase protein and the 35. Trigger is a specific chemical inducer of dimerization (CID). In a preferred embodiment of the method, the gene encodes an inducible caspase protein comprising at least 90% sequence identity with SEQ ID NO: 6. In a more preferred embodiment, the gene encodes an inducible caspase protein comprising the sequence of SEQ ID NO: 6. In a most preferred embodiment, the CID is AP1903. Brief description of the drawings Figure 1A illustrates the basis of the novel hypoimmune pyuripotent cells described herein. Fetuses are protected against rejection during pregnancy by fetomaternal tolerance. These cells exhibit downregulation of MHC class I expression, downregulation of MHC class II expression, and upregulation of CD47. Figure 1B shows that fetomaternal tolerance is mediated by syncytiotrophoblast cells. Figure 1C shows that syncytiotrophoblast cells exhibit low levels of MHC I and II and low levels of CD47. Figure 2 shows murine induced pluripotent stem cells (miPSCs) generated from C57BL / 6 fibroblasts. Pluripotency was demonstrated by reverse transcriptase polymerase chain reaction (rtPCR). Multiple mRNAs associated with pluripotency were detected in extracts of miPSC cells but not in non-induced cells (parental murine fibroblasts). Figure 3 confirms the pluripotency of miPSC cells. C57BL / 6 miPSC cells formed teratomas in syngeneic mice, as well as in BALB / c nude and scid beige mice. No teratomas formed in immunocompetent allogeneic BALB / c mice. Figure 4 shows that when β-2-microglobulin expression is eliminated in miPSC cells, MHC-I expression cannot be induced by IFN-γ stimulation (right panel). As a control, parental miPSC cells stimulated with IFN-γ (left panel) increased their MHC-I expression. Figure 5 shows that the miPSC / B-2-microglobulin knockout further comprising a Ciita expression knockout (double knockout) showed no baseline MHC-II expression and could not be induced by TNF-α to express MHC-II. Figure 6A shows increased expression of Cd47 from a transgene added to the double knockout of αβ-2-microglobulin / αβα (iPSC cells) hypo Figure 6B shows that the C57BL / 6 iPSC cells hyp0 BALB / c cells survive in the allogeneic environment, but the parent cells PSC do not. Figure 7 shows an embodiment of the invention. It shows a schematic diagram of the iPSC engineering that resulted in the hypoimmune pyripotent cells of the invention. To generate hypoimmune stem cells, CRISPR-Cas9 engineering was first used to delete both B2m alleles. Second, CRISPR-Cas9 engineering was used to delete both alleles of the Ciita gene. Third, a lentivirus was used to target a Cd47 gene. Figure 8A schematically shows the role of B2m in the MHC I complex. A B2m knockout reduces MHC I in mice or HLA-I in humans. Figure 8B schematically shows that Ciita is a transcription factor that causes the expression of MHC II in mice or the expression of HLA-II in humans. A Ciita knockout reduces the expression of MHC II or HLA-II. Figures 9A, 9B, and 9C show that iPSCs 82m- / - lack MHC-I expression, iPSCs B2m- / -Ciita- / - lack MHC-I and MHC-II, and iPSCs B2m- / -Ciita- / -Cd47 tg lack MHC-I and MHC-II and overexpress Cd47. Figures 10A, 10B, 10C, 10D, and 10E show mouse models of iPSCs of type "Wild" cells transplanted with hypoimmune PSCs into allogeneic or syngeneic host mice. Here, the iPSCs were formed from C57BL / 6 mice, and the allogeneic mice are BALB / c. In Figure 10A, "wild-type iPSCs" formed teratomas only in the thighs of syngeneic C57BL / 6 mice. In contrast, an immune response was mounted in allogeneic host mice (BALB / c), and no teratomas grew. In Figure 10B, wild-type iPSCs formed teratomas in syngeneic C57BL / 6 mice. In Figure 10C, the immune response prevented teratoma formation in allogeneic BALB / c mice. Figure 10D compares the T cell response (IFN-γ and IL-4) to iPSCs in syngeneic and allogeneic hosts using a point frequency assay (frequency of cells releasing IFN-γ and IL-4). IFN-γ and IL-4 release was very low in C57BL / 6 hosts but increased dramatically in BALB / c hosts. Figure 10E depicts the B cell responses in syngeneic and allogeneic hosts. iPSCs were incubated with serum from host animals that had previously received iPSCs. Bound immunoglobulins were measured using flow cytometry. The mean fluorescence intensity (MFI) was significantly higher in serum taken from BALB / c allogeneic recipient hosts. Figures 11A, 11B, 11C, 11D, and 11E show the partial effect of knocking out the B2m gene in the iPSCs described above. In Figure 11A, B2m- / - iPSCs grew in the thighs of syngeneic C57BL / 6 mice, forming teratomas due to the lack of an immune response, whereas a partial immune response was mounted in allogeneic host mice (BALB / c); for example, some of the transplanted cells survived. In Figure 11B, B2m- / - iPSCs formed teratomas in syngeneic mice. In Figure 11C, partial survival (60%) was achieved in allogeneic hosts. In Figure 11D, the differences in T cell response (IFN-γ and IL-4) between the two hosts showed a weak but detectable T cell response against the B2m- / - iPSCs. Figure 11E shows the B cell responses in the different host mice, demonstrating a weaker immune response compared to wild-type iPSCs. However, there was a significantly stronger immunoglobulin response after allogeneic transplantation of B2m- / - iPSCs into BALB / c compared to syngeneic transplantation into C57BL / 6. Therefore, there was limited survival of the B2m- / - iPSCs in allogeneic recipients. Figures 12A, 12B, 12C, 12D, and 12E show the partial increased effect of B2m and Ciita gene inactivation in iPSCs on cell survival in syngeneic and allogeneic host mice. In Figure 12A, B2m- / -Ciita- / - iPSCs formed teratomas in syngeneic C57BL / 6 mice due to a lack of immune response, whereas a partial (but reduced compared to B2m-A iPSCs) immune response was mounted in allogeneic host mice (BALB / c). In Figure 12B, B2m- / -Ciita- / - iPSCs formed teratomas in syngeneic mice. Figure 12C shows that some cell grafts (91.7%) survived in allogeneic hosts. Figure 12D shows the differences in T cell response (IFN-γ and IL-4) between the two hosts, with a slightly higher IFN-γ response in allogeneic receptors compared to syngeneic ones. Figure 12E represents the B cell responses in the different host mice. Weaker immune response was observed compared to wild-type iPSCs and B2m- / - iPSCs. No significant difference was observed between allogeneic and syngeneic receptors. Overall, there was limited survival of B2m- / - iPSCs on allogeneic receptors, which may be attributed to a moderate immune response. Figures 13A, 13B, 13C, 13D, and 13E show the full effect of inactivating the B2m and Ciita genes and incorporating the Cd47 transgene into iPSCs on cell survival in syngeneic and allogeneic host mice. In Figure 13A, iPSC B2m- / -Ciita- / -Cd47tg teratomas grew in both syngeneic and allogeneic C57BL / 6 mice. All transplanted cell grafts survived. In Figure 13B, iPSC B2m7-Ciita- / -Cd47tg formed teratomas in C57BL / 6 mice. In Figure 13C, 100% of the cell grafts survived in the allogeneic hosts. Figure 13D shows the lack of T cell response (IFN-γ and IL-4) to allogeneic receptors. No difference was observed between the two hosts. Figure 13E shows the lack of B cell response to allogeneic receptors. No difference was observed between the two hosts. Therefore, there was complete survival of the B2m- / - Ciita- / - Cd47tg iPSCs to allogeneic receptors.They were not immunogenic since they did not elicit a T cell or B cell response. Figures 14A, 14B, and 14C show that B2m-A Ciita- / - Cd47tg iPSCs (referred to as non-immunogenic pluripotent (H|P) cells) evaded the host immune system. In Figure 14A, expression of stimulatory NK cell ligands did not increase in HIP cells. A fusion protein that recognizes several ligands of the NK cell transmembrane protein NKG2D was used to assess the level of activating ligands, which can activate cytotoxic NK cell activity. The binding of the fusion protein to iPSCs is therefore a global parameter for their expression of activated NKG2D ligands. In Figure 14B, HIP cells did not cause NK cells to increase their expression of CD107a, a marker for functional NK cell activity. Conversely, the iPSC B2m- / - Ciita - / - induced CD107a expression in NK cells and thus activated their cytophytic function. In Figure 14C, the Elispot assays.IFN-γ induced by syngeneic NK cells purified from C57BL / 6 mouse spleen showed no NK cell response triggered by HIP cells. Therefore, the NK cells were not activated to release IFN-γ. The spot frequency for HIP cells was not different from that of unstimulated NK cells (negative control). Only B2m- / - Ciita- / - PSCs resulted in a significant increase in IFN-γ spot frequencies. Figures 15A and 15B show additional data demonstrating that HIP cells evaded rejection or death by the innate immune system due to the Cd47 transgene. An m vivo NK cell assay involved injecting a 50% iPSC / 50% HIP mixture into the NK cell-rich peritoneum of syngenic C57BL / 6 mice. Here, cytotoxicity was caused by NK cells. After 24 and 48 hours, peritoneal cells were retrieved and sorted. Figure 15A compares iPSCs with B2m- / - Ciita- / - iPSCs (without the Cd47 transgene). The B2m- / - Ciita- / - iPSCs were selectively eliminated by NK cells. Figure 15B compares iPSCs with B2m- / - Ciita- / - Cd47 tg iPSCs (HIP cells). HIP cells were not selectively destroyed by NK cells. The proportion of 50% HIP cells among peripheral iPSCs was maintained, indicating no stimulation of NK cells. Therefore, while inactivation of MHC-I and MHC-II made cells highly susceptible to NK cell death, overexpression of Cd47 eliminated the interaction of stimulatory NK cells. Figure 16 shows that the marine HIP cells of the invention showed a normal murine karyotype. Figures 17A, 17B, and 17C show that the murine HIP cells of the invention retained pluripotency during the engineering process. RT-PCR analyses of generally accepted markers indicating pyuripotency (Nanog, Oci é, Sox2, Esrrb, Tbx3, TcH, and actin as a loading control) are shown. Pluripotent markers were expressed throughout the three-step engineering process. Figure 17A compares B2m- / - iPSCs and marine fibroblasts (negative control). The B2m- / - iPSCs retained the pluripotency genes. Figure 17B shows the same analysis, but the B2m-A Ciita-A iPSCs retained the same pluripotency genes. Figure 17C shows the same analysis, but with the B2rn-A Ciita-7-Cd47 tg iPSCs (HIP cells). These cells retained the same pluripotency genes.Furthermore, histological images of teratomas that developed after HIP cell transplantation in beige SCID mice show that all three cell types associated with ectoderm, mesoderm, and endoderm were identified. Immunofluorescence markers for all three germ layers were detected (data not shown). Cell morphology was consistent with neuroectoderm, mesoderm, and endoderm. Immunofluorescence staining for DAPI, GFAP, cytokeratin 8, and brachyosis confirmed the pluripotency of HIP cells. Figures 18A, 18B, and 18G show HIP cells differentiated into cells of mesodermal lineage and that lost their pluripotent markers. Figure 18A shows that the pluripotent markers in HIP cells (labeled "mHIP") were lost in differentiated murine endothelial cells (labeled "miEC"). Figure 18B shows that the pluripotent markers were retained in HIP cells but not in differentiated murine smooth muscle cells (labeled "miSMC"). Figure 18C shows that the pluripotent markers were retained in HIP cells but not in differentiated murine cardiomyocytes (labeled "miCM"). These results were confirmed by immunohistochemistry (data not shown). Endothelial cells were detected using anti-CD31 and anti-VE-cadherin, smooth muscle cells were detected using anti-SMA and anti-SM22 antibodies.and cardiomyocytes were detected using anti-troponin I antibodies and anti-sarcomeric alpha actintin antibodies). Figures 19A and 19B show that HIP cells differentiated into endodermal islet cells (ilC) that produced C-peptide and insulin. In Figure 19A, differentiation markers were not detected in HIP cells but were present in the induced islet cells. In Figure 19B, the induced islet cells produce insulin. Immunohistochemical staining for C-peptide confirmed these results (data not shown). Figures 20A and 20B show HIP cells differentiated from the ectoderm lineage. Figure 20A shows HIP cells in vitro, and Figure 20B shows differentiated neuron cells. Immunohistochemical staining with the neuroectodermal stem cell markers Nestin and Tuj-1 confirmed these results (data not shown). Figures 21A, 21B, and 21C show that differentiated HIP cells retained the reduced MHC I and II phenotype and Cd47 overexpression. Figure 21A compares MHC-I, MHC-II, and Cd47 expression between induced mouse endothelial cells ("miECs") and B2EC- / -Ciita-A Cd47 tg miECs. Figure 21B compares MHC-I, MHC-II, and Cd47 expression between induced mouse smooth muscle cells ("míSMCs") and B2m- / -Cuta- / -Cd47 tg miSMCs. Figure 21C compares MHC-I, MHC-II, and Cd47 expression between induced mouse cardiomyocytes ("miCMs") and B2M- / -Ciita- / -Cd47 tg miCMs. Figures 22A, 22B, and 22C show that HIP-derived endothelial cells are not immunogenic. Figure 22A shows the transplantation of C56BL / 6 miECs into syngeneic and allogeneic mice. In allogeneic BALB / c recipient mice, miECs elicited a pronounced immune response, but not in syngeneic mice. This was evidenced by the strong IFN-γ and immunoglobulin ELISPO responses (FACS assay) in BALB / c recipients (Figure 22B). In Figure 22C, neither HIPs nor miECs elicited an immune response in either syngeneic or allogeneic recipients. Figures 23A, 23B, and 23C show that mouse-induced muscle cells differentiated from HIP cells are not immunogenic. Figure 23A shows the transplantation of C56BL / 6 miSMC cells into syngeneic and allogeneic mice. In allogeneic BALB / c receptor mice, miSMCs elicited a pronounced immune response, but not in syngeneic mice. This was evidenced by the strong responses in ELISPO IFN-γ and immunoglobulin assays (FACS assays) in BALB / c receptors. In Figure 23C, neither HIP cells nor miSMCs elicited an immune response in either syngeneic or allogeneic receptors. Figure 25 shows that differentiated cells (miECS, miSMC, miCM) derived from HIP cells evaded rejection by the innate immune system. An NK fusion protein assay showed that none of the three differentiated cells had increased expression of NK cell-stimulatory ligands compared to differentiated cells derived from miPSCs. Figures 26A and 26B show that the HIP-derived miECs of the invention evaded an immune reaction and achieved long-term survival in an allogeneic host. In Figure 26A, miEC grafts derived from miPSCs showed long-term survival in syngeneic recipients (C57BL / 6) but were rejected in allogeneic recipients (BALB / c). In Figure 26B, the HIP-derived miECs achieved long-term survival after transplantation in both syngeneic and allogeneic recipients. Figure 27; HIP-derived miECs organized to form vascular structures in allogeneic hosts. After transplantation into a Matrigel matrix, over six weeks, the miECs organized three-dimensionally to form vascular structures. These results were confirmed by immunofluorescence staining for luciferase and VE-oadherin; the miECs were transduced to express luciferase prior to transplantation. Survival was monitored by bioluminescence imaging, and transplanted cells were identified by immunofluorescence staining against luciferase (data not shown). Figure 28 shows that the human HiP cells showed a normal human karyotype! Figure 29 shows that human HIP cells maintained pluripotency during the engineering process. Both hiPSCs (e.g., the starting cells, before the alterations of the invention) and the HIP cells of the invention expressed the pluripotency genes (NANOG, OCT4, SOX2, DPPA4, hTERT, ZFP42, and DEMT3B; G3PDH serves as a loading control) using PCR assays. Immunofluorescent staining confirmed this finding, as the cells expressed the markers TRA-1-60, TRA-1-81, Sox2, Oct4, SSEA-4, and alkaline phosphatase (data not shown). Figures 30A and 30B show that human HIP cells transplanted into humanized allogeneic mice did not elicit an immune response. Figure 3QA shows that T cells did not respond to transplanted HIP cells, as measured by IFN-γ or IL-5 production in ELISPOTS assays. In contrast, transplanted iPSCs did. Figure 30B shows that only iPSCs elicited a strong antibody response by flow cytometry. HIP cells did not. Figures 31A, 31B, 31C, and 31D show that human HIP cells differentiated into the mesodermal lineage. Figure 31A shows the morphology of a human HIP cell. Figure 31B shows HIP-derived endothelial cells stained with CD31, VE-cadherin, and DAPI as a control. Figure 31C shows HIP-derived cardiomyocytes stained with sarcomeric alpha-actinin, troponin I, and DAPI as a control. Figure 31D shows premature vessel formation by HIP-derived endothelial cells. Beating HIP-derived cardiomyocytes were observed (data not shown). Figures 32A and 32B show that transplanted human endothelial cells derived from human HIP cells did not elicit an immune response in allogeneic humanized mice. In Figure 32A, hiECs significantly increased the T cell response in ELISPOTS assays for IFN-γ and IL-5, while hiECs derived from human HIP cells did not. Figure 32B shows the B cell response on flow cytometry. Only hiECs generated significant immunoglobulin binding, as measured by mean fluorescence intensity (MFI). Figures 33A and 33B show that transplantation of human heart myocytes derived from human HIP cells did not result in an immune response in allogeneic humanized mice. Figure 33A shows the differences in T cell responses to "wild-type" hiCMs versus CD47tg B2M- / - CUTA- / - HIP cells in IFN-γ and IL-5 ELISPOTS. Figure 33B shows the B cell response on flow cytometry. Only "wild-type" hiCMs generated a significant hiEC immunoglobulin load, as measured by mean fluorescence intensity (MFI). Figures 34A, 34B, 34C, and 34D show that the human HIP cells of the invention evaded rejection by the innate immune system. NK cells were isolated from BALB / c mice using magnetically activated cell sorting (MACS). 5X10 6 of stimulatory cells (C57BL / 6-derived iPSCs, either iEC, iSMC or iCM and B2M- / - CUTA- / - or B2M-Z- CUTA- / - CD47 tg), were incubated with 5X106 NK cells were classified by MACS on an IFN-γ Elispot plate. After 24 hours, spot frequency was determined using an Elispot reader. The three B2M-Z-CUTA- / - derivatives induced a strong NK cell response. However, the three B2M-Z-CIITA-Z-CD47 tg derivatives did not induce any NK cell response, and their spot frequency was not statistically different from the negative controls (isolated NK cells). (not incubated with a stimulating cell). Figure 34A shows endothelial cells. Figure 34B shows smooth muscle cells. Figure 34C shows cardiomyocytes. Figure 34D shows a positive control of YAC-1 mouse lymphoma. Figures 35A, 35B, and 35C show the innate immune response (or lack thereof). A mixture of 50% by weight of the derivative (5 x 10⁶ cells) and 50% of either C57BL / 6 B2m- / -Ciita- / - or B2m-A Ciita- / - Cd47 tg derivative (5 x 10⁶ cells) was prepared. 6Cells were stained with 10 pM CFSE for 10 minutes and resuspended in 500 pL of saline. The cell mixture was then injected into the NK cell-rich peritoneum of C57BL / 6 (syngenetic) mice. In this syngeneic model, all cytotoxicity is caused by NK cells. After 48 h, peritoneal cells were retrieved, sorted, and their proportion calculated. Cell weight and altered cells were identified by MHCI staining on FACS. Figure 35A shows endothelial cells. Figure 35B shows smooth muscle cells. Figure 35C shows cardiomyocytes. Figures 36A, 36B, and 36C show the genetically engineered human iPSCs verified by FACS. The absence of HLA I and HLA II was confirmed in the B2M-A CUTA- / - hiPSCs. Furthermore, the B2M- / - CUTA- / - CD47 tg showed high CD47 expression. Figure 36A shows the HLA I results. Figure 36B shows the HLA II results. Figure 36C shows the CD47 results. Figures 37A and B show that the immune phenotype was maintained after differentiation of the B2M- / -CUTA- / -CD47 tg iPSCs. When compared to the unmodified weight derivatives, FACS analysis showed that the B2M-Z-CUTA- / -CD47 tg derivatives lacked HLA I and HLA II and overexpressed CD47. Figure 37A shows endothelial cells and Figure 37B shows cardiomyocytes. Detailed description of the invention A. Introduction The invention provides Hypoimmunogenic Pluripotent (HIP) cells that evade host immune responses due to various genetic manipulations as described herein. These cells lack important immune antigens that trigger immune responses and are engineered to avoid phagocytosis. This allows for the derivation of "off-the-shelf" cell products for generating specific tissues and organs. The ability to use human autogeneic HIP cell derivatives in human patients results in significant benefits, including the ability to avoid long-term adjuvant immunosuppressive therapy and the use of medications typically seen in allogeneic transplantation. It also provides significant cost savings, as cell therapies can be used without requiring individualized treatments for each patient.Recently, it was shown that cell products generated from autologous cell sources can undergo immune rejection with few or even a single antigenic mutation. Therefore, autologous cell products are not inherently non-immunogenic. Furthermore, cell engineering and quality control are very labor-intensive and expensive, and autologous cells are not readily available for acute treatment options. Only autogenic cell products will be usable for a larger patient population if this barrier can be overcome. immunological obstacle. HIP cells will serve as a universal cell source for the generation of universally acceptable derivatives. The present invention is directed to the exploitation of fetomaternal tolerance that exists in pregnant women. Although half of a fetus's human leukocyte antigens (HLA) are inherited paternally, and the fetus expresses important mismatched HLA antigens, the maternal immune system does not recognize the fetus as an allogeneic entity and does not initiate an immune response, for example, as seen in a type of "host versus graft" immune reaction. Fetomaternal tolerance is primarily mediated by syncytiotrophoblast cells at the fetal-maternal interface. As shown in Figure 7, syncytiotrophoblast cells exhibit little or no protein from the major histocompatibility complexes I and II (MHC-I and MHC-II), as well as increased expression of CD47, known as the "don't eat me" protein, which suppresses phagocytic innate immune surveillance and the knockout of HLA-deficient cells.Surprisingly, the same tolerogenic mechanisms that prevent rejection of the fetus during pregnancy also allow the HIP cells of the invention to escape rejection and facilitate long-term survival and engraftment of these cells after allogeneic transplantation. These results are strikingly additional in that this fetomaternal tolerance can be introduced with only three genetic modifications (compared to the initial iPSCs, e.g., hiPSCs): two reductions in activity ("knockouts" as described here in more detail) and one increase in activity ("knock in" as described here). In general, other researchers in this field have attempted to suppress iPSC immunogenicity but have only had partial success; see Rong et al., Cell Stem Cell 14: 121–130 (2014) and Gornalusse et al., Nature Biotech doi: 10.1038 / nbt.3860. Therefore, the invention provides for the generation of HIP cells from pluripotent stem cells, and then their maintenance, differentiation and, ultimately, the transplantation of their derivatives into patients who need it. B. Definitions The term "pluripotent cells" refers to cells that can self-renew and proliferate while remaining in an undifferentiated state and that, under appropriate conditions, can be induced to differentiate into specialized cell types. The term "pluripotent cells," as used herein, encompasses embryonic stem cells and other stem cell types, including fetal, amniotic, or somatic stem cells. Exemplary human stem cell lines include the H9 human embryonic stem cell line. Additional exemplary stem cell lines include those available through the National Institutes of Health Human Embryonic Stem Cell Registry and the Howard Hughes Medical Institute's HUES collection (as described in Cowan, C.A. et al., New England Journal of Medicine 350:13 (2004), incorporated herein in its entirety by reference). "Pluripotent stem cells," as used herein, have the potential to differentiate into any of the three germ layers: endoderm (e.g., the joint of the stomach, gastrointestinal tract, lungs, etc.), mesoderm (e.g., muscle, bone, blood, urogenital tissue, etc.), or ectoderm (e.g., epidermal tissues and tissues of the nervous system). The term "pluripotent stem cells," as used herein, also encompasses "induced pluripotent stem cells," or "iPSCs," a type of pluripotent stem cell derived from a non-pluripotent cell. Examples of progenitor cells include somatic cells that have been reprogrammed to induce a pluripotent, undifferentiated phenotype by various means. Such "iPS" or "iPSC" cells can be created by inducing the expression of certain regulatory genes or by the exogenous application of certain proteins. Methods for the induction of iPS cells are known in the art and are described in more detail below. (See, for example, Zhou et al., Stem Cells 27 (11): 2667-74 (2009); Huangfu et al., Nature Biotechnol.26 (7): 795 (2008); Woltjen et al, Nature 458 (7239): 766-770 (2009), and Zhou et al, Cell Stem Cell 8: 381-384 (2009); each of which is incorporated herein by reference in full.) The generation of induced pluripotent stem cells (PSCs) is described below. As used herein, "hiPSC" are human induced pluripotent stem cells, and "miPSC" are mouse induced pluripotent stem cells. The "characteristics of pluripotent stem cells" refer to the characteristics of a cell that distinguish pluripotent stem cells from other cells. The ability to give rise to progeny that can differentiate, under appropriate conditions, into cell types that collectively exhibit characteristics associated with the cell lineages of the three germ layers (endoderm, mesoderm, and ectoderm) is a pluripotent stem cell characteristic. The expression or non-expression of certain combinations of molecular markers are also pluripotent stem cell characteristics. For example, human pluripotent stem cells express at least several, and in some modalities, all of the markers on the following non-exhaustive list: SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49 / 6E, ALP, Sox2, E-cadherin, UTF-1, Oct4, Rex1, and Nanog.The cell morphologies associated with pluripotent stem cells are also pluripotent characteristics of stem cells. As described in this document, cells do not need to undergo pluripotency to be reprogrammed into endodermal progenitor cells and / or hepatocytes. As used in this document, "multipotent" or "multipotent cell" refers to a cell type that can give rise to a limited number of other specific cell types. For example, induced multipotent cells are capable of forming endodermal cells. In addition, multipotent blood stem cells can differentiate into several types of blood cells, including lymphocytes, monocytes, neutrophils, etc. As used herein, the term "oligopotent" refers to the ability of an adult stem cell to differentiate into only a few different cell types. For example, lymphoid or myeloid stem cells are capable of forming cells of the lymphoid or myeloid lineages, respectively. As used herein, the term "unipotent" means the ability of a cell to form only one type of cell. For example, spermatogonial stem cells are only capable of form sperm cells. As used herein, the term "totipotent" means the capacity of a cell to form a complete organism. For example, in mammals, only the zygote and the first stage of blastomere cleavage are totipotent. As used herein, "non-pluripotent cells" refers to mammalian cells that are not pluripotent. Examples of such cells include differentiated cells as well as progenitor cells. Examples of differentiated cells include, but are not limited to, cells from selected tissues such as bone marrow, skin, skeletal muscle, adipose tissue, and peripheral blood. Exemplary cell types include, but are not limited to, fibroblasts, hepatocytes, myoblasts, neurons, osteoblasts, osteoclasts, and T cells. The starting cells used to generate induced pluripotent stem cells, endodermal progenitor cells, and hepatocytes may be non-pluripotent cells. Differentiated cells include, but are not limited to, multipotent cells, oligopotent cells, unipotent cells, progenitor cells, and terminally differentiated cells. In certain modalities, a less potent cell is considered "differentiated" in relation to a more potent cell. A "somatic cell" is a cell that forms the body of an organism. Somatic cells include cells that form the organs, skin, blood, bones, and connective tissue of an organism, but not germ cells. Cells can be from, for example, human or non-human mammals. Examples of non-human mammals include, but are not limited to, mice, rats, cats, dogs, rabbits, guinea pigs, hamsters, sheep, pigs, horses, cattle, and non-human primates. In some modalities, a cell is from an adult human or non-human mammal. In some modalities, a cell is from a neonatal human, an adult human, or a non-human mammal. As used herein, the terms "subject" or "patient" refer to any animal, such as a domesticated animal, a zoo animal, or a human being. The "subject" or "patient" may be a mammal such as a dog, cat, bird, livestock, or a human being. Specific examples of "subjects" and "patients" include, but are not limited to, individuals (particularly humans) with a disease or disorder related to the liver, heart, lung, kidney, pancreas, brain, neural tissue, blood, bones, bone marrow, and the like. Mammalian cells can be from humans or non-human mammals. Exemplary non-human mammals include, but are not limited to, mice, rats, cats, dogs, rabbits, guinea pigs, hamsters, sheep, pigs, horses, cattle, and non-human primates (e.g., chimpanzees, macaques, and apes). A "hypoimmunogenic pluripotent cell" or "HIP cell" is a pluripotent cell that retains its pluripotent characteristics but elicits a reduced immune rejection response when transferred to an allogeneic host. In preferred embodiments, HIP cells do not elicit an immune response. Therefore, "hypoimmunogenic" refers to a significantly reduced or absent immune response compared to the immune response of a normal cell. Parental (i.e., "wild-type") cells prior to immunoengineering as described herein. In many cases, HiP cells are immunologically silent yet retain pluripotent capabilities. Assays for HIP characteristics are described below. The human leukocyte antigen (HLA) complex is a genetic complex that encodes the major proteins of the major histocompatibility complex (MHC) in humans. These cell surface proteins that make up the HLA complex are responsible for regulating the immune response to antigens. In humans, there are two MHC classes, HLA-I and HLA-II. HLA-I includes three proteins, HLA-A, HLA-B, and HLA-C, which present peptides from inside the cell. Antigens presented by the HLA-I complex attract killer T cells (also known as CD8+ T cells or cytotoxic T cells). The HLA-I proteins are associated with p2-microglobulin (β2M). HLA-II includes five proteins, HLA-DP, HLA-DM, HLA-DOB, HLA-DQ, and HLA-DR, which present antigens from outside the cell to T lymphocytes. This stimulates CD4+ cells (also known as T-helper cells).It should be understood that the use of "MHC" or "HLA" is not intended to be restrictive, as it depends on whether the genes are human (HLA) or murine (MHC). Therefore, with regard to mammalian cells, these terms may be used interchangeably in this document. "Gene knockout" in this document refers to a process that renders a particular gene inactive in the host cell in which it resides, resulting in the production of no protein of interest or an inactive form of it. As those skilled in the subject will appreciate, and as will be further described below, this can be achieved in several different ways, including knocking out nucleic acid sequences of a gene, or disrupting the sequence with other sequences, altering the reading frame, or altering the reading frame of regulatory components of the nucleic acid. For example, all or part of a coding region of the gene of interest can be deleted or replaced with "nonsense" sequences, all or part of a regulatory sequence such as a promoter can be deleted or replaced, translation initiation sequences can be deleted or replaced, and so on. Genetic knock-in refers to a process that adds a genetic function to a host cell. This results in increased levels of the encoded protein. As those familiar with the subject will appreciate, this can be achieved in several ways, including adding one or more additional copies of the gene to the host cell or altering a regulatory component of the endogenous gene that increases protein expression. This can be accomplished by modifying the promoter, adding a different promoter, adding an enhancer, or modifying other gene expression sequences. The protein “p-2 microglobulin” or “(32M” or “B2M” refers to the human p2M protein that has the amino acid and nucleic acid sequences shown below; the human gene has the accession number NC_000015.10:44711487-44718159, The "CD47 protein" refers to the human CD47 protein which has the amino acid and nucleic acid sequences shown below: the human gene has the accession number NC_000016,10: 10866208-10941562. The protein "CIITa protein" refers to the human CUTA protein that has the sequences of amino acids and nucleic acids shown below; the human gene has the accession number NC_000003.12: 108043094-108094200. “Wild type” in the context of a cell means a cell found in nature. However, in the context of a pluripotent stem cell, as used herein, it also means an iPSC that may contain nucleic acid changes resulting in pluripotency but has not been subjected to the gene-editing procedures of the invention to achieve hypoimmunogenicity. "Syngenetic" refers to the genetic similarity or identity of a host organism and a cell transplant where there is immunological compatibility: for example, no immune response is generated. "Allogeneic" refers to the genetic dissimilarity of a host organism and a cell transplant where an immune response is generated. "B2MV-" indicates that a diploid cell has had the B2.M gene inactivated on both chromosomes. As described in this document, this can be done in several ways. The term "CUTA- / -" refers to a diploid cell having the CUTA gene inactivated on both chromosomes. As described in this document, this can be done in several ways. "CD47 tg" (which means "transgene") or "CD47+" refers to the fact that the host cell expresses CD47, in some cases by having at least one additional copy of the CD47 gene. An “Oct polypeptide” refers to any of the naturally occurring members of the Octamer family of transcription factors, or variants thereof that maintain transcription factor activity similar to (within at least 50%, 80%, or 90% of activity) that of the closest related naturally occurring family member, or polypeptides comprising at least the DNA-binding domain of the naturally occurring family member, and may further comprise a transcriptional activation domain. Exemplary Oct polypeptides include Oct-1, Oct-2, Oct-3 / 4, Oct-6, Oct-7, Oct-8, Oct-9, and Oct-11. Oct3 / 4 (referred to here as “Oct4”) contains the POU domain, a 150-amino-acid sequence conserved between Pit-1, Oct-1, Oct-2, and uric-86. (See Ryan, AK and Rosenfeld, MG, Genes Dev. 11: 1207–1225) (1997), incorporated in this document as a reference in its entirety).In some forms, the variants have at least 85%, 90%, or 95% amino acid sequence identity throughout their sequence compared to a naturally occurring member of the Oct polypeptide family, such as those listed above or those listed in Genban.k with accession numbers NP-002692.2 (human Oct4) or NP-038661.1 (mouse Oct4). Oct polypeptides (e.g., Oct3 / 4 or Oct4) can be derived from humans, mice, rats, cattle, pigs, or other animals. Generally, the same protein species will be used across all cell species being manipulated. The Oct polypeptide(s) can be a pluripotency factor that can help induce multipotency in non-pluripotent cells. A "K|f polypeptide" refers to any of the naturally occurring members of the Krüppel-like factor (Klfs) family, zinc finger proteins containing amino acid sequences similar to those of the Drosophila embryo pattern regulator Krüppel, or variants of naturally occurring members that maintain similar transcription factor activity (within at least 50%, 80%, or 90% activity) compared to the closest related naturally occurring family member, or polypeptides comprising at least the DNA-binding domain of the naturally occurring family member, and may further comprise a transcriptional activation domain. (See Dang, DT, Pevsner, J., and Yang, VW, Cell Bio! 32: 1103-1121 (2000), incorporated herein by reference in full.) Members of the Klf family include Klf1, Klf2, Klf3, Klf4, Klf5, Klf6, Klf7, Klf8, Klf9, Klf10, Klf1, Klf2, Klf13, Klf14, and Klf15. :Klf16 and Klf17. Klf2 and Klf4 were found to be factors capable of generating iPS cells in mice, and the related genes Klfl and Klf5 also did so, albeit with reduced efficiency. (See Nakagawa et al., Nature Biotechnology 26: 101–106 (2007), incorporated here in full by reference.) In some modalities, the variants have at least 85%, 90%, or 95% amino acid sequence identity along their entire sequence compared to a naturally occurring member of the Klf polypeptide family, such as those listed above or those listed in GenBank accession numbers CAX16088 (mouse Klf4) or CAX14962 (human Klf4). Klf polypeptides (e.g., Klf1, Klf4, and Klf5) can be derived from humans, mice, rats, cattle, pigs, or other animals. Generally, the same protein species will be used across all cell species being manipulated. The Klf polypeptide(s) can be a pluripotency factor.Expression of the Klf4 gene or polypeptide can help induce multipotency in a starting cell or a population of starting cells. A "Myc polypeptide" refers to any of the naturally occurring members of the Myc family. (See, for example, Adhikary, S. & Eilers, M., Nat. Rev. Mol. Cell Biol. 6: 635-645 (2005). Incorporated by reference herein in its entirety.) It also includes variants that maintain similar transcription factor activity compared to the closest related naturally occurring family member (i.e., within at least 50%, 80%, or 90% of activity). It further includes polypeptides comprising at least the DNA-binding domain of a naturally occurring family member and may further comprise a transcriptional activation domain. Exemplary Myc polypeptides include, for example, c-Myc, N-Myc, and L-Myc.In some forms, the variants have at least 85%, 90%, or 95% amino acid sequence identity across their entire sequence compared to a naturally occurring member of the Myc polypeptide family, such as those listed above or those listed in GenBank accession number CAA25015 (human Myc). Myc polypeptides (e.g., c-Myc) can be derived from humans, mice, rats, cattle, pigs, or other animals. Generally, the same protein species will be used across all cell species being manipulated. The Myc polypeptide(s) can be a pluripotency factor. A "Sox polypeptide" refers to any of the naturally occurring members of the SRY-related HMG-box (Sox) transcription factors, characterized by the presence of the high-mobility cluster (HMG) domain, or variants thereof that maintain similar transcription factor activity compared to the closest related natural family member (i.e., within at least 50%, 80%, or 90% of activity). It also includes polypeptides comprising at least the DNA-binding domain of the naturally occurring family member and may further comprise a transcriptional activation domain. (See, for example, Dang, DT et al., Int. J. Biochem. Cell Biol. 32: 1103-1121 (2000), incorporated herein in full by reference.) Exemplary Sox polypeptides include, for example, Sox1, Sox2, Sox3, Sox4, Sox5, Sox6, Sox7, Sox8, Sox9, Sox10, Sox11, Sox12, Sox13, Sox14, Sox15, Sox17, Sox18, Sox-21, and Sox30. Sox1 has been shown to produce iPS cells with similar efficiency to Sox2, and the Sox3, Sox15, and Sox18 genes have also been shown to generate iPS cells, although with somewhat lower efficiency than Sox2 (See Nakagawa, et al., Nature Biotechnology 26: 101-106 (2007), incorporated here in full by reference). In some forms, the variants have at least 85%, 90%, or 95% amino acid sequence identity along their entire sequence compared to a naturally occurring member of the Sox polypeptide family, such as those listed above or those listed under GenBank registration number CAA83435 (human Sox2). Sox polypeptides (e.g., Sox1, Sox2, Sox3, Sox15, or Sox18) can be derived from humans, mice, rats, cattle, pigs, or other sources.In general, the same protein species will be used with the cell species being manipulated. The Sox polypeptide(s) can be a pluripotency factor. As discussed here, SOX2 proteins find particular use in the generation of PSCs. By "differentiated hypoimmunogenic pluripotent cells" 1 In this document, "differentiated HIP cells" or "dHIP cells" refers to iPS cells that have been engineered to possess hypoimmunogenicity (e.g., by B2M and CUTA knockout and CD47 knockout) and are then differentiated into a cell type for definitive transplantation into subjects. Thus, for example, HIP cells can be differentiated into hepatocytes ("dHIP hepaphocytes"), pancreatic beta cells or islet organoids ("dHIP beta cells"), endothelial cells ("dHIP endothelial cells"), etc. The term "identity" percentage, in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that have a specific percentage of nucleotides or amino acid residues that are identical when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to skilled users) or by visual inspection. Depending on the application, the "identity" percentage may exist over a region of the sequence being compared, for example, over a functional domain, or alternatively, it may exist over the entire length of the two sequences being compared. For sequence comparison, one sequence typically acts as a reference sequence against which the test sequences are compared.When using a sequence comparison algorithm, the test and reference sequences are entered into a computer, the subsequence coordinates are designated, if necessary, and the sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the sequence identity percentage for the test sequence relative to the reference sequence, based on the designated program parameters. Optimal sequence alignment for comparison can be performed, for example, using the Smith & Waterman local homology algorithm, Adv. Ap. Mates 2: 482 (1981), the Needleman & Wiinsch homology alignment algorithm, J. Mol. Biol. 48: 443 (1970), or the Pearson & Lipman similarity search method, Proc. Nat. Acad Sci USA 85: 2444 (1988). computerized versions of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Computer Group software package, 575 Science Dr, Madison, Wisconsin), or by visual inspection (see generally Ausubel et al., infra). An example of an algorithm suitable for determining sequence identity and sequence similarity is the BLAST algorithm, described in Altschul et al., J, Mol Biol. 215: 403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.nih.nih.gov / ). Inhibitors, activators, and modulators affect the function or expression of a biologically relevant molecule. The term "modulator" includes both inhibitors and activators. They can be identified using in vitro and in vivo assays for the expression or activity of a target molecule. "Inhibitors" are agents that, for example, inhibit the expression of or bind to target molecules or proteins. They can partially or completely block stimulation or have protease-inhibitory activity. They can reduce, decrease, prevent, or delay activation, including inactivation, desensitization, or downregulation of the target protein's activity. Modulators can be antagonists of the target molecule or protein. Activators are agents that, for example, induce or activate the function or expression of a target molecule or protein. They can bind to, stimulate, enhance, open, activate, or facilitate the activity of the target molecule. Activators can be agonists of the target molecule or protein. Homologs are bioactive molecules that are similar to a reference molecule in their nucleotide, peptide, functional, or structural sequence. Homologs may include sequence derivatives that share a certain percentage of identity with the reference sequence. Thus, in one modality, homologous or derived sequences share at least 70 percent sequence identity. In a specific modality, homologous or derived sequences share at least 80 or 85 percent sequence identity. In a specific modality, homologous or derived sequences share at least 90 percent sequence identity. In a specific modality, homologous or derived sequences share at least 95 percent sequence identity. In a more specific modality, homologous or derived sequences share at least 50, 55, 60, 65, 70, 75, or 85 percent sequence identity.86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity. Homologous or derived nucleic acid sequences can also be defined by their ability to remain bound to a reference nucleic acid sequence under highly stringent hybridization conditions. Homologs that have structural or functional similarity to a reference molecule can be chemical derivatives of the reference molecule. Methods for the detection, generation, and selection of structural and functional homologs, as well as derivatives, are known in the art. Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature. The greater the desired degree of homology between the probe and the hybridizable sequence, the higher the relative temperature that can be used. As a result, it follows that the relative temperatures Higher temperatures tend to make reaction conditions more stringent, while lower temperatures are less so. For further details and an explanation of the stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers (1995), incorporated herein by reference in its entirety. The "rigor" of the hybridization reactions can be easily determined by a technician in the field, and is generally an empirical calculation that depends on the length of the probe, the washing temperature, and the salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. "Strong conditions" or "high-strength conditions," as defined in this document, can be identified by those that: (1) employ a low ionic strength and a high temperature for washing, e.g., 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50°C; (2) employ a denaturing agent, such as formamide, during hybridization, e.g., 50% (v / v) formamide with 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 Mm sodium phosphate buffer at pH 6.5 with 750 Mm sodium chloride, 75 Mm sodium citrate at 42°C; or (3) overnight hybridization in a solution employing 50% formamide. 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 Mm sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated sperm DNA from Simon (5 pL / mL), 0.1% SDS and 10% dextran sulfate at 42°C, with a 10-minute wash at 42°C in 0.2 x of SSC (sodium chloride / sodium citrate) followed by a maximum of 10 minutes of rigorous washing consisting of 0.1 x of SSC containing EDTA at 55°C. It is intended that each maximum numerical limitation given throughout this specification shall include all lower numerical limitations, as if such lower numerical limitations were expressly written in this document. Each minimum numerical limitation given throughout this specification shall include each upper numerical limitation, as if such upper numerical limitations were expressly written in this document. Each numerical range given throughout this specification shall include each narrower numerical range that falls within a wider numerical range, as if such narrower numerical ranges were expressly written herein. As used herein, the term "modification" refers to an alteration that physically differentiates the modified molecule from the parent molecule. In one embodiment, an amino acid change in a variant polypeptide of CD47, HSVtk, EC-CD, or iCasp9 prepared according to the methods described herein differentiates it from the corresponding parent molecule that has not been modified according to the methods described herein, such as wild-type proteins, naturally occurring mutant proteins, or other engineered proteins that do not include the modifications of such variant polypeptide. In another embodiment, a variant polypeptide includes one or more modifications that differentiate the function of the variant polypeptide from the unmodified polypeptide. For example, an amino acid change in a variant polypeptide affects its receptor-binding profile.In other embodiments, a variant polypeptide comprises substitution, deletion, or insertion modifications, or combinations thereof. In another embodiment, a variant polypeptide includes one or more modifications that increase its affinity for a molecule. receptor compared to the affinity of the unmodified polypeptide. In one modality, a variant polypeptide includes one or more substitutions, insertions, or deletions with respect to a corresponding native or main sequence. In certain modalities, a variant polypeptide includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31–40, 41–50, or 51 or more modifications. The term "episomal vector" in this document refers to a genetic vector that can exist and replicate autonomously in the cytoplasm of a cell; for example, one that is not integrated into the genomic DNA of the host cell. Several episomal vectors are known in the art and are described below. "Knockout" in the context of a gene means that the host cell harboring the knockout does not produce a functional protein from the gene. As described herein, knockout can take a variety of forms, from the complete or partial knockout of the coding sequence, the introduction of frameshift mutations so that a functional protein is not produced (either a truncated or nonsense sequence), the knockout or alteration of a regulatory component (e.g., a promoter) such that the gene is not transcribed, preventing translation through mRNA binding, and so on. In general, knockout occurs at the level of the genomic DNA, so that the offspring of the cells also carry the knockout permanently. "Knock-in" in the context of a gene means that the host cell harboring the knock-in has a more functional protein active within the cell. As described herein, a knock-in can be performed in a variety of ways, usually by introducing at least one copy of a transgene (tg) encoding the protein into the cell, although this can also be done by replacing regulatory components, for example, by adding a constitutive promoter to the endogenous gene. In general, knock-in technologies result in the integration of the additional copy of the transgene into the host cell. VII. Cells of invention The invention provides compositions and methodologies for generating HIP cells, starting with wild-type cells, making them pluripotent (e.g., by producing induced pluripotent stem cells, or iPSCs), and then generating HIP cells from the iPSC population. A. Methodologies for genetic alterations The invention includes methods for modifying nucleic acid sequences within cells or under cell-free conditions to generate both pluripotent cells and HIP cells. Exemplary technologies include homologous recombination, knock-in, ZFNs (zinc finger nucleases), TALEN 35 (transcription-activating nucleases), CRISPR (clustered regularly interspecied short palindromic repeats) / Cas9, and other site-specific nuclease technologies. These techniques enable the breakage of double-stranded DNA at desired locus sites. These controlled double-strand breaks promote homologous recombination at the specific locus sites. This process is It focuses on attacking specific sequences of nucleic acid molecules, such as chromosomes, with endonucleases that recognize and bind to the sequences and induce a double-strand break in the nucleic acid molecule. The double-strand break is repaired by error-prone non-homologous end joining (NHEJ) or homologous recombination (HR). As those skilled in the field will appreciate, several different techniques can be used to design the pluripotent cells of the invention, as well as to engineer iPSCs to become hypoimmunogenic as described herein. In general, these techniques can be used individually or in combination. For example, in the generation of HIP cells, CRISPR can be used to reduce the expression of the active protein B2M and / or CUTA in the genetically engineered cells, with viral techniques (e.g., lentiviruses) to block CD47 functionality. Furthermore, as those familiar with the subject will appreciate, although one approach sequentially uses a CRISPR step to remove B2M, followed by a CRISPR step to remove CUTA, with a final lentivirus step to target CD47 functionality, these genes can be manipulated in different orders using different technologies. As described in more detail below, transient expression of reprogramming genes is generally performed to generate / induce pluripotent stem cells a. CRISPR Technologies In one approach, cells are manipulated using clustered regularly interspecied short palindromic repeats / Cas ("CRISPR") technologies, as they are known in the field. CRISPR can be used to generate the initial iPSCs or to generate HIP cells from iPSCs. There are a large number of CRISPR-based techniques; see, for example, Doudna and Charpentier, Science doi: 10.1126 / science.1258096, which is incorporated here by reference. CRISPR techniques and kits are commercially available. b. TALEN Technologies In some embodiments, the HIP cells of the invention are manufactured using transcription activator-like nuclease (TALEN) methodologies. TALENs are restriction enzymes combined with a nuclease that can be engineered to bind to and cut virtually any desired DNA sequence. TALEN kits are commercially available. c. Zinc finger technologies In one approach, cells are manipulated using zinc finger nuclease technologies. Zinc finger nucleases are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleaving domain. Zinc finger domains can be engineered to target specific desired DNA sequences, allowing zinc finger nucleases to target unique sequences within complex genomes. By harnessing the endogenous DNA repair machinery, these reagents can be used to precisely alter the genomes of higher organisms, similar to those of CRISPR and TALEN. d. Virus-based technologies A wide variety of viral techniques can be used to generate the HIP cells of the invention (as well as for the original generation of iPSCs), including, but not limited to, the use of retroviral vectors, lentiviral vectors, adenovirus vectors, and Sendai viral vectors. The episomal vectors used in iPSC generation are described below. e. Downregulation of genes using interfering RNA In other cases, genes encoding proteins used in HLA molecules are downregulated by RNAi technologies. RNA interference (RNAi) is a process in which RNA molecules inhibit gene expression, often by degrading specific mRNA molecules. Two types of RNA molecules, microRNAs (miRNAs) and small interfering RNAs (pRNAs), are central to RNA interference. They bind to target mRNA molecules and either increase or decrease their activity. RNAi helps cells defend themselves against parasitic nucleic acids, such as those of viruses and transposons. RNAi also influences development. sdRNA molecules are a class of asymmetric siRNAs comprising a 19-21 base guide (antisense) strand. They contain a 5' phosphate, a 2' or 2' modified pyrimidine, and six phosphothioates at the 3' positions. They also contain a sense strand containing 3' sterol-conjugated residues, two phosphothioates at the 3' position, and 2' modified pyrimidines. Both strands contain 2' purines with continuous stretches of unmodified purines not exceeding a length of 3. sdRNA is described in U.S. Patent No. 7,796,443, incorporated herein by reference in its entirety. For all these technologies, well-established recombinant techniques are used to generate recombinant nucleic acids, as described herein. In certain modalities, the recombinant nucleic acids (whether encoding a desired polypeptide, e.g., CD47, or stop sequences) can be operationally linked to one or more regulatory nucleotide sequences in an expression construct. The regulatory nucleotide sequences will generally be appropriate for the host cell and the target organism. Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art for a variety of host cells.Typically, one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcription start and termination sequences, translation start and termination sequences, and enhancer or activator sequences. Constitutive or inducible promoters, as they are known in the field, are also considered. Promoters may be natural promoters or hybrid promoters that combine elements of more than one promoter. An expression construct may be present in a cell as an episome, such as a plasmid, or the expression construct may be inserted into a chromosome. In one specific modality, the expression vector includes a selectable marker gene to enable [specific expression]. The selection of transformed host cells. Certain modalities include an expression vector comprising a nucleotide sequence encoding a variant polypeptide operatively linked to at least one regulatory sequence. The regulatory sequence for use herein includes promoters, enhancers, and other expression control elements. In certain modalities, an expression vector is designed for the selection of the host cell to be transformed, the particular variant polypeptide to be expressed, the number of vector copies, the ability to control that copy number, or the expression of any other vector-encoded protein, such as antibiotic markers. Examples of suitable mammalian promoters include, for instance, the promoters of the following genes: the hamster ubiquitin / S27a promoter (document WO 97 / 15664), the early promoter of simian vacuolation virus 40 (SV40), the late major promoter of adenovirus, the mouse metallothyroin-1 promoter, the Rous sarcoma virus (RSV) long terminal repeat region, the mouse mammary tumor virus (MMTV) promoter, the Moloney merine leukemia virus long terminal region, and the early promoter of human cytomegalovirus (CMV). Examples of other heterotomous mammalian promoters include actin, immunoglobulin, and heat shock promoters. In additional embodiments, promoters for use in mammalian host cells can be obtained from viral genomes such as polyomavirus, fowlpox virus (UK 2.211.504 published 5 July 1989), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis virus 8, and simian virus 40 (SV40). In further embodiments, heterotomous mammalian promoters are used. Examples include the actin promoter, an immunoglobulin promoter, and heat shock promoters. The early and late promoters of SV40 are conveniently obtained as an SV40 restriction fragment that also contains the origin of viral replication of SV40. Piers et al., Nature 273: 113-120 (1978), The immediate early promoter of human cytomegalovirus is conveniently obtained as a restriction fragment Hindlll E. Greenaway, PJ et al., Gene 18: 355-360 (1982).The above references are incorporated by reference in their entirety. B. Generation of pluripotent cells. The invention provides methods for producing non-immunogenic pluripotent cells from pluripotent cells. Thus, the first step is to provide the pluripotent stem cells. The generation of mouse and human pluripotent stem cells (generally referred to as iPSCs; miPSCs for murine cells or hiPSCs for human cells) is generally well known in the field. As those familiar with the subject will appreciate, there are a variety of different methods for iPSC generation. The original induction was performed from mouse embryonic or adult fibroblasts using the viral introduction of four transcription factors, Oct3 / 4, Sox2, c-Myc, and Klf4; see Takahashi and Yamanaka Cell 126: 663-676 (2006), which is incorporated here by reference in its entirety and specifically for the techniques described therein. Since then, a series of methods; see Seki et al., World J. Stem Cells 7 (1): 116-125 (2015) for a review, and Lakshmipathy and Vermuri, editors, Methods in Molecular Biology. Pluripotent Stem Cells, Methods and Protocols, Springer 2013, both of which are expressly incorporated herein by reference in their entirety, and in particular for methods for generating hiPSCs (see, for example, Chapter 3 of the last reference). In general, iPSCs are generated by the transient expression of one or more "reprogramming factors" in the host cell, usually introduced using episomal vectors. Under these conditions, small numbers of cells are induced to become iPSCs (generally, the efficiency of this step is low, as no selection markers are used). Once the cells are "reprogrammed" and become pluripotent, they lose the episomal vector(s) and produce the factors using endogenous genes. This loss of the episomal vector(s) results in cells that are called "zero-footprint" cells. This is desirable because the fewer genetic modifications (particularly in the host cell's genome), the better. Therefore, it is preferable that the resulting iPSCs have no permanent genetic modifications. As is also appreciated by experts in the field, the number of reprogramming factors used or that can be used can vary. Typically, when fewer reprogramming factors are used, the efficiency of transforming cells into a pluripotent state decreases, as does the degree of pluripotency itself. For example, a lower quantity of reprogramming factors may result in cells that are not fully pluripotent, but rather capable of differentiating into fewer cell types. In some modalities, a single reprogramming factor, OCT4, is used. In other modalities, two reprogramming factors, OCT4 and KLF4, are used. In other modalities, three reprogramming factors, OCT4, KLF4, and SOX2, are used. In other modalities, four reprogramming factors, OCT4, KLF4, SOX2, and c-Myc, are used. In other modalities, 5, 6, or 7 reprogramming factors may be used, selected from SOKMNLT, SOX2, OCT4 (POU5F1), KLF4, MYC, NANOG, LIN28, and SV40L T antigen. In general, these reprogramming factor genes are provided in episomal vectors, such as those known in the art and commercially available. For example, ThermoFisher / Invitrogen sells a Sendai virus reprogramming kit to generate zero-fingerprint hiPSCs; see catalog number A34546. ThermoFisher also sells EBNA-based systems; see catalog number A14703. In addition, there are a number of commercially available hiPSC lines; see, for example, the Gibco® Episomal hiPSC line, K18945, which is a zero-footprint, viral-impact human iPSC cell line (see also Burridge et al., 2011, supra). In general, as is known in the technique, iPSCs are made from non-pluripotent cells, such as CD34+ umbilical cord blood cells, fibroblasts, etc., transiently expressing reprogramming factors as described in this document. For example, successful iPSCs were also generated using only Oct3 / 4, Sox2, and Kif4, while They omitted the C-Myc, although with reduced reprogramming efficiency. In general, iPSCs are characterized by the expression of certain factors including KLF4, Nanog, OCT4, SOX2, ESRRB, TBX3, c-Myc, and TCL1. The novel or augmented expression of these factors for the purposes of the invention may be achieved through the induction or modulation of an endogenous locus or through the expression of a transgene. For example, murine iPSCs can be generated using the methods of Diecke et al., Sel Rep. 2016 Jan 28; 5: 8081 (doi: 10.1038 / srep08081), which are incorporated here by reference in full and specifically for the methods and reagents for generating the iPSCs. See also, for example, Burridge et al., PLoS One, 2011 6(4): 18293, incorporated here by reference in full and specifically for the methods described therein. In some cases, the pluripotency of cells is measured or confirmed as described in this document, for example, by analyzing reprogramming factors as generally shown in Figure 17 or by performing differentiation reactions as described in this document and in the Examples. C. Generation of hypoimmunogenic pluripotent cells The present invention relates to the generation, manipulation, growth, and transplantation of hypoimmunogenic cells in a patient as defined herein. The generation of HIP cells from pluripotent stem cells is achieved with only three genetic modifications, resulting in minimal disruption of cellular activity while conferring immunosilencing to the cells. As discussed in this document, one approach involves reducing or knocking out the activity of MHC I and II proteins (HLA I and II in human cells). This can be achieved by altering the genes that encode their components. In one approach, the coding region or regulatory sequences of the gene are cleaved using CRISPR. In another approach, gene translation is reduced using RNA interference technologies. The third approach involves altering a gene that regulates susceptibility to macrophage phagocytosis, such as CD47, and this is typically a gene knockout using viral technologies. In some cases, when CRISPR is used for genetic modifications, hiPSC cells containing a Cas9 construct can be used, allowing high-efficiency editing of the cell line; see, for example, the human episomal Cas9 iPSC cell line, A33124, from Life Technologies. 1. Reduction of HLA-I The HIP cells of the invention include a reduction in MHC I function (HLA I when the cells are derived from human cells). As those technically familiar with the subject will appreciate, reduction of function can be achieved in several ways, including knocking out nucleic acid sequences of a gene, interrupting the sequence with other sequences, or altering the regulatory components of the nucleic acid structure. For example, all or part of a coding region of the gene of interest can be deleted or replaced with "nonsense" sequences, frameshift mutations can be introduced, or all or part of A regulatory sequence such as a promoter can be removed or replaced, translation initiation sequences can be removed or replaced, etc. As those skilled in the art will appreciate, successful reduction of MHC I function (HLAI when cells are derived from human cells) in pyuripotent cells can be measured using techniques known in the art and as described below: e.g., FACS techniques using labeled antibodies that bind to the HLA complex; e.g., using commercially available HLA-A, B, C antibodies that bind to the alpha chain of major human class I HLA histocompatibility antigens. a. Alteration of B2m In one embodiment, the reduction of HLA-I activity is achieved by disrupting the expression of the p-2 microglobulin gene in the pluripotent stem cell, the human sequence of which is described herein. This alteration is generally referred to herein as a gene knockout, and in the HIP cells of the invention, it is performed on both alleles in the host cell. The techniques for performing both disruptions are generally the same. One particularly useful approach uses CRISPR technology to alter the gene. In some cases, CRISPR technology is used to introduce small deletions / insertions into the gene's coding region, so that a functional protein is not produced. This is often the result of frameshift mutations that generate stop codons, i.e., truncated codons, resulting in non-functional proteins. Therefore, a useful technique is to use CRISPR sequences designed to target the coding sequence of the B2M gene in mice or the B2M gene in humans. After gene editing, transfected iPSC cultures are dissociated into individual cells. The individual cells are expanded into full-size colonies and analyzed for CRISPR editing by detecting the presence of an aberrant sequence at the CRISPR cleavage site. Clones with deletions in both alleles are selected. Such clones did not express B2M / B2M, as demonstrated by PCR, and did not express HLA-I, as demonstrated by FACS analysis (see Examples 1 and 6, for example). Assays to test whether the B2M gene has been inactivated are known and described herein. In one modality, the assay is a Western blot of cell lysates tested with antibodies against the 82M protein. In another modality, reverse transcriptase polymerase chain reaction (rt-PCR) confirms the presence of the inactivating alteration. In addition, cells can be analyzed to confirm that the HLA I complex is not expressed on the cell surface. This can be assessed using FACS assays with antibodies against one or more HLA cell surface components, as discussed previously. 35 It is worth mentioning that others have had poor results when trying to silence the B2M genes in both alleles. See, for example, Gornalusse et al., Nature Biotech. Doi' 10.1038 / nbt.3860). 2. Reduction of HLA-II In addition to a reduction in HLA I, the HIP cells of the invention also lack function MHC II (HLA II when cells are derived from human cells). As those familiar with the subject will appreciate, reduction of function can be achieved in several ways, including knocking nucleic acid sequences in a gene, adding nucleic acid sequences to a gene, disrupting the reading frame, interrupting the sequence with other sequences, or altering the regulatory components of the nucleic acid. In one modality, all or part of a coding region of the gene of interest can be deleted or replaced with "nonsense" sequences. In another modality, regulatory sequences such as a promoter, translation initiation sequences, etc., can be deleted or replaced. Successful reduction of MHC II function (HLA II when cells are derived from human cells) in pluripotent cells or their derivatives can be measured using techniques known in the field, such as Western blot using antibodies against the protein, FACS techniques, rt-PCR techniques, etc. a. Cutaneous alteration In one embodiment, the reduction of HLA-II activity is achieved by disrupting the expression of the CUTA gene in the pluripotent stem cell, the human sequence of which is shown herein. This alteration is generally referred to herein as a "knock-off" gene, and in the HIP cells of the invention, it is carried out on both alleles in the host cell. The assays to test whether the CUTA gene has been inactivated are known and described herein. In one modality, the assay is a Western blot of cells tested with antibodies against the CUTA protein. In another modality, reverse transcription polymerase chain reaction (rt-PCR) confirms the presence of the inactivating alteration. In addition, cells can be analyzed to confirm that the HLA II complex is not expressed on the cell surface. Again, this assay is performed as is known in the field (see Figure 21, for example) and is generally carried out using Western blots or FACS assays based on commercial antibodies that bind to human HLA-DR class II, DP, and most DQ antigens, as described below. One particularly useful modality uses CRISPR technology to alter the CUTA gene. CRISPRs were engineered to target the coding sequence of the Ciita gene in mice or the CUTA gene in humans, a transcription factor essential for all MHC II molecules. After gene editing, transfected iPSC cultures were dissociated into single cells. These were expanded into full-size colonies and tested for successful CRISPR editing by detecting the presence of an aberrant sequence from the CRISPR cleavage site. Clones with deletions did not express Ciita / CIITA as determined by PCR and did not express MHC II / HLA-H as determined by FACS analysis. 3. Reduction of phagocytosis In addition to the reduction of HLA I and II (or MHC I and II), generally using B2M knockouts and CUTA, the HIP cells of the invention have reduced susceptibility to macrophage phagocytosis and NK cell destruction. The resulting HIP cells "escape" the macrophage immune and innate pathways due to one or more CD47 transgenes. a. Increased CD47 In some modalities, the reduction of macrophage phagocytosis and the susceptibility to NK cell destruction results from the increase of CD47 on the surface of the HIP cell. This is achieved in various ways, as those familiar with "knock-in" or transgenic technologies will appreciate. In some cases, the increased expression of CD47 results from one or more CD47 transgenes. Therefore, in some embodiments, one or more copies of a CD47 gene are added to HIP cells under the control of an inducible or constitutive promoter, the latter being preferred. In some embodiments, a Gentiviral construct is used as described herein or as known in the field. CD47 genes can be integrated into the host cell genome under the control of a suitable promoter as known in the field. HIP cell lines were generated from B2M- / - CUTA- / - iPSCs. Cells containing lentivirus vectors expressing CD47 were selected using a blasticidin marker. The CD47 gene sequence was synthesized and the AON was cloned into the blasticidin-resistant Lentivirus pLent16 / V5 plasmid (Thermo Fisher Scientific, Waltham, MA). In some cases, CD47 gene expression can be increased by altering the regulatory sequences of the endogenous CD47 gene, for example, by replacing the endogenous promoter with a constitutive or a different inducible promoter. This can generally be done using established techniques such as CRISPR. Once altered, the presence of sufficient CD47 expression can be analyzed using known techniques such as those described in the Examples, such as Western blots, ELISA assays, or FACS assays using anti-CD47 antibodies. In general, "sufficiency" in this context means an increase in CD47 expression on the surface of the HIP cell that silences NK cell death. Natural expression levels on the cells are too low to protect them from NK cell lysis once their MHC I is removed. 4. Suicide genes In some embodiments, the invention provides hypoimmunogenic pluripotent cells comprising a "suicide gene" or "suicide switch." These are incorporated to function as a "safety switch" that can cause the death of the hypoimmunogenic pluripotent cells should they grow and divide in an undesirable manner. The "suicide gene" ablation approach includes a suicide gene in a gene transfer vector that encodes a protein that induces cell death only when activated by a specific compound. A suicide gene can encode an enzyme that selectively converts a toxic non-cellular compound into highly toxic metabolites. The result is to specifically eliminate cells that express the enzyme. In some modalities, the suicide gene is the herpesvirus thymidine kinase (HSV-tk) gene and the trigger is ganciclovir. In other modalities, the suicide gene is the Escherichia coli cytosine deaminase (EC-CD) gene and the trigger is 5-fluorocytosine (5-FC) (Barese et al. Mol. Therap. 20 (10): 1932-1943 (2012), Xu et al., Cell Res. 8: 73-8 (1998), both incorporated here in full by reference. In other embodiments, the suicide gene is an inducible caspase protein. An inducible caspase protein comprises at least a portion of a caspase protein capable of inducing apoptosis. In one embodiment, the caspase protein portion is exemplified in SEQ ID NO: 6. In preferred embodiments, the inducible caspase protein is iCasp9. It comprises the sequence of the human FK506-binding protein, FKBP12, with an F36V mutation, connected via a series of amino acids to the gene encoding caspase 9. The FKBP12-F36V sequence binds with high affinity to a small-molecule dilution agent, AP1903. Therefore, the suicide function of iCasp9 in the present invention is triggered by the administration of a chemical dilution inducer (CID). In some embodiments, the CID is the small-molecule drug AP1903. Dimerization causes rapid induction of apoptosis. (See document WQ2011146862; Stasi et al., N. Engl. J. Med 365; 18 (2011); Tey et al., Biol.Blood Marrow Transplant. 13: 913-924 (2007), each of which is incorporated herein by reference in its entirety). 5. HIP phenotype assays and pluripotency retention. Once HIP cells have been generated, their hypoimmunogenicity and / or retention of pluripotency can be analyzed as generally described in this document and in the examples, For example, hypoimmunogenicity is assessed using various techniques, as illustrated in Figures 13 and 15. These techniques include transplantation into allogeneic hosts and monitoring the growth of HIP cells (e.g., teratomas) that evade the host immune system. HIP derivatives are transduced to express luciferase and can then be tracked using bioluminescence imaging. Similarly, the host animal's T cell and / or B cell response to HIP cells is analyzed to confirm that the HIP cells do not elicit an immune reaction in the host animal. T cell function is assessed using ELISpot, ELISA, FACS, POR, or mass cytometry (CYTOF). The B cell or antibody response is assessed using FACS or Luminex.Additionally or alternatively, cells can be tested to determine their ability to evade innate immune responses, for example, the destruction of NK cells, as generally shown in Figure 14. The litholytic activity of NK cells is assessed in vitro or in vivo (as shown in Figure 15). Similarly, the retention of pluripotency is tested in various ways. In one modality, pluripotency is assessed by the expression of certain pluripotency-specific factors as generally described in this document and shown in Figure 29. Additionally or alternatively, HIP cells differentiate into one or more cell types as an indication of pluripotency. D. Preferred embodiments of the invention This document provides hypoimmunogenic pluripotent stem cells ("HIP cells") that exhibit pluripotency but do not elicit a host immune response when transplanted into an allogeneic host such as a human patient, either as HIP cells or as differentiated products of HIP cells. In one modality, human pluripotent stem cells (hiPSCs) are made hypoimmunogenic by a) cleaving the B2M gene at each allele (e.g., B2M- / -), b) cleaving the CUTA gene at each allele (e.g., CUTA- / -), and c) overexpressing the CD47 gene (CD47+, e.g., by introducing one or more additional copies of the CD47 gene or by activating the genomic gene). This makes the B2M- / - CUTA- / - CD47t hiPSC population non-immunogenic. In a preferred modality, the cells are non-immunogenic. In another modality, the HIP cells are made non-immunogenic B2MCIITA, as described above, but are further modified by including an inducible suicide gene that is induced to kill the cells in vivo when needed. E. Maintenance of HIP cells Once generated, HIP cells can be maintained in an undifferentiated state, as is known to maintain iPSCs. For example, HIP cells are cultured in Matrigel using culture media that prevent differentiation and maintain pluripotency. F. Differentiation of HIP cells The invention provides HIP cells that differentiate into various cell types for subsequent transplantation into subjects. As those skilled in the art will appreciate, the differentiation methods depend on the desired cell type and utilize established techniques. The cells are differentiated in suspension and then placed in a gel matrix, such as Matrigel, gelatin, or fibrin / thrombin, to facilitate cell survival. Differentiation is then assessed using standard techniques, typically by evaluating the presence of specific cell markers. In some modalities, HIP cells are differentiated into hepatocytes to treat loss of hepatocyte function or liver cirrhosis. Several techniques can be used to differentiate HIP cells into hepatocytes; see, for example, Pettinato et al., doi: 10.1038 / spre32888, Snykers et al., Methods Mol Biol 698; 305-314 (2011), Si-Tayeb et al., Hepatology 51; 297-305 (2010), and Asgari et al., Stem Cell Rev 493-504 (2013), all of which are expressly incorporated herein by reference in their entirety and specifically for the methodologies and reagents for differentiation. Differentiation is analyzed as is known in the field, generally by evaluating the presence of associated hepatocytes and / or specific markers, which include, among others, albumin, alpha fetoprotein, and fibrinogen.Differentiation can also be measured functionally, such as ammonia metabolism, LDL storage and uptake, ICG uptake and release, and glycogen storage. In some modalities, HIP cells differentiate into beta-like cells or organoids of islets for transplantation to treat type I diabetes mellitus (T1DM). Cell systems are a promising way to address T1DM; see, for example, Ellis et al., doi:10.1038 / nrgastro.2017.93, incorporated here by reference. Additionally, Pagliuca et al. report on the successful differentiation of i3 cells from hiPSCs (see doi:10.106 / j.cell.2014.09.040, which is incorporated here by reference in its entirety and in particular for the methods and reagents described therein for the large-scale production of functional human (3) cells from human pluripotent stem cells). Furthermore, Vegas et al. show the production of human (3) cells from human pluripotent stem cells followed by encapsulation to avoid immune rejection by the host (doi:10.1038 / nm.4030).incorporated herein by reference in its entirety and in particular for the methods and reagents described herein for the large-scale production of cells (3 functional human cells from human pluripotent stem cells). Differentiation is performed as is known in the field, generally by assessing the presence of associated or specific p-cell markers, including, among others, insulin. Differentiation can also be measured functionally, such as by measuring glucose metabolism (see Murare et al., doi: 10.1016 / j.cels.2016.09.002, which is incorporated here in its entirety as a reference, and specifically for the biomarkers described therein). Once dHIP beta cells are generated, they can be transplanted (either as a cell suspension or within a gel matrix as discussed here) into the portal vein / liver, omentum, gastrointestinal mucosa, bone marrow, muscle, or subcutaneous pockets. In some modalities, HIP cells are differentiated into retinal pigment epithelium (RPE) to treat vision-threatening eye diseases. Human pluripotent stem cells have been differentiated into RPE cells using the techniques described in Kamao et al., Stem Cell Reports 2014;2:205-18, incorporated here in full by reference, and in particular for the methods and reagents described here for differentiation techniques and reagents; see also Mandai et al., doi: 10.1056 / NEJMoa1608368, also incorporated in full for techniques for generating RPE cell sheets and transplantation into patients. Differentiation can be attempted as is known in the field, generally by evaluating the presence of RPE-associated and / or specific markers or by measuring them functionally. See, for example, Kamao et al., Doi: 10.1016 / j.stemcr.2013.12.007, incorporated here as a reference in its entirety and specifically for the markers described in the first paragraph of the results section. In some modalities, HIP cells are differentiated into cardiomyocytes to treat cardiovascular diseases. The techniques for differentiating hiPSCs into cardiomyocytes are well-known and are discussed in the examples. Differentiation can be assayed as known in the technique, generally by evaluating the presence of cardiomyocyte markers or specific markers, or by measuring them functionally; see, for example, Loh et al., doi: 10.1016 / j.cell.2016,06.001, incorporated here in its entirety as a reference, specifically for methods of differentiating stem cells, including cardiomyocytes. In some forms, HIP cells differentiate into colony-forming cells Endothelial cells (ECFCs) are used to form new blood vessels to treat peripheral arterial disease. Techniques for differentiating endothelial cells are known. See, for example, Prasain et al., Doi: 10.1038 / nbt.3048. This reference is incorporated in its entirety and specifically for methods and reagents for the generation of endothelial cells from human plunpotent stem cells, and also for transplantation techniques. Differentiation can be assayed as known in the field, generally by evaluating the presence of endothelial cell-associated markers or specific markers, or by measuring them functionally. In some modalities, HIP cells are differentiated into thyroid progenitor cells and thyroid follicular organoids that can secrete thyroid hormones to treat autoimmune thyroiditis. Techniques for differentiating thyroid cells are well known in the field. See, for example, Kurmann et al., Doi: 10.106 / j.stem.2015.09.004, which is expressly incorporated here as a reference in its entirety and specifically for methods and reagents for generating thyroid cells from human pluripotent stem cells, and also for transplantation techniques. Differentiation can be assayed as known in the field, generally by evaluating the presence of specific or thyroid cell-associated markers or by functional measurement. G. Transplantation of differentiated HIP cells As those skilled in the art will appreciate, differentiated HIP derivatives are transplanted using techniques known in the field that depend on both the cell type and the final use of these cells. In general, the dHIP cells of the invention are transplanted intravenously or by injection into specific sites in the patient. When transplanted into specific sites, the cells can be suspended in a gei matrix to prevent dispersion while they settle. To help the invention described herein be more fully understood, the following examples are provided. It should be understood that these examples are for illustrative purposes only and should not be interpreted as limiting the invention in any way. HIV. EXAMPLES A. General Techniques 1. Generation of marine iPSCs These cells were generated using the methods of Diecke et ai, Sci Rep. 2015, January 28; 5: 8081 (doi. 10.1038 / srep08081), incorporated in full herein and specifically for the methods and reagents for the generation of miPSCs. Mouse tail fibroblasts were dissociated and isolated using type IV collagenase (Life Technologies, Grand Island, NY, USA) and maintained in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS). L-glutamine, 4.5 g / L glucose, 100 U / mL penicillin, and 100 pg / mL streptomycin were incubated at 37°C, 20% O₂, and 6% CO₂ in a humidified incubator. They were then reprogrammed 1*10 e murine fibroblasts using a novel codon-optimized mini-intromco piasmid (co-MIP) (10-12 pm of DNA) expressing the four factors of Oct4, KLF4, Sox2, and c-Myc reprogramming were performed using the Neon transfection system. After transfection, fibroblasts were placed on a feeder layer of MEF and maintained in fibroblast medium with the addition of sodium butyrate (0.2 mM) and 50 pg / mL ascorbic acid. When ESC-like colonies appeared, the media were changed to murine iPSC medium containing DMEM, 20% FBS, L-glutamine, non-essential amino acids (NEAAs), p-mercaptoethanol, and 10 ng / mL leukemia inhibitory factor (LIF). After two passages, marine iPSCs were transferred to 0.2% gelatin-coated plates and further expanded. With each passage, iPSCs were sorted for the murine pluripotency marker SSEA-1 using magnetically activated cell sorting (MACS). 2. Generation of human iPSCs The generation of hiPSCs was performed as generally described in Burridge et al., PLoS One, 2011 6 (4). 18293, which is incorporated herein by reference in its entirety and specifically for the methods described in this document. The human episomal iPSC line Gíbco® (catalog number A33124, Thermo Fisher Scientific) was derived from CD34+ cord blood using a triple plasmid, seven-factor (SOKMNLT, SOX2, OCT4 (POU5F1), KLF4, MYC, NANOG, LIN28, and SV40L T antigen) EBNA-based episomal system. This iPSC line is considered zero-footprint because there was no integration into the genome following the reprogramming event. It has been shown to be free of all reprogramming genes. The Gibco® episomal human hiPSC line has a normal karyotype and endogenous expression of pluripotent markers such as Oct4, Sox2, and Nanog (as shown by RT-PCR) and Oct4, SSEA4, TRA-1-60, and TRA-1-81 (as shown by ICC). Whole-genome expression and epigenetic profiling analyses demonstrated that this episomal hiPSC line is molecularly indistinguishable from human embryonic stem cell lines (Burridge et al., 2011). In directed differentiation and teratoma analyses, these hiPSCs retained their differentiation potential for the ectodermal, endodermal, and mesodermal lineages (Burridge et al., 2011). Furthermore, vascular, hematopoietic, neural, and cardiac lineages were obtained with robust efficiencies (Burridge et al., 2011). 3. FACS analysis of surface molecules a. Detection of human surface molecules ROW I Human iPSCs, iCMs, and iECs were seeded in 6-well plates and stimulated with 100 ng / mL human IFN-γ (Peprotech, Rocket Hill, NJ). Cells were harvested and labeled with either HLA-A, B, C conjugated to APC (clone G46_2.6, cat. no. 562006, BD BioSciences, San Jose, CA) or isotype control IgG1 antibody 35 conjugated to APC (clone MOPC-21, cat. no. 555751, BD BioSciences). The HLA-A, B, C antibody specifically binds to the alpha chain of major human class I HLA histocompatibility antigens. Data analysis was performed by flow cytometry (BD BioSciences), and results were expressed as a change in isotype control. 4. Detection of human surface molecules HLAII Human iPSCs, iCMs, and iECs were seeded in 6-well plates and stimulated with 100 ng / mL human IFN-γ (Peprotech, Rocket Hill, NJ). Cells were harvested and labeled with HLA-DR, DP, DP, DQ (clone Tu3a, ca. no. 563591, BD BioSciences, San Jose, CA) labeled with Alexa-fluor647 or with the isotype control antibody lgG2a labeled with Alexa-fluor647 (clone G155-178, ca. no. 557715, BD BioSciences). The HLA-DR, DP, DQ antibody specifically binds to human HLA class II HLA-DR, DP, and most DQ antigens. Data analysis was performed using flow cytometry (BD Bioscience) and results were expressed as a change in isotype control. 5. Detection of human surface molecules CD47 Human iPSCs, iCMs, and iECs were seeded in 6-well plates and stimulated with 100 ng / mL of human IFNγ (Peprotech, Rocket Hill, NJ). Cells were harvested and labeled with either PerCP-Cy5-conjugated CD47 (clone B6H12, cat. no. 561261, BD BioSciences, San Jose, CA) or PerCP-Cy5-conjugated IgG1 isotype control antibody (clone MOPC-21, cat. no. 550795, BD BioSciences). The B6H12 CD47 monoclonal antibody specifically binds to CD47, a 42–52 kDa N-linked glycan protein. Data analysis was performed by flow cytometry (BD Bioscience), and results were expressed as a change in isotype control. 6. Detection of murine surface molecule MHC I For the detection of MHC I surface molecules in miPSCs, miECs, miSMCs, and miCMs, cells were placed in 6-well gelatin-coated plates and stimulated with 100 ng / mL of mouse IFNγ (Peprotech, Rocket Hill, NJ). After collection, cells were labeled with either the PerCP-eFlour710-labeled MHC I antibody (clone AF6-88.5.5.3, catalog no. 46-5958-82, eBioscience, Santa Clara, CA) or the PerCP-eFlour710-labeled lgG2b isotype control antibody (clone eB149 / 10H5, cat. no. 46-4031-80, eBioscience). The MHCi antibody reacts with the H-2Kb MHC class I alloantigen. Data analysis was performed using flow cytometry (BD Bioscience) and results were expressed as a change in isotype control. 30 7. Detection of mouse MHC II surface molecules For the detection of MHC II surface molecules on miPSCs, miECs, miSMCs, and miCMs, cells were placed in 6-well gelatin-coated plates and stimulated with 100 ng / mL mouse TNFα (Peprotech, Rocket Hill, NJ). After collection, cells were labeled with the PerCP-eFlour710-labeled MHC II antibody (clone M5 / 114.15.2, catalog number 46-5321-82, eBioscience, Santa Clara, CA) or the PerCP-eFlour710 labeled lgG2a / K isotype control antibody (clone eBM2a, cat. no. 46-4724-80, eBioscience). The MHC II antibody reacts with the mouse major histocompatibility complex class II on glycoproteins encoded in both lA and lE subregions. Data analysis was performed by flow cytometry (BD Bioscience) and the results were They expressed it as a change in the opposite of isotypes. 8. Detection of mouse Cd47 surface molecules For the detection of Cd47 surface molecules on miPSCs, miECs, miSMCs, and miCMs, cells were placed in 6-well gelatin-coated plates and stimulated with 100 ng / mL mouse TNFκB (Peprotech, Rocket Hill, NJ). After collection, cells were labeled with either the Alexa Fluor 647-labeled Cd47 antibody (clone miap301, Cat. No. 563584, BD BioSciences, San Jose, CA) or the Alexa Fluor 647-labeled IgG2a / K isotype control antibody (clone R35-95, Cat. No. 557690, BD BioSciences). The Cd47 antibody specifically binds to the extracellular domain of CD47, also known as the integrin-associated protein (IAP). Data analysis was performed using flow cytometry (BD Bioscience) and results were expressed as a change in isotype control. 9. Determination of the morphology: of mouse cells in vivo after allogeneic transplantation. Allogeneic mice were placed in an induction chamber and anesthesia was induced with 2% isoflurane (Isothesia, Butler Schein). One million cells, either miPSC-derived cardiomyocytes (iniCM), miPSC-derived smooth muscle cells (miSMC), or miPSC-derived endothelial cells (miEC), were mixed in 250 µL of 0.9% saline with 250 pL of BD Matrigel High Concentration (1:1; BD Biosciences) and injected subcutaneously into the lower back of mice using a 23-G syringe. Matrigel plugs were explanted 1, 2, 3, 4, 5, 6, 8, 10, and 12 weeks after implantation and fixed with 4% paraformaldehyde and 1% glutenaldehyde for 24 h, followed by dehydration and paraffin embedding. 5 µm thick sections were cut and stained with hematoxylin and eosin (H&E). 10 Determination of human cell morphology in vivo after allogeneic transplantation Humanized NSG-SGM3 mice were placed in an induction chamber and anesthesia was induced with 2% isoflurane (Isothesia, Butler Schein). One million cells, either hiPSC-derived cardiomyocytes (hiCM) or hiPSC-derived endothelial cells (hiEC), were mixed in 250 pIL of 0.9% saline containing ZVAD (100 mM benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fluoromethyl ketone, Calbiochem), Bcl-XL BH4 (cell-permeable TAT peptide, 50 nM, Calbiochem), cyclosporine A (200 nM, Sigma), IGF-1 (100 ng / mL, Peprotech), and pinacidil (50 mM, Sigma) with 250 µL of BD Matrigel High Concentration (1:1; BD Biosciences) and injected subcutaneously into the lower back of mice using a 23-G syringe. The Matrigel plugs were explanted 2, 4, 6, 8, 10 and 12 weeks after implantation and were fixed with 4% paraformaldehyde and 1% glutenaldehyde for 24 h, followed by dehydration and paraffin embedding.Sections 5 pm thick were cut and stained with hematoxylin and eosin (HE). B. Example 1. Generation of knowledge out of B-2 Microgiobuin from pluripotent cells in a mouse model Generation of induced pluripotent cells: Hypoimmune pluripotent cells were generated in a mouse modality. Human hypoimmune pluripotent cells are another modality that are generated using the strategies described in this document; Mouse induced pluripotent stem cells (rniPSCs) were generated from C57BL / 6 fibroblasts. Mitomycin-inhibited mouse CF1 embryonic fibroblasts (MEF, Applied Stemcell, CA) were thawed and maintained in DMEM + GlutaMax 31966 (Gibco, Grand Island, NY) with 10% heat-inactivated fetal calf sera (FCS hi), 1% MEM-NEAA, and 1% Pen Strep (Thermo Fisher Scientific-Gibco, Waitham, MA). After the MEF feeder cells formed a 100% confluent monolayer, miPSCs were cultured in MEF on KO DMEM 10829 with 15% KO replacement serum, 1% MEM-NEAA, 1% Pen Strep (Thermo Fisher-Gibco), 1x beta-mercaptoethanol, and 100 LIF units (Millipore, Billerica, MA). Cells were maintained in 10 cm dishes, the medium was changed daily, and cells were passed every 2–3 days using 0.05% fipsin-EDTA (Thermo Fisher-Gibco). miPSCs were cultured on gelatin (Millipore) without feeders using standard media.Cell cultures were regularly examined for mycoplasma infections using the MycoAlert kit (Lonza, Cologne, Germany). Mice: BALB / c (BALB / cAnNCrl, H2d), C57BL / 6 (C57BL / 6J, B6, H2b), BALB / c nude (BALB / c NU / NU. CAnN.CgFoxn1 <nu>Mice (Crl, H2d) and Scid beige (CBySmn.CB17-Prkdcscid / J) (all 6–12 weeks old) were used as recipients for different assays. Mice were purchased from Charles River Laboratories (Sulzfeld, Germany) and received humane treatment in accordance with the Guidelines for the Practice of Laboratory Animals. Animal experiments were approved by the Hamburg Consumer Protection Agency (Amt für Gesundheit une Verbraucherschutz) and were conducted in accordance with local and EU guidelines. Confirmation of pluripotency: Pluripotency was demonstrated by rtPCR. RNA was extracted using the PureLink RNA Mini Kit (Thermo Fisher Scientific). A DNase I step was included to remove contaminating genomic DNA. cDNA was generated using the Applied Biosystems® High-Capacity cDNA Reverse Transcription Kit. Reverse transcriptase-free (RT-free) controls were also generated from all RNA samples. Gene-specific primers were used to amplify target sequences using the AmpliTaq Gold 360 master mix (Thermo Fisher Scientific-Applied Biosystems, Waltham, MA). PCR reactions were visualized on 2% agarose gels. A set of positive control primers amplifying a constitutively expressed maintenance gene (Actb) encoding a cell cytoskeleton protein was included. Results are shown in Figure 2.The pluripotency markers Nanog, Oct4, Sox2, Esrrb, Tbx3, Tcl1 were detected by rtPCR of miPSC cells but not parental fibroblasts. Pluripotency was also assessed by immunofluorescence. miPSCs were seeded in 24-well plates and processed for RT-PCR and immunocytochemical (ICC) analysis 48 h post-seeding. For ICC, cells were fixed, permeabilized, and blocked using the Image-iT Fixation / Permeabilization Kit (Thermo Fisher Scientific, Waitham, MA). Cells were stained overnight at 4°C with primary antibodies for Sox2 and Oct4. After several washes, the cells were The cells were incubated with AlexaFluor488 secondary antibody and NucBIue ReadyProbes fixed-cell reagent (Thermo Fisher Scientific). The stained cells were imaged using a fluorescence microscope and were positive for Sox2 and Oct4. Data not shown. Figure 3 shows further confirmation of pluripotency by a functional assay. 2x10® miPSC cells were injected into the thigh muscle of C57BL / 6 receptor (syngeneic), BALB / c (allogeneic), naked BALB / c (allogeneic but T-cell deficient), and scid beige (immunodeficient) mice. Teratomas formed in all mice except the immunocompetent allogeneic BALB / c mice. B-2 Microglobulin Knockout: CRISPR technology was used to eliminate the B2m gene. To target the coding sequence of the mouse β2-microglobulin (β2m) gene, the CRISPR sequence 5'-TTCGGCTTCCCATTCTCCGG(TGG)-3' was aligned and ligated into all-in-one (AIO) vectors containing the Cas9 expression cassette according to the kit instructions (GeneArt CRISPR Nuclease Vector Kit, Thermo Fischer Scientific, Waltham, MA). (Other CRISPR sequences that worked, but were less effective, were 5'-GTATACTCACGCCACCCAC(CGG)-3' and 5'-GGCGTATGTATCAGTCTCAG(TGG)-3'.) iPSCs were transfected with the AIO vectors using neon electroporation with two 1200 V pulses of 20 ms duration. Transfected iPSC cultures were dissociated into single cells using 0.5% trypsin.0.5% (Gibco) and then sorted with a FACSAriaTM cell sorter (BD Bioscience, Franklin Lakes, NJ) to remove doublets and residues by selective gating on front and side scatter emission. Individual cells were expanded to full-size colonies and CRISPR edits were analyzed by detecting the presence of the aberrant sequence at the CRISPR cleavage site. Briefly, the target sequence was amplified by PCR using AmpliTaq Gold Mastermix (Thermo Fisher-Applied Biosystems, Waitham, MA) and the primers B2m gDNA: F: 5' CTGGATCAGACATATGTGTTGGGA-3j A: 5'-GCAAAGCAGTTTTAAGTCCACACAG-3' After purification of the obtained PCR product (PureLink® Pro 96 PCR Purification Kit, Thermo Fisher Scientific, Waltham, MA), Sanger sequencing was performed using a Personal Genome Machine (PGM™, Thermo Fisher Scientific). For homogeneity identification, a 250 bp region of the B2m gene was PCR-amplified using the following B2m gDNA PGM primers: F: 5'-TTTTCAAAATGTGGGTAGACTTTGG-3' and A: 5'- GGATTTCAATGTGA.GGCGGGT-3 The PCR product was purified as described above and prepared using the Ion PGM Hi Q template kit (Thermo Fisher Scientific). Experiments were performed on the Ion system. PGM™ with the Ion 318™ v2 chip kit (Thermo Fisher Scientific). Pyuripotency analyses were performed again. As shown in Figure 4, β2-microglobulin expression was eliminated in miPSC cells. MHC-I expression was not induced by IFN-γ stimulation (right panel). As a control, parental miPSC cells were stimulated with IFN-γ (left panel). C. Example 2: Generation of double knockout pyuripotent cells of B-2 Microglobulin / Cita. CRIPSR technology was used for the additional knockout of the dita gene. To target the The mouse Ciita gene coding sequence, the CRISPR sequence 5-GGTCCATCTATGGTCATAGAGG (CGG)-3', was aligned and ligated into A1-ln-GnE (A1) vectors containing the Cas9 expression cassette according to the kit instructions (GeneArt CRISPR Nuclease Vector Kit, Thermo Fischer, Waltham, MA). miPSCs were transfected with A1 vectors using the same conditions as for B2m-KO. Transfected miPSC cultures were dissociated into single cells using 0.05% trypsin (Thermo Fischer-Gibco) and then sorted with a FACSAria™ cell sorter (BD Bioscience, Franklin Lakes, NJ) to remove doublets and residues by selective gating on front- and side-scatter emission. Individual cells were expanded into full-size colonies and CRISPR edits were analyzed by detecting the presence of the aberrant sequence at the CRISPR cleavage site.Briefly, the target sequence was amplified by PCR using AmpliTaq Gold Mastermix (Thermo Fisher Applied Biosystems, Darmstadt, Germany) and the Ciita gDNA primers F: 5'-CCCCCAGAACGATGAGCTT-3', R: 5'-TGCAGAAGTCCTGAGAAGGCC-3'. After purification of the PCR product (PureLink® Pro 96 PCR Purification Kit, Thermo Fisher, Waitham, MA), Sanger sequencing was performed. Using the DNA sequence chromatogram, edited clones were identified by the presence of the aberrant sequence at the CRISPR cleavage site. Indel size was calculated using the TIDE tool. PCR and ICC were repeated to verify the pyuripotent status of the cells. Figure 5 confirms the double knockout of miPSC / β-2-microglobulin / Ciita. MHC-II could not be induced by TNF-γ to be expressed. D. Example 3: Generation of the double knockout of B-2 Microglobulin / Cyta-Cd47 + Pyuripotent Cells A Cd47 expression vector was introduced into a previously generated B2m / Ciita double knockout miPSC. The vector was delivered using a lentivirus containing the blasticidin resistance cassette. The Cd47 gene sequence was synthesized, and the DNA was cloned into the lentivirus pLent16 / V5 plasmid (ThermoFisher, Waltham, MA) containing a blasticidin resistance marker. Sanger sequencing was performed to verify that no mutations occurred. Lentivirus generation was performed with a 1x10 reservation title 7 TU / mL. The recombinant vector was transduced in 2x10 s miPSC with double knockout by B2m-KO / Ciita J It was cultured in blasticidin-resistant MEF cells for 72 h with an MOI ratio of 1:10, followed by antibiotic selection with 12.6 pg / mL of blasticidin for 7 days. Selected antibiotic groups were tested by RT-qPCR amplification of Cd47 mRNA and flow cytometry detection of Cd47. After Cd47 expression was confirmed, the cells were expanded and subjected to pluripatent assays. Figure 6A shows increased Cd47 expression from a transgene added to the double knockout of ii-2-microglobulin / Cüta (iPShypo cells). Figure 6B shows that iPShypo C57BL / 6 cells survive in the allogeneic BALB / c environment, but parental iPS cells do not. This new result confirms that hypoimmune pyuripotent cells survive when transplanted into what would otherwise be incompatible hosts. E. Differentiation of mouse cells from mHIP cells Islet cells: mHIP cells were differentiated into islet cells using techniques adapted from Líu et al., Exp. Diabetes Res 2012: 201295 (dot: 10.1155 / 2012 / 201295), which is incorporated herein by reference, and in particular for the differentiation techniques detailed therein. iPS cells were transferred to gelatin-coated flasks for 30 minutes to remove the feed layer and seeded at 1x1 O' 5 Cells per well were placed on collagen-1 coated plates in DMEIWF-12 medium supplemented with 2 mM glutamine, 100 pM non-essential amino acids, 10 ng / mL activin A, 10 mM nicotinamide, and 1 pg / mL laminin with 10% FBS overnight. ES-D3 cells were then exposed to DMEM / F-12 medium supplemented with 2 mM L-glutamine, 100 pM non-essential amino acids, 10 ng / mL activin A, 10 mM nicotinamide, 25 pg / mL insulin, and 1 pg / mL laminin with 2% FBS for 6 days. Neural stem cells: mHIP cells were differentiated into neural cells using techniques adapted from Abraches et al., Doi: 10.1371 / journal.pone.0006286, which are incorporated herein by reference, particularly for the differentiation techniques described herein. To initiate the monolayer protocol, ES cells were seeded in high-density (1.5 x 10⁻⁵) clonal-grade serum-free ESGRO medium (Miliipore). s cells / cm 2 ), After 24 hours, the ES cells were gently dissociated and placed on plates on a tissue culture medium coated with 0.1% (v / v) gelatin at 1*10 4 cells / cm 2 in RHB-A or N2B27 media (StemCeil Science Inc.), changing the media daily. For reseeding on day 4, the cells were dissociated and plated at 2 x 10 4 cells / cm 2 Cells were seeded in a laminin-coated plastic tissue culture dish in RHB-A medium supplemented with 5 ng / mL murine bFGF (Peprotech). From this point, cells were reseeded under the same conditions every 4 days, and the medium was changed every 2 days, for a total of 20 days of culture. To quantify the number of differentiating neurons at each time point, cells were placed on laminin-coated glass plates in 24-well Nunc plates, and 2 days after seeding, the medium was changed to a mixture of RHB-A:Neurobasal:B27 (1:0.02) to allow for better survival of differentiated neurons. Smooth muscle cells: mHIP cells were differentiated into SM cells using techniques adapted from Huang et al., Biochem Biophys Res Commun 2006: 351 (2) 321-7, incorporated herein by reference, particularly for the differentiation techniques described therein. The resuspended iPSCs were The cells were cultured in 6-well gelatin-coated plastic Petri dishes (Falcon, Becton-Dickinson) at 2 million cells per well at 37°C, 5% CO2 in 2 mL of differentiation medium containing 10 pM atRA. The differentiation medium consisted of DMEM, 15% fetal calf serum, 2 mM L-glutamine, 1 mM MTG (Sigma), 1% non-essential amino acids, penicillin, and streptomycin. Culture was continued for 10 days with daily changes of fresh medium. From day 11, the differentiation medium was replaced with serum-free culture medium, which consisted of knockout DMEM, 15% knockout replacement serum, 2 mM L-glutamine, 1 mM MTG, 1% non-essential amino acids, penicillin, and streptomycin. Cultures were continued for another 10 days with daily changes of the serum-free medium. Cardiomyocytes: mHIP cells were differentiated into CM cells using techniques adapted from Kattman et al., Cell Stem Cell 8: 228-240 (2011), which are incorporated herein by reference and, in particular, for the differentiation techniques described herein. Endothelial cells: mHIP cells differentiated into endothelial cells as they are known. F. Example 4: Allogeneic transplantation of HIP cell derivatives shows long-term survival in fully immunocompetent recipients. a. Mice: BALB / c (BALB / cAnNCrl, H2d), C57BL / 6 (C57BU6J, B6, H2b), BALB / c bare (BALB / c NU / NU, CAnN.CgFoxn1 <nu>Mice (Cr!, H2d) and Scid beige (CBySmn.CB17-Prkdcscid / J) (all 6–12 weeks old) were used as recipients for different assays. The number of animals per experimental group is shown in each figure. Mice were purchased from Charles River Laboratories (Sulzfeld, Germany) and received humane treatment in accordance with the Guidelines for the Principles of Laboratory Animals. Animal experiments were approved by the Hamburg Office for Health and Consumer Protection and were conducted in accordance with local and German consumer protection guidelines. b. Pluripotency analysis by RT-PCR and IF miPSCs were seeded in 24-well plates and processed for RT-PCR and immunofluorescence (IF) analysis 48 h post-seeding. For ICC, cells were fixed, permeabilized, and blocked using the Image-iT™ Fixation / Permeabilization Kit (Thermo Fisher Cat. No. R37602). Cells were stained overnight at 4°C with primary antibodies for Sox2, SSEA-1, Oct4, and alkaline phosphatase. After several washes, cells were incubated with AlexaFluor 488 secondary antibody and NucBie Fixed Cell ReadyProbes Reagent (all Thermo Fisher Scientific). Stained cells were imaged using a fluorescence microscope. For RT-PCR, RNA was extracted using the PureLink™ RNA Mini Kit (Thermo Fisher Cat. No. 12183018A). A DNase I step was included to remove contaminating genomic DNA. cDNA was generated using the Applied Biosystems® High-Yield cDNA Reverse Transcription Kit. Reverse transcriptase-free (RT-free) controls were also generated from all RNA samples. Gene-specific primers were used to amplify target sequences using the AmpíiTaq Gol® 360 Master Mix (Thermo Fisher Cat. No. 4398876). PCR reactions were visualized on 2% agarose gels. A set of positive control primers amplifying a constitutively expressed maintenance gene (Acto) encoding a cell cytoskeleton protein was included. c. Editing mouse PSC genes: The miPSCs underwent three gene-editing steps. First, CRISPRs targeting the mouse B2m gene coding sequence were reassociated and ligated into vectors containing the Cas9 expression cassette. The transfected miPSCs were dissociated into single cells, expanded into colonies, sequenced, and tested for homogeneity. Second, these B2m- / - miPSCs were transfected with vectors containing CRISPRs targeting Ciita, the master regulator of MHC II molecules. Expanded single-cell colonies were sequenced, and B2m- / - Ciita- / - clones were identified by the presence of an aberrant CRISPR cleavage site sequence. Third, the Cd47 gene sequence was synthesized, and the DNA was cloned into a blasticidin-resistant lentivirus plasmid. The miPSC B2m- / - Ciita- / - were transfected and grown in the presence of blasticidin.Selected antibiotic groups were tested for Cd47 overexpression, and B2m- / -Ciita- / -Cd47 tg miPSCs were expanded. FACS analyses demonstrated high MHC I expression, modest but detectable MHC II expression, and negligible Cd47 expression in wild-type miPSCs. The lack of MHC I expression, MHC II expression, and Cd47 overexpression were confirmed in the associated engineered miPSC lines. All engineered miPSC lines were tested for pluripotency. This was confirmed in the B2m- / -Ciita- / -Cd47 tg miPSCs after three engineering steps, demonstrating their potential to form cells of all three germ layers. d. Generation of miPSC B2m- / - CR1PSR technology was used to knock out the B2m gene. To target the mouse beta-2-microglobulin (B2m) gene coding sequence, the CRISPR sequence 5'-TTCGGCTTCCCATTCTCCGG(TGG)-3' was aligned and ligated into All in One (AIO) vectors containing the Cas9 expression cassette according to the kit instructions (GeneArt CRISPR Nuclease Vector Kit, Thermo Fischer, Waltham, MA). iPSCs were transfected with the AIO vectors using neon electroporation with two 1200 V pulses of 20 ms duration. Transfected iPSC cultures were dissociated into single cells using 0.05% trypsin (Gibco) and then sorted with a FACSAria cell sorter (BD Bioscience, Franklin Lakes, NJ) to remove doublets and residues by selective gating on front- and side-scatter emission.Individual cells were expanded into full-size colonies and analyzed for CRISPR editing by detecting the presence of an aberrant sequence at the CRISPR cleavage site. Briefly, the target sequence was amplified by PCR using AmpíiTaq Gold Mastermix (Applied Biosystems, Darmstadt, Germany) and B2m primers. gDNA F: 5'CTGGATCAGACATATGTGTTGGGAGA-3', R: 5'-GCAAAGCAGTTTTAAGTAGAGAG-3' After cleaning the obtained PGR product (PureLink® Pro 96 PCR Purification Kit, Thermo Fisher), Sanger sequencing was performed. Using Ion Personal Genome Machine (PGM) sequencing for homogeneity identification, a 250 bp region of the B2m gene was PCR-amplified using B2m gDNA PGM F: 5*-TTTTCAAAATGTGGGGGGGGG 3 primers. The PCR product was purified as described above and prepared using the Ion PGM Hi Q template kit (Thermo Fisher). Experiments were performed on the Ion PGM™ system with the Ion 318™ v2 chip kit (Thermo Fisher). Pluripotency assays were performed again. A reduced growth rate or differentiation capacity of B2m iPSCs was not observed as previously reported in the technique. e. Generation of miPSC B2m- / - and Ciita-A: CRISPR technology was used for further knockout of the dita gene. To target the mouse dita gene coding sequence, the CRISPR sequence 5'-GGTCCATCTATGGTCATAGAGG(CGG)-3' was aligned and ligated into All-In-One (AIO) vectors containing the Cas9 expression cassette according to the kit instructions (GeneArt CRISPR Nuclease Vector Kit, Thermo Fisher, Waltham, MA). miPSCs were transfected with the AIO vectors using the same conditions as for B2m-KO. Transfected miPSC cultures were dissociated into single cells using 0.05% trypsin (Gibco) and then sorted with a FACSAria cell sorter (BD Bioscience, Franklin Lakes, NJ) to remove doublets and residues by selective gating on front- and side-scatter emission. Individual cells were expanded into full-size colonies and analyzed for CRISPR editing by detecting the presence of an aberrant CRISPR cleavage site sequence.Briefly, the target sequence was amplified by PCR using AmpliTaq Gold Mastermix (Applied Biosystems, Darmstadt, Germany) and the Ciita gDNA primers F: 5'-CCCCCAGAACGATGAGCTT-3', R: 5'-TGCAGAAGTCCTGAGAAGGCC-3'. After purification of the PCR product (PureLink® Pro 96 PCR Purification Kit, Thermo Fisher), Sanger sequencing was performed. Using the DNA sequence chromatogram, edited clones were identified by the presence of an aberrant sequence at the CRISPR cleavage site. Indel size was calculated using the TIDE tool. PCR and ICC were performed again to verify the pluripotency status of the cells. f. Generation of the miPSC B2m- / - Cíita-A Cd47 tg: The iPSC cell lines B2m-KO, Ciita-KO, and Cd47-tg were generated through antibiotic resistance selection following lenitvirus-mediated administration of a Cd47 expression vector containing the blasticidin resistance cassette. The Cd47 gene sequence was synthesized, and the DNA was cloned into the blasticidin-resistant lentivirus pLenti6 / V5 (ThermoFisher). Sanger sequencing was performed to verify that no mutations had occurred. Lentivirus generation was carried out with a stock titer of 1 x 10⁻⁶ z TU / mL Transduction was performed in 2x10 5 miPSC B2m- / -Ciita- / -, were cultured in blasticidin-resistant MEF cells for 72 h with a MOl ratio of 1:10. followed by antibiotic selection with 12.5 pg / mL bisticstane for 7 days. The selected antibiotic groups were tested by RT-qPCR amplification of Cd47 mRNA and flow cytometry detection of Cd47. After confirmation of Cd4α, the cells were expanded and confirmed by pluripotency assays. g. Derivation and characterization of iPSC-derived endothelial cells (iEC). iECs were derived using a three-dimensional approach. Briefly, to initiate differentiation, iPSCs were cultured in ultra-low, non-adhesive plates to form embryoid body (EB) aggregates in EBM2 medium (Lonza) in the absence of leukemia inhibitory factor (LIF). After 4 days of suspension culture, the EBs were reattached to 0.2% gelatin-coated plates and cultured in EBM2 medium supplemented with VEGF-A165 (50 ng / mL; PeproTech). After 3 weeks of differentiation, single-cell suspensions were obtained using a cell dissociation regulator (Life Technologies) and labeled with APG-conjugated anti-mouse CD31 antibodies (eBiosciences) and PE-conjugated anti-mouse CD144 antibodies (BD Biosciences). iECs were purified by fluorescence-activated cell sorting (FACS) of the CD31+CD144+ population.IECs were maintained in supplemental EBM2 media with recombinant murine vascular endothelial growth factor (50 ng / mL). Its phenotype was confirmed by immunofluorescence for CD31 and VE cadherin, as well as by PCR and tube-forming assays to demonstrate endothelial function in forming premature vascular structures. Note: Differentiation protocols using confluent iPSC monolayers in 0.1% gelatin or Matrigel have also been successful. Note: Other endothelial cell media have also been used successfully. h. Derivation and characterization of iPSC-derived smooth muscle cells (iSMCs): The resuspended iPSCs were grown in 0.1% gelatin-coated plastic petri dishes with 6 wells (Falcon, Becton-Dickinson) at 2 million cells per well at 37°C, 5% CO₂ 2 in 2 mL of differentiation medium containing 10 pM. The differentiation medium was made from DMEM, 15% fetal calf serum, 2 mM L-glutamine, 1 mM MTG (Sigma), 1% non-essential amino acids, penicillin, and streptomycin. The culture was continued for 10 days with daily changes of the medium. From day 11, the differentiation medium was replaced with a serum udder culture medium from a DMEM knockout: 15% knockout replacement serum, 2 mM L-glutamine, 1 mM MTG, 1% non-essential amino acids, penicillin, and streptomycin. Cultures were continued for another 10 days with daily changes of serum-free medium. The phenotype was confirmed by immunofluorescence and PCR for both SMA and SM22. i. Derivation and characterization of iPSC-derived cardiomyocytes (iCMs): Before differentiation, iPSCs were passed twice through gelatin-coated plates to remove feeder cells. In summary, iPSCs were dissociated with TrypLE (in vitro) and They were cultured at 75,000–100,000 cells / mL without any additional growth factors for 48 h. The 3-day-old EB cells dissociated and differentiated into cardiac cells. In summary, 6 x 10 4 -10x10 4 Cells were inoculated into individual wells of a 96-well flat-bottom plate (Becton Dickenson, Franklin Lakes, NJ) coated with gelatin in StemPro-34 SF medium (Invitrogen), supplemented with 2 mM L-glutamine, 1 mM ascorbic acid (Sigma), human VEGF (5 ng / mL), human DKK1 (150 ng / mL), human bFGF (10 ng / mL), and human FGF10 (12.5 ng / mL) (R&D Systems). Cultures were harvested 4 or 5 days later (a total of 7 or 8 days). Their phenotype was confirmed by IF for troponin I and sarcomeric alpha-actinin, as well as PCR for Gata4 and Mhy6. The cells began beating between 8 and 10 days. This demonstrated their functional differentiation. j. Derivation and characterization of islet cells derived from iPSC (ilC) iPS cells were transferred to gelatin-coated flasks for 30 minutes to remove the feed layer and seeded at 1 x 10⁻⁶ cells per well in collagen I-coated plates in DMEM / F-12 medium supplemented with 2 mM glutamine, 100 pM non-essential amino acids, 10 ng / mL activin A, 10 mM nicotinamide, and 1 pg / mL laminin with 10% FBS overnight. ES-D3 cells were then exposed to DMEM / F-12 medium supplemented with 2 mM L-glutamine, 100 pM non-essential amino acids, 10 ng / mL activin A, 10 mM nicotinamide, 25 pg / mL insulin, and 1 pg / mL laminin with 2% FBS for 6 days. Its phenotype was confirmed by immunofluorescence for c-peptide, PCR for glucagon, Ngn3, amylase, insulin 2, somatostatin, and insulin production. k. Derivation and characterization of iPSC-derived neuronal cells (INC) To initiate the monolayer protocol, iPSCs were gently dissociated and placed on plates onto tissue culture plastic coated with 0.1% gelatin at 1x10 4 cells / cm 2 in RH8-A or N2B27 media (StemCell Science Inc.), changing the media every two days. For reseeding on day 4, the cells were dissociated and plated at 2 x 10 4 cells / cm 2 Cells were seeded in laminin-coated tissue culture plastic in RHB-A medium supplemented with 5 ng / mL of marine bFGF (Peprotech). From this point, cells were reseeded under the same conditions every 4 days, and the medium was changed every 2 days, for a total of 20 days in culture. To quantify the number of differentiating neurons at each time point, cells were placed on laminin-coated glass plates in 24-well Nunc plates, and 2 days after seeding, the medium was changed to a mixture of RHB-A:Neurobasal:B27 (1:1–0.02) to promote the survival of differentiated neurons. Their phenotype was confirmed by immunofluorescence (IF) for Tuj-1 and nestin. l. Elispot Essays For the enzyme-linked ImmunoSpot (Elispot) unidirectional assays, recipient splenocytes were isolated from fresh spleen 5 days after cell injection (miPSC, miPSC B2m- / - or miPSC B2m- / - Ciita- / - or miPSC B2m- / - Ciita- / - Cd47 tg) and used as responder cells. Donor cells (miPSC, miPSC B2m- / - or miPSC B2m- / - Ciita- / - or miPSC B2m-A Ciita- / - Cd47 tg) were inhibited by mitomycin and served as stimulator cells.10 s Stimulating cells were incubated with 5x10 s Responding splenocyte receptors were monitored for 24 hours, and spot frequencies of IFNγ and IL-4 were automatically enumerated using an Elispot plate reader. Assays were performed in quadruplicate. m. Teratoma assays to study the survival of iPSCs in vivo Six-week-old syngeneic or allogeneic mice were used for transplantation of wild-type iPSCs or non-immunogenic iPSCs. One × 10⁻⁵ cells in 100 pL were injected into the right thigh muscle of the mice. Transplanted animals were routinely observed every two days, and tumor growth was measured with calipers. Animals were euthanized after the development of tumors larger than 1.5 cm. 3 or after an observation period of 100 days. n. In vitro NK cell assays CD107 expression in NK cells after co-culture with iPSCs or HIP cells was measured by flow cytometry as a marker of NK cell activation. Using the Elispot principle, NK cells were co-cultured with wild-type iPSCs or HIP cells, and IFN-γ release was measured. According to the "self-loss theory," MHC I-deficient stem cells have been shown to be susceptible to NK cell killing, since murine and human PSCs express ligands to activate NK receptors. Although the expression of activating receptors has been reported to decrease with differentiation, NK cell killing of B2m- / - derivatives has been observed. Although isolated HLA-E or HLA-G expression in human pluripotent stem cells has been used to mitigate the expected innate immune response in HLA IV- cells, there are additional highly effective non-MHC inhibitory ligands among them. The invention provides that Cd47 was found to be a surprisingly potent inhibitor of innate immune clearance. o. Mouse data summary All engineered miPSC lines were transplanted into syngeneic C57BL / 6 and allogeneic BALB / c receptors without immunosuppression. While all genetically modified cells developed teratomas in syngeneic receptors, their survival depended on their level of hypoimmunogenicity in allogeneic receptors. Sixty percent of the B2m- / - miPSCs in BALB / c showed teratoma formation, a subtle Elispot response, and a moderate IgM antibody response was still observed. In miPSC B2m"A Ciita- / - cells, 91.7% teratoma formation occurred in allogeneic BALB / c, with a lower Elispot response and no antibody response. The miPSC B2m- / - Ciita- / - Cd47 tg cell line showed 100% teratoma formation, and there were no Elispot or antibody responses. The contribution of Cd47 overexpression was further evaluated in innate immunity assays by comparing miPSC B2m- / - Ciita- / - with miPSC B2m-A Ciita- / - Cd47 tg.Overexpression of CD47 significantly reduced CD107 expression on NK cells and IFN-γ release from NK cells, thereby mitigating innate immune clearance. In summary, each step of the engineering process has made the miPSCs more hypoimmunogenic. HIP B2m- / - Ciita- / - cells were differentiated into hypoimmunogenic endothelial-like cells (miECs), smooth muscle-like cells (miSMCs), and cardiomyocyte-like cells (miCMs). “Wild-type” miPSC derivatives (i.e., unengineered miPSCs) served as controls. All derivatives exhibited the typical morphology, cell marker immunofluorescence, and gene expression of their predicted mature tissue cell lines. MHC I and II expression in the weight derivatives was generally highly upregulated compared to their parental mPSC line, but varied markedly by cell type. As expected, miECs had by far the highest expression of MHC I and MHC II, miSMCs had moderate expression of MHC I and MHC II, while miCMs had moderate expression of MHC I but very low expression of MHC II.All wild-type derivatives had fairly low Cd47 expression, although slightly higher compared to miPSCs. All B2m- / - Ciita- / - Cd47 tg derivatives appropriately showed a complete lack of MHC I and MHC II and significantly higher Cd47 expression than their weight counterparts. Matrigel stoppers containing 5x10 5 wild-type miEC, miSMC, and miCM were grafted into subcutaneous pockets of allogeneic C57BL / 6 or BALB / c mice. After 5 days, all allogeneic recipients mounted a strong cellular immune response, as well as a strong IgM antibody response, against these differentiated wild-type cell grafts. In contrast, none of the Ciita- / -Cd47 tg (HlP) derivatives showed detectable increases in IFN-γ Elispot frequencies or IgM antibody production. The morphology of the transplanted cells was also confirmed. Allogeneic mice were placed in an induction chamber and anesthesia was induced with 2% isoflurane (Isothesia, Butler Schein). One million cells, either HlP miPSC-derived cardiomyocytes (miCM), HlP miPSC-derived smooth muscle cells (miSMC), or HlP miPSC-derived endothelial cells (miEC), were mixed in 250 pL of 0.9% saline with 250 pL of BD Matrigel High Concentration (1:1; BD Biosciences) and injected subcutaneously into the lower back of mice using a 23-G syringe. Matrigel plugs were explanted 1, 2, 3, 4, 5, 6, 8, 10, and 12 weeks after implantation and fixed with 4% paraformaldehyde and 1% glutenaldehyde for 24 h, followed by dehydration and paraffin embedding. Five-micrometer-thick sections were cut and stained with hematoxylin and eosin (H&E). Histology confirmed morphologically adequate miCM, miSMC, and miEC. G. Example 5: Generation of human PSCs The human episomal iPSC line was derived from CD34+ cord blood (Cat. No. A33T24, Thermo Fisher Scientific) using a Thermo Fisher 3-plasmid, 3-factor EBNA-based episomal system (SOKMNLT, SOX2, OCT4 (POU5F1), KLF4, MYC, NANQG, LIN28, and SV40LT antigen). This iPSC line is considered to have a zero footprint, as there was no integration into the genome following the reprogramming event. It has been shown to be free of all reprogramming genes. iPSCs have a normal XX karyotype and endogenous expression of pluripotent markers such as Oct4, Sox2, Nanog (as shown by RT-PCR), Oct4, SSEA4, TRA-1-60, and TRA-1-81 (as shown by ICC). In directed differentiation and teratoma analyses, these hiPSCs retained their potential to Differentiation for the ectodermal, endodermal, and mesodermal lineages was achieved. In addition, the vascular, endothelial, and cardiac lineages were derived with robust efficiencies. Note: Several gene delivery vehicles were successfully used for IPSC generation, including retroviral vectors, adenoviral vectors, Sendai virus, and virus-free reprogramming methods (using episomal vectors, piggyBac transposon, synthetic mRNA, microRNA, recombinant proteins, and small molecule drugs, etc.). Note: Different factors were successfully used for reprogramming, such as the first reported combination of OCT3 / 4, SQX2, KLF4, and C-MYC, known as the Yamanaka factors. In one modality, only three of these factors were successfully combined, omitting C-MYC, although with reduced reprogramming efficiency. In one modality, L-MYC or GLIS1 instead of C-MYC showed improved reprogramming efficiency. In another modality, the reprogramming factors are not limited to genes associated with pluripotency. a. Statistics All data are expressed as mean ± SD or in box plots showing the mean and minimum-to-maximum range. Intergroup differences were appropriately assessed using unpaired Student's t-test or one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. * p < 0.05, ** p < 0.01. H. Example 6: Generation of human HIP cells The human Cas9 PSC underwent two stages of gene editing. In the first step, CRISPR technology was implemented by combining the coding sequence of the human beta-2-microglobulin (B2M) gene with the CRISPR sequence 5'-CGTGAGTAAACCTGAATCTT-3 > and the coding sequence of the human CUTA gene with the CRISPR sequence 5'-GATATTGGCATAAGCCTCCC-3'. The linearized CRISPR sequence with the T7 promoter was used to synthesize the gRNA according to the kit instructions (MEGAshortscript T7 Transcription Kit, Thermo Fisher). The collected in vitro transcription (IVT) gRNA was then purified using the MEGAciear Transcription Cleanup Kit. For IVT gRNA delivery, single-cell cells were electroporated with 300 ng of IVT gRNA using a Neon electroporation system. After electroporation, the Cas9-edited iPSCs were expanded for single-cell seeding: the iPSC cultures were dissociated into single cells using TrypLE (Gibco) and stained with Tral-60 Alexa Fluor® 488 and propidium iodide (Pl).The Aria FACS cell sorter (BD Biosciences) was used for sorting, and doublets and debris were excluded from seeding by selective emission selection in forward and lateral scattering. Viable pluripotent cells were selected in the absence of pluripotent stem cells (PSCs) and in the presence of Alexa Fluor 488 staining of Tra1-60. Individual cells were then expanded into full-size colonies, after which the colonies were tested for CRISPR editing. CRISPR-mediated cleavage was assessed using the GeneArt genomic cleavage detection kit (Thermo Fisher). Genomic DNA was isolated from 1 x 10⁻⁶ hiPSCs, and the B2M and CUTA genomic DNA regions were amplified by PCR using the Ampl.iTaq Gold 360 master mix. Primer sets F: 5'-TGGGGCCAAATCATGTAGACTC-3' and R: 5'-TCAGTGGGGGGGGTGAATTCAGTGT-3' were used for B2M, as well as F: 5'-CTTAACAGCGATGCTGACCCC-3' and R: 5'-TGGCCTCCATCTCCCCTCTCTT-3' for CUTA. For TIDE analysis, the obtained PCR product was purified (PureLink PCR Purification Kit, Thermo Fisher) and Sanger sequencing was performed to predict indel frequency. After confirmation of B2M / CIITA knockout, the hPSCs were further characterized by karyotype analysis and the TaqMan scoring panel (Thermo Fisher). The PSCs were found to be pluripotent and maintained a normal karyotype (46, XX) throughout the genome editing process. In the second stage, the CD47 gene was synthesized and its DNA cloned into a lentivirus plasmid with an EF1 promoter and puromycin resistance. The cells were transduced with lentiviral stocks of 1 x 10 7 TU / mL and 6 pg / mL of polybrene (Thermo Fisher). The media were changed daily after transduction. Three days post-transduction, the cells were expanded and selected with 0.5 pg / mL puromycin. After 5 days of antibiotic selection, antibiotic-resistant colonies emerged and were expanded to generate stable clusters. CD47 levels were confirmed by qPCR. Pluripotency assay (TaqMan hPSC Scorecard Panel, Thermo Fisher) was performed, and kanotyping was repeated to verify the pyuripotent status of the cells. I. Example 7: differentiation of human HIP cells 1. Differentiation of hHIP cells into human cardiomyocytes This was done using a protocol adapted from Sharma et al., J. Vis Exp. 2015 doi: 10.3791 / 52628, incorporated here in its entirety as a reference, and specifically for techniques to differentiate the cells. hiPSCs were seeded in dilute Matrigel (356231, Corning) in 6-well plates and maintained in Flex 8 medium (Thermo Fisher). After the cells reached 90% confluency, differentiation was initiated, and the medium was changed to 5 mL of RPMI1640 containing 2% B-27 minus insulin (both Gibcó) and 6 pM CHIR-99021 (Selleck Qhem). After 2 days, the medium was changed to RPMI1640 containing 2% B-27 minus insulin without CHIR. On day 3, 5 pL of 1WR1 was added to the media for two more days. On day 5, the medium was changed back to RPMl 1640 containing 2% B-27 less insulin medium, and incubated for 48 hours.On day 7, the media was changed to RPMl 1640 containing B27 plus insulin (Gibco) and replaced every 3 days thereafter with the same media. Spontaneous cardiomyocyte blunting was first observed around day 10 to day 12. Cardiomyocyte purification was performed on day 10 post-differentiation. Briefly, the media was changed to low-glucose media and maintained for 3 days. On day 13, the medium was changed again to RPMl 1640 containing B27 plus insulin. This procedure was repeated on day 14. The remaining cells are highly purified cardiomyocytes. 2. Differentiation of hHIP cells into human endothelial cells hiPSC cells were seeded in dilute Matrigel (356231, Corning) in 6-well plates and maintained in Essential 8 Flex medium (Thermo Fisher). Once the cells reached 60% confluency, differentiation was initiated, and the medium was changed to RPMl 1640 containing 2% B-27. On day 2, the media were changed to reduced media: RPMI1640 containing 2% less insulin (both Gibco) and 5 pM of CHIR-99021 (Selleck Chem). From day 4 to day 7, cells were exposed to RPMI EC, RPMI1640 media containing 2% B-27 less insulin plus 50 ng / mL vascular endothelial growth factor (VEGF; R&D Systems, Minneapolis, MN, USA), 10 ng / mL basic fibroblast growth factor (FGFb; R&D Systems), Y-2763210 pM (Sigma-Aldrich, Saint Louis, MO, USA) and SB 431542 1 pM (Sigma-Aldrich). Endothelial cell clusters were visible from day 7 and the cells were maintained in EGM-2 SingleQuots media (Lonza, Basel, Switzerland) plus 10% FCS hi (Gibco), 25 ng / mL vascular endothelial growth factor (VEGF; R&D Systems, Minneapolis, MN, USA).), 2 ng / mL of basic fibroblast growth factor (FGFb; R&D Systems), Y-27632 10 pM (Sigma-Aldrich, Saint Louis, MO, USA), and SB 431542 1 pM (Sigma-Aldrich). The differentiation process was completed after 14 days, and undifferentiated cells were shed during the process. For purification, cells were processed using MACS according to the manufacturers' protocol using CD31 microbeads (Miltenyi, Auburn, CA). Highly purified EC cells were cultured in EGM-2 SingleQuots medium (Lonza, Basel, Switzerland) with supplements and 10% FCS hi (Gibco). TrypLE was used to process the cells 1:3 every 3 to 4 days. J. Transplantation in Humanized Mice Humanized NSG-SGM3 mice were placed in an induction chamber and anesthesia was induced with 2% isoflurane (Isothesia, Butler Schein). One million cells, either niPSC-derived cardiomyocytes (hiCM) or hiPSC-derived endothelial cells (hiECin 250 pL of 0.9% saline containing ZVAD (100 mM, benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fluoromethyl ketone, Calbiochem), Bcl-XL BH4 (cell-permeating TAT peptide, 50 nM, Calbiochem), cyclosporine A (200 nM, Sigma), IGF-1 (100 ng / mL, Peprotech), and pinacidil (50 mM, Sigma), were mixed with 250 pL of BD Mathgel High Concentration (1:1; BD Biosciences) and injected subcutaneously into the lower back of mice using a 23-G syringe. Matrigel plugs were explanted 2, 4, 6, 8, 10 and 12 weeks after implantation and were fixed with 4% Paraformaldehyde and 1% Glutenaldehyde for 24 h, followed by dehydration and paraffin embedding.A 5 pm thick section was cut and stained with hematoxylin and eosin (HE) and the morphology was confirmed. IX. Example sequences: SEQ ID NO:1 - B-2-Human Microglobulin mrsvalavlallslsgleaiqrtpkiqvysrhpaengksnflncyvsgfhpsdievdllkngeriekvehs DLSFSKDWSFILLYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDI SEQ ID NO:2 - Human CUTA proteins, 160 N-terminal amino acids MRCLAPRPAGSYLSEPQGSSQCATMELGPLEGGYLELLNSDADPLCLYHFYDQMDLAGEEEIELYSEPD tdtincdqfsrllcdmegdeetreayaniaeldqyvfqdsqleglskdifkhigpdevigesmempaevgq KSQKRPFPEELPADLKHWKW SEQ ID NO:3 -Humana CD47 MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTWlPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDG ALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRWSWFSPNE NILIViFPIFAILLFWGQFGIKTLKYRSGGMDECTIALLVAGLVIVIVGAILFVPGEYSLKNATGlGLIVTSTGI LILLHYYVFSTAÍGLTSFVIAILVIQVIAYILAWGLSLCIAACIMHGPLLISGLSILALAQLLGLVYMKFVE SEQ ID N0:4 - Herpes simplex virus (HSV-tk) thymidine kinase MASYPCHQHASAFDQAARSRGHSNRRTALRPRRQQEATEVRLEQKMPTLLRVYIDGPHGMGKTTTTQL LVALGSRDDIVYVPEP1VITYWQVLGASETIANIYTTQHRLDQGEÍSAGDAAVVMTSAQIT™GMPYAVTDAVL APHVGGEAGSSHAPPPALTLIFDRHPIAALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGALPED RHIDRLAKRQRPGERLDLAMLAAIRRVYGLLANTVRYLQGGGSWWEDWGQLSGTAVPPQGAEPQSNAG PRPHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLRPMHVFILDYDQSPAGCRDALLQLTSGMVQ THVTTICGARFPGARFTGEANARTGEANT SEQ ID N0;5 -Cltosina Desaminase de Escherichia co / r (EC-CD) msnnalqtiinarlpgeeglwqihlqdgkisaidaqsgvmpitensldaeqglvippfvephihldttqtag QPNWNQSGTLFEGIERWAERKALLTHDDVKQRAWQTLKWQQQTHIERKALLTHDDVKQRAWQTLKWQQQSDHKVATVK QEVAPWIDLQIVAFPQEGILSYPNGEALLEEALRLGADWGAIPHFEFTREYGVESLHKTFALAQKYDRLID VHCDEIDDEQSRFVETVAALAHHEGIVIGARVTASHTTAMHSYNGAYTSRLFRLLKIVISGINFVANPLVNIHL QGRFDTYPKRRGITRVKEMLESGINVCFGHDDVFDPWYPLGTANMLQVLHMGLHVCQLMGYGQINDGL 20 NLITHHSARTLNLQDYGIAAGNSANUILPAENGFDALRRQVPVRYSVRGGKVIASTQPAQTTVYLEQPEAI DYKR SEQ ID NO:6 - Truncated human caspase 9 9 GFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKG DLTAKKMVLALLELAQQDHGALDCCWVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSL GGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSD1FVSYS TFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS All patent publications and documents disclosed or referenced herein are incorporated by reference in their entirety. The foregoing description is provided for illustrative and descriptive purposes only. This description is not intended to limit the invention to the precise form disclosed. The scope of the invention is intended to be defined by the appended claims.< / nu> < / nu>

Claims

CLAIMS 1. A method for generating a hypoimmunogenic pluripotent stem cell comprising: a) Eliminate the activity of both alleles of a B2M gene in an induced pluripotent stem cell (iPSC): b) eliminate the activity of both alleles of a CUTA gene in said PSC; and c) Increase the expression of CD47 in said IPSC.

2. The method according to claim 1, wherein said iPSC is human, said B2M gene is human, said CUTA gene is human, and said increased expression of CD47 results from the introduction of at least one copy of a human CD47 gene under the control of a promoter in said iPSC cell.

3. The method according to claim 1, wherein said iPSC is murine, said B2m gene is murine, said Ciita gene is murine, and said increased expression of Cd47 results from the introduction of at least one copy of a murine Cd47 gene under the control of a promoter in said iPSC cell.

4. The method according to any of claims 2-3, wherein said promoter is a constitutive promoter.

5. The method according to any of claims 1-3, wherein said interruption in both alleles of said B2M gene results from a Clustered Regularly Interspaced Short Palindromic Repeats / Cas9 (CRISPR) reaction that interrupts both alleles of said B2M gene.

6. The method according to any of claims 1-3, wherein said interruption in both alleles of said CUTA gene results from a CRISPR reaction that interrupts both alleles of said CUTA gene.

7. A human hypo-immunogenic pluripotent (hHIP) stem cell comprising: a) one or more alterations that inactivate both alleles of an endogenous B2M gene; b) one or more alterations that inactivate both alleles of an endogenous CUTA gene; and c) an alteration that causes an increase in the expression of a CD47 gene in said hHIP stem cell; wherein said hHIP stem cell causes a first natural killer (NK) cell response that is less than a second NK cell response caused by an induced pluripotent stem cell (iPSC) comprising said B2M and CUTA alterations but not comprising said increased expression of the CD47 gene and wherein said first and second NK cell responses are measured by determining the IFN-γ levels of NK cells incubated with either said hHIP or iPSC in vitro.

8. A human hypo-immunogenic pluripotent (hHIP) stem cell comprising: a) one or more alterations that inactivate both alleles of an endogenous B2M gene; b) one or more alterations that inactivate both alleles of an endogenous CUTA gene; and c) one or more alterations that cause an increase in the expression of a CD47 gene in said hHiP stem cell: wherein said hHIP stem cell induces a first T cell response in a humanized mouse strain that is lower than a second T cell response in said humanized mouse strain induced by an iPSC, and wherein said first and second T cell responses are measured by determining the IFN-γ levels of said humanized mice in an Elispot assay.

9. A method, comprising transplanting the hHIP stem cell of any of claims 7 or 8 into a human subject.

10. A hypoimmunogenic pyuripotent cell, comprising: a) an endogenous Major Histocompatibility Antigen Class I (HLA-I) function that is reduced when compared to a pyuripotent stem cell: b) an endogenous Major Histocompatibility Antigen Class II (HLA-II) function that is reduced when compared to said pyripotent stem cell; and cj a reduced susceptibility to NK cell destruction compared to said pyuripotent stem cell; where said hypoimmunogenic pyripotent cell is less susceptible to rejection when transplanted into a subject as a result of said reduced HLA-I function, said reduced HLA-Il function and reduced susceptibility to NK cell destruction.

11. The hypoimmunogenic pyuripotent cell according to claim 10, wherein said HLA-I function is reduced by a reduction in the expression of the 13-2 microglobulin protein.

12. The hypoimmunogenic pyuripotent cell according to claim 11, wherein a gene encoding said microglobulin protein is knocked out |3-2.

13. The hypoimmunogenic pyuripotent cell according to claim 12, wherein said B-2 microglobulin protein has at least 90% sequence identity with SEQ ID NO:

1.

14. The hypoimmunogenic pyuripotent cell according to claim 13, wherein said microglobulin B-2 protein has the sequence of SEQ ID NO.

1.

15. The hypoimmunogenic pyuripotent cell according to claim 10, wherein said HLA-I function is reduced by a reduction in the expression of the HLA-A protein.

16. The hypoimmunogenic pyuripotent cell according to claim 15, wherein a knockout of a gene encoding said HLA-A protein is performed.

17. The hypoimmunogenic pyuripotent cell is in accordance with claim 10, wherein said HLA-I function is reduced by a reduction in the expression of the HLA-B protein.

18. The hypoimmunogenic pyuripotent cell according to claim 17, wherein an HLA-B protein knockout is performed.

19. The hypoimmunogenic pyuripotent cell according to claim 10, wherein said HLA-I function is reduced by a reduction in the expression of the HLA-C protein.

20. The hypoimmunogenic pyuripotent cell according to claim 19, wherein a knockout of a gene encoding said HLA-C protein is performed.

21. The hypoimmunogenic pluripotent cell according to any of claims 10-20, wherein said hypoimmunogenic pluripotent cell does not comprise an HLA-I function.

22. The hypoimmunogenic pluripotent cell according to any of claims 10-21, wherein said HLA-II function is reduced by a reduction in the expression of the CUTA protein.

23. The hypoimmunogenic pluripotent cell according to claim 22, wherein a knockout of a gene encoding said CUTA protein is performed.

24. The hypoimmunogenic pluripotent cell according to claim 23, wherein said CUTA protein has at least 90% sequence identity with SEQ ID NO:

2.

25. The hypoimmunogenic pluripotent cell according to claim 24, wherein said CUTA protein has the sequence SEQ ID NO:

2.

26. The hypoimmunogenic pluripotent cell according to any of claims 10-21, wherein said HLA-II function is reduced by a reduction in the expression of the HLA-DP protein.

27. The hypoimmunogenic pluripotent cell according to claim 26, wherein a knockout of a gene encoding said HLA-DP protein is performed.

28. The hypoimmunogenic pluripotent cell according to any of claims 10-21, wherein said HLA-II function is reduced by a reduction in the expression of the HLA-DR protein.

29. The hypoimmunogenic pluripotent cell according to claim 28, wherein a knockout of a gene encoding said HLA-DR protein is performed.

30. The hypoimmunogenic pluripotent cell according to any of claims 10-21, wherein said HLA-II function is reduced by a reduction in the expression of the HLA-DQ protein.

31. The hypoimmunogenic pluripotent cell according to claim 30, wherein a knockout of a gene encoding said HLA-DQ protein is performed.

32. The hypoimmunogenic pluripotent cell according to any of claims 10-31, wherein said hypoimmunogenic pluripotent cell does not comprise an HLA-II function.

33. The hypoimmunogenic pluripotent cell according to any of claims 10-32, wherein said reduced susceptibility to NK cell destruction is caused by an increased expression of a CD47 protein.

34. The hypoimmunogenic pluripotent cell according to claim 33, wherein said increased expression of the CD47 protein results from a modification at a locus of the CD47 gene 0 endogenous.

35. The hypoimmunogenic pluripotent cell according to claim 33, wherein said increased expression of the CD47 protein results from a CD47 transgene.

36. The hypoimmunogenic pluripotent cell according to any of claims 33-35, wherein said CD47 protein has at least 90% sequence identity with SEQ ID NO-3.

37. The hypoimmunogenic pluripotent cell according to claim 27, wherein said CD47 protein has the sequence of SEQ ID NO:

3.

38. The hypoimmunogenic pluripotent cell according to any of the claims 10-37, which further comprises a suicide gene that is triggered by a trigger that causes said hypoimmunogenic pluripotent cell to die.

39. The hypoimmunogenic pluripotent cell according to claim 38, wherein said suicide gene is a herpes simplex virus (HSV-tk) thymidine kinase gene and said activator is ganciclovir.

40. The hypoimmunogenic pluripotent cell according to claim 39, wherein said HSV-tk gene encodes a protein comprising at least 90% sequence identity with SEQ ID NO: 4 41 The hypoimmunogenic pluripotent cell according to claim 40, wherein said HSV-tk gene encodes a protein comprising the sequence of SEQ ID NO:

4. 42 The hypoimmunogenic pluripotent cell according to claim 38, wherein said suicide gene is an Escherichia coli (EC-CD) cytosine desarninase gene and said activator is 5-fluorocytosine (5-FC).

43. The hypoimmunogenic pluripotent cell according to claim 42, wherein said EC-CD gene encodes a protein comprising at least 90% sequence identity with SEQ ID NO.

5.

44. The hypoimmunogenic pluripotent cell according to claim 43, wherein said EC-CD gene encodes a protein comprising the sequence SEQ ID NO:

5.

45. The hypoimmunogenic pluripotent cell according to claim 38, wherein said suicide gene encodes an inducible caspase protein and said trigger is a chemical inducer of dimerization (CID).

46. ​​The hypoimmunogenic pluripotent cell according to claim 45, wherein said gene encodes an inducible caspase protein comprising at least 90% sequence identity with SEQ ID NO: 6 47. The hypoimmunogenic pluripotent cell according to claim 46, wherein said gene encodes an inducible caspase protein comprising the sequence of SEQ ID NO:

6.

48. The hypoimmunogenic pluripotent cell according to any of claims 45-47, wherein said CID is AP1903.

49. A method for producing a hypoimmunogenic pluripotent cell, comprising' a) Reducing an endogenous function of the Major Histocompatibility Antigen Class l (HLA-l) in a pluripotent cell: b) To reduce an endogenous function of the major histocompatibility antigen class II (HLA-II) in a pluripotent cell; and c) Increase the expression of a protein that reduces the susceptibility of said pluripotent cell to death by NK cells 50. The method according to claim 49, wherein said HLA-l function is reduced to reducing the expression of an S-2 microglobulin protein.

51. The method according to claim 50, wherein said protein expression of β-2 microglobulin is reduced by knockout of a gene that codes for this β-2 microglobulin protein.

52. B-2 microglobulin 50, wherein said B-2 microglobulin protein has at least 90% sequence identity with SEQ ID NO:

1.

53. B-2 microglobulin 51, wherein said fi-2 microglobulin protein has the sequence SEQ ID NO.

1. 54 The method according to claim 49, wherein said HLA-I function is reduced by reducing the expression of the HLA-A protein.

55. The method according to claim 54, wherein said HLA-A protein expression is reduced by knocking out a gene encoding said HLA-A protein.

56. The method according to claim 49, wherein said HLA-I function is reduced by reducing the expression of the HLA-B protein.

57. The method according to claim 56, wherein said HLA-B protein expression is reduced by knocking out a gene encoding said HLA-B protein.

58. The method according to claim 49, wherein said HLA-I function is reduced by reducing the expression of the HLA-C protein.

59. The method according to claim 58, wherein said HLA-C protein expression is reduced by knocking out a gene encoding said HLA-C protein.

60. The method according to any of claims 49-59, wherein the hypoimmunogenic pluripotent cell© does not comprise an HLA-I function.

61. The method according to any of claims 49-60, wherein said HLA-II function is reduced by reducing the expression of a CUTA protein.

62. The method according to claim 60, wherein said CUTA protein expression is reduced by knockout of a gene encoding said CUTA protein.

63. The method according to claim 61, wherein dioha protein CUTA has at least 90% sequence identity with SEQ ID NO:

2.

64. The method according to claim 63, wherein said CUTA protein has the sequence of SEQ ID NO:

2.

65. The method according to any of claims 49-60, wherein said HLA-II function is reduced by reducing the expression of an HLA-DP protein.

66. The method according to claim 65, wherein said HLA-DP protein expression is reduced by knocking out a gene encoding said HLA-DP protein.

67. The method according to any of claims 49-60, wherein said HLA-II function is reduced by reducing the expression of an HLA-DR protein.

68. The method according to claim 67, wherein said HLA-DR protein expression is reduced by knack-out of a gene encoding said HLA-DR protein. 69 The method according to any of claims 49-60, wherein said HLA-yl function is reduced by reducing the expression of an HLA-DQ protein.

70. The method according to claim 69, wherein said protein expression HLA-DQ is reduced by knocking out a gene that codes for the HLA-DQ protein.

71. The method according to any of claims 49-70, wherein said hypoimmunogenic pluripotent cell does not comprise an HLA-II function.

72. The method according to any of claims 49-71, wherein said increased expression of a protein that reduces the susceptibility of said pluripotent cell to macrophage phagocytosis results from a modification to an endogenous gene locus.

73. The method according to claim 72, wherein said endogenous gene locus encodes a CD47 protein.

74. The method according to any of claims 49-71, wherein said increased expression of the protein results from the expression of a transgene.

75. The method according to claim 74, wherein said transgene encodes a CD47 protein.

76. The method according to any of claims 73 or 74, wherein said CD47 protein has at least 90% sequence identity with SEQ ID NO:

3.

77. The method according to claim 76, wherein said CD47 protein has the sequence of SEQ ID NO:

3.

78. The method according to any of claims 49-77, further comprising expressing a suicide gene that is activated by a trigger that causes said hypoimmunogenic pluripotent cell to die.

79. The method according to claim 78, wherein said suicide gene is a herpes simplex virus (HSV-tk) thymidine kinase gene and said activator is ganciclovir.

80. The method according to claim 79, wherein said HSV-tk gene encodes a protein comprising at least 90% sequence identity with SEQ ID NO:

4.

81. The method according to claim 80, wherein said HSV-tk gene encodes a protein comprising the sequence of SEQ ID NO:

4.

82. The method according to claim 78, wherein said suicide gene is an Escherichia coli cytosine deaminase gene (EC-CD) and said activator is 5-fluorocytosine (5-FC), 83. The method according to claim 82, wherein said EC-CD gene encodes a protein comprising at least 90% sequence identity with SEQ ID NO:

5.

84. The method according to claim 83, wherein said EC-CD gene encodes a protein comprising the sequence SEQ ID NO:

5.

85. The method according to claim 78, wherein said suicide gene encodes a caspase-inducible protein and said trigger is a specific chemical inducer of dimerization (CID).

86. The method according to claim 85, wherein said gene encodes an inducible caspase protein comprising at least 90% sequence identity with SEQ ID NO:

6.

87. The method according to claim 86, wherein said gene encodes an inducible caspase protein comprising the sequence SEQ ID NO:

6.

88. The method according to any of claims 85-87, wherein said CID is AP1903.