Universal cells and their manufacturing method

Abstract

The present invention discloses universal cells and methods for their production. By inactivating major histocompatibility complex (MHC) class I and class II genes in cells and then overexpressing at least one of SLC35A1, GNE, CMAS, ST3Gal5, ST8SIA1, MUC1, APMAP, SMAGP, and CD43, the resulting human pluripotent stem cells or human cell lines can avoid attack by T cells and further avoid killing by NK cells and macrophages. At the same time, these low-immunogenic pluripotent stem cells retain their stemness and differentiation potential. [Figure 1] JPEG2026501446000009.jpg117169

Description

Detailed Description of the Invention

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and benefit of Chinese invention patent application bearing application number 202211627210.4, filed on December 16, 2022, the entire contents of which are incorporated herein by reference.

[0002] [Technical Field] The present invention relates to the general field of genetic engineering and stem cell technology, and specifically to universal cells and methods for producing the same.

[0003] [Background technology] Although in vitro cell culture or stem cell differentiation induction can regenerate large numbers of healthy functional cells in vitro, and allotransplantation of functional cells can treat diseases, immune incompatibility and immune rejection of transplanted cells are major obstacles to their clinical use. Stem cells are "seed" cells with the ability to self-renew and differentiate into somatic cells with specific functions. Based on differences in stem cell properties, they are broadly classified into totipotent stem cells (TSCs), pluripotent stem cells (PSCs), and adult stem cells. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have the potential for unlimited proliferation, self-renewal, and differentiation into various cell types, and hold promising prospects for use in the treatment of cancer, neurological, cardiovascular, and other related diseases.

[0004] Although autologous cell transplantation therapy can avoid immune rejection, the cost of producing autologous cells from patients is high, the manufacturing process takes a long time (Khera et al., 2013), and the quality and efficacy of cell products derived from individuals are unstable. Evidence suggests that cells in patients' bodies differ from those of the general population, which may affect the therapeutic efficacy of autologous cells.

[0005] Studies have shown that immunosuppressants, HLA matching, and genome editing can reduce immunogenicity and the host immune system's rejection of xenotransplanted cells. Immunosuppressants have significant side effects, including bone marrow suppression, liver toxicity, hair loss, and gastrointestinal adverse events. Currently, HLA-matched iPSC banks have been established in the United States, Japan, and China. However, because HLA antigen genes are the most polymorphic genes in the human genome, establishing and maintaining these banks is costly. Currently, matching tests in each region's iPSC banks cannot reach the majority of the population in each country and can only cover specific populations (Solomon et al., 2015; Turner et al., 2013). Allogeneic cell therapy for large patient populations offers clear advantages over matching banks in terms of economics and construction and operating costs, but suffers from severe immune rejection. Therefore, the creation of universal PSCs with allogeneic immunocompatibility is urgently needed.

[0006] The human major histocompatibility complex (MHC), or human leukocyte antigens (HLA), is responsible for immune incompatibility. The HLA complex is composed of a series of genes classified into class I, class II, and class III. MHC class I genes are expressed in most histiocyte types, but transplanted cells expressing "non-autologous" MHC class I molecules stimulate CD8+ T cell activation and are eliminated. CD4+ helper T cells recognize MHC class II genes on "non-autologous" cells, resulting in immune rejection, whereas class III molecules are not involved in immunity. Genome editing techniques can be used to modify immunogenic elements to generate hypoimmunogenic cells, enabling the mass production of immune-privileged "off-the-shelf" cell therapy products.

[0007] In recent years, previous reports have shown that knockout of genes such as B2M and CIITA can eliminate MHC class I and MHC class II gene expression on the cell surface or in the host, thereby enabling the cells to induce immune tolerance or evade T / B cell-specific immune responses, generating immunocompatible universal PSCs and laying an important foundation for the wider use of cells, tissues, and organs derived from universal PSCs. However, HLA molecules are the main inhibitory ligands for natural killer (NK) cells, and MHC class I-negative cells are susceptible to degradation by natural killer (NK) cells. In vivo and in vitro data have shown that host NK cells inhibit the expression of transplanted B2M - / - It has been shown that donor cells can be eliminated (Flahou et al., 2021). Therefore, previous methods need to be improved to generate better universal donor cells to avoid immune responses.

[0008] Summary of the Invention In response to the shortcomings of the prior art, the present invention provides a new method for modifying cells to achieve low immunogenicity, and for the first time, a large number of genes involved in the expression of sialic acid-related proteins are screened. Through multi-functional experiments, nine new genes, including SLC35A1, GNE, CMAS, ST3Gal5, ST8SIA1, MUC1, APMAP, SMAGP, and CD43, are finally identified, which can significantly reduce or avoid recognition and attack by the immune system, especially attack by natural killer cells and macrophages. The present invention for the first time presents a feasible strategy for the cross-disciplinary application of sialic acid-related protein genes to modify cells to achieve immune privilege effects, and has achieved unexpected technical results.

[0009] The present invention successfully constructed B2M / CIITA biallelic knockout positive clone DKO cells by knocking out beta-2-microglobulin (B2M) in the endoplasmic reticulum of human pluripotent stem cells and CIITA, a positive regulatory factor transcribed by MHC class II genes. Subsequently, lentiviral vectors were used to overexpress the novel genes identified in the present invention, SLC35A1, GNE, CMAS, ST3Gal5, ST8SIA1, MUC1, APMAP, SMAGP, or CD43, in the DKO cells. The resulting human pluripotent stem cells not only avoid T cell attack but also NK cell and macrophage killing. At the same time, these hypoimmunogenic pluripotent stem cells retain important biological functions of pluripotent stem cells, such as stemness and differentiation potential.

[0010] To achieve the above objectives, the present invention adopts the following technical solutions.

[0011] In a first aspect, the present invention provides a universal cell, which, relative to a wild-type cell: 1) reduced or absent expression of MHC class I and / or MHC class II human leukocyte antigens, and 2) The gene is an expression sequence of at least one of SLC35A1, GNE, CMAS, ST3Gal5, ST8SIA1, MUC1, APMAP, SMAGP, and CD43, and the expression level of the gene is increased; These cells can avoid attack by T cells and killing by NK cells and macrophages.

[0012] In some embodiments, the cells comprise under- or non-expressing MHC class I and MHC class II human leukocyte antigens.

[0013] In one embodiment, the cells further comprise modifying them to increase expression of one or more polypeptides, wherein the increased polypeptides are selected from DUX4, CD27, CD35, CD200, HLA-C, PD-L1, CD47, CD24, CD26, CCL21, Mfge8, and SerpinB9.

[0014] In one embodiment, in the above cells, a genome editing tool (e.g., a TALEN and / or CRISPR system) is used to target one or more genes of one or more transcriptional regulators encoding MHC class I and one or more genes of one or more transcriptional regulators encoding MHC class II, thereby achieving reduced or no expression of MHC class I and MHC class II genes.

[0015] In one embodiment, in order to achieve reduced or no expression of MHC class I and MHC class II genes, the MHC class I transcriptional regulatory factor is preferably selected from one or more of B2M, TAP1, TAP2, TAP-related glycoprotein (Tapasin), and NLRC5, and the MHC class II transcriptional regulatory factor is preferably selected from one or more of CIITA, RFXANK, RFX5, and RFXAP; More preferably, the transcriptional regulator is B2M or CIITA.

[0016] In one embodiment, the cell further comprises a genetic modification that targets the CIITA gene with a rare-cutter endonuclease that selectively inactivates the CIITA gene. In one embodiment, the cell further comprises a genetic modification that targets the B2M gene with a rare-cutter endonuclease that selectively inactivates the B2M gene.

[0017] In certain embodiments, the rare-cutter endonuclease is selected from a CAS protein, a TALE-nuclease, a zinc finger nuclease, a meganuclease, and a homing endonuclease.

[0018] In one embodiment, the genetic modification targeting the CIITA gene or the B2M gene with a rare-cutter endonuclease comprises a CAS protein or a polynucleotide encoding a CAS protein, and at least one guide ribonucleic acid sequence for specifically targeting the CIITA gene or the B2M gene.

[0019] In a specific embodiment, the CRISPR / CAS9 system is used to directly knock out the B2M and CIITA exon segments at both ends, respectively, of which the target sequences of the gRNA corresponding to the B2M gene are SEQ ID NO: 22 and SEQ ID NO: 23, and the target sequences of the gRNA corresponding to the CIITA gene are SEQ ID NO: 24 and SEQ ID NO: 25.

[0020] In one aspect, reduced or no expression of MHC class I and / or MHC class II genes is achieved in the cells by introducing gene expression modifier molecules corresponding to one or more genes of one or more transcriptional regulators encoding MHC class I or one or more genes of one or more transcriptional regulators encoding MHC class II, wherein the gene expression modifier molecules include one selected from siRNA, shRNA, microRNA, antisense RNA, and inhibitory molecules mediated by another RNA.

[0021] In one embodiment, the amino acid sequence of SLC35A1, GNE, CMAS, ST3Gal5, ST8SIA1, MUC1, APMAP, SMAGP, or CD43 has 70% or more homology, for example 80% or more homology, further for example 90% or more, 95% or more, 98% or more, or 99% or more homology, to the sequence set forth in SEQ ID NO: 1, 3, 4, 8, 10, 12, 16, 17, or 19, respectively; More preferably, the amino acid sequence of SLC35A1, GNE, CMAS, ST3Gal5, ST8SIA1, MUC1, APMAP, SMAGP, or CD43 is set forth in SEQ ID NO: 1, 3, 4, 8, 10, 12, 16, 17, or 19, respectively; Even more preferably, the universal cell comprises an expression sequence of at least one of the following genes: MUC1, APMAP, and CD43; Most preferably, the universal cell comprises an expression sequence for CD43.

[0022] In some embodiments, the cells are embryonic stem cells.

[0023] In some embodiments, the cells are pluripotent stem cells.

[0024] In certain embodiments, the cells are hypoimmunogenic stem cells.

[0025] In certain embodiments, the cells are human stem cells or human somatic cells.

[0026] In a second aspect, the present invention provides a method for producing a universal cell according to the first aspect, comprising: 1) knocking out one or more genes of one or more transcriptional regulators of MHC class I in a cell, and / or 2) knocking out one or more genes of one or more transcriptional regulators of MHC class II in the cell; 3) introducing into the cell a nucleic acid sequence encoding at least one protein selected from SLC35A1, GNE, CMAS, ST3Gal5, ST8SIA1, MUC1, APMAP, SMAGP, and CD43.

[0027] In one aspect, preferably, the MHC class I transcriptional regulator is selected from one or more of B2M, TAP1, TAP2, TAP-related glycoprotein (Tapasin), or NLRC5, and preferably, the MHC class II transcriptional regulator is selected from one or more of CIITA, RFXANK, RFX5, and RFXAP.

[0028] In one embodiment, the transcriptional regulator is selected from B2M and CIITA.

[0029] In one embodiment, the knockout described in steps 1) and 2) is a genetic modification that targets the CIITA gene or the B2M gene with a rare-cutter endonuclease that selectively inactivates the CIITA gene or the B2M gene.

[0030] Preferably, said rare-cutter endonuclease is selected from CAS proteins, TALE-nucleases, zinc finger nucleases, meganucleases, and homing endonucleases.

[0031] More preferably, the genetic modification targeting the CIITA gene or B2M gene with a rare-cutter endonuclease comprises a CAS protein or a polynucleotide encoding the CAS protein, and at least one guide ribonucleic acid sequence for specifically targeting the CIITA gene or B2M gene.

[0032] In one embodiment, in steps 1) and 2), the CRISPR system is used to directly knock out the B2M and CIITA exon segments at both ends, respectively, in which the target sequences of the gRNA corresponding to the B2M gene are SEQ ID NO: 22 and SEQ ID NO: 23, and the target sequences of the gRNA corresponding to the CIITA gene are SEQ ID NO: 24 and SEQ ID NO: 25.

[0033] In one aspect, the knockout described in step 1) or 2) refers to reducing or eliminating the expression of MHC class I and / or MHC class II genes by introducing gene expression modifiers corresponding to one or more genes of one or more transcriptional regulators encoding MHC class I or one or more genes of one or more transcriptional regulators encoding MHC class II, wherein the gene expression modifiers include one selected from siRNA, shRNA, microRNA, antisense RNA, and other RNA-mediated inhibitory molecules.

[0034] In one aspect, in step 3), a nucleic acid sequence encoding at least one protein selected from SLC35A1, GNE, CMAS, ST3Gal5, ST8SIA1, MUC1, APMAP, SMAGP, and CD43 is introduced into the cell using a gene knock-in method.

[0035] In one aspect, in step 3), a nucleic acid sequence encoding at least one protein selected from SLC35A1, GNE, CMAS, ST3Gal5, ST8SIA1, MUC1, APMAP, SMAGP, and CD43 is introduced into the cell using an expression vector.

[0036] Preferably, the expression vector used in step 3) above is a viral vector.

[0037] In one embodiment, the viral vector utilized in step 3) is a lentivirus.

[0038] In one aspect, in step 3), a nucleic acid sequence encoding at least one protein selected from SLC35A1, GNE, CMAS, ST3Gal5, ST8SIA1, MUC1, APMAP, SMAGP, and CD43 is introduced into a predetermined site of the cell, and preferably, the predetermined site of the cell is a safe harbor gene site.

[0039] In one embodiment, the amino acid sequence of SLC35A1, GNE, CMAS, ST3Gal5, ST8SIA1, MUC1, APMAP, SMAGP, or CD43 has 70% or more homology, for example 80% or more homology, further for example 90% or more, 95% or more, 98% or more, or 99% or more homology, to the sequence set forth in SEQ ID NO: 1, 3, 4, 8, 10, 12, 16, 17, or 19, respectively; More preferably, the amino acid sequence of SLC35A1, GNE, CMAS, ST3Gal5, ST8SIA1, MUC1, APMAP, SMAGP, or CD43 is set forth in SEQ ID NO: 1, 3, 4, 8, 10, 12, 16, 17, or 19, respectively; Even more preferably, in step 3), a nucleic acid sequence encoding at least one protein selected from MUC1, APMAP, and CD43 is introduced into the cell using an expression vector; Most preferably, in step 3), a nucleic acid sequence encoding CD43 is introduced into cells using an expression vector.

[0040] In one embodiment, the universal cell further comprises a second expression vector, which comprises a polynucleotide sequence encoding one or more selected from DUX4, CD27, CD35, CD200, HLA-C, PD-L1, CD47, CD24, CD26, CCL21, Mfge8, and SerpinB9.

[0041] In one embodiment, the second expression vector is an inducible expression vector, and preferably, the second expression vector is a viral vector.

[0042] In a third aspect, the present invention provides a method for producing differentiated universal cells, comprising culturing universal cells produced by the method of the second aspect under differentiation conditions to produce differentiated, hypoimmunogenic cells.

[0043] In some embodiments, the differentiation conditions are applied to differentiate cells into a cell type selected from cardiomyocytes, neurons, glial cells, endothelial cells, T cells, NK cells, NKT cells, macrophages, hematopoietic progenitor cells, mesenchymal cells, islet cells, chondrocytes, retinal pigment epithelial cells, kidney cells, liver cells, thyroid cells, skin cells, blood cells, and epithelial cells.

[0044] In a fourth aspect, the present invention provides a method of treatment, comprising administering to a patient suitable for cell therapy a population of differentiated, hypoimmunogenic cells produced by a method according to the third aspect.

[0045] In a fifth aspect, the present invention provides a composition, said composition comprising a universal cell according to the first aspect.

[0046] In one aspect, the composition comprises a universal cell according to the first aspect and one or more therapeutic agents, including peptides, cytokines, small molecule compounds, large molecules, ADCs, antibodies, nanoparticles, biological analogs, mRNA, herbal medicines, proteins, vaccines, checkpoint inhibitors, mitogens, growth factors, small RNAs, double-stranded RNAs (dsRNAs), monocytes, feeder cells, feeder cell components or replacement factors thereof, vectors comprising one or more polynucleic acids of interest, antibodies, etc.

[0047] In a sixth aspect, the present invention provides a cell that has increased expression of at least one protein selected from SLC35A1, GNE, CMAS, ST3Gal5, ST8SIA1, MUC1, APMAP, SMAGP, and CD43, and has decreased or absent MHC class I and / or MHC class II human leukocyte antigens; Preferably, the cells express at least one of MUC1, APMAP, and CD43 proteins; More preferably, the cells express CD43. In a seventh aspect, the present invention provides a cell that does not express CIITA, has increased expression of at least one protein selected from SLC35A1, GNE, CMAS, ST3Gal5, ST8SIA1, MUC1, APMAP, SMAGP, and CD43, and has decreased or absent expression of MHC class I and / or MHC class II human leukocyte antigens; Preferably, the cells express at least one of MUC1, APMAP, and CD43 proteins; More preferably, the cells express CD43.

[0048] In an eighth aspect, the present invention provides a cell that does not express B2M, has increased expression of at least one protein selected from SLC35A1, GNE, CMAS, ST3Gal5, ST8SIA1, MUC1, APMAP, SMAGP, and CD43, and has reduced or absent MHC class I and / or MHC class II human leukocyte antigens; Preferably, the cells express at least one of MUC1, APMAP, and CD43 proteins; More preferably, the cells express CD43.

[0049] In a ninth aspect, the present invention provides a cell that does not express CIITA and B2M, has increased expression of at least one protein selected from SLC35A1, GNE, CMAS, ST3Gal5, ST8SIA1, MUC1, APMAP, SMAGP, and CD43, and has reduced or absent expression of MHC class I and / or MHC class II human leukocyte antigens; Preferably, the cells express at least one of MUC1, APMAP, and CD43 proteins; More preferably, the cells express CD43.

[0050] In a tenth aspect, the present invention provides a cell that has increased expression of at least one protein selected from SLC35A1, GNE, CMAS, ST3Gal5, ST8SIA1, MUC1, APMAP, SMAGP, and CD43, and at least one polypeptide selected from DUX4, CD27, CD35, CD200, HLA-C, PD-L1, CD47, CD24, CD26, CCL21, Mfge8, and SerpinB9, and has reduced or absent MHC class I and / or MHC class II human leukocyte antigens; Preferably, the cells express at least one of MUC1, APMAP, and CD43 proteins; More preferably, the cells express CD43.

[0051] In an eleventh aspect, the present invention provides a cell that does not express CIITA, but has increased expression of at least one protein selected from SLC35A1, GNE, CMAS, ST3Gal5, ST8SIA1, MUC1, APMAP, SMAGP, and CD43, and at least one polypeptide selected from DUX4, CD27, CD35, CD200, HLA-C, PD-L1, CD47, CD24, CD26, CCL21, Mfge8, and SerpinB9, and has reduced or no expression of MHC class I and / or MHC class II human leukocyte antigens; Preferably, the cells express at least one of MUC1, APMAP, and CD43 proteins; More preferably, the cells express CD43.

[0052] In a twelfth aspect, the present invention provides a cell that does not express B2M, but has increased expression of at least one protein selected from SLC35A1, GNE, CMAS, ST3Gal5, ST8SIA1, MUC1, APMAP, SMAGP, and CD43, and at least one polypeptide selected from DUX4, CD27, CD35, CD200, HLA-C, PD-L1, CD47, CD24, CD26, CCL21, Mfge8, and SerpinB9, and has reduced or no expression of MHC class I and / or MHC class II human leukocyte antigens; Preferably, the cells express at least one of MUC1, APMAP, and CD43 proteins; More preferably, the cells express CD43.

[0053] In a thirteenth aspect, the present invention provides a cell that does not express CIITA and B2M, but has increased expression of any one protein of SLC35A1, GNE, CMAS, ST3Gal5, ST8SIA1, MUC1, APMAP, SMAGP, or CD43, and at least one polypeptide selected from DUX4, CD27, CD35, CD200, HLA-C, PD-L1, CD47, CD24, CD26, CCL21, Mfge8, and SerpinB9, and has reduced or no expression of MHC class I and / or MHC class II human leukocyte antigens, Preferably, the cells express at least one of MUC1, APMAP, and CD43 proteins; More preferably, the cells express CD43.

[0054] In the cells according to the sixth to thirteenth aspects, the cells are selected from stem cells, differentiated cells, pluripotent stem cells, induced pluripotent stem cells, adult stem cells, progenitor cells, somatic cells, primary T cells, and chimeric antigen receptor T cells.

[0055] In a fourteenth aspect, the present invention provides use of a universal cell according to the first aspect, a composition according to the fifth aspect, or a cell according to any one of the sixth to thirteenth aspects in the manufacture of a product used in cell therapy.

[0056] In a fifteenth aspect, the present invention provides use of a universal cell according to the first aspect, a composition according to the fifth aspect, or a cell according to any one of the sixth to thirteenth aspects in the manufacture of a product used in organ transplantation.

[0057] In a sixteenth aspect, the present invention provides use of a universal cell according to the first aspect, a composition according to the fifth aspect, or a cell according to any one of the sixth to thirteenth aspects in constructing a universal PSCs cell bank.

[0058] In a seventeenth aspect, the present invention provides the use of a universal cell according to the first aspect, a composition according to the fifth aspect, or a cell according to any one of the sixth to thirteenth aspects as a gene therapy drug carrier.

[0059] In an eighteenth aspect, the present invention provides a reagent kit for differentiating the universal cells according to the first aspect into hypoimmunogenic cardiomyocytes, wherein the reagent kit comprises a first culture medium and a second culture medium; the first medium is a basal medium containing a GSK-3 inhibitor; The second medium is a basal medium containing a Wnt inhibitor.

[0060] In one embodiment, the reagent kit comprises a basal medium; and / or the GSK-3 inhibitor is selected from CHIR99021; and / or the Wnt inhibitor is selected from any one, two, or more of IWR-1, IWP-2, IWP-4, and C59; and / or the concentration of the GSK-3 inhibitor is 0.5 μM to 10 μM, preferably 6 μM; And / or, the concentration of the Wnt inhibitor is 0.5 μM to 20 μM, preferably 2 μM.

[0061] In one embodiment, the basal medium is selected from RPMI1640 medium, mTESR1 medium, etc., and more preferably RPMI1640 medium with B27 supplemented with insulin.

[0062] In a nineteenth aspect, the present invention provides a method for differentiating a universal cell according to the first aspect into a hypoimmunogenic cardiomyocyte, the method comprising: (a) culturing stem cells in a differentiation medium supplemented with a GSK-3 inhibitor to induce differentiation into cardiomyocytes; (b) continuing to induce differentiation in a differentiation medium; (c) continuing to induce differentiation in a differentiation medium supplemented with a Wnt inhibitor; (d) continuing to induce differentiation in a differentiation medium.

[0063] In one embodiment, the method comprises: (a) on day 0 to 2, culturing stem cells in a differentiation medium supplemented with a GSK-3 inhibitor to induce differentiation into cardiomyocytes; (b) one day after induction, continuing to induce differentiation in basal medium; (c) On days 3 to 5, continuing to induce differentiation in a differentiation medium supplemented with a Wnt inhibitor; (d) continuing to induce differentiation in the basal medium on days 5 to 15.

[0064] In one embodiment, the differentiation medium in steps (a), (b), (c), and (d) comprises RPMI1640 basal medium and B27 supplement without insulin. In one embodiment, the GSK-3 inhibitor is selected from CHIR99021; and / or the Wnt inhibitor is selected from any one, two, or more of IWR-1, IWP-2, IWP-4, and C59; In one embodiment, the concentration of the GSK-3 inhibitor is 0.5 μM to 10 μM, preferably 6 μM; and / or the concentration of the Wnt inhibitor is 0.5 μM to 20 μM, preferably 2 μM; In one embodiment, when the universal cells in step (a) have grown to a confluency of 85 to 98% (for example, 95%), the medium is replaced with a differentiation medium to induce differentiation.

[0065] In one embodiment, in step (d), motile cells can be observed on day 9.

[0066] In certain embodiments, stem cells include pluripotent stem cells, specialized stem cells, and unipotent stem cells.

[0067] In certain embodiments, the stem cells are human pluripotent stem cells, for example, human embryonic stem cells, human induced pluripotent stem cells.

[0068] In certain embodiments, the article of manufacture is selected from a reagent, a composition, a medium, and the like.

[0069] In a twentieth aspect, the present invention provides a cell therapeutic agent for preventing or treating heart disease, the cell therapeutic agent comprising differentiated and mature cardiomyocytes obtained by the above method.

[0070] In certain embodiments, the cardiac disease may be at least one selected from the group consisting of myocardial infarction, angina pectoris, ischemic cardiomyopathy, primary cardiomyopathy, secondary cardiomyopathy, and heart failure, but is not limited to these.

[0071] Beneficial effects of the present invention: The present invention inactivates major histocompatibility complex (MHC) class I and class II genes in stem cells, and then overexpresses at least one protein selected from SLC35A1, GNE, CMAS, ST3Gal5, ST8SIA1, MUC1, APMAP, SMAGP, and CD43. The resulting human pluripotent stem cells can avoid attack by T cells and further avoid killing by NK cells and macrophages. Among these, the relative killing ratios of MUC1 (0.736), APMAP (0.744), and CD43 (0.520) were excellent, all below 0.75. DKO+CD43 cells were optimal for avoiding NK cell killing. At many ratios, the multiple killing rates were all lower than DKO, and the average killing rate was superior to that of the positive control WT and the conventional DKO+CD47 (0.581 and 0.579). iPSC-DKO+CD43 cells clearly avoided MAC cell killing, with an evasive effect superior to that of iPSC-WT and iPSC-DKO cells. This unexpected transformation effect was achieved. At the same time, these low-immunogenic pluripotent stem cells retained their stemness and differentiation potential.

[0072] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] B2M gene knockout strategy (A) in human embryonic stem cell line H1 and human induced pluripotent stem cell line iPSC, and results (B, C).

[0073] [Figure 2] Strategy for knocking out the CIITA gene in human embryonic stem cell line H1 and human induced pluripotent stem cell line iPSC (A) and results (B, C).

[0074] [Figure 3] This is an expression diagram showing the RNA levels of B2M and CIITA detected by RT-qPCR in H1 cells and DKO cells in Example 1.

[0075] FIG. 4 shows the results of detecting B2M protein levels in H1 cells and DKO cells by Western blotting in Example 1.

[0076] [Figure 5] This is a diagram showing the expression of HLA class I / class II in various cells detected by flow cytometry after stimulating wild-type H1 cells WT, iPSC cells WT, and two types of cells DKO with INF-gamma in Example 1. Among them, T cells are a positive control for detecting HLA class I / class II molecules.

[0077] [FIG. 6] Karyotype diagram of the B2M / CIITA biallelic knockout positive clone H1 cell DKO obtained in Example 1.

[0078] [Figure 7] Expression diagram of stemness gene POU5F1 / NANOG / SOX2 at the RNA and protein levels in WT and DKO H1 cells detected by immunofluorescence (A) and RT-qPCR (B) in Example 2. Among them, MSCs served as a negative control.

[0079] [Figure 8] In Example 2, H1 cells and WT and DKO iPSC cells were detected by immunofluorescence and flow cytometry. Figure 8A shows the results of flow cytometry for detecting the expression of stemness genes SSEA-4 and Tra1-81 on the surface of WT H1 cells and DKO H1 cells. Figure 8B shows the results of flow cytometry for detecting the expression of stemness genes SSEA-4, TRA-1-60, TRA-1-81, OCT-4, and SOX2 on the surface of iPSC-WT and iPSC-DKO cells. Figure 8C shows the results of immunofluorescence for detecting the protein levels of stemness genes TRA-1-60, TRA-1-81, OCT-4, SOX2, and NANOG in iPSC-WT cells. Figure 8D shows the results of immunofluorescence for detecting the protein levels of stemness genes TRA-1-60, TRA-1-81, OCT-4, SOX2, and NANOG in iPSC-DKO cells.

[0080] [Figure 9] Using immunohistochemistry, H1 cells and DKO cells can form teratomas with three germ layers: inner, middle, and outer germ layers.

[0081] Figure 10 shows the results of testing the immune functions of WT and DKO cells using RTCA detection in Example 2. The three figures in the first row show the results of RTCA detection of the killing rates of NK cells against H1 WT and H1 DKO cells, i.e., the immune evasion function of H1 WT and H1 DKO cells against NK cells. The three figures in the second row show the results of RTCA detection of the killing rates of T cells against H1 WT and H1 DKO cells, i.e., the immune evasion function of WT and DKO cells against T cells. The three figures in the third row show the results of RTCA detection of the killing rates of NK cells against iPSC-WT and iPSC-DKO cells, i.e., the immune evasion function of iPSC-WT and iPSC-DKO cells against NK cells. The three figures in the fourth row show the results of RTCA detection of the killing rates of T cells against iPSC-WT and iPSC-DKO cells, i.e., the results of detection of the immune evasion function of iPSC-WT and iPSC-DKO cells against T cells.

[0082] [Fig. 11] Schematic diagram of the structure of pGC-EF1a plasmid.

[0083] FIG. 12 is a flow cytometric diagram showing the expression of CD47 in the H1 cell DKO+CD47 cell line constructed in Example 3.

[0084] FIG. 13 shows the results of NK cell killing of the CD47-overexpressing H1 cell DKO+CD47 cell line, H1 cell WT, and H1 cell DKO cells detected by RTCA in Example 3.

[0085] [Figure 14] This shows the results of RTCA detection of NK cell killing against cell lines overexpressing candidate proteins, H1 WT cells (positive control), and H1 DKO cells (negative control) in Example 4.

[0086] [Figure 15] For the H1 DKO+CD43 cell line constructed in Example 5, the overexpression level of mRNA relative to DKO was detected by qPCR.

[0087] [Figure 16] Expression of stemness genes in the H1 cell DKO+CD43 cell line constructed in Example 5 was detected by immunofluorescence (A) and flow cytometry (B).

[0088] [Figure 17] This is a diagram showing the results of immunofluorescence detection in Example 5, demonstrating that H1 DKO+CD43 cells expressed ecto-, meso-, and endodermal marker proteins at the protein level.

[0089] [Figure 18] In Example 5, immunohistochemistry was used to demonstrate that H1 cells DKO+CD43 cells can form teratomas with three germ layers: inner, middle, and outer.

[0090] [Figure 19] In Examples 6 and 8, RTCA was used to detect NK cell killing of the H1 DKO+CD43 cell line, H1 WT cells, H1 DKO cells, H1 DKO+CD47 cells, and H1 DKO+CD24 cells, all of which overexpress CD43 protein (A), and a summary of the results of repeated experiments normalized to the DKO killing at each time (B). Also shown is a summary of the results of killing of WT iPSC cells, DKO iPSC cells, and DKO iPSC+CD43 cell lines (C) and a summary of the results of repeated experiments normalized to the DKO killing at each time (D).

[0091] Figure 20 shows the results of RTCA detection of mixed lymphocyte (PBNK) killing of the CD43 protein-overexpressing H1 DKO+CD43 cell line, H1 WT cells, H1 DKO cells, H1 DKO+CD47 cells, and H1 DKO+CD24 cells in Examples 7 and 8 (A), and a summary of the results of repeated experiments normalized to the DKO killing at each time (B). Also shown is the results of killing of WT iPSC cells, DKO iPSC cells, and DKO iPSC+CD43 cell lines (C), and a summary of the results of repeated experiments normalized to the DKO killing at each time (D).

[0092] [Figure 21] This figure shows the results of Example 7, in which H1 WT cells, H1 DKO cells, H1 DKO cells+CD43 cells, H1 DKO cells+CD47 cells, and H1 DKO cells+CD24 cells were co-cultured with PBNK cells for 24 hours to detect whether they avoided PBNK cell killing.

[0093] [Figure 22] This figure shows the results of Example 7, in which WT H1 cells, DKO H1 cells, DKO H1 cells+CD43, DKO H1 cells+CD47, and DKO H1 cells+CD24 cells were co-cultured with PBNK cells for 24 hours, and then the number of spots formed by stimulating IFN-γ secretion from PBNK cells was detected by Elispot.

[0094] Figure 23 shows the results (A and B) of Example 7, in which the levels of CD107a degranulation in CD8+ T and NK cells in mixed lymphocytes were detected by FACS after co-culturing WT H1 cells, DKO H1 cells, DKO H1 cells+CD43, DKO H1 cells+CD47, and DKO H1 cells+CD24 cells with PBNK cells for 4 hours.

[0095] Figure 24 shows the results of Example 8, in which RTCA was used to detect the avoidance of macrophage cell killing after 24 hours of co-culture of WT iPSCs, DKO iPSCs, and DKO iPSCs + CD43 cells with PBNK cells (A), and a summary of the results of multiple repeated experiments normalized to the DKO killing rate each time (B).

[0096] [Figure 25] In Example 9, (A) shows the results of detecting the evasion of DKO+CD43 differentiated cells against NK cell killing by RTCA, and (B) shows a summary of the results of multiple repeated experiments normalized to the DKO killing each time.

[0097] [Figure 26] In Example 9, (A) shows the results of detecting the evasion of DKO+CD43 differentiated cells from killing by PBNK cells by RTCA, and (B) shows a summary of the results of multiple repeated experiments normalized to the DKO killing each time.

[0098] [Figure 27] In Example 9, (A) shows the results of detecting the evasion of DKO+CD43 differentiated cells against macrophage killing by RTCA, and (B) shows a summary of the results of multiple repeated experiments normalized to the DKO killing each time.

[0099] [Figure 28] Stemness gene protein detection was performed on NHP-iPSCs by immunofluorescence (A), and immunohistochemistry was used to demonstrate that the cells can form teratomas with three germ layers: inner, middle, and outer germ layers (B).

[0100] [Figure 29] This is a diagram showing the expression of HLA class I / class II in various cells detected by flow cytometry after stimulating NHP iPSC-WT and NHP iPSC-DKO cells with INF-gamma in Example 10.

[0101] Description: The labels NHP WT, NHP DKO1, NHP DKO2, and NHP DKO+CD43 in Figures 30 to 33 below correspond to NHP iPSC-WT, NHP iPSC-DKO1, NHP iPSC-DKO2, and NHP iPSC-DKO+CD43.

[0102] [Figure 30] This shows the results of detecting the killing of NHP iPSC-WT and NHP iPSC-DKO cells by monkey T cells using RTCA in Example 10.

[0103] [Figure 31] Example 10: Detection of NHP iPSC-DKO+CD43 overexpression.

[0104] Figure 32 shows the results of RTCA detection of monkey NK cell killing of NHP iPSC-WT and NHP iPSC-DKO cells, as well as the NHP iPSC-DKO+CD43 cell line overexpressing CD43 protein, in Example 10 (A), and a summary of the results of multiple repeated experiments normalized to the DKO killing rate each time (B).

[0105] Figure 33 shows the results of RTCA in Example 10, in which monkey PBMC cells were used to detect killing of NHP iPSC-WT and iPSC-DKO cells, as well as an NHP iPSC-DKO+CD43 cell line overexpressing CD43 protein (A), and a summary of the results of multiple repeated experiments normalized to the DKO killing rate for each experiment (B).

[0106] [Figure 34] Fluorescence imaging results of the transplanted cells of Example 10 in NHP (A) and NOG mice (B).

[0107] Figure 35 shows the results of detecting in vivo immune system activation in NHPs in Example 10, using Elispot to detect the number of spots formed by IFN-γ secretion by monkey immune cells after injection of NHP iPSC-WT and iPSC-DKO cells, and NHP iPSC-DKO+CD43 cells overexpressing CD43 protein into NHPs.

[0108] Figure 36 is a flowchart of hiPSC-CM differentiation, culture, and compound treatment in Example 11. The flowchart includes the time periods for pluripotent stem cell culture, cardiomyocyte differentiation, culture, purification, and compound treatment, as well as the media, additives, and compounds used, and corresponding procedures. Days -2 to 0 are the hiPSC culture stage, in which the culture medium used is mTESR1. Days 0 to 15 are the cardiomyocyte induction stage, in which the culture medium used is RPMI1640 supplemented with insulin-free B27, and cells are induced with the GSK3 inhibitor CHIR99021 and the Wnt inhibitor IWR-1, respectively. Days 15 to 30 are the cardiomyocyte maintenance stage, in which the culture medium used is RPMI1640 supplemented with B27.

[0109] [Figure 37] A is a bright-field schematic diagram and flow cytometry detection results of iCM cells at differentiation day 15 in Example 11, and B is a bright-field schematic diagram and flow cytometry detection results of iCM cells at differentiation day 30 in Example 1. The left side is the bright-field schematic diagram, and the right side is the cTnt positivity rate result detected by flow cytometry.

[0110] [Figure 38] Example 11 compares the expression of iCM and cardiomyocyte-related genes by fluorescent quantitative PCR on day 30 of differentiation.

[0111] [Figure 39] Schematic diagram of iCM calcium transient oscillation in Example 11.

[0112] [Figure 40] Results of echocardiographic function detection in mice with myocardial infarction in Example 11.

[0113] [Figure 41] This is an expression diagram of HLA class I / class II in various cells detected by flow cytometry after stimulating each iCM cell type with INF-gamma in Example 12.

[0114] [Figure 42] This shows the results of detecting NK cell killing of iCM cells differentiated from WT and DKO+CD43 cells by RTCA in Example 12.

[0115] [Figure 43] Photographs showing the killing of iCM cells differentiated from WT and DKO+CD43 cells using PBMCs in Example 12.

[0116] [Figure 44] In Example 12, the CFSE signal level was detected by FACS to evaluate the activation ability of iCM cells differentiated from WT and DKO+CD43 cells toward PBMC cells.

[0117] Example 1. Construction of B2M and CIITA double knockout cell line (DKO) 1. Cell culture reagents:

[0118] [Table 1]

[0119] 2. Methods and Results: The present invention selects human induced pluripotent stem cells (iPSC cells) to construct target cell lines, which may be constructed using human pluripotent stem cell lines H1 (Wicell, WA01) or H9 (Wicell, WA09), and the cell culture and gene knockout reagents used are shown in Table 1. CRISPR / CAS9 is used to knock out beta-2-microglobulin (B2M) in the endoplasmic reticulum so that it does not form functional molecules on the cell surface MHC class I, thereby producing allogeneic CD8 + Avoidance of T cell killing: Avoidance of CD4+ T cell killing is achieved by knocking out CIITA, a positive regulator transcribed by MHC class II genes, thereby reducing the expression of MHC class II molecules.

[0120] The CRISPR / CAS9 gene knockout strategy for B2M, as shown in Figure 1, involved direct knockout of both ends of the B2M exon segment using B2M-gRNA1 and B2M-gRNA2, followed by genome sequence validation using two pairs of PCR primers, B2M-F1 / R1 and B2M-F2 / R2. Figure 1A shows the B2M gene knockout strategy for the B2M and CIITA double knockout cell lines (DKO). Figure 1B shows the PCR validation results for the B2M gene knockout in the H1 cell line. Figure 1C shows the PCR validation results for the B2M gene knockout in iPSC cell lines.

[0121] gRNA sequence: B2M-gRNA1:CGTGAGTAAACCTGAATCTT B2M-gRNA2:AGTCACATGGTTCACACGGC Identification primers B2M-F1:TGGGGCCAAATCATGTAGACTC B2M-R1:TCAGTGGGGGTGAATTCAGTGT B2M-F2+B2M-R2=608bp After knockout: no band B2M-F2:CAGAAGTCCTTGAGAGCCTCC B2M-R2:TGTGCATCAGTATCTCAGCAGG B2M-F2+B2M-R2=812bp After knockout: 569bp.

[0122] The CIITA CRISPR / CAS9 gene knockout strategy was shown in Figure 2. This strategy involved direct knockout of both ends of the CIITA exon segment using CIITA-gRNA1 and CIITA-gRNA2, followed by genome sequence validation using two pairs of PCR primers, CIITA-F1 / R1 and CIITA-F2 / R2. Figure 2A shows the CIITA gene knockout strategy for B2M and CIITA double knockout cell lines (DKO). Figure 2B shows the PCR validation results for the CIITA gene in the H1 cell line. Figure 2C shows the PCR validation results for the CIITA gene in iPSC cell lines.

[0123] gRNA sequence: CIITA-gRNA1:GATATTGGCATAAGCCTCCC CIITA-gRNA2:CATCGCTGTTAAGAAGCTCC Identification primers: CIITA-F1:CTGTGCCTCTACCACTTCTATG CIITA-R1:CCTTCCATGTCACACAACAGCC CIITA-F1+CIITA-R1=368bp After knockout: no band CIITA-F2:TGGAATCCACACTTTCCAGTTC CIITA-R2:TGGAGTCTCCGTTCCTCCAG CIITA-F2+CIITA-R2=889bp After knockout: 459bp The specific operations are as follows: 1) Human pluripotent stem cells were cultured to 80% confluency in a Matrigel-coated 6-well plate using mTeSR1. After digestion with Tryple, they were neutralized with DMEM / F12 and counted. 2 x 10 6 The cells were aspirated into EP tubes, centrifuged, and the supernatant removed.

[0124] 2) Neon transfection system: 100 μL electrotransfection system, 15 μg TrueCut TM Cas9 Protein + 3 μg gRNA (B2MgRNA1 + B2MgRNA2 + CIITA gRNA1 + CIITA gRNA2) was added to form the RNP system, which was then mixed uniformly and left at room temperature for 20 minutes.

[0125] 3) The cells were resuspended in 100 μL of RNP electrotransfection system and electrotransfected using a Neon transfection system with electrotransfection parameters of 1200 V, 30 ms, and 1 pause. After electrotransfection, the cells were quickly added to pre-warmed medium and uniformly seeded into one well of a Matrigel-coated 6-well plate.

[0126] 4) Fresh mTeSR1 medium was replaced daily. After single cell growth, one was picked and cloned into a 48-well plate. After the clone was amplified, a genome sample was collected and subjected to PCR to detect the genome editing status. The PCR results are shown in Figures 1 and 2. PCR-positive clones were sent to the company for further verification by Sanger sequencing.

[0127] 5) The positively identified B2M / CIITA biallelic knockout clone DKO was amplified and cultured, and cryopreserved.

[0128] The RNA expression levels of B2M and CIITA in the B2M / CIITA biallelic knockout clone DKO were detected by qPCR. As shown in Figure 3, knockout was confirmed.

[0129] B2M-F:AAGATGAGTATGCCTGCCGT B2M-R:ATGCGGCATCTTCAAACCTC CIITA-F:CCTGGAGCTTCTTAACAGCGA CIITA-R:TGTGTCGGGTTCTGAGTAGAG Western blot analysis was performed to detect the expression of B2M protein in the B2M / CIITA biallelic knockout clone DKO. As shown in Figure 4, knockout was confirmed.

[0130] WT and DKO cells were stimulated with IFN-gamma. After plating, cells were cultured for 2 days, and IFN-gamma-containing medium was added to the cells during liquid exchange. After 48 hours, the cells were digested and analyzed by flow cytometry for HLA class I / II expression. The results are shown in Figure 5. The B2M / CIITA biallelic knockout stem cell-positive clone H1 and the two iPSC DKO cell lines failed to respond to IFN-gamma stimulation and expressed HLA class I / II molecules. The top panel shows the results for H1 cells, and the bottom panel shows the results for iPSCs. In all panels, the left panel shows HLA class I detection using HLA-ABC, and the right panel shows HLA class II detection using HLA-DR, DQ, and DP. T cells served as a positive control for HLA class I / II detection.

[0131] Karyotype analysis was performed on the resulting B2M / CIITA biallelic knockout H1 cell positive clone (DKO). Chromosome preparations fixed on glass slides were treated with trypsin and then stained with Giemsa stain. The number and morphological structure of metaphase chromosomes were analyzed to determine whether the karyotype matched the normal karyotype. As shown in Figure 6, the karyotype of the H1 DKO cell was normal.

[0132] Example 2. Verification of stemness and immune function of the DKO cell line in Example 1 1. Expression of stemness genes in WT and DKO cells Immunofluorescence detection showed that iPSCs and WT and DKO cells of the H1 cell line expressed the stemness genes POU5F1 and NANOG at the protein level. Cells were plated in 12-well plates and grown to 60-80% confluency. The medium was then aspirated and fixed with 4% paraformaldehyde. After cell membrane disruption, the cells were incubated overnight at 4°C with primary antibodies against POU5F1 and NANOG. After washing out the primary antibodies, the cells were incubated with fluorescently labeled secondary antibodies at room temperature and then photographed under a fluorescent microscope. The results are shown in Figure 7A. RT-qPCR detection showed that WT and DKO H1 cells expressed the stemness genes POU5F1, NANOG, and SOX2 at the RNA level. The results are shown in Figure 7B (MSCs served as a negative control for stemness gene expression).

[0133] The flow cytometry results are shown in Figure 8. Figure 8A shows that the stemness genes SSEA-4 and Tra1-81 were highly expressed on the cell surface of WT H1 cells and DKO H1 cells, with the percentages being 100%, 99.98%, 96.75%, and 99.13%, respectively. Figure 8B shows the expression of the stemness genes SSEA-4, TRA-1-60, TRA-1-81, OCT-4, and SOX2 on the cell surface of WT and DKO iPSCs by flow cytometry. Figure 8C shows the protein levels of the stemness genes TRA-1-60, TRA-1-81, OCT-4, SOX2, and NANOG in WT iPSCs by immunofluorescence. FIG. 8D shows the results of immunofluorescence detection of the protein levels of stemness genes TRA-1-60, TRA-1-81, OCT-4, SOX2, and NANOG in iPSC-DKO cells.

[0134] 2. Differentiation potential of the obtained B2M / CIITA biallelic knockout positive clones (DKO) Immunodeficient mice (SCID Beige) were subcutaneously injected with 100 μL of a suspension containing 5E+5 DKO cells, and teratomas with a volume of 1.5 cm were formed. 3 Once they had grown larger, they were removed and sectioned and stained.

[0135] The resulting B2M / CIITA biallelic knockout positive clones (DKO) formed teratomas in vivo and differentiated into cells representing the three germ layers, endo-, meso-, and ecto-, as shown in Figure 9 .

[0136] 3. Verification of immune function of DKO cells T cell and NK cell killing experiments were performed using the xCELLigence RTCA Instrument. Equal numbers of WT and H1 cells and the iPSC DKO cell line were resuspended in Essential 8 medium containing IL-2 and seeded onto Matrigel-coated 96-well E-plates. Activated T cells or NK cells were added for killing detection. RTCA detection data were analyzed using xCELLigence software to calculate the killing rate and evasion function.

[0137] [Table 2]

[0138] RTCA data shown in Figure 10 indicate that WT cells avoided NK cell killing but were killed by T cells due to their expression of HLA class I. DKO cells were able to avoid T cell killing while at the same time being more susceptible to NK cell killing.

[0139] Example 3. Construction of DKO+CD47 cell line and verification of immune function CD47 (NM_198793) was overexpressed in H1 DKO cells obtained in Example 1 using a lentiviral vector. The amino acid sequence of CD47 is shown in SEQ ID NO: 26. The overexpression sequence cDNA (SEQ ID NO: 27) was constructed in a lentiviral plasmid (pGC-EF1a) primed with EF1a and carrying a puromycin screening marker. The structure of the pGC-EF1a plasmid is shown in Figure 11. After successful ligation, the plasmid was cleaved with BamHI / NheI, and the accuracy of the inserted sequence was verified by Sanger sequencing, followed by viral packaging. H1 DKO human pluripotent stem cells constructed in Example 2 were transfected, and the medium was replaced with puromycin-containing medium 24 hours later. 48 hours later, screening was performed. The results of the constructed stable transformant cell line, H1 DKO+CD47, are shown in Figure 12. After confirming correct expression, cell expansion and subsequent functional testing were performed.

[0140] Referring to Example 2, we used RTCA to examine whether the overexpressing H1 DKO+CD47 cell line could avoid killing by T cells and NK cells at the same time. As shown in Figure 13, NK cells could effectively kill H1 DKO cells, and H1 WT and H1 DKO+CD47 overexpressing cells could avoid killing by NK cells.

[0141] Example 4. Screening of universal cells expressing sialic acid-related molecule proteins Construction strategies and screening of each molecular protein according to the present invention: In this study, a total of 21 target candidates were screened, and their amino acid and cDNA sequences are shown in Table 3. The target candidates included sialic acid synthase genes, sialyltransferase genes, and highly sialylated cell surface protein genes. Sialic acid synthase gene related: SLC35A1, NANS, GNE, CMAS.

[0142] Sialyltransferase gene related: ST3Gal1, ST3Gal2, ST3Gal4, ST3Gal5, ST6Gal1, ST8SIA1, ST6GalNAC1.

[0143] Highly sialylated cell surface protein genes: MUC1, MUC12, MUC16, MUC21, APMAP, SMAGP, PODXL, CD43, CD44, CD45.

[0144] [Table 3]

[0145] The 21 target candidates screened in this study were overexpressed using lentiviral vectors in H1 DKO cells obtained in Example 1. The amino acid sequences of the 21 target candidates are shown in Table 3. The overexpression sequence cDNA (sequences shown in Table 3) was constructed into a lentiviral plasmid (pGC-EF1a) primed with EF1a and carrying a puromycin screening marker. The structure of the pGC-EF1a plasmid is shown in Figure 11. After successful ligation, the plasmid was cleaved with BamHI / NheI, and the accuracy of the inserted sequence was verified by Sanger sequencing, followed by viral packaging. H1 DKO human pluripotent stem cells constructed in Example 2 were transfected, and the medium was replaced with puromycin-containing medium 24 hours later. Screening was performed after 48 hours. Stable transformants of the 21 target candidates were subjected to multiple NK cell in vitro killing experiments (n≧5) to tentatively screen for new target candidates with the best NK cell evasion potential.

[0146] The relative killing ratios for each round were normalized to the negative control H1 cells and DKO cells. A killing ratio of less than 1 indicates evasion of DKO; the lower the ratio, the stronger the evasion ability. The positive controls were WT and DKO+CD47. The average relative killing ratios for multiple rounds were 0.581 and 0.579, respectively, which was significantly different from DKO, demonstrating the stability of the detection platform. The summary of the screening results is shown in Figure 14. Statistical analysis revealed that of the 21 genes screened, nine genes, including SLC35A1 (0.858), GNE (0.852), CMAS (0.903), ST3Gal5 (0.834), ST8SIA1 (0.822), MUC1 (0.736), APMAP (0.744), SMAGP (0.895), and CD43 (0.520), showed significant differences in resistance to DKO, while the remaining 12 genes showed significant differences in resistance to DKO. None of these genes demonstrated favorable evasion ability in in vitro NK cell killing experiments. Among them, the relative killing ratios of MUC1 (0.736), APMAP (0.744), and CD43 (0.520) were excellent, less than 0.75. However, H1 cells DKO+CD43 were the optimal cells for evading NK cell killing. At many ratios, the multiple killing rate was lower than that of DKO, and the average killing rate was superior to that of the positive control H1 cells WT and the prior art H1 cells DKO+CD47, demonstrating an unexpected transformation effect.

[0147] Example 5. Construction of DKO+CD43 cell line and detection of stemness and differentiation potential 1. Construction of H1 cell DKO+CD43 cells and detection of overexpression The nucleic acid sequence (NM_003123.6) encoding CD43 (the amino acid sequence of CD43 is set forth in SEQ ID NO: 19) was directly synthesized and constructed into a lentiviral plasmid (pGC-EF1a) primed with EF1a and carrying a puromycin screening marker. The structure of the pGC-EF1a plasmid is shown in Figure 11. The plasmid was cleaved with BamHI / NheI, and after successful ligation, the accuracy of the inserted sequence was verified by Sanger sequencing, and viral packaging was performed. H1 cell DKO human pluripotent stem cells obtained in Example 2 were transfected and screened by switching to a medium containing puromycin. qPCR was used to detect mRNA overexpression levels in the constructed H1 cell DKO+CD43 cells, with H1 cell DKO cells serving as a negative control. The results are shown in Figure 15.

[0148] CD43 F1:GCTGGTGGTAAGCCCAGAC CD43 R1:GGCTCGCTAGTAGAGACCAAA 2. Expression of stemness genes in H1 DKO+CD43 cells Referring to Example 2, immunofluorescence detection showed that H1 DKO+CD43 cells expressed the stemness genes NANOG, OCT4, SOX2, TRA-1-60, and TRA-1-81 at the protein level. The results are shown in Figure 16A. Flow cytometry detection showed that H1 DKO+CD43 cells highly expressed the stemness genes SSEA-4, TRA-1-60, TRA-1-81, and OCT-4 on their cell surface, with the percentages being 97.72%, 99.85%, 99.35%, and 95.39%, respectively. The results are shown in Figure 16B.

[0149] 3. Detection of the differentiation potential of H1 DKO+CD43 cells into three germ layers DKO+CD43 cells were used for tri-germ differentiation. To generate mesodermal, endodermal, and ectodermal cells, dissociated H1 DKO+CD43 single cells were resuspended in tri-germ medium supplemented with Y27632, and an appropriate amount of cells was attached to a well plate coated with Matrigel and containing cell culture glass. After 24 hours, the medium was replaced with prewarmed differentiation medium (STEMdiff™ Trilineage Differentiation Kit (STEMCELL, Catalog # 05230)), with daily changes. On day 7, mesodermal, endodermal, and ectodermal cells were obtained. Immunofluorescence detection showed that H1 DKO+CD43 cells expressed the ectodermal marker proteins PAX6 and GAD1, the mesodermal marker proteins Brachyury and NCAM, and the endodermal marker proteins SOX17 and FOXA2 at the protein level. The results are shown in Figure 17.

[0150] 4. Detection of teratoma-forming ability of H1 cells DKO+CD43 Referring to Example 2, 100 μL of a suspension containing 5E+5 H1 cells, DKO+43 cells, was subcutaneously injected into an immunodeficient mouse (SCID Beige), and a teratoma with a volume of 1.5 cm 3 After they had grown larger, they were removed and sectioned for staining. The results are shown in Figure 18. H1 cells DKO+CD43 cells can form teratomas in mice and differentiate into cells with three germ layers: internal, medial, and external.

[0151] Example 6. Verification of the NK killing evasion function of the H1 cell DKO+CD43 cell line This example involves the following cell lines: H1 cells DKO+CD47, H1 cells DKO+CD24.

[0152] The H1 DKO+CD47 cells were used in Example 3, and the H1 DKO+CD24 cells were derived from DKO and overexpressed CD24 by lentiviral transfection. The lentivirus construction and overexpression procedures are described in Example 3. The amino acid sequence of CD24 is MGRAMVARLGLGLLLLALLLPTQIYSSETTTGTSSNSSQSTSNSGLAPNPTNATTKAAGGALQSTASLFVVSLSLLHLYS.

[0153] CD47 promotes immune evasion by interacting with signal-regulatory protein alpha (SIRPa) on the surface of immune cells. Overexpression of CD47 in B2M and CIITA double knockout (DKO) cells confers evasion from T and NK cell killing. (PMID:32433947 / PMID:30778232)

[0154] CD24 promotes immune evasion by interacting with the inhibitory receptor sialic acid-binding Ig-like lectin 10 (Siglec-10) on the surface of immune cells (PMID: 31367043). The amino acid sequence of CD24 is shown in SEQ ID NO: 28.

[0155] 1. Detection of evasion function of H1 cell DKO+CD43 cells by RTCA Referring to Example 2, in an NK cell killing experiment using RTCA detection, the DKO cells constructed according to Example 2 were completely killed by NK cells, while H1 cells (WT), H1 cells (DKO+CD43), H1 cells (DKO+CD47), and H1 cells (DKO+CD24) were avoided. RTCA was performed multiple times, and the results were normalized to the DKO killing rate for each replicate. The results showed that H1 cells (DKO+CD43) had the lowest killing rate, and there was a significant difference compared to H1 cells (WT), H1 cells (DKO+CD24), and H1 cells (DKO+CD47), as shown in Figures 19A and 19B.

[0156] Example 7. Verification of the ability of DKO+CD43 cell lines to evade T+NK mixed lymphocyte (PBNK) killing 1. Detecting the PBNK evasion ability of H1 DKO+CD43 cells by RTCA NK activating factors were added to PBMCs in advance to increase the NK ratio and T cell killing capacity. RTCA experiments were then performed using activated mixed lymphocytes (PBNK) as effector cells. (PMID: 33309274) Referring to Example 2, in PBNK cell killing experiments using RTCA detection, H1 cells (WT) and H1 cells (DKO) constructed according to Example 2 were completely killed by NK cells, while H1 cells (DKO+CD43), H1 cells (DKO+CD47), and H1 cells (DKO+CD24) were avoided. RTCA was performed multiple times, and the results were normalized to the killing rate of each DKO cell. The killing rate of H1 cells (DKO+CD43) was found to be the lowest, and significantly different from that of H1 cells (DKO+CD24) and H1 cells (DKO+CD47). This is shown in Figures 20A and 20B.

[0157] 2. Elispot detection of IFN-γ spot secretion from PBNK cells The method described in "Application of Long-term cultured Interferon-γ Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle" (PMID: 26275095) was used to detect IFN-γ spot secretion by NK cells using Elispot to measure the immune evasion function of DKO+CD43 cells. The specific procedure is as follows: H1 cells (WT), H1 cells (DKO), H1 cells (DKO+CD43), H1 cells (DKO+CD47), and H1 cells (DKO+CD24) were plated in a 96-well plate. After 24 hours, the medium was discarded and PBNK cells were added for culture. After 24 hours, the PBNK cells were harvested and IFN-γ secretion was measured. At the same time, the remaining H1 cells (WT), H1 cells (DKO), H1 cells (DKO+CD43), H1 cells (DKO+CD47), and H1 cells (DKO+CD24) were monitored after PBNK cell removal. The results are shown in Figure 21. Compared with H1 cells (WT) and H1 cells (DKO+CD43), H1 cells (DKO+CD47), and H1 cells (DKO+CD24) were killed less by PBNK cells. Therefore, H1 cell DKO+CD43, H1 cell DKO+CD47, and H1 cell DKO+CD24 cells clearly have a higher ability to avoid killing by PBNK cells than WT and DKO cells.

[0158] After 24 h, the collected PBNK cells were plated onto IFN-γ antibody-coated 96-well plates and incubated in a 37°C incubator for 24 h. After adding affinity antibody and streptavidin, incubation was performed, followed by colorimetric detection of IFN-γ secretion spots.

[0159] The Elispot detection results are shown in Figure 22. PBNK-NC is a simple PBNK negative control without co-culture with target cells. After co-culture of H1 DKO+CD43, H1 DKO+CD47, and H1 DKO+CD24 cells with PBNK, the number of spots formed by stimulating IFN-γ secretion from PBNK cells was similar but significantly lower than that of WT and DKO cells. Among these, the number of spots formed by H1 DKO+CD43 cells was the lowest, and significantly lower than that of H1 DKO+CD47 and H1 DKO+CD24 cells. This indicates that overexpression of CD43, CD47, and CD24 can not only prevent T cell activation but also counteract the NK cell activation induced by B2M / CIITA knockout, with CD43 having the most potent effect.

[0160] 3. Measurement of immune evasion function of DKO+CD43 cells by detecting lymphocyte activation.

[0161] The specific operation method is as follows: The degree of CD107a degranulation detected by FACS indicates the activation of CD8+ T and NK cells in mixed lymphocytes by target cells. Target cells were seeded onto a 48-well plate coated with Matrigel. 24 hours after cell adhesion, pre-activated PBNK cells were resuspended at a 1:1 E:T ratio and added to the 48-well plate. At the same time, 1 μL of a Golgi transport inhibitor (Monensin) and 1 μL of BV-421-CD107a antibody were added to each well. After uniform mixing, the cells were placed in a 37°C incubator and continued to incubate.

[0162] After 4 hours of co-culture, the supernatant of PBNK cells was collected, centrifuged, and washed once with PBS to detect CD107a degranulation. The results are shown in Figures 23A and 23B. H1 cells (WT) and H1 cells (DKO) + CD43 cells showed reduced NK cell activation compared to DKO cells.

[0163] Example 8. Immune evasion function of iPSC DKO+CD43 cell line To verify the evasive function of iPSC-derived DKO+CD43 cells against different immune cells, experiments were performed using NK cells, PBMCs (a mixture of T cells and NK cells), and macrophages. RTCA was used to detect the evasive function of iPSC-DKO+CD43 cells against different immune cells. See Examples 2 and 3 for specific detection methods.

[0164] 1. Resistance of iPSC-DKO+CD43 cells to NK cell killing NK cell killing was measured using the XCelligence platform (ACEA BioSciences). Various iPSC cells were resuspended in 100 μl of cell-specific medium and plated onto a 96-well E-plate (ACEA BioSciences) coated with Matrigel (Sigma-Aldrich). After the cell index reached 1, NK cells were added at an E:T ratio of 1:1. Data were normalized and analyzed using RTCA software (ACEA). The results are shown in Figure 19C. iPSC-DKO+CD43 cells clearly evade NK cell killing, demonstrating superior evasive effect compared to iPSC-WT.

[0165] 2. Resistance of iPSC-DKO+CD43 cells to PBMC cell killing NK activators were added to PBMCs in advance to increase the proportion of NK cells and the killing ability of T cells. RTCA experiments were performed using activated mixed lymphocytes (PBMCs) as effector cells to comprehensively evaluate the immune evasion ability of iPSC-DKO+CD43 cells (see literature PMID: 33309274 for methods). Specific procedures were performed to measure PBMC cell killing using the XCelligence platform (ACEA BioSciences). Various iPSC cells were resuspended in 100 μl of cell-specific medium and plated onto a 96-well E-plate (ACEA BioSciences) coated with Matrigel (Sigma-Aldrich). After the cell index reached 1, PBMCs were added at an E:T ratio of 2:1. Data were normalized and analyzed using RTCA software (ACEA). The results are shown in Figure 20C. iPSC-DKO+CD43 cells clearly evaded PBMC killing, with a superior evasion effect compared to iPSC-WT.

[0166] 3. Resistance of iPSC-DKO+CD43 cells to macrophage killing MAC cell killing was measured using the XCelligence platform (ACEA BioSciences). Various iPSC cells were resuspended in 100 μl of cell-specific medium and plated onto a 96-well E-plate (ACEA BioSciences) coated with Matrigel (Sigma-Aldrich). After the cell index reached 1, MAC cells were added at an E:T ratio of 2:1. Data were normalized and analyzed using RTCA software (ACEA). The results are shown in Figure 24. iPSC-DKO+CD43 cells clearly evade MAC cell killing, demonstrating superior evasion compared to iPSC-WT and iPSC-DKO cells.

[0167] Example 9. Verification of immune evasion of DKO+CD43 differentiated cells Cells were plated on Matrigel and, when they reached 40% confluence, switched to differentiation medium (PMID: 22865887, Retinoic Acid-induced Differentiation of hESCs), with daily changes of medium. Once fully grown, they were transferred to 0.1% gelatin-coated culture dishes and passaged every 3 days with daily changes of medium, resulting in a 10-day differentiation cycle.

[0168] 1. RTCA detects the escape of H1 DKO+CD43 differentiated cells from NK cell killing. Referring to Example 2, in the NK cell killing experiments using RTCA detection, H1 cells (WT), H1 cells (DKO+CD43), H1 cells (DKO+CD47), and H1 cells (DKO+CD24) differentiated cells were avoided. RTCA was performed multiple times, and the results of multiple replicate experiments were normalized to the DKO killing rate for each replicate. The killing results, summarized in Figures 25A and 25B, show that H1 cells (DKO+CD43) had the lowest killing rate, and there was a significant difference compared to H1 cells (DKO+CD24) and H1 cells (DKO+CD47).

[0169] 2. RTCA detects the escape of H1 DKO+CD43 differentiated cells from T+NK mixed lymphocyte (PBNK) killing. NK activators were added to PBMCs in advance to increase the NK ratio and T cell killing capacity in PBMCs, and RTCA experiments were performed using activated mixed lymphocytes (PBNK) as effector cells. Referring to Example 2, in the PBNK cell killing experiments using RTCA detection, as shown in Figures 26A and 26B, WT and DKO differentiated cells were completely killed by NK cells, while H1 DKO+CD43, H1 DKO+CD47, and H1 DKO+CD24 differentiated cells were avoided.

[0170] 3. Detection of evasion of DKO+CD43 differentiated cells from macrophage killing by RTCA Macrophages were activated in advance and used as effector cells in the RTCA experiment. Referring to Example 2, in the macrophage killing experiment using RTCA detection, as shown in Figures 27A and 27B, H1 WT and H1 DKO differentiated cells were completely killed by macrophages (MAC), while H1 DKO+CD43 differentiated cells were spared.

[0171] Example 10. Construction and validation of universal NHP cells This example describes investigating the in vivo and in vitro immunogenicity of universal simian stem cells (iPSCs) in a non-human primate (NHP) model.

[0172] 1. Universal NHP Cell Construction a) Construction of NHP iPSC-DKO cells In this study, monkey iPSC-DKO (monkey iPSC cells, produced using the CTS™ CytoTune™-iPS 2.1 Sendai Viral Reprogramming Reagent Kit (product number: A34546)) cells were produced using the method described in Example 1. iPSC stemness gene expression and teratoma formation were detected using the method described in Example 2, and the results are shown in Figure 28.

[0173] NHP-B2M-gRNA1 and NHP-B2M-gRNA2 were used to directly knock out both ends of the B2M exon segment, followed by genome sequence validation using a pair of PCR primers NHP-B2M-F / R. NHP-CIITA-gRNA1 and NHP-CIITA-gRNA2 were used to directly knock out both ends of the CIITA exon segment, followed by genome PCR validation using a pair of PCR primers NHP-CIITA-F / R. Two NHP iPSC-DKOs were selected for further validation.

[0174] NHP-B2M-gRNA1 CGTGAGTAAACCTGAATCTT NHP-B2M-gRNA2 AGTCACATGGTTCACACGGC NHP-CIITA-gRNA1 CATCGCTGTTGAGAAGCTCC NHP-CIITA-gRNA2 GATATTGGCATAAGCCTCCC B2M identification primers NHP-B2M-F CATTTGGCCAGAGTGGAAATG NHP-B2M-R TGGGACTCATTCAGGGTAGTA CIITA identification primers NHP-CIITA-F CTGTGAGGTGACTGAGCATATC NHP-CIITA-R GGCCAGCAATGAGCATACTA NHP iPSC-WT, NHP iPSC-DKO1, and NHP iPSC-DKO2 cells were stimulated with IFN-gamma. Cells were plated in well plates, and on day 2, IFN-gamma-containing medium was added to the cells during liquid exchange. After 48 hours, the cells were digested and HLA class I / II expression was detected by flow cytometry. NHP iPSC-DKO1 and NHP iPSC-DKO2 cells are two different clonal lines produced from the same lot. The results are shown in Figure 29. B2M / CIITA biallelic knockout NHP iPSC-DKO1 and NHP iPSC-DKO2 cells did not express HLA class I / II and failed to respond to IFN-gamma stimulation, upregulating HLA class I / II.

[0175] b) Verification of the ability of NHP iPSC-DKO cells to evade T cell killing T cell killing was measured using the XCelligence platform (ACEA BioSciences). NHP iPSC-WT and iPSC-DKO cells were resuspended in 100 μl of cell-specific medium and plated onto a 96-well E-plate (ACEA BioSciences) coated with Matrigel (Sigma-Aldrich). After the cell index reached 1, T cells were added at an E:T ratio of 2:1. Data were normalized and analyzed using RTCA software (ACEA). The results are shown in Figure 30. NHP DKO cells clearly avoided T cell killing, while NHP iPSC-WT cells were killed.

[0176] 2. In Vitro Detection of Universal NHP Cell Evasion Against Immune System Killing NHP iPSC-DKO+CD43 (the gene sequence of monkey CD43 is shown in XM_005591647.3) cells were produced using the method described above, and overexpression was confirmed by rt-qPCR. The results are shown in Figure 31.

[0177] a) Killing monkeys NK cell killing was measured using the XCelligence platform (ACEA BioSciences). NHP iPSC-WT, NHP iPSC-DKO, and NHP iPSC-DKO+CD43 cells were resuspended in 100 μl of cell-specific medium and plated onto a 96-well E-plate (ACEA BioSciences) coated with Matrigel (Sigma-Aldrich). After the cell index reached 1, T cells were added at an E:T ratio of 1:1. Data were normalized and analyzed using RTCA software (ACEA). The results are shown in Figures 32A and 32B. NHP-WT and NHP iPSC-DKO+CD43 cells clearly evaded monkey NK cell killing, and their ability to resist killing became even more pronounced after CD43 overexpression.

[0178] b) Monkey PBMC killing PBMC cell killing was measured using the XCelligence platform (ACEA BioSciences). NHP iPSC-WT, NHP iPSC-DKO, and NHP iPSC-DKO+CD43 cells were resuspended in 100 μl of cell-specific medium and plated onto a 96-well E-plate (ACEA BioSciences) coated with Matrigel (Sigma-Aldrich). After the cell index reached 1, PBMC cells were added at an E:T ratio of 2:1. Data were normalized and analyzed using RTCA software (ACEA). The results are shown in Figure 33. NHP iPSC-DKO+CD43 cells clearly avoided PBMC killing, while NHP-WT and NHP-DKO cells were killed.

[0179] 3. In Vivo Detection of Universal NHP Cell Evasion Against Immune System Killing a) Survival rate of transplanted cells Throughout the experiment, bioluminescence imaging (BLI) was performed after injection of NHP cells to monitor universal NHP cell viability. For BLI, anesthetized monkeys were intravenously injected with D-luciferin potassium (375 mg / kg) (Biosynth AG) dissolved in sterile PBS (pH 7.4) (Gibco, Invitrogen) (intraperitoneal injection was used in anesthetized mice). Animals were imaged using Largo (Spectral Instruments Imaging, Tucson, AZ). Bioluminescence was measured in a region of interest (ROI) and measured as maximum photons / second / square centimeter / sphericity (p / s / cm). 2 Quantify in units of ( / sr).

[0180] Specifically, NHP iPSC-WT and NHP iPSC-DKO+CD43 cells were infected with luciferase (luc)-transfected lentivirus and injected separately. The luc luminescent substrate, potassium D-luciferin, was then intravenously injected, and the cell fluorescence intensity was monitored using an in vivo imaging system (iVIS spectrum, PerkinElmer) to demonstrate the viability of the cells in vivo. NHP iPSC-DKO+CD43 cells remained viable in vivo at day 14, whereas no signal was detected in NHP-WT cells, as shown in Figure 34A. The same batch of cells was injected into immunodeficient NOG mice and fluorescence was monitored. NHP iPSC-WT and NHP iPSC-DKO+CD43 cells continued to grow in the NOG mice, indicating no difference in their in vivo proliferation abilities, as shown in Figure 34B. The difference in survival time observed in NHPs between NHP iPSC-DKO+CD43 cells and other cells is due to the speed of rejection by the immune system, indicating that NHP iPSC-DKO+CD43 cells can resist immune rejection and have low immunogenicity.

[0181] b) The degree to which transplanted cells activate the body's immune system Referring to the procedures in Example 7, an Elispot experiment was performed to detect the degree of immune activation of the monkey's in vivo immune cells on days 0, 7, and 14 after cell transplantation. The Elispot results are shown in Figure 35. The number of spots formed by stimulating IFN-γ secretion after transplantation of NHP iPSC-DKO+CD43 cells into monkeys was significantly lower than that of NHP iPSC-WT cells, indicating that transplantation of CD43-overexpressing NHP cells did not significantly activate the in vivo immune system.

[0182] Example 11. Differentiation of universal stem cells into iCM cells 1. In vitro differentiation of stem cells into iCM cells The in vitro differentiation method of stem cell iPSCs into iCM cells is shown in Figure 36. When hiPSCs grew to approximately 95% confluence, the cells were first washed with DPBS, and then 1 mL of ethylenediaminetetraacetic acid (versene) was added and digested at 37°C for 5 minutes. The versene was aspirated, and the cells were pipetted into 1 mL of mTeSR1 containing 10 μM Y-27632 to separate into single cells. After counting, 2 × 10 5Cells were seeded at a density of 100 cells / well onto a 12-well plate coated with Matrigel (induction day -2). On day -1, they were cultured for 1 day in fresh mTeSR1 medium without Y-27632. On day 0, when the cells reached approximately 95% confluence, the medium was replaced with cardiomyocyte differentiation medium, which contained RPMI 1640 basal medium and insulin-free B27 supplement. From days 0 to 2 of induction, 5 μM CHIR99021 was added to the medium. On day 2, the medium was replaced with fresh differentiation medium. From days 3 to 7 of induction, 2 μM IWR-1 was added to the medium. On day 7, the medium was replaced with fresh differentiation medium, and the liquid was changed every two days. On day 9 of induction, spontaneous motile cells could be observed. On day 15 of differentiation, the medium was discarded, and 500 μL of TrypleExpress was added to each well and incubated at 37°C for 15 minutes. The cells were pipetted and the cell suspension was collected, then the wells were rinsed with differentiation medium to collect the remaining cells. The cell suspension was then filtered through a 70 μm pore size nylon filter and collected by centrifugation at 200 g for 5 min. The medium was maintained with the cardiomyocytes, and the cell pellet was resuspended and collected at 4 × 10 5 The cells were seeded at a density of 100 cells / well into 12-well plates coated with Matrigel and cultured, with the liquid replaced every two days.

[0183] 2. Confirmation of iCM Cell Labeling Derived from Universal Stem Cells a) FACS detection results Following this differentiation process, clear cell movement was observed around day 11. On days 15 and 30 of differentiation, hiPSC-CMs were digested with Tryple Express for 30 minutes, collected, centrifuged, and washed with PBS. Cells were then fixed with 4% PFA at room temperature for 30 minutes, blocked with antibody for clearing, and incubated at room temperature for 10 minutes. Primary antibody was added to the cells at the dilution indicated in the manufacturer's instructions. After incubation at room temperature for 1 hour, the cells were washed twice with PBS to remove nonspecifically bound primary antibody, and then analyzed by flow cytometry. The results are shown in Figure 37. The results showed that on day 15, the cTnt+ cell ratio in the WT group was 71.91%, and that in the DKO+CD43 iPSC group was 91.55%. On day 30, the cTnt+ cell ratio in the WT iPSC group was 93.03%, and that in the DKO+CD43 iPSC group was 94.11%.

[0184] b) QPCR detection results On day 30 of differentiation, expression of cardiomyocyte-related genes was compared by fluorescent quantitative PCR. The medium was aspirated, washed once with PBS, and 500 μL of Trizol-lysed cells was added to each well of a 12-well plate. The cell lysate was collected in a 1.5 mL RNase-free EP tube. 100 μL of chloroform was added to each tube, vortexed vigorously for 15 s, incubated at room temperature for 5 min, and then centrifuged at 12,000 g for 15 min at 4°C. The upper layer was carefully aspirated into a new 1.5 mL RNase-free EP tube, an appropriate amount of isopropanol was added, and the tubes were gently inverted six times. The upper layer was carefully aspirated at room temperature, an appropriate amount of isopropanol was added, and the tubes were gently inverted six times. The upper layer was then incubated at room temperature for 10 min, and then centrifuged at 12,000 g for 10 min at 4°C. The supernatant was then removed and 500 μL of pre-chilled 75% ethanol was added, rinsed, and centrifuged at 7,500 g for 5 min at 4°C. The ethanol was removed as thoroughly as possible, the tube was left open for 5 minutes, 15-20 μL of DEPC water was added, and the tube was placed on ice. After the RNA was dissolved, the RNA concentration was measured using a Nano Drop concentration meter. Reverse transcription was performed according to the PrimeScript™ RT Master Mix Reagent Kit instruction manual. The reaction system was prepared on ice.

[0185] Reaction system (20 μL):

[0186] [Table 4]

[0187] Reaction process:

[0188] [Table 5]

[0189] The resulting cDNA can be stored at -20°C. A fluorescent quantitative PCR reaction system was prepared using 2x SYBR Green qPCR Master Mix reagent.

[0190] Reaction system (20 μL):

[0191] [Table 6]

[0192] Reaction process:

[0193] [Table 7]

[0194] Based on the Ct values ​​provided by the software, the △△Ct algorithm was used to calculate the fold difference in sample mRNA expression.

[0195] The results are shown in Figure 38. iCMs differentiated from WT iPSC cells and DKO+CD43 iPSC cells expressed cardiomyocyte-related genes, and the relative expression levels of cardiomyocytes differentiated from DKO+CD43 iPSC cells were higher.

[0196] 3. Confirmation of iCM cell function derived from universal stem cells a) Calcium transient detection results of iCM cells derived from universal stem cells Calcium transients are one of the most representative functional parameters of cardiomyocytes, and excitation-contraction coupling in cardiomyocytes is mediated by intracellular calcium ion oscillations. In mature cardiomyocytes, 70% of calcium ion influx is mediated by ryanodine receptor 2 (RYR2) via the sarcoplasm. 2+ Ca induction 2+ Release (Ca 2+ -induced-Ca 2+ -release,CICR). Intracellular calcium ions are released from the sarcoplasmic / endoplasmic reticulum Ca 2+ ATP enzyme (sarcoplasmic / endoplasmic reticulum Ca 2+ATPase, SERCA) or Na+ / Ca 2+ Exchanger (Na+ / Ca 2+ Rhod-2 AM is a fluorescent dye that can pass through the cell membrane. Its fluorescence intensity is very weak before it enters the cell and is cleaved. After entering the cell, it is cleaved by intracellular esterases to form Fluo-4. Rhod-2 cannot pass through the cell membrane and remains intracellularly. Rhod-2 then binds to calcium ions and emits fluorescence, allowing it to indicate dynamic changes in calcium ion concentration in the cell.

[0197] On day 30 of differentiation, the medium was discarded, and medium containing 1 μM Rhod-2 AM was added. The cells were incubated at 37°C for 30 min to allow Fluo-4 AM to penetrate the cell membrane and enter the cells. After washing away excess dye, spontaneous calcium transients were recorded at 37°C using a DeltaVision Ultra live-cell workstation and system. Recordings lasted at least 30 s each time. As shown in Figure 39, calcium transient recordings showed that CMs differentiated from WT and DKO+CD43 iPSCs exhibited regular calcium transients. Among these, iCMs differentiated from DKO+CD43 iPSCs exhibited significantly higher calcium transient frequency and amplitude within 30 s than iCMs differentiated from WT iPSCs.

[0198] 4. Validation of drug effects of iCM cells derived from universal stem cells in a permanently ligated mouse myocardial infarction model Myocardial infarction (MI) and acute coronary heart disease are among the most common causes of death from cardiovascular disease. The mouse MI model, involving permanent ligation of the left anterior descending (LAD) coronary artery, closely resembles human MI. The mouse surgical model of myocardial infarction induced by permanent LAD coronary artery ligation is highly repeatable and is currently a widely used method for creating a mouse myocardial infarction model, providing a stable model basis for subsequent product therapeutic efficacy testing. After successful creation of the MI model, blood flow ceased in a large portion of the left ventricular myocardium. Insufficient oxygen supply to the myocardium leads to ischemic death of cardiomyocytes. This pathological condition triggers a response in ventricular tissue, ultimately resulting in ventricular dysfunction, remodeling, and heart failure. The specific steps for conducting the mouse permanent left anterior descending coronary artery ligation model are as follows: 1) The mice were weighed, and the anesthetic dose and respiratory volume were determined. A heating pad was preheated to 37°C.

[0199] 2) Mice were intraperitoneally injected with 1.25% tribromoethanol at a dose of 10 μL / g.

[0200] 3) Hair was removed from the throat and left side of the chest cavity of the mouse using a hair removal cream.

[0201] 4) The depth of anesthesia was checked by grasping the tail or hind legs, and the mouse was placed supine on a heating pad. A small gauze dressing was placed under the animal's head to prevent the eyes from overheating. Gel was applied to the eye area to prevent dryness.

[0202] 5) The limbs were fixed to the surface of a heating pad with tape. A 5-0 suture was placed around the upper front teeth, and the ends of the ring were attached to the heating pad with tape. The animal's mouth was held open, and the trachea was intubated via the oral cavity.

[0203] 6) The ventilator was set to a ventilation volume of 2 mL and a respiratory rate of 125 breaths / min.

[0204] 7) The left front paw was removed from the tape and the mouse was carefully moved under the right mirror. Once the animal was correctly positioned, the left front paw was fixed.

[0205] 8) The boundary between the left pectoralis minor and major muscles was determined, and an oblique incision was made in the skin along the line with scissors. The pectoral fascia was bluntly separated with blunt fine scissors without incision.

[0206] 9) Open the chest cavity with the ribs between the third and fourth ribs, making sure not to touch the heart or lungs to avoid contacting the arteries in the chest, which could cause severe bleeding.

[0207] 10) Using flexible fine forceps, the pericardium was slowly removed and opened (taking care not to damage the heart and lungs).

[0208] 11) Locate the left anterior descending (LAD) coronary artery. The LAD artery shows a single line extending from the left auricle to the apex of the heart. It is a pale pink line (or may not be visible).

[0209] 12) A 7-0 suture was passed through the left atrium, 2-3 mm below the LAD, using a suture needle. The suture was pulled gently to avoid damaging the cardiac tissue. (This position was designated the high ligation position; the middle ligation position was 1-2 mm below the high ligation position; the low ligation position was 1-2 mm below the middle ligation position. Note: It is important not to insert too deeply or too shallowly into the ventricular cavity.) In the sham-operated animals, the suture was pulled under the LAD and slowly removed, taking care not to damage the tissue. The third rib was clamped with forceps, and two 5-0 sutures were placed under the third and fourth ribs.

[0210] 13) Three drops of 0.9% saline solution were placed in the opening, and the expiratory tube was closed for two or three breathing cycles to allow the lungs to fill normally with air, and then the suture was pulled and fixed.

[0211] 14) The skin of the chest was sutured twice with 5-0 sutures and fixed twice.

[0212] 15) After the mice had woken up, they were returned to their cages and observed periodically.

[0213] 16) Four days after the modeling was completed, a second thoracotomy was performed and cells were injected into three locations around the infarct, with each injection volume being approximately 5-10 μL, for a total of approximately 2 million cells. The chest was then closed at the above steps, and the mouse's condition was observed. Two months after the cell injection, function was detected by echocardiography, and cardiac structure and function were evaluated in vivo using echocardiograms. Related measurements included left ventricular wall thickness, internal diameter, weight, contraction rate, ejection fraction, etc. After the mouse's chest and abdomen were depilated and anesthetized, the specific procedures were as follows: 17) The left ventricle was imaged in the B model next to the sternum in the long axis view.

[0214] 18) Adjust the mouse ultrasound stage to position the long axis of the left ventricle in the same plane as the ultrasound. When taking the image, the aortic valve and left ventricular apex are placed in the same plane as the ultrasound. The aortic valve and apex are fixed points and remain unchanged across consecutive studies in the same animal.

[0215] 19) An M model was performed on the left ventricle to image the ventricular wall and interior.

[0216] 20) After imaging, the relevant features were calculated at selected locations according to the maximum amplitude of ventricular systole and diastole: ejection fraction (EF%), left ventricular intramural fraction (FS%), left ventricular end-systolic volume (LVVol;s), and left ventricular end-diastolic volume (LVVol;d).

[0217] Cardiomyocytes differentiated approximately 30 days prior to differentiation were used to evaluate the efficacy of drugs in mice with myocardial infarction. The therapeutic efficacy of U66- and U68-mutated cardiomyocytes was compared. Three mice were included in the sham-operated group, three in the vehicle group, and three in the iPSC DKO + CD43 differentiated iCM treatment group, using a solvent containing 5% human serum albumin and 95% complex electrolytes. Figure 40 shows that the mutated cardiomyocytes significantly improved cardiac function in mice. Compared to the vehicle group, the iPSC DKO + CD43 differentiated iCM treatment group showed an improvement in EF (from 20.34 to 34.14), FS (from 9.12 to 15.83), a decrease in end-systolic volume (from 74.08 to 51.67), and a decrease in end-diastolic volume (from 59.01 to 34.29).

[0218] Data in this study are presented as mean ± standard deviation (SD). Differences between groups were calculated using Student's t-test, with P < 0.001 indicating a statistically significant difference. All data were plotted and analyzed using Graphpad Prism 8.0 software.

[0219] Example 12. Verification of low immunogenicity of iCM cells derived from universal stem cells 1. Background: Human pluripotent stem cells selected by this invention avoid allogeneic CD8+ T cell killing by knocking out beta-2-microglobulin (B2M) in the endoplasmic reticulum using CRISPR / CAS9, preventing the formation of functional MHC class I molecules on the cell surface. Avoidance of CD4+ T cell killing is achieved by knocking out CIITA, a positive regulatory factor transcribed by the MHC class II gene, thereby reducing the expression of MHC class II molecules. Expression of CD43 in B2M / CIITA biallelic knockout cells (DKO) using a lentiviral method avoids innate immune killing. The successfully constructed cells, iPSC DKO+CD43, are universal stem cells. Cardiomyocytes derived from universal stem cells (iPSC DKO+CD43-iCM) also have the ability to inactivate the immune system and avoid killing by it.

[0220] 2. Verification of low immunogenicity of iCM cells derived from universal stem cells a) Measurement of retention of the hypoimmunogenic phenotype of universally derived cells iCM cells differentiated from WT and DKO+CD43 iPSCs were stimulated with IFN-gamma. Cells were plated in well plates, and on day 2, IFN-gamma-containing medium was added during liquid exchange. After 48 hours, cells were digested and HLA class I / II expression was detected by flow cytometry. The results are shown in Figure 41. B2M / CIITA biallelic knockout iPSC DKO+CD43 stem cells differentiated into iCM positive clones did not express HLA class I / II, and failed to respond to IFN-gamma stimulation, although HLA class I / II expression was upregulated.

[0221] b) In vitro detection of evasion against immune system killing b-1. Verification of NK cell killing evasion function NK cell killing was measured using the XCelligence platform (ACEA BioSciences, San Diego, CA). iCM cells differentiated from WT iPSCs and DKO+CD43 iPSCs were resuspended in 100 μl of cell-specific medium and plated onto a Matrigel-coated 96-well E-plate (ACEA BioSciences). After the cell index reached 1, NK cells were added at an E:T ratio of 1:1. Data were normalized and analyzed using RTCA software (ACEA). The results are shown in Figure 42, indicating that only DKO+CD43 iPSCs evaded NK cell killing.

[0222] b-2. Verification of the ability to avoid killing by T+NK mixed lymphocytes (PBMC) NK activating factors were added to PBMCs in advance to improve and maintain the NK ratio and T cell killing ability in PBMCs. Killing experiments were then conducted using activated mixed lymphocytes as effector cells to comprehensively evaluate the immune evasion ability of iPSC DKO+CD43-iCM cells.

[0223] iCM cells differentiated from WT iPSCs and DKO+CD43 iPSCs pre-labeled with DIL (MCE, HY-D0083) were plated in 12-well plates. After 24 hours, the medium was discarded and PBMCs were added and co-cultured. After PBMC removal, the remaining iCM cells differentiated from WT and DKO+CD43 iPSCs were observed for 24 hours. The results are shown in Figure 43. Brightfield and fluorescent images showed significantly more viable DKO+CD43 iPSCs than WT-iPSCs, indicating that DKO+CD43 iPSCs were more resistant to immune cell killing.

[0224] c. In vitro detection of the degree of immune system activation iCM cells differentiated from WT iPSCs and DKO+CD43 iPSCs were plated in 12-well plates. After 24 hours, the medium was discarded and co-cultured with C-SFE-labeled PBMCs and PHA (PMID: 17122895). PHA is a PBMC activation stimulant. Flow cytometry was performed on day 5 to confirm whether iCM stimulated PBMC proliferation. The results are shown in Figure 44. The PHA-stimulated group stimulated PBMCs at 65.67%, the WT iPSC-iCM+PHA group at 83.55%, and the DKO+CD43-iPSC group at 64.97%. The results showed that WT iPSCs further stimulated PBMC proliferation with PHA, but co-culture of DKO+CD43-iPSCs with PBMCs did not further stimulate PBMC proliferation.

[0225] Although the embodiments of the present invention have been described above, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the scope of the spirit and principles of the present invention should be included within the scope of the claims of the present invention. [Brief explanation of the drawings]

[0226] [Figure 1] FIG. 1 shows the strategy for knocking out the B2M gene in human embryonic stem cell line H1 and human induced pluripotent stem cell line iPSC (A) and the results (B, C). [Figure 2] FIG. 1 shows the strategy for knocking out the CIITA gene in human embryonic stem cell line H1 and human induced pluripotent stem cell line iPSC (A) and the results (B, C). [Figure 3] FIG. 1 is an expression diagram showing the RNA levels of B2M and CIITA detected by RT-qPCR in H1 cells and DKO cells in Example 1. [Figure 4] FIG. 1 shows the results of detecting B2M protein levels in H1 cells and DKO cells by Western blotting in Example 1. [Figure 5]This figure shows the HLA class I / II expression patterns in wild-type H1 cells (WT), iPSC cells (WT), and two DKO cell types stimulated with INF-gamma in Example 1, and then detected by flow cytometry. T cells were used as a positive control for detecting HLA class I / II molecules. [Figure 6] FIG. 1 is a karyotype diagram of the B2M / CIITA biallelic knockout positive clone H1 cell DKO obtained in Example 1. [Figure 7A] Figure 2 shows the expression of stemness gene POU5F1 / NANOG / SOX2 at the RNA and protein levels in WT and DKO H1 cells by immunofluorescence (A) and RT-qPCR (B) in Example 2. MSCs were used as a negative control. [Figure 7B] Figure 2 shows the expression of stemness gene POU5F1 / NANOG / SOX2 at the RNA and protein levels in WT and DKO H1 cells by immunofluorescence (A) and RT-qPCR (B) in Example 2. MSCs were used as a negative control. [Figure 8A] In Example 2, WT and DKO H1 cells and iPSC cells were detected using immunofluorescence and flow cytometry. Figure 8A shows the results of flow cytometry analysis to detect the expression of stemness genes SSEA-4 and Tra1-81 on the cell surface of WT and DKO H1 cells. [Figure 8B] In Example 2, H1 cells and WT and DKO iPSC cells were detected using immunofluorescence and flow cytometry. Figure 8B shows the results of flow cytometry analysis to detect the expression of stemness genes SSEA-4, TRA-1-60, TRA-1-81, OCT-4, and SOX2 on the surface of iPSC-WT and iPSC-DKO cells. [Figure 8C]In Example 2, H1 cells and WT and DKO iPSC cells were detected by immunofluorescence and flow cytometry. Figure 8C shows the results of immunofluorescence detection of the protein levels of stemness genes TRA-1-60, TRA-1-81, OCT-4, SOX2, and NANOG in iPSC-WT cells. [Figure 8D] In Example 2, H1 cells and WT and DKO iPSC cells were detected by immunofluorescence and flow cytometry. Figure 8D shows the results of immunofluorescence detection of the protein levels of stemness genes TRA-1-60, TRA-1-81, OCT-4, SOX2, and NANOG in iPSC-DKO cells. [Figure 9] Using immunohistochemistry, H1 cells DKO cells were able to form teratomas with three germ layers: inner, middle, and outer. [Figure 10] These figures show the results of using RTCA to examine the immune functions of WT and DKO cells in Example 2. The three figures in the first row show the results of RTCA assay of NK cell killing rates against H1 WT and H1 DKO cells, i.e., the immune evasion function of H1 WT and H1 DKO cells against NK cells. The three figures in the second row show the results of RTCA assay of T cell killing rates against H1 WT and H1 DKO cells, i.e., the immune evasion function of WT and DKO cells against T cells. The three figures in the third row show the results of RTCA assay of NK cell killing rates against iPSC-WT and iPSC-DKO cells, i.e., the immune evasion function of iPSC-WT and iPSC-DKO cells against NK cells. The three figures in the fourth row show the results of RTCA detection of the killing rates of T cells against iPSC-WT and iPSC-DKO cells, i.e., the results of detection of the immune evasion function of iPSC-WT and iPSC-DKO cells against T cells. [Figure 11] FIG. 1 is a schematic diagram of the structure of pGC-EF1a plasmid. [Figure 12] FIG. 10 is an expression diagram of CD47 detected by a flow cytometer in the H1 cell DKO+CD47 cell line constructed in Example 3. [Figure 13] FIG. 10 shows the results of detecting NK cell killing of CD47-overexpressing H1 cell DKO+CD47 cell line, H1 cell WT, and H1 cell DKO cells by RTCA in Example 3. [Figure 14] FIG. 1 shows the results of detecting NK cell killing of cell lines overexpressing candidate proteins, H1 WT cells (positive control), and H1 DKO cells (negative control) by RTCA in Example 4. [Figure 15] For the H1 cell DKO+CD43 cell line constructed in Example 5, the overexpression level of mRNA relative to DKO was detected by qPCR. [Figure 16A] The expression of stemness genes in the H1 cell DKO+CD43 cell line constructed in Example 5 was detected by immunofluorescence (A) and flow cytometry (B). [Figure 16B] The expression of stemness genes in the H1 cell DKO+CD43 cell line constructed in Example 5 was detected by immunofluorescence (A) and flow cytometry (B). [Figure 17] FIG. 10 is a diagram showing the results of immunofluorescence detection in Example 5, showing that H1 DKO+CD43 cells expressed ecto-, meso-, and endodermal marker proteins at the protein level. [Figure 18] In Example 5, immunohistochemistry was used to demonstrate that H1 cells DKO+CD43 cells can form teratomas with three germ layers: inner, middle, and outer. [Figure 19A] In Examples 6 and 8, RTCA was used to detect NK cell killing of the H1 DKO+CD43 cell line, H1 WT cells, H1 DKO cells, H1 DKO+CD47 cells, and H1 DKO+CD24 cells, all of which overexpress CD43 protein. (A) This figure shows a summary of the results of repeated experiments normalized to the DKO killing rate at each time (B). (C) This figure shows a summary of the results of repeated experiments normalized to the DKO killing rate at each time (D). [Figure 19B]In Examples 6 and 8, RTCA was used to detect NK cell killing of the H1 DKO+CD43 cell line, H1 WT cells, H1 DKO cells, H1 DKO+CD47 cells, and H1 DKO+CD24 cells, all of which overexpress CD43 protein. (A) This figure shows a summary of the results of repeated experiments normalized to the DKO killing rate at each time (B). (C) This figure shows a summary of the results of repeated experiments normalized to the DKO killing rate at each time (D). [Figure 19C] In Examples 6 and 8, RTCA was used to detect NK cell killing of the H1 DKO+CD43 cell line, H1 WT cells, H1 DKO cells, H1 DKO+CD47 cells, and H1 DKO+CD24 cells, all of which overexpress CD43 protein. (A) This figure shows a summary of the results of repeated experiments normalized to the DKO killing rate at each time (B). (C) This figure shows a summary of the results of repeated experiments normalized to the DKO killing rate at each time (D). [Figure 19D] In Examples 6 and 8, RTCA was used to detect NK cell killing of the H1 DKO+CD43 cell line, H1 WT cells, H1 DKO cells, H1 DKO+CD47 cells, and H1 DKO+CD24 cells, all of which overexpress CD43 protein. (A) This figure shows a summary of the results of repeated experiments normalized to the DKO killing rate at each time (B). (C) This figure shows a summary of the results of repeated experiments normalized to the DKO killing rate at each time (D). [Figure 20A]In Examples 7 and 8, (A) shows the results of RTCA detection of mixed lymphocyte (PBNK) killing of the H1 DKO+CD43 cell line, H1 WT, H1 DKO, H1 DKO+CD47, and H1 DKO+CD24 cells, all of which overexpress CD43 protein, and (B) shows a summary of the results of repeated experiments normalized to the DKO killing at each time. (C) shows the results of killing of WT iPSC, DKO iPSC, and DKO iPSC+CD43 cell lines and (D) shows a summary of the results of repeated experiments normalized to the DKO killing at each time. [Figure 20B] In Examples 7 and 8, (A) shows the results of RTCA detection of mixed lymphocyte (PBNK) killing of the H1 DKO+CD43 cell line, H1 WT, H1 DKO, H1 DKO+CD47, and H1 DKO+CD24 cells, all of which overexpress CD43 protein, and (B) shows a summary of the results of repeated experiments normalized to the DKO killing at each time. (C) shows the results of killing of WT iPSC, DKO iPSC, and DKO iPSC+CD43 cell lines and (D) shows a summary of the results of repeated experiments normalized to the DKO killing at each time. [Figure 20C] In Examples 7 and 8, (A) shows the results of RTCA detection of mixed lymphocyte (PBNK) killing of the H1 DKO+CD43 cell line, H1 WT, H1 DKO, H1 DKO+CD47, and H1 DKO+CD24 cells, all of which overexpress CD43 protein, and (B) shows a summary of the results of repeated experiments normalized to the DKO killing at each time. (C) shows the results of killing of WT iPSC, DKO iPSC, and DKO iPSC+CD43 cell lines and (D) shows a summary of the results of repeated experiments normalized to the DKO killing at each time. [Figure 20D]In Examples 7 and 8, (A) shows the results of RTCA detection of mixed lymphocyte (PBNK) killing of the H1 DKO+CD43 cell line, H1 WT, H1 DKO, H1 DKO+CD47, and H1 DKO+CD24 cells, all of which overexpress CD43 protein, and (B) shows a summary of the results of repeated experiments normalized to the DKO killing at each time. (C) shows the results of killing of WT iPSC, DKO iPSC, and DKO iPSC+CD43 cell lines and (D) shows a summary of the results of repeated experiments normalized to the DKO killing at each time. [Figure 21] This figure shows the results of Example 7, in which H1 WT cells, H1 DKO cells, H1 DKO cells+CD43 cells, H1 DKO cells+CD47 cells, and H1 DKO cells+CD24 cells were co-cultured with PBNK cells for 24 hours to detect whether they avoided PBNK cell killing. [Figure 22] This figure shows the results of Example 7, in which WT H1 cells, DKO H1 cells, DKO H1 cells+CD43, DKO H1 cells+CD47, and DKO H1 cells+CD24 cells were co-cultured with PBNK cells for 24 hours, and then the number of spots formed by stimulating IFN-γ secretion from PBNK cells was detected by Elispot. [Figure 23A] In Example 7, H1 cells WT, H1 cells DKO, H1 cells DKO+CD43, H1 cells DKO+CD47, and H1 cells DKO+CD24 were co-cultured with PBNK cells for 4 hours, and then the degree of CD107a degranulation of CD8+ T and NK cells in the mixed lymphocytes was detected by FACS (Figures A and B). [Figure 23B] In Example 7, H1 cells WT, H1 cells DKO, H1 cells DKO+CD43, H1 cells DKO+CD47, and H1 cells DKO+CD24 were co-cultured with PBNK cells for 4 hours, and then the degree of CD107a degranulation of CD8+ T and NK cells in the mixed lymphocytes was detected by FACS (Figures A and B). [Figure 24A]In Example 8, WT iPSC cells, DKO iPSC cells, and DKO iPSC cells + CD43 cells were co-cultured with PBNK cells for 24 hours, and then the results were detected by RTCA to avoid macrophage cell killing (A). Also, the results of multiple repeated experiments, normalized to the DKO cell killing rate each time (B), are summarized. [Figure 24B] In Example 8, WT iPSC cells, DKO iPSC cells, and DKO iPSC cells + CD43 cells were co-cultured with PBNK cells for 24 hours, and then the results were detected by RTCA to avoid macrophage cell killing (A). Also, the results of multiple repeated experiments, normalized to the DKO cell killing rate each time (B), are summarized. [Figure 25A] FIG. 10 shows the results of detecting the evasion of DKO+CD43 differentiated cells from NK cell killing by RTCA in Example 9 (A), and a summary of the results of multiple repeated experiments normalized to the DKO killing each time (B). [Figure 25B] FIG. 10 shows the results of detecting the evasion of DKO+CD43 differentiated cells from NK cell killing by RTCA in Example 9 (A), and a summary of the results of multiple repeated experiments normalized to the DKO killing each time (B). [Figure 26A] FIG. 10 shows the results of detecting, by RTCA, the escape of DKO+CD43 differentiated cells from killing of PBNK cells in Example 9 (A), and a summary of the results of multiple repeated experiments normalized to the DKO killing each time (B). [Figure 26B] FIG. 10 shows the results of detecting, by RTCA, the escape of DKO+CD43 differentiated cells from killing of PBNK cells in Example 9 (A), and a summary of the results of multiple repeated experiments normalized to the DKO killing each time (B). [Figure 27A] FIG. 10 shows the results of detecting the evasion of DKO+CD43 differentiated cells from macrophage killing by RTCA in Example 9 (A), and a summary of the results of multiple repeated experiments normalized to the DKO killing each time (B). [Figure 27B]FIG. 10 shows the results of detecting the evasion of DKO+CD43 differentiated cells from macrophage killing by RTCA in Example 9 (A), and a summary of the results of multiple repeated experiments normalized to the DKO killing each time (B). [Figure 28A] Immunofluorescence was used to detect stemness gene proteins in NHP-iPSCs (A), and immunohistochemistry was used to demonstrate that the cells can form teratomas with three germ layers: inner, middle, and outer germ layers (B). [Figure 28B] Immunofluorescence was used to detect stemness gene proteins in NHP-iPSCs (A), and immunohistochemistry was used to demonstrate that the cells can form teratomas with three germ layers: inner, middle, and outer germ layers (B). [Figure 29] FIG. 10 shows an expression diagram of HLA class I / class II in various cells detected by flow cytometry after stimulating NHP iPSC-WT and NHP iPSC-DKO cells with INF-gamma in Example 10.

[0227] Description: The labels NHP WT, NHP DKO1, NHP DKO2, and NHP DKO+CD43 in Figures 30 to 33 below correspond to NHP iPSC-WT, NHP iPSC-DKO1, NHP iPSC-DKO2, and NHP iPSC-DKO+CD43. [Figure 30] FIG. 11 shows the results of detecting the killing of NHP iPSC-WT and NHP iPSC-DKO cells by monkey T cells using RTCA in Example 10. [Figure 31] In Example 10, detection of NHP iPSC-DKO+CD43 overexpression. [Figure 32A] In Example 10, (A) shows the results of RTCA detection of monkey NK cell killing of NHP iPSC-WT and NHP iPSC-DKO cells, as well as the NHP iPSC-DKO+CD43 cell line overexpressing CD43 protein, and (B) shows a summary of the results of multiple repeated experiments normalized to the DKO killing rate in each experiment. [Figure 32B]In Example 10, (A) shows the results of RTCA detection of monkey NK cell killing of NHP iPSC-WT and NHP iPSC-DKO cells, as well as the NHP iPSC-DKO+CD43 cell line overexpressing CD43 protein, and (B) shows a summary of the results of multiple repeated experiments normalized to the DKO killing rate in each experiment. [Figure 33A] In Example 10, (A) shows the results of RTCA detection of monkey PBMC cell killing of NHP iPSC-WT and iPSC-DKO cells, as well as the NHP iPSC-DKO+CD43 cell line overexpressing CD43 protein, and (B) shows a summary of the results of multiple repeated experiments normalized to the DKO killing rate for each experiment. [Figure 33B] In Example 10, (A) shows the results of RTCA detection of monkey PBMC cell killing of NHP iPSC-WT and iPSC-DKO cells, as well as the NHP iPSC-DKO+CD43 cell line overexpressing CD43 protein, and (B) shows a summary of the results of multiple repeated experiments normalized to the DKO killing rate for each experiment. [Figure 34] 1 shows the results of fluorescence imaging of the transplanted cells of Example 10 in NHP (A) and NOG mice (B). [Figure 35] This figure shows the results of detecting the activation of the in vivo immune system of NHPs in Example 10, using Elispot to detect the number of spots formed by IFN-γ secretion by monkey immune cells after injecting NHP iPSC-WT and iPSC-DKO cells, and NHP iPSC-DKO+CD43 cells that overexpress CD43 protein into NHPs. [Figure 36]1 shows a flowchart of hiPSC-CM differentiation, culture, and compound treatment in Example 11. The flowchart includes the time periods for pluripotent stem cell culture, cardiomyocyte differentiation, culture, purification, and compound treatment, as well as the media, additives, and compounds used, and corresponding procedures. Days -2 to 0 are the hiPSC culture stage, in which the culture medium used is mTESR1. Days 0 to 15 are the cardiomyocyte induction stage, in which the culture medium used is RPMI1640 supplemented with insulin-free B27, and cells are induced with the GSK3 inhibitor CHIR99021 and the Wnt inhibitor IWR-1, respectively. Days 15 to 30 are the cardiomyocyte maintenance stage, in which the culture medium used is RPMI1640 supplemented with B27. [Figure 37] A is a bright-field schematic diagram and flow cytometry detection results of iCM cells on differentiation day 15 in Example 11, and B is a bright-field schematic diagram and flow cytometry detection results of iCM cells on differentiation day 30 in Example 1. The left side is the bright-field schematic diagram, and the right side is the cTnt positivity rate result detected by flow cytometry. [Figure 38] In Example 11, the expression of iCM and cardiomyocyte-related genes is compared by fluorescent quantitative PCR on day 30 of differentiation. [Figure 39] FIG. 11 is a schematic diagram of iCM calcium transient oscillation in Example 11. [Figure 40] 1 shows the results of echocardiographic functional detection of mice with myocardial infarction in Example 11. [Figure 41] This is an expression diagram of HLA class I / class II in various cells detected by flow cytometry after stimulating each iCM cell with INF-gamma in Example 12. [Figure 42] FIG. 13 shows the results of detecting NK cell killing of iCM cells differentiated from WT and DKO+CD43 cells by RTCA in Example 12. [Figure 43] 12 shows photographs of the killing of iCM cells differentiated from WT and DKO+CD43 cells using PBMCs in Example 12. [Figure 44]In Example 12, the CFSE signal level was detected by FACS to evaluate the activation ability of iCM cells differentiated from WT and DKO+CD43 cells toward PBMC cells.

Claims

1. Universal cells, relative to wild-type cells, 1) reduced or absent expression of MHC class I and / or MHC class II human leukocyte antigens; and 2) the gene is an expression sequence of at least one of SLC35A1, GNE, CMAS, ST3Gal5, ST8SIA1, MUC1, APMAP, SMAGP, and CD43, and the expression level of the gene is increased; It is characterized by being able to avoid attack by T cells and killing by NK cells and macrophages. Universal cells.

2. The cells are characterized in that they contain reduced or absent expression of MHC class I and MHC class II human leukocyte antigens. The universal cell of claim 1 .

3. the cells are further modified to increase expression of one or more polypeptides of DUX4, CD27, CD35, CD200, HLA-C, PD-L1, CD47, CD24, CD26, CCL21, Mfge8, and SerpinB9. The universal cell according to claim 1 or 2.

4. In the cell, using a genome editing tool to target one or more genes of one or more transcriptional regulators encoding MHC class I or one or more genes of one or more transcriptional regulators encoding MHC class II, thereby achieving reduced or no expression of MHC class I and / or MHC class II genes; Preferably, the MHC class I transcriptional regulator is selected from one or more of B2M, TAP1, TAP2, TAP-related glycoprotein (Tapasin), or NLRC5, and the MHC class II transcriptional regulator is selected from one or more of CIITA, RFXANK, RFX5, and RFXAP; More preferably, the transcriptional regulatory factor is B2M or CIITA, The universal cell according to claim 1 or 2.

5. The cell further comprises a genetic modification targeting the CIITA gene with a rare-cutter endonuclease that selectively inactivates the CIITA gene. The universal cell according to claim 4.

6. The cells further comprise a genetic modification that targets the B2M gene with a rare-cutter endonuclease that selectively inactivates the B2M gene. The universal cell according to any one of claims 1 to 5.

7. The rare-cutter endonuclease is selected from the group consisting of a CAS protein, a TALE-nuclease, a zinc finger nuclease, a meganuclease, and a homing endonuclease. The universal cell according to claim 5 or 6.

8. The genetic modification targeting the CIITA gene or the B2M gene with a rare-cutter endonuclease is characterized in that it comprises a CAS protein or a polynucleotide encoding a CAS protein, and at least one guide ribonucleic acid sequence for specifically targeting the CIITA gene or the B2M gene. The universal cell according to claim 7.

9. The CRISPR / CAS9 system is used to directly knock out the B2M and CIITA exon segments at both ends, respectively, and the target sequences of the guide ribonucleic acid sequence gRNA corresponding to the B2M gene are SEQ ID NOs: 22 and 23, and the target sequences of the guide ribonucleic acid sequence gRNA corresponding to the CIITA gene are SEQ ID NOs: 24 and 25; The universal cell according to claim 8.

10. The cell is introduced with a gene expression modifier molecule corresponding to one or more genes of one or more transcriptional regulatory factors encoding MHC class I, or a gene expression modifier molecule corresponding to one or more genes of one or more transcriptional regulatory factors encoding MHC class II, thereby realizing reduced or no expression of MHC class I and / or MHC class II genes, wherein the gene expression modifier molecule comprises one selected from siRNA, shRNA, microRNA, antisense RNA, and an inhibitory molecule mediated by another RNA. The universal cell according to claim 1 or 2.

11. the amino acid sequence of SLC35A1, GNE, CMAS, ST3Gal5, ST8SIA1, MUC1, APMAP, SMAGP, or CD43 has 70% or more homology, for example 80% or more homology, and further for example 90% or more, 95% or more, 98% or more, or 99% or more homology, with the sequence set forth in SEQ ID NO: 1, 3, 4, 8, 10, 12, 16, 17, or 19, respectively; More preferably, the amino acid sequence of SLC35A1, GNE, CMAS, ST3Gal5, ST8SIA1, MUC1, APMAP, SMAGP, or CD43 is set forth in SEQ ID NO: 1, 3, 4, 8, 10, 12, 16, 17, or 19, respectively; Even more preferably, said universal cell comprises an expression sequence of at least one of the following genes: MUC1, APMAP, and CD43; Most preferably, the universal cells contain an expression sequence of CD43. The universal cell according to any one of claims 1 to 10.

12. The cells are embryonic stem cells or pluripotent stem cells, and more preferably human stem cells or human somatic cells. A universal cell according to any one of claims 1 to 11.

13. The method for producing a universal cell according to any one of claims 1 to 12, 1) knocking out one or more genes of one or more transcriptional regulators of MHC class I in stem cells, and / or 2) knocking out one or more genes of one or more transcriptional regulators of MHC class II in stem cells; 3) introducing into the cell a nucleic acid sequence encoding at least one protein selected from SLC35A1, GNE, CMAS, ST3Gal5, ST8SIA1, MUC1, APMAP, SMAGP, and CD43; Method for producing universal cells.

14. The transcriptional regulatory factor of MHC class I is selected from one or more of B2M, TAP1, TAP2, TAP-related glycoprotein (Tapasin), or NLRC5, and the transcriptional regulatory factor of MHC class II is selected from one or more of CIITA, RFXANK, RFX5, and RFXAP, preferably, the transcriptional regulatory factor is selected from B2M and CIITA. The method for producing universal cells according to claim 13 .

15. The knockout described in step 1) or 2) is a genetic modification that targets the CIITA gene or the B2M gene with a rare-cutter endonuclease that selectively inactivates the CIITA gene or the B2M gene; Preferably, the rare-cutter endonuclease is selected from a CAS protein, a TALE-nuclease, a zinc finger nuclease, a meganuclease, and a homing endonuclease; More preferably, the genetic modification targeting the CIITA gene or the B2M gene with a rare-cutter endonuclease comprises a CAS protein or a polynucleotide encoding a CAS protein, and at least one guide ribonucleic acid sequence for specifically targeting the CIITA gene or the B2M gene; Even more preferably, in steps 1) and 2), the CRISPR system is used to directly knock out the B2M and CIITA exon segments at both ends, respectively, wherein the target sequences of the gRNA corresponding to the B2M gene are SEQ ID NOs: 2 and 3, and the target sequences of the gRNA corresponding to the CIITA gene are SEQ ID NOs: 4 and 5; The method according to claim 13 or 14.

16. The knockout described in step 1) or 2) is to realize reduced or no expression of MHC class I and / or MHC class II genes by introducing gene expression modifier molecules corresponding to one or more genes of one or more transcriptional regulators encoding MHC class I or one or more genes of one or more transcriptional regulators encoding MHC class II, wherein the gene expression modifier molecules include one selected from siRNA, shRNA, microRNA, antisense RNA, and other RNA-mediated inhibitory molecules; The method according to claim 13 or 14.

17. In step 3), a nucleic acid sequence encoding at least one protein selected from SLC35A1, GNE, CMAS, ST3Gal5, ST8SIA1, MUC1, APMAP, SMAGP, and CD43 is introduced into cells using an expression vector; Preferably, the expression vector used in step 3) is a viral vector; More preferably, the viral vector is a lentivirus. The manufacturing method according to any one of claims 13 to 16.

18. In step 3), a nucleic acid sequence encoding at least one protein selected from SLC35A1, GNE, CMAS, ST3Gal5, ST8SIA1, MUC1, APMAP, SMAGP, and CD43 is introduced into a predetermined site of the stem cell, and preferably, the predetermined site of the stem cell is a safe harbor gene site. The manufacturing method according to any one of claims 13 to 17.

19. the amino acid sequence of any one of SLC35A1, GNE, CMAS, ST3Gal5, ST8SIA1, MUC1, APMAP, SMAGP, or CD43 has 70% or more homology, for example 80% or more homology, and further for example 90% or more, 95% or more, 98% or more, or 99% or more homology, with the sequence shown in SEQ ID NO: 1, 3, 4, 8, 10, 12, 16, 17, or 19, respectively; More preferably, the amino acid sequence of SLC35A1, GNE, CMAS, ST3Gal5, ST8SIA1, MUC1, APMAP, SMAGP, or CD43 is set forth in SEQ ID NO: 1, 3, 4, 8, 10, 12, 16, 17, or 19, respectively; Even more preferably, in step 3), a nucleic acid sequence encoding at least one protein selected from MUC1, APMAP, and CD43 is introduced into the cell using an expression vector; Most preferably, in step 3), a nucleic acid sequence encoding CD43 is introduced into the cell using an expression vector. The manufacturing method according to any one of claims 13 to 18.

20. The universal stem cell further comprises a second expression vector, which comprises a polynucleotide sequence encoding one or more selected from DUX4, CD27, CD35, CD200, HLA-C, PD-L1, CD47, CD24, CD26, CCL21, Mfge8, and SerpinB9. The manufacturing method according to any one of claims 13 to 19.

21. The second expression vector is an inducible expression vector, preferably a viral vector. The method of claim 20.

22. A method for producing differentiated universal cells, comprising culturing the universal cells according to any one of claims 1 to 12 under differentiation conditions to produce differentiated, low-immunogenic cells. method.

23. The differentiation conditions are applied to differentiate the cells into a cell type selected from cardiomyocytes, neurons, glial cells, endothelial cells, T cells, NK cells, NKT cells, macrophages, hematopoietic progenitor cells, mesenchymal cells, islet cells of Langerhans, chondrocytes, retinal pigment epithelial cells, kidney cells, liver cells, thyroid cells, skin cells, blood cells, and epithelial cells; 23. The method of claim 22.

24. Administering the differentiated, hypoimmunogenic cell population produced by the method of claim 22 or 23 to a patient suitable for cell therapy. Treatment method.

25. The universal cell according to any one of claims 1 to 12, preferably further comprising one or more therapeutic agents, preferably comprising peptides, cytokines, checkpoint inhibitors, mitogens, growth factors, small RNAs, double stranded RNAs (dsRNA), monocytes, feeder cells, feeder cell components or replacement factors thereof, vectors comprising one or more polynucleic acids of interest, antibodies, etc. composition.

26. A cell that has increased expression of at least one protein selected from SLC35A1, GNE, CMAS, ST3Gal5, ST8SIA1, MUC1, APMAP, SMAGP, and CD43, and has decreased or no expression of MHC class I and / or MHC class II human leukocyte antigens, Preferably, the cells express at least one of MUC1, APMAP, and CD43 proteins; More preferably, the cells express CD43. cell.

27. do not express CIITA, have increased expression of at least one protein selected from SLC35A1, GNE, CMAS, ST3Gal5, ST8SIA1, MUC1, APMAP, SMAGP, and CD43, and have decreased or absent expression of MHC class I and / or MHC class II human leukocyte antigens; cell.

28. A cell that does not express B2M, has increased expression of any one protein of SLC35A1, GNE, CMAS, ST3Gal5, ST8SIA1, MUC1, APMAP, SMAGP, or CD43, and has reduced or no expression of MHC class I and / or MHC class II human leukocyte antigens, Preferably, the cells express at least one of MUC1, APMAP, and CD43 proteins; More preferably, the cells express CD43. cell.

29. A cell that does not express CIITA and B2M, has increased expression of at least one protein selected from SLC35A1, GNE, CMAS, ST3Gal5, ST8SIA1, MUC1, APMAP, SMAGP, and CD43, and has reduced or absent MHC class I and / or MHC class II human leukocyte antigens, Preferably, the cells express at least one of MUC1, APMAP, and CD43 proteins; More preferably, the cells express CD43. cell.

30. A cell having increased expression of at least one protein selected from SLC35A1, GNE, CMAS, ST3Gal5, ST8SIA1, MUC1, APMAP, SMAGP, and CD43, and at least one polypeptide selected from DUX4, CD27, CD35, CD200, HLA-C, PD-L1, CD47, CD24, CD26, CCL21, Mfge8, and SerpinB9, and having reduced or absent MHC class I and / or MHC class II human leukocyte antigens, Preferably, the cells express at least one of MUC1, APMAP, and CD43 proteins; More preferably, the cells express CD43. cell.

31. A cell which does not express CIITA, and which has increased expression of at least one protein selected from SLC35A1, GNE, CMAS, ST3Gal5, ST8SIA1, MUC1, APMAP, SMAGP, and CD43, and at least one polypeptide selected from DUX4, CD27, CD35, CD200, HLA-C, PD-L1, CD47, CD24, CD26, CCL21, Mfge8, and SerpinB9, and which has reduced or no expression of MHC class I and / or MHC class II human leukocyte antigens, Preferably, the cells express at least one of MUC1, APMAP, and CD43 proteins; More preferably, the cells express CD43. cell.

32. A cell which does not express B2M, and which has increased expression of at least one protein selected from SLC35A1, GNE, CMAS, ST3Gal5, ST8SIA1, MUC1, APMAP, SMAGP, and CD43, and at least one polypeptide selected from DUX4, CD27, CD35, CD200, HLA-C, PD-L1, CD47, CD24, CD26, CCL21, Mfge8, and SerpinB9, and which has reduced or no expression of MHC class I and / or MHC class II human leukocyte antigens, Preferably, the cells express at least one of MUC1, APMAP, and CD43 proteins; More preferably, the cells express CD43. cell.

33. A cell which does not express CIITA and B2M, and which has increased expression of at least one protein selected from SLC35A1, GNE, CMAS, ST3Gal5, ST8SIA1, MUC1, APMAP, SMAGP, and CD43, and at least one polypeptide selected from DUX4, CD27, CD35, CD200, HLA-C, PD-L1, CD47, CD24, CD26, CCL21, Mfge8, and SerpinB9, and which has reduced or no expression of MHC class I and / or MHC class II human leukocyte antigens, Preferably, the cells express at least one of MUC1, APMAP, and CD43 proteins; More preferably, the cells express CD43. cell.

34. selected from stem cells, differentiated cells, pluripotent stem cells, induced pluripotent stem cells, adult stem cells, progenitor cells, somatic cells, primary T cells, and chimeric antigen receptor T cells; A cell according to any one of claims 26 to 33.

35. In the production of a product used in cell therapy, the universal cells according to any one of claims 1 to 12, the composition according to claim 25, or the cells according to claims 26 to 34, use.

36. In the production of a product used for organ transplantation, the universal cells according to any one of claims 1 to 12, the composition according to claim 25, or the cells according to claims 26 to 34, use.

37. In the construction of a universal PSCs cell bank of the universal stem cells according to any one of claims 1 to 12, the composition according to claim 25, or the cells according to claims 26 to 34, use.

38. The universal stem cells according to any one of claims 1 to 12, the composition according to claim 25, or the cells according to claims 26 to 34 are used as a gene therapy drug carrier. use.

39. A reagent kit for obtaining isolated hypoimmunogenic cardiomyocytes from the universal stem cells according to any one of claims 1 to 12 by in vitro differentiation, comprising a first culture medium and a second culture medium: the first medium is a basal medium containing a GSK-3 inhibitor; The second medium is a basal medium containing a Wnt inhibitor; Reagent kit.

40. the first culture medium or the second culture medium comprises RPMI1640 basal medium and B27 supplement without insulin; and / or the GSK-3 inhibitor is selected from CHIR99021; and / or the Wnt inhibitor is selected from any one, two, or more of IWR-1, IWP-2, IWP-4, and C59; and / or the concentration of the GSK-3 inhibitor is 0.5 μM to 10 μM, preferably 6 μM; and / or the concentration of the Wnt inhibitor is 0.5 μM to 20 μM, preferably 2 μM; 40. The reagent kit of claim 39.

41. A method for obtaining isolated hypoimmunogenic cardiomyocytes from the universal stem cells according to any one of claims 1 to 12 by in vitro differentiation, wherein the gene activity of endogenous beta-2 microglobulin (B2M) and endogenous class II transactivator (CIITA) has been eliminated in the universal stem cells, and the expression of CD43 protein is increased, comprising: (a) culturing stem cells in a basal medium supplemented with a GSK-3 inhibitor to induce differentiation into cardiomyocytes; (b) continuing to induce differentiation in a basal medium; (c) continuing to induce differentiation in a basal medium supplemented with a Wnt inhibitor; (d) continuing to induce differentiation in a basal medium; More preferably, (a) culturing stem cells in a first medium supplemented with a GSK-3 inhibitor on days 0-2 to induce differentiation into cardiomyocytes; (b) one day after induction, continuing to induce differentiation in a basal medium; (c) continuing to induce differentiation in a second medium containing a Wnt inhibitor on days 3 to 5; (d) continuing to induce differentiation in the basal medium from day 5 to day 15, method.

42. the first culture medium and the second culture medium in steps (a), (b), (c), and (d) comprise RPMI1640 basal medium and B27 supplement without insulin; and / or the GSK-3 inhibitor is selected from CHIR99021; and / or the Wnt inhibitor is selected from any one, two, or more of IWR-1, IWP-2, IWP-4, and C59; and / or the concentration of the GSK-3 inhibitor is 0.5 μM to 10 μM, preferably 6 μM; and / or the concentration of the Wnt inhibitor is 0.5 μM to 20 μM, preferably 2 μM; and / or, when the universal stem cells in step (a) have grown to a confluence of 85 to 98% (e.g., 95%), the medium is replaced with the first medium to induce differentiation.

42. The method of claim 41.

43. The basal medium is selected from PRMI1640 medium, mTESR1 medium, etc., and more preferably B27 PRMI1640 medium supplemented with insulin; 42. The method of claim 41.

44. A cell therapy agent for preventing or treating heart disease, comprising differentiated cardiomyocytes produced by the method according to any one of claims 41 to 43. Cellular therapy agents.

45. The cardiac disease may be at least one selected from the group consisting of myocardial infarction, angina pectoris, ischemic cardiomyopathy, primary cardiomyopathy, secondary cardiomyopathy, and heart failure, but is not limited thereto; The cell therapy agent described in claim 44.

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