IMMUNOLOGICALLY COMPATIBLE AND REVERSIBLE UNIVERSAL PLURIPOTENT STEM CELL AND APPLICATION THEREOF

Abstract

Provided is an immunologically compatible and reversible universal pluripotent stem cell or a derivative thereof. An inducible gene expression system and the expression sequence of at least one immunologically compatible molecule are introduced into the genome of the pluripotent stem cell or the derivative thereof. The immunologically compatible molecule is used to regulate the expression of immune response-related genes in the pluripotent stem cell or the derivative thereof. The expression of the immunologically compatible molecule is regulated by the inducible gene expression system. The described method can increase the immunological compatibility between a transplant and a recipient, and can simultaneously reversibly restore the antigen-presenting abilities of transplant cells.

Claims

1. A pluripotent stem cell or a derivative thereof, wherein an inducible gene expression system and an expression sequence of at least one immunologically compatible molecule are introduced into the genome of the pluripotent stem cell or the derivative thereof; the immunologically compatible molecule is used to regulate the expression of immune response-related genes in the pluripotent stem cell or the derivative thereof; and the expression of the immunologically compatible molecule is regulated by the inducible gene expression system.

2. The pluripotent stem cell or the derivative thereof according to claim 1, wherein the to inducible gene expression system is regulated by an exogenous inducer; and the on and off state of the inducible gene expression system is controlled by adjusting the added amount, duration of action, and type of the exogenous inducer so as to control the expression amount of the expression sequence of the immunologically compatible molecule.

3. The pluripotent stem cell or the derivative thereof according to claim 2, wherein the inducible gene expression system includes at least one selected from the group consisting of the Tet-Off system and dimer-induced expression system.

4. The pluripotent stem cell or the derivative thereof according to claim 1, wherein the immune response-related genes include: (1) major histocompatibility complex genes, including at least one selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DQB1, HLA-DPA1 and HLA-DPB1; (2) major histocompatibility complex-related genes, including at least one selected from the group consisting of B2M and CIITA; and (3) T cell surface negative costimulatory molecules, including at least one selected from the group consisting of CTLA4, PD-1 and BTLA.

5. The pluripotent stem cell or the derivative thereof according to claim 1, wherein the immunologically compatible molecule includes at least one selected from the group consisting of: (1) negative costimulatory molecules on the antigen-presenting cell surface; (2) activating antibodies against “negative costimulatory molecules on the T cell surface” located on the transplanted cell surface, wherein the “negative costimulatory molecules on the T cell surface” include at least one selected from the group consisting of CTLA4, PD-1 and BTLA; (3) stimulating ligands against “negative costimulatory molecules on the T cell surface” located on the transplanted cell surface; (4) immune tolerance-related genes; (5) HLA-C class molecules; (6) shRNAs and/or shRNA-miRs for major histocompatibility complex genes; and (7) shRNAs and/or shRNA-miRs for major histocompatibility complex-related genes.

6. The pluripotent stem cell or the derivative thereof according to claim 5, wherein (1) the negative costimulatory molecules on the antigen-presenting cell surface include at least one selected from the group consisting of PD-L1, Siglec-15, B7-H4 and B7-H5; (2) the activating antibodies against the “negative costimulatory molecules on the T cell surface” located on the transplanted cell surface are membrane antibodies; (3) the stimulating ligands against the “negative costimulatory molecules on the T cell surface” located on the transplanted cell surface include at least one selected from the group consisting of PD-L1, Siglec-15, B7-H4 and B7-H5; (4) the immune tolerance-related genes include at least one selected from the group consisting of CD47 and HLA-G; (5) the HLA-C class molecules include HLA-C multiple alleles with a proportion of more than 90% in total in the human population, or fusion protein genes composed of the HLA-C multiple alleles with a proportion of more than 90% in total in the human population and B2M; (6) the major histocompatibility complex genes include at least one selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DQB1, HLA-DPA1 and HLA-DPB1; and (7) the major histocompatibility complex-related genes include at least one selected from the group consisting of B2M and CIITA.

7. The pluripotent stem cell or the derivative thereof according to claim 5, wherein target sequences of the shRNAs and/or shRNA-miRs for the major histocompatibility complex genes are at least one selected from the group consisting of SEQ ID NO. 14 to SEQ ID NO. 23; and target sequences of the shRNAs and/or shRNA-miRs for the major histocompatibility complex-related genes are at least one selected from the group consisting of SEQ ID NO. 1 to SEQ ID NO. 13.

8. The pluripotent stem cell or the derivative thereof according to claim 1, wherein an shRNA and/or miRNA processor complex-related gene and/or an anti-interferon effector molecule are further introduced into the genome of the pluripotent stem cell or the derivative thereof.

9. The pluripotent stem cell or the derivative thereof according to claim 8, wherein the shRNA and/or miRNA processor complex-related gene includes at least one selected from the group consisting of Dhrosha, Ago1, Ago2, Dicer1, Exportin-5, TRBP (TARBP2), PACT (PRKRA) and DGCR8; and the anti-interferon effector molecule is shRNA and/or shRNA-miR for at least one selected from the group consisting of PKR, 2-5As, IRF-3 and IRF-7.

10. The pluripotent stem cell or the derivative thereof according to claim 9, wherein target sequences of the shRNAs and/or shRNA-miRs for PKR, 2-5As, IRF-3 or IRF-7 are at least one selected from the group consisting of SEQ ID NO. 31 to SEQ ID NO. 90.

11. The pluripotent stem cell or the derivative thereof according to claim 7 or 10, wherein a general backbone sequence of the shRNAs or shRNA-miRs for major histocompatibility complex genes, major histocompatibility complex-related genes, and PKR, 2-5As, IRF-3 or IRF-7 is as shown below: (1) general backbone sequence of shRNA: comprising, in sequence from 5′ to 3′, an shRNA target sequence, a stem-loop sequence, and a reverse complement sequence of the shRNA target sequence; wherein the shRNA target sequence is as defined in claim 7 or 10, and the length of the loop sequence is 3-9 bases; and (2) general backbone sequence of shRNA-miR: obtained by replacing a target sequence in microRNA-30 or microRNA-155 with the shRNA-miR target sequence as defined in claim 7 or 10.

12. The pluripotent stem cell or the derivative thereof according to any one of claims 1-11, wherein the inducible gene expression system, the expression sequence of the immunologically compatible molecule, the shRNA and/or miRNA processor complex-related gene, and the anti-interferon effector molecule are introduced by means of viral vector interference, non-viral vector transfection or gene editing.

13. The pluripotent stem cell or the derivative thereof according to claim 12, wherein the gene editing is gene knock-in.

14. The pluripotent stem cell or the derivative thereof according to any one of claims 1-11, wherein introduction loci for the inducible gene expression system, the expression sequence of the immunologically compatible molecule, the shRNA and/or miRNA processor complex-related gene, and the anti-interferon effector molecule are genomic safe loci.

15. The pluripotent stem cell or the derivative thereof according to claim 14, wherein the genomic safe loci include at least one selected from the group consisting of the AAVS1 safe locus, the eGSH safe locus, and the H11 safe locus.

16. The pluripotent stem cell or the derivative thereof according to any one of claims 1-15, wherein the pluripotent stem cell includes an embryonic stem cell, an embryonic germ cell, an embryonic carcinoma cell, or an induced pluripotent stem cell; the derivative includes a pluripotent stem cell-derived three-germ-layer-derived organ, tissue or cell; and the pluripotent stem cell-derived three-germ-layer-derived cell includes a mesenchymal stem cell, a neural stem or progenitor cell, or other adult stem cell.

17. A method for preparing an immunologically compatible and reversible universal pluripotent stem cell or a derivative thereof, comprising by introducing an inducible gene expression system and an expression sequence of at least one immunologically compatible molecule into the genome of the pluripotent stem cell or the derivative thereof; the immunologically compatible molecule is used to regulate the expression of immune response-related genes in the pluripotent stem cell or the derivative thereof.

18. The method for preparing an immunologically compatible and reversible universal pluripotent stem cell or a derivative thereof according to claim 17, wherein the immunologically compatible molecule is as defined in any one of claims 5-7.

19. The method for preparing an immunologically compatible and reversible universal pluripotent stem cell or a derivative thereof according to claim 17, wherein the inducible gene expression system includes at least one selected from the group consisting of the Tet-Off system and dimer-induced expression system.

20. The method for preparing an immunologically compatible and reversible universal pluripotent stem cell or a derivative thereof according to claim 17, wherein an shRNA and/or miRNA processor complex-related gene and/or an anti-interferon effector molecule are further introduced into the genome of the pluripotent stem cell or the derivative thereof, wherein the shRNA and/or miRNA processor complex-related gene is as defined in claim 9; and the anti-interferon effector molecule is as defined in any one of claims 9-11.

21. The method for preparing an immunologically compatible and reversible universal pluripotent stem cell or a derivative thereof according to claim 17, wherein introduction loci for the inducible gene expression system, immunologically compatible molecule, shRNA and/or miRNA processor complex-related gene, and anti-interferon effector molecule are genomic safe loci; and the genomic safe loci include at least one selected from the group consisting of the AAVS1 safe locus, the eGSH safe locus, and the H11 safe locus.

22. Use of the pluripotent stem cell or the derivative thereof according to any one of claims 1-16 in the preparation of a product for cellular therapy.

23. Use of the pluripotent stem cell or the derivative thereof according to any one of claims 1-16 in the preparation of a product for organ transplantation.

24. Use of the pluripotent stem cell or the derivative thereof according to any one of claims 1-16 in the construction of a universal PSC cell bank.

25. Use of the pluripotent stem cell or the derivative thereof according to any one of claims 1-16 as a gene drug carrier.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0090] FIG. 1 shows an “AAVS1 KI Vector (shRNA, shRNA-miR, Gene, Neo)” KI Donor plasmid map.

[0091] FIG. 2 shows an “AAVS1 KI Vector (shRNA, shRNA-miR, Gene, Puro)” KI Donor plasmid map.

[0092] FIG. 3 shows a Cas9(D10A) plasmid map.

[0093] FIG. 4 shows a gRNA clone AAVS1-1 plasmid map.

[0094] FIG. 5 shows a gRNA clone AAVS1-2 plasmid map.

[0095] FIG. 6 shows a gRNA clone eGSH-1 plasmid map.

[0096] FIG. 7 shows a gRNA clone eGSH-2 plasmid map.

[0097] FIG. 8 shows the cell-specific .sup.51Cr release rate (T cell activity) of hPSC-derived EB sphere immunologically compatible cells in Example 2.

[0098] FIG. 9 shows the cell-specific .sup.51Cr release rate (T cell activity) of hPSC-derived EB sphere immunologically compatible cells after a Dox treatment in Example 3.

[0099] FIG. 10 shows the cell-specific .sup.51Cr release rate (NK cell activity) of hPSC-derived EB sphere immunologically compatible cells in Example 4.

[0100] FIG. 11 shows the cell-specific .sup.51Cr release rate (NK cell activity) of hPSC-derived EB sphere immunologically compatible cells after a Dox treatment in Example 5.

[0101] FIG. 12 shows the percentage of CFSE.sup.+PI.sup.+ cells in hPSC-derived EB sphere cells in Example 6.

[0102] FIG. 13 shows the percentage of CFSE.sup.+PI.sup.+ cells in hPSC-derived EB sphere cells after a Dox treatment in Example 7.

[0103] FIG. 14 shows the NK cell cytotoxicity value in Example 8.

[0104] FIG. 15 shows the NK cell cytotoxicity value after a Dox treatment in Example 9.

[0105] FIG. 16 shows .sup.51Cr natural release rate (T cell activity) of hPSC-MSC, NSC and EB immunologically compatible cells in Example 10.

[0106] FIG. 17 shows .sup.51Cr natural release rate (NK cell activity) of hPSC-MSC, NSC and EB immunologically compatible cells in Example 11.

[0107] FIG. 18 shows the percentage of CFSE.sup.+PI.sup.+ cells in hPSC, MSC, NSC and EB immunologically compatible cells in Example 12.

[0108] FIG. 19 shows the NK cell cytotoxicity value in Example 13.

[0109] FIG. 20 shows the cell-specific .sup.51Cr release rate (T cell activity) of hPSC, MSC, NSC and EB immunologically compatible cells in Example 14.

[0110] FIG. 21 shows the MTT values of the hPSC, MSC, NSC and EB immunologically compatible cells in Example 15.

DETAILED DESCRIPTION

[0111] In order to understand the technical content of the present disclosure more clearly, the following examples are specifically given for detailed description in conjunction with the accompanying drawings. It should be understood that these examples are only used to describe the present disclosure, rather than limiting the scope of the present disclosure.

[0112] Experimental methods in which no specific conditions are indicated in the following examples are usually carried out under conventional conditions, for example, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or in accordance with the conditions recommended by the manufacturer. Various common chemical reagents used in the examples are all commercially available products.

Example 1 Method for Preparing Immunologically Compatible and Reversible Universal Pluripotent Stem Cells and Derivatives Thereof

[0113] Provided was a method for preparing an immunologically compatible and reversible universal pluripotent stem cell and a derivative thereof, involving introducing an inducible gene expression system and an expression sequence of at least one immunologically compatible molecule into the genome of the pluripotent stem cell or the derivative thereof, wherein the immunologically compatible molecule was used to regulate the expression of immune response-related genes in the pluripotent stem cell or the derivative thereof; and the expression of the immunologically compatible molecule was regulated by the inducible gene expression system.

[0114] When carrying out immunological compatibility modification, the modification could be carried out on hPSCs first, and after the modification was completed, they were then differentiated into pluripotent stem cell derivatives for use; alternatively, immunological compatibility modification could be carried out after hPSCs were differentiated into pluripotent stem cell derivatives.

[0115] In the method:

[0116] I. Pluripotent Stem Cells and Derivatives Thereof

[0117] Pluripotent stem cells were selected from embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and other forms of pluripotent stem cells, such as hPSCs-MSCs, NSCs, and EB cells. Among them:

[0118] iPSCs: pE3.1-OG—KS and pE3.1-L-Myc—hmiR302 cluster were electrotransfected into somatic cells by using our established third-generation efficient and safe episomal-iPSC induction system (6F/BM1-4C), and cultured in RM1 for 2 days, in BioCISO-BM1 with 2 μM Parnate for 2 days, in BioCISO-BM1 with 2 μM Parnate, 0.25 mM sodium butyrate, 3 μM CHIR99021 and 0.5 μM PD03254901 for 2 days, and in the stem cell medium BioCISO until approximately 17 days, iPSC clones were picked, and the picked iPSC clones were purified, digested, and passaged to obtain stable iPSCs. For the specific construction method, see: Stem Cell Res Ther. 2017 Nov. 2; 8(1): 245.

[0119] hPSCs-MSCs: iPSCs were cultured in a stem cell medium (BioCISO, containing 10 μM TGFβ inhibitor SB431542) for 25 days during which digestion and the passage were carried out at 80-90 confluence (2 mg/mL Dispase digestion), the cells were passaged 1:3 into a Matrigel-coated culture plate and then into an ESC-MSC medium (knockout DMEM medium, containing 10% KSR, NEAA, double antibody, glutamine, β-mercaptoethanol, 10 ng/mL bFGF and SB-431542), wherein the medium was changed every day, the passage was carried out at 80-90 confluence (1:3 passage), and the cells were cultured continuously for 20 days. For the specific construction method, see: Proc Natl Acad Sci USA. 2015; 112(2): 530-535.

[0120] NSCs: iPSCs were cultured in an induction medium (knockout DMEM medium, containing 10% KSR, TGF-β inhibitor, and BMP4 inhibitor) for 14 days, and rosette-shaped nerve cells were picked and cultured in a low-adherence culture plate, wherein the medium used was DMEM/F12 (containing 1% N2, Invitrogen) and Neurobasal medium (containing 2% B27, Invitrogen) at a ratio of 1:1 and further contained 20 ng/ml bFGF and 20 ng/ml EGF, and for digestion, Accutase was used for digestion and passage. For the specific construction method, see: FASEB J. 2014; 28(11): 4642-4656.

[0121] EB cells: iPSCs with a confluence of 95% were digested with BioC-PDE1 for 6 min and then scraped into a mass by a mechanical scraping passage method, cell mass settling occurred, and the settled cell mass was transferred to a low-adherence culture plate and cultured using BioCISO-EB1 for 7 days during which the medium was changed every other day. After 7 days, the cells were transferred to a Matrigel-coated culture plate to continue adherent culture with BioCISO, and after 7 days, embryoid bodies (EBs) with a structure of inner, middle and outer germ layers could be obtained. For the specific construction method, see: Stem Cell Res Ther. 2017 Nov. 2; 8(1): 245.

[0122] The derivative included pluripotent stem cell-derived three-germ-layer-derived organs, tissues or cells; and the pluripotent stem cell-derived three-germ-layer-derived cells included mesenchymal stem cells, neural stem or progenitor cells, or other adult stem cells.

[0123] II. Genomic Safe Locus (Safe Harbor)

[0124] It could be selected from the AAVS1 safe locus, the eGSH safe locus, or other safe loci:

[0125] (1) The AAVS1 Safe Locus

[0126] the AAVS1 locus (alias the “PPP1R2C locus”), located on chromosome 19 of the human genome, is a validated “safe harbor” locus that could ensure the intended function of the transferred DNA fragment. This locus is an open chromosomal structure, which could ensure that the transferred gene could be transcribed normally, and the insertion of an exogenous fragment of interest at this locus has no known side effects on cells.

[0127] (2) The eGSH Safe Locus

[0128] the eGSH safe locus, located on chromosome 1 of the human genome, is another documented validated “safe harbor” locus that could ensure the intended function of the transferred DNA fragment.

[0129] (3) Other Safe Loci

[0130] the H11 safe locus (also called Hipp11), located on human chromosome 22, is a locus between the two genes Eif4enif1 and Drg1. The locus was discovered and named by Simon Hippenmeyer in 2010. Since the H11 locus is located between the two genes, the risk of affecting the expression of endogenous genes after the insertion of an exogenous gene is small. The H11 locus is verified to be an intergenic safe transcription activation region, a new “safe harbor” locus other than the AAVS1 locus and the eGSH locus.

[0131] III. Inducible Gene Expression System

[0132] 1. The Tet-Off System

[0133] In the absence of doxycycline, the tTA protein continued to act on the tet promoter, resulting in continuous gene expression. This system was useful where a transgene was required to be maintained in a state of continuous expression. When doxycycline was added, doxycycline could change the structure of the tTA protein, making it impossible to bind to the promoter, thereby reducing the level of gene expression driven thereby. To keep this system in an “off” state, continuous addition of doxycycline was necessary.

[0134] In the present disclosure, the tet-Off system and one or more immunologically compatible molecule sequences were knocked into the genomic safe locus of pluripotent stem cells, whereby the expression of the immunologically compatible molecule could be accurately turned on or off depending on whether to add doxycycline or not, such that the expression of major histocompatibility complex-related genes in the pluripotent stem cell or the derivative thereof could be reversibly regulated.

[0135] 2. Dimer Turn-Off Expression System

[0136] Dimer-mediated gene expression regulation system: There were many approaches to chemically regulate the transcription of target genes, and the most common one was regulation by an allosteric modulator that affected the activity of transcription factors. One such approach was to use a dimerization inducer or a dimer to reconstitute active transcription factors on an inactive fusion protein. The most commonly used system was the natural product rapamycin or a biologically inactive analog as a dimerization agent. Rapamycin (or an analog thereof) had a high affinity with the homoplasmic protein FKBP12 (an FKBP protein that bound to FK506) and a large serine-threonine protein kinase called FRAP [FRBP-rapamycin-related protein, or mTOR (target of rapamycin in mammalian)], and also had the function of combining with these two proteins, so that these two proteins were brought together as a heterodimer. To regulate the transcription of a target gene, a DNA binding domain was fused to one or more FKBP domains, and a transcription repression domain was fused to the 93 amino acid position of FRAP, called FRB. As such, it was sufficient to bind the FKBP-rapamycin complex. The two fusion proteins could dimerize only in the presence of rapamycin (or an analog thereof). Thus, the transcription of a gene with a binding site to the DNA-binding domain was inhibited.

[0137] IV. Immunologically Compatible Molecule

[0138] The immunologically compatible molecule could regulate the expression of immune response-related genes in a pluripotent stem cell or a derivative thereof. The types and sequences of specific immunologically compatible molecules were shown in Table 1:

TABLE-US-00001 TABLE 1 Immunologically compatible molecules Immunologically The function of compatible Specific immunologically molecule immunologically compatible No. classification compatible molecule molecule 1 shRNA of major B2M (beta-2- Knocking down the histocompatibility microglobulin) expression of B2M. complex gene- shRNA related 2 gene CIITA shRNA Knocking down the expression of CIITA. 3 shRNA-miR of B2M (beta-2- Knocking down the major complex microglobulin) expression of B2M. gene-related gene shRNA-miR 4 histocompatibility CIITA shRNA-miR Knocking down the expression of CIITA. 5 shRNA of major HLA-A shRNA Knocking down the complex gene expression of HLA-A. 6 histocompatibility HLA-B shRNA Knocking down the expression of HLA-B. 7 shRNA-miR of HLA-A shRNA-miR Knocking down the major histocompatibility expression of HLA-A. 8 complex gene HLA-B shRNA-miR Knocking down the expression of HLA-B. 9 Major HLA-C retained HLA-C was a major histocompatibility (preferably retained histocompatibility complex gene homozygous HLA- antigen that could HLA-C C, preferably cause a strong homozygote retained rejection response. homozygous HLA-C There were 12 HLA- from 30 HLA-C C alleles with strains that covered frequency >1%, and more than 90% of the cumulative the human frequency was 90% or population). more. Therefore, it The sequences of could be retained, HLA-C and immunological homozygotes were compatibility could be shown in SEQ ID achieved as long as NO. 107 to SEQ ID matching was NO. 118, satisfied. respectively. 10 Major HLA-C-B2M fusion Direct expression of histocompatibility protein (preferably fully functional HLA- complex gene retained C molecules. HLA-C-B2M homozygous HLA- Immunological fusion gene C, preferably compatibility could be homozygote retained achieved as long as homozygous matching was HLA-C from 30 satisfied. HLA-C strains that covered more than 90% of the human population) 11 Immune tolerance- CD47 Blocking and related genes (Accession number: inhibiting the activity NM_198793.2) of almost all the innate immune responses of macrophages, NK cells, etc., and inhibiting T cell activity through the effect of its negative costimulatory molecules. 12 HLA-G Exerting (Accession number: immunosuppressive NM_002127) effects by directly binding to various inhibitory receptors. 13 Stimulating PD-L1 The binding of PD-1 ligands against (Accession number: with PD-L1 could “negative NM_001314029) transmit an inhibitory costimulatory signal to reduce the molecules on the T proliferation of CD8+ cell surface” T cells in the lymph located on the nodes and cause transplanted cell reduced proliferation surface or apoptosis of T cells themselves, effectively disarming the immune response in the body. 14 Siglec-15 Siglec-15 inhibited (Accession number: the specific immune NM_213602) response of T cells by regulating the growth of T cells, and blocking the immunosuppressive effect of Siglec-15 could restore/improve the anti-tumor immune response in the body. 15 B7H4 B7H4 could (Accession number: negatively regulate NM_001253850) the immune response of T cells by inhibiting T cell proliferation, cytokine production and cell cycle progression. 16 B7H5 B7-H5 inhibited T (Accession number: cell growth, arrested NM_001282559) the cell cycle and down-regulated cytokine production. 17 Activating BTLA activating Blocking the binding antibodies against antibody of BTLA with “negative (SEQ ID NO. 120) HVEM, hindering the costimulatory interaction between molecules on the T HVEM and LIGHT, cell surface” and thereby limiting located on the the proliferation of T transplanted cell and B cells and the surface production of Ig, etc.

[0139] The major histocompatibility complex gene HLA-C homozygote in Table 1 was at least one of HLA-C*01:02 (SEQ ID NO. 107), HLA-C*02:02 (SEQ ID NO. 108), HLA-C*03:03 (SEQ ID NO. 109), HLA-C*03:04 (SEQ ID NO. 110), HLA-C*04:01 (SEQ ID NO. 111), HLA-C*05:01 (SEQ ID NO. 112), HLA-C*06:02 (SEQ ID NO. 113), HLA-C*07:01 (SEQ ID NO. 114), HLA-C*07:02 (SEQ ID NO. 115), HLA-C*08:01 (SEQ ID NO. 116), HLA-C*12:02 (SEQ ID NO. 117) and HLA-C*16:01 (SEQ ID NO. 118).

[0140] The sequence of the HLA-C-B2M fusion gene in Table 1 was shown below:

TABLE-US-00002 (SEQ ID NO. 119) N-TCTGGTGGCGGAGGCTCGGGCGGAGGTGGGTCGGGTGGCGGCGGATC AATGTCTCGCTCCGTGGCCTTAGCTGTGCTCGCGCTACTCTCTCTTTCT GGCCTGGAGGCTATCCAGCGTACTCCAAAGATTCAGGTTTACTCACGTC ATCCAGCAGAGAATGGAAAGTCAAATTTCCTGAATTGCTATGTGTCTGG GTTTCATCCATCCGACATTGAAGTTGACTTACTGAAGAATGGAGAGAGA ATTGAAAAAGTGGAGCATTCAGACTTGTCTTTCAGCAAGGACTGGTCTT TCTATCTCTTGTACTACACTGAATTCACCCCCACTGAAAAAGATGAGTA TGCCTGCCGTGTGAACCATGTGACTTTGTCACAGCCCAAGATAGTTAAG TGGGATCGAGACATGTAA

[0141] Wherein N represented at least one of the HLA-C sequences, shown in SEQ ID NO. 107 to SEQ ID NO. 118 respectively.

[0142] The target sequences of the above shRNA or shRNA-miR for the immunologically compatible molecule were shown in Table 2:

TABLE-US-00003 TABLE 2 Target sequence of shRNA or shRNA-miR No. Start Target sequence (DNA) Region Sequence number B2M  1  634 GGGAGCAGAGAATTCTCTTAT UTR3 SEQ ID NO. 1  2  635 GGAGCAGAGAATTCTCTTATC UTR3 SEQ ID NO. 2  3  636 GAGCAGAGAATTCTCTTATCC UTR3 SEQ ID NO. 3 CIITA  1  231 GCTACCTGGAGCTTCTTAACA ORF SEQ ID NO. 4  2  238 GGAGCTTCTTAACAGCGATGC ORF SEQ ID NO. 5  3  879 GGGTCTCCAGTATATTCATCT ORF SEQ ID NO. 6  4 2656 GCCTCCTGATGCACATGTACT ORF SEQ ID NO. 7  5 2917 GGAAGACCTGGGAAAGCTTGT ORF SEQ ID NO. 8  6 3276 GGCTAAGCTTGTACAATAACT ORF SEQ ID NO. 9  7 3688 GCGGAATGAACCACATCTTGC UTR3 SEQ ID NO. 10  8 3801 GGCCTTCTCTGAAGGACATTG UTR3 SEQ ID NO. 11  9 4354 GGACTCAATGCACTGACATTG UTR3 SEQ ID NO. 12 10 4578 GGTACCCACTGCTCTGGTTAT UTR3 SEQ ID NO. 13 HLA-A  1   96 GCTCCCACTCCATGAGGTATT ORF SEQ ID NO. 14  2  111 GGTATTTCTTCACATCCGTGT ORF SEQ ID NO. 15  3  279 AGGAGACACGGAATGTGAAGG ORF SEQ ID NO. 16  4 1164 CCCTTCCCTTTGTGACTTGAA UTR3 SEQ ID NO. 17 HLA-B  1   95 GCTCCCACTCCATGAGGTATT ORF SEQ ID NO. 18  2  110 GGTATTTCTACACCTCCGTGT ORF SEQ ID NO. 19  3  273 GGACCGGAACACACAGATCTA ORF SEQ ID NO. 20  4  275 ACCGGAACACACAGATCTACA ORF SEQ ID NO. 21  5  278 GGAACACACAGATCTACAAGG ORF SEQ ID NO. 22  6  279 GAACACACAGATCTACAAGGC ORF SEQ ID NO. 23

[0143] The sequence of the above shRNA or shRNA-miR for the immunologically compatible molecule was as follows:

[0144] 1. shRNA Sequence

[0145] (1) General backbone sequence of shRNA: comprising, in sequence from 5′ to 3′, an shRNA target sequence, a stem-loop sequence, and a reverse complement sequence of the shRNA target sequence. Specifically, as follows,

TABLE-US-00004 5′-N.sub.1 . . . N.sub.21TTCAAGAGA N.sub.22 . . . N.sub.42-3′

[0146] wherein:

[0147] a. N represented the base A or T or G or C;

[0148] b. the sequence N1 . . . N21 was the same as the shRNA target sequence;

[0149] c. the sequence N22 . . . N42 was reverse complementary to the sequence N1 . . . N21;

[0150] d. this backbone sequence did not include promoters and terminators, and promoters and terminators were additionally required to regulate the expression of this backbone sequence through inducible expression system elements; and

[0151] (2) shRNA sequence: the sequence N1 . . . N21 and the sequence N22 . . . N42 in the general backbone sequence of shRNA were replaced with the shRNA target sequence of the corresponding gene.

[0152] 2. shRNA-miR Sequence

[0153] (1) General backbone sequence of shRNA-miR: obtained by replacing a target sequence in microRNA-30 with the shRNA-miR target sequence. Specifically, as follows,

TABLE-US-00005 5′-GAGGCTTCAGTACTTTACAGAATCGTTGCCTGCACATCTTGGAAACA CTTGCTGGGATTACTTCTTCAGGTTAACCCAACAGAAGGCTAAAGAAGGT ATATTGCTGTTGACAGTGAGCGM.sub.1N.sub.1 . . . N.sub.21TAGTGAAGCCACAGA TGTAN.sub.22 . . . N.sub.42M.sub.2TGCCTACTGCCTCGGACTTCAAGGGGCTACTT TAGGAGCAATTATCTTGTTTACTAAAACTGAATACCTTGCTATCTCTTTG ATACATTTTTACAAAGCTGAATTAAAATGGTATAAAT-3′

[0154] wherein:

[0155] a. the base M represented the base A or C, and N represented the base A or T or G or C;

[0156] b. the base M1 was complementary to the base M2; and the sequence N1 . . . N21 was reverse complementary to the sequence N22 . . . N42;

[0157] c. if N1 was the base G, then M1 was the base A; otherwise, M1 was the base C;

[0158] d. the sequence N1 . . . N21 was the same as the shRNA target sequence; and

[0159] e. this backbone sequence did not include promoters and terminators, and the promoters and terminators were additionally required to regulate the expression of this backbone sequence through inducible expression system elements.

[0160] (2) shRNA-miR Sequence:

[0161] The sequences N1 . . . N21 and N22 . . . N42 in the backbone sequence of shRNA-miR were replaced with the shRNA target sequence of the corresponding gene.

[0162] In the immunologically compatible molecule, knock-in schemes in Tables 3 to 6 below, the shRNA or shRNA-miR immunologically compatible molecule sequences of each experimental group were all shRNA or shRNA-miR immunologically compatible molecules constructed using the target sequences 1 in Table 2. Those skilled in the art can understand that shRNA or shRNA-miR immunologically compatible molecules constructed using other target sequences can also achieve the technical effects of the present disclosure and all fall within the scope of the claims of the present disclosure.

[0163] V. Knock-In Scheme of the Immunologically Compatible Molecule

[0164] 1. Experimental Scheme and Grouping

[0165] The experimental schemes for knocking in one or more immunologically compatible molecules into genomic safe loci of pluripotent stem cells were shown in Tables 3-6, wherein Aa1-D3 represented experimental groups, and the sign “+” represented the knocked-in immunologically compatible molecule.

TABLE-US-00006 TABLE 3 Knock-in scheme I of the immunologically compatible molecule Immunologically compatible Experimental group molecule Aa1 Aa2 Ab1 Ab2 Ac1 Ac2 Ad1 Ad2 Ae1 Ae2 Af1 B2M (beta-2- + + microglobulin) shRNA CIITA shRNA + + B2M (beta-2- + + microglobulin) shRNA-miR CIITA shRNA- + + miR HLA-A shRNA + + HLA-B shRNA + + HLA-A shRNA- + miR HLA-B shRNA- + miR HLA-C retained (preferably retained homozygous HLA-C, preferably retained homozygous HLC-C from 30 HLA-C strains that covered more than 90% of the human population) HLA-C-B2M fusion protein (preferably retained homozygous HLA-C, preferably retained homozygous HLC-C from 30 HLA-C strains that covered more than 90% of the human population) CD47 HLA-G PD-L1 Siglec-15 B7H4 B7H5 BTLA activating antibody

TABLE-US-00007 TABLE 4 Knock-in scheme II of immunologically compatible molecule Specific immunologically compatible Experimental group molecule Af2 Ag1 Ag2 Ah Ai1 Ai2 Ai3 Ai4 Ai5 Ai6 B1 B2M (beta-2- microglobulin) shRNA CIITA shRNA B2M (beta-2- + (3′UTR) + + + + microglobulin) shRNA-miR CIITA shRNA-miR + + + + + + + + HLA-A shRNA HLA-B shRNA HLA-A shRNA- + + + + + + miR HLA-B shRNA- + + + + + + miR HLA-C retained + + + + + (preferably retained homozygous HLA- C, preferably retained homozygous HLC- C from 30 HLA-C strains that covered more than 90% of the human population). HLA-C*01:02 (SEQ ID NO. 107) was taken as an example here. HLA-C-B2M + fusion protein (preferably retained homozygous HLA- C, preferably retained homozygous HLC- C from 30 HLA-C strains that covered more than 90% of the human population). HLA-C*01:02- B2M fusion gene was taken as an example here. CD47 + + + + + HLA-G + + + + + PD-L1 + Siglec-15 B7H4 B7H5 BTLA activating antibody

TABLE-US-00008 TABLE 5 Knock-in scheme III of immunologically compatible molecule Specific immunologically compatible molecule B2 B3 B4 B5 C1 C2 C3 C4 C5 C6 C7 B2M (beta-2-microglobulin) shRNA CIITA shRNA B2M (beta-2-microglobulin) + + + + + + + + + + + shRNA-miR CIITA shRNA-miR + + + + + + + + + + + HLA-A shRNA HLA-B shRNA HLA-A shRNA-miR HLA-B shRNA-miR HLA-C retained (preferably retained homozygous HLA-C, preferably retained homozygous HLC-C from 30 HLA-C strains that covered more than 90% of the human population) HLA-C-B2M fusion protein (preferably retained homozygous HLA-C, preferably retained homozygous HLC-C from 30 HLA-C strains that covered more than 90% of the human population) CD47 + + + + + + + + + + + HLA-G + + + + + + + + + + + PD-L1 + + + + + + Siglec-15 + + + + + + + B7H4 + + + + + + B7H5 + + + + + + BTLA activating antibody + + + + + + +

TABLE-US-00009 TABLE 6 Knock-in scheme IV of immunologically compatible molecule Specific immunologically compatible molecule D1 D2 D3 B2M (beta-2-microglobulin) shRNA CIITA shRNA B2M (beta-2-microglobulin) shRNA-miR + + CIITA shRNA-miR + + + HLA-A shRNA HLA-B shRNA HLA-A shRNA-miR + HLA-B shRNA-miR + HLA-C retained (preferably retained homozygous HLA-C, + preferably retained homozygous HLA-C from 30 HLA-C strains that covered more than 90% of the human population) HLA-C*02:02 (SEQ ID NO. 108) was taken as an example here. HLA-C-B2M fusion protein (preferably retained homozygous HLA-C, preferably retained homozygous HLA-C from 30 HLA-C strains that covered more than 90% of the human population) CD47 + + HLA-G + PD-L1 + + + Siglec-15 + + + B7H4 + + + B7H5 BTLA activating antibody + + +

[0166] 2. Construction of KI Plasmid in Each Group

[0167] An “AAVS1 KI Vector (shRNA, shRNA-miR, Gene, Neo)” KI Donor plasmid and an “AAVS1 KI Vector (shRNA, shRNA-miR, Gene, Puro)” KI Donor plasmid were constructed. These two plasmids were collectively referred to as the “AAVS1 KI Vector” KI backbone plasmid (the same below), and the plasmid structures were shown in FIGS. 1 and 2.

[0168] Construction Method:

[0169] a. Acquisition of basic backbone of the plasmid: Primers were designed, an Amp(R)-pUC origin fragment was obtained from pUC18 (Takara, Code No. 3218) plasmid by PCR, and the product was then recovered.

[0170] b. Acquisition of recombination arms: Primers were designed, the genomic DNA of a human cell was taken as a template, AAVS1-HR-L (SEQ ID NO. 25) and AAVS1-HR-R (SEQ ID NO. 26) fragments were amplified, and the product was then recovered.

[0171] c. Acquisition of processor complex genes: mRNA was extracted from a human cell and reverse transcribed into cDNA, primers were then designed, the cDNA was used as a template to amplify the genes DROSHA and AGO2, and finally, the product was recovered.

[0172] d. Acquisition of other plasmid elements: Primers were designed, plasmids containing the plasmid elements were directly subcloned, and the product was then recovered.

[0173] e. Plasmid assembly: The various products obtained in the previous steps were ligated into a circular plasmid by overlap PCR or by recombination using a recombinase (Nanjing Vazyme Biotech, C113-01).

[0174] The immunologically compatible molecule was knocked into the “AAVS1 KI Vector” KI backbone plasmid at MCS I (multiple cloning site I) and/or MCS II (multiple cloning site II) and/or at the position of the general backbone of shRNA. The specific knock-in positions were as follows:

[0175] (1) Group Aa1: The immunologically compatible molecule “B2M shRNA” was knocked in at the “general backbone of shRNA”.

[0176] (2) Group Aa2: The immunologically compatible molecule “B2M shRNA-miR” was knocked in at “MCS I”.

[0177] (3) Group Ab1: The immunologically compatible molecule “CIITA shRNA” was knocked in at the “general backbone of shRNA”.

[0178] (4) Group Ab2: The immunologically compatible molecule “CIITA shRNA-miR” was knocked in at “MCS I”.

[0179] (5) Group Ac1: The immunologically compatible molecules “B2M shRNA” and “CIITA shRNA” were knocked in at the “general backbone of shRNA” (the 2 immunologically compatible molecules were seamlessly linked).

[0180] (6) Group Ac2: The immunologically compatible molecules “B2M shRNA-miR” and “CIITA shRNA-miR” were knocked in at “MCS I” (the 2 immunologically compatible molecules were seamlessly linked).

[0181] (7) Group Ad1: The immunologically compatible molecule “HLA-A shRNA” was knocked in at the “general backbone of shRNA”.

[0182] (8) Group Ad2: The immunologically compatible molecule “HLA-A shRNA-miR” was knocked in at “MCS I”.

[0183] (9) Group Ae1: The immunologically compatible molecule “HLA-B shRNA” was knocked in at the “general backbone of shRNA”.

[0184] (10) Group Ae2: The immunologically compatible molecule “HLA-B shRNA-miR” was knocked in at “MCS I”.

[0185] (11) Group Af1: The immunologically compatible molecules “HLA-A shRNA” and “HLA-B shRNA” were knocked in at the “general backbone of shRNA” (the 2 immunologically compatible molecules were seamlessly linked).

[0186] (12) Group Af2: The immunologically compatible molecules “HLA-A shRNA-miR” and “HLA-B shRNA-miR” were knocked in at “MCS I” (the 2 immunologically compatible molecules were seamlessly linked).

[0187] (13) Group Ag1: The immunologically compatible molecules “HLA-A shRNA-miR” and “HLA-B shRNA-miR” were knocked in at “MCS I” (the 2 immunologically compatible molecules were seamlessly linked), and the immunologically compatible molecule “HLA-C” was knocked in at “MCS II”.

[0188] (14) Group Ag2: The immunologically compatible molecule “B2M shRNA-miR 3′UTR” was knocked in at “MCS I”, and the immunologically compatible molecule “HLA-C-B2M” was knocked in at “MCS II”.

[0189] (15) Group Ah: The immunologically compatible molecules “CIITA shRNA-miR”, “HLA-A shRNA-miR” and “HLA-B shRNA-miR” were knocked in at “MCS I” (the 3 immunologically compatible molecules were seamlessly linked), and the immunologically compatible molecule “HLA-C” was knocked in at “MCS II”.

[0190] (16) Group Ai1: The immunologically compatible molecules “B2M shRNA-miR” and “CIITA shRNA-miR” (the 2 immunologically compatible molecules were seamlessly linked) were knocked in at “MCS I”, and the immunologically compatible molecule “CD47” was knocked in at “MCS II”.

[0191] (17) Group Ai2: The immunologically compatible molecules “B2M shRNA-miR” and “CIITA shRNA-miR” were knocked in at “MCS I” (the 2 immunologically compatible molecules were seamlessly linked), and the immunologically compatible molecule “HLA-G” was knocked in at “MCS II”.

[0192] (18) Group Ai3: The immunologically compatible molecules “B2M shRNA-miR” and “CIITA shRNA-miR” were knocked in at “MCS I” (the 2 immunologically compatible molecules were seamlessly linked), and the immunologically compatible molecules “CD47” and “HLA-G” were knocked in at “MCS II” (the 2 immunologically compatible molecules were linked using EMCV IRESwt).

[0193] (19) Group Ai4: The immunologically compatible molecules “CIITA shRNA-miR”, “HLA-A shRNA-miR” and “HLA-B shRNA-miR” were knocked in at “MCS I” (the 3 immunologically compatible molecules were seamlessly linked), and the immunologically compatible molecules “HLA-C” and “CD47” were knocked in at “MCS II” (the 2 immunologically compatible molecules were linked using EMCV IRESwt).

[0194] (20) Group Ai5: The immunologically compatible molecules “CIITA shRNA-miR”, “HLA-A shRNA-miR” and “HLA-B shRNA-miR” were knocked in at “MCS I” (the 3 immunologically compatible molecules were seamlessly linked), and the immunologically compatible molecules “HLA-C” and “HLA-G” were knocked in at “MCS II” (the 2 immunologically compatible molecules were linked using EMCV IRESwt).

[0195] (21) Group Ai6: The immunologically compatible molecules “CIITA shRNA-miR”, “HLA-A shRNA-miR” and “HLA-B shRNA-miR” were knocked in at “MCS I” (the 3 immunologically compatible molecules were seamlessly linked), and the immunologically compatible molecules “HLA-C”, “CD47” and “HLA-G” were knocked in at “MCS II” (the 3 immunologically compatible molecules were linked using EMCV IRESwt).

[0196] (22) Group B1: The immunologically compatible molecules “B2M shRNA-miR” and “CIITA shRNA-miR” were knocked in at “MCS I” (the 2 immunologically compatible molecules were seamlessly linked), and the immunologically compatible molecules “CD47”, “HLA-G” and “PD-L1” were knocked in at “MCS II” (the 3 immunologically compatible molecules were linked using EMCV IRESwt).

[0197] (23) Group B2: The immunologically compatible molecules “B2M shRNA-miR” and “CIITA shRNA-miR” were knocked in at “MCS I” (the 2 immunologically compatible molecules were seamlessly linked), and the immunologically compatible molecules “CD47”, “HLA-G” and “Siglec-15” were knocked in at “MCS II” (the 3 immunologically compatible molecules were linked using EMCV IRESwt).

[0198] (24) Group B3: The immunologically compatible molecules “B2M shRNA-miR” and “CIITA shRNA-miR” were knocked in at “MCS I” (the 2 immunologically compatible molecules were seamlessly linked), and the immunologically compatible molecules “CD47”, “HLA-G” and “B7H4” were knocked in at “MCS II” (the 3 immunologically compatible molecules were linked using EMCV IRESwt).

[0199] (25) Group B4: The immunologically compatible molecule “B2M shRNA-miR” and “CIITA shRNA-miR” were knocked in at “MCS I” (the 2 immunologically compatible molecules were seamlessly linked), and the immunologically compatible molecules “CD47”, “HLA-G” and “B7H5” were knocked in at “MCS II” (the 3 immunologically compatible molecules were linked using EMCV IRESwt).

[0200] (26) Group B5: The immunologically compatible molecules “B2M shRNA-miR” and “CIITA shRNA-miR” were knocked in at “MCS I” (the 2 immunologically compatible molecules were seamlessly linked), and the immunologically compatible molecules “CD47”, “HLA-G” and “BTLA activating antibody” were knocked in at “MCS II” (the 3 immunologically compatible molecules were linked using EMCV IRESwt).

[0201] (27) Group C1: The immunologically compatible molecules “B2M shRNA-miR” and “CIITA shRNA-miR” were knocked in at “MCS I” (the 2 immunologically compatible molecules were seamlessly linked), and the immunologically compatible molecules “CD47”, “HLA-G”, “PD-L1”, “Siglec-15”, “B7H4”, “B7H5” and “BTLA activating antibody” were knocked in at “MCS II” (the 7 immunologically compatible molecules were linked using EMCV IRESwt).

[0202] (28) Group C2: The immunologically compatible molecules “B2M shRNA-miR” and “CIITA shRNA-miR” were knocked in at “MCS I” (the 2 immunologically compatible molecules were seamlessly linked), and the immunologically compatible molecules “CD47”, “HLA-G”, “Siglec-15”, “B7H4”, “B7H5” and “BTLA activating antibody” were knocked in at “MCS II” (the 6 immunologically compatible molecules were linked using EMCV IRESwt).

[0203] (29) Group C3: The immunologically compatible molecules “B2M shRNA-miR” and “CIITA shRNA-miR” were knocked in at “MCS I” (the 2 immunologically compatible molecules were seamlessly linked), and the immunologically compatible molecules “CD47”, “HLA-G”, “PD-L1”, “B7H4”, “B7H5” and “BTLA activating antibody” were knocked in at “MCS II” (the 6 immunologically compatible molecules were linked using EMCV IRESwt).

[0204] (30) Group C4: The immunologically compatible molecules “B2M shRNA-miR” and “CIITA shRNA-miR” were knocked in at “MCS I” (the 2 immunologically compatible molecules were seamlessly linked), and the immunologically compatible molecules “CD47”, “HLA-G”, “PD-L1”, “Siglec-15”, “B7H5” and “BTLA activating antibody” were knocked in at “MCS II” (the 6 immunologically compatible molecules were linked using EMCV IRESwt).

[0205] (31) Group C5: The immunologically compatible molecules “B2M shRNA-miR” and “CIITA shRNA-miR” were knocked in at “MCS I” (the 2 immunologically compatible molecules were seamlessly linked), and the immunologically compatible molecules “CD47”, “HLA-G”, “PD-L1”, “Siglec-15”, “B7H4” and “BTLA activating antibody” were knocked in at “MCS II” (the 6 immunologically compatible molecules were linked using EMCV IRESwt).

[0206] (32) Group C6: The immunologically compatible molecules “B2M shRNA-miR” and “CIITA shRNA-miR” were knocked in at “MCS I” (the 2 immunologically compatible molecules were seamlessly linked), and the immunologically compatible molecules “CD47”, “HLA-G”, “PD-L1”, “Siglec-15” and “BTLA activating antibody” were knocked in at “MCS II” (the 5 immunologically compatible molecules were linked using EMCV IRESwt).

[0207] (33) Group C7: The immunologically compatible molecules “B2M shRNA-miR” and “CIITA shRNA-miR” were knocked in at “MCS I” (the 2 immunologically compatible molecules were seamlessly linked), and the immunologically compatible molecules “CD47”, “HLA-G”, “PD-L1”, “Siglec-15”, “B7H4” and “B7H5” were knocked in at “MCS II” (the 6 immunologically compatible molecules were linked using EMCV IRESwt).

[0208] (34) Group D1: The immunologically compatible molecules “B2M shRNA-miR” and “CIITA shRNA-miR” were knocked in at “MCS I” (the 2 immunologically compatible molecules were seamlessly linked), and the immunologically compatible molecules “CD47”, “PD-L1”, “Siglec-15”, “B7H4” and “BTLA activating antibody” were knocked in at “MCS II” (the 5 immunologically compatible molecules were linked using EMCV IRESwt).

[0209] (35) Group D2: The immunologically compatible molecules “B2M shRNA-miR” and “CIITA shRNA-miR” were knocked in at “MCS I” (the 2 immunologically compatible molecules were seamlessly linked), and the immunologically compatible molecules “HLA-G”, “PD-L1”, “Siglec-15”, “B7H4” and “BTLA activating antibody” were knocked in at “MCS II” (the 5 immunologically compatible molecules were linked using EMCV IRESwt).

[0210] (36) Group D3: The immunologically compatible molecules “B2M shRNA-miR” and “CIITA shRNA-miR” were knocked in at “MCS I” (the 2 immunologically compatible molecules were seamlessly linked), and the immunologically compatible molecules “CD47”, “PD-L1”, “Siglec-15”, “B7H4” and “BTLA activating antibody” were knocked in at “MCS II” (the 5 immunologically compatible molecules were linked using EMCV IRESwt).

[0211] VI. shRNA/miRNA Processor Complex-Related Gene and Anti-Interferon Effector Molecule

[0212] Primary miRNA (pri-miRNA) in the nucleus was microprocessed by the complex Drosha-DGCR8, the pri-miRNA was cleaved into precursor miRNA (pre-miRNA), and at this time, a hairpin structure was formed. Next, the pre-miRNA was transported out of the nucleus via the Exportin-S-Ran-GTP complex. RNase Dicer enzyme, which bound to the double-stranded RNA-binding protein TRBP (TARBP2) in the cytoplasm, cleaved the pre-miRNA into a mature length, and at this time, the miRNA was still in a double-stranded state. Finally, it was transported into AGO2 to form RISC (RNA-induced silencing complex). Ultimately, one strand of the miRNA duplex remained in the RISC complex, while the other was expelled and rapidly degraded. As the main binding protein of Drosha, DGCR8 could bind to pri-miRNA via its two double-stranded RNA binding regions at the C-terminus and recruit and guide Drosha to cleave the pri-miRNA at the correct position to produce pre-miRNA, and the pre-miRNA was further processed and cleaved by Dicer and TRBP/PACT to form mature miRNA. The deletion or abnormal expression of DGCR8 could affect the cleaving activity of Drosha, which affects the activity of miRNA and lead to the occurrence of diseases. TRBP could recruit the Dicer complex and miRNA to form RISC Ago2.

[0213] When the present disclosure utilized gene knock-in technology to knock-in shRNA-miR expression sequences (which were directed at HLA class I molecules and HLA class II molecules and the expression of which could be inducibly turned off) at a genomic safe locus, it was preferable to simultaneously knock in shRNA and/or miRNA processing machinery the expression of which could be inducibly turned off expression, including at least one selected from the group consisting of Dhrosha (Accession number: NM_001100412), Ago1 (Accession number: NM_012199), Ago2 (Accession number: NM_001164623), Dicer1 (Accession number: NM_001195573), Exportin-5 (Accession number: NM_020750), TRBP (Accession number: NM_134323), PACT (Accession number: NM_003690) and DGCR8 (Accession number: NM_022720), such that cell functions and processing for other miRNAs were not affected.

[0214] In addition, during induced IFN production, double-stranded RNA-dependent Protein Kinase (PKR, which was a key factor in the entire cell signal transduction pathway) and 2′,5′-oligoadenylate synthetase (2-5As) were both involved, and these two enzymes were closely related to dsRNA-induced IFN production. PKR could inhibit protein synthesis by phosphorylating eukaryotic cell transcription factors, making cells stagnate in G0/G1 and G2/M phases and inducing apoptosis, while dsRNA could promote 2-5As synthesis, resulting in non-specific activation of an RNase, i.e., Rnase L, causing all mRNAs in the cells to be degraded, leading to cell death. Induction specificity for type I interferon was achieved by members of the IRF transcription factor family. In the absence of the expression of IRF-3 and IRF-7 in cells, type I interferon could not be induced and could not be secreted in many viral infection circumstances. IFN response was absent, and the recovery thereof required co-expression of the above two proteins.

[0215] The present disclosure utilized gene knock-in technology to knock-in an expression sequence of immunologically compatible molecule shRNA-miR at a genomic safe locus; preferably sequences of shRNA and/or shRNA-miR for at least one selected from the group consisting of PKR, 2-5As, IRF-3 and IRF-7 (the expression of at least one selected from the group consisting of PKR, 2-5As, IRF-3 and IRF-7 can be inducibly turned off) is simultaneously knocked in, so as to reduce dsRNA-induced interferon response and avoid cytotoxicity.

[0216] The order of the insertion positions of the shRNA/miRNA processor complex-related gene, the anti-interferon effector molecule, the immunologically compatible molecule at the genomic safe loci was not limited. They could be arranged in any order without interfering with each other or affecting the structures and functions of other genes in the genome.

[0217] The specific target sequences of anti-interferon effector molecules were shown in Table 7.

TABLE-US-00010 TABLE 7 Target sequences of anti-interferon effector molecules Interferon effector molecule No. Start Target sequence (DNA) Region Sequence number PKR  1 1538 GGATGGATTTGATTATGATCC ORF SEQ ID NO. 31  2 1672 GGACCTTGGAACAATGGATTG ORF SEQ ID NO. 32  3 1982 GCTAATTCTTGCTGAACTTCT ORF SEQ ID NO. 33  4 1992 GCTGAACTTCTTCATGTATGT ORF SEQ ID NO. 34  5 2722 GCCTCATCTCTTTGTTCTAAA UTR3 SEQ ID NO. 35  6 3708 GCTCTGGAGAAGATATATTTG UTR3 SEQ ID NO. 36  7 4080 GCTCTTGAGGGAACTAATAGA UTR3 SEQ ID NO. 37  8 4144 GGGACGGCATTAATGTATTCA UTR3 SEQ ID NO. 38  9 4250 GGACAAACATGCAAACTATAG UTR3 SEQ ID NO. 39 10 4270 GCAGCAACCAGCTACCATTCT UTR3 SEQ ID NO. 40 2-5 As  1   49 GCAGTTCTGTTGCCACTCTCT UTR5 SEQ ID NO. 41 (OAS1)  2  363 GGGAGAGTTCATCCAGGAAAT ORF SEQ ID NO. 42  3  364 GGAGAGTTCATCCAGGAAATT ORF SEQ ID NO. 43  4  365 GAGAGTTCATCCAGGAAATTA ORF SEQ ID NO. 44  5  400 GCCTGTCAAAGAGAGAGAGCA ORF SEQ ID NO. 45  6  471 GCTCAGCTTCGTACTGAGTTC ORF SEQ ID NO. 46  7  644 GCTTCACAGAACTACAGAGAG ORF SEQ ID NO. 47  8  884 GCATCTACTGGACAAAGTATT ORF SEQ ID NO. 48  9 1055 GGCTGAATTACCCATGCTTTA ORF SEQ ID NO. 49 10 1056 GCTGAATTACCCATGCTTTAA ORF SEQ ID NO. 50 2-5 As  1  138 GGGTTGGTTTATCCAGGAATA ORF SEQ ID NO. 51 (OAS2)  2  354 GGATCAGAAGAGAAGCCAACG ORF SEQ ID NO. 52  3  467 GGTTCACCATCCAGGTGTTCA ORF SEQ ID NO. 53  4  698 GCTCTCTTCTCTGGAACTAAC UTR3 SEQ ID NO. 54  5 1211 GCTAGAGTGACTCCATCTTAA UTR3 SEQ ID NO. 55  6 1302 GCTGACCACCAATTATAATTG UTR3 SEQ ID NO. 56  7 1322 GCAGAATATTTAAGGCCATAC UTR3 SEQ ID NO. 57  8 1393 GCCCACTTAAAGGCAGCATTA UTR3 SEQ ID NO. 58  9 1442 GGTCATCAATACCACTGTTAA UTR3 SEQ ID NO. 59 10 1958 GCATTCCTCCTTCTCCTTTCT UTR3 SEQ ID NO. 60 2-5 As  1  620 GGAGGAACTTTGTGAACATTC ORF SEQ ID NO. 61 (OAS3)  2  776 GCTGTAAGAAGGATGCTTTCA ORF SEQ ID NO. 62  3 1985 GCTGCAGGCAGGATTGTTTCA ORF SEQ ID NO. 63  4 2619 GCAGTTCGAGGTCAAGTTTGA ORF SEQ ID NO. 64  5 3852 GCCAATTAGCTGAGAAGAATT UTR3 SEQ ID NO. 65  6 3983 GCAGGTTTACAGTGTATATGT UTR3 SEQ ID NO. 66  7 5130 GCCTACAGAGACTAGAGTAGG UTR3 SEQ ID NO. 67  8 5478 GCAGTTGGGTACCTTCCATTC UTR3 SEQ ID NO. 68  9 5573 GCAACTCAGGTGCATGATACA UTR3 SEQ ID NO. 69 10 5993 GCATGGCGCTGGTACGTAAAT UTR3 SEQ ID NO. 70 IRF3  1  148 GCCTCGAGTTTGAGAGCTA UTR5 SEQ ID NO. 71  2  301 AGACATTCTGGATGAGTTA UTR5 SEQ ID NO. 72  3    43 GGGTCTGTTACCCAAAGAA UTR5 SEQ ID NO. 73  4   44 GGTCTGTTACCCAAAGAAT UTR5 SEQ ID NO. 74  5  508 GGAAGGAAGCGGACGCTCA ORF SEQ ID NO. 75  6  275 GGAGGCAGTACTTCTGATA UTR5 SEQ ID NO. 76  7  111 CGCTCTAGAGCTCAGCTGA UTR5 SEQ ID NO. 77  8  834 CCACCACCTCAACCAATAA UTR3 SEQ ID NO. 78  9   78 ATTTCAAGAAGTCGATCAA UTR5 SEQ ID NO. 79 10  488 GAAGATCTGATTACCTTCA ORF SEQ ID NO. 80 IRF-7  1   68 GGACACTGGTTCAACACCTGT UTR5 SEQ ID NO. 81  2   75 GGTTCAACACCTGTGACTTCA UTR5 SEQ ID NO. 82  3   83 ACCTGTGACTTCATGTGTGCG UTR5 SEQ ID NO. 83  4 1098 GCTGGACGTGACCATCATGTA ORF SEQ ID NO. 84  5 1101 GGACGTGACCATCATGTACAA ORF SEQ ID NO. 85  6 1102 GACGTGACCATCATGTACAAG ORF SEQ ID NO. 86  7 1103 ACGTGACCATCATGTACAAGG ORF SEQ ID NO. 87  8 1512 ACGCTATACCATCTACCTGGG ORF SEQ ID NO. 88  9 1694 GCCTCTATGACGACATCGAGT ORF SEQ ID NO. 89 10 1705 GACATCGAGTGCTTCCTTATG ORF SEQ ID NO. 90

[0218] The shRNA or shRNA-miR backbone sequence of the above anti-interferon effector molecules was the same as the shRNA or shRNA-miR backbone sequence of the immunologically compatible molecules.

[0219] In the anti-interferon effector molecule knock-in schemes of Examples 14-16 below, the anti-interferon effector molecule sequences of each experimental group were all shRNAs or shRNA-miRs constructed by using target sequence 1 in Table 7. Those skilled in the art can understand that shRNA or shRNA-miR anti-interferon effector molecules constructed using other target sequences can also achieve the technical effects of the present disclosure and all fall within the scope of protection of the claims of the present disclosure.

[0220] VII. Method for Knocking Exogenous Gene Inducible Expression System (Tet-Off System or Dimer Turn-Off Expression System) and Immunologically Compatible Molecule into Genomic Safe Loci of Pluripotent Stem Cell and Test Method

[0221] (I) Construction of sgRNA

[0222] 1. Plasmid

[0223] Cas9(D10A) plasmid and sgRNA plasmid were used to knock in exogenous genes. The map of the Cas9(D10A) plasmid was shown in FIG. 3, the map of the sgRNA plasmid with the AAVS1 safe locus was shown in FIGS. 4 and 5, and the map of the sgRNA plasmid with the eGSH safe locus was shown in FIGS. 6 and 7.

[0224] 2. Homologous Arms

[0225] (1). The nucleotide sequences of the AAVS1 homology arms AAVS1-HR-L and AAVS1-HR-R were shown in SEQ ID NO. 91 and SEQ ID NO. 92, respectively.

[0226] (2). The nucleotide sequences of the eGSH homology arms eGSH-HR-L and eGSH-HR-R were shown in SEQ ID NO. 93 and SEQ ID NO. 94, respectively.

[0227] 3. sgRNA Sequence

TABLE-US-00011 sgRNA-AAVS1-1: (SEQ ID NO. 95) 5′-TATAAGGTGGTCCCAGCTCGGGG-3′; sgRNA-AAVS1-2: (SEQ ID NO. 96) 5′-AGGGCCGGTTAATGTGGCTCTGG-3′. sgRNA-eGSH-1: (SEQ ID NO. 97) 5′-GGTGGAAGCTTCATTCCAGATGG-3′; sgRNA-eGSH-2: (SEQ ID NO. 98) 5′-GACCTGCCTCATTAAATATCAGG-3′.

[0228] 4. Plasmid Construction Method

[0229] (1) Empty sgRNA vector was digested with the restriction endonuclease BbsI and then recovered.

[0230] (2) sgRNA primers (with vector sticky ends) were synthesized.

[0231] (3) The primers were diluted with water to 10 μM, a reaction system was prepared and boiled in boiling water for 5 min, and the product was then cooled to room temperature to obtain an annealed product.

[0232] Reaction system: upstream primer: 2 μL, downstream primer: 2 μL, and water: 12.8 μL

[0233] (4) A DNA ligation reaction kit (TaKaRa, 6022) was used to ligate the vector and the annealed product in the previous steps to obtain a sgRNA plasmid containing the gene target sequence.

[0234] (II) Gene Editing Process

[0235] 1. Single-Cell Cloning Operating Steps for AAV Gene Knock-In

[0236] (1) Electrotransfection Procedure:

[0237] Donor cell preparation: Human pluripotent stem cells

[0238] Kit: Human Stem Cell Nucleofector® Kit 1

[0239] Instrument: Electrotransfection instrument

[0240] Medium: BioCISO

[0241] Inducible plasmid: s Cas9D10A, sgRNA clone AAVS1-1, sgRNA clone AAVS1-2, AAV S1 neo Vectol, and AAVS1 neo Vector II

[0242] Note: Inducible plasmids used for eGSH gene knock-in: Cas9D10A, sgRNA clone eGSH-1, sgRNA clone eGSH-2, and eGSH-neo/eGSH-puro(donor). Comparing the donor plasmid here with AAVS1, only the left and right recombination arms were different, and the other elements were the same. Since the gene editing process of eGSH was the same as that of AAVS1, it will not be repeated hereinbelow.

[0243] (2) The electrotransfected human pluripotent stem cells were screened in a dual-antibiotic medium containing G418 and puro.

[0244] (3) Single-cell clones were screened and cultured to obtain a single-cell clone strain.

[0245] 2. Culture Reagent of AAV Gene Knock-In Single-Cell Clone Strain

[0246] (1) Medium: BioCISO+300 μg/ml G418+0.5 μg/ml puro

[0247] (It should be placed at room temperature in advance for 30-60 minutes in the dark until it returned to room temperature. Note: BioCISO should not be preheated at 37° C. to avoid reduced biomolecular activity.)

[0248] (2) Matrigel: hESC grade Matrigel

[0249] (Before cell passaging or resuscitation, a Matrigel working solution was added to a cell culture flask or culture dish and shaken until uniform, and it was ensured that the Matrigel completely covered the bottom of the culture flask or culture dish and that the Matrigel at anywhere could not be dried before use. In order to ensure that the cells could better adhere and survive, Matrigel should be placed in an incubator at 37° C. for a coating time, which was not less than 0.5 hours for 1:100× Matrigel and not be less than 2 hours for 1:200×Matrigel.)

[0250] (3) Digestion solution: EDTA was dissolved with DPBS to a final concentration of 0.5 mM, pH 7.4

[0251] (Note: EDTA could not be diluted with water; otherwise, cells would die due to reduced osmotic pressure.)

[0252] (4) Cryopreservative fluid: 60% BioCISO+30% ESCs grade FBS+10% DMSO

[0253] (It was preferred to prepare the cryopreservative fluid immediately before use.)

[0254] 3. Process of Routine Maintenance Subculture

[0255] (1) Optimal Passage Time and Passage Ratio

[0256] a. Optimal time for passage: When the overall cell confluence reached 80% to 90%.

[0257] b. Optimal ratio for passage: 1:4 to 1:7 passage, and the optimal confluence on the next day should be maintained at 20% to 30%.

[0258] (2) Passaging Process

[0259] a. The Matrigel in the coated cell culture flask or culture dish was aspirated and discarded in advance, an appropriate amount of medium (BioCISO+300 μg/ml G418+0.5 μg/ml puro) was added, and the flask or dish was placed in an incubator at 37° C., 5% CO.sub.2;

[0260] b. when the cells met passage requirements, the medium supernatant was aspirated, and an appropriate amount of 0.5 mM EDTA digestion solution was added to the cell flask or culture dish;

[0261] c. the cells were put into an incubator at 37° C., 5% CO.sub.2 and incubated for 5-10 minutes (digested until most of the cells observed under a microscope were shrunk and rounded but not yet floating), and the cells were detached from the wall by gentle pipetting, then the cell suspension was aspirated into a centrifuge tube for centrifugation at 200 g for 5 min);

[0262] d. after centrifugation, the supernatant was discarded, the cells were resuspended with a medium, the cells were repeatedly gently pipetted several times until uniformly mixed, and the cells were then transferred to a Matrigel-coated flask or dish prepared in advance;

[0263] e. after the cells were transferred to the cell flask or culture dish, the flask or dish was shaken horizontally back and forth and from side to side, and after no abnormality was observed under the microscope, the cells were shaken uniform and placed in an incubator for culturing at 37° C., 5% CO.sub.2; and

[0264] f. the next day, the adherence and survival state of the cells were observed, and the medium was aspirated and replaced every day on schedule as normal.

[0265] 4. Cell Cryopreservation

[0266] (1) According to routine passaging operating steps, the cells were digested with 0.5 mM EDTA until most of the cells were shrunk and rounded but not yet floating, the cells were gently pipetted, the cell suspension was collected and centrifuged at 200 g for 5 minutes, the supernatant was discarded, an appropriate amount of cryopreservative fluid was added to resuspend the cells, and the cells were transferred to cryopreservation tubes (it was recommended to cryopreserve one tube for a 6-well plate confluence of 80%, and the volume of the cryopreservative fluid was 0.5 ml/tube);

[0267] (2) the cryopreservation tubes were placed in a programmed cooling box and immediately placed at −80° C. overnight (it was necessary to ensure that the temperature of the cryopreservation tubes decreased by 1° C. per minute); and

[0268] (3) the cells were immediately transferred into liquid nitrogen the next day.

[0269] 5. Cell Resuscitation

[0270] (1) A Matrigel-coated cell flask or culture dish was prepared in advance; and before cell resuscitation, the Matrigel was aspirated, an appropriate amount of BioCISO was added to the cell flask or culture dish, and the cell flask or culture dish was incubated in an incubator at 37° C., 5% CO.sub.2;

[0271] (2) the cryopreservation tubes were quickly taken out from the liquid nitrogen, immediately placed in a water bath at 37° C. and quickly shaken to quickly thaw the cells; and upon careful observation, when ice crystals disappeared completely, shaking was stopped, and the cells were transferred to a biological safety cabinet;

[0272] (3) 10 ml DMEM/F12 (1:1) basal medium was added to a 15 ml centrifuge tube in advance and equilibrated to room temperature, 1 ml DMEM/F12 (1:1) was pipetted by a Pasteur pipette, slowly added to the cryopreservation tube, and gently mixed, and the cell suspension was transferred to the prepared 15 ml centrifuge tube containing DMEM/F12 (1:1), and centrifuged at 200 g for 5 min;

[0273] (4) the supernatant was carefully discarded, an appropriate amount of BioCISO was added, the cells were gently mixed until uniform and seeded in the cell flask or culture dish prepared in advance, the flask or dish was shaken horizontally back and forth, and from side to side, and after no abnormality was observed under the microscope, the cells were shaken uniform and placed in an incubator for culturing at 37° C., 5% CO.sub.2; and

[0274] (5) the next day, the adherence and survival state of the cells were observed, and the medium was replaced every day on schedule as normal. If the adherence was good, BioCISO was replaced with BioCISO+300 μg/ml G418+0.5 μg/ml puro.

[0275] (III) Detection Method of AAVS1 Gene Knock-In

[0276] 1. Detection of Single-Cell Clone AAVS1 Gene Knock-In

[0277] (1) Detection Instructions for AAVS1 Gene Knock-In

[0278] a. Test objective: gene-knock-in-treated cells were detected by PCR to test whether the cells were homozygous. Since the two donor fragments only had differences in terms of resistance gene sequence, in order to determine whether the cells were homozygous (the two chromosomes were respectively knocked in with donor fragments of different resistance genes), it was necessary to detect whether the genome of the cells contained the donor fragments of the two resistance genes, and only cells with dual knock-in were likely to be correct homozygotes.

[0279] b. Test method: First of all, a primer was designed inside the donor plasmid (non-recombination-arm part), and another primer was then designed in the genome PPP1R12C (non-recombination-arm part). If the donor fragments could be inserted correctly in the genome, there would be bands of interest; otherwise, no bands of interest would appear).

[0280] c. The primer sequences and PCR protocols in the test scheme were shown in Table 8:

TABLE-US-00012 TABLE 8 Primer sequences and PCR protocols in the test scheme PCR Primer Sequence Product conditions No. abbreviation Sequence (5′ 3′) number (bp) (Phanta enzyme) 1 F1 CCATAGCTCAGTCTGG SEQ ID NO. 99 1578 Annealing at TCTATC 58° C., 2 R1 TCAGGATGATCTGGAC SEQ ID NO. 100 extension for GAAGAG 1 min, and 30 cycles 3 F2 CCGGTCCTGGACTTTG SEQ ID NO. 101 1728 Annealing at TCTC 62° C., 4 R2 CTCGACATCGGCAAGG SEQ ID NO. 102 extension for TGTG 1 min 30 sec, and 30 cycles 5 F2 CGCATTGGAGTCGCTT SEQ ID NO. 103 1874 Annealing at (Outer) TAAC 60° C., 6 R2 CGAGCTGCAAGAACTC SEQ ID NO. 104 extension for (Outer) TTCCTCAC 1 min 30 sec, and 18 cycles 7 F3 CACGGCACTTACCTGT SEQ ID NO. 105 2151 Annealing at (Outer) GTTCTGG 60° C., 8 R3 CAGTACAGGCATCCCT SEQ ID NO. 106 extension for (Outer) GTGAAAG 1 min 30 sec, and 18 cycles Note: The detection method for eGSH gene knock-in was the same as the detection principle and method for AAVS1 gene knock-in, and will not be described here.

[0281] VIII. Test Method for Gene Knock-In at Genomic Safe Locus

[0282] (1) Test objective: gene-knock-in-treated cells were detected by PCR to test whether the cells were homozygous. Since the two donor fragments only had differences in terms of resistance gene sequence, in order to determine whether the cells were homozygous (the two chromosomes were respectively knocked in with donor fragments of different resistance genes), it was necessary to detect whether the genome of the cells contained the donor fragments of the two resistance genes, and only cells with dual knock-in were likely to be correct homozygotes.

[0283] (2) Test method: First, a primer was designed inside the donor plasmid (non-recombination-arm part), and another primer was then designed in the genome (non-recombination-arm part). If the donor fragments could be inserted correctly in the genome, there would be bands of interest; otherwise, no bands of interest would appear.

[0284] IX. Test Method for Allogeneic Immunological Compatibility Effect (Specific Immunity and Innate Immunity)

[0285] 1. Preparation of Effector Cells

[0286] Blood was drawn from volunteers to isolate T cells and NK cells. Effector cells and immunologically compatible pluripotent stem cells were derived from different people.

[0287] 1) T cell isolation: Human peripheral blood mononuclear cells (PBMC) were isolated using Ficoll-hypaque density gradient centrifugation, and T cells were isolated using Dynabeads™ CD3 kit (Invitrogen™, Cat. No. 11151D). The cells were resuspended in RPMI1640 medium containing 10% FBS, and the cells were counted by trypan blue staining and concentrated to 1×10.sup.7 cells/mL.

[0288] 2) NK cell isolation: NK cells were sorted and isolated using MagniSort™ Human NK cell Enrichment Kit (Invitrogen™, Cat. No. 8804-6819-74). The cells were resuspended in RPMI1640 medium containing 10% FBS, and the cells were counted by trypan blue staining and concentrated to 1×10.sup.7 cells/mL.

[0289] 2. Preparation of Target Cells

[0290] Embryoid body cells prepared from PSCs were taken, digested and resuspended, and the cells were counted by trypan blue staining and prepared into a cell suspension of 1×10.sup.7 cells/mL.

[0291] 3. The .sup.51Cr Release Assay

[0292] When normal cells came into contact with T/NK cells (allogeneic), T/NK attacked the normal cells and caused cell lysis and death. However, if there was good immunological compatibility, no attack by T/NK would occur, that is, immune escape. Therefore, the detection of the amount of .sup.51Cr in the medium could reflect immunological compatibility. The less the amount of .sup.51Cr released into the detection medium, the better the immunological compatibility.

[0293] Cell-mediated cytotoxicity was quantitatively detected, wherein target cells were labeled with the radioisotope .sup.51Cr and co-incubated with effector molecules or cells, and the cytotoxic activity was determined according to the radiation pulse count (cpm) of .sup.51Cr released by target cell lysis.

[0294] 1) The target cells were labeled with 100 μCi (Ci, unit of radioactivity) Na.sup.51CrO.sub.4 at 37° C. for 120 min and shaken every 15 min; after labeling, centrifugation was further carried out 5 times with a washing solution; and finally, the cells were resuspended in a culture solution to 1×10.sup.6 cells/mL for use.

[0295] 2) The target cells and T/NK cells were added to a 96-well culture plate, wherein 100 μl of target cells (2.5×10.sup.3 cells) and 100 μl of effector cells (E/T=1:2, 1:5, or 1:10, E/T was the ratio of target cells to effector cells (T/NK)), and a natural release control well (100 μl target cells+100 μl medium) and a maximum release well (100 μl target cells+100 μl 2% SDS) were established. They were placed at 37° C., 5% CO.sub.2 for 4 h for incubation. After they were taken out, the supernatant was aspirated from each well with a pipette and centrifuged, 100 μl of supernatant was taken to measure the cpm value with a γ counter.

[0296] Note: Generally, the natural release rate of .sup.51Cr was required to be less than 10%.

[0297] 3) Calculation of results: The natural release rate of .sup.51Cr and the activity of T/NK cells were calculated according to the formulas:

[00001]${ }^{51}\mathrm{Cr}\mathrm{natural}\mathrm{release}\mathrm{rate}\left(\%\right)=\frac{\mathrm{cpm}\mathrm{value}\mathrm{of}\mathrm{natural}\mathrm{release}\mathrm{control}\mathrm{well}}{\mathrm{cpm}\mathrm{value}\mathrm{of}\mathrm{maximal}\mathrm{release}\mathrm{control}\mathrm{well}}\times 100\%\mathrm{Activity}\mathrm{of}T/\mathrm{NK}\mathrm{cell}\left(\%\right)=\frac{\mathrm{cpm}\mathrm{value}\mathrm{of}\mathrm{experimental}\mathrm{well}-\mathrm{cpm}\mathrm{value}\mathrm{of}\mathrm{natural}\mathrm{release}\mathrm{control}\mathrm{well}}{\mathrm{cpm}\mathrm{value}\mathrm{of}\mathrm{maximal}\mathrm{release}\mathrm{control}\mathrm{well}-\mathrm{cpm}\mathrm{value}\mathrm{of}\mathrm{natural}\mathrm{release}\mathrm{control}\mathrm{well}}\times 100\%$

[0298] 4. The CFSE Test Assay

[0299] The fluorescent dye CFSE, also known as CFDA SE (5,6-carboxyfluorescein diacetate, succinimidyl ester, hydroxyfluorescein diacetate succinimidyl ester), was a fluorescent dye that could penetrate cell membranes and could be detected by flow cytometry.

[0300] 1) A CFSE working solution (with a final concentration of 5 μmol/L CFSE) was added to the target cells, and the cells were incubated at 37° C., 5% CO.sub.2 for 10 min, and washed twice. After trypan blue staining and counting, the cells were resuspended in a medium and prepared into 1×10.sup.6 cells/mL for later use.

[0301] 2) The target cells and the effector cells were added to 5 ml flow tubes, wherein 100 μl of target cells (1×10.sup.5 ml.sup.−1) and effector cells (E/T=1:2, 1:5, or 1:10, E/T is the ratio of target cells to effector cells (T)) were added to each tube, and the flow tube in which only target cells were added was used as a control. The effector cells and the target cells in the flow tubes were gently mixed until uniform. PI was added, and they were placed at 37° C., 5% CO.sub.2 for 4 h for incubation. The percentage of CFSE.sup.+PI.sup.+ cells (dead target cells) was detected by flow cytometry.

Target cell death rate (%)=death rate of target cells stimulated with T cells (%)−the natural death rate of target cells (%)

[0302] 5. Analysis of CD.sub.107a Expression in NK Cells by Flow Cytometer (FCM)

[0303] When the NK cells killed the target cells, CD.sub.107a molecules were transported to the surface of the cell membrane, and NK cells with positive CD.sub.107a molecule expression could represent NK cells with killing activity.

[0304] 1) The effector cells and the target cells were mixed at a certain ratio (E/NK=3:1, 1:1, or 1:3, E/NK was the ratio of target cells to effector cells (NK)), placed in culture wells, and incubated at 37° C., 5% CO.sub.2 for 2 h, monensin (2 μmol/L) was added for continued incubation for 3.5 h, and PE-Cy5-CD.sub.107a and FITC-CD56 antibodies were then added for incubation for 30 min. After washing three times with PBS buffer, the cells were fixed with 200 μL of 1% paraformaldehyde for flow analysis. As a positive control, the effector cells were simultaneously stimulated with only PMA (2.5 μg/mL) and ionomycin (0.5 μg/mL).

[0305] Note: The natural expression frequency of CD.sub.107a on the surface of the NK cell membrane was very low, about 1.2% to 5.8%.

[0306] 2) Calculation of Results

NK cell cytotoxicity=CD.sub.107a positive rate upon stimulation by target cells (%)−CD.sub.107a natural expression rate (%)

[0307] X. MTT Experiment for Detecting Cell Viability

[0308] After digestion and cell counting, the cells were blown uniform with a corresponding medium and plated into a 96-well plate in which each well was seeded with 3000 cells, with 5 duplicate wells, the wells were then supplemented with the corresponding medium to a final volume of 150 uL, the corresponding medium was changed every day, the cells were placed in an incubator at 37° C., 5% CO.sub.2 for 72 h, then the MTT value was measured.

Example 2

[0309] The Tet-Off system and a combination of immunologically compatible molecules (Tables 3-6) were knocked into the genomic safe locus AAVS1 of hPSC-derived EBs by means of the method of Example 1 to obtain EB sphere immunologically compatible cells.

[0310] .sup.51Cr release assay was used to detect the effect of immunological compatibility between the EB sphere immunologically compatible cells and T cells:

[0311] 1. The EB sphere immunologically compatible cells were digested into individual cells as target cells;

[0312] 2. .sup.51Cr-labeled target cells and T cells were added to a 96-well culture plate at a ratio of 1:5 for post-reaction detection; and

[0313] 3. the cell-specific .sup.51Cr release rate of the EB sphere immunologically compatible cells was detected according to the .sup.51Cr release assay, and the results are shown in Table 9 and FIG. 8.

TABLE-US-00013 TABLE 9 .sup.51Cr release rate of EB sphere immunologically compatible cells Independent Mean .sup.51Cr sample release rate Deviation t-test Group (%) (±) (*p < 0.01) N 60.23 1.82 (control) Aa1 44.80 1.10 * Aa2 43.15 0.41 * Ab1 45.20 1.44 * Ab2 41.89 0.66 * Ac1 42.32 1.24 * Ac2 40.14 1.44 * Ad1 45.75 0.33 * Ad2 44.63 0.30 * Ae1 45.74 0.34 * Ae2 44.79 1.06 * Af1 44.81 0.06 * Af2 43.42 0.38 * Ag1 42.68 0.09 * Ag2 42.74 0.03 * Ah 41.69 0.05 * Ai1 38.71 0.27 * Ai2 40.47 0.86 * Ai3 37.49 0.82 * Ai4 38.85 0.78 * Ai5 39.73 1.04 * Ai6 37.43 0.02 * B1 35.00 1.30 * B2 34.47 0.33 * B3 35.22 0.23 * B4 35.29 0.04 * B5 35.49 0.22 * C1 30.09 0.19 * C2 33.58 0.38 * C3 32.22 0.06 * C4 30.54 0.37 * C5 30.22 0.05 * C6 31.12 0.11 * C7 31.18 1.68 * D1 29.10 0.94 * D2 32.28 1.21 * D3 31.18 0.12 *

[0314] It can be seen in Table 9 and FIG. 8 that the immunological compatibility effect of the hPSC-derived EB sphere cells that we prepared was significant, and especially, Groups C1, C4, C5 and D1 were the most significant.

Example 3

[0315] In this example, where a transplant became diseased, the effect of non-expression of the knocked-in immunologically compatible molecule after adding an inducer (Dox) was tested. The steps were as follows:

[0316] (1) 6 μM of Dox was added to a medium of the EB sphere immunologically compatible cells prepared in Example 2 for 48 h of treatment;

[0317] (2) the Dox-treated EB sphere immunologically compatible cells were digested into individual cells as target cells;

[0318] (3).sup.51Cr-labeled target cells and T cells were added to a 96-well culture plate at a ratio of 1:5 for post-reaction detection; and

[0319] (4) the cell-specific .sup.51Cr release rate of the EB sphere immunologically compatible cells was detected according to the .sup.51Cr release assay, and the results are shown in Table 10 and FIG. 9.

TABLE-US-00014 TABLE 10 .sup.51Cr release rate of Dox-treated EB sphere immunologically compatible cells Mean .sup.51Cr Deviation Group release rate (%) (±) N 58.50 1.22 (control) Aa1 59.10 1.29 Aa2 59.56 0.16 Ab1 58.56 0.42 Ab2 58.89 0.57 Ac1 57.88 2.28 Ac2 59.41 0.84 Ad1 59.92 0.39 Ad2 58.83 0.66 Ae1 58.62 0.78 Ae2 58.70 0.45 Af1 59.52 0.65 Af2 59.63 1.24 Ag1 59.13 0.98 Ag2 59.59 0.37 Ah 58.67 0.71 Ai1 58.77 1.03 Ai2 58.73 0.92 Ai3 58.86 0.60 Ai4 59.11 0.52 Ai5 58.24 0.49 Ai6 58.51 0.53 B1 58.94 0.85 B2 59.26 1.31 B3 58.65 0.83 B4 58.44 1.07 B5 58.63 0.54 C1 59.35 1.04 C2 59.36 1.18 C3 58.99 1.07 C4 58.93 0.23 C5 58.79 1.17 C6 58.53 0.82 C7 58.58 1.94 D1 58.41 0.69 D2 59.51 1.46 D3 58.48 0.99

[0320] It can be seen in Table 10 and FIG. 9 that after the hPSC-derived EB sphere immunologically compatible cells that we prepared were treated with the inducer (Dox), the immunologically compatible molecule sequence knocked therein exhibited non-expression effects. Thus, reversible regulation of immunological compatibility in cells was achieved.

Example 4

[0321] The Tet-Off system and a combination of immunologically compatible molecules (Tables 3-6) were knocked into the genomic safe locus AAVS1 of hPSC-derived EBs by means of the method of Example 1 to obtain EB sphere immunologically compatible cells.

[0322] .sup.51Cr release assay was used to detect the effect of immunological compatibility between the EB sphere immunologically compatible cells and NK cells:

[0323] 1. The EB sphere immunologically compatible cells were digested into individual cells as target cells;

[0324] 2. .sup.51Cr-labeled target cells and T cells were added to a 96-well culture plate at a ratio of 1:5 for post-reaction detection; and

[0325] 3. the cell-specific .sup.51Cr release rate of the EB sphere immunologically compatible cells was detected according to the .sup.51Cr release assay, and the results are shown in Table 11 and FIG. 10.

TABLE-US-00015 TABLE 11 .sup.51Cr release rate of EB sphere immunologically compatible cells Independent Mean .sup.51Cr sample release rate Deviation t-test Group (%) (±) (*p < 0.01) N 58.26 0.22 (control) Aa1 43.76 0.45 * Aa2 42.56 0.60 * Ab1 41.97 0.56 * Ab2 43.01 1.09 * Ac1 41.48 0.21 * Ac2 40.97 0.53 * Ad1 44.66 0.57 * Ad2 43.50 0.26 * Ae1 44.34 1.00 * Ae2 43.12 0.57 * Af1 43.91 0.51 * Af2 42.88 0.11 * Ag1 42.59 0.57 * Ag2 42.26 1.00 * Ah 41.45 0.18 * Ai1 39.69 0.32 * Ai2 39.92 1.07 * Ai3 38.46 0.58 * Ai4 40.28 0.70 * Ai5 39.67 0.17 * Ai6 38.47 0.20 * B1 36.35 0.30 * B2 36.94 0.49 * B3 37.54 0.18 * B4 37.18 1.27 * B5 37.64 0.17 * C1 34.35 0.48 * C2 35.98 0.32 * C3 35.62 0.23 * C4 34.69 0.97 * C5 34.27 0.31 * C6 34.66 0.02 * C7 34.65 0.03 * D1 33.63 0.55 * D2 35.49 0.23 * D3 35.05 0.61 *

[0326] It can be seen in Table 11 and FIG. 10 that the immunological compatibility effect of the EB sphere cells that we prepared was significant, and especially, Groups C1, C4, C5 and D1 were the most significant.

Example 5

[0327] In this example, when a transplant became diseased, the effect of non-expression of the knocked-in immunologically compatible molecule was tested after adding an inducer (Dox). The steps were as follows:

[0328] (1) 6 μM of Dox was added to a medium of the EB sphere immunologically compatible cells prepared in Example 4 for 48 h of treatment;

[0329] (2) the Dox-treated EB sphere immunologically compatible cells were digested into individual cells as target cells;

[0330] (3) .sup.51Cr-labeled target cells and NK cells were added to a 96-well culture plate at a ratio of 1:5 for post-reaction detection; and

[0331] (4) the cell-specific .sup.51Cr release rate of the EB sphere immunologically compatible cells was detected according to the .sup.51Cr release assay, and the results are shown in Table 12 and FIG. 11.

TABLE-US-00016 TABLE 12 .sup.51Cr release rate of Dox-treated EB sphere immunologically compatible cells Mean .sup.51Cr release rate Deviation Group (%) (±) N (control) 57.44 1.03 Aa1 56.90 1.29 Aa2 56.44 0.16 Ab1 57.44 0.42 Ab2 57.11 0.57 Ac1 58.12 2.28 Ac2 56.59 0.84 Ad1 56.08 0.39 Ad2 57.17 0.66 Ae1 57.38 0.78 Ae2 57.30 0.45 Af1 56.48 0.65 Af2 56.37 1.24 Ag1 56.87 0.98 Ag2 56.41 0.37 Ah 57.33 0.71 Ai1 57.23 1.03 Ai2 57.27 0.92 Ai3 57.14 0.60 Ai4 56.89 0.52 Ai5 57.76 0.49 Ai6 57.49 0.53 B1 57.06 0.85 B2 56.74 1.31 B3 57.35 0.83 B4 57.56 1.07 B5 57.37 0.54 C1 56.65 1.04 C2 56.64 1.18 C3 57.01 1.07 C4 57.07 0.23 C5 57.21 1.17 C6 57.47 0.82 C7 57.42 1.94 D1 57.59 0.69 D2 56.49 1.46 D3 57.52 0.99

[0332] It can be seen in Table 12 and FIG. 11 that when the EB sphere immunologically compatible cells that we prepared were treated with the inducer (Dox), the immunologically compatible molecule sequence knocked therein exhibited non-expression effects. Thus, reversible regulation of immunological compatibility in cells was achieved.

Example 6

[0333] In this example, the CFSE test assay was used to detect the immunological compatibility effect of EB sphere immunologically compatible cells:

[0334] 1. The EB sphere immunologically compatible cells prepared in Example 2 were digested into individual cells as target cells;

[0335] 2. CFSE-labeled target cells and T cells were added to a 5 ml flow tube at a ratio of 1:5 for reaction and then detection; and

[0336] 3. the percentage of CFSE.sup.+PI.sup.+ cells (dead target cells) in the EB sphere immunologically compatible cells was detected according to the CFSE test assay, and the results are shown in Table 13 and FIG. 12.

TABLE-US-00017 TABLE 13 CFSE test results of EB sphere immunologically compatible cells Independent Mean death sample rate of target Deviation t-test Group cell (%) (±) (*p < 0.01) N 60.81 1.17 (control) Aa1 44.91 1.08 * Aa2 43.17 0.40 * Ab1 45.20 1.44 * Ab2 42.01 0.77 * Ac1 42.42 1.34 * Ac2 40.19 1.50 * Ad1 45.74 0.28 * Ad2 44.65 0.33 * Ae1 45.84 0.45 * Ae2 44.84 1.16 * Af1 44.87 0.15 * Af2 43.36 0.36 * Ag1 42.72 0.16 * Ag2 42.78 0.06 * Ah 41.73 0.14 * Ai1 38.88 0.16 * Ai2 40.64 0.87 * Ai3 37.54 0.97 * Ai4 39.07 0.96 * Ai5 39.71 1.06 * Ai6 37.40 0.03 * B1 35.07 1.48 * B2 34.53 0.40 * B3 35.33 0.22 * B4 35.31 0.07 * B5 35.48 0.23 * C1 30.18 0.11 * C2 33.70 0.46 * C3 32.31 0.15 * C4 30.63 0.41 * C5 30.27 0.08 * C6 31.13 0.24 * C7 31.23 1.58 * D1 29.16 0.85 * D2 32.35 1.11 * D3 31.33 0.24 *

[0337] It can be seen in Table 13 and FIG. 12 that the immunological compatibility effect of the EB sphere cells that we prepared was significant, and especially, Groups C1, C4, C5 and D1 were the most significant.

Example 7

[0338] In this example, when a transplant became diseased, the effect of non-expression of the knocked-in immunologically compatible molecule after using an inducer (Dox) was tested in the CFSE test assay. The steps were as follows:

[0339] (1) 6 μM of Dox was added to a medium of the EB sphere immunologically compatible cells prepared in Example 2 for 48 h of treatment;

[0340] (2) the Dox-treated EB sphere immunologically compatible cells were digested into individual cells as target cells;

[0341] (3) CFSE-labeled target cells and T cells were added to a 5 ml flow tube at a ratio of 1:5 for reaction and then detection; and

[0342] (4) the percentage of CFSE.sup.+PI.sup.+ cells (dead target cells) in the hPSC-derived EB sphere cells was detected according to the CFSE test assay, and the results are shown in Table 14 and FIG. 13.

TABLE-US-00018 TABLE 14 CFSE test results of Dox-treated EB sphere immunologically compatible cells Mean death rate of target Deviation Group cell (%) (±) N (control) 60.21 1.08 Aa1 59.90 1.35 Aa2 59.56 0.23 Ab1 60.45 0.41 Ab2 60.11 0.54 Ac1 61.23 2.39 Ac2 59.69 0.75 Ad1 59.13 0.46 Ad2 60.16 0.71 Ae1 60.41 0.72 Ae2 60.40 0.51 Af1 59.53 0.66 Af2 59.43 1.15 Ag1 59.81 1.00 Ag2 59.45 0.49 Ah 60.37 0.66 Ai1 60.26 0.94 Ai2 60.44 1.02 Ai3 60.31 0.58 Ai4 59.94 0.43 Ai5 60.98 0.86 Ai6 60.46 0.55 B1 60.03 0.85 B2 59.81 1.46 B3 60.40 0.76 B4 60.67 1.06 B5 60.39 0.63 C1 59.63 1.10 C2 59.73 1.30 C3 60.12 1.16 C4 60.16 0.33 C5 60.30 1.17 C6 60.53 0.83 C7 60.43 1.97 D1 60.64 0.62 D2 59.55 1.46 D3 60.58 0.84

[0343] It can be seen in Table 14 and FIG. 13 that when the hPSC-derived EB sphere immunologically compatible cells that we prepared were treated with the inducer (Dox), the immunologically compatible molecule sequence knocked therein exhibited non-expression effects.

Example 8

[0344] In this example, the expression of CD.sub.107a in NK cells was analyzed using the flow cytometer (FCM) to detect the immunological compatibility effect of the EB sphere immunologically compatible cells prepared in Example 2:

[0345] 1. The EB sphere immunologically compatible cells were digested into individual cells as target cells;

[0346] 2. the target cells and NK cells were added to a 5 ml flow tube at a ratio of 1:1 for reaction and then detection; and

[0347] 3. the cytotoxicity of the NK cells was detected according to the expression of CD.sub.107a in the NK cells, and the results are shown in Table 15 and FIG. 14.

TABLE-US-00019 TABLE 15 Detection results of CD.sub.107a expression in NK cells Mean Independent cytotoxicity sample of NK cell Deviation t-test Group (%) (±) (*p < 0.01) N 55.81 1.17 (control) Aa1 39.22 0.97 * Aa2 36.70 0.40 * Ab1 38.93 1.23 * Ab2 36.39 0.77 * Ac1 35.91 1.16 * Ac2 35.88 1.50 * Ad1 38.87 0.28 * Ad2 37.92 0.33 * Ae1 38.97 0.45 * Ae2 38.45 1.13 * Af1 38.30 0.15 * Af2 36.93 0.36 * Ag1 36.07 0.16 * Ag2 36.31 0.06 * Ah 35.22 0.12 * Ai1 32.65 0.16 * Ai2 32.62 0.86 * Ai3 29.66 0.96 * Ai4 31.97 0.96 * Ai5 33.50 1.06 * Ai6 31.45 0.17 * B1 29.53 1.48 * B2 28.90 0.40 * B3 29.62 0.22 * B4 29.66 0.07 * B5 29.94 0.22 * C1 24.80 0.11 * C2 28.26 0.29 * C3 27.25 0.52 * C4 25.01 0.41 * C5 25.29 0.08 * C6 26.33 0.24 * C7 25.14 1.58 * D1 24.21 0.85 * D2 27.06 1.11 * D3 26.19 0.24 *

[0348] It can be seen in Table 15 and FIG. 14 that the immunological compatibility effect of the hPSC-derived EB sphere cells that we prepared was significant, and especially, Groups C1, C4, C5 and D1 were the most significant.

Example 9

[0349] In this example, when a transplant became diseased, the effect of non-expression of the knocked-in immunologically compatible molecule after using an inducer (Dox) was tested by the analysis of CD.sub.107a expression in NK cells using the flow cytometer (FCM). The steps were as follows:

[0350] (1) 6 μM of Dox was added to a medium of the EB sphere immunologically compatible cells prepared in Example 2 for 48 h of treatment;

[0351] (2) the Dox-treated EB sphere immunologically compatible cells were digested into individual cells as target cells;

[0352] (3) the target cells and NK cells were added to a 5 ml flow tube at a ratio of 1:1 for reaction and then detection; and

[0353] (4) the cytotoxicity of the NK cells was detected according to the expression of CD.sub.107a in the NK cells, and the results are shown in Table 16 and FIG. 15.

TABLE-US-00020 TABLE 16 Detection results of CD.sub.107a expression in Dox-treated NK cells Mean cytotoxicity of Deviation Group NK cell (%) (±) N (control) 55.43 1.08 Aa1 54.68 1.46 Aa2 54.35 0.47 Ab1 55.05 1.06 Ab2 55.32 0.21 Ac1 56.70 2.02 Ac2 54.68 0.75 Ad1 54.99 1.94 Ad2 55.45 0.24 Ae1 55.54 0.61 Ae2 55.76 0.13 Af1 54.61 0.72 Af2 54.28 1.02 Ag1 54.32 0.22 Ag2 54.62 0.62 Ah 55.18 0.85 Ai1 54.69 0.21 Ai2 55.55 0.85 Ai3 55.10 0.70 Ai4 54.59 0.23 Ai5 55.91 0.97 Ai6 55.07 0.37 B1 55.20 0.64 B2 55.47 0.40 B3 55.29 0.57 B4 54.94 0.57 B5 55.53 0.40 C1 55.40 1.25 C2 54.89 1.17 C3 55.59 0.47 C4 55.26 0.35 C5 55.01 1.01 C6 55.99 0.28 C7 54.43 1.21 D1 54.96 1.63 D2 55.55 0.71 D3 55.87 1.11

[0354] It can be seen in Table 16 and FIG. 15 that when the hPSC-derived EB sphere immunologically compatible cells that we prepared were treated with the inducer (Dox), the immunologically compatible molecule sequence knocked therein exhibited non-expression effects.

Example 10

[0355] Through Examples 2-9, it could be basically determined that the immunological compatibility effects of Groups C1, C4, C5 and D1 were the best. Comprehensively considering factors such as the difficulty of constructing immunologically compatible cells, the scheme of Group D1 was selected as an example hereinafter to test the effect of immunological compatibility between the allogeneic hPSCs and hPSC-derived derivatives (hPSCs-MSCs, NSCs and EBs) (treated by the technical solution of the present disclosure) and T cells.

[0356] By means of the technical solution of Example 1, the immunologically compatible molecule of Group D1 was knocked into the genomic safe locus AAVS1 of hPSCs, hPSCs-MSCs, NSCs, and EB cells, and the cells were digested into individual cells as target cells.

[0357] .sup.51Cr release assay was used to detect the immunological compatibility effect of the target cells:

[0358] 1. .sup.51Cr-labeled target cells and T cells were added to a 96-well culture plate at a ratio of 1:5 for post-reaction detection; and

[0359] 2. the cell-specific .sup.51Cr release rate of the target cells was detected according to the .sup.51Cr release assay, and the results are shown in Table 17 and FIG. 16.

TABLE-US-00021 TABLE 17 .sup.51Cr release rates of hPSC-MSC, NSC and EB immunologically compatible cells Mean .sup.51Cr Group release rate (%) Deviation (±) hPSCs 1 52.49 1.55 2 29.60 0.57 3 52.98 0.69 hPSCs-MSCs 1 50.04 0.73 2 27.86 0.69 3 48.89 0.58 NSCs 1 56.74 0.40 2 30.60 1.01 3 56.69 0.90 EBs 1 59.37 0.94 2 29.87 0.62 3 57.84 0.55 Note: Group 1 was a control group (immunologically non-compatible); Group 2 was a constructed immunologically compatible cell group; and Group 3 was an immunologically compatible cell group treated with an inducer (Dox).

[0360] It can be seen in Table 17 and FIG. 16 that the immunological compatibility effects of the allogeneic hPSCs and hPSC-derived derivatives (hPSCs-MSCs, NSCs, and EBs) that we prepared were significant, and after being treated with the inducer (Dox), they could all show an effect of non-expression of the knocked-in immunologically compatible molecule sequence.

Example 11

[0361] In this example, the effect of immunological compatibility between the target cells prepared in Example 10 and NK cells was tested by the .sup.51Cr release assay.

[0362] 1. .sup.51Cr-labeled target cells and NK cells were added to a 96-well culture plate at a ratio of 1:5 for post-reaction detection; and

[0363] 2. the cell-specific .sup.51Cr release rate of the target cells was detected according to the .sup.51Cr release assay, and the results are shown in Table 18 and FIG. 17.

TABLE-US-00022 TABLE 18 .sup.51Cr release rates of hPSC-MSC, NSC and EB immunologically compatible cells Mean .sup.51Cr Group release rate (%) Deviation (±) hPSCs 1 51.71 0.70 2 33.06 0.93 3 50.94 0.98 MSCs 1 47.67 0.86 2 32.19 0.20 3 47.24 0.36 NSCs 1 54.08 0.53 2 34.08 0.02 3 53.78 1.15 EBs 1 57.13 0.96 2 34.56 0.37 3 57.43 0.41 Note: Group 1 was a control group (immunologically non-compatible); Group 2 was a constructed immunologically compatible cell group; and Group 3 was an immunologically compatible cell group treated with an inducer (Dox).

[0364] It can be seen in Table 18 and FIG. 17 that the immunological compatibility effects of the allogeneic hPSCs and hPSC-derived derivatives (hPSCs-MSCs, NSCs, and EBs) that we prepared were significant, and after being treated with the inducer (Dox), they could all show an effect of non-expression of the knocked-in immunologically compatible molecule sequence.

Example 12

[0365] In this example, by the CFSE test assay, the effect of immunological compatibility between the target cells prepared in Example 10 and T cells was tested.

[0366] (1) CFSE-labeled target cells (hPSCs, MSCs, NSCs, and EBs) and T cells were added to a 5 ml flow tube at a ratio of 1:5 for reaction and then detection; and

[0367] (2) the percentage of CFSE.sup.+PI.sup.+ cells (dead target cells) in each kind of cell was detected according to the CFSE test assay, and the results are shown in Table 19 and FIG. 18.

TABLE-US-00023 TABLE 19 CFSE test results of hPSC, MSC, NSC and EB immunologically compatible cells Mean death rate Group of target cell (%) Deviation (±) hPSCs 1 54.73 0.16 2 28.96 0.39 3 53.85 0.88 MSCs 1 50.53 1.33 2 28.44 0.69 3 50.12 0.61 NSCs 1 56.80 0.72 2 30.38 0.23 3 56.61 1.12 EBs 1 60.27 1.26 2 31.38 1.10 3 59.73 1.25 Note: Group 1 was a control group (immunologically non-compatible); Group 2 was a constructed immunologically compatible cell group; and Group 3 was an immunologically compatible cell group treated with an inducer (Dox).

[0368] It can be seen in Table 19 and FIG. 18 that the immunological compatibility effects of the allogeneic hPSCs and hPSC-derived derivatives (hPSCs-MSCs, NSCs, and EBs) that we prepared were significant, and after being treated with the inducer (Dox), they could all show an effect of non-expression of the knocked-in immunologically compatible molecule sequence.

Example 13

[0369] In this example, the expression of CD.sub.107a in NK cells was analyzed using the flow cytometer (FCM) to detect the immunological compatibility effect of the target cells prepared in Example 10:

[0370] 1. the target cells and NK cells were added to a 5 ml flow tube at a ratio of 1:1 for reaction and then detection; and

[0371] 2. the cytotoxicity of the NK cells was detected according to the expression of CD.sub.107a in the NK cells, and the results are shown in Table 20 and FIG. 19.

TABLE-US-00024 TABLE 20 Detection results of CD.sub.107a expression in NK cells Mean death rate Group of NK cell Deviation (±) hPSCs 1 49.07 0.11 2 24.02 0.20 3 49.36 0.27 MSCs 1 44.93 0.73 2 23.90 0.72 3 45.27 0.48 NSCs 1 52.37 0.86 2 25.58 0.13 3 52.05 0.21 EBs 1 55.60 0.77 2 24.33 0.87 3 54.81 1.07 Note: Group 1 was a control group (immunologically non-compatible); Group 2 was a constructed immunologically compatible cell group; and Group 3 was an immunologically compatible cell group treated with an inducer (Dox).

[0372] It can be seen in Table 20 and FIG. 19 that the immunological compatibility effects of the allogeneic hPSCs and hPSC-derived derivatives (hPSCs-MSCs, NSCs, and EBs) that we prepared were significant, and after being treated with the inducer (Dox), they could all show an effect of non-expression of the knocked-in immunologically compatible molecule sequence.

Example 14

[0373] Through Examples 2 to 9, we selected the scheme of Group D1 for subsequent verification experiments. It was confirmed by Examples 10 to 13 that the immunological compatibility effects of the allogeneic hPSCs and hPSC-derived derivatives (hPSCs-MSCs, NSCs, and EBs) that we prepared were significant, and that when the inducer (Dox) was used, the knocked-in sequence all were not expressed. Since an shRNA/shRNA-miR gene manipulation method was used in some of the experimental schemes, the consequences of overuse of shRNA/shRNA-miR had to be considered.

[0374] In this example, the shRNA/miRNA processor complex gene Dhrosha and an anti-interferon effector molecule (PKR-shRNA) were simultaneously inserted into the genomic safe locus of the hPSC, MSC, NSC, and EB immunologically compatible cells prepared in Group D1 of Example 10, wherein Dhrosha was knocked into the position of MCS III in an AAVS1 KI vector, while PKR-shRNA was knocked into the position of the general backbone of shRNA in the AAVS1 KI vector and was seamlessly linked with other shRNAs.

[0375] The target cells (hPSCs, MSCs, NSCs, and EBs) and T cells were added to a 96-well culture plate at a ratio of 1:5 for reaction and then detection; and the cell-specific .sup.51Cr release rates of the hPSC, MSC, NSC and EB immunologically compatible cells were detected according to the .sup.51Cr release assay, and the results are shown in Table 21 and FIG. 20.

TABLE-US-00025 TABLE 21 Cell-specific .sup.51Cr release rates of hPSC, MSC, NSC and EB immunologically compatible cells Group Mean .sup.51Cr release rate Deviation (±) hPSCs A1 52.59 1.44 B1 52.22 0.32 A2 29.60 0.65 B2 29.65 0.27 A3 53.02 0.78 B3 52.96 0.71 MSCs A1 49.65 0.33 B1 50.04 0.73 A2 27.90 0.73 B2 27.86 0.69 A3 49.07 0.67 B3 48.89 0.58 NSCs A1 56.79 0.54 B1 56.74 0.40 A2 30.58 1.03 B2 30.60 1.01 A3 56.76 1.08 B3 56.69 0.90 EBs A1 59.49 0.89 B1 59.37 0.94 A2 29.52 0.57 B2 29.87 0.62 A3 57.84 0.55 B3 56.95 0.66 Note: A represented a group without knock-in of shRNA-miR processor complex genes and anti-interferon effector molecules; and B represented a group with knock-in of shRNA-miR processor complex genes and anti-interferon effector molecules. 1 was a control group (immunologically non-compatible); 2 was an immunologically compatible cell group which was constructed; and 3 was an immunologically compatible cell group treated with an inducer (Dox).

[0376] It can be seen in Table 21 and FIG. 20 that the immunological compatibility effects of the immunologically compatible cells of allogeneic hPSCs and hPSC-derived derivatives (hPSCs-MSCs, NSCs, and EBs) that we prepared were not affected by the knocked-in shRNA-miR processor complex genes and anti-interferon effector molecules.

Example 15

[0377] In this example, the shRNA/miRNA processor complex gene Dhrosha (the expression of which could be inducibly turned off) and an anti-interferon effector molecule (PKR-shRNA) were simultaneously inserted into the genomic safe loci of the hPSC, MSC, NSC, and EB immunologically compatible cells prepared in Group D1 of Example 10, and the effects thereof on the activity of the immunologically compatible cells were detected.

[0378] The MTT method was used to detect cell activity. 3000 cells were separately taken into a 96-well plate and cultured for 72 h before detection. The results are shown in Table 22 and FIG. 21.

TABLE-US-00026 TABLE 22 Cell activity Independent sample t- Mean MTT Deviation test (n = 5, *p < 0.01, Group value (OD490) (±) **p < 0.001) hPSCs 1 1.17 0.05 ** 2 1.38 0.07 MSCs 1 0.78 0.14 * 2 1.12 0.11 NSCs 1 0.77 0.07 * 2 0.91 0.02 EBs 1 0.73 0.04 ** 2 1.00 0.11 Note: 1 represented a group without knock-in of shRNA-miR processor complex genes and anti-interferon effector molecules; and 2 represented a group with knock-in of shRNA-miR processor complex genes and anti-interferon effector molecules.

[0379] It can be seen in Table 22 and FIG. 21 that after the immunologically compatible cells were knocked-in with shRNA/miRNA processor complex genes and related genes and anti-interferon effector molecules, the activity of the immunologically compatible cells was significantly improved. It was indicated that the immunologically compatible cells, which were inserted with shRNA/shRNA-miR immunologically compatible molecules, would take up excess regulatory processor complex genes and anti-interferon effector molecules. Therefore, when preparing immunologically compatible cells, it was preferable to simultaneously knock in shRNA/miRNA processor complex genes and related genes and anti-interferon effector molecules.

Example 16

[0380] The present disclosure further tested the effects of different shRNA/miRNA processor complex genes and anti-interferon effector molecules on the immunological compatibility of immunologically compatible cells.

[0381] The scheme of the immunologically compatible molecules of Group D1 was used for verification test. While knocking in immunologically compatible molecules at the genomic safe locus of EBs (which were representatives of hPSCs derivatives), shRNA/miRNA processor complex genes and/or anti-interferon effector molecules were knocked-in to detect the effects thereof on the activity of the immunologically compatible cells. The experimental groupings are shown in Table 23.

[0382] The shRNA/miRNA processor complex gene was knocked into the position of MCS III in the AAV KI vector, while the anti-interferon effector molecule was knocked into the position of the general backbone of shRNA in the AAVS1 KI vector and was seamlessly linked with other shRNAs.

[0383] The MTT method was used to detect cell activity. 3000 cells were separately taken into a 96-well plate and cultured for 72 h before detection. The detection results are shown in Table 24.

TABLE-US-00027 TABLE 23 Experimental grouping Processor complex genes and anti- interferon effector molecules 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Dhrosha + + + Ago1 + + + Ago2 + + + Dicer1 + + + Exportin-5 + + + TRBP + + + (TARBP2) PACT + + + (PRKRA) DGCR8 + + + anti-PKR + + + shRNA anti-2-5As + + + shRNA anti-IRF-3 + + + shRNA-miR anti-IRF-7 + + + shRNA-miR Note: “+” represented knocked-in shRNA/miRNA processor complex gene and anti-interferon effector molecule. All of Groups 1-16 were knocked in with immunologically compatible molecules.

TABLE-US-00028 TABLE 24 Experiment results Mean MTT Independent sample t-test Group value (OD490) Deviation (±) (*p < 0.01, **<0.001)  1 0.53 0.04 —  2 0.58 0.06  3 0.69 0.07 **  4 0.57 0.07  5 0.53 0.02  6 0.75 0.07 **  7 0.62 0.11  8 0.64 0.05 *  9 0.52 0.11 10 0.68 0.11 * 11 0.66 0.13 12 0.50 0.07 13 0.55 0.05 14 0.78 0.10 ** 15 0.85 0.09 ** 16 1.36 0.07 **

[0384] The experimental results indicated that the simultaneous knock-in of the shRNA/miRNA processor complex genes and/or anti-interferon effector molecules could enhance the activity of the constructed immunologically compatible cells.

Example 17

[0385] The present disclosure further tested the effects of different inducible expression systems and safe loci on the immunological compatibility of immunologically compatible cells.

[0386] The scheme of the immunologically compatible molecules of Group D1 was used for verification experiments. The safe loci and reversible turn-off expression systems were tested on hPSC-derived EBs, and the immunological compatibility thereof with T cells was tested. The experimental groupings are shown in Table 25.

[0387] The prepared target cells and T cells were added to a 96-well culture plate at a ratio of 1:5 for reaction and then detection; and the effects of different safe loci and different inducible gene expression systems on the cell-specific .sup.51Cr release rate of the EB immunologically compatible cells were detected according to the .sup.51Cr release assay, and the detection results are shown in Table 26.

TABLE-US-00029 TABLE 25 Experimental grouping Inducible gene Was inducible Group Safe locus expression system expression system on? 1 A AAVS1 tet-Off No B AAVS1 tet-Off Yes 2 A AAVS1 Dimer No B AAVS1 Dimer Yes 3 A eGSH tet-Off No B eGSH tet-Off Yes 4 A eGSH Dimer No B eGSH Dimer Yes 5 A H11 tet-Off No B H11 tet-Off Yes 6 A H11 Dimer No B H11 Dimer Yes

TABLE-US-00030 TABLE 26 .sup.51Cr release rate of each experimental group Mean .sup.51Cr Independent sample t- Group release rate Deviation (±) test (*p < 0.01) 1 A 30.09 0.19 * B 58.44 1.07 2 A 30.54 0.37 * B 58.63 0.54 3 A 30.22 0.05 * B 59.35 1.04 4 A 31.12 0.11 * B 59.36 1.18 5 A 31.18 1.68 * B 58.99 1.07 6 A 29.10 0.94 * B 58.93 0.23

[0388] The above experimental data indicated that the different safe loci did not affect the expression of the inserted gene, and the different inducible gene expression systems also did not affect the expression of the inserted gene, i.e., not affecting immunological compatibility; in addition, the inducible gene expression systems could play a reversible turn-off function.

Example 18

[0389] In the present disclosure, effects were tested, wherein the concentration of an exogenous inducer was adjusted so that the transplant gradually expresses a low concentration of HLA molecules to stimulate the body. Therefore, the body gradually developed tolerance to the transplant and finally achieved stable tolerance.

[0390] The immunologically compatible EBs of Group D1 in Example 2 were used for a verification test, and the test was carried out in EBs (which were representatives of hPSCs derivatives). The immunologically compatible EBs were divided into 2 groups. The immunologically compatible EBs of Group 2 were co-cultured with T cells at a ratio of 1:1, and the added amount of Dox was gradually increased according to the following table. After the inducer induced the EBs to develop tolerance, the .sup.51Cr release assay and karyotyping assay were performed.

TABLE-US-00031 TABLE 27 Added concentration of Dox Concentration No. of Dox T cells added? Culture time 1 0.1875 uM Yes One week 2 0.375 uM Yes One week 3 0.75 uM Yes One week 4 1.5 uM Yes One week 5 3 uM Yes One week 6 6 uM Yes One week

[0391] By the .sup.51Cr release assay, the effect of immunological compatibility between immunologically compatible EBs and T cells of the two groups was tested. Flow cytometry sorting was performed first, co-cultured T cells were removed, an inducer (Dox, 6 μM) was then added to both the immunologically compatible EBs of Group 1 (which were not induced with Dox for tolerance), and the immunologically compatible EBs of Group 2 (which were gradually induced with Dox over 6 weeks for tolerance), for 48 h of treatment, then the cells were digested into individual cells as target cells; and after labeling the target cells (EBs) with .sup.51Cr, the target cells and T cells were added to a 96-well culture plate at a ratio of 1:5 for reaction and then detection, and whether the immunologically compatible EBs developed immune tolerance was detected according to the .sup.51Cr release assay.

TABLE-US-00032 TABLE 28 Immune tolerance effect of EBs Independent Mean 51Cr Deviation sample t-test Group release rate (±) (*p < 0.01) 1 Immunologically compatible 58.44 1.07 * EBs without Dox-induction for tolerance 2 Immunologically compatible 30.09 0.19 EBs gradually induced with Dox over 6 weeks for tolerance

TABLE-US-00033 TABLE 29 Karyotyping assay Chromosome Karyotype Group mitosis phase abnormality rate 1 Immunologically compatible EBs 100 5% without Dox-induction for tolerance 2 Immunologically compatible EBs 100 0 gradually induced with Dox over 6 weeks for tolerance

[0392] The experimental results indicated that the transplant could develop tolerance by gradually adjusting the amount of the inducer (Dox) used. In addition, after tolerance is developed, it was beneficial for the own immune system to eliminate mutants from the transplant, making the transplant safer.

[0393] The above embodiments are only preferences of the present disclosure, and those skilled in the art can understand that without departing from the principle and gist of the present disclosure, modifications and replacements with equivalent effects on these embodiments all fall within the scope of protection of the claims of the present disclosure.