CN114457033A - Pluripotent stem cell derivative for expressing IL-6 blocker and application thereof - Google Patents
Pluripotent stem cell derivative for expressing IL-6 blocker and application thereof Download PDFInfo
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Abstract
The invention discloses a pluripotent stem cell expressing an IL-6 blocking substance or a derivative thereof and application thereof, wherein the pluripotent stem cell or the derivative thereof comprises at least one of a non-immune compatible pluripotent stem cell expressing the IL-6 blocking substance or a derivative thereof, an immune compatible pluripotent stem cell expressing the IL-6 blocking substance or a derivative thereof, and an immune compatible reversible pluripotent stem cell expressing the IL-6 blocking substance or a derivative thereof. The pluripotent stem cells or derivatives thereof expressing the IL-6 blocker provided by the invention can be used for inducing iPSCs (induced pluripotent stem cells) or differentiating into MSCs (mesenchymal stem cells) which are low-immunogenicity cells for application, can continuously express the IL-6 blocker in vivo, and is used for treating related diseases with high IL-6 expression or treating rheumatoid arthritis, depression and anxiety.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a pluripotent stem cell derivative for expressing an IL-6 blocking substance and application thereof.
Background
Stem cells are "seed" cells with self-renewal ability and differentiation ability into specific functional somatic cells, have the potential to regenerate into various tissues, organs and human bodies, and play a central and irreplaceable role in immune response, aging, tumorigenesis and other important biological activities. Stem cells are mainly classified into: totipotent stem cells (Totipotent stem cells), Pluripotent Stem Cells (PSCs), and adult stem cells (adult stem cells). The typical PSCs mainly include Embryonic Stem Cells (ESCs), Embryonic Germ Cells (EGCs), Embryonic Carcinoma Cells (ECCs), Induced Pluripotent Stem Cells (iPSCs), and the like, and such cells have a very deep and wide application prospect due to their powerful functions and can be restricted to some extent by ethics.
IL-6 is a cytokine produced by various tissue cells with complex physiological functions, and IL-6 has wide biological effects. It can induce B cells to differentiate and produce immunoglobulins; promoting proliferation and growth of T cells; promoting proliferation of bone marrow hematopoietic stem cells; enhancing blood cell differentiation; anti-tumor effects, etc. IL-6 has been widely associated with a number of clinical conditions, with elevated IL-6 levels in patients with rheumatoid arthritis, depression and anxiety.
Therefore, it is of great interest to develop a pluripotent stem cell or a derivative thereof that can express an IL-6 blocker in humans.
However, the conception or establishment of the autologous iPSCs cell bank or the immune matched PSCs cell bank requires great expenditure of money, material resources and manpower. The molecular immunological basis for allogeneic recipient organ, tissue or cell transplantation is based primarily on the matching of the classical major histocompatibility complexes MHC-I and MHC-II (human HLA-I, HLA-II). By 6 months 2019, over 20000 HLA system alleles have been identified and named, and only 5000 allele factors of classical HLA-A, B, C are respectively exceeded, and various possible random combinations of these classical HLA-I/II alleles will be astronomical numbers, and as the number of combinations found for new alleles increases, there is a great obstacle to tissue matching and donor selection before organ, tissue and cell transplantation, and also great difficulty in constructing a PSCs cell library covering the immune match of the human population.
Therefore, the construction of allogeneic immune-compatible universal PSCs is imminent. In recent years, a plurality of reports have been provided that the deletion expression of genes on the cell surfaces of HLA-I and HLA-II or the genes thereof is realized by knocking out genes such as B2M, CIITA and the like, so that the cells have immune tolerance or escape T/B cell specific immune response, and universal PSCs with immune compatibility are generated, thereby laying an important foundation for the application of wider universal PSCs source cells, tissues and organs. Also, cells have been reported to overexpress CTLA4-Ig, PD-L1 and thereby inhibit allogeneic immune rejection. Recently, it has been reported that when B2M and CIITA are knocked out, CD47 is knocked in, so that cells obtain escape specific immune response, and have immune tolerance or escape natural immune response of cells such as NK cells, so that the cells have more comprehensive and stronger immune compatibility characteristics. However, these approaches are either not fully immune compatible, and still allow for immunological rejection of the allografts by other routes; or completely eliminate the allogeneic immune rejection response, but simultaneously make the cells of the donor-derived transplant lose the antigen presenting capability, which brings great risk of diseases such as tumorigenicity and virus infection to the recipient.
Therefore, it is also reported that, when the B2M is not directly knocked out, the HLA-A, HLA-B is knocked out or the CIITA is knocked out together, the HLA-C is kept, 12 HLA-C immune matching antigens covering more than 90% of people are constructed, so that the transplanted cells still have a certain degree of antigen presenting function, and the inherent immune response of NK cells can be inhibited through the HLA-C. However, in the cells, the antigen type presented by HLA-I antigen is reduced by more than two thirds, the integrity of the presented antigen is reduced irreversibly, the presenting of various tumor, virus and other disease antigens has great bias, the risk of diseases such as tumor and virus infection is still kept to a certain extent, and the pathogenic risk is higher under the condition that CIITA is knocked out simultaneously; secondly, 12 high-frequency immune match HLA-C antigen species are very different, and the part of the area can only account for 70 percent by verification and calculation, while the HLA data of large sample size which is not authoritative currently in China, Indian and other big countries is displayed, so that the prepared general PSCs are still subjected to huge match vacancy tests; thirdly, the method can go through repeated gene editing for a plurality of times, at least two rounds of single cell isolation culture meters are needed according to each gene editing, the whole process needs at least more than six rounds of single cell isolation culture, and the processes are inevitable and cause various unpredictable mutations of cells due to multiple times of gene editing off-target or unstable chromatin or due to passage proliferation of a large number of single cells, thereby further inducing various problems of carcinogenesis, metabolic diseases and the like. It follows that such immuno-compatible schemes are also a matter of convenience in the "transition period", and many problems remain that are not better solved.
In addition, inducing killing of the suicide gene after donor tissue and cell disease has been induced, which results in serious tissue necrosis, cytokine storm and other unpredictable disease risk problems, and it is a big problem that proper donor cells, tissues and organs do not exist after the cell death of the design.
Disclosure of Invention
In a first aspect, the present invention provides a pluripotent stem cell or a derivative thereof.
In a second aspect, the present invention provides a use of the pluripotent stem cell or the derivative thereof in the preparation of a medicament for treating diseases.
In a third aspect, the invention provides a preparation comprising the pluripotent stem cells or derivatives thereof.
The technical scheme adopted by the invention is as follows:
in a first aspect of the invention, there is provided a pluripotent stem cell or a derivative thereof comprising an expression sequence of a-6 blocker, wherein the IL-6 blocker is an IL-6 antibody and/or an IL-6 receptor antibody;
preferably, the expression sequence of the IL-6 blocker is inserted in the genome of the pluripotent stem cell or the derivative thereof.
More preferably, the expression sequence of the IL-6 blocker is inserted into a safe site of the genome of the pluripotent stem cell or the derivative thereof.
Further preferably, the genomic safe site comprises one or more of AAVS1 safe site, eGSH safe site, H11 safe site.
In a second aspect of the invention, there is provided a pluripotent stem cell or a derivative thereof comprising an expression sequence of an IL-6 blocker, wherein the IL-6 blocker is an IL-6 antibody and/or an IL-6 receptor antibody; the B2M gene and/or CIITA gene of the genome of the pluripotent stem cell or the derivative thereof is knocked out.
Preferably, the expression sequence of the IL-6 blocker is inserted in the genome of the pluripotent stem cell or the derivative thereof.
More preferably, the expression sequence of the IL-6 blocker is inserted into a safe site of the genome of the pluripotent stem cell or the derivative thereof.
Further preferably, the genomic safe site comprises one or more of AAVS1 safe site, eGSH safe site, H11 safe site.
In a third aspect of the invention, there is provided a pluripotent stem cell or a derivative thereof, comprising an expression sequence of an IL-6 blocker, wherein the IL-6 blocker is an IL-6 antibody and/or an IL-6 receptor antibody;
the pluripotent stem cells or the derivatives thereof also comprise an immune compatible molecule expression sequence, and the immune compatible molecule is used for regulating and controlling the expression of genes related to immune response in the pluripotent stem cells or the derivatives thereof.
Preferably, the expression sequence of the IL-6 blocker, the expression sequence of the immune compatible molecule are inserted in the genome of the pluripotent stem cell or the derivative thereof.
More preferably, the expression sequence of the IL-6 blocker, the expression sequence of the immune compatible molecule are inserted in a safe site of the genome of the pluripotent stem cell or the derivative thereof.
Further preferably, the genomic safe site comprises one or more of AAVS1 safe site, eGSH safe site, H11 safe site.
In a fourth aspect of the invention, there is provided a pluripotent stem cell or a derivative thereof, an expression sequence of an IL-6 blocker, wherein the IL-6 blocker is an IL-6 antibody and/or an IL-6 receptor antibody;
the pluripotent stem cells or the derivatives thereof also comprise an immune compatible molecule expression sequence, and the immune compatible molecule is used for regulating and controlling the expression of genes related to immune response in the pluripotent stem cells or the derivatives thereof;
the pluripotent stem cell or the derivative thereof further comprises an inducible gene expression system.
Preferably, the inducible gene expression system is at least one of a Tet-Off system, a dimer inducible expression system.
Preferably, the expression sequence of the IL-6 blocker, the expression sequence of the immune-compatible molecule and the inducible gene expression system are inserted in the genome of the pluripotent stem cell or the derivative thereof.
More preferably, the expression sequence of the IL-6 blocker, the expression sequence of the immune compatible molecule and the inducible gene expression system are inserted at a safe site in the genome of the pluripotent stem cell or the derivative thereof.
Further preferably, the genomic safe site comprises one or more of AAVS1 safe site, eGSH safe site, H11 safe site.
The pluripotent stem cell or derivative thereof according to the third or fourth aspect of the invention, further wherein the immune-compatible molecule comprises one or more of:
(I) immune tolerance-related genes including CD47 or HLA-G;
(II) HLA-C molecules comprising HLA-C alleles in a proportion of more than 90% in total in the population, or fusion protein genes consisting of more than 90% of HLA-C alleles and B2M;
(III) shRNA and/or shRNA-miR targeting the gene associated with the immune response.
The pluripotent stem cell or the derivative thereof according to the third or fourth aspect of the present invention, further wherein the gene associated with an immune response comprises:
major histocompatibility complex genes including at least one 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-DPB 1;
(II) major histocompatibility complex related genes comprising at least one of B2M and CIITA.
The target sequence of the shRNA and/or shRNA-miR targeting B2M is selected from one of SEQ ID NO. 6-SEQ ID NO. 8;
the target sequence of the shRNA and/or shRNA-miR of the targeting CIITA is selected from one of SEQ ID NO. 9-SEQ ID NO. 18;
the target sequence of the shRNA and/or shRNA-miR of the target HLA-A is selected from one of SEQ ID NO. 19-SEQ ID NO. 21;
the target sequence of the shRNA and/or shRNA-miR targeting HLA-B is selected from one of SEQ ID NO. 22-SEQ ID NO. 27;
the target sequence of the target HLA-C shRNA and/or shRNA-miR is selected from one of SEQ ID NO. 28-SEQ ID NO. 33;
the target sequence of the shRNA and/or shRNA-miR of the target HLA-DRA is selected from one of SEQ ID NO. 34-SEQ ID NO. 43;
the target sequence of the shRNA and/or shRNA-miR of the target HLA-DRB1 is selected from one of SEQ ID NO. 44-SEQ ID NO. 48;
the target sequence of the shRNA and/or shRNA-miR of the target HLA-DRB3 is selected from one of SEQ ID NO. 49-SEQ ID NO. 50;
the target sequence of the shRNA and/or shRNA-miR of the target HLA-DRB4 is selected from one of SEQ ID NO. 51-SEQ ID NO. 60;
the target sequence of the shRNA and/or shRNA-miR of the target HLA-DRB5 is selected from one of SEQ ID NO. 61-SEQ ID NO. 69;
the target sequence of the shRNA and/or shRNA-miR of the target HLA-DQA1 is selected from one of SEQ ID NO. 70-SEQ ID NO. 76;
the target sequence of the shRNA and/or shRNA-miR of the target HLA-DQB1 is selected from one of SEQ ID NO. 77-SEQ ID NO. 86;
the target sequence of the shRNA and/or shRNA-miR of the target HLA-DPA1 is selected from one of SEQ ID NO. 87-SEQ ID NO. 96;
the target sequence of the shRNA and/or shRNA-miR targeting HLA-DPB1 is selected from one of SEQ ID NO. 97-SEQ ID NO. 106.
The pluripotent stem cell or the derivative thereof according to the third or fourth aspect of the present invention, wherein at least one of an shRNA processing complex-related gene, an miRNA processing complex-related gene, and an anti-interferon effector molecule is further introduced into the genome of the pluripotent stem cell or the derivative thereof.
Preferably, the shRNA processing complex related gene and the miRNA processing complex related gene comprise at least one of Drosha, Ago1, Ago2, Dicer1, Exportin-5, TRBP (TARBP2), PACT (PRKRA) and DGCR 8;
the anti-interferon effector molecule is preferably shRNA and/or shRNA-miR targeting at least one of PKR, 2-5As, IRF-3 and IRF-7.
The target sequence of the shRNA and/or shRNA-miR of the target PKR is selected from one of SEQ ID NO. 107-SEQ ID NO. 116;
the target sequence of the shRNA and/or shRNA-miR targeting 2-5As is selected from one of SEQ ID NO. 117-SEQ ID NO. 146;
the target sequence of the shRNA and/or shRNA-miR of the targeted IRF-3 is selected from one of SEQ ID NO. 147-SEQ ID NO. 156;
the target sequence of the shRNA and/or shRNA-miR targeting the IRF-7 is selected from one of SEQ ID NO. 157-SEQ ID NO. 166.
In the pluripotent stem cell or the derivative thereof according to the third or fourth aspect of the invention, further, the shRNA expression framework: the gene sequence sequentially comprises an shRNA target sequence, a stem-loop sequence, a reverse complementary sequence of the shRNA target sequence and Poly T from 5 'to 3';
wherein the shRNA target sequence, the stem-loop sequence and the reverse complementary sequence of the shRNA target sequence form a hairpin structure;
poly T is a transcription terminator of RNA polymerase III;
shRNA-miR expression framework: replacing a target sequence in the microRNA-30 or microRNA-155 with a shRNA-miR target sequence to obtain the target sequence.
More preferably, the length of the stem-loop sequence in the shRNA expression frame is 3-9 bases; the poly T is 5-6 bases in length.
The pluripotent stem cell or the derivative thereof according to the first to fourth aspects of the present invention, further comprising an embryonic stem cell, an embryonic germ cell, an embryonic cancer cell, or an induced pluripotent stem cell.
The pluripotent stem cell or the derivative thereof according to the first to fourth aspects of the present invention, further comprising an adult stem cell, each germ layer cell or tissue into which the pluripotent stem cell is differentiated;
the adult stem cells include mesenchymal stem cells or neural stem cells.
The pluripotent stem cell or the derivative thereof according to the first to fourth aspects of the present invention, further comprising an IL-6 antibody having a heavy chain sequence as set forth in SEQ ID NO.1 and a light chain sequence as set forth in SEQ ID NO. 2.
The pluripotent stem cell or the derivative thereof according to the first to fourth aspects of the present invention, further comprising an IL-6 receptor antibody having a heavy chain sequence as set forth in SEQ ID NO.3 and a light chain sequence as set forth in SEQ ID NO. 4.
It will be appreciated by those skilled in the art that the objects of the invention may be equally achieved using other IL-6 antibody or IL-6 receptor antibody expression sequences.
In a fifth aspect of the present invention, there is provided a use of the pluripotent stem cell or the derivative thereof according to any one of the first to fourth aspects of the present invention for the manufacture of a medicament for the treatment of a disease, the disease being at least one of rheumatoid arthritis, depression and anxiety.
In a sixth aspect of the invention, there is provided a formulation comprising a pluripotent stem cell or a derivative thereof according to any one of the first to fourth aspects.
The invention has the beneficial effects that:
the invention provides a pluripotent stem cell expressing an IL-6 blocker or a derivative thereof, which can be used for inducing iPSCs (induced pluripotent stem cells) or differentiating the iPSCs into MSCs (mesenchymal stem cells) which are low in immunogenicity to be used by autologous cells, can continuously express the IL-6 blocker in vivo, and is used for treating IL-6 high-expression related diseases or rheumatoid arthritis, depression and anxiety.
The invention also provides an immune compatible pluripotent stem cell or a derivative thereof for expressing the IL-6 blocker, wherein B2M and CIITA genes in the pluripotent stem cell or the derivative thereof are knocked out, or an immune compatible molecule expression sequence is introduced into the genome of the pluripotent stem cell or the derivative thereof, so that the immunogenicity of the pluripotent stem cell or the derivative thereof is low, and when the pluripotent stem cell or the derivative thereof is transplanted into a recipient, the problem of allogeneic immune rejection between a donor cell and the recipient can be overcome, so that the donor cell can continuously express the IL-6 blocker in the recipient for a long time.
The invention also provides an inducible gene expression system and an immune compatible molecule expression sequence which are introduced into the genome of the immune compatible reversible pluripotent stem cell or the derivative thereof for expressing the IL-6 blocker. The inducible gene expression system is controlled by an exogenous inducer, and the opening and closing of the inducible gene expression system are controlled by adjusting the addition amount, the continuous action time and the type of the exogenous inducer, so that the expression quantity of the epidemic compatible molecular expression sequence is controlled. While the immune-compatible molecule may regulate the expression of genes associated with an immune response in the pluripotent stem cell or derivative thereof. When the immune compatible molecule is normally expressed, the expression of genes related to the immune response in the pluripotent stem cell or the derivative thereof is suppressed or overexpressed, and the allogeneic immune rejection response between the donor cell and the recipient can be eliminated or reduced, so that the donor cell can continuously express the IL-6 blocker in the recipient for a long time. When the donor cell is diseased, the expression of the immune compatible molecules can be closed by induction of an exogenous inducer, so that the HLA class I molecules can be reversibly re-expressed on the surface of the donor cell, the antigen presenting capability of the donor cell is recovered, and the diseased cell can be eliminated by a receptor, thereby improving the clinical safety of the general pluripotent stem cell or the derivative thereof, and greatly expanding the value of the general pluripotent stem cell in clinical application.
In addition, the addition amount and the lasting action time of the exogenous inducer can be adjusted to ensure that the transplant gradually expresses low-concentration HLA molecules to stimulate the receptor, so that the receptor gradually generates tolerance on the transplant, and finally stable tolerance is achieved. At the moment, even if the HLA class I molecules with unmatched HLA class I molecule expression on the surface of the transplanted cells can be compatible with the recipient immune system, so that after the expression of the immune compatible molecules in the transplanted cells is induced to be closed, the recipient immune system can re-identify the cells with gene mutation presented by the HLA class I molecules in the transplanted cells on one hand, and eliminate diseased cells; on the other hand, the non-mutated part is not cleared by the recipient immune system due to the allogeneic HLA class i molecule tolerance produced by training with the above mentioned inducers. Thus, the recipient immune system can only eliminate the graft with harmful mutation, the graft with normal function is kept, and when the harmful graft is eliminated, the mode of HLA class I molecule silencing on the cell surface of the graft can be transferred. The graft immune tolerance program mediated by exogenous inducer can also be implanted without inducing or inducing in other ways to turn on or off the surface expression of HLA class I molecules after the receptor is completely tolerated
Drawings
FIG. 1, AAVS1 KI Vector (shRNA, constitutive) plasmid map.
FIG. 2, AAVS1 KI Vector (shRNA, inducible) plasmid map.
FIG. 3, AAVS1 KI Vector (shRNA-miR, constitutive) plasmid map.
FIG. 4, AAVS1 KI Vector (shRNA-miR, inducible) plasmid map.
FIG. 5, sgRNA clone B2M-1 plasmid map.
FIG. 6, sgRNA clone B2M-2 plasmid map.
FIG. 7, sgRNA clone CIITA-1 plasmid map.
FIG. 8, sgRNA clone CIITA-2 plasmid map.
Figure 9, Cas9(D10A) plasmid map.
FIG. 10, sgRNA Clone AAVS1-1 plasmid map.
FIG. 11, sgRNA Clone AAVS1-2 plasmid map.
Detailed Description
The present invention will be described in further detail with reference to the following specific embodiments and accompanying drawings. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. The various chemicals used in the examples are commercially available.
1 Experimental materials and methods
1.1 IL-6 blockers
The IL-6 blocking agent may be selected from IL-6 antibody and/or IL-6 receptor antibody.
The Heavy Chain (HC) sequence of the IL-6 antibody is shown as SEQ ID NO.1, and the Light Chain (LC) sequence is shown as SEQ ID NO. 2; the Heavy Chain (HC) sequence of the IL-6 receptor antibody is shown as SEQ ID NO.3, and the Light Chain (LC) sequence is shown as SEQ ID NO. 4.
The front ends of the IL-6 antibody and the IL-6 receptor antibody are provided with signal peptide sequences which are shown in SEQ ID NO. 5.
GAGGTGCAGCTGGTGGAGAGCGGCGGCAAGCTGCTGAAGCCCGGCGGCAGCCTGAAGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTTCGCCATGAGCTGGTTCAGGCAGAGCCCCGAGAAGAGGCTGGAGTGGGTGGCCGAGATCAGCAGCGGCGGCAGCTACACCTACTACCCCGACACCGTGACCGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACACCCTGTACCTGGAGATGAGCAGCCTGAGGAGCGAGGACACCGCCATGTACTACTGCGCCAGGGGCCTGTGGGGCTACTACGCCCTGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCAGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACCGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGAGCTGGAACAGCGGCGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCCGCCCCCGAGCTGCTGGGCGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCCCCCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCAGGGAGCCCCAGGTGTACACCCTGCCCCCCAGCAGGGACGAGCTGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCGGCAAG(SEQ ID NO.1)。
CAGATCGTGCTGATCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCAGCGCCAGCAGCAGCGTGAGCTACATGTACTGGTACCAGCAGAAGCCCGGCAGCAGCCCCAGGCTGCTGATCTACGACACCAGCAACCTGGCCAGCGGCGTGCCCGTGAGGTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGGATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCGGCTACCCCTACACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGAGGACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCAGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGAGCAGCCCCGTGACCAAGAGCTTCAACAGGGGCGAGTGC(SEQ ID NO.2)。
GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCAGGAGCCTGAGGCTGAGCTGCGCCGCCAGCAGGTTCACCTTCGACGACTACGCCATGCACTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGCGGCATCAGCTGGAACAGCGGCAGGATCGGCTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCGAGAACAGCCTGTTCCTGCAGATGAACGGCCTGAGGGCCGAGGACACCGCCCTGTACTACTGCGCCAAGGGCAGGGACAGCTTCGACATCTGGGGCCAGGGCACCATGGTGACCGTGAGCAGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACCGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGAGCTGGAACAGCGGCGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCCGCCCCCGAGCTGCTGGGCGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCCCCCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCAGGGAGCCCCAGGTGACCTACCTGCCCCCCAGCAGGGACGAGCTGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCGGCAAG(SEQ ID NO.3)。
GACATCCAGATGACCCAGAGCCCCAGCAGCGTGAGCGCCAGCGTGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGGGCATCAGCAGCTGGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGGCGCCAGCAGCCTGGAGAGCGGCGTGCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCAGCTACTACTGCCAGCAGGCCAACAGCTTCCCCTACACCTTCGGCCAGGGCACCAAGCTGGAGATCAAGAGGACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCAGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGAGCAGCCCCGTGACCAAGAGCTTCAACAGGGGCGAGTGC(SEQ ID NO.4)。
ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACGAATTCG(SEQ ID NO.5)。
It will be appreciated by those skilled in the art that the objects of the invention may be equally achieved using other IL-6 antibody or IL-6 receptor antibody expression sequences.
1.2 pluripotent Stem cells or derivatives thereof
The pluripotent stem cells can be selected from Embryonic Stem Cells (ESCs), Induced Pluripotent Stem Cells (iPSCs) and other forms of pluripotent stem cells, such as hPSCs-MSCs, NSCs, EBs cells. Wherein:
ESCs: HN4 cells were selected and purchased from Shanghai department of sciences.
And (2) iPSCs: using a third generation highly efficient and safe episomal-iPSCs induction system (6F/BM1-4C) established by us, pE3.1-OG-KS and pE3.1-L-Myc-hmiR 302cluster are transferred into somatic cells through electricity, RM1 is cultured for 2 days, BioCISO-BM1 containing 2uM Parnate is cultured for 2 days, BioCISO-BM1 containing 2uM Parnate, 0.25mM sodium butyrate, 3uM CHIR99021 and 0.5uM PD03254901 is cultured for 2 days, iPSCs clones can be picked up after being cultured to about 17 days by using a dry cell culture medium BioCISO, and the picked iPSCs clones are purified, digested and passaged to obtain stable iPSCs. The specific construction method is as follows: stem Cell Res ther.2017nov 2; 8(1):245.
hPSCs-MSCs: iPSCs were cultured for 25 days using a stem cell medium (BioCISO, containing 10uM TGF β inhibitor SB431542), during which digestion passage was carried out at 80-90 confluence (2mg/mL Dispase digestion), 1:3 passages were carried out on Matrigel-coated plates, followed by culture in ESC-MSC medium (knockkockout DMEM medium, containing 10% KSR, NEAA, diabody, glutamine, β -mercaptoethanol, 10ng/mL bFGF and SB-431542), liquid change every day, passage was carried out at 80-90 confluence (1:3 passages), and continuous culture was carried out for 20 days. The specific construction method is as follows: proc Natl Acad Sci U S A.2015; 112(2):530-535.
NSCs: iPSCs are cultured for 14 days by using an induction medium (a knockout DMEM medium containing 10% KSR, a TGF-beta inhibitor and a BMP4 inhibitor), rose annular nerve cells are picked to a low-adhesion culture plate for culture, the culture medium is cultured by using DMEM/F12 (containing 1% N2 and Invitrogen) and Neurobasal medium (containing 2% B27 and Invitrogen) in a ratio of 1:1 and also contains 20ng/ml bFGF and 20ng/ml EGF, and digestion is carried out by using Accutase for digestion and passage. The specific construction method is as follows: FASEB J.2014; 28(11):4642-4656.
EBs cells: and digesting iPSCs with the confluence of 95% for 6min by using a BioC-PDE1, scraping the cells into blocks by using a mechanical scraping method, settling and reducing cell masses, transferring the settled cell masses into a low-adhesion culture plate, culturing for 7 days by using a BioCISO-EB1, and changing the liquid every other day. After 7 days, the cells were transferred to a Matrigel-coated plate and adherent culture was continued using BioCISO, and Embryoid Bodies (EBs) having an inner, middle and outer mesoderm structure were obtained after 7 days. The specific construction method is as follows: stem Cell Res ther.2017nov 2; 8(1):245.
The pluripotent stem cell derivative also includes adult stem cells, each germ layer cell or tissue, organ into which the pluripotent stem cells are differentiated; the adult stem cells include mesenchymal stem cells or neural stem cells.
1.3 genomic safety sites
As a more preferred approach, to ensure stable expression of the knock-in gene/expression construct, the gene/expression construct may be knocked into a genomic safety site, which may be selected from the AAVS1 safety site, the eGSH safety site, or other safety sites:
(1) AAVS1 safety site
The AAVS1 site (the alias "PPP 1R2C site") is located on chromosome 19 of the human genome and is a verified "safe harbor" site that ensures the desired function of the transferred DNA fragment. The site is an open chromosome structure, can ensure that the transgene can be normally transcribed, and has no known side effect on cells when the exogenous target segment is inserted into the site.
(2) eGSH safe site
The eGSH safe site is located on chromosome 1 of the human genome, and is another 'safe harbor' site which can ensure the expected function of the transferred DNA fragment after the paper verifies.
(3) Other safety sites
The H11 safe site (also called Hipp11) is located on the number 22 chromosome of a human, is a site between two genes Eif4enif1 and Drg1, is discovered and named in 2010 by Simon Hippenmeyer, and has little risk of influencing endogenous gene expression after the insertion of a foreign gene because the H11 site is located between the two genes. The H11 site was verified to be a safe transcription activation region between genes, a new "safe harbor" site outside the AAVS1, eGSH sites.
1.4 inducible Gene expression System
The inducible gene expression system is selected from: the tet-Off system or the dimer switch Off expression system:
(1) tet-Off system
In the absence of tetracycline, the tTA protein continues to act on the tet promoter, resulting in sustained gene expression. This system is very useful in situations where it is desirable to maintain the transgene in a sustained expression state. When tetracycline is added, the tetracycline can change the structure of the tTA protein, so that the tTA protein cannot be combined with a promoter, and the expression level of the gene driven by the tTA protein is reduced. To keep the system in an "off" state, the tetracycline must be added continuously.
Knocking the sequence of the tet-Off system and one or more immune compatible molecules into the genome safe site of the pluripotent stem cell, and precisely turning on or Off the expression of the immune compatible molecules by the addition or non-addition of tetracycline, thereby reversibly regulating the expression of major histocompatibility complex related genes in the pluripotent stem cell or the derivative thereof.
(2) Dimer-switched off expression system
Dimer-mediated gene expression regulation system: there are many ways of chemically regulating transcription of target genes, most commonly regulated using allosteric modulators that influence the activity of transcription factors. One such method is the use of dimerizing inducers or dimers to recombine active transcription factors on inactive fusion proteins. The most commonly used system is rapamycin (rapamydn), a natural product, or an analog that is biologically inactive, as the drug for dimerization. The rapamycin (or analog) sibling protein FKBP12 (the protein to which FKBP binds to FK 506) and a large serine-threonine protein kinase, known as FRAP [ FRBP-rapamycin associated protein, mTOR (mammalian target of rapamycin), have high affinity and function to bind to both proteins, thus bringing them together as a heterologous dimer. To regulate transcription of a target gene, a DNA binding domain is fused to one or more FKBP domains and a transcription repressing domain is fused to amino acid position 93 of FRAP, designated FRB, which is sufficient to bind the FKBP-rapamycin complex. Dimerization of these two fusion proteins can only occur in the presence of rapamycin. Thus inhibiting transcription of genes having sites that bind to the DNA binding region.
1.5 immune compatible molecules
The immune compatible molecule can regulate the expression of allogeneic immune rejection related genes in the pluripotent stem cells or derivatives thereof.
The types and sequences of specific immune-compatible molecules are shown in table 1.
TABLE 1 immune compatible molecules
The target sequences of the shRNA or shRNA-miR immune compatible molecules are shown in Table 2.
TABLE 2 target sequences for shRNA or shRNA-miR
In the scheme of knocking in the immune compatible molecules in the subsequent experiments, the shRNA or shRNA-miR sequences of each experimental group are shRNA or shRNA-miR immune compatible molecules constructed by adopting the target sequence 1 in the table 2. Those skilled in the art will understand that: the shRNA or shRNA-miR immune compatible molecule constructed by other target sequences can also realize the technical effect of the invention and all fall into the protection scope of the claims of the invention.
1.6 shRNA/miRNA processing complex gene and anti-interferon effector molecule
The primary miRNA (pri-miRNA) in the nucleus is microprocessed through the complex Drosha-DGCR8, which cleaves the pri-miRNA into a precursor miRNA (pre-miRNA), which then forms a hairpin. Then, the pre-miRNA is transported out of the nucleus via the Exportin-5-Ran-GTP complex. The RNase Dicer enzyme, which binds to the double-stranded RNA-binding protein TRBP (TARBP2) in the cytoplasm, breaks down the pre-miRNA into mature lengths, at which point the miRNA is still in a double-stranded state. Finally, it is transported into AGO2 to form RISC (RNA-induced silencing complex). Finally, one strand of the miRNA double strand is retained in the RISC complex, and the other strand is eliminated and rapidly degraded. While DGCR8, the main binding protein of Drosha, can bind to pri-miRNA through two double-stranded RNA binding regions at its C-terminal end, recruit and guide Drosha to cut at the right position of pri-miRNA to produce pre-miRNA, which is further cut by Dicer and TRBP/PACT processing to form mature miRNA. Deletion or abnormal expression of DGCR8 affects the cleavage activity of Drosha, which in turn affects the activity of miRNA, leading to disease. TRBP is able to recruit Dicer complex mirnas to form RISC Ago 2.
When the shRNA-miR expression sequences aiming at HLA class I molecules, HLA class II molecules and the like which can be induced to be expressed in a closed mode are knocked in at a genome safe site by using a gene knock-in technology, shRNA and/or miRNA processing machines which can be induced to be expressed in a closed mode preferably comprise Drosha (access number: NM-001100412), Ago1(access number: NM-012199), Ago2(access number: NM-001164623), Dicer1(access number: NM-001195573), export-5 (access number: NM-020750), TRBP (access number: NM-134323), PACT (access number: NM-003690) and DGCR8(access number: NM-022720) so that cells do not occupy the processing of other miRNAs and influence the functions of the cells.
In addition, during IFN induction, double-stranded RNA-dependent Protein Kinase (PKR), which is a key factor of the whole cell signal transduction pathway, and 2 ', 5' Oligoadenylate Synthetase (2,5-Oligoadenylate Synthetase,2-5As), which are closely related to dsRNA-induced IFN, are involved. PKR can inhibit protein synthesis by phosphorylating eukaryotic cell transcription factors, arrest cells in G0/G1 and G2/M phases and induce apoptosis, while dsRNA can promote synthesis of 2-5As, which results in nonspecific activation of RNase, RNaseL, degradation of all mRNA in cells and cell death. The specificity of induction of type I interferons is achieved by members of the IRF transcription factor family, which are not inducible to be secreted in many viral infections in the absence of IRF-3 and IRF-7 expression in cells. Lack of IFN response, in order to recover, requires the two proteins were expressed together.
By utilizing a gene knock-in technology, when an immune compatible molecule shRNA-miR expression sequence is knocked in at a genome safety site, preferably, the shRNA and/or shRNA-miR expression sequence which can induce closed expression and aims at inhibiting PKR, 2-5As, IRF-3 and IRF-7 genes is knocked in at the same time, so that the interferon reaction induced by dsRNA is reduced, and the cytotoxicity is avoided.
The sequence of the insertion positions of the shRNA/miRNA processing complex related gene, the anti-interferon effector molecule and the immune compatible molecule at the genome safety site is not limited, and the shRNA/miRNA processing complex related gene, the anti-interferon effector molecule and the immune compatible molecule can be arranged in any sequence without mutual interference or influence on the structure and the function of other genes of the genome.
Specific target sequences for anti-interferon effector molecules are shown in table 3.
TABLE 3 target sequences for interferon effector molecules
In the subsequent experiment including the scheme of knocking-in of the anti-interferon effector molecules, the target sequences of the anti-interferon effector molecules of each experimental group are the anti-interferon effector molecules constructed by adopting the target sequence 1 in the table 3. Those skilled in the art will understand that:
the anti-interferon effector molecules constructed by other target sequences can also achieve the technical effects of the invention and fall into the protection scope of the claims of the invention.
1.7 Universal framework sequences of the immune compatible molecules, the shRNA or shRNA-miR universal framework immune compatible molecules of the anti-interferon effector molecules, and the shRNA or shRNA-miR universal framework molecules of the anti-interferon effector molecules are as follows:
(1) the constitutive expression framework of shRNA is:
GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGCTAGCGCCACC(SEQ ID NO.167)N1...N21TTCAAGAGA(SEQ ID NO.168)N22...N42TTTTTT；
wherein:
a、N1...N21shRNA target sequence for the corresponding Gene, N22...N42Is a reverse complementary sequence of the shRNA target sequence of the corresponding gene;
b. if the plasmid needs to express shRNAs of a plurality of genes, each gene corresponds to a shRNA expression frame and then is connected seamlessly;
c. constitutive shRNA plasmids with different resistance genes only have different resistance genes and have the same other sequences;
d. n represents A, T, G, C bp;
e. SEQ ID No.167 is the U6 promoter sequence;
f. SEQ ID NO.168 is a stem-loop sequence.
(2) The shRNA inducible expression framework is as follows:
GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGCTCGGTACCCGGGTCGAGGTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTGCTAGCGCCACC(SEQ ID NO.169)N1...N21TTCAAGAGA(SEQ ID NO.168)N22...N42TTTTTT；
wherein:
a、N1...N21shRNA target sequence for the corresponding Gene, N22...N42Is a reverse complementary sequence of the shRNA target sequence of the corresponding gene;
b. if the plasmid needs to express shRNAs of a plurality of genes, each gene corresponds to a shRNA expression frame and then is connected seamlessly;
c. constitutive shRNA plasmids with different resistance genes only have different resistance genes and have the same other sequences;
d. n represents A, T, G, C bp;
e. SEQ ID No.166 is the H1TO promoter sequence;
f. SEQ ID NO.165 is a stem-loop sequence.
(3) The shRNA-miR constitutive or inducible expression framework is as follows:
the shRNA-miR target sequence is used for replacing a target sequence in microRNA-30 to obtain the shRNA-miR target sequence, and the specific sequence is as follows:
GAGGCTTCAGTACTTTACAGAATCGTTGCCTGCACATCTTGGAAACACTTGCTGGGATTACTTCTTCAGGTTAACCCAACAGAAGGCTAAAGAAGGTATATTGCTGTTGACAGTGAGCG(SEQ ID NO.170)M1N1...N21TAGTGAAGCCACAGATGTA(SEQ ID NO.171)N22...N42M2TGCCTACTGCCTCGGACTTCAAGGGGCTACTTTAGGAGCAATTATCTTGTTTACTAAAACTGAATACCTTGCTATCTCTTTGATACATTTTTACAAAGCTGAATTAAAATGGTATAAAT(SEQ ID NO.172)；
wherein:
a、N1...N21shRNA-miR target sequence, N, as a corresponding gene22...N42Is a reverse complementary sequence of shRNA-miR target sequence of a corresponding gene;
b. if the plasmid needs to express shRNA-miR of a plurality of genes, each gene corresponds to a shRNA-miR expression frame and is then connected seamlessly;
c. constitutive shRNA-miR plasmids with different resistance genes only have different resistance genes and have the same other sequences;
d. m is A or C, N is A, T, G, C;
e. if N is present1Is a G base, then M1Is A base; otherwise M1Is a C base;
f、M1base and M2And (3) base complementation.
1.8 Gene editing System, Gene editing method and inspection method
1.8.1 Gene editing System
The gene editing technology adopts a CRISPR-Cas9 gene editing system. The Cas9 protein used by the inventors was Cas9(D10A), Cas9(D10A) bound to sgrnas responsible for specific recognition of the target sequence (genomic DNA), which was then single-stranded cleaved by Cas9 (D10A). Double Strand breaks in genomic DNA (DSB) must occur, and two Cas 9(D10A)/sgRNA must cleave the two strands of genomic DNA separately, and not too far apart. The Cas 9(D10A)/sgRNA scheme has the advantage of higher specificity and lower probability of off-target compared to the Cas 9/sgRNA scheme. The plasmids or Donor fragments used in the gene editing system were: cas9(D10A) plasmid, sgRNA clone plasmid, Donor fragment.
(1) Cas9(D10A) plasmid: a plasmid expressing the Cas9(D10A) protein, specifically single-stranded cleaving genomic DNA under the direction of sgrnas.
(2) sgRNA plasmid: a plasmid for expressing sgRNA, sgRNA (small guide RNA), is a guide RNA (guide RNA, gRNA) responsible for directing targeted cleavage of the expressed Cas9(D10A) protein at gene editing.
(3) Donor fragment: the two ends contain recombination arms which are respectively positioned at the left side and the right side of the breaking position of the genome DNA, and the middle part contains genes, fragments or expression elements needing to be inserted. In the presence of the Donor fragment, the cells undergo a Homologous Recombination (HR) reaction at the site of the genomic break. If the Donor fragment is not added, Non-homologous End Joining-NHEJ reaction occurs at the site of the genomic break in the cell. This fragment was obtained by digesting KI (Knock-in, the same applies hereinafter) Vector plasmid and recovering it.
1.8.2 constitutive and inducible plasmids
Constitutive plasmid: the expression function of the Donor fragment obtained from the constitutive plasmid cannot be regulated after knocking in the genomic DNA.
Inducible plasmids: after knocking in the genomic DNA, the expression function of the Donor fragment obtained from the inducible plasmid can be controlled by adding an inducer, which is equivalent to adding a switch for turning on or off the expression function.
1.8.3 plasmid construction method
(1) Cas9(D10A) plasmid: this Plasmid no longer needs to be constructed and is ordered directly from Addgene (Plasmid 41816, Addgene).
(2) sgRNA plasmid: the original blank Plasmid is ordered from Addge (Plasmid 41824, Addge), then the DNA sequence is input in the website (URL: https:// ccttop. cos. uni-heidelberg. de) to design the target sequence, and finally different target sequences are respectively put into the blank sgRNA Plasmid to complete the construction.
(3) KI (Knock-in, the same applies below) Vector plasmid:
acquisition of Amp (R) -pUC origin fragment: designing PCR primers, and amplifying and recovering the fragment by a PCR method by using a pUC18(Takara, Code No.3218) plasmid as a template and a high fidelity enzyme (Nanjing Novozam organism, P505-d 1);
acquisition of aavs1 or eGSH recombination arms: extracting genome DNA of human cells and designing corresponding primers, and then amplifying and recovering the fragments by using the human genome DNA as a template and using a high fidelity enzyme (Nanjing Novozam organism, P505-d1) through a PCR method;
c. acquisition of the individual plasmid elements: designing PCR amplification primers of each element, and then respectively amplifying and recovering each plasmid element by using a plasmid containing the element as a template and using a high fidelity enzyme (Nanjing NuoZanza, P505-d1) through a PCR method;
d. assembly into a complete plasmid: the fragments obtained in the previous step were ligated together using a multi-fragment recombinase (Nanjing Nozam, C113-02) to form a complete plasmid.
1.8.4 Gene editing Process
One, single cell cloning operation step of AAVS1 gene knock-in
(1) Electric transfer program:
donor cell preparation: human pluripotent stem cells.
The kit comprises: human Stem Cell
Kit 1。
The instrument comprises: an electrotransformation instrument.
Culture medium: BioCISO.
Induction of plasmid: cas9D10A, sgRNA clone AAVS1-1, sgRNA clone AAVS1-2, AAVS1neo Vector I and AAVS1neo Vector II.
Note: induction plasmid used for the knock-in of the eGSH gene: cas9D10A, sgRNA clone eGSH-1, sgRNA clone eGSH-2, eGSH-neo/eGSH-puro (donor) comparison of the donor plasmid with AAVS1 shows that only the right and left recombination arms are different, and the other elements are the same. Since the gene editing process of eGSH is the same as that of AAVS1, the following description will not be repeated.
(2) The transformed human pluripotent stem cells are screened in a double antibiotic medium containing G418 and puro.
(3) And (4) carrying out single cell clone screening and culture to obtain a single cell clone strain.
Second, AAVS1 gene knock-in single cell clone strain culture reagent
(1) Culture medium: BioCISO + 300. mu.g/mL G418+ 0.5. mu.g/mL puro (should be placed at room temperature in advance, protected from light for 30-60 minutes until room temperature is restored. Note that BioCISO should not be placed at 37 ℃ for preheating to avoid reduction of the activity of the biomolecule.).
(2) Matrix glue: hESC grade Matrigel (before passage or cell recovery, the Matrigel working solution is added into a cell culture bottle dish and is shaken up to ensure that the Matrigel completely sinks to the bottom of the culture bottle dish and any Matrigel cannot be dried before use. to ensure that the cells can be attached to the wall and survive better, the Matrigel is put into a 37 ℃ culture box for 1:100X Matrigel cannot be less than 0.5 hour and 1:200X Matrigel cannot be less than 2 hours.).
(3) Digestion solution: EDTA was dissolved using DPBS to a final concentration of 0.5mM, pH7.4 (note: EDTA cannot be diluted with water, otherwise the cells die due to reduced osmotic pressure).
(4) Freezing and storing liquid: 60% BioCISO + 30% ESCs grade FBS + 10% DMSO (the frozen stock is preferably ready for use).
Thirdly, the conventional maintenance subculture process
(1) Optimal time of passage and passage ratio
a. The best passage time: the overall confluency of the cells reaches 80 to 90 percent;
b. the optimal ratio of passage: the optimal confluence degree of the passage is maintained at 20-30% in the next day after passage of 1: 4-1: 7.
(2) Passage process
a. The Matrigel in the coated cell culture flask dishes was previously aspirated away, and an appropriate amount of medium (BioCISO + 300. mu.g/mL G418+ 0.5. mu.g/mL puro) was added to the flask and placed at 37 ℃ in 5% CO2Incubation in an incubator;
b. when the cells meet the requirement of passage, sucking the supernatant of the culture medium, and adding a proper amount of 0.5mM EDTA digestive solution into a cell bottle dish;
c. the cells were incubated at 37 ℃ with 5% CO2Incubating in an incubator for 5-10 minutes (digesting until most cells are observed to shrink and become round under a microscope but not float, gently blowing the cells to separate the cells from the wall, sucking the cell suspension into a centrifugal tube, and centrifuging for 5 minutes at 200 g;
d. centrifuging, discarding the supernatant, suspending the cells by using a culture medium, gently and repeatedly blowing the cells for several times until the cells are uniformly mixed, and then transferring the cells to a bottle dish prepared for coating Matrigel in advance;
e. after the cells were transferred to the cell flask, the cells were horizontally shaken up all around, observed under a mirror to be free from abnormality, and then shaken up and placed at 37 ℃ with 5% CO2Culturing in an incubator;
f. observing the adherent survival state of the cells the next day, and normally and regularly changing the culture medium every day by sucking off the culture medium.
Fourthly, freezing and storing cells
(1) According to the conventional passage operation steps, digesting the cells by using 0.5mM EDTA until most cells shrink and become round but do not float, gently blowing and beating the cells, collecting cell suspension, centrifuging for 5 minutes at 200g, removing supernatant, adding a proper amount of freezing medium to resuspend the cells, and transferring the cells to a freezing tube (suggesting that one frozen cell with 80% confluence degree of a six-well plate and 0.5 mL/cell of freezing medium is frozen);
(2) placing the freezing tube in a programmed cooling box, and immediately placing the freezing tube at-80 ℃ overnight (ensuring that the temperature of the freezing tube is reduced by 1 ℃ per minute);
(3) the next day the cells were immediately transferred into liquid nitrogen.
Fifth, cell recovery
(1) Preparing a Matrigel-coated cell bottle dish in advance, sucking out the Matrigel before recovering the cells, adding a proper amount of BioCISO into the cell bottle dish, placing at 37 ℃ and 5% CO2Incubating in an incubator;
(2) taking out the cryopreservation tube from liquid nitrogen quickly, immediately putting the tube into a 37 ℃ water bath kettle for quick shaking to quickly melt the cells, carefully observing, stopping shaking after the ice crystals completely disappear, and transferring the cells to a biological safety cabinet;
(3) adding 10mL of DMEM/F12(1:1) basal medium into a 15mL centrifuge tube in advance, balancing to room temperature, sucking 1mL of DMEM/F12(1:1) by using a Pasteur pipette, slowly adding the DMEM/F12(1:1) into a freezing tube, gently mixing, transferring the cell suspension into a prepared 15mL centrifuge tube containing DMEM/F12(1:1), and centrifuging for 5 minutes at 200 g;
(4) carefully removing supernatant, adding appropriate amount of BioCISO, gently mixing cells, seeding into a cell bottle dish prepared in advance, shaking up horizontally, and observing under the mirror, shaking up, and standing at 37 deg.C and 5% CO2Culturing in an incubator;
(5) the adherent survival state of the cells is observed the next day, and the liquid is normally changed on time every day. If the adherence is good, the BioCISO is changed to BioCISO + 300. mu.g/mL G418+ 0.5. mu.g/mL puro.
1.8.5AAVS1 gene knock-in detection method
Single cell clone AAVS1 gene knock-in detection
(1) AAVS1 Gene knock-in assay
a. The purpose of the test is as follows: detecting the cells subjected to the gene knock-in treatment by PCR, and testing whether the cells are homozygotes; since the two Donor fragments only have difference in the sequences of the resistance genes, in order to determine whether the cell is homozygous (the two chromosomes knock in the Donor fragments of different resistance genes respectively), it is necessary to determine whether the genome of the cell contains the Donor fragments of the two resistance genes, and only the double-knocked-in cell is likely to be the correct homozygous;
b. the test method comprises the steps of designing a primer in a Donor plasmid (non-recombinant arm part), and designing another primer in a genome PPP1R12C (non-recombinant arm part); if the Donor fragment can be correctly inserted into the genome, a target band appears, otherwise no target band appears);
c. test protocol primer sequences and PCR protocols are shown in Table 4.
TABLE 4 test protocol primer sequences and PCR protocol
Secondly, the detection method of the eGSH gene knock-in is the same as the detection principle and the detection method of the AAVS1 gene knock-in.
1.8.6 inspection method of knock-in Gene method at genomic safety site
(1) The purpose of the test is as follows: the cells treated by knock-in were tested for homozygote by PCR. Since the two Donor fragments have differences only in the sequences of the resistance genes, it is necessary to determine whether the cell is homozygous (the two chromosomes knock in the Donor fragments of different resistance genes), and it is only possible that the cell with double knockin will be the correct homozygous by examining whether the genome of the cell contains the Donor fragments of the two resistance genes.
(2) The test method comprises the following steps: first, one primer was designed inside the Donor plasmid (non-recombinant arm portion), and then the other primer was designed in the genome (non-recombinant arm portion). If the Donor fragment is inserted correctly in the genome, the target band will appear, otherwise no target band will appear.
1.9 method for measuring IL-6 blocking substance expression of pluripotent Stem cells
Detection of IL-6 antibody: the detection of anti-IL-6 antibodies expressed by pluripotent stem cells and derivatives thereof was performed using ELISA (double antigen sandwich assay). Collecting culture supernatant of the pluripotent stem cells expressing the anti-IL-6 antibody and the derivatives thereof, loading the culture supernatant on an ELISA plate coated with the human IL-6 antigen, loading sample diluent 40ul into a sample hole to be detected, then loading 10ul of the sample to be detected, loading culture supernatant of the pluripotent stem cells not expressing the anti-IL-6 antibody and the derivatives thereof into a control group, and gently mixing the culture supernatants. Placing the plate on a sealing plate, incubating for 30min at 37 ℃, washing for 5 times, adding 50ul enzyme-labeled IL-6 antigen reagent, placing the plate on the sealing plate, incubating for 30min at 37 ℃, washing for 5 times, adding color development liquid, developing for 15min, adding 50ul stop solution, reading, measuring the absorbance value at 450nm, wherein the expression quantity of the IL-6 antibody is in positive correlation with the color depth.
IL-6 receptor antibody detection: detection of IL-6 receptor antibodies expressed by pluripotent stem cells and derivatives thereof was performed using ELISA (competitive assay). Collecting culture supernatant of the pluripotent stem cells expressing the IL-6 receptor antibody and the derivatives thereof, diluting the culture supernatant by 5 times by using sample diluent, mixing the dilution with the enzyme-labeled IL-6 antigen (1:1), loading the mixture on an enzyme-labeled plate coated with the IL-6 receptor antigen, adding the culture supernatant of the pluripotent stem cells not expressing the IL-6 receptor antibody and the derivatives thereof to a control group, and gently mixing the culture supernatants. Sealing the plate, incubating at 37 deg.C for 30min, washing for 5 times, adding color developing solution, developing for 15min, adding stop solution 50ul, reading, and measuring absorbance value at 450nm, wherein the expression level of IL-6 receptor antibody is inversely related to color depth.
1.10 mouse rheumatoid arthritis model detection of therapeutic Effect of IL-6 blockers
In humanized NSG mice (The Jackson Laboratory (JAX)), human immune cells from The same donor are injected to reconstitute The immune system of The mice. After 2 weeks, collagen is used for inducing the arthritis model of the mouse to be established, firstly, bovine type II collagen and complete adjuvant are mixed and emulsified, 4-6 points of the back of the mouse are taken for injection, 200 ul/mouse, after 3 weeks, incomplete adjuvant and bovine type II collagen are mixed and emulsified, 3-5 points of the tail root of the mouse are injected, and 100 ul/mouse are used. After the mice show arthritic symptoms, the mice with the same symptoms are selected for grouping and then the experiment is carried out. A200 uL PBS (containing 106 pluripotent stem cell derivatives expressing IL-6 blocking substances, which are derived from the same donor as human immunocytes) was injected into the tail vein for rheumatoid arthritis treatment. And then scoring the rheumatoid arthritis treatment condition, and performing difference statistical analysis.
1.11 testing the therapeutic Effect of IL-6 blockers in the mouse Depression model
In humanized NSG mice (The Jackson Laboratory (JAX)), human immune cells from The same donor are injected to reconstitute The immune system of The mice. Mice were modelled using chronic unpredictable mild stress, stress factors such as: clamping tail for 1min, clicking sole for 10s, swimming with 4 deg.C ice water for 5min, reversing day and night, fasting for 24h, prohibiting water for 24h, wetting cage for 24h, and feeding at 45 ° for 24 h. 1-2 stress modes are selected daily, and repetition is avoided as much as possible for 6 weeks. And (4) judging whether the molding is successful or not by using a sweet water preference test. After the model is successfully made, 200uL PBS (containing 10 percent) is injected into tail vein6The pluripotent stem cell derivative expressing an IL-6 blocker, which is derived from the same donor as the human immune cell) for the treatment of depression. The effect of treatment of depressive symptoms was then tested using the sugar water preference test.
Anhedonia is a typical symptom in depressed patients, while symptoms in depressed mice show a significant decrease in sugar water preference. We examined the change of the sugar water preference degree of depressed mice by using a sugar water preference test to judge whether the mice have depression symptoms.
Sugar water preference test: the mice are trained for 48h first, so that the mice are suitable for drinking sugar water. All mice were housed in cages, each cage containing 2 vials, one vial containing 1% sugar water solution, the other vial containing pure water. Bottle positions were changed 1 time every 12 h. After training, animals were fasted for 12h without water, and then each cage of mice was given 2 bottles of water weighed beforehand: 1% sugar water solution and pure water, and drinking water freely for 1 h. . After the test is finished, weighing the weight of the drinking bottle to calculate the consumption of the sugar water and the consumption of the pure water,
and calculating the preference percentage of the mouse sugar water according to a formula: the percentage of preference (%) for animals for sugar water is sugar water consumption (g)/total liquid consumption (sugar water consumption (g) + pure water consumption (g)) x 100%.
1.12 testing the therapeutic Effect of IL-6 blockers in the mouse anxiety model
In humanized NSG mice (The Jackson Laboratory (JAX)), human immune cells from The same donor are injected to reconstitute The immune system of The mice. After 2 weeks, tail vein injection of 200uL PBS (containing 10)6The pluripotent stem cell derivative expressing an IL-6 blocking substance,the pluripotent stem cell derivative and the human immune cell are from the same donor) for treating anxiety disorder. The mice were then tested for anxiety using the elevated plus maze test.
The elevated plus maze has a pair of open arms and a pair of closed arms, rodents tend to move in the closed arms due to darkness but move in the open arms due to curiosity and exploratory property, and when facing a novel stimulus, the animals simultaneously generate an exploratory impulse and fear, which cause conflicting exploration and avoidance behaviors, thereby generating anxiety psychology. The anxiolytic can obviously increase the times and time for entering the open arm, and the cross maze is higher from the ground, which is equivalent to a human standing on the cliff, so that the experimental subject generates fear and uneasy mind. The frequency of the mice entering the open arms and the retention time are negatively related to the anxiety of the rats, and the less the frequency of the mice entering the open arms and the shorter the retention time are, the more serious the anxiety of the mice is.
2. Experimental protocol
First, the gene expressing the IL-6 blocker was knocked into a genomic safe site (safe harbour) to achieve expression of the IL-6 blocker by the pluripotent stem cell derivative. Thereby enabling the pluripotent stem cell derivative to be applied to disease treatment. Then, the derivative (the pluripotent stem cell derivative expressing the IL-6 blocker) can be modified by a gene editing technology to be a constitutive immune-compatible universal pluripotent stem cell derivative and an immune-compatible reversible universal pluripotent stem cell derivative, so that disease treatment can be performed in an allogeneic way.
Specifically, the following genetic manipulations were carried out in pluripotent stem cell derivatives expressing IL-6 blockers (IL-6 receptor antibodies, IL-6 antibodies) to achieve immunological compatibility of allogeneic hPSCs and hPSCs-derived derivatives (hPSCs-MSCs, NSCs, EBs).
The experimental protocol for knocking in genes expressing IL-6 blockers, one or more immune compatible molecules, genes related to shRNA and/or miRNA processing complex, anti-interferon effector molecules into safe loci in the genome of pluripotent stem cells is shown in tables 5 and 6, wherein the "+" sign indicates knocking in a gene or nucleic acid sequence and the "-" sign indicates gene knock out.
The following vectors were constructed according to the plasmid construction method in 1.8.3, respectively:
TABLE 5 constitutive expression protocol
The plasmids selected and the specific knock-in positions were as follows:
the IL-6 antibody is a blocking antibody, and the structure of the antibody is as follows: signal peptide + light chain (containing stop codon) + IRESWT (SEQ ID NO.181) + signal peptide + heavy chain (containing stop codon), the stop codon is generally TGA.
The IL-6 receptor antibody is a blocking antibody, and the structure of the antibody is as follows: the signal peptide + light chain (containing a stop codon) + IRESWt (SEQ ID NO.181) + signal peptide + heavy chain (containing a stop codon), the stop codon being generally TGA.
The signal peptide sequence was ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCAC TTGTCACGAATTCG (SEQ ID NO. 5).
General principle: the IL-6 acceptor antibody sequence and the anti-IL-6 antibody sequence are placed at the position of MCS2 of the corresponding plasmid, the shRNA is placed in a shRNA expression frame of the corresponding plasmid, the shRNAMIR is placed in a shRNAMIR expression frame of the corresponding plasmid, and other genes are placed at the position of MCS1 of the corresponding plasmid. Maps of the respective plasmids are shown in FIGS. 1to 11.
Wherein the sgRNA clone B2M plasmid comprises sgRNA clone B2M-1 and sgRNA clone B2M-2 plasmids. The sgRNA clone CIITA plasmid comprises sgRNA clone CIITA-1 and sgRNA clone CIITA-2 plasmids.
(1) Aa1 subgroup (IL-6 receptor antibody)
The MCS2 of the AAVS1 KI Vector (shRNA, constitutive) plasmid was placed into the IL-6 receptor antibody sequence (LC light chain and HC heavy chain sequences of the antibody were connected using EMCV IRESWT in the following order: LC light chain, EMCV IRESWT, HC heavy chain, the same below).
(2) Aa2 subgroup (IL-6 receptor antibody, shRNA, Gene)
The IL-6 receptor antibody sequence was placed into MCS2 of the AAVS1 KI Vector (shRNA, constitutive) plasmid. The shRNA expression framework places the shRNA target sequence (if multiple shrnas are present, they are seamlessly joined). MCS1 was placed into the gene sequence (if multiple genes were present, they were ligated using EMCV IRESWT).
(3) Aa3 subgroup (IL-6 receptor antibody, shRNA-miR, gene)
The IL-6 receptor antibody sequence was placed into MCS2 of AAVS1 KI Vector (shRNA-miR, constitutive) plasmid. The shRNA-miR expression framework places shRNA target sequences (if multiple shRNA-mirs are present, they are seamlessly connected). MCS1 was placed into the gene sequence (if multiple genes were present, they were ligated using EMCV IRESWT).
(4) Aa4 grouping (IL-6 receptor antibody, B2M and CIITA double knockout, Gene)
The IL-6 receptor antibody sequence was placed into MCS2 of the AAVS1 KI Vector (shRNA, constitutive) plasmid. MCS1 was placed into the gene sequence (if multiple genes were present, they were ligated using EMCV IRESWT).
The target sequence of the sgRNA clone B2M plasmid was placed into the sgRNA target sequence of B2M (SEQ ID NO.182 and SEQ ID NO.183), and the target sequence of the sgRNA clone CIITA plasmid was placed into the sgRNA target sequence of CIITA (SEQ ID NO.184 and SEQ ID NO. 185).
(5) Aa5 subgroup (IL-6 receptor antibody, shRNA, Gene)
A method grouped with Aa 2.
(6) Aa6 subgroup (IL-6 receptor antibody, shRNA-miR, gene)
A method grouped with Aa 3.
(7) Ab1 grouping (IL-6 antibody)
MCS2 of AAVS1 KI Vector (shRNA, constitutive) plasmid was put into LC light and HC heavy chain sequences of antibody, and ligated together using EMCV IRESwt, in the following order: LC light chain, EMCV IRESwt, HC heavy chain.
(8) Ab2 subgroup (IL-6 antibody, shRNA, gene)
MCS2 of AAVS1 KI Vector (shRNA, constitutive) plasmid was put into LC light and HC heavy chain sequences of antibody, and ligated together using EMCV IRESwt, in the following order: LC light chain, EMCV IRESwt, HC heavy chain. The shRNA expression frame is placed into the shRNA target sequence (seamlessly joined if multiple shrnas are present). MCS1 was placed into the gene sequence (if multiple genes were present, they were ligated using EMCV IRESWT).
(9) Ab3 grouping (IL-6 antibody, shRNA-miR, gene)
The MCS2 of AAVS1 KI Vector (shRNA-miR, constitutive) plasmid was put into the LC light chain and HC heavy chain sequences of the antibody, and the middle was connected using EMCV IRESWht, and the ordering was as follows: LC light chain, EMCV IRESwt, HC heavy chain. The shRNA-miR expression framework places shRNA target sequences (if multiple shRNA-mirs are present, they are seamlessly connected). MCS1 was placed into the gene sequence (if multiple genes were present, they were ligated using EMCV IRESWT).
(10) Ab4 grouping (IL-6 antibody, B2M and CIITA double knockout, Gene)
MCS2 of AAVS1 KI Vector (shRNA, constitutive) plasmid was put into LC light and HC heavy chain sequences of antibody, and ligated together using EMCV IRESwt, in the following order: LC light chain, EMCV IRESwt, HC heavy chain. MCS1 was placed into the gene sequence (if multiple genes were present, they were ligated using EMCV IRESWT).
The target sequence of the sgRNA clone B2M plasmid was placed into the sgRNA target sequence of B2M (SEQ ID NO.182 and SEQ ID NO.183), and the target sequence of the sgRNA clone CIITA plasmid was placed into the sgRNA target sequence of CIITA (SEQ ID NO.184 and SEQ ID NO. 185).
(11) Ab5 grouping (IL-6 antibody, shRNA, gene)
Methods grouped with Ab 2.
(12) Ab6 grouping (IL-6 antibody, shRNA-miR, gene)
Methods grouped with Ab 3.
TABLE 6 Experimental protocol for inducible expression
(1) Ba1 subgroup (IL-6 receptor antibody, shRNA, gene)
The IL-6 receptor antibody sequence was placed into MCS2 of the AAVS1 KI Vector (shRNA, inducible) plasmid. The shRNA expression framework places the shRNA target sequence (if multiple shrnas are present, they are seamlessly joined). MCS1 was placed into the gene sequence (if multiple genes were present, they were ligated using EMCV IRESWT).
(2) Ba2 grouping (IL-6 receptor antibody, shRNA-miR, gene)
IL-6 receptor antibody sequences were placed into MCS2 of AAVS1 KI Vector (shRNA-miR, inducible) plasmid. The shRNA-miR expression framework places shRNA target sequences (if multiple shRNA-mirs are present, they are seamlessly connected). MCS1 was placed into the gene sequence (if there were multiple genes then ligated using EMCV IRESWT).
(3) Ba3 subgroup (IL-6 receptor antibody, shRNA, gene)
Method grouped with Ba 1.
(4) Ba4 grouping (IL-6 receptor antibody, shRNA-miR, gene)
Method grouped with Ba 2.
(5) Bb1 grouping (IL-6 antibody, shRNA, Gene)
The anti-IL-6 antibody sequence was placed into MCS2 of the AAVS1 KI Vector (shRNA, inducible) plasmid. The shRNA expression framework places the shRNA target sequence (if multiple shrnas are present, they are seamlessly joined). MCS1 was placed into the gene sequence (if there were multiple genes then ligated using EMCV IRESWT).
(6) Bb2 grouping (IL-6 antibody, shRNA-miR, gene)
anti-IL-6 antibody sequences were placed into MCS2 of AAVS1 KI Vector (shRNA-miR, inducible) plasmid. The shRNA-miR expression framework places shRNA target sequences (if multiple shRNA-mirs are present, they are seamlessly connected). MCS1 was placed into the gene sequence (if multiple genes were present, they were ligated using EMCV IRESWT).
(7) Bb3 grouping (IL-6 antibody, shRNA, Gene)
Methods grouped with Bb 1.
(8) Bb4 grouping (IL-6 antibody, shRNA-miR, gene)
Methods grouped with Bb 2.
Example 1 detection of pluripotent Stem cell derivatives expressing IL-6 receptor antibodies
The experimental group protocols in tables 5 and 6 were knocked into the genome safety sites of iPSCs, EBs, MSCs and NSCs cells at 37 ℃ with 0.5% CO2Culturing in an incubator, collecting culture supernatant of the pluripotent stem cells expressing the IL-6 receptor antibody and the derivatives thereof, diluting by 5 times with sample diluent, mixing with the enzyme-labeled IL-6 antigen (1:1), loading on an enzyme-labeled plate which already holds the IL-6 receptor antigen, adding culture supernatant of the pluripotent stem cells not expressing the IL-6 receptor antibody and the derivatives thereof to a control group, and gently mixing. After sealing the plate, the plate was incubated at 37 ℃ for 30min, washed 5 times, then developed by adding developing solution for 15min, added with stop solution 50ul, and read to measure absorbance at 450nm, with the results shown in Table 7.
TABLE 7 ELISA detection of IL-6 receptor antibody expressed by each experimental group
As can be seen from the above table, the pluripotent stem cells or derivatives thereof prepared according to the present invention can efficiently express IL-6 receptor antibodies. And the expression level is relatively constant in each group, so that the IL-6 receptor antibody expressed by the pluripotent stem cell derivative is not influenced by cell differentiation morphology and other exogenous genes (immune compatibility modification).
Example 2 detection of pluripotent Stem cell derivatives expressing IL-6 antibody
The experimental group protocols in tables 5 and 6 were knocked into the genome safety sites of iPSCs, EBs, MSCs and NSCs cells at 37 ℃ with 0.5% CO2Culturing in an incubator, collecting culture supernatant of the pluripotent stem cells expressing the IL-6 antibody and the derivatives thereof, loading the culture supernatant on an enzyme label plate which already contains the human IL-6 antigen, adding 40ul of sample diluent into a sample hole to be detected, then adding 10ul of sample to be detected, adding culture supernatant of the pluripotent stem cells not expressing the IL-6 antibody and the derivatives thereof into a control group, and gently mixing the culture supernatants. After sealing the plate, placing the plate at 37 ℃ for incubation for 30min, washing for 5 times, adding 50ul enzyme-labeled IL-6 antigen reagent, after sealing the plate, placing the plate at 37 ℃ for incubation for 30min, washing for 5 times, adding developing solution for developing for 15min, adding 50ul stop solution, reading and measuring the absorbance value at 450nm, wherein the results are shown in Table 8.
TABLE 8 ELISA detection of IL-6 antibody expressed by each experimental group
As can be seen from the above table, the pluripotent stem cells or derivatives thereof prepared by the present invention can efficiently express IL-6 antibody. And the expression level is relatively constant in each group, so that the IL-6 antibody expressed by the pluripotent stem cell derivative is not influenced by cell differentiation morphology and other exogenous genes (immune compatibility modification).
Example 3 Effect of IL-6 blockers in the treatment of rheumatoid arthritis
The inventors selected cells (MSCs) that only expressed the blocker protocol group (Aa1, Ab1) for testing. In the humanized NSG mouse rheumatoid arthritis model, hPSCs and hPSCs-derived derivatives (hPSCs-MSCs, hPSCs-NSCs, hPSCs-EBs) capable of expressing IL-6 blockers (IL-6 receptor antibody, IL-6 antibody) were injected into mice, and the effect of treating rheumatoid arthritis was observed, and the results are shown in Table 9.
Note: to avoid the problem of immune compatibility, the immunocytes used are derived from the same person as the hPSCs and derivatives of hPSCs.
TABLE 9 Effect of IL-6 blocking substance expression in each experimental group on rheumatoid arthritis treatment
Through the experiment, the stem cell expressing the IL-6 blocking substance or the derivative thereof prepared by the invention can be proved to have the effect of effectively treating rheumatoid arthritis.
Example 4 Effect of IL-6 blockers in the treatment of Depression
The inventors selected cells (MSCs) that only expressed the blocker protocol group (Aa1, Ab1) for testing. In the humanized NSG mouse depression model, hPSCs and hPSCs-derived derivatives (hPSCs-MSCs, hPSCs-NSCs and hPSCs-EBs) capable of expressing IL-6 blockers (IL-6 receptor antibody and IL-6 antibody) were injected into mice, and the effect of treating depression was observed.
Note: to avoid the problem of immune compatibility, the immunocytes used are derived from the same person as the hPSCs and derivatives of hPSCs.
TABLE 10 Effect of various experimental groups expressing IL-6 blockers on treating depression
Through the experiment, the stem cell expressing the IL-6 blocker or the derivative thereof prepared by the invention can be proved to have the effect of effectively treating depression.
Example 5 Effect of IL-6 blockers in the treatment of anxiety disorders
The inventors selected cells (MSCs) that only expressed the blocker protocol group (Aa1, Ab1) for testing. In the humanized NSG mouse anxiety model, hPSCs and hPSCs-derived derivatives (hPSCs-MSCs, hPSCs-NSCs, hPSCs-EBs) capable of expressing IL-6 blockers (IL-6 receptor antibody, IL-6 antibody) were injected into mice, and the effect of treating depression was observed, and the results are shown in Table 11.
Note: to avoid the problem of immune compatibility, the immunocytes used are derived from the same person as the hPSCs and derivatives of hPSCs.
TABLE 11 Effect of each experimental group expressing IL-6 blockers on treating anxiety disorders
Note: p <0.01 compared to control.
Through the experiments, the stem cells expressing the IL-6 blocker or the derivatives thereof prepared by the invention can play an anxiolytic role and can be used for treating anxiety disorder.
Example 6 Effect of immune-compatible molecule-inducible expression sets in the treatment of rheumatoid arthritis
Through the above examples, hPSCs and hPSCs derived derivatives expressing IL-6 blockers are effective in treating rheumatoid arthritis, depression and anxiety.
Furthermore, the problem of immune compatibility of hPSCs and derivatives of hPSCs must be considered. The inventors therefore selected a suitable combination to test for immune compatibility. Based on the characteristic of low immunogenicity of MSCs, in a humanized NSG mouse disease model, hPSCs-derived immuno-compatible MSCs capable of expressing IL-6 blockers (IL-6 antibodies) were injected into mice, and the effect of treatment of rheumatoid arthritis was observed, with the results shown in table 12.
Note: the immunocytes used are derived from non-identical persons with the MSCs of hPSCs source.
The control group refers to the NSG mouse disease model without MSCs cell injection.
The process of adding the Dox group is: mice were fed with 0.5mg/mL Dox in the mouse diet, and the mice were used from the time of injection of the expression blocker cells until the end of the experiment.
TABLE 12 reversible expression test results for immune-compatible molecule-inducible expression sets
The above experiments show that: in the treatment of diseases. MSCs that express only blockers (group 2), which have low immunogenicity and can exist within a foreign body for a certain period of time, can exert a certain therapeutic effect, while those that are immuno-compatibly engineered (groups 3-11, including constitutive and reversible inducible immuno-compatibility), which have better immuno-compatibility effects, are present in vivo for a longer time (or can coexist for a long period of time) than MSCs that have not been immuno-compatibly engineered, which exert better therapeutic effects, whereas group 5 is the B2M and CIITA gene knock-out groups, which completely eliminate the effects of HLA-I and HLA-II molecules, and thus have the best therapeutic effects. However, there are group 8-15 protocols set up due to their constitutive immune compatible modifications (knock-in/knock-out) which cannot be cleared when the graft becomes mutated or otherwise unwanted. In groups 12-15, the mice injected with the expression blocker cells will be abolished from their immune compatibility by the use of Dox inducer (always used) simultaneously with the injection of the expression blocker cells into the mice, and will be present in vivo for a time period comparable to that of the MSCs without immune compatibility engineering, and will be treated with a therapeutic effect comparable to that of the MSCs without immune compatibility engineering.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
SEQUENCE LISTING
<110> future Chile regenerative medicine institute (Guangzhou) Co., Ltd
Wang Linli
<120> pluripotent stem cell derivative expressing IL-6 blocking substance and application thereof
<130>
<160> 185
<170> PatentIn version 3.5
<210> 1
<211> 1347
<212> DNA
<213> Artificial sequence
<400> 1
gaggtgcagc tggtggagag cggcggcaag ctgctgaagc ccggcggcag cctgaagctg 60
agctgcgccg ccagcggctt caccttcagc agcttcgcca tgagctggtt caggcagagc 120
cccgagaaga ggctggagtg ggtggccgag atcagcagcg gcggcagcta cacctactac 180
cccgacaccg tgaccggcag gttcaccatc agcagggaca acgccaagaa caccctgtac 240
ctggagatga gcagcctgag gagcgaggac accgccatgt actactgcgc caggggcctg 300
tggggctact acgccctgga ctactggggc cagggcacca gcgtgaccgt gagcagcgcc 360
agcaccaagg gccccagcgt gttccccctg gcccccagca gcaagagcac cagcggcggc 420
accgccgccc tgggctgcct ggtgaaggac tacttccccg agcccgtgac cgtgagctgg 480
aacagcggcg ccctgaccag cggcgtgcac accttccccg ccgtgctgca gagcagcggc 540
ctgtacagcc tgagcagcgt ggtgaccgtg cccagcagca gcctgggcac ccagacctac 600
atctgcaacg tgaaccacaa gcccagcaac accaaggtgg acaagaaggt ggagcccaag 660
agctgcgaca agacccacac ctgccccccc tgccccgccc ccgagctgct gggcggcccc 720
agcgtgttcc tgttcccccc caagcccaag gacaccctga tgatcagcag gacccccgag 780
gtgacctgcg tggtggtgga cgtgagccac gaggaccccg aggtgaagtt caactggtac 840
gtggacggcg tggaggtgca caacgccaag accaagccca gggaggagca gtacaacagc 900
acctacaggg tggtgagcgt gctgaccgtg ctgcaccagg actggctgaa cggcaaggag 960
tacaagtgca aggtgagcaa caaggccctg cccgccccca tcgagaagac catcagcaag 1020
gccaagggcc agcccaggga gccccaggtg tacaccctgc cccccagcag ggacgagctg 1080
accaagaacc aggtgagcct gacctgcctg gtgaagggct tctaccccag cgacatcgcc 1140
gtggagtggg agagcaacgg ccagcccgag aacaactaca agaccacccc ccccgtgctg 1200
gacagcgacg gcagcttctt cctgtacagc aagctgaccg tggacaagag caggtggcag 1260
cagggcaacg tgttcagctg cagcgtgatg cacgaggccc tgcacaacca ctacacccag 1320
aagagcctga gcctgagccc cggcaag 1347
<210> 2
<211> 639
<212> DNA
<213> Artificial sequence
<400> 2
cagatcgtgc tgatccagag ccccgccatc atgagcgcca gccccggcga gaaggtgacc 60
atgacctgca gcgccagcag cagcgtgagc tacatgtact ggtaccagca gaagcccggc 120
agcagcccca ggctgctgat ctacgacacc agcaacctgg ccagcggcgt gcccgtgagg 180
ttcagcggca gcggcagcgg caccagctac agcctgacca tcagcaggat ggaggccgag 240
gacgccgcca cctactactg ccagcagtgg agcggctacc cctacacctt cggcggcggc 300
accaagctgg agatcaagag gaccgtggcc gcccccagcg tgttcatctt cccccccagc 360
gacgagcagc tgaagagcgg caccgccagc gtggtgtgcc tgctgaacaa cttctacccc 420
agggaggcca aggtgcagtg gaaggtggac aacgccctgc agagcggcaa cagccaggag 480
agcgtgaccg agcaggacag caaggacagc acctacagcc tgagcagcac cctgaccctg 540
agcaaggccg actacgagaa gcacaaggtg tacgcctgcg aggtgaccca ccagggcctg 600
agcagccccg tgaccaagag cttcaacagg ggcgagtgc 639
<210> 3
<211> 1338
<212> DNA
<213> Artificial sequence
<400> 3
gaggtgcagc tggtggagag cggcggcggc ctggtgcagc ccggcaggag cctgaggctg 60
agctgcgccg ccagcaggtt caccttcgac gactacgcca tgcactgggt gaggcaggcc 120
cccggcaagg gcctggagtg ggtgagcggc atcagctgga acagcggcag gatcggctac 180
gccgacagcg tgaagggcag gttcaccatc agcagggaca acgccgagaa cagcctgttc 240
ctgcagatga acggcctgag ggccgaggac accgccctgt actactgcgc caagggcagg 300
gacagcttcg acatctgggg ccagggcacc atggtgaccg tgagcagcgc cagcaccaag 360
ggccccagcg tgttccccct ggcccccagc agcaagagca ccagcggcgg caccgccgcc 420
ctgggctgcc tggtgaagga ctacttcccc gagcccgtga ccgtgagctg gaacagcggc 480
gccctgacca gcggcgtgca caccttcccc gccgtgctgc agagcagcgg cctgtacagc 540
ctgagcagcg tggtgaccgt gcccagcagc agcctgggca cccagaccta catctgcaac 600
gtgaaccaca agcccagcaa caccaaggtg gacaagaagg tggagcccaa gagctgcgac 660
aagacccaca cctgcccccc ctgccccgcc cccgagctgc tgggcggccc cagcgtgttc 720
ctgttccccc ccaagcccaa ggacaccctg atgatcagca ggacccccga ggtgacctgc 780
gtggtggtgg acgtgagcca cgaggacccc gaggtgaagt tcaactggta cgtggacggc 840
gtggaggtgc acaacgccaa gaccaagccc agggaggagc agtacaacag cacctacagg 900
gtggtgagcg tgctgaccgt gctgcaccag gactggctga acggcaagga gtacaagtgc 960
aaggtgagca acaaggccct gcccgccccc atcgagaaga ccatcagcaa ggccaagggc 1020
cagcccaggg agccccaggt gacctacctg ccccccagca gggacgagct gaccaagaac 1080
caggtgagcc tgacctgcct ggtgaagggc ttctacccca gcgacatcgc cgtggagtgg 1140
gagagcaacg gccagcccga gaacaactac aagaccaccc cccccgtgct ggacagcgac 1200
ggcagcttct tcctgtacag caagctgacc gtggacaaga gcaggtggca gcagggcaac 1260
gtgttcagct gcagcgtgat gcacgaggcc ctgcacaacc actacaccca gaagagcctg 1320
agcctgagcc ccggcaag 1338
<210> 4
<211> 642
<212> DNA
<213> Artificial sequence
<400> 4
gacatccaga tgacccagag ccccagcagc gtgagcgcca gcgtgggcga cagggtgacc 60
atcacctgca gggccagcca gggcatcagc agctggctgg cctggtacca gcagaagccc 120
ggcaaggccc ccaagctgct gatctacggc gccagcagcc tggagagcgg cgtgcccagc 180
aggttcagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cctgcagccc 240
gaggacttcg ccagctacta ctgccagcag gccaacagct tcccctacac cttcggccag 300
ggcaccaagc tggagatcaa gaggaccgtg gccgccccca gcgtgttcat cttccccccc 360
agcgacgagc agctgaagag cggcaccgcc agcgtggtgt gcctgctgaa caacttctac 420
cccagggagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag 480
gagagcgtga ccgagcagga cagcaaggac agcacctaca gcctgagcag caccctgacc 540
ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccagggc 600
ctgagcagcc ccgtgaccaa gagcttcaac aggggcgagt gc 642
<210> 5
<211> 60
<212> DNA
<213> Artificial sequence
<400> 5
atgtacagga tgcaactcct gtcttgcatt gcactaagtc ttgcacttgt cacgaattcg 60
<210> 6
<211> 21
<212> DNA
<213> human
<400> 6
gggagcagag aattctctta t 21
<210> 7
<211> 21
<212> DNA
<213> human
<400> 7
ggagcagaga attctcttat c 21
<210> 8
<211> 21
<212> DNA
<213> human
<400> 8
gagcagagaa ttctcttatc c 21
<210> 9
<211> 21
<212> DNA
<213> human
<400> 9
gctacctgga gcttcttaac a 21
<210> 10
<211> 21
<212> DNA
<213> human
<400> 10
ggagcttctt aacagcgatg c 21
<210> 11
<211> 21
<212> DNA
<213> human
<400> 11
gggtctccag tatattcatc t 21
<210> 12
<211> 21
<212> DNA
<213> human
<400> 12
gcctcctgat gcacatgtac t 21
<210> 13
<211> 21
<212> DNA
<213> human
<400> 13
ggaagacctg ggaaagcttg t 21
<210> 14
<211> 21
<212> DNA
<213> human
<400> 14
ggctaagctt gtacaataac t 21
<210> 15
<211> 21
<212> DNA
<213> human
<400> 15
gcggaatgaa ccacatcttg c 21
<210> 16
<211> 21
<212> DNA
<213> human
<400> 16
ggccttctct gaaggacatt g 21
<210> 17
<211> 21
<212> DNA
<213> human
<400> 17
ggactcaatg cactgacatt g 21
<210> 18
<211> 21
<212> DNA
<213> human
<400> 18
ggtacccact gctctggtta t 21
<210> 19
<211> 21
<212> DNA
<213> human
<400> 19
gctcccactc catgaggtat t 21
<210> 20
<211> 21
<212> DNA
<213> human
<400> 20
ggtatttctt cacatccgtg t 21
<210> 21
<211> 21
<212> DNA
<213> human
<400> 21
aggagacacg gaatgtgaag g 21
<210> 22
<211> 21
<212> DNA
<213> human
<400> 22
gctcccactc catgaggtat t 21
<210> 23
<211> 21
<212> DNA
<213> human
<400> 23
ggtatttcta cacctccgtg t 21
<210> 24
<211> 21
<212> DNA
<213> human
<400> 24
ggaccggaac acacagatct a 21
<210> 25
<211> 21
<212> DNA
<213> human
<400> 25
accggaacac acagatctac a 21
<210> 26
<211> 21
<212> DNA
<213> human
<400> 26
ggaacacaca gatctacaag g 21
<210> 27
<211> 21
<212> DNA
<213> human
<400> 27
gaacacacag atctacaagg c 21
<210> 28
<211> 21
<212> DNA
<213> human
<400> 28
ttcttacttc cctaatgaag t 21
<210> 29
<211> 21
<212> DNA
<213> human
<400> 29
aagttaagaa cctgaatata a 21
<210> 30
<211> 21
<212> DNA
<213> human
<400> 30
aacctgaata taaatttgtg t 21
<210> 31
<211> 21
<212> DNA
<213> human
<400> 31
acctgaatat aaatttgtgt t 21
<210> 32
<211> 21
<212> DNA
<213> human
<400> 32
aagcgttgat ggattaatta a 21
<210> 33
<211> 21
<212> DNA
<213> human
<400> 33
agcgttgatg gattaattaa a 21
<210> 34
<211> 21
<212> DNA
<213> human
<400> 34
gggtctggtg ggcatcatta t 21
<210> 35
<211> 21
<212> DNA
<213> human
<400> 35
ggtctggtgg gcatcattat t 21
<210> 36
<211> 21
<212> DNA
<213> human
<400> 36
gcatcattat tgggaccatc t 21
<210> 37
<211> 21
<212> DNA
<213> human
<400> 37
gcacatggag gtgatggtgt t 21
<210> 38
<211> 21
<212> DNA
<213> human
<400> 38
ggaggtgatg gtgtttctta g 21
<210> 39
<211> 21
<212> DNA
<213> human
<400> 39
gagaagatca ctgaagaaac t 21
<210> 40
<211> 21
<212> DNA
<213> human
<400> 40
gctttaatgg ctttacaaag c 21
<210> 41
<211> 21
<212> DNA
<213> human
<400> 41
ggctttacaa agctggcaat a 21
<210> 42
<211> 21
<212> DNA
<213> human
<400> 42
gctttacaaa gctggcaata t 21
<210> 43
<211> 21
<212> DNA
<213> human
<400> 43
gctccgtact ctaacatcta g 21
<210> 44
<211> 21
<212> DNA
<213> human
<400> 44
gatgaccaca ttcaaggaag a 21
<210> 45
<211> 21
<212> DNA
<213> human
<400> 45
gaccacattc aaggaagaac t 21
<210> 46
<211> 21
<212> DNA
<213> human
<400> 46
gctttcctgc ttggcagtta t 21
<210> 47
<211> 21
<212> DNA
<213> human
<400> 47
ggcagttatt cttccacaag a 21
<210> 48
<211> 21
<212> DNA
<213> human
<400> 48
gcagttattc ttccacaaga g 21
<210> 49
<211> 21
<212> DNA
<213> human
<400> 49
gcgtaagtct gagtgtcatt t 21
<210> 50
<211> 21
<212> DNA
<213> human
<400> 50
gacaatttaa ggaagaatct t 21
<210> 51
<211> 21
<212> DNA
<213> human
<400> 51
ggccatagtt ctccctgatt g 21
<210> 52
<211> 21
<212> DNA
<213> human
<400> 52
gccatagttc tccctgattg a 21
<210> 53
<211> 21
<212> DNA
<213> human
<400> 53
gcagatgacc acattcaagg a 21
<210> 54
<211> 21
<212> DNA
<213> human
<400> 54
gatgaccaca ttcaaggaag a 21
<210> 55
<211> 21
<212> DNA
<213> human
<400> 55
gaccacattc aaggaagaac c 21
<210> 56
<211> 21
<212> DNA
<213> human
<400> 56
gctttgtcag gaccaggttg t 21
<210> 57
<211> 21
<212> DNA
<213> human
<400> 57
gaccaggttg ttactggttc a 21
<210> 58
<211> 21
<212> DNA
<213> human
<400> 58
gaagcctcac agctttgatg g 21
<210> 59
<211> 21
<212> DNA
<213> human
<400> 59
gatggcagtg cctcatcttc a 21
<210> 60
<211> 21
<212> DNA
<213> human
<400> 60
ggcagtgcct catcttcaac t 21
<210> 61
<211> 21
<212> DNA
<213> human
<400> 61
gcagcaggat aagtatgagt g 21
<210> 62
<211> 21
<212> DNA
<213> human
<400> 62
gcaggataag tatgagtgtc a 21
<210> 63
<211> 21
<212> DNA
<213> human
<400> 63
ggttcctgca cagagacatc t 21
<210> 64
<211> 21
<212> DNA
<213> human
<400> 64
gcacagagac atctataacc a 21
<210> 65
<211> 21
<212> DNA
<213> human
<400> 65
gagacatcta taaccaagag g 21
<210> 66
<211> 21
<212> DNA
<213> human
<400> 66
gagtactgga acagccagaa g 21
<210> 67
<211> 21
<212> DNA
<213> human
<400> 67
gctttcctgc ttggctctta t 21
<210> 68
<211> 21
<212> DNA
<213> human
<400> 68
ggctcttatt cttccacaag a 21
<210> 69
<211> 21
<212> DNA
<213> human
<400> 69
gctcttattc ttccacaaga g 21
<210> 70
<211> 21
<212> DNA
<213> human
<400> 70
ggatgtggaa cccacagata c 21
<210> 71
<211> 21
<212> DNA
<213> human
<400> 71
gatgtggaac ccacagatac a 21
<210> 72
<211> 21
<212> DNA
<213> human
<400> 72
gtggaaccca cagatacaga g 21
<210> 73
<211> 21
<212> DNA
<213> human
<400> 73
ggaacccaca gatacagaga g 21
<210> 74
<211> 21
<212> DNA
<213> human
<400> 74
gagccaactg tattgcctat t 21
<210> 75
<211> 21
<212> DNA
<213> human
<400> 75
agccaactgt attgcctatt t 21
<210> 76
<211> 21
<212> DNA
<213> human
<400> 76
gccaactgta ttgcctattt g 21
<210> 77
<211> 21
<212> DNA
<213> human
<400> 77
gggtagcaac tgtcaccttg a 21
<210> 78
<211> 21
<212> DNA
<213> human
<400> 78
ggatttcgtg ttccagttta a 21
<210> 79
<211> 21
<212> DNA
<213> human
<400> 79
gcatgtgcta cttcaccaac g 21
<210> 80
<211> 21
<212> DNA
<213> human
<400> 80
gcgtcttgtg accagataca t 21
<210> 81
<211> 21
<212> DNA
<213> human
<400> 81
gcttatgcct gcccagaatt c 21
<210> 82
<211> 21
<212> DNA
<213> human
<400> 82
gcaggaaatc actgcagaat g 21
<210> 83
<211> 21
<212> DNA
<213> human
<400> 83
gctcagtgca ttggccttag a 21
<210> 84
<211> 21
<212> DNA
<213> human
<400> 84
ggtgagtgct gtgtaaataa g 21
<210> 85
<211> 21
<212> DNA
<213> human
<400> 85
gacatatata gtgatccttg g 21
<210> 86
<211> 21
<212> DNA
<213> human
<400> 86
ggaaagtcac atcgatcaag a 21
<210> 87
<211> 21
<212> DNA
<213> human
<400> 87
gctcacagtc atcaattata g 21
<210> 88
<211> 21
<212> DNA
<213> human
<400> 88
gccctgaaga cagaatgttc c 21
<210> 89
<211> 21
<212> DNA
<213> human
<400> 89
gcggaccatg tgtcaactta t 21
<210> 90
<211> 21
<212> DNA
<213> human
<400> 90
ggaccatgtg tcaacttatg c 21
<210> 91
<211> 21
<212> DNA
<213> human
<400> 91
gcgtttgtac agacgcatag a 21
<210> 92
<211> 21
<212> DNA
<213> human
<400> 92
ggctggctaa cattgctata t 21
<210> 93
<211> 21
<212> DNA
<213> human
<400> 93
gctggctaac attgctatat t 21
<210> 94
<211> 21
<212> DNA
<213> human
<400> 94
ggaccaggtc acatgtgaat a 21
<210> 95
<211> 21
<212> DNA
<213> human
<400> 95
ggaaaggtct gaggatattg a 21
<210> 96
<211> 21
<212> DNA
<213> human
<400> 96
ggcagattag gattccattc a 21
<210> 97
<211> 21
<212> DNA
<213> human
<400> 97
gcctgatagg acccatattc c 21
<210> 98
<211> 21
<212> DNA
<213> human
<400> 98
gcatccaata gacgtcattt g 21
<210> 99
<211> 21
<212> DNA
<213> human
<400> 99
gcgtcactgg cacagatata a 21
<210> 100
<211> 21
<212> DNA
<213> human
<400> 100
gctgtcacat aataagctaa g 21
<210> 101
<211> 21
<212> DNA
<213> human
<400> 101
gctaaggaag acagtatata g 21
<210> 102
<211> 21
<212> DNA
<213> human
<400> 102
gggatttcta aggaaggatg c 21
<210> 103
<211> 21
<212> DNA
<213> human
<400> 103
ggagttgaag agcagagatt c 21
<210> 104
<211> 21
<212> DNA
<213> human
<400> 104
gccagtgaac acttaccata g 21
<210> 105
<211> 21
<212> DNA
<213> human
<400> 105
gcttctctga agtctcattg a 21
<210> 106
<211> 21
<212> DNA
<213> human
<400> 106
ggctgcaact aacttcaaat a 21
<210> 107
<211> 21
<212> DNA
<213> human
<400> 107
ggatggattt gattatgatc c 21
<210> 108
<211> 21
<212> DNA
<213> human
<400> 108
ggaccttgga acaatggatt g 21
<210> 109
<211> 21
<212> DNA
<213> human
<400> 109
gctaattctt gctgaacttc t 21
<210> 110
<211> 21
<212> DNA
<213> human
<400> 110
gctgaacttc ttcatgtatg t 21
<210> 111
<211> 21
<212> DNA
<213> human
<400> 111
gcctcatctc tttgttctaa a 21
<210> 112
<211> 21
<212> DNA
<213> human
<400> 112
gctctggaga agatatattt g 21
<210> 113
<211> 21
<212> DNA
<213> human
<400> 113
gctcttgagg gaactaatag a 21
<210> 114
<211> 21
<212> DNA
<213> human
<400> 114
gggacggcat taatgtattc a 21
<210> 115
<211> 21
<212> DNA
<213> human
<400> 115
ggacaaacat gcaaactata g 21
<210> 116
<211> 21
<212> DNA
<213> human
<400> 116
gcagcaacca gctaccattc t 21
<210> 117
<211> 21
<212> DNA
<213> human
<400> 117
gcagttctgt tgccactctc t 21
<210> 118
<211> 21
<212> DNA
<213> human
<400> 118
gggagagttc atccaggaaa t 21
<210> 119
<211> 21
<212> DNA
<213> human
<400> 119
ggagagttca tccaggaaat t 21
<210> 120
<211> 21
<212> DNA
<213> human
<400> 120
gagagttcat ccaggaaatt a 21
<210> 121
<211> 21
<212> DNA
<213> human
<400> 121
gcctgtcaaa gagagagagc a 21
<210> 122
<211> 21
<212> DNA
<213> human
<400> 122
gctcagcttc gtactgagtt c 21
<210> 123
<211> 21
<212> DNA
<213> human
<400> 123
gcttcacaga actacagaga g 21
<210> 124
<211> 21
<212> DNA
<213> human
<400> 124
gcatctactg gacaaagtat t 21
<210> 125
<211> 21
<212> DNA
<213> human
<400> 125
ggctgaatta cccatgcttt a 21
<210> 126
<211> 21
<212> DNA
<213> human
<400> 126
gctgaattac ccatgcttta a 21
<210> 127
<211> 21
<212> DNA
<213> human
<400> 127
gggttggttt atccaggaat a 21
<210> 128
<211> 21
<212> DNA
<213> human
<400> 128
ggatcagaag agaagccaac g 21
<210> 129
<211> 21
<212> DNA
<213> human
<400> 129
ggttcaccat ccaggtgttc a 21
<210> 130
<211> 21
<212> DNA
<213> human
<400> 130
gctctcttct ctggaactaa c 21
<210> 131
<211> 21
<212> DNA
<213> human
<400> 131
gctagagtga ctccatctta a 21
<210> 132
<211> 21
<212> DNA
<213> human
<400> 132
gctgaccacc aattataatt g 21
<210> 133
<211> 21
<212> DNA
<213> human
<400> 133
gcagaatatt taaggccata c 21
<210> 134
<211> 21
<212> DNA
<213> human
<400> 134
gcccacttaa aggcagcatt a 21
<210> 135
<211> 21
<212> DNA
<213> human
<400> 135
ggtcatcaat accactgtta a 21
<210> 136
<211> 21
<212> DNA
<213> human
<400> 136
gcattcctcc ttctcctttc t 21
<210> 137
<211> 21
<212> DNA
<213> human
<400> 137
ggaggaactt tgtgaacatt c 21
<210> 138
<211> 21
<212> DNA
<213> human
<400> 138
gctgtaagaa ggatgctttc a 21
<210> 139
<211> 21
<212> DNA
<213> human
<400> 139
gctgcaggca ggattgtttc a 21
<210> 140
<211> 21
<212> DNA
<213> human
<400> 140
gcagttcgag gtcaagtttg a 21
<210> 141
<211> 21
<212> DNA
<213> human
<400> 141
gccaattagc tgagaagaat t 21
<210> 142
<211> 21
<212> DNA
<213> human
<400> 142
gcaggtttac agtgtatatg t 21
<210> 143
<211> 21
<212> DNA
<213> human
<400> 143
gcctacagag actagagtag g 21
<210> 144
<211> 21
<212> DNA
<213> human
<400> 144
gcagttgggt accttccatt c 21
<210> 145
<211> 21
<212> DNA
<213> human
<400> 145
gcaactcagg tgcatgatac a 21
<210> 146
<211> 21
<212> DNA
<213> human
<400> 146
gcatggcgct ggtacgtaaa t 21
<210> 147
<211> 19
<212> DNA
<213> human
<400> 147
gcctcgagtt tgagagcta 19
<210> 148
<211> 19
<212> DNA
<213> human
<400> 148
agacattctg gatgagtta 19
<210> 149
<211> 19
<212> DNA
<213> human
<400> 149
gggtctgtta cccaaagaa 19
<210> 150
<211> 19
<212> DNA
<213> human
<400> 150
ggtctgttac ccaaagaat 19
<210> 151
<211> 19
<212> DNA
<213> human
<400> 151
ggaaggaagc ggacgctca 19
<210> 152
<211> 19
<212> DNA
<213> human
<400> 152
ggaggcagta cttctgata 19
<210> 153
<211> 19
<212> DNA
<213> human
<400> 153
cgctctagag ctcagctga 19
<210> 154
<211> 19
<212> DNA
<213> human
<400> 154
ccaccacctc aaccaataa 19
<210> 155
<211> 19
<212> DNA
<213> human
<400> 155
atttcaagaa gtcgatcaa 19
<210> 156
<211> 19
<212> DNA
<213> human
<400> 156
gaagatctga ttaccttca 19
<210> 157
<211> 21
<212> DNA
<213> human
<400> 157
ggacactggt tcaacacctg t 21
<210> 158
<211> 21
<212> DNA
<213> human
<400> 158
ggttcaacac ctgtgacttc a 21
<210> 159
<211> 21
<212> DNA
<213> human
<400> 159
acctgtgact tcatgtgtgc g 21
<210> 160
<211> 21
<212> DNA
<213> human
<400> 160
gctggacgtg accatcatgt a 21
<210> 161
<211> 21
<212> DNA
<213> human
<400> 161
ggacgtgacc atcatgtaca a 21
<210> 162
<211> 21
<212> DNA
<213> human
<400> 162
gacgtgacca tcatgtacaa g 21
<210> 163
<211> 21
<212> DNA
<213> human
<400> 163
acgtgaccat catgtacaag g 21
<210> 164
<211> 21
<212> DNA
<213> human
<400> 164
acgctatacc atctacctgg g 21
<210> 165
<211> 21
<212> DNA
<213> human
<400> 165
gcctctatga cgacatcgag t 21
<210> 166
<211> 21
<212> DNA
<213> human
<400> 166
gacatcgagt gcttccttat g 21
<210> 167
<211> 253
<212> DNA
<213> Artificial sequence
<400> 167
gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60
ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120
aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180
atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240
cgctagcgcc acc 253
<210> 168
<211> 9
<212> DNA
<213> Artificial sequence
<400> 168
ttcaagaga 9
<210> 169
<211> 686
<212> DNA
<213> Artificial sequence
<400> 169
gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60
ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120
aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180
atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240
ctttaccact ccctatcagt gatagagaaa agtgaaagtc gagtttacca ctccctatca 300
gtgatagaga aaagtgaaag tcgagtttac cactccctat cagtgataga gaaaagtgaa 360
agtcgagttt accactccct atcagtgata gagaaaagtg aaagtcgagt ttaccactcc 420
ctatcagtga tagagaaaag tgaaagtcga gtttaccact ccctatcagt gatagagaaa 480
agtgaaagtc gagtttacca ctccctatca gtgatagaga aaagtgaaag tcgagctcgg 540
tacccgggtc gaggtaggcg tgtacggtgg gaggcctata taagcagagc tcgtttagtg 600
aaccgtcaga tcgcctggag acgccatcca cgctgttttg acctccatag aagacaccgg 660
gaccgatcca gcctgctagc gccacc 686
<210> 170
<211> 119
<212> DNA
<213> Artificial sequence
<400> 170
gaggcttcag tactttacag aatcgttgcc tgcacatctt ggaaacactt gctgggatta 60
cttcttcagg ttaacccaac agaaggctaa agaaggtata ttgctgttga cagtgagcg 119
<210> 171
<211> 19
<212> DNA
<213> Artificial sequence
<400> 171
tagtgaagcc acagatgta 19
<210> 172
<211> 119
<212> DNA
<213> Artificial sequence
<400> 172
tgcctactgc ctcggacttc aaggggctac tttaggagca attatcttgt ttactaaaac 60
tgaatacctt gctatctctt tgatacattt ttacaaagct gaattaaaat ggtataaat 119
<210> 173
<211> 22
<212> DNA
<213> Artificial sequence
<400> 173
ccatagctca gtctggtcta tc 22
<210> 174
<211> 22
<212> DNA
<213> Artificial sequence
<400> 174
tcaggatgat ctggacgaag ag 22
<210> 175
<211> 20
<212> DNA
<213> Artificial sequence
<400> 175
ccggtcctgg actttgtctc 20
<210> 176
<211> 20
<212> DNA
<213> Artificial sequence
<400> 176
ctcgacatcg gcaaggtgtg 20
<210> 177
<211> 20
<212> DNA
<213> Artificial sequence
<400> 177
cgcattggag tcgctttaac 20
<210> 178
<211> 24
<212> DNA
<213> Artificial sequence
<400> 178
cgagctgcaa gaactcttcc tcac 24
<210> 179
<211> 23
<212> DNA
<213> Artificial sequence
<400> 179
cacggcactt acctgtgttc tgg 23
<210> 180
<211> 23
<212> DNA
<213> Artificial sequence
<400> 180
cagtacaggc atccctgtga aag 23
<210> 181
<211> 590
<212> DNA
<213> Artificial sequence
<400> 181
cccctctccc tccccccccc ctaacgttac tggccgaagc cgcttggaat aaggccggtg 60
tgcgtttgtc tatatgttat tttccaccat attgccgtct tttggcaatg tgagggcccg 120
gaaacctggc cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg 180
aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct ctggaagctt cttgaagaca 240
aacaacgtct gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct 300
ctgcggccaa aagccacgtg tataagatac acctgcaaag gcggcacaac cccagtgcca 360
cgttgtgagt tggatagttg tggaaagagt caaatggctc tcctcaagcg tattcaacaa 420
ggggctgaag gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggtg 480
cacatgcttt acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg 540
ggacgtggtt ttcctttgaa aaacacgatg ataatatggc cacaaccatg 590
<210> 182
<211> 23
<212> DNA
<213> Artificial sequence
<400> 182
cgcgagcaca gctaaggcca cgg 23
<210> 183
<211> 23
<212> DNA
<213> Artificial sequence
<400> 183
actctctctt tctggcctgg agg 23
<210> 184
<211> 23
<212> DNA
<213> Artificial sequence
<400> 184
acccagcagg gcgtggagcc agg 23
<210> 185
<211> 23
<212> DNA
<213> Artificial sequence
<400> 185
gtcagagccc caaggtaaaa agg 23
Claims (21)
1. A pluripotent stem cell or a derivative thereof comprising an expression sequence for an IL-6 blocker,
the IL-6 blocker is an IL-6 antibody and/or an IL-6 receptor antibody;
the expression sequence of the IL-6 blocker is preferably inserted in the genome of the pluripotent stem cell or a derivative thereof.
2. A pluripotent stem cell or a derivative thereof comprising an expression sequence of an IL-6 blocker,
the IL-6 blocker is an IL-6 antibody and/or an IL-6 receptor antibody;
the expression sequence of the IL-6 blocker is preferably inserted in the genome of the pluripotent stem cell or the derivative thereof;
the B2M gene and/or CIITA gene of the genome of the pluripotent stem cell or the derivative thereof is knocked out.
3. A pluripotent stem cell or a derivative thereof comprising an expression sequence for an IL-6 blocker,
the IL-6 blocker is an IL-6 antibody and/or an IL-6 receptor antibody;
the expression sequence of the IL-6 blocker is preferably inserted in the genome of the pluripotent stem cell or the derivative thereof;
the pluripotent stem cells or the derivatives thereof also comprise an immune compatible molecule expression sequence, and the immune compatible molecule is used for regulating and controlling the expression of genes related to immune response in the pluripotent stem cells or the derivatives thereof;
the immune compatible molecule expression sequence is preferably inserted in the genome of the pluripotent stem cell or a derivative thereof.
4. A pluripotent stem cell or a derivative thereof comprising an expression sequence for an IL-6 blocker,
the IL-6 blocker is an IL-6 antibody and/or an IL-6 receptor antibody;
the expression sequence of the IL-6 blocker is preferably inserted in the genome of the pluripotent stem cell or the derivative thereof;
the pluripotent stem cells or the derivatives thereof also comprise an immune compatible molecule expression sequence, and the immune compatible molecule is used for regulating and controlling the expression of genes related to immune response in the pluripotent stem cells or the derivatives thereof;
the immune compatible molecule expression sequence is preferably inserted into the genome of the pluripotent stem cell or a derivative thereof;
the pluripotent stem cell or the derivative thereof further comprises an inducible gene expression system;
the inducible gene expression system is preferably inserted into the genome of the pluripotent stem cell or a derivative thereof.
5. The pluripotent stem cell or the derivative thereof according to claim 4, wherein the inducible gene expression system is at least one of a Tet-Off system and a dimer inducible expression system.
6. The pluripotent stem cell or derivative thereof of claim 3 or 4, wherein the immune-compatible molecule comprises one or more of:
(I) immune tolerance-related genes including CD47 or HLA-G;
(II) HLA-C molecules comprising HLA-C alleles in a proportion of more than 90% in total in the population, or fusion protein genes consisting of more than 90% of HLA-C alleles and B2M;
(III) shRNA and/or shRNA-miR targeting the gene associated with the immune response.
7. The pluripotent stem cell or the derivative thereof according to claim 3 or 4, wherein the genes associated with the immune response comprise:
major histocompatibility complex genes including at least one 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-DPB 1;
(II) major histocompatibility complex related genes comprising at least one of B2M and CIITA.
8. The pluripotent stem cell or the derivative thereof according to claim 6,
the target sequence of the shRNA and/or shRNA-miR targeting B2M is selected from one of SEQ ID NO. 6-SEQ ID NO. 8;
the target sequence of the shRNA and/or shRNA-miR of the targeting CIITA is selected from one of SEQ ID NO. 9-SEQ ID NO. 18;
the target sequence of the shRNA and/or shRNA-miR of the target HLA-A is selected from one of SEQ ID NO. 19-SEQ ID NO. 21;
the target sequence of the shRNA and/or shRNA-miR of the target HLA-B is selected from one of SEQ ID NO. 22-SEQ ID NO. 27;
the target sequence of the target HLA-C shRNA and/or shRNA-miR is selected from one of SEQ ID NO. 28-SEQ ID NO. 33;
the target sequence of the shRNA and/or shRNA-miR of the target HLA-DRA is selected from one of SEQ ID NO. 34-SEQ ID NO. 43;
the target sequence of the shRNA and/or shRNA-miR of the target HLA-DRB1 is selected from one of SEQ ID NO. 44-SEQ ID NO. 48;
the target sequence of the shRNA and/or shRNA-miR of the target HLA-DRB3 is selected from one of SEQ ID NO. 49-SEQ ID NO. 50;
the target sequence of the shRNA and/or shRNA-miR of the target HLA-DRB4 is selected from one of SEQ ID NO. 51-SEQ ID NO. 60;
the target sequence of the shRNA and/or shRNA-miR of the target HLA-DRB5 is selected from one of SEQ ID NO. 61-SEQ ID NO. 69;
the target sequence of the shRNA and/or shRNA-miR of the target HLA-DQA1 is selected from one of SEQ ID NO. 70-SEQ ID NO. 76;
the target sequence of the shRNA and/or shRNA-miR of the target HLA-DQB1 is selected from one of SEQ ID NO. 77-SEQ ID NO. 86;
the target sequence of the shRNA and/or shRNA-miR of the target HLA-DPA1 is selected from one of SEQ ID NO. 87-SEQ ID NO. 96;
the target sequence of the shRNA and/or shRNA-miR of the target HLA-DPB1 is selected from one of SEQ ID NO. 97-SEQ ID NO. 106.
9. The pluripotent stem cell or the derivative thereof according to claim 3 or 4, wherein at least one of an shRNA processing complex-associated gene, an miRNA processing complex-associated gene, and an anti-interferon effector molecule is further introduced into the genome of the pluripotent stem cell or the derivative thereof.
10. The pluripotent stem cell or the derivative thereof according to claim 9, wherein the shRNA-processing complex-associated gene or miRNA-processing complex-associated gene comprises at least one of Drosha, Ago1, Ago2, Dicer1, Exportin-5, TRBP (TARBP2), PACT (PRKRA), DGCR 8; the anti-interferon effector molecule is preferably shRNA and/or shRNA-miR targeting at least one of PKR, 2-5As, IRF-3 and IRF-7.
11. The pluripotent stem cell or the derivative thereof according to claim 10,
the target sequence of the shRNA and/or shRNA-miR of the target PKR is selected from one of SEQ ID NO. 107-SEQ ID NO. 116;
the target sequence of the shRNA and/or shRNA-miR targeting 2-5As is selected from one of SEQ ID NO. 117-SEQ ID NO. 146;
the target sequence of the shRNA and/or shRNA-miR of the targeted IRF-3 is selected from one of SEQ ID NO. 147-SEQ ID NO. 156;
the target sequence of the IRF-7-targeting shRNA and/or shRNA-miR is selected from one of SEQ ID NO. 157-SEQ ID NO. 166.
12. The pluripotent stem cell or the derivative thereof according to claim 6 or 9, wherein the pluripotent stem cell or the derivative thereof,
the shRNA expression framework is as follows: the gene sequence sequentially comprises an shRNA target sequence, a stem-loop sequence, a reverse complementary sequence of the shRNA target sequence and Poly T from 5 'to 3';
wherein the shRNA target sequence, the stem-loop sequence and the reverse complementary sequence of the shRNA target sequence form a hairpin structure; poly T is a transcription terminator of RNA polymerase III;
shRNA-miR expression framework: replacing a target sequence in the microRNA-30 or microRNA-155 with a shRNA-miR target sequence to obtain the target sequence.
13. The pluripotent stem cell or the derivative thereof according to claim 12, wherein the stem-loop sequence in the shRNA expression framework is 3 to 9 bases in length; the length of the Poly T is 5-6 bases.
14. The pluripotent stem cell or the derivative thereof according to any one of claims 1to 4, wherein the IL-6 blocker expression sequence, the immune-compatible molecule expression sequence or the inducible gene expression system is inserted into a safe site in the genome of the pluripotent stem cell or the derivative thereof.
15. The pluripotent stem cell or the derivative thereof of claim 14, wherein the genomic safety site comprises one or more of an AAVS1 safety site, an eGSH safety site, and an H11 safety site.
16. The pluripotent stem cell or the derivative thereof according to any one of claims 1to 4, wherein the pluripotent stem cell comprises an embryonic stem cell, an embryonic germ cell, an embryonic carcinoma cell, or an induced pluripotent stem cell.
17. The pluripotent stem cell or the derivative thereof according to any one of claims 1to 4, wherein the derivative of the pluripotent stem cell comprises an adult stem cell, each germ layer cell or tissue into which the pluripotent stem cell is differentiated;
the adult stem cells include mesenchymal stem cells or neural stem cells.
18. The pluripotent stem cell or the derivative thereof according to any one of claims 1to 4, wherein the IL-6 antibody has a heavy chain sequence as shown in SEQ ID No.1 and a light chain sequence as shown in SEQ ID No. 2.
19. The pluripotent stem cell or the derivative thereof according to any one of claims 1to 4, wherein the IL-6 receptor antibody has a heavy chain sequence as shown in SEQ ID No.3 and a light chain sequence as shown in SEQ ID No. 4.
20. Use of the pluripotent stem cell or the derivative thereof according to any one of claims 1to 19 for the preparation of a medicament for the treatment of a disease, wherein the disease is at least one of rheumatoid arthritis, depression and anxiety.
21. A formulation comprising the pluripotent stem cells or derivatives thereof of any of claims 1to 19.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104471059A (en) * | 2012-07-12 | 2015-03-25 | 爱姆斯坦生物技术公司 | Mesenchymal-like stem cells derived from human embryonic stem cells, methods and uses thereof |
| CN108368520A (en) * | 2015-11-04 | 2018-08-03 | 菲特治疗公司 | The genome project of pluripotent cell is transformed |
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2020
- 2020-10-30 CN CN202011190618.0A patent/CN114457033A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104471059A (en) * | 2012-07-12 | 2015-03-25 | 爱姆斯坦生物技术公司 | Mesenchymal-like stem cells derived from human embryonic stem cells, methods and uses thereof |
| CN108368520A (en) * | 2015-11-04 | 2018-08-03 | 菲特治疗公司 | The genome project of pluripotent cell is transformed |
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