Induced pluripotent stem cell and application thereof

Abstract

The invention discloses an induced pluripotent stem cell and application thereof, IPEX-iPSCs cell (1) carries a specific mutation site of an IPEX syndrome patient, namely carries an FOXP3 specific mutation site (c.694Tgt; c, p.C232R), which can reflect disease characteristics; (2) infinite culture amplification and passage in vitro can be realized, so that research and use are facilitated; and (3) insertion of an exogenous reprogramming vector is not contained, so that a guarantee is provided for the safety of clinical application in the future.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to an induced pluripotent stem cell and an application thereof. Background Art

[0002] IPEX syndrome is a rare X-linked genetic disease that combines immune dysfunction, polyendocrinopathy, and enteropathy. The disease is characterized by abnormal regulation of the immune system, dysfunction of multiple endocrine glands, and inflammatory lesions of the intestine. According to a study published by Dotta and Vendrame in 2002, IPEX syndrome is usually associated with polyendocrinopathy in the neonatal period. The disease is mostly found in male infants under six months old, and its clinical symptoms include severe watery diarrhea, type 1 diabetes (requiring insulin treatment), and dermatitis. As the disease progresses, patients may develop systemic autoimmune problems. Usually, three main symptoms including severe watery diarrhea, eczematous dermatitis, and endocrine abnormalities (especially type 1 diabetes) can be used as indicators for the diagnosis of this disease.

[0003] IPEX syndrome is a rare X-linked genetic disease that combines immune regulation, polyendocrinopathy, and enteropathy, manifesting as abnormal regulation of the immune system, dysfunction of multiple endocrine glands, and inflammatory damage to the intestine. According to an article published by Dotta and Vendrame in 2002, this syndrome has been generally regarded as a syndrome caused by polyendocrinopathy in the neonatal period. It is suspected that the occurrence of this syndrome is mostly clinical symptoms presented in male infants less than six months old, including severe watery diarrhea, insulin-dependent type 1 diabetes mellitus (IDDM), and dermatitis during this growth stage. This disease will develop systemic autoimmunity problems clinically. Generally speaking, three symptoms can be observed as indicative considerations: severe watery diarrhea, eczematous dermatitis, and endocrine abnormalities, which are mostly regarded as insulin-dependent diabetes. Most IPEX syndrome patients also have other autoimmune phenomena, including Coombs positive anemia, autoimmune thrombocytopenia, autoimmune low neutrophilia, and tubular nephropathy. Currently, IPEX syndrome is mainly supported by immunosuppressants and control of diabetes and autoimmune thyroid disease in clinical treatment, but the prognosis of patients is not ideal with these treatments alone. Hematopoietic stem cell transplantation is the only possible means of cure, but patients must face the risk of rejection after bone marrow transplantation and the possibility that only a few patients can survive for a long time. Summary of the invention

[0004] The present invention is accomplished based on the following findings of the inventors:

[0005] Reprogramming human somatic cells into induced pluripotent stem cells (iPSCs) is a new strategy for studying human development, the pathological mechanisms of genetic diseases, and future personalized stem cell therapy in medicine. However, the establishment of iPSCs specific to IPEX syndrome patients has not been reported so far, so there is no ideal in vitro model to study the progression and pathological mechanisms of this genetic disease. For future personalized stem cell therapy in medicine, there is also a lack of cell sources and treatment plans specific to individual IPEX syndrome patients.

[0006] It has been proven that FOXP3 gene defects cause abnormalities in the number and function of Treg cells, but the specific mechanism is still unclear and cannot reflect the specific pathophysiological process of the gene in autoimmunity, endocrine diseases, and enteropathy. IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) syndrome is a rare hereditary immune dysregulation disease caused by FOXP3 gene mutations.

[0007] The present invention aims to solve one of the technical problems in the related art at least to a certain extent. To this end, the present invention provides an induced pluripotent stem cell and its application.

[0008] An induced pluripotent stem cell of the present invention has been deposited in the China Center for Type Culture Collection (CCTCC), address: Wuhan University, Wuhan, China, with a deposit date of December 12, 2024, a deposit number of CCTCC NO: C2024138, and a classification name: human pluripotent stem cells IPEX-iPSCs Homo sapiens.

[0009] In a first aspect of the present invention, the present invention provides an induced pluripotent stem cell, wherein the induced pluripotent stem cell carries a mutation site specific to an IPEX syndrome patient.

[0010] Preferably, the IPEX syndrome patient-specific mutation site is a FOXP3-specific mutation site.

[0011] Preferably, the specific mutation sites are c.694T>C, p.C232R.

[0012] Preferably, the nucleotide sequence of FOXP3 carried by the induced pluripotent stem cells is as shown in SEQ ID NO:1.

[0013] Preferably, the amino acid sequence of FOXP3 carried by the induced pluripotent stem cells is as shown in SEQ ID NO:2.

[0014] Preferably, the induced pluripotent stem cells are derived from human blood cells.

[0015] Preferably, the induced pluripotent stem cells are derived from PBMC cells.

[0016] In a second aspect of the present invention, the present invention provides an induced pluripotent stem cell, wherein the induced pluripotent stem cell carries a FOXP3-specific mutation site.

[0017] Preferably, the induced pluripotent stem cells are IPEX syndrome patient-specific cells.

[0018] Preferably, the specific mutation sites are c.694T>C, p.C232R.

[0019] Preferably, the nucleotide sequence of FOXP3 carried by the induced pluripotent stem cells is as shown in SEQ ID NO:1.

[0020] Preferably, the amino acid sequence of FOXP3 carried by the induced pluripotent stem cells is as shown in SEQ ID NO:2.

[0021] Preferably, the induced pluripotent stem cells are derived from human blood cells.

[0022] Preferably, the induced pluripotent stem cells are derived from PBMC cells.

[0023] In the third aspect of the present invention, the present invention provides the following applications of the above-mentioned induced pluripotent stem cells:

[0024] Application in building disease models;

[0025] Application in the preparation of drugs for treating diseases;

[0026] Application in screening candidate drugs for treating diseases;

[0027] or in the evaluation of drugs for the treatment of diseases;

[0028] Application in cell therapy of diseases;

[0029] Applications in cell transplantation and tissue repair;

[0030] Application in organ regeneration;

[0031] Application in the preparation of preparations targeting tumor lesions in vivo;

[0032] Or its application in anti-tumor therapy with immune cells.

[0033] In the fourth aspect of the present invention, the present invention proposes an IPEX syndrome in vitro model cell, which includes induced pluripotent stem cells, wherein the induced pluripotent stem cells carry FOXP3-specific mutation sites, and the specific mutation sites are c.694T>C, p.C232R.

[0034] The IPEX-iPSCs cells of the present invention (1) carry IPEX syndrome patient-specific mutation sites, namely, carry FOXP3-specific mutation sites (c.694T>C, p.C232R), which can reflect the characteristics of the disease; (2) can be cultured, amplified and passaged indefinitely in vitro, which is convenient for research and use; (3) do not contain the insertion of exogenous reprogramming vectors, which provides a guarantee for the safety of future clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the morphology of IPEX-iPSCs cells;

[0036] Figure 2 The results of sequencing the FOXP3 gene mutation site c.694T>C (p.C232R) in IPEX-iPSCs cells, where a is the sequencing result of normal iPSCs cells and b is the sequencing result of IPEX-iPSCs cells;

[0037] Figure 3 This is the result of immunofluorescence staining of IPEX-iPSCs cells;

[0038] Figure 4 is the mRNA expression of pluripotency markers of healthy control-iPSc and IPES-iPSc;

[0039] Figure 5 This is the result of chromosome karyotype analysis of IPEX-iPSCs cells;

[0040] Figure 6 The three-germ layer differentiation results of teratomas obtained from IPEX-iPSCs cells and teratoma sections stained, where a is IPEX-iPSCs teratoma, b is endoderm, c is mesoderm, and d is cortex. DETAILED DESCRIPTION

[0041] The present invention is further described below in conjunction with specific examples, which are only used to explain the present invention and are not to be construed as limiting the present invention. It will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to these embodiments without departing from the principles and purposes of the present invention, and that the scope of the present invention is defined by the claims and their equivalents. The experimental methods for which specific conditions are not specified in the following examples are usually tested under conventional conditions or under conditions recommended by the manufacturer.

[0042] The present invention obtains IPEX syndrome-specific induced pluripotent stem cells (IPEX-iPSCs cells for short) by reprogramming human blood cells, especially PBMCs derived from IPEX patients. The IPEX-iPSCs cells of the present invention carry FOXP3-specific mutation sites (c.694T>C, p.C232R), and the IPEX-iPSCs cells of the present invention have many applications, including but not limited to applications in constructing disease models, applications in preparing drugs for treating diseases, applications in screening candidate drugs for treating diseases, or applications in drug evaluation for treating diseases, applications in cell therapy for diseases, applications in cell transplantation and tissue repair, applications in organ regeneration, applications in preparing preparations targeting tumor lesions in vivo, or applications in immune cell anti-tumor therapy.

[0043] In particular, the IPEX syndrome in vitro model cell of the present invention includes an induced pluripotent stem cell, which carries a FOXP3 specific mutation site, and the specific mutation site is c.694T>C, p.C232R. The IPEX-iPSCs cell of the present invention is used as an in vitro model cell of IPEX syndrome, and is used as a basic research material for IPEX syndrome in vitro. The characteristics of IPEX-iPSCs that can be infinitely amplified in vitro and induce hematopoietic differentiation under appropriate conditions can be used to study the disease progression process and pathological mechanism of human IPEX syndrome in vitro. Compared with the traditional method of obtaining disease-specific cells from patients, it brings great convenience to scientific research materials, and provides a new platform and new perspective for studying the pathogenesis of IPEX. In addition, the IPEX-iPSCs of the present invention can also provide support for individual-specific cell sources and treatment plans for IPEX syndrome patients during medical personalized stem cell therapy.

[0044] The reagents and consumables used in Examples 1-5 of the present invention are as follows:

[0045] iPSCs reprogramming kit (manufacturer: Beijing Saibei, catalog number: CA5002002), which includes reprogramming additive I, reprogramming additive II, reprogramming additive III, Reproeasy human cell reprogramming medium, PGM1 human pluripotent stem cell medium, PSCeasy human pluripotent stem cell recovery medium, PSCeasy human pluripotent stem cell digestion solution, PSCeasy human pluripotent stem cell base working solution, and human induced pluripotent stem cells;

[0046] Pluripotent stem cell 4-marker immunocytochemistry kit (Invitrogen, catalog number: A24881);

[0047] Virus genomic DNA / RNA extraction kit (TIANGEN, catalog number: #DP315);

[0048] SCF (stem cell factor) (manufacturer: PeproTech, catalog number: 300-07-50UG);

[0049] FLT-3 (Flt3 ligand) (Manufacturer: PeproTech, Catalog No.: 300-19-50UG);

[0050] IL-3 (interleukin 3) (manufacturer: Biolegend, catalog number: 578002);

[0051] IL-6 (interleukin 6) (manufacturer: PeproTech, catalog number: 200-06);

[0052] TB Green (Takara, catalog number: RR820A);

[0053] RNase free water (Takara, product number: RT121-02)

[0054] Example 1 Preparation of IPEX-iPSCs cells

[0055] The reprogramming technology of the present invention is not limited to the use of free vectors for reprogramming, Sendai virus (SeV) for reprogramming, and the like. In this Example 1, a reprogramming kit is used to reprogram PBMC cells from IPEX patients to obtain IPEX-iPSCs cells through reprogramming technology.

[0056] The PBMCs used in the present invention are derived from the blood of an IPEX patient, and the specific information of the IPEX patient is as follows: male, 7 years old, diagnosed with IPEX syndrome.

[0057] The IPEX-iPSCs cell preparation process of the present invention is as follows:

[0058] (1) Day-4: Count the number of PBMCs from the patient and adjust the number of cells to 1×10 using PBMCs culture medium. 6 / mL, inoculated in 1 well of a 24-well plate;

[0059] (2) Day-3 to Day-1: Observe the cell status and change half of the PBMCs culture medium daily;

[0060] Among them, the PBMCs culture medium is: Reproeasy human cell reprogramming medium + 100ng / mL SCF + 100ng / mL FLT-3 + 20ng / mL IL-3 + 20ng / mL IL-6; for example, to prepare 1mL of PBMCs culture medium: take 1mL of Reproeasy human cell reprogramming medium, 100ng SCF, 100ng FLT-3, 20ng IL-3, 20ng IL-6, and mix well;

[0061] (3) Day 0: Observe the cells under a microscope to ensure that the cells are in good condition. After counting the cells, take 5×10 5 The PBMCs were resuspended in 200 μL reprogramming medium A, mixed evenly and seeded into 96-well plates (1×10 4 -2×10 4 ), and returned to the 37°C constant temperature incubator for culture;

[0062] Among them, reprogramming medium A is: 2mL Reproeasy human cell reprogramming medium + 2μL reprogramming additive I + 200ng / mL SCF + 200ng / mL FLT-3 + 40ng / mL IL-3 + 40ng / mL IL-6. For example, to prepare about 2mL of reprogramming medium A: take 2mL Reproeasy human cell reprogramming medium, 2μL reprogramming additive I, 400ng SCF, 400ng FLT-3, 40ng IL-3, 40ng IL-6, and mix well;

[0063] (4) Day 1: Observe the cells under a microscope, carefully aspirate the supernatant, and add 200 μL of reprogramming medium A to continue culturing;

[0064] (5) Day 2: Add 5 μL of reprogramming additive II to 100 μL of reprogramming medium A, mix well to make reprogramming medium B, add reprogramming medium B to a 96-well plate where the cell nucleus has enlarged and changed from round to spindle-shaped, and place the culture plate back into the incubator;

[0065] (6) Day 4: Prepare reprogramming medium C: 2 mL Reproeasy human cell reprogramming medium + 1 μL reprogramming supplement III. After the cells have grown to full size in the 96-well plate, transfer them to one well of a 12-well plate (with Matrigel laid in advance) to try to form single cells attached to the wall;

[0066] (7) Day 5: Remove Reprogramming Medium C and replace with 1 ml of fresh Reproeasy Human Cell Reprogramming Medium (without cytokines) and continue culturing;

[0067] (8) Day 6: Observe the cells under a microscope, carefully discard 500 μL of the original culture medium, and add 500 μL of fresh Reproeasy human cell reprogramming medium (without cytokines) to continue culturing;

[0068] (9) Day 7: Observe the cells under a microscope, carefully discard the original culture medium, add 500 μL of fresh Reproeasy human cell reprogramming medium (without cytokines) + 500 μL PGM1 human pluripotent stem cell medium and continue culturing;

[0069] (10) Day 8-20: Observe the cells under a microscope every day and replace with fresh PGM1 human pluripotent stem cell culture medium. After the cell clones grow, pick out single clones for expansion culture and cryopreservation.

[0070] The morphology of the IPEX-iPSCs cells of the present invention after stable passage and culture for 3 days is shown in the figure below. Figure 1 shown.

[0071] Gene sequencing of FOXP3 of normal iPSCs cells and IPEX-iPSCs cells of the present invention is performed, such as Figure 2 As shown, Figure 2 a in the figure is the sequencing result of normal iPSCs cells, and b is the sequencing result of IPEX-iPSCs cells; Figure 2 It can be seen from the figures that the IPEX-iPSCs cells of the present invention carry IPEX syndrome patient-specific mutation sites, specifically, the FOXP3 gene mutation site of the IPEX-iPSCs cells of the present invention is c.694T>C (p.C232R), that is, the 694th nucleotide in the nucleotide sequence mutates from thymine (T) to cytosine (C), and in the protein sequence, the 232nd amino acid mutates from cysteine ​​(C) to arginine (R).

[0072] The full-length nucleotide sequence of FOXP3 carried by the IPEX-iPSCs cells of the present invention is shown in SEQ ID NO: 1:

[0073] AGTTTCCCACAAGCCAGGCTGATCCTTTTCTGTCAGTCCACTTCACCAAGC

[0074] CTGCCCTTGGACAAGGACCCGATGCCCAACCCCAGGCCTGGCAAGCCCTC

[0075] GGCCCCTTCCTTGGCCCTTGGCCATCCCCAGGAGCCCTCGCCCAGCTGGAG

[0076] GGCTGCACCCAAAGCCTCAGACCTGCTGGGGGCCCGGGGCCCAGGGGGA

[0077] ACCTTCCAGGGCCGAGATCTTCGAGGCGGGGCCCATGCCTCCTCTTCTTCC

[0078] TTGAACCCCATGCCACCATCGCAGCTGCAGCTCTCAACGGTGGATGCCCAC

[0079] GCCCGGACCCCTGTGCTGCAGGTGCACCCCCTGGAGAGCCCAGCCATGAT

[0080] CAGCCTCACACCACCCACCACCGCCACTGGGGTCTTCTCCCTCAAGGCCC

[0081] GGCCTGGCCTCCCACCTGGGATCAACGTGGCCAGCCTGGAATGGGTGTCC

[0082] AGGGAGCCGGCACTGCTCTGCACCTTCCCAAATCCCAGTGCACCCAGGAA

[0083] GGACAGCACCCTTTCGGCTGTGCCCCAGAGCTCCTACCCACTGCTGGCAAA

[0084] TGGTGTCTGCAAGTGGCCCGGATGTGAGAAGGTCTTCGAAGAGCCAGAGG

[0085] ACTTCCTCAAGCACTGCCAGGCGGACCATCTTCTGGATGAGAAGGGCAGG

[0086] GCACAA C GTCTCCTCCAGAGAGAGATGGTACAGTCTCTGGAGCAGCAGCT

[0087] GGTGCTGGAGAAGGAGAAGCTGAGTGCCATGCAGGCCCACCTGGCTGGG

[0088] AAAATGGCACTGACCAAGGCTTCATCTGTGGCATCATCCGACAAGGGCTCC

[0089] TGCTGCATCGTAGCTGCTGGCAGCCAAGGCCCTGTCGTCCCAGCCTGGTCT

[0090] GGCCCCCGGGAGGCCCCTGACAGCCTGTTTGCTGTCCGGAGGCACCTGTG

[0091] GGGTAGCCATGGAAACAGCACATTCCCAGAGTTCCTCCACAACATGGACTA

[0092] CTTCAAGTTCCACAACATGCGACCCCCTTTCACCTACGCCACGCTCATCCG

[0093] CTGGGCCATCCTGGAGGCTCCAGAGAAGCAGCGGACACTCAATGAGATCT

[0094] ACCACTGGTTCACACGCATGTTTGCCTTCTTCAGAAACCATCCTGCCACCT

[0095] GGAAGAACGCCATCCGCCACAACCTGAGTCTGCACAAGTGCTTTGTGCGG

[0096] GTGGAGAGCGAGAAGGGGGCTGTGTGGACCGTGGATGAGCTGGAGTTCC

[0097] GCAAGAAACGGAGCCAGAGGCCCAGCAGGTGTTCCAACCCTACACCTGGC

[0098] CCCTGACCTCAAGATCAAGGAAAGGAGGATGGACGAACAGGGGCCAAAC

[0099] TGGTGGGAGGCAGAGGTGGTGGGGGCAGGGATGATAGGCCCTGGATGTGC

[0100] CCACAGGGACCAAGAAGTGAGGTTTCCACTGTCTTGCCTGCCAGGGCCCC

[0101] TGTTCCCCCGCTGGCAGCCACCCCCTCCCCCATCATATCCTTTGCCCCAAGG

[0102] CTGCTCAGAGGGGCCCCGGTCCTGGCCCCAGCCCCCACCTCCGCCCCAGA

[0103] CACACCCCCCAGTCGAGCCCTGCAGCCAAACAGAGCCTTCACAACCAGCC

[0104] ACACAGAGCCTGCCTCAGCTGCTCGCACAGATTACTTCAGGGCTGGAAAA

[0105] GTCACACAGACACACAAAATGTCACAATCCTGTCCCTCACTCAACACAAA

[0106] CCCCAAAACACAGAGAGCCTGCCTCAGTACACTCAAACAACCTCAAAGCT

[0107] GCATCATCACACAATCACACACAAGCACAGCCCTGACAACCCACACACCC

[0108] CAAGGCACGCACCCACAGCCAGCCTCAGGGCCCACAGGGGCACTGTCAA

[0109] CACAGGGGTGTGCCCAGAGGCCTACACAGAAGCAGCGTCAGTACCCTCAG

[0110] GATCTGAGGTCCCAACACGTGCTCGCTCACACACACGGCCTGTTAGAATTC

[0111] ACCTGTGTATCTCACGCATATGCACACGCACAGCCCCCCAGTGGGTCTCTT

[0112] GAGTCCCGTGCAGACACACACAGCCACACACACTGCCTTGCCAAAAATAC

[0113] CCCGTGTCTCCCCTGCCACTCACCTCACTCCCATTCCCTGAGCCCTGATCCA

[0114] TGCCTCAGCTTAGACTGCAGAGGAACTACTCATTTATTTGGGATCCAAGGC

[0115] CCCCAACCCACAGTACCGTCCCCAATAAACTGCAGCCGAGTCCCCA

[0116] The full-length amino acid sequence is shown in SEQ ID NO: 2:

[0117] MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRG

[0118] GAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQ

[0119] LSTVDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLE

[0120] WVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPE

[0121] DFLKHCQADHLLDEKGRAQ R LLQREMVQSLEQQLVLEKEKLSAMQAHLAG

[0122] KMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGS

[0123] HGNSTFPEFLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEIYHWFT

[0124] RMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKRSQR

[0125] PSRCSNPTPGP

[0126] Example 2 Immunofluorescence identification

[0127] 1. Experimental methods

[0128] The IPEX-iPSCs of the present invention were seeded on a cell slide with a diameter of 1.5 cm pre-coated with matrix gel, with a seeding density of about 20% of the bottom area. When the cell fusion rate reached 60%-70%, immunofluorescence staining was performed using a pluripotent stem cell 4-marker immunocytochemistry kit.

[0129] 2. Experimental results

[0130] Immunofluorescence results (see Figure 3 ) showed that IPEX-iPSCs positively expressed multiple pluripotency marker proteins (including SOX2, OCT4, SSEA4 and TRA-1-60), indicating that the IPEX-iPSCs cells of the present invention have pluripotency.

[0131] Example 3 q-PCR detection

[0132] 1. Experimental methods

[0133] (1) cDNA of IPEX-iPSCs: reverse transcribed from RNA extracted using a viral genomic DNA / RNA extraction kit;

[0134] (2) qPCR reaction was performed using cDNA of IPEX-iPSCs. The primers were designed and synthesized by Shanghai Bioengineering. The sequences are shown in Table 1. The reaction system was prepared according to Table 2.

[0135] Table 1 Primer sequences for stem cell pluripotency markers

[0136]

[0137]

[0138] Table 2 qPCR reaction system

[0139]

[0140] The qPCR program is shown in Table 3:

[0141] Table 3 qPCR program

[0142]

[0143] (2) Data analysis: -ΔΔCt The relative expression level of the target gene was calculated by this method.

[0144] 2. Experimental results

[0145] mRNA expression of pluripotency markers in healthy control-iPSc and IPEX-iPSCs of the present invention Figure 4 As shown in the figure, qPCR results showed that multiple pluripotency marker genes (including SOX2, OCT4, DMT3B, ZFP42 and GABRB3) were positively expressed in IPEX-iPSCs. Among them, SOX2 and OCT4 are pluripotency transcription factors, DNMT3B is DNA methyltransferase 3B; ZFP42: zinc finger protein 42; GABRB3 is γ-aminobutyric acid receptor β3 subunit.

[0146] Example 4 Karyotype Detection

[0147] 1. Experimental methods

[0148] Collect IPEX-iPSCs cells, digest and centrifuge, and take about 1×10 6 The cells were resuspended in 10 mL PGM1 culture medium and transferred to a centrifuge tube; the samples were sent for external inspection at the Molecular Center of the Children's Hospital Affiliated to Chongqing Medical University.

[0149] 2. Experimental results

[0150] The results of karyotype detection of IPEX-iPSCs cells are shown in Figure 5 The test results showed that the chromosome number was normal and there was no abnormality in the chromosome structure, indicating that IPEX-iPSCs cells did not have chromosomal aberrations and all showed normal karyotypes.

[0151] Example 5 Identification of teratoma formation

[0152] 1. Experimental methods

[0153] (1) Prepare 1×10 IPEX-iPSCs cells 6 cells / mouse, injected subcutaneously in the groin of mice, with an injection volume of 100 μL / spot / mouse, and observed for 4 weeks after injection. The observation contents included the tumor formation at the transplanted site of the mice and the general condition of the mice;

[0154] (2) After the animals were euthanized, the tumors were removed, samples were collected, the tumors were weighed, and pathological histological examinations were performed on the tumors.

[0155] 2. Experimental results

[0156] The results of IPEX-iPSCs teratoma section staining are shown in Figure 6 , indicating that IPEX-iPSCs also have the potential to differentiate into the three germ layers in vivo.

[0157] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0158] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. An induced pluripotent stem cell, characterized in that The induced pluripotent stem cells carry IPEX syndrome patient-specific mutation sites.

2. The induced pluripotent stem cell according to claim 1, characterized in that The IPEX syndrome patient-specific mutation site is a FOXP3-specific mutation site.

3. The induced pluripotent stem cell according to claim 2, characterized in that The specific mutation sites are c.694T>C, p.C232R.

4. The induced pluripotent stem cell according to claim 2 or 3, characterized in that The nucleotide sequence of the FOXP3 carried by the induced pluripotent stem cells is shown as SEQ ID NO: 1; or the amino acid sequence of the FOXP3 carried by the induced pluripotent stem cells is shown as SEQ ID NO:

2.

5. The induced pluripotent stem cell according to any one of claims 1 to 3, characterized in that: The induced pluripotent stem cells are derived from human blood cells.

6. The induced pluripotent stem cell according to claim 5, characterized in that The induced pluripotent stem cells are derived from PBMC cells.

7. An induced pluripotent stem cell, characterized in that: The induced pluripotent stem cells carry a FOXP3-specific mutation site.

8. The induced pluripotent stem cell according to claim 7, characterized in that The induced pluripotent stem cells are IPEX syndrome patient-specific cells.

9. The use of the induced pluripotent stem cell according to any one of claims 1 to 8, characterized in that: Application in building disease models; Application in the preparation of drugs for treating diseases; Application in screening candidate drugs for treating diseases; or in the evaluation of drugs for the treatment of diseases; Application in cell therapy of diseases; Applications in cell transplantation and tissue repair; Application in organ regeneration; Application in the preparation of preparations targeting tumor lesions in vivo; Or its application in anti-tumor therapy with immune cells.

10. An in vitro model cell for IPEX syndrome, characterized in that: It includes induced pluripotent stem cells, which carry FOXP3 specific mutation sites, and the specific mutation sites are c.694T>C, p.C232R.

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