NFAT1-based induced pluripotent stem cell construction method and application of NFAT1-based induced pluripotent stem cell in smoke disease

Abstract

The invention discloses a construction method of induced pluripotent stem cells based on NFAT1 and application of the induced pluripotent stem cells in smoke diseases. The invention provides a construction method of induced pluripotent stem cells for preparing a smoke disease model, which comprises the following steps of: reprogramming CD34 + cells in in-vitro human PBMCs (OCT4 gene, SOX2 gene, KLF4 gene, c-MYC gene and NFAT1 gene are jointly introduced into the CD34 + cells); the obtained induced pluripotent stem cells are the induced pluripotent stem cells for preparing the smoke disease model. The invention constructs a pluripotent stem cell model capable of accurately simulating the pathological characteristics of smoke disease by introducing NFAT1. The model provides a brand new technical means for pathogenesis research, drug screening and personalized treatment of smoke diseases, and has important application value in the fields of disease model construction, tissue repair, precision medical treatment and the like.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to a method for constructing induced pluripotent stem cells based on NFAT1 and its application in moyamoya disease. Background Art

[0002] Moyamoya disease is a chronic cerebrovascular occlusive disease, the main characteristics of which are the slow progressive stenosis or occlusion of the bilateral internal carotid artery termini and / or the origins of the anterior cerebral artery and middle cerebral artery, accompanied by the formation of abnormal capillary networks at the base of the skull. The etiology and pathogenesis of this disease have not been fully elucidated, and there are many problems and deficiencies in the establishment of existing disease models related to moyamoya disease. Although current research on moyamoya disease can effectively induce pluripotency, it fails to fully simulate its specific pathological characteristics, especially the abnormalities related to angiogenesis. This limitation affects the application effect of iPSCs in the study of the mechanism of moyamoya disease.

[0003] NFAT1 is a member of the nuclear factor of activated T cell family and plays an important regulatory role in the body's immune response, gene transcription, etc. The NFAT1 protein is activated under the stimulation of calcium / calcineurin (Ca 2+ / calcineurin), translocates to the nucleus after dephosphorylation and binds to specific sequences of DNA, regulating the gene expression of cells and participating in the regulation of various biological functions such as tumor immunity, myocardial function regulation, and apoptosis. Under physiological conditions, NFAT1 plays a key role in the regulation and homeostasis of the body. However, when there is a p.R4810K mutation at the susceptibility gene locus for moyamoya disease RNF213 , the ubiquitination ability of the RNF213 protein, which is an E3 ubiquitin ligase, is weakened, resulting in a decrease in the ubiquitination and degradation of NFAT1 and an increase in the nuclear NFAT1 level; after the ubiquitination is restored, the NFAT1 level returns to normal. The prior art has not explored RNF213 the impact of this interaction with NFAT1 on the pathogenesis of moyamoya disease, resulting in insufficient in-depth research. Based on the above background, there is an urgent need to develop an improved method for constructing iPSCs, by introducing the NFAT1 regulatory mechanism, to better simulate the molecular pathological characteristics of moyamoya disease, so as to provide a new scientific basis for the research and treatment of the disease.

[0004] Difficulties in the prior art: The complexity of multi-factor interaction is a significant challenge. RNF213The interaction with NFAT1 involves a complex signaling network, and it is difficult for the prior art to accurately simulate this complexity in in vitro models. In addition, while improving the reprogramming efficiency of iPSCs, how to ensure that the constructed model has a high degree of disease specificity remains a technical problem. More importantly, current technologies are difficult to create an iPSC model that fully reflects the characteristics of moyamoya disease, especially the insufficient simulation of key pathological features such as abnormal angiogenesis, which significantly limits the progress of disease mechanism research and drug screening. Summary of the Invention

[0005] The object of the present invention is to provide a method for constructing induced pluripotent stem cells based on NFAT1 and its application in moyamoya disease.

[0006] In a first aspect, the present invention claims to protect a method for constructing induced pluripotent stem cells for preparing a moyamoya disease model.

[0007] The method for constructing induced pluripotent stem cells for preparing a moyamoya disease model claimed by the present invention may include the following steps: reprogramming CD34 + cells in isolated human PBMCs, and the obtained induced pluripotent stem cells are the induced pluripotent stem cells for preparing a moyamoya disease model; The reprogramming is to OCT4 gene, SOX2 gene, KLF4 gene, c-MYC gene and NFAT1 gene are co-introduced into the CD34 + cells and cultured until the induced pluripotent stem cells are obtained.

[0008] Further, in the method, OCT4 gene, SOX2 gene, KLF4 gene, c-MYC gene and NFAT1 gene are introduced into the CD34 + cells in the form of a recombinant vector; the recombinant vector is five recombinant plasmids obtained by cloning OCT4 gene, SOX2 gene, KLF4 gene, c-MYC gene and NFAT1 gene into the pCXWB-EBNA1 vector respectively. Specifically, the recombinant vector is to OCT4 gene, SOX2 gene, KLF4 gene, c-MYC gene and NFAT1Five recombinant plasmids obtained by separately replacing small fragments between two EcoR I sites in the pCXWB-EBNA1 vector with genes.

[0009] Furthermore, the CD34 + cells can be obtained by the following method: inoculating ex vivo human PBMCs into a CD34 + cell expansion medium for culture (changing the medium every 2 days regularly) to promote the proliferation of CD34 + cells; the CD34 + cell expansion medium is StemSpan SFEM Ⅱ.

[0010] Even further, after co-introducing the OCT4 gene, SOX2 gene, KLF4 gene, c-MYC gene, and NFAT1 gene into the CD34 + cells, the cells are cultured by the following method: on the 1st day after transfection, transfer the treated CD34 + cells to a culture plate pre-coated with laminin and culture them with mTeSR™1 medium, changing the medium daily until induced pluripotent stem cells (iPSCs) are obtained. Among them, iPSC-like cells appear in about 2 - 3 days and clone formation occurs in about 1 week.

[0011] In the above method, the human PBMCs can be either PBMCs from healthy individuals or PBMCs from moyamoya disease patients.

[0012] In a second aspect, the present invention claims the induced pluripotent stem cells prepared by the method described in the first aspect above.

[0013] In a third aspect, the present invention claims the application of the method described in the first aspect above or the induced pluripotent stem cells described in the second aspect above in the preparation of a moyamoya disease model.

[0014] The moyamoya disease model may include but is not limited to: endothelial cells, smooth muscle cells, organoid cell compositions, and organoid models for simulating moyamoya disease prepared from the induced pluripotent stem cells. It also includes T cells, Treg cells, etc. with moyamoya disease characteristics prepared from the induced pluripotent stem cells.

[0015] In an embodiment of the present invention, the moyamoya disease model is a moyamoya disease cerebral microvascular endothelial cell model.

[0016] In a fourth aspect, the present invention claims a method for preparing a moyamoya disease cerebral microvascular endothelial cell model.

[0017] The method for preparing a cerebral microvascular endothelial cell model of moyamoya disease, which is claimed by the present invention, may include the following steps: (A1)Induced pluripotent stem cells are prepared by using the method described in the first aspect above; (A2)The induced pluripotent stem cells are directionally induced to differentiate into cerebral microvascular endothelial cells, and thus the cerebral microvascular endothelial cell model of moyamoya disease is obtained.

[0018] In an embodiment of the present invention, in step (A2), the induced pluripotent stem cells are directionally induced to differentiate into cerebral microvascular endothelial cells by a method including the following steps: (a1)Before induction of differentiation, the induced pluripotent stem cells are inoculated on a culture plate pre-coated with Matrigel, and then 10 μM Y-27632 is added to the culture system of the induced pluripotent stem cells, and cultured for 3 days; (a2)The induced pluripotent stem cells treated in (a1) are cultured in DeSR1 medium supplemented with 6 μM CHIR99021 for 24 hours; The DeSR1 medium is composed of MEM non-essential amino acid solution (100X), GlutaMAX, β-mercaptoethanol and basal medium 1; in the DeSR1 medium, the final concentration of the MEM non-essential amino acid solution (100X) is 1% by volume, the final concentration of GlutaMAX is 0.5% by volume, the final concentration of β-mercaptoethanol is 0.1 mM, and the balance is the basal medium 1; Further, the basal medium 1 may be DMEM / F12 medium.

[0019] (a3)The medium of the induced pluripotent stem cells treated in (a2) is replaced with DeSR2 medium and cultured for 5 days, and the fresh DeSR2 medium is replaced every day during the period; The DeSR2 medium is obtained by adding B27 to the DeSR1 medium; the final concentration of B27 in the DeSR2 medium is 1% by volume.

[0020] (a4)The medium of the induced pluripotent stem cells treated in (a3) is replaced with hECSR1 medium and cultured for 2 days; The hECSR1 medium is composed of basic fibroblast growth factor (bFGF), retinoic acid (RA), B27, and basal medium 2; in the hECSR1 medium, the final concentration of the basic fibroblast growth factor (bFGF) is 20 ng / mL, the final concentration of the retinoic acid (RA) is 10 μM, the final concentration of β-mercaptoethanol is 0.1 mM, and the balance is the basal medium 2; Further, the basal medium 2 can be human endothelial SFM medium.

[0021] (a5) Transfer the induced pluripotent stem cells treated in (a4) to a culture plate coated with Matrigel, and replace the medium with hECSR2 medium on the second day. After culturing for 1 day, the induced pluripotent stem cells are directed to differentiate into cerebral microvascular endothelial cells; The hECSR2 medium is the medium obtained by removing fibroblast growth factor (bFGF) and retinoic acid (RA) from the hECSR1 medium.

[0022] In the fifth aspect, the present invention claims to protect the moyamoya disease cerebral microvascular endothelial cell model prepared by the method described in the fourth aspect above.

[0023] In the sixth aspect, the present invention claims to protect the application of the method described in the fourth aspect above or the moyamoya disease cerebral microvascular endothelial cell model described in the fifth aspect above in any of the following: (B1) Drug screening and / or drug evaluation for the treatment and / or prevention of moyamoya disease; (B2) Studying the pathogenesis of moyamoya disease; (B3) Evaluating gene therapy strategies for moyamoya disease; (B4) Preparing products for drug screening and / or drug evaluation for the treatment and / or prevention of moyamoya disease; (B5) Preparing products for studying the pathogenesis of moyamoya disease; (B6) Preparing products for evaluating gene therapy strategies for moyamoya disease.

[0024] Experimental evidence shows that the present invention constructs a pluripotent stem cell model that can accurately simulate the pathological characteristics of moyamoya disease by introducing NFAT1 This model provides a new technical means for studying the pathogenesis of moyamoya disease, drug screening, and personalized treatment, and has important application values in the fields of disease model construction, tissue repair, and precision medicine.

[0025] The innovation points and technical advantages of the present invention are as follows: ① Disease specificity of the model: Adding the NFAT1 factor, combined with RNF213The mutant background significantly enhances the simulation ability of the iPSCs model for specific pathological features of moyamoya disease. ② Balance between efficiency and safety: The optimized circular plasmid system not only ensures integration-free safety but also significantly improves the reprogramming efficiency. ③ In-depth mechanism research: Reveal RNF213 The interaction with NFAT1 in the pathogenesis of moyamoya disease provides a new perspective for the study of disease mechanisms. ④ Broad application prospects: The tube formation ability of the cerebral microvascular endothelial cells differentiated from the constructed iPSCs model is similar to that of moyamoya disease patients, and can be used for drug screening, gene therapy research and the development of precision medicine programs for moyamoya disease. Through the above technical solutions, the present invention provides an important tool for the study of the pathogenesis and potential treatment of moyamoya disease, and opens up a new path for the exploration of personalized treatment of complex diseases. Brief Description of the Drawings

[0026] Figure 1 The process of inducing the reprogramming of iPSC cells from PBMCs of healthy individuals by the pCXWB-EBNA1 plasmid ( OCT4 , SOX2 , KLF4 , c-MYC ), and the process of inducing the reprogramming of iPSC cells from PBMCs of healthy individuals by the pCXWB-EBNA1 plasmid ( OCT4 , SOX2 , KLF4 , c-MYC , NFAT1 ). In the figure, D represents the number of days.

[0027] Figure 2 The specific antibody staining of stem cell markers for the iPSC cells induced by the pCXWB-EBNA1 plasmid ( OCT4 , SOX2 , KLF4 , c-MYC ) from PBMCs of healthy individuals (A), the specific antibody staining of stem cell markers for the iPSC cells induced by the pCXWB-EBNA1 plasmid ( OCT4 , SOX2 , KLF4 , c-MYC ) from PBMCs of moyamoya disease patients (B), and the specific antibody staining of stem cell markers for the iPSC cells induced by the pCXWB-EBNA1 plasmid ( OCT4 , SOX2 , KLF4 , c-MYC , NFAT1 ) from PBMCs of healthy individuals (C).

[0028] Figure 3 The process of inducing the reprogramming of iPSC cells from PBMCs of healthy individuals by the pCXWB-EBNA1 plasmid (OCT4 , SOX2 , KLF4 , c-MYC , NFAT1 ) Karyotype analysis of iPSC cells induced by reprogramming showed no abnormality in chromosome number (A) and no abnormality in chromosome structure (B).

[0029] Figure 4 PBMCs from healthy individuals were used to induce the differentiation of iPSC cells by the pCXWB-EBNA1 plasmid ( OCT4 , SOX2 , KLF4 , c-MYC ) Specific antibody staining of markers of endothelial cells differentiated from iPSC cells induced by reprogramming (A), PBMCs from moyamoya disease patients were used to induce the differentiation of iPSC cells by the pCXWB-EBNA1 plasmid ( OCT4 , SOX2 , KLF4 , c-MYC ) Specific antibody staining of markers of endothelial cells differentiated from iPSC cells induced by reprogramming (B), and PBMCs from healthy individuals were used to induce the differentiation of iPSC cells by the pCXWB-EBNA1 plasmid ( OCT4 , SOX2 , KLF4 , c-MYC , NFAT1 ) Specific antibody staining of markers of endothelial cells differentiated from iPSC cells induced by reprogramming (C).

[0030] Figure 5 Representative images (A) and tube formation statistics (B) of cerebral microvascular endothelial cells differentiated from iPSC cells reprogrammed from healthy individuals without NFAT1, moyamoya disease patients without NFAT1, and moyamoya disease patients with NFAT1 at 6 hours. In the figure, HC represents the group of healthy individuals without NFAT1 (i.e., cerebral microvascular endothelial cells differentiated from iPSC cells induced by the four-gene pCXWB-EBNA1 plasmid containing OCT4 , SOX2 , KLF4 and c-MYC ); MMD represents the group of moyamoya disease patients without NFAT1 (i.e., cerebral microvascular endothelial cells differentiated from iPSC cells induced by the four-gene pCXWB plasmid containing OCT4 , SOX2 , KLF4 and c-MYC ); HC+NFAT1 represents the group of healthy individuals with NFAT1 (i.e., cerebral microvascular endothelial cells differentiated from iPSC cells induced by the four-gene pCXWB-EBNA1 plasmid containing OCT4 , SOX2 , KLF4 , c-MYC and NFAT1Brain microvascular endothelial cells induced by five-gene pCXWB-EBNA1 plasmid for reprogramming iPSC differentiation. * indicates P<0.05, ** indicates P<0.01. Specific implementation manners

[0031] The present invention will be further described in detail below in conjunction with specific implementation manners. The provided examples are only for clarifying the present invention and not for limiting the scope of the present invention. The following provided examples can be used as a guide for those of ordinary skill in the art to make further improvements and do not constitute any limitation to the present invention in any way.

[0032] The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0033] Example 1: Construction method of induced pluripotent stem cells based on NFAT1 and its application in moyamoya disease 1. Sample preparation Collect 5-10 ml of peripheral venous blood samples from healthy donors and patients diagnosed with moyamoya disease (without RNF213 p.R4810K mutation), ensure that the donors have no other serious medical history, and collect them by venipuncture using aseptic techniques. At the same time, add anticoagulant (EDTA) during blood collection to prevent blood coagulation. Subsequently, isolate the peripheral blood mononuclear cells (PBMCs) in the whole blood sample using Ficoll-Paque density gradient solution. The specific operation is as follows: Slowly add the whole blood to the upper layer of the pre-prepared Ficoll-Paque density gradient solution, avoid stirring, and then centrifuge at 500×g for 20 minutes (room temperature, no braking). After centrifugation, collect the mononuclear cell layer at the interface and transfer it to a new centrifuge tube. Then, place the isolated PBMCs cells in the dedicated CD34 + cell expansion medium StemSpan SFEM Ⅱ (STEMCELL; 09605), and culture them in a constant temperature incubator at 37°C and 5% CO 2 . Replace the medium every 2 days regularly, and observe the cell morphology using an inverted microscope to keep the cell density within an appropriate range and avoid overcrowding to promote the effective expansion of CD34 + cells. After culturing for 6-9 days, the CD34 + cells reach the peak, and the cells can be electroporated without purification.

[0034] 2. Design of reprogramming factors Select the episomal vector pCXWB-EBNA1 (Addgene, #37624), andOCT4 Genes (positions 63 - 1145 of NCBI ReferenceSequence: NM_002701.6, see NCBI database, Update: PRI 09 - JUL - 2024), SOX2 Genes (positions 437 - 1390 of NCBI Reference Sequence: NM_003106.4, see NCBI database, Update: PRI 12 - NOV - 2024), KLF4 Genes (positions 598 - 2037 of NCBI Reference Sequence: NM_004235.6, see NCBI database, Update: PRI 24 - OCT - 2024), c-MYC Genes (positions 364 - 1728 of NCBI ReferenceSequence: NM_002467.6 see NCBI database, Update: PRI 11 - DEC - 2024) and NFAT1 Genes (positions 221 - 2998 of NCBI Reference Sequence: NM_173091.4, see NCBI database, Update: PRI 08 - FEB - 2025) were used to replace small fragments between two EcoRⅠ restriction sites of the pCXWB - EBNA1 vector to obtain 5 recombinant plasmids. According to the different foreign genes, the 5 recombinant plasmids were named pCXWB - OCT4, pCXWB - SOX2, pCXWB - KLF4, pCXWB - c - MYC, and pCXWB - NFAT1 respectively. Subsequently, the Qiagen Maxi Kit method was used to extract endotoxin - free high - purity plasmid DNA to ensure the removal of endotoxin and other impurities. Next, the Lonza Nucleofector 2B system was used for electroporation to efficiently introduce the constructed circular plasmids into CD34 + cells, thus achieving stable expression of reprogramming factors. There were two reprogramming situations, involving the introduction of 4 recombinant plasmids, namely pCXWB - OCT4, pCXWB - SOX2, pCXWB - KLF4, and pCXWB - c - MYC, and the introduction of 5 recombinant plasmids, namely pCXWB - OCT4, pCXWB - SOX2, pCXWB - KLF4, pCXWB - c - MYC, and pCXWB - NFAT1.

[0035] 3. Cell culture and screening On the 1st day after transfection, the transfected CD34 +The cells were transferred to a culture plate pre-coated with laminin, and the medium was changed daily using mTeSR™1 medium (STEMCELL; 85850) to maintain an appropriate cell density. Photos were taken and observed on the 6th, 11th, 19th, and 28th days after electroporation. The number of formed clones in the group with NFAT1 added was relatively large ( Figure 1 ). The formed iPSC clones were observed under a microscope, and clones with regular morphology and clear boundaries were selected for picking. The selected clones were transferred to a new culture plate and continued to be cultured in mTeSR™1 medium to ensure cell homogeneity and pluripotency.

[0036] For iPSCs from healthy individuals and Moyamoya disease patients constructed with single-gene expression vectors of OCT4 , SOX2 , KLF4 and c-MYC genes, and iPSCs from healthy individuals constructed with single-gene expression vectors of OCT4 , SOX2 , KLF4 , c-MYC and NFAT1 genes, immunofluorescence staining was performed using specific antibodies of pluripotent stem cell markers TRA-1-60 (S) (CST; 4746T), Sox2 (CST; 23064T), SSEA4 (CST; 4755T), and OCT-4A (CST; 2890T). The results showed positive staining of pluripotent stem cell-specific markers in the constructed iPSC cells, demonstrating the successful construction of iPSCs ( Figure 2 ). At the same time, karyotype analysis was performed on iPSCs from healthy individuals constructed with single-gene expression vectors of OCT4 , SOX2 , KLF4 , c-MYC and NFAT1 genes. The results showed that there were no abnormalities in the chromosome number and morphological structure of the constructed iPSCs ( Figure 3 ). The above results indicate that the karyotype of the constructed iPSC cells did not change during the reprogramming process.

[0037] 4. Disease model validation (1) Differentiation of iPSCs into brain microvascular endothelial cells Before differentiation, iPSCs were made single using Accutase and seeded at a density of 25,000 to 50,000 cells per square centimeter on a culture plate pre-coated with Matrigel. The medium was supplemented with 10 μM Rho-associated protein kinase (ROCK) inhibitor Y-27632, and cultured at 37°C, 5% CO 2Amplify and culture for 3 days under the conditions. Differentiation was initiated on day 0 by adding 6 μM CHIR99021 to DeSR1 medium. The composition of DeSR1 medium is as follows: DMEM / F12 (Gibco; 11320033) as the basal medium, 1% (v / v) MEM non-essential amino acid solution (100X) (Gibco; 11140050), 0.5% (v / v) GlutaMAX™ supplement (Gibco; 35050061), and 0.1 mM β-mercaptoethanol (each concentration is the final concentration in the medium). After 24 hours, the medium was changed to DeSR2 medium, which is DeSR1 medium supplemented with 1% (v / v) B27 (Gibco; 17504044) at a final concentration, and the medium was changed daily for the next 5 days. On day 6, the medium was changed to hECSR1 medium. The composition of hECSR1 medium is as follows: human endothelial SFM medium (Gibco; 11111044) supplemented with 20 ng / ml basic fibroblast growth factor (bFGF), 10 μM retinoic acid (RA), and 1% (v / v) B27 (Gibco; 17504044) (each concentration is the final concentration in the medium). After culturing in hECSR1 medium for 2 days, the cells on day 8 were digested with Accutase and seeded at a density of 1,000,000 cells per square centimeter on a 48-well tissue culture plate coated with 100 μg / ml Matrigel. On day 9, the medium was changed to hECSR2 medium, which is hECSR1 medium without RA and bFGF, to ensure that the cells formed a confluent monolayer and the differentiation of brain microvascular endothelial cells was completed.

[0038] (2) Confirmation of the differentiation of brain microvascular endothelial cells The endothelial cells differentiated in step (1) were immunofluorescently stained with antibodies against endothelial-specific markers CD31 (CST; 3528T), ZO-1 (CST; 15652T), VE-Cadherin (CST; 2500T), and Claudin-5 (CST; 49564T) to confirm the endothelial cell phenotype and prove the success of differentiation. The results are as Figure 4 shown. The results showed that the differentiated cells were positive for endothelial-specific marker staining, indicating that the differentiation of brain microvascular endothelial cells was completed.

[0039] (3) Detection of angiogenesis ability Lumen formation assay: The differentiated cerebral microvascular endothelial cells described above were suspended in Matrigel matrix and cultured at 37 °C for 6 hours. The formation of tubular structures by the cells in the matrix was observed and recorded to evaluate the angiogenesis ability. The experiment was set up with 3 replicates, and 2 sub-wells were set in each group for each replicate. ImageJ software was used to measure the node count of tube formation.

[0040] The experimental results showed that: Using OCT4 , SOX2 , KLF4 , c-MYC and NFAT1 The cerebral microvascular endothelial cells differentiated from healthy human iPSCs constructed with single-gene expression vectors of five genes had a tube formation ability close to that of the cerebral vascular endothelial cells differentiated from iPSCs of patients with moyamoya disease constructed with single-gene expression vectors of OCT4 , SOX2 , KLF4 and c- MYC four genes, which was closer to the disease state ( Figure 5 ). This result indicates that adding NFAT1 during the construction of iPSCs can simulate the disease state of moyamoya disease to a certain extent.

[0041] The present invention has been described in detail above. For those skilled in the art, without departing from the gist and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modification, use or improvement of the present invention, including changes made by using conventional techniques known in the art that depart from the scope disclosed in this application.

Claims

1. A method for constructing induced pluripotent stem cells for preparing a moyamoya disease model, comprising the following steps: + The cells are reprogrammed, and the resulting induced pluripotent stem cells are the induced pluripotent stem cells used to prepare a moyamoya disease model; The reprogramming is OCT4 Gene, SOX2 Gene, KLF4 Gene, c-MYC Genes and NFAT1 Gene co-introduction into the CD34 + cells, and cultured until the induced pluripotent stem cells are obtained.

2. The method according to claim 1, characterized in that: In the method, OCT4 Gene, SOX2 Gene, KLF4 Gene, c-MYC Genes and NFAT1 The gene is introduced into the CD34 + The recombinant vector is OCT4 Gene, SOX2 Gene, KLF4 Gene, c-MYC Genes and NFAT1 The genes were cloned into pCXWB-EBNA1 vector to obtain 5 recombinant plasmids.

3. The method according to claim 1 or 2, characterized in that: The human PBMCs are PBMCs from healthy people or PBMCs from patients with moyamoya disease.

4. Induced pluripotent stem cells prepared by the method according to any one of claims 1 to 3.

5. Use of the method according to any one of claims 1 to 3 or the induced pluripotent stem cells according to claim 4 in preparing a moyamoya disease model.

6. The use according to claim 5, characterized in that: The moyamoya disease model is a moyamoya disease cerebral microvascular endothelial cell model.

7. A method for preparing a moyamoya disease cerebral microvascular endothelial cell model, comprising the following steps: (A1) preparing induced pluripotent stem cells using the method according to any one of claims 1 to 3; (A2) Directly inducing the differentiation of the induced pluripotent stem cells into brain microvascular endothelial cells, thereby obtaining the moyamoya disease brain microvascular endothelial cell model.

8. The method according to claim 7, characterized in that: In step (A2), the induced pluripotent stem cells are directed to differentiate into brain microvascular endothelial cells according to a method comprising the following steps: (a1) before inducing differentiation, adding 10 μM Y-27632 to the culture system of the induced pluripotent stem cells and culturing for 3 days; (a2) culturing the induced pluripotent stem cells treated in (a1) in DeSR1 medium supplemented with 6 μM CHIR99021 for 24 hours; The DeSR1 culture medium is composed of MEM non-essential amino acid solution, GlutaMAX, β-mercaptoethanol and basal culture medium; in the DeSR1 culture medium, the final concentration of the MEM non-essential amino acid solution is 1% by volume, the final concentration of the GlutaMAX is 0.5% by volume, the final concentration of the β-mercaptoethanol is 0.1 mM, and the remainder is the basal culture medium; Furthermore, the basal culture medium is DMEM / F12 culture medium; (a3) replacing the culture medium of the induced pluripotent stem cells treated in (a2) with DeSR2 culture medium, and culturing for 5 days, during which time the DeSR2 culture medium is replaced with fresh one every day; The DeSR2 medium is obtained by adding B27 to the DeSR1 medium; the final concentration of B27 in the DeSR2 medium is 1% by volume; (a4) replacing the culture medium of the induced pluripotent stem cells treated in (a3) ​​with hECSR1 culture medium and culturing for 2 days; The hECSR1 culture medium is composed of basic fibroblast growth factor, retinoic acid, B27 and a basic culture medium; in the hECSR1 culture medium, the final concentration of the basic fibroblast growth factor is 20 ng / mL, the final concentration of the retinoic acid is 10 μM, the final concentration of the β-mercaptoethanol is 0.1 mM, and the remainder is the basic culture medium; Further, the basal culture medium is human endothelial SFM culture medium; (a5) transferring the induced pluripotent stem cells treated in (a4) to a culture plate coated with matrix gel, replacing the culture medium with hECSR2 culture medium on the next day, and achieving directed induction and differentiation of the induced pluripotent stem cells into brain microvascular endothelial cells after culturing for 1 day; The hECSR2 culture medium is a culture medium obtained by removing fibroblast growth factor and retinoic acid from the hECSR1 culture medium.

9. The moyamoya disease cerebral microvascular endothelial cell model prepared by the method of claim 7 or 8.

10. Use of the method according to claim 7 or 8 or the moyamoya disease cerebral microvascular endothelial cell model according to claim 9 in any of the following: (B1) Drug screening and / or drug evaluation for the treatment and / or prevention of moyamoya disease; (B2) Study the pathogenesis of moyamoya disease; (B3) Evaluate gene therapy strategies for moyamoya disease; (B4) Preparation of products for drug screening and / or drug evaluation for the treatment and / or prevention of moyamoya disease; (B5) Preparation of products for studying the pathogenesis of moyamoya disease; (B6) Preparing products for evaluating gene therapy strategies for moyamoya disease.

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