Method and application of iPSC-induced differentiation into vascular endothelial cells

Abstract

The present invention provides a method and application of inducing differentiation of iPSC into vascular endothelial cells. Furthermore, the monoclonal antibody specifically targeting ALK5 provided by the present invention can effectively improve the efficiency of inducing skin fibroblasts to generate iPSCs; and the induced iPSCs have good ability to differentiate into vascular endothelial cells, and have good application prospects.

Description

Technical Field

[0001] The present application relates to the biological field, specifically to a method for preparing vascular endothelial cells, and more specifically to a method and application of iPSC-induced differentiation of vascular endothelial cells. Background Art

[0002] The main function of blood vessels is to transport oxygen and nutrients to various tissues in the body and to transport waste products from tissues to the kidneys, liver and intestines for metabolism. Endothelial cells line the inner wall of blood vessels in contact with blood and maintain vascular integrity. Endothelial dysfunction is associated with a variety of disease types such as atherosclerosis, coronary artery disease, hypertension, diabetes, congestive heart failure, etc. The key role of healthy functional endothelial cells in the repair and regeneration of damaged tissues is a focus of regenerative medicine.

[0003] At present, endothelial cells can be isolated from adipose tissue, blood or obtained by differentiation of induced pluripotent stem cells. The limitation of endothelial cells isolated in vivo is that the number of divisions before cell senescence is limited, and it is difficult to obtain tissue-specific endothelial cells, and the quantity and quality of endothelial cells obtained are limited. In contrast, pluripotent stem cells have multidirectional differentiation potential and maintain proliferation ability during dozens of passages. Endothelial cells produced by directional induction of pluripotent stem cells have become an alternative and renewable cell source. Pluripotent stem cells include embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). At present, a variety of endothelial cell efficient differentiation technologies have been gradually established, and the differentiation efficiency is claimed to be more than 70%, but there are various difficulties in actual operation, and the differentiation efficiency generally does not exceed 50%. Differentiated endothelial cells showed blood circulation reconstruction ability after transplantation into mouse ischemic disease models, which confirmed the value of endothelial cells derived from pluripotent stem cells in regenerative medicine research.

[0004] Early methods for differentiating pluripotent stem cells into endothelial cells included co-culturing with stromal cells (usually mouse bone marrow-derived stromal cell lines such as OP9) or differentiating into endothelial cells through the formation of embryoid bodies. These two differentiation methods recreate the early embryonic development process, but the differentiation efficiency is often low, and endothelial cells are mixed with other cell types. Current differentiation strategies mainly induce stem cells to transform into endothelial cell fate by adding small molecule active substances to stem cells in monolayer culture. Most protocols using this strategy can be divided into mesoderm differentiation stage and endothelial cell differentiation stage. In the first stage, the signal that manipulates PSCs to differentiate into mesoderm is the common point of these monolayer differentiation systems, and growth factors such as activin A and BMP4 are commonly used. In the second stage, bFGF and VEGF are the most commonly used growth factors. GSK-3 inhibitors, such as CHIR99021, are also often added to the culture medium to regulate the Wnt signaling pathway, which promotes the differentiation of mesoderm into the endothelial lineage. Inhibition of the TGF-β signaling pathway is essential for the endothelial lineage specification of mesoderm cells and the maintenance of endothelial cells (avoiding the transformation of endothelial cells to mesenchymal cells). Therefore, SB431542, an inhibitor of the TGF-β signaling pathway, is also often included in the differentiation protocol. In order to further improve the differentiation efficiency of endothelial cells, researchers have screened key transcription factors in the endothelial differentiation process. For example, Elcheva et al. conducted a comprehensive screening and found 27 genes related to the formation of mesoderm and vascular progenitor cells. Among them, ETV2 plays an important role in directly inducing the fate of endothelial cells. ETV2 is an important transcription factor for the development of cardiac, endothelial and hematopoietic lineages. Overexpression of ETV2 by RNA transfection or gene editing can effectively induce stem cells to differentiate into endothelial cells.

[0005] The key to effectively obtaining vascular endothelial cells is to have sufficient induced stem cells. In 2007, scientists from Japan and the United States reported that four transcription factors, KLF4, OCT4, SOX2 and C-MYC, could be introduced into human fibroblasts to reversely differentiate them into cells with embryonic stem cell characteristics, and named them induced pluripotent stem cells (iPSC). In this article published in the journal Cell, the researchers selected 24 genes related to embryonic stem cell signal transduction as candidate genes, infected human fibroblasts, and performed genome integration, using cell morphological characteristics, embryonic stem cell key gene expression, and teratoma formation as iPSC identification indicators. Finally, the four transcription factors, Oct4, Sox2, Klf4 and c-Myc, were determined, which can transform normal somatic cells into pluripotent stem cells. The pluripotent stem cells produced by this gene induction are called iPSC cells. This method of obtaining stem cells directly from differentiated cells has brought conceptual innovations to the field of embryonic stem cells and even the entire field of life sciences. We can use the patient's somatic cells as a source to obtain patient-specific iPSC cells, which can then differentiate into vascular endothelial cells for disease treatment. This application method can avoid immune compatibility and ethical issues. Since then, the emergence of iPSC cell induction technology has also attracted great attention to its biomedical applications. However, the current application of iPSC cells still faces the problem of low induction efficiency.

[0006] Transforming growth factor β-1 (TGF-β1) is a cytokine that is involved in the disease process of many different organs, such as the kidney, liver, lung, skin, heart or blood vessels. The classic TGF-β1 signaling pathway requires binding to the TGF-β type II receptor (TβRII), followed by the recruitment of the TGF-β type I receptor (TβRI) with serine / threonine kinase activity, which phosphorylates into Smads proteins. Smads enter the cell nucleus as transcription factors to activate or inhibit the expression of specific genes. Among the different TβRIs, the main ones are activin receptor-like kinase 1 and activin receptor-like kinase 5 (ALK5). Studies have found that in the process of preparing iPSCs, type I transforming growth factor receptor (ALK5) inhibitors can efficiently produce induced pluripotent stem cells (iPSCs). However, at present, there are not many effective ALK5 inhibitors, especially those that are not toxic to iPSC cells. Summary of the invention

[0007] The present invention provides a method for inducing differentiation of iPSCs into vascular endothelial cells.

[0008] Furthermore, the present invention also provides a method for efficiently preparing iPSC cells, wherein the method comprises using a transforming growth factor receptor type I (ALK5) inhibitor.

[0009] Furthermore, the transforming growth factor receptor type I (ALK5) inhibitor is a monoclonal antibody specific for ALK5.

[0010] Specifically, the light chain variable region sequence of the monoclonal antibody specific for ALK5 is shown in SEQ ID NO: 1, and the heavy chain variable region sequence is shown in SEQ ID NO: 2.

[0011] Further, the present invention provides a method for inducing iPSC cells using fibroblasts, wherein the method comprises using HS27 skin fibroblasts to transfect plasmids pCXLE-hOCT3 / 4-shp53-F, pCXLE-hSK, pCXLE-hUL and pCXWB-EBNA1 nuclear transfection, the cells are first seeded into a 6-well plate covered with gelatin, and then the cells are inoculated into a 6-well plate covered with gelatin. The cells were cultured in human fibroblast complete medium (with monoclonal antibody ALK5-G2 added at a final concentration of 50 μg / mL) for 6 days, then transferred to a mouse fibroblast (MEF) feeder layer and cultured in human embryonic stem cell medium (with monoclonal antibody ALK5-G2 added at a final concentration of 50 μg / mL) until iPSC clones appeared. iPSC clones were collected with a glass needle and inoculated into a 6-well plate covered with Matrigel and amplified with mTeSR1 medium. TrypLE was used every 3 days. TM iPSCs were isolated by digestion with Express enzyme (1X) and passaged at a ratio of 1:3.

[0012] Furthermore, the present invention also provides a method for inducing differentiation of iPSC cells into vascular endothelial cells, comprising taking human induced pluripotent stem cells at 1.0×10 5 / well were inoculated into a 12-well plate pretreated with matrix gel (1:30 dilution ratio), the culture medium was BIOCISO (containing 10μmol / LY27632 and 10μmol / LCHIR99021), and cultured for 24h; 24h later, differentiation began: 100μg / L activin A was used for 1d, the basic culture medium was RPMI / 1640 (containing 1×B27- and 100-fold diluted matrix gel); replaced with RPMI / 1640 culture medium (containing 1umol / LCHIR99021 and 5μg / L bone morphogenetic protein 4) for 1d. Replace the StmePro-34SFM medium (containing 0.4μmol / L thioglycerol, 2mmol / L L-glutamine and 50mg / L ascorbic acid) containing 300μg / L vascular endothelial growth factor, 10μg / L bone morphogenetic protein 4 and 54g / L basic fibroblast growth factor for 3 days. Use cell digestion solution (0.25% Trypsin / 0.02% EDTA) to digest and inoculate into 0.1% gelatin-coated well plates at a ratio of 1:3. The culture medium is EGM containing 20μg / L vascular endothelial growth factor, 20μg / L basic fibroblast growth factor and 1μmol / L CHIR99021. Change the culture medium once every 2 days. Culture until the differentiated cells are harvested 15 days after the start of differentiation, which is the vascular endothelial cells.

[0013] Furthermore, the present invention also provides the use of a monoclonal antibody specific for ALK5 in improving the efficiency of inducing skin fibroblasts to generate iPSCs; the light chain variable region sequence of the monoclonal antibody for ALK5 is shown in SEQ ID NO: 1, and the heavy chain variable region sequence is shown in SEQ ID NO: 2.

[0014] Beneficial Effects

[0015] The present invention provides an innovative method and application of inducing differentiation of iPSC into vascular endothelial cells. Furthermore, the monoclonal antibody specifically targeting ALK5 provided by the present invention can effectively improve the efficiency of inducing skin fibroblasts to generate iPSCs; and the induced iPSCs have good ability to differentiate into vascular endothelial cells, and have good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Monoclonal antibody ALK5-G2 specificity identification results

[0017] Figure 2 Western bolt detection of ALK5 protein expression in cells DETAILED DESCRIPTION

[0018] The specific embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although the specific embodiments of the present invention are shown in the accompanying drawings, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present invention and to enable the scope of the present invention to be fully communicated to those skilled in the art.

[0019] Example 1 Preparation of monoclonal antibodies against ALK5

[0020] BALB / c female mice aged 4-6 weeks were selected and immunized intraperitoneally with 80 μg ALK5 recombinant protein (CB414141087, TargetMol China) mixed with an equal volume of Freund's adjuvant. Immunization was performed once every 14 days for a total of 3 times (the first time was with Freund's complete adjuvant, and the second and third times were with Freund's incomplete adjuvant). Two weeks after the last immunization, mouse serum was collected and the serum titer of the immunized mice was detected by indirect ELISA. The serum dilution factor corresponding to the minimum P value when P≥0.2 and P / N≥2.1 was used as the serum titer, where P and N were the D450 values ​​of the positive and negative serum wells, respectively. The serum titer of mouse No. 2 was as high as 1:128000. Therefore, mouse No. 3 was selected for cell fusion and hybridoma cell screening. The mouse with the highest serum titer was selected and boosted with 80 μg (0.1 mL) ALK5 recombinant protein intraperitoneally, and cell fusion was performed 3 days later. Splenocytes of BALB / c mice aged 4-6 weeks were used as feeder cells, and SP2 / 0 cells (1×10 7 ) and spleen cells of BALB / c mice after immunization (1×10 8 ) were fused under the action of 50% PEG1500, and the cells were added to 96-well plates and cultured in a 37°C, 5% CO2 incubator. When the cell colonies grew to 1 / 3 of the well, the culture supernatant was collected, and the ALK5 recombinant protein was used as the coating antigen to detect the hybridoma cell lines that secreted specific antibodies by indirect ELISA. 13 fusion cells with higher absorbance (D450) values ​​were selected, and subcloned for 3 generations by limiting dilution method to obtain a hybridoma cell line that can stably secrete mAb, named ALK5-G2.

[0021] After clonal growth, cells with a positive rate of 100% were expanded and stored in liquid nitrogen.

[0022] Six 8-week-old BALB / c female mice that had been adaptively raised for one week were selected and each mouse was pretreated by intraperitoneal injection of 0.5 mL of pristane. Ten days later, 5×10 hybridoma cells ALK5-G2 were inoculated intraperitoneally. 5 / mouse. Observe the swelling of the abdomen of the mouse. When the swelling affects the movement, collect the ascites, centrifuge at 10000r / min for 10min, remove the fat tissue and precipitate, and aspirate the supernatant into a new centrifuge tube. Purify the monoclonal antibody ALK5-G2 from the ascites by the octanoic acid-ammonium sulfate method. The purity is good after SDS-PAGE detection. The mass concentration of the monoclonal antibody is 2.5mg / mL determined by the BCA method. After packaging, store it in a -80℃ refrigerator for later use.

[0023] Example 2 Specificity Identification of Monoclonal Antibody ALK5-G2

[0024] Western blot was used to identify the binding of monoclonal antibody ALK5-G2 to ALK5 recombinant protein and mouse serum. 15% PAGE gel was selected for electrophoresis at 120V voltage. After the end, it was transferred to PVDF membrane using full wet transfer at 110V voltage for 100min. The transferred PVDF membrane was blocked with 1% BSA at room temperature for 2h, and mAb diluted in TBST was used as the primary antibody, incubated at room temperature for 1h, and then washed with TBST 3 times. Goat anti-mouse IgG (H+L) labeled with horseradish peroxidase was used as the secondary antibody, diluted 1:5000 and incubated at room temperature for 1h, and then washed with TBST 3 times. The color developing solution was added and incubated for 15min under light-proof conditions for development and observation. The results are as follows Figure 1 shown.

[0025] The results showed that the monoclonal antibody ALK5-G2 could react with ALK5 recombinant protein but not with mouse serum, indicating that the monoclonal antibody had good specificity.

[0026] Example 3 Affinity detection of monoclonal antibody ALK5-G2

[0027] The affinity of humanized antibodies was detected by ForteBio technology, and FAB2G was selected as the binding sensor. The mass concentration of the fixed antibody was 10 μg / mL, and the ALK5-His protein was diluted to 200, 100, 50, 25, 12.5 and 6.25 nmol / L as the analyte for affinity detection. BlitzPro1.2 software was used for data analysis to obtain the binding rate constant (Kon), dissociation rate constant (Koff) and KD. As shown in Table 1.

[0028] Table 1 Affinity of antibodies

[0029]

[0030]

[0031] As can be seen from Table 1, the affinity of monoclonal antibody ALK5-G2 is relatively high, K D The value is 1.58×10-9 mol / L.

[0032] Example 3: Sequencing of the variable regions of monoclonal antibody ALK5-G2

[0033] The following primers were synthesized based on the constant region sequence of the antibody gene:

[0034] A:5′-GGGGATATCCACCATGRACTTCGGGYTGAGCTKGGTTTT-3′

[0035] B:5′-GACHGATGGGGSTGTYGTGCTAGCTGNRGAGACDGTGA-3′

[0036] C:5′-GGGGATATCCACCATGGAGACAGACACACTCCTGCTAT-3′

[0037] D:5′-GGATACAGTTGGTGCAGTCGACTTACGTTTKATTTCCARCTT-3′

[0038] 5×10 6 Total RNA of hybridoma cell ALK5-G2 was reverse transcribed into cDNA. A and B were used as primers for PCR amplification of the heavy chain variable region of the monoclonal antibody, and C and D were used as primers for PCR amplification of the light chain variable region of the monoclonal antibody. The PCR reactions were all hot-started, and the reaction conditions were: 94°C for 5 minutes; 94°C for 45 seconds, 60°C for 45 seconds, 72°C for 1 minute and 10 seconds, 30 cycles; 72°C for 7 minutes. The PCR products were separated by 1% agarose gel electrophoresis and the target fragments were recovered and purified. The clones were cloned into the pGEM-T (Promega) vector, transformed into Escherichia coli TGl cells, and screened on IPTGIX-gal plates. The white plaques were inoculated in LB liquid culture medium containing ampicillin for amplification. The positive clones were screened, and the plasmids were extracted and sequenced using QIAGEN's plasmid extraction kit. The sequences of the light chain and heavy chain variable regions of the monoclonal antibody ALK5-G2 were determined to be shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0039] Example 4: Validation of the efficacy of monoclonal antibody ALK5-G2 in the preparation of iPSC cells

[0040] Control group:

[0041] Plasmids pCXLE-hOCT3 / 4-shp53-F (plasmid #27077), pCXLE-hSK (plasmin #27078), pCXLE-hUL (plasmid #27080) and pCXWB-EBNAl (plasmin #37624) (Addgene) (https: / / www.addgene.org / ). Electroporation was performed using the Amaxa Basic Nucleofector kit with the T-020 program (250 V, 10 ms, 1, 0.4 mm cuvette). 1x10 6 HS27 skin fibroblasts (Cat. No. XY-XB-2473, ScienCell Cell) were transfected with 1 μg of plasmids pCXLE-hOCT3 / 4-shp53-F, pCXLE-hSK, pCXLE-hUL, and pCXWB-EBNA1. The transfected cells were first seeded into a 6-well plate covered with gelatin. The cells were cultured in complete human fibroblast medium for 6 days, then transferred to a mouse fibroblast (MEF) feeder layer and cultured in human embryonic stem cell medium until iPSC clones appeared. iPSC clones were collected with a glass needle and inoculated into a 6-well plate covered with Matrigel and expanded with mTeSR1 medium. TrypLE was used every 3 days. TM iPSCs were isolated by digestion with Express enzyme (1X) and passaged at a ratio of 1:3.

[0042] Experimental Group:

[0043] Plasmids pCXLE-hOCT3 / 4-shp53-F (plasmid #27077), pCXLE-hSK (plasmin #27078), pCXLE-hUL (plasmid #27080) and pCXWB-EBNAl (plasmin #37624) (Addgene) (https: / / www.addgene.org / ). Electroporation was performed using the Amaxa Basic Nucleofector kit with the T-020 program (250 V, 10 ms, 1, 0.4 mm cuvette). 1x10 6 HS27 skin fibroblasts (Cat. No. XY-XB-2473, ScienCell Cell) were transfected with 1 μg of plasmids pCXLE-hOCT3 / 4-shp53-F, pCXLE-hSK, pCXLE-hUL, and pCXWB-EBNA1. The transfected cells were first seeded into a 6-well plate covered with gelatin. The cells were cultured in human fibroblast complete medium (with monoclonal antibody ALK5-G2 added at a final concentration of 50 μg / mL) for 6 days, then transferred to a mouse fibroblast (MEF) feeder layer and cultured in human embryonic stem cell medium (with monoclonal antibody ALK5-G2 added at a final concentration of 50 μg / mL) until iPSC clones appeared. iPSC clones were collected with a glass needle and inoculated into a 6-well plate covered with Matrigel and amplified with mTeSR1 medium. TrypLE was used every 3 days. TM iPSCs were isolated by digestion with Express enzyme (1X) and passaged at a ratio of 1:3.

[0044] The newly emerged iPSC clones in the experimental group were compared with an equal number of untransfected HS27 skin fibroblasts for Western blot detection of intracellular ALK5 protein expression. The results are as follows: Figure 2 As shown. Figure 2 It can be seen that the expression of ALK5 protein in iPSC clones was significantly inhibited.

[0045] Using Molecular Probes TM The human pluripotent stem cell (PSC) live cell imaging kit was used to identify the 12th generation iPSC cells prepared by the above two methods, and the proportion of positive clones was observed under an inverted fluorescence microscope. The results are shown in Table 2.

[0046] Table 2 Proportion of positive clones in each group

[0047] Group Positive clone ratio Control group 12.53±0.21 Experimental Group 43.89±0.42*

[0048] From the results in Table 2, it can be seen that the ratio of iPSC-positive cells obtained from the transfected cells treated with the monoclonal antibody ALK5-G2 was significantly increased, and the difference was significant compared with the control group (P<0.05).

[0049] After collecting the iPSC positive cells prepared in the two groups, further testing revealed that the induced pluripotent stem cell proliferation marker Ki67 was specifically expressed and strongly positively localized in the cell nucleus, further proving that iPSC positive cells were prepared.

[0050] Example 5 Differentiation of iPSC cells into vascular endothelial cells

[0051] The two types of human induced pluripotent stem cells (passage 30) collected in Example 4 were respectively cultured at 1.0×10 5 / well were inoculated into a 12-well plate pretreated with matrix gel (1:30 dilution ratio), the culture medium was BIOCISO (containing 10μmol / LY27632 and 10μmol / LCHIR99021), and cultured for 24h; 24h later, differentiation began: 100μg / L activin A was used for 1d, the basic culture medium was RPMI / 1640 (containing 1×B27- and 100-fold diluted matrix gel); replaced with RPMI / 1640 culture medium (containing 1umol / LCHIR99021 and 5μg / L bone morphogenetic protein 4) for 1d. Replace the StmePro-34SFM medium (containing 0.4μmol / L thioglycerol, 2mmol / L L-glutamine and 50mg / L ascorbic acid) containing 300μg / L vascular endothelial growth factor, 10μg / L bone morphogenetic protein 4 and 54g / L basic fibroblast growth factor for 3 days. Use cell digestion solution (0.25% Trypsin / 0.02% EDTA) to digest and inoculate into 0.1% gelatin-coated well plates at a ratio of 1:3. The culture medium is EGM containing 20μg / L vascular endothelial growth factor, 20μg / L basic fibroblast growth factor and 1μmol / L CHIR99021. Change the culture medium once every 2 days. Culture until the differentiated cells are harvested 15 days after the start of differentiation, which is the vascular endothelial cells.

[0052] Immunofluorescence was used to detect the expression of endothelial cell markers. The induced vascular endothelial cells were fixed: 40g / L paraformaldehyde was used to fix the cells, and the cells were treated at room temperature for 10min, and then washed with DPBS 3 times, each time for 5min; blocked and punched: the punching blocking solution (1xPBS / 5% BSA / 0.3% Tritonx-100) was added, and the cells were kept at room temperature for 30min; primary antibody incubation: the primary antibody anti-human VE-Cadherin monoclonal antibody (1:400) and mouse anti-human CD31 monoclonal antibody (1:200) were added, and the cells were incubated at 4℃ overnight; secondary antibody incubation: DPBS was washed 3 times, each time for 5min, and the secondary antibody was added, and the cells were kept away from light for 1h at room temperature; sealing and imaging: DAPI antifade agent was used for counterstaining and sealing, and the cells were observed and photographed under a confocal fluorescence microscope. The results showed that the induced and differentiated vascular endothelial cells in the two groups highly expressed VE-Cadherin and CD31, two endothelial cell membrane-specific markers, and were located on the cell membrane, indicating that the endothelial cells were successfully induced and differentiated.

[0053] The ring formation experiment was used to detect the angiogenesis ability of endothelial cells in vitro: low growth factor matrix collagen solution was added to the pre-cooled 24-well plate, 350 μL / well, and the matrix gel was allowed to solidify at room temperature for 1 h; the endothelial cells prepared by the above-mentioned induced differentiation were digested into single cells using cell digestion solution (0.25% Trypsin / 0.02% EDTA), and the cells were counted and resuspended and inoculated into the above-mentioned well plate, with the number of cells inoculated being 50,000 / cm 2 The culture medium was EGM culture medium containing 200μg / L mass concentration of vascular endothelial growth factor. After 12h, the iPSC cells prepared in the experimental group differentiated into vascular endothelial cells and took pictures. The results showed that the iPSC cells prepared in the experimental group had better vascular ring-forming ability than those in the control group, and the ring-forming ability was improved by (25.46±0.21)%, indicating that the iPSC cells in the experimental group had better cell activity.

[0054] Although the embodiments of the present invention are described above in conjunction with the accompanying drawings, the present invention is not limited to the above specific embodiments and application fields, and the above specific embodiments are only illustrative and instructive, rather than restrictive. A person of ordinary skill in the art can also make many forms under the guidance of this specification and without departing from the scope of protection of the claims of the present invention, all of which belong to the protection of the present invention.

Claims

1. A monoclonal antibody specific for ALK5, characterized in that The light chain variable region sequence of the monoclonal antibody is shown in SEQ ID NO: 1, and the heavy chain variable region sequence is shown in SEQ ID NO:

2.

2. Use of a monoclonal antibody specific for ALK5 in improving the efficiency of inducing iPSCs from skin fibroblasts in vitro; the light chain variable region sequence of the monoclonal antibody specific for ALK5 is shown in SEQ ID NO: 1, and the heavy chain variable region sequence is shown in SEQ ID NO:

2.

3. A method for inducing iPSC cells to differentiate into vascular endothelial cells, characterized in that iPSC cells are obtained by transfecting human skin fibroblasts with transcription factors and then culturing them in a culture medium supplemented with a monoclonal antibody specific for ALK5; vascular endothelial cells are obtained by inducing the iPSC cells using a conventional induction method, and the light chain variable region sequence of the monoclonal antibody specific for ALK5 is shown in SEQ ID NO: 1, and the heavy chain variable region sequence is shown in SEQ ID NO: 2.

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