Method for inducing iPSCs (induced pluripotent stem cells) to directionally differentiate into vascular endothelial cells by small molecule compound and cytokine composition

Abstract

The invention discloses a method for inducing iPSCs (induced pluripotent stem cells) to directionally differentiate into vascular endothelial cells by using a small molecule compound and cell factor composition, which is characterized in that iPSCs cells are used as starting cells, and a vascular endothelial cell differentiation scheme completely dependent on chemical small molecule and cell factor stimulation is established. An E6 culture medium containing CHIR-99021, BMP4 and Activin A is utilized to induce mesoderm progenitor cells, an E6 culture medium containing VEGFA (vascular endothelial growth factor A), FGF-2, 8-Bromoadenosine 3 ', 5'-cyclic monophosphate monophosphate, SB431542, Forskolin and Melatonin is utilized to induce the endothelial cells, and an E6 culture medium containing VEGFA, FGF-2 and Hydrocortisone is utilized to further induce and differentiate the endothelial cells into the vascular endothelial cells. By using the scheme, the differentiation efficiency can exceed 99%, and differentiated cells have phenotypes and functions of vascular endothelial cells and do not express pluripotent related markers SSEA-4, TRA-1-60 and TRA-1-81.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for inducing iPSCs to differentiate into vascular endothelial cells by combining small molecule compounds and cytokine compositions, belonging to the field of directional differentiation. BACKGROUND

[0002] Vascular endothelial cells are one of the main components of blood vessels, located at the innermost side of the lumen, in direct contact with the liquid inside the blood vessels. The main function of vascular endothelial cells is the transport and exchange of nutrients and metabolites, and also participates in a large number of important physiological processes such as blood clotting, lymphocyte activation, blood pressure regulation, tissue and organ regeneration, etc. In addition, vascular endothelial cells produce a large number of cytokines, collectively known as vascular secretory factors, which play an important role in the homeostasis and regeneration of different tissues and organs. The disorder of endothelial cell function can directly or indirectly lead to cardiovascular diseases including atherosclerosis, hypertension, thrombosis, myocardial infarction, stroke, etc. According to the China Cardiovascular Health and Disease Report 2021, the number of cardiovascular disease patients in China is about 330 million, including 245 million cases of hypertension, 45.3 million cases of lower extremity arterial disease, 13 million cases of stroke, and 11.39 million cases of coronary heart disease. Cardiovascular diseases account for about 45% of the overall mortality. Overall, cardiovascular and cerebrovascular diseases are closely related to endothelial cell dysfunction, and these diseases cannot be fundamentally solved. Drugs for vascular endothelial cells have broad application prospects.

[0003] There are two main ways to obtain human vascular endothelial cells at present: one is to isolate primary endothelial cells from human tissues, including umbilical cord vein endothelial cells, peripheral blood endothelial progenitor cells and umbilical cord blood endothelial progenitor cells, etc. The heterogeneity of the donor, the HLA matching and the expansion process limit the clinical application. The other way is to transform other human cells into vascular endothelial cells, including the directional differentiation of pluripotent stem cells and the direct transformation of somatic cells. Induced pluripotent stem cells (iPSCs) can differentiate into all cell types and have almost unlimited proliferation capacity. The induction of differentiation to obtain vascular endothelial cells that meet the clinical application is currently considered to be the most ideal technical path. The efficiency of iPSCs directional differentiation into vascular endothelial cells is the key to this technical path. Low differentiation efficiency not only increases the difficulty of purifying the target cells, but often also accompanies unexpected differentiation and residual iPSCs. In particular, the tumorigenicity of residual iPSCs is a major risk in clinical application. The currently reported scalable iPSCs differentiation into vascular endothelial cells has unsatisfactory differentiation efficiency and cannot well meet the needs of clinical application. In addition, the highest efficiency of iPSCs differentiation into vascular endothelial cells reported so far is achieved by delivering mRNA encoding ETV2 transcription factor into mesodermal progenitor cells through electroporation, and the efficiency of iPSCs differentiation into vascular endothelial cells can reach 95%.

[0004] Although the differentiation scheme based on cell transfection has a high efficiency in small-scale tests, however, because of the introduction of the electroporation process, such a scheme has great difficulty in applying to large-scale production, and its disadvantages mainly manifest in the following aspects. First, the electroporation process is difficult to guarantee consistent efficiency under the conditions of small-scale and large-scale production, and has a natural disadvantage in system amplification; second, the endothelial cells produced by the scheme containing the electroporation process have poor expansion capacity, because the cell damage caused by electroporation; third, the introduction of the electroporation process not only increases the cost of materials and equipment, but also increases the complexity of operation. SUMMARY

[0005] In order to overcome the above-mentioned defects, the purpose of the present application is to provide a method for inducing iPSCs directional differentiation into vascular endothelial cells by a combination of small molecule compounds and cytokine composition. To improve the differentiation efficiency and simplify the operation.

[0006] In order to achieve the above purpose, the technical scheme adopted by the present application is: a method for inducing iPSCs directional differentiation into vascular endothelial cells by a combination of small molecule compounds and cytokine composition, characterized in that the method comprises the following steps: (1) inoculating iPSCs in an inoculation medium; (2) Replace the old iPSCs seeding medium with mesoderm cell differentiation induction medium I; (3) Replace the old mesoderm cell differentiation induction medium I with E6 medium; (4) Replace the old E6 medium with endothelial cell differentiation induction medium I; (5) Replace the old endothelial cell differentiation induction medium I with endothelial cell differentiation induction medium II; (6) Obtain the vascular endothelial cells after the treatment of endothelial cell differentiation induction medium II.

[0007] Compared with the prior art, the beneficial effects of the present application are: taking iPSCs cells as the starting cells, a vascular endothelial cell differentiation scheme completely dependent on chemical small molecules and cytokine stimulation is established. The differentiation process is controlled within one week from the beginning to the end, and the vascular endothelial cell differentiation efficiency calculated according to the CD31 and CD144 double positive cells is more than 99%, which is better than the results of the prior art scheme. The differentiated cells have the phenotype and function of vascular endothelial cells and do not express pluripotency related markers SSEA-4, TRA-1-60 and TRA-1-81. The differentiation scheme completely based on chemical small molecules and cytokine stimulation has a good linear relationship in the conversion process from small-scale production to large-scale production, and can better ensure the consistency of efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0008] Figure 1 Flow chart of iPSCs differentiation into vascular endothelial cells; Figure 2 Cell morphology during the differentiation of iPSCs into vascular endothelial cells; Figure 3 Flow cytometry results of vascular endothelial cells differentiated under different iPSCs seeding conditions; Figure 4 Flow cytometry results of vascular endothelial cells differentiated under different mesoderm progenitor cell induction medium I conditions; Figure 5 Flow cytometry results of vascular endothelial cells differentiated under different endothelial cell differentiation induction medium I conditions; Figure 6 Flow cytometry results of vascular endothelial cells differentiated under different endothelial cell induction medium II conditions; Figure 7 Flow cytometry results of pluripotency related markers of iPSCs derived vascular endothelial cells; Figure 8 Cell morphology of expanded iPSCs derived vascular endothelial cells at P1 generation; Figure 9Flow cytometry results of endothelial cell markers of iPSCs-derived vascular endothelial cells at P1, P2 and P3 generations; Figure 10 DiI-Ac-LDL uptake test results of iPSCs-derived vascular endothelial cells; Figure 11 NO production test results of iPSCs-derived vascular endothelial cells; Figure 12 Results of 2D-tube formation experiment of iPSCs-derived vascular endothelial cells in vitro. DETAILED DESCRIPTION

[0009] The preferred embodiments of the present application are described in detail below with reference to the accompanying drawings, so that the advantages and features of the present application can be more easily understood by those skilled in the art, and the scope of protection of the present application is more clearly defined.

[0010] Figure 1 is a process flow chart of iPSCs differentiation into vascular endothelial cells, the time point of differentiation initiation is defined as day 0, and the core process flow is from day -1 inoculation to day 6 differentiation end, with a total time consumption of 1 week.

[0011] Figure 2 Cell morphologies corresponding to time points during the process of iPSCs differentiation into vascular endothelial cells are shown. The iPSCs are the morphology of the iPSCs used for differentiation before digestion, and all images are obtained under 10x objective, with a scale bar of 200 μm.

[0012] Figure 3 Flow cytometry results of vascular endothelial cells differentiated under different iPSCs inoculation amounts. Examples 1-6 are shown, different densities of iPSCs were inoculated on day -1, and vascular endothelial cell differentiation was initiated and completed under the same conditions on day 0. After differentiation, the cells were co-stained with CD144-PE and CD31-APC antibodies, and the proportion of CD144-PE and CD31-APC double positive cells was detected on a flow cytometer. The results show that the differentiation result is best when the cell inoculation amount is 18000 per square centimeter. The negative control is the flow cytometry result of the cells after differentiation without antibody staining treatment, and the isotype control is the flow cytometry result of the cells after differentiation co-stained with CD144-PE and CD31-APC antibodies.

[0013] Figure 4Flow cytometry results of vascular endothelial cells differentiated under different mesoderm progenitor induction medium I conditions. Shown are examples 7-12, iPSCs were seeded at a density of 18000 per square centimeter on day -1, and vascular endothelial cell differentiation was initiated on day 0 with different mesoderm progenitor induction medium I, and differentiation was completed under otherwise consistent conditions. Cells at the end of differentiation were co-stained with CD144-PE and CD31-APC antibodies, and the proportion of CD144-PE and CD31-APC double positive cells was detected on a flow cytometer. The results show that the best differentiation results were obtained when 25 ng / mL of BMP4 and 50 ng / mL of Activin A were included in the mesoderm progenitor induction medium I. The negative control is the flow cytometry results of cells at the end of differentiation without antibody staining, and the isotype control is the flow cytometry results of cells at the end of differentiation co-stained with non-specific antibodies homologous to CD144-PE and CD31-APC antibodies.

[0014] Figure 5 Flow cytometry results of vascular endothelial cells differentiated under different endothelial induction medium I conditions. Based on the iPSCs seeding density and the preferred process of mesoderm progenitor induction medium I described in the foregoing examples, the only difference in examples 13-18 is the composition of the endothelial induction medium I, and differentiation was completed under otherwise consistent conditions. Cells at the end of differentiation were co-stained with CD144-PE and CD31-APC antibodies, and the proportion of CD144-PE and CD31-APC double positive cells was detected on a flow cytometer. The results show that the best differentiation results were obtained when 10 μΜ of SB431542 and 2 μΜ of forskolin were added to the basal endothelial induction medium I. The negative control is the flow cytometry results of cells at the end of differentiation without antibody staining, and the isotype control is the flow cytometry results of cells at the end of differentiation co-stained with non-specific antibodies homologous to CD144-PE and CD31-APC antibodies.

[0015] Figure 6Flow cytometry results of iPSCs-derived vascular endothelial cells differentiated under different conditions of endothelial induction medium II. Based on the preferred process of iPSCs seeding density, mesoderm progenitor induction medium I and endothelial induction medium I described in the preceding examples, the only difference in the process of Example 19-12 is the composition of endothelial induction medium II, the rest of the process is kept consistent. After the differentiation, the cells were co-stained with CD144-PE and CD31-APC antibodies and the proportion of CD144-PE and CD31-APC double positive cells was detected by flow cytometry. The results showed that the best differentiation result was obtained when 50 ng / mL VEGFA was added to the basic endothelial induction medium II. The negative control is the flow cytometry result of the cells without antibody staining after the differentiation, and the isotype control is the flow cytometry result of the cells co-stained with CD144-PE and CD31-APC antibodies after the differentiation.

[0016] Figure 7 Flow cytometry results of pluripotency related markers of iPSCs-derived vascular endothelial cells. The iPSCs and the cells obtained after the differentiation of Example 21 were fixed and immunostained with antibodies of pluripotency related cell surface markers SSEA4, TRA-1-60 and TRA-1-81. After completion, the proportion of positive cells was detected by flow cytometry. The results showed that the cell product obtained by differentiation almost did not express pluripotency related markers, suggesting that there was no pluripotent cell residue and the completeness of differentiation.

[0017] Figure 8 Cell morphology of expanded iPSCs-derived vascular endothelial cells at P1 generation. The results of microscopic imaging of the cells obtained after the differentiation of Example 21, which were seeded into endothelial cell medium after digestion, showed uniform typical endothelial cell morphology. All images were obtained under 4x objective, and the scale bar is 500 μm.

[0018] Figure 9 Flow cytometry results of endothelial cell markers of iPSCs-derived vascular endothelial cells at P1, P2 and P3 generations. The results of Example 21 are shown. After the differentiation, the cells obtained were seeded into endothelial cell medium for continuous expansion, and after each expansion, the collected cells were co-stained with CD144-PE and CD31-APC antibodies and the proportion of CD144-PE and CD31-APC double positive cells was detected by flow cytometry. The results showed that the endothelial cells obtained by differentiation had a stable endothelial cell phenotype of CD144 and CD31 double positivity when expanded in vitro. The negative control is the flow cytometry result of the cells without antibody staining, and the isotype control is the flow cytometry result of the cells co-stained with CD144-PE and CD31-APC antibodies.

[0019] Figure 10 Acetylated low density lipoprotein uptake of iPSCs-derived vascular endothelial cells. Dil-Ac-LDL labeled human acetylated low density lipoprotein was added to the culture medium of iPSCs, mesoderm progenitor cells and endothelial cells differentiated from P1 and P2, and imaged under a fluorescence microscope after 6h of incubation, using a 10x objective, with a scale bar of 200μm. The results showed that the differentiated endothelial cells had strong fluorescent signals in the cells, indicating that their acetylated low density lipoprotein uptake was positive, consistent with the functional characteristics of endothelial cells.

[0020] Figure 11 NO production of iPSCs-derived vascular endothelial cells. NO probe DAF-FM DA was added to the culture medium of iPSCs, mesoderm progenitor cells and endothelial cells differentiated from P1 and P2, and imaged under a fluorescence microscope after 30min of incubation, using a 10x objective, with a scale bar of 200μm. The results showed that the differentiated endothelial cells had strong fluorescent signals, indicating that their NO production was positive, consistent with the functional characteristics of endothelial cells.

[0021] Figure 12 2D-tube formation of iPSCs-derived vascular endothelial cells in vitro. P1 iPSCs-derived vascular endothelial cells were seeded in Matrigel pre-coated culture plates, and 24h later Calcein-AM was added to 1μM, and imaged under a fluorescence microscope after 10min of incubation, using a 4x objective, with a scale bar of 1000μm. The results showed that iPSCs-derived vascular endothelial cells could form a continuous network structure, consistent with the in vitro function of endothelial cells. Example 1

[0022] 1.1 Matrigel-coated culture vessels (Day -2) 1.1.1 Matrigel dilution factor: According to the Matrigel batch number received, query the dilution factor of this batch of Matrigel, such as this batch recommends a dilution factor of 310μL, which means that 310μL can coat 4 6-well plates, i.e. 6-well plates per well is 12.9μL.

[0023] 1.1.2 Place 15mL centrifuge tubes, DMEM / F-12, 6-well plates and 10mL pipettes in the 2-8℃ refrigerator for at least 30min.

[0024] 1.1.3 Add 6mL of pre-cooled DMEM / F-12 to the 15mL centrifuge tube and place it on the ice-water mixture.

[0025] 1.1.4 Pre-cool the 200 pL tip in 6 mL of DMEM / F-12 by pipetting a few times, add the thawed Matrigel into the pre-cooled DMEM / F-12 (in a 15 mL centrifuge tube), mix well.

[0026] 1.1.5 The diluted Matrigel solution must be used immediately for coating the culture dishes, gently shake the culture dishes to evenly coat the surface of the culture dishes with the Matrigel solution.

[0027] 1.1.6 Incubate at least 1 hour at room temperature (15 - 25 °C) before use, do not allow the Matrigel to evaporate.

[0028] 1.2 iPSCs single cell seeding (Day -1) 1.2.1 Single cell digestion: wash the iPSCs at the time of passage in a 6-well plate with 2 mL PBS per well for 2 times, add 1.0 mL TrypLE Select per well, incubate at 37 °C in a 5% CO2 incubator for 7 min.

[0029] 1.2.2 Terminate digestion: terminate the digestion by adding 1.0 mL of TeSR-E8 complete medium containing 10 pM Y-27632 per well (1:1 dilution with the digestion solution), tap the edge of the culture plate to dissociate the iPSCs, transfer the cell suspension to a 15 mL centrifuge tube.

[0030] 1.2.3 Centrifugation: centrifuge at 270 g for 4 min at room temperature.

[0031] 1.2.4 Resuspension: after discarding the supernatant, resuspend the cell pellet in each well with 1.0 mL of TeSR-E8 complete medium containing 10 pM Y-27632, use a 1000 pL tip to gently pipette for 30 times to disperse the cells.

[0032] 1.2.5 Cell filtration: after mixing well, use a 40 pm filter to filter the cell suspension to obtain the iPSCs single cell suspension.

[0033] 1.2.6 Cell counting: after filtration, mix 20 pL of the iPSCs single cell suspension with 20 pL of AO / PI staining solution, count the cells, and record the live cell concentration and cell viability.

[0034] 1.2.7 Pre-coat the culture dish before use: before use, gently tilt one side of the culture dish to allow the excess Matrigel solution to pool on the edge of one side, remove the excess Matrigel solution, and ensure that the coated surface is not scratched.

[0035] 1.2.8 Immediately add TeSR-E8 complete medium containing 10 mM Y-27632 (2.0 mL per well for 6-well plate), and shake crossly.

[0036] 1.2.9 Cell seeding: Seed the iPSCs single cell suspension into the pre-coated 6-well plate at a density of 10,000 cells / cm 2 2 in terms of viable cells, and shake crossly, then place in 37°C, 5% CO2 incubator.

[0037] 1.3 Mesoderm cell differentiation induction (Day 0-2) 1.3.1 Change mesoderm cell differentiation induction medium I (Day 0-1): On day 0, if no abnormality is observed, completely discard the old medium, and immediately supplement the mesoderm induction medium (E6 medium + 8 mM CHIR-99021) into the culture system at a ratio of 3 mL medium per 10 cm 2 2 culture area, shake crossly, and then place in 37°C, 5% CO2 incubator for 24 h.

[0038] 1.3.2 Change mesoderm cell differentiation induction medium II (Day 1-2): On day 1, if no abnormality is observed, completely discard the old medium, and immediately supplement E6 medium into the culture system at a ratio of 3 mL medium per 10 cm 2 2 culture area, shake crossly, and then place in 37°C, 5% CO2 incubator for 24 h.

[0039] 1.4 Endothelial cell differentiation induction (Day 2-6) 1.4.1 Change endothelial cell differentiation induction medium I (Day 2-4): On day 2, if no abnormality is observed, completely discard the old medium, and immediately supplement endothelial cell differentiation induction medium I (E6 medium + 300 ng / mL VEGFA + 200 ng / mL FGF-2 + 1 mM 8-Bromoadenosine 3', 5'-cyclic monophosphate sodium salt monohydrate + 50 mM Melatonin) into the culture system at a ratio of 6 mL medium per 10 cm 2 2 culture area, shake crossly, and then place in 37°C, 5% CO2 incubator for 48 h, and examine under a microscope every 24 h.

[0040] 1.4.2 Change endothelial cell differentiation induction medium II (Day 4-6): On day 4, if no abnormality is observed, completely discard the old medium, and immediately supplement E6 medium into the culture system at a ratio of 6 mL medium per 10 cm 2Add endothelial induction medium II (E6 medium + 10 ng / mL VEGFA + 10 ng / mL FGF-2 + 10 μM Hydrocortisone) to the culture system at a ratio of 6 mL / cm², shake well, and incubate at 37°C in a 5% CO2 incubator for 48 h. Examine the culture system under a microscope every 24 h.

[0041] 1.5 Detection of the efficiency of iPSCs differentiating into vascular endothelial cells (day 6) 1.5.1 Cell digestion: per 10cm 2 The culture area was washed twice with 2.0 mL PBS, and then divided into groups of 10 cm². 2 Add 1.0 mL of TrypLE Select to the culture medium and incubate in a CO2 incubator for 9 min for digestion.

[0042] 1.5.2 Termination of digestion: Per 10cm 2 Add 1.0 mL of Advanced DMEM / F12 basal medium to the culture plate to stop digestion, tap the edge of the culture plate to dissociate the cells, and transfer the cell suspension to a 15 mL centrifuge tube.

[0043] 1.5.3 Centrifugation: Centrifuge at 400g for 5 min, RT.

[0044] 1.5.4 After discarding the supernatant, use Advanced DMEM / F12 basal medium (per 10 cm³). 2 The cells collected from the culture area were resuspended in 1.0 mL of water, and a single-cell suspension was obtained by gentle pipetting 35 times with a 1000 μL pipette tip.

[0045] 1.5.5 Cell counting: Mix 20 μL of single-cell suspension with 20 μL of AO / PI staining solution and count the cells. Record the concentration of viable cells and cell viability.

[0046] 1.5.6 Flow cytometry: 1 million cells were taken from each sample and divided into 3 equal parts. The first part was left untreated. The second part was stained with Mouse IgG1 kappa Isotype Control APC and Mouse IgG1 Isotype Control PE. The third part was stained with anti-human CD31 Antibody APC and anti-human CD144 Monoclonal Antibody PE. After staining, the proportion of CD31 and CD144 positive cells was detected by flow cytometer. Example 2

[0047] The difference from Example 1 is that the cell seeding density is 12000 cells / cm2 in live cell count 2 The rest is the same as Example 1. Example 3

[0048] The difference from Example 1 is that the cell seeding density is 14000 cells / cm2 in live cell count 2 The rest is the same as Example 1. Example 4

[0049] The difference from Example 1 is that the cell seeding density is 16000 cells / cm2 in live cell count 2 The rest is the same as Example 1. Example 5

[0050] The difference from Example 1 is that the cell seeding density is 18000 cells / cm2 in live cell count 2 The rest is the same as Example 1. Example 6

[0051] The difference from Example 1 is that the cell seeding density is 20000 cells / cm2 in live cell count 2 The rest is the same as Example 1. Example 7

[0052] The difference from Example 5 is that the composition of Mesoderm Induction Medium 1 is E6 medium added with 8 μΜ CHIR-99021 and 10 ng / mL hBMP4, and the rest is the same as Example 5. Example 8

[0053] The difference from Example 5 is that the composition of Mesoderm Induction Medium 1 is E6 medium added with 8 μΜ CHIR-99021, 25 ng / mL hBMP4, and the rest is the same as Example 5. Example 9

[0054] The difference from Example 5 is that the composition of Mesoderm Induction Medium 1 is E6 medium added with 8 μΜ CHIR-99021, 50 ng / mL hBMP4, and the rest is the same as Example 5. Example 10

[0055] The difference from Example 8 is that the composition of Mesoderm Induction Medium 1 is E6 medium added with 8 μΜ CHIR-99021, 25 ng / mL hBMP4 and 25 ng / mL Activin A, and the rest is the same as Example 8. Example 11

[0056] The difference from Example 8 is that the composition of the mesodermal induction medium 1 is E6 medium supplemented with 8 μM CHIR-99021, 25 ng / mL hBMP4 and 50 ng / mL Activin A, and the rest is the same as in Example 8. Example 12

[0057] The difference from Example 8 is that the composition of the mesodermal induction medium 1 is E6 medium supplemented with 8 μM CHIR-99021, 25 ng / mL hBMP4 and 100 ng / mL Activin A, and the rest is the same as in Example 8. Example 13

[0058] The difference from Example 11 is that the endothelial induction medium I is composed of E6 medium supplemented with 300 ng / mL VEGFA, 200 ng / mL FGF-2, 1 mM 8-Bromoadenosine 3′,5′-cyclic monophosphate sodium salt monohydrate, 50 μM Melatonin and 5 μM SB431542, the rest being the same as in Example 11. Example 14

[0059] The difference from Example 11 is that the endothelial induction medium I is composed of E6 medium supplemented with 300 ng / mL VEGFA, 200 ng / mL FGF-2, 1 mM 8-Bromoadenosine 3′,5′-cyclic monophosphate sodium salt monohydrate, 50 μM Melatonin and 10 μM SB431542, and the rest is the same as in Example 11. Example 15

[0060] The difference from Example 11 is that the endothelial induction medium I is composed of E6 medium supplemented with 300 ng / mL VEGFA, 200 ng / mL FGF-2, 1 mM 8-Bromoadenosine 3′,5′-cyclic monophosphate sodium salt monohydrate, 50 μM Melatonin and 20 μM SB431542, and the rest is the same as in Example 11. Example 16

[0061] The difference from Example 14 is that the composition of Endothelial Induction Medium I is E6 medium supplemented with 300 ng / mL VEGFA, 200 ng / mL FGF-2, 1 mM 8-Bromoadenosine 3',5'-cyclic monophosphate sodium salt monohydrate, 50 µM Melatonin, 10 µM SB431542 and 1 µM forskolin, the rest is the same as Example 14. Example 17

[0062] The difference from Example 14 is that the composition of Endothelial Induction Medium I is E6 medium supplemented with 300 ng / mL VEGFA, 200 ng / mL FGF-2, 1 mM 8-Bromoadenosine 3',5'-cyclic monophosphate sodium salt monohydrate, 50 µM Melatonin, 10 µM SB431542 and 2 µM forskolin, the rest is the same as Example 14. Example 18

[0063] The difference from Example 14 is that the composition of Endothelial Induction Medium I is E6 medium supplemented with 300 ng / mL VEGFA, 200 ng / mL FGF-2, 1 mM 8-Bromoadenosine 3',5'-cyclic monophosphate sodium salt monohydrate, 50 µM Melatonin, 10 µM SB431542 and 4 µM forskolin, the rest is the same as Example 14. Example 19

[0064] The difference from Example 17 is that the composition of Endothelial Induction Medium II is E6 medium supplemented with 5 ng / mL VEGFA, 10 ng / mL FGF-2 and 10 µM Hydrocortisone, the rest is the same as Example 17. Example 20

[0065] The difference from Example 17 is that the composition of Endothelial Induction Medium II is E6 medium supplemented with 20 ng / mL VEGFA, 10 ng / mL FGF-2 and 10 µM Hydrocortisone, the rest is the same as Example 17. Example 21

[0066] The difference from Example 17 is that the composition of endothelial induction medium II is E6 medium added with E6 medium added with 50 ng / mL VEGFA, 10 ng / mL FGF-2 and 10 μΜ Hydrocortisone, and the rest is the same as Example 17. Example 22

[0067] The difference from Example 17 is that the composition of endothelial induction medium II is E6 medium added with E6 medium added with 100 ng / mL VEGFA, 10 ng / mL FGF-2 and 10 μΜ Hydrocortisone, and the rest is the same as Example 17. Example 23

[0068] Residual detection of pluripotency markers 23.1 Preparation of cells to be tested 23.1.1 Preparation of vascular endothelial cells from iPSCs to be tested: cells at the end of the 6th day of the differentiation scheme described in Example 21 were selected for testing, and the operation of collecting and counting them was the same as in Example 21.

[0069] 23.1.2 Preparation of iPSCs to be tested: iPSCs about to be passaged were selected for testing, and the operation of collecting and counting them was the same as in Example 21.

[0070] 23.2 Staining of surface markers of pluripotency 23.2.1 Cell fixation: after the counting of the cells to be tested was completed, centrifugation was performed at 400 g for 3 min, the supernatant was removed, and the cell pellet was resuspended with 4% paraformaldehyde (1.0 mL per 1E+06 cells), and incubated at room temperature for 10-15 min for fixation.

[0071] 23.2.2 Cell washing: after fixation was completed, centrifugation was performed at 500 g for 3 min, the cell pellet was resuspended with PBS (2.0 mL per 1E+06 cells) after the supernatant was removed, and centrifugation was performed at 500 g for 3 min after standing for 5 min, and the operation was repeated twice.

[0072] 23.2.3 Cell aliquoting: the cells were aliquoted into 1.5 mL EP tubes at 1E+06 / tube.

[0073] 23.2.4 Incubation of primary antibodies: after aliquoting was completed, centrifugation was performed at 500 g for 3 min, the supernatant was removed, and the cell pellet was resuspended with 250 μL of primary antibody staining solution (containing Anti-SSEA-4 4 Antibody, Anti-TRA-1-60 Antibody, Anti-TRA-1-81 Antibody, respectively), and incubated overnight in a 4°C medical refrigerator.

[0074] 23.2.5 Primary antibody washing: After the primary antibody incubation is complete, centrifuge at 500g for 3min, discard the supernatant, add 750μl of washing buffer to each tube, shake and resuspend, let stand for 5min, centrifuge at 500g for 3min, and repeat twice.

[0075] 23.2.6 Secondary antibody incubation: After washing with primary antibody, add 250 μL of secondary antibody staining solution to each tube to resuspend the cell pellet and incubate overnight at 4°C.

[0076] 23.2.7 Secondary antibody washing: After the secondary antibody incubation is completed, centrifuge at 500g for 3min, discard the supernatant, add 750μl of washing buffer to each tube, shake and resuspend, let stand for 5min, centrifuge at 500g for 3min, and repeat twice.

[0077] 23.2.8 Flow cytometry: After washing with secondary antibody, 50 μL of PBS was added to each tube to resuspend the cell pellet, and the pluripotency marker signal of each cell was immediately detected on a flow cytometer.

[0078] 23.3 Results of residual pluripotency marker detection in iPSC-derived vascular endothelial cells: Compared to iPSC cells, vascular endothelial cells derived from a compound- and cytokine-dominated differentiation protocol showed significant negativity on pluripotency-related surface markers, such as... Figure 7 As shown, this is consistent with the extremely high differentiation efficiency, indicating that the differentiation is complete. Example 24

[0079] Expansion of iPSC-derived vascular endothelial cells and phenotypic detection of expanded cells 24.1 gelatin-coated culture dishes: One day before subculture, dilute the 2% gelatin solution to 0.2% with PBS, add 1.0 mL of 0.2% gelatin solution per 10 cm2 culture area, and incubate at 37°C for later use.

[0080] 24.2 Collection and Counting of Vascular Endothelial Cells Derived from P0 Generation iPSCs 24.2.1 Cell digestion: For cells that have completed differentiation, digest every 10 cm³. 2 The culture area was washed twice with 2.0 mL PBS, and then divided into groups of 10 cm². 2 Add 1.0 mL of TrypLE Select to the culture medium and incubate in a CO2 incubator for 9 min for digestion.

[0081] 24.2.2 Termination of digestion: Per 10cm 2 Add 1.0 mL of EGM-2 complete culture medium to the culture plate to stop digestion, tap the edge of the culture plate to dissociate the cells, and transfer the cell suspension to a 15 mL centrifuge tube.

[0082] 24.2.3 Centrifugation: 400g for 5 min, RT.

[0083] 24.2.4 Discard the supernatant and resuspend the cell pellet with 1.0 mL of EGM-2 complete medium (per 10 cm 2 Discard the supernatant and resuspend the cell pellet with 1.0 mL of EGM-2 complete medium (per 10 cm

[0084] 24.2.5 Cell counting: Take 20 μL of the single cell suspension and mix with 20 μL of AO / PI staining solution. Count the cells and record the viable cell concentration and cell viability.

[0085] 24.3 Seeding and culturing of P1 iPSCs-derived vascular endothelial cells 24.3.1 Remove the liquid in the 0.2% gelatin pre-coated culture dish and add 2.0 mL of fresh EGM-2 complete medium (per 10 cm 2 Discard the supernatant and resuspend the cell pellet with 1.0 mL of EGM-2 complete medium (per 10 cm

[0086] 24.3.2 According to the counting results of the P0 iPSCs-derived vascular endothelial cells collected, seed 12,000 cells / cm 2 of viable cells into the 0.2% gelatin pre-coated culture dish, record as P1, and shake well. Then, place the dish in a 37°C, 5% CO2 incubator for culture. Replace the medium every 2 days until complete confluence. 24.4 The seeding and culturing of P2 and P3 iPSCs-derived vascular endothelial cells are consistent with P1.

[0087] 24.5 Phenotype detection of P1, P2 and P3 iPSCs-derived vascular endothelial cells 24.5.1 Cell digestion: Wash the culture dish twice with 2.0 mL of PBS (per 10 cm 2 Discard the supernatant and resuspend the cell pellet with 1.0 mL of EGM-2 complete medium (per 10 cm 2 Discard the supernatant and resuspend the cell pellet with 1.0 mL of EGM-2 complete medium (per 10 cm

[0088] 24.5.2 Termination of digestion: Add 1.0 mL of EGM-2 complete medium (per 10 cm 2 Termination of digestion: Add 1.0 mL of EGM-2 complete medium (per 10 cm

[0089] 24.5.3 Centrifugation: 400g for 5 min, RT.

[0090] 24.5.4 After discarding the supernatant, the cells were incubated with EGM-2 complete medium (1.0 mL per 10 cm2 of culture area) 2 The collected cells were added to 1.0 mL of resuspension buffer, and a single cell suspension was obtained by gently pipetting 1000 μL of the cell pellet 35 times with a pipette tip.

[0091] 24.5.5 Cell counting: 20 μL of the single cell suspension was mixed with 20 μL of AO / PI staining solution, and the number of viable cells was counted. The concentration of viable cells and the cell viability were recorded.

[0092] 24.5.6 Flow cytometry: 1 million cells were taken from each sample and divided into three equal parts. The first part was not treated, the second part was stained with Mouse IgG1 kappa Isotype Control APC and Mouse IgG1 Isotype Control PE, and the third part was stained with anti-human CD31 Antibody APC and anti-human CD144 Monoclonal Antibody PE. After staining, the proportion of CD31 and CD144 positive cells was detected by flow cytometry.

[0093] 24.6 Results of the phenotype detection of the iPSC-derived vascular endothelial cells after expansion: The vascular endothelial cells generated by the compound and cytokine-based differentiation protocol can be stably expanded in vitro and have a typical vascular endothelial cell morphology, as shown in FIG. 6A. After 3 generations of expansion culture, the total expansion ratio was about 100 times, and the surface markers of the vascular endothelial cells did not change, as shown in FIG. 6B. Figure 8 Figure 9 Example 25

[0094] Detection of the low-density lipoprotein uptake capacity of iPSC-derived vascular endothelial cells 25.1 Preparation of cells to be tested 25.1.1 Preparation of iPSC-derived vascular endothelial cells to be tested: P1 and P2 iPSC-derived vascular endothelial cells were selected for testing, and the method of inoculation and culture was the same as in Example 24.

[0095] 25.1.2 Preparation of iPSC cells to be tested: iPSCs from the day before passage were selected for testing.

[0096] 25.1.3 Preparation of mesodermal progenitor cells to be tested: cells after the end of the second day of the differentiation protocol were selected for testing, and the differentiation process was the same as in Example 11.

[0097] 25.2 Incubation and observation of low-density lipoprotein ​​25.2.1 For the vascular endothelial cells derived from iPSCs, on day 3 of P1 and P2 generation, add Human Dil-Ac-LDL to 15 μg / mL to each well of a 12-well plate, and after gentle mixing, incubate at 37°C in a 5% CO2 incubator for 6 h. For the iPSCs cells, on the day before passage, add Human Dil-Ac-LDL to 15 μg / mL to each well of a 12-well plate, and after gentle mixing, incubate at 37°C in a 5% CO2 incubator for 6 h. For the mesodermal progenitor cells, on the day after the end of the second day of the differentiation protocol, add Human Dil-Ac-LDL to 15 μg / mL to each well of a 12-well plate, and after gentle mixing, incubate at 37°C in a 5% CO2 incubator for 6 h.

[0098] 25.2.2 After incubation, discard the medium in the 12-well plate, and wash each well 4 times with 0.5 mL of medium (EGM-2 complete medium for the vascular endothelial cells derived from iPSCs, E8 medium for the iPSCs cells, and E6 medium for the mesodermal progenitor cells), and then add 1.0 mL of medium (EGM-2 complete medium for the vascular endothelial cells derived from iPSCs, E8 medium for the iPSCs cells, and E6 medium for the mesodermal progenitor cells) to each well, and immediately take a fluorescent image under a fluorescence microscope.

[0099] 25.2.3 The results of the low-density lipoprotein uptake detection of the vascular endothelial cells derived from iPSCs: the vascular endothelial cells produced by the differentiation protocol directed by the compounds and cytokines have a significant low-density lipoprotein uptake capacity, as shown in Figure 10 which is consistent with the function of typical vascular endothelial cells. Example 26

[0100] Detection of the NO production capacity of the vascular endothelial cells derived from iPSCs 26.1 Preparation of the cells to be tested 26.1.1 Preparation of the vascular endothelial cells derived from iPSCs to be tested: select the vascular endothelial cells derived from iPSCs of P1 and P2 generation for testing, and perform the inoculation and culture according to Example 24.

[0101] 24.1.2 Preparation of the iPSCs cells to be tested: select the iPSCs on the day before passage for testing.

[0102] 26.1.3 Preparation of the mesodermal progenitor cells to be tested: select the cells after the end of the second day of the differentiation protocol for testing, and perform the differentiation according to Example 11.

[0103] 26.2 Incubation and observation of the NO probe 26.2.1 For the cells to be tested, add L-NAME HCl to 5 mM in the culture system of iPSCs-derived vascular endothelial cells at P1 and P2, mesodermal progenitor cells at the end of day 1 of differentiation protocol, and shake well before incubation at 37 °C, 5% CO2 overnight.

[0104] 26.2.2 For the cells to be tested, add DAF-FM DA to 1 mM in the culture system of iPSCs-derived vascular endothelial cells at P1 and P2, mesodermal progenitor cells at day 2 of differentiation protocol, and shake well before incubation at 37 °C, 5% CO2.

[0105] 26.2.3 After 30 min of adding DAF-FM DA, discard the old medium, wash each culture system with PBS for 3 times, and then add the corresponding fresh medium (iPSCs-derived vascular endothelial cells use EGM-2 complete medium, iPSCs use E8 medium, and mesodermal progenitor cells use E6 medium), and directly detect the FITC channel fluorescence signal under a fluorescence microscope.

[0106] 26.3 The results of detecting the NO production ability of iPSCs-derived vascular endothelial cells: compared with iPSCs and mesodermal progenitor cells, the vascular endothelial cells produced by the differentiation protocol dominated by compounds and cytokines have a significant NO production ability, as shown in Figure 11 , which is consistent with the function of typical vascular endothelial cells. Example 27

[0107] 2D tube formation ability test of iPSCs-derived vascular endothelial cells 27.1 Preparation of cells to be tested: select iPSCs-derived vascular endothelial cells at P1 for testing, and the method of inoculation and culture is the same as that in Example 24.

[0108] 27.2 Matrigel coating: 1 h before the end of the culture of iPSCs-derived vascular endothelial cells at P1, add 350 μL of Matrigel (Corning, 354234) stock solution to 2 wells of a 12-well plate in a biological safety cabinet, respectively, and wait for it to spread flat. After completion, transfer to a 37 °C, 5% CO2 incubator and wait for it to gel.

[0109] 27.3 Collect iPSCs-derived vascular endothelial cells at P1 and count them, which is the same as in Example 24.

[0110] 27.4 2D-tube vascular endothelial cell seeding: according to the counting results, the density of iPSCs-derived vascular endothelial cells was adjusted to 70,000 / mL with EGM-2 complete medium, 1 mL of iPSCs-VECs single cell suspension was inoculated into each well of the pre-paved 12-well plate, and then it was placed in a 37°C, 5% CO2 incubator for 24 hours after shaking.

[0111] 27.5 Live cell dye incubation: after 24 hours of 2D-tube vascular endothelial cell seeding, Calcein, AM was added to each culture system at 1 μM, and then it was placed in a 37°C, 5% CO2 incubator for 10 minutes after shaking.

[0112] 27.6 Dye washing: after 10 minutes, the culture medium was aspirated, each well was washed twice with 1.0 mL of EGM-2 complete medium, and then 1.0 mL of fresh EGM-2 complete medium was added to each well.

[0113] 27.7 2D-tube morphology observation and imaging: the fluorescence image under the FITC / GFP channel was taken under the fluorescence microscope.

[0114] 2D tube formation ability test results of iPSCs-derived vascular endothelial cells: the vascular endothelial cells generated by the compound and cytokine-dominated differentiation scheme can form typical 2D tubes in vitro, as shown in Figure 12 , which is consistent with the reported vascular endothelial function.

[0115] Summary table of results of each embodiment: Efficiency of differentiation of vascular endothelial cells calculated from CD31 and CD144 double positive cells Whether having the phenotype of vascular endothelial cells Whether having the function of vascular endothelial cells Whether the differentiated cells express pluripotency related marker SSEA-4 Whether the differentiated cells express pluripotency related marker TRA-1-60 Whether the differentiated cells express pluripotency related marker TRA-1-81 Low density lipoprotein uptake capacity of vascular endothelial cells NO production capacity of vascular endothelial cells 2D tube formation capacity of vascular endothelial cells Example 1 72.1% Yes Yes No No No Positive Positive Positive Example 2 77.4% Yes Yes No No No Positive Positive Positive Example 3 86.6% Yes Yes No No No Positive Positive Positive Example 4 89.9% Yes Yes No No No Positive Positive Positive Example 5 92.1% Yes Yes No No No Positive Positive Positive Example 6 91.5% Yes Yes No No No Positive Positive Positive Example 7 93.2% Yes Yes No No No Positive Positive Positive Example 8 94.0% Yes Yes No No No Positive Positive Positive Example 9 93.5% Yes Yes No No No Positive Positive Positive Example 10 95.1% Yes Yes No No No Positive Positive Positive Example 11 96.3% Yes Yes No No No Positive Positive Positive Example 12 96.1% Yes Yes No No No Positive Positive Positive Example 13 97.5% Yes Yes No No No Positive Positive Positive Example 14 98.4% Yes Yes No No No Positive Positive Positive Example 15 98.3% Yes Yes No No No Positive Positive Positive Example 16 98.5% Yes Yes No No No Positive Positive Positive Example 17 98.8% Yes Yes No No No Positive Positive Positive Example 18 98.5% Yes Yes No No No Positive Positive Positive Example 19 99.0% Yes Yes No No No Positive Positive Positive Example 20 99.2% Yes Yes No No No Positive Positive Positive Example 21 99.3% Yes Yes No No No Positive Positive Positive Example 22 99.1% Yes Yes No No No Positive Positive Positive The sources of the reagents and other materials mentioned in this patent application are shown in the following table: Material name Manufacturer Part number TeSR-E8 Complete Kit STEMCELL 05990 E6 medium Life technologies A1516401 ReLeSR STEMCELL 5872 AO / PI staining solution Countstar H42021503 TrypLE Select Gibco 12563-029 Matrigel Corning 354277 DMEM / F12 Life technologies 11320-033 Y-27632 Selleck S6390 PBS Without Ca++ and Mg++ BI 02-024-1ACS 8-Bromo-cAMP sodium salt MCE HY-12306 Melatonin Sigma-Aldrich M5250 hydrocortisone selleck S1696 CHIR-99021 Selleck S1263 Recombinant Human BMP-4 R&D CR93 Activin A R&D GMP-C687 VEGF-A R&D C083 FGF-2 R&D GMP-C046 SB431542 Selleck S1067 Forskolin selleck S2449 Mouse IgG1 kappa Isotype Control APC eBioscienc 17-4714-82 Mouse IgG1 Isotype Control PE ThermoFisher MG104 APC anti-human CD31 Antibody Biolegend 303116 PE anti-humanCD144 Monoclonal Antibody Thermo Fisher Scientific 14-1449-82 Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor488 Thermo Fisher Scientific A-21206 Recombinant Anti-Brachyury Antibody Abcam ab209665 The above embodiments are only for illustrating the technical concept and characteristics of the present application, and the purpose is to enable those skilled in the art to understand the content of the present application and implement it, and it cannot limit the protection scope of the present application, and any equivalent changes or modifications made according to the spirit and essence of the present application should be covered within the protection scope of the present application.

Claims

1. A method of inducing iPSCs to differentiate directionally into vascular endothelial cells by a combination of a small molecule compound and a cytokine composition, characterized by, The method comprises the following steps: (1) inoculating iPSCs in an inoculation medium; (2) replacing the old iPSC inoculation medium with mesoderm cell differentiation induction medium I; (3) replacing the old mesoderm cell differentiation induction medium I with E6 medium; (4) replacing the old E6 medium with endothelial cell differentiation induction medium I; (5) replacing the old endothelial cell differentiation induction medium I with endothelial cell differentiation induction medium II; (6) obtaining vascular endothelial cells after treatment with endothelial cell differentiation induction medium II.

2. The method of inducing the directed differentiation of iPSCs into vascular endothelial cells from a small molecule compound and cytokine composition according to claim 1, characterized in that The differentiation efficiency of the vascular endothelial cells is more than 99% according to CD144 and CD31 double positive cells.

3. The method of inducing the directed differentiation of iPSCs into vascular endothelial cells by a small molecule compound and a cytokine composition according to claim 1, characterized in that, The replacement of the old iPSC inoculation medium with mesoderm cell differentiation induction medium I to initiate the iPSC differentiation process is defined as day 0 of the scheme, the iPSC inoculation is completed on day-1, the iPSC inoculation medium treatment time is day-1 to day 0, the replacement of the old iPSC inoculation medium with mesoderm cell differentiation induction medium I is completed on day 0, the mesoderm cell differentiation induction medium I treatment time is day 0-1, the replacement of the old mesoderm cell differentiation induction medium with E6 medium is completed on day 1, the E6 medium treatment time is day 1-2, the replacement of the old E6 medium with endothelial cell differentiation induction medium I is completed on day 2, the endothelial cell differentiation induction medium I treatment time is day 2-4, the replacement of the old endothelial cell differentiation induction medium I with endothelial cell differentiation induction medium II is completed on day 4, and the endothelial cell differentiation induction medium II treatment time is day 4-6.

4. The method of inducing the directed differentiation of iPSCs into vascular endothelial cells by a small molecule compound and a cytokine composition according to claim 1, characterized in that, The iPSCs inoculated in the step 1 are single-cell-digested iPSCs, and the cell inoculation density of the iPSCs is 10,000 cells / cm 2 - 20,000 cells / cm 2 .

5. The method of claim 1, wherein the small molecule compound and cytokine composition are used to induce the directional differentiation of iPSCs into vascular endothelial cells. The iPSC inoculation medium in step 1 is TeSR-E8 medium containing 10 μM Y-27632.

6. The method of inducing the directed differentiation of iPSCs into vascular endothelial cells from a small molecule compound and cytokine composition according to claim 1, characterized in that, The base medium of the mesoderm cell differentiation induction medium I is E6 medium, and the additives include one or a mixture of WNT signal activator, hBMP4 and Activin-A, wherein the WNT signal activator is CHIR-99021, and the addition amount of CHIR-99021, hBMP4 and Activin-A is 8 μM, 10-50 ng / mL and 25-100 ng / mL, respectively.

7. The method of claim 1, wherein the small molecule compound and cytokine composition are used to induce the directional differentiation of iPSCs into vascular endothelial cells. The basal medium of the endothelial cell differentiation induction medium I is E6 medium, and the additives include VEGFA, FGF-2, Melatonin and a cAMP analogue, and further include one of a TGF-beta signal inhibitor, a cAMP signal activator or a mixture thereof, wherein the cAMP analogue is 8-Bromoadenosine 3',5'-cyclic monophosphate sodium salt monohydrate, the TGF-beta signal inhibitor is SB431542, and the cAMP signal activator is Forskolin, and the added amounts of VEGFA, FGF-2, SB431542, Forskolin, Melatonin and 8-Bromoadenosine 3',5'-cyclic monophosphate sodium salt monohydrate are 300 ng / mL, 200 ng / mL, 5-20 µM, 1-4 µM, 50 µM and 1 mM respectively.

8. The method of claim 1, wherein the small molecule compound and cytokine composition are used to induce the directional differentiation of iPSCs into vascular endothelial cells. The basal medium of the endothelial cell differentiation induction medium II is E6 medium, and the additives include VEGFA, FGF-2 and Hydrocortisone, and the added amounts of VEGFA, FGF-2 and Hydrocortisone are 5-100 ng / mL, 10 ng / mL and 10 µM respectively.

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