Application of Yoda1 in promoting differentiation of iPSC-ECs

Abstract

The invention relates to the technical field of biological medicines, and discloses an application of Yoda1 in promoting differentiation of iPSC-ECs. The application of the Yoda1 in promoting the differentiation of the iPSC-ECs is specifically as follows: adding the Yoda1 into a culture medium in a mesoderm differentiation stage and an endothelial differentiation stage, and intervening the differentiation of the iPSC-ECs. According to the application of the Yoda1 in promoting the differentiation of the iPSC-ECs, provided by the invention, after the Yoda1 is added to a culture medium in a mesoderm differentiation stage and an endothelial differentiation stage, the iPSC-ECs formed by differentiation have relatively high positive cell rate, relatively high mRNA levels of CD144, eNOS and Piezo1 and relatively high protein levels of CD144 and Piezo1, which indicates that the Yoda1 promotes the differentiation efficiency of the iPSC-ECs; according to the application of the Yoda1 in promoting differentiation of the iPSC-ECs provided by the invention, after the Yoda1 is added to a culture medium in a mesoderm differentiation stage and an endothelial differentiation stage, the iPSC-ECs formed by differentiation have relatively high total number of tube forming intersection points and total area of a tube cavity, which indicates that the Yoda1 promotes cell tube forming of the iPSC-ECs.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an application of Yoda1 in promoting the differentiation of iPSC-ECs. Background Art

[0002] Induced pluripotent stem cells (iPSCs) are a type of pluripotent stem cells with similar pluripotency characteristics to embryonic stem cells, with the potential for self-renewal and multidirectional differentiation; iPSCs can be induced to differentiate into various specific cell types, among which iPSC-derived endothelial cells (iPSC-ECs) are an important cell type; iPSC-ECs have shown great potential and application value in promoting wound healing, angiogenesis, and treating cardiovascular diseases; in view of this, how to effectively promote the differentiation of iPSC-ECs has become a technical problem that technicians in this field urgently need to solve. Summary of the invention

[0003] The purpose of the present invention is to provide an application of Yoda1 in promoting the differentiation of iPSC-ECs.

[0004] To achieve the above object, the present invention provides the following technical solutions:

[0005] An application of Yoda1 in promoting the differentiation of iPSC-ECs.

[0006] Furthermore, the application is specifically: adding Yoda1 to the culture medium in the mesoderm differentiation stage and the endothelial differentiation stage to intervene in the differentiation of iPSC-ECs.

[0007] Furthermore, the intervention concentration of Yoda1 is 5 μM.

[0008] Furthermore, the intervention duration of Yoda1 is 1 hour.

[0009] Compared with the prior art, the present invention has the following beneficial effects:

[0010] The present invention provides an application of Yoda1 in promoting the differentiation of iPSC-ECs. After Yoda1 is added to the culture medium in the mesoderm differentiation stage and the endothelial differentiation stage, the differentiated iPSC-ECs have a higher CD144 positive cell rate, higher CD144, eNOS and Piezo1 mRNA levels, and higher CD144 and Piezo1 protein levels, which indicates that Yoda1 promotes the differentiation efficiency of iPSC-ECs.

[0011] The present invention provides an application of Yoda1 in promoting the differentiation of iPSC-ECs. After Yoda1 is added to the culture medium in the mesoderm differentiation stage and the endothelial differentiation stage, the differentiated iPSC-ECs form a higher total number of tube intersections and a total tube lumen area in an in vitro tube formation experiment, which indicates that Yoda1 promotes the cell tube formation of iPSC-ECs. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the detailed description of the preferred embodiments below. The accompanying drawings are only for the purpose of illustrating the preferred embodiments and are not to be considered as limiting the present invention. Moreover, the same reference symbols are used throughout the accompanying drawings to represent the same components. In the accompanying drawings:

[0013] Figure 1 The results of calcium influx detection are shown in Figure 1, where A is the intracellular Ca2+ in iPSCs induced by 10 μM Yoda1 group, 5 μM Yoda group and DMSO control group. 2+ Level changes; B is the difference comparison of calcium influx peak values ​​among 10μM Yoda1 group, 5μM Yoda group and DMSO control group;

[0014] Figure 2 This is the CCK8 detection result diagram;

[0015] Figure 3 The results of the CD144 positive cell rate detection of iPSC-ECs are shown in Figure 1, where A is the normal control group (67.3%), B is the Yoda1 5μM 1h / d intervention group (84.5%), and C is the Yoda1 5μM continuous intervention group (60.5%);

[0016] Figure 4 Figure 2 is the mRNA level of CD144, eNOS and Piezo1 in iPSC-ECs;

[0017] Figure 5 The protein expression levels of CD144 and Piezo1 in iPSC-ECs are shown in Figure 1, where A is the protein band from the western blot experiment, and B is the result of quantifying and comparing the differences of the protein bands using Image J software;

[0018] Figure 6 The photos of the iPSC-ECs cell tube formation experiment after CD144+ sorting, A is the normal control group, B is the Yoda1 5μM 1h / d intervention group;

[0019] Figure 7This is the result of quantifying the cell tube formation experiment using Image J software and performing one-way analysis of variance, where A is the quantification and statistical analysis of the total lumen area, and B is the quantification and statistical analysis of the total number of tube intersections. DETAILED DESCRIPTION

[0020] Now, various exemplary embodiments of the present invention are described in detail, and this detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present invention. It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention.

[0021] In addition, for the numerical range in the present invention, it is understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0022] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0023] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0024] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0025] In the following examples, the iPSCs (induced pluripotent stem cells) are hiPSCs (human induced pluripotent stem cells) purchased from Beijing Saibei Biotechnology Co., Ltd.;

[0026] In the following examples, the drugs, reagents and consumables are all commercially available, as shown in Table 1;

[0027] Table 1 Drugs, reagents and consumables

[0028]

[0029]

[0030]

[0031] In the following examples, the experimental instruments are all commercially available, as shown in Table 2;

[0032] Table 2 Experimental instruments

[0033]

[0034]

[0035] Example 1

[0036] Culture of iPSCs

[0037] 1)Planning:

[0038] Add 1 mL of human pluripotent stem cell substrate solution to each well of a six-well plate and incubate overnight at 37°C;

[0039] 2) Cell recovery:

[0040] The frozen iPSCs cells were taken out of liquid nitrogen and thawed in a water bath at 37°C. The thawed iPSCs cells were then transferred to a 15 mL centrifuge tube containing 5 mL of human pluripotent stem cell culture medium and centrifuged at 1200 rpm for 5 min at room temperature.

[0041] After the centrifugation is completed, the supernatant is aspirated without touching the cell aggregates, and then 1 mL of human pluripotent stem cell culture medium is added to the centrifuge tube to resuspend the cells to obtain a cell suspension;

[0042] The human pluripotent stem cell substrate solution in the six-well plate in step 1) was aspirated, and then the cell suspension was seeded in the six-well plate, and 2 mL of human pluripotent stem cell culture medium containing 5 μM Y27632 was added to each well of the six-well plate, and cultured in an incubator at 37° C., and the human pluripotent stem cell culture medium was changed every day;

[0043] 3) Cell passaging:

[0044] When the cell density in the six-well plate in step 2) reaches 80%, the medium in the six-well plate is aspirated and washed once with DPBS;

[0045] Add 0.5 mL of human pluripotent stem cell digestion solution to each well of the six-well plate, and culture at 37°C for 3-4 minutes. When the cell edges are rolled up, remove the digestion solution and add 2 mL of human pluripotent stem cell culture medium containing 5 μM Y27632. Gently pipette to form a cell suspension.

[0046] According to the subculture ratio of 1:6, the culture medium of human pluripotent stem cells containing 5 μM Y27632 was supplemented to 2 mL and cultured in an incubator at 37°C.

[0047] The optimal intervention concentration of Yoda1 was determined by detecting calcium influx levels using a cellular calcium ion fluorescent probe (Fura-2, AM)

[0048] 1) Probe loading: Cells were cultured in 96-well plates and divided into three groups: 10 μM Yoda1 group, 5 μM Yoda1 group, and DMSO control group. Each group had 6 wells, for a total of 18 wells. 6×10 6 iPSCs cells were cultured for 24 hours and loaded with probes after the cells were confluent.

[0049] The culture medium was removed by aspiration, and the cells were washed once with SBS solution, and 50 μL SBS solution containing 4 μM Fura-2 AM and 0.02% Pluronic F127 was added, and the cells were cultured in the dark for 1 h; the cells were washed twice with SBS solution, and 200 μL SBS solution was added;

[0050] 2) Detection using a desktop calcium flow detection workstation: The system was set to automatically add different concentrations of Yoda1 solution 30 seconds after the start of the detection, so that the final concentrations of Yoda1 in the cell buffer SBS were 10 μM, 5 μM, and 0, respectively. Fura-2 was excited at 340 nm and 380 nm wavelengths, and the fluorescence intensity of the emitted light at 510 nm was detected. The intracellular calcium ion concentration was calculated by the ratio of the fluorescence intensity F340 / F380 corresponding to the two excitations;

[0051] The results of calcium ion influx test are as follows Figure 1 As shown, A is the intracellular Ca2+ of iPSCs induced by 10 μM Yoda1 group, 5 μM Yoda group and DMSO control group. 2+ Level changes; B is the difference comparison of calcium influx peak values ​​among 10μM Yoda1 group, 5μM Yoda group and DMSO control group;

[0052] Depend on Figure 1 It can be seen that compared with the DMSO control group, both the 10μM Yoda1 group and the 5μM Yoda group could induce Ca 2+ The levels of Yoda1 increased (P<0.001), but there was no significant difference between the 10μM Yoda1 group and the 5μM Yoda group (P>0.05); therefore, in order to reduce the effect of Yoda1 on the viability of iPSCs cells, 5μM Yoda1 was determined to be the optimal intervention concentration.

[0053] Determining the optimal intervention duration of Yoda1 based on CCK8 detection

[0054] iPSCs in the logarithmic growth phase were digested and 100 μL of 5000 cells were added to each well. The digested PSCs were added to a 96-well plate and incubated at 37°C and 5% CO. 2 The cells were cultured in a cell culture incubator; after adhesion, the cells were grouped into 5 μM Yoda1 0h group, 1h group, 2h group, 12h group, 24h group, 36h group, and DMSO control 0h group, 1h group, 2h group, 12h group, 24h group, and 36h group, with 5 wells in each group, for a total of 60 wells; 10 μL CCK-8 solution was added to each well for 1 h before detection, and the absorbance value at a wavelength of 450 nm was detected with an enzyme marker (A); cell viability = (A Tn / A T0 )×100%;

[0055] CCK8 test results Figure 2 As shown;

[0056] Depend on Figure 2 It can be seen that 5μM Yoda1 can reduce the proliferation of PSCs after intervening in iPSCs for 12h, 24h or 36h, while intervening for 1h or 2h will not have a significant effect on the proliferation of iPSCs. Therefore, 1 to 2h is determined to be the optimal intervention time for Yoda1.

[0057] Example 2

[0058] Induction of differentiation of iPSCs into ECs (Yoda1 5μM 1h / d)

[0059] 1) Preparation of Yoda1 solution:

[0060] Add 0.66 mg of Yoda1 to 37.155 μL of DMSO to obtain a Yoda1 solution with a concentration of 5 μM;

[0061] 2) Mesoderm differentiation stage 1 (day 0-2):

[0062] On the first day, DMEM containing 25 ng / mL BMP4, 12 ng / mL Actin-A, 6 μM CHIR99021 and 5 μM Yoda1 was used as a differentiation medium to induce differentiation of the iPSCs cells cultured in Example 1 in a 37°C incubator for 1 h, and then the medium was replaced with DMEM medium containing 25 ng / mL BMP4, 12 ng / mL Actin-A and 6 μM CHIR99021 to continue inducing differentiation. On the second day, the operation of day 1 was repeated.

[0063] 3) Mesoderm differentiation stage 2 (day 3-4):

[0064] On the third day, DMEM containing 25 ng / mL BMP4, 12 ng / mL activin A, 3 μM CHIR99021 and 5 μM Yoda1 was used as differentiation medium. The iPSCs cells induced to differentiate in step 2) were induced to differentiate for 1 hour in a 37°C incubator. After that, the medium was replaced with DMEM medium containing 25 ng / mL BMP4, 12 ng / mL Actin-A and 3 μM CHIR99021 to continue inducing differentiation. On the fourth day, the operation of day 3 was repeated.

[0065] 4) Endothelial differentiation stage (days 5 to 8):

[0066] On day 5, the iPSCs cells induced to differentiate in step 3) were induced to differentiate for 1 h in an incubator at 37°C using ECM containing 50 ng / mL VEGF, 25 ng / mL FGF-basic, 10 μM SB431542 and 5 μM Yoda1 as differentiation medium. The medium was then replaced with ECM containing 50 ng / mL VEGF, 25 ng / mL FGF-basic and 10 μM SB431542 to continue inducing differentiation. The operation of day 5 was repeated on days 6, 7 and 8.

[0067] Example 3

[0068] Purification of iPSC-ECs

[0069] 1) Digestion of cells:

[0070] After the induction of differentiation was completed on the 8th day in Example 2, the culture medium used for inducing differentiation in the six-well plate was aspirated and washed with DPBS three times;

[0071] Add 0.5 mL of TrypLE Express digestion enzyme to each well of the six-well plate and digest for 1 min. Then add 1 mL of ECM medium to neutralize the enzyme activity and gently pipette to form a cell suspension.

[0072] The cell suspension was screened using a 40 μM cell sieve to obtain a single cell suspension, which was then centrifuged at 1200 rpm for 5 min at 4 °C;

[0073] 2) Magnetic beads labeled CD144:

[0074] After the centrifugation in step 1) is completed, the supernatant is removed and the cells are counted. 780 μL of 5% BSA solution was added to each cell to resuspend the cells, and then 20 μL of microbeads containing mouse anti-human FcR blocking monoclonal antibody and 20 μL of mouse anti-human CD144 monoclonal antibody were added, vortexed to mix, and incubated at 4°C for 15 min; 5% BSA solution was added, and the mixture was centrifuged at 1200 rpm at 4°C for 5 min, the supernatant was removed, and 500 μL of 5% BSA solution was added to resuspend the cells to obtain a cell suspension;

[0075] 3) Magnetic separation of CD144+ cells:

[0076] Add 500 μL of the cell suspension obtained in step 2) to the sorting column, and when the liquid in the sorting column is emptied, wash the sorting column three times with 3 mL of 5% BSA solution, then remove the sorting column from the magnetic sorter and place it in a 15 mL conical tube, add 3 mL of ECM culture medium to the sorting column, push the sorting column piston, and wash the cells marked with magnetic beads to obtain a cell suspension;

[0077] The cell suspension was centrifuged at 1200 rpm for 5 min at room temperature, the supernatant was removed, and the ECM medium was added to resuspend the cells to obtain a cell suspension;

[0078] 0.2% gelatin was added to each well of the six-well plate and incubated at 37°C for 30 min. After that, the 0.2% gelatin used for plating in the six-well plate was aspirated and 3×10 5 The cell suspension was inoculated into a six-well plate with the inoculation number of cells / well.

[0079] When the cell density reaches 90%, the cell suspension is subcultured at a subculture ratio of 1:3 using ECM culture medium as the culture medium, referring to step 1) of Example 3.

[0080] Comparative Example

[0081] Induction of differentiation of iPSCs into ECs (Yoda1 5μM continuous)

[0082] 1) Preparation of Yoda1 solution:

[0083] Add 0.66 mg of Yoda1 to 37.155 μL of DMSO to obtain a Yoda1 solution with a concentration of 5 μM;

[0084] 2) Mesoderm differentiation stage 1 (day 0-2):

[0085] The iPSCs cells cultured in Example 1 were induced to differentiate in a 37° C. incubator using DMEM containing 25 ng / mL BMP4, 12 ng / mL Actin-A, 6 μM CHIR99021, and 5 μM Yoda1 as a culture medium;

[0086] 3) Mesoderm differentiation stage 2 (day 3-4):

[0087] The iPSCs cells induced to differentiate in step 2) were induced to differentiate in a 37° C. incubator using a DMEM medium containing 25 ng / mL BMP4, 12 ng / mL activin A, 3 μM CHIR99021, and 5 μM Yoda1 as a culture medium;

[0088] 4) Endothelial differentiation stage (days 5 to 8):

[0089] The iPSCs cells induced to differentiate in step 3) were induced to differentiate in an incubator at 37° C. using an ECM culture medium containing 50 ng / mL VEGF, 25 ng / mL FGF-basic, 10 μM SB431542 and 5 μM Yoda1 as the culture medium.

[0090] Effect verification

[0091] 1. Effect of Yoda1 on the differentiation efficiency of iPSC-ECs

[0092] To investigate the role of Yoda1 in promoting the differentiation of iPSC-ECs, the CD144-positive cell rate of iPSC-ECs was detected by flow cytometry, the mRNA levels of CD144, eNOS, and Piezo1 of iPSC-ECs were detected by PCR, and the protein expression levels of CD144 and Piezo1 of iPSC-ECs were detected by WB;

[0093] 1. Flow cytometry

[0094] 10 7 ~10 8 Each iPSC-ECs was digested with TrypLE Express enzyme for 2 min, then 1.5 mL of ECM medium was added to neutralize the activity of TrypLE Express enzyme, the mixture was gently pipetted, centrifuged at 1200 rpm at 4°C for 5 min, the supernatant was removed, 2 mL of 1% FBS was added to resuspend the cells, the mixture was centrifuged at 1200 rpm at 4°C for 5 min, the supernatant was removed, and finally 300 μL of 1% FBS was added to obtain a cell suspension;

[0095] Transfer 100 μL of cell suspension to a 1.5 mL EP tube, add PE-CD144 antibody to the EP tube, and incubate at 4°C in the dark for 30 min;

[0096] After the dark-protected culture, 1 mL of 1% FBS was added to the EP tube, vortexed, centrifuged at 1200 rpm for 5 min at 4°C, the liquid in the EP tube was poured out, inverted for 30 s, 1 mL of 1% FBS was added, vortexed, centrifuged at 1200 rpm for 5 min at 4°C, the liquid in the EP tube was poured out, 100 μL of 1% FBS was added, vortexed, and the CD144-positive cell rate of iPSC-ECs was detected using a Beckman CytoFLEX S analytical flow cytometer;

[0097] The results of CD144 positive cell rate detection of iPSC-ECs are as follows Figure 3 As shown, A is the normal control group, B is the Yoda1 5μM 1h / d intervention group, and C is the Yoda1 5μM continuous intervention group;

[0098] The normal control group refers to the group without adding Yoda1 to the ECM culture medium, the Yoda1 5 μM 1h / d intervention group refers to Example 2, and the Yoda1 5 μM continuous intervention group refers to the comparative example;

[0099] Depend on Figure 3 It can be seen that the CD144 positive cell rate of the normal control group was 67.3%, the CD144 positive cell rate of the Yoda1 5μM 1h / d intervention group was 84.5%, and the CD144 positive cell rate of the Yoda1 5μM continuous intervention group was 60.5%. Compared with the CD144 positive cell rate of the normal control group, the CD144 positive cell rate of the Yoda1 5μM 1h / d intervention group increased, and the CD144 positive cell rate of the Yoda15μM continuous intervention group decreased;

[0100] 2. PCR

[0101] 1) RNA extraction and concentration determination

[0102] The total RNA was extracted using the Trizol method, which included the following steps:

[0103] ① Collect iPSC-ECs into 1.5 mL centrifuge tubes, 5×10 6 ~6×10 6 Add 1 mL of Trizol to each iPSC-ECs, mix well by pipetting, centrifuge at 1200 rpm for 10 min at 4°C, and transfer the supernatant to a new enzyme-free EP tube;

[0104] ② Add chloroform to the supernatant obtained in step ① according to the ratio of 1 mL Trizol to 0.2 mL chloroform, shake the tube for 15 seconds, centrifuge at 1200 rpm for 15 minutes at 4°C, and transfer the aqueous phase from the RNA to a new enzyme-free EP tube;

[0105] ③ Add isopropanol to the aqueous phase obtained in step ② according to the ratio of 1 mL Trizol to 0.5 mL isopropanol. Centrifuge at 1200 rpm for 10 min at 4°C. A white precipitate, i.e. RNA, is visible. Aspirate the supernatant.

[0106] ④ Add 75% ethanol to the white precipitate obtained in step ③ according to the ratio of 1 mL Trizol to 1 mL 75% ethanol, gently blow, centrifuge at 7500 rpm at 4°C for 5 min, and remove the supernatant;

[0107] ⑤Invert the enzyme-free EP tube after removing the supernatant in step ④, then add 20 μL of enzyme-free water and pipette to dissolve the RNA;

[0108] ⑥ Use Nanodrop One ultra-micro spectrophotometer to measure the concentration of RNA;

[0109] 2) Reverse transcription of RNA into cDNA

[0110] Reverse transcription of RNA into cDNA using the HiFiScript cDNA Synthesis Kit from Cosmed includes the following steps:

[0111] ① Prepare the reverse transcription reaction system according to the reagents listed in Table 3. The total volume of the reverse transcription reaction system is 13 μL;

[0112] Table 3 Reverse transcription reaction system

[0113]

[0114] ② Mix the reverse transcription reaction system obtained in step ①, centrifuge briefly, incubate at 70°C for 10 min, then quickly ice bath for 2 min, centrifuge briefly, and collect the liquid on the tube wall to the bottom of the tube;

[0115] ③ Add 4 μL 5xRT Buffer, 2 μL 0.1M DTT, and 1 μL 200U / μL HiFiScript to the liquid obtained in step ②, mix well, centrifuge briefly, incubate at 50°C for 15 min, then incubate at 85°C for 15 min, centrifuge briefly, and place on ice for later use, or store in a -20°C refrigerator;

[0116] 3) Real-time fluorescence quantification

[0117] Real-time fluorescence quantitative detection was performed using the Kangwei Century UltraSYBR Mixture kit;

[0118] The reaction system is shown in Table 4;

[0119] Table 4 Reaction system

[0120]

[0121] The reaction conditions are shown in Table 5;

[0122] Table 5 Reaction conditions

[0123]

[0124] The primer sequences are shown in Table 6;

[0125] Table 6 Primer sequences

[0126]

[0127]

[0128] The CT value obtained after the real-time fluorescence quantitative detection was used with GAPDH as the internal reference. -△△CT The relative mRNA levels of Oct4, Nanog, CD144, and eNOS in iPSC-ECs were compared by the method;

[0129] The mRNA levels of CD144, eNOS, and Piezo1 in iPSC-ECs were as shown in Figure 4 As shown;

[0130] Depend on Figure 4 It can be seen that the mRNA levels of CD144, eNOS and Piezo1 in the Yoda1 5μM 1h / d intervention group increased, and the mRNA levels of CD144, eNOS and Piezo1 in the Yoda1 5μM continuous intervention group decreased, and the differences were statistically significant (P<0.05). Therefore, it can be determined that Yoda1 5μM 1h / d has a promoting effect on the induced differentiation of iPSC-ECs;

[0131] 3. WB

[0132] 1) Preparation of cell protein samples:

[0133] After the induction of differentiation on the 8th day, the cells were rinsed with ice PBS for 3 times, and then 80 μL of RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors was added to each well of the six-well plate. The cells on the six-well plate were repeatedly scraped with a cell scraper. Finally, the cell suspension in the six-well plate was transferred to a 1.5 mL EP tube, lysed on ice for 30 min, vortexed every 10 min, centrifuged at 12000 rpm at 4 ° C for 30 min, and the protein supernatant was collected and stored at -80 ° C for later use;

[0134] 2) Determination of protein concentration by BCA method:

[0135] ① Preparation of protein standard: add 0.8 mL of protein standard preparation solution to 20 mg of protein standard (BSA) and fully dissolve to obtain 25 mg / mL protein standard solution; take 20 μL of 25 mg / mL protein standard solution and dissolve it in 980 μL of lysate to dilute it to 0.5 mg / mL protein standard solution;

[0136] ② Preparation of BCA working solution: Mix BCA reagent A solution and BCA reagent B solution at a volume ratio of 50:1;

[0137] ③ Preparation of standard curve samples: add 0.5 mg / mL standard to a 96-well plate at 0, 1 μL, 2 μL, 4 μL, 8 μL, 12 μL, 16 μL, and 20 μL, and add PBS to make up to 20 μL, that is, the concentrations of the standard are 0, 0.025 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, and 0.5 mg / mL respectively;

[0138] ④BCA working solution incubation: Add 200 μL BCA working solution to each well of the standard curve sample and protein sample, and incubate at 37°C for 30 min;

[0139] ⑤ Concentration determination and calculation: Use a full-wavelength microplate reader to measure the absorbance at a wavelength of 562nm, and calculate the protein concentration of the sample based on the standard curve and the sample volume used;

[0140] 3) Protein denaturation

[0141] The proteins of different groups were adjusted to the same mass, concentration and volume, and 5× SDS-PAGE protein loading buffer was added, and the mixture was placed in an iron bath at 100°C for 10 min;

[0142] 4) Protein gel electrophoresis

[0143] ① Prepare separation gel: Wash the glass plate, dry it and fix it on the gel preparation rack, and prepare 2 pieces of 6% separation gel and 2 pieces of 10% separation gel according to the reagents listed in Table 7;

[0144] Table 7 Separation Gel

[0145]

[0146] ② Filling the separation gel: fully mix the separation gel prepared in step ①, gently pour it from one side of the glass plate with a 1mL pipette tip, seal it with ultrapure water to make the liquid level, and let it stand at room temperature for 30 minutes to gel;

[0147] ③ Prepare 4 5% concentrated gels according to the reagents listed in Table 8;

[0148] Table 8 Concentrated Gel

[0149]

[0150]

[0151] ④ Filling concentrated gel: After the separation gel in step ② is gelled, pour out the upper layer of ultrapure water and absorb it with filter paper. Then, fully mix the concentrated gel prepared in step ③ and pour the concentrated gel from one side of the glass plate. After filling, immediately add the comb vertically and let it stand at room temperature for 30 minutes to gel;

[0152] ⑤ Loading: Add 30.3g Tris base, 144.0g glycine and 10g 10% SDS into distilled water, make up to 1000mL, stir the magnetic beads thoroughly, and dilute to 1×;

[0153] Except for Piezo1 protein, which was loaded at 40 μg, the loading mass of other proteins was 25 μg. The loading volume was determined by the protein concentration. 1L 1× electrophoresis solution was added to the electrophoresis tank. The comb was pulled out, and pre-stained markers were added to the left and right sides respectively. The corresponding proteins were added to the middle well according to the grouping.

[0154] ⑥ Gel running: Set the voltage of the concentrated gel to 80V. After the protein sample enters the separation gel and the pre-stained marker is separated, adjust the voltage to 120V and stop the electrophoresis when it reaches the bottom of the concentrated gel.

[0155] ⑦ Transfer: Prepare 8cm x 6cm PVDF membrane for each gel, and soak the PVDF membrane in methanol. Open the transfer clip, with the blackboard facing down, and place the fiber pad, two layers of thick filter paper, gel, PVDF membrane, two layers of thick filter paper, and fiber pad in order from top to bottom. Close the transfer clip, put the transfer clip into the transfer tank, put an ice cube in the transfer tank, pour in 1L 1× electrotransfer solution, put the transfer tank into ice for transfer, voltage 100V, transfer 1.5~2h, the specific time is determined by the molecular weight of the protein;

[0156] ⑧ Blocking: After the transfer is completed, take out the PVDF membrane from the transfer clip and put the membrane into 5% skim milk powder for blocking for 1 hour;

[0157] ⑨ Primary antibody incubation: Use primary antibody diluent to dilute the primary antibody in an appropriate ratio. After blocking, cut the membrane according to the molecular weight of the protein and incubate with the corresponding primary antibody at 4°C overnight;

[0158] ⑩ Secondary antibody culture: dilute the secondary antibody with 5% BSA at an appropriate ratio. The next day, recover the primary antibody and wash the membrane 5 times with 1×TBST, 5 minutes each time. After washing, place the membrane in the secondary antibody for culture at room temperature for 1 hour. After culture, wash the membrane 5 times with 1×TBST, 5 minutes each time.

[0159] Development: Prepare ECL developer in a darkroom, mix equal volumes of solution A and solution B, drop an appropriate amount of developer onto the film for development and exposure, and analyze the grayscale value of the bands for statistical analysis.

[0160] The protein expression levels of CD144 and Piezo1 in iPSC-ECs were as follows Figure 5 As shown, A is the protein band of western blot experiment, and B is the result of quantifying and comparing the differences of protein bands using Image J software;

[0161] Depend on Figure 5 It can be seen that the CD144 and Piezo1 protein levels in iPSC-ECs in the Yoda1 5μM 1h / d intervention group were higher than those in the normal control group, and the difference was statistically significant (P<0.05). The above results show that after Piezo1 is activated by Yoda1, the differentiation efficiency of iPSC-ECs is increased.

[0162] 2. Effect of Yoda1 on cell tube formation after iPSC-ECs differentiation

[0163] In order to study the tube formation effect of iPSC-ECs after Yoda1 intervention, a cell tube formation experiment was performed on iPSC-ECs;

[0164] 1) Preparation before the experiment:

[0165] Thaw Matrigel in a 4°C refrigerator before use, and pre-cool the 96-well plate and pipette tip in a -20°C refrigerator;

[0166] 2) Glue laying:

[0167] After Matrigel is thawed, gently blow and mix, add 50 μL Matrigel to each well of the 96-well plate, and in order to further make the gel even, the 96-well plate with gel can be placed on a shaker and shaken evenly for 5 minutes; after shaking, place the 96-well plate with gel in a 37°C incubator and culture for 30 minutes;

[0168] 3) Inoculation of cells:

[0169] While the gel is waiting, digest the cells that have grown in the six-well plate to prepare a cell suspension. Add 50 μL of the cell suspension to one well of the 96-well plate for about 10 seconds. 4 cells / well; after the Matrigel culture was completed for 30 min, the cell suspension was added to the 96-well plate;

[0170] 4) Take photos:

[0171] It starts to form a tube after 4-6 hours, forms a tube within 18 hours, and the tubular structure begins to disintegrate after 24 hours. The photo can be taken at the appropriate time.

[0172] 5) Data processing:

[0173] Image analysis and processing were performed using Image J software, and the tube formation effect was determined by calculating the total tube intersection points and the total tubular area;

[0174] Cell tube formation experiment photo Figure 6 As shown, A is the normal control group, B is the Yoda15μM 1h / d intervention group;

[0175] The results of quantification of cell tube formation experiment images and one-way ANOVA analysis using Image J software are shown in the figure below. Figure 7 As shown, A is the quantification and statistical analysis of the total lumen area, and B is the quantification and statistical analysis of the total number of tube intersections.

[0176] Depend on Figure 6 and Figure 7 It can be seen that compared with the normal control group, the total number of tube intersections and the total lumen area in the Yoda1 5μM 1h / d intervention group were increased, and the difference was statistically significant (P<0.05). This confirmed that the tube formation effect of iPSC-ECs after Yoda1 intervention was better than that of the normal group.

[0177] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that the specific implementation methods of the present invention can still be modified or replaced by equivalents. Any modification or equivalent replacement that does not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. An application of Yoda1 in promoting the differentiation of iPSC-ECs.

2. The use of Yoda1 in promoting differentiation of iPSC-ECs according to claim 1, characterized in that: The application is specifically: adding Yoda1 to the culture medium in the mesoderm differentiation stage and the endothelial differentiation stage to intervene in the differentiation of iPSC-ECs.

3. The use of Yoda1 in promoting the differentiation of iPSC-ECs according to claim 2, characterized in that: The intervention concentration of Yoda1 was 5 μM.

4. The use of Yoda1 in promoting iPSC-ECs differentiation according to claim 2, characterized in that: The intervention duration of Yoda1 is 1 hour.

Images