Establishment method and application of iPSCs-derived valvular interstitial cell calcification model
By optimizing the iPSC induction and differentiation protocol, phenotypically stable iPSC-derived valvular interstitial cells were constructed, solving the problem of difficulty in obtaining human primary cells. This enabled the construction of an efficient valvular calcification model and a drug screening platform, promoting basic research and treatment progress in valvular heart disease.
Patent Information
- Application Number
- CN202511405483.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-29
- Publication Date
- 2025-12-05
AI Technical Summary
In the current technology, obtaining human primary valvular interstitial cells is difficult, ethical restrictions exist, and there are large batch-to-batch differences, resulting in a lack of effective drug intervention for the treatment of valvular heart disease. Furthermore, the current technology makes it difficult to construct standardized and reproducible valvular calcification models.
By optimizing the induction differentiation protocol, mature valvular interstitial cells were constructed using iPSCs. Using specific culture media and inducing factors such as CHIR99021, they were differentiated into phenotypically stable iPSC-derived valvular interstitial cells and cultured in osteogenic induction medium to form an efficient calcification model.
The obtained iPSCs-derived valvular interstitial cells have a phenotype highly similar to human primary cells, strong proliferative capacity, and are suitable for large-scale production. They can stably construct in vitro models of calcified valvular heart disease and provide a high-quality drug screening platform.
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Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of cell model construction, and particularly relates to a method for establishing an iPSCs-derived valve interstitial cell calcification model and application. BACKGROUND
[0002] Valvular heart disease is one of the major cardiovascular diseases in the world, and its treatment still mainly relies on surgical intervention, including valve repair or replacement. However, there are still many limitations in related technologies, such as recurrence of lesions, calcification of biological valves, and lifelong anticoagulation for mechanical valves. In particular, as the core pathological process of valve calcification, there is currently no effective drug intervention method, and it is urgent to develop specific anti-calcification drugs. Valve interstitial cells (VICs) play a key role in the calcification process and are an ideal cell source for constructing in vitro calcification models and drug screening. However, human primary valve interstitial cells (hVICs) have problems such as difficulty in obtaining, ethical restrictions, and batch-to-batch differences. Induced pluripotent stem cells (iPSCs) have self-renewal and multi-directional differentiation potential, can efficiently differentiate into VICs with stable phenotype, provide a new solution for constructing standardized and repeatable valve calcification models, and can be applied to high-throughput screening and mechanism research of new anti-calcification drugs, which has broad research and clinical translation prospects. SUMMARY
[0003] The application can not only provide high-quality seed cells for tissue engineering valve construction, but also solve the problem of the lack of good human primary valve interstitial cell substitutes. The valve interstitial cells constructed by the application have the advantages of easy acquisition, high differentiation efficiency, strong proliferation ability, obvious valve interstitial cell phenotype, and almost no immune rejection. In particular, the iPSCs-derived valve interstitial cells constructed by the application can effectively replace human primary valve interstitial cells and be used as a calcification model for calcification mechanism research, thereby promoting the basic research and drug screening of calcific valve disease.
[0004] The application is to construct mature valve interstitial cells, which has great significance for the treatment of valvular heart disease. Since valve interstitial cells account for 80%-90% of valve cells, and almost all pathological origins of heart valve disease are valve interstitial cells, it is difficult to obtain and proliferate human primary valve interstitial cells, which affects the research progress in the field of valves.
[0005] The application provides a method for constructing an iPSCs-derived valve interstitial cell calcification model, comprising the following steps: (1) Preparation of osteogenic induction medium: containing 1-3% fetal bovine serum, 8-12 mmol·L -1 β-glycerophosphate sodium, 0.08-0.12 μmol·L -1 dexamethasone and 48-52 μg·mL -1Ascorbic acid, prepared in a DMEM high-sugar medium-based medium; (2) The iPSCs-derived valve interstitial cells are cultured in the osteogenic induction medium in step (1) for at least 3 days at 37 DEG C and under a 5% CO2 condition.
[0006] In an embodiment of the present application, the content of the fetal bovine serum in step (1) is 2%.
[0007] In an embodiment of the present application, the content of the sodium beta-glycerophosphate in step (1) is 10 mmol / L. -1 .
[0008] In an embodiment of the present application, the content of the dexamethasone in step (1) is 0.1 μmol / L. -1 .
[0009] In an embodiment of the present application, the content of the ascorbic acid in step (1) is 50 μg / mL. -1 .
[0010] The present application also provides the iPSCs-derived valve interstitial cell calcification model obtained by the construction method.
[0011] The present application also provides the application of the iPSCs-derived valve interstitial cell calcification model in the preparation of an anti-calcification drug screening product.
[0012] In an embodiment of the present application, the product is a kit Compared with the prior art, the present application has the following beneficial effects: (1) The mature valve interstitial cells obtained by optimizing the induction and differentiation scheme in the present application are highly close to human primary valve interstitial cells in phenotype and function, have higher acquisition efficiency and stronger proliferation capacity, and are suitable for large-scale production and stable application.
[0013] (2) The iPSCs-derived mature valve interstitial cells obtained in the present application can be used as a substitute for human primary cells, and are widely applied to the construction of tissue engineering valves and related in-vitro function and mechanism research, and break through the technical bottleneck of limited primary cell resources.
[0014] (3) The cells obtained in the present application are more likely to exhibit a typical calcification phenotype under osteogenic induction culture conditions, and can stably construct an in-vitro cell model of calcific aortic valve disease, thereby providing a high-quality experimental platform for the screening and mechanism research of anti-calcification drugs. BRIEF DESCRIPTION OF DRAWINGS
[0015] Figure 1An example of the optimized post-differentiation strategy of the present application, using magnetic bead sorting to obtain CD144-negative cells, followed by the addition of 8 μΜ CHIR99021 to promote their differentiation into valve mesenchymal cells.
[0016] Figure 2 Results of the evaluation of cell proliferation activity by the CCK-8 kit (Cell Counting Kit-8, Dojindo) in Example 1.
[0017] Figure 3 Results of the evaluation of cell proliferation activity by the 5-ethynyl-2'-deoxyuridine (EdU) kit (BeyoClick™ EdU-488 Cell Proliferation Kit, Beyotime) in Example 1.
[0018] Figure 4 Microscopic morphological comparison chart in Example 1, showing that the intervention of CHIR99021 promotes the differentiation of iPSCs into human primary valve mesenchymal cells.
[0019] Figure 5 Effect of different culture conditions and CHIR99021 concentrations on the differentiation of iPSCs into mature valve mesenchymal cells in Example 1.
[0020] Figure 6 Quantitative real-time polymerase chain reaction to quantify the markers of human primary valve mesenchymal cells, iPSCs-derived valve mesenchymal cells cultured in high-glucose DMEM medium, and iPSCs-derived valve mesenchymal cells cultured in KnockOut TM Comparison results of markers of iPSCs-derived valve mesenchymal cells cultured in DMEM medium.
[0021] Figure 7 Detection of the calcification ability of valve mesenchymal cells from different sources under osteogenic induction medium conditions by Western blot in Example 2.
[0022] Figure 8 Evaluation of the calcification ability of valve mesenchymal cells from different sources under osteogenic induction medium conditions by alizarin red S staining in Example 2.
[0023] Figure 9 Effect of three candidate anti-calcification drugs on the expression of key osteogenic proteins during the induction of calcification of iPSCs-derived mature valve mesenchymal cells in Example 3.
[0024] Figure 10 Effect of three candidate anti-calcification drugs on alizarin red staining during the induction of calcification of iPSCs-derived mature valve mesenchymal cells in Example 3. Detailed Implementation
[0025] Table 1. Chinese-English Bilingual Table
[0026]
[0027] Example 1: Differentiation and maturation of iPSC-derived valve interstitial cells (1) Seeding and culture of iPSCs ① Dilute Matrigel with DMEM medium at a volume ratio of 1:1000 and add 4.5 ml of diluted Matrigel to each T25 culture flask. Transfer the culture flask to an incubator and let it stand for 0.5 h so that a suitable Matrigel environment for the survival and proliferation of iPSCs can be formed at the bottom of T25.
[0028] ② Remove the T25 culture flask containing iPSCs at approximately 80% confluence, discard the old culture medium, wash twice with 2 ml DPBS, add 2 ml Versene, and incubate for 10 min of static digestion. During this time, before use, discard the diluted matrix gel used for coating in step ① of the T25 flask, gently rinse once with PBS, and add 3 ml of mTeSR1 medium containing 10 μM Y27632 for later use. After digestion, add an equal volume of mTeSR1 medium to neutralize (i.e., add the cell culture medium digested with Versene to the prepared T25 culture flask containing mTeSR1), transfer to a 15 ml centrifuge tube, centrifuge at 1000 rpm for 5 min, and discard the supernatant. Resuspend the cells in mTeSR1 medium containing 10 μM Y27632 and count them at 4 w / cm². 2 The cells were inoculated at a density of approximately 100% into T25 culture flasks pre-filled with 3 ml of mTeSR1 medium and cultured until the next day.
[0029] (2) iPSC seed cells differentiate into mature valve interstitial cells Observe the status of iPSCs on the second day of culture to determine whether to proceed to the next stage of differentiation (e.g. Figure 1 ); ①D1-D3 differentiation process: Discard the old culture medium, wash twice with 2ml DPBS, and replace the medium with N2-B27 medium containing 8μM CHIR99021 and 25ng / ml BMP4. Add 4.5ml of this medium to each T25 culture flask, and change the medium daily for three consecutive days. N2-B27 medium is a commercially available complementary product, and CHIR99021 and BMP4 are both commercially available inducing factors.
[0030] D4-D6 days of differentiation, discard old medium, wash twice with 2 ml DPBS, replace medium with StemPro-34 SFM medium containing 1 μM Forskolin and 100 ng / mL VEGF-165, add 4.5 ml medium per T25 flask, change medium every day for three days. Stempro-34 SFM medium is a commercially available complete product, and Forskolin and VEGF-165 are commercially available inducers.
[0031] D7 days of differentiation, discard old medium, wash twice with 2 ml DPBS, replace medium with KnockOut DMEM medium containing 8 μM CHIR99021, add 4.5 ml medium per flask, induce for one day, and on D8 and thereafter replace medium with KnockOut DMEM medium without CHIR99021. TM KnockOut DMEM medium, obtain iPSCs-derived valve mesenchymal cells (i.e. iVIC-K8). TM
[0032] Isolate iPSCs-derived valve cells, including valve endothelial cells (CD144 positive) and valve mesenchymal cells (CD144 negative) after step 2 D4-D6 days of differentiation, use CD144 magnetic bead sorting to remove CD144 positive cells, and seed CD144 negative cells in a 96-well plate at a cell count of 5 x 10 3 each well, and culture in DMEM high glucose medium (DMEM high glucose type (4.5 g / L glucose) item number: 11965118), DMEM low glucose medium (DMEM low glucose type (1 g / L glucose) item number: 11885092) and KnockOut™ DMEM medium (stem cell culture medium), each medium is divided into 0, 2, 4, 8, 16, a total of five concentration gradients of CHIR99021 content. Add 10 μL CCK-8 reagent at 24 hours, continue to incubate for 2 hours, then use a microplate reader to measure the absorbance value (OD) of each well at 450 nm wavelength to reflect the cell activity level. The results show that the iPSCs-derived mature valve mesenchymal cells cultured in KnockOut™ DMEM medium (stem cell culture medium) containing 8 μM CHIR99021 have the strongest proliferation activity compared with high glucose medium and low glucose medium, see Figure 2 .
[0033] The iPSCs-derived valve cells after differentiation for 4-6 days in step ii were sorted out for CD144-negative cells using magnetic beads and seeded in 24-well plates in DMEM high glucose medium, DMEM low glucose medium and KnockOut™ DMEM medium containing 8 μM CHIR99021 (stem cell culture medium). After the cells stably adhered and grew, 10 μM of EdU was added for 2 hours of incubation. Then, cell fixation, membrane permeation and EdU fluorescence staining were performed according to the kit instructions, and finally the proportion of EdU-positive cells was counted using a fluorescence microscope for imaging to evaluate the cell proliferation capacity. The results showed that the iPSCs-derived mature valve interstitial cells in the KnockOut™ DMEM medium containing 8 μM CHIR99021 had the strongest proliferation activity, and the results are shown in FIG. 2. TM DMEM medium (stem cell culture medium) had the strongest proliferation activity, and the results are shown in FIG. 2. Figure 3 .
[0034] Figures 2-3 It can be seen that the iPSCs-VIC (iPSCs-derived valve interstitial cells) had the strongest proliferation potential in the KnockOut™ DMEM medium containing 8 μM CHIR99021, and the results are shown in FIG. 2. TM DMEM medium can significantly improve the proliferation potential of iPSCs-VIC (iPSCs-derived valve interstitial cells), providing a basis for large-scale drug screening.
[0035] Figure 4 It can be seen that the cell population obtained by inducing human iPSCs was obtained by removing CD144+ (endothelial) cells through magnetic bead sorting, and further induced to form mature valve interstitial cells (iVIC-K8) with stable morphology under specific culture conditions (stem cell culture medium containing 8 μM CHIR99021). The morphology of three types of cells, including human primary valve interstitial cells (hVIC), CD144- cells and iVIC-K8 cells, was observed under an inverted microscope, and a morphological comparison was made.
[0036] Figure 5The proportion of CD31 and VIM (vimentin) double-labeled positive cells was analyzed by flow cytometry, and the specific process was as follows: the CD144 negative cells obtained by magnetic bead sorting from the iPSCs-derived valve cells differentiated on D4-D6 in step ② were cultured in DMEM high-sugar medium, DMEM low-sugar medium and KnockOut™ DMEM medium, and different concentrations (0, 2, 4, 8 μM) of CHIR99021 were added to each medium for 1 day, followed by culture in the above three media without CHIR99021 for 2 days. The induced differentiated cells were gently digested from the culture dish and single-cellized, resuspended with PBS, and then subjected to antibody staining. Anti-human CD31-FITC and Vimentin-APC antibodies were used for surface and intracellular staining, respectively. After staining, the cells were detected by a BD flow cytometer, and the data were analyzed using FlowJo software. It can be seen that the proportion of CD31⁻VIM⁺ cells treated with different concentrations of CHIR99021 in stem cell culture medium remains at a high level (more than 95%), indicating that the differentiation efficiency of valve interstitial cells is almost not affected by the concentration of CHIR99021. On the contrary, in high-sugar and low-sugar medium, when the concentration of CHIR99021 is increased to 8 μM, the proportion of CD31 positive cells increases significantly (such as 67.9% vs. 44.8%), and the non-stem cell culture medium may inhibit the differentiation of iPSCs into interstitial cells, resulting in a decrease in the proportion of interstitial cells. The experiment shows that different concentrations of CHIR99021 have the least effect on the proportion of CD31⁻VIM⁺ cells in stem cell culture medium, indicating that this condition more stably induces interstitial cells and avoids endothelial contamination, thereby providing technical support for obtaining uniform iVIC-K8 cells.
[0037] Figure 6Comparison of fibrosis / interstitial marker gene expression levels of mature iVIC-K8 cells, primary hVIC cells and iVIC cells under other induction conditions. Total RNA was extracted from iVIC cells induced under different conditions and primary hVIC cells, and qRT-PCR was performed after reverse transcription to detect the expression of interstitial-related markers such as COL1A2, COL3A1, a-SMA and VIM. Each group in the figure includes: primary valve interstitial cells (hVIC), iVIC-H0 induced by DMEM high-sugar medium (without CHIR99021 factor), and iVIC-K0 / K2 / K4 / K8 (K0: 0 μM CHIR99021; K2: 2 μM CHIR99021; K4: 4 μM CHIR99021; K8: 8 μM CHIR99021) induced by adding different concentrations of CHIR99021 in stem cell medium, etc. The results show that iVIC-K8 is closest to primary valve interstitial cells at the molecular expression level, verifying the effectiveness and reliability of the induction strategy (8 μM CHIR99021 + stem cell medium) for obtaining mature functional iVIC cells.
[0038] Example 2 Establishment of iPSCs-derived valve interstitial cell calcification model (1) Establishment of iPSCs-derived valve interstitial cell calcification model ① Preparation of osteogenic induction medium: containing 2% fetal bovine serum, 10 mmol·L -1 β-glycerophosphate sodium, 0.1 μmol·L -1 Dexamethasone and 50 μg·mL -1 Ascorbic acid-containing osteogenic induction medium (OM medium, based on DMEM high-sugar medium).
[0039] ② At step ③ in Example 1, 9 days after differentiation, discard the culture medium, wash twice with DPBS, replace with 4.5 ml / bottle of OM medium, and culture at 37°C, 5% CO2 for 3 days. Replace the medium every 48 h. Short-term (3-day) induction can be analyzed by WB detection of up-regulated calcification markers, and 28-day culture can be stained with alizarin red.
[0040] Three groups were compared: ① iPSCs-derived valve interstitial cells induced by adding 8 μM CHIR99021, ② iPSCs-derived valve interstitial cells without CHIR99021, and ③ primary valve interstitial cells. All three groups of cells were cultured according to the above step ② for 3 days. RUNX2 and BMP2 were used as calcification-related markers for detection. The results are as follows Figure 7As shown, CHIR99021 treatment significantly up-regulated the protein expression levels of RUNX2 and BMP2 in iPSCs-derived VICs, indicating that the calcification ability of iPSCs-derived VICs was significantly enhanced, close to or exceeding that of primary VICs. This suggests that CHIR99021 can effectively promote the osteogenic differentiation potential of iPSCs-derived VICs.
[0041] Three groups were set up in the experiment: ① iPSCs-derived VICs induced by adding 8 μM CHIR99021, ② iPSCs-derived VICs without adding CHIR99021, and ③ primary VICs. The three groups of cells were cultured for 3 days by the above step ②, and the results are shown in Figure 8 As shown, CHIR99021 treatment significantly enhanced the calcification ability of iPSCs-derived VICs, with a significantly stronger staining degree than the untreated group, close to or exceeding the calcification level of primary VICs. This suggests that CHIR99021 not only helps to enhance osteogenic differentiation, but also can improve the functional performance of iPSCs-derived VICs under calcification conditions.
[0042] Figures 7-8 It can be seen that the VICs of the present scheme have the same calcification ability as primary VICs.
[0043] Example 3 Evaluation of the application potential of iPSCs-derived VIC calcification model in drug research and development To further evaluate the application potential of the iPSCs-VIC calcification model in drug research and development, three representative anti-calcification candidate molecules, ERRa inverse agonist (XCT-790) (10 μM), Dooku-1 (10 μM), and Andrographolide (AGP, 20 μM), were selected for intervention experiments. iPSCs-derived VICs induced by adding 8 μM CHIR99021 were added to the above drugs together with OM medium, and each set up a parallel control group. The protein expression of calcification markers RUNX2 and BMP-2 was detected on the 3rd day of induction, Figure 9 It can be shown that the iPSCs-derived mature VIC calcification model has good responsiveness to anti-calcification drugs, and is suitable for subsequent high-throughput drug screening and mechanism research.
[0044] iPSCs-derived VICs induced by adding 8 μM CHIR99021 were added to the above drugs together with OM medium, and each set up a parallel control group. The protein expression of calcification markers RUNX2 and BMP-2 was detected on the 3rd day of induction, Figure 10 It can be shown that the iPSCs-derived mature VIC calcification model has good responsiveness to anti-calcification drugs.
[0045] The above described embodiments are only to illustrate the preferred modes of the present application, and are not intended to limit the scope of the present application. Any modification and improvement made by those skilled in the art to the technical solutions of the present application without departing from the design spirit of the present application shall fall within the protection scope of the present application.
Claims
1. A method for constructing an iPSCs-derived valvular interstitial cell calcification model, characterized in that, comprising the following steps: (1) Osteogenic induction medium preparation: containing 1-3% fetal bovine serum, 8-12 mmol·L -1 β-glycerophosphate sodium, 0.08-0.12 μmol·L -1 Dexamethasone and 48-52 μg·mL -1 Ascorbic acid, based on DMEM high glucose medium (2) culturing the iPSCs-derived valve mesenchymal cells in the osteogenic induction medium in step (1) for at least 3 days at 37℃ and 5% CO2.
2. The construction method of claim 1, wherein, The content of fetal bovine serum in step (1) is 2%.
3. The construction method of claim 2, wherein, The content of sodium β-glycerophosphate in step (1) is 10 mmol·L -1 .
4. The construction method according to claim 3, characterized in that, The content of dexamethasone in step (1) is 0.1 μmol·L -1 .
5. The construction method according to claim 4, characterized in that, The content of ascorbic acid in step (1) is 50 μg·mL -1 .
6. The iPSCs-derived valve mesenchymal cell calcification model obtained by the construction method of any one of claims 1-5.
7. The use of the iPSCs-derived valve mesenchymal cell calcification model of claim 6 in the preparation of an anti-calcification drug screening product.
8. Use according to claim 7, characterized in that, The product is a kit.