CN112680410B - Method for inducing pluripotent stem cells to culture heart fibroblasts in differentiation mode and culture solution thereof - Google Patents
Method for inducing pluripotent stem cells to culture heart fibroblasts in differentiation mode and culture solution thereof Download PDFInfo
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Abstract
The invention discloses a method for inducing pluripotent stem cells to differentiate and culture heart fibroblasts and a culture solution thereof, wherein the method comprises the following steps: carrying out directed differentiation culture on the induced pluripotent stem cells by using a culture solution for promoting cardiac mesoderm progenitor cells to obtain cardiac mesoderm progenitor cells; then, continuously carrying out differentiation culture by using a second heart field progenitor cell culture solution to obtain second heart field progenitor cells; finally, the purified mature cardiac fibroblasts are obtained by culturing with a cardiac fibroblast culture solution. In the culture method, the materials are available, the culture time is short, the differentiation efficiency is high, and the obtained cardiac fibroblasts can be used for clinical treatment of heart failure, in-vitro disease modeling research, drug screening and other biomedical applications.
Description
Technical Field
The invention belongs to a stem cell differentiation technology in the technical field of stem cell treatment, and particularly relates to a method for inducing pluripotent stem cells to differentiate and culture heart fibroblasts and a culture solution thereof.
Background
Cardiac fibroblasts have two important functions: the production, remodeling of extracellular matrix, and the secretion of complex mixtures of growth factors and regulatory molecules in extracellular vesicles have the effect of regulating cardiac function. When the heart is damaged, cardiac fibroblasts can be activated to repair damaged cardiomyocytes. Transplantation of cardiac fibroblasts is necessary to restore impaired cardiac function, while cardiac fibroblasts are tissue-specific cells distinct from other fibroblasts in the body, and the limited number of available donors is a significant and critical limitation for clinical transplantation. One of the methods with great clinical application potential is to induce the differentiation of pluripotent stem cells into cardiac fibroblasts in vitro, and further to be used for transplantation therapy of human heart damage. The technology does not need to use egg cells or embryonic cells, and avoids the ethical problem brought by research and application of embryonic stem cells; meanwhile, the cardiac fibroblasts generated by the method can be proliferated in a large amount in the culture process, and can provide an unlimited source of isogenic cardiac fibroblasts for patients. The cardiac fibroblast obtained by induced differentiation of induced pluripotent stem cells derived from patient autologous cell reprogramming can effectively avoid immunological rejection when applied to individual treatment, and can improve clinical treatment effect to the maximum extent.
Studies of cardiac developmental mechanisms have shown that the epicardium supports cardiomyocyte proliferation early in development and provides fibroblasts and vascular smooth muscle cells to the developing heart. The existing methods for differentiating pluripotent stem cells into cardiac fibroblasts mainly focus on inducing differentiation from human pluripotent stem cells to produce epicardial cells, followed by differentiation into cardiac fibroblasts. For example, in a recent patent (CN105518125A), KDR + and PDFG alpha + cardiovascular mesoderm progenitor cell populations are obtained by treating human pluripotent stem cells under the action of BMP4, bFGF and activin A for 3-5 days, and are osmotically contacted with WNT inhibitor and BMP4 to generate WT1+ cardiovascular progenitor cell populations, which are then cultured in a maturation cocktail containing a VEGF component for several days to generate epicardial lineage cells expressing WT1 and EPDC. The method has long culture period, and the obtained cardiac fibroblasts have low purity and contain a large amount of non-fibroblast populations, such as cardiovascular smooth muscle lineage cell populations and epicardial lineage cell populations expressing retinol dehydrogenase. Another patent (CN108148802A) is to induce differentiation of pluripotent stem cells into mesodermal cells by activating WNT signaling pathway, then to convert the mesodermal cells into cardiomyocyte precursor cells by inhibiting WNT signaling pathway, then to convert the obtained cardiomyocyte precursor cells into pre-epicardial cells that are both TBX18 and WT1 positive by activating fibroblast growth factor signaling pathway and WNT signaling pathway, then to prepare epicardial cells from the pre-epicardial cells, to treat the epicardial cells with bFGF, and then to treat the cells with bFGF plus fetal calf serum to obtain cardiac fibroblasts. The differentiation system is quite complicated in procedure, and the used reagent is complex and contains animal-derived components, so that the differentiation system is unfavorable for later clinical treatment application. Therefore, although there is a method of inducing differentiation of pluripotent stem cells into cardiac exogenous cells and further producing cardiac fibroblasts, an effective differentiation method for obtaining cardiac fibroblasts with good characteristics has not been described.
In conclusion, there is an urgent need to develop an effective culture solution and method for generating cardiac fibroblasts from pluripotent stem cells, so that the cardiac fibroblasts obtained in vitro can be effectively applied to disease mechanism research, drug screening and clinical cell therapy.
Disclosure of Invention
The method takes induced pluripotent stem cells as raw materials, adopts WNT, BMP and FGF signal to modulate sequentially to generate cardiac mesoderm progenitor cells, further differentiates the cardiac mesoderm progenitor cells into second cardiac field progenitor cells, and finally effectively generates cardiac fibroblasts. The invention provides a method for culturing cardiac fibroblasts by induced pluripotent stem cells, which can greatly shorten the preparation period of the cardiac fibroblasts, and the culture solution has definite components and does not contain animal-derived components, so that the obtained cardiac fibroblasts can be applied to clinical treatment.
The method for inducing the pluripotent stem cells to differentiate and culture the cardiac fibroblasts comprises the following steps:
step S1, performing directed differentiation culture on the induced pluripotent stem cells by using the culture solution of the cardiac mesoderm progenitor cells to obtain cardiac mesoderm progenitor cells;
step S2, continuously carrying out differentiation culture on the cardiac mesoderm progenitor cells by using a second cardiac field progenitor cell culture solution to obtain second cardiac field progenitor cells;
and step S3, digesting the second cardiac field progenitor cells, and culturing the second cardiac field progenitor cells with a cardiac fibroblast culture solution to obtain purified mature cardiac fibroblasts.
In a preferred embodiment of the present invention,
step S1, specifically, selecting 5% CO at 35-39 deg.C, preferably 37 deg.C, 3-7% CO 2 Under the condition, performing directed differentiation culture on the induced pluripotent stem cells for 1-3 days, preferably 2 days, by using a cardiac mesoderm progenitor cell culture solution, removing nonadherent cells, and performing directed differentiation to obtain cardiac mesoderm progenitor cells;
step S2, specifically, the cardiac mesoderm progenitor cells are treated at 35-39 deg.C, preferably 37 deg.C, and 3-7% CO, preferably 5% CO 2 Under the condition, continuously culturing the second heart field progenitor cells by using a second heart field progenitor cell culture solution for 3-5 days, preferably 4 days to obtain second heart field progenitor cells, and replacing the culture solution every 1-2 days, preferably 2 days, during the continuous culture period;
step S3, specifically, the second cardiac field progenitor cells obtained in step S2 are digested with TryplE at 3000- 2 Density inoculation at 35-39 deg.C, preferably 37 deg.C, 3-7% CO, preferably 5% 2 Under the condition, culturing the mature cardiac fibroblasts for 5-7 days by using a cardiac fibroblast culture solution to obtain purified mature cardiac fibroblasts, and carrying out subculture.
In the present invention, the induced pluripotent stem cells are obtained by peripheral blood mononuclear cell electroporation.
The invention also provides a culture solution for promoting the cardiac mesoderm progenitor cells, which comprises the following components: RPMI1640 basal medium, Human Serum Albumin (HSA), L-ascorbyl acid 2-phosphate, Polyvinyl alcohol (PVA), Wnt activator, BMP antagonist and FGF activator.
The Wnt activator is CHIR99021, the BMP antagonist is BMP4, and the FGF activator is FGF 2.
In the cardiomesoderm progenitor cell culture solution, the HSA concentration is 1-5 mg/mL, preferably 2.5mg/mL, and the L-ascorbic acid 2-phosphate concentration is 50-100 μ g/mL, preferably 75 μ g/mL; the concentration of the PVA is 0.1-10%, preferably 2.5%; the concentration of the Wnt activator is 4-8 mu mol/L, preferably 6 mu mol/L; the concentration of the BMP antagonist is 1-10 ng/mL, preferably 5 ng/mL; the concentration of the FGF activator is 1-10 ng/mL, and 5ng/mL is preferred.
The invention also provides a cardiac fibroblast culture solution, which comprises: DMEM high glucose medium (DMEM high glucose with GlutaMAX supplement, manufacturer: Thermo Fisher Cat # 10566016) 10% KnockOut TM SR XenoFree CTSTM (manufacturer: Thermo Fisher Cat: 12618013) and MEM Non-Essential Amino Acids Solution,100X (MEM Non-Essential Amino Acids Solution, manufacturer: Thermo Fisher Cat: 11140050).
The invention also provides a culture solution for promoting second heart field progenitor cells, which comprises the heart fiber culture solution, beta-Mercaptoethanol (beta-Mercaptoethanol, beta-Me) and FGF activator.
In the culture solution for promoting the second cardiac field progenitor cells, the concentration of the beta-mercaptoethanol is 0.1-10 mmol/L, preferably 1 mmol/L; the FGF activator is FGF2, and the concentration of the FGF activator is 25-100 ng/mL, preferably 50 ng/mL.
The cardiac fibroblasts differentiated by the method can be used for myocardial interstitial reconstruction, clinical cell replacement transplantation treatment of heart failure diseases, in-vitro disease modeling research, drug screening and other biomedical applications.
The components in the 3 groups of culture solution can act synergistically to promote the differentiation of induced pluripotent stem cells into cardiac fibroblasts. In the cardiomesoderm progenitor cell culture solution, RPMI1640 is used as a basic culture medium, so that the cardiomesoderm progenitor cell culture solution is rich in nutrient components and can provide nutrient substances required by metabolism for cardiac differentiation; HSA plays multiple roles in the formulation, such as acting as an antidote/buffer by binding lipids and excess proteins, maintaining culture stability by binding hormones and growth peptides, and reducing oxidative damage to cells by binding free radicals; l-ascorbyl acid 2-phosphate is an antioxidant, has good effect of improving cardiac differentiation, and can promote the mass proliferation of cardiovascular progenitor cells by increasing collagen synthesis. The combination of the RPMI1640 basic culture medium, HSA and L-ascorbyl acid 2-phosphate can effectively promote the differentiation of the induced pluripotent stem cells to the cardiac mesodermal progenitor cells. In addition, PVA as a high molecular polymer has good stability, and the addition of PVA with a certain concentration into the culture solution for promoting the cardiac mesoderm progenitor cells can effectively promote the proliferation and differentiation of cardiac fibroblasts, and obviously reduce the culture cost. The WNT activator is CHIR 99021; the BMP antagonist is BMP4 and can promote the expression of mesoderm marker PDGFR alpha; meanwhile, during development, signaling from cardiac fibroblasts is essential to stimulate cardiac fibroblasts to differentiate and proliferate, and the FGF transduction pathway activating molecule FGF2 contributes to the generation of cardiac mesodermal progenitors. The three components of CHIR99021, BMP4 and FGF2 cooperate with each other and can effectively enhance the induction of the differentiation of the pluripotent stem cells to the cardiac mesoderm progenitor cells under the effective action of PVA.
The second cardiac field progenitor cell culture solution comprises a cardiac fibroblast culture solution, beta-Me and FGF activator fibroblast growth factor FGF 2. Wherein, the beta-Me is used as a strong and effective reducing agent, can neutralize oxygen free radicals accumulated in a culture solution and plays a role in inducing cell proliferation and differentiation. FGF2 acts as a Fibroblast Growth Factor (FGF) signal that contributes to the generation of cardiac mesodermal progenitors and exerts a dominant effect on BMP signaling, directing cardiac mesodermal progenitors to non-cardiomyocyte fates, generating secondary cardiac field progenitors.
The components of the heart fibroblast culture solution are DMEM high glucose with GlutaMAX supplement and 10% KnockOut TM SR XenoFree CTS TM And MEM Non-Essential Amino Acids Solution,100X, is rich in nutrition, does not contain animal-derived components, and can be used for effectively treating diabetesMaintain the growth, proliferation and maturation of cardiac fibroblasts.
Counting the cardiac fibroblasts obtained by culture, performing activity statistics, and identifying cell types, wherein the cell types comprise cardiac fibroblast characteristic protein expression and surface marker expression, and the cardiac fibroblasts can be stored at low temperature or used for medical research.
The positive progress effects of the invention are as follows: the culture solution disclosed by the invention is simple and clear in components, does not contain animal-derived components, and can effectively promote the induced pluripotent stem cells to differentiate towards the direction of heart fibroblasts due to the addition of some components. For example, 2.5% of PVA is added into the culture solution for promoting the cardiac mesoderm progenitor cells, so that the culture cost is reduced, the generation of the cardiac mesoderm progenitor cells is obviously improved, and the culture solution has an obvious positive progress; FGF2 is selected as an FGF activator in the culture solution, so that the induced pluripotent stem cells can be effectively promoted to be progenitor cells of cardiac mesoderm, and then to be differentiated to progenitor cells of a second cardiac field, and the non-cardiac myocyte fate differentiation of the cells to cardiac fibroblasts is effectively promoted; the culture solution for promoting the second cardiac field progenitor cells comprises DMEM high glucose with GlutaMAXsuppplemen, 10% KnockOut TM SR XenoFree CTS TM MEM nongenetic amino acids 100X, beta-Me and FGF2, and the culture solution only selects key components, and can simply and effectively promote the differentiation and proliferation of progenitor cells in the second heart field. The method adopts the culture solution, differentiates the induced pluripotent stem cells into second cardiac field progenitor cells by controlling a key signal path, and further generates the cardiac fibroblasts. According to the method, on one hand, the raw material induced pluripotent stem cells can be converted from the mononuclear cells of the blood of a patient by electricity, the number of the raw material induced pluripotent stem cells is not limited, and the generated cardiac fibroblasts can be used for autologous treatment of the patient and can effectively avoid immunological rejection; on the other hand, the system differentiation scheme has a period of 12 days, can obtain a large amount of mature cardiac fibroblasts in a short time, has high purity, complete functions and clinical practicability, and is an effective method for obtaining the cardiac fibroblasts.
Drawings
FIG. 1 is a photomicrograph of induced pluripotent stem cells;
FIG. 2 shows different culture media each at 1X 10 6 The number of pluripotent stem cells obtained;
FIGS. 3A to 3D are photomicrographs of cardiac fibroblasts obtained from experimental groups 1 to 4, respectively;
FIG. 4 is a photograph showing the results of immunofluorescence assay of protein expression characteristic to cardiac fibroblasts;
FIG. 5 is a photograph of flow identification of cardiac fibroblast surface markers;
Detailed Description
The present invention will be further described with reference to specific examples.
The directional differentiation culture steps of the heart fibroblast are as follows:
induced pluripotent stem cell plating: obtaining induced pluripotent stem cells by peripheral blood mononuclear cell plasmid electrotransformation method, dissociating human induced pluripotent stem cells with 1mL/well Versene solution (Gibco) at 37 deg.C for 5min at density of 0.25-0.6 × 10 6 cells/cm 2 The cells were seeded on 12-well plates and the matrigel was LN521(BioLamina) supplemented with 10. mu. mol/L of ROCK inhibitor (Y-27632) (Tocris). Cells were cultured in mTeSR1 medium for 5 days, with media changed daily until 100% confluence was reached at the beginning of differentiation. The cell state was observed to be good and no differentiation was observed. The microphotographs of the induced pluripotent stem cells are shown in FIG. 1.
Inducing pluripotent stem cells to differentiate directionally: the 12-well plate full of induced pluripotent stem cells was placed in a sterile operating table, the 12-well plate was gently shaken, the medium was aspirated using an electric aspirator, and dead cells as well as non-adherent cells were removed with a 2mL pipette. And slowly adding fresh culture solution for promoting cardiac mesoderm progenitor cells into the culture medium per well (2 mL/well if the culture medium is stored in a refrigerator at 4 ℃, the culture medium needs to be taken out in advance and preheated for 20 minutes at room temperature), placing the culture medium into an incubator, and culturing for 2 days to perform directional differentiation on the induced pluripotent stem cells.
Taking out the 12-hole plate after 2 days, placing the 12-hole plate in an aseptic operation table, slightly shaking the 12-hole plate, sucking out the culture solution by using an electric suction apparatus, removing dead cells and nonadherent cells by using a 2mL pipette, slowly adding a fresh culture solution for promoting the second cardiac field progenitor cells along the hole wall by using a 5mL pipette, continuously culturing, replacing the culture medium solution once every 2 days during continuous culture, and continuously culturing the culture solution in an incubator for 10 days, wherein the culture medium is 2.5 mL/hole;
after 6 days, the 12-well plate culture medium was removed and placed in a sterile operating platform, and the cells in the plate were digested with TryplE at 3000- 2 Inoculating the culture medium to a new 12-hole plate culture plate at a density of 2 mL/hole, continuously putting the culture medium into an incubator for culturing for 5-7 days, and replacing the culture medium every 2 days. Cells are then collected and tested.
Effect example 1 Effect of partial Components in culture solution on differentiation efficiency of cardiac fibroblasts
Experiment groups 1 to 4 study the influence of the small molecular compounds such as CHIR99021, BMP4, PVA and FGF2 on the differentiation efficiency of the cardiac fibroblasts by designing whether the components such as CHIR99021, BMP4, PVA and FGF2 are added into the culture solution or not and adopting the directional differentiation culture method of the cardiac fibroblasts. The components and contents of the culture medium added to each experimental group are shown in table 1.
TABLE 1 composition and content of culture broth
FIG. 2 summarizes each 1X 10 of the different experimental groups 6 The number of cells obtained from the plurality of induced pluripotent stem cells; FIGS. 3A to 3D are bright field photographs of cardiac fibroblast microscopes obtained from experimental groups 1 to 4, respectively. As can be seen from fig. 2 and fig. 3A to 3D, when CHIR99021, BMP4, PVA, and FGF2 were sequentially added to the culture solution at appropriate concentrations, the differentiation efficiency of cardiac fibroblasts was gradually increased, indicating that each component had the effect of promoting cardiac fibroblast differentiation. FIGS. 3A to 3D from morphological observation, the cells obtained in the experimental groups 3 and 4 were obviousThe morphology of the cardiac fibroblasts shows that the addition of FGF2 in the culture solution of the cardiac mesoderm progenitor cells has the function of directionally promoting the differentiation of induced pluripotent stem cells into the cardiac fibroblasts. The number of cells obtained in experimental group 4 was significantly greater than that obtained in experimental groups 1, 2 and 3, because 2.5% PVA was added to experimental group 4, indicating that PVA added to the culture medium had significant efficacy in promoting differentiation of induced pluripotent stem cells into cardiac cells. In conclusion, the culture solution is added with components such as CHIR99021, BMP4, PVA, FGF2 and the like at a certain concentration, so that the differentiation of the induced pluripotent stem cells to the cardiac fibroblasts can be effectively promoted.
Effect example 2 immunofluorescence assay results for protein expression characteristic of cardiac fibroblasts
The immunofluorescence treatment step comprises: taking the cultured 12-pore plate, sucking out the culture solution, slowly adding 1 × PBS along the wall of the 12-pore plate, and washing for 2 times; then 4% PFA was slowly added along the plate walls, the cells were fixed by standing at room temperature for 18 minutes, the PFA was gently aspirated, and the cells were washed 3 times with 1 XPBS. Adding 0.3% Triton X100, and incubating at 37 deg.C for 30 min; blocking with 5% BSA and incubating at 37 ℃ for 30 min; primary antibody was added directly to the blocking solution in an amount of 20. mu.L/well, incubated at 37 ℃ for 1 hour, and washed 3 times with 1 XPBS. Adding a secondary antibody diluted by 1% BSA (dilution ratio of 1:200), incubating at 37 ℃ in the dark for 30 minutes, absorbing the secondary antibody, adding 1 XPBS, and washing for 3 times, each time for 5 minutes; then adding 1 XPBS prepared DAPI with final concentration of 1 μ g/mL, staining for 1 min, removing DAPI, adding 1 XPBS and washing for 2 times; finally, 1 × PBS was added, and the mixture was observed under a fluorescent microscope and photographed.
FIG. 4 shows the results of immunofluorescence assay of cells cultured in the culture medium and the culture method described in Experimental group 4. The characteristic protein of cardiac fibroblast is FIBROBLAST, and the cardiac fibroblast has the function of secreting FIBRONECTIN (FIBRONOECTIN), and whether the generated cells are cardiac FIBROBLASTS can be identified by detecting FIBROBLAST and FIBRONECTIN. Immunofluorescence identification shows that the generated cells can express FIBROBLASTS and can powerfully generate FIBRONOCTIN, and the generated cells are cardiac FIBROBLASTS and have good functionality. The result shows that the culture solution can effectively obtain the cardiac fibroblasts with good functionality by the method.
Effect example 3 flow assay of cardiac fibroblast surface antigens
The flow type processing steps are as follows: cell samples were centrifuged at 300rcf for 2min, the supernatant removed, washed once with 5mL PBS, and the cells diluted to 2X 10 with FACS buffer 7 Blowing, beating and uniformly mixing; preparing special flow tubes, adding 50 mu L of cell sample suspension into each special flow tube, adding 50 mu L of corresponding antibody into sample cells, uniformly mixing, and incubating for 30 minutes in a dark place; then adding 2mL of FACS buffer into the cells incubated by the antibody, uniformly mixing by using a vortex mixer, centrifuging for 3 minutes at room temperature at 300rcf, removing supernatant, adding a secondary antibody in a 100 mu L centrifuge tube into the flow tube, and incubating for 15 minutes at room temperature in a dark place; and finally, adding 2mL of flow buffer into the flow tube, uniformly mixing by vortex oscillation, centrifuging for 2min at room temperature at 300rcf, removing supernatant, adding 300 mu L of flow buffer into the flow tube, uniformly mixing by vortex oscillation, and detecting the positive rate of the marker by selecting a proper channel in a BD FACSCaliur flow cytometer.
FIG. 5 shows the flow identification results of cell surface markers obtained from the culture solution and the method described in Experimental group 4. The result of the detection of the surface marker clone TE-7 of the cardiac fibroblast shows that the positive expression rate of clone TE-7 obtained by the method is 99.58 percent, and the result not only shows that the cell obtained by the method is the cardiac fibroblast, but also shows that the purity is higher and reaches more than 99 percent. Therefore, the culture solution and the culture method can efficiently obtain the human heart fibroblasts, and the culture solution does not contain animal-derived components, so that the invention has clinical practicability.
The invention can obtain induced pluripotent stem cells by a monocyte plasmid electrotransformation method in blood of a heart disease patient, and obtains the heart fibroblasts by directional differentiation by adopting the culture solution and the method. The culture solution contains WNT activator CHIR99021, BMP antagonist BMP4 and FGF conduction path activating molecule FGF2, and through controlling key signal path and under the action of 2.5% PVA, the culture solution effectively promotes the induced pluripotent stem cells to differentiate towards cardiac mesoderm, then under the continuous action of beta-Me and FGF2, second cardiac progenitor cells are generated, and further culture and induction are carried out to obtain mature cardiac fibroblasts. The culture solution has simple and clear components, can effectively promote the generation of cardiac fibroblasts, does not contain animal-derived components, and has clinical applicability. Meanwhile, the method develops a new method, adopts the human induced pluripotent stem cells to obtain the cardiac fibroblasts through the second heart field progenitor cells, has simple procedure, only needs 12 days for the culture period, greatly improves the preparation speed of the cardiac fibroblasts, reduces the preparation cost, is an effective method for obtaining the cardiac fibroblasts, and can be used for the mechanism research of cardiac diseases, the drug screening and the clinical cell treatment.
Claims (7)
1. A method for inducing pluripotent stem cells to differentiate and culture heart fibroblasts is characterized by comprising the following steps:
step S1, performing directed differentiation culture on the induced pluripotent stem cells by using the culture solution of the cardiac mesoderm progenitor cells to obtain cardiac mesoderm progenitor cells; specifically, the reaction is carried out at 35-39 ℃ and 3-7% CO 2 Under the condition, performing directed differentiation culture on the induced pluripotent stem cells for 1-3 days by using a culture solution for promoting cardiac mesoderm progenitor cells, removing nonadherent cells, and performing directed differentiation to obtain the cardiac mesoderm progenitor cells;
the composition of the culture solution for promoting the cardiac mesoderm progenitor cells comprises: RPMI1640 basic culture medium, human serum albumin, L-ascorbic acid 2 phosphoric acid, polyvinyl alcohol, Wnt activator, BMP antagonist and FGF activator; the concentration of the human serum albumin is 1-5 mg/mL, and the concentration of the L-ascorbic acid 2 phosphoric acid is 50-100 mug/mL; the mass concentration of the polyvinyl alcohol is 0.1-10%; the concentration of the Wnt activator is 4-8 mu mol/L; the concentration of the BMP antagonist is 1-10 ng/mL; the concentration of the FGF activator is 1-10 ng/mL;
step S2, continuously carrying out differentiation culture on the cardiac mesoderm progenitor cells by using a second cardiac field progenitor cell culture solution to obtain second cardiac field progenitor cells; specifically, the heart mesoderm progenitor cells are cultured at 35-39 DEG C、3～7％CO 2 Continuously culturing the second heart field progenitor cells for 3-5 days by using a second heart field progenitor cell culture solution under the condition, and replacing the culture solution every 1-2 days during continuous culture; the second cardiac field progenitor cell culture solution comprises a cardiac fibroblast culture solution, beta-mercaptoethanol and an FGF activator; the concentration of the beta-mercaptoethanol in the heart fiber culture solution is 0.1-10 mmol/L; the concentration of the FGF activator in the heart fiber culture solution is 25-100 ng/mL;
step S3, digesting the second cardiac field progenitor cells, and culturing the second cardiac field progenitor cells with a cardiac fibroblast culture solution to obtain purified mature cardiac fibroblasts; specifically, the second cardiac field progenitor cells obtained in step S2 are digested by TryplE at 3000- 2 Density inoculation, 3-7% CO at 35-39 deg.C 2 Under the condition, culturing the mature cardiac fibroblasts for 5 to 7 days by using a cardiac fibroblast culture solution to obtain purified mature cardiac fibroblasts, and carrying out subculture for 5 to 10 times; the heart fibroblast culture solution comprises: DMEM high-sugar medium DMEM high glucose with GlutaMAX supplement, KnockOut TM SR XenoFree CTS TM And MEM Non-Essential Amino Acids Solution; the KnockOut TM SR XenoFree CTS TM The concentration is 5% -15%; the concentration of the MEM Non-Essential Amino Acids Solution is 0.5-2%.
2. The method of claim 1, wherein:
step S1, specifically, 5% CO at 37 ℃ 2 Under the condition, performing directed differentiation culture on the induced pluripotent stem cells for 2 days by using a culture solution for promoting cardiac mesoderm progenitor cells, removing nonadherent cells, and performing directed differentiation to obtain the cardiac mesoderm progenitor cells;
step S2, specifically, the cardiac mesodermal progenitors were incubated at 37 deg.C with 5% CO 2 Under the condition, continuously culturing the second heart field progenitor cells by using a second heart field progenitor cell culture solution for 4 days to obtain second heart field progenitor cells, and replacing the culture solution every 2 days during the continuous culture period;
step S3, specifically, the second cardiac field progenitor cells obtained in step S2 are digested with TryplE at 3000- 2 Density inoculation, 5% CO at 37 ℃ 2 Under the condition, culturing the mature cardiac fibroblasts for 5 to 7 days by using a cardiac fibroblast culture solution to obtain purified mature cardiac fibroblasts, and carrying out subculture for 5 to 10 times.
3. The method of claim 1, wherein the induced pluripotent stem cells are obtained by peripheral blood mononuclear cell electroporation.
4. The method of claim 1, wherein the Wnt activator is CHIR99021, the BMP antagonist is BMP4, and the FGF activator is FGF 2.
5. The method of claim 1, wherein the human serum albumin concentration is 2.5mg/mL and the L-ascorbic acid 2 phosphate concentration is 75 μ g/mL; the mass concentration of the polyvinyl alcohol is 2.5%; the concentration of the Wnt activator is 6 mu mol/L; the concentration of the BMP antagonist is 5 ng/mL; the concentration of the FGF activator is 5 ng/mL.
6. The method of claim 1, wherein the KnockOut TM SR XenoFree CTS TM The concentration is 10%; the concentration of the MEM Non-Essential Amino Acids Solution is 1 percent.
7. The method of claim 1, wherein the concentration of said β -mercaptoethanol in said cardiac fibroblast culture fluid is 1 mmol/L; the FGF activator is FGF2, and the concentration of the FGF activator in the heart fiber culture solution is 50 ng/mL.
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