Culture medium and method for transdifferentiating fibroblasts into heart valve endothelial-like cells and application of culture medium and method

Abstract

The invention relates to a culture medium and method for transdifferentiating fibroblasts into heart valve endothelial-like cells and application of the culture medium and relates to the field of cell differentiation, and the culture medium comprises an induction culture medium I, an induction culture medium II, an induction culture medium III and an induction culture medium IV; according to the method, the human skin fibroblasts can be directly transdifferentiated into the heart valve endothelial-like cells in the induction culture media I-IV, a stem cell stage is not needed, a virus vector is not used for introducing transcription factors in the transdifferentiation process, and only small molecule compounds and cell factors are used; therefore, the technology has very high safety and a very wide application prospect.

Description

Technical Field

[0001] The present invention relates to the field of cell differentiation, and particularly to a culture medium, a method for transdifferentiating fibroblasts into cardiac valve endothelial-like cells, and their applications. Background Art

[0002] Cardiac valve disease is a highly prevalent disease worldwide, often causing severe cardiac dysfunction. Valve replacement surgery is the most effective treatment method. The commonly seen valves on the market are biological valves and mechanical valves, but both have some defects that limit their service life. Therefore, it is particularly necessary to construct a new type of tissue-engineered valve with growth and regeneration ability.

[0003] Valve endothelial cells (VECs) are an important component of natural valves. How to obtain these cells quickly and efficiently is a topic worthy of exploration. Currently, induced pluripotent stem cell (iPSC) technology enables somatic cells to be reprogrammed into primitive undifferentiated cells and then differentiated into valve endothelial-like cells, which seems to be a good choice. This method provides the possibility for the source of seed cells for new type tissue-engineered valves. For example, Patent CN113073076 A discloses a method for differentiating pluripotent stem cells into valve endothelial cells and valve interstitial cells, including the following steps: preparing a culture flask or dish for differentiation; preparing iPSCs; inoculating cells on D0; differentiating on D1 - D4; differentiating on D5 - D6; magnetic bead sorting of CD144-positive cells; culturing and subculturing valve endothelial cells and valve interstitial cells. Uniform valve endothelial cells and valve interstitial cells can be stably obtained, which can meet the requirements of seed cells for tissue-engineered valves applied clinically. However, the low differentiation efficiency and potential tumorigenic risk hinder its further development and application. How to obtain valve endothelial cells efficiently and safely remains a major challenge.

[0004] Although existing iPSCs and ESCs can be differentiated into valve endothelial cells, during the formation of iPSC cells, transcription factors need to be introduced into cells through viral vectors, and the final acquisition efficiency of iPSCs is generally less than 1%. Therefore, the efficiency of obtaining valve endothelial cells is also relatively low. Moreover, due to the use of viral vectors, the insertion of foreign genes, and the existence of stem cell state, there is a relatively high risk of tumorigenesis; the source of ESC cells is very limited, and there are certain ethical issues. The existence of stem cell state also has potential safety hazards of tumorigenic risk. Therefore, in view of the limited research on differentiating induced pluripotent stem cells and ESCs into valve endothelial-like cells, the low efficiency, and the major safety hazard of tumorigenicity, the present invention provides a culture medium, a method for transdifferentiating fibroblasts into cardiac valve endothelial-like cells, and their applications. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a culture medium, a method and an application for transdifferentiating fibroblasts into cardiac valve endothelial-like cells. The object of the present invention is to directly transdifferentiate human dermal fibroblasts (HDF) into cardiac valve endothelial-like cells without going through the stem cell stage, and without using viral vectors or first reprogramming the cells into pluripotent stem cells during the transdifferentiation process. Only by using small molecule compounds and cytokines can cardiac valve endothelial-like cells be obtained safely and efficiently, providing a reliable cell source for novel tissue engineering valves.

[0006] The technical solution of the present invention to solve the above technical problem:

[0007] The first object is to provide a culture medium for transdifferentiating fibroblasts into cardiac valve endothelial-like cells, including induction medium I, induction medium II, induction medium III and induction medium IV;

[0008] The induction medium I includes the following components in the following amounts: 25 - 35 μM 5-Azacytidine;

[0009] The induction medium II includes the following components in the following amounts: 20 - 30 ng / mL BMP-4, 1 - 9 μM CHIR99021;

[0010] The induction medium III includes the following components in the following amounts: 45 - 55 ng / mL VEGF, 7 - 13 μM DAPT, 20 - 30 ng / mL PLGF, 35 - 45 ng / mL HGF, 95 - 105 μM 8-Br-cAMP;

[0011] The induction medium IV includes the following components in the following amounts: 45 - 55 ng / mL VEGF, 1 - 9 μM SB431542, 95 - 105 μM 8-Br-cAMP.

[0012] The beneficial effect of the present invention is that in the above culture medium, human dermal fibroblasts can be directly transdifferentiated into cardiac valve endothelial-like cells without going through the stem cell stage, and without using viral vectors to introduce transcription factors during the transdifferentiation process. Only small molecule compounds and cytokines are used, which makes this technology have high safety and broad application prospects.

[0013] On the basis of the above technical solution, the present invention can also be improved as follows.

[0014] Further, the induction medium I includes the following components in the following amounts: 27 - 33 μM 5-Azacytidine;

[0015] The induction medium II comprises components in the following dosages: 22 - 28 ng / mL BMP-4, 3 - 7 μM CHIR99021;

[0016] The induction medium III comprises components in the following dosages: 46 - 54 ng / mL VEGF, 8 - 12 μM DAPT, 23 - 27 ng / mL PLGF, 37 - 43 ng / mL HGF, 97 - 102 μM 8-Br-cAMP;

[0017] The induction medium IV comprises components in the following dosages: 47 - 53 ng / mL VEGF, 3 - 7 μM SB431542, 97 - 103 μM 8-Br-cAMP.

[0018] Furthermore, the induction medium I comprises components in the following dosage: 30 μM 5-Azacytidine;

[0019] The induction medium II comprises components in the following dosages: 25 ng / mL BMP-4, 5 μM CHIR99021;

[0020] The induction medium III comprises components in the following dosages: 50 ng / mL VEGF, 10 μM DAPT, 25 ng / mL PLGF, 40 ng / mL HGF, 100 μM 8-Br-cAMP;

[0021] The induction medium IV comprises components in the following dosages: 50 ng / mL VEGF, 5 μM SB431542, 100 μM 8-Br-cAMP.

[0022] The beneficial effect of adopting the above further scheme is that: the conversion rate of fibroblasts into heart valve endothelial-like cells by using the above induction medium I - induction medium IV is more excellent.

[0023] Furthermore, the induction medium I further comprises HDF complete medium;

[0024] The induction medium II further comprises any one of TeSR TM -E6 basal medium, N2B27 basal medium;

[0025] The induction medium III further comprises any one of TeSR TM -E6 basal medium, StemPro-34 basal medium;

[0026] The induction medium IV further comprises EGM-2 complete medium.

[0027] The second object is to provide a kit for transdifferentiating fibroblasts into cardiac valve endothelial-like cells, including induction medium I, induction medium II, induction medium III, and induction medium IV in the medium for transdifferentiating fibroblasts into cardiac valve endothelial-like cells described above.

[0028] The beneficial effect of adopting the above solution is that by making induction medium I to induction medium IV into a kit, it is convenient to transdifferentiate fibrotic cells into cardiac valve endothelial-like cells.

[0029] The third object is to provide a method for transdifferentiating fibroblasts into cardiac valve endothelial-like cells, including the following steps:

[0030] (1) Incubate fibroblasts with induction medium I in the medium for transdifferentiating fibroblasts into cardiac valve endothelial-like cells described above to obtain incubated fibroblasts;

[0031] (2) Continuously culture the incubated fibroblasts with induction medium II in the medium for transdifferentiating fibroblasts into cardiac valve endothelial-like cells described above to obtain induced fibroblasts;

[0032] (3) Continuously culture the induced fibroblasts with induction medium III in the medium for transdifferentiating fibroblasts into cardiac valve endothelial-like cells described above to obtain hiVEC cells;

[0033] (4) Inoculate the hiVEC cells on PGG-DAV (PGG cross-linked acellular valve) and co-culture with induction medium IV in the medium for transdifferentiating fibroblasts into cardiac valve endothelial-like cells described above to obtain cardiac valve endothelial-like cells.

[0034] Among them, the method for obtaining the fibroblasts is as follows: Separate foreskin tissue into pieces, digest with trypsin solution, and then neutralize with FBS with a volume 1.5 to 2 times that of the trypsin solution. Rinse the suspended tissue with PBS solution, and then resuspend it in a mixed solution containing DMEM complete medium, FBS, and type IV collagenase with a volume ratio of (8 - 12):(0.5 - 1.5):(3 - 7). Stir evenly at room temperature, aspirate the tissue, and wash it with PBS solution to obtain fibroblasts.

[0035] The beneficial effect of adopting the above further solution is that the culture method of the present invention can directly transdifferentiate human skin fibroblasts into cardiac valve endothelial-like cells without going through the stem cell stage, and no viral vector is used to introduce transcription factors during the transdifferentiation process.

[0036] Further, the incubation conditions in step (1) are: 37°C, 5% CO2, and the time is 1 - 3 days;

[0037] The culture conditions in step (2) are: 37 °C, 5% CO2, and the time is 2 - 6 days;

[0038] The culture conditions in step (3) are: 37 °C, 5% CO2, and the time is 6 - 10 days;

[0039] The co - culture conditions in step (4) are: 37 °C, 5% CO2, and the time is 5 - 9 days.

[0040] Furthermore, the specific steps for collecting cells by magnetic bead sorting in step (3) are: subjecting the mixture obtained by further culturing in induction medium III to digestion with digestive enzymes, magnetic bead incubation, rinsing, sieving, rinsing of the sorting column, and magnetic bead sorting, and collecting hiVEC cells.

[0041] Furthermore, the magnetic beads used in the magnetic bead incubation are magnetic beads against CD144 or / and magnetic beads against CD31.

[0042] The fourth objective is to provide an application of cardiac valve endothelial - like cells, using the cardiac valve endothelial - like cells prepared by the method of trans - differentiating the fibroblasts into cardiac valve endothelial - like cells in the fabrication of artificial heart valves.

[0043] The beneficial effect of adopting the above - mentioned further scheme is: by using the cardiac valve endothelial - like cells prepared by the present invention in the fabrication of artificial heart valves, the technology has high safety and broad application prospects. Brief Description of the Drawings

[0044] Figure 1 It is the flow cytometry diagram of endothelial markers CD31 and CD144 of Isotype, HDF, and PDMS10 of the present invention;

[0045] Figure 2 It is the flow cytometry diagram of endothelial markers CD31 and CD144 of Isotype and HDF - hiVEC of the present invention;

[0046] Figure 3 It is the flow cytometry diagram of endothelial markers CD31 and CD144 of Isotype and HDF - hiVEC - PGG - DHV of the present invention;

[0047] Figure 4 It is the RNA - seq component analysis diagram of HDF - hiVEC - PGG - DHV of the present invention. Detailed Embodiments

[0048] The principles and features of the present invention are described below. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0049] Description of the sources of materials and reagents:

[0050] Trypsin was purchased from Gibco; type IV collagenase was purchased from Gibco; DMEM was purchased from Gibco; Fibronectin solution was purchased from Sigma-aldrich; HDF complete medium was purchased from Gibco; 5-Azacytidine was purchased from Selleck; TeSR TM -E6 was purchased from Stemcell; N2B27 basal medium was purchased from CellArtis, BMP-4 was purchased from PeproTech; CHIR99021 was purchased from Selleck; VEGF was purchased from PeproTech; DAPT was purchased from Selleck; PLGF (protein accession number: P49764 (V19-P158), gene ID: 18654) was purchased from PeproTech; HGF was purchased from PeproTech; 8-Br-cAMP (molecular formula: C10H10BrN5NaO6P, CAS number: 76939-46-3) was purchased from Selleck; EDTA was purchased from ThermoScientific; anti-CD144 magnetic beads were purchased from Miltenyi; anti-CD31 magnetic beads were purchased from Miltenyi; EGM-2 complete medium was purchased from Lonza; SB431542 (molecular formula: C22H16N4O3, CAS number: 301836-41-9) was purchased from Selleck; PGG cross-linked decellularized valve (PGG-DAV) was prepared according to the method in the reference (reference: W.H. Qiao, P. Liu, D. Hu, M. Al Shirbini, X.M. Zhou, N.G. Dong. Sequential hydrophile and lipophile solubilization as an efficient method for decellularization of porcine aortic valve leaflets: Structure, mechanical property and biocompatibility study. Journal of Tissue Engineering and Regenerative Medicine, 2018, 12(2): e828-e840.

[0051] L.N.Sierad,A.Simionescu,C.Albers,J.Chen,J.Maivelett,M.E.Tedder,etal.Design and Testing of a Pulsatile Conditioning System for DynamicEndothelialization of Polyphenol-Stabilized Tissue Engineered HeartValves.Cardiovascular Engineering and Technology,2010,1(2):138-153.)。

[0052] Example

[0053] This example relates to a culture medium for transdifferentiating fibroblasts into heart valve endothelial-like cells, including induction medium I, induction medium II, induction medium III, and induction medium IV;

[0054] The induction medium I includes the following components in the following amounts: 25 - 35 μM 5-Azacytidine;

[0055] The induction medium II includes the following components in the following amounts: 20 - 30 ng / mL BMP-4, 1 - 9 μM CHIR99021;

[0056] The induction medium III includes the following components in the following amounts: 45 - 55 ng / mL VEGF, 7 - 13 μM DAPT, 20 - 30 ng / mL PLGF, 35 - 45 ng / mL HGF, 95 - 105 μM 8-Br-cAMP;

[0057] The induction medium IV includes the following components in the following amounts: 45 - 55 ng / mL VEGF, 1 - 9 μM SB431542, 95 - 105 μM 8-Br-cAMP.

[0058] Preferably, in this example, the induction medium I includes the following components in the following amounts: 27 - 33 μM 5-Azacytidine;

[0059] The induction medium II includes the following components in the following amounts: 22 - 28 ng / mL BMP-4, 3 - 7 μM CHIR99021;

[0060] The induction medium III comprises the following components in the following amounts: 46 - 54 ng / mL VEGF, 8 - 12 μM DAPT, 23 - 27 ng / mL PLGF, 37 - 43 ng / mL HGF, 97 - 102 μM 8-Br-cAMP;

[0061] The induction medium IV comprises the following components in the following amounts: 47 - 53 ng / mL VEGF, 3 - 7 μM SB431542, 97 - 103 μM 8-Br-cAMP.

[0062] Specifically in this example, the induction medium I comprises the following component in the following amount: 30 μM 5-Azacytidine;

[0063] The induction medium II comprises the following components in the following amounts: 25 ng / mL BMP-4, 5 μM CHIR99021;

[0064] The induction medium III comprises the following components in the following amounts: 50 ng / mL VEGF, 10 μM DAPT, 25 ng / mL PLGF, 40 ng / mL HGF, 100 μM 8-Br-cAMP;

[0065] The induction medium IV comprises the following components in the following amounts: 50 ng / mL VEGF, 5 μM SB431542, 100 μM 8-Br-cAMP.

[0066] Preferably in this example, the induction medium I further comprises HDF complete medium;

[0067] The induction medium II further comprises any one of TeSR TM -E6 basal medium, N2B27 basal medium;

[0068] The induction medium III further comprises any one of TeSR TM -E6 basal medium, StemPro-34 basal medium;

[0069] The induction medium IV further comprises EGM-2 complete medium.

[0070] This example also relates to a kit for transdifferentiating fibroblasts into heart valve endothelial-like cells, comprising the induction medium I, induction medium II, induction medium III, and induction medium IV in the medium for transdifferentiating fibroblasts into heart valve endothelial-like cells described above.

[0071] This example also relates to a method for transdifferentiating fibroblasts into heart valve endothelial-like cells, comprising the following steps:

[0072] (1) Incubate the fibroblasts with Induction Medium I in the medium for transdifferentiating fibroblasts into heart valve endothelial-like cells to obtain the incubated fibroblasts;

[0073] (2) Continuously culture the incubated fibroblasts with Induction Medium II in the medium for transdifferentiating fibroblasts into heart valve endothelial-like cells to obtain the induced fibroblasts;

[0074] (3) Continuously culture the induced fibroblasts with Induction Medium III in the medium for transdifferentiating fibroblasts into heart valve endothelial-like cells to obtain hiVEC cells;

[0075] (4) Seed the hiVEC cells on PGG-DAV and co-culture them with Induction Medium IV in the medium for transdifferentiating fibroblasts into heart valve endothelial-like cells to obtain heart valve endothelial-like cells.

[0076] Preferably in this example, the incubation conditions in step (1) are: 37 °C, 5% CO2, and the time is 1 - 3 days;

[0077] The culture conditions in step (2) are: 37 °C, 5% CO2, and the time is 2 - 6 days;

[0078] The culture conditions in step (3) are: 37 °C, 5% CO2, and the time is 6 - 10 days;

[0079] The co-culture conditions in step (4) are: 37 °C, 5% CO2, and the time is 5 - 9 days.

[0080] Preferably in this example, the specific steps for collecting cells by magnetic bead sorting in step (3) are: subject the mixture obtained by continuous culture with Induction Medium III to digestion with digestive enzymes, magnetic bead incubation, rinsing, sieving, rinsing of the sorting column, and magnetic bead sorting to collect hiVEC cells.

[0081] Preferably in this example, the magnetic beads used in the magnetic bead incubation are magnetic beads against CD144 or / and magnetic beads against CD31.

[0082] The method for transdifferentiating fibroblasts into heart valve endothelial-like cells provided in this example includes the following specific steps:

[0083] Step 1: Obtaining and culturing human dermal fibroblasts (HDF):

[0084] After circumcision, human dermal fibroblasts are isolated. The foreskin tissue is separated into 0.2 × 0.2 cm 2The fragments were digested with 5 mL of trypsin solution and neutralized with 10 mL of FBS. The suspended tissue was rinsed with PBS solution and then resuspended in a solution containing 20 mL of DMEM, 2 mL of FBS, and 10 mg of type IV collagenase. The mixture was stirred at a constant speed at room temperature. The tissue was gently aspirated, washed with PBS solution, resuspended in 10 mL of complete DMEM medium, transferred to a culture dish, and further cultured in an incubator (5% CO2, 37 °C).

[0085] Step 2: The prepared PDMS well plate with a hardness of 2.1 MPa was first cleaned with ultrapure water, immersed in 75% medical alcohol for 30 min for disinfection, then taken out, the remaining alcohol was poured out, placed in a biosafety cabinet, irradiated with ultraviolet light for 30 min, and then rinsed 3 times with sterile PBS for standby.

[0086] Step 3: Take out the previously prepared and aliquoted 1 mg / mL Fibronectin solution from the -80 °C ultra-low temperature refrigerator, place it in an ice box and melt for 30 - 45 min. After disinfection, dilute it to 20 μg / mL with sterile PBS in a biosafety cabinet. This is the working solution. The recommended concentration of Fibronectin is 5 μg / cm 2 , calculate the required volume according to the bottom area of the well plate or culture dish, add it and incubate at room temperature for at least 45 min. After the coating is completed, recover it with a pipette gun and store it in a 4 °C refrigerator. It can be used repeatedly for many times.

[0087] Step 4: Digest and passage the HDF cells. After centrifugation and resuspension, count the cells with a cell counting plate. According to the cell density, add an appropriate amount of cell suspension so that the number of cells in each well of the 12-well PDMS-covered well plate reaches 2×10 4 cells (the number of cells added to other culture plates or culture dishes is calculated according to the bottom area ratio). Add 2 mL of HDF complete medium to each well of the 12-well plate. After adding the cells, gently shake the plate 10 - 15 times up, down, left, and right in the cross direction. Observe under an optical inverted microscope to ensure that the cells are evenly distributed and not adhered. After spraying and disinfecting with 75% medical alcohol, place it back in the CO2 incubator and culture overnight (usually 12 h) to obtain the passaged fibroblasts.

[0088] Step 5: The next day, aspirate the old HDF complete medium, and add freshly prepared induction medium I (30 μM 5-Azacytidine is added to the complete medium) to each well. Incubate the medium for 1 day to obtain the incubated fibroblasts.

[0089] Step 6: After 1 day, aspirate the induction medium I and replace it with freshly prepared induction medium II (TeSR TM- Add 25 ng / mL BMP-4 (recombinant human bone morphogenetic protein 4) and 5 μM CHIR99021 to the E6 or N2B27 basal medium and continue culturing for 4 days, changing the fresh culture medium every day to obtain induced fibroblasts. CHIR99021 is an aminopyrimidine derivative, which is an effective inhibitor of GSK3, inhibiting GSK3β (IC50 = 6.7 nM) and GSK3α (IC50 = 10 nM), acting as a WNT activator.

[0090] Step 7: After 4 days, aspirate the induction medium II and replace it with freshly prepared induction medium III (TeSR TM - Add 50 ng / mL VEGF (vascular endothelial growth factor), 10 μM DAPT ((3,5-difluorophenylacetyl)-L-alanyl-L-2-phenylglycine tert-butyl ester), 25 ng / mL PLGF (protein accession number: P49764 (V19 - P158), gene ID: 18654), 40 ng / mL HGF (hepatocyte growth factor) and 100 μM 8-Br-cAMP (molecular formula: C 10 H 10 BrN5NaO6P, CAS number: 76939 - 46 - 3) to the E6 or StemPro-34 basal medium and continue culturing for 8 days, changing the fresh culture medium every day.

[0091] Step 8: After 8 days, some cells showed a cobblestone-like morphology. Digest the cells with digestive enzymes. After neutralizing the digestion of the target cells and centrifuging to discard the supernatant, resuspend the cells with sterile PBS and then centrifuge (set the rotation speed to 2000 rpm and the centrifugation time to 3 min), and discard the supernatant after centrifugation.

[0092] Magnetic bead incubation: Ensure that the number of cells collected for each sample is greater than or equal to 1.0 × 10 7 cells. Dilute 20 μL of anti-CD144 or CD31 magnetic beads 5-fold with 80 μL of separation buffer (PBS supplemented with 0.5% BSA and 2 mM EDTA (ethylenediaminetetraacetic acid)), add the magnetic beads to the target cells, pipette and mix well repeatedly, place on a shaker in a 4°C refrigerator at a rotation speed of 100 rpm, and incubate for 3 min.

[0093] Cell rinsing: Add 1 mL of separation buffer to each sample tube, transfer the sample tube to a centrifuge, set the rotation speed to 300 × g and the centrifugation time to 10 min, and centrifuge after balancing. First, slowly aspirate most of the supernatant with a 1 mL pipette, and carefully aspirate the remaining part with a 100 μL pipette, taking care not to aspirate the cell mass.

[0094] Cell sieving: Slowly add 0.5 mL of separation buffer along the inner wall of each sample tube, pipette thoroughly to mix evenly, and pass the cells through a 30-μm nylon mesh sieve to remove cell clumps to avoid clogging the sorting column.

[0095] Sorting column rinsing: Add 3 - 5 mL of buffer (prepared by diluting Diluting MACS BSA Stock Solution 20-fold with Rinsing Solution) to the sorting column and rinse it 3 times to improve the permeability of the sorting column.

[0096] Magnetic bead sorting (MACS sorting): Install the rinsed sorting column onto the quadro MACS separator, add the cell suspension to the sorting column, and the cell suspension in the column flows through the sorting column due to gravity. Unlabeled cells flow into the centrifuge tube below. Rinse 3 times with 0.5 mL of buffer to collect the cells fully. Cell collection: Take the sorting column out of the magnetic field, quickly add 1 mL of buffer, and rapidly push the liquid through the column with the syringe piston. The target cells flow into the centrifuge tube below to obtain hiVEC cells. Replace it with freshly prepared induction medium Ⅳ (EGM-2 complete medium supplemented with 50 ng / mL VEGF, 5 μM SB431542 (molecular formula: C 22 H 16 N4O3, CAS number: 301836 - 41 - 9), 100 μM 8-Br-cAMP) and continue the culture for subsequent experiments.

[0097] Step 9: Seed the hiVEC cells obtained by MACS sorting at a density of 2×10 5 cells onto the PGG crosslinked acellular valve (PGG-DAV) and co-culture for 7 days. The medium is induction medium Ⅳ and is changed daily to obtain heart valve endothelial-like cells (HDF-hiVEC-PGG-DHV). Finally, evaluate the induction efficiency by flow cytometry.

[0098] Control example

[0099] The decellularized porcine aortic valve was prepared according to the method reported in the literature (reference: W.H. Qiao, P. Liu, D. Hu, M. Al Shirbini, X.M. Zhou, N.G. Dong. Sequential hydrophile and lipophile solubilization as an efficient method for decellularization of porcine aortic valve leaflets: Structure, mechanical property and biocompatibility study. Journal of Tissue Engineering and Regenerative Medicine, 2018, 12(2): e828 - e840); subsequently, the decellularized valve (PGG - DAV) was cross - linked with PGG and cultured to improve its mechanical properties, and the decellularized porcine aortic valve (Isotype) was obtained as a control.

[0100] Experimental examples

[0101] (1) Flow cytometry detection:

[0102] The results are as Figures 1 to 3 shown. Among them, Figure 1 are the flow cytometry plots of endothelial markers CD31 and CD144 of Isotype, HDF, and PDMS10. It can be seen from Figure 1 that the efficiency of HDF transdifferentiating into endothelial cells is 9.96%; Figure 2 are the flow cytometry plots of endothelial markers CD31 and CD144 of Isotype and HDF - hiVEC. It can be seen from Figure 2 that the purity of endothelial cells sorted by magnetic beads is 78.63%; Figure 3 are the flow cytometry plots of endothelial markers CD31 and CD144 of Isotype and HDF - hiVEC - PGG - DHV. It can be seen from Figure 3 that after sorting by magnetic beads and continuing to culture the cells on the PGG - cross - linked decellularized valve for 7 days, the purity of endothelial cells is 92.51%.

[0103] (2) RNA - seq component analysis:

[0104] Figure 4 is the RNA - seq component analysis chart. It can be seen from Figure 4 that after culturing on the PGG - cross - linked decellularized valve for 7 days, the overall similarity between HDF - hiVEC and natural valve VEC cells (HAVEC) reaches more than 98%.

[0105] In summary, it can be seen that the present invention directly transdifferentiates human skin fibroblasts into cardiac valve endothelial-like cells, and the overall similarity with natural valve VEC cells reaches more than 98%. The cell source is very rich, without going through the stem cell stage, and no viral vector is used to introduce transcription factors during the transdifferentiation process. Only small molecule compounds and cytokines are used, which makes this technology have high safety and broad application prospects.

[0106] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A culture medium for transdifferentiation of fibroblasts into heart valve endothelial-like cells, characterized in that: It includes induction medium I, induction medium II, induction medium III and induction medium IV; The induction medium I includes the following components in the following amounts: 25-35 μM 5-Azacytidine; The induction medium II includes the following components in the following amounts: 20-30 ng / mL BMP-4, 1-9 μM CHIR99021; The induction medium III includes the following components: 45-55 ng / mL VEGF, 7-13 μM DAPT, 20-30 ng / mL PLGF, 35-45 ng / mL HGF, 95-105 μM 8-Br-cAMP; The induction medium IV includes the following components: 45-55 ng / mL VEGF, 1-9 μM SB431542, and 95-105 μM 8-Br-cAMP.

2. The culture medium for transdifferentiation of fibroblasts into heart valve endothelial-like cells according to claim 1, characterized in that: The induction medium I includes the following components in the following amounts: 27-33 μM 5-Azacytidine; The induction medium II includes the following components in the following amounts: 22-28 ng / mL BMP-4, 3-7 μM CHIR99021; The induction medium III includes the following components: 46-54 ng / mL VEGF, 8-12 μM DAPT, 23-27 ng / mL PLGF, 37-43 ng / mL HGF, 97-102 μM 8-Br-cAMP; The induction medium IV includes the following components in the following amounts: 47-53 ng / mL VEGF, 3-7 μM SB431542, and 97-103 μM 8-Br-cAMP.

3. The culture medium for transdifferentiation of fibroblasts into heart valve endothelial-like cells according to claim 1, characterized in that: The induction medium I includes the following components in the following amounts: 30 μM 5-Azacytidine; The induction medium II includes the following components in the following amounts: 25 ng / mL BMP-4, 5 μM CHIR99021; The induction medium III includes the following components: 50 ng / mL VEGF, 10 μM DAPT, 25 ng / mL PLGF, 40 ng / mL HGF, 100 μM 8-Br-cAMP; The induction medium IV includes the following components: 50 ng / mL VEGF, 5 μM SB431542, and 100 μM 8-Br-cAMP.

4. The culture medium for transdifferentiation of fibroblasts into heart valve endothelial-like cells according to claim 1, characterized in that: The induction medium I also includes HDF complete medium; The induction medium II also includes TeSR TM -E6 basic culture medium, N2B27 basic culture medium; The induction medium III also includes TeSR TM -E6 basal medium, StemPro-34 basal medium, any one; The induction medium IV also includes EGM-2 complete medium.

5. A kit for transdifferentiation of fibroblasts into heart valve endothelial-like cells, characterized in that: The invention comprises the induction medium I, induction medium II, induction medium III and induction medium IV in the culture medium for transdifferentiation of fibroblasts into heart valve endothelial-like cells as described in any one of claims 1 to 4.

6. A method for transdifferentiating fibroblasts into heart valve endothelial-like cells, characterized in that: The steps include: (1) incubating fibroblasts with the induction medium I in the medium for transdifferentiation of fibroblasts into heart valve endothelial-like cells according to any one of claims 1 to 4 to obtain incubated fibroblasts; (2) continuing to culture the incubated fibroblasts using the induction medium II in the medium for transdifferentiation of fibroblasts into heart valve endothelial-like cells according to any one of claims 1 to 4 to obtain induced fibroblasts; (3) continuing to culture the induced fibroblasts using the induction medium III in the medium for transdifferentiation of fibroblasts into heart valve endothelial-like cells according to any one of claims 1 to 4 to obtain hiVEC cells; (4) The hiVEC cells are inoculated on PGG-DAV and co-cultured with the induction medium IV in the culture medium for transdifferentiation of fibroblasts into heart valve endothelial-like cells as described in any one of claims 1 to 4 to obtain heart valve endothelial-like cells.

7. The method for transdifferentiation of fibroblasts into heart valve endothelial-like cells according to claim 6, characterized in that: The incubation conditions in step (1) are: 37° C., 5% CO 2 , and the time is 1-3 days; The culture conditions in step (2) are: 37° C., 5% CO 2 , and the culture time is 2-6 days; The culture conditions in step (3) are: 37° C., 5% CO 2 , and the culture time is 6-10 days; The co-culture conditions in step (4) are: 37° C., 5% CO 2 , and the duration is 5-9 days.

8. The method for transdifferentiation of fibroblasts into heart valve endothelial-like cells according to claim 6, characterized in that: The specific steps of collecting cells by magnetic bead sorting in step (3) are as follows: the mixture obtained by continuing to culture the induction medium III is digested with digestive enzymes, incubated with magnetic beads, rinsed, screened, rinsed with sorting columns and sorted with magnetic beads to collect hiVEC cells.

9. The method for transdifferentiation of fibroblasts into heart valve endothelial-like cells according to claim 8, characterized in that: The magnetic beads used in the magnetic bead incubation are anti-CD144 magnetic beads and / or anti-CD31 magnetic beads.

10. Use of heart valve endothelial-like cells, characterized in that: The heart valve endothelial-like cells prepared by the method for transdifferentiating fibroblasts into heart valve endothelial-like cells as described in any one of claims 6 to 9 are used to prepare artificial heart valves.

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