Method for inducing human pluripotent stem cells to differentiate into myocardial cells

Abstract

The invention belongs to the technical field of cell culture, and particularly relates to a method for inducing differentiation of human pluripotent stem cells into myocardial cells, glycidyl methacrylate is found and applied as a key induction factor for the first time, and the optimal concentration of glycidyl methacrylate in a differential culture medium is 0.5-5.1% by volume. According to the method, human pluripotent stem cells can be efficiently induced into cardiac muscle cells with an autonomous pulsation function within 7-10 days by utilizing a chemical component determination system containing specific concentration of transferrin, L-ascorbic acid and other additives through a definite induced differentiation stage and a culture maintenance stage, and the proportion of cTnT positive cells is stably higher than 80% within 9 days. According to the scheme, the components are clear, the batch stability is good, the differentiation efficiency is remarkably superior to that of a traditional method, and a high-quality cell source is provided for heart disease research, drug screening and cell therapy.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cell culture, and particularly relates to a method for inducing human pluripotent stem cells to differentiate into cardiomyocytes. BACKGROUND

[0002] Cardiac diseases, such as myocardial infarction and heart failure, are one of the leading causes of human death and disability worldwide. Cardiomyocytes in adult mammals are terminally differentiated cells with extremely limited or even completely lost proliferative capacity. When the heart is damaged due to ischemia, hypoxia, or other reasons, the necrotic myocardial tissue cannot be effectively repaired by self-renewal of cells, and is eventually replaced by fibrous scar tissue, leading to progressive and irreversible decline in cardiac function. Currently, drug therapy, interventional surgery and other means in clinical practice primarily aim to alleviate symptoms and delay disease progression, but cannot achieve regeneration and functional reconstruction of myocardial tissue. Therefore, heart transplantation is the only radical treatment for patients with end-stage heart failure. However, the severe shortage of donor hearts, immune rejection, and high medical costs greatly limit its widespread application.

[0003] Regenerative medicine based on cell replacement brings new hope for the fundamental treatment of cardiac diseases. Human pluripotent stem cells, including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), have the potential for self-renewal and multi-directional differentiation, and can be induced to differentiate into various functional cells, including cardiomyocytes, in vitro, thereby providing a potential and unlimited source of cells for cardiac repair, disease modeling, drug screening, and toxicity evaluation.

[0004] However, how to efficiently and stably direct the differentiation of human pluripotent stem cells into high-purity functional cardiomyocytes remains a key technical bottleneck in the current field. Although existing differentiation strategies have made some progress, they still have the following outstanding problems: 1. The cardiomyocyte population obtained by differentiation often contains other types of cells (such as fibroblasts and endothelial cells), and the proportion of cTnT and other myocardial-specific marker-positive cells fluctuates greatly between different experimental batches, making it difficult to obtain a purity higher than 80%, which seriously affects the reliability and consistency of subsequent applications.

[0005] 2. It often takes as long as 15 days or even longer from the start of stem cells to obtain cardiomyocytes with autonomous beating function, and the long cycle increases the cost of culture and the risk of contamination.

[0006] 3. Many differentiation systems rely on serum, serum substitutes or undefined animal-derived components, which not only introduce potential pathogen risks and zoonosis risks, but also lead to unclear culture medium chemical composition, large batch-to-batch differences, and serious hindrance to clinical application and standardized drug screening.

[0007] Therefore, it is of great significance to develop a method for the directional differentiation of human pluripotent stem cells into cardiomyocytes with clear chemical composition, no or less animal-derived components, batch stability, high differentiation efficiency and short differentiation period, and a matching culture medium, for promoting the basic research and clinical translation of cardiac regenerative medicine. SUMMARY

[0008] To solve the above problems, the present application first discovers and applies glycidyl methacrylate as a key inducing factor in the differentiation medium.

[0009] The specific scheme is as follows: the present application provides a method for inducing human pluripotent stem cells to differentiate into cardiomyocytes, which comprises the following steps: S1: culturing human pluripotent stem cells in a feeder-free culture system, using culture vessels coated with Matrigel and / or human pluripotent stem cell culture medium, and culturing until the cell density reaches 70%-90%; S2: treating the human pluripotent stem cells with an induction differentiation medium containing glycidyl methacrylate to induce them to differentiate into cardiomyocytes, wherein the concentration of glycidyl methacrylate in the induction differentiation medium is 0.5%-5.1% by volume; S3: continuing to culture with a maintenance medium containing no glycidyl methacrylate (GMA) until cardiomyocytes with beating function are obtained.

[0010] Further, in S2, the induction differentiation medium comprises a basic medium and an induction differentiation additive, and the basic medium is selected from one or more of RPMI 1640 medium, DMEM / F12 medium or IMDM medium.

[0011] Further, the induction differentiation additive comprises one or more of transferrin, L-ascorbic acid, sodium selenite, ethanolamine, L-carnitine, putrescine, linoleic acid and biotin.

[0012] Further, the concentration ranges of each component in the induction differentiation additive are as follows: transferrin: 10 ng / mL-30 ng / mL, L-ascorbic acid: 100-500 μg / mL, sodium selenite: 10-20 ng / mL, ethanolamine: 1-3 μg / mL, L-carnitine: 1-5 μg / mL, putrescine: 10 ng / mL-20 ng / mL, linoleic acid: 0.5-2 μg / mL, and biotin: 0.1 μg / mL-0.5 μg / mL.

[0013] Further, in S1, the human pluripotent stem cells are inoculated into a culture vessel coated with Matrigel at a density of 1×10 4 -6×10 4 cells / cm².

[0014] Further, the culture time for inducing the human pluripotent stem cells to differentiate into cardiomyocytes is 2-5 days, and the time for continuing the culture in the maintenance medium is 5-15 days.

[0015] In another aspect, the present application protects the use of glycidyl methacrylate in the preparation of an induction differentiation medium for inducing human pluripotent stem cells to differentiate into cardiomyocytes, and the concentration of the glycidyl methacrylate in the induction differentiation medium is 3.4%-5.1% by volume.

[0016] In addition, an induction differentiation medium for inducing human pluripotent stem cells to differentiate into cardiomyocytes comprises a basic medium, glycidyl methacrylate, and an induction differentiation additive, wherein the concentration of the glycidyl methacrylate is 0.5%-5.1% by volume, preferably 3.4%-5.1%, the basic medium is RPMI1640 medium, and the induction differentiation additive comprises transferrin, L-ascorbic acid, sodium selenite, ethanolamine, L-carnitine, putrescine, linoleic acid, and biotin; the concentration of each component is as follows: transferrin: 20 ng / mL, L-ascorbic acid: 300 μg / mL, sodium selenite: 15 ng / mL, ethanolamine: 2 μg / mL, L-carnitine: 3 μg / mL, putrescine: 15 ng / mL, linoleic acid: 1 μg / mL, and biotin: 0.3 μg / mL.

[0017] The present application has the beneficial effect that a high concentration of glycidyl methacrylate is found to induce human pluripotent stem cells to differentiate into cardiomyocytes with high efficiency in a specific medium, and the human pluripotent stem cells can be differentiated into beating human cardiomyocytes in about 10 days by adding folic acid, and the differentiation efficiency can reach 80% in 9 days. DETAILED DESCRIPTION

[0018] The present application is described below by specific embodiments. Unless otherwise specified, the technical means used in the present application are methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative, not limiting the scope of the present application, the essence and scope of the present application are only limited by the claims. For those skilled in the art, various changes or modifications to the composition and amount of the materials in these embodiments without departing from the essence and scope of the present application also fall within the scope of the present application.

[0019] Example 1 S1 Human pluripotent stem cell culture stage: The Matrigel diluted at a ratio of 1:150 uniformly covers the bottom of the culture dish, and it is incubated at 37°C for 12h. Human pluripotent stem cells are inoculated in the culture dish coated with Matrigel, and when the cells grow to 70%-80% confluence, the original stem cell culture medium is removed. Then, the cells are washed with PBS buffer solution without calcium and magnesium ions for 3 times, and the cells are passaged by using 0.5mmol / L EDTA solution, and then the EDTA solution is discarded. When the cell morphology is observed under a microscope, the cell morphology is retracted and the intercellular space is obviously increased, the digestion process is immediately terminated. Then 5 mL of complete culture medium is added, and the bottle wall is gently blown in sequence with a pipette to ensure that all adherent cells are collected. Pay attention to the moderate blowing strength to avoid foam and mechanical damage to the cells. Count the cells using a cell counting plate and calculate the cell density. After adjusting to the appropriate concentration with complete culture medium, it is divided into new culture bottles and placed at 37°C for 3-5 minutes. Then add fresh stem cell culture medium, blow the cells into a uniform suspension, and inoculate them into a 96-well culture plate at a ratio of 1:4. Place the culture plate in a biological safety cabinet, cover the plate cover, and place it at room temperature for 1 hour to ensure uniform and complete Matrigel coating. Finally, the cell culture plate is moved to a 37°C constant temperature incubator containing 5% CO2, the shaking table cover is closed, and the shaking speed is controlled below 200r / min.

[0020] S2 Induced differentiation stage: After the human pluripotent stem cells were grown to 80% confluency using stem cell medium, the medium was changed to induction differentiation medium. The induction differentiation medium comprises basal medium (RPMI 1640), glycidyl methacrylate (3.4% by volume), and induction differentiation additives, wherein the induction differentiation additives include transferrin (20 ng / mL), L-ascorbic acid (300 μg / mL), sodium selenite (15 ng / mL), ethanolamine (2 μg / mL), L-carnitine (3 μg / mL), putrescine (15 ng / mL), linoleic acid (1 μg / mL), and biotin (0.3 μg / mL). The human pluripotent stem cells were induced to differentiate into mesodermal precursor cells by culturing in a 37°C incubator with 5% CO2for 48 hours.

[0021] S3 Maintenance culture stage: The induction differentiation medium was changed to maintenance medium, which is the same basal medium without glycidyl methacrylate and the induction differentiation additives. The culture was continued in a 37°C incubator with 5% CO2, and the maintenance medium was changed every two days. The culture was continued for 5-10 days until spontaneous beating of the cells was observed, and functional cardiomyocytes were obtained.

[0022] Example 2 The method for inducing human pluripotent stem cells to differentiate into cardiomyocytes is as follows: S1 Human pluripotent stem cell culture stage: The same as the S1 step of Example 1.

[0023] S2 Induction differentiation stage: After the human pluripotent stem cells were grown to 80% confluency using stem cell medium, the medium was changed to induction differentiation medium. The induction differentiation medium comprises basal medium (DMEM / F12), glycidyl methacrylate (5.1% by volume), and induction differentiation additives, wherein the induction differentiation additives include transferrin (10 ng / mL), L-ascorbic acid (500 μg / mL), sodium selenite (20 ng / mL), ethanolamine (3 μg / mL), L-carnitine (5 μg / mL), putrescine (20 ng / mL), linoleic acid (2 μg / mL), and biotin (0.5 μg / mL). The human pluripotent stem cells were induced to differentiate into mesodermal precursor cells by culturing in a 37°C incubator with 5% CO2for 72 hours.

[0024] S3 Maintenance culture stage: The same as the S3 step of Example 1, and the culture was continued until the cells beat obviously, and high-purity cardiomyocytes were obtained.

[0025] Example 3 A method for inducing human pluripotent stem cells to differentiate into cardiomyocytes, comprising the following steps: S1: human pluripotent stem cell culture stage: The same as the S1 step of Example 1.

[0026] S2: induction differentiation stage: After the human pluripotent stem cells are cultured to 80% confluence using the stem cell culture medium, the induction differentiation medium is used for replacement. The induction differentiation medium comprises a base culture medium (IMDM), glycidyl methacrylate (4.2% by volume), and an induction differentiation additive, wherein the induction differentiation additive comprises transferrin (15 ng / mL), L-ascorbic acid (400 μg / mL), sodium selenite (18 ng / mL), ethanolamine (2.5 μg / mL), L-carnitine (4 μg / mL), putrescine (18 ng / mL), linoleic acid (1.5 μg / mL), and biotin (0.4 μg / mL). The human pluripotent stem cells are induced to differentiate into mesodermal precursor cells by being cultured in a 37°C constant-temperature incubator with 5% CO2 for 60 hours.

[0027] S3: maintenance culture stage: The same as the S3 step of Example 1, and the cells are cultured until the cell beating is stable, and mature cardiomyocytes are obtained.

[0028] Effect verification The expression of the cardiomyocyte marker cTnT is detected by flow cytometry. In Examples 1-3, the proportion of cTnT-positive cardiomyocytes is higher than 80% within 7-10 days, and the cell beating is powerful and stable. The addition of glycidyl methacrylate significantly improves the differentiation efficiency and cell purity, and the repeatability between batches is good.

[0029] S1: human pluripotent stem cell culture stage (the same as in the foregoing examples): The culture dish is coated with a 1:150 dilution of Matrigel working solution and is left to stand at 37°C overnight. The human pluripotent stem cells are inoculated into the coated culture dish, and the stem cell culture medium (such as TeSR-E8) is used for culture until the confluence is 70%-80%. The culture medium is aspirated, and the cells are washed with calcium-free and magnesium-free PBS for 1-3 times. The cells are passaged and digested using 0.5 mmol / L EDTA, and the cell layer is gently shaken until it is loose. Then, 5 mL of complete culture medium is added, and the cells are gently blown into a suspension. After counting, the cell concentration is adjusted, and the cells are inoculated into a 96-well plate at a ratio of 1:4. The Matrigel is uniformly coated after the cells are left to stand at room temperature for 1 hour, and then the cells are placed in a 5% CO2, 37°C incubator, with the shaking speed controlled to be ≤200 r / min.

[0030] S2: induction differentiation stage: When the cell confluence reaches 80%, the induction differentiation medium is used for replacement.

[0031] Control group: basal medium + induction-differentiation additives, without glycidyl methacrylate.

[0032] Experimental group 1: basal medium + induction-differentiation additives + glycidyl methacrylate (0.5% by volume).

[0033] Experimental group 2: basal medium + induction-differentiation additives + glycidyl methacrylate (3.4% by volume).

[0034] Experimental group 3: basal medium + induction-differentiation additives + glycidyl methacrylate (5.1% by volume).

[0035] Incubation in a 5% CO2, 37°C incubator for 48 hours to induce differentiation of cells into mesodermal precursors.

[0036] S3: Maintenance culture phase: Change to maintenance medium (basal medium + induction-differentiation additives, without glycidyl methacrylate). Change every two days until day 9. Observe cell beating every day and periodically check cTnT expression by flow cytometry.

[0037] The results are as follows: Table 1. Influence of different GMA on differentiation efficiency Group GMA concentration (V / V%) 3-day differentiation efficiency 6-day differentiation efficiency 9-day differentiation efficiency Control group 0 15.2 ± 2.1 25.6 ± 3.2 32.4 ± 3.8 Experimental group 1 0.5% 15.8 ± 2.2 24.9 ± 1.6 43.4 ± 1.8 Experimental group 2 3.4% 25.7 ± 2.8 52.1 ± 3.5 88.9 ± 4.0 Experimental group 3 5.1% 24.5 ± 3.1 48.1 ± 1.6 80.1 ± 3.7

[0038] Note: Differentiation efficiency is expressed as the percentage of cTnT-positive cells. Each data point is the average of three replicates.

[0039] The time of first appearance of beating cardiomyocytes in experimental group 2 was on day 8, while in the control group it was on day 10.5, indicating that glycidyl methacrylate accelerates the differentiation process. At all time points, the differentiation efficiency of the experimental groups was higher than that of the control group. In particular, on day 9, experimental group 2 reached an efficiency of 88.9%, while the control group only reached 32.4%, demonstrating that glycidyl methacrylate significantly improves the efficiency of cardiomyocyte generation. This indicates that glycidyl methacrylate at a concentration of 3.4% effectively promotes the differentiation of human pluripotent stem cells into cardiomyocytes, optimizing the differentiation time and efficiency, making it suitable for applications in cardiac cell regeneration medicine.

Claims

1. A method of inducing differentiation of human pluripotent stem cells into cardiomyocytes, characterized by, The method comprises the following steps: S1: culturing human pluripotent stem cells in a feeder-free culture system, using a culture vessel coated with Matrigel and a human pluripotent stem cell culture medium, and culturing to a cell density of 70%-90%; S2: treating the human pluripotent stem cells with an induction differentiation medium containing glycidyl methacrylate, and inducing differentiation into myocardial cells, wherein the concentration of glycidyl methacrylate in the induction differentiation medium is 0.5%-5.1% by volume; S3: continuing to culture using a maintenance medium without glycidyl methacrylate until myocardial cells with beating function are obtained.

2. The method of claim 1, wherein, In S2, the induction differentiation medium comprises a basic medium selected from one or more of RPMI 1640 medium, DMEM / F12 medium, or IMDM medium, and an induction differentiation additive.

3. The method of claim 2, wherein, The induction differentiation additive comprises one or more of transferrin, L-ascorbic acid, sodium selenite, ethanolamine, L-carnitine, putrescine, linoleic acid, and biotin.

4. The method of claim 3, wherein, The concentrations of the components in the induction differentiation additive are as follows: transferrin: 10-30 ng / mL, L-ascorbic acid: 100-500 μg / mL, sodium selenite: 10-20 ng / mL, ethanolamine: 1-3 μg / mL, L-carnitine: 1-5 μg / mL, putrescine: 10-20 ng / mL, linoleic acid: 0.5-2 μg / mL, and biotin: 0.1-0.5 μg / mL.

5. The method of claim 1, wherein, In S1, the human pluripotent stem cells are seeded at a density of 1 x 10 4 ~ 6 x 10 4 cells / cm2 into culture vessels coated with Matrigel.

6. The method of claim 1, wherein, The culture time for inducing the human pluripotent stem cells to differentiate into myocardial cells is 2-5 days, and the time for continuing to culture using the maintenance medium is 5-15 days.

7. Use of glycidyl methacrylate for the preparation of an induction differentiation medium for the differentiation of human pluripotent stem cells into cardiomyocytes, characterized in that, The concentration of glycidyl methacrylate in the induction differentiation medium is 0.5%-5.1% by volume.

8. An induction differentiation medium for differentiation of human pluripotent stem cells into cardiomyocytes, characterized by, The induction differentiation medium comprises a basic medium, glycidyl methacrylate, and an induction differentiation additive, wherein the concentration of glycidyl methacrylate is 0.5%-5.1% by volume.

9. The induction differentiation medium of claim 8, wherein, The basic medium is RPMI 1640 medium, and the induction differentiation additive comprises transferrin, L-ascorbic acid, sodium selenite, ethanolamine, L-carnitine, putrescine, linoleic acid, and biotin.

10. The induction differentiation medium of claim 9, wherein, The concentrations of the components in the induction differentiation additive are as follows: transferrin: 20 ng / mL, L-ascorbic acid: 300 μg / mL, sodium selenite: 15 ng / mL, ethanolamine: 2 μg / mL, L-carnitine: 3 μg / mL, putrescine: 15 ng / mL, linoleic acid: 1 μg / mL, and biotin: 0.3 μg / mL.