A method for inducing stem cells to differentiate into cardiomyocytes and a culture medium combination thereof

Abstract

The present invention discloses a method for inducing stem cells to differentiate into cardiomyocytes and a culture medium combination thereof, which relates to the technical field of cell differentiation. The characteristic of this method is to change cell fate by adjusting the pH value, including using acidic conditions to induce stem cells to differentiate into cardiomyocytes. The present invention elaborates in detail the specific operation steps of this method, and conducts identification and functional analysis on the differentiated cardiomyocytes. It provides new ideas for the generation of cardiomyocytes derived from human pluripotent stem cells.

Description

Technical Field

[0001] The present invention relates to the technical field of cell differentiation, and in particular, to a method for inducing stem cells to differentiate into cardiomyocytes and a culture medium combination thereof. Background Art

[0002] Human pluripotent stem cells (hPSCs) can differentiate into all cell types in the human body and are important model systems for studying human embryonic development. Specific cell types generated by hPSC differentiation have broad application prospects in cell therapy, drug screening, and disease model research.

[0003] Cardiomyocytes are non-renewable. Therefore, cardiomyocytes generated by hPSC differentiation provide materials for the regenerative repair of heart diseases and drug screening. Currently, the commonly used method of inducing cardiomyocytes with small molecule inhibitors has unstable efficiency and high costs, which is not conducive to large-scale production.

[0004] In view of this, the present invention is specifically proposed. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for inducing stem cells to differentiate into cardiomyocytes and a culture medium combination thereof.

[0006] The present invention is implemented as follows:

[0007] In a first aspect, an embodiment of the present invention provides a method for inducing stem cells to differentiate into cardiomyocytes, which includes the following steps: inducing and differentiating stem cells into the mesoderm using a first differentiation medium; inducing and differentiating the mesoderm using a second differentiation medium, wherein 6 - 10 mmol / L of an acid is added to the second differentiation medium or an acid is added during the induction and differentiation process such that the final concentration of the acid in the second differentiation medium is 6 - 10 mmol / L.

[0008] In a second aspect, the present invention provides a culture medium combination for inducing stem cells to differentiate into cardiomyocytes, which includes a first differentiation medium and a second differentiation medium; the first differentiation medium includes a basic differentiation medium and a basic differentiation medium containing 1 - 10 μmol / L of a WNT pathway activator; the second differentiation medium is a basic differentiation medium containing 6 - 10 mmol / L of an acid.

[0009] The present invention has the following beneficial effects:

[0010] The method provided by this application for inducing stem cells to differentiate into cardiomyocytes by adjusting the pH value can provide an effective and feasible new method for the generation of cardiomyocytes derived from stem cells, with stable efficiency, low cost, and being conducive to large-scale production. The obtained cardiomyocytes have high survival rate and purity, and broad application prospects, and can provide materials for the regenerative repair of heart diseases and drug screening. Controlling cell fate by pH regulation also provides new ideas for the differentiation of other cell types and has broad application prospects. Brief Description of the Drawings

[0011] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, other relevant drawings can be obtained based on these drawings without creative efforts.

[0012] Figure 1 Acidic conditions promote the differentiation of H1 cells into cardiomyocytes; among them, A. The effect of acidic conditions on the RNA expression level; B. After induction of differentiation with hydrochloric acid, the proportion of TNNT2-positive cells was analyzed by flow cytometry and compared with the traditional WNT inhibitor (IWP-2) method; and on the basis of the WNT inhibitor (IWP-2) method, hydrochloric acid was added and compared with the method without adding acid; C. After induction of differentiation with different concentrations of hydrochloric acid or lactic acid, the proportion of TNNT2-positive cells was analyzed by flow cytometry;

[0013] Figure 2 Acidic conditions promote the differentiation of H9 and NL4 iPSC cells into cardiomyocytes, and the proportion of TNNT2-positive cells was analyzed by flow cytometry; among them, A is H9; B is NL4 iPSC. Detailed Embodiments

[0014] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Those not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments not specified by the manufacturer are all conventional products that can be obtained through commercial purchase.

[0015] Through a series of creative efforts, the inventors found that the method of changing cell fate by adjusting the pH value has low cost, simple operation, and non-toxic components. Specifically, acidic culture medium can induce the generation of high-purity cardiomyocytes, which is conducive to technological development and clinical application. The present invention confirmed the specific operation steps of the method constructed based on the above concept, and identified and analyzed the functions of the differentiated cardiomyocytes, providing a new approach for the preparation of cardiomyocytes derived from stem cells.

[0016] An embodiment of the present invention provides a method for inducing stem cells to differentiate into cardiomyocytes, which includes the following steps: inducing and differentiating stem cells into mesoderm using a first differentiation medium; inducing and differentiating the mesoderm using a second differentiation medium, wherein an acid with any concentration in the range of 6-10 mmol / L is added to the second differentiation medium or an acid is added during the process of inducing differentiation such that the final concentration of the acid in the second differentiation medium is 6-10 mmol / L, and the final concentration of the acid can be any concentration in, for example, 6 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L, and 10 mmol / L or the range between any two of them.

[0017] It should be noted that the "first" and "second" in the first differentiation medium and the second differentiation medium are not intended to limit the number, composition, or type of the medium, but rather to distinguish different culture stages. The first differentiation medium is the differentiation medium used in the first stage, and the second differentiation medium is the differentiation medium used in the second stage. Without specific limitation on its composition, it can be understood as a common differentiation medium, and the same applies to the subsequent third differentiation medium.

[0018] Adding an acid with a concentration of 6-10 mmol / L to the second differentiation medium means that an acid is pre-added to the basic differentiation medium such that the final concentration of the acid is 6-10 mmol / L.

[0019] Preferably, a WNT inhibitor with a final concentration of 3-5 μmol / L can be added to the second differentiation medium, or a WNT inhibitor is added during the process of inducing and culturing cells in the second differentiation medium such that the concentration of the WNT inhibitor reaches any value in the range of 3-5 μmol / L, specifically, it can be any one of 3 μmol / L, 3.5 μmol / L, 4 μmol / L, 4.5 μmol / L, and 5 μmol / L or the range between any two of them. Preferably, the time for the second differentiation medium to induce and differentiate the mesoderm is 3-7 days, and the specific duration can be any one of 3 days, 4 days, 5 days, 6 days, and 7 days or the range between any two of them, preferably 6 days. When it is 6 days and the culture time of the first differentiation medium is the 1st-2nd day, the culture time of the second differentiation medium is the 3rd-8th day. During the period of culturing with the second differentiation medium, it is possible to choose to replace the fresh second differentiation medium every day, or choose to supplement the acid in the differentiation medium such that the pH in the second differentiation medium is always maintained between 6.2 and 6.9. The pH value in this range can enable the stem cells to differentiate in the cardiomyocyte direction.

[0020] Preferably, when the method is to add an acid during the induction of differentiation so that the final concentration of the acid in the second differentiation medium is 6-10 mmol / L, the addition time of the acid is the 1st to 6th day of inducing the mesoderm with the second differentiation medium;

[0021] When the method is to add a WNT inhibitor during the process of inducing and culturing cells with the second differentiation medium so that the concentration of the WNT inhibitor reaches 3-5 μmol / L, the addition time of the WNT inhibitor is the 1st to 3rd day of inducing the mesoderm with the second differentiation medium.

[0022] Preferably, the WNT inhibitor is any one of IWP-2, IWR-1 and XAV939.

[0023] Preferably, when the WNT inhibitor is IWP-2, the final concentration of the WNT inhibitor in the second differentiation medium is 3-5 μmol / L.

[0024] In a preferred embodiment, the acid includes at least one of hydrochloric acid and lactic acid. Compared with other acids, these two acids have no toxic and side effects on cells and can effectively induce and promote the differentiation of cells into the cardiomyocyte direction.

[0025] In a preferred embodiment, the step of inducing the stem cells to differentiate into the mesoderm includes: inducing human pluripotent stem cells with a cell density of 60%-80% with the first differentiation medium and differentiating them into the mesoderm.

[0026] Preferably, the first differentiation medium includes: a basal differentiation medium and a basal differentiation medium supplemented with a WNT pathway activator, or only includes a basal differentiation medium;

[0027] When the first differentiation medium includes a basal differentiation medium and a basal differentiation medium supplemented with a WNT pathway activator, the step of inducing the stem cells to differentiate into the mesoderm includes: first culturing the stem cells with the basal differentiation medium supplemented with a WNT pathway activator for 1-2 days, preferably 1 day, and then replacing it with the basal differentiation medium for culturing for 1-2 days, preferably 1 day, to obtain the mesoderm.

[0028] When the first differentiation medium only includes a basal differentiation medium, the step of inducing the stem cells to differentiate into the mesoderm includes: culturing the stem cells with the basal differentiation medium for 1-2 days and adding a WNT pathway activator during the process, and then replacing it with the basal differentiation medium for culturing to obtain the mesoderm.

[0029] WNT pathway activators can induce the differentiation of human pluripotent stem cells into mesoderm by activating the Wnt / β-catenin pathway. Preferably, the final concentration of the WNT pathway activator in the basal differentiation medium is any value between 1 and 10 μmol / L, such as any one of 1 μmol / L, 2 μmol / L, 3 μmol / L, 4 μmol / L, 5 μmol / L, 6 μmol / L, 7 μmol / L, 8 μmol / L, 9 μmol / L or 10 μmol / L, or the range between any two of them, and preferably 5 μmol / L. Preferably, the WNT pathway activator is a GSK3β inhibitor. Preferably, the GSK3β inhibitor is CHIR99021 and / or CHIR99021 HCl.

[0030] Preferably, after inducing the differentiation of the mesoderm with the second differentiation medium, the cells are cultured and differentiated with the third differentiation medium, and the third differentiation medium is a basal differentiation medium without added acid.

[0031] Preferably, the time for culturing and differentiating the cells with the third differentiation medium is 2 to 4 days.

[0032] Preferably, the third differentiation medium is the basal differentiation medium.

[0033] The basal differentiation medium described in any of the foregoing embodiments can be selected to purchase an existing commercial differentiation medium for culturing stem cells, or prepared by oneself based on the existing public content.

[0034] Preferably, the components of the basal differentiation medium include at least one of DMEM / F12, L-ascorbic acid-2-phosphate magnesium, sodium selenite, transferrin, chemically defined lipid concentrate, and penicillin-streptomycin, and more preferably include all of them.

[0035] Preferably, in the basic differentiation medium, the final concentration of L-ascorbic acid-2-phosphate magnesium is 50-80 mg / L, specifically, it can be any one of 50 mg / L, 60 mg / L, 70 mg / L or 80 mg / L, or the range between any two of them; the final concentration of sodium selenite is 10-20 μg / L, specifically, it can be any one of 10 μg / L, 15 μg / L or 20 μg / L, or the range between any two of them; the final concentration of transferrin is 1-20 mg / L, specifically, it can be any one of 1 mg / L, 5 mg / L, 10 mg / L, 15 mg / L or 20 mg / L, or the range between any two of them; the final concentration of penicillin-streptomycin is 1-20 U / mL, specifically, it can be any one of 1 U / mL, 5 U / mL, 10 U / mL, 15 U / mL or 20 U / mL, or the range between any two of them.

[0036] In an alternative embodiment, the stem cells are pluripotent stem cells.

[0037] Preferably, the pluripotent stem cells are selected from at least one of human embryonic stem cells (stem cells isolated or obtained from human embryos within 14 days after fertilization without in vivo development) and human induced pluripotent stem cells.

[0038] The embodiment of the present invention also provides a medium combination for inducing stem cells to differentiate into cardiomyocytes, which includes a first differentiation medium and a second differentiation medium; the first differentiation medium includes a basic differentiation medium; the second differentiation medium is a basic differentiation medium containing 6-10 mmol / L of an acid.

[0039] Preferably, the acid is at least one of hydrochloric acid and lactic acid.

[0040] Preferably, the second differentiation medium further contains 3-5 μmol / L of a WNT inhibitor.

[0041] Preferably, the first differentiation medium further includes a basic differentiation medium containing 1-10 μmol / L of a WNT pathway activator.

[0042] Preferably, the medium combination further includes the third differentiation medium described in any of the foregoing embodiments.

[0043] It can be understood that the first to third differentiation media can be the same as the first to third differentiation media described in any corresponding foregoing embodiments, and will not be elaborated herein.

[0044] The features and properties of the present invention will be further described in detail below with reference to the embodiments.

[0045] Example 1

[0046] To verify the role of pH in myocardial differentiation, hydrochloric acid and lactic acid were used to create an acidic condition, which was applied from day 3 to day 8 of differentiation to detect its effect on myocardial differentiation.

[0047] The specific implementation plan is as follows: Human embryonic stem cell line H1 was cultured in E8 medium, and the fresh medium was changed daily. When the cell density reached 70%-80%, subculture was carried out. First, it was washed twice with DPBS-EDTA, incubated at room temperature for 5 minutes for the third time, the DPBS-EDTA was aspirated, and E8 medium containing 5 μmol / L ROCK inhibitor was added. After resuspending the cells, they were seeded into a 12-well plate pre-coated with Matrigel at a density of 1:6. After the cells adhered to the wall for 1 day, the E8 medium was changed and the cells were cultured for another day.

[0048] D1-2: When the cells grew to 60%-70%, differentiation was started. On the first day, a basic differentiation medium supplemented with 5 μmol / L CHIR99021-HCl was used. The basic differentiation medium included DMEM / F12, L-ascorbic acid-2-phosphate magnesium (final concentration 64 mg / L), sodium selenite (final concentration 13.6 μg / L), transferrin (final concentration 10 mg / L), Chemical defined lipid concentrate (final concentration 1×), and penicillin-streptomycin (final concentration 10 U / mL). Subsequently, the new basic differentiation medium was replaced and the cells were cultured for another day.

[0049] D3-8: From the third day to the eighth day, the cells were cultured in a second differentiation medium supplemented with hydrochloric acid (HCl) or lactic acid. The second differentiation medium was obtained by adding hydrochloric acid or lactic acid to the basic differentiation medium. The final concentrations of hydrochloric acid and lactic acid in the second differentiation medium were both 8 mmol / L. During this period, the fresh acid-containing second differentiation medium was changed daily.

[0050] D9: On the ninth day of differentiation, the basic differentiation medium was replaced and the cells were continuously cultured, and the medium was changed daily during this period.

[0051] D11-13: The expression levels of myocardial markers (NKX2-5 and TNNT2) in the differentiated cells were detected on the eleventh to thirteenth days.

[0052] In addition, 2 more groups were set as controls. Control group 1 used the traditional WNT inhibitor (IWP-2) method to induce myocardial differentiation, and IWP-2 with a final concentration of 3-5 μmol / L was added from the third day to the fifth day of differentiation to induce myocardial differentiation.

[0053] The control group 2 used a traditional WNT inhibitor (IWP-2) method combined with acid treatment to induce cardiomyocyte differentiation. IWP-2 with a final concentration of 3-5 μmol / L was added from the third day to the fifth day of differentiation to induce cardiomyocyte differentiation, and hydrochloric acid with a final concentration of 8 mmol / L was added from the third day to the eighth day of differentiation.

[0054] The results are shown in Figure 1 , and the results showed that both hydrochloric acid (HCl) and lactic acid (LA) increased the expression levels of the cardiomyocyte marker genes NKX2-5 and TNNT2. TNNT2 is a cardiomyocyte-specific marker, and both hydrochloric acid and lactic acid treatments were able to increase the proportion of TNNT2-positive cells, indicating that acidic conditions can induce stem cells to differentiate into cardiomyocytes.

[0055] As a control, the cardiomyogenic efficiency of the traditional WNT inhibitor (IWP-2) method for inducing cardiomyocyte differentiation was similar to that induced by acidic conditions, indicating that the efficiency of inducing stem cells to differentiate into cardiomyocytes under acidic conditions is comparable to the traditional method.

[0056] In addition, Figure 1 as shown in B, compared with the traditional WNT inhibitor (IWP-2) method, the combination of acidic conditions and IWP-2 increased the efficiency of cardiomyocyte differentiation, indicating that acidic conditions can be combined with other induction methods to improve the efficiency of cardiomyocyte differentiation.

[0057] Example 2

[0058] Verify the effects of different concentrations of hydrochloric acid and lactic acid on the differentiation of cells into cardiomyocytes. The experimental procedure was referred to Example 1, except that hydrochloric acid and lactic acid at different concentrations (3 mmol / L, 6 mmol / L, 8 mmol / L, 10 mmol / L, 11 mmol / L) were added to the basic differentiation medium from the 3rd to the 8th day to detect the effects of different concentrations of hydrochloric acid and lactic acid on cardiomyocyte differentiation. The results are referred to Figure 1 C in

[0059] Example 3

[0060] Verify the effects of hydrochloric acid on the differentiation of H9 and NL4 iPSC cells into cardiomyocytes.

[0061] The experimental method was generally the same as that in Example 1, except that:

[0062] (1) Replace human embryonic stem cell H1 with H9 and NL4 iPSC cells;

[0063] (2) From the 3rd to the 8th day of cell differentiation, the cells were induced and cultured with a basic differentiation medium containing 8 mmol / L hydrochloric acid.

[0064] The proportion of TNNT2-positive cells was analyzed by flow cytometry. The results are shown in Appendix Figure 2 . It can be seen from the results that hydrochloric acid treatment can increase the proportion of TNNT2-positive cells, indicating that acidic conditions can promote the differentiation of H9 and NL4 iPSC cells into cardiomyocytes.

[0065] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for inducing stem cells to differentiate into cardiomyocytes, characterized in that, It includes the following steps: Induce and differentiate stem cells into mesoderm using a first differentiation medium; the first differentiation medium includes: a basal differentiation medium and a basal differentiation medium supplemented with a WNT pathway activator, or only includes the basal differentiation medium; Induce and differentiate the mesoderm using a second differentiation medium on the 3rd to 8th day of differentiation, and 6 - 10 mmol / L acid is added to the second differentiation medium; the pH of the second differentiation medium is 6.2 - 6.9, and the acid includes at least one of hydrochloric acid and lactic acid. The second differentiation medium is prepared by adding the acid to the basal differentiation medium; The stem cells are pluripotent stem cells, and the pluripotent stem cells are selected from at least one of human embryonic stem cells and human induced pluripotent stem cells. The human embryonic stem cells are stem cells isolated or obtained from a human embryo within 14 days of fertilization without in vivo development.

2. The method for inducing stem cells to differentiate into cardiomyocytes according to claim 1, wherein A WNT inhibitor is further added to the second differentiation medium, or a WNT inhibitor is added during the process of inducing and culturing cells with the second differentiation medium.

3. The method for inducing stem cells to differentiate into cardiomyocytes according to claim 2, characterized in that, The WNT inhibitor is any one of IWP-2, IWR-1, and XAV939.

4. The method for inducing stem cells to differentiate into cardiomyocytes according to claim 3, wherein When the WNT inhibitor is IWP-2, the final concentration of the WNT inhibitor in the second differentiation medium is 3 - 5 μmol / L.

5. The method for inducing stem cells to differentiate into cardiomyocytes according to any one of claims 2 to 4, characterized in that, When the method is to add a WNT inhibitor during the process of inducing and culturing cells with the second differentiation medium, the addition time of the WNT inhibitor is the 1st to 3rd day of inducing and differentiating the mesoderm using the second differentiation medium.

6. The method for inducing stem cells to differentiate into cardiomyocytes according to claim 1, characterized in that, The components of the basal differentiation medium include: DMEM / F12, L-ascorbic acid-2-phosphate magnesium, sodium selenite, transferrin, chemically defined lipid concentrate, and penicillin-streptomycin.

7. The method for inducing stem cells to differentiate into cardiomyocytes according to claim 6, characterized in that, In the basal differentiation medium, the final concentration of L-ascorbic acid-2-phosphate magnesium is 50 - 80 mg / L, the final concentration of sodium selenite is 10 - 20 μg / L, the final concentration of transferrin is 1 - 20 mg / L, and the final concentration of penicillin-streptomycin is 1 - 20 U / mL.

8. The method for inducing stem cells to differentiate into cardiomyocytes according to any one of claims 1 to 7, characterized in that, When the first differentiation medium includes a basal differentiation medium and a basal differentiation medium supplemented with a WNT pathway activator, the step of inducing and differentiating stem cells into mesoderm includes: first culturing stem cells with the basal differentiation medium supplemented with a WNT pathway activator for 1 - 2 days, and then replacing it with the basal differentiation medium for culturing to obtain the mesoderm; When the first differentiation medium only includes the basal differentiation medium, the step of inducing and differentiating stem cells into mesoderm includes: adding a WNT pathway activator during the process of culturing stem cells with the basal differentiation medium for 1 - 2 days, and then replacing it with the basal differentiation medium for culturing to obtain the mesoderm.

9. The method for inducing stem cells to differentiate into cardiomyocytes according to claim 8, characterized in that, The final concentration of the WNT pathway activator in the basal differentiation medium is 1 - 10 μmol / L.

10. The method for inducing stem cells to differentiate into cardiomyocytes according to claim 8, characterized in that, The WNT pathway activator is a GSK3β inhibitor.

11. The method for inducing stem cells to differentiate into cardiomyocytes according to claim 10, wherein The GSK3β inhibitor is CHIR99021 and / or CHIR99021 HCl.

12. The method for inducing stem cells to differentiate into cardiomyocytes according to any one of claims 1 to 7, characterized in that, After inducing and differentiating the mesoderm with the second differentiation medium, the cells are cultured and differentiated with the third differentiation medium, and the third differentiation medium is a basic differentiation medium without added acid.

13. The method for inducing stem cells to differentiate into cardiomyocytes according to claim 12, characterized in that, The duration of culturing and differentiating the cells with the third differentiation medium is 2 to 4 days.

14. A culture medium combination for inducing stem cells to differentiate into cardiomyocytes, characterized in that, It includes: a first differentiation medium and a second differentiation medium; The first differentiation medium includes a basic differentiation medium and a basic differentiation medium containing 1 to 10 μmol / L of a WNT pathway activator; the WNT pathway activator is CHIR99021 and / or CHIR99021 HCl; The second differentiation medium is a basic differentiation medium containing 6 to 10 mmol / L of an acid; the acid is at least one of hydrochloric acid and lactic acid; The stem cells are pluripotent stem cells, and the pluripotent stem cells are selected from at least one of human embryonic stem cells and human induced pluripotent stem cells, and the human embryonic stem cells are stem cells isolated or obtained from a human embryo within 14 days of fertilization without in vivo development.

15. The medium combination for inducing stem cells to differentiate into cardiomyocytes according to claim 14, characterized in that, The second differentiation medium also contains a WNT inhibitor.

16. The medium combination for inducing stem cells to differentiate into cardiomyocytes according to claim 15, characterized in that, The WNT inhibitor is IWP-2; the final concentration of IWP-2 in the second differentiation medium is 3 to 5 μmol / L.

17. The medium combination for inducing stem cells to differentiate into cardiomyocytes according to any one of claims 14 to 16, characterized in that The medium combination also includes a third differentiation medium, and the third differentiation medium is a basic differentiation medium.

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