CN112813023A

CN112813023A - Method and medicament for inducing stem cells to differentiate towards myocardial cells and passaging and purifying myocardial cells

Info

Publication number: CN112813023A
Application number: CN202110221043.2A
Authority: CN
Inventors: 陈国凯; 孟雅
Original assignee: University of Macau
Current assignee: University of Macau
Priority date: 2021-02-26
Filing date: 2021-02-26
Publication date: 2021-05-18

Abstract

The invention discloses a method and a medicament for inducing stem cells to differentiate towards myocardial cells and passaging and purifying the myocardial cells, and belongs to the technical field of regulation and maintenance of differentiation of human pluripotent stem cells towards the myocardial cells. The method for inducing the stem cells to differentiate towards the myocardial cells comprises the following steps: culturing the human pluripotent stem cells in a first medium containing nicotinamide, wherein the first medium is E5 medium. The method for passaging the myocardial cells comprises the following steps: culturing the cardiomyocytes in a second medium comprising nicotinamide, wherein the second medium is E6 medium. The method for purifying the myocardial cells comprises the following steps: culturing the passaged cardiomyocytes in a third medium containing nicotinamide, wherein the third medium is E6 medium. The method can provide an effective and feasible new method for the generation and subculture of the human pluripotent stem cell-derived cardiomyocyte, has stable efficiency and lower cost, and is beneficial to large-scale production. The corresponding medicament has wide application prospect.

Description

Method and medicament for inducing stem cells to differentiate towards myocardial cells and passaging and purifying myocardial cells

Technical Field

The invention relates to the technical field of differentiation regulation and maintenance of human pluripotent stem cells to cardiac muscle cells, in particular to a method and a medicament for inducing differentiation of stem cells to the cardiac muscle cells and passage and purification of the cardiac muscle cells.

Background

Human pluripotent stem cells (hPSCs) can differentiate into all cell types in our body and are an important model system for studying human embryonic development. The specific cell types generated by differentiation of hpscs have great utility in cell therapy, drug screening, and disease model studies. The cardiomyocytes are non-regenerative, so the cardiomyocytes generated by hPSC differentiation provide materials for regenerative repair of heart diseases and drug screening. The method for inducing the myocardial cells by the conventional small-molecule inhibitor has unstable efficiency and higher cost, and is not beneficial to large-scale production.

In addition, there are still difficulties in how to culture and maintain hPSC-induced differentiated cardiomyocytes. For example, the hPSC-induced differentiated cardiomyocytes are difficult to passage and have low cell survival rate. And the purity of the myocardial cells can be reduced by long-term culture, and the use of the myocardial cells for regeneration and repair and drug screening is not facilitated. Therefore, the development of an economical and effective differentiation method, the subsequent generation of the myocardial cells and the maintenance of the purity of the myocardial cells have great significance for the application of the method.

In view of this, the invention is particularly proposed.

Disclosure of Invention

One object of the present invention includes providing a method for inducing differentiation of stem cells into cardiomyocytes.

Another object of the present invention is to provide a method for passaging cardiomyocytes.

Another object of the present invention is to provide a method for purifying cardiomyocytes.

The fourth object of the present invention is to provide a pharmaceutical agent containing the cardiomyocytes obtained by the above method.

The application can be realized as follows:

in a first aspect, the present application provides a method for inducing differentiation of stem cells into cardiomyocytes, comprising the steps of: culturing the human pluripotent stem cells in a first medium containing nicotinamide, wherein the first medium is E5 medium.

In an alternative embodiment, the concentration of nicotinamide in the first culture medium is not less than 10 mmol/L.

In an alternative embodiment, the time of action of nicotinamide on human pluripotent stem cells is 4-6 days.

In an alternative embodiment, the human pluripotent stem cells are differentiated into mesoderm and nicotinamide is added to induce differentiation into myocardium.

In alternative embodiments, differentiation of human pluripotent stem cells into mesoderm is induced by opening the Wnt/β -catenin pathway.

In alternative embodiments, the Wnt/β -catenin pathway is opened using the GSK3 β inhibitor CHIR 99021.

In an alternative embodiment, differentiation into mesoderm is performed with human pluripotent stem cells having a cell density of 60-80%.

In an alternative embodiment, the medium used for the differentiation process to mesoderm is E5 medium without TGF β, FGF2 and insulin but containing lipid concentrate.

In an alternative embodiment, further comprising continuing the culture differentiation with insulin-containing E5 medium after culturing with the first medium comprising nicotinamide.

In a second aspect, the present application provides a method for passaging cardiomyocytes, comprising the steps of: culturing the cardiomyocytes in a second medium comprising nicotinamide, wherein the second medium is E6 medium.

In an alternative embodiment, the concentration of nicotinamide in the second culture medium is not less than 10 mmol/L.

In an alternative embodiment, the cardiomyocytes are obtained by the above-described method for inducing stem cell differentiation into cardiomyocytes.

In an alternative embodiment, the cardiomyocytes are human pluripotent stem cells after day 10-15 of differentiation into cardiomyocytes.

In an alternative embodiment, the cardiomyocytes are human pluripotent stem cells induced by IWP-2 or nicotinamide.

In an alternative embodiment, nicotinamide promotes cardiomyocyte survival by inhibiting the ROCK pathway.

In an alternative embodiment, the cardiomyocytes after digestion into single cells are placed in a second medium comprising nicotinamide, and the culture is continued after 1-2 days by changing to a second medium not comprising nicotinamide.

In an alternative embodiment, TrypLE is used for digestion, followed by neutralization with bovine serum albumin, solid-liquid separation, and the cardiomyocytes in the single cell state after removal of the digestive fluid are placed in a second medium containing nicotinamide.

In a third aspect, the present application provides a method for purifying cardiomyocytes, comprising the steps of: culturing the passaged cardiomyocytes in a third medium containing nicotinamide, wherein the third medium is E6 medium.

In an alternative embodiment, the concentration of nicotinamide in the third culture medium is not less than 10 mmol/L.

In an alternative embodiment, the method of passaging is as described above.

In an alternative embodiment, the passaging is performed at a ratio of 1:3 to 1: 4.

In alternative embodiments, a ROCK inhibitor or nicotinamide is added during passaging.

In an alternative embodiment, the ROCK inhibitor is Y27632.

In an alternative embodiment, the ROCK inhibitor is added at a concentration of 5-20. mu. mmol/L and the nicotinamide is added at a concentration of 10-20 mmol/L.

In an alternative embodiment, the cardiomyocytes are cultured in the nicotinamide-containing third medium for 4-6 days, with a new nicotinamide-containing third medium being replaced every 2-3 days.

In a fourth aspect, the present application provides a pharmaceutical agent comprising cardiomyocytes obtained by the above-described method for inducing stem cell differentiation into cardiomyocytes.

In a fifth aspect, the present application provides a pharmaceutical agent comprising the cardiomyocytes obtained by the above-described method for passaging cardiomyocytes.

In a sixth aspect, the present application provides a pharmaceutical agent comprising cardiomyocytes obtained by the above method for purifying cardiomyocytes.

The beneficial effect of this application includes:

the method for inducing the stem cells to differentiate towards the direction of the myocardial cells and carrying out the passage and purification of the myocardial cells, which is provided by the application, can provide an effective and feasible new method for the generation and the passage culture of the myocardial cells derived from the human pluripotent stem cells, has stable efficiency and lower cost, and is beneficial to large-scale production. The survival rate and purity of the obtained myocardial cells are high, and the medicament containing the myocardial cells obtained by the method has wide application prospect, and can provide materials for regenerative repair and drug screening of heart diseases.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a graph of the effect of nicotinamide treatment at different concentrations and at different times on differentiation of hPSC H1 cells towards the myocardium;

FIG. 2 is a graph of nicotinamide-induced differentiation of H1 cells towards cardiomyocytes;

FIG. 3 is a cytoskeleton and electrophysiological function analysis of cardiomyocytes;

FIG. 4 is a graph of nicotinamide-induced differentiation of hPSC H9 cells and iPSC NL4 into the myocardium;

FIG. 5 is a graph of nicotinamide promoting cardiomyocyte survival after passaging;

FIG. 6 is a graph of survival of nicotinamide induced differentiation promoted cardiomyocytes after passage;

FIG. 7 is a graph of nicotinamide enhancing purity of cardiomyocytes.

Detailed Description

In order to make the objects, 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. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The following is a detailed description of the method and the pharmaceutical composition for inducing differentiation of stem cells into cardiomyocytes and passaging and purification of cardiomyocytes provided in the present application.

The application provides a method for inducing stem cells to differentiate towards myocardial cells, which comprises the following steps: culturing the human pluripotent stem cells in a first medium containing nicotinamide, wherein the first medium is E5 medium.

Among them, human pluripotent stem cells (hpscs) include human embryonic stem cells and human induced pluripotent stem cells.

By reference, the composition of a common nicotinamide-free E5 medium may be: DMEM/F12, holo-transferrin (holo-transferrin)10 μ g/mL, sodium selenite (sodium selenite)14ng/mL, magnesium ascorbyl phosphate sesquimagnesium salt (L-Ascorbic acid 2-phosphate magnesium salt) 64 μ g/mL and lipid concentrate (chemical defined lipid concentrate), wherein the lipid concentrate is prepared in a ratio of 1: the concentration of 100 was used for dilution.

In an alternative embodiment, the concentration of nicotinamide in the first culture medium is not less than 10mmol/L (which may be abbreviated as 10mM, the same applies hereinafter), and for example, may be 10mmol/L (10mM), 15mmol/L (15mM), or 20mmol/L (20mM), and the like.

In alternative embodiments, the time period of action of nicotinamide on human pluripotent stem cells may be 4-6 days, such as 4 days, 5 days, or 6 days, etc.

In an alternative embodiment, the human pluripotent stem cells are differentiated into mesoderm, and nicotinamide is added to induce differentiation into myocardium. Preferably, differentiation into mesoderm is performed with human pluripotent stem cells having a cell density of 60 to 80%.

In practice, differentiation of human pluripotent stem cells into mesoderm can be induced by opening the Wnt/β -catenin pathway. As a reference, opening the Wnt/β -catenin pathway can be achieved using the GSK3 β inhibitor CHIR 99021. The medium used for the differentiation process into mesoderm was E5 medium without TGF β, FGF2 and insulin but containing Lipid Concentrate (chemical Defined Lipid Concentrate).

Further, it comprises culturing and differentiating with an insulin-containing E5 medium after culturing with the first medium containing nicotinamide, and the myocardial cell begins to beat approximately at 9-13 days of differentiation, at which time the expression of the myocardial marker TNNT2 can be detected.

In summary, the following steps can be referred to: hPSCs are cultured until the cell density is 60-80% (such as 60%, 70% or 80%), the Wnt/beta-catenin pathway is opened by GSK3 beta inhibitor CHIR99021 to induce hPSCs to differentiate towards mesoderm, and then nicotinamide is added to induce cells to differentiate towards myocardium. After 4-6 days (e.g., 4, 5 or 6 days) of treatment, the culture is continued until day 9-13 of differentiation, and the expression of the cardiac marker TNNT2 is detected.

In particular, reference may be made to: culturing human pluripotent stem cells in E8 culture medium, replacing with fresh culture medium every day, and passaging when cell density reaches 70-80% (such as 70%, 75% or 80%). First washed 2 times with DPBS-EDTA, incubated for a third 5 minutes at room temperature, pipetted DPBS-EDTA, added to E8 medium containing 5 μ M ROCK inhibitor, resuspended cells, and then incubated at 1:3-1: 6 (e.g., 1:3, 1:4, 1: 5, or 1: 6, etc.) were placed in a 12-well plate previously coated with matrigel. The culture medium of E8 was changed 1 day after the cells were attached to the wall. When the cells grow to 60-80% (e.g., 60%, 70%, or 80%), differentiation is initiated by adding E5 differentiation medium containing 5. mu.M CHIR99021 the first day. E5 differentiation medium containing 10mM or 20mM nicotinamide was added after 1 day and treated for 4 days, during which time E5 medium containing nicotinamide was changed daily. On day 6 of differentiation, medium E5 was changed and culture was continued for 2 days with daily changes. The differentiated cells were then continued to be cultured in E5 medium containing insulin, and the cardiomyocytes began to beat approximately 9-13 days after differentiation.

Further, the present application also provides a method for passaging cardiomyocytes, comprising the steps of: culturing the cardiomyocytes in a second medium comprising nicotinamide, wherein the second medium is E6 medium. The cardiomyocytes can be obtained by, but not limited to, the method for inducing differentiation of stem cells into cardiomyocytes described above.

By reference, the composition of a common nicotinamide-free E6 medium may be: DMEM/F12, holo-transferrin (holo-transferrin) 10. mu.g/mL, sodium selenite (sodium selenite)14ng/mL, magnesium sesquimagnesium ascorbate phosphate (L-Ascorbic acid 2-phosphate sesquimagnesium stearate) 64. mu.g/mL, lipid concentrate (used diluted at a concentration of 1: 100) and insulin 10. mu.g/mL.

In an alternative embodiment, the concentration of nicotinamide in the second culture medium is not less than 10mmol/L (10mM), for example 10mmol/L (10mM), 15mmol/L (15mM), or 20mmol/L (20mM), and the like.

Preferably, the cardiomyocytes used for the passaging are cardiomyocytes obtained 10-15 days after the human pluripotent stem cells are differentiated in the direction of the cardiomyocytes. Preferably, the cardiomyocytes are human pluripotent stem cells induced by IWP-2 or nicotinamide.

In an alternative embodiment, the cardiomyocytes after digestion into single cells are placed in a second medium comprising nicotinamide, and the culture is continued after 1-2 days by changing to a second medium not comprising nicotinamide. In the process, nicotinamide can promote survival of the myocardial cells by inhibiting a ROCK pathway, so that the survival rate of the myocardial cells during passage can be obviously improved.

It is referred to that the digestion treatment is carried out by TrypLE, followed by neutralization reaction with bovine serum albumin, solid-liquid separation, and the cardiomyocytes in the single cell state after removal of the digestive juice are placed in the second medium containing nicotinamide.

In particular, reference may be made to: culturing human pluripotent stem cells in E8 culture medium, replacing fresh culture medium every day, and carrying out passage when the cell density reaches 70-80%. First washed 2 times with DPBS-EDTA, incubated for a third 5 minutes at room temperature, pipetted DPBS-EDTA, added to E8 medium containing 5 μ M ROCK inhibitor, resuspended cells, and then incubated at 1:3-1: 6 density was spread on 12 well plates previously coated with matrigel. Culture was continued 1 day after cell attachment by changing the culture medium E8 (e.g., Essential 8 medium from Saimer Feishell science Life sciences). Wherein, the composition of the E8 culture medium can be 10 mug/mL of holo-transferrin (holo-transferrin), 14ng/mL of sodium selenite (sodium selenite), 64 mug/mL of L-Ascorbic acid 2-phosphate sesquimagnesium stearate, 10 mug/mL of insulin, 1.74ng/mL of TGF beta and 2100 ng/mL.

When the cells grew to 60-80%, differentiation was initiated by adding E5 differentiation medium containing 5. mu.M CHIR99021 the first day. The next day, the culture was changed to fresh E5 differentiation medium for 1 day. E5 differentiation medium containing 3. mu.M IWP-2 was added on the third day, and treated for 3 days, during which the E5 medium containing IWP-2 was changed every day. On day 6 of differentiation, medium E5 was changed and culture was continued for 2 days with daily changes. On day 8 of differentiation, the differentiated cells were further cultured in E5 medium containing insulin (E6 medium) and the culture medium was changed every 1 to 2 days. After culturing for 10-15 days (e.g., 10 days, 11 days, 12 days, 13 days, 14 days or 15 days), the cardiomyocytes were digested with TrypLE, neutralized with BSA, centrifuged to remove the digest, and the cardiomyocytes were resuspended in E6 medium containing ROCK inhibitor Y2763210 μ M, or containing nicotinamide 0.1, 0.5, 1, 5, 10, 20 mM.

Further, the present application also provides a method for purifying cardiomyocytes, which comprises the following steps: culturing the passaged cardiomyocytes in a third medium containing nicotinamide, wherein the third medium can be E6 medium. The passaging treatment may be, but is not limited to, a passaging method with reference to the cardiomyocytes described above.

In an alternative embodiment, the concentration of nicotinamide in the third culture medium is also not less than 10 mmol/L.

In an alternative embodiment, the cardiomyocytes for passaging are human pluripotent stem cells after 10-15 days of differentiation into cardiomyocytes.

In alternative embodiments, passaging may be performed at a ratio of 1:3 to 1: 4.

Preferably, a ROCK inhibitor or nicotinamide is added during the passage.

By reference, the ROCK inhibitor may be, but is not limited to, Y27632.

Preferably, the concentration of ROCK inhibitor added may be 5-20. mu. mmol/L (preferably 10. mu. mmol/L), and the concentration of nicotinamide added may be 10-20 mmol/L.

Preferably, the cardiomyocytes are cultured in the nicotinamide-containing third medium for 4-6 days, with a new nicotinamide-containing third medium being replaced every 2-3 days.

In summary, the method for inducing stem cell differentiation toward cardiomyocytes and the method for passaging and purifying cardiomyocytes provided by the present application can effectively culture and maintain the hPSC-induced cardiomyocytes, facilitate the passage of the hPSC-induced cardiomyocytes, increase the survival rate of the obtained cardiomyocytes, and maintain the purity of the cardiomyocytes after long-term culture at a high level. Moreover, the method has low cost and is beneficial to large-scale production.

Accordingly, the present application also provides a pharmaceutical agent comprising a cardiomyocyte obtained by the method for inducing stem cell differentiation into a cardiomyocyte, or by the method for passaging a cardiomyocyte, or by the method for purifying a cardiomyocyte. The medicament has wide application prospect and can provide materials for regenerative repair and drug screening of heart diseases.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

Nicotinamide promotes differentiation of human embryonic stem cells H1 towards cardiac muscle cells

Human embryonic stem cells H1 were cultured in E8 medium, and were passaged at a cell density of 70% by changing fresh medium every day. First washed 2 times with DPBS-EDTA, incubated for a third 5 minutes at room temperature, pipetted DPBS-EDTA, added to E8 medium containing 5 μ M ROCK inhibitor, resuspended cells, and then incubated at 1:3 density into 12-well plates previously coated with matrigel. The culture medium of E8 was changed 1 day after the cells were attached to the wall. When the cells grew to 60% differentiation was initiated, E5 differentiation medium containing 5. mu.M CHIR99021 was added on the first day. E5 differentiation medium containing 10mM or 20mM nicotinamide was added after 1 day and treated for 4 days, during which time E5 medium containing nicotinamide was changed daily. On day 6 of differentiation, medium E5 was changed and culture was continued for 2 days with daily changes. The differentiated cells were then continued to be cultured in E5 medium containing insulin and the cardiomyocytes began to beat approximately 9-10 days after differentiation.

At the same time, the concentration and time of nicotinamide treatment were optimized in this example, and the results are shown in fig. 1. The figure shows that 10mM or 20mM nicotinamide significantly promotes the expression of the marker gene of the cardiomyocytes, and the hPSC can be significantly promoted to differentiate towards the cardiomyocytes after being treated for 4-6 days.

In FIG. 1, A is a schematic diagram of differentiation of hPSC toward cardiac muscle. Wherein CHIR represents CHIR99021 treatment concentration of 5. mu.M, and IWP-2 is used as positive control treatment concentration of 3. mu.M for myocardial differentiation. hPSCs were cultured in E8 medium 2 days before the start of differentiation (D-2), cells were cultured in E5 basal medium 7 days before differentiation (D0-D7), and after 7 days (D7-end), cells were cultured in E5 medium with insulin for 10 days to examine the gene expression of markers of cardiomyocytes.

B is a graph of the effect of different concentrations of nicotinamide (Nam) on the expression of the genes for markers NKX2-5 (top) and TNNT2 (bottom) of cardiomyocytes. Wherein p <0.05, indicates a significant difference compared to the control (Nam 0) without nicotinamide.

C is a graph of gene expression of markers NKX2-5 (upper) and TNNT2 (lower) in cardiomyocytes assayed by the addition of nicotinamide (Nam 10mM or 20mM) at different times during differentiation.

As can be seen from FIG. 1, nicotinamide is preferably treated at a concentration of at least 10mM for a period of at least 3 days, preferably for a period of 1-5 days or 1-7 days of differentiation (FIG. 1).

Further, the positive control group was treated with IWP-23. mu.M for 4 days during days 1-5 of differentiation and for 3 days during days 2-5 of differentiation according to the results of FIG. 1. After 13 days of differentiation, the proportion of cardiomyocytes positive to TNNT2 and the expression of markers NKX2-5 and TNNT2 of the cardiomyocytes were examined, which indicates that nicotinamide can effectively induce hPSC to produce cardiomyocytes. Among them, TNNT2 is a cardiomyocyte-specific marker, and TNNT 2-positive cells indicate cardiomyocytes among differentiated cells.

In fig. 2, a is a scale chart of TNNT2 positive cardiomyocytes analyzed by flow cytometry. Light gray lines indicate isotype antibody negative controls. The upper left panel represents a negative control group, i.e., an induction group without the addition of IWP-2 or nicotinamide; the upper right panel represents the IWP-2 induced differentiation group; the bottom left panel represents the nicotinamide 10mM induced differentiation group; the lower right panel represents the nicotinamide 20mM differentiation-inducing group.

B is a picture of cardiomyocytes induced to differentiate using antibodies against NKX2-5 and TNNT2 labeled with 3. mu.M IWP2 and 20mM nicotinamide. The left panel shows the result of NKX2-5 antibody staining, the middle panel shows the result of TNNT2 antibody staining, and the right panel shows the combined result of NKX2-5, TNNT2 and DAPI staining. Scale 50 μm.

As can be seen from fig. 2, the TNNT2 positive cells were detected on days 12 to 15 of differentiation, and the results showed that the ratio of TNNT2 positive cells in nicotinamide-induced differentiated cells was more than 90%, indicating that nicotinamide induced more than 90% of H1 cells to differentiate into cardiomyocytes.

In addition, nicotinamide 20mM was treated for 4 days during days 1-5 of differentiation, the positive control group was treated for 3 days during days 2-5 of differentiation using IWP-23 μ M, and cytoskeleton and electrophysiological functions of cardiomyocytes were analyzed 30 days after differentiation, as shown in fig. 3, showing that nicotinamide induced differentiation cardiomyocytes had mature cytoskeletal structure, higher levels of cardiomyocyte marker gene expression, and normal electrophysiological signals of atrial cells and ventricular cells.

In FIG. 3, A is the expression of cytoskeletal protein α -actin in IWP2 or nicotinamide-induced cardiomyocytes, with bars representing α -actin and ovals representing DAPI. Scale 10 μm.

B is a length chart of the muscle segments marked by the statistical alpha-actin. The results show that the nicotinamide induced cardiomyocytes have longer sarcomere length, which indicates that the cardiomyocytes have higher maturity.

C is a graph comparing the expression of the myocardial-associated genes of differentiated cells of different treatment groups. The light color indicates that the gene expression level is higher, and the dark color indicates that the gene expression level is lower. From this result, it was seen that nicotinamide-induced cardiomyocytes expressed higher levels of cardiomyocyte-associated genes such as MYL2, MYH7, ACTN2, RYR2, TNNT2, and MYL7, while lower levels of pericardium cell genes WT1 and TBX18 were expressed.

D is the action potential diagram of the myocardial cells induced by the analysis of nicotinamide. The myocardial cells are shown to have the action potentials of normal atrial cells and ventricular cells, and the myocardial cells are shown to have the electrophysiological functions of the normal myocardial cells.

Analysis of cardiomyocytes derived from nicotinamide-induced differentiation according to figure 3 can lead to: the cells show regular sarcomere structures at 25-30 days of differentiation, highly express a plurality of cardiomyocyte-specific genes, and have action potentials of atrial myocytes and ventricular myocytes. The nicotinamide induced differentiation of the myocardial cells is proved to have the cytoskeleton structure and the electrophysiological function of the normal human myocardial cells.

Example 2

Nicotinamide promotes differentiation of various human pluripotent stem cells to myocardial cells

Human embryonic stem cells H9 and human induced pluripotent stem cells NL4 were cultured in E8 medium, and the medium was replaced with fresh medium every day, and passaged until the cell density reached 70%. First washed 2 times with DPBS-EDTA, incubated for a third 5 minutes at room temperature, pipetted DPBS-EDTA, added to E8 medium containing 5 μ M ROCK inhibitor, resuspended cells, and then incubated at 1:3 density into 12-well plates previously coated with matrigel. The culture medium of E8 was changed 1 day after the cells were attached to the wall. When the cells grew to 60% differentiation was initiated, E5 differentiation medium containing 5. mu.M CHIR99021 was added on the first day. E5 differentiation medium containing 10mM or 20mM nicotinamide was added after 1 day and treated for 4 days, during which time E5 medium containing nicotinamide was changed daily. On day 6 of differentiation, medium E5 was changed and culture was continued for 2 days with daily changes. The differentiated cells were then cultured with continued insulin-containing E5 medium and started beating approximately 9-10 days after differentiation, at which time the cells were tested for expression of the higher levels of the cardiomyocyte marker genes NKX2-5 and TNNT 2. The proportion of TNNT2 positive cardiomyocytes detected on days 12-15 of differentiation was over 60% (see FIG. 4).

Specifically, nicotinamide was used for 4 days during days 1-5 of differentiation, and IWP-23. mu.M was used for 3 days during days 2-5 of differentiation in the positive control group. After differentiation for 10-13 days, the gene expression of markers NKX2-5 and TNNT2 of the cardiomyocytes and the proportion of TNNT 2-positive cardiomyocytes were examined. The results show that nicotinamide can effectively induce various hPSCs to produce myocardial cells.

In FIG. 4, A is a graph of nicotinamide (Nam) induced H9 cell to differentiate into cardiomyocytes, and the expression of NKX2-5 and TNNT2 genes of the cardiomyocytes is analyzed. P <0.05, indicating a significant difference compared to the control without added nicotinamide.

B is nicotinamide (Nam) inducing NL4 cells to differentiate into myocardial cells, and the expression profiles of NKX2-5 and TNNT2 genes of the myocardial cells are analyzed. P <0.05, indicating a significant difference compared to the control without added nicotinamide.

C is a proportion diagram of TNNT2 positive cells of differentiated cells detected after nicotinamide (Nam) induces H9 cells to differentiate. The light grey line in the figure indicates the isotype antibody negative control, which is, from left to right: negative control group, i.e. induction group without adding IWP-2 or nicotinamide; IWP-2 induced differentiation group; nicotinamide 10mM induced the differentiation group.

D is a proportion of TNNT2 positive cells detected from differentiated cells after nicotinamide (Nam) induced differentiation of NL4 cells. The light grey line in the figure indicates the isotype antibody negative control, which is, from left to right: negative control group, i.e. induction group without adding IWP-2 or nicotinamide; IWP-2 induced differentiation group; nicotinamide 10mM induced differentiation group; nicotinamide 20mM induced the differentiation group.

Example 3:

nicotinamide increases the survival rate of IWP-2 induced cardiomyocytes at passage

Human embryonic stem cells H1 were cultured in E8 medium, and were passaged at a cell density of 70% by changing fresh medium every day. First washed 2 times with DPBS-EDTA, incubated for a third 5 minutes at room temperature, pipetted DPBS-EDTA, added to E8 medium containing 5 μ M ROCK inhibitor, resuspended cells, and then incubated at 1:3 density into 12-well plates previously coated with matrigel. The culture medium of E8 was changed 1 day after the cells were attached to the wall. When the cells grew to 60% differentiation was initiated, E5 differentiation medium containing 5. mu.M CHIR99021 was added on the first day. The next day, the culture was changed to fresh E5 differentiation medium for 1 day. E5 differentiation medium containing 3. mu.M IWP-2 was added on the third day, and treated for 3 days, during which the E5 medium containing IWP-2 was changed every day. On day 6 of differentiation, medium E5 was changed and culture was continued for 2 days with daily changes. On day 8 of differentiation, the differentiated cells were further cultured in E5 medium containing insulin (E6 medium) and the culture medium was changed every 2 days. After 10 days of culture, the cardiomyocytes were digested with TrypLE, neutralized with BSA, centrifuged to remove the digest and the cardiomyocytes were resuspended in E6 medium under different conditions, including negative control (no reagent added), containing ROCK inhibitor Y2763210 μ M, or E6 medium containing nicotinamide 0.1, 0.5, 1, 5, 10, 20 mM.

Referring to fig. 5, it can be seen that the survival rate of cells of the control group without addition of ROCK inhibitor or nicotinamide was low when H1 cells were passaged after induced differentiation into cardiomyocytes by IWP-2, whereas addition of ROCK inhibitor (ROCK) or nicotinamide (Nam) promoted survival of cardiomyocytes and increased the ratio of cardiomyocytes.

In fig. 5, a is a graph of the effect of different concentrations of nicotinamide (Nam) on cell viability after passage of cardiomyocytes. The results show that ROCK inhibitor Y27632(10 μ M) or nicotinamide (>5mM) significantly improves the survival rate of cardiomyocytes 24 hours after passaging. P <0.05, indicating a significant difference compared to the control group.

B is a graph of the effect of different concentrations of nicotinamide (Nam) on the MLC phosphorylation levels of cardiomyocytes after 1 hour of passaging. The results indicate that ROCK inhibitor Y27632(10 μ M) or nicotinamide (>5mM) inhibits phosphorylation of MLC.

C is a statistical scale of phosphorylation levels of MLC versus total MLC protein levels. The results indicate that ROCK inhibitor Y27632(10 μ M) or nicotinamide (>5mM) inhibits phosphorylation of MLC.

D is a comparison graph of different cell states before and after the myocardial cell passage under an optical microscope. The results show that: pre-passaging cardiomyocytes (Before); negative Control (Control) 24 hours after passage; adding ROCK inhibitor Y2763210 mu M (ROCKi) after passage; nicotinamide 10mM (Nam 10mM) was added after passage; nicotinamide 20mM (Nam 20mM) was added after passage. The scale bar is 100 μm.

E is a proportion diagram of TNNT2 positive cells before and after the passage of the myocardial cells. The light grey line in the figure indicates the isotype antibody negative control. From left to right are: pre-passaged cardiomyocytes (beform); negative Control cardiomyocytes (Control) cultured for 24 hours after passaging; adding ROCK inhibitor Y2763210 mu M to culture cardiomyocytes (ROCKi) for 24 hours after passage; cardiomyocytes cultured for 24 hours after passage with nicotinamide 10mM added (Nam 10 mM); after passage, nicotinamide 20mM was added to the cardiomyocytes (Nam 20mM) which were cultured for 24 hours.

In summary, the results in fig. 5 show that addition of ROCK inhibitor Y2763210 μ M or

nicotinamide

5, 10 or 20mM significantly improves survival of cardiomyocytes 24 hours after passaging (fig. 5A). Preferred working concentrations of nicotinamide are 10 or 20 mM. Myosin Light Chain (MLC) is a downstream substrate for ROCK, and its phosphorylation level affects the process of cytoskeletal contraction, thereby accelerating cell death at passage. Phosphorylation levels of MLC were measured 1 hour after cardiomyocyte passage and it was found that ROCK inhibitor or high concentration of nicotinamide (>5mM) reduced the phosphorylation levels of MLC (fig. 5B). It is speculated that ROCK inhibitors or high concentrations of nicotinamide promote cardiomyocyte survival by inhibiting MLC phosphorylation. The survival cardiomyocytes at 24 hours after passage were analyzed by flow cytometry, and the proportion of TNNT2 positive cells was significantly increased by adding ROCK inhibitor Y2763210 μ M or nicotinamide 10mM or 20mM (fig. 5D-E). Indicating that nicotinamide promotes survival of cardiomyocytes after passaging by inhibiting the ROCK pathway.

Example 4

Nicotinamide increases survival of nicotinamide-induced cardiomyocytes upon passaging

Reference example 1 induction of differentiation of H1 cells into cardiomyocytes with nicotinamide, after 10 days of culture, cardiomyocytes were digested with TrypLE, neutralized with BSA, the digest was removed by centrifugation, and the cardiomyocytes were resuspended in E6 medium of various conditions, including negative control group (without any reagent added), E6 medium containing ROCK inhibitor Y2763210 μ M, or containing nicotinamide 0.1, 0.5, 1, 5, 10, 20 mM.

Referring to fig. 6, H1 cells were induced to differentiate into cardiomyocytes using nicotinamide and then passaged, which indicated that nicotinamide also increased the survival rate of such cardiomyocytes, indicating that nicotinamide was effective in survival of cardiomyocytes induced by a variety of differentiation methods.

In FIG. 6, A is a graph of the effect of different concentrations of nicotinamide (Nam) on the survival of nicotinamide induced differentiated cardiomyocytes. H1 cell differentiation is induced by nicotinamide, the obtained myocardial cells are passaged after 10 days, and the survival rate of the cells is detected after 24 hours. P <0.05, indicating a significant difference compared to the control group.

And B is a comparison graph of different cell states before and after passage of nicotinamide induced differentiation myocardial cells observed under an optical microscope. Pre-passaging cardiomyocytes (Before); negative Control (Control) 24 hours after passage; adding ROCK inhibitor Y2763210 mu M (ROCKi) after passage; nicotinamide 10mM (Nam 10mM) was added after passage; nicotinamide 20mM (Nam 20mM) was added after passage. The scale bar is 100 μm.

In summary, fig. 6 shows that: the survival rate of 24 hours after the passage of the myocardial cells is obviously improved by adding ROCK inhibitor Y2763210 mu M or 1, 5, 10 or 20mM nicotinamide. Preferred working concentrations of nicotinamide are 10 or 20 mM. Thus indicating that the nicotinamide can improve the survival rate of the passage of the myocardial cells obtained by different differentiation methods.

Example 5

Nicotinamide promotes cardiomyocyte purification

Reference example 3 induced the differentiation of H1 cells into cardiomyocytes, and the addition of ROCK inhibitor Y27632 for passaging 10 days after differentiation. Followed by 6 days of incubation under different incubation conditions, wherein the negative Control group (Control) was supplemented with no reagents in E6 medium, and the other two groups were supplemented with 10mM or 20mM nicotinamide in E6 medium, respectively. After 6 days of culture, the medium was changed every 2 days with or without nicotinamide, and the expression of genes related to myocardium was analyzed in nicotinamide-treated or untreated cells, indicating that nicotinamide increased the expression of various myocardial characteristic genes. The proportion of TNNT 2-labeled cardiomyocytes was further examined by flow cytometry. The results show that the proportion of cardiomyocytes reached 90% after nicotinamide treatment, increasing the purity of cardiomyocytes (fig. 7). These results indicate that nicotinamide promotes the purification of cardiomyocytes derived from the differentiation of H1 cells.

Specifically, the cardiomyocytes obtained by differentiating the H1 cells were passaged with nicotinamide or ROCK inhibitor, and cultured for 4-6 days by adding nicotinamide at various concentrations.

In FIG. 7, A is a diagram showing the observation of cardiomyocytes under different culture conditions under an optical microscope. The results show that the addition of nicotinamide was significantly more cardiomyocytes. The scale bar is 100 μm.

B is a scale chart for detecting myocardial cells positive to TNNT2 under different culture conditions. The results show that nicotinamide 10mM or 20mM treatment group TNNT2 positive cytosis, indicating that nicotinamide increased cardiomyocyte purity.

And C is a graph for detecting the influence of different culture conditions on the expression of the myocardial related genes. The light color indicates that the gene expression level is higher, and the dark color indicates that the gene expression level is lower. The results show that the expression of the cardiomyocyte-associated genes RYR2, TBX5, ACTN2, MYH7, ATP2A2, NPPA, TNNT2 and MYH7 in the nicotinamide 10mM or 20mM treated group is increased. And the expression of pericardium cell related genes WT1 and TBX18 is reduced.

Example 6

This example provides a method for inducing stem cells to differentiate towards cardiomyocytes, which comprises the following steps:

human pluripotent stem cells were cultured in E8 medium, replaced with fresh medium every day, and passaged until the cell density reached 75%. First washed 2 times with DPBS-EDTA, incubated for a third 5 minutes at room temperature, pipetted DPBS-EDTA, added to E8 medium containing 5 μ M ROCK inhibitor, resuspended cells, and then incubated at 1:4 density plates were placed in 12 well plates previously coated with matrigel. The culture medium of E8 was changed 1 day after the cells were attached to the wall. When the cells grew to 60% differentiation was initiated, E5 differentiation medium containing 5. mu.M CHIR99021 was added on the first day. E5 differentiation medium containing 10mM nicotinamide was added after 1 day and treated for 4 days, during which time E5 medium containing nicotinamide was changed daily. On day 6 of differentiation, medium E5 was changed and culture was continued for 2 days with daily changes. The differentiated cells were then continued to be cultured in E5 medium containing insulin until the cardiomyocytes began to beat. The other conditions were the same as in example 1.

Example 7

human pluripotent stem cells were cultured in E8 medium, replaced with fresh medium every day, and passaged when the cell density reached 80%. First washed 2 times with DPBS-EDTA, incubated for a third 5 minutes at room temperature, pipetted DPBS-EDTA, added to E8 medium containing 5 μ M ROCK inhibitor, resuspended cells, and then incubated at 1: 5 density was placed in 12 well plates previously coated with matrigel. The culture medium of E8 was changed 1 day after the cells were attached to the wall. When the cells grew to 80% differentiation was initiated, E5 differentiation medium containing 5. mu.M CHIR99021 was added on the first day. E5 differentiation medium containing 10mM nicotinamide was added after 1 day and treated for 4 days, during which time E5 medium containing nicotinamide was changed daily. On day 6 of differentiation, medium E5 was changed and culture was continued for 2 days with daily changes. The differentiated cells were then continued to be cultured in E5 medium containing insulin until the cardiomyocytes began to beat. The other conditions were the same as in example 1.

In conclusion, the method for inducing the stem cells to differentiate towards the direction of the myocardial cells and carrying out the passage and purification of the myocardial cells, which is provided by the application, can provide an effective and feasible new method for the generation and the passage culture of the myocardial cells derived from human pluripotent stem cells, has stable efficiency and lower cost, and is beneficial to large-scale production. The survival rate and purity of the obtained myocardial cells are high, and the medicament containing the myocardial cells obtained by the method has wide application prospect, and can provide materials for regenerative repair and drug screening of heart diseases.

The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and various modifications and changes will occur to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method of inducing differentiation of stem cells toward cardiomyocytes comprising the steps of: culturing human pluripotent stem cells in a first medium comprising nicotinamide, said first medium being E5 medium;

preferably, the concentration of said nicotinamide in said first culture medium is not less than 10 mmol/L;

preferably, said nicotinamide has an effect on said human pluripotent stem cells for a period of 4 to 6 days.

2. The method according to claim 1, wherein said human pluripotent stem cells are differentiated into mesoderm, and said nicotinamide is added to induce differentiation into myocardium;

preferably, the differentiation of said human pluripotent stem cells into mesoderm is induced by opening the Wnt/β -catenin pathway;

preferably, the Wnt/β -catenin pathway is opened using the GSK3 β inhibitor CHIR 99021;

preferably, differentiation into mesoderm is performed with human pluripotent stem cells having a cell density of 60-80%;

preferably, the medium used for the differentiation process to mesoderm is E5 medium without TGF β, FGF2 and insulin but containing lipid concentrate.

3. The method of claim 1 or 2, further comprising continuing culture differentiation with insulin-containing E5 medium after culturing with said first medium comprising said nicotinamide.

4. A method for passaging cardiomyocytes, comprising the steps of: culturing the cardiomyocytes in a second medium comprising nicotinamide, wherein the second medium is E6 medium;

preferably, the concentration of said nicotinamide in said second culture medium is not less than 10 mmol/L;

preferably, the cardiomyocytes are obtained by the method for inducing the stem cells to differentiate into cardiomyocytes according to any one of claims 1 to 3;

preferably, the cardiac muscle cell is the cardiac muscle cell 10-15 days after the human pluripotent stem cell is differentiated to the cardiac muscle cell;

preferably, the cardiac muscle cell is a cardiac muscle cell induced by human pluripotent stem cells through IWP-2 or nicotinamide;

preferably, said nicotinamide promotes said cardiomyocyte survival by inhibiting the ROCK pathway.

5. The method of passaging according to claim 4, wherein the cardiomyocytes digested into single cells are placed in the second medium containing nicotinamide, and the culture is continued after 1-2 days by changing to the second medium containing no nicotinamide;

preferably, the digestion treatment is carried out by TrypLE, followed by neutralization reaction with bovine serum albumin, solid-liquid separation, and the cardiomyocytes in the single cell state after removal of the digestive juice are placed in the second medium containing nicotinamide.

6. A method for purifying cardiomyocytes, comprising the steps of: culturing the passaged cardiomyocytes in a third medium containing nicotinamide, wherein the third medium is E6 medium;

preferably, the concentration of said nicotinamide in said third medium is not less than 10 mmol/L;

preferably, the method of passaging is the method of passaging of claim 4 or 5.

7. Purification process according to claim 6, characterized in that passage is carried out in a ratio of 1:3 to 1: 4;

preferably, a ROCK inhibitor or nicotinamide is added in the process of passage;

preferably, the ROCK inhibitor is Y27632;

preferably, the addition concentration of the ROCK inhibitor is 5-20 mu mmol/L, and the addition concentration of the nicotinamide is 10-20 mmol/L;

preferably, said cardiomyocytes are cultured in said third medium comprising nicotinamide for 4-6 days, with a new third medium comprising nicotinamide being replaced every 2-3 days.

8. A pharmaceutical preparation comprising the cardiomyocytes obtained by the method for inducing stem cell differentiation into cardiomyocytes according to any one of claims 1 to 3.

9. A pharmaceutical preparation comprising the cardiomyocytes produced by the method for passaging the cardiomyocytes according to claim 4 or 5.

10. A pharmaceutical preparation comprising the cardiomyocytes obtained by the method for purifying the cardiomyocytes according to claim 6 or 7.