CN116121180A

CN116121180A - Media and methods for producing cardiomyocytes from pluripotent stem cells

Info

Publication number: CN116121180A
Application number: CN202211713170.5A
Authority: CN
Inventors: 俞君英; 吴文青; 张颖
Original assignee: Nuwacell Ltd
Current assignee: Nuwacell Ltd
Priority date: 2022-12-27
Filing date: 2022-12-27
Publication date: 2023-05-16

Abstract

The present invention provides media and methods for producing cardiomyocytes from pluripotent stem cells. The culture medium is serum-free and animal-derived component-free, and comprises basal medium, nicotinamide compound, human Platelet Lysate (PLT), heparin compound, insulin, human transferrin and Human Serum Albumin (HSA). By using the medium and the method, high-purity and high-number myocardial cells can be efficiently provided.

Description

Media and methods for producing cardiomyocytes from pluripotent stem cells

Technical Field

The present invention relates to the field of stem cell biology, in particular to a medium, kit and method for generating Cardiomyocytes (CM) from pluripotent stem cells (also called pluripotent stem cells, PSCs), and a cardiomyocyte population thereof.

Background

Coronary heart disease (Coronary Artery Disease, CAD) is the second largest cardiovascular disease next to hypertension, and the incidence and mortality rates are very high worldwide, and the incidence rate is also rising in china in recent years. At present, the main clinical treatment methods of coronary heart disease such as drug therapy, percutaneous Coronary Intervention (PCI) and Coronary Artery Bypass Grafting (CABG) cannot obviously recover the function of the heart after damage. Because human heart muscle does not have regeneration ability, once the heart is damaged and dead due to myocardial infarction and the like, once the critical point is exceeded, the heart can enter the vicious circle for compensation, and finally, the heart can irreversibly develop into heart failure. According to epidemiological investigation in China, the prevalence rate of chronic heart failure in the population is 0.9%, at least 1000 thousands of patients, and the total mortality rate is 32%.

Induced Pluripotent Stem Cells (iPSC) have all differentiation capacities of Embryonic Stem Cells (ESC), can be directionally differentiated into myocardial cells due to easy expansion, and have no immune rejection and ethical problems, so that a new idea for treating coronary heart disease is provided. Recent studies have found that after transplantation of cardiomyocytes produced by human ESCs/iPSCs, the cardiac function of the myocardial pigs is significantly improved, and both morphological and physiological results indicate that the transplanted myocardium integrates into the host heart and forms an effective contractile function unit (Kawamura et al 2012; shiba et al 2012).

Although the process of differentiating the pluripotent stem cells into the myocardial cells in vitro is clear in principle, and various induced differentiation schemes exist at present, the existing differentiation schemes still have the problems of long culture time, low efficiency, high difficulty in cell separation, incapability of achieving clinical use requirements on the number and quality of the obtained myocardial cells, and the like.

Thus, there remains a need in the art for new approaches to the induction of cardiomyocytes from pluripotent stem cells that are more robust and efficient.

Disclosure of Invention

Through intensive studies, the present inventors have developed a serum-free and animal-derived component-free cardiomyocyte medium that can be used as a basal medium for a cardiomyocyte maintenance medium to enhance the differentiation efficiency (e.g., increase the proportion and number of cardiomyocytes) of cardiomyocytes at the differentiation stage of generating cardiomyocytes from pluripotent stem cells. Furthermore, the present inventors have further found that the cardiomyocyte culture medium can also be used as a cardiomyocyte maturation medium to promote further maturation of cardiomyocytes. On this basis, the invention also proposes a method for generating Induced Cardiomyocytes (iCM) from Pluripotent Stem Cells (PSC). The culture medium and the method can be used for efficiently providing high-purity and high-number myocardial cells.

Accordingly, in a first aspect, the present invention provides a serum-free and animal-derived component-free cardiomyocyte culture medium comprising a basal medium and nicotinamide compound, human Platelet Lysate (PLT), heparin compound, insulin, human transferrin, and Human Serum Albumin (HSA).

In one or more embodiments, the nicotinamide compound is nicotinamide, a derivative or analog thereof, or a salt thereof. Most preferably, the nicotinamide compound is nicotinamide.

In one or more embodiments, the heparin-like compound is heparin, a derivative or analog thereof, or a salt thereof. Most preferably, the heparin-like compound is heparin or heparin sodium.

In one or more embodiments, the nicotinamide compound is at a concentration of 0.5-20mM, preferably 1-10mM, e.g., 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, or 10mM, based on the total cardiomyocyte medium.

In one or more embodiments, the heparin-based compound is present at a concentration of 0.1-100. Mu.g/mL, preferably 0.1-50. Mu.g/mL, e.g., 0.1. Mu.g/mL, 0.5. Mu.g/mL, 1. Mu.g/mL, 3. Mu.g/mL, 5. Mu.g/mL, 7. Mu.g/mL, 9. Mu.g/mL, 10. Mu.g/mL, 12. Mu.g/mL, 14. Mu.g/mL, 15. Mu.g/mL, 16. Mu.g/mL, 18. Mu.g/mL, 19. Mu.g/mL, 20. Mu.g/mL, 22. Mu.g/mL, 24. Mu.g/mL, 26. Mu.g/mL, 28. Mu.g/mL, 30. Mu.g/mL, 32. Mu.g/mL, 34. Mu.g/mL, 36. Mu.g/mL, 38. Mu.g/mL, 40. Mu.g/mL, 42. Mu.g/mL, 44. Mu.g/mL, 46. Mu.g/mL, 48. Mu.g/mL, 50. Mu.g/mL, or 50. Mu.g/mL, based on the total amount of the cardiomyocyte medium.

In one or more embodiments, the concentration of PLT is 0.1-10% by volume, preferably 0.1-5% by volume, e.g., 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5% by volume, based on the total cardiomyocyte medium.

In one or more embodiments, the concentration of insulin is 0.1-20 μg/mL, preferably 1-15 μg/mL, e.g., 1 μg/mL, 2 μg/mL, 3 μg/mL, 4 μg/mL, 5 μg/mL, 6 μg/mL, 7 μg/mL, 8 μg/mL, 9 μg/mL, 10 μg/mL, 11 μg/mL, 12 μg/mL, 13 μg/mL, 14 μg/mL, or 15 μg/mL, based on the total cardiomyocyte medium.

In one or more embodiments, the concentration of human transferrin is 1-100 μg/mL, preferably 1-20 μg/mL, e.g., 1 μg/mL, 5 μg/mL, 10 μg/mL, 20 μg/mL, 25 μg/mL, 30 μg/mL, 35 μg/mL, 40 μg/mL, 45 μg/mL, 50 μg/mL, 55 μg/mL, 60 μg/mL, 65 μg/mL, 70 μg/mL, 75 μg/mL, 80 μg/mL, 85 μg/mL, 90 μg/mL, 95 μg/mL, or 100 μg/mL, based on the total amount of cardiomyocyte medium.

In one or more embodiments, the concentration of HSA is 0.2-20mg/mL, preferably 1-10mg/mL, e.g., 1mg/mL, 2mg/mL, 3mg/mL, 4mg/mL, 5mg/mL, 6mg/mL, 7mg/mL, 8mg/mL, 9mg/mL, or 10mg/mL, based on the total cardiomyocyte medium.

In one or more embodiments, the cardiomyocyte culture medium further comprises one or more or all of the components selected from the group consisting of: thioglycerol (MTG); pyruvate; ethanolamine; ascorbic acid, derivatives thereof or salts thereof; selenite; and glutamine or a derivative thereof.

In a preferred embodiment, the cardiomyocyte culture medium comprises:

(i)0.1-20mg/mL HSA；

(ii)10-200μM MTG；

(iii) 50-100 μg/mL ascorbic acid;

(iv) 1-10mM nicotinamide;

(v) 50-200 mug/mL pyruvate;

(vi) 1-15 μg/mL insulin;

(vii) 1-20 μg/mL human transferrin;

(viii) 5-40ng/mL selenite;

(ix) 5-50 mu M ethanolamine;

(x) 0.1-5% by volume PLT;

(xi) Heparin sodium of 0.1-50 mug/mL; and

(xii) 1.0-5.0% by volume Glutamax.

In one or more embodiments, the basal medium is a chemically defined serum-free medium, and is preferably selected from the group consisting of: DMEM, DMEM/F12, ham's F, RPMI-1640, IMDM, BME, IMDM/F12, alpha-MEM or combinations thereof. In a more preferred embodiment, the basal medium consists of 1:1 IMDM and DMEM/F12.

In a second aspect, the present invention provides a cardiomyocyte maintenance medium comprising a cardiomyocyte culture medium according to the present invention as a basal medium and comprising a WNT signaling pathway activator as a supplement.

In one or more embodiments, the WNT signal pathway activator is a GSK3 inhibitor selected from the group consisting of CHIR99021, CHIR98014, NP031112, TWS119, AZD2858, AZD1080, SB415286, LY2090314, AR-A014418, SB216763, (5-methyl-1H-pyrazol-3-yl) - (2-phenylquinazolin-4-yl) amine, 2-thio (3-iodobenzyl) -5- (1-pyridinyl) [1,3,4] -oxadiazole, alpha-4-dibromoacetophenone, 3- (1- (3-hydroxypropyl) -1H-pyrrolo [2,3-b ] pyridin-3-yl ] -4-pyrazin-2-yl-pyrrole-2, 5-dione, 2-chloro-1- (4, 5-dibromothiophen-2-yl) -ethanone, and GF 109203X.

In one or more embodiments, the WNT signaling pathway activator is at a concentration of 0.5-6. Mu.M, preferably 0.5-4. Mu.M, e.g., 0.5. Mu.M, 0.7. Mu.M, 0.8. Mu.M, 0.9. Mu.M, 1.1. Mu.M, 1.2. Mu.M, 1.3. Mu.M, 1.4. Mu.M, 1.5. Mu.M, 1.6. Mu.M, 1.7. Mu.M, 1.8. Mu.M, 1.9. Mu.M, 2.0. Mu.M, 2.1. Mu.M, 2.2. Mu.M, 2.3. Mu.M, 2.4. Mu.M, 2.5. Mu.M, 2.6. Mu.M, 2.7. Mu.M, 2.8. Mu.M, 2.9. Mu.M, 3.0. Mu.M, 3.5. Mu.M, or 4. Mu.M, based on the total amount of myocardial cell maintenance medium.

In a third aspect, the invention provides a method of producing Induced Cardiomyocytes (iCM) from Pluripotent Stem Cells (PSC), the method comprising:

(a) Culturing pluripotent stem cells in a cardiomyocyte differentiation medium to induce differentiation thereof, thereby forming cardiomyocyte precursor cells;

(b) Culturing the cardiomyocyte precursor cells obtained in step (a) in a cardiomyocyte maintenance medium according to the present invention as described above, thereby forming immature cardiomyocytes; and optionally

(c) Culturing the immature cardiomyocytes obtained in step (b) in a cardiomyocyte maturation medium to produce mature cardiomyocytes.

In one or more embodiments, the pluripotent stem cells are induced pluripotent stem cells (ipscs) or Embryonic Stem Cells (ESCs).

In one or more embodiments, the step (a) comprises: in a cardiomyocyte differentiation medium, PSC is first cultured for a period of time in the presence of WNT signaling pathway activator, and then PSC is cultured for a period of time in the presence of WNT signaling pathway inhibitor.

In one or more embodiments, the WNT signaling pathway activator is CHIR99021 and the WNT signaling pathway inhibitor is XAV939. Preferably, the concentration of CHIR99021 is 1-10 μm, preferably 2-7 μm, for example 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, based on the total cardiomyocyte differentiation medium. Preferably, the concentration of XAV939 is 1-10. Mu.M, preferably 2-7. Mu.M, e.g.1. Mu.M, 2. Mu.M, 3. Mu.M, 4. Mu.M, 5. Mu.M, 6. Mu.M, 7. Mu.M, 8. Mu.M, 9. Mu.M, 10. Mu.M, based on the total amount of cardiomyocyte differentiation medium.

In one or more embodiments, in step (b), the cardiomyocyte precursor cells are present in a 1 to 5x10 ratio ⁴ Individual cells/cm ² For example, 1X10 ⁴ Individual cells/cm ² 、1.5x10 ⁴ Individual cells/cm ² 、2x10 ⁴ Individual cells/cm ² 、2.5x10 ⁴ Individual cells/cm ² 、3x10 ⁴ Individual cells/cm ² 、3.5x10 ⁴ Individual cells/cm ² 、4x10 ⁴ Individual cells/cm ² 、4.5x10 ⁴ Individual cells/cm ² 、5x10 ⁴ Individual cells/cm ² Is a density inoculation of (3).

In one or more embodiments, in step (c), the immature cardiomyocytes are seeded onto a fibronectin matrix.

In one or more embodiments, in step (c), the cardiomyocyte maturation medium is a cardiomyocyte medium according to the present invention as described above.

In one or more embodiments, the method is performed under two-dimensional (2D) culture conditions.

In a fourth aspect, the invention provides a kit comprising a cardiomyocyte culture medium according to the invention as described above.

In one or more embodiments, the kits of the invention further comprise a WNT signaling pathway activator, wherein the WNT signaling pathway activator and the cardiomyocyte medium are provided in separate containers, respectively.

In a fifth aspect, the invention provides a population of cells obtained according to the method of the invention.

In one or more embodiments, greater than 95% of the cells in the population of cells are ctnt+ cardiomyocytes without purification or enrichment.

Advantageous effects

Compared with the prior art, the culture medium and the method have at least the following advantages:

first, the time required for inducing differentiation (about 30-45 days) is greatly shortened, the differentiation efficiency is higher (the proportion or purity of cardiomyocytes can be more than 90%, even more than 95%, the number of cardiomyocytes is large, and the amplification factor is more than 30 times, for example 35-40 times (the amplification factor is usually less than 5 times in the prior art);

secondly, the culture system used in the differentiation process does not contain serum and animal source components, and is suitable for the production of subsequent clinical-grade cell preparations;

thirdly, the process is simple and convenient to operate, high-purity myocardial cells can be obtained without purification, and the method has little damage to cells and is very suitable for clinical and scientific research applications;

fourth, the maturation of cardiomyocytes can be promoted, thereby obtaining a large number of functional cardiomyocytes of high purity and maturity.

Drawings

Fig. 1 shows an exemplary flow of the method according to the invention.

Fig. 2 shows the cell morphology changes during cardiomyocyte production from ipscs according to example 1 of the present invention, showing the cell morphology of hiPSC differentiated on day 0, cardiomyocyte precursor cells (D9 cells) on day 9, immature cardiomyocytes (D30 cells) on day 30, and mature cardiomyocytes (D40 cells) on day 40, respectively.

FIG. 3 shows the increased expression of specific markers of myocardial precursor cells (D9 cells) obtained on day 9 of differentiation according to example 1 of the present invention.

FIG. 4 shows the proportion of CTNT+ phenotype of myocardial precursor cells (D9 cells) in a differentiated cell population obtained on day 9 of differentiation (D9) using a flow cytometer according to example 1 of the present invention.

FIG. 5 shows pictures of cardiomyocytes at different differentiation times (day 10, day 16, day 22 and day 30) according to example 1 of the present invention, showing that cardiomyocyte expansion was evident after addition of cardiomyocyte maintenance medium.

FIG. 6 shows the variation of the expression of specific markers (NKX2.5+/KCNH2+/MYH2+/MYH2 V+/MYH2+) of cardiomyocytes at different differentiation times (day 9 and day 30) according to example 1, demonstrating that cardiomyocyte maintenance medium promotes specific differentiation of cardiomyocyte precursors to immature cardiomyocytes.

FIG. 7 shows the proportion of CTNT+ phenotype (i.e., purity of cardiomyocytes) of central myocytes of the differentiated cell population on day 30 of differentiation (D30) as measured using a flow cytometer according to example 1 of the present invention.

FIG. 8 shows the CTNT+/alpha-actinin+ and MLC2V+ phenotypes of cardiomyocytes differentiated on day 30 (D30) using cytoimmunofluorescent staining according to example 1 of the present invention.

FIG. 9 shows the expansion ratio of cardiomyocytes (represented by P1 and P2, respectively) at day 16 and day 30 of differentiation relative to cells (represented by P0) at day 9 of differentiation, according to example 1 of the present invention.

FIG. 10 shows that the specific marker (MYH 7 and MLC 2V) expression of D45 cells (mature cardiomyocytes) was increased relative to D30 cells (immature cardiomyocytes) after addition of cardiomyocyte maturation medium according to example 1 of the present invention.

FIG. 11 shows the detection of spontaneous action potentials of D45 cells (mature cardiomyocytes) using patch clamp according to example 1 of the present invention.

FIG. 12 shows the effect of cardiomyocyte maintenance media (media 1-3) using different basal media on differentiation and expansion of immature cardiomyocytes according to example 2 of the present invention, wherein basal media 1 (DMEM/F12 medium supplemented with B27 supplement) was used for media 1; basal medium 2 (RPMI 1640 medium supplemented with B27 supplement) was used as medium 2; whereas basal medium 3 (exemplary cardiomyocyte medium CM-SFM) was used for medium 3.

FIG. 13 shows the effect of different CHIR concentrations in cardiomyocyte maintenance medium (i.e. medium 1-5) on cardiomyocyte differentiation and expansion, according to example 3 of the present invention.

FIG. 14 shows the effect of 0.1% gelatin, VTN and fibronectin matrix on cell differentiation according to example 4 of the invention.

Detailed Description

Definition of the definition

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of the present invention, the following terms are defined below.

The term "about" when used in conjunction with a numerical value is intended to encompass numerical values within a range having a lower limit of 5% less than the specified numerical value and an upper limit of 5% greater than the specified numerical value.

The term "and/or" is understood to mean any one of the selectable items or a combination of any two or more of the selectable items.

As used herein, the terms "comprises" or "comprising" are intended to include the stated element, value, or step, but do not exclude any other elements, values, or steps. In this document, the terms "comprises" or "comprising" when used herein, unless otherwise indicated, also encompass the circumstance of consisting of the recited elements, values, or steps. For example, when referring to a cell culture medium comprising a basal medium and one or more supplements or supplements (e.g., differentiation factors), it is also intended to encompass that the cell culture medium consists of a basal medium and one or more supplements or supplements (e.g., differentiation factors).

Herein, references to the concentration of factors (including various differentiation factors, growth factors, and the like supplements) added to the medium refer to the concentration based on the total amount of the medium referred to.

The expressions "pluripotent stem cells" and "multipotent stem cells" are used interchangeably herein to refer to stem cells having the potential to differentiate into cell types that produce three germ layers (endodermal, ectodermal, mesodermal), also abbreviated herein as PSC. In the present invention, pluripotent stem cells include, for example, embryonic Stem Cells (ESCs) and Induced Pluripotent Stem Cells (iPSCs), etc.

Embryonic stem cells are undifferentiated pluripotent cells originally derived from an embryo, which may be, for example, established embryonic stem cell lines (e.g., human embryonic stem cell line MA09 cells), embryonic stem cells isolated or obtained from human embryos within 14 days of fertilization without in vivo development, or embryonic stem cells produced from adult cells by artificial means such as nuclear transfer with differentiation into any cell type. It should be noted, however, that embryonic stem cells do not refer to, and should not refer to, cells produced by disrupting a human embryo developed in vivo, nor to cells isolated from a human embryo fertilized for greater than 14 days. ES cells that may be used include, but are not limited to, e.g., H1, H7, H9, H14, and ACT30 ES cells.

Induced pluripotent stem cells (ipscs) are cells generated from somatic cells by expressing or inducing expression of cytokines, having the potential to differentiate into various germ layers. A variety of reprogramming methods known in the art may be used to reprogram somatic cells into induced pluripotent stem cells. See, for example, akram Al Abbar et Al Induced Pluripotent Stem Cells: reprogramming Platforms and Applications in Cell Replacement Therapy,28Apr 2020, https:// doi.org/10.1089/bins.2019.0046. Typically, reprogramming somatic cells to a dedifferentiated or pluripotent state involves expression of reprogramming factors (including transcription factors). The related transcription factor may comprise, consist of, or consist essentially of one or more of the following: OCT4, SOX2, KLF4, and MYC (OSKM); SOX2, KLF4, and OCT4 (SKO); OCT4, SOX2, KLF4 and GLIS1 (OSKG); OCT4, SOX2, NANOG, and LIN28 (OSNL); or OCT4, SOX2, KLF4, c-MYC, NANOG and LIN28 (OKSMNL). The reprogramming factors or nucleic acids encoding these reprogramming factors are then introduced into somatic cells to induce the production of ipscs.

As used herein, "cardiomyocytes" refer to the basic cells that make up heart tissue, and have their specific marker expression and/or function and activity. Specific markers for cardiomyocytes typically include NKX2.5, MYH6, KCNH2, MLC2V, and MYH7.

Herein, the expression "induced cardiomyocytes" refers to cardiomyocytes (including cardiomyocytes of various degrees of maturation) induced by pluripotent stem cells, such as embryonic stem cells (ES) or induced pluripotent stem cells (ipscs), also abbreviated herein as CM or iCM. Cardiomyocytes induced by pluripotent stem cells are also abbreviated herein as PSC-CM or iPSC-CM.

As used herein, "cardiomyocyte precursor" refers to cardiomyocytes that (without dedifferentiation or reprogramming) can form functional cardiomyocytes, including immature and mature (end stage) cardiomyocytes.

As used herein, "immature cardiomyocytes" refers to cardiomyocytes that have been mature cultured to form mature (end stage) cardiomyocytes, which have a smaller volume and lower MLC2V and MYH7 expression levels than mature cardiomyocytes.

In this context, "mature cardiomyocytes" refer to cardiomyocytes with contractile function at the end stage, which have higher MLC2V and MYH7 expression levels than immature cardiomyocytes.

In this context, an "expression marker" or "marker" may be used to determine the identity of a cell type. The DNA sequence of a cell-specific gene can be transcribed into mRNA and is typically subsequently translated into a protein (the gene product thereof) that performs a specific function in the cell. Expression of the marker may be detected and quantified at the RNA level or protein level by methods known in the art. iPSC cell markers are known in the art and include, but are not limited to, TRA-1-60, TRA-1-81, ecat1, nanog, oct4/POU5F1, sox2, rex1/Zfp-42 and UTF1, or any combination thereof. Cardiomyocyte-specific markers include, but are not limited to, NKX2.5, KCNH2, MYH6, MLC2V, MYH7, MLC2 a, and CTNT. Cardiomyocytes at different differentiation stages, such as cardiomyocyte precursors, immature cardiomyocytes and mature cardiomyocytes, can be identified/distinguished by the presence and/or expression level of cardiomyocyte-specific markers; and determining the maturity of cardiomyocytes differentiated from the PSCs.

In this context, the term "differentiation" refers to the process of converting undifferentiated or less differentiated cells into more differentiated cells, such as the process of differentiating ipscs into iCM. Ipscs can be induced to differentiate towards iCM by adding supplements such as differentiation factors to the cell basal medium.

Herein, differentiation factors include, but are not limited to, cytokines or small molecule compounds that cause cell differentiation. Herein, the time "Dn such as D30" associated with the directed differentiation of PSCs into cardiomyocytes refers to the number of days that pluripotent stem cells (e.g., ESCs or ipscs) having the potential for tricermal differentiation were placed under differentiation conditions.

As used herein, "culture medium" refers to a medium capable of supporting the survival, growth, propagation, maintenance and/or differentiation of cells in an in vitro environment. The medium may have or include a basal medium and one or more supplements or supplements.

As used herein, "cardiomyocyte differentiation medium" or "specific cardiomyocyte differentiation medium" or "differentiation medium" are used interchangeably to refer to a medium that can be used to induce the directional differentiation of pluripotent stem cells into cardiomyocyte precursor cells.

As used herein, "cardiomyocyte maintenance medium" or "specific cardiomyocyte maintenance medium" or "maintenance medium" are used interchangeably to refer to a medium that can be used to differentiate and maintain expansion of PSC-derived cardiomyocyte precursors into immature cardiomyocytes.

As used herein, "cardiomyocyte maturation medium" or "specific cardiomyocyte maturation medium" or "maturation medium" refers to a medium that can be used to further mature PSC-derived immature cardiomyocytes into mature cardiomyocytes.

As used herein, "basal medium" refers to a basal component or matrix of a medium (e.g., differentiation medium, maintenance medium, or maturation medium) as opposed to a supplement or supplement to the medium. The basal medium in the medium can act as a source of nutrients, hormones, and/or factors that aid in cell proliferation, maintenance, or differentiation. The basal medium typically comprises about 95 to 99% by volume of the medium.

"supplement" is used interchangeably herein with "supplement" and refers to an added or supplemented component of a medium (e.g., differentiation, maintenance or maturation medium) relative to its basal medium. The supplement or supplement is not typically added to the basal medium in advance, but is added as needed at the time of use. Of course, the supplement or supplement may also be added to the basal medium prior to use.

Herein, "ROCK inhibitor" refers to a substance that inhibits the Rho kinase (ROCK) signaling pathway, such as: y-27632, HA100, HA1152, and Blebbbistatin. In some embodiments, ROCK inhibitors are preferably added to the differentiation medium used after dissociation during the differentiation of CM from PSCs. For example, in a 2-dimensional differentiation culture regimen, ROCK inhibitors may be added to the differentiation medium to promote cell attachment. The concentration of ROCK inhibitor used may be any suitable concentration. In one or more embodiments, the ROCK inhibitor is Y-27632 or Blebbistatin, and the concentration of Y-27632 or Blebbistatin in the medium may be, for example, 1-50 μm, such as 1 μm, 1.5 μm, 2 μm, 2.5 μm, 3 μm, 3.5 μm, 4 μm, 4.5 μm, 5 μm, 5.5 μm, 6 μm, 6.5 μm, 7 μm, 7.5 μm, 8 μm, 9 μm, 10 μm, 15 μm, 20 μm, 30 μm, 50 μm, but is not limited thereto, preferably 10 μm.

Herein, the expression "embryoid body" or "EB" refers to a multicellular three-dimensional aggregate with differentiation potential formed by pluripotent stem cells. For example, ipscs can be suspension cultured under low adhesion conditions under conditions that favor agglomeration of the ipscs to produce 3-dimensional iPSC spheres, which are embryoid bodies with the potential to differentiate into three germ layers (including inner embryo, mesoderm, and ectoderm) and to differentiate into various adult specialized cell populations.

The various aspects of the invention are described in further detail below.

I. Culture medium

The invention provides a myocardial cell culture medium and a myocardial maintenance medium containing the same as a basal medium and WNT signal pathway activator as a supplement.

(1) Cardiomyocyte culture medium

The cardiomyocyte culture medium according to the present invention comprises basal medium, nicotinamide compound, human Platelet Lysate (PLT), heparin compound, insulin, human transferrin, and Human Serum Albumin (HSA). The cardiomyocyte culture medium of the invention is serum-free and free of animal-derived components.

Herein, the expression "nicotinamide-based compound" means nicotinamide, a derivative or analogue thereof, or a salt thereof. Nicotinamide is an amide form of niacin, both belonging to the vitamin B3 family, a precursor to Nicotinamide Adenine Dinucleotide (NAD). NAD acts as a coenzyme in a number of cellular processes including energy metabolism and DNA repair. Nicotinamide can be converted to Nicotinamide Mononucleotide (NMN) by nicotinamide riboside transferase (NAMPT) and then converted to NAD by nicotinamide mononucleotide adenyltransferase (NMNAT) ⁺ . Examples of nicotinamide-type compounds that may be mentioned herein include, but are not limited to, nicotinamide salts (e.g., hydrochloride salts), nicotinamide analogs, nicotinamide derivatives, and metabolites of nicotinamide or nicotinamide analogs, such as NAD, NADH, and NADPH. As used herein, the term "nicotinamide analog" refers to any molecule known to function similarly to nicotinamide. Examples of nicotinamide analogs include, but are not limited to, thionicotinamide (niacinamide) and niacin. Examples of nicotinamide derivatives include, but are not limited to, substituted nicotinamide and thionicotinamide, such as N-substituted nicotinamide and thionicotinamide. Salts of the above compounds include, for exampleTheir hydrochloride salts.

As used herein, the term "heparin-like compound" refers to heparin, derivatives or analogs thereof, or salts thereof. Examples of heparin analogues and derivatives include, but are not limited to, substituted heparin such as low molecular heparin derivatives such as enoxaparin, dalteparin, tinzaparin. Salts that may be mentioned include, but are not limited to, heparin sodium and lithium salts, calcium salts and salts of substituted heparin. In some preferred embodiments, the nicotinamide compound is nicotinamide. In some preferred embodiments, the heparin-like compound is heparin or heparin sodium.

In some embodiments, the cardiomyocyte culture medium according to the present invention further optionally comprises at least one component selected from the group consisting of: glutamine or its derivatives (such as glutamine, glutaMaX-1, L-glutamine and L-alanyl-L-glutamine), ascorbic acid, its derivatives or salts thereof (such as ascorbic acid, magnesium ascorbate salt, sodium ascorbate salt, ascorbyl glucoside, 3-ethyl ascorbic acid, ascorbyl tetraisopalmitate, ascorbyl palmitate, ascorbyl phosphate, etc.), pyruvate (such as sodium pyruvate or calcium pyruvate), selenite (such as sodium selenite or calcium selenite), thioglycerol (MTG), and ethanolamine.

In some preferred embodiments, the cardiomyocyte culture medium according to the present invention comprises a basal medium and a nicotinamide compound, human Platelet Lysate (PLT), heparin compound, insulin, human transferrin, human Serum Albumin (HSA), and any one or more components selected from the group consisting of: thioglycerol (MTG), pyruvate, ethanolamine, ascorbic acid, selenite, glutamax.

In some more preferred embodiments, the cardiomyocyte culture medium according to the present invention comprises a basal medium and the following components: nicotinamide, human Platelet Lysate (PLT), heparin or heparin sodium, insulin, human transferrin, human Serum Albumin (HSA), thioglycerol (MTG), pyruvate, ethanolamine, ascorbic acid, selenite, glutamax; or consist of the above components.

The cardiomyocyte culture medium according to the present invention comprises a basal medium. Basal media known in the art to be suitable for maintaining cardiomyocytes can be used in the present invention. Preferably, the basal medium is a serum-free and animal-derived component-free medium with defined chemical components, such as DMEM, DMEM/F12, ham's F12, RPMI-1640, IMDM, BME, IMDM/F12, alpha-MEM or combinations thereof. In a preferred embodiment, the basal medium is a 1:1 mixture of DMEM/F12 and IMDM.

The concentration of the supplement ingredients other than the basal medium is not particularly limited as long as it does not interfere with the effects of the present invention.

In one or more embodiments, the concentration of nicotinamide may be, for example, 0.5-20mM, such as about 0.5mM, about 1mM, about 2mM, about 3mM, about 4mM, about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, about 11mM, about 12mM, about 13mM, about 14mM, about 15mM, about 16mM, about 17mM, about 18mM, about 19mM, about 20mM, or may be used at a concentration falling within a range between any two of the recited values, such as 5-20mM, 10-20mM, or preferably at about 1-20mM, 1-10mM, 1-8mM, or more preferably at 1-5mM, based on the total amount of cardiomyocyte media of the present invention. When other nicotinamide-based compounds are used, the concentration of the compounds may be an equivalent concentration determined in comparison to the nicotinamide concentration described above.

In one or more embodiments, the concentration of heparin or heparin sodium may be, for example, 0.1-100 μg/mL, such as about 0.1 μg/mL, about 0.5 μg/mL, about 1 μg/mL, about 5 μg/mL, about 10 μg/mL, about 15 μg/mL, about 20 μg/mL, about 25 μg/mL, about 30 μg/mL, about 35 μg/mL, about 40 μg/mL, about 45 μg/mL, about 50 μg/mL, about 55 μg/mL, about 60 μg/mL, about 65 μg/mL, about 70 μg/mL, about 75 μg/mL, about 80 μg/mL, about 85 μg/mL, about 90 μg/mL, about 95 μg/mL, or about 100 μg/mL, or may fall within a range of, for example, between any two of the foregoing values of about 10 μg/mL, preferably between about 10 μg/mL, or between about 0.50 μg/mL. When other heparin-like compounds are used, the compound may be used at an equivalent concentration determined in comparison to the heparin or heparin sodium concentration described above.

In one or more embodiments, the concentration of human Platelet Lysate (PLT) may be, for example, 0.1% -10% v/v, e.g., about 0.1%, about 0.5%, about 1.1%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 3.5%, about 4.0%, about 4.5%, about 5.0%, about 5.5%, about 6.0%, about 6.5%, about 7.0%, about 7.5%, about 8.0%, about 8.5%, about 9.0%, about 9.5%, about 9.6%, about 9.8%, about 9.9% or 10% (v/v), or may be a concentration falling within a range between any of the two values, e.g., 0.5-7% (v/v) or 1-5% (v/v), preferably about 4.5-v).

In one or more embodiments, the concentration of insulin may be, for example, 0.1-20 μg/mL, e.g., about 0.1 μg/mL, about 0.2 μg/mL, about 0.3 μg/mL, about 0.4 μg/mL, about 0.5 μg/mL, about 0.7 μg/mL, about 0.8 μg/mL, about 0.9 μg/mL, about 1 μg/mL, about 2 μg/mL, about 3 μg/mL, about 4 μg/mL, about 5 μg/mL, about 6 μg/mL, about 7 μg/mL, about 8 μg/mL, about 9 μg/mL, about 10 μg/mL, about 11 μg/mL, about 12 μg/mL, about 13 μg/mL, about 14 μg/mL, about 15 μg/mL, or any value falling between about 5 μg/mL, preferably between about 15 μg/mL, about 17 μg/mL, about 15 μg/mL, or about 20 μg/mL, or any value may fall between about 1 μg/mL, about 15 μg/mL, about 17 μg/mL, or about 17 μg/mL.

In one or more embodiments, the concentration of human transferrin may be, for example, 1-100 μg/mL, preferably 1-20 μg/mL, for example, about 1 μg/mL, about 5 μg/mL, about 10 μg/mL, about 15 μg/mL, about 20 μg/mL, or may be a concentration falling in a range between any two of the foregoing values, for example 5-15 μg/mL, preferably about 1-15 μg/mL, based on the total amount of cardiomyocyte media of the present invention.

In one or more embodiments, the concentration of Human Serum Albumin (HSA) may be, for example, 0.2-20mg/mL, based on the total amount of cardiomyocyte media of the present invention, e.g., about 0.2mg/mL, about 1.0mg/mL, about 1.6mg/mL, about 1.8mg/mL, about 2.2mg/mL, about 2.8mg/mL, about 3.0mg/mL, about 3.6mg/mL, about 4.0mg/mL, about 4.6mg/mL, about 5.0mg/mL, about 5.6mg/mL, about 6.0mg/mL, about 6.6mg/mL, about 7.0mg/mL, about 7.6mg/mL, about 8.0mg/mL, about 8.6mg/mL, about 9.0mg/mL, about 9.6mg/mL, about 10.0mg/mL, about 10.6mg/mL, about 11.0mg/mL, about 11.6mg/mL, about 12.0mg/mL, about 12.6mg/mL, about 13.0mg/mL, about 13.6mg/mL, about 16.16 mg, about 15 mg/mL, about 16.0mg/mL, about 15 mg/mL, about 16.16 mg/mL, about 15 mg/mL, about 15.0mg/mL, about 15 mg/mL, about 16.0mg/mL, about 15 mg/mL, about 15.0mg/mL, about 15 mg/mL, about 15.0mg/mL, about 16.0mg/mL, about 17.0mg/mL, or the two of the drug.

In one or more embodiments, the concentration of thioglycerol (MTG) can be, for example, 1-200. Mu.M, for example, about 1. Mu.M, about 5. Mu.M, about 10. Mu.M, about 15. Mu.M, about 20. Mu.M, about 25. Mu.M, about 30. Mu.M, about 35. Mu.M, about 40. Mu.M, about 45. Mu.M, about 50. Mu.M, about 55. Mu.M, about 60. Mu.M, about 80. Mu.M, about 100. Mu.M, about 110. Mu.M, about 120. Mu.M, about 130. Mu.M, about 140. Mu.M, about 150. Mu.M, about 160. Mu.M, about 170. Mu.M, about 180. Mu.M, about 190. Mu.M, about 200. Mu.M, or a concentration falling within a range between any two of the foregoing values, for example, 10-200. Mu.M or 50-150. Mu.M, based on the total amount of cardiomyocyte media of the present invention.

In one or more embodiments, the concentration of ascorbic acid or a derivative thereof or salt thereof may be, for example, 10-100 μg/mL, for example, about 10 μg/mL, about 20 μg/mL, about 30 μg/mL, about 40 μg/mL, about 50 μg/mL, about 60 μg/mL, about 70 μg/mL, about 80 μg/mL, about 90 μg/mL, about 100 μg/mL, or may be a concentration falling within a range between any two of the above, for example, 50-100 μg/mL, especially about 80 μg/mL, based on the total amount of cardiomyocyte culture medium of the present invention.

In one or more embodiments, pyruvate (e.g., sodium salt) may be used at a concentration of, for example, 10-200 μg/mL, e.g., about 40 μg/mL, about 50 μg/mL, about 60 μg/mL, about 70 μg/mL, about 80 μg/mL, about 90 μg/mL, about 100 μg/mL, about 110 μg/mL, about 120 μg/mL, about 130 μg/mL, about 140 μg/mL, about 150 μg/mL, about 160 μg/mL, about 170 μg/mL, about 180 μg/mL, about 190 μg/mL, about 200 μg/mL, or at a concentration falling within a range between any two of the foregoing values, e.g., 50-200 μg/mL, especially about 110 μg/mL, based on the total amount of cardiomyocyte media of the present invention.

In one or more embodiments, the concentration of selenite (e.g., sodium salt) may be, for example, 1-50ng/mL, e.g., about 1ng/mL, about 5ng/mL, about 10ng/mL, about 15ng/mL, about 20ng/mL, about 25ng/mL, about 30ng/mL, about 35ng/mL, about 40ng/mL, about 45ng/mL, about 46ng/mL, about 47ng/mL, about 48ng/mL, about 49ng/mL, or about 50ng/mL, or may be a concentration falling within a range between any two of the foregoing values, e.g., 5-40ng/mL, based on the total amount of cardiomyocyte media of the present invention.

In one or more embodiments, the concentration of ethanolamine may be, for example, 1 to 50 μm, based on the total amount of cardiomyocyte culture medium of the present invention, e.g., about 5. Mu.M, about 6. Mu.M, about 7. Mu.M, about 8. Mu.M, about 9. Mu.M, about 10. Mu.M, about 11. Mu.M, about 12. Mu.M, about 13. Mu.M, about 14. Mu.M, about 15. Mu.M, about 16. Mu.M, about 17. Mu.M, about 18. Mu.M, about 19. Mu.M, about 20. Mu.M, about 21. Mu.M, about 22. Mu.M, about 23. Mu.M, about 24. Mu.M, about 25. Mu.M, about 26. Mu.M, about 27. Mu.M, about 28. Mu.M, about 29. Mu.M, about 30. Mu.M, about 31. Mu.M, about 32. Mu.M, about 33. Mu.M, about 34. Mu.M, about 35. Mu.M, about 36. Mu.M, about 37. Mu.M, about 38. Mu.M, about 39. Mu.M, about 40. Mu.M, about 41. Mu.M, about 42. Mu.M, about 43. Mu.M, about 44. Mu.M, about 45. Mu.M, about 50. Mu.M, or any of these may fall between the two values in the range of about 50. Mu.M, about 50. M, or about 50. Mu.M.

In one or more embodiments, the concentration of glutamine or a derivative thereof (such as Glutamax) based on the total amount of cardiomyocyte media of the present invention can be, for example, 0.5-5.0% v/v, e.g., 0.5% v/v, 1.0% v/v, 1.2% v/v, 1.4% v/v, 1.6% v/v, 1.8% v/v, 2.0% v/v, 2.2% v/v, 2.4% v/v, 2.6% v/v, 2.8% v/v, 3.0% v/v, 3.2% v/v, 3.4% v/v, 3.6% v/v, 4.0% v/v, 4.2% v/v, 4.6% v/v, 4.8% v/v, approximately 5.0% v, or any value falling between any two of these values, e.g., 0.0-5% v or 0.0.2% v.

(2) Cardiomyocyte maintenance medium

The invention also provides a cardiomyocyte maintenance medium comprising a cardiomyocyte culture medium as a basal medium and comprising a WNT signaling pathway activator as a supplement.

The cardiomyocyte culture medium used in the cardiomyocyte maintenance medium of the present invention may be a cardiomyocyte culture medium according to the present invention as described in any of the embodiments herein (including the above).

Herein, the term "WNT signaling pathway activator" refers to an agonist of the WNT signaling pathway. WNT signaling pathway activators may include GSK3 inhibitors. GSK3 inhibitors may include, for example, but are not limited to, polynucleotides, polypeptides, and small molecules. Exemplary GSK3 inhibitors include, for example, but are not limited to, kenpullone, 1-Azakenpaullone, CHIR99021, CHIR98014, NP031112, TWS119, AZD2858, AZD1080, SB415286, LY2090314, AR-A014418, CT 99021, CT 20026, SB216763, AR-A014418, TDZD-8, BIO-Acetoxime (5-methyl-1H-pyrazol-3-yl) - (2-phenylquinazolin-4-yl) amine, pyridocarbazole-cyclopentadienyl ruthenium complex, TDZD-8 (4-benzyl-2-methyl-1, 2, 4-thiadiazolidine-3, 5-dione), 2-thio (3-iodobenzyl) -5- (1-pyridinyl) [1,3,4] -oxadiazole, OTZT, alpha-4-dibromoacetophenone, AR-AO 144-18, 3- (1- (3-hydroxypropyl) -1H-pyrrolo [2,3-b ] pyridin-3-yl ] -4-pyrazin-2-yl-2, 5-dione, TWS119 (pyrrolopyrimidine compound), L (H-KEAPPAPPQSPP-NH 2) or a myristylated form thereof, 2-chloro-1- (4, 5-dibromothiophen-2-yl) -ethyl ketone, OTD-10920, and preferably, ZT-10920, GSK3 inhibitor is Kenpullone, 1-Azakenpaullone, CHIR99021, CHIR98014, NP031112, TWS119, AZD2858 AZD1080, SB415286, LY2090314, AR-A014418, SB216763, AR-A014418, BIO-Acetoxime, most preferably, the GSK3 inhibitor is CHIR99021, a (5-methyl-1H-pyrazol-3-yl) - (2-phenylquinazolin-4-yl) amine, 2-thio (3-iodobenzyl) -5- (1-pyridinyl) [1,3,4] -oxadiazole, α -4-dibromoacetophenone, AR-AO 144-18, 3- (1- (3-hydroxypropyl) -1H-pyrrolo [2,3-b ] pyridin-3-yl ] -4-pyrazin-2-yl-2, 5-dione, 2-chloro-1- (4, 5-dibromothiophen-2-yl) -ethanone, and gf109203x.

In any of the embodiments described above, the WNT signaling pathway activator may be added to the myocardial maintenance medium in accordance with the invention at any suitable concentration that promotes cardiomyocyte differentiation and proliferation. The concentration of WNT signaling pathway activator may be 0.5-6. Mu.M, such as 1. Mu.M, 2. Mu.M, 3. Mu.M, 4. Mu.M, 5. Mu.M, 6. Mu.M, based on the total amount of cardiomyocyte maintenance medium. Preferably, the cardiomyocyte maintenance medium according to the present invention comprises 0.5-6. Mu.M of GSK-3 inhibitor, more preferably 0.5-4. Mu.M.

Methods for producing cardiomyocytes from PSC

In yet another aspect, the invention also provides a method for producing Induced Cardiomyocytes (iCM) from Pluripotent Stem Cells (PSC). The method of the invention comprises the following steps:

(b) Culturing the cardiomyocyte precursor cells obtained in step (a) in a cardiomyocyte maintenance medium according to the present invention, thereby forming immature cardiomyocytes; and optionally

The method according to the invention may be performed under two-dimensional (2D) or three-dimensional (3D) culture conditions. The method according to the present invention is preferably performed under two-dimensional (2D) culture conditions because of the 2D culture mode of adherence induced differentiation, which is more advantageous in handling and observing the state of cells, and in culturing in an immobilized state, the efficiency of induced differentiation can be further improved.

The steps of the method of the present invention may be carried out under suitable culture temperature conditions. Suitable culture temperatures can be routinely determined by those skilled in the art. For example, step a, step b and/or step c may be performed at, for example, about 35-37 ℃.

The pluripotent stem cells useful in the methods of the invention may be any stem cell having the potential to differentiate to produce three germ layers (endoderm, ectoderm, mesoderm). Pluripotent stem cells are also abbreviated herein as PSC. In the method of the present invention, preferably, the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.

Ipscs for use in the methods of the invention may also be obtained from commercially available cell lines, or may also be prepared from: bone marrow cells, epithelial cells, endothelial cells, fibroblasts, peripheral blood mononuclear cells, hematopoietic cells, keratinocytes, hepatocytes, intestinal cells, mesenchymal cells, bone marrow precursor cells, and spleen cells.

In some embodiments, the pluripotent stem cells used in the methods of the invention include Embryonic Stem Cells (ESCs) or Induced Pluripotent Stem Cells (iPSCs), and may be of any suitable mammalian origin, including human, as well as mouse, pig, non-human primate, etc., particularly human-derived iPSCs. In some embodiments, the pluripotent stem cells used in the invention may be genetically modified or not before or after inducing differentiation according to the methods of the invention.

In other embodiments, multipotential stem cells may be multipotential validated prior to use in the present invention. Methods for PSC pluripotency verification are known in the art, including, but not limited to, detection of expression of pluripotency markers and the ability to form teratomas comprising inner, middle, and outer three germ layers in vivo.

Fig. 1 shows an exemplary flow of the method according to the invention. As shown in fig. 1, the method according to the invention comprises: hpscs differentiate first into cardiomyocytes (also referred to herein as pre-differentiation and P0 phase), which then differentiate further into immature cardiomyocytes (also referred to herein as P1 and P2 phase), which mature further into mature cardiomyocytes (also referred to herein as P3 phase).

Early differentiation (pluripotent stem cell culture)

Prior to initiation of differentiation (e.g., about D-2 to D0 days of differentiation), an adherent PSC culture or embryoid body for initiating the differentiation process can be obtained by suitable two-dimensional (2D) or three-dimensional (3D) cell culture conditions.

In some embodiments, the undifferentiated PSC cells are subjected to 2D culture to obtain an adherent PSC cell culture prior to initiation of differentiation. The PSC cell seeding density for 2D culture can be any suitable density. In some preferred embodiments, the inoculation density is 1-5x 10 ⁴ /cm ² . Undifferentiated PSCs can be inoculated onto feeder cells-free substrates for 2D culture. Suitable matrices include, but are not limited to, laminin, fibronectin, vitronectin, proteoglycans, entactin, collagen I, collagen IV, collagen VIII, heparan sulfate, matrigel ^TM Or any combination thereof. Matrigel ^TM Available from commercial sources, such as BD Biosciences. Laminin (including recombinant human laminin) may be used as Matrigel ^TM Is an alternative to (c). In some casesIn a preferred embodiment, PSC cells are seeded in Matrigel ^TM 2D culture was performed thereon. The incubation time of the undifferentiated PSC on the substrate after inoculation can be of any suitable length, but is typically 1-3 days, e.g., about 2 days, to obtain an adherent PSC culture of suitable density (e.g., about 50-70% confluence, preferably about 60% confluence).

In other embodiments, the undifferentiated PSC cells are 3D cultured to obtain Embryoid Bodies (EBs) prior to initiation of differentiation. Devices for three-dimensional culture include, for example, culture flasks, spinner flasks, shake bioreactors, or cell culture bags. Suspension culture conditions suitable for the formation of three-dimensional embryoid bodies from single cell ipscs are known in the art. In one or more embodiments, single cell PSCs are shake-suspended in culture flasks (e.g., T25 flasks). In some embodiments, the oscillation speed is 10-100rpm. In some embodiments, the incubation time is 8-32 hours. The size and morphology of the EB obtained can be monitored during the culture and the culture time adjusted as appropriate. In some preferred embodiments, T25 flasks are used to inoculate a cell density of 0.1X10 ⁶ -5×10 ⁶ PSC cells were cultured for 8-32 hours at 10-20 rpm/mL to obtain embryoid bodies.

Where the PSCs obtained are adherent cells grown as colonies or larger aggregates prior to the 2D or 3D culture described above, the PSCs are preferably dissociated from the solid surface or dispersed from the aggregates to facilitate subsequent differentiation uniformity of the PSC cells. Methods that can be used for PSC harvesting include, but are not limited to, enzymatic digestion, non-enzymatic cleavage, and mechanical harvesting. In some embodiments, PSCs are harvested using enzymatic digestion. Collagenase, dispase, or a combination thereof can be used to obtain PSC minipellets. Alternatively, a trypsin-like enzyme (e.g., tripLE) or accutase treatment can be used to obtain PSC single cell suspensions. In the method according to the invention, preferably the harvested PSC is an agglomerate having no more than 200 cells, 10-20 cells, or no more than 6-10 cells, more preferably a single cell suspension.

The medium suitable for pre-differentiation PSC culture can be any suitable pluripotent stem cell maintenance medium including, but not limited to, teSR1, essential 8. In some embodiments, a suitable small molecule compound Rock inhibitor may be added to the medium to promote cell attachment. ROCK inhibitors useful in the present invention include, but are not limited to: blebbistatin, thiazovivin, Y27632, fabauil, AR122-86, Y27632H-1152, Y-30141, wf-536, HA-1077, hydroxy-HA-1077, GSK269962A, SB-772077-B, N- (4-pyridyl) -N' - (2, 4, 6-trichlorophenyl) urea, 3- (4-pyridyl) -1H-indole, and (R) - (+) -trans-N- (4-pyridyl) -4- (1-aminoethyl) -cyclohexanecarboxamide. Preferably, the ROCK inhibitor used in the present invention is Blebbistatin or Y27632. The ROCK inhibitor may be added to the medium at any suitable concentration, for example, 0.5-20 μm,1-15 μm,5-15 μm, preferably about 10 μm Blebbbistatin.

In some preferred embodiments, a single cell suspension of undifferentiated PSCs is produced about 1-3 days prior to differentiation and at about 1-5x 10 ⁴ /cm ² To obtain an adherent PSC cell culture for differentiation, preferably said culture has a confluency of about 50-70%, for example about 60%.

Myocardial precursor cell differentiation stage (also called P0 stage)

After obtaining an adherent undifferentiated PSC culture or embryoid body of suitable density, pluripotent stem cells are induced to differentiate toward cardiomyocyte precursor cells in a cardiomyocyte differentiation medium according to the method of the present invention. The cardiomyocyte differentiation medium may be a conventional medium. For example, a medium containing a basal medium, ascorbic acid, and HSA (human serum albumin) can be used. Suitable basal media include, but are not limited to, DMEM, DMEM/F12, ham's F12, RPMI-1640, IMDM, BME, IMDM/F12, alpha-MEM or combinations thereof. In some preferred embodiments, the basal medium is DMEM/F12. The differentiation medium may or may not contain insulin. In the presence of insulin, insulin may be added at a pre-stage of the differentiation phase of the cardiac precursor cells, such as day 1. The cardiomyocyte differentiation medium may further comprise heparin sodium. In some embodiments, the differentiation medium comprises one or more, or preferably all, selected from heparin sodium, ascorbic acid, human serum albumin, and insulin. For the concentration of ascorbic acid, HSA and insulin, one skilled in the art can readily determine, for example, from 10 to 100. Mu.g/mL ascorbic acid, from 0.1 to 2mg/mL, preferably from 0.1 to 1mg/mL HSA and from 0.1 to 20. Mu.g/mL insulin. In some embodiments, the concentration of heparin sodium may be in the range of 0.5-10 μg/ml. To enhance the directed differentiation towards myocardial precursor cells, WNT signaling pathway activators and WNT signaling pathway inhibitors may be used in combination in the methods of the invention to provide fine control of the differentiation process.

In this cardiomyocyte precursor cell differentiation stage, a WNT signaling pathway activator that may be used may be GSK3 inhibitors, such as, but not limited to, CHIR99021, CHIR98014, SB216763, AT7519, AZD2858, and the like. Preferably CHIR99021 is used. The concentration of the GSK3 inhibitor in the differentiation medium is not particularly limited as long as it can block GSK 3. For example, the concentration of CHIR99021 in the differentiation medium may be, for example, 1 to 10 μm, such as 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, and is not limited thereto, preferably about 2 to 6 μm.

The WNT inhibitor used in this differentiation stage of cardiac muscle precursor cells is a substance that can inhibit WNT signaling pathway. Examples of WNT inhibitors that may be used include, but are not limited to, IWP2, IWR1, KY02111, XAV939, and the like. Preferably, the WNT inhibitor used is XAV939. The concentration of the WNT inhibitor in the differentiation medium is not particularly limited as long as it can block the WNT signaling pathway. For example, the concentration of XAV939 in the differentiation medium may be, for example, 1 to 10. Mu.M, such as 1. Mu.M, 2. Mu.M, 3. Mu.M, 4. Mu.M, 5. Mu.M, 6. Mu.M, 7. Mu.M, 8. Mu.M, 9. Mu.M, 10. Mu.M, and is not limited thereto, preferably about 2 to 6. Mu.M.

In some embodiments, step (a) of the method according to the invention comprises: in a cardiomyocyte differentiation medium, PSC is first cultured for a period of time in the presence of WNT signaling pathway activator, and then PSC is cultured for a period of time in the presence of WNT signaling pathway inhibitor. Herein, the period of time in which the WNT signaling pathway activator is present in the differentiation medium is also referred to as the "WNT signaling pathway activation period"; the period of time in which the WNT signaling pathway inhibitor is present in the differentiation medium is also referred to as "WNT signaling pathway inhibition period". In general, the WNT signaling pathway activator does not overlap with the WNT signaling pathway inhibitor's time of presence in the differentiation medium. In some embodiments, there is no interval between the WNT signaling pathway activation period and the WNT signaling pathway inhibition period, or in other embodiments about 1-2 days, e.g., about 1 day, between the two. The WNT signaling pathway activation period and WNT signaling pathway inhibition period may be of any suitable length. In some embodiments, the WNT signaling pathway is activated for a period of 1-2 days. In other embodiments, the WNT signaling pathway inhibition period is 2-3 days. In a preferred embodiment, the WNT signaling pathway activator is added to the differentiation medium on days D0-D1 of differentiation; and WNT inhibitor was added to the differentiation medium at D2-D4 days. In addition, the timing of the addition of WNT signaling pathway activators and WNT signaling pathway inhibitors may be adjusted as appropriate by monitoring cells in the differentiation process. Thus, other suitable timing schemes for the addition of WNT signaling pathway activators and WNT signaling pathway inhibitors are also within the contemplation of the invention.

Maintenance period (period P1 to period P2)

The cardiomyocyte precursor cells obtained through the aforementioned differentiation stage are further specialized and expanded to immature cardiomyocytes in the cardiomyocyte-maintaining medium according to the present invention.

In some embodiments according to the invention, the cardiomyocyte culture for the maintenance phase comprises culturing on a substrate, e.g., culturing the cells for at least about 18-22 days, and preferably including at least one dissociation and re-seeding step therebetween, to control the cell culture at a density suitable for cell expansion. In one or more embodiments, the culturing comprises the steps of: (i) Myocardial precursor cells (e.g., cells differentiated for about 9 days) are dissociated to obtain a single cell suspension, seeded on feeder-free substrates, and cultured for a period of time, e.g., 6-7 days. In one or more embodiments, the culturing further comprises the steps of: (ii) Cells that were pre-cultured (e.g., cells that differentiated for about 15 days to about 16 days, preferably for about 16 days) were dissociated to obtain a single cell suspension, seeded on feeder-free substrates, and cultured for a period of time, e.g., for about 14 days to about 15 days.

Suitable matrices for maintenance-phase cardiomyocyte culture include, but are not limited to, laminin, fibronectin, vitronectin, proteoglycans, entactin, collagen I, collagen IV, collagen VIII, heparan sulfate, or any combination thereof, such as VTN or Matrigel ^TM 。

In one embodiment, the cardiomyocytes are cultured on Vitronectin (VTN) plates. In one or more embodiments, steps (i) and (ii) comprise dissociating the cells with a cell dissociation solution. In one or more embodiments, the cell dissociation solution is Accutase or TrypLE. In one or more embodiments, the seeding density in steps (i) and (iii) is preferably a low density, e.g. 1-5x 10 ⁴ Individual cells/cm ² . In one or more embodiments, steps (i) and (iii) comprise seeding the cells in the presence of a ROCK inhibitor. In one or more embodiments, the ROCK inhibitor is Blebbistatin, and preferably the concentration of ROCK inhibitor in the cardiomyocyte maintenance medium is 5-20 μm, e.g. 10 μm.

In one or more embodiments, cardiomyocyte maintenance medium according to the present invention is subjected to a pipetting operation every time interval (e.g. 1 or 2 days) during the maintenance period.

The purity of the differentiated cell population center myocytes can be determined by analyzing the CTNT positive phenotype ratio of the cells using, for example, flow cytometry as described in the examples. In some embodiments, the purity of the cardiomyocytes in the obtained population of immature cardiomyocytes (e.g. D30 cells) is greater than 90%, 91%, 92%, 93% or 95%.

The multiple of cardiomyocyte expansion can be determined using conventional methods for determining the proportion of cardiomyocyte expansion. In some embodiments, the number of immature cardiomyocytes (e.g. D30 cells) is up to a factor of at least 30, e.g. 35-40, times the number of cardiomyocyte precursors (e.g. D9 cells).

Maturity (P3 stage)

At this stage of culture, the immature cardiomyocytes can be further matured in a cardiomyocyte maturation medium. The cardiomyocyte maturation medium used at this stage is not particularly limited. For example, conventional cardiomyocyte maturation media or other suitable cardiomyocyte maturation media may be used. Conventional cardiomyocyte maturation media include, for example, basal media supplemented with B27, such as DMEM/F12 media supplemented with B27, wherein the concentration of B27 can be 0.5-2 vol%. In a preferred embodiment, the cardiomyocyte culture medium according to the present invention may be used as a cardiomyocyte maturation medium for this stage. According to this embodiment, the maturation of cardiomyocytes can be further promoted.

In some embodiments, the cell culture at the maturation stage comprises culturing the cells in maturation medium for, e.g., 10-15 days. The degree of maturation of the induced cardiomyocytes can be determined by cell morphology or myocardial marker expression levels. Depending on factors such as maturity and/or cell number, the incubation time may be prolonged or shortened as appropriate.

In one or more embodiments, cardiomyocyte maturation medium is subjected to a pipetting operation at intervals (e.g. 1 or 2 days) during the maturation period.

In some embodiments, the mature cell culture comprises culturing cardiomyocytes on a substrate. In some embodiments, the culturing comprises: immature cardiomyocytes (e.g. cells differentiated for about 30 days) were dissociated to obtain a single cell suspension, seeded on a substrate and cultured for a period of time, e.g. 10-15 days. The matrix for the maturation period may be the same as the matrix for the maintenance period. Particularly preferred is the use of fibronectin as a maturation matrix, since fibronectin may enhance the maturation and biological functions of cardiomyocytes compared to other maturation matrices. In one or more embodiments, the single cell suspension of immature cardiomyocytes is obtained by dissociating cells using a cell dissociation solution. In one or more embodiments, the cell dissociation solution is Accutase or TrypLE. In one or more embodiments, the inoculation density in the culturing step is preferably low density, e.g., 1-5x 10 ⁴ Individual cells/cm ² . At one or more ofIn embodiments, the cells are seeded in the presence of a ROCK inhibitor. In one or more embodiments, the ROCK inhibitor is Blebbistatin, and preferably the ROCK inhibitor is used at a concentration of 5 to 20 μm, e.g., 10 μm, based on the total volume of medium.

Furthermore, it should be understood that although the foregoing stages of the differentiation and/or maturation process involved in the methods of the present invention, such stages of division do not preclude the possibility of two or more differentiation and/or maturation processes occurring simultaneously in the same stage.

III kit

In yet another aspect, the invention provides a kit useful for producing CM from PSCs, the kit comprising a cardiomyocyte culture medium of the invention. In some embodiments, the kit further comprises a WNT signaling pathway activator. The WNT signaling pathway activator may be placed in a separate container from the cardiomyocyte culture medium of the invention, or may be placed in the same container as a mixture or composition. Preferably, the WNT signaling pathway activator is provided in a separate container from the cardiomyocyte medium, as this provides greater flexibility (the cardiomyocyte medium can be used as the myocardial maturation medium alone or in combination with the WNT signaling pathway activator as the myocardial maintenance medium). In some embodiments, the invention also provides a kit comprising a myocardial maintenance medium of the invention.

In some embodiments, the cardiomyocyte culture medium or myocardial maintenance medium of the present invention may be contained in the same container in a kit in the form of a mixture or in separate containers in the kit in the form of the components they contain. As will be apparent to those of skill in the art, the components included in each individual container of the kits of the invention may be in any suitable form, for example, liquid or powder form.

Cell population

In yet another aspect, the invention also provides a population of cells. The cell population, without purification or enrichment, has an induced cardiomyocyte purity of greater than 90% (e.g. 91%, 92%, 93%, 94%) or even greater than 95%, or in other words, greater than 90%, preferably greater than 95% of the cells in the cell population are ctnt+ cardiomyocytes without purification or enrichment. Such cell populations may suitably be generated from pluripotent stem cells by a method according to the invention. In some embodiments, the cardiomyocyte expresses NKX2.5, MYH6, KCNH2, MLC2V, and MYH7. In some embodiments, the cardiomyocyte expresses NKX2.5, MYH6, KCNH2, MLC2V, MYH, and MLC2α.

Because the cell populations of the present invention have high CM purity, they are suitable for use in a variety of applications including, but not limited to, use in drug screening, teratogen and toxic compound assays, or therapeutic applications, such as in the preparation of cellular or pharmaceutical compositions.

The PSC-derived CM cells according to the present invention can be identified and characterized in a variety of ways, including cell morphology, expression of various cardiomyocyte markers, action potential, calcium transients associated with contractions, and in particular regular spontaneous beating, to thereby determine the cardiomyocyte characteristics, purity, maturity, etc. of the produced CM. The functional cells=or mature cardiomyocytes obtained according to the present invention are numerous and have a high degree of maturation.

In one embodiment, qPCR may be used to detect cell marker expression of a population of PSC-derived CM cells, including, but not limited to, common markers for cardiomyocytes (NKX 2.5 and MYH 6); markers for myocardial precursor cells (KCNH 2 and MLC2 a); and markers for mature cardiomyocytes (MLC 2V and MYH 7).

In one embodiment, the purity of cardiomyocytes can be characterized by detecting the expression of cell surface cardiac troponin T (CTNT) using flow cytometry. Flow cytometry evaluation is generally performed using antibodies specific for troponin T (CTNT, also known as TNNT 2) (clone 13-11). The PSC-derived cardiomyocyte population obtained by the method of the invention can reach a purity of more than 90%, even more than 95%, without purification or enrichment, as detected by flow cytometry.

In one embodiment, the cardiomyocyte expansion fold may be determined using a cardiomyocyte expansion ratio assay.

In one embodiment, the cardiomyocyte purity of the resulting iCM cell population can be identified using cellular immunofluorescent staining, e.g., by specific markers CTNT and/or actin staining; and/or determining the maturity of the cardiomyocytes by staining for cell surface specific markers MLC2V and MYH 7.

In another embodiment, the purity of the central myocytes of the cell population can also be determined by detecting the proportion of ctnt+ phenotype characteristic of the central myocytes of the cell population using flow cytometry on the obtained PSC-derived CM.

In another embodiment, the action potential of the iCM cells obtained may be detected by using patch clamp detection experiments to determine the functionality of the induced cardiomyocytes.

Examples

Characterization method

Cell morphology observations

Images CellSens Dimension were obtained using an Olympus IX73 microscope and cell morphology of PSCs at different times during differentiation was recorded.

Myocardial cell expansion ratio determination

Cells to be examined (such as D15-D16 (P1) and D30 (P2) cells) are digested into single cells, resuspended in a culture medium, and the number of cells is detected by a cell-taking-up machine (Vicell) and compared with the number of cells inoculated with cells (such as P0 (D9) cells) before expansion according to the detected number of cells.

RT-qPCR

Expression of cell center muscle markers differentiated from pluripotent stem cells PSC at different differentiation times was detected using RT-qPCR. Specifically, RNA was extracted using Kit RNAprep Pure Cell/Bacteria Kit (TIANGEN, cat. DP 430); reverse transcription was performed using kit Reverse Transcriptase (Vazyme, cat. Number R223-01); RT-qPCR was performed using a kit TransStart Top Green qPCR SuperMix (Transgen, cat. AQ 131) to determine the relative expression level of the cardiac marker genes tested.

Flow cytometry

Cell phenotypes were examined using flow cytometry using anti-CTNT antibodies (DSHB, #ct3). Cells grown by adherence were treated for flow cytometry detection. Cells were fixed with a fixation buffer of 2% paraformaldehyde (Sigma-Aldrich) at room temperature for 10 min, then permeabilized with 0.2% triton x100 (Sigma-Aldrich) for 10 min, FACS buffer washed once, the supernatant removed, and then cells were incubated with mouse monoclonal anti-cardiac troponin T (CTNT) antibody (DSHB) at 1:1000 dilution in FACS buffer for 1 hour at room temperature. After incubation they were washed once with FACS buffer, then incubated with donkey anti-mouse Alexa Fluor 647 (Invitrogen, carlsbad, CA) at a 1:1000 dilution for 1 hour, again washed 1 time with FACS buffer, and then analyzed for CTNT expression using a Beckman Cytoflex flow cytometer.

Cell immunofluorescence assay

Cell phenotype was detected by immunofluorescent staining, using the following immunofluorescent antibodies: anti-CTNT antibodies (DSHB, #ct3); anti-alpha-Actinin antibodies (abcam, #ab 137346); anti-MLC 2V antibody (Proteintech, # 10906-1-AP); alexa Fluor 488Donkey Anti-Mouse IgG (H+L) Anti-body (thermo filter, #A 11029); alexa Fluor594Donkey Anti-Rab IgG (H+L) Anti-body (Thermofiser, #A11012).

Taking cells to be detected, removing the complete culture medium, and washing the cells once by using PBS; then fixed with 2% paraformaldehyde (Sigma-Aldrich) for 10 min at room temperature, rinsing 3 cells with PBS at room temperature for 5 min each; the cells were permeabilized with 0.1% TritonX100 (Sigma-Aldrich) for 10 min, rinsed 3 times with PBS for 5 min at room temperature; blocking with PBS containing 2% sheep serum (Invitrogen) for 1 hour at room temperature; adding an anti-CTNT antibody (DSHB, #ct3); anti-alpha-Actinin antibodies (abcam, #ab 137346); anti-MLC 2V antibody (Proteintech, # 10906-1-AP) antibody 1:1000, incubating for 1 hour at room temperature; rinsing the cells 3 times with PBS at room temperature for 5 minutes each; adding the corresponding fluorescent secondary antibody 1:1000, incubating for 1 hour at room temperature in a dark place; the secondary antibody was blotted off and the cells were rinsed 3 times with PBS for 5 minutes at room temperature; DAPI staining solution 1 was added: incubation for 10 min at 5000 min at room temperature in the absence of light, rinsing the cells with PBS at room temperature for 5 min each time; the tablets were capped with a capping agent, and the results were observed under a fluorescence microscope (Olympus IX 73) and photographed.

Patch clamp detection

Part of the cells were seeded on a 48-well slide (pre-coated fibreonectin) at D30, cultured in an incubator until D45 was reached, and the cell slide was removed. Opening a patch clamp instrument for preheating, putting a cell climbing sheet into a patch clamp device, immersing a reference electrode in a dish, adjusting the distance under a low power mirror, straightening a proper electrode, flushing an electrode internal liquid, finding the electrode under a microscope after the electrode is filled with the liquid, adjusting the electrode to the cell side for sealing, adjusting parameters, slightly sucking a cell membrane by a mouth, clamping by voltage after membrane rupture, and recording the cell potential. Results were summarized using the Clampfit analysis software.

Example 1:

d-2 to D0 (early differentiation): pluripotent stem cell culture

Pluripotent stem cells were prepared according to the method of CN 108373998B. The pluripotent stem cells were subjected to pluripotent verification prior to differentiation culture, and tested for the expression of various pluripotent markers thereof, and for the ability to form teratomas comprising inner, middle and outer germ layers in immunodeficient mice. The validated pluripotent stem cells were routinely cultured in E8 maintenance medium.

About day-2 (D-2) prior to differentiation culture, undifferentiated pluripotent stem cells with a confluency of 70-80% were digested to complete single cells with TrypLE or Acceutase, resuspended in a suitable volume of pluripotent stem cell maintenance medium E8. Only 10. Mu.M Rock inhibitor Blebbbistatin (used to promote cell attachment) was added to the medium on day 1. At a cell seeding density of 2x 10 ⁴ /cm ² The cell suspension was seeded on Matrigel plates and placed at 37℃with 5% CO ₂ Culturing in an incubator with saturated humidity, and changing liquid every day.

D0 to D9 (phase P0): directional differentiation of myocardial precursor cells

When pluripotent stem cells were cultured to a confluency of 60%, PSC maintenance medium was aspirated, and cardiomyocyte differentiation medium having the composition shown in Table 1 below was added thereto, followed by further culturing at 37℃with 5% CO ₂ The culture was carried out in an incubator at a concentration and saturated humidity, and fresh medium was changed every day. CulturingThe amount of the base used is about 0.2-0.3mL/cm ² (culture dish bottom area). The small molecule GSK3 inhibitor CHIR99021 was added to the differentiation medium at a concentration of 5. Mu.M at days D0-D1. The small molecule WNT inhibitor XAV939 was added to the differentiation medium at a concentration of 5 μm at D2-D4 days.

Table 1: composition of cardiomyocyte differentiation medium

For a pair ofP0Characterization of phase differentiated cells

Characterization of the P0 phase differentiated cells shows that the differentiation of the pluripotent stem cells to the cardiomyocyte precursor cells can be accelerated and the effect is stable after the cardiomyocyte differentiation medium is added and the GSK3 inhibitor and the WNT inhibitor are used in combination on days 0-9 of differentiation.

Briefly, after addition of cardiomyocyte differentiation medium and combined use of GSK3 inhibitor and WNT inhibitor, specific marker expression of cardiomyocyte precursors was detected by RT-qPCR at day 9 and compared to PSC cells at day 0. The results are shown in FIG. 2 (upper left and upper right panels) and FIG. 3 (where NKX2.5, MYH6 are common markers for cardiomyocytes, KCNH2 is a marker for cardiomyocytes, and MLC2V, MYH7 is a marker for mature cardiomyocytes). The results demonstrate that the use of a cardiomyocyte differentiation medium in combination with a GSK3 inhibitor and a WNT inhibitor on days 0-9 induced pluripotent stem cells in a directed manner significantly increases the differentiation efficiency.

Flow cytometry examined the cell phenotype on day 9 of differentiation (D9) and compared to control ipscs. The results (see FIG. 4) indicate that the cells at day 9 of differentiation exhibited positive CTNT, a marker characteristic of cardiomyocytes, and the differentiation efficiency reached 87.27%. This means that the purity of the myocardial precursor cells in the cell population obtained by differentiation is high.

D9 to D30 (phase P1 andp2 phase): immature cardiomyocyte differentiation and expansion

The cells of D9 were digested into single cells and resuspended at a density in a suitable volume of cardiomyocyte-maintaining medium. Rock inhibitors were added to the medium only on day 1. The cell suspension was seeded on Matrigel plates (or VTN plates) and placed at 37℃in 5% CO ₂ The cardiomyocyte is cultured in an incubator with saturated humidity for 6-7 days, and the cardiomyocyte maintenance medium is changed every 2 days. The Rock inhibitor is Blebb statin at a concentration of 10. Mu.M; cell density of 1-5x 10 ⁴ /cm ² . The culture medium is used in an amount of 0.2-0.3mL/cm ² (culture dish bottom area).

At D15 to D16, the P1 phase cells were digested into single cells and resuspended at a density in a suitable volume of specific myocardial maintenance medium. Rock inhibitors were added to the medium only on day 1. The cell suspension was seeded on Matrigel plates (or VTN plates) and placed at 37℃in 5% CO ₂ The cardiomyocyte is cultured in an incubator with saturated humidity for 14-15 days, and the cardiomyocyte maintenance medium is changed every 2 days. The Rock inhibitor is Blebb statin at a concentration of 10. Mu.M; cell density of 1-5x 10 ⁴ /cm ² . The culture medium is used in an amount of 0.2-0.3mL/cm ² (culture dish bottom area).

The myocardial cell maintenance medium comprises basal medium CM-SFM and 2 mu MCHIR99021, wherein the CM-SFM comprises the following components: 50% (IMDM) (Sigma); 50% DMEM/F12 (1:1) (Gibco); 0.5-10mg/mL human serum albumin (HSA, sinopharm, CN); 10-150. Mu.M (e.g., 50. Mu.M) 1-thioglycerol (MTG, sigma); 80 μg/mL ascorbic acid (Sigma); 1-5mM (1 mM, 2mM, 3mM, 4mM or 5 mM) nicotinamide (NAM, sigma); 110 μg/mL sodium pyruvate (Sigma); 1-15 μg/mL insulin (baiting, CN); 1-20 μg/mL transferrin (Sigma); 5-40ng/mL sodium selenite (Sigma); 20. Mu.M ethanolamine (Sigma); 0.5% -4% (v/v) (0.5%, 1%, 2%, 3% or 4%) human platelet lysate (PLT, BI); 0.5-50 μg/mL (0.5, 1, 3, 10, 30, or 50 μg/mL) heparin sodium (Thermo); 1% (v/v) GlutaMAX-1 (Invitrogen).

For a pair ofP1-P2Characterization of phase differentiated cells

Cell morphology observations from day 9 to day 30 of differentiation showed that the precursor cells were specialized to immature cardiomyocytes, and that cell morphology became clearer, the number was significantly increased, and the beating was more regular from day 30. The results are shown in FIG. 5, which demonstrates the change in cell morphology and number on day 10, day 16, day 22 and day 30 of differentiation.

The expression level of specific markers of cardiomyocytes differentiated on day 9 and day 30 was detected by RT-qPCR and compared with pluripotent stem cell ipscs. The results are shown in FIG. 6 (where NKX2.5 and MYH6 are common markers for cardiomyocytes, KCNH2 is a marker for cardiomyocytes, and MLC2V, MYH is a marker for mature cardiomyocytes). The results showed that, after the addition of the cardiomyocyte maintenance medium, the precursor cells were promoted to differentiate specifically toward the immature cardiomyocytes on days 9-30 of differentiation.

Flow cytometry examined the cell phenotype on day 30 of differentiation (D30) and compared to control ipscs. The results (see FIG. 7) indicate that the cells at day 30 of differentiation exhibited positive CTNT, a marker characteristic of cardiomyocytes, and the differentiation efficiency reached 95% or more. This means that the purity of the myocytes in the center of the cell population obtained by differentiation was high.

Immunofluorescent staining of the cells at day 30 (D30) of differentiation (see fig. 8) demonstrated the phenotype of the resulting differentiated cells as: ctnt+/α -actinin+, MLC2v+. The overlap ratio of the first two specific markers reaches more than 95%, which further indicates that the differentiated cell population has high myocardial cell purity. The cellular immunofluorescent staining of the latter specific marker also shows that cardiomyocytes in the differentiated cell population have a high degree of maturation.

Further, the cardiomyocyte expansion ratio was measured from day 9 to day 30 of differentiation. Briefly, the number of cultured cells was determined as described in the characterization methods section above, at stage P0 before addition of cardiomyocyte maintenance medium (i.e. day 9 of differentiation) and at stages P1 and P2 after addition of cardiomyocyte maintenance medium; and the change in the expansion ratio of the cells at the differentiation stage P1 and the differentiation stage P2 was calculated with respect to the number of cells at day 9. As shown in fig. 9, by day D30, the cell expansion factor reached about 37 times relative to day D9.

The above results indicate that the use of the cardiomyocyte culture medium of the present invention as a basal medium for the cardiomyocyte maintenance medium in the P1 phase and the P2 phase significantly promotes the differentiation and expansion of cardiomyocyte precursors into immature cardiomyocytes, thereby obtaining a large number of immature cardiomyocytes with high purity (or high cell ratio).

D30 to D45 (P3 phase): further maturation of immature cardiomyocytes

The cells of D30 were digested into single cells with TrypLE or Ackutase, resuspended in a defined volume of myocardial maturation medium at a defined density, plated onto fibronectin (fibronectin) plates, and Rock inhibitor was added to the medium. Cells seeded on fibronectin plates were placed at 37℃with 5% CO ₂ Culturing in an incubator with saturated humidity for 10-15 days. The myocardial maturation medium was changed every 2 days during the culture. The Rock inhibitor is Blebb statin at a concentration of 10. Mu.M; cell density of 1-5x 10 ⁴ /cm ² . The culture medium is used in an amount of 0.2-0.3mL/cm ² (culture dish bottom area). The composition of the cardiomyocyte maturation medium described above is identical to the CM-SFM used in the differentiation and expansion stages of immature cardiomyocytes.

For a pair ofP2Characterization of mature cells

After addition of serum-free cardiomyocyte maturation medium (including serum substitutes), cardiomyocyte specific marker expression was detected by RT-qPCR at day 30 and day 45 of differentiation. The results are shown in FIG. 10 (where MLC2V and MYH7 are markers of mature cardiomyocytes; where pluripotent stem cells served as controls).

As can be seen from FIG. 10, on days 30-45, a significant increase in the expression level or maturity of the mature cardiomyocyte markers was obtained in a very short time using the cardiomyocyte culture medium of the present invention as a cardiomyocyte maturation medium. This shows that the use of the cardiomyocyte maturation medium of the present invention significantly promotes cardiomyocyte maturation.

The spontaneous action potential of the cardiomyocytes on day 45 was examined using patch clamp (see fig. 11), indicating that the differentiated mature cardiomyocytes had contractile function and were functional cardiomyocytes.

The experimental results show that after the cardiomyocyte maturation medium is added in the cardiomyocyte maturation stage, the cardiomyocyte maturation can be obviously promoted, so that the cardiomyocytes with high quantity, high maturation degree and functionality can be obtained.

Example 2:

to examine the effect of cardiomyocyte maintenance media using different basal media on the differentiation and expansion of immature cardiomyocytes, cardiomyocyte maintenance media were formulated containing 2 μm GSK3 inhibitor CHIR99021 and 3 different basal media, respectively. Cardiomyocyte maintenance medium 1 (control 1) contained CHIR99021 and basal medium 1 (DMEM/F12 medium supplemented with B27 supplement (1% by volume). Medium 2 (control 2) contained CHIR99021 and basal medium 2 (RPMI 1640 medium supplemented with B27 supplement (1% by volume). Medium 3 (i.e., the cardiomyocyte maintenance medium of the present invention as shown in example 1) comprises CHIR99021 and basal medium 3 (i.e., the cardiomyocyte culture medium CM-SFM of the present invention as shown in example 1).

Cardiomyocytes were generated from ipscs in a similar manner as described in example 1. Specifically, D9 cells were generated from ipscs. Thereafter, after digestion of the D9 cells into single cells, the cells were then grown at approximately 2X 10 ⁴ /cm ² Is added to the above 3 different media. It was found at day 7 after inoculation (see fig. 12) that the cardiomyocyte maintenance medium of the present invention using the cardiomyocyte culture medium of the present invention as a basal medium significantly promoted expansion of cardiomyocytes relative to the control medium (medium 1 and medium 2) using basal media 1 and 2; while the cardiomyocytes cultured in medium 1 and medium 2 had very limited expansion, although medium 2 using basal medium 2 was slightly better than medium 1 using basal medium 1. The results show that the cardiomyocyte culture medium provided by the invention can be used as a basic culture medium of a cardiomyocyte maintenance medium to remarkably promote the differentiation and expansion of cardiomyocyte precursors to immature cardiomyocytes.

Example 3:

to examine the effect of adding different concentrations of the GSK3 inhibitor CHIR99021 on the differentiation and expansion of immature cardiomyocytes to the same cardiomyocyte maintenance medium, 5 different media of different concentrations of the GSK3 inhibitor CHIR99021 were prepared. Medium 1 was CM-SFM medium without addition of CHIR 99021. Medium 2 was CM-SFM medium supplemented with 2. Mu.M CHIR 99021. Medium 3 was CM-SFM medium supplemented with 3. Mu.M CHIR 99021. Medium 4 was CM-SFM medium supplemented with 4. Mu. MCHIR 99021. Medium 5 was CM-SFM medium supplemented with 6. Mu.M CHIR 99021.

Cardiomyocytes were generated from ipscs in a similar manner as described in example 1. Specifically, D9 cells were generated from ipscs. Thereafter, after digestion of the D9 cells into single cells, the cells were then grown at approximately 1X 10 ⁴ /cm ² Is added to the above 5 different media. On day 7 after inoculation (see FIG. 13), CM-SFM medium without CHIR99021 (Medium 1) was found to be very limited for cardiomyocyte expansion, with medium 2-5 culturing more immature cardiomyocytes, and especially medium 2 culturing the most immature cardiomyocytes, significantly promoting cardiomyocyte expansion. The results indicate that lower concentrations of GSK3 inhibitor in the cardiomyocyte maintenance medium will generally favor cardiomyocyte differentiation and expansion.

Example 4:

to examine the effect of the matrix used for culture on cardiomyocyte maturation, 3 different matrices were tested at the myocardial maturation stage: 0.1% gelatin, vitronectin (VTN), fibronectin.

Cardiomyocytes were generated from ipscs in a similar manner as described in example 1. Specifically, D30 cells were generated from ipscs. The cells of D30 were digested into single cells with TrypLE or Ackutase, resuspended in an appropriate volume of the myocardial maturation medium CM-SFM of the invention at a density, plated on 0.1% gelatin, vitronectin, or fibronectin plates, and Rock inhibitor was added to the medium. Cells seeded on fibronectin plates were placed at 37℃with 5% CO ₂ Culturing in an incubator with saturated humidity for 10-15 days. The myocardial maturation medium of the present invention was changed every 2 days during the culture. The Rock inhibitor is Blebb statin at a concentration of 10. Mu.M; cell density of 1-5x 10 ⁴ /cm ² . CulturingThe amount of the base is 0.2-0.3mL/cm ² (culture dish bottom area). As a result (see FIG. 14), it was found that only on the fibronectin plate, the mature cardiomyocytes were long-spindle-shaped and continuously beating was evident.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims

1. A serum-free and animal-derived component-free cardiomyocyte culture medium comprising a basal medium and nicotinamide compound, human Platelet Lysate (PLT), heparin compound, insulin, human transferrin, and Human Serum Albumin (HSA).

2. The cardiomyocyte culture medium of claim 1 wherein the nicotinamide compound is nicotinamide, a derivative or analog thereof, or a salt thereof.

3. The cardiomyocyte culture medium of claim 1 wherein the heparin-like compound is heparin, a derivative or analog thereof, or a salt thereof.

4. A cardiomyocyte culture medium according to any of claims 1-3, wherein the concentration of said nicotinamide compound is 0.5-20mM, preferably 1-10mM, based on the total cardiomyocyte culture medium.

5. A cardiomyocyte culture medium according to any of claims 1-3, wherein the concentration of the heparin-like compound is 0.1-100 μg/mL, preferably 0.1-50 μg/mL, based on the total cardiomyocyte culture medium.

6. A cardiomyocyte culture medium according to any of claims 1-3, wherein the concentration of PLT is 0.1-10 vol.%, preferably 0.1-5 vol.%, based on the total cardiomyocyte culture medium.

7. A cardiomyocyte culture medium according to any of claims 1-3, wherein the concentration of insulin is 0.1-20 μg/mL, preferably 1-15 μg/mL, based on the total cardiomyocyte culture medium.

8. A cardiomyocyte culture medium according to any of claims 1-3 wherein the concentration of human transferrin is 1-100 μg/mL, preferably 1-20 μg/mL, based on the total cardiomyocyte culture medium.

9. The cardiomyocyte culture medium of any of claims 1-3, wherein the concentration of HSA is 0.2-20mg/mL, preferably 1-10mg/mL, based on the total cardiomyocyte culture medium.

10. The cardiomyocyte culture medium of any of claims 1-3, wherein the cardiomyocyte culture medium further comprises one or more or all of the components selected from the group consisting of: thioglycerol (MTG); pyruvate; ethanolamine; ascorbic acid, derivatives thereof or salts thereof; selenite; and glutamine or a derivative thereof.

11. The cardiomyocyte culture medium of claim 10 wherein the cardiomyocyte culture medium comprises:

(i)1-10mg/mL HSA；

(ii)10-200μM MTG；

(iii) 50-100 μg/mL ascorbic acid;

(iv) 1-10mM nicotinamide;

(v) 50-200 mug/mL pyruvate;

(vi) 1-15 μg/mL insulin;

(vii) 1-20 μg/mL human transferrin;

(viii) 5-40ng/mL selenite;

(ix) 5-50 mu M ethanolamine;

(x) 0.1-5% by volume PLT;

(xi) Heparin sodium of 0.1-50 mug/mL; and

(xii) 1.0-5.0% by volume Glutamax.

12. A cardiomyocyte culture medium according to any of claims 1 to 3 wherein the basal medium is selected from the group consisting of: DMEM, DMEM/F12, ham's F, RPMI-1640, IMDM, BME, IMDM/F12, alpha-MEM or combinations thereof.

13. A cardiomyocyte maintenance medium comprising the cardiomyocyte culture medium of any of claims 1-12 as a basal medium and comprising WNT signaling pathway activator as a supplement.

14. The cardiomyocyte maintenance medium of claim 13 wherein the WNT signaling pathway activator is a GSK3 inhibitor selected from the group consisting of CHIR99021, CHIR98014, NP031112, TWS119, AZD2858, AZD1080, SB415286, LY2090314, AR-a014418, SB216763, (5-methyl-1H-pyrazol-3-yl) - (2-phenylquinazolin-4-yl) amine, 2-thio (3-iodobenzyl) -5- (1-pyridinyl) [1,3,4] -oxadiazole, α -4-dibromoacetophenone, 3- (1- (3-hydroxypropyl) -1H-pyrrolo [2,3-b ] pyridin-3-yl ] -4-pyrazin-2-yl-pyrrole-2, 5-dione, 2-chloro-1- (4, 5-dibromothiophen-2-yl) -ethanone, and GF 109203X.

15. The cardiomyocyte maintenance medium of any of claims 13 to 14 wherein the concentration of WNT signaling pathway activator is 0.5-6 μm, preferably 0.5-4 μm, based on the total cardiomyocyte maintenance medium.

16. A method of producing Induced Cardiomyocytes (iCM) from Pluripotent Stem Cells (PSCs), the method comprising:

(b) Culturing the cardiomyocyte precursor cells obtained in step (a) in a cardiomyocyte maintenance medium according to any of claims 13-15, thereby forming immature cardiomyocytes; and optionally

17. The method of claim 16, wherein the cardiomyocyte maturation medium in step (c) is the cardiomyocyte medium of any of claims 1-12.

18. The method of claim 16 or 17, wherein the pluripotent stem cells are induced pluripotent stem cells (ipscs) or Embryonic Stem Cells (ESCs).

19. The method of claim 16 or 17, wherein step (a) comprises: in a cardiomyocyte differentiation medium, PSC is first cultured for a period of time in the presence of WNT signaling pathway activator, and then PSC is cultured for a period of time in the presence of WNT signaling pathway inhibitor.

20. The method of claim 19, wherein the WNT signaling pathway activator is CHIR99021 and the WNT signaling pathway inhibitor is XAV939.

21. The method of claim 16 or 17, wherein the cardiomyocyte precursors in step (b) are present at 1-5x10 ⁴ Individual cells/cm ² Is used for the inoculation of the cell density of the strain.

22. The method of claim 16 or 17, wherein the immature cardiomyocytes in step (c) are seeded on a fibronectin matrix.

23. The method of claim 16 or 17, wherein the method is performed under two-dimensional (2D) culture conditions.

24. A kit comprising the cardiomyocyte culture medium of any of claims 1 to 12.

25. The kit of claim 24, further comprising a WNT signaling pathway activator, wherein the WNT signaling pathway activator and the cardiomyocyte medium are provided in separate containers.

26. A population of cells obtained by the method of any one of claims 16-23.

27. The cell population of claim 26, wherein, without purification or enrichment, greater than 95% of the cells in the cell population are ctnt+ cardiomyocytes.