WO2024041645A1 - 用于制备心肌细胞的化合物及其用途 - Google Patents

用于制备心肌细胞的化合物及其用途 Download PDF

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WO2024041645A1
WO2024041645A1 PCT/CN2023/115013 CN2023115013W WO2024041645A1 WO 2024041645 A1 WO2024041645 A1 WO 2024041645A1 CN 2023115013 W CN2023115013 W CN 2023115013W WO 2024041645 A1 WO2024041645 A1 WO 2024041645A1
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structural
cdk8
cardiomyocytes
stem cells
pluripotent stem
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French (fr)
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赵扬
杨晓淳
岑梓牧
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北京大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

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  • the present invention relates to the field of biomedicine, especially the field of regenerative medicine. Specifically, the present invention relates to a compound for preparing cardiomyocytes and its use, as well as a method of using the compound to differentiate pluripotent stem cells, such as induced pluripotent stem cells, into cardiomyocytes.
  • Heart disease is one of the leading causes of death in humans, and cardiac cell therapy, in vitro myocardial disease modeling or drug development all require the support of large numbers of cardiomyocytes.
  • cardiomyocytes as terminally differentiated cells, cannot be regenerated in vivo, and primary cardiomyocytes are difficult to culture and expand in vitro. Therefore, the source of cardiomyocytes becomes a major issue.
  • induced pluripotent stem cells hiPSC
  • hiPSC-CM cardiomyocytes
  • hiPSC-CM The efficient, stable, and large-scale production of hiPSC-CM is crucial for its practical application, but the efficiency and quality of the current differentiation system are still unstable. Therefore, there remains a need in the art for new compounds and methods that can be used to efficiently generate functional cardiomyocytes from stem cells, such as induced pluripotent stem cells, through reprogramming, thereby treating cardiac diseases such as heart failure.
  • stem cells such as induced pluripotent stem cells
  • Embodiment 1 A method for preparing cardiomyocytes by differentiation of pluripotent stem cells, the method comprising:
  • ii) Cultivate the pluripotent stem cells in a basal medium supplemented with a CDK8 inhibitor and a WNT signaling pathway activator for approximately 24h to approximately 48h, and then culture the pluripotent stem cells in a basal medium without the addition of the CDK8 inhibitor and WNT signaling pathway activator. Continue to culture the cells for approximately 24 hours;
  • step iii) Culturing the cells obtained in step ii) in a basal medium supplemented with a WNT signaling pathway inhibitor for approximately 48 hours, and then continuing to culture the cells in a basal medium without the addition of a WNT signaling pathway inhibitor for approximately 24 hours;
  • step iv) culturing the cells obtained in step iii) in basal medium supplemented with insulin for approximately 3-6 days;
  • Embodiment 2 The method of embodiment 1, wherein the pluripotent stem cells are selected from embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC).
  • ESC embryonic stem cells
  • iPSC induced pluripotent stem cells
  • Embodiment 3 The method of embodiment 1 or 2, wherein the pluripotent stem cells in step i) are cultured in multi-well plates.
  • Embodiment 4 The method of any one of embodiments 1-3, wherein the pluripotent stem cells in step i) are grown to approximately 80-90% confluence.
  • Embodiment 5 The method of any one of embodiments 1-4, wherein the basal medium is RPMI medium and A combination of insulin-free B27 cell culture supplements.
  • Embodiment 6 The method of any one of embodiments 1-6, wherein the basal medium added with insulin in step iv) is a combination of RPMI medium (such as RPMI1640 medium) and B27 cell culture additives.
  • RPMI medium such as RPMI1640 medium
  • Embodiment 7 The method of any one of embodiments 1-6, wherein the CDK8 inhibitor is selected from the group consisting of BI-1347 or a structural analog thereof, MSC2530818 or a structural analog thereof, AS2863619 or a structural analog thereof, Senexin A or Its structural analogs, CCT-251921 or its structural analogs, Senexin C or its structural analogs, JH-XVI-178 or its structural analogs, CCT251545 or its structural analogs, BRD6989 or its structural analogs, JH-XI -10-02 or its structural analogues, SEL120-34A or its structural analogues, LY2857785 or its structural analogues, CDK8-IN-1 or its structural analogues, CDK8-IN-5 or its structural analogues, CDK8- IN-3 or its structural analogues, CDK8-IN-4 or its structural analogues, CDK8-IN-6 or its structural analogues, CDK8-IN-7
  • Embodiment 8 The method of any one of embodiments 1-7, wherein the WNT signaling pathway activator is CHIR99021 or a structural analog thereof, CHIR98014 or a structural analog thereof, or SB212763 or a structural analog thereof.
  • Embodiment 9 The method of any one of embodiments 1-8, wherein the WNT signaling pathway inhibitor is IWR1 or a structural analog thereof, IWP2 or a structural analog thereof, or IWP4 or a structural analog thereof.
  • Embodiment 10 The method of any one of embodiments 1-9, wherein the insulin is human insulin.
  • Embodiment 11 The method of any one of embodiments 1-10, wherein the final concentration of the CDK8 inhibitor, such as BI-1347 or a structural analog thereof, is about 0.001 ⁇ M to about 5 ⁇ M, preferably about 0.004 ⁇ M to about 3 ⁇ M, For example, about 0.1 ⁇ M, about 0.2 ⁇ M, about 0.3 ⁇ M, about 0.4 ⁇ M, about 0.5 ⁇ M, about 0.6 ⁇ M, about 0.7 ⁇ M, about 0.8 ⁇ M, about 0.9 ⁇ M, about 1 ⁇ M, about 2 ⁇ M, about 3 ⁇ M, about 4 ⁇ M, or about 5 ⁇ M.
  • the final concentration of the CDK8 inhibitor such as BI-1347 or a structural analog thereof.
  • Embodiment 12 The method of any one of embodiments 1-11, wherein the final concentration of the WNT signaling pathway activator such as CHIR99021 or a structural analog thereof is about 1 ⁇ M to about 25 ⁇ M or higher, such as about 1 ⁇ M, about 2 ⁇ M , about 3 ⁇ M, about 4 ⁇ M, about 5 ⁇ M, about 6 ⁇ M, about 7 ⁇ M, about 8 ⁇ M, about 9 ⁇ M, about 10 ⁇ M, about 11 ⁇ M, about 12 ⁇ M, about 13 ⁇ M, greater than 14 ⁇ M, about 15 ⁇ M, about 16 ⁇ M, about 17 ⁇ M, about 18 ⁇ M, about 19 ⁇ M, about 20 ⁇ M, about 21 ⁇ M, about 22 ⁇ M, about 23 ⁇ M, about 24 ⁇ M, about 25 ⁇ M or higher.
  • the final concentration of the WNT signaling pathway activator such as CHIR99021 or a structural analog thereof is about 1 ⁇ M to about 25 ⁇ M or higher,
  • Embodiment 13 The method of any one of embodiments 1-12, wherein the concentration of the WNT signaling pathway inhibitor is about 1 ⁇ M to about 10 ⁇ M or higher, such as about 1 ⁇ M, about 2 ⁇ M, about 3 ⁇ M, about 4 ⁇ M, about 5 ⁇ M, about 6 ⁇ M, about 7 ⁇ M, about 8 ⁇ M, about 9 ⁇ M, about 10 ⁇ M or higher.
  • Embodiment 14 The method of any one of embodiments 1-13, wherein the pluripotent stem cells are human pluripotent stem cells and the cardiomyocytes are human cardiomyocytes.
  • Embodiment 15 The method of any one of embodiments 1-14, wherein the cardiomyocytes are functional cardiomyocytes, eg, beating cardiomyocytes.
  • Embodiment 16 The method of any one of embodiments 1-15, wherein the cardiomyocyte expresses one or more cardiomyocyte-specific proteins selected from the group consisting of: cTNT, cTNI, ⁇ -ACTININ, MEF2C, and NKX2.5.
  • Embodiment 17 A cardiomyocyte prepared by the method of any one of embodiments 1-16.
  • Embodiment 18 A pharmaceutical composition comprising cardiomyocytes prepared by the method of any one of embodiments 1-16 and a pharmaceutically acceptable carrier.
  • Embodiment 19 Use of the cardiomyocytes of embodiment 17 or the pharmaceutical composition of embodiment 18 for the preparation of a medicament for the treatment of cardiac disease in a subject in need thereof.
  • Embodiment 20 A method of treating cardiac disease in a subject in need thereof, comprising administering to the subject the cardiomyocytes of Embodiment 17 or the pharmaceutical composition of Embodiment 18.
  • Embodiment 21 The use of embodiment 19 or the method of embodiment 20, wherein the subject is a human.
  • Embodiment 22 Use of a CDK8 inhibitor in the preparation of a reagent for preparing cardiomyocytes by pluripotent stem cell differentiation.
  • Embodiment 23 The use of embodiment 22, wherein the CDK8 inhibitor is selected from BI-1347 or a structural analog thereof, MSC2530818 or a structural analog thereof, AS2863619 or a structural analog thereof, Senexin A or a structural analog thereof, CCT -251921 or its structural analogues, Senexin C or its structural analogues, JH-XVI-178 or its structural analogues, CCT251545 or its structural analogues, BRD6989 or its structural analogues, JH-XI-10-02 or its Structural analogues, SEL120-34A or its structural analogues, LY2857785 or its structural analogues, CDK8-IN-1 or its structural analogues, CDK8-IN-5 or its structural analogues, CDK8-IN-3 or its structure Analogs, CDK8-IN-4 or its structural analogs, CDK8-IN-6 or its structural analogs, CDK8-IN-7 or its structural
  • Embodiment 24 The use of embodiment 22 or 23, wherein the agent is used in combination with a WNT signaling pathway activator, a WNT signaling pathway inhibitor, and/or insulin to prepare cardiomyocytes from pluripotent stem cell differentiation.
  • Embodiment 25 The use of any one of embodiments 22-24, wherein the agent is used to increase the efficiency of producing cardiomyocytes from differentiation of pluripotent stem cells.
  • Embodiment 26 The use of any one of embodiments 22-25, wherein the agent is used to increase the efficiency of preparing cardiomyocytes from pluripotent stem cell differentiation in the presence of a high concentration of WNT signaling pathway activator.
  • Embodiment 27 Use of any one of embodiments 22-26, wherein the reagent is for preparing cardiomyocytes by the method of any one of embodiments 1-16.
  • FIG. 1 The differentiation process from human stem cells to cardiomyocytes used in this experiment.
  • the whole differentiation process is divided into 4 stages: hiPSC stage, the first stage of differentiation into mesoderm, the second stage of differentiation into cardiac progenitor cells, and the third stage of differentiation into cardiomyocytes, mainly using activators (CHIR) and inhibitors of the WNT signaling pathway agent (IWR1) is completed, the colored arrow indicates that in the first stage of differentiation, the use time and concentration of small molecule CHIR have an important impact on differentiation efficiency.
  • hiPSC stage the first stage of differentiation into mesoderm
  • the second stage of differentiation into cardiac progenitor cells mainly using activators (CHIR) and inhibitors of the WNT signaling pathway agent (IWR1)
  • the colored arrow indicates that in the first stage of differentiation, the use time and concentration of small molecule CHIR have an important impact on differentiation efficiency.
  • IWR1 WNT signaling pathway agent
  • FIG. 2 Adding BI-1347 from 0 to 48 hours in the high-dose CHIR group can efficiently differentiate cardiomyocytes.
  • Figure 3 Verification of the effect of BI-1347 on stabilizing and optimizing the cardiomyocyte system.
  • (a) Effects of using different concentrations of CHIR in the first stage, with or without BI-1347 (0-48h, 0.5 ⁇ M), on the differentiation efficiency of hiPSC-M cell lines.
  • (b) The effect of using different concentrations of CHIR in the first stage, with or without BI-1347 (0-48h, 0.5 ⁇ M), on the differentiation efficiency of hiPSC-F cell lines.
  • (d) Effect of different doses of BI-1347 on differentiation efficiency.
  • the first phase uses high-dose CHIR concentrations while treating cells with Compound A for 0-48 h.
  • Figure 4 Identification of transcriptional expression of hiPSC-CM-related genes after BI-1347 optimized system.
  • (b) Genome-wide comparison of iPSCs, iPSC-cm, and BI-1347-treated (0-48h) iPSC-cm without metabolic purification. The heatmap shows log2(FPKM+1) normalized z-scores over multiple samples. Hierarchical clustering was performed on 19,999 genes.
  • FIG. 5 hiPSC-CM qPCR identification after BI-1347 optimized system.
  • (b) Current-voltage diagram of calcium current in iPSC-CM cultured with and without BI-1347. Data are expressed as mean ⁇ SEM. n 15-16.
  • the present invention provides a CDK8 inhibitor for use in the differentiation of pluripotent stem cells to prepare cardiomyocytes.
  • the CDK8 inhibitor is used to differentiate cardiomyocytes from pluripotent stem cells in vitro.
  • the invention provides the use of a CDK8 inhibitor described herein in the preparation of a reagent for preparing cardiomyocytes from differentiation of pluripotent stem cells.
  • the reagents are used to prepare cardiomyocytes from pluripotent stem cells in vitro.
  • the pluripotent stem cells described herein may be pluripotent stem cells from mammals, such as mice, rats, non-human primates or humans.
  • the pluripotent stem cells described herein are human pluripotent stem cells.
  • Pluripotent stem cells as described herein include, but are not limited to, embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC).
  • ESC embryonic stem cells
  • iPSC induced pluripotent stem cells
  • the pluripotent stem cells described herein are isolated pluripotent stem cells.
  • the embryonic stem cells (ESCs) described herein can be derived from humans or non-human animals.
  • Human embryonic stem cells (ESC) are limited to stem cells isolated from human embryos within 14 days of fertilization that have not undergone in vivo development.
  • the iPSCs described herein can be derived from any cell type.
  • the starting cell type may be keratinocytes, fibroblasts, hematopoietic cells, mesenchymal cells, hepatocytes or gastric cells.
  • the induction of iPSCs places no restrictions on the degree of cell differentiation or the age of the subject from whom the cells are collected; even undifferentiated progenitor cells (including adult stem cells) and terminally differentiated mature cells can be used as a source of iPSCs herein.
  • reprogramming factors can be used to generate pluripotent stem cells from somatic cells.
  • pluripotent stem cells can be induced from somatic cells through a combination of reprogramming factors and small molecule compounds.
  • the cardiomyocytes are human cardiomyocytes. In some embodiments of aspects herein, the cardiomyocytes are functional cardiomyocytes. In some embodiments of aspects herein, the cardiomyocytes are beating cardiomyocytes. In some embodiments of aspects herein, the cardiomyocytes express one or more cardiomyocyte-specific proteins selected from: cTNT, cTNI, ⁇ -ACTININ, MEF2C, and NKX2.5.
  • the CDK8 inhibitor or the agent is used in combination with a WNT signaling pathway activator, a WNT signaling pathway inhibitor, and/or insulin to differentiate cardiomyocytes from pluripotent stem cells.
  • the CDK8 inhibitor or the agent is used to increase the efficiency of cardiomyocyte differentiation from pluripotent stem cells.
  • the CDK8 inhibitor or the agent is used to increase the concentration of a WNT signaling pathway activator, such as CHIR99021, in the presence of a high concentration of a WNT signaling pathway activator (e.g., from about 8 ⁇ M to about 25 ⁇ M or a higher concentration of a WNT signaling pathway activator such as CHIR99021).
  • a WNT signaling pathway activator such as CHIR99021
  • the concentration of the WNT signaling pathway activator is about 1 ⁇ M to about 25 ⁇ M or higher, such as about 1 ⁇ M, about 2 ⁇ M, about 3 ⁇ M, about 4 ⁇ M, about 5 ⁇ M, about 6 ⁇ M, about 7 ⁇ M, about 8 ⁇ M, About 9 ⁇ M, about 10 ⁇ M, about 11 ⁇ M, about 12 ⁇ M, about 13 ⁇ M, greater than 14 ⁇ M, about 15 ⁇ M, about 16 ⁇ M, about 17 ⁇ M, about 18 ⁇ M, about 19 ⁇ M, about 20 ⁇ M, about 21 ⁇ M, about 22 ⁇ M, about 23 ⁇ M, about 24 ⁇ M, about 25 ⁇ M or higher.
  • the CDK8 inhibitor is used at a concentration of about 0.001 ⁇ M to about 5 ⁇ M, preferably about 0.004 ⁇ M to about 3 ⁇ M, such as about 0.1 ⁇ M, about 0.2 ⁇ M, about 0.3 ⁇ M, about 0.4 ⁇ M , about 0.5 ⁇ M, about 0.6 ⁇ M, about 0.7 ⁇ M, about 0.8 ⁇ M, about 0.9 ⁇ M, about 1 ⁇ M, about 2 ⁇ M, about 3 ⁇ M, about 4 ⁇ M, or about 5 ⁇ M.
  • the concentration of the WNT signaling pathway inhibitor is about 1 ⁇ M to about 10 ⁇ M or higher, such as about 1 ⁇ M, about 2 ⁇ M, about 3 ⁇ M, about 4 ⁇ M, about 5 ⁇ M, about 6 ⁇ M, about 7 ⁇ M , about 8 ⁇ M, about 9 ⁇ M, about 10 ⁇ M or higher.
  • the efficiency of differentiation of pluripotent stem cells into cardiomyocytes using the CDK8 inhibitor is compared to not using the CDK8 inhibitor (especially when using about 8 ⁇ M to about 25 ⁇ M or more High concentrations of WNT signaling pathway activators such as CHIR99021) increase at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 400%, at least about 500% or higher.
  • the CDK8 inhibitor includes, but is not limited to, BI-1347 or a structural analog thereof, MSC2530818 or a structural analog thereof, AS2863619 or a structural analog thereof, Senexin A or a structural analog thereof, CCT-251921 or its structural analog, Senexin C or its structural analog, JH-XVI-178 or its structural analog, CCT251545 or its structural analog, BRD6989 or its structural analog, JH-XI-10-02 or Its structural analogs, SEL120-34A or its structural analogs, LY2857785 or its structural analogs, CDK8-IN-1 or its structural analogs, CDK8-IN-5 or its structural analogs, CDK8-IN-3 or its Structural analogs, CDK8-IN-4 or structural analogs thereof, CDK8-IN-6 or structural analogs thereof, CDK8-IN-7 or structural analogs thereof, CDK8/19-IN-1 or structural analogs thereof, DS96432529 or its structural
  • CDK8 inhibitors suitable for the present invention please refer to the literature: Yu, Mingfeng, et al. "Potent and orally bioavailable CDK8 inhibitors: Design, synthesis, structure-activity relationship analysis and biological evaluation.” European Journal of Medicinal Chemistry 214(2021): 113248; Bhurta, Deendyal, and Sandip B. Bharat. "Analyzing the scaffold diversity of cyclin-dependent kinase inhibitors and revisiting the clinical and preclinical pipeline.” Medicinal Research Reviews 42.2(2022):654-709; and Hatcher, John M. , et al. "Development of Highly Potent and Selective Pyrazolopyridine Inhibitor of CDK8/19.” ACS Medicinal Chemistry Letters 12.11(2021):1689-1693.
  • the WNT signaling pathway activator includes, but is not limited to, CHIR99021 or a structural analog thereof, CHIR98014 or a structural analog thereof, SB212763 or a structural analog thereof.
  • the WNT signaling pathway inhibitor includes, but is not limited to, IWR1 or a structural analog thereof, IWP2 or a structural analog thereof, IWP4 or a structural analog thereof.
  • the insulin is human insulin.
  • CDK8 inhibitors, WNT signaling pathway activators and/or WNT signaling pathway inhibitors in the present invention all encompasses their salt forms.
  • the CDK8 inhibitor is used to prepare cardiomyocytes from differentiation of pluripotent stem cells by a method comprising:
  • step iii) Cultivate the cells obtained in step ii) in a basal medium supplemented with a WNT signaling pathway inhibitor;
  • step iv) culturing the cells obtained in step iii) in a basal medium supplemented with insulin;
  • step ii) includes culturing the pluripotent stem cells for a first period of time in basal medium supplemented with a CDK8 inhibitor and a WNT signaling pathway activator, and then without the addition of the CDK8 inhibitor and WNT signaling pathway activator. Continue culturing the cells in the activator's basal medium for a second period of time.
  • the first period of time is from about 24 hours (h) to about 48 hours (h).
  • the second period of time is approximately 24 hours (h).
  • step iii) includes culturing the cells obtained in step ii) for a third period of time in basal medium supplemented with a WNT signaling pathway inhibitor, and then continuing in basal medium without the addition of a WNT signaling pathway inhibitor.
  • the cells were cultured for a fourth period of time.
  • the third period of time is from about 24 hours (h) to about 48 hours (h).
  • the fourth period of time is approximately 24 hours (h).
  • step iv) includes culturing the cells obtained in step iii) in basal medium supplemented with insulin for a fifth period of time.
  • the fifth period of time is from about 3 days to about 6 days.
  • the method includes:
  • step iii) Culturing the cells obtained in step ii) in a basal medium adding the WNT signaling pathway inhibitor for approximately 48 hours, and then continuing to culture the cells in a basal medium without adding the WNT signaling pathway inhibitor for approximately 24 hours;
  • step iv) culturing the cells obtained in step iii) in a basal medium supplemented with insulin for approximately 3-6 days;
  • the pluripotent stem cells in step i) are cultured in multi-well plates. In some embodiments, the pluripotent stem cells in step i) are grown to approximately 80-90% confluence.
  • the basal medium is a combination of RPMI medium (such as RPMI1640 medium) and insulin-free B27 cell culture supplement (B27minus insulin); or CDM3 medium (Paul W Burridge et al., Chemically defined and albumin-free generation of human atrial and ventricular myocytes from combination of human pluripotent stem cells.Stem Cell Research, Volume 19, March 2017, Pages 94-103).
  • the basal medium supplemented with insulin in step iv) is a combination of RPMI medium (such as RPMI1640 medium) and B27 cell culture additives, or a combination of CDM3 medium and S12 cell culture additives.
  • the volume ratio of RPMI medium and B27 cell culture supplement (with or without insulin) in the basal medium is approximately 50:1.
  • the medium in step iii) does not contain the CDK8 inhibitor and WNT signaling pathway activator. In some embodiments, the medium in step iv) does not comprise the CDK8 inhibitor, the WNT signaling pathway activator and the WNT signaling pathway inhibitor.
  • the cells obtained in step ii) are predominantly cardiac mesoderm cells. In some embodiments, the cells obtained in step iii) are predominantly cardiac progenitor cells. In some embodiments, the cells obtained in step iv) are predominantly cardiomyocytes.
  • the present invention provides a method for preparing cardiomyocytes by differentiation of pluripotent stem cells, the method comprising:
  • step iii) Cultivate the cells obtained in step ii) in a basal medium supplemented with a WNT signaling pathway inhibitor;
  • step iv) culturing the cells obtained in step iii) in a basal medium supplemented with insulin;
  • step ii) includes culturing the pluripotent stem cells for a first period of time in basal medium supplemented with a CDK8 inhibitor and a WNT signaling pathway activator, and then without the addition of the CDK8 inhibitor and WNT signaling pathway activator. Continue culturing the cells in the activator's basal medium for a second period of time.
  • the first period of time is from about 24 hours (h) to about 48 hours (h).
  • the second period of time is approximately 24 hours (h).
  • step iii) includes culturing in basal medium supplemented with a WNT signaling pathway inhibitor The cells obtained in step ii) are cultured for a third period of time in a basal medium without adding a WNT signaling pathway inhibitor for a fourth period of time.
  • the third period of time is from about 24 hours (h) to about 48 hours (h).
  • the fourth period of time is approximately 24 hours (h).
  • step iv) includes culturing the cells obtained in step iii) in basal medium supplemented with insulin for a fifth period of time.
  • the fifth period of time is from about 3 days to about 6 days.
  • the method includes:
  • step iii) Culturing the cells obtained in step ii) in a basal medium adding a WNT signaling pathway inhibitor for approximately 48 hours, and then continuing to culture the cells in a basal medium without adding a WNT signaling pathway inhibitor for approximately 24 hours;
  • step iv) culturing the cells obtained in step iii) in a basal medium supplemented with insulin for approximately 3-6 days;
  • the pluripotent stem cells in step i) are cultured in multi-well plates. In some embodiments, the pluripotent stem cells in step i) are grown to approximately 80-90% confluence.
  • the basal medium is a combination of RPMI medium (eg, RPMI1640 medium) and insulin-free B27 cell culture additives; or a combination of CDM3 medium and insulin-free S12 cell culture additives.
  • the basal medium supplemented with insulin in step iv) is a combination of RPMI medium (such as RPMI1640 medium) and B27 cell culture additives, or a combination of CDM3 medium and S12 cell culture additives.
  • the cells obtained in step ii) are predominantly cardiac mesoderm cells. In some embodiments, the cells obtained in step iii) are predominantly cardiac progenitor cells. In some embodiments, the cells obtained in step iv) are predominantly cardiomyocytes.
  • the CDK8 inhibitor includes, but is not limited to, BI-1347 or a structural analog thereof, MSC2530818 or a structural analog thereof, AS2863619 or a structural analog thereof, Senexin A or a structural analog thereof, CCT-251921 or Its structural analogues, Senexin C or its structural analogues, JH-XVI-178 or its structural analogues, CCT251545 or its structural analogues, BRD6989 or its structural analogues, JH-XI-10-02 or its structural analogues , SEL120-34A or its structural analogs, LY2857785 or its structural analogs, CDK8-IN-1 or its structural analogs, CDK8-IN-5 or its structural analogs, CDK8-IN-3 or its structural analogs, CDK8-IN-4 or its structural analogues, CDK8-IN-6 or its structural analogues, CDK8-IN-7 or its structural analogues, CDK8/19-
  • the CDK8 inhibitor is BI-1347 or a structural analog thereof.
  • the WNT signaling pathway activator includes, but is not limited to, CHIR99021 or a structural analog thereof, CHIR98014 or a structural analog thereof, SB212763 or a structural analog thereof.
  • the WNT signaling pathway inhibitor includes, but is not limited to, IWR1 or a structural analog thereof, IWP2 or a structural analog thereof, IWP4 or a structural analog thereof.
  • the insulin is human insulin.
  • the final concentration of the CDK8 inhibitor is about 0.001 ⁇ M to about 5 ⁇ M, preferably about 0.004 ⁇ M to about 3 ⁇ M, such as about 0.1 ⁇ M, about 0.2 ⁇ M, about 0.3 ⁇ M, about 0.4 ⁇ M, about 0.5 ⁇ M, about 0.6 ⁇ M, about 0.7 ⁇ M, about 0.8 ⁇ M, about 0.9 ⁇ M, about 1 ⁇ M, about 2 ⁇ M, about 3 ⁇ M, about 4 ⁇ M, or about 5 ⁇ M.
  • the final concentration of the WNT signaling pathway activator is about 1 ⁇ M to about 25 ⁇ M or higher, such as about 1 ⁇ M, about 2 ⁇ M, about 3 ⁇ M, about 4 ⁇ M, about 5 ⁇ M, about 6 ⁇ M, about 7 ⁇ M, about 8 ⁇ M, about 9 ⁇ M, about 10 ⁇ M, about 11 ⁇ M, about 12 ⁇ M, about 13 ⁇ M, greater than 14 ⁇ M, about 15 ⁇ M, about 16 ⁇ M, about 17 ⁇ M, about 18 ⁇ M, about 19 ⁇ M, about 20 ⁇ M, about 21 ⁇ M, about 22 ⁇ M, About 23 ⁇ M, about 24 ⁇ M, about 25 ⁇ M or higher.
  • the concentration of the WNT signaling pathway inhibitor is about 1 ⁇ M to about 10 ⁇ M or higher, such as about 1 ⁇ M, about 2 ⁇ M, about 3 ⁇ M, about 4 ⁇ M, about 5 ⁇ M, about 6 ⁇ M, about 7 ⁇ M, about 8 ⁇ M, About 9 ⁇ M, about 10 ⁇ M or higher.
  • the invention provides a cardiomyocyte prepared by the method of the invention.
  • the invention provides a pharmaceutical composition comprising cardiomyocytes prepared by the method of the invention and a pharmaceutically acceptable carrier.
  • the invention also provides the use of cardiomyocytes prepared by the method of the invention or the pharmaceutical composition of the invention in the preparation of a medicament for the treatment of cardiac disease in a subject in need thereof.
  • the heart disease especially myocardial disease, includes but is not limited to heart failure, myocardial infarction, etc.
  • the invention also provides a method of treating cardiac disease in a subject in need thereof, the method comprising administering to the subject cardiomyocytes prepared by the method of the invention or a pharmaceutical composition of the invention.
  • a "subject" may be a mammal or a non-mammal.
  • the subject may be a human, or a non-human mammal such as a mouse or rat or a non-human primate, preferably a human.
  • the present invention provides a culture medium for preparing cardiomyocytes from differentiation of pluripotent stem cells, the culture medium comprising the CDK8 inhibitor and the WNT signaling pathway activator.
  • the culture medium further comprises a basal medium, for example, the basal medium is a combination of RPMI medium (such as RPMI1640 medium) and insulin-free B27 cell culture additives; or is a CDM3 medium and insulin-free S12 cell culture supplement.
  • the present invention provides a culture medium system for preparing cardiomyocytes through differentiation of pluripotent stem cells, the culture medium system comprising
  • a first culture medium which contains the CDK8 inhibitor described herein and the WNT signaling pathway activator;
  • the first, second and/or third culture medium further comprises a basal culture medium
  • the basal culture medium is RPMI medium (such as RPMI1640 medium) and insulin-free B27 cells Culture addition or a combination of CDM3 medium and insulin-free S12 cell culture supplements.
  • the culture medium system further comprises a fourth culture medium for culturing and/or expanding pluripotent stem cells.
  • the fourth culture medium may be any culture medium that can be used to culture and/or expand pluripotent stem cells, which is readily available to those skilled in the art.
  • the culture medium or culture medium system is used in the methods of the invention.
  • the invention provides a kit for preparing cardiomyocytes from differentiation of pluripotent stem cells, the kit comprising at least a CDK8 inhibitor as defined herein.
  • the kit includes a culture medium or culture medium system of the invention.
  • FIG. 1 The main process of differentiation of human stem cells into cardiomyocytes is shown in Figure 1 (refer to Aguilar et al., 2015). It involves culturing human hiPSC cells in a monolayer and starting to differentiate when their confluence reaches about 80%. In the first stage (0-72h), the WNT signaling pathway activator CHIR99021 (CHIR) is used; in the second stage, the WNT signaling pathway inhibitor IWR1 is used. 48h; in the third stage, insulin is added to the basal differentiation medium, and the cells can spontaneously differentiate into beating cardiomyocytes.
  • CHIR99021 CHIR99021
  • hiPSC stem cells
  • Cardiac mesoderm Cardiac mesoderm, Stage I
  • CPC cardiac progenitor cells
  • CM cardiomyocytes
  • the hiPSCs used in this experiment were routinely cultured in 6-well plates, passaged once in about 4 days, and placed in a cell incubator with a constant temperature of 37°C and 5% CO2. The passage steps are detailed as follows:
  • Matrigel needs to be operated on ice throughout the process.
  • the original matrigel is diluted 50 times with pre-cooled DMEM/F-12 and added to the well plate. The amount added is based on the amount that can cover the bottom of the plate (taking a 6-well plate as an example, 850uL/well).
  • After spreading place it in the incubator 37 Incubate at °C for 30 minutes, and absorb the liquid before use;
  • hiPSCs are isolated into CDM medium at a ratio of 1:10 or 1:12.
  • the isolation steps are consistent with the above passage steps.
  • Y27632 (5 ⁇ M) needs to be added to the CDM medium, which is recorded as day -3;
  • RPMI+B27 Use RPMI+B27 for continuous culture and change the medium every 3 days.
  • the cells will spontaneously differentiate into beating hiPSC-CM within 3-6 days, which is the third stage of differentiation. Cell beating can be observed as early as day 7-8.
  • CHIR99021 (CHIR) in the first stage (from hiPSC to cardiac mesoderm, 0-72h) plays a decisive role in the success of differentiation.
  • CHIR99021 the dose of CHIR99021 (CHIR) in the first stage (from hiPSC to cardiac mesoderm, 0-72h)
  • the optimal concentration of the WNT pathway activator CHIR used in the first stage of differentiation is unstable and has significant differences between cell lines or batches.
  • the optimal concentration range of CHIR is very narrow, and often only a concentration range of 2-4 ⁇ M can achieve high differentiation efficiency.
  • the above problems make the differentiation system very unstable and make the large-scale production of cardiomyocytes challenging.
  • BI-1347 has stable and outstanding effects on the same cell lines in multiple batches of experiments.
  • the effective concentration test results show that BI-1347 has significant effects within a wide concentration range (0.004-3 ⁇ M) ( Figure 3c, Figure 3d).
  • CDK8 inhibitor CDK8 inhibitor
  • two other compounds with the same target MSC2530818, AS2863619
  • MSC2530818, AS2863619 two other compounds with the same target
  • RNA-seq results showed that differentiated cardiomyocytes treated with or without BI-1347 were similar, and myocardial-specific marker genes were significantly expressed (Figure 4a-c).
  • Immunofluorescence results showed that the myocardial-specific expression of sarcomere-related proteins cTNT, cTNI, and ⁇ -ACTININ were positive, and the myocardial-specific expression of transcription factors MEF2C and NKX2.5 were also positive, consistent with normal cardiomyocytes (Figure 4d).
  • the electrophysiological identification results also showed that the action potential, calcium flow and other characteristics of cardiomyocytes differentiated by BI-1347 were relatively consistent, and the proportions of each subtype of differentiated myocardium were similar ( Figure 5a-j).
  • adding CDK8 inhibitors such as BI-1347 in the first stage of differentiation of stem cells into cardiomyocytes from 0 to 48 hours can expand the applicable range of CHIR concentration and time, and improve the stability of the myocardial differentiation system between different cell lines or between different batches. efficiency and stability.

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Abstract

涉及生物医药领域，特别是再生医学领域。具体而言，涉及一种用于制备心肌细胞的化合物及其用途，以及利用所述化合物将多能干细胞例如诱导多能干细胞分化成心肌细胞的方法。

Description

用于制备心肌细胞的化合物及其用途 技术领域
本发明涉及生物医药领域，特别是再生医学领域。具体而言，本发明涉及一种用于制备心肌细胞的化合物及其用途，以及利用所述化合物将多能干细胞例如诱导多能干细胞分化成心肌细胞的方法。
发明背景
心脏病是人类最主要致死原因之一，并且心脏细胞治疗、体外心肌疾病建模或药物研发都需要大量心肌细胞的支持。但心肌细胞作为终末分化细胞体内不可以再生，且原代心肌细胞难以在体外培养扩增。因此心肌细胞来源成为重大问题。目前，可以通过重编程人类体细胞获取诱导多能干细胞(hiPSC)，从而定向分化出多种功能性细胞，包括心肌细胞(hiPSC-CM)，这为心肌细胞来源提供了新思路。而hiPSC-CM的高效、稳定、大规模生产对其实际应用至关重要，但目前分化体系效率与质量仍存在不稳定的问题。因此，本领域仍然需要新的化合物和方法，其能够用于通过重编程高效地从干细胞例如诱导型多能干细胞产生功能性心肌细胞，从而治疗心脏疾病例如心力衰竭。
发明概述
本发明至少提供以下实施方案：
实施方案1.一种由多能干细胞分化制备心肌细胞的方法，所述方法包括：
i)提供多能干细胞；
ii)在添加了CDK8抑制剂和WNT信号通路激活剂的基础培养基中培养所述多能干细胞大约24h-大约48h，然后在不添加所述CDK8抑制剂和WNT信号通路激活剂的基础培养基中继续培养所述细胞大约24h；
iii)在添加了WNT信号通路抑制剂的基础培养基中培养步骤ii)所获得的细胞大约48h，然后在不添加WNT信号通路抑制剂的基础培养基中继续培养所述细胞大约24h；
iv)在添加了胰岛素的基础培养基中培养步骤iii)所获得的细胞大约3-6天；和
v)任选地，收获所获得的心肌细胞。
实施方案2.实施方案1的方法，其中所述多能干细胞选自胚胎干细胞(ESC)或诱导性多能干细胞(iPSC)。
实施方案3.实施方案1或2的方法，其中步骤i)中的多能干细胞在多孔板中培养。
实施方案4.实施方案1-3中任一项的方法，其中步骤i)中的多能干细胞生长至大约80-90％的汇合度。
实施方案5.实施方案1-4中任一项的方法，其中所述基础培养基是RPMI培养基和 不含胰岛素的B27细胞培养添加剂的组合。
实施方案6.实施方案1-6中任一项的方法，其中步骤iv)中所述添加胰岛素的基础培养基是RPMI培养基(如RPMI1640培养基)和B27细胞培养添加剂的组合。
实施方案7.实施方案1-6中任一项的方法，其中所述CDK8抑制剂选自BI-1347或其结构类似物、MSC2530818或其结构类似物、AS2863619或其结构类似物、Senexin A或其结构类似物、CCT-251921或其结构类似物、Senexin C或其结构类似物、JH-XVI-178或其结构类似物、CCT251545或其结构类似物、BRD6989或其结构类似物、JH-XI-10-02或其结构类似物、SEL120-34A或其结构类似物、LY2857785或其结构类似物、CDK8-IN-1或其结构类似物、CDK8-IN-5或其结构类似物、CDK8-IN-3或其结构类似物、CDK8-IN-4或其结构类似物、CDK8-IN-6或其结构类似物、CDK8-IN-7或其结构类似物、CDK8/19-IN-1或其结构类似物、DS96432529或其结构类似物、Wogonin或其结构类似物，优选地，所述CDK8抑制剂是BI-1347或其结构类似物。
实施方案8.实施方案1-7中任一项的方法，其中所述WNT信号通路激活剂是CHIR99021或其结构类似物、CHIR98014或其结构类似物、或SB212763或其结构类似物。
实施方案9.实施方案1-8中任一项的方法，其中所述WNT信号通路抑制剂是IWR1或其结构类似物、IWP2或其结构类似物、或IWP4或其结构类似物。
实施方案10.实施方案1-9中任一项的方法，其中所述胰岛素为人胰岛素。
实施方案11.实施方案1-10中任一项的方法，其中所述CDK8抑制剂例如BI-1347或其结构类似物的终浓度为大约0.001μM-大约5μM，优选大约0.004μM-大约3μM，例如大约0.1μM、大约0.2μM、大约0.3μM、大约0.4μM、大约0.5μM、大约0.6μM、大约0.7μM、大约0.8μM、大约0.9μM、大约1μM、大约2μM、大约3μM、大约4μM或大约5μM。
实施方案12.实施方案1-11中任一项的方法，其中所述WNT信号通路激活剂如CHIR99021或其结构类似物的终浓度为大约1μM-大约25μM或更高，例如大约1μM、大约2μM、大约3μM、大约4μM、大约5μM、大约6μM、大约7μM、大约8μM、大约9μM、大约10μM、大约11μM、大约12μM、大约13μM、大于14μM、大约15μM、大约16μM、大约17μM、大约18μM、大约19μM、大约20μM、大约21μM、大约22μM、大约23μM、大约24μM、大约25μM或更高。
实施方案13.实施方案1-12中任一项的方法，其中所述WNT信号通路抑制剂的浓度为大约1μM-大约10μM或更高，例如大约1μM、大约2μM、大约3μM、大约4μM、大约5μM、大约6μM、大约7μM、大约8μM、大约9μM、大约10μM或更高。
实施方案14.实施方案1-13中任一项的方法，其中所述多能干细胞是人多能干细胞，且所述心肌细胞是人心肌细胞。
实施方案15.实施方案1-14中任一项的方法，其中所述心肌细胞是功能性心肌细胞，例如能够跳动的心肌细胞。
实施方案16.实施方案1-15中任一项的方法，其中所述心肌细胞表达选自以下的一或多种心肌特异性蛋白：cTNT、cTNI、α-ACTININ、MEF2C和NKX2.5。
实施方案17.一种通过实施方案1-16中任一项的方法制备的心肌细胞。
实施方案18.药物组合物，其包含通过实施方案1-16中任一项的方法制备的心肌细胞和药学上可接受的载体。
实施方案19.通过实施方案17的心肌细胞或实施方案18的药物组合物在制备用于在有需要的对象中治疗心脏疾病的药物中的用途。
实施方案20.一种在有需要的对象中治疗心脏疾病的方法，所述方法包括给所述对象施用实施方案17的心肌细胞或实施方案18的药物组合物。
实施方案21.实施方案19的用途或实施方案20的方法，其中所述对象是人。
实施方案22.CDK8抑制剂在制备用于通过多能干细胞分化制备心肌细胞的试剂中的用途。
实施方案23.实施方案22的用途，其中所述CDK8抑制剂选自BI-1347或其结构类似物、MSC2530818或其结构类似物、AS2863619或其结构类似物、Senexin A或其结构类似物、CCT-251921或其结构类似物、Senexin C或其结构类似物、JH-XVI-178或其结构类似物、CCT251545或其结构类似物、BRD6989或其结构类似物、JH-XI-10-02或其结构类似物、SEL120-34A或其结构类似物、LY2857785或其结构类似物、CDK8-IN-1或其结构类似物、CDK8-IN-5或其结构类似物、CDK8-IN-3或其结构类似物、CDK8-IN-4或其结构类似物、CDK8-IN-6或其结构类似物、CDK8-IN-7或其结构类似物、CDK8/19-IN-1或其结构类似物、DS96432529或其结构类似物、Wogonin或其结构类似物，优选地，所述CDK8抑制剂是BI-1347或其结构类似物。
实施方案24.实施方案22或23的用途，其中所述试剂用于与WNT信号通路激活剂、WNT信号通路抑制剂和/或胰岛素组合来由多能干细胞分化制备心肌细胞。
实施方案25.实施方案22-24中任一项的用途，其中所述试剂用于提高由多能干细胞分化制备心肌细胞的效率。
实施方案26.实施方案22-25中任一项的用途，其中所述试剂用于在高浓度WNT信号通路激活剂存在下提高由多能干细胞分化制备心肌细胞的效率。
实施方案27.实施方案22-26中任一项的用途，其中所述试剂用于通过实施方案1-16中任一项的方法制备心肌细胞。
附图简述
图1.本实验所使用的从人类干细胞到心肌细胞分化流程。分化全过程分为4个阶段：hiPSC阶段、第一阶段分化为中胚层、第二阶段分化为心脏祖细胞、第三阶段分化为心肌细胞，主要使用WNT信号通路的激活剂(CHIR)和抑制剂(IWR1)完成，彩色箭头表示在分化第一阶段，小分子CHIR的使用时间和浓度对分化效率有重要影响。
图2.高剂量CHIR组0-48h加入BI-1347可高效分化心肌细胞。(a)CHIR高剂量组 (0-48h CHIR 20μM)，第一阶段加BI-1347和不加BI-1347的分化效率差异，绿色为cTNT免疫荧光染色阳性的心肌细胞，蓝色为细胞核染料Hoechst。(b)不同CHIR浓度下，BI-1347和其他两种拥有相同靶点的化合物(MSC2530818、AS2863619)的心肌分化效率统计结果。三者结果相似，在高剂量下，分化效率显著高于对照组。(c)标尺为1mm。
图3.验证BI-1347对稳定优化心肌细胞体系的作用。(a)第一阶段使用不同浓度CHIR，有无BI-1347(0-48h，0.5μM)对hiPSC-M细胞系分化效率的影响。(b)第一阶段使用不同浓度CHIR，有无BI-1347(0-48h，0.5μM)对hiPSC-F细胞系分化效率的影响。(c)不同BI-1347浓度处理下的分化效率。(d)不同剂量的BI-1347对分化效率的影响。第一阶段使用高剂量CHIR浓度，同时使用化合物A在0-48h内处理细胞。
图4.BI-1347优化体系后的hiPSC-CM相关基因转录表达鉴定。(a)iPSC、iPSC-cm-BI-1347和iPSC-cm+BI-1347(0.5μM,48h)组bulk RNA-seq的主成分分析。每个点代表一个RNA-seq样本。N＝3。(b)iPSC、iPSC-cm和BI-1347处理(0-48h)未进行代谢纯化的iPSC-cm的全基因组比较。热图显示了多个样本上log2(FPKM+1)归一化的z值。对19,999个基因进行了分层聚类。(c)iPSC、未加BI-1347处理的iPSC-cm和加BI-1347(0.5μM,48h)处理的iPSC-cm(第12天，均未进行代谢纯化)的基因表达数据。热图显示了多个样本上log2(FPKM+1)归一化的z分数。(d)第一阶段加入BI-1347分化第12天的hiPSC-CM免疫荧光鉴定结果，从左至右依次为：红色为cTNT、绿色为MEF2C、蓝色为核染料Hoechst；红色为NKX2.5、绿色为cTNI、蓝色为核染料Hoechst；绿色为α-ACTININ、蓝色为核染料Hoechst。以上3张图比例尺为100μm。
图5.BI-1347优化体系后的hiPSC-CM qPCR鉴定。(a)加BI-1347培养(0.5μmol/L,0-48h)与不加BI-1347培养的iPSC-CM中的动作电位。(b)加BI-1347培养与不加BI-1347培养的iPSC-CM中钙电流的电流-电压图，数据表示为mean±SEM。n＝15-16。(c)比较加BI-1347培养组(n＝25cells)与不加BI-1347培养组(n＝25cells)中动作电位的最大舒张电压(MDP),峰值电压,动作电位幅度(APA),和动作电位复极化到不同水平(90％和50％)的时间。(d)记录的程序和电压钳模式下加BI-1347培养组与不加BI-1347培养组中记录到的钙电流例图。(e)钳制电压为-10mV时，两组iPSC-CMs中记录到的钙电流密度。(f)分别在加BI-1347组(n＝25cells)和不加BI-1347组(n＝25cells)中记录到的三种iPSC-CM亚型的动作电位图。(g)iPSC-CMs亚型的百分比。(h)比较加BI-1347培养的心室肌样细胞组(n＝19cells),不加BI-1347培养的心室肌样细胞组(n＝21cells),加BI-1347培养的心房肌样细胞组(n＝3cells),不加BI-1347培养的心房肌样细胞组(n＝2cells),加BI-1347培养的窦房结样细胞组(n＝3cells),不加BI-1347培养的窦房结样细胞组(n＝2cells)中动作电位的最大舒张电压(MDP),峰值电压,动作电位幅度(APA),和动作电位复极化到不同水平(90％和50％)的时间。(i)比较加BI-1347培养组(n＝39cells)与不加BI-1347培养组(n＝40cells)中自发钙瞬变荧光强度变化，上升到最大值的时间，半高宽，频率，上升到最大值50％的时间和钙瞬变分别衰减到50％和90％的时间。(j)加BI-1347培养与不加BI-1347培养的iPSC-CM中自发钙瞬变的荧光 图像(上)和时间过程(下)。
具体实施方式
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的蛋白质和核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。例如，本发明中使用的标准重组DNA和分子克隆技术为本领域技术人员熟知，并且在如下文献中有更全面的描述：Sambrook，J.，Fritsch，E.F.和Maniatis，T.，Molecular Cloning：A Laboratory Manual；Cold Spring Harbor Laboratory Press：Cold Spring Harbor，1989(下文称为“Sambrook”)。
如本文所用，术语“和/或”涵盖由该术语连接的项目的所有组合，应视作各个组合已经单独地在本文列出。例如，“A和/或B”涵盖了“A”、“A和B”以及“B”。例如，“A、B和/或C”涵盖“A”、“B”、“C”、“A和B”、“A和C”、“B和C”以及“A和B和C”。
在一方面，本发明提供一种CDK8抑制剂，其用于由多能干细胞分化制备心肌细胞。在一些实施方案中，所述CDK8抑制剂用于在体外由多能干细胞分化制备心肌细胞。
在一方面，本发明提供本文所述CDK8抑制剂在制备用于由多能干细胞分化制备心肌细胞的试剂中的用途。在一些实施方案中，所述试剂用于在体外由多能干细胞分化制备心肌细胞。
本文所述的多能干细胞可以是来自哺乳动物，例如是小鼠、大鼠、非人灵长类动物或人的多能干细胞。优选地，本文所述的多能干细胞是人多能干细胞。本文所述的多能干细胞包括但不限于胚胎干细胞(ESC)或诱导性多能干细胞(iPSC)。优选地，本文所述的多能干细胞是诱导性多能干细胞(iPSC)。优选地，本文所述多能干细胞是分离的多能干细胞。
本文所述的胚胎干细胞(ESC)，其来源可以是人或非人动物。对于人胚胎干细胞(ESC)仅限于利用未经过体内发育的受精14天以内的人胚胎分离获取的干细胞。
本文所述的iPSC可以来源于任何细胞类型。例如，起始细胞类型可以是角质形成细胞、成纤维细胞、造血细胞、间充质细胞、肝细胞或胃细胞。iPSC的诱导对细胞分化的程度或收集细胞的对象的年龄没有限制；甚至未分化的祖细胞(包括成体干细胞)和最终分化的成熟细胞可以用作本文的iPSC的来源。本领域技术人员可以容易地施用本领域已知的方法产生诱导的多能干细胞。例如，可以使用重编程因子用于从体细胞产生多能干细胞。然而，也可能使用小分子化合物从体细胞诱导多能干细胞。或者，可以通过重编程因子和小分子化合物的组合从体细胞诱导多能干细胞。
在本文各方面的一些实施方能中，所述心肌细胞是人心肌细胞。在本文各方面的一些实施方案中，所述心肌细胞是功能性心肌细胞。在本文各方面的一些实施方案中，所述心肌细胞是能够跳动的心肌细胞。在本文各方面的一些实施方案中，所述心肌细胞表达选自以下的一或多种心肌特异性蛋白：cTNT、cTNI、α-ACTININ、MEF2C和NKX2.5。
在本文各方面的一些实施方案中，所述CDK8抑制剂或所述试剂用于与WNT信号通路激活剂、WNT信号通路抑制剂和/或胰岛素组合来由多能干细胞分化制备心肌细胞。
在本文各方面的一些实施方案中，所述CDK8抑制剂或所述试剂用于提高由多能干细胞分化制备心肌细胞的效率。在一些实施方案中，所述CDK8抑制剂或所述试剂用于在高浓度WNT信号通路激活剂(例如大约8μM-大约25μM或更高浓度的WNT信号通路激活剂如CHIR99021)存在下提高由多能干细胞分化制备心肌细胞的效率。
在一些实施方案中，所述WNT信号通路激活剂的浓度为大约1μM-大约25μM或更高，例如大约1μM、大约2μM、大约3μM、大约4μM、大约5μM、大约6μM、大约7μM、大约8μM、大约9μM、大约10μM、大约11μM、大约12μM、大约13μM、大于14μM、大约15μM、大约16μM、大约17μM、大约18μM、大约19μM、大约20μM、大约21μM、大约22μM、大约23μM、大约24μM、大约25μM或更高。
在本文各方面的一些实施方案中，所述CDK8抑制剂的使用浓度为大约0.001μM-大约5μM，优选大约0.004μM-大约3μM，例如大约0.1μM、大约0.2μM、大约0.3μM、大约0.4μM、大约0.5μM、大约0.6μM、大约0.7μM、大约0.8μM、大约0.9μM、大约1μM、大约2μM、大约3μM、大约4μM或大约5μM。
在本文各方面的一些实施方案中，所述WNT信号通路抑制剂的浓度为大约1μM-大约10μM或更高，例如大约1μM、大约2μM、大约3μM、大约4μM、大约5μM、大约6μM、大约7μM、大约8μM、大约9μM、大约10μM或更高。
在本文各方面的一些实施方案中，与不使用所述CDK8抑制剂相比，使用所述CDK8抑制剂能够将由多能干细胞分化制备心肌细胞的效率(特别是在使用大约8μM-大约25μM或更高浓度的WNT信号通路激活剂如CHIR99021时)提高至少约10％、至少约20％、至少约30％、至少约40％、至少约50％、至少约60％、至少约70％、至少约80％、至少约90％、至少约100％、至少约150％、至少约200％、至少约250％、至少约300％、至少约400％、至少约500％或更高。
在本文各方面的一些实施方案中，所述CDK8抑制剂包括但不限于BI-1347或其结构类似物、MSC2530818或其结构类似物、AS2863619或其结构类似物、Senexin A或其结构类似物、CCT-251921或其结构类似物、Senexin C或其结构类似物、JH-XVI-178或其结构类似物、CCT251545或其结构类似物、BRD6989或其结构类似物、JH-XI-10-02或其结构类似物、SEL120-34A或其结构类似物、LY2857785或其结构类似物、CDK8-IN-1或其结构类似物、CDK8-IN-5或其结构类似物、CDK8-IN-3或其结构类似物、CDK8-IN-4或其结构类似物、CDK8-IN-6或其结构类似物、CDK8-IN-7或其结构类似物、CDK8/19-IN-1或其结构类似物、DS96432529或其结构类似物、Wogonin或其结构类似物。优选地，所述CDK8抑制剂是BI-1347或其结构类似物。
更多的适用于本发明的CDK8抑制剂例如可以参见文献：Yu,Mingfeng,et al."Potent and orally bioavailable CDK8 inhibitors:Design,synthesis,structure-activity relationship analysis and biological evaluation."European Journal of Medicinal Chemistry 214(2021): 113248；Bhurta,Deendyal,and Sandip B.Bharate."Analyzing the scaffold diversity of cyclin‐dependent kinase inhibitors and revisiting the clinical and preclinical pipeline."Medicinal Research Reviews 42.2(2022):654-709；和Hatcher,John M.,et al."Development of Highly Potent and Selective Pyrazolopyridine Inhibitor of CDK8/19."ACS Medicinal Chemistry Letters 12.11(2021):1689-1693.
在本文各方面的一些实施方案中，所述WNT信号通路激活剂包括但不限于CHIR99021或其结构类似物、CHIR98014或其结构类似物、SB212763或其结构类似物。在一些实施方案中，所述WNT信号通路抑制剂包括但不限于IWR1或其结构类似物、IWP2或其结构类似物、IWP4或其结构类似物。在一些实施方案中，所述胰岛素为人胰岛素。
本发明中使用CDK8抑制剂、WNT信号通路激活剂和/或WNT信号通路抑制剂均涵盖其盐形式。
在本文各方面的一些实施方案中，所述CDK8抑制剂用于通过包含以下步骤的方法由多能干细胞分化制备心肌细胞：
i)提供多能干细胞；
ii)在添加CDK8抑制剂和WNT信号通路激活剂的基础培养基中培养所述多能干细胞；
iii)在添加WNT信号通路抑制剂的基础培养基中培养步骤ii)所获得的细胞；
iv)在添加胰岛素的基础培养基中培养步骤iii)所获得的细胞；和
v)任选地，收获所获得的心肌细胞。
在一些实施方案中，步骤ii)包括在添加CDK8抑制剂和WNT信号通路激活剂的基础培养基中培养所述多能干细胞第一时间段，然后在不添加所述CDK8抑制剂和WNT信号通路激活剂的基础培养基中继续培养所述细胞第二时间段。在一些实施方案中，所述第一时间段是大约24小时(h)-大约48小时(h)。在一些实施方案中，所述第二时间段是大约24小时(h)。
在一些实施方案中，步骤iii)包括在添加WNT信号通路抑制剂的基础培养基中培养步骤ii)所获得的细胞第三时间段，然后在不添加WNT信号通路抑制剂的基础培养基中继续培养所述细胞第四时间段。在一些实施方案中，所述第三时间段是大约24小时(h)-大约48小时(h)。在一些实施方案中，所述第四时间段是大约24小时(h)。
在一些实施方案中，步骤iv)包括在添加胰岛素的基础培养基中培养步骤iii)所获得的细胞第五时间段。在一些实施方案中，所述第五时间段是大约3天-大约6天。
在一些实施方案中，所述方法包括：
i)提供多能干细胞；
ii)在添加所述CDK8抑制剂和WNT信号通路激活剂的基础培养基中培养所述多能干细胞大约24h-大约48h，然后在不添加所述CDK8抑制剂和WNT信号通路激活剂的基础培养基中继续培养所述细胞大约24h；
iii)在添加所述WNT信号通路抑制剂的基础培养基中培养步骤ii)所获得的细胞大约48h，然后在不添加WNT信号通路抑制剂的基础培养基中继续培养所述细胞大约24h；
iv)在添加胰岛素的基础培养基中培养步骤iii)所获得的细胞大约3-6天；和
v)任选地，收获所获得的心肌细胞。
在一些实施方案中，步骤i)中的多能干细胞在多孔板中培养。在一些实施方案中，步骤i)中的多能干细胞生长至大约80-90％的汇合度。
在一些实施方案中，所述基础培养基是RPMI培养基(如RPMI1640培养基)和不含胰岛素的B27细胞培养添加剂(B27minus insulin)的组合；或者是CDM3培养基(Paul W Burridge et al.,Chemically defined generation of human cardiomyocytes.Nature Methods volume 11,pages855–860(2014))和不含胰岛素的S12细胞培养添加剂(Fei Pei et al.,Chemical-defined and albumin-free generation of human atrial and ventricular myocytes from human pluripotent stem cells.Stem Cell Research,Volume 19,March 2017,Pages 94-103)的组合。在一些实施方案中，步骤iv)中所述添加胰岛素的基础培养基是RPMI培养基(如RPMI1640培养基)和B27细胞培养添加剂的组合，或者是CDM3培养基和S12细胞培养添加剂的组合。在一些实施方案中，所述基础培养基中RPMI培养基和B27细胞培养添加剂(有或无胰岛素)的体积比是大约50:1。
在一些实施方案中，步骤iii)中的培养基不包含所述CDK8抑制剂和WNT信号通路激活剂。在一些实施方案中，步骤iv)中的培养基不包含所述CDK8抑制剂、所述WNT信号通路激活剂和所述WNT信号通路抑制剂。
在一些实施方案中，步骤ii)获得的细胞主要为心肌中胚层细胞。在一些实施方案中，步骤iii)获得的细胞主要是心脏祖细胞。在一些实施方案中，步骤iv)获得的细胞主要是心肌细胞。
在另一方面，本发明提供一种由多能干细胞分化制备心肌细胞的方法，所述方法包括：
i)提供多能干细胞；
ii)在添加CDK8抑制剂和WNT信号通路激活剂的基础培养基中培养所述多能干细胞；
iii)在添加WNT信号通路抑制剂的基础培养基中培养步骤ii)所获得的细胞；
iv)在添加胰岛素的基础培养基中培养步骤iii)所获得的细胞；和
v)任选地，收获所获得的心肌细胞。
在一些实施方案中，步骤ii)包括在添加CDK8抑制剂和WNT信号通路激活剂的基础培养基中培养所述多能干细胞第一时间段，然后在不添加所述CDK8抑制剂和WNT信号通路激活剂的基础培养基中继续培养所述细胞第二时间段。在一些实施方案中，所述第一时间段是大约24小时(h)-大约48小时(h)。在一些实施方案中，所述第二时间段是大约24小时(h)。
在一些实施方案中，步骤iii)包括在添加WNT信号通路抑制剂的基础培养基中培养 步骤ii)所获得的细胞第三时间段，然后在不添加WNT信号通路抑制剂的基础培养基中继续培养所述细胞第四时间段。在一些实施方案中，所述第三时间段是大约24小时(h)-大约48小时(h)。在一些实施方案中，所述第四时间段是大约24小时(h)。
在一些实施方案中，步骤iv)包括在添加胰岛素的基础培养基中培养步骤iii)所获得的细胞第五时间段。在一些实施方案中，所述第五时间段是大约3天-大约6天。
在一些实施方案中，所述方法包括：
i)提供多能干细胞；
ii)在添加CDK8抑制剂和WNT信号通路激活剂的基础培养基中培养所述多能干细胞大约24小时(h)-大约48小时(h)，然后在不添加所述CDK8抑制剂和WNT信号通路激活剂的基础培养基中继续培养所述细胞大约24h；
iii)在添加WNT信号通路抑制剂的基础培养基中培养步骤ii)所获得的细胞大约48h，然后在不添加WNT信号通路抑制剂的基础培养基中继续培养所述细胞大约24h；
iv)在添加胰岛素的基础培养基中培养步骤iii)所获得的细胞大约3-6天；和
v)任选地，收获所获得的心肌细胞。
在一些实施方案中，步骤i)中的多能干细胞在多孔板中培养。在一些实施方案中，步骤i)中的多能干细胞生长至大约80-90％的汇合度。
在一些实施方案中，所述基础培养基是RPMI培养基(如RPMI1640培养基)和不含胰岛素的B27细胞培养添加剂的组合；或者是CDM3培养基和不含胰岛素的S12细胞培养添加剂的组合。在一些实施方案中，步骤iv)中所述添加胰岛素的基础培养基是RPMI培养基(如RPMI1640培养基)和B27细胞培养添加剂的组合，或者是CDM3培养基和S12细胞培养添加剂的组合。
在一些实施方案中，步骤ii)获得的细胞主要为心肌中胚层细胞。在一些实施方案中，步骤iii)获得的细胞主要是心脏祖细胞。在一些实施方案中，步骤iv)获得的细胞主要是心肌细胞。
在一些实施方案中，所述CDK8抑制剂包括但不限于BI-1347或其结构类似物、MSC2530818或其结构类似物、AS2863619或其结构类似物、Senexin A或其结构类似物、CCT-251921或其结构类似物、Senexin C或其结构类似物、JH-XVI-178或其结构类似物、CCT251545或其结构类似物、BRD6989或其结构类似物、JH-XI-10-02或其结构类似物、SEL120-34A或其结构类似物、LY2857785或其结构类似物、CDK8-IN-1或其结构类似物、CDK8-IN-5或其结构类似物、CDK8-IN-3或其结构类似物、CDK8-IN-4或其结构类似物、CDK8-IN-6或其结构类似物、CDK8-IN-7或其结构类似物、CDK8/19-IN-1或其结构类似物、DS96432529或其结构类似物、Wogonin或其结构类似物。优选地，所述CDK8抑制剂是BI-1347或其结构类似物。在一些实施方案中，所述WNT信号通路激活剂包括但不限于CHIR99021或其结构类似物、CHIR98014或其结构类似物、SB212763或其结构类似物。在一些实施方案中，所述WNT信号通路抑制剂包括但不限于IWR1或其结构类似物、IWP2或其结构类似物、IWP4或其结构类似物。在一些实施方案中， 所述胰岛素为人胰岛素。
在一些实施方案中，所述CDK8抑制剂例如BI-1347或其结构类似物的终浓度为大约0.001μM-大约5μM，优选大约0.004μM-大约3μM，例如大约0.1μM、大约0.2μM、大约0.3μM、大约0.4μM、大约0.5μM、大约0.6μM、大约0.7μM、大约0.8μM、大约0.9μM、大约1μM、大约2μM、大约3μM、大约4μM或大约5μM。
在一些实施方案中，所述WNT信号通路激活剂如CHIR99021或其结构类似物的终浓度为大约1μM-大约25μM或更高，例如大约1μM、大约2μM、大约3μM、大约4μM、大约5μM、大约6μM、大约7μM、大约8μM、大约9μM、大约10μM、大约11μM、大约12μM、大约13μM、大于14μM、大约15μM、大约16μM、大约17μM、大约18μM、大约19μM、大约20μM、大约21μM、大约22μM、大约23μM、大约24μM、大约25μM或更高。
在一些实施方案中，所述WNT信号通路抑制剂的浓度为大约1μM-大约10μM或更高，例如大约1μM、大约2μM、大约3μM、大约4μM、大约5μM、大约6μM、大约7μM、大约8μM、大约9μM、大约10μM或更高。
在一方面，本发明提供一种通过本发明的方法制备的心肌细胞。
在一方面，本发明提供一种药物组合物，其包含通过本发明的方法制备的心肌细胞和药学上可接受的载体。
在一方面，本发明还提供通过本发明的方法制备的心肌细胞或本发明的药物组合物在制备用于在有需要的对象中治疗心脏疾病的药物中的用途。所述心脏疾病特别是心肌疾病，包括但不限于心力衰竭、心肌梗死等。
在一方面，本发明还提供一种在有需要的对象中治疗心脏疾病的方法，所述方法包括给所述对象施用通过本发明的方法制备的心肌细胞或本发明的药物组合物。
如本文所用，“对象”可以是哺乳动物或非哺乳动物。所述对象可以是人，或非人哺乳动物例如小鼠或大鼠或非人灵长类动物，优选是人。
在另一方面，本发明提供一种用于由多能干细胞分化制备心肌细胞的培养基，所述培养基包含所述CDK8抑制剂和所述WNT信号通路激活剂。在一些实施方案中，所述培养基还包含基础培养基，例如，所述基础培养基是RPMI培养基(如RPMI1640培养基)和不含胰岛素的B27细胞培养添加剂的组合；或者是CDM3培养基和不含胰岛素的S12细胞培养添加剂的组合。
在另一方面，本发明提供一种用于由多能干细胞分化制备心肌细胞的培养基系统，所述培养基系统包含
1)第一培养基，其包含本文所述CDK8抑制剂和所述WNT信号通路激活剂；
2)第二培养基，其包含本文所述WNT信号通路抑制剂；和
3)第三培养基，其包含胰岛素。
在一些实施方案中，所述第一、第二和/或第三培养基还包含基础培养基，例如，所述基础培养基是RPMI培养基(如RPMI1640培养基)和不含胰岛素的B27细胞培养添加 剂的组合；或者是CDM3培养基和不含胰岛素的S12细胞培养添加剂的组合。
在一些实施方案中，所述培养基系统还包含用于培养和/或扩增多能干细胞的第四培养基。所述第四培养基可以是任何可用于培养和/或扩增多能干细胞培养基，其是本领域技术人员容易获得的。
在一些实施方案中，所述培养基或培养基系统用于本发明的方法。
在一方面，本发明提供一种用于由多能干细胞分化制备心肌细胞的试剂盒，所述试剂盒至少包含本文所定义的CDK8抑制剂。在一些实施方案中，所述试剂盒包含本发明的培养基或培养基系统。
实施例
为了便于理解本发明，下面将参照相关具体实施例及附图对本发明进行更全面的描述。附图中给出了本发明的较佳实施例。但是，本发明可以以许多不同的形式来实现，并不限于本文所描述的实施例。相反地，提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
实施例1.人干细胞分化成心肌细胞
人干细胞分化成心肌细胞的主要流程如图1所示(参考Aguilar et al.,2015)。包括将人类hiPSC细胞单层培养，待其汇合度至80％左右开始分化，第一阶段(0-72h)使用WNT信号通路激活剂CHIR99021(CHIR)；第二阶段使用WNT信号通路抑制剂IWR1处理48h；第三阶段在基础分化培养基中加入胰岛素，细胞可自发分化为跳动的心肌细胞。整个过程经历干细胞(hiPSC)、心肌中胚层(Cardiac mesoderm，Stage I)、心脏祖细胞(CPC，Stage II)、心肌细胞(CM，Stage III)四个阶段，通常情况下7-10天即可在显微镜下观察到跳动的心肌细胞。更详细的实验步骤如下：
实验所用主要试剂见下表：

1.1干细胞的传代与培养
本实验使用的hiPSC常规培养于6孔板中，4天左右传代1次，置于恒温37℃、5％CO2的细胞培养箱。其传代步骤详述如下：
1)显微镜下观察细胞密度，细胞增殖至总面积80％左右准备传代；
2)传代前于孔板中提前铺Matrigel。Matrigel全程需在冰上操作。原始matrigel使用预冷DMEM/F-12稀释50倍后加入孔板中，加入量以能铺满皿底为标准(以6孔板为例，850uL/孔)，铺好后置于培养箱37℃孵育30min，使用前吸干液体；
3)将干细胞培养基PGM1或CDM(视后续实验目的决定)、DPBS和Versene提前于37℃预热，干细胞培养基加入Y27632(5μM)；
4)从培养箱取出细胞后吸干培养基，每孔加入1ml DPBS清洗剩余培养基后吸干，加入1mL Versene，37℃消化3min；
5)取出后细胞应底尚未脱落板底，立刻吸出Versene，使用1ml PGM1培养基吹打皿底细胞3-4次，使细胞从皿底脱落；
6)将细胞悬液与剩余培养基混合后，加入已铺好Matrigel的新孔板。传代比例为1:6-1:12，视起始细胞数量可稍有调整；
7)于传代后12-24h更换新的PGM1以撤去Y27632，每天观察细胞状态与密度。
1.2心肌细胞定向分化
干细胞至心肌细胞分化常规在24、96或384孔板中完成。步骤详述如下(图3.1)：
1)hiPSCs以1:10或1:12的比例分离至CDM培养基中，分离步骤与上述传代步骤一致，CDM培养基需加入Y27632(5μM)，记为第-3天；
2)于传代后12-24h换液以撤去Y27632，仍然使用CDM培养基培养，每天观察细胞状态与密度；
3)当hiPSC达到80-90％的汇合度时，将培养基更换为RPMI+B27minus(50ml培养基RPMI1640+1ml B27minus)，并加不同浓度CHIR，同时加入或不加入不同浓度BI-1347，记为分化第0天，hiPSC阶段结束；
注意：不加BI-1347时，CHIR使用剂量灵活且不稳定，细胞系不同、批次不同、操作者不同等均会导致分化结果较大的差异；
4)24-48h后将培养基更换为RPMI+B27minus；
5)72h时，将培养基更换为RPMI+B27minus并加入小分子IWR1 5μM，记为分化第3天，此时细胞分化为中胚层阶段，第一阶段结束；
6)IWR1加入48h后将培养基更换为RPMI+B27minus，撤去IWR1，记为分化第5天；
7)使用RPMI+B27minus培养24h，记为分化第6天，此时细胞分化为hiPSC-CPC，随后将培养基更换为RPMI+B27，第二阶段结束；
8)使用RPMI+B27持续培养，每3天换液一次，细胞将在3-6天内自发分化为跳动的hiPSC-CM，为分化第三阶段。最早可以于第7-8天观察到细胞跳动。
实施例2.CHIR过高剂量下利用小分子BI-1347提高hiPSC心肌分化效率
本发明人令人惊讶地发现，从人干细胞分化成心肌细胞的过程中，第一阶段(从hiPSC到心脏中胚层，0-72h)的CHIR99021(CHIR)剂量对分化是否成功起决定性作用。在起始细胞密度合适的前提下，分化第一阶段使用的WNT通路激活剂CHIR的最佳浓度不稳定，细胞系间或批次间有明显差异。且CHIR的最佳浓度范围很窄，往往只有2-4μM的浓度区域可以达到较高的分化效率。上述问题使分化体系十分不稳定，也使心肌细胞的大规模生产充满挑战。
本发明人更令人惊讶地发现，在第一阶段(0-48h)加入小分子BI-1347，可有效提高多种hiPSC细胞系在不同CHIR剂量组特别是高CHIR剂量组中的心肌分化效率(图2a、图2b，图3a、图3b)，使CHIR的有效作用浓度显著拓宽，分化约7-10天可见跳动的心肌细胞。加入BI-1347后，相同细胞系不同批次或不同细胞系，心肌分化效率标准差明显减小、均值显著提升，极少发生分化完全失败的批次(图2c、图2d)。
BI-1347对相同细胞系多批次实验中效果稳定且突出。有效浓度测试结果显示，BI-1347在较宽的浓度范围(0.004-3μM)内均有显著效果(图3c、图3d)。此外，由于BI-1347靶点(CDK8抑制剂)已知，还测试了具有相同靶点的另外两种化合物(MSC2530818、AS2863619)，其对心肌分化效率的影响与BI-1347相似(图2b)，因此这些小分子可通过对CDK8的抑制发挥作用。
在此基础上，对BI-1347优化体系后分化的心肌细胞(hiPSC-CM)进行鉴定。RNA-seq结果显示，使用或不使用BI-1347处理分化的心肌细胞相近，心肌特异性标记基因均显著表达(图4a-c)。免疫荧光结果显示心肌特异表达肌节相关蛋白cTNT、cTNI、α-ACTININ为阳性，心肌特异表达转录因子MEF2C、NKX2.5也呈阳性，与正常心肌细胞一致(图4d)。电生理鉴定结果也显示采用BI-1347分化的心肌细胞，动作电位、钙流等特征较为一致，分化心肌各个亚型的比例相似(图5a-j)。
综上所述，在干细胞分化为心肌细胞的第一阶段0-48h加入CDK8抑制剂如BI-1347可拓展CHIR浓度和时间的适用范围，提高不同细胞系间或不同批次间的心肌分化体系的效率和稳定性。

Claims (27)

  1. 一种由多能干细胞分化制备心肌细胞的方法，所述方法包括：
    i)提供多能干细胞；
    ii)在添加了CDK8抑制剂和WNT信号通路激活剂的基础培养基中培养所述多能干细胞大约24h-大约48h，然后在不添加所述CDK8抑制剂和WNT信号通路激活剂的基础培养基中继续培养所述细胞大约24h；
    iii)在添加了WNT信号通路抑制剂的基础培养基中培养步骤ii)所获得的细胞大约48h，然后在不添加WNT信号通路抑制剂的基础培养基中继续培养所述细胞大约24h；
    iv)在添加了胰岛素的基础培养基中培养步骤iii)所获得的细胞大约3-6天；和
    v)任选地，收获所获得的心肌细胞。
  2. 权利要求1的方法，其中所述多能干细胞选自胚胎干细胞(ESC)或诱导性多能干细胞(iPSC)。
  3. 权利要求1或2的方法，其中步骤i)中的多能干细胞在多孔板中培养。
  4. 权利要求1-3中任一项的方法，其中步骤i)中的多能干细胞生长至大约80-90％的汇合度。
  5. 权利要求1-4中任一项的方法，其中所述基础培养基是RPMI培养基和不含胰岛素的B27细胞培养添加剂的组合。
  6. 权利要求1-6中任一项的方法，其中步骤iv)中所述添加胰岛素的基础培养基是RPMI培养基(如RPMI1640培养基)和B27细胞培养添加剂的组合。
  7. 权利要求1-6中任一项的方法，其中所述CDK8抑制剂选自BI-1347或其结构类似物、MSC2530818或其结构类似物、AS2863619或其结构类似物、Senexin A或其结构类似物、CCT-251921或其结构类似物、Senexin C或其结构类似物、JH-XVI-178或其结构类似物、CCT251545或其结构类似物、BRD6989或其结构类似物、JH-XI-10-02或其结构类似物、SEL120-34A或其结构类似物、LY2857785或其结构类似物、CDK8-IN-1或其结构类似物、CDK8-IN-5或其结构类似物、CDK8-IN-3或其结构类似物、CDK8-IN-4或其结构类似物、CDK8-IN-6或其结构类似物、CDK8-IN-7或其结构类似物、CDK8/19-IN-1或其结构类似物、DS96432529或其结构类似物、Wogonin或其结构类似物，优选地，所述CDK8抑制剂是BI-1347或其结构类似物。
  8. 权利要求1-7中任一项的方法，其中所述WNT信号通路激活剂是CHIR99021或其结构类似物、CHIR98014或其结构类似物或SB212763或其结构类似物。
  9. 权利要求1-8中任一项的方法，其中所述WNT信号通路抑制剂是IWR1或其结构类似物、IWP2或其结构类似物或IWP4或其结构类似物。
  10. 权利要求1-9中任一项的方法，其中所述胰岛素为人胰岛素。
  11. 权利要求1-10中任一项的方法，其中所述CDK8抑制剂例如BI-1347或其结构类似物的终浓度为大约0.001μM-大约5μM，优选大约0.004μM-大约3μM，例如大约0.1 μM、大约0.2μM、大约0.3μM、大约0.4μM、大约0.5μM、大约0.6μM、大约0.7μM、大约0.8μM、大约0.9μM、大约1μM、大约2μM、大约3μM、大约4μM或大约5μM。
  12. 权利要求1-11中任一项的方法，其中所述WNT信号通路激活剂如CHIR99021或其结构类似物的终浓度为大约1μM-大约25μM或更高，例如大约1μM、大约2μM、大约3μM、大约4μM、大约5μM、大约6μM、大约7μM、大约8μM、大约9μM、大约10μM、大约11μM、大约12μM、大约13μM、大于14μM、大约15μM、大约16μM、大约17μM、大约18μM、大约19μM、大约20μM、大约21μM、大约22μM、大约23μM、大约24μM、大约25μM或更高。
  13. 权利要求1-12中任一项的方法，其中所述WNT信号通路抑制剂的浓度为大约1μM-大约10μM或更高，例如大约1μM、大约2μM、大约3μM、大约4μM、大约5μM、大约6μM、大约7μM、大约8μM、大约9μM、大约10μM或更高。
  14. 权利要求1-13中任一项的方法，其中所述多能干细胞是人多能干细胞，且所述心肌细胞是人心肌细胞。
  15. 权利要求1-14中任一项的方法，其中所述心肌细胞是功能性心肌细胞，例如能够跳动的心肌细胞。
  16. 权利要求1-15中任一项的方法，其中所述心肌细胞表达选自以下的一或多种心肌特异性蛋白：cTNT、cTNI、α-ACTININ、MEF2C和NKX2.5。
  17. 一种通过权利要求1-16中任一项的方法制备的心肌细胞。
  18. 药物组合物，其包含通过权利要求1-16中任一项的方法制备的心肌细胞和药学上可接受的载体。
  19. 通过权利要求17的心肌细胞或权利要求18的药物组合物在制备用于在有需要的对象中治疗心脏疾病的药物中的用途。
  20. 一种在有需要的对象中治疗心脏疾病的方法，所述方法包括给所述对象施用权利要求17的心肌细胞或权利要求18的药物组合物。
  21. 权利要求19的用途或权利要求20的方法，其中所述对象是人。
  22. CDK8抑制剂在制备用于通过多能干细胞分化制备心肌细胞的试剂中的用途。
  23. 权利要求22的用途，其中所述CDK8抑制剂选自BI-1347或其结构类似物、MSC2530818或其结构类似物、AS2863619或其结构类似物、Senexin A或其结构类似物、CCT-251921或其结构类似物、Senexin C或其结构类似物、JH-XVI-178或其结构类似物、CCT251545或其结构类似物、BRD6989或其结构类似物、JH-XI-10-02或其结构类似物、SEL120-34A或其结构类似物、LY2857785或其结构类似物、CDK8-IN-1或其结构类似物、CDK8-IN-5或其结构类似物、CDK8-IN-3或其结构类似物、CDK8-IN-4或其结构类似物、CDK8-IN-6或其结构类似物、CDK8-IN-7或其结构类似物、CDK8/19-IN-1或其结构类似物、DS96432529或其结构类似物、Wogonin或其结构类似物，优选地，所述CDK8抑制剂是BI-1347或其结构类似物。
  24. 权利要求22或23的用途，其中所述试剂用于与WNT信号通路激活剂、WNT 信号通路抑制剂和/或胰岛素组合来由多能干细胞分化制备心肌细胞。
  25. 权利要求22-24中任一项的用途，其中所述试剂用于提高由多能干细胞分化制备心肌细胞的效率。
  26. 权利要求22-25中任一项的用途，其中所述试剂用于在高浓度WNT信号通路激活剂存在下提高由多能干细胞分化制备心肌细胞的效率。
  27. 权利要求22-26中任一项的用途，其中所述试剂用于通过权利要求1-16中任一项的方法制备心肌细胞。
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