Method for inducing pluripotent stem cells to differentiate into ventricular myocytes in vitro

Abstract

Provided in the present invention is a method for inducing pluripotent stem cells to differentiate into ventricular myocytes in vitro, which is achieved by maintaining, amplifying and culturing pluripotent stem cells in vitro, adding a substance capable of activating the Smad1/5/8 signaling pathway directly or indirectly into the culture medium when pluripotent stem cells are in the middle stage of myocardial differentiation, i.e. the period of differentiating into cardiac muscle cells from mesoderm cells or myocardial precursor cells, which enables stem cells to differentiate into ventricular myocytes directionally. Ventricular myocytes with biological activity and function are obtained successfully by means of the method of the present invention, which reveals the regulatory mechanism during differentiation of myocardial precursor cells into ventricular myocytes; moreover, the human ventricular myocytes obtained via differentiation can be widely used in treating myocardial infarction by cell transplantation, in toxicological analysis of the heart and in the development of heart-related drugs.

Claims

1. A method for promoting stem cell differentiation into a ventricular cardiomyocyte, the method comprising: 1) activating the Smad1/5/8 signaling pathway in a mesodermal cell or a cardiac progenitor cell that is differentiated from a stem cell; and 2) inhibiting the Wnt signaling pathway in the mesodermal cell or cardiac progenitor cell, and 3) without purification, obtaining a cardiomyocyte cell population differentiated from the mesodermal cell or cardiac progenitor cell, wherein at least 80% of the cardiomyocyte population are ventricular cardiomyocytes, wherein said mesodermal cell or cardiac progenitor cell is simultaneously contacted with an exogenous activator of the Smad1/5/8 signaling pathway and an exogenous inhibitor of the Wnt signaling pathway in a same cell culture medium, thereby activating said Smad1/5/8 signaling pathway and inhibiting said Wnt signaling pathway in said mesodermal cell or cardiac progenitor cell.

2. The method of claim 1, wherein the stem cell is a pluripotent stem cell, a totipotent stem cell, a multipotent stem cell, an oligopotent stem cell, a unipotent stem cell, an embryonic stem cell, an induced pluripotent stem cell, a fetal stem cell, or an adult stem cell.

3. The method of claim 1, wherein the stem cell is a mammalian stem cell.

4. The method of claim 3, wherein the stem cell is a human stem cell, a human embryonic stem cell, or a human induced pluripotent stem cell.

5. The method of claim 1, wherein the stem cell has differentiated to form the mesodermal cell by contacting the stem cell with one or more of basic fibroblast growth factor (bFGF), bone morphogenetic protein 2 (BMP 2), bone morphogenetic protein 4 (BMP 4), activin A, a BMP antagonist, a BMP pathway inhibitor, and a Wnt3a pathway activator.

6. The method of claim 5, wherein the BMP antagonist is a BMP 4 antagonist, the BMP pathway inhibitor is a small molecule BMP pathway inhibitor, and/or the Wnt3a pathway activator is a small molecule Wnt3a pathway activator.

7. The method of claim 6, wherein the BMP 4 antagonist is Noggin, the small molecule BMP pathway inhibitor is Dorsomorphin, and/or the small molecule Wnt3a pathway activator is an ATP-competitive inhibitor of GSK-3α/β, a cell-permeable bis-indolo (indirubin) compound, or 6-bromoindirubin-3′-oxime (BIO).

8. The method of claim 1, wherein the Smad1/5/8 pathway is activated by: contacting the mesodermal cell with a BMP family member; contacting the mesodermal cell with an agonist of retinoic acid receptor γ (RARγ), wherein the mesodermal cell is cultured in a medium that does not comprise retinoic acid or a precursor thereof; or contacting the mesodermal cell with an antagonist of retinoic acid receptor α (RARα) and/or retinoic acid receptor β (RARβ), wherein the mesodermal cell is cultured in a medium comprising retinoic acid or a precursor thereof.

9. The method of claim 8, wherein: the BMP family member comprises BMP 2 and/or BMP 4, and the BMP 2 and/or BMP 4 is used at a final concentration of 0.01-1200 ng/ml; the retinoic acid precursor is vitamin A, and/or the RARγ agonist is BMS961 (3-Fluoro-4-[[2-hydroxy-2-(5,5,8,8-tetramethyl-5,6,7,8,-tetrahydro-2-naphthalenyl)acetyl]amino]-benzoic acid), Palovarotene (4-[(E)-2-[5,5,8,8-tetramethyl-3-(1H-pyrazol-1-ylmethyl)-5,6,7,8-tetrahydronaphthalen-2-yl]ethenyl]benzoic acid), or CD 437 (6-(4-Hydroxy-3-tricyclo[3.3.1.13,7]dec-1-ylphenyl)-2-naphthalenecarboxylic acid), and/or the RARγ agonist is used at a final concentration of 0.001-100 μM; or the antagonist of RARα is Ro41-5253, BMS195614, or ER50891, and/or the antagonist of RARβ is LE135, and/or the antagonist of RARα and/or RARβ is used at a final concentration of 0.001-100 μM.

10. The method of claim 1, wherein the inhibition of the Wnt signaling pathway comprises contacting the mesodermal cell with a Wnt inhibitor to differentiate the mesodermal cell into a ventricular cardiomyocyte.

11. The method of claim 10, wherein the Wnt inhibitor comprises at least one of dickkopf homolog 1 (DKK1), IWP, and an inhibitor of Wnt response (IWR).

12. The method of claim 10, wherein the Wnt inhibitor is used at a final concentration of 0.01-1200 ng/ml.

13. A method for inducing stem cell differentiation into a ventricular cardiomyocyte, comprising: differentiating a stem cell to a mesodermal cell or a cardiac progenitor cell; activating the Smad1/5/8 signaling pathway in the mesodermal cell or cardiac progenitor cell; and inhibiting the Wnt signaling pathway in the mesodermal cell or cardiac progenitor cell, and without purification, obtaining a cardiomyocyte cell population differentiated from the mesodermal cell or cardiac progenitor cell in which at least 80% of the cardiomyocyte population are ventricular cardiomyocytes, wherein said mesodermal cell or cardiac progenitor cell is simultaneously contacted with an exogenous activator of the Smad1/5/8 signaling pathway and an exogenous inhibitor of the Wnt signaling pathway in a same cell culture medium, thereby activating said Smad1/5/8 signaling pathway and inhibiting said Wnt signaling pathway in said mesodermal cell or cardiac progenitor cell.

14. The method of claim 13, wherein the stem cell is differentiated to the mesodermal cell or cardiac progenitor cell by adding one or more factors that promote differentiation to cardiomyocyte in a culture medium of the stem cell.

15. The method of claim 14, wherein the one or more factors that promote differentiation to cardiomyocyte comprise at least one of BMP4, bFGF, Activin A, Noggin, Dorsomorphin, and a Wnt3a pathway activator.

16. The method of claim 13, wherein the Wnt signaling pathway is inhibited by adding an inhibitor of the Wnt signaling pathway in a culture medium of the mesodermal cell or cardia progenitor cell.

17. The method of claim 16, wherein the inhibitor of the Wnt signaling pathway comprises at least one of dickkopf homolog 1 (DKK1), IWP, and an inhibitor of Wnt response (IWR).

18. The method of claim 13, wherein the Smad1/5/8 signaling pathway is activated by adding in a culture medium of the mesodermal cell or cardia progenitor cell: i) a BMP family member; ii) an activator of retinoic acid receptor (RARγ), wherein the culture medium does not contain retinoic acid or a precursor thereof; or iii) an antagonist of RARα and/or RARβ, wherein the culture medium contains retinoic acid or a precursor thereof.

19. The method of claim 18, wherein the BMP family member comprises BMP 2 and/or BMP 4.

20. A method for generating a ventricular cardiomyocyte from a stem cell, which method comprises: 1) contacting a stem cell with an agent to initiate stem cell differentiation; 2) differentiating the stem cell treated by the agent to form a mesodermal cell; 3) activating the Smad1/5/8 pathway in the mesodermal cell to promote ventricular cardiomyocyte formation; 4) contacting the mesodermal cell with one or more of DKK1, IWP, and an inhibitor of Wnt response (IWR) to differentiate the mesodermal cell into a ventricular cardiomyocyte; and 5) without purification, obtaining a cardiomyocyte cell population differentiated from the stem cell, wherein at least 80% of the cardiomyocyte population are ventricular cardiomyocytes, wherein said mesodermal cell or cardiac progenitor cell is simultaneously contacted with an exogenous activator of the Smad1/5/8 signaling pathway and an exogenous one or more of DKK1, IWP and an inhibitor of Wnt response (IWR) in a same cell culture medium, thereby activating said Smad1/5/8 signaling pathway and inhibiting said Wnt signaling pathway in said mesodermal cell or cardiac progenitor cell.

21. The method of claim 20, wherein the Smad1/5/8 signaling pathway is activated by: (i) contacting the mesodermal cell with a BMP family member; (ii) contacting the mesodermal cell with an agonist of RARγ, wherein the mesodermal cell is cultured in a medium that does not comprise retinoic acid or a precursor thereof; or (iii) contacting the mesodermal cell with an antagonist of RARα and/or RARβ, wherein the mesodermal cell is cultured in a medium comprising retinoic acid or a precursor thereof.

22. The method of claim 21, wherein the BMP family member comprises BMP 2 and/or BMP 4.

23. The method of claim 1, wherein the exogenous activator of the Smad1/5/8 signaling pathway comprises a BMP family member and the exogenous inhibitor of the Wnt signaling pathway comprises DKK1, and the same cell culture medium comprises both DKK1 and the BMP family member.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows the expression of molecules involved in the BMP signaling pathway during the middle stage of cardiac differentiation of stem cells, and the effects on expression of the ventricle-specific marker gene IRX-4. FIG. 1A shows reverse transcription-polymerase chain reaction (RT-PCR) analysis of the expression of BMP2, BMP4, and their receptors at 5 and 6 days of cardiac differentiation. FIG. 1B shows western blot analysis of downstream signaling molecules [phosphorylated Smad1/5/8 (P-Smad1/5/8)] of the BMP pathway. T-Smad1/5/8 represents total Smad1/5/8 proteins. β-actin served as an internal loading control. The histogram in FIG. 1C presents the experimental results of quantitative RT-PCR analysis of IRX-4 gene expression levels at 14 days of differentiation. The results show IRX-4 expression levels in cells treated with 1 μM retinoic acid and 200 ng/mL BMP4 during different stages of differentiation. Connected line indicates the cardiac differentiation efficiency of the stem cells under the corresponding inductive conditions. N represents noggin, B represents BMP4; NVa represents differentiated cells cultured in vitamin A-free medium; RA represents retinoic acid; and numbers represent the concentrations (Unit for BMP4 is ng/mL). Data are expressed as relative values compared with the expression level of glyceraldehyde-3-phosphate dehydrogenase (GADPH).

(2) FIG. 2 presents quantitative RT-PCR analysis of the expression levels of the ventricle-specific early marker gene IRX-4 at day 14 in differentiated cultures with various treatments. FIG. 2A shows that, after the addition of BMP4 with various concentrations to the cultures, the IRX-4 expression level is elevated with increasing concentrations of BMP4. However, the expression level is reduced by addition of a BMP antagonist, noggin. FIG. 2B illustrates that the IRX-4 expression level is effectively reduced by addition of various doses of noggin to the medium without the retinoic acid precursor, vitamin A. FIG. 2C shows that in the presence of 1 μM retinoic acid, the IRX-4 expression level is elevated with increasing concentrations of BMP4 added to the cultures. FIG. 2D shows that the elevation of the IRX-4 expression level by BMP4 in the presence of retinoic acid is reduced with additions of increasing concentrations of noggin in the cultures. N represents noggin; B represents BMP4, NVa represents differentiated cells treated in vitamin A-free medium; RA represents retinoic acid; and numbers represents the concentrations (Unit is ng/mL). The results of quantitative RT-PCR are indicated as relative values compared with the expression levels of GADPH.

(3) FIG. 3 presents quantitative RT-PCR analysis of IRX-4 gene expression levels at day 14 of differentiation. The results show that by treatment of the stem cells at days 5-8 of cardiac differentiation, other members of the BMP family also effectively antagonized the inhibitory effect of retinoic acid on IRX-4 expression. The antagonistic effect is enhanced with increasing doses of BMP family member growth factors. RA represents retinoic acid; numbers represent the concentrations of the growth factor (Unit is ng/mL); the concentration of retinoic acid is 1 μM. The results of quantitative RT-PCR are indicated as relative values compared with the expression levels of GADPH.

(4) FIG. 4 displays ventricle-specific MLC-2v expression in long term cultures that differently treated with retinoic acid, BMP4, and noggin. FIG. 4A shows western blot analysis of MLC-2v expression in stem cell-derived CMs at day 90 of differentiation treated with retinoic acid, noggin and BMP4 with different combinations. FIG. 4B presents the results of double immunofluorescence staining of cTNT and MLC-2v in CMs at day 90 of differentiation after retinoic acid, BMP4, and noggin treatments. Letter B in the figures represents BMP4, NVa represents differentiated cells cultured in vitamin A-free medium; RA represents 1 μM retinoic acid; and numbers represent the concentrations (ng/mL).

(5) FIG. 5 presents images from confocal laser scanning microscopy and simultaneous recordings of APs of calcium activity in differentiated CMs and the classification of the differentiated CMs according to the specific calcium activity patterns in the various types of CMs. FIG. 5A shows the features of Ca.sup.2+ sparks in CMs with ventricular-like APs, Ca.sup.2+ transients in cells with atrial-like APs, and Ca.sup.2+ oscillations in cells with nodal-like APs. FIG. 5B presents the proportions of CMs with Ca.sup.2+ sparks, Ca.sup.2+ transients, and Ca.sup.2+ oscillations in different treatments as classified by calcium signaling patterns of the various subtypes of CMs in A. The vertical axis represents the proportions of CMs with the three different calcium activities; RA represents 1 μM retinoic acid; Letter B represents BMP4; NVa represents vitamin A-free medium; N represents noggin; and numbers represent the concentrations (ng/mL).

(6) FIG. 6 shows quantitative RT-PCR analysis of BMP2 expression in cells treated with a RARγ activator at day 6 of differentiation. The results of quantitative RT-PCR are indicated as relative values compared with those of GADPH. NVa represents vitamin A-free medium.

(7) FIG. 7 shows quantitative RT-PCR analysis of ventricle-specific IRX-4 expression at day 14 of stem cell differentiation after the addition of various regulators (a RAR activator or inhibitor) to vitamin A-free medium during middle stage of cardiac differentiation of stem cells. RA represents retinoic acid; RAi represents a retinoic acid inhibitor, BMS189453; NVa represents vitamin A-free medium; The RAR pan-antagonist is BMS493. The results of quantitative RT-PCR are expressed as relative values compared with cTNT expression

(8) FIG. 8 shows the proportions of cardiomyocytes with different AP characteristics, and MLC-2v (a mature VM-specific marker gene) expressing cells in the total cardiomyocyte population differentiated under various differentiation conditions. FIG. 8A shows the proportion of cells with atrial-, ventricular-, and nodal-like APs in CMs differentiated under various induction conditions (n>30). FIG. 8B shows flow cytometric analyses of the proportions of MLC-2v-expressing cells in the total cardiomyocytes population (cTNT-positive cells) among 90 day cultures treated under various conditions. RA represents 1 μM retinoic acid; B represents BMP4; NVa represents vitamin A-free medium; N represents noggin; and numbers represent the concentrations (Unit is ng/mL). The RARγ concentration is 0.1 μM.

(9) FIG. 9 illustrates the process of inducing differentiation of PSCs into VMs in vitro in Example 2 (infra) of the present invention.

(10) FIG. 10 illustrates the process of inducing differentiation of PSCs into VMs in vitro in Example 3 (infra) of the present invention.

(11) FIG. 11 illustrates the process of inducing differentiation of PSCs into VMs in vitro in Example 4 (infra) of the present invention.

(12) FIG. 12 presents the results of double immunofluorescence staining of cTnT and MLC-2v in differentiated CMs after retinoic acid treatment.

(13) FIG. 13 presents the results of double immunofluorescence staining of cTnT and MLC-2v in differentiated CMs after treatment with retinoic acid and 200 ng/mL BMP4.

(14) FIG. 14 presents the results of double immunofluorescence staining of cTnT and MLC-2v in differentiated CMs after treatment with 1200 ng/mL noggin in vitamin A-free medium.

(15) FIG. 15 presents the results of double immunofluorescence staining of cTnT and MLC-2v in differentiated CMs acquired from Example 2 (infra).

(16) FIG. 16 presents the results of double immunofluorescence staining of cTnT and MLC-2v in differentiated CMs after culture in vitamin A-free medium.

(17) FIG. 17 presents the results of double immunofluorescence staining of cTnT and MLC-2v in differentiated CMs acquired from Example 3 (infra).

(18) In FIGS. 12-17, “*” indicates non-ventricular CMs and “{circumflex over ( )}” indicates MLC-2v-expressing VMs.

EXAMPLES

(19) The following examples are provided to describe the present invention, but do not restrict the scope of the present invention. Unless specified otherwise, the technical terms used in the embodiments are conventional terms known to those individuals skilled in the procedures using materials that are commercially available.

(20) In the following examples, the human ESC line H7 was purchased from WiCell Research Institute, USA; B27 supplement and RPMI1640 medium were purchased from Invitrogen; Activin A, bFGF, DKK1, BMP4, and noggin were purchased from R&D systems.

Example 1

Role of BMP/Smad1/5/8 Signaling Pathways in Inducing Differentiation of Cardiac Progenitor Cells into VMs

(21) 1) Previous research indicated that during cardiac differentiation of stem cells, addition of retinoic acid or its precursors (e.g., vitamin A) to the culture medium at the differentiation stage that determines the subtype of CMs induces directed differentiation of stem cells into AMs. On the other side, addition of a retinoic acid inhibitor to the culture medium or exclusion of vitamin A in the culture medium induces directed differentiation of stem cells into VMs. See, Zhang Q et al., Cell Res., 2011, 21:579-587. Using RT-PCR, the expression of BMP2/4 and their corresponding receptor in differentiated human ESCs was analyzed during the middle stage of cardiac differentiation. As shown in FIG. 1, both the ligands and receptors of the BMP pathway are expressed in the cultured cells. Western blot analysis of BMP2/4 downstream signaling molecules (phosphorylated Smad1/5/8) showed that under the culture condition with retinoic acid addition and in the absence of retinoic acid or vitamin A, Smad1/5/8 molecules are phosphorylated (FIG. 1). These results demonstrated that activation of the BMP pathway during the middle stage of cardiac differentiation of stem cells. These results indicated that during days 5-8 of stem cell differentiation, the BMP pathway is involved in regulating the differentiation of cardiac progenitor cells into VMs.

(22) 2) The role of the BMP pathway in directed differentiation of CM subtypes was analyzed further during cardiac differentiation of stem cells. IRX-4 is a marker gene expressed during early differentiation of VMs. Thus, the IRX-4 expression level was measured to further study the role of the BMP pathway in differentiation of CM subtypes.

(23) As indicated in FIG. 2, in vitamin A-free medium, the IRX-4 expression level was effectively reduced by addition of a BMP2/4 pathway inhibitor, noggin, during day 5 to day 8 of differentiation. The expression level of IRX-4 decreased with increasing doses of noggin (300, 600, and 1200 ng/mL).

(24) 3) IRX-4 expression level was repressed by addition of retinoic acid during day 5 to day 8 of differentiation. Furthermore, when retinoic acid was added simultaneously with various doses of BMP4 during day 5 to day 8 of differentiation, and the measurement of the expression levels of IRX-4 by quantitative RT-PCR showed that the IRX-4 expression level in retinoic acid-treated culture was elevated by addition of BMP. As the dose of BMP4 increased, the expression level of IRX-4 increased correspondingly (FIG. 2).

(25) Additionally, other members of the BMP family antagonize the inhibitory effect of retinoic acid on IRX-4 expression. Most BMP family members have similar functions. Quantitative RT-PCR analysis (FIG. 3) showed that during middle stage of cardiac differentiation, the IRX-4 expression level was elevated to various degrees by other BMP family members in the presence of 1 μM retinoic acid. The expression level of IRX-4 increased with increasing concentrations of those BMP family members added to the medium.

(26) In summary, the analysis of early specific IRX-4 expression in differentiated cultures indicated that the BMP signaling pathway effectively improves IRX-4 expression in differentiated CMs. This finding shows that during differentiation of stem cells, the BMP signaling pathway is involved in their early differentiation into VMs and plays a role in promoting this process.

(27) 4) To verify the role of the BMP pathway in regulating the differentiation of VMs from stem cells, calcium activities of cardiomyocytes were recorded with confocal laser scanning microscopy at 60-90 days of differentiation. Calcium activity in VMs clearly differs from that in AMs and nodel cells. Calcium activity in VMs has a higher imaging frequency, known as Ca.sup.+ sparks. Imaging of the AM calcium activity showed a lower frequency with large signals, called Ca.sup.+ transients, whereas imaging of calcium activity in nodel cells demonstrated obvious periodicity called Ca.sup.+ oscillations. First, using the single-cell patch clamp technique in conjunction with confocal laser scanning microscopy, it was found that patch clamp-recorded calcium activities in 20 cells with ventricular-like APs exclusively shows Ca.sup.+ sparks. Patch clamp-recorded calcium activities in 20 cells with atrial-like APs exclusively showed Ca.sup.+ transients, whereas patch clamp-recorded calcium activities in 20 cells with nodal-like APs exclusively shows Ca.sup.+ oscillations. Thus, comparing the image pattern of calcium activities in CMs is an effective method to distinguish VMs from AMs and nodel cells. Calcium imaging data showed that the majority of differentiated CMs with retinoic acid treatment had Ca.sup.+ transients, while the proportion of cells with Ca.sup.+ transients decreased with increasing concentrations of BMP4. In contrast, the proportion of cells with Ca.sup.+ sparks among differentiated CMs increased with increasing concentrations of BMP4. This result indicates that activation of the BMP signaling pathway effectively induces stem cells to differentiate into VMs (FIG. 5).

(28) 5) As mentioned above, during early differentiation of CMs, IRX-4 is an important gene with specific expression in differentiated VMs. As CMs mature, VMs begin to specifically express the MLC-2v gene. Thus, the MLC-2v expression level was measured in differentiated cells at day 90 of culture after treatment with various growth factors. Western blot analysis showed that the MLC-2v protein expression level in differentiated cells at day 90 also increased with increasing concentrations of BMP4 (FIG. 4). Moreover, flow cytometry was performed to determine the proportion of MLC-2v-expressing VMs among differentiated CMs (cTNT-expressing cells) at day 90. The results (FIG. 8) showed that addition of BMP4 to differentiated cells after retinoic acid treatment effectively increased the proportion of MLC-2v-expressing cells among differentiated CMs, with the highest proportion obtained by BMP4 treatment alone. The most classical method to identify the subtypes of CMs is to measure the APs of the CMs. The proportions of cells with atrial-, ventricular-, and nodal-like APs among differentiated CMs were analyzed after treatment with retinoic acid and various doses of BMP4 (FIG. 8). Among differentiated CMs after retinoic acid treatment, the proportion of cells with ventricular-like APs increased significantly with increasing doses of BMP4. Among differentiated cells treated with BMP4 alone, more than 90% of CMs had ventricular-like APs, indicating that more than 90% of CMs were VMs.

(29) 6) Addition of BMP2/4, activin A, bFGF and/or noggin during early cardiac differentiation of ESCs and growth factors such as DKK1 during the middle stage of cardiac differentiation efficiently induced stem cells to differentiate into CMs. Quantitative RT-PCR analysis showed that ventricle-specific IRX-4 expression was reduced by addition of RARα and RARβ activators along with DKK1 during middle stage of cardiac differentiation (FIG. 7). However, high expression of BMP2 and IRX-4 was induced by addition of DKK1 with a RARγ activator (FIGS. 6 and 7). Additionally, in vitamin A-containing medium, early specific IRX-4 expression in VMs was activated by addition of DKK1 and antagonists of RARα and RAR. This indicated induced differentiation of stem cells into VMs. Because retinoic acid has three RARs receptors (RARα, RARβ, and RARγ), simultaneous inhibition of RARα and RARβ in the presence of vitamin A or retinoic acid has a similar mechanism and effect as that of independent activation of RARγ alone.

(30) Flow cytometric analysis (FIG. 8) demonstrated that the proportion of MLC-2v-expressing cardiomyocytes in cardiomyocytes population (cTNT-expressing cells) reached up to 80% at 90 days of differentiation induced by RARγ. Additionally, electrophysiological identification of APs indicated that 92% of RARγ-induced, differentiated CMs at 90 days had ventricular-like APs.

Example 2

Inducing Differentiation of PSCs into VMs In Vitro (Technical Solution I)

(31) The human ESC line H7 was cultured on gelatin-coated petri dishes in RPMI 1640 medium supplemented with B27 at 37° C. in a CO.sub.2 incubator. The process of cardiac differentiation is presented in FIG. 9. During the first 3 days of differentiation, the differentiation medium contained activin A (10 ng/mL), BMP4 (6 ng/mL), and bFGF (6 ng/mL). At the end of day 3, the medium was exchanged with a BMP2/4 inhibitor, noggin (300 ng/mL) added to the medium. At the end of day 5, the medium was replaced with vitamin A-free, B27 supplemented RPMI1640 medium. A Wnt3a inhibitor, DKK1 (300 ng/mL) and BMP4 (10 ng/mL) were also added to the medium. At the end of day 8, the medium was replaced with medium containing 300 ng/mL DKK1 only. At the end of day 10, the medium was replaced with growth factor-free medium. Thereafter, the medium was replaced with B27-containing RPMI1640 medium every 3 days. A large number of beating CMs was observed at day 14 of differentiation. The workflow of the technical solution is shown in FIG. 9.

Example 3

Inducing Differentiation of PSCs into VMs In Vitro (Technical Solution II)

(32) The human ESC line H7 was cultured on gelatin-coated petri dishes in RPMI 1640 medium with 1×B27 at 37° C. in a CO.sub.2 incubator. The process of cardiac differentiation is presented in FIG. 10. During the first 3 days of differentiation, the differentiation medium contained activin A (10 ng/mL), BMP4 (6 ng/mL), and bFGF (6 ng/mL). At the end of day 3, the medium was exchanged with differentiation medium containing a BMP2/4 inhibitor, noggin (300 ng/mL). At the end of day 5, the medium was replaced with vitamin A-free, B27-containing RPMI 1640 medium containing a Wnt3a inhibitor, DKK1 (300 ng/mL), and RARγ activator, BMS961 (0.1 μM, Tocris). At the end of day 8, the medium was replaced with medium containing 300 ng/mL DKK1 only. At the end of day 10, the medium was replaced with growth factor-free medium. Thereafter, the medium was replaced with B27-containing RPMI 1640 medium every 3 days. A large number of beating CMs was observed at day 14 of differentiation. The workflow of the technical solution is shown in FIG. 10.

Example 4

Inducing Differentiation of PSCs into VMs In Vitro (Technical Solution III)

(33) The human ESC line H7 was cultured on gelatin-coated petri dishes in 1×B27-containing RPMI 1640 medium at 37° C. in a CO.sub.2 incubator. The process of cardiac differentiation is presented in FIG. 11. During the first 3 days of differentiation, the differentiation medium contained activin A (10 ng/mL), BMP4 (6 ng/mL), and bFGF (6 ng/mL). At the end of day 3, the medium was exchanged with differentiation medium containing a BMP2/4 inhibitor, noggin (300 ng/mL). At the end of day 5, the medium was replaced with vitamin A-free, B27-containing RPMI1640 medium containing a Wnt3a inhibitor, DKK1 (300 ng/mL), as well as antagonists of RARα and RARβ, BMS195614 (0.1 μM) and LE135 (0.5 μM), respectively. At the end of day 8, the medium was replaced with medium containing 300 ng/mL DKK1 only. At the end of day 10, the culture medium was replaced with growth factor-free medium. Thereafter, the medium was replaced with B27-containing RPMI 1640 medium every 3 days. A large number of beating CMs was observed at day 14 of differentiation. The workflow of the technical solution is shown in FIG. 11.

(34) FIG. 12 shows the results of double immunofluorescence staining of cTnT and MLC-2v in differentiated CMs after retinoic acid treatment.

(35) FIG. 13 shows the results of double immunofluorescence staining of cTnT and MLC-2v in differentiated CMs after treatment with retinoic acid and 200 ng/mL BMP4.

(36) FIG. 14 shows the results of double immunofluorescence staining of cTnT and MLC-2v in differentiated CMs after treatment with 1200 ng/mL Noggin in vitamin A-free medium.

(37) FIG. 15 shows the results of double immunofluorescence staining of cTnT and MLC-2v in differentiated CMs acquired from Example 2.

(38) FIG. 16 shows the results of double immunofluorescence staining of cTnT and MLC-2v in differentiated CMs after culture in vitamin A-free medium.

(39) FIG. 17 shows the results of double immunofluorescence staining of cTnT and MLC-2v in differentiated CMs acquired from Example 3.

(40) In FIGS. 12-17, “*” indicates non-ventricular CMs and “{circumflex over ( )}” indicates MLC-2v-expressing VMs.

(41) Confocal laser scanning microscopy was performed to analyze calcium activities in differentiated CMs acquired from Example 2 and 3 at 60-90 days. The results of calcium imaging are shown in FIG. 5, from which the proportion of cells with Ca.sup.2+ sparks among total differentiated CMs can be calculated directly.

(42) In the above examples, the effective ranges of the final concentration for the relevant additives in the medium are 0.01-1200 ng/mL for growth factors and 0.001-100 μM for small molecules.

(43) In some aspects, the methods disclosed in the present invention successfully generate biologically active and functional VMs. These methods can be used to reveal the regulatory mechanisms of CPC differentiation into VMs, whereas the resulting differentiated human VMs have extensive applications in cell transplantation therapy of myocardial infarction, toxicological analysis of cardiac drugs, and cardiac drug development.

(44) Although the present invention has been fully described with general instructions and specific embodiments, it is noted that various changes and modifications will become apparent to those skilled in the procedures. Therefore, such changes and modifications made to the invention without departing from its essence are being protected within the scope of the present invention as claimed.

INDUSTRIAL APPLICABILITY

(45) In some aspects, the present invention provides a method to induce differentiation of PSCs into VMs in vitro, which successfully generates biologically active and functional VMs. It can not only reveal the regulatory mechanisms underlying differentiation of VMs from CSCs, but also produce human VMs that have broad applications in cell transplantation therapy of myocardial infarction, as well as cardiac-toxicological analysis of drug safety, and drug development for heart diseases.