Method for Inducing Pluripotent Stem Cells to Differentiate into Ventricular Cardiomyocytes 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.

1 - 11 . (canceled) 
     
     
         12 . A method for promoting pluripotent stem cell differentiation into ventricular cardiomyocyte(s), the method comprises activating the Smad1/5/8 signaling pathway in a pluripotent stem cell at the middle stage of pluripotent stem cell differentiation, wherein the middle stage is when a mesodermal cell or a cardiac progenitor cell differentiates into a cardiomyocyte. 
     
     
         13 . The method of  claim 12 , characterized in that the stem cell is a pluripotent stem cell, a totipotent stem cell, a multipotent stem cell, an oligopotent stem cell, or a unipotent stem cell. 
     
     
         14 . The method of  claim 12 , characterized in that the stem cell is an embryonic stem cell, an induced pluripotent stem cell, a fetal stem cell, or an adult stem cell. 
     
     
         15 . The method of  claim 12 , characterized in that the stem cell is a mammalian stem cell. 
     
     
         16 . The method of  claim 15 , characterized in that the mammalian stem cell is a human stem cell. 
     
     
         17 . The method of  claim 12 , characterized in that the stem cell is a human embryonic stem cell or a human induced pluripotent stem cell. 
     
     
         18 . The method of  claim 12 , characterized in that the stem cell has differentiated to form mesoderm by contacting an undifferentiated 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. 
     
     
         19 - 27 . (canceled) 
     
     
         28 . The method of  claim 12 , characterized in that the Smad1/5/8 pathway is activated by contacting the stem cell with BMP 2 and/or BMP 4. 
     
     
         29 . The method of  claim 28 , characterized in that the BMP 2 and/or BMP 4 is used at a final concentration of 0.01-1200 ng/ml. 
     
     
         30 . The method of  claim 12 , characterized in that the Smad1/5/8 pathway is activated by culturing the stem cell in a medium that does not comprise retinoic acid or its precursor and contacting the stem cell with an agonist of retinoic acid receptor 7 (RARγ). 
     
     
         31 . The method of  claim 30 , characterized in that the retinoic acid precursor is vitamin A. 
     
     
         32 . The method of  claim 30 , characterized in that the RARγ agonist is BMS961, Palovarotene, or CD437 from SIGMA-ALDRICH. 
     
     
         33 . The method of  claim 30 , characterized in that the RARγ agonist is used at a final concentration of 0.001-100 μM. 
     
     
         34 . The method of  claim 12 , characterized in that the Smad1/5/8 pathway is activated by contacting the stem cell with an antagonist of retinoic acid receptor α (RARα) and/or retinoic acid receptor β (RARβ). 
     
     
         35 . The method of  claim 34 , characterized in that the antagonist of RARα is Ro41-5253, BMS195614, or ER50891, and the antagonist of RARβ is LE135. 
     
     
         36 . The method of  claim 33 , characterized in that the antagonist of RARα and/or RARβ is used at a final concentration of 0.001-100 μM. 
     
     
         37 - 51 . (canceled) 
     
     
         52 . A pharmaceutical composition for treating a cardiac injury or disorder, which pharmaceutical composition comprises an effective amount of the ventricular cardiomyocytes produced by the method of  claim 12 , and a pharmaceutically acceptable carrier or excipient. 
     
     
         53 . A method for treating a cardiac injury or disorder in a subject, which method comprises administering, to a subject to which such treatment is needed or desirable, an effective amount of the pharmaceutical composition of  claim 52 . 
     
     
         54 - 59 . (canceled)