Methods and compositions for preparing cardiomyocytes from stem cells and uses thereof

• US 10,590,386 B2
• Filed: 10/28/2015
• Issued: 03/17/2020
• Est. Priority Date: 06/13/2010
• Status: Active Grant

• Current Assignee: Institute of Biophysics Chinese Academy of Sciences (Chinese Academy of Sciences)
• Original Assignee: Institute of Biophysics Chinese Academy of Sciences (Chinese Academy of Sciences)
• Inventors: Ma, Yue
• Primary Examiner(s): Moloye, Titilayo
• Application Number:
  US14/925,829
• Publication Number:
  US 20160122719A1
• Time in Patent Office: 1,602 Days
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/385 of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0657 Cardiomyocytes; Heart cells

Abstract

The present invention discloses novel compositions and methods for enhancing cardiac differentiation efficiency of stem cells or promoting ventricular and atrial cardiomyocytes formation from stem cells. The present invention also discloses the atrial and ventricular cardiomyocytes formed from the stem cells, and the uses of the cardiomyocytes for repairing cardiac injuries and screening for new medicaments for treating cardiac injuries.

Claims

1. A composition comprising a mesodermal cell prepared in vitro and an exogenous agent that inhibits the retinoic acid signaling pathway in the mesodermal cell, wherein the retinoic acid signaling pathway of the mesodermal cell is inhibited by the exogenous agent thereby promoting ventricular cardiomyocyte formation from the mesodermal cell.

2. The composition of claim 1, wherein the mesodermal cell is differentiated in vitro from a pluripotent stem cell, a totipotent stem cell, a multipotent stem cell, an oligopotent stem cell, or a unipotent stem cell.

3. The composition of claim 1, wherein the mesodermal cell is differentiated in vitro from a mammalian stem cell.

4. The composition of claim 1, wherein the mesodermal cell is differentiated in vitro from a human stem cell.

5. The composition of claim 1, wherein the mesodermal cell is prepared in vitro by contacting a stem cell with at least one of basic fibroblast growth factor (bFGF), BMP 4 and activin A.

6. The composition of claim 1, wherein the mesodermal cell is prepared in vitro by contacting a stem cell with Wnt-3a, Bio, or CHIR99021.

7. The composition of claim 1, wherein the exogenous agent is selected from the group consisting of a retinoic acid antagonist, a retinoic acid receptor antagonist, and a retinoic X receptor antagonist.

8. The composition of claim 1, wherein the retinoic acid signaling pathway of the mesodermal cell is inhibited by contacting the mesodermal cell with the exogenous agent that reduces vitamin A, or by contacting the mesodermal cell with the exogenous agent that does not comprise vitamin A.

11. The composition of claim 1, wherein the mesodermal cell is differentiated in vitro from an embryonic stem cell, an induced pluripotent stem cell, a fetal stem cell, or an adult stem cell.

12. The composition of claim 1, wherein the mesodermal cell is differentiated in vitro from a human embryonic stem cell or a human induced pluripotent stem cell.

13. The composition of claim 1, wherein the exogenous agent is a pan-retinoic acid receptor antagonist.

14. The composition of claim 1, wherein the exogenous agent is an inhibitor of retinoic acid synthesis.

15. The composition of claim 1, wherein the exogenous agent is selected from the group consisting of BMS-189453, AGN194310, AGN193109, Ro41-5253, SR11335, 9-cis-retinoic acid, disulfiram, and citral.

16. The composition of claim 1, wherein the exogenous agent is BMS-189453.

17. The composition of claim 1, further comprising a BMP antagonist.

18. The composition of claim 17, wherein the BMP antagonist is selected from the group consisting of a BMP 4 antagonist, Noggin, Chordin, Tsg, a BMP soluble receptor, BMPRIA, BMPRIB, and Dorsomorphin.

19. The composition of claim 1, further comprising a Wnt inhibitor.

20. The composition of claim 19, wherein the Wnt inhibitor is dickkopf homolog 1 (DKK1).

21. The composition of claim 1, further comprising a BMP antagonist and a Wnt inhibitor.

22. The composition of claim 21, wherein the exogenous agent is BMS-189453, the BMP antagonist is Noggin, and the Wnt inhibitor is dickkopf homolog 1 (DKK1).

23. The composition of claim 1, further comprising a culture medium with reduced or depleted vitamin A.

24. The composition of claim 1, wherein the mesodermal cell is prepared in vitro by culturing said mesodermal cell on a cell culture plate.

9. A cell culture medium, which comprises a population of cells differentiated in vitro from a mesodermal cell whose retinoic acid signaling pathway has been inhibited by an exogenous agent, wherein, without enrichment or isolation, at least 60% of the cells in the population are cardiomyocytes, and wherein at least 80% of the cardiomyocytes are ventricular cardiomyocytes.

10. The cell culture medium of claim 9, wherein at least 90%, at least 95%, at least 99%, or 100% of the cardiomyocytes in the cell population are ventricular cardiomyocytes.