Methods for making and using sinoatrial node-like pacemaker cardiomyocytes and ventricular-like cardiomyocytes

Patent metadata

Field	Value
Publication number	US-10561687-B2
Application number	US-201615550572-A
Country	US
Kind code	B2
Filing date	Feb 17, 2016
Priority date	Feb 17, 2015
Publication date	Feb 18, 2020
Grant date	Feb 18, 2020

Abstract

Provided are methods for producing compositions comprising a population of cardiomyocytes enriched for or substantially devoid of sinoatrial node-like pacemaker cardiomyocytes (SANLCM) from human pluripotent stem cells (hPSCs), and methods of use thereof.

First claim

The invention claimed is:

1. A method of producing a population of cardiomyocytes, the method comprising:
(a) incubating cardiovascular mesoderm cells in a cardiac induction medium comprising a WNT inhibitor, VEGF, or both for a period of time to generate cardiovascular progenitor cells that express NKX2-5; and
(b) incubating the cardiovascular progenitor cells in a basic medium comprising VEGF for a period of time to generated a population of cardiomyocytes that are enriched for NKX2-5 pos , cTNT pos cells.

2. The method of claim 1 , wherein the cardiovascular mesoderm cells are generated from pluripotent stem cells (PSCs) according to the method:
(a) incubating the PSCs in an embryoid body medium comprising a BMP component for a period of time to generate embryoid bodies; and
(b) incubating the embryoid bodies in a mesoderm induction medium comprising a BMP component and an activin component for a period of time to generate cardiovascular mesoderm cells.

3. The method of claim 2 , wherein the PSCs are human PSCs (hPSCs).

4. The method of claim 3 , wherein the hPSCs are induced pluripotent stem cells (iPSCs) or human embryonic stem cells (hESCs).

5. The method of claim 3 , wherein the hPSCs are hPSCs comprising a NKX2-5 reporter construct.

6. The method of claim 5 , further comprising isolating a population of ventricular-like cardiomyocytes (VLCM) from the population of cardiomyocytes comprising
selecting NKX2-5 positive cardiotnyocytes according to NKX2-5 reporter construct expression.

7. The method of claim 5 , wherein the NKX2-5 reporter construct is a fluorescent NKX2-5 reporter construct.

8. The method of claim 1 , wherein the cardiac induction medium comprises a WNT inhibitor.

9. The method of claim 8 , wherein the cardiac induction medium further comprises VEGF.

10. The method of claim 9 , wherein the WNT inhibitor is IWP2.

11. The method of claim 10 , wherein the cardiac induction medium further comprises at least one of a FGF component and an activin/nodal inhibitor.

12. The method of claim 10 , wherein the cardiac induction medium further comprises SR-431542.

13. The method of claim 10 , further comprising the step of isolating the population of cardiomyocytes using a cardiomyocyte-specific surface marker or markers.

14. The method of claim 13 wherein the step of isolating the population of cardiomyocytes uses the markers signal-regulatory protein alpha (SIRPA) and thymocyte differentiation antigen 1 (THY-1/CD90).

15. The method of claim 2 , wherein the embryoid body medium BMP component is BMP4 and the mesoderm induction medium BMP component is BMP4.

16. The method of claim 15 , wherein the embryoid body medium further comprises a Rho-associated protein kinase (ROCK) inhibitor, the mesoderm induction medium activin component is Activin A, and the mesoderm induction medium further comprises bFGF.

17. The method of claim 12 , wherein the cardiovascular mesoderm cells are generated from pluripotent stem cells (PSCs) according to the method:
(a) incubating the PSCs in an embryoid body medium comprising a BMP component for a period of time to generate embryoid bodies; and
(b) incubating the embryoid bodies in a mesoderm induction medium comprising a BMP component and an activin component for a period of time to generate cardiovascular mesoderm cells.

18. The method of claim 17 , wherein the embryoid body medium BMP component is BMP4 and the mesoderm induction medium BMP component is BMP4.

19. The method of claim 18 , wherein the embryoid body medium further comprises a Rho-associated protein kinase (ROCK) inhibitor, the mesoderm induction medium activin component is Activin A, and the mesoderm induction medium further comprises bFGF.

20. The method of claim 19 , wherein the PSCs are human PSCs (hPSCs).

21. The method of claim 20 , wherein the hPSCs are human induced pluripotent stem cells (hiPSCs) or human embryonic stem cells (hESCs).

22. The method of claim 7 , wherein the fluorescent NKX2-5 reporter construct is a NKX2-5:GFP reporter construct.

Assignees

• Univ Health Network

Inventors

• Keller Gordon
• Protze Stephanie

Classifications

• A61P9/00

  Drugs for disorders of the cardiovascular system · CPC title

• C12N2506/02

  from embryonic cells · CPC title

• C12N2501/16

  Activin; Inhibin; Mullerian inhibiting substance · CPC title

• C12N2501/999

  Small molecules not provided for elsewhere · CPC title

• C12N2501/15

  Transforming growth factor beta (TGF-β) · CPC title