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

• US 10,561,687 B2
• Filed: 02/17/2016
• Issued: 02/18/2020
• Est. Priority Date: 02/17/2015
• Status: Active Grant

• Current Assignee: University Health Network
• Original Assignee: University Health Network
• Inventors: Keller, Gordon, Protze, Stephanie
• Primary Examiner(s): Noble, Marcia S
• Application Number:
  US15/550,572
• Publication Number:
  US 20180028572A1
• Time in Patent Office: 1,462 Days
• CPC Class Codes: A61K 35/34 Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes vascular smooth muscle A61K35/44
• 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 2501/15 Transforming growth factor beta (TGF-β)
• 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 2501/165 Vascular endothelial growth factor [VEGF]
• 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 2501/727 Kinases (EC 2.7.)
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2506/45 from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 5/0657 Cardiomyocytes; Heart cells
• CPC Class Codes: C12N 5/10 Cells modified by introduction of foreign genetic material

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.

Claims

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.