Cardiomyocytes and methods of producing and purifying cardiomyocytes

• US 8,153,427 B2
• Filed: 03/17/2008
• Issued: 04/10/2012
• Est. Priority Date: 03/15/2007
• Status: Expired due to Fees

• Current Assignee: NeoStem Oncology, LLC (Lisata Therapeutics, Inc.)
• Original Assignee: California Stem Cell, Inc. (Lisata Therapeutics, Inc.)
• Inventors: Nistor, Gabriel
• Primary Examiner(s): Afremova, Vera
• Application Number:
  US12/050,050
• Publication Number:
  US 20090155831A1
• Time in Patent Office: 1,485 Days
• Field of Search: None
• US Class Current: 435/377
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/34 Sugars
• CPC Class Codes: C12N 2500/38 Vitamins
• 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 2501/385 of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
• CPC Class Codes: C12N 2501/395 Thyroid hormones
• CPC Class Codes: C12N 2501/70 Enzymes
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 5/0657 Cardiomyocytes; Heart cells

Abstract

The invention provides methods for producing a culture of cardiomyocytes and cultures of cardiomyocytes. Exemplary methods of producing and cultures of cardiomyocytes include a population of cells including cells having spontaneous and periodic electrical activity, and/or including nodal, sino-atrial or pacemaker cells; immature cardiomyocytes (cardiomyoblasts); mature contractile cardiomyocytes; or a mixed population of two or more of such cells.

Claims

1. A method for producing a culture of cardiomyocytes, comprising:

A) Providing a culture of stem cells that are at least 90% confluent or the cells have overgrown to form multiple layers of cells, or proliferating stem cells until the cells are at least 90% confluent or the cells have overgrown to form multiple layers of cells;

B) Inducing formation of mesoderm by contacting the overgrown stem cells with a bone morphogenic protein (BMP) receptor ligand and an fibroblast growth factor (FGF) receptor ligand for a period of about 2 to 15 days; and

C) promoting cardiomyocyte formation by contacting mesoderm cells with neural cells, or a neural cell conditioned culture supernatant, for a period of about 1 to 21 days, thereby producing a culture of cardiomyocytes.

2. The method of claim 1, wherein the cardiomyocytes comprise a population of cells comprising cells having spontaneous and periodic electrical activity.

3. The method of claim 2, wherein the cells having spontaneous and periodic electrical activity comprise nodal, sino-atrial or pacemaker cells;

immature cardiomyocytes (cardiomyoblasts);

mature contractile cardiomyocytes;

or a mixed population thereof.

4. The method of claim 1, wherein the stem cells of step A) comprise a cell monolayer with 100% or more confluency.

5. The method of claim 1, wherein the stem cells of step A) form a multilayer culture.

6. The method of claim 1, wherein the stem cells of step A) have a density of about 150,000 to about 250,000 cells/cm 2.

7. The method of claim 1, wherein step B) further comprises addition of an acid.

8. The method of claim 7, wherein the acid comprises lactic, citric, fumaric, ascorbic, folic, malic, succinic, oxalacetic, or ketoglutaric acid.

9. The method of claim 1, wherein step B) further comprises subjecting the mesoderm cells to hypoxia.

10. The method of claim 1, wherein the stem cells, mesoderm cells or cardiomyocytes are subjected to an oxygen concentration of about 0.5 to 2.0%.

11. The method of claim 1, wherein step B) further comprises addition of an agent that inhibits cell death or apoptosis.

12. The method of claim 11, wherein the agent comprises selenium, lithium;

ascorbic acid or ascorbate or superoxide dismutase (SOD).

13. The method of claim 12, wherein the selenium comprises sodium selenite.

14. The method of claim 12, wherein the selenium is at a concentration of 1 to 20 ng/ml, or 0.5 to 50 ng/ml.

15. The method of claim 12, wherein the lithium comprises lithium carbonate.

16. The method of claim 12, wherein the lithium is at a concentration of 1 to 20 ng/ml, or 0.5-50 ng/ml.

17. The method of claim 12, wherein the ascorbic acid or ascorbate comprises sodium or calcium ascorbate.

18. The method of claim 12, wherein the ascorbic acid or ascorbate is at a concentration of 0.1 to 20 ug/ml, or 1 ug/ml.

19. The method of claim 12, wherein the superoxide dismutase (SOD) is at a concentration of 1 to 100 u/ml, or 1 to 10 u/ml.