CARDIOMYOCYTES AND METHODS OF PRODUCING AND PURIFYING CARDIOMYOCYTES

• US 20090155831A1
• Filed: 03/17/2008
• Published: 06/18/2009
• Est. Priority Date: 03/15/2007
• Status: Active Grant

• Current Assignee: NeoStem Oncology, LLC (Lisata Therapeutics, Inc.)
• Original Assignee: California Stem Cell, Inc. (Lisata Therapeutics, Inc.)
• Inventors: NISTOR, GABRIEL
• Granted Patent: US 8,153,427 B2
• Time in Patent Office: Days
• US Class Current: 435/29
• 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; and

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;

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

70. A cardiomyocyte population produced by the method of claim 1.

71. A cardiomyocyte population comprising nodal, sino-atrial or pacemaker cells, mature contractile cardiomyocytes, immature cardiomyocytes (cardioblasts), or a mixed population thereof produced by the method of claim 1.

73. A method for identifying a cardioactive agent, comprising:

A) contacting a cardiomyocyte population produced by the method of claim 1 with a test agent; and

B) determining if the test agent modulates an activity or function of cardiomyocytes within the population, wherein modulating an activity or function of cardiomyocytes within the population identifies the test agent as a cardioactive agent.

2-69. -69. (canceled)

72. (canceled)

74-80. -80. (canceled)

81. A cell culture of immature cardiomyocytes (cardiomyblasts), wherein 50% or more of said culture comprises immature cardiomyocyte (cardiomyblast) cells, and wherein no more than about 30% of said cells beat or contract.

82. The cell culture of claim 81, wherein 60% or more of said culture comprises immature cardiomyocyte (cardiomyblast) cells.

83-90. -90. (canceled)

91. A cell culture of immature cardiomyocytes (cardiomyblasts), wherein 50% or more of said cell culture comprises immature cardiomyocyte (cardiomyblast) cells, and wherein 75% or more of said immature cardiomyocyte (cardiomyblast) cells survive anaerobic conditions for 30, 60 or more minutes.

92. A cell culture of immature cardiomyocytes (cardiomyblasts), wherein 50% or more of said culture comprises immature cardiomyocyte (cardiomyblast) cells, and wherein no more than about 25% of said immature cardiomyocyte (cardiomyblast) cells die when subjected to anaerobic conditions for 30, 60 or more minutes.

93-102. -102. (canceled)