DIRECT DIFFERENTIATION OF CARDIOMYOCYTES FROM HUMAN EMBRYONIC STEM CELLS

• US 20090202498A1
• Filed: 12/22/2006
• Published: 08/13/2009
• Est. Priority Date: 12/22/2005
• Status: Active Grant

• Current Assignee: ES Cell International Pte Ltd. (Lineage Cell Therapeutics, Inc.)
• Original Assignee: ES Cell International Pte Ltd. (Lineage Cell Therapeutics, Inc.)
• Inventors: Mummery, Christine Lindsay, Sun, William, Zweigerdt, Robert, Graichen, Ralph Eberhard, Davidson, Bruce Paul, Xu, Xiuqin
• Granted Patent: US 8,318,489 B2
• Time in Patent Office: Days
• US Class Current: 424/93.700
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• 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 2500/99 Serum-free medium
• CPC Class Codes: C12N 2501/02 Compounds of the arachidonic acid pathway, e.g. prostaglandins, leukotrienes
• CPC Class Codes: C12N 2501/105 Insulin-like growth factors [IGF]
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/125 Stem cell factor [SCF], c-kit ligand [KL]
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/33 Insulin together with transferrin C12N2500/25; Insulin-like growth factors C12N2501/105
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• CPC Class Codes: C12N 2502/30 tumour cells
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 5/0043 Medium free of human- or animal-derived components
• CPC Class Codes: C12N 5/0657 Cardiomyocytes; Heart cells
• CPC Class Codes: G01N 33/502 for testing non-proliferative effects
• CPC Class Codes: G01N 33/5061 Muscle cells

Abstract

The present invention relates to the induction of differentiation in stem cells to cardiomyocytes and factors such as prostaglandin alone or in combination with other factors including essential minerals selected from the group including transferrin and selenium, small molecules selected from the group including a p38 MAPK inhibitor such as SB203580 and protein growth factors of the FGF, IGF and BMP families such as but not limited to IGF1, FGF2, BMP2, BMP4 and BMP6. and insulin that influence the process of differentiation to cardiomyocytes. Media that is appropriate for the induction of differentiation of cardiomyocytes from stem cells is also provided wherein the media contains these factors. The use of cardiomyocytes and cardiac progenitors produced by the directed differentiation in transplantation and screening for cardiac compounds is also provided.

Claims

1-74. -74. (canceled)

75. A method of inducing or enhancing cardiomyocyte differentiation of a stem cell, the method comprising culturing the stem cell in the presence of prostacyclin (PGI2), or an analogue or functional equivalent thereof, alone or in combination with an essential mineral, a small molecule or a protein growth factor of the FGF, IGF or BMP family.

78. A method according to claim 75 wherein the protein growth factor of the FGF, IGF or BMP family is selected from the group comprising FGF-2, IGF-1, BMP2, BMP4, BMP6 and homologues and functional equivalents thereof.

79. A method according to claim 75, wherein the small molecule is a p38 MAPK inhibitor.

80. A method according to claim 79, wherein the p38 MAPK inhibitor is SB203580.

81. A method according to claim 75, 76 or 77, further comprising culturing the stem cell in the presence of transferrin or selenium.

82. A method according to claim 75, 76 or 77, further comprising co-culturing the stem cell with an embryonic cell.

83. A method according to claim 82, wherein the embryonic cell is a visceral endoderm or visceral endoderm-like cell.

84. A method according to claim 82, wherein the embryonic cell is an END2 cell.

85. A method according to claim 75, 76 or 77, further comprising co-culturing the stem cell with an embryonic cell and wherein the embryonic cell expresses a gene product expressed by a gene listed in Table 1.

86. A method according to claim 75 or 76, further comprising culturing the stem cell in a culture medium in which the effect caused by insulin, or an analogue thereof is reduced.

87. A method according to claim 86, wherein the effect caused by insulin, or an analogue thereof, is reduced by culturing the stem cell in the absence of insulin, or an analogue thereof, or by depleting insulin, or an analogue thereof, from the culture medium.

88. A method according to claim 75, further comprising culturing the stem cell in the presence of a gene product expressed by a gene listed in Table 1.

89. A cardiomyocyte or a cardiac progenitor prepared by a method according to any one of claims 75, 76 or 77.

92. A conditioned media produced by culturing a stem cell by a method according to claim 75, 76 or 77.

93. A cell composition comprising a cardiomyocyte or a cardiac progenitor prepared by a method according to claim 75, 76 or 77 and a carrier.

94. A method for preventing or treating a cardiac condition the method including transplanting a cardiomyocyte or cardiac progenitor cell prepared by a method according to claim 75, 76 or 77 into damaged cardiac tissue of a subject.

95. A method for screening compounds which affect cardiomyocyte activity, said method comprising:

obtaining a cardiomyocyte or cardiac progenitor cell prepared by a method according to claim 75, 76 or 77;

subjecting the cardiomyocyte or cardiac progenitor to a compound for screening; and

determining an effect on the cardiac activity of the cardiomyocyte or cardiac progenitor before and after subjecting the compound to the cardiomyocyte or cardiac progenitor.

76. A method of inducing or enhancing cardiomyocyte differentiation of a stem cell, the method comprising culturing the stem cell in the presence of a gene product expressed by a gene listed in Table 1.

77. A method of inducing or enhancing cardiomyocyte differentiation in a stem cell said method comprising culturing the stem cell in a culture medium in which the effect caused by insulin, or an analogue thereof, is reduced.

90. A conditioned media from a culture of an embryonic cell or a defined media when used to induce or enhance cardiomyocyte differentiation of a stem cell, wherein said media:

(i) comprises a gene product expressed from a gene listed in Table 1;

(ii) comprises PGI2, or an analogue or functional equivalent thereof, alone or in combination with an essential mineral, a small molecule or a protein growth factor of the FGF, IGF or BMP family;

(iii) comprises transferrin;

(iv) comprises selenium;

or

(v) has a reduced amount of insulin, or an analogue thereof.

91. A conditioned media according to claim 90, wherein the embryonic cell is an END2 cell.