Direct differentiation of cardiomyocytes from human embryonic stem cells
First Claim
1. A method of inducing cardiomyocyte differentiation of a stem cell, the method comprising culturing the stem cell in the presence of a prostaglandin, analogue or functional equivalent thereof alone or in combination with essential minerals, small molecules and protein growth factors of the FGF, IGF and BMP families.
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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.
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Citations
74 Claims
- 1. A method of inducing cardiomyocyte differentiation of a stem cell, the method comprising culturing the stem cell in the presence of a prostaglandin, analogue or functional equivalent thereof alone or in combination with essential minerals, small molecules and protein growth factors of the FGF, IGF and BMP families.
- 7. A culture media when used for the induction of cardiomyocyte differentiation in stem cells, said media comprising a prostaglandin, analogue or functional equivalent thereof alone or in combination with essential minerals, small molecules and protein growth factors of the FGF, IGF and BMP families.
- 13. A method of inducing cardiomyocyte differentiation of a stem cell, the method comprising culturing the cells in the presence of a gene product expressed from a gene listed in Table 1.
- 16. A culture media when used for the induction of cardiomyocyte differentiation in stem cells, said media comprising a gene product expressed from a gene listed in Table 1.
- 19. A culture media when used for the induction of cardiomyocyte differentiation in stem cells, said media comprising at least one of Fgf9, Bmp6, Bmp4, Scf, Igf2, Ptgis or Ptgs1.
- 23. A method of enhancing induction of cardiomyocyte differentiation from a co-culture of a stem cell and an embryonic cell, said method comprising inducing expression of a gene product expressed from a gene listed in Table 1.
- 27. A method for enhancing induction of cardiomyocyte differentiation of a conditioned media from a culture of an embryonic cell, said method comprising culturing the stem cell and the conditioned media in the presence of a gene product expressed from a gene listed in Table 1.
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30. A method of enhancing induction of cardiomyocyte differentiation by a conditioned media from a culture of an embryonic cell, said method comprising culturing the stem cell and the conditioned media in the presence of Fgf9, Bmp6, Bmp4, Scf, Igf2, Ptgis or Ptgs1.
- 31. A method for enhancing induction of cardiomyocyte differentiation by a conditioned media from a culture of an embryonic cell or a defined media, said method comprising culturing the stem cell and the conditioned media or the defined media in the presence any one of the factors selected from the group consisting of growth factors of the FGF, BMP and IGF families, prostaglandin, p38 MAPK inhibitor, transferrin and selenium.
- 37. A conditioned media from a culture of an embryonic cell or a defined media when used for enhancing induction of cardiomyocyte differentiation comprising any one of the factors selected from the group consisting of Fgf9, Bmp6, Bmp4, Scf, Igf2, Ptgis, Ptgs1, growth factors of the FGF and IGF family, prostaglandin, p38 MAPK inhibitor, transferrin or selenium.
- 42. A method of progressing cardiomyocyte differentiation in a stem cell culture said method comprising reducing an effect caused by insulin or an analogue thereof on the stem cell in culture.
- 44. A method of enabling cardiomyocyte differentiation in a stem cell culture said method comprising culturing the stem cell in the presence of a defined medium supplemented with essential minerals selected from the group including Transferrin and Selenium, small molecules selected from the group including SB203580 and PGI2 and protein growth factors of the FGF, IGF and BMP families selected from the group comprising IGF1 and FGF2, BMP2, BMP4 and BMP6, a homologue or functional equivalent thereof.
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48. Use of insulin or an analogue thereof for the inhibition of cardiomyocyte differentiation in stem cells.
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64. A method for preparing a genetically modified cardiomyocyte or cardiac progenitor from a hES cell said method comprising:
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introducing a gene into the hES cell;
differentiating the hES cell into a cardiomyocyte or cardiac progenitor by a method according to any one of claims 1, 13, 23, and 42.
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65. A method according to claim 64 wherein the gene is a reporter gene.
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66. A method according to claim 64 wherein the gene is selected from Table 1.
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67. A method according to any one of claims 64 wherein the gene is selected from the group consisting of Bmp6, Fgf9, Bmp4, Scf, Igf2, Ptgis and Ptgs1.
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68. A genetically modified cardiomyocyte or cardiac progenitor prepared by a method according to any one of claims 64.
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69. A method for preparing an enriched population of cardiomyocytes and cardiac progenitors differentiated from a hES cell in culture said method comprising:
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preparing a genetically modified cardiomyocyte or cardiac progenitor according to claim 67 with a reporter gene; and
selecting for the cardiomyocytes and cardiac progenitors with or without the reporter gene to enrich the population for cardiomyocytes and cardiac progenitors.
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70. An enriched population of cardiomyocytes and cardiac progenitors differentiated from a hES cell prepared by a method according to claim 69.
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71. A myocardial model for testing the ability of stem cells that have differentiated into cardiomyocytes or cardiac progenitors for cardiac transplantation said model comprising;
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a host animal for receiving the cardiomyocytes or cardiac progenitors of claims 53 by transplantation; and
a measurable parameter of cardiac function to measure activity of the transplanted cardiomyocytes and cardiac progenitors.
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72. A method for screening compounds which affect cardiomyocyte activity, said method comprising:
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obtaining a cardiomyocyte or cardiac progenitor according to claim 53 and 67;
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.
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73. A method according to claim 72 wherein the effect is determined physiologically.
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74. A method according to claim 72 wherein the effect is determined electrophysiologically.
Specification