초록 ▼

Methods for culturing the pluripotent stem cells to undergo epithelial-to-mesenchymal transition and for generating high-yield, high-purity cardiomyocyte cultures from pluripotent stem cells are described. Pluripotent stem cells are cultured on a support with an overlaid matrix and, optionally, expo

Methods for culturing the pluripotent stem cells to undergo epithelial-to-mesenchymal transition and for generating high-yield, high-purity cardiomyocyte cultures from pluripotent stem cells are described. Pluripotent stem cells are cultured on a support with an overlaid matrix and, optionally, exposed to one or more factors to induce epithelial-to-mesenchymal transition and cardiogenesis.

대표청구항 ▼

1. A method for culturing pluripotent stem cells to obtain foci of cells undergoing an epithelial-to-mesenchymal transition, the method comprising the steps of: providing pluripotent stem cells as a monolayer on a support;applying a matrix layer to the apical surface of pluripotent stem cells of the

1. A method for culturing pluripotent stem cells to obtain foci of cells undergoing an epithelial-to-mesenchymal transition, the method comprising the steps of: providing pluripotent stem cells as a monolayer on a support;applying a matrix layer to the apical surface of pluripotent stem cells of the monolayer, wherein both the support and the matrix layer contact the pluripotent stem cells; andculturing the pluripotent stem cells contacted by the matrix layer to induce the contacted pluripotent stem cells to form multilayered foci of cells undergoing the epithelial-to-mesenchymal transition, wherein culturing comprises providing Activin A to the pluripotent stem cells contacted by the matrix layer, and wherein cells undergoing the epithelial-to-mesenchymal transition comprise N-cadherin-positive mesenchymal cells. 2. The method of claim 1, further comprising the step of culturing the foci to form a differentiated cell population comprising cardiomyocytes. 3. The method of claim 1, wherein Activin A is provided in the matrix layer. 4. The method of claim 1, wherein culturing comprises providing 100 ng/ml Activin A and at least one factor selected from the group consisting of 0-10 ng/ml BMP4 and 0-25 ng/ml bFGF to the pluripotent stem cell. 5. The method of claim 1, wherein the Activin A is provided to the pluripotent stem cells for only one day. 6. The method of claim 1, wherein the matrix layer comprises extracellular matrix proteins. 7. The method of claim 1, wherein the pluripotent stem cells are cultured in defined medium. 8. The method of claim 7, wherein the defined medium comprises a serum-free growth supplement. 9. The method of claim 7, wherein the defined medium is substantially free of insulin. 10. The method of claim 7, further comprising the step of adding insulin to the defined medium on day 5 of culture. 11. The method of claim 2, wherein the differentiated cell population comprises 30-90% cardiac troponinT (cTnT)-positive cardiomyocytes. 12. The method of claim 1, wherein the foci have a mean diameter of about 100 μm to about 200 μm. 13. The method of claim 1, wherein the cells do not form embryoid bodies. 14. The method of claim 1, further comprising the step of detecting a mesenchymal marker. 15. The method of claim 14, wherein the marker is N-cadherin surface expression. 16. The method of claim 1, further comprising the step of contacting the pluripotent stem cells to an agent that modulates foci formation. 17. A culture composition comprising: a support;a monolayer of pluripotent stem cells on the support;a matrix layer, wherein the support and the matrix layer contact the monolayer of pluripotent stem cells, wherein the matrix layer contacts the apical surface of pluripotent stem cells of the monolayer, and wherein at least some of the pluripotent stem cells form multilayered foci comprising N-cadherin-positive cells. 18. A method for administering in vitro-generated cardiomyocytes to a human or non-human animal, the method comprising the steps of: providing a monolayer of pluripotent stem cells on a support in vitro;applying a matrix layer to the apical surface of pluripotent stem cells of the monolayer, wherein both the support and the matrix layer contact the pluripotent stem cells;culturing pluripotent stem cells contacted by the matrix layer in vitro to form a differentiated cell population comprising cardiomyocytes, wherein culturing comprises providing Activin A to the pluripotent stem cells contacted by the matrix layer; andadministering cardiomyocytes from the differentiated cell population to the human or non-human animal. 19. The method of claim 18, wherein cardiomyocytes from the differentiated cell population are administered to the heart of the human or non-human animal. 20. A method of producing cardiomyocytes, the method comprising the steps of: (a) providing vector-free iPS cells in a monolayer on a support in vitro;(b) applying a matrix layer to the apical surface of the vector-free iPS cells, wherein both the support and the matrix layer contact the vector-free iPS cells;(c) culturing the vector-free iPS cells contacted by the matrix layer in vitro whereby the vector-free iPS cells differentiate to form cardiomyocytes, wherein culturing comprises providing Activin A to the pluripotent stem cells contacted by the matrix lam; and(d) isolating the cardiomyocytes from the vector-free iPS cell culture to obtain a population of cardiomyocytes. 21. The method of claim 1, further comprising providing at least one factor selected from the group consisting of bone morphogenetic protein 4 (BMP4) and basic fibroblast growth factor (bFGF) to the pluripotent stem cells contacted by the matrix layer. 22. The method of claim 21, wherein the Activin A and the at least one factor selected from the group consisting of bone morphogenetic protein 4 (BMP4) and basic fibroblast growth factor (bFGF) are added sequentially. 23. The method of claim 21, wherein the at least one factor is provided in the matrix layer.