Cardiac differentiation of human pluripotent stem cells under defined conditions using matrix overlay methods
AssigneeWisconsin Alumni Research Foundation
InventorsTimothy J. Kamp, Sean Palecek, Jianhua Zhang, Samira Azarin, Xiaojun Lian
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Abstract
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.
Claims23
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;
- and
- culturing 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.
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.
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;
- and
- administering cardiomyocytes from the differentiated cell population to the human or non-human animal.
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.
Dependencies are inferred from the claim text.