Differentiation of pluripotent stem cells and cardiac progenitor cells into striated cardiomyocyte fibers using laminins LN-511, LN-521 and LN-221
The present disclosure describes methods of differentiating cardiomyocyte progenitor cells and mature cardiomyocyte cells from pluripotent stem cells. The methods may include differentiating pluripotent stems cells on a substrate including (i) laminin-511 or 521 and (ii) laminin-221. The cardiomyocyte progenitor cells and mature cardiomyocyte cells produced by the methods may form a human heart muscle cell line for use in regenerative cardiology. Also described are methods of identifying functional cardiomyocyte progenitor cells and their use in therapeutic applications.
1 . A method for treating injured human cardiac muscle, comprising:
(a) culturing human pluripotent stem cells on a culture plate coated with a substrate in a medium containing basic fibroblast growth factor (bFGF), wherein the substrate comprises a mixture of two laminins consisting of (i) recombinant LN-521 or recombinant LN-511 and (ii) recombinant LN-221;
(b) culturing the pluripotent stem cells on the substrate in the presence of a differentiation medium containing an effective amount of a GSK-3 inhibitor to stimulate Wnt signaling and obtain brachyury positive cells;
(c) culturing the brachyury positive cells from step (b) on the substrate in a differentiation medium containing an effective amount of a Wnt inhibitor to promote cardiac mesodermal specification;
(d) removing the differentiation medium containing the Wnt inhibitor from step (c) and replacing with a basal medium to culture the cells on the substrate to produce human cardiomyocyte progenitor cells that express Islet-1 and NKX2.5 transcription factor; and
(e) transplanting the human cardiomyocyte progenitor cells into the injured human cardiac muscle.
2 . The method of claim 1 , wherein:
the pluripotent stem cells are cultured in the presence of the differentiation medium containing an effective amount of a GSK-3 inhibitor for a first time period of about 12 hours to about 48 hours;
the brachyury positive cells are cultured in a cell culture medium devoid of inhibitors for a second time period of about 12 hours to about 48 hours before being cultured in the differentiation medium containing an effective amount of a Wnt inhibitor; and
the brachyury positive cells are cultured in the differentiation medium containing an effective amount of a Wnt inhibitor for a third time period of about 12 hours to about 48 hours.
3 . The method of claim 2 , wherein the cardiomyocyte progenitor cells are obtained by the fifth day after the beginning of differentiation.
4 . The method of claim 2 , wherein the cells in step (d) are cultured for up to 2 days in the basal medium.
5 . The method of claim 1 , wherein the step (a) has a duration of about 4 days.
6 . The method of claim 1 , wherein the differentiation media and the basal medium are chemically defined and xeno-free.