US 10,888,588 B2
Directed cardiomyocyte differentiation and ventricular specification of stem cells
Roger Joseph Hajjar, Tenafly, NJ (US); and Camie W. Chan, Hong Kong (CN)
Assigned to ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI, New York, NY (US); and THE UNIVERSITY OF HONG KONG, Hong Kong (CN)
Appl. No. 15/30,325
Filed by ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI, New York, NY (US); and THE UNIVERSITY OF HONG KONG, Pokfulam, Hong Kong (CN)
PCT Filed Oct. 17, 2014, PCT No. PCT/US2014/061193
§ 371(c)(1), (2) Date Apr. 18, 2016,
PCT Pub. No. WO2015/058117, PCT Pub. Date Apr. 23, 2015.
Claims priority of provisional application 61/892,923, filed on Oct. 18, 2013.
Prior Publication US 2016/0271183 A1, Sep. 22, 2016
Int. Cl. A61K 35/34 (2015.01); G01N 33/50 (2006.01); C12N 5/077 (2010.01)
CPC A61K 35/34 (2013.01) [C12N 5/0657 (2013.01); G01N 33/5061 (2013.01); C12N 2500/02 (2013.01); C12N 2501/155 (2013.01); C12N 2501/16 (2013.01); C12N 2501/415 (2013.01); C12N 2501/727 (2013.01); C12N 2501/73 (2013.01); C12N 2501/999 (2013.01); C12N 2506/02 (2013.01); C12N 2506/45 (2013.01)] 13 Claims
 
1. A method of generating a cardiogenic embryoid body comprising at least one ventricular-like cardiomyocyte, said method comprising incubating a non-terminally differentiated human cell in suspension under serum-free, feeder-free culture conditions, wherein said incubating comprises the following sequential steps:
(a) culturing the non-terminally differentiated human cell for 24 to 72 hours in a suspension consisting essentially of basement membrane preparation extracted from Engelbreth-Holm-Swarm mouse sarcoma, a culture medium for cells that have not terminally differentiated, bone morphogenetic protein 4 (BMP4), and Rho kinase inhibitor;
(b) incubating the culture for 3 to 5 days in a suspension consisting essentially of the basement membrane preparation, a serum-free medium formulated to support the growth of human hematopoietic progenitor cells, ascorbic acid, L-alanyl-L-glutamine, BMP4, and activin-A; and
(c) growing the culture for at least four days in a suspension consisting essentially of the basement membrane preparation, the serum-free medium, ascorbic acid, L-alanyl-L-glutamine, BMP4, activin-A, and Inhibitor of Wnt Response 1 (IWR-1) to generate a culture of cardiomyocytes with a yield of at least 90% ventricular-like cardiomyocytes.