1. Department of Gene and Cell Medicine, The Black Family Stem Cell Institute, Mount Sinai School of Medicine, 1425 Madison Avenue, New York, New York 10029, USA
2. Wells Center for Pediatric Research, Indiana University School of Medicine, 1044 West Walnut Street, Indiana 46202, USA
3. Greenberg Division of Cardiology, Departments of Medicine and Pharmacology, Weill Medical College of Cornell University, 520 East 70th Street, New York, New York 10021, USA
4. McEwen Centre for Regenerative Medicine, University Health Network, 101 College Street, Toronto, Ontario M5G 1L7, Canada
5. Department of Infectious Diseases, King's College London, London SE1 9RT, UK
6. VistaGen Therapeutics Inc., 384 Oyster Point Boulevard, Suite 8, San Francisco, California 94080, USA

Correspondence to: Gordon M. Keller^1,4 Correspondence and requests for materials should be addressed to G.M.K. (Email: gkeller@uhnresearch.ca).

Abstract

The functional heart is comprised of distinct mesoderm-derived lineages including cardiomyocytes, endothelial cells and vascular smooth muscle cells. Studies in the mouse embryo and the mouse embryonic stem cell differentiation model have provided evidence indicating that these three lineages develop from a common Flk-1⁺ (kinase insert domain protein receptor, also known as Kdr) cardiovascular progenitor that represents one of the earliest stages in mesoderm specification to the cardiovascular lineages¹. To determine whether a comparable progenitor is present during human cardiogenesis, we analysed the development of the cardiovascular lineages in human embryonic stem cell differentiation cultures. Here we show that after induction with combinations of activin A, bone morphogenetic protein 4 (BMP4), basic fibroblast growth factor (bFGF, also known as FGF2), vascular endothelial growth factor (VEGF, also known as VEGFA) and dickkopf homolog 1 (DKK1) in serum-free media, human embryonic-stem-cell-derived embryoid bodies generate a KDR^low/C-KIT(CD117)^neg population that displays cardiac, endothelial and vascular smooth muscle potential in vitro and, after transplantation, in vivo. When plated in monolayer cultures, these KDR^low/C-KIT^neg cells differentiate to generate populations consisting of greater than 50% contracting cardiomyocytes. Populations derived from the KDR^low/C-KIT^neg fraction give rise to colonies that contain all three lineages when plated in methylcellulose cultures. Results from limiting dilution studies and cell-mixing experiments support the interpretation that these colonies are clones, indicating that they develop from a cardiovascular colony-forming cell. Together, these findings identify a human cardiovascular progenitor that defines one of the earliest stages of human cardiac development.

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