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 KDRlow/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 KDRlow/C-KITneg cells differentiate to generate populations consisting of greater than 50% contracting cardiomyocytes. Populations derived from the KDRlow/C-KITneg 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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Acknowledgements
We thank M. Oza for MEA (multi-electrode arrays) measurement and members of the Keller laboratory for critically reading this manuscript. G.M.K., S.J.K. and G.W.A. are supported by the National Institutes of Health/National Heart Lung and Blood Institute.
Author Contributions L.Y. carried out most of the experiments; L.Y., S.J.K. and G.M.K. designed the study; L.Y. and G.M.K. analysed the data and wrote the manuscript; M.H.S. and L.J.F. performed the transplantation and differentiation study in the normal hearts; E.D.A. was responsible for the transplantation and analyses of the infracted hearts; T.K.R. and G.W.A. carried out the patch-clamp study; E.H. and R.M.L. generated the AAV (adeno-associated virus)–GFP–hES2 cells; M.K. provided advice on experimental design and analysed data; and L.Y. and K.B. performed the field potential recording.
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The file contains Supplementary Methods, Supplementary Figures 1-5 with Legends and Legends to Supplementary Movies S1-S4. (PDF 4882 kb)
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The file contains Supplementary Movie S1 showing day 14 EBs with contracting cells derived from hES2 cells. (MOV 317 kb)
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The file contains Supplementary Movie S2 showing aggregates with contracting cells generated from day 6 EB-derived KDRlow/C-KITneg cells. Aggregates were cultured in the low cluster plates for 10 days (MOV 592 kb)
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The file contains Supplementary Movie S3 showing monolayer of contracting cells generated from day 6 EB-derived KDRlow/C-KITneg cells. The sorted cells were cultured on a gelatin coated well for 10 days (MOV 753 kb)
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The file contains Supplementary Movie S4 showing a cardiac colony generated from day 6 EB-derived KDRlow/C-KITneg cells. The colony was maintained in methylcellulose cultures for 10 days. (MOV 336 kb)
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Yang, L., Soonpaa, M., Adler, E. et al. Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population. Nature 453, 524–528 (2008). https://doi.org/10.1038/nature06894
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DOI: https://doi.org/10.1038/nature06894
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