Differentiation of human embryonic stem cells (hESCs) into cardiac progenitors is a powerful approach to dissect the molecular control of early human cardiogenesis. Cardiomyocytes can be derived from hESCs via the formation of embryoid bodies (EBs), which are cellular aggregates containing derivatives of all three germ layers that arise from differentiating hESCs. In order to identify, purify and characterize emerging cardiac progenitor cells (hESC-CPCs), we engineered a hESC line, NKX2-5GFP/w, in which GFP replaced the coding region of the conserved cardiac transcription factor NKX2-5. Using this line we have optimized a protocol for the differentiation of hESC-CPCs and cardiomyocytes (hESC-CMs) from hESCs in a 96-well plate format in serum free, chemically defined media that enables the precise and reproducible manipulation of differentiation conditions. EBs are formed by centrifugation (spin EBs). The combination of Activin A, bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF) and WNT 3a drives mesoderm formation until day three. Subsequently, the cytokine cocktail is replaced with media without growth factors and the EBs endogenous signaling networks control the specification of cardiovascular progenitors. Contractile foci are found in almost all EBs and all beating areas are GFP positive. Flow cytometric analysis for GFP demonstrates that up to 30 % of cells within the EB are NKX2-5 positive. The production of GFP+ cells is temporally restricted to day six and day nine of EB differentiation; after this time no further cardiac cells are specified. Electrophysiological studies show that the GFP+ CMs have a fetal phenotype. Expression analysis supports this finding demonstrating that NKX2-5+ hESC-CPCs and CMs constitute developmentally distinct populations with both having gene expression profiles closer to fetal heart than adult heart. Clonal analysis demonstrates that NKX2-5+ cells are capable of giving rise to the three major lineages in the heart, namely cardiomyocytes, smooth muscle and endothelium. Together, these data demonstrate that NKX2-5GFP/w hESC lines facilitate the identification, isolation and culture of human cardiovascular progenitors.

David A Elliott1*, Stefan R Braam2, Louise Lagerqvist3, Katerina Koutsis1, Rob Jenny1, Magdaline Costa1, Elizabeth Ng1, Tanya Hatzistavrou1, Rhys JP Skelton, Claire E Hirst1, Cissy Yu1, Ouda Khammy4, Xuleing Li1, Sue Mei Lim1, Richard P Davis2, Adam L Goulburn1, Robert Passier2, John M Haynes3, Colin W Pouton3, David M Kaye4, Christine L Mummery2, Andrew G Elefanty1 and Edouard G Stanley1.

1 Monash Immunology and Stem Cell Laboratories, Monash University, Victoria, 3800, Australia 2 Department of Anatomy and Embryology, Leiden University Medical Centre, Leiden, The Netherlands 3 Monash Institute of Pharmaceutical Science, Monash University, Parkville, Victoria 3052, Australia 4 Baker IDI Heart & Diabetes Institute, Melbourne, Victoria, 3004, Australia


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  • Received: 05 March 2012
  • Accepted: 28 March 2012
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