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The cardiac progenitor cells (CPCs) in the anterior heart field (AHF) are located in the pharyngeal mesoderm (PM), where they expand, migrate and eventually differentiate into major cell types found in the heart, including cardiomyocytes. The mechanisms by which these progenitors are able to expand within the PM microenvironment without premature differentiation remain largely unknown. Through in silico data mining, genetic loss-of-function studies, and in vivo genetic rescue studies, we identified N-cadherin and interaction with canonical Wnt signals as a critical component of the microenvironment that facilitates the expansion of AHF-CPCs in the PM. CPCs in N-cadherin mutant embryos were observed to be less proliferative and undergo premature differentiation in the PM. Notably, the phenotype of N-cadherin deficiency could be partially rescued by activating Wnt signaling, suggesting a delicate functional interaction between the adhesion role of N-cadherin and Wnt signaling in the early PM microenvironment. This study suggests a new mechanism for the early renewal of AHF progenitors where N-cadherin provides additional adhesion for progenitor cells in the PM, thereby allowing Wnt paracrine signals to expand the cells without premature differentiation.

Distinct families of multipotent heart progenitors play a central role in the generation of diverse cardiac, smooth muscle and endothelial cell lineages during mammalian cardiogenesis. The identification of precise paracrine signals that drive the cell-fate decision of these multipotent progenitors, and the development of novel approaches to deliver these signals in vivo, are critical steps towards unlocking their regenerative therapeutic potential. Herein, we have identified a family of human cardiac endothelial intermediates located in outflow tract of the early human fetal hearts (OFT-ECs), characterized by coexpression of Isl1 and CD144/vWF. By comparing angiocrine factors expressed by the human OFT-ECs and non-cardiac ECs, vascular endothelial growth factor (VEGF)-A was identified as the most abundantly expressed factor, and clonal assays documented its ability to drive endothelial specification of human embryonic stem cell (ESC)-derived Isl1+ progenitors in a VEGF receptor-dependent manner. Human Isl1-ECs (endothelial cells differentiated from hESC-derived ISL1+ progenitors) resemble OFT-ECs in terms of expression of the cardiac endothelial progenitor- and endocardial cell-specific genes, confirming their organ specificity. To determine whether VEGF-A might serve as an in vivo cell-fate switch for human ESC-derived Isl1-ECs, we established a novel approach using chemically modified mRNA as a platform for transient, yet highly efficient expression of paracrine factors in cardiovascular progenitors. Overexpression of VEGF-A promotes not only the endothelial specification but also engraftment, proliferation and survival (reduced apoptosis) of the human Isl1+ progenitors in vivo. The large-scale derivation of cardiac-specific human Isl1-ECs from human pluripotent stem cells, coupled with the ability to drive endothelial specification, engraftment, and survival following transplantation, suggest a novel strategy for vascular regeneration in the heart.