Dual reporter MESP1 mCherry/w-NKX2-5 eGFP/w hESCs enable studying early human cardiac differentiation.

Understanding early differentiation events leading to cardiogenesis is crucial for controlling fate of human pluripotent stem cells and developing protocols that yield sufficient cell numbers for use in regenerative medicine and drug screening. Here, we develop a new tool to visualize patterning of early cardiac mesoderm and cardiomyocyte development in vitro by generating a dual MESP1(mCherry/w)-NKX2-5(eGFP/w) reporter line in human embryonic stem cells (hESCs) and using it to examine signals that lead to formation of cardiac progenitors and subsequent differentiation. MESP1 is a pivotal transcription factor for precardiac mesoderm in the embryo, from which the majority of cardiovascular cells arise. Transcription factor NKX2-5 is expressed upon cardiac crescent formation. Induction of cardiac differentiation in this reporter line resulted in transient expression of MESP1-mCherry, followed by continuous expression of NKX2-5-eGFP. MESP1-mCherry cells showed increased expression of mesodermal and epithelial-mesenchymal-transition markers confirming their mesodermal identity. Whole-genome microarray profiling and fluorescence-activated cell sorting analysis of MESP1-mCherry cells showed enrichment for mesodermal progenitor cell surface markers PDGFR-α, CD13, and ROR-2. No enrichment was found for the previously described KDR+PDGFR-α+ progenitors. MESP1-mCherry derivatives contained an enriched percentage of NKX2-5-eGFP and Troponin T expressing cells, indicating preferential cardiac differentiation; this was enhanced by inhibition of the Wnt-pathway. Furthermore, MESP1-mCherry derivatives harbored smooth muscle cells and endothelial cells, demonstrating their cardiac and vascular differentiation potential under appropriate conditions. The MESP1-NKX2-5 hESC reporter line allows us to identify molecular cues crucial for specification and expansion of human cardiac mesoderm and early progenitors and their differentiation to specific cardiovascular derivatives.