Selection of a common multipotent cardiovascular stem cell using the 3.4-kb MesP1 promoter fragment.

Common cardiovascular progenitor cells are characterized and induced by expression of the transcription factor MesP1. To characterize this population we used a 3.4-kb promoter fragment previously described by our group. This served to isolate MesP1-positive cells from differentiating ES stem cells via magnetic cell sorting based on a truncated CD4 surface marker. As this proximal promoter fragment omits a distal non-cardiovasculogenic enhancer region, we were able to achieve a synchronized fraction of highly enriched cardiovascular progenitors. These led to about 90% of cells representing the three cardiovascular lineages: cardiomyocytes, endothelial cells and smooth muscle cells as evident from protein and mRNA analyses. In addition, electrophysiological and pharmacological parameters of the cardiomyocytic fraction show that almost all correspond to the multipotent early/intermediate cardiomyocyte subtype at day 18 of differentiation. Further differentiation of these cells was not impaired as evident from strong and synchronous beating at later stages. Our work contributes to the understanding of the earliest cardiovasculogenic events and may become an important prerequisite for cell therapy, tissue engineering and pharmacological testing in the culture dish using pluripotent stem cell-derived as well as directly reprogrammed cardiovascular cell types. Likewise, these cells provide an ideal source for large-scale transcriptome and proteome analyses.

Induction of MesP1 by Brachyury(T) generates the common multipotent cardiovascular stem cell.

AIMS: Our recent work demonstrated that common cardiovascular progenitor cells are characterized and induced by the expression of the transcription factor mesoderm posterior1 (MesP1) in vertebrate embryos and murine embryonic stem cells. As the proliferative potential of stem cell-derived cardiomyocytes is limited, it is crucial to understand how MesP1 expression is mediated in order to achieve reasonable and reliable yields for novel stem cell-based therapeutic options. As potential upstream regulators of MesP1, we therefore analysed Eomes and Brachyury(T), which had been controversially discussed as being crucial for cardiovasculogenic lineage formation. METHODS AND RESULTS: Wild-type and transgenic murine embryonic stem cell lines, mRNA analyses, embryoid body formation, and cell sorting revealed that the MesP1 positive population emerges from the Brachyury(T) positive fraction. In situ hybridizations using wild-type mouse embryos confirmed that Brachyury(T) colocalises with MesP1 in vivo. Likewise, shRNA-based loss of Brachyury(T) causes a dramatic decrease in MesP1 expression accompanied by reduced cardiac markers in differentiating embryonic stem cells, which is reflected in vivo via in situ hybridizations using Brachyury(T) knock-out embryos where MesP1 mRNA is greatly abolished. We finally defined a 3.4 kb proximal MesP1-promoter fragment which is directly bound and activated by Brachyury(T) via a T responsive element as shown via bandshift, chromatin immuneprecipitation, and reporter assays. CONCLUSION: Our work contributes to the understanding of the earliest cardiovasculogenic events and may become an important prerequisite for cell therapy, tissue engineering, and pharmacological testing in the culture dish using pluripotent stem cell-derived as well as directly reprogrammed cardiovascular cell types.