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Fischer, Evelyn (2010): Gezielte Differenzierung („Forward Programming“) von pluripotenten Stammzellen zu verschiedenen kardiovaskulären Zelltypen. Dissertation, LMU München: Medizinische Fakultät



AIMS: The proliferative potential of pluripotent stem cell-derived cardiomyocytes is limited, and reasonable yields for novel therapeutic options have yet to be achieved. In addition, various clinical applications will require the generation of specific cardiac cell types. Whereas early cardiovascular precursors appear to be important for novel approaches such as reseeding decellularized hearts, direct cell transplantation may require ventricular cells. Our recent work demonstrated that MesP1 represents a master regulator sufficient to induce cardiovasculogenesis in pluripotent cells. This led to our hypothesis that 'forward programming' towards specific subtypes may be feasible via overexpression of distinct early cardiovascular transcription factors. METHODS AND RESULTS: Here we demonstrate that forced expression of Nkx2.5 similar to MesP1 is sufficient to enhance cardiogenesis in murine embryonic stem cells (mES). In comparison to control transfected mES cells, a five-fold increased appearance of beating foci was observed as well as upregulated mRNA and protein expression levels. In contrast to MesP1, no increase of the endothelial lineage within the cardiovasculogenic mesoderm was observed. Likewise, Flk-1, the earliest known cardiovascular surface marker, was not induced via Nkx2.5 as opposed to MesP1. Detailed patch clamping analyses showed electrophysiological characteristics corresponding to all subtypes of cardiac ES cell differentiation in Nkx2.5 as well as MesP1 programmed embryoid bodies, but fractions of cardiomyocytes had distinct characteristics: MesP1 forced the appearance of early/intermediate type cardiomyocytes in comparison to control transfected ES cells whereas Nkx2.5 led to preferentially differentiated ventricular cells. CONCLUSION: Our findings show proof of principle for cardiovascular subtype-specific programming of pluripotent stem cells and confirm the molecular hierarchy for cardiovascular specification initiated via MesP1 with differentiation factors such as Nkx2.5 further downstream.