Gezielte Differenzierung („Forward Programming“) von pluripotenten Stammzellen zu verschiedenen kardiovaskulären Zelltypen
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vor 13 Jahren
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.
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.
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