Abstract 3821: Mesp1 Mediates Cardiovascular Differentiation By Dkk-1 Driven Inhibition Of Wnt-signaling And Is A Suitable Tool For Isolation Of Es Cell Derived Cardiovascular Progenitors
We have recently shown that MesP1 is a cardiac key transcription factor sufficient to induce ectopic beating heart tissue. MesP1 overexpression in ES cells results in significantly increased cardiovasculogenesis. Our results revealed a prominent function of MesP1 within a gene regulatory cascade causing Dkk-1 mediated blockage of wnt signalling. MesP1 may become a tool to preprogram ES cells for cardiac cell therapy and tissue engineering. In addition to directed differentiation, future cardiac cell therapy strategies will require protocols for a high-yield isolation of specific cell types to avoid the hazard of teratoma and tumor formation. Therefore we have developed a method for magnetic cell sorting (MACS) of ES cells expressing a human CD4 surface marker lacking its intracellular domain (ΔCD4) under control of transgenic promoters. Here we show that the MACS system can successfully be transferred to a MesP1 promoter-ΔCD4 construct. After transfection into murine ES cells several stable clones were obtained in which the time course of ΔCD4-expression revealed a peak at days two and three of differentiation. Using MACS we were able to enrich this population to purities of over 97%. The MACS-positive fractions showed a strong enrichment of the endogenous murine MesP1 mRNA confirming the functionality of our construct. In reaggregation experiments after MACS we verified the viability of the sorted cells which could be further propagated in their primitive differentiation state in the presence of LIF. After LIF withdrawal subsequent spontaneous beating activity was approximately 10 fold increased. This effect is reflected by an upregulation of cardiovascular mRNAs and on the protein level (FACS) by a 3 to 10 fold increased appearance of TroponinI, α-MHC and CD31 expressing cells. The cardiomyocytes revealed normal expression patterns of sarcomeric proteins. Whole Cell Patch Clamp analyses showed action potentials characteristic for early type cardiomyocytes corresponding to embryonic hearts at ED 10. These results suggest that cardiovascular progenitor cells can be selected from ES cells via MACS purification based on MesP1 promoter driven ΔCD4 expression. These cells are predestined for cell transplantation and tissue engineering approaches.