MesP1 dependent signalling pathways during the formation of common cardiovascular progenitors in vivo and in vitro
Zusammenfassung der Projektergebnisse
Our work demonstrated that common cardiovascular progenitor cells are characterized and induced by the expression of the transcription factor mesoderm posterior 1 (Mesp1) in vertebrate embryos and murine embryonic stem cells. 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. The extension of this project by programming embryonic stem cells with the transcription factor Tbx3 which is important for the development and function of cardiac pacemaker cells represents a further excellent example for the specific function of a transcription factor during the differentiation of stem cells and the cardiogenesis. The functionality of a permanent Tbx3 expression, we have investigated in forward programming murine embryonic stem cells with the nodal cell inducer Tbx3 plus an additional Myh6-promotor-based antibiotic selection leading to cellular aggregates consisting of >80% physiologically and pharmacologically functioning pacemaker-like cells characterized through their electrophysiological features, Ca2+-handling and their ability to stimulate adult myocardial tissue. These induced sinoatrial bodies (iSABs) exhibited highly increased beating rates (300-400 bpm), coming close to those found in mouse hearts. We could identify the functionality of membrane channels and the intracellular Ca-store involving in the modulation of the beating frequency. To ultimately test the functional pacemaker activity of the iSABs, we took advantage of an ex vivo model based on cultivated murine heart slices. iSABs increased the number of active slice regions and the frequency of slice contractions. The direct interaction between iSABs and slice cells could be proved by a calcein transfer from the calcein-labelled iSABs into the surrounding region of the slice via Cx connections including stimulation of contractions in this region. Moreover, we found a functional coupling between iSABs and slice cells by stimulation of Ca2+-transients in the slice caused by the rhythmical active ES-derived pacemaker-like cells. Our study presented for the first time generated pacemaker-like cells derived from embryonic stem cells with functioning membrane- and Ca2+-clock the typical phenomena for cardiac pacemaker cells. Moreover, these iSABs had also the ability to stimulate adult myocardium. This project has produced results extending the understanding about specific transcription factors which are responsible for the development into different heart cells. Thereby, the successful performed projects provide the basic for further research projects investigating the enrichment of into cardiac linage differentiating stem cells. This is important for the application of generated cardiac cells for the heart regeneration. Our generated stem cellderived cardiac cells linages can be furthermore used as model for the fundamental research or as drug testing model. It is consequently possible that our published data will be cited in international journals.
Projektbezogene Publikationen (Auswahl)
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Induction of MesP1 by Brachyury(T) generatest the common multipotent cardiovascular stem cell. Cardiovasc Res 92 (2011), 115-122
David R., Jarsch V.B., Schwarz F., Nathan P., Gegg M., Lickert H., Franz W.M.
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Selection of a common multipotent cardiovascular stem cell using the 3.4-kb MesP1 promotor fragment. Basic Res Cardiol 108 (2013), 312
David R., Schwarz F., Rimmbach C., Nathan P., Jung J., Brenner C., Jarsch V., Stieber J., Franz W.M.
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Programming and isolation of highly pure physiological and pharmacologically functional sinus-nodal bodies from pluripotent stem cells. Stem Cell Reports 2 (2014), 1-14
Jung J., Husse B., Rimmbach C., Krebs S., Stieber J., Steinhoff G., Dendorfer A., Franz WM., David R.
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Generation of cardiac pacemaker cells by programming and differentiation. Biochim Biophys Acta 1863 (2016), 1948-1952
Husse B., Franz W.M.
