Characterization, expansion and differentiation of induced cardiac progenitor cells (iCPCs)

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Direct reprogramming or transdifferentiation of cardiac resident fibroblasts into mesodermal or cardiac progenitor cells currently represents one of the most promising therapeutic approaches. In our previous work we identified a transcritption factor cocktail consisting of at least four factors that allow a reproducible induction of cardiac progenitor cells (iCPCs). We identified this appropriate gene cocktail by applying transgenic fibroblasts that express yellow fluorescent protein (YFP) under the control of an Nkx2.5 cardiac enhancer. The arising iCPCs express YFP, grow colony-like, and further exhibit a mesodermal gene expression profile (e.g. Eomes, Ets2). Simultaneously, typical fibroblast markers become down-regulated in the iCPCs.In the applied project we first plan to characterize these iCPCs in detail, then achieve a stable expansion by specific cultivation conditions, and finally differentiate them into the main cardiac lineages.In the first part of the project (aim 1) the iCPCs will be analyzed in detail at the single cell level by single cell RNA sequencing (RNASeq). We assume that iCPCs exhibit several different transdifferentiation stages, similar to the generation process of induced pluripotent stem cells (iPSCs). Single cell RNASeq will help to understand the heterogeneity of iCPCs. In addition, iCPCs will be compared to cardiac progenitor cells of different developmental stages (e.g. primitive streak or mesodermal progenitor cells). Thereby iCPCs can be mapped to a specific stage of cardiogenesis.The second part of the project (aim 2) will analyze epigenetics of iCPCs. They will be compared to in vivo collected progenitor cells of sufficient transgenic mouse models which should reveal differences concerning histon modifications and the global methylation state. Aim 3 targets the optimization of appropriate cultivation and expansion conditions by preserving the specific phenotype of iCPCs. In support of this, iPSCs of sufficient transgenic mouse models (e.g. tagging primitive streak or mesodermal progenitor cells) will be generated to evaluate sufficient culture conditions for different developmental stages avoiding further differentiation. The maintenance of the various stages will each be observed by the expression of a fluorescent reporter protein (no Cre-models will be used).In the fourth part of the project (aim 4) we will establish appropriate and efficient conditions for establishing cardiovascular differentiation of the iCPCs. Optimally, we will be able to differentiate the iCPCs into cardiomyocytes as well as endothelial cells and smooth muscle cells.The last part of the project (aim 5) aims to alter the currently used lentiviral reprogramming technique into a non-integrative method. Therefore, we plan to use Sendai virus, which is already applied on a regular basis for iPSC generation. This should serve as a first translational step to direct the technique to a potential in vivo administration.