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

将心脏常驻成纤维细胞直接重编程或转分化为中胚层细胞或心脏祖细胞是目前最有前途的治疗方法之一。在我们之前的工作中，我们发现了一种转录因子鸡尾酒，由至少四个因子组成，可以重复诱导心脏祖细胞（iCPCs）。我们通过在Nkx2.5心脏增强子的控制下，应用表达黄色荧光蛋白（YFP）的转基因成纤维细胞，鉴定了这种合适的基因鸡尾酒。产生的iCPCs表达YFP，生长成集落样，并进一步表现出中胚层基因表达谱（如Eomes， Ets2）。同时，典型的成纤维细胞标记物在iCPCs中下调。在应用项目中，我们首先计划对这些iCPCs进行详细的表征，然后通过特定的培养条件实现稳定的扩增，最后将其分化为主要的心脏谱系。在项目的第一部分（目标1）中，将通过单细胞RNA测序（RNASeq）在单细胞水平上详细分析iCPCs。我们假设iCPCs表现出几个不同的转分化阶段，类似于诱导多能干细胞（iPSCs）的生成过程。单细胞RNASeq将有助于理解iCPCs的异质性。此外，将iCPCs与不同发育阶段的心脏祖细胞（如原始条纹细胞或中胚层祖细胞）进行比较。因此，iCPCs可以映射到心脏发生的特定阶段。该项目的第二部分（目标2）将分析iCPCs的表观遗传学。它们将与体内收集的足够转基因小鼠模型的祖细胞进行比较，这将揭示组蛋白修饰和整体甲基化状态的差异。目的3旨在通过保留iCPCs的特定表型来优化合适的培养和扩增条件。为了支持这一点，将产生足够的转基因小鼠模型的iPSCs（例如标记原始条纹或中胚层祖细胞），以评估不同发育阶段的足够培养条件，避免进一步分化。每个阶段的维持将通过荧光报告蛋白的表达来观察（不使用cre模型）。在项目的第四部分（目标4），我们将为建立iCPCs的心血管分化建立适当和有效的条件。在理想情况下，我们将能够将iCPCs分化为心肌细胞、内皮细胞和平滑肌细胞。该项目的最后一部分（目标5）旨在将目前使用的慢病毒重编程技术转变为非整合方法。因此，我们计划使用仙台病毒，这种病毒已经被常规应用于iPSC的生成。这应该是将该技术转化为潜在体内给药的第一步。

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.