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

诱导心脏祖细胞 (iCPC) 的表征、扩增和分化

基本信息

项目摘要

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.
将心脏固有成纤维细胞直接重编程或转分化为中胚层或心脏祖细胞是目前最有前途的治疗方法之一。在我们以前的工作中,我们确定了一个转录因子鸡尾酒组成的至少四个因素,使心脏祖细胞(iCPCs)的可重复诱导。我们通过应用在Nkx2.5心脏增强子控制下表达黄色荧光蛋白(YFP)的转基因成纤维细胞来鉴定这种适当的基因鸡尾酒。产生的iCPC表达YFP,生长为集落样,并进一步表现出中胚层基因表达谱(例如Eomes,Ets2)。同时,典型的成纤维细胞标志物在iCPC中下调。在应用项目中,我们首先计划详细描述这些iCPC,然后通过特定的培养条件实现稳定扩增,最后将其分化为主要的心脏谱系。在项目的第一部分(目的1)中,将通过单细胞RNA测序(RNASeq)在单细胞水平上详细分析iCPC。我们假设iCPC表现出几个不同的转分化阶段,类似于诱导多能干细胞(iPSC)的生成过程。单细胞RNASeq将有助于了解iCPC的异质性。此外,将iCPC与不同发育阶段的心脏祖细胞(例如原条或中胚层祖细胞)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(目的2)将分析iCPCs的表观遗传学。将它们与足够的转基因小鼠模型的体内收集的祖细胞进行比较,所述转基因小鼠模型应当揭示关于希斯顿修饰和整体甲基化状态的差异。目的3通过保留iCPCs的特异性表型,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(例如标记原条或中胚层祖细胞)的iPSC,以评估不同发育阶段的足够培养条件,避免进一步分化。各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(目的4)中,我们将建立适当和有效的条件来建立iCPC的心血管分化。最理想的是,我们将能够分化为心肌细胞以及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改变为非整合方法。因此,我们计划使用仙台病毒,该病毒已经定期用于iPSC生成。这应该作为第一个翻译步骤,以指导该技术的潜在体内给药。

项目成果

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Professor Dr. Markus Krane其他文献

Professor Dr. Markus Krane的其他文献

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{{ truncateString('Professor Dr. Markus Krane', 18)}}的其他基金

Nanoflare-based isolation of cardiac progenitor cells derived from murine and human pluripotent stem cells
基于 Nanoflare 的小鼠和人类多能干细胞来源的心脏祖细胞的分离
  • 批准号:
    262640928
  • 财政年份:
    2014
  • 资助金额:
    --
  • 项目类别:
    Research Grants
Konstruktion von drei-dimensionalen kardiomyozytären Gewebekonstrukten differenziert aus murinen induzierten pluripotenten Stammzellen
小鼠诱导多能干细胞分化的三维心肌细胞组织构建体的构建
  • 批准号:
    149546944
  • 财政年份:
    2009
  • 资助金额:
    --
  • 项目类别:
    Research Fellowships
Unbiased mapping of non-myocyte cell (NMC) populations in the human heart – Cellular composition & single cell transcriptome analysis considering age, anatomical location and cardiac pathologies
人类心脏中非肌细胞 (NMC) 群体的无偏绘图 â 细胞组成
  • 批准号:
    434373242
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:
    Research Grants

相似国自然基金

基于Riemann-Hilbert方法的相关问题研究
  • 批准号:
    11026205
  • 批准年份:
    2010
  • 资助金额:
    3.0 万元
  • 项目类别:
    数学天元基金项目

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