Rbm24在胚胎干细胞分化成心肌细胞的作用及机制

Human embryonic stem cells (ESC) with their ability to differentiate into cardiomyocytes in culture hold a great potential for replacement therapies. However, directed differentiation of ESCs towards cardiomyocytes is still a poorly defined process. ESC differentiation in vitro recapitulates the developmental programs that orchestrates cell fate decisions in the cardiogenesis. Elucidating the basic mechanisms of cardiac differentiation will provide insight to understanding of heart development,and help develop strategies for in vitro manipulating hESC differentiation..We previously employed a transcriptome analysis on enriched cardiomyocytes derived from ESC and discovered a large number of yet uncharacterized genes with little or no information on their function during heart development. Rbm24 (RNA-binding motif protein 24) is one of the genes up-regulated in cardiomyocytes. The temporal expression Rbm24 analysis in mouse embryos and zebra-fish indicated that specific expression of Rbm24 in the early cardiac mesoderm, in parallel with expression of the early cardiomyocyte-specific transcription factors Nkx2.5 and Gata4. This suggests that Rbm24 may be required for early cardiac lineage specification. Moreover, our in vivo studies by loss-of-function phenotype analysis in zebra-fish model confirmed that Rbm24 is required for cardiogenesis and proper heart function during early heart development. Based on these results, we hypothesize that Rbm24 may play an important role in ESC cardiac differentiation. .The current proposal is focused on the role of Rbm24 in ESC cardiac differentiation with the aim to understand cellular and the molecular mechanism by which Rbm24 acts during cardiac specification. Firstly, a lentiviral-mediated RNAi will be employed to knockdown Rbm24 gene in ES cells, followed by functional analysis of molecular and electrophysiological phenotypes of cardiomyocytes derived from ESC. To determine whether Rbm24 directly promotes cardiac specification during cardiac cell-fate specification, we will generate a recombinant ESC line in which the expression of Rbm24 can be temporally and specifically induced upon doxycylin (Dox) addition. Taking advantage of this inducible system for conditional expression of Rbm24 during ESC differentiation, we can monitor the temporal regulation of down-stream gene expression by Rbm24. Finally, Rbm24-expressing GFP tagged cells will be transplanted in mouse myocardial infarction (MI) model for preliminary evaluation of function improvement of myocardial ischemia. .This study will provide significant insight to regulatory network in early development and contribute to a better understanding mechanism of ES cardiac differentiation. Results will guide better ways to in vitro manipulate ESC differentiation towards cardiac lineage, with ultimate goal to develop therapeutic application for heart disease patients.

胚胎干细胞（ESC）因可分化为心肌细胞而具备用于心脏病替代治疗的巨大潜能，但其分化的调控机制尚不明确。我们通过对人源 ESC分化的心肌细胞转录谱分析，发现一个在心肌分化早期上调表达的基因Rbm24 （RNA binding motif 24）。动物模型的功能缺失表型提示Rbm24对心脏发育和维持正常心功能有重要作用。本项目着眼于研究Rbm24在ESC心肌分化中的作用机制。首先运用shRNA干扰分析Rbm24沉默对ESC心肌细胞分化的影响。进一步利用Cre /loxP基因重组系统与Tet-on可诱导调控系统相结合，对Rbm24基因在ESC心肌分化过程进行可诱导特异性表达，探索Rbm24在分化过程中的作用环节，及与相关信号通路调控的联系；最后在小鼠心肌梗塞模型中对Rbm24 阳性心肌细胞移植对心肌缺血功能改善进行初步评价。预期研究结果将为揭示ESC心肌分化调节机制提供新的理论依据。

胚胎干细胞（ESC）因可分化为心肌细胞而具备用于心脏病替代治疗的巨大潜能，但其分化的调控机制尚不明确。我们通过对人源 ESC 分化的心肌细胞转录谱分析，发现一个在心肌分化早期上调表达的基因 Rbm24 （RNA binding motif 24）。动物模型的功能缺失表型提示Rbm24对心脏发育和维持正常心功能有重要作用。.本项目着眼于研究Rbm24在ESC 心肌分化中的作用机制。利用 Cre /loxP 基因重组系统与 Tet-on 可诱导调控系统相结合，对 Rbm24 基因在 ESC 心肌分化过程进行可诱导特异性表达，探索 Rbm24 在分化过程中的作用环节，及与相关信号通路调控的联系。进而运用 shRNA 干扰分析 Rbm24 沉默对 ESC 心肌细胞分化的影响。 .该研究发现干细胞分化过程中，RNA结合蛋白Rbm24可以通过调节mRNA可变剪接，精准调控mRNA选择性表达组织特异性的转录异构体，进而调控干细胞向心肌分化。首次证明了RNA结合蛋白Rbm24通过调节可变剪切促进干细胞向心肌分化，开拓了干细胞分化调控的视野，为干细胞再生医学转化提供应用前景。