Identification of novel molecules involved in cardiac differentiation of embryonic stem cells

本课题旨在阐明胚胎干细胞（embryonic stem cells，ES细胞）体外分化为心肌细胞的分子机制，提高心肌细胞的分化效率，并建立ES细胞源性心肌细胞的分离方法。为此，采取了多种方法。首先，我们分析了Smads及其特异性配体在ES细胞心脏分化过程中的作用，发现Smad 2在心脏分化的早期和晚期被双峰激活。Smad 2在早期被Nodal/Cripto激活，这对于内胚层和中胚层诱导以及随后的心脏分化是不可或缺的。TGF-β和Activin负责晚期Smad 2激活，其通过调节心肌细胞的增殖和分化来负性调节心肌发生。其次，我们对心肌细胞进行了信号序列捕获，并鉴定了几种跨膜分子，这些分子尚未被认为在心肌细胞中表达。其中四种在ES细胞和P19 CL 6细胞的心脏分化过程中被诱导表达，这些分子在心脏分化中的作用正在分析中。我们还确定了RNA适体，特异性结合心肌细胞，通过系统进化的配体指数富集（SELEX）。从分化的ES细胞中分离心肌细胞的方法目前正在用鉴定的适体开发。

The aims of this project are to clarify the molecular mechanisms involved in in vitro differentiation of embryonic stem (ES) cells into cardiac myocytes, to improve the efficiency of cardiac differentiation, and to develop the isolation method of ES cell-derived cardiac myocytes. For those purposes, the multiple approaches have been taken. First, we analyzed the role of Smads and their specific ligands during cardiac differentiation of ES cells, and found that Smad2 was activated bimodally in the early and late phases of cardiac differentiation. Smad2 is activated by Nodal/Cripto in the early phase, which is indispensable for endodermal and mesodermal induction and subsequent cardiac differentiation. TGF-β and Activin are responsible for the late Smad2 activation, which negatively regulates cardiomyogenesis by modulating proliferation and differentiation of cardiac myocytes. Second, we performed signal sequence trap on cardiac myocytes, and identified several transmembrane molecules, which have not been recognized as being expressed in cardiac myocytes. Four of these are induced to be expressed during cardiac differentiation of ES cells and Pl9CL6 cells, and the role of these molecules in cardiac differentiation is now being analyzed. We also identified RNA aptamers, which specifically bind to cardiac myocytes, by systematic evolution of ligands by exponential enrichment (SELEX). The isolation method of cardiac myocytes from differentiated ES cells are now being developed with the identified aptamers.