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

本课题旨在阐明胚胎干细胞体外分化为心肌细胞的分子机制，提高心脏分化效率，发展胚胎干细胞源性心肌细胞的分离方法。为此目的，采取了多种办法。首先，我们分析了smad及其特异性配体在ES细胞心脏分化过程中的作用，发现Smad2在心脏分化的早期和晚期被双峰激活。Smad2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必不可少的。TGF-β和Activin负责Smad2的晚期激活，Smad2通过调节心肌细胞的增殖和分化负向调节心肌发生。其次，我们对心肌细胞进行了信号序列陷阱，并鉴定了几个未被识别为在心肌细胞中表达的跨膜分子。其中4个在ES细胞和Pl9CL6细胞的心脏分化过程中被诱导表达，目前正在分析这些分子在心脏分化中的作用。我们还通过指数富集（SELEX）的配体系统进化发现了RNA适体，它可以特异性地结合到心肌细胞上。从分化的胚胎干细胞中分离心肌细胞的方法正在研究中。

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.