Xiong Yiqin, Zhou Bin, Chang Ching-Pin
Department of Medicine, Division of Cardiovascular Medicine, Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA, USA.
Methods Mol Biol. 2012;843:101-9. doi: 10.1007/978-1-61779-523-7_10.
Malformations of heart valves are one of the most common serious congenital defects. Heart valves are developed from endocardial cushions of the heart. The endocardial cushion in early heart development consists of two cell layers: an outer myocardial cell layer and an inner endocardial cell layer with abundant extracellular matrix (cardiac jelly) in between. Endocardial cells of the cushion, triggered by signals from myocardial cells, delaminate from the surface of the endocardial cushion and undergo transdifferentiation into mesenchymal cells. This process of endocardial-to-mesenchymal transformation (EMT) begins in the atrioventricular canal at embryonic day 9 (E9) and in the cardiac outflow tract at E10 of mouse development. Once formed by the EMT, the mesenchymal cells invade the cardiac jelly, proliferate, and populate the endocardial cushion. The cellularized endocardial cushion then undergoes morphological remodeling; it lengthens and matures into a thin elongated valve leaflet. Here we describe a method to culture endocardial cushions and measure EMT ex vivo. EMT can thus be analyzed independent of other concurrent developmental defects in mice. This culture method also enables ex vivo manipulations of signaling or gene function during EMT to delineate molecular pathways essential for heart valve development.
心脏瓣膜畸形是最常见的严重先天性缺陷之一。心脏瓣膜由心脏的心内膜垫发育而来。心脏早期发育过程中的心内膜垫由两层细胞组成：外层的心肌细胞层和内层的心内膜细胞层，两者之间有丰富的细胞外基质（心胶）。心内膜垫的心内膜细胞在心肌细胞发出的信号触发下，从心内膜垫表面分层，并转分化为间充质细胞。这种心内膜到间充质的转化（EMT）过程在小鼠胚胎发育第9天（E9）的房室管以及E10的心脏流出道开始。一旦通过EMT形成，间充质细胞就会侵入心胶，增殖并填充心内膜垫。细胞化的心内膜垫随后进行形态重塑；它延长并成熟为薄而细长的瓣膜小叶。在这里，我们描述了一种体外培养心内膜垫并测量EMT的方法。因此，可以独立于小鼠中其他并发的发育缺陷来分析EMT。这种培养方法还能够在EMT过程中对信号传导或基因功能进行体外操作，以描绘心脏瓣膜发育所必需的分子途径。
