Development of hepatocytes from ES cells

描述(由申请人提供)： 最终内胚层的发育及其随后的规范导致许多主要器官的形成，包括肝、胰腺、肺、甲状腺和肠道。在适当的培养条件下，小鼠胚胎干细胞(ES)分化为类胚体(EBS)，其中含有所有三种胚层的衍生物。这些研究大多集中在中胚层和外胚层衍生物的发育上。造血、血管、肌肉和神经元谱系的发育与早期胚胎的发育相似，从而验证了ES系统用于早期发育研究的有效性。最近，研究提供了ES分化培养中内胚层发育的证据，并证明了胰岛素表达细胞和具有肝细胞特征的细胞的产生。ES研究尚未建立有效诱导内胚层或其对肝脏的规范的条件。 这项建议通过研究特定的发育步骤来解决这些问题，包括(1)诱导ES细胞形成内胚层命运，(2)将内胚层指定为肝细胞前体，称为肝母细胞，以及(3)肝母细胞成熟为功能肝细胞。因此，肝脏作为内胚层来源的组织在这一应用中是重点。早期内胚层宿命细胞以及肝母细胞将通过细胞分选分析从ES细胞培养中鉴定出来。将在两种肝功能衰竭的小鼠模型中分析内胚层来源细胞的肝脏再繁殖潜力。该项目还将寻求翻译小鼠ES细胞分化程序，以优化人类肝细胞的生成。因此，这些研究的最终目标是开发一种可行的方法来从ES细胞中产生有功能的肝细胞，并了解从ES细胞中协调肝细胞发育的机制。

DESCRIPTION (provided by applicant): The development of definitive endoderm and its subsequent specification leads to the formation of many of the major organs including the liver, pancreas, lungs, thyroid, and intestines. Under appropriate culture conditions, mouse embryonic stem (ES) cells differentiate into embryoid bodies (EBs) that contain derivatives of all three germ layers. Most of these studies have focused on the development of mesoderm and ectoderm derivatives. The development of hematopoietic, vascular, muscle and neuronal lineages were found to parallel that in the early embryo, thus validating the ES system for early developmental studies. More recently, studies have provided evidence for endoderm development in ES differentiation cultures and demonstrated the generation of insulin expressing cells and cells with hepatocyte characteristics. ES studies have not yet established conditions for the efficient induction of endoderm or its specification to liver. This proposal addresses these issues by investigating specific steps of development including (1) induction of ES cells to an endoderm fate, (2) specification of endoderm into a hepatocyte progenitor, called the hepatoblast and (3) maturation of the hepatoblast into a functional hepatocyte. Thus, the liver is the focus in this application as an endoderm-derived tissue. Early endoderm fated cells as well as the hepatoblast will be characterized from ES cell cultures using cell sorting analysis. The liver repopulation potential of endoderm-derived cells will be analyzed in two mouse models of liver failure. The project will also seek to translate the murine ES cell differentiation program to optimize the generation of human hepatocytes. Therefore, the ultimate goal of these studies is to develop a feasible approach to generate functional hepatocytes from ES ells and to understand the mechanisms that orchestrate the hepatocyte development from ES cells.