Differentiating Human ESC Towards Hepatocytes

描述（由申请人提供）：肝脏疾病的原位肝移植（OLT）治疗具有相当高的发病率和死亡率。此外，由于器官短缺，每年有成千上万的人死于器官移植。因此，更安全、更方便的替代疗法将使许多需要肝移植的人受益。一种可能解决这个问题的方法是开发一种表达肝脏特异性基因的增殖细胞系，这种细胞系可以用于细胞移植或生物人工肝。从人类胚胎干细胞（hESC）中培养这样的细胞系也将为药理学和毒理学研究提供有价值的细胞。具体目的：1)确定体外诱导hESC向肝细胞分化的条件；2)对分化hESC进行表征；3)建立NOD-SCID小鼠体内hESC电位；4)在非人灵长类动物体内建立hESC潜能。方法：将对多种条件进行经验性分析，以确定沿肝细胞谱系分化hESC的最有效方法。这将包括优化培养基、细胞外基质和生长因子的实证研究。细胞的纯度将提高转导肝脏特异性借给病毒载体。纯化的细胞将通过肝脏特异性基因产物、生长因子反应性和潜在致癌性的测定来表征。为了确定细胞在移植后是否能够移植、增殖和发挥功能，将在免疫缺陷NOD- SCID小鼠肝细胞损伤模型和非人灵长类动物中进行研究。创新的成像方法将被用来评估细胞在体内的生存能力和增殖能力。与健康的关系：如果研究成功进行，它将提供分化的人肝细胞的无限来源，可用于毒理学和药理学研究，并可用于人体细胞治疗。

DESCRIPTION (provided by applicant): Treatment of liver disease with orthotopic liver transplantation (OLT) carries considerable morbidity and mortality. Moreover, due to organ shortages, thousands of people die each year without getting transplanted. Therefore, safer and more convenient alternative therapies will benefit many people requiring liver transplantation. An approach that might address this problem is the development of a proliferative cell line that expresses liver-specific genes which could be employed for cell transplantation or for a bioartificial liver. Developing such a line from human embryonic stem cells (hESC) would also provide cells valuable for pharmacology and toxicology studies. Specific Aims: 1) to determine conditions for directing the hESC to differentiate into hepatocytes in vitro; 2) to characterize the differentiated hESC; 3) to establish the in vivo potential of hESC in NOD-SCID mice; and 4) to establish the in vivo potential or hESC in nonhuman primates. Methods: A variety of conditions will be empirically assayed to delineate the most effective approach to differentiate the hESC along a hepatocyte lineage. This will include empiric studies of optimizing media, extracellular matrix, and growth factors. The purity of the cells will be enhanced with transduction of liver-specific lent virus vectors. The purified cells will be characterized with assays of liver-specific gene products, growth factor responsiveness, and potential oncogenicity. To determine whether the cells can engraft, proliferate, and function after transplantation, studies will be conducted in immunodeficient NOD- SCID mouse models of liver cell injury and in nonhuman primates. Innovative imaging approaches will be utilized to assess the viability and proliferation of the cells over time in vivo. Health Relatedness: If the studies are successfully undertaken, it will provide for the development of an unlimited source of differentiated human hepatocytes that can be used for toxicology and pharmacology studies, and can be employed in cell-based therapeutics in man.