Ruiz Joseph C
Transposagen Biopharmaceuticals, Inc., Lexington, KY, USA.
Methods Mol Biol. 2011;702:249-60. doi: 10.1007/978-1-61737-960-4_18.
The demand for primary human hepatocytes to test the toxicity of new drug candidates and to develop cell therapies for liver disease far exceed the number of hepatocytes that can be isolated from donated tissues. Less than 700 whole livers per year are available for research applications. The ability to utilize nonhepatic progenitor cells, such as adipose stromal cells (ASCs), to generate derivatives that mimic primary human hepatocytes would enable the scale-up production of cell products for bioartifical liver-assist devices, cell therapy, and drug discovery applications. ASC hepatogenesis is a rapidly evolving field with improved protocols continually being reported in the literature. In this chapter, current and effective protocols for the expansion, hepatic differentiation, and functional characterization of ASC-derived hepatic cells are outlined. Two major features distinguish optimized methodologies: (a) cytokine-mediated "reprogramming" of mesenchymal ASCs to enable transdifferentiation into endodermal cell lineages, and (b) treatment with sequential media formulations containing factors/extracellular matrices that mimic the temporal expression profiles seen during fetal liver development. Criteria for success are acquisition of hepatic functional activities, such as albumin/urea production and p450 CYP activities, at levels that approach those observed in primary human hepatocyte controls.
用于测试新药候选物毒性以及开发肝病细胞疗法的原代人肝细胞的需求，远远超过了从捐赠组织中分离得到的肝细胞数量。每年可用于研究用途的全肝不到700个。利用非肝祖细胞，如脂肪基质细胞（ASC），来生成模拟原代人肝细胞的衍生物，将能够扩大用于生物人工肝辅助装置、细胞治疗和药物发现应用的细胞产品的生产规模。ASC向肝细胞的分化是一个快速发展的领域，文献中不断报道有改进的方案。在本章中，概述了用于ASC来源的肝细胞的扩增、肝分化和功能表征的当前有效方案。优化方法有两个主要特点：（a）细胞因子介导的间充质ASC的“重编程”，以使其能够转分化为内胚层细胞谱系；（b）用含有模拟胎儿肝脏发育过程中观察到的时间表达谱的因子/细胞外基质的顺序培养基配方进行处理。成功的标准是获得肝脏功能活性，如白蛋白/尿素生成和p450 CYP活性，其水平接近在原代人肝细胞对照中观察到的水平。
