Department of Human Genetics and South Texas Diabetes and Obesity Institute, University of Texas Rio Grande Valley School of Medicine, McAllen, TX, USA.
Department of Human Genetics and South Texas Diabetes and Obesity Institute, University of Texas Rio Grande Valley School of Medicine, Brownsville, TX, USA.
Methods Mol Biol. 2022;2549:85-101. doi: 10.1007/7651_2021_375.
In vitro hepatocyte cell models are being used to study the pathogenesis of liver disease and in the discovery and preclinical stages of drug development. The culture of hepatic cell lines and primary hepatocytes as in vitro cell models has been carried out for several decades. However, hepatic cell lines (hepatic carcinoma generated or immortalized) have limited accuracy when recapitulating complex physiological functions of the liver. Additionally, primary hepatocytes sourced from human cadavers or medical biopsies are difficult to obtain due to sourcing limitations, particularly for large-scale population studies or in applications requiring large number of cells. Hepatocyte cultures differentiated from human embryonic stem cells (ESCs) and induced pluripotent stem cell (iPSCs) overcome in large part the limitations of traditional hepatocyte in vitro models. In this chapter, we described an efficient protocol routinely used in our laboratory to differentiate human iPSCs into functional hepatocyte cultures for in vitro modeling of liver function and disease. The protocol uses a three-stage differentiation strategy to generate functional hepatocytes from human iPSCs. The differentiated cells show characteristic hepatocyte morphology including flat and polygonal shape, distinct round nuclei, and presence of biliary canaliculi and they express hepatic markers alpha-fetoprotein (AFP), albumin (ALB), E-cadherin (CHD1), hepatocyte nuclear factor 4 alpha (HNF4α), and actin.
体外肝细胞模型正被用于研究肝脏疾病的发病机制，以及在药物开发的发现和临床前阶段。几十年来，人们一直在培养肝细胞系和原代肝细胞作为体外细胞模型。然而，肝肿瘤生成或永生化的肝细胞系在重现肝脏复杂生理功能方面的准确性有限。此外，由于来源有限，尤其是在需要大量细胞的大规模人群研究或应用中，从人体尸体或医学活检中获取的原代肝细胞较为困难。从人类胚胎干细胞（ESC）和诱导多能干细胞（iPSC）分化而来的肝细胞克服了传统体外肝细胞模型的大部分局限性。在本章中，我们描述了一种在我们实验室中常规使用的有效方案，用于将人类 iPSC 分化为功能性肝细胞培养物，用于体外肝脏功能和疾病建模。该方案使用三阶段分化策略从人类 iPSC 中生成功能性肝细胞。分化后的细胞表现出典型的肝细胞形态，包括扁平多角形、明显的圆形核、胆管的存在，并且表达肝标志物甲胎蛋白（AFP）、白蛋白（ALB）、E-钙黏蛋白（CHD1）、肝细胞核因子 4α（HNF4α）和肌动蛋白。
