Efficient Generation of Functional Hepatocytes from Human Induced Pluripotent Stem Cells for Disease Modeling and Disease Gene Discovery.
Efficient Generation of Functional Hepatocytes from Human Induced Pluripotent Stem Cells for Disease Modeling and Disease Gene Discovery.
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从人类诱导多能干细胞中高效生成功能性肝细胞，用于疾病建模和疾病基因发现。
DOI：
10.1007/7651_2021_375
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发表时间：
2022
期刊：
影响因子：
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通讯作者：
Blangero,John
中科院分区：
文献类型：
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作者：
Kumar,Satish;Curran,JoanneE;Williams-Blangero,Sarah;Blangero,John
体外肝细胞模型用于研究肝病的发病机制以及药物开发的发现和临床前阶段。肝细胞系和原代肝细胞作为体外细胞模型的培养已经进行了几十年。然而，肝细胞系（肝癌产生或永生化）在重现肝脏复杂的生理功能时具有有限的准确性。此外，由于来源限制，很难获得来源于人尸体或医学活检的原代肝细胞，特别是对于大规模群体研究或需要大量细胞的应用。从人胚胎干细胞（ESC）和诱导多能干细胞（iPSC）分化的肝细胞培养物在很大程度上克服了传统肝细胞体外模型的局限性。在这一章中，我们描述了一个有效的协议，常规使用在我们的实验室分化成功能性肝细胞培养人iPSCs在体外模拟肝功能和疾病。该方案使用三阶段分化策略从人iPSC产生功能性肝细胞。分化的细胞表现出特征性的肝细胞形态，包括扁平和多边形形状，明显的圆形核，存在胆小管，并且它们表达肝标志物甲胎蛋白（AFP）、白蛋白（ALB）、E-钙粘蛋白（CHD 1）、肝细胞核因子4 α（HNF 4 α）和肌动蛋白。
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.