Life Science R&D Division, FUJIFILM Cellular Dynamics, Inc., 525 Science Drive, Madison, WI 53711, USA.
Life Science R&D Division, FUJIFILM Cellular Dynamics, Inc., 525 Science Drive, Madison, WI 53711, USA
Biol Open. 2020 Dec 16;9(12):bio055087. doi: 10.1242/bio.055087.
Non-alcoholic fatty liver disease (NAFLD) affects 30-40% of adults and 10% of children in the US. About 20% of people with NAFLD develop non-alcoholic steatohepatitis (NASH), which may lead to cirrhosis and liver cancer, and is projected to be a leading cause of liver transplantation in the near future. Human induced pluripotent stem cells (iPSC) from NASH patients are useful for generating a large number of hepatocytes for NASH modeling applications and identification of potential drug targets. We developed a novel defined differentiation process to generate cryopreservable hepatocytes using an iPSC panel of NASH donors and apparently healthy normal (AHN) controls. iPSC-derived hepatocytes displayed stage specific phenotypic markers, hepatocyte morphology, with bile canaliculi. Importantly, both fresh and cryopreserved definitive endoderm and hepatoblasts successfully differentiated to pure and functional hepatocytes with increased CYP3A4 activity in response to rifampicin and lipid accumulation upon fatty acid (FA) treatment. End-stage hepatocytes integrated into three-dimensional (3D) liver organoids and demonstrated increased levels of albumin secretion compared to aggregates consisting of hepatocytes alone. End-stage hepatocytes derived from NASH donors demonstrated spontaneous lipidosis without FA supplementation, recapitulating a feature of NASH hepatocytes Cryopreserved hepatocytes generated by this protocol across multiple donors will provide a critical cell source to facilitate the fundamental understanding of NAFLD/NASH biology and potential high throughput screening applications for preclinical evaluation of therapeutic targets.
非酒精性脂肪性肝病（NAFLD）影响美国 30-40%的成年人和 10%的儿童。大约 20%的 NAFLD 患者发展为非酒精性脂肪性肝炎（NASH），可能导致肝硬化和肝癌，预计在不久的将来成为肝移植的主要原因。来自 NASH 患者的人诱导多能干细胞（iPSC）可用于生成大量肝细胞，用于 NASH 建模应用和鉴定潜在的药物靶点。我们开发了一种新颖的定义明确的分化过程，使用 NASH 供体和明显健康的正常（AHN）对照的 iPSC 面板生成可冷冻保存的肝细胞。iPSC 衍生的肝细胞显示出阶段特异性表型标志物、肝细胞形态和胆小管。重要的是，新鲜和冷冻保存的明确内胚层和肝母细胞都成功分化为纯和功能性肝细胞，对利福平的 CYP3A4 活性增加，并在脂肪酸（FA）处理时脂质积累。终末期肝细胞整合到三维（3D）肝类器官中，并与仅由肝细胞组成的聚集物相比，表现出更高水平的白蛋白分泌。来自 NASH 供体的终末期肝细胞在没有 FA 补充的情况下自发发生脂肪变性，再现了 NASH 肝细胞的一个特征。该方案生成的冷冻保存的肝细胞将跨越多个供体提供关键的细胞来源，以促进对 NAFLD/NASH 生物学的基本理解和治疗靶点的高通量筛选应用的临床前评估。
