Collins Daniel P, Hapke Joel H, Aravalli Rajagopal N, Steer Clifford J
Cytomedical Design Group, LLC, Saint Paul, MN 55127, USA.
Department of Electrical and Computer Engineering, College of Science and Engineering, University of Minnesota, Minneapolis, MN 55455, USA.
Hepat Med. 2020 Jun 11;12:79-92. doi: 10.2147/HMER.S245916. eCollection 2020.
Research directed towards drug development, metabolism, and liver functions often utilize primary hepatocytes (PH) for preliminary in vitro studies. Variability in the in vitro functionality of PH and the unsuitability of hepatocarcinoma cells for these studies have driven researchers to look to ESC, iPS, and other stem cell types using differentiation protocols to provide more reliable and available cells. This study describes the development of hepatocyte-like cells through the in vitro differentiation of human TERT-immortalized cord blood-derived multi-lineage progenitor cells (MLPC). The E12 clonal cell line derived from polyclonal TERT-transfected cells was used throughout the study.
E12 MLPC were subjected to a three-step differentiation protocol using alternating combinations of growth factors, cytokines, and maturational factors. Cells at various stages of differentiation were analyzed for consistency with PH by morphology, immunohistochemistry, urea production, and gene expression.
E12 MLPC were shown to significantly change morphology with each stage of differentiation. Coincidental with the morphological changes in the cells, immunohistochemistry data documented the differentiation to committed endoderm by the expression of SOX-17 and GATA-4; the progression to committed hepatocyte-like cells by the expression of a large number of markers including α-fetoprotein and albumin; and the final differentiation by the expression of nuclear and cytoplasmic HNF4. Fully differentiated cells demonstrated gene expression, urea production, and immunohistochemistry consistent with PH. A methodology and medium formulation to continuously expand the E12-derived hepatocyte-like cells is described.
The availability of immortalized hepatocyte-like cell lines could provide a consistent tool for the study of hepatic diseases, drug discovery, and the development of cellular therapies for liver disorders. Utilization of these techniques could provide a basis for the development of bridge therapies for liver failure patients awaiting transplant.
针对药物开发、代谢和肝功能的研究通常利用原代肝细胞（PH）进行初步体外研究。原代肝细胞体外功能的变异性以及肝癌细胞不适用于这些研究，促使研究人员寻求使用分化方案的胚胎干细胞（ESC）、诱导多能干细胞（iPS）和其他干细胞类型，以提供更可靠且可用的细胞。本研究描述了通过人端粒酶逆转录酶（TERT）永生化脐血来源的多谱系祖细胞（MLPC）的体外分化来生成肝细胞样细胞。在整个研究中使用了源自多克隆TERT转染细胞的E12克隆细胞系。
使用生长因子、细胞因子和成熟因子的交替组合，对E12 MLPC进行三步分化方案。通过形态学、免疫组织化学、尿素生成和基因表达分析分化各阶段的细胞与原代肝细胞的一致性。
E12 MLPC在分化的每个阶段均显示出明显的形态变化。与细胞形态变化一致，免疫组织化学数据表明，通过SOX - 17和GATA - 4的表达分化为定型内胚层；通过包括甲胎蛋白和白蛋白在内的大量标志物的表达进展为定型肝细胞样细胞；通过核和细胞质肝细胞核因子4（HNF4）的表达实现最终分化。完全分化的细胞在基因表达、尿素生成和免疫组织化学方面与原代肝细胞一致。描述了一种持续扩增源自E12的肝细胞样细胞的方法和培养基配方。
永生化肝细胞样细胞系的可用性可为肝病研究、药物发现以及肝脏疾病细胞疗法开发提供一致的工具。利用这些技术可为等待移植的肝衰竭患者开发过渡治疗提供基础。
