Pharmacological Induction of a Progenitor State for the Efficient Expansion of Primary Human Hepatocytes. Homo sapiens

Transplantation of genetically corrected hepatocytes is an attractive alternative to liver transplantation but is hampered by the low amplification potential of these cells in vitro. Here, we describe a method for generating proliferative hepatic progenitor cells (iHPC) from human hepatocytes as an expandable cell source for liver therapy. Dedifferentiation of primary hepatocytes to iHPC was achieved in less than 7 days by culturing the cells in medium with a cocktail of growth factors and small molecules. In culture, iHPC expressed a combination of endoderm hepatic progenitor and mesenchymal stem cell markers and proliferated vigorously, allowing for their expansion by at least 104 times. RNA sequencing of iHPC demonstrated that they displayed far more subtle changes in both transcriptome and transposcriptome, compared to hepatocyte-derived iPSC. Finally, transplantation of iHPC into the liver of immuno-deficient mice showed iHPC differentiation potential in vivo, without triggering detectable tumor development. Overall design: Transcriptome profiling of human primary hepatocytes from two different pediatric donors, de-differentiation timepoints to induced hepatic progenitor cells (iHPC), one iHPC clone per donor at different passages and two differentiation timepoints to hepatocyte-like cells (HLC).

基因校正肝细胞移植是肝移植极具吸引力的替代方案，但受限于这些细胞在体外的扩增潜能低下。本研究报道了一种可从人肝细胞中制备诱导性肝祖细胞（induced hepatic progenitor cells, iHPC）的方法，将其作为可扩增细胞源用于肝脏治疗。通过将原代肝细胞置于含生长因子与小分子复合物的培养基中培养，可在7天内将其定向去分化为iHPC。在培养过程中，iHPC可共表达内胚层肝祖细胞与间充质干细胞标志物，并具有旺盛的增殖能力，可实现至少104倍的细胞扩增。对iHPC进行RNA测序结果显示，与肝细胞来源的诱导多能干细胞（induced pluripotent stem cells, iPSC）相比，iHPC的转录组与转座子转录组（transposcriptome）仅发生极细微的表达变化。最后，将iHPC移植至免疫缺陷小鼠肝脏内的实验证实，iHPC在体内具有分化潜能，且未引发可检测到的肿瘤发生。实验整体设计：对来自2名不同儿科供者的人原代肝细胞、其去分化为iHPC过程中的多个时间点样本、每名供者的1株iHPC克隆在不同传代代次的样本，以及其向肝细胞样细胞（hepatocyte-like cells, HLC）分化过程中的2个时间点样本进行转录组谱分析。