Levy Gahl, Bomze David, Heinz Stefan, Ramachandran Sarada Devi, Noerenberg Astrid, Cohen Merav, Shibolet Oren, Sklan Ella, Braspenning Joris, Nahmias Yaakov
Alexander Grass Center for Bioengineering, The Hebrew University of Jerusalem, Jerusalem, Israel.
Upcyte Technologies GmbH, Hamburg, Germany.
Nat Biotechnol. 2015 Dec;33(12):1264-1271. doi: 10.1038/nbt.3377. Epub 2015 Oct 26.
Hepatocytes have a critical role in metabolism, but their study is limited by the inability to expand primary hepatocytes in vitro while maintaining proliferative capacity and metabolic function. Here we describe the oncostatin M (OSM)-dependent expansion of primary human hepatocytes by low expression of the human papilloma virus (HPV) genes E6 and E7 coupled with inhibition of epithelial-to-mesenchymal transition. We show that E6 and E7 expression upregulates the OSM receptor gp130 and that OSM stimulation induces hepatocytes to expand for up to 40 population doublings, producing 10 to 10 cells from a single human hepatocyte isolate. OSM removal induces differentiation into metabolically functional, polarized hepatocytes with functional bile canaliculi. Differentiated hepatocytes show transcriptional and toxicity profiles and cytochrome P450 induction similar to those of primary human hepatocytes. Replication and infectivity of hepatitis C virus (HCV) in differentiated hepatocytes are similar to those of Huh7.5.1 human hepatoma cells. These results offer a means of expanding human hepatocytes of different genetic backgrounds for research, clinical applications and pharmaceutical development.
肝细胞在新陈代谢中起着关键作用，但其研究受到体外无法扩增原代肝细胞同时维持增殖能力和代谢功能的限制。在此，我们描述了通过人乳头瘤病毒（HPV）基因E6和E7的低表达并结合上皮-间质转化的抑制，实现原代人肝细胞在抑瘤素M（OSM）依赖下的扩增。我们表明，E6和E7的表达上调了OSM受体gp130，并且OSM刺激可诱导肝细胞扩增多达40次群体倍增，从单个分离的人肝细胞产生10至10个细胞。去除OSM可诱导分化为具有功能性胆小管的代谢功能正常、极化的肝细胞。分化的肝细胞显示出与原代人肝细胞相似的转录和毒性谱以及细胞色素P450诱导情况。丙型肝炎病毒（HCV）在分化肝细胞中的复制和感染性与Huh7.5.1人肝癌细胞相似。这些结果为扩增不同遗传背景的人肝细胞用于研究、临床应用和药物开发提供了一种方法。
