Weiand Matthias, Ballmaier Paula, Niemietz Christoph, Schmidt Hartmut, Zibert Andree
Medizinische Klinik B, Universitätsklinikum Münster, Münster, Germany.
Biochem Biophys Rep. 2022 Jul 16;31:101308. doi: 10.1016/j.bbrep.2022.101308. eCollection 2022 Sep.
Human primary cells, including urine-derived cells (UCs), are an excellent source for generation of pluripotent stem cells (iPSCs) to model disease. However, replicative senescence starts early and shortens the time window for generation of iPSCs. We addressed the question whether combinations of transgenes allows efficient immortalization of UCs, iPSC generation, and differentiation into hepatocyte-like cells (HLCs). Retroviral transfer of three gene cassettes HPVE6E7 (H), hTERT/p53DD (T), cyclinD1/CDK4R24C (C) encoding five genes was established in primary UCs. Long-term cell proliferation was observed in cells carrying transgenes H, HT, HC, and HCT, whereas cells carrying transgenes C, T and CT showed early senescence similar to UCs. iPSCs could be exclusively generated from immortalized UCs transduced with transgenes HCT and HC. iPSC colonies appeared however later and in smaller number as compared to UCs. Using an established hepatic differentiation protocol, HLCs were obtained with high efficacy. Of note, a high expression of individual transgenes was observed in immortalized UCs, which was down-regulated after reprogramming in four out of five genes. One transgene was re-expressed in HLCs as compared to iPSCs. Our data suggest that individual transgene combinations result in advanced growth rates of immortalized cells and do not prevent iPSC formation and HLC differentiation. Retroviral transgene expression is mostly silenced in iPSCs but can be rarely re-expressed after hepatic differentiation. An extended time window for iPSC establishment can be proposed that allows straightforward functional analyses of differentiated cells.
人类原代细胞，包括尿液来源细胞（UCs），是生成用于疾病建模的多能干细胞（iPSCs）的优质来源。然而，复制性衰老出现得早，缩短了生成iPSCs的时间窗口。我们探讨了转基因组合是否能使UCs高效永生化、生成iPSCs并分化为肝细胞样细胞（HLCs）这一问题。在原代UCs中建立了编码五个基因的三个基因盒HPVE6E7（H）、hTERT/p53DD（T）、细胞周期蛋白D1/细胞周期蛋白依赖性激酶4R24C（C）的逆转录病毒转导。在携带转基因H、HT、HC和HCT的细胞中观察到长期细胞增殖，而携带转基因C、T和CT的细胞则表现出与UCs相似的早期衰老。iPSCs只能从用转基因HCT和HC转导的永生化UCs中产生。然而，与UCs相比，iPSC集落出现得更晚且数量更少。使用既定的肝脏分化方案，高效获得了HLCs。值得注意的是，在永生化UCs中观察到单个转基因的高表达，在重编程后五个基因中有四个基因的表达下调。与iPSCs相比，有一个转基因在HLCs中重新表达。我们的数据表明，单个转基因组合导致永生化细胞的生长速度加快，且不阻碍iPSC形成和HLC分化。逆转录病毒转基因表达在iPSCs中大多沉默，但在肝脏分化后很少能重新表达。可以提出一个延长的iPSC建立时间窗口，这允许对分化细胞进行直接的功能分析。
