源自人诱导多能干细胞(hiPSC)的肝细胞和肝类器官(HO)是有前途的基于细胞的肝病疗法。重编程转基因的去除可以影响hiPSC向三个胚层的分化潜力,但不会影响晚期发育阶段的肝细胞和肝类器官。在这里,我们使用基于Cre重组酶介导的loxP侧翼重编程盒去除的可切除多顺反子慢病毒载体,从正常人成纤维细胞中产生了hiPSCs.比较具有相同遗传背景的转基因携带和无转基因hiPSCs的特性,所有hiPSC的多能状态非常相似,如多能标记的表达所示,胚胎身体形成,和体外三谱系分化。然而,体外分化为肝细胞后,无转基因的hiPSC优于转基因残留的hiPSC。有趣的是,通过从hiPSCs中消除转基因,可以显着增强人肝类器官(hHOs)的生成和肝分化,如通过上调胎儿肝脏(CK19,SOX9和ITGA6)和功能性肝细胞(白蛋白,ASGR1,HNF4α,CYP1A2,CYP3A4和AAT)在分化培养基中培养后的标记。因此,消除重编程转基因有助于hiPSC分化为肝细胞样细胞和具有肝祖细胞特性的肝类器官。因此,我们的发现为iPSC衍生的肝类器官的特征提供了重要的见解。
Hepatocytes and hepatic organoids (HOs) derived from human induced pluripotent stem cells (hiPSCs) are promising cell-based therapies for liver diseases. The removal of reprogramming transgenes can affect hiPSC differentiation potential into the three germ layers but not into hepatocytes and hepatic organoids in the late developmental stage. Herein, we generated hiPSCs from normal human fibroblasts using an excisable polycistronic lentiviral vector based on the Cre recombinase-mediated removal of the loxP-flanked reprogramming cassette. Comparing the properties of transgene-carrying and transgene-free hiPSCs with the same genetic background, the pluripotent states of all hiPSCs were quite similar, as indicated by the expression of pluripotent markers, embryonic body formation, and tri-lineage differentiation in vitro. However, after in vitro differentiation into hepatocytes, transgene-free hiPSCs were superior to the transgene-residual hiPSCs. Interestingly, the generation and hepatic differentiation of human hepatic organoids (hHOs) were significantly enhanced by transgene elimination from hiPSCs, as observed by the upregulated fetal liver (CK19, SOX9, and ITGA6) and functional hepatocyte (albumin, ASGR1, HNF4α, CYP1A2, CYP3A4, and AAT) markers upon culture in differentiation media. Thus, the elimination of reprogramming transgenes facilitates hiPSC differentiation into hepatocyte-like cells and hepatic organoids with properties of liver progenitor cells. Our findings thus provide significant insights into the characteristics of iPSC-derived hepatic organoids.