Jeong Jaemin, Kim Tae Hun, Kim Myounghoi, Jung Yun Kyung, Kim Kyeong Sik, Shim Sehwan, Jang Hyosun, Jang Won Il, Lee Seung Bum, Choi Dongho
Department of Surgery, Hanyang University College of Medicine, Seoul 04763, Korea.
Laboratory of Radiation Exposure & Therapeutics, National Radiation Emergency Medical Center, Korea Institute of Radiological & Medical Science, Seoul 01812, Korea.
Biology (Basel). 2022 Mar 23;11(4):493. doi: 10.3390/biology11040493.
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 (, 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.
源自人诱导多能干细胞（hiPSC）的肝细胞和肝类器官（HO）是治疗肝脏疾病很有前景的基于细胞的疗法。重编程转基因的去除会影响hiPSC向三个胚层的分化潜能，但在发育后期不会影响其向肝细胞和肝类器官的分化。在此，我们使用基于Cre重组酶介导去除loxP侧翼重编程盒的可切除多顺反子慢病毒载体，从正常人成纤维细胞生成了hiPSC。比较具有相同遗传背景的携带转基因和无转基因hiPSC的特性，所有hiPSC的多能状态非常相似，这通过多能性标志物的表达、胚状体形成和体外三系分化得以体现。然而，在体外分化为肝细胞后，无转基因hiPSC优于残留转基因的hiPSC。有趣的是，从hiPSC中消除转基因显著增强了人肝类器官（hHO）的生成和肝分化，在分化培养基中培养时，可观察到胎儿肝脏（CK19、SOX9和ITGA6）和功能性肝细胞（ASGR1、HNF4α、CYP1A2、CYP3A4和AAT）标志物上调。因此，重编程转基因的消除促进了hiPSC分化为具有肝祖细胞特性的肝细胞样细胞和肝类器官。我们的研究结果为iPSC衍生肝类器官的特性提供了重要见解。
