Elimination of Reprogramming Transgenes Facilitates the Differentiation of Induced Pluripotent Stem Cells into Hepatocyte-like Cells and Hepatic Organoids

SIMPLE SUMMARY: The elimination of reprogramming transgenes affects the differentiation potential of human induced pluripotent stem cells (hiPSCs) at the early embryonic development stage, but not during the late stage of development into hepatocytes and hepatic organoids. Using an excisable polycistronic lentiviral system (STEMCCA, Cre-loxP system), we generated both transgene-carrying and transgene-free hiPSCs from human fibroblasts and demonstrated that the elimination of transgenes enhances the differentiation potential of iPSCs toward hepatocyte-like cells and the generation of hepatic organoids, exhibiting efficient hepatic differentiation. Our findings thus provide significant insights into the characteristics of iPSC-derived hepatic organoids. ABSTRACT: 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.