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.

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Cholangiopathy is a diverse spectrum of chronic progressive bile duct disorders with limited treatment options and dismal outcomes. Scaffold-and stem cell-based tissue engineering technologies hold great promise for reconstructive surgery and tissue repair. Here, we report a combined application of 3D scaffold fabrication and reprogramming of patient-specific human hepatocytes to produce implantable artificial tissues that imitate the mechanical and biological properties of native bile ducts. The human chemically derived hepatic progenitor cells (hCdHs) were generated using two small molecules A83-01 and CHIR99021 and seeded inside the tubular scaffold engineered as a synergistic combination of two layers. The inner electrospun fibrous layer was made of nanoscale-macroscale polycaprolactone fibers acting to promote the hCdHs attachment and differentiation, while the outer microporous foam layer served to increase mechanical stability. The two layers of fiber and foam were fused robustly together thus creating coordinated mechanical flexibility to exclude any

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Background: Artificial tissueoid has tissue-like properties that consist of multicellular component and three-dimension microenvironments. This model recapitulated the in situ environment interactions by providing structure, physiology, and arrangement of individual cells. Here we introduce a novel artificial liver tissueoid platform which can provide in vivo liver multicellular microenvironments and modulate hepatocytes behavior. Methods: The liver tissueoid was hierarchically assembled with cell-laden sheets of which base structures were composed of extracellular matrix (ECM) mimicking electrospun fiber mats. To generate liver tissueoid, human chemically-derived hepatic progenitors (hCdHs) are generated by small molecules as our previously reported. The microfabricated edge-framework for the fiber mat enabled not only stable culture of the functionalized cells but also multilayering construction of cell-laden complexes even including heterogeneous composition of assembly. Results: The heterogenous multilayer stacking, assembling multi-layer stacking with both hCdHs and endothelial cells (HUVECs), significantly enhanced the functional properties of hCdHs to differentiate into hepatocytes and affect survival of mice in acute injury model. Especially, the differentiation potential of hCdHs was associated with OSM downstream signaling pathways. Interestingly, single layered liver tissueoid without HUVECs additionally more activated OSM-downstream signaling pathways, whereas double and triple layered liver tissueoid with HUVECs included both OSM-dependent and OSM-independent pathway which was mediated by selective activation of AKT signaling. Conclusions: Overall, our results suggested that liver tissueoid showed efficient hepatic differentiation capacity of hCdHs and exhibited therapeutic effect after transplantation into liver injury mouse.