Fig. 3

Single donor cell-derived LOs generated from single donor-derived hiPSC-endoderm, ECs, and MCs. a Schematic representation of the protocol for LO generation and differentiation from single donor-derived hiPSC-endoderm, ECs, and MCs. b Morphology of single donor cell-derived LOs (SDC-LOs) on days 1 and 15. Scale bars, 100 μm. c Immunocytochemical detection of ALB (red) and A1AT (green) expression in differentiated SDC-LOs on day 15. Scale bars, 100 μm. d Expression of common hepatic genes (ALB, G6PC, RBP4, TAT, and TDO2), urea/ammonia metabolism-related genes (CPS1 and OTC), and cytochrome P450-encoding genes (CYP2C9, CYP2C19, CYP3A4, CYP3A5, and CYP3A7) in EC-iPSC-HLCs (blue, n = 6) and differentiated SDC-LOs (red, n = 6), determined by qPCR. e ALB production in EC-iPSC-HLCs (n = 6), differentiated SDC-LOs (n = 6), and primary human hepatocytes (PHHs, n = 4); data normalized to 1 million cells. f Urea production in EC-iPSC-HLCs (n = 3), differentiated SDC-LOs (n = 3), and PHHs (n = 4); data normalized to 1 million cells. g Quantification of CYP3A4 activity in EC-iPSC-HLCs (n = 4) and SDC-LOs (n = 4) with or without rifampicin (25 μM) induction; data normalized to 1 million cells. ***p < 0.001. ns not significant, ALB albumin, EC endothelial cell, HGF hepatocyte growth factor, hiPSC human induced pluripotent stem cell, HLC hepatic-like cell, LO liver organoid, MC mesenchymal cell, PHH primary human hepatocyte, UC umbilical cord, CYP cytochrome P450, G6PC glucose-6-phosphatase catalytic subunit, TDO2 tryptophan 2,3-dioxygenase