Scientist STEMCELL Technologies Inc. Vancouver, British Columbia, Canada
Abstract: Functionally relevant human hepatocyte models are critical for drug safety and efficacy screening, cell therapy, and disease modeling. However, currently available in vitro models, such as primary human hepatocytes and immortalized cell lines, often rapidly de-differentiate or lack metabolic maturity. Human pluripotent stem cell (hPSC)-derived hepatocytes and liver organoids represent convenient, scalable, and patient-representative alternatives to conventional models. STEMdiff™ Hepatocyte Kit supports efficient and reproducible generation of hPSC-derived hepatocyte-like cells (HLCs) over 21 days. hPSCs are patterned to definitive endoderm cells, then differentiated to hepatic progenitors, and finally matured to HLCs. The resulting HLCs exhibited hepatic marker expression (66 ± 8% ALB+A1AT+; mean ± SD) and mature functionality, including CYP3A4 enzymatic activity and albumin secretion (n = 2 - 15). HLCs were also replated in Matrigel® domes to establish passageable liver organoids amenable to cryopreservation using STEMdiff™ Hepatic Organoid Growth Medium (n = 11). These organoids were further matured in 3D culture using STEMdiff™ Hepatic Organoid Differentiation Medium (ODM), resulting in significant downregulation of fetal hepatocyte gene AFP (5.7-fold decrease; p < 0.0001), and increased expression of several mature hepatic genes, including ALB, ASGR1, CYP3A4, CYP2C9, UGT1A1, and OTC (n = 2 - 20). ODM-differentiated cultures also exhibited increased albumin secretion (2.9-fold increase; p = 0.0199) as well as CYP3A4 activity that was modulated using ketoconazole (average 2.0-fold decrease) and calcitriol (average 2.5-fold increase). HLC-derived organoids were sensitive to ketoconazole-, rifampicin-, and acetaminophen-induced hepatotoxicity (average ODM-differentiated organoid IC50 = 58 µM, 527 µM, and 1.8x104 µM, respectively; n = 2), and could be used to generate viability data that successfully distinguished between low and high liver injury-causing compounds that are structurally similar. These results demonstrate the utility of STEMdiff™ Hepatocyte Kit and STEMdiff™ Hepatic Organoid Media in hPSC-derived liver modeling and drug screening applications.
