3-D culture and endothelial cells improve maturity of human pluripotent stem cell-derived hepatocytes

Human-induced pluripotent stem cell (hiPSC)-derived hepatocytes (iHEP) offer an attractive alternative to primary human hepatocytes (PHH) for drug toxicity studies, as PHHs are limited in supply, vary in their metabolic activity between donors, and rapidly lose their functionality in vitro. However, one of the major drawbacks with iHEP cells in drug safety studies is their decreased phenotypic maturity, with lower liver specific enzyme activity compared with that of PHH. Here we evaluated the effects of 3D culture and non-parenchymal cells on the maturation of iHEPs. We describe a serum-free, chemically defined 3D in vitro model using iHEP cells, which is compatible with automation and conventional assay plates. The iHEP cells cultured in this model form polarized aggregates with functional bile canaliculi and strongly increased expression of albumin, urea and genes encoding phase I and II drug metabolism enzymes and bile transporters. Cytochrome P450-mediated metabolism is significantly higher in 3D iHEP aggregates compared to 2D iHEP culture. Furthermore, addition of human liver sinusoidal endothelial cells (sECs) and iPS-derived endothelial cells (iECs) improved mature hepatocyte function and CYP450 enzyme activity. Also, ECs formed endothelial networks within the hepatic 3D cultures, mimicking aspects of an in vivo architecture. Collectively, these results suggest that the iHEP/EC aggregates described here may have the potential to be used for many applications, including as an in vitro model to study liver diseases associated with sinusoidal endothelial cells. STATEMENT OF SIGNIFICANCE: iPS-derived hepatocytes provide an inexhaustible source of cells for drug screening, toxicology studies and cell-based therapies, but lack mature phenotype of adult primary human hepatocytes (PHH). Herein, we show that 3D culture of iPS-derived hepatocytes and their co-culture with human sinusoidal endothelial cells (sECs) to improve their maturity.