Abstract
Generating the proliferation of differentiated normal adult human hepatocytes is a major challenge and an expected central step in understanding the microenvironmental conditions that regulate the phenotype of human hepatocytes in vitro. In this work, we described optimized 3D culture conditions of primary human hepatocytes (PHH) to trigger two waves of proliferation and we identified matrix stiffness and cell-cell interactions as the main actors necessary for this proliferation. We demonstrated that DNA replication and overexpression of cell cycle markers are modulate by the matrix stiffness while PHH cultured in 3D without prior cellular interactions did not proliferate. Besides, we showed that PHH carry out an additional cell cycle after transient inhibition of MAPK MER1/2-ERK1/2 signaling pathway. Collagen cultured hepatocytes are organized as characteristic hollow spheroids able to maintain survival, cell polarity and hepatic differentiation for long-term culture periods of at least 28 days. Remarkably, we demonstrated by transcriptomic analysis and functional experiments that proliferating cells are mature hepatocytes with high detoxication capacities. In conclusion, the advanced 3D model described here, named Hepoid, is particularly relevant for obtaining normal human proliferating hepatocytes. By allowing concomitant proliferation and differentiation, it constitutes a promising tool for many pharmacological and biotechnological applications.