Differentiation Of Human Pluripotent Stem Cells Into Hepatic Cells And Development Of A Liver Tissue On A Chip

The therapeutic potential of human pluripotent stem (hPS) cells is threatened by the difficulty to homogeneously direct cell differentiation into specific lineages. Aims: a) to efficiently derive mature hepatic cells from hPS cells; b) to integrate the specific lineages into a microfluidic platform to obtain a functional liver tissue on a chip. Methods: Human embryonic stem cells (cell line HES2, National Stem Cell Bank, Madison WI) and induced hPS cells (cell line ADHF#1, iCEMS, Kyoto University) were grown on mouse embryonic fibroblasts (Chemicon, Temecula, CA) and were differentiated on matrigel. Then we developed a multi-stage microfluidic technology to derive mature cells from stem cells. Obtained cells have been characterized both with hepatic markers (alpha-fetoprotein, cytokeratins 18, 19, albumin, CYP3A) and functional tests (glycogen storage, indocyanine green uptake, albumin secretion). Results: We efficiently differentiated both human embryonic and induced pluripotent stem cells. Two hepatic lineages were obtained: hepatocyte- and cholangiocyte-like cells showing high CYP3A expression, indocyanine uptake, glycogen storage and albumin secretion over a 14-day period. This technology allowed to accurately control hPS cells expansion and fate toward early endoderm commitment, hepatic development and functional maturation on a chip. Compared to conventional culture, microfluidic platform allowed shortening of the time required for differentiation and enhanced functional activity. The proportion between hepatocyte- and cholangiocyte-like cells was 3:1. In particular, we obtained 75% of cells with glycogen storage capacity, whereas the number of CYP3A-positive cells resulted in a 59% of the total, with a 20% increase compared to the standard hepatocytes differentiation. Albumin production was about 40% higher than standard conditions. Conclusions: The engineerization of pluripotent cell differentiation into hepatic lineages will allow us to further understand the mechanisms involved in tissue development. Moreover, mature hepatic cells fully integrated on a chip could be directly used for temporal-defined toxicological assays and drug screening