Two-dimensionally cultured functional hepatocytes generated from human induced pluripotent stem cell-derived hepatic organoids for pharmaceutical research

Human induced pluripotent stem (iPS) cell-derived hepatocyte-like cells (HLCs) are expected to replace primary human hepatocytes (PHHs) as a new stable source of hepatocytes for pharmaceutical researches. However, HLCs have lower hepatic functions than PHHs, take a long time to be differentiated and cannot be prepared in large quantities due to not proliferating after their terminal differentiation. To overcome these problems, we have established hepatic organoids (iHOs) from HLCs. The iHOs could proliferate approximately 10^20-fold by more than 10 passages and expressed most hepatic genes higher than HLCs. Furthermore, in order to enable the widespread use of iHOs for in vitro drug discovery research, we have developed the two-dimensional culture protcol for them. Two-dimensional cutured iHOs (2D-iHOs) expressed most of major hepatocyte marker genes and proteins much higher than HLC, iHOs, and even PHHs. The 2D-iHOs had glycogen storage capacity, the capacity to uptake and release indocyanine green (ICG), albumin and urea secretion, and the capacity of bile canaliculi formation. Importantly, the 2D-iHOs had the activity of major drug-metabolizing enzymes and responded to hepatotoxic drugs, comparable to PHHs. Thus, 2D-iHOs overcome the limitation of the current models and promise to provide robust and reproducible pharmaceutical assays.