Cell Aggregation Culture Induces Functional Differentiation of Induced Hepatocyte-like Cells through Activation of Hippo Signaling

Recent progress in direct lineage reprogramming has enabled the generation of induced hepatocyte-like (iHep) cells and revealed their potential as an alternative to hepatocytes for medical applications. However, the hepatic functions of iHep cells are insufficient compared with those of primary hepatocytes. Here, we show that cell-aggregate formation can rapidly induce growth arrest and hepatic maturation of iHep cells through activation of Hippo signaling. During formation of iHep cell aggregates, Yap inactivation is induced by actin reorganization and intercellular adhesion, leading to upregulation of Hnf1 alpha expression in the absence of the Yap/Tead/Chd4 transcriptional repressor unit. Hnf1 alpha then acts as a central transcription factor that regulates liver-enriched gene expression in iHep cell aggregates and induces functional differentiation of iHep cells. Moreover, iHep cell aggregates efficiently reconstitute injured liver tissues and support hepatic function after transplantation. Thus, iHep cell aggregates may provide insights into basic research and potential therapies for liver diseases.