Hyperbaric Oxygen Treatment Enhances Definitive Endoderm Differentiation and Hepatic Maturation of Human-Induced Pluripotent Stem Cells in Association with mTORC2-Dependent AKT Phosphorylation

Hepatocyte-like cells derived from human-induced pluripotent stem cells (hiPSCs) are widely used in disease modeling, drug screening, and regenerative medicine. However, efficient generation of mature hepatocyte-like cells from hiPSCs remains challenging. In this study, we investigated whether hyperbaric oxygen treatment (HBOT) during the definitive endoderm (DE) differentiation process enhances DE induction and subsequent hepatocyte maturation. hiPSCs were exposed to HBOT 1∼3 times during a 4-day DE differentiation protocol. Among these, two HBOT sessions most effectively increased the expression of DE-specific markers, including SRY-box transcription factor 17, GATA binding protein 4, forkhead box A2, and C-X-C motif chemokine receptor 4 (CXCR4). Flow cytometry revealed that HBOT increased both the proportion of CXCR4-positive DE cells and the mean CXCR4 fluorescence intensity. Mechanistically, HBOT-induced phosphorylation of protein kinase B (AKT) at serine 473, which was not completely inhibited by phosphoinositide 3-kinase but was reduced by Torin2 treatment. This suggests that AKT activation induced by HBOT is associated with a mechanistic target of rapamycin complex 2-dependent pathway. Furthermore, DE cells generated under HBOT conditions produced hepatocyte-like cells exhibiting increased expression of hepatic markers, such as albumin, alpha-fetoprotein, hepatocyte nuclear factor 4 alpha, and asialoglycoprotein receptor 1. Collectively, these results demonstrate that HBOT enhances DE induction efficiency during the early differentiation stage and promotes the generation of more mature hiPSC-derived hepatocyte-like cells. The findings suggest that HBOT can be a useful physical regulatory strategy to improve hiPSC-based hepatocyte differentiation protocols.