Fate of long-term culture on hepatocyte-like cells generated from human adipose-derived mesenchymal stem cells.

BACKGROUND: We previously established a 21-day protocol to generate hepatocyte-like cells (HLCs) from adipose-derived stem cells and are conducting preclinical studies toward their clinical application in liver failure. This study examined how long HLCs maintain viability and hepatocyte-specific functions after differentiation. METHODS: Adipose-derived stem cells (2.0 × 106) were differentiated into HLCs using a 3-step protocol for 21 days. After differentiation, cells were either transferred to transplantation medium (TM), used clinically, or maintained in step-3 differentiation medium (DM). Cell viability, hepatocyte-specific gene expression, and functional assays were performed on days 24 and 28. Because bile acid synthesis may induce cytotoxicity during extended culture, the expression of bile acid-related genes and transporters was evaluated on day 35. RESULTS: Viability markedly decreased when HLCs were transferred to TM, whereas cells maintained in DM preserved viability up to day 28. The expression of mature hepatocyte markers (AAT, ALB, CPS1, and OTC) remained comparable between days 21 and 28. Hepatocyte functions, including CYP3A4 activity and ammonia metabolism, were also sustained in DM-cultured HLCs. Importantly, genes involved in bile acid synthesis and transport, which could contribute to cytotoxicity during prolonged culture, were undetectable even after 28 days. CONCLUSION: HLCs generated using our protocol maintained viability and hepatocyte-specific functions for at least 28 days when cultured in DM, without detectable bile acid-related gene expression. These findings suggest that transplantation timing can be flexibly adjusted for at least 1 week after the completion of differentiation.