Differentiation of embryoid-body cells derived from embryonic stem cells into hepatocytes in alginate microbeads in vitro.

AIM: Embryonic stem (ES) cells are being widely investigated as a promising source of hepatocytes with their proliferative, renewable, and pluripotent capacities. However, controlled and scalable ES cell differentiation culture into functional hepatocytes is challenging. In this study, we examined the differentiating potential of embryoid-body cells derived from ES cells into hepatocytes in alginate microbeads containing exogenous growth factors in vitro.

METHODS: Embryoid bodies were formed from ES cells by suspension methods. Embryoid bodies cultured for 5 d were treated with trypsin-EDTA. The disaggregated cells were encapsulated in alginate microbeads and stimulated with exogenous growth factors to induce hepatic differentiation. In the course of cell differentiation, cell morphology and viability were observed, and the expression patterns of some genes of the hepatocyte were confirmed by RT-PCR. An immunofluorescence analysis revealed the expression of albumin (ALB) and cytokeratin-18 (CK18). Hepatocyte functional assays were confirmed by the secretion of ALB and urea.

RESULTS: We showed that embryoid-body cells could maintain cell viability in alginate microbeads in vitro. We also found that directed differentiated cells expressed several hepatocyte genes including alpha-fetoprotein (AFP), ALB, Cyp7a1, CK18, transthyretin (TTR) and tyrosine aminotransferase (TAT) and produced ALB and urea in alginate microbeads. The directed differentiated cells expressed ALB and CK18 proteins on d 14. However, embryoid-body cells could not form hepatocytes without exogenous growth factors in alginate microbeads.

CONCLUSION: The differentiation of embryoid-body cells into hepatocytes containing exogenous growth factors in alginate microbeads gives rise to functional hepatocytes and may develop scalable stem cell differentiation strategies for bioartificial livers and hepatocyte transplantation.