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


CONCLUSIONS
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.
Fang
Sheng
S
Department of Hepatobiliary Surgery, Affiliated Drum Tower Hospital, Medical College of Nanjing University, Nanjing 210008, China.
Qiu
Yu-dong
YD
Mao
Liang
L
Shi
Xiao-lei
XL
Yu
De-cai
DC
Ding
Yi-tao
YT
D000464:Alginates / Q000737:chemistry*; D000818:Animals; D001483:Base Sequence; D002454:Cell Differentiation; D017931:DNA Primers; D053595:Embryonic Stem Cells / Q000166:cytology*; D005455:Fluorescent Antibody Technique; D020723:Glucuronic Acid / Q000737:chemistry; D022781:Hepatocytes / Q000166:cytology*; D006603:Hexuronic Acids / Q000737:chemistry; D066298:In Vitro Techniques; D051379:Mice; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000464:Alginates; D017931:DNA Primers; D006603:Hexuronic Acids; D020723:Glucuronic Acid