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

AbstractAim: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 α-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.

[1]  K. Sidhu,et al.  Differentiation of Encapsulated Embryonic Stem Cells After Transplantation , 2006, Transplantation.

[2]  Martin Yarmush,et al.  Alginate‐PLL microencapsulation: Effect on the differentiation of embryonic stem cells into hepatocytes , 2006, Biotechnology and bioengineering.

[3]  Yu Shu,et al.  Mouse Embryonic Stem Cells: Advances in Technology , 2006 .

[4]  Wei Wang,et al.  Proliferation and Differentiation of Mouse Embryonic Stem Cells in APA Microcapsule: A Model for Studying the Interaction between Stem Cells and Their Niche , 2006, Biotechnology progress.

[5]  Hossein Baharvand,et al.  Differentiation of human embryonic stem cells into hepatocytes in 2D and 3D culture systems in vitro. , 2006, The International journal of developmental biology.

[6]  N. Benvenisty,et al.  Study of hepatocyte differentiation using embryonic stem cells , 2005, Journal of cellular biochemistry.

[7]  D. Choi,et al.  In Vitro Differentiation of Mouse Embryonic Stem Cells: Enrichment of Endodermal Cells in the Embryoid Body , 2005, Stem cells.

[8]  T. Ochiya,et al.  Direct hepatic fate specification from mouse embryonic stem cells , 2005, Hepatology.

[9]  P. Zandstra,et al.  Scalable production of embryonic stem cell-derived cells. , 2005, Methods in molecular biology.

[10]  K. Sasaki,et al.  Embryonic stem cell-derived embryoid bodies in three-dimensional culture system form hepatocyte-like cells in vitro and in vivo. , 2004, Tissue engineering.

[11]  S. Arii,et al.  Expression of the liver‐specific gene Cyp7a1 reveals hepatic differentiation in embryoid bodies derived from mouse embryonic stem cells , 2004, Genes to cells : devoted to molecular & cellular mechanisms.

[12]  J. Itskovitz‐Eldor,et al.  Three‐dimensional porous alginate scaffolds provide a conducive environment for generation of well‐vascularized embryoid bodies from human embryonic stem cells , 2004, Biotechnology and bioengineering.

[13]  Xiao-Qing He,et al.  High‐ratio differentiation of embryonic stem cells into hepatocytes in vitro , 2004, Liver international : official journal of the International Association for the Study of the Liver.

[14]  Gordon Keller,et al.  Development of definitive endoderm from embryonic stem cells in culture , 2004, Development.

[15]  A. Bishop,et al.  Embryonic stem cells , 2004, Cell proliferation.

[16]  M. Manns,et al.  Quantitative gene expression profiling reveals a fetal hepatic phenotype of murine ES-derived hepatocytes. , 2004, The International journal of developmental biology.

[17]  Gabriela Kania,et al.  Generation of glycogen- and albumin-producing hepatocyte-like cells from embryonic stem cells , 2004, Biological chemistry.

[18]  Shu Dong Xiao,et al.  Generation of hepatocytes from cultured mouse embryonic stem cells , 2003, Liver transplantation : official publication of the American Association for the Study of Liver Diseases and the International Liver Transplantation Society.

[19]  Smadar Cohen,et al.  Liver tissue engineering within alginate scaffolds: effects of cell-seeding density on hepatocyte viability, morphology, and function. , 2003, Tissue engineering.

[20]  Jian Wang,et al.  Induction of hepatic differentiation in embryonic stem cells by co-culture with embryonic cardiac mesoderm. , 2003, Surgery.

[21]  Y. Ouji,et al.  In vitro differentiation of hepatocyte-like cells from embryonic stem cells promoted by gene transfer of hepatocyte nuclear factor 3 beta. , 2003, Hepatology research : the official journal of the Japan Society of Hepatology.

[22]  Wai-Hee Lo,et al.  Chondrogenesis of human mesenchymal stem cells encapsulated in alginate beads. , 2003, Journal of biomedical materials research. Part A.

[23]  M. Salto‐Tellez,et al.  AFP+, ESC‐Derived Cells Engraft and Differentiate into Hepatocytes in Vivo , 2002, Stem cells.

[24]  J. Neuberger,et al.  A Bioartificial Liver--State of the Art , 2002, Science.

[25]  J. Ijzermans,et al.  Prospects for the temporary treatment of acute liver failure. , 2002, European journal of gastroenterology & hepatology.

[26]  J C Perriard,et al.  Mass Production of Embryoid Bodies in Microbeads , 2001, Annals of the New York Academy of Sciences.

[27]  Naohiro Terada,et al.  Hepatic maturation in differentiating embryonic stem cells in vitro , 2001, FEBS letters.

[28]  M. Goosen.  Physico‐chemical and Mass Transfer Considerations in Microencapsulation , 1999, Annals of the New York Academy of Sciences.

[29]  H. Niwa,et al.  Endoderm-specific gene expression in embryonic stem cells differentiated to embryoid bodies. , 1996, Experimental cell research.

[30]  A. Globerson,et al.  In vitro differentiation of mouse embryonic yolk sac cells. , 1987, Differentiation; research in biological diversity.

[31]  M. Frain,et al.  Differential expression of albumin and α-fetoprotein genes in fetal tissues of mouse and rat , 1984 .

[32]  M. Frain,et al.  Differential expression of albumin and alpha-fetoprotein genes in fetal tissues of mouse and rat. , 1984, Developmental biology.