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Journal of Biochemistry Advance Access originally published online on January 2, 2008
Journal of Biochemistry 2008 143(4):497-503; doi:10.1093/jb/mvm244
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© 2008 The Japanese Biochemical Society.

Induction of Efficient Differentiation and Survival of Porcine Neonatal Pancreatic Cell Clusters Using an EBV-based Plasmid Expressing HGF

Min Sun Kim, Ji-Won Kim, Chenglin Sun, Sang Taek Oh, Kun Ho Yoon and Suk Kyeong Lee*

Research Institute of Immunobiology, Department of Biomedical Sciences, Catholic University of Korea, Seoul 137-701, Republic of Korea

*To whom correspondence should be addressed. Tel: + 82 2 590 2396, Fax: + 82 2 532 0575, E-mail: sukklee{at}catholic.ac.kr

Received October 11, 2007; Accepted December 16, 2007


  Abstract

Porcine neonatal pancreatic cell clusters (NPCCs) have been actively studied as a source of pancreatic stem cell transplantation for the treatment of diabetes. In this study, the hepatocyte growth factor (HGF) gene was cloned in an Epstein–Barr virus (EBV)-based plasmid vector (pEBVHGF) and the effects of the HGF expression on the survival and differentiation of NPCCs were analysed. For comparison, pHGF was constructed by deleting EBNA-1 and OriP from pEBVHGF. The expression of HGF, as measured by ELISA, lasted longer when pEBVHGF was used than when pHGF was used. C-Met phosphorylation co-related with the expression of HGF in the transfected NPCCs. Immunocytochemistry experiments showed that NPCCs showed a higher and longer expression of insulin when they were transfected with pEBVHGF than with pHGF. Moreover, a greater number of NPCCs survived for a longer period after they were transfected with pEBVHGF than when they were transfected with pHGF. Taken together, these results indicate that transfecting NPCCs with the HGF gene using an EBV-based plasmid is a more effective method of inducing differentiation to beta cells and enhancing survival than using a conventional plasmid. Therefore, it may be possible to use EBV-based plasmids to modify pancreatic stem cells for xenotransplantation.

Key Words: Epstein–Barr virus-based plasmids, hepatocyte growth factor, insulin, porcine neonatal pancreatic cell clusters

Abbreviations: DAPI, 4'6-diamidino-2-phenylindole; DMEM, Dulbeco's; Modified Eagle Medium; EBNA-1, EBV nuclear antigen 1; EBV-based plasmid, Epstein-Barr virus-based plasmids; ELISA, enzyme-linked immunosorbent assay; FBS, fetal bovine serum; GFP, green fluorescence protein; HBSS, Hank's; balanced salt solution; HGF, hepatocyte growth factor; IFA, immuno-fluorescence assay; NPCCs, porcine neonatal pancreatic cell clusters; OriP, EBV replication origin


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