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

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.

Repeated Gene Transfection Impairs the Engraftment of Transplanted Porcine Neonatal Pancreatic Cells

“…EcoRV and Stu I were used in order to remove the oriP and EBNA-1 sequences from pEBVHGF, resulting in pHGF [20]. An empty EBV-based plasmid, pCEP4, was also used as a control [20].…”

“…An empty EBV-based plasmid, pCEP4, was also used as a control [20]. Plasmid (5 µg) and lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) were mixed each with 500 µL Opti-MEM (GIBCO, Grand Island, NY, USA) at room temperature; the two liquids were then carefully mixed together and incubated for 30 minutes.…”

“…In our previous study, HGF gene was cloned in an Epstein-Barr virus (EBV)-based plasmid (pEBVHGF) and transfected to monolayer cultured pancreatic cells [20]. HGF was expressed about seven times longer in those cells than in the cells transfeced with a conventional plasmid (pHGF).…”

“…HGF was expressed about seven times longer in those cells than in the cells transfeced with a conventional plasmid (pHGF). Phosphorylation of the HGF receptor, c-MET, was observed three days after the transfection of pEBVHGF [20]. When pEBVHGF was transfected to pancreatic cells, an increase in insulin secretion was also seen compared with the conventional plasmid transfected cells [20].…”

“…Phosphorylation of the HGF receptor, c-MET, was observed three days after the transfection of pEBVHGF [20]. When pEBVHGF was transfected to pancreatic cells, an increase in insulin secretion was also seen compared with the conventional plasmid transfected cells [20]. EBV-based plasmid contains EBNA-1 and oriP sequences derived from EBV, and these two were confirmed to help the plasmid replicate and remain in the transfected pancreatic cells during cell proliferation [21].…”