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

Abstract

BackgroundPreviously, we reported that neonatal porcine pancreatic cells transfected with hepatocyte growth factor (HGF) gene in an Epstein-Barr virus (EBV)-based plasmid (pEBVHGF) showed improved proliferation and differentiation compared to those of the control. In this study, we examined if pancreatic cells transfected repeatedly with pEBVHGF can be successfully grafted to control blood glucose in a diabetes mouse model.MethodsNeonatal porcine pancreatic cells were cultured as a monolayer and were transfected with pEBVHGF every other day for a total of three transfections. The transfected pancreatic cells were re-aggregated and transplanted into kidney capsules of diabetic nude mice or normal nude mice. Blood glucose level and body weight were measured every other day after transplantation. The engraftment of the transplanted cells and differentiation into beta cells were assessed using immunohistochemistry.ResultsRe-aggregation of the pancreatic cells before transplantation improved engraftment of the cells and facilitated neovascularization of the graft. Right before transplantation, pancreatic cells that were transfected with pEBVHGF and then re-aggregated showed ductal cell marker expression. However, ductal cells disappeared and the cells underwent fibrosis in a diabetes mouse model two to five weeks after transplantation; these mice also did not show controlled blood glucose levels. Furthermore, pancreatic cells transplanted into nude mice with normal blood glucose showed poor graft survival regardless of the type of transfected plasmid (pCEP4, pHGF, or pEBVHGF).ConclusionFor clinical application of transfected neonatal porcine pancreatic cells, further studies are required to develop methods of overcoming the damage for the cells caused by repeated transfection and to re-aggregate them into islet-like structures.

Glycated Albumin Causes Pancreatic β-Cells Dysfunction Through Autophagy Dysfunction

“…Pancreatic islets were isolated from male Sprague-Dawley rats (250 -300 g) by collagenase digestion and separated by discontinuous gradient purification as previously described (2,23). The islets and INS-1 cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (Invitrogen, Carlsbad, California), 100 IU/mL penicillin, 100 g/mL streptomycin, and 10mM HEPES at 37°C in a humidified incubator containing 5% CO 2 and 95% air.…”

Successful xenotransplantation with re‐aggregated and encapsulated neonatal pig liver cells for treatment of mice with acute liver failure

“…Second, the cell re‐aggregation method was adapted. Previous studies reporting on three‐dimensional culture systems that allow cell clusters to maintain their spheroid structure demonstrated a good influence on cell viability and function compared to the monolayer culture system . Fewer cells detached from the clusters and died after re‐aggregation.…”

“…Encapsulated adult pig hepatocytes have been evaluated as a xenogeneic cell source . Encapsulation is a well‐established tool for xenogeneic cell transplantation that has already been applied to pig islet transplantation in humans . However, some modifications are required to improve the survival of transplanted cells.…”