Diabetes Metab J.  2011 Feb;35(1):72-79. 10.4093/dmj.2011.35.1.72.

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

• Seo MK
• Sun CL
• Kim JW
• Yoon KH
• Lee SK

• KMID: 2281495
• DOI: http://doi.org/10.4093/dmj.2011.35.1.72

Abstract

BACKGROUND
Previously, 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.
METHODS
Neonatal 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.
RESULTS
Re-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).
CONCLUSION
For 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.

Keyword

Diabetes mellitus; Hepatocyte growth factor; Porcine neonatal pancreatic cells; Transfection

MeSH Terms

Animals
Blood Glucose
Body Weight
Capsules
Diabetes Mellitus
Fibrosis
Graft Survival
Hepatocyte Growth Factor
Herpesvirus 4, Human
Kidney
Mice
Mice, Nude
Plasmids
Transfection
Transplants
Blood Glucose
Capsules
Hepatocyte Growth Factor

Figure

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  Fig. 1 Successful transplantation of neonatal porcine pancreatic cell clusters (NPCCs) in normal mice. NPCCs were harvested and transplanted immediately into the kidneys of normal nude mice without any manipulation. Two and eight weeks after transplantation, transplanted NPCCs were examined using immunohistochemistry. Insulin is stained red and pancytokeratin is stained green, while nuclear staining is shown in blue.

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  Fig. 2 Effect of re-aggregation on in vivo survival of pancreatic cells after transplantation. Single-cell state or re-aggregated pancreatic cells were transplanted in the kidney capsule of normal mice. The graft was examined following hematoxylin staining or immunohistochemistry two weeks after transplantation. The black arrow point to blood vessels within the graft. Insulin is shown in red and pancytokeratin is shown in green, while nuclear staining is shown in blue. NPCCs, neonatal porcine pancreatic cell clusters.

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  Fig. 3 Effect of repeated transfection on cell viability. Pancreatic cells were transfected repeatedly with pCEP4, pHGF, or pEBVHGF every other day up to three times. Twenty-four hours after each transfection, viable cell count was analyzed using a CCK-8 kit. Cell viabilities are expressed as percent of untransfected control cells. Data are presented as the mean ± standard error of three independent experiments.

• 

  Fig. 4 Concentrations of blood glucose and body weight following transplantation of pancreatic cells transfected with pHGF or pEBVHGF. Pancreatic cells were transfected with pHGF or pEBVHGF, and re-aggregated. The re-aggregated cells were transplanted into the type 1 diabetes mouse model. Blood glucose level (A) and body weight (B) were measured every other day. Time 0 indicates the day of pancreatic cell transplantation.

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  Fig. 5 Pancytokeratin and insulin expressions in the pancreatic cells transfected three times with pEBVHGF and re-aggregated. The cells were fixed and stained right after transplantation into the diabetes mouse model. Insulin is shown in red and pancytokeratin is shown in green, while nuclear staining is shown in blue. Imaging was performed with a confocal microscope (400×objective).

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  Fig. 6 No expression of insulin or pancytokeratin in the transplanted pancreatic cells in a diabetes mouse model. Pancreatic cells were transfected three times with pHGF or pEBVHGF, re-aggregated, and transplanted into a type 1 diabetes mice. Survival and differentiation of the transplanted cells were examined using immunohistochemistry two and five weeks after transplantation. Insulin is stained red and pancytokeratin is stained green, while nuclear staining is shown in blue.

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  Fig. 7 Loss of pancreatic cells transplanted into normal mice. Pancreatic cells were transfected three times with pCEP4, pHGF, or pEBVHGF and then re-aggregated. Normal nude mice were transplanted with the re-aggregated pancreatic cells in the kidney capsule. The transplanted cells were examined using immunohistochemistry one, two, and eight weeks after transplantation. Insulin is shown in red and pancytokeratin is shown in green, while nuclear staining is shown in blue.

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