Diabetes Metab J.  2011 Apr;35(2):119-129. 10.4093/dmj.2011.35.2.119.

Adenoviruses Expressing PDX-1, BETA2/NeuroD and MafA Induces the Transdifferentiation of Porcine Neonatal Pancreas Cell Clusters and Adult Pig Pancreatic Cells into Beta-Cells

• You YH
• Ham DS
• Park HS
• Rhee M
• Kim JW
• Yoon KH

• KMID: 2281710
• DOI: http://doi.org/10.4093/dmj.2011.35.2.119

Abstract

BACKGROUND
A limitation in the number of insulin-producing pancreatic beta-cells is a special feature of diabetes. The identification of alternative sources for the induction of insulin-producing surrogate beta-cells is a matter of profound importance. PDX-1/VP16, BETA2/NeuroD, and MafA overexpression have been shown to influence the differentiation and proliferation of pancreatic stem cells. However, few studies have been conducted using adult animal pancreatic stem cells.
METHODS
Adult pig pancreatic cells were prepared from the non-endocrine fraction of adult pig pancreata. Porcine neonatal pancreas cell clusters (NPCCs) were prepared from neonatal pigs aged 1-2 days. The dispersed pancreatic cells were infected with PDX-1/VP16, BETA2/NeuroD, and MafA adenoviruses. After infection, these cells were transplanted under the kidney capsules of normoglycemic nude mice.
RESULTS
The adenovirus-mediated overexpression of PDX-1, BETA2/NeuroD and MafA induced insulin gene expression in NPCCs, but not in adult pig pancreatic cells. Immunocytochemistry revealed that the number of insulin-positive cells in NPCCs and adult pig pancreatic cells was approximately 2.6- and 1.1-fold greater than those in the green fluorescent protein control group, respectively. At four weeks after transplantation, the relative volume of insulin-positive cells in the grafts increased in the NPCCs, but not in the adult porcine pancreatic cells.
CONCLUSION
These data indicate that PDX-1, BETA2/NeuroD, and MafA facilitate the beta-cell differentiation of NPCCs, but not adult pig pancreatic cells. Therefore PDX-1, BETA2/NeuroD, and MafA-induced NPCCs can be considered good sources for the induction of pancreatic beta-cells, and may also have some utility in the treatment of diabetes.

Keyword

Ad-BETA2/NeuroD; Ad-MafA; Ad-PDX-1/VP16; Beta-cell; Neonatal pig cell clusters; Pancreatic exocrine cell; Transdifferentiation; Transplantation

MeSH Terms

Adenoviridae
Adult
Aged
Animals
Capsules
Gene Expression
Humans
Immunohistochemistry
Insulin
Kidney
Pancreas
Stem Cells
Swine
Transplants
Capsules
Insulin

Figure

• 

  Fig. 1 Schematic of the experimental design. The general scheme of the pancreatic cell culture system was separated into two groups: porcine neonatal pancreatic cell clusters (NPCCs) and adult pig pancreatic cells. In monolayer cultures, we employed adenoviruses for PDX-1, BETA2/NeuroD, and MafA expression. Ad-GFP was used as an adenoviral control. On day 0, Ad-GFP or adenovirus-infected NPCCs and adult pig pancreatic cells were transplanted under the kidney capsule of normoglycemic nude mice. Finally, all grafts were harvested at 4 weeks.

• 

  Fig. 2 Adenovirus-mediated expression of green fluorescent protein (GFP) and PDX-1+BETA2+MafA in the neonatal pancreatic cell clusters (NPCCs) and adult pig pancreatic cells. The NPCCs (A) and adult pig pancreatic cells (B) were visible 48 hours after infection with Ad-GFP (left) and Ad-PDX-1/VP16+BETA2/NeuroD+MafA (right).

• 

  Fig. 3 The effect of adenovirus infection on beta-cell-specific gene expression in neonatal pancreatic cell clusters (NPCCs) (A)and adult pig pancreatic cells (B). (C) The bar graphs illustrates the quantification of the RT-PCR results. Results are expressed as means±standard deviation for n=10 independent experiments. GFP, green fluorescent protein. aP≤0.05, bP≤0.001.

• 

  Fig. 4 Immunocytochemical staining of insulin and cytokeratin expression levels in neonatal pancreatic cell clusters (NPCCs) (A) and adult pig pancreatic cells (B). Immunocytochemical staining of insulin and cytokeratin in NPCCs and adult pig pancreatic cells was performed at 7th day after transduction. GFP, green fluorescent protein.

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  Fig. 5 Glucose-stimulated insulin secretion (GSIS) was observed between neonatal pancreatic cell clusters (NPCCs) and adult pig pancreatic cells. GFP, green fluorescent protein. aP≤0.05.

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  Fig. 6 Insulin immunohistochemical staining in neonatal pancreatic cell clusters (NPCCs) (A) and adult pig pancreatic cell (B) grafts. Immunohistochemical staining for insulin on Ad-GFP (left) and Ad-PDX-1/VP16+BETA2+MafA (right) grafts was performed at 4 weeks.

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