Diabetes Metab J.  2017 Oct;41(5):405-416. 10.4093/dmj.2017.41.5.405.

Generation of Insulin-Expressing Cells in Mouse Small Intestine by Pdx1, MafA, and BETA2/NeuroD

• Lee SH
• Rhee M
• Kim JW
• Yoon KH

• KMID: 2392484
• DOI: http://doi.org/10.4093/dmj.2017.41.5.405

Abstract

BACKGROUND
To develop surrogate insulin-producing cells for diabetes therapy, adult stem cells have been identified in various tissues and studied for their conversion into β-cells. Pancreatic progenitor cells are derived from the endodermal epithelium and formed in a manner similar to gut progenitor cells. Here, we generated insulin-producing cells from the intestinal epithelial cells that induced many of the specific pancreatic transcription factors using adenoviral vectors carrying three genes: PMB (pancreatic and duodenal homeobox 1 [Pdx1], V-maf musculoaponeurotic fibrosarcoma oncogene homolog A [MafA], and BETA2/NeuroD).
METHODS
By direct injection into the intestine through the cranial mesenteric artery, adenoviruses (Ad) were successfully delivered to the entire intestine. After virus injection, we could confirm that the small intestine of the mouse was appropriately infected with the Ad-Pdx1 and triple Ad-PMB.
RESULTS
Four weeks after the injection, insulin mRNA was expressed in the small intestine, and the insulin gene expression was induced in Ad-Pdx1 and Ad-PMB compared to control Ad-green fluorescent protein. In addition, the conversion of intestinal cells into insulin-expressing cells was detected in parts of the crypts and villi located in the small intestine.
CONCLUSION
These data indicated that PMB facilitate the differentiation of mouse intestinal cells into insulin-expressing cells. In conclusion, the small intestine is an accessible and abundant source of surrogate insulin-producing cells.

Keyword

Adenoviridae; Beta cells; Differentiation; Injections; Intestine, small; Transcription factors

MeSH Terms

Adenoviridae
Adult Stem Cells
Animals
Endoderm
Epithelial Cells
Epithelium
Fibrosarcoma
Gene Expression
Genes, Homeobox
Insulin
Intestine, Small*
Intestines
Mesenteric Arteries
Mice*
Oncogenes
RNA, Messenger
Stem Cells
Transcription Factors
Insulin
RNA, Messenger
Transcription Factors

Figure

• 

  Fig. 1 The experimental design and characterization of the small intestine. (A) The adenovirus combinations were composed of three distinct groups: adenoviruse (Ad)-green fluorescent protein (GFP; control); Ad-pancreatic and duodenal homeobox 1 (Pdx1); and Ad-PMB (Pdx1, V-maf musculoaponeurotic fibrosarcoma oncogene homolog A [MafA], and BETA2/NeuroD). Day 0 indicated the day of adenoviral induction through the intestinal arteries in the C57BL/6 mice. On day 5, we confirmed the overexpression of the adenovirus-mediated ectopic genes, and then all the mice were harvested at 4 weeks after viral injection. The small intestine was characterized with the intestinal markers (B) Pdx-1, (C) chromogranin A, (D) cytokeratin 19, and (E) Lgr5 by immunohistochemical staining (black allows) in the mouse duodenum (×200).

• 

  Fig. 2 Ectopic gene expression in the mouse intestine 5 days after viral injection. (A) Signal from the green fluorescent protein (GFP) was observed in the adenoviruse (Ad)-GFP, Ad-pancreatic and duodenal homeobox 1 (Pdx1), and Ad-PMB (Pdx1, V-maf musculoaponeurotic fibrosarcoma oncogene homolog A [MafA], and BETA2/NeuroD) groups, but not in the sham-operated group (saline injection, ×100). (B) mRNA expression levels of exogenous transcription factors (PMB) were increased at 5 days after infection with Ad-Pdx1 (gray bar) and Ad-PMB (black bar) compared to Ad-GFP (white bar) (n=2). The mean±standard error values are presented (error bars, standard error). DAPI, 2-(4-amidinophenyl)-1H-indole-6-carboxamidine. aP≤0.05, bP≤0.02.

• 

  Fig. 3 Expression of insulin mRNA in mouse intestine at 4 weeks. (A) Endogenous insulin, PMB (pancreatic and duodenal homeobox 1 [Pdx1], V-maf musculoaponeurotic fibrosarcoma oncogene homolog A [MafA], and BETA2/NeuroD) transcription factors were evaluated by reverse transcription polymerase chain reaction analysis in the adenoviruse (Ad)-green fluorescent protein (GFP), Ad-Pdx1, and Ad-PMB groups. Mouse islets were used as positive controls. (B) Insulin, PMB transcription factors were analyzed by quantitative real-time polymerase chain reaction in the Ad-GFP (n=4, white bar), Ad-Pdx1 (n=5, gray bar), and Ad-PMB (n=4, black bar) injected groups. The mean±standard error values are presented (error bars, standard error). aP≤0.05, bP≤0.02, cP≤0.005.

• 

  Fig. 4 Production of insulin+ cells within the small intestinal cells located in the villus and crypt. (A) Insulin+ cells (yellow arrows) and (B) C-peptide+ cells (white arrows) were detected in the adenoviruse (Ad)-pancreatic and duodenal homeobox 1 (Pdx1) and Ad-PMB (Pdx1, V-maf musculoaponeurotic fibrosarcoma oncogene homolog A [MafA], and BETA2/NeuroD) groups but not in the Ad-green fluorescent protein (GFP) group (×400) by immunofluorescence analysis. (C) Quantification of insulin+ cells in the villus and crypt cells. DAPI, 2-(4-amidinophenyl)-1H-indole-6-carboxamidine.

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