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. 2005 Oct 18;102(42):15116-21.
doi: 10.1073/pnas.0507567102. Epub 2005 Oct 6.

Lineage tracing and characterization of insulin-secreting cells generated from adult pancreatic acinar cells

Kohtaro Minami  1 , Masaaki Okuno, Kazumasa Miyawaki, Akinori Okumachi, Katsuhiko Ishizaki, Kazunobu Oyama, Miho Kawaguchi, Nobuko Ishizuka, Toshihiko Iwanaga, Susumu Seino
Affiliations

Lineage tracing and characterization of insulin-secreting cells generated from adult pancreatic acinar cells

Kohtaro Minami et al. Proc Natl Acad Sci U S A. .

Abstract

Although several studies have suggested that insulin-secreting cells can be generated in vitro from cells residing in adult exocrine pancreas, neither the origin of these cells nor their precise insulin secretory properties was obtained. We show here that insulin-secreting cells can be derived from adult mouse pancreatic exocrine cells by suspension culture in the presence of EGF and nicotinamide. The frequency of insulin-positive cells was only 0.01% in the initial preparation and increased to approximately 5% in the culture conditions. Analysis by the Cre/loxP-based direct cell lineage tracing system indicates that these newly made cells originate from amylase/elastase-expressing pancreatic acinar cells. Insulin secretion is stimulated by glucose, sulfonylurea, and carbachol, and potentiation by glucagon-like peptide-1 also occurs. Insulin-containing secretory granules are present in these cells. In addition, we found that the enzymatic dissociation of pancreatic acini itself leads to activation of EGF signaling, and that inhibition of EGF receptor kinase blocks the transdifferentiation. These data demonstrate that pancreatic acinar cells can transdifferentiate into insulin-secreting cells with secretory properties similar to those of native pancreatic beta cells, and that activation of EGF signaling is required in such transdifferentiation.

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Figures

Fig. 1.
Fig. 1.
Generation of insulin-producing cells from adult mouse exocrine pancreas. (A) Quantitative real-time RT-PCR analysis for insulin genes. Insulin expression in the culture was gradually increased. (B) Immunostaining of exocrine pancreas-derived cells. Insulin-positive cells are increased by the culture. (Scale bars, 50 μm.) (C) Double immunostaining of insulin and C-peptide. The insulin-positive cells are also positive for C-peptide. (Scale bar, 20 μm.)
Fig. 2.
Fig. 2.
Cell lineage tracing by the Cre/loxP-based system. (A) The scheme of pancreatic acinar cell specific cell marking. In cells from the R26R-ECFP mouse, expression of the fluorescent protein (ECFP) is activated through the action of Cre recombinase to remove a transcriptional “stop” sequence. When amylase/elastase-expressing acinar cells are infected with adenovirus expressing Cre recombinase under control of either amylase or elastase promoter, the cells are labeled permanently with ECFP. (B–D) Lineage tracing of labeled acinar cells. Pancreatic acinar cells from R26R-ECFP were labeled by infection of Ad-pAmy-Cre at ≈50% efficiency (B). Because fluorescence of ECFP is diminished after fixation, ECFP expression was detected by using anti-GFP antibody. Cells positive for insulin (arrowheads), ECFP (arrows), and both insulin and ECFP (asterisks) are observed (B). Photographs of higher magnification of insulin/ECFP double-positive cells are shown (C). CK/ECFP double-positive cells are also observed (D). (Scale bars, 20 μm.)
Fig. 3.
Fig. 3.
Insulin secretory properties of pancreatic acinar-derived cells. (A) Temporal gene expression analysis by RT-PCR. Representative photographs from four independent experiments are shown. Genes involved in glucose-induced insulin secretion were induced by the culture. Note that the expression pattern becomes similar to that of the pancreatic islets and the clonal β cells line MIN6-m9. GK, glucokinase; HK, hexokinase, Stx1a, syntaxin 1a. (B) Electron microscopic analysis of pancreatic acinar-derived cells. Cells containing small secretory granules with crystalline structure were often found. (Scale bar, 500 nm.) (C) Immunoelectron microscopic analysis for insulin. Insulin immunoreactivities were detected in the secretory granules. (Scale bar, 200 nm.) (D and E) Insulin secretion in pancreatic acinar-derived cells. Insulin secretion was stimulated by 30 mM KCl/0.1 μM glibenclamide (Glib)/0.1 mM carbachol (CCh), or increased concentrations of glucose (G3, 3 mM; G10, 10 mM; G20, 20 mM) for 60 min. Potentiation by glucagon-like peptide-1 (7–36 amide) (100 nM) is also shown. Data are means ± SE of three to seven independent experiments. (F and G) Insulin secretion in native mouse pancreatic islets.
Fig. 4.
Fig. 4.
Expressions of molecules involved in pancreatic development. (A) Temporal gene expression analysis by RT-PCR. Transcription factors involved in pancreas development and β cell functions were induced in the acinar-derived cells, but Pax4 was not induced. Expression of PGP9.5, a potential endocrine progenitor marker, was detected. Glucagon, somatostatin (Sst), and pancreatic polypeptide (PP) was induced by the culture. (B) Protein expression determined by immunoblotting. Pdx1, Foxa2, Ptf1a, and PGP9.5 were induced or increased by the culture. (C) Double immunostaining for insulin and PGP9.5. Almost all of the insulin-positive cells were also positive for PGP9.5. PGP9.5-positive cells negative for insulin were frequently found. (Scale bars, 50 μm.)
Fig. 5.
Fig. 5.
Involvement of EGF signaling in acinar cell transdifferentiation. (A) Tyrosine phosphorylation of cellular protein. In dissociated pancreatic cells (+), tyrosine phosphorylation was increased compared with undissociated pancreas (–). Antiphosphotyrosine antibody (PY20) was used for detection. (B) Phosphorylation of EGFR. Tyrosine phosphorylation of the EGFR was detected in dissociated pancreatic cells. (C) Activation of intracellular signaling pathways. After dissociation of pancreas, phosphorylated forms of extracellular signal-regulated protein kinase 1/2 (ERK1/2), c-Jun kinase (JNK), and Akt were increased. (D) Effect of EGFR kinase inhibitor AG1478 on intracellular signaling. AG1478 attenuated activation of ERK1/2 and Akt. Effects on p38 mitogen-activated protein kinase and JNK were unclear. (E) Effect of AG1478 on acinar cell transdifferentiation. AG1478 strongly inhibited induction of genes indicating transdifferentiation.
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