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First published online 14 July 2005
doi: 10.1242/dev.01925

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Pancreatic epithelial plasticity mediated by acinar cell transdifferentiation and generation of nestin-positive intermediates

¹ Department of Surgery, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
² Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
³ Department of Surgery, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA
⁴ Division of Gastroenterology, Department of Genetics and Abramson Cancer Center University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA
⁵ Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
⁶ Division of Endocrinology, Diabetes and Metabolism, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA
⁷ Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA
⁸ Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA

View larger version (101K): [in a new window]   Fig. 1. Acinar-to-ductal metaplasia in primary explant cultures of mouse pancreas. (A,B) Phase contrast images of freshly harvested (day 0) non-transgenic pancreatic explant. (C) Hematoxylin and Eosin-stained section of non-transgenic day 0 explant. (D) Untreated non-transgenic day 5 explant. (E) Untreated MT-TGF day 5 explant cultured in the absence of exogenous growth factors. (F) Hematoxylin and Eosin-stained section of expanded duct-like epithelium arising from day 5 MT-TGF pancreas. (G) Non-transgenic day 5 explant cultured with 50 ng/ml rhTGF. (H) Non-transgenic day 5 explant cultured with 50 ng/ml rhHGF. (I) Non-transgenic day 14 explant cultured with 50 ng/ml rhTGF. (J) Quantification of duct-like structures formed following culture for 5 days with variable concentrations of either rhHGF (dark bars) or rhTGF (light bars). (K) Effect of EGF receptor-specific tyrosine kinase inhibitor EKI-785 on expansion of duct-like epithelium by either 50 ng/ml rhHGF (dark bars) or 50 ng/ml rhTGF (light bars). Values indicate mean±s.e.m. for three separate experiments; – – indicates no ductal structures observed.

View larger version (148K): [in a new window]   Fig. 2. Acinar cell-specific recombination of the R26R reporter allele in Villin-Cre pancreas. (A) Low magnification view of neonatal pancreas from a Villin-Cre; R26R pup stained for ß-gal activity (blue) and counterstained with Eosin. (B) High-magnification view showing lack of ß-gal activity in islet and large duct. (C) High-magnification view showing an intralobular duct with no ß-gal activity and all cells with a clear acinar morphology positive for ß-gal. (D) High magnification view showing no ß-gal staining in terminal intercalated ducts that are most closely associated with acini. ß-Gal activity is lacking in the large blood vessel in the upper right corner. In all panels, ß-gal activity is present in all acinar cells. Scale bars: 10 µm. Arrows indicate pancreatic ducts. a, acinus; i, islet; v, blood vessel.

View larger version (115K): [in a new window]   Fig. 3. Villin-Cre-based lineage tracing reveals that acinar cells transdifferentiate into ductal cells. Pancreatic epithelium from Villin-Cre;R26R mice was isolated and fixed immediately after plating (A,C,E) or after culture in the presence of TGF for 5 days (B,D,F). On day 0, most cells (A) contained ß-gal activity (blue) and were amylase positive (C) and cytokeratin negative (E), confirming that ß-gal expression was confined to acinar cells. A small percentage of cells did not display ß-gal activity, and most of these cells were positive for the ductal cytokeratins (arrows in C and E). Following 5 days of TGF treatment, most cells were still ß-gal positive (B). No intact amylase-positive cells were observed (D) and ß-gal activity was present in cells expressing ductal cytokeratins (F), indicating that the ß-gal-expressing acinar cells had transdifferentiated into ductal cells. Scale bars: 100 µm in A,B; 10 µm in C-F.

View larger version (150K): [in a new window]   Fig. 4. Ela-CreERT2-based lineage tracing confirms acinar cell origin of metaplastic epithelium. (A,B) Phase-contrast images of X-gal stained Ela-CreERT2; R26R epithelium on initiation of explant culture, demonstrating mosaic expression of ß-gal reporter in acinar cells. (C-F) Phase-contrast (C,D) and bright-field (E,F) images of X-gal stained Ela-CreERT2; R26R epithelium on day 4 of culture. ß-Gal reporter marks acinar cell origin of metaplastic epithelium.

View larger version (34K): [in a new window]   Fig. 5. Activation of nestin expression during TGF-induced acinar-to-ductal metaplasia. (A) Semi-quantitative RT-PCR demonstrating induction of nestin expression relative to ß-actin and GAPDH loading controls. Lane numbers indicate days in culture. (B) Quantification of RT-PCR results from three separate experiments. Values on x-axis indicate days in culture.

View larger version (52K): [in a new window]   Fig. 6. TGF-induced acinar-to-ductal metaplasia proceeds through nestin-positive intermediates. Images from day 0 (A,D,G,J), day 2 (B,E,H,K) and day 5 (C,F,I,L) TGF-treated non-transgenic pancreatic explants, double immunolabeled for: (A-C) nestin (green) and amylase (red); (D-F) nestin (green) and ductal cytokeratins (red). Owing to high cytokeratin and low nestin expression, composite color is orange rather than yellow (arrowheads); unmerged images are provided in Fig. S2 in the supplementary material. (G-I) Ductal cytokeratins (green) and amylase (red); (J-L) carbonic anhydrase II (green) and amylase (red). Nuclei are labeled in blue. On day 0, there were no nestin-positive cells and no cells co-expressing acinar and ductal markers. Following only 2 days of culture, cells had reduced levels of amylase and were beginning to acquire ductal cytokeratins. Following 5 days of culture, amylase protein was no longer detected, and cells had acquired expression of both ductal cytokeratins and carbonic anhydrase II. All images are presented at identical magnifications.

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