Figure 1. Targeting strategy with the Nestin-Cre transgene. (A) Schematic of the conditional KrasG12D allele. In the absence of the Cre recombinase, only WT Kras is expressed; once Cre is expressed, the stop region is excised and KrasG12D becomes expressed. (B–E) In Nestin-Cre/ R26R mice, LacZ staining is observed only in acinar cells (B), and is absent in ductal cells (arrowheads) and islets (I). (C) Overlay of LacZ immunostaining (E) with CK19 (D) demonstrates that ductal cells are not targeted. (F) Pdx1-Cre leads to the expression of KrasG12D in all pancreatic cell types while Nestin-Cre drives the expression of KrasG12D only in acinar cells. Scale bar: 20 mm. doi:10.1371/journal.pone.0027725.g001 two episodes of caerulein-induced AP, rapidly develop high-grade PanINs that have the capacity to progress to PDAC. This study confirms that acute pancreatic injury has the potential to contribute to PDAC development in Kras-based mouse models and support the hypothesis that PDAC can arise in the Nestin cell lineage. Results Nestin progenitor cells do not give rise to ductal cells We have previously shown that embryonic Nestin-expressing pancreatic progenitors give rise to acinar cells and not to endocrine or centroacinar cells, while a potential contribution to ductal cells was not completely excluded [6]. To resolve this issue, multiple lineage tracing experiments were done by breeding the mouse line that expresses the Cre recombinase under the control of Nestin regulatory elements [17] with the reporter mouse line R26R. This last line carries the b-Galactosidase (LacZ) gene under the control of the ubiquitous promoter R26 and a transcriptional stop region surrounded by LoxP sites (LoxP-Stop-LoxP or LSL) [18]. In the double transgenic mice, LacZ expression is activated following Cre-mediated recombination in embryonic Nestin- expressing cells (similar approach to the one described for Kras in Fig. 1A). Subsequently, by genetic transmission, all cells derived from the Nestin-expressing progenitors express LacZ. Sections Acute Pancreatitis and Pancreatic Cancer across the pancreata of Nestin-Cre/R26R animals at 2 months of age were stained for LacZ activity. As observed before, the Nestin- Cre transgene allowed for the targeting of ,70% of acinar cells (Fig. 1B). LacZ staining was observed only in acinar structures, never in the ductal, endocrine or centroacinar cells (Fig. 1B) and co-immunofluorescence with the ductal marker cytokeratin 19 (CK19) confirmed this observation (Fig. 1C–E). These results demonstrate that embryonic pancreatic Nestin-expressing progen- itors do not give rise to ductal cells (Fig. 1F). Expression of LacZ was also rarely observed in pancreatic non-parenchymal cell types such as endothelial and mesenchymal cells (not shown). Acute pancreatitis dramatically accelerates lesion progression in N/K pancreata Between 2 to 6 months, N/K mice developed low grade PanINs in limited numbers (2–10 isolated lesions or small foci per section), as well as limited areas of acinar to ductal metaplasia (ADM) (Fig. 2A–B, G) similar to what is observed in Pdx1-Cre; LSL- KrasG12D (P/K) mice [4,14]. While with time P/K mice can develop PDAC but at very low frequency, N/K mutant mice rarely survived past 6 months due to severe neurological problems, preventing us from following a possible progression to pancreatic cancer. We have previously shown that two episodes of acute pancreatitis (AP) induced by caerulein accelerated PanIN progression from low- to high-grade and increased PDAC frequency in the P/K mouse model [14]. To test the possibility that mutated Kras activation in the Nestin lineage could lead to PDAC, a cohort of 17 2-month-old N/K mice were subjected to caerulein treatment. Caerulein is a cholecystokinin (CCK) analog that binds to and activates the CCK receptor [19,20]. While the drug dosage we used is low compared to the ones used by other groups [21,22], extensive loss of acinar cells was observed in wild type mice at 24 and 48 hours following AP, concomitant with a dramatic inflammatory infiltration and interstitial expansion (Fig.S1) as described by others [21,22,23]. One week following AP, nearly complete regeneration of the pancreas was observed at the histological level, with rare residual inflammatory infiltrates while proliferation levels remained high as shown by Ki67 expression in acinar cells (Fig.S1A–D). These results are consistent with another published report [15]. To evaluate the possibility that following pancreatic injury, Nestin expression could be detected in different pancreatic cell types, short term lineage tracing experiments were done. LacZ staining in Nestin-Cre/R26R pancreata, 1 week post caerulein treatment, was observed only in acinar structures and no staining was detected in endocrine, centroacinar or ductal cells (not shown). All 17 caerulein-treated N/K mice started getting sick when they were about 4 month-old, 2 months post-treatment, and had to be sacrificed. Six animals were lost without necropsy. By contrast, only 3 out of 9 untreated N/K mice died and none of the others were sacrificed due to sickness during this period. P/K mice when subjected to AP did not have shorter life expectancy than untreated mice at least over a period of 8 months (not shown). These observations suggested that the decrease in overall survival of N/K mice from 6 to 4 months was not directly related to pancreatic insufficiency. Four month-old N/K pancreata exhib- ited few PanIN-1A and -1B and rare ADM foci (less than 5% of the section) (Fig. 2A, B and G). Two months post caerulein- induced AP extensive pancreatic dysplasia was observed in N/K pancreata characterized by a mix of low- and high-grade PanIN and large ADM foci (Fig. 2C–F). The increase in lesion grade and the extensive ADM were documented by morphometric analyses that compared the presence and grade of lesions between sections PLoS ONE | www.plosone.org 2 November 2011 | Volume 6 | Issue 11 | e27725