All animal studies were reviewed and approved by the Ethics Committee of Nanjing Medical University in accordance with the established standards of the humane handling of research animals. Male Sprague-Dawley rats (150–300 g) were obtained from Vital River Laboratories (Beijing, China). Rats were kept in standard laboratory conditions with light-dark cycles of 12–12 h and free access to chow and water. Rats were sacrificed by decapitation, before undergoing laparotomy. Rat pancreas was dissected and placed into cold 0.9% NaCl solution.

Quiescent PSCs were isolated from rat pancreas with enzyme digestion and Nycodenz gradient centrifugation as described previously (15). Briefly, after connective tissue and blood vessels were removed, pancreatic tissue was slowly infused with 10 ml enzyme solution (mixture of collagenase P, protease and DNase) until all lobules were swollen and well infused. The pancreas tissue was pre-incubated, finely minced and incubated again, in order to obtain a cell suspension. After washing, PSCs were obtained by ladder centrifugation with Nycodenz solution. Cells were cultured in DMEM/F12 (Wisent, Canada) supplemented with 10% fetal bovine serum (FBS) (Wisent, Canada), 4 mM L-glutamine (Sigma-Aldrich, St. Louis, MO, USA) and 1% penicillin-streptomycin (Thermo Scientific Hyclone, Waltham, MA, USA) at 37°C with 5% CO₂. Cells were subcultured at 90% confluence in the following passages.

Morphological appearances of isolated cells were assessed under a contrast-phase microscope with blue-green autofluorescence at 320 nm from lipid droplets within the cytoplasm. PSCs were evaluated by immunocytochemistry/immunofluorescence (ICC/IF) and immunocytochemistry (ICC) using antibodies specific for PSC markers, α-SMA, desmin, vimentin, and GFAP. Antibodies for epithelium and macrophage markers (pan-CK, CK19, CD68) were used as negative controls to rule out possible cell contamination. Cells were seeded and stained in a 24-well plate (Corning Inc., NY, USA). ICC/IF and ICC were performed with standard procedures. The primary antibodies and their working dilutions were as follows: rabbit anti-α-SMA 1:100 (Abcam, Cambridge, MA, USA); rabbit anti-desmin 1:200 (Abcam); rabbit anti-vimentin 1:100 (Cell Signaling Technology, Beverly, MA, USA); mouse anti-GFAP 1:500 (Sigma-Aldrich); rabbit anti-CK19 1:300 (Abcam); mouse anti-CD68 1:100 (Abcam); and mouse anti-pan-CK 1:100 (Thermo Fisher Scientific, USA). DAPI and hematoxylin were used for nuclear counterstaining in ICC/IF and ICC, respectively. Cells were observed under a fluorescence microscope (Nikon Ti, Japan).

Cell migration assays were carried out in 24-well transwell chambers with polycarbonate filters (pore size, 8 μm; Merck Millipore, Billerica, MA, USA). A total of 2×10⁴ cultured cells, at 40 h and on day 10 after isolation, were plated in serum-free medium in the upper chamber, and media containing 10% FBS alone was added to the lower chamber. After 40 h of incubation, residual medium in the upper chamber was removed carefully with a pipette. Cells on the surface of the filter membrane were fixed and stained with crystal violet staining solution (Beyotime, Shanghai, China) according to the manufacturer's instructions. Penetrated cells on the lower surface of the membrane were examined and counted under the microscope, before being removed with a cotton swab. Non-penetrated cells were assessed via the same method. Migration rates were calculated with the following formula: number of penetrated cells divided by the total number of both penetrated and non-penetrated cells.

For cell motility studies of PSC, a live cell imaging system was used. There are many essential elements for this system. Firstly, a camera was mounted on the pressurized chamber on the stage of a Nikon TE 2000-E inverted optical microscope. Secondly, the microscope employed a number of computer controlled motorized systems in order to rapidly reconfigure the microscope during automated image acquisition. Finally, a small cell incubator on the microscope was used to control the cell environment and maintain our samples in a humidified and CO₂ enriched atmosphere. The system was kept in a darkened room with ambient temperature maintained close to 25°C. The entire process was controlled by NIH-Elements AR software, and the following image processing was performed using Image-Pro Plus 6.0. Cells were cultured in 6-well plate and single cell movements was tracked with automatic record of a series of continuous images at an interval of 2 min under 100 magnified vision over 72 h. Cell motility capacity was assessed with the length of cell movement track over a fixed time.

Total RNA was extracted from quiescent and activated PSCs cultured in 6-cm Petri dishes with Qiagen RNeasy Micro kit (Qiagen, Mannheim, Germany) according to the manufacturer's instructions. Agarose gel electrophoresis was used for quality control of the isolated RNA. Reverse transcription was conducted using PrimeScript RT Master Mix (Takara, Dalian, China), while quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed using FastStart Universal SYBR Green Master (ROX) (Roche, Germany) on a StepOne Plus Real-Time-PCR System (Applied Biosystems, Waltham, MA, USA) according to the manufacturer's instructions. The PCR program consisted of an initial enzyme activation step at 95°C for 10 min, followed by 45 cycles of amplification with 95°C for 15 sec, followed by incubation at an appropriate temperature for 1 min. Finally, a melting curve profile was set at 95°C (15 sec), 60°C (1 min), and 95°C (15 sec). β-actin was used as an endogenous control to which each gene of interest was normalized. Relative quantification was calculated by the ΔΔCT method and normalized based on the designated control. Primer sequences used in qRT-PCR are shown in Table I.

Total proteins were prepared with a protein extraction kit (Beyotime) and quantified by the bicinchoninic acid assay (BCA) method (Beyotime) according to the manufacturer's instructions. Polyvinylidene fluoride (PVDF) membranes (Millipore) were blocked in 5% non-fat dried milk in phosphate-buffered saline with Tween-20 (PBST) and incubated overnight at 4°C with appropriate primary antibodies [rabbit anti-α-SMA, dilution 1:200 (Abcam); rabbit anti-vimentin, dilution 1:1,000 (Cell Signaling Technology); rabbit anti-S100A4, dilution 1:250 (Abcam); rabbit anti-N-cadherin, dilution 1:200 (Santa Cruz Biotechnology, Dallas, TX, USA); mouse anti-E-cadherin, dilution 1:1,000 (Abcam); rabbit anti-BMP7, dilution 1:1,000 (Abcam); rabbit anti-Slug, dilution 1:700 (Abcam); and rabbit anti-Snail, dilution 1:1,000 (Cell Signaling Technology)]. Membranes were then washed and incubated for 2 h at room temperature with secondary horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG (Cell Signaling Technology). All protein expression levels were normalized to β-actin (Abcam).

Statistical analysis was performed using SPSS, version 13.0 (SPSS, Inc., Chicago, IL, USA). All experiments were repeated in triplicate, and the most representative data are shown in this report. Numeric data were expressed as mean ± standard deviation (SD). Statistical significance between two groups was determined by the Student's t-test, with P-values <0.05 considered statistically significant.

The yield of rat quiescent PSCs was ~4 to 7 million cells per gram of pancreatic tissue. Freshly isolated PSCs remained quiescent within the first 48 h of primary culture as indicated by their typical round or polygon shape and autofluorescence from lipid droplets in their cytoplasm (15). After 48 h, they began to transform to an ‘activated’ form, acquiring fibroblastic characteristics and losing their lipid droplets. Apte et al previously demonstrated that freshly isolated PSCs cultured on plastic were gradually activated after 48 h and fully activated after 7 days in vitro (15). For this reason, we chose cells cultured for 40 h after isolation as representative of the quiescent state and cells cultured for 10 days as representing the fully activated state (Fig. 1). Primarily cultured cells were positive for PSC marker (α-SMA, vimentin, GFAP and desmin) staining in ICC and ICC/IF (Fig. 2). To exclude possible contamination with macrophages and epithelial cells, the purity of PSCs were confirmed with negative immunostaining with CK19, CD68, and pan-CK (Fig. 3).

PSC migration ability was assessed using transwell chambers with 40-h incubation time. More cells migrated to the lower surface of the membrane 10 days after isolation compared with those isolated after only 40 h. In order to rule out possible differences in cell adherence and proliferation ability after activation, the cell migration rate was calculated. Increased cell migration rate was observed after PSC activation (Fig. 4).

Cell motility capacity was assessed by live cell imaging system. PSC motility capacity was increased after cell activation, as the activated PSCs moved a longer distance during a fixed culture time. The real-time images of randomly chosen monitored cells are showed in Fig. 5. At the same time, PSCs underwent characteristic morphological changes in vitro, including loss of lipid droplets, increased cell volume and transition to a myofibroblastic phenotype, which is in accordance with our previous findings (15,16).

To determine whether PSC activation involve an EMT-like process, we analyzed expression of epithelial markers E-cadherin, bone morphogenetic protein 7 (BMP7), and desmoplakin, and the mesenchymal markers N-cadherin, fibroblast-specific protein 1 (S100A4), vimentin, fibronectin1, and collagen1α1, during the PSC activation process by qRT-PCR. E-cadherin, BMP7 and desmoplakin were significantly downregulated, while N-cadherin, S100A4, vimentin, fibronectin1 and collagen1α1 were upregulated in activated PSCs after 10 days of in vitro culture compared with quiescent PSCs (Fig. 6).

Since the Snail transcription factor family, such as Snail and Slug (also known as Snail1 and Snail2), is reported to play a crucial role during the EMT process, we used qRT-PCR to examine the expression of Snail1 and Slug during PSC activation. Both Snail1 and Slug mRNA levels increased significantly in activated PSCs compared to quiescent PSCs. We also examined the expression of EMT-related genes at the protein level by immunoblotting. Western blot analysis confirmed that the decreased protein expression of epithelial markers, including E-cadherin and BMP-7, was accompanied by increased expression of mesenchymal markers (i.e., N-cadherin, vimentin, S100A4) after PSCs were activated (Fig. 7). Furthermore, the two EMT-related transcription factors, Snail and Slug, also showed increased expression at the protein level during PSC activation.