Isolation of Murine Pancreatic Stellate Cells and the Establishment of a New ex-vivo Activation Model

Abstract

Background: Pancreatic stellate cells (PSCs) are critical in the development of pancreatic fibrosis. In vitro, cell attachment itself can promote cell activation. Currently, there is a lack of methods for isolating activated PSCs that are unaffected by cell attachment. This study aims to identify effective methods for isolating quiescent and activated murine PSCs (mPSCs) and to evaluate the potential of caerulein in inducing mPSC activation in an ex vivo model.

Methods: Pancreatic tissue from mice was digested with collagenase P (1.17 U/mL), Pronase (0.5 mg/mL), and DNase I (0.01 mg/mL). Quiescent and activated mPSCs were isolated using a Nycodenz gradient. Immunostaining for α-smooth muscle actin (α-SMA), Desmin, glial fibrillary acidic protein (GFAP), vimentin, CK19, and CD68 was performed to confirm cell purity. Real-time quantitative PCR (RT-PCR) and RNA sequencing assessed the activation phenotype following caerulein treatment.

Results: Quiescent and activated mPSCs were successfully isolated using the Nycodenz gradient, with cells exhibiting typical stellate morphology and positive staining for α-SMA, Desmin and vimentin. Oil Red O staining confirmed lipid droplets in quiescent mPSCs. In the caerulein-treated group, mPSC activation was significantly greater than in the saline-treated control group. RT-PCR revealed progressive upregulation of acta2 (**p<0.01, d4 compared to d2, ^## p<0.01,d7 compared to d4,**p<0.01,d7 compared to d2), col1a (**p<0.01, d4 compared to d2,**p<0.01,d7 compared to d2), and actg2 (**p<0.01, d4 compared to d2, ^## p<0.01,d7 compared to d4, **p<0.01,d7 compared to d2) mRNA levels at 2, 4, and 7 days post-adhesion. Fibroblast markers were also upregulated, and KEGG and GO enrichment analyses identified key pathways involved in ECM-receptor interactions, cell cycle regulation, PI3K-Akt signaling, and extracellular matrix remodeling.

Conclusion: The Nycodenz gradient efficiently isolates quiescent mPSCs, and short-term caerulein treatment effectively activates mPSCs ex vivo, providing a valuable model for studying mPSC activation and related signaling pathways.

Keywords: chronic pancreatitis; pancreatic fibrosis; pancreatic stellate cells.

Figure 1

Isolation methods of primary mPSCs. (A) The flow chart of ladder centrifugation methods for mPSCs. (B) The enzyme mixture solution was injected into the biliopancreatic duct of murine. (C) The pancreatic tissue was fully swollen and digested. (D) The white and thin cell layer in the middle isolated by ladder centrifugation was mPSCs.

Figure 2

Verification of isolated cells. (A) The mPSCs isolated from ladder centrifugation method and cultured for 2 days (×100). (B) The mPSCs isolated from ladder centrifugation method and cultured for 2 days (×200). (C) Representative immunofluorescence staining images of α-SMA and Desmin in mPSCs (×400). (D) Representative immunofluorescence staining images of Vimentin and GFAP in mPSCs (×400). (E) Representative immunofluorescence staining images of CK19 and CD68 in mPSCs (×400). (F) The oil red O staining of mPSCs (×200). (G) The mRNA level of acta2, col1a1 and actg2 in isolated mPSCs cultured for 2 days, 4 days and 7 days. Data are expressed as mean ± SEM, n=5 in (G). (**p<0.01, compared to d2 group, ^##p<0.01, compared to d4 group).

Figure 3

Short-term intraperitoneal injection of caerulein is an effective method to establish mPSCs activation model. (A) Activated mPSCs model was established by short-term intraperitoneal injection of caerulein or NS as control group. (B) The cell number of mPSCs in caerulein-injected group was greater than in NS-injected group. (C) The mPSCs in caerulein-injected group showed larger and flatter shape compared to NS-injected group. (D) The mPSCs in caerulein-injected group showed higher expression level of fibroblast markers, including acta2 and col1a1 compared to NS-injected group. (E) The mPSCs in caerulein-injected group showed higher level of α-SMA protein expression level. (F) Quantification of immunofluorescence (α-SMA). Data are expressed as mean ± SEM, n=8 in (B), n=4 in (D), n=5 in (E). (*p<0.05, ****p<0.0001).

Figure 4

Altered mPSCs activated pattern was verified by transcriptomics analysis. (A) PCA analysis of mPSCs in caerulein-treated group and NS-treated group. The red dots represented the quiescent cells, and the blue dots represented the activated samples. The two major principal components explained 52.71% and 14.01% of the total variance, respectively. (B) The volcanic plot presenting the distribution of genes (|log2FoldChange| >1 and padj < 0.05). (C) The heatmap hierarchical clustering presenting the different distribution of two groups. (D) KEGG enrichment analysis of differentially expressed genes. (E) GO enrichment analysis of differentially expressed genes. (F) The GSEA figure of cell cycle, AMPK signaling pathway and fatty acid metabolism pathway. (G) Heatmap of ECM-receptor interaction and cell cycle - related gene expression profiles based on the RNA-seq data. n = 4–5 per group.