Isolation and characterization of islet stellate cells in rat

Abstract

The central role of PSCs in pancreatic fibrogenesis is well established. However, the mechanism responsible for the islet fibrosis presenting in the late stage of T2DM has not been fully elucidated. This study was designed to determine whether the endocrine pancreatic islets contain cells resembling PSCs. PSCs were isolated from pancreas using standard explants techniques. A similar method was used to acquire ISCs. Adherent ISCs with a stellate, angular morphology migrated from the edge of cultured islets within 48 h of primary culture. ISCs contained fewer lipid droplets than equivalent PSCs, and their rapid disappearance accompanied by the increased expression of α-SMA suggested that ISCs were more rapidly activated than PSCs in vitro. They expressed α-SMA, vimentin, GFAP and were positive for ECM components col-I, col-III and FN, all of which are characteristics of classical PSCs. However, ISCs differed from PSCs by having reduced rates of proliferation and migration in vitro. Our in vitro study shows that isolated islets contain a population of stellate cells which are phenotypically similar but not identical to PSCs. In view of the established role of PSCs in pancreatic fibrosis, we suggest that these may contribute to islet fibrosis in T2DM.

Keywords: ISC; PSC; islet; islet stellate cell; pancreatic stellate cell.

Figure 1. Isolation and culture of ISCs from isolated rat islets. Islets were characterized in the pancreatic digest after DTZ staining as red-stained clusters among the unstained exocrine digest (a). Isolated islets adhered to the bottom of culture dish, and acted as explant cultures from which there was a progressive outgrowth of proliferating cells from the edge of the islets (b: 0 h; c: 36 h; d: 48 h; e:72 h; f and g: 7 d). After 72 h in primary culture, we immune-stained the islet and the proliferating cells with antibodies against insulin (h: insulin; i: DAPI). Islet β-cells showed positive insulin immunoreactivity, but the proliferating adherent cell outgrowth did not express insulin.

Figure 2. Lipid droplets (A) and expression of α-SMA (B) in ISC and in classical PSCs populations were compared. (a-f: Pancreas block and the proliferating PSCs; g-l: Islet and the proliferating ISCs) (A) Lipid droplets in cytoplasm were detected by Oil red O staining. PSCs carried numerous lipid droplets when they migrated from pancreas blocks after 48 h (a and b), which persisted unchanged during 72 h in culture (c and d). After 72 h in culture, immunofluorescent staining for α-SMA showed PSCs were negative for α-SMA (e: α-SMA; f: DAPI). In contrast, after 48 h culture ISCs containing lipid droplets were very rarely observed (g and h), and all the lipid droplets were gone by 72 h (i and j), when ISCs were positive for α-SMA (k and j).

Figure 3. Immunofluorescence visualization of markers for PSCs was used to characterize the ISCs after passage 3. Immunofluorescent staining for α-SMA, GFAP, vimentin was performed on PSCs (a-c) and ISCs (d-f) respectively. Both populations of stellate cells were positive for α-SMA (a and d), vimentin (b and e) and GFAP (c and f).

Figure 4. Immunofluorescence visualization of ECM components in PSCs and ISCs. Immunofluorescent staining for Col-I, Col-III and FN was performed on PSCs (a-c) and ISCs (d-f) respectively. Both PSCs and ISCs were immunopositive for col-I (a and d), col-III (b and e) and FN (c and f), respectively.

Figure 5. Proliferation of ISCs and PSCs in vitro. Cell proliferation was determined with the CCK-8 kit. By 36 h in culture the number of PSCs was significantly greater than that of ISCs, and this difference was further extended after 72 h in culture, demonstrating that the ISCs proliferated at a significantly slower rate than the PSCs. Data were expressed as mean ± SE (n = 15), ** P < 0.01, ISCs vs. PSCs. (The error bars were smaller than the symbols).

Figure 6. Migration of ISCs and PSCs in vitro. (A) In wound healing assay, 24 h after the wound formation, the number of ISCs migrating into the cell-free area was significantly less than that of PSCs. Data were expressed as mean ± SE (n = 5), ** P < 0.01, ISCs vs. PSCs. (B) In transwell migration assay, cells adhering to the lower side of the insert membrane were purple after crystal violet staining. After 24 h the number of ISCs migrating through the membrane was significantly less than that of PSCs (n = 5), ** P < 0.01, ISCs vs. PSCs. The brown dots are pores in the membrane.