ISOLATION OF QUIESCENT (NORMAL) HUMAN PANCREATIC STELLATE CELLS (HPSCS)

Abstract

Activated pancreatic stellate cells (PSCs) play a central role in pancreatic fibrosis. Most of our knowledge of PSC biology has been obtained from rat PSC cultures, with studies of human PSCs (hPSCs) being limited to pre-activated cancer- or inflammation-associated cells isolated by the outgrowth method from resected pancreatic specimens. Thus, an in vitro model ofhPSCs in their normal, quiescent state has been lacking. The aims of this study were: i) to isolate quiescent hPSCs using density gradient centrifugation; ii) to assess their morphology and their expression of stellate cell selective markers; and iii) to evaluate their response to the mitogen platelet derived growth factor (PDGF). Methods: hPSCs were isolated from normal pancreatic tissue (n = 4, obtained during resections for non-malignant and non-inflammatory pancreatic pathologies), using a Nycodenz density gradient. Freshly isolated and cultured cells were examined by phase contrast microscopy. Cells were immunostained for the stellate cell selective markers [glial fibrillary acidic protein (GFAP) and desmin] and α smooth muscle actin (an activation marker). hPSCs were incubated for 24h with and without PDGF (20ng/ml) and cell proliferation assessed by measuring 3H-thymidine incorporation into cellular DNA. PDGF receptor β expression by hPSCs was assessed by western blotting. Results: Freshly isolated hPSCs displayed a polygonal appearance with multiple refringent lipid droplets in the cytoplasm. These droplets were progressively lost in culture although ~10% of cells retained their droplets until the first passage at 10 days. Human PSCs expressed GFAP, desmin, αSMA and PDGF receptor β. Notably, hPSCs responded to PDGF by significantly increased proliferation [data expressed as % of control, mean ± SE: 138.3 ± 9.9; p < 0.05; n = 3 separate hPSC preparations]. Conclusions: Isolation of (normal) quiescent human PSCs is feasible. Freshly isolated and early culture hPSCs exhibit the presence of lipid droplets similar to quiescent rat PSCs and display the same markers as their rodent and human cancer-associated counterparts. In addition, they respond toPDGF in a manner similar to rat PSCs. Implication: The ability to isolate normal human PSCs from normal pancreatic tissue provides a useful in vitro tool to study their biology in their physiological, non-activated state.