Generation and characterization of immortalized rat pancreatic stellate cells

Abstract

Pancreatic stellate cells (PSCs) are involved in, among other things, the pathogenesis of pancreatic fibrosis. Here, we present the generation of immortalized PSCs 7 and 14 days after isolation by retroviral gene transfer of the SV40 large T antigen encoding region. Propagated cell lines [large T immortalized cells (LTC)-7, LTC-14] retained characteristics of primary cells in terms of morphology, responsiveness to mediators regulating cellular functions such as proliferation, and expression profile of a number of investigated genes. Whereas LTC-14 kept the morphological features of the differentiation status of the primary cells they were made of, LTC-7 appeared similar to an earlier stage. Thus the established cell lines represent a versatile tool to investigate various aspects of PSC biology.

Bone marrow-derived pancreatic stellate cells in rats

“…The activation process of PSCs has been shown to be accompanied by a gradual loss of lipid droplets and rising expression of α-SMA [1,2,16]. Figure 4A and 4B summarize the characteristic course of both markers in PSCs obtained from the restored pancreas, reflecting the typical transition from resting cells into the activated myofibroblastic phenotype during in vitro cultivation.…”

“…The purification procedure based on npg the high fat content of quiescent PSCs ensured the nonactivated phenotype of freshly prepared stellate cells [16].…”

“…We investigated isolated PSCs obtained from untreated control rats as well as from the restored organ following an episode of acute pancreatitis induced by dibutyltin dichloride (DBTC). The isolation method for PSC was based upon fat content, thus addressing the quiescent PSC population [16].…”

TGF-β-induced stromal CYR61 promotes resistance to gemcitabine in pancreatic ductal adenocarcinoma through downregulation of the nucleoside transporters hENT1 and hCNT3

“…L3.6p cells were provided by Dr Isaiah Fidler (MD Anderson) (16). RLT-PSC human pancreatic stellate cells (PSCs) were provided by Dr Ralf Jesenofsky (University of Heidelberg) (17); HPSC-T human PSCs were provided by Dr Rosa Hwang (MD Anderson) (18); LTC-14 rat PSCs were provided by Dr Gisele Sparman (University Hospital of Rostock) (19) and imPSC mouse PSCs were provided by Dr Raul Urrutia (Mayo Clinic) (20). Both human and murine PSCs were obtained directly from the labs that isolated the cells and were functionally validated by their expression patterns in the indicated studies.…”

“…Consistent with this hypothesis, in the microarray dataset that analyzed gene expression in whole-tissue PDAC samples, CYR61 expression significantly correlated with expression of the TGFB1 ligand and also with the well-established TGF-β target genes SERPINE1 (PAI-1) and SMAD7 ( Figure 5A-C). Moreover, in the rat PSC cell line LTC-14 (19) and the mouse PSC cell line imPSC (20), which were isolated from normal pancreas and have low basal CYR61 expression, TGF-β induced CYR61 expression in a dosedependent fashion ( Figure 5D). TGF-β induced CYR61 protein expression as early as 6 h post treatment and mRNA expression as early as 3 h post treatment, suggesting that the induction of CYR61 by TGF-β was a direct effect and not via induction of other growth factors ( Figure 5E and F).…”