The stimulation of human biliary tree stem/progenitor cells (hBTSCs) in peribiliary glands (PBGs) have been demonstrated in several cholangiopathies, such as Primary Sclerosing Cholangitis (PSC) and Cholangiocarcinoma (CCA). hBTSCs also display features of EMT, senescence and dysplasia in these pathologies. Our aim was to investigate which effects putative agents of human cholangiopathies reproduce in primary cultures of hBTSCs. hBTSCs were isolated from donor organs (N=6), grown in self-renewal control conditions (Kubota's Medium) and exposed (from 24h to 10 days) to concentrations of lipopolysaccharides (LPS), oxysterols, hydrophobic biliary salts, or high glucose. Viability (MTS assay), Population Doubling (PD), the expression (RT-qPCR) of pluripotency, transit-amplifying compartment, EMT, pNF-κB and HDAC6 genes and senescence associated secreted phenotype (SASP) by IF and ELISA were investigated. Glycochenodeoxycholate (0.5mM), LPS (200ng/ml), oxysterols [(+)-4-Cholesten-3-one (0.14mM), Cholesta–4,6–dien-3-one (0.14mM), 5α-Cholestan-3-one (0.14mM)] or high concentrations of Glucose (28mM) did not affected viability of hBTSCs. LPS, Cholesta-4,6-dien-3-one and Glucose induced a significant increase of cell proliferation after 10 days of exposure (p<0.01). The MTS assay confirmed a high proliferation rate after chronic exposure to LPS, oxysterols, or high glucose (p<0.01). A significant increase of markers of transit-amplifying compartment and EMT (p<0.05) occurred in presence of Cholesta-4,6-dien-3-one while, LPS and high Glucose effect was an increased expression of PCNA and EMT genes. Increased senescent cells after 10 days of exposure to each of the investigated factor compared to controls (p<0.01) were observed. LPS, Cholesta-4,6-dien-3-one and high Glucose induced enhanced IL-6 secretion compared to controls (p<0.01), high levels of pNF-κB and HDAC6 (p<0.01), and traces of LC3 protein, suggesting inflammation-induced autophagy.
The exposure of primary cultures of human biliary tree stem/progenitor cells (hBTSCs) to different micro-environmental factors induces proliferation, epithelial-mesenchymal transition (EMT) and senescence, which are typical pathological features of human cholangiopathies
L. Nevi;
2018-01-01
Abstract
The stimulation of human biliary tree stem/progenitor cells (hBTSCs) in peribiliary glands (PBGs) have been demonstrated in several cholangiopathies, such as Primary Sclerosing Cholangitis (PSC) and Cholangiocarcinoma (CCA). hBTSCs also display features of EMT, senescence and dysplasia in these pathologies. Our aim was to investigate which effects putative agents of human cholangiopathies reproduce in primary cultures of hBTSCs. hBTSCs were isolated from donor organs (N=6), grown in self-renewal control conditions (Kubota's Medium) and exposed (from 24h to 10 days) to concentrations of lipopolysaccharides (LPS), oxysterols, hydrophobic biliary salts, or high glucose. Viability (MTS assay), Population Doubling (PD), the expression (RT-qPCR) of pluripotency, transit-amplifying compartment, EMT, pNF-κB and HDAC6 genes and senescence associated secreted phenotype (SASP) by IF and ELISA were investigated. Glycochenodeoxycholate (0.5mM), LPS (200ng/ml), oxysterols [(+)-4-Cholesten-3-one (0.14mM), Cholesta–4,6–dien-3-one (0.14mM), 5α-Cholestan-3-one (0.14mM)] or high concentrations of Glucose (28mM) did not affected viability of hBTSCs. LPS, Cholesta-4,6-dien-3-one and Glucose induced a significant increase of cell proliferation after 10 days of exposure (p<0.01). The MTS assay confirmed a high proliferation rate after chronic exposure to LPS, oxysterols, or high glucose (p<0.01). A significant increase of markers of transit-amplifying compartment and EMT (p<0.05) occurred in presence of Cholesta-4,6-dien-3-one while, LPS and high Glucose effect was an increased expression of PCNA and EMT genes. Increased senescent cells after 10 days of exposure to each of the investigated factor compared to controls (p<0.01) were observed. LPS, Cholesta-4,6-dien-3-one and high Glucose induced enhanced IL-6 secretion compared to controls (p<0.01), high levels of pNF-κB and HDAC6 (p<0.01), and traces of LC3 protein, suggesting inflammation-induced autophagy.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
