Different micro-environtmental factors induce proliferation, epithelial-mesenchymal transition (EMT) and senescence of primary cultures of human biliary tree stem/progenitor cells (hBTSCs), recapitulating the pathological features typical of human cholangiopathies

Background and Aims: The activation of human biliary tree stem/progenitor cells (hBTSCs) located in peribiliary glands (PBGs) havebeen recently described in different cholangiopathies, including Primary Sclerosing Cholangitis (PSC) and Cholangiocarcinoma (CCA). In these pathologies, hBTSCs also display features of EMT, senescence and dysplasia. The aim of the present study was to investigate putative agents reproducing in primary cultures of hBTSCs the pathologic features observed in PBGs of human cholangiopathies. Method: hBTSCs were isolated from donor organs (n = 6), cultured in self-renewal control conditions (Kubota’s Medium, KM) and exposed (from 24 hours to 10 days) to increasing 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-kB and HDAC6 genes and senescence associated secreted phenotype (SASP) by IF and ELISAwere examined. Results: Glycochenodeoxycholate (0.5 mM), LPS (200 ng/ml), oxysterols [(+)-4-Cholesten-3-one (0.14 mM), Cholesta-4,6-dien-3-one (0.14 mM), 5α-Cholestan-3-one (0.14 mM) or high concentrations of Glucose (28 mM) did not affected the 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 (10 days) to LPS, oxysterols, or high glucose (p < 0.01). Cholesta-4, 6-dien-3-one determined a significant increase of markers of transit-amplifying compartment and EMT (p < 0.05) while, bacterial LPS and high Glucose determined an increased expression of PCNA and EMTgenes. Senescent cells increased after 10 days of exposure to each of the investigated factor compared to controls (p < 0.01). LPS, Cholesta-4,6-dien-3-one and high Glucose induced enhanced IL-6 secretion compared to controls (p < 0.01). In addition, conditioned cell cultures expressed high levels of pNF-kB and HDAC6 (p < 0.01), and traces of LC3 protein suggesting inflammation-induced autophagy. Conclusion: We identified different micro-environmental factors capable to induce in hBTSCs proliferation, enhanced expression of pluripotency and transit-amplifying genes, EMT and senescence, recapitulating the pathologic features described in PBGs of human cholangiopathies. IL-6, the overexpression of pNF-kB and HDAC6 and activation of autophagy are involved in these pathologic changes.