P.04.6 PRIMARY HUMAN BILIARY TREE STEM/PROGENITOR CELLS (HBTSCS) EXPOSED TO MICROENVIRONMENTAL FACTORS SHOWED PROLIFERATION, EPITHELIAL-MESENCHYMAL TRANSITION (EMT) AND SENESCENCE, RECAPITULATING THE PATHOLOGICAL FEATURES TYPICAL OF HUMAN CHOLANGIOPATHIES

Gravity plays a key role in regulating cell processes such as proliferation, differentiation and cell function. The aim of the study was to evaluate the effects of microgravity on differentiation and exo-metabolome profile of human hepatic and biliary tree stem/progenitor cells. Simulated weightless conditions were obtained by the Rotary Cell Culture System (Synthecon). Primary cultures of human biliary tree stem cells (hBTSCs) and immortalized human hepatic cell line (HepG2, used as controls) were cultured in microgravity or in normogravity conditions. Self replication and differentiation toward mature cells were determined, respectively, by culturing in Kubota’s Medium (KM) and hormonally defined medium (HDM) tailored for hepatocyte differentiation. RT-qPCR was used to evaluate gene expression and NMR to analyze the cell exo-metabolome profile. Microgravity determined a stable increase of stemness genes (OCT4, SOX17, PDX1) in hBTSCs (p< 0.05 vs normogravity). hBTSCs cultured in microgravity conditions showed an impaired capacity to differentiate toward mature hepatocytes (HDM media), since the expression of hepatocyte lineage genes (ALB, ASBT and CYP3A4) was significantly lower with respect to normogravity (p< 0.05). In HepG2, the microgravity caused a lower (p< 0.05 vs normogravity) expression of CYP3A4, a terminal differentiation gene expressed in lobular zone 3. The NMR Principal Component Analysis of the exo-metabolome cell profile evidenced that, in microgravity, both cell lines presented higher glucose consumption and lower consumption of pyruvate and glutamate with respect to normogravity (p< 0.05), with formation of fermentation (lactate, acetate) and ketogenesis products (B-hydroxybutyrate). Interestingly, while in normogravity the differentiation of hBTSCs toward mature hepatocytes was associated with increased oxidative phosphorylation (p< 0.05), this was prevented by microgravity in association with the impaired cell differentiation. A glycolytic metabolism has been associated with a pluripotency state of human embryonic stem cells, in different studies. Our results demonstrated significant combined biologic and metabolomic effects of microgravity on hepatic stem/progenitor cells with several implications. From one side, these effects of microgravity should be taken into consideration for space medicine programs but, from the other side, they could be of interest for the generation of devices based on stem/progenitor cells.