P.10.4: The Differentiation and Metabolism of Human Hepatic and Biliary Tree Stem/Progenitor Cells can be Significantly Modulated by Microgravity

Background and Aims: Gravity plays a key role in regulating cellprocesses such as proliferation, differentiation and cell function. Theaim of the study was to evaluate the effects of microgravity ondifferentiation and exo-metabolome profile of human hepatic andbiliary tree stem/progenitor cells.Methods: Simulated weightless conditions were obtained by theRotary Cell Culture System (Synthecon). Primary cultures of humanbiliary tree stem cells (hBTSCs) and immortalized human hepatic cellline (HepG2, used as controls) were cultured in microgravity or innormogravity conditions. Self replication and differentiation towardmature cells were determined, respectively, by culturing in Kubota’sMedium (KM) and hormonally defined medium (HDM) tailored forhepatocyte differentiation. RT-qPCR was used to evaluate geneexpression and NMR to analyze the cell exo-metabolome profile.Results: Microgravity determined a stable increase of stemness genes(OCT4, SOX17, PDX1) in hBTSCs (p < 0.05 vs normogravity). hBTSCscultured in microgravity conditions showed an impaired capacity todifferentiate toward mature hepatocytes (HDM media), since theexpression of hepatocyte lineage genes (ALB, ASBT and CYP3A4) wassignificantly 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 lobularzone 3. The NMR Principal Component Analysis of the exometabolome cell profile evidenced that, in microgravity, both celllines presented higher glucose consumption and lower consumptionof pyruvate and glutamate with respect to normogravity (p < 0.05),with formation of fermentation (lactate, acetate) and ketogenesisproducts (B-hydroxybutyrate). Interestingly, while in normogravitythe differentiation of hBTSCs toward mature hepatocytes wasassociated with increased oxidative phosphorylation (p < 0.05), thiswas prevented by microgravity in association with the impaired celldifferentiation.Conclusions: A glycolytic metabolism has been associated with apluripotency state of human embryonic stem cells, in differentstudies. Our results demonstrated significant combined biologic andmetabolomic effects of microgravity on hepatic stem/progenitor cellswith several implications. From one side, these effects of microgravityshould be taken into consideration for space medicine programs but,from the other side, they could be of interest for the generation ofdevices based on stem/progenitor cells.