Microgravity maintains stemness and enhance glycolytic metabolism in human hepatic and biliary tree stem/progenitor cells

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 microgravityon differentiation and exo-metabolome profile of human hepaticand biliary tree stem/progenitor cells.Simulated weightless conditions were obtained by the RCCS(Synthecon). Primary cultures of human biliary tree stem cells(hBTSCs) and immortalized human hepatic cell line (HepG2)were cultured in microgravity or in normogravity conditions.Self-replication and differentiation toward mature cells weredetermined, respectively, by culturing in Kubota’s Medium andhormonally defined medium tailored for hepatocyte differentiation. RT-qPCR was used to evaluate gene expression and NMR toanalyze the cell exo-metabolome profile.Microgravity determined an increase of stemness genes (OCT4,SOX17, PDX1) in hBTSCs (p < 0.05 vs normogravity). hBTSCs cultured in microgravity showed an impaired capacity to differentiatetoward mature hepatocytes, since the expression of hepatocytelineage genes (ALB, ASBT and CYP3A4) was significantly lowerwith respect to normogravity (p < 0.05). In HepG2, the microgravitycaused a lower (p < 0.05 vs normogravity) expression of CYP3A4,a terminal differentiation gene expressed in lobular zone 3. TheNMR PCA of the exo-metabolome cell profile evidenced that, inmicrogravity, both cell lines presented higher glucose consumptionand lower consumption of pyruvate and glutamate with respectto normogravity (p < 0.05), with formation of fermentation andketogenesis products. Interestingly, while in normogravity thedifferentiation of hBTSCs toward mature hepatocytes was associated with increased oxidative phosphorylation (p < 0.05), this wasprevented by microgravity in association with the impaired celldifferentiation.Our results demonstrated significant combined biologic andmetabolomic effects of microgravity on hepatic stem/progenitorcells with several implications. From one side, these effects ofmicrogravity should be taken into consideration for space medicineprograms but, from the other side, they could be interesting for thegeneration of devices based on stem/progenitor cells.