Background/Objectives:Homo-cultured human hepatocytes undergo de-differentiation and loss of liver-specific metabolic functionin vitro, whereas mesenchymal stem cells (MSCs) preserve the morphology and functionality of hepatocytes in heterotypic co-culture [1]. AsMSCs normally reside in a hypoxic niche [2], we hypothesised that hypoxic preconditioning (HPc) could optimise the co-culturehepatotrophic effect of adipose-derived stem cells (ADSCs), a subtype of MSCs,and HPc-induced potentiative effect depended on intra-ADSCs activity of reactive oxygen species (ROS), a pivotal signalling factor implicated in oxidative stress [3]. Methods:Human ADSCs with or without theaddition of10-mM N-acetylcysteine (NAC), an antioxidant against ROS,were subjected to 2% O₂ HPc, with 20% O₂ normoxia-preconditioned (NPc) cells as control, for 24 hours. Intra-ADSCs ROS activity was measured using flow cytometry with chloromethyl 2',7'-dichlorodihydrofluorescein diacetate, and normalised to percentage of mean fluorescence intensity of control cells. Fresh human hepatocytes were seeded onto media-refreshed ADSCs monolayers ata ratio of 2.5:1and co-cultured for 7 days, with hepatocyte mono-culture as control. Indirect hepatocyte co-culture in ADSCs-conditioned media was also performed to determine the role ofADSCs-derived paracrine factors in the co-culture hepatotrophic effect. Albumin, caspase-cleaved cytokeratin 18(CCK18), and cytokeratin 18 (CK18) in culture supernatant were measured using enzyme-linked immunosorbent assays to determine the effects of ADSCs co-culture on liver-specific synthetic metabolism, caspase-mediated apoptosis, and total death of co-cultured hepatocytes, respectively; all data were shown as mean±SD, and normalised to one million seeded viable hepatocytes. Results:HPc significantly increased intra-ADSCs ROS activity (190.0±22.2%, P<0.05),while 10-mM NAC reduced ROS activity in HPc-ADSCs (109.6±28.8%, P<0.05). HPc-ADSCs co-cultureimproved albumin synthesis up to day 7 (HPc vs NPc vs control, 5.4±0.3 vs 4.7±0.2 vs 1.4±0.2 μg, P<0.01), and also reduced the release of soluble CCK18 (17.3±2.6 vs 30.4±3.4 vs 42.0±2.0 U, P<0.01) and CK18 (91.3±13.2 vs 179.0±14.0 vs 364.8±15.6 U, P<0.01) from hepatocytes up to day 4. ROS inhibition reducedhepatotrophic (albumin, 4.5±0.4 μg, P<0.01), antiapoptotic (CCK18, 30.5±2.4 U, P<0.01), and prosurvival effects (CK18, 176.0±13.2 U, P<0.01) of HPc-ADSCsco-culture. ADSCs indirect co-culture had minimal hepatotrophic (1.4±0.1 vs 1.2±0.1 vs 1.4±0.2 μg, P>0.05), antiapoptotic (44.4±3.3 vs 46.4±5.3 vs 42.0±2.0 U, P>0.05), and prosurvival effects (368.4±13.2 vs 353.9±21.2 vs 364.8±15.6 U, P>0.05) on hepatocytes.Conclusions:HPc-induced oxidative stress potentiatesthe hepatotrophic, antiapoptotic, and prosurvival effects of MSCs co-culture on hepatocytes by a non-paracrine mechanism dependent on intra-MSCs ROS activity.