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Oral Communications 4
9.1 - Hypoxic preconditioning potentiates hepatotrophic, antiapoptotic, and prosurvival effects of mesenchymal stem cells on co-cultured human hepatocytes by a reactive oxygen species dependent, non-paracrine mechanism
Presenter: Hong, Qin, London, United Kingdom Authors: Harry H. Qin1, Celine Filippi 1, Mitry M. Ragai1, Anil Dhawan1, Robin D. Hughes1
Hypoxic preconditioning potentiates hepatotrophic, antiapoptotic, and prosurvival effects of mesenchymal stem cells on co-cultured human hepatocytes by a reactive oxygen species dependent, non-paracrine mechanism
Harry H. Qin1, Celine Filippi 1, Mitry M. Ragai1, Anil Dhawan1, Robin D. Hughes1
1Institute of Liver Studies, King's College London School of Medicine at King's College Hospital, London, United Kingdom
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 . AsMSCs normally reside in a hypoxic niche , 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 . 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.
Gu J, Shi X, Zhang Y, Ding Y: Heterotypic interactions in the preservation of morphology and functionality of porcine hepatocytes by bone marrow mesenchymal stem cells in vitro. J Cell Physiol 2009, 219:100-108.
Das R, Jahr H, van Osch GJ, Farrell E: The role of hypoxia in bone marrow-derived mesenchymal stem cells: considerations forregenerative medicine approaches. Tissue Eng Part B Rev2010:159-168.
Kim JH, Park SH, Park SG, Choi JS, Xia Y, Sung JH: The pivotal role of reactive oxygen species generation in the hypoxia-induced stimulation of adipose derivedstem cells. Stem Cells Dev2011:1753-1761.
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