title: Hypoxic preconditioning potentiates the trophic effects of mesenchymal stem cells on co-cultured human primary hepatocytes.
abstract: BACKGROUND
  Mesenchymal stem/stromal cells (MSCs) improve the metabolic function of co-cultured hepatocytes. The present study aimed to further enhance the trophic effects of co-culture with hepatocytes using hypoxic preconditioning (HPc) of the MSCs and also to investigate the underlying molecular mechanisms involved.
  
  
  METHODS
  Human adipose tissue-derived MSCs were subjected to hypoxia (2 % O2; HPc) or normoxia (20 % O2) for 24 h and then co-cultured with isolated human hepatocytes. Assays of metabolic function and apoptosis were performed to investigate the hepatotrophic and anti-apoptotic effects of co-culture. Indirect co-cultures and co-culture with MSC-conditioned medium investigated the role of paracrine factors in the hepatotrophic effects of co-culture. Reactive oxygen species (ROS) activity was antagonised with N-acetylcysteine to investigate whether HPc potentiated the effects of MSCs by intracellular ROS-dependent mechanisms. Tumour necrosis factor (TNF)-α, transforming growth factor (TGF)-β1, and extracellular collagen production was determined and CASP9 and BAX/BCL-2 signalling pathways analysed to investigate the role of soluble factors, extracellular matrix deposition, and apoptosis-associated gene signalling in the effects of co-culture.
  
  
  RESULTS
  HPc potentiated the hepatotrophic and anti-apoptotic effects of co-culture by ROS-dependent mechanisms. There was increased MSC TGF-β1 production, and enhanced MSC deposition of extracellular collagen, with reduced synthesis of TNF-α, as well as a downregulation of the expression of pro-apoptotic CASP9, BAX, BID and BLK genes and upregulated expression of anti-apoptotic BCL-2 in hepatocytes.
  
  
  CONCLUSIONS
  HPc potentiated the trophic and anti-apoptotic effects of MSCs on hepatocytes via mechanisms including intracellular ROS, autocrine TGF-β, extracellular collagen and caspase and BAX/BCL-2 signalling pathways.
authors.affiliation: Dhawan Lab, Institute of Liver Studies, King's College London and King's College Hospital NHS Foundation Trust, London, SE5 9PJ, UK. hong.qin@outlook.com.
authors.forename: Harry H
authors.initials: HH
authors.lastname: Qin
authors.affiliation: Dhawan Lab, Institute of Liver Studies, King's College London and King's College Hospital NHS Foundation Trust, London, SE5 9PJ, UK. celine.filippi@kcl.ac.uk.|NIHR Biomedical Research Centre at Guy's and St Thomas NHS Foundation Trust and King's College London, London, SE1 9RT, UK. celine.filippi@kcl.ac.uk.
authors.forename: Céline
authors.initials: C
authors.lastname: Filippi
authors.affiliation: Dhawan Lab, Institute of Liver Studies, King's College London and King's College Hospital NHS Foundation Trust, London, SE5 9PJ, UK. sunsongmed@163.com.
authors.forename: Song
authors.initials: S
authors.lastname: Sun
authors.affiliation: Dhawan Lab, Institute of Liver Studies, King's College London and King's College Hospital NHS Foundation Trust, London, SE5 9PJ, UK. sharon.lehec@nhs.net.
authors.forename: Sharon
authors.initials: S
authors.lastname: Lehec
authors.affiliation: Dhawan Lab, Institute of Liver Studies, King's College London and King's College Hospital NHS Foundation Trust, London, SE5 9PJ, UK. anil.dhawan@kcl.ac.uk.|Paediatric Liver, GI and Nutrition Centre, King's College Hospital Denmark Hill, London, SE5 9RS, UK. anil.dhawan@kcl.ac.uk.
authors.forename: Anil
authors.initials: A
authors.lastname: Dhawan
authors.affiliation: Dhawan Lab, Institute of Liver Studies, King's College London and King's College Hospital NHS Foundation Trust, London, SE5 9PJ, UK. robin.hughes@kcl.ac.uk.
authors.forename: Robin D
authors.initials: RD
authors.lastname: Hughes
mesh_terms: D017209:Apoptosis / Q000235:genetics; D015687:Cell Hypoxia; D018920:Coculture Techniques; D003094:Collagen / Q000378:metabolism; D017077:Culture Media, Conditioned; D015536:Down-Regulation; D019254:Genes, bcl-2; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism* / Q000637:transplantation; D006801:Humans; D019194:Ischemic Preconditioning; D045164:Mesenchymal Stem Cell Transplantation; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism*; D019899:Paracrine Communication; D017382:Reactive Oxygen Species / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000235:genetics
chemical_list: C494826:BAX protein, human; D017077:Culture Media, Conditioned; D017382:Reactive Oxygen Species; D053773:Transforming Growth Factor beta1; D014409:Tumor Necrosis Factor-alpha; D051028:bcl-2-Associated X Protein; D003094:Collagen