title: Mass spectrometry analysis of adipose-derived stem cells reveals a significant effect of hypoxia on pathways regulating extracellular matrix.
abstract: BACKGROUND
  Adipose-derived stem cells (ASCs) are being increasingly recognized for their potential to promote tissue regeneration and wound healing. These effects appear to be partly mediated by paracrine signaling pathways, and are enhanced during hypoxia. Mass spectrometry (MS) is a valuable tool for proteomic profiling of cultured ASCs, which may help to reveal the identity of the factors secreted by the cells under different conditions. However, serum starvation which is essentially required to obtain samples compatible with secretome analysis by MS can have a significant influence on ASCs. Here, we present a novel and optimized culturing approach based on the use of a clinically relevant serum-free formulation, which was used to assess the effects of hypoxia on the ASC proteomic profile.
  
  
  METHODS
  Human ASCs from three human donors were expanded in StemPro® MSC SFM XenoFree medium. Cells were cultured for 24 h in serum- and albumin-free supplements in either normoxic (20 %) or hypoxic (1 %) atmospheres, after which the cells and conditioned medium were collected, subfractionated, and analyzed using MS. Prior to analysis, the secreted proteins were further subdivided into a secretome (>30 kDa) and a peptidome (3-30 kDa) fraction.
  
  
  RESULTS
  MS analysis revealed the presence of 342, 98, and 3228 proteins in the normoxic ASC secretome, peptidome, and proteome, respectively. A relatively small fraction of the proteome (9.6 %) was significantly affected by hypoxia, and the most regulated proteins were those involved in extracellular matrix (ECM) synthesis and cell metabolism. No proteins were found to be significantly modulated by hypoxic treatment across all cultures for the secretome and peptidome samples.
  
  
  CONCLUSIONS
  This study highlights ECM remodeling as a significant mechanism contributing to the ASC regenerative effect after hypoxic preconditioning, and further underscores considerable inter-individual differences in ASC response to hypoxia. The novel culture paradigm provides a basis for future proteomic studies under conditions that do not induce a stress response, so that the best responders can be accurately identified for prospective therapeutic use. Data are available via ProteomeXchange with identifier PXD003550.
authors.affiliation: Department of Health Science and Technology, Laboratory for Stem Cell Research, Aalborg University, Fredrik Bajers Vej 3B, Aalborg, 9220, Denmark.
authors.forename: Simone
authors.initials: S
authors.lastname: Riis
authors.affiliation: Department of Health Science and Technology, Laboratory for Medical Mass Spectrometry, Aalborg University, Aalborg, Denmark.
authors.forename: Allan
authors.initials: A
authors.lastname: Stensballe
authors.affiliation: Department of Health Science and Technology, Laboratory for Stem Cell Research, Aalborg University, Fredrik Bajers Vej 3B, Aalborg, 9220, Denmark.
authors.forename: Jeppe
authors.initials: J
authors.lastname: Emmersen
authors.affiliation: Department of Health Science and Technology, Laboratory for Stem Cell Research, Aalborg University, Fredrik Bajers Vej 3B, Aalborg, 9220, Denmark.
authors.forename: Cristian Pablo
authors.initials: CP
authors.lastname: Pennisi
authors.affiliation: Department of Health Science and Technology, Laboratory for Medical Mass Spectrometry, Aalborg University, Aalborg, Denmark.
authors.forename: Svend
authors.initials: S
authors.lastname: Birkelund
authors.affiliation: Department of Health Science and Technology, Laboratory for Stem Cell Research, Aalborg University, Fredrik Bajers Vej 3B, Aalborg, 9220, Denmark.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Zachar
authors.affiliation: Department of Health Science and Technology, Laboratory for Stem Cell Research, Aalborg University, Fredrik Bajers Vej 3B, Aalborg, 9220, Denmark. trinef@hst.aau.dk.
authors.forename: Trine
authors.initials: T
authors.lastname: Fink
mesh_terms: D017667:Adipocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D000273:Adipose Tissue / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D015687:Cell Hypoxia; D049109:Cell Proliferation / Q000187:drug effects; D017077:Culture Media, Conditioned / Q000737:chemistry; D030562:Databases, Protein; D005109:Extracellular Matrix / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000032:analysis / Q000378:metabolism*; D063990:Gene Ontology; D006801:Humans; D033181:Information Dissemination; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D058977:Molecular Sequence Annotation; D010100:Oxygen / Q000494:pharmacology*; D061251:Primary Cell Culture; D020543:Proteome / Q000032:analysis* / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D017077:Culture Media, Conditioned; D016326:Extracellular Matrix Proteins; D020543:Proteome; D010100:Oxygen
keywords: ASCs; ECM; Hypoxia; Mass spectrometry; Proteomics; Secretome; Serum-free culture