title: Functional and differential proteomic analyses to identify platelet derived factors affecting ex vivo expansion of mesenchymal stromal cells.
abstract: BACKGROUND
  Multilineage differentiation, immunomodulation and secretion of trophic factors render mesenchymal stromal cells (MSC) highly attractive for clinical application. Human platelet derivatives such as pooled human platelet lysate (pHPL) and thrombin-activated platelet releasate in plasma (tPRP) have been introduced as alternatives to fetal bovine serum (FBS) to achieve GMP-compliance. However, whereas both pHPL and tPRP support similar proliferation kinetics of lipoaspirate-derived MSC (LA-MSC), only pHPL significantly accelerates bone marrow-derived MSC (BM-MSC) expansion. To identify functionally bioactive factors affecting ex vivo MSC expansion, a differential proteomic approach was performed and identified candidate proteins were evaluated within a bioassay.
  
  
  RESULTS
  Two dimensional difference gel electrophoresis (2D-DIGE), MALDI-TOF analyses and complementary Western blotting revealed 20 differential protein species. 14 candidate proteins occured at higher concentrations in pHPL compared to tPRP and 6 at higher concentrations in tPRP. The candidate proteins fibrinogen and apolipoprotein A1 differentially affected LA- and BM-MSC proliferation.In a second set of experiments, reference cytokines known to foster proliferation in FBS were tested for their effects in the human supplements. Interestingly although these cytokines promoted proliferation in FBS, they failed to do so when added to the humanized system.
  
  
  CONCLUSIONS
  The differential proteomic approach identified novel platelet derived factors differentially acting on human MSC proliferation. Complementary testing of reference cytokines revealed a lack of stimulation in the human supplements compared to FBS. The data describe a new coherent approach to combine proteomic technologies with functional testing to develop novel, humanized, GMP-compliant conditions for MSC expansion.
authors.forename: Sven
authors.initials: S
authors.lastname: Kinzebach
authors.forename: Lisa
authors.initials: L
authors.lastname: Dietz
authors.forename: Harald
authors.initials: H
authors.lastname: Klüter
authors.affiliation: Institute of Transfusion Medicine and Immunology, Medical Faculty Mannheim, Heidelberg University; German Red Cross Blood Service Baden-Württemberg, Friedrich-Ebert-Str, 107, Mannheim, Hessen D-68167, Germany. thierse@immunoproteomics.de.
authors.forename: Hermann-Josef
authors.initials: HJ
authors.lastname: Thierse
authors.forename: Karen
authors.initials: K
authors.lastname: Bieback
mesh_terms: D000273:Adipose Tissue / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D016632:Apolipoprotein A-I / Q000302:isolation & purification / Q000494:pharmacology*; D001792:Blood Platelets / Q000737:chemistry* / Q000378:metabolism; D001854:Bone Marrow Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D002417:Cattle; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D016207:Cytokines / Q000302:isolation & purification / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional; D005340:Fibrinogen / Q000302:isolation & purification / Q000494:pharmacology*; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D010982:Platelet-Derived Growth Factor / Q000302:isolation & purification / Q000494:pharmacology*; D040901:Proteomics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D016632:Apolipoprotein A-I; D016207:Cytokines; D010982:Platelet-Derived Growth Factor; D005340:Fibrinogen