title: Long-Term Expansion in Platelet Lysate Increases Growth of Peripheral Blood-Derived Endothelial-Colony Forming Cells and Their Growth Factor-Induced Sprouting Capacity.
abstract: BACKGROUND
  Efficient implementation of peripheral blood-derived endothelial-colony cells (PB-ECFCs) as a therapeutical tool requires isolation and generation of a sufficient number of cells in ex vivo conditions devoid of animal-derived products. At present, little is known how the isolation and expansion procedure in xenogeneic-free conditions affects the therapeutical capacity of PB-ECFCs.
  
  
  RESULTS
  The findings presented in this study indicate that human platelet lysate (PL) as a serum substitute yields twice more colonies per mL blood compared to the conventional isolation with fetal bovine serum (FBS). Isolated ECFCs displayed a higher proliferative ability in PL supplemented medium than cells in FBS medium during 30 days expansion. The cells at 18 cumulative population doubling levels (CPDL) retained their proliferative capacity, showed higher sprouting ability in fibrin matrices upon stimulation with FGF-2 and VEGF-A than the cells at 6 CPDL, and displayed low β-galactosidase activity. The increased sprouting of PB-ECFCs at 18 CPDL was accompanied by an intrinsic activation of the uPA/uPAR fibrinolytic system. Induced deficiency of uPA (urokinase-type plasminogen activator) or uPAR (uPA receptor) by siRNA technology completely abolished the angiogenic ability of PB-ECFCs in fibrin matrices. During the serial expansion, the gene induction of the markers associated with inflammatory activation such as VCAM-1 and ICAM-1 did not occur or only to limited extent. While further propagation up to 31 CPDL proceeded at a comparable rate, a marked upregulation of inflammatory markers occurred in all donors accompanied by a further increase of uPA/uPAR gene induction. The observed induction of inflammatory genes at later stages of long-term propagation of PB-ECFCs underpins the necessity to determine the right time-point for harvesting of sufficient number of cells with preserved therapeutical potential.
  
  
  CONCLUSIONS
  The presented isolation method and subsequent cell expansion in platelet lysate supplemented culture medium permits suitable large-scale propagation of PB-ECFC. For optimal use of PB-ECFCs in clinical settings, our data suggest that 15-20 CPDL is the most adequate maturation stage.
authors.affiliation: Dept. of Physiology, ICaR-VU, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Dimitar
authors.initials: D
authors.lastname: Tasev
authors.affiliation: Dept. of Physiology, ICaR-VU, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Michiel H
authors.initials: MH
authors.lastname: van Wijhe
authors.affiliation: Dept. of Physiology, ICaR-VU, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Ester M
authors.initials: EM
authors.lastname: Weijers
authors.affiliation: Dept. of Physiology, ICaR-VU, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Victor W M
authors.initials: VW
authors.lastname: van Hinsbergh
authors.affiliation: Dept. of Physiology, ICaR-VU, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Pieter
authors.initials: P
authors.lastname: Koolwijk
mesh_terms: D000328:Adult; D015415:Biomarkers; D001792:Blood Platelets / Q000378:metabolism*; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D000066673:Cell Self Renewal; D066026:Endothelial Progenitor Cells / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D005260:Female; D005337:Fibrin / Q000378:metabolism; D006801:Humans; D016130:Immunophenotyping; D018836:Inflammation Mediators / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology*; D008077:Lipoproteins, LDL / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D018919:Neovascularization, Physiologic; D010641:Phenotype; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism; D055293:Receptors, Urokinase Plasminogen Activator / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism; D055815:Young Adult
chemical_list: D015415:Biomarkers; D018836:Inflammation Mediators; D036341:Intercellular Signaling Peptides and Proteins; D008077:Lipoproteins, LDL; D017395:Plasminogen Activator Inhibitor 1; D055293:Receptors, Urokinase Plasminogen Activator; D005337:Fibrin; D014568:Urokinase-Type Plasminogen Activator