title: Isolation method and xeno-free culture conditions influence multipotent differentiation capacity of human Wharton's jelly-derived mesenchymal stem cells.
abstract: BACKGROUND
  Human Wharton's jelly (WJ) has become a preferred source of mesenchymal stem cells (MSCs) whose clinical applications are limited by the use of adequate xeno-free (XF), in vitro manipulation conditions. Therefore, the objective of our study was to characterize WJ-derived MSCs (WJ-MSCs), isolated by different methods and cultured in a commercially available, MSC XF medium, not least of all by investigating their endothelial differentiation capacity.
  
  
  METHODS
  WJ explants and enzymatically dissociated WJ cells were cultured in a defined, XF medium for MSCs. Adherent cells at passages 2 and 5 were characterized as MSCs by flow cytometry, MTT, real-time quantitative reverse transcription PCR, and functional multipotent differentiation assays. The endothelial differentiation capacity of MSCs isolated and expanded until passage 2 in the MSC XF medium, and then subcultured for five passages in a commercially available endothelial growth medium (group A), was assessed over serial passages, as compared to adherent WJ-derived cells isolated and expanded for five consecutive passages in the endothelial medium (group B).
  
  
  RESULTS
  The MSC phenotype of WJ explant- and pellet-derived cells, isolated and expanded in the MSC XF medium, was proven based on the expression of CD44/CD73/CD90/CD105 surface markers and osteo-/adipo-/chondrogenic multipotent differentiation potential, which differed according to the isolation method and/or passage number. Upon exposure to endothelial differentiation cues, cells belonging to group A did not exhibit endothelial cell characteristics over serial passages; by contrast, WJ pellet-derived cells belonging to group B expressed endothelial characteristics at gene, protein and functional levels, potentially due to culture conditions favoring the isolation of other stem/progenitor cell types than MSCs, able to give rise to an endothelial progeny.
  
  
  CONCLUSIONS
  The use of defined, MSC XF media for isolation and expansion of human WJ-MSCs is a prerequisite for the establishment of their real endothelial differentiation capacity, as candidates for clinical therapy applications. Thus, the standardization of WJ-MSCs isolation and culture expansion techniques in defined, MSC XF media, for their accurate characterization, would be a priority in the stem cell research field.
authors.forename: Maria Cristina
authors.initials: MC
authors.lastname: Corotchi
authors.forename: Mirel Adrian
authors.initials: MA
authors.lastname: Popa
authors.forename: Anca
authors.initials: A
authors.lastname: Remes
authors.forename: Livia Elena
authors.initials: LE
authors.lastname: Sima
authors.forename: Ilinca
authors.initials: I
authors.lastname: Gussi
authors.forename: Marilena
authors.initials: M
authors.lastname: Lupu Plesu
mesh_terms: D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D049109:Cell Proliferation; D042783:Endothelial Cells / Q000378:metabolism*; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000378:metabolism*; D059467:Transcriptome; D014470:Umbilical Cord / Q000166:cytology*