title: Effects of different serum conditions on osteogenic differentiation of human adipose stem cells in vitro.
abstract: BACKGROUND
  Currently, human adipose stem cells (hASCs) are differentiated towards osteogenic lineages using culture medium supplemented with L-ascorbic acid 2-phosphate (AsA2-P), dexamethasone (Dex) and beta-glycerophosphate (β-GP). Because this osteogenic medium (OM1) was initially generated for the differentiation of bone marrow-derived mesenchymal stem cells, the component concentrations may not be optimal for the differentiation of hASCs. After preliminary screening, two efficient osteogenic media (OM2 and OM3) were chosen to be compared with the commonly used osteogenic medium (OM1). To further develop the culture conditions towards clinical usage, the osteo-inductive efficiencies of OM1, OM2 and OM3 were compared using human serum (HS)-based medium and a defined, xeno-free medium (RegES), with fetal bovine serum (FBS)-based medium serving as a control.
  
  
  METHODS
  To compare the osteo-inductive efficiency of OM1, OM2 and OM3 in FBS-, HS- and RegES-based medium, the osteogenic differentiation was assessed by alkaline phosphatase (ALP) activity, mineralization, and expression of osteogenic marker genes (runx2A, DLX5, collagen type I, osteocalcin, and ALP).
  
  
  RESULTS
  In HS-based medium, the ALP activity increased significantly by OM3, and mineralization was enhanced by both OM2 and OM3, which have high AsA2-P and low Dex concentrations. ALP activity and mineralization of hASCs was the weakest in FBS-based medium, with no significant differences between the OM compositions due to donor variation. However, the qRT-PCR data demonstrated significant upregulation of runx2A mRNA under osteogenic differentiation in FBS- and HS-based medium, particularly by OM3 under FBS conditions. Further, the expression of DLX5 was greatly stimulated by OM1 to 3 on day 7 when compared to control. The regulation of collagen type I, ALP, and osteocalcin mRNA was modest under induction by OM1 to 3. The RegES medium was found to support the proliferation and osteogenic differentiation of hASCs, but the composition of the RegES medium hindered the comparison of OM1, OM2 and OM3.
  
  
  CONCLUSIONS
  Serum conditions affect hASC proliferation and differentiation significantly. The ALP activity and mineralization was the weakest in FBS-based medium, although osteogenic markers were upregulated on mRNA level. When comparing the OM composition, the commonly used OM1 was least effective. Accordingly, higher concentration of AsA2-P and lower concentration of Dex, as in OM2 and OM3, should be used for the osteogenic differentiation of hASCs in vitro.
authors: Laura
authors: L
authors: Kyllönen
authors: Suvi
authors: S
authors: Haimi
authors: Bettina
authors: B
authors: Mannerström
authors: Heini
authors: H
authors: Huhtala
authors: Kristiina M
authors: KM
authors: Rajala
authors: Heli
authors: H
authors: Skottman
authors: George K
authors: GK
authors: Sándor
authors: Susanna
authors: S
authors: Miettinen
mesh_terms: D017667:Adipocytes / Q000378:metabolism / Q000502:physiology*; D000273:Adipose Tissue / Q000378:metabolism / Q000502:physiology*; D000469:Alkaline Phosphatase / Q000378:metabolism; D002454:Cell Differentiation / Q000502:physiology*; D049109:Cell Proliferation / Q000502:physiology; D002478:Cells, Cultured; D024042:Collagen Type I / Q000378:metabolism; D050659:Core Binding Factor Alpha 1 Subunit / Q000378:metabolism; D003470:Culture Media / Q000378:metabolism; D005260:Female; D018398:Homeodomain Proteins / Q000378:metabolism; D006801:Humans; D008875:Middle Aged; D015675:Osteocalcin / Q000378:metabolism; D010012:Osteogenesis / Q000502:physiology*; D044967:Serum / Q000378:metabolism*; D013234:Stem Cells / Q000378:metabolism / Q000502:physiology*; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D024042:Collagen Type I; D050659:Core Binding Factor Alpha 1 Subunit; D003470:Culture Media; C100596:DLX5 protein, human; D018398:Homeodomain Proteins; C493709:RUNX2 protein, human; D014157:Transcription Factors; D015675:Osteocalcin; D000469:Alkaline Phosphatase