title: Gene expression-based enrichment of live cells from adipose tissue produces subpopulations with improved osteogenic potential.
abstract: BACKGROUND
  Mesenchymal stem cells have been increasingly used for cell-based therapies. Adipose-derived stem/stromal cells (ASCs) from the stromal vascular fraction (SVF) of fat tissue are a particularly attractive option for cell based therapy given their accessibility and relative abundance. However, their application in both clinical and basic science investigations is complicated by the isolation of differentiable cells within the SVF. Current enrichment strategies, such as monolayer passaging and surface marker-based sorting, can be time-consuming or overly stringent. Ideally, a population of cells with great regenerative capacity could be isolated with high yields so that extensive in vitro manipulation is not necessary. The objective of this study was to determine whether SVF cells sorted based on expression of alkaline phosphatase liver/bone/kidney (ALPL) resulted in populations with increased osteogenic differentiation potential.
  
  
  METHODS
  SVF samples were obtained from four, human donors and processed to isolate initial, heterogeneous cell populations. These SVF cells underwent a four day osteogenic priming period, after which they were treated with a fluorescent, oligodeoxynucleotide molecular beacon probe specific for ALPL mRNA. Cells were separated into positive and negative groups using fluorescence-activated cell sorting (FACS) then differentiated down the osteogenic lineage. Differentiation was assessed by measuring calcified matrix production in each sample.
  
  
  RESULTS
  Cells positive for ALPL expression (ALPL+) represented approximately 34% of the gated population, while cells negative for ALPL expression (ALPL-) represented approximately 18%. ALPL+ cells produced 3.7-fold and 2.1-fold more calcified matrix than ALPL- and unsorted SVF cells, respectively, indicating a significant improvement in osteogenic differentiation. Further, ALPL+ cells showed increases in metabolite production for both adipogenesis and chondrogenesis, suggesting that the enrichment process yields an enhanced multipotent phenotype. Osteogenic differentiation response and cell yields for ALPL+ cells were markedly improved over surface marker-sorted samples.
  
  
  CONCLUSIONS
  This study demonstrates a novel method to enrich heterogeneous SVF cells for increased osteogenic potential. The procedure requires less time and results in higher yields of therapeutically useful cells than other existing approaches. Gene expression-based sorting of MSCs is a potentially paradigm-shifting approach that could benefit applications spanning from basic science to clinical therapy.
authors.forename: Hetal D
authors.initials: HD
authors.lastname: Marble
authors.forename: Bryan A
authors.initials: BA
authors.lastname: Sutermaster
authors.forename: Manisha
authors.initials: M
authors.lastname: Kanthilal
authors.forename: Vera C
authors.initials: VC
authors.lastname: Fonseca
authors.forename: Eric M
authors.initials: EM
authors.lastname: Darling
mesh_terms: D000273:Adipose Tissue / Q000166:cytology* / Q000201:enzymology / Q000502:physiology*; D000469:Alkaline Phosphatase / Q000096:biosynthesis / Q000235:genetics; D002454:Cell Differentiation; D002469:Cell Separation / Q000379:methods; D005260:Female; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000201:enzymology / Q000502:physiology*; D010012:Osteogenesis / Q000235:genetics*
chemical_list: D000469:Alkaline Phosphatase