title: CD90 (Thy-1)-positive selection enhances osteogenic capacity of human adipose-derived stromal cells.
abstract: BACKGROUND
  Stem cell-based bone tissue engineering with adipose-derived stromal cells (ASCs) has shown great promise for revolutionizing treatment of large bone deficits. However, there is still a lack of consensus on cell surface markers identifying osteoprogenitors. Fluorescence-activated cell sorting has identified a subpopulation of CD105(low) cells with enhanced osteogenic differentiation. The purpose of the present study was to compare the ability of CD90 (Thy-1) to identify osteoprogenitors relative to CD(105).
  
  
  METHODS
  Unsorted cells, CD90(+), CD90(-), CD105(high), and CD105(low) cells were treated with an osteogenic differentiation medium. For evaluation of in vitro osteogenesis, alkaline phosphatase (ALP) staining and alizarin red staining were performed at 7 days and 14 days, respectively. RNA was harvested after 7 and 14 days of differentiation, and osteogenic gene expression was examined by quantitative real-time polymerase chain reaction. For evaluation of in vivo osteogenesis, critical-sized (4-mm) calvarial defects in nude mice were treated with the hydroxyapatite-poly(lactic-co-glycolic acid) scaffold seeded with the above-mentioned subpopulations. Healing was followed using micro-CT scans for 8 weeks. Calvaria were harvested at 8 weeks postoperatively, and sections were stained with Movat's Pentachrome.
  
  
  RESULTS
  Transcriptional analysis revealed that the CD90(+) subpopulation was enriched for a more osteogenic subtype relative to the CD105(low) subpopulation. Staining at day 7 for ALP was greatest in the CD90(+) cells, followed by the CD105(low) cells. Staining at day 14 for alizarin red demonstrated the greatest amount of mineralized extracellular matrix in the CD90(+) cells, again followed by the CD105(low) cells. Quantification of in vivo healing at 2, 4, 6, and 8weeks postoperatively demonstrated increased bone formation in defects treated with CD90(+) ASCs relative to all other groups. On Movat's Pentachrome-stained sections, defects treated with CD90(+) cells showed the most robust bony regeneration. Defects treated with CD90(-) cells, CD105(high) cells, and CD105(low) cells demonstrated some bone formation, but to a lesser degree when compared with the CD90(+) group.
  
  
  CONCLUSIONS
  While CD105(low) cells have previously been shown to possess an enhanced osteogenic potential, we found that CD90(+) cells are more capable of forming bone both in vitro and in vivo. These data therefore suggest that CD90 may be a more effective marker than CD105 to isolate a highly osteogenic subpopulation for bone tissue engineering.
authors.affiliation: Hagey Laboratory for Pediatric Regenerative Medicine, Plastic and Reconstructive Surgery Division, Department of Surgery, Stanford University School of Medicine, Stanford, CA 94305-5148, USA.
authors.forename: Michael T
authors.initials: MT
authors.lastname: Chung
authors.forename: Chunjun
authors.initials: C
authors.lastname: Liu
authors.forename: Jeong S
authors.initials: JS
authors.lastname: Hyun
authors.forename: David D
authors.initials: DD
authors.lastname: Lo
authors.forename: Daniel T
authors.initials: DT
authors.lastname: Montoro
authors.forename: Masakazu
authors.initials: M
authors.lastname: Hasegawa
authors.forename: Shuli
authors.initials: S
authors.lastname: Li
authors.forename: Michael
authors.initials: M
authors.lastname: Sorkin
authors.forename: Robert
authors.initials: R
authors.lastname: Rennert
authors.forename: Michael
authors.initials: M
authors.lastname: Keeney
authors.forename: Fan
authors.initials: F
authors.lastname: Yang
authors.forename: Natalina
authors.initials: N
authors.lastname: Quarto
authors.forename: Michael T
authors.initials: MT
authors.lastname: Longaker
authors.forename: Derrick C
authors.initials: DC
authors.lastname: Wan
mesh_terms: D000273:Adipose Tissue / Q000166:cytology*; D000328:Adult; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002454:Cell Differentiation; D048429:Cell Size; D005260:Female; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D008875:Middle Aged; D010012:Osteogenesis; D012886:Skull / Q000473:pathology; D013194:Staining and Labeling; D017154:Stromal Cells / Q000166:cytology / Q000201:enzymology; D018800:Thy-1 Antigens / Q000378:metabolism*
chemical_list: D018800:Thy-1 Antigens; D000469:Alkaline Phosphatase