title: Isolation and characterization of human osteoblasts from needle biopsies without in vitro culture.
abstract: CONCLUSIONS
  We isolate and characterize osteoblasts from humans without in vitro culture. These techniques should be broadly applicable to studying the pathogenesis of osteoporosis and other bone disorders.
  
  
  BACKGROUND
  There is currently no data regarding the expression of specific genes or pathways in human osteoblasts that have not been subjected to extensive in vitro culture. Thus, we developed methods to rapidly isolate progressively enriched osteoblast populations from humans and characterized these cells.
  
  
  METHODS
  Needle bone biopsies of the posterior iliac crest were subjected to sequential collagenase digests. The cells from the second digest were stained with an alkaline phosphatase (AP) antibody, and the AP+ cells were isolated using magnetic cell sorting.
  
  
  RESULTS
  Relative to AP- cells, the AP+ cells contained virtually all of the mineralizing cells and were enriched for key osteoblast marker genes. The AP+ cells were further purified by depletion of cells expressing CD45, CD34, or CD31 (AP+/CD45/34/31- cells), which represented a highly enriched human osteoblast population devoid of hematopoietic/endothelial cells. These cells expressed osteoblast marker genes but very low to undetectable levels of SOST. We next used high-throughput RNA sequencing to compare the transcriptome of the AP+/CD45/34/31- cells to human fibroblasts and identified genes and pathways expressed only in human osteoblasts in vivo, but not in fibroblasts, including 448 genes unique to human osteoblasts.
  
  
  CONCLUSIONS
  We provide a detailed characterization of highly enriched human osteoblast populations without in vitro culture. These techniques should be broadly applicable to studying the pathogenesis of osteoporosis and other bone disorders.
authors.affiliation: College of Medicine, Mayo Clinic, Rochester, MN, USA.
authors.forename: K
authors.initials: K
authors.lastname: Fujita
authors.forename: M M
authors.initials: MM
authors.lastname: Roforth
authors.forename: E J
authors.initials: EJ
authors.lastname: Atkinson
authors.forename: J M
authors.initials: JM
authors.lastname: Peterson
authors.forename: M T
authors.initials: MT
authors.lastname: Drake
authors.forename: L K
authors.initials: LK
authors.lastname: McCready
authors.forename: J N
authors.initials: JN
authors.lastname: Farr
authors.forename: D G
authors.initials: DG
authors.lastname: Monroe
authors.forename: S
authors.initials: S
authors.lastname: Khosla
mesh_terms: D000328:Adult; D000368:Aged; D000469:Alkaline Phosphatase / Q000378:metabolism; D001707:Biopsy, Needle / Q000379:methods; D002469:Cell Separation / Q000379:methods; D005786:Gene Expression Regulation; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D007085:Ilium / Q000473:pathology; D008297:Male; D008875:Middle Aged; D010006:Osteoblasts / Q000378:metabolism / Q000473:pathology*; D010024:Osteoporosis / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D055114:X-Ray Microtomography / Q000379:methods; D055815:Young Adult
chemical_list: D000469:Alkaline Phosphatase