Isolation and characterization of human osteoblasts from needle biopsies without in vitro culture

K. Fujita, M. M. Roforth, E. J. Atkinson, J. M. Peterson, Matthew M Drake, L. K. McCready, Joshua Farr, David G Monroe, Sundeep Khosla

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

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. Introduction: 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.

Original languageEnglish (US)
Pages (from-to)887-895
Number of pages9
JournalOsteoporosis International
Volume25
Issue number3
DOIs
StatePublished - Mar 2014

Fingerprint

Needle Biopsy
Osteoblasts
Alkaline Phosphatase
Bone and Bones
Genes
Osteoporosis
In Vitro Techniques
Fibroblasts
Population
High-Throughput Nucleotide Sequencing
Collagenases
Transcriptome
Endothelial Cells
Gene Expression

Keywords

  • Bone biopsy
  • Osteoblasts
  • Osteoporosis

ASJC Scopus subject areas

  • Endocrinology, Diabetes and Metabolism

Cite this

Isolation and characterization of human osteoblasts from needle biopsies without in vitro culture. / Fujita, K.; Roforth, M. M.; Atkinson, E. J.; Peterson, J. M.; Drake, Matthew M; McCready, L. K.; Farr, Joshua; Monroe, David G; Khosla, Sundeep.

In: Osteoporosis International, Vol. 25, No. 3, 03.2014, p. 887-895.

Research output: Contribution to journalArticle

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