Published in:
01-03-2014 | Original Article
Isolation and characterization of human osteoblasts from needle biopsies without in vitro culture
Published in: Osteoporosis International | Issue 3/2014
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Summary
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.