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

Koji Fujita, Matthew Roforth, James Peterson, Salman Kirmani, Matthew Drake, Louise McCready, David Monroe, Sundeep Khosla

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There is currently no data regarding the expression of specific genes or pathways in purified human osteoblasts that have not been subjected to extensive in vitro culture. Since the latter is likely to alter the RNA expression profile of these cells, we have developed methods to obtain progressively enriched human osteoblast populations within 2-4 hr of obtaining a small needle biopsy (1-2 mm x 1 cm) and coupled this to high-throughput RNA sequencing (RNAseq) and focused QPCR analyses. Needle biopsies were obtained from the posterior iliac crest of 8 human male subjects and subjected to serial collagenase digests. Because the 1st30 minute digest(~100 million cells) contained surface hematopoietic and loosely adherent cells, we used the 2nd60 minute digest (~10 million cells), which contained mineralizing cells and was highly enriched for osteoblast marker genes (col1a1, 5-fold; osteocalcin [ocn],8-fold;bsp, 11-fold; osteopontin [opn], 10-fold). The 2ndfraction was then stained with an AP antibody and the AP+ cells (~1 million cells) were rapidly isolated (within ~2 hr from biopsy) using magnetic activated cell sorting. Relative to AP- cells, the AP+ cells contained all of the mineralizing cells and were further enriched for osteoblast marker genes (AP, 10-fold;col1a1, 1000-fold;ocn, 300-fold;bsp, 300-fold;opn, 50-fold). We then further purified the AP+ cells by depletion of cells expressing CD45, CD34, orCD31 by FACS to obtain AP+/CD45/34/31- (AP+/—) cells (within ~4 hr from biopsy), which represented a highly enriched human osteoblast preparation devoid of hematopoietic/endothelial cells. In addition to in vitro mineralization, AP+/— cells expressed very low to undetectable levels of osteocyte marker genes, including E11,dmp-1, phex, fgf23,andsost.Finally, we used high-throughput RNAseq analysis to compare the transcriptome of the AP+/— cells (n = 6) to human fibroblasts (n = 3). By Ingenuity PathwayAnalysis, we identified a set of unique pathways reflecting genes expressed only inhuman osteoblasts (AP+/— cells)in vivo but not in fibroblasts, including (all P,0.05)Osteoblast Related, VDR/RXR Activation, LXR/RXR Activation, NF-kB Signaling,CXCR4 Signaling, and GPCR Signaling.In summary, we describe a novel approach to isolate and interrogate highly enriched human osteoblast populations without in vitro culture which should bebroadly applicable to studying the pathogenesis of osteoporosis and other metabolic bone diseases.

Original languageUndefined/Unknown
JournalDepartment of Paediatrics and Child Health
Publication statusPublished - 1 Feb 2013

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