title: High throughput, quantitative analysis of human osteoclast differentiation and activity.
abstract: Osteoclasts are multinuclear cells that degrade bone under both physiological and pathophysiological conditions. Osteoclasts are therefore a major target of osteoporosis therapeutics aimed at preserving bone. Consequently, analytical methods for osteoclast activity are useful for the development of novel biomarkers and/or pharmacological agents for the treatment of osteoporosis. The nucleation state of an osteoclast is indicative of its maturation and activity. To date, activity is routinely measured at the population level with only approximate consideration of the nucleation state (an 'osteoclast population' is typically defined as cells with ≥3 nuclei). Using a fluorescent substrate for tartrate-resistant acid phosphatase (TRAP), a routinely used marker of osteoclast activity, we developed a multi-labelled imaging method for quantitative measurement of osteoclast TRAP activity at the single cell level. Automated image analysis enables interrogation of large osteoclast populations in a high throughput manner using open source software. Using this methodology, we investigated the effects of receptor activator of nuclear factor kappa-B ligand (RANK-L) on osteoclast maturation and activity and demonstrated that TRAP activity directly correlates with osteoclast maturity (i.e. nuclei number). This method can be applied to high throughput screening of osteoclast-targeting compounds to determine changes in maturation and activity.
authors.affiliation: Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052, Australia.
authors.forename: Natalie A
authors.initials: NA
authors.lastname: Diepenhorst
authors.affiliation: Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052, Australia.
authors.forename: Cameron J
authors.initials: CJ
authors.lastname: Nowell
authors.affiliation: Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052, Australia.
authors.forename: Patricia
authors.initials: P
authors.lastname: Rueda
authors.affiliation: Biomarkers & Research, Nordic Bioscience, Herlev, Denmark.
authors.forename: Kim
authors.initials: K
authors.lastname: Henriksen
authors.affiliation: Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052, Australia.
authors.forename: Tracie
authors.initials: T
authors.lastname: Pierce
authors.affiliation: Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052, Australia.
authors.forename: Anna E
authors.initials: AE
authors.lastname: Cook
authors.affiliation: Therapeutic Innovation Pole of Rheumatology, Institut de Recherches Servier, Suresnes, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Pastoureau
authors.affiliation: Therapeutic Innovation Pole of Rheumatology, Institut de Recherches Servier, Suresnes, France.
authors.forename: Massimo
authors.initials: M
authors.lastname: Sabatini
authors.affiliation: Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052, Australia.
authors.forename: William N
authors.initials: WN
authors.lastname: Charman
authors.affiliation: Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052, Australia.
authors.forename: Arthur
authors.initials: A
authors.lastname: Christopoulos
authors.affiliation: Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052, Australia.
authors.forename: Roger J
authors.initials: RJ
authors.lastname: Summers
authors.affiliation: Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052, Australia.
authors.forename: Patrick M
authors.initials: PM
authors.lastname: Sexton
authors.affiliation: Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052, Australia. Electronic address: chris.langmead@monash.edu.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Langmead
mesh_terms: D002454:Cell Differentiation; D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods; D010010:Osteoclasts / Q000166:cytology* / Q000378:metabolism*; D053245:RANK Ligand / Q000378:metabolism*; D059010:Single-Cell Analysis / Q000379:methods*; D000071681:Tartrate-Resistant Acid Phosphatase / Q000378:metabolism*
chemical_list: D053245:RANK Ligand; C505688:TNFSF11 protein, human; D000071681:Tartrate-Resistant Acid Phosphatase
keywords: Bone resorption; High throughput; ImageJ; Osteoclast; Quantitative image analysis; Tartrate-resistant acid phosphatase (TRAP)