Differentiation of functional osteoclasts from human peripheral blood CD14+ monocytes

Riedlova, P. , Sood, S., Goodyear, C. S. and Ansalone, C. (2023) Differentiation of functional osteoclasts from human peripheral blood CD14+ monocytes. Journal of Visualized Experiments(191), e64698. (doi: 10.3791/64698) (PMID:36779608)

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Osteoclasts (OCs) are bone-resorbing cells that play a pivotal role in skeletal development and adult bone remodeling. Several bone disorders are caused by increased differentiation and activation of OCs, so the inhibition of this pathobiology is a key therapeutic principle.Two key factors drive the differentiation of OCs from myeloid precursors: macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL). Human circulating CD14+ monocytes have long been known to differentiate into OCs in vitro. However, the exposure time and the concentration of RANKL influence the differentiation efficiency. Indeed, protocols for the generation of human OCs in vitro have been described, but they often result in a poor and lengthy differentiation process. Herein, a robust and standardized protocol for generating functionally active mature human OCs in a timely manner is provided. CD14+ monocytes are enriched from human peripheral blood mononuclear cells (PBMCs) and primed with M-CSF to upregulate RANK. Subsequent exposure to RANKL generates OCs in a dose- and time-dependent manner. OCs are identified and quantified by staining with tartrate acid-resistant phosphatase (TRAP) and light microscopy analysis. Immunofluorescence staining of nuclei and F-actin is used to identify functionally active OCs. In addition, OSCAR+CD14− mature OCs are further enriched via flow cytometry cell sorting, and OC functionality quantified by mineral (or dentine/bone) resorption assays and actin ring formation. Finally, a known OC inhibitor, rotenone, is used on mature OCs, demonstrating that adenosine triphosphate (ATP) production is essential for actin ring integrity and OC function. In conclusion, a robust assay for differentiating high numbers of OCs is established in this work, which in combination with actin ring staining and an ATP assay provides a useful in vitro model to evaluate OC function and to screen for novel therapeutic compounds that can modulate the differentiation process.

Item Type:Articles
Glasgow Author(s) Enlighten ID:Riedlova, Ms Patricia and Sood, Dr Shatakshi and Ansalone, Dr Cecilia and Goodyear, Professor Carl
Authors: Riedlova, P., Sood, S., Goodyear, C. S., and Ansalone, C.
College/School:College of Medical Veterinary and Life Sciences > School of Infection & Immunity
Research Centre:College of Medical Veterinary and Life Sciences > School of Infection & Immunity > Centre for Immunobiology
Journal Name:Journal of Visualized Experiments
Publisher:MyJove Corporation
ISSN (Online):1940-087X
Copyright Holders:Copyright © 2023 JoVE
First Published:First published in Journal of Visualized Experiments 191: e64698
Publisher Policy:Reproduced under a Creative Commons License

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