Characterization of osteoclasts derived from CD14+ monocytes isolated from peripheral blood

Abstract

Bone resorption is solely mediated by osteoclasts. Therefore, a pure osteoclast population is of high interest for the investigation of biological aspects of the osteoclasts, such as the direct effect of growth factors and hormones, as well as for testing and characterizing inhibitors of bone resorption. We have established a pure, stable, and reproducible system for purification of human osteoclasts from peripheral blood. We isolated CD14-positive (CD14+) monocytes using anti-CD14-coated beads. After isolation, the monocytes are differentiated into mature osteoclasts by stimulation with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappaB ligand (RANKL). Osteoclast formation was only observed in the CD14+ population, not in the CD14- population, and only in the presence of both M-CSF and RANKL, confirming that the CD14+ system is a pure population of osteoclast precursors. No expression of osteoclast markers was observed in the absence of RANKL, whereas RANKL dose-dependently induced the expression of cathepsin K, tartrate-resistant acid phosphatase (TRACP), and matrix metallo proteinase (MMP)-9. Furthermore, morphological characterization of the cells demonstrated that actin rings were only formed in the presence of RANKL. Moreover, the osteoclasts were capable of forming acidic resorption lacunae, and inhibitors of lysosomal acidification attenuated this process. Finally, we measured the response to known bone resorption inhibitors, and found that the osteoclasts were sensitive to these and thereby provided a robust and valid method for interpretation of the effect of antiresorptive compounds. In conclusion, we have established a robust assay for developing osteoclasts that can be used to study several biological aspects of the osteoclasts and which in combination with the resorption marker CTX-I provides a useful tool for evaluating osteoclast function in vitro.

A 3D in vitro bone organ model using human progenitor cells

“…In the present study, we aimed at the further development of the model as a bone organotypic culture by introducing an osteoclastic cell source, namely human peripheral blood-derived monocytes (Komano et al, 2006;Sorensen et al, 2007). The multi-cell co-culture system was assessed for the presence and functionality of its different cellular components.…”

Directed differentiation of hematopoietic precursors and functional osteoclasts from human ES and iPS cells

“…Although it is known that osteoclast precursors reside in the CD14 ϩ monocyte fraction from PBMCs, high donor variability, sample heterogeneity, low precursor frequency, and unpredictable responsiveness to different batches of serum for in vitro culture have provided the biggest obstacles to establishing defined, efficient, and reproducible systems for studying osteoclast differentiation and for screening novel antiresorptive compounds. [8][9][10] Moreover, the currently available approaches for studying osteoclasts are not suitable for studying the ontogeny of osteoclasts during human embryonic development or for understanding the pathophysiology of genetic diseases affecting osteoclasts or changes in bone metabolism during aging.…”

“…Although it is known that osteoclast precursors reside in the CD14 ϩ monocyte fraction from PBMCs, high donor variability, sample heterogeneity, low precursor frequency, and unpredictable responsiveness to different batches of serum for in vitro culture have provided the biggest obstacles to establishing defined, efficient, and reproducible systems for studying osteoclast differentiation and for screening novel antiresorptive compounds. [8][9][10] Moreover, the currently available approaches for studying osteoclasts are not suitable for studying the ontogeny of osteoclasts during human embryonic development or for understanding the pathophysiology of genetic diseases affecting osteoclasts or changes in bone metabolism during aging.The differentiation of both murine and human embryonic stem cells (ESCs) into multiple hematopoietic lineages is now well established as a powerful tool for studying early embryonic hematopoiesis and lineage restriction and for generating unlimited numbers of cell populations for transplantation and in vitro study. [11][12][13][14][15][16] The exciting recent discoveries in generating induced pluripotent stem cells (iPSCs) by reprogramming somatic cells to an ESC-like state also has enabled further understanding of the ontogeny of specific embryonic lineages and has opened the door to generating patient-specific stem cells.…”

Induction of osteoclast progenitors in inflammatory conditions: key to bone destruction in arthritis

“…Several monocyte markers were used, such as CD11b, CD14, CD16 or CD51/61, to purify human peripheral OCPs and test their osteoclastogenic potential in vitro, upon treatment with cytokines (including RANKL, M-CSF, TNF-α, IL-6, IL-17 and IL-32), synovial fibroblasts or synovial fluid from arthritic patients [47][48][49][50][51][52][53][54][55][56][57]. The osteoclast forming capacity of CD14 + cells has been observed to be higher compared with CD11b + or CD61 + cells [53], and to correspond to the expression level of CD16 and DC-STAMP (dendritic cell-specific transmembrane protein) [54].…”