Name: differentiation of functional osteoclasts from human peripheral blood cd14+ monocytes
Uploaded: 2023-01-27T13:15:00
Duration: 11 min 52 s
Description: Watch this Scientific Journal Video about Differentiation of Functional Osteoclasts from Human Peripheral Blood CD14+ Monocytes at JoVE.com

The authors gratefully acknowledge the Flow Core Facility and the Glasgow Imaging Facility (GIF) within the School of Infection and Immunity for their support and assistance in this work.

The authors declare that they have no competing interests.

The easy culture and isolation of large numbers of functional OCs in vitro are important for advancing the understanding of bone biology and OC-mediated diseases. Classically, OCs were generated in co-cultures with osteoblasts or stromal cells and hematopoietic cells from the spleen or bone marrow38,39. A significant breakthrough in the understanding of osteoclastogenesis was the identification of RANKL as the major regulator of OC formation, differentiation, and survival40. Early protocols of RANKL-dependent culture systems utilized PBMCs for OC generation21,41,42. However, these mixed cultures are lengthy and present many confounding factors that limit the ability to test the direct effects on OC differentiation and function. This protocol describes an efficient and reliable in vitro model of osteoclastogenesis from human peripheral CD14+ monocytes in which optimal osteoclastogenesis can be obtained within 7 days (Figure 1 and Figure 2), which is considerably faster when compared to some other protocols43,44,45,46. The main distinguishing features of this protocol are (1) the use of purified CD14+ monocytes, (2) the priming of the monocytes with M-CSF prior to exposure to RANKL, (3) the length of the culture (&#60;7 days), and (4) the reliable detection of the inhibition of OC formation (TRAP staining) and function (resorption, ATP production, actin ring reorganization) with inhibitors. 
During the optimization of the methodology, several critical points were identified. It has been observed that the in vitro differentiation of OCs is largely dependent on the seeding density of the CD14+ monocytes. Thus, in this protocol, the cells are seeded at a high density (1 x 105 cells/well of a 96-well plate, in 100 &#181;L of medium), as it is essential for the cells to be able to interact with each other and to be in proximity to fuse and become mature OCs. Similarly, seeding cells at a density that is too high limits their differentiation and growth due to medium limitations and a lack of the required space. Furthermore, to achieve maximum success with this protocol, it is important to perform the density gradient separation carefully and to ensure that the enriched population of CD14+ cells is as pure as possible. For example, inadequate washing steps result in a lack of removal of platelets, which consequently inhibits OC differentiation47,48. Similarly, the presence of minor T cell contamination in isolated CD14+ preparations stimulated with M-CSF alone can result in OC differentiation, potentially via RANKL secretion by T cells49. Therefore, it is important to include an M-CSF control for every experiment. A routine purity check, especially when using a new isolation kit, is also recommended to ensure the purity of the sample.
Optimum OC numbers (range: ~200-1,600 OCs/well) are achieved using &#945;-MEM medium enriched with nucleosides and L-glutamine. Other conventional culture media, including Dulbecco&#39;s modified eagle medium (DMEM) and Roswell Park Memorial Institute (RPMI) 1640 medium, affect the OC yield. The source of FBS can also influence osteoclastogenesis. Different batches of FBS can lead to reduced RANK-L-derived osteoclastogenesis, as well as the appearance of low numbers of TRAP+ multinucleated cells in the M-CSF controls (Supplementary Figure 3). Therefore, to achieve consistent results, it is advised to test new FBS batches before use and to continue with the same batch throughout the experiments to minimize variations in the differentiation process. Additionally, donor-to-donor variability, in terms of total numbers of differentiated OCs obtained at the end time point, constitutes a limitation when using this protocol to compare, for instance, healthy donors to patients. In these cases, it is imperative to use exactly the same conditions and the same lot of medium, FBS, and other reagents.
Another necessary step for optimum OC differentiation and maturation is priming the monocytes with M-CSF before the RANKL addition. The exposure of the cells to M-CSF 18-24 h prior to RANKL primes the monocytes to upregulate RANK expression15,26. The addition of RANKL at this time point ensures optimal OC differentiation in a dose-dependent manner. The degree of OC differentiation varies from donor to donor; however, 25 ng/mL RANKL is usually sufficient to differentiate a high number of OCs in most donors. Additionally, 25 ng/mL RANKL can be used in assays for the initial screening of compounds, as it facilitates the evaluation of both the enhancing and inhibitory effects of the test compounds. Other culture systems have used longer M-CSF pre-incubation times prior to RANKL addition, but this results in a longer culturing time for osteoclastogenesis50. In addition, leaving the primed monocytes to incubate overnight allows them to attach to the plate, albeit not in a fully adherent state. Therefore, when RANKL is introduced for the first time, the medium must be half-changed very carefully rather than completely changed to prevent the detachment and loss of the primed monocytes. The medium also needs to be refreshed every 3-4 days to avoid medium depletion and prevent cell death. Moreover, due to the low volume used in this assay (100 &#181;L/well in a 96-well plate), it is of the utmost importance to have a frame of empty wells that are filled with an aqueous solution (i.e., sterile distilled H2O or PBS) around the assay wells. This prevents medium evaporation and edge effects.
Finally, for metabolic assays (e.g., ATP assays), it is imperative that the cells are viable to avoid huge standard deviation between replicates (Figure 5). High viability of the cells is also important for sorting the cells and for the further culturing of the sorted OCs (Figure 4). This method, however, has several limitations. Fully mature OCs are very adherent and difficult to detach from the plates. The larger OCs are often impossible to detach, which can lead to a lower cell yield. Therefore, the cells need to be counted after sorting and prior to plating at the required concentration. Furthermore, in the present protocol, a non-enzymatic method (accutase) to detach the OCs is used to prevent membrane alterations in downstream surface staining for flow cytometry. The use of cell scrapers (both with soft or hard endings) was also tested and led to high cell death. Enzymatic detachment using 0.05% Trypsin/EDTA solutions can be used for a higher yield of detached OCs when membrane integrity is not required for downstream applications. Additionally, to prevent the OCs from clumping together, the use of a high concentration of EDTA in all the buffers following cell detachment, as well as appropriate filtering prior to flow cytometry acquisition, are highly recommended. It is important to note that OC cultures are a heterogeneous population of cells consisting of mature OCs, OC precursors, and macrophages. Macrophages can be easily distinguished from OCs, although both mononuclear pre-OCs and multinuclear OCs express OSCAR and cannot be distinguished with the present method (Figure 4). Indeed, this latter issue constitutes the main limitation of this method. In addition, a low expression of OSCAR is also present in M-CSF cultures (Figure 4B) and might indicate macrophages that are primed for OC lineage commitment. It is important to set the gate for OSCAR+ cells based on the FMO staining signal, as shown in Figure 4B.
In summary, this protocol describes an optimized and robust method for the efficient production of active and functionally mature OCs from circulating primary human monocytes. The strength of this protocol is its ability to generate OCs in a short time duration and yield high numbers of differentiated OCs. This method opens the way for investigating the basic mechanisms underlying OC differentiation and function.

Osteoclasts (OCs) are multinucleated giant cells of hematopoietic lineage with a unique capacity to resorb bone. They are responsible for the development and continuous remodeling of the skeleton1,2. In the skeletal phases of development, OCs and tissue-resident macrophages are derived from erythro-myeloid progenitors and colonize the bone niche and organ tissues. In physiological conditions, erythro-myeloid progenitors are required for normal bone development and tooth eruption, while the influx of circulating blood monocytes into the bone niche provides postnatal maintenance of the OCs, bone mass, and bone marrow cavity3. Under pathological conditions, monocytes are recruited to sites of active inflammation and can contribute to pathological bone destruction4,5.
Patients with several forms of arthritis experience joint inflammation, leading to progressive joint destruction caused by OCs6. For instance, in rheumatoid arthritis (RA), overactivated OCs are responsible for pathological bone erosion and joint destruction7,8, and current treatments often do not improve or stop the bone damage9,10,11. Alterations in circulating monocytes both in terms of population distribution and transcriptomic and epigenetic signatures have been reported in RA patients12,13,14. Moreover, it has been reported that altered monocyte responses to inflammatory stimulation affect osteoclastogenesis in RA patients with active disease15,16,17.
The differentiation of OCs is a complex multistep process comprising the commitment of myeloid precursor cells to differentiation into OC precursors. During osteoclastogenesis, OCs become giant and multinucleated through cell-cell fusion, incomplete cytokinesis, and a nuclear recycling process described as fission and fusion18,19,20. The ability to differentiate OCs in vitro has allowed for significant advances in the understanding of bone biology21. OCs differentiate from precursors upon exposure to macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL). The latter is essential for the normal development and function of OCs in vitro and in vivo, even under inflammatory conditions6,22,23. RANKL is presented by osteoblasts and osteocytes, as well as by activated T cells and fibroblasts in the inflamed RA synovium2,24,25. During the OC differentiation process, monocytes exposed to M-CSF upregulate receptor activator of nuclear factor kappa-B (RANK) expression on their cell membrane and, under subsequent stimulation with RANKL, differentiate into tartrate-resistant acid phosphatase (TRAP)-positive mononuclear pre-OCs and then into multinucleated OCs15,26. OCs produce several enzymes, the chief among them is TRAP, which enables the degradation of phosphoproteins within bone27. A regulator and marker of OC differentiation is the OC-associated receptor (OSCAR). It is upregulated early in precursor cells committing to the OC lineage28. Mature giant multinucleated OCs can degrade (resorb) the skeletal matrix by generating a large sealing zone, which is made of an actin ring surrounding a ruffled border21,29,30. The bone resorption capability of OCs requires cytoskeleton reorganization and the consequent polarization and formation of a convoluted membrane, which is the so-called ruffled border. The ruffled border is surrounded by a large circular band of an F-actin-rich structure, which is the actin ring or sealing zone. Actin ring integrity is essential for OCs to resorb bone both in vitro and in vivo, and defective ruffled border formation is associated with lower vacuolar adenosine triphosphatase (V-ATPase) expression31,32,33. Moreover, OCs are mitochondria-rich cells, and adenosine triphosphate (ATP) associates with mitochondrial-like structures in OCs localized at the ruffled border31,32,33. Rotenone acts as a strong inhibitor of the mitochondrial complex I and impacts ATP production. Rotenone has also been shown to inhibit OC differentiation and function34.
This protocol describes an efficient and optimized method of in vitro osteoclastogenesis&#160;from human peripheral blood samples. In human peripheral blood, CD14+ monocytes are the main source of OCs15,35,36. In this protocol, the kinetics of exposure and the concentrations of M-CSF and RANKL have been adjusted for optimum osteoclastogenesis. Mononuclear cells are first separated from the erythrocytes and granulocytes present in whole blood by&#160;density gradient; they are then enriched for CD14+ monocytes using positive selection by magnetic beads. The isolated CD14+ monocytes are then incubated overnight with M-CSF. This primes the monocytes to upregulate the expression of RANK15,26. The subsequent addition of RANKL induces osteoclastogenesis and multinucleation in a time-dependent manner. Active-resorbing OCs show the characteristic distribution of F-actin rings at the edge of the cell membrane30,32 and staining for TRAP. Mature OCs are analyzed by quantifying TRAP+ multinucleated (more than three nuclei) cells. The functional capacity of mature OCs can be assessed by their resorption, actin ring integrity, and ATP production. Furthermore, differentiated CD14&#8722; OSCAR+ OCs can be enriched and used to assess the effects of certain compounds on OC functionality via mineral (or dentine) resorption and F-actin organization. Additionally, in this work, a known OC inhibitor, rotenone, is used as an exemplar of a compound that affects the functionality of OCs. Reduced OC resorption activity under rotenone is associated with reduced ATP production and actin ring fragmentation. In conclusion, this protocol establishes a robust assay that can be used as a reference method to study several biological aspects of OC differentiation and function in vitro. 
This methodology can be used to assess (1) the potential of circulating monocytes to differentiate into OCs in health and diseases, as well as (2) the impact of therapeutic candidates on OC differentiation and function. This robust osteoclastogenesis protocol enables the determination of the efficacy and mechanisms of bone-targeted therapies on both OC differentiation from precursor cells and the function of mature OCs.

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			<td>&#181;-Slide 18 well chamber slides</td>
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			<td>8-well glass chamber slides&#160;</td>
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			<td>80807&#160;</td>
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			<td>96-well TC plate&#160;</td>
			<td>Corning&#160;</td>
			<td>3596&#160;</td>
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			<td>96-well osteo assay stripwell plate&#160;&#160;</td>
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			<td>3989&#160;</td>
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			<td>Acetate solution</td>
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			<td>386-3</td>
			<td>from kit Cat No. 387A-1KT</td>
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			<td>Acetone&#160;</td>
			<td>VWR&#160;</td>
			<td>20066.330&#160;</td>
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			<td>Acid phosphatase, Leukocyte (TRAP) kit&#160;</td>
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			<td>387A-1KT&#160;</td>
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			<td>Alexa Fluor 488 Phalloidin</td>
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			<td>A12379&#160;</td>
			<td>AF488&#160;&#160;&#160;</td>
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			<td>Alexa Fluor 647 Phalloidin</td>
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			<td>A22287</td>
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			<td>Alfa Aesar 2-Deoxy-D-glucose&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>11321867&#160;</td>
			<td>2DG, 98%&#160;</td>
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			<td>Alpha minimum essential medium&#160;</td>
			<td>gibco&#160;</td>
			<td>22571-020&#160;</td>
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			<td>ATPlite 1step&#160;</td>
			<td>PerkinElmer&#160;</td>
			<td>6016731&#160;</td>
			<td>Luminiscence ATP detection assay system&#160;</td>
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			<td>BD FACSAria III cell sorter</td>
			<td>BD Biosciences</td>
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			<td>Bovine serum albumin (BSA)&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>A9418-100G&#160;</td>
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			<td>Cell culture microplate, 96-well, PS, F-bottom&#160;</td>
			<td>Greiner bio-one&#160;</td>
			<td>655083&#160;</td>
			<td>White-bottom plates&#160;</td>
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			<td>Citrate solution</td>
			<td>Sigma Aldrich</td>
			<td>91-5</td>
			<td>from kit Cat No. 387A-1KT</td>
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			<td>Corning 6ml round-bottom polystyrene test tubes</td>
			<td>Fisher Scientific</td>
			<td>352054</td>
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			<td>Corning osteo assay surface multiple well plate</td>
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			<td>CLS3989</td>
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			<td>Corning osteo assay Surface multiple well plate 1 x 8 stripwell</td>
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			<td>EasySep human CD14 positive selection kit&#160;</td>
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			<td>EasySep red blood cell lysis buffer (10x)</td>
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			<td>eBioscience fixable viability dye eFluor 780</td>
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			<td>EVOS FL auto imaging system</td>
			<td>Thermo Fisher</td>
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			<td>Falcon round-bottom polypropylene test tubes with cap</td>
			<td>Fisher Scientific</td>
			<td>10314791</td>
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			<td>Falcon tubes 15 mL</td>
			<td>Corning&#160;</td>
			<td>430790&#160;</td>
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			<td>Falcon tubes 50 mL&#160;</td>
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			<td>430828&#160;</td>
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			<td>Fast Garnet GBC base solution</td>
			<td>Sigma Aldrich</td>
			<td>387-2</td>
			<td>from kit Cat No. 387A-1KT</td>
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			<td>Fetal bovine serum&#160;</td>
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			<td>Ficoll-Paque Plus&#160;</td>
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			<td>Formaldehyde&#160;</td>
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			<td>F-8775&#160;</td>
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			<td>Human sRANK ligand&#160;</td>
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			<td>Receptor activator of nuclear factor kappa-B ligand&#160;(RANKL)</td>
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			<td>ImageJ Image analysis software</td>
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			<td>L-glutamine&#160;</td>
			<td>gibco&#160;</td>
			<td>25030-024&#160;</td>
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			<td>Lithium heparin tubes (9 mL)&#160;</td>
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			<td>Macrophage colony-stimulating factor&#160;</td>
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			<td>Napthol AS-BI phosphoric acid solution</td>
			<td>Sigma Aldrich</td>
			<td>387-1</td>
			<td>from kit Cat No. 387A-1KT</td>
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			<td>Neubauer hemacytometer counting chamber&#160;</td>
			<td>Camlab</td>
			<td>SKU 1127885</td>
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			<td>Oligomycin from Streptomyces Diastatochromogenes&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>Q4876-5MG&#160;</td>
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			<td>OSCAR Antibody, anti-human, Vio Bright FITC, REAfinit</td>
			<td>Miltenyi Biotec</td>
			<td>130-107-661 and 130-107-617</td>
			<td>Clone REA494</td>
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			<td>PE/Cyanine7 anti-human CD14 antibody</td>
			<td>Biolegend</td>
			<td>325618</td>
			<td>Clone HCD14&#160;</td>
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			<td>Penicilin/streptomycin&#160;</td>
			<td>SIGMA&#160;</td>
			<td>P0781&#160;</td>
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			<td>PHERAstar machine and software</td>
			<td>BMG LABTECH</td>
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			<td></td>
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			<td>Phosphate-buffered saline (DPBS, 1x)&#160;</td>
			<td>gibco&#160;</td>
			<td>14190-094&#160;</td>
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			<td>REA control antibody (S), human IgG1, Vio Bright FITC, REAfinity</td>
			<td>Miltenyi Biotec</td>
			<td>130-113-443</td>
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			<td>Sodium hypochlorite solution&#160;&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>425044-1L&#160;</td>
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			<td>Sodium nitrite solution</td>
			<td>Sigma Aldrich</td>
			<td>91-4</td>
			<td>from kit Cat No. 387A-1KT</td>
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			<td>Tartrate solution</td>
			<td>Sigma Aldrich</td>
			<td>387-3</td>
			<td>from kit Cat No. 387A-1KT</td>
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			<td>Triton X-100&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>9002-93-1&#160;</td>
			<td></td>
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		<tr>
			<td>Trypan blue&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>T8154-100ML&#160;</td>
			<td></td>
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differentiation of functional osteoclasts from human peripheral blood cd14+ monocytes

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&#8722; 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&#160;provides a useful in vitro model to evaluate OC function and to screen for novel therapeutic compounds that can modulate the differentiation process.

OC generation from CD14+ monocytes 
This method aimed to easily differentiate a large number of OCs from human peripheral blood CD14+ monocytes in vitro, typically in 1 week. Firstly, CD14+ monocytes were enriched from PBMCs and primed with M-CSF overnight to upregulate RANK, as previously reported15. Following monocyte priming, to determine the optimum concentration of RANKL for OC differentiation and maturation, RANKL concentrations of 1 ng/mL, 25 ng/mL, 50 ng/mL, and 100 ng/mL, along with 25ng/mL M-CSF, were used. The addition of RANKL produced increasing numbers of large TRAP-positive multinucleated OCs in a dose-dependent manner, and this was assessed using TRAP staining. Mature OCs are defined as TRAP-positive cells with multiple nuclei (typically more than three; Figure 2A,B and Supplementary Figure 1). Furthermore, the kinetics of OC differentiation from monocytes were investigated using TRAP staining and light microscopy over a 2-14 day culture period. In this instance, OC differentiation using an intermediate concentration of 50 ng/mL RANKL was chosen to assess how fast OCs differentiated in culture. In these culture conditions, multinucleated OCs were visible from day 5 onward, and optimal differentiation was reached on day 7 (Figure 2C). The prolonged incubation of cultures beyond 10 days on plastics resulted in abnormally giant fused cells. In this protocol, days 6-8 are usually used as the optimal endpoint of OC generation. The OCs can be quantified or used for downstream assays.
Functional assessment of differentiated OCs 
To determine the functional activity of the generated OCs, we examined their resorptive activity by differentiating the OCs on a mineralized surface. As large OCs are only generated after a 7 day culture period, and to allow sufficient time to resorb the mineral substrate, the cultures were maintained until day 10. The formation of round holes, or resorption pits, was observed only on the mineralized surfaces of wells containing cells that had been treated with both M-CSF and RANKL (Figure 3). Thus, the percentage of dissolved mineralized surface (resorption pits) allows for determining the OC resorptive capacity. Additionally, the OCs differentiated following this protocol up to day 7, both on plastic and glass chamber slides, displayed a well-organized actin ring structure that could be visualized by immunofluorescent staining (Supplementary Figure 2).
Effect of an inhibitor on mature OC function 
The above mentioned culturing conditions were utilized to determine the functional capability of the in vitro generated OCs&#160;in the presence of the known OC inhibitor, rotenone34. The OCs were differentiated for 6-8 days, and CD14&#8722;OSCAR+ OCs and OC precursors were enriched via flow cytometry (Figure 4). The enriched cells were then plated at 50,000 cells/per well onto a mineral-coated 96-well plate in pro-osteoclastogenic medium (25 ng/mL M-CSF and RANKL) for 3 days. Treatment with rotenone (Figure 5A,B) dose-dependently inhibited the resorption of the mineralized surface in comparison with the untreated control well, consistent with previous studies34. Additionally, OC functionality was assessed via ATP production and actin ring formation. The rotenone-dependent inhibition of OC resorption was associated with the inhibition of ATP production (Figure 5C). Resorbing OCs are highly polarized cells that regulate their resorptive capacity by promoting cytoskeletal organization. Alexa fluor 647 conjugated phalloidin was used to label the F-actin cytoskeleton of the mature OCs cultured in the presence or absence of rotenone. Rotenone caused the fragmentation of the RANKL-derived actin ring of the mature OCs (Figure 5D).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64698/64698fig02.jpg" /> 
Figure 2: OCs efficiently differentiating from CD14+ monocyte precursors. CD14+ monocytes were magnetically enriched, plated at 1 x 105 cells/well in 96-well plates, and incubated overnight with 25 ng/mL M-CSF. (A) M-CSF-primed monocytes were stimulated with increasing concentrations of RANKL (1 ng/mL, 25 ng/mL, 50 ng/mL, and 100 ng/mL), fixed, and stained for TRAP on day 7. Images were acquired, and the TRAP+ multinucleated cells (MNCs) were counted. Representative images of TRAP staining are shown in Supplementary Figure 1. The error bars show mean &#177; SD (n = 3). The data were analyzed with a one-way ANOVA and Holm-Sidak&#39;s multiple comparisons test for paired data; * P &#8804; 0.05 and ** P &#8804; 0.005.&#160;(B) Representative image of a TRAP-stained well of a 96-well plate showing the typical amount of OCs/well expected and their morphology under 25 ng/mL RANK-L in comparison to M-CSF-derived macrophages at day 7. Scale bars: 1000 &#181;m. (C) Representative images of OC formation under 50 ng/mL RANKL assessed via TRAP staining from day 2 to day 14. OCs are visible from day 5 onward. Giant abnormally fused OCs are present after 10 days. Scale bars: 200 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64698/64698fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64698/64698fig03.jpg" /> 
Figure 3: Resorptive OCs differentiated from CD14+ monocytes. CD14+ cells isolated from PBMCs were differentiated for 10 days into OCs in the presence of 25 ng/mL M-CSF (M) and RANKL (R) on mineral assay surface (osteo-assay) plates. (A) Images of representative reconstructed wells taken at 10x magnification to analyze the resorption on day 10 (mineral substrate in gray; resorption pits in white). Scale bars: 1000 &#181;m. (B) Quantification of the percentage of resorbed area. The resorption data were analyzed with a Wilcoxon paired analysis. The error bars show mean &#177; SD (n = 7). <a href="https://www.jove.com/files/ftp_upload/64698/64698fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64698/64698fig04.jpg" /> 
Figure 4: Flow cytometry enrichment of CD14&#8722;OSCAR+ OCs. CD14+ monocytes were enriched from PBMCs, and the OCs were differentiated as previously described. Adherent OC cultures were detached with accutase and stained for flow cytometry. (A-C) OCs at day 8 were sorted based on CD14 and OSCAR expression. (A) Representative sorting gating strategy. The cells were gated as singlets, negative for dead staining, and the CD14+ OSCAR+ (red) and CD14&#8722; OSCAR+ (blue) subsets were sorted. (B) Representative plots showing the overlapping OSCAR staining of RANKL-derived OCs (cyan) and control M-CSF-derived macrophages (orange). In red is the OSCAR isotype-stained control of RANKL-derived OCs. (C) The sorted populations were plated on plastic and allowed to adhere for 2 h in pro-OC medium (25 ng/mL M-CSF and 50 ng/mL RANKL), followed by TRAP staining and visualization. The representative images show a lack of TRAP+ cells in the CD14+ subset (red) and mono- and multinucleated TRAP+ pre-OCs and OCs in the CD14&#8722; subset (blue). Scale bars: 200 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64698/64698fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64698/64698fig05.jpg" /> 
Figure 5: Assays to assess the function of mature OCs. To evaluate the function of mature OCs, CD14+ cells isolated from PBMCs were cultured either with M-CSF (M) alone or combined with RANKL (R) for 7 days, the OCs were enriched via flow cytometry, and the OCs were then treated with the inhibitor rotenone for 24 h. (A) Mature OCs were sorted via flow cytometry (CD14&#8722;OSCAR+) and were cultured on a mineral assay surface in the presence or absence of rotenone for 3 days, after which the cells were bleached and imaged at 10x to reveal the resorbed area (resorption pits in white). (A) Representative reconstructed images of wells. Scale bars: 1000 &#181;m. (B) The quantification of the percentage of resorbed area. The data in (B) were analyzed with a one-way ANOVA with Dunn&#39;s multiple comparisons test (n = 7); * P &#8804; 0.05 and** P &#8804; 0.01. The error bars show the mean &#177; SD. (C) Total intracellular ATP content of undifferentiated and day 7 differentiated mature OCs differentiated with RANKL and treated with either vehicle or rotenone (10 nM and 30 nM). Here, 2DG and oligomycin were used as positive controls for the assay and were added 30 min prior to cell lysis and ATP quantification. The error bars show the mean &#177; SD (n = 4). The data were analyzed with a one-way ANOVA and Dunnett&#39;s multiple comparison test for paired data. ** P &#8804; 0.01. (D) A representative 20x image of mature OCs stained for actin ring formation (red) and nuclei (blue), showing the loss of the actin ring with the inhibitor. Scale bars: 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64698/64698fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Plate format</td>
			<td>96 well-plate</td>
			<td>48 well-plate</td>
			<td>24 well-plate</td>
			<td>12 well-plate</td>
			<td>6 well-plate</td>
		</tr>
		<tr>
			<td>volume</td>
			<td>100 &#181;L</td>
			<td>225&#8211;250 &#181;L</td>
			<td>450&#8211;500 &#181;L</td>
			<td>0.8&#8211;1 mL</td>
			<td>1.8&#8211;2 mL</td>
		</tr>
	</tbody>
</table>
Table 1: Volume of cell suspension for different plate formats. The volumes are calculated starting from a 1 x 106 cells/mL solution and provide an optimum density for cell-cell fusion.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Fluorophore, clone&#160;&#160;</td>
			<td>Volume (&#956;L) per 106 cells</td>
		</tr>
		<tr>
			<td>CD14</td>
			<td>PE/Cyanine7, HCD14&#160;</td>
			<td>5 &#956;L</td>
		</tr>
		<tr>
			<td>OSCAR</td>
			<td>FITC, REA494</td>
			<td>10 &#956;L&#160;</td>
		</tr>
		<tr>
			<td>Cell sorting buffer&#160;</td>
			<td></td>
			<td>80 &#956;L&#160;</td>
		</tr>
	</tbody>
</table>
Table 2 : Antibody master mix solution. 
Supplementary Figure 1: TRAP staining of the RANKL dose response. CD14+ monocytes were magnetically enriched, plated at 1 x 105 cells/well in 96-well plates, and incubated overnight with 25 ng/mL M-CSF, as in Figure 2. Representative images of TRAP staining show MCSF-primed monocytes stimulated with increasing concentrations of RANKL (1 ng/mL, 25 ng/mL, 50 ng/mL, and 100 ng/mL), fixed, and stained for TRAP on day 7. Scale bars: 400 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64698/Supl. Figure 1.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 2: Acting ring staining in fully differentiated OCs. (A) A 10x magnification of OCs differentiated on TC plastic and stained with AF647 phalloidin (in red). Scale bar: 400 &#181;m. (B) A 40x magnification of OCs differentiated on glass chamber slides and stained with AF488 phalloidin (in yellow). Scale bar: 100 &#181;m.The nuclei are stained with DAPI, shown in blue in (A) and in cyan in (B). <a href="https://www.jove.com/files/ftp_upload/64698/Supl.Figure 2.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 3: Effect of different FBS batches on OC differentiation efficiency. OCs were differentiated from CD14+ monocytes in the presence of 25 ng/mL M-CSF and 50 ng/mL RANKL (MR) for 7 days. The control wells had M-CSF only (M). (A) Representative 10x magnifications (scale bars: 400 &#181;m) and (B) quantification of TRAP-stained OCs differentiated from one donor in two different batches of FBS. The error bars show the mean &#177; SD of three technical replicates. <a href="https://www.jove.com/files/ftp_upload/64698/suppl.Figure 3.pdf" target="_blank">Please click here to download this File.</a>

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