This work was supported in part by a JSPS KAKENHI grant from the Japan Society for the Promotion of Science (No. 16K11776 to H. K., No. 17K17306 to K. S., No. 16K20637 to K. K., No. 16K20636 to M. S., No. 18K09862 to I. M.).&#160;

All animal procedures and animal care were performed according to Tohoku University rules and regulations.
1. Murine Hindlimb Dissection and Handling
<ol>
	<li>Before dissection, fill a plate with approximately 50 mL of &#945;-MEM depending on the size of the container and place it on ice. This will serve as a collection container until the dissection of all bones is complete. For this and subsequent experiments, use &#945;-MEM containing 10% fetal bovine serum (FBS), 100 IU/mL penicillin G, and 100 &#956;g/mL streptomycin.</li>
	<li>Euthanize C57Bl/6J mouse by inhalation of an overdose of 5% isoflurane.</li>
	<li>Fix the mouse in a supine position using pins pierced through the palms and feet and spray thoroughly with 70% ethanol.</li>
	<li>Make a skin incision using sterile surgical scissors and tweezers at the level of the hip and extend it down to the feet exposing the muscles.</li>
	<li>Cut the tendon attaching the quadriceps muscle and the tendon attaching the hamstring muscle to the bone. Peel away the muscles exposing both hip and knee joints. Locating these tendons will ease freeing both muscles from their attachments without leaving soft tissue tags. Do not cut into the bone.</li>
	<li>Position the scissors between the head of the femur and the joint space and cut into it freeing the femur but keeping the bone intact. This will enable detachment of the bones without the risk of exposing the bone marrow.</li>
	<li>Similarly cut away the muscles attaching to the tibia. Cut the tibia free from its attachment at the ankle joint keeping the bone intact to avoid contamination.</li>
	<li>Scrape any remaining soft tissue from the femur and tibia using the scissors blades and kimwipe and place it in a plate with &#945;-MEM placed on ice.</li>
</ol>
2. Murine Hindlimb Bone Marrow Isolation
<ol>
	<li>Fill a 30 G needle syringe with &#945;-MEM.</li>
	<li>Disconnect the tibia and femur away from the knee joint.</li>
	<li>Cut the ends of the long bone from both sides using scissors.</li>
	<li>Hold the bone from the shaft using tweezers, insert the needle into the marrow space of the bone and slowly flush the content of the marrow into a 6 cm culture dish. The bone will appear white, indicating the extraction of all bone marrow content. Repeat for all bones.</li>
	<li>Strain the bone marrow extract using a 40 &#956;m nylon cell strainer into a 50 mL conical centrifuge tube and centrifuge at 300 x g for 5 min.</li>
	<li>Discard the supernatant, wash with 5 mL of &#945;-MEM, and centrifuge at 300 x g for 5 min. Repeat wash for a total of 2 times.</li>
	<li>Resuspend the pellet in 10 mL of &#945;-MEM and perform a cell count using a hemocytometer as follows:
	<ol>
		<li>Make a 1:1 dilution of cell suspension by adding 10 &#956;L of cell suspension to 10 &#956;L of 0.4% trypan blue in a 1.5 mL tube and pipette once.</li>
		<li>Cover the hemocytometer chamber with a slide cover and transfer the dye suspension to the hemocytometer. It is important not to overfill or underfill the chamber and allow the suspension to fill the chamber by capillary action.</li>
		<li>Place the hemocytometer on a microscope stage. Using 10x objective, focus on the center square. Count all cells bound by 3 lines. To make sure that cells are not counted twice, count the upper and right corners of each square. Dead cells will appear dark blue, these are excluded from the count.</li>
	</ol>
	</li>
	<li>Depending on the downstream application, seed the cells into an appropriate culture vessel. For this particular experiment, follow section 3.</li>
</ol>
3. Generation of BMM
<ol>
	<li>Seed 1 x 107 cells/10 mL in a 10 cm culture dish and add M-CSF 100 ng/mL. The final concentration of M-CSF is 100 ng/mL of culture medium. Incubate the culture at 37 &#176;C, 5% CO2 for 3 days.</li>
	<li>After 3 days, remove the culture medium, wash the cells vigorously with 10 mL of phosphate buffered saline (PBS) twice to remove non-adherent cells, add 5 mL of room temperature 0.02% trypsin-EDTA in PBS and incubate at 37 &#176;C, 5% CO2 for 5 min.</li>
	<li>Detach the cells by thorough pipetting. Make sure that the cells have been detached by observing the culture under a microscope (the cells should appear rounded and floating in media). If the cells remain attached, repeat pipetting thoroughly to detach them. When the cells have been detached, add 5 mL of &#945;-MEM to inactivate the reaction.</li>
	<li>Collect the cells into a 50 mL conical tube and centrifuge at 300 x g for 5 min. Discard the supernatant, and wash with 5 mL of &#945;-MEM.</li>
	<li>Centrifuge at 300 x g for 5 min and resuspend the pellet in 10 mL of &#945;-MEM.</li>
	<li>Perform a cell count as previously described in step 2.7.1.</li>
	<li>Seed 1 x 106 cells/10 mL in a 10 cm culture dish and add M-CSF 100 ng/mL. The final concentration of M-CSF is 100 ng/mL of culture medium. Incubate the culture at 37 &#176;C, 5% CO2 for 3 days.</li>
</ol>
4. Generation of Osteoclasts from BMM as Osteoclast Precursors
<ol>
	<li>After 3 days, harvest the attached cells which represent BMM as osteoclast precursors, follwoing steps 3.2-3.7.</li>
	<li>Seed BMM at 5 x 104 cells/200 &#956;L of &#945;-MEM in a 96 well plate. Add RANKL for a final concentration of 50 ng/mL or TNF-&#945; for a final concentration of 100 ng/mL. Add M-CSF for a final concentration of 100 ng/mL to each well.</li>
	<li>Add anti-c-fms antibody (final concentrations of 0, 1, 10, 100, 1000 ng/mL) to each well.</li>
	<li>Incubate at 37 &#176;C, 5% CO2 for 4 days. Change media every other day for 4 days.</li>
</ol>
5. Tartrate-resistant Acid Phosphatase (TRAP) Stain
<ol>
	<li>After 4 days of incubation, gently aspirate the culture medium of each well. Wash each well once with 200 &#956;L of room temperature 1x PBS.</li>
	<li>Add 200 &#956;L of 10% formalin to each well for fixation and incubate for 1 h at room temperature. Aspirate the formalin and wash each well 3 times with deionized water.</li>
	<li>Add 200 &#956;L of 0.2% Triton X-100 in PBS to each well for permeabilization and incubate for 1 h at room temperature. Aspirate the Triton X-100 and wash each well 3 times with deionized water.</li>
	<li>Add 200 &#956;L of TRAP stain solution to each well. Visualize the stain under the microscope. It will take from 10-30 min for the stain to develop properly.</li>
	<li>Aspirate the stain and wash each well with deionized water 3 times, and air dry. Keep at room temperature to use in further observation.</li>
</ol>
6. Proliferation Assay
<ol>
	<li>Seed BMM as osteoclast precursors at 5 x 103 cells/200 &#956;L of &#945;-MEM in a 96 well plate. Add M-CSF for a final concentration of 100 ng/mL to each well.</li>
	<li>Add anti-c-fms antibody (final concentrations of 0, 1, 10, 100, 1,000 ng/mL) and incubate at 37 &#176;C, 5% CO2 for 3 days without medium change.</li>
	<li>After 3 days, wash the cells with 1x PBS. Add 100 &#956;L of &#945;-MEM to each well.</li>
	<li>Add 10 &#956;L of cell counting kit solution and incubate at 37 &#176;C, 5% CO2 for 2 h and determine the absorbance of each well at 450 nm using a microplate reader.</li>
</ol>

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W., Takano-Yamamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-c-Fms+antibody+inhibits+lipopolysaccharide-induced+osteoclastogenesis+in+vivo.">Anti-c-Fms antibody inhibits lipopolysaccharide-induced osteoclastogenesis in vivo.</a> FEMS Immunology and Medical Microbiology. 64 (2), 219-227 (2012).</li><li>Kimura, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+anti-c-Fms+antibody+inhibits+osteoclastogenesis+in+a+mouse+periodontitis+model.">An anti-c-Fms antibody inhibits osteoclastogenesis in a mouse periodontitis model.</a> Oral Diseases. 20 (3), 319-324 (2014).</li><li>Kitaura, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+M-CSF+receptor+c-Fms+antibody+inhibits+mechanical+stress-induced+root+resorption+during+orthodontic+tooth+movement+in+mice.">An M-CSF receptor c-Fms antibody inhibits mechanical stress-induced root resorption during orthodontic tooth movement in mice.</a> Angle Orthodontist. 79 (5), 835-841 (2009).</li><li>Kitaura, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+anti-c-Fms+antibody+inhibits+orthodontic+tooth+movement.">An anti-c-Fms antibody inhibits orthodontic tooth movement.</a> Journal of Dental Research. 87 (4), 396-400 (2008).</li><li>Bettina, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M-CSF+Mediates+Host+Defense+during+Bacterial+Pneumonia+by+Promoting+the+Survival+of+Lung+and+Liver+Mononuclear+Phagocytes.">M-CSF Mediates Host Defense during Bacterial Pneumonia by Promoting the Survival of Lung and Liver Mononuclear Phagocytes.</a> Journal of Immunology. 196 (12), 5047-5055 (2016).</li><li>Dougall, W. 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The authors have nothing to disclose.

In this study, we investigated the effect of anti-c-fms antibody on RANKL-induced osteoclast formation, TNF-&#945;-induced osteoclast formation and M-CSF-induced osteoclast precursor proliferation. We found that the effective amount of anti-c-fms antibody for the inhibition of osteoclastogenesis among RANKL-induced osteoclast formation, TNF-&#945; induced osteoclast formation and M-CSF-induced proliferation of osteoclast precursors is different.
RANKL mediates osteoclastogenesis in the presenc...

Osteoclasts are highly specialized cells, and they differentiate from hematopoietic stem cells through the fusion of multiple osteoclast precursors. They are essential for healthy bone remodeling and contribute to pathologic bone resorption associated with inflammatory osteolytic diseases such as rheumatoid arthritis and periodontal disease1.
Osteoclastogenesis and osteoclast function are mediated by two key factors; macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL). Both M-CSF and RANKL are important for osteoclast differentiation2. Another factor which has been shown to induce osteoclast formation from bone marrow macrophages in vitro is tumor necrosis factor alpha (TNF-&#945;)3,4,5. TNF-&#945; mediated osteoclast formation has been shown to be critical for osteolysis in destructive bone diseases such as rheumatoid arthritis6, periodontal disease7, and postmenopausal osteoporosis8.
C-fms is the membrane receptor of M-CSF and mediates its action. The role of c-fms is well documented in the literature as it was demonstrated that the administration of an antibody against c-fms (anti-c-fms antibody) completely arrested osteoclastogenesis in an arthritis model as well as in TNF-&#945; induced bone erosions while osteoclastogenesis distant from the inflamed joint was still robust9. Administration of anti-c-fms antibody inhibited osteoclast formation and bone resorption induced by lipopolysaccharide (LPS) in mouse calvariae10 as well as in LPS-induced&#160; periodontitis model11. Moreover, anti-c-fms antibody inhibited mechanical stress-induced root resorption during orthodontic tooth movement12, as well as orthodontic tooth movement and bone resorption associated with orthodontic tooth movement13.
M-CSF has been reported to have other functions which are essential to the host immune response. The absence of M-CSF in op/op mice with bacterial pneumonia lead to the increase of bacterial burden and bacterial dissemination to the liver with hepatic necrosis14. Therefore, M-CSF is important for immunological response in the protection from infection.
This study demonstrates the effect of anti-c-fms antibody on M-CSF and RANKL or TNF-&#945; induced osteoclast formation in vitro and M-CSF induced proliferation of osteoclast precursors. This protocol demonstrates the isolation of murine bone marrow cells from long bones, and the steps to generate bone marrow macrophages (BMM) which are regarded as osteoclast precursors and to induce the differentiation of BMM into multinuclear osteoclasts by two methods; either RANKL or TNF-&#945;. The protocol also compares the arrest of osteoclastogenesis in both methods using anti-c-fms antibody.
The process by which bone marrow is extracted and subsequently used to generate osteoclast precursors is reliable in producing large quantities of pure osteoclast cultures which can be used in multiple downstream applications such as drug testing. The use of either RANKL or TNF-&#945; to induce osteoclastogenesis in this protocol distinguishes osteoclastogenesis as a physiologic process (RANKL) from osteoclastogenesis as a pathologic process (TNF-&#945;), which in turn provides two alternative procedures to guide the decision of which one is superior in terms of the reagents to be used in downstream applications. This protocol also provides a method by which we can determine a suitable concentration of anti-c-fms antibody that can be safely used for later studies involved in bone resorption and osteolytic diseases.

<table><tbody><tr><td>Anti-c-Fms antibody</td><td></td><td></td><td>AFS98, a rat monoclonal, antimurine, c-Fms antibody (IgG2a)</td></tr><tr><td>TNF-&amp;alpha;</td><td></td><td></td><td>Recombinant murine TNF-&amp;alpha; prepared in our laboratory. TNF-&amp;alpha; cDNA fragment cloned by RT-PCR and cloned into a pGEX-6P-I (Amersham Biosciences, Piscataway, NJ) to generate a GST-fusion protein. GST-TNF-&amp;alpha; was expressed in Escherichia coli BL21 cells cells (Stratagene, La Jolla, CA). The cells were lysed under nondenaturing conditions and GST-TNF-&amp;alpha; was purified over a glutathione-Sepharose column. GST was cleaved off by PreScission Protease (Amersham Biosciences) by manufacturer&amp;rsquo;s directions and was removed by a glutathione-Sepharose column.</td></tr><tr><td>RANKL</td><td>PEPROTECH</td><td>315-11</td><td>Recombinant Murine sRANK Ligand, Source: &lt;em&gt;E. coli&lt;/em&gt;</td></tr><tr><td>M-CSF</td><td></td><td></td><td>Recombinant human M-CSF. 1/10 vol of CMG14&amp;ndash;12 cell line culture supernatant at 5x10&lt;sup&gt;6&lt;/sup&gt;&amp;nbsp;cells in a 10 cm suspension culture dish.</td></tr><tr><td>&amp;alpha;-MEM</td><td>Wako</td><td></td><td>with L-Glutamine and phenol red</td></tr><tr><td>Fetal Bovine Serum</td><td>Biowest</td><td>s1820-500</td><td>Fetal Bovine Serum French Origin</td></tr><tr><td>Culture dish</td><td>Corning</td><td></td><td>100 mm x 20 mm style dish</td></tr><tr><td>96-well plate</td><td>Thermofischer Scientific</td><td></td><td>Nun clon Delta surface</td></tr><tr><td>Cell counting kit-8</td><td>Dojindo, Kumamoto, Japan</td><td></td><td></td></tr><tr><td>Microplate reader</td><td>Sunrise REMOTE; Tekan Japan, Kawasaki, Japan</td><td></td><td></td></tr><tr><td>Cell strainer</td><td>Corning</td><td></td><td>40 &amp;mu;m Nylon</td></tr><tr><td>Centrifuge tube</td><td>Corning</td><td></td><td>50 mL CentriStar cap</td></tr><tr><td>Triton X-100</td><td>Wako</td><td></td><td>Polyoxyethylene (10) Octyphenyl ether</td></tr></tbody></table>

effect of anti-c-fms antibody on osteoclast formation and proliferation of osteoclast precursor in vitro

Bone remodeling is a complex process and it involves periods of deposition and resorption. Bone resorption is a process by which bone is broken down by osteoclasts in response to different stimuli. Osteoclast precursors differentiate into multinuclear osteoclasts in response to macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor Kappa-B ligand (RANKL). Under pathologic conditions, the cytokine profile is different and involves a mixture of inflammatory cytokines. Tumor necrosis factor alpha (TNF-&#945;) is one of the most important cytokines as it is found in large amounts in areas involved with inflammatory osteolysis. The purpose of this protocol is to provide a method by which murine bone marrow is isolated to generate osteoclasts through induction with M-CSF and either RANKL or TNF-&#945; which will be subsequently inhibited by increasing doses of anti-c-fms antibody, the receptor for M-CSF. This experiment highlights the therapeutic value of anti-c-fms antibody in diseases of inflammatory bone resorption.

The purpose of this protocol is to evaluate the effect of anti-c-fms antibody on osteoclast formation in the presence of RANKL or TNF-&#945;, and to determine the effect of M-CSF on osteoclast precursors proliferation. In this protocol, we have provided a reliable process by which large quantities of pure osteoclast cultures are generated. We have also provided a way to test anti-c-fms antibody suitable concentration for inhibiting osteoclast formation under different culture conditions.<...

Watch this Scientific Journal Video about Effect of Anti-c-fms Antibody on Osteoclast Formation and Proliferation of Osteoclast Precursor In Vitro at JoVE.com

Effect of Anti-c-fms Antibody on Osteoclast Formation and Proliferation of Osteoclast Precursor In Vitro

This protocol includes dissection of murine long bones and bone marrow isolation, to generate bone marrow macrophages and produce osteoclasts using macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor Kappa-B ligand (RANKL) or tumor necrosis factor alpha (TNF-&#945;) and their arrest by anti-c-fms antibody, M-CSF receptor.

Bone remodeling is a complex process and it involves periods of deposition and resorption. Bone resorption is a process by which bone is broken down by ...

effect-anti-c-fms-antibody-on-osteoclast-formation-proliferation

Research

JoVE Journal

Immunology and Infection

5.9K Views.  Tohoku University Graduate School of Dentistry. This protocol includes dissection of murine long bones and bone marrow isolation, to generate bone marrow macrophages and produce osteoclasts using macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor Kappa-B ligand (RANKL) or tumor necrosis factor alpha (TNF-α) and their arrest by anti-c-fms antibody, M-CSF receptor.

Video: Effect of Anti-c-fms Antibody on Osteoclast Formation and Proliferation of Osteoclast Precursor In Vitro

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We developed an in vitro fully human preclinical model of a co-culture of breast cancer cells and monocytes undergoing differentiation towards osteoclasts. We optimized a model of osteoclastogenesis starting from a sample of peripheral blood collected from healthy donors. Peripheral blood mononuclear cells (PBMCs) were first separated by density gradient centrifugation, seeded at a high density and induced to differentiate by adding two growth factors (GFs): receptor activator of nuclear factor-&#954;B ligand (RANKL) and macrophage colony-stimulating factor (MCSF). The cells were left in culture for 14 days and then fixed and analyzed by downstream analysis. In osteolytic bone metastases, one of the effects of cancer cell arrival in bone is the induction of osteoclastogenesis. We thus challenged our model with co-cultures of breast cancer cells to study the differentiation power of cancer cells with respect to GFs. A straightforward way of studying cancer cell-osteoclast interaction is to perform indirect co-cultures based on the use of conditioned medium collected from breast cancer cell cultures and mixed with fresh medium. This mixture is then used to induce osteoclast differentiation. We also optimized a method of direct co-culture in which cancer cells and monocytes undergoing differentiation share the medium and exchange secreted factors. This is a significant improvement over the original indirect co-culture method as researchers can observe the reciprocal interactions of the two cell types and perform downstream analyses for both cancer cells and osteoclasts. This method enables us to study the effect of drugs on the metastatic bone microenvironment and to seed cell lines other than those derived from breast cancer. The model can also be used to study other diseases such as osteoporosis or other bone conditions.

This protocol describes development of an in vitro human preclinical model of osteoclastogenesis from peripheral blood monocytes cultured with breast cancer cell lines to mimic the cancer cell-osteoclast interaction. The model could be used to further our understanding of bone metastasis formation and improve therapeutic options.

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We ...

Cancer Research

Stimulation of Notch Signaling in Mouse Osteoclast Precursors

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro investigation of its varying and sometimes contradictory roles a challenge. As a component of direct cell-cell communication with both receptors and ligands bound to the plasma membrane, Notch signaling cannot be activated in vitro by simple addition of ligands to culture media, as is possible with many other signaling pathways. Instead, Notch ligands must be presented to cells in an immobilized state. 
Variations in methods of Notch signaling activation can lead to different outcomes in cultured cells. In osteoclast precursors, in particular, differences in methods of Notch stimulation and osteoclast precursor culture and differentiation have led to disagreement over whether Notch signaling is a positive or negative regulator of osteoclast differentiation. While closer comparisons of osteoclast differentiation under different Notch stimulation conditions in vitro and genetic models have largely resolved the controversy regarding Notch signaling and osteoclasts, standardized methods of continuous and temporary stimulation of Notch signaling in cultured cells could prevent such discrepancies in the future.
This protocol describes two methods for stimulating Notch signaling specifically in cultured mouse osteoclast precursors, though these methods should be applicable to any adherent cell type with minor adjustments. The first method produces continuous stimulation of Notch signaling and involves immobilizing Notch ligand to a tissue culture surface prior to the seeding of cells. The second, which uses Notch ligand bound to agarose beads allows for temporary stimulation of Notch signaling in cells that are already adhered to a culture surface. This protocol also includes methods for detecting Notch activation in osteoclast precursors as well as representative transcriptional markers of Notch signaling activation.

Notch signaling is a form of cellular communication that relies upon direct contact between cells. To properly induce Notch signaling in vitro, Notch ligands must be presented to cells in an immobilized state. This protocol describes methods for in vitro stimulation of Notch signaling in mouse osteoclast precursors.

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro ...

Developmental Biology

A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result in a series of bone metabolic diseases, including osteoporosis. To develop pharmaceutical targets for the prevention of pathological bone mass loss, the mechanisms by which osteoclasts differentiate from precursors must be understood. The ability to isolate and culture a large number of osteoclasts in vitro is critical in order to determine the role of specific genes in osteoclast differentiation. Inactivation of the mammalian/mechanistic target of rapamycin complex 1 (TORC1) in osteoclasts can decrease osteoclast number and increase bone mass; however, the underlying mechanisms require further study. In the present study, a RANKL-based protocol to isolate and culture osteoclasts from mouse bone marrow and to study the influence of mTORC1 inactivation on osteoclast formation is described. This protocol successfully resulted in a large number of giant osteoclasts, typically within one week. Deletion of Raptor impaired osteoclast formation and decreased the activity of secretory tartrate-resistant acid phosphatase, indicating that mTORC1 is critical for osteoclast formation.

This manuscript describes a protocol to isolate and culture osteoclasts in vitro from mouse bone marrow, and to study the role of the mammalian/mechanistic target of rapamycin complex 1 in osteoclast formation.

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result ...

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone resorption. Osteoclasts can be generated in vitro from monocyte/macrophage precursor cells in the presence of certain cytokines, which promote survival and differentiation. Here, both in vivo and in vitro techniques are demonstrated, which allow scientists to study different cytokine contributions towards osteoclast differentiation, signaling, and activation. The minicircle DNA delivery gene transfer system provides an alternative method to establish an osteoporosis-related model is particularly useful to study the efficacy of various pharmacological inhibitors in vivo. Similarly, in vitro culturing protocols for producing osteoclasts from human precursor cells in the presence of specific cytokines enables scientists to study osteoclastogenesis in human cells for translational applications. Combined, these techniques have the potential to accelerate drug discovery efforts for osteoclast-specific targeted therapeutics, which may benefit millions of osteoporosis and arthritis patients worldwide.

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described.

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone ...

Medicine

Differentiation and Characterization of Osteoclasts from Human Induced Pluripotent Stem Cells

This protocol details the propagation and passaging of human iPSCs and their differentiation into osteoclasts. First, iPSCs are dissociated into a single-cell suspension for further use in embryoid body induction. Following mesodermal induction, embryoid bodies undergo hematopoietic differentiation, producing a floating hematopoietic cell population. Subsequently, the harvested hematopoietic cells undergo a macrophage colony-stimulating factor maturation step and, finally, osteoclast differentiation. After osteoclast differentiation, osteoclasts are characterized by staining for TRAP in conjunction with a methyl green nuclear stain. Osteoclasts are observed as multinucleated, TRAP+ polykaryons. Their identification can be further supported by Cathepsin K staining. Bone and mineral resorption assays allow for functional characterization, confirming the identity of bona fide osteoclasts. This protocol demonstrates a robust and versatile method to differentiate human osteoclasts from iPSCs and allows for easy adoption in applications requiring large quantities of functional human osteoclasts. Applications in the areas of bone research, cancer research, tissue engineering, and endoprosthesis research could be envisioned.

This protocol presents the differentiation of human osteoclasts from induced pluripotent stem cells (iPSCs) and describes methods for the characterization of osteoclasts and osteoclast precursors.

This protocol details the propagation and passaging of human iPSCs and their differentiation into osteoclasts. First, iPSCs are dissociated into a ...

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.

Differentiation of Functional Osteoclasts from Human Peripheral Blood CD14+ Monocytes

Osteoclasts are key bone-resorbing cells in the body. This protocol describes a reliable method for the in vitro differentiation of osteoclasts from human peripheral blood monocytes. This method can be used as an important tool to further understand osteoclast biology in homeostasis and in diseases.

Osteoclasts (OCs) are bone-resorbing cells that play a pivotal role in skeletal development and adult bone remodeling. Several bone disorders are caused ...

Osteoclast Derivation from Mouse Bone Marrow

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the organic and inorganic matrices of bone means that they play a key role in regulating skeletal remodeling. Together, osteoblasts and osteoclasts are responsible for the dynamic coupling process that involves both bone resorption and bone formation acting together to maintain the normal skeleton during health and disease.
As the principal bone-resorbing cell in the body, changes in osteoclast differentiation or function can result in profound effects in the body. Diseases associated with altered osteoclast function can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis, to more commonly observed pathologies such as osteoporosis, in which excessive osteoclastic bone resorption predisposes to fracture formation.
An ability to isolate osteoclasts in high numbers in vitro has allowed for significant advances in the understanding of the bone remodeling cycle and has paved the way for the discovery of novel therapeutic strategies that combat these diseases. 
Here, we describe a protocol to isolate and cultivate osteoclasts from mouse bone marrow that will yield large numbers of osteoclasts.

Osteoclasts are the principal bone-resorbing cell in the body. An ability to isolate osteoclasts in large numbers has resulted in significant advances in the understanding of osteoclast biology. In this protocol, we describe a method for isolation, cultivating and quantifying osteoclast activity in vitro.

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the ...

Biology

A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro

Mature osteoclasts are multinucleated cells that can degrade bone through the secretion of acids and enzymes. They play a crucial role in various diseases (e.g., osteoporosis and bone cancer) and are therefore important objects of research. In vitro, their activity can be analyzed by the formation of resorption pits. In this protocol, we describe a simple pit assay method using calcium phosphate (CaP) coated cell culture plates, which can be easily visualized and quantified. Osteoclast precursors derived from human peripheral blood mononuclear cells (PBMCs) were cultured on the coated plates in the presence of osteoclastogenic stimuli. After 9 days of incubation, osteoclasts were fixed and stained for fluorescence imaging while the CaP coating was counterstained by calcein. To quantify the resorbed area, the CaP coating on plates was stained with 5% AgNO3 and visualized by brightfield imaging. The resorption pit area was quantified using ImageJ.

A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro

Here, we present a simple and effective assay procedure for resorption pit assays using calcium phosphate coated cell culture plates.

Mature osteoclasts are multinucleated cells that can degrade bone through the secretion of acids and enzymes. They play a crucial role in various diseases ...

Isolation of Monocyte-Macrophage Lineage Cells from Rat Bones by Secondary Adherence Method

With a decrease of bone mineral density, bones are more likely to fracture, thus negatively affecting a patient's quality of life. The growth and development of bones are mainly regulated by osteoblasts and osteoclasts. It has been widely accepted that osteoclasts are derived from bone marrow monocyte-macrophage cells (BMMs). BMMs and other hematopoietic stem cells are located in the bone marrow cavity. Therefore, isolating single stable BMMs from different and heterogeneous cell populations is a huge challenge. Here we present a protocol for the isolation of BMMs from SD rats, called the secondary adherence method. Adherent cells cultured for 24-48 h in primary culture were collected. Flow cytometric analysis showed that approximately 37.94% of the cells were CD11b/c+ (monocyte-macrophage surface antigen). Tartrate resistant acid phosphatase (TRAP) staining and western blot analysis demonstrated that BMMs could differentiate into osteoclasts in vitro. The above findings suggested that the secondary adherence cells could be considered as a suitable cellular model for osteoclast differentiation research.

Here we present a protocol for the isolation of BMMs from SD rats, called the secondary adherence method.

With a decrease of bone mineral density, bones are more likely to fracture, thus negatively affecting a patient's quality of life. The growth and ...

Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or macrophage precursors. Excessive bone resorption is one the most significant cellular mechanisms leading to osteolytic diseases, including osteoporosis, periodontitis, and periprosthetic osteolysis. The main physiological function of osteoclasts is to absorb both the hydroxyapatite mineral component and the organic matrix of bone, generating the characteristic resorption appearance on the surface of bones. There are relatively few osteoclasts compared to other cells in the body, especially in adult bones. Recent studies have focused on how to obtain more mature osteoclasts in less time, which has always been a problem. Several improvements in the isolation and culture techniques have developed in laboratories in order to obtain more mature osteoclasts. Here, we introduce a method that isolates bone marrow in less time and with less effort compared to the traditional procedure, using a special and simple device. With the use of density gradient centrifugation, we obtain large amounts of fully differentiated osteoclasts from rat bone marrow, which are identified by classical methods.

Osteoclasts are tissue-specific macrophage polykaryons derived from the monocyte-macrophage lineage of hematopoietic stem cells. This protocol describes how to isolate bone marrow cells so that large quantities of osteoclasts are obtained while reducing the risk of accidents found in traditional methods.

Effect of Anti-c-fms Antibody on Osteoclast Formation and Proliferation of Osteoclast Precursor In Vitro

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Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines

Stimulation of Notch Signaling in Mouse Osteoclast Precursors

A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation and Characterization of Osteoclasts from Human Induced Pluripotent Stem Cells

Differentiation of Functional Osteoclasts from Human Peripheral Blood CD14⁺ Monocytes

Osteoclast Derivation from Mouse Bone Marrow

A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro

Isolation of Monocyte-Macrophage Lineage Cells from Rat Bones by Secondary Adherence Method

Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow