This work was supported by the University of Pennsylvania Center for Musculoskeletal Disorders (5 P30 AR050950-09), a grant from the AO Foundation (S-16-12A), the Philadelphia VA Medical Center Translational Musculoskeletal Research Center, and an intramural orthopaedic surgery departmental research development fund. JWA is supported by the University of Pennsylvania Postdoctoral Opportunities in Research and Teaching (PENN-PORT) fellowship funded by the National Institute of General Medical Sciences Institutional Research and Career Development Award (IRACDA; 5 K12 GM081259-08).

All research involving vertebrate animals was performed in accordance with protocols approved by the University of Pennsylvania Institutional Animal Care and Use Committee (IACUC).
1. Culture Media Preparation
<ol>
	<li>Prepare &#945;-MEM by dissolving minimum essential medium (MEM) powder and 1.9 g sodium bicarbonate in 900 ml H2O and supplement with 2 mM L-glutamine, 100 units/ml penicillin, 100 &#956;g/ml streptomycin, and 100 ml heat-inactivated fetal bovine serum. Sterilize using a 0.22 &#181;m polyethersulfone (PES) filter.</li>
	<li>Prepare macrophage medium by supplementing &#945;-MEM with 35 ng/ml recombinant mouse Macrophage-Colony Stimulating Factor (M-CSF)</li>
	<li>Prepare osteoclast medium by supplementing macrophage medium with 100 ng/ml RANKL</li>
</ol>
2. Bone Marrow Cell Isolation
<ol>
	<li>Fill one 10 ml syringe with a 25&#8541; gauge needle per mouse with 10 ml &#945;-MEM.</li>
	<li>Euthanize mice via 10 min exposure to CO2 at a flow rate of 1.3 L/min. Ensure animal death by following CO2 exposure with cervical dislocation before proceeding with dissection.</li>
	<li>Using clean technique, dissect out and separate the tibiae and femora. Disinfect mouse skin with 70 % ethanol before making an incision along the anterior surface of each hind limb to expose the tibia and femur.
	<ol>
		<li>Cut through the knee joint to free the distal end of the femur, and cut through the femur as close to the pelvis as possible. Remove the femur and clean as much soft tissue as possible. To remove the tibia, cut through the ankle and remove as much soft tissue as possible.</li>
	</ol>
	</li>
	<li>Hold the bone with forceps and flush marrow into a 15 ml conical tube with 2.5 ml room temperature &#945;-MEM combining marrow flushes from a single mouse into a single tube. 
	NOTE: A successful marrow flush will change the coloration of the bone from red to beige.</li>
	<li>Allow debris to settle to the bottom of the tube and transfer supernatant to a clean 15 ml conical tube taking care to avoid transferring any debris. 
	NOTE: Some cells may also settle with the marrow debris. If a greater number of cells is required, marrow flushes can be filtered through a 70 &#181;m cell strainer into a clean 15 ml tube.</li>
	<li>Centrifuge tube(s) at 300 x g for 5min at room temperature to pellet cells.</li>
	<li>Vacuum aspirate the supernatant and resuspend cell pellet in 0.5 ml Ammonium-Chloride-Potassium (ACK) lysing buffer and incubate at 37 &#176;C for 3 min to lyse red blood cells.</li>
	<li>Add 10 ml PBS directly to ACK-cell solution, and centrifuge at 300 x g for 5 min. 
	NOTE: The previously red cell pellet should now be beige.</li>
	<li>Vacuum aspirate supernatant and resuspend pellet in 10 ml &#945;-MEM, and plate in a 100 mm tissue culture-treated dish. Incubate cells overnight at 37 &#176;C in a humidified tissue culture incubator. 
	NOTE: Counting cells is not necessary for this step. During this time, adherent cells from the bone marrow will attach to the culture surface; non-adherent bone marrow cell populations will remain in suspension. MCSF is not present in the culture medium during this initial incubation.</li>
</ol>
3. Enrichment of Osteoclast Precursors
<ol>
	<li>After overnight incubation of marrow flushes, transfer culture supernatant containing non-adherent cells to a 50 ml conical tube. 
	NOTE: Culture dishes with adherent cells, which include bone marrow mesenchymal progenitor cells, can be discarded or fed with fresh &#945;-MEM, if desired for other experiments. 
	NOTE: Osteoclasts can be differentiated directly from these non-adherent bone marrow cells by plating them into tissue culture-treated dishes at a density of 4 x 105 cells/cm2 in osteoclast medium and refreshing medium every other day for 5 days.</li>
	<li>Add &#945;-MEM to non-adherent cell solution to a final volume of 18 ml and add M-CSF to a final concentration of 35 ng/ml.</li>
	<li>Add 3 ml of cell suspension each to 6 60 mm suspension culture dishes.</li>
	<li>Incubate plates overnight at 37 &#176;C in a humidified tissue culture incubator. During this time, M-CSF will stimulate differentiation of macrophages, which can adhere even to non-tissue culture-treated surfaces.</li>
	<li>After incubating overnight, re-feed osteoclast precursors with 3 ml fresh macrophage medium. This will remove non-adherent cells that haven&#39;t differentiated into macrophages.</li>
	<li>Continue to culture osteoclast precursors at 37 &#176;C and 5 % CO2 for 2 days or until cultures reach roughly 70% confluence.</li>
</ol>
4. Osteoclast Differentiation
<ol>
	<li>Wash osteoclast precursors with 5 ml PBS and treat cells with 1 ml marine-origin proteolytic/collagenolytic enzyme at room temperature until the cells can be dislodged by gentle tapping of the plate. 
	NOTE: Osteoclast precursors can only be lifted if they are cultured in suspension dishes; precursors attach too firmly to tissue culture-treated surfaces to be lifted. Trypsin can activate osteoclast precursors and should be avoided.</li>
	<li>Add 4 ml &#945;-MEM to each dish and transfer cells to a 50 ml conical tube. Count cells using a hemocytometer and dilute cells to a density of 5 x 104 cells/ml and add M-CSF to a final concentration of 35 ng/ml and RANKL to a final concentration of 100 ng/ml.</li>
	<li>Seed cells at a density of 2.6 x 104 cells/cm2 into tissue culture-treated plates and incubate at 37 &#176;C in a humidified tissue culture incubator for 2 days. Differentiation is typically carried out in 24-well plates, where 5 x 104 cells are seeded into each well.</li>
	<li>2 days after the start of the differentiation, re-feed cells by replacing medium with 1 ml fresh osteoclast medium. 
	NOTE: Osteoclast differentiation will usually be complete within 3 days. Resulting osteoclasts can be TRAP-stained for easy quantification and morphological analysis.</li>
</ol>
5. Continuous Stimulation of Notch Signaling with Jagged1-coated Surface
<ol>
	<li>Suspend goat anti-human IgG Fc antibody in PBS at a concentration of 10 &#956;g/ml (1:1,000 dilution of 10 mg/ml stock). Add antibody solution to culture surface at a density of 1.3 &#956;g/cm2 and incubate at room temperature for 1 hr. In the case of 24-well plates, add 250 &#956;l (2.5 &#956;g) per well.</li>
	<li>Vacuum aspirate wells and refill with 1 ml PBS. Repeat this step 2 times.</li>
	<li>Suspend recombinant Jagged1-Fc fusion protein in PBS at a concentration of 10 &#956;g/ml. Add Jagged1-FC solution to the antibody-coated culture surface at a density of 1.3 &#956;g/cm2 and incubate at room temperature for 2 hr. Culture surfaces coated with antibody only can be used as controls.</li>
	<li>Vacuum aspirate wells and refill with 1 ml PBS. Repeat this step 2 times. Keep PBS in wells until just prior to cell seeding to prevent them from drying.</li>
	<li>Seed 2.6 x 104 osteoclast precursors/cm2 onto coated surfaces. Notch signaling will be continuously stimulated for at least 3 days.</li>
</ol>
6. Temporary Stimulation of Notch Signaling with Jagged1-coated Beads
<ol>
	<li>Combine the following in an appropriately sized tube: 0.5 &#956;g/cm2 Jagged1-Fc, 21 &#956;l/cm2 protein G agarose beads, and PBS to a final volume of 1.5 ml. To prepare an adequate number of Jagged1 beads for 1 well of a 24 well plate, combine 1 &#956;g Jagged1-Fc, 40 &#956;l protein G agarose beads, and PBS to 1.5 ml.</li>
	<li>Incubate tube with rotation at 4 &#176;C for 2 hr.</li>
	<li>Centrifuge tube at 300 x g for 1 min to pellet beads. Resuspend beads in 1.5 ml PBS. Repeat this wash 2 more times.</li>
	<li>Resuspend Jagged1 beads in 130 &#956;l/cm2 culture medium and apply to cultured cells.</li>
	<li>Incubate cells with beads at 37 &#176;C and 5 % CO2 for desired period. Remove beads with several washes of PBS or culture medium.</li>
	<li>Verify Notch signaling activation via measurement of Notch target gene transcripts10.</li>
</ol>

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D., Shimada, I. S., Sherwin, C., Spees, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch1-STAT3-ETBR+signaling+axis+controls+reactive+astrocyte+proliferation+after+brain+injury.">Notch1-STAT3-ETBR signaling axis controls reactive astrocyte proliferation after brain injury.</a> Proc Natl Acad Sci U S A. 112 (28), 8726-8731 (2015).</li><li>Kostianovsky, A. M., Maier, L. M., Baecher-Allan, C., Anderson, A. C., Anderson, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Up-regulation+of+gene+related+to+anergy+in+lymphocytes+is+associated+with+Notch-mediated+human+T+cell+suppression.">Up-regulation of gene related to anergy in lymphocytes is associated with Notch-mediated human T cell suppression.</a> J Immunol. 178 (10), 6158-6163 (2007).</li><li>Teitelbaum, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoclasts:+what+do+they+do+and+how+do+they+do+it?.">Osteoclasts: what do they do and how do they do it?.</a> Am J Pathol. 170 (2), 427-435 (2007).</li><li>Ma, J., 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=Disruption+of+the+transcription+factor+RBP-J+results+in+osteopenia+attributable+to+attenuated+osteoclast+differentiation.">Disruption of the transcription factor RBP-J results in osteopenia attributable to attenuated osteoclast differentiation.</a> Mol Biol Rep. 40 (3), 2097-2105 (2013).</li><li>Bai, S., 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=NOTCH1+regulates+osteoclastogenesis+directly+in+osteoclast+precursors+and+indirectly+via+osteoblast+lineage+cells.">NOTCH1 regulates osteoclastogenesis directly in osteoclast precursors and indirectly via osteoblast lineage cells.</a> J Biol Chem. 283 (10), 6509-6518 (2008).</li><li>Yamada, T., 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=Regulation+of+osteoclast+development+by+Notch+signaling+directed+to+osteoclast+precursors+and+through+stromal+cells.">Regulation of osteoclast development by Notch signaling directed to osteoclast precursors and through stromal cells.</a> Blood. 101 (6), 2227-2234 (2003).</li><li>Sethi, N., Dai, X., Winter, C. G., Kang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-derived+JAGGED1+promotes+osteolytic+bone+metastasis+of+breast+cancer+by+engaging+notch+signaling+in+bone+cells.">Tumor-derived JAGGED1 promotes osteolytic bone metastasis of breast cancer by engaging notch signaling in bone cells.</a> Cancer Cell. 19 (2), 192-205 (2011).</li><li>Sekine, C., 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=Differential+regulation+of+osteoclastogenesis+by+Notch2/Delta-like+1+and+Notch1/Jagged1+axes.">Differential regulation of osteoclastogenesis by Notch2/Delta-like 1 and Notch1/Jagged1 axes.</a> Arthritis Res Ther. 14 (2), R45 (2012).</li><li>Ashley, J. W., Ahn, J., Hankenson, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+Signaling+Promotes+Osteoclast+Maturation+and+Resorptive+Activity.">Notch Signaling Promotes Osteoclast Maturation and Resorptive Activity.</a> J Cell Biochem. 116 (11), 2598-2609 (2015).</li><li>Fukushima, 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=The+association+of+Notch2+and+NF-kappaB+accelerates+RANKL-induced+osteoclastogenesis.">The association of Notch2 and NF-kappaB accelerates RANKL-induced osteoclastogenesis.</a> Mol Cell Biol. 28 (20), 6402-6412 (2008).</li><li>Canalis, E., Schilling, L., Yee, S. P., Lee, S. 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The authors declare that they have no competing financial interests.

Critical steps within the protocol
Culture and in vitro differentiation of osteoclast precursors provides a useful platform for investigation of molecular mechanisms of osteoclastogenesis and identification of therapeutic targets for bone regeneration and preservation of bone mass. When culturing mouse osteoclast precursors, the most critical element is maintenance of precursors in a na&#239;ve state. As macrophage-like cells, osteoclast precursors are primed to respond to bacterial comp...

The mammalian Notch signaling pathway is homologous to the same pathway in Drosophila melanogaster and consists of four transmembrane Notch receptors (Notch1-4) and five membrane-bound ligands of the Jagged (JAG1 &#38; JAG2) and Delta-like (DLL1, 3,&#38; 4) families1. Notch signaling is initiated when Notch receptor on a receiving cell is bound by ligand on a transmitting cell2. During this trans-activation, the membrane-bound Notch ligand produces a stretching force on the membrane-bound Notch receptor3,4. The stretching force of ligand binding induces conformational changes in the Notch receptor that facilitate extracellular cleavage of the receptor by TNFalpha converting enzyme (TACE) followed by an intracellular cleavage event mediated by a presenilin-containing gamma-secretase complex (&#947;-secretase). &#947;-secretase releases the Notch intracellular domain (NICD) which translocates into the nucleus where it forms a transcriptional activation complex with CBF-1-Su(H)-Lag-1 (CSL), mastermind-like (MAML), and cell type-specific factors to drive expression of target genes5.
The mechanical elements of Notch signaling activation result in the need for unique methods of Notch pathway activation in vitro. Soluble Notch ligands can bind to Notch receptors, but fail to produce stretching forces necessary for NICD release while at the same time competitively inhibiting binding of cell-associated Notch ligands. Thus, addition of soluble Notch ligands to culture medium can attenuate normal Notch signaling6,7. Fortunately, Notch ligands can induce NICD release if they are fixed to a suitably rigid substrate5,8,9,10. Seeding cells on ligand-coated culture substrates or applying ligand-coated beads to cells can both activate Notch signaling, and the choice between them depends primarily on the desired timing of Notch stimulation. For immediate, temporary Notch signaling activation, as would be desired during the midpoint of a functional or differentiation assay, Notch ligand can be bound to agarose beads, applied to cultured cells, and washed out at any time. For more sustained Notch signaling from the beginning of a culture period, tissue culture plates can be coated with ligand prior to cell seeding.
For the purposes of this protocol, methods are carried out using mouse osteoclast precursors, but the methods and variations on the methods described here are applicable to a wide variety of cell types6,11,12,13,14. Osteoclasts are terminally differentiated hematopoietic linage cells that are responsible for the resorption of bone tissue, and they are implicated in multiple disorders of bone loss15. Thus, in vitro study of the differentiation of osteoclasts from their monocyte/macrophage-lineage precursors and the molecular mechanisms controlling their function is essential to better understanding of osteoclasts and development of new bone-regenerating therapies. While it is now generally accepted that Notch signaling plays a positive role in the differentiation and function of osteoclasts, variations in both Notch signaling stimulation and osteoclast precursor culture and differentiation led to initially contradictory findings16,17,18,19. Closer examination of the differences in methods and use of genetic models have greatly clarified the role of Notch signaling in osteoclastogenesis, but application of standardized Notch stimulation and culture methods could prevent such controversies in future studies of Notch signaling in other cell types20,21,22,23.
There are multiple methods for culturing and differentiating mouse osteoclast precursors, and, as with varying methods for stimulating Notch signaling, the best method will depend upon the experimental question. Herein, our preferred method of culturing adherent and non-adherent fractions of marrow cells flushed from mouse long bones will be presented. This method has the advantage of requiring essentially no specialized equipment and producing cells that are applicable to a variety of differentiation methods.

<table><tbody><tr><td>Recombinant mouse M-CSF</td><td>Biolegend</td><td>576402</td><td>Available from multiple suppliers, test activity before experiments</td></tr><tr><td>Recombinant mouse RANKL</td><td>Shenandoah Biotechnology</td><td>200-04</td><td>Available from multiple suppliers, test activity before experiments</td></tr><tr><td>Recombinant human Jagged1-Fc</td><td>R&amp;amp;D Systems</td><td>1277-JG-050</td><td>Available from multiple suppliers, test activity before experiments</td></tr><tr><td>Protein G agarose beads</td><td>InvivoGen</td><td>gel-agg-2</td><td></td></tr><tr><td>Goat anti-human IgG Fc</td><td>Jackson ImmunoResearch</td><td>109-001-008</td><td></td></tr><tr><td>Minimum Essential Medium powder</td><td>Sigma-Aldrich</td><td>M0894</td><td></td></tr><tr><td>Accutase cell dissociation reagent</td><td>ThermoFisher</td><td>A1110501</td><td>Used to lift osteoclast precursors</td></tr><tr><td>Acid Phosphatase, Leukocyte (TRAP) Kit</td><td>Sigma-Aldrich</td><td>387A-1KT</td><td>Used to stain differentiated osteoclasts</td></tr></tbody></table>

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.

The aim of this method is to culture and stimulate Notch signaling in osteoclast precursors. When properly cultured, osteoclast precursors exhibit a primarily elongated spindle-shaped morphology with smooth cytoplasm (Figure 1A). Care should be taken to avoid immunological activation of the osteoclast precursors. Upon activation, precursors spread and become flattened with foamy cytoplasm (Figure 1B). These &#34;fried egg&#34; cells are resistant to RANK ...

Watch this Scientific Journal Video about Stimulation of Notch Signaling in Mouse Osteoclast Precursors at JoVE.com

Stimulation of Notch Signaling in Mouse Osteoclast Precursors

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 ...

stimulation-of-notch-signaling-in-mouse-osteoclast-precursors

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7.8K Views.  Perelman School of Medicine, University of Pennsylvania. 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.

Video: 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

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

Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and molecular complexity of skeletal elements increases with development. Tissue heterogeneity, such as at the interface of cartilaginous and osseous elements with each other or with surrounding tissues, is one obstacle to the study of developing cartilage and bone. Our protocol provides a rapid method of tissue processing and isolation of cartilage and bone that yields high quality RNA for gene expression analysis. Fresh frozen tissues of mouse embryos are sectioned and brief cresyl violet staining is used to visualize cartilage and bone with colors distinct from surrounding tissues. Slides are then rapidly dehydrated, and cartilage and bone are isolated subsequently by LCM. The minimization of exposure to aqueous solutions during this process maintains RNA integrity. Mouse Meckel&rsquo;s cartilage and mandibular bone at E16.5 were successfully collected and gene expression analysis showed differential expression of marker genes for osteoblasts, osteocytes, osteoclasts, and chondrocytes. High quality RNA was also isolated from a range of tissues and embryonic ages. This protocol details sample preparation for LCM including cryoembedding, sectioning, staining and dehydrating fresh frozen tissues, and precise isolation of cartilage and bone by LCM resulting in high quality RNA for transcriptomic analysis.

This protocol describes laser capture microdissection for the isolation of cartilage and bone from fresh frozen sections of the mouse embryo. Cartilage and bone can be rapidly visualized by cresyl violet staining and collected precisely to yield high quality RNA for transcriptomic analysis.

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and ...

Isolation of Whole Cell Protein Lysates from Mouse Facial Processes and Cultured Palatal Mesenchyme Cells for Phosphoprotein Analysis

Mammalian craniofacial development is a complex morphological process during which multiple cell populations coordinate to generate the frontonasal skeleton. These morphological changes are initiated and sustained through diverse signaling interactions, which often include protein phosphorylation by kinases. Here, two examples of physiologically-relevant contexts in which to study phosphorylation of proteins during mammalian craniofacial development are provided: mouse facial processes, in particular E11.5 maxillary processes, and cultured mouse embryonic palatal mesenchyme cells derived from E13.5 secondary palatal shelves. To overcome the common barrier of dephosphorylation during protein isolation, adaptations and modifications to standard laboratory methods that allow for isolation of phosphoproteins are discussed. Additionally, best practices are provided for proper analysis and quantification of phosphoproteins following western blotting of whole cell protein lysates. These techniques, particularly in combination with pharmacological inhibitors and/or murine genetic models, can be used to gain greater insight into the dynamics and roles of various phosphoproteins active during craniofacial development.

The protocol presents a method for isolating whole cell protein lysates from dissected mouse embryo facial processes or cultured mouse embryonic palatal mesenchyme cells and performing subsequent western blotting to assess phosphorylated protein levels.

Mammalian craniofacial development is a complex morphological process during which multiple cell populations coordinate to generate the frontonasal ...

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 ...

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 ...

Analyzing Alveolar Bone Remodeling to Explore Skeletal Mechanical Biology

Using Inducible Osteoblastic Lineage-Specific Stat3 Knockout Mice to Study Alveolar Bone Remodeling During Orthodontic Tooth Movement

The alveolar bone, with a high turnover rate, is the most actively-remodeling bone in the body. Orthodontic tooth movement (OTM) is a common artificial process of alveolar bone remodeling in response to mechanical force, but the underlying mechanism remains elusive. Previous studies have been unable to reveal the precise mechanism of bone remodeling in any time and space due to animal model-related restrictions. The signal transducer and activator of transcription 3 (STAT3) is important in bone metabolism, but its role in osteoblasts during OTM is unclear. To provide in vivo evidence that STAT3 participates in OTM at specific time points and in particular cells during OTM, we generated a tamoxifen-inducible osteoblast lineage-specific Stat3 knockout mouse model, applied orthodontic force, and analyzed the alveolar bone phenotype. 
Micro-computed tomography (Micro-CT) and stereo microscopy were used to access OTM distance. Histological analysis selected the area located within three roots of the first molar (M1) in the cross-section of the maxillary bone as the region of interest (ROI) to evaluate the metabolic activity of osteoblasts and osteoclasts, indicating the effect of orthodontic force on alveolar bone. In short, we provide a protocol for using inducible osteoblast lineage-specific Stat3 knockout mice to study bone remodeling under orthodontic force and describe methods for analyzing alveolar bone remodeling during OTM, thus shedding new light on skeletal mechanical biology.

Author Spotlight: Comparing Alveolar and Long Bone Remodeling to Explore OTM Model Potential 

This study provides a protocol for using inducible osteoblast lineage-specific Stat3 knockout mice to study bone remodeling under orthodontic force and describes methods for analyzing alveolar bone remodeling during orthodontic tooth movement, thus shedding light on skeletal mechanical biology.

The alveolar bone, with a high turnover rate, is the most actively-remodeling bone in the body. Orthodontic tooth movement (OTM) is a common artificial ...

Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model

Transgenic mouse models are powerful for understanding the critical genes controlling osteoclast differentiation and activity, and for studying mechanisms and pharmaceutical treatments of osteoporosis. Cathepsin K (Ctsk)-Cre mice have been widely used for functional studies of osteoclasts. The signal transducer and activator of transcription 3 (STAT3) is relevant in bone homeostasis, but its role in osteoclasts in vivo remains poorly defined. To provide the in vivo evidence that STAT3 participates in osteoclast differentiation and bone metabolism, we generated an osteoclast-specific Stat3 deletion mouse model (Stat3 fl/fl; Ctsk-Cre) and analyzed its skeletal phenotype. Micro-CT scanning and 3D reconstruction implied increased bone mass in the conditional knockout mice. H&#38;E staining, calcein and alizarin red double staining, and tartrate-resistant acid phosphatase (TRAP) staining were performed to detect bone metabolism. In short, this protocol describes some canonical methods and techniques to analyze skeletal phenotype and to study the critical genes controlling osteoclast activity in vivo.

Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model

This protocol describes a canonical method to understand the critical genes controlling osteoclast activity in vivo. This method uses a transgenic mouse model and some canonical techniques to analyze skeletal phenotype.

Transgenic mouse models are powerful for understanding the critical genes controlling osteoclast differentiation and activity, and for studying mechanisms ...

Notch Signaling Pathway

The Notch signaling pathway is a major intracellular signaling pathway that is highly conserved over a broad spectrum of metazoan species. It stands unique from other intracellular signaling mechanisms in animals because notch protein itself acts as the receptor as well as the primary signaling molecule.
The Notch gene came into the limelight in 1914 after the discovery that its mutation in Drosophila melanogaster leads to a serrated (or &#34;notched&#34;) wing margin phenotype. It was not until 1985 that the gene was shown to encode a transmembrane surface receptor, the core signaling component of the pathway.
<h4>The Notch Signaling Mechanism</h4>
The notch receptor present on the target cell surface undergoes two proteolytic cleavages after its direct interaction with the Delta/Serrate ligand expressed on the surface of the signaling cell. These proteolytic cleavages - sequentially mediated by ADAM metalloproteinases and the &#947;-Secretase complex - release the cytosolic part of the receptor, called the Notch intracellular domain (NICD), from the membrane into the cell. This NICD is the active signaling molecule, which then translocates to the nucleus where it directly participates in the regulation of the expression of Notch target genes.
<h4>Regulation of Signaling</h4>
The notch signaling is a complex signaling circuitry regulated at various levels:
<ol>
	<li>The ligand specificity of the notch receptors is regulated by the O-glycosylation of the Notch protein prior to membrane translocation.</li>
	<li>The expression of the ligand/receptor ratio on the cells determines its role as a signaling cell or a responding cell.</li>
	<li>The signaling is only activated by the trans-interaction between the ligand and the receptor expressed on two different cells. The cis-interaction between the ligand and the receptor expressed on the same cell is inhibitory.</li>
	<li>The extent of the cell to cell contact in tissues can also affect the strength and frequency of notch signaling.</li>
</ol>
<h4>Notch Signaling Functions and Associated Diseases</h4>
Recent studies on vertebrates and invertebrates indicate that besides driving multiple aspects of metazoan development, the notch signaling pathway also controls stem cell fate and maintenance in adult tissues. However, the pathway is gene dosage-sensitive, and the appropriate level of signaling is vital for normal differentiation and tissue homeostasis. Any dysregulation in the pathway, either by direct mutations or altered signaling output, may lead to disease states. These include vascular dementia, and developmental disorders like Adams-Oliver syndrome, or Alagille syndrome.

Regulation of Notch Signaling Pathway and Stem Cells&#039; Fate

The Notch signaling pathway is a major intracellular signaling pathway that is highly conserved over a broad spectrum of metazoan species. It stands ...

Stimulation of Notch Signaling in Mouse Osteoclast Precursors

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Chapters in this video

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

Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis

Isolation of Whole Cell Protein Lysates from Mouse Facial Processes and Cultured Palatal Mesenchyme Cells for Phosphoprotein Analysis

Differentiation and Characterization of Osteoclasts from Human Induced Pluripotent Stem Cells

Osteoclast Derivation from Mouse Bone Marrow

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

Using Inducible Osteoblastic Lineage-Specific Stat3 Knockout Mice to Study Alveolar Bone Remodeling During Orthodontic Tooth Movement

Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model

Notch Signaling Pathway