This work was supported by the National Institutes of Health (R01 AR061164-01A1).

All experimental procedures were approved by the Institutional Animal Care and Use Committee at the Maine Medical Center Research Institute.
1. Collection Tubes Preparation
<ol>
	<li>Make the BMSC culture media by supplementing Minimum Essential Medium &#945; (MEM &#945;) with 10% Fetal Bovine Serum and 1% of Penicillin/Streptomycin.</li>
	<li>Place 100 &#181;L of BMSC culture media in 1.5 mL microcentrifuge tubes. 3 bones will usually fit in one single tube so prepare accordingly.</li>
	<li>Cut the ends of 200 &#181;L pipette tips enough so that they fit in the centrifuge tubes. Insert the tips inside the tubes and ensure that the lids can close before placing the tubes on ice. 
	NOTE: Alternatively, 0.75 mL microcentrifuge tubes with the ends cut can be inserted into the 1.5 mL microcentrifuge tube.</li>
</ol>
2. Harvest of Bones from Mice
<ol>
	<li>Euthanize the animals using CO2 followed by cervical dislocation and spray them with 70% ethanol. 
	NOTE: Ensure that animals from the same sex and preferably the same age are used. We routinely use animals aged 7 to 8 weeks. We recommend pooling cells from at least 3 animals per experimental groups to yield a better representative and reproducible cell population.</li>
	<li>Place the euthanized mouse on its back on a dissecting board. Use forceps to create a tent of skin on the abdomen. Make a small incision (around 1 cm) using sterile dissecting scissors. Peel back the skin towards the limbs and the feet from the cut in order to expose the lower abdomen and the legs.
	<ol>
		<li>Cut below the ankle joint to remove the foot. Cut along the iliac crest at the hip to separate the femur head from the hipbone. While the mouse is still on its back, make an incision in the middle of the hipbone to separate the two iliac bones.</li>
	</ol>
	</li>
	<li>Using lint-free wipes, carefully remove the muscle from the femurs, tibias, and iliac bones. 
	NOTE: Cutting as much of the tendons and muscle from the bone as possible makes the cleaning faster and easier with the lint-free wipes (e.g., kimwipes). Use care when manipulating the bones as they tend to break easily and their fragility may be increased with certain genotypes.</li>
	<li>Once all the samples have been dissected, place them in PBS on ice and move to a sterile culture hood for the remaining steps of isolation. 
	NOTE: Working aseptically as much as possible will reduce the potential for contamination in the cultures.</li>
	<li>Make small cuts (approximately 1 to 2 mm) at both the proximal and distal ends of the bones before placing them in the sectioned tips within the centrifuge tubes. 
	Note: Ensure to cut a sufficient amount so that marrow can be centrifuged out; however, care must be taken to avoid cutting too much as cell populations tend to vary based on proximity within the marrow space.</li>
	<li>Centrifuge at 10,000 x g for 15 s at room temperature. Ensure that all the marrow is flushed and pelleted at the bottom of the 1.5 mL tube while the bones are inside the inserts (either 200 &#181;L pipet tip or 0.75 mL microcentrifuge tube). Once all the marrow is collected, remove the inserts with the bones from the collection tubes. 
	NOTE: If all the marrow is flushed, the bones will appear white. However, if marrow remains in certain bones, try cutting ends again and centrifuge.</li>
</ol>
3. Cell Suspension
<ol>
	<li>Using a 25 G needle, pull the cell pellets up and down slowly to break up clumps and combine all the samples into a single 15 mL conical tube. Add 10 mL of BMSC Culture Media per 500 &#181;L of samples.</li>
	<li>Place a 70 &#181;m filter on top of a 50 mL conical tube and pass the cell suspension through this filter in order to remove possible bone fragments.</li>
</ol>
4. Plating and Culture of Mixed Populations of Bone Marrow Cells (Total BMCs)
NOTE: Cells are cultured at 37 &#176;C in an incubator with 5% CO2.
<ol>
	<li>Calculate the number of cells and plate them at 1.0 x 106 cells/cm2 in culture media.Allow 72 h of mixed culture for cells to attach. 
	NOTE: We recommend diluting the cell suspension 20x in culture media and diluting again in trypan blue before counting the cells using a hemocytometer. For 7-week-old male C57Bl6/J mice, we routinely yield a cell number of around 50 x 106 cells but age, sex, and strain can affect cell number greatly. 
	NOTE: A mix population of cells should attach at the bottom of the culture well.</li>
	<li>After 72 h, remove the media and replace it with fresh media. Continue changing the media every other day and check for confluency. When the cells reach 80-100% confluency, they are ready for differentiation. 
	NOTE: Because this culture is mixed with both BMSCs and HSCs, it can be directly used for osteoblastogenesis, adipogenesis as well as osteoclastogenesis.</li>
</ol>
5. Plating and Culture of the Split Population of BMSCs (Adherent BMSCs)
NOTE: Cells are cultured at 37 &#176;C in an incubator with 5% CO2.
<ol>
	<li>Plate all the cells collected from the femurs, tibias, and iliac bones of one mouse into a 10 cm culture dish (55 cm2 of culture area). 
	NOTE: When pooling 2-3 C57BL/6J mice, we typically plate this &#39;total&#39; bone marrow population in a 150 cm2 flask.</li>
	<li>After 48 h of mixed culture, remove the culture media (containing the non-adherent HSCs). If osteoclastogenesis experiments are to be performed, set this population of non-adherent cells aside (see section 7), if not, aspirate and discard these cells.</li>
	<li>Wash the plate/flask with PBS carefully as to not disturb the attached cells.</li>
	<li>Remove PBS and lift the cells by adding 0.25% trypsin and incubating at 37 &#176;C for 1-3 min. Quench the trypsin by adding cell media to the cell suspension. At this point, BMSCs, which are now lifted, can be counted (as previously done in step 4.1) and plated for future experiments.</li>
	<li>Calculate the number of cells needed for plating. Centrifuge the required volume of cell suspension at 1,000 x g for 5 min at room temperature. 
	Note: Cells are typically plated based on end-point experiments. For example, if protein/RNA is to be isolated we typically plate at a higher density (~1.0-2.0 x 106 cells/well in 6-well plate).</li>
	<li>Remove the media and resuspend the cell pellet in the volume of culture media needed for plating. Plate the cells in culture media according to the experiments and allow them to attach for a few days. When the BMSCs reach confluency, proceed to perform differentiation.</li>
</ol>
6. Differentiation of BMSCs
<ol>
	<li>Osteoblasts
	<ol>
		<li>Make osteoblast differentiation media by adding 800 &#181;L of 1 M &#946;-glycerol phosphate in PBS and 200 &#181;L of 0.5 M ascorbic acid in PBS to 99 mL of BMSC culture media (see step 1.1 for the composition of the BMSC culture media).
		<ol>
			<li>Once BMSCs cultures (either total BMCs from step 4.3 or adherent BMSCs from step 5.6) are confluent, differentiate them into osteoblasts by switching the culture media to osteoblast differentiation media.</li>
		</ol>
		</li>
		<li>Replace the differentiation media every other day. Cells start producing white nodules of mineralization. Visualize them with the naked eye on the bottom of the well; this process can occur within a few days to 3 weeks.
		<ol>
			<li>In order to assess osteoblastogenesis, perform alkaline phosphatase staining and/or Von Kossa staining on the wells as described in section 8. 
			NOTE: Change the media carefully by tilting the plates at a slight angle in order to avoid disruption of the cell monolayer.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Adipocytes
	<ol>
		<li>Make adipocyte base media by mixing together 90 mL of Dulbecco Modified Eagle Medium (DMEM) High Glucose, 10 mL of Fetal Bovine Serum, 1 mL of Penicillin/Streptomycin, 100 &#181;L 20 mM Rosiglitazone and 100 &#181;L of 2 mM Insulin. If needed, store this media at 4 &#176;C for the week during the differentiation experiment.</li>
		<li>Make adipocyte induction media by adding 200 &#181;L of 62.5 mM 3-isobutyl-1-methylxanthine (IBMX) and 25 &#181;L of 1 mM Dexamethasone to 25 mL of Adipocyte Base Media.</li>
		<li>Once BMSCs cultures (either total BMCs from step 4.3 or adherent BMSCs from step 5.6) are confluent, change the media to adipocyte induction media. Consider this Day 0 of differentiation.</li>
		<li>After 48 h, remove the media and refresh the culture with adipocyte induction media.</li>
		<li>On Day 4, switch to adipocyte base media. 
		NOTE: Lipid droplets can start appearing as early as Day 2 or Day 4.</li>
		<li>On Day 7, observe that cells have accumulated maximum lipid droplets and display a phenotype similar to a mature adipocyte. Do not culture past this point, as it may result cell death/detachment.</li>
	</ol>
	</li>
</ol>
7. Differentiation of HSCs into Osteoclasts
<ol>
	<li>Make Osteoclast Differentiation Media by adding 50 &#181;L of 25 &#181;g/mL of Receptor Activator of NFKB Ligand (RANKL) and 15 &#181;L of 25 &#181;g/mL of macrophage Colony Stimulating Factor (mCSF) to 25 mL of BMSC Culture Media.</li>
	<li>Once the total BMCs (from step 4.3) or the non-adherent cells (from step 5.2) are attached to the well, culture media can be switched to Osteoclast Differentiation Media. Replenish differentiation media every other day.</li>
	<li>Observe the osteoclast cultures every day under a microscope at 10X magnification. Observe that osteoclasts fuse and form multinucleated cells, typically around Day 5-7 of differentiation.</li>
</ol>
8. Staining
<ol>
	<li>Osteoblasts 
	NOTE: For the Alkaline Phosphatase (AP) staining, we often use an AP staining kit (see Table of Materials) according to the guidelines.
	<ol>
		<li>Perform AP staining by following kit manufacturer&#39;s guidelines.
		<ol>
			<li>Aspirate media and rinse cells with PBS. Fix the cells with 4% Paraformaldehyde (PFA) in PBS for 12 min at room temperature.</li>
			<li>In the meantime, make the AP solution using the reagents from the AP staining kit by adding 500 &#181;L Sodium Nitrite to 500 &#181;L FRV-Alkaline solution and mixing gently; let stand for 2 min. Add 22.5 mL dH2O and 500 &#181;L Naphthol AS-BI Alkaline. Mix gently. 
			Caution: PFA is toxic and must be handled with care.</li>
			<li>Once the cells are fixed, rinse with PBS and add AP solution to each well (1 to 2 mL per well). Cover with aluminum foil and let stain for 30 min at room temperature protected from light. Aspirate the solution, wash with distilled water, and allow the plate to dry.</li>
		</ol>
		</li>
		<li>Acquire images of stained colonies using a camera in order to observe alkaline phosphatase positive cells.</li>
		<li>Alternatively perform Von Kossa instead of alkaline phosphatase staining. For this, wash wells thoroughly with distilled water as any remaining PBS will precipitate with silver nitrate.
		<ol>
			<li>Fix the cells with 4% PFA in PBS for 12 min at room temperature. Then, add enough 5% silver nitrate solution to cover the well and expose to strong light for 1 h at room temperature.</li>
		</ol>
		</li>
		<li>Remove the silver nitrate solution and rinse each well with distilled water. Add 5% sodium thiosulfate solution to the cells and let stand for 3 min.</li>
		<li>Aspirate the sodium thiosulfate and wash with distilled water. Let the plate dry and observe the mineral deposit stained in dark brown/black with naked eye.</li>
	</ol>
	</li>
	<li>Adipocytes
	<ol>
		<li>For Oil Red O (ORO) staining, fix the cells using 10% Neutral Buffered Formalin for 1 h at room temperature.</li>
		<li>In the meantime, make the ORO Stock Solution by dissolving 0.035 g in 10 mL of isopropanol. This is sufficient for a 6-well plate. Mix and pass the solution using one filter paper (pore size 11 &#181;m).</li>
		<li>Make the ORO Working Solution by diluting 9 mL of ORO stock solution with 6 mL of distilled water. Leave the solution on a rocker until ready for use.</li>
		<li>Prepare 60% isopropanol in distilled water (make enough to cover each well).</li>
		<li>Once the cells are fixed, remove the fixative and wash once with distilled water. Replace the water with the 60% isopropanol solution for 1 min. 
		NOTE: Add liquid slowly while tipping the plate; adipocytes dislodge very easily.</li>
		<li>Remove the isopropanol and add enough ORO Working Solution to cover all the cells. Incubate for 15 min on a low-speed rocker at room temperature. Remove ORO and wash twice with distilled water. 
		NOTE: At this point, ORO-stained lipid droplets can be visualized under the microscope.</li>
		<li>To quantify ORO, destain the ORO by adding 1 mL of 100% isopropanol to each well and by rocking slowly for 10 min.</li>
		<li>In the meantime, prepare a dilution series to create a standard curve based on the following 8 standards using ORO Working Solution (2,100 &#181;g/mL) and isopropanol as a diluent: 500, 350, 300, 250, 150, 100, 75, and 0 &#181;g/mL.</li>
		<li>Load 200 &#181;L of the standards and the destained samples in triplicates on a plate and read the absorbance at 490 nm.
		<ol>
			<li>Determine concentration of ORO for each sample based on the standard curve. 
			Note: We recommend normalizing the concentration ORO to cell number. Cell number can be quantified by crystal violet staining or DNA quantity.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Osteoclasts 
	NOTE: When ready, we suggest staining osteoclasts for Tartrate-Resistant Acid Phosphatase (TRAP) using the TRAP staining kit (see Table of Materials).
	<ol>
		<li>For TRAP staining, fix cells for 30 min at room temperature with 2.5% glutaraldehyde in PBS. In the meantime, make the TRAP solution by mixing together 50 &#181;L of Fast Garnets Base Solution, 50 &#181;L of Sodium Nitrite, 4.55 mL of distilled water, 50 &#181;L of Napthol AS-Biphosphate, 200 &#181;L of Acetate solution and 100 &#181;L Tartrate solution.</li>
		<li>Aspirate off fixative, and rinse the cells with PBS twice before adding TRAP solution for 1 h at 37 &#176;C.</li>
		<li>Wash with distilled water before counting osteoclasts under a microscope (magnification 10X). The TRAP+ cells appear purple with 3 or more nuclei.</li>
	</ol>
	</li>
</ol>

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	<li>Friedenstein, A. J., Latzinik, N. W., Grosheva, A. G., Gorskaya, U. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marrow+microenvironment+transfer+by+heterotopic+transplantation+of+freshly+isolated+and+cultured+cells+in+porous+sponges.">Marrow microenvironment transfer by heterotopic transplantation of freshly isolated and cultured cells in porous sponges.</a> Exp Hematol. 10 (2), 217-227 (1982).</li><li>Chen, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+growth+and+differentiation+of+osteoprogenitors+in+mouse+bone+marrow+stromal+cell+cultures+by+increased+donor+age+and+glucocorticoid+treatment.">Inhibition of growth and differentiation of osteoprogenitors in mouse bone marrow stromal cell cultures by increased donor age and glucocorticoid treatment.</a> Bone. 35 (1), 83-95 (2004).</li><li>Kokabu, 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=BMP3+suppresses+osteoblast+differentiation+of+bone+marrow+stromal+cells+via+interaction+with+Acvr2b.">BMP3 suppresses osteoblast differentiation of bone marrow stromal cells via interaction with Acvr2b.</a> Mol Endocrinol. 26 (1), 87-94 (2012).</li><li>Ghali, O., 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=Dexamethasone+in+osteogenic+medium+strongly+induces+adipocyte+differentiation+of+mouse+bone+marrow+stromal+cells+and+increases+osteoblast+differentiation.">Dexamethasone in osteogenic medium strongly induces adipocyte differentiation of mouse bone marrow stromal cells and increases osteoblast differentiation.</a> BMC Cell Biol. 16, 9 (2015).</li><li>Kretlow, J. D., 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=Donor+age+and+cell+passage+affects+differentiation+potential+of+murine+bone+marrow-derived+stem+cells.">Donor age and cell passage affects differentiation potential of murine bone marrow-derived stem cells.</a> BMC Cell Biol. 9, 60 (2008).</li><li>DeMambro, V. E., 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=Igfbp2+Deletion+in+Ovariectomized+Mice+Enhances+Energy+Expenditure+but+Accelerates+Bone+Loss.">Igfbp2 Deletion in Ovariectomized Mice Enhances Energy Expenditure but Accelerates Bone Loss.</a> Endocrinology. 156 (11), 4129-4140 (2015).</li><li>Maridas, D. E., 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=IGFBP-4+regulates+adult+skeletal+growth+in+a+sex-specific+manner.">IGFBP-4 regulates adult skeletal growth in a sex-specific manner.</a> J Endocrinol. 233 (1), 131-144 (2017).</li><li>Nadri, 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=An+efficient+method+for+isolation+of+murine+bone+marrow+mesenchymal+stem+cells.">An efficient method for isolation of murine bone marrow mesenchymal stem cells.</a> Int J Dev Biol. 51 (8), 723-729 (2007).</li><li>Sreejit, P., Dilip, K. B., Verma, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+mesenchymal+stem+cell+lines+from+murine+bone+marrow.">Generation of mesenchymal stem cell lines from murine bone marrow.</a> Cell Tissue Res. 350 (1), 55-68 (2012).</li><li>Abdallah, B. M., 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=CD34+defines+an+osteoprogenitor+cell+population+in+mouse+bone+marrow+stromal+cells.">CD34 defines an osteoprogenitor cell population in mouse bone marrow stromal cells.</a> Stem Cell Res. 15 (3), 449-458 (2015).</li><li>Akiyama, 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=Characterization+of+bone+marrow+derived+mesenchymal+stem+cells+in+suspension.">Characterization of bone marrow derived mesenchymal stem cells in suspension.</a> Stem Cell Res Ther. 3 (5), 40 (2012).</li><li>Lin, C. S., Ning, H., Lin, G., Lue, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+CD34+truly+a+negative+marker+for+mesenchymal+stromal+cells?.">Is CD34 truly a negative marker for mesenchymal stromal cells?.</a> Cytotherapy. 14 (10), 1159-1163 (2012).</li></ol>

The authors disclose that they have no financial conflict of interests.

In this article, two methods of culture of BMSCs are presented with their advantages and limitations. Isolating cells coming from the bone marrow is a relatively effortless process. However, obtaining a cell population representative of the mesenchymal stem cells or osteoclastic progenitors can be challenging due to the diverse cellular environment of the marrow cavity.
Culturing the entirety of the bone marrow contents provides a close representation of the in vivo microenvironment. ...

Primary murine BMSCs are commonly used as an in vitro model of mesenchymal stem cells since their discovery in the early 1980s1. Indeed, cultures of plastic-adherent cells flushed from the bone marrow cavity of long bones maintain the capacity to be differentiated into osteoblasts, osteoclasts, chondrocytes, or adipocytes in many studies2,3,4,5. However, the bone marrow is a unique tissue composed of many different cell populations including, but not limited to, BMSCs, HSCs, endothelial, and immune cells. Thus, isolation and culture techniques can yield cell populations with different homogeneity. Using such techniques to test the differentiation potential from cells can be challenging. For example, when comparing cells from mice with different genotypes, starting with a mixed cell population limits the interpretation of the data. Conversely, obtaining homogenous populations of BMSCs and HSCs can be technically difficult and may not be as representative of an ex vivo model.
In our laboratory, we are primarily interested in the utilization of BMSCs due to their potential to be differentiated into osteoblasts, osteoclasts, and adipocytes. Here, we present techniques of BMSCs and HSCs isolation and culture used to assess osteoblastogenesis or adipogenesis in vitro, as well as cultures of HSCs to differentiate into osteoclasts. One method uses a mixed population of Bone Marrow Cells (BMCs) containing BMSCs and HSCs directly suitable for adipogenesis, osteoblastogenesis, and osteoclastogenesis (called Total BMCs). This method is a closer ex vivo representation of the heterogeneity found amongst the cells of the bone marrow microenvironment. Another method separates adherent from non-adherent cells in an attempt to culture &#34;purer&#34; populations of BMSCs and HSCs (called Adherent BMSCs). The later method allows the cell culture experiments to start with a more accurate number of BMSCs or HSCs and reduces the potential of complex indirect effects of other cell populations that remain in the culture. Both methods have been previously published and used to address different research questions6,7,8,9.

<table><tbody><tr><td>200&amp;mu;L pipet tips&amp;nbsp;</td><td>Rainin</td><td>17014401</td><td></td></tr><tr><td>1.5 mL centrifuge tubes</td><td>USA Scientific</td><td>1615-5500</td><td></td></tr><tr><td>15 mL conical tube&amp;nbsp;</td><td>VWR</td><td>89039-668</td><td></td></tr><tr><td>50 mL conical tube</td><td>VWR</td><td>89039-660</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>21-040-CM</td><td></td></tr><tr><td>Ethanol</td><td>Fisher Science</td><td>04-355-451</td><td></td></tr><tr><td>Dissection tools</td><td></td><td></td><td></td></tr><tr><td>70 &amp;mu;m filters</td><td>BD Falcon</td><td>352350</td><td></td></tr><tr><td>0.25% trypsin</td><td>Gibco</td><td>25200</td><td></td></tr><tr><td>Kimwipe</td><td>VWR</td><td>82003-820</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Electron Microscopy Science</td><td>15710</td><td></td></tr><tr><td>Alkaline Phosphatase kit</td><td>Sigma-Aldrich</td><td>86R</td><td></td></tr><tr><td>Silver Nitrate</td><td>Sigma-Aldrich</td><td>S6506</td><td></td></tr><tr><td>Sodium Thiosulfate</td><td>Sigma-Aldrich</td><td>S7026</td><td></td></tr><tr><td>Oil Red O</td><td>Sigma-Aldrich</td><td>O0625</td><td></td></tr><tr><td>Isopropanol</td><td>Sigma-Aldrich</td><td>190764</td><td></td></tr><tr><td>10% Neutral Buffered Formalin</td><td>Sigma-Aldrich</td><td>F5554-4L</td><td></td></tr><tr><td>Whatman filter Grade 1</td><td>Sigma-Aldrich</td><td>Z274852</td><td></td></tr><tr><td>Tartrate-Resistant Acid Phosphatase kit</td><td>Sigma-Aldrich</td><td>387A</td><td></td></tr><tr><td>Glutaraldehyde</td><td>Electron</td><td>16220</td><td></td></tr><tr><td>MEM&amp;alpha;</td><td>Gibco</td><td>12571</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>VWR</td><td>97068-085</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>&amp;beta;-glycerol phosphate</td><td>Sigma-Aldrich</td><td>G9891</td><td></td></tr><tr><td>Ascorbic acid</td><td>Sigma-Aldrich</td><td>A4544</td><td></td></tr><tr><td>DMEM High Glucose&amp;nbsp;</td><td>Sigma-Aldrich</td><td>D5796</td><td></td></tr><tr><td>Rosiglitazone</td><td>Cayman Chemical</td><td>717410</td><td></td></tr><tr><td>Insulin</td><td>Sigma-Aldrich</td><td>I6634</td><td></td></tr><tr><td>IBMX</td><td>Sigma-Aldrich</td><td>I5879</td><td></td></tr><tr><td>Dexamethasone</td><td>Sigma-Aldrich</td><td>D4902</td><td></td></tr><tr><td>RANKL</td><td>Peprotech</td><td>310-01</td><td></td></tr><tr><td>mCSF</td><td>Peprotech</td><td>315-02</td><td></td></tr><tr><td>Axio Observer inverter microscope</td><td>Zeiss</td><td></td><td></td></tr></tbody></table>

isolation, culture, and differentiation of bone marrow stromal cells and osteoclast progenitors from mice

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within the bone marrow cavity alongside hematopoietic stem cells (HSCs), which can give rise to red blood cells, immune progenitors, and osteoclasts. Thus, extractions of cell populations from the bone marrow results in a very heterogeneous mix of various cell populations, which can present challenges in experimental design and confound data interpretation. Several isolation and culture techniques have been developed in laboratories in order to obtain more or less homogeneous populations of BMSCs and HSCs invitro. Here, we present two methods for isolation of BMSCs and HSCs from mouse long bones: one method that yields a mixed population of BMSCs and HSCs and one method that attempts to separate the two cell populations based on adherence. Both methods provide cells suitable for osteogenic and adipogenic differentiation experiments as well as functional assays.

Overview of the Cell Cultures 
The two culture techniques allow the assessment of differentiation on a mixed population of BMCs comprised of BMSCs and HSCs (Total BMCs, Figure 1A) or a population of BMSCs split from HSCs (Adherent BMSCs, Figure 1B). 48 hr after the cells from the marrow are isolated and plated (Figure 1C), they rapidly attach to the bottom of the plastic culture...

Watch this Scientific Journal Video about Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice at JoVE.com

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

In this article, we present methods to isolate and differentiate bone marrow stromal cells and hematopoietic stem cells from mouse long bones. Two different protocols are presented yielding different cell populations suitable for expansion and differentiation into osteoblasts, adipocytes, and osteoclasts.

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

isolation-culture-differentiation-bone-marrow-stromal-cells

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Developmental Biology

30.8K Views.  Maine Medical Center Research Institute. In this article, we present methods to isolate and differentiate bone marrow stromal cells and hematopoietic stem cells from mouse long bones. Two different protocols are presented yielding different cell populations suitable for expansion and differentiation into osteoblasts, adipocytes, and osteoclasts.

Video: Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

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 ...

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of &#34;contaminating&#34; progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for ...

Immunology and Infection

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Biology

Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method for isolating DCs primarily involves inducing bone marrow (BM) differentiation into DCs by injecting large doses (&#62;10 ng/mL) of granulocyte-macrophage colony-stimulating factor/interleukin-4 (GM-CSF/IL-4), making the procedure complex and expensive. In this protocol, using all BM cells cultured in 10 ng/mL GM-CSF/IL-4 medium, after 3-4 half-culture exchanges, up to 2.7 x 107 CD11c+ cells&#160;(DCs) per mouse (two femurs) were harvested with a purity of 80%-95%. After 10 days in culture, the expression of CD11c, CD80, and MHC II increased, whereas the number of cells decreased. The number of cells peaked after 7 days of culture. Moreover, this method only took 10 min to harvest all bone marrow cells, and a high number of DCs were obtained after 1 week of culture.

Here, we present an economical and efficient method to isolate and generate high-purity bone marrow-derived dendritic cells from mice after 7 days of culture with 10 ng/mL GM-CSF/IL-4.

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method ...

Isolation and Culture of Bone Marrow-Derived Macrophages from Mice

Macrophages have important effector functions in homeostasis and inflammation. These cells are present in every tissue in the body and have the important ability to change their profile according to the stimuli present in the microenvironment. Cytokines can profoundly affect macrophage physiology, especially IFN-&#947; and interleukin 4, generating M1 and M2 types respectively. Because of the versatility of these cells, the production of a population of bone marrow-derived macrophages can be a basic step in many experimental models of cell biology. The aim of this protocol is to help researchers in the isolation and culture of macrophages derived from bone marrow progenitors. Bone marrow progenitors from pathogen-free C57BL/6 mice are transformed into macrophages upon exposure to macrophage colony-stimulating factor (M-CSF) that, in this protocol, is obtained from the supernatant of the murine fibroblast lineage L-929. After incubation, mature macrophages are available for use from the 7th to the 10th day. A single animal can be the source of approximately 2 x 107 macrophages. Therefore, it is an ideal protocol for obtaining large amounts of primary macrophages using basic methods of cell culture.

The present protocol describes the isolation and culture of bone marrow-derived macrophages from mice.

Macrophages have important effector functions in homeostasis and inflammation. These cells are present in every tissue in the body and have the important ...

Optimizing Stromal Cell Isolation from Thymus and Bone Marrow

Stromal Cell Isolation From Hematopoietic Organs

Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens through which to study previously unresolved heterogeneity at steady state, as well as changes in cell type representation induced by extrinsic stresses or during aging. Here, we present step-wise protocols for the isolation of high-quality stromal cell populations from murine and human thymus, as well as murine bone and bone marrow. Cells isolated through these protocols are suitable for generating high-quality single-cell multiomics datasets. The impacts of sample digestion, hematopoietic lineage depletion, FACS analysis/sorting, and how these factors influence compatibility with single-cell sequencing are discussed here. With examples of FACS profiles indicating successful and inefficient dissociation and downstream stromal cell yields in post-sequencing analysis, recognizable pointers for users are provided. Considering the specific requirements of stromal cells is crucial for acquiring high-quality and reproducible results that can advance knowledge in the field.

Author Spotlight: Advancing Hematopoietic Research Using Stromal Cell Isolation for Single Cell Sequencing

Here we present protocols that enable isolation of stromal cells from murine bone, bone marrow, thymus and human thymic tissue compatible with single-cell multiomics.

Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens ...

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 ...

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.

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or ...

Utilizing Low Oxygen Tension to Reduce Hematopoietic Cells in Murine Bone Marrow Stromal Cell Cultures

Currently, there remains a lack of universally accepted markers to prospectively isolate a homogeneous population of skeletal stem cells (SSCs). For this reason, BMSCs, which support hematopoiesis and contribute to all the functions of the skeleton, continue to be widely used to study multipotent mesenchymal progenitors (MMPs) and to infer SSC function. Moreover, given the breadth of transgenic murine models used to study musculoskeletal diseases, the use of BMSCs also serves as a powerful tool to examine the molecular mechanisms regulating MMPs and SSCs. However, common isolation procedures for murine BMSCs result in over 50% of recovered cells being of hematopoietic origins, potentially hindering the interpretation of the data generated during these studies. Here, we describe a method using low oxygen tension or hypoxia for the selective elimination of CD45+ cells in BMSC cultures. Importantly, this method can be easily implemented to not only reduce hemopoietic contaminants but to also enhance the percentage of MMPs and putative SSCs in BMSC cultures.

A routine culture of bone marrow stromal cells (BMSCs) leads to the isolation of heterogeneous cell populations, with many cells being of hematopoietic origins. Here, we describe a method that utilizes low oxygen tension to greatly reduce hematopoietic contaminants in murine BMSC cultures.

Currently, there remains a lack of universally accepted markers to prospectively isolate a homogeneous population of skeletal stem cells (SSCs). For this ...

In Vitro Mouse Bone Marrow Stromal Cell Differentiation into Adipocytes

Source: Maridas, D. E. et al., <a href="https://app.jove.com/v/56750">Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice.</a>&#160;J. Vis. Exp.&#160;(2018)
This video demonstrates the differentiation of bone marrow stromal cells (BMSCs) into adipocytes. In the two-step process, in the presence of specific growth factors and hormones, undifferentiated BMSCs first differentiate into preadipocytes. The second step involving treatment with glucose and insulin leads to the formation of mature adipocytes with multiple lipid droplets.

Source: Maridas, D. E. et al., Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice.&#160;J. Vis. ...