Name: isolation, culture, and differentiation of bone marrow stromal cells and osteoclast progenitors from mice
Uploaded: 2018-01-06T14:00:00
Description: Watch this Scientific Journal Video about Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice at JoVE.com

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...

     

<ol>
	<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>

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.

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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">200&#956;L pipet tips&#160;</td>
			<td style="width:135px;">Rainin</td>
			<td style="width:119px;">17014401</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.5mL centrifuge tubes</td>
			<td style="width:135px;">USA Scientific</td>
			<td>1615-5500</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">15mL conical tube&#160;</td>
			<td style="width:135px;">VWR</td>
			<td>89039-668</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50mL conical tube</td>
			<td style="width:135px;">VWR</td>
			<td>89039-660</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffered Saline (PBS)&#160;</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">21-040-CM</td>
			<td></td>
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			<td height="21" style="height:21px;">Ethanol</td>
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			<td>04-355-451</td>
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			<td height="21" style="height:21px;">Dissection tools</td>
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			<td height="21" style="height:21px;">0.25% trypsin</td>
			<td style="width:135px;">Gibco</td>
			<td style="width:119px;">25200</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Kimwipe</td>
			<td style="width:135px;">VWR</td>
			<td>82003-820</td>
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		<tr height="40">
			<td height="40" style="height:40px;">Paraformaldehyde (PFA)</td>
			<td style="width:135px;">Electron Microscopy Science</td>
			<td style="width:119px;">15710</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alkaline Phosphatase kit</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">86R</td>
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			<td height="21" style="height:21px;">Silver Nitrate</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">S6506</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Sodium Thiosulfate</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">S7026</td>
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			<td style="width:119px;">O0625</td>
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			<td height="21" style="height:21px;">Isopropanol</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td>190764</td>
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			<td height="21" style="height:21px;">10% Neutral Buffered Formalin</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td>F5554-4L</td>
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			<td height="21" style="height:21px;">Whatman filter Grade 1</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z274852</td>
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			<td height="21" style="height:21px;">Tartrate-Resistant Acid Phosphatase kit</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">387A</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Glutaraldehyde</td>
			<td style="width:135px;">Electron</td>
			<td style="width:119px;">16220</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MEM&#945;</td>
			<td style="width:135px;">Gibco</td>
			<td style="width:119px;">12571</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:135px;">VWR</td>
			<td style="width:119px;">97068-085</td>
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			<td height="21" style="height:21px;width:216px;">Penicillin/Streptomycin</td>
			<td style="width:135px;">Invitrogen</td>
			<td style="width:119px;">15140-122</td>
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			<td height="21" style="height:21px;width:216px;">&#946;-glycerol phosphate</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">G9891</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ascorbic acid</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">A4544</td>
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			<td height="21" style="height:21px;width:216px;">DMEM High Glucose&#160;</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">D5796</td>
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			<td height="21" style="height:21px;width:216px;">Rosiglitazone</td>
			<td style="width:135px;">Cayman Chemical</td>
			<td style="width:119px;">717410</td>
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			<td height="21" style="height:21px;width:216px;">Insulin</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">I6634</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">IBMX</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">I5879</td>
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			<td height="21" style="height:21px;width:216px;">Dexamethasone</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">D4902</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RANKL</td>
			<td style="width:135px;">Peprotech</td>
			<td style="width:119px;">310-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">mCSF</td>
			<td style="width:135px;">Peprotech</td>
			<td style="width:119px;">315-02</td>
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		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Axio Observer inverter microscope</td>
			<td style="width:135px;">Zeiss</td>
			<td style="width:119px;"></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 ...

The overall goal of this procedure is to isolate and culture bone marrow stromal cells, or BMSCs. This method can help answer key questions in the bone and adipocyte fields about progenitor cells. The main advantage of this technique is that BMSCs can be isolated in a relatively quick, repeatable, and inexpensive manner.Visual demonstration of this method is useful, as the dissection steps can be difficult to explain with text alone. Before beginning the bone harvest procedure, add 100 microliters of bone marrow stem cell culture medium into one 1.5 milliliter microcentrifuge tube for every three bones to be harvested. And cut the end from one 200 microliter pipette tip per microcentrifuge tube, so that each tip can fit in a single tube with the lids closed.Next, place the first 78-week-old euthanized mouse in the supine position on a dissection board and spray the animal with 70%ethanol. Use forceps to create a tent of skin on the abdomen, and use sterile dissection scissors to make an approximately one centimeter skin incision. Peel back the skin to expose the lower abdomen and legs.Cut along the iliac crest of each hip to separate the femur heads from the pelvis, and make an incision through the midline of the pelvis to separate the iliac bones, and cut below the ankle joints to remove the feet. Then, separate the tibia from the femur by cutting at the knee joint. Then, use lint-free wipes to carefully remove the muscle from the femurs, tibias, and iliac bones.When all of the bones have been collected, place the samples in PBS on ice, and transfer the samples to a sterile culture hood. Using sterile technique, make one to two millimeter cuts at both the proximal and distal ends of each bone before placing three bones into each modified micropipette tip in the microcentrifuge tubes. Harvest the bone marrow from the bones by centrifugation, confirming if the marrow has been completely pelleted to the bottom of each microcentrifuge tube at the end of the spin.If all of the marrow has been collected, the bones will appear white. Remove the micropipette tips and the bones from the collection tubes for proper biosafety disposal. To plate the bone marrow cells, first use a one milliliter syringe equipped with a 25 gauge needle to slowly re-suspend the pellets and to break up any clumps, and pool the samples into a single 15 milliliter conical tube.Add 10 milliliters of bone marrow stem cell culture medium per 500 microliters of sample, and filter the cell suspension through a 70 micron filter into a 50 milliliter conical tube to remove any bone fragments. After counting, seed the bone marrow cells at one times 10 to the sixth cells per square centimeter density in fresh culture medium for a 72 hour incubation at 37 degrees Celsius and 5%CO2. On day three, replace the supernatant with fresh medium, changing the medium every two days thereafter until the cells reach 80%to 100%confluency.To plate a split bone marrow cell population culture, plate the harvested bone marrow cells from one mouse into a 10 centimeter cell culture dish for 48 hours in fresh culture medium in a cell culture incubator. On day two of culture, remove the non-adherent hematopoietic stem cell-containing supernatant, and carefully wash the plate with PBS, without disturbing the adherent bone marrow cells. Replace the PBS with 0.25%trypsin.After one to three minutes at 37 degrees Celsius, quench the reaction with fresh cell culture medium, and count the cells in the resulting cell suspension. Centrifuge the appropriate number of cells for plating, and re-suspend the pellet in enough fresh culture medium for the desired plating density. Then, place the plates in the cell culture incubator until confluency.To induce bone marrow stem cell osteoblast differentiation, replace the cell culture medium with differentiation medium every two days, until the cells begin to produce white nodules of mineralization that can be observed macroscopically. To induce bone marrow stem cell adipocyte differentiation, replace the cell culture medium of a confluent bone marrow cell culture with adipocyte induction medium. After two days of culture, replace the supernatant with fresh adipocyte induction medium, switching to adipocyte base medium on day four.On day seven, confirm that cells have accumulated lipid droplets, and that they display a phenotype similar to mature adipocytes. To differentiate the hematopoietic stem cells into osteoclasts, when a total bone marrow cell culture or non-adherent cell culture becomes adherent, replace the cell culture medium with osteoclast differentiation medium. Change the medium every two days, and check for osteoclast differentiation daily under a light microscope at 10x magnification until the osteoclasts fuse and form multi-nucleated cells, typically around days five to seven of differentiation.Confluent cultures of BMSCs can be differentiated into osteoblasts using an osteogenic medium composed of ascorbic acid and beta-glycerophosphate. After a few days of differentiation, the osteoblasts begin expressing alkaline phosphatase. And further differentiation will trigger osteoid matrix production, and ultimately the mineralization of this matrix.Interestingly, only a proportion of the cells will differentiate into osteoblasts in vitro, as revealed by crystal violet staining. Whether mixed populations or split bone marrow cell populations are used, only a proportion of the cells will differentiate into adipocytes, which appear as round cells with multiple lipid vacuoles that can be stained with oil red O dye. Human stem cell cultures supplemented with murine cell stimulating factor and RANKL will fuse rapidly to form multi-nucleated cells that stain positive for TRAP.These osteoclasts are capable of resorbing mineral matrix in vitro, and can be used to assess osteoclastic activity. Once mastered, this technique can be completed in less than an hour, if it is performed properly, depending on the number of mice. While attempting this procedure, it's important to remember to sterilize the tools and to prepare the media beforehand, so as to be able to work as quickly and as aseptically as possible.After watching this video, you should have a good understanding of how to isolate and culture bone marrow stromal cells.

Research

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