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>

<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>

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. Yet, isolating cells from different groups of mice (genotypes, treatments, ages, etc.) can result in a very different number of BMSCs or HSCs at the inception of the in vitro experiment. Therefore, separating BMSCs and HSCs and re-plating them allow a better control over the cell number at the start of the differentiation experiment. However, reducing the heterogeneity of the cell culture might affect the outcomes of the BMSCs or HSCs as both secrete factors that influence mesenchymal and osteoclastic differentiation. It is important to consider those limitations when designing the isolation and the culture of the BMSCs and HSCs.
The multitude of cell populations present in the bone marrow can introduce challenges in the interpretation of the data. In addition to HSCs and BMSCs, the bone marrow microenvironment also contains differentiated immune cells, red blood cells, endothelial cells,etc. Those populations may remain in the adherent or non-adherent fraction and thus can affect the experimental outcomes. Sorting BMSCs can be an option in order to achieve a &#34;purer&#34; cell population. Indeed, BMSCs are thought to express CD34 while HSCs do not10. However, it was also suggested that the lack of CD34 on the surface of BMSCs may be the result of culture and a study has shown that a non-adherent subset of CD34+ BMSCs could differentiate into osteoblast, adipocytes and chondrocytes11,12. Therefore, when considering sorting to isolate BMSCs, an array of well-defined markers should be designed beforehand. Other obstacles to cell sorting are timing and cost. Indeed, adding a sorting step could increase the time of the isolation protocol considerably as well as a significant increase in cost. In summary, sorting cells present technical challenges that may reduce the yield and quality of BMSCs.

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 border="0" cellpadding="0" cellspacing="0" width="636">
	<tbody>
		<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>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol</td>
			<td style="width:135px;">Fisher Science</td>
			<td>04-355-451</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissection tools</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70&#956;m filters</td>
			<td style="width:135px;">BD Falcon</td>
			<td style="width:119px;">352350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.25% trypsin</td>
			<td style="width:135px;">Gibco</td>
			<td style="width:119px;">25200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kimwipe</td>
			<td style="width:135px;">VWR</td>
			<td>82003-820</td>
			<td></td>
		</tr>
		<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>
			<td></td>
		</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>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silver Nitrate</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">S6506</td>
			<td></td>
		</tr>
		<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>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oil Red O</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">O0625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isopropanol</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td>190764</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10% Neutral Buffered Formalin</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td>F5554-4L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Whatman filter Grade 1</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z274852</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glutaraldehyde</td>
			<td style="width:135px;">Electron</td>
			<td style="width:119px;">16220</td>
			<td></td>
		</tr>
		<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>
			<td></td>
		</tr>
		<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>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin/Streptomycin</td>
			<td style="width:135px;">Invitrogen</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<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>
			<td></td>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rosiglitazone</td>
			<td style="width:135px;">Cayman Chemical</td>
			<td style="width:119px;">717410</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">IBMX</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">I5879</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</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 plate and tend to form a monolayer. Once the cells reach confluence (Figure 1D), they can be used for differentiation assays.
BMSCs Differentiated into Osteoblasts 
Confluent cultures of BMSCs can be differentiated into osteoblasts using an osteogenic media composed of ascorbic acid and &#946;-glycerol phosphate (Figure 2A, B). After a few days of differentiation, osteoblasts start expressing alkaline phosphatase (Figure 2C). Further differentiation will trigger osteoid matrix production and ultimately the mineralization of this matrix (Figure 2D). Interestingly, only a proportion of the cells will differentiate into osteoblasts in vitro. Indeed, staining with crystal violet (Figure 2E) reveals that not all cells express alkaline phosphatase or mineralize the plate.
BMSCs Differentiated into Adipocytes 
Adipogenesis can be tested on cultures of mixed BMCs as well as on the more homogenous cell population. As observed with osteoblast differentiation, whether the mixed populations (Figure 3A) or the split populations (Figure 3B) are used, only a proportion of the cells are adipogenic. Adipocytes appear round with multiple lipid vacuoles that can be stained in red with ORO (Figure 3C). In our experience, in vitro adipocytes always appear multi-vacuolated unlike in vivo white adipocytes. Differences in genotype or culture conditions might increase adipogenesis to a point where adipocytes detach and float in the culture plate. To resolve this, the adipogenesis experiment can be stopped at an earlier time point.
HSCs Differentiated into Osteoclasts 
Osteoclastogenesis can be induced in the cultures by supplementing the media with mCSF and RANKL. HSCs start to fuse rapidly to form multinucleated cells that stain positive for TRAP (Figure 4A). Those osteoclasts are capable of resorbing mineral matrix in vitro and thus can be used to assess osteoclastic activity (Figure 4B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56750/56750fig1.jpg" /> 
Figure 1: Schematic timeline of the protocols for the culture of BMSCs and HSCs. Different culture techniques to yield Total BMCs composed of a mixed population of cells (A) or Adherent BMSCs (B). Representative image of BMSCs culture isolated from C57B6/J mice 48 hr after their plating (C) and after they are split from HSCs (D). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56750/56750fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56750/56750fig2.jpg" /> 
Figure 2: Images of wells of BMSCs cultures differentiated into osteoblasts. BMSCs before (A) and after (B) being placed in osteogenic media. Osteoblasts can be stained for alkaline phosphatase expression in pink (C) and for mineral deposit in brown (D). Crystal violet can be used in order to represent cell number after differentiation (E). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56750/56750fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56750/56750fig3.jpg" /> 
Figure 3: Representative images of the cultures of BMSCs differentiated into adipocytes. ORO-stained adipocytes obtained from a mixed population of BMSCs (A) and a split culture (B) after 7 days in adipogenic media. Image of an adipocyte stained with ORO (C). Quantification of ORO after 7 days in adipogenic media before (D) and after (E) normalization to crystal violet absorbance. Data are presented as means with error bars representing standard error of the mean. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56750/56750fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56750/56750fig4.jpg" /> 
Figure 4: HSCs cultures differentiated into osteoclasts. TRAP staining of osteoclasts appearing as large purple-pink multinucleated cells (A). Mineralized surface stained in black by Von Kossa with resorption pits left by osteoclasts differentiated from HSCs after 7 days (B). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56750/56750fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Nanyang Technological University

Research

JoVE Journal

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

Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the process by which they must be harvested can be associated with significant donor site morbidity. In contrast, adipose-derived stromal cells (ASCs) are more readily abundant and more easily harvested, making them an appealing alternative to BM-MSCs. Like BM-MSCs, ASCs can differentiate into osteogenic lineage cells and can be used in tissue engineering applications, such as seeding onto scaffolds for use in craniofacial skeletal defects. ASCs are obtained from the stromal vascular fraction (SVF) of digested adipose tissue, which is a heterogeneous mixture of ASCs, vascular endothelial and mural cells, smooth muscle cells, pericytes, fibroblasts, and circulating cells. Flow cytometric analysis has shown that the surface marker profile for ASCs is similar to that for BM-MSCs. Despite several published reports establishing markers for the ASC phenotype, there is still a lack of consensus over profiles identifying osteoprogenitor cells in this heterogeneous population. This protocol describes how to isolate and use a subpopulation of ASCs with enhanced osteogenic capacity to repair critical-sized calvarial defects.

The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction.

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the ...

An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade clots in the bone marrow and release MSCs for further use. This protocol provides a rapid and inexpensive alternative to bone marrow resampling. Bone marrow is a major source of MSCs, which are interesting for tissue engineering and autologous stem cell therapies. Upon withdrawal bone marrow may clot, as it comprises all of the hematopoietic system. The resulting clots contain also MSCs that are lost for expansion culture or direct stem cell therapy. We experienced that 74% of canine bone marrow samples contained clots and yielded less than half of the stem cell number expected from unclotted samples. Thus, we developed a protocol for enzymatic digestion of those clots to avoid labor-intense and costly bone marrow resampling. Urokinase - a clinically approved and readily available thrombolytic drug &#8211; clears away the bone marrow clots almost completely. As a consequence, treated bone marrow aspirates yield similar numbers of MSCs as unclotted samples. Also, after urokinase treatment the cells kept their metabolic activity and the ability to differentiate into chondrogenic, osteogenic and adipogenic lineages. Our protocol salvages clotted blood and bone marrow samples without affecting the quality of the cells. This obsoletes resampling, considerably reduces sampling costs and enables the use of clotted samples for research or therapy.

Mesenchymal stem cells are usually obtained from bone marrow and require expansion culture. When samples clot before processing, a protocol using the (enzymatic) thrombolytic drug urokinase can be applied to degrade the clot. Thus, cells are released and available for expansion culture. This protocol provides a rapid and inexpensive alternative to resampling.

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade ...

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. Although numerous immortalized adipogenic cell lines have been established, adipose-derived stem cells from the stromal vascular fraction of subcutaneous white adipose tissues provide a reliable cellular system ex vivo much closer to adipose development in vivo. Pig adipose-derived stem cells (pADSC) are isolated from 7- to 9-day old piglets. The dorsal white fat depot of porcine subcutaneous adipose tissues is sliced, minced and collagenase digested. These pADSC exhibit strong potential to differentiate into adipocytes. Moreover, the pADSC also possess multipotency, assessed by selective stem cell markers, to differentiate into various mesenchymal cell types including adipocytes, osteocytes, and chondrocytes. These pADSC can be used for clarification of molecular switches in regulating classical adipocyte differentiation or in direction to other mesenchymal cell types of mesodermal origin. Furthermore, extended lineages into cells of ectodermal and endodermal origin have recently been achieved. Therefore, pADSC derived in this protocol provide an abundant and assessable source of adult mesenchymal stem cells with full multipotency for studying adipose development and application to tissue engineering of regenerative medicine.

This protocol describes the isolation of pig adipose-derived stem cells (pADSC) from subcutaneous adipose tissues with examination of multipotency. The multipotent pADSC are used to delineate processes of adipocyte differentiation and study transdifferentiation into multiple cell lineages of mesodermal mesenchyme or further lineages of ectoderm and endoderm for regenerative studies.

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Isolating Mesangiogenic Progenitor Cells (MPCs) from Human Bone Marrow

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel cell population specifically selected for growth in human serum supplemented medium. These cells are characterized by morphological, phenotypic, and molecular features distinct from MSCs and we named them Mesodermal Progenitor Cells (MPCs). MPCs are round, with a thick highly refringent core region; they show strong, trypsin resistant adherence to plastic. Failure to expand MPCs directly revealed that they are slow in cycling. This is as also suggested by Ki-67 negativity. On the other hand, culturing MPCs in standard medium designed for MSC expansion, gave rise to a population of exponentially growing MSC-like cells. Besides showing mesenchymal differentiation capacity MPCs retained angiogenic potential, confirming their multiple lineage progenitor nature. Here we describe an optimized highly reproducible protocol to isolate and characterize hBM-MPCs by flow cytometry (CD73, CD90, CD31, and CD45), nestin expression, and F-actin organization. Protocols for mesengenic and angiogenic differentiation of MPCs are also provided. Here we also suggest a more appropriate nomenclature for these cells, which has been re-named as "Mesangiogenic Progenitor Cells".

Isolating Mesangiogenic Progenitor Cells MPCs from Human Bone Marrow

Here we describe an optimized, highly reproducible protocol to isolate Mesodermal Progenitor Cells (MPCs) from human bone marrow (hBM). MPCs were characterized by flow cytometry and nestin expression. They showed the ability to give rise to exponentially growing MSC-like cell cultures while retaining their angiogenic potential.

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. Although many efforts have been made to reveal the underlying mechanisms of decidualization, the exact signaling between the epithelial cells that are in contact with the embryo and the underlying stromal cells remains poorly understood. Therefore, studying decidualization in a way that takes both the epithelial and stromal cells into account could improve our knowledge about the molecular details of decidualization. For this purpose, in vivo models of artificial decidualization are physiologically the most relevant; however, manipulation of intercellular communication is limited. Currently, in vitro cultures of endometrial stromal cells are being used to investigate the modulation of decidualization by several signaling molecules. Conventionally, human or mouse endometrial stromal cells are used. However, the availability of human samples is very often limited. Furthermore, the use of murine tissues is accompanied with variety in the method of culturing. This study presents a validated and standardized method to obtain pure Endometrial Epithelial Cell (EEC) and Stromal Cell (ESC) cultures using adult intact mice treated with estrogen for three consecutive days. The protocol is optimized to improve the yield, viability, and purity of the cells and was further extended in order to study decidualization in a coculture of EEC and ESC. This model may be suitable to exploit the importance of both cell types in decidualization and to evaluate the contribution of significant signaling molecules secreted by EEC or ESC during the intercellular communication.

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

This study presents a standardized and validated method for the isolation and culture of primary mouse endometrial stromal and epithelial cells, which can be used in a coculture system to study in vitro decidualization.

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

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 ...