We acknowledge the support of NIH grants R01 DE021683, R01 DE019434, U01 HL099776, The Oak Foundation and The Hagey Laboratory for Pediatric Regenerative Medicine.

NOTE: Ethical statement: All research involving vertebrate animals was performed in accordance protocols approved by the Stanford Administrative Panel on Laboratory Animal Care (APLAC).
1. Preparation
<ol>
	<li>Allow 10 ml of commercially available density gradient cell separation media (which contains polysucrose and sodium diatrizoate, adjusted to a density of 1.077 g/ml) to come to RT in a 50 ml conical tube.</li>
	<li>Prepare flow cytometry (FACS) buffer with 1x phosphate buffered saline (PBS) and 2% fetal calf serum (FCS). Take a 50 ml aliquot and maintain this aliquot at RT for use during the density gradient cell separation media step.</li>
	<li>Have a bucket of ice ready to maintain isolated tissue and cells in during the isolation procedure.</li>
</ol>
2. Prepare Culture Media
<ol>
	<li>Prepare basal media: MEM (Minimal Essential Medium), without phenol red (500 ml), glutamate (1x), FCS (10x), 10,000 units/ml penicillin (1x).</li>
	<li>Filter basal media with a 0.22 &#956;m filter.</li>
	<li>Prepare bone marrow macrophage induction media:
	<ol>
		<li>Take 50 ml of the basal media prepared in Step 2.1.</li>
		<li>Add Prostaglandin E2 (PGE2, Mr 352.465 g/mol) at 10-7 M final concentration.</li>
		<li>Add Macrophage-Colony Stimulating Factor (M-CSF) at 10 ng/ml.</li>
		<li>Do not filter the final solution of bone marrow macrophage stimulating media.</li>
	</ol>
	</li>
	<li>Prepare osteoclast induction media:
	<ol>
		<li>Take 50 ml of the basal media prepared in Step 2.1.</li>
		<li>Add Prostaglandin E2 (PGE2, Mr 352.465 g/mol) at 10-7 M final concentration</li>
		<li>Add Macrophage-Colony Stimulating Factor (M-CSF) at 10 ng/ml.</li>
		<li>Add RANKL at 10 ng/ml.</li>
		<li>Do not filter the final solution of osteoclast induction media.</li>
	</ol>
	</li>
</ol>
3. Bone Marrow Isolation
<ol>
	<li>Euthanize one C57BL/6 mouse using carbon dioxide inhalation method, followed by cervical dislocation. 
	NOTE: Rodents must be euthanized by trained personnel using appropriate technique, equipment and agents.</li>
	<li>Position and secure the mouse on a dissection board and spray with 70% alcohol.</li>
	<li>Dissect out the femurs, tibiae, humeri and spine.
	<ol>
		<li>Begin with the mouse in supine position. Make an incision vertically along the lower limb and begin by dissecting along the length of the femur and tibia by dividing soft tissue attachments. Finally, dislocate bones from the knee and hip joint to free bones fully from the skeleton.</li>
		<li>Proceed to make an incision along the length of the upper limb to isolate the humerus. Dislocate the humerus at the shoulder and elbow joints by dissecting soft tissue capsular attachment.</li>
		<li>Finally, turn the mouse over, so that the mouse lies prone. Make a skin incision along the length of the spine, in the midline. Observe the paravertebral soft tissue on each side of the spine and incise along the bony edge of the spine, dissecting through the paravertebral soft tissue mass. Using a scissors, make a horizontal cut across the spine at the base of the neck, and at the end of the spine, just proximal to the tail insertion, freeing the spine from soft tissue attachment.</li>
	</ol>
	</li>
	<li>Once the bones are harvested, keep them in FACS buffer on ice.</li>
	<li>Use tissue paper to clean muscle and soft tissue from the bones.</li>
	<li>Place the cleaned bones into a pestle and mortar with 3 ml of FACS buffer.</li>
	<li>Crush the bones gently in the pestle and mortar.</li>
	<li>Aspirate off the blood-stained fluid and transfer into a 50 ml conical tube by passing the solution through a 70 &#956;m cell strainer.</li>
	<li>Add 5 ml FACS buffer to the crushed bone and crush further. Again, remove the fluid using a pipette and transfer to the 50 ml conical tube through a 70 &#956;m strainer.</li>
	<li>Typically crush the bones twice to three times in fresh FACS buffer, using a mortar and pestle, each time adding a further 5 ml of fresh FACS buffer, until the fluid does not stain red.</li>
	<li>Centrifuge the bone marrow solution at 200 x g for 5 min at 4 &#176;C to yield a cell pellet.</li>
</ol>
4. Gradient Separation of Bone Marrow Cells
<ol>
	<li>Aspirate the supernatant and resuspend in 10 ml of FACS buffer (maintained at RT).</li>
	<li>Take the 50 ml conical tube containing 10 ml of commercially available density gradient cell separation media (RT).</li>
	<li>Layer the bone marrow solution onto the density gradient cell separation media. Do this slowly using an electric pipette. Angle the pipette tip against the inner surface near the top of the conical tube and tilt the conical tube to about 30&#176;. Using a slow speed setting on the pipette, gently expel the bone marrow solution so as not to disturb the density gradient cell separation media. 
	NOTE: Ultimately there will be a clear definition between the two solutions (cellular solution and density gradient cell separation media solution).</li>
	<li>Using a RT centrifuge, centrifuge the density gradient cell separation media and cell solution in a balanced centrifuge at 200 x g for 15 min. In addition, remove acceleration and deceleration on the centrifuge to ensure adequate phase separation using the density gradient cell separation media gradient.</li>
	<li>After centrifugation, aspirate off the cloudy middle layer which contains the bone marrow cells of interest.</li>
	<li>Transfer these cells into a new conical tube. Add 20 ml of additional FACS buffer (kept on ice) to wash the cells.</li>
	<li>Centrifuge at 200 x g for 5 min at 4 &#176;C to yield a cell pellet.</li>
	<li>Resuspend the final cell pellet in 1ml of macrophage stimulating media to allow for cell counting using a hemocytometer.</li>
</ol>
5. Cell Counting
<ol>
	<li>Take 10 &#181;l of the cell solution and mix with 10 &#956;l of trypan blue. Load 10 &#181;l of the resultant mix onto a hemocytometer and obtain the cell count per ml of solution.</li>
</ol>
6. Cell Culturing
<ol>
	<li>Place 2 ml of macrophage stimulating media into each well of a 24-well plate.</li>
	<li>Add 200,000 cells to each well. 
	NOTE: In our experience, cell-plating density is one of the most crucial requirements for adequate in vitro culturing of osteoclasts.</li>
	<li>Gently agitate the plate and place in a regular incubator at 37 &#176;C.</li>
	<li>Do not change the media for 3 days.</li>
	<li>After 3 days in macrophage stimulating media, change the media to osteoclast media.</li>
	<li>Hereafter, change the media daily for 5&#8211;7 days, at which point, large, multinucleated osteoclasts should be visible on the culture plate.</li>
</ol>
7. Staining with Tartrate Resistant Acid Phosphatase (TRAP)
<ol>
	<li>After 5&#8211;7 days of in vitro culture, aspirate the media from the culture well.</li>
	<li>Wash the wells gently with 1x PBS three times.</li>
	<li>Fix the cells by adding 4% paraformaldehyde solution and leave for 1 hr at 4 &#176;C.</li>
	<li>During this time, prepare the TRAP stain (which is commercially available) by pre-warming to 37 &#176;C.</li>
	<li>After 1 hr in paraformaldehyde, aspirate off paraformaldehyde and wash gently 3 times with 1x PBS.</li>
	<li>Add 1 ml of pre-warmed TRAP stain into each well of the plate to be stained.</li>
	<li>Place the plate in an incubator at 37 &#176;C for 1 hr, shielded from light.</li>
	<li>After 1 hr, aspirate off the TRAP stain.</li>
	<li>Wash the wells 3 times with pre-warmed deionized water.</li>
	<li>Counterstain the well with Gill&#8217;s Hematoxylin (CAUTION) for 1&#8211;2 min.</li>
	<li>Image osteoclasts using bright field microscopy.</li>
	<li>Wash wells with deionized water until an adequate color intensity of the stain is achieved, typically when nuclei appear blue.</li>
</ol>
8. Osteoclast Resorption Assay
<ol>
	<li>Use a commercially available 24-well polystyrene culture dish that is pre-coated with either bone substrate or an inorganic crystalline calcium phosphate coating.</li>
	<li>Place 2 ml of macrophage stimulating media into each well.</li>
	<li>Add 200,000 freshly isolated bone marrow cells obtained at the end of step 2 to each well.</li>
	<li>Gently agitate the plate prior to placing it in an incubator at 37 &#176;C for 3 days.</li>
	<li>On day 3, change the media to osteoclast differentiation media.</li>
	<li>Perform daily media changes with osteoclast differentiation media for 5&#8211;7 days.</li>
</ol>
9. Quantification of Osteoclast Resorption Activity
<ol>
	<li>After 5&#8211;7 days of in vitro culture in osteoclast differentiation media on a mineralized substrate, analyze the mineralized surface, as described below, to assess the ability of osteoclasts to form resorption pits, an indicator of osteoclastic activity.</li>
	<li>Aspirate media from the resorption assay plate and add 100 &#956;l of 10% bleach solution to each well.</li>
	<li>Incubate the cells with the 10% bleach solution for 15 min at RT.</li>
	<li>Wash the wells 3 times with distilled water.</li>
	<li>Aspirate off any remaining water and shake off excess water from the plate and allow to air dry for 3&#8211;5 hr.</li>
	<li>Counterstain the plate with a commercially available Von Kossa staining kit to allow for easier visualization of the un-resorbed substrate.</li>
	<li>Use bright field microscopy to take images of representative parts of the resorbed surface.</li>
	<li>Using image analysis software, calculate the percentage of the resorbed surface to allow for quantification of osteoclastic resorptive activity.</li>
</ol>

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+resorption+restored+in+osteopetrotic+mice+by+transplants+of+normal+bone+marrow+and+spleen+cells.">Bone resorption restored in osteopetrotic mice by transplants of normal bone marrow and spleen cells.</a> Science. 190, 784-785 (1975).</li><li>Burger, E. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+formation+of+osteoclasts+from+long-term+cultures+of+bone+marrow+mononuclear+phagocytes.">In vitro formation of osteoclasts from long-term cultures of bone marrow mononuclear phagocytes.</a> The Journal of experimental medicine. 156, 1604-1614 (1982).</li><li>Underwood, J. 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L., 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=Hyperglycemia+induced+and+intrinsic+alterations+in+type+2+diabetes-derived+osteoclast+function.">Hyperglycemia induced and intrinsic alterations in type 2 diabetes-derived osteoclast function.</a> Oral diseases. 19, 303-312 (2013).</li><li>Schueler, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intratibial+injection+of+human+multiple+myeloma+cells+in+NOD/SCID+IL-2Rgamma(null)+mice+mimics+human+myeloma+and+serves+as+a+valuable+tool+for+the+development+of+anticancer+strategies.">Intratibial injection of human multiple myeloma cells in NOD/SCID IL-2Rgamma(null) mice mimics human myeloma and serves as a valuable tool for the development of anticancer strategies.</a> PloS one. 8, (2013).</li><li>Xing, L., Boyce, B. 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None of the authors has disclosure or conflict of interest to declare.

An ability to easily isolate and cultivate large numbers of osteoclasts in vitro has been responsible for helping to advance understanding of bone biology and osteoclast-mediated diseases. It was the identification of RANKL that lead to this, when it was recently identified as the major regulator of osteoclast formation, differentiation and survival16-18.
It has been our experience that the in vitro cultivation of osteoclasts from bone marrow is largely dependent o...

Bone remodeling is dynamic and involves the coupling of bone formation with bone resorption1. This tightly regulated process is responsible for maintaining the skeleton during normal homeostasis, and in response to injury and disease.
Osteoclasts are unique, multinucleated cells that are capable of resorbing both the organic and inorganic matrices of bone. Osteoclasts are derived from the monocyte/macrophage lineage of the bone marrow2-5. Abnormalities in the function or formation of osteoclasts can result in a variety of clinical pathologies, including common conditions like osteoporosis.
The ability to generate osteoclasts in vitro has allowed for significant advances in our understanding of bone biology6. As a result, new therapeutic agents are emerging to treat osteoclast-related diseases which are responsible for significant morbidities and mortalities7. Homeostatic maintenance of bone mass and strength requires the concerted action of bone-forming osteoblasts and bone-resorbing osteoclasts8,9. Bone homeostasis is altered in a number of diseases, including post-menopausal osteoporosis, in which increased osteoclast activity leads to pathogenic loss of bone mass and density10. With increasing availability of transgenic murine models of human disease, there is more opportunity to decipher the role of the osteoclasts in human bone disease11-13.
Numerous protocols for osteoclast culturing techniques appear in the literature, with many variations described9,12,14. Xing and colleagues describe similar methodology to the protocol described below, in their description of osteoclastogenic assays from murine bone marrow cells. However to release the bone marrow cells following long bone harvest, Xing et al. flush the marrow cavity with &#945;-MEM complete media14. Catalfamo examines the effect of hyperglycemia on osteoclast function and describes a method in which all cells mobilized by bone marrow flushing are cultured for 24 hr, at which point the non-adherent cells are discarded12, a technique also used by Boyle et al.9 These previously published protocols necessitate the practice of flushing the bone marrow, a tedious practice, which also introduces the risk of a needle stick injury and loss of valuable bone marrow, as one must cut both ends of the bone. The protocol, which we describe, implements the use of a mortar and pestle to isolate osteoclasts, which is similar to the method of macrophage isolation described by Weischenfeldt et al.15 
Our experience, however, is that osteoclast isolation and in vitro culture using previously published techniques results in variable outcomes in terms of osteoclast production, often resulting in an inability to cultivate osteoclasts. Therefore, we have devised a protocol that allows for the consistent isolation of mouse bone marrow to produce large numbers of multinucleated osteoclasts in vitro, with an approximate yield of 70-80% of cells initially plated forming macrophages and subsequently osteoclasts, in the presence of osteoclast induction media.

<table border="0" cellpadding="0" cellspacing="0" width="1017">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:503px;">Name of the Material/Equipment</td>
			<td style="width:165px;">Company</td>
			<td style="width:175px;">Catalog Number</td>
			<td style="width:175px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM, no glutamine, no phenol red</td>
			<td>Gibco</td>
			<td>51200-038</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">M-CSF, recombinant mouse</td>
			<td>Gibco</td>
			<td>PMC2044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Recombinant Mouse TRANCE/RANK L/TNFSF11 (E. coli expressed)</td>
			<td>R&#38;D Systems</td>
			<td>462-TEC-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Prostaglandin E2</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Histopaque-1077</td>
			<td>Sigma-Aldrich</td>
			<td>10771</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acid Phosphatase, Lekocyte (TRAP) kit</td>
			<td>Sigma-Aldrich</td>
			<td>387A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Osteoassay bone resorption plates, 24 well plates</td>
			<td>Corning Life Sciences</td>
			<td>3987</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The aim of this method was to easily isolate large numbers of osteoclasts in vitro, typically in one week. Successful isolation of large numbers of osteoclasts was confirmed using tartrate-resistant acid phosphatase staining (Figure 1A). Large osteoclasts are visualized as large purple cells with multiple nuclei (typically &#8805; 3 nuclei). Using this protocol, it is common to isolate osteoclasts with as many as 30 nuclei per osteoclast (Figure 1B).
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Osteoclast Derivation from Mouse Bone Marrow

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 ...

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24.0K Views.  Stanford University School of Medicine. 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.

Video: Osteoclast Derivation from Mouse Bone Marrow

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. 

This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Homing of Hematopoietic Cells to the Bone Marrow

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow. Various chemomkines and receptors are involved in the homing of hematopoietic stem cells. [1, 2]
This paper outlines the classic homing protocol used in hematopoietic stem cell studies. In general this involves isolating the cell population whose homing needs to be investigated, staining this population with a dye of interest and injecting these cells into the blood stream of a recipient animal. The recipient animal is then sacrificed at a pre-determined time after injection and the bone marrow evaluated for the percentage or absolute number of cells which are positive for the dye of interest. In one of the most common experimental schemes, the homing efficiency of hematopoietic cells from two genetically distinct animals (a wild type animal and the corresponding knock-out) is compared. This article describes the hematopoietic cell homing protocol in the framework of such as experiment.

This article describes a protocol used to study the homing of hematopoietic cells to their niches in the bone marrow.

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow.  Various ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the trophectoderm mediates implantation and generates the fetal part of the placenta. As a result, the development of this lineage is essential for embryo survival. Derivation of trophoblast stem (TS) cells from mouse blastocysts was first described by Tanaka et al. 1998. The ability of TS cells to preserve the trophoblast specific property and their expression of stage- and cell type-specific markers after proper stimulation provides a valuable model system to investigate trophoblast lineage development whereby recapitulating early placentation events. Furthermore, trophoblast cells are one of the few somatic cell types undergoing natural genome amplification. Although the molecular pathways underlying trophoblast polyploidization have begun to unravel, the physiological role and advantage of trophoblast genome amplification remains largely elusive. The development of diploid stem cells into polyploid trophoblast cells in culture makes this ex vivo system an excellent tool for elucidating the regulatory mechanism of genome replication and instability in health and disease. Here we describe a protocol based on previous reports with modification published in Chiu et al. 2008.

In this video, we demonstrate the isolation of mouse blastocysts and the derivation of trophoblast stem cells from blastocysts. We also describe conditions for maintenance of the stem cell property as well as induction of differentiation in culture.

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation of an adaptive immune response. Experimental work with these cells is rather challenging. Their abundance in organs and tissues is relatively low. As a result, they cannot be isolated in large numbers. They are also difficult to transfect with cDNA constructs. In the murine model, these problems can be partially overcome by in vitro differentiation from bone marrow progenitors in the presence of M-CSF for macrophages or GM-CSF for dendritic cells. In this way, it is possible to obtain large amounts of these cells from very few animals. Moreover, bone marrow progenitors can be transduced with retroviral vectors carrying cDNA constructs during early stages of cultivation prior to their differentiation into bone marrow derived dendritic cells and macrophages. Thus, retroviral transduction followed by differentiation in vitro can be used to express various cDNA constructs in these cells. The ability to express ectopic proteins substantially extends the range of experiments that can be performed on these cells, including live cell imaging of fluorescent proteins, tandem purifications for interactome analyses, structure-function analyses, monitoring of cellular functions with biosensors and many others. In this article, we describe a detailed protocol for retroviral transduction of murine bone marrow derived dendritic cells and macrophages with vectors coding for fluorescently-tagged proteins. On the example of two adaptor proteins, OPAL1 and PSTPIP2, we demonstrate its practical application in flow cytometry and microscopy. We also discuss the advantages and limitations of this approach.

In this article, we provide a detailed protocol for the expression of fluorescent fusion proteins in murine bone marrow derived dendritic cells and macrophages. The method is based on the transduction of bone marrow progenitors with retroviral constructs followed by differentiation into macrophages and dendritic cells in vitro.

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation ...

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.