This work was supported by the Ronald Peter Griggs Memorial Fellowship in ALS Research (to KP) and a Neuromuscular Research Partnership Program grant from the CIHR, the ALS Society of Canada, and Muscular Dystrophy Canada (JNM-69682) to CK and FMR. Additional support was also provided by the ALS Society of America (57969). We would like to thank Dr. R. Keith Humphries from the Terry Fox Laboratory/Department of Pathology, BC Cancer Agency, Vancouver, Canada and the staff at the Simon Fraser University Animal Care Unit.

NOTE: Ethics Statement: This protocol has been reviewed and approved by the Animal Care Committee of Simon Fraser University (UACC; permit numbers 1037K-12 and 1060K-03) and is in compliance with the Canadian Council on Animal Care, the NIH Guide for the Care and Use of Laboratory Animals, and the EEC Council Directive.
1. Special Considerations
NOTE: Busulfan is cytotoxic. Handle and dispose according to the material safety data sheet and institutional guidelines.
<ol>
	<li>Perform all techniques aseptically in a laminar flow hood.</li>
	<li>Sterilize instruments prior to use by wrapping in an appropriate packaging material, such as a peel package, and autoclaving.</li>
	<li>While the risk for infection is lower with busulfan conditioning compared to irradiation, handle animals only in a laminar flow hood for the first 2 weeks following transplantation.</li>
</ol>
2. Conditioning of Recipient Mice
<ol>
	<li>Dilute busulfan to 3 mg/ml with sterile phosphate buffered saline (PBS). 
	NOTE: It is important to make a fresh dilution of busulfan just prior to use each day of injection.</li>
	<li>Administer 20 mg/kg of diluted busulfan to recipient mice via IP injection daily.</li>
	<li>Repeat daily IP injections of 20 mg/kg busulfan until a total dose of 60-100 mg/kg has been delivered (i.e., for an 80 mg/kg total dose, administer 20 mg/kg of busulfan for 4 consecutive days).</li>
</ol>
3. Isolation of Donor Bone Marrow Cells
NOTE: This protocol has been successfully used for isolating and preparing BM cells from up to 5 donor mice. Typically the cell yield per mouse is approximately 30-40 million BMDCs, which is sufficient to transplant 12-16 recipient mice. If more donor mice are needed the protocol may need to be adjusted accordingly.
<ol>
	<li>Following the last day of busulfan conditioning euthanize a GFP donor mouse (one to six months old) using CO2 (or by other euthanasia procedure accepted at institution). To avoid graft complications use syngeneic donors that are the same sex as the recipients.</li>
	<li>Spray mouse with 70% ethanol. Lift skin at the abdomen and using surgical scissors make an incision through the skin from the abdominal cavity up the leg towards the ankle. Holding the foot, firmly pull the skin from the ankle towards the hip exposing the leg tissue.</li>
	<li>Trim away muscle and fat tissue from the femur to expose the hip joint.</li>
	<li>While gently pulling on the foot to extend the leg, press the scissors against the hip joint. Cut just above the head of the femur taking care not to cut the femur itself.</li>
	<li>To help maintain sterility, hold the leg by the foot and clean any remaining tissue from the bones by rubbing the bone surface with autoclaved tissues.</li>
	<li>Separate the femur and tibia by cutting through the knee joint and place the femur in a culture dish containing sterile PBS. Incubate on ice.</li>
	<li>Remove and discard the fibula by cutting at the points where the fibula connects to the tibia. Place the tibia in the culture dish with the femur and incubate on ice.</li>
	<li>Repeat steps 3.2-3.7 for the other leg, and if necessary, additional donor mice. Following removal of the bones, sterilize the surgical tools with a hot bead sterilizer&#160;or use a new set of sterile tools for the subsequent steps.</li>
	<li>For the femurs, hold the femur with forceps and using surgical scissors carefully &#8216;shave&#8217; the distal ends off the bone. Remove as little of the bone as necessary to expose the BM cavity.</li>
	<li>Fill a syringe with 3 ml of sterile PBS and attach a 23 G&#160;needle. Carefully bore the needle into the BM cavity and flush the BM into a sterile culture dish. Be sure to scrape the medullary cavity with the needle point to ensure removal of all desired cells. Following extraction, ensure that the red BM is no longer visible and the bone now appears white.</li>
	<li>Repeat steps 3.9-3.10 for subsequent femurs, pooling all of the BM in the same culture dish.</li>
	<li>For the tibiae, hold the tibia with forceps and carefully &#8216;shave&#8217; the end where the tibia was attached to the knee to expose the BM cavity. Make a second cut along the bone where the visible red BM ends.</li>
	<li>Fill a syringe with 3 ml of sterile PBS and attach a 25 G&#160;needle. Gently insert the needle into the BM cavity (from the end that was attached to the knee) and flush the BM into the culture dish containing the BM from the femurs. Scrape the walls of the BM cavity with the needle to remove all cells. Following extraction, ensure that the red BM is no longer visible and the bone now appears white.</li>
	<li>Repeat steps 3.12-3.13 for subsequent tibiae, pooling all of the BM in the same culture dish.</li>
	<li>Gently triturate the BM with a 1 ml pipette tip to dissociate the cells.</li>
	<li>Pass the cell suspension through a 40 &#956;M basket filter into a sterile 50 mL centrifuge tube. Rinse the dish with PBS to get any remaining cells and transfer this through the filter into the centrifuge tube.</li>
	<li>Centrifuge for 5 min at 450 x g at 4 &#176;C. Remove and discard the supernatant.</li>
	<li>Re-suspend the pellet in 3 ml of erythrocyte lysing buffer. Incubate on ice for 8.5 min.</li>
	<li>Add ~30 ml of sterile PBS to quench the lysing buffer.</li>
	<li>Centrifuge for 5 min at 450 x g at 4 &#176;C. Remove and discard the supernatant.</li>
	<li>Re-suspend the pellet in 1 ml of sterile PBS. Keep on ice.</li>
	<li>Take a small aliquot of the cell homogenate and dilute with PBS (1:100 for 1 mouse; 1:200 for 2 mice, etc.) to quantify the cell number using a haemocytometer.</li>
	<li>Dilute the cell suspension to 8 x 106 cells/ml and incubate on ice until injection.</li>
</ol>
4. Bone Marrow Transplantation
<ol>
	<li>Fill a 0.5 ml syringe (with a 28 G&#160;insulin needle) with 300 &#956;l of diluted cell suspension for each mouse to be injected. Be sure to remove any air bubbles present in the syringe.</li>
	<li>Place the mouse cage containing the conditioned recipient mice on a heating pad and allow the tail veins to dilate.</li>
	<li>Take a recipient mouse and place in the restraining device. Wipe the tail with surgical gauze soaked with 70% ethanol.</li>
	<li>Firmly hold the tail and with the bevel up, gently insert the needle into one of the lateral tail veins and inject the cell suspension.</li>
	<li>Hold surgical gauze on the injection site until bleeding stops prior to introducing the mouse into a new clean cage.</li>
</ol>
5. Estimating the Extent of BM Chimerism
NOTE: Chimerism levels are typically variable in the peripheral blood at 1 week post-BMT, and as such chimerism levels are normally estimated at least 2 weeks post-BMT. Levels of chimerism should reach &#62;80% GFP+ cells in the peripheral blood within 3-4 weeks post-BMT.
<ol>
	<li>Prepare FACS buffer (2 mM EDTA + 2% fetal bovine serum in PBS). 
	NOTE: FACS buffer can be stored at 4 &#176;C for several weeks.</li>
	<li>Place the mouse in a restraining tube and shave the fur on the posterior leg to expose the lateral saphenous vein.</li>
	<li>Thinly coat the leg with petroleum jelly and pierce the saphenous vein with a 25 G&#160;needle.</li>
	<li>Collect approximately 50 &#956;l of blood with a heparin coated capillary tube. Add the blood to a micro-centrifuge tube containing 1 ml of FACS buffer. Mix the tube by inversion and store on ice.</li>
	<li>Hold surgical gauze on the injection site until bleeding stops before returning mouse back to the cage.</li>
	<li>Centrifuge the blood samples for 5 min at 900 x g. Remove and discard the supernatant.</li>
	<li>Re-suspend the pellet in 500 &#956;l of erythrocyte lysing buffer. Incubate on ice for 8.5 min.</li>
	<li>Add 1 ml of FACS buffer to quench the lysing buffer.</li>
	<li>Centrifuge the samples for 5 min at 900 x g. Remove and discard the supernatant.</li>
	<li>Perform a second lysis step in 500 &#956;l of erythrocyte lysing buffer. Incubate on ice for 8.5 min.</li>
	<li>Add 1 ml of FACS buffer to quench the lysing buffer.</li>
	<li>Centrifuge the samples for 5 min at 900 x g. Remove and discard the supernatant. Check that the pellet is now white. 
	NOTE: If the pellet still appears red, an additional lysis step may be needed before proceeding to the next step.</li>
	<li>Re-suspend the pellet in 1 ml of FACS buffer. Centrifuge the samples for 5 min at 900 x g. Remove and discard the supernatant.</li>
	<li>Re-suspend the pellet in 300 &#956;l of FACS buffer and quantify GFP+ cells using flow cytometry (NOTE: At this point any desired immunostaining of the cells can be performed, using standard techniques, prior to flow cytometric analysis).</li>
</ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+bone+marrow+transplantation+(BMT):+providing+optimal+veterinary+and+husbandry+care+to+irradiated+mice+in+BMT+studies.">Principles of bone marrow transplantation (BMT): providing optimal veterinary and husbandry care to irradiated mice in BMT studies.</a> Journal of the American Association for Laboratory Animal Science: JAALAS. 48 (1), 11-22 (2009).</li><li>Jimenez, M., Ercilla, G., Martinez, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+reconstitution+after+allogeneic+stem+cell+transplantation+with+reduced-intensity+conditioning+regimens.">Immune reconstitution after allogeneic stem cell transplantation with reduced-intensity conditioning regimens.</a> Leukemia. 21 (8), 1628-1637 (2007).</li><li>Ellen Nath, C., John Shaw, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Busulphan+in+blood+and+marrow+transplantation:+dose,+route,+frequency+and+role+of+therapeutic+drug+monitoring.">Busulphan in blood and marrow transplantation: dose, route, frequency and role of therapeutic drug monitoring.</a> Current clinical pharmacology. 2 (1), 75-91 (2007).</li><li>Galaup, A., Paci, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacology+of+dimethanesulfonate+alkylating+agents:+busulfan+and+treosulfan.">Pharmacology of dimethanesulfonate alkylating agents: busulfan and treosulfan.</a> Expert Opinion on Drug Metabolism &amp; Toxicology. 9 (3), 333-347 (2013).</li><li>Lewis, C. -. 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The authors have nothing to disclose.

Busulfan conditioning allows for the generation of high-level BM chimerism in mice with hematopoietic cells that are readily detectable. This conditioning can be performed without the need for irradiation facilities. Moreover, myelosuppression with the doses of busulfan outlined in this protocol is well tolerated, minimizing toxic side effects caused by myeloablative doses of irradiation, and thus may be a more suitable technique to generate BM chimerism in young, old, or diseased mice that are more susceptible to myeloa...

Recently, there has been considerable interest in the roles of microglia and bone marrow (BM)-derived cells (BMDCs) in the central nervous system (CNS), both during disease as well as with normal aging. Microglia, the resident immune cells of the CNS, are now known to develop in the CNS following the entry of primitive myeloid progenitors during embryogenesis1. Microglia retain aspects of their myelomonocytic lineage well into adult life. While evidence suggests that the contribution of BMDCs to the microglial pool is minimal in healthy adult animals1, the role BMDCs play in the progression of various neurodegenerative diseases remains unclear. This uncertainty is compounded by the fact that it is difficult to distinguish BMDCs that accumulate within the CNS from endogenous resident microglia, as no universally accepted discriminating immunohistochemical marker has been identified. In order to monitor BMDCs in vivo, we employ a BM transplantation (BMT) protocol whereby endogenous BM cells of a recipient mouse are replaced with those of a donor mouse ubiquitously expressing green fluorescent protein (GFP) under control of the &#946;-actin promoter. This protocol permits the determination of both the localization and morphology of GFP+ BMDCs within the recipient mouse using immunohistochemistry, and facilitates further characterization of these cells using fluorescence-activated cell sorting (FACS) followed by subsequent biochemical assays. 
For a successful BMT, recipient BM cells need to be ablated (termed myeloablation) in order to generate niche space within the recipient BM to allow for donor cell engraftment. Often, myeloablation is achieved using total body gamma irradiation which induces double strand breaks in DNA that leads to cell death, particularly in actively dividing cells such as hematopoietic progenitor cells2,3. The irradiation protocols are done to induce sufficient BM cell death such that animal lethality results if the recipient animal does not achieve adequate donor cell engraftment from BMT (so called &#8216;lethal irradiation&#8217;). However, irradiation requires a specialized facility and equipment, along with veterinary and animal husbandry resources not available to researchers at all institutions. Furthermore, myeloablative irradiation can cause potentially lethal damage to other tissues, and due to immunosuppresion, irradiated animals are more susceptible to secondary infections3. As such, reduced intensity conditioning (so called &#8216;RIC&#8217; protocols) regimens have been developed that are intended to minimize potentially toxic side effects of the conditioning protocol in patients, particularly for use in at risk populations such as children and the elderly4. 
Some RIC protocols rely upon chemotherapeutics such as busulfan in order to condition the BM compartment. Busulfan is a bifunctional DNA alkylating agent often used clinically as an alternative to irradiation5,6. Notably, busulfan can be safely administered to mice by intraperitoneal (IP) injection and does not require the specialized facilities and equipment necessary to irradiate mice. Busulfan conditioning has been used extensively in our lab7, as well as in several other recent publications8&#8211;11. When doses of 60-100 mg/kg are employed, high degrees of stable chimerism (>80% GFP+ cells) can be established in the peripheral blood and BM7. Importantly, at these doses myeloablation is not complete and as such mice are able to survive without receiving support BM cells (K. Peake, J. Manning, C. Lewis, and C. Krieger, unpublished observations). Moreover, with myelosuppressive busulfan conditioning there is rapid reconstitution of peripheral blood myelomonocytic cells by donor cells. However, the replacement of peripheral blood lymphocytes by donor cells is slower, highlighting the lack of immunosuppression that occurs with 60-100 mg/kg doses of busulfan compared to total body irradiation7. 
We have successfully used busulfan-induced chimerism to investigate BMDCs in wild-type mice, as well as in mouse models of the neurodegenerative disorders amyotrophic lateral sclerosis (ALS)7 and Alzheimer disease. It has been observed that under certain conditions significant numbers of GFP+ BMDCs accumulate within the CNS7,12. Importantly, like myeloablative conditioning with irradiation, busulfan conditioning followed by BMT is sufficient to allow for GFP+ BMDC accumulation within the CNS in both wild-type mice, and mice with neurological disorders7&#8211;11. As a major obstacle in treating neurodegenerative disorders is the ability to get therapeutic molecules from the circulation into the CNS where they can exert beneficial effects, this raises the possibility that BMDCs could be engineered to express therapeutic molecules, and subsequently used as a vehicle to deliver therapeutics to the CNS of busulfan conditioned recipients, a mechanism we are actively investigating. Although we have found that myeloablative conditioning with irradiation leads to greater accumulation of GFP+ BMDCs in the CNS compared to busulfan conditioning7, irradiation may have considerable toxicity in patients with neurodegenerative disorders and in murine models of CNS disease.
While we, and many others, have used BMT models to study BMDCs in neurodegenerative disorders, the ability to largely replace the BM compartment of a recipient mouse with a distinctly identifiable population of BM cells is an invaluable tool for studying various aspects of the hematopoietic system. This includes a wide array of research topics such as hematopoietic lineage development, leukemia, organ transplantation, graft-versus-host disease, and immunobiology.

<table border="0" cellpadding="0" cellspacing="0" width="726">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name of Reagent/ Equipment</td>
			<td style="width:224px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Busulfex</td>
			<td style="width:224px;">Otsuka Canada Pharmaceutical Inc.</td>
			<td style="width:119px;">DIN 02240602</td>
			<td>Dilute busulfan to 3 mg/mL with sterile PBS just prior to use. Busulfan is cytotoxic. Handle and dispose according to the MSDS and institutional guidelines.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ACK Lysing Buffer</td>
			<td style="width:224px;">Life Technologies</td>
			<td style="width:119px;">A10492-01</td>
			<td>Other lysis buffer could be substituted but incubation times may need to be adjusted accordingly</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:224px;">Life Technologies</td>
			<td>12483-020</td>
			<td></td>
		</tr>
	</tbody>
</table>

busulfan as a myelosuppressive agent for generating stable high-level bone marrow chimerism in mice

Bone marrow transplantation (BMT) is often used to replace the bone marrow (BM) compartment of recipient mice with BM cells expressing a distinct biomarker isolated from donor mice. This technique allows for identification of donor-derived hematopoietic cells within the recipient mice, and can be used to isolate and characterize donor cells using various biochemical techniques. BMT typically relies on myeloablative conditioning with total body irradiation to generate niche space within the BM compartment of recipient mice for donor cell engraftment. The protocol we describe here uses myelosuppressive conditioning with the chemotherapeutic agent busulfan. Unlike irradiation, which requires the use of specialized facilities, busulfan conditioning is performed using intraperitoneal injections of 20 mg/kg busulfan until a total dose of 60-100 mg/kg has been administered. Moreover, myeloablative irradiation can have toxic side effects and requires successful engraftment of donor cells for survival of recipient mice. In contrast, busulfan conditioning using these doses is generally well tolerated and mice survive without donor cell support. Donor BM cells are isolated from the femurs and tibiae of mice ubiquitously expressing green fluorescent protein (GFP), and injected into the lateral tail vein of conditioned recipient mice. BM chimerism is estimated by quantifying the number of GFP+ cells within the peripheral blood following BMT. Levels of chimerism&nbsp;>80% are typically observed in the peripheral blood 3-4 weeks post-transplant and remain established for at least 1 year. As with irradiation, conditioning with busulfan and BMT allows for the accumulation of donor BM-derived cells within the central nervous system (CNS), particularly in mouse models of neurodegeneration. This busulfan-mediated CNS accumulation may be more physiological than total body irradiation, as the busulfan treatment is less toxic and CNS inflammation appears to be less extensive. We hypothesize that these cells can be genetically engineered to deliver therapeutics to the CNS.

The administration of 60-100 mg/kg of busulfan is generally well tolerated by mice. However, animals typically lose weight during the conditioning phase (~5-10%) and thus the diet may need to be supplemented with treats such as irradiated sunflower seeds to encourage eating. Levels of chimerism of &#62;80% GFP+ cells in the peripheral blood and BM should be consistently achievable using 60-100 mg/kg busulfan7. Our laboratory has successfully used this protocol to generate over 100 chimeric mice with &#62;80% G...

Watch this Scientific Journal Video about Busulfan as a Myelosuppressive Agent for Generating Stable High-level Bone Marrow Chimerism in Mice at JoVE.com

Busulfan as a Myelosuppressive Agent for Generating Stable High-level Bone Marrow Chimerism in Mice

We describe a protocol whereby busulfan conditioning permits the bone marrow of a recipient mouse to be replaced with bone marrow cells from donor mice ubiquitously expressing green fluorescent protein, in the absence of irradiation. This technique is useful to study bone marrow cell accumulation in the central nervous system.

Bone marrow transplantation (BMT) is often used to replace the bone marrow (BM) compartment of recipient mice with BM cells expressing a distinct ...

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13.2K 조회수.  Simon Fraser University. We describe a protocol whereby busulfan conditioning permits the bone marrow of a recipient mouse to be replaced with bone marrow cells from donor mice ubiquitously expressing green fluorescent protein, in the absence of irradiation. This technique is useful to study bone marrow cell accumulation in the central nervous system.

비디오: Busulfan as a Myelosuppressive Agent for Generating Stable High-level Bone Marrow Chimerism in Mice

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro and in vivo and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds.

Receptor tyrosine kinases are ectopically expressed in many cancers and have been identified as therapeutic targets in acute leukemia. This manuscript describes an efficient strategy for pre-clinical evaluation of tyrosine kinase inhibitors for the treatment of acute leukemia.

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are ...

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Urinary Bladder Distention Evoked Visceromotor Responses as a Model for Bladder Pain in Mice

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized by increased urgency and frequency of urination, as well as nocturia and general pelvic pain, especially upon bladder filling or voiding. Despite years of research, the cause of IC/BPS remains elusive and treatment strategies are unable to provide complete relief to patients. In order to study nervous system contributions to the condition, many animal models have been developed to mimic the pain and symptoms associated with IC/BPS. One such murine model is urinary bladder distension (UBD). In this model, compressed air of a specific pressure is delivered to the bladder of a lightly anesthetized animal over a set period of time. Throughout the procedure, wires in the superior oblique abdominal muscles record electrical activity from the muscle. This activity is known as the visceromotor response (VMR) and is a reliable and reproducible measure of nociception. Here, we describe the steps necessary to perform this technique in mice including surgical manipulations, physiological recording, and data analysis. With the use of this model, the coordination between primary sensory neurons, spinal cord secondary afferents, and higher central nervous system areas involved in bladder pain can be unraveled. This basic science knowledge can then be clinically translated to treat patients suffering from IC/BPS.

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized in part by pelvic pain. In order to study nervous system contributions to the condition, a physiological model of urinary bladder pain is used in mice and rats.

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition ...

Femoral Bone Marrow Aspiration in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.

This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant cells with their microenvironment, their rarity and the overwhelming excess of hematopoietic cells. Towards this end, we developed an in vitro 2D clonogenic model of dormancy of estrogen-sensitive breast cancer cells in the bone marrow. The model consists of a few key elements necessary for dormancy. These include 1) the use of estrogen sensitive breast cancer cells, which are the type likely to remain dormant for extended periods, 2) incubation of cells at clonogenic density, where the structural interaction of each cell is primarily with the substratum, 3) fibronectin, a key structural element of the marrow and 4) FGF-2, a growth factor abundantly synthesized by bone marrow stromal cells and heavily deposited in the extracellular matrix. Cells incubated with FGF-2 form dormant clones after 6 days, which consist of 12 or less cells that have a distinct flat appearance, are significantly larger and more spread out than growing cells and have large cytoplasm to nucleus ratios. In contrast, cells incubated without FGF-2 form primarily growing colonies consisting of >30 relatively small cells. Perturbations of the system with antibodies, inhibitors, peptides or nucleic acids on day 3 after incubation can significantly affect various phenotypic and molecular aspects of the dormant cells at 6 days and can be used to assess the roles of membrane-localized or intracellular molecules, factors or signaling pathways on the dormant state or survival of dormant cells. While recognizing the in vitro nature of the assay, it can function as a highly useful tool to glean significant information about the molecular mechanisms necessary for establishment and survival of dormant cells. This data can be used to generate hypotheses to be tested in vivo models.

An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

We developed an in vitro model of dormancy in the bone marrow for estrogen-sensitive breast cancer cells. The goal of this protocol is to demonstrate use of the model for the study of the molecular and cellular biology of dormancy and for generation of hypotheses for subsequent testing in vivo.

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant ...

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest improved outcome measures in both experimental and clinical studies. Although the BMMSC are implanted into the tendon lesion in large numbers (usually 10 - 20 million cells), only a relatively small number survive (&#60;10%) although these can persist for up to 5 months after implantation. This appears to be a common observation in other species where BMMSC have been implanted into other tissues and it is important to understand when this loss occurs, how many survive the initial implantation process and whether the cells are cleared into other organs. Tracking the fate of the cells can be achieved by radiolabeling the BMMSC prior to implantation which allows non-invasive in vivo imaging of cell location and quantification of cell numbers.
This protocol describes a cell labeling procedure that uses Technetium-99m (Tc-99m), and tracking of these cells following implantation into injured flexor tendons in horses. Tc-99m is a short-lived (t1/2 of 6.01 hr) isotope that emits gamma rays and can be internalized by cells in the presence of the lipophilic compound hexamethylpropyleneamine oxime (HMPAO). These properties make it ideal for use in nuclear medicine clinics for the diagnosis of many different diseases. The fate of the labeled cells can be followed in the short term (up to 36 hr) by gamma scintigraphy to quantify both the number of cells retained in the lesion and distribution of the cells into lungs, thyroid and other organs. This technique is adapted from the labeling of blood leukocytes and could be utilized to image implanted BMMSC in other organs.

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

This protocol describes the radiolabeling of equine mesenchymal stem cells and their implantation into tendon injuries in the horse in order to determine cell survival and tissue distribution using gamma scintigraphy.

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest ...

Database-guided Flow-cytometry for Evaluation of Bone Marrow Myeloid Cell Maturation

A working group initiated within the French Cytometry Association (AFC) was developed in order to harmonize the application of multiparameter flow cytometry (MFC) for myeloid disease diagnosis in France. The protocol presented here was agreed-upon and applied between September 2013 and November 2015 in six French diagnostic laboratories (University Hospitals of Saint-Etienne, Grenoble, Clermont-Ferrand, Nice, and Lille and Institut Paoli-Calmettes in Marseille) and allowed the standardization of bone marrow sample preparation and data acquisition. Three maturation databases were developed for neutrophil, monocytic, and erythroid lineages with bone marrow from "healthy" donor individuals (individuals without any evidence of a hematopoietic disease). A robust method of analysis for each myeloid lineage should be applicable for routine diagnostic use. New cases can be analyzed in the same manner and compared against the usual databases. Thus, quantitative and qualitative phenotypic abnormalities can be identified and those above 2SD compared with data of normal bone marrow samples should be considered indicative of pathology. The major limitation is the higher variability between the data achieved using the monoclonal antibodies obtained with the methods based on hybridoma technologies and currently used in clinical diagnosis. Setting criteria for technical validation of the data acquired may help improve the utility of MFC for MDS diagnostics. The establishment of these criteria requires analysis against a database. The reduction of investigator subjectivity in data analysis is an important advantage of this method.

The MDS diagnosis is difficult in the absence of morphological criteria or non-informative cytogenetics. MFC could help refine the MDS diagnostic process. To become useful for clinical practice, the MFC analysis must be based on parameters with sufficient specificity and sensitivity, and data should be reproducible between different operators.

A working group initiated within the French Cytometry Association (AFC) was developed in order to harmonize the application of multiparameter flow ...

Combining Volumetric Capnography And Barometric Plethysmography To Measure The Lung Structure-function Relationship

Tools to measure lung and airways volume are critical for pulmonary researchers interested in evaluating the impact of disease or novel therapies on the lung. Barometric plethysmography is a classic technique to evaluate the lung volume with a long history of clinical use. Volumetric capnography utilizes the profile of exhaled carbon dioxide to determine the volume of the conducting airways, or dead space, and provides an index of airways homogeneity. These techniques may be used independently, or in combination to evaluate the dependence of airways volume and homogeneity on lung volume. This paper provides detailed technical instructions to replicate these techniques and our representative data demonstrates that the airways volume and homogeneity are highly correlated to lung volume. We also provide a macro for the analysis of capnographic data, which can be modified or adapted to fit different experimental designs. The advantage of these measures is that their advantages and limitations are supported by decades of experimental data, and they can be made repeatedly in the same subject without expensive imaging equipment or technically advanced analysis algorithms. These methods may be particularly useful for investigators interested in perturbations that change both the functional residual capacity of the lung and airways volume.

Here, we describe two measures of pulmonary function &#8211; barometric plethysmography, which allows the measurement of lung volume, and volumetric capnography, a tool to measure the anatomic dead space and airways uniformity. These techniques may be used independently or combined to assess airways function at different lung volumes.

Tools to measure lung and airways volume are critical for pulmonary researchers interested in evaluating the impact of disease or novel therapies on the ...