Name: Competitive Transplants to Evaluate Hematopoietic Stem Cell Fitness (Video) | JoVE Journal
Uploaded: 2016-08-31T13:00:00.000+00:00
Duration: 8 min 53 s
Description: Learn competitive bone marrow transplant assays to evaluate hematopoietic stem cell fitness in vivo without cell sorting. This method reveals self-renewal mechanisms and requires minimal animals.

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5 minutes

We are grateful to Roxann H&#233;tu-Arbour for assistance with the figure design and demonstration of the procedures. Research in the lab was supported by a Transition award from the Cole Foundation, Discovery grant no. 419226-2012 from the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Canada Foundation for Innovation (CFI Leaders Fund grant no. 31377). KMH is a Chercheur-Boursier Junior for the Fonds de recherche du Qu&#233;bec - Sant&#233; (FRQS).

All procedures described in this protocol have been approved by the institutional animal ethics committee and follow the Canadian Council on Animal Care guidelines.
Note: To maintain sterile/specific pathogen-free housing conditions, conduct all procedures involving direct handling of live mice inside a biological safety cabinet or a laminar flow hood. Clean or sterilize cages, restraining devices, housing materials, chow and water provided to the animals appropriately. Use only sterile, disposable needles for the injections and for blood sampling. Aseptic technique is crucial during preparation of the graft.
1. Preparation of Recipient Mice
<ol>
	<li>Identify recipient mice with ear notches (or other method approved by the local animal ethics committee8) to enable individual follow up of peripheral blood and bone marrow reconstitution over time. Weigh the mice for post-transplant follow-up. Use recipient mice that are between 7 and 12 weeks old (less than 25 g). 
	Note: In the provided example, CD45.1+ CD45.2+ recipient mice were bred in house by crossing C57Bl/6 mice with congenic B6.SJL mice.</li>
	<li>Irradiate recipient mice with two doses of 450 rad to destroy hematopoietic activity. Give the first dose the day before transplantation and the second 1-2 hr before transplantation. 
	Note: X-ray or gamma rays can both be used depending on the availability of appropriate facilities.</li>
</ol>
2. Preparation of Donor and Competitor Bone Marrow Cells
<ol>
	<li>Euthanize donor (test and control; CD45.2+) and competitor (CD45.1+; B6.SJL) mice by CO2 asphyxiation followed by cervical dislocation or using methods approved by the local animal ethics committee.</li>
	<li>Place mice on their back, legs open wide, in an empty petri dish or on a sterile gauze (to keep the work surface clean) inside a biological safety cabinet. Wet the skin and fur with 70% ethanol/30% H2O (v/v).</li>
	<li>Cut off the foot with a surgical blade or sharp scissors. Cut open the skin along the leg and pull away the skin using serrated forceps. Cut away excess muscle.</li>
	<li>Dislocate femur by pulling on the leg bone and using scissor blades against the pelvic bones as a counter force. Detach tibia and femur from the kneecap and remove remaining bits of muscle and tendons. Place the bones in 2-3 ml sterile phosphate buffered saline (PBS) in a 6-well tissue culture plate.</li>
	<li>Collect bone marrow cells by flushing the bones with 5 ml sterile PBS.
	<ol>
		<li>Hold the bone gently with forceps and insert a 25 G needle attached to a filled 1 ml syringe to one end of the bone. Press the plunger and collect the cells in a 15 ml conical tube. Repeat as needed until the center of the bone is white.</li>
	</ol>
	</li>
	<li>Dissociate the cells by repeated passing through the needle to obtain a single cell suspension. Avoid bubbles and excess force. Note: Use a slightly larger needle (22 G) to improve cell viability at this step.</li>
	<li>Pass the cells through a 70 &#181;m nylon strainer to ensure uniform cell suspension and to remove debris and clumps.</li>
	<li>Remove an aliquot for cell counting using a hemocytometer. Centrifuge the remaining cell suspension at 200 x g for 5 min at 4 &#176;C. Readjust cell density as required in sterile PBS (108 cells/ml). Keep the cells on ice.</li>
</ol>
3. Establishing Donor Cell HSC Equivalents
<ol>
	<li>Transfer the equivalent of 3 x 106 cells (30 &#181;l) into a 5 ml round bottom polystyrene tube for flow cytometry staining. Add an equal volume of unlabeled antibody against CD16/CD32 to block non-specific staining (diluted in staining buffer (PBS supplemented with 0.1% bovine serum albumin (BSA) and 1 mM EDTA) for a final concentration of 2.5 &#181;g/ml). Incubate 5 min at RT.</li>
	<li>Add 90 &#181;l fluorochrome-conjugated antibody master mix prepared in staining buffer: biotinylated Lineage cocktail (B220, CD3e, CD11b, GR1, Ter119), Sca1, CD117, CD135, CD150. Incubate on ice for 30 min, protected from light. 
	Note: Appropriate dilutions should be determined for each lot of antibody. See Table of Materials for dilutions used in this study.</li>
	<li>Add 2 ml staining buffer to the cells, vortex and centrifuge at 200 x g for 5 min at 4 &#176;C to remove unbound antibody. Decant supernatant and resuspend pellet by flicking.</li>
	<li>Add 10 &#181;l fluorochrome-conjugated streptavidin diluted in staining buffer (see Materials Table). Incubate on ice for 20 min, protected from light. Wash as in step 3.3 to remove unbound streptavidin.</li>
	<li>Acquire the cell samples using a flow cytometer with at least six detectors9. Determine the frequency of Lin- Sca1+ CD117+ CD135- CD150+ cells (estimated HSCs) in each sample using the analysis template shown in Figure 2 and as previously reported10,11. 
	Note: The frequency of functional HSCs within that population can be estimated between 1/3 and 1/52,12.</li>
	<li>Establish estimated HSC equivalents for all samples11, using one sample (typically, the competitor, B6.SJL CD45.1+ in this example) as baseline as detailed below and in Figure 2.
	<ol>
		<li>Calculate the numbers of cells required using the following formulas: 
		#transplanted HSCs / recipient = 5 x 105 cells x estimated HSC frequency (as determined for baseline sample; this number is usually between 75 and 125 for wildtype C57Bl/6 mice)10-12. 
		Note: In Figure 2, for Sample A, the result is 139 HSCs. 
		total # transplanted cells (for other samples; e.g. Sample B in Figure 2) = # transplanted HSCs/HSC frequency. 
		Note: In Figure 2, for Sample B, the result is 197,826 (or approximately 2 x 105) total bone marrow cells.</li>
	</ol>
	</li>
	<li>Calculate the total numbers of cells required per graft recipient for each sample using the results obtained above. 
	Note: This number should be 5 x 105 for the baseline sample (competitor).
	<ol>
		<li>Multiply the number obtained from step 3.7 by the number of recipient mice and add enough cells for at least two additional mice to compensate for the dead volume in the syringe.</li>
	</ol>
	</li>
	<li>Mix competitor (e.g., CD45.1+) and test cells (CD45.2+) in 1:1 HSC equivalent ratio as calculated in step 3.6 and adjust the final volume for 200 &#181;l per injection using sterile PBS. 
	Note: For a group of five recipient mice the suggested volume would thus be at least 1.4 ml, containing 3.5 x 106 competitor bone marrow cells (or 973 estimated HSCs for Sample A in Figure 2), and the equivalent number of test cells (in Figure 2, the equivalent for Sample B would be 197,826 x 7 = 1,384,782 bone marrow cells).</li>
</ol>
4. Bone Marrow Transplants
<ol>
	<li>Warm up the recipient mice irradiated in step 1.2 with a red heat lamp to ensure dilation of the blood vessels and to make the lateral tail veins more visible.</li>
	<li>Prepare a 1 ml tuberculin syringe equipped with a 27 G needle (or smaller). Aspirate approximately 750 &#181;l of the prepared cell suspension (for 3 recipient mice). Make sure there are no air bubbles in the syringe.</li>
	<li>Insert the mouse into a restraining device. Examine the tail and look for the lateral tail veins that should be clearly visible on both sides of the tail. Wipe the injection site with ethanol. Insert the needle bevel up, parallel to the skin and gently press the plunger. Injection should be easy. If force is required, the needle is not in the vein. 
	Note: Older mice have thicker skin, which may make finding the vein more difficult. If the needle is not in the vein, reinsert the needle proximal to the initial injection site.</li>
	<li>Remove the needle and press the injection site with sterile gauze for a few seconds to stop bleeding. Transfer the mouse into a clean cage. 
	Note: Use the same syringe and needle for three injections, after which the needle may become too dull.</li>
	<li>For post-transplant follow-up, inject 1 ml sterile PBS subcutaneously to rehydrate the mice for the first five days. Add antibiotics in drinking water to prevent infections (if required; optional). Weigh mice every two to three days for the first three weeks and euthanize mice that have lost more than 15-20% their pre-transplant body weight (or as determined by the local animal ethics committee).</li>
</ol>
5. Analysis of Peripheral Blood
<ol>
	<li>Collect a drop of blood (approx. 50-100 &#181;l) into EDTA tubes.
	<ol>
		<li>To collect blood from the mandibular vein, use a lancet or a 22 G needle to pierce the facial skin near the hairless spot located under the jawbone and place the collecting tube to receive the drop of blood13. Collect blood at 4, 8, 12 and 16 weeks after transplant to follow multi-lineage reconstitution.</li>
	</ol>
	</li>
	<li>Add 1 ml freshly prepared RT red blood cell lysis buffer (9 parts 0.16 M NH4Cl + 1 part 0.17 M Tris-HCl pH 7.65) to the drop of blood collected in step 5.1.1. Mix and transfer sample to a 5 ml round-bottom polystyrene tube suitable for flow cytometry. Let stand for 4 min at RT.</li>
	<li>Add 4 volumes of ice-cold PBS. Mix by turning end-to-end and transfer immediately on ice. Centrifuge 10 min at 200 x g at 4 &#176;C.</li>
	<li>Decant supernatant and resuspend pellet by flicking. Supernatant should be clear and red. Add 2 ml staining buffer and vortex briefly. Centrifuge 5 min at 200 x g at 4 &#176;C.</li>
	<li>Decant supernatant and resuspend pellet by flicking.</li>
	<li>Proceed with flow cytometry staining using the general protocol detailed in steps 3.1 to 3.3 and the following antibodies for the master mix: CD45.1, CD45.2, CD3e, CD19, GR1.</li>
	<li>Acquire the cell samples using a flow cytometer with at least six detectors9. Determine the frequency of donor-derived cells for each lineage (CD19+ B lymphocytes, CD3e+ T lymphocytes, GR1bright granulocytes) in each sample using the analysis template provided in Figure 3 and as published10,11.</li>
</ol>
6. Analysis of Bone Marrow Reconstitution
<ol>
	<li>Collect bone marrow cells as detailed in section 2. Stain the cells for flow cytometry analysis using the general protocol detailed in steps 3.1 to 3.3 and the following antibodies for the master mix: Lineage cocktail, Sca1, CD117, CD135, CD150, CD45.1, CD45.2. Detect lineage cocktail antibodies using fluorochrome-conjugated streptavidin as described in step 3.4.</li>
	<li>Acquire cell samples using a flow cytometer with at least eight detectors9. If only six are available, add CD135 to the same channel as lineage panel as CD135+ cells will be excluded. Determine the frequency of donor-derived Lin- Sca1+ CD117+ CD135- CD150+ HSCs in each sample using the analysis template provided in Figure 4 and as published10.</li>
</ol>

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<li>Oguro, H., Ding, L., Morrison, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SLAM+family+markers+resolve+functionally+distinct+subpopulations+of+hematopoietic+stem+cells+and+multipotent+progenitors%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">SLAM family markers resolve functionally distinct subpopulations of hematopoietic stem cells and multipotent progenitors.</a> Cell Stem Cell. 13 (1), 102-116 (2013).</li>
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</ol>

The authors declare that they have no competing financial interests.

The protocol described here is designed to evaluate the relative fitness of donor (test) HSCs against known competitor HSCs. The situation of competition increases the relative sensitivity of the assay (more likely to detect moderate decreases in stem cell fitness) and provides an internal technical control for the efficacy of irradiation and injection. However, it should not be used as an absolute measure of HSC fitness; a decrease in competitive reconstitution does not automatically mean that the HSCs would not perform...

Hematopoiesis is a regenerative process that ensures the replenishing of blood cells that have been lost through injury, radiation and cell death. This process is ensured by hematopoietic stem cells (HSC) that largely reside in the adult bone marrow. In addition, hematopoietic stem cells can be used for therapeutic purposes in autoimmune disorders, hematological malignancies and immunodeficiencies1. There is thus a need to better understand the mechanisms that regulate HSC function, including their proliferative expansion and their ability to reach and engraft the recipient bone marrow after transplant. Although recent studies have reported several cell surface markers, including the SLAM family members CD150 and CD48, to prospectively enrich adult HSCs and fetal HSCs to approximately 50% purity2-4, the gold standard measure for functional HSCs remains an in vivo repopulating assay to determine their ability to re-establish blood cell production in an irradiated host5.
The in vivo clonal repopulating assay was initially developed by Till and McCulloch6 and has since been refined and expanded. As originally defined, HSCs ensure lifelong blood cell production through self-renewal and differentiation. The transfer of HSCs into an irradiated recipient thus allows us to assess: their ability to differentiate through the analysis of the different blood cell lineages (T lymphocytes, B lymphocytes, granulocytes, monocytes) and their capacity for self-renewal through serial transplantation. The assay would usually involve the comparison of the functionality and/or quantity of two populations of HSCs, e.g., cells coming from two mice of different genotypes or cells that have been treated or untreated with different factors that could influence the maintenance or expansion of HSCs in culture. Donor chimerism, or the contribution of transferred donor HSCs to blood cell production can then be determined by flow cytometry analysis in the peripheral blood and bone marrow using cell surface markers or other methods that will distinguish donor cells from the recipient, or host. The most widely used markers are certainly the two alleles for the gene Ptprc or CD45 leukocyte antigen7 that we have chosen for the examples provided below.
The clonal repopulation assay can be either competitive or non-competitive. In a non-competitive setting, control and test HSCs are transferred into separate recipient mice and the outcome for each cell type will be independent of the other. In a competitive setting, the function of both test and control HSCs is measured against a population of competitor HSCs. The protocol described here uses the competitive setting but can also be adapted for non-competitive situations. Both approaches have their advantages and limitations, and we will compare them in detail in the discussion. We also describe different approaches to ensure equity in the number of transplanted HSCs, explain how to adapt the assay for the quantification of HSCs by limiting dilution assay (LDA), and provide examples of both successful and unsuccessful transplants for the interpretation of results.

<table><tbody><tr><td>Microtainer tubes with K&lt;sub&gt;2&lt;/sub&gt;EDTA</td><td>BD Biosciences</td><td>365974</td><td></td></tr><tr><td>20 G needle</td><td>BD Syringe</td><td></td><td>For blood sampling from the mandibular vein</td></tr><tr><td>LabQuake Shaker rotisserie</td><td>Thermo&amp;nbsp; Scientific</td><td>C415110</td><td></td></tr><tr><td>Purified anti-mouse CD16/CD32 (clone 2.4G2, Fc Block)</td><td>BD Biosciences</td><td>553142</td><td>2.50 &amp;mu;g/ml</td></tr><tr><td>Pe-Cy7-conjugated anti-mouse CD3e (clone 145-2C11)</td><td>eBioscience</td><td>25-0031</td><td>0.25 &amp;mu;g/ml</td></tr><tr><td>PE-conjugated anti-mouse CD19 (clone 1D3)</td><td>eBioscience</td><td>12-0193</td><td>0.25 &amp;mu;g/ml</td></tr><tr><td>APC-eFluor780 (APC-Cy7 equivalent)-conjugated anti-mouse GR1 (clone RB6-8C5)</td><td>eBioscience</td><td>47-5931</td><td>0.25 &amp;mu;g/ml</td></tr><tr><td>FITC-conjugate anti-mouse CD45.1 (clone A20)</td><td>eBioscience</td><td>11-0453</td><td>2.50 &amp;mu;g/ml</td></tr><tr><td>eFluor450-conjugated anti-mouse CD45.2 (clone 104)</td><td>eBioscience</td><td>48-0454</td><td>1.00 &amp;mu;g/ml</td></tr><tr><td>Biotinylated anti-human/mouse CD45R (B220) (clone RA3-6B2)</td><td>eBioscience</td><td>13-0452</td><td>1.25 &amp;mu;g/ml</td></tr><tr><td>Biotinylated anti-mouse CD3e (clone 145-2C11)</td><td>eBioscience</td><td>13-0031</td><td>1.25 &amp;mu;g/ml</td></tr><tr><td>Biotinylated anti-mouse CD11b (clone M1/70)</td><td>eBioscience</td><td>13-0112</td><td>1.25 &amp;mu;g/ml</td></tr><tr><td>Biotinylated anti-mouse GR1 (clone RB6-8C5)</td><td>eBioscience</td><td>13-5931</td><td>1.25 &amp;mu;g/ml</td></tr><tr><td>Biotinylated anti-mouse TER119 (clone TER119)</td><td>eBioscience</td><td>13-5921</td><td>0.625 &amp;mu;g/ml</td></tr><tr><td>V500 streptavidin</td><td>BD Biosciences</td><td>56149</td><td>0.5 &amp;mu;g/ml</td></tr><tr><td>PE-conjugated anti-mouse CD117 (clone 2B8)</td><td>BD Biosciences</td><td>553355</td><td>0.25 &amp;mu;g/ml</td></tr><tr><td>PE-Cy7-conjugated anti-mouse Ly6A/E (Sca1) (clone D7)</td><td>BD Biosciences</td><td>558162</td><td>0.25 &amp;mu;g/ml</td></tr><tr><td>PerCP-eFluor710-conjugated anti-mouse CD135 (clone A2F10)</td><td>eBioscience</td><td>46-1351</td><td>0.5 &amp;mu;g/ml</td></tr><tr><td>Alexa fluor 647-conjugated anti-mouse CD150 (clone TC15-12F12.2)</td><td>Biolegend</td><td>115918</td><td>0.625 &amp;mu;g/ml
BD Biosciences and eBioscience do not carry the same clone</td></tr><tr><td>1 ml tuberculin syringe with 27 G needle</td><td>BD Syringe</td><td>309623</td><td></td></tr><tr><td>1 ml tuberculin syringe with 25 G needle</td><td>BD Syringe</td><td>309626</td><td></td></tr><tr><td>70 &amp;mu;m cell strainer</td><td>BD Falcon</td><td>352350</td><td></td></tr></tbody></table>

competitive transplants to evaluate hematopoietic stem cell fitness

Competitive Transplants to Evaluate Hematopoietic Stem Cell Fitness

The gold standard definition of a hematopoietic stem cell (HSC) is a cell that when transferred into an irradiated recipient will have the ability to reestablish blood cell production for the lifespan of the recipient. This protocol explains how to set up a functional assay to compare the HSC capacities of two different populations of cells, such as bone marrow from mice of two different genotypes, and how to analyze the recipient mice by flow cytometry. The protocol uses HSC equivalents rather than cell sorting for standardization and discusses the advantages and disadvantages of both approaches. We further discuss different variations to the basic protocol, including serial transplants, limiting dilution assays, homing assays and non-competitive transplants, including the advantages and preferred uses of these varied approaches. These assays are central for the study of HSC function and could be used not only for the investigation of fundamental HSC intrinsic aspects of biology but also for the development of preclinical assays for bone marrow transplant and HSC expansion in culture.

A general description of the competitive transplant setting, including secondary transplants (discussed further below) can be found in Figure 1. A representative analysis for pre-transplant bone marrow HSCs can be found in Figure 2. More detailed information on the exclusion of doublets and dead cells can be found elsewhere9.
Figures 3 and 4 provide e...

This protocol provides step-by-step guidelines for setting up competitive mouse bone marrow transplant experiments to study hematopoietic stem/progenitor cell function without prior purification of stem cells by cell sorting.

The gold standard definition of a hematopoietic stem cell (HSC) is a cell that when transferred into an irradiated recipient will have the ability to ...

The overall goal of this experiment is to determine the fitness of bone marrow hematopoietic stem cells in vivo using a competitive transplant assay. So this method can help answer key questions in the field of stem cell biology and hematopoietic cell transplantation about the impact of various manipulations on stem cell self-renewal after transplant. The main advantages of this technique are that it requires a limited number of animals and that it does not involve cell sorting.Though this method can provide insight into stem cell function, it can be also useful in other fields such as immunology, through the use of mixed bone marrow chimeras. The initial development of this technique in the 1960s paved the way for researchers in the field of stem cell biology to explore the identity and function of hematopoietic stem cells in humans and mice. Demonstrating the procedure will be Roxanne Hetu-Arbour, a Master's student in the lab, and Edward Owusu Kwarteng, a PhD student.Begin by placing the mouse inside a biological safety cabinet on a surgical platform and wet the skin and fur with 70%ethanol. Next, take hold of a hind foot and cut the skin up along the leg. Proceed to pull away the skin and cut away the excess muscle.Then, with the scissor blades against the pelvic bones as a counter-force, pull on the leg bone to dislocate the femur. Detach the tibia and the femur from the kneecap, then remove the foot, and remove the remaining bits of muscle and tendons. Then, place the bones in two to three milliliters of sterile PBS in one well of a six-well tissue culture plate.When all of the bones have been harvested, grasp the bones one at a time with a forceps, and insert a 25-gauge needle attached to a one-milliliter syringe filled with sterile PBS into one end of the bone. Depress the plunger, collecting the cells in a 15-milliliter conical tube until the center of each bone is white. Using a 22-gauge needle, dissociate the cells into a single cell suspension by repeatedly passing them through the needle tip.After all of the bone marrow has been collected, filter the cells through a 70-micron nylon strainer and count the resulting cell suspensions. Then, centrifuge the bone marrow cells, remove the supernatant, and dilute the samples to a one times ten to the eighth cells per milliliter concentration in fresh PBS, on ice. To establish donor hematopoietic stem cell equivalents, transfer 30 microliter aliquots of cells into five-milliliter round-bottomed polystyrene tubes and add an equal volume of unlabeled antibody against CD16 and CD32 to the samples for five minutes at room temperature to block any non-specific staining.Next, incubate the cells in 90 microliters of the appropriate fluorochrome conjugated primary antibody master mix on ice, protected from light. After 30 minutes, add two milliliters of staining buffer to the cells and vortex the tubes. Follow this with centrifugation.Decant the supernatants and resuspend the pellets by flicking. Then, label the cells with 10 microliters of fluorochrome conjugated streptavidin on ice for 20 minutes in the dark. At the end of the incubation, wash the cells in two more milliliters of buffer and analyze the samples by flow cytometry.Using one sample as a baseline, establish the estimated hematopoietic stem cell equivalents for each of the samples. After calculating the number of hematopoietic stem cells per sample as outlined in the text, mix the competitor and test hematopoietic stem cells at a one-to-one ratio and adjust the final volume of the cells to 200 microliters per injection in sterile PBS. For adoptive transfer of the hematopoietic stem cells, warm up an irradiated recipient mouse with a heat lamp.Then aspirate 750 microliters of cells into one one-milliliter tuberculin syringe equipped with a 27-gauge needle and remove the bubbles. Next, with the mouse in the appropriate restraining device, wipe the injection site with ethanol. Insert the needle into a lateral tail vein, bevel side up and parallel to the skin, and gently depress the plunger.When all of the cells have been injected, remove the needle and compress the injection site with sterile gauze for a few seconds to stop the bleeding. Then place the mouse in a clean cage. To analyze the peripheral blood, pierce the facial skin near the hairless spot located under the jawbone and collect one to two drops of blood in an EDTA tube.Place the tube in a rotator to prevent clotting while waiting to proceed. Next, mix one milliliter of freshly-prepared, room temperature, red blood cell lysis buffer with the blood and transfer the sample to a five-milliliter, round-bottomed polystyrene tube. After four minutes at room temperature, add four volumes of ice-cold PBS to each tube and cover the opening with a cap.Mix the tube a few times by inversion and immediately place the cells on ice. After centrifugation, decant the clear red supernatants and resuspend the pellets by flicking. Then add two milliliters of staining buffer to the cells and briefly vortex them.Follow this with a a second centrifugation. Resuspend the pellets by flicking and proceed with flow cytometry staining as detailed in the text. The competitor-derived cells should be present in all three of the myeloid, B-lymphoid, and T-lymphoid lineages.The competitor and donor cells are also found within both lineage negative and hematopoietic stem cell bone marrow cell populations. When the donor hematopoietic stem cells are functionally equivalent to the competitor cells, the proportions of the donor and the competitor-derived cells are equal. The residual host cells are typically almost exclusively T lymphocytes, as these cells are more radioresistant.When the donor cells are observed at a much lower proportion of peripheral blood leukocytes than the competitor cells, some aspects of hematopoietic stem cell function are defective, and warrant further investigation. When the donor and competitor cells are both present in low numbers and the host cells represent the majority of peripheral blood cells, the transplant is considered unsuccessful, and no conclusions can be drawn about the relative functionality of the donor versus the competitor cells. Once mastered, the bone marrow preparation and the transplantation steps can be completed in six to seven hours if they are performed properly.While attempting this procedure, it is important to remember to maintain sterile conditions during the cell preparation and the animal manipulation steps. Following this procedure, other methods, like a limiting dilution assay, can be performed to answer additional questions regarding the functional stem cell numbers. After watching this video, you should have a good understanding of how to perform competitive bone marrow transplants.Don't forget that working with irradiation can be extremely hazardous, and that the appropriate precautions should always be taken while performing this procedure.

competitive-transplants-to-evaluate-hematopoietic-stem-cell-fitness

Research

JoVE Journal

Developmental Biology

This manuscript provides a description of the methodology used to establish transduction-transplantation mouse models. A detailed account is given of technical errors to avoid when performing bone marrow transplants. A clear understanding should be gained of the importance of high viral titer, transfection/transduction, and irradiation.

Transduction-Transplantation Mouse Model of Myeloproliferative Neoplasm

Quantification of donor-derived cells is required to monitor engraftment after stem cell transplantation in patients with hemoglobinopathies. A combination of flow cytometry-based cell sorting, colony formation assay, and subsequent analysis of short tandem repeats may be used to assess the proliferation and differentiation of progenitors in the erythroid compartment.

Detection of Residual Donor Erythroid Progenitor Cells after Hematopoietic Stem Cell Transplantation for Patients with Hemoglobinopathies

We describe the use of hematopoietic stem cell transplantation (HSCT) to assess the malignant potential of genetically engineered hematopoietic cells. HSCT is useful for evaluating various malignant hematopoietic cells in vivo as well as generating a large cohort of mice with myelodysplastic syndromes (MDS) or leukemia to evaluate novel therapies.

Use of Hematopoietic Stem Cell Transplantation to Assess the Origin of Myelodysplastic Syndrome

The goal of this protocol is to isolate nonhuman primate CD34+ cells from primed bone marrow, to gene-modify these cells with lentiviral vectors, and to prepare a product for infusion into the autologous host. The total protocol length is approximately 48 h.

Preparation and Gene Modification of Nonhuman Primate Hematopoietic Stem and Progenitor Cells

Described are protocols for the highly efficient genome editing of murine hematopoietic stem and progenitor cells (HSPC) by the CRISPR/Cas9 system to rapidly develop mouse model systems with hematopoietic system-specific gene modifications.

Lentiviral CRISPR/Cas9-Mediated Genome Editing for the Study of Hematopoietic Cells in Disease Models

Here we describe a reliable method to measure mitochondrial mass and membrane potential in ex vivo cultured hematopoietic stem cells and T cells.

Measurement of Mitochondrial Mass and Membrane Potential in Hematopoietic Stem Cells and T-cells by Flow Cytometry

We describe three methods of bone marrow transplantation (BMT): BMT with total-body irradiation, BMT with shielded irradiation, and BMT method with no pre-conditioning (adoptive BMT) for the study of clonal hematopoiesis in mouse models.

Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis

This protocol enables the maintenance of quiescent human hematopoietic stem cells in vitro by mimicking the bone marrow microenvironment using abundant fatty acids, cholesterol, lower concentrations of cytokines, and hypoxia.

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

This protocol describes the intrafemoral injection of a few hematopoietic or leukemic stem cells, including gene-edited cells, in murine xenograft models, which will not only enable the quick and safe transplantation of cells but also serial analyses of the bone marrow.

Simplified Intrafemoral Injections Using Live Mice Allow for Continuous Bone Marrow Analysis

Intrafemoral injections allow for the engraftment of a small number of hematopoietic stem and progenitor cells (HSPCs), by placing the cells directly in the bone marrow cavity. Here we describe an experimental protocol of intrafemoral injection of human HSPCs into immunodeficient mice.

Intrafemoral Injection of Human Hematopoietic Stem and Progenitor Cells into Immunocompromised Mice

Here, we present a protocol that integrates CRISPR/Cas9 technology with the Cell Counting Kit-8 (CCK-8) assay to identify candidate genes that are crucial for the abnormal proliferative capacity of hematopoietic stem and progenitor cells.

Evaluation of Abnormal Growth-related Genes of Hematopoietic Stem and Progenitor Cells by Combining CRISPR/Cas9 Technology with Cell Counting

Learn competitive bone marrow transplant assays to evaluate hematopoietic stem cell fitness in vivo without cell sorting. This method reveals self-renewal mechanisms and requires minimal animals.

Competitive Transplants to Evaluate Hematopoietic Stem Cell Fitness

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