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, dispo...

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Z., 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=Optimal+protocol+for+total+body+irradiation+for+allogeneic+bone+marrow+transplantation+in+mice.">Optimal protocol for total body irradiation for allogeneic bone marrow transplantation in mice.</a> Bone Marrow Transplant. 30 (12), 843-849 (2002).</li><li>Benz, C., 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=Hematopoietic+Stem+Cell+Subtypes+Expand+Differentially+during+Development+and+Display+Distinct+Lymphopoietic+Programs.">Hematopoietic Stem Cell Subtypes Expand Differentially during Development and Display Distinct Lymphopoietic Programs.</a> Cell Stem Cell. 10 (3), 273-283 (2012).</li><li>Eppert, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+gene+expression+programs+influence+clinical+outcome+in+human+leukemia.">Stem cell gene expression programs influence clinical outcome in human leukemia.</a> Nat Med. 17 (9), 1086-1093 (2011).</li><li>McIntosh, B. 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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 border="0" cellpadding="0" cellspacing="0" width="703">
	<tbody>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">Microtainer tubes with K2EDTA</td>
			<td style="width:89px;">BD Biosciences</td>
			<td style="width:112px;"></td>
			<td style="width:119px;">365974</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">20G needle</td>
			<td style="width:89px;">BD Syringe</td>
			<td style="width:112px;"></td>
			<td style="width:119px;"></td>
			<td>For blood sampling from the mandibular vein</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">LabQuake Shaker rotisserie</td>
			<td style="width:89px;">Thermo&#160; Scientific</td>
			<td style="width:112px;"></td>
			<td style="width:119px;">C415110</td>
			<td>Any other rotating mixer will work as well to prevent coagulation of blood samples</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Purified anti-mouse CD16/CD32 (clone 2.4G2, Fc Block)</td>
			<td style="width:89px;">BD Biosciences</td>
			<td style="width:112px;">2.50</td>
			<td style="width:119px;">553142</td>
			<td>Alternatively use clone 93 from eBioscience (cat # 14-0161) or Biolegend (cat# 101310)&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pe-Cy7-conjugated anti-mouse CD3e (clone 145-2C11)</td>
			<td style="width:89px;">eBioscience</td>
			<td style="width:112px;">0.25</td>
			<td style="width:119px;">25-0031</td>
			<td>For most flow cytometry antibodies, the clone is important but the colours and companies can vary depending on the available equipment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PE-conjugated anti-mouse CD19 (clone 1D3)</td>
			<td style="width:89px;">eBioscience</td>
			<td style="width:112px;">0.25</td>
			<td style="width:119px;">12-0193</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">APC-eFluor780 (APC-Cy7 equivalent)-conjugated anti-mouse GR1 (clone RB6-8C5)</td>
			<td style="width:89px;">eBioscience</td>
			<td style="width:112px;">0.25</td>
			<td style="width:119px;">47-5931</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">FITC-conjugate anti-mouse CD45.1 (clone A20)</td>
			<td style="width:89px;">eBioscience</td>
			<td style="width:112px;">2.50</td>
			<td style="width:119px;">11-0453</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">eFluor450-conjugated anti-mouse CD45.2 (clone 104)</td>
			<td style="width:89px;">eBioscience</td>
			<td style="width:112px;">1.00</td>
			<td style="width:119px;">48-0454</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Biotinylated anti-human/mouse CD45R (B220) (clone RA3-6B2)</td>
			<td style="width:89px;">eBioscience</td>
			<td style="width:112px;">1.25</td>
			<td style="width:119px;">13-0452</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Biotinylated anti-mouse CD3e (clone 145-2C11)</td>
			<td style="width:89px;">eBioscience</td>
			<td style="width:112px;">1.25</td>
			<td style="width:119px;">13-0031</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Biotinylated anti-mouse CD11b (clone M1/70)</td>
			<td style="width:89px;">eBioscience</td>
			<td style="width:112px;">1.25</td>
			<td style="width:119px;">13-0112</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Biotinylated anti-mouse GR1 (clone RB6-8C5)</td>
			<td style="width:89px;">eBioscience</td>
			<td style="width:112px;">1.25</td>
			<td style="width:119px;">13-5931</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Biotinylated anti-mouse TER119 (clone TER119)</td>
			<td style="width:89px;">eBioscience</td>
			<td style="width:112px;">0.63</td>
			<td style="width:119px;">13-5921</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">V500 streptavidin</td>
			<td style="width:89px;">BD Biosciences</td>
			<td style="width:112px;">0.50</td>
			<td style="width:119px;">561419</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PE-conjugated anti-mouse CD117 (clone 2B8)</td>
			<td style="width:89px;">BD Biosciences</td>
			<td style="width:112px;">0.25</td>
			<td style="width:119px;">553355</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PE-Cy7-conjugated anti-mouse Ly6A/E (Sca1) (clone D7)</td>
			<td style="width:89px;">BD Biosciences</td>
			<td style="width:112px;">0.25</td>
			<td style="width:119px;">558162</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">PerCP-eFluor710-conjugated anti-mouse CD135 (clone A2F10)</td>
			<td style="width:89px;">eBioscience</td>
			<td style="width:112px;">0.50</td>
			<td style="width:119px;">46-1351</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Alexa fluor 647-conjugated anti-mouse CD150 (clone TC15-12F12.2)</td>
			<td style="width:89px;">Biolegend</td>
			<td style="width:112px;">0.63</td>
			<td style="width:119px;">115918</td>
			<td>BD Biosciences and eBioscience do not carry the same clone</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1ml tuberculin syringe with 27G needle</td>
			<td style="width:89px;">BD Syringe</td>
			<td style="width:112px;"></td>
			<td style="width:119px;">309623</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1ml tuberculin syringe with 25G needle</td>
			<td style="width:89px;">BD Syringe</td>
			<td style="width:112px;"></td>
			<td style="width:119px;">309626</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">70 um cell strainer</td>
			<td style="width:89px;">BD Falcon</td>
			<td style="width:112px;"></td>
			<td style="width:119px;">352350</td>
			<td></td>
		</tr>
	</tbody>
</table>

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...

Watch this Scientific Journal Video about Competitive Transplants to Evaluate Hematopoietic Stem Cell Fitness at JoVE.com

Competitive Transplants to Evaluate Hematopoietic Stem Cell Fitness

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

15.0K Views.  Institut National de la Recherche Scientifique. 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 ...