Name: competitive transplants to evaluate hematopoietic stem cell fitness
Uploaded: 2016-08-31T13:00:00.000+00:00
Duration: 8 min 53 s
Description: Watch this Scientific Journal Video about Competitive Transplants to Evaluate Hematopoietic Stem Cell Fitness at JoVE.com

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>

<ol>
	<li>Li, H. W., Sykes, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+concepts+in+haematopoietic+cell+transplantation.">Emerging concepts in haematopoietic cell transplantation.</a> Nat Rev Immunol. 12 (6), 403-416 (2012).</li><li>Kiel, M. J., Yilmaz, O. H., Iwashita, T., Terhorst, C., Morrison, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SLAM+family+receptors+distinguish+hematopoietic+stem+and+progenitor+cells+and+reveal+endothelial+niches+for+stem+cells.">SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells.</a> Cell. 121 (7), 1109-1121 (2005).</li><li>Kim, I., He, S., Yilmaz, O. H., Kiel, M. J., Morrison, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+purification+of+fetal+liver+hematopoietic+stem+cells+using+SLAM+family+receptors.">Enhanced purification of fetal liver hematopoietic stem cells using SLAM family receptors.</a> Blood. 108 (2), 737-744 (2006).</li><li>Mayle, A., Luo, M., Jeong, M., Goodell, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+analysis+of+murine+hematopoietic+stem+cells.">Flow cytometry analysis of murine hematopoietic stem cells.</a> Cytometry A. 83 (1), 27-37 (2013).</li><li>Rossi, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Less+Is+More:+Unveiling+the+Functional+Core+of+Hematopoietic+Stem+Cells+through+Knockout+Mice.">Less Is More: Unveiling the Functional Core of Hematopoietic Stem Cells through Knockout Mice.</a> Cell Stem Cell. 11 (3), 302-317 (2012).</li><li>Till, J. E., McCulloch, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+direct+measurement+of+the+radiation+sensitivity+of+normal+mouse+bone+marrow+cells.">A direct measurement of the radiation sensitivity of normal mouse bone marrow cells.</a> Radiat Res. 14, 213-222 (1961).</li><li>Shen, F. W., 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=Cloning+of+Ly-5+cDNA.">Cloning of Ly-5 cDNA.</a> Proc Natl Acad Sci U S A. 82 (21), 7360-7363 (1985).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+GM+mice.">Identification of GM mice.</a> Laboratory Animals. 37 (suppl 1), 33-35 (2003).</li><li>Rundberg Nilsson, A., Bryder, D., Pronk, C. J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequency+determination+of+rare+populations+by+flow+cytometry:+A+hematopoietic+stem+cell+perspective.">Frequency determination of rare populations by flow cytometry: A hematopoietic stem cell perspective.</a> Cytometry Part A. 83A (8), 721-727 (2013).</li><li>Abidin, B. M., Owusu Kwarteng, E., Heinonen, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frizzled-6+Regulates+Hematopoietic+Stem/Progenitor+Cell+Survival+and+Self-Renewal.">Frizzled-6 Regulates Hematopoietic Stem/Progenitor Cell Survival and Self-Renewal.</a> J Immunol. 195 (5), 2168-2176 (2015).</li><li>Heinonen, K. M., Vanegas, J. R., Lew, D., Krosl, J., Perreault, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt4+enhances+murine+hematopoietic+progenitor+cell+expansion+through+a+planar+cell+polarity-like+pathway.">Wnt4 enhances murine hematopoietic progenitor cell expansion through a planar cell polarity-like pathway.</a> PLoS One. 6 (4), e19279 (2011).</li><li>Oguro, H., Ding, L., Morrison, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SLAM+family+markers+resolve+functionally+distinct+subpopulations+of+hematopoietic+stem+cells+and+multipotent+progenitors.">SLAM family markers resolve functionally distinct subpopulations of hematopoietic stem cells and multipotent progenitors.</a> Cell Stem Cell. 13 (1), 102-116 (2013).</li><li>Golde, W. T., Gollobin, P., Rodriguez, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid,+simple,+and+humane+method+for+submandibular+bleeding+of+mice+using+a+lancet.">A rapid, simple, and humane method for submandibular bleeding of mice using a lancet.</a> Lab Anim (NY). 34 (9), 39-43 (2005).</li><li>Santaguida, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=JunB+protects+against+myeloid+malignancies+by+limiting+hematopoietic+stem+cell+proliferation+and+differentiation+without+affecting+self-renewal.">JunB protects against myeloid malignancies by limiting hematopoietic stem cell proliferation and differentiation without affecting self-renewal.</a> Cancer Cell. 15 (4), 341-352 (2009).</li><li>Czechowicz, A., Kraft, D., Weissman, I. L., Bhattacharya, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+transplantation+via+antibody-based+clearance+of+hematopoietic+stem+cell+niches.">Efficient transplantation via antibody-based clearance of hematopoietic stem cell niches.</a> Science. 318 (5854), 1296-1299 (2007).</li><li>Zhang, C. C., Lodish, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+hematopoietic+stem+cells+change+their+surface+phenotype+during+ex+vivo+expansion.">Murine hematopoietic stem cells change their surface phenotype during ex vivo expansion.</a> Blood. 105 (11), 4314-4320 (2005).</li><li>Benveniste, P., 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=Intermediate-Term+Hematopoietic+Stem+Cells+with+Extended+but+Time-Limited+Reconstitution+Potential.">Intermediate-Term Hematopoietic Stem Cells with Extended but Time-Limited Reconstitution Potential.</a> Cell Stem Cell. 6 (1), 48-58 (2010).</li><li>Fazekasde St Groth, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evaluation+of+limiting+dilution+assays.">The evaluation of limiting dilution assays.</a> J Immunol Methods. 49 (2), R11-R23 (1982).</li><li>Louis, I., Heinonen, K. M., Chagraoui, J., Vainio, S., Sauvageau, G., Perreault, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+signaling+protein+Wnt4+enhances+thymopoiesis+and+expands+multipotent+hematopoietic+progenitors+through+beta-catenin-independent+signaling.">The signaling protein Wnt4 enhances thymopoiesis and expands multipotent hematopoietic progenitors through beta-catenin-independent signaling.</a> Immunity. 29 (1), 57-67 (2008).</li><li>Cui, Y. 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. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonirradiated+NOD,B6.SCID+Il2rgamma-/-+Kit(W41/W41)+(NBSGW)+mice+support+multilineage+engraftment+of+human+hematopoietic+cells.">Nonirradiated NOD,B6.SCID Il2rgamma-/- Kit(W41/W41) (NBSGW) mice support multilineage engraftment of human hematopoietic cells.</a> Stem Cell Reports. 4 (2), 171-180 (2015).</li></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

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...

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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 ...

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

Research

JoVE Journal

Developmental Biology

15.2K Views.  Institut National de la Recherche Scientifique. 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. 

Video: Competitive Transplants to Evaluate Hematopoietic Stem Cell Fitness

Transduction-Transplantation Mouse Model of Myeloproliferative Neoplasm

Transduction-transplantation is a quick and efficient way to model human hematologic malignancies in mice. This technique results in expression of the gene of interest in hematopoietic cells and can be used to study the gene's role in normal and/or malignant hematopoiesis. This protocol provides a detailed description on how to perform transduction-transplantation using calreticulin (CALR) mutations recently identified in myeloproliferative neoplasm (MPN) as an example. In this protocol whole bone marrow cells from 5-flurouracil (5-FU) treated donor mice are transduced with a retrovirus encoding mutant CALR and transplanted into lethally irradiated syngeneic hosts. Donor cells expressing mutant CALR are marked with green fluorescent protein (GFP). Transplanted mice develop an MPN phenotype including elevated platelets in the peripheral blood, expansion of megakaryocytes in the bone marrow, and bone marrow fibrosis. We provide a step-by-step account of how to generate retrovirus, calculate viral titer, transduce whole bone marrow cells, and transplant into irradiated recipient mice.

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 is a quick and efficient way to model human hematologic malignancies in mice. This technique results in expression of the ...

Cancer Research

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 ...

Medicine

Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early embryo and the lack of assays that functionally identify the long-term multilineage engraftment potential of individual putative HSC precursors. Here, we describe methodology that enables the isolation and characterization of functionally validated HSC precursors at the single cell level. First, we utilize index sorting to catalog the precise phenotypic parameter of each individually sorted cell, using a combination of phenotypic markers to enrich for HSC precursors with additional markers for experimental analysis. Second, each index-sorted cell is co-cultured with vascular niche stroma from the aorta-gonad-mesonephros (AGM) region, which supports the maturation of non-engrafting HSC precursors to functional HSC with multilineage, long-term engraftment potential in transplantation assays. This methodology enables correlation of phenotypic properties of clonal hemogenic precursors with their functional engraftment potential or other properties such as transcriptional profile, providing a means for the detailed analysis of HSC precursor development at the single cell level.

Here we present methodology for the clonal analysis of hematopoietic stem cell precursors during murine embryonic development. We combine index sorting of single cells from the embryonic aorta-gonad-mesonephros region with endothelial cell co-culture and transplantation to characterize the phenotypic properties and engraftment potential of single hematopoietic precursors.

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early ...

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

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood cytopenias, dysplasia, and a propensity for transformation to acute leukemia. NUP98-HOXD13 (NHD13) transgenic mice recapitulate human MDS in terms of peripheral blood cytopenias, dysplasia, and transformation to acute leukemia. We previously demonstrated that MDS could be transferred from a genetically engineered mouse with MDS to wild-type recipients by transplanting MDS bone marrow nucleated cells (BMNC). To more clearly understand the MDS cell of origin, we have developed approaches to transplant specific, immunophenotypically defined hematopoietic subsets. In this article, we describe the process of isolating and transplanting specific populations of hematopoietic stem and progenitor cells. Following transplantation, we describe approaches to assess the efficiency of transplantation and persistence of the donor MDS cells.

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.

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood ...

Engineering Oncogenic Heterozygous Gain-of-Function Mutations in Human Hematopoietic Stem and Progenitor Cells

Throughout their lifetime, hematopoietic stem and progenitor cells (HSPCs) acquire somatic mutations. Some of these mutations alter HSPC functional properties such as proliferation and differentiation, thereby promoting the development of hematologic malignancies. Efficient and precise genetic manipulation of HSPCs is required to model, characterize, and better understand the functional consequences of recurrent somatic mutations. Mutations can have a deleterious effect on a gene and result in loss-of-function (LOF) or, in stark contrast, may enhance function or even lead to novel characteristics of a particular gene, termed gain-of-function (GOF). In contrast to LOF mutations, GOF mutations almost exclusively occur in a heterozygous fashion. Current genome-editing protocols do not allow for the selective targeting of individual alleles, hampering the ability to model heterozygous GOF mutations. Here, we provide a detailed protocol on how to engineer heterozygous GOF hotspot mutations in human HSPCs by combining CRISPR/Cas9-mediated homology-directed repair and recombinant AAV6 technology for efficient DNA donor template transfer. Importantly, this strategy makes use of a dual fluorescent reporter system to allow for the tracking and purification of successfully heterozygously edited HSPCs. This strategy can be employed to precisely investigate how GOF mutations affect HSPC function and their progression toward hematological malignancies.

Novel strategies to faithfully model somatic mutations in hematopoietic stem and progenitor cells (HSPCs) are necessary to better study hematopoietic stem cell biology and hematological malignancies. Here, a protocol to model heterozygous gain-of-function mutations in HSPCs by combining the use of CRISPR/Cas9 and dual rAAV donor transduction is described.

Throughout their lifetime, hematopoietic stem and progenitor cells (HSPCs) acquire somatic mutations. Some of these mutations alter HSPC functional ...

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

Hematopoietic stem cells possess the ability for long-term self-renewal and the potential to differentiate into various types of mature blood cells. However, the accumulation of cancerous mutations in hematopoietic stem and progenitor cells (HSPCs) can block normal differentiation, induce aberrant proliferation, and ultimately lead to leukemogenesis. To identify and/or evaluate the cancerous mutations, we integrated CRISPR/Cas9 technology with the Cell Counting Kit-8 (CCK-8) assay to investigate a model gene Trp53, that is essential for abnormal proliferative ability of HSPCs. Specifically, bone marrow cells enriched for HSPCs from Cas9 mice were harvested and then subjected to viral transfection with single-guide RNAs (sgRNAs) targeting one or several candidate genes to introduce genetic alterations in HSPCs. Then, a CCK-8 assay was performed to investigate the proliferative capacity of transfected HSPCs. The sgRNA targeting efficiency was confirmed by a Tracking of Indels by Decomposition assay. These identified genes may play crucial roles in leukemogenesis and could serve as potential therapeutic targets.

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.

Hematopoietic stem cells possess the ability for long-term self-renewal and the potential to differentiate into various types of mature blood cells. ...

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

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting from advances in genome editing technology and lentivirus-mediated transgene delivery systems, an efficient and economical method is described here that establishes mice in which genes are manipulated specifically in hematopoietic stem cells. Lentiviruses are used to transduce Cas9-expressing lineage-negative bone marrow cells with a guide RNA (gRNA) targeting specific genes and a red fluorescence reporter gene (RFP), then these cells are transplanted into lethally-irradiated C57BL/6 mice. Mice transplanted with lentivirus expressing non-targeting gRNA are used as controls. Engraftment of transduced hematopoietic stem cells are evaluated by flow cytometric analysis of RFP-positive leukocytes of peripheral blood. Using this method, ~90% transduction of myeloid cells and ~70% of lymphoid cells at 4 weeks after transplantation can be achieved. Genomic DNA is isolated from RFP-positive blood cells, and portions of the targeted site DNA are amplified by PCR to validate the genome editing. This protocol provides a high-throughput evaluation of hematopoiesis-regulatory genes and can be extended to a variety of mouse disease models with hematopoietic cell involvement.

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.

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting ...

Immunology and Infection

Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor cells (HSPCs). Mutations in driver genes, that confer cellular fitness, can lead to the development of expanding HSPC clones that increasingly give rise to progeny leukocytes harboring the somatic mutation. Because clonal hematopoiesis has been associated with heart disease, stroke, and mortality, the development of experimental systems that model these processes is key to understanding the mechanisms that underly this new risk factor. Bone marrow transplantation procedures involving myeloablative conditioning in mice, such as total-body irradiation (TBI), are commonly employed to study the role of immune cells in cardiovascular diseases. However, simultaneous damage to the bone marrow niche and other sites of interest, such as the heart and brain, is unavoidable with these procedures. Thus, our lab has developed two alternative methods to minimize or avoid possible side effects caused by TBI: 1) bone marrow transplantation with irradiation shielding and 2) adoptive BMT to non-conditioned mice. In shielded organs, the local environment is preserved allowing for the analysis of clonal hematopoiesis while the function of resident immune cells is unperturbed. In contrast, the adoptive BMT to non-conditioned mice has the additional advantage that both the local environments of the organs and the hematopoietic niche are preserved. Here, we compare three different hematopoietic cell reconstitution approaches and discuss their strengths and limitations for studies of clonal hematopoiesis in cardiovascular disease.

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.

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor ...

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

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production of all mature blood cells. In recent years cellular metabolism has emerged as a crucial regulator of HSC function and fate. We have previously demonstrated that modulation of mitochondrial metabolism influences HSC fate. Specifically, by chemically uncoupling the electron transport chain we were able to maintain HSC function in culture conditions that normally induce rapid differentiation. However, limiting HSC numbers often precludes the use of standard assays to measure HSC metabolism and therefore predict their function. Here, we report a simple flow cytometry assay that allows reliable measurement of mitochondrial membrane potential and mitochondrial mass in scarce cells such as HSCs. We discuss the isolation of HSCs from mouse bone marrow and measurement of mitochondrial mass and membrane potential post ex vivo culture. As an example, we show the modulation of these parameters in HSCs via treatment with a metabolic modulator. Moreover, we extend the application of this methodology on human peripheral blood-derived T cells and human tumor infiltrating lymphocytes (TILs), showing dramatic differences in their mitochondrial profiles, possibly reflecting different T cell functionality. We believe this assay can be employed in screenings to identify modulators of mitochondrial metabolism in various cell types in different contexts.

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

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production ...

Biology

Ex Vivo Expansion and Genetic Manipulation of Mouse Hematopoietic Stem Cells in Polyvinyl Alcohol-Based Cultures

Self-renewing&#160;multipotent&#160;hematopoietic stem cells (HSCs) are an important cell type due to their abilities to support hematopoiesis throughout life and reconstitute the entire blood system following transplantation. HSCs are used clinically in stem cell transplantation therapies, which represent curative treatment for a range of blood diseases. There is substantial interest in both understanding the mechanisms that regulate HSC activity and hematopoiesis, and developing new HSC-based therapies. However, the stable culture and expansion of HSCs ex vivo has been a major barrier in studying these stem cells in a tractable ex vivo system. We recently developed a polyvinyl alcohol-based culture system that can support the long-term and large-scale expansion of transplantable mouse HSCs and methods to genetically edit them. This protocol describes methods to culture and genetically manipulate mouse HSCs via electroporation and lentiviral transduction. This protocol is expected to be useful to a wide range of experimental hematologists interested in HSC biology and hematopoiesis.

Ex Vivo Expansion and Genetic Manipulation of Mouse Hematopoietic Stem Cells in Polyvinyl Alcohol-Based Cultures

Presented here is a protocol to initiate, maintain, and analyze mouse hematopoietic stem cell cultures using ex vivo polyvinyl alcohol-based expansion, as well as methods to genetically manipulate them by lentiviral transduction and electroporation.

Self-renewing&#160;multipotent&#160;hematopoietic stem cells (HSCs) are an important cell type due to their abilities to support hematopoiesis throughout ...

Competitive Transplants to Evaluate Hematopoietic Stem Cell Fitness

Summary

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Chapters in this video

Transduction-Transplantation Mouse Model of Myeloproliferative Neoplasm

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

Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture

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

Engineering Oncogenic Heterozygous Gain-of-Function Mutations in Human 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

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

Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis

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

Ex Vivo Expansion and Genetic Manipulation of Mouse Hematopoietic Stem Cells in Polyvinyl Alcohol-Based Cultures