Competitive Transplants to Evaluate Hematopoietic Stem Cell Fitness

Summary

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.

Abstract

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.

Introduction

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% purity^2-4, 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.

Access restricted. Please log in or start a trial to view this content.

Protocol

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

1. 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.
2. 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.

2. Preparation of Donor and Competitor Bone Marrow Cells

1. 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.
2. 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).
3. 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.
4. 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.
5. Collect bone marrow cells by flushing the bones with 5 ml sterile PBS.
   1. 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.
6. 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.
7. Pass the cells through a 70 µm nylon strainer to ensure uniform cell suspension and to remove debris and clumps.
8. Remove an aliquot for cell counting using a hemocytometer. Centrifuge the remaining cell suspension at 200 x g for 5 min at 4 °C. Readjust cell density as required in sterile PBS (10⁸ cells/ml). Keep the cells on ice.

3. Establishing Donor Cell HSC Equivalents

1. Transfer the equivalent of 3 x 10⁶ cells (30 µl) 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 µg/ml). Incubate 5 min at RT.
2. Add 90 µl 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.
3. Add 2 ml staining buffer to the cells, vortex and centrifuge at 200 x g for 5 min at 4 °C to remove unbound antibody. Decant supernatant and resuspend pellet by flicking.
4. Add 10 µl 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.
5. 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 reported^10,11.
   Note: The frequency of functional HSCs within that population can be estimated between 1/3 and 1/5^2,12.
6. 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.
   1. 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.
7. 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).
   1. 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.
8. 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 µl 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).

4. Bone Marrow Transplants

1. 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.
2. Prepare a 1 ml tuberculin syringe equipped with a 27 G needle (or smaller). Aspirate approximately 750 µl of the prepared cell suspension (for 3 recipient mice). Make sure there are no air bubbles in the syringe.
3. 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.
4. 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.
5. 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).

5. Analysis of Peripheral Blood

1. Collect a drop of blood (approx. 50-100 µl) into EDTA tubes.
   1. 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.
2. 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.
3. 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 °C.
4. 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 °C.
5. Decant supernatant and resuspend pellet by flicking.
6. 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.
7. 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, GR1^bright granulocytes) in each sample using the analysis template provided in Figure 3 and as published^10,11.

6. Analysis of Bone Marrow Reconstitution

1. 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.
2. 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.

Access restricted. Please log in or start a trial to view this content.

Results

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...

Access restricted. Please log in or start a trial to view this content.

Discussion

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...

Access restricted. Please log in or start a trial to view this content.

Materials

Name	Company	Catalog Number	Comments
Microtainer tubes with K2EDTA	BD Biosciences	365974
20 G needle	BD Syringe		For blood sampling from the mandibular vein
LabQuake Shaker rotisserie	Thermo  Scientific	C415110
Purified anti-mouse CD16/CD32 (clone 2.4G2, Fc Block)	BD Biosciences	553142	2.50 μg/ml
Pe-Cy7-conjugated anti-mouse CD3e (clone 145-2C11)	eBioscience	25-0031	0.25 μg/ml
PE-conjugated anti-mouse CD19 (clone 1D3)	eBioscience	12-0193	0.25 μg/ml
APC-eFluor780 (APC-Cy7 equivalent)-conjugated anti-mouse GR1 (clone RB6-8C5)	eBioscience	47-5931	0.25 μg/ml
FITC-conjugate anti-mouse CD45.1 (clone A20)	eBioscience	11-0453	2.50 μg/ml
eFluor450-conjugated anti-mouse CD45.2 (clone 104)	eBioscience	48-0454	1.00 μg/ml
Biotinylated anti-human/mouse CD45R (B220) (clone RA3-6B2)	eBioscience	13-0452	1.25 μg/ml
Biotinylated anti-mouse CD3e (clone 145-2C11)	eBioscience	13-0031	1.25 μg/ml
Biotinylated anti-mouse CD11b (clone M1/70)	eBioscience	13-0112	1.25 μg/ml
Biotinylated anti-mouse GR1 (clone RB6-8C5)	eBioscience	13-5931	1.25 μg/ml
Biotinylated anti-mouse TER119 (clone TER119)	eBioscience	13-5921	0.625 μg/ml
V500 streptavidin	BD Biosciences	56149	0.5 μg/ml
PE-conjugated anti-mouse CD117 (clone 2B8)	BD Biosciences	553355	0.25 μg/ml
PE-Cy7-conjugated anti-mouse Ly6A/E (Sca1) (clone D7)	BD Biosciences	558162	0.25 μg/ml
PerCP-eFluor710-conjugated anti-mouse CD135 (clone A2F10)	eBioscience	46-1351	0.5 μg/ml
Alexa fluor 647-conjugated anti-mouse CD150 (clone TC15-12F12.2)	Biolegend	115918	0.625 μg/ml BD Biosciences and eBioscience do not carry the same clone
1 ml tuberculin syringe with 27 G needle	BD Syringe	309623
1 ml tuberculin syringe with 25 G needle	BD Syringe	309626
70 μm cell strainer	BD Falcon	352350