Generation of Bone Marrow Chimeras

Abstract

Bone marrow chimeras are widely used in immunological studies, to dissect the contributions of hematopoietic and non-hematopoietic cells in immune cell development or functions, to quantify the impact of a given mutation, or in preclinical studies for hematopoietic stem cell transplantation. Here we describe a set of procedures for the generation of bone marrow chimeras.

1. Introduction

Immune and blood cells develop from hematopoietic stem cells (HSC) located in the bone marrow or fetal liver, in adult and fetal mice, respectively. Because HSC, the immature progenitors, and most mature cells derived thereof are more radio-sensitive than most other tissues, including non-hematopoietic thymic stromal components, it is possible to use irradiation doses consistent with animal survival that ablate the immune and hematopoietic system. Developmental “niches” emptied by radiation-induced myeloablation can be filled by HSC transplanted by intravenous injection, generating chimeric animals in which most or all hematopoietic and immune cells are derived from donor-derived HSC, whereas non-hematopoietic cells remain of host origin. The generation of bone marrow chimeras, on which this chapter focuses, has been a powerful tool to study T cell development, and continues to be invaluable to study a variety of immune processes and immune cell functions [1]. In addition to its use in biomedical research, HSC transplantation is used routinely in the clinic, with indications spanning from treatment of genetic blood disorders to cancer therapy; experimental bone marrow chimeras are important preclinical tools for such studies, although this aspect will not be addressed further here.

Despite its broad applicability, bone marrow chimera generation is inefficient for studies of specific cell types that develop exclusively or principally from fetal HSC or to study the development of cells from mutant mice that carry mutations incompatible with postnatal development or survival. Such situations can benefit from the generation of fetal liver chimera that use a similar procedure to transplant fetal HSC [2] and is not described here.

In the specific field of T cell development studies, HSC transplantation is mostly used in two distinct circumstances. First, it can distinguish, among the phenotypic consequences of a given mutation, those that are intrinsic to the hematopoietic component of the thymus (generally the developing thymocytes) and those that primarily affect the host thymic environment, especially the thymic epithelium. Specific applications of this approach study the impact of various signaling components of the thymic epithelium, including MHC haplotypes, antigenic peptides, and stromal cytokines, on thymocyte development. Second, “mixed” bone marrow chimera, in which irradiated hosts are reconstituted with HSC of two distinct genotypes (e.g., mutant and wild-type) are highly sensitive tools to identify and quantify incomplete developmental defects. Varying the respective proportions of each component is useful to identify indirect effects [3].

Although the generation of bone marrow chimeric mice does not involve any particularly challenging step, it requires careful logistics for animal procedures and a specialized infrastructure, especially for irradiation. After transplantation, myeloid and B cell reconstitution precedes that of the T cell compartment, which is not complete before 8 weeks, even though donor-derived thymocytes can generally be detected 3–4 weeks after transplantation [1].

Because usual myeloablative irradiation doses do not completely ablate the host immune system, allelic markers are used to distinguish donor from recipient cells at time of analysis. Commonly used surface molecules for this purpose are CD45 or CD90 (Thy-1). Congenic mice carrying variants of these molecules recognized by allele-specific antibodies (e.g., CD45.1 and CD45.2 or CD90.1 and CD90.2) are available from commercial providers. Despite their names, genetic differences between such “congenic” lines and the reference strain (e.g., C57BL/6) are extensive and often include genes not genetically linked to the locus encoding the allelic markers. Recent studies have shown the potential consequences of such extensive variations [4], and new mouse strains are being developed to address these issues. Transgenic expression of fluorescent protein, e.g., green fluorescent protein (GFP), can be used as an alternative to track donor-derived versus host cells.

2. Materials

2.1. Preparation of Recipient Mice (See Note 1)

1. 8–12-week-old recipient mice (e.g., C57BL/6 congenic CD45.1), preferentially gender matched to donor mice (see Notes 2 and 3).

2. Sulfamethoxazole-trimethoprim suspension (e.g., Sulfatrim pediatric suspension).

2.2. Donor Bone Marrow Preparation

1. 6–12-week-old donor mice (e.g., C57BL/6 CD45.2).

2. Refrigerated cell culture centrifuge.

3. Hemocytometer (for cell counting).

4. Light microscope.

5. Sterilized scissors and forceps for dissection.

6. 27Gx1/2″ needles

7. 60 ×15 mm tissue culture dishes.

8. 70 μm cell strainers.

9. 50 mL conical sterile polypropylene centrifuge tubes.

10. 3 mL syringes, single use only.

11. Phosphate-buffered saline (PBS), pH 7.4.

12. Flush medium: HBSS supplemented with 5% FCS and 1% penicillin/streptomycin/glutamine (working concentrations: 100 units/mL penicillin G sodium, 100 μg/mL streptomycin sulfate and 2 mM L-glutamine). Medium must be made fresh.

13. ACK (ammonium-chloride-potassium) lysing buffer (containing 155 mM ammonium chloride, 10 mM potassium bicarbonate and 0.1 mM EDTA), commercially available.

2.3. T Cell Depletion (Optional; See Note 3)

1. Magnetic beads for depletion of T cells (e.g., Dynabeads Mouse Pan T (Thy1.2), Thermo Fisher Scientific).

2. Magnet.

3. Rocker.

4. 15 mL and 50 mL conical sterile polypropylene centrifuge tubes.

5. Fluorochrome-conjugated antibody specific for TCRβ (clone H57–597).

2.4. Bone Marrow Injection

1. Heat lamp.

2. Alcohol prep pads (saturated with 70% isopropyl alcohol).

3. 27Gx1/2″ needles.

4. 1 mL syringes, single use only.

3. Method

3.1. Prepare Recipient Mice

1. Put recipient mice on Sulfatrim water [containing 400 mg/L sulfamethoxazole and 80 mg/L trimethoprim (final concentrations) in autoclaved water] 7 days prior to irradiation and maintained 2 weeks after transplantation to avoid opportunistic infections. Change Sulfatrim water every 7 days (see Note 4).

2. Irradiate recipient mice with 9.5 Gy 4–8 h prior bone marrow injection (see Note 5).

3.2. Prepare Bone Marrow Cell Suspension from Donor Mice

1. Prepare 12 mL syringes tipped with 27Gx1/2″ needles and filled with 10 mL of flush medium.

2. Euthanize donor mice following local animal regulations. Dissect long bones (femur and tibia) from surrounding tissue, and place stripped bones in 5 mL ice-cold PBS in a 60 ×15 mm tissue culture dish on ice. Keep all cells on ice (see Note 6).

3. Cut bone extremities with scissors, and flush marrow cavity with 2–3 mL flush medium per bone into a 50 mL conical tube, and let cells pass through the 70 μm cells strainer by 12 mL syringe tipped with 27Gx1/2″ needle. If flushed bone marrow columns are too big to pass through the strainer, gently dissociate them by rubbing with pestle of the 3 mL syringe, and wash with flush medium.

4. Spin bone marrow cells in cell culture centrifuge, at 500 g, for 10 min at 4°C.

5. Resuspend bone marrow cells in 2 mL flush medium/mouse (see Note 7).

6. Count the cells (see Note 8).

7. Resuspend the cells at 10 million/mL in flush medium. Set aside one million cells for analysis.

3.3. T Cell Depletion (Optional)

1. Resuspend the Dynabeads in the vial (i.e., vortex for >30 sec, or tilt and rotate for 5 min).

2. Use 30 μL Dynabeads for 10 million cells for depletion. Transfer the desired volume of Dynabeads in a 15 mL or 50 mL conical tubes, depending on the volume of the bone marrow cell suspension to be processed (from Subheading 3.2, step 7), and add the same volume of flush medium (e.g., add 1.5 mL flush medium into 1.5 mL Dynabeads) or 1 mL flush medium if the volume of the Dynabeads is less than 1 mL.

3. Place the tubes in a magnet for 2 min and discard the supernatant.

4. Add bone marrow cells from Subheading 3.2, step 7, to the washed Dynabeads, and incubate for 30 min at 2–8°C with gentle tilting and rotation on a rocker.

5. Place the tube on magnet. After 2 min, gently pipet the supernatant containing the T cell-depleted bone marrow cells, and transfer to a 15 mL or 50 mL tube. Discard bead-containing tube.

6. Add 10 mL PBS into bone marrow cells and convert tubes two to three times.

7. Spin the cells in cell culture centrifuge, at 500 g, for 10 min at 4°C.

8. Discard the supernatants. Resuspend bone marrow cells in 10 mL PBS, and centrifuge cells at 500 g, for 10 min at 4°C.

9. Resuspend bone marrow cells in 1 mL PBS/mouse and count the cells (see Note 8).

10. Resuspend bone marrow cells in PBS at about 50–100 million cells per mL. Set aside one million cells for analysis.

11. Check T cell depletion efficiency. Stain one million cells before and after T cell depletion (Fig. 1) (see Note 9). Use DAPI to distinguish dead cells from live cells.

Fig. 1.

Representative flow cytometry profiles of T cell depletion. Bone marrow cells before and after T cell depletion were stained with a fluorochrome-conjugated antibody specific for TCRβ. Numbers indicate αβ T cell percentages in live bone marrow cells

3.4. Bone Marrow Injection

1. Warm the irradiated recipient mice under a heating lamp for 1–2 min, and wipe tails with alcohol prep pads to make tail veins visible.

2. Intravenously inject 200 μL of bone marrow cells (10–20 million cells) to each recipient mouse 4–8 h after irradiation (see Notes 10 and 11).

3. Analyze T cell development 8 weeks after transplantation (see Notes 12 and 13).

4. Notes

1. Care must be taken to follow local animal use regulations and to obtain necessary approvals for the proposed procedures.

2. By convention, the “.2” allelic designation (e.g., CD45.2) refers to the variant carried by C57BL/6 mice. The “.1” allele origin depends on the specific gene (e.g., SJL strain for CD45.1). Practically, most mouse mutants were generated in C57BL/6 or 129 mice or ES cells and therefore are CD45.2. Given the timeline of these experiments, it is good practice to verify allelic markers on each strain (and if in doubt each donor and recipient mouse) before proceeding.

3. Although matching of donor and recipient genders is highly preferable to avoid immune reaction of female cells against male determinants, it is not always possible or can be impractical. In such cases, efficient T cell depletion generally allows transplantation of female cells into male donors. It is not recommended to transfer male bone marrow cells into female recipient mice.

4. If the recipient mice are imported, rest the mice in the animal facility for 3–4 days before starting the experiments.

5. A total of 9.5 Gy irradiation in one continuous cycle is generally sufficient to eliminate HSC, bone marrow progenitors, and most immune cells; higher doses are more efficient but result in greater toxicity and can lead to animal death despite efficient transplantation. Pilot experiments can be performed with local equipment if needed. Alternatively, to attenuate side effects, myeloablation can be achieved with two cycles of irradiation, each of 5.5–7 Gy at a 3–4-h interval [5]. To avoid overstressing of recipient mice, the intravenous injection should be performed 4–8 h postirradiation. If the recipient mice are irradiated properly, their fur frequently turns gray. Although myeloablative irradiation at the doses indicated in this chapter eliminates most host immune cells in the thymus and secondary lymphoid organs, ablation is often incomplete for mature T cells in lymphoid organs and generally partial for T cells at mucosal sites. Even though remaining host cells can be identified with allele-specific antibodies, there are circumstances where their persistence is detrimental. In such cases, host mice carrying mutations in the Rag1 or Rag2 gene [6, 7] and therefore lacking B and T cells are a useful alternative.

6. Optional: Bones from the front legs could also be taken to increase yield.

7. Do not lyse red blood cells.

8. Take 10 μL cell suspension in the Eppendorf tubes, and add 10 μL ACK lysing buffer at room temperature for 2 min. Add 180 uL trypan blue and count cells.

9. Cell staining is performed as described in Chapter 7. As a general rule, distinct antibodies must be used for T cell depletion and for detection of remaining T cells. If anti-CD90 (Thy1) is used for T cell depletion, anti-TCRb or anti-CD3 can be used to detect T cells remaining in the bone marrow after T cells depletion. Additional staining antibodies can be included to quantify other populations depending on study objectives.

10. Each recipient mouse should receive at least five million bone marrow cells to consistently achieve T cell reconstitution after 6 weeks. However, lower donor cell numbers (down to one million cells) are generally sufficient to ensure hematopoietic reconstitution and mouse survival. Note that these numbers are valid for transplantation of whole or T cell-depleted bone marrow but do not apply to transplantation of purified HSC.

11. Bone marrow cells should be filtered through 70 μm Nylon before loading the syringes for injection. In addition to tail vein injection, bone marrow cells can also be injected retro-orbitally into anesthetized mice.

12. If the transplantation fails, the recipient mice die generally within 14 days after irradiation. Thus, chimeric mice should be regularly examined according to local regulations during the two weeks following transplantation.

13. Four weeks after transplantation, blood from the recipient mice can be sampled to detect donor-derived myeloid and B cells. This would help to check the colonization of the donor cells.