Bone Marrow and Fetal Liver Radiation Chimeras

Abstract

Radiation chimeras are prepared by subjecting recipient mice to sublethal or lethal dose of irradiation and injecting them with hematopoietic stem cells (HSC) from untreated donor mice. HSC can be obtained from bone marrow or fetal liver. This technique is a powerful tool when coupled with gene targeting strategies to investigate function of HSCs, thymocyte development, and T cell function. This protocol describes how to produce bone marrow or fetal liver chimeras.

1. Introduction

The presence of hematopoietic stem cells (HSCs) in bone marrow was inferred from experiments that arose from studies on the effects of irradiation on mammals early in the nuclear age [1]. HSCs are defined by their ability to support long-term reconstitution of all mature blood cell types. Bone marrow transplantation (BMT) has become a valuable research tool to study immune cell development and function especially in mouse gene knockout models. In cases where gene targeting results in late embryonic death, fetal liver cells can be used as a source of HSC for transplantation in wild-type adult mice to allow study of the effects of mutations specific to immune cell development and function. Based on the work done in animal models, HSC transplantation has evolved into an effective treatment for hematopoietic diseases and as an increasingly important component of anticancer therapy.

HSC transplantation can be used to differentiate between cell intrinsic and environmental effects on immune cell development and function. For example, a targeted mutation that causes a severe defect in T cell development can be traced to lymphoid cells if the phenotype is observed in wild-type mice transplanted with bone marrow from the mutated mouse. Conversely, a defect in thymic epithelial cell function would be evidenced by defective T cell development in spite of transfer of wild-type HSC into the mutant mouse. Competitive transplantation includes a mixture of HSC from different mouse strains to assess the ability of mutant cells to function in competition. This technique allows characterization of reduced function due to mutations that may not be evident in the absence of competition. In HCS transplantation experiments it is often critical to be able to differentiate host from donor cells. This can be done using antibodies that recognize different congenic alleles (such as CD45.1 and CD45.2), or the use of transgenic markers such as EGFP. These markers can be used to assess reconstitution using blood samples or harvested tissues at experimental endpoints. The timing for reconstitution varies but one should expect to be able to observe donor cell development in the thymus within 2–3 weeks and stable thymic reconstitution within 8–9 weeks.

2. Materials

Use sterile technique throughout and work in a laminar flow hood when possible or practical. Dissection of leg bones can be done outside of hood.

Water bath, ice, centrifuge, microscope, trypan blue, and hemocytometer or automated cell counter are used in the procedures listed below.

2.1. Host Mice (See Note 1)

1. C57BL/6 CD45.2 congenic mice 6–12 weeks old, weight at least 20 g (see Notes 2 and 3).

2. Drinking water acidified with hydrochloric acid to pH 2.5–3.0.

3. Antibiotics: Amoxicillin or other.

2.2. Bone Marrow Preparation

1. C57BL/6 CD45.1 congenic mice 6–12 weeks old.

2. 10 ml syringes tipped with 25 g needles.

3. Flushing media: RPMI 1640, 2 % fetal calf serum, 1 % penicillin/streptomycin, 1 % HEPES, and 1 % l -glutamine.

4. ACK lysing buffer.

2.3. Optional T Cell Depletion

1. Wash media: RPMI 1640, 1 % penicillin/streptomycin.

2. Low-Tox rabbit or guinea pig complement.

3. Antibodies (clone name): Anti-CD90.2 (HO-13–4), anti-CD8 (83–12-5), anti-CD4 (C3PO) (see Note 4).

4. DNase—2 mg/ml stock.

2.4. Fetal Liver Preparation

1. Flushing media (see above).

2. Sterilized dissecting tools (curved forceps, tweezers, small and large scissors), dissecting microscope.

3. Timed pregnant mice days E12.5–18.5 (see Note 5).

2.5. Analysis

1. Flow cytometer and software to analyze data.

2. 5 ml polystyrene tubes required for flow cytometer.

3. FACS buffer: 1× PBS, 0.5–1.0 % BSA, 0.1 % (W/V) sodium azide (see Note 6).

4. Antibodies (clone name): Anti-CD45.1-FITC (A20), anti-CD45.2-PE (104), anti-B220-APC (RA3–6B2), anti-CD90-APC (53–2.1), anti-CD16 (2.4G2) antibodies.

5. Commercial 1-step fix/lyse solution.

3. Methods

3.1. Prepare Host Mice

1. One week before irradiation, the recipient mice are given acidified drinking water (to prevent growth of Pseudomonas species) supplemented with antibiotics such as amoxicillin (0.5 mg/ml).

3.2. Prepare Bone Marrow Suspension from Donor Mice

1. Prepare 10 ml syringes with 10 ml of flushing media.

2. Sacrifice donor mice following institutional guidelines and soak fur with 70 % ethanol. Snip skin at the base of tail and peel over legs and up the body to contain hair inside the inverted skin. Dissect long bones (femur, tibia), and strip off tissue using sterile forceps, scissors, and gauze (see Note 7). Place stripped bones in Petri dishes containing ice-cold flushing media and keep on ice.

3. Snip both ends of bones with sharp scissor or bone clippers to allow easy entry of needle into one end. Use needle to ensure that bone is open on both ends if needed. The red marrow should be easily visible in the bone. Insert needle into one end of the bone and using some force, flush marrow cavity with 2–3 ml of flushing media into 50 ml conical tubes on ice. In most cases, the marrow comes out as an intact tube of red tissue.

4. Agitate gently, and then spin marrow cells at 1000 × g for 5 min at 4 °C.

5. Carefully remove supernatant from the loosely pelleted cells and resuspend in complete flushing medium using about 1 ml for each mouse used for marrow collection.

6. Process cells by pipetting up and down several times and then run through a cell strainer. Use a sterile forceps or 5 ml syringe plunger to mash marrow clumps and rinse the strainer with 3 ml complete flushing media. Place marrow cells on ice.

7. Remove 5 μl of the marrow cell suspension for counting and place in an Eppendorf tube. Add 95 μl of ACK lysis buffer to the 5 μl cell sample in the Eppendorf tube, mix well, and count the cells.

3.3. Optional T Cell Depletion of Marrow

T cell depletion of marrow eliminates resident T cells in the graft which allows analysis of T cell function of cells derived from engrafted HSC and prevents graft-versus-host disease (GVHD) if MHC mismatches are present (see Note 2).

1. Place counted bone marrow cells in 50 ml tube and fill with washing media, spin down cells, and remove media (see Note 8).

2. Prepare bone marrow cell suspension at 2.0 × 10⁷ cells/ml in RPMI/PSG without FCS.

3. Add antibodies at predetermined concentrations and mix gently (see Note 4).

4. Place on ice for 15 min.

5. Wash bone marrow cells with RPMI/PSG without FCS. Do not resuspend pellet.

6. Add diluted guinea pig complement to bring cell number to 2.0 × 10⁷ /ml (see Note 9). Add DNase to 40 μg/ml of cell suspension. Mix well.

7. Place in 37 °C water bath × 40 min.

8. Wash with RPMI/PSG and resuspend cells in 50 % volume used in step 1. Count cells and resuspend at 5.0 × 10⁷ /ml in sterile PBS (see Note 10).

3.4. Prepare Liver Cell Suspension

1. Harvest E14–E15 embryos and place in flushing media in a 15 cm tissue culture dish on ice (see Note 11).

2. Once cooled, rinse embryo in flushing media, place on a piece of gauze, and euthanize the following institutional guidelines. Remove a piece of tissue (tail, foot) and freeze for genotyping if needed (see Note 12).

3. Dissect liver and place in sterile Eppendorf tube with 1 ml flushing media. Be sure that liver is numbered the same as genotyping biopsy.

4. Prepare cell suspension by mashing and pipetting the liver up and down first with a P1000 tip and then with a P200 tip.

5. Allow cells to settle for 5 min on ice. Transfer cell suspension to a new sterile Eppendorf tube taking care to avoid debris that has settled to the bottom of the tube (see Note 13).

6. Strain cells with a 40 μM filter. Count viable cells and adjust to 0.25–1 × 10⁷ cells/ml in sterile PBS for injection.

3.5. Inject HSC into Mice

1. At least 4–6 h before injection, irradiate recipient mice using 9.5 Gy (see Note 14). It is advisable to wait ~2 h after the last irradiation dose before injecting cells.

2. Inject 1.0 × 10⁷ depleted BM cells in 200 μl sterile PBS in the tail vein of the irradiated recipient mouse. Alternatively inject 0.5–2 × 10⁶ fetal liver cells in 200 μl sterile PBS (see Note 15).

3. Keep mice on acidified water containing antibiotics and in autoclaved cages for 2 weeks after injection of stem cell inoculum.

4. Wait for 1 month before testing for T cell chimerism. Peripheral T cell reconstitution is complete after approximately 8 weeks.

3.6. Analysis of Chimera Reconstitution

Immune reconstitution can be monitored using blood samples. If bone marrow was T cell-depleted, the presence of donor B cells can be used to quickly determine if transplant was successful.

1. Obtain 100 μl heparinized blood samples from transplanted mice and divide into two FACS tubes. For controls, obtain 150 μl blood samples from a CD45.2 mouse and divide into three FACS tubes (see Note 16). Obtain 50 μl blood from a CD45.1 mouse and place into one FACS tube. Add 0.5 μl anti-CD16/CD32 antibody to block antibody binding by the Fc receptor and incubate on ice for 10 min.

2. Add antibody mixes (CD45.1, CD45.2, and B220 or CD45.1, CD45.2, and CD90) to blood from transplanted mice at predetermined concentrations (0.25–1.0 μl/sample should work). Add single antibodies to the four control tubes. Incubate on ice for 15 min.

3. Add 1 ml 1-step fix/lyse solution to each FACS tube, mix gently, and incubate for 15 min minimum (see Note 17).

4. Spin down cells at 500 × g for 5 min at room temperature. Remove supernatant and resuspend cells in 1 ml FACS buffer, spin down again, remove supernatant, and add 500 μl FACS buffer.

5. Use single-color controls to set up compensation on cytometer and run samples. If successful, a population of donor CD45.1positive cells should be readily apparent. Gate on the donor cells to determine levels of B cell (B220) and T cell (CD90) chimerism. Depending on results, proceed to experimental endpoints (see Note 18).

4. Notes

1. Ensure that institutional guidelines are followed for all animal housing, handling, procedures, and euthanasia.

2. It is important to match MHC antigens of donor and host mice to avoid complications due to GVHD or host-versus-graft disease (HVGD) unless BMT is being used to study these syndromes in animal models [2].

3. Here we use allelic differences in CD45 to identify host and donor cells. Another option is the use of a transgenic marker, such as EGFP expression by donor mouse cells.

4. We use supernatants from hybridoma cultures as an antibody source. These are IgM antibodies to enhance complement lysis. Other antibodies and isotypes (IgG) can be used but all antibodies should be titrated beforehand to optimize efficiency. Titrations ranging from 0.25 to 5.0 μg/ml should be sufficient.

5. Timed pregnant mice are commercially available for commonly used strains. Alternatively, any strain of mice can be used to produce timed pregnant mice following procedures outlined by Mader et al. [3].

6. Fetal bovine serum can also be used at 3–10 % instead of BSA. Sodium azide is used as a preservative; its addition to the buffer is optional.

7. Bone marrow from humerus, hips, and vertebral bodies can also be used to increase the number of cells. Vertebral bodies and hips can be crushed and bone fragments filtered out.

8. Ensure that cells are thoroughly washed with at least 10 volumes to eliminate serum proteins.

9. Nonspecific cell loss is variable, but can be substantial using complement treatment. The following suggestions can help to minimize nonspecific cell lysis. Ensure that cells, centrifuge, and buffers are kept cool or on ice at all times. Use care to gently resuspend cell pellets, especially after washes. Antibodies should be titrated. New lots of complement should be prepared according to the specification sheet and dilutions tested for efficient lysis and minimal nonspecific killing. Typically a 1:5–1:20 dilution should be sufficient. Low-toxicity rabbit complement may work better if IgG antibodies are used, or may be used instead or in combination with guinea pig complement to improve depletion or reduce unwanted toxicity. There are several reliable commercially available alternative methods to effectively deplete defined cell populations. For example commercially available kits can be used to deplete various populations using antibodies coupled with a magnetic bead capture strategy. Although convenient (especially for small-scale depletions), these kits and the needed equipment can be costly. For reference, bone marrow of normal mice should contain about 5 % T cells and about 12–20 million marrow cells/mouse after T cell depletion.

10. Use flow cytometry (Subheading 3.6) to assess the efficiency of T cell depletion using anti-CD90 or similar antibodies. If needed, another cycle of complement depletion can be done. If using two cycles, results may be improved if different antibodies are used in each cycle.

11. Hematopoiesis peaks in liver at days E14–E15 making this an ideal time to harvest HSC. However, HSC can be harvested from fetal liver from days E12.5–E18.5 if needed [4].

12. Use care to avoid cross contamination between embryos and avoid contamination with mother’s blood.

13. Fetal liver cells can be frozen at this point in FCS with 10 % DMSO and stored in liquid nitrogen. To prepare cells for injection, thaw cells, add 50 ml cold PBS, incubate for 10 min, spin down cells, and repeat cold PBS wash to eliminate DMSO. Filter cells, count, and adjust as described in procedure.

14. C57BL/6 mice are relatively resistant to radiation effects and can be given a single 9.5–10.0 Gy dose. Irradiation can be divided into two 4.75 Gy treatments 2–3 h apart to reduce potential radiation injury. Immunodeficient mice such as Rag2 knockout can be injected without irradiation or should only be given 6.0 Gy. BALB/c mice are radiation sensitive and should be given two 4.50 Gy doses.

15. Fewer cells can be injected, down to about 1 × 10⁶ per mouse, but may delay reconstitution. Bone marrow cells can also be injected retro-orbitally into anesthetized mice. Newborn mice can be injected intraperitoneally.

16. This procedure works equally well with heparin- or EDTA- treated blood.

17. Cells can be stored for up to 48 h in the 1-step fix/lyse solution following the manufacturer’s recommendations.

18. The choices for antibodies to stain cells are great and are dictated by experimental design. In general, consider using fluorochromes (such as Pacific-Blue) that have minimal overlap in other channels for gating on donor cells. Preliminary experiments should be done to optimize fluorochrome choices and antibody concentrations. Be sure to include appropriate controls to allow accurate gate setting for data analysis.