Balb/c (H‐2Kd, CD45.2), C57BL/6 (H‐2Kb, CD45.2; referred to as B6), and C57BL/6‐Ly5.2 (H‐2Kb, CD45.1; referred to as B6Ly5.2) mice were purchased from Charles River Laboratories (Wilmington, MA, USA). CBA/J (H‐2Kk, CD45.2) mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA). A minimum of five mice were assessed per group in the individual arms of the experiments to allow for statistical analysis. Fewer then five mice were used to compare chimerism levels following engraftment enhancement in those mice with initial chimerism levels following IUHCT of less than 1% because of the limitation in engraftment enhancement in this group. Animals were housed in the Animal Laboratory Facility of the Abramson Research Center at The Children’s Hospital of Philadelphia. The experimental protocols were approved by the Institutional Animal Care and Use Committee at The Children’s Hospital of Philadelphia and followed guidelines set forth by the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Donor bone marrow harvest and T‐Cell depletion
Adult donor bone marrow cells were harvested from 6‐ to 8‐weekold adult female mice after sacrifice by cervical dislocation by flushing the tibias and femurs with Ca++/Mg++ free phosphate‐buffered saline (PBS; Gibco, Grand Island, NY, USA) using a 26‐gauge needle. The cells were processed into a single‐cell suspension by gentle passage through the 26‐gauge needle and subsequently filtered through a 70‐µm nylon mesh filter and layered over Ficoll (Histopaque 1077, Sigma, St Louis, MO, USA). After centrifugation at 600g for 15 minutes at room temperature, the light‐density mononuclear cell (LDMC) layer was removed and washed with sterile PBS. CD3+ T‐cell depletion (TCD) was performed by labeling the bone marrow LDMC with fluorescein isothiocyanate (FITC)‐conjugated, anti‐CD3 monoclonal antibody (mAb; Pharmingen, San Diego, CA, USA) followed by incubation with anti‐FITC microbeads (Miltenyi Biotec, Auburn, CA, USA) and subsequent passage through a VarioMACs magnetic cell sorter (Miltenyi Biotec, Auburn, CA, USA). A CD3+ cell fraction of less than 0.5% of the donor bone marrow cells after depletion was confirmed by flow cytometry on a FACScan (Becton Dickinson, Mountain View, CA). On average, CD3 depletion resulted in a loss of approximately 30% of the cells from our donor population. Cells were counted prior to transplantation and more than 95% viability was confirmed by trypan blue exclusion.
In utero BMT
Time‐dated E14 Balb/c mice were injected as previously described [5
]. Briefly, under methoxyflurane (Medical Developments Australia, Australia) anesthesia and sterile technique, a midline laparotomy was performed and the uterine horns exposed. All fetuses were injected intraperitoneally using a 100‐µm glass beveled pipette with 5 × 106
TCD B6 bone marrow cells in 5 µL of PBS. The fetus and uterine horns were returned to the abdomen and the abdominal cavity was closed after administering a 1 mL intraperitoneal bolus of sterile PBS, which replaced fluid lost during the procedure. Control animals received 5 µL of PBS instead of donor cells. A total of 25 fetuses were injected with PBS and 466 fetuses were injected with donor cells. Pups were carried to term and weaned at 3 weeks of age.
At 4 or 5 weeks of life and either 1 or 7 days after treatment with BU, chimeric and control mice were injected with 30 × 106 TCD B6Ly5.2 bone marrow cells in 200 µL of PBS via the lateral tail vein. A total of seven, five, and five mice were transplanted with donor cells 1 day after treatment with 5 mg/kg, 15 mg/kg, and 35 mg/kg BU, respectively. Similarly, a total of seven, six, and five mice were transplanted with donor cells 7 days after treatment with 5 mg/kg, 15 mg/kg, and 35 mg/kg BU, respectively. These numbers were chosen based on the number of chimeric mice born after IUHCT and to allow for adequate statistical comparisons between groups.
BU (Sigma) was reconstituted as follows: 500 mg or 150 mg of BU was dissolved in 2.0 mL dimethylsulfoxide (DMSO; Sigma); 100 µL of this solution was then mixed with 900 µL DMSO and 4 mL warm (37°C) PBS (pH 7.4) to create a 5 mg/mL or 1.5 mg/mL solution. Mice were weighed and one of three doses of BU (35 mg/kg, 15 mg/kg, or 5 mg/kg) was injected intraperitoneally. A total of 19, 16, and 23 mice were treated with 5 mg/kg, 15 mg/kg, and 35 mg/kg BU, respectively. These doses were chosen based on studies demonstrating that 15 mg/kg of BU is approximately one‐fifth the LD50/30
in adult mice as well as the finding that 35 mg/kg of BU was the minimal effective dose required to rescue the twitcher mouse in a congenic hematopoietic cell transplantation system [15
Flow cytometric analysis
The FITC‐conjugated mAbs included antibodies against H‐2Kb, CD45, and Mouse IgG2a. Phycoerythrin (PE)‐conjugated mAbs included antibodies against H‐2Kd, CD45.1, and Mouse IgG2a. For lineage analysis biotinylated antibodies against CD3, B220, CD11b, Gr1, Ter119, rat IgG2a, rat IgG2b, and hamster IgG1 were developed with streptavidin‐PE. Nonspecific Fcγ receptor binding was blocked by the mAb against mouse Fcγ receptor 2.4G2. Conjugated antibodies with irrelevant specificities listed above served as negative controls. Propidium iodide staining was used to exclude dead cells in dual‐color flow cytometry. A minimum of 10,000 events were assessed for each individual flow cytometric analysis. All antibodies were purchased from Pharmingen and flow cytometry was performed on a FACScan (Becton Dickinson).
Blood sampling and donor chimerism and multilineage engraftment assessment
Chimerism was assessed at 4 or 5 weeks of life in recipients of IUHCT prior to treatment with BU and postnatal BMT. Chimerism was analyzed by dual‐color flow cytometry for H‐2Kb (donor cells) and H‐2Kd (recipient cells) every week for the first 4 weeks following postnatal BMT in chimeric and control mice. Levels were then assessed every other week for the next 8 weeks and then every month. The contribution to engraftment of postnatal and prenatal donor cells was distinguished by dual‐color flow cytometry for CD45.1 (present only on the postnatal donor cells) at the same time points. All mice were analyzed at each time point (1 day post‐BU: 5, 15, 35 mg/kg chimeric and 35 mg/kg naïve groups, n = 7, 5, 5, and 7, respectively; 7 days post‐BU: 5, 15, 35 mg/kg chimeric and 35 mg/kg naïve groups, n = 7, 6, 5, and 5, respectively) and followed for at least 6 months after postnatal BMT. Donor cell lineage analysis was performed at 2 and 6 months after postnatal BMT by dual‐color flow cytometry for H‐2Kb (donor cells) and CD3, B220, and CD11b lineage markers. For each analysis, approximately 200 µL of peripheral blood was collected in heparinized capillary tubes via retro‐orbital vein puncture and diluted to 10 mL with heparinized PBS. The samples were layered over a Ficoll gradient. The LDMCs were collected after centrifugation at 600g for 15 minutes and subsequently washed in PBS. A minimum of 10,000 events was analyzed for each determination.
Mixed lymphocyte reaction
Donor‐specific tolerance was assessed by mixed lymphocyte reaction (MLR) in chimeric mice after IUHCT and postnatal BU‐conditioned BMT. Splenocyte responder cells were harvested by hemisplenectomy and subjected to mixed lymphocyte culture by standard methods. Briefly, 2 × 105 splenic responder cells were cultured in triplicate at 37°C in 5% CO2 for 4 days with 4 × 105 mitomycin C‐treated stimulator cells (host, donor, and third‐party) in RPMI 1640 medium (Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal calf serum (Life Technologies), 50 mM 2‐mercaptoethanol (Sigma), and antibiotics (penicillin, 100 U/mL; streptomycin, 100 mg/mL; Life Technologies). Eighteen hours prior to harvesting, cells were pulsed with [³H] thymidine. Radioactivity was counted using a liquid scintillation counter at the time of harvest. Stimulation indices (SIs) were calculated by dividing mean counts per minute (c.p.m.) by mean background c.p.m.
Assessment of hematologic parameters after BU treatment
Naïve (non‐in utero transplanted) 4‐week‐old Balb/c mice were treated with 0 mg/kg, 5 mg/kg, 15 mg/kg, or 35 mg/kg of BU injected intraperitoneally (n = 5, 5, 5, and 6, respectively, chosen to allow for statistical comparison between groups). Peripheral blood (50 µL) was collected in ethylene‐diamine‐tetraacetic acid tubes (Kabe Labortechnik GMBH)by retro‐orbital vein puncture at 3 days before BU treatment and 1, 4, 7, 11, 14, 21, and 28 days after treatment. Hematologic parameters (white blood cell count, hemoglobin, and platelets) were assessed with a Hema Vet CBC machine (Mascot, CDC Technologies, Oxford, CT, USA). The hematologic parameters of mice receiving 35 mg/kg were assessed every month for 6 months following BU injection due to prolonged thrombocytopenia.
Assessment of the effect of BU treatment on stem/progenitor cells
In a separate experiment, 4‐week‐old naïve Balb/c mice were treated with 0 mg/kg, 5 mg/kg, 15 mg/kg, or 35 mg/kg of BU injected intraperitoneally. Bone marrow cells were harvested and the LDMCs isolated as described above at 1 and 7 days after BU treatment (n = 6 for each harvest time point and dose of BU administered, n = 13 for naïve non‐BU‐treated control mice). The effect of BU treatment on stem/progenitor cells was assessed by colony‐forming unit assays with LDMCs from treated and nontreated mice according to standard protocol. Briefly, 9 × 10³ LDMCs were resuspended in 300 µL Dulbecco’s modified Eagle’s medium supplemented with 30 U/mL human epogen and mixed with 2.7 mL methylcellulose. The cells were vortexed vigorously and subsequently plated in duplicate at 3 × 10³ cells/well and cultured for 14 days at 37°C in 5% CO2. On days 10 and 14 of culture, the frequency of colony‐forming unit cells per 105 nucleated cells and the number of cells in the three biggest colonies were counted.
Assessment of graft‐versus‐host disease
Mice were weighed prior to receipt of postnatal BMT and weekly following transplantation. Mice were also monitored twice a week for clinical signs of graft‐versus‐host disease (GVHD) including runting, fur loss, and serositis and for side effects of BU treatment such as persistent alopecia.
Data are represented as the mean of the respective group ± 1 standard deviation. A two‐tailed Student’s t test for comparison of means with unequal variance was used for statistical analysis. A p value less than 0.05 was considered statistically significant.