|
|
Blood, Vol. 105, Issue 4, 1456-1466, February 15, 2005
HOXB6 overexpression in murine bone marrow immortalizes a myelomonocytic precursor in vitro and causes hematopoietic stem cell expansion and acute myeloid leukemia in vivo
Blood Fischbach et al.
105: 1456
Supplemental materials for: Fischbach et al, Vol 105, Issue 4, 1456-1466
Materials and methods for supplemental figures:
Bone marrow transduction and generation of transplant chimeras. High-titer retrovirus was produced by transfecting 293-T cells with the appropriate retroviral vector and the pCL Ecotropic packaging plasmid using a calcium phosphate transfection protocol (Invitrogen, Carlsbad, CA).34 Retroviral supernatants were harvested at 48 and 72 hours after transfection. Donor bone marrow (Ly 5.1+) was harvested from mice 5 days after intraperitoneal injection with 150 mg/kg 5-fluorouracil (Pharmacia & Upjohn, Kalamazoo, MI), dispersed into a single-cell suspension, and cultured in 24-well plates in prestimulatory media consisting of StemSpan SFEM supplemented with 15% heat-inactivated fetal calf serum, 100 ng/mL recombinant murine (rm) stem cell factor, 50 ng/mL rm IL-6, and 10 ng/mL rm IL-3 (StemCell Technologies, Vancouver, BC, Canada) for 48 hours. Marrow cells were infected with high-titer retrovirus on 2 consecutive days by spinoculation in the presence of 4 µg/mL polybrene (Sigma). At 24 hours after the second spinoculation, bone marrow cells were analyzed by FACS analysis for transduction efficiency (EGFP+ or YFP+). Transplant recipients received 5 × 105 bone marrow cells by tail vein injection after lethal irradiation (950 cGY) from a 137Cs source (J. L. Shepherd and Associates, San Fernando, CA). CFC dose per animal was retrospectively calculated by performing CFC assays on freshly transduced marrow. Transduced CFC dose was also calculated by multiplying total CFC injected per animal by the transduction efficiency determined by FACS analysis. In some experiments, CFC transduction was confirmed by direct examination of colonies with fluorescence microscopy.
CFC and serial replating assays. CFC assays were performed using freshly transduced marrow cells or single-cell suspensions of marrow and spleen from primary transplant chimeras sacrificed 1 to 2 months after transplantation by plating in Methocult M3434 media; 1.0 mg/mL methylcellulose in IMDM supplemented with 15% FBS, 1% BSA, recombinant human (rh) insulin, human transferrin, 10-4 M 2-mercaptoethanol, 2mM L-glutamine, rm SCF, rm IL-3, rh IL-6, and rh erythropoietin (StemCell Technologies). Cultures were scored for colony formation on day 8. For serial replating assays, contents of an entire dish were aspirated and replated in M3434 media. Selected colonies from primary cultures were plucked for Wright-Giemsa–stained cytospins. Following 4 rounds of serial replating, cells from individual dishes were placed in liquid culture in growth media (GM); DME-H21 supplemented with 10% heat inactivated FCS, 10 ng/mL rm SCF, 5 ng/mL IL-6, and 1% B16-GM-CSF conditioned media (cytokines from StemCell Technologies).
Competitive repopulation assays. Competitive repopulation unit (CRU) assays were performed as previously described by Szilvassy et al.36 Briefly, serial bone marrow dilutions from primary transplant chimeras 1 to 2 months after transplantation were injected into lethally irradiated C57BL/6 hosts. 1 X 105 Ly 5.2+ helper bone marrow cells from C57/BL6 donors were mixed with the experimental bone marrow to ensure short-term bone marrow reconstitution. Recipient mice were bled 12 weeks after transplantation. A mouse was judged to have received at least 1 CRU if greater than 2% peripheral blood leukocytes were Ly5.1+ or EGFP+. Myeloid and lymphoid engraftment were verified with Mac-1 and B-220 staining, respectively. Poisson statistics were applied to calculate the CRU frequencies (L-Calc 1.0 software, StemCell Technologies).
Files in this Data Supplement:
- Figure S1. HOXB6 overexpression produces an increase in marrow MAC-1+ and c-kit+ cells and a decrease in B220+ cells (JPG, 727 KB)
-
(A,B) Representative FACS plots of marrow (A) and spleen (B) from MIG control and HOXB6 chimeras 2 months after transplant. (C) Relative percentage (mean ± SD) of TER-119+ cells in the ungated and EGFP+ (ie, retrovirally transduced) populations of bone marrow (n = 4 and n = 3 mice for HOXB6 and MIG, respectively; *P < .05). (D) Relative percentage (mean ± SD) of c-kit+ cells in the ungated and EGFP+ populations of marrow (n = 5 and n = 2 mice for HOXB6 and MIG, respectively; *P < .05). See Figure 5B,C for quantification of MAC-1+ and B220+ populations.
- Figure S2. HOXB6 overexpression perturbs T-lymphopoiesis (JPG, 494 KB)
-
Representative FACS plots from peripheral blood (A) and thymus (B) from MIG and HOXB6 transplant chimeras 2 months after transplantation. (C) HOXB6 overexpression produces a decrease in single-positive CD4+/CD8- and CD4-/CD8+ thymocytes, accompanied by an increase in CD4+/CD8+ thymocytes. Results are mean percentages ± SD (P = .06, .09, and .007 for CD4+/CD8+, CD4+/CD8-, and CD4-/CD8+ populations, respectively; n = 2 mice each for MIG and HOXB6).
- Figure S3. HOXB6 transplant chimeras have a myelodysplastic phenotype before developing AML (JPG, 537 KB)
-
Wright-Giemsa–stained peripheral blood, bone marrow, and spleen specimens from a representative HOXB6 transplant chimera that developed AML. (A) Peripheral blood at 2 months after transplant is notable for relatively normal-appearing granulocytic and lymphoid forms (left panel) as well as dysplastic myeloid forms (right panel). When the mouse with AML was killed, peripheral blood (B), bone marrow (C), and spleen (D) were notable for abundant blast forms. HOXB6-induced AMLs have varying degrees of monocytic differentiation (solid arrows) and can be classified as acute monocytic leukemia or acute myelomonocytic leukemia by the Bethesda classification of hematopoietic neoplasms.
- Figure S4. AML in HOXB6/MEIS1-transfected mice express both HOXB6 and MEIS1 (JPG, 178 KB)
-
(A) Representative FACS analysis of a HOXB6/MEIS mouse with AML demonstrating expression of both HOXB6 (GFP) and MEIS1 (YFP). The top left panel shows HL-60 cells used as negative control. The top right panel shows the HOXB6-immortalized bone marrow cell line used as a positive control for GFP. The bottom left panel shows 3T3 cells transduced with MEIS1/YFP as a positive control for MEIS1 (YFP). Data were acquired on a FACSCalibur machine with a 510/20–nm band-pass filter to detect GFP and a 550/30-nm band-pass filter to detect YFP. (B) FACS analysis of a representative HOXB6/MEIS mouse with AML. The immunophenotype is indistinguishable from AML induced by HOXB6 alone. (C) Southern blot analysis demonstrating proviral integration of MEIS1 in HOXB6/MEIS1 mice with AML. Genomic DNA isolated from marrow suspensions of HOXB6/MEIS1-induced mice with AML was digested with EcoR1 to release the full-length MEIS1 cDNA (1.1 kb) and probed with labeled EcoRI fragment from the MEIS1 retroviral plasmid DNA. Genomic DNA isolated from wild-type bone marrow (B) and spleen (S), as well as 3T3 cells transduced with the control MIY retroviral vector, was used as a negative control while genomic DNA from 3T3 cells transduced with the MEIS1 retroviral vector was used as a positive control. (D) Photomicrographs of peripheral blood (left panel), bone marrow (middle panel), and spleen (right panel) from a representative mouse with HOXB6.
|
|
