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Blood, Vol. 113, Issue 20, 4856-4865, May 14, 2009
Conditional deletion of STAT5 in adult mouse hematopoietic stem cells causes loss of quiescence and permits efficient nonablative stem cell replacement
Blood Wang et al.
113: 4856
Supplemental materials for: Wang et al
Files in this Data Supplement:
- Table S1. Hematology of STAT5 conditional mice two weeks after the last dose of pl:pC treatment (PDF, 15.4 KB)
- Figure S1. Deletion of one allele of STAT5 impairs multi-lineage competitive repopulating ability (JPG, 69.5 KB)
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Total BM cells from STAT5ab+∕ΔN, STAT5ab+∕null or wild type mice (CD45.2) were mixed at a 1:1 donor equivalent ratio with BoyJ BM cells (CD45.1). The BM mixes were injected into lethally irradiated BoyJ recipients (1100 rads). (A) Percentage of donor-derived CD45.2 chimerism in the peripheral blood of each recipient mouse 16 weeks following transplantation. (B) Percentage of donor-derived chimerism in Gr-1, B220, Ter119, and CD4 lineages. Results shown are mean ± SD from 5 recipients of each group. For panels A and B, p<0.01 for STAT5ab+∕ΔN or STAT5ab+∕Null relative to wild-type. (C) The absolute number of both CD34neg and Flk2neg KLS cells representing LT-HSC are shown for wild-type and STAT5ab+∕null mice (N=3). (D) The percentage of Annexin V positive/DAPI negative CD34 negative KLS cells is shown for both wild-type and STAT5ab+∕null mice (N=6). (E) Cell cycle analysis was performed on LT-HSC (CD34 negative KLS) isolated from wild-type and STAT5ab+∕null mice and shown is the percentage of cells within G0, G1, or S/G2/M phases of the cell cycle (N=3).
- Figure S2. Generation of hematopoietic chimeras following transplantation of mutant STAT5 fetal liver cells (JPG, 45 KB)
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(A) Overall donor CD45.2 positive cells are shown for wild-type control and STAT5abΔN∕ΔN or STAT5abnull∕null fetal liver transplantation from 2 separate injection dates. Recipient mice were analyzed 12–16 weeks following lethal irradiation. (B) The same mice as in panel A were analyzed for multilineage repopulation in the hematopoietic cell lineages expressing Gr-1, B220, and CD4. The numbers of mice injected for each group were as follows: WT (N=9), STAT5abΔN∕ΔN (N=7), STAT5abnull∕null (N=5).
- Figure S3. Conditional deletion of STAT5 affects peripheral blood as well as BM cells 16 weeks after pI:pC treatment (JPG, 49.6 KB)
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Mx1-Cre/+STAT5ab+∕null (N=4) and Mx1-Cre/+STAT5abflox∕null (N=3) mice were treated with three doses of pI:pC at day 1, 3, and 5, and monitored up to 16 weeks after the initial treatment. (A) The absolute number of Gr-1, Ter119, and NK1.1 cells in the peripheral blood from each treated mouse. (B) The absolute number of Gr-1 and B220 cells in the BM of each treated mouse.
- Figure S4. Percentage of STAT5 deletion in Gr-1+ cells pre- and post-BM transplantation and percentage of donor chimerism up to 40 weeks following transplantation (JPG, 69.3 KB)
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(A) STAT5 conditional mice were treated with three doses of pI:pC every other day. Host Gr-1 positive cells were sorted prior to BM transplantation and 40 weeks after the transplantation. STAT5 deletion was analyzed by real time PCR. (B–F) Up to 40 weeks after the transplantation, donor chimerism was measured by flow cytometry. Mice 1–5 are the same as the specific mice denoted as 1–5 in Fig. 4B.
- Figure S5. Multi-lineage analysis of STAT5 KO donor engrafted mice (JPG, 78.6 KB)
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(A) Representative dot plot of secondary transplanted recipients (STAT5 KO donor engrafted mice listed in Fig. 4C as the donor) 16 weeks after BM transplantation and mean ± SD of donor chimerism in each lineage is shown. Left, lower level engrafted mice and Right, higher level engrafted mice. (B) Representative dot plots of multi-lineage analysis for the engrafted mice as listed in Fig. 4E and mean ± SD of donor chimerism in each lineage is shown.
- Figure S6. The order of deletion/donor cell injection was critical for HSC engraftment (JPG, 97.2 KB)
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(A) Experimental outline, representative flow cytometry analysis and donor chimerism pre- and post-transplantation with 5 × 106 GFP transgenic BM cells. (B) A representative dot plot of multi-lineage analysis of the donor engrafted mice and mean ± SD of donor chimerism in each lineage is shown.
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