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Blood, Vol. 108, Issue 4, 1208-1215, August 15, 2006
Unique effects of Stat3 on the early phase of hematopoietic stem cell regeneration
Blood Chung et al.
108: 1208
Supplemental materials for: Chung et al
Files in this Data Supplement:
- Table S1. The number of bone marrow cells in recipients transplanted with STAT3-C—transduced cells (PDF, 42.6 KB) -
At the indicated times after transplantation of MIG— or STAT3-C—transduced bone marrow cells, the total cellularity of the marrow of the recipients was determined by multiplying the number of nucleated cells measured in both femurs and both tibiae by 4 based on the assumption that these 4 bones contain 25% of the total bone marrow40. Values shown are the mean ± SEM (n = the number of mice analyzed in each group).
- Table S2. Normal marrow-seeding efficiency of STAT3-C—transduced CRUs (PDF, 42 KB) -
Aliquots of freshly transduced adult mouse bone marrow cells were assayed for their content of GFP+ CRUs to calculate the numbers of CRUs injected into the group of “experimental” mice transplanted with the same cells but in larger numbers (values shown as “Input”). Twenty hours later, bone marrow cells were harvested from both femurs and tibiae of these experimental mice, pooled and aliquots assayed for their content of GFP+ CRUs in secondary mice. The total number of GFP+ CRUs recovered in the bone marrow of these mice 20 hours post-transplant (values shown as “Output”) was then calculated assuming the contents of 2 tibiae and 2 femurs represent 25% of the total bone marrow population in the mouse40. Finally the percentage of injected GFP+ CRUs that had seeded to the bone marrow within 20 hours (last column) was calculated by expressing the output value as a percent of the input value.
- Table S3. Data used to calculate CRU expansion during serial reconstitution (Figure 3) (PDF, 50.6 KB) -
Limiting dilution assays were performed after transduction of bone marrow (BM) cells with MIG or STAT3-C to determine the frequency of GFP+ CRUs present in these cell suspensions (Figure 3A). These CRU frequencies were then calculated from the percentages of negative mice (mice not positively repopulated with GFP+ cells) as described in the Methods. In parallel, aliquots of the same transduced cells (containing the GFP+ CRU numbers indicated, as later determined from the results of the assays shown in the top table) were transplanted into primary (1°) recipients. Forty weeks after transplanting these primary mice, they were sacrificed and the total number of GFP+ CRUs regenerated in their bone marrow was determined by performing CRU assays on these cells in secondary (2°) mice. The fold expansion of the number of GFP+ CRUs injected into the primary mice after 40 weeks was calculated by dividing the number determined to be present after 40 weeks by the number injected. Similarly, 48 weeks after transplanting bone marrow cells from the primary mice (marked with thick box) into the secondary mice, the secondary mice were sacrificed and their bone marrow cells were then transplanted into tertiary (3°) mice. From the percentages of these tertiary mice that were repopulated with GFP+ cells, the frequencies and hence total numbers of GFP+ CRUs regenerated in the secondary mice, and their fold expansion over the number of GFP+ CRUs initially injected into the secondary mice, was also determined. The overall cumulative expansion of GFP+ CRUs was calculated by multiplying the expansion values from both the primary and secondary mice.
- Table S4. The effect of transplant cell dose on cell output per STAT3-C—transduced CRU (PDF, 42.6 KB) -
Eight weeks after transplanting mice with STAT3-C—transduced cells, the bone marrow (BM) cells of the recipients were harvested and assessed for their content of GFP+ CFCs and total GFP+ cells (as well as GFP+ CRUs, see Figure 5A.). The total marrow content of these cell types and the output of these cells per STAT3-C CRU initially injected were then calculated assuming 2 femurs and 2 tibiae represent 25% of the total marrow of the mouse40.
*Assuming 1 STAT3-C—transduced CRU per 8 × 104 cells transplanted.
- Figure S1. Efficiency transduction 5-FU bone marrow cells (PDF, 70 KB) -
5-FU—pretreated bone marrow cells were transduced with MIG, dnSTAT3 or STAT3-C and transplanted into irradiated mice and analyzed for %GFP+, donor-derived (Ly5.1+) cells in the blood 16 weeks later. Additional aliquots of the transduced cells were cultured for another 48 hours and then analyzed for GFP expression. Shown are the representative of the cells. (A) Representative flow cytometry plots of cells from the experiments shown in Figure 1C. (B) Representative flow cytometry plots of cells from the experiments shown in Figure 2A. (C) Gene transfer data for the 48 hour post-transduction measurements from 7 experiments ( mean ± SD). (D) Gene transfer data for CFCs (% GFP+ colonies determined by visual examination under fluorescent microscope; mean ± SD from 3 experiments). (E) Lineage distribution of donor-derived (Ly5.1+) nontransduced (GFP—) WBCs to match the GFP+ data shown in Figure 1E. Values shown are the mean ± SEM.
- Figure S2. Persistent expression of the intact STAT3-C transgene in repopulated mice (PDF, 54.2 KB) -
(A) 2 × 105 5-FU—pretreated bone marrow cells were transduced with STAT3-C and transplanted into irradiated mice. The % of GFP+ WBCs in the peripheral blood of individual recipient mice is shown. (B) Seven weeks after transplantation, expression of the transgene was analyzed by RT-PCR using specific primers (indicated in the upper pannel). Results are from a representative experiment.
- Figure S3. Effect of STAT3-C on the in vitro colony-forming ability of transduced bone marrow cells (PDF, 9.96 KB) -
Bone marrow cells from 5-FU—pretreated mice were transduced with the MIG or the STAT3-C vector and immediately plated in semi-solid methylcellulose cultures to assay the number of CFCs present. Shown are the number of GFP+ colonies per 104 GFP+ cells assayed (mean ± SEM from 6 experiments).
- Figure S4. Multi-lineage reconstitution of mice by STAT3-C—transduced bone marrow cells (PDF, 13.5 KB) -
Single cell suspensions were prepared from the bone marrow (BM), thymus, spleen and peripheral blood (PB) of mice that had been transplanted with MIG— or STAT3-C—transduced bone marrow cells 36 weeks previously. The mean ± SEM relative proportions of GFP+ myeloid (GM), B-lymphoid (B) and T cells in the GFP+ WBCs are shown (4 mice/group).
- Figure S5. STAT3-C—mediated enhancing effect does not involve a paracrine mechanism (PDF, 60.6 KB) -
(A) Schematic illustration of the experimental design. Ly5.1+ bone marrow cells from day 4 5-FU pretreated Ly5.1+ mice were transduced with the indicated retroviral vector, mixed with nontransduced Ly5.2+ cells and then transplanted into Ly5.1+ recipients. (B) The % Ly5.2+ WBCs (produced from the co-transplanted nontransduced cells) was then determined (6 mice in each group).
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