Figure 4.
Ex vivo treatment of WT HSCs or systemic treatment of C57BL/6 mice with rAREG impairs repopulation, leading to HSC exhaustion. (A) Schematic presentation of the experimental design. Ex vivo culture of BM SLAM cells from C57BL/6 mice treated with rAREG or vehicle, followed by BMT or flow cytometry analysis of total HSCs and mbHSCs. (B) Time course of the proportion of total HSCs (left) and mbHSCs (right) during 10 days of culture of cells described in panel A (n = 6-7 assays per group). (C) rAREG treatment impairs WT HSC repopulation (CD45.2+) in that mice that received transplantation (CD45.1+). SLAM cells cultured with or without 500 ng/mL rAREG for 5 days, along with 2 × 105 competing CD45.1+ WT BM cells, were transplanted into lethally irradiated BoyJ recipients. Donor CD45.2+ cells engraftment levels (frequencies at different time points after BMT and absolute numbers at 16-week after BMT) in recipient CD45.1+ mice over time after transplantation were determined by flow cytometry (n = 6). (D) rAREG compromises long-term HSC function. WBMCs from the primary recipients described in panel C were transplanted into sublethally irradiated BoyJ recipients. Donor CD45.2+ cells engraftment levels (frequencies and absolute numbers) in recipient CD45.1+ mice were determined using flow cytometry 16 weeks after BMT (n = 6). (E) rAREG treatment causes HSC exhaustion, as determined using a limited dilution transplant assay. Graded numbers of SLAM cells (CD45.2+) from the ex vivo culture plus 2 × 105 radio-protector BM cells were transplanted into lethally irradiated BoyJ recipients (CD45.1+). Plotted are the percentages of recipients containing less than 1% donor (CD45.2+) blood nucleated cells at 16 weeks after transplantation. The frequency of functional HSCs was calculated according to Poisson statistics (competitive repopulating units: vehicle, 1/108.3 and rAREG, 1/246.8). (F) Systemic rAREG treatment alters hematopoiesis in WT mice. C57BL/6 mice were treated with increasing doses (0, 5, 10, and 15 μg) of rAREG every other day for 10 days, followed by analysis of BM cell counts, total HSCs, and mbHSCs 4 weeks later by flow cytometry (n = 6). Frequencies (left) and absolute numbers (right) are shown. Results are presented as mean ± standard deviation of 3 independent experiments. Two-tailed unpaired t test or Wilcoxon rank sum test was performed to compare the vehicle vs rAREG at different time points (0, 1, 2, 5, or 10 days after treatment) in panel B; vehicle vs rAREG at different time points (4, 8, 12, or 16 weeks after BMT) in panel C; vehicle vs rAREG in panels D and E. For panel F, one-way ANOVA, followed by t tests was performed to compare different treatments (0, 5, 10, or 15 μg of rAREG). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001.

Ex vivo treatment of WT HSCs or systemic treatment of C57BL/6 mice with rAREG impairs repopulation, leading to HSC exhaustion. (A) Schematic presentation of the experimental design. Ex vivo culture of BM SLAM cells from C57BL/6 mice treated with rAREG or vehicle, followed by BMT or flow cytometry analysis of total HSCs and mbHSCs. (B) Time course of the proportion of total HSCs (left) and mbHSCs (right) during 10 days of culture of cells described in panel A (n = 6-7 assays per group). (C) rAREG treatment impairs WT HSC repopulation (CD45.2+) in that mice that received transplantation (CD45.1+). SLAM cells cultured with or without 500 ng/mL rAREG for 5 days, along with 2 × 105 competing CD45.1+ WT BM cells, were transplanted into lethally irradiated BoyJ recipients. Donor CD45.2+ cells engraftment levels (frequencies at different time points after BMT and absolute numbers at 16-week after BMT) in recipient CD45.1+ mice over time after transplantation were determined by flow cytometry (n = 6). (D) rAREG compromises long-term HSC function. WBMCs from the primary recipients described in panel C were transplanted into sublethally irradiated BoyJ recipients. Donor CD45.2+ cells engraftment levels (frequencies and absolute numbers) in recipient CD45.1+ mice were determined using flow cytometry 16 weeks after BMT (n = 6). (E) rAREG treatment causes HSC exhaustion, as determined using a limited dilution transplant assay. Graded numbers of SLAM cells (CD45.2+) from the ex vivo culture plus 2 × 105 radio-protector BM cells were transplanted into lethally irradiated BoyJ recipients (CD45.1+). Plotted are the percentages of recipients containing less than 1% donor (CD45.2+) blood nucleated cells at 16 weeks after transplantation. The frequency of functional HSCs was calculated according to Poisson statistics (competitive repopulating units: vehicle, 1/108.3 and rAREG, 1/246.8). (F) Systemic rAREG treatment alters hematopoiesis in WT mice. C57BL/6 mice were treated with increasing doses (0, 5, 10, and 15 μg) of rAREG every other day for 10 days, followed by analysis of BM cell counts, total HSCs, and mbHSCs 4 weeks later by flow cytometry (n = 6). Frequencies (left) and absolute numbers (right) are shown. Results are presented as mean ± standard deviation of 3 independent experiments. Two-tailed unpaired t test or Wilcoxon rank sum test was performed to compare the vehicle vs rAREG at different time points (0, 1, 2, 5, or 10 days after treatment) in panel B; vehicle vs rAREG at different time points (4, 8, 12, or 16 weeks after BMT) in panel C; vehicle vs rAREG in panels D and E. For panel F, one-way ANOVA, followed by t tests was performed to compare different treatments (0, 5, 10, or 15 μg of rAREG). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001.

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