rhTPO Ameliorates Radiation-Induced Long-Term Hematopoietic Stem Cell Injury in Mice

Figures

• 

  Figure 1. Effects of recombinant human thrombopoietin on survival and hematopoietic cell recovery in irradiated mice. (A) Survival of C57BL/6J mice was monitored for 30 days after 9.2 Gy TBI. Kaplan–Meier survival rate curves of 5 groups (n = 8 per group) administered with phosphate-buffered saline (PBS; vehicle) or indicated doses of rhTPO 2 h after IR. (B) Peripheral blood cell counts of white blood cells (WBCs), red blood cells (RBCs), platelets (PLTs), and neutrophils (NEs) of mice (n = 6 per group) administered with PBS (vehicle) or rhTPO (100 μg/kg) 2 h after IR. Data are reported as the mean ± standard deviation. * p < 0.05, ** p < 0.01, *** p < 0.001 versus the normal group. ## p < 0.01, ### p < 0.001 versus irradiation treated with the vehicle group.

• 

  Figure 2. The influence of rhTPO on the frequencies and numbers of long-term hematopoietic cells and BMNCs and on the BM colony-forming ability one and two months after TBI. Sham-irradiated mice were administered phosphate-buffered saline (PBS) and shielded from irradiation. The vehicle and rhTPO groups were administered PBS or 100 μg/kg rhTPO, respectively, at 2 h after 6.5 Gy TBI (n = 6 per group). BM cells were harvested at one and two months post-irradiation. (A) Bone marrow mononuclear cell (BMNC) count. (B) A representative gating strategy of LT-HSC, ST-HSC, and MPP was analyzed by flow cytometry. The frequencies of (C) HSC (Lin−Scal+c-kit+), (E) HPC (Lin−Scal−c-kit+), (G) LT-HSC (CD34−CD135−LSK), (I) ST-HSC (CD34+CD135−LSK), and (K) MPP (CD34+CD135+LSK) in BMNCs (%). The numbers of (D) HSCs, (F) HPC, (H) LT-HSCs, (J) ST-HSCs, and (L) MPPs per mouse. Colony-forming units of the mice (n = 5 per group) treated with PBS or rhTPO at (M) one month and (N) two months after 6.5 Gy TBI. Data are reported as the mean ± standard deviation. * p < 0.05, ** p < 0.01, *** p < 0.001.

• 

  Figure 3. Injection of rhTPO enhances long-term and multilineage engraftment of irradiated HSCs after BM transplantation. The mice were sham-irradiated as a Normal control or subjected to IR and then treated with vehicle or rhTPO as described in the text. (A) Schematic of the experimental procedure. (B) Representative FACS plots and (C,D) percentages of donor (CD45.2) chimeras from Normal, IR + vehicle, and IR + rhTPO group mice in peripheral blood of recipient mice (CD45.1) (n = 6 per group) at 4, 8, 12, and 16 weeks after transplantation. (E,F) Frequencies of Normal, IR + vehicle, and IR + rhTPO group donor (CD45.2)-derived myeloid cells (CD45.2+ CD11b+ and/or Gr-1+) and T (CD45.2+CD3+) and B (CD45.2+ B220+) lymphocytes 12 weeks after transplantation in peripheral blood of recipient mice (CD45.1). Data are reported as the mean ± standard deviation. * p < 0.05, ** p < 0.01, *** p < 0.001.

• 

  Figure 4. Injection of rhTPO attenuates TBI-induced residual BM injury in part via inhibition of HSC proliferation. Mice were sham-irradiated as controls or subjected to IR and then treated with vehicle or rhTPO one and two months after TBI as described above. (A) Representative FACS plots of HSC proliferation. The frequency of cell-cycle distribution (Ki67/7-AAD staining) in LSK cells was analyzed by flow cytometry (B) one month and (C) two months after TBI. (D) Representative FACS plots of HSC apoptosis and (E) the frequency of cell apoptosis (Annexin V/7-AAD staining) in LSK cells were analyzed by flow cytometry. Data are reported as the mean ± standard deviation (n = 5 per group). * p < 0.05, ** p < 0.01, *** p < 0.001.

• 

  Figure 5. Effects of rhTPO on ROS, DNA damage, and cell senescence in HSCs after irradiation. Mice were sham-irradiated as controls or subjected to IR and then treated with vehicle or rhTPO as described above. The mice were euthanized one and two months after exposure to TBI to harvest BMNCs. (A) Representative analysis of ROS levels in HSCs by flow cytometry. (B) Percentages of DCF+ cells in LSKs from non-IR, vehicle, or rhTPO-treated mice (n = 5 per group), presented as the mean ± standard error. (C) Representative analysis of γH2AX expression in HSCs by flow cytometry. (D) Percentages of γH2AX+ cells in LSKs from non-IR, vehicle, or rhTPO-treated mice (n = 5 per group) were presented as the mean ± standard error. HSCs were isolated from BMNCs by cell sorting and analyzed for the expression of p16, p53, and p21 mRNA by real-time PCR. (E–G) Expression levels of p16, p53, and p21 mRNA in HSCs. The levels of p16, p53, and p21mRNA expression are expressed as the mean ± standard error of fold changes compared to the control (n = 8). * p < 0.05, ** p < 0.01, *** p < 0.001.