Figure 3
Figure 3. Gadd45a−/− HSCs show enhanced reconstitution ability. (A) Single CD34−LSK and CD34+LSK cells from young mice were sorted in a 96-well plate and cultured for 14 days in vitro. The percentage of colonies was calculated by dividing the number of colonies by the original number of single cells seeded (n = 3). (B) Ten thousand CD34−LSK cells from young mice were sorted and expanded in liquid culture. Cell numbers were counted at various time points (n = 3). (C) BM cells from young mice were mixed in a 1:1 ratio with competitors (CD45.1) and injected into lethally irradiated recipients (CD45.1/2). Subsequently, the chimeric BM cells were retransplanted into the secondary recipient mice (CD45.1/2) 4 months later. The chimerism in PB was shown 16 weeks after transplantation (first, n = 10; second, n = 5). (D) One hundred fifty CD34−LSK cells along with 1 × 106 BM cells (competitor) were injected into lethally irradiated recipients. Four months later, 150 donor-derived CD34−LSK cells were purified and retransplanted with fresh competitors into secondary recipients. The chimerism in PB is shown at 16 weeks after transplantation (first: G+/+, n = 5; G−/−, n = 6; second: G+/+, n = 11; G−/−, n = 10). (E) Three-round serial transplantation was conducted using 4000 purified LSK cells along with 1 × 106 fresh competitors each time. Chimerism in PB was shown at the indicated time after transplantation (first, n = 5; second, n = 6; third, n = 3). (F) The absolute number of donor-derived CD34−LSK cells was calculated 3 or 4 months after transplantation. The number of Gadd45a−/− donor-derived CD34−LSK cells was divided by that of Gadd45a+/+ donor-derived CD34−LSK cells (first, n = 5; second, n = 6; third, n = 3). (G) The ratio of PB cells/HSCs was calculated as the PB contribution divided by the absolute number of donor-derived CD34−LSK cells (first, n = 5; second, n = 6; third, n = 3). (H) Cell cycle analysis was performed on donor-derived HSCs 3 weeks after the second round of transplantation by staining with DAPI. The percentage of cycling cells in SG2M phases is shown (G+/+, n = 8; G−/−, n = 10) (*P < .05, **P < .01, ***P < .001). N.S., not significant.

Gadd45a−/−HSCs show enhanced reconstitution ability. (A) Single CD34−LSK and CD34+LSK cells from young mice were sorted in a 96-well plate and cultured for 14 days in vitro. The percentage of colonies was calculated by dividing the number of colonies by the original number of single cells seeded (n = 3). (B) Ten thousand CD34−LSK cells from young mice were sorted and expanded in liquid culture. Cell numbers were counted at various time points (n = 3). (C) BM cells from young mice were mixed in a 1:1 ratio with competitors (CD45.1) and injected into lethally irradiated recipients (CD45.1/2). Subsequently, the chimeric BM cells were retransplanted into the secondary recipient mice (CD45.1/2) 4 months later. The chimerism in PB was shown 16 weeks after transplantation (first, n = 10; second, n = 5). (D) One hundred fifty CD34−LSK cells along with 1 × 106 BM cells (competitor) were injected into lethally irradiated recipients. Four months later, 150 donor-derived CD34−LSK cells were purified and retransplanted with fresh competitors into secondary recipients. The chimerism in PB is shown at 16 weeks after transplantation (first: G+/+, n = 5; G−/−, n = 6; second: G+/+, n = 11; G−/−, n = 10). (E) Three-round serial transplantation was conducted using 4000 purified LSK cells along with 1 × 106 fresh competitors each time. Chimerism in PB was shown at the indicated time after transplantation (first, n = 5; second, n = 6; third, n = 3). (F) The absolute number of donor-derived CD34−LSK cells was calculated 3 or 4 months after transplantation. The number of Gadd45a−/− donor-derived CD34−LSK cells was divided by that of Gadd45a+/+ donor-derived CD34−LSK cells (first, n = 5; second, n = 6; third, n = 3). (G) The ratio of PB cells/HSCs was calculated as the PB contribution divided by the absolute number of donor-derived CD34−LSK cells (first, n = 5; second, n = 6; third, n = 3). (H) Cell cycle analysis was performed on donor-derived HSCs 3 weeks after the second round of transplantation by staining with DAPI. The percentage of cycling cells in SG2M phases is shown (G+/+, n = 8; G−/−, n = 10) (*P < .05, **P < .01, ***P < .001). N.S., not significant.

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