American Society of Hematology

Figure 6

$Figure 6. Accumulation of DNA damage impairs HSCs maintenance in old Gadd45a−/− HSCs. (A) Gadd45a expression (relative to GAPDH or β-actin) in young (2 months old) and old LT-HSCs (24 months old) was measured by qRT-PCR (SYBR green assay) (n = 4). (B) The absolute number of LT-HSCs, ST-HSCs, MPPs, and HPCs in old mice was measured by FACS (n = 8). (C) Twenty thousand BM cells from old mice were cultured with cytokines in methylcellulose semisolid medium for 9 days. The number of colonies were counted (n = 4). The colony forming unit-granulocyte/macrophage colonies include the colony forming unit-granulocyte and colony forming unit-macrophage colonies. (D) Single CD34−LSK and CD34+LSK cells were sorted in a 96-well plate and cultured for 14 days in vitro. Colonies were counted in each group (G+/+, n = 6; G−/−, n = 8). (E) Ten thousand CD34−LSK cells from old mice were cultured in SFEM with stem cell factor (50 ng/mL) and Thrombopoietin (50 ng/mL). Cell numbers were counted at particular time points (n = 4). (F) Competitive transplantation of aging LT-HSCs was conducted by injecting 500 CD34−LSK cells along with 1 × 106 competitor cells into lethally irradiated recipients. Four months later, the chimeric BM cells were retransplanted into the secondary recipient mice. The chimerism in PB is shown at 4 or 16 weeks after transplantation (first, n = 7-8; second, n = 7). (G) The absolute number of donor-derived CD34−LSK cells was calculated 4 months after the first transplantation using FACS (n = 6). (H-I) LSK cells were isolated from old mice and subjected to the comet assay and γ-H2AX staining. DAPI was used to stain the nucleus. (J) Cell cycle analysis was performed on CD34−CD48−CD150+LSK, CD34−LSK, and LSK fractions with Ki67/DAPI staining. The percentages of cells in the G0, G1, and S/G2/M phases were analyzed using flow cytometry (G+/+, n = 4; G−/−, n = 5). (K) Single CD34−LSK cells were sorted in a 96-well plate and were exposed to 1Gy IR; colonies were counted after 14-day culture. The percentage of colonies (determined by dividing the number of colonies by the original number of seeded single cells) in each group is shown (G+/+, n = 3; G−/−, n = 4) (*P < .05, **P < .01, ***P < .001). N.S., not significant.$

Accumulation of DNA damage impairs HSCs maintenance in old Gadd45a^−/−HSCs. (A) Gadd45a expression (relative to GAPDH or β-actin) in young (2 months old) and old LT-HSCs (24 months old) was measured by qRT-PCR (SYBR green assay) (n = 4). (B) The absolute number of LT-HSCs, ST-HSCs, MPPs, and HPCs in old mice was measured by FACS (n = 8). (C) Twenty thousand BM cells from old mice were cultured with cytokines in methylcellulose semisolid medium for 9 days. The number of colonies were counted (n = 4). The colony forming unit-granulocyte/macrophage colonies include the colony forming unit-granulocyte and colony forming unit-macrophage colonies. (D) Single CD34⁻LSK and CD34⁺LSK cells were sorted in a 96-well plate and cultured for 14 days in vitro. Colonies were counted in each group (G^+/+, n = 6; G^−/−, n = 8). (E) Ten thousand CD34⁻LSK cells from old mice were cultured in SFEM with stem cell factor (50 ng/mL) and Thrombopoietin (50 ng/mL). Cell numbers were counted at particular time points (n = 4). (F) Competitive transplantation of aging LT-HSCs was conducted by injecting 500 CD34⁻LSK cells along with 1 × 10⁶ competitor cells into lethally irradiated recipients. Four months later, the chimeric BM cells were retransplanted into the secondary recipient mice. The chimerism in PB is shown at 4 or 16 weeks after transplantation (first, n = 7-8; second, n = 7). (G) The absolute number of donor-derived CD34⁻LSK cells was calculated 4 months after the first transplantation using FACS (n = 6). (H-I) LSK cells were isolated from old mice and subjected to the comet assay and γ-H2AX staining. DAPI was used to stain the nucleus. (J) Cell cycle analysis was performed on CD34⁻CD48⁻CD150⁺LSK, CD34⁻LSK, and LSK fractions with Ki67/DAPI staining. The percentages of cells in the G0, G1, and S/G2/M phases were analyzed using flow cytometry (G^+/+, n = 4; G^−/−, n = 5). (K) Single CD34⁻LSK cells were sorted in a 96-well plate and were exposed to 1Gy IR; colonies were counted after 14-day culture. The percentage of colonies (determined by dividing the number of colonies by the original number of seeded single cells) in each group is shown (G^+/+, n = 3; G^−/−, n = 4) (*P < .05, **P < .01, ***P < .001). N.S., not significant.

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