Maintenance of hematopoietic cells by SCD MSCs. (A-B) Lineage-negative hematopoietic cells from B6 mice were cultured alone (control) or together with MSCs from SS or SA mice. After 6 days of coculture, we determined the number of Lin^–Sca1⁺c-Kit⁺ population (N = 9-10 cocultures from 5 mice) (A) and number of Lin^–Sca1⁺c-Kit⁺CD48^-CD150⁺ HSC population (N = 9-10 cocultures from 5 mice) (B) present within the culture system by flow cytometry. MSCs were treated with NAC or vehicle (control) for 8 hours followed by coculture with lineage-negative hematopoietic cells for 3 days. (C) The fold change difference in Lin^–Sca1⁺c-Kit⁺ population after NAC treatment (N = 5 cocultures from 5 mice) is presented relative to that of the MSC-only group. (D) The fold-change difference in Lin^–Sca1⁺c-Kit⁺CD48^–CD150⁺ HSC population after NAC treatment (N= cocultures from 5 mice) is presented relative to that of the MSC-only group. HSC maintenance gene expression among MSCs was assessed by RT-PCR after 8 hours of NAC treatment of Scf (E), Opn (F), and Cxcl12 (G) (N = 5 cocultures from 5 mice). HSCs cocultured with SS or SA MSCs were transplanted into CD45.1 mice and analyzed after 16 weeks. (H) Bone marrow HSC donor engraftment frequency (N = 3-4 mice). (I) Mean fluorescence intensity (MFI) of ROS content in engrafted HSCs (N = 3-4 mice). (J) MFI of γH2AX in HSCs (N = 3-4 mice). Data are presented as mean ± standard error of the mean. Data were analyzed by using 1-way analysis of variance with Tukey multiple comparison test (A-B), or a 2-tailed, unpaired Student t test (C-J). *P ≤ .05, **P ≤ .01, ***P ≤ .001, ****P ≤ .0001. N.S., not significant.