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. 2022 Aug 17;31(1):230–248. doi: 10.1016/j.ymthe.2022.08.011

Figure 4.

Figure 4

MSCs accelerate DDR foci resolution, preventing the cell-cycle delay and reducing the level of apoptosis in GE-HSPCs

(A) Confocal images of 53BP1-positive foci (red), γH2AX-positive foci (purple), and DAPI-positive nuclei (blue) in HSPCs gene edited according to standard procedures (GE CD34+) or in the presence of MSCs (GE CD34+ + MSCs) at 24 and 72 h upon gene editing. Asterisks indicate positive foci. Scale bar represents 20 μm. (B and C) Quantification of 53BP1-γH2AX co-localizing foci (B) and all γH2AX (C) foci from (A); 24 h (53BP1): n = 4; 72 h (53BP1): n = 4; 24 h (γH2AX): n = 4; 72 h (γH2AX): n = 3. Each error bar shows means ± SEM. p values were determined by Mann-Whitney test (ns p > 0,05; ∗p < 0.05). (D) Quantification of 53BP1- and γH2AX-positive foci from (A)–(C). Positive cells were divided in subgroups according to the number of foci identified per cell (from 1 to more than 5). 24 h (53BP1): n = 4; 72 h (53BP1): n = 4; 24 h (γH2AX): n = 4; 72 h (γH2AX): n = 3. Each error bar shows means ± SEM. p values were determined by Mann-Whitney test for each subgroup (only significant comparisons were reported; ∗p ≤ 0.05). (E) qPCR expression analysis of p21 in untreated (UT) and gene-edited (GE) HSPCs co-cultured with MSCs or in plastic dishes for 24 and 72 h. Each error bar shows means ± SEM (n ≥ 3). Gene expression was calculated as 2−ΔCT relative to GUSB gene. (F) Percentage of GE-HSPCs (distinguished between primitive cells and progenitors) in the indicated cell-cycle phases measured at 24 (n = 7) and 72 h (n = 6) after editing. Significance was calculated for each time point comparing GE CD34+ + MSCs with GE CD34+. Each error bar shows means ± SEM. p values were determined by Mann-Whitney test for each cell-cycle phase (only significant comparisons were reported; ∗p ≤ 0.05). (G) Percentage of apoptotic cells within different GE-HSPC subsets (primitive cells and progenitors) at the indicated time points after gene editing. Live: Annexin V−, 7AAD−; early apoptotic: Annexin V+, 7AAD−; late apoptotic: Annexin V+, 7AAD+; necrotic: Annexin V−, 7AAD+. n = 3 for each time point analyzed.