Autophagy prevents irradiation injury and maintains stemness through decreasing ROS generation in mesenchymal stem cells

Abstract

Stem cells were characterized by their stemness: self-renewal and pluripotency. Mesenchymal stem cells (MSCs) are a unique type of adult stem cells that have been proven to be involved in tissue repair, immunoloregulation and tumorigenesis. Irradiation is a well-known factor that leads to functional obstacle in stem cells. However, the mechanism of stemness maintenance in human MSCs exposed to irradiation remains unknown. We demonstrated that irradiation could induce reactive oxygen species (ROS) accumulation that resulted in DNA damage and stemness injury in MSCs. Autophagy induced by starvation or rapamycin can reduce ROS accumulation-associated DNA damage and maintain stemness in MSCs. Further, inhibition of autophagy leads to augment of ROS accumulation and DNA damage, which results in the loss of stemness in MSCs. Our results indicate that autophagy may have an important role in protecting stemness of MSCs from irradiation injury.

Figure 1

The effect of irradiation injury on stemness of MSCs in vitro. MSCs were irradiated at 6 Gy and collected at 6 h after irradiation. (a) MSCs surface markers in different groups were detected by flow cytometry. (b) CFU-F assays. The number of colonies was determined after 14 days of culture. (c) Real-time PCR and western blotting were employed to examine the expression of stemness markers Nanog, Oct4 and Sox2 of MSCs exposed to irradiation compared with control groups. (d) Osteogenic differentiation of MSCs was detected by Alizarin Red stain. (e) The quantitative expression of osteogenic marker genes ALPL, OGN and RUNX2 were measured by real-time PCR at 0, 7 and 14 days. (f) Adipogenic differentiation of MSCs was detected by Oil red-O. (g) The quantitative expression of adipogenesis marker genes LPL, CFD and PPAR-γ were measured by real-time PCR at 0, 7 and 14 days. The data presented are from three replicates as mean±S.E. *P<0.05

Figure 2

MSCs pretreated with starvation or rapamycin maintained stemness after irradiation. (a) CFU-F assays. The number of colonies was determined after 14 days of culture. (b) The expression of stemness markers Nanog, Oct4 and Sox2 of irradiated MSCs pretreated with starvation or rapamycin measured by real-time PCR and western blotting. (c) Osteogenic differentiation of irradiated MSCs pretreated with starvation or rapamycin was detected by Alizarin Red stain. (d) The quantitative expression of osteogenic marker genes ALPL, OGN and RUNX2 were measured by real-time PCR at 0, 7 and 14 days. (e) Adipogenic differentiation of irradiated MSCs pretreated with starvation or rapamycin was detected by Oil red-O. (f) The quantitative expression of adipogenesis marker genes LPL, CFD and PPAR-γ were measured by real-time PCR at 0, 7 and 14 days. The data presented are from three replicates as mean ±S.E. *P<0.05

Figure 3

Examination of autophagy in MSCs pretreated with starvation or rapamycin exposed to irradiation. (a) Total protein extracts were analyzed by western blotting with antibody against LC3. GAPDH expression was used as control. (b) Electron micrographs exhibited numerous vacuoles with cytoplasmic content in irradiated MSCs pretreated with starvation or rapamycin; most of them were clearly identified as being autophagosomes. Scale bar: 2 μm

Figure 4

Autophagy decreases ROS generation and alleviates DNA damage in irradiated MSCs. (a) Irradiated MSCs pretreated with starvation or rapamycin were stained with DCF-DA to determine ROS levels measured by immunofluorescence. Cell nucleus was stained with Hoechst 33258. (b) MSCs were stained with γ-H2A.X antibody to determine DNA damage. Cell nucleus was stained with DAPI. Images were captured with fluorescence microscope, magnification × 100. (c and d) Mitochondrial ROS staining with MitoSOX was measured by FACS and immunofluorescence. (e and f) MMP staining with rhodamine 123 was measured by FACS and immunofluorescence

Figure 5

Inhibition autophagy results in stemness injury and ROS increase in irradiated MSCs. (a) LC3 expression was detected by western blotting assay in irradiated MSCs pretreated with starvation by autophagy inhibitor 3-MA or CQ. (b) Irradiated MSCs pretreated with starvation were transfected with shRNAs to knockdown the autophagy-associated genes ATG7 and Beclin1. The bottom panel is a GAPDH-loading control. (c) CFU-F assays. The number of colonies was determined after 14 days of culture. (d) The expression of stemness markers Nanog, Oct4 and Sox2 of irradiated MSCs pretreated with starvation by inhibiting autophagy were measured by real-time PCR and western blotting. (e) Osteogenic differentiation of irradiated MSCs pretreated with starvation by autophagy inhibitors was stained with Alizarin Red S. (f) The expression of osteogenic markers ALPL, OGN and RUNX2 were measured by real-time PCR. (g) Adipogenic differentiation of irradiated MSCs pretreated with starvation by autophagy inhibitors were stained with Oil red-O. (h) The expression of adipogenic markers LPL, CFD and PPAR-γ were measured by real-time PCR. (i) Irradiated MSCs pretreated with starvation by autophagy inhibitors stained with DCF-DA to detect ROS level were measured by immunofluorescence. Cell nucleus was stained with Hoechst 33258. (j) Irradiated MSCs pretreated with starvation by autophagy inhibitors were stained with γ-H2A.X antibody to determine DNA damage. Cell nucleus was stained with DAPI. The data presented are from three replicates as mean±S.D. *P<0.05