Effect of Differently Polarized Macrophages on Proliferation and Differentiation of Ependymal Cells from Adult Spinal Cord

Ependymal cells (EpCs) are a kind of multi-potent stem cells in the central canal of adult spinal cord, which proliferate following spinal cord injury (SCI). Although they can differentiate into functional neurons in vitro, EpC progeny differentiate mainly into astrocytes after SCI, and the mechanism remains unclear. The present study aimed to explore whether neuroinflammation induced by classically activated macrophages (M1) or alternatively activated macrophages (M2) had an effect on EpC proliferation and/or differentiation. EpCs were isolated from intact spinal cord of adult mice and co-cultured with M1 or M2, respectively, in vitro. EpC proliferation was detected using a Cell Counting Kit-8 (CCK8) assay and Ki67 staining. Expression of Sox2 (SRY-box 2) in EpCs derived from different groups was detected by immunofluorescence and western blotting. Also explored was whether the mitogen activated protein kinase (MAPK) signaling pathway was involved in EpC proliferation. Immunofluorescence staining of beta III-tubulin and MAP2 were performed to assess the differentiation direction of EpCs in different culture conditions. Immunofluorescence and western blotting assays showed much more Sox2-positive EpCs in the group EpCs-M1 than the group EpCs-M2 in vitro (p < 0.01). The percentage of EpCs with positive Sox2 staining was decreased after tumor necrosis factor alpha (TNF alpha) antibody was added into the medium of EpCs-M1. Correspondingly, fewer Sox2-positive staining cells were observed in the central canal of TNF alpha-deficient mice with SCI. M1 co-culture promoted EpC proliferation significantly, which could be downregulated by Sox2 gene silencing (p < 0.01). Interestingly, M1 regulated the expression of Sox2 through the MAPK signaling pathway, especially the activation of ERK and p38 kinase. Co-culture in M2 conditioned medium obviously increased the proportion of beta III-tubulin-positive cells (p < 0.01). Small amounts of MAP2-positive neurons could be detected on day 7 in the M2 group and the control group. M1 conditioned medium could promote EpC proliferation in response to SCI through the TNF alpha-MAPK-Sox2 signaling pathway; M2 conditioned medium favors EpCs differentiating toward neurons.