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. 2011 Nov 10;21(11):1838–1851. doi: 10.1089/scd.2011.0137

FIG. 3.

FIG. 3.

Cell identity: marker expression profile of pretreated proliferating NPCs. NPCs were dissociated after the 3 weeks exposure to MSC-CM or control conditions, and cells were seeded overnight under serum-free conditions. The marker expression profile was analyzed by immunocytochemistry and quantitative RT-PCR. Illustrative fluorescence images of the immunostainings are shown for: Nestin (red) and DAPI (blue) (A); A2B5 (red) and DAPI (blue) (C); PDGFRα (green) and DAPI (blue) (E); NG2 (green) and DAPI (G); O4 (red) and DAPI (blue) (I); RIP (red) and DAPI (blue) (K); CNPase (red) and DAPI (blue) (M); GalC (green) and DAPI (blue) (O); MBP (red) and DAPI (blue) (Q); GFAP (green) and DAPI (blue) (S); DCX (green) and DAPI (blue) (U). Scale bar=10 μm. Quantitative analysis show the percentage of positive cells for each marker (B, D, F, H, J, L, N, P, R, T, and V). Note that the number of RIP positive cells was significantly increased after MSC-CM treatments compared with control condition, while there was no significant change for the other tested markers after MSC-CM stimulation. (W) Quantitative RT-PCR for glial fate determinants and stem/progenitor cell markers. Delta delta Ct method was used for analysis considering glyceraldehyde 3-phosphate dehydrogenase as normalizer gene and control condition as calibrator. Relative expression levels of Nestin, NG2, PDGFRα, Olig1, Olig2, CNPase, and MBP after treatment with 100% MSC-CM compared with control condition (dashed line) are shown. After 3 weeks of pretreatment with MSC-CM there was a significant increase in the expression of CNPase and MBP, while there was a slight decrease in the expression levels of NG2 and PDGFRα. All experiments were performed at least in triplicate in 3 independent experiments. Data are shown as mean±SD. For statistical analysis 1-way ANOVA-Tukey post hoc was performed. Asterisks above individual columns indicate significant difference compared with control. Asterisks above a line spanning 2 columns indicate significant difference between MSC-CM treatments. *P<0.05; ***P<0.001. RT-PCR, real-time-polymerase chain reaction; DAPI, 4′, 6′-diamidino-2-phenylindole dihydrochloride; GFAP, glial fibrillary acidic protein; DCX, doublecortin.