Figure 6.

Immunocytochemical analyses of transplanted NCSC-derived NSCs or predifferentiated NCSC-derived NSCs into demyelinated mouse organotypic cerebellar slice cultures. (A,B) Immunostaining against MBP showing untreated control and 0.5 µM H₂O₂ treated cerebellar mouse slices. Confocal images show a clear decrease in MBP fluorescence at 0.5 µM H₂O₂ compared to control in a representative area. Insets show a central area of A and B respectively under higher magnification. Undifferentiated or predifferentiated NCSC-derived NSCs (treated with PTXF for 3 days, +PTXF) were transplanted into the slices and further cultivated for 2 weeks to determine their ability to differentiate and produce myelin in this demyelination model. (C) Immunostaining against MBP and huNu showing a 0.5 µM H₂O₂ treated cerebellar mouse slice transplanted with predifferentiated NCSC-derived NSCs (+PTXF-treated cells). White arrowheads indicate huNu⁺MBP⁺ double positive cells, representing cells of human origin which express MBP, human oligodendrocytes. Inset shows a clear huNu⁺MBP⁺ cell indicating its human origin. (D) huNu⁺MBP⁺ cell from the inset in C, at higher magnification showing this cell from human origin expressed MBP at the protein level. (E) Immunostaining against MBP and huNu showing a 0.5 µM H₂O₂ treated cerebellar mouse slice transplanted with untreated NCSC-derived NSCs (−PTXF-untreated cells). Magenta arrowheads indicate huNu⁺ cells of human origin. Inset shows a huNu positive cell indicating its human origin. (F) Inset indicated in E, at higher magnification showing a huNu⁺MBP⁻ NCSC-derived NSCs. (G) Quantification showing the percentage of huNu⁺MBP⁺ double positive cells in H₂O₂-treated cerebellar mouse slices transplanted with untreated NCSC-derived NSCs (−PTXF) or with predifferentiated NCSC-derived NSCs (+PTXF), showing the ratio of MBP⁺ cells of human origin present after 2 weeks of transplantation. Nonparametric Kruskal-Wallis test (* p < 0.05) shows a significant increase of MBP⁺ cells of human origin in slices transplanted with predifferentiated NSCs (10.87% ± 0.73%, +PTXF), compared to slices transplanted with untreated NSCs (5.63% ± 0.64%, −PTXF). (H) Quantification of huNu⁺ cells in H₂O₂-treated cerebellar mouse slices transplanted with untreated NCSC-derived NSCs (31.71% ± 16.02%, −PTXF) or with predifferentiated NCSC-derived NSCs (67.87% ± 7.23%, +PTXF), indicating the proportion of cells from human origin present after 2 weeks of transplantation. No significant differences were observed in the ratio of huNu⁺ cells according to nonparametric Kruskal-Wallis test p = 0.1266. NCSCs: neural crest-derived stem cells, NSCs: neural stem cells, MBP: myelin basic protein, H₂O₂: hydrogen peroxide, PTXF: pentoxifylline, huNu: human Nuclei.