Figure 3.
(a) Phase contrast microscopy and immunofluorescence staining of glial-restricted progenitors (grown in oligomedium and specific growth factors for 2 weeks) and premyelinating oligodendrocytes (5 days after withdrawal of 4 growth factors: Shh, NT-3, bFGF, and PDGF-AA). Three experimental conditions were evaluated (control, collagen 50 μg/ml, and collagen 100 μg/ml). For neural progenitor-derived oligodendrocytes culture system protocol details see Monaco et al., J. Vis. Exp 2012.11
- Glial-restricted precursors: in comparison to control, collagen I induced apoptosis (fragmented or pyknotic nuclei), bizarre cellular morphology (enlarged soma, loss of bipolar morphology, thick processes) and increased number of GFAP+/nestin- cells (b,c,d,e).
- Premyelinating oligodendrocytes: in comparison to control, collagen I induced lower cell counts (c), reduced ramification of cellular processes (b) and increased number of GFAP+/O4- cells (d).
(b) Single-cell fractal dimension (D) analysis. Representative cells at different differentiation stages, selected for the fractal dimension analysis, are shown with corresponding D values. D values quantify the degree of cellular morphological complexity (low in bipolar cells, high in ramified cells).
See the text for statistical analysis; Tukey’s post-hoc comparison *p<0.05; **p<0.01