Culture and Differentiation of Oligodendrocyte Progenitor Cells (OPCs).

<p>A) OPCs cultured for 3 days after extraction on poly-L-lysine coated culture dishes. Numerous astrocytes are present. B) OPCs after passaging and expanding in the presence of FGF2 and PDGF-AA, to induce proliferation and maintenance of the OPCs. C) OPCs cultured in differentiation inducing medium for 7 days. Mature oligodendrocytes appear in the culture. D) OPCs cultured for 7 days in the differentiating medium and FGF8. All images taken at 100X magnification. E–G) Immunocytochemistry of OPCs in undifferentiating medium (E), differentiation inducing medium (F), and in the presence of FGF8 (G), staining for Olig2 (in green) and DAPI for nuclear staining (in blue). H) Histogram presenting the percentage of Olig2+ cells in the various cultures observed in E–G. Percentages are with respect to the total number of cells, calculated by DAPI staining. I–K) Immunocytochemistry of NG2+/Brdu+ cell in undifferentiating medium (I), differentiation inducing medium (J), and in the presence of FGF8 (K). L) Histogram presenting the percentage of NG2+/BrdU+ cells in the different cultures, with respect to the total number of cells. *p<0.05 (paired t-test), n = 4. Scale Bar indicates 150 µm for A–D and 100 µm for E–G and I–K.</p

Matrigel cultures of OPCs.

<p>A) Oligodendrocyte progenitor cell matrigel culture where a PBS-soaked bead was placed 0.5 mm away from the cell cluster. Image taken at 40X magnification. B–C) OPCs where a cluster of cells migrating towards a FGF8-soaked bead can be observed. D) Histogram depicting the oligodendrocyte progenitor cell proximal/distal rate in control cultures and where FGF8 was included. *p<0.01 (paired t-test, n = 4). (E–G) Matrigel cultures where on one side a FGF8-soaked bead was placed and on the other side a FGF8 inhibitor. (F, G) Close-up images of the areas near the inhibitor-soaked bead (F) or near the FGF8-soaked bead (G). H) OPCs where a FGF8-soaked bead was placed and on the other side a PBS-soaked bead, as control of the inhibitor. Scale bar indicates 500 µm for (A–C, D, H) and 200 µm for (F, G). All images were taken 48 hours after culture.</p

Oligodendrocyte progenitor cell differentiation with and without FGF8.

<p>A) Immunocytochemistry of NG2+/Olig2+, PDGFR-A+/Olig2+ and O4+ cells in the undifferentiated, differentiating and FGF8 culture media. In all images, blue staining corresponds to the nuclei (DAPI). Scale bar indicates 50 µm. B) Histogram depicting the percentage of cells in the different cell cultures staining for markers analyzed in A. Percentages are with respect to the total number of cells. C) Histogram depicting the results from the quantitative PCR analysis of the different cell cultures. *p<0.05, **p<0.01, ***p<0.001 (paired t-test in B, one way-ANOVA in C), n = 4.</p

In Vivo Transplantation of FGF8-soaked beads in Chronically Demyelinated Mice.

<p>A) FGF8-soaked heparin beads (seen in green) surrounded by NG2-positive cells (in red. B) Control experiment where PBS-soaked beads were transplanted. C) Histogram depicting the percentage of NG2 immunoreactivity in the area injected with the heparin beads. The red bar indicates the percentage in the case FGF8-soaked beads was used, while the blue bar indicates the sham control. D) NG2 immunoreactivity in the corpus callsoum and cortex in the area near the PBS-soaked beads. E) NG2 immunoreactivity in the corpus callsoum and cortex in the area near the FGF8-soaked beads. F, G) MBP immunoreactivity in the area near the PBS or FGF8-soaked beads, respectively. H) Histogram depicting the percentage of MBP immunoreactivity in the area injected with the PBS- (blue bar) or FGF8- (red bar) soaked heparin beads. Scale bar measures 50 µm. *p<0.01 (paired t-test), n = 4.</p