Figure 2
The effect of astrocyte conditioned media on motor neuron-progenitors and motor neurons derived from differentiation of mouse embryonic stem cells. (a-i) Cultures grown in wt non-Tg astrocyte CM. (a) Immunolabelling for Olig2 (red), Hb9:eGFP (green) and β3-tubulin (light blue) showing Hb9:eGFP+β3-tubulin+ cells (white arrows) and Olig2+ cells (red arrows). Scale bar 50 μm. (b) High-magnification of white boxed area in (a), showing co-expression of Hb9:eGFP and β3-tubulin (white arrow). Scale bar 33 μm. (c, d) High-magnification channel views of red boxed area in (a). Olig2+ cells (red arrows) do not co-express β3-tubulin. Scale bar 20 μm. (e) Immunolabelling for Olig2 (red), Hb9:eGFP (green), Islet1 (white) and nuclei counterstained with DAPI (blue), showing Hb9:eGFP+Islet1+ cells (white arrows), Olig2+ cells (red arrows) and Olig2+Hb9:eGFP+Islet1+ cell (green arrow). Scale bar 50 μm. (f-i) High-magnification channel views of white boxed area in (e). MNs show co-expression of Islet1 and Hb9:eGFP (white arrows, f,g). Most Olig2+ progenitors do not co-express Islet1 or Hb9:eGFP (red arrows, i). Very rarely, an Olig2+Hb9:GFP+Islet1+ cell is detected (green arrow, f-i). Scale bar 25 μm. (j) A higher proportion of MNs (percentage of total cells) differentiated in wt non-Tg versus control or Tg SOD1G93A media (P<0.001) and, in Tg SOD1G93A CM versus control (P<0.01). (k) A higher proportion of MN-progenitors differentiated in wt non-Tg versus control or Tg SOD1G93A media (P<0.001). Comparable proportions of MN-progenitors differentiated in control and Tg SOD1G93A media. (l) Comparable proportions of MNs differentiated in wt non-Tg and Tg WTSOD1 CM and these were significantly higher versus control (P<0.001). (m) Slightly more MN-progenitors differentiated in wt non-Tg versus Tg WTSOD1 CM (P<0.05). Comparable proportions of MN-progenitors differentiated in control and Tg WTSOD1 CM. Values represent means ± s.e.m from n=4 or n=3 independent experiments (indicated in graphs), performed in triplicate, analysed by one-way ANOVA followed by Tukey’s post-test. *P<0.05, **P<0.01, ***P<0.001.