Supplementary Figure 8: Functional validation of selected candidate factors

a-d: OPC proliferation assay (neuron/oligodendrocyte cocultures):

(a) Representative images of spinal cord coculture (25 days in vitro) treated with recombinant proteins and immunostained for Olig2 (in green). Cell nuclei counterstained with DAPI (pseudocolored in red). Oligodendrocyte lineage cells appear yellow (quantified in b). Images are representative of 3 to 5 similar experiments.

(b) Quantitation of Olig2+ cells after treatment of cocultures with the indicated factors between DIV 12 and DIV 25 (n=3 to 4 independent coculture experiments; Pdgf: p=0.0113; Sparcl1: p=0.9947; Vegfc: p=0.3451; Fgf1: p=0.0179; Pleiotrophin: p=0.4774; Tmsb4x: p=0.3125; Timp3: p=0.1766; Activin A: p=0.0869). The percentage of Olig2+ cells was calculated relative to the cell number determined by DAPI and normalized to vehicle treated cultures (set to 100%, dashed lines). The known OPC mitogen Pdgf was used as positive control. Note the increase in the number of Olig2+ cells after treatment with Fgf1.

(c,d) Confirmation of Fgf1 as a mitogen for OPCs. Representative images of cocultures treated with vehicle (top) or Fgf1 (bottom) and costained for Olig2 (red, in c) and Ki67 (green, in c). Percentage of Ki67+, Olig2+ cells after Fgf1 treatment is quantitated in d (n=4 independent experiments per condition, p= p=0.0153, t=5.013, d.f.=3).

e,f: Oligodendrocyte differentiation assay (neuron-free mixed glial cultures):

(e) Representative images of primary oligodendrocytes in a mixed glial culture co-immunostained for MBP and GFAP (left) or Olig2 and Apc-CC1 (right) 3 days after plating. Images are representative of 5 similar experiments.

(f) Quantitation of the percentage of CC1 expressing oligodendrocytes in relation to Olig2+ cells after 3 (Vehicle vs. Klotho, p=0.5403, Vehicle vs. Pleiotrophin, p=0.2397, Vehicle vs. Timp3, p=0.5629, Vehicle vs. Activin, p=0.0003) and 5 days (Vehicle vs. Klotho, p=0.6483, Vehicle vs. Pleiotrophin, p=0.8878, Vehicle vs. Timp3, p=0.7308, Vehicle vs. Activin, p=0.0026) treatment with either vehicle or one of the indicated recombinant proteins (n=3-5 independent experiments each group). Note the activity of Activin A as a differentiation factor.

g,h: Myelination assay (neuron/oligodendrocyte cocultures):

(g) Representative image of a myelinating spinal cord coculture (25 days in vitro) immunostained for MBP (in green) and Smi31 (in red). The image is representative of >10 similar experiments.

(h) The ensheathment/myelination index was calculated by dividing the MBP+ area of myelin by the Smi31+ area of axons (using single channel images) and defining this ratio as 1.0 in vehicle treated controls (dashed line). Note the pro-myelinating activity of 5 factors in this assay, which was not optimized for concentrations and treatment times. Number of independent experiments per condition: n=3 for Vegfc (p=0.8806), and Tmsb4x (p=0.025), n=5 for Sparcl1 (p=0.5781) and Fgf1 (p=0.0498), n=11 for Pdgf (p=0.0049), Ptn (p=0.0005), Timp3 (p=0.0264) and Activin A (p=0.0132).

(i) Mixed neuron-free glial cultures treated for 3 days with Activin A in combination with either 2.5 μM SIS3 (SI), 0.5 μM LY294002 (LY) or 10 μM UO126 (UO) were immunostained as in (e) and statistically analyzed for the ratio of CC1 to Olig2+ cells (n=3 to 6 independent experiments per condition). Note the requirement of MAPK (ERK 1/2) (UO) in Activin A-stimulated oligodendrocyte differentiation (Vehicle vs. Vehicle+SI: p=0.8215, t=0.2828; Vehicle vs. Vehicle+LY: p=0.1509, t=1.953; Vehicle vs. Vehicle + UO: p=0.0674, t=2.193; Activin A vs. Activin A+SI: p=0.3050, t=1.01; Activin A vs. Activin A+LY: p=0.9959, t=0.0521; Activin A vs. Activin A+UO: p=0.0005 t=4.751; d.f.=29; F=12.47)

Data are means ± s.e.m. *p<0.05; **p<0.01; ***p<0.001, Wilcoxon matched pairs test (in b,d,f,h), or one-way analysis of variance (ANOVA) followed by Bonferroni test (in i).