Fig. 4

Effect of MSC-conditioned medium containing high and low levels of PDGF-AA on oligodendrocyte precursor cells exposed to OGD. A The diagram shows the protocol used. After the suspension phases (neurosphere and oligosphere), NSC-derived OPCs were seeded in adhesion as undifferentiated precursors ( − 3 DIV). After 2 DIVs, cultures were exposed to OGD for 3 h, followed by a further DIV differentiation induced by addition of T3. Cells were cultured in standard medium (ctrl), mesenchymal stem cell medium (MSC med), or conditioned medium obtained from donor samples 5919 (low PDGF-AA) or 6111 (high PDGF-AA), following OGD exposure throughout the differentiation. At 12 DIVs after differentiation induction, cells were processed for immunocytochemistry and cell-based HCS analysis. B Graph shows the percentage of condensed nuclei measured in OPC cultures exposed to normoxia control conditions or OGD. Cells were cultured in standard medium (ctrl), mesenchymal stem cell medium (MSC med), or conditioned medium obtained from donor samples 5919 (low PDGF-AA) or 6111 (high PDGF-AA). C, D Graphs show the percentage of NG2- (A) or MBP- (B) positive cells in OPC cultures exposed to normoxia control conditions or OGD. Cells were cultured in standard medium (ctrl), mesenchymal stem cell medium (MSC med), or conditioned medium obtained from donor samples 5919 (low PDGF-AA) or 6111 (high PDGF-AA). E Representative images of OPC cultures exposed to normoxia under control conditions, or to OGD under control conditions, with the culture medium of mesenchymal stem cells (MSC med), or conditioned media. The following stainings were used: NG2 (green; OPC), MBP (red; mature oligodendrocytes) Hoechst (blue; nuclei). Images were modified increasing brightness for visualization purposes, but High Content Screening software process the raw images independently from post-acquisition modifications. The same brightness/contrast manipulation was applied to the whole image set. Scale bar: 200 µm. Statistical analysis. Columns represent the mean + SEM. One-way ANOVA followed by Tukey’s post-test. Asterisks represent the differences between the indicated groups (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001)