Fig. 6

Primary adult gmASTRs and wmASTRs responding to TLR3 agonist Poly(I:C) distinctly modulate OPC behavior. a–d Primary adult grey matter astrocytes (gmASTRs) and white matter ASTRs (wmASTRs) were cultured for 24 h in the presence or absence of TLR3 agonist Poly(I:C), after which conditioned medium (ACM) was collected in the next 24 h. For extracellular matrix proteins (ECM), ASTRs were cultured for 48 h in the presence or absence of Poly(I:C) after which the cells were lysed, ECM was collected and used as a coating. Oligodendrocyte progenitor cells (OPCs) were cultured in the presence of ACM or on ECM coatings, for 1 day in the presence of PDGF-AA and FGF-2 to assess proliferation (a, % Ki67-positive cells of A2B5-positive cells), or differentiated for six days after growth factor withdrawal to assess metabolic activity (b, MTT), differentiation (c, % MBP-positive cells of total cells), and myelin membrane formation (d, % myelin membranes formed by MBP-positive cells). Bars represent relative means to their respective untreated control ACM or ECM coatings, which were set to 1 in each independent experiment. Individual data points (black dots) represent independent OPC culture preparations with three to five different batches of ACM or ECM. Error bars indicate standard error of the mean. Statistical analyses are performed using column statistics with a one-sample t test (*p < 0.05) to assess differences with untreated control ACM or ECM coatings, and with an unpaired two-sided t test (^##p < 0.01) to assess differences between response to ACM or ECM coatings of Poly(I:C)-treated gmASTRs and ACM or ECM coatings of Poly(:C)-treated wmASTRs Absolute values are 42.5 ± 13.9% proliferation, 34.9 ± 7.6% differentiation, and 58.4 ± 9.1% myelin membrane formation with control wmACM; 38.4 ± 14.1% proliferation, 35.3 ± 12.2% differentiation, and 67.5 ± 10.1% myelin membrane formation with control gmACM; 21.6 ± 4.0% proliferation, 31.8 ± 5.7% differentiation, and 81.1 ± 10.1% myelin membrane formation on control wmECM coatings and 31.4 ± 5.8% proliferation, 37.5 ± 5.3% differentiation, and 75.8 ± 18.6% myelin membrane formation on control gmECM coatings. Note that OPC proliferation is decreased in the presence of ACM of Poly(I:C)-treated wmASTRs (^*p = 0.027) and increased on ECM coatings of Poly(I:C)-treated wmASTRs (^*p = 0.012), but not on ECM-coatings of gmASTRs (^##p = 0.0016 compared to ECM from Poly(I:C)-treated wmASTRs), while myelin membrane formation is decreased in the presence of ACM of Poly(I:C)-treated gmASTRs (p = 0.016)