Fig. 3

Secreted factors from wmASTRs inhibit myelin membrane formation. a–f Oligodendrocyte progenitors cells (OPCs) were cultured with PDGF-AA and FGF2 for 24 h to assess proliferation (% Ki67-positive cells of A2B5-positive cells, schematic representation in a), or differentiated into mature oligodendrocytes (OLGs) for 6 days after growth factor withdrawal to assess metabolic activity (MTT), cytotoxicity (LDH), differentiation (% MBP-positive cells), and myelin membrane formation (% myelin membranes formed by MBP-positive cells) (schematic representation in b). Assays were performed in the presence or absence of astrocyte (ASTR)-conditioned medium (ACM) or ASTR-derived extracellular matrix (ECM) coatings obtained from primary adult grey matter (gm) or white matter (wm) ASTRs. Representative images of MBP-positive OLGs (red, arrow indicates a MBP-positive cell with myelin membrane, arrowhead a MBP-positive cell without myelin membrane) in the presence of non-conditioned medium (NCM) or ACM are shown in c; representative images of proliferation on no ECM (PLL) or ECM coatings stained for Ki67 (red) and A2B5 (black) are shown in d. Quantification of assays of 4–12 independent cell culture preparations (black dots) with 4–12 different batches of ACM (e) and ECM coatings (f) are shown. Bars represent the relative means to NCM (e) or no ECM (f), which were set to 1 in each independent experiment. Error bars indicate the standard error of the mean. Statistical analyses are performed using column statistics with a one-sample t test (*p < 0.05) to assess differences between treatments and control, while a paired two-sided t test (*p < 0.05) is used to examine differences between effects of gmACM or gmECM coatings versus respectively wmACM or wmECM coatings. Absolute values of NCM are for proliferation 37.5 ± 22.4%, cytotoxicity 25.9 ± 2.9%, differentiation 48.6 ± 17.6%, and myelin membrane formation 68.0 ± 14.4% and of no ECM for proliferation 17.8 ± 4.8%, differentiation 33.3 ± 4.7%, and myelin membrane formation 65.3 ± 3.8%. Note that upon exposure to wmACM, myelin membrane formation is decreased compared to NCM (p = 0.005), while metabolic activity is increased upon gmACM treatment compared to NCM (*p = 0.016) and wmACM treatment (^#p = 0.033) (c, d). In addition, OPC proliferation is higher on gmECM coatings than on no ECM (p = 0.041) (e, f). Scale bars are 25 μm