FIGURE 1.

Astrocyte-conditioned medium from human TG2 expressing cells does not significantly affect OPC maturation. (A) Wild-type human TG2 (LV TG2), GFP and empty vector (MOCK) were lentivirally expressed in astrocytes and subjected to western blot analyses. Note that lentiviral expression of wild type human TG2 (LV TG2) in primary rat astrocytes results in an additional band indicative of the human TG2 (hTG2, arrow), which has a slightly higher molecular weight than endogenous rat TG2 (rTG2; 78 kDa, arrowhead). (B–F) Schematic representation of the experimental set up is shown in B. Astrocyte conditioned medium (ACM) derived from lentivirally transduced astrocytes (MOCK, GFP or LV TG2) is directly added to oligodendrocyte progenitor cells (OPCs, monocultures) and analyzed at the immature oligodendrocyte stage (imOLG, “I,” D) or added both at the OPC stage and the imOLG stage and analyzed at the mature OLG stage (mOLG, “II,” E,F). OPC differentiation (D,E) and myelin membrane formation (F) are, respectively, assessed as the percentage of myelin basic protein (MBP)-positive cells and the percentage of myelin membrane bearing MBP-positive cells. Representative images of MBP (myelin marker, red)-positive cells without (left) and with myelin membranes (right, arrow) are shown in C. Scale bar is 100 μm. Data are shown as mean + SEM, calculated as average percentage compared to OPCs that received non-conditioned medium (CTRL), which was set to 100% in each independent experiment (n = 4). The percentage of MBP-positive cells in control cells is 1.6 ± 0.2% at the imOLG stage (D) and 16.7 ± 0.2% at the mOLG stage (E), the percentage of myelin membranes at the mOLG stage is 35.6 ± 18.0% (F). Statistical analyses were performed using a one-sample t-test (not significant).