Apposing small arrows (in A, B, … Modification of E16 spinal cord culture for cerebral cortex derived culture Our next goal was to utilize E16 rat cortex for the co-culture model. Surprisingly, simply adopting the spinal cord culture protocol to the cerebral cortex failed to produce discernable myelin see more formation (Fig. 5E), which was in great contrast to rather abundant myelin formed in the spinal cord derived culture (Fig. 5F). The density Inhibitors,research,lifescience,medical of pNF-labeled axons appeared comparable between cultures from these two CNS sources, suggesting

that neurons may already well developed in the cortex-derived culture. However, very few MBP+ mature OLs, if any, were found in the cortex-derived culture (Fig. 5C). We then assessed the cell phenotype, and the data showed that total number of OL lineage cells (Olig2+) in the cortex-derived cultures were slightly higher than that from the spinal cord (31.0% vs. 28.3%), indicating OLs in the cortex-derived cultures may fail to mature. This issue was re-examined in cultures at DIV17 (the onset of myelination in the Inhibitors,research,lifescience,medical spinal cord derived cultures), and the result revealed that most OL lineages in the cortex-derived cultures Inhibitors,research,lifescience,medical remained as either NG2+ early progenitors or O4+ late progenitors (Fig. 5A). In contrast, much fewer OL progenitor cells (Fig. 5B) and more mature OLs (Fig. 5D) were found in the spinal cord-derived culture at this stage. Figure 5 Comparison

between the spinal cord and cortex-derived co-cultures. The cortex-derived culture Inhibitors,research,lifescience,medical contained more OL progenitor cells (A, NG2+ and O4+) but much fewer mature OLs (C, myelin basic protein [MBP]+) than from the spinal cord (B and D, respectively). … To accelerate OL maturation, we included T3 (60 ng/mL) in the medium starting at DIV10. As expected, Inhibitors,research,lifescience,medical OLs matured quickly as shown by a markedly decrease in early OL progenitor cells (NG2+) but an increase of late progenitor cells

(O4+) and mature OLs (MBP+) three days after T3 was introduced to the medium (Fig. 6A–C). At DIV26, active myelination was noted (Fig. 6D). At DIV40, myelin segments were in abundance (Fig. 6E) and the nodes of Ranvier were detected (Fig. 6F). Finally, the cell phenotypes next in the cortex-derived culture were also determined and compared to that from the spinal cord. In general, the cortex-derived culture contained less neurons (22.3% vs. 38.5%), slightly more OL lineage (31.0% vs. 28.3%), but very few microglia/macrophage (less than 2% vs. 10%) compared to that from the spinal cord. Figure 6 Myelination was significantly increased in the cortex-derived culture by accelerating OL maturation. Three days after T3 was introduced into the medium, the number of NG2+ (A) was reduced while O4+ (B) and myelin basic protein (MBP)+ OLs (C) increased … Quantification of myelination An important aspect of the co-culture models is the feasibility to quantify myelin formation at a specified stage.