Together, our data shows that differences between MD-astrocytes and IP-astrocytes cannot be explained by serum exposure alone and that serum exposure causes lasting gene expression changes that persist after serum withdrawal. The much closer match of cultured IP-astrocyte gene profiles to those of acutely purified astrocytes indicates that IP-astrocyte cultures are better models of astrocytes than are MD-astrocytes. We therefore assessed whether IP-astrocytes exhibited well-characterized astrocytic functions in culture. MD-astrocytes promote CNS neuron survival selleck compound in culture (Banker, 1980 and Wagner

et al., 2006). We asked if the cultured IP-astrocytes could similarly promote CNS neuronal survival. We purified P5 retinal ganglion cells (RGCs) by immunopanning as described in Barres et al. (1998) and added conditioned media (CM) from P1 (IP-astrocytes P1 ACM) or P7 astrocytes (IP-astrocytes P7 ACM). RGC growth media (RGC

GM) and MD-astrocytes CM (MD-ACM) were used as positive controls. In the absence of any growth factors or astrocyte-derived media, fewer than 5% of RGCs survive. Both P1 learn more ACM and P7 ACM (∗p < 0.05, ∗∗p < 0.01), were as strongly effective at promoting RGC survival for 3 days in culture as was MD-ACM (Figures 5A and 5B). Astrocytes are known to secrete many proteins that have been shown to be important in the CNS for instance apolipoprotein E (APOE), amyloid precursor protein (APP) and thrombospondin 2 (TSP2) (Farber et al., 1995, Mauch et al., 2001 and Christopherson many et al., 2005). We verified with western blotting that ACM from MD-astrocytes and IP-astros P1 and P7 contained these three proteins. A Coomassie stain was used to verify that equivalent amounts of

protein were loaded (Figure 5C). Both P1 ACM and P7 ACM contained APOE and APP. However, only P7 ACM contained TSP2. This differential protein expression at different astrocyte ages shows that we can use this new culture system to tease apart the roles of astrocytes at different developmental time points based on our ability to purify astrocytes at different ages. Interestingly, MD-ACM contained much higher levels of APP, TSP2, and APOE, molecules known to be critical regulators of synapse formation and function (Figures 5D–5F). These results questioned whether IP-astrocytes were as capable as MD-astrocytes at inducing the formation of structural and functional synapses in culture. To directly address this question, we next tested the ability of IP-astrocytes to induce structural synapses by exposing RGCs to feeder layers of P1, P7 IP-astrocytes, MD-astrocytes or a control with no astrocytes. Neuronal cultures were stained for bassoon, a presynaptic marker and homer, a postsynaptic marker (Figure 5G). The number of colocalized puncta in each condition were quantified and we have plotted the number of colocalized puncta as a fold change over control (Figure 5H). There were significant increases in synapse number over control with MD-astrocytes (fold change = 3.12, p < 0.