Death of astrocytes shortly after their generation and the elevated expression of hbegf mRNA in endothelial cells compared to astrocytes ( Cahoy et al., 2008 and Daneman et al., 2010) support the hypothesis that astrocytes may require vascular cell-derived trophic support. MD-astrocytes show remarkable proliferative ability and can be passaged repeatedly over many months.

In contrast, most astrocyte proliferation in vivo is largely complete by P14 (Skoff and Knapp, 1991). To directly compare the proliferative capacities of MD and IP-astrocytes P7, we plated dissociated single cells at low density in a defined, serum-free 3MA media containing HBEGF and counted clones at 1, 3, and 7DIV (Figures S1Q–S1S). MD-astrocytes displayed a much higher proliferative capacity, 75% of them dividing once every 1.4 days by 7DIV. In contrast, 71% of IP-astrocytes divided less than once every 3 days (Figure S1S). Thus IP-astrocytes have a more modest ability to divide compared with MD-astrocytes, this is more in line with what is expected in vivo (Skoff and Knapp 1991). Using gene profiling, we determined if gene expression of cultured IP-astrocytes was more similar to that of acutely purified astrocytes, compared to MD-astrocytes. Total RNA was isolated from acutely purified astrocytes from P1 and P7 rat brains (IP-astrocytes P1 and

P7) and from acutely isolated cells cultured for 7DIV with HBEGF (IP-astrocytes P1 and P7 7DIV, respectively) and from MD-astrocytes (McCarthy Adenylyl cyclase and de Vellis, 1980). RT-PCR with cell-type specific primers was used to assess the purity of the isolated learn more RNA. We used GFAP, brunol4,

MBP, occludin, CX3CR1 as mentioned above, as well as chondroitin proteoglycan sulfate 4 (CSPG4) for OPCs and pericytes. MD-astrocytes consistently had some neuron contamination because of the high percentage of contaminating neural stem cells ( Hildebrand et al., 1997; Figure 4A). This was not observed in IP-astrocyte cultures. IP-astrocytes P1 and P7 7DIV cells had an expression profile resembling their acutely isolated counterparts, where only 118 and 54 genes respectively differed significantly (p < 0.05). In contrast, MD-astrocyte expression profiles were significantly different from that of acutely purified cells (Table 1; Figure 4B). With a very stringent statistical test (moderated t test) and posttest (Bonferroni correction) to identify the most significant changes, we found that 547 and 729 genes were significantly different (p < 0.05) between acute IP-astrocytes P1 or P7 cells and MD-astrocytes, respectively. These results strongly suggest that by gene expression, cultured IP-astrocytes are more similar to cortical astrocytes in vivo. Only 54 genes out of over 31,000 genes differed significantly between acute IP-astrocytes P7 and IP-astrocytes P7 7DIV (p < 0.05).