Figure 2. RMS astrocytes regulate the formation and growth of BVs A, Micrographs depicting PECAM+ BVs (red) and GFAP+ astrocytes (green) at the beginning of postnatal development (P3, top) and in adulthood (P60, bottom). Note that at the early developmental periods, astrocytes are positioned at the outer border of the RMS, whereas in adults they are located inside of the migratory stream. RMS is indicated by the dashed white line. B, In vitro method for studying the role of astrocytes on BV formation and growth in cultures. Astrocytes were prepared from the different brain regions of P3 pups or from adult SVZ, and the ring of aorta was taken from adult mice. The conditioning medium (CM) derived from astrocytes was added to a 3D cultured aorta's rings every second day. C, Confocal image of cultured astrocytes. Astrocytes were stained with GFAP antibodies (green), and the cell nuclei were depicted by DAPI staining (red). Note that almost every DAPI-positive cell is colocalizing with GFAP, indicating high purity of astrocyte cultures. D, Quantification of cell density and purity of astrocyte cultures prepared from cortex, cerebellum, and SVZ-RMS. The first graph shows that there is no difference in the cell number of cultured astrocytes derived from different brain regions. The second graph shows the high purity of astrocyte cultures. E, Representative images showing BV growth from the aortic ring in the absence (control) and presence of conditioning medium derived from astrocytes of different regions. Arrows indicate newly formed BVs. F, Formation and growth of new blood vessels from the aortic ring after addition of conditioning medium from cortical, cerebellar, and SVZ astrocyte cultures. Note that only conditioning medium from P3 SVZ astrocytes potentiates formation and growth of new blood vessels. G, Western blot analysis of astrocyte culture-derived conditioning medium (CM) showing that in contrast to P3 cortex, P3 cerebellum, and adult SVZ-RMS, P3 SVZ-RMS-derived astrocytes secrete VEGF. Since adult astrocytes require longer culturing to reach the confluence, we have also immunoblotted conditioning medium of 14 d cultured adult SVZ astrocytes. Astrocyte cultures from which conditioning medium was derived were also immunoblotted for GFAP to ascertain an equal amount of astrocytes used for experiment.