Interactions between rat neural precursor and brain endothelial cells

Lim, JC, Wolpaw AJ, Hladky SB, Caldwell MA* and Barrand, MA. Department of Pharmacology, University of Cambridge, Cambridge CB2 1PD and * Cambridge Centre for Brain Repair, Forvie Site, Robinson Way, Cambridge CB2 2PY, UK.

Barrier properties are known to exist in blood vessels supplying the brain at a relatively immature stage of development (Engelhardt 2003), even before differentiated astrocytes appear. Thus it has been suggested that neural precursor cells might be involved in inducing many of the blood-brain barrier (BBB) properties that appear in the microvascular endothelium. The primary aim of this study was to examine the interactions between neural precursor cells (NPC) and brain capillary endothelial cells (RBEC) grown together in a co-culture system, comparing them with those found during co-culture of RBEC with C6 glioma cells. RBEC cells were grown from microvessels isolated from the brains of male Wistar rats of 200 gm in weight as described (Barrand et al 1995) and used between passage 1 and 4.

NPCs were isolated from the brains of day 14 embryonic rats and cultured as free floating neurospheres in Dulbecco’s modified essential medium and Ham’s F12 (3:1) containing growth factors as described (Caldwell and Svendsen 1998) until co-culture when the growth factors were replaced in the medium by 75 µg ml-1 of endothelial cell growth supplement and 10% fetal calf serum. Under these conditions, the neurospheres become adherent and the cells start to differentiate into GFAP-staining glial cells and beta-tubulin III staining neuronal cells. Observations were made of cell proliferation and level of expression of BBB characteristics ie alkaline phosphatase activity (ALP), P-glycoprotein (Pgp) expression and transendothelial electrical resistance (TEER) in RBECs grown for up to 9 days as monolayers on filter inserts in the absence or presence of NPCs or C6 glioma cells. Statistical analysis was performed using ANOVA followed by Dunnett’s multiple comparison test. With RBECs co-cultured with differentiating NPCs, there was little difference in cell proliferative state from that seen in monoculture whereas RBECs co-cultured with C6 glioma cells showed a rapid increase in cell number (n=6, p<0.05) and in ALP activity (n = 6; p < 0.05). There was a significantly higher TEER across monolayers of RBECs co-cultured (n = 5; p < 0.001) for 9 days with NPCs compared to those grown in monoculture and those co-cultured with C6 glioma cells exhibited a decrease in TEER (n = 5; p < 0.001). Real time RT-PCR was used to compare expression of the genes encoding Pgp ie mdr1a and mdr1b in the different co-culture systems. The relative expression of mdr1a in RBECs co-cultured with differentiating NPCs was significantly increased to 2.0 ± 0.27 (n = 4; p < 0.05) compared to RBECs grown in monoculture. The upregulation of Pgp, increased TEER, and relatively non-proliferative state of RBECs co-cultured with NPCs suggest that NPCs could influence induction and maintenance of blood-brain barrier properties in brain vasculature. The factors secreted by NPCs that bring about these effects have yet to be identified but are clearly distinct from those produced by glioma cells that encourage RBEC proliferation and reduce BBB integrity.

Barrand MA, Robertson KJ and von Weikersthal SF (1995) FEBS Lett 374, 179-183.
Caldwell MA and Svendsen CN (1998) Exp Neurol152, 1-10.
Engelhardt B (2003) Cell Tissue Res314, 119-129.

This work was supported by a grant from BBSRC. JL holds Gates and ORS Scholarships.