title: In vitro modeling of the neurovascular environment by coculturing adult human brain endothelial cells with human neural stem cells.
abstract: Brain and vascular cells form a functionally integrated signalling network that is known as the neurovascular unit (NVU). The signalling (autocrine, paracrine and juxtacrine) between different elements of this unit, especially in humans, is difficult to disentangle in vivo. Developing representative in vitro models is therefore essential to better understand the cellular interactions that govern the neurovascular environment. We here describe a novel approach to assay these cellular interactions by combining a human adult cerebral microvascular endothelial cell line (hCMEC/D3) with a fetal ganglionic eminence-derived neural stem cell (hNSC) line. These cell lines provide abundant homogeneous populations of cells to produce a consistently reproducible in vitro model of endothelial morphogenesis and the ensuing NVU. Vasculature-like structures (VLS) interspersed with patches of differentiating neural cells only occurred when hNSCs were seeded onto a differentiated endothelium. These VLS emerged within 3 days of coculture and by day 6 were stabilizing. After 7 days of coculture, neuronal differentiation of hNSCs was increased 3-fold, but had no significant effect on astrocyte or oligodendrocyte differentiation. ZO1, a marker of adherens and tight junctions, was highly expressed in both undifferentiated and differentiated endothelial cells, but the adherens junction markers CD31 and VE-cadherin were significantly reduced in coculture by approximately 20%. A basement membrane, consisting of laminin, vitronectin, and collagen I and IV, separated the VLS from neural patches. This simple assay can assist in elucidating the cellular and molecular signaling involved in the formation of VLS, as well as the enhancement of neuronal differentiation through endothelial signaling.
authors.affiliation: University of Pittsburgh, McGowan Institute for Regenerative Medicine, Pittsburgh, Pennsylvania, United States of America; Kings College London, Institute of Psychiatry, Department of Neuroscience, London, United Kingdom; Tri-service General Hospital, Department of Neurology, National Defense Medical Centre, Taipei, Taiwan.
authors.forename: Chung-Hsing
authors.initials: CH
authors.lastname: Chou
authors.affiliation: ReNeuron Ltd, Guildford, Surrey, United Kingdom.
authors.forename: John D
authors.initials: JD
authors.lastname: Sinden
authors.affiliation: INSERM U1016, Institut Cochin, Paris, France; CNRS, UMR8104, Paris, France; Université Paris Descartes, Sorbonne Paris Cité, Paris, France.
authors.forename: Pierre-Olivier
authors.initials: PO
authors.lastname: Couraud
authors.affiliation: University of Pittsburgh, McGowan Institute for Regenerative Medicine, Pittsburgh, Pennsylvania, United States of America; University of Pittsburgh, Department of Radiology, Pittsburgh, Pennsylvania, United States of America; University of Pittsburgh, Department of Bioengineering, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Michel
authors.initials: M
authors.lastname: Modo
mesh_terms: D001253:Astrocytes / Q000166:cytology; D001921:Brain / Q000166:cytology*; D002454:Cell Differentiation; D002460:Cell Line; D002478:Cells, Cultured; D018920:Coculture Techniques; D042783:Endothelial Cells / Q000166:cytology*; D006801:Humans; D058953:Neural Stem Cells / Q000166:cytology*