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Peeyush Kumar T.,Harvard University | Peeyush Kumar T.,Angiogenesis and Brain Development Laboratory | Vasudevan A.,Harvard University | Vasudevan A.,Angiogenesis and Brain Development Laboratory
Journal of Visualized Experiments | Year: 2014

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1 coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis. © JoVE 2006-2014. All Rights Reserved.

Li S.,Harvard University | Li S.,Angiogenesis and Brain Development Laboratory | Joshee S.,Harvard University | Joshee S.,Angiogenesis and Brain Development Laboratory | And 2 more authors.
Biomolecular Concepts | Year: 2014

Midbrain GABA neurons, endowed with multiple morphological, physiological and molecular characteristics as well as projection patterns are key players interacting with diverse regions of the brain and capable of modulating several aspects of behavior. The diversity of these GABA neuronal populations based on their location and function in the dorsal, medial or ventral midbrain has challenged efforts to rapidly uncover their developmental regulation. Here we review recent developments that are beginning to illuminate transcriptional control of GABA neurons in the embryonic midbrain (mesencephalon) and discuss its implications for understanding and treatment of neurological and psychiatric illnesses. © De Gruyter 2014.

Won C.,Harvard University | Won C.,Angiogenesis and Brain Development Laboratory | Lin Z.,Harvard University | Kumar T. P.,Harvard University | And 9 more authors.
Nature Communications | Year: 2013

Gamma-aminobutyric acid neurons, born in remote germinative zones in the ventral forebrain (telencephalon), migrate tangentially in two spatially distinct streams to adopt their specific positions in the developing cortex. The cell types and molecular cues that regulate this divided migratory route remains to be elucidated. Here we show that embryonic vascular networks are strategically positioned to fulfil the task of providing support as well as critical guidance cues that regulate the divided migratory routes of gamma-aminobutyric acid neurons in the telencephalon. Interestingly, endothelial cells of the telencephalon are not homogeneous in their gene expression profiles. Endothelial cells of the periventricular vascular network have molecular identities distinct from those of the pial network. Our data suggest that periventricular endothelial cells have intrinsic programs that can significantly mould neuronal development and uncovers new insights into concepts and mechanisms of central nervous system angiogenesis from both developmental and disease perspectives. © 2013 Macmillan Publishers Limited. All rights reserved.

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