The "Brain Milking" Method for the Isolation of Neural Stem Cells and Oligodendrocyte Progenitor Cells from Live Rats.

Tissue-specific neural stem cells (NSCs) remain active in the mammalian postnatal brain. They reside in specialized niches, where they generate new neurons and glia. One such niche is the subependymal zone (SEZ; also called the ventricular-subventricular zone), which is located across the lateral walls of the lateral ventricles, adjacent to the ependymal cell layer. Oligodendrocyte progenitor cells (OPCs) are abundantly distributed throughout the central nervous system, constituting a pool of proliferative progenitor cells that can generate oligodendrocytes. Both NSCs and OPCs exhibit self-renewal potential and quiescence/activation cycles. Due to their location, the isolation and experimental investigation of these cells is performed postmortem. Here, we describe in detail "brain milking", a method for the isolation of NSCs and OPCs, amongst other cells, from live animals. This is a two-step protocol designed for use in rodents and tested in rats. First, cells are "released" from the tissue via stereotaxic intracerebroventricular (i.c.v.) injection of a "release cocktail". The main components are neuraminidase, which targets ependymal cells and induces ventricular wall denudation, an integrin-ß1-blocking antibody, and fibroblast growth factor-2. At a second "collection" step, liquid biopsies of cerebrospinal fluid are performed from the cisterna magna, in anesthetized rats without the need of an incision. Results presented here show that isolated cells retain their endogenous profile and that NSCs of the SEZ preserve their quiescence. The denudation of the ependymal layer is restricted to the anatomical level of injection and the protocol (release and collection) is tolerated well  by the animals. This novel approach paves the way for performing longitudinal studies of endogenous neurogenesis and gliogenesis in experimental animals.

Isolation of neural stem and oligodendrocyte progenitor cells from the brain of live rats.

Postnatal brain neural stem and progenitor cells (NSPCs) cluster in anatomically inaccessible stem cell niches, such as the subependymal zone (SEZ). Here, we describe a method for the isolation of NSPCs from live animals, which we term "milking." The intracerebroventricular injection of a release cocktail, containing neuraminidase, integrin-ß1-blocking antibody, and fibroblast growth factor 2, induces the controlled flow of NSPCs in the cerebrospinal fluid, where they are collected via liquid biopsies. Isolated cells retain key in vivo self-renewal properties and their cell-type profile reflects the cell composition of their source area, while the function of the niche is sustained even 8 months post-milking. By changing the target area more caudally, we also isolate oligodendrocyte progenitor cells (OPCs) from the corpus callosum. This novel approach for sampling NSPCs and OPCs paves the way for performing longitudinal studies in experimental animals, for more in vivo relevant cell culture assays, and for future clinical neuro-regenerative applications.

Dystroglycan Suppresses Notch to Regulate Stem Cell Niche Structure and Function in the Developing Postnatal Subventricular Zone.

While the extracellular matrix (ECM) is known to regulate neural stem cell quiescence in the adult subventricular zone (SVZ), the function of ECM in the developing SVZ remains unknown. Here, we report that the ECM receptor dystroglycan regulates a unique developmental restructuring of ECM in the early postnatal SVZ. Dystroglycan is furthermore required for ependymal cell differentiation and assembly of niche pinwheel structures, at least in part by suppressing Notch activation in radial glial cells, which leads to the increased expression of MCI, Myb, and FoxJ1, transcriptional regulators necessary for acquisition of the multiciliated phenotype. Dystroglycan also regulates perinatal radial glial cell proliferation and transition into intermediate gliogenic progenitors, such that either acute or constitutive loss of function in dystroglycan results in increased oligodendrogenesis. These findings reveal a role for dystroglycan in orchestrating both the assembly and function of the SVZ neural stem cell niche.

The extracellular matrix protein laminin α2 regulates the maturation and function of the blood-brain barrier.

Laminins are major constituents of the gliovascular basal lamina of the blood-brain barrier (BBB); however, the role of laminins in BBB development remains unclear. Here we report that Lama2(-/-) mice, lacking expression of the laminin α2 subunit of the laminin-211 heterotrimer expressed by astrocytes and pericytes, have a defective BBB in which systemically circulated tracer leaks into the brain parenchyma. The Lama2(-/-) vascular endothelium had significant abnormalities, including altered integrity and composition of the endothelial basal lamina, inappropriate expression of embryonic vascular endothelial protein MECA32, substantially reduced pericyte coverage, and tight junction abnormalities. Additionally, astrocytic endfeet were hypertrophic and lacked appropriately polarized aquaporin4 channels. Laminin-211 appears to mediate these effects at least in part by dystroglycan receptor interactions, as preventing dystroglycan expression in neural cells led to a similar set of BBB abnormalities and gliovascular disturbances, which additionally included perturbed vascular endothelial glucose transporter-1 localization. These findings provide insight into the cell and molecular changes that occur in congenital muscular dystrophies caused by Lama2 mutations or inappropriate dystroglycan post-translational modifications, which have accompanying brain abnormalities, including seizures. Our results indicate a novel role for laminin-dystroglycan interactions in the cooperative integration of astrocytes, endothelial cells, and pericytes in regulating the BBB.