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

Summary 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-β1-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

Reversal of Postnatal Brain Astrocytes and Ependymal Cells towards a Progenitor Phenotype in Culture.

Peer reviewed: TrueAcknowledgements: This work was supported by a Erasmus+ traineeship to DK and the authors are grateful to Benedikt Berninger (Johannes Gutenberg University, Mainz, Germany) for his support. The contribution of Filippos Katsaitis in the scientific and artistic task of preparation of the graphical abstract, using the BioRender basic plan application, are also gratefully acknowledged.Publication status: PublishedAstrocytes and ependymal cells have been reported to be able to switch from a mature cell identity towards that of a neural stem/progenitor cell. Astrocytes are widely scattered in the brain where they exert multiple functions and are routinely targeted for in vitro and in vivo reprogramming. Ependymal cells serve more specialized functions, lining the ventricles and the central canal, and are multiciliated, epithelial-like cells that, in the spinal cord, act as bi-potent progenitors in response to injury. Here, we isolate or generate ependymal cells and post-mitotic astrocytes, respectively, from the lateral ventricles of the mouse brain and we investigate their capacity to reverse towards a progenitor-like identity in culture. Inhibition of the GSK3 and TGFβ pathways facilitates the switch of mature astrocytes to Sox2-expressing, mitotic cells that generate oligodendrocytes. Although this medium allows for the expansion of quiescent NSCs, isolated from live rats by "milking of the brain", it does not fully reverse astrocytes towards the bona fide NSC identity; this is a failure correlated with a concomitant lack of neurogenic activity. Ependymal cells could be induced to enter mitosis either via exposure to neuraminidase-dependent stress or by culturing them in the presence of FGF2 and EGF. Overall, our data confirm that astrocytes and ependymal cells retain a high capacity to reverse to a progenitor identity and set up a simple and highly controlled platform for the elucidation of the molecular mechanisms that regulate this reversal

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-β1-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