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

Abstract

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-b1-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 selfrenewal 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

“…In order to assess if the failure to reverse the astrocytes up to the level of ID3 expression, which would be compatible with an endogenous quiescent NSC identity, could be due to the inability of the NSC-stemness medium to foster a bona fide NSC identity, we tested this medium on rat NSPCs obtained by brain milking. This method allows the direct isolation of NSPCs from the brains of live adult rats, without the use of aggressive, tissue-dissociation protocols, thus allowing the cells to retain in higher fidelity their endogenous properties [36,37]. We confirmed the presence of cells phenotypically compatible with endogenous, quiescent NSCs (GFAP+, ID3+, EGFR-) in the samples obtained after milking (Figure 3A).…”

“…As with the astroglial cells cultured in 10% FBS, we did not detect any mitotic cells throughout the 5 days. The milking of the brain protocol involves the compromise of the integrity of the ependymal cell layer at the level of the lateral ventricles, induced by the administration of neuraminidase [36,37]. Even though the areas of ependymal damage are later characterized by the formation of astroglial scars, in some cases, we also observed that some long-term, surviving ependymal cells were expressing markers of mitosis (Supplementary Figure S2).…”

“…Original images have been produced and archived from tissue used and processed as described in previous publications. For the post-brain milking tissue shown in Figure S2, see [37].…”

“…Mixed cell cultures, which included approximately 25% healthy ependymal cells, could be prepared only by dissecting and dissociating periventricular areas of the lateral ventricles that were subsequently cultured in DMEM complemented with 10% FBS. Ependymal cells were identified by the combined expression of at least two of the following markers: S100β (a marker of astrocytes and ependymal cells) [37], β-catenin [22], pericentrin (PCNT, a protein that marks basal bodies, for which we did not quantify the staining but followed an on/off approach), and acetylated α-tubulin (to mark directly the cilia) (Figure 4A-C). The cells were kept in culture for 5 days, with the percentage of ependymal cells resting stable (Figure 4D).…”

Heterogeneity of quiescent and active neural stem cells in the postnatal brain

“…Moreover, the CSF supplies the SEZ, in an age-dependent manner, both with proquiescence factors, such as Neurotrophin3 or Platelet-Derived Growth Factor-D, and activating signals, such as Insulin-like Growth Factor-2 (Delgado et al, 2014;Silva-Vargas et al, 2016;Delgado et al, 2021). The in vitro culture of NSCs, isolated from live rats using the method of milking the SEZ, revealed the separate regulation of self-renewal and of exit from quiescence, since choroid plexus-conditioned media significantly enhanced the latter and at the same time significantly decreased the former (McClenahan et al, 2021). Interestingly, NT3 is acting on NSCs both from the CSF and blood vessels.…”