Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Abstract

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G 1 phase due to a G 1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G 1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.

Concurrent MEK targeted therapy prevents MAPK pathway reactivation during BRAFV600E targeted inhibition in a novel syngeneic murine glioma model

“…Cell cycle analyses were conducted by treating cells with Hoechst 33342 at a final concentration of 2 ng/ml and DNA content (2n versus 4 n) was determined as described [26]. Analyses were conducted using the FACS ARIA II (BD Biosciences).…”

Neural stem cell heterogeneity through time and space in the ventricular-subventricular zone

“…whether a cell was in G 0 /G 1 or in S/G 2 -M) did reveal that EGFR + populations were more frequently in S/G 2 -M phases than EGFR − populations (Pastrana et al, 2009). Recently, a protocol utilizing FUCCI (fluorescence ubiquitination cell cycle inhibitor) mice tested whether V-SVZ cells were in G 1 phase of the cell cycle vs. S/G 2 /M (Daynac et al, 2015). This sorting strategy has potential applications which include the detection of NSC division, as the fluorescence intensity in cells from FUCCI mice increases when a cell starts to divide (Roccio et al, 2013).…”

Live-cell time-lapse imaging and single-cell tracking of in vitro cultured neural stem cells – Tools for analyzing dynamics of cell cycle, migration, and lineage selection

“…The majority of these have been performed in rodent primary NSC cultures [17,18,20,2,13,25,12] spanning 4–14 days of time-lapse image acquisition. A subset of these studies have investigated cell cycle by single-cell tracking of Fucci- cell cycle reporter expressing mouse NSC [22,21,3]; however, analyses of Fucci -mouse NSC ( Fucci -mNSC) in vitro have focused on short term experiments (24–72 h). In parallel, few studies have investigated single-cell tracking in time-lapse imaging of human NSC (hNSC) [1,7], and these have again been constrained to short term experiments (24–48 h).…”