Isolation and Expansion of Neurospheres from Postnatal (P1−3) Mouse Neurogenic Niches

1. J Vis Exp. 2020 May 23;(159). doi: 10.3791/60822.

Isolation and Expansion of Neurospheres from Postnatal (P1-3) Mouse Neurogenic
Niches.

Soares R(1), Ribeiro FF(2), Lourenço DM(2), Rodrigues RS(2), Moreira JB(2),
Sebastião AM(2), Morais VA(3), Xapelli S(4).

Author information:
(1)Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina,
Universidade de Lisboa; Instituto de Farmacologia e Neurociências, Faculdade de
Medicina, Universidade de Lisboa; Instituto de Biologia Molecular, Faculdade de
Medicina, Universidade de Lisboa.
(2)Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina,
Universidade de Lisboa; Instituto de Farmacologia e Neurociências, Faculdade de
Medicina, Universidade de Lisboa.
(3)Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina,
Universidade de Lisboa; Instituto de Biologia Molecular, Faculdade de Medicina,
Universidade de Lisboa.
(4)Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina,
Universidade de Lisboa; Instituto de Farmacologia e Neurociências, Faculdade de
Medicina, Universidade de Lisboa; .

The neurosphere assay is an extremely useful in vitro technique for studying the
inherent properties of neural stem/progenitor cells (NSPCs) including
proliferation, self-renewal and multipotency. In the postnatal and adult brain,
NSPCs are mainly present in two neurogenic niches: the subventricular zone (SVZ)
lining the lateral ventricles and the subgranular zone of the hippocampal
dentate gyrus (DG). The isolation of the neurogenic niches from postnatal brain
allows obtaining a higher amount of NSPCs in culture with a consequent advantage
of higher yields. The close contact between cells within each neurosphere
creates a microenvironment that may resemble neurogenic niches. Here, we
describe, in detail, how to generate SVZ- and DG-derived neurosphere cultures
from 1-3-day-old (P1-3) mice, as well as passaging, for neurosphere expansion.
This is an advantageous approach since the neurosphere assay allows a fast
generation of NSPC clones (6-12 days) and contributes to a significant reduction
in the number of animal usage. By plating neurospheres in differentiative
conditions, we can obtain a pseudomonolayer of cells composed of NSPCs and
differentiated cells of different neural lineages (neurons, astrocytes and
oligodendrocytes) allowing the study of the actions of intrinsic or extrinsic
factors on NSPC proliferation, differentiation, cell survival and
neuritogenesis.

DOI: 10.3791/60822
PMID: 32510488 [Indexed for MEDLINE]

Auteurs Bordeaux Neurocampus