Soares Rita, Ribeiro Filipa F, Lourenço Diogo M, Rodrigues Rui S, Moreira João B, Sebastião Ana M, Morais Vanessa A, Xapelli Sara
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.
Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa; Instituto de Farmacologia e Neurociências, Faculdade de Medicina, Universidade de Lisboa.
J Vis Exp. 2020 May 23(159). doi: 10.3791/60822.
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.
神经球分析是一种极其有用的体外技术，用于研究神经干细胞/祖细胞（NSPCs）的内在特性，包括增殖、自我更新和多能性。在出生后和成年大脑中，NSPCs主要存在于两个神经发生微环境中：侧脑室衬里的脑室下区（SVZ）和海马齿状回（DG）的颗粒下区。从出生后大脑中分离神经发生微环境能够在培养中获得更多的NSPCs，从而具有更高产量的优势。每个神经球内细胞之间的紧密接触创造了一个可能类似于神经发生微环境的微环境。在这里，我们详细描述了如何从1 - 3日龄（P1 - 3）小鼠中生成源自SVZ和DG的神经球培养物，以及如何传代以扩大神经球。这是一种有利的方法，因为神经球分析能够快速生成NSPC克隆（6 - 12天），并有助于显著减少动物使用数量。通过将神经球接种在分化条件下，我们可以获得由NSPCs和不同神经谱系（神经元、星形胶质细胞和少突胶质细胞）的分化细胞组成的假单层细胞，从而能够研究内在或外在因素对NSPC增殖、分化、细胞存活和神经突发生的作用。
