One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

Abstract

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

GDF-15 Secreted from Human Umbilical Cord Blood Mesenchymal Stem Cells Delivered Through the Cerebrospinal Fluid Promotes Hippocampal Neurogenesis and Synaptic Activity in an Alzheimer's Disease Model

“…The established hUCB-MSC lines were maintained in a cell factory in accordance with good manufacturing practice guidelines at MEDIPOST Co., Ltd., and were tested according to the criteria of the International Society of Cell Therapy for surface antigen expression, mesodermal differentiation, and proliferation rate. The mouse NSCs were cultured according to previously described methods [19]. Briefly, 6-month-old C57BL/6 mice were sacrificed, and the subventricular zone (SVZ) and hippocampi were collected.…”

A co-culture model of the hippocampal neurogenic niche reveals differential effects of astrocytes, endothelial cells and pericytes on proliferation and differentiation of adult murine precursor cells

“…For isolation of hippocampal precursor cells the dentate gyri were microdissected (Walker and Kempermann, 2014) from actin-GFP mice. The tissue was enzymatically digested using the Neural Tissue Dissociation Kit (Miltenyi) according to the manufacturer's instructions.…”

Gelatin Directly Enhances Neurogenic Differentiation Potential in Bone Marrow-Derived Mesenchymal Stem Cells Without Stimulation of Neural Progenitor Cell Proliferation

“…When culturing NPCs, EGF and bFGF play important roles in stimulating NPC proliferation (Fiorelli et al, 2013;Mothe and Tator, 2013) and neurosphere formation (Mothe and Tator, 2013;Walker and Kempermann, 2014). The absence of these mitotic growth factors in the MSC culture system significantly contributes to inducing inhibition of NPC FIG.…”