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. 2017 Nov 5;7(21):e2603.
doi: 10.21769/BioProtoc.2603.

Isolation, Culture and Differentiation of Adult Hippocampal Precursor Cells

Stefanie N Bernas  1   2 , Odette Leiter  1   2 , Tara L Walker  1   2 , Gerd Kempermann  1   2
Affiliations

Isolation, Culture and Differentiation of Adult Hippocampal Precursor Cells

Stefanie N Bernas et al. Bio Protoc. .

Abstract

There are two neurogenic niches in the adult mammalian brain: the subventricular zone of the lateral ventricle and the subgranular zone of the hippocampal dentate gyrus. Cells from these areas can be isolated and maintained in vitro, using two different culture systems to assess their potential regarding proliferation and differentiation in a reductionist model. While the neurosphere assay is primarily performed to directly study the proliferative and differentiation potential of cells in individual brains, the monolayer culture allows single cell analysis in a rather homogeneous cell population. Here, we describe the isolation, culturing methods and differentiation of neural precursor cells in both systems.

Keywords: Adherent monolayer; Adult mouse; Dentate gyrus; Differentiation; Neuroscience; Neurosphere; Precursor cell; Subventricular zone.

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Figures

Figure 1.
Figure 1.. Isolation of neural precursor cells from the SVZ and the DG.
A. Required tools; B. Isolated brain; C. Coronal cut through the brain at the area of the optic chiasm using a scalpel; D. Cut along the longitudinal fissure; E. Dissected SVZ tissue; F. Dissected DG; G. Mincing of the dissected tissue using a scalpel; H. Resuspended SVZ tissue; I. DG tissue after enzymatic incubation.
Figure 2.
Figure 2.. Neural precursor cells from the adult mouse brain can be cultured as adherent monolayer cultures (A) or as neurospheres (B).
Scale bars = 50 µm.
Figure 3.
Figure 3.. Monolayer cultures under proliferation conditions.
A. Monolayer cultures under proliferation conditions express cellular markers for progenitor cells of the nervous system such as Nestin (green) and Sox2 (magenta). B. Progenitor cells can be labeled to assess their proliferation capacity by examining the fraction of cells undergoing S-phase (BrdU label, green) relative to the overall number of cells (Hoechst 33342, blue). Scale bars = 50 µm.
Figure 4.
Figure 4.. Monolayer cultures as well as neurospheres can be differentiated.
A. Differentiated cells of a monolayer culture with GFAP-positive astrocytes (green) and Map2ab-positive neurons (magenta). B. Differentiated neurosphere culture with β-III-tubulin-positive neurons (magenta) and GFAP-positive astrocytes (green). C. Differentiated neurosphere culture with O4-positive Oligodendrocytes (red). Scale bars = 50 µm.
See this image and copyright information in PMC

References

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