Isolation of Neural Stem Cells from the embryonic mouse hippocampus for in vitro growth or engraftment into a host tissue

Oksana Rybachuk; Olga Kopach; Tetyana Pivneva; Vitaliy Kyryk

doi:10.21769/BioProtoc.3165

Isolation of Neural Stem Cells from the embryonic mouse hippocampus for in vitro growth or engraftment into a host tissue

Bio Protoc. 2019 Feb 20;9(4):e3165. doi: 10.21769/BioProtoc.3165.

Authors

Oksana Rybachuk^{1

2}, Olga Kopach^{1

3}, Tetyana Pivneva^{1

2}, Vitaliy Kyryk²

Affiliations

¹ Department of Sensory Signaling, Bogomoletz Institute of Physiology, Kyiv, Ukraine.
² State Institute of Genetic and Regenerative Medicine, Kyiv, Ukraine.
³ Queen Square Institute of Neurology, University College London, London WC1N 3BG, UK.

Abstract

For both stem cell research and treatment of the central nervous system disorders, neural stem/progenitor cells (NSPCs) represent an important breakthrough tool. In the expanded stem cell-based therapy use, NSPCs not only provide a powerful cell source for neural cell replacement but a useful model for developmental biology research. Despite numerous approaches were described for isolation of NSPCs from either fetal or adult brain, the main issue remains in extending cell survival following isolation. Here we provide a simple and affordable protocol for making viable NSPCs from the fetal mouse hippocampi, which are capable of maintaining the high viability in a 2D monolayer cell culture or generating 3D neuro-spheroids of cell aggregates. Further, we describe the detailed method for engraftment of embryonic NSPCs onto a host hippocampal tissue for promoting multilinear cell differentiation and maturation within endogenous environment. Our experimental data demonstrate that embryonic NSPCs isolated using this approach show the high viability (above 88%). Within a host tissue, these cells were capable of differentiating to the main neural subpopulations (principal neurons, oligodendrocytes, astroglia). Finally, NSPC-derived neurons demonstrated matured functional properties (electrophysiological activity), becoming functionally integrated into the host hippocampal circuits within a couple of weeks after engraftment.

Keywords: 3D neuro-spheroids; Adherent 2D monolayer cell culture; Cell viability; Embryonic neural stem/progenitor cells (NSPCs); Mouse hippocampus; Organotypic hippocampal tissue.