Isolation of Radial Glia-Like Neural Stem Cells from Fetal and Adult Mouse Forebrain via Selective Adhesion to a Novel Adhesive Peptide-Conjugate

Károly Markó,Emília Madarász,Nóra Hádinger,Márta Jelitai,Tímea Kőhidi,Gábor Mező

doi:10.1371/journal.pone.0028538

Károly Markó, Emília Madarász + Show 4 more

Open Access

https://doi.org/10.1371/journal.pone.0028538

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Abstract

Preferential adhesion of neural stem cells to surfaces covered with a novel synthetic adhesive polypeptide (AK-cyclo[RGDfC]) provided a unique, rapid procedure for isolating radial glia-like cells from both fetal and adult rodent brain. Radial glia-like (RGl) neural stem/progenitor cells grew readily on the peptide-covered surfaces under serum-free culture conditions in the presence of EGF as the only growth factor supplement. Proliferating cells derived either from fetal (E 14.5) forebrain or from different regions of the adult brain maintained several radial glia-specific features including nestin, RC2 immunoreactivity and Pax6, Sox2, Blbp, Glast gene expression. Proliferating RGl cells were obtained also from non-neurogenic zones including the parenchyma of the adult cerebral cortex and dorsal midbrain. Continuous proliferation allowed isolating one-cell derived clones of radial glia-like cells. All clones generated neurons, astrocytes and oligodendrocytes under appropriate inducing conditions. Electrophysiological characterization indicated that passive conductance with large delayed rectifying potassium current might be a uniform feature of non-induced radial glia-like cells. Upon induction, all clones gave rise to GABAergic neurons. Significant differences were found, however, among the clones in the generation of glutamatergic and cathecolamine-synthesizing neurons and in the production of oligodendrocytes.

Highlights

Proliferating cells with potential to generate more than one neural cell types can be isolated from the mammalian CNS at any ages [1]
Diverse cell populations corresponding to the criteria of ‘‘neural stemness’’ exist in the entire lifespan of mammals starting from the early embryonic neural plate [2] up to the neurogenic regions of the adult brain [3,4]
Beside resident stem cells in the adult neurogenic zones, the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the hippocampus, quiescent and active progenitor cells seem to persist in the brain parenchyma [5], as well

Summary

Introduction

Proliferating cells with potential to generate more than one neural cell types can be isolated from the mammalian CNS at any ages [1]. Radial glia-like neural stem/progenitor cells adhered rapidly to AK-cyclo[RGDfC]-coated surfaces in serum-free culture conditions. The mRNA profile of all embryo-derived RGl clones revealed the characteristics of radial glia-like neural stem cells. They expressed Pax, Sox, Olig, Glast and Blbp, while from pluripotency genes, Nanog mRNA was detected occasionally at low level and Oct was not transcribed (Figure 2f). In embryo-derived RGl cultures, withdrawal of EGF did not initiate the formation of GFAP-immunoreactive cells In these cultures, differentiating neurons resided on the top of RC2immunpositive substrate-attached cells (Figure 5b). In contrast to fetal RGl cultures, substrate-attached cells displayed GFAP-immunoreactivity (Figure 5e) in all adult-derived clones with or without induction.

Discussion

Findings

Materials and Methods