Adult Rodent Neurogenic Regions: The Ventricular Subependyma Contains Neural Stem Cells, But the Dentate Gyrus Contains Restricted Progenitors

A, Comparison of the numbers of primary spheres generated from different neurogenic regions in the adult mouse brain. The data are expressed as the mean (+SEM) number of spheres generated per 10,000 cells plated (i.e., per well; density = 20 cells per microliter). Tissue from each region (aLV, anterior lateral ventricle; pLV, posterior lateral ventricle; 3rd V, third ventricle;HV, hippocampal arch; DG, dentate gyrus) was dissociated into single cells and plated in serum-free media containing EGF + FGF2 and B27 supplement; spheres were counted at 7 DIV. Note that the aLV cells generate neurospheres at a rate ∼120-fold higher than the DG cells (0.54 ± 0.1), on the basis of data from n > 120 animals and 10 separate experiments. B, Comparison of the number of spheres generated from adult dentate gyrus at different rostrocaudal levels. Rostral, middle, and caudal sections through the DG yield a similar number of sphere colonies. Tissue from each individual 500 μm section (n = 85 sections from >20 animals) through the DG was separately dissociated into single cells and cultured at 20 cells per microliter; spheres were counted at 7 DIV.

A, B, Comparison of the self-renewal ability of adult anterior lateral ventricle (subependymal) and dentate gyrus spheres. A, The data are expressed as the mean (+SEM) number of spheres generated per single sphere dissociation. At each passage, individual spheres (n > 60 spheres per condition) were dissociated per tissue culture well, and the number of sphere colonies that formed was counted after 7 DIV. Although the adult aLV neurosphere-initiating cells demonstrated self-renewal by giving rise to secondary and tertiary neurospheres (as did the adult pLV, 3rd V, and HA neurospheres; data not shown), the adult DG sphere-initiating cells did not demonstrate self-renewal and did not give rise to secondary sphere colonies. Individual spheres were dissociated and replated in identical media conditions as were used for primary culture (EGF + FGF2 with B27 supplement). B, The data are expressed as the percentage of individual spheres that passaged to generate new spheres. Procedures were followed as described for A. Note that nearly 100% of adult aLV spheres give rise to new spheres at each passage.

A, B, Comparison of the size of primary spheres derived from adult anterior lateral ventricle (subependyma) (A) and dentate gyrus (B). Note that the DG spheres are smaller than the aLV spheres. Scale bars, 100 μm.C–G, Comparison of the ability of adult aLV- and DG-derived primary spheres to contribute to different neural cell lineages. Primary adult aLV neurospheres generate both GFAP⁺ astrocytes (C) and βIII-tubulin⁺ neurons (E), whereas adult DG spheres generate only GFAP⁺astrocytes (D). Small adult DG clumps generate only βIII-tubulin⁺ neurons (F). Note that although both cell types can be derived from adult aLV or DG cultures, the aLV-derived neurons and glia are generated by a common precursor, whereas the DG-derived neurons and glia are derived from separate progenitors. Individual spheres or clumps were plated on MATRIGEL basement membrane matrix in 1% FBS for 7–8 DIV and then processed for immunocytochemistry. Scale bars, 50 μm. G, Neuronal and glial gene expression were confirmed using RT-PCR. RNA was isolated from differentiated aLV and DG sphere colonies. Primers were used to detect GFAP (150 bp) and NF-H (452 bp). Sphere colonies derived from subependymal tissue (aLV) generate both neuronal (NF-H) and glial (GFAP) progeny, whereas colonies derived from dentate gyrus cells (DG) generate differentiated progeny that express GFAP but do not express NF-H. Data are representative of at least three separate experiments.

Comparison of the sphere-forming ability of primary tissue from adult rat neurogenic regions and a clonal cell line of adult hippocampal progenitors in the presence and absence of growth factors (GF). The data are expressed as the mean (+SEM) number of spheres generated per 10,000 cells plated (i.e., per well; density = 20 cells per microliter). Cells were plated in either serum-free media (no GF) or serum-free media with EGF and FGF2 (GF). AHPs were compared with primary adult rat tissue (DG, dentate gyrus;aLV, anterior lateral ventricle). Resultant spheres were counted at 7 DIV. Note that both AHPs and primary aLV spheres (12 ± 2) were generated in no GF, whereas zero spheres arose from adult DG in the absence of growth factors (0.0 ± 0.0). A small number of DG spheres were generated in GF(1.5 ± 0.3).