Susan K. Goderie

Adult SVZ stem cells lie in a vascular niche: A quantitative analysis of niche cell-cell interactions

There is an emerging understanding of the importance of the vascular system within stem cell niches. Here, we examine whether neural stem cells (NSCs) in the adult subventricular zone (SVZ) lie close to blood vessels, using three-dimensional whole mounts, confocal microscopy, and automated computer-based image quantification. We found that the SVZ contains a rich plexus of blood vessels that snake along and within neuroblast chains. Cells expressing stem cell markers, including GFAP, and proliferation markers are closely apposed to the laminin-containing extracellular matrix (ECM) surrounding vascular endothelial cells. Apical GFAP+ cells are admixed within the ependymal layer and some span between the ventricle and blood vessels, occupying a specialized microenvironment. Adult SVZ progenitor cells express the laminin receptor alpha6beta1 integrin, and blocking this inhibits their adhesion to endothelial cells, altering their position and proliferation in vivo, indicating that it plays a functional role in binding SVZ stem cells within the vascular niche.

Timing of CNS Cell Generation: A Programmed Sequence of Neuron and Glial Cell Production from Isolated Murine Cortical Stem Cells

Multipotent stem cells that generate both neurons and glia are widespread components of the early neuroepithelium. During CNS development, neurogenesis largely precedes gliogenesis: how is this timing achieved? Using clonal cell culture combined with long-term time-lapse video microscopy, we show that isolated stem cells from the embryonic mouse cerebral cortex exhibit a distinct order of cell-type production: neuroblasts first and glioblasts later. This is accompanied by changes in their capacity to make neurons versus glia and in their response to the mitogen EGF. Hence, multipotent stem cells alter their properties over time and undergo distinct phases of development that play a key role in scheduling production of diverse CNS cells.

Swelling-induced release of glutamate, aspartate, and taurine from astrocyte cultures

Swelling of primary astrocyte cultures by exposing them to hypotonic media caused release of label after the cells had been allowed to accumulate 3H-L-glutamate, 3H-D-aspartate, or 3H-taurine. Comparable release of endogenous L-glutamate or taurine, as measured by high- pressure liquid chromatography (HPLC), was also found. Release of label was not affected by treating the cells with cytochalasin B, indicating that microfilament polymerization was not significantly involved. Hypotonic-induced release did not appear to principally involve reversal of the Na(+)-dependent uptake system since increasing external K+ to depolarize the cells by replacement of external Na+, thus maintaining isotonic conditions, increased release to a lesser extent. Threo beta-hydroxyaspartate, a potent 3H-L-glutamate uptake blocker, added externally stimulated efflux of 3H-L-glutamate independently of the swelling-induced efflux. Upon restoration of swollen cells to isotonic medium they showed an unimpaired ability to take up 3H-L- glutamate. The swelling-induced release of label was inhibited by a number of anion transport inhibitors, one of which has been shown to significantly improve outcome in an experimental brain trauma/hypoxia model in which astrocyte swelling is an early event.

FGF2 Concentration Regulates the Generation of Neurons and Glia from Multipotent Cortical Stem Cells

The embryonic cerebral cortex contains a population of stem-like founder cells capable of generating large, mixed clones of neurons and glia in vitro. We report that the default state of early cortical stem cells is neuronal, and that stem cells are heterogeneous in the number of neurons that they generate. In low fibroblast growth factor (FGF2) concentrations, most maintain this specification, generating solely neuronal progeny. Oligodendroglial production within these clones is stimulated by a higher, threshold level of FGF2, and astrocyte production requires additional environmental factors. Because most cortical neurons are born before glia in vivo, these data support a model in which the scheduled production of cortical cells involves an intrinsic neuronal program in the early stem cells and exposure to environmental, glia-inducing signals.

Adult SVZ Lineage Cells Home to and Leave the Vascular Niche via Differential Responses to SDF1/CXCR4 Signaling

Neural progenitor cells (NPCs) in the adult subventricular zone (SVZ) are associated with ependymal and vasculature niches, which regulate stem cell self-renewal and differentiation. Activated Type B stem cells and their progeny, the transit-amplifying type C cells, which express EGFR, are most highly associated with vascular cells, indicating that this niche supports lineage progression. Here, we show that proliferative SVZ progenitor cells home to endothelial cells in a stromal-derived factor 1 (SDF1)- and CXC chemokine receptor 4 (CXCR4)-dependent manner. We show that SDF1 strongly upregulates EGFR and alpha6 integrin in activated type B and type C cells, enhancing their activated state and their ability to bind laminin in the vascular niche. SDF1 increases the motility of type A neuroblasts, which migrate from the SVZ toward the olfactory bulb. Thus, differential responses to SDF1 can regulate progenitor cell occupancy of and exit from the adult SVZ vascular niche.

Multipotent Stem Cells from the Mouse Basal Forebrain Contribute GABAergic Neurons and Oligodendrocytes to the Cerebral Cortex during Embryogenesis

During CNS development, cell migrations play an important role, adding to the cellular complexity of different regions. Earlier studies have shown a robust migration of cells from basal forebrain into the overlying dorsal forebrain during the embryonic period. These immigrant cells include GABAergic neurons that populate the cerebral cortex and hippocampus. In this study we have examined the fate of other basal forebrain cells that migrate into the dorsal forebrain, identifying basal cells using an antibody that recognizes both early (dlx1/2) and late (dlx 5/6) members of the dlx homeobox gene family. We found that a subpopulation of cortical and hippocampal oligodendrocytes are also ventral-derived. We traced the origin of these cells to basal multipotent stem cells capable of generating both GABAergic neurons and oligodendrocytes. A clonal analysis showed that basal forebrain stem cells produce significantly more GABAergic neurons than dorsal forebrain stem cells from the same embryonic age. Moreover, stem cell clones from basal forebrain are significantly more likely to contain both GABAergic neurons and oligodendrocytes than those from dorsal. This indicates that forebrain stem cells are regionally specified. Whereas dlx expression was not detected within basal stem cells growing in culture, these cells produced dlx-positive products that are capable of migration. These data indicate that the developing cerebral cortex incorporates both neuronal and glial products of basal forebrain and suggest that these immigrant cells arise from a common progenitor, a dlx-negative basal forebrain stem cell.

Asymmetric Distribution of EGFR Receptor during Mitosis Generates Diverse CNS Progenitor Cells

It has been debated whether asymmetric distribution of cell surface receptors during mitosis could generate asymmetric cell divisions by yielding daughters with different environmental responsiveness and, thus, different fates. We have found that in mouse embryonic forebrain ventricular and subventricular zones, the EGFR can distribute asymmetrically during mitosis in vivo and in vitro. This occurs during divisions yielding two Nestin+ progenitor cells, via an actin-dependent mechanism. The resulting sibling progenitor cells respond differently to EGFR ligand in terms of migration and proliferation. Moreover, they express different phenotypic markers: the EGFRhigh daughter usually has radial glial/astrocytic markers, while its EGFRlow sister lacks them, indicating fate divergence. Lineage trees of cultured cortical glioblasts reveal repeated EGFR asymmetric distribution, and asymmetric divisions underlie formation of oligodendrocytes and astrocytes in clones. These data suggest that asymmetric EGFR distribution contributes to forebrain development by creating progenitors with different proliferative, migratory, and differentiation responses to ligand.

Astrocytic Swelling Due to Hypotonic or High K+ Medium Causes Inhibition of Glutamate and Aspartate Uptake and Increases Their Release

Astrocytic swelling occurs readily in ischemia and traumatic brain injury (TBI) as part of the cytotoxic or cellular edema response. Ischemia is known to produce large extracellular increases in both [K+] and excitatory amino acids (EAA) in vivo, and astrocytic swelling in vitro leads to marked release of EAA. In this study we compared the effect of swelling due to hypotonic media and high K+ medium on the uptake and release of EAA by rat primary astrocyte cultures in vitro. In both cases, there was a significant inhibition of uptake of [3H]l-glutamate and [3H]d-aspartate, and increased release of preloaded [3H]d-aspartate. The kinetics of the increased efflux was very different in response to hypotonic or high K+ media. In hypotonic medium there was a rapid initial release followed by a decline in the rate of release over time. This release was independent of whether Na+ was present. Upon exposure to high K+ medium there was a slow progressive increase in release of [3H]d-aspartate, which never showed any subsequent decline until the media was returned to normal [K+]. In high K+ media there was also an initial transient increase in [3H]d-aspartate release, which we attribute to reversal of the amino acid uptake system. The increased release due to hypotonic medium was not affected by a drop in temperature from 37 to 26°C, while the increased release due to high K+ medium was completely inhibited. The decreased uptake and increased release of EAA when astrocytes swell will short-circuit a presumed important, protective uptake system for EAA and may contribute to the increased levels of extracellular EAA seen during ischemia, TBI, and other pathologic states.

Automated Cell Lineage Construction: A Rapid Method to Analyze Clonal Development Established with Murine Neural Progenitor Cells

Understanding cell lineage relationships is fundamental to understanding development, and can shed light on disease etiology and progression. We present a method for automated tracking of lineages of proliferative, migrating cells from a sequence of images. The method is applicable to image sequences gathered either in vitro or in vivo. Currently, generating lineage trees from progenitor cells over time is a tedious, manual process, which limits the number of cell measurements that can be practically analyzed. In contrast, the automated method is rapid and easily applied, and produces a wealth of measurements including the precise position, shape, cell-cell contacts, motility and ancestry of each cell in every frame, and accurate timings of critical events, e.g. mitosis and cell death. Furthermore, it automatically produces graphical output that is immediately accessible. Application to clonal development of mouse neural progenitor cells growing in cell culture reveals complex changes in cell cycle rates during neuron and glia production. The method enables a level of quantitative analysis of cell behavior over time that was previously infeasible.

Methylmercury uptake in rat primary astrocyte cultures: the role of the neutral amino acid transport system.

The significance of the dense labeling pattern of methylmercury (MeHg) over astrocytes in areas of damaged cortex remains obscure, and the extent to which individual neurons are altered by MeHg accumulation in astrocytes is unknown. As a first step in understanding the relationship between the astrocyte and the mechanisms of MeHgs neurotoxicity, studies were directed at how MeHg is transported into cultured astrocytes. Uptake of [203Hg]MeHg in primary astrocyte cultures from neonatal rat cerebral cortex following incubations with MeHgCl conformed to a simple diffusion process. Uptake of [203Hg]MeHg by astrocytes exhibited the kinetic criteria of a specific transport system when added to the media as the L-cysteine conjugate. Saturation kinetics, substrate specificity and inhibition, and trans-stimulation were demonstrated in the presence of this SH-containing amino acid. Cysteine-mediated uptake of MeHg was inhibited by the coadministration of L-methionine, and 2-aminobicyclo-[2,2,1]-heptane-2-carboxylic acid. 2-Methylaminoisobutyric acid was ineffective in inhibiting the uptake of the MeHg-cysteine conjugate. Preloading of the astrocytes with glutamate was moderately effective in trans-stimulating the uptake of MeHg-cysteine conjugates, while in the absence of cysteine, uptake of [203Hg]MeHg was unchanged. These results indicate the presence in astrocytes of a neutral amino acid carrier transport System L, capable of selectively mediating cysteine-MeHg uptake. The substrate specificity and high affinity of this transport system resemble the properties of the System L neutral amino acid transport across the blood-brain barrier in the rat. Cellular uptake of MeHg-cysteine conjugates was not inhibited by preincubation of astrocytes with 100 microM N-ethylmaleimide or NaF.(ABSTRACT TRUNCATED AT 250 WORDS)