Interaction between astrocytes and adult subventricular zone precursors stimulates neurogenesis - PubMed (original) (raw)

Interaction between astrocytes and adult subventricular zone precursors stimulates neurogenesis

D A Lim et al. Proc Natl Acad Sci U S A. 1999.

Abstract

Neurogenesis continues in the mammalian subventricular zone (SVZ) throughout life. However, the signaling and cell-cell interactions required for adult SVZ neurogenesis are not known. In vivo, migratory neuroblasts (type A cells) and putative precursors (type C cells) are in intimate contact with astrocytes (type B cells). Type B cells also contact each other. We reconstituted SVZ cell-cell interactions in a culture system free of serum or exogenous growth factors. Culturing dissociated postnatal or adult SVZ cells on astrocyte monolayers-but not other substrates-supported extensive neurogenesis similar to that observed in vivo. SVZ precursors proliferated rapidly on astrocytes to form colonies containing up to 100 type A neuroblasts. By fractionating the SVZ cell dissociates with differential adhesion to immobilized polylysine, we show that neuronal colony-forming precursors were concentrated in a fraction enriched for type B and C cells. Pure type A cells could migrate in chains but did not give rise to neuronal colonies. Because astrocyte-conditioned medium alone was not sufficient to support SVZ neurogenesis, direct cell-cell contact between astrocytes and SVZ neuronal precursors may be necessary for the production of type A cells.

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Figures

Figure 1

Figure 1

Astrocyte monolayers support the proliferation and differentiation of SVZ cells. (A and C) Differential interference contrast (DIC) images of typical colonies from postnatal SVZ precursors. (B and D) Epifluorescent images of the postnatal SVZ precursors depicted in A and C, respectively. (B) Colonies are immunopositive for the neuronal marker, neuron-specific tubulin (Tuj1; red), and have incorporated BrdUrd (green) between 4 and 5 days in vitro (DIV). (D) Colonies are also immunopositive for neuronal marker MAP-2. (E) Time course of SVZ neurogenesis. Tuj1+ cell density in coculture vs. culture on different substrates is reported at 2-day intervals. Error bars = SEM of triplicate cultures. Astrocyte monolayers alone did not produce Tuj1+ cells at any time point. (F) DIC image of a neuronal colony from the adult SVZ. (G) Respective epifluorescence showing BrdUrd incorporation (yellow-green) and Tuj1 immunoreactivity (red). All above cultures were photographed at 5 DIV. (Bars = 10 μm.) (Inset) Schematic of SVZ cellular architecture as seen in a coronal section. The ventricular cavity would be to the right of the ependymal cell layer (gray). Type A migratory neuroblasts (red) migrate through glial tubes formed by type B cells (blue). The direction of migration would be perpendicular to the plane of the page. Type C cells (green) also are in intimate contact with type B astrocytes. Type A, B2, and C are mitotic.

Figure 2

Figure 2

Fractionation of SVZ cells by differential adhesion yields populations of cells with distinct characteristics. (A–C) Fraction 1, type A cells. See Results and Fig. 3 for details. (A) DIC image of isolated type A cells. (B) Epifluorescent image showing Tuj1 staining (red). (C) Purified type A cells are PSA-NCAM+ (red) and can migrate in chains. (D) Aggregate of type A cells immediately after they were embedded in Matrigel. (E) Chain migration from aggregates after 6 h in culture. The culture in E was double-stained for Tuj1 (F) and GFAP (G). (H) A positive control for GFAP staining (green) in Matrigel culture. (I–K) Fraction 4, B/C cells. Most of the adherent cells had a flattened, spread, phase-dark appearance, and ≈30% of these cells were GFAP+ (see Fig. 3 Inset). Nuclei are counterstained with Hoechst 33258 (blue). (Bar = 10 μm.)

Figure 3

Figure 3

The four fractions of SVZ cells have different yields and abilities to generate Tuj1+ colonies. (A) Fractionation yield. Fraction 4 was collected after treatment with trypsin. (B) Equal numbers of cells from the different fractions in A were plated onto astrocyte monolayers. Cultures were fixed at 5 DIV. Clusters of more than four Tuj1+ cells were counted as colonies. Error bars = SEM of triplicate cultures. (Inset) Immunocytochemistry of fraction 1 (type A cells) and fraction 4 (type B/C cells) purity. For every SVZ fractionation, an aliquot of each fraction was plated for immunostaining for type A markers (Tuj1 and PSA-NCAM) and a type B marker (GFAP). Between 500 and 1,000 cells were counted in each fraction. Standard deviation is indicated in parentheses. Note that in fraction 4, almost half of the cells were immunonegative for all three markers; these are putative type C cells. There is no marker specific for type C cells at present.

Figure 4

Figure 4

Fraction 1 (type A cells) and fraction 4 (type B/C cells) in astrocyte coculture. (A) Time course of a cell–astrocyte coculture vs. culture on PDK. Error bars = SEM of triplicate cultures. (B) Tuj1+ cells stained with diaminobenzidine in a cell–astrocyte coculture at 5 DIV. (C) Time course of B/C cell–astrocyte coculture vs. culture on PDK. (D) Typical colony of Tuj1+ cells stained with diaminobenzidine in astrocyte coculture at 5 DIV. (E and F) Cocultures were exposed to BrdUrd from 2 to 4 DIV. (E) DIC image of a neuronal colony. (F) Same colony double-stained for Tuj1 (red) and BrdUrd (green). DIC (G) and epifluorescent (H) images of a neuronal colony stained for PSA-NCAM. (Bars = 10 μm.)

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