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. 1999 May 1;19(9):3287-97.
doi: 10.1523/JNEUROSCI.19-09-03287.1999.

Epidermal and Fibroblast Growth Factors Behave as Mitogenic Regulators for a Single Multipotent Stem Cell-Like Population From the Subventricular Region of the Adult Mouse Forebrain

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Epidermal and Fibroblast Growth Factors Behave as Mitogenic Regulators for a Single Multipotent Stem Cell-Like Population From the Subventricular Region of the Adult Mouse Forebrain

A Gritti et al. J Neurosci. .
Free PMC article

Abstract

The subventricular zone (SVZ) of the adult mammalian forebrain contains kinetically distinct precursor populations that contribute new neurons to the olfactory bulb. Because among forebrain precursors there are stem-like cells that can be cultured in the presence of mitogens such as epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2), we asked whether distinct subsets of stem-like cells coexist within the SVZ or whether the proliferation of a single type of SVZ stem-like cell is controlled by several GFs. We show that the latter is the case. Thus cells isolated from the SVZ coexpress the EGF and FGF receptors; by quantitative analysis, the number of stem-like cells isolated from the SVZ by either FGF2 or EGF is the same, whereas no additive effect occurs when these factors are used together. Furthermore, short-term administration of high-dose [3H]thymidine in vivo depletes both the EGF- and FGF2-responsive stem-like cell populations equally, showing they possess closely similar proliferation kinetics and likely belong to the constitutively proliferating SVZ compartment. By subcloning and population analysis, we demonstrate that responsiveness to more than one GF endows SVZ cells with an essential stem cell feature, the ability to vary self-renewal, that was until now undocumented in CNS stem-like cells. The multipotent stem cell-like population that expands slowly in the presence of FGF2 in culture switches to a faster growth mode when exposed to EGF alone and expands even faster when exposed to both GFs together. Analogous responses are observed when the GFs are used in the reverse order, and furthermore, these growth rate modifications are fully reversible.

Figures

Fig. 1.
Fig. 1.
Cells from mouse SVZ express both EGF and FGF type-1 receptors. A, EGFR and FGFR1 transcripts detected in SVZ explants. Bands of the expected size are visible in the ethidium bromide-stained gel after RT-PCR on RNA extracts. L, Ladder; lane 1, EGFR, 320 bp; lane 2, FGFR1, 500 bp. B, C, Cells from SVZ explants processed for double immunofluorescence labeling using antibodies against the FGFR1 (B) and EGFR (C) 1 hr after tissue dissociation. Virtually all the SVZ cells contain the FGFR1 (B), whereas only a subset displays IR to both receptors (B,C, arrows). Although cells expressing only FGFR1 were observed (B, C,arrowheads), cells displaying only EGFR were never seen. Scale bar, 20 μm.
Fig. 2.
Fig. 2.
EGF- and FGF2-responsive CNS stem-like cells derive from the same adult SVZ precursor population that constitutively proliferates in vivo. Cells dissociated from SVZ tissue and from striatal tissue (excluding the SVZ) were plated in the presence of either EGF or FGF2 at 20 ng/ml. To determine proportions of single cells, doublets, and triplets in the dissociated cells, we plated the cells onto coverslips and labeled the cells with the fluorescent nuclear-staining DAPI. A, The percentages of single cells, doublets, and triplets of all cells in culture are shown, determined soon after plating and 48 hr later. These data show that the vast majority (99%) of the cells that will eventually give rise to spheres in these cultures are single cells. B, Because the vast majority of spheres are formed from single cells, we were justified in using the number of spheres per well formed after 8–10 DIV as a measure of the number of stem-like cells (see also Gritti et al., 1996). Spheres were generated exclusively from dissociated SVZ explants. Closely similar numbers of spheres were formed in response to EGF and FGF2. Data are the mean ± SE of four independent experiments in triplicate. C, Freshly dissociated SVZ cells were plated in the presence of either EGF or FGF2 at 20 ng/ml or both. The cell populations that give rise to EGF- and FGF2-responsive cells are the same, because no increase in the number of clonal spheres per well was observed in the presence of both GFs compared with cultures exposed to either EGF or FGF2. Data are the mean ± SE of four independent experiments in triplicate. D, The number of spheres per well was assessed in SVZ cultures established from animals injected with high doses of [3H]thymidine or saline (control) over 12 hr. [3H]thymidine caused a significant and equal decrease in the numbers of spheres generated in the presence of EGF and FGF2, showing that the SVZ precursors from which these spheres derive possess closely similar proliferation kinetics in vivo. Data are the mean ± SE of three independent experiments in quadruplicate (p < 0.01 vs saline, Student’s t test).
Fig. 3.
Fig. 3.
SVZ stem-like cells retain simultaneous expression of EGFR and FGFR1 after long-term subculturing and expansion by means of either EGF or FGF2. SVZ stem-like cell cultures serially subcultured for at least 2 months with either EGF or FGF2 contain mRNA transcripts of both EGFR and FGFR1. A, After RT-PCR on RNA extract from SVZ cultures grown in EGF, the transcripts of EGFR (lane 1) and FGFR1 (lane 2) are present. Both receptor transcripts are also present in cells cultured with FGF2 (lane 3, EGFR; lane 4, FGFR1). L, Ladder. B–E, Cells coexpressing EGFR (B,D) and FGFR1 (C, E) are detected by double immunofluorescence assay on cells serially subcultured in the presence of either EGF (B,C, arrows) or FGF2 (D,E, arrows). As in primary SVZ cultures, cells bearing FGFR1 but not EGFR were found in EGF- (B,C, arrowheads) and FGF2-cultured SVZ cells (D, E, arrowheads); cells displaying only EGFR-IR were never detected. Scale bar,BE, 25 μm.
Fig. 4.
Fig. 4.
EGF and FGF2 are interchangeable mitogenic regulators for the same SVZ stem-like cell population in culture. Cell growth curves were obtained by assessing the total cell number after each subculturing step, under the various growth conditions analyzed. The rate of doubling (reciprocal of the doubling time; seeinsets) ± SE for the growth curves was calculated after cell growth had stabilized under the new conditions (inbold in the insets) after each GF switch. The data best fitted the equation: y =a · 2sx, wherey is the total number of cells, x is the time (DIV), s is the rate of doubling, anda is a constant. A, After serial subculturing in the continuous presence of FGF2, stem-like cells were dissociated, with one-half replated (arrow) in the presence of EGF (FGF2EGF; open circles) and one-half replated in the presence of FGF2 (FGF2; open squares) for further serial subculturing. FGF2-responsive stem-like cells continued to proliferate and expand in number in the presence of EGF, faster than they did in response to FGF2 (see inset). After serial subculturing in EGF, stem-like cells were replated in FGF2 medium (arrowhead), in which they resumed their slow growth mode (see inset;FGF2EGFFGF2;filled squares). B, Stem-like cells serially subcultured in the presence of EGF (open circles) continued to grow and expand when exposed to FGF2 (arrow; EGFFGF2;open squares). The cells proliferated at a slower rate in FGF2 than in EGF. However, the faster growth mode was restored when the cells were returned to EGF medium (arrowhead;EGFFGF2EGF;filled circles; see inset). At various times, cells underwent clonal analysis to confirm the retention of multipotentiality (A, B,asterisks; examples shown in Figs. 6, 7). The data are from one of three representative experiments yielding closely similar results.
Fig. 5.
Fig. 5.
The expansion rate of the stem-like cell population is faster in the presence of EGF and FGF2 together than in the presence of either GF alone. Cell growth curves were obtained by assessing the total cell number after each subculturing step, under the various growth conditions. The rate of doubling (reciprocal of the doubling time; see insets) ± SE for the growth curves was calculated after cell growth had stabilized under the new conditions (in bold in the insets) after each GF switch. The data were best fitted to the following equation:y = a · 2sx, where y is the total number of cells, x is the time (DIV), sis the rate of doubling, and a is a constant.A, B, Cells isolated and serially passaged in the presence of EGF (A, open circles) or FGF2 (B, open squares) were dissociated and replated either in the presence of the initial GF or with both EGF and FGF2 (A,B, arrows). The expansion rate is much higher in the presence of both GFs, regardless of whether the cultures were established by EGF or FGF2 (EGF orFGF2EGF+FGF2; filled triangles). The original expansion rate was resumed when the cultures were replated in medium containing the original GF (A, arrowhead;EGFEGF+FGF2EGF;filled circles; B,arrowhead;FGF2FGF2+EGFFGF2;filled squares; see insets for rates of doubling). At various times cells underwent clonal analysis to confirm retention of multipotentiality (A, B,asterisks; examples shown in Figs. 6, 7). The data are from one of three experiments yielding closely similar results.
Fig. 6.
Fig. 6.
SVZ stem-like cells retain multipotentiality after multiple sequential changes in culturing conditions: FGF2→EGF→FGF2. After each GF switch, samples were taken from the culture (indicated byasterisks in Figs. 4, 5) and used to establish clonal spheres. In this case, the culture was initially established with FGF2, grown for ∼1 month in EGF, then returned to FGF2, and cultured for ∼2 months in this GF. Single cells were transferred to single wells (1 cell/well) by micromanipulation and followed by time-lapse microphotography. A mark was incised on the vessel to mark the field containing the cell. The progeny of a single cell was plated onto glass coverslips and allowed to differentiate for 5 DIV by removal of growth factors. The cells retained multipotentiality under all the growth conditions exemplified in Figures 4 and 5. A,B, The single cell shown in A formed a clone by 10 DIV (B). C–E, Progeny of this cell included neuronal (MAP2-IR; C,arrow), astroglial (GFAP-IR; D,arrowhead), and oligodendroglial (O4-IR;E) type cells. Scale bars: A,B, 25 μm; CE, 10 μm.
Fig. 7.
Fig. 7.
SVZ stem-like cells retain multipotentiality after multiple sequential changes in culturing conditions: EGF→FGF2→EGF. After each GF switch, samples were taken from the culture (indicated by asterisks in Figs. 4, 5) and used to establish clonal spheres. In this case, the culture was initially established in EGF, grown in the presence of FGF2 for almost 60 d, and returned to EGF for 2 months. Single cells were transferred to single wells (1 cell/well) by micromanipulation and followed by time-lapse microphotography. A mark was incised on the vessel to mark the field containing the cell. The progeny of a single cell was plated onto glass coverslips and allowed to differentiate for 5 DIV by removal of growth factors. The cells retained multipotentiality under all the growth conditions exemplified in Figures 4 and 5. A,B, The single cell shown in A formed a clone by 7 DIV (B). C–E, Progeny of this cell included neuronal (MAP2-IR; C,arrows), astroglial (GFAP-IR; D,arrowhead), and oligodendroglial (O4-IR;E) type cells. Scale bars: A,B, 12 μm; CE, 10 μm.

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