Stem cell factor stimulates neurogenesis in vitro and in vivo

Abstract

Cerebral ischemia stimulates neurogenesis in proliferative zones of the rodent forebrain. To identify the signaling factors involved, cerebral cortical cultures prepared from embryonic mouse brains were deprived of oxygen. Hypoxia increased bromodeoxyuridine (BrdU) incorporation into cells that expressed proliferation markers and immature neuronal markers and that lacked evidence of DNA damage or caspase-3 activation. Hypoxia-conditioned medium and stem cell factor (SCF), which was present in hypoxia-conditioned medium at increased levels, also stimulated BrdU incorporation into normoxic cultures. The SCF receptor, c-kit, was expressed in neuronal cultures and in neuroproliferative zones of the adult rat brain, and in vivo administration of SCF increased BrdU labeling of immature neurons in these regions. Cerebral hypoxia and ischemia may stimulate neurogenesis through trophic factors, including SCF.

Figure 1

Hypoxia increases BrdU incorporation in cerebral cortical cultures. (a) Cortical cultures were stained with Ab’s against the indicated markers and counterstained with the nuclear stain DAPI (blue). (b) The percentage of cells expressing each marker is shown, indicating that most cells at this stage in culture are immature neurons. (c) Cultures were treated with BrdU and deprived of oxygen for up to 24 hours, and BrdU was visualized by immunocytochemistry and quantified by cell counting. (d) The hypoxia-induced increase in BrdU incorporation was reduced by the cell cycle inhibitors aphidicolin, cytosine arabinoside, and hydroxyurea. Data shown are representative fields (a) or mean ± SEM, n = 3 (b–d).

Figure 2

Hypoxia induces BrdU incorporation into uninjured cells that coexpress markers of proliferating cells and of immature, but not mature, neurons. Cerebral cortical cultures were treated with BrdU, exposed to hypoxia for 8 hours, and labeled with an Ab against BrdU and another marker, and nuclei were counterstained with DAPI. Most cells incorporating BrdU showed no evidence of DNA damage as assayed by PANT labeling (a), Klenow labeling (b), or TUNEL (c), or of caspase activation measured with an Ab against the 17- to 20-kDa caspase-3 cleavage product (d). BrdU incorporation colocalized with the cell proliferation markers PCNA (e) and phospho-histone-H3 (f), with retroviral infectivity reported by a GFP-expressing vector (g), and with the “replication-licensing” protein CDC47 (h). As shown in g, not all pFB-hrGFP–infected cells incorporated BrdU, which may be related to differences in labeling efficiency between the two markers. BrdU incorporation also colocalized with nestin (i) and to a large extent with E-NCAM (j), but not with the mature neuronal markers NeuN (k) and MAP-2 (l). Data are representative fields from at least three experiments per row.

Figure 3

HCM transfers hypoxic stimulation of neurogenesis to normoxic cultures. Cerebral cortical cultures were exposed to normoxia or hypoxia for 8 hours, medium was removed, and normoxia-conditioned control medium (none), whole HCM (all), or HCM fractions were added to normoxic cultures, together with BrdU and, in some cases, pFB-hrGFP, for 24–72 hours (a). Incubation for 72 hours with HCM fractions of more than 30 kDa increased the number of cells showing BrdU incorporation and GFP fluorescence (b). HCM fractions of less than 30 kDa had no effect on BrdU or pFB-hrGFP labeling, but reduced cell viability at 24–72 hours as measured by MTT absorbance (c). To characterize BrdU-labeled cells, cultures were exposed to hypoxia for 8 hours, medium was removed, and HCM was added to normoxic cultures, together with BrdU, and in some cases, pFB-hrGFP (d) for 72 hours. Some cultures were also stained with Ab’s against nestin (e) or MAP-2 (f). BrdU incorporation colocalized with pFB-hrGFP infectivity and with nestin, but not MAP-2 immunostaining. Data are mean ± SEM, n = 3 (b), mean values that varied by less than 10%, n = 3 (c), or representative fields from three experiments (d–f).

Figure 4

Hypoxia induces expression of SCF and FGF-2 in vitro. Cerebral cortical cultures were exposed to hypoxia for up to 24 hours, and cellular expression of growth factors and cytokines was measured by Western blotting (a), which showed increased expression of SCF and FGF-2 at 4–24 hours. SCF expression was quantified by computer densitometry (b). SCF levels were also increased after the indicated periods of hypoxia (h) in culture supernatants (c). Enhanced expression of SCF and FGF-2 was due to transcriptional activation, because RT-PCR showed increases in SCF and FGF-2 mRNA levels (d). Data are representative blots from three experiments (a, c, d) or mean ± SEM, n = 3 (b).

Figure 5

SCF and FGF-2 stimulate BrdU incorporation in vitro. Treatment of normoxic cultures for 24 hours with SCF increased both the number of cells incorporating BrdU and the number of viable cells measured by MTT absorbance (a). FGF-2 also increased BrdU incorporation in these cultures (b), but the effects of maximally effective concentrations (10 ng/ml) of SCF and FGF-2 were not additive (c). Ab’s against SCF and FGF-2 each reduced the component of BrdU labeling stimulated by unfractionated HCM (all) by approximately 40%, but these effects were not additive (d). Anti-SCF and the combination of anti-SCF plus anti–FGF-2 (but not anti–FGF-2 alone) reduced BrdU labeling stimulated by the 30- to 50-kDa fraction of HCM by approximately 75%, consistent with the preferential localization of immunoreactive SCF to this fraction on Western blots. Anti–FGF-2 and the combination of anti–FGF-2 plus anti-SCF (but not anti-SCF alone) reduced BrdU labeling stimulated by the 10- to 30-kDa fraction of HCM by approximately 70%, consistent with the M_r value of 17.2 kDa for FGF-2. Data are mean ± SEM, n = 3, or representative blots from three experiments (inset to d). Asterisks in d indicate that the percentage of inhibition of BrdU labeling by a given Ab or combination of Ab’s is significantly different in that HCM fraction than in unfractionated HCM (P < 0.05; ANOVA and post hoc Student-Newman-Keuls tests).

Figure 6

The c-kit expression in vitro and in SVZ and SGZ of normal and ischemic rat brain in vivo. Normoxic cerebral cortical cultures (a) and cultures deprived of oxygen for 8 hours (b) were stained with Ab’s against c-kit and SCF and with DAPI. Brain sections through SVZ (c–f) and SGZ of DG (g–j) were also immunostained with an Ab against c-kit, which was visualized with DAB. c-kit was expressed in SVZ (c and e) and SGZ (g and i) of normal brain (Con), and expression was increased 24 hours after MCA occlusion (Isch) in the ipsilateral SVZ (d and f) and SGZ (h and j). (c, d, g, and h) ×200. (e, f, i, and j) ×400 (HP). Data are representative fields from at least three experiments per panel.

Figure 7

Intraventricular SCF stimulates BrdU incorporation in SGZ and SVZ in vivo. (a) Brain sections through SGZ and SVZ were immunostained with anti-BrdU Ab 1 week after intraventricular infusion of SCF or aCSF vehicle (n = 6). Compared with aCSF (control), SCF increased the number of BrdU-positive cells in SGZ and SVZ. Proliferation was more pronounced on the cannula side, as compared with the contralateral side. (b) BrdU-labeled cells in SGZ (top) and SVZ (bottom) were counted in control brain (n = 6) and both ipsilateral (cannula side) and contralateral to SCF infusion (n = 6). In some experiments, SCF was infused together with anti–c-kit Ab (n = 4). Bars (left to right) represent control; SCF-treated, cannula side; SCF-treated, control side; and SCF- and anti–c-kit-treated, cannula side. BrdU-positive cells were increased in SVZ on both the infused and contralateral sides and in SGZ on the infused side.The effect of SCF was partially blocked by coadministration of anti–c-kit Ab (*P < 0.05, Student t test). (c) Rat brain sections through SGZ, obtained 1 week after SCF infusion, were double-labeled for BrdU (red) and NeuN or Neuro D (green). Merged images show that BrdU labeling colocalized with Neuro D and, in some cases, NeuN. Data shown are representative fields from the number of experiments given above (a and c), or mean ± SEM (n = 3) (b).