Immature neurons from CNS stem cells proliferate in response to platelet-derived growth factor

Abstract

Identifying external signals involved in the regulation of neural stem cell proliferation and differentiation is fundamental to the understanding of CNS development. In this study we show that platelet-derived growth factor (PDGF) can act as a mitogen for neural precursor cells. Multipotent stem cells from developing CNS can be maintained in a proliferative state under serum-free conditions in the presence of fibroblast growth factor-2 (FGF2) and induced to differentiate into neurons, astrocytes, and oligodendrocytes on withdrawal of the mitogen. PDGF has been suggested to play a role during the differentiation into neurons. We have investigated the effect of PDGF on cultured stem cells from embryonic rat cortex. The PDGF alpha-receptor is constantly expressed during differentiation of neural stem cells but is phosphorylated only after PDGF-AA treatment. In contrast, the PDGF beta-receptor is hardly detectable in uncommitted cells, but its expression increases during differentiation. We show that PDGF stimulation leads to c-fos induction, 5'-bromo-2'deoxyuridine incorporation, and an increase in the number of immature cells stained with antibodies to neuronal markers. Our findings suggest that PDGF acts as a mitogen in the early phase of stem cell differentiation to expand the pool of immature neurons.

Fig. 1.

Western blot analysis was used to study PDGF receptor expression and phosphorylation in CNS stem cells during differentiation. Proliferating cells (FGF2) were compared with differentiating cells after FGF2 withdrawal (No add.). All cell lysates were immunoprecipitated with a PDGF α-receptor antibody. For immunoprecipitation of the PDGF β-receptor, the supernatant from the α-receptor precipitation was further incubated with a PDGF β-receptor antibody. Anti-PDGF α-receptor antibody, anti-PDGF β-receptor antibody, or anti-phosphotyrosine antibody was used for immunoblotting. Lysates were made from cortical stem cells grown in the presence of FGF2, at the start of the experiment, and from cells grown 1, 2, 4, and 6 d after FGF2 withdrawal. Cells treated with PDGF-AA for 24 hr were included as a positive control for phosphorylated PDGF α-receptor.

Fig. 2.

Western blot analysis of PDGF receptor expression and phosphorylation in CNS stem cells after PDGF-AA stimulation. Immunoprecipitation of cell lysates was performed using antibodies to PDGF α-receptor and PDGF β-receptor. Anti-PDGF α-receptor antibody, anti-PDGF β-receptor antibody, or anti-phosphotyrosine antibody was used for immunoblotting. Lysates were made from cortical stem cells grown in the presence of FGF2 (at the start of the experiment), from cells treated continuously with PDGF-AA after the withdrawal of FGF2 (A), and from cells stimulated with a single dose of PDGF-AA at the time of FGF2 withdrawal (B).

Fig. 3.

Northern blot analysis of c-fosexpression in cortical stem cells after PDGF-AA stimulation. Cell cultures were incubated without FGF2 for 3 hr, stimulated with PDGF-AA, and harvested for RNA preparation after 10 min, 30 min, 1 hr, and 2 hr. The levels of RNA expression are given as the ratios of c-fos to GAPDH pixels.

Fig. 4.

BrdU labeling of PDGF-AA-stimulated cortical stem cells. Parallel stem cell cultures were untreated, treated once with PDGF-AA, or treated continuously with PDGF-AA for 2, 4, and 6 d. Before fixation the cells were exposed to BrdU for 14 hr. Incorporation of BrdU was detected using anti-BrdU antibodies. Stained cells (duplicate dishes) were counted (in seven parallel fields; 200× magnification) and plotted as the ratio of BrdU-positive cells to the total cell number. *** denotes p < 0.001.

Fig. 5.

Total cell number in cortical cultures after PDGF-AA treatment. FGF2-treated cells were harvested and counted at the start of the experiment. After FGF2 withdrawal, parallel stem cell cultures were untreated, treated with a single dose of PDGF-AA, or treated continuously with PDGF-AA for 2, 4, 6, and 8 d. The total cell number was measured using a Coulter Z1 cell counter. * denotesp < 0.05, ** p < 0.01, and *** p < 0.001.

Fig. 6.

Staining of PDGF-AA-treated cortical stem cells with neuronal markers. After fixation, the cells were stained with MAP2 antibodies. Control cells, grown in the presence of FGF2, were fixed at the start of the experiment (A). Cells were grown for 2 d (B–D), 4 d (E–G), or 6 d (H–J) without FGF2 in the absence or presence of PDGF. Parallel cultures were untreated (B, E, H), received a single dose of PDGF-AA (C, F,I), or PDGF-AA was added daily (D,G, J). The cells were photographed at 200× magnification.

Fig. 7.

MAP2 and BrdU double staining of PDGF-AA-treated cortical cells. Stem cell cultures were untreated (A,B) or treated with PDGF-AA (C,D) for 2 d after FGF2 withdrawal. The cells were exposed to BrdU for 14 hr, fixed, and stained with MAP2 antibodies (A, C). Defined fields of the culture dish were photographed using a computerized microscope. The cultures were subsequently stained for BrdU, and the same fields were photographed again (B, D).

Fig. 8.

Cell survival studies of PDGF-AA-treated cortical stem cells. A TUNEL assay was performed on cells grown for 2 and 4 d in the presence or absence (no addition, no add.) of PDGF-AA. Cells grown in the presence of FGF2 were included as a control. Cells going through apoptosis were counted using a fluorescence microscope and plotted as the ratio to total number of cells (A). *** denotes p < 0.001. Western blot analysis was used to study PKB/c-Akt phosphorylation in CNS stem cells after FGF2 and PDGF-AA stimulation compared with untreated cells (no add.) (B). Total cell lysates were used for immunoblotting with anti-PKB/c-Akt antibody (Akt) and anti-phosphorylated PKB/c-Akt antibody (p-Akt).