Interplay between FGF2 and BMP controls the self-renewal, dormancy and differentiation of rat neural stem cells

Abstract

Results Establishment of rat foetal NS cell culturesWe followed the protocol established for foetal mouse tissue described in Conti et al. (Conti et al., 2005). Dissociated cells from E13.5 rat foetal cortex or spinal cord were plated onto laminincoated tissue culture plastic in medium supplemented with FGF2 and epidermal growth factor (EGF). Many cells attached within 24 hours. The primary cultures were heterogeneous; they contained nestin-positive precursor cells, TuJ1-positive neurons and a small Summary Mouse and human central nervous system progenitor cells can be propagated extensively ex vivo as stem cell lines. For the rat, however, in vitro expansion has proven to be problematic owing to proliferation arrest and differentiation. Here, we analyse the establishment, in adherent culture, of undifferentiated tripotent neural stem (NS) cell lines derived from rat foetal brain and spinal cord. Rat NS cells invariably undergo growth arrest and apparent differentiation after several passages; however, conditioned medium from proliferating cultures can overcome this block, enabling continuous propagation of undifferentiated rat NS cells. We found that dormancy is induced by autocrine production of bone morphogenetic proteins (BMPs). Accordingly, the BMP antagonist noggin can replace conditioned medium to sustain continuous self-renewal. Noggin can also induce dormant cells to re-enter the cell cycle, upon which they reacquire neurogenic potential. We further show that fibroblast growth factor 2 (FGF2) is required to suppress terminal astrocytic differentiation and maintain stem cell potency during dormancy. These findings highlight an extrinsic regulatory network, comprising BMPs, BMP antagonists and FGF2 signals, that governs the proliferation, dormancy and differentiation of rat NS cells and which can be manipulated to enable long-term clonogenic self-renewal.

Epigenomic enhancer annotation reveals a key role for NFIX in neural stem cell quiescence

“…Analysis of the gene expression profile of cell cycle-arrested NS cells showed that these cells are very significantly enriched for genes induced by fibroblasts and different types of adult tissue stem cells when they enter quiescence, thus suggesting that different cell types, including NS cells, employ overlapping quiescence gene expression programs. In contrast, these cells were not significantly similar to parenchymal astrocytes, in agreement with a recent report showing that while BMP signaling alone promotes terminal astrocytic differentiation, exposure to both BMP and FGF2 maintains the stem cell character of NS cells (Sun et al 2011). …”

“…BMP signaling promotes quiescence in not only NS cell cultures (Mira et al 2010;Sun et al 2011; this study), but also the adult hippocampus, where perturbation of BMP signaling results in excessive proliferation and eventual depletion of hippocampal stem cells (Mira et al 2010). BMPs have also been implicated in the quiescent state of other types of adult stem cells, including hair follicle, intestinal, and hematopoietic stem cells (Kobielak et al 2007;Li and Clevers 2010;Lien et al 2011).…”

“…To model NSC quiescence in culture, we replaced the mitogen EGF with BMP4 in the culture medium of NS cells, which also contains FGF2 (Conti et al 2005;Mira et al 2010;Sun et al 2011). We monitored cell proliferation by staining for the proliferation marker Ki67 and measuring incorporation of the thymidine analog EdU.…”

“…NSCs are routinely maintained in culture (NS cell cultures) in the presence of high concentrations of mitogens and are highly proliferative (Pastrana et al 2009;Ehm et al 2010;Mira et al 2010;Sun et al 2011). However, BMP ligands have recently been shown to promote cell cycle arrest in adherent cultures of mouse and rat NS cells (Mira et al 2010;Sun et al 2011).…”

Characterization of the neural stem cell gene regulatory network identifies OLIG2 as a multifunctional regulator of self-renewal

“…To address this question, we treated NS cells with three different growth factor regimes to induce cell cycle exit and stimulate differentiation or quiescence, and we used microarrays to detect significantly up-and down-regulated genes. We used BMP4 to induce astrocytic differentiation (Sun et al 2011a), treatment with B27-and FGF2-containing medium to induce an early neuronal progenitor fate (Spiliotopoulos et al 2009), and BMP4 + FGF2 to induce NS cell quiescence (Sun et al 2011a;Martynoga et al 2013). In all three experiments, 1340-2054 genes were significantly down-regulated and 1442-2054 genes were up-regulated, showing the extent of transcriptional rewiring.…”