Neural stem cells (NSCs) represent a unique type of precursor cells that are capable of self-renewal and differentiation into multiple neural cell types, including neurons and glia [1–3]. During early brain development, NSCs in the germinal region generate numerous progeny in a highly organized manner to construct the nervous system. Adult mammalian brains also harbour a population of adult NSCs that are primarily located in the subventricular zone of the lateral ventricle and the dentate gyrus of the hippocampus to maintain regional ongoing neurogenesis [4–7]. Advances in NSC biology have highlighted the promise of NSCs in stem cell-based therapies for neurological disorders [8–11]. Understanding molecular mechanisms regulating the behaviour of NSCs, including their proper expansion in vitro with multipotentiality but not tumorigenicity, is a critical step towards these goals.
As the defining hallmark of stem cells, self-renewal refers to the process by which stem cells expand to generate at least one of the two daughter cells with the same range of developmental potentials as its parental cell [12, 13]. Stem cell self-renewal is critical for both embryonic development and adult homeostatic tissue maintenance. In the mammalian brain, NSCs are subject to tight and complex regulation in different regions and at different stages of development. The earliest neuroepithilial NSCs, for example, self-renew and expand rapidly to produce a vast number of progeny in order to meet the need of brain histogenesis. Whereas most adult stem cells in vivo usually reside in a micro-environment (niche) and remain relatively quiescent , they engage in active self-renewal upon injury signals or under certain physiologic conditions that demand rapid production of new progeny. Due to the complex nature of self-renewal in vivo, stem cells in culture provide a better-defined system to investigate how self-renewal is controlled by intrinsic and extrinsic mechanisms.
Emerging evidence suggests that self-renewal is regulated by diverse mechanisms in different stem cells [13, 15]. In the case of NSCs, it has long been noted that cell expansion is promoted by the growth factor FGF-2, although little is known about the underlying cytoplasmic signalling mechanism [16–20]. NSCs isolated from different regions of the brain or different stages of development, grown as either "neurosphere" or adherent monolayer culture, all undergo robust proliferation when supplemented with FGF-2 in serum-free defined medium [21–25]. Self-renewal entails not only proliferation but also maintenance of the stem cell state. Cellular sub-cloning experiments showed that the clonal progeny of NSCs still preserved multipotentiality after expansion by FGF-2 [23, 26], and in vitro expanded adult NSCs retained multipotentiality in vivo even after serial transplantation . Genetic ablation of FGF-2 locus in mice resulted in severe defects in the maintenance of a slow-dividing stem cell pool, providing in vivo evidence that FGF-2 is necessary for normal NSC self-renewal . Interestingly, FGF-2 is present in normal adult NSC niches, can be induced by diverse types of pathological conditions, and is functionally capable of enhancing the inherently limited self-renewal of endogenous NSCs after ischemic stroke [29–35]. Under different biological contexts, FGF-2 may additionally act in coordination with many other types of extrinsic signalling molecules to exquisitely control adult NSC self-renewal in response to changes of cell physiological milieu, tissue homeostatic states and diverse environmental stimuli [5, 10, 36–41].
FGF-2 receptors (FGFRs) belong to the family of receptor tyrosine kinases [42, 43]. The ligand binding, which is facilitated by heparin, leads to dimerization and autophosphorylation of FGFRs. Consequently, various phosphorylated tyrosine residues on the receptor serve as docking sites for adaptor or enzymatic proteins that link the receptor to downstream intracellular signalling pathways. Previous studies have implicated multiple pathways downstream of FGFRs, including the canonical MAPK (Extracellular signal-regulated kinase, Erk1/2) and phospholipase C (PLC) signalling [42, 44]. However, it is unknown whether any of these pathways function in adult NSC self-renewal despite genetic evidence that has clearly implicated the role of FGFR1 in regulating adult NSC proliferation and neurogenesis [32, 45, 46]. Erk1/2 activation, for instance, has been shown to be important for myoblast proliferation, whereas its suppression promotes self-renewal of mouse embryonic stem cells [47, 48]. These findings suggest that signalling pathways are largely conserved, yet their effects are context-dependent . Thus, it is necessary to analyze the specific role of a given pathway in a particular cellular process.
In this study, we aim to gain molecular understanding on the role and mechanism of FGFR signalling in regulation of adult NSC self-renewal. Choosing the well-established rat hippocampal adult NSCs as our model system, we undertook multiple experimental strategies to assess whether specific FGFR signalling is sufficient to promote the self-renewal of adult NSCs, and further dissect out the functional requirement and cooperation of MAPK, PLC pathways in FGF-2-dependent self-renewal of adult NSCs.