Neural Stem Cells and Their Manipulation

Prithi Rajan; Evan Snyder

Introduction

Neural stem cells (NSCs) may be isolated from embryonic and adult brains, and are defined by their dual properties of self-renewal and their capacity to differentiate into the fates characteristic of the adult nervous system. The resident population of NSCs in the developing brain peaks before embryonic days 12–14 (E12–E14) in the rat, and gradually diminishes because of differentiation into neurons initially, followed by astrocytes and then oligodendrocytes. Neurogenesis is maximal around E14, followed by gliogenesis, which peaks around E19 (Caviness et al., 1995; Frederiksen and McKay, 1988). Neuronal architecture and glial differentiation continue to occur postnatally, especially synaptic pruning and myelination. NSCs are isolated with ease from all areas of the embryonic central nervous system, including cerebral cortex, hippocampus, striatum, mid-brain including the substantia nigra, cerebellum, and spinal cord. In the adult there are structural zones to which these cells are restricted; this is discussed in relative detail below. NSCs have also been isolated from the neural crest and retina, and have been used as biological platforms to study the mechanisms of regulation of proliferation, survival, and differentiation. Neural crest stem cells (NCSCs) are isolated from chick, mouse, or rat neural tube explants and give rise to tissue derivatives of the neural crest including neurons and glia from the peripheral nervous system and smooth muscle (Shah and Anderson, 1997). Retinal stem cells have been isolated from the ciliary margin zone of the adult eyes of amphibians and fish, and have also been cultured from pigmented ciliary margin of mouse retinas (Moshiri et al., 2004; Tropepe et al., 2000).

The most obvious practical advantage of NSCs is their potential to be an unrestricted source of neurons for replacement therapies. In addition to these transplantation therapies other important uses of stem cells lie in the creation of platforms for drug discovery (Rajan et al., 2006). To make these processes more efficient by the successful manipulation of these cells, it is necessary to study the biology of NSCs in in vitro culture systems that are used to maintain and propagate them, and to determine the signaling pathways that control the proliferation, differentiation, and survival of these cells in culture. Although the population of NSCs in the adult brain is appreciably less than in the embryonic brain, NSCs exist in localized regions called niches. The biology of the adult NSCs within their niches also needs to be studied in detail for two reasons: in addition to replacement therapies in which cells from allogeneic donors may be transplanted, resident stem cells may also be mobilized in order to harness their therapeutic potential. The use of fetal and adult NSCs for transplantation therapies may also benefit by knowledge of the in vivo survival and differentiation requirements of these cells.

In this review we attempt to summarize the signals controlling some aspects of neural stem cell biology including the in vivo niches in which neural stem cells reside in the adult and during development in the embryo, and the signals that are known to orchestrate the proliferation, survival, and differentiation of NSCs in vitro. Inclusion of the details of each pathway is beyond the scope of this review, but may be found in several excellent reviews focusing on individual pathways (Bieberich, 2004; Blaise et al., 2005; Cross and Templeton, 2004; Kleber and Sommer, 2004; Louvi and Artavanis-Tsakonas, 2006; McMahon, 2000; Polster and Fiskum, 2004; Stupack, 2005; Sela- Donenfeld and Wilkinson, 2005). Depending on the laboratory in which these studies were performed, NSC cultures have been derived from rodent or human tissues, and cultured under conditions that vary in three-dimensional structure (monolayer or neurosphere), and culture additives [serum, B27, epidermal growth factor (EGF), or leukemia inhibitory factor (LIF)]. Admittedly, these parameters confer significant differences to the NSC cultures generated. For example, neurospheres, which are NSC cultures maintained in suspended balls of cells, generate cultures of vastly different local density and extracellular matrix composition when compared with NSCs generated in monolayers. In addition, NSC cultures prepared from tissue derived from identical brain regions but from different ages of embryo differ in their responses to growth factors, attesting to the idea that the response elicited from a cell by an impinging ligand is affected by the context in which the signal is received, which in turn is dictated by cell-intrinsic and extracellular cues. For these contextual reasons, only those results that have been obtained in mammalian, and preferably neural-related, systems are considered here. Finally, some conclusions have been drawn by overexpression of signaling proteins and transcription factors, which may lead to spurious effects. However, in this review we consider data generated using all these paradigms in addition to in vivo studies to generate a heuristic model that will undoubtedly undergo refinements as our understanding of stem cell biology progresses.

Adult Niches for Stem Cells In Vivo

The adult mammalian brain has two prominent zones wherein stem cells reside. Stem cells are known to exist in the subventricular zone (SVZ), which extends anatomically around the ventricle in the cerebral cortex. These stem cells participate in the rostral migratory stream (RMS) in rodents, generating interneurons in the olfactory bulb; however, there is little evidence for the presence of a migrating stream similar to the RMS in humans (Sanai et al., 2004). The second concentration of stem cells in the adult occurs in the subgranular zone (SGZ) in the hippocampus, from which neurons are generated in the dentate gyrus (Gage, 2000). Although the stem cells that reside in the SVZ were originally thought to comprise the layer of ependymal cells that line the ventricle (Johansson et al., 1999), the current consensus suggests instead that astrocytes serve as functional stem cells (Alvarez-Buylla et al., 2002), whereas the ependymal cells participate in regulating the niche that these cells occupy (Lim et al., 2000). The current model suggests that the niche comprises three types of cell: the stem cell/astrocyte, which contacts the basal lamina and is capable of self-renewal (B cell); the preneuronal cell (C cell); and the young migrating neuron (A cell). B cells express glial fibrillary acidic protein (GFAP), a mature astrocytic marker, whereas stage-specific embryonic antigen 1 (SSEA1), which is considered characteristic of rodent embryonic stem cells, is present on a subset of astrocytes in vivo (Alvarez-Buylla and Lim, 2004). C cells may also be considered stem cells and respond to EGF as a mitogen in vitro to give rise to NSC cultures. A similar hierarchy of cells is present in the SGZ in the adult hippocampus. In this case GFAP-positive astrocytes give rise to neuronal precursors, which mature into granule cells that populate the dentate gyrus.

In addition, there is some evidence for the presence of restricted progenitors, distributed in the white matter of the cerebral cortex, which have been designated white matter precursor cells (WMPCs) (Goldman and Sim, 2005). These are largely glial progenitors and less restricted multipotential progenitors that are scattered in the SVZ and throughout the parenchyma of the brain. WMPCs express platelet-derived growth factor receptor α(PDGFRα) and the A2B5 epitope and thus appear to be oligodendrocyte precursors (Scolding et al., 1998, 1999), but have the capacity to generate all neural phenotypes when cultured in vitro (Nunes et al., 2003). When sorted WMPCs were analyzed for their gene expression profiles they exhibited some characteristics of neural progenitor cells such as HES1, musashi, doublecortin, and MASH1 (Goldman and Sim, 2005). Surprisingly, about 4% of the dissociated white matter from human surgical biopsies was shown to comprise these A2B5-positive cells.

Although the biology by which the WMPCs are maintained in the brain remains to be elucidated, a picture is emerging of the SVZ and SGZ niches, mostly by in vivo transgenic and gene ablation studies. The participation of extracellular matrix (ECM), basal lamina, and blood vessels and the paracrine effects of the cells themselves are important...