Investigating radial glia in vitro

Progress in Neurobiology 83 (2007) 53–67 www.elsevier.com/locate/pneurobio

Investigating radial glia in vitro Steven M. Pollard a,*, Luciano Conti b,** b

a Wellcome Trust Centre for Stem Cell Research, University of Cambridge,Tennis Court Road, Cambridge CB2 1QR, United Kingdom Department of Pharmacological Sciences and Centre for Stem Cell Research, University of Milano, Via Balzaretti 9, 20133 Milano, Italy

Received 6 November 2006; received in revised form 25 January 2007; accepted 20 February 2007

Abstract During mammalian neurogenesis newly born neurons migrate radially along the extended bipolar process of cells termed radial glia. Our views of radial glia as a ‘static’ support/guide cell have changed over recent years. It is now clear that within the developing cortex, and possibly the entire central nervous system (CNS), radial glia actively divide, producing daughter cells that include both neurons and glia. A subset of forebrain radial glia may serve as the founders of adult forebrain neural stem cells and genetic disruption of normal radial glia function can result in tumorigenesis or congenital neurological disorders. Elucidating the cell intrinsic and environmental cues that regulate radial glia behaviour is therefore essential for a full understanding of mammalian CNS development and physiology. Here, we review those studies in which radial glia have been investigated in vitro following isolation from foetal tissues or differentiation of embryonic stem (ES) cells. We discuss how these approaches, together with an ability to expand radial glia-like neural stem (NS) cell lines, may offer unique opportunities in basic and applied neurobiology. # 2007 Elsevier Ltd. All rights reserved. Keywords: Radial glia; Neural stem cells; In vitro cell culture; Embryonic stem cells; EGF and FGF-2; Neurogenesis

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Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sources of radial glia . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Foetal central nervous system . . . . . . . . . . . . . . 2.2. Adult CNS and brain tumours . . . . . . . . . . . . . . 2.3. Embryonic stem cells . . . . . . . . . . . . . . . . . . . . Isolation and expansion of foetal radial glia in vitro. . . . 3.1. Strategies for isolating foetal radial glia . . . . . . . 3.2. Expanding foetal and adult radial glia-like cells . . Radial glia from ES cells . . . . . . . . . . . . . . . . . . . . . . 4.1. Differentiation of ES cells to radial glia . . . . . . . 4.2. Expansion of radial glia-like cells from ES cells . Benefits and applications of in vitro approaches . . . . . . 5.1. Model system for stem cell biology . . . . . . . . . . 5.2. Cell-based disease modelling and drug screening . 5.3. Cell replacement strategies . . . . . . . . . . . . . . . .

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Abbreviations: BLBP, brain lipid-binding protein; BMP, bone morphogenetic protein; CNS, central nervous system; DRG, dorsal root ganglia; EB, embryoid body; EC cells, embryonic carcinoma cells; EGF, epidermal growth factor; ES cells, embryonic stem cells; FACS, fluorescence-activated cell sorting; FGF, fibroblast growth factor; GABA, gamma-aminobutyric acid; GFAP, glial fibrillary acid protein; GFP, green fluorescent protein; GLAST, astrocyte-specific glutamate transporter; HD, Huntington’s disease; ICM, inner cell mass; LIF, leukemia inhibitor factor; NEP, neuroepithelial progenitor; PDGFRa, platelet derived growth factor receptor a; PNS, peripheral nervous system; RA, retinoic acid; RBP-1, retinol binding protein 1; Shh, sonic hedgehog; SVZ, sub ventricular zone * Corresponding author. Tel.: +44 1223 760281. ** Corresponding author. Tel.: +39 02 5031 8403; fax: +39 02 5031 8284. E-mail addresses: [email protected] (S.M. Pollard), [email protected] (L. Conti). 0301-0082/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.pneurobio.2007.02.008

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Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Overview Understanding how the mammalian brain is generated is one of the most exciting and daunting challenges facing scientists. Although extraordinarily complex, both in structure and function, scientists have made progress in uncovering some of the cellular changes and underlying biochemical mechanisms responsible for the construction of this organ (Jessell and Sanes, 2000). Studies in developmental biology using various model organisms, as well as recently completed genome sequencing projects, have revealed a remarkable functional and genetic conservation of transcriptional regulators and signalling pathways across the animal kingdom. We are therefore faced with the task of understanding how interactions between these intrinsic and extrinsic signals can co-ordinately regulate cell behaviour within the developing mammalian CNS such that the correct numbers and types of mature cells are generated at the right place and right time. Radial glia are highly abundant within the developing CNS. They have a dual function as a support for migrating neurons and as a progenitor population. In the following sections, we outline the key properties of radial glia and discuss studies in which they have been cultured in vitro. More detailed accounts of radial glia function in vivo can be found elsewhere, including this issue (Campbell and Gotz, 2002; Chanas-Sacre et al., 2000; Ever and Gaiano, 2005; Gotz, 2003; Hevner, 2006; Morest and Silver, 2003). In later sections, we discuss recent work, including our own, which suggests that cells with similarities to radial glia can be isolated from foetal tissues, or generated from embryonic stem cells, and then subsequently maintained indefinitely as tissue specific neural stem cell lines (Bibel et al., 2004; Conti et al., 2005; Liour and Yu, 2003; Pollard et al., 2006c). 2. Sources of radial glia In order to isolate radial glia in vitro it is first necessary to identify those tissues from which they could be derived. As mammalian radial glia are a transient foetal cell type, it might seem clear that the developing foetal CNS would be the only tissue from which these cells could be isolated. However, there is also evidence that cells with similarities to radial glia can be generated in adult tissues through activation or reprogramming events that occur following injury and disease or within the cell culture environment. Also, the ability to differentiate embryonic stem cells to neural lineages provides an alternative way to generate radial glia in unlimited numbers in vitro. We discuss each of these potential sources below. 2.1. Foetal central nervous system At early developmental stages in mammals a population of cells becomes specified to the neural lineage. The molecular

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basis of this neural induction has been studied extensively in many vertebrate model organisms and seems largely conserved across phyla. Induction of the neuroectoderm is promoted through antagonism of the bone morphogenetic protein (BMP) signalling pathway and in some species pro-neural fibroblast growth factor (FGF) signals (reviewed in: (Munoz-Sanjuan and Brivanlou, 2002; Stern, 2005; Weinstein and HemmatiBrivanlou, 1999). All neural cells of the mature CNS are descended, either directly or indirectly, from this neuroepithelial cell population. Here, we refer to these cells as progenitors rather than stem cells as continuous self-renewal has not been demonstrated for this population. Neuroepithelial progenitors (NEPs) undergo interkinetic nuclear migration, a process in which the nucleus oscillates between the apical and basal surfaces co-ordinately with cell cycle progression, leading to the formation of a pseudostratified epithelium (Sauer, 1935). The earliest known molecular marker specifically expressed within this population is the transcription factor Sox1 (Pevny et al., 1998). The related and functionally redundant proteins Sox2 and Sox3 are also expressed in these cells, but have broader roles in the development of non-neural tissues such as the epiblast and extraembryonic ectoderm (Avilion et al., 2003; Wood and Episkopou, 1999). The NEPs comprise the neural plate, which undergoes a striking morphogenetic movement that results in formation of the neural tube. Concurrent with these events, signalling molecules secreted by adjacent tissues, such as somite-derived retinoic acid (RA), BMP signals from overlying ectoderm, and notochord-derived sonic hedgehog (Shh), act on the neuroepithelium. These signals lead to activation of various classes of transcription factors, which together convey a positional ‘code’ establishing sub-regions with the CNS and specifying cells as distinct neuronal and glial subtypes (Briscoe and Ericson, 2001). It is at this point, approximately embryonic day 9.5–10.5 in mouse, and concurrent with the onset of neurogenesis, that a second morphologically and antigenically distinct cell type, termed radial glia, arises in the neuroepithelial tissue (Gotz and Huttner, 2005; Misson et al., 1988). Radial glia were originally named epithelial cells, spongioblasts, radial cells, or fetal ependymal cells, by investigators around the late 19th century (reviewed in: (Bentivoglio and Mazzarello, 1999; Rakic, 2003). These cells have a bipolar morphology, with one extension and broad endfoot sited at the luminal surface and a longer process extending in the opposite direction through to the basement membrane adjacent to the pia mater. Similar to NEPs, they also exhibit an ovoid cell body and have a nucleus situated in the ventricular zone, adjacent to the lumen that undergoes interkinetic nuclear migration. Ultrastructural studies performed using electron microscopy revealed that radial glia function as a substrate/guide upon which newly generated immature neurons migrate (Rakic, 1971a,b). Radial glia display

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astrocyte characteristics, such as electron lucent processes, abundant intermediate filaments, and glycogen granules condensed at their end-feet (Choi, 1981; Rakic, 1971a). They also express several astrocyte markers, such as astrocytespecific glutamate transporter (GLAST) (Shibata et al., 1997), brain lipid-binding protein (BLBP) (Feng et al., 1994), and glial fibrillary acid protein (GFAP) (Levitt and Rakic, 1980), although in rodents GFAP is not expressed by radial glia (Choi, 1981; Sancho-Tello et al., 1995). Radial glia are also immunoreactive to the RC2 and Vimentin antibodies (Houle and Fedoroff, 1983; Misson et al., 1988). Together these astrocytic features distinguish radial glia from their NEP ancestors. Recently, our views of radial glia function have been extended following evidence that they actively divide and are capable of generating neurons and astrocytes. A widely used neural stem/progenitor marker is the intermediate filament protein nestin, which was originally identified following expression cloning using the monoclonal antibody Rat-401 (Lendahl et al., 1990). This antibody was initially characterised as a marker of radial glia in the developing rat brain (Hockfield and McKay, 1985). Since cells cultured in vitro which displayed nestin immunoreactivity act as neuronal progenitors, these studies provide the earliest clue that radial glia could function as neuronal progenitors (Frederiksen and McKay, 1988). However, as nestin is also expressed by NEPs from E7.5 in the neural plate, these neurogenic cells could not be clearly assigned radial glia identity (Lothian and Lendahl, 1997). In vivo studies of adult songbird ventricular zone identified proliferation ‘hotspots’ associated with radial cells linked to neurogenic regions (Alvarez-Buylla et al., 1990), while in vivo fate mapping studies using retroviruses in chick and rat provided further data that radial glia might act as neural progenitors (Gray and Sanes, 1992; Halliday and Cepko, 1992). Despite these studies, the general view remained that a separate neurogenic progenitor or self-renewing stem cell population co-exists with radial glia in the ventricular zone. Since 2000, this view has been challenged by both in vitro and in vivo data that suggested radial glia are neurogenic progenitors (Hartfuss et al., 2001; Malatesta et al., 2000; Miyata et al., 2001; Noctor et al., 2001, 2002). In vitro isolation of radial glia by cell sorting using a transgenic GFP-reporter driven by the human GFAP (hGFAP) promoter (which is active in mouse radial glia unlike the endogenous murine promoter), and subsequent cell culture, revealed a neuronal differentiation capacity (see Section 2.2) (Malatesta et al., 2000). Moreover, within the developing cortex all dividing precursors have a radial glia phenotype, as revealed by both morphology and molecular markers (e.g. BLBP, GLAST, RC2 and Vimentin) (Hartfuss et al., 2001; Noctor et al., 2002). Crucially, in vivo experiments in which single cells were transduced with GFP-expressing retrovirus in utero revealed that radial glia and newborn neurons are clonally related (Noctor et al., 2001). To identify whether radial glia are used throughout the nervous system as neuronal progenitors, genetic fate mapping experiments have been carried out in which transgenic mouse strains are created that express the recombinase Cre under the

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control of the human GFAP promoter. This promoter drives expression in mouse radial glia, in contrast to the endogenous mouse GFAP promoter which is not active in radial glia. When using this promoter element only cortical neurons and not those in ventral regions were labelled following crossing of mice to a reporter strain activated by Cre excision (Malatesta et al., 2003). By contrast using a 1.6 kb fragment of the BLBP promoter Anthony et al. found that all neurons, regardless of their regional identity, derive from radial glia (Anthony et al., 2004). It is presently unclear whether the hGFAP promoter is activated too late in development to mark ventral radial glia, or the BLBP is ‘leaky’ and activated in earlier neuroepithelial cells. The discrepancy between these studies may also reflect the difficulty of defining when a ‘true’ radial glia phenotype emerges. Radial glia are not a single uniform cell population and are located not only within the cortex but throughout the developing brain and spinal cord. Region-specific expression of transcription factors in radial glia is likely to determine the fate of progeny (Kriegstein and Gotz, 2003). For example, dorsally within the cortex at E13.5 nearly all of the Pax6 positive cells co-express RC2, and these account for around half of the total cell population. By contrast, ventrally within the ganglionic eminence, Pax6-expressing cells represent only 5% of the total population (Gotz et al., 1998), with the majority of radial cells in this region expressing the retinol binding protein 1 (RBP-1). The presence or absence of Pax6 and RBP-1 may confer neuronal subtype identity to the radial glia progeny; glutamatergic projection neuron or GABAergic interneuron, respectively (Stoykova et al., 2000). Similar heterogeneity has been noted in the spinal cord where genes such as Pax7 and Nkx2.2 are expressed by radial glia within specific regions (Ogawa et al., 2005). Alongside the regional heterogeneity, there are also temporal changes in radial glia marker expression. This has been most clearly demonstrated for the markers RC2, BLBP and GLAST, which when analysed in primary dissociated cells by immunocytochemistry show a dynamic expression pattern within the mouse cortex though E12.5–E16.5 (Hartfuss et al., 2001). Radial glia cells may also regulate cell fate cell nonautonomously through the local release of molecules that drive specific neuronal differentiation. For example, radial glia within the mesencephalon, but not in other CNS regions, produce Wnt5a that helps in establishment of the midbrain dopaminergic phenotype (Castelo-Branco et al., 2006). Similarly, sonic hedgehog or retinoic acid released by radial glial cells of the ventrolateral telencephalon may regulate the generation of striatal projection neurons. A further functional consequence resulting from radial glia heterogeneity might be the formation of cellular boundaries at the junctions between distinct radial glia phenotypes. This may help prevent cell mixing, thereby maintaining discrete cellular compartments (Kriegstein and Gotz, 2003; Stoykova et al., 1997). Thus, it is likely that spatial and temporal differences in radial glia are generating the diversity of cellular phenotypes within the nervous system. As they are present through neurogenic phases of development in all regions of the CNS there is a

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wide window of opportunity to isolate these cells for in vitro studies. 2.2. Adult CNS and brain tumours Following the completion of cortical neurogenesis radial glia retract their radial processes and convert into multipolar astrocytes, and in rodents activate GFAP expression (Mori et al., 2005; Schmechel and Rakic, 1979). It may therefore seem unwise to suggest isolation of radial glia from the adult brain for in vitro studies. However, some specialised radial glia do persist into adulthood in certain regions of the CNS. There are also circumstances in which the radial glia phenotype may be wholly or partly re-acquired, such as following injury, reprogramming/dedifferentiation in vitro, or genetic/epigenetic disruptions that occur during tumorigenesis. Radial glia that persist into adulthood in mammalian species include Bergmann glia and Muller glia, which reside within adult cerebellum and retina, respectively. Bergmann glia provide a guide for migrating granule neurons during cerebellar development, and their ablation, through activation of a toxin specifically within GFAP expressing cells, results in granular neuron degeneration (Bellamy, 2006; Cui et al., 2001;). Interactions of Bergmann glia with immature neurons have been monitored in vitro using time-lapse video microscopy studies (Hatten, 1984; Hatten et al., 1984). In adult mouse brain Bergmann glia express the transcription factors Sox1 and Sox2, and may be expandable in vitro as stem cells following exposure to the growth factors epidermal growth factor (EGF) and FGF-2 (Klein et al., 2005; Sottile et al., 2006). Muller glia, located within the adult retina, have been shown to guide migration of microglia (Sanchez-Lopez et al., 2004), and conditions for isolating and culturing them have been established for rat (Hicks and Courtois, 1990). Similar to cortical radial glia, isolation and differentiation of Muller glia has revealed a latent neuronal differentiation capacity, which may be physiologically relevant in disease or injury conditions (Das et al., 2006). Radial glia are also present within the adult hippocampus. Here, a vimentin immunoreactive radial glia-like cell with astrocytic features, termed a type 1 cell, may function as a continuously self-renewing stem cell population (Cameron et al., 1993; Eckenhoff and Rakic, 1984; Kempermann et al., 2004; Rickmann et al., 1987; Seri et al., 2001). In non-mammalian vertebrates, such as fish and amphibians, radial glia persist in large numbers throughout the CNS (Zupanc and Clint, 2003). These cells may provide a stem cell population responsible for the continued CNS growth and regenerative capacity seen in such species. Such widespread neurogenesis does not occur within the adult mammalian brain. However, our views of the adult mammalian brain as a tissue devoid of stem cell activity have changed following studies in vivo and in vitro in the 1990s (reviewed in Gage, 2000). New neurons are continually generated in both mouse and human adult brain, and arise from stem cells located within both the hippocampus and the sub ventricular zone (SVZ) of the forebrain. Fate-mapping experiments in mice have shown that these SVZ stem cells (termed type B cells) arise from a

subpopulation of radial glia present within the developing striatum (Merkle et al., 2004). These radial glia-derived SVZ astrocytes express GFAP and platelet derived growth factor receptor a (PDGFRa) (Jackson et al., 2006), and may have characteristics intermediate between ‘normal’ astrocytes and radial glia (Liu et al., 2006). Therefore, NEPs, radial glia and adult SVZ astrocytes represent a continuous lineage exhibiting multipotent neural differentiation potential (Alvarez-Buylla et al., 2001; Merkle et al., 2004). Dedifferentiation of adult cells to a radial glial phenotype occurs in vitro following exposure of cells to EGF and FGF-2 (see Section 3.2). There are also reports of radial glia markers being reacquired in vivo following disease or injury in the adult such as in rats following spinal cord injury (Shibuya et al., 2002,2003). A further circumstance whereby cells with radial glia properties may be re-acquired in vivo is during tumorigenesis. Within the CNS a variety of primary tumours can develop which are usually classified according to histological criteria that identify the major cell type present (Greenberg et al., 1999). Gliomas are the most common type of adult brain tumour, and these can be further classified as astrocytomas, oligodendrogliomas or ependymomas, depending on the resemblance of tumour biopsies to astrocytes, oligodendrocytes or ependymal cells, respectively. The most common and lethal type of brain tumour in adults is glioblastoma multiforme (GBM)—also termed high grade astrocytoma which is highly malignant. In children the most common tumour arises within the cerebellum and is known as medulloblastoma. Recently, evidence in support of a cancer stem cell hypothesis has emerged for many of these solid tumours within the CNS. This hypothesis proposes the existence of a hierarchical rather than stochastic organisation of cell lineage in tumours, such that only a subpopulation of cells, termed ‘‘cancer stem cells’’, are responsible for tumour expansion (Singh et al., 2004a,b). Neural progenitor markers such as nestin and vimentin are expressed by cell populations within a wide range of different brain tumours. Singh and colleagues isolated a cancer stem cell population by a cell sorting procedure based on the CD133 antigen (also known as prominin). They showed that transplantation of one hundred CD133-positive cells into immunocompromised mice results in formation of a tumour with similar features to the donor tumour. By contrast, no tumours are produced when the CD133-negative population is transplanted, even when 105 cells were transplanted, indicating the presence of a cancer stem cell population in the CD133positive population (Singh et al., 2004a,b). Recognition that radial glia are the ancestors of adult forebrain SVZ stem cells and can act as neural stem cells in vitro (see Section 3.2) raises the prospect that this cell type may also represent the cancer stem cell population. Previously, it has been assumed that GFAP immunoreactivity observed in astrocytomas indicated an astrocytic cell state. However, GFAP could also indicate a radial glia or a specific adult SVZ astrocyte phenotype. A recent study has shown that PDGFRa, which is commonly upregulated in certain gliomas, is expressed by adult SVZ stem cells and its forced expression in this region results in abnormal cell proliferation (Jackson et al., 2006). It

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should be extremely informative to analyse glioma biopsies for markers specific to each of these cell types. In a recent report, Taylor and co-workers have identified a radial glia-like population (CD133+/Nestin+/RC2+/BLBP+) as the candidate stem cell fraction within ependymomas (Taylor et al., 2005). Deciphering the relationship between adult stem cells, radial glia and cancer stem cells will have important clinical implications, as the molecular events controlling self-renewal in the cancer stem cells represent a key therapeutic target. Indeed, the practical consequence of the cancer stem cell hypothesis is that effective therapies will need to eradicate or prevent the cancer stem cell from self-renewing. Therapies directed to progenies of the cancer stem cell may not be successful. 2.3. Embryonic stem cells ES cells are derived from the isolated inner cell mass (ICM) of blastocyst stage embryos. Remarkably, they can be clonally expanded in culture as pluripotent cell lines, retaining a capacity to differentiate into all cell types of the adult organism (Smith, 2001). The derivation of ES cells from human blastocysts has created considerable excitement as these may provide a route to generate unlimited quantities of human cell types and tissues for use in basic biology and medicine. A major goal in ES cell biology is to develop protocols that enable lineage specific control of differentiation. Over the past 10 years protocols have been developed for conversion of mouse and human ES cells to the neural lineage (reviewed in Stavridis and Smith, 2003). In vitro differentiation of ES cells provides a route to identifying mechanisms involved in the first stages of nervous system formation which are difficult to study in early mammalian embryos. Many laboratories are now investigating how we can control human ES cell differentiation so that desirable terminally differentiated cell types, such as dopaminergic neurons, can be routinely generated for trials in clinical applications. During neuronal differentiation, ES cells undergo progressive lineage restrictions similar to those observed during normal foetal development, and a range of distinct progenitors can be obtained (Xian and Gottlieb, 2001). Accordingly, ES cell neural differentiation protocols provide a means to isolate distinct neural precursor populations, such as neuroepithelial cells, radial glia and neuroblasts, and are a unique tool to study molecular and cellular events during those transitions between neuroepithelial-radial glia and radial glia-neuron. The generation of neural precursors in vitro from ES cells also has the advantage that unlimited amounts of material can be generated. The accessibility of the cell culture environment also permits direct microscopic inspection of cellular changes as well as providing a simple means to test the biological effects of soluble factors through their direct addition to the culture media. ES cells can also be readily engineered using gene targeting with a view to performing genetic loss- and gain-of-function studies with candidate gene regulators. We will discuss studies in which radial glia have been differentiated from ES cells later in this article.

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3. Isolation and expansion of foetal radial glia in vitro 3.1. Strategies for isolating foetal radial glia Initial studies of radial glia function focussed on monitoring of cells in vivo using histological and microscopic observation in order to determine their function. However, the availability of freshly isolated foetal radial glia cells in vitro provides a complementary approach to understanding these cells with significant experimental advantages. Studies carried out through the 1980s using glial progenitors from the rat optic nerve provide a clear example of how in vitro approaches can be used to successfully identify details of the biochemical mechanisms that regulate progenitor cell behaviour (Barres and Raff, 1994; Raff et al., 1983). Similar strategies should prove informative for the radial glia lineage. Radial glia can be obtained from dissociation of foetal CNS tissues. However, a major hurdle to subsequent in vitro characterisation of these cells is the heterogeneity of primary cultures, which comprise multiple immature and differentiated cell types, as well as distinct radial glia subtypes. The tools enabling clear identification of radial glia in mixed primary cultures came following the development of cell type specific monoclonal antibodies such as RC1. For example, RC1 and morphological criteria were used to identify radial glia in culture that differentiate to astrocytes over the course of 4 days in culture (Culican et al., 1990). Routine use of fluorescent activated cell sorting (FACS) technologies provides a means to isolate specific subpopulations of cells in mixed cultures. Isolation of radial glia cells using FACS relies on the existence of antibodies that bind specific cell surface markers. To date no such cell surface epitope specific to radial glia has been identified. However, some cell surface markers enabling enrichment have been identified, such as CD15 (also called SSEA1/LeX) or CD133 (Capela and Temple, 2006; Uchida et al., 2000). More recently an antibody, 473HD, which binds the chondroitin sulphate epitope has been shown to label radial glia, and can be used to enrich for them by immunopanning strategies (von Holst et al., 2006). Identification of additional cell surface markers that can be used instead of, or in combination with, these should enable isolation of radial glia or radial glia subpopulations. An alternative, although less flexible method, for isolating radial glia is to utilise mouse transgenic and gene targeting technologies. Reporter mice can be generated in which either a transgene or an endogenous radial glia promoter drive expression of reporters, either fluorescent or enzyme-based. Cells with activated reporter expression can then be isolated using FACS. Such a strategy was followed by Malatesta et al. who used a transgenic mouse line expressing the green fluorescent protein (GFP) under the human GFAP promoter (Malatesta et al., 2000). These mice were previously shown to exhibit transgene expression in astrocytes in the mature brain and radial glia within the ventricular zone of the developing cortex (Zhuo et al., 1997). Malatesta et al. found that in the E1418 cortex of hGFAP-eGFP mice, the transgene was expressed at high levels in cells that were radial glia by both morphology and

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antigenic properties. Isolation of these cells by FACS and their culture in vitro in basal media resulted in differentiated clones, predominantly with mixed composition (neurons and astroglia), with other clones failing to proliferate and producing a single neuron. This study provides the first clear data that radial glia have a neuronal differentiation capacity, at least in vitro. A similar approach was described by Anthony et al. that used transgenic mice expressing GFP under the regulation of the 1.6 kb BLBP promoter (Anthony et al., 2004). Analysis of these transgenic mice demonstrated that the onset and pattern of GFP expression in the developing forebrain is essentially identical to endogenous BLBP and that GFP expression is restricted to radial glia. FACS of radial glia cells from the brain of these BLBP-GFP mice led to the similar conclusion that radial glia can generate neurons—although in this study neurogenic radial glia were also identified in ventral tissues (Anthony et al., 2004). In summary, both cell surface markers and transgenic reporters provide a means to isolate or enrich for radial glia from primary cell cultures. 3.2. Expanding foetal and adult radial glia-like cells Primary cell culture provides a useful way to isolate and characterise radial glia cells isolated directly from neural tissues. However, an extension of these approaches would be the expansion of cells in vitro following exposure to mitogens. Immature cells that can be expanded indefinitely in vitro while retaining a multipotent differentiation capacity are stem cell lines, and such neural stem cells would offer a valuable tool for molecular and biochemical studies, and for human cells certain clinical applications (Gottlieb, 2002). The use of cell lines to model events of normal development became widespread in the early 1980s when the first tumorderived neural cell lines were derived. Among these were those derived from neuroblastomas, glioblastomas and the rat pheochromocytoma PC12 (reviewed in: (Gottlieb, 2002). PC12 cells have proven invaluable to model processes such as neurotransmitter release and signalling pathways, while the rat C6 glioma line may provide a model system to decipher essential mechanisms within cancer stem cells (Kondo et al., 2004). Noteworthy, a subclone of C6 (C6-R) has morphological similarities to radial glia (Friedlander et al., 1998). However, an inevitable limitation of tumor-derived cell lines is that they contain significant genetic and epigenetic alterations. As an alternative to study more physiologically relevant cells, a number of laboratories developed strategies that exploit forced transgenic expression of oncogenes to promote continuous cell division. Rodent and human cells produced by this approach show phenotypes closely resembling those of normal immature neural cells (Cattaneo and Conti, 1998). However these approaches also have the caveat that the genetic manipulations may disrupt normal biology. In 1992, Reynolds and Weiss made the key discovery that cells from foetal mouse CNS can continuously divide in suspension culture as floating aggregates of cells termed neurospheres following exposure to the growth factor EGF (Reynolds et al., 1992). Removal of exogenous EGF results in

differentiation to astrocyte, neuronal and oligodendrocyte lineages. A drawback of the neurosphere culture paradigm is that they are heterogeneous, and self-renewing stem cells also undergo a degree of commitment and differentiation which makes it difficult to draw firm conclusions from population based experiments e.g. transplantation or gene expression profiling studies (Singec et al., 2006; Suslov et al., 2002). Neurospheres can be generated from tissues throughout the foetal CNS, and at a variety of developmental stages (reviewed in Gage, 2000). Large numbers of radial glia are present in these primary dissociated cell populations. At the time of these studies a role for radial glia as a progenitor population was still unappreciated but they now represent a candidate for the stem cell that maintains self-renewal of long-term expanded neurospheres in vitro. Indeed cells expressing radial glia markers are present in large numbers within neurospheres, as determined by analysis of RC2, BLBP and GLAST immunoreactivity (Hartfuss et al., 2001). Recently, we have determined conditions that enable expansion of foetal mouse neural stem (NS) cells as homogeneous cultures in the absence of commitment, differentiation and death (Conti et al., 2005). The key conditions seem to be culture upon an adherent substrate and exposure to both EGF and FGF-2. Using these conditions cell lines can be generated either directly from primary dissociated neural tissue, or from long-term expanded neurospheres. Similar cell lines were also derived from human foetal forebrain, although their long-term expandability and subsequent neuronal differentiations have yet to be fully defined. Characterisation of marker genes reveals that all cells express the markers BLBP, RC2, GLAST, Vimentin, Nestin, and Sox2. Moreover, the cells undergo dynamic morphological changes, including nuclear migration reminiscent of radial glia, and can exhibit bipolar extensions with broad end-feet similar to radial glia (Conti et al., 2005). Analogous NS cell lines, identical in all key characteristics to those derived from foetal neural tissues, can be isolated from both short-term and long-term expanded neurospheres or directly from primary cell cultures using adherent conditions. We found that those radial glia-like cells present in neurospheres are likely the founders of NS cells and that established NS cells can readily generate neurospheres when cultured in suspension (Pollard, unpublished). These observations indicate that NS cells/radial glia cells are the neurosphere forming stem cells. In contrast to adherent cultures, stem cells constitute only a fraction of the entire cell population in neurospheres as differentiation occurs in aggregates. Are NS cells in vitro similar to a specific in vivo radial glial subpopulation? To address this issue it will be necessary to define more rigorously gene expression patterns in vivo and in vitro. It is currently difficult to draw firm conclusions as few markers exist and these are often shared with related NEPs or astrocytes. Global expression profiling of directly isolated radial glia and in vitro expanded NS cells should address this issue. It is also possible that the artificial nature of the tissue culture environment may result in a unique or synthetic cell state

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without a direct in vivo counterpart. In fact we find that NS cells express combinations of transcription factors not noted during normal development (Pollard et al., 2006c). The combination of FGF-2 plus EGF may create a synthetic cell state with an appropriate balance of these key transcription factors to suppress lineage commitment and allow self-maintaining divisions (Pollard et al., 2006c). Nevertheless, as those best characterised markers, together with morphological criteria and functional attributes (neurogenesis), are similar between NS cells and radial glia it is fair to term them radial glia-like NS cells. Future studies should reveal the extent of changes that occur upon exposure to high levels of FGF and EGF and how physiologically relevant the resulting in vitro phenotype is. Also, although possibly the most parsimonious explanation, it is still not clear whether radial glia are the founders/cell-oforigin for in vitro expandable NS cells. Prospective isolation of radial glia and subsequent assays for NS cell colony forming activity have not yet been carried out. Neurospheres have also been established from adult forebrain SVZ (Reynolds and Weiss, 1992), and we have found that similar to foetal cells, culture in the presence of both EGF and FGF-2 adherently enables expansion of pure populations of adult NS cells in the absence of cell differentiation (Pollard et al., 2006c). Adult NS cell lines show an identical marker profile and behaviour in vitro to those foetal derived cells. Interestingly, adult-derived NS cells do not express GFAP, a marker that distinguishes adult SVZ astrocytes (type B cells) from their radial glia ancestors. Thus, adult SVZ cells and/or derivatives such as the transit amplifying descendants may respond to in vitro conditions by reestablishing features of radial glia through a process of dedifferentiation (Doetsch et al., 2002; Pollard et al., 2006c). 4. Radial glia from ES cells 4.1. Differentiation of ES cells to radial glia Mouse ES cells can be maintained in vitro in the absence of differentiation through a combination of cell extrinsic signals (LIF/gp130 and BMP), and cell intrinsic determinants, such as the transcription factors Sox2, Oct4 and Nanog (Chambers and Smith, 2004). In order to differentiate ES cells, the cell culture medium is altered such that LIF and BMP/serum are replaced by alternative inductive signals. An example of this is the differentiation to mesendoderm lineage, which is achieved through replacement of LIF with Nodal-related growth factors (Kubo et al., 2004). For differentiation to neural lineages initial reports showed that neurons can be efficiently generated through the exposure of ES cells to retinoic acid and serum in suspension culture, following LIF withdrawal (Bain et al., 1995; Strubing et al., 1995; Zupanc and Clint, 2003). Modifications to this protocol were developed soon after in which exposure to retinoic acid is not required and neural precursor populations could be induced and subsequently enriched by selective survival in a serum free basal media (Okabe et al., 1996). Other distinct protocols have developed which rely on co-culture with a stromal cell line PA6, or

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exposure to conditioned media (Kawasaki et al., 2000; Rathjen et al., 1999), and details of these various approaches are reviewed elsewhere (Lang et al., 2004; Stavridis and Smith, 2003). ES cells provide a unique tool to investigate molecular events occurring during the transition of NEP to radial glia, and from radial glia to differentiated neural cells (neuron, astrocyte or oligodendrocyte). The default model of neural induction proposes that the key event is the removal of BMP signalling, and that there are no ‘positive’ inductive signals (MunozSanjuan and Brivanlou, 2002). This hypothesis led investigators to search for further simplification of ES cell differentiation media. Tropepe et al. developed a protocol in which low efficiency (0.2%) neural induction occurs following direct suspension culture of ES cells (Tropepe et al., 2001). The massive cell death occurring during the first few days in culture makes it unclear whether this protocol is truly reflective of a neural induction, or simply selection of pre-differentiated cells which can arise spontaneously during ES cell propagation. An improved protocol has been developed by Ying et al. in which Sox1 expressing neuroepithelial cells can be generated in adherent monolayer at high efficiencies (>60%) (Ying et al., 2003). The development of this adherent monolayer protocol provides several experimental advantages. Fully defined media and the absence of heterologous cell interactions provide consistent results between experiments and enables simple isolation of a large amount of protein and nucleic acid for molecular profiling approaches (Pollard et al., 2006a). Furthermore, researchers can directly monitor the cellular transitions occurring during early neural induction. The developmental path of neural differentiation of ES cells in vitro may recapitulate neural development in vivo (Bain et al., 1995). Thus, differentiation to neuronal and glial subtypes from ES cells could occur via conversion to transient neural progenitors (Fig. 1). Given that radial glia function as a progenitor cell for neurons and glia within the CNS (Hartfuss et al., 2001; Malatesta et al., 2000; Miyata et al., 2001; Noctor et al., 2001, 2002), Liour and colleagues investigated whether this cell type is also present in differentiating ES cell cultures (Liour et al., 2006; Liour and Yu, 2003). Using both ES cells and embryonal carcinoma (EC) cell lines the authors carried out neural differentiation experiments by embryoid body (EB) formation and retinoic acid exposure. They found that when attached to treated tissue culture plastic for 7 days EB undergo differentiation to MAP2 immunoreactive neurons, which migrate out from the EB. To search for radial glia ‘scaffolds’ that might account for this migratory behaviour, Liour and coworkers performed an immunocytochemistry analysis with the RC2 antibody. In this way, they identified a subpopulation of RC2 positive cells, which co-stained for nestin and were in close proximity to clusters of migrating neurons. Morphologically, the RC2 immunoreactive cells are bipolar and contain long unbranched processes. These functional and phenotypic features are consistent with a radial glia identity. Subsequent studies using the adherent monolayer differentiation protocols have noted an analogous RC2 and BLBP positive cell population emerging from neuroepithelial rosettes (Fig. 1) concomitant with

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Fig. 1. Pictures of cultures of ES cells at different stages of monolayer neural differentiation (Ying et al., 2003). Cultures of undifferentiated ES cells immunoreactive for the ES cell marker Oct4 (red; a). Following neural differentiation the cultures drop Oct4 expression (b) and form neural rosette structures containing neuroepithelial cells immunoreactive for nestin (red; c) and showing the green signal due to activation of the GFP reporter by the sox1 promoter in 46C ES cell line (Ying et al., 2003; d). After 8 days of exposure to neural differentiation conditions, RC2 immunoreactive cells appear in the culture at the edge of the neural rosettes (red; e). After 14 days of differentiation, the cultures are highly in enriched neurons expressing the b3-tubulin marker (red; f). Nuclei show the blue DAPI staining (a,b,e).

neurogenesis (Liour et al., 2006; Lowell et al., 2006). These results have also been confirmed by other groups using a range of different parental ES cell lines and neural differentiation protocols, suggesting that the transition of neuroepithelial cells to RC2 immunoreactive radial glia-like cells may be a general occurrence of ES cell neural differentiation. A drawback of current ES cell neural differentiation protocols is that a contaminating population of non-neural cells and residual ES cells also exist within the cultures, likely maintained by paracrine LIF signalling. A strategy developed to overcome this incomplete conversion is known as ‘‘lineage selection’’, in which a reporter or drug selectable marker is ‘knocked-in’ through gene targeting, or expressed as a transgene under cell-type specific promoter elements (Li et al., 2001). For example, the Sox1-GFP reporter mice provide a tool to isolate NEPs using FACS (Aubert et al., 2003). However, homogeneous differentiation of ES cells can also be achieved through optimisation of cell culture conditions. Bibel and co-workers showed homogeneous cultures of cortical glutamatergic neurons can be efficiently generated from ES cells using an optimised EB and retinoic acid based protocol (Bibel et al., 2004; Plachta et al., 2004). Interestingly, during this differentiation procedure an apparently uniform population of Pax6 immunoreactive radial glia population is generated, circumventing the need for genetic lineage selection strategies for this lineage. The proposed induction of a cortical pyramidal neuronal type might seem in contrast with the wellcharacterized posteriorizing effect that RA exerts during neural

development, and with the broad spectrum of neuronal and glial phenotypes generated using other RA-based ES cell differentiation protocols. Indeed, several groups have used RA to induce posteriorization of ES-cell-derived neural precursors and to promote differentiation of motoneurons and GABAergic interneurons (Wichterle et al., 2002). The success of this protocol in promoting a cortical radial-glia-like fate could be due to particularly favourable cell culture conditions operating during the re-plating of the NEPs, such as RA concentration, window of application and differentiation state of the starting ES cell population. Alternatively, these cells may not correspond to any particular positional identity but may have certain changes imposed by the artificial cell culture environment. Transplants of these cells have revealed some developmental restrictions (Plachta et al., 2004). Upon injection in chick embryos they generate neurons in the spinal cord and dorsal root ganglia (DRG). However, only in the spinal cord do they acquire an appropriate regional identity. This contrasts with the injection of non RA-treated EBs that were able to generate both spinal cord neurons and DRG neurons (Plachta et al., 2004). Thus, the in vitro differentiation protocol used appears to restrict the global CNS–PNS differentiation potential of ES cell derived progenitors to a CNS fate. Further experiments are necessary to confirm the proposed radial glial and cortical pyramidal neuron phenotypes of the RA-induced cells. Unresolved issues are the exact requirements for RA as an inductive signal and whether a variety of neuronal and glial subtypes arise from radial glia progenitors.

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4.2. Expansion of radial glia-like cells from ES cells Those strategies developed to efficiently generate neural cell types from mouse ES cells have led to a growing knowledge of factors and molecular mechanisms regulating neural induction. However, it would be desirable for both basic and translational biology to isolate tissue specific stem cells and expand these as distinct cell lines. Exposure to FGF-2 and/ or EGF has been used to sustain transient expansion of foetal and adult cells, with maintenance of neuronal differentiation potential (Gage, 2000). We recently described that ES cellderived neural precursor cells, normally fated to rapidly differentiate to neurons and glia, can be readily expanded as adherent clonal stem cell lines using the growth factors EGF and FGF-2, and importantly undergo symmetrical stem cell divisions without accompanying differentiation (Conti et al., 2005). The initial cultures highly enriched in ES-derived Sox1 positive neuroephitelial cells, re-plated in a completely defined serum-free basal medium in the presence of FGF-2 and EGF, lead to the appearance of a population of bipolar cells. The early heterogeneity of the population reduces in a couple of passages, giving rise to morphologically homogeneous cells

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(Fig. 2). These cultures can be efficiently expanded for over 100 passages and are in effect immortal. These cells show no appreciable expression of the pluripotency factors Oct4 and Nanog, or the NEP cell marker Sox1. However, they do express the same phenotypic markers as those NS cell lines obtained from foetal and adult tissues, namely, nestin, BLBP, GLAST, RC2 and Sox2 (Fig. 2). ES cell-derived radial glia-like NS cells retain neurogenic potential after expansion for over 100 passages (Fig. 3), and are also capable of efficiently producing astrocytes and oligodendrocytes (Conti et al., 2005; Glaser et al., 2007). We have found these protocols robust and reproducible across a range of parental ES cells and using both adherent monolayer differentiations and EB formation with RA exposure. The ability to generate stable and homogeneous radial glia-like NS cells from ES cells clearly opens several experimental opportunities due to the ability to use available genetically engineered ES cell lines with a view to performing genetic loss- and gain-of-function studies with candidate gene regulators. ES cells can generate a wide spectrum of neurons when differentiating directly (i.e., with no intermediate expansion of the neural progenitors) using empirically determined protocols,

Fig. 2. NS cell lines show antigenic properties of radial glia-like cells. NS cell cultures propagated in EGF and FGF-2 grow in adhesion conditions and show a typical bipolar morphology (a). Immunocytochemistry analysis of the cultures shows uniform expression of the neural precursor markers Nestin (b) and sox 2 (c), of the radial glia antigens RC2 (d) and BLBP (e) and the immunoreactivity to the transcription factors pax6 (f) and Olig2 (g). NS cells do not show expression of neuronal (TuJ-1; h) or astrocyte (GFAP; i) antigens, Immunreactivity is shown in red; DAPI nuclear staining is shown in blue.

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Fig. 3. NS cells can generate neurons and astrocytes in vitro. Cultures of NS cells exposed to neuronal differentiation conditions efficiently generate neurons (a) that show Map2 immunostaining (red; b). Upon addition of serum, NS cells can be converted into astrocytes (c) that exhibit GFAP immunoreactivity (green; d). Nuclei show the blue DAPI staining.

or conditions designed to mimic developmental inductive events (Barberi et al., 2003; Wichterle et al., 2002). An important issue is the potential of NS cells to differentiate to different neuronal subtypes. Our results indicate that all NS cell lines, regardless of their source (Fig. 4), largely differentiate into GABAergic neurons (Conti et al., 2005) similar to

neurospheres. In contrast, without exposure to mitogens radial glia precursors generated from ES cells differentiate into glutamatergic neurons (Bibel et al., 2004). Thus, it appears that in vitro expansion of NS cells somehow restricts their neuronal subtype differentiation. It may be necessary to devise modified protocols that enable expansion of cell lines with alternative

Fig. 4. Schematic representation of the sources of NS cells. NS cell lines can be generated both from ES cell lines (a) (deriving from the ICM of the blastocysts; a schematic section of a blastocysts is present in upper part of the figure), from the germinative areas of the fetal brain (b) and from the sub ventricular zone (c; SVZ) of the adult brain. (b) and (c) represent schematic coronal sections of a fetal (b) and adult (c) brain, respectively; in blue are indicated the ventricles and in green the germinative area (b) and the SVZ (c).

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regional phenotypes e.g. midbrain dopaminergic neurons. Further studies are required to address whether cell culture conditions of long-term expanded NS cells can be altered such that cells with distinct regional identities can be re-established. 5. Benefits and applications of in vitro approaches Identification of those molecular principles underlying radial glia cell fate choice, self-renewal and differentiation within the simplified tissue culture environment provides a foundation which enables the identification of similar mechanisms in vivo—either in the developing embryo and foetus, or within the adult. Radial glia-like NS cell lines also enable cell based biomedical applications such as modelling of diseases, pharmaceutical drug screening and cell-based medicine (Pollard et al., 2006b).

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maintenance and differentiation and delivery of siRNA or overexpression vectors to the cells is straightforward. Furthermore, heterologous cell interactions, such as the attachment and migration of neurons along radial glia can be monitored. Additionally, NS cells provide a suitable context to perform promoter analysis in order to uncover cis regulatory elements controlling radial glia specific genes. Gene manipulation can be introduced either through random integration of vector DNA into the genome, or transduction and integration of viral vectors (Conti et al., 2005). Additionally, NS cells can be derived from wild type and engineered ES cell lines as well as from foetal or adult tissues, and so previously engineered transgenic mouse strains can be exploited. Each of those technologies developed for manipulation of ES cells, such as gene targeting, gene trap, RNAi knockdown and functional genetic screening should be readily transferable to NS cell lines.

5.1. Model system for stem cell biology 5.2. Cell-based disease modelling and drug screening Mechanisms regulating stem cells are currently being investigated intensively. The large amounts of material required to study biochemical changes can be difficult to obtain from mammalian embryos. Expansion of radial glia in vitro in unlimited numbers provides a means to obtain sufficient material for such studies. Equally the accessibility of differentiating ES cell cultures enables a simple live monitoring of the transitions through each phase in the formation of the nervous system, from neural induction, transitions from NEPs to radial glia and phases of neurogenesis and gliogenesis. NS cells can be derived from multiple sources (Fig. 4) and are expanded in defined serum-free basal media, dividing through symmetrical self-renewal with little spontaneous differentiation or cell death. Similar to ES cells, NS cells are amenable to genetic manipulation enabling screening and creation of stable transgenic cell lines. DNA microarrays and proteomic approaches are also straightforward when working with NS cells due to the homogeneity and simple differentiation protocols. The utility of NS cells as a tool to study epigenetic restrictions and commitment has recently been illustrated in studies of reprogramming by either cell fusion or nuclear transfer (Blelloch et al., 2006; Silva et al., 2006). Several studies in mouse have shown that Notch signals promote radial glia identity, directly controlling expression of the radial glia marker BLBP in vivo, and that ErbB2 and FGF2 signalling might modulate this event (Ever and Gaiano, 2005; Gaiano et al., 2000; Hatakeyama et al., 2004; Lowell et al., 2006; Patten et al., 2006). This pathway, together with FGF can be studied in detail using NS cells to dissect downstream events. When Notch signalling is activated in ES cells it triggers both an increased neural lineage commitment and subsequent maintenance of a BLBP immunoreactive progenitor populations (Lowell et al., 2006). The downstream events of Notch, ErbB2 and FGF signalling and their cross regulatory interactions remain unclear and are likely to be complex (Ever and Gaiano, 2005). Investigating these signalling pathways in vitro is attractive, as pharmacological agents or recombinant proteins can be directly assessed for effects on radial glia

Cellular models of neurodegenerative diseases have been mostly based on engineered immortalized cell lines (PC12, neuroblastoma, etc.) or, when possible, on primary neural cultures from patients/animal models. These model systems have significant limitations, thus impacting their effectiveness for understanding molecular mechanisms of disease. For example, primary cultures have an intrinsic heterogeneity in addition to the limited accessibility (for cells from patients) while immortalized cell lines may have a poor physiologic relevance to the disease. NS cells offer a versatile cellular setting for creating models of nervous system diseases. Modelling disease in homogeneous neural stem cell systems has the benefit that the primary events responsible for disease can be studied in the context of human cells, which is currently difficult due to their pre-symptomatic nature. In principle, using cell fusion or reprogramming technologies, it should be possible to generate ES cells and then NS cells from humans bearing the disease gene. In addition disease models can be engineered directly in already established wild type mouse or human NS cells (Pollard et al., 2006b). Huntington’s disease (HD) is an example in which modelling of the disease using NS cells may lead to improved knowledge of pathological mechanisms and a resource for drug discovery purposes. HD is an inherited neurological disorder caused by an excessive repeating of the CAG trinucleotide in exon 1 of the Huntingtin gene. This mutation leads to the production of a protein showing an expanded polyglutamine tract in its N-terminus that results in loss of protective function of the wt gene and neural toxicity due to gain of function. This causes the characteristic neurodegeneration of medium spiny projection neurons in the corpus striatum (Cattaneo et al., 2002;). Accordingly, HD patients show signs of motor dysfunction and impairments of cognitive and psychiatric faculties. Discovery of the disease-linked gene in 1993 has provided the possibility of performing a precise diagnosis; however the disease still remains incurable. The complex

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pathogenetic mechanisms have not been fully elucidated and no drugs are available to treat the pathology (Cattaneo et al., 2005;). Several cellular models of HD have been produced by inserting the full length or an N-terminal portion of the gene in immortalized cell lines from different sources. By virtue of their homogeneity, stability and capability to generate GABAergic neurons in vitro, NS cells may be a useful system to produce novel and useful cellular models of HD (Sipione and Cattaneo, 2002). Cell lines can be generated from ES cellderived NS cells harbouring the human mutant Huntingtin gene or directly from foetal brain areas of R6/2 mice, which model HD (Mangiarini et al., 1996; Conti, unpublished). These cell lines may be used for studies on self-renewal or for biological, biochemical and pharmacological analyses on their GABAergic neuronal derivates. The characteristics of NS cells are particularly well suited to high-throughput chemical and genetic screens aimed at identifying small molecules capable of directing stem cell behaviour. These compounds might be used in the lab to produce cell types of interest or in patients in order to stimulate regenerative and reparative events. Such small molecule screens will become widespread as more advanced protocols for generating pure cell populations of interest (both neuronal subtypes, astrocytes and oligodendrocytes, and their precursors) become available. Tools for engineering both ES and NS cells enable the design of convenient reporters for multiplex screening assays for fully automated detection of lead compounds. Phenotypic assays of differentiation or signalling pathway-specific screens can be devised using luminescent, fluorescent or enzyme-based colorimetric reporters. Assays could be established to screen for factors that affect a range of processes, including stem cell self-renewal, promotion of differentiation to specific fates, neuronal survival, axon guidance and synaptogenesis. 5.3. Cell replacement strategies Cell replacement approaches represent a new approach to treating a broad spectrum of neurodegenerative diseases (Conti et al., 2006). The aim is to replace diseased or injured tissue with healthy cells grown in the lab which would engraft following transplantation and function to either halt, slow, or reverse pathogenic events. Using stem cells and their derivatives for clinical applications is attractive as it could impact on a range of medical needs. Even though sometimes oversold as a panacea, stem cell transplantation is already used in the clinic, e.g. bone marrow transplantation, skin grafting and corneal transplant. Although replacement of damaged neurons by cell transplantation is being enthusiastically explored as a potential treatment for many neurodegenerative diseases, stroke and traumatic brain injury, application of regenerative medicine to brain diseases is still far from a reality. NS cells may represent a potential unlimited source of cells for regenerative medicine. Transplantation studies have shown they can survive and differentiate in both foetal and adult brain environments, and unlike ES cells, they do not generate teratomas. Further studies have to be performed in order to test

their ability to generate various neuronal lineages in vitro and in vivo and to assess their long-term stability and functional in vivo reconstitution. 6. Conclusions Our views of radial glia have been radically altered by the realization that they are progenitors capable of generating neurons. Various challenges lay ahead as we look for a deeper understanding of how these cells are established, maintained and transformed into neurons and glia. The complexity and challenges of understanding nervous system ontogeny should benefit tremendously from cell culture studies, which offer a simplified setting to deeply dissect basic molecular, cellular and developmental processes. Expansion of radial glia-like (NS) cells in vitro provides a unique cellular model system for making fundamental progress in radial glia biology. While there are several experimental advantages of working with cells in culture, there is of course the caveat that in vitro findings must be validated by experiments in vivo before drawing conclusions of normal physiology. However, for future regenerative medicines and other biomedical applications relying on stem and progenitor cells, the artificial nature of the cell culture environment is not of primary concern and may even offer unique opportunities to bypass physiological constraints and generate useful cellular phenotypes (Anderson, 2001; Joseph and Morrison, 2005). Acknowledgements We thank Gillian Morrison, Austin Smith, Yirui Sun, Sandra Gomez Lopez, Evangelia Papadimou, Catarina Ramos and Elena Cattaneo for helpful comments during preparation of the manuscript. Our apologies to all whose works were not cited due to space limitations. S. Pollard is supported by the Biotechnology and Biological Sciences Research Council (BBSRC) of the United Kingdom. L. Conti is supported by the Italian Ministry of Research. Each author is involved in the European Commission Framework VI Integrated Project ‘‘EuroStemCell.’’ L. Conti is involved in the European Commission Framework VI STREP Project ‘‘Neuroscreen.’’ References Alvarez-Buylla, A., Garcia-Verdugo, J.M., Tramontin, A.D., 2001. A unified hypothesis on the lineage of neural stem cells. Nat. Rev. Neurosci. 2, 287– 293. Alvarez-Buylla, A., Theelen, M., Nottebohm, F., 1990. Proliferation ‘‘hot spots’’ in adult avian ventricular zone reveal radial cell division. Neuron 5, 101–109. Anderson, D.J., 2001. Stem cells and pattern formation in the nervous system: the possible versus the actual. Neuron 30 (1), 19–35. Anthony, T.E., Klein, C., Fishell, G., Heintz, N., 2004. Radial glia serve as neuronal progenitors in all regions of the central nervous system. Neuron 41, 881–890. Aubert, J., Stavridis, M.P., Tweedie, S., O’Reilly, M., Vierlinger, K., Li, M., Ghazal, P., Pratt, T., Mason, J.O., Roy, D., Smith, A., 2003. Screening for mammalian neural genes via fluorescence-activated cell sorter purification

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