The stem-cell menagerie


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The stem-cell menagerie

  1. 1. The stem-cell menagerie Larysa Pevny1 and Mahendra S. Rao2 1 Neuroscience Center, Department of Genetics, University of North Carolina, Chapel Hill, NC 27599, USA 2 Laboratory of Neurosciences, National Institute on Aging, Baltimore, MD 21224, USA The numbers, types and locations of stem cells in the nervous system have been the subject of much discus- sion. This review summarizes data on the types of stem cell present at different stages of development and in the adult brain, and the markers suggested to dis- tinguish between the various possibilities that have been reported. We present evidence that more than one class of stem cell is present in the developing and adult nervous systems, and that it might be possible to dis- tinguish between stem-cell populations and to localize the cell of origin of a particular neurosphere, based on markers that persist in culture and by using universal stem-cell markers prospectively to identify stem cells in vivo. The early forming neural tube comprises a relatively homogenous population of cells. These cells appear to lack markers of neurons, radial glia, astrocytes and oligo- dendrocytes and can be dissociated and maintained in clonal culture. Upon differentiation, individual neuroe- pithelial cells can differentiate into neurons, astrocytes and oligodendrocytes in vitro and in vivo after transplan- tation. These cells have been termed neuroepithelial stem cells and their properties have been characterized. A large body of evidence suggests that the survival and prolifer- ation of neuroepithelial stem cells are regulated by basic fibroblast growth factor (bFGF) [1,2]. Localization of stem cells at this stage of development is within the ventricular zone and at least four populations of cells are derived directly from these cells, including radial glia, neural-crest stem cells, neurons and subventricular zone (SVZ) cells. As development progresses, the rapid proliferation of the neuroepithelium and the early differentiation of neurons and radial glial cells leads to an elaboration of the simple neuroepithelial structure in the cortex and caudal neural tube. The earliest step involves the creation of a marginal zone, into which early-born neurons migrate when they exit the ventricular zone. Around mid-embry- ogenesis, the ventricular zone is much reduced in size and additional zones of mitotically active precursors can be identified. Mitotically active cells derived from the ventricular zone that accumulate adjacent to this zone have been termed SVZ cells (Fig. 1). The SVZ is later called the subependymal zone, as the ventricular zone diminishes in size to a single layer of ependymal cells. The SVZ is prominent in the forebrain and can be identified as far caudally as the fourth ventricle. No SVZ can be detected in more caudal regions of the brain and, if it exists, the SVZ in such regions is likely to comprise a very small population of cells. An additional germinal matrix that is derived from the rhombic lip of the fourth ventricle, called the external granule layer, generates the granule cells of the cerebellum. Around the time when the SVZ can be clearly demarcated, an additional stem-cell population can be isolated and propagated in culture. This population was first described by Weiss and colleagues [3,4] and well over 1000 papers have been published describing and/or using these cells. Cells within this second stem-cell population have been termed the epidermal growth factor (EGF)- responsive stem cells, although both fibroblast growth factor (FGF) and EGF might be able to support their proliferation [5]. EGF-dependent stem cells grow in suspen- sioncultureandsequentialclonalanalysissuggeststhatthis stem-cell population constitutes a fraction of any sphere of cells that undergoes self-renewal and differentiates into neurons, astrocytes and oligodendrocytes in vitro and after Fig. 1. Two linearly related neural stem-cell populations are present during devel- opment. Ventricular zone (VZ) cells have been termed neuroepithelial (NEP) stem cells to distinguish them from the neurosphere-forming subventricular zone (SVZ) stem cells (one of which is shown in yellow; the turquoise cells around the SVZ cell are those derived from it that can, in the context of a neurosphere, become dif- ferentiated cells or progenitors that can give rise to neurons, astrocytes or oligo- dendrocytes). A lineage relationship between the earlier-born ventricular-zone cells and the ventricular-zone-derived SVZ cells has been convincingly demon- strated in vitro and in vivo by a variety of experimental techniques [7,28,69]. Early- born ventricular-zone stem cells can give rise directly to radial glia and ependyma, which are thought to give rise to adult stem cells. Ventricular-zone stem cells can also give rise to neural-crest stem cells (NCSC) and PNS stem cells, and to cells that form neurons, astrocytes or oligodendrocytes independently of neurosphere- forming SVZ cells. Cell types in bold are the commonly described neural stem cell populations. TRENDS in Neurosciences NEP/VZ stem cell Neurosphere-forming SVZ stem cell Neuron Astrocyte Oligodendrocyte Radial glia NCSC/PNS stem cell Differentiated cells or progenitors Ependyma Adult stem cells Adult neurosphere PNS cells Corresponding authors: Larysa Pevny (, Mahendra S. Rao ( Review TRENDS in Neurosciences Vol.26 No.7 July 2003 351 0166-2236/03/$ - see front matter. Published by Elsevier Science Ltd. doi:10.1016/S0166-2236(03)00169-3
  2. 2. transplantation. EGF-dependent stem cells can be isolated from the entire rostrocaudal axis from embryonic-day (E)14 onwards and such stem cells have also been isolated from adult cadavers [4,6,7]. Several lines of evidence suggest that in the developing embryo, the neurosphere-forming stem-cell population is likely to reside in the SVZ in regions where a defined SVZ exists and to reside outside the ventricular zone in more caudal brain regions, including the spinal cord. Retroviral labeling experiments indicate that subpopulations of cells within the SVZ are multipotent. High levels of EGF and EGF-receptor expression are seen in the SVZ but not in the early ventricular zone [8,9]. EGF-receptor-knockout mice do not display altered size or prominence of the ventricular zone that contains FGF-dependent neuroepithelial stem cells, although their later neuronal and glial survival is affected [10,11]. By contrast, FGF-null and FGF-receptor- null mutations altered the size of the ventricular domain, in addition to affecting neuronal and glial populations [12–14]. Furthermore, it has been difficult to isolate neurospheres from regions of the brain in which an SVZ cannot be morphologically identified using EGF alone, and EGF-dependent neurosphere-forming stem cells cannot be isolated at stages before formation of the SVZ. Moreover, microdissection experiments have shown that the region containing the greatest neurosphere-forming ability includes the SVZ and its immediate environs [15]. Thus, a neurosphere-forming stem-cell population that depends on EGF is present during late embryonic development and is likely to be localized to the subventricular zone or to an equivalent region throughout the rostrocaudal axis. Larger numbers of EGF-dependent neurosphere-forming stem cells are present in cranial regions where the SVZ is large, and smaller numbers are present in more caudal regions. Two populations of stem cells during late embryogenesis As development proceeds, the ventricular zone becomes much diminished in size, while the SVZ continues to expand. However, both domains contain dividing cells, suggesting that two populations of neural stem cells co- exist for a period of time during development. Culture experiments have shown that this is, indeed, true. Both FGF and EGF can be used to derive neurosphere-forming stem cells at later stages of development. In general, FGF and EGF in combination generate more neurospheres than either factor alone [7,16–18]. Population and statistical analyses show that these represent two distinct populations of cells. Transition from an FGF-dependent to an EGF-dependent stem cell can be demonstrated using chimeric animals [16] and stem cells can be isolated from regions in which an SVZ cannot be identified [17]. Thus, during early neurogenesis, a single population of stem cells is present and is localized to the ventricular zone. It should be noted that, although this review describes these cells as homogenous in appearance, growth-factor dependence and cytokine-response pattern- ing influences have already further subdivided this population in terms of its differentiation into subclasses of neurons [19]. During the second half of embryogenesis, however, two populations of stem cells can be isolated, with the predominant proliferating population being localized to the SVZ and a smaller population of cells localized to the diminishing ventricular zone. Ventricular-zone-derived neuroepithelial cells and SVZ-derived neurosphere-form- ing stem cells are morphologically distinct and can be distinguished based on their response to factors and the cell types that they generate in vivo (Table 1). The predominant differences between these populations appear to be their growth-factor responsiveness, their positional-marker expression and the subtypes of neuron that they generate. Both populations, however, express nestin, can grow as neurospheres and lack expression of markers characteristic of differentiated cells. In addition, both populations are likely to express the ATP-binding- cassette protein ABCG2, have high levels of telomerase activity, and synthesize detectable levels of telomerase reverse transcriptase (TERT) and telomerase-repeat- binding factors 1 and 2 (TRF1 and TRF2) [20,21]. Multiple stem-cell candidates during the early postnatal period and in the adult During late embryonic development and the early post- natal period, proliferation ceases and the SVZ diminishes in size in proportion to the entire brain. At this stage, neurogenesis has mostly ceased, with exceptions being in the olfactory bulb, hippocampus and external granular layer of the cerebellum. In the adult, neurogenesis can be seen predominantly in the olfactory bulb and the hippo- campus, and perhaps to a very small extent in the cortex (reviewed in Refs [22–24]). Additional dividing popu- lations can be detected throughout the brain, as assessed by bromodeoxyuridine (BRDU) incorporation, including Table 1. Differences between ventricular zone (VZ)- and subventricular zone (SVZ)-derived stem cellsa Property VZ neural stem cells SVZ neural stem cells Refs EGF-receptor expression Low or undetectable High and essential for isolation [10,40] Cytokine dependency FGF is required for proliferation EGF is necessary and sufficient for proliferation [1,2,4,5,14,16] Presence of a single cilium Absent Present on a subpopulation [37] Interkinetic nuclear movement Feature of normal development Not observed in vivo [35,56,68] Neurotransmitter response Glutamate causes proliferation Glutamate inhibits proliferation [69] GFAP expression Not observed Might define a stem-cell population in vivo [33,44–46] Type of neuron generated Projection neurons Primarily interneurons [20,69] Radial glial differentiation Normal aspect of development Ability to generate radial glia unknown [33,41] Neural crest differentiation Demonstrated in vitro and in vivo Unknown [19] Positional markers Differ between VZ and SVZ Differ between VZ and SVZ [83,84] a Note that the two populations of cells have several similarities, including the expression of nestin, Sox-1 and Sox-2, and the absence of most lineage-specific markers. Abbreviations: EGF, epidermal growth factor; FGF, fibroblast growth factor; GFAP, glial fibrillary acid protein. Review TRENDS in Neurosciences Vol.26 No.7 July 2003352
  3. 3. astrocytes or their precursors, oligodendrocyte precursors or other glial precursors, endothelial cells in the develop- ing blood vessels, and circulating dividing hematopoietic cells. Retroviral lineage analysis has not, for the most part, detected multipotential cells, even in regions of ongoing neurogenesis (e.g. Refs [15,25,26]). This suggests that stem-cell populations, if they exist, represent a very small proportion of the total progenitor pool, or are largely quiescent or restricted in their differentiation potential in vitro [27]. Evidence that stem cells are present, albeit in a quiescent state, has come from multiple reports of isolation of a multipotent self-renewing neurosphere-forming cells (Fig. 2). Investigators have reported isolation of stem cells from the entire rostrocaudal axis and from different stages of postnatal development, including from cadavers. Clonal analysis of sequentially passaged neurospheres has suggested that individual cells are multipotent and can generate neurons, astrocytes and oligodendrocytes in vitro and in vivo after transplantation [17,20,28]. The identity in vivo of the cell that possesses the ability to generate neurosphere-forming cells in vitro remains unknown, although several candidates have been pro- posed (Fig. 2). These include perinatal astrocytes, radial glial cells, embryonic neural cell-adhesion molecule (ENCAM)-positive population of multipotent stem cells and, possibly, transdifferentiated cells. Ependymal cells Ependymal cells are the remnants of the proliferating ventricular zone and are, therefore, a logical candidate site for an adult multipotent stem cell. Ependymal cells are relatively quiescent in vivo but can enter the cell cycle and respond to injury by proliferation. Infusion of FGF and EGF can cause a proliferation of ependymal cells and retroviral lineage analysis has suggested that individual cells can generate astrocytes and neurons in at least some regions of the brain [29,30]. Ependymomal tumors express both neuronal and glial markers. Thus, it is reasonable to assume that ependymal cells are multipotent. Whether they possess sufficient self-renewal ability, however, has been questioned and several investigators have discounted these cells as stem cell candidates. Van der Kooy and colleagues have pointed out that most neurospheres do not consist of ciliated cells [31]. Assuming that LeX/SSEA-1 is a marker of adult neural stem cells, Capela and Temple provide evidence that purified ependymal cells, which are LeX/SSEA-1-negative, do not make neurospheres [32]. A proposed resolution is that the dividing ependymal cells that maintain stem cell properties are SVZ type-B cells, which contact the ventricle [33]. Neurospheres derived from ciliated ependymal cells do not undergo significant self-renewal and neurospheres that undergo self-renewal can be isolated from other regions of the brain. Thus, if ependymal cells represent an adult stem cell population they can at best represent a minority population of the neurosphere-generating cells. SVZ cells The SVZ is the site of a second population of stem cells present in early development (see preceding section) and undifferentiated cells can be identified in the SVZ even at late adult stages. Cells with the ability to form neuro- spheres that self-renew and are multipotent in culture can be isolated from the cortical SVZ. Retroviral labeling of SVZ cells has, however, suggested regional heterogeneity and shown that the SVZ consists of a mixture of stem and progenitor cells [25,34–36] (reviewed in Ref. [24]). Tritiated-thymidine injections to kill actively dividing cells at later stages of SVZ development have suggested that ,1% of the cells are slowly dividing stem cells [27] that can regenerate the remaining cells in the SVZ. It is not clear, however, which cell in this heterogeneous population equates to the neurosphere-forming stem-cell. Different groups have suggested different locations and different properties. In the adult, neurosphere-forming stem cells might be localized in the SVZ to the type-B glial- fibrillary-acid protein (GFAP)-positive astrocytic cells, to the type-C GFAP-negative cells (reviewed in Refs [37,38]) or to a distinct population that has not been clearly defined [32,39]. Recent compelling experiments demonstrate that the majority of EGF-responsive cells in the adult SVZ that generate neurospheres are actually derived from the rapidly dividing transit-amplifying type-C cells [409]. Thus, at least one population of multipotent stem cells exists in the adult SVZ and this population is relatively quiescent, although it can enter the cell cycle and participate in repair and regeneration in the olfactory bulb and hippocampus and can generate neurospheres. Other potential stem-cell populations Radial glia as stem cells Radial glial cells, as defined by RC1 and RC2 immunor- eactivity, can be identified at as early as E10–E11 in mice and at E14 in rats and can then be distinguished from the proliferating ventricular-zone stem cells and from SVZ cells by their characteristic morphology and antigen expression [41]. Radial glia persist until late perinatal ages and transform into astrocytes as a normal process of development [42,43]. Recent in vivo studies have suggested that radial glia form the majority of progenitor cells that give rise to neurons of the cortical germinal zone Fig. 2. Potential stem cells during neural development. The different types of potential stem cell present at different developmental ages are listed. Cells ident- ified by radial glial antigens are not detected in adult tissue and glial-fibrillary-acid protein (GFAP)-positive cells are not seen before embryonic-day (E)16. However, green-fluorescent protein (GFP) expression driven by the human GFAP promoter leads to expression of GFP in radial glia, ependymal cells and subsets of cells in the ventricular zone (VZ), a property that has been exploited by some investigators and has lead to controversy in the field. Abbreviations: ENCAM, embryonic neural cell-adhesion molecule; P, postnatal day, SVZ, subventricular zone. TRENDS in Neurosciences E9 Adult E14 SVZ stem cell Type-B (GFAP+) Type-C (GFAP-) Ependymal cells Parenchymal ENCAM+ cells Transdifferentiated cells Oligodendrocytes, neurons, progenitors, other TSCs P0 VZ SVZ Radial glia Astrocyte VZ SVZ VZ Stem cells during neural development Review TRENDS in Neurosciences Vol.26 No.7 July 2003 353
  4. 4. and might also function as a self-renewing multipotent population [44–46]. Moreover, it has recently been hypothesized that adult SVZ cells might be derived from embryonic radial glia that retain neuroepithelial stem-cell characteristics into adulthood [33]. Astrocytes as stem cells Steindler and colleagues have argued that, in addition to GFAP-expressing SVZ cells and GFAP-expressing radial glia, at least during perinatal stages, a subset of astrocytes is multipotent. Laywell and colleagues have shown that astrocytes can be cultured in vitro and induced to generate neurospheres that can then differentiate into neurons, astrocytes and oligodendrocytes [6,39]. Consistent with the idea that astrocytes might be stem cells at least in vitro are experiments showing that glial cells do not undergo senescence [47,48]. Moreover, these cells express high levels of TERT and are spontaneously immortal. Thus, at least some astrocytes fulfill the criteria of stem cells [20,21]. It should be emphasized, however, that not all astrocytes are stem cells, because the frequency of neurosphere generation from astrocyte cultures is not robust and neurospheres cannot be generated from astrocytes isolated from adult brain. Furthermore, most early neurospheres do not express GFAP in culture. This suggests that astrocytes are likely to undergo a kind of transformation in culture to dedifferentiate or transdifferentiate into stem cells and that they are not likely to behave as stem cells in vivo. Stem cells as tissue culture artifacts or transdifferentiated cells Given the evidence that few stem cells can be identified in vivo and that a stem-cell response is not an important aspect of neural repair and regeneration, it is important to consider whether the cells that form neurospheres in vitro represent transformed cells that do not possess stem-cell characteristics in vivo. It is possible, for example, that manipulation in culture of differentiated or partially differentiated neural cells can induce dedifferentiation into a stem-cell state. Indeed, experiments described by Brewer [49] and Raff [50] suggest that this is possible and is a robust occurrence. Brewer and colleagues [49] described how postmitotic neurons could be induced to dedifferentiate into dividing progenitor cells, and Kondo and Raff [51] showed that glial progenitor cells can be dedifferentiated and up to 80% of the population can then be induced to differentiate into neurons. Other investigators have suggested that stem cells that normally contribute to other tissues might be able to contribute to neurogenesis. Such transdifferentiation has been described in multiple reports [52,53]. Furthermore, Weismann and colleagues showed in an impressive set of experiments that circulating hematopoietic progenitors can reside in many tissues, including the brain, indicating that non-neural stem cells could be present in the brain in small numbers [54]. More recently, Doetsch et al. have provided compelling evidence that progenitor cells retain stem-cell properties [40]. Specifically, they showed that after exposure to high concentrations of EGF, type-C amplifying progenitors of the SVZ function as stem cells in vitro. The idea that neural stem cells themselves do not exist in large numbers in the adult brain, but that neurospheres can be generated from cells that undergo transformation in culture, does not contradict the avail- able data, although the process of transdifferentiation and its frequency is relatively controversial [55]. Studying neurosphere-forming stem cells The problem of studying stem cells in general has been compounded by the difficulty of propagating a pure population of stem cells in vitro. Neurosphere cultures in a defined medium and high concentrations of EGF have allowed investigators to propagate and maintain neural stem-cell populations and represent a major technical advance. Most investigators have, however, been unable to maintain stem cells as a pure population of cells. Most neurospheres in culture comprise a heterogeneous popu- lation of cells with the number of self-renewing stem cells being a fraction of the total population (often ,5%). The inability to purify a homogenous population of stem cells has precluded the use of large-scale comparative analysis that has been used so successfully in other systems. The divergence of opinion on the identity of the stem- cell population in vivo and its precise location has further hampered our ability to analyze stem-cell development in vivo. It has not been possible to harness transgenic technology, long-term slice cultures, retroviral labeling techniques or real-time visualization strategies to examine directly fundamental biological issues such as symmetric and asymmetric division, differentiation and cell-cycle regulation, or to combine these observations with large- scale genomic analysis techniques to define the process of stem-cell self-renewal and differentiation in detail and, thus, to manipulate it to enhance repair and regeneration. To surmount these difficulties in studying neural stem- cell populations, investigators have begun to assess several strategies. Selection strategies to identify stem- cell populations prospectively have been developed and markers that can be used to localize stem cells in vivo have begun to be assessed. These results are exciting and offer the potential for visualizing stem cells in vivo and following their development in transgenic animals and in purified culture assays. Some of the recently described markers are discussed in the following sections. Universal stem-cell markers and stem-cell-subtype markers The cellular properties used to define a neural stem population – the ability to form neurospheres, the ability to self-renew and the ability of single cells to differentiate into neurons, astrocytes and oligodendrocytes – might correlate with expression of general molecular markers, which are referred to here as ‘universal stem-cell markers’ [56–63] (Table 2). The word ‘universal’, rather than ‘specific’, is used here because so far no marker that is exclusive to stem cells has been identified. However, markers that can be expressed by many potential stem-cell candidates or transdifferentiated cells have been categor- ized as universal. In addition to already identified universal markers (see following discussion), several laboratories have begun using large-scale analysis Review TRENDS in Neurosciences Vol.26 No.7 July 2003354
  5. 5. techniques to compare stem-cell populations. Results from these analyses suggest that there might be additional universal markers that characterize all stem cells [64–67]. Our laboratory, for example, compared the expression of 500 genes by stem cells and has identified eleven genes related to the cell cycle and apoptosis that might be unique to early-developing neural stem cells [68]. These studies also raise the possibility that stem cells from different tissues might be more closely related than previously assumed and could share common molecular regulators. Indeed, several investigators have argued for the concept of ‘stemness’ or a molecular signature that is universal for stem-cell populations, irrespective of the tissue from which they are identified. Several investi- gations have profiled gene expression in different stem-cell populations and have found that embryonic, hematopoie- tic and neural stem cells share many similarities at the transcriptional level [65–67]. Negative selection strategies This review briefly summarizes a number of approaches that take advantage of universal stem-cell markers to isolate, characterize and manipulate neural stem cells by both prospective positive and negative selection strategies. Several groups have proposed a negative selection criterion that takes advantage of the observation that stem cells do not express markers characteristic of differentiated cells. Rao and colleagues have used the absence of expression of neuronal, astrocytic and oligo- dendroglial markers to enrich samples for stem cells from late fetal stages [20,69]. Whether these data can be extrapolated to adult stem cells remains to be determined. Bartlett and colleagues [70], in similar experiments, have suggested two potential markers that can be used to enrich for neural stem cells in adults. The authors showed that low levels of staining for peanut agglutinin (PNA) and heat-stable antigen (HSA) can, when combined with size selection, be used to select for stem-cell populations from neurosphere cultures. Using a similar negative selection strategy, Maric et al. used surface ganglioside epitopes emerging on differentiating CNS cells to isolate neural progenitors from E13 rat telencephalon by fluorescence- activated cell sorting (FACS) [71]. Positive selection strategy Even though it is possible to identify and isolate stem cells prospectively from a mixed population, it is difficult to use an absence of expression of markers to localize stem cells in vivo, given the multiplicity of markers required. Therefore, parallel approaches have identified positive selection markers that can be used to identify neural stem cells. Weissman and colleagues have suggested that AC133 might be an additional neural stem-cell marker [72], although it is currently useful only in human tissue. Quesenberry and colleagues have shown that within a neurosphere derived from adult tissue, populations of cells that display low levels of staining with Hoechst and Rhodamine-123 are enriched for stem cells [73,74]. The efflux is likely to be mediated by ABCG2, a member of the multi-drug-resistance family of transporters that is pre- sent on neural stem cells during development and is downregulated in differentiated cells [20,75]. More recently, it has been demonstrated that the LeX/SSEA-1 antigen is expressed by a subset of cells in the adult SVZ but not in the ependymal zone. Using this cell-surface antigen for FACS sorting, Capella and colleagues were able to isolate cells that formed multipotent neurospheres from the adult brain [32]. An alternative approach to select positively and prospectively for neural stem cells is to generate mouse lines in which the expression of a drug-selection marker or green-fluorescent protein (GFP) is driven by regulatory elements of a universal neural stem-cell marker [76–78]. For example, the SOXB1 subfamily of transcription Table 2. Potential markers for neural stem-cell populationsa Potential universal stem-cell marker Comments Refs Nestin expression Probably expressed in all dividing neural populations [56–58] Musashi expression Also expressed by progenitor populations [60,61] Hu expression Also expressed by progenitor populations [86] Neuralstemnin expression Recently described factor that might be relatively specific to dividing populations [63] Sox-1 expression Appears relatively specific to neural stem-cell populations, although it persists (transiently) in some progenitor-cell populations [80,87,88] Sox-2 expression Appears to be expressed by all neurosphere-forming cells, with localization to regions rich in stem cells [79,82,88] LeX/SSEA-1 expression Has been used prospectively to identify stem-cell populations [32] Response to ACh Has been used prospectively to identify stem-cell populations [20] Telomerase activity and TERT expression Present in all stem-cell populations tested but also present in non- stem-cell populations [20,21] Low levels of Hoechst and Rhodamine staining Appear to be specific for quiescent stem-cell populations but do not identify rapidly-dividing stem-cell populations [20,73,74] ABCG2 expression Appears relatively specific in vivo and in vitro [20,75,89] Aldefluor labeling Might be a non-specific method of prospectively identifying stem-cell populations [90–92] Absence of differentiation markers Has been used successfully to enrich for stem-cell populations at multiple stages of development [20,69,70] a Markers that are known to be relatively specific for stem-cell populations are listed. Current data do not conclusively show whether any of these markers has the requisite specificity and sensitivity to be used as a single marker for all potential stem-cell populations in the adult brain. Combinations of markers might, however, uniquely specify all neural stem-cell populations. Abbreviation: TERT, telomerase reverse transcriptase. Review TRENDS in Neurosciences Vol.26 No.7 July 2003 355
  6. 6. factors, which includes SOX1, SOX2 and SOX3, represents one group of conserved pan-neural markers. Sox1, Sox2 and Sox3 are coexpressed in proliferating neural progeni- tors throughout rodent embryogenesis and into adulthood [79–81]. Zappone et al., using a Sox2 promoter to direct expression of b-gal, have shown that telencephalic stem cells express Sox2 in vivo and that this expression persists in neurospheres derived from the telencephalon for at least 40 generations [82]. More recently, to develop an in vivo system for analyzing neurogenesis, Pevny and colleagues generated transgenic mice expressing an enhanced GFP (EGFP) under the control of the regulatory regions of the Sox2 gene. In this mouse line, EGFP expression is confined to progenitor-cell populations during early development and persists in selected popu- lations in the adult (Ellis et al., unpublished; Fig. 3). Moreover, prospective clonal analysis of SOX2–EGFP- positive cells demonstrates that multipotential stem cells isolated from both the embryonic CNS and the adult CNS all express SOX2–EGFP. Thus, positive and negative selection criteria can be used to define populations of stem cells at any stage of development. These markers, either singly or in concert, might help to localize stem cells in vivo, and their expression in neurospheres could help to define whether a particular neurosphere contains a multipotent stem cell. Universal stem-cell markers provide a means to identify cells that fulfill the basic criteria of a stem cell – self-renewal and multipotential differentiation – and, thus, define shared features when the cells are removed from their environmental milieu. They cannot, however, distinguish between types of stem cell. However, it has been previously shown that positional markers that define the rostrocaudal identity of stem cells persist over multiple generations in vivo [82–84]. It is, therefore, reasonable to assume that markers characteristic of the cell that generates a neurosphere in vitro will persist, at least over initial passages. Thus, if markers exist that dis- tinguish between the potential stem-cell candidates, it might be possible to determine whether all neurospheres are derived from the same population or from a hetero- geneous population, the properties of which depend on the particular stem-cell population that generated them. Potential markers that might distinguish the cell of origin of neurosphere-forming cells are listed in Table 3. This table is by no means complete but serves as an important starting point. If, for example, most first- generation neurospheres contained predominantly cells Fig. 3. SOX2-EGFP expression [i.e. expression of enhanced green-fluorescent protein (EGFP) under control of regulatory elements of the Sox-2 gene] identifies stem-cell populations of the embryonic and adult nervous systems. (a) EGFP expression in an embryonic-day (E)10 embryo from the SOX2–EGFP mouse line. EGFP is expressed throughout the neuroepithelium. (b) Coronal section through adult forebrain showing expression of EGFP in the lateral ventricle (LV) and (c) EGFP-expressing cells con- fined to the subgranular zone (SGZ) of the hippocampus. (d) Transverse section through adult spinal cord showing expression of EGFP confined to ependymal cells sur- rounding the central canal. (e–h) A single EGFP-expressing cell [shown in bright field (e) and by EGFP fluorescence (f)] isolated from the adult subventricular zone (SVZ) can give rise to a multipotential neurosphere (g). (h) Immunolabeling for glial-fibrillary-acid protein (GFAP) and tubulin (using the TuJ antibody), indicating that a single EGFP-positive cell is multipotent. Scale bars, 100 mm (a–d) and 20 mm (e–h). From Ellis et al. (unpublished). TRENDS in Neurosciences Overview of SOX2–EGFP expression in embryonic and adult CNS SGZ of adult hippocampus Ependyma of adult central canalEpendyma and SVZ of adult LVEGFP expression at E10 Multipotential neurospheres arise from SOX2–EGFP+ single cells from adult LV GFAP-TuJEGFP+ neurosphereEGFP+ single cell EGFP+ single cell (a) (b) (c) (d) (f)(e) (g) (h) Review TRENDS in Neurosciences Vol.26 No.7 July 2003356
  7. 7. that expressed polysialic-acid-bearing neural cell- adhesion molecule (PSA-NCAM) and were self-renewing, then one could reasonably presume that PSA-NCAM- positive cells present in vivo generate most of the neuro- spheres seen in culture. If, by contrast, cells were predominantly GFAP-positive (and PSA-NCAM-nega- tive), then one would assume that a population of GFAP-expressing cells (i.e. astrocytes, SVZ type-B cells or radial glia) generates neurosphere-forming cells. Double-labeling experiments using additional markers, such as S-100b, RC2 and the glutamate-transporter GLAST, might further distinguish between these possibilities. The presence of a cilium [31] and the coexpression of neuro–glial markers might identify ependymal cells as the predominant neurosphere- forming population. The presence of a heterogeneous population of neurospheres would suggest that mul- tiple stem-cell populations are present. Indeed, such heterogeneity of neurosphere populations has already been postulated on anatomical criteria [28,85]. None of the markers listed in Table 3 is unique to stem cells and expression of these markers is also seen in differentiated cells. It might, therefore, be necessary to combine potential cell-type-specific markers with univer- sal stem-cell markers in double-labeling or cell-isolation experiments. For example, if a GFAP-positive cell that formed a neurosphere coexpressed TERT, had high telomerase activity, expressed Sox1 and Sox2 and had high ABCG2 expression and activity, then it would be reasonable to assume in subsequent experiments that this represented a stem cell. The coexpression of GFAP with such a universal cell marker would allow this particular stem cell to be distinguished from an RC1-positive stem- cell population and to be localized in vivo. It is crucial, however, that apart from determining the expression of markers in early-passage neurospheres, the properties of the cells are also tested rigorously by passaging and single- cell cloning. Equally important would be to demonstrate the absence of markers that define dividing populations of cells that are not multipotent but are more restricted; such markers include A2B5, CD24 and CD44 (see preceding discussion). Experiments along similar lines in the hematopoietic system have helped to define in vivo the stem-cell population that has the highest self-renewing capacity and has allowed investigators to begin to probe fundamental aspects of stem-cell biology [54]. A potential problem with this approach is the possibility that neurospheres might not contain only a stem-cell population. Stem cells differentiate in response to environmental signals and it is difficult to ensure homogeneity of the microenvironment in a neurosphere-type culture, and it should be emphasized that prospective identification of cells by a combination of markers must be validated (at least initially) by rigorous single-cell clonal analysis. Summary Initial investigations suggest that multiple classes of stem cells exist during early development and in the adult. At least one cell type in the SVZ, and at least one additional population of cells in regions of the brain where there is no SVZ, must be stem cells. The non-SVZ stem cell might be a radial glial cell, an astrocyte or a transformed cell, and it might be possible to distinguish between these possibi- lities by marker expression. Staining with universal stem- cell markers, combined with markers that distinguish between stem-cell populations in early-passage neuro- sphere cultures, will determine the cell of origin of the neurosphere-forming cell. Persistence of these specific stem-cell-type markers in vitro would suggest that the cell does not transform or alter its properties, and changes in expression of these markers would suggest that the properties of a stem cell in culture do not reflect its properties in vivo. Recent breakthroughs in identifying stem-cell markers suggest that it will be possible to localize stem cells in vivo and to correlate the proper- ties of the stem cell with its in vitro neurosphere counterpart using universal and cell-type-specific mar- kers. Identifying universal and cell-type-specific markers will allow comparison of results between laboratories that grow neurospheres but use different isolation procedures. Furthermore, the ability to examine the stem-cell niche in vivo, to obtain relatively large numbers of potential stem cells, and to propagate stem cells for prolonged periods in vitro are relatively unique to the study of neural stem-cell populations. Clearly additional experiments are needed, but it is exciting that techniques and markers have begun to be identified that allow investigators an unprecedented view of stem-cell biology. Table 3. Potential markers that distinguish between neurosphere-forming stem-cell populationsa Marker Comments Refs GFAP Might distinguish between stem cells in the VZ and cortex and those derived from radial glia and astrocytes [22–24,41–43] S-100b and GLAST Might distinguish between stem cells in the VZ and cortex and those derived from radial glia and astrocytes [41–46] MAP2 and b-111 tubulin Might identify transdifferentiating neuronal cells. Might also recognize ependymal stem cells [20] RC1, RC2 and vimentin Might identify radial-glia-derived stem cells [44–46] PSA-NCAM Might distinguish cortical and parenchymal stem cells from other stem-cell populations [40] Might also recognize ependymal stem-cells A2B5 Might identify transdifferentiating glial-precursor cells and distinguish between glial precursors and other dividing progenitor-cell populations [20,69] a The listed markers are known to be relatively specific for subsets of stem-cell populations. However, in some cases, combinations of markers might uniquely specify a neural stem-cell population, which can then be distinguished from differentiated cells that express the same marker by the additional expression of unique stem-cell markers. Abbreviations: GFAP, glial fibrillary acid protein; GLAST, a glutamate transporter; MAP2, microtubule-associated-protein 2; PSA-NCAM, neural cell-adhesion molecule bearing polysialic acid; VZ, ventricular zone. 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