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    The stem-cell menagerie The stem-cell menagerie Document Transcript

    • Review TRENDS in Neurosciences Vol.26 No.7 July 2003 351The stem-cell menagerieLarysa Pevny1 and Mahendra S. Rao21 Neuroscience Center, Department of Genetics, University of North Carolina, Chapel Hill, NC 27599, USA2 Laboratory of Neurosciences, National Institute on Aging, Baltimore, MD 21224, USAThe numbers, types and locations of stem cells in the can be detected in more caudal regions of the brain and, ifnervous system have been the subject of much discus- it exists, the SVZ in such regions is likely to comprise asion. This review summarizes data on the types of stem very small population of cells. An additional germinalcell present at different stages of development and in matrix that is derived from the rhombic lip of the fourththe adult brain, and the markers suggested to dis- ventricle, called the external granule layer, generates thetinguish between the various possibilities that have granule cells of the cerebellum.been reported. We present evidence that more than one Around the time when the SVZ can be clearlyclass of stem cell is present in the developing and adult demarcated, an additional stem-cell population can benervous systems, and that it might be possible to dis- isolated and propagated in culture. This population wastinguish between stem-cell populations and to localize first described by Weiss and colleagues [3,4] and well overthe cell of origin of a particular neurosphere, based on 1000 papers have been published describing and/or usingmarkers that persist in culture and by using universal these cells. Cells within this second stem-cell populationstem-cell markers prospectively to identify stem cells have been termed the epidermal growth factor (EGF)-in vivo. responsive stem cells, although both fibroblast growth factor (FGF) and EGF might be able to support theirThe early forming neural tube comprises a relatively proliferation [5]. EGF-dependent stem cells grow in suspen-homogenous population of cells. These cells appear to lack sion culture and sequential clonal analysis suggests that thismarkers of neurons, radial glia, astrocytes and oligo- stem-cell population constitutes a fraction of any sphere ofdendrocytes and can be dissociated and maintained in cells that undergoes self-renewal and differentiates intoclonal culture. Upon differentiation, individual neuroe- neurons, astrocytes and oligodendrocytes in vitro and afterpithelial cells can differentiate into neurons, astrocytesand oligodendrocytes in vitro and in vivo after transplan- NEP/VZ stem cell NCSC/PNS stem celltation. These cells have been termed neuroepithelial stem PNS cellscells and their properties have been characterized. A largebody of evidence suggests that the survival and prolifer-ation of neuroepithelial stem cells are regulated by basic Neurosphere-formingfibroblast growth factor (bFGF) [1,2]. Localization of stem SVZ stem cellcells at this stage of development is within the ventricular Adult neurospherezone and at least four populations of cells are deriveddirectly from these cells, including radial glia, neural-creststem cells, neurons and subventricular zone (SVZ) cells. Radial glia Ependyma As development progresses, the rapid proliferation of Differentiated cells or progenitorsthe neuroepithelium and the early differentiation ofneurons and radial glial cells leads to an elaboration of Adult stem cellsthe simple neuroepithelial structure in the cortex andcaudal neural tube. The earliest step involves the creation Neuron Astrocyte Oligodendrocyteof a marginal zone, into which early-born neurons migrate TRENDS in Neuroscienceswhen they exit the ventricular zone. Around mid-embry-ogenesis, the ventricular zone is much reduced in size and Fig. 1. Two linearly related neural stem-cell populations are present during devel-additional zones of mitotically active precursors can be opment. Ventricular zone (VZ) cells have been termed neuroepithelial (NEP) stemidentified. Mitotically active cells derived from the 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 SVZventricular zone that accumulate adjacent to this zone cell are those derived from it that can, in the context of a neurosphere, become dif-have been termed SVZ cells (Fig. 1). The SVZ is later called ferentiated cells or progenitors that can give rise to neurons, astrocytes or oligo- dendrocytes). A lineage relationship between the earlier-born ventricular-zonethe subependymal zone, as the ventricular zone cells and the ventricular-zone-derived SVZ cells has been convincingly demon-diminishes in size to a single layer of ependymal cells. strated in vitro and in vivo by a variety of experimental techniques [7,28,69]. Early-The SVZ is prominent in the forebrain and can be 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 canidentified as far caudally as the fourth ventricle. No SVZ 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- Corresponding authors: Larysa Pevny (larysa_pevny@med.unc.edu), forming SVZ cells. Cell types in bold are the commonly described neural stem cellMahendra S. Rao (raomah@grc.nia.nih.gov). populations.http://tins.trends.com 0166-2236/03/$ - see front matter. Published by Elsevier Science Ltd. doi:10.1016/S0166-2236(03)00169-3
    • 352 Review TRENDS in Neurosciences Vol.26 No.7 July 2003transplantation. EGF-dependent stem cells can be isolated than either factor alone [7,16– 18]. Population andfrom the entire rostrocaudal axis from embryonic-day statistical analyses show that these represent two distinct(E)14 onwards and such stem cells have also been isolated populations of cells. Transition from an FGF-dependent tofrom adult cadavers [4,6,7]. an EGF-dependent stem cell can be demonstrated using Several lines of evidence suggest that in the developing chimeric animals [16] and stem cells can be isolated fromembryo, the neurosphere-forming stem-cell population is regions in which an SVZ cannot be identified [17].likely to reside in the SVZ in regions where a defined SVZ Thus, during early neurogenesis, a single population ofexists and to reside outside the ventricular zone in more stem cells is present and is localized to the ventricularcaudal brain regions, including the spinal cord. Retroviral zone. It should be noted that, although this reviewlabeling experiments indicate that subpopulations of cells describes these cells as homogenous in appearance,within the SVZ are multipotent. High levels of EGF and growth-factor dependence and cytokine-response pattern-EGF-receptor expression are seen in the SVZ but not in the ing influences have already further subdivided thisearly ventricular zone [8,9]. EGF-receptor-knockout mice population in terms of its differentiation into subclassesdo not display altered size or prominence of the ventricular of neurons [19]. During the second half of embryogenesis,zone that contains FGF-dependent neuroepithelial stem however, two populations of stem cells can be isolated, withcells, although their later neuronal and glial survival is the predominant proliferating population being localizedaffected [10,11]. By contrast, FGF-null and FGF-receptor- to the SVZ and a smaller population of cells localized to thenull mutations altered the size of the ventricular domain, diminishing ventricular zone. Ventricular-zone-derivedin addition to affecting neuronal and glial populations neuroepithelial cells and SVZ-derived neurosphere-form-[12 – 14]. Furthermore, it has been difficult to isolate ing stem cells are morphologically distinct and can beneurospheres from regions of the brain in which an SVZ distinguished based on their response to factors and thecannot be morphologically identified using EGF alone, and cell types that they generate in vivo (Table 1). TheEGF-dependent neurosphere-forming stem cells cannot be predominant differences between these populationsisolated at stages before formation of the SVZ. Moreover, appear to be their growth-factor responsiveness, theirmicrodissection experiments have shown that the region positional-marker expression and the subtypes of neuroncontaining the greatest neurosphere-forming ability that they generate. Both populations, however, expressincludes the SVZ and its immediate environs [15]. Thus, nestin, can grow as neurospheres and lack expression ofa neurosphere-forming stem-cell population that depends markers characteristic of differentiated cells. In addition,on EGF is present during late embryonic development and both populations are likely to express the ATP-binding-is likely to be localized to the subventricular zone or to an cassette protein ABCG2, have high levels of telomeraseequivalent region throughout the rostrocaudal axis. activity, and synthesize detectable levels of telomeraseLarger numbers of EGF-dependent neurosphere-forming reverse transcriptase (TERT) and telomerase-repeat-stem cells are present in cranial regions where the SVZ is binding factors 1 and 2 (TRF1 and TRF2) [20,21].large, and smaller numbers are present in more caudalregions. Multiple stem-cell candidates during the early postnatal period and in the adultTwo populations of stem cells during late During late embryonic development and the early post-embryogenesis natal period, proliferation ceases and the SVZ diminishesAs development proceeds, the ventricular zone becomes in size in proportion to the entire brain. At this stage,much diminished in size, while the SVZ continues to neurogenesis has mostly ceased, with exceptions being inexpand. However, both domains contain dividing cells, the olfactory bulb, hippocampus and external granularsuggesting that two populations of neural stem cells co- layer of the cerebellum. In the adult, neurogenesis can beexist for a period of time during development. Culture seen predominantly in the olfactory bulb and the hippo-experiments have shown that this is, indeed, true. Both campus, and perhaps to a very small extent in the cortexFGF and EGF can be used to derive neurosphere-forming (reviewed in Refs [22 – 24]). Additional dividing popu-stem cells at later stages of development. In general, FGF lations can be detected throughout the brain, as assessedand EGF in combination generate more neurospheres by bromodeoxyuridine (BRDU) incorporation, includingTable 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.http://tins.trends.com
    • Review TRENDS in Neurosciences Vol.26 No.7 July 2003 353astrocytes or their precursors, oligodendrocyte precursors been questioned and several investigators have discountedor other glial precursors, endothelial cells in the develop- these cells as stem cell candidates. Van der Kooy anding blood vessels, and circulating dividing hematopoietic colleagues have pointed out that most neurospheres do notcells. Retroviral lineage analysis has not, for the most part, consist of ciliated cells [31]. Assuming that LeX/SSEA-1 isdetected multipotential cells, even in regions of ongoing a marker of adult neural stem cells, Capela and Templeneurogenesis (e.g. Refs [15,25,26]). This suggests that provide evidence that purified ependymal cells, which arestem-cell populations, if they exist, represent a very small LeX/SSEA-1-negative, do not make neurospheres [32]. Aproportion of the total progenitor pool, or are largely proposed resolution is that the dividing ependymal cellsquiescent or restricted in their differentiation potential in that maintain stem cell properties are SVZ type-B cells,vitro [27]. Evidence that stem cells are present, albeit in a which contact the ventricle [33]. Neurospheres derivedquiescent state, has come from multiple reports of isolation from ciliated ependymal cells do not undergo significantof a multipotent self-renewing neurosphere-forming cells self-renewal and neurospheres that undergo self-renewal(Fig. 2). Investigators have reported isolation of stem cells can be isolated from other regions of the brain. Thus, iffrom the entire rostrocaudal axis and from different stages ependymal cells represent an adult stem cell populationof postnatal development, including from cadavers. Clonal they can at best represent a minority population of theanalysis of sequentially passaged neurospheres has neurosphere-generating cells.suggested that individual cells are multipotent and cangenerate neurons, astrocytes and oligodendrocytes in vitro SVZ cellsand in vivo after transplantation [17,20,28]. The SVZ is the site of a second population of stem cells The identity in vivo of the cell that possesses the ability present in early development (see preceding section) andto generate neurosphere-forming cells in vitro remains undifferentiated cells can be identified in the SVZ even atunknown, although several candidates have been pro- late adult stages. Cells with the ability to form neuro-posed (Fig. 2). These include perinatal astrocytes, radial spheres that self-renew and are multipotent in culture canglial cells, embryonic neural cell-adhesion molecule be isolated from the cortical SVZ. Retroviral labeling of(ENCAM)-positive population of multipotent stem cells SVZ cells has, however, suggested regional heterogeneityand, possibly, transdifferentiated cells. and shown that the SVZ consists of a mixture of stem and progenitor cells [25,34 –36] (reviewed in Ref. [24]).Ependymal cells Tritiated-thymidine injections to kill actively dividingEpendymal cells are the remnants of the proliferating cells at later stages of SVZ development have suggestedventricular zone and are, therefore, a logical candidate site that , 1% of the cells are slowly dividing stem cells [27]for an adult multipotent stem cell. Ependymal cells are that can regenerate the remaining cells in the SVZ.relatively quiescent in vivo but can enter the cell cycle and It is not clear, however, which cell in this heterogeneousrespond to injury by proliferation. Infusion of FGF and population equates to the neurosphere-forming stem-cell.EGF can cause a proliferation of ependymal cells and Different groups have suggested different locations andretroviral lineage analysis has suggested that individual different properties. In the adult, neurosphere-formingcells can generate astrocytes and neurons in at least some stem cells might be localized in the SVZ to the type-B glial-regions of the brain [29,30]. Ependymomal tumors express fibrillary-acid protein (GFAP)-positive astrocytic cells, toboth neuronal and glial markers. Thus, it is reasonable to the type-C GFAP-negative cells (reviewed in Refs [37,38])assume that ependymal cells are multipotent. Whether or to a distinct population that has not been clearly definedthey possess sufficient self-renewal ability, however, has [32,39]. Recent compelling experiments demonstrate that the majority of EGF-responsive cells in the adult SVZ that Stem cells during neural development generate neurospheres are actually derived from the rapidly dividing transit-amplifying type-C cells [409]. Adult Thus, at least one population of multipotent stem cells P0 SVZ stem cell exists in the adult SVZ and this population is relatively Type-B (GFAP+) quiescent, although it can enter the cell cycle and E14 VZ Type-C (GFAP-) E9 participate in repair and regeneration in the olfactory VZ SVZ Ependymal cells VZ bulb and hippocampus and can generate neurospheres. SVZ Radial glia Parenchymal ENCAM+ cells Astrocyte Transdifferentiated cells Oligodendrocytes, neurons, Other potential stem-cell populations progenitors, other TSCs Radial glia as stem cells Radial glial cells, as defined by RC1 and RC2 immunor- TRENDS in Neurosciences eactivity, can be identified at as early as E10 –E11 in mice and at E14 in rats and can then be distinguished from theFig. 2. Potential stem cells during neural development. The different types of proliferating ventricular-zone stem cells and from SVZpotential stem cell present at different developmental ages are listed. Cells ident- cells by their characteristic morphology and antigenified by radial glial antigens are not detected in adult tissue and glial-fibrillary-acidprotein (GFAP)-positive cells are not seen before embryonic-day (E)16. However, expression [41]. Radial glia persist until late perinatalgreen-fluorescent protein (GFP) expression driven by the human GFAP promoter ages and transform into astrocytes as a normal process ofleads to expression of GFP in radial glia, ependymal cells and subsets of cells in development [42,43]. Recent in vivo studies havethe ventricular zone (VZ), a property that has been exploited by some investigatorsand has lead to controversy in the field. Abbreviations: ENCAM, embryonic neural suggested that radial glia form the majority of progenitorcell-adhesion molecule; P, postnatal day, SVZ, subventricular zone. cells that give rise to neurons of the cortical germinal zonehttp://tins.trends.com
    • 354 Review TRENDS in Neurosciences Vol.26 No.7 July 2003and might also function as a self-renewing multipotent in vitro. The idea that neural stem cells themselves do notpopulation [44 – 46]. Moreover, it has recently been exist in large numbers in the adult brain, but thathypothesized that adult SVZ cells might be derived from neurospheres can be generated from cells that undergoembryonic radial glia that retain neuroepithelial stem-cell transformation in culture, does not contradict the avail-characteristics into adulthood [33]. able data, although the process of transdifferentiation and its frequency is relatively controversial [55].Astrocytes as stem cellsSteindler and colleagues have argued that, in addition to Studying neurosphere-forming stem cellsGFAP-expressing SVZ cells and GFAP-expressing radial The problem of studying stem cells in general has beenglia, at least during perinatal stages, a subset of astrocytes compounded by the difficulty of propagating a pureis multipotent. Laywell and colleagues have shown that population of stem cells in vitro. Neurosphere cultures inastrocytes can be cultured in vitro and induced to generate a defined medium and high concentrations of EGF haveneurospheres that can then differentiate into neurons, allowed investigators to propagate and maintain neuralastrocytes and oligodendrocytes [6,39]. Consistent with stem-cell populations and represent a major technicalthe idea that astrocytes might be stem cells at least in vitro advance. Most investigators have, however, been unable toare experiments showing that glial cells do not undergo maintain stem cells as a pure population of cells. Mostsenescence [47,48]. Moreover, these cells express high neurospheres in culture comprise a heterogeneous popu-levels of TERT and are spontaneously immortal. Thus, lation of cells with the number of self-renewing stem cellsat least some astrocytes fulfill the criteria of stem cells being a fraction of the total population (often ,5%). The[20,21]. It should be emphasized, however, that not all inability to purify a homogenous population of stem cellsastrocytes are stem cells, because the frequency of has precluded the use of large-scale comparative analysisneurosphere generation from astrocyte cultures is not that has been used so successfully in other systems.robust and neurospheres cannot be generated from The divergence of opinion on the identity of the stem-astrocytes isolated from adult brain. Furthermore, cell population in vivo and its precise location has furthermost early neurospheres do not express GFAP in hampered our ability to analyze stem-cell development inculture. This suggests that astrocytes are likely to undergo vivo. It has not been possible to harness transgenica kind of transformation in culture to dedifferentiate or technology, long-term slice cultures, retroviral labelingtransdifferentiate into stem cells and that they are not techniques or real-time visualization strategies to examinelikely to behave as stem cells in vivo. directly fundamental biological issues such as symmetric and asymmetric division, differentiation and cell-cycleStem cells as tissue culture artifacts or transdifferentiated regulation, or to combine these observations with large-cells scale genomic analysis techniques to define the process ofGiven the evidence that few stem cells can be identified in stem-cell self-renewal and differentiation in detail and,vivo and that a stem-cell response is not an important thus, to manipulate it to enhance repair and regeneration.aspect of neural repair and regeneration, it is important to To surmount these difficulties in studying neural stem-consider whether the cells that form neurospheres in vitro cell populations, investigators have begun to assessrepresent transformed cells that do not possess stem-cell several strategies. Selection strategies to identify stem-characteristics in vivo. It is possible, for example, that cell populations prospectively have been developed andmanipulation in culture of differentiated or partially markers that can be used to localize stem cells in vivo havedifferentiated neural cells can induce dedifferentiation begun to be assessed. These results are exciting and offerinto a stem-cell state. Indeed, experiments described by the potential for visualizing stem cells in vivo andBrewer [49] and Raff [50] suggest that this is possible and following their development in transgenic animals andis a robust occurrence. Brewer and colleagues [49] in purified culture assays. Some of the recently describeddescribed how postmitotic neurons could be induced to markers are discussed in the following sections.dedifferentiate into dividing progenitor cells, and Kondoand Raff [51] showed that glial progenitor cells can be Universal stem-cell markers and stem-cell-subtypededifferentiated and up to 80% of the population can then markersbe induced to differentiate into neurons. The cellular properties used to define a neural stem Other investigators have suggested that stem cells that population – the ability to form neurospheres, the abilitynormally contribute to other tissues might be able to to self-renew and the ability of single cells to differentiatecontribute to neurogenesis. Such transdifferentiation has into neurons, astrocytes and oligodendrocytes – mightbeen described in multiple reports [52,53]. Furthermore, correlate with expression of general molecular markers,Weismann and colleagues showed in an impressive set of which are referred to here as ‘universal stem-cell markers’experiments that circulating hematopoietic progenitors [56 –63] (Table 2). The word ‘universal’, rather thancan reside in many tissues, including the brain, indicating ‘specific’, is used here because so far no marker that isthat non-neural stem cells could be present in the brain in exclusive to stem cells has been identified. However,small numbers [54]. More recently, Doetsch et al. have markers that can be expressed by many potential stem-cellprovided compelling evidence that progenitor cells retain candidates or transdifferentiated cells have been categor-stem-cell properties [40]. Specifically, they showed that ized as universal. In addition to already identifiedafter exposure to high concentrations of EGF, type-C universal markers (see following discussion), severalamplifying progenitors of the SVZ function as stem cells laboratories have begun using large-scale analysishttp://tins.trends.com
    • Review TRENDS in Neurosciences Vol.26 No.7 July 2003 355Table 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 [63] dividing populations Sox-1 expression Appears relatively specific to neural stem-cell populations, [80,87,88] although it persists (transiently) in some progenitor-cell populations Sox-2 expression Appears to be expressed by all neurosphere-forming cells, [79,82,88] with localization to regions rich in stem cells 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 [20,21] non- stem-cell populations Low levels of Hoechst and Rhodamine staining Appear to be specific for quiescent stem-cell populations but [20,73,74] do not identify rapidly-dividing stem-cell populations ABCG2 expression Appears relatively specific in vivo and in vitro [20,75,89] Aldefluor labeling Might be a non-specific method of prospectively identifying [90 –92] stem-cell populations Absence of differentiation markers Has been used successfully to enrich for stem-cell populations [20,69,70] at multiple stages of developmenta 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 requisitespecificity 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 allneural stem-cell populations. Abbreviation: TERT, telomerase reverse transcriptase.techniques to compare stem-cell populations. Results from selection, be used to select for stem-cell populations fromthese analyses suggest that there might be additional neurosphere cultures. Using a similar negative selectionuniversal markers that characterize all stem cells [64– 67]. strategy, Maric et al. used surface ganglioside epitopesOur laboratory, for example, compared the expression of emerging on differentiating CNS cells to isolate neural500 genes by stem cells and has identified eleven genes progenitors from E13 rat telencephalon by fluorescence-related to the cell cycle and apoptosis that might be unique activated cell sorting (FACS) [71].to early-developing neural stem cells [68]. These studies also raise the possibility that stem cells Positive selection strategyfrom different tissues might be more closely related than Even though it is possible to identify and isolate stem cellspreviously assumed and could share common molecular prospectively from a mixed population, it is difficult to useregulators. Indeed, several investigators have argued for an absence of expression of markers to localize stem cellsthe concept of ‘stemness’ or a molecular signature that is in vivo, given the multiplicity of markers required.universal for stem-cell populations, irrespective of the Therefore, parallel approaches have identified positivetissue from which they are identified. Several investi- selection markers that can be used to identify neural stemgations have profiled gene expression in different stem-cell cells. Weissman and colleagues have suggested thatpopulations and have found that embryonic, hematopoie- AC133 might be an additional neural stem-cell markertic and neural stem cells share many similarities at the [72], although it is currently useful only in human tissue.transcriptional level [65 –67]. Quesenberry and colleagues have shown that within a neurosphere derived from adult tissue, populations of cellsNegative selection strategies that display low levels of staining with Hoechst andThis review briefly summarizes a number of approaches Rhodamine-123 are enriched for stem cells [73,74]. Thethat take advantage of universal stem-cell markers to efflux is likely to be mediated by ABCG2, a member of theisolate, characterize and manipulate neural stem cells by multi-drug-resistance family of transporters that is pre-both prospective positive and negative selection strategies. sent on neural stem cells during development and isSeveral groups have proposed a negative selection downregulated in differentiated cells [20,75]. Morecriterion that takes advantage of the observation that recently, it has been demonstrated that the LeX/SSEA-1stem cells do not express markers characteristic of antigen is expressed by a subset of cells in the adult SVZdifferentiated cells. Rao and colleagues have used the but not in the ependymal zone. Using this cell-surfaceabsence of expression of neuronal, astrocytic and oligo- antigen for FACS sorting, Capella and colleagues weredendroglial markers to enrich samples for stem cells from able to isolate cells that formed multipotent neurosphereslate fetal stages [20,69]. Whether these data can be from the adult brain [32].extrapolated to adult stem cells remains to be determined. An alternative approach to select positively andBartlett and colleagues [70], in similar experiments, have prospectively for neural stem cells is to generate mousesuggested two potential markers that can be used to enrich lines in which the expression of a drug-selection marker orfor neural stem cells in adults. The authors showed that green-fluorescent protein (GFP) is driven by regulatorylow levels of staining for peanut agglutinin (PNA) and elements of a universal neural stem-cell marker [76 – 78].heat-stable antigen (HSA) can, when combined with size For example, the SOXB1 subfamily of transcriptionhttp://tins.trends.com
    • 356 Review TRENDS in Neurosciences Vol.26 No.7 July 2003factors, which includes SOX1, SOX2 and SOX3, represents expression in neurospheres could help to define whetherone group of conserved pan-neural markers. Sox1, Sox2 a particular neurosphere contains a multipotent stem cell.and Sox3 are coexpressed in proliferating neural progeni- Universal stem-cell markers provide a means totors throughout rodent embryogenesis and into adulthood identify cells that fulfill the basic criteria of a stem cell –[79 – 81]. Zappone et al., using a Sox2 promoter to direct self-renewal and multipotential differentiation – and,expression of b-gal, have shown that telencephalic stem thus, define shared features when the cells are removedcells express Sox2 in vivo and that this expression persists from their environmental milieu. They cannot, however,in neurospheres derived from the telencephalon for at distinguish between types of stem cell. However, it hasleast 40 generations [82]. More recently, to develop an been previously shown that positional markers that definein vivo system for analyzing neurogenesis, Pevny and the rostrocaudal identity of stem cells persist over multiplecolleagues generated transgenic mice expressing an generations in vivo [82 – 84]. It is, therefore, reasonable toenhanced GFP (EGFP) under the control of the regulatory assume that markers characteristic of the cell thatregions of the Sox2 gene. In this mouse line, EGFP generates a neurosphere in vitro will persist, at leastexpression is confined to progenitor-cell populations over initial passages. Thus, if markers exist that dis-during early development and persists in selected popu- tinguish between the potential stem-cell candidates, itlations in the adult (Ellis et al., unpublished; Fig. 3). might be possible to determine whether all neurospheresMoreover, prospective clonal analysis of SOX2 –EGFP- are derived from the same population or from a hetero-positive cells demonstrates that multipotential stem cells geneous population, the properties of which depend on theisolated from both the embryonic CNS and the adult CNS particular stem-cell population that generated them.all express SOX2 – EGFP. Potential markers that might distinguish the cell of Thus, positive and negative selection criteria can be origin of neurosphere-forming cells are listed in Table 3.used to define populations of stem cells at any stage of This table is by no means complete but serves as andevelopment. These markers, either singly or in concert, important starting point. If, for example, most first-might help to localize stem cells in vivo, and their generation neurospheres contained predominantly cells Overview of SOX2–EGFP expression in embryonic and adult CNS SGZ of adult hippocampus (a) (b) (c) (d) EGFP expression at E10 Ependyma and SVZ of adult LV Ependyma of adult central canal Multipotential neurospheres arise from SOX2–EGFP+ single cells from adult LV (e) (f) (g) (h) EGFP+ single cell EGFP+ single cell EGFP+ neurosphere GFAP-TuJ TRENDS in NeurosciencesFig. 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-cellpopulations 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 expressedthroughout 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 singleEGFP-positive cell is multipotent. Scale bars, 100 mm (a– d) and 20 mm (e– h). From Ellis et al. (unpublished).http://tins.trends.com
    • Review TRENDS in Neurosciences Vol.26 No.7 July 2003 357Table 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 [22– 24,41–43] astrocytes S-100b and GLAST Might distinguish between stem cells in the VZ and cortex and those derived from radial glia and [41– 46] astrocytes 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 [20,69] other dividing progenitor-cell populationsa 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 neuralstem-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 moleculebearing polysialic acid; VZ, ventricular zone.that expressed polysialic-acid-bearing neural cell- capacity and has allowed investigators to begin to probeadhesion molecule (PSA-NCAM) and were self-renewing, fundamental aspects of stem-cell biology [54].then one could reasonably presume that PSA-NCAM- A potential problem with this approach is thepositive cells present in vivo generate most of the neuro- possibility that neurospheres might not contain onlyspheres seen in culture. If, by contrast, cells were a stem-cell population. Stem cells differentiate inpredominantly GFAP-positive (and PSA-NCAM-nega- response to environmental signals and it is difficulttive), then one would assume that a population of to ensure homogeneity of the microenvironment in aGFAP-expressing cells (i.e. astrocytes, SVZ type-B cells neurosphere-type culture, and it should be emphasizedor radial glia) generates neurosphere-forming cells. that prospective identification of cells by a combinationDouble-labeling experiments using additional markers, of markers must be validated (at least initially) bysuch as S-100b, RC2 and the glutamate-transporter rigorous single-cell clonal analysis.GLAST, might further distinguish between thesepossibilities. The presence of a cilium [31] and the Summarycoexpression of neuro – glial markers might identify Initial investigations suggest that multiple classes of stemependymal cells as the predominant neurosphere- cells exist during early development and in the adult. Atforming population. The presence of a heterogeneous least one cell type in the SVZ, and at least one additionalpopulation of neurospheres would suggest that mul- population of cells in regions of the brain where there is no SVZ, must be stem cells. The non-SVZ stem cell might be atiple stem-cell populations are present. Indeed, such radial glial cell, an astrocyte or a transformed cell, and itheterogeneity of neurosphere populations has already might be possible to distinguish between these possibi-been postulated on anatomical criteria [28,85]. lities by marker expression. Staining with universal stem- None of the markers listed in Table 3 is unique to stem cell markers, combined with markers that distinguishcells and expression of these markers is also seen in between stem-cell populations in early-passage neuro-differentiated cells. It might, therefore, be necessary to sphere cultures, will determine the cell of origin of thecombine potential cell-type-specific markers with univer- neurosphere-forming cell. Persistence of these specificsal stem-cell markers in double-labeling or cell-isolation stem-cell-type markers in vitro would suggest that the cellexperiments. For example, if a GFAP-positive cell that does not transform or alter its properties, and changes informed a neurosphere coexpressed TERT, had high expression of these markers would suggest that thetelomerase activity, expressed Sox1 and Sox2 and had properties of a stem cell in culture do not reflect itshigh ABCG2 expression and activity, then it would be properties in vivo. Recent breakthroughs in identifyingreasonable to assume in subsequent experiments that this stem-cell markers suggest that it will be possible torepresented a stem cell. The coexpression of GFAP with localize stem cells in vivo and to correlate the proper-such a universal cell marker would allow this particular ties of the stem cell with its in vitro neurospherestem cell to be distinguished from an RC1-positive stem- counterpart using universal and cell-type-specific mar-cell population and to be localized in vivo. It is crucial, kers. Identifying universal and cell-type-specific markershowever, that apart from determining the expression of will allow comparison of results between laboratories thatmarkers in early-passage neurospheres, the properties of grow neurospheres but use different isolation procedures.the cells are also tested rigorously by passaging and single- Furthermore, the ability to examine the stem-cell niche incell cloning. Equally important would be to demonstrate vivo, to obtain relatively large numbers of potential stemthe absence of markers that define dividing populations of cells, and to propagate stem cells for prolonged periods incells that are not multipotent but are more restricted; such vitro are relatively unique to the study of neural stem-cellmarkers include A2B5, CD24 and CD44 (see preceding populations. Clearly additional experiments are needed,discussion). Experiments along similar lines in the but it is exciting that techniques and markers have begunhematopoietic system have helped to define in vivo the to be identified that allow investigators an unprecedentedstem-cell population that has the highest self-renewing view of stem-cell biology.http://tins.trends.com
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