Neural stem cells

Journal of Pathology J Pathol 2002; 197: 536–550. Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002 / path.1189

Review Article

Neural stem cells Nigel L. Kennea and Huseyin Mehmet* Weston Laboratory, Institute of Reproductive and Developmental Biology, Division of Paediatrics, Obstetrics and Gynaecology, Imperial College of Science, Technology and Medicine, London W12 0NN, UK

* Correspondence to: Huseyin Mehmet, Weston Laboratory, Institute of Reproductive and Developmental Biology, Division of Paediatrics, Obstetrics and Gynaecology, Imperial College of Science, Technology and Medicine, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, UK. E-mail: [email protected]

Abstract Neural stem cells (NSCs) have the ability to self-renew, and are capable of differentiating into neurones, astrocytes and oligodendrocytes. Such cells have been isolated from the developing brain and more recently from the adult central nervous system. This review aims to provide an overview of the current research in this evolving area. There is now increasing knowledge of the factors controlling the division and differentiation of NSCs during normal brain development. In addition, the cues for differentiation in vitro, and the possibility of transdifferentiation are reviewed. The discovery of these cells in the adult brain has encouraged research into their role during neurogenesis in the normal mature brain and after injury. Lastly other sources of neural precursors are discussed, and the potential for stem cells to be used in cell replacement therapy for brain injury or degenerative brain diseases with a particular emphasis on cerebral ischaemia and Parkinson’s disease. Copyright # 2002 John Wiley & Sons, Ltd. Keywords:

brain development; stem cell; differentiation; neurogenesis; brain injury

What is a neural stem cell? Pluripotent stem cells are usually derived from embryonic tissues. At least three different types of mammalian pluripotent stem cell have been identified: embryonal nor carcinoma (EC) cells, embryonic stem (ES) cells derived from the inner cell mass of blastocysts, and embryonic germ (EG) cells obtained from postimplantation embryos. In the early 1990s, several groups reported the existence of a subset of stem cells found in the central nervous system (CNS). These cells, derived from fetal rat or mouse brain, grew in culture, displaying an almost unlimited lifespan. However, they were more restricted in their differentiation potential, compared with ES cells, giving rise predominantly to the three major cell types of the CNS: neurones, astrocytes, and oligodendrocytes, and were therefore named NSCs. Such cells have also been isolated from the adult CNS, although it is still not clear whether these dividing cells are truly multipotent, or whether their fates have become more restricted through development [1]. For the purposes of this review, NSCs have been defined as neural cells with the potential to selfrenew and to generate all the different cell types of the nervous system following differentiation.

An overview of brain development during embryogenesis Neural differentiation is an early event in mammalian embryogenesis that occurs soon after germ layer differentiation. The tissues of the CNS are derived from a clearly defined neuroectoderm, the neural plate, which lies along the dorsal midline of the embryo. It appears Copyright # 2002 John Wiley & Sons, Ltd.

that the neural plate arises by the local suppression or avoidance of signals that induce non-neural differentiation. Examples of such signals are from the bone morphogenetic proteins (BMPs) that instruct epidermal differentiation around the start of gastrulation, and other transforming growth factor-b (TGF-b) superfamily molecules [2,3]. Neural fate is suppressed by BMPs [4] and, in vivo, several molecules that promote neural differentiation such as noggin, follistatin, and chordin are BMP antagonists. While noggin antagonizes BMP signalling, it is not essential for induction of early neurogenesis since knockout mice are normal at embryonic day 8.5 (although they die at birth). These data suggest that other BMP antagonists may compensate in the absence of noggin expression and illustrate the concept of redundant signalling pathways during embryonic development [5]. The actions of BMPs are further complicated by the fact that there are two BMP receptors, the ubiquitously expressed BMPR-1A and BMPR-1B, which does not appear until embryonic day 9. Expression of BMPR-1A induces BMPR-1B expression, a process that is inhibited by sonic hedgehog [6].

CNS stem cells The further development of the neural plate into the cells of the mature CNS is clearly and precisely regulated with both spatial and temporal patterns of differentiation. Growth and proliferation of the cells in the early neural plate eventually result in the closure of the developing neural groove to form the hollow neural tube. The cavity of the neural tube later gives rise to the ventricular system, while the epithelial layer contains the stem cells that will give rise to the neuronal

Neural stem cells

and glial cells of the CNS. One of the central questions in developmental neurobiology has been the mechanism by which the simple neuroepithelium, which is only a single cell thick, can give rise to the diverse cell types that make up the mammalian CNS. There is now a wealth of research identifying both the intrinsic factors and the extrinsic soluble signals that affect this regional patterning and specific neural differentiation. One example of this in vertebrate neurogenesis is the opposing soluble signals that impart dorsal and ventral patterning: sonic hedgehog (Shh) and the BMP antagonists, chordin and noggin, are secreted from the floor plate [7], while other signals originate from the roof plate to form gradients of signal concentration. The precise concentration and ratio of each signal within the neural tube are central to the development of specific neuronal phenotypes at different points along the gradients. For example, there is a concentrationdependent induction of patterning genes within progenitor cells that encode homeodomain transcription factors. Differences in such expression patterns create groups of neurones with different patterns of division and differentiation (and ultimately distinct phenotypes) along the dorso-ventral axis of the plate [8,9]. In the human, the neural tube is formed during the third and fourth weeks of gestation. Initially, the neuroepithelial lining consists of morphologically similar NSCs in a single layer. These cells then divide symmetrically to enrich the NSC pool, or asymmetrically to generate more differentiated progeny from which mature cells of neuronal and glial lineages develop. The signals that determine symmetrical or asymmetrical division are not fully understood [10], although presumably the BMP family of molecules is again involved. Retroviral labelling studies have been used to identify dividing cells in the ventricular zone. Cai et al. demonstrated that about 48% of labelled cells remained in colonies within the ventricular zone, suggesting selfrenewal at this site [11]. It is likely that in humans and other mammals this active proliferation of progenitors will be balanced by apoptosis in order to maintain a steady population, but the exact mechanisms are yet to be determined [12,13]. As development continues, neurones migrate away, guided in part by radially oriented glial processes, and the ventricular zone diminishes in size. Neural stem cells remain attached to the basal lamina. It has been demonstrated that these cells may divide asymmetrically and more differentiated progeny migrate away from the ventricular zone towards the overlying cortex. Such cells can further divide and can be distinguished from neural stem cells by the acquisition of specific phenotypic markers [14]. Neurones first, then glia

During CNS development, temporal patterning results in the generation of neuronal cell types prior to oligodendrocytes [15,16]. In the spinal cord, it seems that these two cell populations can arise from common precursors, and the final fate decision depends on Copyright # 2002 John Wiley & Sons, Ltd.

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extrinsic signals, and specific patterns of transcriptional activation [8,17]. There are two main classes of transcription factor that can determine neuronal or glial fate. These are the homeodomain factors, an example of which is NKx2.2 [18], and the basic helix-loop-helix family of transcription factors that include Olig1 and Olig2 [19,20]. The expression of the Olig transcription factors is regulated by extrinsic signals such as Shh and their expression has been shown to define clearly cells that become oligodendrocytes. However, in the mouse, Olig1 and Olig2 are expressed from E9, well before the presence of oligodendrocyte precursors. At this early stage, Olig2 is known to be central to neuronal development in a specific region of the ventral spinal cord [21] and localizes to cells that can be tracked to motor neurone fate. The importance of Olig2 in neurodevelopment has been demonstrated in gain-offunction experiments [22,23]. As development proceeds, Olig1 and Olig2 expression persists and begins to overlap with the homeodomain transcription factor NKx2.2. These double-positive progenitor cells migrate away from the ventral midline and mature into oligodendrocytes. Recently Zhou and Anderson developed Olig1 and Olig2 double-mutant mice [24]. These animals lack oligodendrocytes and also display a considerable loss of motor neurones. In the Olig1/Olig2 double mutants, the progeny of stem cells in the pMN region of the developing cord, which normally develop into motor neurones and then oligodendrocytes, instead form V2 inter-neurones and subsequently astrocytes. These results suggest that the expression of combinations of transcription factors determines the fate of pools of pluripotent cells in the developing embryo. This does not, however, preclude the existence of more restricted dividing cell populations. The expression of pro-gliogenic transcription factors may be regulated through cell surface receptors such as notch [25]. It has been shown that jagged-1 (a notch ligand) signalling by neurones suppresses the oligodendrocyte phenotype [26]. Presumably, when the neuronal numbers are sufficient, jagged-1 is downregulated and pro-oligodendrocyte signals (one of which may be an increase in electrical activity) trigger myelination [27]. Once a neural precursor has made a fate commitment to the oligodendrocyte lineage, the final step to the formation of a myelin-forming cell requires the presence of the transcription factor Sox 10. Indeed, NSCs lacking this transcription factor fail to myelinate retinal ganglion axons following transplantation [28]. At this point, neurotrophic signals such as fibroblast growth factor (FGF), thyroid hormone, and plateletderived growth factor (PDGF) are important players in the promotion of oligodendrocyte formation [29]. Several other trophic factors and their receptors are also involved in fate choice, both in vivo and in vitro. As outlined earlier, members of the TGF-b superfamily are central [30] and play specialized roles in the maturation of specific cell types. Thus, BMP-2 induces the basic helix-loop-helix transcription factor Mash-1 J Pathol 2002; 197: 536–550.

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and is involved in neurogenesis, while BMP-4 and BMP-7 enhance adrenergic sympathetic neurones in neural crest cell cultures. Other growth factors such as glial-derived neurotrophic factor (GDNF) are also involved. GDNF-deficient mice have deficits in dorsal root ganglia and sympathetic neurones, as well as lacking an enteric neural network [31–33]. A similar phenotype is observed in mice lacking the GDNF receptor [34] and other growth factor receptors including Ret and ErbB [35]. The importance of extrinsic signals in the decision process has been demonstrated by Temple and colleagues [36], who observed that FGF concentrations also increase during fetal development and that this specifically promotes glial differentiation, possibly explaining the switch from neuronal to glial fate. Significantly, this study showed that the intrinsic ability of neural precursors to generate neurones was not diminished with time, but rather that the environment became progressively more gliogenic.

Neural crest stem cells (NCSCs) At the margins of the neural plate, an additional group of neural stem cells reside within the neural crest and give rise to progeny that form the peripheral nervous system as well as craniofacial mesenchyme and melanocytes [37]. There are several stages to the development of NCSCs, including the early specification of such cells from more multipotent cells of the neural plate, proliferation in the dorsal neural tube, and finally, separation from the tube [38], at which point they become recognized as a specific and clearly defined cell population. The process of delamination from the neural tube has been studied in detail and involves the down-regulation of several key neural adhesion molecules including NCAM, N-cadherin, and cadherin 6b [39]. These changes are thought to be necessary for cell migration away from the neural tube, since ectopic overexpression of such molecules prevents neural crest delamination [40]. Like CNS stem cells, NCSCs have the capacity to self-renew and develop into mature cell types with neural and non-neural phenotypes. They differ from CNS stem cells by their extraordinary ability to migrate long distance and by their ability to differentiate into mesenchymal lineages. It is likely, however, that the NCSCs and CNS stem cells share a common FGF-dependent progenitor [41] and that NCSCs are a heterogeneous population of multipotent cells varyingly committed to neural or mesenchymal fates.

Neurogenesis in the adult mammalian brain Ongoing neurogenesis in the adult was identified as early as 1962 by Altman, who demonstrated [3H]thymidine incorporation in the adult rat brain [42,43]. Because of the very limited recovery of brain tissue from CNS injury, it seemed unlikely that an endogenous pool of precursor cells could exact functional Copyright # 2002 John Wiley & Sons, Ltd.

N. L. Kennea and H. Mehmet

regeneration. Nevertheless, populations of dividing cells with the potential to self-renew and differentiate into mature neural phenotypes have been isolated from two main areas of the adult brain [44,45]. Dividing cells continue to be seen in the subventricular zone (SVZ), a remnant of the embryonic ventricular zones, and persist throughout life. In vivo, these progenitors give rise to migrating cells that eventually differentiate into inter-neurones within the olfactory bulb [46]. A similar population of progenitor cells has also been identified in the subgranular zone of the hippocampus that contributes to ongoing neurogenesis in the dentate gyrus [44,47,48].

Neurogenesis in the adult subventricular zone (SVZ) The SVZ lies along the lateral walls of the lateral ventricles and contains actively proliferating cells throughout adult life [49]. As outlined earlier, antagonism of BMP signals favour neurogenesis in the embryo; a similar function has been identified in the adult SVZ, where noggin, expressed by ependymal cells, can also block BMP signalling. In the absence of noggin, BMP favours gliogenesis over the production of neurones. In this way, a specific gliogenic niche is created in the SVZ [50]. Proliferation of SVZ stem cells has been demonstrated by [3H]thymidine and bromodeoxyuridine (BrdU) incorporation into dividing cells. In these labelling experiments, the progeny have been demonstrated to migrate forward along the rostral migratory tract into the olfactory bulb, where they develop into inter-neurones [46,51–54]. In the rodent, it takes only 2–6 days for the cells to cover this distance of up to 8 mm. These migrating neuronal precursor cells, also known as type A cells, form long chains and migrate without the guidance of axons or radial glia. These chains are ensheathed by layers of specialized GFAP-positive astrocytes (type B cells), of which there are at least two forms, based on whether they contact the ependyma (type B1) or not (type B2) [55]. Chain migration has been demonstrated using explant cultures and has been shown to be severalfold faster than the equivalent migration along radial glia. Type A cells express nestin (a neural precursor marker), TuJ1 (a neurone-specific tubulin), and polysialylated NCAM (PSA-NCAM). This polysialylated form of NCAM is thought to be very important in chain migration, since NCAM null mice [56,57] and those in which the PSA form is degraded [58] show impaired cell migration and smaller olfactory bulbs. There are at least two other cell types in the SVZ: type C cells, initially thought to be rapidly proliferating immature precursors that form clumps adjacent to migrating type A cells [59], and the ependymal cells (type E cells) that line the ventricular cavity. There has been considerable debate as to which cells in the SVZ are the multipotent ‘stem cells’ that give rise to the migrating neuronal type A cells. Experiments measuring [3H]thymidine and BrdU incorporation have J Pathol 2002; 197: 536–550.

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been unhelpful, since considerable uptake has been observed in many of the SVZ cell types. Johansson et al. [60] have suggested that the ependymal cells are the SVZ stem cells. They demonstrated that Dil (a fluorescent dye)-labelled ependymal cells could divide in vivo and over time, labelled cells were seen to appear as neurones in the olfactory bulb. Co-culture experiments with labelled and unlabelled cells showed that the dye was unlikely to be transferred between cell types. Dil-labelled cells increased after experimental injury and many cells became GFAP-positive astrocytes. The assumption that ependymal cells are the stem cells that generate type A cells has been challenged by other groups, based on their very slow rate of division and the possibility of Dil labelling occurring in small proportions of other cell types in the SVZ uptake experiments. Indeed, the work of Alvarez-Buylla’s group suggested that type A cells do not self-renew [61], but can be generated from cultures enriched for type B and C cells. In later studies by the same group, following the specific ablation of type A and type C cells, it was found that type B cells, the astrocytes, gave rise to new type C cells which in turn differentiated into the migrating neural cells [62]. This was confirmed by specifically labelling type B cells with retroviral vectors and tracking their migration to the olfactory bulb. Finally, purified SVZ-derived astrocytes cultured in the presence of FGF and epidermal growth factor (EGF) are able to form expanding neurospheres [63] and differentiate into neuronal phenotypes [62]. This result clearly challenges the traditional dogma that NSCs give rise to neurones first, then glia. In fact, it is a clear example of how glial cells might generate neurones! Further evidence for this intriguing possibility was obtained very recently, when it was shown that in the hippocampus, astrocytes are capable of regulating neurogenesis by instructing the stem cells to adopt a neuronal fate and may themselves be the hippocampal stem cells [64].

Neurogenesis in the adult hippocampus In addition to the SVZ, active neural cell proliferation has been discovered in the dentate gyrus in rodents. The dividing cells arise in the subgranular zone and then migrate into the granule cell layer, where they differentiate to acquire neuronal morphology and phenotype. Gage and co-workers have demonstrated a similar population of dividing cells in a group of oncology patients who had BrdU infusion for diagnostic purposes and later died [65]. In all treated patients, BrdUlabelled cells were present in the granule layer and co-expressed neuronal markers. Very recently, it has been claimed that while neurogenesis occurs in both the dentate gyrus and the subependymal zone, NSCs reside only in the latter region. Seaberg and Van der Kooy elegantly microdissected and cultured cells from both brain regions and observed that only committed neuronal and glial progenitors were present in the dentate gyrus, while true NSCs Copyright # 2002 John Wiley & Sons, Ltd.

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were present in the olfactory bulb. One caveat of this study is that cells were studied in culture and so it cannot be excluded that an important factor necessary for plasticity of dentate gyrus NSCs is absent in vitro [66]. On the other hand, using retroviral GFP labelling of clonally derived NSCs from the adult (rat) hippocampus, Gage and co-workers showed that these cells could develop into neurones or astrocytes [67]. Post-mitotic GFP-labelled neurones displayed action potentials and functional synaptic inputs [68]. However, it was not shown whether a single NSC could give rise to both astrocytes and neurones in culture and so committed neural progenitors for each lineage may have been present in the starting material.

A role for neurogenesis in healthy adults? Since it is now evident that new neurones are constantly being generated in the adult, why does the mature brain display only a very limited capacity to repair? Perhaps this is a reflection not of the cell population that resides in adult neurogenic regions, but rather that the environment is not conducive to promote stem cell proliferation and terminal differentiation. Does adult neurogenesis represent an endogenous attempt to replace lost or damaged cells, or is it a more active process? In the avian brain, the song system, in particular the high vocal centre in canaries and zebra finches, has been well researched [69]. It seems that neurones are produced during adulthood to replace others [70]. Although the total number of neurones in this brain region remains constant, singing has been shown to increase the levels of brain-derived neurotrophic factor (BDNF). Both BDNF expression and the survival of newborn neurones are proportional to the number of songs in a given unit of time [71]. In this system, neurogenesis seems necessary for the learning of new song patterns and neuronal cell death is likely to be as important as new nerve formation [72]. There has been much interest in external influences on neurogenesis in the adult hippocampus, particularly since it is of primary importance in long-term potentiation of memory. In adult mice, neurogenesis in the hippocampus has been shown to be regulated by physical exercise, with subjects exposed to enhanced environments displaying improved performance in learning tasks such as the Morris water maze [73]. Similarly, Kempermann et al. have demonstrated an increase in the neuronal number in the dentate gyrus and in the size of the granule layer in adult mice housed in an enriched environment compared with litter-mate controls [74], while van Praag et al. reported increased neurogenesis in the dentate gyrus in mice given access to a running wheel [73]. These observations suggest that in healthy animals, ongoing neurogenesis in the adult hippocampus may contribute to behaviour, learning, and memory. The role of other external factors has also been studied. Gould and Tanapat have recently reviewed the effects of glucocorticoids and stress on hippocampal J Pathol 2002; 197: 536–550.

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neurogenesis [75]. Adrenal glucocorticoids appear to have an inhibitory effect on dentate cell proliferation in the rat that is most evident shortly after birth (the socalled stress hyporesponsive time), when endogenous steroids decline to low levels and granule cell neurogenesis is rapid. Experimental increases in glucocorticoids at this time markedly reduce cell proliferation. Similarly, it has been shown that exposure to stressful environments during this critical period also results in reduced cell division in the dentate gyrus, while repeated stress leads to profound and prolonged suppression.

Isolation, culture, and differentiation of neural stem cells Rodent neural cell precursors have been identified in many areas of the developing brain and also in specific regions of the adult CNS. Such cells have been shown by clonal analysis [76,77], labelling [78] or transplant experiments to have the potential for differentiation into all three neural cell types: neurones, oligodendrocytes, and astrocytes. It is important to recognize, however, that cells from different sites are not identical, displaying different growth characteristics, trophic factor requirements, and specific patterns of differentiation [14,79,80]. This suggests that more restricted progenitors arise through the developmental programme.

Are there more restricted NSC compartments? Pluripotent progenitors clearly differ in their potential according to the developmental stage at which they were isolated and the site from where they were obtained [14]. There is also good evidence that committed glial and neuronal precursors exist in the pool, as well as more primitive stem cells; studies of neural stem cell clones have demonstrated that there are single cells that can only give rise to cells of neuronal phenotype, and even cells whose progeny are single specific neuronal subtypes. Many investigators have now maintained pools of cells, from a variety of brain regions that are committed neuronal progenitors, but many experiments used mixed populations of cells. Much more recently, isolation of neuronal precursors has been successfully achieved by cell sorting of cells, transfected with green fluorescent protein under the early neuronal promoter of a1 tubulin, from the embryonic forebrain [81]. These cells did not give rise to glial populations. Similarly, glial precursors can be isolated from E13 in mice and E14.5 in rats [82]. Such cells can be identified by A2B5 immunoreactivity and purified by immunopanning using this antibody. These cells also express NKx2.2 and PLP-DM20, while other markers of more differentiated cells, such as PDGF receptor a, are absent [83]. Rao et al. have termed these cells glial-restricted precursors and have demonstrated their differentiation into oligodendrocytes and astrocytes both in vivo and in vitro, suggesting a common precursor. Other groups have isolated more restricted Copyright # 2002 John Wiley & Sons, Ltd.

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precursors. Raff et al. isolated and characterized the oligodendrocyte type-2 astrocyte precursor cell (O2A). This more committed cell type was initially discovered in the rat optic nerve [84], but has subsequently been isolated from other areas, and does not appear to differentiate into type 1 astrocytes in vivo. The differentiation of such cells into mature oligodendrocytes has been extensively studied and the stage of development defined by combinations of phenotypic markers. Astrocyte precursors have also been identified [85] that give rise to type 1 astrocytes, but not oligodendrocytes. These cells can be isolated from the rat optic nerve up to postnatal day 1, are A2B5+, and up-regulate glial fibrillary acidic protein (GFAP) upon differentiation into astrocytes.

Neural stem cells from embryonic stem cells Stem cells from the inner cell mass of the blastocyst are truly totipotent and give rise to all tissues, including those of the nervous system. ES cells can be maintained in long-term culture as floating aggregates, known as embryoid bodies, and retain their ability to differentiate into cell types of all three germ layers. Several groups have now proposed culture conditions that enrich neural progenitors from murine ES cells [86–88]. Protocols have been published suggesting that exposure to retinoids leads to the development of neural progenitors [89], which can be further differentiated to mature cell types. Okabe et al. developed elaborate sequential culture conditions that differentiate the ES cells towards a nestin-positive neural phenotype in medium supplemented with insulin, transferrin, selenium, and fibronectin, and then can maintain these more committed progenitors with the mitogen bFGF [86]. Growth factor withdrawal then triggers differentiation into neuronal and glial phenotypes. Other groups have successfully purified NSCs from ES cultures by selecting cells expressing the surface markers A2B5 or PSANCAM [87], or the transcription factors Sox 1 and Sox 2 [90]. In embryogenesis, Sox 1 is confined to the neuroepithelium of the neural plate, whereas Sox 2 is found in the early neural crest too. This approach allows enrichment of cells expressing the neurogenic basic helix-loop-helix transcription factors Mash 1 and Mash 4A as well as Pax 3 and Pax 6, which are known to be important in dorso-ventral neural tube patterning. More recently, with the increased availability of human ES cells [91], groups have shown that such cells can be propagated in a similar way to murine ES cells and can differentiate into neural lineages. Carpenter et al. have demonstrated such differentiation using combinations of growth factors and retinoic acid [92]. As in murine ES studies, they demonstrated that more committed neural progenitors could be enriched with antibody selection for A2B5 and PSA-NCAM using immunopanning or magnetic bead sorting. Other groups have confirmed these findings and have shown J Pathol 2002; 197: 536–550.

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that enriched neural precursors from ES cells can incorporate and differentiate in vivo [93]. Zhang et al. transplanted enriched neural precursors into the lateral ventricles of newborn mice and observed migration to multiple brain regions, followed by differentiation into cells with mature neuronal and astrocytic phenotypes. Interestingly, no mature oligodendrocytes were identified [94]. Such in vivo studies are important as they suggest that transplanted cells have the potential to populate the brain [95,96]. This has led to work in injury models to see whether transplanted cells can integrate and functionally improve outcome following defined CNS insults where multiple cell types are lost [97,98].

Culture conditions for neural stem cells Growth factor requirements define at least two major classes of stem cell in the CNS. One group of cells requires high levels of EGF [99] and can be expanded as floating cell aggregates, called neurospheres (Figure 1), for many passages without apparent phenotypic change, although they eventually become FGF-responsive [100,101]. These neurospheres are a heterogeneous clonal group of cells that can be propagated by mechanical dissociation. Such multipotent cells are immunoreactive for nestin and readily incorporate [3H]thymidine or BrdU. Only a small proportion of the cells within a neurosphere are likely to be multipotent and can normally be identified by their ability to form further neurospheres. Weiss and co-workers have demonstrated that EGF-dependent cells cannot be isolated before E14.5 in the rat, some time after the beginning of neurogenesis [102].

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A second group of precursor cells isolated from the CNS are FGF-dependent. These cells can be grown as floating colonies like EGF-dependent cells [77], but can also be propagated as adherent cultures [103]. FGFdependent stem cells do not respond to EGF and indeed do not express the EGF receptor either in vivo or in vitro. It has been suggested that EGF-dependent cells may arise from FGF-dependent precursors, but this remains unproven. Both types of neural precursor have been isolated in multiple brain regions and appear to co-exist. Both can self-renew and can differentiate in vitro into the three neural cell types. Differentiation in culture can be driven by growth factor withdrawal or by specific inductive signals. Both labelled EGF and FGF-dependent neural stem cells have also been successfully transplanted into animal models and both seem to differentiate unambiguously into all three neural phenotypes. The roles of EGF and FGF themselves have been investigated using intraventricular infusions. In animal models, these growth factors cause an increase in the size of the ventricular zone and promote cell division defined by BrdU incorporation. In the EGF experiments, vigorous division of EGF-responsive cells is followed by differentiation into predominantly glial cells [104]. In contrast, FGF directs modest cell division and promotes a more neuronal phenotype [104,105]. It thus seems likely that distinct stem cell populations are programmed to differentiate into specific cell types during development in response to growth factor signals. Much of the above work has been performed in rodent models, although similar multipotent cells have been identified in the human [106,107]. Uchida et al.

Figure 1. Human neurosphere clones derived from post-mortem brain tissue of an 8-week-old human embryo. Dissociated neural cells were cultured in 10% fetal calf serum and the medium was changed every 2 days. After 4 days, the cells aggregated to form neurospheres (left). After a further 2 days in culture, the spheres flattened out and cells with neural morphology (arrow) began to emerge (right). Bar=50 mm. Copyright # 2002 John Wiley & Sons, Ltd.

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have described the isolation of neural stem cells, and subsequent neurosphere cultures, from fresh human fetal tissue. The cells were obtained from dissociated brain and spinal cord, and enriched by fluorescence activated cell sorting. Subsequently, it was demonstrated that single CD133+, CD34x, CD45x, CD24x/lo, 5E12+ cells could generate neurospheres, self-renew, and differentiate into neurones and glia. When these cells were injected into the lateral ventricles of immunodeficient NOD/SCID new-born mice, they showed engraftment, migration, and region-specific neuronal differentiation on examination up to 7 months later [108]. Clear differences have been found between rodent- and human-derived neurosphere cultures. For example, the survival of human neurospheres in culture is greatly enhanced by the neurotrophic factors NT3 and NT4, together with PDGF [109]. With the identification of adult neurogenesis, it was only a matter of time before neural stem cells were cultured from the adult. In fact, such cells have now been cultured from cadavers up to 5 days after death [110]. There are clearly similarities between the in vivo and culture properties of neural stem cells from rodents and man, but there are notable differences as well. One of the most evident is that differentiation of rat neurospheres generates large quantities of oligodendrocytes, whereas similar experiments in human cells generate very few. The reason for this is not clear and more work is needed to determine what signals influence the cell phenotype and, indeed, what are the specific cues for terminal differentiation.

Transdifferentiation To confuse matters further, it has been reported that NSCs can dedifferentiate to less committed progenitors and even into cell types normally arising from the other two germ layers (endoderm and mesoderm). Normal diploid cells in the mature organism contain almost identical DNA and it is the selective transcription or repression of specific genes that determines the cell type. It is now becoming increasingly clear that any somatic cell has the potential to be reprogrammed to a less differentiated state [111], although the mechanisms underlying this process are not yet understood. Cell fusion studies have been used to determine that pluripotent stem cells contain factors that can reprogramme the nuclei of host somatic cells to become less restricted in their differentiation potential. For example, the fusion of thymocytes with embryonal carcinoma cells reactivated the (inactive) X-chromosome in the malederived thymocytes [112]. Similarly, co-culture of SVZ NSCs with muscle cells can induce their differentiation into myocytes expressing specific muscle markers [113]. This plasticity is not restricted to NSCs and there are numerous reports of non-neural stem cells undergoing transdifferentiation to a pro-neural form (reviewed in [ref. 114]). More intriguingly, a population of Copyright # 2002 John Wiley & Sons, Ltd.

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mesenchymal stem cells has been isolated from bone marrow stroma that can be induced to generate neurones [115–118], oligodendrocytes [119,120], and astrocytes [117,119–122]. There is also evidence of growth factor-mediated dedifferentiation in vivo. Kondo and Raff caused purified oligodendrocyte progenitors to become NSCs by treating them with BMPs [29]. The resulting cells were now capable of generating neurones and astrocytes, although the existence of minute numbers of contaminating stem cells in the original optic nerve preparation was not formally ruled out. In a separate study of patients treated with human EGF to aid the healing of leg ulcers, the regenerating epidermis contained cells expressing b-1 integrin and keratin 19, markers of epidermal stem cells [123]. However, these experiments did not exclude the possibility that existing stem cells were recruited to the wound site. However, the concept of stem cell transdifferentiation is not universally accepted [124]. In one study, Bjornson et al. intravenously injected clonally derived NSCs into the tail veins of irradiated mice and found that they generated significant numbers of haematopoietic cells [125]. However, these results could not be reproduced in a study by Van der Kooy and coworkers, where 1 million neurosphere cells were injected into the tail vein of each of 128 host animals. Unlike Bjornson et al., they failed to detect significant numbers of haematopoietic cells arising from the labelled neurospheres and it was suggested that long-term culture of stem cells in the earlier experiments may have accounted for the difference in the results [126]. More recently, two independent studies have provided an alternative explanation for transdifferentiation. In the first, by co-culturing GFP-labelled neurones with hygromycin-resistant ES cells, undifferentiated hybrid cells with molecular characteristics of both neurones and ES cells were obtained [127]. In the second study, co-culture of bone marrow cells (labelled with GFP) with ES cells resulted in fused cells that were GFP-positive and expressed a number of ES cell genes. Significantly the hybrid cells were predominantly tetraploid, demonstrating that bone marrow cells can fuse spontaneously with other cell types, adopting their phenotype [128]. Of course, ethically, the transdifferentiation debate is of particular interest. It has been argued that if adult stem cells can be induced to differentiate into any cell type, then the need for embryonic stem cell research is reduced [129]. However, a large body of research still suggests that fetal stem cells are significantly more plastic than their adult counterparts.

Neurogenesis and brain injury Much research is now focused on understanding neurogenesis in degenerative diseases and following cerebral injury. This research both investigates the potential for endogenous repair and aims to learn more about the J Pathol 2002; 197: 536–550.

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permissive and restrictive cues in the damaged brain that will be crucially important if cell replacement therapy is considered in the future. It is now clear that injury to the CNS does indeed result in the proliferation of neural precursors. A number of studies have used BrdU labelling in conjunction with neurone-specific markers to demonstrate the proliferation and differentiation of endogenous neural precursors following experimental stroke [130]. For example, using a gerbil model of cerebral ischaemia, the proliferation, migration, and differentiation of NSCs were investigated using BrdU together with PSA-NCAM and NeuN to label NSCs and neurones, respectively. BrdU-labelled NSCs were first detected in the subgranular zone of the dentate gyrus 20 days after injury, with NeuN-positive neurones increasing up to 60 days following injury [131]. Similarly, neurogenesis was found to increase ten-fold in the subgranular zone of the dentate gyrus after global ischaemia in the gerbil [132]. Endogenous repair in response to stroke can also involve the proliferation of neural progenitor cells in the SVZ. Following middle cerebral artery occlusion, injection of BrdU specifically labelled cells in the ependymal and subependymal layers. Interestingly, the cells stained as astrocytes immediately after stroke but later acquired the characteristic antigenic markers of neurones after injury [133], lending further support to Alvarez-Buylla’s hypothesis of SVZ stem cells. In a separate model, employing chemically induced seizures in the rodent, a pronounced increase in the generation of neuronal precursors in the SVZ and their subsequent migration and integration towards the olfactory bulb were reported [134]. Proliferation was demonstrated using BrdU labelling and migration was confirmed by retroviral tracing. While it was proposed that ischaemia-induced neurogenesis might contribute to the specific recovery of memory function lost following injury, a high proportion of the dividing cells were lost over the weeks following injury. It remains to be demonstrated whether such responses are specific or represent a generic global response that occurs in areas that already have ongoing adult neurogenesis. The demonstration of production and survival of neural cell types following injury has led to interest into whether exogenous cells could be used for cell replacement in disease and injury where cells are lost.

Repairing the injured nervous system with stem cells As outlined above, there is exciting evidence that neural cell proliferation and differentiation occur in the healthy adult brain and can be further stimulated by injury. Neural cells, however, have a limited capacity to regenerate and the small population of endogenous neural stem cells seem unable to reconstitute fully and restore function after damage. This has Copyright # 2002 John Wiley & Sons, Ltd.

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led several groups to examine the potential of cell replacement therapy after cerebral injury [135,136]. These studies leave little doubt that under appropriate conditions, cellular grafts can integrate and function in the brain. There are three main sources of cells for replacement: neural tissue from fetuses; immortalized cell lines; and ES cells. Fetal neural cell transplantation has been used in several brain injury models. Fetal cortical grafts survive in the infarct area following focal forebrain ischaemic injury in adult rats and appear to receive connections from the surrounding brain, with a resulting improvement in motor function [137], spatial learning, and memory [138]. Experience already exists of fetal neural cell transfer in humans suffering neurodegenerative disorders. Over 300 patients with Parkinson’s disease have now received fetal mesencephalic precursors grafted into the striatum. These grafts are spontaneously active and can restore dopamine release to near-normal levels with symptomatic improvement. There is a downside, however; in a recent clinical trial in Denver and New York, 15% of grafted patients developed unacceptable dyskinesias [139]. Moreover, the supply of fetal neural tissue is limited and consequently only small numbers of neurones are available. This may be partially overcome by in vitro expansion. Encouragingly, embryonic day 12 NSCs propagated in culture retain the capacity to differentiate into dopaminergic neurones and to improve outcome in a rat model of Parkinson’s disease [140]. Immune rejection and subsequent failure of the graft can also be a problem, particularly in the xenotransplantation of fetal dopaminergic neurones, where there is poor graft survival and no clinical improvement [141]. One proposed advantage of embryonic cells is that they can be modified using cloning technology to express the patient’s own genotype. This technology could be used to provide a source of immune-compatible cells for transplantation. However, it has been argued that far from being rejected as transplants, the capacity for unlimited growth in ES cell cultures poses a real risk of tumour formation in recipients. Thus, there may be more than ethical reasons for using adult stem cells rather than those derived from embryos [142]. As an alternative to fetal tissue, immortalized cell lines have been employed in animal studies of brain injury. Neurones grafted from a human teratocarcinoma cell line into rats with focal ischaemia resulted in histological integration and functional improvement [143], as did grafting a hippocampal neuroepithelial cell line into damaged hippocampus in the mouse [144]. A number of studies have also demonstrated the successful transplantation of oligodendrocyte progenitor cells (OPCs) for demyelinating diseases. OPCs transplanted into the spinal cord of rats prior to experimental autoimmune encephalomyelitis (EAE) induced by injection of myelin basic protein survived for long periods of time [145]. Impressively, the cell line used (CG4) was shown to migrate as far as 6 cm from J Pathol 2002; 197: 536–550.

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the site of injection, but did not integrate into the white matter tracts of healthy rats, and did not proliferate uncontrollably, despite being transformed. The close association of the transplanted progenitors with reactive astrocytes suggests that contrary to the current dogma, inflammatory signals within the injured CNS actually promote integration. These results agree with those of Franklin et al. [146], who found that CG4 cells can also integrate into ethidium bromide-induced demyelinating lesions in the spinal cord. In this study too, the cells migrated away from the injection site to repopulate areas of demyelination and were again unable to penetrate uninjured white matter tissue. There are, however, considerable fears that immortalized cell lines are prone to tumourigenesis and that they are unable to reconstitute the wide variety of cell types lost in cerebral injury. This makes them of only limited use in clinical applications. Embryonic stem cells provide the most promising alternative source of cells for therapeutic transfer. They are pluripotent, can be propagated in vitro, and can be engineered to express therapeutic genes. They migrate and differentiate into regionally appropriate cell types, and do not appear to interfere with normal brain development [95]. The first demonstration that mouse ES cells can be induced to express multiple neural phenotypes in culture was reported by Bain et al. [89], who used retinoic acid as the trigger for differentiation. The newly formed neurones were capable of generating action potentials and expressed numerous ion channels. ES cells expanded and differentiated in vitro into oligodendrocyte precursors can effectively myelinate host axons in animal models of human myelin disease [147,148]. Cortical neurones undergoing injury-induced apoptosis can be replaced by transplanted embryonic neural stem cells and, importantly, multipotent neural progenitors implanted into lesioned hippocampus, neocortex or striatum in adult rodents demonstrate appropriate differentiation in situ into region-specific neuronal and glial subtypes [149]. In the developing brain, stable clones of neural stem cells can participate in aspects of normal brain development when injected into the germinal zones of newborn mice.

Factors affecting transplant success A number of factors can influence the success of cellbased therapies. Not only are environmental factors important, but also the source of stem cells, their developmental stage, natural programmed cell death (by apoptosis) processes, and the receptiveness of the host environment will all contribute to the ultimate survival of grafted cells. For example, it appears that neural precursors cultured from different areas of the brain have different properties. In a recent study, Svendsen’s laboratory found that cells isolated from the developing rat brain, propagated as neurospheres, have different growth properties and give rise to cells with distinct phenotypes depending on their site of origin [150]. The stage of differentiation of the Copyright # 2002 John Wiley & Sons, Ltd.

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transplanted cells may also be a factor. For demyelinating diseases, the use of multipotential neural precursors, rather than more restricted oligodendrocyte precursors, seems to be more useful [151–153]. The developmental age of the NSCs used for transplantation may also be a significant factor in determining successful outcome. Embryonic or adult NSCs?

Comparisons of fetal stem cells with those derived from defined developmental niches in the mature brain seem to indicate that NSC plasticity decreases with developmental age. Although the precise mechanisms are not fully understood, there is evidence that many of the fate decisions may be cell-intrinsic [154]. For example, EGF receptor expression increases in cortical stem cells with increased passage number and this may result in an increased responsiveness to this ligand [155]. Human fetal NSCs have been successfully isolated from fresh post-mortem brain tissue by exploiting the unique expression of the cell surface marker CD133 together with the absence of CD34 and CD45. Not only can single cells with this antigenic phenotype form neurosphere cultures that differentiate into both neurones and glia, but they can also be successfully transplanted into brains of immune-deficient neonatal mice [108]. The importance of such studies is illustrated by the fact that they can be extrapolated to non-human primates. Using BrdU-labelled human NSCs, Snyder and co-workers observed that transplantation into the developing primate forebrain resulted in the integration of the donor cells into both the mature cerebral cortex and the subventricular zone, where, presumably, they remained until needed for further neurogenesis or post-injury repair [156]. One further advantage of fetal NSCs is that they can be rapidly propagated in vitro with little or no change in their plasticity. In one study, human neural progenitors isolated from embryonic forebrain were expanded for up to a year in culture using EGF, FGF, and leukaemia inhibitory factor (LIF). Subsequent injection of these cell lines into the developing rat brain showed extensive migration and integration [157]. For many, the thought of using fetal tissue for stem cell transplantation is morally unacceptable. Coupled with the spectre of couples conceiving with the sole purpose of obtaining aborted brain tissue for the treatment of one or other of the parents, scientists have turned to the most unlikely sources for NSCs. Indeed, investigators have claimed to have isolated functional NSCs from adult post-mortem brain tissue as late as 20 h after death [110]. Although it is suspected that adult NSCs have a more limited ability to form all the neural subtypes, they may have an even broader potential than first thought. Using a chick mouse chimera approach, adult stem cells were reported to give rise readily to cells in all the germ layers, demonstrating a high degree of plasticity and indicating that their application may go beyond the treatment of CNS J Pathol 2002; 197: 536–550.

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disorders [158]. Although it is known that stem cells in the subventricular zone of the adult mammalian brain can proliferate, migrate to the olfactory bulb, and ultimately differentiate into mature neurones, are these cells functional? In an elegant study from Frisen’s laboratory, labelled stem cells were shown to respond to a specific odour-induced signal (by up-regulating expression of the c-Fos proto-oncogene) in the olfactory bulb. These results indicate that newly formed adult neurones can functionally integrate into the synaptic circuitry of the mature brain [159]. Survival of grafts

Areas of adult neurogenesis have been shown to contain high numbers of apoptotic cells [160]. Presumably, during normal adult neurogenesis, programmed cell death plays an important regulatory function by eliminating excess cells from neurogenic regions, similar to that observed in the embryo [161]. Apoptosis of transplanted NSCs may also be a major factor in determining the successful outcome of cell therapy. Inhibition of caspases can significantly increase the survival of nigral transplants in a model of Parkinson’s disease [162]. Survival of dopaminergic grafts into the striatum of Parkinsonian rats is also significantly increased if the transplants are spiked with a small population of fibroblasts expressing FGF. Presumably this growth factor both acts as a survival signal and enhances neuronal differentiation [163]. Similarly, stem cell factor (SCF) is important in the survival of defined stem cell populations, although it is not clear whether this is true for NSCs [164]. In the generation of dopaminergic neurones derived from cultures of pluripotent mouse ES cells, increased numbers of tyrosine hydroxylase (TH, the first and rate-limiting enzyme in dopamine synthesis) and dopamine-producing neurones were obtained in the presence of neurotrophins in combination with defined survival-promoting factors, including IL-1b and GDNF [165]. In parallel experiments, the mRNA levels of the anti-apoptotic gene bcl-2 were also increased in these cultures. However, the role of apoptosis in graft survival is not straightforward, since the anti-apoptotic gene bcl-2 only protects against apoptosis in culture, while NSCs isolated from transgenic mice overexpressing Bcl-2 do not show increased survival on grafting; however, they do, rather intriguingly, display improved fibre outgrowth [166]. One should also consider that engineering NSCs to express pro-survival genes could increase the risk of tumourigenesis. Clearly, more work is needed to determine the role of apoptosis in the survival of stem cell transplants. Cell environment

Cell–cell interactions may be important in determining the correct terminal differentiation of NSCs. While the culture of either embryonic striatal precursors or adult subependymal cells in the presence of FGF yielded only small numbers of neurones producing TH, the inclusion of conditioned medium from glial Copyright # 2002 John Wiley & Sons, Ltd.

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cell cultures increased the yield of TH-positive neurones by more than 17-fold. Embryonic striatal precursors were significantly more responsive to the differentiation environment, further indicating that stem cells from earlier developmental sources may provide more successful transplants [167]. The importance of the host environment has been demonstrated by transplantation into the cerebral ventricles of embryonic hosts in utero. Not only do the donor cells differentiate into neurones, but they also acquire the phenotype of the surrounding cells. McKay and co-workers found that cells incorporated into the host hippocampus assumed morphologies resembling granule and pyramidal neurones, while those that integrated into the inferior colliculus resembled tectal neurones that reside in this region (reviewed in [168]). The precise site of injection can also influence the fate of transplanted NSCs. By marking transplanted cells with GFP, human NSCs have been shown to differentiate into post-mitotic neurones throughout the brain, while differentiation into glial cell types occurred predominantly at the site of injection [157]. Such outcomes may be overridden by employing genetically modified donor cells that offer the advantage of combining cell replacement with gene therapy [169]. For instance, transplants into a refractory environment (e.g. where activated microglia are present) would have an increased chance of success if the cells transferred contained a gene to counteract this hostile environment. For example, it has been suggested that ectopic expression of the neural cell adhesion molecule L1 in astrocytes can increase the speed and efficiency of innervation of branching axons, thus improving the transplant success of grafted NSCs [170]. Stem cell therapy has been successfully employed for CNS lesions where there is significant cell loss; for example, to repair focal infarctions resulting from stroke. However, even small numbers of neurones undergoing apoptosis can be replaced by human fetal neural precursors. It seems that the brain can detect and respond to even small changes in cell number or subtle perturbations in normal function by providing the appropriate cues for available stem cells to differentiate and repair the damage [149]. At the other extreme, what would be the outcome of grafts in a situation where cell loss was so extensive that tissue structure was significantly disrupted? In an important new development, transplantation of a polymer scaffold seeded with NSCs may offer a significant improvement in motor function in a severe traumatic spinal cord injury model in rats [171].

Future perspectives There are issues that remain to be clarified about stem cell transplantation into injured brains, such as the ongoing debate as to which cells are best suited for transplantation therapy [172]. In an ideal world, one would be able to stimulate the proliferation and J Pathol 2002; 197: 536–550.

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appropriate differentiation of endogenous stem cells. In fact, a number of growth factor-based therapies probably work, at least in part, through this mechanism. Early experiments in stem cell transplantation suggested that embryonic tissue was significantly more plastic than that derived from the adult. Although subsequent research has indicated that adult stem cells possess a broader developmental potential than was first thought, they have a more limited lifespan than ES cells. Clearly, at this point in time, research should not be restricted to a particular cell type or a particular developmental age. It cannot be denied that human fetal stem cells, especially when derived as a result of therapeutic cloning, are ethically controversial. Cell replacement strategies must also contend with the fact that differentiation of multipotential cells is strongly influenced by environmental signals. In several models of adult brain injury, some transplants are prone to gliosis and apoptosis for prolonged periods after transfer and so clinical improvement may only be temporary [97]. Considerable work is therefore needed in order to identify the triggers for specific neural cell proliferation and integration, and to determine further how the environment of the injured brain may be manipulated to become more permissive for effective repair.

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