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Making and repairing the mammalian brain—signaling toward neurogenesis and gliogenesis
Seminars in Cell & Developmental Biology 14 (2003) 161–168
Making and repairing the mammalian brain—signaling toward neurogenesis and gliogenesis Y.E. Sun∗ , K. Martinowich, W. Ge Departments of Psychiatry and Behavioral Sciences and Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, NPI 48-149, 760 Westwood Plaza, Los Angeles, CA 90024, USA
Abstract Neural stem cells (NSCs) are subscribed extraordinary potential in repair of the damaged nervous system. However, the molecular identity of NSCs has not been established. Most NSC cultures contain large numbers of multipotent, bipotent, and lineage restricted neural progenitors, the majority of which appear to lose neurogenic potential after expansion. This review first discusses the neurogenic to gliogenic switch that is characteristic of progenitor development in vivo and in NSC cultures, and then the cell intrinsic and extrinsic mechanisms regulating the sequential differentiation of neurons and glia. Finally, we discuss potential methods for maintaining the neurogenic potential of NSC cultures after expansion. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: bHLH factors; Methylation; Gliogenesis; Neurogenesis; Stem cells
1. Introduction Neural stem cell (NSC) research has recently garnered significant public interest due to the extraordinary potential subscribed to NSCs in the repair of the damaged nervous system. However, the neurogenic potential of nearly all current mouse and rat NSC preparations decreases during in vitro expansion, which may present an obstacle in obtaining a stable source of NSCs. This change in neurogenic potential is recapitulated in the developing central nervous system (CNS), whereby multipotent neural progenitors (MNPs) first produce neurons then differentiate into glia. In this review, we will first discuss an important although frequently overlooked issue: the heterogeneity of most NSC cultures. Through this discussion, the myths portraying NSC preparations as being composed of homogeneous NSCs with stable self-renewing and multipotential features should be abandoned. We will then discuss some of the recently discovered cell intrinsic and extrinsic signals that serve to regulate the sequential onset of neuronal and glial differentiation. Finally, we discuss the potential ways to maintain the neurogenic potential of NSC cultures after in vitro expansion. The recent discovery of the oligo1/2 genes has provided many new insights into the development of the oligodendroglial lineage.
∗ Corresponding
author. Tel.: +1-310-267-0438; fax: +1-310-260-5050. E-mail address: [email protected] (Y.E. Sun).
Related work has been extensively reviewed by Stiles and co-workers [1], and therefore, will not be re-discussed here.
2. Neural stem cells and multipotent neural progenitors NSCs in the mammalian CNS are defined as those cells having the ability to self-renew as well as to maintain the potential of generating all three major cell types of the CNS: neurons as well as two types of glial cells, astrocytes and oligodendrocytes [2–4]. These two characteristics of NSCs, however, can only be manifested in vitro when NSCs are cultured as free-floating neurospheres or as monolayers adhered to the proper substrate. A number of important issues, including whether the NSCs are able to carry out unlimited cycles of self-renewal in vitro and in vivo, whether they are fast proliferating or relatively quiescent, and whether the characteristic division of NSCs is symmetric or asymmetric, still remain to be addressed. MNPs are usually defined as the very early neuroepithelial progenitors of the developing CNS. These cells essentially give rise to all neuronal, astroglial and oligodendroglial cells in the CNS. During development in vivo, MNPs are thought to first go through limited cycles of expansion through symmetric divisions [5–7]. At the point when neurogenesis begins, cell divisions become asymmetric. In the region of the cerebral cortex during the neurogenic period, MNPs have recently been found to be in the form of radial glia [8–10].
1084-9521/03/$ – see front matter © 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1084-9521(03)00007-7
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Fig. 1. MNPs switch from a neurogenic to a gliogenic state both in vivo and in vitro. In vivo, cerebral cortical MNPs first undergo symmetric divisions resulting in self-renewal. When cortical neurogenesis peaks at mouse embryonic day12 (E12) they undergo asymmetric divisions, and then switch back to symmetric divisions when astrogliogenesis starts at ∼E18. (NSCs may be generated only at certain points during development.) In vitro, progenitor cells isolated from the cortex at various developmental stages can proliferate and give rise to neurons, then astrocytes, and finally to oligodendrocytes. These two types of glial lineages may segregate from each other before they segregate from the neuronal lineage. The difference between MNP and NSC could be that NSC can undergo unlimited cycles of self-renewal, whereas MNP proliferate and subsequently differentiate.
Asymmetric divisions of the MNPs/radial glia generate neurons, which migrate along the radial glial fibers to populate the cortical plate. Towards the end of neurogenesis cortical progenitors switch back to symmetrical divisions. At this time a portion of the remaining progenitors finish up the last round of neurogenesis while some start to produce glial cells (Fig. 1). Work from Temple and co-workers has suggested that progenitor cells undergo prolonged series of symmetric divisions to give rise to astrocytes [11]. Interestingly, unlike astroglia, which can arise indiscriminately from many areas of the CNS, oligodendroglia appear to hail only from specific locations within the CNS, typically the more ventral regions of the neural tube [12,13]. Following differentiation, oligodendrocytes migrate into the white matter of the CNS to myelinate axons. Although in culture, both oligodendrocytes and type 2 astrocytes can arise from a common O-2A progenitor, it appears that in vivo the astrocytic and oligodendrocytic lineages segregate from each other before they segregate from the neuronal lineage [14]. As a result, in addition to multipotent progenitors, which can give rise to all three lineages, bipotential progenitors for astrocytes and neurons or oligodendrocytes and neurons are readily apparent in the developing CNS. On the contrary, common glial progenitors retaining the ability to give rise to both astrocytes and oligodendrocytes are rarely observed [1].
It is interesting to speculate that NSCs could be slowly dividing cells, which are only generated from MNPs in vivo at certain points during development. In culture, it appears that only a small portion of the MNPs isolated at any time during development have the potential of becoming the sphere-forming multipotent NSCs [15]. Bartlett’s group recently claimed that the low peanut agglutinin binding (PNAlow ) and low heat-stable antigen (HSAlow ) expressing, relatively large diameter cells (>12 m) purified from the adult CNS, are the sphere-forming multipotent NSCs [16]. As high as 80% of these cells appear able to form neural spheres that can be differentiated into all three neural lineages. It is necessary that more research be done to further validate this method of NSC isolation. Interestingly, although neural sphere formation is an artificial criterion, it does seem to be tightly associated with multipotency and self-renewal properties. It is worth noting that almost all the currently existing in vitro expanded preparations of NSC are heterogeneous. In those ‘NSC’ cultures, either in the form of neural spheres or as monolayers, only a minority of the nestin positive cells fit the stem cell criteria, i.e. the ability to both self-renew and maintain multipotency [4]. It is possible that the majority of the cells in the monolayered ‘NSC’ cultures are undifferentiated neuroblasts, glioblasts, bipotential neuro-glial progenitors or MNPs, and only a small portion are the sphere-forming NSCs (see below for the distinction between MNPs and NSCs). The cellular composition is even more diverse in cultured neural spheres, especially those larger ones, where in addition to all the undifferentiated progenitors, differentiated neuronal and glial cells, as well as cells with apoptotic features also exist. Furthermore, due to the three-dimensional association among the heterogeneous cell populations, a greater degree of cell–cell interactions likely occur, affecting the biological characteristics of various populations in the neural sphere including NSCs. Indeed, the neurogenic potential appears to be slightly higher in expanded neural spheres than in expanded NSCs maintained as a monolayer (Sun et al., unpublished observations, [15]). Surprisingly in the case of human fetal NSCs maintained as neural spheres, the neurogenic potential (20% of total cells become neurons upon differentiation) remains the same for up to 251 days [17]. Although the neurogenic potential of human NSCs cultured as monolayers has not been reported, the sustained neurogenic activities in human neural spheres as compared to those of rodent probably reflect the much more prolonged neurogenic period during human CNS development. Despite the fact that neural spheres may help maintain the neurogenic potential to a certain extent, the heterogeneity and the complicated cell–cell interactions in neural spheres present a major obstacle for dissecting out the molecular mechanisms by which proliferation and differentiation are regulated. To date most people have considered MNPs and NSCs as one and the same, but we believe that MNPs are distinguishable from NSCs based on their sphere formation
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(self-renewal) abilities. Only a small portion (<10%) of the early neuroepithelial cells (MNPs) isolated from the CNS can actually form neural spheres [62], whereas by definition, all NSCs should retain this capability. In addition, whether neuronal or glial fate restricted progenitors really do exist should be reconsidered, since more and more recent findings suggest that glial and even neuronal progenitor cells can have plastic potentials. When provided the optimal conditions, these cells are able to return to multi- or bipotent progenitor states, and subsequently produce both neurons and glia [18–20]. It is agreed that neuronal, astroglial, and oligodendroglial progenitors have different gene expression patterns from each other and from those of the multi- and bipotent progenitors. At which point the gene expression pattern becomes irreversibly established or whether there is a point at which no return is possible remains to be answered. Whether GFAP positive cells are really NSCs has been a hotly contested subject. One entertainable idea is that GFAP positive astrocytes, unlike neurons and oligodendrocytes, are not terminally differentiated cells. Although the gene expression program of these cells are likely different from that of MNPs, under appropriate conditions, these cells might be able to reprogram their gene expression profile to match that of MNPs or even that of a neuroblast to eventually produce neurons.
3. Sequential onset of neuronal and glial differentiation from neural progenitors Throughout the developing CNS, neuronal production almost always precedes the generation of glial cells. This sequence is conserved throughout the CNS with the exception of two regions, the subventricular zone of the forebrain and the hippocampal dentate gyrus. In these areas ongoing neurogenesis persists throughout the adult life. Unique, although currently unknown signals in these two regions must provide a niche that is conducive and nurturing for sustained neurogenesis. Similar to MNP in vivo, when isolated from the developing CNS and expanded in ‘NSC’ cultures, most neural progenitors appear to initially be more neurogenic and then become gliogenic. During in vivo development, differentiated neurons are proposed to communicate with progenitors in a negative feedback manner to instruct progenitors to switch from a neurogenic to a gliogenic state [21]. In expanded ‘NSC’ cultures maintained as a monolayer, however, differentiation is limited due to the continuous presence of the mitogen, bFGF. Therefore, in these cultures the feedback mechanism may not apply. Interestingly, progenitors in ‘NSC’ cultures still gradually switch from neurogenic to gliogenic. This suggests the possible existence of an intrinsic ‘clock’ that controls the differentiation course of neural progenitors, i.e. first neurogenic, then switch to gliogenic [1,11]. The presence of the ‘clock’ is further supported by the following findings. It is known that the totipotent
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embryonic stem (ES) cells, when allowed to differentiate spontaneously, produce neurons before the differentiation of any glia. Recently, many reports have described that ‘NSC’ cultures can be derived from ES cells. After a sphere (embryoid body, EB)-formation process which takes about 4–7 days, neural epithelial cells (neural progenitors) from partially differentiated ES cell cultures can be selectively expanded in standard ‘NSC’ culture conditions [22,23]. Interestingly, in ES cell-derived ‘NSC’ cultures, where neuronal and glial differentiation is suppressed from passage to passage in the presence of bFGF, progenitors still first enter a neurogenic period and then switch to a gliogenic phase (Fig. 2). The ES-‘NSC’ culture conditions are essentially the same through passages. Therefore, these findings support the existence of a cell intrinsic ‘clock’ for neural progenitor differentiation.
4. Changes in cell intrinsic properties allowing neural progenitor to switch from neurogenic to gliogenic Little is known about the mechanisms underlying the temporal switch made by neural progenitors from neurogenic to gliogenic either in vivo or in vitro. Both cell intrinsic and extrinsic factors may control this neurogenic to gliogenic switch. Although detailed gene expression profiles of progenitors in the neurogenic period versus the gliogenic period have not yet been established, a number of genes have been reported as being differentially expressed. These include the pro-neural basic helix–loop–helix (bHLH) transcription factors [24], suppressors of cytokine signaling 2 (socs2) [25], and the EGF receptor [26]. Both the pro-neural bHLH factors and socs2 are highly expressed in progenitors during the neurogenic period. EGF receptors, on the other hand, are expressed only in late progenitors. It is unclear whether the expression of EGF receptors is involved in the termination of neurogenesis and the triggering of glial differentiation. But, it is known that expression of the pro-neural bHLH genes is important for promoting neurogenesis as well as for suppressing gliogenesis during the neurogenic period [20,27]. The socs2 expression is thought to be important in maintaining pro-neural bHLH gene expression via inhibition of STAT5 activation [25]. The pro-neural bHLH genes are different from other neurogenic bHLH factors in that the pro-neural factors are expressed exclusively in dividing neural progenitors [28]. So far, four genes have been found in this category: neurogenin1 (ngn1), ngn2, mash1 and math1. Loss of expression of these genes leads to severe defects in neurogenesis and a precocious progenitor fate switch from neurogenic to gliogenic in the developing CNS of mutant mice [20,29–33]. Ngn1 not only promotes neurogenesis but also inhibits astroglial differentiation by different mechanisms [27]. A mutation of Ngn1, which abolishes its ability to bind DNA, is unable to induce neuronal differentiation, yet it can still suppress astrogliogenesis. It was proposed that Ngn1 suppresses the expression of astroglial genes in
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Fig. 2. ES-NSCs switch from being neurogenic to gliogenic upon expansion. ES cells were cultured in suspension for 1 week in the presence of LIF. After that, these cells (passage 0, P0) were moved onto polyornithine/fibronectin-coated dishes and cultured in bFGF-containing serum-free medium (P1). When cells became 80% confluent, they were passed onto new dishes for continuous expansion (P2, 3, 4, and 5). At each passage (shown for P2, 3, 4, and 5), a cohort of cells were allowed to differentiate for 4 days upon bFGF withdrawal. They were then subjected to immunostaining. Tuj1 (red) stains neurons, GFAP (green) marks astrocytes.
part by sequestering the transcription coactivating complex p300/CBP-Smad1 away from the STATs, which are known to act upon astroglial gene targets [27]. In addition, Ngn1 expression also inhibits the activation of the astrogliogenic JAK-STAT pathway in the developing CNS [27]. Later on during development, as the expression of the proneural bHLH factors declines, the JAK-STAT pathway and STATs’ function become enhanced, leading to subsequent glial differentiation (Fig. 3). Due to the action of Notch signaling, not all neural progenitors in the developing ventricular zone express the
pro-neural bHLH genes during neurogenesis [34]. What then are the factors that suppress precocious gliogenesis in cells that do not express detectable levels of bHLH factors? Recently, another cell intrinsic property was discovered, which is involved also in inhibiting precocious astroglial differentiation during development. This property was shown to be cytosine DNA methylation of glial genes [35,36]. Cytosine methylation at a CpG dinucleotide site is one of the major epigenetic modifications occurring in the mammalian genome [37], and is usually involved in gene silencing. During CNS development, it was reported
Fig. 3. Cell intrinsic properties inhibit precocious glial differentiation during the neurogenic period. Extracellular factors LIF, BMPs and activation of Notch signaling lead to glial differentiation in late neural progenitor cells. Their gliogenic function is suppressed during neurogenesis due to the expression of pro-neural bHLH facts and DNA methylation of glial genes. The pro-neural bHLH factors are highly expressed during neurogenesis, which inhibit glial differentiation by sequestering smad1-CBP away from glial-specific genes and by inhibiting activation of STATs. DNA methylation of glial genes during the neurogenic period also inhibits glial differentiation by suppressing glial gene transcription and inhibiting STAT1/3 phosphorylation.
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that one of the astroglial marker genes, GFAP, is heavily methylated within its promoter region during the neurogenic period [35,36,38]. It becomes demethylated at late embryonic stages slightly before the onset of glial differentiation. Recently, it was shown that methylation of the STAT-binding element within the GFAP promoter inhibits its association with activated/phosphorylated STAT1/3, and therefore is involved in silencing of this glial marker gene during neurogenesis [35,36]. Additional studies showed that a number of CpG sites surrounding the STAT-binding element also undergo demethylation at the time when progenitors become gliogenic [36]. Methylation of some of these sites has the ability to inhibit activation of the promoter as well. Such inhibition is likely mediated via the binding of DNA methyl-binding proteins such as MeCP2, which might trigger heterochromatin establishment through the recruitment of histone deacteylases (HDACs) and histone lysine methyl transferases [36,39–43]. In addition to the GFAP gene, another astroglial marker gene, s100β, undergoes demethylation at late developmental stages (Fan and Sun, unpublished observations). More importantly, the expression of the astrogliogenic transcription factor, stat1, is in itself regulated by DNA methylation. Methylation of particular CpG sites within the stat1 promoter are inhibitory for its transcription during the neurogenic period (Fan and Sun, unpublished observation). These sites become demethylated at late embryonic stages rendering the promoter more active. The role of DNA methylation in suppressing precocious astrogliogenesis in vivo is further supported by gene knockout studies. In a CNS-specific DNA methylatransferase 1 (dnmt1) knockout mouse, the CNS becomes prematurely demethylated resulting in precocious astroglial differentiation [36]. It is worth noting that the STAT-binding elements in both the s100β and stat1 promoters do not contain any CpG sites (Fan and Sun, unpublished observations). Therefore, methylation of the STAT-binding element to reduce STAT association may not be a general silencing mechanism. Alternatively, binding of repressional methyl-binding proteins to methylated promoters of various glial genes could result in remodeling of the chromatin structure, leading to gene repression. Moreover, DNA methylation appears to inhibit leukemia inhibitory factor (LIF) triggered STAT3 phosphorylation via inhibition of the LIF receptor gp130 expression, which leads to reduced activation of the tyrosine kinase JAK1, and consequently results in decreased STAT1/3 phosphorylation [36] (Fan and Sun, unpublished observations) (Fig. 3).
5. The diverse, cell context-dependent effects of extracellular factors on neuro- and gliogenesis In contrast to the limited work reported on the cell intrinsic factors affecting neuronal and glial differentiation, extracellular factors have been, for the past several years, at the center of research on NSC differentiation in vitro. It was proposed that platelet-derived growth factor (PDGF)
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can promote neurogenesis [44,45]. Alternatively, LIF and its family members, cilliary neurotrophic factor (CNTF) and interleukin-6 (IL-6), as well as activation of the Notch signaling pathway can promote astroglial differentiation [44,46–48]. Finally, thyroid hormone (T3) and Sonic hedgehog (Shh) enhance oligodendroglial differentiation [44,49]. The bone morphogenetic proteins (BMPs) can either enhance neurogenesis or glial differentiation depending on the age of the neural progenitors [50–52]. BMPs act on early, ngn1-expressing neural progenitors to promote neuronal differentiation [27] (Fig. 4). Alternatively, they work on late, or non ngn1-expressing progenitors to enhance the differentiation of the opposite astrocytic fate [27,52] (Fig. 4). In fact, the cell context-dependent effect of extracellular factors can be extended to almost all the factors mentioned above. For example, PDGF promotes neuronal differentiation only in early progenitor cells, likely through the preferential enhancement of neuroblast proliferation, which ultimately results in increased neuronal production upon their differentiation into neurons. In expanded late neural progenitors, PDGF is unable to promote neurogenesis, but still enhances the proliferation of progenitors. Interestingly, even the potent astroglial-inducing factor, LIF, is capable of enhancing neuronal differentiation in ngn1 expressing cells in vitro [53]. While LIF and CNTF use the JAK-STAT pathway, and the PI3-kinase-AKT pathway to promote gliogenesis [46,54], they use a different downstream pathway (likely, PKC) to enhance the transcription activation function of Ngn1, hence promoting neurogenesis (Hasan and Sun, unpublished observations). In addition, Notch signaling can only induce astroglial differentiation in late progenitors, but not in early ones [48]. It remains to be determined whether Shh and T3 act differentially on early versus late NSC cultures. Together, the above findings are reminiscent of a recurring theme in development; i.e. the same extracellular factors can be used repetitively during development to regulate very different developmental aspects. In order to achieve this goal those factors must be able to utilize various cell context-dependent molecular mechanisms to turn on different sets of gene expression programs.
6. Regulation of cell intrinsic properties Cell intrinsic factors can influence the actions of various extracellular factors. What then regulates those intrinsic factors? For example, what turns on the expression of neurogenic bHLH factors and what shuts them off at the end of neurogenesis? What triggers demethylation of various glial genes at the end of neurogenesis? All these questions are extremely important for understanding the biological ‘clock’ that controls the timing of neuronal and glial differentiation. The downstream Notch target genes hes1/5 and the negative HLH regulators, including the Id factors have been proposed to inhibit neurogenesis [55,56] as well as the expression and function of neurogenic bHLH factors
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Fig. 4. BMPs can either enhance neurogenesis or glial differentiation depending on the age of the neural progenitors. BMP stimulation of neural progenitors leads to smad1 phosphorylation. Smad1 subsequently associates with CBP/p300 to form a potent transcriptional coactivating complex. When Ngn is present, the coactivating complex will associate with Ngn to induce neurogenesis in early progenitors. The absence of Ngn in late neural progenitors leaves the coactivating complex free to associate with STATs to activate glial differentiation.
[47,57–59]. However, the action of these factors appears to be in existence throughout development. Not understanding the stage-dependent actions of these factors makes theorizing on how they regulate the neurogenic to gliogenic switch a daunting task. The mitogen bFGF is itself thought to inhibit neurogenesis, resulting in the gliogenic switch [60,61]. One difficulty in understanding the mechanisms underlying bFGF action is the fact that bFGF acts only gradually to convert neurogenic cells to a gliogenic state. Currently, no signaling mechanisms can explain such a gradual mode of regulation.
7. Conclusion Recently, much progress had been made towards understanding the molecular control of neuronal and glial differentiation in NSC cultures. Most currently existing mouse and rat NSC culture preparations have a tendency to lose their neurogenic potential upon expansion in vitro. Understanding the regulation of neurogenesis during development could help generate methods to maintain the neurogenic potential of NSCs even after expansion. For example, forced expression of exogenous neurogenic bHLH factors allows even late NSCs to become neurogenic (Sun, unpublished observations). However, an inducible system to control the expression of the exogenous pro-neural bHLH factors would be required to allow expansion of the progenitors containing the exogenous bHLH gene. Understanding the regulatory mechanisms for pro-neural bHLH gene expression may also allow us to come up with a means to switch on and off the endogenous pro-neural bHLH genes in NSC cultures to allow neurogenesis or progenitor expansion. Finally, it needs to be emphasized that nearly all extracellular fac-
tors have cell context-dependent actions. Understanding the detailed intracellular mechanisms of the extracellular factors in various cellular contexts will equip us with better methods to intervene or control the differentiation process of neural progenitors. Invariant control of the differentiation process will be critical for making NSC useful for medical applications.
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Making and repairing the mammalian brain—signaling toward neurogenesis and gliogenesis Y.E. Sun∗ , K. Martinowich, W. Ge Departments of Psychiatry and Behavioral Sciences and Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, NPI 48-149, 760 Westwood Plaza, Los Angeles, CA 90024, USA
Abstract Neural stem cells (NSCs) are subscribed extraordinary potential in repair of the damaged nervous system. However, the molecular identity of NSCs has not been established. Most NSC cultures contain large numbers of multipotent, bipotent, and lineage restricted neural progenitors, the majority of which appear to lose neurogenic potential after expansion. This review first discusses the neurogenic to gliogenic switch that is characteristic of progenitor development in vivo and in NSC cultures, and then the cell intrinsic and extrinsic mechanisms regulating the sequential differentiation of neurons and glia. Finally, we discuss potential methods for maintaining the neurogenic potential of NSC cultures after expansion. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: bHLH factors; Methylation; Gliogenesis; Neurogenesis; Stem cells
1. Introduction Neural stem cell (NSC) research has recently garnered significant public interest due to the extraordinary potential subscribed to NSCs in the repair of the damaged nervous system. However, the neurogenic potential of nearly all current mouse and rat NSC preparations decreases during in vitro expansion, which may present an obstacle in obtaining a stable source of NSCs. This change in neurogenic potential is recapitulated in the developing central nervous system (CNS), whereby multipotent neural progenitors (MNPs) first produce neurons then differentiate into glia. In this review, we will first discuss an important although frequently overlooked issue: the heterogeneity of most NSC cultures. Through this discussion, the myths portraying NSC preparations as being composed of homogeneous NSCs with stable self-renewing and multipotential features should be abandoned. We will then discuss some of the recently discovered cell intrinsic and extrinsic signals that serve to regulate the sequential onset of neuronal and glial differentiation. Finally, we discuss the potential ways to maintain the neurogenic potential of NSC cultures after in vitro expansion. The recent discovery of the oligo1/2 genes has provided many new insights into the development of the oligodendroglial lineage.
∗ Corresponding
author. Tel.: +1-310-267-0438; fax: +1-310-260-5050. E-mail address: [email protected] (Y.E. Sun).
Related work has been extensively reviewed by Stiles and co-workers [1], and therefore, will not be re-discussed here.
2. Neural stem cells and multipotent neural progenitors NSCs in the mammalian CNS are defined as those cells having the ability to self-renew as well as to maintain the potential of generating all three major cell types of the CNS: neurons as well as two types of glial cells, astrocytes and oligodendrocytes [2–4]. These two characteristics of NSCs, however, can only be manifested in vitro when NSCs are cultured as free-floating neurospheres or as monolayers adhered to the proper substrate. A number of important issues, including whether the NSCs are able to carry out unlimited cycles of self-renewal in vitro and in vivo, whether they are fast proliferating or relatively quiescent, and whether the characteristic division of NSCs is symmetric or asymmetric, still remain to be addressed. MNPs are usually defined as the very early neuroepithelial progenitors of the developing CNS. These cells essentially give rise to all neuronal, astroglial and oligodendroglial cells in the CNS. During development in vivo, MNPs are thought to first go through limited cycles of expansion through symmetric divisions [5–7]. At the point when neurogenesis begins, cell divisions become asymmetric. In the region of the cerebral cortex during the neurogenic period, MNPs have recently been found to be in the form of radial glia [8–10].
1084-9521/03/$ – see front matter © 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1084-9521(03)00007-7
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Fig. 1. MNPs switch from a neurogenic to a gliogenic state both in vivo and in vitro. In vivo, cerebral cortical MNPs first undergo symmetric divisions resulting in self-renewal. When cortical neurogenesis peaks at mouse embryonic day12 (E12) they undergo asymmetric divisions, and then switch back to symmetric divisions when astrogliogenesis starts at ∼E18. (NSCs may be generated only at certain points during development.) In vitro, progenitor cells isolated from the cortex at various developmental stages can proliferate and give rise to neurons, then astrocytes, and finally to oligodendrocytes. These two types of glial lineages may segregate from each other before they segregate from the neuronal lineage. The difference between MNP and NSC could be that NSC can undergo unlimited cycles of self-renewal, whereas MNP proliferate and subsequently differentiate.
Asymmetric divisions of the MNPs/radial glia generate neurons, which migrate along the radial glial fibers to populate the cortical plate. Towards the end of neurogenesis cortical progenitors switch back to symmetrical divisions. At this time a portion of the remaining progenitors finish up the last round of neurogenesis while some start to produce glial cells (Fig. 1). Work from Temple and co-workers has suggested that progenitor cells undergo prolonged series of symmetric divisions to give rise to astrocytes [11]. Interestingly, unlike astroglia, which can arise indiscriminately from many areas of the CNS, oligodendroglia appear to hail only from specific locations within the CNS, typically the more ventral regions of the neural tube [12,13]. Following differentiation, oligodendrocytes migrate into the white matter of the CNS to myelinate axons. Although in culture, both oligodendrocytes and type 2 astrocytes can arise from a common O-2A progenitor, it appears that in vivo the astrocytic and oligodendrocytic lineages segregate from each other before they segregate from the neuronal lineage [14]. As a result, in addition to multipotent progenitors, which can give rise to all three lineages, bipotential progenitors for astrocytes and neurons or oligodendrocytes and neurons are readily apparent in the developing CNS. On the contrary, common glial progenitors retaining the ability to give rise to both astrocytes and oligodendrocytes are rarely observed [1].
It is interesting to speculate that NSCs could be slowly dividing cells, which are only generated from MNPs in vivo at certain points during development. In culture, it appears that only a small portion of the MNPs isolated at any time during development have the potential of becoming the sphere-forming multipotent NSCs [15]. Bartlett’s group recently claimed that the low peanut agglutinin binding (PNAlow ) and low heat-stable antigen (HSAlow ) expressing, relatively large diameter cells (>12 m) purified from the adult CNS, are the sphere-forming multipotent NSCs [16]. As high as 80% of these cells appear able to form neural spheres that can be differentiated into all three neural lineages. It is necessary that more research be done to further validate this method of NSC isolation. Interestingly, although neural sphere formation is an artificial criterion, it does seem to be tightly associated with multipotency and self-renewal properties. It is worth noting that almost all the currently existing in vitro expanded preparations of NSC are heterogeneous. In those ‘NSC’ cultures, either in the form of neural spheres or as monolayers, only a minority of the nestin positive cells fit the stem cell criteria, i.e. the ability to both self-renew and maintain multipotency [4]. It is possible that the majority of the cells in the monolayered ‘NSC’ cultures are undifferentiated neuroblasts, glioblasts, bipotential neuro-glial progenitors or MNPs, and only a small portion are the sphere-forming NSCs (see below for the distinction between MNPs and NSCs). The cellular composition is even more diverse in cultured neural spheres, especially those larger ones, where in addition to all the undifferentiated progenitors, differentiated neuronal and glial cells, as well as cells with apoptotic features also exist. Furthermore, due to the three-dimensional association among the heterogeneous cell populations, a greater degree of cell–cell interactions likely occur, affecting the biological characteristics of various populations in the neural sphere including NSCs. Indeed, the neurogenic potential appears to be slightly higher in expanded neural spheres than in expanded NSCs maintained as a monolayer (Sun et al., unpublished observations, [15]). Surprisingly in the case of human fetal NSCs maintained as neural spheres, the neurogenic potential (20% of total cells become neurons upon differentiation) remains the same for up to 251 days [17]. Although the neurogenic potential of human NSCs cultured as monolayers has not been reported, the sustained neurogenic activities in human neural spheres as compared to those of rodent probably reflect the much more prolonged neurogenic period during human CNS development. Despite the fact that neural spheres may help maintain the neurogenic potential to a certain extent, the heterogeneity and the complicated cell–cell interactions in neural spheres present a major obstacle for dissecting out the molecular mechanisms by which proliferation and differentiation are regulated. To date most people have considered MNPs and NSCs as one and the same, but we believe that MNPs are distinguishable from NSCs based on their sphere formation
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(self-renewal) abilities. Only a small portion (<10%) of the early neuroepithelial cells (MNPs) isolated from the CNS can actually form neural spheres [62], whereas by definition, all NSCs should retain this capability. In addition, whether neuronal or glial fate restricted progenitors really do exist should be reconsidered, since more and more recent findings suggest that glial and even neuronal progenitor cells can have plastic potentials. When provided the optimal conditions, these cells are able to return to multi- or bipotent progenitor states, and subsequently produce both neurons and glia [18–20]. It is agreed that neuronal, astroglial, and oligodendroglial progenitors have different gene expression patterns from each other and from those of the multi- and bipotent progenitors. At which point the gene expression pattern becomes irreversibly established or whether there is a point at which no return is possible remains to be answered. Whether GFAP positive cells are really NSCs has been a hotly contested subject. One entertainable idea is that GFAP positive astrocytes, unlike neurons and oligodendrocytes, are not terminally differentiated cells. Although the gene expression program of these cells are likely different from that of MNPs, under appropriate conditions, these cells might be able to reprogram their gene expression profile to match that of MNPs or even that of a neuroblast to eventually produce neurons.
3. Sequential onset of neuronal and glial differentiation from neural progenitors Throughout the developing CNS, neuronal production almost always precedes the generation of glial cells. This sequence is conserved throughout the CNS with the exception of two regions, the subventricular zone of the forebrain and the hippocampal dentate gyrus. In these areas ongoing neurogenesis persists throughout the adult life. Unique, although currently unknown signals in these two regions must provide a niche that is conducive and nurturing for sustained neurogenesis. Similar to MNP in vivo, when isolated from the developing CNS and expanded in ‘NSC’ cultures, most neural progenitors appear to initially be more neurogenic and then become gliogenic. During in vivo development, differentiated neurons are proposed to communicate with progenitors in a negative feedback manner to instruct progenitors to switch from a neurogenic to a gliogenic state [21]. In expanded ‘NSC’ cultures maintained as a monolayer, however, differentiation is limited due to the continuous presence of the mitogen, bFGF. Therefore, in these cultures the feedback mechanism may not apply. Interestingly, progenitors in ‘NSC’ cultures still gradually switch from neurogenic to gliogenic. This suggests the possible existence of an intrinsic ‘clock’ that controls the differentiation course of neural progenitors, i.e. first neurogenic, then switch to gliogenic [1,11]. The presence of the ‘clock’ is further supported by the following findings. It is known that the totipotent
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embryonic stem (ES) cells, when allowed to differentiate spontaneously, produce neurons before the differentiation of any glia. Recently, many reports have described that ‘NSC’ cultures can be derived from ES cells. After a sphere (embryoid body, EB)-formation process which takes about 4–7 days, neural epithelial cells (neural progenitors) from partially differentiated ES cell cultures can be selectively expanded in standard ‘NSC’ culture conditions [22,23]. Interestingly, in ES cell-derived ‘NSC’ cultures, where neuronal and glial differentiation is suppressed from passage to passage in the presence of bFGF, progenitors still first enter a neurogenic period and then switch to a gliogenic phase (Fig. 2). The ES-‘NSC’ culture conditions are essentially the same through passages. Therefore, these findings support the existence of a cell intrinsic ‘clock’ for neural progenitor differentiation.
4. Changes in cell intrinsic properties allowing neural progenitor to switch from neurogenic to gliogenic Little is known about the mechanisms underlying the temporal switch made by neural progenitors from neurogenic to gliogenic either in vivo or in vitro. Both cell intrinsic and extrinsic factors may control this neurogenic to gliogenic switch. Although detailed gene expression profiles of progenitors in the neurogenic period versus the gliogenic period have not yet been established, a number of genes have been reported as being differentially expressed. These include the pro-neural basic helix–loop–helix (bHLH) transcription factors [24], suppressors of cytokine signaling 2 (socs2) [25], and the EGF receptor [26]. Both the pro-neural bHLH factors and socs2 are highly expressed in progenitors during the neurogenic period. EGF receptors, on the other hand, are expressed only in late progenitors. It is unclear whether the expression of EGF receptors is involved in the termination of neurogenesis and the triggering of glial differentiation. But, it is known that expression of the pro-neural bHLH genes is important for promoting neurogenesis as well as for suppressing gliogenesis during the neurogenic period [20,27]. The socs2 expression is thought to be important in maintaining pro-neural bHLH gene expression via inhibition of STAT5 activation [25]. The pro-neural bHLH genes are different from other neurogenic bHLH factors in that the pro-neural factors are expressed exclusively in dividing neural progenitors [28]. So far, four genes have been found in this category: neurogenin1 (ngn1), ngn2, mash1 and math1. Loss of expression of these genes leads to severe defects in neurogenesis and a precocious progenitor fate switch from neurogenic to gliogenic in the developing CNS of mutant mice [20,29–33]. Ngn1 not only promotes neurogenesis but also inhibits astroglial differentiation by different mechanisms [27]. A mutation of Ngn1, which abolishes its ability to bind DNA, is unable to induce neuronal differentiation, yet it can still suppress astrogliogenesis. It was proposed that Ngn1 suppresses the expression of astroglial genes in
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Fig. 2. ES-NSCs switch from being neurogenic to gliogenic upon expansion. ES cells were cultured in suspension for 1 week in the presence of LIF. After that, these cells (passage 0, P0) were moved onto polyornithine/fibronectin-coated dishes and cultured in bFGF-containing serum-free medium (P1). When cells became 80% confluent, they were passed onto new dishes for continuous expansion (P2, 3, 4, and 5). At each passage (shown for P2, 3, 4, and 5), a cohort of cells were allowed to differentiate for 4 days upon bFGF withdrawal. They were then subjected to immunostaining. Tuj1 (red) stains neurons, GFAP (green) marks astrocytes.
part by sequestering the transcription coactivating complex p300/CBP-Smad1 away from the STATs, which are known to act upon astroglial gene targets [27]. In addition, Ngn1 expression also inhibits the activation of the astrogliogenic JAK-STAT pathway in the developing CNS [27]. Later on during development, as the expression of the proneural bHLH factors declines, the JAK-STAT pathway and STATs’ function become enhanced, leading to subsequent glial differentiation (Fig. 3). Due to the action of Notch signaling, not all neural progenitors in the developing ventricular zone express the
pro-neural bHLH genes during neurogenesis [34]. What then are the factors that suppress precocious gliogenesis in cells that do not express detectable levels of bHLH factors? Recently, another cell intrinsic property was discovered, which is involved also in inhibiting precocious astroglial differentiation during development. This property was shown to be cytosine DNA methylation of glial genes [35,36]. Cytosine methylation at a CpG dinucleotide site is one of the major epigenetic modifications occurring in the mammalian genome [37], and is usually involved in gene silencing. During CNS development, it was reported
Fig. 3. Cell intrinsic properties inhibit precocious glial differentiation during the neurogenic period. Extracellular factors LIF, BMPs and activation of Notch signaling lead to glial differentiation in late neural progenitor cells. Their gliogenic function is suppressed during neurogenesis due to the expression of pro-neural bHLH facts and DNA methylation of glial genes. The pro-neural bHLH factors are highly expressed during neurogenesis, which inhibit glial differentiation by sequestering smad1-CBP away from glial-specific genes and by inhibiting activation of STATs. DNA methylation of glial genes during the neurogenic period also inhibits glial differentiation by suppressing glial gene transcription and inhibiting STAT1/3 phosphorylation.
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that one of the astroglial marker genes, GFAP, is heavily methylated within its promoter region during the neurogenic period [35,36,38]. It becomes demethylated at late embryonic stages slightly before the onset of glial differentiation. Recently, it was shown that methylation of the STAT-binding element within the GFAP promoter inhibits its association with activated/phosphorylated STAT1/3, and therefore is involved in silencing of this glial marker gene during neurogenesis [35,36]. Additional studies showed that a number of CpG sites surrounding the STAT-binding element also undergo demethylation at the time when progenitors become gliogenic [36]. Methylation of some of these sites has the ability to inhibit activation of the promoter as well. Such inhibition is likely mediated via the binding of DNA methyl-binding proteins such as MeCP2, which might trigger heterochromatin establishment through the recruitment of histone deacteylases (HDACs) and histone lysine methyl transferases [36,39–43]. In addition to the GFAP gene, another astroglial marker gene, s100β, undergoes demethylation at late developmental stages (Fan and Sun, unpublished observations). More importantly, the expression of the astrogliogenic transcription factor, stat1, is in itself regulated by DNA methylation. Methylation of particular CpG sites within the stat1 promoter are inhibitory for its transcription during the neurogenic period (Fan and Sun, unpublished observation). These sites become demethylated at late embryonic stages rendering the promoter more active. The role of DNA methylation in suppressing precocious astrogliogenesis in vivo is further supported by gene knockout studies. In a CNS-specific DNA methylatransferase 1 (dnmt1) knockout mouse, the CNS becomes prematurely demethylated resulting in precocious astroglial differentiation [36]. It is worth noting that the STAT-binding elements in both the s100β and stat1 promoters do not contain any CpG sites (Fan and Sun, unpublished observations). Therefore, methylation of the STAT-binding element to reduce STAT association may not be a general silencing mechanism. Alternatively, binding of repressional methyl-binding proteins to methylated promoters of various glial genes could result in remodeling of the chromatin structure, leading to gene repression. Moreover, DNA methylation appears to inhibit leukemia inhibitory factor (LIF) triggered STAT3 phosphorylation via inhibition of the LIF receptor gp130 expression, which leads to reduced activation of the tyrosine kinase JAK1, and consequently results in decreased STAT1/3 phosphorylation [36] (Fan and Sun, unpublished observations) (Fig. 3).
5. The diverse, cell context-dependent effects of extracellular factors on neuro- and gliogenesis In contrast to the limited work reported on the cell intrinsic factors affecting neuronal and glial differentiation, extracellular factors have been, for the past several years, at the center of research on NSC differentiation in vitro. It was proposed that platelet-derived growth factor (PDGF)
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can promote neurogenesis [44,45]. Alternatively, LIF and its family members, cilliary neurotrophic factor (CNTF) and interleukin-6 (IL-6), as well as activation of the Notch signaling pathway can promote astroglial differentiation [44,46–48]. Finally, thyroid hormone (T3) and Sonic hedgehog (Shh) enhance oligodendroglial differentiation [44,49]. The bone morphogenetic proteins (BMPs) can either enhance neurogenesis or glial differentiation depending on the age of the neural progenitors [50–52]. BMPs act on early, ngn1-expressing neural progenitors to promote neuronal differentiation [27] (Fig. 4). Alternatively, they work on late, or non ngn1-expressing progenitors to enhance the differentiation of the opposite astrocytic fate [27,52] (Fig. 4). In fact, the cell context-dependent effect of extracellular factors can be extended to almost all the factors mentioned above. For example, PDGF promotes neuronal differentiation only in early progenitor cells, likely through the preferential enhancement of neuroblast proliferation, which ultimately results in increased neuronal production upon their differentiation into neurons. In expanded late neural progenitors, PDGF is unable to promote neurogenesis, but still enhances the proliferation of progenitors. Interestingly, even the potent astroglial-inducing factor, LIF, is capable of enhancing neuronal differentiation in ngn1 expressing cells in vitro [53]. While LIF and CNTF use the JAK-STAT pathway, and the PI3-kinase-AKT pathway to promote gliogenesis [46,54], they use a different downstream pathway (likely, PKC) to enhance the transcription activation function of Ngn1, hence promoting neurogenesis (Hasan and Sun, unpublished observations). In addition, Notch signaling can only induce astroglial differentiation in late progenitors, but not in early ones [48]. It remains to be determined whether Shh and T3 act differentially on early versus late NSC cultures. Together, the above findings are reminiscent of a recurring theme in development; i.e. the same extracellular factors can be used repetitively during development to regulate very different developmental aspects. In order to achieve this goal those factors must be able to utilize various cell context-dependent molecular mechanisms to turn on different sets of gene expression programs.
6. Regulation of cell intrinsic properties Cell intrinsic factors can influence the actions of various extracellular factors. What then regulates those intrinsic factors? For example, what turns on the expression of neurogenic bHLH factors and what shuts them off at the end of neurogenesis? What triggers demethylation of various glial genes at the end of neurogenesis? All these questions are extremely important for understanding the biological ‘clock’ that controls the timing of neuronal and glial differentiation. The downstream Notch target genes hes1/5 and the negative HLH regulators, including the Id factors have been proposed to inhibit neurogenesis [55,56] as well as the expression and function of neurogenic bHLH factors
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Fig. 4. BMPs can either enhance neurogenesis or glial differentiation depending on the age of the neural progenitors. BMP stimulation of neural progenitors leads to smad1 phosphorylation. Smad1 subsequently associates with CBP/p300 to form a potent transcriptional coactivating complex. When Ngn is present, the coactivating complex will associate with Ngn to induce neurogenesis in early progenitors. The absence of Ngn in late neural progenitors leaves the coactivating complex free to associate with STATs to activate glial differentiation.
[47,57–59]. However, the action of these factors appears to be in existence throughout development. Not understanding the stage-dependent actions of these factors makes theorizing on how they regulate the neurogenic to gliogenic switch a daunting task. The mitogen bFGF is itself thought to inhibit neurogenesis, resulting in the gliogenic switch [60,61]. One difficulty in understanding the mechanisms underlying bFGF action is the fact that bFGF acts only gradually to convert neurogenic cells to a gliogenic state. Currently, no signaling mechanisms can explain such a gradual mode of regulation.
7. Conclusion Recently, much progress had been made towards understanding the molecular control of neuronal and glial differentiation in NSC cultures. Most currently existing mouse and rat NSC culture preparations have a tendency to lose their neurogenic potential upon expansion in vitro. Understanding the regulation of neurogenesis during development could help generate methods to maintain the neurogenic potential of NSCs even after expansion. For example, forced expression of exogenous neurogenic bHLH factors allows even late NSCs to become neurogenic (Sun, unpublished observations). However, an inducible system to control the expression of the exogenous pro-neural bHLH factors would be required to allow expansion of the progenitors containing the exogenous bHLH gene. Understanding the regulatory mechanisms for pro-neural bHLH gene expression may also allow us to come up with a means to switch on and off the endogenous pro-neural bHLH genes in NSC cultures to allow neurogenesis or progenitor expansion. Finally, it needs to be emphasized that nearly all extracellular fac-
tors have cell context-dependent actions. Understanding the detailed intracellular mechanisms of the extracellular factors in various cellular contexts will equip us with better methods to intervene or control the differentiation process of neural progenitors. Invariant control of the differentiation process will be critical for making NSC useful for medical applications.
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