YEXNR-11854; No. of pages: 5; 4C: Experimental Neurology xxx (2014) xxx–xxx
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Experimental Neurology journal homepage: www.elsevier.com/locate/yexnr
Commentary
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Cell replacement therapy: Lessons from teleost fish
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Günther K.H. Zupanc ⁎, Ruxandra F. Sîrbulescu
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Laboratory of Neurobiology, Department of Biology, Northeastern University, Boston, MA 02115, USA
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Keywords: Cell replacement therapy Regeneration Adult stem cells Microenvironment Teleost fish
Many disorders of the CNS are characterized by a massive loss of neurons. A promising therapeutic strategy to cure such conditions is based on the activation of endogenous stem cells. Implementation of this strategy will benefit from a better understanding of stem cell dynamics and the local CNS microenvironment in regeneration-competent vertebrate model systems. Using a spinal cord injury paradigm in zebrafish larvae, Briona and Dorsky (2014) have provided evidence for the existence of two distinct neural stem cell populations. One population has the characteristics of radial glia and expresses the homeobox transcription factor Dbx. The other lacks Dbx but expresses Olig2. These results are placed in the context of other studies that also support the notion of heterogeneity of adult stem cells in the CNS. The implication that differences among stem cell populations, in combination with specific factors from the local cellular microenvironment, might have a decisive impact on the fate choices of the new cells, is discussed. Reviewed evidence suggests that rather few modifications in the signaling pathways involved in the control of stem cell behavior have led, in the course of evolution, to the pronounced differences between mammals and regeneration-competent organisms. As a consequence, rather minor pharmacological manipulations may be sufficient to reactivate the hidden neurogenic potential of the mammalian CNS, and thus make it available for therapeutic applications. © 2014 Published by Elsevier Inc.
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Article history: Received 13 May 2014 Revised 6 September 2014 Accepted 11 October 2014 Available online xxxx
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Brain injuries caused by trauma and stroke, as well as spinal cord injuries, are complex disorders of the central nervous system (CNS) associated with massive loss of neurons. It has been estimated that globally every year over 10 million people suffer a traumatic brain injury (Hyder et al., 2007), approximately 17 million a stroke (Feigin et al., 2014), and up to 500,000 a spinal cord injury (Bickenbach et al., 2013). While current pharmacological treatments and rehabilitative therapies can lead to modest improvements in neurological functions in some individuals (Behrman et al., 2006; El-Kheir et al., 2014; Fehlings and Baptiste, 2005; Young, 2014), no cures exist yet for either of these conditions. A promising strategy toward restoration of function is based on harnessing the intrinsic potential of the CNS for self-repair through activation of endogenous neural stem/progenitor cells. The feasibility of such a strategy is supported by the observation that after experimental injuries proliferation is stimulated among both active and latent stem/ progenitor cells (Greenberg, 2007; Obermair et al., 2008; Ohab and Carmichael, 2008; Panayiotou and Malas, 2013; Parent, 2003;
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Brain and spinal cord injuries: curable through activation of endogenous stem cells?
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⁎ Corresponding author. Fax: +1 617 373 8592. E-mail address: [email protected] (G.K.H. Zupanc).
Richardson et al., 2007; Zhang et al., 2005). Cerebral ischemia, for example, evokes the mobilization of endogenous neural stem/progenitor cells in the two neurogenic niches of the adult mammalian brain, the subventricular zone of the lateral ventricles and the subgranular zone of the dentate gyrus of the hippocampus (Arvidsson et al., 2002; Jin et al., 2001; Liu et al., 1998; Rueger et al., 2010; Takasawa et al., 2002; Zhang et al., 2008). In addition, under pathological conditions, including focal and global ischemia, new neurons may arise in certain brain regions from adult stem cells that are quiescent in the healthy brain (Ohira, 2011). Similarly, traumatic injuries to the spinal cord can recruit ependymal cell populations which are normally quiescent (presumably post-mitotic) in the intact spinal cord (Spassky et al., 2005), and induce long-lasting ependymal proliferation within a large area around the lesion site (Lacroix et al., 2014; Lee et al., 2014; Meletis et al., 2008). An important property of the neural progenitors generated in response to injury is their ability to deviate from a normal pattern of development to migrate to the sites of injury. Such a specific re-routing of migration is commonly observed after stroke when neuroblasts generated in the subventricular zone (from where they migrate under healthy conditions via the rostral migratory stream into the olfactory bulb) change their path to migrate to the ischemic boundary zone (Arvidsson et al., 2002; Jin et al., 2003; Kojima et al., 2010; Parent et al., 2002; Yamashita et al., 2006). This area, which defines the border between infarcted and non-infarcted tissue, is characterized by pronounced angiogenesis (Ohab et al., 2006; Thored et al., 2007).
http://dx.doi.org/10.1016/j.expneurol.2014.10.006 0014-4886/© 2014 Published by Elsevier Inc.
Please cite this article as: Zupanc, G.K.H., Sîrbulescu, R.F., Cell replacement therapy: Lessons from teleost fish, Exp. Neurol. (2014), http:// dx.doi.org/10.1016/j.expneurol.2014.10.006
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The limited success in the implementation of therapies based on endogenous stem cells in the CNS of mammals makes obvious the need for a better understanding of the dynamics of adult stem cells both in normal development and under pathological conditions. Although the mammalian CNS makes clear attempts for self-repair after injury or neurodegeneration, these attempts do not lead to successful structural repair and functional recovery. An attractive research strategy is, therefore, to study non-mammalian model systems with an intrinsic potential for regeneration of nervous tissue. Despite the wide variation in regenerative potential among vertebrates, such a strategy is viable because the capacity for regeneration manifests itself on a continuum, with regeneration competence and regeneration incompetence representing just the two extremes of this spectrum. Most importantly, throughout this range of possibilities, animals share many mechanistic principles that underlie the regenerative processes (Stoick-Cooper et al., 2007). These shared that molecular signaling pathways make it possible to extract general principles from the study of regeneration-
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Teleost fish: models for regeneration competency
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competent organisms, and, based on the insights obtained, to develop therapeutic strategies for regeneration-incompetent vertebrates, including humans. Among the regeneration-competent organisms, several species of teleost fish are particularly well examined. Historically, three milestones were critical toward the establishment of teleost fish as a model of regeneration competency: In 1922, Koppányi and Weiss reported that 60 days after spinal cord transection European carp regained normal swimming behavior (Koppányi and Weiss, 1922). In 1935, Tuge and Hanzawa showed that the functional regeneration after spinal cord injury is closely linked to the structural repair of lesioned spinal cord tissue (Tuge and Hanzawa, 1935). Finally, in 1961 Kirsche and Kirsche demonstrated that the successful regeneration critically depends on the presence of ‘matrix zones’ (Kirsche and Kirsche, 1961), a synonym for neurogenic niches. If these areas of high mitotic activity are destroyed through injury, regeneration fails. Since these early studies, a much more comprehensive picture of the developmental events underlying the generation of new neurons has emerged, in both the intact and the injured teleostean CNS (Chapouton et al., 2007; Kaslin et al., 2008; Sîrbulescu and Zupanc, 2011, 2013; Zupanc and Sîrbulescu, 2011, 2013). Although only a dozen or so species out of an estimated 30,000 teleosts have been studied in detail thus far, the multitude of features shared among diverse lineages suggests that adult neurogenesis and neuronal regeneration, including their underlying molecular pathways, are evolutionarily conserved traits of many, if not all, teleosts. In each of the species examined, new neurons are generated during adulthood in dozens of proliferation zones of the brain as well as in the spinal cord. These proliferation zones are distinguished by a high concentration of adult stem/progenitor cells. The number of their progeny, relative to the total number of brain cells, is at least one, if not two, orders of magnitude higher than in the mammalian brain or spinal cord. The continuous generation of new cells, combined with the long-term persistence of roughly half of the adult-born cells, results in the growth of the teleostean brain beyond embryonic and juvenile stages of development. Injuries induce the replacement of lost cells by new cells arising both from the neurogenic niches active in the intact brain and from population(s) of quiescent stem cells spread over wide areas of the brain. This structural repair results most often in complete functional recovery.
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Neurogenesis and angiogenesis in this region are closely linked in that subventricular zone cells increase expression of genes involved in angiogenesis after stroke (Liu et al., 2007), while endothelial cells produce factors that promote neurogenesis, neuronal differentiation, and survival of the new neurons (Jin et al., 2002; Leventhal et al., 1999; Louissaint et al., 2002; Meng et al., 2006). Despite these encouraging findings of recent years, a cell replacement therapy based on the activation of endogenous stem/progenitor cells is still far from its clinical implementation. Major hurdles that need to be overcome toward this goal include the activation of a sufficient number of stem/progenitor cells, the targeted migration of the neuroblasts to areas of focal damage, the controlled development of their progeny into the correct, functional types of cells, and the promotion of long-term survival of the new cells. Neither of these critical issues has been resolved in animal models thus far. For instance, after experimentally induced stroke immature neurons born in the subventricular zone migrate to the striatal area damaged by the ischemic insult, and even start to express markers for striatal medium-sized spiny neurons (the phenotype most severely affected by ischemia). However, the majority of them die within a few weeks after the stroke (Arvidsson et al., 2002). Thus, it appears that the local environment is unable to support the long-term survival of new neurons. Equally significant, the total number of new neuronal cells is far too low to replace the degenerated ones. In this study, the number of new neurons generated in response to injury totaled just 1600, while an estimated 800,000 such cells were lost. In the mammalian spinal cord, the situation appears even more severe, since although contusion injury promotes massive proliferation of local progenitor cells, their progeny invariably differentiates into astrocytes and, more rarely, into oligodendrocytes, but not into neurons (Lacroix et al., 2014; Meletis et al., 2008). The importance of better understanding how different aspects of the molecular microenvironment support the development of neural progenitor cells in regeneration-competent organisms is underscored as the number of studies using stem cell implantations increases. A recent study elegantly pointed out current caveats of stem cell replacement therapy in mammals. Kumamaru et al. (2012) performed a detailed analysis of the expression profiles of neural stem/progenitor cells transplanted into the injured spinal cord of mice. They found that, while the grafted stem cells had a positive immediate effect, reducing levels of apoptosis and inflammation, in the long term they failed to properly differentiate. In addition, these grafted cells showed increased expression of brain tumor-specific alternative splicing variants in a number of genes. Interestingly, the microenvironment of the injured spinal cord appeared to have a distinct inhibitory effect on overall stem cell gene expression, much more so than the unlesioned tissue or cell culture environments (Kumamaru et al., 2012).
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Dbx-expressing progenitors: stem cells in the zebrafish spinal cord 185 What is it that enables fish — very much in contrast to mammals — to successfully regenerate CNS tissue? One of the key aspects mentioned above — stem cell dynamics — has been addressed in a report published in a recent issue of Experimental Neurology (Briona and Dorsky, 2014). Using a dbx1a:GFP transgenic reporter line, the authors showed that this gene was preferentially expressed in a slowly dividing progenitor population in the spinal cord of embryonic and larval zebrafish. Dbx genes encode a family of homeodomain-containing proteins of the Drosophila H2.0 class (Lu et al., 1992). During mouse embryogenesis, the Dbx gene is expressed in many regions of the CNS, including the telencephalon, diencephalon, mesencephalon, cerebellum, and spinal cord. However, within each of these regions, its expression is highly restricted to areas of mitotic activity (Lu et al., 1992, 1994). Dbx-expressing precursors generate a subset of interneurons, as well as astrocytes and a subpopulation of oligodendrocytes (Fogarty et al., 2005). In the spinal cord, a discrete subset of commissural interneurons, whose fate is controlled by the activity of Dbx1, plays a critical role in the regulation of left-right alternation of firing in motoneurons innervating hindlimb muscles, and thus in the control of proper walking movements (Lanuza et al., 2004). Co-labeling of the cells expressing the dbx1a:GFP transgene in the zebrafish spinal cord with glial fibrillary acidic protein (GFAP) identified them as radial glia. These cells continue to generate neurons beyond embryogenesis during larval stages of development, but are distinct from progenitors expressing Olig2, which have been proposed as neural
Please cite this article as: Zupanc, G.K.H., Sîrbulescu, R.F., Cell replacement therapy: Lessons from teleost fish, Exp. Neurol. (2014), http:// dx.doi.org/10.1016/j.expneurol.2014.10.006
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A pronounced effect of the cellular microenvironment on activity of adult stem cells and development of their progeny is well established for the mammalian brain. In their classic study, Reynolds and Weiss (1992) isolated stem cells from the striatum of the adult mouse brain that, although quiescent in vivo, can be induced to proliferate in vitro by epidermal growth factor in the presence of a non-adhesive substrate (Reynolds and Weiss, 1992). The inability of these stem cells to proliferate in vivo may be due either to the effect of inhibitory factors, or to the absence of permissive factors, in the microenvironment of the adult mammalian CNS. A corollary to this hypothesis predicts that the abolishment of inhibitory factors, or the provision of proper permissive factors, will activate the quiescent stem cells and initiate the development of their progeny. Such a hypothesis is in line with observations in teleost fish. The molecular microenvironment in the teleostean CNS has several distinct characteristics of major importance for their neurogenic and regenerative capacity, including the absence of key inhibitory factors as commonly found in mammals, increased expression of growth factors which promote cell proliferation and axonal growth, and the absence of a barrier-like glial scar formation in response to injury [for reviews, see (Diaz Quiroz and Echeverri, 2013; Sîrbulescu and Zupanc, 2013; Zupanc and Sîrbulescu, 2013)]. For example, in zebrafish, Nogo-A (reticulon 4), one of the main inhibitory proteins in mammalian myelin, lacks its key N-terminal domain (Diekmann et al., 2005), and the expression of its receptors, NgRs, in the adult spinal cord is very low (Klinger et al., 2004). Complementarily, the expression of fibroblast growth factors (FGFs) and several receptors of the retinoic acid pathway increases after spinal cord lesion in zebrafish, and has been proposed to play a role in motoneuron regeneration (Reimer et al., 2009). Increased FGF levels also induce the proliferation and polarization of GFAP-positive astroglia, which migrate towards the site of the lesion and bridge it, facilitating axonal regeneration across the injury (Goldshmit et al., 2012). Thus, in the teleostean spinal cord, unlike in the mammalian one, gliosis at the injury site serves an exclusively regenerative function, without the inhibitory effects of scar formation (Diaz Quiroz and Echeverri, 2013; Silver and Miller, 2004). A comparative analysis of adult neurogenesis also supports the hypothesis that the local microenvironment plays the defining role in specifying the fate of neural progenitor cells. In teleost fish, the cerebellum generates more adult-born neurons than any other structure in the brain (Teles et al., 2012; Zupanc et al., 2005; Zupanc and Horschke, 1995). By contrast, adult neurogenesis appears to be absent from the cerebellum of most mammalian species, including laboratory rats (Altman and Das, 1966). Remarkably, in the molecular layer of the cerebellum of rabbits, precursors of interneurons immunoreactive for GABA are generated throughout adulthood (Ponti et al., 2008), indicating the persistence of neurogenic potential beyond early postnatal stages of development in the cerebellum of at least some mammalian species. In line with this notion, astrocytic neural stem cells expressing nestin and the cell surface marker CD15 have been identified in the adult mammalian cerebellum (Walton et al., 2013). Like the adult stem cells in the teleostean cerebellum, many of these cells reside within the molecular layer. Although they are quiescent in vivo, such stem cells are able to form neurospheres in vitro, can be propagated, and give rise to each of the major neuronal subtypes in the cerebellum
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The question whether more than one population of adult stem cells exists in a given region of the brain or the spinal cord, as suggested by the study of Briona and Dorsky (2014), is of fundamental importance for understanding stem cell dynamics and the development of their progeny. Yet, the fact that a convincing answer is still remote even for the most common model systems indicates the complexity of this problem. In teleost fish, one of the best-studied brain regions is a subdivision of the cerebellum, the corpus cerebelli. Here, new cells are predominantly generated in the molecular layer and in a small, medial structure which in transverse sections appears arrowhead-shaped, and thus is referred to as the ‘dorsal tip’ (Teles et al., 2012; Zupanc et al., 2005; Zupanc and Horschke, 1995). The latter is located at the midline next to a largely obliterated lumen at the interface between the granule cell layer and the dorsal molecular layer. This lumen is thought to be derived from a displacement of the fourth ventricle during early postembryonic development (Kaslin et al., 2009). Based on the expression of the intermediate filament protein nestin, the same authors suggested that the stem cells in the dorsal tip are neuroepithelial cells, and that the neuroepithelial identity of adult stem cells in the brain distinguishes non-mammalian vertebrates from mammals, which generate new cells from astrocytic stem cells (Bonaguidi et al., 2011; Doetsch et al., 1999; Laywell et al., 2000; Seri et al., 2001). The glial vs. epithelial identity of adult stem cells is not yet clearly resolved. First, it is still a matter of debate whether the neural stem cells in the adult mammalian CNS are exclusively astrocytes [for a detailed discussion of this controversy, see (Chojnacki et al., 2009)]. Second, a recent detailed analysis of the stem cell characteristics in the dorsal tip, as well as in other parts of the teleostean corpus cerebelli, has pointed to an unexpected complexity (Sîrbulescu et al., in press). Immunostaining against various glial markers (such as S100β, GFAP, and vimentin) in combination with stem cell markers (such as Sox2, Islet1, Meis 1/2/3, and Pax6) and cell proliferation markers (such as BrdU and PCNA) revealed a heterogenous stem cell population. Approximately half of them express glial markers, whereas the others do not. The stem cells that lack glial markers are preferentially located in the immediate vicinity of the ventricular lumen, while the ones that express S100β, GFAP, and/or vimentin are predominantly subventricular. It is possible that the stem cells that lack expression of glial proteins and are located in direct contact with the ventricular lumen correspond to the epithelial cells described by Kaslin et al. (2009). Although the coexistence of various cell types in the ventricular and subventricular zones has been described for reptiles, birds, and mammals, it is widely assumed that only one cell type exerts neurogenic function (GarcíaVerdugo et al., 2002). The proposed heterogeneity of stem cells in the adult teleostean CNS raises an interesting question — whether the repertoire of fate-choices differs between the different stem cell populations. Using identical experimental protocols, a recent investigation in knifefish identified the vast majority of adult stem cells in a brain stem area as glia. Remarkably, in this region gliogenesis dominates (Sîrbulescu et al., 2014), while in the cerebellum most adult-born cells develop into neurons, specifically cerebellar granule cells. This difference in the cellular identity of the progeny may be related to the difference in the identity of the stem cells. It will, therefore, be important to analyze the fates of the various stem cells under in vivo conditions. Transplantation experiments could help elucidate whether possible differences in cellular
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fate are attributable to intrinsic properties of the stem cells, or to the local microenviroment from which these cells receive molecular signals. In vitro experiments have shown that the development of adult stem cells isolated from various neurogenic niches of the teleost fish brain, as well as the differentiation of their progeny, is largely dependent on extracellular matrix proteins and serum-derived growth factors (Hinsch and Zupanc, 2006).
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stem cells in post-embryonic zebrafish (Park et al., 2007; Reimer et al., 2008). In the study of Briona and Dorsky (2014), the population of dbx1a:GFP + radial glia increased its rate of neurogenesis in response to spinal cord transection performed on larval zebrafish at 5 days post fertilization, suggesting that the dbx1a + progenitors can serve as stem cells for the regeneration of injured interneurons.
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Please cite this article as: Zupanc, G.K.H., Sîrbulescu, R.F., Cell replacement therapy: Lessons from teleost fish, Exp. Neurol. (2014), http:// dx.doi.org/10.1016/j.expneurol.2014.10.006
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The above observations indicate that rather minor modifications in the processes that regulate stem cell maintenance and progeny development have led, in the course of evolution, to fundamental differences in the extent of adult neurogenesis and neuronal regeneration between teleost fish and mammals. Importantly, these processes and their underlying molecular signaling pathways are highly conserved among vertebrates. As a consequence, rather minor pharmacological manipulations, such as stimulation of quiescent stem cells with proper growth factors, appear to be sufficient to restore the developmental and regenerative programs in the adult mammalian CNS. A promising experimental strategy to identify the permissive characteristics of the CNS is to induce traumatic brain injury or spinal cord injury in regeneration-competent organisms, and to examine changes in gene expression and protein synthesis after the injury. Depending on the chosen post-lesion time point, degenerative processes, such as apoptotic cell death, or repair processes, such as cell proliferation and neuronal differentiation, dominate. Combination of such lesion paradigms with high-throughput methods has proven to be a powerful approach to identify gene or protein candidates involved in neuronal regeneration (Ilieş et al., 2012; Zupanc et al., 2006). Several lesion paradigms have been developed over the past century, including amputation of the caudal part of the spinal cord, cord transection at various levels, application of mechanical lesions to the telencephalon, optic tectum, and cerebellum, or lesioning of the retina and optic nerve [for review, see (Sîrbulescu and Zupanc, 2013)]. Traditionally, these lesion paradigms have been applied to adult fish. Briona and Dorsky (2014) used an alternate approach, application of spinal cord injury in larval zebrafish. The use of larvae instead of adult individuals has several advantages. Most significant is the overall shorter time scale: In larval zebrafish whose spinal cord is injured at 5 days post fertilization, newly generated neuronal somata can be found at the injury site 4 days later, compared to 4 weeks post injury in adult zebrafish (Hui et al., 2010). Moreover, in the larval zebrafish, sensory and motor function also recover within 4–5 days, as opposed to 6–8 weeks post injury in the adult (Reimer et al., 2008). Two further practical features make larvae well suited for high-throughput testing of pharmacological compounds, or genetic and morpholino oligonucleotide screens, designed to identify targets of drugs that may modulate regenerative processes: their transparency, which enables investigators to carry out non-invasive in vivo imaging; and their tremendously reduced space requirement, compared to adults, which makes it possible to fit numerous fish into multi-well assay plates. Capitalizing on these advantages, larval and juvenile zebrafish have been used in several diseaserelated areas for in vivo drug discovery [for reviews, see (Novodvorsky et al., 2013; Zon and Peterson, 2005)]. Nevertheless, such experiments need to be complemented by investigations in adult fish, since the larval CNS shows a significantly higher degree of intrinsic neural plasticity, and the ultimate goal remains the discovery of drugs that significantly improve regeneration after traumatic brain injury or spinal cord injury in adult individuals. Undoubtedly, the use of such experimental paradigms, both in adult and larval models, will greatly facilitate the identification of permissive factors that enable teleost fish to regenerate structure and to regain function after CNS injury. In turn, a better understanding of the molecular correlates of the regenerative capability of fish could guide the
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Please cite this article as: Zupanc, G.K.H., Sîrbulescu, R.F., Cell replacement therapy: Lessons from teleost fish, Exp. Neurol. (2014), http:// dx.doi.org/10.1016/j.expneurol.2014.10.006
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Commentary
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Cell replacement therapy: Lessons from teleost fish
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Günther K.H. Zupanc ⁎, Ruxandra F. Sîrbulescu
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Laboratory of Neurobiology, Department of Biology, Northeastern University, Boston, MA 02115, USA
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Keywords: Cell replacement therapy Regeneration Adult stem cells Microenvironment Teleost fish
Many disorders of the CNS are characterized by a massive loss of neurons. A promising therapeutic strategy to cure such conditions is based on the activation of endogenous stem cells. Implementation of this strategy will benefit from a better understanding of stem cell dynamics and the local CNS microenvironment in regeneration-competent vertebrate model systems. Using a spinal cord injury paradigm in zebrafish larvae, Briona and Dorsky (2014) have provided evidence for the existence of two distinct neural stem cell populations. One population has the characteristics of radial glia and expresses the homeobox transcription factor Dbx. The other lacks Dbx but expresses Olig2. These results are placed in the context of other studies that also support the notion of heterogeneity of adult stem cells in the CNS. The implication that differences among stem cell populations, in combination with specific factors from the local cellular microenvironment, might have a decisive impact on the fate choices of the new cells, is discussed. Reviewed evidence suggests that rather few modifications in the signaling pathways involved in the control of stem cell behavior have led, in the course of evolution, to the pronounced differences between mammals and regeneration-competent organisms. As a consequence, rather minor pharmacological manipulations may be sufficient to reactivate the hidden neurogenic potential of the mammalian CNS, and thus make it available for therapeutic applications. © 2014 Published by Elsevier Inc.
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Article history: Received 13 May 2014 Revised 6 September 2014 Accepted 11 October 2014 Available online xxxx
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Brain injuries caused by trauma and stroke, as well as spinal cord injuries, are complex disorders of the central nervous system (CNS) associated with massive loss of neurons. It has been estimated that globally every year over 10 million people suffer a traumatic brain injury (Hyder et al., 2007), approximately 17 million a stroke (Feigin et al., 2014), and up to 500,000 a spinal cord injury (Bickenbach et al., 2013). While current pharmacological treatments and rehabilitative therapies can lead to modest improvements in neurological functions in some individuals (Behrman et al., 2006; El-Kheir et al., 2014; Fehlings and Baptiste, 2005; Young, 2014), no cures exist yet for either of these conditions. A promising strategy toward restoration of function is based on harnessing the intrinsic potential of the CNS for self-repair through activation of endogenous neural stem/progenitor cells. The feasibility of such a strategy is supported by the observation that after experimental injuries proliferation is stimulated among both active and latent stem/ progenitor cells (Greenberg, 2007; Obermair et al., 2008; Ohab and Carmichael, 2008; Panayiotou and Malas, 2013; Parent, 2003;
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Brain and spinal cord injuries: curable through activation of endogenous stem cells?
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⁎ Corresponding author. Fax: +1 617 373 8592. E-mail address: [email protected] (G.K.H. Zupanc).
Richardson et al., 2007; Zhang et al., 2005). Cerebral ischemia, for example, evokes the mobilization of endogenous neural stem/progenitor cells in the two neurogenic niches of the adult mammalian brain, the subventricular zone of the lateral ventricles and the subgranular zone of the dentate gyrus of the hippocampus (Arvidsson et al., 2002; Jin et al., 2001; Liu et al., 1998; Rueger et al., 2010; Takasawa et al., 2002; Zhang et al., 2008). In addition, under pathological conditions, including focal and global ischemia, new neurons may arise in certain brain regions from adult stem cells that are quiescent in the healthy brain (Ohira, 2011). Similarly, traumatic injuries to the spinal cord can recruit ependymal cell populations which are normally quiescent (presumably post-mitotic) in the intact spinal cord (Spassky et al., 2005), and induce long-lasting ependymal proliferation within a large area around the lesion site (Lacroix et al., 2014; Lee et al., 2014; Meletis et al., 2008). An important property of the neural progenitors generated in response to injury is their ability to deviate from a normal pattern of development to migrate to the sites of injury. Such a specific re-routing of migration is commonly observed after stroke when neuroblasts generated in the subventricular zone (from where they migrate under healthy conditions via the rostral migratory stream into the olfactory bulb) change their path to migrate to the ischemic boundary zone (Arvidsson et al., 2002; Jin et al., 2003; Kojima et al., 2010; Parent et al., 2002; Yamashita et al., 2006). This area, which defines the border between infarcted and non-infarcted tissue, is characterized by pronounced angiogenesis (Ohab et al., 2006; Thored et al., 2007).
http://dx.doi.org/10.1016/j.expneurol.2014.10.006 0014-4886/© 2014 Published by Elsevier Inc.
Please cite this article as: Zupanc, G.K.H., Sîrbulescu, R.F., Cell replacement therapy: Lessons from teleost fish, Exp. Neurol. (2014), http:// dx.doi.org/10.1016/j.expneurol.2014.10.006
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The limited success in the implementation of therapies based on endogenous stem cells in the CNS of mammals makes obvious the need for a better understanding of the dynamics of adult stem cells both in normal development and under pathological conditions. Although the mammalian CNS makes clear attempts for self-repair after injury or neurodegeneration, these attempts do not lead to successful structural repair and functional recovery. An attractive research strategy is, therefore, to study non-mammalian model systems with an intrinsic potential for regeneration of nervous tissue. Despite the wide variation in regenerative potential among vertebrates, such a strategy is viable because the capacity for regeneration manifests itself on a continuum, with regeneration competence and regeneration incompetence representing just the two extremes of this spectrum. Most importantly, throughout this range of possibilities, animals share many mechanistic principles that underlie the regenerative processes (Stoick-Cooper et al., 2007). These shared that molecular signaling pathways make it possible to extract general principles from the study of regeneration-
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competent organisms, and, based on the insights obtained, to develop therapeutic strategies for regeneration-incompetent vertebrates, including humans. Among the regeneration-competent organisms, several species of teleost fish are particularly well examined. Historically, three milestones were critical toward the establishment of teleost fish as a model of regeneration competency: In 1922, Koppányi and Weiss reported that 60 days after spinal cord transection European carp regained normal swimming behavior (Koppányi and Weiss, 1922). In 1935, Tuge and Hanzawa showed that the functional regeneration after spinal cord injury is closely linked to the structural repair of lesioned spinal cord tissue (Tuge and Hanzawa, 1935). Finally, in 1961 Kirsche and Kirsche demonstrated that the successful regeneration critically depends on the presence of ‘matrix zones’ (Kirsche and Kirsche, 1961), a synonym for neurogenic niches. If these areas of high mitotic activity are destroyed through injury, regeneration fails. Since these early studies, a much more comprehensive picture of the developmental events underlying the generation of new neurons has emerged, in both the intact and the injured teleostean CNS (Chapouton et al., 2007; Kaslin et al., 2008; Sîrbulescu and Zupanc, 2011, 2013; Zupanc and Sîrbulescu, 2011, 2013). Although only a dozen or so species out of an estimated 30,000 teleosts have been studied in detail thus far, the multitude of features shared among diverse lineages suggests that adult neurogenesis and neuronal regeneration, including their underlying molecular pathways, are evolutionarily conserved traits of many, if not all, teleosts. In each of the species examined, new neurons are generated during adulthood in dozens of proliferation zones of the brain as well as in the spinal cord. These proliferation zones are distinguished by a high concentration of adult stem/progenitor cells. The number of their progeny, relative to the total number of brain cells, is at least one, if not two, orders of magnitude higher than in the mammalian brain or spinal cord. The continuous generation of new cells, combined with the long-term persistence of roughly half of the adult-born cells, results in the growth of the teleostean brain beyond embryonic and juvenile stages of development. Injuries induce the replacement of lost cells by new cells arising both from the neurogenic niches active in the intact brain and from population(s) of quiescent stem cells spread over wide areas of the brain. This structural repair results most often in complete functional recovery.
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Neurogenesis and angiogenesis in this region are closely linked in that subventricular zone cells increase expression of genes involved in angiogenesis after stroke (Liu et al., 2007), while endothelial cells produce factors that promote neurogenesis, neuronal differentiation, and survival of the new neurons (Jin et al., 2002; Leventhal et al., 1999; Louissaint et al., 2002; Meng et al., 2006). Despite these encouraging findings of recent years, a cell replacement therapy based on the activation of endogenous stem/progenitor cells is still far from its clinical implementation. Major hurdles that need to be overcome toward this goal include the activation of a sufficient number of stem/progenitor cells, the targeted migration of the neuroblasts to areas of focal damage, the controlled development of their progeny into the correct, functional types of cells, and the promotion of long-term survival of the new cells. Neither of these critical issues has been resolved in animal models thus far. For instance, after experimentally induced stroke immature neurons born in the subventricular zone migrate to the striatal area damaged by the ischemic insult, and even start to express markers for striatal medium-sized spiny neurons (the phenotype most severely affected by ischemia). However, the majority of them die within a few weeks after the stroke (Arvidsson et al., 2002). Thus, it appears that the local environment is unable to support the long-term survival of new neurons. Equally significant, the total number of new neuronal cells is far too low to replace the degenerated ones. In this study, the number of new neurons generated in response to injury totaled just 1600, while an estimated 800,000 such cells were lost. In the mammalian spinal cord, the situation appears even more severe, since although contusion injury promotes massive proliferation of local progenitor cells, their progeny invariably differentiates into astrocytes and, more rarely, into oligodendrocytes, but not into neurons (Lacroix et al., 2014; Meletis et al., 2008). The importance of better understanding how different aspects of the molecular microenvironment support the development of neural progenitor cells in regeneration-competent organisms is underscored as the number of studies using stem cell implantations increases. A recent study elegantly pointed out current caveats of stem cell replacement therapy in mammals. Kumamaru et al. (2012) performed a detailed analysis of the expression profiles of neural stem/progenitor cells transplanted into the injured spinal cord of mice. They found that, while the grafted stem cells had a positive immediate effect, reducing levels of apoptosis and inflammation, in the long term they failed to properly differentiate. In addition, these grafted cells showed increased expression of brain tumor-specific alternative splicing variants in a number of genes. Interestingly, the microenvironment of the injured spinal cord appeared to have a distinct inhibitory effect on overall stem cell gene expression, much more so than the unlesioned tissue or cell culture environments (Kumamaru et al., 2012).
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Dbx-expressing progenitors: stem cells in the zebrafish spinal cord 185 What is it that enables fish — very much in contrast to mammals — to successfully regenerate CNS tissue? One of the key aspects mentioned above — stem cell dynamics — has been addressed in a report published in a recent issue of Experimental Neurology (Briona and Dorsky, 2014). Using a dbx1a:GFP transgenic reporter line, the authors showed that this gene was preferentially expressed in a slowly dividing progenitor population in the spinal cord of embryonic and larval zebrafish. Dbx genes encode a family of homeodomain-containing proteins of the Drosophila H2.0 class (Lu et al., 1992). During mouse embryogenesis, the Dbx gene is expressed in many regions of the CNS, including the telencephalon, diencephalon, mesencephalon, cerebellum, and spinal cord. However, within each of these regions, its expression is highly restricted to areas of mitotic activity (Lu et al., 1992, 1994). Dbx-expressing precursors generate a subset of interneurons, as well as astrocytes and a subpopulation of oligodendrocytes (Fogarty et al., 2005). In the spinal cord, a discrete subset of commissural interneurons, whose fate is controlled by the activity of Dbx1, plays a critical role in the regulation of left-right alternation of firing in motoneurons innervating hindlimb muscles, and thus in the control of proper walking movements (Lanuza et al., 2004). Co-labeling of the cells expressing the dbx1a:GFP transgene in the zebrafish spinal cord with glial fibrillary acidic protein (GFAP) identified them as radial glia. These cells continue to generate neurons beyond embryogenesis during larval stages of development, but are distinct from progenitors expressing Olig2, which have been proposed as neural
Please cite this article as: Zupanc, G.K.H., Sîrbulescu, R.F., Cell replacement therapy: Lessons from teleost fish, Exp. Neurol. (2014), http:// dx.doi.org/10.1016/j.expneurol.2014.10.006
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A pronounced effect of the cellular microenvironment on activity of adult stem cells and development of their progeny is well established for the mammalian brain. In their classic study, Reynolds and Weiss (1992) isolated stem cells from the striatum of the adult mouse brain that, although quiescent in vivo, can be induced to proliferate in vitro by epidermal growth factor in the presence of a non-adhesive substrate (Reynolds and Weiss, 1992). The inability of these stem cells to proliferate in vivo may be due either to the effect of inhibitory factors, or to the absence of permissive factors, in the microenvironment of the adult mammalian CNS. A corollary to this hypothesis predicts that the abolishment of inhibitory factors, or the provision of proper permissive factors, will activate the quiescent stem cells and initiate the development of their progeny. Such a hypothesis is in line with observations in teleost fish. The molecular microenvironment in the teleostean CNS has several distinct characteristics of major importance for their neurogenic and regenerative capacity, including the absence of key inhibitory factors as commonly found in mammals, increased expression of growth factors which promote cell proliferation and axonal growth, and the absence of a barrier-like glial scar formation in response to injury [for reviews, see (Diaz Quiroz and Echeverri, 2013; Sîrbulescu and Zupanc, 2013; Zupanc and Sîrbulescu, 2013)]. For example, in zebrafish, Nogo-A (reticulon 4), one of the main inhibitory proteins in mammalian myelin, lacks its key N-terminal domain (Diekmann et al., 2005), and the expression of its receptors, NgRs, in the adult spinal cord is very low (Klinger et al., 2004). Complementarily, the expression of fibroblast growth factors (FGFs) and several receptors of the retinoic acid pathway increases after spinal cord lesion in zebrafish, and has been proposed to play a role in motoneuron regeneration (Reimer et al., 2009). Increased FGF levels also induce the proliferation and polarization of GFAP-positive astroglia, which migrate towards the site of the lesion and bridge it, facilitating axonal regeneration across the injury (Goldshmit et al., 2012). Thus, in the teleostean spinal cord, unlike in the mammalian one, gliosis at the injury site serves an exclusively regenerative function, without the inhibitory effects of scar formation (Diaz Quiroz and Echeverri, 2013; Silver and Miller, 2004). A comparative analysis of adult neurogenesis also supports the hypothesis that the local microenvironment plays the defining role in specifying the fate of neural progenitor cells. In teleost fish, the cerebellum generates more adult-born neurons than any other structure in the brain (Teles et al., 2012; Zupanc et al., 2005; Zupanc and Horschke, 1995). By contrast, adult neurogenesis appears to be absent from the cerebellum of most mammalian species, including laboratory rats (Altman and Das, 1966). Remarkably, in the molecular layer of the cerebellum of rabbits, precursors of interneurons immunoreactive for GABA are generated throughout adulthood (Ponti et al., 2008), indicating the persistence of neurogenic potential beyond early postnatal stages of development in the cerebellum of at least some mammalian species. In line with this notion, astrocytic neural stem cells expressing nestin and the cell surface marker CD15 have been identified in the adult mammalian cerebellum (Walton et al., 2013). Like the adult stem cells in the teleostean cerebellum, many of these cells reside within the molecular layer. Although they are quiescent in vivo, such stem cells are able to form neurospheres in vitro, can be propagated, and give rise to each of the major neuronal subtypes in the cerebellum
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The question whether more than one population of adult stem cells exists in a given region of the brain or the spinal cord, as suggested by the study of Briona and Dorsky (2014), is of fundamental importance for understanding stem cell dynamics and the development of their progeny. Yet, the fact that a convincing answer is still remote even for the most common model systems indicates the complexity of this problem. In teleost fish, one of the best-studied brain regions is a subdivision of the cerebellum, the corpus cerebelli. Here, new cells are predominantly generated in the molecular layer and in a small, medial structure which in transverse sections appears arrowhead-shaped, and thus is referred to as the ‘dorsal tip’ (Teles et al., 2012; Zupanc et al., 2005; Zupanc and Horschke, 1995). The latter is located at the midline next to a largely obliterated lumen at the interface between the granule cell layer and the dorsal molecular layer. This lumen is thought to be derived from a displacement of the fourth ventricle during early postembryonic development (Kaslin et al., 2009). Based on the expression of the intermediate filament protein nestin, the same authors suggested that the stem cells in the dorsal tip are neuroepithelial cells, and that the neuroepithelial identity of adult stem cells in the brain distinguishes non-mammalian vertebrates from mammals, which generate new cells from astrocytic stem cells (Bonaguidi et al., 2011; Doetsch et al., 1999; Laywell et al., 2000; Seri et al., 2001). The glial vs. epithelial identity of adult stem cells is not yet clearly resolved. First, it is still a matter of debate whether the neural stem cells in the adult mammalian CNS are exclusively astrocytes [for a detailed discussion of this controversy, see (Chojnacki et al., 2009)]. Second, a recent detailed analysis of the stem cell characteristics in the dorsal tip, as well as in other parts of the teleostean corpus cerebelli, has pointed to an unexpected complexity (Sîrbulescu et al., in press). Immunostaining against various glial markers (such as S100β, GFAP, and vimentin) in combination with stem cell markers (such as Sox2, Islet1, Meis 1/2/3, and Pax6) and cell proliferation markers (such as BrdU and PCNA) revealed a heterogenous stem cell population. Approximately half of them express glial markers, whereas the others do not. The stem cells that lack glial markers are preferentially located in the immediate vicinity of the ventricular lumen, while the ones that express S100β, GFAP, and/or vimentin are predominantly subventricular. It is possible that the stem cells that lack expression of glial proteins and are located in direct contact with the ventricular lumen correspond to the epithelial cells described by Kaslin et al. (2009). Although the coexistence of various cell types in the ventricular and subventricular zones has been described for reptiles, birds, and mammals, it is widely assumed that only one cell type exerts neurogenic function (GarcíaVerdugo et al., 2002). The proposed heterogeneity of stem cells in the adult teleostean CNS raises an interesting question — whether the repertoire of fate-choices differs between the different stem cell populations. Using identical experimental protocols, a recent investigation in knifefish identified the vast majority of adult stem cells in a brain stem area as glia. Remarkably, in this region gliogenesis dominates (Sîrbulescu et al., 2014), while in the cerebellum most adult-born cells develop into neurons, specifically cerebellar granule cells. This difference in the cellular identity of the progeny may be related to the difference in the identity of the stem cells. It will, therefore, be important to analyze the fates of the various stem cells under in vivo conditions. Transplantation experiments could help elucidate whether possible differences in cellular
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fate are attributable to intrinsic properties of the stem cells, or to the local microenviroment from which these cells receive molecular signals. In vitro experiments have shown that the development of adult stem cells isolated from various neurogenic niches of the teleost fish brain, as well as the differentiation of their progeny, is largely dependent on extracellular matrix proteins and serum-derived growth factors (Hinsch and Zupanc, 2006).
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stem cells in post-embryonic zebrafish (Park et al., 2007; Reimer et al., 2008). In the study of Briona and Dorsky (2014), the population of dbx1a:GFP + radial glia increased its rate of neurogenesis in response to spinal cord transection performed on larval zebrafish at 5 days post fertilization, suggesting that the dbx1a + progenitors can serve as stem cells for the regeneration of injured interneurons.
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Altman, J., Das, G.D., 1966. Autoradiographic and histological studies of postnatal neurogenesis. I. A longitudinal investigation of the kinetics, migration and transformation of cells incorporating tritiated thymidine in neonate rats, with special reference to postnatal neurogenesis in some brain regions. J. Comp. Neurol. 126, 337–389. Arvidsson, A., Collin, T., Kirik, D., Kokaia, Z., Lindvall, O., 2002. Neuronal replacement from endogenous precursors in the adult brain after stroke. Nat. Med. 8, 963–970. Behrman, A.L., Bowden, M.G., Nair, P.M., 2006. Neuroplasticity after spinal cord injury and training: an emerging paradigm shift in rehabilitation and walking recovery. Phys. Ther. 86, 1406–1425. Bickenbach, J., Bodine, C., Brown, D., Burns, A., Campbell, R., Cardenas, D., Charlifue, S., Chen, Y., Gray, D., Li, L., Officer, A., Post, M., Shakespeare, T., Sinnott, A., von Groote, P., Xiong, X. (Eds.), 2013. International Perspectives on Spinal Cord Injury. World Health Organization, Geneva. Bonaguidi, M.A., Wheeler, M.A., Shapiro, J.S., Stadel, R.P., Sun, G.J., Ming, G.-L., Song, H., 2011. In vivo clonal analysis reveals self-renewing and multipotent adult neural stem cell characteristics. Cell 145, 1142–1155. Briona, L.K., Dorsky, R.I., 2014. Radial glial progenitors repair the zebrafish spinal cord following transection. Exp. Neurol. 256C, 81–92. Chapouton, P., Jagasia, R., Bally-Cuif, L., 2007. Adult neurogenesis in non-mammalian vertebrates. Bioessays 29, 745–757. Chojnacki, A.K., Mak, G.K., Weiss, S., 2009. Identity crisis for adult periventricular neural stem cells: subventricular zone astrocytes, ependymal cells or both? Nat. Rev. Neurosci. 10, 153–163. Diaz Quiroz, J.F., Echeverri, K., 2013. Spinal cord regeneration: where fish, frogs and salamanders lead the way, can we follow? Biochem. J. 451, 353–364. Diekmann, H., Klinger, M., Oertle, T., Heinz, D., Pogoda, H.M., Schwab, M.E., Stuermer, C.A.O., 2005. Analysis of the reticulon gene family demonstrates the absence of the neurite growth inhibitor Nogo-A in fish. Mol. Biol. Evol. 22, 1635–1648. Doetsch, F., Caillé, I., Lim, D.A., García-Verdugo, J.M., Alvarez-Buylla, A., 1999. Subventricular zone astrocytes are neural stem cells in the adult mammalian brain. Cell 97, 703–716. El-Kheir, W.A., Gabr, H., Awad, M.R., Ghannam, O., Barakat, Y., Farghali, H.A., El Maadawi, Z.M., Ewes, I., Sabaawy, H.E., 2014. Autologous bone marrow-derived cell therapy combined with physical therapy induces functional improvement in chronic spinal cord injury patients. Cell Transplant. 23, 729–745. Fehlings, M.G., Baptiste, D.C., 2005. Current status of clinical trials for acute spinal cord injury. Injury 36 (Suppl. 2), B113–B122. Feigin, V.L., Forouzanfar, M.H., Krishnamurthi, R., Mensah, G.A., Connor, M., Bennett, D.A., Moran, A.E., Sacco, R.L., Anderson, L., Truelsen, T., O'Donnell, M., Venketasubramanian, N., Barker-Collo, S., Lawes, C.M., Wang, W., Shinohara, Y., Witt, E., Ezzati, M., Naghavi, M., Murray, C., 2014. Global and regional burden of stroke during 1990–2010: findings from the Global Burden of Disease Study 2010. Lancet 383, 245–254. Fogarty, M., Richardson, W.D., Kessaris, N., 2005. A subset of oligodendrocytes generated from radial glia in the dorsal spinal cord. Development 132, 1951–1959. García-Verdugo, J.M., Ferrón, S., Flames, N., Collado, L., Desfilis, E., Font, E., 2002. The proliferative ventricular zone in adult vertebrates: a comparative study using reptiles, birds, and mammals. Brain Res. Bull. 57, 765–775. Goldshmit, Y., Sztal, T.E., Jusuf, P.R., Hall, T.E., Nguyen-Chi, M., Currie, P.D., 2012. Fgfdependent glial cell bridges facilitate spinal cord regeneration in zebrafish. J. Neurosci. 32, 7477–7492. Greenberg, D.A., 2007. Neurogenesis and stroke. CNS Neurol. Disord. Drug Targets 6, 321–325. Guo, Z., Wang, X., Xiao, J., Wang, Y., Lu, H., Teng, J., Wang, W., 2013. Early postnatal GFAPexpressing cells produce multilineage progeny in cerebrum and astrocytes in cerebellum of adult mice. Brain Res. 1532, 14–20. Hinsch, K., Zupanc, G.K.H., 2006. Isolation, cultivation, and differentiation of neural stem cells from adult fish brain. J. Neurosci. Methods 158, 75–88. Hui, S.P., Dutta, A., Ghosh, S., 2010. Cellular response after crush injury in adult zebrafish spinal cord. Dev. Dyn. 239, 2962–2979. Hyder, A.A., Wunderlich, C.A., Puvanachandra, P., Gururaj, G., Kobusingye, O.C., 2007. The impact of traumatic brain injuries: a global perspective. NeuroRehabilitation 22, 341–353. Ilieş, I., Zupanc, M.M., Zupanc, G.K.H., 2012. Proteome analysis reveals protein candidates involved in early stages of brain regeneration of teleost fish. Neuroscience 219, 302–313. Jin, K., Minami, M., Lan, J.Q., Mao, X.O., Batteur, S., Simon, R.P., Greenberg, D.A., 2001. Neurogenesis in dentate subgranular zone and rostral subventricular zone after focal cerebral ischemia in the rat. Proc. Natl. Acad. Sci. U. S. A. 98, 4710–4715. Jin, K., Zhu, Y., Sun, Y., Mao, X.O., Xie, L., Greenberg, D.A., 2002. Vascular endothelial growth factor (VEGF) stimulates neurogenesis in vitro and in vivo. Proc. Natl. Acad. Sci. U. S. A. 99, 11946–11950. Jin, K., Sun, Y., Xie, L., Peel, A., Mao, X.O., Batteur, S., Greenberg, D.A., 2003. Directed migration of neuronal precursors into the ischemic cerebral cortex and striatum. Mol. Cell. Neurosci. 24, 171–189. Kaslin, J., Ganz, J., Brand, M., 2008. Proliferation, neurogenesis and regeneration in the non-mammalian vertebrate brain. Philos. Trans. R. Soc. Lond. B Biol. Sci. 363, 101–122. Kaslin, J., Ganz, J., Geffarth, M., Grandel, H., Hans, S., Brand, M., 2009. Stem cells in the adult zebrafish cerebellum: initiation and maintenance of a novel stem cell niche. J. Neurosci. 29, 6142–6153.
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The above observations indicate that rather minor modifications in the processes that regulate stem cell maintenance and progeny development have led, in the course of evolution, to fundamental differences in the extent of adult neurogenesis and neuronal regeneration between teleost fish and mammals. Importantly, these processes and their underlying molecular signaling pathways are highly conserved among vertebrates. As a consequence, rather minor pharmacological manipulations, such as stimulation of quiescent stem cells with proper growth factors, appear to be sufficient to restore the developmental and regenerative programs in the adult mammalian CNS. A promising experimental strategy to identify the permissive characteristics of the CNS is to induce traumatic brain injury or spinal cord injury in regeneration-competent organisms, and to examine changes in gene expression and protein synthesis after the injury. Depending on the chosen post-lesion time point, degenerative processes, such as apoptotic cell death, or repair processes, such as cell proliferation and neuronal differentiation, dominate. Combination of such lesion paradigms with high-throughput methods has proven to be a powerful approach to identify gene or protein candidates involved in neuronal regeneration (Ilieş et al., 2012; Zupanc et al., 2006). Several lesion paradigms have been developed over the past century, including amputation of the caudal part of the spinal cord, cord transection at various levels, application of mechanical lesions to the telencephalon, optic tectum, and cerebellum, or lesioning of the retina and optic nerve [for review, see (Sîrbulescu and Zupanc, 2013)]. Traditionally, these lesion paradigms have been applied to adult fish. Briona and Dorsky (2014) used an alternate approach, application of spinal cord injury in larval zebrafish. The use of larvae instead of adult individuals has several advantages. Most significant is the overall shorter time scale: In larval zebrafish whose spinal cord is injured at 5 days post fertilization, newly generated neuronal somata can be found at the injury site 4 days later, compared to 4 weeks post injury in adult zebrafish (Hui et al., 2010). Moreover, in the larval zebrafish, sensory and motor function also recover within 4–5 days, as opposed to 6–8 weeks post injury in the adult (Reimer et al., 2008). Two further practical features make larvae well suited for high-throughput testing of pharmacological compounds, or genetic and morpholino oligonucleotide screens, designed to identify targets of drugs that may modulate regenerative processes: their transparency, which enables investigators to carry out non-invasive in vivo imaging; and their tremendously reduced space requirement, compared to adults, which makes it possible to fit numerous fish into multi-well assay plates. Capitalizing on these advantages, larval and juvenile zebrafish have been used in several diseaserelated areas for in vivo drug discovery [for reviews, see (Novodvorsky et al., 2013; Zon and Peterson, 2005)]. Nevertheless, such experiments need to be complemented by investigations in adult fish, since the larval CNS shows a significantly higher degree of intrinsic neural plasticity, and the ultimate goal remains the discovery of drugs that significantly improve regeneration after traumatic brain injury or spinal cord injury in adult individuals. Undoubtedly, the use of such experimental paradigms, both in adult and larval models, will greatly facilitate the identification of permissive factors that enable teleost fish to regenerate structure and to regain function after CNS injury. In turn, a better understanding of the molecular correlates of the regenerative capability of fish could guide the
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(Klein et al., 2005). Despite their multilineage potential, in vivo, the endogenous stem cells in the adult cerebellum differentiate exclusively into astrocytes, including Bergmann glia (Guo et al., 2013). However, when adult cerebellar-derived neurospheres are transplanted into the perinatal cerebellum (which is thought to provide a permissive environment), they generate multiple cell types, including cerebellar neurons (Klein et al., 2005).
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Please cite this article as: Zupanc, G.K.H., Sîrbulescu, R.F., Cell replacement therapy: Lessons from teleost fish, Exp. Neurol. (2014), http:// dx.doi.org/10.1016/j.expneurol.2014.10.006
