Investigating the use of primary adult subventricular zone neural precursor cells for neuronal replacement therapies

Brain Research Bulletin, Vol. 57, No. 6, pp. 759 –764, 2002 Copyright © 2002 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/02/$–see front matter

PII S0361-9230(01)00768-7

Investigating the use of primary adult subventricular zone neural precursor cells for neuronal replacement therapies Daniel A. Lim,1 Nuria Flames,2 Lucı´a Collado2 and Daniel G. Herrera1* 1

Department of Psychiatry, Weill Medical College of Cornell University, New York, NY, USA; and 2 Departamento de Biologı´a Celular, Universidad de Valencia, Valencia, Spain

ABSTRACT: With the relatively recent discovery that neurogenesis persists throughout life in restricted regions of the adult mammalian brain, including those of human beings, there has been great interest in the use of adult-derived neural stem cells for neuronal replacement. There are many great hurdles that must be overcome in order for such replacement strategies to succeed. In this review, we outline some of these hurdles and discuss recent experiments that investigate the potential of using neural precursor cells found in the subventricular zone of the adult brain for brain repair. © 2002 Elsevier Science Inc.

(SVZ) and rostral migratory stream [48]. Subsequently, these cells are incorporated at their final positions where they differentiate and are inserted into functional circuits [4,49,54,58]. Neurons produced postnatally for the olfactory bulb (OB) in the vertebrate brain are generated within or close to the walls of cerebral ventricles. For the hippocampus and cerebellum, postnatal neurogenic centers are localized in situ [2,9], where the precursor cells of the new neurons are found [3,49,64]. Recently, there has been intense research on the adult SVZ or subependymal layer [61] as the site of origin of neural stem cells and on the potential of these cells to be used for neuronal replacement. While there has been much interest and work performed investigating the use of fetal or cultured stem cells, there are several distinct advantages to employing resident adult stem cells for neuronal replacement strategies. Non-autologous tissue grafting always has the potential for immunologic rejection, possibly requiring the long-term, if not life-long, use of immunosuppressive agents; adult-derived, autologous stem cells would avoid this difficulty. Furthermore, as has been recently discussed in many reviews [14,16,20,25], there is currently a great controversy regarding the ethical considerations of embryonic-derived tissues; stem cells derived from the graft recipient would alleviate most ethical concerns. The success of SVZ stem cell neuronal replacement depends upon understanding the developmental potential of the resident stem cells, the molecular signals that determine differentiation, migration, survival, and integration into functional circuits, and the potential signals of host target tissue that may affect these cellular processes. Here, we discuss these many hurdles that must be overcome and briefly review some of the experiments utilizing primary adult SVZ neuronal precursors that approach these difficulties.

KEY WORDS: Graft, Adult neurogenesis, Neural precursors, Brain repair, SVZ.

INTRODUCTION The cause of neuronal death in Parkinson’s, Alzheimer’s, and other neurodegenerative diseases is not known, except in some hereditary forms of these disorders in which a mutated gene has been identified. Even in these cases, the molecular mechanisms that underlie the loss of specific populations of neurons have not been determined. Some of the biochemical events that occur during neuronal death have been elucidated, but there are few suitable strategies to regenerate the lost neurons. Embryonic neural cell transplantation has been investigated intensely to achieve neuronal regeneration [12]. Fewer studies have focused on the use of adult brain-derived cells in neuronal replacement strategies. Neurogenesis in the vertebrate brain was once thought to be a phenomenon exclusive to embryonic development; however, in relatively recent years, postnatal neurogenesis has been found in fishes [11], amphibians [15,64], reptiles [28,33,49,62], and birds [3,5,34,55]. Neurogenesis in adult mammals has been shown to exist in the olfactory bulbs [1,8,18,19,47] and hippocampus of rodents [2,31,41] and primates [36], including humans [26]. Recent evidence suggests that neurogenesis occurs in the cortex of adult primates and rodents under specific circumstances as well [37,52]. New neurons migrate to their final destinations either radially, guided presumably by radial processes of radial glial cells as it has been described for the radial migration of neuroblasts in the developing mammalian brain [65], tangentially to the brain surface [59], or in chains as in the adult mammalian subventricular zone

SOME TECHNICAL ISSUES: IDENTIFICATION OF SVZ-GRAFTED CELLS When fetal tissue is transplanted into an adult host, grafted neurons tend to remain together, forming a relatively easy to identify “neonucleus.” However, when using SVZ cells that can potentially migrate long distances [18,46,50], methods designed to distinguish between graft and host tissue are an absolute requirement. Following the review by Cadusseau and Peschanski [13] on the identification of embryonic grafted cells, we can classify the

* Address for correspondence: Daniel G. Herrera, Department of Psychiatry, Weill Cornell Medical College, 1300 York Avenue, Box 244, New York, NY 10021, USA. Fax: ⫹1-(212)-746-8529; E-mail: [email protected]

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grafted cell markers into four groups: nuclear, cytoplasmic, cytoplasmic membrane-linked, or exogenous markers of cell processes (e.g., horseradish peroxidase injections into the transplant). Nuclear markers have been the most widely used in SVZ grafts. In recent studies, SVN cells from adult male mice were transplanted into the cerebral cortex of adult female mice and then hybridized in situ with a Y-chromosome-specific probe [39], therefore, using an intrinsic nuclear marker to detect the SVZ-derived cells and their progeny. Genetic modification of donor cells, either by viral vector transfection or using transgenic donors, allows effective detection of grafted cells and their progeny as well (see Fig. 1). Exogenous nuclear markers that are incorporated into DNA during its synthesis (e.g., BrdU and tritiated thymidine) can be used to follow cells after transplantation. In contrast to intrinsic nuclear markers or genetic modifications of donor cells, exogenous nuclear markers are diluted with cell division, which may occurs after SVZ transplantation, thereby weakening the signal. Several transplantation studies have used intrinsic cytoplasmic markers to identify grafted cells, but these cells need to be phenotypically different from those located in the host tissue surrounding the graft. Such markers could be molecules or enzymes specific to a discrete neuronal population. SVZ cells, in vivo, give rise to GABAergic and dopaminergic neurons [46,70]. The presence of dopaminergic cell bodies at a site grafted with SVZ cells where no dopaminergic cell bodies are found in the host suggest that these aminergic neurons are derived from the transplant. However, there remains the possibility that the dopaminergic phenotype is induced, directly or indirectly, by the graft itself. Exogenous cytoplasmic markers are compounds that are rapidly and avidly incorporated by cells in vivo and have been used to label cells before transplantation. Among these compounds are enzymes (horseradish peroxidase), lectins, fluorescent dyes, and gold particles. The techniques of cytoplasmic labeling has raised controversy on many occasions due to several potential problems: (1) the possibility of artefactual labeling of host cells, (2) the sometimes unpredictable labeling of grafted cells, and (3) the fading of signal with time. For example, fluoro-gold is incorporated into fetal cells but is not well retained after transplantation into the adult brains [69]; leakage and subsequent uptake of the tracer by host tissue has been noted, which can lead to erroneous interpretations [13]. Markers linked to cytoplasmic membranes can be used when studying neural transplants between species or strains of mice. For instance, specific glycoproteins are present on the cell surface of specific strains of mice, and grafts derived from these mice can therefore be identified in hosts that are of a different strain or species. Unfortunately, the majority of cross-species transplants do not survive without immunosuppressive treatment. We prefer the use of genetic markers [29] that allow identification of grafted SVZ cells even after cell division; some of these markers even allow for electron microscopic (EM) identification of grafted cells (Fig. 1). EM provides information about the ultrastructure of the grafted cells, which aids their classification, the precise intercellular arrangement of the graft site, and the presence of synaptic contacts that suggest participation in neuronal circuits. CELL TYPES AND ARCHITECTURE OF THE ADULT SVZ As mentioned above, neural stem cells are maintained in the SVZ of the adult mammalian brain. The majority of cells in the adult SVZ are neuroblasts (type A cells) [51] that migrate through an extensive network of tangentially oriented pathways within the lateral wall of the lateral ventricle [21,48]. Type A cells traverse long distances through this network at high speeds by means of chain migration [21,48,73]. Cells in the SVZ network enter the

rostral migratory stream (RMS) and migrate anteriorly into the OB, where they differentiate into interneurons [32,46,48,73]. The chains of type A cells are ensheathed by slowly proliferating astrocytes (type B cells), the second most common cell type in this germinal layer. The most actively proliferating cells in the SVZ, type C, form small clusters dispersed throughout the network. These foci of proliferating type C cells are in close proximity to chains of type A cells [23]. There is evidence that the SVZ lineage progresses from the type B cell (the stem cell) to the rapidlydividing type C cell (transient amplifying intermediate) to the migratory type A cell (migratory neuroblast) [22,24]. TRANSPLANTATION EXPERIMENTS TO DETERMINE THE POTENTIAL OF SVZ-DERIVED PRECURSORS Although the structure of the postnatal SVZ is likely different from the adult, postnatal SVZ cells transplanted to the adult mouse SVZ migrate to the OB and differentiate into interneurons. This result suggests that postnatal SVZ cells respond to local microenvironmental cues in a manner similar to adult SVZ cells. To test the developmental potential of SVZ cells, neonatal SVZ has been grafted into the adult brain [44,74]. The SVZ is a population of neuronal progenitor cells situated in a well-delineated region of the anterior part of the neonatal SVN [50,51], the SVZ, which is comprised mostly of cells similar to type A cells in the adult, likely represents a transient collection of neuroblasts migrating en mass to the OB. SVZ cells from postnatal day 0 –2 rats were prelabeled by intraperitoneal injections of the cell proliferation marker BrdU. These cells were then implanted into the striatum of adult rats approximately 1 month after unilateral denervation by 6-OHDA [74]. The recipient brains were processed at different postimplantation times, and grafted cells identified by their BrdU-positive nuclei. According to this study, at all intervals, the majority of the surviving SVZ cells exhibited a neuronal phenotype. Interestingly, they could be distinguished from the cells of the host striatum because they resembled the intrinsic granule cells of the OB, their usual fate. At longer times, a greater number of the transplanted SVZ cells had migrated from their site of implantation, often towards an outlying blood vessel, and the density of cells within the core of the transplant was reduced. There were rarely any signs of transplant rejection or a glial scar surrounding the transplant. In the core of the transplant there were low numbers of glial fibrillary acidic protein-positive cells. One interpretation of these results is that the majority of grafted SVZ cells are similar to the adult type A cells (migratory neuroblasts). Type A cells already express neuronal markers, including the transcription factor Dlx-2, which has been shown to be important for the generation of a certain class of interneurons in embryonic development [6]; hence, it is possible that type A cells already have a genetic program initiated that “commits” them to a particular neuronal phenotype. It is also possible that the adult striatum lacks developmental cues that would reveal the full developmental potential of SVZ cells. For instance, perhaps SVZ cells can respond to extracellular signals instructing development of striatal neurons but that these signals are not present in the adult brain. To test this notion, postnatal mouse SVZ precursors were grafted into the ventricles of the embryonic day 15 brains [44]. Graft-derived cells were found at multiple levels of the neuraxis, including septum, thalamus, hypothalamus, and in large numbers in the midbrain inferior colliculus. There was no integration into the cortex or hippocampus. Neuronal differentiation of graft-derived cells was demonstrated by expression of the neuron-specific transgene, double-staining with neuron-specific ␤-tubulin antibodies, and the dendritic arbors

ADULT STEM CELLS IN BRAIN REPAIR

FIG. 1. Strategies in neural stem cell grafting. Transgenic mice carrying genetic markers can be used as donors of neural stem cells in transplants into the adult brain, thus allowing detection of donor-derived cells and their progeny. (A) Micrograph shows semi-thin section of adult striatum transplanted with median ganglionic eminence (MGE)-derived cells from neuron-specific enolase promoter (NSE:LacZ) mice. Differentiated neurons express the ␤-galactosidase gene, that with proper histochemical processing results in typical perinuclear blue precipitates. 1 cm ⫽ 10 ␮m. Arrow points to the cell shown at the electron microscopic (EM) level in (B). EM analysis of the blue precipitate shown in (A) reveals the deposit of electrodense crystals (arrow). This typical morphology allows easy identification of graft-derived cells that had differentiated into mature neurons. 1 cm ⫽ 2 ␮m. (C) Grafts derived from transgenic mice carrying the human placental alkaline phosphatase are clearly seen here. MGE cells obtained from transgenic mice were transplanted into adult striatum. The morphology of transplanted cells can be clearly determined. 1 cm ⫽ 100 ␮m.

revealed by a lipophilic dye. This study suggests that postnatal SVZ cells can migrate through and differentiate into neurons within multiple embryonic brain regions other than the OB. Similar to the study by Zigova et al. [74], SVZ-derived grafted cells were only observed to differentiate into interneurons, although specific neuronal phenotypes were not intensively studied in either study. As previously indicated, there are a large number of proliferating neural precursors that persists in the adult brain SVZ. We transplanted explants of adult SVZ carrying ␤-galactosidase under the control of neuron-specific enolase promoter (NSE:LacZ) [30]

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FIG. 2. Subventricular zone (SVZ) derived grafts transplanted into the adult brain. Experiments were carried out using neuron-specific enolase promoter (NSE:LacZ) transgenic mice [30]. These animals carry the ␤-galactosidase gene, which allows the detection of these cells with histochemical processing, attached to the NSE promoter (a neuronal marker). Therefore, cells derived from these mice will be detected as carrying a “blue dot” when they differentiate into neurons. The SVZ of adult NSE:LacZ mice was dissected out into small explants and transplanted into three different brain regions: cortex, striatum, and the olfactory bulb. SVZ cell grafted into the striatum or cortex migrated very little outside of the graft and rarely differentiated into fully mature neurons. Grafts into the olfactory bulb, the target of SVZ born young neurons, dispersed throughout the olfactory bulb and differentiated into bulb granular neurons. These results suggested that adult SVZ-derived cells had limited differentiation and migration potential when transplanted into a non-neurogenic area of the brain.

into the striatum, cortex, hippocampus, and OB of adult mice [40] (Fig. 2). Two to eight weeks after transplantation, grafted cells were present in all recipient regions. Interestingly, SVZ explants grafted to the cortex and striatum maintained the typical cytoarchitecture found normally in situ (e.g., type A cells remained associated to type B, type C, and ependymal cells). SVZ explants transplanted to the OB consistently migrated extensively from the graft site and differentiated into mature interneurons; in fact, the migration was so complete that what remained of the explant was difficult to observe. Electron microscopy confirmed that most graft-derived cells in the OB were neurons. Only very few graftderived cells migrated away from explants in the striatum and cortex, suggesting that these regions do not support the migration of SVZ cells. Furthermore, the majority of SVZ cells that migrated from the explants in the striatum and cortex were found to be astrocytes by EM analysis; only very few NSE:LacZ positive cells could be confirmed to be neurons. The dentate gyrus of the hippocampus is, like the OB, a neurogenic area of the adult brain. Interestingly, SVZ explants grafted to the dentate gyrus did not support the formation of SVZ-derived neurons. While it is possible that the neurogenic microenvironment of the dentate gyrus is distinct from that of the SVZ and that SVZ cells are unable to interpret the cues, it is also possible that the use of explants impaired the interaction of SVZ cells with local developmental

762 signals. The tight intercellular interactions of cells within the explants may not have allowed graft-derived cells to position in the precise neurogenic region of the dentate gyrus. It is possible that intimate contact between precursors and cells of the dentate gyrus is required in order for the precursors to differentiate into neurons [71]. Recently, another group has shown that there is a survival of adult SVZ-derived precursors grafted into the adult brain [39]. However, this study did not fully characterize the phenotype of the surviving cells. The graft sites of neural cell transplants likely contain a multitude of extracellular signals that influence the proliferation, migration, differentiation, survival, and functional integration of the transplanted cells. The SVZ of adult brains appears to contain microenvironmental cues that can direct the neuronal differentiation of not only SVZ cells but also neural stem cells cultured from the adult hippocampus [70]. Hence, it will be important to elucidate the molecular signals native to the adult SVZ; recreating aspects of the SVZ neurogenic microenvironment at graft sites may enhance current neuronal replacement transplantation strategies. We showed that the bone morphogenetic protein (BMP) antagonist Noggin is expressed by ependymal cells adjacent to the SVZ [45]. SVZ cells were found to express both BMPs as well as their cognate receptors. BMPs potently inhibited neurogenesis both in vitro and in vivo. BMPinduced signaling blocked the production of neurons by SVZ precursors by directing their glial differentiation. Purified mouse Noggin protein promoted neurogenesis in vitro and inhibited glial cell differentiation. Using a viral vector, Noggin was overexpressed in the adult striatum, which promoted neuronal differentiation of dissociated SVZ cells grafted to this region [45]. These data together suggests that ependymal Noggin production creates a neurogenic environment in the adjacent SVZ by blocking endogenous BMP signaling. These data also suggest that BMP signaling may be a part of the signaling that induces the glial differentiation of grafted SVZ cells in our earlier study [40] and that antagonizing this signaling may promote neuronal differentiation of SVZ precursors at a diversity of transplant sites. It is important to note that while Noggin promoted the neuronal differentiation of grafted SVZ cells, the migration in the ectopic location of the striatum was very limited. It is also important to realize that the injury that results from the grafting itself may induce the expression of extracellular signals that may influence the fate of transplanted cells [7,35]. Hence, it will be important to further reveal not only the neurogenic microenvironment of the SVZ but also to determine the changes induced in the transplant site itself. Recently, Steve Goldman’s lab demonstrated that the adenoviral vector induced overexpression of brain-derived neurotrophic factor (BDNF) from the ependymal cell layer resulted not only in the expected increase production of OB interneurons, but also in the generation of new neurons in the striatum [10]. Intriguingly, the control adenoviral vector (that carried only the green fluorescent protein (GFP) marker gene) also induced some neurogenesis for the striatum. The authors suggest that the adenoviral vector itself may have induced the expression of particular cytokines, altering the microenvironment of either the SVZ or the striatum, allowing for the observed striatal neurogenesis. This study suggests that the striatal neurogenesis has its origin in the adult SVZ, however, direct evidence for this is lacking. Similar to the results from the Goldman laboratory, studies from Marla Luskin’s group demonstrate that infusion of BDNF directly into the ventricles also appears to induce neurogenesis in the striatum as well as the septum and hypothalamus [60]. It is not clear where these new neurons were born; the authors suggest that they might have their origin in the SVZ but may also be born from

LIM ET AL. stem cells residing locally. Lesioning of dopaminergic neurons in the substantia nigra (SN) followed by administration of transforming growth factor (TGF)␣ into the putamen resulted in the generation of new dopaminergic neurons in the striatum [27]. These studies together suggest that a combination of gene therapy, trophic factors, and manipulation of neural stem cells from the adult brain—in or ex situ—may be the key to brain repair following traumatic or neurodegenerative injury. NEURONAL PRECURSORS IN THE ADULT HIPPOCAMPUS Reviewing the use of neural stem cells derived from the adult hippocampus and its use in transplantations is not a goal of this paper; however, there are very interesting findings that we would like to mention since the first successful adult-derived neural cell grafts were done with hippocampal stem cells [66]. Neurogenesis persists in the adult dentate gyrus of rodents throughout the life of the organism. The factors regulating proliferation, survival, migration, and differentiation of neuronal progenitors are now being extensively studied. Similar to SVZ cells, cells from the adult hippocampus can be propagated, cloned in vitro, and induced to differentiate into neurons and glial cells [31]. Cells cultured from the adult rodent hippocampus can be genetically marked and transplanted back to the adult brain where they survive and differentiate into mature neurons and glial cells [30]. Primary embryonic hippocampal neurons can develop morphologically and functionally in culture but do not survive more than a few weeks. Basic fibroblast growth factor (bFGF), in a dose-dependent manner, can induce the survival (50 pg to 1 ng/ml) and proliferation (10 –20 ng/ml) of embryonic and adult hippocampal progenitor neurons in vitro [30]. In serum-free medium containing high concentrations of bFGF, neuronal precursors could be passaged and grown as continuous cell lines and differentiated into neurons. The neuronal nature of the differentiated cells was positively established by immunostaining with several different neuron-specific markers and by ultrastructural analyses [67]. Cultured adult rat hippocampal progenitors (AHPs) grafted to adult rat hippocampus showed site-specific neuronal differentiation. When grafting AHPs into homotypic (hippocampus) or heterotypic (the rostral migratory pathway) neurogenic sites or a heterotypic, non-neurogenic site (the cerebellum) it was found that grafts into neurogenic— but not non-neurogenic sites—showed neuronal differentiation [70]. Furthermore, AHPs grafted in the rostral migratory pathway migrated into the OB, differentiating into tyrosine-hydroxylase-positive neurons, a non-hippocampus phenotype. These results reveal that AHP populations can respond to persistent neuronal differentiation cues in the adult central nervous system [70]. POSSIBLE STRATEGIES TO ACHIEVE BRAIN REPAIR USING ADULT SVZ CELLS The success of SVZ-derived grafts into the brain has been limited. Possible strategies to improve neuronal replacement in non-neurogenic sites by adult SVZ cells include, but are not limited to, modifying the neural stem cells, altering the host environment to better accept the SVZ cells, or both. SVZ cells could be modified prior to grafting and exposed to molecules that have an effect on the proliferation, migration, differentiation, survival, and/or functional integration of the transplanted cells into the host tissue. Some of the molecules that have been shown to have a role on SVZ cells in vivo or in vitro include among others BDNF [10,42,60], epidermal growth factor [53,67], fibroblast growth factor [38,57], TGF␣ [27,72], ephrins [17], or Noggin [45] and exposure to these factors could result in increased

ADULT STEM CELLS IN BRAIN REPAIR potential of SVZ cells to replace lost neurons. Another strategy to replace neurons would consist in not only exposing SVZ cells to these factors but also genetically modifying these cells. We need to understand further the biology of SVZ neural stem cells and how and when to possibly use them to restore brain function. For instance, it would be a profound advance in grafting technology to understand the molecular basis of tangential chain migration so that we may direct the dispersal of grafted precursors. Specific populations of SVZ cells could be selected for transplantation and modified to restore neurological function in specific areas or neuronal populations. Neurogenesis has been shown to exist in the adult human brain, and a recent paper suggests that neural stem cells are located in the adult human OBs [56], which would perhaps allow relatively facile surgical access for the isolation of stem cells for autograft; temporal lobe resection samples from humans also have been found to harbor neuronal precursors [43, 63,68]. Treatment of neurological disorders with adult stem cells may soon be possible if progress in this field of research continues at the pace of the last few years.

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