Glial influences on neural stem cell development: cellular niches for adult neurogenesis

Neural stem cells continually generate new neurons in very limited regions of the adult mammalian central nervous system. In the neurogenic regions there are unique and highly specialized microenvironments (niches) that tightly regulate the neuronal development of adult neural stem cells. Emerging evidence suggests that glia, particularly astrocytes, have key roles in controlling multiple steps of adult neurogenesis within the niches, from proliferation and fate specification of neural progenitors to migration and integration of the neuronal progeny into pre-existing neuronal circuits in the adult brain. Identification of specific niche signals that regulate these sequential steps during adult neurogenesis might lead to strategies to induce functional neurogenesis in other brain regions after injury or degenerative neurological diseases.     

Olfactory bulb neurogenesis and its neurological impact

Contrary to the long-held dogma according to which the adult mammalian brain does not produce neurons anymore, neuronal turnover has been reported in two discrete areas of the adult brain: the hippocampus and the olfactory bulb. Adult-generated neurons are produced from neural stem cells located in the hippocampal subgranular zone and the subventricular zone of the lateral ventricles. Recently, number of genetic and epigenetic factors that modulate proliferation of stem cells, migration, differentiation and survival of newborn neurons have been characterized. We know that neurogenesis increases in the diseased brain, after stroke or after traumatic brain injury. Importantly, progenitors from the subventricular zone, but not from the subgranular zone, are incorporated at the sites of injury, where they replace some of the degenerated neurons. Thus, the central nervous system has the capacity to regenerate itself after injury and, today, researchers develop strategies aimed at promoting neurogenesis in diseased areas. This basic research is attracting a lot of attention because of the hope that it will lead to regeneration and reconstruction therapy for the damaged brain. In this review, we discuss major findings concerning the organization of the neurogenic niche located in the subventricular zone and examine both intrinsic and extrinsic factors that regulate adult neurogenesis. Then, we present evidences for the intrinsic capability of the adult brain for cell replacement, and shed light on recent works demonstrating that one can greatly enhance appropriate brain cell replacement by using molecular cues known to endogenously control proliferation, migration, differentiation and/or survival of subventricular zone progenitors. Finally, we review some of the advantages and limits of strategies aimed at using endogenous progenitors and their relevance to human clinics.     

Region-specific generation of functional neurons from naive embryonic stem cells in adult brain

Embryonic stem (ES) cells are multipotent progenitors with unlimited developmental potential, and in vitro differentiated ES cell-derived neuronal progenitors can develop into functional neurons when transplanted in the central nervous system. As the capacity of naive primary ES cells to integrate in the adult brain and the role of host neural tissue therein are yet largely unknown, we grafted low densities of undifferentiated mouse ES (mES) cells in adult mouse brain regions associated with neurodegenerative disorders; and we demonstrate that ES cell-derived neurons undergo gradual integration in recipient tissue and acquire morphological and electrophysiological properties indistinguishable from those of host neurons. Only some brain areas permitted survival of mES-derived neural progenitors and formed instructive environments for neuronal differentiation and functional integration of naive mES cells. Hence, region-specific presence of microenvironmental cues and their pivotal involvement in controlling ES cell integration in adult brain stress the importance of recipient tissue characteristics in formulating cell replacement strategies for neurodegenerative disorders.     

Cell replacement therapies for nervous system regeneration

The adult brain was thought to be a slowly decaying organ, a sophisticated but flawed machine condemned to inevitable decline. Today we know that the brain is more plastic than previously assumed, as most prominently demonstrated by the constitutive birth of new neurons that occurs in selected regions of the adult brain, even in humans. However, the overall modest capacity for endogenous repair of the central nervous system (CNS) has sparked interest in understanding the barriers to neuronal regeneration and in developing novel approaches to enable neuronal and circuit repair for therapeutic benefit in neurodegenerative disorders and traumatic injuries. Scientists recently assembled in Baeza, a picturesque town in the south of Spain, to discuss aspects of CNS regeneration. The picture that emerged shows how an integrated view of developmental and adult neurogenesis may inform the manipulation of neural progenitors, differentiated cells, and pluripotent stem cells for therapeutic benefit and foster new understanding of the inner limits of brain plasticity.     

Isolation and culture of adult neurons and neurospheres

Here we present a protocol for extraction and culture of neurons from adult rat or mouse CNS. The method proscribes an optimized protease digestion of slices, control of osmolarity and pH outside the incubator with Hibernate and density gradient separation of neurons from debris. This protocol produces yields of millions of cortical, hippocampal neurons or neurosphere progenitors from each brain. The entire process of neuron isolation and culture takes less than 4 h. With suitable growth factors, adult neuron regeneration of axons and dendrites in culture proceeds over 1-3 weeks to allow controlled studies in pharmacology, electrophysiology, development, regeneration and neurotoxicology. Adult neurospheres can be collected in 1 week as a source of neuroprogenitors ethically preferred over embryonic or fetal sources. This protocol emphasizes two differences between neuron differentiation and neurosphere proliferation: adhesion dependence and the differentiating power of retinyl acetate.     

Antibodies against pax6 immunostain amacrine and ganglion cells and neuronal progenitors,but not rod precursors,in the normal and regenerating retina of the goldfish

Pax6 is a developmental regulatory gene that plays a key role in the development of the embryonic brain, eye, and retina. This gene is also expressed in discrete groups of neurons within the adult brain. In this study, antibodies raised against a fusion protein from a zebra fish pax6 cDNA were used to investigate the expression of the pax6 gene in the mature, growing, and regenerating retina of the goldfish. On western blots of retinal proteins, the pax6 antibodies recognize a single band at the approximate size of the zebra fish pax6 protein. In retinal sections, the antibodies label the nuclei of mature amacrine and some ganglion cells. At the retinal margin, where neurogenesis and cellular differentiation continually occur in goldfish, the antibodies label neuronal progenitors and the newly postmitotic neurons. Following injury and during neuronal regeneration, the antibodies label mitotically active progenitors of regenerating neurons. Rod precursors, proliferating cells that normally give rise solely to rod photoreceptors and are the presumed antecedents of the injury-stimulated neuronal progenitors, are not immunostained by antibodies to the pax6 protein. The results of this study document the identity of pax6-expressing cells in the mature retina and demonstrate that in the goldfish pax6 is expressed in neuronal progenitors during both retinal growth and regeneration. © 1996 John Wiley & Sons, Inc.     

Aging impairs the essential contributions of non‐glial progenitors to neurorepair in the dorsal telencephalon of the Killifish Nothobranchius furzeri

The aging central nervous system (CNS) of mammals displays progressive limited regenerative abilities. Recovery after loss of neurons is extremely restricted in the aged brain. Many research models fall short in recapitulating mammalian aging hallmarks or have an impractically long lifespan. We established a traumatic brain injury model in the African turquoise killifish (Nothobranchius furzeri), a regeneration‐competent vertebrate that evolved to naturally age extremely fast. Stab‐wound injury of the aged killifish dorsal telencephalon unveils an impaired and incomplete regeneration response when compared to young individuals. In the young adult killifish, brain regeneration is mainly supported by atypical non‐glial progenitors, yet their proliferation capacity clearly declines with age. We identified a high inflammatory response and glial scarring to also underlie the hampered generation of new neurons in aged fish. These primary results will pave the way to unravel the factor age in relation to neurorepair, and to improve therapeutic strategies to restore the injured and/or diseased aged mammalian CNS.     

The receptor for Granulocyte-colony stimulating factor (G-CSF) is expressed in radial glia during development of the nervous system

Background  

Granulocyte colony-stimulating (G-CSF) factor is a well-known hematopoietic growth factor stimulating the proliferation and differentiation of myeloid progenitors. Recently, we uncovered that G-CSF acts also as a neuronal growth factor in the brain, which promotes adult neural precursor differentiation and enhances regeneration of the brain after insults. In adults, the receptor for G-CSF is predominantly expressed in neurons in many brain areas. We also described expression in neurogenic regions of the adult brain, such as the subventricular zone and the subgranular layer of the dentate gyrus. In addition, we found close co-localization of the G-CSF receptor and its ligand G-CSF. Here we have conducted a systematic expression analysis of G-CSF receptor and its ligand in the developing embryo.     

Laminin-immunoreactive glia distinguish regenerative adult CNS systems from non-regenerative ones.

Most regions of the adult mammalian central nervous system (CNS) do not support axonal growth and regeneration. Laminin, expressed by cultured astrocytes and known to promote neurite outgrowth of cultured neurons, is normally present in brain basement membranes, and only transiently induced in adult brain astrocytes by injury. Here I provide three lines of evidence which suggest that the continued expression of laminin by astrocytes may be a prerequisite for axonal growth and regeneration in adult CNS. Firstly, laminin is continuously present in astrocytes of adult rat olfactory bulb apparently in close association with the olfactory nerve axons. Secondly, laminin is continuously expressed by astrocytes in adult frog brain, and sectioning of the optic tract further increases laminin immunoreactivity in astrocytes of the optic tectum during the period of axonal regeneration. Lastly, laminin appears normally in astrocytes of the frog and goldfish optic nerves which regenerate, but not in astrocytes of the rat or chick optic nerves which do not regenerate. The selective association of laminin with axons that undergo growth and regeneration in vivo is consistent with the possibility that astrocytic laminin provides these central nervous systems with their regenerative potential.     

Neural progenitors from human embryonic stem cells.

The derivation of neural progenitor cells from human embryonic stem (ES) cells is of value both in the study of early human neurogenesis and in the creation of an unlimited source of donor cells for neural transplantation therapy. Here we report the generation of enriched and expandable preparations of proliferating neural progenitors from human ES cells. The neural progenitors could differentiate in vitro into the three neural lineages--astrocytes, oligodendrocytes, and mature neurons. When human neural progenitors were transplanted into the ventricles of newborn mouse brains, they incorporated in large numbers into the host brain parenchyma, demonstrated widespread distribution, and differentiated into progeny of the three neural lineages. The transplanted cells migrated along established brain migratory tracks in the host brain and differentiated in a region-specific manner, indicating that they could respond to local cues and participate in the processes of host brain development. Our observations set the stage for future developments that may allow the use of human ES cells for the treatment of neurological disorders.     

Characterization of Proliferating Neural Progenitors after Spinal Cord Injury in Adult Zebrafish

Zebrafish can repair their injured brain and spinal cord after injury unlike adult mammalian central nervous system. Any injury to zebrafish spinal cord would lead to increased proliferation and neurogenesis. There are presences of proliferating progenitors from which both neuronal and glial loss can be reversed by appropriately generating new neurons and glia. We have demonstrated the presence of multiple progenitors, which are different types of proliferating populations like Sox2⁺ neural progenitor, A2B5⁺ astrocyte/ glial progenitor, NG2⁺ oligodendrocyte progenitor, radial glia and Schwann cell like progenitor. We analyzed the expression levels of two common markers of dedifferentiation like msx-b and vimentin during regeneration along with some of the pluripotency associated factors to explore the possible role of these two processes. Among the several key factors related to pluripotency, pou5f1 and sox2 are upregulated during regeneration and associated with activation of neural progenitor cells. Uncovering the molecular mechanism for endogenous regeneration of adult zebrafish spinal cord would give us more clues on important targets for future therapeutic approach in mammalian spinal cord repair and regeneration.     

Brain regeneration in anuran amphibians

Urodele amphibians are highly regenerative animals. After partial removal of the brain in urodeles, ependymal cells around the wound surface proliferate, differentiate into neurons and glias and finally regenerate the lost tissue. In contrast to urodeles, this type of brain regeneration is restricted only to the larval stages in anuran amphibians (frogs). In adult frogs, whereas ependymal cells proliferate in response to brain injury, they cannot migrate and close the wound surface, resulting in the failure of regeneration. Therefore frogs, in particular Xenopus, provide us with at least two modes to study brain regeneration. One is to study normal regeneration by using regenerative larvae. In this type of study, the requirement of reconnection between a regenerating brain and sensory neurons was demonstrated. Functional restoration of a regenerated telencephalon was also easily evaluated because Xenopus shows simple responses to the stimulus of a food odor. The other mode is to compare regenerative larvae and non-regenerative adults. By using this mode, it is suggested that there are regeneration-competent cells even in the non-regenerative adult brain, and that immobility of those cells might cause the failure of regeneration. Here we review studies that have led to these conclusions.     

In vivo fate analysis reveals the multipotent and self-renewal features of embryonic AspM expressing cells

Radial Glia (RG) cells constitute the major population of neural progenitors of the mouse developing brain. These cells are located in the ventricular zone (VZ) of the cerebral cortex and during neurogenesis they support the generation of cortical neurons. Later on, during brain maturation, RG cells give raise to glial cells and supply the adult mouse brain of Neural Stem Cells (NSC). Here we used a novel transgenic mouse line expressing the CreER(T2) under the control of AspM promoter to monitor the progeny of an early cohort of RG cells during neurogenesis and in the post natal brain. Long term fate mapping experiments demonstrated that AspM-expressing RG cells are multi-potent, as they can generate neurons, astrocytes and oligodendrocytes of the adult mouse brain. Furthermore, AspM descendants give also rise to proliferating progenitors in germinal niches of both developing and post natal brains. In the latter--i.e. the Sub Ventricular Zone--AspM descendants acquired several feature of neural stem cells, including the capability to generate neurospheres in vitro. We also performed the selective killing of these early progenitors by using a Nestin-GFP(flox)-TK allele. The forebrain specific loss of early AspM expressing cells caused the elimination of most of the proliferating cells of brain, a severe derangement of the ventricular zone architecture, and the impairment of the cortical lamination. We further demonstrated that AspM is expressed by proliferating cells of the adult mouse SVZ that can generate neuroblasts fated to become olfactory bulb neurons.     

Towards brain repair: Insights from teleost fish

In the adult mammalian brain, the ability to minimize secondary cell death after injury, and to repair nervous tissue through generation of new neurons, is severely compromised. By contrast, certain taxa of non-mammalian vertebrates possess an enormous potential for regeneration. Examination of one of these taxa, teleost fish, has revealed a close link between this phenomenon and constitutive adult neurogenesis. Key factors mediating successful regeneration appear to be: elimination of damaged cells by apoptosis, instead of necrosis; activation of mechanisms that prevent the occurrence of secondary cell death; increased production of new neurons that replace neurons lost to injury; and activation of developmental mechanisms that mediate directed migration of the new cells to the site of injury, the differentiation of the young cells, and their integration into the existing neural network. Comparative analysis has suggested that constitutive adult neurogenesis is a primitive vertebrate trait, the main function of which has been to ensure a numerical matching between muscle fibers/sensory receptor cells and central elements involved in motor control/processing of sensory information associated with these peripheral elements. It is hypothesized that, when in the course of the evolution of mammals a major shift in the growth pattern from hyperplasia to hypertrophy took place, the number of neurogenic brain regions and new neurons markedly decreased. As a consequence, the potential for neuronal regeneration was greatly reduced, but remnants of neurogenic areas have persisted in the adult mammalian brain in form of quiescent stem cells. It is likely that the study of regeneration-competent taxa will provide important information on how to activate intrinsic mechanisms for successful brain regeneration in humans.     

Transplanted neurons form both normal and ectopic projections in the adult brain

Transplantation of embryonic or stem cell derived neurons has been proposed as a potential therapy for several neurological diseases. Previous studies reported that transplanted embryonic neurons extended long-distance projections through the adult brain exclusively to appropriate targets. We transplanted E14 lateral ganglionic eminence (LGE) and E15 cortical precursors from embryonic mice into the intact adult brain and analyzed the projections formed by transplanted neurons. In contrast to previous studies, we found that transplanted embryonic neurons formed distinct long-distance projections to both appropriate and ectopic targets. LGE neurons transplanted into the adult striatum formed projections not only to the substantia nigra, a normal target, but also to the claustrum and through all layers of fronto-orbital cortex, regions that do not normally receive striatal input. In some cases, inappropriate projections outnumbered appropriate projections. To examine the relationship between the donor cells and host brain in establishing the pattern of projections, we transplanted cortical precursors into the adult striatum. Despite their heterotopic location, cortical precursors not only predominantly formed projections appropriate for cortical neurons, but they also formed projections to inappropriate targets. Transplantation of GFP-expressing cells into beta-galactosidase-expressing mice confirmed that the axonal projections were not created by the fusion of donor and host cells. These results suggest that repairing the brain using transplantation may be more complicated than previously expected, because exuberant ectopic projections could result in brain dysfunction. Understanding the signals regulating axonal extension in the adult brain will be necessary to harness stem cells or embryonic neurons for effective neuronal-replacement therapies.     

Neural stem cells in mammalian development

Neural stem cells (NSCs) are primary progenitors that give rise to neurons and glia in the embryonic, neonatal and adult brain. In recent years, we have learned three important things about these cells. First, NSCs correspond to cells previously thought to be committed glial cells. Second, embryonic and adult NSCs are lineally related: they transform from neuroepithelial cells into radial glia, then into cells with astroglial characteristics. Third, NSCs divide asymmetrically and often amplify the number of progeny they generate via symmetrically dividing intermediate progenitors. These advances challenge our traditional perceptions of glia and stem cells, and provide the foundation for understanding the molecular basis of mammalian NSC behavior.     

Using the Tg(nrd:egfp)/albino zebrafish line to characterize in vivo expression of neurod

In this study, we used a newly-created transgenic zebrafish, Tg(nrd:egfp)/albino, to further characterize the expression of neurod in the developing and adult retina and to determine neurod expression during adult photoreceptor regeneration. We also provide observations regarding the expression of neurod in a variety of other tissues. In this line, EGFP is found in cells of the developing and adult retina, pineal gland, cerebellum, olfactory bulbs, midbrain, hindbrain, neural tube, lateral line, inner ear, pancreas, gut, and fin. Using immunohistochemistry and in situ hybridization, we compare the expression of the nrd:egfp transgene to that of endogenous neurod and to known retinal cell types. Consistent with previous data based on in situ hybridizations, we show that during retinal development, the nrd:egfp transgene is not expressed in proliferating retinal neuroepithelium, and is expressed in a subset of retinal neurons. In contrast to previous studies, nrd:egfp is gradually re-expressed in all rod photoreceptors. During photoreceptor regeneration in adult zebrafish, in situ hybridization reveals that neurod is not expressed in Müller glial-derived neuronal progenitors, but is expressed in photoreceptor progenitors as they migrate to the outer nuclear layer and differentiate into new rod photoreceptors. During photoreceptor regeneration, expression of the nrd:egfp matches that of neurod. We conclude that Tg(nrd:egfp)/albino is a good representation of endogenous neurod expression, is a useful tool to visualize neurod expression in a variety of tissues and will aid investigating the fundamental processes that govern photoreceptor regeneration in adults.     

Migration of bone marrow progenitor cells in the adult brain of rats and rabbits

Neurogenesis takes place in the adult mammalian brain in three areas:Subgranular zone of the dentate gyrus(DG);subventricular zone of the lateral ventricle;olfactory bulb.Different molecular markers can be used to characterizethe cells involved in adult neurogenesis.It has been recently suggested that a population of bone marrow(BM)progenitor cells may migrate to the brain and differentiate into neuronal lineage.To explore this hypothesis,we injected recombinant SV40-derived vectors into the BM and followed the potential migration of the transduced cells.Long-term BM-directed gene transfer using recombinant SV40-derived vectors leads to expression of the genes delivered to the BM firstly in circulating cells,then after several months in mature neurons and microglial cells,and thus without central nervous system(CNS)lesion.Most of transgene-expressing cells expressed NeuN,a marker of mature neurons.Thus,BM-derived cells may function as progenitors of CNS cells in adult animals.The mechanism by which the cells from the BM come to be neurons remains to be determined.Although the observed gradual increase in transgene-expressing neurons over 16mo suggests that the pathway involved differentiation of BM-resident cells into neurons,cell fusion as the principal route cannot be totally ruled out.Additional studies using similar viral vectors showed that BM-derived progenitor cells migrating in the CNS express markers of neuronal precursors or immature neurons.Transgene-positive cells were found in the subgranular zone of the DG of the hippocampus 16 mo after intramarrow injection of the vector.In addition to cells expressing markers of mature neurons,transgene-positive cells were also positive for nestin and doublecortin,molecules expressed by developing neuronal cells.These cells were actively proliferating,as shown by short term BrdU incorporation studies.Inducing seizures by using kainic acid increased the number of BM progenitor cells transduced by SV40vectors migrating to the hippocampus,and these cells were seen at earlier time points in the DG.We show that the cell membrane chemokine receptor,CCR5,and its ligands,enhance CNS inflammation and seizure activity in a model of neuronal excitotoxicity.SV40-based gene delivery of RNAi targeting CCR5 to the BM results in downregulating CCR5 in circulating cells,suggesting that CCR5 plays an important role in regulating traffic of BM-derived cells into the CNS,both in the basal state and in response to injury.Furthermore,reduction in CCR5 expression incirculating cells provides profound neuroprotection from excitotoxic neuronal injury,reduces neuroinflammation,and increases neuronal regeneration following this type of insult.These results suggest that BM-derived,transgeneexpressing,cells can migrate to the brain and that they become neurons,at least in part,by differentiating into neuron precursors and subsequently developing into mature neurons.