Postnatal neural precursor cell regions in the rostral subventricular zone, hippocampal subgranular zone and cerebellum of the dog (Canis lupus familiaris)

Identification of neural stem and progenitor cells (NPCs) in vitro and in vivo is essential to the use of developmental and disease models of neurogenesis. The dog is a valuable large animal model for multiple neurodegenerative diseases and is more closely matched to humans than rodents with respect to brain organization and complexity. It is therefore important to determine whether immunohistochemical markers associated with NPCs in humans and rodents are also appropriate for the dog. The NPC markers CD15, CD133, nestin, GFAP and phosphacan (DSD-1) were evaluated in situ in the canine rostral telencephalon, hippocampal dentate gyrus, and cerebellum at different postnatal time-points. Positive staining results were interpreted in the context of region and cellular morphology. Our results showed that neurospheres and cells within the rostral subventricular zone (SVZ), dentate gyrus subgranular zone (SGZ), and white matter tracts of the cerebellum were immunopositive for CD15, nestin and GFAP. Neurospheres and the cerebellum were immunonegative for CD133, whereas CD133 staining was present in the postnatal rostral SVZ. Anti-phosphacan antibody staining delineated the neurogenic niches of the rostral lateral ventricle SVZ and the hippocampal SGZ. Positive staining for phosphacan was also noted in white matter tracts of the cerebellum and within the Purkinje layer. Our results showed that in the dog these markers were associated with regions shown to be neurogenic in rodents and primates.     

Hypothalamic Subependymal Niche: A Novel Site of the Adult Neurogenesis

The discovery of undifferentiated, actively proliferating neural stem cells (NSCs) in the mature brain opened a brand new chapter in the contemporary neuroscience. Adult neurogenesis appears to occur in specific brain regions (including hypothalamus) throughout vertebrates’ life, being considered an important player in the processes of memory, learning, and neural plasticity. In the adult mammalian brain, NSCs are located mainly in the subgranular zone (SGZ) of the hippocampal dentate gyrus and in the subventricular zone (SVZ) of the lateral ventricle ependymal wall. Besides these classical regions, hypothalamic neurogenesis occurring mainly along and beneath the third ventricle wall seems to be especially well documented. Neurogenic zones in SGZ, SVZ, and in the hypothalamus share some particular common features like similar cellular cytoarchitecture, vascularization pattern, and extracellular matrix properties. Hypothalamic neurogenic niche is formed mainly by four special types of radial glia-like tanycytes. They are characterized by distinct expression of some neural progenitor and stem cell markers. Moreover, there are numerous suggestions that newborn hypothalamic neurons have a significant ability to integrate into the local neural pathways and to play important physiological roles, especially in the energy balance regulation. Newly formed neurons in the hypothalamus can synthesize and release food intake regulating neuropeptides and they are sensitive to the leptin. On the other hand, high-fat diet positively influences hypothalamic neurogenesis in rodents. The nature of this intriguing new site of adult neurogenesis is still so far poorly studied and requires further investigations.     

Locating and labeling neural stem cells in the brain

The phenomenon of adult neurogenesis has been demonstrated in most mammals including humans. At least two regions of the adult brain maintain stem cells throughout life; the subgranular zone (SGZ) of the hippocampal dentate gyrus, and the subventricular zone (SVZ) of the lateral ventricle wall. Both regions continuously produce neurons that mature and become integrated into functional networks that are involved in learning and memory and odor discrimination, respectively. Apart from these well‐studied regions neurogenesis has been reported in a number of other brain regions, such as amygdala and cortex. However, these studies have been contested and there is currently no well‐postulated function for non‐SVZ/SGZ neurogenesis. The studies of the regional localization of neurogenesis in the brain have been made possible due to several methods for detecting adult neurogenesis including; bromodeoxyuridine labeling (BrdU) together with markers of mature neurons, genetic labeling, by mouse transgenesis, or with the use of viral vectors. These techniques are already put to creative use and will be essential for the discovery of the nature of the adult neural stem cells. In this mini‐review, we will discuss the localization of neural stem/progenitor cells in the brain and their implications as well as discussing the pro's and con's of stem cell labeling techniques. J. Cell. Physiol. 226: 1–7, 2010. © 2010 Wiley‐Liss, Inc.     

Brain ischemia, neurogenesis, and neurotrophic receptor expression in primates

Generation of new neurons persists in the normal adult mammalian brain, with neural stem/progenitor cells residing in at least two brain regions: the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus (DG). Adult neurogenesis is well documented in the rodent, and has also been demonstrated in vivo in nonhuman primates and humans. Brain injuries such as ischemia affect neurogenesis in adult rodents as both global and focal ischemic insults enhance the proliferation of progenitor cells residing in SGZ or SVZ. We addressed the issue whether an injury triggered activation of endogenous neuronal precursors also takes place in the adult primate brain. We found that the ischemic insult increased the number of progenitor cells in monkey SGZ and SVZ, and caused gliogenesis in the ischemia-prone hippocampal CA1 sector. To better understand the mechanisms regulating precursor cell division and differentiation in the primate, we analyzed the expression at protein level of a panel of potential regulatory molecules, including neurotrophic factors and their receptors. We found that a fraction of mitotic progenitors were positive for the neurotrophin receptor TrkB, while immature neurons expressed the neurotrophin receptor TrkA. Astroglia, ependymal cells and blood vessels in SVZ were positive for distinctive sets of ligands/receptors, which we characterized. Thus, a network of neurotrophic signals operating in an autocrine or paracrine manner may regulate neurogenesis in adult primate SVZ. We also analyzed microglial and astroglial proliferation in postischemic hippocampal CA1 sector. We found that proliferating postischemic microglia in adult monkey CA1 sector express the neurotrophin receptor TrkA, while activated astrocytes were labeled for nerve growth factor (NGF), ligand for TrkA, and the tyrosine kinase TrkB, a receptor for brain derived neurotrophic factor (BDNF). These results implicate NGF and BDNF as regulators of postischemic glial proliferation in adult primate hippocampus.     

For the long run: maintaining germinal niches in the adult brain

The adult mammalian brain retains neural stem cells that continually generate new neurons within two restricted regions: the subventricular zone (SVZ) of the lateral ventricle and the dentate gyrus subgranular zone (SGZ) of the hippocampus. Though these cellular populations are spatially isolated and subserve different brain systems, common themes begin to define adult neurogenic niches: (1) astrocytes serve as both stem cell and niche cell, (2) a basal lamina and concomitant vasculogenesis may be essential components of the niche, and (3) "embryonic" molecular morphogens and signals persist in these niches and play critical roles for adult neurogenesis. The adult neurogenic niches can be viewed as "displaced" neuroepithelium, pockets of cells and local signals that preserve enough embryonic character to maintain neurogenesis for life.     

Neural differentiation of human embryonic stem cells as an in vitro tool for the study of the expression patterns of the neuronal cytoskeleton during neurogenesis

The neural differentiation of human embryonic stem cells (ESCs) is a potential tool for elucidating the key mechanisms involved in human neurogenesis. Nestin and β-III-tubulin, which are cytoskeleton proteins, are marker proteins of neural stem cells (NSCs) and neurons, respectively. However, the expression patterns of nestin and β-III-tubulin in neural derivatives from human ESCs remain unclear. In this study, we found that neural progenitor cells (NPCs) derived from H9 cells express high levels of nestin and musashi-1. In contrast, β-III-tubulin was weakly expressed in a few NPCs. Moreover, in these cells, nestin formed filament networks, whereas β-III-tubulin was distributed randomly as small particles. As the differentiation proceeded, the nestin filament networks and the β-III-tubulin particles were found in both the cell soma and the cellular processes. Moreover, the colocalization of nestin and β-III-tubulin was found mainly in the cell processes and neurite-like structures and not in the cell soma. These results may aid our understanding of the expression patterns of nestin and β-III-tubulin during the neural differentiation of H9 cells.     

PDGFR alpha-positive B cells are neural stem cells in the adult SVZ that form glioma-like growths in response to increased PDGF signaling

Neurons and oligodendrocytes are produced in the adult brain subventricular zone (SVZ) from neural stem cells (B cells), which express GFAP and have morphological properties of astrocytes. We report here on the identification B cells expressing the PDGFRalpha in the adult SVZ. Specifically labeled PDGFRalpha expressing B cells in vivo generate neurons and oligodendrocytes. Conditional ablation of PDGFRalpha in a subpopulation of postnatal stem cells showed that this receptor is required for oligodendrogenesis, but not neurogenesis. Infusion of PDGF alone was sufficient to arrest neuroblast production and induce SVZ B cell proliferation contributing to the generation of large hyperplasias with some features of gliomas. The work demonstrates that PDGFRalpha signaling occurs early in the adult stem cell lineage and may help regulate the balance between oligodendrocyte and neuron production. Excessive PDGF activation in the SVZ in stem cells is sufficient to induce hallmarks associated with early stages of tumor formation.     

The Olfactory Bulb in Newborn Piglet Is a Reservoir of Neural Stem and Progenitor Cells

The olfactory bulb (OB) periventricular zone is an extension of the forebrain subventricular zone (SVZ) and thus is a source of neuroprogenitor cells and neural stem cells. While considerable information is available on the SVZ-OB neural stem cell (NSC)/neuroprogenitor cell (NPC) niche in rodents, less work has been done on this system in large animals. The newborn piglet is used as a preclinical translational model of neonatal hypoxic-ischemic brain damage, but information about the endogenous sources of NSCs/NPCs in piglet is needed to implement endogenous or autologous cell-based therapies in this model. We characterized NSC/NPC niches in piglet forebrain and OB-SVZ using western blotting, histological, and cell culture methods. Immunoblotting revealed nestin, a NSC/NPC marker, in forebrain-SVZ and OB-SVZ in newborn piglet. Several progenitor or newborn neuron markers, including Dlx2, musashi, doublecortin, and polysialated neural cell adhesion molecule were also detected in OB-SVZ by immunoblotting. Immunohistochemistry confirmed the presence of nestin, musashi, and doublecortin in forebrain-SVZ and OB-SVZ. Bromodeoxyuridine (BrdU) labeling showed that the forebrain-SVZ and OB-SVZ accumulate newly replicated cells. BrdU-positive cells were immunolabeled for astroglial, oligodendroglial, and neuronal markers. A lateral migratory pathway for newly born neuron migration to primary olfactory cortex was revealed by BrdU labeling and co-labeling for doublecortin and class III β tubulin. Isolated and cultured forebrain-SVZ and OB-SVZ cells from newborn piglet had the capacity to generate numerous neurospheres. Single cell clonal analysis of neurospheres revealed the capacity for self-renewal and multipotency. Neurosphere-derived cells differentiated into neurons, astrocytes, and oligodendrocytes and were amenable to permanent genetic tagging with lentivirus encoding green fluorescent protein. We conclude that the piglet OB-SVZ is a reservoir of NSCs and NPCs suitable to use in autologous cell therapy in preclinical models of neonatal/pediatric brain injury.     

Genetic visualization of neurogenesis

Neurons are generated from stem or progenitor cells in discrete areas in the adult brain. The exact temporal and spatial distribution of adult neurogenesis has, however, been difficult to establish because of inherent limitations with the currently used techniques, and there are numerous controversies with regard to whether neurons are generated in specific regions or in response to insults. We describe here the generation of transgenic mice that express conditionally active Cre recombinase under the control of a nestin enhancer element. These mice allow the recombination of reporter alleles specifically in neural stem and progenitor cells and the visualization of their progeny in the adult brain. This offers a simple and efficient way to visualize live adult born neurons without the caveats of currently used techniques.     

Role of the nuclear receptor Tailless in adult neural stem cells

Tailless (Tlx) is an orphan nuclear receptor which is specifically expressed in the neural stem cells of the two largest germinal neurogenesis zones in the adult mouse brain, the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus. By interacting with its cofactors, Tlx represses its target genes and plays an important role in the maintenance of adult NSCs. This review provides a snapshot of current knowledge about Tlx function in adult NSCs.     

Functional evaluation of neural stem cell differentiation by single cell calcium imaging

Neurogenesis in the adult mammalian brain occurs in two specific brain areas, the subventricular zone (SVZ) bordering the lateral ventricles and the subgranular zone (SGZ) of the hippocampus. Although these regions are prone to produce new neurons, cultured cells from these neurogenic niches tend to be mixed cultures, containing both neurons and glial cells. Several reports highlight the potential of the self-healing capacity of the brain following injury. Even though much knowledge has been produced on the neurogenesis itself, brain repairing strategies are still far away from patients cure. Here we review general concepts in the neurogenesis field, also addressing the methods available to study neural stem cell differentiation. A major problem faced by research groups and companies dedicated to brain regenerative medicine resides on the lack of good methods to functionally identify neural stem cell differentiation and novel drug targets. To address this issue, we developed a unique single cell calcium imaging-based method to functionally discriminate different cell types derived from SVZ neural stem cell cultures. The unique functional profile of each SVZ cell type was correlated at the single cell level with the immunodetection of specific phenotypic markers. This platform was raised on the basis of the functional response of neurons, oligodendrocytes and immature cells to depolarising agents, to thrombin and to histamine, respectively. We also outline key studies in which our new platform was extremely relevant in the context of drug discovery and development in the area of brain regenerative medicine.     

The dynamic role of bone morphogenetic proteins in neural stem cell fate and maturation

The bone morphogenetic proteins (BMPs) are a group of powerful morphogens that are critical for development of the nervous system. The effects of BMP signaling on neural stem cells are myriad and dynamic, changing with each stage of development. During early development inhibition of BMP signaling differentiates neuroectoderm from ectoderm, and BMP signaling helps to specify neural crest. Thus modulation of BMP signaling underlies formation of both the central and peripheral nervous systems. BMPs secreted from dorsal structures then form a gradient which helps pattern the dorsal-ventral axis of the developing spinal cord and brain. During forebrain development BMPs sequentially induce neurogenesis and then astrogliogenesis and participate in neurite outgrowth from immature neurons. BMP signaling also plays a critical role in maintaining adult neural stem cell niches in the subventricular zone (SVZ) and subgranular zone (SGZ). BMPs are able to exert such diverse effects through closely regulated temporospatial expression and interaction with other signaling pathways.     

Neurogenesis and progenitor cells in the adult human brain: a comparison between hippocampal and subventricular progenitor proliferation

For more than a decade, we have known that the human brain harbors progenitor cells capable of becoming mature neurons in the adult human brain. Since the original landmark article by Eriksson et al. in 1998 (Nat Med 4:1313-1317), there have been many studies investigating the effect that depression, epilepsy, Alzheimer's disease, Huntington's disease, and Parkinson's disease have on the germinal zones in the adult human brain. Of particular interest is the demonstration that there are far fewer progenitor cells in the hippocampal subgranular zone (SGZ) compared with the subventricular zone (SVZ) in the human brain. Furthermore, the quantity of progenitor cell proliferation in human neurodegenerative diseases differs from that of animal models of neurodegenerative diseases; there is minimal progenitor proliferation in the SGZ and extensive proliferation in the SVZ in the human. In this review, we will present the data from a range of human and rodent studies from which we can compare the amount of proliferation of cells in the SVZ and SGZ in different neurodegenerative diseases.     

Glycogen Synthase Kinase-3β Regulates Equilibrium Between Neurogenesis and Gliogenesis in Rat Model of Parkinson’s Disease: a Crosstalk with Wnt and Notch Signaling

Neurogenesis involves generation of functional newborn neurons from neural stem cells (NSCs). Insufficient formation or accelerated degeneration of newborn neurons may contribute to the severity of motor/nonmotor symptoms of Parkinson’s disease (PD). However, the functional role of adult neurogenesis in PD is yet not explored and whether glycogen synthase kinase-3β (GSK-3β) affects multiple steps of adult neurogenesis in PD is still unknown. We investigated the possible underlying molecular mechanism of impaired adult neurogenesis associated with PD. Herein, we show that single intra-medial forebrain bundle (MFB) injection of 6-hydroxydopamine (6-OHDA) efficiently induced long-term activation of GSK-3β and reduced NSC self-renewal, proliferation, neuronal migration, and neuronal differentiation accompanied with increased astrogenesis in subventricular zone (SVZ) and hippocampal dentate gyrus (DG). Indeed, 6-OHDA also delayed maturation of neuroblasts in the DG as witnessed by their reduced dendritic length and arborization. Using a pharmacological approach to inhibit GSK-3β activation by specific inhibitor SB216763, we show that GSK-3β inhibition enhances radial glial cells, NSC proliferation, self-renewal in the SVZ, and the subgranular zone (SGZ) in the rat PD model. Pharmacological inhibition of GSK-3β activity enhances neuroblast population in SVZ and SGZ and promotes migration of neuroblasts towards the rostral migratory stream and lesioned striatum from dorsal SVZ and lateral SVZ, respectively, in PD model. GSK-3β inhibition enhances dendritic arborization and survival of granular neurons and stimulates NSC differentiation towards the neuronal phenotype in DG of PD model. The aforementioned effects of GSK-3β involve a crosstalk between Wnt/β-catenin and Notch signaling pathways that are known to regulate NSC dynamics.     

Characterization of Neural Stem/Progenitor Cells Expressing VEGF and its Receptors in the Subventricular Zone of Newborn Piglet Brain

Neural stem/progenitor cell (NSP) biology and neurogenesis in adult central nervous system (CNS) are important both towards potential future therapeutic applications for CNS repair, and for the fundamental function of the CNS. In the present study, we report the characterization of NSP population from subventricular zone (SVZ) of neonatal piglet brain using in vivo and in vitro systems. We show that the nestin and vimentin-positive neural progenitor cells are present in the SVZ of the lateral ventricles of neonatal piglet brain. In vitro, piglet NSPs proliferated as neurospheres, expressed the typical protein of neural progenitors, nestin and a range of well-established neurodevelopmental markers. Upon dissociation and subculture, piglet NSPs differentiated into neurons and glial cells. Clonal analysis demonstrates that piglet NSPs are multipotent and retain the capacity to generate both glia and neurons. These cells expressed VEGF, VEGFR1, VEGFR2 and Neuropilin-1 and -2 mRNAs. Real time PCR revealed that SVZ NSPs from newborn piglet expressed total VEGF and all VEGF splice variants. These findings show that piglet NSPs may be helpful to more effectively design growth factor based strategies to enhance endogenous precursor cells for cell transplantation studies potentially leading to the application of this strategy in the nervous system disease and injury.     

Longterm quiescent cells in the aged human subventricular neurogenic system specifically express GFAP‐δ

A main neurogenic niche in the adult human brain is the subventricular zone (SVZ). Recent data suggest that the progenitors that are born in the human SVZ migrate via the rostral migratory stream (RMS) towards the olfactory bulb (OB), similar to what has been observed in other mammals. A subpopulation of astrocytes in the SVZ specifically expresses an assembly‐compromised isoform of the intermediate filament protein glial fibrillary acidic protein (GFAP‐δ). To further define the phenotype of these GFAP‐δ expressing cells and to determine whether these cells are present throughout the human subventricular neurogenic system, we analysed SVZ, RMS and OB sections of 14 aged brain donors (ages 74‐93). GFAP‐δ was expressed in the SVZ along the ventricle, in the RMS and in the OB. The GFAP‐δ cells in the SVZ co‐expressed the neural stem cell (NSC) marker nestin and the cell proliferation markers proliferating cell nuclear antigen (PCNA) and Mcm2. Furthermore, BrdU retention was found in GFAP‐δ positive cells in the SVZ. In the RMS, GFAP‐δ was expressed in the glial net surrounding the neuroblasts. In the OB, GFAP‐δ positive cells co‐expressed PCNA. We also showed that GFAP‐δ cells are present in neurosphere cultures that were derived from SVZ precursors, isolated postmortem from four brain donors (ages 63‐91). Taken together, our findings show that GFAP‐δ is expressed in an astrocytic subpopulation in the SVZ, the RMS and the OB. Importantly, we provide the first evidence that GFAP‐δ is specifically expressed in longterm quiescent cells in the human SVZ, which are reminiscent of NSCs.     

Transplanted neuronal precursors migrate and differentiate in the devel-oping mouse brain

The subventricular zone (SVZ), lining the lateral ventricle in forebrain, retains a population of neuronal precursors with the ability of proliferation in adult mammals. To test the potential of neuronal precursors in adult mice, we transplanted adult SVZ cells labeled with fluorescent dye PKH26 into the lateral ventricle of the mouse brain in different development stages. The preliminary results indicated that the grafted cells were able to survive and migrate into multiple regions of the recipient brain, including SVZ, the third ventricle, thalamus, superior colliculus, inferior colliculus, cerebellum and olfactory bulb etc; and the amount of survival cells in different brain regions was correlated with the development stage of the recipient brain. Immunohistochemical studies showed that most of the grafted cells migrating into the specific target could express neuronal or astrocytic marker. Our results revealed that the neuronal precursors in adult SVZ still retained immortality and ability of prolife     

Transplanted neuronal precursors migrate and differentiate in the developing mouse brain

The subventricular zone (SVZ), lining the lateral ventricle in forebrain, retains a population of neuronal precursors with the ability of proliferation in adult mammals. To test the potential of neuronal precursors in adult mice, we transplanted adult SVZ cells labeled with fluorescent dye PKH26 into the lateral ventricle of the mouse brain in different development stages. The preliminary results indicated that the grafted cells were able to survive and migrate into multiple regions of the recipient brain, including SVZ, the third ventricle, thalamus, superior colliculus, inferior colliculus, cerebellum and olfactory bulb etc; and the amount of survival cells in different brain regions was correlated with the development stage of the recipient brain. Immunohistochemical studies showed that most of the grafted cells migrating into the specific target could express neuronal or astrocytic marker. Our results revealed that the neuronal precursors in adult SVZ still retained immortality and ability of proliferation, which is likely to be induced by some environmental factors.