Endogenous factors derived from embryonic cortex regulate proliferation and neuronal differentiation of postnatal subventricular zone cell cultures

In rodents, the subventricular zone (SVZ) harbours neural stem cells that proliferate and produce neurons throughout life. Previous studies showed that factors released by the developing cortex promote neurogenesis in the embryonic ventricular zone. In the present report, we studied in the rat the possible involvement of endogenous factors derived from the embryonic cortex in the regulation of the development of postnatal SVZ cells. To this end, SVZ neurospheres were maintained with explants or conditioned media (CM) prepared from embryonic day (E) 13, E16 or early postnatal cortex. We demonstrate that early postnatal cortex-derived factors have no significant effect on SVZ cell proliferation or differentiation. In contrast, E13 and E16 cortex release diffusible, heat-labile factors that promote SVZ cell expansion through increased proliferation and reduced cell death. In addition, E16 cortex-derived factors stimulate neuronal differentiation in both early postnatal and adult SVZ cultures. Fibroblast growth factor (FGF)-2- but not epidermal growth factor (EGF)-immunodepletion drastically reduces the mitogenic effect of E16 cortex CM, hence suggesting a major role of endogenous FGF-2 released by E16 cortex in the stimulation of SVZ cell proliferation. The evidence we provide here for the regulation of SVZ cell proliferation and neuronal differentiation by endogenous factors released from embryonic cortex may be of major importance for brain repair research.     

Pax6 regulates the epidermal growth factor-responsive neural stem cells of the subventricular zone

Neural stem/progenitor cells transit from fibroblast growth factor-responsive to epidermal growth factor (EGF)-responsive stem cells in the subventricular zone (SVZ). Here, we provide evidences that Pax6 plays a crucial role in the regulation of the EGF-responsive stem cell pool of the SVZ. Using Pax6 homozygous mutant mice (E18.5d), we found that the neurospheres were formed less than that from the wild-type mice, and the expression of EGF receptor in these neurospheres is downregulated very much. The amount of EGF-responsive cells in the mutant dorsal cortex SVZ (E18.5d) was also decreased from 16.8 (wild) to 4.5% (mutant), by flow cytometry method. Immunostaining of the cortex showed a downregulation of EGF receptor expression. All these results suggest that Pax6 regulate the EGF-responsive stem cells in the SVZ.     

Epidermal and fibroblast growth factors behave as mitogenic regulators for a single multipotent stem cell-like population from the subventricular region of the adult mouse forebrain.

The subventricular zone (SVZ) of the adult mammalian forebrain contains kinetically distinct precursor populations that contribute new neurons to the olfactory bulb. Because among forebrain precursors there are stem-like cells that can be cultured in the presence of mitogens such as epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2), we asked whether distinct subsets of stem-like cells coexist within the SVZ or whether the proliferation of a single type of SVZ stem-like cell is controlled by several GFs. We show that the latter is the case. Thus cells isolated from the SVZ coexpress the EGF and FGF receptors; by quantitative analysis, the number of stem-like cells isolated from the SVZ by either FGF2 or EGF is the same, whereas no additive effect occurs when these factors are used together. Furthermore, short-term administration of high-dose [3H]thymidine in vivo depletes both the EGF- and FGF2-responsive stem-like cell populations equally, showing they possess closely similar proliferation kinetics and likely belong to the constitutively proliferating SVZ compartment. By subcloning and population analysis, we demonstrate that responsiveness to more than one GF endows SVZ cells with an essential stem cell feature, the ability to vary self-renewal, that was until now undocumented in CNS stem-like cells. The multipotent stem cell-like population that expands slowly in the presence of FGF2 in culture switches to a faster growth mode when exposed to EGF alone and expands even faster when exposed to both GFs together. Analogous responses are observed when the GFs are used in the reverse order, and furthermore, these growth rate modifications are fully reversible.     

Dose‐dependent effect of EGF on migration and differentiation of adult subventricular zone astrocytes

Adult neural stem cells (NSCs) are located in the subventricular zone (SVZ), a specialized brain niche located on the walls of the lateral ventricle. Under physiological conditions, NSCs generate a large number of young neurons and some oligodendrocytes, however the mechanisms controlling cell proliferation and migration are unclear. In vitro, epidermal growth factor (EGF) signaling has been shown to be an important mediator of cell proliferation and migration in the adult brain; however, the primary SVZ progenitors that respond to EGF are not well known. In this study, we isolated SVZ type‐B astrocytes and cultured them under different EGF concentrations. We found a dose‐dependent effect of EGF on proliferation rates and migration of SVZ type‐B astrocytes. We found that GFAP+ type‐B astrocytes gave rise to highly migratory and proliferating cells that expressed Olig2 and NG2. After EGF withdrawal, a significant number of EGF‐stimulated cells differentiated into S100β+/O4+ oligodendrocytes. This study provides new insights about the production of oligodendrocytes derived from the astrocyte NSCs residing in the adult SVZ. To be able to manipulate the endogenous adult progenitors, it is crucial to identify and isolate the responding primary precursors and determine the extracellular signals that regulate their cell division, migration, and fate. © 2010 Wiley‐Liss, Inc.     

Dopaminergic lesion enhances growth factor-induced striatal neuroblast migration

Adult neurogenesis persists in the subventricular zone and is decreased in Parkinson disease (PD). The therapeutic potential of neurogenesis in PD requires understanding of mechanisms of 1) neural stem cell generation; 2) their guidance to the lesion site; and 3) the environment that enables neuronal differentiation, survival, and functional integration. We examined the combined intraventricular infusion of epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF-2) in a 6-hydroxydopamine-induced rodent model of PD. Epidermal growth factor and FGF-2 induced a massive increase in cell proliferation and in numbers of doublecortin-expressing neuroblasts in the subventricular zone. These growth factors also increased dopaminergic neurogenesis in the olfactory bulb and promoted the migration of newly generated neuroblasts from the subventricular zone into the adjacent striatum. The effects of EGF and FGF-2 were present in unlesioned animals but were dramatically enhanced in 6-hydroxydopamine-lesioned animals.These findings suggest that newly generated neuroblasts may be redirected to the region of dopaminergic deficit, and that EGF and FGF-2 can enhance dopaminergic neurogenesis in the olfactory bulb but not in the striatum. Similar mechanisms may be involved in the increased numbers of dopaminergic neurons observed in the olfactory bulbs of PD patients and their functional olfactory deficits.     

Relationship of gliomas to the ventricular walls

The role of neural stem cells in gliomagenesis remains controversial. The aim of this study was to determine the anatomic relationship of human gliomas to the lining of the ventricular walls, known as the subventricular zone, an area replete with neural stem cells. We performed a retrospective radiographic analysis of 100 consecutive patients with gliomas and sought to determine the relationship of the lesions to the ventricular walls as seen on their MRI scans. Our results indicated that in 93% of cases the lesions contacted at least one region of the lateral ventricular wall. Contact with the ventricular wall was independent of the glioma size or mass effect. These findings were correlated to cytoarchitectural studies of the human subventricular zone. Our findings lend further support that there is an intimate association between gliomas and the subventricular zone.     

Endothelial cell-derived bone morphogenetic proteins control proliferation of neural stem/progenitor cells

Neurogenesis persists in the adult brain subventricular zone where neural stem/progenitor cells (NSPCs) lie close to brain endothelial cells (BECs). We show in mouse that BECs produce bone morphogenetic proteins (BMPs). Coculture of embryonic and adult NSPCs with BECs activated the canonical BMP/Smad pathway and reduced their proliferation. We demonstrate that coculture with BECs in the presence of EGF and FGF2 induced a reversible cell cycle exit of NSPCs (LeX⁺) and an increase in the amount of GFAP/LeX-expressing progenitors thought to be stem cells. Levels of the phosphatidylinositol phosphatase PTEN were upregulated in NSPCs after coculture with BECs, or treatment with recombinant BMP4, with a concomitant reduction in Akt phosphorylation. Silencing Smad5 with siRNA or treatment with Noggin, a BMP antagonist, demonstrated that upregulation of PTEN in NSPCs required BMP/Smad signaling and that this pathway regulated cell cycle exit of NSPCs. Therefore, BECs may provide a feedback mechanism to control the proliferation of NSPCs.     

Controlling neural stem cell division within the adult subventricular zone: an APPealing job

For years, scientists investigating amyloid precursor protein (APP) have focused on its pathogenetic role in the brains of Alzheimer's disease patients. Now, a study by Caille et al. adds new sites of action and new physiological functions for APP. They show that there are binding sites for secreted N-terminal nonamyloidogenic APP (sAPP) on epidermal growth factor (EGF)-responsive neural stem cells in the subventricular zone of the adult brain, where sAPP acts as an EGF cofactor to stimulate proliferation of these cells. This result opens the hypothesis that changes in the levels of sAPP could influence activity of the neurogenic regions of the adult brain in normal and pathological conditions.     

EGF and NGF injected into the brain of old mice enhance BDNF and ChAT in proliferating subventricular zone

The response of cells localized in the brain subventricular zone (SVZ) to growth factor stimulation has been largely described for development and adult life, whereas no information on their behavior during aging is available. To address the question of whether the cells in the SVZ of old mice respond to the intracerebroventricular administration of epidermal growth factor (EGF) and nerve growth factor (NGF), we studied the distribution of proliferating cells and the effects on ChAT and brain-derived neurotrophic factor (BDNF) synthesis in forebrain and SVZ. It was found that the conjoint administration of EGF + NGF produced a major increase in ChAT expression in both forebrain and SVZ. The ChAT mRNA levels and the number of ChAT positive cells localized in the ventricular border and in the parenchyma of SVZ area were also increased significantly in the mice receiving EGF + NGF. Enhanced numbers of SVZ cells expressing proliferative markers were also discovered in EGF + NGF treated mice and some of these cells expressed cholinergic markers, as demonstrated by double immunostaining. In addition, EGF and NGF treatments significantly upregulate BDNF protein and mRNA levels in this brain region. The present study demonstrates that cells localized in SVZ of aged mouse brain retain the capacity to respond to EGF and NGF and that after stimulation with these two growth factors, the synthesis of ChAT and BDNF also increases. The implication that cells of the SVZ remain a reservoir of cholinergic and BDNF-positive neurons in aged brain opens a new perspective for understanding the role of growth factors during neurodegenerative disorders associated with aging.     

The subventricular zone releases factors which can be protective in oxygen/glucose deprivation-induced cortical damage: an organotypic study

A number of studies have already established the role of the subventricular zone in sustaining adult neurogenesis in different brain regions and under different pathological conditions, but nothing is reported about the role of this germinal area in preserving cell viability. In this work, we developed an organotypic culture model of the forebrain structures that comprise the neocortex, striatum, subventricular zone, and corpus callosum. With this model, we investigated the role of the subventricular zone in modulating cell viability in the cortex after oxygen/glucose deprivation. Here we have demonstrated that soluble heat-labile factors released by the subventricular zone in the media can lead to protection specifically in the cortical area. No protection was observed when medium, conditioned with factors released during the insult was administered to the hippocampal slices. Moreover, the use of different modifications of the slice cultures showed that the removal of the subventricular zone increased the cellular damage induced by oxygen/glucose deprivation. Furthermore, by using pharmacological experiments to investigate the possible mechanisms that regulate this subventricular function, we found evidence of purinergic involvement. We postulate that extracellular ATP signaling in the subventricular zone exacerbates cortical damage induced by hypoxia/hypoglycemia. For the first time, we demonstrate in vitro that the germinal subventricular zone can release factors that can be protective after exposure to a metabolic stressor. These released factors are not yet characterized but we identified in the extracellular ATP a factor that may interfere with the protective role of the subventricular zone during metabolic cortical damage.     

Oligodendrogenesis in the subventricular zone and the role of epidermal growth factor

Demyelinating diseases are characterized by an extensive loss of oligodendrocytes and myelin sheaths from axolemma. These neurological disorders are a common cause of disability in young adults, but so far, there is no effective treatment against them. It has been suggested that neural stem cells (NSCs) may play an important role in brain repair therapies. NSCs in the adult subventricular zone (SVZ), also known as Type-B cells, are multipotential cells that can self-renew and give rise to neurons and glia. Recent findings have shown that cells derived from SVZ Type-B cells actively respond to epidermal-growth-factor (EGF) stimulation becoming highly migratory and proliferative. Interestingly, a subpopulation of these EGF-activated cells expresses markers of oligodendrocyte precursor cells (OPCs). When EGF administration is removed, SVZ-derived OPCs differentiate into myelinating and pre-myelinating oligodendrocytes in the white matter tracts of corpus callosum, fimbria fornix and striatum. In the presence of a demyelinating lesion, OPCs derived from EGF-stimulated SVZ progenitors contribute to myelin repair. Given their high migratory potential and their ability to differentiate into myelin-forming cells, SVZ NSCs represent an important endogenous source of OPCs for preserving the oligodendrocyte population in the white matter and for the repair of demyelinating injuries.     

Agonistic encounters in aged male mouse potentiate the expression of endogenous brain NGF and BDNF: possible implication for brain progenitor cells' activation

The condition of dominance or submission following agonistic encounters in the adult male mouse is known to differentially affect brain nerve growth factor, a neurotrophin playing a role in brain remodeling, in the fine tuning of behaviour and in the regulation of the basal forebrain cholinergic neurons. During development and adult life nerve growth factor regulates brain expression of neurotransmitters and the stimulation of progenitor cells (stem cells) which, under different external stimuli, may differentiate into neuronal and/or glial cells promoting the recovery of the injured brain. However, little information is available for the aged brain. Thus in the present study we investigated the effect of the social status ('dominance' vs. 'submission') in the aged mouse on the presence of nerve growth factor, brain-derived neurotrophic factor, choline acetyltransferase, neuropeptide Y and progenitor cells of selected brain regions. We found that aged dominant mice showed increased brain-derived neurotrophic factor in the subventricular zone and hippocampus and increased choline acetyltransferase in the septum and basal nuclei, which were associated with increased presence of progenitor cells in the subventricular zone. Conversely, in aged subordinate mice the data showed a marked brain increase in nerve growth factor in the subventricular zone and hippocampus, choline acetyltransferase in the septum and basal nuclei and neuropeptide Y in the hippocampus and parietal cortex. The possible functional implications of these findings are discussed.     

Using endogenous neural stem cells to enhance recovery from ischemic brain injury

The use of cell-based therapy may be a valid therapeutic approach to ischemic brain injury. Stem cells have been proposed as a new form of cell based therapy in a variety of disorders, including acute and degenerative brain diseases. Up to date most efforts have concentrated on transplantation of embryonic stem cells (ESC) or neural stem cells (NSCs) obtained from immortalized cell lines into the diseased brain. These procedures require harvesting the appropriate stem cell, expansion in vitro and transplantation. Endogenous NSCs have been identified in the central nervous system where they reside largely in the subventricular zone and in the subgranular zone of the hippocampus. Endogenous NSCs may be capable of self-renewal and differentiation into functional glia and neurons. Manipulation of endogenous NSCs may bypass the need to use ESC as a form of therapy thus avoiding the complex ethical and biological issues involved with ES cells or immortalized cell lines. This review summarizes the evidence recently gathered in support of a therapeutic role for endogenous NSCs in acute experimental stroke.     

成年大鼠脑室下区神经干细胞上N-甲基-D-天冬氨酸受体亚单位1的超微分布

以免疫电镜和免疫组化及免疫金-银双重标记技术,从超微结构方面对N-甲基-D-天冬氨酸受体亚单位1在神经干细胞上进行准确定位,结果证实脑室下区存在神经干细胞,是成年哺乳动物中枢神经系统重要的发生区,而N-甲基-D-天冬氨酸受体亚单位1的表达比Nestin高,且分布于神经干细胞胞膜、胞浆、以及粗面内质网、高尔基复合体上。     

Neural progenitor cells transplanted into the uninjured brain undergo targeted migration after stroke onset

Endogenous neural stem cells normally reside in their niche, the subventricular zone, in the uninjured rodent brain. Upon stroke, these cells become more proliferative and migrate away from the subventricular zone into the surrounding parenchyma. It is not known whether this stroke-induced behavior is due to changes in the niche or introduction of attractive cues in the infarct zone, or both. A related question is how transplanted neural stem cells respond to subsequent insults, including whether exogenous stem cells have the plasticity to respond to subsequent injuries after engraftment. We addressed this issue by transplanting neural progenitor cells (NPCs) into the uninjured brain and then subjecting the animal to stroke. We were able to follow the transplanted NPCs in vivo by labeling them with superparamagnetic iron oxide particles and imaging them via high-resolution magnetic resonance imaging (MRI) during engraftment and subsequent to stroke. We find that transplanted NPCs that are latent can be activated in response to stroke and exhibit directional migration into the parenchyma, similar to endogenous neural NPCs, without a niche environment.     

The role of hypoxia in stem cell regulation of the central nervous system: From embryonic development to adult proliferation

Hypoxia is involved in the regulation of various cell functions in the body, including the regulation of stem cells. The hypoxic microenvironment is indispensable from embryonic development to the regeneration and repair of adult cells. In addition to embryonic stem cells, which need to maintain their self-renewal properties and pluripotency in a hypoxic environment, adult stem cells, including neural stem cells (NSCs), also exist in a hypoxic microenvironment. The subventricular zone (SVZ) and hippocampal dentate gyrus (DG) are the main sites of adult neurogenesis in the brain. Hypoxia can promote the proliferation, migration, and maturation of NSCs in these regions. Also, because most neurons in the brain are non-regenerative, stem cell transplantation is considered as a promising strategy for treating central nervous system (CNS) diseases. Hypoxic treatment also increases the effectiveness of stem cell therapy. In this review, we firstly describe the role of hypoxia in different stem cells, such as embryonic stem cells, NSCs, and induced pluripotent stem cells, and discuss the role of hypoxia-treated stem cells in CNS diseases treatment. Furthermore, we highlight the role and mechanisms of hypoxia in regulating adult neurogenesis in the SVZ and DG and adult proliferation of other cells in the CNS.     

Self-renewal and differentiation of reactive astrocyte-derived neural stem/progenitor cells isolated from the cortical peri-infarct area after stroke

In response to stroke, subpopulations of cortical reactive astrocytes proliferate and express proteins commonly associated with neural stem/progenitor cells such as glial fibrillary acidic protein (GFAP) and Nestin. To examine the stem cell-related properties of cortical reactive astrocytes after injury, we generated GFAP-CreER(TM);tdRFP mice to permanently label reactive astrocytes. We isolated cells from the cortical peri-infarct area 3 d after stroke, and cultured them in neural stem cell medium containing epidermal growth factor and basic fibroblast growth factor. We observed tdRFP-positive neural spheres in culture, suggestive of tdRFP-positive reactive astrocyte-derived neural stem/progenitor cells (Rad-NSCs). Cultured Rad-NSCs self-renewed and differentiated into neurons, astrocytes, and oligodendrocytes. Pharmacological inhibition and conditional knock-out mouse studies showed that Presenilin 1 and Notch 1 controlled neural sphere formation by Rad-NSCs after stroke. To examine the self-renewal and differentiation potential of Rad-NSCs in vivo, Rad-NSCs were transplanted into embryonic, neonatal, and adult mouse brains. Transplanted Rad-NSCs were observed to persist in the subventricular zone and secondary Rad-NSCs were isolated from the host brain 28 d after transplantation. In contrast with neurogenic postnatal day 4 NSCs and adult NSCs from the subventricular zone, transplanted Rad-NSCs differentiated into astrocytes and oligodendrocytes, but not neurons, demonstrating that Rad-NSCs had restricted differentiation in vivo. Our results indicate that Rad-NSCs are unlikely to be suitable for neuronal replacement in the absence of genetic or epigenetic modification.     

Human neural stem cells promote proliferation of endogenous neural stem cells and enhance angiogenesis in ischemic rat brain

Transplantation of human neural stem cells into the dentate gyrus or ventricle of rodents has been reportedly to enhance neurogenesis. In this study, we examined endogenous stem cell proliferation and angiogenesis in the ischemic rat brain after the transplantation of human neural stem cells. Focal cerebral ischemia in the rat brain was induced by middle cerebral artery occlusion. Human neural stem cells were transplanted into the subventricular zone. The behavioral performance of human neural stem cells-treated ischemic rats was significantly improved and cerebral infarct volumes were reduced compared to those in untreated animals. Numerous transplanted human neural stem cells were alive and preferentially localized to the ipsilateral ischemic hemisphere. Furthermore, 5-bromo-2′-deoxyuridine-labeled endogenous neural stem cells were observed in the subventricular zone and hippocampus, where they differentiated into cells immunoreactive for the neural markers doublecortin, neuronal nuclear antigen Neu N, and astrocyte marker glial fibrillary acidic protein in human neural stem cells-treated rats, but not in the untreated ischemic animals. The number of 5-bromo-2′-deoxyuridine-positive ? anti-von Willebrand factor-positive proliferating endothelial cells was higher in the ischemic boundary zone of human neural stem cells-treated rats than in controls. Finally, transplantation of human neural stem cells in the brains of rats with focal cerebral ischemia promoted the proliferation of endogenous neural stem cells and their differentiation into mature neural-like cells, and enhanced angiogenesis. This study provides valuable insights into the effect of human neural stem cell transplantation on focal cerebral ischemia, which can be applied to the development of an effective therapy for stroke.     

In vivo labeling of neuroblasts in the subventricular zone of rats

The studies of neural stem cell fate, as well as the possibility to genetically manipulate them, represent important tools for modern neuroscience research. Furthermore, the potential use of these cells in treatment of neurological disorders makes these methods valuable for the development of new treatment paradigms. Here we report a method to genetically mark and modify neuroblasts and progenitor cells in the subventricular zone of post-natal rats using retroviral vectors. Using GFP as a marker gene we were able to follow the cells as they migrate and differentiate into olfactory interneurons. The cells were found in the olfactory bulb already 1 week after injection of the vector and after 3 weeks all cells had reached this area. There was a higher efficiency of the labeling of cells in neonatal rats compared to adults but injecting directly into the subventricular zone could to some extent counteract this effect. However, the cell types generated by the GFP positive cells were the same in neonatal and adult animals. This method will be a powerful tool to study the genetic interplay involved in neural stem cell differentiation and may be instrumental in finding a way to instruct these cells to participate in brain repair in the adult central nervous system.