Beta 1 integrin-mediated effects of tenascin-R domains EGFL and FN6-8 on neural stem/progenitor cell proliferation and differentiation in vitro

Neural stem/progenitor cells (NSCs) have the capacity for self-renewal and differentiation into major classes of central nervous system cell types, such as neurons, astrocytes, and oligodendrocytes. The determination of fate of NSCs appears to be regulated by both intrinsic and extrinsic factors. Mounting evidence has shown that extracellular matrix molecules contribute to NSC proliferation and differentiation as extrinsic factors. Here we explore the effects of the epidermal growth factor-like (EGFL) and fibronectin type III homologous domains 6-8 (FN6-8) of the extracellular matrix molecule tenascin-R on NSC proliferation and differentiation. Our results show that domain FN6-8 inhibited NSC proliferation and promoted NSCs differentiation into astrocytes and less into oligodendrocytes or neurons. The EGFL domain did not affect NSC proliferation, but promoted NSC differentiation into neurons and reduced NSC differentiation into astrocytes and oligodendrocytes. Treatment of NSCs with beta 1 integrin function-blocking antibody resulted in attenuation of inhibition of the effect of FN6-8 on NSC proliferation. The influence of EGFL or FN6-8 on NSCs differentiation was inhibited by beta 1 integrin antibody application, implicating beta 1 integrin in proliferation and differentiation induced by EGFL and FN6-8 mediated triggering of NSCs.     

Jagged1 signals in the postnatal subventricular zone are required for neural stem cell self-renewal

Neural stem cells (NSCs) in the postnatal mammalian brain self-renew and are a source of neurons and glia. To date, little is known about the molecular and cellular mechanisms regulating the maintenance and differentiation of these multipotent progenitors. We show that Jagged1 is required by mitotic cells in the subventricular zone (SVZ) and stimulates self-renewal of multipotent epidermal growth factor-dependent NSCs. Jagged1-expressing cells line the adult SVZ and are juxtaposed to Notch1-expressing cells, some of which are putative NSCs. In vitro, endogenous Jagged1 acts through Notch1 to promote NSC maintenance and multipotency. In vivo, reducing Jagged1/Notch1 signaling decreases the number of proliferating cells in the SVZ. In addition, soluble Jagged1 promotes self-renewal and neurogenic potential of multipotent neural progenitors in vitro. Our findings suggest a central role for Jagged1 in the NSC niche in the SVZ for maintaining a population of NSCs in the postnatal brain.     

Tauroursodeoxycholic Acid Enhances Mitochondrial Biogenesis,Neural Stem Cell Pool,and Early Neurogenesis in Adult Rats

Although neurogenesis occurs in restricted regions of the adult mammalian brain, neural stem cells (NSCs) produce very few neurons during ageing or after injury. We have recently discovered that the endogenous bile acid tauroursodeoxycholic acid (TUDCA), a strong inhibitor of mitochondrial apoptosis and a neuroprotective in animal models of neurodegenerative disorders, also enhances NSC proliferation, self-renewal, and neuronal conversion by improving mitochondrial integrity and function of NSCs. In the present study, we explore the effect of TUDCA on regulation of NSC fate in neurogenic niches, the subventricular zone (SVZ) of the lateral ventricles and the hippocampal dentate gyrus (DG), using rat postnatal neurospheres and adult rats exposed to the bile acid. TUDCA significantly induced NSC proliferation, self-renewal, and neural differentiation in the SVZ, without affecting DG-derived NSCs. More importantly, expression levels of mitochondrial biogenesis-related proteins and mitochondrial antioxidant responses were significantly increased by TUDCA in SVZ-derived NSCs. Finally, intracerebroventricular administration of TUDCA in adult rats markedly enhanced both NSC proliferation and early differentiation in SVZ regions, corroborating in vitro data. Collectively, our results highlight a potential novel role for TUDCA in neurologic disorders associated with SVZ niche deterioration and impaired neurogenesis.     

The Molecular Profiles of Neural Stem Cell Niche in the Adult Subventricular Zone

Neural stem cells (NSCs) reside in a unique microenvironment called the neurogenic niche and generate functional new neurons. The neurogenic niche contains several distinct types of cells and interacts with the NSCs in the subventricular zone (SVZ) of the lateral ventricle. While several molecules produced by the niche cells have been identified to regulate adult neurogenesis, a systematic profiling of autocrine/paracrine signaling molecules in the neurogenic regions involved in maintenance, self-renewal, proliferation, and differentiation of NSCs has not been done. We took advantage of the genetic inducible fate mapping system (GIFM) and transgenic mice to isolate the SVZ niche cells including NSCs, transit-amplifying progenitors (TAPs), astrocytes, ependymal cells, and vascular endothelial cells. From the isolated cells and microdissected choroid plexus, we obtained the secretory molecule expression profiling (SMEP) of each cell type using the Signal Sequence Trap method. We identified a total of 151 genes encoding secretory or membrane proteins. In addition, we obtained the potential SMEP of NSCs using cDNA microarray technology. Through the combination of multiple screening approaches, we identified a number of candidate genes with a potential relevance for regulating the NSC behaviors, which provide new insight into the nature of neurogenic niche signals.     

Neuronal-glial interactions in central nervous system neurogenesis: the neural stem cell perspective

Essentially, three neuroectodermal-derived cell types make up the complex architecture of the adult CNS: neurons, astrocytes and oligodendrocytes. These elements are endowed with remarkable morphological, molecular and functional heterogeneity that reaches its maximal expression during development when stem/progenitor cells undergo progressive changes that drive them to a fully differentiated state. During this period the transient expression of molecular markers hampers precise identification of cell categories, even in neuronal and glial domains. These issues of developmental biology are recapitulated partially during the neurogenic processes that persist in discrete regions of the adult brain. The recent hypothesis that adult neural stem cells (NSCs) show a glial identity and derive directly from radial glia raises questions concerning the neuronal-glial relationships during pre- and post-natal brain development. The fact that NSCs isolated in vitro differentiate mainly into astrocytes, whereas in vivo they produce mainly neurons highlights the importance of epigenetic signals in the neurogenic niches, where glial cells and neurons exert mutual influences. Unravelling the mechanisms that underlie NSC plasticity in vivo and in vitro is crucial to understanding adult neurogenesis and exploiting this physiological process for brain repair. In this review we address the issues of neuronal/glial cell identity and neuronal-glial interactions in the context of NSC biology and NSC-driven neurogenesis during development and adulthood in vivo, focusing mainly on the CNS. We also discuss the peculiarities of neuronal-glial relationships for NSCs and their progeny in the context of in vitro systems.     

Microenvironmental determinants of adult neural stem cell proliferation and lineage commitment in the healthy and injured central nervous system

The discovery of neural stem cells (NSC) which ensure continuous neurogenesis in the adult mammalian brain, has led to a conceptual revolution in basic neuroscience and to high hopes for clinical nervous tissue repair. However, several research issues remain to address before neural stem cells can be harnessed for regenerative therapies. The presence of NSC in a nervous structure is demonstrated in vitro by primary culture of dissociated adult nervous tissue in the presence of the specific mitogens EGF and bFGF. This leads to spherical masses of proliferating cells endowed with capacities for self-renewal and, after growth factor removal, differentiation into the three characteristic cell types of nervous tissue (neurons, astrocytes, oligodendrocytes). In vivo, neurogenesis per se, i.e. production of new neurons, occurs only in a small subset of NSC-endowed structures. The production of oligodendrocytes, i.e. myelinating glial cells, is similarly restricted. Such in vivo restrictions were formally demonstrated to arise from the tissular microenvironnement, which led to the emerging concept of "neurogenic niche". In this context, major challenges now consist in identifying the nature of tissue-specific extracellular signals that determine lineage commitment of NSC progeny, understanding why NSCs display weak in vivo reactivity to lesions compared to other stem cell types in adults, and identifying the factors behind the very high resistance to tumorigenesis displayed by NSCs. Altogether, the current data offer hope for the future use of adult NSCs in regenerative therapies, provided that tissue-specific signals are identified in view of counteracting the intrinsic repression of new cell genesis and/or stimulating endogenous NSC recruitment to lesion sites.     

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.     

肿瘤坏死因子-α对大鼠中脑神经干细胞分化和寡突胶质细胞增殖的影响

为观察肿瘤坏死因子对神经干细胞(NSCs)分化的影响,本研究应用体外扩增的新生大鼠中脑NSCs,使用免疫组织化学技术,观察了肿瘤坏死因子—α(TNF—α)对NSCs分化及其后代细胞的影响。结果显示：(1)TNF—α可提高中脑NSCs后代中神经元和寡突胶质细胞所占的比例;(2)TNF—α可明显诱导由NSCs分化的寡突胶质细胞增殖,但对星形胶质细胞的增殖作用不明显。上述观察结果提示TNF—α对NSCs的应用具有重要影响。     

神经干细胞分离培养方法的介绍

在成体的许多组织中发现了多能干细胞,这些干细胞可以进行自我复制,参与组织的正常修复。神经干细胞在体外能分化为神经元、星形胶质细胞和少突胶质细胞,并具有多向分化潜能。成体神经干细胞和胚胎干细胞都能分化成成体神经系统中的各种神经细胞。神经干细胞具有自我更新能力,因此神经干细胞可以应用于神经损伤或者神经疾病的修复。本文概述了神经干细胞体外分离培养的方法及其生长影响因子。     

Phosphatidylglucoside: a novel marker for adult neural stem cells

We investigated the expression of a novel glycophospholipid, phosphatidylglucoside (PtdGlc), in adult mouse brains. Immunohistochemical analysis with DIM21 antibody, a monoclonal anti-PtdGlc antibody, revealed robust PtdGlc staining in the two primary neurogenic regions of the adult rodent brain, the subventricular zone (SVZ) lining the lateral ventricle and the subgranular zone of the dentate gyrus. Intriguingly, the staining pattern of PtdGlc appeared to overlap that of glial fibrillary acidic protein, an adult neural stem cell marker in these regions. Further immunohistochemical analysis revealed that PtdGlc expression on the cell membranes of adult SVZ neural stem cells significantly overlapped with other proposed adult neural stem cell markers. Moreover, PtdGlc(+) cells isolated from adult mouse SVZs by fluorescence-activated cell sorting with anti-PtdGlc antibody efficiently generated neurospheres in cell culture. These cells differentiated into neurons, astrocytes, and oligodendrocytes in vitro, directly demonstrating that PtdGlc-expressing cells possessed multipotency. Our data suggest that PtdGlc could be a useful adult stem cell marker.     

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.     

Blocking BE301622 gene expression by RNAi initiates differentiation of neural stem cells in rat

Neural stem cells (NSCs) are capable of differentiating into neurons, astrocytes and oligodendrocytes. However, the molecular mechanisms regulating NSCs differentiation are not well understood. Our previous research by microarray analysis certified that a lot of genes are differentially expressed in the course of NSC differentiation. In this study we report the function of one of these genes, BE301622, by RNAi techniques. To silence the BE301622 gene, a long, double-stranded RNA (dsRNA) was synthesized by using a kit (Ambion T7 MegaScript) and transformed into NSCs. Expression of mRNA was tested through RT-PCR. The result showed the expression of BE301622 gene was specificially suppressed. This finding effectively validated that BE301622 is involved in the differentiation of NSCs.     

Human Mesenchymal Stem Cells Signals Regulate Neural Stem Cell Fate

Neural stem cells (NSCs) differentiate into neurons, astrocytes and oligodendrocytes depending on their location within the central nervous system (CNS). The cellular and molecular cues mediating end-stage cell fate choices are not completely understood. The retention of multipotent NSCs in the adult CNS raises the possibility that selective recruitment of their progeny to specific lineages may facilitate repair in a spectrum of neuropathological conditions. Previous studies suggest that adult human bone marrow derived mesenchymal stem cells (hMSCs) improve functional outcome after a wide range of CNS insults, probably through their trophic influence. In the context of such trophic activity, here we demonstrate that hMSCs in culture provide humoral signals that selectively promote the genesis of neurons and oligodendrocytes from NSCs. Cell–cell contacts were less effective and the proportion of hMSCs that could be induced to express neural characteristics was very small. We propose that the selective promotion of neuronal and oligodendroglial fates in neural stem cell progeny is responsible for the ability of MSCs to enhance recovery after a wide range of CNS injuries. Special issue dedicated to Anthony Campagnoni.     

Epidermal Growth Factor in the CNS: A Beguiling Journey from Integrated Cell Biology to Multiple Sclerosis. An Extensive Translational Overview

This article reviews the wealth of papers dealing with the different effects of epidermal growth factor (EGF) on oligodendrocytes, astrocytes, neurons, and neural stem cells (NSCs). EGF induces the in vitro and in vivo proliferation of NSCs, their migration, and their differentiation towards the neuroglial cell line. It interacts with extracellular matrix components. NSCs are distributed in different CNS areas, serve as a reservoir of multipotent cells, and may be increased during CNS demyelinating diseases. EGF has pleiotropic differentiative and proliferative effects on the main CNS cell types, particularly oligodendrocytes and their precursors, and astrocytes. EGF mediates the in vivo myelinotrophic effect of cobalamin on the CNS, and modulates the synthesis and levels of CNS normal prions (PrP^Cs), both of which are indispensable for myelinogenesis and myelin maintenance. EGF levels are significantly lower in the cerebrospinal fluid and spinal cord of patients with multiple sclerosis (MS), which probably explains remyelination failure, also because of the EGF marginal role in immunology. When repeatedly administered, EGF protects mouse spinal cord from demyelination in various experimental models of autoimmune encephalomyelitis. It would be worth further investigating the role of EGF in the pathogenesis of MS because of its multifarious effects.

     

溶血磷脂酸对离体培养的大鼠胚胎神经干细胞向神经胶质细胞分化的影响

本研究用免疫细胞化学荧光双标技术观察了溶血磷脂酸（lysophosphatidic acid,LPA）对大鼠胚胎神经干细胞（neural stem cells,NSCs）分化为少突胶质细胞（galactocerebroside—positive,Gal-C阳性）和星形胶质细胞（grim fibrillary acidic protein-positive,GFAP阳性）的影响,并且用RT-PCR技术对NSCs可能表达的LPA受体进行分析。结果显示：（1）加入不同浓度（0．010．0μmol／L）LPA,第7天进行检测时,少突胶质细胞数量呈明显的剂量依赖性增加,峰值出现在1．0μmol／LLPA组,少突胶质细胞所占百分比从对照组的8．5％增加到32．6％;（2）星形胶质细胞的分化几乎不受LPA的影响,第7天时各LPA处理组星形胶质细胞百分比与对照组相比均无显著性差异;（3）RT-PCR结果显示,大鼠胚胎NSCs的LPA1和LPA3受体表达明显,而LPA3受体表达很弱。以上结果表明,较低浓度的LPA可能作为细胞外信号,通过LPA1和LPA3受体促进大鼠胚胎NSCs向少突胶质细胞分化和生成,但对星形胶质细胞的分化过程无明显影响。     

Neonatal Subventricular Zone Electroporation

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal cells¹. Understanding the molecular pathways responsible for NSC self-renewal, commitment, and differentiation is critical for harnessing their unique potential to repair the brain and better understand central nervous system disorders. Previous methods for the manipulation of mammalian systems required the time consuming and expensive endeavor of genetic engineering at the whole animal level². Thus, the vast majority of studies have explored the functions of NSC molecules in vitro or in invertebrates.Here, we demonstrate the simple and rapid technique to manipulate neonatal NPCs that is referred to as neonatal subventricular zone (SVZ) electroporation. Similar techniques were developed a decade ago to study embryonic NSCs and have aided studies on cortical development^3,4 . More recently this was applied to study the postnatal rodent forebrain^5-7. This technique results in robust labeling of SVZ NSCs and their progeny. Thus, postnatal SVZ electroporation provides a cost and time effective alternative for mammalian NSC genetic engineering.