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.     

Over-Expression of hNGF in Adult Human Olfactory Bulb Neural Stem Cells Promotes Cell Growth and Oligodendrocytic Differentiation

The adult human olfactory bulb neural stem/progenitor cells (OBNC/PC) are promising candidate for cell-based therapy for traumatic and neurodegenerative insults. Exogenous application of NGF was suggested as a promising therapeutic strategy for traumatic and neurodegenerative diseases, however effective delivery of NGF into the CNS parenchyma is still challenging due mainly to its limited ability to cross the blood–brain barrier, and intolerable side effects if administered into the brain ventricular system. An effective method to ensure delivery of NGF into the parenchyma of CNS is the genetic modification of NSC to overexpress NGF gene. Overexpression of NGF in adult human OBNSC is expected to alter their proliferation and differentiation nature, and thus might enhance their therapeutic potential. In this study, we genetically modified adult human OBNS/PC to overexpress human NGF (hNGF) and green fluorescent protein (GFP) genes to provide insight about the effects of hNGF and GFP genes overexpression in adult human OBNS/PC on their in vitro multipotentiality using DNA microarray, immunophenotyping, and Western blot (WB) protocols. Our analysis revealed that OBNS/PC-GFP and OBNS/PC-GFP-hNGF differentiation is a multifaceted process involving changes in major biological processes as reflected in alteration of the gene expression levels of crucial markers such as cell cycle and survival markers, stemness markers, and differentiation markers. The differentiation of both cell classes was also associated with modulations of key signaling pathways such MAPK signaling pathway, ErbB signaling pathway, and neuroactive ligand-receptor interaction pathway for OBNS/PC-GFP, and axon guidance, calcium channel, voltage-dependent, gamma subunit 7 for OBNS/PC-GFP-hNGF as revealed by GO and KEGG. Differentiated OBNS/PC-GFP-hNGF displayed extensively branched cytoplasmic processes, a significant faster growth rate and up modulated the expression of oligodendroglia precursor cells markers (PDGFRα, NG2 and CNPase) respect to OBNS/PC-GFP counterparts. These findings suggest an enhanced proliferation and oligodendrocytic differentiation potential for OBNS/PC-GFP-hNGF as compared to OBNS/PC-GFP.     

骨髓间充质干细胞体外趋化神经前体细胞的机制

骨髓间充质干细胞(BMSC)和神经前体细胞(NPC)移植于脑组织损伤动物的实验证明这两类细胞移植后均能在体内迁徙,与周围细胞整合,促进神经功能修复。BMSC促进神经功能修复的机制之一被认为与其分泌一些细胞因子和趋化因子有关,但具体机制不十分明确。为从基质细胞衍生因子-1α(SDF-1α)及其唯一的受体CXCR4这对分子相互作用的机制上探讨BMSC移植的可能治疗作用,实验采用ELISA法检测了体外培养的BMSC上清液中SDF-1α的含量,体外微孔隔离室迁移实验发现NPC能在BMSC分泌的培养上清液中SDF-1α的作用下发生定向迁移,特异性抗CXCR4单抗能有效阻断NPC的定向迁移效应,证实了BMSC分泌的SDF-1α促进表达CXCR4的NPC向病灶处迁移可能是促进神经功能修复的机制之一,从而为干细胞移植治疗神经功能缺损提供了一个新的思路。     

Olfactory Enrichment Influences Adult Neurogenesis Modulating GAD67 and Plasticity-Related Molecules Expression in Newborn Cells of the Olfactory Bulb

The olfactory bulb (OB) is a highly plastic region of the adult mammalian brain characterized by continuous integration of inhibitory interneurons of the granule (GC) and periglomerular cell (PGC) types. Adult-generated OB interneurons are selected to survive in an experience-dependent way but the mechanisms that mediate the effects of experience on OB neurogenesis are unknown. Here we focus on the new-generated PGC population which is composed by multiple subtypes. Using paradigms of olfactory enrichment and/or deprivation combined to BrdU injections and quantitative confocal immunohistochemical analyses, we studied the effects of olfactory experience on adult-generated PGCs at different survival time and compared PGC to GC modulation. We show that olfactory enrichment similarly influences PGCs and GCs, increasing survival of newborn cells and transiently modulating GAD67 and plasticity-related molecules expression. However, PGC maturation appears to be delayed compared to GCs, reflecting a different temporal dynamic of adult generated olfactory interneuron integration. Moreover, olfactory enrichment or deprivation do not selectively modulate the survival of specific PGC phenotypes, supporting the idea that the integration rate of distinct PGC subtypes is independent from olfactory experience.     

Characterization of TLX Expression in Neural Stem Cells and Progenitor Cells in Adult Brains

TLX has been shown to play an important role in regulating the self-renewal and proliferation of neural stem cells in adult brains. However, the cellular distribution of endogenous TLX protein in adult brains remains to be elucidated. In this study, we used immunostaining with a TLX-specific antibody to show that TLX is expressed in both neural stem cells and transit-amplifying neural progenitor cells in the subventricular zone (SVZ) of adult mouse brains. Then, using a double thymidine analog labeling approach, we showed that almost all of the self-renewing neural stem cells expressed TLX. Interestingly, most of the TLX-positive cells in the SVZ represented the thymidine analog-negative, relatively quiescent neural stem cell population. Using cell type markers and short-term BrdU labeling, we demonstrated that TLX was also expressed in the Mash1+ rapidly dividing type C cells. Furthermore, loss of TLX expression dramatically reduced BrdU label-retaining neural stem cells and the actively dividing neural progenitor cells in the SVZ, but substantially increased GFAP staining and extended GFAP processes. These results suggest that TLX is essential to maintain the self-renewing neural stem cells in the SVZ and that the GFAP+ cells in the SVZ lose neural stem cell property upon loss of TLX expression.Understanding the cellular distribution of TLX and its function in specific cell types may provide insights into the development of therapeutic tools for neurodegenerative diseases by targeting TLX in neural stem/progenitors cells.     

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.     

Efficient Derivation of Multipotent Neural Stem/Progenitor Cells from Non-Human Primate Embryonic Stem Cells

The common marmoset (Callithrix jacchus) is a small New World primate that has been used as a non-human primate model for various biomedical studies. We previously demonstrated that transplantation of neural stem/progenitor cells (NS/PCs) derived from mouse and human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) promote functional locomotor recovery of mouse spinal cord injury models. However, for the clinical application of such a therapeutic approach, we need to evaluate the efficacy and safety of pluripotent stem cell-derived NS/PCs not only by xenotransplantation, but also allotransplantation using non-human primate models to assess immunological rejection and tumorigenicity. In the present study, we established a culture method to efficiently derive NS/PCs as neurospheres from common marmoset ESCs. Marmoset ESC-derived neurospheres could be passaged repeatedly and showed sequential generation of neurons and astrocytes, similar to that of mouse ESC-derived NS/PCs, and gave rise to functional neurons as indicated by calcium imaging. Although marmoset ESC-derived NS/PCs could not differentiate into oligodendrocytes under default culture conditions, these cells could abundantly generate oligodendrocytes by incorporating additional signals that recapitulate in vivo neural development. Moreover, principal component analysis of microarray data demonstrated that marmoset ESC-derived NS/PCs acquired similar gene expression profiles to those of fetal brain-derived NS/PCs by repeated passaging. Therefore, marmoset ESC-derived NS/PCs may be useful not only for accurate evaluation by allotransplantation of NS/PCs into non-human primate models, but are also applicable to analysis of iPSCs established from transgenic disease model marmosets.     

RhoC Involved in the Migration of Neural Stem/Progenitor Cells

Alzheimer’s disease (AD) is characterized by deposition of beta-amyloid peptides (Aβ) and progressive loss of neurons. Neural stem/progenitor cells (NSPCs) can proliferate and produce immature neurons even in the brain of AD patients. However, Aβ₄₂ significantly decreased the expression of RhoC in NSPCs during the co-incubation (P < 0.01). Treating with RhoC siRNA prevented membrane from protrusion and led to a significant reduction in cell migration in responses to SDF-1. Compared with wild-type mice, the numbers of RhoC-immunoreactive cells in hippocampus and cortex were significantly down-regulated in APP/PS1 mice aged 9 months. The results suggest that Aβ₄₂ down-regulates the expression of RhoC in NSPCs in vitro and in vivo; down-regulated RhoC expression results in decreased migration of NSPCs.     

Differential Effects of Isoxazole-9 on Neural Stem/Progenitor Cells,Oligodendrocyte Precursor Cells,and Endothelial Progenitor Cells

Adult mammalian brain can be plastic after injury and disease. Therefore, boosting endogenous repair mechanisms would be a useful therapeutic approach for neurological disorders. Isoxazole-9 (Isx-9) has been reported to enhance neurogenesis from neural stem/progenitor cells (NSPCs). However, the effects of Isx-9 on other types of progenitor/precursor cells remain mostly unknown. In this study, we investigated the effects of Isx-9 on the three major populations of progenitor/precursor cells in brain: NSPCs, oligodendrocyte precursor cells (OPCs), and endothelial progenitor cells (EPCs). Cultured primary NSPCs, OPCs, or EPCs were treated with various concentrations of Isx-9 (6.25, 12.5, 25, 50 μM), and their cell numbers were counted in a blinded manner. Isx-9 slightly increased the number of NSPCs and effectively induced neuronal differentiation of NSPCs. However, Isx-9 significantly decreased OPC number in a concentration-dependent manner, suggesting cytotoxicity. Isx-9 did not affect EPC cell number. But in a matrigel assay of angiogenesis, Isx-9 significantly inhibited tube formation in outgrowth endothelial cells derived from EPCs. This potential anti-tube-formation effect of Isx-9 was confirmed in a brain endothelial cell line. Taken together, our data suggest that mechanisms and targets for promoting stem/progenitor cells in the central nervous system may significantly differ between cell types.     

The Neural Stem/Progenitor Cell Marker Nestin Is Expressed in Proliferative Endothelial Cells,but Not in Mature Vasculature

Nestin is an intermediate filament protein that is known as a neural stem/progenitor cell marker. It is expressed in undifferentiated central nervous system (CNS) cells during development, but also in normal adult CNS and in CNS tumor cells. Additionally, nestin is expressed in endothelial cells (ECs) of CNS tumor tissues and of adult tissues that replenish by angiogenesis. However, the regulation of nestin expression in vascular endothelium has not been analyzed in detail. This study showed that nestin expression was observed in proliferating endothelial progenitor cells (EPCs), but not in mature ECs. In adherent cultured cells derived from bone marrow cells, EPCs that highly expressed nestin also expressed the endothelial marker CD31 and the proliferation marker Ki67. ECs cultured without growth factors showed attenuated nestin immunoreactivity as they matured. Transgenic mice that carried the enhanced green fluorescent protein under the control of the CNS-specific second intronic enhancer of the nestin gene showed no reporter gene expression in EPCs. This indicated that the mechanisms of nestin gene expression were different in EPCs and CNS cells. Immunohistochemistry showed nestin expression in neovascular cells from two distinct murine models. Our results demonstrate that nestin can be used as a marker protein for neovascularization. (J Histochem Cytochem 58:721–730, 2010)     

Japanese Encephalitis Virus Induce Immuno-Competency in Neural Stem/Progenitor Cells

Background

The low immunogenicity of neural stem/progenitor cells (NSPCs) coupled with negligible expression of MHC antigens has popularized their use in transplantation medicine. However, in an inflammatory environment, the NSPCs express costimulatory molecules and MHC antigens, and also exhibit certain immunomodulatory functions. Since NSPCs are the cellular targets in a number of virus infections both during postnatal and adult stages, we wanted to investigate the immunological properties of these stem cells in response to viral pathogen.

Methodology/Principal Findings

We utilized both in vivo mouse model and in vitro neurosphere model of Japanese encephalitis virus (JEV) infection for the study. The NSPCs residing in the subventricular zone of the infected brains showed prominent expression of MHC-I and costimulatory molecules CD40, CD80, and CD86. Using Flow cytometry and fluorescence microscopy, we observed increased surface expression of co-stimulatory molecule and MHC class I antigen in NSPCs upon progressive JEV infection in vitro. Moreover, significant production of pro-inflammatory cyto/chemokines was detected in JEV infected NSPCs by Cytokine Bead Array analysis. Interestingly, NSPCs were capable of providing functional costimulation to allogenic T cells and JEV infection resulted in increased proliferation of allogenic T cells, as detected by Mixed Lymphocyte reaction and CFSE experiments. We also report IL-2 production by NSPCs upon JEV infection, which possibly provides mitogenic signals to T cells and trigger their proliferation.

Conclusion/Significance

The in vivo and in vitro findings clearly indicate the development of immunogenicity in NSPCs following progressive JEV infection, in our case, JEV infection. Following a neurotropic virus infection, NSPCs possibly behave as immunogenic cells and contribute to both the innate and adaptive immune axes. The newly discovered immunological properties of NSPCs may have implications in assigning a new role of these cells as non-professional antigen presenting cells in the central nervous system.     

Integrin-Associated Protein Promotes Neuronal Differentiation of Neural Stem/Progenitor Cells

Neural stem/progenitor cells (NSPCs) proliferate and differentiate depending on their intrinsic properties and local environment. During the development of the mammalian nervous system, NSPCs generate neurons and glia sequentially. However, little is known about the mechanism that determines the timing of switch from neurogenesis to gliogenesis. In this study, we established a culture system in which the neurogenic potential of NSPCs is decreased in a time-dependent manner, so that short-term-cultured NSPCs differentiate into more neurons compared with long-term-cultured NSPCs. We found that short-term-cultured NSPCs express high levels of integrin-associated protein form 2 (IAP2; so-called CD47) mRNA using differential display analysis. Moreover, IAP2 overexpression in NSPCs induced neuronal differentiation of NSPCs. These findings reveal a novel mechanism by which IAP2 induces neuronal differentiation of NSPCs.     

NEUROD1 Intrinsically Initiates Differentiation of Induced Pluripotent Stem Cells into Neural Progenitor Cells

Cell type specification is a delicate biological event in which every step is under tight regulation. From a molecular point of view, cell fate commitment begins with chromatin alteration, which kickstarts lineage-determining factors to initiate a series of genes required for cell specification. Several important neuronal differentiation factors have been identified from ectopic over-expression studies. However, there is scarce information on which DNA regions are modified during induced pluripotent stem cell (iPSC) to neuronal progenitor cell (NPC) differentiation, the cis regulatory factors that attach to these accessible regions, or the genes that are initially expressed. In this study, we identified the DNA accessible regions of iPSCs and NPCs via the Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq). We identified which chromatin regions were modified after neuronal differentiation and found that the enhancer regions had more active histone modification changes than the promoters. Through motif enrichment analysis, we found that NEUROD1 controls iPSC differentiation to NPC by binding to the accessible regions of enhancers in cooperation with other factors such as the Hox proteins. Finally, by using Hi-C data, we categorized the genes that directly interacted with the enhancers under the control of NEUROD1 during iPSC to NPC differentiation.     

Microparticle Shedding from Neural Progenitor Cells and Vascular Compartment Cells Is Increased in Ischemic Stroke

Purpose

Ischemic stroke has shown to induce platelet and endothelial microparticle shedding, but whether stroke induces microparticle shedding from additional blood and vascular compartment cells is unclear. Neural precursor cells have been shown to replace dying neurons at sites of brain injury; however, if neural precursor cell activation is associated to microparticle shedding, and whether this activation is maintained at long term and associates to stroke type and severity remains unknown. We analyzed neural precursor cells and blood and vascular compartment cells microparticle shedding after an acute ischemic stroke.

Methods

Forty-four patients were included in the study within the first 48h after the onset of stroke. The cerebral lesion size was evaluated at 3–7 days of the stroke. Circulating microparticles from neural precursor cells and blood and vascular compartment cells (platelets, endothelial cells, erythrocytes, leukocytes, lymphocytes, monocytes and smooth muscle cells) were analyzed by flow cytometry at the onset of stroke and at 7 and 90 days. Forty-four age-matched high cardiovascular risk subjects without documented vascular disease were used as controls.

Results

Compared to high cardiovascular risk controls, patients showed higher number of neural precursor cell- and all blood and vascular compartment cell-derived microparticles at the onset of stroke, and after 7 and 90 days. At 90 days, neural precursor cell-derived microparticles decreased and smooth muscle cell-derived microparticles increased compared to levels at the onset of stroke, but only in those patients with the highest stroke-induced cerebral lesions.

Conclusions

Stroke increases blood and vascular compartment cell and neural precursor cell microparticle shedding, an effect that is chronically maintained up to 90 days after the ischemic event. These results show that stroke induces a generalized blood and vascular cell activation and the initiation of neuronal cell repair process after stroke. Larger cerebral lesions associate with deeper vessel injury affecting vascular smooth muscle cells.     

Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative neurology. This directive has arisen from the extensive evidence that brain repair is achieved after focal or systemic NPC transplantation in several preclinical models of neurological diseases.These experimental data have identified the cell delivery route as one of the main hurdles of restorative stem cell therapies for brain diseases that requires urgent assessment. Intraparenchymal stem cell grafting represents a logical approach to those pathologies characterized by isolated and accessible brain lesions such as spinal cord injuries and Parkinson''s disease. Unfortunately, this principle is poorly applicable to conditions characterized by a multifocal, inflammatory and disseminated (both in time and space) nature, including multiple sclerosis (MS). As such, brain targeting by systemic NPC delivery has become a low invasive and therapeutically efficacious protocol to deliver cells to the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system (CNS).This alternative method of cell delivery relies on the NPC pathotropism, specifically their innate capacity to (i) sense the environment via functional cell adhesion molecules and inflammatory cytokine and chemokine receptors; (ii) cross the leaking anatomical barriers after intravenous (i.v.) or intracerebroventricular (i.c.v.) injection; (iii) accumulate at the level of multiple perivascular site(s) of inflammatory brain and spinal cord damage; and (i.v.) exert remarkable tissue trophic and immune regulatory effects onto different host target cells in vivo.Here we describe the methods that we have developed for the i.v. and i.c.v. delivery of syngeneic NPCs in mice with experimental autoimmune encephalomyelitis (EAE), as model of chronic CNS inflammatory demyelination, and envisage the systemic stem cell delivery as a valuable technique for the selective targeting of the inflamed brain in regenerative neurology.     

外周输入依赖的嗅球颗粒细胞的突触结构可塑性

活动依赖的突触结构可塑性是学习和记忆的基础.哺乳动物,尤其是啮齿类动物,具有高度发达的嗅觉系统和惊人的气味学习和记忆能力.本研究以CNGA2敲除而导致外周输入缺失的小鼠为模型,研究嗅球内活动依赖的突触结构可塑性.利用特异性的突触前和突触后标记物,发现外周输入缺失减少了突触标记蛋白突触素(synaptophysin)和抑制性突触标记蛋白桥蛋白(gephyrin)在嗅球外网状层和颗粒细胞层中的表达;兴奋性突触标记蛋白囊泡谷氨酸转运蛋白1(VGluT1)的表达水平只在外网状层中有显著下降,而在颗粒细胞层中没有明显变化.进一步通过活体质粒电转标记嗅球颗粒细胞后发现,CNGA2敲除小鼠颗粒细胞上位于外网状层中的远端树突棘密度显著减小,而位于颗粒细胞层中的近端树突棘密度没有明显变化.这些结果表明颗粒细胞上的树-树突触具有对外周活动依赖的结构可塑性,而轴-树突触则无.