Characterization Of Neural Stem Cells Research Articles

Single-Factor SOX2 Mediates Direct Neural Reprogramming of Human Mesenchymal Stem Cells via Transfection of In Vitro Transcribed mRNA

Neural stem cells (NSCs) are a prominent cell source for understanding neural pathogenesis and for developing therapeutic applications to treat neurodegenerative disease because of their regenerative capacity and multipotency. Recently, a variety of cellular reprogramming technologies have been developed to facilitate in vitro generation of NSCs, called induced NSCs (iNSCs). However, the genetic safety aspects of established virus-based reprogramming methods have been considered, and non-integrating reprogramming methods have been developed. Reprogramming with in vitro transcribed (IVT) mRNA is one of the genetically safe reprogramming methods because exogenous mRNA temporally exists in the cell and is not integrated into the chromosome. Here, we successfully generated expandable iNSCs from human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) via transfection with IVT mRNA encoding SOX2 (SOX2 mRNA) with properly optimized conditions. We confirmed that generated human UCB-MSC-derived iNSCs (UM-iNSCs) possess characteristics of NSCs, including multipotency and self-renewal capacity. Additionally, we transfected human dermal fibroblasts (HDFs) with SOX2 mRNA. Compared with human embryonic stem cell-derived NSCs, HDFs transfected with SOX2 mRNA exhibited neural reprogramming with similar morphologies and NSC-enriched mRNA levels, but they showed limited proliferation ability. Our results demonstrated that human UCB-MSCs can be used for direct reprogramming into NSCs through transfection with IVT mRNA encoding a single factor, which provides an integration-free reprogramming tool for future therapeutic application.

Reduction of Movement in Neurological Diseases: Effects on Neural Stem Cells Characteristics

Both astronauts and patients affected by chronic movement-limiting pathologies face impairment in muscle and/or brain performance. Increased patient survival expectations and the expected longer stays in space by astronauts may result in prolonged motor deprivation and consequent pathological effects. Severe movement limitation can influence not only the motor and metabolic systems but also the nervous system, altering neurogenesis and the interaction between motoneurons and muscle cells. Little information is yet available about the effect of prolonged muscle disuse on neural stem cells characteristics. Our in vitro study aims to fill this gap by focusing on the biological and molecular properties of neural stem cells (NSCs). Our analysis shows that NSCs derived from the SVZ of HU mice had shown a reduced proliferation capability and an altered cell cycle. Furthermore, NSCs obtained from HU animals present an incomplete differentiation/maturation. The overall results support the existence of a link between reduction of exercise and muscle disuse and metabolism in the brain and thus represent valuable new information that could clarify how circumstances such as the absence of load and the lack of movement that occurs in people with some neurological diseases, may affect the properties of NSCs and contribute to the negative manifestations of these conditions.

Human-Induced Pluripotent Stem Cells Manufactured Using a Current Good Manufacturing Practice-Compliant Process Differentiate Into Clinically Relevant Cells From Three Germ Layers.

The discovery of reprogramming and generation of human-induced pluripotent stem cells (iPSCs) has revolutionized the field of regenerative medicine and opened new opportunities in cell replacement therapies. While generation of iPSCs represents a significant breakthrough, the clinical relevance of iPSCs for cell-based therapies requires generation of high-quality specialized cells through robust and reproducible directed differentiation protocols. We have recently reported manufacturing of human iPSC master cell banks (MCB) under current good manufacturing practices (cGMPs). Here, we describe the clinical potential of human iPSCs generated using this cGMP-compliant process by differentiating them into the cells from all three embryonic germ layers including ectoderm, endoderm, and mesoderm. Most importantly, we have shown that our iPSC manufacturing process and cell culture system is not biased toward a specific lineage. Following controlled induction into a specific differentiation lineage, specialized cells with morphological and cellular characteristics of neural stem cells, definitive endoderm, and cardiomyocytes were developed. We believe that these cGMP-compliant iPSCs have the potential to make various clinically relevant products suitable for cell therapy applications.

Redundant regulation of localization and protein stability of DmPar3.

Apical-basal polarity is an important characteristic of epithelia and Drosophila neural stem cells. The conserved Par complex, which consists of the atypical protein kinase C and the scaffold proteins Baz and Par6, is a key player in the establishment of apical-basal cell polarity. Membrane recruitment of Baz has been reported to be accomplished by several mechanisms, which might function in redundancy, to ensure the correct localization of the complex. However, none of the described interactions was sufficient to displace the protein from the apical junctions. Here, we dissected the role of the oligomerization domain and the lipid-binding motif of Baz in vivo in the Drosophila embryo. We found that these domains function in redundancy to ensure the apical junctional localization of Baz: inactivation of only one domain is not sufficient to disrupt the function of Baz during apical-basal polarization of epithelial cells and neural stem cells. In contrast, mutation of both domains results in a strongly impaired protein stability and a phenotype characterized by embryonic lethality and an impaired apical-basal polarity in the embryonic epithelium and neural stem cells, resembling a baz-loss of function allele. Strikingly, the binding of Baz to the transmembrane proteins E-Cadherin, Echinoid, and Starry Night was not affected in this mutant protein. Our findings reveal a redundant function of the oligomerization and the lipid-binding domain, which is required for protein stability, correct subcellular localization, and apical-basal cell polarization.

Characterization of neural stem cells modified with hypoxia/neuron-specific VEGF expression system for spinal cord injury.

Spinal cord injury (SCI) is an incurable disease causing an ischemic environment and functional defect, thus a new therapeutic approach is needed for SCI treatment. Vascular endothelial growth factor (VEGF) is a potent therapeutic gene to treat SCI via angiogenesis and neuroprotection, and both tissue-specific gene expression and high gene delivery efficiency are important for successful gene therapy. Here we design the hypoxia/neuron dual-specific gene expression system (pEpo-NSE) and efficient gene delivery platform can be achieved by the combination ex vivo gene therapy with erythropoietin (Epo) enhancer, neuron-specific enolase (NSE) promoter and neural stem cells (NSCs). An in vitro model, NSCs transfected with pEpo-NSE were consistently and selectively overexpressing therapeutic genes in response to neural differentiation and hypoxic conditions. Also, in SCI model, ex vivo gene therapy using pEpo-NSE system with NSCs significantly enhanced gene delivery efficiency compared with pEpo-NSE system gene therapy alone. However, microarray analysis reveals that introducing exogenous pEpo-NSE and VEGF triggers biological pathways in NSCs such as glycolysis and signaling pathways such as Ras and mitogen-activated protein kinase, leading to cell proliferation, differentiation and apoptosis. Collectively, it indicates that the pEpo-NSE gene expression system works stably in NSCs and ex vivo gene therapy using pEpo-NSE system with NSCs improves gene expression efficiency. However, exogenously introduced pEpo-NSE system has an influence on gene expression profiles in NSCs. Therefore, when we consider ex vivo gene therapy for SCI, the effects of changes in gene expression profiles in NSCs on safety should be investigated.

Brain-derived Neurotrophic Factor Promotes Growth of Neurons and Neural Stem Cells Possibly by Triggering the Phosphoinositide 3-Kinase/ AKT/Glycogen Synthase Kinase-3β/β-catenin Pathway.

Brain-derived neurotrophic factor (BDNF) plays a crucial role in promoting survival and differentiation of neurons and neural stem cells (NSCs), but the downstream regulating mechanisms remain poorly understood. We investigated whether BDNF exerts its effect by triggering the phosphoinositide 3-kinase (PI3K), protein kinase B, PKB (AKT), glycogen synthase kinase-3β (GSK-3β) and β-catenin signaling pathway in cultured neurons and NSCs derived from the rat embryonic spinal cord. Immunocytochemistry was used to detect neuronal and NSCs characteristics. RT-PCR was used to detect PI3K/AKT/GSK3β/β-catenin pathway expression. Neurons and NSCs were successfully separated and cultured from Sprague-Dawley rat embryonic spinal cord and were respectively labeled using immunocytochemistry. Neuron-specific nuclear protein, neuronal class III β-tubulin, and neurofilament expression were detected in neurons; nestin, glial fibrillary acidic protein, microtubule-associated protein 2 and chondroitin sulfate glycosaminoglycan expression were detected in the NSCs. BDNF promoted significant neuronal growth (number, soma size, and average neurite length), as well as NSCs proliferation and differentiation, but BDNF antibody decreased neuronal growth and NSCs proliferation and differentiation. RT-PCR was used to detect changes in BDNF signal pathway components, showing that BDNF upregulated tropomyosin receptor kinase B, phosphoinositide 3-kinase (PI3K), AKT and β-catenin, but downregulated GSK-3β in the neurons and NSCs. BDNF antibody downregulated BDNF, tropomyosin receptor kinase B, PI3K, AKT, β-catenin and cellular-myelocytomatosis viral oncogene, but upregulated GSK- 3β, in the neurons and NSCs. Our findings suggested that BDNF contributed to neuronal growth and proliferation and differentiation of NSCs in vitro by stimulating PI3K/AKT/GSK3β/β-catenin pathways.

Transcription analysis of cochlear development in minipigs

Conclusions: The critical stage for cochlear gene regulation in the embryo is E49–E56. These data provide a valuable resource to help elucidate gene regulation in cochlear development and will benefit ear-related studies, especially those that focus on gene and stem cell therapies in hearing loss.Objectives: To investigate gene regulation in cochlear development and provide reference information for ear-related research.Methods: Total RNA extraction from the Bama miniature pig cochlea was performed at five time points that covered most of the cochlea development process (E42, E49, E56, E91, P13). Transcriptome sequencing was performed by Illumina HiSeq 2500. Transcriptome data were mapped to the genome using TopHat2. Gene expression was quantified using the Cuffquant and Cuffnorm modules. GO and KEGG pathway-enrichment analysis were in progress about the differentially expressed genes (DEGs) by Perl programs.Results: Sequencing of 1778643560000 paired reads revealed 35.9 G of data, and 19,432 expression genes were detected. The ratio of these pairs to the Ensembl genome database was approximately 71% in all samples. The stages E49–E91 and, especially, E49–E56 included the most rapid changes in gene regulation and differentiation for the cochlea. The stem cell characteristics of neural stem cells decreased most rapidly during this period.

Neural stem cell heterogeneity through time and space in the ventricular-subventricular zone.

The origin and classification of neural stem cells (NSCs) has been a subject of intense investigation for the past two decades. Efforts to categorize NSCs based on their location, function and expression have established that these cells are a heterogeneous pool in both the embryonic and adult brain. The discovery and additional characterization of adult NSCs has introduced the possibility of using these cells as a source for neuronal and glial replacement following injury or disease. To understand how one could manipulate NSC developmental programs for therapeutic use, additional work is needed to elucidate how NSCs are programmed and how signals during development are interpreted to determine cell fate. This review describes the identification, classification and characterization of NSCs within the large neurogenic niche of the ventricular-subventricular zone (V-SVZ). A literature search was conducted using Pubmed including the keywords "ventricular-subventricular zone," "neural stem cell," "heterogeneity," "identity" and/or "single cell" to find relevant manuscripts to include within the review. A special focus was placed on more recent findings using single-cell level analyses on neural stem cells within their niche(s). This review discusses over 20 research articles detailing findings on V-SVZ NSC heterogeneity, over 25 articles describing fate determinants of NSCs, and focuses on 8 recent publications using distinct single-cell analyses of neural stem cells including flow cytometry and RNA-seq. Additionally, over 60 manuscripts highlighting the markers expressed on cells within the NSC lineage are included in a chart divided by cell type. Investigation of NSC heterogeneity and fate decisions is ongoing. Thus far, much research has been conducted in mice however, findings in human and other mammalian species are also discussed here. Implications of NSC heterogeneity established in the embryo for the properties of NSCs in the adult brain are explored, including how these cells may be redirected after injury or genetic manipulation.

Electrical Property Characterization of Neural Stem Cells in Differentiation.

Electrical property characterization of stem cells could be utilized as a potential label-free biophysical approach to evaluate the differentiation process. However, there has been a lack of technology or tools that can quantify the intrinsic cellular electrical markers (e.g., specific membrane capacitance (Cspecific membrane) and cytoplasm conductivity (σcytoplasm)) for a large amount of stem cells or differentiated cells. In this paper, a microfluidic platform enabling the high-throughput quantification of Cspecific membrane and σcytoplasm from hundreds of single neural stem cells undergoing differentiation was developed to explore the feasibility to characterize the neural stem cell differentiation process without biochemical staining. Experimental quantification using biochemical markers (e.g., Nestin, Tubulin and GFAP) of neural stem cells confirmed the initiation of the differentiation process featured with gradual loss in cellular stemness and increased cell markers for neurons and glial cells. The recorded electrical properties of neural stem cells undergoing differentiation showed distinctive and unique patterns: 1) in the suspension culture before inducing differentiation, a large distribution and difference in σcytoplasm among individual neural stem cells was noticed, which indicated heterogeneity that may result from the nature of suspension culture of neurospheres; and 2) during the differentiation in adhering monolayer culture, significant changes and a large difference in Cspecific membrane were located indicating different expressions of membrane proteins during the differentiation process, and a small distribution difference in σcytoplasm was less significant that indicated the relatively consistent properties of cytoplasm during the culture. In summary, significant differences in Cspecific membrane and σcytoplasm were observed during the neural stem cell differentiation process, which may potentially be used as label-free biophysical markers to monitor this process.

Differentiation of Spiral Ganglion-Derived Neural Stem Cells into Functional Synaptogenetic Neurons.

Spiral ganglion neurons (SGNs) are usually damaged in sensorineural hearing loss. SGN-derived neural stem cells (NSCs) have been identified and proposed to differentiate into neurons to replace damaged SGNs. However, it remains obscure whether SGN-NSC-derived neurons (ScNs) are electrophysiologically functional and possess the capability to form neural connections. Here, we found that SGN-derived cells demonstrated NSC characteristics and differentiated into SGN-like glutamatergic neurons. Neurotrophins significantly increased neuronal differentiation and neurite length of ScNs. Patch clamp recording revealed that ScNs possessed SGN-like NaV and HCN channels, suggesting electrophysiological function. FM1-43 staining and synaptic protein immunofluorescence showed ScNs possess the ability to form neural connections. Astrocyte-conditioned medium was able to stimulate ScNs to express synaptic proteins. These data suggested that neurotrophins are able to stimulate postnatal SGN-NSCs to differentiate into functional glutamatergic ScNs with the capability to form synaptic connections in vitro.

Effect of Nano Silver on Inducement of Differentiation of Neural Stem Cells of SD Rats

Objective: To explore the effect of nanosilver on inducement of differentiation of neural stem cells of SD rats. Method: nanosilver film and newborn SD rat neural stem cell co-culture method is adopted to perform primary and subculture on SD rat neural stem cells and to induce differentiation. Nestin staining is adopted to identify the characteristics of neural stem cells. Immunohistochemistry method (β-III-tubulin, GFAP staining) is adopted to detect the status of differentiation from neural stem cells into neurons and neurogliocytes. Result: The neural stem cells of newborn SD rats can form Nestin positive cell balls in case of no serum culture. After induction of differentiation, NF-positive cells and GFAP-positive cells can be seen through immunofluorescence staining, wherein for nanosilver intervention group, the proportion of hippocampal NSCs differentiating to group neurons is smaller than that of the control group, with significantly reduced impact on astrocyte differentiation. Conclusion: nanosilver has adverse effect on normal differentiation of hippocampal NSCs to neurons and astrocytes.

Abstract A03: The effects of the cellular microenvironment on medulloblastoma metastasis

Abstract Medulloblastoma (MB) is the most common pediatric brain tumor, known to be highly malignant and invasive within the brain as well as extraneural locations. The mechanisms inducing MB metastasis and dissemination are not completely understood. Astrocytes, the most abundant cell type in the brain, have been shown in our lab to secrete factors that induce breast cancer brain metastasis (Wang et al., 2013). In addition to astrocytes, many brain cell types have also been implicated in the malignancy of brain tumors, including microglia, pericytes, and endothelial cells as part of the neurovascular unit. The overall goal of this study is to determine the effects of cells in the tumor microenvironment on MB metastasis. Our hypothesis is that astrocytes and/or other cell types in the neurovascular unit and microenvironment secrete factors that bring upon phenotypic changes in MB cells, leading to increased metastasis. To investigate this hypothesis, astrocyte conditioned media (ACM) from neonatal rat astrocyte cultures were used in in vitro assays to determine functional changes to Daoy MB cells. We observed that ACM (both serum free and serum depleted) in the lower chamber of a Boyden chamber Matrigel invasion assay induced invasion of Daoy cells more than 50-fold in comparison to DMEM in the lower well. Brain endothelial cell conditioned media also increased invasion of Daoy cells, which was comparable to ACM. ACM did not significantly affect proliferation or apoptosis of Daoy cells, as tested by BrdU incorporation and Caspase-3/-7 activity, respectively. When cultured appropriately in serum free ACM (+bFGF and EGF), Daoy cells were able to form larger and more proliferative neurospheres in comparison to DMEM, indicative of an increase in neural or cancer stem cell characteristics. To determine the effects of ACM on MB metastasis in vivo, we developed a zebrafish xenotransplant model. Daoy-GFP cells were injected into the hindbrain of 48 hour post fertilization Absolut FLK-mCherry zebrafish embryos. Using time-lapse confocal microscopy, we are able to image the cells and observe their movement within the microenvironment after injection. With this method we are working to determine changes in cell morphology and movement in Daoy-GFP cells that were cultured in ACM prior to injection. In order to further elucidate the mechanism in which ACM increases invasion and neurosphere formation, microarray studies are underway to pin-point a factor(s) or pathway(s) involved. Together, these data indicate that cells comprising the neurovascular unit, namely endothelial cells and astrocytes, contribute in a paracrine manner to MB metastasis and dissemination. Citation Format: Emily Gronseth, Ling Wang, Chris Koceja, Ramani Ramchandran. The effects of the cellular microenvironment on medulloblastoma metastasis. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr A03.

Isolation and characterization of neural stem cells from dystrophic mdx mouse

The blood-brain barrier (BBB) is altered in mdx mouse, an animal model to study Duchenne muscular dystrophy (DMD). Our previous work demonstrated that perivascular glial endfeet control the selective exchanges between blood and neuropil as well as the BBB development and integrity; the alterations of dystrophin and dystrophin-associated protein complex (DAPs) in the glial cells of mdx mouse, parallel damages of the BBB and increase in vascular permeability. The aim of this study was to improve our knowledge about brain cellular components in the mdx mouse through the isolation, for the first time, of the adult neural stem cells (ANSCs). We characterized them by FACS, electron microscopy, confocal immunofluorescence microscopy, Real Time-PCR and western blotting, and we studied the expression of the DAPs aquaporin-4 (AQP4), potassium channel Kir4.1, α- and β-dystroglycan (αDG, βDG), α-syntrophin (αSyn), and short dystrophin isoform Dp71 proteins. The results showed that the mdx ANSCs expressed CD133 and Nestin receptor as the control ones, but showed a reduction in Notch receptor and altered cell proliferation with an increment in the apoptotic nuclei. Ultrastructurally, they appeared 50% size reduced compared to control ones, with a few cytoplasmic organelles. Moreover, the mdx ANSCs are devoid in full length dystrophin 427, and they expressed post-transcriptional reduction in the Dp71 in parallel with the ubiquitin proteasome activation, and decrement of DAPs proteins which appeared diffused in the cytoplasm and not polarized on the stem cells plasmamembrane, as prevalently observed in the controls. Overall, these results indicate that structural and molecular alterations affect the neural stem cells in the dystrophic brain, whose increased apoptosis and reduced Dp71 and DAPs proteins expression, together with loss in Dp427 dystrophin, could be responsible of the altered mdx glial maintenance and differentiation and consequent failure in the vessels barrier control occurring in the adult dystrophic brain.

Blocking mPTP on Neural Stem Cells and Activating the Nicotinic Acetylcholine Receptor α7 Subunit on Microglia Attenuate Aβ-Induced Neurotoxicity on Neural Stem Cells.

β-Amyloid (Aβ) can stimulate microglia to release a variety of proinflammatory cytokines and induce neurotoxicity. Nicotine has been reported to inhibit TNF-α, IL-1, and ROS production in microglia. Mitochondrial permeability transition pore (mPTP) plays an important role in neurotoxicity as well. Here, we investigated whether activating the microglial α7-nAChR has a neuroprotective role on neural stem cells (NSCs) and the function of mPTP in NSCs in this process. The expression of α7-nAChR in rat NSCs was detected by immunocytochemistry and RT-PCR. The viability of microglia and NSCs was examined by MTT assay. The mitochondrial membrane potential (ΔΨm) and morphological characteristics of NSCs was measured by JC-1 staining and transmission electron microscopy respectively. The distribution of cytochrome c in the subcellular regions of NSCs was visualized by confocal laser scanning microscopy, and the expression levels of cyclophilin D and cleaved caspase-3 were assayed by western blot. The apoptotic rate of NSCs was measured by flow cytometry. The expression of α7-nAChR was detected in microglial cells, but no expression was found in NSCs. The viability of rat microglial cells and NSCs was not affected by reagents or coculture itself. Aβ1-42-mediated microglial activation impaired the morphology and the ΔΨm of mitochondria of NSCs as well as increased cell apoptosis. However, the damage was attenuated when the α7-nAChRs on microglial cells were activated or the mPTPs on NSCs were blocked. Blockade of mPTPs on NSCs and activation of α7-nAChRs on microglia exhibit neuroprotective roles in Aβ-induced neurotoxicity of NSCs.

Proliferation and differentiation characteristics of neural stem cells during course of cerebral cortical histogenesis.

Recent advancements in the research field of stem cell biology have enabled the realization of regenerative medicine in various systems of the body, including the central nervous system. However, fundamental knowledge regarding how neural stem cells divide and generate young neurons in mammals, especially in vivo, is still inadequate. In this article, we shall summarize the concept of cell cycle/division of neural stem cells that generate projection neurons in the murine cerebral cortex. We shall also review the molecular mechanisms that modulate the critical parameters related to the cell cycle regulatory mechanisms, with special reference to the cell cycle regulatory protein p27(Kip1) , an inhibitor of progression of the cell cycle at the G1 phase. A better understanding of the mechanisms controlling cell cycle progression is expected to contribute to the development of novel strategies to increase the efficiency of neural cell/tissue production, both in vivo and in vitro.

New insight into LSD1 function in human cortical neurogenesis.

The cerebral cortex of primates has evolved massively and intricately in comparison to that of other species. Accumulating evidence indicates that this is caused by changes in cell biological features of neural stem cells (NSCs), which differentiate into neurons and glial cells during development. The fate of NSCs during rodent cortical development is stringently regulated by epigenetic factors, such as histone modification enzymes, but the role of these factors in human corticogenesis is largely unknown. We have recently discovered that a lysine-specific demethylase 1 (LSD1), which catalyzes the demethylation of methyl groups in the histone tail, plays a unique role in human fetal NSCs (hfNSCs). We show that, unlike the role previously reported in mice, LSD1 in hfNSCs is necessary for neuronal differentiation and controls the expression of HEYL, one of the NOTCH target genes, by modulating the methylation level of histones on its promoter region. Interestingly, LSD1-regulation of Heyl expression is not observed in mouse NSCs. Furthermore, we first demonstrated that HEYL is able to maintain the undifferentiated state of hfNSCs. Our findings provide a new insight indicating that LSD1 may be a key player in the development and characterization of the evolved cerebral cortex.

Direct Reprogramming of Human Amniotic Fluid Stem Cells by OCT4 and Application in Repairing of Cerebral Ischemia Damage.

Amniotic fluid stem cells (AFSCs) are a type of fetal stem cell whose stemness encompasses both embryonic and adult stem cells, suggesting that they may be easily and efficiently reprogrammed into induced pluripotent stem cells (iPSCs). To further simplify the reprogramming process, the creation of AFSC-derived iPSCs using a single factor is desirable. Here we report the generation of one-factor human AFSC-iPSCs (AiPSCs) from human AFSCs by ectopic expression of the transcription factor OCT4. Just like human embryonic stem cells, AiPSCs exhibited similar epigenetic status, global gene expression profiles, teratoma formation and in vitro & in vivo pluripotency. Our results indicate that the OCT4 is necessary and sufficient to directly reprogram human AFSCs into pluripotent AiPSCs. Moreover, reflecting the similar memory characteristics of AFSCs and neural stem cells, we show that AiPSC membrane-derived vesicles (MVs) repair cerebral ischemia damage. We anticipate that the successful generation of one-factor AiPSCs will facilitate the creation of patient-specific pluripotent stem cells without the need for transgenic expression of oncogenes. Moreover, MVs from tissue-specific AiPSCs have potential in tissue repair, representing a novel application of iPSCs.

Insights into the Biology and Therapeutic Applications of Neural Stem Cells.

The cerebral cortex is essential for our higher cognitive functions and emotional reasoning. Arguably, this brain structure is the distinguishing feature of our species, and yet our remarkable cognitive capacity has seemingly come at a cost to the regenerative capacity of the human brain. Indeed, the capacity for regeneration and neurogenesis of the brains of vertebrates has declined over the course of evolution, from fish to rodents to primates. Nevertheless, recent evidence supporting the existence of neural stem cells (NSCs) in the adult human brain raises new questions about the biological significance of adult neurogenesis in relation to ageing and the possibility that such endogenous sources of NSCs might provide therapeutic options for the treatment of brain injury and disease. Here, we highlight recent insights and perspectives on NSCs within both the developing and adult cerebral cortex. Our review of NSCs during development focuses upon the diversity and therapeutic potential of these cells for use in cellular transplantation and in the modeling of neurodevelopmental disorders. Finally, we describe the cellular and molecular characteristics of NSCs within the adult brain and strategies to harness the therapeutic potential of these cell populations in the treatment of brain injury and disease.

Characterization of neural stem cells and their progeny in the sensory circumventricular organs of adult mouse.

Although evidence has accumulated that neurogenesis and gliogenesis occur in the subventricular zone (SVZ) and subgranular zone (SGZ) of adult mammalian brains, recent studies indicate the presence of neural stem cells (NSCs) in adult brains, particularly the circumventricular regions. In the present study, we aimed to determine characterization of NSCs and their progenitor cells in the sensory circumventricular organs (CVOs), including organum vasculosum of the lamina terminalis, subfornical organ, and area postrema of adult mouse. There were two types of NSCs: tanycyte-like ependymal cells and astrocyte-like cells. Astrocyte-like NSCs proliferated slowly and oligodendrocyte progenitor cells (OPCs) and neural progenitor cells (NPCs) actively divided. Molecular marker protein expression of NSCs and their progenitor cells were similar to those reported in the SVZ and SGZ, except that astrocyte-like NSCs expressed S100β. These circumventricular NSCs possessed the capacity to give rise to oligodendrocytes and sparse numbers of neurons and astrocytes in the sensory CVOs and adjacent brain regions. The inhibition of vascular endothelial growth factor (VEGF) signaling by using a VEGF receptor-associated tyrosine kinase inhibitor AZD2171 largely suppressed basal proliferation of OPCs. A single systemic administration of lipopolysaccharide attenuated proliferation of OPCs and induced remarkable proliferation of microglia. The present study indicates that sensory circumventricular NSCs provide new neurons and glial cells in the sensory CVOs and adjacent brain regions.

EphB2 activation is required for ependymoma development as well as inhibits differentiation and promotes proliferation of the transformed cell.

Our intracranial implantation mouse model of ependymoma clearly demonstrates overexpression of the ephrin receptor EphB2 in Ink4a/Arf(−/−) supratentorial embryonic neural stem cells (STeNSCs) to be essential for transformation and disease development; however the requirement for and consequence of receptor activation on transformation and neural stem cell function were not examined. We definitively illustrate the necessity for receptor activation in cellular transformation and the importance of implantation site and microenvironment in directing ependymoma development. In vitro assays of EphB2 overexpressing Ink4a/Arf(−/−) STeNSCs showed no changes in their neural stem cell characteristics (stem cell marker expression and self-renewal) upon receptor activation, but EphB2 driven tumor cells were inhibited significantly in differentiation and exhibited increased tumorsphere formation and cellular proliferation in response to ephrin-B ligand mediated receptor activation. Additionally, we observed substantial differences in the phosphorylation state of several key proteins involved in Ras and p38 MAPK signaling when comparing EphB2 overexpressing Ink4a/Arf(−/−) STeNSCs and tumor cells with relatively little change in total protein levels. We propose that EphB2 mediated ependymoma development is a multifactorial process requiring microenvironment directed receptor activation, resulting in changes in the phosphorylation status of key regulatory proteins, maintenance of a stem-like state and cellular proliferation.