Showing papers on "Neurosphere published in 2013"
TL;DR: Evidence is presented that differentiated airway epithelial cells can revert into stable and functional stem cells in vivo, and this capacity of committed cells to dedifferentiate into stem cells may have a more general role in the regeneration of many tissues and in multiple disease states, notably cancer.
Abstract: Cellular plasticity contributes to the regenerative capacity of plants, invertebrates, teleost fishes and amphibians. In vertebrates, differentiated cells are known to revert into replicating progenitors, but these cells do not persist as stable stem cells. Here we present evidence that differentiated airway epithelial cells can revert into stable and functional stem cells in vivo. After the ablation of airway stem cells, we observed a surprising increase in the proliferation of committed secretory cells. Subsequent lineage tracing demonstrated that the luminal secretory cells had dedifferentiated into basal stem cells. Dedifferentiated cells were morphologically indistinguishable from stem cells and they functioned as well as their endogenous counterparts in repairing epithelial injury. Single secretory cells clonally dedifferentiated into multipotent stem cells when they were cultured ex vivo without basal stem cells. By contrast, direct contact with a single basal stem cell was sufficient to prevent secretory cell dedifferentiation. In analogy to classical descriptions of amphibian nuclear reprogramming, the propensity of committed cells to dedifferentiate is inversely correlated to their state of maturity. This capacity of committed cells to dedifferentiate into stem cells may have a more general role in the regeneration of many tissues and in multiple disease states, notably cancer.
571 citations
TL;DR: It is shown that transplanted human fibroblasts and human astrocytes, which are engineered to express inducible forms of neural reprogramming genes, convert into neurons when reprograming genes are activated after transplantation.
Abstract: Cellular reprogramming is a new and rapidly emerging field in which somatic cells can be turned into pluripotent stem cells or other somatic cell types simply by the expression of specific combinations of genes. By viral expression of neural fate determinants, it is possible to directly reprogram mouse and human fibroblasts into functional neurons, also known as induced neurons. The resulting cells are nonproliferating and present an alternative to induced pluripotent stem cells for obtaining patient- and disease-specific neurons to be used for disease modeling and for development of cell therapy. In addition, because the cells do not pass a stem cell intermediate, direct neural conversion has the potential to be performed in vivo. In this study, we show that transplanted human fibroblasts and human astrocytes, which are engineered to express inducible forms of neural reprogramming genes, convert into neurons when reprogramming genes are activated after transplantation. Using a transgenic mouse model to specifically direct expression of reprogramming genes to parenchymal astrocytes residing in the striatum, we also show that endogenous mouse astrocytes can be directly converted into neural nuclei (NeuN)-expressing neurons in situ. Taken together, our data provide proof of principle that direct neural conversion can take place in the adult rodent brain when using transplanted human cells or endogenous mouse cells as a starting cell for neural conversion.
380 citations
TL;DR: These findings provide a molecular basis for how cells with neural stem cell lineage emerge at sites of brain injury and imply that the high levels of SHH known to enter the brain from extraneural sources after invasive injury can trigger this response.
331 citations
TL;DR: A chemically defined xeno‐free medium culture system for rapidly generating astrocytes from neural stem cells derived from PSC is described and it is shown thatAstrocyte development in vitro, mimics normal development in vivo, and also passes through a CD44+ astroCyte precursor stage.
Abstract: Astrocytes can be generated from various tissue sources including human pluripotent stem cells (PSC). In this manuscript, we describe a chemically defined xeno-free medium culture system for rapidly generating astrocytes from neural stem cells derived from PSC. We show that astrocyte development in vitro, mimics normal development in vivo, and also passes through a CD44(+) astrocyte precursor stage. Astrocytes generated by our method display similar gene expression patterns, morphological characteristics and functional properties to primary astrocytes, and they survive and integrate after xenotransplantation. Whole genome expression profiling of astrocyte differentiation was performed at several time points of differentiation, and the results indicate the importance of known regulators and identify potential novel regulators and stage-specific lineage markers.
225 citations
TL;DR: The proliferation dynamics of B1, C, and A cells in vivo are determined using whole mounts of the walls of the lateral ventricles of adult mice and three cell cycle analysis methods using thymidine analogs to provide essential information on the dynamics of adult progenitor cell proliferation in the V-SVZ.
Abstract: Proliferating neural stem cells and intermediate progenitors persist in the ventricular-subventricular zone (V-SVZ) of the adult mammalian brain. This extensive germinal layer in the walls of the lateral ventricles is the site of birth of different types of interneurons destined for the olfactory bulb. The cell cycle dynamics of stem cells (B1 cells), intermediate progenitors (C cells), and neuroblasts (A cells) in the V-SVZ and the number of times these cells divide remain unknown. Using whole mounts of the walls of the lateral ventricles of adult mice and three cell cycle analysis methods using thymidine analogs, we determined the proliferation dynamics of B1, C, and A cells in vivo. Achaete-scute complex homolog (Ascl)1+ C cells were heterogeneous with a cell cycle length (TC) of 18–25 h and a long S phase length (TS) of 14–17 h. After C cells, Doublecortin+ A cells were the second-most common dividing cell type in the V-SVZ and had a TC of 18 h and TS of 9 h. Human glial fibrillary acidic protein (hGFAP)::GFP+ B1 cells had a surprisingly short Tc of 17–18 h and a TS of 4 h. Progenitor population analysis suggests that following the initial division of B1 cells, C cells divide three times and A cells once, possibly twice. These data provide essential information on the dynamics of adult progenitor cell proliferation in the V-SVZ and how large numbers of new neurons continue to be produced in the adult mammalian brain.
224 citations
TL;DR: The results imply that CD133 could be used as a therapeutic target in GBMs, since it appears in an interconvertible state, changing its subcellular localization between the cytoplasm and the plasmamembrane of neurosphere cells.
Abstract: The role of the cell surface CD133 as a cancer stem cell marker in glioblastoma (GBM) has been widely investigated, since it identifies cells that are able to initiate neurosphere growth and form heterogeneous tumors when transplanted in immune-compromised mice. However, evidences of CD133-negative cells exhibiting similar properties have also been reported. Moreover, the functional role of CD133 in cancer stem/progenitor cells remains poorly understood. We studied the biological effects of CD133 downregulation in GBM patient-derived neurospheres. Our results indicate that there is not a hierarchical relation between CD133-positive and CD133-negative cells composing the neurospheres. Indeed, CD133 appears in an interconvertible state, changing its subcellular localization between the cytoplasm and the plasmamembrane of neurosphere cells. Silencing of CD133 in human GBM neurospheres using lentivirus-mediated short hairpin RNA impairs the self-renewal and tumorigenic capacity of neurosphere cells. These results imply that CD133 could be used as a therapeutic target in GBMs.
207 citations
TL;DR: It is reported that the cells survive and differentiate upon transplant into newborn rat brain and can self-renew and can differentiate into multiple functional neuronal subtypes and glial cells in vitro.
Abstract: Human neural stem cells hold great promise for research and therapy in neural disease. We describe the generation of integration-free and expandable human neural progenitor cells (NPCs). We combined an episomal system to deliver reprogramming factors with a chemically defined culture medium to reprogram epithelial-like cells from human urine into NPCs (hUiNPCs). These transgene-free hUiNPCs can self-renew and can differentiate into multiple functional neuronal subtypes and glial cells in vitro. Although functional in vivo analysis is still needed, we report that the cells survive and differentiate upon transplant into newborn rat brain.
190 citations
TL;DR: After completion of this protocol, NCSCs can be used for numerous applications, including the generation of sufficient cell numbers to perform drug screens, for the development of cell therapeutics on an industrial scale and to provide a robust model for human disease.
Abstract: Multipotent neural crest stem cells (NCSCs) have the potential to generate a wide range of cell types including melanocytes; peripheral neurons; and smooth muscle, bone, cartilage and fat cells. This protocol describes in detail how to perform a highly efficient, lineage-specific differentiation of human pluripotent cells to a NCSC fate. The approach uses chemically defined media under feeder-free conditions, and it uses two small-molecule compounds to achieve efficient conversion of human pluripotent cells to NCSCs in ~15 d. After completion of this protocol, NCSCs can be used for numerous applications, including the generation of sufficient cell numbers to perform drug screens, for the development of cell therapeutics on an industrial scale and to provide a robust model for human disease. This protocol can be also be applied to patient-derived induced pluripotent stem cells and thus used to further the knowledge of human disease associated with neural crest development, for example, Treacher-Collins Syndrome.
175 citations
TL;DR: The data indicate that FOXM1 signaling through its direct interaction with MELK regulates key mitotic genes in GSCs in a PLK1‐dependent manner and thus, this protein complex is a potential therapeutic target for GBM.
Abstract: Glioblastoma multiforme (GBM) is a life-threatening brain tumor. Accumulating evidence suggests that eradication of glioma stem-like cells (GSCs) in GBM is essential to achieve cure. The transcription factor FOXM1 has recently gained attention as a master regulator of mitotic progression of cancer cells in various organs. Here, we demonstrate that FOXM1 forms a protein complex with the mitotic kinase MELK in GSCs, leading to phosphorylation and activation of FOXM1 in a MELK kinase-dependent manner. This MELK-dependent activation of FOXM1 results in a subsequent increase in mitotic regulatory genes in GSCs. MELK-driven FOXM1 activation is regulated by the binding and subsequent trans-phosphorylation of FOXM1 by another kinase PLK1. Using mouse neural progenitor cells (NPCs), we found that transgenic expression of FOXM1 enhances, while siRNA-mediated gene silencing diminishes neurosphere formation, suggesting that FOXM1 is required for NPC growth. During tumorigenesis, FOXM1 expression sequentially increases as cells progress from NPCs, to pretumorigenic progenitors and GSCs. The antibiotic Siomycin A disrupts MELK-mediated FOXM1 signaling with a greater sensitivity in GSC compared to neural stem cell. Treatment with the first-line chemotherapy agent for GBM, Temozolomide, paradoxically enriches for both FOXM1 (+) and MELK (+) cells in GBM cells, and addition of Siomycin A to Temozolomide treatment in mice harboring GSC-derived intracranial tumors enhances the effects of the latter. Collectively, our data indicate that FOXM1 signaling through its direct interaction with MELK regulates key mitotic genes in GSCs in a PLK1-dependent manner and thus, this protein complex is a potential therapeutic target for GBM.
172 citations
TL;DR: A function of p21 is identified in the direct regulation of the expression of pluripotency factor Sox2, a key regulator of the specification and maintenance of neural progenitors and a regulation of NSC expansion driven by a p21/Sox2/p53 axis.
171 citations
TL;DR: A highly efficient serum‐free pluripotent stem cell neural induction medium is reported that can induce hPSCs into primitive NSCs (pNSCs) in 7 days, obviating the need for time‐consuming, laborious embryoid body generation or rosette picking.
Abstract: Human pluripotent stem cells (hPSCs), including human embryonic stem cells and human induced pluripotent stem cells, are unique cell sources for disease modeling, drug discovery screens, and cell therapy applications. The first step in producing neural lineages from hPSCs is the generation of neural stem cells (NSCs). Current methods of NSC derivation involve the time-consuming, labor-intensive steps of an embryoid body generation or coculture with stromal cell lines that result in low-efficiency derivation of NSCs. In this study, we report a highly efficient serum-free pluripotent stem cell neural induction medium that can induce hPSCs into primitive NSCs (pNSCs) in 7 days, obviating the need for time-consuming, laborious embryoid body generation or rosette picking. The pNSCs expressed the neural stem cell markers Pax6, Sox1, Sox2, and Nestin; were negative for Oct4; could be expanded for multiple passages; and could be differentiated into neurons, astrocytes, and oligodendrocytes, in addition to the brain region-specific neuronal subtypes GABAergic, dopaminergic, and motor neurons. Global gene expression of the transcripts of pNSCs was comparable to that of rosette-derived and human fetal-derived NSCs. This work demonstrates an efficient method to generate expandable pNSCs, which can be further differentiated into central nervous system neurons and glia with temporal, spatial, and positional cues of brain regional heterogeneity. This method of pNSC derivation sets the stage for the scalable production of clinically relevant neural cells for cell therapy applications in good manufacturing practice conditions.
TL;DR: Peptide functionalization with cell adhesion and cell differentiation motifs show superior cell survival and differentiation properties compared to those observed upon culturing neural cells in non-modified peptide hydrogels.
TL;DR: Findings show that transplanted enteric neural progenitor cells can generate functional enteric neurons in the postnatal bowel and advances the notion that cell therapy is a promising strategy for enteric neuropathies.
Abstract: Cell therapy has the potential to treat gastrointestinal motility disorders caused by diseases of the enteric nervous system. Many studies have demonstrated that various stem/progenitor cells can give rise to functional neurons in the embryonic gut; however, it is not yet known whether transplanted neural progenitor cells can migrate, proliferate, and generate functional neurons in the postnatal bowel in vivo. We transplanted neurospheres generated from fetal and postnatal intestinal neural crest-derived cells into the colon of postnatal mice. The neurosphere-derived cells migrated, proliferated, and generated neurons and glial cells that formed ganglion-like clusters within the recipient colon. Graft-derived neurons exhibited morphological, neurochemical, and electrophysiological characteristics similar to those of enteric neurons; they received synaptic inputs; and their neurites projected to muscle layers and the enteric ganglia of the recipient mice. These findings show that transplanted enteric neural progenitor cells can generate functional enteric neurons in the postnatal bowel and advances the notion that cell therapy is a promising strategy for enteric neuropathies.
TL;DR: The effects of a range of intrinsic and extrinsic factors that regulate neural stem/precursor cell functions are covered, highlighting those that have already shown evidence of preclinical effectiveness and discussing others that warrant further preclinical investigation.
Abstract: Neural stem/precursor cells in the adult brain reside in the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the dentate gyrus in the hippocampus. These cells primarily generate neuroblasts that normally migrate to the olfactory bulb (OB) and the dentate granule cell layer respectively. Following brain damage, such as traumatic brain injury, ischemic stroke or in degenerative disease models, neural precursor cells from the SVZ in particular, can migrate from their normal route along the rostral migratory stream (RMS) to the site of neural damage. This neural precursor cell response to neural damage is mediated by release of endogenous factors, including cytokines and chemokines produced by the inflammatory response at the injury site, and by the production of growth and neurotrophic factors. Endogenous hippocampal neurogenesis is frequently also directly or indirectly affected by neural damage. Administration of a variety of factors that regulate different aspects of neural stem/precursor biology often leads to improved functional motor and/or behavioral outcomes. Such factors can target neural stem/precursor proliferation, survival, migration and differentiation into appropriate neuronal or glial lineages. Newborn cells also need to subsequently survive and functionally integrate into extant neural circuitry, which may be the major bottleneck to the current therapeutic potential of neural stem/precursor cells. This review will cover the effects of a range of intrinsic and extrinsic factors that regulate neural stem/precursor cell functions. In particular it focuses on factors that may be harnessed to enhance the endogenous neural stem/precursor cell response to neural damage, highlighting those that have already shown evidence of preclinical effectiveness and discussing others that warrant further preclinical investigation.
TL;DR: The effects of the principal growth and neurotrophic factors as well as their intracellular signal transduction on neurogenesis, fate determination and neuroprotective mechanisms are discussed.
Abstract: The identification and isolation of multipotent neural stem and progenitor cells in the brain, giving rise to neurons, astrocytes, and oligodendrocytes initiated many studies in order to understand basic mechanisms of endogenous neurogenesis and repair mechanisms of the nervous system and to develop novel therapeutic strategies for cellular regeneration therapies in brain disease. A previous review (Trujillo et al., Cytometry A 2009;75:38-53) focused on the importance of extrinsic factors, especially neurotransmitters, for directing migration and neurogenesis in the developing and adult brain. Here, we extend our review discussing the effects of the principal growth and neurotrophic factors as well as their intracellular signal transduction on neurogenesis, fate determination and neuroprotective mechanisms. Many of these mechanisms have been elucidated by in vitro studies for which neural stem cells were isolated, grown as neurospheres, induced to neural differentiation under desired experimental conditions, and analyzed for embryonic, progenitor, and neural marker expression by flow and imaging cytometry techniques. The better understanding of neural stem cells proliferation and differentiation is crucial for any therapeutic intervention aiming at neural stem cell transplantation and recruitment of endogenous repair mechanisms.
TL;DR: It is shown that transplanted autologous rhesus monkey iPSC-derived neural progenitors survive for up to 6 months and differentiate into neurons, astrocytes, and myelinating oligodendrocyte in the brains of MPTP-induced hemiparkinsonian rhesUS monkeys with a minimal presence of inflammatory cells and reactive glia.
TL;DR: Adipose- and bone marrow-derived mesenchymal stem cells have similar in vitro glioma tropism, and adipose tissue may provide a more efficient source of MSCs for research and clinical applications, while minimizing patient morbidity during cell harvesting.
Abstract: Introduction
Glioblastoma is the most common primary malignant brain tumor, and is refractory to surgical resection, radiation, and chemotherapy. Human mesenchymal stem cells (hMSC) may be harvested from bone marrow (BMSC) and adipose (AMSC) tissue. These cells are a promising avenue of investigation for the delivery of adjuvant therapies. Despite extensive research into putative mechanisms for the tumor tropism of MSCs, there remains no direct comparison of the efficacy and specificity of AMSC and BMSC tropism towards glioma.
Methods
Under an IRB-approved protocol, intraoperative human Adipose MSCs (hAMSCs) were established and characterized for cell surface markers of mesenchymal stem cell origin in conjunction with the potential for tri-lineage differentiation (adipogenic, chondrogenic, and osteogenic). Validated experimental hAMSCs were compared to commercially derived hBMSCs (Lonza) and hAMSCs (Invitrogen) for growth responsiveness and glioma tropism in response to glioma conditioned media obtained from primary glioma neurosphere cultures.
Results
Commercial and primary culture AMSCs and commercial BMSCs demonstrated no statistically significant difference in their migration towards glioma conditioned media in vitro. There was statistically significant difference in the proliferation rate of both commercial AMSCs and BMSCs as compared to primary culture AMSCs, suggesting primary cultures have a slower growth rate than commercially available cell lines.
Conclusions
Adipose- and bone marrow-derived mesenchymal stem cells have similar in vitro glioma tropism. Given the well-documented ability to harvest larger numbers of AMSCs under local anesthesia, adipose tissue may provide a more efficient source of MSCs for research and clinical applications, while minimizing patient morbidity during cell harvesting.
TL;DR: The results show that postnatal survival, overall brain growth, and specific aspects of brain development critically depend on mTORC1 function.
Abstract: The mammalian target of rapamycin (mTOR) regulates cell growth in response to various intracellular and extracellular signals. It assembles into two multiprotein complexes: the rapamycin-sensitive mTOR complex 1 (mTORC1) and the rapamycin-insensitive mTORC2. In this study, we inactivated mTORC1 in mice by deleting the gene encoding raptor in the progenitors of the developing CNS. Mice are born but never feed and die within a few hours. The brains deficient for raptor show a microcephaly starting at E17.5 that is the consequence of a reduced cell number and cell size. Changes in cell cycle length during late cortical development and increased cell death both contribute to the reduction in cell number. Neurospheres derived from raptor-deficient brains are smaller, and differentiation of neural progenitors into glia but not into neurons is inhibited. The differentiation defect is paralleled by decreased Stat3 signaling, which is a target of mTORC1 and has been implicated in gliogenesis. Together, our results show that postnatal survival, overall brain growth, and specific aspects of brain development critically depend on mTORC1 function.
TL;DR: It is shown that the Notch ligand Delta-like 1 (Dll1) is required to maintain quiescent NSCs in the adult mouse subventricular zone and suggests a model in which N SCs produce their own niche cells for their maintenance through asymmetric Dll1 inheritance at mitosis.
Abstract: Stem cells often divide asymmetrically to produce one stem cell and one differentiating cell, thus maintaining the stem cell pool. Although neural stem cells (NSCs) in the adult mouse subventricular zone have been suggested to divide asymmetrically, intrinsic cell fate determinants for asymmetric NSC division are largely unknown. Stem cell niches are important for stem cell maintenance, but the niche for the maintenance of adult quiescent NSCs has remained obscure. Here we show that the Notch ligand Delta-like 1 (Dll1) is required to maintain quiescent NSCs in the adult mouse subventricular zone. Dll1 protein is induced in activated NSCs and segregates to one daughter cell during mitosis. Dll1-expressing cells reside in close proximity to quiescent NSCs, suggesting a feedback signal for NSC maintenance by their sister cells and progeny. Our data suggest a model in which NSCs produce their own niche cells for their maintenance through asymmetric Dll1 inheritance at mitosis.
TL;DR: A simple method to generate and expand multipotent, self-renewing pre-rosette neural stem cells from both human embryonic stem cells and human induced pluripotent stem cells without utilizing embryoid body formation, manual selection techniques, or complex combinations of small molecules is developed.
TL;DR: The use of cell-penetrating O2 probes based on phosphorescent Pt-porphyrins to perform high-resolution 2D and 3D mapping of O2 in spheroid structures by live cell fluorescence imaging technique is described.
TL;DR: It is found that GD3 is colocalized and interacts with the mitogen receptor EGFR in the microdomain structure of the plasma membrane and that the interaction is responsible for sustaining the expression of EGFR and its downstream signaling to maintain the self-renewal capability of NSCs.
Abstract: Mounting evidence supports the notion that gangliosides serve regulatory roles in neurogenesis; little is known, however, about how these glycosphingolipids function in neural stem cell (NSC) fate determination. We previously demonstrated that ganglioside GD3 is a major species in embryonic mouse brain: more than 80% of the NSCs obtained by the neurosphere method express GD3. To investigate the functional role of GD3 in neurogenesis, we compared the properties of NSCs from GD3-synthase knockout (GD3S-KO) mice with those from their wild-type littermates. NSCs from GD3S-KO mice showed decreased self-renewal ability compared with those from the wild-type animals, and that decreased ability was accompanied by reduced expression of EGF receptor (EGFR) and an increased degradation rate of EGFR and EGF-induced ERK signaling. We also showed that EGFR switched from the low-density lipid raft fractions in wild-type NSCs to the high-density layers in the GD3S-KO NSCs. Immunochemical staining revealed colocalization of EGFR and GD3, and EGFR could be immunoprecipitated from the NSC lysate with an anti-GD3 antibody from the wild-type, but not from the GD3S-KO, mice. Tracking the localization of endocytosed EGFR with endocytosis pathway markers indicated that more EGFR in GD3S-KO NSCs translocated through the endosomal−lysosomal degradative pathway, rather than through the recycling pathway. Those findings support the idea that GD3 interacts with EGFR in the NSCs and that the interaction is responsible for sustaining the expression of EGFR and its downstream signaling to maintain the self-renewal capability of NSCs.
TL;DR: Size-controllable networked neurospheres comprised of cerebral cortical neuronal cells that mimics the cytoarchitecture of the cortical region of the brain are developed and are a potential in vitro model for Alzheimer's disease studies.
TL;DR: It is suggested that integrin α3 contributes to the invasive nature of GSCs via ERK1/2, which renders integrinα3 a prime candidate for anti-invasion therapy for GBM.
Abstract: Gliomas are the most common primary tumours of the central nervous system, with glioblastomas (GBMs) being the most malignant entity The poor prognosis of GBM patients is largely due to the highly invasive nature of these tumours These invading cells are extremely resistant to radiation and chemotherapy, and currently, there are no anti-invasive therapies available (Nakada et al, 2007, 2013) A better understanding of the glioma invasion mechanism will help in developing therapeutic strategies to combat GBM
An increasing body of evidence suggests that a subpopulation of tumour stem-like cell properties in glioma, called either glioma stem-like cells (GSCs) or glioma-initiating cells, is responsible for tumour formation, maintenance, and malignant progression (Singh et al, 2003; Tamase et al, 2009; Natsume et al, 2011) These rare tumour cells are characterised by their strong tumourigenic properties and self-renewal ability It is critical to understand how the properties of GSCs make them particularly difficult to eradicate From our data and data from other studies, it is clear that GSCs are primarily responsible for invasion (Liu et al, 2006; Beier et al, 2007; Tamase et al, 2009) However, the molecular features of GSCs that orchestrate the invasion process remain to be elucidated If we can identify the responsible molecules that mediate GSC invasion, these molecules may represent promising targets for the development of novel anti-invasive therapies
In GSCs, the expression of some neural stem markers such as nestin, SOX2, and Musashi-1 has been reported (Ignatova et al, 2002) In addition, CD133 has been evaluated as an enrichment marker for GSCs; however, several studies have demonstrated their limitations as specific markers (Beier et al, 2007; Wang et al, 2008) To date, there is no perfect GSC marker that can isolate these cells alone GSCs tend to form neurospheres in the specific culture medium containing various kinds of growth factors, such as epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) The neurosphere assay is one of the reasonable methods for isolating GSCs However, a previous report showed that the microenvironment in the neurosphere induces cell differentiation, suggesting that the neurosphere cannot maintain the stem cell properties on its own (Pollard et al, 2009) Therefore, it is difficult to obtain pure cell groups of GSC However, it is reasonable to investigate the molecular characteristics of GSCs using cell groups that containing high numbers of GSCs In this study, we used neurosphere methods and cell sorting with CD133 as a marker in order to investigate the biology of GSCs
Integrins are cell surface migration-promoting receptor glycoproteins that mediate various intracellular signals through interaction with the extracellular matrix (ECM) Integrins also have a significant role in the attachment of cells to the ECM, through the formation of cell adhesion complexes, consisting of integrins and many cytoplasmic proteins In particular, in GBMs, integrins participate in the regulation of complex processes, such as invasion, tumour growth, and angiogenesis by interacting with the ECM in the brain (Nakada et al, 2007) A phase II clinical trial demonstrated that combining the integrin inhibitor cilengitide with standard chemoradiation improved the survival of patients with newly diagnosed GBMs (Nabors et al, 2012) Currently, cilengitide is under phase III clinical trials for patients with GBM and the results will be reported soon (Kurozumi et al, 2012)
In this study, we report that GSCs attach to fibronectin and laminin and highly express integrin α3 The immunohistochemistry demonstrated that integrin α3 is localised in GBM cells, especially in invading cells and cells surrounding vessels in vivo Additionally, overexpression of integrin α3 increases glioma migration and invasion, whereas downregulation of integrin α3 inhibits glioma invasion concomitant with a change in the phosphorylation level of the extracellular signal–regulated kinase (ERK) 1/2 pathway These results suggest that integrin α3 have significant roles in the invasive behavior of GSCs through the activation of ERK1/2
TL;DR: A human embryonic stem cell (hESC)-derived 3-dimensional in vitro model that allows for testing of potential developmental neurotoxicants is developed and suggests that the 3-D hESC-derived model could be used to test for Nano-DNT.
Abstract: Nanoparticles (NPs) have been shown to accumulate in organs, cross the blood–brain barrier and placenta, and have the potential to elicit developmental neurotoxicity (DNT). Here, we developed a human embryonic stem cell (hESC)-derived 3-dimensional (3-D) in vitro model that allows for testing of potential developmental neurotoxicants. Early central nervous system PAX6+ precursor cells were generated from hESCs and differentiated further within 3-D structures. The 3-D model was characterized for neural marker expression revealing robust differentiation toward neuronal precursor cells, and gene expression profiling suggested a predominantly forebrain-like development. Altered neural gene expression due to exposure to non-cytotoxic concentrations of the known developmental neurotoxicant, methylmercury, indicated that the 3-D model could detect DNT. To test for specific toxicity of NPs, chemically inert polyethylene NPs (PE-NPs) were chosen. They penetrated deep into the 3-D structures and impacted gene expression at non-cytotoxic concentrations. NOTCH pathway genes such as HES5 and NOTCH1 were reduced in expression, as well as downstream neuronal precursor genes such as NEUROD1 and ASCL1. FOXG1, a patterning marker, was also reduced. As loss of function of these genes results in severe nervous system impairments in mice, our data suggest that the 3-D hESC-derived model could be used to test for Nano-DNT.
TL;DR: A concept that emerged from developmental studies may hold the clue for neural repair because adult stem cells in the neurogenic niches glial in nature, but also glial cells outside these niches display greater potential when reacting to brain injury.
Abstract: The discovery in the year 2000 that radial glial cells act as neural stem and progenitor cells in development has led to a change in the concept of neural stem cells in the adult brain. Not only are adult stem cells in the neurogenic niches glial in nature, but also glial cells outside these niches display greater potential when reacting to brain injury. Thus, a concept that emerged from developmental studies may hold the clue for neural repair.
TL;DR: The controversies surrounding the origin and the identification of GBM stem cells are discussed and the micro-environment impact on their biology is highlighted, as GICs also require a specific micro- environment to maintain their “stem cell” properties.
Abstract: Glioblastoma multiforme (GBM, WHO grade IV) is the most common and lethal subtype of primary brain tumor with a median overall survival of 15 months from the time of diagnosis. The presence in GBM of a cancer population displaying neural stem cell (NSC) properties as well as tumor-initiating abilities and resistance to current therapies suggests that these glioblastoma-initiating cells (GICs) play a central role in tumor development and are closely related to NSCs. However, it is nowadays still unclear whether GICs derive from NSCs, neural progenitor cells or differentiated cells such as astrocytes or oligodendrocytes. On the other hand, NSCs are located in specific regions of the adult brain called neurogenic niches that have been shown to control critical stem cell properties, to nourish NSCs and to support their self-renewal. This "seed-and-soil" relationship has also been adapted to cancer stem cell research as GICs also require a specific micro-environment to maintain their "stem cell" properties. In this review, we will discuss the controversies surrounding the origin and the identification of GBM stem cells and highlight the micro-environment impact on their biology.
TL;DR: It is shown that, following ionizing radiation-induced DNA damage, NSCs enter irreversible proliferative arrest with features of cellular senescence, characterized by increased cytokine secretion, loss of stem cell markers, and astrocytic differentiation.
Abstract: The consequences of DNA damage generation in mammalian somatic stem cells, including neural stem cells (NSCs), are poorly understood despite their potential relevance for tissue homeostasis. Here, we show that, following ionizing radiation-induced DNA damage, NSCs enter irreversible proliferative arrest with features of cellular senescence. This is characterized by increased cytokine secretion, loss of stem cell markers, and astrocytic differentiation. We demonstrate that BMP2 is necessary to induce expression of the astrocyte marker GFAP in irradiated NSCs via a noncanonical signaling pathway engaging JAK-STAT. This is promoted by ATM and antagonized by p53. Using a SOX2-Cre reporter mouse model for cell-lineage tracing, we demonstrate irradiation-induced NSC differentiation in vivo. Furthermore, glioblastoma assays reveal that irradiation therapy affects the tumorigenic potential of cancer stem cells by ablating self-renewal and inducing astroglial differentiation.
TL;DR: The critical points that researchers have to keep in mind before extrapolating results or translating experimental transplantation of neurosphere‐derived cells to the clinical setting are largely highlighted.
Abstract: The possibility of obtaining large numbers of cells with potential to become functional neurons implies a great advance in regenerative medicine. A source of cells for therapy is the subventricular zone (SVZ) where adult neural stem cells (NSCs) retain the ability to proliferate, self-renew, and differentiate into several mature cell types. The neurosphere assay, a method to isolate, maintain, and expand these cells has been extensively utilized by research groups to analyze the biological properties of aNSCs and to graft into injured brains from animal models. In this review we briefly describe the neurosphere assay and its limitations, the methods to optimize culture conditions, the identity and the morphology of aNSC-derived neurospheres (including new ultrastructural data). The controversy regarding the identity and “stemness” of cells within the neurosphere is revised. The fine morphology of neurospheres, described thoroughly, allows for phenotypical characterization of cells in the neurospheres and may reveal slight changes that indirectly inform about cell integrity, cell damage, or oncogenic transformation. Along this review we largely highlight the critical points that researchers have to keep in mind before extrapolating results or translating experimental transplantation of neurosphere-derived cells to the clinical setting. Anat Rec, 296:1435-1452, 2013. © 2013 Wiley Periodicals, Inc.
TL;DR: It is shown that MEK/ERK signaling contributes to the cyclical activity of spermatogonial stem cells, and that ERK1/2 is periodically activated in Sertoli cells during the stem cell self‐renewal/proliferation phase, and MeK/ ERK signaling is required for the stage‐related expression of the critical niche factor GDNF.
Abstract: Coordination of stem cell fate is regulated by extrinsic niche signals and stem cell intrinsic factors. In mammalian testes, spermatogonial stem cells maintain constant production of abundant spermatozoa by alternating between self-renewal and differentiation at regular intervals according to a periodical program known as the seminiferous epithelial cycle. Although retinoic acid (RA) signaling has been suggested to direct the cyclical differentiation of spermatogonial stem cells, it remains largely unclear how their cycle-dependent self-renewal/proliferation is regulated. Here, we show that MEK/ERK signaling contributes to the cyclical activity of spermatogonial stem cells. We found that ERK1/2 is periodically activated in Sertoli cells during the stem cell self-renewal/proliferation phase, and that MEK/ERK signaling is required for the stage-related expression of the critical niche factor GDNF. In addition, ERK1/2 is activated in GFRα1-positive spermatogonial stem cells under the control of GDNF and prevent them from being differentiated. These results suggest that MEK/ERK signaling directly and indirectly maintains spermatogonial stem cells by mediating a signal that promotes their periodical self-renewal/proliferation. Conversely, RA signaling directly and indirectly induces differentiation of spermatogonial stem cells. We propose that temporally regulated activations of RA signaling and a signal regulating MEK/ERK antagonistically coordinates the cycle-related activity of spermatogonial stem cells.