Showing papers on "Neurosphere published in 2012"
TL;DR: An 80-d, three-stage process that recapitulates cortical development is demonstrated, in which human PSCs (hPSCs) first differentiate to cortical stem and progenitor cells that then generate cortical projection neurons in a stereotypical temporal order before maturing to actively fire action potentials, undergo synaptogenesis and form neural circuits in vitro.
Abstract: Efficient derivation of human cerebral neocortical neural stem cells (NSCs) and functional neurons from pluripotent stem cells (PSCs) facilitates functional studies of human cerebral cortex development, disease modeling and drug discovery. Here we provide a detailed protocol for directing the differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) to all classes of cortical projection neurons. We demonstrate an 80-d, three-stage process that recapitulates cortical development, in which human PSCs (hPSCs) first differentiate to cortical stem and progenitor cells that then generate cortical projection neurons in a stereotypical temporal order before maturing to actively fire action potentials, undergo synaptogenesis and form neural circuits in vitro. Methods to characterize cortical neuron identity and synapse formation are described.
670 citations
TL;DR: Self-renewable and multipotent iNSCs without tumorigenic potential can be generated directly from fibroblasts by reprogramming with a single factor, Sox2.
509 citations
TL;DR: A detailed mammosphere assay protocol is described for the assessment of stem cell activity and self-renewal, and how data generated by the assay can be analysed and interpreted is discussed.
Abstract: Since the discovery that neural tissue contains a population of stem cells that form neurospheres in vitro, sphere-forming assays have been adapted for use with a number of different tissue types for the quantification of stem cell activity and self-renewal. One tissue type widely used for stem cell investigations is mammary tissue, and the mammosphere assay has been used in both normal tissue and cancer. Although it is a relatively simple assay to learn, it can be difficult to master. There are methodological and analytical aspects to the assay which require careful consideration when interpreting the results. We describe here a detailed mammosphere assay protocol for the assessment of stem cell activity and self-renewal, and discuss how data generated by the assay can be analysed and interpreted.
318 citations
TL;DR: It is proposed that lt-NES cells provide an interesting tool for studying human neurodevelopment and may serve as a standard system to facilitate comparative analyses of hESC and hiPSC-derived neural cells from control and diseased genetic backgrounds.
Abstract: Human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC) provide new prospects for studying human neurodevelopment and modeling neurological disease. In particular, iPSC-derived neural cells permit a direct comparison of disease-relevant molecular pathways in neurons and glia derived from patients and healthy individuals. A prerequisite for such comparative studies are robust protocols that efficiently yield standardized populations of neural cell types. Here we show that long-term self-renewing neuroepithelial-like stem cells (lt-NES cells) derived from 3 hESC and 6 iPSC lines in two independent laboratories exhibit consistent characteristics including i) continuous expandability in the presence of FGF2 and EGF; ii) stable neuronal and glial differentiation competence; iii) characteristic transcription factor profile; iv) hindbrain specification amenable to regional patterning; v) capacity to generate functionally mature human neurons. We further show that lt-NES cells are developmentally distinct from fetal tissue-derived radial glia-like stem cells. We propose that lt-NES cells provide an interesting tool for studying human neurodevelopment and may serve as a standard system to facilitate comparative analyses of hESC and hiPSC-derived neural cells from control and diseased genetic backgrounds.
274 citations
TL;DR: This review describes the identification, characterization and isolation of NC stem and progenitor cells from different tissues in both embryo and adult organisms and discusses their specific properties and their potential application in cell-based tissue and disease-specific repair.
Abstract: Neural crest (NC) cells are a migratory cell population synonymous with vertebrate evolution. They generate a wide variety of cell and tissue types during embryonic and adult development including cartilage and bone, connective tissue, pigment and endocrine cells as well as neurons and glia amongst many others. Such incredible lineage potential combined with a limited capacity for self-renewal, which persists even into adult life, demonstrates that NC cells bear the key hallmarks of stem and progenitor cells. In this review, we describe the identification, characterization and isolation of NC stem and progenitor cells from different tissues in both embryo and adult organisms. We discuss their specific properties and their potential application in cell-based tissue and disease-specific repair.
240 citations
TL;DR: It is shown that Lgr5, a marker for adult stem cells, was expressed in a subset of supporting cells in the newborn and adult murine cochlea, and the responsiveness to Wnt of cells with a capacity for division and sensory cell formation suggests a potential route to new hair cell generation in the adult coChlea.
Abstract: Auditory hair cells are surrounded on their basolateral aspects by supporting cells, and these two cell types together constitute the sensory epithelium of the organ of Corti, which is the hearing apparatus of the ear. We show here that Lgr5, a marker for adult stem cells, was expressed in a subset of supporting cells in the newborn and adult murine cochlea. Lgr5-expressing supporting cells, sorted by flow cytometry and cultured in a single-cell suspension, compared with unsorted cells, displayed an enhanced capacity for self-renewing neurosphere formation in response to Wnt and were converted to hair cells at a higher (>10-fold) rate. The greater differentiation of hair cells in the neurosphere assay showed that Lgr5-positive cells had the capacity to act as cochlear progenitor cells, and lineage tracing confirmed that Lgr5-expressing cells accounted for the cells that formed neurospheres and differentiated to hair cells. The responsiveness to Wnt of cells with a capacity for division and sensory cell formation suggests a potential route to new hair cell generation in the adult cochlea.
231 citations
TL;DR: Current and future work, in which in vivo lineage studies and the use of injury models will complement the in vitro culture analysis, should help in unraveling the properties and function of neural crest-derived progenitors in development and disease.
226 citations
TL;DR: The use of TGF-β inhibitors with radiation to improve therapeutic response in patients with GBM is motivated by data that suggest GIC promotes effective DNA damage response and self-renewal, which creates microenvironment-mediated resistance.
Abstract: The poor prognosis of glioblastoma (GBM) routinely treated with ionizing radiation (IR) has been attributed to the relative radioresistance of glioma-initiating cells (GIC). Other studies indicate that although GIC are sensitive, the response is mediated by undefined factors in the microenvironment. GBM produce abundant transforming growth factor-β (TGF-β), a pleotropic cytokine that promotes effective DNA damage response. Consistent with this, radiation sensitivity, as measured by clonogenic assay of cultured murine (GL261) and human (U251, U87MG) glioma cell lines, increased by approximately 25% when treated with LY364947, a small-molecule inhibitor of TGF-β type I receptor kinase, before irradiation. Mice bearing GL261 flank tumors treated with 1D11, a pan-isoform TGF-β neutralizing antibody, exhibited significantly increased tumor growth delay following IR. GL261 neurosphere cultures were used to evaluate GIC. LY364947 had no effect on the primary or secondary neurosphere-forming capacity. IR decreased primary neurosphere formation by 28%, but did not reduce secondary neurosphere formation. In contrast, LY364947 treatment before IR decreased primary neurosphere formation by 75% and secondary neurosphere formation by 68%. Notably, GL261 neurospheres produced 3.7-fold more TGF-β per cell compared with conventional culture, suggesting that TGF-β production by GIC promotes effective DNA damage response and self-renewal, which creates microenvironment-mediated resistance. Consistent with this, LY364947 treatment in irradiated GL261 neurosphere-derived cells decreased DNA damage responses, H2AX and p53 phosphorylation, and induction of self-renewal signals, Notch1 and CXCR4. These data motivate the use of TGF-β inhibitors with radiation to improve therapeutic response in patients with GBM.
222 citations
TL;DR: Findings provide the first direct evidence that endogenous microglia can exert a dual and opposing influence on neural precursor cell activity within the hippocampus, and that signaling through the CX3CL1–CX3CR1 axis critically contributes toward this process.
Abstract: Exercise has been shown to positively augment adult hippocampal neurogenesis; however, the cellular and molecular pathways mediating this effect remain largely unknown. Previous studies have suggested that microglia may have the ability to differentially instruct neurogenesis in the adult brain. Here, we used transgenic Csf1r-GFP mice to investigate whether hippocampal microglia directly influence the activation of neural precursor cells. Our results revealed that an exercise-induced increase in neural precursor cell activity was mediated via endogenous microglia and abolished when these cells were selectively removed from hippocampal cultures. Conversely, microglia from the hippocampi of animals that had exercised were able to activate latent neural precursor cells when added to neurosphere preparations from sedentary mice. We also investigated the role of CX(3)CL1, a chemokine that is known to provide a more neuroprotective microglial phenotype. Intraparenchymal infusion of a blocking antibody against the CX(3)CL1 receptor, CX(3)CR1, but not control IgG, dramatically reduced the neurosphere formation frequency in mice that had exercised. While an increase in soluble CX(3)CL1 was observed following running, reduced levels of this chemokine were found in the aged brain. Lower levels of CX(3)CL1 with advancing age correlated with the natural decline in neural precursor cell activity, a state that could be partially alleviated through removal of microglia. These findings provide the first direct evidence that endogenous microglia can exert a dual and opposing influence on neural precursor cell activity within the hippocampus, and that signaling through the CX(3)CL1-CX(3)CR1 axis critically contributes toward this process.
187 citations
TL;DR: These findings reveal, for the first time, that the tumor suppressor activity of miR-124 could be partly due to its inhibitory effects on glioma stem-like traits and invasiveness through SNAI2.
172 citations
TL;DR: An increase in the expression of the autophagy genes Atg7, Becn1, Ambra1 and LC3 in vivo in the mouse embryonic olfactory bulb during the initial period of neuronal differentiation at E15.5, along with a parallel increase in neuronal markers is reported, revealing new roles for autophagic-related molecules Atg5 and Ambra 1 during early neuronal differentiation of stem/progenitor cells.
Abstract: Neuroepithelial cells undergoing differentiation efficiently remodel their cytoskeleton and shape in an energy-consuming process. The capacity of autophagy to recycle cellular components and provide energy could fulfill these requirements, thus supporting differentiation. However, little is known regarding the role of basal autophagy in neural differentiation. Here we report an increase in the expression of the autophagy genes Atg7, Becn1, Ambra1 and LC3 in vivo in the mouse embryonic olfactory bulb (OB) during the initial period of neuronal differentiation at E15.5, along with a parallel increase in neuronal markers. In addition, we observed an increase in LC3 lipidation and autophagic flux during neuronal differentiation in cultured OB-derived stem/progenitor cells. Pharmacological inhibition of autophagy with 3-MA or wortmannin markedly decreased neurogenesis. These observations were supported by similar findings in two autophagy-deficient genetic models. In Ambra1 loss-of-function homozygous mice (gt/gt) the expression of several neural markers was decreased in the OB at E13.5 in vivo. In vitro, Ambra1 haploinsufficient cells developed as small neurospheres with an impaired capacity for neuronal generation. The addition of methylpyruvate during stem/progenitor cell differentiation in culture largely reversed the inhibition of neurogenesis induced by either 3-MA or Ambra1 haploinsufficiency, suggesting that neural stem/progenitor cells activate autophagy to fulfill their high energy demands. Further supporting the role of autophagy for neuronal differentiation Atg5-null OB cells differentiating in culture displayed decreased TuJ1 levels and lower number of cells with neurites. These results reveal new roles for autophagy-related molecules Atg5 and Ambra1 during early neuronal differentiation of stem/progenitor cells.
TL;DR: The results indicate that Rad-NSCs are unlikely to be suitable for neuronal replacement in the absence of genetic or epigenetic modification.
Abstract: In response to stroke, subpopulations of cortical reactive astrocytes proliferate and express proteins commonly associated with neural stem/progenitor cells such as glial fibrillary acidic protein (GFAP) and Nestin To examine the stem cell-related properties of cortical reactive astrocytes after injury, we generated GFAP-CreERTM;tdRFP mice to permanently label reactive astrocytes We isolated cells from the cortical peri-infarct area 3 d after stroke, and cultured them in neural stem cell medium containing epidermal growth factor and basic fibroblast growth factor We observed tdRFP-positive neural spheres in culture, suggestive of tdRFP-positive reactive astrocyte-derived neural stem/progenitor cells (Rad-NSCs) Cultured Rad-NSCs self-renewed and differentiated into neurons, astrocytes, and oligodendrocytes Pharmacological inhibition and conditional knock-out mouse studies showed that Presenilin 1 and Notch 1 controlled neural sphere formation by Rad-NSCs after stroke To examine the self-renewal and differentiation potential of Rad-NSCs in vivo, Rad-NSCs were transplanted into embryonic, neonatal, and adult mouse brains Transplanted Rad-NSCs were observed to persist in the subventricular zone and secondary Rad-NSCs were isolated from the host brain 28 d after transplantation In contrast with neurogenic postnatal day 4 NSCs and adult NSCs from the subventricular zone, transplanted Rad-NSCs differentiated into astrocytes and oligodendrocytes, but not neurons, demonstrating that Rad-NSCs had restricted differentiation in vivo Our results indicate that Rad-NSCs are unlikely to be suitable for neuronal replacement in the absence of genetic or epigenetic modification
TL;DR: BT142 is the first brain tumor cell line with an endogenous R132H IDH1 mutation and detectable 2-HG production both in vitro and in vivo, which provides a unique model for studying the biology of IDH 1-mutant glioma and in vitro validation of compounds targetingIDH1-mutants cells.
Abstract: Somatic mutations in the catalytic domain of isocitrate dehydrogenase (IDH) 1/2 and accumulation of the oncometabolite 2-hydroxyglutarate (2-HG) appear to be among the earliest events in gliomagenesis and may contribute to malignant transformation. The lack of cell lines with endogenous mutations has been one of the major challenges in studying IDH1/2-mutant glioma and developing novel therapeutics for these tumors. Here, we describe the isolation of a glioma brain tumor stem cell line (BT142) with an endogenous R132H mutation in IDH1, aggressive tumor-initiating capacity, and 2-HG production. The neurosphere culture method was used to establish a brain tumor stem cell line from an IDH1-mutant anaplastic oligoastrocytoma sample, and an orthotopic xenograft system was developed to allow its rapid expansion. Production of 2-HG by glioma cells with endogenous IDH1 mutations was confirmed by mass spectrometry. BT142 retained an endogenous R132H IDH1 mutation in culture and possessed aggressive tumor-initiating capacity, allowing it to be readily propagated in orthotopic xenografts of nonobese diabetic/severe combined immune deficiency (NOD SCID) mice. Endogenous 2-HG production by BT142 was detectable in both cell culture medium and xenograft animal serum. BT142 is the first brain tumor cell line with an endogenous IDH1 mutation and detectable 2-HG production both in vitro and in vivo, which thus provides a unique model for studying the biology of IDH1-mutant glioma and in vivo validation of compounds targeting IDH1-mutant cells.
TL;DR: It was shown that NC cells could be efficiently induced from hESCs by a combination of growth factors in medium conditioned on stromal cells and that NC stem cells (NCSCs) could be purified by p75 using fluorescence‐activated cell sorting (FACS), the first report on myelination by hESC‐ or iPSC‐derived Schwann cells.
Abstract: The neural crest (NC) is a transient, multipotent, migratory cell population unique to vertebrates that gives rise to diverse cell lineages. Much of our knowledge of NC development comes from studies of organisms such as chicken and zebrafish because human NC is difficult to obtain because of its transient nature and the limited availability of human fetal cells. Here we examined the process of NC induction from human pluripotent stem cells, including human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). We showed that NC cells could be efficiently induced from hESCs by a combination of growth factors in medium conditioned on stromal cells and that NC stem cells (NCSCs) could be purified by p75 using fluorescence-activated cell sorting (FACS). FACS-isolated NCSCs could be propagated in vitro in five passages and cryopreserved while maintaining NCSC identity characterized by the expression of a panel of NC markers such as p75, Sox9, Sox10, CD44, and HNK1. In vitro-expanded NCSCs were able to differentiate into neurons and glia (Schwann cells) of the peripheral nervous system, as well as mesenchymal derivatives. hESC-derived NCSCs appeared to behave similarly to endogenous embryonic NC cells when injected in chicken embryos. Using a defined medium, we were able to generate and propagate a nearly pure population of Schwann cells that uniformly expressed glial fibrillary acidic protein, S100, and p75. Schwann cells generated by our protocol myelinated rat dorsal root ganglia neurons in vitro. To our knowledge, this is the first report on myelination by hESC- or iPSC-derived Schwann cells.
TL;DR: Dedifferentiation of human cortical astrocytes is shown into the neural stem/progenitor phenotype to obtain progenitor and mature cells with a neural fate and methylation analysis showed that epigenetic modifications underlie this process.
TL;DR: It is suggested that CD90 is not only a potential prognostic marker for high-grade gliomas but also a marker for CSCs withingliomas, and it resides within endothelial niche and may also play a critical role in the generation of tumor vasculatures via differentiation into endothelial cells.
TL;DR: It is demonstrated that Sertoli cells derived from mesoderm can be directly converted into a multipotent state that possesses neural stem/progenitor cell properties and is capable of self-renewal and differentiating into glia and electrophysiologically functional neurons.
Abstract: Multipotent neural stem/progenitor cells hold great promise for cell therapy. The reprogramming of fibroblasts to induced pluripotent stem cells as well as mature neurons suggests a possibility to convert a terminally differentiated somatic cell into a multipotent state without first establishing pluripotency. Here, we demonstrate that Sertoli cells derived from mesoderm can be directly converted into a multipotent state that possesses neural stem/progenitor cell properties. The induced neural stem/progenitor cells (iNSCs) express multiple NSC-specific markers, exhibit a global gene-expression profile similar to normal NSCs, and are capable of self-renewal and differentiating into glia and electrophysiologically functional neurons. iNSC-derived neurons stain positive for tyrosine hydroxylase (TH), γ-aminobutyric acid, and choline acetyltransferase. In addition, iNSCs can survive and generate synapses following transplantation into the dentate gyrus. Generation of iNSCs may have important implications for disease modeling and regenerative medicine.
TL;DR: Astrocytes negatively regulate neurogenesis through the Notch pathway, and endocytosis of Notch ligand Jagged1 in astroCytes and Notch signaling from astrocyte to neural stem/progenitor cells depends on the intermediate filament proteins GFAP and vimentin.
Abstract: Adult neurogenesis is regulated by a number of cellular players within the neurogenic niche. Astrocytes participate actively in brain development, regulation of the mature central nervous system (CNS), and brain plasticity. They are important regulators of the local environment in adult neurogenic niches through the secretion of diffusible morphogenic factors, such as Wnts. Astrocytes control the neurogenic niche also through membrane-associated factors, however, the identity of these factors and the mechanisms involved are largely unknown. In this study, we sought to determine the mechanisms underlying our earlier finding of increased neuronal differentiation of neural progenitor cells when cocultured with astrocytes lacking glial fibrillary acidic protein (GFAP) and vimentin (GFAP−/−Vim−/−). We used primary astrocyte and neurosphere cocultures to demonstrate that astrocytes inhibit neuronal differentiation through a cell–cell contact. GFAP−/−Vim−/− astrocytes showed reduced endocytosis of Notch ligand Jagged1, reduced Notch signaling, and increased neuronal differentiation of neurosphere cultures. This effect of GFAP−/−Vim−/− astrocytes was abrogated in the presence of immobilized Jagged1 in a manner dependent on the activity of γ-secretase. Finally, we used GFAP−/−Vim−/− mice to show that in the absence of GFAP and vimentin, hippocampal neurogenesis under basal conditions as well as after injury is increased. We conclude that astrocytes negatively regulate neurogenesis through the Notch pathway, and endocytosis of Notch ligand Jagged1 in astrocytes and Notch signaling from astrocytes to neural stem/progenitor cells depends on the intermediate filament proteins GFAP and vimentin. STEM Cells2012;30:2320–2329
TL;DR: It is found that expression of the MET oncogene was associated with neurospheres expressing the gene signature of mesenchymal and proneural subtypes of glioblastoma and is a candidate target for identification and therapy of a subset of glooblastomas.
Abstract: The existence of treatment-resistant cancer stem cells contributes to the aggressive phenotype of glioblastoma. However, the molecular alterations that drive stem cell proliferation in these tumors remain unknown. In this study, we found that expression of the MET oncogene was associated with neurospheres expressing the gene signature of mesenchymal and proneural subtypes of glioblastoma. Met expression was almost absent from neurospheres expressing the signature of the classical subtype and was mutually exclusive with amplification and expression of the EGF receptor (EGFR) gene. Met-positive and Met-negative neurospheres displayed distinct growth factor requirements, differentiated along divergent pathways, and generated tumors with distinctive features. The Met(high) subpopulation within Met-pos neurospheres displayed clonogenic potential and long-term self-renewal ability in vitro and enhanced growth kinetics in vivo. In Met(high) cells, the Met ligand HGF further sustained proliferation, clonogenicity, expression of self-renewal markers, migration, and invasion in vitro. Together, our findings suggest that Met is a functional marker of glioblastoma stem cells and a candidate target for identification and therapy of a subset of glioblastomas.
TL;DR: Given the efficacy findings and lack of adverse events in the RCS rat in combination with the results from ongoing clinical investigations, HuCNS‐SC appear to be a well‐suited candidate for cell therapy in retinal degenerative conditions.
Abstract: Stem cells derived from the human brain and grown as neurospheres (HuCNS-SC) have been shown to be effective in treating central neurodegenerative conditions in a variety of animal models. Human safety data in neurodegenerative disorders are currently being accrued. In the present study, we explored the efficacy of HuCNS-SC in a rodent model of retinal degeneration, the Royal College of Surgeons (RCS) rat, and extended our previous cell transplantation studies to include an in-depth examination of donor cell behavior and phenotype post-transplantation. As a first step, we have shown that HuCNS-SC protect host photoreceptors and preserve visual function after transplantation into the subretinal space of postnatal day 21 RCS rats. Moreover, cone photoreceptor density remained relatively constant over several months, consistent with the sustained visual acuity and luminance sensitivity functional outcomes. The novel findings of this study include the characterization and quantification of donor cell radial migration from the injection site and within the subretinal space as well as the demonstration that donor cells maintain an immature phenotype throughout the 7 months of the experiment and undergo very limited proliferation with no evidence of uncontrolled growth or tumor-like formation. Given the efficacy findings and lack of adverse events in the RCS rat in combination with the results from ongoing clinical investigations, HuCNS-SC appear to be a well-suited candidate for cell therapy in retinal degenerative conditions.
TL;DR: Corneolimbal stem/progenitor cells are significantly reduced after depletion of sensory nerves, suggesting a critical role of innervation in maintaining stem cells and/or the stem cell niche.
Abstract: Corneolimbal epithelial stem/progenitor cells are a small subpopulation of oligopotent cells located primarily in the basal epithelial layer of the limbus. They produce undifferentiated progeny with limited proliferative potential that migrate centripetally from the periphery of the cornea to replace cells desquamating during normal life.1–7 Corneal limbal stem cells reside primarily in the palisades of Vogt in a niche which maintains their “stemness” by producing a unique anatomic and functional milieu.8,9 Although the exact anatomic location of the niche is thought to be the limbus in humans, it has recently been proposed that epithelial stem/progenitor cells of equal potency are distributed throughout the entire ocular surface in other mammals.10
Detection of corneal epithelial stem cells is the object of controversy between many groups, as there is still no universal consensus over which marker(s) should be used. Side population (SP) cell detection is a commonly used method to quantify these cells. Stem cells express the SP phenotype based on the ability to efflux the DNA-binding dye Hoechst 33342.11–13 SP cells have also been identified in the hematopoietic compartments of different species, and have been isolated from various other adult tissues.12,13 These findings suggest that the SP phenotype represents a common feature of adult tissue-specific stem cells, and the same method has been used to isolate stem cells from human, rabbit, rat, and mouse limbi.14–17
The cornea is the most densely innervated tissue in the body, being 400 times more sensitive than the skin.18 Corneal nerves, in addition to their well known sensory function, help maintain the integrity of the ocular surface by releasing epitheliotrophic substances that promote corneal surface health.19 The limbus, where stem cells reside, is densely innervated; however, the role of these nerves is poorly understood.20 It has been suggested that various nerve-secreted factors and neuropeptides such as nerve growth factor (NGF), substance P, acetylcholine, and brain-derived neurotrophic factor (BDNF) could influence corneal stem cells, and play a role in maintaining epithelial integrity, and promote epithelial proliferation.19 It has also been shown that human corneal stem cells express TrkA which is a high affinity receptor for NGF.21–23 Moreover, corneal limbal stem cells grown in the presence of both epidermal growth factor (EGF) and nerve-secreted factors show the highest rate of colony expansion in vitro compared with EGF alone.24 However, the influence of corneal denervation on epithelial stem/progenitor cells has not yet been studied in vivo.
The purpose of the present study was to elucidate the relationship between corneal stem/progenitor cells and trigeminal nerves in vivo using a mouse model of denervated cornea. Herein, we hypothesize that corneal stem/progenitor cell survival and/or function is dependent on intact corneal innervation. Our data demonstrate that sensory nerve deprivation of cornea affects stem cell homeostasis and lead to a significant decrease in both the frequency and the function of corneolimbal stem/progenitor cells.
TL;DR: A novel, readily accessible source of multipotent human NCSCs for potential cell-replacement therapy is described and a separation strategy based on magnetic cell sorting of p75(NTR) positive ITSCs that formed larger neurospheres and proliferated faster than p75-NTR negative ITSCs is developed.
Abstract: Adult human neural crest-derived stem cells (NCSCs) are of extraordinary high plasticity and promising candidates for the use in regenerative medicine Here we describe for the first time a novel neural crest-derived stem cell population within the respiratory epithelium of human adult inferior turbinate In contrast to superior and middle turbinates, high amounts of source material could be isolated from human inferior turbinates Using minimally-invasive surgery methods isolation is efficient even in older patients
Within their endogenous niche, inferior turbinate stem cells (ITSCs) expressed high levels of nestin, p75NTR, and S100 Immunoelectron microscopy using anti-p75 antibodies displayed that ITSCs are of glial origin and closely related to nonmyelinating Schwann cells Cultivated ITSCs were positive for nestin and S100 and the neural crest markers Slug and SOX10 Whole genome microarray analysis showed pronounced differences to human ES cells in respect to pluripotency markers OCT4, SOX2, LIN28, and NANOG, whereas expression of WDR5, KLF4, and c-MYC was nearly similar ITSCs were able to differentiate into cells with neuro-ectodermal and mesodermal phenotype Additionally ITSCs are able to survive and perform neural crest typical chain migration in vivo when transplanted into chicken embryos However ITSCs do not form teratomas in severe combined immunodeficient mice Finally, we developed a separation strategy based on magnetic cell sorting of p75NTR positive ITSCs that formed larger neurospheres and proliferated faster than p75NTR negative ITSCs Taken together our study describes a novel, readily accessible source of multipotent human NCSCs for potential cell-replacement therapy
TL;DR: It is shown that mammalian target of rapamycin (mTOR) signaling is pivotal in determining proliferation versus quiescence in the adult forebrain neural stem cell (NSC) niche, and mTORC1 activity and progenitor proliferation decline within the quiescent NSC niche of the aging brain.
Abstract: Adult forebrain neurogenesis is dynamically regulated. Multiple families of niche-derived cues have been implicated in this regulation, but the precise roles of key intracellular signaling pathways remain vaguely defined. Here, we show that mammalian target of rapamycin (mTOR) signaling is pivotal in determining proliferation versus quiescence in the adult forebrain neural stem cell (NSC) niche. Within this niche, mTOR complex-1 (mTORC1) activation displays stage specificity, occurring in transiently amplifying (TA) progenitor cells but not in GFAP+ stem cells. Inhibiting mTORC1 depletes the TA progenitor pool in vivo and suppresses epidermal growth factor (EGF)-induced proliferation within neurosphere cultures. Interestingly, mTORC1 inhibition induces a quiescence-like phenotype that is reversible. Likewise, mTORC1 activity and progenitor proliferation decline within the quiescent NSC niche of the aging brain, while EGF administration reactivates the quiescent niche in an mTORC1-dependent manner. These findings establish fundamental links between mTOR signaling, proliferation, and aging-associated quiescence in the adult forebrain NSC niche.
TL;DR: Ectopic transfection of miR-17 prolonged glioblastoma cell survival when the cells were deprived with nutrition or treated with chemotherapeutic drugs and added a new layer of functional mechanism for the well-studied miRNA miR -17.
Abstract: // Haoran Li 1,2 , and Burton B Yang 1,2 1. Sunnybrook Research Institute, Sunnybrook Health Sciences Centre, Toronto 2. Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto Correspondence: Burton B Yang, email: // Keywords : microRNA, stem cell, miR-17, stress response, drug resistance Received : December 28, 2012, Accepted : December 31, 2012, Published : December 31, 2012 Abstract Tumor development not only destroys the homeostasis of local tissues but also the whole body, and thus the tumor cells have to face the body’s defense system, a shortage of nutrition and oxygen, and chemotherapeutic drug treatment. In response to these stresses, tumor cells often alter gene expression and microRNA levels to facilitate survival. We have demonstrated that glioblastoma cells deprived of nutrition or treated with chemotherapeutics drugs expressed increased levels of miR-17. Ectopic transfection of miR-17 prolonged glioblastoma cell survival when the cells were deprived with nutrition or treated with chemotherapeutic drugs. Expression of miR-17 also promoted cell motility, invasion, and tube-like structure formation. We found that these phenotypes were the results of miR-17 targeting PTEN. As a consequence, HIF1α and VEGF were up-regulated. Ectopic expression of miR-17 was found to facilitate enrichment of stem-like tumor cells, since the cells became drug-resistant, showed increased capacity to form colonies and neurospheres, and expressed higher levels of CD133, a phenotype similar to ectopic expression of HIF1α. To further confirm the phenotypic property of stem cells, we demonstrated that glioblastoma cells transfected with miR-17 proliferated slower in different nutritional conditions by facilitating more cells staying in the G1 phase than the control cells. Finally, we demonstrated that miR-17 could repress MDM2 levels, resulting in decreased cell proliferation and drug-resistance. Our results added a new layer of functional mechanism for the well-studied miRNA miR-17.
TL;DR: The present results emphasise the consequences of an inflammatory environment during NPC development, and indicate that strategies to inhibit IL-1β signalling may be necessary to facilitate effective cell transplantation approaches or in conditions where endogenous hippocampal neurogenesis is impaired.
TL;DR: It is found that the seed sequence of microRNA‐9 (miR‐9) is complementary to the 3′‐UTR sequence of Hes1 mRNA, which regulates the proliferation and differentiation of neural stem cells by controlling the dynamics of Hes 1 expression in the developing brain.
Abstract: Earlier studies show that Hes1 expression is oscillatory in neural stem cells but sustained and high in the roof plate and the floor plate, and that such different dynamics of Hes1 expression (oscillatory versus sustained) regulate different proliferation and differentiation characteristics of these cells (active in neural stem cells but rather dormant in roof/floor plate cells). The mechanism of how different dynamics of Hes1 expression is controlled remains to be determined. Here, we found that the seed sequence of microRNA-9 (miR-9) is complementary to the 3'-UTR sequence of Hes1 mRNA. MiR-9 is highly expressed in the ventricular zone of the developing brain, which contains neural stem cells, but it is not expressed in the roof plate or the floor plate. Over-expression of miR-9 negatively regulates the Hes1 protein expression by interacting with the 3'-UTR of Hes1 mRNA, thereby inducing cell cycle exit and neuronal differentiation. Conversely, knockdown of miR-9 inhibits neuronal differentiation. Furthermore, knockdown of miR-9 inhibits the oscillatory expression of Hes1 mRNA in neural stem cells. These results indicate that miR-9 regulates the proliferation and differentiation of neural stem cells by controlling the dynamics of Hes1 expression in the developing brain.
TL;DR: It is revealed that BDNF might contribute to proliferation and neuronal and oligodendrocytic differentiation of NSCs in vitro, most possibly by triggering the Wnt/β‐catenin signaling pathway.
Abstract: Brain-derived neurotrophic factor (BDNF) has critical functions in promoting survival, expansion, and differentiation of neural stem cells (NSCs), but its downstream regulation mechanism is still not fully understood. The role of BDNF in proliferation and differentiation of NSCs through Wnt/β-catenin signaling was studied via cell culture of cortical NSCs, Western blotting, immunocytochemistry, and TOPgal (Wnt reporter) analysis in mice. First, BDNF stimulated NSC proliferation dose dependently in cultured neurospheres that exhibited BrdU incorporation and neuronal and glial differentiation abilities. Second, BDNF effectively enhanced cell commitment to neuronal and oligodendrocytic fates, as indicated by increased differentiation marker Tuj-1 (neuronal marker), CNPase (oligodendrocyte marker), and neuronal process extension. Third, BDNF upregulated expression of Wnt/β-catenin signaling (Wnt1 and free β-catenin) molecules. Moreover, these promoting effects were significantly inhibited by application of IWR1, a Wnt signaling-specific blocker in culture. The TOPgal mouse experiment further confirmed BDNF-triggered Wnt signaling activation by β-gal labeling. Finally, an MEK inhibition experiment showed a mediating role of the microtubule-associated protein kinase pathway in BDNF-triggered Wnt/β-catenin signaling cascades. This study overall has revealed that BDNF might contribute to proliferation and neuronal and oligodendrocytic differentiation of NSCs in vitro, most possibly by triggering the Wnt/β-catenin signaling pathway. Nevertheless, determining the exact cross-talk points at which BDNF might stimulate Wnt/β-catenin signaling pathway in NSC activity requires further investigation.
TL;DR: This review discusses the sources of adult neural crest-derived stem cells (NCSCs) derived from the cranium, as well as their differentiation potential and expression of key stem cell markers, and the expression of marker genes associated with embryonic stem cells.
Abstract: During the process of development, neural crest cells migrate out from their niche between the newly formed ectoderm and the neural tube. Thereafter, they give rise not only to ectodermal cell types, but also to mesodermal cell types. Cell types with neural crest ancestry consequently comprise a number of specialized varieties, such as ectodermal neurons, melanocytes and Schwann cells, as well as mesodermal osteoblasts, adipocytes and smooth muscle cells. Numerous recent studies suggest that stem cells with a neural crest origin persist into adulthood, especially within the mammalian craniofacial compartment. This review discusses the sources of adult neural crest-derived stem cells (NCSCs) derived from the cranium, as well as their differentiation potential and expression of key stem cell markers. Furthermore, the expression of marker genes associated with embryonic stem cells and the issue of multi- versus pluripotency of adult NCSCs is reviewed. Stringent tests are proposed, which, if performed, are anticipated to clarify the issue of adult NCSC potency. Finally, current pre-clinical and clinical data are discussed in light of the clinical impact of adult NCSCs.
TL;DR: It is proposed that the neural stem cell and early neuroblast divisions drive dentate gyrus neurogenesis and not the amplification of type-2a intermediate progenitors as was previously thought.
Abstract: Neural stem/progenitor cells generate neurons in the adult hippocampus. Neural stem cells produce transient intermediate progenitors (type-2 cells), which generate neuroblasts (type-3 cells) that exit the cell cycle, and differentiate into neurons. The precise dynamics of neuron production from the neural stem cells remains controversial. Here we lineage trace Notch-dependent neural stem cells in the dentate gyrus and show that over 7-21 days, the progeny of the neural stem cells progress through an Ascl1(high) intermediate stage (type-2a) to neuroblasts. However, contrary to predictions, this Ascl1(high) population is not an amplifying intermediate, but it differentiates into mitotic Tbr2(+) early neuroblasts, which in turn expand the lineage. After 100 days, the majority of the neural stem cell progeny are neuroblasts or postmitotic neurons. Hence, the neural stem cells require many weeks to generate differentiated neurons. On the basis of this temporal delay in differentiation and population expansion, we propose that the neural stem cell and early neuroblast divisions drive dentate gyrus neurogenesis and not the amplification of type-2a intermediate progenitors as was previously thought.
TL;DR: CDNA microarray analysis identified a number of relevant genes for glioma biology that are differentially expressed in adherent cells and neurospheres derived from the same tumor and found that down-regulation of FABP7 expression in NS by small interfering RNAs significantly reduced cell proliferation and migration.
Abstract: Glioblastoma multiforme (GBM) is among the most deadly cancers. A number of studies suggest that a fraction of tumor cells with stem cell features (Glioma Stem-like Cells, GSC) might be responsible for GBM recurrence and aggressiveness. GSC similarly to normal neural stem cells, can form neurospheres (NS) in vitro, and seem to mirror the genetic features of the original tumor better than glioma cells growing adherently in the presence of serum. Using cDNA microarray analysis we identified a number of relevant genes for glioma biology that are differentially expressed in adherent cells and neurospheres derived from the same tumor. Fatty acid-binding protein 7 (FABP7) was identified as one of the most highly expressed genes in NS compared to their adherent counterpart. We found that down-regulation of FABP7 expression in NS by small interfering RNAs significantly reduced cell proliferation and migration. We also evaluated the potential involvement of FABP7 in response to radiotherapy, as this treatment may cause increased tumor infiltration. Migration of irradiated NS was associated to increased expression of FABP7. In agreement with this, in vivo reduced tumorigenicity of GBM cells with down-regulated expression of FABP7 was associated to decreased expression of the migration marker doublecortin. Notably, we observed that PPAR antagonists affect FABP7 expression and decrease the migration capability of NS after irradiation. As a whole, the data emphasize the role of FABP7 expression in GBM migration and provide translational hints on the timing of treatment with anti-FABP7 agents like PPAR antagonists during GBM evolution.