Tabla espec. clave
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|Ch, H, M, R||ICC, IHC||M||Purified||Monoclonal Antibody|
|Presentation||Purified immunoglobulin in 0.05M Potassium phosphate buffer, pH 8.0 with 0.3M NaCl and 0.05% sodium azide.|
|Safety Information according to GHS|
|Material Size||50 µg|
Ficha datos de seguridad (MSDS)
|Cargo||Número de lote|
|Anti-O4, clone 81 - 2138970||2138970|
|Anti-O4, clone 81 - 2455696||2455696|
|Anti-O4, clone 81 - 2141806||2141806|
|Anti-O4, clone 81 - 2266482||2266482|
|Anti-O4, clone 81 - 2289146||2289146|
|Anti-O4, clone 81 -2517799||2517799|
|Anti-O4, clone 81 -2571196||2571196|
|Anti-O4, clone 81 -2594292||2594292|
|Anti-O4, clone 81 -2691341||2691341|
|Anti-O4, clone 81 -2727278||2727278|
|Anti-O4, clone 81 -2779497||2779497|
|Anti-p75 Low Affinity Nerve Growth -2568539||2568539|
|MOUSE ANTI-OLIGODENDROCYTE MARKER O4 MONOCLONAL ANTIBODY - 1994372||1994372|
Referencias bibliográficas | 92 Disponible | Ver todas las referencias
|Visión general referencias||Aplicación||Especie||Pub Med ID|
|Analysing human neural stem cell ontogeny by consecutive isolation of Notch active neural progenitors. |
Edri, R; Yaffe, Y; Ziller, MJ; Mutukula, N; Volkman, R; David, E; Jacob-Hirsch, J; Malcov, H; Levy, C; Rechavi, G; Gat-Viks, I; Meissner, A; Elkabetz, Y
Nature communications 6 6500 2015
Decoding heterogeneity of pluripotent stem cell (PSC)-derived neural progeny is fundamental for revealing the origin of diverse progenitors, for defining their lineages, and for identifying fate determinants driving transition through distinct potencies. Here we have prospectively isolated consecutively appearing PSC-derived primary progenitors based on their Notch activation state. We first isolate early neuroepithelial cells and show their broad Notch-dependent developmental and proliferative potential. Neuroepithelial cells further yield successive Notch-dependent functional primary progenitors, from early and midneurogenic radial glia and their derived basal progenitors, to gliogenic radial glia and adult-like neural progenitors, together recapitulating hallmarks of neural stem cell (NSC) ontogeny. Gene expression profiling reveals dynamic stage-specific transcriptional patterns that may link development of distinct progenitor identities through Notch activation. Our observations provide a platform for characterization and manipulation of distinct progenitor cell types amenable for developing streamlined neural lineage specification paradigms for modelling development in health and disease.
|The adhesion G protein-coupled receptor GPR56 is a cell-autonomous regulator of oligodendrocyte development. |
Giera, S; Deng, Y; Luo, R; Ackerman, SD; Mogha, A; Monk, KR; Ying, Y; Jeong, SJ; Makinodan, M; Bialas, AR; Chang, BS; Stevens, B; Corfas, G; Piao, X
Nature communications 6 6121 2015
Mutations in GPR56, a member of the adhesion G protein-coupled receptor family, cause a human brain malformation called bilateral frontoparietal polymicrogyria (BFPP). Magnetic resonance imaging (MRI) of BFPP brains reveals myelination defects in addition to brain malformation. However, the cellular role of GPR56 in oligodendrocyte development remains unknown. Here, we demonstrate that loss of Gpr56 leads to hypomyelination of the central nervous system in mice. GPR56 levels are abundant throughout early stages of oligodendrocyte development, but are downregulated in myelinating oligodendrocytes. Gpr56-knockout mice manifest with decreased oligodendrocyte precursor cell (OPC) proliferation and diminished levels of active RhoA, leading to fewer mature oligodendrocytes and a reduced number of myelinated axons in the corpus callosum and optic nerves. Conditional ablation of Gpr56 in OPCs leads to a reduced number of mature oligodendrocytes as seen in constitutive knockout of Gpr56. Together, our data define GPR56 as a cell-autonomous regulator of oligodendrocyte development.
|The adhesion GPCR Gpr56 regulates oligodendrocyte development via interactions with Gα12/13 and RhoA. |
Ackerman, SD; Garcia, C; Piao, X; Gutmann, DH; Monk, KR
Nature communications 6 6122 2015
In the vertebrate central nervous system, myelinating oligodendrocytes are postmitotic and derive from proliferative oligodendrocyte precursor cells (OPCs). The molecular mechanisms that govern oligodendrocyte development are incompletely understood, but recent studies implicate the adhesion class of G protein-coupled receptors (aGPCRs) as important regulators of myelination. Here, we use zebrafish and mouse models to dissect the function of the aGPCR Gpr56 in oligodendrocyte development. We show that gpr56 is expressed during early stages of oligodendrocyte development. In addition, we observe a significant reduction of mature oligodendrocyte number and myelinated axons in gpr56 zebrafish mutants. This reduction results from decreased OPC proliferation, rather than increased cell death or altered neural precursor differentiation potential. Finally, we show that these functions are mediated by Gα12/13 proteins and Rho activation. Together, our data establish Gpr56 as a regulator of oligodendrocyte development.
|Non-aggregating tau phosphorylation by cyclin-dependent kinase 5 contributes to motor neuron degeneration in spinal muscular atrophy. |
Miller, N; Feng, Z; Edens, BM; Yang, B; Shi, H; Sze, CC; Hong, BT; Su, SC; Cantu, JA; Topczewski, J; Crawford, TO; Ko, CP; Sumner, CJ; Ma, L; Ma, YC
The Journal of neuroscience : the official journal of the Society for Neuroscience 35 6038-50 2015
Mechanisms underlying motor neuron degeneration in spinal muscular atrophy (SMA), the leading inherited cause of infant mortality, remain largely unknown. Many studies have established the importance of hyperphosphorylation of the microtubule-associated protein tau in various neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. However, tau phosphorylation in SMA pathogenesis has yet to be investigated. Here we show that tau phosphorylation on serine 202 (S202) and threonine 205 (T205) is increased significantly in SMA motor neurons using two SMA mouse models and human SMA patient spinal cord samples. Interestingly, phosphorylated tau does not form aggregates in motor neurons or neuromuscular junctions (NMJs), even at late stages of SMA disease, distinguishing it from other tauopathies. Hyperphosphorylation of tau on S202 and T205 is mediated by cyclin-dependent kinase 5 (Cdk5) in SMA disease condition, because tau phosphorylation at these sites is significantly reduced in Cdk5 knock-out mice; genetic knock-out of Cdk5 activating subunit p35 in an SMA mouse model also leads to reduced tau phosphorylation on S202 and T205 in the SMA;p35(-/-) compound mutant mice. In addition, expression of the phosphorylation-deficient tauS202A,T205A mutant alleviates motor neuron defects in a zebrafish SMA model in vivo and mouse motor neuron degeneration in culture, whereas expression of phosphorylation-mimetic tauS202E,T205E promotes motor neuron defects. More importantly, genetic knock-out of tau in SMA mice rescues synapse stripping on motor neurons, NMJ denervation, and motor neuron degeneration in vivo. Altogether, our findings suggest a novel mechanism for SMA pathogenesis in which hyperphosphorylation of non-aggregating tau by Cdk5 contributes to motor neuron degeneration.
|Targeting endothelial junctional adhesion molecule-A/ EPAC/ Rap-1 axis as a novel strategy to increase stem cell engraftment in dystrophic muscles. |
Giannotta, Monica, et al.
EMBO Mol Med, 6: 239-58 (2014) 2014
Muscular dystrophies are severe genetic diseases for which no efficacious therapies exist. Experimental clinical treatments include intra-arterial administration of vessel-associated stem cells, called mesoangioblasts (MABs). However, one of the limitations of this approach is the relatively low number of cells that engraft the diseased tissue, due, at least in part, to the sub-optimal efficiency of extravasation, whose mechanisms for MAB are unknown. Leukocytes emigrate into the inflamed tissues by crossing endothelial cell-to-cell junctions and junctional proteins direct and control leukocyte diapedesis. Here, we identify the endothelial junctional protein JAM-A as a key regulator of MAB extravasation. We show that JAM-A gene inactivation and JAM-A blocking antibodies strongly enhance MAB engraftment in dystrophic muscle. In the absence of JAM-A, the exchange factors EPAC-1 and 2 are down-regulated, which prevents the activation of the small GTPase Rap-1. As a consequence, junction tightening is reduced, allowing MAB diapedesis. Notably, pharmacological inhibition of Rap-1 increases MAB engraftment in dystrophic muscle, which results into a significant improvement of muscle function offering a novel strategy for stem cell-based therapies.
|Systemic injection of neural stem/progenitor cells in mice with chronic EAE. |
Donegà, M; Giusto, E; Cossetti, C; Schaeffer, J; Pluchino, S
Journal of visualized experiments : JoVE 2014
Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative neurology. This directive has arisen from the extensive evidence that brain repair is achieved after focal or systemic NPC transplantation in several preclinical models of neurological diseases. These experimental data have identified the cell delivery route as one of the main hurdles of restorative stem cell therapies for brain diseases that requires urgent assessment. Intraparenchymal stem cell grafting represents a logical approach to those pathologies characterized by isolated and accessible brain lesions such as spinal cord injuries and Parkinson's disease. Unfortunately, this principle is poorly applicable to conditions characterized by a multifocal, inflammatory and disseminated (both in time and space) nature, including multiple sclerosis (MS). As such, brain targeting by systemic NPC delivery has become a low invasive and therapeutically efficacious protocol to deliver cells to the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system (CNS). This alternative method of cell delivery relies on the NPC pathotropism, specifically their innate capacity to (i) sense the environment via functional cell adhesion molecules and inflammatory cytokine and chemokine receptors; (ii) cross the leaking anatomical barriers after intravenous (i.v.) or intracerebroventricular (i.c.v.) injection; (iii) accumulate at the level of multiple perivascular site(s) of inflammatory brain and spinal cord damage; and (i.v.) exert remarkable tissue trophic and immune regulatory effects onto different host target cells in vivo. Here we describe the methods that we have developed for the i.v. and i.c.v. delivery of syngeneic NPCs in mice with experimental autoimmune encephalomyelitis (EAE), as model of chronic CNS inflammatory demyelination, and envisage the systemic stem cell delivery as a valuable technique for the selective targeting of the inflamed brain in regenerative neurology.
|Analysis of Mll1 deficiency identifies neurogenic transcriptional modules and Brn4 as a factor for direct astrocyte-to-neuron reprogramming. |
Potts, MB; Siu, JJ; Price, JD; Salinas, RD; Cho, MJ; Ramos, AD; Hahn, J; Margeta, M; Oldham, MC; Lim, DA
Neurosurgery 75 472-82; discussion 482 2014
Mixed lineage leukemia-1 (Mll1) epigenetically regulates gene expression patterns that specify cellular identity in both embryonic development and adult stem cell populations. In the adult mouse brain, multipotent neural stem cells (NSCs) in the subventricular zone generate new neurons throughout life, and Mll1 is required for this postnatal neurogenesis but not for glial cell differentiation. Analysis of Mll1-dependent transcription may identify neurogenic genes useful for the direct reprogramming of astrocytes into neurons.To identify Mll1-dependent transcriptional modules and to determine whether genes in the neurogenic modules can be used to directly reprogram astrocytes into neurons.We performed gene coexpression module analysis on microarray data from differentiating wild-type and Mll1-deleted subventricular zone NSCs. Key developmental regulators belonging to the neurogenic modules were overexpressed in Mll1-deleted cells and cultured cortical astrocytes, and cell phenotypes were analyzed by immunocytochemistry and electrophysiology.Transcriptional modules that correspond to neurogenesis were identified in wild-type NSCs. Modules related to astrocytes and oligodendrocytes were enriched in Mll1-deleted NSCs, consistent with their gliogenic potential. Overexpression of genes selected from the neurogenic modules enhanced the production of neurons from Mll1-deleted cells, and overexpression of Brn4 (Pou3f4) in nonneurogenic cortical astroglia induced their transdifferentiation into electrophysiologically active neurons.Our results demonstrate that Mll1 is required for the expression of neurogenic but not gliogenic transcriptional modules in a multipotent NSC population and further indicate that specific Mll1-dependent genes may be useful for direct reprogramming strategies.
|A phenotypic culture system for the molecular analysis of CNS myelination in the spinal cord. |
Davis, H; Gonzalez, M; Stancescu, M; Love, R; Hickman, JJ; Lambert, S
Biomaterials 35 8840-5 2014
Studies of central nervous system myelination lack defined in vitro models which would effectively dissect molecular mechanisms of myelination that contain cells of the correct phenotype. Here we describe a co-culture of purified motoneurons and oligodendrocyte progenitor cells, isolated from rat embryonic spinal cord using a combination of immunopanning techniques. This model illustrates differentiation of oligodendrocyte progenitors into fully functional mature oligodendrocytes that myelinate axons. It also illustrates a contribution of axons to the rate of oligodendrocyte maturation and myelin gene expression. The defined conditions used allow molecular analysis of distinct stages of myelination and precise manipulation of inductive cues affecting axonal-oligodendrocyte interactions. This phenotypic in vitro myelination model can provide valuable insight into our understanding of demyelinating disorders, such as multiple sclerosis and traumatic diseases such as spinal cord injury where demyelination represents a contributing factor to the pathology of the disorder.
|Pten loss in Olig2 expressing neural progenitor cells and oligodendrocytes leads to interneuron dysplasia and leukodystrophy. |
Maire, CL; Ramkissoon, S; Hayashi, M; Haidar, S; Ramkissoon, L; DiTomaso, E; Ligon, KL
Stem cells (Dayton, Ohio) 32 313-26 2014
Therapeutic modulation of phosphatidylinositol 3-kinase (PI3K)/PTEN signaling is currently being explored for multiple neurological indications including brain tumors and seizure disorders associated with cortical malformations. The effects of PI3K/PTEN signaling are highly cell context dependent but the function of this pathway in specific subsets of neural stem/progenitor cells generating oligodendroglial lineage cells has not been fully studied. To address this, we created Olig2-cre:Pten(fl/fl) mice that showed a unique pattern of Pten loss and PI3K activation in Olig2-lineage cells. Olig2-cre:Pten(fl/fl) animals progressively developed central nervous system white matter hypermyelination by 3 weeks of age leading to later onset leukodystrophy, chronic neurodegeneration, and death by 9 months. In contrast, during immediate postnatal development, oligodendroglia were unaffected but abnormal and accelerated differentiation of lateral subventricular zone stem cells produced calretinin-positive interneuron dysplasia. Neural stem cells isolated from Olig2-cre:Pten(fl/fl) mice also exhibited accelerated differentiation and proliferation into calretinin-positive interneurons and oligodendrocytes indicating such effects are cell autonomous. Opposition of the pathway by treatment of human primary neural progenitor cells (NPCs) with the PI3K inhibitor, NVP-BKM120, blocked in vitro differentiation of neurons and oligodendroglia indicating PI3K/PTEN effects on NPCs can be bidirectional. In summary, our results suggest Pten is a developmental rheostat regulating interneuron and oligodendroglial differentiation and support testing of PI3K modulating drugs as treatment for developmental and myelination disorders. However, such agents may need to be administered at ages that minimize potential effects on early stem/progenitor cell development.
|Generation of highly purified neural stem cells from human adipose-derived mesenchymal stem cells by Sox1 activation. |
Feng, N; Han, Q; Li, J; Wang, S; Li, H; Yao, X; Zhao, RC
Stem cells and development 23 515-29 2014
Neural stem cells (NSCs) are ideal candidates in stem cell-based therapy for neurodegenerative diseases. However, it is unfeasible to get enough quantity of NSCs for clinical application. Generation of NSCs from human adipose-derived mesenchymal stem cells (hAD-MSCs) will provide a solution to this problem. Currently, the differentiation of hAD-MSCs into highly purified NSCs with biological functions is rarely reported. In our study, we established a three-step NSC-inducing protocol, in which hAD-MSCs were induced to generate NSCs with high purity after sequentially cultured in the pre-inducing medium (Step1), the N2B27 medium (Step2), and the N2B27 medium supplement with basic fibroblast growth factor and epidermal growth factor (Step3). These hAD-MSC-derived NSCs (adNSCs) can form neurospheres and highly express Sox1, Pax6, Nestin, and Vimentin; the proportion was 96.1% ± 1.3%, 96.8% ± 1.7%, 96.2% ± 1.3%, and 97.2% ± 2.5%, respectively, as detected by flow cytometry. These adNSCs can further differentiate into astrocytes, oligodendrocytes, and functional neurons, which were able to generate tetrodotoxin-sensitive sodium current. Additionally, we found that the neural differentiation of hAD-MSCs were significantly suppressed by Sox1 interference, and what's more, Step1 was a key step for the following induction, probably because it was associated with the initiation and nuclear translocation of Sox1, an important transcriptional factor for neural development. Finally, we observed that bone morphogenetic protein signal was inhibited, and Wnt/β-catenin signal was activated during inducing process, and both signals were related with Sox1 expression. In conclusion, we successfully established a three-step inducing protocol to derive NSCs from hAD-MSCs with high purity by Sox1 activation. These findings might enable to acquire enough autologous transplantable NSCs for the therapy of neurodegenerative diseases in clinic.
|Differentiation of human breast-milk stem cells to neural stem cells and neurons. |
Hosseini, SM; Talaei-Khozani, T; Sani, M; Owrangi, B
Neurology research international 2014 807896 2014
Objectives. Human breast milk contains a heterogeneous population of cells that have the potential to provide a noninvasive source of cells for cell therapy in many neurodegenerative diseases without any ethical concern. The objectives of this study were to differentiate the breast milk-derived stem cells (BMDSC) toward neural stem cells and then into the neurons and neuroglia. Materials and Methods. To do this, the BMDSC were isolated from human breast milk and cultured in Dulbecco's modified Eagle medium/F12 (DMEM/F12) containing fibroblast growth factor (bFGF). The cells were then characterized by evaluation of the embryonic and stem cell markers. Then, the cells were exposed to culture medium containing 1% B27 and 2% N2 for 7-10 days followed by medium supplemented with B27, N2, bFGF 10 µg/mL, and endothelial growth factor (EGF) 20 µg/mL. Then, the sphere-forming assay was performed. The spheres were then differentiated into three neural lineages by withdrawing growth factor in the presence of 5% FBS (fetal bovine serum). The immunofluorescence was done for β-tubulin III, O4, and GFAP (glial fibrillary acidic protein). Results. The results indicated that the cells expressed both embryonic and mesenchymal stem cell (MSC) markers. They also showed neurospheres formation that was nestin-positive. The cells were also differentiated into all three neural lineages. Conclusion. The BMDSC can behave in the same way with neural stem cells. They were differentiated into oligodendrocytes, and astrocytes as well as neurons.
|Treatment with Anti-EGF Ab Ameliorates Experimental Autoimmune Encephalomyelitis via Induction of Neurogenesis and Oligodendrogenesis. |
Amir-Levy, Y; Mausner-Fainberg, K; Karni, A
Multiple sclerosis international 2014 926134 2014
Background. The neural stem cells (NSCs) migrate to the damaged sites in multiple sclerosis (MS) and in experimental autoimmune encephalomyelitis (EAE). However, the differentiation into neurons or oligodendrocytes is blocked. Epidermal growth factor (EGF) stimulates NSC proliferation and mobilization to demyelinated lesions but also induces astrogenesis and glial scar. Objective. To examine the clinical and histopathological effects of EGF neutralization on EAE. Methods. EAE-induced SJL mice were intravenously treated with either anti-EGF neutralizing antibody (Ab) or isotype control or PBS. On day 9 after immunization, 3 mice of each group were daily treated for 9 days with BrdU and then sacrificed for immunohistochemical analysis. Results. Treatment with anti-EGF Ab significantly ameliorated EAE symptoms during the second relapse. Anti-EGF Ab induced a shift from BrdU(+)GFAP(+) NSCs to BrdU(+)DCX(+) neuroblasts in the subventricular zone (SVZ), increased BrdU(+)NeuN(+) neurons in the granular cell layer of the dentate gyrus, and increased BrdU(+)O4(+) oligodendrocytes in the SVZ. There was no change in the inflammatory infiltrates in response to anti-EGF Ab. Conclusions. Therapy with anti-EGF Ab ameliorates EAE via induction of neurogenesis and oligodendrogenesis. No immunosuppressive effect was found. Further investigation is needed to support these notions of beneficial effect of anti-EGF Ab in MS.
|Designing and troubleshooting immunopanning protocols for purifying neural cells. |
Cold Spring Harbor protocols 2014 1342-7 2014
Purifying and culturing cells from the central nervous system (CNS) has proved to be an incredibly powerful tool for dissecting fundamental neuron and glial properties, and especially powerful in understanding neuronal-glial interactions. In a series of detailed protocols, we have provided step-by-step instructions for purifying and culturing specific types of neurons, glia, and vascular cells from the CNS by immunopanning. This article discusses common pitfalls and errors as well as important design considerations for the immunopanning procedure.
|Effects of sleep and wake on oligodendrocytes and their precursors. |
Bellesi, M; Pfister-Genskow, M; Maret, S; Keles, S; Tononi, G; Cirelli, C
The Journal of neuroscience : the official journal of the Society for Neuroscience 33 14288-300 2013
Previous studies of differential gene expression in sleep and wake pooled transcripts from all brain cells and showed that several genes expressed at higher levels during sleep are involved in the synthesis/maintenance of membranes in general and of myelin in particular, a surprising finding given the reported slow turnover of many myelin components. Other studies showed that oligodendrocyte precursor cells (OPCs) are responsible for the formation of new myelin in both the injured and the normal adult brain, and that glutamate released from neurons, via neuron-OPC synapses, can inhibit OPC proliferation and affect their differentiation into myelin-forming oligodendrocytes. Because glutamatergic transmission is higher in wake than in sleep, we asked whether sleep and wake can affect oligodendrocytes and OPCs. Using the translating ribosome affinity purification technology combined with microarray analysis in mice, we obtained a genome-wide profiling of oligodendrocytes after sleep, spontaneous wake, and forced wake (acute sleep deprivation). We found that hundreds of transcripts being translated in oligodendrocytes are differentially expressed in sleep and wake: genes involved in phospholipid synthesis and myelination or promoting OPC proliferation are transcribed preferentially during sleep, while genes implicated in apoptosis, cellular stress response, and OPC differentiation are enriched in wake. We then confirmed through BrdU and other experiments that OPC proliferation doubles during sleep and positively correlates with time spent in REM sleep, whereas OPC differentiation is higher during wake. Thus, OPC proliferation and differentiation are not perfectly matched at any given circadian time but preferentially occur during sleep and wake, respectively.
|Administration of dexamethasone to neonatal rats induces hypomyelination and changes in the morphology of oligodendrocyte precursors. |
Kim, JW; Kim, YJ; Chang, YP
Comparative medicine 63 48-54 2013
To examine whether hypomyelination in neonatal rats might be related to apoptosis of oligodendrocyte progenitors, we administered dexamethasone (0.5 mg/kg SC) to neonatal rats on postnatal (P) days 1 through 5. Immunofluorescent staining and Western blotting for myelin basic protein (MBP) were performed on P14. Morphologic changes associated with apoptotic death of oligodendrocyte progenitors were assessed by using immunofluorescent staining on P5 of surface markers present at different developmental stages of oligodendrocyte progenitors (O4 and O1) and by double-staining with terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick end-labeling (TUNEL) and O4 or O1. Administration of dexamethasone to neonatal rats reduced the expression of MBP in the white matter by P14. In addition, dexamethasone reduced the expression of O4-positive cells, presumably preoligodendrocytes, in the corpus callosum and induced degenerative changes, such as cytoplasmic condensation and fragmented, tortuous processes, in oligodendrocyte progenitors, and increased the number of TUNEL-positive pyknotic nuclei of oligodendrocyte progenitors. These findings suggest that the dexamethasone-induced decreased expression of MBP in the cerebral hemispheres of the neonatal rats is due to apoptotic degeneration of oligodendrocyte progenitors. Administration of dexamethasone during the critical period of brain development may increase the risk of apoptosis in oligodendrocyte progenitors, subsequently resulting in hypomyelination.
|Murine neural stem cells model Hunter disease in vitro: glial cell-mediated neurodegeneration as a possible mechanism involved. |
Fusar Poli, E; Zalfa, C; D'Avanzo, F; Tomanin, R; Carlessi, L; Bossi, M; Nodari, LR; Binda, E; Marmiroli, P; Scarpa, M; Delia, D; Vescovi, AL; De Filippis, L
Cell death & disease 4 e906 2013
Mucopolysaccharidosis type II (MPSII or Hunter Syndrome) is a lysosomal storage disorder caused by the deficit of iduronate 2-sulfatase (IDS) activity and characterized by progressive systemic and neurological impairment. As the early mechanisms leading to neuronal degeneration remain elusive, we chose to examine the properties of neural stem cells (NSCs) isolated from an animal model of the disease in order to evaluate whether their neurogenic potential could be used to recapitulate the early phases of neurogenesis in the brain of Hunter disease patients. Experiments here reported show that NSCs derived from the subventricular zone (SVZ) of early symptomatic IDS-knockout (IDS-ko) mouse retained self-renewal capacity in vitro, but differentiated earlier than wild-type (wt) cells, displaying an evident lysosomal aggregation in oligodendroglial and astroglial cells. Consistently, the SVZ of IDS-ko mice appeared similar to the wt SVZ, whereas the cortex and striatum presented a disorganized neuronal pattern together with a significant increase of glial apoptotic cells, suggesting that glial degeneration likely precedes neuronal demise. Interestingly, a very similar pattern was observed in the brain cortex of a Hunter patient. These observations both in vitro, in our model, and in vivo suggest that IDS deficit seems to affect the late phases of neurogenesis and/or the survival of mature cells rather than NSC self-renewal. In particular, platelet-derived growth factor receptor-α-positive (PDGFR-α+) glial progenitors appeared reduced in both the IDS-ko NSCs and in the IDS-ko mouse and human Hunter brains, compared with the respective healthy controls. Treatment of mutant NSCs with IDS or PDGF throughout differentiation was able to increase the number of PDGFR-α+ cells and to reduce that of apoptotic cells to levels comparable to wt. This evidence supports IDS-ko NSCs as a reliable in vitro model of the disease, and suggests the rescue of PDGFR-α+ glial cells as a therapeutic strategy to prevent neuronal degeneration.
|Loss of Usp9x disrupts cortical architecture, hippocampal development and TGFβ-mediated axonogenesis. |
Stegeman, S; Jolly, LA; Premarathne, S; Gecz, J; Richards, LJ; Mackay-Sim, A; Wood, SA
PloS one 8 e68287 2013
The deubiquitylating enzyme Usp9x is highly expressed in the developing mouse brain, and increased Usp9x expression enhances the self-renewal of neural progenitors in vitro. USP9X is a candidate gene for human neurodevelopmental disorders, including lissencephaly, epilepsy and X-linked intellectual disability. To determine if Usp9x is critical to mammalian brain development we conditionally deleted the gene from neural progenitors, and their subsequent progeny. Mating Usp9x(loxP/loxP) mice with mice expressing Cre recombinase from the Nestin promoter deleted Usp9x throughout the entire brain, and resulted in early postnatal lethality. Although the overall brain architecture was intact, loss of Usp9x disrupted the cellular organization of the ventricular and sub-ventricular zones, and cortical plate. Usp9x absence also led to dramatic reductions in axonal length, in vivo and in vitro, which could in part be explained by a failure in Tgf-β signaling. Deletion of Usp9x from the dorsal telencephalon only, by mating with Emx1-cre mice, was compatible with survival to adulthood but resulted in reduction or loss of the corpus callosum, a dramatic decrease in hippocampal size, and disorganization of the hippocampal CA3 region. This latter phenotypic aspect resembled that observed in Doublecortin knock-out mice, which is an Usp9x interacting protein. This study establishes that Usp9x is critical for several aspects of CNS development, and suggests that its regulation of Tgf-β signaling extends to neurons.
|p53 regulates neural stem cell proliferation and differentiation via BMP-Smad1 signaling and Id1. |
Liu, H; Jia, D; Li, A; Chau, J; He, D; Ruan, X; Liu, F; Li, J; He, L; Li, B
Stem cells and development 22 913-27 2013
Neural stem cells (NSCs) play essential roles in nervous system development and postnatal neuroregeneration and their deregulation underlies the development of neurodegenerative disorders. Yet how NSC proliferation and differentiation are controlled is not fully understood. Here we present evidence that tumor suppressor p53 regulates NSC proliferation and differentiation via the bone morphogenetic proteins (BMP)-Smad1 pathway and its target gene inhibitor of DNA binding 1 (Id1). p53 deficiency led to increased neurogenesis in vivo, and biased neuronal differentiation and augmented NSC proliferation of ex vivo NSCs. This is accompanied by elevated Smad1 expression/activation in the brain and NSC, which contributes to accelerated neuronal differentiation of p53(-/-) NSCs. p53 deficiency also leads to upregulation of Id1, whose expression is repressed by p53 in BMP-Smad1-dependent and -independent manners. Elevated Id1 expression contributes to augmented proliferation and, unexpectedly, accelerated neuronal differentiation of p53(-/-) NSCs as well. This study reveals a molecular mechanism by which tumor suppressor p53 controls NSC proliferation and differentiation and establishes a connection between p53 and Id1.
|The responses of neural stem cells to the level of GSK-3 depend on the tissue of origin. |
Holowacz, T; Alexson, TO; Coles, BL; Doble, BW; Kelly, KF; Woodgett, JR; Van Der Kooy, D
Biology open 2 812-21 2013
Neural stem cells (NSCs) can be obtained from a variety of sources, but not all NSCs exhibit the same characteristics. We have examined how the level of glycogen synthase kinase-3 activity regulates NSCs obtained from different sources: the mouse embryonic striatum, embryonic hippocampus, and mouse ES cells. Growth of striatal NSCs is enhanced by mild inhibition of GSK-3 but not by strong inhibition that is accompanied by Wnt/TCF transcriptional activation. In contrast, the growth of hippocampal NSCs is enhanced by both mild inhibition of GSK-3 as well as stronger inhibition. Active Wnt/TCF signaling, which occurs normally in the embryonic hippocampus, is required for growth of neural stem and progenitor cells. In the embryonic striatal germinal zone, however, TCF signaling is normally absent and its activation inhibits growth of NSCs from this region. Using a genetic model for progressive loss of GSK-3, we find that primitive ES cell-derived NSCs resemble striatal NSCs. That is, partial loss of GSK-3 alleles leads to an increase in NSCs while complete ablation of GSK-3, and activation of TCF-signaling, leads to their decline. Furthermore, expression of dominant negative TCF-4 in the GSK-3-null background was effective in blocking expression of Wnt-response genes and was also able to rescue neuronal gene expression. These results reveal that GSK-3 regulates NSCs by divergent pathways depending on the tissue of origin. The responses of these neural precursor cells may be contingent on baseline Wnt/TCF signaling occurring in a particular tissue.
|Involvement of miRNAs in the differentiation of human glioblastoma multiforme stem-like cells. |
Aldaz, B; Sagardoy, A; Nogueira, L; Guruceaga, E; Grande, L; Huse, JT; Aznar, MA; Díez-Valle, R; Tejada-Solís, S; Alonso, MM; Fernandez-Luna, JL; Martinez-Climent, JA; Malumbres, R
PloS one 8 e77098 2013
Glioblastoma multiforme (GBM)-initiating cells (GICs) represent a tumor subpopulation with neural stem cell-like properties that is responsible for the development, progression and therapeutic resistance of human GBM. We have recently shown that blockade of NFκB pathway promotes terminal differentiation and senescence of GICs both in vitro and in vivo, indicating that induction of differentiation may be a potential therapeutic strategy for GBM. MicroRNAs have been implicated in the pathogenesis of GBM, but a high-throughput analysis of their role in GIC differentiation has not been reported. We have established human GIC cell lines that can be efficiently differentiated into cells expressing astrocytic and neuronal lineage markers. Using this in vitro system, a microarray-based high-throughput analysis to determine global expression changes of microRNAs during differentiation of GICs was performed. A number of changes in the levels of microRNAs were detected in differentiating GICs, including over-expression of hsa-miR-21, hsa-miR-29a, hsa-miR-29b, hsa-miR-221 and hsa-miR-222, and down-regulation of hsa-miR-93 and hsa-miR-106a. Functional studies showed that miR-21 over-expression in GICs induced comparable cell differentiation features and targeted SPRY1 mRNA, which encodes for a negative regulator of neural stem-cell differentiation. In addition, miR-221 and miR-222 inhibition in differentiated cells restored the expression of stem cell markers while reducing differentiation markers. Finally, miR-29a and miR-29b targeted MCL1 mRNA in GICs and increased apoptosis. Our study uncovers the microRNA dynamic expression changes occurring during differentiation of GICs, and identifies miR-21 and miR-221/222 as key regulators of this process.
|Development of a functional schwann cell phenotype from autologous porcine bone marrow mononuclear cells for nerve repair. |
Rutten, MJ; Janes, MA; Chang, IR; Gregory, CR; Gregory, KW
Stem cells international 2012 738484 2012
Adult bone marrow mononuclear cells (BM-MNCs) are a potential resource for making Schwann cells to repair damaged peripheral nerves. However, many methods of producing Schwann-like cells can be laborious with the cells lacking a functional phenotype. The objective of this study was to develop a simple and rapid method using autologous BM-MNCs to produce a phenotypic and functional Schwann-like cell. Adult porcine bone marrow was collected and enriched for BM-MNCs using a SEPAX device, then cells cultured in Neurobasal media, 4 mM L-glutamine and 20% serum. After 6-8 days, the cultures expressed Schwann cell markers, S-100, O4, GFAP, were FluoroMyelin positive, but had low p75(NGF) expression. Addition of neuregulin (1-25 nM) increased p75(NGF) levels at 24-48 hrs. We found ATP dose-dependently increased intracellular calcium [Ca(2+)](i), with nucleotide potency being UTP = ATP greater than ADP greater than AMP greater than adenosine. Suramin blocked the ATP-induced [Ca(2+)](i) but α, β,-methylene-ATP had little effect suggesting an ATP purinergic P2Y2 G-protein-coupled receptor is present. Both the Schwann cell markers and ATP-induced [Ca(2+)](i) sensitivity decreased in cells passaged greater than 20 times. Our studies indicate that autologous BM-MNCs can be induced to form a phenotypic and functional Schwann-like cell which could be used for peripheral nerve repair.
|Cornichons modify channel properties of recombinant and glial AMPA receptors. |
Coombs, ID; Soto, D; Zonouzi, M; Renzi, M; Shelley, C; Farrant, M; Cull-Candy, SG
The Journal of neuroscience : the official journal of the Society for Neuroscience 32 9796-804 2012
Ionotropic glutamate receptors, which underlie a majority of excitatory synaptic transmission in the CNS, associate with transmembrane proteins that modify their intracellular trafficking and channel gating. Significant advances have been made in our understanding of AMPA-type glutamate receptor (AMPAR) regulation by transmembrane AMPAR regulatory proteins. Less is known about the functional influence of cornichons-unrelated AMPAR-interacting proteins, identified by proteomic analysis. Here we confirm that cornichon homologs 2 and 3 (CNIH-2 and CNIH-3), but not CNIH-1, slow the deactivation and desensitization of both GluA2-containing calcium-impermeable and GluA2-lacking calcium-permeable (CP) AMPARs expressed in tsA201 cells. CNIH-2 and -3 also enhanced the glutamate sensitivity, single-channel conductance, and calcium permeability of CP-AMPARs while decreasing their block by intracellular polyamines. We examined the potential effects of CNIHs on native AMPARs by recording from rat optic nerve oligodendrocyte precursor cells (OPCs), known to express a significant population of CP-AMPARs. These glial cells exhibited surface labeling with an anti-CNIH-2/3 antibody. Two features of their AMPAR-mediated currents-the relative efficacy of the partial agonist kainate (I(KA)/I(Glu) ratio 0.4) and a greater than fivefold potentiation of kainate responses by cyclothiazide-suggest AMPAR association with CNIHs. Additionally, overexpression of CNIH-3 in OPCs markedly slowed AMPAR desensitization. Together, our experiments support the view that CNIHs are capable of altering key properties of AMPARs and suggest that they may do so in glia.
|Progenitor-like traits contribute to patient survival and prognosis in oligodendroglial tumors. |
Ng, FS; Toh, TB; Ting, EH; Koh, GR; Sandanaraj, E; Phong, M; Wong, SS; Leong, SH; Kon, OL; Tucker-Kellogg, G; Ng, WH; Ng, I; Tang, C; Ang, BT
Clinical cancer research : an official journal of the American Association for Cancer Research 18 4122-35 2012
Patient-derived glioma-propagating cells (GPC) contain karyotypic and gene expression profiles that are found in the primary tumor. However, their clinical relevance is unclear. We ask whether GPCs contribute to disease progression and survival outcome in patients with glioma by analyzing gene expression profiles.We tapped into public sources of GPC gene expression data and derived a gene signature distinguishing oligodendroglial from glioblastoma multiforme (GBM) GPCs. By adapting a method in glioma biology, the Connectivity Map, we interrogated its strength of association in public clinical databases. We validated the top-ranking signaling pathways Wnt, Notch, and TGFβ, in GPCs and primary tumor specimens.We observed that patients with better prognosis correlated with oligodendroglial GPC features and lower tumor grade, and this was independent of the current clinical indicator, 1p/19q status. Patients with better prognosis had proneural tumors whereas the poorly surviving cohort had mesenchymal tumors. In addition, oligodendroglial GPCs were more sensitive to Wnt and Notch inhibition whereas GBM GPCs responded to TGFβR1 inhibition.We provide evidence that GPCs are clinically relevant. In addition, the more favorable prognosis of oligodendroglial tumors over GBM could be recapitulated transcriptomically at the GPC level, underscoring the relevance of this cellular model. Our gene signature detects molecular heterogeneity in oligodendroglial tumors that cannot be accounted for by the 1p/19q status alone, indicating that stem-like traits contribute to clinical status. Collectively, these data highlight the limitation of morphology-based histologic analyses in tumor classification, consequently impacting on treatment decisions.
|Small Molecule Induction of Human Umbilical Stem Cells into MBP-positive Oligodendrocytes in a Defined Three-Dimensional Environment. |
Davis, H; Guo, X; Lambert, S; Stancescu, M; Hickman, JJ
ACS chemical neuroscience 3 31-39 2012
Stem cells from umbilical cord would be a favorable alternative to embryonic stem cells for therapeutic applications. In this study, human multipotent progenitor cells (MLPCs) from umbilical cord were differentiated into oligodendrocytes by exposure to a range of microenvironmental chemical and physical cues. Chemical cues were represented by a novel defined differentiation medium containing the neurotransmitter norepinephrine (NE). In traditional 2 dimensional (2D) conditions, the MLPCs differentiated into oligodendrocyte precursors, but did not progress further. However, in a 3 dimensional (3D) environment, the MLPCs differentiated into committed oligodendrocytes that expressed MBP. The apparent method of interaction of NE in stimulating the differentiation process was identified to occur through the adenergic pathway while all prior differentiation methods have used other routes. This novel method of obtaining functional human oligodendrocytes from MLPCs would eliminate many of the difficulties associated with their differentiation from embryonic stem cells.
|IKKβ/NF-κB disrupts adult hypothalamic neural stem cells to mediate a neurodegenerative mechanism of dietary obesity and pre-diabetes. |
Li, J; Tang, Y; Cai, D
Nature cell biology 14 999-1012 2012
Adult neural stem cells (NSCs) are known to exist in a few regions of the brain; however, the entity and physiological/disease relevance of adult hypothalamic NSCs (htNSCs) remain unclear. This work shows that adult htNSCs are multipotent and predominantly present in the mediobasal hypothalamus of adult mice. Chronic high-fat-diet feeding led to not only depletion but also neurogenic impairment of htNSCs associated with IKKβ/NF-κB activation. In vitro htNSC models demonstrated that their survival and neurogenesis markedly decreased on IKKβ/NF-κB activation but increased on IKKβ/NF-κB inhibition, mechanistically mediated by IKKβ/NF-κB-controlled apoptosis and Notch signalling. Mouse studies revealed that htNSC-specific IKKβ/NF-κB activation led to depletion and impaired neuronal differentiation of htNSCs, and ultimately the development of obesity and pre-diabetes. In conclusion, adult htNSCs are important for the central regulation of metabolic physiology, and IKKβ/NF-κB-mediated impairment of adult htNSCs is a critical neurodegenerative mechanism for obesity and related diabetes.
|Querkopf is a key marker of self-renewal and multipotency of adult neural stem cells. |
Sheikh, BN; Dixon, MP; Thomas, T; Voss, AK
Journal of cell science 125 295-309 2012
Adult neural stem cells (NSCs) reside in the subventricular zone (SVZ) and produce neurons throughout life. Although their regenerative potential has kindled much interest, few factors regulating NSCs in vivo are known. Among these is the histone acetyltransferase querkopf (QKF, also known as MYST4, MORF, KAT6B), which is strongly expressed in a small subset of cells in the neurogenic subventricular zone. However, the relationship between Qkf gene expression and the hierarchical levels within the neurogenic lineage is currently unknown. We show here that the 10% of SVZ cells with the highest Qkf expression possess the defining NSC characteristics of multipotency and self-renewal and express markers previously shown to enrich for NSCs. A fraction of cells expressing Qkf at medium to high levels is enriched for multipotent progenitor cells with limited self-renewal, followed by a population containing migrating neuroblasts. Cells low in Qkf promoter activity are predominantly ependymal cells. In addition, we show that mice deficient for Bmi1, a central regulator of NSC self-renewal, show an age-dependent decrease in the strongest Qkf-expressing cell population in the SVZ. Our results show a strong relationship between Qkf promoter activity and stem cell characteristics, and a progressive decrease in Qkf gene activity as lineage commitment and differentiation proceed in vivo.
|Rat Cortical Oligodendrocyte-Embryonic Motoneuron Co-Culture: An In Vitro Axon-Oligodendrocyte Interaction Model. |
Davis, H; Gonzalez, M; Bhargava, N; Stancescu, M; Hickman, JJ; Lambert, S
Journal of biomaterials and tissue engineering 2 206-214 2012
Mechanisms that control the differentiation and function of oligodendrocytes in the central nervous system are complex and involve multiple inputs from the surrounding environment, including localized concentrations of growth factors and the extracellular matrix. Dissection and analysis of these inputs are key to understanding the pathology of central nervous system demyelinating diseases such as multiple sclerosis, where the differentiation of myelinating oligodendrocytes from their precursors underlies the remission phase of the disease. In vitro co-culture models provide a mechanism for the study of factors that regulate differentiation of oligodendrocyte precursors but have been difficult to develop due to the complex nature of central nervous system myelination. This study describes development of an in vitro model that merges a defined medium with a chemically modified substrate to study aspects of myelination in the central nervous system. We demonstrate that oligodendrocyte precursors co-cultured with rat embryonic motoneurons on non-biological substrate (diethylenetriamine trimethoxy-silylpropyldiethylenetriamine), can be induced to differentiate into mature oligodendrocytes that express myelin basic protein, using a serum-free medium. This defined and reproducible model of in vitro myelination could be a valuable tool for the development of treatments for demyelinating diseases such as multiple sclerosis.
|Critical period of axoglial signaling between neuregulin-1 and brain-derived neurotrophic factor required for early Schwann cell survival and differentiation. |
Ma, Z; Wang, J; Song, F; Loeb, JA
The Journal of neuroscience : the official journal of the Society for Neuroscience 31 9630-40 2011
During peripheral nervous system development, successful communication between axons and Schwann cells is required for proper function of both myelinated and nonmyelinated nerve fibers. Alternatively spliced proteins belonging to the neuregulin1 (NRG1) gene family of growth and differentiation factors are essential for Schwann cell survival and peripheral nerve development. Although recent studies have strongly implicated membrane-bound NRG1 forms (type III) in the myelination at late stages, little is known about the role of soluble, heparin-binding forms of NRG1 (type I/II) in regulating early Schwann cell development in vivo. These forms are rapidly released from axons in vitro by Schwann-cell-secreted neurotrophic factors and, unlike membrane-bound forms, have a unique ability to diffuse and adhere to heparan sulfate-rich cell surfaces. Here, we show that axon-derived soluble NRG1 translocates from axonal to Schwann cell surfaces in the embryonic chick between days 5 and 7, corresponding to the critical period of Schwann cell survival. Downregulating endogenous type I/II NRG1 signaling either with a targeted NRG1 antagonist or by shRNA blocks their differentiation from precursors into immature Schwann cells and increases programmed cell death, whereas upregulating NRG1 rescues Schwann cells. Exogenous BDNF also promotes Schwann cell survival through promoting the local release of axonal NRG1. Consistently, increased Schwann cell death occurs both in trkB knock-out mice and after knocking down axonal trkB in chick embryos, which can then be rescued with soluble NRG1. These findings suggest a localized, axoglial feedback loop through soluble NRG1 and BDNF critical for early Schwann cell survival and differentiation.
|Differentiation of neuronal cells from NIH3T3 fibroblasts under defined conditions. |
Wang Z, Sugano E, Isago H, Hiroi T, Tamai M, Tomita H.
Development, growth & differentiation 53 357-65 2011
We attempted to test whether the differentiated NIH/3T3 fibroblasts could be differentiated into neuronal cells without any epigenetic modification. First, a neurosphere assay was carried out, and we successfully generated neurosphere-like cells by floating cultures of NIH/3T3 fibroblasts in neural stem cell medium. These spheres have the ability to form sub-spheres after three passages, and express the neural progenitor markers Nestin, Sox2, Pax6, and Musashi-1. Second, after shifting to a differentiating medium and culturing for an additional 8 days, cells in these spheres expressed the neuronal markers β-tubulin and neurofilament 200 and the astrocytic marker glial fibrillary acidic protein (GFAP). Finally, after treating the spheres with all-trans retinoic acid and taurine, the expression of β-tubulin was increased and the staining of photoreceptor markers rhodopsin and recoverin was observed. The present study shows that NIH/3T3 fibroblasts can generate neurosphere-like, neuron-like, and even photoreceptor-like cells under defined conditions, suggesting that the differentiated non-neuronal cells NIH/3T3 fibroblasts, but not pluripotent cells such as embryonic stem cells or induced pluripotent stem cells, may have the potential to be transdifferentiated into neuronal cells without adding any epigenetic modifier. This transdifferentiation may be due to the possible neural progenitor potential of NIH/3T3 fibroblasts that remains dormant under normal conditions.
|Blockade of the NFκB pathway drives differentiating glioblastoma-initiating cells into senescence both in vitro and in vivo. |
Nogueira L, Ruiz-Ontañon P, Vazquez-Barquero A, Lafarga M, Berciano MT, Aldaz B, Grande L, Casafont I, Segura V, Robles EF, Suarez D, Garcia LF, Martinez-Climent JA, Fernandez-Luna JL.
Oncogene 30 3537-48 2011
Glioblastoma multiforme is one of the most devastating cancers and presents unique challenges to therapy because of its aggressive behavior. Cancer-initiating or progenitor cells have been described to be the only cell population with tumorigenic capacity in glioblastoma. Therefore, effective therapeutic strategies targeting these cells or the early precursors may be beneficial. We have established different cultures of glioblastoma-initiating cells (GICs) derived from surgical specimens and found that, after induction of differentiation, the NFκB transcriptional pathway was activated, as determined by analyzing key proteins such as p65 and IκB and the upregulation of a number of target genes. We also showed that blockade of nuclear factor (NF)κB signaling in differentiating GICs by different genetic strategies or treatment with small-molecule inhibitors, promoted replication arrest and senescence. This effect was partly mediated by reduced levels of the NFκB target gene cyclin D1, because its downregulation by RNA interference reproduced a similar phenotype. Furthermore, these results were confirmed in a xenograft model. Intravenous treatment of immunodeficient mice bearing human GIC-derived tumors with a novel small-molecule inhibitor of the NFκB pathway induced senescence of tumor cells but no ultrastructural alterations of the brain parenchyma were detected. These findings reveal that activation of NFκB may keep differentiating GICs from acquiring a mature postmitotic phenotype, thus allowing cell proliferation, and support the rationale for therapeutic strategies aimed to promote premature senescence of differentiating GICs by blocking key factors within the NFκB pathway.
|Spatiotemporal pattern of neuronal injury induced by dFP in rats: a Model for delayed neuronal cell death following acute OP intoxication. |
Li Y, Lein PJ, Liu C, Bruun DA, Tewolde T, Ford G, Ford BD
Toxicology and applied pharmacology 253 261-9. Epub 2011 Apr 12. 2011
Organophosphate (OP) neurotoxins cause acute cholinergic toxicity and seizures resulting in delayed brain damage and persistent neurological symptoms. Testing novel strategies for protecting against delayed effects of acute OP intoxication has been hampered by the lack of appropriate animal models. In this study, we characterize the spatiotemporal pattern of cellular injury after acute intoxication with the OP diisopropylfluorophosphate (DFP). Adult male Sprague-Dawley rats received pyridostigmine (0.1 mg/kg, im) and atropine methylnitrate (20mg/kg, im) prior to DFP (9 mg/kg, ip) administration. All DFP-treated animals exhibited moderate to severe seizures within minutes after DFP injection but survived up to 72 h. AChE activity was significantly depressed in the cortex, hippocampus, subcortical brain tissue and cerebellum at 1h post-DFP injection and this inhibition persisted for up to 72 h. Analysis of neuronal injury by Fluoro-Jade B (FJB) labeling revealed delayed neuronal cell death in the hippocampus, cortex, amygdala and thalamus, but not the cerebellum, starting at 4h and persisting until 72 h after DFP treatment, although temporal profiles varied between brain regions. At 24h post-DFP injection, the pattern of FJB labeling corresponded to TUNEL staining in most brain regions, and FJB-positive cells displayed reduced NeuN immunoreactivity but were not immunopositive for astrocytic (GFAP), oligodendroglial (O4) or macrophage/microglial (ED1) markers, demonstrating that DFP causes a region-specific delayed neuronal injury mediated in part by apoptosis. These findings indicate the feasibility of this model for testing neuroprotective strategies, and provide insight regarding therapeutic windows for effective pharmacological intervention following acute OP intoxication.Copyright © 2011 Elsevier Inc. All rights reserved.
|Functional neural stem cell isolation from brains of adult mutant SOD1 (SOD1(G93A)) transgenic amyotrophic lateral sclerosis (ALS) mice. |
Lee JC, Jin Y, Jin J, Kang BG, Nam DH, Joo KM, Cha CI
Neurol Res 33 33-7. Epub 2010 Aug 31. 2011
OBJECTIVES: The aim of present study is to investigate more functional neural stem cells (NSCs) could be isolated from brains with amyotrophic lateral sclerosis (ALS) and expanded in vitro, based on previous reports demonstrating de novo neurogenesis is enhanced to replace degenerating neural tissue.METHODS: Thirteen- or eighteen-week-old mutant human Cu/Zn superoxide dismutase (SOD1(G93A)) transgenic ALS and wild-type SOD1 transgenic control mice were utilized. Changes in numbers of NSCs in the dentate gyrus were analyzed by immunohistochemistry against nestin and CD133. NSCs were primarily cultured from hippocampus of ALS or control mice. Expression of NSC markers, in vitro expansion capacity, and differentiating potential were compared.RESULTS: Hippocampus of 13-week-old pre-symptomatic ALS mice harbor more cells that can be propagated for more than 12 passages in vitro, compared with same age control mice. Primarily-cultured cells formed neurospheres in the NSC culture medium, expressed NSC markers, and differentiated into cells with differentiated neural cell characteristics in the differentiation condition confirming that they are NSCs. In contrast, long-term expansible NSCs could not be derived from brains of 18-week-old symptomatic ALS mice with the same experimental techniques, although they had comparable nestin-immunoreactive cells in the dentate gyrus.DISCUSSION: These results would suggest that increased neuroregeneration in early phase of ALS could be translated to regenerative approaches; however, long-term exposure to ALS microenvironments could abolish functional capacities of NSCs.
|Neural stem cell-like gene expression in a mouse ependymoma cell line transformed by human BK polyomavirus. |
Takako Aizawa,Koichi Hasegawa,Tsuyoshi Ohkumo,Seiichi Haga,Kazuhiko Ikeda,Kazuaki Yoshikawa
Cancer science 102 2011
Ependymomas often show characteristics similar to those of neural stem cells in vivo and in vitro. However, few ependymoma cell lines that exhibit neural stem cell-like properties have been reported. In this study, we have characterized a novel cell line, designated Vn19, established from ependymoma that arose in mice inoculated intracerebrally with human BK polyomavirus. Transplanted Vn19 cells in nude mice ubiquitously expressed viral large T antigen in the nucleus and coexpressed neuronal and glial marker proteins in vivo. Remarkably, individual Vn19 cells in dispersed cultures simultaneously expressed marker proteins of neural stem cells (nestin, Bmi1, CD133), neurons (?III tubulin, neurofilament-M) and glial cells (glial fibrillary acidic protein, A2B5, S100?, O4). Ubiquitous and homogenous expression of these multilineage marker proteins was also observed in cloned Vn19 cells. The Vn19 cells formed neurosphere-like aggregates when cultured in the presence of growth factors. Quantitative RT-PCR analysis revealed that expression of mRNA for nestin, neurofilament-H and glial fibrillary acidic protein significantly increased in Vn19 cells cultured under growth factor-deprived conditions. Among MAGE (melanoma antigen) family genes, MAGE-A (A1-8), MAGE-B (B1-3), MAGE-D1, MAGE-E1, MAGE-G1 (necdin-like 2) and MAGE-H1 were expressed in the Vn19 cells, in which neither necdin nor MAGEL2 was detectable. These results suggest that this murine ependymoma cell line recapitulates the gene expression profile in ependymal cells undergoing malignant transformation.
|Identification of a peptide that interacts with Nestin protein expressed in brain cancer stem cells. |
Beck S, Jin X, Yin J, Kim SH, Lee NK, Oh SY, Jin X, Kim MK, Kim EB, Son JS, Kim SC, Nam DH, Kim SH, Kang SK, Kim H, Choi YJ.
Biomaterials 32 8518-28 2011
Glioma stem cells (GSCs) are presumably major culprits for brain tumor initiation, progression, and recurrence after conventional therapies. Thus, selective targeting and eradication of GSCs may provide a promising and effective therapeutic approach. Here, we isolated a GSC-targeting (GSCT) peptide that demonstrated selective binding affinity for many undifferentiated GSCs using in vitro phage display technology. This GSCT peptide binds to isotypes of Nestin proteins specifically expressed in GSCs, enabling it to target Nestin-positive cells in human glioblastoma tissues. In human glioblastoma tissue specimens, the fluorescence-conjugated GSCT peptide could visualize putative GSC populations, showing its possible use as a diagnostic agent. GSCT peptide is also internalized into undifferentiated GSCs specifically in vitro, and moreover, intravenously injected GSCT peptide effectively penetrated into tissues, specifically accumulated in gliomas that arise from subcutaneous and orthotopic implantation, and predominantly targeted Nestin-positive cells in these tumors. Thus, our GSCT peptide may be useful for the development of more promising therapeutic and diagnostic modalities that target GSCs in brain tumors.
|Bidirectional plasticity of calcium-permeable AMPA receptors in oligodendrocyte lineage cells. |
Zonouzi, M; Renzi, M; Farrant, M; Cull-Candy, SG
Nature neuroscience 14 1430-8 2011
Oligodendrocyte precursor cells (OPCs), a major glial cell type that gives rise to myelinating oligodendrocytes in the CNS, express calcium-permeable AMPA receptors (CP-AMPARs). Although CP-AMPARs are important for OPC proliferation and neuron-glia signaling, they render OPCs susceptible to ischemic damage in early development. We identified factors controlling the dynamic regulation of AMPAR subtypes in OPCs from rat optic nerve and mouse cerebellar cortex. We found that activation of group 1 mGluRs drove an increase in the proportion of CP-AMPARs, reflected by an increase in single-channel conductance and inward rectification. This plasticity required the elevation of intracellular calcium and used PI3K, PICK-1 and the JNK pathway. In white matter, neurons and astrocytes release both ATP and glutamate. Unexpectedly, activation of purinergic receptors in OPCs decreased CP-AMPAR expression, suggesting a capacity for homeostatic regulation. Finally, we found that stargazin-related transmembrane AMPAR regulatory proteins, which are critical for AMPAR surface expression in neurons, regulate CP-AMPAR plasticity in OPCs.
|Glypican-1, phosphacan/receptor protein-tyrosine phosphatase-zeta/beta and its ligand, tenascin-C, are expressed by neural stem cells and neural cells derived from embryonic stem cells. |
Abaskharoun M, Bellemare M, Lau E, Margolis RU
ASN Neuro 2 e00039. 2010
The heparan sulfate proteoglycan glypican-1, the chondroitin sulfate proteoglycan phosphacan/RPTP (receptor protein-tyrosine phosphatase)-zeta/beta and the extracellular matrix protein tenascin-C were all found to be expressed by neural stem cells and by neural cells derived from them. Expression of proteoglycans and tenascin-C increased after retinoic acid induction of SSEA1-positive ES (embryonic stem) cells to nestin-positive neural stem cells, and after neural differentiation, proteoglycans and tenascin-C are expressed by both neurons and astrocytes, where they surround cell bodies and processes and in certain cases show distinctive expression patterns. With the exception of tenascin-C (whose expression may decrease somewhat), expression levels do not change noticeably during the following 2 weeks in culture. The significant expression, by neural stem cells and neurons and astrocytes derived from them, of two major heparan sulfate and chondroitin sulfate proteoglycans of nervous tissue and of tenascin-C, a high-affinity ligand of phosphacan/RPTP-zeta/beta, indicates that an understanding of their specific functional roles in stem cell neurobiology will be important for the therapeutic application of this new technology in facilitating nervous tissue repair and regeneration.Artículo Texto completo
|BMP-induced REST regulates the establishment and maintenance of astrocytic identity. |
Kohyama J, Sanosaka T, Tokunaga A, Takatsuka E, Tsujimura K, Okano H, Nakashima K
J Cell Biol 189 159-70. Epub 2010 Mar 29. 2010
Once they have differentiated, cells retain their individual character and repress genes that are specifically expressed in other cell lineages, but how alternative fate choice is restricted during and/or after differentiation remains unclear. In the mammalian central nervous system, neurons, astrocytes, and oligodendrocytes are generated throughout life from common tripotent neural progenitor cells (NPCs). Bone morphogenetic proteins (BMPs) are well-known astrocyte-inducing cytokines. We show here that the expression of a transcriptional repressor, RE1 silencer of transcription (REST)/neuron-restrictive silencer factor (NRSF), is up-regulated and sustained by BMP signal activation in the course of astrocytic differentiation of NPCs, and restricts neuronal differentiation. We further show that, in differentiated astrocytes, endogenous REST/NRSF associates with various neuronal genes and that disruption of its function resulted in their derepression, thereby explaining how ectopic neuronal gene expression is prevented in cells with astrocytic traits. Collectively, our results suggest that REST/NRSF functions as a molecular regulator of the nonneuronal phenotype in astrocytes.Artículo Texto completo
|Imperfect oligodendrocytic and neuronal differentiation of glioblastoma cells. |
WolaÅ„czyk M, HuÅ‚as-Bigoszewska K, Witusik-Perkowska M, Papierz W, JaskÃ³lski D, Liberski PP, Rieske P
Folia Neuropathol 48 27-34 2010
Previously, we have reported that glioblastoma (GBM) cells can be differentiated into cells showing neuronal, glial and non-neural (mesenchymal) phenotypes. Before the differentiation the GBM cells co-expressed GFAP, CD44, Beta III tubulin, MAP2, Vimentin, Nestin and SOX-2, whereas during the exposure to a neural differentiation medium the differentiation process was arrested at the early stages and the GBM cells presented features of four phenotypes: multi-lineage, non-neural (mesenchymal), intermediate of neuronal cells and glial cells. Currently, we decided to check if changes in expression of: TH (tyrosine hydroxylase, marker of catecholaminergic cells) and GABA (neurotransmitter of GABAergic neurons) and markers of oligodendrocytic cells (O4, CNP) occur during the exposure of GBM cells to the differentiation medium. After exposure to the PDGF alpha and thyroid hormones (oligodendrocytic differentiation medium 10-30 days) features of oligodendrocytic differentiation were presented by 0.2-2.4% of analyzed cells. During the prolonged neural differentiation (GDNF, bFGF 20-30 days) only few cells showed expression of GABA. Moreover, in our cell cultures, there were not cells expressing markers of catecholaminergic neurons - TH. Our work confirmed that the neuronal differentiation of GBM was inhibited at the stage of the neuronal intermediate phenotype. Moreover, we showed that the oligodendrocytic differentiation of GBM cells is very inefficient.
|A synthetic cannabinoid agonist promotes oligodendrogliogenesis during viral encephalitis in rats. |
Solbrig, MV; Fan, Y; Hermanowicz, N; Morgese, MG; Giuffrida, A
Experimental neurology 226 231-41 2010
Chronic CNS infection by several families of viruses can produce deficits in prefrontal cortex (PFC) and striatal function. Cannabinoid drugs have been long known for their anti-inflammatory properties and their ability to modulate adult neuro and gliogenesis. Therefore, we explored the effects of systemic administration of the cannabinoid agonist WIN55,212-2(WIN) on prefrontal cortex (PFC) and striatal cytogenesis in a viral model of CNS injury and inflammation based on Borna Disease (BD) virus encephalitis. Active BrdU(+) progenitor populations were significantly decreased 1 week after BrdU labeling in BD rats [pless than 0.001 compared to uninfected (NL) controls] while less than 5% of BrdU(+) cells colabeled for BDV protein. Systemic WIN (1mg/kg i.p. twice daily×7 days) increased the survival of BrdU(+) cells in striatum (pless than 0.001) and PFC of BD rats, with differential regulation of labeled oligodendroglia precursors vs microglia/macrophages. WIN increased the percentage of BrdU(+) oligodendrocyte precursor cells and decreased BrdU(+) ED-1-labeled phagocytic cells, without producing pro- or antiviral effects. BDV infection decreased the levels of the endocannabinoid anandamide (AEA) in striatum (pless than 0.05 compared to NL rats), whereas 2-AG levels were unchanged. Our findings indicate that: 1) viral infection is accompanied by alterations of AEA transmission in the striatum, but new cell protection by WIN appears independent of its effect on endocannabinoid levels; and 2) chronic WIN treatment alters the gliogenic cascades associated with CNS injury, promoting oligodendrocyte survival. Limiting reactive gliogenesis and macrophage activity in favor of oliogodendroglia development has significance for demyelinating diseases. Moreover, the ability of cannabinoids to promote the development of biologically supportive or symbiotic oligodendroglia may generalize to other microglia-driven neurodegenerative syndromes including NeuroAIDS and diseases of aging.Artículo Texto completo
|Folic acid remodels chromatin on Hes1 and Neurog2 promoters during caudal neural tube development. |
Ichi, S; Costa, FF; Bischof, JM; Nakazaki, H; Shen, YW; Boshnjaku, V; Sharma, S; Mania-Farnell, B; McLone, DG; Tomita, T; Soares, MB; Mayanil, CS
The Journal of biological chemistry 285 36922-32 2010
The mechanism(s) behind folate rescue of neural tube closure are not well understood. In this study we show that maternal intake of folate prior to conception reverses the proliferation potential of neural crest stem cells in homozygous Splotch embryos (Sp(-/-)) via epigenetic mechanisms. It is also shown that the pattern of differentiation seen in these cells is similar to wild-type (WT). Cells from open caudal neural tubes of Sp(-/-) embryos exhibit increased H3K27 methylation and decreased expression of KDM6B possibly due to up-regulation of KDM6B targeting micro-RNAs such as miR-138, miR-148a, miR-185, and miR-339-5p. In our model, folate reversed these epigenetic marks in folate-rescued Sp(-/-) embryos. Using tissue from caudal neural tubes of murine embryos we also examined H3K27me2 and KDM6B association with Hes1 and Neurog2 promoters at embryonic day E10.5, the proliferative stage, and E12.5, when neural differentiation begins. In Sp(-/-) embryos compared with WT, levels of H3K27me2 associated with the Hes1 promoter were increased at E10.5, and levels associated with the Neurog2 promoter were increased at E12.5. KDM6B association with Hes1 and Neurog2 promoters was inversely related to H3K27me2 levels. These epigenetic changes were reversed in folate-rescued Sp(-/-) embryos. Thus, one of the mechanisms by which folate may rescue the Sp(-/-) phenotype is by increasing the expression of KDM6B, which in turn decreases H3K27 methylation marks on Hes1 and Neurog2 promoters thereby affecting gene transcription.
|Expression of hyaluronan and the hyaluronan-binding proteoglycans neurocan, aggrecan, and versican by neural stem cells and neural cells derived from embryonic stem cells. |
Abaskharoun M, Bellemare M, Lau E, Margolis RU
Brain Res 1327 6-15. Epub 2010 Feb 20. 2010
We have examined the expression and localization patterns of hyaluronan and hyaluronan-binding chondroitin sulfate proteoglycans in neural stem cells and differentiated neural cells derived from mouse embryonic stem cells. Expression of proteoglycans and hyaluronan was weak in the SSEA1-positive embryonic stem cells but increased noticeably after retinoic acid induction to nestin-positive neural stem cells. After subsequent plating, the hyaluronan-binding chondroitin sulfate proteoglycans aggrecan, neurocan, and versican are expressed by cells in both the astrocytic and neuronal lineages. During the time period that hyaluronan was present, it co-localized with each of the hyaluronan-binding proteoglycans studied and was found to be clearly associated with beta-III tubulin-expressing neurons and oligodendrocytes expressing the O4 sulfatide marker. Although proteoglycan expression levels increased to varying degrees following neural differentiation, they did not change noticably during the following 2 weeks in culture, but there was a significant decrease in hyaluronan expression. Our studies therefore demonstrate the expression by neural stem cells and neural cells derived from them of hyaluronan and its associated proteoglycans, thereby providing a necessary foundation for integrating their specific properties into developing strategies for therapeutic applications.Artículo Texto completo
|Human embryonic stem cell neural differentiation and enhanced cell survival promoted by hypoxic preconditioning. |
Francis, KR; Wei, L
Cell death & disease 1 e22 2010
Transplantation of neural progenitors derived from human embryonic stem cells (hESCs) provides a potential therapy for ischemic stroke. However, poor graft survival within the host environment has hampered the benefits and applications of cell-based therapies. The present investigation tested a preconditioning strategy to enhance hESC tolerance, thereby improving graft survival and the therapeutic potential of hESC transplantation. UC06 hESCs underwent neural induction and terminal differentiation for up to 30 days, becoming neural lineage cells, exhibiting extensive neurites and axonal projections, generating synapses and action potentials. To induce a cytoprotective phenotype, hESC-derived neurospheres were cultured at 0.1% oxygen for 12 h, dissociated and plated for terminal differentiation under 21% oxygen. Immunocytochemistry and electrophysiology demonstrated the 'hypoxic preconditioning' promoted neuronal differentiation. Western blotting revealed significantly upregulated oxygen-sensitive transcription factors hypoxia-inducible factor (HIF)-1α and HIF-2α, while producing a biphasic response within HIF targets, including erythropoietin, vascular endothelial growth factor and Bcl-2 family members, during hypoxia and subsequent reoxygenation. This cytoprotective phenotype resulted in a 50% increase in both total and neural precursor cell survival after either hydrogen peroxide insult or oxygen-glucose deprivation. Cellular protection was maintained for at least 5 days and corresponded to upregulation of neuroprotective proteins. These results suggest that hypoxic preconditioning could be used to improve the effectiveness of human neural precursor transplantation therapies.Artículo Texto completo
|Intra-operatively obtained human tissue: protocols and techniques for the study of neural stem cells. |
Chaichana, KL; Guerrero-Cazares, H; Capilla-Gonzalez, V; Zamora-Berridi, G; Achanta, P; Gonzalez-Perez, O; Jallo, GI; Garcia-Verdugo, JM; Quiñones-Hinojosa, A
Journal of neuroscience methods 180 116-25 2009
The discoveries of neural (NSCs) and brain tumor stem cells (BTSCs) in the adult human brain and in brain tumors, respectively, have led to a new era in neuroscience research. These cells represent novel approaches to studying normal phenomena such as memory and learning, as well as pathological conditions such as Parkinson's disease, stroke, and brain tumors. This new paradigm stresses the importance of understanding how these cells behave in vitro and in vivo. It also stresses the need to use human-derived tissue to study human disease because animal models may not necessarily accurately replicate the processes that occur in humans. An important, but often underused, source of human tissue and, consequently, both NSCs and BTSCs, is the operating room. This study describes in detail both current and newly developed laboratory techniques, which in our experience are used to process and study human NSCs and BTSCs from tissue obtained directly from the operating room.Artículo Texto completo
|Neurogenic potential of isolated precursor cells from early post-gastrula somitic tissue. |
Tropepe V, Alton K, Sachewsky N, Cheng V, Kuo C, Morshead CM
Stem cells and development 18 1533-1542 2009
Adult tissues are known to contain rare populations of stem cells with multilineage differentiation potential that are distinct from other resident tissue-specific stem cells. However, whether multilineage stem cells are involved in tissue development is uncertain, primarily because the identification and characterization of these cells in embryonic tissue primordia is not well established. We tested whether stem cells with multilineage potential are present within the early post-gastrula somite tissue. We show that clonally derived precursor cells generate colonies with self-renewal capacity and have both neurogenic and myogenic lineage potential. Somite colonies contain cells that express Sox2, nestin, and Sca1, but do not express genes indicative of somitic mesoderm specification. Furthermore, we demonstrate that this multilineage potential is not due to colony cells with a pluripotent epiblast identity or the selection of p75 receptor-positive neural crest stem cells. Despite utilizing a highly undifferentiated tissue source, colony formation was not enhanced relative to reported estimates of multilineage stem cells from adult muscle, a derivative of the embryonic somite. Thus, our findings suggest that a permissive in vitro environment is sufficient for the isolation of a discrete population of stem cells in the embryonic somite that may represent the earliest developmental precursor to adult muscle multilineage stem cells.
|Human oligodendrocytes from embryonic stem cells: conserved SHH signaling networks and divergent FGF effects. |
Bao-Yang Hu, Zhong-Wei Du, Xue-Jun Li, Melvin Ayala, Su-Chun Zhang, Bao-Yang Hu, Zhong-Wei Du, Xue-Jun Li, Melvin Ayala, Su-Chun Zhang, Bao-Yang Hu, Zhong-Wei Du, Xue-Jun Li, Melvin Ayala, Su-Chun Zhang
Development (Cambridge, England) 136 1443-52 2009
Human embryonic stem cells (hESCs) offer a platform to bridge what we have learned from animal studies to human biology. Using oligodendrocyte differentiation as a model system, we show that sonic hedgehog (SHH)-dependent sequential activation of the transcription factors OLIG2, NKX2.2 and SOX10 is required for sequential specification of ventral spinal OLIG2-expressing progenitors, pre-oligodendrocyte precursor cells (pre-OPCs) and OPCs from hESC-derived neuroepithelia, indicating that a conserved transcriptional network underlies OPC specification in human as in other vertebrates. However, the transition from pre-OPCs to OPCs is protracted. FGF2, which promotes mouse OPC generation, inhibits the transition of pre-OPCs to OPCs by repressing SHH-dependent co-expression of OLIG2 and NKX2.2. Thus, despite the conservation of a similar transcriptional network across vertebrates, human stem/progenitor cells may respond differently to those of other vertebrates to certain extrinsic factors.
|DNA-damage response, survival and differentiation in vitro of a human neural stem cell line in relation to ATM expression. |
Carlessi, L; De Filippis, L; Lecis, D; Vescovi, A; Delia, D
Cell death and differentiation 16 795-806 2009
Ataxia-telangiectasia (A-T) is a neurodegenerative disorder caused by defects in the ATM kinase, a component of the DNA-damage response (DDR). Here, we employed an immortalized human neural stem-cell line (ihNSC) capable of differentiating in vitro into neurons, oligodendrocytes and astrocytes to assess the ATM-dependent response and outcome of ATM ablation. The time-dependent differentiation of ihNSC was accompanied by an upregulation of ATM and DNA-PK, sharp downregulation of ATR and Chk1, transient induction of p53 and by the onset of apoptosis in a fraction of cells. The response to ionizing radiation (IR)-induced DNA lesions was normal, as attested by the phosphorylation of ATM and some of its substrates (e.g., Nbs1, Smc1, Chk2 and p53), and by the kinetics of gamma-H2AX nuclear foci formation. Depletion in these cells of ATM by shRNA interference (shATM) attenuated the differentiation-associated apoptosis and response to IR, but left unaffected the growth, self-renewal and genomic stability. shATM cells generated a normal number of MAP2/beta-tubulin III+ neurons, but a reduced number of GalC+ oligodendrocytes, which were nevertheless more susceptible to oxidative stress. Altogether, these findings highlight the potential of ihNSCs as an in vitro model system to thoroughly assess, besides ATM, the role of DDR genes in neurogenesis and/or neurodegeneration.
|Pluripotency of mouse spermatogonial stem cells maintained by IGF-1- dependent pathway. |
Yen-Hua Huang, Cheng-Chieh Chin, Hong-Nerng Ho, Chuan-Kai Chou, Chia-Ning Shen, Hung-Chih Kuo, Tsai-Jung Wu, Yu-Chih Wu, Yu-Ching Hung, Chih-Cheng Chang, Thai-Yen Ling, Yen-Hua Huang, Cheng-Chieh Chin, Hong-Nerng Ho, Chuan-Kai Chou, Chia-Ning Shen, Hung-Chih Kuo, Tsai-Jung Wu, Yu-Chih Wu, Yu-Ching Hung, Chih-Cheng Chang, Thai-Yen Ling, Yen-Hua Huang, Cheng-Chieh Chin, Hong-Nerng Ho, Chuan-Kai Chou, Chia-Ning Shen, Hung-Chih Kuo, Tsai-Jung Wu, Yu-Chih Wu, Yu-Ching Hung, Chih-Cheng Chang, Thai-Yen Ling
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology 23 2076-87 2009
Recent studies indicate that neonatal spermatogonial stem cells (SSCs) possess pluripotency. However, the mechanisms that regulate the pluripotent differentiation capacity of SSCs remain unclear. Here, we describe a new method to clonally derive pluripotent SSCs from neonatal mouse testis. By coculturing with testicular stromal cells, SSCs can be maintained and expanded in serum-free conditions. Unlike endogenous SSCs, these in vitro expanded SSCs showed strong alkaline phosphatase (AP) activity and displayed characteristics of embryonic stem cells and primordial germ cells, which were therefore designated as AP(+) germline stem cells (AP(+)GSCs). The pluripotency of AP(+)GSCs was confirmed by in vitro differentiation toward hepatic and neuronal lineages and formation of embryonic chimeras after injection into blastocysts. Further investigation revealed that insulin-like growth factor-1 (IGF-1) secreted from Leydig cells was a key factor involved in maintaining the pluripotency of AP(+)GSCs. The blockage of IGF-1 receptor phosphorylation and its downstream PI3K pathway by PPP or LY294002 dramatically reduced their AP activity and expression of pluripotent genes, such as Oct-4, Blimp1, and Nanog. In conclusion, the present study demonstrated that IGF-1 secreted by testicular Leydig cells plays an important role in maintaining the pluripotency of SSCs in culture, which provides an insight into the molecular mechanism underlying germ cell pluripotency.
|Tissue-specific stem cells: friend or foe? |
Joerg Huelsken, Joerg Huelsken, Joerg Huelsken
Cell research 19 279-81 2009
|Intermediate neuronal progenitors (basal progenitors) produce pyramidal-projection neurons for all layers of cerebral cortex. |
Kowalczyk, T; Pontious, A; Englund, C; Daza, RA; Bedogni, F; Hodge, R; Attardo, A; Bell, C; Huttner, WB; Hevner, RF
Cerebral cortex (New York, N.Y. : 1991) 19 2439-50 2009
The developing cerebral cortex contains apical and basal types of neurogenic progenitor cells. Here, we investigated the cellular properties and neurogenic output of basal progenitors, also called intermediate neuronal progenitors (INPs). We found that basal mitoses expressing transcription factor Tbr2 (an INP marker) were present throughout corticogenesis, from embryonic day 10.5 through birth. Postnatally, Tbr2(+) progenitors were present in the dentate gyrus, subventricular zone (SVZ), and posterior periventricle (pPV). Two morphological subtypes of INPs were distinguished in the embryonic cortex, "short radial" in the ventricular zone (VZ) and multipolar in the SVZ, probably corresponding to molecularly defined INP subtypes. Unexpectedly, many short radial INPs appeared to contact the apical (ventricular) surface and some divided there. Time-lapse video microscopy suggested that apical INP divisions produced daughter INPs. Analysis of neurogenic divisions (Tis21-green fluorescent protein [GFP](+)) indicated that INPs may produce the majority of projection neurons for preplate, deep, and superficial layers. Conversely, proliferative INP divisions (Tis21-GFP(-)) increased from early to middle corticogenesis, concomitant with SVZ growth. Our findings support the hypothesis that regulated amplification of INPs may be an important factor controlling the balance of neurogenesis among different cortical layers.Artículo Texto completo
|Convergence of cells from the progenitor fraction of adult olfactory bulb tissue to remyelinating glia in demyelinating spinal cord lesions. |
Markakis, EA; Sasaki, M; Lankford, KL; Kocsis, JD
PloS one 4 e7260 2009
Progenitor cells isolated from adult brain tissue are important tools for experimental studies of remyelination. Cells harvested from neurogenic regions in the adult brain such as the subependymal zone have demonstrated remyelination potential. Multipotent cells from the progenitor fraction have been isolated from the adult olfactory bulb (OB) but their potential to remyelinate has not been studied.We used the buoyant density gradient centrifugation method to isolate the progenitor fraction and harvest self-renewing multipotent neural cells grown in monolayers from the adult green-fluorescent protein (GFP) transgenic rat OB. OB tissue was mechanically and chemically dissociated and the resultant cell suspension fractionated on a Percoll gradient. The progenitor fraction was isolated and these cells were plated in growth media with serum for 24 hrs. Cells were then propagated in N2 supplemented serum-free media containing b-FGF. Cells at passage 4 (P4) were introduced into a demyelinated spinal cord lesion. The GFP(+) cells survived and integrated into the lesion, and extensive remyelination was observed in plastic sections. Immunohistochemistry revealed GFP(+) cells in the spinal cord to be glial fibrillary acidic protein (GFAP), neuronal nuclei (NeuN), and neurofilament negative. The GFP(+) cells were found among primarily P0(+) myelin profiles, although some myelin basic protein (MBP) profiles were present. Immuno-electron microscopy for GFP revealed GFP(+) cell bodies adjacent to and surrounding peripheral-type myelin rings.We report that neural cells from the progenitor fraction of the adult rat OB grown in monolayers can be expanded for several passages in culture and that upon transplantation into a demyelinated spinal cord lesion provide extensive remyelination without ectopic neuronal differentiation.
|Immunohistochemical markers for quantitative studies of neurons and glia in human neocortex. |
Lyck, L; Dalmau, I; Chemnitz, J; Finsen, B; Schrøder, HD
The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 56 201-21 2008
Reproducible visualization of neurons and glia in human brain is essential for quantitative studies of the cellular changes in neurological disease. However, immunohistochemistry in human brain specimens is often compromised because of prolonged fixation. To select cell lineage-specific antibodies for quantitative studies of neurons and the major types of glia, we used 29 different antibodies, different epitope retrieval methods, and different detection systems to stain tissue arrays of formalin-fixed human brain. The screening pointed at CD45/leukocyte common antigen (LCA), CD68(KP1), 2',3' cyclic nucleotide phosphatase (CNPase), glial fibrillary acidic protein (GFAP), HLA-DR, Ki67, neuronal nuclei (NeuN), p25alpha-antigen, and S100beta as candidates for future cell counting purposes, because these markers visualized specific neuronal and glial cell bodies. However, significant negative correlation between staining result and formalin fixation was observed by blinded scoring of staining for CD45/LCA, CNPase, GFAP, and NeuN in brain specimens fixed by immersion and stored up to 10 years in 4% formalin solution at room temperature, independent of donor sex and postmortem interval. In contrast, improved preservation of NeuN and CNPase staining, and full preservation of GFAP and CD45/LCA staining in tissue fixed by perfusion and stored for up to 3 years in 0.1% paraformaldehyde solution at 4C, indicated that immunohistochemistry can be performed in well-preserved biobank material.Artículo Texto completo
|Immortalization of human neural stem cells with the c-myc mutant T58A. |
De Filippis, L; Ferrari, D; Rota Nodari, L; Amati, B; Snyder, E; Vescovi, AL
PloS one 3 e3310 2008
Human neural stem cells (hNSC) represent an essential source of renewable brain cells for both experimental studies and cell replacement therapies. Their relatively slow rate of proliferation and physiological senescence in culture make their use cumbersome under some experimental and pre-clinical settings. The immortalization of hNSC with the v-myc gene (v-IhNSC) has been shown to generate stem cells endowed with enhanced proliferative capacity, which greatly facilitates the study of hNSCs, both in vitro and in vivo. Despite the excellent safety properties displayed by v-IhNSCs--which do not transform in vitro and are not tumorigenic in vivo--the v-myc gene contains several mutations and recombination elements, whose role(s) and effects remains to be elucidated, yielding unresolved safety concerns. To address this issue, we used a c-myc T58A retroviral vector to establish an immortal cell line (T-IhNSC) from the same hNSCs used to generate the original v-IhNSCs and compared their characteristics with the latter, with hNSC and with hNSC immortalized using c-myc wt (c-IhNSC). T-IhNSCs displayed an enhanced self-renewal ability, with their proliferative capacity and clonogenic potential being remarkably comparable to those of v-IhNSC and higher than wild type hNSCs and c-IhNSCs. Upon growth factors removal, T-IhNSC promptly gave rise to well-differentiated neurons, astrocytes and most importantly, to a heretofore undocumented high percentage of human oligodendrocytes (up to 23%). Persistent growth-factor dependence, steady functional properties, lack of ability to generate colonies in soft-agar colony-forming assay and to establish tumors upon orthotopic transplantation, point to the fact that immortalization by c-myc T58A does not bring about tumorigenicity in hNSCs. Hence, this work describes a novel and continuous cell line of immortalized human multipotent neural stem cells, in which the immortalizing agent is represented by a single gene which, in turn, carries a single and well characterized mutation. From a different perspective, these data report on a safe approach to increase human neural stem cells propagation in culture, without altering their basic properties. These T-IhNSC line provides a versatile model for the elucidation of the mechanisms involved in human neural stem cells expansion and for development of high throughput assays for both basic and translational research on human neural cell development. The improved proclivity of T-IhNSC to generate human oligodendrocytes propose T-IhNSC as a feasible candidate for the design of experimental and, perhaps, therapeutic approaches in demyelinating diseases.Artículo Texto completo
|Glial progenitor-like phenotype in low-grade glioma and enhanced CD133-expression and neuronal lineage differentiation potential in high-grade glioma. |
Rebetz, J; Tian, D; Persson, A; Widegren, B; Salford, LG; Englund, E; Gisselsson, D; Fan, X
PloS one 3 e1936 2008
While neurosphere- as well as xenograft tumor-initiating cells have been identified in gliomas, the resemblance between glioma cells and neural stem/progenitor cells as well as the prognostic value of stem/progenitor cell marker expression in glioma are poorly clarified.Viable glioma cells were characterized for surface marker expression along the glial genesis hierarchy. Six low-grade and 17 high-grade glioma specimens were flow-cytometrically analyzed for markers characteristics of stem cells (CD133); glial progenitors (PDGFRalpha, A2B5, O4, and CD44); and late oligodendrocyte progenitors (O1). In parallel, the expression of glial fibrillary acidic protein (GFAP), synaptophysin and neuron-specific enolase (NSE) was immunohistochemically analyzed in fixed tissue specimens. Irrespective of the grade and morphological diagnosis of gliomas, glioma cells concomitantly expressed PDGFRalpha, A2B5, O4, CD44 and GFAP. In contrast, O1 was weakly expressed in all low-grade and the majority of high-grade glioma specimens analyzed. Co-expression of neuronal markers was observed in all high-grade, but not low-grade, glioma specimens analyzed. The rare CD133 expressing cells in low-grade glioma specimens typically co-expressed vessel endothelial marker CD31. In contrast, distinct CD133 expression profiles in up to 90% of CD45-negative glioma cells were observed in 12 of the 17 high-grade glioma specimens and the majority of these CD133 expressing cells were CD31 negative. The CD133 expression correlates inversely with length of patient survival. Surprisingly, cytogenetic analysis showed that gliomas contained normal and abnormal cell karyotypes with hitherto indistinguishable phenotype.This study constitutes an important step towards clarification of lineage commitment and differentiation blockage of glioma cells. Our data suggest that glioma cells may resemble expansion of glial lineage progenitor cells with compromised differentiation capacity downstream of A2B5 and O4 expression. The concurrent expression of neuronal markers demonstrates that high-grade glioma cells are endowed with multi-lineage differentiation potential in vivo. Importantly, enhanced CD133 expression marks a poor prognosis in gliomas.Artículo Texto completo
|Establishment of autologous embryonic stem cells derived from preantral follicle culture and oocyte parthenogenesis. |
Seung Tae Lee, Mun Hwan Choi, Eun Ju Lee, Seung Pyo Gong, Mi Jang, Sang Hyun Park, Hyang Jee, Dae Yong Kim, Jae Yong Han, Jeong Mook Lim
Fertility and sterility 90 1910-20 2008
OBJECTIVE: To evaluate whether autologous embryonic stem cells can be established without generating clone embryos. DESIGN: Prospective model study. SETTING: Gamete and stem cell biotechnology laboratory in Seoul National University, Seoul, Korea. ANIMAL(S): F1 hybrid B6D2F1 mice. INTERVENTION(S): Preantral follicles were cultured, and oocytes matured in the follicles were parthenogenetically activated. MAIN OUTCOME MEASURE(S): Preimplantation development and stem cell characterization. RESULT(S): More intrafollicular oocytes that were retrieved from secondary follicles matured and developed into blastocysts after parthenogenesis than those that were retrieved from primary follicles. Of those 35 blastocysts derived from 193 parthenotes, one line of colony-forming cells was established from the culturing of early secondary follicles. The established cells were positive for embryonic stem cell-specific markers and had normal diploid karyotype and telomerase activity. They differentiated into embryoid bodies in vitro and teratomas in vivo. Inducible differentiation of the established cells into neuronal lineage cells also was possible. CONCLUSION(S): Autologous embryonic stem cells can be established by preantral follicle culture and oocyte parthenogenesis. A combined technique of follicle culture and oocyte parthenogenesis that does not use developmentally competent oocytes has the potential to replace somatic cell nuclear transfer for autologous cell therapy.
|Differentiation of human embryonic stem cells to regional specific neural precursors in chemically defined medium conditions. |
Erceg, Slaven, et al.
PLoS ONE, 3: e2122 (2008) 2008
|Opalin, a transmembrane sialylglycoprotein located in the central nervous system myelin paranodal loop membrane. |
Yoshikawa, Fumio, et al.
J. Biol. Chem., 283: 20830-40 (2008) 2008
In contrast to compact myelin, the series of paranodal loops located in the outermost lateral region of myelin is non-compact; the intracellular space is filled by a continuous channel of cytoplasm, the extracellular surfaces between neighboring loops keep a definite distance, but the loop membranes have junctional specializations. Although the proteins that form compact myelin have been well studied, the protein components of paranodal loop membranes are not fully understood. This report describes the biochemical characterization and expression of Opalin as a novel membrane protein in paranodal loops. Mouse Opalin is composed of a short N-terminal extracellular domain (amino acid residues 1-30), a transmembrane domain (residues 31-53), and a long C-terminal intracellular domain (residues 54-143). Opalin is enriched in myelin of the central nervous system, but not that of the peripheral nervous system of mice. Enzymatic deglycosylation showed that myelin Opalin contained N- and O-glycans, and that the O-glycans, at least, had negatively charged sialic acids. We identified two N-glycan sites at Asn-6 and Asn-12 and an O-glycan site at Thr-14 in the extracellular domain. Site-directed mutations at the glycan sites impaired the cell surface localization of Opalin. In addition to the somata and processes of oligodendrocytes, Opalin immunoreactivity was observed in myelinated axons in a spiral fashion, and was concentrated in the paranodal loop region. Immunogold electron microscopy demonstrated that Opalin was localized at particular sites in the paranodal loop membrane. These results suggest a role for highly sialylglycosylated Opalin in an intermembranous function of the myelin paranodal loops in the central nervous system.
|Immunohistochemistry (Tissue)||Mouse, Rat||18490449|
|Differentiation of postnatal neural stem cells into glia and functional neurons on laminin-coated polymeric substrates. |
Cristina Martínez-Ramos, Sergio Lainez, Francisco Sancho, M Angeles García Esparza, Rosa Planells-Cases, José Manuel García Verdugo, José Luis Gómez Ribelles, Manuel Salmerón Sánchez, Manuel Monleón Pradas, Juan Antonio Barcia, José Miguel Soria
Tissue engineering. Part A 14 1365-75 2008
A series of polymeric biomaterials, including poly(methyl acrylate), chitosan, poly(ethyl acrylate) (PEA), poly(hydroxyethyl acrylate) (PHEA), and a series of random copolymers containing ethyl acrylate, hydroxyethyl acrylate, and methyl acrylate were tested in vitro as culture substrates and compared for their effect on the differentiation of neural stem cells (NSCs) obtained from the subventricular zone of postnatal rats. Immunocytochemical assay for specific markers and scanning electron microscopy techniques were employed to determine the adhesion of the cultured NSCs to the different biomaterials and the respective neuronal differentiation. The functional properties and the membrane excitability of differentiated NSCs were investigated using a patch-clamp. The results show that the substrate's surface chemistry influences cell attachment and neuronal differentiation, probably through its influence on adsorbed laminin, and that copolymers based on PEA and PHEA in a narrow composition window are suitable substrates to promote cell attachment and differentiation of adult NSCs into functional neurons and glia.
|Legume lectin FRIL preserves neural progenitor cells in suspension culture in vitro. |
Yao, H; Xie, X; Li, Y; Wang, D; Han, S; Shi, S; Nan, X; Bai, C; Wang, Y; Pei, X
Clinical & developmental immunology 2008 531317 2008
In vitro maintenance of stem cells is crucial for many clinical applications. Stem cell preservation factor FRIL (Flt3 receptor-interacting lectin) is a plant lectin extracted from Dolichos Lablab and has been found preserve hematopoietic stem cells in vitro for a month in our previous studies. To investigate whether FRIL can preserve neural progenitor cells (NPCs), it was supplemented into serum-free suspension culture media. FRIL made NPC grow slowly, induced cell adhesion, and delayed neurospheres formation. However, FRIL did not initiate NPC differentiation according to immunofluorescence and semiquantitive RT-PCR results. In conclusion, FRIL could also preserve neural progenitor cells in vitro by inhibiting both cell proliferation and differentiation.
|Activation of the Wnt/beta-catenin signaling reporter in developing mouse olfactory nerve layer marks a specialized subgroup of olfactory ensheathing cells. |
Wang, YZ; Molotkov, A; Song, L; Li, Y; Pleasure, DE; Zhou, CJ
Developmental dynamics : an official publication of the American Association of Anatomists 237 3157-68 2008
Wnt reporter TOPgal mice carry a beta-galactosidase (betagal) gene under the control of the Wnt/beta-catenin signaling responsive elements. We found that the intensely immunolabeled betagal+ cells were co-immunolabeled with Nestin and formed a tangentially oriented single-cell layer in the "connecting or docking zone" where the olfactory sensory axons attached to the brain surface during mid-gestation. During early postnatal development, betagal+ cells were located in the inner olfactory nerve layer (ONLi) and co-labeled with olfactory ensheathing cell (OEC) markers S100beta and NPY but not with lineage-specific markers for neurons, oligodendrocytes, astrocytes, and microglia, demonstrating that the TOPgal marked a subpopulation of OECs. By confocal microscopy, we found that TOPgal activated processes extended along the developing glomerulus and formed multiple tunnel-like structures that ensheathe and bridge olfactory sensory axonal bundles from ONLi to the glomerulus, which may play a key role in glomerulus formation and convergent sorting of the peripheral olfactory axons.
|Olig2-induced neural stem cell differentiation involves downregulation of Wnt signaling and induction of Dickkopf-1 expression. |
Ahn, SM; Byun, K; Kim, D; Lee, K; Yoo, JS; Kim, SU; Jho, EH; Simpson, RJ; Lee, B
PloS one 3 e3917 2008
Understanding stem cell-differentiation at the molecular level is important for clinical applications of stem cells and for finding new therapeutic approaches in the context of cancer stem cells. To investigate genome-wide changes involved in differentiation, we have used immortalized neural stem cell (NSC) line (HB1.F3) and Olig2-induced NSC differentiation model (F3.Olig2). Using microarray analysis, we revealed that Olig2-induced NSC differentiation involves downregulation of Wnt pathway, which was further confirmed by TOPflash/FOPflash reporter assay, RT-PCR analysis, immunoblots, and immunocytochemistry. Furthermore, we found that Olig2-induced differentiation induces the expression of Dickkopf-1(Dkk1), a potent antagonist of Wnt signaling. Dkk1 treatment blocked Wnt signaling in HB1.F3 in a dosage-dependent manner, and induced differentiation into astrocytes, oligodendrocytes, and neurons. Our results support cancer stem cell hypothesis which implies that signaling pathway for self-renewal and proliferation of stem cells is maintained till the late stage of differentiation. In our proposed model, Dkk1 may play an important role in downregulating self-renewal and proliferation pathway of stem cells at the late stage of differentiation, and its failure may lead to carcinogenesis.
|Genesis of neuronal and glial progenitors in the cerebellar cortex of peripuberal and adult rabbits. |
Ponti, G; Peretto, P; Bonfanti, L
PloS one 3 e2366 2008
Adult neurogenesis in mammals is restricted to some brain regions, in contrast with other vertebrates in which the genesis of new neurons is more widespread in different areas of the nervous system. In the mammalian cerebellum, neurogenesis is thought to be limited to the early postnatal period, coinciding with end of the granule cell genesis and disappearance of the external granule cell layer (EGL). We recently showed that in the rabbit cerebellum the EGL is replaced by a proliferative layer called 'subpial layer' (SPL) which persists beyond puberty on the cerebellar surface. Here we investigated what happens in the cerebellar cortex of peripuberal rabbits by using endogenous and exogenously-administered cell proliferation antigens in association with a cohort of typical markers for neurogenesis. We show that cortical cell progenitors extensively continue to be generated herein. Surprisingly, this neurogenic process continues to a lesser extent in the adult, even in the absence of a proliferative SPL. We describe two populations of newly generated cells, involving neuronal cells and multipolar, glia-like cells. The genesis of neuronal precursors is restricted to the molecular layer, giving rise to cells immunoreactive for GABA, and for the transcription factor Pax2, a marker for GABAergic cerebellar interneuronal precursors of neuroepithelial origin that ascend through the white matter during early postnatal development. The multipolar cells are Map5+, contain Olig2 and Sox2 transcription factors, and are detectable in all cerebellar layers. Some dividing Sox2+ cells are Bergmann glia cells. All the cortical newly generated cells are independent from the SPL and from granule cell genesis, the latter ending before puberty. This study reveals that adult cerebellar neurogenesis can exist in some mammals. Since rabbits have a longer lifespan than rodents, the protracted neurogenesis within its cerebellar parenchyma could be a suitable model for studying adult nervous tissue permissiveness in mammals.
|Developing oligodendrocytes express functional GABA(B) receptors that stimulate cell proliferation and migration. |
Karen Luyt, Timothy P Slade, Jienchi J Dorward, Claire F Durant, Yue Wu, Ryuichi Shigemoto, Stuart J Mundell, Anikó Váradi, Elek Molnár
Journal of neurochemistry 100 822-40 2007
GABA(B) receptors (GABA(B)Rs) are involved in early events during neuronal development. The presence of GABA(B)Rs in developing oligodendrocytes has not been established. Using immunofluorescent co-localization, we have identified GABA(B)R proteins in O4 marker-positive oligodendrocyte precursor cells (OPCs) in 4-day-old mouse brain periventricular white matter. In culture, OPCs, differentiated oligodendrocytes (DOs) and type 2 astrocytes (ASTs) express both the GABA(B1abcdf) and GABA(B2) subunits of the GABA(B)R. Using semiquantitative PCR analysis with GABA(B)R isoform-selective primers we found that the expression level of GABA(B1abd) was substantially higher in OPCs or ASTs than in DOs. In contrast, the GABA(B2) isoform showed a similar level of expression in OPCs and DOs, and a significantly higher level in ASTs. This indicates that the expression of GABA(B1) and GABA(B2) subunits are under independent control during oligodendroglial development. Activation of GABA(B)Rs using the selective agonist baclofen demonstrated that these receptors are functionally active and negatively coupled to adenylyl cyclase. Manipulation of GABA(B)R activity had no effect on OPC migration in a conventional agarose drop assay, whereas baclofen significantly increased OPC migration in a more sensitive transwell microchamber-based assay. Exposure of cultured OPCs to baclofen increased their proliferation, providing evidence for a functional role of GABA(B)Rs in oligodendrocyte development. The presence of GABA(B)Rs in developing oligodendrocytes provides a new mechanism for neuronal-glial interactions during development and may offer a novel target for promoting remyelination following white matter injury.
|A novel, immortal, and multipotent human neural stem cell line generating functional neurons and oligodendrocytes. |
Lidia De Filippis, Giuseppe Lamorte, Evan Y Snyder, Antonio Malgaroli, Angelo L Vescovi
Stem cells (Dayton, Ohio) 25 2312-21 2007
The discovery and study of neural stem cells have revolutionized our understanding of the neurogenetic process, and their inherent ability to adopt expansive growth behavior in vitro is of paramount importance for the development of novel therapeutics based on neural cell replacement. Recent advances in high-throughput assays for drug development and gene discovery dictate the need for rapid, reproducible, long-term expansion of human neural stem cells (hNSCs). In this view, the complement of wild-type cell lines currently available is insufficient. Here we report the establishment of a stable human neural stem cell line (immortalized human NSCs [IhNSCs]) by v-myc-mediated immortalization of previously derived wild-type hNSCs. These cells demonstrate three- to fourfold faster proliferation than wild-type cells in response to growth factors but retain rather similar properties, including multipotentiality. By molecular biology, biochemistry, immunocytochemistry, fluorescence microscopy, and electrophysiology, we show that upon growth factor removal, IhNSCs completely downregulate v-myc expression, cease proliferation, and differentiate terminally into three major neural lineages: astrocytes, oligodendrocytes, and neurons. The latter are functional, mature cells displaying clear-cut morphological and physiological features of terminally differentiated neurons, encompassing mostly the GABAergic, glutamatergic, and cholinergic phenotypes. Finally, IhNSCs produce bona fide oligodendrocytes in fractions up to 20% of total cell number. This is in contrast to the negligible propensity of hNSCs to generate oligodendroglia reported so far. Thus, we describe an immortalized hNSC line endowed with the properties of normal hNSCs and suitable for developing the novel, reliable assays and reproducible high-throughput gene and drug screening that are essential in both diagnostics and cell therapy studies.
|Increased number and differentiation of neural precursor cells in the brainstem of superoxide dismutase 1(G93A) (G1H) transgenic mouse model of amyotrophic lateral sclerosis. |
Liu Juan,Zang Dawei,Atkin D Julie
Neurological research 29 2007
The superoxide dismutase 1(G93A G1H) (SOD1(G93A G1H)) transgenic mouse is a model of familial human amyotrophic lateral sclerosis (ALS) that has progressive neurodegeneration within the spinal cord and brainstem. In this study, we investigated the number and differentiation of neural precursor cells (NPCs). Nestin-positive NPCs were rarely seen in the nervous system of wild type controls or pre-disease mice at post-natal days 30 and 60. With disease onset on post-natal day 90, nestin labeled NPCs proliferated preferentially in the brainstem with maximal number and density at post-natal day 120. NPCs did not double-label with CNPase or O(4) markers of oligodendrocytes. The majority of the NPCs co-labeled with the astrocyte maker glial fibrillary acidic protein (GFAP) and a small number with the neuronal marker NeuN. At disease onset, 73 and 10% of NPCs co-expressed GFAP and NeuN respectively while at severe disease stage, 80 and 8% of the NPCs co-expressed GFAP and NeuN. Proliferating cell nuclear antigen (PCNA) was used to confirm that at least some of these cells undergo mitosis. Future studies could be directed at controlling the differentiation of these endogenous NPCs into neurons and astrocytes in order to ameliorate the degeneration within the brainstem of the ALS mouse.
|Neural precursor cells from a fatal human motoneuron disease differentiate despite aberrant gene expression. |
Niklas Pakkasjärvi, Laura Kerosuo, Heidi Nousiainen, Massimiliano Gentile, Juha Saharinen, Satu Suhonen, Hannu Sariola, Leena Peltonen, Marjo Kestilä, Kirmo Wartiovaara, Niklas Pakkasjärvi, Laura Kerosuo, Heidi Nousiainen, Massimiliano Gentile, Juha Saharinen, Satu Suhonen, Hannu Sariola, Leena Peltonen, Marjo Kestilä, Kirmo Wartiovaara
Developmental neurobiology 67 270-84 2007
Precursor cells of the human central nervous system can be cultured in vitro to reveal pathogenesis of diseases or developmental disorders. Here, we have studied the biology of neural precursor cells (NPCs) from patients of lethal congenital contracture syndrome (LCCS), a severe motoneuron disease leading to prenatal death before the 32nd gestational week. LCCS fetuses are immobile because of a motoneuron defect, seen as degeneration of the anterior horn and descending tracts of the developing spinal cord. The genetic defect for the syndrome is unknown. We show that NPCs isolated postmortem from LCCS fetuses grow and are maintained in culture, but display increased cell cycle activity. Global transcript analysis of undifferentiated LCCS precursor cells present with changes in EGF-related signaling when compared with healthy age-matched human controls. Further, we show that LCCS-derived NPCs differentiate into cells of neuronal and glial lineage and that the initial differentiation is not accompanied by overt apoptosis. Cells expressing markers Islet-1 and Hb9 are also generated from the LCCS NPCs, suggesting that the pathogenic mechanism of LCCS does not directly affect the differentiation capacity or survival of the cells, but the absence of motoneurons in LCCS may be caused by a noncell autonomous mechanism.
|Astrocytes promote myelination in response to electrical impulses |
Ishibashi, T. et al.
Neuron, 49(6):823-832 (2006) 2006
|A subpial, transitory germinal zone forms chains of neuronal precursors in the rabbit cerebellum |
Ponti, Giovanna, et al
Dev Biol, 294:168-80 (2006) 2006
|Scaled-up production of mammalian neural precursor cell aggregates in computer-controlled suspension bioreactors. |
Jane A Gilbertson, Arindom Sen, Leo A Behie, Michael S Kallos
Biotechnology and bioengineering 94 783-92 2006
The clinical use of neural precursor cells (NPCs) for the treatment of neurological diseases, such as Parkinson's disease and Huntington's disease, requires overcoming the scarcity of these cells through controlled expansion. The main objective of the present study was to develop a large-scale computer-controlled bioprocess for the expansion of mammalian NPCs in suspension culture by scaling up existing reactor protocols. In order to support the oxygen demands of the maximum cell densities achieved, the volumetric mass transfer coefficient was kept above 1.10/h while scaling-up from small-scale 125 mL vessels to large-scale 500 mL bioreactors. In addition, the maximum shear stress at the impeller tip was maintained between 0.30 and 0.75 Pa to reduce damage to the cells. The resulting large-scale bioprocess achieved maximum viable cell densities of 1.2 x 10(6) cells/mL and a batch multiplication ratio of 9.1. Moreover, the process successfully maintained the NPC characteristics observed in small-scale studies.
|Differentiated human neural stem cells: a new ex vivo model to study HHV-6 infection of the central nervous system. |
Lidia De Filippis, Chiara Foglieni, Sara Silva, Angelo L Vescovi, Paolo Lusso, Mauro S Malnati
Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology 37 Suppl 1 S27-32 2006
BACKGROUND: HHV-6 is the etiologic agent of exanthem subitum, a pediatric illness that may be associated with clinical and laboratory signs of central nervous system involvement. The absence of suitable experimental models has so far hampered the elucidation of the mechanisms of HHV-6-mediated neural cell damage. Recently, the growing knowledge in neurobiology has permitted the establishment of long-term cultures of human neural stem cells (hNSC) that, by virtue of their self-renewal capacity and multipotentiality, provide a valuable tool for the study of neurodegenerative disorders. OBJECTIVES AND STUDY DESIGN: We studied the effects of HHV-6 infection in differentiated cultures of hNSC derived from the telencephalic and diencephalic regions of a 13.5 week post conception (pcw) fetal brain. The prototypic HHV-6 strain GS (subgroup A) was used. RESULTS: hNSC were differentiated ex vivo to obtain mixed cultures encompassing astrocytes, neurons and oligodendrocytes. These differentiated hNSC cultures were found to be susceptible to productive HHV-6A infection, resulting in the formation of syncytia associated with phenotypic alterations. CONCLUSION: These results demonstrate that hNSC may provide a physiologically relevant model to investigate the pathogenic role of HHV-6 in central nervous system disorders.
|Limited influence of olanzapine on adult forebrain neural precursors in vitro. |
J H Councill, E S Tucker, G T Haskell, T M Maynard, D W Meechan, R M Hamer, J A Lieberman, A-S LaMantia
Neuroscience 140 111-22 2006
We evaluated the activity of the atypical antipsychotic drug olanzapine on differentiation and gene expression in adult neural precursor cells in vitro. Neural precursors obtained from forebrain subventricular zone (SVZ)-derived neurospheres express a subset (13/24) of receptors known to bind olanzapine at high to intermediate affinities; in contrast, all 24 are expressed in the SVZ. In the presence of 10 nM, 100 nM or 1 microM olanzapine, there is no significant change in the frequency of oligodendrocytes, neurons, GABAergic neurons and astrocytes generated from neurosphere precursors. In parallel, there is no apparent change in cell proliferation in response to olanzapine, based upon bromodeoxyuridine incorporation. There are no major changes in cytological differentiation in response to the drug; however, at one concentration (10 nM) there is a small but statistically significant increase in the size of glial fibrillary acidic protein-labeled astrocytes derived from neurosphere precursors. In addition, olanzapine apparently modulates expression of one serotonin receptor -- 5HT2A -- in differentiating neurosphere cultures; however, it does not modify expression of several other receptors or schizophrenia vulnerability genes. Thus, olanzapine has a limited influence on differentiation and gene expression in adult neural precursor cells in vitro.
|Glioblastoma-induced attraction of endogenous neural precursor cells is associated with improved survival. |
Glass, Rainer, et al.
J. Neurosci., 25: 2637-46 (2005) 2005
|Neuro-glial differentiation of human bone marrow stem cells in vitro. |
Bossolasco, P, et al.
Exp. Neurol., 193: 312-25 (2005) 2005
Bone marrow (BM) is a rich source of stem cells and may represent a valid alternative to neural or embryonic cells in replacing autologous damaged tissues for neurodegenerative diseases. The purpose of the present study is to identify human adult BM progenitor cells capable of neuro-glial differentiation and to develop effective protocols of trans-differentiation to surmount the hematopoietic commitment in vitro. Heterogeneous cell populations such as whole BM, low-density mononuclear and mesenchymal stem (MSCs), and several immunomagnetically separated cell populations were investigated. Among them, MSCs and CD90+ cells were demonstrated to express neuro-glial transcripts before any treatment. Several culture conditions with the addition of stem cell or astroblast conditioned media, different concentrations of serum, growth factors, and supplements, used alone or in combinations, were demonstrated to alter the cellular morphology in some cell subpopulations. In particular, MSCs and CD90+ cells acquired astrocytic and neuron-like morphologies in specific culture conditions. They expressed several neuro-glial specific markers by RT-PCR and glial fibrillary acid protein by immunocytochemistry after co-culture with astroblasts, both in the absence or presence of cell contact. In addition, floating neurosphere-like clones have been observed when CD90+ cells were grown in neural specific media. In conclusion, among the large variety of human adult BM cell populations analyzed, we demonstrated the in vitro neuro-glial potential of both the MSC and CD90+ subset of cells. Moreover, unidentified soluble factors provided by the conditioned media and cellular contacts in co-culture systems were effective in inducing the neuro-glial phenotype, further supporting the adult BM neural differentiative capability.
|Human embryonic stem cell-derived oligodendrocyte progenitor cell transplants remyelinate and restore locomotion after spinal cord injury. |
Keirstead, Hans S, et al.
J. Neurosci., 25: 4694-705 (2005) 2005
|Maturational effects of lipopolysaccharide on white-matter injury in fetal sheep. |
Pernilla Svedin, Ingmar Kjellmer, Anna-Karin Welin, Sofia Blad, Carina Mallard
Journal of child neurology 20 960-4 2005
White-matter damage has been associated with the development of cerebral palsy in children born both prematurely and at term, and it has been suggested that intrauterine infection can contribute to the brain injury. However, the relative importance of age on white-matter injury following infectious exposure in utero remains unclear. In this study, fetal sheep were exposed to systemic endotoxemia by administration of Escherichia coli lipopolysaccharide (88.7 +/- 7.7 ng/kg) at 65% or 85% of gestation. These gestational ages approximately correspond to human brain development in preterm and near-term infants respectively. White-matter injury was evaluated 3 days after lipopolysaccharide exposure with regard to microglia activation and loss of neurofilament and myelin basic protein. The expression of oligodendrocytes at different maturational stages was demonstrated in preterm and near-term fetuses with the oligodendroglial markers O4 and 2 ,3 -cyclic nucleotide 3 -phospodiesterase. Forty percent of the fetuses in the preterm group and 22% in the near-term group died within 8 hours of the endotoxin exposure. Three of six preterm and two of seven near-term surviving fetuses demonstrated pathologic changes in the brain with regard to increased microglia activation and loss of neurofilament staining. The number of activated microglia was enhanced in the subcortical white matter in both the preterm lipopolysaccharide-exposed fetuses (lipopolysaccharide: 235 +/- 64 cells/mm2; control: 72 +/- 28 cells/mm2; P = .0374) and the near-term fetuses (lipopolysaccharide: 180 +/- 40 cells/mm2; control 23 +/- 16 cells/mm2; P = .0152). There was a loss of neurofilament staining in both preterm fetuses (lipopolysaccharide: 2.20 +/- 0.77 pixel units; control: 0.20 +/- 0.10 pixel units; P = .0306) and near-term fetuses (lipopolysaccharide: 1.15 +/- 0.48 pixel units; control: 0.06 +/- 0.06 pixel units; P = .0285). O4-positive cells were detected at both gestational ages, whereas 2,3-cyclic nucleotide 3-phospodiesterase-positive cells and myelin basic protein staining were mainly detected in the near-term fetuses. In summary, we found white-matter injury in a proportion of both preterm and near-term fetuses after administration of lipopolysaccharide. These results are in agreement with clinical evidence suggesting that both preterm and term infants are at risk of periventricular leukomalacia in association with intrauterine infection.
|Meteorin: a secreted protein that regulates glial cell differentiation and promotes axonal extension |
Nishino, J. et al.
EMBO J. , 23(9):1998-2008 (2004) 2004
|Neural stem cells transplanted into intact brains as neurospheres form solid grafts composed of neurons, astrocytes and oligodendrocyte precursors. |
Karbanová, Jana, et al.
Biomedical papers of the Medical Faculty of the University Palacký, Olomouc, Czechoslovakia, 148: 217-20 (2004) 2004
Neural stem cells (NSCs) are tissue-specific stem cells with self-renewal potential that can give rise to neurons and glia in vivo and in vitro. The aim of this study was to transplant NSCs as whole neurospheres into intact brain and assess the fate and phenotype of their progeny generated in vivo. We isolated NSCs from E14 foetal rat forebrains and cultured them in basic fibroblast and epidermal growth factor-supplemented serum-free medium in the form of neurospheres in vitro. Neurospheres were transplanted into the intact brains of 2 Wistar rats and after a period of 3 weeks, grafted brains were examined immunohistochemically. Neurospheres formed solid grafts that were found in the lateral ventricle and in the velum interpositum under the hippocampus. The majority of cells in the transplanted tissue were identified as beta-III-tubulin(+), NeuN(+), PanNF(+) and synaptophysin(+) neurons and were accumulated throughout the graft centre. GFAP(+) astrocytes were scattered throughout the entire graft and astrocyte processes delimited the outer and perivascular surfaces. A great number of NG2(+) oligodendrocyte precursors was detected. Nestin(+) endothelial cells were found to line capillaries growing in the transplant. These data indicate that nestin(+) NSCs prevailing in neurospheres differentiate following transplantation into nestin(-) neuronal and glial cells which confirms the multipotency of NSCs. Three weeks posttransplantation neuronal and astrocyte cells reached terminal differentiation (formation of synaptic vesicles and superficial and perivascular limiting membranes) while elements of oligodendroglial cell lineage remained immature. Grafting stem cells as non-dissociated neurospheres provide cells with favourable conditions which facilitate cell survival, proliferation and differentiation. However, in the intact brain, grafted neurosphere cells were not found to integrate with the brain parenchyma and formed a compact structure demarcated from its surroundings.
|Implication of gamma-secretase in neuregulin-induced maturation of oligodendrocytes. |
Lai, Chen and Feng, Linyin
Biochem. Biophys. Res. Commun., 314: 535-42 (2004) 2004
|Unique astrocyte ribbon in adult human brain contains neural stem cells but lacks chain migration. |
Sanai, Nader, et al.
Nature, 427: 740-4 (2004) 2004
The subventricular zone (SVZ) is a principal source of adult neural stem cells in the rodent brain, generating thousands of olfactory bulb neurons every day. If the adult human brain contains a comparable germinal region, this could have considerable implications for future neuroregenerative therapy. Stem cells have been isolated from the human brain, but the identity, organization and function of adult neural stem cells in the human SVZ are unknown. Here we describe a ribbon of SVZ astrocytes lining the lateral ventricles of the adult human brain that proliferate in vivo and behave as multipotent progenitor cells in vitro. This astrocytic ribbon has not been observed in other vertebrates studied. Unexpectedly, we find no evidence of chains of migrating neuroblasts in the SVZ or in the pathway to the olfactory bulb. Our work identifies SVZ astrocytes as neural stem cells in a niche of unique organization in the adult human brain.
|Distinct neural precursors in the developing human spinal cord. |
Walder, S; Ferretti, P
The International journal of developmental biology 48 671-4 2004
Both embryonic and adult central nervous system have been shown to contain multipotent neural stem cells, but it is not yet clear whether they consist of a single or distinct populations of neural precursors. Since embryonic human neural precursors, particularly in the spinal cord, have not been extensively characterized, we have studied their behaviour at different days of gestation and in different culture conditions. Depending on dissociation and culture conditions, neurospheres which contain nestin- and vimentin-positive or only vimentin-positive neural precursors can be isolated. Whereas the former can be isolated only at early developmental stages, the latter appear to be present at all the stages examined, between 45 and 89 days of gestation. Furthermore, comparison of the effect of FGF, EGF and the two factors in combination on colony formation shows an additive effect of the two growth factors, indicating the existence of more than one type of neural precursor. Overall our results suggest that the human spinal cord contains distinct and dynamic populations of neural precursors which are developmentally regulated.
|p107 regulates neural precursor cells in the mammalian brain. |
Vanderluit, JL; Ferguson, KL; Nikoletopoulou, V; Parker, M; Ruzhynsky, V; Alexson, T; McNamara, SM; Park, DS; Rudnicki, M; Slack, RS
The Journal of cell biology 166 853-63 2004
Here we show a novel function for Retinoblastoma family member, p107 in controlling stem cell expansion in the mammalian brain. Adult p107-null mice had elevated numbers of proliferating progenitor cells in their lateral ventricles. In vitro neurosphere assays revealed striking increases in the number of neurosphere forming cells from p107(-/-) brains that exhibited enhanced capacity for self-renewal. An expanded stem cell population in p107-deficient mice was shown in vivo by (a) increased numbers of slowly cycling cells in the lateral ventricles; and (b) accelerated rates of neural precursor repopulation after progenitor ablation. Notch1 was up-regulated in p107(-/-) neurospheres in vitro and brains in vivo. Chromatin immunoprecipitation and p107 overexpression suggest that p107 may modulate the Notch1 pathway. These results demonstrate a novel function for p107 that is distinct from Rb, which is to negatively regulate the number of neural stem cells in the developing and adult brain.
|ABC transporter inhibitors that are substrates enhance lentiviral vector transduction into primitive hematopoietic progenitor cells. |
Davis, BM; Humeau, L; Slepushkin, V; Binder, G; Korshalla, L; Ni, Y; Ogunjimi, EO; Chang, LF; Lu, X; Dropulic, B
Blood 104 364-73 2004
High gene transfer efficiencies have been difficult to achieve in hematopoietic progenitor cells (HPCs) but are important to therapeutic success of HPC gene therapy. Efficient gene transfer is especially challenging with use of column-purified vector for clinical application, as opposed to centrifuged vector commonly used for research. We investigated novel approaches to increase transduction by using a clinically applicable protocol and quantities of column-purified lentiviral vector. Recognizing the association of adenosine 5'-triphosphate (ATP)-binding cassette (ABC) transporters with HPC biology, we investigated the effect of transporter inhibitors on transduction. We found the ABC transporter inhibitor verapamil improved transduction efficiency 2- to 6-fold into CD34(+) cells isolated from mobilized peripheral blood, bone marrow, and cord blood. Verapamil also improved transduction in human SCID (severe combined immunodeficient) repopulating cell (SRC) transduction 3- to 4-fold, resulting in 80% to 90% transduction levels in mice receiving primary and secondary transplants without alterations in multilineage reconstitution. Additional ABC transporter substrate inhibitors like quinidine, diltiazem, and ritonavir also enhanced transduction 2- to 3-fold, although ABC transporter inhibitors that are not substrates did not. Enhanced transduction was not observed in mature hematopoietic cells, neurospheres, mesenchymal stem cells, or hepatocytes. Enhancement of transduction in HPCs was observed with vesicular stomatitis virus-G (VSV-G)-pseudotyped lentiviral vector but not with vector pseudotyped with RD114. Thus, we present a new approach for efficient delivery to primitive HPCs by VSV-G-pseudotyped lentiviral vectors.
|Analysis of Neurons Created from Wild-Type and Alzheimer's Mutation Knock-In Embryonic Stem Cells by a Highly Efficient Differentiation Protocol |
Abe Y., Kouyama K., Tomita T., Ban N., Nawa M., Matsuoka M., Niikura T., Aiso S., Kita Y., Iwatsubo T., & Nishimoto I.
J. Neurosci., 23 :8513-8525 (2003) 2003
|Purification of embryonic stem cell-derived neurons by immunoisolation. |
Jüngling, Kay, et al.
FASEB J., 17: 2100-2 (2003) 2003
The pluripotency and high proliferative capacity of embryonic stem (ES) cells (1-3) makes them an attractive source of different cell types for biomedical research and cell replacement therapies. A major prerequisite for these applications is the availability of a homogeneous population of the desired cell type. However, ES cell-derived material contains, for example, undifferentiated cells, which can cause tumor formation after transplantation into the brain (4). To avoid such unwanted side effects, effective purification of distinct types of cells needs to be developed. Here, we describe an immunoisolation procedure to purify neurons from in vitro differentiated mouse ES cells using an antibody against the neuronal cell adhesion molecule L1 (5, 6). Our procedure yields a pure population of differentiated neurons, which are electrically excitable and form excitatory, glutamatergic, and inhibitory GABAergic synapses. The ability to highly purify ES cell-derived neurons will boost their molecular characterization and the further exploration of their therapeutic potential.
|Neural subtype specification of fertilization and nuclear transfer embryonic stem cells and application in parkinsonian mice |
Barberi, T. et al.
Nat. Neurosci., 21(10):1200-1207 (2003) 2003
|Cancerous stem cells can arise from pediatric brain tumors |
Hemmati, Houman D, et al.
Proc Natl Acad Sci USA, 100:15178-15183 (2003) 2003
|Hematopoietic progenitors express neural genes. |
Goolsby, J; Marty, MC; Heletz, D; Chiappelli, J; Tashko, G; Yarnell, D; Fishman, PS; Dhib-Jalbut, S; Bever, CT; Pessac, B; Trisler, D
Proceedings of the National Academy of Sciences of the United States of America 100 14926-31 2003
Bone marrow, or cells selected from bone marrow, were reported recently to give rise to cells with a neural phenotype after in vitro treatment with neural-inducing factors or after delivery into the brain. However, we showed previously that untreated bone marrow cells express products of the neural myelin basic protein gene, and we demonstrate here that a subset of ex vivo bone marrow cells expresses the neurogenic transcription factor Pax-6 as well as neuronal genes encoding neurofilament H, NeuN (neuronal nuclear protein), HuC/HuD (Hu-antigen C/Hu-antigen D), and GAD65 (glutamic acid decarboxylase 65), as well as the oligodendroglial gene encoding CNPase (2',3' cyclic nucleotide 3'-phosphohydrolase). In contrast, astroglial glial fibrillary acidic protein (GFAP) was not detected. These cells also were CD34+, a marker of hematopoietic stem cells. Cultures of these highly proliferative CD34+ cells, derived from adult mouse bone marrow, uniformly displayed a phenotype comparable with that of hematopoietic progenitor cells (CD45+, CD34+, Sca-1+, AA4.1+, cKit+, GATA-2+, and LMO-2+). The neuronal and oligodendroglial genes expressed in ex vivo bone marrow also were expressed in all cultured CD34+ cells, and GFAP was not observed. After CD34+ cell transplantation into adult brain, neuronal or oligodendroglial markers segregated into distinct nonoverlapping cell populations, whereas astroglial GFAP appeared, in the absence of other neural markers, in a separate set of implanted cells. Thus, neuronal and oligodendroglial gene products are present in a subset of bone marrow cells, and the expression of these genes can be regulated in brain. The fact that these CD34+ cells also express transcription factors (Rex-1 and Oct-4) that are found in early development elicits the hypothesis that they may be pluripotent embryonic-like stem cells.
|Thyroid hormone activates oligodendrocyte precursors and increases a myelin-forming protein and NGF content in the spinal cord during experimental allergic encephalomyelitis. |
Calza, L; Fernandez, M; Giuliani, A; Aloe, L; Giardino, L
Proceedings of the National Academy of Sciences of the United States of America 99 3258-63 2002
Remyelination in the adult central nervous system has been demonstrated in different experimental models of demyelinating diseases. However, there is no clear evidence that remyelination occurs in multiple sclerosis, the most diffuse demyelinating disease. In this article, we explore the possibility of promoting myelination in experimental allergic encephalomyelitis, a widely used experimental model of multiple sclerosis, by recruiting progenitors and channeling them into oligodendroglial lineage through administration of thyroid hormone (T4). A large number of proliferating cells (BrdUrd uptake and Ki67-IR) and the expression of markers for undifferentiated precursors (nestin) increased in the subventricular zone and spinal cord of experimental allergic encephalomyelitis animals. T4 administration reduces proliferation and nestin-immunoreactivity and up-regulates expression of markers for oligodendrocyte progenitors [polysialylated-neural cell adhesion molecule (PSA-NCAM), O4, A2B5] and mature oligodendrocytes (myelin basic protein) in the spinal cord, olfactory bulb, and subventricular zone.Artículo Texto completo
|Chronic ischemia preferentially causes white matter injury in the neonatal rat brain. |
Cai, Z, et al.
Brain Res., 898: 126-35 (2001) 2001
|Distribution, differentiation, and survival of intravenously administered neural stem cells in a rat model of amyotrophic lateral sclerosis. |
Mitrecić D, Nicaise C, Gajović S, Pochet R
Cell Transplant 19 537-48. Epub 2010 Mar 26. 2001
The transplantation of neural stem cells (NSCs) is a challenging therapeutic strategy for the treatment of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). To provide insight into the potential of the intravenous delivery of NSCs, we evaluated the delivery of NSCs marked with green fluorescent protein to the central nervous system (CNS) via intravenous tail vein injections in an ALS model. The injected cell fates were followed 1, 3, and 7 days after transplantation. The highest efficiency of cell delivery to the CNS was found in symptomatic ALS (up to 13%), moderate in presymptomatic ALS (up to 6%), and the lowest in wild-type animals (up to 0.3%). NSCs injected into ALS animals preferentially colonized the motor cortex, hippocampus, and spinal cord, and their differentiation was characterized by a decrease of nestin expression and the appearance of MAP2-, GFAP-, O4-, and CD68-positive cells. Tumor necrosis factor (TNF) administration increased the CNS delivery of transplanted cells in wild-type and presymptomatic, but not ALS symptomatic animals. Moreover, a TNF-related increase in NSC differentiation and survival was detected. Apoptosis was detected as the main cause of the loss of transplanted cells and it was influenced by TNF. Although 3 days after TNF treatment cell death was accelerated, TNF slowed down apoptosis after 7 days. This study provides elementary facts about the process occurring after NSCs leave the blood stream and enter the nervous tissue affected by inflammation/degeneration, which should help facilitate the planning of future bench-to-bedside translational projects.
|Monoclonal antibodies (O1 to O4) to oligodendrocyte cell surfaces: an immunocytological study in the central nervous system |
Sommer, I. and Schachner, M.
Dev. Biol., 83:311-327 (1981) 1981
|Developmental expression in central and peripheral nervous system of oligodendrocyte cell surface antigens (O antigens) recognized by monoclonal antibodies |
Schachner, M. et al.
Dev. Biol., 83:328-338 (1981) 1981
|Immuno-electron-microscopic identification of O-antigen-bearing oligodendroglial cells in vitro |
Berg, G and Schachner, M
Cell Tissue Res, 219:313-25 (1981) 1981
Licencias necesarias e Información técnica
|Derivation of oligodendrocyte progenitor cells from a human neural stem cell line|
|Anti-O4, clone 81 (also referred to in the literature as mAB O4) - Data Sheet|
Newsletters / Publications
|Cellutions - The newsletter for Cell Biology Researchers Volume 3: 2011|