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  • Function of mouse embryonic stem cell-derived supporting cells in neural progenitor cell maturation and long term expansion. 23342136

    In the differentiation of mouse embryonic stem (ES) cells into neurons using the 5-stage method, cells in stage 4 are in general used as neural progenitors (NPs) because of their ability to give rise to neurons. The choice of stage 4 raises several questions about neural progenitors such as the type of cell types that are specifically considered to be neural progenitors, the exact time when these progenitors become capable of neurogenesis and whether neurogenesis is an independent and autonomous process or the result of an interaction between NP cells and the surrounding cells.In this study, we found that the confluent monolayer cells and neural sphere like cell clusters both appeared in the culture of the first 14 days and the subsequent 6 weeks. However, only the sphere cells are neural progenitors that give rise to neurons and astrocytes. The NP cells require 14 days to mature into neural lineages fully capable of differentiation. We also found that although the confluent monolayer cells do not undergo neurogenesis, they play a crucial role in the growth, differentiation, and apoptosis of the sphere cells, during the first 14 days and long term culture, by secreted factors and direct cell to cell contact.The sphere cells in stage 4 are more committed to developing into neural progenitors than monolayer cells. Interaction between the monolayer cells and sphere cells is important in the development of stage 4 cell characteristics.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB1637
    Nombre del producto:
    Anti-Tubulin Antibody, beta III isoform, CT, clone TU-20 (Similar to TUJ1)

  • Differentiation of neuronal cells from NIH3T3 fibroblasts under defined conditions. 21477161

    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.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB345
    Nombre del producto:
    Anti-O4 Antibody, clone 81

  • Generation of neural crest cells and peripheral sensory neurons from human embryonic stem cells. 19907983

    Peripheral somatic sensory neurons (PSNs) are responsible for the critical function of transmitting multiple modalities of information from the outside world, including heat, touch, and pain, as well as the position of muscles required for coordinated voluntary movement to the central nervous system. Many peripheral neuropathies exist, including hereditary neurodegeneration in Familial Dysautonomia, infections of PSNs by viruses such as Varicella zoster and damage to PSNs and/or their process resulting from other disease conditions such as diabetes. Understanding of the etiology of these diseases and development of treatments is hampered by the lack of normal and healthy human PSNs for study, which are only available from abortuses or rare surgical procedures.Human embryonic stem cells (hESCs) are an ideal source of cells for generating normal PSNs for study of disease and drug development, since they can be grown virtually indefinitely in tissue culture and have the potential to form any cell type in the body. Several years ago, we generated human neurons with the molecular characteristics of PSNs from hESCs at low (less than 1%) yields (Pomp et al., Stem Cells 23:923-930, 2005). The present chapter details our most recently improved method that uses 2 rounds of PA6-induction to rapidly generate PSNs at more than 25% purity (Pomp et al., Br. Res. 1230: 50-60, 2008).The neural crest (NC) is a transient multipotent embryonic stem cell population that is the source of PSNs. NC cells give rise to diverse and important tissues in man, but human NC has not been studied because of the difficulty in obtaining 3-5 week human embryos. The methods described in this chapter can also be used to quickly generate large numbers of human NC for study.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB1585
    Nombre del producto:
    Anti-Brn-3a Antibody, POU-domain protein, clone 5A3.2

  • Could MDMA Promote Stemness Characteristics in Mouse Embryonic Stem Cells via mGlu5 Metabotropic Glutamate Receptors? 23508940

    Ecstasy, or 3, 4 (±) methylenedioxymethamphetamine (MDMA), is a potent neurotoxic drug. One of the mechanisms for its toxicity is the secondary release of glutamate. Mouse embryonic stem cells (mESCs) express only one glutamate receptor, the metabotropic glutamate receptor 5 (mGlu5), which is involved in the maintenance and self-renewal of mESCs. This study aims to investigate whether MDMA could influence self-renewal via the mGlu5 receptor in mESCs.In this expremental study, we used immunocytochemistry and reverse transcription-polymerase chain reaction (RT-PCR) to determine the presence of the mGlu5 receptor in mESCs. The expression of mGlu5 was evaluated after MDMA was added to mESCs throughout neural precursor cell formation as group 1 and during neural precursor cell differentiation as group 2. The stemness characteristic in treated mESCs by immunofluorescence and flow cytometry was studied. Finally, caspase activity was evaluated by fluorescence staining in the treated group. One-way ANOVA or repeated measure of ANOVA according to the experimental design was used for statistical analyses.In this study mGlu5 expression was shown in mESCs. In terms of neuronal differentiation, MDMA affected mGlu5 expression during neural precursor cell formation (group 1) and not during neural precursor differentiation (group 2). MDMA (450 µM) induced a significant increment in self-renewal properties in mESCs but did not reverse 2-methyl-6(phenylethynyl) pyridine (MPEP, 1 µM), a non-competitive selective mGlu5 antagonist. Fluorescence staining with anti-caspase 3 showed a significant increase in the number of apoptotic cells in the MDMA group.WE OBSERVED A DUAL ROLE FOR MDMA ON MESCS: reduced proliferation and maintenance of self-renewal. The lack of decreasing stemness characteristic in presence of MPEP suggests that MDMA mediates its role through a different mechanism that requires further investigation. In conclusion, despite being toxic, MDMA maintains stemness characteristics.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB4301
    Nombre del producto:
    Anti-Stage-Specific Embryonic Antigen-1 Antibody, clone MC-480

  • Titration of GLI3 repressor activity by sonic hedgehog signaling is critical for maintaining multiple adult neural stem cell and astrocyte functions. 24174682

    Sonic hedgehog (SHH), a key regulator of embryonic neurogenesis, signals directly to neural stem cells (NSCs) in the subventricular zone (SVZ) and to astrocytes in the adult mouse forebrain. The specific mechanism by which the GLI2 and GLI3 transcriptional activators (GLI2(A) and GLI3(A)) and repressors (GLI2(R) and GLI3(R)) carry out SHH signaling has not been addressed. We found that the majority of slow-cycling NSCs express Gli2 and Gli3, whereas Gli1 is restricted ventrally and all three genes are downregulated when NSCs transition into proliferating progenitors. Surprisingly, whereas conditional ablation of Smo in postnatal glial fibrillary acidic protein-expressing cells results in cell-autonomous loss of NSCs and a progressive reduction in SVZ proliferation, without an increase in glial cell production, removal of Gli2 or Gli3 does not alter adult SVZ neurogenesis. Significantly, removing Gli3 in Smo conditional mutants largely rescues neurogenesis and, conversely, expression of a constitutive GLI3(R) in the absence of normal Gli2 and Gli3 abrogates neurogenesis. Thus unattenuated GLI3(R) is a primary inhibitor of adult SVZ NSC function. Ablation of Gli2 and Gli3 revealed a minor role for GLI2(R) and little requirement for GLI(A) function in stimulating SVZ neurogenesis. Moreover, we found that similar rules of GLI activity apply to SHH signaling in regulating SVZ-derived olfactory bulb interneurons and maintaining cortical astrocyte function. Namely, fewer superficial olfactory bulb interneurons are generated in the absence of Gli2 and Gli3, whereas astrocyte partial gliosis results from an increase in GLI3(R). Thus precise titration of GLI(R) levels by SHH is critical to multiple functions of adult NSCs and astrocytes.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Transplantation of primed or unprimed mouse embryonic stem cell-derived neural precursor cells improves cognitive function in Alzheimerian rats. 19616885

    Alzheimer's disease (AD) is a neurodegenerative disorder that is characterized by progressive and irreversible decline of memory. Neuropathological features include the progressive degeneration of cholinergic neurons in the forebrain cholinergic projection system especially nucleus basalis of Meynert (nbM). New cell therapeutic approaches for the replacement of degenerated cells are being researched. The aim of this study was to investigate the production of cholinergic neurons from mouse embryonic stem cells (ESCs) and potential for utilizing ESC-derived neuronal precursor cells (NPCs) and primed NPCs (PNPCs) for cell restorative therapy in a rodent model of AD. NPCs were produced by growth factor-mediated selection under serum-free conditions and differentiated better into cholinergic neurons when NPCs primed with Shh (approximately 22%) in comparison with different cholinergic promoting factors. Behavioral assessment of unilateral nbM ibotenic acid-lesioned rats by Morris water maze and spatial probe test revealed a significant behavioral improvement in memory deficits following transplantation with NPCs and/or PNPCs. Immunohistochemical analysis revealed that the majority (approximately 70%) of the NPCs and/or PNPCs retained neuronal phenotype and approximately 40% of them had a cholinergic cell phenotype following transplantation with no tumor formation, indicating that these may be safe for transplantation. This experimental study has important implications as it suggests that the transplantation of mouse ESC-derived NPCs and/or following commitment to a cholinergic cell phenotype can promote behavioral recovery in a rodent model of AD.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AP106F
    Nombre del producto:
    Rabbit Anti-Goat IgG Antibody, FITC conjugate