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  • BMP4 Signaling Acts via dual-specificity phosphatase 9 to control ERK activity in mouse embryonic stem cells. 22305567

    Extrinsic BMP and LIF signaling collaboratively maintain mouse embryonic stem cell (ESC) pluripotency, whereas appropriate ERK activity is essential for ESC fate commitment. However, how the extrinsic signals restrain appropriate ERK activity remains elusive. Here, we show that, whereas LIF sustains relatively high ERK activity, BMP4 can steadily attenuate ERK activity by upregulating ERK-specific dual-specificity phosphatase 9 (DUSP9). This upregulation requires Smad1/5 and Smad4 and specifically occurs to DUSP9, but not other DUSPs, and only in ESCs. Through DUSP9-mediated inhibition of ERK activity, BMP signaling reinforces the self-renewal status of mouse ESCs together with LIF. Upon LIF withdrawal, ESCs spontaneously undergo neural differentiation, during which process DUSP9 can partially mediate BMP inhibition on neural commitment. Collectively, our findings identify DUSP9 as a critical mediator of BMP signaling to control appropriate ERK activity critical for ESC fate determination.
    Tipo de documento:
    Referencia
    Referencia del producto:
    SCR004
    Nombre del producto:
    Alkaline Phosphatase Detection Kit

  • Injury-induced neural stem/progenitor cells in post-stroke human cerebral cortex. 20104652

    Increasing evidence points to accelerated neurogenesis after stroke, and support of such endogenous neurogenesis has been shown to improve stroke outcome in experimental animal models. The present study analyses post-stroke cerebral cortex after cardiogenic embolism in autoptic human brain. Induction of nestin- and musashi-1-positive cells, potential neural stem/progenitor cells, was observed at the site of ischemic lesions from day 1 after stroke. These two cell populations were present at distinct locations and displayed different temporal profiles of marker expression. However, no surviving differentiated mature neural cells were observed by 90 days after stroke in the previously ischemic region. Consistent with recent reports of neurogenesis in the cerebral cortex after induction of stroke in rodent models, the present current data indicate the presence of a regional regenerative response in human cerebral cortex. Furthermore, observations underline the potential importance of supporting survival and differentiation of endogenous neural stem/progenitor cells in post-stroke human brain.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB5326
    Nombre del producto:
    Anti-Nestin Antibody, clone 10C2

  • The human AC133 hematopoietic stem cell antigen is also expressed in epithelial cells and targeted to plasma membrane protrusions. 10681530

    The human AC133 antigen and mouse prominin are structurally related plasma membrane proteins. However, their tissue distribution is distinct, with the AC133 antigen being found on hematopoietic stem and progenitor cells and prominin on various epithelial cells. To determine whether the human AC133 antigen and mouse prominin are orthologues or distinct members of a protein family, we examined the human epithelial cell line Caco-2 for the possible expression of the AC133 antigen. By both immunofluorescence and immunoprecipitation, the AC133 antigen was found to be expressed on the surface of Caco-2 cells. Interestingly, immunoreactivity for the AC133 antigen, but not its mRNA level, was down-regulated upon differentiation of Caco-2 cells. The AC133 antigen was specifically located at the apical rather than basolateral plasma membrane. An apical localization of the AC133 antigen was also observed in various human embryonic epithelia including the neural tube, gut, and kidney. Electron microscopy revealed that, within the apical plasma membrane of Caco-2 cells, the AC133 antigen was confined to microvilli and absent from the planar, intermicrovillar regions. This specific subcellular localization did not depend on an epithelial phenotype, because the AC133 antigen on hematopoietic stem cells, as well as that ectopically expressed in fibroblasts, was selectively found in plasma membrane protrusions. Hence, the human AC133 antigen shows the features characteristic of mouse prominin in epithelial and transfected non-epithelial cells, i.e. a selective association with apical microvilli and plasma membrane protrusions, respectively. Conversely, flow cytometry of murine CD34(+) bone marrow progenitors revealed the cell surface expression of prominin. Taken together, the data strongly suggest that the AC133 antigen is the human orthologue of prominin.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB4310X
    Nombre del producto:
    Anti-CD133 Antibody, clone 13A4, Alexa Fluor® 488 conjugated

  • 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

  • Decitabine alters the expression of Mecp2 isoforms via dynamic DNA methylation at the Mecp2 regulatory elements in neural stem cells. 24238559

    Aberrant MeCP2 expression in brain is associated with neurodevelopmental disorders including autism. In the brain of stressed mouse and autistic human patients, reduced MeCP2 expression is correlated with Mecp2/MECP2 promoter hypermethylation. Altered expression of MeCP2 isoforms (MeCP2E1 and MeCP2E2) is associated with neurological disorders, highlighting the importance of proper regulation of both isoforms. While known regulatory elements (REs) within the MECP2/Mecp2 promoter and intron 1 are involved in MECP2/Mecp2 regulation, Mecp2 isoform-specific regulatory mechanisms are unknown. We hypothesized that DNA methylation at these REs may impact the expression of Mecp2 isoforms.We used a previously characterized in vitro differentiating neural stem cell (NSC) system to investigate the interplay between Mecp2 isoform-specific expression and DNA methylation at the Mecp2 REs. We studied altered expression of Mecp2 isoforms, affected by global DNA demethylation and remethylation, induced by exposure and withdrawal of decitabine (5-Aza-2'-deoxycytidine). Further, we performed correlation analysis between DNA methylation at the Mecp2 REs and the expression of Mecp2 isoforms after decitabine exposure and withdrawal.At different stages of NSC differentiation, Mecp2 isoforms showed reciprocal expression patterns associated with minor, but significant changes in DNA methylation at the Mecp2 REs. Decitabine treatment induced Mecp2e1/MeCP2E1 (but not Mecp2e2) expression at day (D) 2, associated with DNA demethylation at the Mecp2 REs. In contrast, decitabine withdrawal downregulated both Mecp2 isoforms to different extents at D8, without affecting DNA methylation at the Mecp2 REs. NSC cell fate commitment was minimally affected by decitabine under tested conditions. Expression of both isoforms negatively correlated with methylation at specific regions of the Mecp2 promoter, both at D2 and D8. The correlation between intron 1 methylation and Mecp2e1 (but not Mecp2e2) varied depending on the stage of NSC differentiation (D2: negative; D8: positive).Our results show the correlation between the expression of Mecp2 isoforms and DNA methylation in differentiating NSC, providing insights on the potential role of DNA methylation at the Mecp2 REs in Mecp2 isoform-specific expression. The ability of decitabine to induce Mecp2e1/MeCP2E1, but not Mecp2e2 suggests differential sensitivity of Mecp2 isoforms to decitabine and is important for future drug therapies for autism.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • 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