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  • Comparative proteome and transcriptome analyses of embryonic stem cells during embryoid body-based differentiation. 19862760

    Gene expression analyses of embryonic stem cells (ESCs) will help to uncover or further define signaling pathways and molecular mechanisms involved in the maintenance of self-renewal and pluripotency. We employed a 2-DE-based proteomics approach to analyze human ESC line, Royan H5, in undifferentiated cells and different stages of spontaneous differentiation (days 3, 6, 12, and 20) by embryoid body formation. Out of 945 proteins reproducibly detected on gels, the expression of 96 spots changed during differentiation. Using MS, 87 ESC-associated proteins were identified including several proteins involved in cell proliferation, cell apoptosis, transcription, translation, mRNA processing, and protein folding. Transcriptional changes accompanying differentiation of Royan H5 were also analyzed using microarrays. We developed a comprehensive data set that shows the use of human ESC lines in vitro to mimic gastrulation and organogenesis. Our results showed that proteomics and transcriptomics data are complementary rather than duplicative. Although regulation of many genes during differentiation were observed only at transcript level, modulation of several proteins was revealed only by proteome analysis.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AP308F
    Nombre del producto:
    Goat Anti-Mouse IgG Antibody, (H+L) FITC conjugate

  • The C. elegans sex-determining GLI protein TRA-1A is regulated by sex-specific proteolysis. 17084364

    TRA-1A is the sole representative in Caenorhabditis elegans of the Gli transcription factor family. Its activity is required to specify all somatic female cell fates in XX hermaphrodites. We have found that TRA-1 protein levels are much higher in hermaphrodites than in males, and that the difference is attributable to the predominance in hermaphrodites of C-terminally truncated isoforms that are nearly undetectable in males. Our results support a model in which TRA-1A is negatively regulated by male-specific proteolysis that depends upon specific TRA-1A protein sequences and upon the activity of the fem genes. C-terminally truncated TRA-1 isoforms are stable and can inappropriately feminize XO males, suggesting that they escape this negative regulation. Thus, although C. elegans appears to lack a Hedgehog-signaling pathway, our results indicate that proteolytic processing and degradation of Gli family transcription factors, commonly seen during Hedgehog signaling in other organisms, also control C. elegans sex determination.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB8864

  • Selecting and isolating colonies of human induced pluripotent stem cells reprogrammed from adult fibroblasts. 22370855

    Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate (1-3). We used this method to reprogram late passage (>p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged (4,5). These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals( 6), human newborn fibroblasts, as well as human keratinocytes.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB4360
    Nombre del producto:
    Anti-TRA-1-60 Antibody, clone TRA-1-60

  • Reprogrammed keratinocytes from elderly type 2 diabetes patients suppress senescence genes to acquire induced pluripotency. 22308265

    Nuclear reprogramming enables patient-specific derivation of induced pluripotent stem (iPS) cells from adult tissue. Yet, iPS generation from patients with type 2 diabetes (T2D) has not been demonstrated. Here, we report reproducible iPS derivation of epidermal keratinocytes (HK) from elderly T2D patients. Transduced with human OCT4, SOX2, KLF4 and c-MYC stemness factors under serum-free and feeder-free conditions, reprogrammed cells underwent dedifferentiation with mitochondrial restructuring, induction of endogenous pluripotency genes - including NANOG, LIN28, and TERT, and down-regulation of cytoskeletal, MHC class I- and apoptosis-related genes. Notably, derived iPS clones acquired a rejuvenated state, characterized by elongated telomeres and suppressed senescence-related p15INK4b/p16INK4a gene expression and oxidative stress signaling. Stepwise guidance with lineage-specifying factors, including Indolactam V and GLP-1, redifferentiated HK-derived iPS clones into insulin-producing islet-like progeny. Thus, in elderly T2D patients, reprogramming of keratinocytes ensures a senescence-privileged status yielding iPS cells proficient for regenerative applications.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Three monoclonal antibodies defining distinct differentiation antigens associated with different high molecular weight polypeptides on the surface of human embryonal carc ... 6396197

    Two monoclonal antibodies (TRA-1-60 and TRA-1-81) recognizing distinct cell surface antigens on human embryonal carcinoma (EC) cells were produced and characterized. These antibodies reacted strongly with undifferentiated human EC cells in indirect radioimmunoassays (RIA) and immunofluorescence (IF) assays, but only weakly or not at all with cells derived from pluripotent EC cells differentiating in vitro or in xenograft tumors, nor with other germ cell tumor cell lines that did not also express the typical features of human EC cells. They did not react with murine teratocarcinoma cell lines. A survey of other human tumor cell lines and normal human tissues disclosed that molecules recognized by these antibodies are not confined to human EC cells but that cross-reacting epitopes appear on several neoplastic and normal tissues, although in a different anatomical pattern for each antibody. Both antibodies immunoprecipitated a major polypeptide (apparent molecular weight approximately 240,000) and a minor polypeptide (apparent molecular weight approximately 415,000) from lysates of 125I surface-labeled human EC cells, in this respect resembling another monoclonal antibody, 8-7D, previously described by Blaineau et al. (1,2) However, sequential immunoprecipitation revealed that each of the three antibodies reacted with different molecules of slightly different molecular weights. The epitopes defined by the present antibodies differ from those recognized by the other human EC cell-specific monoclonal antibodies that have been described and provide new markers for studying the differentiation of pluripotent human EC cells.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Reprogramming of human fibroblasts to induced pluripotent stem cells under xeno-free conditions. 19890879

    The availability of induced pluripotent stem cells (iPSCs) has created extraordinary opportunities for modeling and perhaps treating human disease. However, all reprogramming protocols used to date involve the use of products of animal origin. Here, we set out to develop a protocol to generate and maintain human iPSC that would be entirely devoid of xenobiotics. We first developed a xeno-free cell culture media that supported the long-term propagation of human embryonic stem cells (hESCs) to a similar extent as conventional media containing animal origin products or commercially available xeno-free medium. We also derived primary cultures of human dermal fibroblasts under strict xeno-free conditions (XF-HFF), and we show that they can be used as both the cell source for iPSC generation as well as autologous feeder cells to support their growth. We also replaced other reagents of animal origin (trypsin, gelatin, matrigel) with their recombinant equivalents. Finally, we used vesicular stomatitis virus G-pseudotyped retroviral particles expressing a polycistronic construct encoding Oct4, Sox2, Klf4, and GFP to reprogram XF-HFF cells under xeno-free conditions. A total of 10 xeno-free human iPSC lines were generated, which could be continuously passaged in xeno-free conditions and maintained characteristics indistinguishable from hESCs, including colony morphology and growth behavior, expression of pluripotency-associated markers, and pluripotent differentiation ability in vitro and in teratoma assays. Overall, the results presented here demonstrate that human iPSCs can be generated and maintained under strict xeno-free conditions and provide a path to good manufacturing practice (GMP) applicability that should facilitate the clinical translation of iPSC-based therapies.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Biochemical and genetic analysis of the OKa blood group antigen. 3356449

    The monoclonal antibody TRA-1-85 recognizes a cell surface antigen which is expressed by all human cell types tested, including red blood cells (RBCs), but not by mouse cells. All the human RBCs tested were TRA-1-85 positive except those with the rare phenotype Ok(a-). Oka is a blood group antigen of very high frequency and only three unrelated Ok(a-) people are known. The red cells of all three propositi were negative with the TRA-1-85 antibody. To confirm the relationship between the TRA-1-85 antibody and anti-Oka, the immune antibody found in the serum of Ok(a-) individuals, Western blot analysis was used: the TRA-1-85 antibody and anti-Oka gave identical but complex patterns of reactivity in Western blot analysis of human cell lysates or membranes. This suggests that the anti-Oka and TRA-1-85 antibodies recognize the same cell-surface determinant and implies that Oka is not restricted in its expression to the surface of RBCs but is expressed on white blood cells (WBCs) of Ok(a+) individuals and all human cell lines tested to date. WBCs from one of the Ok(a-) propositi were tested and found to be negative with the TRA-1-85 antibody. Finally, the species specificity of the TRA-1-85 antibody has been exploited by the use of somatic cell hybrids and DNA transfection techniques to examine the genetic control of the Oka antigen defined by the TRA-1-85 antibody. We report that the determinant is controlled by a single gene OK present on human chromosome 19.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB4385
    Nombre del producto:
    Anti-TRA-1-85 Antibody, blood group Antigen Ok(a), clone TRA-1-85

  • A simple and efficient cryopreservation method for feeder-free dissociated human induced pluripotent stem cells and human embryonic stem cells. 19602515

    An essential prerequisite for the future widespread application of human induced pluripotent (hiPSCs) and embryonic stem cells (hESCs) is the development of efficient cryopreservation methods to facilitate their storage and transportation.We developed a simple and effective freezing/thawing method of single dissociated hESCs and hiPSCs in a feeder-free culture in the presence of Rho-associated kinase (ROCK) inhibitor Y-27632.Exposure to ROCK inhibitor Y-27632 in freezing solution alone does not significantly enhance the post-thaw survival rate of single dissociated hESCs and hiPSCs. However, when ROCK inhibitor was added to both pre- and post-thaw culture media, there was an enhancement in the survival rate, which further increased when ROCK inhibitor was added to Matrigel as well. Under these treatments, hESCs and hiPSCs retained typical morphology, stable karyotype, expression of pluripotency markers and the potential to differentiate into derivatives of all three germ layers after long-term culture.This method is an effective cryopreservation procedure for single dissociated hESCs in feeder-free culture, which is also applicable for single dissociated hiPSCs using a ROCK inhibitor. The cloning efficiency of hiPSCs and hESCs improves when ROCK inhibitor is added both in Matrigel and in medium in comparison with conventional addition to medium. Therefore, we believe this method would be useful for current and future applications of the pluripotent stem cells.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • MOUSE TRA-1-85 MONOCLONAL ANTIBODY -2782648

    Tipo de documento:
    Certificado de análisis
    Número de lote:
    2782648
    Referencia del producto:
    MAB4385
    Nombre del producto:
    Anti-TRA-1-85 Antibody, blood group Antigen Ok(a), clone TRA-1-85

  • MOUSE TRA-1-85 MONOCLONAL ANTIBODY -2608422

    Tipo de documento:
    Certificado de análisis
    Número de lote:
    2608422
    Referencia del producto:
    MAB4385
    Nombre del producto:
    Anti-TRA-1-85 Antibody, blood group Antigen Ok(a), clone TRA-1-85