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  • H/ACA Box Small Nucleolar RNA 7A Promotes the Self-Renewal of Human Umbilical Cord Mesenchymal Stem Cells. 27573912

    Human umbilical cord blood derived mesenchymal stem cells (uMSC) are pluripotent cells that have been now considered as a promising candidate for various cell-based therapies. However, their limited in vitro proliferation ability and the gradual loss of pluripotency set barricades for further usages. Emerging evidence suggests that small nucleolar RNAs (snoRNA) are actively involved in cell proliferation especially in tumor cells, but their roles in stem cells are largely unknown. In this study, we demonstrated that H/ACA box small nucleolar RNA 7A (SNORA7A) is inversely correlated to the decreased proliferation rate during in vitro passaging of uMSC. Further investigations indicate that SNORA7A overexpression can promote uMSC proliferation and self-renewal. The inhibition of SNORA7A using antisense oligonucleotides significantly reduces the expression and the binding of SNORA7A to DKC1, core protein that essential to form small nucleolar ribonucleo-particles (snoRNP) complex and catalyze pseudouridines in 28S RNA. And the inhibition also significantly suppresses uMSC proliferation and self-renewal. Moreover, overexpression of SNORA7A transcripts with mutations of binding regions for snoRNP core proteins and 28S RNA did not induce proliferation and self-renewal. Besides, SNORA7A also suppresses both the osteogenic and adipogenic differentiation, strengthening its self-renewal maintaining roles in uMSC. Taken together, our study for the first time showed that H/ACA box snoRNAs are actively involved in MSC proliferation as well as pluripotency control, and we identify SNORA7A as one of the critical snoRNAs that regulate the proliferation and self-renewal of uMSC through snoRNP recruiting. Stem Cells 2017;35:222-235.
    Tipo de documento:
    Referencia
    Referencia del producto:
    17-701
    Nombre del producto:
    EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit

  • Optimization of Protocols for Derivation of Mouse Embryonic Stem Cell Lines from Refractory Strains, Including the Non Obese Diabetic Mouse. 21933027

    The derivation of pluripotent embryonic stem cells (ESCs) from a variety of genetic backgrounds remains a desirable objective in the generation of mice functionally deficient in genes of interest and the modeling of human disease. Nevertheless, disparity in the ease with which different strains of mice yield ESC lines has long been acknowledged. Indeed, the generation of bona fide ESCs from the non obese diabetic (NOD) mouse, a well-characterized model of human type I diabetes, has historically proved especially difficult to achieve. Here, we report the development of protocols for the derivation of novel ESC lines from C57Bl/6 mice based on the combined use of high concentrations of leukemia inhibitory factor and serum-replacement, which is equally applicable to fresh and cryo-preserved embryos. Further, we demonstrate the success of this approach using Balb/K and CBA/Ca mice, widely considered to be refractory strains. CBA/Ca ESCs contributed to the somatic germ layers of chimeras and displayed a very high competence at germline transmission. Importantly, we were able to use the same protocol for the derivation of ESC lines from nonpermissive NOD mice. These ESCs displayed a normal karyotype that was robustly stable during long-term culture, were capable of forming teratomas in vivo and germline competent chimeras after injection into recipient blastocysts. Further, these novel ESC lines efficiently formed embryoid bodies in vitro and could be directed in their differentiation along the dendritic cell lineage, thus illustrating their potential application to the generation of cell types of relevance to the pathogenesis of type I diabetes.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Efficient feeder-free episomal reprogramming with small molecules. 21390254

    Genetic reprogramming of human somatic cells to induced pluripotent stem cells (iPSCs) could offer replenishable cell sources for transplantation therapies. To fulfill their promises, human iPSCs will ideally be free of exogenous DNA (footprint-free), and be derived and cultured in chemically defined media free of feeder cells. Currently, methods are available to enable efficient derivation of footprint-free human iPSCs. However, each of these methods has its limitations. We have previously derived footprint-free human iPSCs by employing episomal vectors for transgene delivery, but the process was inefficient and required feeder cells. Here, we have greatly improved the episomal reprogramming efficiency using a cocktail containing MEK inhibitor PD0325901, GSK3? inhibitor CHIR99021, TGF-?/Activin/Nodal receptor inhibitor A-83-01, ROCK inhibitor HA-100 and human leukemia inhibitory factor. Moreover, we have successfully established a feeder-free reprogramming condition using chemically defined medium with bFGF and N2B27 supplements and chemically defined human ESC medium mTeSR1 for the derivation of footprint-free human iPSCs. These improvements enabled the routine derivation of footprint-free human iPSCs from skin fibroblasts, adipose tissue-derived cells and cord blood cells. This technology will likely be valuable for the production of clinical-grade human iPSCs.
    Tipo de documento:
    Referencia
    Referencia del producto:
    SCR004
    Nombre del producto:
    Alkaline Phosphatase Detection Kit

  • Histone content increases in differentiating embryonic stem cells. 25221520

    Mouse Embryonic Stem Cells (ESCs) are pluripotent mammalian cells derived from the Inner Cell Mass (ICM) of mouse blastocysts, which give rise to all three embryonic germ layers both in vivo and in vitro. Mouse ESCs have a distinct epigenetic landscape and a more decondensed chromatin compared to differentiated cells. Numerous studies have shown that distinct histone modifications in ESCs serve as hallmarks of pluripotency. However, so far it is still unknown whether the total histone content (as opposed to histone modifications) remains the same in cells of different developmental stage and differentiation capacity. In this work we show that total histone content differs between pluripotent and differentiated cells. In vitro spontaneous differentiation from ESCs to Embryoid Bodies (EBs) and directed differentiation toward neuronal and endodermal cells entails an increase in histone content. Primary MEFs also contain more histones than ESCs. We suggest that the difference in histone content is an additional hallmark of pluripotency, in addition to and besides histone modifications.
    Tipo de documento:
    Referencia
    Referencia del producto:
    07-146
    Nombre del producto:
    Anti-Histone H2A (acidic patch) Antibody

  • Indolactam V/GLP-1-mediated differentiation of human iPS cells into glucose-responsive insulin-secreting progeny. 21048796

    Nuclear reprogramming of somatic tissue enables derivation of induced pluripotent stem (iPS) cells from an autologous, non-embryonic origin. The purpose of this study was to establish efficient protocols for lineage specification of human iPS cells into functional glucose-responsive, insulin-producing progeny. We generated human iPS cells, which were then guided with recombinant growth factors that mimic the essential signaling for pancreatic development. Reprogrammed with four stemness factors, human fibroblasts were here converted into authentic iPS cells. Under feeder-free conditions, fate specification was initiated with activin A and Wnt3a that triggered engagement into definitive endoderm, followed by priming with fibroblast growth factor 10 (FGF10) and KAAD-cyclopamine. Addition of retinoic acid, boosted by the pancreatic endoderm inducer indolactam V (ILV), yielded pancreatic progenitors expressing pancreatic and duodenal homeobox 1 (PDX1), neurogenin 3 (NGN3) and neurogenic differentiation 1 (NEUROD1) markers. Further guidance, under insulin-like growth factor 1 (IGF-1), hepatocyte growth factor (HGF) and N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), was enhanced by glucagon-like peptide-1 (GLP-1) to generate islet-like cells that expressed pancreas-specific markers including insulin and glucagon. Derived progeny demonstrated sustained expression of PDX1, and functional responsiveness to glucose challenge secreting up to 230 pM of C-peptide. A pancreatogenic cocktail enriched with ILV/GLP-1 offers a proficient means to specify human iPS cells into glucose-responsive hormone-producing progeny, refining the development of a personalized platform for islet-like cell generation.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Nuclear reprogramming with a non-integrating human RNA virus. 25889591

    Advances in the field of stem cells have led to novel avenues for generating induced pluripotent stem cells (iPSCs) from differentiated somatic cells. iPSCs are typically obtained by the introduction of four factors--OCT4, SOX2, KLF4, and cMYC--via integrating vectors. Here, we report the feasibility of a novel reprogramming process based on vectors derived from the non-integrating vaccine strain of measles virus (MV).We produced a one-cycle MV vector by substituting the viral attachment protein gene with the green fluorescent protein (GFP) gene. This vector was further engineered to encode for OCT4 in an additional transcription unit.After verification of OCT4 expression, we assessed the ability of iPSC reprogramming. The reprogramming vector cocktail with the OCT4-expressing MV vector and SOX2-, KLF4-, and cMYC-expressing lentiviral vectors efficiently transduced human skin fibroblasts and formed iPSC colonies. Reverse transcription-polymerase chain reaction and immunostaining confirmed induction of endogenous pluripotency-associated marker genes, such as SSEA-4, TRA-1-60, and Nanog. Pluripotency of derived clones was confirmed by spontaneous differentiation into three germ layers, teratoma formation, and guided differentiation into beating cardiomyocytes.MV vectors can induce efficient nuclear reprogramming. Given the excellent safety record of MV vaccines and the translational capabilities recently developed to produce MV-based vectors now used for cancer clinical trials, our MV vector system provides an RNA-based, non-integrating gene transfer platform for nuclear reprogramming that is amenable for immediate clinical translation.
    Tipo de documento:
    Referencia
    Referencia del producto:
    07-633
    Nombre del producto:

  • Epigenetic regulation of Nanog expression by Ezh2 in pluripotent stem cells. 21490431

    Nanog levels in pluripotent stem cells are heterogeneous and this is thought to reflect two different and interchangeable cell states, respectively poised to self-renew (Nanog-high subpopulation) or to differentiate (Nanog-low subpopulation). However, little is known about the mechanisms responsible for this pattern of Nanog expression. Here, we have examined the impact of the histone methyltransferase Ezh2 on pluripotent stem cells and on Nanog expression. Interestingly, induced pluripotent stem (iPS) cells lacking Ezh2 presented higher levels of Nanog due to a relative expansion of the Nanog-high subpopulation, and this was associated to severe defects in differentiation. Moreover, we found that the Nanog promoter in embryonic stem (ES) cells and iPS cells coexists in two alternative univalent chromatin configurations, either H3K4me3 or H3K27me3, the latter being dependent on the presence of functional Ezh2. Finally, the levels of expression of Ezh2, as well as the amount of H3K27me3 present at the Nanog promoter, were higher in the Nanog-low subpopulation of ES/iPS cells. Together, these data indicate that Ezh2 directly regulates the epigenetic status of the Nanog promoter affecting the balance of Nanog expression in pluripotent stem cells and, therefore, the equilibrium between self-renewal and differentiation.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Retinoid uptake, processing, and secretion in human iPS-RPE support the visual cycle. 24255038

    Retinal pigmented epithelium derived from human induced pluripotent stem (iPS) cells (iPS-RPE) may be a source of cells for transplantation. For this reason, it is essential to determine the functional competence of iPS-RPE. One key role of the RPE is uptake and processing of retinoids via the visual cycle. The purpose of this study is to investigate the expression of visual cycle proteins and the functional ability of the visual cycle in iPS-RPE.iPS-RPE was derived from human iPS cells. Immunocytochemistry, RT-PCR, and Western blot analysis were used to detect expression of RPE genes lecithin-retinol acyl transferase (LRAT), RPE65, cellular retinaldehyde-binding protein (CRALBP), and pigment epithelium-derived factor (PEDF). All-trans retinol was delivered to cultured cells or whole cell homogenate to assess the ability of the iPS-RPE to process retinoids.Cultured iPS-RPE expresses visual cycle genes LRAT, CRALBP, and RPE65. After incubation with all-trans retinol, iPS-RPE synthesized up to 2942 ± 551 pmol/mg protein all-trans retinyl esters. Inhibition of LRAT with N-ethylmaleimide (NEM) prevented retinyl ester synthesis. Significantly, after incubation with all-trans retinol, iPS-RPE released 188 ± 88 pmol/mg protein 11-cis retinaldehyde into the culture media.iPS-RPE develops classic RPE characteristics and maintains expression of visual cycle proteins. The results of this study confirm that iPS-RPE possesses the machinery to process retinoids for support of visual pigment regeneration. Inhibition of all-trans retinyl ester accumulation by NEM confirms LRAT is active in iPS-RPE. Finally, the detection of 11-cis retinaldehyde in the culture medium demonstrates the cells' ability to process retinoids through the visual cycle. This study demonstrates expression of key visual cycle machinery and complete visual cycle activity in iPS-RPE.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB5428
    Nombre del producto:
    Anti-Retinal Pigment Epithelium 65 Antibody

  • Inhibition of endothelial progenitor cell glycogen synthase kinase-3beta results in attenuated neointima formation and enhanced re-endothelialization after arterial injur ... 19454488

    AIMS: Endothelial progenitor cells (EPCs) are circulating pluripotent vascular cells capable of enhancing re-endothelialization and diminishing neointima formation following arterial injury. Glycogen synthase kinase (GSK)-3beta is a protein kinase that has been implicated in the regulation of progenitor cell biology. We hypothesized that EPC abundance and function could be enhanced with the use of an inhibitor of GSK-3beta (GSKi), thereby resulting in improved arterial repair. METHODS AND RESULTS: Human EPCs were expanded ex vivo, treated with a specific GSKi, and then assessed for both yield and functional characteristics by in vitro assays for adherence, apoptosis, and survival. In vivo functionality of treated human EPCs was assessed in immune-tolerant mice subjected to femoral artery wire injury. Re-endothelialization was assessed at 72 h and neointima formation at 7 and 14 days following injury. GSKi treatment resulted in an improvement in the yield of EPCs and a reduction in apoptosis in cells derived from both healthy controls and patients with coronary artery disease. Treatment also increased vascular endothelial growth factor secretion, up-regulated expression of mRNA for the alpha-4 integrin subunit, and improved adhesion, an effect which could be abrogated with an alpha-4 integrin blocking antibody. EPCs without or with ex vivo GSKi treatment enhanced re-endothelialization 72 h following injury as well as reduced neointima formation at 7 days (e.g. endothelial coverage: 7.2 +/- 1.7% vs. 70.7 +/- 5.8% vs. 87.2 +/- 4.1%; intima to media ratios: 1.05 +/- 0.19 vs. 0.39 +/- 0.08 vs. 0.14 +/- 0.02; P 0.05 for all comparisons), an effect that was persistent at 14 days. CONCLUSION: GSKi improves the functional profile of EPCs and is associated with improved re-endothelialization and reduced neointima formation following injury.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB16983
    Nombre del producto:
    Anti-Integrin α4 Antibody, clone P1H4

  • iPS cell-derived cardiogenicity is hindered by sustained integration of reprogramming transgenes. 25077947

    Nuclear reprogramming inculcates pluripotent capacity by which de novo tissue differentiation is enabled. Yet, introduction of ectopic reprogramming factors may desynchronize natural developmental schedules. This study aims to evaluate the effect of imposed transgene load on the cardiogenic competency of induced pluripotent stem (iPS) cells.Targeted inclusion and exclusion of reprogramming transgenes (c-MYC, KLF4, OCT4, and SOX2) was achieved using a drug-inducible and removable cassette according to the piggyBac transposon/transposase system. Pulsed transgene overexpression, before iPS cell differentiation, hindered cardiogenic outcomes. Delayed in counterparts with maintained integrated transgenes, transgene removal enabled proficient differentiation of iPS cells into functional cardiac tissue. Transgene-free iPS cells generated reproducible beating activity with robust expression of cardiac α-actinin, connexin 43, myosin light chain 2a, α/β-myosin heavy chain, and troponin I. Although operational excitation-contraction coupling was demonstrable in the presence or absence of transgenes, factor-free derivatives exhibited an expedited maturing phenotype with canonical responsiveness to adrenergic stimulation.A disproportionate stemness load, caused by integrated transgenes, affects the cardiogenic competency of iPS cells. Offload of transgenes in engineered iPS cells ensures integrity of cardiac developmental programs, underscoring the value of nonintegrative nuclear reprogramming for derivation of competent cardiogenic regenerative biologics.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AB1728
    Nombre del producto:
    Anti-Connexin 43 Antibody, CT, cytosolic