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  • The differentiation of distal lung epithelium from embryonic stem cells. 18785171

    The potential for embryonic stem (ES) cells to differentiate into cells with a distal lung epithelial phenotype has been demonstrated using different in vitro culture methods. Three separate protocols are described here that utilize both murine and human ES cells. The distal lung epithelial phenotype is induced through the use of embryonic distal lung mesenchyme in coculture systems with differentiating embryoid bodies or the use of soluble factors in defined media to maximize definitive endoderm formation and select and maintain the desired phenotype. Phenotypic analysis is demonstrated using immunocytochemistry and SP-C promoter-eGFP reporter gene expression in transgenic ES cells. These methods provide an increased efficiency of distal lung epithelial derivation from ES cells and, therefore, they provide the foundation for the development of a cell replacement product to treat chronic lung disease or a useful in vitro model for the study of lung disease and development.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AB3786
    Nombre del producto:
    Anti-Prosurfactant Protein C (proSP-C) Antibody

  • Retinoic acid inhibits leukemia inhibitory factor signaling pathways in mouse embryonic stem cells. 14603524

    Retinoic acid (RA) induces the differentiation of murine embryonic stem (ES) cells to cell types resembling those found in the early embryo. When cultured in the presence of leukemia inhibitory factor (LIF), ES cells are maintained in an undifferentiated (self-renewing) state. Addition of RA to the culture media overrides the self-renewing effects of LIF to induce ES cell differentiation. Therefore, we hypothesized that RA-induced differentiation of ES cells may be accomplished by antagonism of LIF-induced signaling pathways. We demonstrate that RA-induced differentiation of CCE ES cells is associated with (1) downregulation of the LIF receptor (LIFR); (2) decreased tyrosine phosphorylation of signal transducer and activator of transcription 3 protein (Stat3); and (3) increased activation of extracellular regulated kinase (Erk1/2). We conclude that RA induces CCE ES cell differentiation in the presence of LIF, in part, by disrupting signaling between the LIFR/gp130 receptor and nuclear targets that are required to prevent ES cell differentiation. Our data indicate that RA-induced inhibition of LIF signaling does not involve Erk1/2-dependent actions.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Bioprinted amniotic fluid-derived stem cells accelerate healing of large skin wounds. 23197691

    Stem cells obtained from amniotic fluid show high proliferative capacity in culture and multilineage differentiation potential. Because of the lack of significant immunogenicity and the ability of the amniotic fluid-derived stem (AFS) cells to modulate the inflammatory response, we investigated whether they could augment wound healing in a mouse model of skin regeneration. We used bioprinting technology to treat full-thickness skin wounds in nu/nu mice. AFS cells and bone marrow-derived mesenchymal stem cells (MSCs) were resuspended in fibrin-collagen gel and "printed" over the wound site. At days 0, 7, and 14, AFS cell- and MSC-driven wound closure and re-epithelialization were significantly greater than closure and re-epithelialization in wounds treated by fibrin-collagen gel only. Histological examination showed increased microvessel density and capillary diameters in the AFS cell-treated wounds compared with the MSC-treated wounds, whereas the skin treated only with gel showed the lowest amount of microvessels. However, tracking of fluorescently labeled AFS cells and MSCs revealed that the cells remained transiently and did not permanently integrate in the tissue. These observations suggest that the increased wound closure rates and angiogenesis may be due to delivery of secreted trophic factors, rather than direct cell-cell interactions. Accordingly, we performed proteomic analysis, which showed that AFS cells secreted a number of growth factors at concentrations higher than those of MSCs. In parallel, we showed that AFS cell-conditioned media induced endothelial cell migration in vitro. Taken together, our results indicate that bioprinting AFS cells could be an effective treatment for large-scale wounds and burns.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AB7356
    Nombre del producto:
    Anti-von Willebrand Factor Antibody

  • The muscle regulatory transcription factor MyoD participates with p53 to directly increase the expression of the pro-apoptotic Bcl2 family member PUMA. 28918507

    The muscle regulatory transcription factor MyoD is a master regulator of skeletal myoblast differentiation. We have previously reported that MyoD is also necessary for the elevated expression of the pro-apoptotic Bcl2 family member PUMA, and the ensuing apoptosis, that occurs in a subset of myoblasts induced to differentiate. Herein, we report the identification of a functional MyoD binding site within the extended PUMA promoter. In silico analysis of the murine PUMA extended promoter revealed three potential MyoD binding sites within 2 kb of the transcription start site. Expression from a luciferase reporter construct containing this 2 kb fragment was enhanced by activation of MyoD in both myoblasts and fibroblasts and diminished by silencing of MyoD in myoblasts. Experiments utilizing truncated versions of this promoter region revealed that the potential binding site at position - 857 was necessary for expression. Chromatin immunoprecipitation (ChIP) analysis confirmed binding of MyoD to the DNA region encompassing position - 857. The increase in MyoD binding to the PUMA promoter as a consequence of culture in differentiation media (DM) was comparable to the increase in MyoD binding at the myogenin promoter and was diminished in myoblasts silenced for MyoD expression. Finally, ChIP analysis using an antibody specific for the transcription factor p53 demonstrated that, in myoblasts silenced for MyoD expression, p53 binding to the PUMA promoter was diminished in response to culture in DM. These data indicate that MyoD plays a direct role in regulating PUMA expression and reveal functional consequences of MyoD expression on p53 mediated transcription of PUMA.
    Tipo de documento:
    Referencia
    Referencia del producto:
    17-371
    Nombre del producto:
    EZ-ChIP™

  • Improved derivation efficiency and pluripotency of stem cells from the refractory inbred C57BL/6 mouse strain by small molecules. 25211343

    The ability of small molecules to maintain self-renewal and to inhibit differentiation of pluripotent stem cells has been well-demonstrated. Two widely used molecules are PD 98059 (PD), an inhibitor of extracellular-signal-regulated kinase 1 (ERK), and SC1 (Pluripotin), which inhibits the RasGAP and ERK pathways. However, no studies have been conducted to compare their effects on the pluripotency and derivation of embryonic stem (ES) cells from inbred mice C57BL/6, an important mouse strain frequently used to model behavior, cognitive functions, immune system, and metabolic disorders in humans and also the first mouse strain chosen to be sequenced for its entire genome. We found significantly increased derivation efficiency of ES cells from in vivo fertilized embryos (fES) of C57BL/6 with the use of PD (71.4% over the control of 35.3%). Because fES and ES from cloned embryos (ntES) are not distinguishable in transcription or translation profiles, we used ntES cells to compare the effect of small molecules on their in vitro characteristics, in vitro differentiation ability, and the ability to generate full-term ntES-4N pups by tetraploid complementation. NtES cells exhibited typical ES characteristics and up-regulated Sox2 expression in media with either small-molecule. Higher rates of full term ntES-4N pup were generated by the supplementation of PD or SC1. We obtained the highest efficiency of ntES-4N pup generation ever reported from this strain by supplementing ES medium with SC1. Lastly, we compared the pluripotency of fES, ntES and induced pluripotent stem (iPS) cells of C57BL/6 background using the tetraploid complementation assay. A significant increase in implantation sites and the number of full-term pups were obtained when fES, ntES, and iPS cells were cultured with SC1 compared to the control ES medium. In conclusion, supplementing ES cell culture medium with PD and SC1 increases the derivation efficiency and pluripotency, respectively, of stem cells derived from the refractory inbred C57BL/6 strain.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Dopamine receptors in human embryonic stem cell neurodifferentiation. 23286225

    We tested whether dopaminergic drugs can improve the protocol for in vitro differentiation of H9 human embryonic stem cells (hESCs) into dopaminergic neurons. The expression of 5 dopamine (DA) receptor subtypes (mRNA and protein) was analyzed at each protocol stage (1, undifferentiated hESCs; 2, embryoid bodies [EBs]; 3, neuroepithelial rosettes; 4, expanding neuroepithelium; and 5, differentiating neurons) and compared to human fetal brain (gestational week 17-19). D2-like DA receptors (D2, D3, and D4) predominate over the D1-like receptors (D1 and D5) during derivation of neurons from hESCs. D1 was the receptor subtype with the lowest representation in each protocol stage (Stages 1-5). D1/D5-agonist SKF38393 and D2/D3/D4-agonist quinpirole (either alone or combined) evoked Ca(2+) responses, indicating functional receptors in hESCs. To identify when receptor activation causes a striking effect on hESC neurodifferentiation, and what ligands and endpoints are most interesting, we varied the timing, duration, and drug in the culture media. Dopaminergic agonists or antagonists were administered either early (Stages 1-3) or late (Stages 4-5). Early DA exposure resulted in more neuroepithelial colonies, more neuronal clusters, and more TH(+) clusters. The D1/D5 antagonist SKF83566 had a strong effect on EB morphology and the expression of midbrain markers. Late exposure to DA resulted in a modest increase in TH(+) neuron clusters (∼75%). The increase caused by DA did not occur in the presence of dibutyryl cAMP (dbcAMP), suggesting that DA acts through the cAMP pathway. However, a D2-antagonist (L741) decreased TH(+) cluster counts. Electrophysiological parameters of the postmitotic neurons were not significantly affected by late DA treatment (Stages 4-5). The mRNA of mature neurons (VGLUT1 and GAD1) and the midbrain markers (GIRK2, LMX1A, and MSX1) were lower in hESCs treated by DA or a D2-antagonist. When hESCs were neurodifferentiated on PA6 stromal cells, DA also increased expression of tyrosine hydroxylase. Although these results are consistent with DA's role in potentiating DA neurodifferentiation, dopaminergic treatments are generally less efficient than dbcAMP alone.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Role of Slug transcription factor in human mesenchymal stem cells. 21645238

    The pathways that control mesenchymal stem cells (MSCs) differentiation are not well understood, and although some of the involved transcription factors (TFs) have been characterized, the role of others remains unclear. We used human MSCs from tibial plateau (TP) trabecular bone, iliac crest (IC) bone marrow and Wharton's jelly (WJ) umbilical cord demonstrating a variability in their mineral matrix deposition, and in the expression levels of TFs including Runx2, Sox9, Sox5, Sox6, STAT1 and Slug, all involved in the control of osteochondroprogenitors differentiation program. Because we reasoned that the basal expression level of some TFs with crucial role in the control of MSC fate may be correlated with osteogenic potential, we considered the possibility to affect the hMSCs behaviour by using gene silencing approach without exposing cells to induction media. In this study we found that Slug-silenced cells changed in morphology, decreased in their migration ability, increased Sox9 and Sox5 and decreased Sox6 and STAT1 expression. On the contrary, the effect of Slug depletion on Runx2 was influenced by cell type. Interestingly, we demonstrated a direct in vivo regulatory action of Slug by chromatin immunoprecipitation, showing a specific recruitment of this TF in the promoter of Runx2 and Sox9 genes. As a whole, our findings have important potential implication on bone tissue engineering applications, reinforcing the concept that manipulation of specific TF expression levels may elucidate MSC biology and the molecular mechanisms, which promote osteogenic differentiation.
    Tipo de documento:
    Referencia
    Referencia del producto:
    06-195

  • Non-canonical role for the TRAIL receptor DR5/FADD/caspase pathway in the regulation of MyoD expression and skeletal myoblast differentiation. 19523746

    We report herein that the TRAIL receptor DR5/FADD/caspase pathway plays a role in skeletal myoblast differentiation through modulation of the expression of the muscle regulatory transcription factor MyoD. Specifically, treatment with the selective caspase 3 inhibitor DEVD-fmk or the selective caspase 8 inhibitor IETD-fmk in growth media (GM), prior to culture in differentiation media (DM), inhibited differentiation. Further, this treatment resulted in decreased levels of MyoD message and protein. We next explored a role for the TRAIL receptor DR5/FADD pathway. We found that expression of either dominant negative (dn) FADD or dominant negative (dn) DR5 also resulted in decreased levels of MyoD mRNA and protein and blocked differentiation. This decreased level of MyoD mRNA was not a consequence of altered stability. Treatment with TSA, an inhibitor of histone deacetylases (HDACs), allowed MyoD expression in myoblasts expressing dnDR5. Finally, acetylation of histones associated with the distal regulatory region (DRR) enhancer of MyoD was decreased in myoblasts expressing dnDR5. Thus, our data suggests a non-canonical role for the TRAIL receptor/FADD pathway in the regulation of MyoD expression and skeletal myoblast differentiation.
    Tipo de documento:
    Referencia
    Referencia del producto:
    17-371
    Nombre del producto:
    EZ-ChIP™

  • In vitro differentiation of human amniotic fluid-derived cells: augmentation towards a neuronal dopaminergic phenotype. 20388119

    Amniotic fluid is known to yield a number of cell types which are multipotent, ethically derived, genetically stable, easily grown, expanded and possess favourable immunogenicity, which has resulted in an increasing interest for use in various neuronal disorders such as Parkinson's disease. The neuronal potential of cells derived from the adherent fraction of amniotic fluid, routinely taken by amniocentesis, are least explored. The aim of the present study was to investigate the capacity of these cells for neuronal and dopaminergic differentiation using in vitro differentiation protocols with canonical inductive factors not previously tested. To do this, samples derived from multiple donors were grown under four conditions: standard serum-containing media, NB (neurobasal) media designed specifically for propagation and maintenance of neuronal cells, NB media with addition of retinoic acid and BDNF (brain-derived neurotrophic factor) for NI (neuronal induction), and NB media with addition of FGF8 (fibroblast growth factor-8) and Shh (sonic hedgehog) after NI. Our results showed the presence of multiple neuronal markers after growth in serum-containing medium [TUJ1, MAP2, NF-M, TH (tyrosine hydroxylase)], which was significantly up-regulated after serum withdrawal in NB medium alone with induction of NeuN (neuronal nuclei) and NSE (neuron-specific enolase). NI and DA.I (dopaminergic induction) was accompanied by further increases in expression and a distinct transition to a sustained neuronal morphology. Western blot analysis confirmed increasing TH expression and NURR1, expressed in base serum-containing media, found to be down-regulated after induction. In conclusion, these cells possess a highly favourable base neuronal and dopaminergic prepotential, which may easily be accentuated by standard induction protocols.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AB1987
    Nombre del producto:
    Anti-Neurofilament M (145 kDa) Antibody, CT

  • Optimization and integration of expansion and neural commitment of mouse embryonic stem cells. 17596123

    To harness the potential of ES (embryonic stem) cells for human therapy, technology to develop the large-scale expansion and differentiation of these cells is required. In the present study, we tested various conditions for the expansion and neural commitment of mouse ES cells, using a cell line with a fluorescent reporter, which allows the monitoring of these processes by flow cytometry. The expansion of the 46C ES cell line in the presence of two different media [serum-free ESGRO Completetrade mark and DMEM (Dulbecco's modified Eagle's medium) containing 10% (v/v) fetal bovine serum] was compared. Both media yielded similar cell fold increases at two different initial cell densities and were able to maintain neural commitment potential during expansion. The influence of inocula concentration in the presence of two different media on cell proliferation and efficiency of neural commitment was evaluated. Two different chemically defined serum-free media were tested: the more conventional N2B27 and the second-generation medium RHB-A (Stem Cell Sciences, Edinburgh, Scotland, U.K.). The kinetics of neural commitment was followed during 8 days in the presence of both media. Our results show that inocula concentrations between 5x10(3) and 10(4) cells/cm(2) are the most appropriate to achieve a better cell growth and more efficient neural commitment. We also show that cell culture in RHB-A medium results in higher rates of cell proliferation and neural commitment of ES cells, when compared with N2B27.
    Tipo de documento:
    Referencia
    Referencia del producto:
    SCM012
    Nombre del producto:
    NDiff Neuro-2 Medium Supplement (200x)