搜索结果: 'methocult media formulations for human hematopoietic cells serum containing'

As known from model organisms,such as frog,fish,mouse and chicken,the anterior-posterior patterning of the definitive endoderm (DE) into distinct domains is controlled by a variety of signaling interactions between the DE and its surrounding mesoderm. This includes Wnt/FGFs and BMPs in the posterior half and all-trans-retinoic acid,TGF-$$-ligands,Wnt- and BMP-inhibitors in the anterior half of the DE sheet. However,it is currently unclear how these embryonic tissue interactions can be translated into a defined differentiation protocol for human embryonic stem cells. Activin A has been proposed to direct DE into a SOX2-positive foregut-like cell type. Due to the pleiotropic nature of SOX2 in pluripotency and developing cells of the foregut we purified DE-cells by magnetic cell sorting and tested the effects of anteriorizing and posteriorizing factors on pure endoderm. We show in contrast to previous studies that the generation of the foregut marked by SOX2/FOXA2 double-positive cells does not depend on activin A/TGF-$$-signaling but is mediated by the inhibition of Wnt- and BMP-signaling. Retinoic acid can posteriorize and at the same time dorsalize the foregut towards a PDX1-positive pancreatic duodenal cell type whereas active Wnt/beta-catenin signaling synergistically with FGF-2,BMP-4 and RA induces the formation of CDX2-positive posterior endoderm. Thus,these results provide new insights into the mechanisms behind cell specification of human DE derived from pluripotent stem cells. This article is protected by copyright. All rights reserved. View Publication

Activation of the transcription factor,nuclear factor-kappaB (NF-kappaB),results in up-regulation of not only antiapoptotic genes but also proapoptotic genes,including death receptor 4 (DR4) and death receptor 5 (DR5). Therefore,NF-kappaB activation either suppresses or promotes apoptosis depending on the type of stimulus or cell context. We showed previously that the synthetic retinoid,6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437),effectively induces apoptosis particularly in androgen-independent prostate carcinoma cells. This effect was associated with the ability of CD437 to induce the expression of DR4 and DR5. In the present study,we examined the hypothesis that NF-kappaB activation plays a role in CD437-induced death receptor expression and apoptosis. Treatment of DU145 cells with CD437 resulted in a rapid decrease (textgreater or = 3 hours) of IkappaBalpha,which was accompanied by increased translocation of the NF-kappaB subunit p65 from the cytoplasm to the nucleus and increased NF-kappaB DNA-binding activity (textgreater or = 4 hours). The NF-kappaB inhibitor,helenalin,inhibited CD437-induced IkappaBalpha reduction and p65 nuclear translocation. Accordingly,it also abrogated CD437-induced up-regulation of DR4,activation of caspase-8 and caspase-3,and increased DNA fragmentation. Overexpression of an IkappaBalpha dominant-negative mutant blocked not only CD437-induced p65 nuclear translocation but also DR4 up-regulation,caspase activation,and DNA fragmentation. CD437 was unable to decrease IkappaBalpha protein levels and up-regulate DR4 expression in CD437-resistant DU145 cells. Moreover,knockdown of Fas-associated death domain,caspase-8,and DR4,respectively,suppressed CD437-induced apoptosis. Collectively,these results indicate that CD437 activates NF-kappaB via decreasing IkappaBalpha protein and thereby induces DR4 expression and subsequent apoptosis in DU145 cells. View Publication

Increasing occurrence of moderate to severe intrauterine adhesion (IUA) is seriously affecting the quality of human life. The aim of the study was to establish IUA models in nonhuman primates and to explore the dual repair effects of human umbilical cord-derived mesenchymal stem cells (huMSCs) loaded on autocrosslinked hyaluronic acid gel (HA-GEL) on endometrial damage and adhesion. Here,we recorded the menstrual cycle data in detail with uterine cavities observed and endometrial tissues detected after intervention,and the thicker endometria,decreased amount of fibrotic formation,increased number of endometrium glands,etc.,suggested that both HA-GEL and huMSC/HA-GEL complexes could partially repair IUA caused by mechanical injury,but huMSC/HA-GEL complex transplantation had notable dual repair effects: a reliable antiadhesion property and the promotion of endometrial regeneration. View Publication

An efficient system was developed that induced the differentiation of embryonic stem (ES) cells into blood cells of erythroid,myeloid,and B cell lineages by coculture with the stromal cell line OP9. This cell line does not express functional macrophage colony-stimulating factor (M-CSF). The presence of M-CSF had inhibitory effects on the differentiation of ES cells to blood cells other than macrophages. Embryoid body formation or addition of exogenous growth factors was not required,and differentiation was highly reproducible even after the selection of ES cells with the antibiotic G418. Combined with the ability to genetically manipulate ES cells,this system will facilitate the study of molecular mechanisms involved in development and differentiation of hematopoietic cells. View Publication

Growth of human embryonic stem (hES) cells as a pluripotent population requires a balance between survival,proliferation and self-renewal signals. Here we demonstrate that hES cells express receptors of the tropomyosin-related kinase (TRK) family,which mediate antiapoptotic signals. We show that three TRK ligands,brain-derived neurotrophic factor,neurotrophin 3 and neurotrophin 4,are survival factors for hES cells. Addition of neurotrophins to hES cell cultures effects a 36-fold improvement in their clonal survival. hES cell cultures maintained in medium containing neurotrophins remain diploid and retain full developmental potency. In the presence of neurotrophins,TRK receptors in hES cells are phosphorylated; TRK receptor inhibition leads to hES cell apoptosis. The survival activity of neurotrophins in hES cells is mediated by the phosphatidylinositol-3-kinase pathway but not the mitogen-activated protein kinase pathway. Neurotrophins improve hES cell survival and may facilitate their manipulation and the development of high-throughput screens to identify factors responsible for hES cell differentiation. View Publication

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We have compared the transcriptomic profiles of human induced pluripotent stem cells after their differentiation in hepatocytes like cells in plates and microfluidic biochips. The biochips provided a 3D and dynamic support during the cell differentiation when compared to the 2D static cultures in plates. The microarray have demonstrated the up regulation of important pathway related to liver development and maturation during the culture in biochips. Furthermore,the results of the transcriptomic profile,coupled with immunostaining,and RTqPCR analysis have shown typical biomarkers illustrating the presence of responders of biliary like cells,hepatocytes like cells,and endothelial like cells. However,the overall tissue still presented characteristic of immature and foetal patterns. Nevertheless,the biochip culture provided a specific micro-environment in which a complex multicellular differentiation toward liver could be oriented. View Publication

STAT3 is constitutively activated in many cancer types,including lung cancer,and can induce cancer cell proliferation and cancer stem cell (CSC) maintenance. STAT3 is activated by tyrosine kinases,such as JAK and SRC,but the mechanism by which STAT3 maintains its activated state in cancer cells remains unclear. Here,we show that PRMT5 directly methylates STAT3 and enhances its activated tyrosine phosphorylation in non-small cell lung cancer (NSCLC) cells. PRMT5 expression is also induced by STAT3,suggesting the presence of a positive feedback loop in cancer cells. Furthermore,methylation of STAT3 at arginine 609 by PRMT5 is important for its transcriptional activity and support of tumour growth and CSC maintenance. Indeed,NSCLC cells expressing the STAT3 mutant which R609 was replaced to alanine (R609K) show significantly impaired tumour growth in nude mice. Overall,our study reveals a mechanism by which STAT3 remains activated in NSCLC and provides a new target for cancer therapeutic approaches. Subject terms: Oncogenes,Non-small-cell lung cancer,Growth factor signalling View Publication

Human hematopoiesis originates in a population of stem cells with transplantable lympho-myeloid reconstituting potential,but a method for quantitating such cells has not been available. We now describe a simple assay that meets this need. It is based on the ability of sublethally irradiated immunodeficient nonobese diabetic-scid/scid (NOD/SCID) mice to be engrafted by intravenously injected human hematopoietic cells and uses limiting dilution analysis to measure the frequency of human cells that produce both CD34(-)CD19(+) (B-lymphoid) and CD34(+) (myeloid) colony-forming cell progeny in the marrow of such recipients 6 to 8 weeks post-transplant. Human cord blood (CB) contains approximately 5 of these competitive repopulating units (CRU) per ml that have a similar distribution between the CD38(-) and CD38(+) subsets of CD34(+) CB cells as long-term culture-initiating cells (LTC-IC) (4:1 vs. 2:1). Incubation of purified CD34(+)CD38(-) human CB cells in serum-free medium containing flt-3 ligand,Steel factor,interleukin 3,interleukin 6,and granulocyte colony-stimulating factor for 5-8 days resulted in a 100-fold expansion of colony-forming cells,a 4-fold expansion of LTC-IC,and a 2-fold (but significant,P textless 0.02) increase in CRU. The culture-derived CRU,like the original CB CRU,generated pluripotent,erythroid,granulopoietic,megakaryopoietic,and pre-B cell progeny upon transplantation into NOD/SCID mice. These findings demonstrate an equivalent phenotypic heterogeneity amongst human CB cells detectable as CRU and LTC-IC. In addition,their similarly modest response to stimulation by a combination of cytokines that extensively amplify LTC-IC from normal adult marrow underscores the importance of ontogeny-dependent changes in human hematopoietic stem cell proliferation and self-renewal. View Publication

Human induced pluripotent stem (hiPS) cell lines with tissue-specific or ubiquitous reporter genes are extremely useful for optimizing in vitro differentiation conditions as well as for monitoring transplanted cells in vivo. The adeno-associated virus integration site 1 (AAVS1) locus has been used as a safe harbor" locus for inserting transgenes because of its open chromatin structure View Publication