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

The active movement of cells from subendothelial compartments into the bloodstream (intravasation) has been recognized for several decades by histologic and physiologic studies,yet the molecular effectors of this process are relatively uncharacterized. For extravasation,studies based predominantly on static transwell assays support a general model,whereby transendothelial migration (TEM) occurs via chemoattraction toward increasing chemokine concentrations. However,this model of chemotaxis cannot readily reconcile how chemokines influence intravasation,as shear forces of blood flow would likely abrogate luminal chemokine gradient(s). Thus,to analyze how T cells integrate perivascular chemokine signals under physiologic flow,we developed a novel transwell-based flow chamber allowing for real-time modulation of chemokine levels above (luminal/apical compartment) and below (abluminal/subendothelial compartment) HUVEC monolayers. We routinely observed human T cell TEM across HUVEC monolayers with the combination of luminal CXCL12 and abluminal CCL5. With increasing concentrations of CXCL12 in the luminal compartment,transmigrated T cells did not undergo retrograde transendothelial migration (retro-TEM). However,when exposedto abluminal CXCL12,transmigrated T cells underwent striking retro-TEM and re-entered the flow stream [corrected]. This CXCL12 fugetactic (chemorepellant) effect was concentration-dependent,augmented by apical flow,blocked by antibodies to integrins,and reduced by AMD3100 in a dose-dependent manner. Moreover,CXCL12-induced retro-TEM was inhibited by PI3K antagonism and cAMP agonism. These findings broaden our understanding of chemokine biology and support a novel paradigm by which temporospatial modulations in subendothelial chemokine display drive cell migration from interstitial compartments into the bloodstream. View Publication

Extracorporeal photochemotherapy (ECP) is widely used to treat cutaneous T-cell lymphoma,graft-versus-host disease,and allografted organ rejection. Its clinical and experimental efficacy in cancer immunotherapy and autoreactive disorders suggests a novel mechanism. This study reveals that ECP induces a high percentage of processed monocytes to enter the antigen-presenting dendritic cell (DC) differentiation pathway,within a single day,without added cytokines,as determined by enhanced expression of relevant genes. The resulting DCs are capable of processing and presentation of exogenous and endogenous antigen and are largely maturationally synchronized,as assessed by the level of expression of costimulatory surface molecules. Principal component analysis of the ECP-induced monocyte transcriptome reveals that activation or suppression of more than 1100 genes produces a reproducible distinctive molecular signature,common to ECP-processed monocytes from normal subjects,and those from patients. Because ECP induces normal monocytes to enter the DC differentiation pathway,this phenomenon is independent of disease state. The efficiency with which ECP stimulates new functional DCs supports the possibility that these cells participate prominently in the clinical successes of the treatment. Appropriately modified by future advances,ECP may potentially offer a general source of therapeutic DCs. View Publication

OBJECTIVE: Various preparative protocols have been proposed for the acquisition and cultivation of mesenchymal stem cells (MSC). Whereas surface antigen markers have failed to precisely define this population,microarray analysis might provide a better tool for characterization of MSC. METHODS: In this study,we have analyzed global gene expression profiles of human MSC isolated from adipose tissue (AT),from umbilical cord blood (CB),and from bone marrow (BM) under two growth conditions and have compared them to terminally differentiated human fibroblasts (HS68). Profiles were compared using our Human Genome Microarray representing 51.144 different cDNA clones. RESULTS: Cultured with the appropriate conditions,osteogenic and adipogenic differentiation could be confirmed in all MSC preparations but not in fibroblasts. No phenotypic differences were observed by flow cytometry using a panel of 22 surface antigen markers. Whereas MSC derived from different donors using the same culture procedure yielded a consistent and reproducible gene expression profile,many genes were differentially expressed in MSC from different ontogenetic sources or from different culture conditions. Twenty-five genes were overlapping and upregulated in all MSC preparations from AT,CB,and BM as compared to HS68 fibroblasts. These genes included fibronectin,ECM2,glypican-4,ID1,NF1B,HOXA5,and HOXB6. Many genes upregulated in MSC are involved in extracellular matrix,morphogenesis,and development,whereas several inhibitors of the Wnt pathway (DKK1,DKK3,SFRP1) were highly expressed in fibroblasts. CONCLUSION: Our results have provided a foundation for a more reproducible and reliable quality control using genotypic analysis for defining MSC. View Publication

BACKGROUND AND PURPOSE: Teratogenic substances induce adverse effects during the development of the embryo. Multilineage differentiation of human embryonic stem cells (hESCs) mimics the development of the embryo in vitro. Here,we propose a transcriptomic approach in hESCs for monitoring specific toxic effects of compounds as an alternative to traditional time-consuming and cost-intensive in vivo tests requiring large numbers of animals. This study was undertaken to explore the adverse effects of cytosine arabinoside (Ara-C) on randomly differentiated hESCs.backslashnbackslashnEXPERIMENTAL APPROACH: Human embryonic stem cells were used to investigate the effects of a developmental toxicant Ara-C. Sublethal concentrations of Ara-C were given for two time points,day 7 and day 14 during the differentiation. Gene expression was assessed with microarrays to determine the dysregulated transcripts in presence of Ara-C.backslashnbackslashnKEY RESULTS: Randomly differentiated hESCs were able to generate the multilineage markers. The low concentration of Ara-C (1 nM) induced the ectoderm and inhibited the mesoderm at day 14. The induction of ectodermal markers such as MAP2,TUBB III,PAX6,TH and NESTIN was observed with an inhibition of mesodermal markers such as HAND2,PITX2,GATA5,MYL4,TNNT2,COL1A1 and COL1A2. In addition,no induction of apoptosis was observed. Gene ontology revealed unique dysregulated biological process related to neuronal differentiation and mesoderm development. Pathway analysis showed the axon guidance pathway to be dysregulated.backslashnbackslashnCONCLUSIONS AND IMPLICATIONS: Our results suggest that hESCs in combination with toxicogenomics offer a sensitive in vitro developmental toxicity model as an alternative to traditional animal experiments. View Publication

The lipophilic statin lovastatin decreases cholesterol synthesis and is a safe and effective treatment for the prevention of cardiovascular diseases. Growing evidence points at antitumor potential of lovastatin. Therefore,understanding the molecular mechanism of lovastatin function in different cell types is critical to effective therapy design. In this study,we investigated the effects of lovastatin on the differentiation potential of human embryonic stem (hES) cells (H9 cell line). Multiparameter flow cytometric assay was used to detect changes in the expression of transcription factors characteristic of hES cells. We found that lovastatin treatment delayed NANOG downregulation during ectodermal and endodermal differentiation. Likewise,expression of ectodermal (SOX1 and OTX2) and endodermal (GATA4 and FOXA2) markers was higher in treated cells. Exposure of hES cells to lovastatin led to a minor decrease in the expression of SSEA-3 and a significant reduction in CD133 expression. Treated cells also formed fewer embryoid bodies than control cells. By analyzing hES with and without CD133,we discovered that CD133 expression is required for proper formation of embryoid bodies. In conclusion,lovastatin reduced the heterogeneity of hES cells and impaired their differentiation potential. View Publication

Two monoclonal antibodies,BA16 and BA17,have been developed using a detergent-insoluble extract of human mammary epithelial organoids as immunogen. Indirect immunofluorescent staining of cultured cells showed that the component reacting with the antibodies was filamentous and the intensity of staining was stronger in mitotic cells. Immunoblotting of cell extracts showed that both antibodies react with only one band of 40 X 10(3) molecular weight,which was present in keratin-enriched extracts of cells or organoids. Furthermore,the tissue distribution of the component reacting with the antibodies was that predicted for human keratin 19. The antibodies showed differences in the intensity of staining of cells or tissue sections fixed and prepared in different ways indicating that they reacted with different epitopes. The pattern of expression of the 40 X 10(3) Mr keratin by normal mammary epithelial cells was investigated by immunoperoxidase staining of tissue sections,cultured milk cells,and organoids of different sizes cultured in collagen gels. It was found that basal or myoepithelial cells did not express this keratin. Some heterogeneity of expression of this component was seen in luminal epithelial cells,found almost exclusively in the smaller structures. These cells did,however,express other keratins characteristic of luminal cells. The distribution in the mammary tree of the luminal cells that did not express the 40 X 10(3) Mr keratin appears to be similar to that expected for cells with the proliferative potential to produce new terminal ductal lobular units or an increase in branching of existing terminal ductal lobular units. It is shown that these cells have considerable proliferative potential by the fact that they form large colonies in milk cell cultures. View Publication

Pluripotent stem cells display significant heterogeneity in gene expression,but whether this diversity is an inherent feature of the pluripotent state remains unknown. Single-cell gene expression analysis in cell subsets defined by surface antigen expression revealed that human embryonic stem cell cultures exist as a continuum of cell states,even under defined conditions that drive self-renewal. The majority of the population expressed canonical pluripotency transcription factors and could differentiate into derivatives of all three germ layers. A minority subpopulation of cells displayed high self-renewal capacity,consistently high transcripts for all pluripotency-related genes studied,and no lineage priming. This subpopulation was characterized by its expression of a particular set of intercellular signaling molecules whose genes shared common regulatory features. Our data support a model of an inherently metastable self-renewing population that gives rise to a continuum of intermediate pluripotent states,which ultimately become primed for lineage specification. ?? 2014 The Authors. View Publication

Various strategies have been published enabling cardiomyocyte differentiation of human induced pluripotent stem (iPS) cells. However the complex nature of signaling pathways involved as well as line-to-line variability compromises the application of a particular protocol to robustly obtain cardiomyocytes from multiple iPS lines. Hence it is necessary to identify optimized protocols with alternative combinations of specific growth factors and small molecules to enhance the robustness of cardiac differentiation. Here we focus on systematic modulation of BMP and WNT signaling to enhance cardiac differentiation. Moreover,we improve the efficacy of cardiac differentiation by enrichment via lactate. Using our protocol we show efficient derivation of cardiomyocytes from multiple human iPS lines. In particular we demonstrate cardiomyocyte differentiation within 15 days with an efficiency of up to 95 % as judged by flow cytometry staining against cardiac troponin T. Cardiomyocytes derived were functionally validated by alpha-actinin staining,transmission electron microscopy as well as electrophysiological analysis. We expect our protocol to provide a robust basis for scale-up production of functional iPS cell-derived cardiomyocytes that can be used for cell replacement therapy and disease modeling. View Publication

The Mesenchymal-to-Epithelial Transition (MET) has been recognized as a crucial step for successful reprogramming of fibroblasts to induced pluripotent stem cells (iPSCs). Thus,it has been demonstrated,that the efficiency of reprogramming can be enhanced by promoting an epithelial expression program in cells,with a concomitant repression of key mesenchymal genes. However,a detailed characterization of the epithelial transition associated with the acquisition of a pluripotent phenotype is still lacking to this date. Here,we integrate a panel of morphological approaches with gene expression analyses to visualize the dynamics of episomal reprogramming of human fibroblasts to iPSCs. We provide the first ultrastructural analysis of human fibroblasts at various stages of episomal iPSC reprogramming,as well as the first real-time live cell visualization of a MET occurring during reprogramming. The results indicate that the MET manifests itself approximately 6-12. days after electroporation,in synchrony with the upregulation of early pluripotency markers,and resembles a reversal of the Epithelial-to-Mesenchymal Transition (EMT) which takes place during mammalian gastrulation. View Publication

BackgroundThe simultaneous differentiation of human pluripotent stem cells (hPSCs) into both endodermal and mesodermal lineages is crucial for developing complex,vascularized tissues,yet poses significant challenges. This study explores a method for co-differentiation of mesoderm and endoderm,and their subsequent differentiation into pancreatic progenitors (PP) with endothelial cells (EC).MethodsTwo hPSC lines were utilized. By manipulating WNT signaling,we optimized co-differentiation protocols of mesoderm and endoderm through adjusting the concentrations of CHIR99021 and mTeSR1. Subsequently,mesoderm and endoderm were differentiated into vascularized pancreatic progenitors (vPP) by adding VEGFA. The differentiation characteristics and potential of vPPs were analyzed via transcriptome sequencing and functional assays.ResultsA low-dose CHIR99021 in combination with mTeSR1 yielded approximately 30% mesodermal and 70% endodermal cells. Introduction of VEGFA significantly enhanced EC differentiation without compromising PP formation,increasing the EC proportion to 13.9%. Transcriptomic analyses confirmed the effectiveness of our protocol,showing up-regulation of mesodermal and endothelial markers,alongside enhanced metabolic pathways. Functional assays demonstrated that vPPs could efficiently differentiate into insulin-producing ?-cells,as evidenced by increased expression of ?-cell markers and insulin secretion.ConclusionOur findings provide a robust method for generating vPPs,which holds significant promise for regenerative medicine applications,particularly in diabetes treatment.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-024-04120-5. View Publication

Tuberous Sclerosis Complex (TSC) is a debilitating developmental disorder characterized by a variety of clinical manifestations. While benign tumors in the heart,lungs,kidney,and brain are all hallmarks of the disease,the most severe symptoms of TSC are often neurological,including seizures,autism,psychiatric disorders,and intellectual disabilities. TSC is caused by loss of function mutations in the TSC1 or TSC2 genes and consequent dysregulation of signaling via mechanistic Target of Rapamycin Complex 1 (mTORC1). While TSC neurological phenotypes are well-documented,it is not yet known how early in neural development TSC1/2-mutant cells diverge from the typical developmental trajectory. Another outstanding question is the contribution of homozygous-mutant cells to disease phenotypes and whether such phenotypes are also seen in the heterozygous-mutant populations that comprise the vast majority of cells in patients. Using TSC patient-derived isogenic induced pluripotent stem cells (iPSCs) with defined genetic changes,we observed aberrant early neurodevelopment in vitro,including misexpression of key proteins associated with lineage commitment and premature electrical activity. These alterations in differentiation were coincident with hundreds of differentially methylated DNA regions,including loci associated with key genes in neurodevelopment. Collectively,these data suggest that mutation or loss of TSC2 affects gene regulation and expression at earlier timepoints than previously appreciated,with implications for whether and how prenatal treatment should be pursued. View Publication

Regulation of the homeostasis of vascular endothelium is critical for the processes of vascular remodeling and angiogenesis under physiological and pathological conditions. Here we show that doxorubicin (Dox),a drug used in antitumor therapy,triggered a marked accumulation of p53 and induced CD95 gene expression and apoptosis in proliferating human umbilical vein endothelial cells (HUVECs). Transfection and site-directed mutagenesis experiments using the CD95 promoter fused to an intronic enhancer indicated the requirement for a p53 site for Dox-induced promoter activation. Furthermore,the p53 inhibitor pifithrin-alpha (PFT-alpha) blocked both promoter inducibility and protein up-regulation of CD95 in response to Dox. Up-regulated CD95 in Dox-treated cells was functional in eliciting apoptosis upon incubation of the cells with an agonistic CD95 antibody. However,Dox-mediated apoptosis was independent of CD95/CD95L interaction. The analysis of apoptosis in the presence of PFT-alpha and benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone revealed that both p53 and caspase activation are required for Dox-mediated apoptosis of HUVECs. Finally,Dox triggered Bcl-2 down-regulation,cytochrome c release from mitochondria,and the activation of caspases 9 and 3,suggesting the involvement of a mitochondrially operated pathway of apoptosis. These results highlight the role of p53 in the response of primary endothelial cells to genotoxic drugs and may reveal a novel mechanism underlying the antitumoral properties of Dox,related to its ability to induce apoptosis in proliferating endothelial cells. View Publication