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

Peripheral blood was collected from a clinically characterized female Kleefstra syndrome patient with a heterozygous,de novo,premature termination codon (PTC) mutation (NM024757.4(EHMT1):c.3413GtextgreaterA; p.Trp1138Ter). Peripheral blood mononuclear cells (PBMCs) were reprogrammed with the human OSKM transcription factors using the Sendai-virus (SeV) delivery system. The pluripotency of transgene-free iPSC line was verified by the expression of pluripotency-associated markers and by in vitro spontaneous differentiation towards the 3 germ layers. Furthermore,the iPSC line showed normal karyotype. Our model might offer a good platform to study the pathomechanism of Kleefstra syndrome,also for drug testing,early biomarker discovery and gene therapy studies. View Publication

Despite the global prevalence of gastric disease,there are few adequate models in which to study the fundus epithelium of the human stomach. We differentiated human pluripotent stem cells (hPSCs) into gastric organoids containing fundic epithelium by first identifying and then recapitulating key events in embryonic fundus development. We found that disruption of Wnt/β-catenin signalling in mouse embryos led to conversion of fundic to antral epithelium,and that β-catenin activation in hPSC-derived foregut progenitors promoted the development of human fundic-type gastric organoids (hFGOs). We then used hFGOs to identify temporally distinct roles for multiple signalling pathways in epithelial morphogenesis and differentiation of fundic cell types,including chief cells and functional parietal cells. hFGOs are a powerful model for studying the development of the human fundus and the molecular bases of human gastric physiology and pathophysiology,and also represent a new platform for drug discovery. View Publication

Neutrophils are the most abundant WBCs and have an essential role in the clearance of pathogens. Tight regulation of neutrophil numbers and their recruitment to sites of inflammation is critical in maintaining a balanced immune response. In various inflammatory conditions,such as rheumatoid arthritis,vasculitis,cystic fibrosis,and inflammatory bowel disease,increased serum G-CSF correlates with neutrophilia and enhanced neutrophil infiltration into inflamed tissues. We describe a fully human therapeutic anti-G-CSFR antibody (CSL324) that is safe and well tolerated when administered via i.v. infusion to cynomolgus macaques. CSL324 was effective in controlling G-CSF-mediated neutrophilia when administered either before or after G-CSF. A single ascending-dose study showed CSL324 did not alter steady-state neutrophil numbers,even at doses sufficient to completely prevent G-CSF-mediated neutrophilia. Weekly infusions of CSL324 (%10 mg/kg) for 3 wk completely neutralized G-CSF-mediated pSTAT3 phosphorylation without neutropenia. Moreover,repeat dosing up to 100 mg/kg for 12 wk did not result in neutropenia at any point,including the 12-wk follow-up after the last infusion. In addition,CSL324 had no observable effect on basic neutrophil functions,such as phagocytosis and oxidative burst. These data suggest that targeting G-CSFR may provide a safe and effective means of controlling G-CSF-mediated neutrophilia as observed in various inflammatory diseases. View Publication

Murine embryonic stem cells can differentiate in vitro to form cystic embryoid bodies (CEB) that contain different structures and cell types. The blood islands are one such structure that consist of immature hematopoietic cells surrounded by endothelial cells,the first identifiable vascular cells. CEBs differentiated in vitro developed blood islands initially,and subsequently these blood islands matured to form vascular channels containing hematopoietic cells. Phase contrast microscopy demonstrated the presence of channels in mature CEBs grown in suspension culture,and high resolution light and electron microscopy showed that the cells lining these channels were endothelial cells. The channels appeared less organized than the vasculature of the mature yolk sac. The hematopoietic cells were occasionally seen 'flowing' through the CEB channels,although their numbers were reduced relative to the yolk sac. Analysis of primary CEB cultures showed the presence of cells with two characteristics of endothelial cells: approximately 30% of the cells labelled with fluorescent acetylated low density lipoprotein and a small number of cells were positive for von Willebrand's factor by immunostaining. Thus we conclude that a primitive vasculature forms in CEBs differentiated in vitro,and that not only primary differentiation of endothelial cells but also some aspects of vascular maturation are intrinsic to this cell culture system. CEBs are therefore a useful model for the study of developmental blood vessel formation. View Publication

The presentation of proteins on surfaces is fundamental to numerous cell culture and tissue engineering applications. While a number of physisorption and cross-linking methods exist to facilitate this process,few avoid denaturation of proteins or allow control over protein orientation,both of which are critical to the functionality of many signal proteins and ligands. Often recombinant protein sequences include a poly-histidine tag to facilitate purification. We utilize this sequence to anchor proteins to biosurfaces via a peptide bonded to the surface which conjugates with the poly-histidine tag in the presence of zinc rather than nickel,which is more traditionally used to conjugate poly-histidine tags to surfaces. We demonstrate that this strategy enables the display of proteins on 2D and 3D surfaces without compromising protein function through direct cross-linking or physisorption. View Publication

The epigenetic background of pluripotent stem cells can influence transcriptional and functional behavior. Most of these data have been obtained in standard monolayer cell culture systems. In this study,we used exome sequencing,array comparative genomic hybridization (CGH),miRNA array,DNA methylation array,three-dimensional (3D) tissue engineering,and immunostaining to conduct a comparative analysis of two induced pluripotent stem cell (iPSC) lines used in engineering of 3D human epidermal equivalent (HEE),which more closely approximates epidermis. Exome sequencing and array CGH suggested that their genome was stable following 3 months of feeder-free culture. While the miRNAome was also not affected,≈7% of CpG sites were differently methylated between the two lines. Analysis of the epidermal differentiation complex,a region on chromosome 1 that contains multiple genes involved in skin barrier maturation (including trichohyalin,TCHH),found that in one of the iPSC clones (iKCL004),TCHH retained a DNA methylation signature characteristic of the original somatic cells,whereas in other iPSC line (iKCL011),the TCHH methylation signature matched that of the human embryonic stem cell line KCL034. The difference between the two iPSC clones in TCHH methylation did not have an obvious effect on its expression in 3D HEE,suggesting that differentiation and tissue formation may mitigate variations in the iPSC methylome. View Publication

Structural cardiac remodeling,accompanying cytoskeletal reorganization of cardiac cells,is a major clinical outcome of diastolic heart failure. A highly local Ca(2+) influx across the plasma membrane has been suggested to code signals to induce Rho GTPase-mediated fibrosis,but it is obscure how the heart specifically decodes the local Ca(2+) influx as a cytoskeletal reorganizing signal under the conditions of the rhythmic Ca(2+) handling required for pump function. We found that an inhibition of transient receptor potential canonical 3 (TRPC3) channel activity exhibited resistance to Rho-mediated maladaptive fibrosis in pressure-overloaded mouse hearts. Proteomic analysis revealed that microtubule-associated Rho guanine nucleotide exchange factor,GEF-H1,participates in TRPC3-mediated RhoA activation induced by mechanical stress in cardiomyocytes and transforming growth factor (TGF) β stimulation in cardiac fibroblasts. We previously revealed that TRPC3 functionally interacts with microtubule-associated NADPH oxidase (Nox) 2,and inhibition of Nox2 attenuated mechanical stretch-induced GEF-H1 activation in cardiomyocytes. Finally,pharmacological TRPC3 inhibition significantly suppressed fibrotic responses in human cardiomyocytes and cardiac fibroblasts. These results strongly suggest that microtubule-localized TRPC3-GEF-H1 axis mediates fibrotic responses commonly in cardiac myocytes and fibroblasts induced by physico-chemical stimulation. View Publication

Summary Rett syndrome (RTT) is caused by mutations of MECP2,a methyl CpG binding protein thought to act as a global transcriptional repressor. Here we show,using an isogenic human embryonic stem cell model of RTT,that MECP2 mutant neurons display key molecular and cellular features of this disorder. Unbiased global gene expression analyses demonstrate that MECP2 functions as a global activator in neurons but not in neural precursors. Decreased transcription in neurons was coupled with a significant reduction in nascent protein synthesis and lack of MECP2 was manifested as a severe defect in the activity of the AKT/mTOR pathway. Lack of MECP2 also leads to impaired mitochondrial function in mutant neurons. Activation of AKT/mTOR signaling by exogenous growth factors or by depletion of PTEN boosted protein synthesis and ameliorated disease phenotypes in mutant neurons. Our findings indicate a vital function for MECP2 in maintaining active gene transcription in human neuronal cells. View Publication

Increasing evidence supports the importance of cell extrinsic regulation in stem cell fate control. Hematopoietic stem cells (HSC) are responsive to local signals from their niche and to systemic feedback from progenitors and mature cells. The Notch ligand Delta-1 (DL1),a key component of the stem cell niche,regulates human hematopoietic lineage development in a dose-dependent manner and has been used clinically for primitive progenitor expansion. How DL1 acts to regulate HSC fate and whether these actions are related to its lineage skewing effects are poorly understood. Here we demonstrate that,although DL1 activates signal transducer and activator of transcription 3 signaling similarly to the gp130-activating cytokine interleukin-6 (IL-6),it has opposite effects on myeloid cell production. Mechanistically,these different outcomes are attributable to a DL1-mediated reduction in membrane (m)-bound IL-6 receptor (R) expression,converting progenitor cells from being directly IL-6 responsive to requiring both IL-6 and soluble (s) IL-6R for activation. Concomitant reduction of both mIL-6R (by DL1 supplementation) and sIL-6R (using dynamically fed cultures) reduced myeloid cell production and led to enhanced outputs of human HSCs. This work describes a new mode of cytokine action in which DL1 changes cytokine receptor distributions on hematopoietic cells,altering feedback networks and their impact on stem cell fate. View Publication