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

CD4+CD25+ regulatory T cells play an important role in peripheral tolerance. Upon T cell receptor (TCR)-mediated activation,the cells fail to proliferate but are induced to have a suppressor function. The intracellular signaling events that lead to their responses have not been elucidated. In this study,we have examined the proximal TCR signaling events in freshly isolated human CD4+CD25+ regulatory T cells after TCR ligation. In contrast to CD4+CD25- T cells,TCR ligation of CD4+CD25+ regulatory T cells by anti-CD3 cross-linking resulted in a lower calcium influx and extracellular signal-regulated kinase 1/2 phosphorylation. Examination of the CD3zeta chain phosphorylation status indicated that CD4+CD25+ regulatory T cells have poor phosphorylation of the protein and consequently,reduced recruitment of zeta-associated protein-70 to the TCR immunoreceptor tyrosine motif. The adaptor protein,Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa,which relays signals to downstream signaling components,also showed reduced phosphorylation,which correlated with reduced VAV guanine nucleotide exchange factors association. Consistent with other findings,the defect is accompanied with impaired actin cap formation,implicating a failure of actin remodeling of the cells. Together,our results demonstrate that CD4+CD25+ regulatory T cells have altered TCR proximal signaling pathways,which could be critical for inducing the distinct behavior of these cells. View Publication

B cell-activating factor belonging to the TNF family (BAFF) plays a critical role in B cell maturation,yet its precise role in B cell differentiation into Ig-secreting cells (ISCs) remains unclear. In this study,we find that upon isolation human naive and memory B (MB) cells have prebound BAFF on their surface,whereas germinal center (GC) B cells lack detectable levels of prebound BAFF. We attribute their lack of prebound BAFF to cell activation,because we demonstrate that stimulation of naive and MB cells results in the loss of prebound BAFF. Furthermore,the absence of prebound BAFF on GC B cells is not related to a lack of BAFF-binding receptors or an inability to bind exogenous BAFF. Instead,our data suggest that accessibility to soluble BAFF is limited within GCs,perhaps to prevent skewing of the conventional B cell differentiation program. In support of this concept,whereas BAFF significantly enhances ISC differentiation in response to T cell-dependent activation,we report for the first time the ability of BAFF to considerably attenuate ISC differentiation of MB cells in response to CpG stimulation,a form of T cell-independent activation. Our data suggest that BAFF may be providing regulatory signals during specific T cell-independent events,which protect the balance between MB cells and ISCs outside GCs. Taken together,these data define a complex role for BAFF in humoral immune responses and show for the first time that BAFF can also play an inhibitory role in B cell differentiation. View Publication

This study investigated the effect of mechanical load on human mesenchymal stem cell (hMSC) differentiation under different exogenous transforming growth factor-beta1 (TGF-beta(1)) concentrations (0,1 or 10 ng/ml).The role of the TGF-beta signalling pathway in this process was also studied. Human MSCs were seeded into fibrin-biodegradable polyurethane scaffolds at a cell density of 5 x 10(6) cells per scaffold and stimulated using our bioreactor. One hour of surface motion superimposed on cyclic compression was applied once a day over seven consecutive days. Scaffolds were analysed for gene expression,DNA content and glycosaminoglycan amount. Addition of TGF-beta(1) in the culture medium was sufficient to induce chondrogenesis of hMSCs. Depending on the TGF-beta(1) concentration of the culture medium,mechanical load stimulated chondrogenesis of hMSCs compared to the unloaded scaffolds,with a much stronger effect on gene expression at lower TGF-beta(1) concentrations. With TGF-beta(1) absent in the culture medium,mechanical load stimulated gene transcripts and protein synthesis of TGF-beta(1) and TGF-beta(3). TGF-beta type I receptor inhibitor LY364947 blocked the up-regulation on TGF-beta(1) and TGF-beta(3) production stimulated by mechanical load,and also blocked the chondrogenesis of hMSCs. Taken together,these findings suggest that mechanical load promotes chondrogenesis of hMSCs through TGF-beta pathway by up-regulating TGF-beta gene expression and protein synthesis. View Publication

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by defined factors. However,the low efficiency and slow kinetics of the reprogramming process have hampered progress with this technology. Here we report that a natural compound,vitamin C (Vc),enhances iPSC generation from both mouse and human somatic cells. Vc acts at least in part by alleviating cell senescence,a recently identified roadblock for reprogramming. In addition,Vc accelerates gene expression changes and promotes the transition of pre-iPSC colonies to a fully reprogrammed state. Our results therefore highlight a straightforward method for improving the speed and efficiency of iPSC generation and provide additional insights into the mechanistic basis of the reprogramming process. View Publication

Human embryonic stem cells (hESC) present a novel platform for in vitro investigation of the early embryonic cellular response to ionizing radiation. Thus far,no study has analyzed the genome-wide transcriptional response to ionizing radiation in hESCs,nor has any study assessed their ability to form teratomas,the definitive test of pluripotency. In this study,we use microarrays to analyze the global gene expression changes in hESCs after low-dose (0.4 Gy),medium-dose (2 Gy),and high-dose (4 Gy) irradiation. We identify genes and pathways at each radiation dose that are involved in cell death,p53 signaling,cell cycling,cancer,embryonic and organ development,and others. Using Gene Set Enrichment Analysis,we also show that the expression of a comprehensive set of core embryonic transcription factors is not altered by radiation at any dose. Transplantation of irradiated hESCs to immune-deficient mice results in teratoma formation from hESCs irradiated at all doses,definitive proof of pluripotency. Further,using a bioluminescence imaging technique,we have found that irradiation causes hESCs to initially die after transplantation,but the surviving cells quickly recover by 2 weeks to levels similar to control. To conclude,we show that similar to somatic cells,irradiated hESCs suffer significant death and apoptosis after irradiation. However,they continue to remain pluripotent and are able to form all three embryonic germ layers. Studies such as this will help define the limits for radiation exposure for pregnant women and also radiotracer reporter probes for tracking cellular regenerative therapies. View Publication

Pluripotency and self-renewal of human embryonic stem cells (hESCs) is mediated by a complex interplay between extra- and intracellular signaling pathways,which regulate the expression of pluripotency-specific transcription factors. The homeodomain transcription factor NANOG plays a central role in maintaining hESC pluripotency,but the precise role and regulation of NANOG are not well defined. View Publication

Sampling various compartments within the lower airways to examine human bronchial epithelial cells (HBEC) is essential for understanding numerous lung diseases. Conventional methods to identify HBEC in bronchoalveolar lavage (BAL) and wash (BW) have throughput limitations in terms of efficiency and ensuring adequate cell numbers for quantification. Flow cytometry can provide high-throughput quantification of cell number and function in BAL and BW samples,while requiring low cell numbers. To date,a flow cytometric method to identify HBEC recovered from lower human airway samples is unavailable. In this study we present a flow cytometric method identifying HBEC as CD45 negative,EpCAM/pan-cytokeratin (pan-CK) double-positive population after excluding debris,doublets and dead cells from the analysis. For validation,the HBEC panel was applied to primary HBEC resulting in 98.6% of live cells. In healthy volunteers,HBEC recovered from BAL (2.3% of live cells),BW (32.5%) and bronchial brushing samples (88.9%) correlated significantly (p = 0.0001) with the manual microscopy counts with an overall Pearson correlation of 0.96 across the three sample types. We therefore have developed,validated,and applied a flow cytometric method that will be useful to interrogate the role of the respiratory epithelium in multiple lung diseases. View Publication

CD34 is a transmembrane phosphoglycoprotein used to selectively enrich bone marrow in hematopoietic stem cells for transplantation. Treating rats with CD34+ cells derived from human umbilical cord blood before or after heat stroke has been shown to promote survival. We investigated whether CD34- human placenta-derived stem cells (PDMSCs) could improve survival following heat stroke in rats. Rats were subjected to heat stress (42°C for 98 min) to induce heat stroke. Intravenous administration of PDMSCs 1 day before or immediately after the onset of heat stroke improved survival by 60{\%} and 20{\%},respectively. Pre-treatment with CD34- PDMSCs protected against heat stroke injury more effectively than that treatment after injury. PDMSCs treatment attenuated cerebrovascular dysfunction,the inflammatory response,and lipid peroxidation. These data suggest human PDMSCs protect against heat stroke injury in rats. Moreover,these effects do not require the presence of CD34+ cells. View Publication

Vascularization of engineered human skin constructs is crucial for recapitulation of systemic drug delivery and for their long-term survival,functionality,and viable engraftment. In this study,the latest microfabrication techniques are used and a novel bioengineering approach is established to micropattern spatially controlled and perfusable vascular networks in 3D human skin equivalents using both primary and induced pluripotent stem cell (iPSC)-derived endothelial cells. Using 3D printing technology makes it possible to control the geometry of the micropatterned vascular networks. It is verified that vascularized human skin equivalents (vHSEs) can form a robust epidermis and establish an endothelial barrier function,which allows for the recapitulation of both topical and systemic delivery of drugs. In addition,the therapeutic potential of vHSEs for cutaneous wounds on immunodeficient mice is examined and it is demonstrated that vHSEs can both promote and guide neovascularization during wound healing. Overall,this innovative bioengineering approach can enable in vitro evaluation of topical and systemic drug delivery as well as improve the potential of engineered skin constructs to be used as a potential therapeutic option for the treatment of cutaneous wounds. View Publication

Congenital heart defects are the most common birth defect. The limiting factor in tissue engineering repair strategies is an autologous source of functional cardiomyocytes. Amniotic fluid contains an ideal cell source for prenatal harvest and use in correction of congenital heart defects. This study aims to investigate the potential of amniotic fluid-derived stem cells (AFSC) to undergo non-viral reprogramming into induced pluripotent stem cells (iPSC) followed by growth-factor-free differentiation into functional cardiomyocytes. AFSC from human second trimester amniotic fluid were transfected by non-viral vesicle fusion with modified mRNA of OCT4,KLF4,SOX2,LIN28,cMYC and nuclear GFP over 18 days,then differentiated using inhibitors of GSK3 followed 48 hours later by inhibition of WNT. AFSC-derived iPSC had high expression of OCT4,NANOG,TRA-1-60,and TRA-1-81 after 18 days of mRNA transfection and formed teratomas containing mesodermal,ectodermal,and endodermal germ layers in immunodeficient mice. By Day 30 of cardiomyocyte differentiation,cells contracted spontaneously,expressed connexin 43 and β-myosin heavy chain organized in sarcomeric banding patterns,expressed cardiac troponin T and β-myosin heavy chain,showed upregulation of NKX2.5,ISL-1 and cardiac troponin T with downregulation of POU5F1,and displayed calcium and voltage transients similar to those in developing cardiomyocytes. These results demonstrate that cells from human amniotic fluid can be differentiated through a pluripotent state into functional cardiomyocytes. View Publication

Tumorigenicity of human pluripotent stem cells is a major threat limiting their application in cell therapy protocols. It remains unclear,however,whether suppression of tumorigenic potential can be achieved without critically affecting pluripotency. A previous study has identified hyperexpressed genes in cancer stem cells,among which is E2F2,a gene involved in malignant transformation and stem cell self-renewal. Here we tested whether E2F2 knockdown would affect the proliferative capacity and tumorigenicity of human embryonic stem cells (hESC). Transient E2F2 silencing in hESC significantly inhibited expression of the proto-oncogenes BMI1 and HMGA1,in addition to proliferation of hESC,indicated by a higher proportion of cells in G1,fewer cells in G2/M phase,and a reduced capacity to generate hESC colonies in vitro. Nonetheless,E2F2-silenced cells kept expression of typical pluripotency markers and displayed differentiation capacity in vitro. More importantly,E2F2 knockdown in hESC significantly inhibited tumor growth in vivo,which was considerably smaller than tumors generated from control hESC,although displaying typical teratoma traits,a major indicator of pluripotency retention in E2F2-silenced cells. These results suggest that E2F2 knockdown can inhibit hESC proliferation and tumorigenicity without significantly harming stemness,providing a rationale to future protocols aiming at minimizing risks related to therapeutic application of cells and/or products derived from human pluripotent cells. View Publication

Amyotrophic lateral sclerosis is a progressive disease characterized by the loss of upper and lower motor neurons,leading to paralysis of voluntary muscles. About 10% of all ALS cases are familial (fALS),among which 15-20% are linked to Cu/Zn superoxide dismutase (SOD1) mutations,usually inherited in an autosomal dominant manner. To date only one FDA approved drug is available which increases survival moderately. Our understanding of ALS disease mechanisms is largely derived from rodent model studies,however due to the differences between rodents and humans,it is necessary to have humanized models for studies of disease pathogenesis as well as drug development. Therefore,we generated a comprehensive library of a total 22 of fALS patient-specific induced pluripotent stem cell (iPSC) lines. These cells were thoroughly characterized before being deposited into the library. The library of cells includes a variety of C9orf72 mutations,sod1 mutations,FUS,ANG and FIG4 mutations. Certain mutations are represented with more than one line,which allows for studies of variable genetic backgrounds. In addition,these iPSCs can be successfully differentiated to astroglia,a cell type known to play a critical role in ALS disease progression. This library represents a comprehensive resource that can be used for ALS disease modeling and the development of novel therapeutics. View Publication