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

The self-renewal and differentiation capacities of human pluripotent stem cells (hPSCs) make them a promising source of material for cell transplantation therapy,drug development,and studies of cellular differentiation and development. However,the large numbers of cells necessary for many of these applications require extensive expansion of hPSC cultures,a process that has been associated with genetic and epigenetic alterations. We have performed a combinatorial study on both hESCs and hiPSCs to compare the effects of enzymatic vs. mechanical passaging,and feeder-free vs. mouse embryonic fibroblast feeder substrate,on the genetic and epigenetic stability and the phenotypic characteristics of hPSCs. In extensive experiments involving over 100 continuous passages,we observed that both enzymatic passaging and feeder-free culture were associated with genetic instability,higher rates of cell proliferation,and persistence of OCT4/POU5F1-positive cells in teratomas,with enzymatic passaging having the stronger effect. In all combinations of culture conditions except for mechanical passaging on feeder layers,we noted recurrent deletions in the genomic region containing the tumor suppressor gene TP53,which was associated with decreased mRNA expression of TP53,as well as alterations in the expression of several downstream genes consistent with a decrease in the activity of the TP53 pathway. Among the hESC cultures,we also observed culture-associated variations in global gene expression and DNA methylation. The effects of enzymatic passaging and feeder-free conditions were also observed in hiPSC cultures. Our results highlight the need for careful assessment of the effects of culture conditions on cells intended for clinical therapies. View Publication

Human engineered heart tissues have potential to revolutionize cardiac development research,drug-testing,and treatment of heart disease; however,implementation is limited by the need to use pre-differentiated cardiomyocytes (CMs). Here we show that by providing a 3D poly(ethylene glycol)-fibrinogen hydrogel microenvironment,we can directly differentiate human pluripotent stem cells (hPSCs) into contracting heart tissues. Our straight-forward,ontomimetic approach,imitating the process of development,requires only a single cell-handling step,provides reproducible results for a range of tested geometries and size scales,and overcomes inherent limitations in cell maintenance and maturation,while achieving high yields of CMs with developmentally appropriate temporal changes in gene expression. We demonstrate that hPSCs encapsulated within this biomimetic 3D hydrogel microenvironment develop into functional cardiac tissues composed of self-aligned CMs with evidence of ultrastructural maturation,mimicking heart development,and enabling investigation of disease mechanisms and screening of compounds on developing human heart tissue. View Publication

Alterations in DNA damage response and repair have been observed in Huntington's disease (HD). We generated induced pluripotent stem cells (iPSC) from primary dermal fibroblasts of 5 patients with HD and 5 control subjects. A significant fraction of the HD iPSC lines had genomic abnormalities as assessed by karyotype analysis,while none of our control lines had detectable genomic abnormalities. We demonstrate a statistically significant increase in genomic instability in HD cells during reprogramming. We also report a significant association with repeat length and severity of this instability. Our karyotypically normal HD iPSCs also have elevated ATM-p53 signaling as shown by elevated levels of phosphorylated p53 and H2AX,indicating either elevated DNA damage or hypersensitive DNA damage signaling in HD iPSCs. Thus,increased DNA damage responses in the HD genotype is coincidental with the observed chromosomal aberrations. We conclude that the disease causing mutation in HD increases the propensity of chromosomal instability relative to control fibroblasts specifically during reprogramming to a pluripotent state by a commonly used episomal-based method that includes p53 knockdown. View Publication

Electrospun scaffolds with varied stiffness promote distinct colony morphology of human induced pluripotent stem cells,which affects their subsequent differentiation. On soft scaffolds,induced pluripotent stem cells develop 3D colonies due to the pliability of the electrospun fibrous networks,leading to greater differentiation tendency to ectodermal lineage. View Publication

Mechanisms determining intrinsic differentiation bias inherent to human pluripotent stem cells (hPSCs) toward cardiogenic fate remain elusive. We evaluated the interplay between ErbB4 and EGFR in determining cardiac differentiation in vitro as these receptor tyrosine kinases (RTKs) are key to heart and brain development in vivo. Our results demonstrate that during cardiac differentiation,cell fate biases exist in hPSCs due to cardiac/neuroectoderm divergence post cardiac mesoderm stage. Stage-specific up-regulation of EGFR in concert with persistent Wnt3a signaling post cardiac mesoderm favors commitment towards neural progenitor cells (NPCs). Inhibition of EGFR abrogates these effects with enhanced (textgreater2-fold) cardiac differentiation efficiencies by increasing proliferation of Nkx2-5 expressing cardiac progenitors while reducing proliferation of Sox2 expressing NPCs. Forced overexpression of ErbB4 rescued cardiac commitment by augmenting Wnt11 signaling. Convergence between EGFR/ErbB4 and canonical/non-canonical Wnt signaling determines cardiogenic fate in hPSCs. This article is protected by copyright. All rights reserved. View Publication

This study evaluated the hypothesis that smoke from harm reduction cigarettes impedes attachment and proliferation of H9 human embryonic stem cells (hESCs). Smoke from three harm reduction brands was compared with smoke from a conventional brand. Doses of smoke were measured in puff equivalents (PE) (1 PE = the amount of smoke in one puff that dissolves in 1 ml of medium). Cytotoxic doses were determined using morphological criteria and trypan blue staining,and apoptosis was confirmed using Magic Red staining. Attachment and proliferation of hESC were followed at a noncytotoxic dose in time-lapse videos collected using BioStation technology. Data were mined from videos either manually or using video bioinformatics subroutines developed with CL-Quant software. Mainstream (MS) and sidestream (SS) smoke from conventional and harm reduction cigarettes induced apoptosis in hESC colonies at 1 PE. At 0.1 PE (noncytotoxic),SS smoke from all brands inhibited attachment of hESC colonies to Matrigel with the strongest inhibition occurring in harm reduction brands. At 0.1 PE,SS smoke,but not MS smoke,from all brands inhibited hESC growth,and two harm reduction brands were more potent than the conventional brand. In general,hESC appeared more sensitive to smoke than their mouse ESC counterparts. Although harm reduction cigarettes are often marketed as safer than conventional brands,our assays show that SS smoke from harm reduction cigarettes was at least as potent or in some cases more potent than smoke from a conventional brand and that SS smoke was more inhibitory than MS smoke in all assays. View Publication

Retinoids are important molecules involved in the development and homeostasis of the nervous system. As such,various retinoid derivatives are often found in culture media and supplement formulations to support the growth and maintenance of neural cells. However,all-trans-retinoic acid (ATRA) and its associated derivatives are light sensitive and are highly susceptible to isomerisation. This can lead to variability in retinoid concentrations and the nature of the retinoid species present in culture solutions which in turn can influence biological activity and introduce inconsistency. We have previously described the development of the synthetic retinoid derivative,EC23,as a chemically and light stable alternative that does not degrade and has biological activity similar to ATRA. In this study we demonstrate that the addition of exogenous retinoid can significantly enhance neuronal differentiation of both human neuroprogenitor and human embryonic stem cells. In the former,both ATRA and EC23 induced increased maturation and stabilisation of the axonal cytoskeleton. However,EC23 was particularly potent at lower nanomolar concentrations resulting in significantly greater neurogenesis than ATRA. In ES cells enhanced motor neuron marker expression was also detected in response to both retinoids when incorporated into an established protocol for neuronal differentiation. We propose that synthetic retinoid EC23 represents a valuable addition to the formulation of new and existing culture supplements to enhance neuronal differentiation whilst enabling improved consistency. View Publication

With regard to human induced pluripotent stem cells (hiPSCs),in which adult cells are reprogrammed into embryonic-like cells using defined factors,their functional and transcriptional expression pattern during endothelial differentiation has yet to be characterized. In this study,hiPSCs and human embryonic stem cells (hESCs) were differentiated using the embryoid body method,and CD31(+) cells were sorted. Fluorescence activated cell sorting analysis of hiPSC-derived endothelial cells (hiPSC-ECs) and hESC-derived endothelial cells (hESC-ECs) demonstrated similar endothelial gene expression patterns. We showed functional vascular formation by hiPSC-ECs in a mouse Matrigel plug model. We compared the gene profiles of hiPSCs,hESCs,hiPSC-ECs,hESC-ECs,and human umbilical vein endothelial cells (HUVECs) using whole genome microarray. Our analysis demonstrates that gene expression variation of hiPSC-ECs and hESC-ECs contributes significantly to biological differences between hiPSC-ECs and hESC-ECs as well as to the distances" among hiPSCs� View Publication

Associations between chronic antigen stimulation,T cell dysfunction,and the expression of various inhibitory receptors are well characterized in several mouse and human systems. During chronic hepatitis B virus (HBV) infection (CHB),T cell responses are blunted with low frequencies of virus-specific T cells observed,making these parameters difficult to study. Here,using mass cytometry and a highly multiplexed combinatorial peptide-major histocompatibility complex (pMHC) tetramer strategy that allows for the detection of rare antigen-specific T cells,we simultaneously probed 484 unique HLA-A*1101-restricted epitopes spanning the entire HBV genome on T cells from patients at various stages of CHB. Numerous HBV-specific T cell populations were detected,validated,and profiled. T cells specific for two epitopes (HBVpol387 and HBVcore169) displayed differing and complex heterogeneities that were associated with the disease progression,and the expression of inhibitory receptors on these cells was not linearly related with their extent of T cell dysfunction. For HBVcore169-specific CD8+ T cells,we found cellular markers associated with long-term memory,polyfunctionality,and the presence of several previously unidentified public TCR clones that correlated with viral control. Using high-dimensional trajectory analysis of these cellular phenotypes,a pseudo-time metric was constructed that fit with the status of viral infection in corresponding patients. This was validated in a longitudinal cohort of patients undergoing antiviral therapy. Our study uncovers complex relationships of inhibitory receptors between the profiles of antigen-specific T cells and the status of CHB with implications for new strategies of therapeutic intervention. View Publication

The transient receptor potential canonical (TRPC) family channels are proposed to be essential for store-operated Ca2+ entry in endothelial cells. Ca2+ signaling is involved in NF-kappaB activation,but the role of store-operated Ca2+ entry is unclear. Here we show that thrombin-induced Ca2+ entry and the resultant AMP-activated protein kinase (AMPK) activation targets the Ca2+-independent protein kinase Cdelta (PKCdelta) to mediate NF-kappaB activation in endothelial cells. We observed that thrombin-induced p65/RelA,AMPK,and PKCdelta activation were markedly reduced by knockdown of the TRPC isoform TRPC1 expressed in human endothelial cells and in endothelial cells obtained from Trpc4 knock-out mice. Inhibition of Ca2+/calmodulin-dependent protein kinase kinase beta downstream of the Ca2+ influx or knockdown of the downstream Ca2+/calmodulin-dependent protein kinase kinase beta target kinase,AMPK,also prevented NF-kappaB activation. Further,we observed that AMPK interacted with PKCdelta and phosphorylated Thr505 in the activation loop of PKCdelta in thrombin-stimulated endothelial cells. Expression of a PKCdelta-T505A mutant suppressed the thrombin-induced but not the TNF-alpha-induced NF-kappaB activation. These findings demonstrate a novel mechanism for TRPC channels to mediate NF-kappaB activation in endothelial cells that involves the convergence of the TRPC-regulated signaling at AMPK and PKCdelta and that may be a target of interference of the inappropriate activation of NF-kappaB associated with thrombosis. View Publication

Protein delivery allows a clinical effect to be directly realized without genetic modification of the host cells. We have developed a cationic bolaamphiphile as a non-viral vector for protein delivery application. The relatively low toxicity and efficient protein delivery by the cationic bolaamphiphile prompted us to test the system for the generation of induced pluripotent stem cells (iPSCs) as an alternative to the conventional vector-based genetic approach. Studies on the kinetics and cytotoxicity of the protein delivery system led us to use an optimized cationic bolaamphiphile-protein complex ratio of 7:1 (wt/wt) and a 3 h period of incubation with human fibroblasts,to ensure complete and non-toxic protein delivery of the reprogramming proteins. The reprogrammed cells were shown to exhibit the characteristics of embryonic stem cells,including expression of pluripotent markers,teratoma formation in SCID mice,and ability to be differentiated into a specific lineage,as exemplified by neuronal differentiation. View Publication

BACKGROUND We previously generated induced pluripotent stem cells by reprograming adipose stem cells through the introduction of microRNAs targeting four transcription factors (Oct3/4,Sox2,c-Myc,and Klf4). In this study,we aimed to reprogram cancer cells using microRNAs to explore their therapeutic potential. METHODS Mature microRNAs (mir-302a-d,369-3p and 5p,and mir-200c,as needed) were introduced into colon cancer cells (DLD-1,RKO,and HCT116) using lipofection. RESULTS The transfected cells exhibited an embryonic stem cell-like morphology and expressed the undifferentiated marker genes Nanog,Oct3/4,SOX2,and Klf4,as well as tumor-related antigen-1-60. These cells expressed neurogenic or adipogenic markers,indicating that reprogramming of the cancer cells was partially successful. Moreover,we found that miRNA-expressing DLD-1 cells showed low proliferative activity in vitro and in vivo,accompanied by increased expression of the tumor suppressor genes p16 (ink4a) and p21 (waf1) . miRNA-expressing DLD-1 cells also exhibited enhanced sensitivity to 5-fluorouracil,possibly through the downregulation of multidrug-resistant protein 8. The reprogrammed cells from DLD-1,RKO,and HCT116 cells exhibited reduced c-Myc expression,in contrast to the high c-Myc expression in the induced pluripotent cancer cells that were generated using four transcription factors. CONCLUSIONS Our cancer reprogramming method employing simple lipofection of mature microRNAs is safe and well suited for clinical application,because it avoids integration of exogenous genes into the host genome and allows escape from augmentation of c-Myc gene expression. View Publication