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

Teratogens,substances that may cause fetal abnormalities during development,are responsible for a significant number of birth defects. Animal models used to predict teratogenicity often do not faithfully correlate to human response. Here,we seek to develop a more predictive developmental toxicity model based on an in vitro method that utilizes both human embryonic stem (hES) cells and metabolomics to discover biomarkers of developmental toxicity. We developed a method where hES cells were dosed with several drugs of known teratogenicity then LC-MS analysis was performed to measure changes in abundance levels of small molecules in response to drug dosing. Statistical analysis was employed to select for specific mass features that can provide a prediction of the developmental toxicity of a substance. These molecules can serve as biomarkers of developmental toxicity,leading to better prediction of teratogenicity. In particular,our work shows a correlation between teratogenicity and changes of greater than 10% in the ratio of arginine to asymmetric dimethylarginine levels. In addition,this study resulted in the establishment of a predictive model based on the most informative mass features. This model was subsequently tested for its predictive accuracy in two blinded studies using eight drugs of known teratogenicity,where it correctly predicted the teratogenicity for seven of the eight drugs. Thus,our initial data shows that this platform is a robust alternative to animal and other in vitro models for the prediction of the developmental toxicity of chemicals that may also provide invaluable information about the underlying biochemical pathways. ?? 2010 Elsevier Inc. View Publication

Mesenchymal stromal cells (MSCs) may be derived from a variety of tissues,with human umbilical cord (UC) providing an abundant and noninvasive source. Human UC-MSCs share similar in vitro immunosuppressive properties as MSCs obtained from bone marrow and cord blood. However,the mechanisms and cellular interactions used by MSCs to control immune responses remain to be fully elucidated. In this paper,we report that suppression of mitogen-induced T cell proliferation by human UC-,bone marrow-,and cord blood-MSCs required monocytes. Removal of monocytes but not B cells from human adult PBMCs (PBMNCs) reduced the immunosuppressive effects of MSCs on T cell proliferation. There was rapid modulation of a number of cell surface molecules on monocytes when PBMCs or alloantigen-activated PBMNCs were cultured with UC-MSCs. Indomethacin treatment significantly inhibited the ability of UC-MSCs to suppress T cell proliferation,indicating an important role for PGE(2). Monocytes purified from UC-MSC coculture had significantly reduced accessory cell and allostimulatory function when tested in subsequent T cell proliferation assays,an effect mediated in part by UC-MSC PGE(2) production and enhanced by PBMNC alloactivation. Therefore,we identify monocytes as an essential intermediary through which UC-MSCs mediate their suppressive effects on T cell proliferation. View Publication

Chemokine stromal derived factor 1 (SDF-1) is involved in trafficking of hematopoietic stem cells (HSCs) from the bone marrow (BM) to peripheral blood (PB) and has been found to enhance postischemia angiogenesis. This study was aimed at investigating whether SDF-1 plays a role in differentiation of BM-derived c-kit(+) stem cells into endothelial progenitor cells (EPCs) and in ischemia-induced trafficking of stem cells from PB to ischemic tissues. We found that SDF-1 enhanced EPC number by promoting alpha(2),alpha(4),and alpha(5) integrin-mediated adhesion to fibronectin and collagen I. EPC differentiation was reduced in mitogen-stimulated c-kit(+) cells,while cytokine withdrawal or the overexpression of the cyclin-dependent kinase (CDK) inhibitor p16(INK4) restored such differentiation,suggesting a link between control of cell cycle and EPC differentiation. We also analyzed the time course of SDF-1 expression in a mouse model of hind-limb ischemia. Shortly after femoral artery dissection,plasma SDF-1 levels were up-regulated,while SDF-1 expression in the bone marrow was down-regulated in a timely fashion with the increase in the percentage of PB progenitor cells. An increase in ischemic tissue expression of SDF-1 at RNA and protein level was also observed. Finally,using an in vivo assay such as injection of matrigel plugs,we found that SDF-1 improves formation of tubulelike structures by coinjected c-kit(+) cells. Our findings unravel a function for SDF-1 in increase of EPC number and formation of vascular structures by bone marrow progenitor cells. View Publication

Human mesenchymal stem cells (hMSCs) are capable of differentiating into several cell types including adipocytes,osteoblasts,and chondrocytes,under appropriate culture conditions. We found that SP600125,an inhibitor of Jun N-terminal kinase (JNK),promoted adipogenesis whereas it repressed osteogenesis from hMSCs. SP600125 increased the expression of adipogenic transcription factors,CCAAT/enhancer-binding proteins alpha and beta as well as peroxisome proliferator-activated receptor gamma2,which suggested that the chemical acted on the early steps of transcriptional regulatory cascade in adipogenesis. A gene reporter assay showed that SP600125 and a dominant negative JNK promoted a transcriptional activity dependent on the cAMP-response element (CRE). Thus,JNK represses adipogenesis from hMSCs probably by,at least in part,inhibiting the transactivating function of CRE-binding protein. Another action of JNK,phosphorylation at Ser(307) of insulin receptor substrate-1,was also predicted to contribute to the repression of adipogenesis. View Publication

Valproic acid (VPA) has been used as an anticonvulsant agent for the treatment of epilepsy,as well as a mood stabilizer for the treatment of bipolar disorder,for several decades. The mechanism of action for these effects remains to be elucidated and is most likely multifactorial. Recently,VPA has been reported to inhibit histone deacetylase (HDAC) and HDAC has been reported to play roles in differentiation of mammalian cells. In this study,the effects of HDAC inhibitors on differentiation and proliferation of human adipose tissue-derived stromal cells (hADSC) and bone marrow stromal cells (hBMSC) were determined. VPA increased osteogenic differentiation in a dose dependent manner. The pretreatment of VPA before induction of differentiation also showed stimulatory effects on osteogenic differentiation of hMSC. Trichostatin A (TSA),another HDAC inhibitor,also increased osteogenic differentiation,whereas valpromide (VPM),a structural analog of VPA which does not possess HDAC inhibitory effects,did not show any effect on osteogenic differentiation on hADSC. RT-PCR and Real-time PCR analysis revealed that VPA treatment increased osterix,osteopontin,BMP-2,and Runx2 expression. The addition of noggin inhibited VPA-induced potentiation of osteogenic differentiation. VPA inhibited proliferation of hADSC and hBMSC. Our results suggest that VPA enhance osteogenic differentiation,probably due to inhibition of HDAC,and could be useful for in vivo bone engineering using hMSC. View Publication

Maintenance of pluripotency is crucial to the mammalian embryo's ability to generate the extra-embryonic and embryonic tissues that are needed for intrauterine survival and foetal development. The recent establishment of embryonic stem cells from human blastocysts (hESCs) provides an opportunity to identify the factors supporting pluripotency at early stages of human development. Using this in vitro model,we have recently shown that Nodal can block neuronal differentiation,suggesting that TGFbeta family members are involved in cell fate decisions of hESCs,including preservation of their pluripotency. Here,we report that Activin/Nodal signalling through Smad2/3 activation is necessary to maintain the pluripotent status of hESCs. Inhibition of Activin/Nodal signalling by follistatin and by overexpression of Lefty or Cerberus-Short,or by the Activin receptor inhibitor SB431542,precipitates hESC differentiation. Nevertheless,neither Nodal nor Activin is sufficient to sustain long-term hESC growth in a chemically defined medium without serum. Recent studies have shown that FGF2 can also maintain long-term expression of pluripotency markers,and we find that inhibition of the FGF signalling pathway by the tyrosine kinase inhibitor SU5402 causes hESC differentiation. However,this effect of FGF on hESC pluripotency depends on Activin/Nodal signalling,because it is blocked by SB431542. Finally,long-term maintenance of in-vitro pluripotency can be achieved with a combination of Activin or Nodal plus FGF2 in the absence of feeder-cell layers,conditioned medium or Serum Replacer. These findings suggest that the Activin/Nodal pathway maintains pluripotency through mechanism(s) in which FGF acts as a competence factor and therefore provide further evidence of distinct mechanisms for preservation of pluripotency in mouse and human ESCs. View Publication

The prospect of manipulating endogenous neural stem cells to replace damaged tissue and correct functional deficits offers a novel mechanism for treating a variety of CNS disorders. The aim of this study was to investigate pathways controlling neurite outgrowth in human neural precursor cells,in particular in response to platelet-derived growth factor (PDGF). PDGF-AA,-AB and -BB were found to initiate calcium signalling and produce robust increases in neurite outgrowth. PDGF-induced outgrowth of Tuj1-positive precursors was abolished by the addition of EGTA,suggesting that calcium entry is a critical part of the signalling pathway. Wortmannin and PD098059 failed to inhibit PDGF-induced outgrowth. Clostridium Toxin B increased the amount of PDGF-induced neurite branching but had no effect on basal levels. In contrast,WHI-P154,an inhibitor of Janus protein tyrosine kinase (JAK3),Hck and Syk,prevented PDGF-induced neurite outgrowth. PDGF activates multiple signalling pathways with considerable potential for cross-talk. This study has highlighted the complexity of the pathways leading to neurite outgrowth in human neural precursors,and provided initial evidence to suggest that calcium entry is critical in producing the morphological changes observed. View Publication

Tunneling nanotubes (TnTs) are long,non-adherent,actin-based cellular extensions that act as conduits for transport of cellular cargo between connected cells. The mechanisms of nanotube formation and the effects of the tumor microenvironment and cellular signals on TnT formation are unknown. In the present study,we explored exosomes as potential mediators of TnT formation in mesothelioma and the potential relationship of lipid rafts to TnT formation. Mesothelioma cells co-cultured with exogenous mesothelioma-derived exosomes formed more TnTs than cells cultured without exosomes within 24-48. h; and this effect was most prominent in media conditions (low-serum,hyperglycemic medium) that support TnT formation (1.3-1.9-fold difference). Fluorescence and electron microscopy confirmed the purity of isolated exosomes and revealed that they localized predominantly at the base of and within TnTs,in addition to the extracellular environment. Time-lapse microscopic imaging demonstrated uptake of tumor exosomes by TnTs,which facilitated intercellular transfer of these exosomes between connected cells. Mesothelioma cells connected via TnTs were also significantly enriched for lipid rafts at nearly a 2-fold higher number compared with cells not connected by TnTs. Our findings provide supportive evidence of exosomes as potential chemotactic stimuli for TnT formation,and also lipid raft formation as a potential biomarker for TnT-forming cells. textcopyright 2014 Elsevier Inc. View Publication

The generation of functional retinal pigment epithelium (RPE) is of great therapeutic interest to the field of regenerative medicine and may provide possible cures for retinal degenerative diseases,including age-related macular degeneration (AMD). Although RPE cells can be produced from either embryonic stem cells or induced pluripotent stem cells,direct cell reprogramming driven by lineagedetermining transcription factors provides an immediate route to their generation. By monitoring a human RPE specific Best1::GFP reporter,we report the conversion of human fibroblasts into RPE lineage using defined sets of transcription factors. We found that Best1::GFP positive cells formed colonies and exhibited morphological and molecular features of early stage RPE cells. Moreover,they were able to obtain pigmentation upon activation of Retinoic acid (RA) and Sonic Hedgehog (SHH) signaling pathways. Our study not only established an ideal platform to investigate the transcriptional network regulating the RPE cell fate determination,but also provided an alternative strategy to generate functional RPE cells that complement the use of pluripotent stem cells for disease modeling,drug screening,and cell therapy of retinal degeneration. View Publication

Human induced pluripotent stem cells (iPSC) can be used to understand the pathological mechanisms of human disease. These cells are a promising source for cell-replacement therapy. However,such studies require genetically defined conditions. Such genetic manipulations can be performed using the novel Transcription Activator-Like Effector Nucleases (TALENs),which generate site-specific double-strand DNA breaks (DSBs) with high efficiency and precision. Combining the TALEN and iPSC methods,we developed two iPS cell lines by generating the point mutation A5768G in the SCN1A gene,which encodes the voltage-gated sodium channel Nav1.1 α subunit. The engineered iPSC maintained pluripotency and successfully differentiated into neurons with normal functional characteristics. The two cell lines differ exclusively at the epilepsy-susceptibility variant. The ability to robustly introduce disease-causing point mutations in normal hiPS cell lines can be used to generate a human cell model for studying epileptic mechanisms and for drug screening. View Publication

Down's syndrome (DS),caused by trisomy of human chromosome 21,is the most common genetic cause of intellectual disability. Here we use induced pluripotent stem cells (iPSCs) derived from DS patients to identify a role for astrocytes in DS pathogenesis. DS astroglia exhibit higher levels of reactive oxygen species and lower levels of synaptogenic molecules. Astrocyte-conditioned medium collected from DS astroglia causes toxicity to neurons,and fails to promote neuronal ion channel maturation and synapse formation. Transplantation studies show that DS astroglia do not promote neurogenesis of endogenous neural stem cells in vivo. We also observed abnormal gene expression profiles from DS astroglia. Finally,we show that the FDA-approved antibiotic drug,minocycline,partially corrects the pathological phenotypes of DS astroglia by specifically modulating the expression of S100B,GFAP,inducible nitric oxide synthase,and thrombospondins 1 and 2 in DS astroglia. Our studies shed light on the pathogenesis and possible treatment of DS by targeting astrocytes with a clinically available drug. View Publication

INTRODUCTION The control of differentiation of mesenchymal stromal/stem cells (MSCs) is crucial for tissue engineering strategies employing MSCs. The purpose of this study was to investigate whether the transcriptional co-factor Yes-associated protein (YAP) regulates chondrogenic differentiation of MSCs. METHODS Expression of total YAP,its paralogue transcriptional co-activator with PDZ-binding motif (TAZ),and individual YAP transcript variants during in vitro chondrogenesis of human MSCs was determined by quantitative reverse transcription polymerase chain reaction (RT-PCR). YAP expression was confirmed by western blotting. To determine the effect of high YAP activity on chondrogenesis,C3H10T1/2 MSC-like cells were transduced with human (h)YAP and treated in micromass with bone morphogenetic protein-2 (BMP-2). Chondrogenic differentiation was assessed by alcian blue staining and expression of chondrocyte-lineage genes. BMP signalling was determined by detection of pSmad1,5,8 by western blotting and expression of BMP target genes by quantitative RT-PCR. Finally,YAP and pYAP were detected in mouse embryo hindlimbs by immunohistochemistry. RESULTS YAP,but not TAZ,was downregulated during in vitro chondrogenesis of human MSCs. One of the YAP transcript variants,however,was upregulated in high-density micromass culture. Overexpression of hYAP in murine C3H10T1/2 MSCs inhibited chondrogenic differentiation. High YAP activity in these cells decreased Smad1,5,8 phosphorylation and expression of the BMP target genes Inhibitor of DNA binding/differentiation (Id)1,Id2 and Id3 in response to BMP-2. In developing mouse limbs,Yap was nuclear in the perichondrium while mostly phosphorylated and cytosolic in cells of the cartilage anlage,suggesting downregulation of Yap co-transcriptional activity during physiological chondrogenesis in vivo. CONCLUSIONS Our findings indicate that YAP is a negative regulator of chondrogenic differentiation of MSCs. Downregulation of YAP is required for chondrogenesis through derepression of chondrogenic signalling. Therapeutic targeting of YAP to promote cartilage repair and prevent secondary osteoarthritis is an exciting prospect in rheumatology. View Publication