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

Neural progenitor cells (NPCs) derived from human embryonic stem cells (hESCs) have great potential in cell therapy,drug screening and toxicity testing of neural degenerative diseases. However,the molecular regulation of their proliferation and apoptosis,which needs to be revealed before clinical application,is largely unknown. MicroRNA miR-195 is known to be expressed in the brain and is involved in a variety of proapoptosis or antiapoptosis processes in cancer cells. Here,we defined the proapoptotic role of miR-195 in NPCs derived from two independent hESC lines (human embryonic stem cell-derived neural progenitor cells,hESC-NPCs). Overexpression of miR-195 in hESC-NPCs induced extensive apoptotic cell death. Consistently,global transcriptional microarray analyses indicated that miR-195 primarily regulated genes associated with apoptosis in hESC-NPCs. Mechanistically,a small GTP-binding protein ADP-ribosylation factor-like protein 2 (ARL2) was identified as a direct target of miR-195. Silencing ARL2 in hESC-NPCs provoked an apoptotic phenotype resembling that of miR-195 overexpression,revealing for the first time an essential role of ARL2 for the survival of human NPCs. Moreover,forced expression of ALR2 could abolish the cell number reduction caused by miR-195 overexpression. Interestingly,we found that paraquat,a neurotoxin,not only induced apoptosis but also increased miR-195 and reduced ARL2 expression in hESC-NPCs,indicating the possible involvement of miR-195 and ARL2 in neurotoxin-induced NPC apoptosis. Notably,inhibition of miR-195 family members could block neurotoxin-induced NPC apoptosis. Collectively,miR-195 regulates cell apoptosis in a context-dependent manner through directly targeting ARL2. The finding of the critical role of ARL2 for the survival of human NPCs and association of miR-195 and ARL2 with neurotoxin-induced apoptosis have important implications for understanding molecular mechanisms that control NPC survival and would facilitate our manipulation of the neurological pathogenesis. View Publication

Human macrophages are specialised hosts for HIV-1,dengue virus,Leishmania and Mycobacterium tuberculosis. Yet macrophage research is hampered by lack of appropriate cell models for modelling infection by these human pathogens,because available myeloid cell lines are,by definition,not terminally differentiated like tissue macrophages. We describe here a method for deriving monocytes and macrophages from human Pluripotent Stem Cells which improves on previously published protocols in that it uses entirely defined,feeder- and serum-free culture conditions and produces very consistent,pure,high yields across both human Embryonic Stem Cell (hESC) and multiple human induced Pluripotent Stem Cell (hiPSC) lines over time periods of up to one year. Cumulatively,up to ∼3×10(7) monocytes can be harvested per 6-well plate. The monocytes produced are most closely similar to the major blood monocyte (CD14(+),CD16(low),CD163(+)). Differentiation with M-CSF produces macrophages that are highly phagocytic,HIV-1-infectable,and upon activation produce a pro-inflammatory cytokine profile similar to blood monocyte-derived macrophages. Macrophages are notoriously hard to genetically manipulate,as they recognise foreign nucleic acids; the lentivector system described here overcomes this,as pluripotent stem cells can be relatively simply genetically manipulated for efficient transgene expression in the differentiated cells,surmounting issues of transgene silencing. Overall,the method we describe here is an efficient,effective,scalable system for the reproducible production and genetic modification of human macrophages,facilitating the interrogation of human macrophage biology. View Publication

How tumor suppressor p53 selectively responds to specific signals,especially in normal cells,is poorly understood. We performed genome-wide profiling of p53 chromatin interactions and target gene expression in human embryonic stem cells (hESCs) in response to early differentiation,induced by retinoic acid,versus DNA damage,caused by adriamycin. Most p53-binding sites are unique to each state and define stimulus-specific p53 responses in hESCs. Differentiation-activated p53 targets include many developmental transcription factors and,in pluripotent hESCs,are bound by OCT4 and NANOG at chromatin enriched in both H3K27me3 and H3K4me3. Activation of these genes occurs with recruitment of p53 and H3K27me3-specific demethylases,UTX and JMJD3,to chromatin. In contrast,genes associated with cell migration and motility are bound by p53 specifically after DNA damage. Surveillance functions of p53 in cell death and cell cycle regulation are conserved in both DNA damage and differentiation. Comparative genomic analysis of p53-targets in mouse and human ESCs supports an inter-species divergence in p53 regulatory functions during evolution. Our findings expand the registry of p53-regulated genes to define p53-regulated opposition to pluripotency during early differentiation,a process highly distinct from stress-induced p53 response in hESCs. View Publication

An immunohistological study in the human breast and the rodent breast (from inbred Ludwig/Wistar/Olac rats) was conducted with the use of a murine monoclonal antibody,which reacts with the common acute lymphoblastic antigen,a glycosylated polypeptide of a molecular weight of 100,000. The epitope,as recognized by this antibody,is expressed on myoepithelial cells of the normal human and rat breasts and was studied in the developing rodent mammary gland. Ultrastructural studies in the normal human breast clearly demonstrated the presence of the antigen on the lateral membrane of the myoepithelial cells with no staining of luminal cells,blood vessels,or stromal elements. The antigen survived prolonged enzymatic digestion of human breast tissue and could be demonstrated on myoepithelial cells in single-cell suspensions of human breast where it stained approximately 3-14% of the total cell population. The presence of this antigen on myoepithelial cells is discussed in the context of myoepithelial differentiation in the breast and the potential utility of the antibodies for cell separation. View Publication

Human embryonic stem cells (hESCs) have the ability to form cells derived from all three germ layers,and as such have received significant attention as a possible source for insulin-secreting pancreatic beta-cells for diabetes treatment. While considerable advances have been made in generating hESC-derived insulin-producing cells,to date in vitro-derived glucose-responsive beta-cells have remained an elusive goal. With the objective of increasing the in vitro formation of pancreatic endocrine cells,we examined the effect of varying initial cell seeding density from 1.3 x 104 cells/cm2 to 5.3 x 104 cells/cm2 followed by a 21-day pancreatic endocrine differentiation protocol. Low density-seeded cells were found to be biased toward the G2/M phases of the cell cycle and failed to efficiently differentiate into SOX17-CXCR4 co-positive definitive endoderm cells leaving increased numbers of OCT4 positive cells in day 4 cultures. Moderate density cultures effectively formed definitive endoderm and progressed to express PDX1 in approximately 20% of the culture. High density cultures contained approximately double the numbers of PDX1 positive pancreatic progenitor cells and also showed increased expression of MNX1,PTF1a,NGN3,ARX,and PAX4 compared to cultures seeded at moderate density. The cultures seeded at high density displayed increased formation of polyhormonal pancreatic endocrine cell populations co-expressing insulin,glucagon and somatostatin. The maturation process giving rise to these endocrine cell populations followed the expected cascade of pancreatic progenitor marker (PDX1 and MNX1) expression,followed by pancreatic endocrine specification marker expression (BRN4,PAX4,ARX,NEUROD1,NKX6.1 and NKX2.2) and then pancreatic hormone expression (insulin,glucagon and somatostatin). Taken together these data suggest that initial cell seeding density plays an important role in both germ layer specification and pancreatic progenitor commitment,which precedes pancreatic endocrine cell formation. This work highlights the need to examine standard culture variables such as seeding density when optimizing hESC differentiation protocols. View Publication

There is a great deal of uncertainty on how low (≤ 0.1 Gy) doses of ionizing radiation (IR) affect human cells,partly due to a lack of suitable experimental model systems for such studies. The uncertainties arising from low-dose IR human data undermine practical societal needs to predict health risks emerging from diagnostic medical tests' radiation,natural background radiation,and environmental radiological accidents. To eliminate a variability associated with remarkable differences in radioresponses of hundreds of differentiated cell types,we established a novel,human embryonic stem cell (hESC)-based model to examine the radiobiological effects in human cells. Our aim is to comprehensively elucidate the gene expression changes in a panel of various hESC lines following low IR doses of 0.01; 0.05; 0.1 Gy; and,as a reference,relatively high dose of 1 Gy of IR. Here,we examined the dynamics of transcriptional changes of well-established IR-responsive set of genes,including CDKN1A,GADD45A,etc. at 2 and 16 h post-IR,representing early" and "late" radioresponses of hESCs. Our findings suggest the temporal- and hESC line-dependence of stress gene radioresponses with no statistically significant evidence for a linear dose-response relationship within the lowest doses of IR exposures." View Publication

Hemophilia A,one of the most common genetic bleeding disorders,is caused by various mutations in the blood coagulation factor VIII (F8) gene. Among the genotypes that result in hemophilia A,two different types of chromosomal inversions that involve a portion of the F8 gene are most frequent,accounting for almost half of all severe hemophilia A cases. In this study,we used a transcription activator-like effector nuclease (TALEN) pair to invert a 140-kbp chromosomal segment that spans the portion of the F8 gene in human induced pluripotent stem cells (iPSCs) to create a hemophilia A model cell line. In addition,we reverted the inverted segment back to its normal orientation in the hemophilia model iPSCs using the same TALEN pair. Importantly,we detected the F8 mRNA in cells derived from the reverted iPSCs lines,but not in those derived from the clones with the inverted segment. Thus,we showed that TALENs can be used both for creating disease models associated with chromosomal rearrangements in iPSCs and for correcting genetic defects caused by chromosomal inversions. This strategy provides an iPSC-based novel therapeutic option for the treatment of hemophilia A and other genetic diseases caused by chromosomal inversions. View Publication

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

Neurodegenerative diseases are characterized by chronic and progressive structural or functional loss of neurons. Limitations related to the animal models of these human diseases have impeded the development of effective drugs. This emphasizes the need to establish disease models using human-derived cells. The discovery of induced pluripotent stem cell (iPSC) technology has provided novel opportunities in disease modeling,drug development,screening,and the potential for patient-matched" cellular therapies in neurodegenerative diseases. In this study View Publication

The hepatitis C virus (HCV) infects ∼200 million people worldwide. The majority of infected individuals develop persistent infection,resulting in chronic inflammation and liver disease,including cirrhosis and hepatocellular carcinoma. The ability of HCV to establish persistent infection is partly due to its ability to evade the immune response through multiple mechanisms,including suppression of NK cells. NK cells control HCV replication during the early phase of infection and regulate the progression to chronic disease. In particular,IFN-$\gamma$ produced by NK cells limits viral replication in hepatocytes and is important for the initiation of adaptive immune responses. However,NK cell function is significantly impaired in chronic HCV patients. The cellular and molecular mechanisms responsible for impaired NK cell function in HCV infection are not well defined. In this study,we analyzed the interaction of human NK cells with CD33(+) PBMCs that were exposed to HCV. We found that NK cells cocultured with HCV-conditioned CD33(+) PBMCs produced lower amounts of IFN-$\gamma$,with no effect on granzyme B production or cell viability. Importantly,this suppression of NK cell-derived IFN-$\gamma$ production was mediated by CD33(+)CD11b(lo)HLA-DR(lo) myeloid-derived suppressor cells (MDSCs) via an arginase-1-dependent inhibition of mammalian target of rapamycin activation. Suppression of IFN-$\gamma$ production was reversed by l-arginine supplementation,consistent with increased MDSC arginase-1 activity. These novel results identify the induction of MDSCs in HCV infection as a potent immune evasion strategy that suppresses antiviral NK cell responses,further indicating that blockade of MDSCs may be a potential therapeutic approach to ameliorate chronic viral infections in the liver. 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

textlessptextgreater Induced pluripotent stem cells (iPSC) are a most promising approach to the development of a hepatocyte transplantable mass sufficient to induce long-term correction of inherited liver metabolic diseases,thus avoiding liver transplantation. Their intrinsic self-renewal ability and potential to differentiate into any of the three germ layers identify iPSC as the most promising cell-based therapeutics,but also as drivers of tumor development. Teratoma development currently represents the gold standard to assess iPSC pluripotency. We analyzed the tumorigenic potential of iPSC generated from human hepatocytes (HEP-iPSC) and compared their immunohistochemical profiles to that of tumors developed from fibroblast and hematopoietic stem cell-derived iPSC. HEP-iPSC generated tumors significantly presented more malignant morphological features than reprogrammed fibroblasts or CD34+ iPSC. Moreover,the protooncogene textlessitalictextgreatermyctextless/italictextgreater showed the strongest expression in HEP-iPSC,compared to only faint expression in the other cell subsets. Random integration of transgenes and the use of potent protooncogenes such as textlessitalictextgreatermyctextless/italictextgreater might be a risk factor for malignant tumor development if hepatocytes are used for reprogramming. Nonviral vector delivery systems or reprogramming of cells obtained from less invasive harvesting methods would represent interesting options for future developments in stem cell-based approaches for liver metabolic diseases. textless/ptextgreater View Publication