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

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

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

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

PURPOSE: To investigate the distribution of CD44(+)/CD24(-) cells in breast cancers in relation to tumor size before and after the administration of neoadjuvant chemotherapy. METHODS: CD44(+)/CD24(-) tumor cells obtained from breast cancer specimens were characterized in vivo and in vitro using tumor formation assays and mammosphere generation assays,respectively. The distribution of CD44+/CD24- tumor cells in 78 breast cancer specimens following administration of neoadjuvant chemotherapy was also evaluated using immunofluorescence assays,and this distribution was compared with the extent of tumor invasion predicted by Response Evaluation Criteria in Solid Tumours (RECIST). RESULTS: In 27/78 cases,complete remission (CR) was identified using RECIST. However,18 of these CR cases were associated with a scattered distribution of tumor stem cells in the outline of the original tumor prior to neoadjuvant chemotherapy. After neoadjuvant chemotherapy,24 cases involved cancer cells that were confined to the tumor outline,and 21 cases had tumor cells or tumor stem cells overlapping the tumor outline. In addition,there were 6 patients who were insensitive to chemotherapy,and in these cases,both cancer cells and stem cells were detected outside the contours of the tumor volume imaged prior to chemotherapy. CONCLUSION: CD44+/CD24- tumor cells may be an additional parameter to evaluate when determining the extent of breast cancer invasion. View Publication

Induced pluripotent stem (iPS) cells are generated from somatic cells by the forced expression of a defined set of pluripotency-associated transcription factors. Human iPS cells can be propagated indefinitely,while maintaining the capacity to differentiate into all cell types in the body except for extra-embryonic tissues. This technology not only represents a new way to use individual-specific stem cells for regenerative medicine but also constitutes a novel method to obtain large amounts of disease-specific cells for biomedical research. Despite their great potential,the long reprogramming process (up to 1. month) remains one of the most significant challenges facing standard virus-mediated methodology. In this study,we report the accelerated generation of human iPS cells from adipose-derived stem (ADS) cells,using a new combination of chemical inhibitors under a setting of physiological hypoxia in conjunction with retroviral transduction of Oct4,Sox2,Klf4,and L-Myc. Under optimized conditions,we observed human embryonic stem (ES)-like cells as early as 6. days after the initial retroviral transduction. This was followed by the emergence of fully reprogrammed cells bearing Tra-1-81-positive and DsRed transgene-silencing properties on day 10. The resulting cell lines resembled human ES cells in many respects including proliferation rate,morphology,pluripotency-associated markers,global gene expression patterns,genome-wide DNA methylation states,and the ability to differentiate into all three of the germ layers,both in vitro and in vivo. Our method,when combined with chemical inhibitors under conditions of physiological hypoxia,offers a powerful tool for rapidly generating bona fide human iPS cells and facilitates the application of iPS cell technology to biomedical research. textcopyright 2011 Elsevier Inc. View Publication

Neuron-specific in vitro screening strategies have the potential to accelerate the evaluation of chemicals for neurotoxicity. We examined neurite outgrowth as a measure of neuronal response with a commercially available rat cortex progenitor cell model,where cells were exposed to a chemical during a period of cell differentiation. In control cultures,the fraction of beta-III-tubulin positive neurons and their neurite length increased significantly with time,indicating differentiation of the progenitor cells. Expression of glial fibrillary acidic protein,an astrocyte marker,also increased significantly with time. By seeding progenitor cells at varying densities,we demonstrated that neurite length was influenced by cell-cell spacing. After ten days,cultures seeded at densities of 1000 cells/mm(2) or lower had significantly shorter neurites than cultures seeded at densities of 1250 cells/mm(2) or higher. Progenitor cells were exposed to lithium,a neuroactive chemical with diverse modes of action. Cultures exposed to 30 mmol/L or 10 mmol/L lithium chloride (LiCl) had significantly lower metabolic activity than control cultures,as reported by adenosine triphosphate content,and no neurons were observed after ten days of exposure. Cultures exposed to 3 mmol/L,1 mmol/L,or 0.3 mmol/L LiCl,which encompass lithium's therapeutic range,had metabolic activity similar to control cultures. These cultures exhibited concentration-dependent decreases in neurite outgrowth after ten days of LiCl exposure. Neurite outgrowth results were relatively robust,regardless of the evaluation methodology. This work demonstrates that measurement of neurite outgrowth in differentiating progenitor cell cultures can be a sensitive endpoint for neuronal response under non-cytotoxic exposure conditions. View Publication

Efficient derivation of neural cells from human embryonic stem cells (hESCs) remains an unmet need for the treatment of neurological disorders. The limiting factors for current methods include being labor-intensive,time-consuming and expensive. In this study,we hypothesize that the substrate topography,with optimal geometry and dimension,can modulate the neural fate of hESCs and enhance the efficiency of differentiation. A multi-architectural chip (MARC) containing fields of topographies varying in geometry and dimension was developed to facilitate high-throughput analysis of topography-induced neural differentiation in vitro. The hESCs were subjected to direct differentiation"� View Publication