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

There is increasing evidence that many cancers,including breast cancer,contain populations of cells that display stem-cell properties. These breast cancer stem cells,by virtue of their relative resistance to radiation and cytotoxic chemotherapy,may contribute to treatment resistance and relapse. The elucidation of pathways that regulate these cells has led to the identification of potential therapeutic targets. A number of agents capable of targeting breast cancer stem cells in preclinical models are currently entering clinical trials. Assessment of the efficacy of the agents will require development of innovative clinical trial designs with appropriate biologic and clinical end points. The effective targeting of breast cancer stem cells has the potential to significantly improve outcome for women with both early-stage and advanced breast cancer. View Publication

Characterization of pluripotent stem cells is required for the registration of stem cell lines and allows for an impartial and objective comparison of the results obtained when generating multiple lines. It is therefore crucial to establish specific,fast and reliable protocols to detect the hallmarks of pluripotency. Such protocols should include immunocytochemistry (takes 2 d),identification of the three germ layers in in vitro-derived embryoid bodies by immunocytochemistry (immunodetection takes 3 d) and detection of differentiation markers in in vivo-generated teratomas by immunohistochemistry (differentiation marker detection takes 4 d). Standardization of the immunodetection protocols used ensures minimum variations owing to the source,the animal species,the endogenous fluorescence or the inability to collect large amounts of cells,thereby yielding results as fast as possible without loss of quality. This protocol provides a description of all the immunodetection procedures necessary to characterize mouse and human stem cell lines in different circumstances. View Publication

The generation of endothelial progenitor cells (EPCs) from blood monocytes has been propagated as a novel approach in the diagnosis and treatment of cardiovascular diseases. Low-density lipoprotein (LDL) uptake and lectin binding together with endothelial marker expression are commonly used to define these EPCs. Considerable controversy exists regarding their nature,in particular,because myelomonocytic cells share several properties with endothelial cells (ECs). This study was performed to elucidate whether the commonly used endothelial marker determination is sufficient to distinguish supposed EPCs from monocytes. We measured endothelial,hematopoietic,and progenitor cell marker expression of monocytes before and after angiogenic culture by fluorescence microscopy,flow cytometry,and real-time reverse transcription-polymerase chain reaction. The function of primary monocytes and monocyte-derived supposed EPCs was investigated during vascular network formation and EC colony-forming unit (CFU-EC) development. Monocytes cultured for 4 to 6 days under angiogenic conditions lost CD14/CD45 and displayed a commonly accepted EPC phenotype,including LDL uptake and lectin binding,CD31/CD105/CD144 reactivity,and formation of cord-like structures. Strikingly,primary monocytes already expressed most tested endothelial genes and proteins at even higher levels than their supposed EPC progeny. Neither fresh nor cultured monocytes formed vascular networks,but CFU-EC formation was strictly dependent on monocyte presence. LDL uptake,lectin binding,and CD31/CD105/CD144 expression are inherent features of monocytes,making them phenotypically indistinguishable from putative EPCs. Consequently,monocytes and their progeny can phenotypically mimic EPCs in various experimental models. View Publication

Human embryonic stem cells (hESCs) offer exciting potential in regenerative medicine for the treatment of a host of diseases including cancer,Alzheimer's and Parkinson's disease. They also provide insight into human development and disease and can be used as models for drug discovery and toxicity analyses. The key properties of hESCs that make them so promising for medical use are that they have the ability to self-renew indefinitely in culture and they are pluripotent,which means that they can differentiate into any of more than 200 human cell types. Since proteins are the effectors of cellular processes,it is important to investigate hESC expression at the protein level as well as at the transcript level. In addition,post-translational modifications,such as phosphorylation,may influence the activity of pivotal proteins in hESCs,and this information can only be determined by studying the proteome. In this review,we summarize the results obtained from several proteomics analyses of hESCs that have been reported in the last few years. View Publication

Cryopreservation is the use of low temperatures to preserve structurally intact living cells. The cells that survive the thermodynamic journey from the 37 °C incubator to the -196 °C liquid nitrogen storage tank are free from the influences of time. Thus,cryopreservation is a critical component of cell culture and cell manufacturing protocols. Successful cryopreservation of human cells requires that the cells be derived from patient samples that are collected in a standardized manner,and carefully handled from blood draw through cell isolation. Furthermore,proper equipment must be in place to ensure consistency,reproducibility,and sterility. In addition,the correct choice and amount of cryoprotectant agent must be added at the correct temperature,and a controlled rate of freezing (most commonly 1 °C/min) must be applied prior to a standardized method of cryogenic storage. This appendix describes how human primary cells can be frozen for long-term storage and thawed for growth in a tissue culture vessel. View Publication

The self-renewing epithelium of the small intestine is ordered into stem/progenitor crypt compartments and differentiated villus compartments. Recent evidence indicates that the Wnt cascade is the dominant force in controlling cell fate along the crypt-villus axis. Here we show a rapid,massive conversion of proliferative crypt cells into post-mitotic goblet cells after conditional removal of the common Notch pathway transcription factor CSL/RBP-J. We obtained a similar phenotype by blocking the Notch cascade with a gamma-secretase inhibitor. The inhibitor also induced goblet cell differentiation in adenomas in mice carrying a mutation of the Apc tumour suppressor gene. Thus,maintenance of undifferentiated,proliferative cells in crypts and adenomas requires the concerted activation of the Notch and Wnt cascades. Our data indicate that gamma-secretase inhibitors,developed for Alzheimer's disease,might be of therapeutic benefit in colorectal neoplastic disease. View Publication

BACKGROUND: Induced pluripotent stem (iPS) cells have the capability to undergo self-renewal and differentiation into all somatic cell types. Since they can be produced through somatic cell reprogramming,which uses a defined set of transcription factors,iPS cells represent important sources of patient-specific cells for clinical applications. However,before these cells can be used in therapeutic designs,it is essential to understand their genetic stability. METHODOLOGY/PRINCIPAL FINDINGS: Here,we describe DNA damage responses in human iPS cells. We observe hypersensitivity to DNA damaging agents resulting in rapid induction of apoptosis after γ-irradiation. Expression of pluripotency factors does not appear to be diminished after irradiation in iPS cells. Following irradiation,iPS cells activate checkpoint signaling,evidenced by phosphorylation of ATM,NBS1,CHEK2,and TP53,localization of ATM to the double strand breaks (DSB),and localization of TP53 to the nucleus of NANOG-positive cells. We demonstrate that iPS cells temporary arrest cell cycle progression in the G(2) phase of the cell cycle,displaying a lack of the G(1)/S cell cycle arrest similar to human embryonic stem (ES) cells. Furthermore,both cell types remove DSB within six hours of γ-irradiation,form RAD51 foci and exhibit sister chromatid exchanges suggesting homologous recombination repair. Finally,we report elevated expression of genes involved in DNA damage signaling,checkpoint function,and repair of various types of DNA lesions in ES and iPS cells relative to their differentiated counterparts. CONCLUSIONS/SIGNIFICANCE: High degrees of similarity in DNA damage responses between ES and iPS cells were found. Even though reprogramming did not alter checkpoint signaling following DNA damage,dramatic changes in cell cycle structure,including a high percentage of cells in the S phase,increased radiosensitivity and loss of DNA damage-induced G(1)/S cell cycle arrest,were observed in stem cells generated by induced pluripotency. View Publication

Pluripotent stem cells exist in naive and primed states,epitomized by mouse embryonic stem cells (ESCs) and the developmentally more advanced epiblast stem cells (EpiSCs; ref. 1). In the naive state of ESCs,the genome has an unusual open conformation and possesses a minimum of repressive epigenetic marks. In contrast,EpiSCs have activated the epigenetic machinery that supports differentiation towards the embryonic cell types. The transition from naive to primed pluripotency therefore represents a pivotal event in cellular differentiation. But the signals that control this fundamental differentiation step remain unclear. We show here that paracrine and autocrine Wnt signals are essential self-renewal factors for ESCs,and are required to inhibit their differentiation into EpiSCs. Moreover,we find that Wnt proteins in combination with the cytokine LIF are sufficient to support ESC self-renewal in the absence of any undefined factors,and support the derivation of new ESC lines,including ones from non-permissive mouse strains. Our results not only demonstrate that Wnt signals regulate the naive-to-primed pluripotency transition,but also identify Wnt as an essential and limiting ESC self-renewal factor. View Publication

Precise,robust and scalable directed differentiation of pluripotent stem cells is an important goal with respect to disease modeling or future therapies. Using the AggreWell™400 system we have standardized the differentiation of human embryonic and induced pluripotent stem cells to a neuronal fate using defined conditions. This allows reproducibility in replicate experiments and facilitates the direct comparison of cell lines. Since the starting point for EB formation is a single cell suspension,this protocol is suitable for standard and novel methods of pluripotent stem cell culture. Moreover,an intermediate population of neural precursor cells,which are routinely textgreater95% NCAM(pos) and Tra-1-60(neg) by FACS analysis,may be expanded and frozen prior to differentiation allowing a convenient starting point for downstream experiments. View Publication

Developing efficacious vaccines against enteric diseases is a global challenge that requires a better understanding of cellular recruitment dynamics at the mucosal surfaces. The current paradigm of T cell homing to the gastrointestinal (GI) tract involves the induction of $$4$$7 and CCR9 by Peyer's patch and mesenteric lymph node (MLN) dendritic cells (DCs) in a retinoic acid-dependent manner. This paradigm,however,cannot be reconciled with reports of GI T cell responses after intranasal (i.n.) delivery of antigens that do not directly target the GI lymphoid tissue. To explore alternative pathways of cellular migration,we have investigated the ability of DCs from mucosal and nonmucosal tissues to recruit lymphocytes to the GI tract. Unexpectedly,we found that lung DCs,like CD103(+) MLN DCs,up-regulate the gut-homing integrin $$4$$7 in vitro and in vivo,and induce T cell migration to the GI tract in vivo. Consistent with a role for this pathway in generating mucosal immune responses,lung DC targeting by i.n. immunization induced protective immunity against enteric challenge with a highly pathogenic strain of Salmonella. The present report demonstrates novel functional evidence of mucosal cross talk mediated by DCs,which has the potential to inform the design of novel vaccines against mucosal pathogens. View Publication

The incidence of cardiovascular disease represents a significant and growing health-care challenge to the developed and developing world. The ability of native heart muscle to regenerate in response to myocardial infarct is minimal. Tissue engineering and regenerative medicine approaches represent one promising response to this difficulty. Here,we present methods for the construction of a cell-seeded cardiac patch with the potential to promote regenerative outcomes in heart muscle with damage secondary to myocardial infarct. This method leverages iPS cells and a fibrin-based scaffold to create a simple and commercially viable tissue-engineered cardiac patch. Human-induced pluripotent stem cells (hiPSCs) can,in principle,be differentiated into cells of any lineage. However,most of the protocols used to generate hiPSC-derived endothelial cells (ECs) and cardiomyocytes (CMs) are unsatisfactory because the yield and phenotypic stability of the hiPSC-ECs are low,and the hiPSC-CMs are often purified via selection for expression of a promoter-reporter construct. In this chapter,we describe an hiPSC-EC differentiation protocol that generates large numbers of stable ECs and an hiPSC-CM differentiation protocol that does not require genetic manipulation,single-cell selection,or sorting with fluorescent dyes or other reagents. We also provide a simple but effective method that can be used to combine hiPSC-ECs and hiPSC-CMs with hiPSC-derived smooth muscle cells to engineer a contracting patch of cardiac cells. View Publication