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

Human embryonic stem cells (hESCs) are a promising cell source for tissue engineering and regenerative medicine,but before they can be used in therapies,we must be able to accurately identify the state and progeny of hESCs. One of the most commonly used methods for identification is flow cytometry. Many flow cytometry applications use antibodies to detect the amount of antigen present on/in a cell. This allows for the identification of unique cell populations or the tracking of expression changes within a population during differentiation. The results are typically presented as a percentage of positively expressing cells (%Pos) for a marker of choice,relative to a negative control. However,this reporting term is vulnerable to distortion from outliers and inaccuracy from loss of information about the population's fluorescence intensity. In this article,we describe an alternate strategy that uses the normalized median fluorescence intensity (nMFI),in which the MFI of the stained sample is normalized to the MFI of the negative control,as the reporting term to more accurately describe a population of cells in culture. We observed that nMFI provides a more accurate representation for the quality of a starting population and comparing data of different experimental runs. In addition,we demonstrated that the nMFI is a more sensitive measure of pluripotent and differentiation markers expression changes during hESC differentiation into three germ layer lineages. View Publication

Effector cells derived from central memory CD8(+) T cells were reported to engraft and survive better than those derived from effector memory populations,suggesting that they are superior for use in adoptive immunotherapy studies. However,previous studies did not evaluate the relative efficacy of effector cells derived from naïve T cells. We sought to investigate the efficacy of tumor-specific effector cells derived from naïve or central memory T-cell subsets using transgenic or retrovirally transduced T cells engineered to express a tumor-specific T-cell receptor. We found that naïve,rather than central memory T cells,gave rise to an effector population that mediated superior antitumor immunity upon adoptive transfer. Effector cells developed from naïve T cells lost the expression of CD62L more rapidly than those derived from central memory T cells,but did not acquire the expression of KLRG-1,a marker for terminal differentiation and replicative senescence. Consistent with this KLRG-1(-) phenotype,naïve-derived cells were capable of a greater proliferative burst and had enhanced cytokine production after adoptive transfer. These results indicate that insertion of genes that confer antitumor specificity into naïve rather than central memory CD8(+) T cells may allow superior efficacy upon adoptive transfer. View Publication

BACKGROUND Heterogeneity of endothelial cells (ECs) is a hallmark of the vascular system which may impact the development and management of vascular disorders. Despite the tremendous progress in differentiation of human embryonic stem cells (hESCs) towards endothelial lineage,differentiation into arterial and venous endothelial phenotypes remains elusive. Additionally,current differentiation strategies are hampered by inefficiency,lack of reproducibility,and use of animal-derived products. METHODS To direct the differentiation of hESCs to endothelial subtypes,H1- and H9-hESCs were seeded on human plasma fibronectin and differentiated under chemically defined conditions by sequential modulation of glycogen synthase kinase-3 (GSK-3),basic fibroblast growth factor (bFGF),bone morphogenetic protein 4 (BMP4) and vascular endothelial growth factor (VEGF) signaling pathways for 5 days. Following the initial differentiation,the endothelial progenitor cells (CD34(+)CD31(+) cells) were sorted and terminally differentiated under serum-free conditions to arterial and venous ECs. The transcriptome and secretome profiles of the two distinct populations of hESC-derived arterial and venous ECs were characterized. Furthermore,the safety and functionality of these cells upon in vivo transplantation were characterized. RESULTS Sequential modulation of hESCs with GSK-3 inhibitor,bFGF,BMP4 and VEGF resulted in stages reminiscent of primitive streak,early mesoderm/lateral plate mesoderm,and endothelial progenitors under feeder- and serum-free conditions. Furthermore,these endothelial progenitors demonstrated differentiation potential to almost pure populations of arterial and venous endothelial phenotypes under serum-free conditions. Specifically,the endothelial progenitors differentiated to venous ECs in the absence of VEGF,and to arterial phenotype under low concentrations of VEGF. Additionally,these hESC-derived arterial and venous ECs showed distinct molecular and functional profiles in vitro. Furthermore,these hESC-derived arterial and venous ECs were nontumorigenic and were functional in terms of forming perfused microvascular channels upon subcutaneous implantation in the mouse. CONCLUSIONS We report a simple,rapid,and efficient protocol for directed differentiation of hESCs into endothelial progenitor cells capable of differentiation to arterial and venous ECs under feeder-free and serum-free conditions. This could offer a human platform to study arterial-venous specification for various applications related to drug discovery,disease modeling and regenerative medicine in the future. View Publication

The neurosphere assay (NSA) is one of the most frequently used methods to isolate,expand and also calculate the frequency of neural stem cells (NSCs). Furthermore,this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied,overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay,the neural-colony forming cell assay (N-CFCA),which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential,and thus provides a method to enumerate NSC frequency. In the N-CFCA,colonies ≥2 mm in diameter are derived from cells that meet all the functional criteria of a NSC,while colonies textless 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are textless 2mm and the ones that are ≥2mm in reference to the number of cells that were initially plated. View Publication

Malignant gliomas are the most common and deadly primary malignant brain tumors. In vivo orthotopic models could doubtless represent an appropriate tool to test novel treatment for gliomas. However,methods commonly used to monitor the growth of glioma inside the mouse brain are time consuming and invasive. We tested the reliability of a minimally invasive procedure,based on a secreted luciferase (Gaussia luciferase),to frequently monitor the changes of glioma size. Gluc activity was evaluated from blood samples collected from the tail tip of mice twice a week,allowing to make a growth curve for the tumors. We validated the correlation between Gluc activity and tumor size by analysing the tumor after brain dissection. We found that this method is reliable for monitoring human glioma transplanted in immunodeficient mice,but it has strong limitation in immunocompetent models,where an immune response against the luciferase is developed during the first weeks after transplant. View Publication

Multigene delivery and subsequent cellular expression is emerging as a key technology required in diverse research fields including,synthetic and structural biology,cellular reprogramming and functional pharmaceutical screening. Current viral delivery systems such as retro- and adenoviruses suffer from limited DNA cargo capacity,thus impeding unrestricted multigene expression. We developed MultiPrime,a modular,non-cytotoxic,non-integrating,baculovirus-based vector system expediting highly efficient transient multigene expression from a variety of promoters. MultiPrime viruses efficiently transduce a wide range of cell types,including non-dividing primary neurons and induced-pluripotent stem cells (iPS). We show that MultiPrime can be used for reprogramming,and for genome editing and engineering by CRISPR/Cas9. Moreover,we implemented dual-host-specific cassettes enabling multiprotein expression in insect and mammalian cells using a single reagent. Our experiments establish MultiPrime as a powerful and highly efficient tool,to deliver multiple genes for a wide range of applications in primary and established mammalian cells. View Publication

Germ line mutations in the Adenomatous polyposis coli tumor suppressor gene cause a hereditary form of intestinal tumorigenesis in both mice and man. Here we show that in Apc(Min/+) mice,which carry a heterozygous germ line mutation at codon 850 of Apc,there is progressive loss of immature and mature thymocytes from approximately 80 days of age with complete regression of the thymus by 120 days. In addition,Apc(Min/+) mice show parallel depletion of splenic natural killer (NK) cells,immature B cells,and B progenitor cells in bone marrow due to complete loss of interleukin 7 (IL-7)-dependent B-cell progenitors. Using bone marrow transplantation experiments into wild-type recipients,we have shown that the capacity of transplanted Apc(Min/+) bone marrow cells for T- and B-cell development appears normal. In contrast,although the Apc(Min/+) bone marrow microenvironment supported short-term reconstitution with wild-type bone marrow,Apc(Min/+) animals that received transplants subsequently underwent lymphodepletion. Fibroblast colony-forming unit (CFU-F) colony assays revealed a significant reduction in colony-forming mesenchymal progenitor cells in the bone marrow of Apc(Min/+) mice compared with wild-type animals prior to the onset of lymphodepletion. This suggests that an altered bone marrow microenvironment may account for the selective lymphocyte depletion observed in this model of familial adenomatous polyposis. View Publication

Fate allocation in the gastrulating embryo is spatially organized as cells differentiate into specialized cell types depending on their positions with respect to the body axes. There is a need for in vitro protocols that allow the study of spatial organization associated with this developmental transition. Although embryoid bodies and organoids can exhibit some spatial organization of differentiated cells,methods that generate embryoid bodies or organoids do not yield consistent and fully reproducible results. Here,we describe a micropatterning approach in which human embryonic stem cells are confined to disk-shaped,submillimeter colonies. After 42 h of BMP4 stimulation,cells form self-organized differentiation patterns in concentric radial domains,which express specific markers associated with the embryonic germ layers,reminiscent of gastrulating embryos. Our protocol takes 3 d; it uses commercial microfabricated slides (from CYTOO),human laminin-521 (LN-521) as extracellular matrix coating,and either conditioned or chemically defined medium (mTeSR). Differentiation patterns within individual colonies can be determined by immunofluorescence and analyzed with cellular resolution. Both the size of the micropattern and the type of medium affect the patterning outcome. The protocol is appropriate for personnel with basic stem cell culture training. This protocol describes a robust platform for quantitative analysis of the mechanisms associated with pattern formation at the onset of gastrulation. View Publication