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

Embryonic stem (ES) cells are permanent pluripotent stem cell lines established from pre-implantation mouse embryos. There is currently great interest in the potential therapeutic applications of analogous cells derived from human embryos. The isolation of ES cells is commonly presented as a straightforward transfer of cells in the early embryo into culture. In reality,however,continuous expansion of pluripotent cells does not occur in vivo,and in vitro is the exception rather than the norm. Both genetic and epigenetic factors influence the ability to derive ES cells. We have tracked the expression of a key marker and determinant of pluripotency,the transcription factor Oct-4,in primary cultures of mouse epiblasts and used this to assay the effect of experimental manipulations on the maintenance of a pluripotent cell compartment. We find that expression of Oct-4 is often lost prior to overt cytodifferentiation of the epiblast. The rate and extent of Oct-4 extinction varies with genetic background. We report that treatment with the MAP kinase/ERK kinase inhibitor PD98059,which suppresses activation of the mitogen-activated protein kinases Erk1 and Erk2,results in increased persistence of Oct-4-expressing cells. Oct-4 expression is also relatively sustained in cultures of diapause embryos and of isolated inner cell masses. Combination of all three conditions allowed the derivation of germline-competent ES cells from the normally refractory CBA mouse strain. These findings suggest that the genesis of an ES cell is a relatively complex process requiring epigenetic modulation of key gene expression over a brief time-window. Procedures that extend this time-window and/or directly regulate the critical genes should increase the efficiency of ES cell derivation. View Publication

The human pre-T-cell receptor alpha (TCRalpha; pTalpha) gene encodes a polypeptide which associates with the TCRbeta chain and CD3 molecules to form the pre-TCR complex. The surface expression of the pre-TCR is pTalpha dependent,and signaling through this complex triggers an early alphabeta T-cell developmental checkpoint inside the thymus,known as beta-selection. E2A transcription factors,which are involved at multiple stages of T-cell development,regulate the transcription of the pTalpha gene. Here we show that the regulatory protein Tax of the human T-cell leukemia virus type 1 (HTLV-1) efficiently suppresses the E47-mediated activation of the pTalpha promoter. Furthermore,we report that in Tax lentivirally transduced human MOLT-4 T cells,which constitutively express the pTalpha gene,the amount of pTalpha transcripts decreases. Such a decrease is not observed in MOLT-4 cells transduced by a vector encoding the Tax mutant K88A,which is unable to interact with p300. These data underline that Tax inhibits pTalpha transcription by recruiting this coactivator. Finally,we show that the expression of Tax in human immature thymocytes results in a decrease of pTalpha gene transcription but does not modify the level of E47 transcripts. These observations indicate that Tax,by silencing E proteins,down-regulates pTalpha gene transcription during early thymocyte development. They further provide evidence that Tax can interfere with an important checkpoint during T-cell differentiation in the thymus. View Publication

Vascular tissue engineering aims at creating self-renewing,anti-thrombogenic,vascular grafts,which can be based on endothelial progenitor cells (EPC). EPC harbor essential features such as plasticity and longevity. Unfortunately,the archetype CD34(+) EPC is rare in peripheral blood. Monocytes,i.e. CD14(+) cells also have the ability to differentiate into endothelial-like cells and are by far more abundant in peripheral blood than are CD34(+) EPC. Therefore,CD14(+) cells would seem appropriate candidates for tissue engineering of small-diameter blood vessels. In this study,we investigated the differentiation of CD14(+) cells on three biodegradable biomaterials under angiogenic conditions. Morphological analyses,gene transcript analyses,endothelial marker (i.e. VE-Cadherin and eNOS) and macrophage marker (i.e. CD68 and CD163) expression analyses,revealed that a small fraction (15-25%) of cultured CD14(+) cells differentiated into macrophages after 21 days of culture. The majority of CD14(+) cells (textgreater75%) differentiated into endothelial-like cells (ELC) on all biomaterials used. The expression of endothelial markers was similar to their expression on HUVEC. Since CD14(+) cells are present in high numbers in adult peripheral blood,easy to isolate and because they easily differentiate into ELC on biomaterials,we conclude that CD14(+) cells are a suitable cell source for progenitor-based vascular tissue engineering. View Publication

There is little information on the function of epididymal basal cells. These cells secrete prostaglandins,can metabolize radical oxygen species,and have apical projections that are components of the blood-epididymis barrier. The objective of this study was to develop a reproducible protocol to isolate rat epididymal basal cells and to characterize their function by gene expression profiling. Integrin-alpha6 was used to isolate a highly purified population of basal cells. Microarray analysis indicated that expression levels of 552 genes were enriched in basal cells relative to other cell types. Among these genes,45 were expressed at levels of 5-fold or greater. These highly expressed genes coded for proteins implicated in cell adhesion,cytoskeletal function,ion transport,cellular signaling,and epidermal function,and included proteases and antiproteases,signal transduction,and transcription factors. Several highly expressed genes have been reported in adult stem cells,suggesting that basal cells may represent an epididymal stem cell population. A basal cell culture was established that showed that these basal cells can differentiate in vitro from keratin (KRT) 5-positive cells to cells that express KRT8 and connexin 26,a marker of columnar cells. These data provide novel information on epididymal basal cell gene expression and suggest that these cells can act as adult stem cells. View Publication

BACKGROUND Adoptive T cell transfer (ACT) therapy improves outcomes in patients with advanced malignancies,yet many individuals relapse due to the infusion of T cells with poor function or persistence. Toll-like receptor (TLR) agonists can invigorate antitumor T cell responses when administered directly to patients,but these responses often coincide with toxicities. We posited that TLR agonists could be repurposed ex vivo to condition T cells with remarkable potency in vivo,circumventing TLR-related toxicity. METHODS In this study we investigated how tumor-specific murine CD8+ T cells and human tumor infiltrating lymphocytes (TILs) are impacted when expanded ex vivo with the TLR9 agonist CpG. RESULTS Herein we reveal a new way to reverse the tolerant state of adoptively transferred CD8+ T cells against tumors using TLR-activated B cells. We repurposed the TLR9 agonist,CpG,commonly used in the clinic,to bolster T cell-B cell interactions during expansion for ACT. T cells expanded ex vivo from a CpG-treated culture demonstrated potent antitumor efficacy and prolonged persistence in vivo. This antitumor efficacy was accomplished without in vivo administration of TLR agonists or other adjuvants of high-dose interleukin (IL)-2 or vaccination,which are classically required for effective ACT therapy. CpG-conditioned CD8+ T cells acquired a unique proteomic signature hallmarked by an IL-2R$\alpha$highICOShighCD39low phenotype and an altered metabolic profile,all reliant on B cells transiently present in the culture. Likewise,human TILs benefitted from expansion with CpG ex vivo,as they also possessed the IL-2R$\alpha$highICOShighCD39low phenotype. CpG fostered the expansion of potent CD8+ T cells with the signature phenotype and antitumor ability via empowering a direct B-T cell interaction. Isolated B cells also imparted T cells with the CpG-associated phenotype and improved tumor immunity without the aid of additional antigen-presenting cells or other immune cells in the culture. CONCLUSIONS Our results demonstrate a novel way to use TLR agonists to improve immunotherapy and reveal a vital role for B cells in the generation of potent CD8+ T cell-based therapies. Our findings have immediate implications in the clinical treatment of advanced solid tumors. View Publication

Neural tissue formation is induced by growth factors that activate networks of signal transduction cascades that ultimately lead to the expression of early neural genes,including transcription factors of the SoxB family. Here,we report that fibroblast growth factor (FGF)-induced Erk1/2 (Mapk3 and Mapk1,respectively) mitogen-activated protein kinase (MAPK),but not phosphatidylinositol 3'-OH kinase (PI3K,Pik3r1),signalling is required for neural specification in mouse embryonic stem (ES) cells and in the chick embryo. Further,blocking Erk1/2 inhibits the onset of key SoxB genes in both mouse ES cells (Sox1) and chick embryos (Sox2 and Sox3) and,in both contexts,Erk1/2 signalling is required during only a narrow time window,as neural specification takes place. In the absence of Erk1/2 signalling,differentiation of ES cells stalls following Fgf5 upregulation. Using differentiating ES cells as a model for neural specification,we demonstrate that sustained Erk1/2 activation controls the transition from an Fgf5-positive,primitive ectoderm-like cell state to a neural progenitor cell state without attenuating bone morphogenetic protein (BMP) signalling and we also define the minimum period of Erk1/2 activity required to mediate this key developmental step. Together,these findings identify a conserved,specific and stage-dependent requirement for Erk1/2 signalling downstream of FGF-induced neural specification in higher vertebrates and provide insight into the signalling dynamics governing this process. View Publication