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

The quantitative analysis of cardiomyocyte function is essential for stem cell-based approaches for the in vitro study of human cardiac physiology and pathophysiology. We present a method to comprehensively assess the function of single human pluripotent stem cell-derived cardiomyocyte (hPSC-CMs) through simultaneous quantitative analysis of contraction kinetics,force generation,and electrical activity. We demonstrate that statistical analysis of movies of contracting hPSC-CMs can be used to quantify changes in cellular morphology over time and compute contractile kinetics. Using a biomechanical model that incorporates substrate stiffness,we calculate cardiomyocyte force generation at single-cell resolution and validate this approach with conventional traction force microscopy. The addition of fluorescent calcium indicators or membrane potential dyes allows the simultaneous analysis of contractility and calcium handling or action potential morphology. Accordingly,our approach has the potential for broad application in the study of cardiac disease,drug discovery,and cardiotoxicity screening. View Publication

The prostate transmembrane protein,androgen-induced 1 (PMEPA1) has been previously shown to promote solid malignancies in a variety of cancers,but the role and mechanisms of PMEPA1 in breast cancer has not been fully addressed. Here,we found that PMEPA1 was upregulated in breast cancer cell lines as well as in a set of clinical invasive breast ductal carcinomas. Interestingly,depletion of PMEPA1 decreased breast cancer stem cell (CSC)-enriched populations,while ectopic overexpression of PMEPA1 increased breast CSC-enriched populations. Furthermore,transforming growth factor-$$ (TGF-$$) treatment was also found to upregulate PMEPA1 expression and the CSC-enriched populations in triple-negative breast cancer cell lines. TGF-$$-induced PMEPA1 expression partially contributed to TGF-$$-induced breast CSC maintenance. These findings suggest that TGF-$$-PMEPA1 axis might provide new diagnosis and therapeutic targets for breast cancer treatment. View Publication

Self-renewal of human embryonic stem cells (ESCs) is promoted by FGF and TGFbeta/Activin signaling,and differentiation is promoted by BMP signaling,but how these signals regulate genes critical to the maintenance of pluripotency has been unclear. Using a defined medium,we show here that both TGFbeta and FGF signals synergize to inhibit BMP signaling; sustain expression of pluripotency-associated genes such as NANOG,OCT4,and SOX2; and promote long-term undifferentiated proliferation of human ESCs. We also show that both TGFbeta- and BMP-responsive SMADs can bind with the NANOG proximal promoter. NANOG promoter activity is enhanced by TGFbeta/Activin and FGF signaling and is decreased by BMP signaling. Mutation of putative SMAD binding elements reduces NANOG promoter activity to basal levels and makes NANOG unresponsive to BMP and TGFbeta signaling. These results suggest that direct binding of TGFbeta/Activin-responsive SMADs to the NANOG promoter plays an essential role in sustaining human ESC self-renewal. View Publication

Imatinib is currently the first line therapy for newly diagnosed patients with chronic myeloid leukemia. However,20-25% of patients do not achieve durable complete cytogenetic responses. The mechanism underlying this primary resistance is unknown,but variations in BCR-ABL1 kinase activity may play a role and can be investigated by measuring the autophosphorylation levels of BCR-ABL1 or of a surrogate target such as Crkl. In this study we used flow cytometry to investigate the in vitro inhibition of Crkl phosphorylation by imatinib in CD34(+) cells in diagnostic samples from two groups of patients distinguished by their cytogenetic response. No difference in inhibition of Crkl phosphorylation was observed in the two groups. The observation that increasing the dose of imatinib in vivo did not increase the level of cytogenetic response in some non-responders suggests that in at least a proportion of patients imatinib resistance may be due to activation of BCR-ABL1-independent pathway. View Publication

Human embryonic stem (hES) cells hold great promise for application of human cell and tissue replacement therapy. However,the overwhelming majority of currently available hES cell lines have been directly or indirectly exposed to materials containing animal-derived components during their derivation,propagation,and cryopreservation. Unlike feeder based cultures,which require the simultaneous growth of feeder and stem cells,resulting in mixed cell populations,stem cells grown on feeder-free systems are easily separated from the surface,presenting a pure population of cells for downstream applications. In this study we have developed a novel method to expand hES cells in xeno-free,feeder-free conditions using two different matrices derived from xeno-free human foreskin fibroblasts (XF-HFFs). Using XF-HFF-derived extracellular matrix,together with 100ng/ml recombinant bFGF supplemented HEScGRO Basal Medium,long term xeno-free expansion of hES cells is possible. Resulting hES cells were subjected to stringent tests and were found to maintain ES cell features,including morphology,pluripotency,stable karyotype,and expression of cell surface markers,for at least 20 passages. Xeno-free culturing practices are essential for the translation of basic hES cell research into the clinic. Therefore,the method presented in this study demonstrates that hES cells can be cultured in complete xeno-free conditions without the loss of pluripotency and furthermore,without the possibility of contamination from exogenous sources. View Publication

During digestive organogenesis,the primitive gut tube (PGT) undergoes dramatic elongation and forms a lumen lined by a single-layer of epithelium. In Xenopus,endoderm cells in the core of the PGT rearrange during gut elongation,but the morphogenetic mechanisms controlling their reorganization are undetermined. Here,we define the dynamic changes in endoderm cell shape,polarity,and tissue architecture that underlie Xenopus gut morphogenesis. Gut endoderm cells intercalate radially,between their anterior and posterior neighbors,transforming the nearly solid endoderm core into a single layer of epithelium while concomitantly eliciting radially convergent" extension within the gut walls. Inhibition of Rho/ROCK/Myosin II activity prevents endoderm rearrangements and consequently perturbs both gut elongation and digestive epithelial morphogenesis. Our results suggest that the cellular and molecular events driving tissue elongation in the PGT are mechanistically analogous to those that function during gastrulation� View Publication

The immunodeficiency disorder,X-linked agammaglobulinemia (XLA),results from mutations in the gene encoding Bruton tyrosine kinase (Btk). Btk is required for pre-B cell clonal expansion and B-cell antigen receptor signaling. XLA patients lack mature B cells and immunoglobulin and experience recurrent bacterial infections only partially mitigated by life-long antibody replacement therapy. In pursuit of definitive therapy for XLA,we tested ex vivo gene therapy using a lentiviral vector (LV) containing the immunoglobulin enhancer (Emu) and Igbeta (B29) minimal promoter to drive B lineage-specific human Btk expression in Btk/Tec(-/-) mice,a strain that reproduces the features of human XLA. After transplantation of EmuB29-Btk-LV-transduced stem cells,treated mice showed significant,albeit incomplete,rescue of mature B cells in the bone marrow,peripheral blood,spleen,and peritoneal cavity,and improved responses to T-independent and T-dependent antigens. LV-treated B cells exhibited enhanced B-cell antigen receptor signaling and an in vivo selective advantage in the peripheral versus central B-cell compartment. Secondary transplantation showed sustained Btk expression,viral integration,and partial functional responses,consistent with long-term stem cell marking; and serial transplantation revealed no evidence for cellular or systemic toxicity. These findings strongly support pursuit of B lineage-targeted LV gene therapy in human XLA. View Publication

Activating mutations in signaling molecules,such as JAK2-V617F,have been associated with myeloproliferative neoplasms (MPNs). Mice lacking the inhibitory adaptor protein Lnk display deregulation of thrombopoietin/thrombopoietin receptor signaling pathways and exhibit similar myeloproliferative characteristics to those found in MPN patients,suggesting a role for Lnk in the molecular pathogenesis of these diseases. Here,we showed that LNK levels are up-regulated and correlate with an increase in the JAK2-V617F mutant allele burden in MPN patients. Using megakaryocytic cells,we demonstrated that Lnk expression is regulated by the TPO-signaling pathway,thus indicating an important negative control loop in these cells. Analysis of platelets derived from MPN patients and megakaryocytic cell lines showed that Lnk can interact with JAK2-WT and V617F through its SH2 domain,but also through an unrevealed JAK2-binding site within its N-terminal region. In addition,the presence of the V617F mutation causes a tighter association with Lnk. Finally,we found that the expression level of the Lnk protein can modulate JAK2-V617F-dependent cell proliferation and that its different domains contribute to the inhibition of multilineage and megakaryocytic progenitor cell growth in vitro. Together,our results indicate that changes in Lnk expression and JAK2-V617F-binding regulate JAK2-mediated signals in MPNs. View Publication

The expression of an enzyme,GnT-V,that catalyzes a specific posttranslational modification of a family of glycoproteins,namely a branched N-glycan,is transcriptionally up-regulated during breast carcinoma oncogenesis. To determine the molecular basis of how early events in breast carcinoma formation are regulated by GnT-V,we studied both the early stages of mammary tumor formation by using 3D cell culture and a her-2 transgenic mouse mammary tumor model. Overexpression of GnT-V in MCF-10A mammary epithelial cells in 3D culture disrupted acinar morphogenesis with impaired hollow lumen formation,an early characteristic of mammary neoplastic transformation. The disrupted acinar morphogenesis of mammary tumor cells in 3D culture caused by her-2 expression was reversed in tumors that lacked GnT-V expression. Moreover,her-2-induced mammary tumor onset was significantly delayed in the GnT-V null tumors,evidence that the lack of the posttranslational modification catalyzed by GnT-V attenuated tumor formation. Inhibited activation of both PKB and ERK signaling pathways was observed in GnT-V null tumor cells. The proportion of tumor-initiating cells (TICs) in the mammary tumors from GnT-V null mice was significantly reduced compared with controls,and GnT-V null TICs displayed a reduced ability to form secondary tumors in NOD/SCID mice. These results demonstrate that GnT-V expression and its branched glycan products effectively modulate her-2-mediated signaling pathways that,in turn,regulate the relative proportion of tumor initiating cells and the latency of her-2-driven tumor onset. View Publication

BACKGROUND: Activity through NMDA type glutamate receptors sculpts connectivity in the developing nervous system. This topic is typically studied in the visual system in vivo,where activity of inputs can be differentially regulated,but in which individual synapses are difficult to visualize and mechanisms governing synaptic competition can be difficult to ascertain. Here,we develop a model of NMDA-receptor dependent synaptic competition in dissociated cultured hippocampal neurons. METHODOLOGY/PRINCIPAL FINDINGS: GluN1 -/- (KO) mouse hippocampal neurons lacking the essential NMDA receptor subunit were cultured alone or cultured in defined ratios with wild type (WT) neurons. The absence of functional NMDA receptors did not alter neuron survival. Synapse development was assessed by immunofluorescence for postsynaptic PSD-95 family scaffold and apposed presynaptic vesicular glutamate transporter VGlut1. Synapse density was specifically enhanced onto minority wild type neurons co-cultured with a majority of GluN1 -/- neighbour neurons,both relative to the GluN1 -/- neighbours and relative to sister pure wild type cultures. This form of synaptic competition was dependent on NMDA receptor activity and not conferred by the mere physical presence of GluN1. In contrast to these results in 10% WT and 90% KO co-cultures,synapse density did not differ by genotype in 50% WT and 50% KO co-cultures or in 90% WT and 10% KO co-cultures. CONCLUSIONS/SIGNIFICANCE: The enhanced synaptic density onto NMDA receptor-competent neurons in minority coculture with GluN1 -/- neurons represents a cell culture paradigm for studying synaptic competition. Mechanisms involved may include a retrograde 'reward' signal generated by WT neurons,although in this paradigm there was no 'punishment' signal against GluN1 -/- neurons. Cell culture assays involving such defined circuits may help uncover the rules and mechanisms of activity-dependent synaptic competition in the developing nervous system. View Publication

The identification of succinate dehydrogenase (SDH),fumarate hydratase (FH) and isocitrate dehydrogenase (IDH) mutations in human cancers has rekindled the idea that altered cellular metabolism can transform cells. Inactivating SDH and FH mutations cause the accumulation of succinate and fumarate,respectively,which can inhibit 2-oxoglutarate (2-OG)-dependent enzymes,including the EGLN prolyl 4-hydroxylases that mark the hypoxia inducible factor (HIF) transcription factor for polyubiquitylation and proteasomal degradation. Inappropriate HIF activation is suspected of contributing to the pathogenesis of SDH-defective and FH-defective tumours but can suppress tumour growth in some other contexts. IDH1 and IDH2,which catalyse the interconversion of isocitrate and 2-OG,are frequently mutated in human brain tumours and leukaemias. The resulting mutants have the neomorphic ability to convert 2-OG to the (R)-enantiomer of 2-hydroxyglutarate ((R)-2HG). Here we show that (R)-2HG,but not (S)-2HG,stimulates EGLN activity,leading to diminished HIF levels,which enhances the proliferation and soft agar growth of human astrocytes. These findings define an enantiomer-specific mechanism by which the (R)-2HG that accumulates in IDH mutant brain tumours promotes transformation and provide a justification for exploring EGLN inhibition as a potential treatment strategy. View Publication

The anti-diabetic drug metformin is rapidly emerging as a potential anti-cancer agent. Metformin,effective in treating type 2 diabetes and the insulin resistance syndromes,improves insulin resistance by reducing hepatic gluconeogenesis and by enhancing glucose uptake by skeletal muscle. Epidemiological studies have consistently associated metformin use with decreased cancer incidence and cancer-related mortality. Furthermore,numerous preclinical and clinical studies have demonstrated anti-cancer effects of metformin,leading to an explosion of interest in evaluating this agent in human cancer. The effects of metformin on circulating insulin levels indicate a potential efficacy towards cancers associated with hyperinsulinaemia; however,metformin may also directly inhibit tumour growth. In this review,we describe the mechanism of action of metformin and summarise the epidemiological,clinical and preclinical evidence supporting a role for metformin in the treatment of cancer. In addition,the challenges associated with translating preclinical results into therapeutic benefit in the clinical setting will be discussed. View Publication