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

Cancer stem cells (CSCs) are a small subset of cancer cells with indefinite potential for self-renewal and the capacity to drive tumorigenesis. Brefeldin A (BFA) is an antibiotic that is known to block protein transport and induce endoplasmic reticulum (ER) stress in eukaryotic cells,but its effects on colorectal CSCs are unknown. We investigated the inhibitory effect of BFA on human colorectal cancer Colo 205 cells. We found that BFA effectively reduced the survival of suspension Colo 205 cells (IC₅₀ = ˜15 ng/mL) by inducing apoptosis,and inhibited the clonogenic activity of Colo 205 CSCs in tumorsphere formation assay and soft agar colony formation assay in the same nanogram per milliliter range. We also discovered that at such low concentrations,BFA effectively induced endoplasmic reticulum (ER) stress response as indicated by the increased mRNA expression of ER stress-related genes,such as glucose-regulated protein 78 (GRP78),X-box binding protein 1 (XBP1),and C/EBP homologous protein (CHOP). Finally,we found that BFA reduced the activity of matrix metallopeptidase 9 (MMP-9). These findings suggest that BFA can effectively suppress the progression of colorectal cancer during the tumorigenesis and metastasis stages. These results may lead to the development of novel therapies for the treatment of colorectal cancer. View Publication

SummaryLineage-specific differentiation of human induced pluripotent stem cells (hiPSCs) relies on complex interactions between biochemical and physical cues. Here we investigated the ability of hiPSCs to undergo lineage commitment in response to inductive signals and assessed how this competence is modulated by substrate stiffness. We showed that Activin A-induced hiPSC differentiation into mesendoderm and its derivative,definitive endoderm,is enhanced on gel-based substrates softer than glass. This correlated with changes in tight junction formation and extensive cytoskeletal remodeling. Further,live imaging and biophysical studies suggested changes in cell motility and interfacial contacts underlie hiPSC layer reshaping on soft substrates. Finally,we repurposed an ultra-soft silicone gel,which may provide a suitable substrate for culturing hiPSCs at physiological stiffnesses. Our results provide mechanistic insight into how epithelial mechanics dictate the hiPSC response to chemical signals and provide a tool for their efficient differentiation in emerging stem cell therapies. Graphical abstract Highlights•Tuning of substrate stiffness can enhance mesendoderm/endoderm hiPSC differentiation•Altered tight junction formation drives increased differentiation on soft substrates•Changes in cell motility and interfacial contacts underlie hiPSC layer remodeling Mechanobiology; Stem cells research; Biophysics View Publication

Wound healing in adults largely depends on the functional state of multipotent mesenchymal stromal cells (MSCs). Human fetal tissues at the early stages of development are known to heal quickly with a full-quality restoration of the original structure. The differences in the molecular mechanisms that determine the functional activity of mesodermal cells in fetuses and adults remain virtually unknown. Using two independent human induced pluripotent stem cell (iPSC) lines,we examined the effects of the initial WNT and BMP activation on the differentiation of iPSCs via mesodermal progenitors into MSCs and highlighted the functions of these cells that are altered by the proinflammatory microenvironment. The WNT-induced mesoderm commitment of the iPSCs enhanced the expression of paraxial mesoderm (PM)-specific markers,while the BMP4-primed iPSCs exhibited increased levels of lateral mesoderm (LM)-specific genes. The inflammatory status and migration rate of the isogenic iPSC-derived mesoderm cells were assessed via gene expression analysis and scratch assay under the receptor-dependent activation of the proinflammatory IFN-γ or TNF-α signaling pathway. Reduced IDO1 and ICAM1 expression levels were detected in the WNT- and BMP-induced MSC progenitors compared to the isogenic MSCs in response to stimulation with IFN-γ and TNF-α. The WNT- and BMP-induced MSC progenitors exhibited a higher migration rate than isogenic MSCs upon IFN-γ exposure. The established isogenic cellular model will provide new opportunities to elucidate the mechanisms of regeneration and novel therapeutics for wound healing. View Publication

Robust control of human induced pluripotent stem cell (hIPSC) differentiation is essential to realize its patient-tailored therapeutic potential. Here,we demonstrate a novel application of Y-27632,a small molecule Rho-associated protein kinase (ROCK) inhibitor,to significantly influence the differentiation of hIPSCs in a lineage-specific manner. The application of Y-27632 to hIPSCs resulted in a decrease in actin bundling and disruption of colony formation in a concentration and time-dependent manner. Such changes in cell and colony morphology were associated with decreased expression of E-cadherin,a cell-cell junctional protein,proportional to the increased exposure to Y-27632. Interestingly,gene and protein expression of pluripotency markers such as NANOG and OCT4 were not downregulated by an exposure to Y-27632 up to 36h. Simultaneously,epithelial-to-mesenchymal (EMT) transition markers were upregulated with an exposure to Y-27632. These EMT-like changes in the cells with longer exposure to Y-27632 resulted in a significant increase in the subsequent differentiation efficiency towards mesendodermal lineage. In contrast,an inhibitory effect was observed when cells were subjected to ectodermal differentiation after prolonged exposure to Y-27632. Collectively,these results present a novel method for priming hIPSCs to modulate their differentiation potential with a simple application of Y-27632. View Publication

BACKGROUND: Infection with HIV-1 has been shown to alter expression of a large array of host cell genes. However,previous studies aimed at investigating the putative HIV-1-induced modulation of host gene expression have been mostly performed in established human cell lines. To better approximate natural conditions,we monitored gene expression changes in a cell population highly enriched in human primary CD4+ T lymphocytes exposed to HIV-1 using commercial oligonucleotide microarrays from Affymetrix. RESULTS: We report here that HIV-1 influences expression of genes related to many important biological processes such as DNA repair,cellular cycle,RNA metabolism and apoptosis. Notably,expression of the p53 tumor suppressor and genes involved in p53 homeostasis such as GADD34 were up-regulated by HIV-1 at the mRNA level. This observation is distinct from the previously reported p53 phosphorylation and stabilization at the protein level,which precedes HIV-1-induced apoptosis. We present evidence that the HIV-1-mediated increase in p53 gene expression is associated with virus-mediated induction of type-I interferon (i.e. IFN-alpha and IFN-beta). CONCLUSION: These observations have important implications for our understanding of HIV-1 pathogenesis,particularly in respect to the virus-induced depletion of CD4+ T cells. View Publication

Glioblastoma,the most malignant type of primary brain tumor,is one of the solid cancers where cancer stem cells have been isolated,and studies have suggested resistance of those cells to chemotherapy and radiotherapy. Here,we report the establishment of CSC-enriched cultures derived from human glioblastoma specimens. They grew as neurospheres in serum-free medium with epidermal growth factor and fibroblast growth factor 2,varied in the level of CD133 expression and very efficiently formed highly invasive and/or vascular tumors upon intracerebral implantation into immunodeficient mice. As a novel therapeutic strategy for glioblastoma-derived cancer stem-like cells (GBM-SC),we have tested oncolytic herpes simplex virus (oHSV) vectors. We show that although ICP6 (UL39)-deleted mutants kill GBM-SCs as efficiently as wild-type HSV,the deletion of gamma34.5 significantly attenuated the vectors due to poor replication. However,this was significantly reversed by the additional deletion of alpha47. Infection with oHSV G47Delta (ICP6(-),gamma34.5(-),alpha47(-)) not only killed GBM-SCs but also inhibited their self-renewal as evidenced by the inability of viable cells to form secondary tumor spheres. Importantly,despite the highly invasive nature of the intracerebral tumors generated by GBM-SCs,intratumoral injection of G47Delta significantly prolonged survival. These results for the first time show the efficacy of oHSV against human GBM-SCs,and correlate this cytotoxic property with specific oHSV mutations. This is important for designing new oHSV vectors and clinical trials. Moreover,the new glioma models described in this study provide powerful tools for testing experimental therapeutics and studying invasion and angiogenesis. View Publication

A robust method for culturing human pluripotent stem (hPS) cells under chemically defined and xeno-free conditions is an important tool for stem cell research and for the development of regenerative medicine. Here,we describe a protocol for monolayer culturing of Oct-4-positive hPS cells on a specific laminin-521 (LN-521) isoform,under xeno-free and chemically defined conditions. The cells are dispersed into single-cell suspension and then plated on LN-521 isoform at densities higher than 5,000 cells per cm²,where they attach,migrate and survive by forming small monolayer cell groups. The cells avidly divide and expand horizontally until the entire dish is covered by a confluent monolayer. LN-521,in combination with E-cadherin,allows cloning of individual hPS cells in separate wells of 96-well plates without the presence of rho-associated protein kinase (ROCK) inhibitors or any other inhibitors of anoikis. Characterization of cells maintained for several months in culture reveals pluripotency with a minimal degree of genetic abnormalities. View Publication

Regeneration of human cartilage is inherently inefficient; an abundant autologous source,such as human induced pluripotent stem cells (hiPSCs),is therefore attractive for engineering cartilage. We report a growth factor-based protocol for differentiating hiPSCs into articular-like chondrocytes (hiChondrocytes) within 2 weeks,with an overall efficiency textgreater90%. The hiChondrocytes are stable and comparable to adult articular chondrocytes in global gene expression,extracellular matrix production,and ability to generate cartilage tissue in vitro and in immune-deficient mice. Molecular characterization identified an early SRY (sex-determining region Y) box (Sox)9(low) cluster of differentiation (CD)44(low)CD140(low) prechondrogenic population during hiPSC differentiation. In addition,2 distinct Sox9-regulated gene networks were identified in the Sox9(low) and Sox9(high) populations providing novel molecular insights into chondrogenic fate commitment and differentiation. Our findings present a favorable method for generating hiPSC-derived articular-like chondrocytes. The hiChondrocytes are an attractive cell source for cartilage engineering because of their abundance,autologous nature,and potential to generate articular-like cartilage rather than fibrocartilage. In addition,hiChondrocytes can be excellent tools for modeling human musculoskeletal diseases in a dish and for rapid drug screening. View Publication

B1 cells differ in many ways from conventional B cells,most prominently in the production of natural immunoglobulin,which is vitally important for protection against pathogens. B1 cells have also been implicated in the pathogenesis of autoimmune dyscrasias and malignant diseases. It has been impossible to accurately study B1 cells during health and illness because the nature of human B1 cells has not been successfully defined. This has produced controversy regarding the existence of human B1 cells. Here,we determined the phenotype of human B1 cells by testing sort-purified B cell fractions for three fundamental B1 cell functions based on mouse studies: spontaneous IgM secretion,efficient T cell stimulation,and tonic intracellular signaling. We found that a small population of CD20(+)CD27(+)CD43(+) cells present in both umbilical cord and adult peripheral blood fulfilled these criteria and expressed a skewed B cell receptor repertoire. These B cells express little or no surface CD69 and CD70,both of which are markedly up-regulated after activation of CD20(+)CD27(-)CD43(-) (naive) and CD20(+)CD27(+)CD43(-) (memory) B cells. This work identifies human B1 cells as CD20(+)CD27(+)CD43(+)CD70(-). We determined that the proportion of B1 cells declines with age,which may contribute to disease susceptibility. Identification of human B1 cells provides a foundation for future studies on the nature and role of these cells in human disease. View Publication

Human pluripotent stem cells (hPSCs),including embryonic stem cells and induced pluripotent stem cells,are potentially useful in regenerative therapies for heart disease. For medical applications,clinical-grade cardiac cells must be produced from hPSCs in a defined,cost-effective manner. Cell-based screening led to the discovery of KY02111,a small molecule that promotes differentiation of hPSCs to cardiomyocytes. Although the direct target of KY02111 remains unknown,results of the present study suggest that KY02111 promotes differentiation by inhibiting WNT signaling in hPSCs but in a manner that is distinct from that of previously studied WNT inhibitors. Combined use of KY02111 and WNT signaling modulators produced robust cardiac differentiation of hPSCs in a xeno-free,defined medium,devoid of serum and any kind of recombinant cytokines and hormones,such as BMP4,Activin A,or insulin. The methodology has potential as a means for the practical production of human cardiomyocytes for regeneration therapies. View Publication

It has been shown that Notch signaling mediated by ligands of both Jagged and Delta families expands the hematopoietic stem cell compartment while blocking or delaying terminal myeloid differentiation. Here we show that Delta1- and Jagged1-expressing stromal cells have distinct effects on the clonogenic and differentiation capacities of human CD34(+) CD38(+) cells. Jagged1 increases the number of bipotent colony-forming unit-granulocyte macrophage (CFU-GM) and unipotent progenitors (CFU-granulocytes and CFU-macrophages),without quantitatively affecting terminal cell differentiation,whereas Delta1 reduces the number of CFU-GM and differentiated monocytic cells. Expression analysis of genes coding for Notch receptors,Notch targets,and Notch signaling modulators in supernatant CD34(+) cells arising upon contact with Jagged1 and Delta1 shows dynamic and differential gene expression profiles over time. At early time points,modest upregulation of Notch1,Notch3,and Hes1 was observed in Jagged1-CD34(+) cells,whereas those in contact with Delta1 strikingly upregulated Notch3 and Hes1. Later,myeloid progenitors with strong clonogenic potential emerging upon contact with Jagged1 upregulated Notch1 and Deltex and downregulated Notch signaling modulators,whereas T/NK progenitors originated by Delta1 strikingly upregulated Notch3 and Deltex and,to a lesser extent,Hes1,Lunatic Fringe,and Numb. Together,the data unravel previously unrecognized expression patterns of Notch signaling-related genes in CD34(+) CD38(+) cells as they develop in Jagged1- or Delta1-stromal cell environments,which appear to reflect sequential maturational stages of CD34(+) cells into distinct cell lineages. View Publication

During drug development,measurement of suitable pharmacodynamic biomarkers is key to establishing in vivo drug activity. Binding of monoclonal antibody (mAb) therapeutics to soluble target proteins often results in elevated serum levels of their target antigen,and measuring total (free and bound) concentration of the target antigen can be an important means of demonstrating that the mAb has reached its specific target. However,accurately measuring soluble circulating antigen in preclinical or clinical samples in the presence of a therapeutic mAb presents a bioanalytical challenge. Particularly in the case of low molecular weight and/or multimeric targets,epitopes for capture and detection of the target by reagent antibodies can be obscured by bound therapeutic mAb. Lymphotoxin-alpha (LTα) is a cytokine in the TNF superfamily that has been implicated in the pathophysiology of autoimmune disease,and is a therapeutic target for neutralizing mAb. During preclinical safety studies in cynomolgus macaques,we encountered difficulties in measuring total LTα in serum of dosed animals. When serum LTα trimer was saturated with the anti-LTα mAb,binding of two reagent antibodies,as required for a classic sandwich ELISA,was not feasible,and dissociation methods were also found to be unsuitable. We therefore developed an approach in which excess anti-LTα mAb was added to the in vitro assay system to fully saturate all binding sites,and an anti-idiotypic antibody was used to detect bound therapeutic antibody. Using this method,total LTα could be accurately measured in cynomolgus macaque serum,and was observed to increase with increasing anti-LTα therapeutic mAb dose. Additional in vitro studies demonstrated that the method worked equally well in human serum. This assay strategy will be useful for quantifying total concentrations of other small and/or multimeric target proteins in the presence of a therapeutic antibody. View Publication