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

Imatinib mesylate has shown remarkable efficacy in the treatment of patients in the chronic phase of chronic myeloid leukemia. However,despite an overall significant hematological and cytogenetic response,imatinib therapy may favor the emergence of drug-resistant clones,ultimately leading to relapse. Some imatinib resistance mechanisms had not been fully elucidated yet. In this study we used sensitive and resistant sublines from a Bcr-Abl positive cell line to investigate the putative involvement of telomerase in the promotion of imatinib resistance. We showed that sensitivity to imatinib can be partly restored in imatinib-resistant cells by targeting telomerase expression,either by the introduction of a dominant-negative form of the catalytic protein subunit of the telomerase (hTERT) or by the treatment with all-trans-retinoic acid,a clinically used drug. Furthermore,we showed that hTERT overexpression favors the development of imatinib resistance through both its antiapoptotic and telomere maintenance functions. Therefore,combining antitelomerase strategies to imatinib treatment at the beginning of the treatment should be promoted to reduce the risk of imatinib resistance development and increase the probability of eradicating the disease. View Publication

Many solid cancers display cellular hierarchies with self-renewing,tumorigenic stemlike cells,or cancer-initiating cells (CICs) at the apex. Whereas CICs often exhibit relative resistance to conventional cancer therapies,they also receive critical maintenance cues from supportive stromal elements that also respond to cytotoxic therapies. To interrogate the interplay between chemotherapy and CICs,we investigated cellular heterogeneity in human colorectal cancers. Colorectal CICs were resistant to conventional chemotherapy in cell-autonomous assays,but CIC chemoresistance was also increased by cancer-associated fibroblasts (CAFs). Comparative analysis of matched colorectal cancer specimens from patients before and after cytotoxic treatment revealed a significant increase in CAFs. Chemotherapy-treated human CAFs promoted CIC self-renewal and in vivo tumor growth associated with increased secretion of specific cytokines and chemokines,including interleukin-17A (IL-17A). Exogenous IL-17A increased CIC self-renewal and invasion,and targeting IL-17A signaling impaired CIC growth. Notably,IL-17A was overexpressed by colorectal CAFs in response to chemotherapy with expression validated directly in patient-derived specimens without culture. These data suggest that chemotherapy induces remodeling of the tumor microenvironment to support the tumor cellular hierarchy through secreted factors. Incorporating simultaneous disruption of CIC mechanisms and interplay with the tumor microenvironment could optimize therapeutic targeting of cancer. View Publication

Mammary epithelial stem cells are fundamental to maintain tissue integrity. Cancer stem cells (CSCs) are implicated in both treatment resistance and disease relapse,and the molecular bases of their malignant properties are still poorly understood. Here we show that both normal stem cells and CSCs of the breast are controlled by the prolyl-isomerase Pin1. Mechanistically,following interaction with Pin1,Notch1 and Notch4,key regulators of cell fate,escape from proteasomal degradation by their major ubiquitin-ligase Fbxw7$$. Functionally,we show that Fbxw7$$ acts as an essential negative regulator of breast CSCs' expansion by restraining Notch activity,but the establishment of a Notch/Pin1 active circuitry opposes this effect,thus promoting breast CSCs self-renewal,tumor growth and metastasis in vivo. In human breast cancers,despite Fbxw7$$ expression,high levels of Pin1 sustain Notch signaling,which correlates with poor prognosis. Suppression of Pin1 holds promise in reverting aggressive phenotypes,through CSC exhaustion as well as recovered drug sensitivity carrying relevant implications for therapy of breast cancers. View Publication

GANT61 is a small-molecule inhibitor of glioma-associated oncogene 1 (GLI1)- and GLI2-mediated transcription at the nuclear level that exerts its effect by preventing DNA binding. It has been demonstrated to induce cell death against Ewing's sarcoma family tumor (ESFT) cell lines in a dose-dependent manner. The most sensitive cell line was SK-N-LO,which expresses the EWS-FLI1 fusion gene. SK-N-LO cells treated with GANT61 showed cellular and nuclear morphological changes,including cell shrinkage,chromatin condensation and nuclear fragmentation,in a concentration-dependent manner,as visualized by Hoechst 33342 staining. Furthermore,annexin V-propidium iodide (PI) double-staining revealed a significant increase in the number of late apoptotic cells. GANT61 induced a significant decrease in the proportion of cells in the S phase. Significant decrease of the protein levels of GLI2,survivin,cyclin A and claspin,and significant increase of p21 expression was also observed in the cells treated with GANT61. Moreover,poly (ADP-ribose) polymerase (PARP) cleavage was observed,but no cleavage of caspase-3 or -7,or any change in the expressions of Bcl-2 or p53 were observed. These findings suggest that GANT61 induces cell death of SK-N-LO cells in a caspase-independent manner,by inhibiting DNA replication in the S phase. View Publication

Characterization of molecules with tightly controlled expression patterns during differentiation represents an approach to understanding regulation of hematopoietic stem cell commitment. The multidrug resistance-1 (MDR1) gene product,P-glycoprotein,and the breast cancer resistance protein (BCRP) are expressed differentially during hematopoiesis,with the highest levels in primitive bone marrow stem cell populations that are CD34(low) and CD34(-),respectively. Roles for ATP-binding cassette (ABC) transporter superfamily members in conferring drug resistance have been extensively described. However,recent hematopoietic overexpression studies have begun to reveal previously unknown roles for ABC transporter function in normal and malignant hematopoiesis. Expression of MDR1 and BCRP transporters in the myeloid lineage has been reported in blasts from acute myeloid leukemia,but very low to undetectable in normal myelomonocytic cells. Retroviral-mediated dysregulated expression of the MDR1 transporter resulted in increased hematopoietic repopulating activity and myeloproliferative disease in mice. A distinct functional role for the BCRP transporter as a negative regulator of hematopoietic repopulating activity has recently been demonstrated using the same approach. Additionally,the presence of BCRP expression specifically on hematopoietic side-population stem cells and neural stem/progenitors,makes BCRP an attractive candidate marker for isolation of stem cells with the ability to respond to diverse environmental cues. Regulation of stem cell biology by ABC transporters has emerged as an important new field of investigation. In light of these findings,it will be critical to further characterize this family of proteins in hematopoietic lineage-restricted stem cells and in pluripotent stem cells capable of crossing lineage barriers. View Publication

BACKGROUND Embryonic stem (ES) cells are capable of self-renewal and differentiation into cellular derivatives of all 3 germ layers. In appropriate culture conditions,ES cells can differentiate into specialized cells,including cardiac myocytes,but the efficiency is typically low and the process is incompletely understood. METHODS AND RESULTS We evaluated a chemical library for its potential to induce cardiac differentiation of ES cells in the absence of embryoid body formation. Using ES cells stably transfected with cardiac-specific alpha-cardiac myosin heavy chain (MHC) promoter-driven enhanced green fluorescent protein (EGFP),880 compounds approved for human use were screened for their ability to induce cardiac differentiation. Treatment with ascorbic acid,also known as vitamin C,markedly increased the number of EGFP-positive cells,which displayed spontaneous and rhythmic contractile activity and stained positively for sarcomeric myosin and alpha-actinin. Furthermore,ascorbic acid induced the expression of cardiac genes,including GATA4,alpha-MHC,and beta-MHC in untransfected ES cells in a developmentally controlled manner. This effect of ascorbic acid on cardiac differentiation was not mimicked by the other antioxidants such as N-acetylcysteine,Tiron,or vitamin E. CONCLUSIONS Ascorbic acid induces cardiac differentiation in ES cells. This study demonstrates the potential for chemically modifying the cardiac differentiation program of ES cells. View Publication

T helper 17 (Th17) cells are pathogenic in many inflammatory diseases,but also support the integrity of the intestinal barrier in a non-inflammatory manner. It is unclear what distinguishes inflammatory Th17 cells elicited by pathogens and tissue-resident homeostatic Th17 cells elicited by commensals. Here,we compared the characteristics of Th17 cells differentiating in response to commensal bacteria (SFB) to those differentiating in response to a pathogen (Citrobacter rodentium). Homeostatic Th17 cells exhibited little plasticity towards expression of inflammatory cytokines,were characterized by a metabolism typical of quiescent or memory T cells,and did not participate in inflammatory processes. In contrast,infection-induced Th17 cells showed extensive plasticity towards pro-inflammatory cytokines,disseminated widely into the periphery,and engaged aerobic glycolysis in addition to oxidative phosphorylation typical for inflammatory effector cells. These findings will help ensure that future therapies directed against inflammatory Th17 cells do not inadvertently damage the resident gut population. View Publication

There is growing interest in developing drugs that specifically target glioblastoma tumor-initiating cells (TICs). Current cell culture methods,however,cannot cost-effectively produce the large numbers of glioblastoma TICs required for drug discovery and development. In this paper we report a new method that encapsulates patient-derived primary glioblastoma TICs and grows them in 3 dimension thermoreversible hydrogels. Our method allows long-term culture (˜50 days,10 passages tested,accumulative ˜>10(10)-fold expansion) with both high growth rate (˜20-fold expansion/7 days) and high volumetric yield (˜2.0%A-%10(7)%cells/ml) without the loss of stemness. The scalable method can be used to produce sufficient,affordable glioblastoma TICs for drug discovery. View Publication

RATIONALE: Human embryonic stem cells can form cardiomyocytes when cultured under differentiation conditions. Although the initiating step of mesoderm formation is well characterized,the subsequent steps that promote for cardiac lineages are poorly understood and limit the yield of cardiomyocytes. OBJECTIVE: Our aim was to develop a human embryonic stem cell-based high-content screening assay to discover small molecules that drive cardiogenic differentiation after mesoderm is established to improve our understanding of the biology involved. Screening of libraries of small-molecule pathway modulators was predicted to provide insight into the cellular proteins and signaling pathways that control stem cell cardiogenesis. METHODS AND RESULTS: Approximately 550 known pathway modulators were screened in a high-content screening assay,with hits being called out by the appearance of a red fluorescent protein driven by the promoter of the cardiac-specific MYH6 gene. One potent small molecule was identified that inhibits transduction of the canonical Wnt response within the cell,which demonstrated that Wnt inhibition alone was sufficient to generate cardiomyocytes from human embryonic stem cell-derived mesoderm cells. Transcriptional profiling of inhibitor-treated compared with vehicle-treated samples further indicated that inhibition of Wnt does not induce other mesoderm lineages. Notably,several other Wnt inhibitors were very efficient in inducing cardiogenesis,including a molecule that prevents Wnts from being secreted by the cell,which confirmed that Wnt inhibition was the relevant biological activity. CONCLUSIONS: Pharmacological inhibition of Wnt signaling is sufficient to drive human mesoderm cells to form cardiomyocytes; this could yield novel tools for the benefit of pharmaceutical and clinical applications. View Publication

Copy number variation (CNV) is a common chromosomal alteration that can occur during in vitro cultivation of human cells and can be accompanied by the accumulation of mutations in coding region sequences. We describe here a systematic application of current molecular technologies to provide a detailed understanding of genomic and sequence profiles of human embryonic stem cell (hESC) lines that were derived under GMP-compliant conditions. We first examined the overall chromosomal integrity using cytogenetic techniques to determine chromosome count,and to detect the presence of cytogenetically aberrant cells in the culture (mosaicism). Assays of copy number variation,using both microarray and sequence-based analyses,provide a detailed view genomic variation in these lines and shows that in early passage cultures of these lines,the size range and distribution of CNVs are entirely consistent with those seen in the genomes of normal individuals. Similarly,genome sequencing shows variation within these lines that is completely within the range seen in normal genomes. Important gene classes,such as tumor suppressors and genetic disease genes,do not display overtly disruptive mutations that could affect the overall safety of cell-based therapeutics. Complete sequence also allows the analysis of important transplantation antigens,such as ABO and HLA types. The combined application of cytogenetic and molecular technologies provides a detailed understanding of genomic and sequence profiles of GMP produced ES lines for potential use as therapeutic agents. View Publication

BACKGROUND Human papillomavirus 16 (HPV16) is a major risk factor for the development of head and neck squamous cell carcinoma (HNSCC),particularly the development of oropharyngeal squamous cell carcinoma (OPSCC). Cancer stem cells (CSCs) are resistant to conventional therapies,and it is postulated that they are responsible for disease recurrence and/or progression. Because the prognoses of patients with HPV16-positive and HPV-negative HNSCC are distinct,the authors sought to determine whether differences in the number of CSCs could account for this clinical observation. METHODS CSC populations in HPV16-positive and HPV-negative HNSCC were assessed using a proprietary assay based on expression of the enzyme aldehyde dehydrogenase (ALDH),an in vitro tumorsphere formation assay,and an in vivo limiting cell dilution in nonobese diabetic/severe combined immunodeficiency mice. A high-density tissue microarray was stained with ALDH1,a CSC marker,to determine the association between CSCs and HPV16-positive/HPV-negative OPSCC. RESULTS HPV16-positive HNSCC had a greater intrinsic CSC pool than HPV-negative HNSCC. Inactivation of p53 has been identified as a major mechanism for the elevated CSC population in HPV16-positive HNSCC. In vivo limiting cell dilution experiments using tumors from patients with HPV16-positive and HPV-negative OPSCC indicated that the CSC frequency was 62.5-fold greater in an HPV16-positive OPSCC tumor than in an HPV-negative OPSCC tumor. Primary tumors from patients with HPV16-positive OPSCC were associated with elevated tumor ALDH1 staining,further extending the association between HPV16 and CSCs. CONCLUSIONS The current data and the clinical observation that patients with HPV16-positive HNSCC respond more favorably to current treatment paradigms than patients with HPV-negative HNSCC support the suggestion that CSC phenotype is not homogeneous. Therefore,the reliance on the CSC number may be insufficient to accurately assess the potential of a particular tumor for disease recurrence and/or progression. View Publication

DNA methylation regulates gene expression in normal and malignant cells. The possibility to reactivate epigenetically silenced genes has generated considerable interest in the development of DNA methyltransferase inhibitors. Here,we provide a detailed characterization of RG108,a novel small molecule that effectively blocked DNA methyltransferases in vitro and did not cause covalent enzyme trapping in human cell lines. Incubation of cells with low micromolar concentrations of the compound resulted in significant demethylation of genomic DNA without any detectable toxicity. Intriguingly,RG108 caused demethylation and reactivation of tumor suppressor genes,but it did not affect the methylation of centromeric satellite sequences. These results establish RG108 as a DNA methyltransferase inhibitor with fundamentally novel characteristics that will be particularly useful for the experimental modulation of epigenetic gene regulation. View Publication