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

Human pluripotent stem cells (HPSCs) cultured in conditions that maintain pluripotency via FGF and TGFβ signaling have been described as being in a primed state. These cells have been shown to exhibit characteristics more closely related to mouse epiblast-derived stem cells than to so called naïve mouse PSCs said to possess a more ground state pluripotency that mimics the early mouse embryo inner cell mass. Initial attempts to create culture conditions favorable for generation of naïve HPSCs from primed HPSCs has required the use of mouse embryonic fibroblasts as a feeder layer to support this transition. A protocol for the routine derivation and maintenance of naïve HPSCs in completely defined conditions is highly desirable for stem cell researchers to enhance the study and clinical translation of naïve HPSCs. Here we describe a standard protocol for transitioning primed HPSCs to a naïve state using commercial RSet media and xeno-free recombinant vitronectin. View Publication

Retinoid-related molecules (RRM) are novel agents with tumor-selective cytotoxic/antiproliferative activity,a different mechanism of action from classic retinoids and no cross-resistance with other chemotherapeutics. ST1926 and CD437 are prototypic RRMs,with the former currently undergoing phase I clinical trials. We show here that ST1926,CD437,and active congeners cause DNA damage. Cellular and subcellular COMET assays,H2AX phosphorylation (gamma-H2AX),and scoring of chromosome aberrations indicate that active RRMs produce DNA double-strand breaks (DSB) and chromosomal lesions in NB4,an acute myeloid leukemia (AML) cell line characterized by high sensitivity to RRMs. There is a direct quantitative correlation between the levels of DSBs and the cytotoxic/antiproliferative effects induced by RRMs. NB4.437r blasts,which are selectively resistant to RRMs,do not show any sign of DNA damage after treatment with ST1926,CD437,and analogues. DNA damage is the major mechanism underlying the antileukemic activity of RRMs in NB4 and other AML cell lines. In accordance with the S-phase specificity of the cytotoxic and antiproliferative responses of AML cells to RRMs,increases in DSBs are maximal during the S phase of the cell cycle. Induction of DSBs precedes inhibition of DNA replication and is associated with rapid activation of ataxia telangectasia mutated,ataxia telangectasia RAD3-related,and DNA-dependent protein kinases with subsequent stimulation of the p38 mitogen-activated protein kinase. Inhibition of ataxia telangectasia mutated and DNA-dependent protein kinases reduces phosphorylation of H2AX. Cells defective for homologous recombination are particularly sensitive to ST1926,indicating that this process is important for the protection of cells from the RRM-dependent DNA damage and cytotoxicity. View Publication

The mTOR protein is a master regulator of cell growth and proliferation,and inhibitors of its kinase activity have the potential to become new class of anticancer drugs. Starting from quinoline 1,which was identified in a biochemical mTOR assay,we developed a tricyclic benzonaphthyridinone inhibitor 37 (Torin1),which inhibited phosphorylation of mTORC1 and mTORC2 substrates in cells at concentrations of 2 and 10 nM,respectively. Moreover,Torin1 exhibits 1000-fold selectivity for mTOR over PI3K (EC(50) = 1800 nM) and exhibits 100-fold binding selectivity relative to 450 other protein kinases. Torin1 was efficacious at a dose of 20 mg/kg in a U87MG xenograft model and demonstrated good pharmacodynamic inhibition of downstream effectors of mTOR in tumor and peripheral tissues. These results demonstrate that Torin1 is a useful probe of mTOR-dependent phenomena and that benzonaphthridinones represent a promising scaffold for the further development of mTOR-specific inhibitors with the potential for clinical utility. View Publication

BACKGROUND There is an unmet need for developing faithful animal models for preclinical evaluation of immunotherapy. The current approach to generate preclinical models for immunotherapy evaluation has been to transplant CD34+ cells from umbilical cord blood into immune-deficient mice followed by implantation of patient derived tumor cells. However,current models are associated with high tumor rejection rate secondary to the allograft vs. tumor response from human leukocyte antigen (HLA) mismatches. We herein report the first development of a novel,humanized patient-derived xenograft (PDX) model using autologous CD34+ cells from bone marrow aspirate obtained from a patient with metastatic clear cell renal cell carcinoma (mRCC) from whom a PDX had been developed. CASE DESCRIPTION This is a 68-year-old Caucasian man diagnosed with mRCC with metastasis to the liver in 2014. He was treated with sunitinib +/- AGS-003 and underwent a cytoreductive right nephrectomy,left adrenalectomy and partial liver resection. PDX was generated using resected nephrectomy specimen. After surgery,patient received multiple lines of standard of care therapy including sunitinib,axitinib,bevacizumab,everolimus and cabozantinib. While progressing on cabozantinib,he was treated with nivolumab. Seven years after initiation of nivolumab,and 4 years after stopping systemic therapy,he remains in complete remission. To generate autologous PDX model,bone marrow aspirate was performed and CD34+ hematopoietic stem/progenitor cells (HSPCs) were isolated and injected into 150 rad irradiated non-obese diabetic scid gamma null (NSG) mice. At 11 weeks post-transplant,the matched patient PDX was injected subcutaneously into the humanized mice and the mice were treated with nivolumab. CONCLUSIONS Our case represents successful therapy of nivolumab in mRCC. Furthermore,HPSCs obtained from a single bone marrow aspirate were able to reconstitute an immune system in the mice that allowed nivolumab to inhibit the tumor growth of PDX and recapitulated the durable remission observed in the patient with nivolumab. We observed the reconstitution of human T cells,B cells and natural killer (NK) cells and unlike the humanized mouse model using cord blood,our model system eliminates the tumor rejection from mis-matched HLA. Our autologous humanized renal cell carcinoma (RCC) PDX model provides an effective tool to study immunotherapy in a preclinical setting. View Publication

Recent methodological advances have improved the ease and efficiency of generating human induced pluripotent stem cells (hiPSCs),but this now typically results in a greater number of hiPSC clones being derived than can be wholly characterized. It is therefore imperative that methods are developed which facilitate rapid selection of hiPSC clones most suited for the downstream research aims. Here we describe a combination of procedures enabling the simultaneous screening of multiple clones to determine their genomic integrity as well as their cardiac differentiation potential within two weeks of the putative reprogrammed colonies initially appearing. By coupling splinkerette-PCR with Ion Torrent sequencing,we could ascertain the number and map the proviral integration sites in lentiviral-reprogrammed hiPSCs. In parallel,we developed an effective cardiac differentiation protocol that generated functional cardiomyocytes within 10 days without requiring line-specific optimization for any of the six independent human pluripotent stem cell lines tested. Finally,to demonstrate the scalable potential of these procedures,we picked 20 nascent iPSC clones and performed these independent assays concurrently. Before the clones required passaging,we were able to identify clones with a single integrated copy of the reprogramming vector and robust cardiac differentiation potential for further analysis. View Publication

In the context of pancreatic cancer,metastasis remains the most critical determinant of resectability,and hence survival. The objective of this study was to determine whether Hedgehog (Hh) signaling plays a role in pancreatic cancer invasion and metastasis because this is likely to have profound clinical implications. In pancreatic cancer cell lines,Hh inhibition with cyclopamine resulted in down-regulation of snail and up-regulation of E-cadherin,consistent with inhibition of epithelial-to-mesenchymal transition,and was mirrored by a striking reduction of in vitro invasive capacity (P textless 0.0001). Conversely,Gli1 overexpression in immortalized human pancreatic ductal epithelial cells led to a markedly invasive phenotype (P textless 0.0001) and near total down-regulation of E-cadherin. In an orthotopic xenograft model,cyclopamine profoundly inhibited metastatic spread; only one of seven cyclopamine-treated mice developed pulmonary micrometastases versus seven of seven mice with multiple macrometastases in control animals. Combination of gemcitabine and cyclopamine completely abrogated metastases while also significantly reducing the size of primary" tumors. Gli1 levels were up-regulated in tissue samples of metastatic human pancreatic cancer samples compared with matched primary tumors. Aldehyde dehydrogenase (ALDH) overexpression is characteristic for both hematopoietic progenitors and leukemic stem cells; cyclopamine preferentially reduced "ALDH-high" cells by approximately 3-fold (P = 0.048). We confirm pharmacologic Hh pathway inhibition as a valid therapeutic strategy for pancreatic cancer and show for the first time its particular efficacy against metastatic spread. By targeting specific cellular subpopulations likely involved in tumor initiation at metastatic sites� View Publication

Poor recovery of cryopreserved human embryonic stem (hES) cells and induced pluripotent stem (iPS) cells is a significant impediment to progress with pluripotent stem cells. In this study,we demonstrate that Y-27632,a specific inhibitor of Rho kinase (ROCK) activity,significantly enhances recovery of hES cells from cryopreserved stocks when cultured with or without a growth inactivated feeder layer. Furthermore,treatment with the ROCK inhibitor for several days increased the number of colonies and colony size of hES cells compared to shorter exposures. Remarkably,hES cells that had formed relatively few colonies 5 days after thawing exhibited rapid growth upon addition of Y-27632. Additionally,we determined that Y-27632 significantly improves the recovery of cryopreserved human iPS cells and their growth upon subculture. Thus,Y-27632 provides a means to kick-start" slow-growing human pluripotent stem cells� View Publication

Brain-derived microvascular endothelial cells (BMECs),which play a central role in blood brain barrier (BBB),can be used for the evaluation of drug transport into the brain. Although human BMEC cell lines have already been reported,they lack original properties such as barrier integrity. Pluripotent stem cells (PSCs) can be used for various applications such as regenerative therapy,drug screening,and pathological study. In the recent study,an induction method of BMECs from PSCs has been established,making it possible to more precisely study the in vitro human BBB function. Here,using induced pluripotent stem (iPS) cell-derived BMECs,we examined the effects of oxygen-glucose deprivation (OGD) and OGD/reoxygenation (OGD/R) on BBB permeability. OGD disrupted the barrier function,and the dysfunction was rapidly restored by re-supply of the oxygen and glucose. Interestingly,TNF-α,which is known to be secreted from astrocytes and microglia in the cerebral ischemia,prevented the restoration of OGD-induced barrier dysfunction in an apoptosis-independent manner. Thus,we could establish the in vitro BBB disease model that mimics the cerebral ischemia by using iPS cell-derived BMECs. View Publication

The coding region determinant-binding protein/insulin-like growth factor II mRNA-binding protein (CRD-BP/IMP1) is an RNA-binding protein specifically recognizing c-myc,leader 3' IGF-II and tau mRNAs,and the H19 RNA. CRD-BP/IMP1 is predominantly expressed in embryonal tissues but is de novo activated and/or overexpressed in various human neoplasias. To address the question of whether CRD-BP/IMP1 expression characterizes certain cell types displaying distinct proliferation and/or differentiation properties (i.e. stem cells),we isolated cell subpopulations from human bone marrow,mobilized peripheral blood,and cord blood,all sources known to contain stem cells,and monitored for its expression. CRD-BP/IMP1 was detected only in cord blood-derived CD34(+) stem cells and not in any other cell type of either adult or cord blood origin. Adult BM CD34(+) cells cultured in the presence of 5'-azacytidine expressed de novo CRD-BP/IMP1,suggesting that epigenetic modifications may be responsible for its silencing in adult non-expressing cells. Furthermore,by applying the short interfering RNA methodology in MCF-7 cells,we observed,subsequent to knocking down CRD-BP/IMP1,decreased c-myc expression,increased IGF-II mRNA levels,and reduced cell proliferation rates. These data 1) suggest a normal role for CRD-BP/IMP1 in pluripotent stem cells with high renewal capacity,like the CB CD34(+) cells,2) indicate that altered methylation may directly or indirectly affect its expression in adult cells,3) imply that its de novo activation in cancer cells may affect the expression of c-Myc and insulin-like growth factor II,and 4) indicate that the inhibition of CRD-BP/IMP1 expression might affect cancer cell proliferation. View Publication

Recent studies have shown that cellular bioenergetics may be involved in stem cell differentiation. Considering that during cancerogenesis cells acquire numerous properties of stem cells,it is possible to assume that the energy metabolism in tumorigenic cells might be differently regulated. The aim of this study was to compare the mitochondrial bioenergetic profile of normal pluripotent human embryonic stem cells (hESC) and relatively nullipotent embryonal carcinoma cells (2102Ep cell line). We examined three parameters related to cellular bioenergetics: phosphotransfer system,aerobic glycolysis,and oxygen consumption. Activities and expression levels of main enzymes that facilitate energy transfer were measured. The oxygen consumption rate studies were performed to investigate the respiratory capacity of cells. 2102Ep cells showed a shift in energy distribution towards adenylate kinase network. The total AK activity was almost 3 times higher in 2102Ep cells compared to hESCs (179.85±5.73 vs 64.39±2.55mU/mg of protein) and the expression of AK2 was significantly higher in these cells,while CK was downregulated. 2102Ep cells displayed reduced levels of oxygen consumption and increased levels of aerobic glycolysis compared to hESCs. The compromised respiration of 2102Ep cells is not the result of increased mitochondrial mass,increased proton leak,and reduced respiratory reserve capacity of the cells or impairment of respiratory chain complexes. Our data showed that the bioenergetic profile of 2102Ep cells clearly distinguishes them from normal hESCs. This should be considered when this cell line is used as a reference,and highlight the importance of further research concerning energy metabolism of stem cells. View Publication