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

Drug-induced gastrointestinal toxicity (GIT) is a common treatment-emergent adverse event that can negatively impact dosing,thereby limiting efficacy and treatment options for patients. An in vitro assay of GIT is needed to address patient variability,mimic the microphysiology of the gut,and accurately predict drug-induced GIT. Primary human ileal organoids (termed 'enteroids') have proven useful for stimulating intestinal stem cell proliferation and differentiation to multiple cell types present in the gut epithelium. Enteroids have enabled characterization of gut biology and the signaling involved in the pathogenesis of disease. Here,enteroids were differentiated from four healthy human donors and assessed for culture duration-dependent differentiation status by immunostaining for gut epithelial markers lysozyme,chromogranin A,mucin,and sucrase isomaltase. Differentiated enteroids were evaluated with a reference set of 31 drugs exhibiting varying degrees of clinical incidence of diarrhea,a common manifestation of GIT that can be caused by drug-induced thinning of the gut epithelium. An assay examining enteroid viability in response to drug treatment demonstrated 90{\%} accuracy for recapitulating the incidence of drug-induced diarrhea. The human enteroid viability assay developed here presents a promising in vitro model for evaluating drug-induced diarrhea. View Publication

Prader-Willi syndrome (PWS) is a genomic imprinting disorder caused by the loss of function of the paternal chromosome 15q11-13,resulting in a spectrum of symptoms associated with hypothalamic dysfunction. PWS patients lack the expression of paternally expressed genes (PEGs) in the 15q11-13 locus but possess an epigenetically silenced set of these genes in the maternal allele. Thus,activation of these silenced genes can serve as a therapeutic target for PWS. Here,we leverage CRISPR-based epigenome editing system to modulate the DNA methylation status of the PWS imprinting control region (PWS-ICR) in induced pluripotent stem cells (iPSCs) derived from PWS patients. Successful demethylation in the PWS-ICR restores the PEG expression from the maternal allele and reorganizes the methylation patterns in other PWS-associated imprinted regions beyond the PWS-ICR. Remarkably,these corrected epigenomic patterns and PEG expression are maintained following the differentiation of these cells into hypothalamic organoids. Finally,the single-cell transcriptomic analysis of epigenome-edited organoids demonstrates a partial restoration of the transcriptomic dysregulation observed in PWS. This study highlights the utility of epigenome editing technology as a therapeutic approach in addressing PWS and potentially other imprinting disorders. The authors develop CRISPR-based epigenome editing strategy to reactivate silenced maternally inherited genes for Prader-Willi syndrome in human iPSC and hypothalamic organoid models,highlighting its potential for treating imprinting disorders. View Publication

Human embryonic stem (hES) cells represent a potential source for cell replacement therapy of many degenerative diseases. Most frequently,hES cell lines are derived from surplus embryos from assisted reproduction cycles,independent of their quality or morphology. Here,we show that hES cell lines can be obtained from poor-quality blastocysts with the same efficiency as that obtained from good- or intermediate-quality blastocysts. Furthermore,we show that the self-renewal,pluripotency,and differentiation ability of hES cell lines derived from either source are comparable. Finally,we present a simple and reproducible embryoid body-based protocol for the differentiation of hES cells into functional cardiomyocytes. The five new hES cell lines derived here should widen the spectrum of available resources for investigating the biology of hES cells and advancing toward efficient strategies of regenerative medicine. View Publication

ATP citrate lyase (ACL) knockdown (KD) causes tumor suppression and induces differentiation. We have previously reported that ACL KD reverses epithelial-mesenchymal transition (EMT) in lung cancer cells. Because EMT is often associated with processes that induce stemness,we hypothesized that ACL KD impacts cancer stem cells. By assessing tumorsphere formation and expression of stem cell markers,we showed this to be the case in A549 cells,which harbor a Ras mutation,and in two other non-small-cell lung cancer cell lines,H1975 and H1650,driven by activating EGFR mutations. Inducible ACL KD had the same effect as stable ACL KD. Similar effects were noted in another well-characterized Ras-induced mammary model system (HMLER). Moreover,treatment with hydroxycitrate phenocopied the effects of ACL KD,suggesting that the enzymatic activity of ACL was critical. Indeed,acetate treatment reversed the ACL KD phenotype. Having previously established that ACL KD impacts signaling through the phosphatidylinositol 3-kinase (PI3K) pathway,not the Ras-mitogen-activated protein kinase (MAPK) pathway,and that EMT can be reversed by PI3K inhibitors,we were surprised to find that stemness in these systems was maintained through Ras-MAPK signaling,and not via PI3K signaling. Snail is a downstream transcription factor impacted by Ras-MAPK signaling and known to promote EMT and stemness. We found that snail expression was reduced by ACL KD. In tumorigenic HMLER cells,ACL overexpression increased snail expression and stemness,both of which were reduced by ACL KD. Furthermore,ACL could not initiate either tumorigenesis or stemness by itself. ACL and snail proteins interacted and ACL expression regulated the transcriptional activity of snail. Finally,ACL KD counteracted stem cell characteristics induced in diverse cell systems driven by activation of pathways outside of Ras-MAPK signaling. Our findings unveil a novel aspect of ACL function,namely its impact on cancer stemness in a broad range of genetically diverse cell types. View Publication

Identification of agents that target human leukemia stem cells is an important consideration for the development of new therapies. The present study demonstrates that rocaglamide and silvestrol,closely related natural products from the flavagline class of compounds,are able to preferentially kill functionally defined leukemia stem cells,while sparing normal stem and progenitor cells. In addition to efficacy as single agents,flavaglines sensitize leukemia cells to several anticancer compounds,including front-line chemotherapeutic drugs used to treat leukemia patients. Mechanistic studies indicate that flavaglines strongly inhibit protein synthesis,leading to the reduction of short-lived antiapoptotic proteins. Notably though,treatment with flavaglines,alone or in combination with other drugs,yields a much stronger cytotoxic activity toward leukemia cells than the translational inhibitor temsirolimus. These results indicate that the underlying cell death mechanism of flavaglines is more complex than simply inhibiting general protein translation. Global gene expression profiling and cell biological assays identified Myc inhibition and the disruption of mitochondrial integrity to be features of flavaglines,which we propose contribute to their efficacy in targeting leukemia cells. Taken together,these findings indicate that rocaglamide and silvestrol are distinct from clinically available translational inhibitors and represent promising candidates for the treatment of leukemia. View Publication

Schizophrenia has been considered a devastating clinical syndrome rather than a single disease. Nevertheless,the mechanisms behind the onset of schizophrenia have been only partially elucidated. Several studies propose that levels of trace elements are abnormal in schizophrenia; however,conflicting data generated from different biological sources prevent conclusions being drawn. In this work,we used synchrotron radiation X-ray microfluorescence spectroscopy to compare trace element levels in neural progenitor cells (NPCs) derived from two clones of induced pluripotent stem cell lines of a clozapine-resistant schizophrenic patient and two controls. Our data reveal the presence of elevated levels of potassium and zinc in schizophrenic NPCs. Neural cells treated with valproate,an adjunctive medication for schizophrenia,brought potassium and zinc content back to control levels. These results expand the understanding of atomic element imbalance related to schizophrenia and may provide novel insights for the screening of drugs to treat mental disorders. ?? 2014 Elsevier B.V. View Publication

High aldehyde dehydrogenase (ALDH) activity is a marker commonly used to isolate stem cells,particularly breast cancer stem cells (CSCs). Here,we determined that ALDH1A1 activity is inhibited by acetylation of lysine 353 (K353) and that acetyltransferase P300/CBP-associated factor (PCAF) and deacetylase sirtuin 2 (SIRT2) are responsible for regulating the acetylation state of ALDH1A1 K353. Evaluation of breast carcinoma tissues from patients revealed that cells with high ALDH1 activity have low ALDH1A1 acetylation and are capable of self-renewal. Acetylation of ALDH1A1 inhibited both the stem cell population and self-renewal properties in breast cancer. Moreover,NOTCH signaling activated ALDH1A1 through the induction of SIRT2,leading to ALDH1A1 deacetylation and enzymatic activation to promote breast CSCs. In breast cancer xenograft models,replacement of endogenous ALDH1A1 with an acetylation mimetic mutant inhibited tumorigenesis and tumor growth. Together,the results from our study reveal a function and mechanism of ALDH1A1 acetylation in regulating breast CSCs. View Publication

The bacterial CRISPR-Cas9 system has emerged as an effective tool for sequence-specific gene knockout through non-homologous end joining (NHEJ),but it remains inefficient for precise editing of genome sequences. Here we develop a reporter-based screening approach for high-throughput identification of chemical compounds that can modulate precise genome editing through homology-directed repair (HDR). Using our screening method,we have identified small molecules that can enhance CRISPR-mediated HDR efficiency,3-fold for large fragment insertions and 9-fold for point mutations. Interestingly,we have also observed that a small molecule that inhibits HDR can enhance frame shift insertion and deletion (indel) mutations mediated by NHEJ. The identified small molecules function robustly in diverse cell types with minimal toxicity. The use of small molecules provides a simple and effective strategy to enhance precise genome engineering applications and facilitates the study of DNA repair mechanisms in mammalian cells. View Publication

The expression and function of the NK cell receptor KIR2DL4 are controversial. Two common alleles of the transmembrane domain of KIR2DL4 exist. The 10A allele with 10 adenines at the end of the transmembrane exon encodes a full length receptor,whereas the 9A allele has only 9 adenines resulting in a frame shift which in turn generates a stop codon early in the first cytoplasmic exon. The possibility that the 10A and 9A alleles might result in differences in expression and function of KIR2DL4 was explored using mAbs to KIR2DL4. Transfection experiments with cDNA from the 10A and 9A alleles revealed significant membrane expression only with the protein encoded by the 10A allele. Analysis of peripheral blood NK cells demonstrated that only in subjects with at least one 10A allele was cell surface expression of KIR2DL4 detectable,and then only on the minor CD56(bright) NK cell subset. The major CD56(dim) NK cell subset did not cell surface express KIR2DL4 but,interestingly,did so after in vitro culture. Functional analysis using cultured NK cells in redirected lysis assays demonstrated that KIR2DL4 is an activating receptor for NK cells with at least one 10A allele. No significant activity was detected for NK cells generated from subjects homozygous for the 9A allele. These data show that genotype influences cell surface expression and function of KIR2DL4 which may account for reported differences in KIR2DL4 expression and function. View Publication

The advent of human induced pluripotent stem cell (hiPSC) technology has produced patient-specific hiPSC derived cardiomyocytes (hiPSC-CMs) that can be used as a platform to study cardiac diseases and to explore new therapies.The ability to genetically manipulate hiPSC-CMs not only is essential for identifying the structural and/or functional role of a protein but can also provide valuable information regarding therapeutic applications. In this chapter,we describe protocols for culture,maintenance,and cardiac differentiation of hiPSCs. Then,we provide a basic procedure to transduce hiPSC-CMs. View Publication

We report for the first time a microdevice that enables the selective enrichment,culture,and identification of tumor-initiating cells on native polydimethylsiloxane (PDMS). For nearly a decade,researchers have identified tumor-initiating breast cancer cells within heterogeneous populations of breast cancer cells by utilizing low-attachment serum-free culture conditions,which lead to the formation of spheroidal colonies (mammospheres) that are enriched for tumor-initiating cells. However,the utility of this assay has been limited by difficulties in combining this culture-plate-based technique with other cellular and molecular analyses. Integrating the mammosphere technique into a microsystem can enable it to be combined directly with a number of functions,such as cell sorting,drug screens,and molecular assays. In this work,we demonstrate mammosphere culture within a PDMS microdevice. We first prove that a native hydrophobic PDMS surface is as effective as commercial low-attachment plates at selectively promoting the formation of mammospheres. We then experimentally assess the PDMS microdevice. Time-lapse images of mammosphere formation within the microdevice show that mammospheres form from single cells or small clusters of cells. Following formation of the mammospheres,it is desirable to evaluate the cells within the spheroids for enrichment of tumor initiating cells. To perform assays such as this (which require the loading and rinsing of reagents) without flushing the cells (which are in suspension) from the device,the culture chamber is separated from a reagent reservoir by a commercially available microporous membrane,and thus reagents are exchanged between the reservoir and the culture chamber by diffusion only. Using this capability,we verify that the mammospheres are enriched for tumor initiating cells by staining aldehyde dehydrogenase activity,a cancer stem cell marker. To the best of our knowledge,this is the first assay that enables the direct observation of tumor-initiating cells within a suspended mammosphere. View Publication

A chronic antigenic stimulation is believed to sustain the leukemogenic development of chronic lymphocytic leukemia (CLL) and most of lymphoproliferative malignancies developed from mature B cells. Reproducing a proliferative stimulation ex vivo is critical to decipher the mechanisms of leukemogenesis in these malignancies. However,functional studies of CLL cells remains limited since current ex vivo B cell receptor (BCR) stimulation protocols are not sufficient to induce the proliferation of these cells,pointing out the need of mandatory BCR co-factors in this process. Here,we investigated benefits of several BCR co-stimulatory molecules (IL-2,IL-4,IL-15,IL-21 and CD40 ligand) in multiple culture conditions. Our results demonstrated that BCR engagement (anti-IgM ligation) concomitant to CD40 ligand,IL-4 and IL-21 stimulation allowed CLL cells proliferation ex vivo. In addition,we established a proliferative advantage for ZAP70 positive CLL cells,associated to an increased phosphorylation of ZAP70/SYK and STAT6. Moreover,the use of a tri-dimensional matrix of methylcellulose and the addition of TLR9 agonists further increased this proliferative response. This ex vivo model of BCR stimulation with T-derived cytokines is a relevant and efficient model for functional studies of CLL as well as lymphoproliferative malignancies. View Publication