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

The T-cell surface molecule TIGIT is an immune checkpoint molecule that inhibits T-cell responses,but its roles in cancer are little understood. In this study,we evaluated the role TIGIT checkpoint plays in the development and progression of gastric cancer. We show that the percentage of CD8 T cells that are TIGIT+ was increased in gastric cancer patients compared with healthy individuals. These cells showed functional exhaustion with impaired activation,proliferation,cytokine production,and metabolism,all of which were rescued by glucose. In addition,gastric cancer tissue and cell lines expressed CD155,which bound TIGIT receptors and inactivated CD8 T cells. In a T cell-gastric cancer cell coculture system,gastric cancer cells deprived CD8 T cells of glucose and impaired CD8 T-cell effector functions; these effects were neutralized by the additional glucose or by TIGIT blockade. In gastric cancer tumor cells,CD155 silencing increased T-cell metabolism and IFNγ production,whereas CD155 overexpression inhibited T-cell metabolism and IFNγ production; this inhibition was neutralized by TIGIT blockade. Targeting CD155/TIGIT enhanced CD8 T-cell reaction and improved survival in tumor-bearing mice. Combined targeting of TIGIT and PD-1 further enhanced CD8 T-cell activation and improved survival in tumor-bearing mice. Our results suggest that gastric cancer cells inhibit CD8 T-cell metabolism through CD155/TIGIT signaling,which inhibits CD8 T-cell effector functions,resulting in hyporesponsive antitumor immunity. These findings support the candidacy of CD155/TIGIT as a potential therapeutic target in gastric cancer. Cancer Res; 77(22); 6375-88. textcopyright2017 AACR. View Publication

A major challenge for clinical application of pluripotent stem cell therapy for Parkinson's disease (PD) is large-scale manufacturing and cryopreservation of neurons that can be efficiently prepared with minimal manipulation. To address this obstacle,midbrain dopamine neurons were derived from human induced pluripotent stem cells (iPSC-mDA) and cryopreserved in large production lots for biochemical and transplantation studies. Cryopreserved,post-mitotic iPSC-mDA neurons retained high viability with gene,protein,and electrophysiological signatures consistent with midbrain floor-plate lineage. To test therapeutic efficacy,cryopreserved iPSC-mDA neurons were transplanted without subculturing into the 6-OHDA-lesioned rat and MPTP-lesioned non-human-primate models of PD. Grafted neurons retained midbrain lineage with extensive fiber innervation in both rodents and monkeys. Behavioral assessment in 6-OHDA-lesioned rats demonstrated significant reversal in functional deficits up to 6 months post transplantation with reinnervation of the host striatum and no aberrant growth,supporting the translational development of pluripotent cell-based therapies in PD. View Publication

Human embryonic stem cell line BJNhem20-TetR was generated using non-viral method. The construct pCAG-TetRnls was transfected using microporation procedure. BJNhem20-TetR can subsequently be transfected with any vector harbouring a TetO (Tet operator) sequence to generate doxycycline based inducible line. For example,in human embryonic stem cells,the pSuperior based TetO system has been transfected into a TetR containing line to generate OCT4 knockdown cell line (Zafarana et al.,2009). Thus BJNhem20-TetR can be used as a tool to perturb gene expression in human embryonic stem cells. View Publication

The U1 small nuclear (sn)RNA (U1) is a multifunctional ncRNA,known for its pivotal role in pre-mRNA splicing and regulation of RNA 3' end processing events. We recently demonstrated that a new class of human U1-like snRNAs,the variant (v)U1 snRNAs (vU1s),also participate in pre-mRNA processing events. In this study,we show that several human vU1 genes are specifically upregulated in stem cells and participate in the regulation of cell fate decisions. Significantly,ectopic expression of vU1 genes in human skin fibroblasts leads to increases in levels of key pluripotent stem cell mRNA markers,including NANOG and SOX2. These results reveal an important role for vU1s in the control of key regulatory networks orchestrating the transitions between stem cell maintenance and differentiation. Moreover,vU1 expression varies inversely with U1 expression during differentiation and cell re-programming and this pattern of expression is specifically de-regulated in iPSC-derived motor neurons from Spinal Muscular Atrophy (SMA) type 1 patient's. Accordingly,we suggest that an imbalance in the vU1/U1 ratio,rather than an overall reduction in Uridyl-rich (U)-snRNAs,may contribute to the specific neuromuscular disease phenotype associated with SMA. View Publication

The chromatin landscape and cellular metabolism both contribute to cell fate determination,but their interplay remains poorly understood. Using genome-wide siRNA screening,we have identified prohibitin (PHB) as an essential factor in self-renewal of human embryonic stem cells (hESCs). Mechanistically,PHB forms protein complexes with HIRA,a histone H3.3 chaperone,and stabilizes the protein levels of HIRA complex components. Like PHB,HIRA is required for hESC self-renewal. PHB and HIRA act together to control global deposition of histone H3.3 and gene expression in hESCs. Of particular note,PHB and HIRA regulate the chromatin architecture at the promoters of isocitrate dehydrogenase genes to promote transcription and,thus,production of α-ketoglutarate,a key metabolite in the regulation of ESC fate. Our study shows that PHB has an unexpected nuclear role in hESCs that is required for self-renewal and that it acts with HIRA in chromatin organization to link epigenetic organization to a metabolic circuit. View Publication

Mesoderm is the developmental precursor to myriad human tissues including bone,heart,and skeletal muscle. Unravelling the molecular events through which these lineages become diversified from one another is integral to developmental biology and understanding changes in cellular fate. To this end,we developed an in vitro system to differentiate human pluripotent stem cells through primitive streak intermediates into paraxial mesoderm and its derivatives (somites,sclerotome,dermomyotome) and separately,into lateral mesoderm and its derivatives (cardiac mesoderm). Whole-population and single-cell analyses of these purified populations of human mesoderm lineages through RNA-seq,ATAC-seq,and high-throughput surface marker screens illustrated how transcriptional changes co-occur with changes in open chromatin and surface marker landscapes throughout human mesoderm development. This molecular atlas will facilitate study of human mesoderm development (which cannot be interrogated in vivo due to restrictions on human embryo studies) and provides a broad resource for the study of gene regulation in development at the single-cell level,knowledge that might one day be exploited for regenerative medicine. View Publication

In view of recent reports documenting pervasive translation outside of canonical protein-coding sequences,we wished to determine the proportion of major histocompatibility complex (MHC) class I-associated peptides (MAPs) derived from non-canonical reading frames. Here we perform proteogenomic analyses of MAPs eluted from human B cells using high-throughput mass spectrometry to probe the six-frame translation of the B-cell transcriptome. We report that ∼ 10% of MAPs originate from allegedly noncoding genomic sequences or exonic out-of-frame translation. The biogenesis and properties of these 'cryptic MAPs' differ from those of conventional MAPs. Cryptic MAPs come from very short proteins with atypical C termini,and are coded by transcripts bearing long 3'UTRs enriched in destabilizing elements. Relative to conventional MAPs,cryptic MAPs display different MHC class I-binding preferences and harbour more genomic polymorphisms,some of which are immunogenic. Cryptic MAPs increase the complexity of the MAP repertoire and enhance the scope of CD8 T-cell immunosurveillance. View Publication

The high mobility group A1 gene (HMGA1) has been previously implicated in breast carcinogenesis,and is considered an attractive target for therapeutic intervention because its expression is virtually absent in normal adult tissue. Other studies have shown that knockdown of HMGA1 reduces the tumorigenic potential of breast cancer cells in vitro. Therefore,we sought to determine if silencing HMGA1 can affect breast cancer development and metastatic progression in vivo. We silenced HMGA1 expression in the human breast cancer cell line MDA-MB-231 using an RNA interference vector,and observed a significant reduction in anchorage-independent growth and tumorsphere formation,which respectively indicate loss of tumorigenesis and self-renewal ability. Moreover,silencing HMGA1 significantly impaired xenograft growth in immunodeficient mice,and while control cells metastasized extensively to the lungs and lymph nodes,HMGA1-silenced cells generated only a few small metastases. Thus,our results show that interfering with HMGA1 expression reduces the tumorigenic and metastatic potential of breast cancer cells in vivo,and lend further support to investigations into targeting HMGA1 as a potential treatment for breast cancer. View Publication

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

AC133 antigen is a novel marker for human hematopoietic stem/progenitor cells. In this study,we examined the expression and proliferation potential of AC133(+) cells obtained from steady-state peripheral blood (PB). The proportion of AC133(+) cells in the CD34(+) subpopulation of steady-state PB was significantly lower than that of cord blood (CB),although that of cytokine-mobilized PB was higher than that of CB. The proliferation potential of AC133(+)CD34(+) and AC133(-)CD34(+) cells was examined by colony-forming analysis and analysis of long-term culture-initiating cells (LTC-IC). Although the total number of colony-forming cells was essentially the same in the AC133(+)CD34(+) fraction as in the AC133(-)CD34(+) fraction,the proportion of LTC-IC was much higher in the AC133(+)CD34(+) fraction. Virtually no LTC-IC were detected in the AC133(-)CD34(+) fraction. In addition,the features of the colonies grown from these two fractions were quite different. Approximately 70% of the colonies derived from the AC133(+)CD34(+) fraction were granulocyte-macrophage colonies,whereas more than 90% of the colonies derived from the AC133(-)CD34(+) fraction were erythroid colonies. Furthermore,an ex vivo expansion study observed expansion of colony-forming cells only in the AC133(+)CD34(+) population,and not in the AC133(-)CD34(+) population. These findings suggest that to isolate primitive hematopoietic cells from steady-state PB,selection by AC133 expression is better than selection by CD34 expression. View Publication