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

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 culture conditions,human induced-pluripotent stem cells (hiPSC)-derived neurons form synaptic connections with other cells and establish neuronal networks,which are expected to be an in vitro model system for drug discovery screening and toxicity testing. While early studies demonstrated effects of co-culture of hiPSC-derived neurons with astroglial cells on survival and maturation of hiPSC-derived neurons,the population spiking patterns of such hiPSC-derived neurons have not been fully characterized. In this study,we analyzed temporal spiking patterns of hiPSC-derived neurons recorded by a multi-electrode array system. We discovered that specific sets of hiPSC-derived neurons co-cultured with astrocytes showed more frequent and highly coherent non-random synchronized spike trains and more dynamic changes in overall spike patterns over time. These temporally coordinated spiking patterns are physiological signs of organized circuits of hiPSC-derived neurons and suggest benefits of co-culture of hiPSC-derived neurons with astrocytes. View Publication

There is compelling evidence for the role of the leucine-rich repeat kinase 2 (LRRK2) and in particular its kinase function in Parkinson's disease. Orally bioavailable,brain penetrant and potent LRRK2 kinase inhibitors are in the later stages of clinical development. Here,we describe a facile and robust assay to quantify LRRK2 kinase pathway activity by measuring LRRK2-mediated phosphorylation of Rab10 in human peripheral blood neutrophils. We use the selective MJFF-pRab10 monoclonal antibody recognising the Rab10 Thr73 phospho-epitope that is phosphorylated by LRRK2. We highlight the feasibility and practicability of using our assay in the clinical setting by studying a few patients with G2019S LRRK2 associated and sporadic Parkinson's as well as healthy controls. We suggest that peripheral blood neutrophils are a valuable resource for LRRK2 research and should be considered for inclusion in Parkinson's bio-repository collections as they are abundant,homogenous and express relatively high levels of LRRK2 as well as Rab10. In contrast,the widely used peripheral blood mononuclear cells are heterogeneous and only a minority of cells (monocytes and contaminating neutrophils) express LRRK2. While our LRRK2 kinase pathway assay could assist in patient stratification based on LRRK2 kinase activity,we envision that it may find greater utility in pharmacodynamic and target engagement studies in future LRRK2 inhibitor trials. View Publication

Many neuropsychiatric disorders are thought to result from subtle changes in neural circuit formation. We used human embryonic stem cells and induced pluripotent stem cells (hiPSCs) to model mature,post-mitotic excitatory neurons and examine effects of fibroblast growth factor 2 (FGF2). FGF2 gene expression is known to be altered in brain regions of major depressive disorder (MDD) patients and FGF2 has anti-depressive effects in animal models of depression. We generated stable inducible neurons (siNeurons) conditionally expressing human neurogenin-2 (NEUROG2) to generate a homogenous population of post-mitotic excitatory neurons and study the functional as well as the transcriptional effects of FGF2. Upon induction of NEUROG2 with doxycycline,the vast majority of cells are post-mitotic,and the gene expression profile recapitulates that of excitatory neurons within 6 days. Using hES cell lines that inducibly express NEUROG2 as well as GCaMP6f,we were able to characterize spontaneous calcium activity in these neurons and show that calcium transients increase in the presence of FGF2. The FGF2-responsive genes were determined by RNA-Seq. FGF2-regulated genes previously identified in non-neuronal cell types were up-regulated (EGR1,ETV4,SPRY4,and DUSP6) as a result of chronic FGF2 treatment of siNeurons. Novel neuron-specific genes were also identified that may mediate FGF2-dependent increases in synaptic efficacy including NRXN3,SYT2,and GALR1. Since several of these genes have been implicated in MDD previously,these results will provide the basis for more mechanistic studies of the role of FGF2 in MDD. View Publication

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

We describe a new procedure for large-scale CB processing in the collection bag,thus minimizing the risk of CB contamination. A solution of 6% hydroxyethyl starch (HES) was added directly to the CB containing bag. After RBC sedimentation at 4 degrees C,the WBC-rich supernatant was collected in a satellite bag and centrifuged. After supernatant removal,the cell pellet was resuspended and the percent recovery of total WBC,CD34+ progenitor cells,CFU-GM and cobblestone area-forming cells (CAFC) evaluated. Results obtained with three different types of CB collection bags (300,600 and 1000 ml) were analyzed and compared with those of an open system in 50 ml tubes. CB processing procedures in 300 and 1000 ml bags were associated with better WBC,CFU,CD34+ cell and CAFC recovery (83-93%). This novel CB processing procedure appears to be easy,effective and particularly suitable for large-scale banking under GMP conditions. View Publication