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

Consistent and robust manufacturing is essential for the translation of cell therapies,and the utilisation automation throughout the manufacturing process may allow for improvements in quality control,scalability,reproducibility and economics of the process. The aim of this study was to measure and establish the comparability between alternative process steps for the culture of hiPSCs. Consequently,the effects of manual centrifugation and automated non-centrifugation process steps,performed using TAP Biosystems' CompacT SelecT automated cell culture platform,upon the culture of a human induced pluripotent stem cell (hiPSC) line (VAX001024c07) were compared. This study,has demonstrated that comparable morphologies and cell diameters were observed in hiPSCs cultured using either manual or automated process steps. However,non-centrifugation hiPSC populations exhibited greater cell yields,greater aggregate rates,increased pluripotency marker expression,and decreased differentiation marker expression compared to centrifugation hiPSCs. A trend for decreased variability in cell yield was also observed after the utilisation of the automated process step. This study also highlights the detrimental effect of the cryopreservation and thawing processes upon the growth and characteristics of hiPSC cultures,and demonstrates that automated hiPSC manufacturing protocols can be successfully transferred between independent laboratories. View Publication

An increasing number of genetic variants have been implicated in autism spectrum disorders (ASDs),and the functional study of such variants will be critical for the elucidation of autism pathophysiology. Here,we report a de novo balanced translocation disruption of TRPC6,a cation channel,in a non-syndromic autistic individual. Using multiple models,such as dental pulp cells,induced pluripotent stem cell (iPSC)-derived neuronal cells and mouse models,we demonstrate that TRPC6 reduction or haploinsufficiency leads to altered neuronal development,morphology and function. The observed neuronal phenotypes could then be rescued by TRPC6 complementation and by treatment with insulin-like growth factor-1 or hyperforin,a TRPC6-specific agonist,suggesting that ASD individuals with alterations in this pathway may benefit from these drugs. We also demonstrate that methyl CpG binding protein-2 (MeCP2) levels affect TRPC6 expression. Mutations in MeCP2 cause Rett syndrome,revealing common pathways among ASDs. Genetic sequencing of TRPC6 in 1041 ASD individuals and 2872 controls revealed significantly more nonsynonymous mutations in the ASD population,and identified loss-of-function mutations with incomplete penetrance in two patients. Taken together,these findings suggest that TRPC6 is a novel predisposing gene for ASD that may act in a multiple-hit model. This is the first study to use iPSC-derived human neurons to model non-syndromic ASD and illustrate the potential of modeling genetically complex sporadic diseases using such cells.Molecular Psychiatry advance online publication,11 November 2014; doi:10.1038/mp.2014.141. View Publication

The combination of in vitro multi-electrode arrays (MEAs) and the neuronal differentiation of stem cells offers the capability to study human neuronal networks from patient or engineered human cell lines. Here,we use MEA-based assays to probe synaptic function and network interactions of hiPSC-derived neurons. Neuronal network behaviour first emerges at approximately 30 days of culture and is driven by glutamate neurotransmission. Over a further 30 days,inhibitory GABAergic signalling shapes network behaviour into a synchronous regular pattern of burst firing activity and low activity periods. Gene mutations in L-type voltage gated calcium channel subunit genes are strongly implicated as genetic risk factors for the development of schizophrenia and bipolar disorder. We find that,although basal neuronal firing rate is unaffected,there is a dose-dependent effect of L-type voltage gated calcium channel inhibitors on synchronous firing patterns of our hiPSC-derived neural networks. This demonstrates that MEA assays have sufficient sensitivity to detect changes in patterns of neuronal interaction that may arise from hypo-function of psychiatric risk genes. Our study highlights the utility of in vitro MEA based platforms for the study of hiPSC neural network activity and their potential use in novel compound screening. View Publication

In order to overcome the challenges associated with a limited number of airway epithelial cells that can be obtained from clinical sampling and their restrained capacity to divide ex vivo,miniaturization of respiratory drug discovery assays is of pivotal importance. Thus,a 96-well microplate system was developed where primary human small airway epithelial (hSAE) cells were cultured at an air-liquid interface (ALI). After four weeks of ALI culture,a pseudostratified epithelium containing basal,club,goblet and ciliated cells was produced. The 96-well ALI cultures displayed a cellular composition,ciliary beating frequency,and intercellular tight junctions similar to 24-well conditions. A novel custom-made device for 96-parallelized transepithelial electric resistance (TEER) measurements,together with dextran permeability measurements,confirmed that the 96-well culture developed a tight barrier function during ALI differentiation. 96-well hSAE cultures were responsive to transforming growth factor $\beta$1 (TGF-$\beta$1) and tumor necrosis factor $\alpha$ (TNF-$\alpha$) in a concentration dependent manner. Thus,the miniaturized cellular model system enables the recapitulation of a physiologically responsive,differentiated small airway epithelium,and a robotic integration provides a medium throughput approach towards pharmaceutical drug discovery,for instance,in respect of fibrotic distal airway/lung diseases. View Publication

Human embryonic stem cells (hESCs) serve as a valuable in vitro model for studying early human developmental processes due to their ability to differentiate into all three germ layers. Here,we present a comprehensive multi-omics dataset generated by differentiating hESCs into cardiomyocytes via the mesodermal lineage,collecting samples at 10 distinct time points. We measured mRNA levels by mRNA sequencing (mRNA-seq),translation levels by ribosome profiling (Ribo-seq),and protein levels by quantitative mass spectrometry-based proteomics. Technical validation confirmed high quality and reproducibility across all datasets,with strong correlations between replicates. This extensive dataset provides critical insights into the complex regulatory mechanisms of cardiomyocyte differentiation and serves as a valuable resource for the research community,aiding in the exploration of mammalian development and gene regulation. View Publication

SUMMARY Elevated interferon (IFN) signaling is associated with kidney diseases including COVID-19,HIV,and apolipoprotein-L1 (APOL1) nephropathy,but whether IFNs directly contribute to nephrotoxicity remains unclear. Using human kidney organoids,primary endothelial cells,and patient samples,we demonstrate that IFN-? induces pyroptotic angiopathy in combination with APOL1 expression. Single-cell RNA sequencing,immunoblotting,and quantitative fluorescence-based assays reveal that IFN-?-mediated expression of APOL1 is accompanied by pyroptotic endothelial network degradation in organoids. Pharmacological blockade of IFN-? signaling inhibits APOL1 expression,prevents upregulation of pyroptosis-associated genes,and rescues vascular networks. Multiomic analyses in patients with COVID-19,proteinuric kidney disease,and collapsing glomerulopathy similarly demonstrate increased IFN signaling and pyroptosis-associated gene expression correlating with accelerated renal disease progression. Our results reveal that IFN-? signaling simultaneously induces endothelial injury and primes renal cells for pyroptosis,suggesting a combinatorial mechanism for APOL1-mediated collapsing glomerulopathy,which can be targeted therapeutically. In brief Juliar et al. address interferon signaling in kidney disease. Organoids,primary cells,and clinical datasets reveal that interferon signaling simultaneously induces APOL1 expression and endothelial cell pyroptosis. This suggests a combinatorial mechanism for APOL1-mediated collapsing glomerulopathy,which can be targeted therapeutically. The findings may also be relevant in other organs. Graphical Abstract View Publication

ABSTRACTLoss of Cdx2 in vivo leads to stunted development of the allantois,an extraembryonic mesoderm-derived structure critical for nutrient delivery and waste removal in the early embryo. Here,we investigate how CDX2 dose-dependently influences the gene regulatory network underlying extraembryonic mesoderm development. By engineering human induced pluripotent stem cells (hiPSCs) consisting of wild-type (WT),heterozygous (CDX2-Het),and homozygous null CDX2 (CDX2-KO) genotypes,differentiating these cells in a 2D gastruloid model,and subjecting these cells to single-nucleus RNA and ATAC sequencing,we identify several pathways that are dose-dependently regulated by CDX2 including VEGF and non-canonical WNT. snATAC-seq reveals that CDX2-Het cells retain a WT-like chromatin accessibility profile,suggesting accessibility alone is not sufficient to drive this variability in gene expression. Because the loss of CDX2 or TBXT phenocopy one another in vivo,we compared differentially expressed genes in our CDX2-KO to those from TBXT-KO hiPSCs differentiated in an analogous experiment. This comparison identifies several communally misregulated genes that are critical for cytoskeletal integrity and tissue permeability. Together,these results clarify how CDX2 dose-dependently regulates gene expression in the extraembryonic mesoderm and reveal pathways that may underlie the defects in vascular development and allantoic elongation seen in vivo. Summary: Using 2D human gastruloids,CDX2 is shown to dose-dependently influence genes related to tissue permeability,cell-cell adhesions,and cytoskeletal architecture during extraembryonic mesoderm development. View Publication

This study presents a comprehensive transcriptomic analysis of feeder-free extended pluripotent stem cells (ffEPSCs) and their parental human embryonic stem cells (ESCs),providing new insights into understanding human early development and cellular heterogeneity of pluripotency. Leveraging Smart-seq2-based single-cell RNA sequencing (scRNA-seq),we have compared gene expression profiles between ESCs and ffEPSCs and uncovered distinct subpopulations within both groups. Through pseudotime analysis,we have mapped the transition process from ESCs to ffEPSCs,revealing critical molecular pathways involved in the shift from a primed pluripotency to an extended pluripotent state. Additionally,we have employed repeat sequence analysis based on the latest T2T database and identified the stage-specific repeat elements contributing to regulating pluripotency and developmental transitions. This dataset deepens our understanding on early pluripotency and highlights the role of repeat sequences in early embryonic development. Our findings thus offer valuable resources for researchers in stem cell biology,pluripotency,early embryonic development,and potential cell therapy and regenerative medical applications. View Publication

In recent years,Raman spectroscopy has garnered growing interest in the field of biomedical research. It offers a non-invasive and label-free approach to defining the molecular fingerprint of immune cells. We utilized Raman spectroscopy on optically trapped immune cells to investigate their molecular compositions. While numerous immune cell types have been studied in the past,the characterization of living human CD3/CD28-stimulated T cell subsets remains incomplete. In this study,we demonstrate the capability of Raman spectroscopy to readily distinguish between naïve and stimulated CD4 and CD8 cells. Additionally,we compared these cells with monocytes and discovered remarkable similarities between stimulated T cells and monocytes. This paper contributes to expanding our knowledge of Raman spectroscopy of immune cells and serves as a launching point for future clinical applications. View Publication