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

This study describes the characterization of one induced pluripotent stem cell line (iPSC) from a healthy female. It is crucial to use iPSCs derived from healthy individuals as controls in genetic disease studies. Thus,we established a human iPSC cell line derived from healthy people. The iPSC cell line was generated in our lab from the peripheral blood mononuclear cells (PBMCs) of a 28-year-old girl. The generated hiPSC line is free of episomal vectors,has a normal karyotype,expresses pluripotency markers and can differentiate into three germ layers in vivo. View Publication

Neutrophils are the most abundant leukocytes in human blood and are essential components of innate immunity. Until recently,neutrophils were considered homogeneous and transcriptionally inactive cells,but both concepts are being challenged. Single-cell RNA sequencing (scRNA-seq) offers an unbiased view of cells along a continuum of transcriptional states. However,the use of scRNA-seq to characterize neutrophils has proven technically difficult,explaining in part the paucity of published single-cell data on neutrophils. We have found that modifications to the data analysis pipeline,rather than to the existing scRNA-seq chemistries,can significantly increase the detection of human neutrophils in scRNA-seq. We have then applied a modified pipeline to the study of human peripheral blood neutrophils. Our findings indicate that circulating human neutrophils are transcriptionally heterogeneous cells,which can be classified into one of four transcriptional clusters that are reproducible among healthy human subjects. We demonstrate that peripheral blood neutrophils shift from relatively immature (Nh0) cells,through a transitional phenotype (Nh1),into one of two end points defined by either relative transcriptional inactivity (Nh2) or high expression of type I IFN-inducible genes (Nh3). Transitions among states are characterized by the expression of specific transcription factors. By simultaneously measuring surface proteins and intracellular transcripts at the single-cell level,we show that these transcriptional subsets are independent of the canonical surface proteins that are commonly used to define and characterize human neutrophils. These findings provide a new view of human neutrophil heterogeneity,with potential implications for the characterization of neutrophils in health and disease. 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

The development of patient-derived cardiac disease models has advanced rapidly due to the progress of human-induced pluripotent stem cell (hiPSC) technologies. Many protocols detail individual parts of the entire workflow,from handling hiPSCs and differentiating them into cardiomyocytes to live contraction imaging via widefield/phase-contrast and fluorescence microscopy. Here,we propose a streamlined protocol that guides users through hiPSC culture,differentiation,expansion,and functional imaging of hiPSC cardiomyocytes. First,hiPSC maintenance and handling procedures are outlined. Differentiation occurs over a two-week period,followed by selective expansion to increase the yield of hiPSC cardiomyocytes. Comprehensive characterization and quantification enable detailed contraction profiling of these cells. Designed to be low-cost,this protocol is suited for applications in drug discovery,screening,and clinical testing of patient-specific phenotypes. The addition of cardiomyocyte expansion and automated analysis distinguishes our protocol from current approaches. View Publication

SummaryApolipoprotein E4 (APOE4) is the leading genetic risk factor for Alzheimer’s disease. While most studies examine the role of APOE4 in aging,APOE4 causes persistent changes in brain structure as early as infancy and is associated with altered functional connectivity that extends beyond adolescence. Here,we used human induced pluripotent stem cell-derived cortical and ganglionic eminence organoids (COs and GEOs) to examine APOE4’s influence during the development of cortical excitatory and inhibitory neurons. We show that APOE4 reduces cortical neurons and increases glia by promoting gliogenic transcriptional programs. In contrast,APOE4 increases proliferation and differentiation of GABAergic progenitors resulting in early and persistent increases in GABAergic neurons. Multi-electrode array recordings in assembloids revealed that APOE4 disrupts neural network function resulting in heightened excitability and synchronicity. Together,our data provide new insights on how APOE4 influences cortical neurodevelopmental processes and the establishment of functional networks. Highlights•APOE4 accelerates differentiation and maturation at developmental time points•APOE4 results in a loss of cortical neurons and increase in astrocytes and outer radial glia•APOE4 enhances proliferation,differentiation,and maturation of GABAergic neurons•APOE4 alters GABA-related genes,linked to increased GABA response and synchronicity Meyer-Acosta et al. reveal that Alzheimer’s disease genetic risk factor APOE4 decreases cortical neurons and increases glia in cortical organoids and enhances GABAergic neuron maturation in ganglionic eminence organoids derived from iPSCs. These cellular changes result in heightened excitability and synchronicity in APOE4-fused organoids,providing insight into the cellular processes that may underlie altered brain structure observed in APOE4 infants. 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

Autoantibodies targeting cytosolic 5′-nucleotidase 1A (cN1A) are found in several autoimmune diseases,including inclusion body myositis (IBM),Sjögren’s syndrome,and systemic lupus erythematosus. While they have diagnostic relevance for IBM,little is known about the autoreactive B cells that produce these antibodies. To address this,we developed a robust protocol for the expression and site-specific biotinylation of recombinant human cN1A in Escherichia coli. The resulting antigen is suitable for generating double-labelled fluorescent baits for the isolation and characterisation of cN1A-specific B cells by flow cytometry. Site-specific biotinylation was achieved using the AviTag and BirA ligase,preserving the protein’s structure and immunoreactivity. Western blot analysis confirmed that the biotinylated cN1A was recognised by both human and rabbit anti-cN1A antibodies. Compared to conventional chemical biotinylation,this strategy minimises structural alterations that may affect antigen recognition. This approach provides a reliable method for producing biotinylated antigens for use in immunological assays. While demonstrated here for cN1A,the protocol can be adapted for other autoantigens to support studies of antigen-specific B cells in autoimmune diseases. View Publication

We describe humans with rare biallelic loss-of-function PTCRA variants impairing pre-TCRα expression. Low circulating naïve αβ T cell counts at birth persisted over time,with normal memory αβ and high γδ T cell counts. Their TCRα repertoire was biased,suggesting that noncanonical thymic differentiation pathways can rescue αβ T cell development. Only a minority of these individuals were sick,with infection,lymphoproliferation,and/or autoimmunity. We also report that 1 in 4000 individuals from the Middle East and South Asia are homozygous for a common hypomorphic PTCRA variant. They had normal circulating naïve αβ T cell counts but high γδ T cell counts. Although residual pre-TCRα expression drove the differentiation of more αβ T cells,autoimmune conditions were more frequent in these patients than in the general population. View Publication

Expansion of the glutamine tract (poly-Q) in the protein huntingtin (HTT) causes the neurodegenerative disorder Huntington’s disease (HD). Emerging evidence suggests that mutant HTT (mHTT) disrupts brain development. To gain mechanistic insights into the neurodevelopmental impact of human mHTT,we engineered male induced pluripotent stem cells to introduce a biallelic or monoallelic mutant 70Q expansion or to remove the poly-Q tract of HTT. The introduction of a 70Q mutation caused aberrant development of cerebral organoids with loss of neural progenitor organization. The early neurodevelopmental signature of mHTT highlighted the dysregulation of the protein coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2),a transcription factor involved in mitochondrial integrated stress response. CHCHD2 repression was associated with abnormal mitochondrial morpho-dynamics that was reverted upon overexpression of CHCHD2. Removing the poly-Q tract from HTT normalized CHCHD2 levels and corrected key mitochondrial defects. Hence,mHTT-mediated disruption of human neurodevelopment is paralleled by aberrant neurometabolic programming mediated by dysregulation of CHCHD2,which could then serve as an early interventional target for HD. Subject terms: Huntington's disease,Mechanisms of disease,Stem cells View Publication

If iPS cells can be established easily and efficiently using freshly collected blood cells,it will enhance regenerative and personalized medicine. While reports of iPS derivation from blood-derived endothelial progenitor cells using RNA have been documented,none have been reported from peripheral blood-derived mononuclear cells (PBMCs). In this study,we established a method to generate iPS cells from PBMCs using synthetic RNAs and found that MDM4,which suppresses p53,improved reprogramming efficiency. Subject terms: Reprogramming,Induced pluripotent stem cells View Publication

Blood oxygen saturation (SaO2) is one of the most important environmental factors in clinical heart protection. This study used human heart samples and human induced pluripotent stem cell-cardiomyocytes (iPSC-CMs) to assess how SaO2 affects human CM cell cycle activities. The results showed that there were significantly more cell cycle markers in the moderate hypoxia group (SaO2: 75{\%} to 85{\%}) than in the other 2 groups (SaO2 {\textless}75{\%} or {\textgreater}85{\%}). In iPSC-CMs 15{\%} and 10{\%} oxygen (O2) treatment increased cell cycle markers,whereas 5{\%} and rapid change of O2 decreased the markers. Moderate hypoxia is beneficial to the cell cycle activities of post-natal human CMs. View Publication