20 research outputs found
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Cellular deconvolution of GTEx tissues powers discovery of disease and cell-type associated regulatory variants.
The Genotype-Tissue Expression (GTEx) resource has provided insights into the regulatory impact of genetic variation on gene expression across human tissues; however, thus far has not considered how variation acts at the resolution of the different cell types. Here, using gene expression signatures obtained from mouse cell types, we deconvolute bulk RNA-seq samples from 28 GTEx tissues to quantify cellular composition, which reveals striking heterogeneity across these samples. Conducting eQTL analyses for GTEx liver and skin samples using cell composition estimates as interaction terms, we identify thousands of genetic associations that are cell-type-associated. The skin cell-type associated eQTLs colocalize with skin diseases, indicating that variants which influence gene expression in distinct skin cell types play important roles in traits and disease. Our study provides a framework to estimate the cellular composition of GTEx tissues enabling the functional characterization of human genetic variation that impacts gene expression in cell-type-specific manners
Subtle changes in chromatin loop contact propensity are associated with differential gene regulation and expression.
While genetic variation at chromatin loops is relevant for human disease, the relationships between contact propensity (the probability that loci at loops physically interact), genetics, and gene regulation are unclear. We quantitatively interrogate these relationships by comparing Hi-C and molecular phenotype data across cell types and haplotypes. While chromatin loops consistently form across different cell types, they have subtle quantitative differences in contact frequency that are associated with larger changes in gene expression and H3K27ac. For the vast majority of loci with quantitative differences in contact frequency across haplotypes, the changes in magnitude are smaller than those across cell types; however, the proportional relationships between contact propensity, gene expression, and H3K27ac are consistent. These findings suggest that subtle changes in contact propensity have a biologically meaningful role in gene regulation and could be a mechanism by which regulatory genetic variants in loop anchors mediate effects on expression
High-Throughput and Cost-Effective Characterization of Induced Pluripotent Stem Cells.
Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) offers the possibility of studying the molecular mechanisms underlying human diseases in cell types difficult to extract from living patients, such as neurons and cardiomyocytes. To date, studies have been published that use small panels of iPSC-derived cell lines to study monogenic diseases. However, to study complex diseases, where the genetic variation underlying the disorder is unknown, a sizable number of patient-specific iPSC lines and controls need to be generated. Currently the methods for deriving and characterizing iPSCs are time consuming, expensive, and, in some cases, descriptive but not quantitative. Here we set out to develop a set of simple methods that reduce cost and increase throughput in the characterization of iPSC lines. Specifically, we outline methods for high-throughput quantification of surface markers, gene expression analysis of in vitro differentiation potential, and evaluation of karyotype with markedly reduced cost
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Allele-specific NKX2-5 binding underlies multiple genetic associations with human electrocardiographic traits.
The cardiac transcription factor (TF) gene NKX2-5 has been associated with electrocardiographic (EKG) traits through genome-wide association studies (GWASs), but the extent to which differential binding of NKX2-5 at common regulatory variants contributes to these traits has not yet been studied. We analyzed transcriptomic and epigenomic data from induced pluripotent stem cell-derived cardiomyocytes from seven related individuals, and identified ~2,000 single-nucleotide variants associated with allele-specific effects (ASE-SNVs) on NKX2-5 binding. NKX2-5 ASE-SNVs were enriched for altered TF motifs, for heart-specific expression quantitative trait loci and for EKG GWAS signals. Using fine-mapping combined with epigenomic data from induced pluripotent stem cell-derived cardiomyocytes, we prioritized candidate causal variants for EKG traits, many of which were NKX2-5 ASE-SNVs. Experimentally characterizing two NKX2-5 ASE-SNVs (rs3807989 and rs590041) showed that they modulate the expression of target genes via differential protein binding in cardiac cells, indicating that they are functional variants underlying EKG GWAS signals. Our results show that differential NKX2-5 binding at numerous regulatory variants across the genome contributes to EKG phenotypes
Ultra-Sharp Nanowire Arrays Natively Permeate, Record, and Stimulate Intracellular Activity in Neuronal and Cardiac Networks
Intracellular access with high spatiotemporal resolution can enhance our
understanding of how neurons or cardiomyocytes regulate and orchestrate network
activity, and how this activity can be affected with pharmacology or other
interventional modalities. Nanoscale devices often employ electroporation to
transiently permeate the cell membrane and record intracellular potentials,
which tend to decrease rapidly to extracellular potential amplitudes with time.
Here, we report innovative scalable, vertical, ultra-sharp nanowire arrays that
are individually addressable to enable long-term, native recordings of
intracellular potentials. We report large action potential amplitudes that are
indicative of intracellular access from 3D tissue-like networks of neurons and
cardiomyocytes across recording days and that do not decrease to extracellular
amplitudes for the duration of the recording of several minutes. Our findings
are validated with cross-sectional microscopy, pharmacology, and electrical
interventions. Our experiments and simulations demonstrate that individual
electrical addressability of nanowires is necessary for high-fidelity
intracellular electrophysiological recordings. This study advances our
understanding of and control over high-quality multi-channel intracellular
recordings, and paves the way toward predictive, high-throughput, and low-cost
electrophysiological drug screening platforms.Comment: Main manuscript: 33 pages, 4 figures, Supporting information: 43
pages, 27 figures, Submitted to Advanced Material
iPSCORE: A Resource of 222 iPSC Lines Enabling Functional Characterization of Genetic Variation across a Variety of Cell Types.
Large-scale collections of induced pluripotent stem cells (iPSCs) could serve as powerful model systems for examining how genetic variation affects biology and disease. Here we describe the iPSCORE resource: a collection of systematically derived and characterized iPSC lines from 222 ethnically diverse individuals that allows for both familial and association-based genetic studies. iPSCORE lines are pluripotent with high genomic integrity (no or low numbers of somatic copy-number variants) as determined using high-throughput RNA-sequencing and genotyping arrays, respectively. Using iPSCs from a family of individuals, we show that iPSC-derived cardiomyocytes demonstrate gene expression patterns that cluster by genetic background, and can be used to examine variants associated with physiological and disease phenotypes. The iPSCORE collection contains representative individuals for risk and non-risk alleles for 95% of SNPs associated with human phenotypes through genome-wide association studies. Our study demonstrates the utility of iPSCORE for examining how genetic variants influence molecular and physiological traits in iPSCs and derived cell lines
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In vitro Differentiation of Human iPSC-derived Retinal Pigment Epithelium Cells (iPSC-RPE).
Induced Pluripotent Stem Cells (iPSCs) serve as an excellent model system for studying the molecular underpinnings of tissue development. Human iPSC-derived retinal pigment epithelium (iPSC-RPE) cells have fetal-like molecular profiles. Hence, biobanks like iPSCORE, which contain iPSCs generated from hundreds of individuals, are an invaluable resource for examining how common genetic variants exert their effects during RPE development resulting in individuals having different propensities to develop Age-related Macular Degeneration (AMD) as adults. Here, we present an optimized, cost-effective and highly reproducible protocol for derivation of human iPSC-RPE cells using small molecules under serum-free condition and for their quality control using flow cytometry and immunofluorescence. While most previous protocols have required laborious manual selection to enrich for iPSC-RPE cells, our protocol uses whole culture passaging and yields a large number of iPSC-RPE cells with high purity (88-98.1% ZO-1 and MiTF double positive cells). The simplicity and robustness of this protocol would enable its adaption for high-throughput applications involving the generation of iPSC-RPE samples from hundreds of individuals
In vitro Differentiation of Human iPSC-derived Cardiovascular Progenitor Cells (iPSC-CVPCs).
Induced pluripotent stem cell derived cardiovascular progenitor cells (iPSC-CVPCs) provide an unprecedented platform for examining the molecular underpinnings of cardiac development and disease etiology, but also have great potential to play pivotal roles in the future of regenerative medicine and pharmacogenomic studies. Biobanks like iPSCORE ( Stacey et al., 2013 ; Panopoulos et al., 2017 ), which contain iPSCs generated from hundreds of genetically and ethnically diverse individuals, are an invaluable resource for conducting these studies. Here, we present an optimized, cost-effective and highly standardized protocol for large-scale derivation of human iPSC-CVPCs using small molecules and purification using metabolic selection. We have successfully applied this protocol to derive iPSC-CVPCs from 154 different iPSCORE iPSC lines obtaining large quantities of highly pure cardiac cells. An important component of our protocol is Cell confluency estimates (ccEstimate), an automated methodology for estimating the time when an iPSC monolayer will reach 80% confluency, which is optimal for initiating iPSC-CVPC derivation, and enables the protocol to be readily used across iPSC lines with different growth rates. Moreover, we showed that cellular heterogeneity across iPSC-CVPCs is due to varying proportions of two distinct cardiac cell types: cardiomyocytes (CMs) and epicardium-derived cells (EPDCs), both of which have been shown to have a critical function in heart regeneration. This protocol eliminates the need of iPSC line-to-line optimization and can be easily adapted and scaled to high-throughput studies or to generate large quantities of cells suitable for regenerative medicine applications
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Author Correction: Cellular deconvolution of GTEx tissues powers discovery of disease and cell-type associated regulatory variants.
An amendment to this paper has been published and can be accessed via a link at the top of the paper