20 research outputs found

    Subtle changes in chromatin loop contact propensity are associated with differential gene regulation and expression.

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    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.

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    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

    Ultra-Sharp Nanowire Arrays Natively Permeate, Record, and Stimulate Intracellular Activity in Neuronal and Cardiac Networks

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    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.

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    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

    In vitro Differentiation of Human iPSC-derived Cardiovascular Progenitor Cells (iPSC-CVPCs).

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    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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