scholarly journals Chromatin compartment dynamics in a haploinsufficient model of cardiac laminopathy

2019 ◽  
Author(s):  
Alessandro Bertero ◽  
Paul A. Fields ◽  
Alec S. T. Smith ◽  
Andrea Leonard ◽  
Kevin Beussman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Large Scale ◽  
Induced Pluripotent Stem Cell ◽  
Lamin A ◽  
Chromosome Conformation ◽  
Human Cardiomyocytes ◽  
Nuclear Lamins ◽  
Genome Wide ◽  
Induced Pluripotent ◽  
Gene Expression Alterations

AbstractPathogenic mutations in A-type nuclear lamins cause dilated cardiomyopathy, which is postulated to result from dysregulated gene expression due to changes in chromatin organization into active and inactive compartments. To test this, we performed genome-wide chromosome conformation analyses (Hi-C) in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with a haploinsufficient mutation for lamin A/C. Compared to gene-corrected cells, mutant hiPSC-CMs have marked electrophysiological and contractile alterations, with modest gene expression changes. While large-scale changes in chromosomal topology are evident, differences in chromatin compartmentalization are limited to a few hotspots that escape inactivation during cardiogenesis. These regions exhibit upregulation of multiple non-cardiac genes including CACNA1A, encoding for neuronal P/Q-type calcium channels. Pharmacological inhibition of the resulting current partially mitigates the electrical alterations. On the other hand, A/B compartment changes do not explain most gene expression alterations in mutant hiPSC-CMs. We conclude that global errors in chromosomal compartmentation are not the primary pathogenic mechanism in heart failure due to lamin A/C haploinsufficiency.SummaryBertero et al. observe that lamin A/C haploinsufficiency in human cardiomyocytes markedly alters electrophysiology, contractility, gene expression, and chromosomal topology. Contrary to expectations, however, changes in chromatin compartments involve just few regions, and most dysregulated genes lie outside these hotspots.Condensed titleGenomic effects of lamin A/C haploinsufficiency

scholarly journals Chromatin compartment dynamics in a haploinsufficient model of cardiac laminopathy

2019 ◽  
Vol 218 (9) ◽  
pp. 2919-2944 ◽  
Author(s):  
Alessandro Bertero ◽  
Paul A. Fields ◽  
Alec S.T. Smith ◽  
Andrea Leonard ◽  
Kevin Beussman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Large Scale ◽  
Induced Pluripotent Stem Cell ◽  
Nuclear Lamina ◽  
Lamin A ◽  
Chromosome Conformation ◽  
Nuclear Lamins ◽  
Genome Wide ◽  
Induced Pluripotent ◽  
Gene Expression Alterations

Mutations in A-type nuclear lamins cause dilated cardiomyopathy, which is postulated to result from dysregulated gene expression due to changes in chromatin organization into active and inactive compartments. To test this, we performed genome-wide chromosome conformation analyses in human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) with a haploinsufficient mutation for lamin A/C. Compared with gene-corrected cells, mutant hiPSC-CMs have marked electrophysiological and contractile alterations, with modest gene expression changes. While large-scale changes in chromosomal topology are evident, differences in chromatin compartmentalization are limited to a few hotspots that escape segregation to the nuclear lamina and inactivation during cardiogenesis. These regions exhibit up-regulation of multiple noncardiac genes including CACNA1A, encoding for neuronal P/Q-type calcium channels. Pharmacological inhibition of the resulting current partially mitigates the electrical alterations. However, chromatin compartment changes do not explain most gene expression alterations in mutant hiPSC-CMs. Thus, global errors in chromosomal compartmentation are not the primary pathogenic mechanism in heart failure due to lamin A/C haploinsufficiency.

scholarly journals Genome-wide molecular effects of the neuropsychiatric 16p11 CNVs in an iPSC-to-iN neuronal model

2020 ◽  
Author(s):  
Thomas R. Ward ◽  
Xianglong Zhang ◽  
Louis C. Leung ◽  
Bo Zhou ◽  
Kristin Muench ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Cell Model ◽  
Copy Number Variants ◽  
Induced Pluripotent Stem Cell ◽  
Neuronal Model ◽  
Autism Spectrum ◽  
Genome Wide ◽  
Induced Pluripotent ◽  
Methylation Patterns

AbstractCopy number variants (CNVs), either deletions or duplications, at the 16p11.2 locus in the human genome are known to increase the risk for autism spectrum disorders (ASD), schizophrenia, and for several other developmental conditions. Here, we investigate the global effects on gene expression and DNA methylation using a 16p11.2 CNV patient-derived induced pluripotent stem cell (iPSC) to induced neuron (iN) cell model system. This approach revealed genome-wide and cell-type specific alterations to both gene expression and DNA methylation patterns and also yielded specific leads on genes potentially contributing to some of the known 16p11.2 patient phenotypes. PCSK9 is identified as a possible contributing factor to the symptoms seen in carriers of the 16p11.2 CNVs. The protocadherin (PCDH) gene family is found to have altered DNA methylation patterns in the CNV patient samples. The iPSC lines used for this study are available through a repository as a resource for research into the molecular etiology of the clinical phenotypes of 16p11.2 CNVs and into that of neuropsychiatric and neurodevelopmental disorders in general.

Abstract 111: Conjoining Gene Expression And Cell Organization With Imaging To Explore Cell States In Hipsc-derived Cardiomyocytes

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Ruwanthi Gunawardane
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Structural Organization ◽  
Large Scale ◽  
Single Cells ◽  
Induced Pluripotent Stem Cell ◽  
Mrna Abundance ◽  
Automated Classification ◽  
Subcellular Organization ◽  
Induced Pluripotent

The Allen Institute for Cell Science is developing a state space of stem cell structural signatures to study changes in cellular organization of human induced pluripotent stem cells (hiPSCs) and other cell states through differentiation. Towards this goal, we have used CRISPR/Cas9 to generate a collection of ~50 endogenous fluorescently tagged hiPSC lines (www.allencell.org), each expressing a monoallelic EGFP-tagged protein that localizes to a particular cellular structure or organelle. In this study, we focuson hiPSC-derived cardiomyocytes and compare the relationship between sarcomeric structural organization and gene expression signatures at large scale. We developed several tools and novel quantitative approaches to achieve this: 1) scarless GFP-tagging of cardiac genes such as ACTN2 to study the organization and morphogenesis of the contractile apparatus; 2) a robust protocol for differentiation of hiPSCs into cardiomyocytes and methods for preparing cells for imaging; and 3) a quantitative, image-based platform for the systematic and automated classification of subcellular organization in single cells. We use these approaches to quantify subcellular organization and gene expression in >30,000 individual human induced pluripotent stem cell-derived cardiomyocytes, producing a publicly available dataset that describes the population distributions of local and global sarcomere organization, mRNA abundance, and correlations between these traits. While the mRNA abundance of some phenotypically important genes correlates with subcellular organization (e.g., MYH7), these two cellular metrics are heterogeneous and often uncorrelated, which suggests that geneexpression alone is not sufficient to classify cell states. Instead, we posit thatcell state should be defined by observing full distributions of quantitative, multidimensional traits in single cells that also account for space, time, and function. This platform provides a multidimensional approach to classify hiPSC-derived cardiomyocytes based on structural organization and gene expression in single cells.

scholarly journals Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells Provide Insights on Parenteral Nutrition Associated Cholestasis in the Immature Liver

2021 ◽  
Author(s):  
T. Hang Nghiem-Rao ◽  
Courtney Pfeifer ◽  
Michelle Asuncion ◽  
Joshua Nord ◽  
Daniel Schill ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Bile Acid ◽  
Parenteral Nutrition ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Computational Techniques ◽  
Adult Liver ◽  
Induced Pluripotent ◽  
The Impact

Abstract Parenteral nutrition-associated cholestasis (PNAC) significantly limits the safety of intravenous parenteral nutrition (PN). Critically ill infants are highly vulnerable to PNAC-related morbidity and mortality, however the impact of hepatic immaturity on PNAC is poorly understood. We examined developmental differences between fetal/infant and adult livers, and used human induced pluripotent stem cell-derived hepatocyte-like cells (iHLC) to gain insights into the contribution of development to altered sterol metabolism and PNAC. We used RNA-sequencing and computational techniques to compare gene expression patterns in human fetal/infant livers, adult liver, and iHLC. We identified distinct gene expression profiles between the human feta/infant livers compared to adult liver, and close resemblance of iHLC to human developing livers. Compared to adult, both developing livers and iHLC had significant downregulation of xenobiotic, bile acid, and fatty acid metabolism; and lower expression of the sterol metabolizing gene ABCG8. When challenged with stigmasterol, a plant sterol found in intravenous soy lipids, lipid accumulation was significantly higher in iHLC compared to adult-derived HepG2 cells. Our findings provide insights into altered bile acid and lipid metabolizing processes in the immature human liver, and support the use of iHLC as a relevant model system of developing liver to study lipid metabolism and PNAC.

scholarly journals Batch effects and the effective design of single-cell gene expression studies

2016 ◽  
Author(s):  
Po-Yuan Tung ◽  
John D. Blischak ◽  
Chiaowen Joyce Hsiao ◽  
David A. Knowles ◽  
Jonathan E. Burnett ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Induced Pluripotent Stem Cell ◽  
Expression Levels ◽  
Amplification Bias ◽  
Expression Studies ◽  
Cell Gene Expression ◽  
Gene Expression Studies ◽  
Induced Pluripotent ◽  
Gene Expression Levels

AbstractSingle cell RNA sequencing (scRNA-seq) can be used to characterize variation in gene expression levels at high resolution. However, the sources of experimental noise in scRNA-seq are not yet well understood. We investigated the technical variation associated with sample processing using the single cell Fluidigm C1 platform. To do so, we processed three C1 replicates from three human induced pluripotent stem cell (iPSC) lines. We added unique molecular identifiers (UMIs) to all samples, to account for amplification bias. We found that the major source of variation in the gene expression data was driven by genotype, but we also observed substantial variation between the technical replicates. We observed that the conversion of reads to molecules using the UMIs was impacted by both biological and technical variation, indicating that UMI counts are not an unbiased estimator of gene expression levels. Based on our results, we suggest a framework for effective scRNA-seq studies.

scholarly journals Ion Channel Expression and Electrophysiology of Singular Human (Primary and Induced Pluripotent Stem Cell-Derived) Cardiomyocytes

Cells ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 3370
Author(s):  
Christina Schmid ◽  
Najah Abi-Gerges ◽  
Michael Georg Leitner ◽  
Dietmar Zellner ◽  
Georg Rast
Keyword(s):  
Stem Cell ◽  
Single Cell ◽  
Pluripotent Stem Cell ◽  
Quantitative Polymerase Chain Reaction ◽  
Drug Effects ◽  
Induced Pluripotent Stem Cell ◽  
Ionic Currents ◽  
Human Cardiomyocytes ◽  
Ventricular Cells ◽  
Induced Pluripotent

Subtype-specific human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are promising tools, e.g., to assess the potential of drugs to cause chronotropic effects (nodal hiPSC-CMs), atrial fibrillation (atrial hiPSC-CMs), or ventricular arrhythmias (ventricular hiPSC-CMs). We used single-cell patch-clamp reverse transcriptase-quantitative polymerase chain reaction to clarify the composition of the iCell cardiomyocyte population (Fujifilm Cellular Dynamics, Madison, WI, USA) and to compare it with atrial and ventricular Pluricytes (Ncardia, Charleroi, Belgium) and primary human atrial and ventricular cardiomyocytes. The comparison of beating and non-beating iCell cardiomyocytes did not support the presence of true nodal, atrial, and ventricular cells in this hiPSC-CM population. The comparison of atrial and ventricular Pluricytes with primary human cardiomyocytes showed trends, indicating the potential to derive more subtype-specific hiPSC-CM models using appropriate differentiation protocols. Nevertheless, the single-cell phenotypes of the majority of the hiPSC-CMs showed a combination of attributes which may be interpreted as a mixture of traits of adult cardiomyocyte subtypes: (i) nodal: spontaneous action potentials and high HCN4 expression and (ii) non-nodal: prominent INa-driven fast inward current and high expression of SCN5A. This may hamper the interpretation of the drug effects on parameters depending on a combination of ionic currents, such as beat rate. However, the proven expression of specific ion channels supports the evaluation of the drug effects on ionic currents in a more realistic cardiomyocyte environment than in recombinant non-cardiomyocyte systems.

scholarly journals Histone H3K9 methylation promotes formation of genome compartments inCaenorhabditis elegansvia chromosome compaction and perinuclear anchoring

2020 ◽  
Vol 117 (21) ◽  
pp. 11459-11470 ◽  
Author(s):  
Qian Bian ◽  
Erika C. Anderson ◽  
Qiming Yang ◽  
Barbara J. Meyer
Keyword(s):  
Gene Expression ◽  
Nuclear Periphery ◽  
Chromosome Conformation ◽  
C Elegans ◽  
Genome Wide ◽  
Central Regions ◽  
In Trans ◽  
Genomic Regions ◽  
In Cis ◽  
Gene Expression Levels

Genomic regions preferentially associate with regions of similar transcriptional activity, partitioning genomes into active and inactive compartments within the nucleus. Here we explore mechanisms controlling genome compartment organization inCaenorhabditis elegansand investigate roles for compartments in regulating gene expression. Distal arms ofC. eleganschromosomes, which are enriched for heterochromatic histone modifications H3K9me1/me2/me3, interact with each other bothin cisandin trans,while interacting less frequently with central regions, leading to genome compartmentalization. Arms are anchored to the nuclear periphery via the nuclear envelope protein CEC-4, which binds to H3K9me. By performing genome-wide chromosome conformation capture experiments (Hi-C), we showed that eliminating H3K9me1/me2/me3 through mutations in the methyltransferase genesmet-2andset-25significantly impaired formation of inactive Arm and active Center compartments.cec-4mutations also impaired compartmentalization, but to a lesser extent. We found that H3K9me promotes compartmentalization through two distinct mechanisms: Perinuclear anchoring of chromosome arms via CEC-4 to promote theircisassociation, and an anchoring-independent mechanism that compacts individual chromosome arms. In bothmet-2 set-25andcec-4mutants, no dramatic changes in gene expression were found for genes that switched compartments or for genes that remained in their original compartment, suggesting that compartment strength does not dictate gene-expression levels. Furthermore, H3K9me, but not perinuclear anchoring, also contributes to formation of another prominent feature of chromosome organization, megabase-scale topologically associating domains on X established by the dosage compensation condensin complex. Our results demonstrate that H3K9me plays crucial roles in regulating genome organization at multiple levels.

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