scholarly journals Dynamic effects of genetic variation on gene expression revealed following hypoxic stress in cardiomyocytes

2020 ◽  
Author(s):  
Michelle C. Ward ◽  
Nicholas E. Banovich ◽  
Abhishek Sarkar ◽  
Matthew Stephens ◽  
Yoav Gilad
Keyword(s):  
Gene Expression ◽  
Complex Traits ◽  
Induced Pluripotent Stem Cell ◽  
Chromatin Accessibility ◽  
Regulatory Changes ◽  
Life Threatening ◽  
Culturing Conditions ◽  
Induced Pluripotent

AbstractOne life-threatening outcome of cardiovascular disease is myocardial infarction, where cardiomyocytes are deprived of oxygen. To study inter-individual differences in response to hypoxia, we established an in vitro model of induced pluripotent stem cell-derived cardiomyocytes from 15 individuals. We measured gene expression levels, chromatin accessibility, and methylation levels in four culturing conditions that correspond to normoxia, hypoxia and short or long-term re-oxygenation. We characterized thousands of gene regulatory changes as the cells transition between conditions. Using available genotypes, we identified 1,573 genes with a cis expression quantitative locus (eQTL) in at least one condition, as well as 367 dynamic eQTLs, which are classified as eQTLs in at least one, but not in all conditions. A subset of genes with dynamic eQTLs is associated with complex traits and disease. Our data demonstrate how dynamic genetic effects on gene expression, which are likely relevant for disease, can be uncovered under stress.

scholarly journals Dynamic effects of genetic variation on gene expression revealed following hypoxic stress in cardiomyocytes

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Michelle C Ward ◽  
Nicholas E Banovich ◽  
Abhishek Sarkar ◽  
Matthew Stephens ◽  
Yoav Gilad
Keyword(s):  
Gene Expression ◽  
Complex Traits ◽  
Induced Pluripotent Stem Cell ◽  
Chromatin Accessibility ◽  
Regulatory Changes ◽  
Life Threatening ◽  
Vitro Model ◽  
Culturing Conditions ◽  
Induced Pluripotent

One life-threatening outcome of cardiovascular disease is myocardial infarction, where cardiomyocytes are deprived of oxygen. To study inter-individual differences in response to hypoxia, we established an in vitro model of induced pluripotent stem cell-derived cardiomyocytes from 15 individuals. We measured gene expression levels, chromatin accessibility, and methylation levels in four culturing conditions that correspond to normoxia, hypoxia, and short- or long-term re-oxygenation. We characterized thousands of gene regulatory changes as the cells transition between conditions. Using available genotypes, we identified 1,573 genes with a cis expression quantitative locus (eQTL) in at least one condition, as well as 367 dynamic eQTLs, which are classified as eQTLs in at least one, but not in all conditions. A subset of genes with dynamic eQTLs is associated with complex traits and disease. Our data demonstrate how dynamic genetic effects on gene expression, which are likely relevant for disease, can be uncovered under stress.

scholarly journals Application of Human Induced Pluripotent Stem Cell-Derived Cellular and Organoid Models for COVID-19 Research

2021 ◽  
Vol 9 ◽  
Author(s):  
Yumei Luo ◽  
Mimi Zhang ◽  
Yapei Chen ◽  
Yaoyong Chen ◽  
Detu Zhu
Keyword(s):  
Induced Pluripotent Stem Cell ◽  
Cell Types ◽  
Life Cycles ◽  
Global Public Health ◽  
Health Crisis ◽  
Infection Mechanisms ◽  
Induced Pluripotent ◽  
The Brain

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its rapid international spread has caused the coronavirus disease 2019 (COVID-19) pandemics, which is a global public health crisis. Thus, there is an urgent need to establish biological models to study the pathology of SARS-CoV-2 infection, which not only involves respiratory failure, but also includes dysregulation of other organs and systems, including the brain, heart, liver, intestines, pancreas, kidneys, eyes, and so on. Cellular and organoid models derived from human induced pluripotent stem cells (iPSCs) are ideal tools for in vitro simulation of viral life cycles and drug screening to prevent the reemergence of coronavirus. These iPSC-derived models could recapitulate the functions and physiology of various human cell types and assemble the complex microenvironments similar with those in the human organs; therefore, they can improve the study efficiency of viral infection mechanisms, mimic the natural host-virus interaction, and be suited for long-term experiments. In this review, we focus on the application of in vitro iPSC-derived cellular and organoid models in COVID-19 studies.

scholarly journals Development of Physiologically Responsive Human iPSC-Derived Intestinal Epithelium to Study Barrier Dysfunction in IBD

2020 ◽  
Vol 21 (4) ◽  
pp. 1438 ◽  
Author(s):  
John P. Gleeson ◽  
Hannah Q. Estrada ◽  
Michifumi Yamashita ◽  
Clive N. Svendsen ◽  
Stephan R. Targan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Intestinal Permeability ◽  
Intestinal Epithelium ◽  
Adherens Junction ◽  
Induced Pluripotent Stem Cell ◽  
Barrier Dysfunction ◽  
Junction Protein ◽  
Vitro Model ◽  
Induced Pluripotent

In inflammatory bowel disease (IBD), the intestinal epithelium is characterized by increased permeability both in active disease and remission states. The genetic underpinnings of this increased intestinal permeability are largely unstudied, in part due to a lack of appropriate modelling systems. Our aim is to develop an in vitro model of intestinal permeability using induced pluripotent stem cell (iPSC)-derived human intestinal organoids (HIOs) and human colonic organoids (HCOs) to study barrier dysfunction. iPSCs were generated from healthy controls, adult onset IBD, and very early onset IBD (VEO-IBD) patients and differentiated into HIOs and HCOs. EpCAM+ selected cells were seeded onto Transwell inserts and barrier integrity studies were carried out in the presence or absence of pro-inflammatory cytokines TNFα and IFNγ. Quantitative real-time PCR (qRT-PCR), transmission electron microscopy (TEM), and immunofluorescence were used to determine altered tight and adherens junction protein expression or localization. Differentiation to HCO indicated an increased gene expression of CDX2, CD147, and CA2, and increased basal transepithelial electrical resistance compared to HIO. Permeability studies were carried out in HIO- and HCO-derived epithelium, and permeability of FD4 was significantly increased when exposed to TNFα and IFNγ. TEM and immunofluorescence imaging indicated a mislocalization of E-cadherin and ZO-1 in TNFα and IFNγ challenged organoids with a corresponding decrease in mRNA expression. Comparisons between HIO- and HCO-epithelium show a difference in gene expression, electrophysiology, and morphology: both are responsive to TNFα and IFNγ stimulation resulting in enhanced permeability, and changes in tight and adherens junction architecture. This data indicate that iPSC-derived HIOs and HCOs constitute an appropriate physiologically responsive model to study barrier dysfunction and the role of the epithelium in IBD and VEO-IBD.

scholarly journals Functional dynamic genetic effects on gene regulation are specific to particular cell types and environmental conditions

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Anthony S Findley ◽  
Alan Monziani ◽  
Allison L Richards ◽  
Katherine Rhodes ◽  
Michelle C Ward ◽  
...  
Keyword(s):  
Gene Expression ◽  
Complex Traits ◽  
Induced Pluripotent Stem Cell ◽  
Cell Types ◽  
Cell System ◽  
Genetic Effects ◽  
Evolutionary Features ◽  
Multiple Cell ◽  
Multiple Treatments ◽  
Induced Pluripotent

Genetic effects on gene expression and splicing can be modulated by cellular and environmental factors; yet interactions between genotypes, cell type and treatment have not been comprehensively studied together. We used an induced pluripotent stem cell system to study multiple cell types derived from the same individuals and exposed them to a large panel of treatments. Cellular responses involved different genes and pathways for gene expression and splicing, and were highly variable across contexts. For thousands of genes, we identified variable allelic expression across contexts and characterized different types of gene-environment interactions, many of which are associated with complex traits. Promoter functional and evolutionary features distinguished genes with elevated allelic imbalance mean and variance. On average half of the genes with dynamic regulatory interactions were missed by large eQTL mapping studies, indicating the importance of exploring multiple treatments to reveal previously unrecognized regulatory loci that may be important for disease.

scholarly journals Cultivation and characterization of human midbrain organoids in sensor integrated microfluidic chips

2019 ◽  
Author(s):  
Sarah Spitz ◽  
Cristian Zanetti ◽  
Silvia Bolognin ◽  
Mudiwa Nathasia Muwanigwa ◽  
Lisa Smits ◽  
...  
Keyword(s):  
Induced Pluripotent Stem Cell ◽  
Microfluidic Chips ◽  
Interstitial Flow ◽  
Precise Control ◽  
Non Invasive ◽  
Dynamic Cultivation ◽  
Induced Pluripotent

1.ABSTRACTWith its ability to emulate microarchitectures and functional characteristics of native organs in vitro, induced pluripotent stem cell (iPSC) technology has enabled the generation of a plethora of organotypic constructs, including that of the human midbrain. However, reproducibly engineering and differentiating such human midbrain organoids (hMOs) under a biomimetic environment favorable for brain development still remains challenging. This study sets out to address this problem by combining the potential of iPSC technology with the advantages of microfluidics, namely its precise control over fluid flow combined with sensor integration. Here, we present a novel sensor-integrated platform for the long-term cultivation and non-invasive monitoring of hMOs under an interstitial flow regime. Our results show that dynamic cultivation of iPSC-derived hMOs maintains high cellular viabilities and dopaminergic neuron differentiation over prolonged cultivation periods of up to 50 days.

scholarly journals Induction of Pluripotent Stem Cell-Derived Cardiomyocyte Toxicity by Supernatant of Long Term-Stored Red Blood Cells in Vitro

2018 ◽  
Vol 46 (3) ◽  
pp. 1230-1240 ◽  
Author(s):  
Fengyan Fan ◽  
Yang Yu ◽  
Liping Sun ◽  
Shufang Wang ◽  
Rui Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Gene Expression Array ◽  
Expression Array

Background/Aims: Preserved red blood cells (RBCs) in vitro undergo a series of morphological, functional and metabolic changes during storage. RBC metabolites accumulate over time during storage, the toxicity of the supernatants of RBCs (SSRBCs) on tissue cells is largely unknown. Here, we aimed to study cardiomyocyte toxicity by supernatant of long term-stored RBCs in vitro and to discover elements involved in the mechanism. Methods: Using human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) and real-time cell analyzing (RTCA), we analyzed the cardiotoxicity of d0, d14 and d35 SSRBCs. To analyze the cardiotoxicity of potassium (K) and lactic acid (LA) in SSRBCs, solutions containing the same concentrations of K and LA were respectively prepared and co-cultured with hiPS-CMs. Immunofluorescence and Gene Expression Array of hiPS-CMs were performed to evaluate the effects of d35 K and d35 SSRBCs. Results: The beating of hiPS-CM was stopped by d14, d35 SSRBCs, or d35 K solution. Beating resumed within 48 hours in the presence of d14 SSRBC or d35 K but not d35 SSRBC; d0, d14 and d35 LA solution had no effect on beating patterns. At 48h after treatment, the immunofluorescence results showed that the integrity of the filament and sarcomere were intact. Gene Expression Array results found 14 differentially expressed genes which were likely to play an important role in the cytotoxic effect. Conclusion: Our results demonstrated cardiomyocyte toxicity by long term-stored SSRBCs in vitro. Besides high K-induced cardiotoxicity, there must be other unknown components in long term-stored SSRBCs that are cytotoxic to hiPS-CMs.

scholarly journals Comprehensive preclinical evaluation of how cardiac safety profiles of potential COVID-19 drugs are modified by disease associated factors.

2021 ◽  
Author(s):  
Clifford TeBay ◽  
Jeffrey McArthur ◽  
Melissa Mangala ◽  
Nicholas Kerr ◽  
STEWART Heitmann ◽  
...  
Keyword(s):  
Experimental Approach ◽  
Induced Pluripotent Stem Cell ◽  
Diverse Populations ◽  
Cardiac Safety ◽  
Preclinical Evaluation ◽  
Cardiac Repolarisation ◽  
Proarrhythmic Risk ◽  
Induced Pluripotent

Background and Purpose: Hydroxychloroquine and chloroquine, alone or in combination with azithromycin, have been proposed as therapies for COVID-19. However, there is currently scant and inconsistent data regarding their proarrhythmic potential in these patients. Moreover, their risk profile in the setting of altered physiological states encountered in patients with COVID-19 (i.e. febrile state, electrolyte imbalances, and/or acidosis) is unknown. Experimental approach: Potency of hERG block was measured using high-throughput electrophysiology in the presence of variable environmental factors. These potencies informed simulations to predict population risk profiles. Effects on cardiac repolarisation were verified in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) from three separate individuals. Key Results: Chloroquine and hydroxychloroquine blocked hERG with IC50 of 1.47±0.07 µM and 3.78±0.17 µM respectively, indicating proarrhythmic risk at concentrations effective against SARS-CoV-2 in vitro and proposed in COVID-19 clinical trials. Hypokalaemia and hypermagnesemia increased potency of chloroquine and hydroxychloroquine, indicating increased proarrhythmic risk. Acidosis significantly reduced potency of all drugs (i.e. reduced proarrhythmic risk), whereas increased temperature decreased potency of chloroquine and hydroxychloroquine but increased potency for azithromycin. In silico simulations across genetically diverse populations predicted that 17% of individuals exhibit action potential durations >500 ms at the highest proposed therapeutic levels, equating to significant QT prolongation. Conclusion and Implications: Significant proarrhythmic risk is predicted for hydroxychloroquine and chloroquine at doses proposed to treat COVID-19. Clinicians should carefully consider the risk of such treatments, and implement long term QT interval monitoring in trials, particularly in patients with electrolyte imbalances.

scholarly journals Spatiotemporal mosaic patterning of pluripotent stem cells using CRISPR interference

2018 ◽  
Author(s):  
Ashley R.G. Libby ◽  
David A. Joy ◽  
Po-Lin So ◽  
Mohammad A. Mandegar ◽  
Jonathon M. Muncie ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Symmetry Breaking ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Cell Populations ◽  
Form Complex ◽  
Induced Pluripotent ◽  
Cell Cell

ABSTRACTMorphogenesis results from the interactions of asymmetric cell populations to form complex multicellular patterns and structures comprised of distinct cell types. However, current methods to model morphogenic events offer little control over parallel cell type co-emergence and do not offer the capability to selectively perturb gene expression in specific subpopulations of cells. We have developed an in vitro system that can spatiotemporally interrogate cell-cell interactions and multicellular organization within human induced pluripotent stem cell (hiPSC) colonies. We examined the effects of independently knocking down molecular regulators of cortical tension and cell-cell adhesion using inducible CRISPRi: Rho-associated kinase-1 (ROCK1) and E-cadherin (CDH1), respectively. Induced mosaic knockdown of ROCK1 or CDH1 in hiPSC populations resulted in differential patterning events within hiPSC colonies indicative of cell-driven population organization. Patterned colonies retained an epithelial phenotype and nuclear expression of pluripotency markers. Gene expression within each of the mixed populations displayed a transient wave of differential expression with induction of knockdown that stabilized in coordination with intrinsic pattern formation. Mosaic patterning of hiPSCs enables the genetic interrogation of emergent multicellular properties of pluripotent cells, leading to a greater mechanistic understanding of the specific molecular pathways regulating the dynamics of symmetry breaking events that transpire during developmental morphogenesis.SIGNIFICANCEHuman embryonic development entails a series of multicellular morphogenic events that lead to primitive tissue formation. Attempts to study human morphogenic processes experimentally have been limited due to divergence from model organisms and the inability of current human in vitro models to accurately control the coincident emergence of heterogeneous cell populations in the spatially controlled manner necessary for proper tissue structure. We developed a human induced pluripotent stem cell (iPSC) in vitro model that enables temporal control over the emergence of heterotypic subpopulations of cells. We examined mosaic knockdown of two target molecules to create predictable and robust cell-patterning events within hiPSC colonies. This method allows for dynamic interrogation of intrinsic cell mechanisms that initiate symmetry breaking events and provides direct insight(s) into tissue developmental principles.

scholarly journals Applicability of hiPSC-Derived Neuronal Cocultures and Rodent Primary Cortical Cultures for In Vitro Seizure Liability Assessment

2020 ◽  
Vol 178 (1) ◽  
pp. 71-87
Author(s):  
Anke M Tukker ◽  
Fiona M J Wijnolts ◽  
Aart de Groot ◽  
Remco H S Westerink
Keyword(s):  
Microelectrode Array ◽  
Activity Patterns ◽  
Primary Cultures ◽  
Induced Pluripotent Stem Cell ◽  
Network Activity ◽  
Neuronal Network Activity ◽  
Cortical Cultures ◽  
Life Threatening ◽  
Induced Pluripotent

Abstract Seizures are life-threatening adverse drug reactions which are investigated late in drug development using rodent models. Consequently, if seizures are detected, a lot of time, money and animals have been used. Thus, there is a need for in vitro screening models using human cells to circumvent interspecies translation. We assessed the suitability of cocultures of human-induced pluripotent stem cell (hiPSC)-derived neurons and astrocytes compared with rodent primary cortical cultures for in vitro seizure liability assessment using microelectrode arrays. hiPSC-derived and rodent primary cortical neuronal cocultures were exposed to 9 known (non)seizurogenic compounds (pentylenetetrazole, amoxapine, enoxacin, amoxicillin, linopirdine, pilocarpine, chlorpromazine, phenytoin, and acetaminophen) to assess effects on neuronal network activity using microelectrode array recordings. All compounds affect activity in hiPSC-derived cocultures. In rodent primary cultures all compounds, except amoxicillin changed activity. Changes in activity patterns for both cell models differ for different classes of compounds. Both models had a comparable sensitivity for exposure to amoxapine (lowest observed effect concentration [LOEC] 0.03 µM), linopirdine (LOEC 1 µM), and pilocarpine (LOEC 0.3 µM). However, hiPSC-derived cultures were about 3 times more sensitive for exposure to pentylenetetrazole (LOEC 30 µM) than rodent primary cortical cultures (LOEC 100 µM). Sensitivity of hiPSC-derived cultures for chlorpromazine, phenytoin, and enoxacin was 10-30 times higher (LOECs 0.1, 0.3, and 0.1 µM, respectively) than in rodent cultures (LOECs 10, 3, and 3 µM, respectively). Our data indicate that hiPSC-derived neuronal cocultures may outperform rodent primary cortical cultures with respect to detecting seizures, thereby paving the way towards animal-free seizure assessment.

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