A simple, cost-effective but highly efficient system for deriving ventricular cardiomyocytes from human pluripotent stem cells

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Engineering micro-alignments of 2- and 3-D hESC-derived ventricular tissues to reproduce anisotropic properties of the native heart: an accurate arrhythmias model for cardiotoxicity screening

DMM 2011 entitled: Re-engineering Regenerative MedicinePoster Session - Heart Regeneration: no. 26In the native heart, ventricular CMs are aligned in a highly organized structured manner such that the conduction of electrical signals is anisotropic for directional and coordinated contractions to effectively pump blood. In other words, electrical conduction is asymmetrical (i.e. anisotropy) with distinct transverse and longitudinal velocities. Unlike the native ventricle, clusters of hESC-CMs differentiated using either the EB formation or directed differentiation are random structures with NO obvious organization and anisotropy as we previously published. Using a microgroove technology, here we engineered organized 2- and 3-D hESC-derived ventricular strips, followed by high-resolution optical mapping recordings …postprin

A comprehensive TALEN-based knockout library for generating human induced pluripotent stem cell-based models for cardiovascular diseases

Rationale: Targeted genetic engineering using programmable nucleases such as transcription activator-like effector nucleases (TALENs) is a valuable tool for precise, site-specific genetic modification in the human genome. Objective: The emergence of novel technologies such as human induced pluripotent stem cells (iPSCs) and nuclease-mediated genome editing represent a unique opportunity for studying cardiovascular diseases in vitro. Methods and Results: By incorporating extensive literature and database searches, we designed a collection of TALEN constructs to knockout (KO) eighty-eight human genes that are associated with cardiomyopathies and congenital heart diseases. The TALEN pairs were designed to induce double-strand DNA break near the starting codon of each gene that either disrupted the start codon or introduced a frameshift mutation in the early coding region, ensuring faithful gene KO. We observed that all the constructs were active and disrupted the target locus at high frequencies. To illustrate the general utility of the TALEN-mediated KO technique, six individual genes (TNNT2, LMNA/C, TBX5, MYH7, ANKRD1, and NKX2.5) were knocked out with high efficiency and specificity in human iPSCs. By selectively targeting a dilated cardiomyopathy (DCM)-causing mutation (TNNT2 p.R173W) in patient-specific iPSC-derived cardiac myocytes (iPSC-CMs), we demonstrated that the KO strategy ameliorates the DCM phenotype in vitro. In addition, we modeled the Holt-Oram syndrome (HOS) in iPSC-CMs in vitro and uncovered novel pathways regulated by TBX5 in human cardiac myocyte development. Conclusions: Collectively, our study illustrates the powerful combination of iPSCs and genome editing technology for understanding the biological function of genes and the pathological significance of genetic variants in human cardiovascular diseases. The methods, strategies, constructs and iPSC lines developed in this study provide a validated, readily available resource for cardiovascular research

Correction of human phospholamban R14del mutation associated with cardiomyopathy using targeted nucleases and combination therapy

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iPSC modeling of RBM20-deficient DCM identifies upregulation of RBM20 as a therapeutic strategy

Recent advances in induced pluripotent stem cell (iPSC) technology and directed differentiation of iPSCs into cardiomyocytes (iPSC-CMs) make it possible to model genetic heart disease in vitro. We apply CRISPR/Cas9 genome editing technology to introduce three RBM20 mutations in iPSCs and differentiate them into iPSC-CMs to establish an in vitro model of RBM20 mutant dilated cardiomyopathy (DCM). In iPSC-CMs harboring a known causal RBM20 variant, the splicing of RBM20 target genes, calcium handling, and contractility are impaired consistent with the disease manifestation in patients. A variant (Pro633Leu) identified by exome sequencing of patient genomes displays the same disease phenotypes, thus establishing this variant as disease causing. We find that all-trans retinoic acid upregulates RBM20 expression and reverts the splicing, calcium handling, and contractility defects in iPSC-CMs with different causal RBM20 mutations. These results suggest that pharmacological upregulation of RBM20 expression is a promising therapeutic strategy for DCM patients with a heterozygous mutation in RBM20

Role of ER Stress in Ventricular Contractile Dysfunction in Type 2 Diabetes

BACKGROUND: Diabetes mellitus (DM) is associated with an increased risk of ischemic heart disease and of adverse outcomes following myocardial infarction (MI). Here we assessed the role of endoplasmic reticulum (ER) stress in ventricular dysfunction and outcomes after MI in type 2 DM (T2DM). METHODOLOGY AND PRINCIPAL FINDINGS: In hearts of OLETF, a rat model of T2DM, at 25∼30 weeks of age, GRP78 and GRP94, markers of ER stress, were increased and sarcoplasmic reticulum calcium ATPase (SERCA)2a protein was reduced by 35% compared with those in LETO, a non-diabetic control. SERCA2a mRNA levels were similar, but SERCA2a protein was more ubiquitinated in OLETF than in LETO. Left ventricular (LV) end-diastolic elastance (Eed) was higher in OLETF than in LETO (53.9±5.2 vs. 20.2±5.6 mmHg/µl), whereas LV end-systolic elastance and positive inotropic responses to β-adrenergic stimulation were similar in OLETF and LETO. 4-Phenylbutyric acid (4-PBA), an ER stress modulator, suppressed both GRP up-regulation and SERCA2a ubiquitination and normalized SERCA2a protein level and Eed in OLETF. Sodium tauroursodeoxycholic acid, a structurally different ER stress modulator, also restored SERCA2a protein level in OLETF. Though LV dysfunction was modest, mortality within 48 h after coronary occlusion was markedly higher in OLETF than in LETO (61.3% vs. 7.7%). Telemetric recording showed that rapid progression of heart failure was responsible for the high mortality rate in OLETF. ER stress modulators failed to reduce the mortality rate after MI in OLETF. CONCLUSIONS: ER stress reduces SERCA2a protein via its augmented ubiquitination and degradation, leading to LV diastolic dysfunction in T2DM. Even at a stage without systolic LV dysfunction, susceptibility to lethal heart failure after infarction is markedly increased, which cannot be explained by ER stress or change in myocardial response to sympathetic nerve activation

Using the delphi method to evaluate the appropriateness of urban freight transport solutions

Before implementing an Urban Freight Transport (UFT) solution, certainty is required about the effectiveness of the considered alternatives. Selecting an effective solution necessitates the engagement of all stakeholders involved in the management of the UFT system. The aim of the study is the formulation of a common assessment platform for facilitating the selection of the most appropriate UFT solution, taking into account the solutions’ effectiveness and the stakeholder perceptions and consensus. Solution maturity, social acceptance, and user uptake, which are considered as the main drivers of stakeholders’ engagement, are evaluated based on a real time Delphi survey, in parallel with solutions’ sustainability dimensions (economy and energy, environment, society, transport, and mobility). The Delphi method emerges as a suitable tool in this direction as stakeholders’ subjective judgments, and not analytical techniques, are required. The platform is demonstrated through the assessment of ten UFT solutions by 184 stakeholders (public authorities, supply chain operators, and other interested groups) who reside in cities across the world. The results of the demonstration showed that Intelligent Transportation Systems (ITS) for freight monitoring and electric vehicles are the highest rated solutions, while drone deliveries are the lowest, reaching respectively the highest and lowest consensus levels. © 2020 by the authors. Licensee MDPI, Basel, Switzerland

Validating the Simulated Impacts of Urban Freight Transport

This paper provides a comprehensive guide for assessing the impacts of an urban freight transport system through traffic simulation software. Based on a calibrated and validated city wide urban traffic model that integrates the freight trips of six freight vehicles in the examined day, aim of the paper is to establish an impact assessment framework that evaluates model’s environmental and transport impacts. The analysis of the freight trips showed that they account for 22009.2 g of CO2 emissions and 84.17 h of total network delays on a daily basis. Other impact assessment indicators’ values such as average vehicle speed, first attempt successful deliveries, freight vehicles’ number of roundtrips, load factor, utilization factor, emissions, traffic throughput, etc. were also computed. © 2021, The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG