An initial map of chromosomal segmental copy number variations in the chicken

<p>Abstract</p> <p>Background</p> <p>Chromosomal segmental copy number variation (CNV) has been recently recognized as a very important source of genetic variability. Some CNV loci involve genes or conserved regulatory elements. Compelling evidence indicates that CNVs impact genome functions. The chicken is a very important farm animal species which has also served as a model for biological and biomedical research for hundreds of years. A map of CNVs in chickens could facilitate the identification of chromosomal regions that segregate for important agricultural and disease phenotypes.</p> <p>Results</p> <p>Ninety six CNVs were identified in three lines of chickens (Cornish Rock broiler, Leghorn and Rhode Island Red) using whole genome tiling array. These CNVs encompass 16 Mb (1.3%) of the chicken genome. Twenty six CNVs were found in two or more animals. Whereas most small sized CNVs reside in none coding sequences, larger CNV regions involve genes (for example prolactin receptor, aldose reductase and zinc finger proteins). These results suggest that chicken CNVs potentially affect agricultural or disease related traits.</p> <p>Conclusion</p> <p>An initial map of CNVs for the chicken has been described. Although chicken genome is approximately one third the size of a typical mammalian genome, the pattern of chicken CNVs is similar to that of mammals. The number of CNVs detected per individual was also similar to that found in dogs, mice, rats and macaques. A map of chicken CNVs provides new information on genetic variations for the understanding of important agricultural traits and disease.</p

Combinatorial Polymer Electrospun Matrices Promote Physiologically-Relevant Cardiomyogenic Stem Cell Differentiation

Myocardial infarction results in extensive cardiomyocyte death which can lead to fatal arrhythmias or congestive heart failure. Delivery of stem cells to repopulate damaged cardiac tissue may be an attractive and innovative solution for repairing the damaged heart. Instructive polymer scaffolds with a wide range of properties have been used extensively to direct the differentiation of stem cells. In this study, we have optimized the chemical and mechanical properties of an electrospun polymer mesh for directed differentiation of embryonic stem cells (ESCs) towards a cardiomyogenic lineage. A combinatorial polymer library was prepared by copolymerizing three distinct subunits at varying molar ratios to tune the physicochemical properties of the resulting polymer: hydrophilic polyethylene glycol (PEG), hydrophobic poly(ε-caprolactone) (PCL), and negatively-charged, carboxylated PCL (CPCL). Murine ESCs were cultured on electrospun polymeric scaffolds and their differentiation to cardiomyocytes was assessed through measurements of viability, intracellular reactive oxygen species (ROS), α-myosin heavy chain expression (α-MHC), and intracellular Ca2+ signaling dynamics. Interestingly, ESCs on the most compliant substrate, 4%PEG-86%PCL-10%CPCL, exhibited the highest α-MHC expression as well as the most mature Ca2+ signaling dynamics. To investigate the role of scaffold modulus in ESC differentiation, the scaffold fiber density was reduced by altering the electrospinning parameters. The reduced modulus was found to enhance α-MHC gene expression, and promote maturation of myocyte Ca2+ handling. These data indicate that ESC-derived cardiomyocyte differentiation and maturation can be promoted by tuning the mechanical and chemical properties of polymer scaffold via copolymerization and electrospinning techniques

Human cardiomyocytes are more susceptible to irreversible electroporation by pulsed electric field than human esophageal cells

Abstract Pulse electric field‐based (PEF) ablation is a technique whereby short high‐intensity electric fields inducing irreversible electroporation (IRE) are applied to various tissues. Here, we implemented a standardized in vitro model to compare the effects of biphasic symmetrical pulses (100 pulses, 1–10 μs phase duration (d), 10–1000 Hz pulse repetition rate (f)) using two different human cellular models: human‐induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs) and human esophageal smooth muscle cells (hESMCs) cultured in monolayer format. We report the PEF‐induced irreversibly electroporated cell monolayer areas and the corresponding electric field thresholds (EFTs) for both cardiac and esophageal cultures. Our results suggest marked cell type specificity with EFT estimated to be 2–2.5 times lower in hiPSC‐CMs than in hESMCs when subjected to identical PEF treatments (e.g., 0.90 vs 1.85 kV/cm for the treatment of 100 pulses with d = 5 μs, f = 10 Hz, and 0.65 vs 1.67 kV/cm for the treatment of 100 pulses with d = 10 μs, f = 10 Hz). PEF treatment can result in increased temperature around the stimulating electrodes and lead to unanticipated thermal tissue damage that is proportional to the peak temperature rise and to the duration of the PEF‐induced elevated temperatures. In our study, temperature increases ranged from less than 1°C to as high as 30°C, however, all temperature changes were transient and quickly returned to baseline and the highest observed ∆T returned to 50% of its maximum recorded temperature in tens of seconds

Production of Single Contracting Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes: Matrigel Mattress Technique

This unit describes the published Matrigel mattress method. Briefly, we describe the preparation of the mattress, replating of the human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) on the Matrigel mattress, and hiPSC-CM mattress maintenance. Adherence to this protocol will yield individual, robustly shortening hiPSC-CMs, which can be used for downstream applications

International Multisite Study of Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes for Drug Proarrhythmic Potential Assessment

Summary: To assess the utility of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as an in vitro proarrhythmia model, we evaluated the concentration dependence and sources of variability of electrophysiologic responses to 28 drugs linked to low, intermediate, and high torsades de pointes (TdP) risk categories using two commercial cell lines and standardized protocols in a blinded multisite study using multielectrode array or voltage-sensing optical approaches. Logistical and ordinal linear regression models were constructed using drug responses as predictors and TdP risk categories as outcomes. Three of seven predictors (drug-induced arrhythmia-like events and prolongation of repolarization at either maximum tested or maximal clinical exposures) categorized drugs with reasonable accuracy (area under the curve values of receiver operator curves ∼0.8). hiPSC-CM line, test site, and platform had minimal influence on drug categorization. These results demonstrate the utility of hiPSC-CMs to detect drug-induced proarrhythmic effects as part of the evolving Comprehensive In Vitro Proarrhythmia Assay paradigm. : Blinova et al. tested human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for improving torsades de pointes arrhythmia risk prediction of drugs in the Comprehensive In Vitro Proarrhythmia Assay (CiPA) initiative. This validation study confirms their utility based on electrophysiologic responses to 28 blinded drugs, with minimal influence from cell lines, test sites, and electrophysiological platforms. Keywords: comprehensive in vitro proarrhythmia assay, CiPA, human-induced pluripotent stem cell-derived cardiomycotes, hiPSC-CM, drug-induced ventricular arrhythmia Torsade de Pointes, microelectrode array, voltage-sensitive dye

Schematic representation of polymer synthesis PEG-PCL block copolymers were first synthesized by ring-opening polymerization.

<p>CPCL was then randomly added within the PCL subunit. Polymers are abbreviated <i>x</i>%PEG-<i>y</i>%PCL-<i>z</i>%CPCL.</p

EB differentiation on polymer fiber scaffolds in the presence of DMH1.

<p>EBs with and without DMH1 treatment were stained for SERCA2a, an indicator of active Ca²⁺ transient. EBs on 4%PEG-86%PCL-10%CPCL exhibited enhanced expression of SERCA2a, compared to untreated EBs on PCL scaffolds or glass alone. Scale bars = 50 µm.</p

EB attachment and protein expression.

<p>(<b>a</b>) Phase contrast and fluorescence images of EBs are shown at day 10. Cells were cultured on gelatin coated glass cover slips with and without 4%PEG-86%PCL-10%CPCL copolymer scaffolds. Cells grown on 4%PEG-86%PCL-10%CPCL maintained more adhered, circular EBs and (<b>b</b>) exhibited higher α-MHC protein expression. Scale bars = 10 µm. * <i>p</i><0.0005 versus control.</p

Characterization of polymer properties.

<p>M_n was measured by GPC according to dn/dc light scattering values. Wet and dry moduli were calculated from stress/strain measurements.</p>a<p>Molecular weight measured by GPC in THF,</p>b<p>Measured on a uniaxial Bose ElectroForce 3100 mechanical tester,</p>c<p>Measured by DMA.</p