ABSTRACT
Hypertrophic cardiomyopathy (HCM) is a common cardiovascular disease. However, effective methods of its therapy have not been developed so far. To date patient-specific induced pluripotent stem cell-derived cardiomyocytes are supposed to be a useful tool for studying HCM molecular mechanisms and to help find new approaches to HCM therapy. Using non-integrating episomal vectors, we generated an iPSC line from peripheral blood mononuclear cells of an HCM patient carrying a heterozygous p.N515del mutation in MYBPC3. The iPSC line expressed pluripotency markers, gave rise to derivatives of three germ layers during spontaneous differentiation, had normal karyotype, and retained the patient-specific mutation.
Subject(s)
Cardiomyopathy, Hypertrophic , Induced Pluripotent Stem Cells , Cardiomyopathy, Hypertrophic/genetics , Cell Differentiation , Heterozygote , Humans , Leukocytes, Mononuclear , MutationABSTRACT
Hypertrophic cardiomyopathy (HCM) is a frequent cardiovascular pathology caused by a huge number of mutations in sarcomere-associated proteins. This genetic diversity leads to differences in pathogenetic mechanisms and hampers HCM therapy. Cardiomyocytes derived from patient-specific induced pluripotent stem cells give new opportunities for studying underlying HCM mechanisms. We generated an iPSC line from peripheral blood mononuclear cells of an HCM patient with a heterozygous p.E510Q mutation in HADHA using non-integrating episomal vectors. The iPSC line showed typical morphology, expression of pluripotency markers, capacity to be differentiated into derivatives of three germ layers, and presence of the patient-specific mutation.
Subject(s)
Cardiomyopathy, Hypertrophic , Induced Pluripotent Stem Cells , Cardiomyopathy, Hypertrophic/genetics , Heterozygote , Humans , Leukocytes, Mononuclear , Mitochondrial Trifunctional Protein, alpha Subunit , MutationABSTRACT
Hypertrophic cardiomyopathy (HCM) is one of the most frequent cardiovascular diseases but no methods to prevent its progression have been developed. Cardiomyocytes derived from patient-specific induced pluripotent stem cells can become a platform to study pathogenesis of the disease and to search for more effective therapy methods. We generated two iPSC lines from peripheral blood mononuclear cells of an HCM patient with heterozygous p.M659I mutation in MYH7 using episomal vectors. The iPSC lines expressed pluripotency markers, demonstrated ability to spontaneously differentiate into derivatives of three germ layers, and retained the mutation.
Subject(s)
Cardiomyopathy, Hypertrophic , Induced Pluripotent Stem Cells , Cardiac Myosins/genetics , Cardiomyopathy, Hypertrophic/genetics , Cell Differentiation , Humans , Leukocytes, Mononuclear , Mutation , Myosin Heavy Chains/geneticsABSTRACT
Erythromycin is an antibiotic that prolongs the QT-interval and causes Torsade de Pointes (TdP) by blocking the rapid delayed rectifying potassium current (IKr) without affecting either the slow delayed rectifying potassium current (IKs) or inward rectifying potassium current (IK1). Erythromycin exerts this effect in the range of 1.5-100 µM. However, the mechanism of action underlying its cardiotoxic effect and its role in the induction of arrhythmias, especially in multicellular cardiac experimental models, remain unclear. In this study, the re-entry formation, conduction velocity, and maximum capture rate were investigated in a monolayer of human-induced pluripotent stem cell (iPSC)-derived cardiomyocytes from a healthy donor and in a neonatal rat ventricular myocyte (NRVM) monolayer using the optical mapping method under erythromycin concentrations of 15, 30, and 45 µM. In the monolayer of human iPSC-derived cardiomyocytes, the conduction velocity (CV) varied up to 12 ± 9% at concentrations of 15-45 µM as compared with that of the control, whereas the maximum capture rate (MCR) declined substantially up to 28 ± 12% (p < 0.01). In contrast, the tests on the NRVM monolayer showed no significant effect on the MCR. The results of the arrhythmogenicity test provided evidence for a "window" of concentrations of the drug (15-30 µM) at which the probability of re-entry increased.
Subject(s)
Action Potentials/drug effects , Anti-Bacterial Agents/toxicity , Erythromycin/toxicity , Heart Rate/drug effects , Induced Pluripotent Stem Cells/drug effects , Myocytes, Cardiac/drug effects , Torsades de Pointes/chemically induced , Toxicity Tests , Voltage-Sensitive Dye Imaging , Animals , Animals, Newborn , Cardiotoxicity , Cell Line , Humans , Induced Pluripotent Stem Cells/metabolism , Myocytes, Cardiac/metabolism , Rats , Rats, Sprague-Dawley , Risk Assessment , Time Factors , Torsades de Pointes/metabolism , Torsades de Pointes/physiopathologyABSTRACT
Generation of patient-specific induced pluripotent stem cells (iPSCs) and their subsequent differentiation into cardiomyocytes opened new opportunities for studying pathogenesis of inherited cardiovascular diseases. One of these diseases is hypertrophic cardiomyopathy (HCM) for which no efficient therapy methods have been developed so far. In this study, the approach based on patient-specific iPSCs was applied to create a model of the disease. Genetic analysis of a hypertrophic cardiomyopathy patient revealed R326Q mutation in the MYBPC3 gene. iPSCs of the patient were generated and characterized. The cells were differentiated into cardiomyocytes together with the control iPSCs from a healthy donor. The patient's iPSC-derived cardiomyocytes exhibited early HCM features, such as abnormal calcium handling and increased intracellular calcium concentration. Therefore, cardiomyocytes obtained by directed differentiation of iPSCs from the HCM patient can be used as a model system to study HCM pathogenesis.
Subject(s)
Cardiomyopathy, Hypertrophic/metabolism , Induced Pluripotent Stem Cells/metabolism , Models, Biological , Adult , Calcium/metabolism , Cardiomyopathy, Hypertrophic/diagnosis , Cardiomyopathy, Hypertrophic/genetics , Cell Differentiation , Cells, Cultured , Humans , Induced Pluripotent Stem Cells/pathology , MaleABSTRACT
We performed transcriptome analysis of some human induced pluripotent stem cells, embryonic stem cells, and human somatic cells using DNA microarrays. PluriTest bioinformatic system was used for evaluation of cell pluripotency. Changes in the genome structure and status of X-chromosome gene expression was analyzed using microarray technology.
Subject(s)
Embryonic Stem Cells/physiology , Genes, X-Linked , Induced Pluripotent Stem Cells/physiology , Transcriptome , Cells, Cultured , DNA/genetics , Embryonic Stem Cells/cytology , Gene Expression , Gene Expression Profiling , Humans , Induced Pluripotent Stem Cells/cytology , Oligonucleotide Array Sequence AnalysisABSTRACT
The isolation and study of autologous human stem cells remain among the most urgent problems in cell biology and biomedicine to date. Induced pluripotent stem cells can be derived from human somatic cells by the overexpression of a number of genes. In this study we reprogrammed fetal human skin fibroblasts by transduction with retroviral vectors carrying murine Oct4 , Sox2 , Klf4 , and c-Myc cDNAs. As a result, cells with the protein expression and gene transcription pattern characteristic of human embryonic stem cells were derived. These induced pluripotent cells are capable of differentiation in vitro into the ectoderm, mesoderm, and endoderm derivatives.
ABSTRACT
Sex chromosome evolution is accompanied by significant divergence in morphology and gene content and results in most genes of one of the sex chromosomes being present in two dosages in one sex and in one dosage in the other. To eliminate the difference in the expression levels of these genes between sexes and to restore equal expression levels of the genes between sex chromosomes and autosomes, mechanisms of dosage compensation have appeared. Studies of three classical objects,Drosophila melanogaster,Caenorhabditis elegans, and mammals, have shown that dosage compensation of X-linked genes can be achieved through completely different chromosome-wide mechanisms. New data on sex chromosome gene expression demonstrating that many sex chromosome genes can be expressed at different levels in males and females were recently obtained from birds and butterflies. In this review, dosage compensation mechanisms inD. melanogaster,C. elegans, and mammals are considered and the data on sex chromosome gene expression in birds and butterflies, and their influence on our view of dosage compensation, are discussed.