Generation of an induced pluripotent stem cell line, ICGi028-A, by reprogramming peripheral blood mononuclear cells of a patient suffering from hypertrophic cardiomyopathy and carrying a heterozygous p.E510Q mutation in HADHA.

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.

Generation of an induced pluripotent stem cell line, ICGi029-A, by reprogramming peripheral blood mononuclear cells of a patient suffering from hypertrophic cardiomyopathy and carrying a heterozygous p.N515del mutation in MYBPC3.

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.

Generation of two clonal iPSC lines, ICGi019-A and ICGi019-B, by reprogramming peripheral blood mononuclear cells of a patient suffering from hypertrophic cardiomyopathy and carrying a heterozygous p.M659I mutation in MYH7.

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.

The Use of iPSC-Derived Cardiomyocytes and Optical Mapping for Erythromycin Arrhythmogenicity Testing.

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.

Applying Patient-Specific Induced Pluripotent Stem Cells to Create a Model of Hypertrophic Cardiomyopathy.

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.

[The comparison of laboratory tests Quantiferon, Tubinferon and Diaskintest in children with tuberculosis infection.]

The study was carried out to compare results of three tests on the basis of antigens CFP-10 and ESAT-6: laboratory tests QuantiFERON (QFT) and Tubinferon (TIF) and intra-cutaneous test with allergen tuberculous recombinant - Diaskintest (DST). Tubinferon is a Russian analog of test QuantiFERON and is based on quantitative analysis of interferon-gamma after incubation of samples of whole blood in the presence of antigens of Mycobacterium tuberculosis (MBT) - tuberculin and recombinant antigens ESAT-6 and САЗ-10. The results QFT, TIF and DST are examined on 63 patients aged from 7 months to 15 years at parallel setup. According complex examination 63 children were divided at three groups: group I - 29 children infected with MBT and without disease; group II - 8 children non-infected with MBT; group III - 26 children with active forms of tuberculosis. It is established that at high rate of coincidence of results of all three tests (68.3%) in particular situations they can react differently, providing thereby additional information. The results of tests QFT and TIF coincide in 74.6% of all cases. The tests in vitro (QFT, TFT) occurred more sensitive under immuno-pathologic conditions in comparison with DST test. The TFT test demonstrated higher sensitivity under infection with MBT (58.6%) as compared with DST (34.5%) and QFT (34.5%) but lesser than in children with tuberculosis: TFT - 61.5%, QFT - 92.3%, DST - 80.8%. The conclusion is made that all three tests provide positive result more often under tuberculosis infection than in case of infected with MBT. The Tubinferon test is recommended to application and especially for children infected with MBT prior development of disease.

Incidence of cytogenetic aberrations in two B lineage subpopulations in multiple myeloma patients analyzed by combination of whole-genome profiling and FISH.

Multiple myeloma (MM) is an incurable malignant disease of the terminal developmental stage of B-lymphocytes. While genetic heterogeneity of MM is widely described, little is known about its genetic basis as well as primary damage during plasma cells (PC) development. In this study, we focused on genome-wide screening of DNA copy number changes using oligonucleotide-based array-CGH together with I-FISH of the IgH locus rearrangements in pair samples of bone marrow B-cells (CD19+) and CD138+ PC from newly diagnosed MM patients. The IgH disruption was found in 8.9% (4/45) of CD19+ samples and in 57.8% (26/45) of CD138+ samples. The genomic profiling using array-CGH identified copy number alterations (CNAs) in 10% (2/20) of CD19+ samples in regions known to be important for MM pathogenesis. In contrast, we found CNAs in 100% (16/16) of CD138+ samples. Most common chromosomal abnormalities were trisomies of odd-numbered chromosomes (3, 5, 7, 9, 11, 15, 19 and 21), gain 1q, gain Xq and monosomy of chromosome 13. We did not find any correlation between incidence of CNAs in CD19+ and CD138+ cells. In conclusion, effective utilization of FISH and array-CGH can identify genetic lesions in premalignant stages leading to better understanding and characterization of MM.

Characteristics of induced human pluripotent stem cells using DNA microarray technology.

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.

[Molecular basis of Waldenström macroglobulinemia]. / Molekulární podstata Waldenströmovy makroglobulinemie.

Waldenström macroglobulinemia is a rare lymphoproliferative disease that is currently classified into lymphomas with incidence of 3 cases per million. This disease comprises about 1-2% of hematological malignancies and is characterized by infiltration of malignant B cells into the bone marrow and presence of monoclonal immunoglobulin IgM in serum. WM is still an incurable disease with median survival of 5 years. Molecular basis of this disease remains unclear even though deletion of 6q, trisomy of chromosomes 4 and 8, deletion of 13q and increased expression of IL-6 seem to be typical for this disease. The most important changes of microRNA are increased expression of miR-155 and decreased expression of miR-9*. This work aims to describe current knowledge about the molecular basis of this disease.

Visualization of numerical centrosomal abnormalities by immunofluorescent staining.

The presence of multiple centrosomes in tumor cells is associated with the formation of multipolar mitotic spindles and results in aneuploidy of both daughter cells. Centrosome amplification is a feature of all cancer cells. We have previously described centrosome amplification in abnormal B cells. Further studies of centrosome amplification in different stages of B lineage development could provide important information about multiple myeloma pathogenesis.

Centrosome amplification as a possible marker of mitotic disruptions and cellular carcinogenesis in multiple myeloma.

Centrosome amplification (CA) as a potential marker of mitotic disruptions in multiple myeloma (MM) was investigated in two populations of B-cell lineage: B-cells and plasma cells (PCs). Using immunofluorescent staining, it was shown that CA in B-cells is present in 3.2+/-2.5% in healthy donors versus 9.9+/-7.9% in MM patients (p<0.0001). Based on the calculated threshold value of CA in B-cells, 37% (14/38) of MM patients were positive. There was no significant correlation between CA-positive MM cases (based on PC samples evaluation) and the occurrence of cytogenetic abnormalities in PCs, including del(13)(q14), del(17)(p13), gain(1)(q21) and hyperdiploidy.

Derivation of induced pluripotent stem cells from fetal human skin fibroblasts.

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.

Dosage compensation of sex chromosome genes in eukaryotes.

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.