Modulation of Notch Signaling at Early Stages of Differentiation of Human Induced Pluripotent Stem Cells to Dopaminergic Neurons.

Elaboration of protocols for differentiation of human pluripotent stem cells to dopamine neurons is an important issue for development of cell replacement therapy for Parkinson's disease. A number of protocols have been already developed; however, their efficiency and specificity still can be improved. Investigating the role of signaling cascades, important for neurogenesis, can help to solve this problem and to provide a deeper understanding of their role in neuronal development. Notch signaling plays an essential role in development and maintenance of the central nervous system after birth. In our study, we analyzed the effect of Notch activation and inhibition at the early stages of differentiation of human induced pluripotent stem cells to dopaminergic neurons. We found that, during the first seven days of differentiation, the cells were not sensitive to the Notch inhibition. On the contrary, activation of Notch signaling during the same time period led to significant changes and was associated with an increase in expression of genes, specific for caudal parts of the brain, a decrease of expression of genes, specific for forebrain, as well as a decrease of expression of genes, important for the formation of axons and dendrites and microtubule stabilizing proteins.

Different Expressions of Pericardial Fluid MicroRNAs in Patients With Arrhythmogenic Right Ventricular Cardiomyopathy and Ischemic Heart Disease Undergoing Ventricular Tachycardia Ablation.

Introduction: Pericardial fluid is enriched with biologically active molecules of cardiovascular origin including microRNAs. Investigation of the disease-specific extracellular microRNAs could shed light on the molecular processes underlying disease development. Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited heart disease characterized by life-threatening arrhythmias and progressive heart failure development. The current data about the association between microRNAs and ARVC development are limited. Methods and Results: We performed small RNA sequence analysis of microRNAs of pericardial fluid samples obtained during transcutaneous epicardial access for ventricular tachycardia (VT) ablation of six patients with definite ARVC and three post-infarction VT patients. Disease-associated microRNAs of pericardial fluid were identified. Five microRNAs (hsa-miR-1-3p, hsa-miR-21-5p, hsa-miR-122-5p, hsa-miR-206, and hsa-miR-3679-5p) were found to be differentially expressed between patients with ARVC and patients with post-infarction VT. Enrichment analysis of differentially expressed microRNAs revealed their close linkage to cardiac diseases. Conclusion: Our data extend the knowledge of pericardial fluid microRNA composition and highlight five pericardial fluid microRNAs potentially linked to ARVC pathogenesis. Further studies are required to confirm the use of pericardial fluid RNA sequencing in differential diagnosis of ARVC.

Transfer of Synthetic Human Chromosome into Human Induced Pluripotent Stem Cells for Biomedical Applications.

AlphoidtetO-type human artificial chromosome (HAC) has been recently synthetized as a novel class of gene delivery vectors for induced pluripotent stem cell (iPSC)-based tissue replacement therapeutic approach. This HAC vector was designed to deliver copies of genes into patients with genetic diseases caused by the loss of a particular gene function. The alphoidtetO-HAC vector has been successfully transferred into murine embryonic stem cells (ESCs) and maintained stably as an independent chromosome during the proliferation and differentiation of these cells. Human ESCs and iPSCs have significant differences in culturing conditions and pluripotency state in comparison with the murine naïve-type ESCs and iPSCs. To date, transferring alphoidtetO-HAC vector into human iPSCs (hiPSCs) remains a challenging task. In this study, we performed the microcell-mediated chromosome transfer (MMCT) of alphoidtetO-HAC expressing the green fluorescent protein into newly generated hiPSCs. We used a recently modified MMCT method that employs an envelope protein of amphotropic murine leukemia virus as a targeting cell fusion agent. Our data provide evidence that a totally artificial vector, alphoidtetO-HAC, can be transferred and maintained in human iPSCs as an independent autonomous chromosome without affecting pluripotent properties of the cells. These data also open new perspectives for implementing alphoidtetO-HAC as a gene therapy tool in future biomedical applications.