Budash G. Evaluation of morphological and functional characteristics of pluripotent stem cells and their in vitro differentiation towards cardiomyocytes in cell culture

Pluripotent stem cells such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) due to their ability to expand in culture and development potential can be the main source of cardiomyocytes. There are a lot of protocols available for cardiac differentiation from pluripotent stem cells, although the efficiency still might be improved. Low yield of cardiomyocytes is the major hindrance for use of PSCs in developmental studies, drug safety or discovery research, exploration of disease in dish models and tissue repair. The aim of the research was to define the characteristics of proliferation and differentiation of embryonic and induced pluripotent stem cells into murine cardiomyocytes under the influence of various differentiation factors. Transgenic murine iPSC line and ESC line were were genetically modified and express IRES-flanked enhanced green fluorescent protein (GFP) under the control of cardiac alpha myosin heavy chain promoter. We applied flow cytometry and fluorescence microscopy to test the efficiency of the differentiation processes. We investigated the influence of cell seeding density, the time when embryoid bodies were attached to the surface and the mobility of cells in suspension when the embryoid bodies were formed on the process of differentiation. We found that modifying such parameter as siding density of cells before the formation of embryoid bodies can beneficially effect differentiation of pluripotent stem cells into cardiomyocytes. Among the embryoid bodies formed within range from 250 cells to 2000 cells per embryoid body the highest percentage of GFP+ cells was obtained from embryoid bodies formed with 500 cells. However, this method prolonged the process of differentiation for iPSC and failed to differentiate ESC into cardiomyocytes. Amount of embryoid bodies with beating areas produced with hanging drop method is significantly higher compared to the mass culture method, but this method is very time- and labor-consuming, and could not be applied for large scale production of cardiomyocytes. Attachment of embryoid bodies on early stages of differentiation prolongs the whole process of differentiation. Mass culture method proved to produce the most robust yield of cardiomyocytes. Ascorbic acid and QS11 enhanced cardiac differentiation of murine ESCs and iPSCs greatly when they were applied together. We got the priority data with the optimal timing of cyclosporine applying. The scheme of efficient differentiation of pluripotent cells into cardiomyocytes was obtained.