Characterization of the cardiogenesis of embryonic stem cells

Abstract

Cardiovascular diseases persist as the leading cause of mortality worldwide. Stem cell therapy, aimed to restore contractility and proper vasculature, has gained considerable attention as an attractive therapeutic option. However, proper cell differentiation, survival and integration in an infarcted zone remain elusive. This thesis aims to utilize in vitro techniques to obtain a systematic characterization of how individual stimulations can affect the cardiogenesis process of embryonic stem cells. First, a compliant microfluidic system was developed to study the individual and combined effects of culture dimensions and uniaxial cyclic stretch on the differentiation process. A smaller culture dimension, with a characteristic length scale of hundreds of micrometers, dramatically enhanced differentiation partly due to an accumulation of cell-secreted and cardiogenic BMP2. Uniaxial cyclic stretch, on the other hand, inhibited differentiation. With this microfluidic platform and a GFP-reporting differentiation cell line, effects of various external stimuli on differentiation were systematically studied. Next, the effects of collagen I and cell alignment, two biophysical signatures of the adult myocardium, on promoting phenotypic changes of isolated embryonic stem cell derived cardiomyocytes (ESCDMs) were investigated. Effects of collagen I depended on how it was presented to the cells and overlaying collagen gel impeded cell elongation. Binucleation. characteristic of maturing cardiomyocytes, was reduced with soluble collagen supplement and nanoscale topography and was associated with an increase in cytokinesis. Both nanoscale topography and microcontact printing resulted in aligned cardiomyocyte monolayers but produced different morphologies. Lastly, the lessons learned from studying the aforementioned processes were applied to test the utility of ESCDMs as biological actuators. Three proof-of-concept experiments were conducted: ESCDM monolayers were able to contract synchronously as a cell-assemble, force generated by the cell monolayer was estimated to be comparable to that by neonatal myocytes and lastly, the direction of contraction could be controlled with surface patterning. This work advances our understanding on the cardiogenic potential of murine embryonic stem cells and elucidated complex biological questions with well-characterized and controlled tissue engineering techniques.