A novel system for perfusion and real-time analysis of single hESC-derived cardiomyocytes

About one week after fertilization between egg and sperm, the cells of the developing human embryo are primed to start forming all the cell types in the body. Scientists have shown that cells can be isolated at this early stage of development and then grown in the laboratory to produce large numbers of human embryonic stem cells (hESCs). Even in the laboratory, these cells can also be induced to make all cell types in the body. This may provide new opportunities to study early human development, understand more about genetic disorders or provide new sources of transplantable cells for conditions such as cardiovascular or Parkinsons disease. While hESCs could potentially have significant impact on our understanding of developmental biology and treatment of disease, the cell types derived from hESCs have not been studied in detail. For example, beating heart cells (cardiomyocytes) have been produced from hESCs but at best only limited information is available as to how they respond to different drugs or to the effect disruption of specific genes may have. Equally, no-one knows whether the characteristics of hESC-cardiomyocytes produced one day will be comparable to those produced the next. We have demonstrated that sufficient numbers of hESC-derived cardiomyocytes can be produced for detailed analysis. We now aim to engineer a new system that can simultaneously administer and analyze the effect of a variety of cardioactive drugs / gene modulatory agents. This will enable rapid development of hESC-cardiomyocytes as a tool for understanding human development and disease, as well as providing a platform to evaluate the utility of these cells for future medical application.

Although many labs can successfully culture and spontaneously differentiate human embryonic stem cells (hESCs), the fundamental biology of undifferentiated and differentiated cells is poorly understood. To develop hESCs as scientific and therapeutic modalities, it will be vital to functionally characterize the differentiated cell types, preferably at the single cell level, to ensure reproducible preparations can be made. Thus, the aim of this proposal is to establish a platform technology to evaluate whether cohorts of single hESC-cardiomyocytes of comparable function can be produced both from different preparations using the same hESC line and from preparations using independently-derived lines Recently, we have standardized culture and cardiac differentiation between three independently-derived hESC lines, HUES-7, BG01 and NOTT1. We have now developed a high throughput differentiation protocol that utilizes growth factor induction to rapidly generate up to 45% spontaneously beating embryoid bodies (EBs). Beating areas can be readily disaggregated to single beating cardiomyocytes that are amenable to electrophysiology, calcium imaging and video edge detection, thus providing outputs to measure cardiomyocyte function and response In this proposal, we have designed and will engineer a semi-automated perfusion system that will interface with Multi Electrode Array, video edge and single cell fluorecence detection equipment to produce simultaneous real-time readouts for electrical and contractile activity, and fluorescence. This will facilitate rapid evaluation of functionality and reproducibility of different hESC-cardiomyocyte preparations as well as responsiveness to challenge with chronotropic agents. Finally, we will provide proof of principle that the system is amenable to delivery of lentiviral vectors designed for gene overexpression or knockdown in hESC-cardiomyocytes, thus establishing a novel route to studying gene function within these cells