188. Functional Analysis of Human Induced Pluripotent Stem Cell-Derived Cardiac Myocytes

Abstract

Recent and exciting advances in stem cell biology have made it possible to quickly and efficiently derive induced pluripotent stem cells (iPSCs) from human somatic cell types, as well as differentiate pluripotent cells to committed lineages, such as cardiac myocytes (hiPSC-CMs). The nature of these cells makes them ideal potential candidates for cell therapy treatments, such as implantation of hiPSC-CMs following myocardial infarction. Use in therapy requires optimization of these cells physiologically, which in turn requires appropriate assays to determine cell function beyond gene expression profiles. Difficulty in accurately characterizing hiPSC-CM function comes from large variability in cell size and shape, as well as isotropy of axis of contraction. We utilize here micropatterning techniques, whereby single cell-sized (2500 μm2) rectangles of fibronectin are applied to glass cover slips. The hiPSC-CMs are then cultured on the cover slip at a low density such that one cell occupies the rectangular shaped space. This persuades the cells to assume a rectangular shape, similar to that of an adult myocyte, and causes them to contract along the long axis of the rectangle. By normalizing size, shape, and alignment of hiPSC-CMs, microscopy can be used to determine and compare kinetics of contraction and relaxation, utilizing the IonOptix software and an inverted microscope. Additionally, this technique can be used for traction force microscopy. Here we use fibronectin patterns that are micropatterned onto a polyacrylamide hydrogel, and the hiPSC-CMs are cultured on top of it. Next, 0.2 μm diameter fluorescent beads are embedded in the hydrogel such that when the cell contracts, it pulls on the hydrogel, and movement of the beads is then tracked using confocal microscopy. An ImageJ program analyzes movement of the beads and uses the known Young's modulus of the hydrogel to calculate traction force generated by the individual cells. Preliminary results show that iPSC-CMs will morphologically take the shape of the fibronectin stamp and will contract along the long axis of the rectangle. Additional quantification of these data will be presented and discussed.