Human iPSC-Derived Cardiac Myocytes: Toward an In Vitro Model of Cardiac Physiology

Abstract

Cardiovascular Disease is a growing public health issue in the modern world, with a high incidence rate that continues to increase, and poor mortality rates. Recent technological advances have made it possible to efficiently derive cardiac myocytes from human induced pluripotent stem cells (hiPSC-CMs). These have been seen as a model for human heart disease, as well as a potential source for cellular transplantation into failing diseased heart tissue. Many laboratories have devoted substantial effort to examining the functional properties of hiPSC-CMs, including electrophysiology, intracellular calcium handling, and gene/protein expression and force. In the first part of this thesis, we utilize traction force microscopy (TFM) to determine the maximum force production of isolated hiPSC-CMs under varied culture and assay conditions. We elucidate here the relationship between cell morphology and force production, and find a significant relationship between cell size and force. HiPSC-CMs developing in culture for two weeks produce significantly less force than cells cultured from one to three months and hiPSC-CMs cultured for three months resemble the cell morphology of neonatal rat ventricular myocytes. Unexpectedly, hiPSC-CMs produce less force when assayed on increasingly stiff substrates, and generate less strain energy. Finally, hiPSC-CMs cultured in conditions of physiologic calcium concentrations are larger and produce more force than cells cultured in standard media. In the second part of this thesis, we address the concept of immaturity in hiPSC-CMs, and attempt to accelerate maturation. We use genome editing to engineer hiPSC-CMs that contain an inducible gene expression cassette, in order to overexpress two proteins associated with maturity: SERCA2a and cardiac troponin I (cTnI). We find that we are able to overexpress both proteins in differentiated hiPSC-CMs after two weeks of treatment with doxycycline. SERCA2a-overexpressing cells showed significant alterations in physiologic function, including increased chronotropy and decreased time to peak in calcium transients following treatment with isoproterenol, a β-adrenergic agonist. Furthermore, using an impedance-measuring system to track contractility kinetics, we found that SERCA2a-overexpressing cells had shortened time to peak and time to baseline after gene induction, with continued response to isoproterenol. As a sign of maturation, SERCA cells also expressed increased cTnI, a key marker of maturity. Using RNAseq, we found that cTnI-overexpressing cells had marked, global changes in their gene expression profile. Key findings include upregulation of genes associated with cardiac contractility and development, such as cardiac myomesin and tropomyosin and ryanodine receptor, and downregulation of genes associated with pacemaker and ventricular cell types, such as HCN and GREM2, and genes associated with skeletal myocytes, such as skeletal muscle actin. Overall, our findings show that hiPSC-CMs have physiologic function similar to that of immature cardiac myocytes, but that we are able to induce maturation by overexpression of genes associated with maturity.