Background: From the first successful differentiation of human stem cells to cardiomyocytes, stem cells have held promise in the field of cardiac regeneration. These cells provide an indefinite source of de novo cardiomyocytes and potentially unlimited tissue for human transplantation. Yet the mammalian heart is a complex organ, comprised of four chambers, a highly coordinated electrical conduction system, and a heterogeneous mix of cell types, which may not match the differentiated tissues produced in vitro. Despite efforts focused on increasing cardiomyocyte differentiation yield, relatively little progress has been made in differentiating function- specific cardiomyocytes, such as pacemaker cells. Differentiation of nodal pacemaker cells would provide a new cell source for biological pacing of the heart. Recently, retinoic acid (Ra) signaling has been shown to play a critical role in the differentiation of atrial and pacemaker cells, mimicking the cells found in the native pacemaker. I hypothesize that specific differentiation of pacemaker cells with Ra would provide superior biological pacemaking function both in vitro and in vivo, compared to conventional, heterogeneous stem cell-derived populations. The proposed work seeks to fully characterize the genetic profile, functional electrophysiological properties, and biological pacemaker potential, both in vitro and in vivo, of Ra-derived cardiomyocytes. Approach: To derive pacemakers, human induced pluripotent stem cells (hiPSC) will be differentiated as monolayers over a 14-day protocol with and without Ra. Our preliminary data indicates that Ra treatment enriches molecular and genetic expression profiles to that of native pacemaker cells.
Aim 1 of this work seeks apply our Ra differentiation protocol on 6 different hiPSC lines (half male / half female) derived from umbilical cord blood. Thus, allowing us to examine the applicability of this method for pediatric patients.
In Aim 2, hiPSC- derived cells will be aggregated into spheroids, pacing units. The size of these pacing units will be optimized for maximum spontaneous beating with minimal cell death. Pacemaker function of these spheres will be tested with our in vitro engraftment model.
In Aim 3, optimized pacing units will be engrafted to rat ventricular myocardium in vivo to record spontaneous beating induced by the hiPSC-pacing units. The major readouts are i) RT-qPCR analysis of gene expression, ii) single-cell intracellular potential recordings from patch-clamp, iii) macro-scale, multi-electrode array measurements of field potentials, iv) high-resolution optical mapping of monolayers and ex vivo whole hearts with a voltage-sensitive dye, v) 24/7 telemetry biopotential recordings of ECG in vivo, and vi) echocardiographic measurements. Successful completion of this project will lead to the first in-depth study of the biological pacemaker potential of Ra-derived cardiomyocytes, in terms of optimized differentiation, dynamic dose range, safety margin, and therapeutic effect in vivo. The proposed work will be conducted under the supervision of Dr. Hee Cheol Cho at Emory University & Georgia Tech.
Traditional cardiac pacemaker devices are not designed for pediatric patients and present many limitations for quality of life of the patient. This project seeks to develop biological pacemakers, which are free of invasive and indwelling hardware, and made up of newly formed pacemaker cells from patient-derived pluripotent stem cells. Successful outcomes of this proposal will provide the first insights into required doses, optimal delivery mechanisms, and safety margin of this new therapeutic, as an alternative or bridge-to-device to electronic pacemaker devices. 1
