Using Rabbit Induced Pluripotent Stem Cell-Derived Cardiomyocytes to Investigate Drug-Induced Fetal Heart Malformations

Abstract

Introduction: Teratogenic events such as fetal heart malformations are serious and tragic drug-induced toxicities. Preclinical studies conducted during drug safety evaluation are intended to identify teratogenic risks using rodent and nonrodent models, largely based on the experience with thalidomide, which is teratogenic in humans and rabbits but refractory in rodents. Methods: To study the teratogenic properties of thalidomide in rabbit, a novel method was designed to develop cardiomyocytes (CMs) from rabbit induced pluripotent stem cells (rbiPSCs). Throughout the course of differentiation, rbiPSC-CMs were examined by real time polymerase chain reaction, high-content analysis, calcium flux measurements, and electrophysiology to confirm the cell's mature CM phenotype. The effects of thalidomide were then measured in each of the endpoints when present throughout differentiation. Results: As early as differentiation day (DD) 3 of the differentiation, rbiPSCs begin to express early CM markers (e.g., NXK2.5) and mesodermal markers (e.g., FOXF1). By DD 21, rbiPSC-CMs expressed messenger RNA (mRNA) and protein of mature CM genes, including TNNT2, HCN4, and KCNJ2. Spontaneous beating was observed by microscopy and electrophysiology as early as DD 8, and live cell calcium probes demonstrated pulsatile calcium flux. Thalidomide treatment to rbiPSC-CMs downregulated mRNA and protein of early and mature cardiac markers, suggesting suppressed differentiation. In addition, thalidomide treatment abrogated the onset of spontaneously beating cells. Conclusions: Taken together, these data indicate that rbiPSC-CMs can recapitulate thalidomide-induced cardiac teratogenicity observed in rabbits. Moving forward, this model can be deployed to investigate the underlying mechanisms of rabbit teratogenicity and better understand species-specific differences in drug-induced fetal heart malformations.