AAV-mediated conversion of human pluripotent stem cell-derived pacemaker.

Malfunction of nodal pacemaker (Pm) cardiomyocytes (CMs) due to diseases or aging leads to rhythm generation disorders, necessitating electronic Pm implantation. We functionally reprogrammed human pluripotent stem cell (hPSC) derived-ventricular (V) CMs into -PmCMs via recombinant adeno-associated virus serotype 9 (rAAV9)-mediated overexpression of engineered HCN1 channel (HCN1ΔΔΔ) whose S3-S4 linker has been strategically deleted by design to promote cardiac pacemaking. rAAV9-HCN1ΔΔΔ-reprogrammed hPSC-PmCMs converted from -VCMs showed automaticity and action potential parameters typical of native nodal PmCMs. Implantation of rAAV9-HCN1ΔΔΔ-based BPm in a preclinical porcine model of complete heart block significantly reduced the dependence on device-supported pacing and generated spontaneous heart rhythms from the BPm. Collectively, these results have further laid the groundwork on BPm for future translation.

Human Cardiac Ventricular-Like Organoid Chambers and Tissue Strips From Pluripotent Stem Cells as a Two-Tiered Assay for Inotropic Responses.

Traditional drug discovery is an inefficient process. Human pluripotent stem cell-derived cardiomyocytes can potentially fill the gap between animal and clinical studies, but conventional two-dimensional cultures inadequately recapitulate the human cardiac phenotype. Here, we systematically examined the pharmacological responses of engineered human ventricular-like cardiac tissue strips (hvCTS) and organoid chambers (hvCOC) to 25 cardioactive compounds covering various drug classes. While hvCTS effectively detected negative and null inotropic effects, the sensitivity to positive inotropes was modest. We further quantified the predictive capacity of hvCTS in a blinded screening, with accuracies for negative, positive, and null inotropic effects at 100%, 86%, and 80%, respectively. Interestingly, hvCOC, with a pro-maturation milieu that yields physiologically complex parameters, displayed enhanced positive inotropy. Based on these results, we propose a two-tiered screening system for avoiding false positives and negatives. Such an approach would facilitate drug discovery by leading to better overall success.

Bioengineering an electro-mechanically functional miniature ventricular heart chamber from human pluripotent stem cells.

Tissue engineers and stem cell biologists have made exciting progress toward creating simplified models of human heart muscles or aligned monolayers to help bridge a longstanding gap between experimental animals and clinical trials. However, no existing human in vitro systems provide the direct measures of cardiac performance as a pump. Here, we developed a next-generation in vitro biomimetic model of pumping human heart chamber, and demonstrated its capability for pharmaceutical testing. From human pluripotent stem cell (hPSC)-derived ventricular cardiomyocytes (hvCM) embedded in collagen-based extracellular matrix hydrogel, we engineered a three-dimensional (3D) electro-mechanically coupled, fluid-ejecting miniature human ventricle-like cardiac organoid chamber (hvCOC). Structural characterization showed organized sarcomeres with myofibrillar microstructures. Transcript and RNA-seq analyses revealed upregulation of key Ca2+-handling, ion channel, and cardiac-specific proteins in hvCOC compared to lower-order 2D and 3D cultures of the same constituent cells. Clinically-important, physiologically complex contractile parameters such as ejection fraction, developed pressure, and stroke work, as well as electrophysiological properties including action potential and conduction velocity were measured: hvCOC displayed key molecular and physiological characteristics of the native ventricle, and showed expected mechanical and electrophysiological responses to a range of pharmacological interventions (including positive and negative inotropes). We conclude that such "human-heart-in-a-jar" technology could facilitate the drug discovery process by providing human-specific preclinical data during early stage drug development.

Gene Delivery for the Generation of Bioartificial Pacemaker.

Electronic pacemakers have been used in patients with heart rhythm disorders for device-supported pacing. While effective, there are such shortcomings as limited battery life, permanent implantation of catheters, the lack of autonomic neurohumoral responses, and risks of lead dislodging. Here we describe protocols for establishing porcine models of sick sinus syndrome and complete heart block, and the generation of bioartificial pacemaker by delivering a strategically engineered form of hyperpolarization-activated cyclic nucleotide-gated pacemaker channel protein via somatic gene transfer to convert atrial or ventricular muscle cardiomyocytes into nodal-like cells that rhythmically fire action potentials.