Engineering the Building Blocks of a Biological Pacemaker

Abstract

Conventional electronic pacemakers have long been the standard of care for cardiac conduction disorders. However, these devices carry significant limitations including lead-related complications, possibility of infection, finite battery life, and an inability to mature alongside pediatric patients. Biological pacemakers are a compelling alternative, as these tissue-based constructs derived from human stem cells have the potential to restore native electromechanical function without a dependence on hardware. This thesis presents a systems-oriented approach to constructing the foundational elements of a biological pacemaker using cardiac organoids derived from human-induced pluripotent stem cells. Three primary aims were explored in this work: the transdifferentiation of three-dimensional His Purkinje-like organoids, the development of sinoatrial node-like organoids, and the design and validation of a catheter-compatible implantation strategy. The His Purkinje specification was pursued through the iterative adaptation of a foundational two-dimensional differentiation protocol, using a combination of different small molecules referred to as a PURK-Cocktail to drive cardiomyocytes to a specialized fate. This protocol was initially applied to whole three-dimensional organoids, and then later returned to a two-dimensional format to optimize drug and cell density parameters for later downstream reaggregation. We identified 5105 cells at 0.5X to be the most favorable conditions for upregulation of conduction markers ETV1, CX40, and HCN4. The sinoatrial nodal specification was studied through iterative adaptation of a NODAL inhibition-based differentiation protocol, using SB431542 alongside the standard differentiation protocol for cardiomyocytes. A combination of 12 μM IWR and 1 μM SB431542 in mini spinner reactors indicated sinoatrial specification through the preservation of cardiac identity, upregulation of key markers like SHOX2 and HCN4, and significant increase in beat frequency when compared to ventricular myocytes. For the implantation strategy, a nitinol coronary stent coated with PDMS was loaded with a mixed population of cardiac and vascular organoids in a Matrigel matrix. These organoids remained viable after one week within the stent, and confocal imaging suggested early integration. To further explore vascularization, cardiac and endothelial single cells were co-aggregated at defined ratios to yield composite organoids with evident colocalization between the two target populations. Together, these results establish a foundational systems-level framework for engineering and implanting a biological pacemaker.