Combinatorial maturation strategy for disease modeling and phenotypic drug screening of Duchenne muscular dystrophy cardiomyopathy
Macadangdang, Jesse Radcliff
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Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) offer great promise for regenerative medicine, preclinical drug screening, and cardiac disease modeling applications. One of the most significant hurdles towards adoption of hPSC-CM technologies, however, is cardiomyocyte developmental immaturity. Current differentiation methods produce hPSC-CMs with structural and functional characteristics most closely resembling fetal cardiomyocytes, which significantly hinders our ability to predict patient drug responses or model adult-onset cardiomyopathies. The following dissertation addresses this challenge with the goal of engineering structurally and functionally mature cardiac tissues from hPSC-CMs for in vitro disease modeling and drug screening applications. Here, we present the development of a bio-inspired, combinatorial method for enhancing the maturation of hPSC-CMs that incorporates distinct physical, biochemical, and genetics cues. We began by investigating the role of surface nanotopography on hPSC-CM development and found that, similar to primary cardiomyocytes, hPSC-CMs exhibited a nanotopographic size-dependent phenotype. Utilizing the optimal nanotopographic surfaces dimensions for promoting maturation, we tested whether this maturation cue alone could improve our ability to model the cardiomyopathy associated with Duchenne Muscular Dystrophy (DMD). Although we were able to measure a blunted cytoskeletal response to the nanotopography in dystrophin-null hPSC-CMs, this difference was mild and we were unable to detect a functional disease phenotype. We therefore explored more comprehensive methods for inducing hPSC-CM maturation and developed our combinatorial maturation (ComboMat) protocol. The ComboMat protocol incorporates biomimetic nanotopography, thyroid hormone T3, and Let7i microRNA overexpression to produce hPSC-CMs with enhanced sarcomere development, improved electrophysiological and contractile function, improved mitochondrial respiratory capacity, and a transcriptome upregulated for metabolic and muscle development. When the ComboMat protocol is applied to a CRISPR-edited dystrophin knockout (KO) model of DMD cardiomyopathy, a distinctive, endogenously occurring disease phenotype emerges. Mature dystrophin KO hPSC-CMs exhibit greater propensity for arrhythmia with a higher resting cytosolic calcium content compared to Normal hPSC-CM controls. A phenotypic drug screen of dystrophin KO hPSC-CMs using the ComboMat protocol identified compounds that mitigated arrhythmogenic behavior. The ComboMat protocol can be applied to other cardiac disease models, cardiotoxicity studies, or cardiac tissue engineering applications. In vitro screening assays must predict the response of the human heart with high fidelity in order to be adopted. Taken together, this research demonstrates the utility of bioengineering strategies to mature hPSC-CMs in order to develop more biomimetic, adult-like cardiac tissues for preclinical screening applications.
- Bioengineering