Poster H15 - Organoids as an Optimized In Vitro Models of Hypertrophic Cardiomyopathy

Friday, October 25, 2024

3:30 PM – 4:30 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Sara Kaufman

Charles R. Westgate Scholar
Johns Hopkins University
Hollywood, Florida, United States

Primary Investigator(s)

DK

Deok-Ho Kim

Professor
Johns Hopkins University
Ellicott City, Maryland, United States

Co-Author(s)

DL

Dong-Ik I. Lee, PhD

Faculty
Johns Hopkins University, United States
JC

Joseph Criscione

PhD Candidate
Johns Hopkins University
Bernards, New Jersey, United States
FY

Feiyu Yang

Introduction: This project aims to simulate hypertrophic cardiomyopathy in cardiac organoids using endothelin-1 (ET-1) and phenylephrine (PE). Cardiomyopathy has long been studied using animal models, but concerns regarding the organisms’ veracity in comparison to pathogenic manifestations in humans and ethical concerns have shifted focus to in-vitro disease models. Though hypertrophic cardiomyopathy has been simulated in two-dimensional environments, three-dimensional cell culture allows for more accurate modeling of cell-to-cell interactions and pathogenic behavior. To establish these models, cardiac organoids are first grown from WTC11 hiPSCs. The organoids are considered viable once they start beating spontaneously, on day 15. They are then treated with various concentrations of ET-1 (100, 300, and 1000 µM) and PE (30, 100, and 300 µM) until their functionality begins to decline (functional assays taken at days 3, 7, and 10). The effects of the vasoconstrictor peptide and 𝛂-receptor agonist are subsequently characterized using qPCR and functional testing. Assays include AlphaSMA, SERCA2a, and Myh7 to analyze organoid maturity; ANP, BNP, Col1a, Col3a, and TGF to quantify pathogenic remodeling; and contractility conduction velocity to measure the cardiac organoids’ functional phenotypes. In a pilot study, organoids were successfully generated and size measurements were collected on days 3, 7, and 10, and 15 to evaluate organoid maturity and viability. This ongoing project shows advantages in creating optimized, mature cardiac organoids that model hypertrophy. It can have widespread impacts in clinical research regarding heart failure, a global leading cause of death, by facilitating drug discovery and enhancing personalized medicine.

Materials and

Methods: This project comprises two aims. (1) Development of mature cardiac organoid from hPSCs following protocol from Lewis-Israeli, et al., (confirmed with AlphaSMA, SERCA2a, and Myh7 qPCR assays). (2) Development of hypertrophic cardiac disease model using cardiac organoids (induced by ET-1 and PE, characterized with the following hypertrophic and fibrotic markers: ANP, BNP, Col1a, Col3a, TGF by qPCR and Western blot analysis; contractility conduction velocity functional assay).

The following materials are necessary. Endothelin-1 (ET-1): Vasoconstrictor peptide used in experimental groups. Phenylephrine (PE): α-receptor agonist used in experimental groups. Standard cells, cell maintenance culture equipment, and facility: WTC11 hiPSCs (used to create organoids), E8 and RPMI media, PBS, cell incubator, biological hood, culture wells/flasks, pipettes; necessary to culture the hPSCs prior to and during differentiation. Stem cell differentiation and organoid upkeep supplies: CHIR99021, BMP4, Activin A, Wnt-C59, B27, and stem cell culture room/facility. Supplies for qPCR and Western analyses of gene and protein expression (hypertrophic markers ANP, BNP, and Myh7, AlphaSMA, SERCA2a, Col1a, Col3a, TGF): qPCR reagent kit, thermal cycler, fluorimeter, Western gels, filter paper, electrophoresis system. Supplies for contractile conduction velocity assay: CuriBio Mantarray.

As shown in Figure 1, cardiac organoids are first created from WTC11 hCMECs. This process takes 15 days and involves the supplies listed above. Then, as seen in Figure 2, analysis is conducted on days 15, 18, 22, and 25 of the experiment. These assays include contractility measurements, qPCR, and Western blots to quantify AlphaSMA, SERCA2a, Col1a, Col3a, TGF, Myh7, ANP, BNP, and conduction velocity.

Results, Conclusions, and Discussions: Hypertrophic cardiomyopathy (HCM) is a genetic heart disease that affects 1 in 500 patients and often leads to heart failure (HF) (Kramer et al., 2015). Heart disease is a global leading cause of death, and HF patients have a survival rate of ~50% (Ponzoni et al., 2023).

3D cell culture offers a promising future for drug testing that offers higher fidelity than both 2D culture and animal models. A leading form of 3D culture is currently cardiac organoids. However, cardiac organoids were only recently developed in 2020, and methods to induce heart failure in them are still in progress. Researchers recently attempted to model HCM in cardiac organoids using endothelin-1 (ET-1) but faced shortcomings that prevented development of a sound model.

To expand upon these previous findings, this study uses higher doses of ET-1, incorporates the use of PE, and involves a more thorough analysis of the organoids. This project is still ongoing. In a pilot test, organoids were successfully created, as shown in Figure 3. The organoids beat spontaneously and survived 15 days of culture post formation.

The organoids created in this study can be higher-fidelity models of HCM, providing increased clinical relevance. If successful in simulating HCM, this project will create a platform on which to expand the treatment of heart failure, the global leading cause of death. It can help streamline drug discovery and enable personalized medicine approaches, potentially helping save lives.

Acknowledgements (Optional): I am grateful for support from the Kim Laboratory at Johns Hopkins School of Medicine.