Poster AA19 - Differentiation of hPSC-derived Cardiac Organoids with Consistent Chamber Formation

Introduction: Cardiovascular diseases (CVDs) are a major global health issue, and better strategies for in vitro modeling of CVDs are urgently needed. Compared with the traditional 2D cell culture models, which are not close to the human heart physiology in structure and function, the 3D human pluripotent stem cell (hPSC)-derived cardiac organoids offer great promises for cardiovascular disease modeling and drug testing. However, the current method for differentiating hPSC-derived cardiac organoids cannot meet the general requirement of recapitulating heart morphology with robust and consistent cardiac chamber formation. Establishing a differentiation protocol for obtaining chamber-forming cardiac organoids reproducibly becomes essential and significant. Our hypothesis posits that following the physiological pathway of early heart development via cell self-organization and Wnt signaling, specifically the size of embryonic body (EB) and CHIR99201 concentration, we can modulate the cardiogenic differentiation and foster the development of chamber-like structures in the cardiac organoids.

Materials and

Methods: Triple reporter hPSC line (3R) expressing fluorescent proteins linked to cardiac marker genes (TNNT2: GFP, CDH5: mOrange, TAGLN: CFP) was utilized for cardiac organoid differentiation. Cells were cultured in Matrigel-coated plates with E8 medium supplemented with ROCK inhibitor. Subsequently, passaged cells were seeded into ultra-low attachment (ULA) plates for organoid formation, with seeding densities of 2500 or 5000 cells per well. Mesoderm formation was induced using CHIR99021, followed by differentiation into cardiac organoids over 15 days. Imaging of organoids was done by Keyence fluorescence microscope and confocal microscope, focusing on organoid area and fluorescence signals. Image analysis was performed using BioLabel and Organlysis software, while contractility was analyzed using ImageJ macro "MUSCLEMOTION."

Results, Conclusions, and Discussions:

Results: The cardiac organoid differentiation was robust and consistent across all sizes of EB and CHIR concentration with regular contractions. There was 89% EB successfully differentiated into cardiac organoids (Fig.1A). Chamber-forming cardiac organoids were identified in Fig.1B by confocal microscope imaging and cryosectioning. The large EB size (5000 cells) yielded over 80% chamber formation in comparison to the small EB size (2500 cells) in only 147 out of 400 organoids (Fig.1C) along with varied cardiogenesis at different CHIR concentrations (Fig.1D). Longer and more obvious contractions were observed in chamber-forming cardiac organoids compared to non-chamber-forming cardiac organoids (Fig.1D)

Our study demonstrates the intricate impact of CHIR99021 concentrations and EB size on cardiac chamber formation with longer and more pronounced contractions in hPSC-derived cardiac organoids. Future research will explore the relationship between chamber formation and cardiac biomechanics and further corresponding molecular mechanisms.

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