Poster U6 - Enhanced Vascularization in hPSC-derived Cardiac Organoids by Dynamic Culture

Introduction: Human pluripotent stem cells (hPSC)-derived cardiac organoids (COs) have gained increasing interest in biotechnology due to their characteristics like diverse cardiovascular cell types, 3D complex structure, and function that recapitulates the human heart in early development. However, the lack of an essential vascular network in the human heart remains a major challenge for enabling further applications of COs in cardiovascular research. In this study, we explored the possibility of vascularization enhancement by controlled dynamic culture of hPSC directly differentiated COs.

Materials and

Methods: Two conditions were created to compare vascularization in COs: 1) dynamic culturing (Dyn) and 2) static culturing (St). Firstly, hPSCs were cultured for two days in ultra-low attachment plates to develop spheroids. The spheroids were then transferred to pillar plates followed by direct cardiac differentiation the next day. Dynamic culturing is applied on day 7 of cardiac differentiation. Live-cell imaging was used to monitor the process of vascularization for up to 16 days and the corresponding fluorescence intensity of each cell type from its reporter expression was analyzed. The cardiac function was measured by video-based contractility analysis. Data were presented as mean +/- standard error of the mean (SEM). The student’s t-test was used for the statistical analysis.

Results, Conclusions, and Discussions: Cardiomyocytes (CMs) and endothelial cells (ECs) were imaged on pillars under the two conditions shown in Figs. 1A and B. Successful cardiac differentiation was present, displaying hPSC-CM contraction on day 12 and onwards. Higher intensity values of hPSC-CMs under Dyn were observed in all four days compared to St. The hPSC-ECs intensity displayed greater intensity values compared to St, demonstrating enhanced vascularization in VCOs due to the active Dyn as shown in Figs. 1C and D. The functionality was improved in Dyn as indicated with higher contraction velocity as shown in Fig. 1E. We also observed consistent chamber-like formation in the Dyn group.

This study demonstrates the effects of incorporating dynamic culture for enhancing cardiogenesis and vasculogenesis in hPSC-derived COs, which hold great promise for biomedical applications in modeling early cardiovascular development, congenital heart disease, and serving more advanced platform for cardiovascular pharmaceutical discoveries.

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