BS44 Generation of coronary smooth muscle cells from human embryonic stem cells for translational approaches in cardiac regeneration

Introduction Smooth muscle cells (SMCs) are important for coronary artery formation, which is crucial for the blood supply of the heart. Dysfunction in coronary SMCs (cSMCs), seen in conditions like atherosclerosis, contributes to heart attacks. Generating these cells from heart multipotent progenitors in vitro offers promise for disease modeling and regenerative therapies, given the unique characteristics of heart-specific vessel cells. Methods Previously, our laboratory developed a protocol for generating epicardial-derived SMCs from human embryonic stem cells (hESCs) using PDGF-B (P) and TGF-beta (T) signaling. We assessed coronary-specific marker expression using immunohistochemistry, RT-qPCR and a reporter cell line that we generated using CRISPR technology. We focused on PLA2G5, the most specific cSMC marker identified from our fetal heart atlas. Alongside PT conditions, we investigated the potential of RepSox, a small molecule that promoted SMC differentiation via NOTCH signaling, to potentially enhance cSMC marker expression. Results Human embryonic stem cell (hESC)-derived SMCs were differentiated from epicardial cells using PT and RepSox conditions. Epicardial cell seeding density was optimised using flow cytometry. The condition that led to the highest expression of SMC general markers, such as calponin and transgelin, was selected. Approximately 30% of the cell population co-expressed PLA2G5 alongside general SMC markers, as revealed by immunofluorescence staining. Using RepSox instead of TGF-beta signaling resulted in morphological differences, exhibiting a more rounded and proliferative phenotype compared to the PT condition. Furthermore, using the reporter cell line, epicardial cells exposed to PT or RepSox were positive for the red PLA2G5 signal from day 5 onwards, with an increased signal intensity observed in the RepSox condition until day 10 of differentiation. Conclusion We successfully differentiated SMCs from epicardial cells, co-expressing heart-specific coronary markers, yet the potential of RepSox to enhance Pla2G5 expression compared to PT requires further investigation. Moreover, the function of SMCs co-expressing coronary specific markers needs to be investigated to finally validate them for translational purposes. Conflict of Interest No