Epi-SANOs (organoids integrated with epicardial cells) yielded beat-to-beat periods of 389±24ms vs 422±25ms in controls (p=0.36) and reduced short-term variability (9.0 vs 23.5ms, p=0.09).
Integrating epicardial cells into hiPSC-derived pacemaker organoids enhances cellular heterogeneity and stabilizes beat-to-beat regularity, providing a more accurate in vitro model of the native sinoatrial node.
Absolute Event Rate: 389% vs 422%
p-value: p=0.36
Introduction: The sinoatrial node (SAN) is heterogeneous and comprises pacemaker cells (PCs), atrial cardiomyocytes (ACMs), transitional zone (TZ) cells, and stromal cells. Epicardial cells (Epics) serve as a major source of nonmyocytes, contributing to sinoatrial node (SAN) development and function. The objective of this study is to generate a SAN tissue model from human induced pluripotent stem cells. Hypothesis: Integrating SAN-like organoids with Epics (Epi-SANOs) recapitulates heterogeneity of SAN and enables stable pacing with proper response to autonomic stimuli. Approach Epi-SANOs were generated by combining ACMs and PCs with WTC11-derived Epics at a 1:1 ratio at d15. Control SANOs were generated from WTC11-derived ACMs and PCs. Single-cell RNA sequencing was performed at d20, d25, and d30. The beating period, variability, and the chronotropic response to autonomic stimuli were analyzed using a multi-electrode array. Optical mapping with membrane potential-specific dye detects action potentials of the pacemaker from Epi-SANOs and those paced by Epi-SANOs. Results: The content of PCs marked by SHOX2+/cTNT+ cells was higher in Epi-SANOs (20%) than in control SANOs (16%) at d30. Fibroblast (FB) population was higher in Epi-SANOs than in control SANOs at d20 and d30, suggesting commitment of Epics to PCs and FBs. Native SAN is delineated into the head, tail, and TZ cells. Compared to the native SAN single-cell transcriptome, most PCs in control SANOs were mapped to TZ cells. In contrast, PCs in Epi-SANOs were identified as head and tail cells. To understand whether heterogenous SAN cells mimic SAN-like pacing, we assessed the beat-to-beat periods of Epi-SANOs and control SANOs; 389±24ms and 422±25ms, respectively (p=0.36, n=9). Analysis of beat-to-beat variability using Poincaré plots revealed that the SD1 (short-term variability) was smaller in Epi-SANOs than in control-SANOs (9.0±2.6 23.5±7.5ms, p=0.09, n=9). This result showed that Epics did not affect the mean pacing frequencies, however, Epi-SANOs tended to have more regular beat-to-beat automaticity compared to control SANOs. Following isoproterenol (sympathetic agonist) and carbachol (parasympathetic agonist) treatment, the beating rates of Epi-SANOs were changed with a wider dynamic range than control SANOs. Additionally, Epi-SANOs responded to Ivabradine, an I(f) channel blocker, by decreasing their beating rates. This data showed Epi-SANOs exhibit the ability to rate-adapt to physiological stimuli. Conclusion: Epics contribute to the heterogeneity of PCs in hiPSC-derived pacemaker organoids and stabilize beat-to-beat regularity. Our data support the potential of Epi-SANOs as organoid models of the native SAN. Funding sources AHA 25POST1366457 to MNK; NHLBI to HCC; Children’s Heart Foundation to HCC; Maryland Stem Cell Research Fund to HCC This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.
Koakutsu et al. (Fri,) conducted a other in Sinoatrial node tissue modeling (n=9). Epicardial cells integrated with SAN-like organoids (Epi-SANOs) vs. Control SANOs (without epicardial cells) was evaluated on Beat-to-beat periods (ms) (p=0.36). Epi-SANOs (organoids integrated with epicardial cells) yielded beat-to-beat periods of 389±24ms vs 422±25ms in controls (p=0.36) and reduced short-term variability (9.0 vs 23.5ms, p=0.09).