Yoshinori Yoshida

Engineered heart tissues (EHTs) have been used in cardiovascular research for nearly three decades, addressing limitations of animal models and oversimplified two-dimensional single-cell systems in recapitulating cardiac (patho) physiology. Recently, epicardium-integrated EHTs have gained attention as therapeutic tools for remuscularizing the failing heart. The epicardium, the outermost mesothelial layer of the heart, plays a crucial role in cardiac development and disease by serving as a source of progenitor cells and by providing essential paracrine signalling. Hutchinson–Gilford progeria syndrome (HGPS) is a laminopathy caused by a point mutation in the LMNA gene, leading to expression of progerin and resulting in premature aging with a severe cardiovascular phenotype. We developed an epicardium-integrated EHT model to enable more physiologically relevant in vitro modelling of HGPS. Epicardial cells were incorporated into the EHTs and maintained in long-term culture. Cellular differentiation, progerin expression, transcriptomic changes, and functional responses to electrical pacing were assessed over time. Epicardial cells were observed to differentiate into cardiac fibroblasts and vascular smooth muscle cells, enabling incorporation of key non-myocyte populations relevant to HGPS pathology. The epicardium-integrated EHTs exhibited progerin expression and developed hallmarks of HGPS within 60 d of culture. Gene set enrichment analysis revealed upregulation of pathways associated with atherosclerosis, apoptosis, fibrosis, and oxidative stress, which are processes linked to both HGPS and physiological aging. Additionally, a culture time-dependent decline in the contractile response of EHTs to pacing was observed. In conclusion, our epicardium-integrated EHT model recapitulates key molecular, cellular, and functional features of HGPS-associated CVD. This platform provides a promising tool for investigating HGPS disease mechanisms and evaluating therapeutic strategies and may be adaptable for modelling other CVDs and regenerative applications.

NUP98 rearrangements represent a distinct, high-risk subtype of acute myeloid leukemia (AML), particularly in pediatric patients. However, their prevalence, genetic features, and clinical implications in adult AML remain poorly characterized. Using targeted-capture sequencing, we identified 41 cases with NUP98 rearrangements among 1569 AML cases, representing the majority of 44 NUP98-rearranged cases detected across 4753 myeloid neoplasms. Fifteen distinct fusion partners were detected, with NUP98::NSD1 and NUP98::HOXA9 being the most frequent. Notably, two novel fusions—NUP98::MEOX2 and NUP98::HOXA6—were identified. Co-mutations were relatively infrequent; FLT3-ITD and WT1 mutations were the most common, while NPM1 mutations were exclusive. FLT3-ITD was significantly enriched in NUP98::NSD1 cases, whereas TET2 mutations were more frequent in NUP98::HOXA9 cases. Clonal hierarchy analysis suggested that NUP98 rearrangements occur early in leukemogenesis. NUP98-rearranged AML exhibited higher relapse rates and shorter event-free survival. Specifically, NUP98::NSD1 was associated with a poor induction response, whereas NUP98::HOXA9 and NUP98 fusions with other partners showed higher remission rates but frequent relapse. Allogeneic hematopoietic stem cell transplantation was associated with better survival, underscoring its significance. These findings reveal the genetic and clinical heterogeneity of NUP98-rearranged AML in adults and support its classification as a distinct entity, highlighting the need for fusion partner-specific therapeutic strategies.

Reactivating the human epicardium post-cardiac injury holds promise for cardiac tissue regeneration. Despite successful differentiation protocols yielding pure, self-renewing epicardial cells from induced pluripotent stem cells (iPSCs), these cells maintain an embryonic, proliferative state, impeding adult epicardial reactivation investigation. We introduce an optimized method that employs mammalian target of rapamycin (mTOR) signaling inhibition in embryonic epicardium, inducing a quiescent state that enhances multi-step epicardial maturation. This yields functionally mature epicardium, valuable for modeling adult epicardial reactivation. Furthermore, we assess cardiac organoids with cardiomyocytes and mature epicardium, probing molecular mechanisms governing epicardial quiescence during cardiac maturation. Our results highlight iPSC-derived mature epicardium’s potential in investigating adult epicardial reactivation, pivotal for effective cardiac regeneration. Additionally, the cardiac organoid model offers insight into intricate cardiomyocyte-epicardium interactions in cardiac development and regeneration.

NUP98 rearrangements represent a distinct, high-risk subtype of acute myeloid leukemia (AML), particularly in pediatric patients. However, their prevalence, genetic features, and clinical implications in adult AML remain poorly characterized. Using targeted-capture sequencing, we identified 41 cases with NUP98 rearrangements among 1569 AML cases, representing the majority of 44 NUP98 -rearranged cases detected across 4,753 myeloid neoplasms. Fifteen distinct fusion partners were detected, with NUP98::NSD1 and NUP98::HOXA9 being the most frequent. Notably, two novel fusions— NUP98::MEOX2 and NUP98::HOXA6 —were identified. Co-mutations were relatively infrequent; FLT3 -ITD and WT1 mutations were the most common, while NPM1 mutations were exclusive. FLT3 -ITD was significantly enriched in NUP98::NSD1 cases, whereas TET2 mutations were more frequent in NUP98::HOXA9 cases. Clonal hierarchy analysis suggested that NUP98 rearrangements occur early in leukemogenesis. NUP98 -rearranged AML exhibited higher relapse rates and shorter event-free survival. Specifically, NUP98::NSD1 was associated with a poor induction response, whereas NUP98::HOXA9 and NUP98 fusions with other partners showed higher remission rates but frequent relapse. Allogeneic hematopoietic stem cell transplantation was associated with better survival, underscoring its significance. These findings reveal the genetic and clinical heterogeneity of NUP98 -rearranged AML in adults and support its classification as a distinct entity, highlighting the need for fusion partner-specific therapeutic strategies.

Numerous reports investigating channelopathies, including Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT), have successfully reproduced using cardiomyocytes (CMs) differentiated from human induced pluripotent stem cells (hiPSCs). However, the relationship between action potentials (AP) and calcium transient waveforms—especially after drug treatment—remains unclear. In this study, we simultaneously loaded a membrane potential dye FluoVolt and the new calcium indicator Calbryte^TM 590 AM and optimized stimulation and detection of both dyes to successfully obtain a higher signal-to-noise (S/N) ratio than the conventional membrane potential dye-red fluorescence Ca²⁺ dye combination, thus enabling the simultaneous recording of both AP and calcium transient waveforms in single hiPSC-CMs, which continued even after gradual increases in drug concentration. In drug-loading experiments on CPVT1 (RyR2-I4587V) hiPSC-derived ventricular-like CMs, carvedilol and flecainide demonstrated some effectiveness, while JTV519 at 3 µM exhibited both efficacy and alterations in AP waveforms. The Ca²⁺/calmodulin-dependent serine-threonine protein kinase II (CaMKII) inhibitor KN-93 at 1 µM was highly effective (93%) at reducing Ca²⁺ transient abnormalities without altering AP waveforms.

Engineered heart tissues (EHTs) using human induced pluripotent stem cells provide a valuable in vitro platform for assessing pharmacological and toxicological effects on cardiac functions. EHT devices offer a feasible approach to readily evaluate drug responses on contractile properties, including contractile force and relaxation, by measuring the moving distance of pillars attached to EHTs. However, the absorption of small molecule compounds by polydimethylsiloxane (PDMS), the material commonly used to construct EHT devices, hinders the accurate evaluation of the pharmacodynamic properties of drug candidates on contractility. Here, we developed a low-absorption EHT device using polystyrene (PS) to address this issue. Moreover, we generated an original Python-based analysis program to avoid analytical bias when tracking pillar positions and analyzing the contractile waveform drawn from EHT movements. This analytical platform enables the detection of increased contractile force during EHT maturation and the negative inotropic effects of diltiazem and blebbistatin on EHT contractile functions. Moreover, EHTs with PS-based devices suppressed the absorption of a cardiotoxic drug, doxorubicin, thus allowing the detection of cardiotoxic effects even at low concentrations compared to EHTs grown on PDMS-based devices. Together, our EHT system represents a useful in vitro platform for accurate evaluations of drug responses by the human heart.