Kenji Miki

Pathological remodeling after myocardial infarction (MI) involves multifactorial mechanisms, highlighting the need for combinatorial therapeutic strategies. Synthetic mRNAs offer design flexibility and represent a modality particularly suitable for such approaches. To investigate this, five genes ( Hgf , Igf1 , Pdgfb , Cxcl12 , and Tgfβ1 ) were selected from a model in which extracellular vesicles secreted by human iPSC‐derived cardiomyocytes restored cardiac function. Synthetic mRNAs encoding these genes were delivered via polyplex nanomicelles by direct myocardial administration in mice with MI‐induced heart failure. Nanomicelles have been shown to provide stable encapsulation, enhanced local expression, and prolonged persistence. This treatment promoted angiogenesis via the PI3K–Akt–ETV4 axis, suppressed fibrosis via inhibition of the JNK/FOXO3 pathway, and enhanced repair by activating ERK signaling, together yielding multifaceted benefits, including tissue regeneration, improved contractility, and extended survival. These findings establish the therapeutic potential of multigene mRNA cocktail therapy for post‐MI heart failure and mark an important step toward developing new interventions for diseases characterized by complex remodeling.

Dilated cardiomyopathy (DCM) is the most prevalent pediatric cardiomyopathy and has a poor prognosis. Although heart transplantation is the only curative option, the severe donor shortage in Japan underscores an urgent need for alternative therapies. Here, we generated induced pluripotent stem cell (iPSC) lines from two unrelated children with the same TNNT2‐R151W mutation. We then investigated the cellular characteristics of the cardiomyocytes derived from these iPSC lines (R151W iPSC‐CMs) and evaluated their contractile function in pillar‐based engineered heart tissue (EHT). We also assessed the therapeutic potential of overexpressing wild‐type TNNT2 in patient‐derived iPSCs (wtTNNT2‐OE‐iPSCs). R151W‐iPSC‐CMs exhibited pronounced sarcomere disarray, attenuated Ca ²⁺ transient amplitude, prolonged time to peak, and delayed decay tau. These characteristics are indicative of failing myocardium and were restored in wtTNNT2‐OE‐iPSC‐CMs. Pillar‐based EHT assays revealed a substantial decrease in contractile force in R151W‐EHTs compared to wtTNNT2‐OE‐EHTs, which is suggestive of the systolic dysfunction observed clinically in DCM. Collectively, these results provide the first functional evidence that the TNNT2‐R151W mutation leads to pediatric DCM by causing sarcomere insufficiency and disturbances in Ca ²⁺ handling. Our patient‐specific iPSC‐EHT platform faithfully recapitulates key features of pediatric DCM and could offer a robust system for mechanistic studies and drug testing. Furthermore, phenotypic rescue upon overexpression of wild‐type TNNT2 suggests that this allele is amenable to a gene replacement approach aimed at restoring wild‐type function in TNNT2‐mutant cardiomyopathies.

Current differentiation protocols for human induced pluripotent stem cells (hiPSCs) produce heterogeneous cardiomyocytes (CMs). Although chamber-specific CM selection using cell surface antigens enhances biomedical applications, a cell surface marker that accurately distinguishes between hiPSC-derived atrial CMs (ACMs) and ventricular CMs (VCMs) has not yet been identified. We have developed an approach for obtaining functional hiPSC-ACMs and -VCMs based on CD151 expression. For ACM differentiation, we found that ACMs are enriched in the CD151^low population and that CD151 expression is correlated with the expression of Notch4 and its ligands. Furthermore, Notch signaling inhibition followed by selecting the CD151^low population during atrial differentiation leads to the highly efficient generation of ACMs as evidenced by gene expression and electrophysiology. In contrast, for VCM differentiation, VCMs exhibiting a ventricular-related gene signature and uniform action potentials are enriched in the CD151^high population. Our findings enable the production of high-quality ACMs and VCMs appropriate for hiPSC-derived chamber-specific disease models and other applications.

Research on cardiomyopathy models using engineered heart tissue (EHT) created from disease‐specific induced pluripotent stem cells (iPSCs) is advancing rapidly. However, the study of restrictive cardiomyopathy (RCM), a rare and intractable cardiomyopathy, remains at the experimental stage because there is currently no established method to replicate the hallmark phenotype of RCM, particularly diastolic dysfunction, in vitro. In this study, we generated iPSCs from a patient with early childhood‐onset RCM harboring the TNNI3 R170W mutation (R170W‐iPSCs). The properties of R170W‐iPSC‐derived cardiomyocytes (CMs) and EHTs were evaluated and compared with an isogenic iPSC line in which the mutation was corrected. Our results indicated altered calcium kinetics in R170W‐iPSC‐CMs, including prolonged tau, and an increased ratio of relaxation force to contractile force in R170W‐EHTs. These properties were reversed in the isogenic line, suggesting that our model recapitulates impaired relaxation of RCM, i.e., diastolic dysfunction in clinical practice. Furthermore, overexpression of wild‐type TNNI3 in R170W‐iPSC‐CMs and ‐EHTs effectively rescued impaired relaxation. These results highlight the potential efficacy of EHT, a modality that can accurately recapitulate diastolic dysfunction in vitro, to elucidate the pathophysiology of RCM, as well as the possible benefits of gene therapies for patients with RCM.

Epicardial formation is necessary for normal myocardial morphogenesis. Here, we show that differentiating hiPSC-derived lateral plate mesoderm with BMP4, RA and VEGF (BVR) can generate a premature form of epicardial cells (termed pre-epicardial cells, PECs) expressing WT1, TBX18, SEMA3D, and SCX within 7 days. BVR stimulation after Wnt inhibition of LPM demonstrates co-differentiation and spatial organization of PECs and cardiomyocytes (CMs) in a single 2D culture. Co-culture consolidates CMs into dense aggregates, which then form a connected beating syncytium with enhanced contractility and calcium handling; while PECs become more mature with significant upregulation of UPK1B, ITGA4, and ALDH1A2 expressions. Our study also demonstrates that PECs secrete IGF2 and stimulate CM proliferation in co-culture. Three-dimensional PEC-CM spheroid co-cultures form outer smooth muscle cell layers on cardiac micro-tissues with organized internal luminal structures. These characteristics suggest PECs could play a key role in enhancing tissue organization within engineered cardiac constructs in vitro.

One of the earliest maturation steps in cardiomyocytes (CMs) is the sarcomere protein isoform switch between TNNI1 and TNNI3 (fetal and neonatal/adult troponin I). Here, we generate human induced pluripotent stem cells (hiPSCs) carrying a TNNI1^EmGFP and TNNI3^mCherry double reporter to monitor and isolate mature sub-populations during cardiac differentiation. Extensive drug screening identifies two compounds, an estrogen-related receptor gamma (ERRγ) agonist and an S-phase kinase-associated protein 2 inhibitor, that enhances cardiac maturation and a significant change to TNNI3 expression. Expression, morphological, functional, and molecular analyses indicate that hiPSC-CMs treated with the ERRγ agonist show a larger cell size, longer sarcomere length, the presence of transverse tubules, and enhanced metabolic function and contractile and electrical properties. Here, we show that ERRγ-treated hiPSC-CMs have a mature cellular property consistent with neonatal CMs and are useful for disease modeling and regenerative medicine.

Specific cell lineages differentiated from human induced pluripotent stem cells (iPSCs) are promising sources for cell replacement therapy and are useful biomedical research tools for research on disease mechanisms and drug discovery. Among the different lineages, cardiac lineage has been one of the most efficiently differentiated through several established protocols. In this chapter, we describe our reproducible and highly efficient methodology for differentiating iPSCs into cardiomyocytes using embryoid bodies. We also describe methods to dissociate iPSC-derived cardiomyocytes and to evaluate iPSC-derived cardiomyocytes.

Transplant of human induced pluripotent stem cell derived cardiomyocytes (hiPS-CMs) cell-sheet is a promising approach for treating ischemic cardiomyopathy (ICM). However, poor blood supply to the transplanted cell-sheet is a concern related to the effectiveness and durability of the treatment. Herein, we hypothesized that the combined the omentum flap might enhance survival and the therapeutic effects of hiPS-CM cell-sheet transplant for ICM treatment. Treatment by Wnt signaling molecules in hiPS cells produced hiPS-CMs, which were magnetically labeled by superparamagnetic iron oxide (SPIO), followed by culture in the thermoresponsive dishes to generate hiPS-CMs cell-sheets. A porcine ICM model included 4 groups; sham operation, omentum flap only, cell-sheet only, or combination therapy. Ejection fraction (EF) was significantly greater in the cell-sheet only and combination group compared to the other groups during the follow-up period. At 3 months, the EF of the combination group was significantly greater than that of the cell-sheet only group. Consistently, the survival rate of the SPIO-labeled hiPS-CMs, as assessed by MRI, was significantly greater in the combination group than in the cell-sheet only group. This cell delivery system would be useful in optimizing the hiPS-CM cell-sheet transplant for treating severe heart failure.