Tani Hidenori

Human pluripotent stem cell-derived cardiomyocyte (hPSC-CM) differentiation can improve using chemical compounds which mimic early heart development. However, variations in hPSC-CM differentiation efficiency and its poor reproducibility have remained a challenge. Here, we report a unique metabolic method to promote hPSC-CM differentiation that involves marked suppression of the mitochondrial oxidative phosphorylation from the mesendoderm to the cardiac mesoderm, which is regulated by PHGDH, a rate-limiting enzyme in the serine synthesis pathway. Mechanistically, PHGDH inhibition impairs mitochondrial respiration by blocking the electron transport chain, resulting in elevated ROS levels and promoting the cardiomyocyte lineage specification by disrupting the cardiopharyngeal mesoderm lineage differentiation. Additionally, antioxidant supplementation can scavenge ROS and eliminate the effects of PHGDH inhibition. Collectively, our findings show that serine synthesis pathway can regulate cardiomyocyte lineage specification and have implications in providing a cellular source for transplantation and elucidating the potential mechanisms of heart development and pathogenesis of heart disease.

Three-dimensional cultures mimic in vivo environments better than two-dimensional cultures and are often used in drug discovery research. Herein, we present a protocol for producing homogeneous induced pluripotent stem cell (iPSC) spheroids and microtissues using the suction technique. We describe steps for preparing the suction device, preparing and seeding cells, and suction sedimentation of cells. We then detail procedures for self-assembly and evaluation of spheroids. For complete details on the use and execution of this protocol, please refer to Moriwaki et al.1.

Recently human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have become an attractive platform to evaluate drug responses for cardiotoxicity testing and disease modeling. Moreover, three-dimensional (3D) cardiac models, such as engineered heart tissues (EHTs) developed by bioengineering approaches, and cardiac spheroids (CSs) formed by spherical aggregation of hPSC-CMs, have been established as useful tools for drug discovery and transplantation. These 3D models overcome many of the shortcomings of conventional 2D hPSC-CMs, such as immaturity of the cells. Cardiac organoids (COs), like other organs, have also been studied to reproduce structures that resemble a heart in vivo more closely and optimize various culture conditions. Heart-on-a-chip (HoC) developed by a microfluidic chip-based technology that enables real-time monitoring of contraction and electrical activity, provides multifaceted information that is essential for capturing natural tissue development in vivo. Recently, 3D experimental systems have been developed to study organ interactions in vitro. This review aims to discuss the developments and advancements of hPSC-CMs and 3D cardiac tissues.

There are high expectations regarding heart regeneration for refractory heart failure (HF). Transplantation of human pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs) is expected to replace CMs lost due to HF, and various studies have been conducted to apply this therapy clinically. Though issues such as arrhythmias and immune rejection remain, the mass production of purified hPSC-derived CMs, their efficient transplantation, and methods to improve their engraftment pushed up the transplantation of hPSC-derived CMs to the clinical stage. In contrast, a direct cardiac reprogramming method has been developed, where cardiac fibroblasts are directly converted into CM-like cells without undergoing PSCs by overexpressing reprogramming factors. Although many challenges still remain in the clinical application of direct cardiac reprogramming, this can be a novel treatment which overcomes issues of transplantation of hPSC-derived CMs.

Cardiac regenerative therapy using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) has been applied in clinical settings. Herein, we aimed to investigate the in vivo metabolic profiles of hiPSC-CM grafts. RNA sequencing and imaging mass spectrometry were performed in the present study, which revealed that hiPSC-CM grafts matured metabolically over time after transplantation. Glycolysis, which was active in the hiPSC-CM grafts immediately after transplantation, shifted to fatty acid oxidation. Additionally, we examined the metabolic profile of teratomas that may form when non-CMs, including undifferentiated human induced pluripotent stem cells (hiPSCs), remain in transplanted cells. The upregulated gene expression of amino acid transporters and the high accumulation of amino acids, such as methionine and aromatic amino acids, were observed in the teratomas. We show that subcutaneous teratomas derived from undifferentiated hiPSCs can be detected in vivo using positron emission tomography with [18F]fluorophenylalanine ([18F]fPhe). These results provided insights into the clinical application of cardiac regenerative therapy.

Abstract Here an electrical stimulation system is described for maturing microfiber‐shaped cardiac tissue (cardiac microfibers, CMFs). The system enables stable culturing of CMFs with electrical stimulation by placing the tissue between electrodes. The electrical stimulation device provides an electric field covering whole CMFs within the stimulation area and can control the beating of the cardiac microfibers. In addition, CMFs under electrical stimulation with different frequencies are examined to evaluate the maturation levels by their sarcomere lengths, electrophysiological characteristics, and gene expression. Sarcomere elongation (14% increase compared to control) is observed at day 10, and a significant upregulation of electrodynamic properties such as gap junction protein alpha 1 (GJA1) and potassium inwardly rectifying channel subfamily J member 2 (KCNJ2) (maximum fourfold increase compared to control) is observed at day 30. These results suggest that electrically stimulated cultures can accelerate the maturation of microfiber‐shaped cardiac tissues compared to those without electrical stimulation. This model will contribute to the pathological research of unexplained cardiac diseases and pharmacologic testing by stably constructing matured CMFs.

BACKGROUND: The clinical application of human induced pluripotent stem cell-derived cardiomyocytes (CMs) for cardiac repair commenced with the epicardial delivery of engineered cardiac tissue; however, the feasibility of the direct delivery of human induced pluripotent stem cell-derived CMs into the cardiac muscle layer, which has reportedly induced electrical integration, is unclear because of concerns about poor engraftment of CMs and posttransplant arrhythmias. Thus, in this study, we prepared purified human induced pluripotent stem cell-derived cardiac spheroids (hiPSC-CSs) and investigated whether their direct injection could regenerate infarcted nonhuman primate hearts. METHODS: We performed 2 separate experiments to explore the appropriate number of human induced pluripotent stem cell-derived CMs. In the first experiment, 10 cynomolgus monkeys were subjected to myocardial infarction 2 weeks before transplantation and were designated as recipients of hiPSC-CSs containing 2×107 CMs or the vehicle. The animals were euthanized 12 weeks after transplantation for histological analysis, and cardiac function and arrhythmia were monitored during the observational period. In the second study, we repeated the equivalent transplantation study using more CMs (6×107 CMs). RESULTS: Recipients of hiPSC-CSs containing 2×107 CMs showed limited CM grafts and transient increases in fractional shortening compared with those of the vehicle (fractional shortening at 4 weeks after transplantation: 26.2±2.1%; 19.3±1.8%; P<0.05), with a low incidence of posttransplant arrhythmia. Transplantation of increased dose of CMs resulted in significantly greater engraftment and long-term contractile benefits (fractional shortening at 12 weeks after transplantation: 22.5±1.0%; 16.6±1.1%; P<0.01, left ventricular ejection fraction at 12 weeks after transplantation: 49.0±1.4%; 36.3±2.9%; P<0.01). The incidence of posttransplant arrhythmia slightly increased in recipients of hiPSC-CSs containing 6×107 CMs. CONCLUSIONS: We demonstrated that direct injection of hiPSC-CSs restores the contractile functions of injured primate hearts with an acceptable risk of posttransplant arrhythmia. Although the mechanism for the functional benefits is not fully elucidated, these findings provide a strong rationale for conducting clinical trials using the equivalent CM products.

Cardiac regenerative therapy using human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is expected to become an alternative to heart transplantation for severe heart failure. It is now possible to produce large numbers of human pluripotent stem cells (hPSCs) and eliminate non-cardiomyocytes, including residual undifferentiated hPSCs, which can cause teratoma formation after transplantation. There are two main strategies for transplanting hPSC-CMs: injection of hPSC-CMs into the myocardium from the epicardial side, and implantation of hPSC-CM patches or engineered heart tissues onto the epicardium. Transplantation of hPSC-CMs into the myocardium of large animals in a myocardial infarction model improved cardiac function. The engrafted hPSC-CMs matured, and microvessels derived from the host entered the graft abundantly. Furthermore, as less invasive methods using catheters, injection into the coronary artery and injection into the myocardium from the endocardium side have recently been investigated. Since transplantation of hPSC-CMs alone has a low engraftment rate, various methods such as transplantation with the extracellular matrix or non-cardiomyocytes and aggregation of hPSC-CMs have been developed. Post-transplant arrhythmias, imaging of engrafted hPSC-CMs, and immune rejection are the remaining major issues, and research is being conducted to address them. The clinical application of cardiac regenerative therapy using hPSC-CMs has just begun and is expected to spread widely if its safety and efficacy are proven in the near future.

Three-dimensional (3D) cultures are known to more closely mimic in vivo conditions compared with 2D cultures. Cardiac spheroids (CSs) and organoids (COs) are useful for 3D tissue engineering and are advantageous for their simplicity and mass production for regenerative therapy and drug discovery. Herein, we describe a large-scale method for producing homogeneous human induced pluripotent stem cell (hiPSC)-derived CSs (hiPSC-CSs) and COs without scaffolds using a porous 3D microwell substratum with a suction system. Our method has many advantages, such as increased efficiency and improved functionality, homogeneity, and sphericity of hiPSC-CSs. Moreover, we have developed a substratum on a clinically relevant large scale for regenerative therapy and have succeeded in producing approximately 40,000 hiPSC-CSs with high sphericity at once. Furthermore, we efficiently produced a fused CO model consisting of hiPSC-derived atrial and ventricular cardiomyocytes localized on opposite sides of one organoid. This method will facilitate progress toward hiPSC-based clinical applications.

Cardiomyocytes (CMs) have little regenerative capacity. After myocardial infarction (MI), scar formation and myocardial remodeling proceed in the infarct and non-infarct areas, respectively, leading to heart failure (HF). Prolonged activation of cardiac fibroblasts (CFs) and inflammatory cells may contribute to this process; however, therapies targeting these cell types remain lacking. Cardiac reprogramming converts CFs into induced CMs, reduces fibrosis, and improves cardiac function in chronic MI through the overexpression of Mef2c/Gata4/Tbx5/Hand2 (MGTH). However, whether cardiac reprogramming reduces inflammation in infarcted hearts remains unclear. Moreover, the mechanism through which MGTH overexpression in CFs affects inflammatory cells remains unknown. Here, we showed that inflammation persists in the myocardium until three months after MI, which can be reversed with cardiac reprogramming. Single-cell RNA sequencing demonstrated that CFs expressed pro-inflammatory genes and exhibited strong intercellular communication with inflammatory cells, including macrophages, in chronic MI. Cardiac reprogramming suppressed the inflammatory profiles of CFs and reduced the relative ratios and pro-inflammatory signatures of cardiac macrophages. Moreover, fluorescence-activated cell sorting analysis (FACS) revealed that cardiac reprogramming reduced the number of chemokine receptor type 2 (CCR2)-positive inflammatory macrophages in the non-infarct areas in chronic MI, thereby restoring myocardial remodeling. Thus, cardiac reprogramming reduced the number of inflammatory macrophages to exacerbate cardiac function after MI.

Monitoring cardiac differentiation and maturation from human pluripotent stem cells (hPSCs) and detecting residual undifferentiated hPSCs are indispensable for the development of cardiac regenerative therapy. MicroRNA (miRNA) is secreted from cells into the extracellular space, and its role as a biomarker is attracting attention. Here, we performed an miRNA array analysis of supernatants during the process of cardiac differentiation and maturation from hPSCs. We demonstrated that the quantification of extracellular miR-489-3p and miR-1/133a-3p levels enabled the monitoring of mesoderm and cardiac differentiation, respectively, even in clinical-grade mass culture systems. Moreover, extracellular let-7c-5p levels showed the greatest increase with cardiac maturation during long-term culture. We also verified that residual undifferentiated hPSCs in hPSC-derived cardiomyocytes (hPSC-CMs) were detectable by measuring miR-302b-3p expression, with a detection sensitivity of 0.01%. Collectively, we demonstrate that our method of seamlessly monitoring specific miRNAs secreted into the supernatant is non-destructive and effective for the quality evaluation of hPSC-CMs.

Although the extracellular matrix (ECM) plays essential roles in heart tissue engineering, the optimal ECM components for heart tissue organization have not previously been elucidated. Here, we focused on the main ECM component, fibrillar collagen, and analyzed the effects of collagens on heart tissue engineering, by comparing the use of porcine heart-derived collagen and other organ-derived collagens in generating engineered heart tissue (EHT). We demonstrate that heart-derived collagen induces better contraction and relaxation of human induced pluripotent stem cell-derived EHT (hiPSC-EHT) and that hiPSC-EHT with heart-derived collagen exhibit more mature profiles than those with collagens from other organs. Further, we found that collagen fibril formation and gel stiffness influence the contraction, relaxation, and maturation of hiPSC-EHT, suggesting the importance of collagen types III and type V, which are relatively abundant in the heart. Thus, we demonstrate the effectiveness of organ-specific collagens in tissue engineering and drug discovery.

BACKGROUND: Because adult cardiomyocytes have little regenerative capacity, resident cardiac fibroblasts (CFs) synthesize extracellular matrix after myocardial infarction (MI) to form fibrosis, leading to cardiac dysfunction and heart failure. Therapies that can regenerate the myocardium and reverse fibrosis in chronic MI are lacking. The overexpression of cardiac transcription factors, including Mef2c/Gata4/Tbx5/Hand2 (MGTH), can directly reprogram CFs into induced cardiomyocytes (iCMs) and improve cardiac function under acute MI. However, the ability of in vivo cardiac reprogramming to repair chronic MI with established scars is undetermined. METHODS: We generated a novel Tcf21iCre/reporter/MGTH2A transgenic mouse system in which tamoxifen treatment could induce both MGTH and reporter expression in the resident CFs for cardiac reprogramming and fibroblast lineage tracing. We first tested the efficacy of this transgenic system in vitro and in vivo for acute MI. Next, we analyzed in vivo cardiac reprogramming and fusion events under chronic MI using Tcf21iCre/Tomato/MGTH2A and Tcf21iCre/mTmG/MGTH2A mice, respectively. Microarray and single-cell RNA sequencing were performed to determine the mechanism of cardiac repair by in vivo reprogramming. RESULTS: We confirmed the efficacy of transgenic in vitro and in vivo cardiac reprogramming for acute MI. In chronic MI, in vivo cardiac reprogramming converted ≈2% of resident CFs into iCMs, in which a majority of iCMs were generated by means of bona fide cardiac reprogramming rather than by fusion with cardiomyocytes. Cardiac reprogramming significantly improved myocardial contraction and reduced fibrosis in chronic MI. Microarray analyses revealed that the overexpression of MGTH activated cardiac program and concomitantly suppressed fibroblast and inflammatory signatures in chronic MI. Single-cell RNA sequencing demonstrated that resident CFs consisted of 7 subclusters, in which the profibrotic CF population increased under chronic MI. Cardiac reprogramming suppressed fibroblastic gene expression in chronic MI by means of conversion of profibrotic CFs to a quiescent antifibrotic state. MGTH overexpression induced antifibrotic effects partly by suppression of Meox1, a central regulator of fibroblast activation. CONCLUSIONS: These results demonstrate that cardiac reprogramming could repair chronic MI by means of myocardial regeneration and reduction of fibrosis. These findings present opportunities for the development of new therapies for chronic MI and heart failure.

Heart transplantation (HT) is the only definitive treatment available for patients with end-stage heart failure who are refractory to medical and device therapies. However, HT as a therapeutic option, is limited by a significant shortage of donors. To overcome this shortage, regenerative medicine using human pluripotent stem cells (hPSCs), such as human embryonic stem cells and human-induced pluripotent stem cells (hiPSCs), has been considered an alternative to HT. Several issues, including the methods of large-scale culture and production of hPSCs and cardiomyocytes, the prevention of tumorigenesis secondary to contamination of undifferentiated stem cells and non-cardiomyocytes, and the establishment of an effective transplantation strategy in large-animal models, need to be addressed to fulfill this unmet need. Although post-transplantation arrhythmia and immune rejection remain problems, the ongoing rapid technological advances in hPSC research have been directed toward the clinical application of this technology. Cell therapy using hPSC-derived cardiomyocytes is expected to serve as an integral component of realistic medicine in the near future and is being potentially viewed as a treatment that would revolutionize the management of patients with severe heart failure.

Background: Because adult cardiomyocytes have little regenerative capacity, resident cardiac fibroblasts (CFs) synthesize extracellular matrix after myocardial infarction (MI) to form fibrosis, leading to cardiac dysfunction and heart failure. Therapies that can regenerate the myocardium and reverse fibrosis in chronic MI are lacking. The overexpression of cardiac transcription factors, including Mef2c/Gata4/Tbx5/Hand2 (MGTH), can directly reprogram CFs into induced cardiomyocytes (iCMs) and improve cardiac function under acute MI. However, the ability of in vivo cardiac reprogramming to repair chronic MI with established scars is undetermined. Methods: We generated a novel Tcf21iCre/reporter/MGTH2A transgenic mouse system in which tamoxifen treatment could induce both MGTH and reporter expression in the resident CFs for cardiac reprogramming and fibroblast lineage tracing. We first tested the efficacy of this transgenic system in vitro and in vivo for acute MI. Next, we analyzed in vivo cardiac reprogramming and fusion events under chronic MI using Tcf21iCre/Tomato/MGTH2A and Tcf21iCre/mTmG/MGTH2A mice, respectively. Microarray and single-cell RNA sequencing were performed to determine the mechanism of cardiac repair by in vivo reprogramming. Results: We confirmed the efficacy of transgenic in vitro and in vivo cardiac reprogramming for acute MI. In chronic MI, in vivo cardiac reprogramming converted ≈2% of resident CFs into iCMs, in which a majority of iCMs were generated by means of bona fide cardiac reprogramming rather than by fusion with cardiomyocytes. Cardiac reprogramming significantly improved myocardial contraction and reduced fibrosis in chronic MI. Microarray analyses revealed that the overexpression of MGTH activated cardiac program and concomitantly suppressed fibroblast and inflammatory signatures in chronic MI. Single-cell RNA sequencing demonstrated that resident CFs consisted of 7 subclusters, in which the profibrotic CF population increased under chronic MI. Cardiac reprogramming suppressed fibroblastic gene expression in chronic MI by means of conversion of profibrotic CFs to a quiescent antifibrotic state. MGTH overexpression induced antifibrotic effects partly by suppression of Meox1, a central regulator of fibroblast activation. Conclusions: These results demonstrate that cardiac reprogramming could repair chronic MI by means of myocardial regeneration and reduction of fibrosis. These findings present opportunities for the development of new therapies for chronic MI and heart failure.

In vitro reconstruction of highly mature engineered heart tissues (EHTs) is attempted for the selection of cardiotoxic drugs suitable for individual patients before administration. Mechanical contractile force generated in the EHTs is known to be a critical indicator for evaluating the EHT response. However, measuring contractile force requires anchoring the EHT in a tailored force-sensing cell culture chamber, causing technical difficulties in the stable evaluation of contractile force in long-term culture. This paper proposes a hydrogel-sheathed human induced pluripotent stem cell (hiPSC)-derived heart microtissue (H3M) that can provide an anchor-free contractile force measurement platform in commonly used multi-well plates. The contractile force associated with tissue formation and drug response is calculated by motion tracking and finite element analysis on the bending angle of the hydrogel sheath. From the experiment of the drug response, H3M is an excellent drug screening platform with high sensitivity and early testing capability compared to conventionally anchored EHT. This unique platform would be useful and versatile for regenerative therapy and drug discovery research in EHT.

The emergence of human induced pluripotent stem cells (hiPSCs) and efficient differentiation of hiPSC-derived cardiomyocytes (hiPSC-CMs) induced from diseased donors have the potential to recapitulate the molecular and functional features of the human heart. Although the immaturity of hiPSC-CMs, including the structure, gene expression, conduct, ion channel density, and Ca2+ kinetics, is a major challenge, various attempts to promote maturation have been effective. Three-dimensional cardiac models using hiPSC-CMs have achieved these functional and morphological maturations, and disease models using patient-specific hiPSC-CMs have furthered our understanding of the underlying mechanisms and effective therapies for diseases. Aside from the mechanisms of diseases and drug responses, hiPSC-CMs also have the potential to evaluate the safety and efficacy of drugs in a human context before a candidate drug enters the market and many phases of clinical trials. In fact, novel drug testing paradigms have suggested that these cells can be used to better predict the proarrhythmic risk of candidate drugs. In this review, we overview the current strategies of human engineered heart tissue models with a focus on major cardiac diseases and discuss perspectives and future directions for the real application of hiPSC-CMs and human engineered heart tissue for disease modeling, drug development, clinical trials, and cardiotoxicity tests.

The emergence of human induced pluripotent stem cells (hiPSCs) has revealed the potential for curing end-stage heart failure. Indeed, transplantation of hiPSC-derived cardiomyocytes (hiPSC-CMs) may have applications as a replacement for heart transplantation and conventional regenerative therapies. However, there are several challenges that still must be overcome for clinical applications, including large-scale production of hiPSCs and hiPSC-CMs, elimination of residual hiPSCs, purification of hiPSC-CMs, maturation of hiPSC-CMs, efficient engraftment of transplanted hiPSC-CMs, development of an injection device, and avoidance of post-transplant arrhythmia and immunological rejection. Thus, we developed several technologies based on understanding of the metabolic profiles of hiPSCs and hiPSC derivatives. In this review, we outline how to overcome these hurdles to realize the transplantation of hiPSC-CMs in patients with heart failure and introduce cutting-edge findings and perspectives for future regenerative therapy.

The severe shortage of donor hearts hampered the cardiac transplantation to patients with advanced heart failure. Therefore, cardiac regenerative therapies are eagerly awaited as a substitution. Human induced pluripotent stem cells (hiPSCs) are realistic cell source for regenerative cardiomyocytes. The hiPSC-derived cardiomyocytes are highly expected to help the recovery of heart. Avoidance of teratoma formation and large-scale culture of cardiomyocytes are definitely necessary for clinical setting. The combination of pure cardiac spheroids and gelatin hydrogel succeeded to recover reduced ejection fraction. The feasible transplantation strategy including transplantation device for regenerative cardiomyocytes are established in this study.

Cell Biology; Metabolomics; Stem Cell Research

In this article, Ieda and colleagues showed that a soft matrix, which is comparable with native myocardium, efficiently promoted cardiac reprogramming. This soft matrix enhanced cardiac reprogramming via inhibition of integrin, Rho/ROCK, actomyosin, and YAP/TAZ signaling and subsequent suppression of fibroblast programs, which were activated on conventional rigid substrates, thus demonstrating that mechanotransduction plays a critical role in cardiac reprogramming.

Background: Patients presenting with acute coronary syndrome (ACS) from left main (LM) disease are at a high risk for mortality despite recent advancement in devices and techniques during percutaneous coronary interventions (PCI). We aimed to evaluate patient characteristics, clinical presentations, and key clinical characteristics associated with adverse in-hospital outcomes among ACS patients undergoing LM-PCI. Methods: We retrospectively identified 280 LM-ACS patients (3.7 %) from 7608 ACS patients in the prospective multicenter Japan Cardiovascular Database-Keio Inter-Hospital Cardiovascular Studies registry from March 2009 to May 2016 and divided them into those with/without PCI/coronary artery bypass grafting. We compared baseline demographics, coronary lesion characteristics, PCI details, and short-term outcomes, including in-hospital mortality and periprocedural complications, between the two groups. Results: Among LM-ACS patients, 38.6 % presented with ST elevation myocardial infarction, 29.6 % with cardiogenic shock (CS), and 15.4 % with cardiac arrest. The observed in-hospital mortality rate was 18.9 % with presence of CS [odds ratio (OR): 10.16, 95 % confidence interval (CI): 4.51–22.91, p < 0.001] and absence of prior revascularization (de novo patients; OR: 4.31, 95 % CI: 1.43–12.94, p = 0.009) was independently associated with higher incidence of in-hospital mortality. Notably, the observed mortality rate was substantially higher among de novo patients than the predicted mortality rate with a contemporary risk model (observed: 25.1 %; predicted: 11.6 %). Conclusions: Prior revascularization act as a protective factor among LM-ACS patients in the contemporary era of PCI. Further studies to detect those at higher risk for LM coronary lesion progression are needed to fully implement these findings into clinical practice.

Direct cardiac reprogramming from fibroblasts can be a promising approach for disease modeling, drug screening, and cardiac regeneration in pediatric and adult patients. However, postnatal and adult fibroblasts are less efficient for reprogramming compared with embryonic fibroblasts, and barriers to cardiac reprogramming associated with aging remain undetermined. In this study, we screened 8400 chemical compounds and found that diclofenac sodium (diclofenac), a non-steroidal anti-inflammatory drug, greatly enhanced cardiac reprogramming in combination with Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Hand2. Intriguingly, diclofenac promoted cardiac reprogramming in mouse postnatal and adult tail-tip fibroblasts (TTFs), but not in mouse embryonic fibroblasts (MEFs). Mechanistically, diclofenac enhanced cardiac reprogramming by inhibiting cyclooxygenase-2, prostaglandin E2/prostaglandin E receptor 4, cyclic AMP/protein kinase A, and interleukin 1β signaling and by silencing inflammatory and fibroblast programs, which were activated in postnatal and adult TTFs. Thus, anti-inflammation represents a new target for cardiac reprogramming associated with aging.

The mesoderm arises from pluripotent epiblasts and differentiates into multiple lineages; however, the underlying molecular mechanisms are unclear. Tbx6 is enriched in the paraxial mesoderm and is implicated in somite formation, but its function in other mesoderms remains elusive. Here, using direct reprogramming-based screening, single-cell RNA-seq in mouse embryos, and directed cardiac differentiation in pluripotent stem cells (PSCs), we demonstrated that Tbx6 induces nascent mesoderm from PSCs and determines cardiovascular and somite lineage specification via its temporal expression. Tbx6 knockout in mouse PSCs using CRISPR/Cas9 technology inhibited mesoderm and cardiovascular differentiation, whereas transient Tbx6 expression induced mesoderm and cardiovascular specification from mouse and human PSCs via direct upregulation of Mesp1, repression of Sox2, and activation of BMP/Nodal/Wnt signaling. Notably, prolonged Tbx6 expression suppressed cardiac differentiation and induced somite lineages, including skeletal muscle and chondrocytes. Thus, Tbx6 is critical for mesoderm induction and subsequent lineage diversification. Sadahiro et al. show that Tbx6 is critical for mesoderm induction and subsequent lineage diversification from pluripotent stem cells (PSCs). Transient Tbx6 expression induced nascent mesoderm and cardiovascular lineages from mouse and human PSCs, whereas prolonged Tbx6 expression suppressed cardiac differentiation and induced somite lineages, including skeletal muscle and chondrocytes.

Cardiac diseases are among the most common causes of death globally. Cardiac muscle has limited proliferative capacity, and regenerative therapies are highly in demand as a new treatment strategy. Although pluripotent reprogramming has been developed, it has obstacles, such as a potential risk of tumor formation, poor survival of the transplanted cells, and high cost. We previously reported that fibroblasts can be directly reprogrammed to cardiomyocytes by overexpressing a combination of three cardiac-specific transcription factors (Gata4, Mef2c, Tbx5 (together, GMT)). We and other groups have promoted cardiac reprogramming by the addition of certain miRNAs, cytokines, and epigenetic factors, and unraveled new molecular mechanisms of cardiac reprogramming. More recently, we discovered that Sendai virus (SeV) vector expressing GMT could efficiently and rapidly reprogram fibroblasts into integration-free cardiomyocytes in vitro via robust transgene expression. Gene delivery of SeV-GMT also improves cardiac function and reduces fibrosis after myocardial infarction in mice. Through direct cardiac reprogramming, new cardiomyocytes can be generated and scar tissue reduced to restore cardiac function, and, thus, direct cardiac reprogramming may serve as a powerful strategy for cardiac regeneration. Here, we provide an overview of the previous reports and current challenges in this field.

Direct cardiac reprogramming holds great promise for regenerative medicine. We previously generated directly reprogrammed induced cardiomyocyte-like cells (iCMs) by overexpression of Gata4, Mef2c, and Tbx5 (GMT) using retrovirus vectors. However, integrating vectors pose risks associated with insertional mutagenesis and disruption of gene expression and are inefficient. Here, we show that Sendai virus (SeV) vectors expressing cardiac reprogramming factors efficiently and rapidly reprogram both mouse and human fibroblasts into integration-free iCMs via robust transgene expression. SeV-GMT generated 100-fold more beating iCMs than retroviral-GMT and shortened the duration to induce beating cells from 30 to 10 days in mouse fibroblasts. In vivo lineage tracing revealed that the gene transfer of SeV-GMT was more efficient than retroviral-GMT in reprogramming resident cardiac fibroblasts into iCMs in mouse infarct hearts. Moreover, SeV-GMT improved cardiac function and reduced fibrosis after myocardial infarction. Thus, efficient, non-integrating SeV vectors may serve as a powerful system for cardiac regeneration. Ieda and colleagues show that non-integrating Sendai virus (SeV) vectors expressing cardiac reprogramming factors efficiently reprogrammed mouse and human fibroblasts into induced cardiomyocyte-like cells. In vivo delivery of SeV vectors enhanced in vivo cardiac reprogramming compared to conventional retrovirus vectors, improved cardiac function, and reduced fibrosis after myocardial infarction.

The coronary vascular system is critical for myocardial growth and cardiomyocyte survival. However, the molecular mechanism regulating coronary angiogenesis remains elusive. Vascular endothelial growth factor (VEGF) regulates angiogenesis by binding to the specific receptors Flk1 and Flt1, which results in different functions. Despite the importance of Flk1 and Flt1, their expression in the coronary vasculature remains largely unknown due to the lack of appropriate antibodies for immunostaining. Here, we analyzed multiple reporter mice including Flk1-GFP BAC transgenic (Tg), Flk1-LacZ knock-in, Flt1-DsRed BAC Tg, and Flk1-GFP/Flt1-DsRed double Tg animals to determine expression patterns in mouse hearts during cardiac growth and after myocardial infarction (MI). We found that Flk1 was expressed in endothelial cells (ECs) with a pattern of epicardial-to-endocardial transmural gradients in the neonatal mouse ventricle, which was downregulated in adult coronary vessels with development. In contrast, Flt1 was homogeneously expressed in the ECs of neonatal mouse hearts and expression was maintained until adulthood. After MI, expression of both Flk1 and Flt1 was induced in the regenerating coronary vessels at day 7. Intriguingly, Flk1 expression was downregulated thereafter, whereas Flt1 expression was maintained in the newly formed coronary vessels until 30 days post-MI, recapitulating their expression kinetics during development. This is the first report demonstrating the spatiotemporal expression patterns of Flk1 and Flt1 in the coronary vascular system during development and after MI; thus, this study suggests that these factors have distinct and important functions in coronary angiogenesis.

Direct reprogramming is a promising approach in regenerative medicine. Overexpression of the cardiac transcription factors Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Hand2 (GHMT) directly reprogram fibroblasts into cardiomyocyte-like cells (iCMs). However, the critical timing of transgene expression and the molecular mechanisms for cardiac reprogramming remain unclear. The conventional doxycycline (Dox)-inducible temporal transgene expression systems require simultaneous transduction of two vectors (pLVX-rtTA/pLVX-cDNA) harboring the reverse tetracycline transactivator (rtTA) and the tetracycline response element (TRE)-controlled transgene, respectively, leading to inefficient cardiac reprogramming. Herein, we developed a single-construct-based polycistronic Dox-inducible vector (pDox-cDNA) expressing both the rtTA and TRE-controlled transgenes. Fluorescence activated cell sorting (FACS) analyses, quantitative RT-PCR, and immunostaining revealed that pDox-GMT increased cardiac reprogramming three-fold compared to the conventional pLVX-rtTA/pLVX-GMT. After four weeks, pDox-GMT-induced iCMs expressed multiple cardiac genes, produced sarcomeric structures, and beat spontaneously. Co-transduction of pDox-Hand2 with retroviral pMX-GMT increased cardiac reprogramming three-fold compared to pMX-GMT alone. Temporal Dox administration revealed that Hand2 transgene expression is critical during the first two weeks of cardiac reprogramming. Microarray analyses demonstrated that Hand2 represses cell cycle-promoting genes and enhances cardiac reprogramming. Thus, we have developed an efficient temporal transgene expression system, which could be invaluable in the study of cardiac reprogramming.