Heart-derived collagen promotes maturation of engineered heart tissue.

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.

Cardiac Regenerative Therapy Using Human Pluripotent Stem Cells for Heart Failure: A State-of-the-Art Review.

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.

Scalable manufacturing of clinical-grade differentiated cardiomyocytes derived from human-induced pluripotent stem cells for regenerative therapy.

Basic research on human pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs) for cardiac regenerative therapy is one of the most active and complex fields to achieve this alternative to heart transplantation and requires the integration of medicine, science, and engineering. Mortality in patients with heart failure remains high worldwide. Although heart transplantation is the sole strategy for treating severe heart failure, the number of donors is limited. Therefore, hPSC-derived CM (hPSC-CM) transplantation is expected to replace heart transplantation. To achieve this goal, for basic research, various issues should be considered, including how to induce hPSC proliferation efficiently for cardiac differentiation, induce hPSC-CMs, eliminate residual undifferentiated hPSCs and non-CMs, and assess for the presence of residual undifferentiated hPSCs in vitro and in vivo. In this review, we discuss the current stage of resolving these issues and future directions for realizing hPSC-based cardiac regenerative therapy.

The Present State and Future Perspectives of Cardiac Regenerative Therapy Using Human Pluripotent Stem Cells.

The number of patients with heart failure (HF) is increasing with aging in our society worldwide. Patients with HF who are resistant to medication and device therapy are candidates for heart transplantation (HT). However, the shortage of donor hearts is a serious issue. As an alternative to HT, cardiac regenerative therapy using human pluripotent stem cells (hPSCs), such as human embryonic stem cells and induced pluripotent stem cells, is expected to be realized. Differentiation of hPSCs into cardiomyocytes (CMs) is facilitated by mimicking normal heart development. To prevent tumorigenesis after transplantation, it is important to eliminate non-CMs, including residual hPSCs, and select only CMs. Among many CM selection systems, metabolic selection based on the differences in metabolism between CMs and non-CMs is favorable in terms of cost and efficacy. Large-scale culture systems have been developed because a large number of hPSC-derived CMs (hPSC-CMs) are required for transplantation in clinical settings. In large animal models, hPSC-CMs transplanted into the myocardium improved cardiac function in a myocardial infarction model. Although post-transplantation arrhythmia and immune rejection remain problems, their mechanisms and solutions are under investigation. In this manner, the problems of cardiac regenerative therapy are being solved individually. Thus, cardiac regenerative therapy with hPSC-CMs is expected to become a safe and effective treatment for HF in the near future. In this review, we describe previous studies related to hPSC-CMs and discuss the future perspectives of cardiac regenerative therapy using hPSC-CMs.

A Method for Cardiac Differentiation, Purification, and Cardiac Spheroid Production of Human Induced Pluripotent Stem Cells.

Human induced pluripotent stem cells (hiPSCs) are one of the most promising cell sources for regenerative medicine. To realize the promise of hiPSCs for cardiac regenerative therapy, three major obstacles must be overcome: the first is the achievement of large-scale production of cardiomyocytes, the second is the successful elimination of non-cardiac cells containing residual pluripotent stem cells (PSCs) to prevent tumor formation, and the third is the achievement of high engraftment efficiency of transplanted cardiomyocytes. In this chapter, we introduce our protocols for cardiac differentiation, purification, and preparation of cardiac spheroids for safe and effective regenerative medicine.

Intramyocardial Transplantation of Human iPS Cell-Derived Cardiac Spheroids Improves Cardiac Function in Heart Failure Animals.

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.

Tryptophan Metabolism Regulates Proliferative Capacity of Human Pluripotent Stem Cells.

Human pluripotent stem cells (hPSCs) have a unique metabolic signature for maintenance of pluripotency, self-renewal, and survival. Although hPSCs could be potentially used in regenerative medicine, the prohibitive cost associated with large-scale cell culture presents a major barrier to the clinical application of hPSC. Moreover, without a fully characterized metabolic signature, hPSC culture conditions are not optimized. Here, we performed detailed amino acid profiling and found that tryptophan (TRP) plays a key role in the proliferation with maintenance of pluripotency. In addition, metabolome analyses revealed that intra- and extracellular kynurenine (KYN) is decreased under TRP-supplemented conditions, whereas N-formylkynurenine (NFK), the upstream metabolite of KYN, is increased thereby contributing to proliferation promotion. Taken together, we demonstrate that TRP is indispensable for survival and proliferation of hPSCs. A deeper understanding of TRP metabolism will enable cost-effective large-scale production of hPSCs, leading to advances in regenerative medicine.

Metabolic Regulation of Cardiac Differentiation and Maturation in Pluripotent Stem Cells: A Lesson from Heart Development.

The heart, one of the more complex organs, is composed from a number of differentiated cells. In general, researchers consider that the cardiac cells are derived from the same origin as mesodermal cells, except neural crest cells. However, as the developmental stages proceed, cardiac mesodermal cells are differentiated into various types of cells via cardiac progenitors and demonstrate different programming in transcriptional network and epigenetic regulation in a spatiotemporal manner. In fact, the metabolic feature also changes dramatically during heart development and cardiac differentiation. Researchers reported that each type of cell exhibits different metabolic features that can be used to specifically identify them. Metabolism is a critical process for generating energy and biomass in all living cells and organisms and has been long regarded as a passenger, rather than an active driver, for intracellular status. However, recent studies revealed that metabolism influences self-renewal and cell fate specification via epigenetic changes directly or indirectly. Metabolism mirrors the physiological status of the cell and endogenous cellular activity; therefore, understanding the metabolic signature of each cell type serves as a guide for innovative methods of selecting and differentiating desired cell types. Stem cell biology and developmental biology hold great promise for cardiac regenerative therapy, for which, successful strategy depends on the precise translation of the philosophy of cardiac development in the early embryo to the cell production system. In this review, we focus on the metabolism during heart development and cardiac differentiation and discuss the next challenge to unlock the potential of cell biology for regenerative therapy based on metabolism.

Fatty Acid Synthesis Is Indispensable for Survival of Human Pluripotent Stem Cells.

The role of lipid metabolism in human pluripotent stem cells (hPSCs) is poorly understood. We have used large-scale targeted proteomics to demonstrate that undifferentiated hPSCs express different fatty acid (FA) biosynthesis-related enzymes, including ATP citrate lyase and FA synthase (FASN), than those expressed in hPSC-derived cardiomyocytes (hPSC-CMs). Detailed lipid profiling revealed that inhibition of FASN resulted in significant reduction of sphingolipids and phosphatidylcholine (PC); moreover, we found that PC was the key metabolite for cell survival in hPSCs. Inhibition of FASN induced cell death in undifferentiated hPSCs via mitochondria-mediated apoptosis; however, it did not affect cell survival in hPSC-CMs, neurons, or hepatocytes as there was no significant reduction of PC. Furthermore, we did not observe tumor formation following transplantation of FASN inhibitor-treated cells. Our findings demonstrate the importance of de novo FA synthesis in the survival of undifferentiated hPSCs and suggest applications for FASN inhibition in regenerative medicine.

Concise Review: Genetic and Epigenetic Regulation of Cardiac Differentiation from Human Pluripotent Stem Cells.

Human pluripotent stem cells (hPSCs), including both embryonic stem cells and induced pluripotent stem cells, are the ideal cell sources for disease modeling, drug discovery, and regenerative medicine. In particular, regenerative therapy with hPSC-derived cardiomyocytes (CMs) is an unmet medical need for the treatment of severe heart failure. Cardiac differentiation protocols from hPSCs are made on the basis of cardiac development in vivo. However, current protocols have yet to yield 100% pure CMs, and their maturity is low. Cardiac development is regulated by the cardiac gene network, including transcription factors (TFs). According to our current understanding of cardiac development, cardiac TFs are sequentially expressed during cardiac commitment in hPSCs. Expression levels of each gene are strictly regulated by epigenetic modifications. DNA methylation, histone modification, and noncoding RNAs significantly influence cardiac differentiation. These complex circuits of genetic and epigenetic factors dynamically affect protein expression and metabolic changes in cardiac differentiation and maturation. Here, we review cardiac differentiation protocols and their molecular machinery, closing with a discussion of the future challenges for producing hPSC-derived CMs. Stem Cells 2019;37:992-1002.

Sall1 transiently marks undifferentiated heart precursors and regulates their fate.

Cardiac progenitor cells (CPCs) are a crucial source of cells in cardiac development and regeneration. However, reported CPCs are heterogeneous, and no gene has been identified to transiently mark undifferentiated CPCs throughout heart development. Here we show that Spalt-like gene 1 (Sall1), a zing-finger transcription factor, is expressed in undifferentiated CPCs giving rise to both left and right ventricles. Sall1 was transiently expressed in precardiac mesoderm contributing to the first heart field (left ventricle precursors) but not in the field itself. Similarly, Sall1 expression was maintained in the second heart field (outflow tract/right ventricle precursors) but not in cardiac cells. In vitro, high levels of Sall1 at mesodermal stages enhanced cardiomyogenesis, whereas its continued expression suppressed cardiac differentiation. Our study demonstrates that Sall1 marks CPCs in an undifferentiated state and regulates cardiac differentiation. These findings provide fundamental insights into CPC maintenance, which can be instrumental for CPC-based regenerative medicine.