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.