田野崎 翔

Cardioplegia is often used prior to acquisition of human cardiac tissue to minimize warm ischemia time, which can severely confound studies of cardiac metabolism. However, there are several choices of cardioplegia solutions, and whether these solutions differentially impact tissue metabolism or metabolomic studies is not known. Here, we perform untargeted metabolomics, using both liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry, on a large cohort of hearts transplanted for cardiomyopathy or from gift-of-life donors, and who have received different cardioplegia solutions. We show that different cardioplegia solutions distinctly impact cardiac metabolism and tissue metabolomic studies. Notably, these differences are mild relative to those seen comparing failing to nonfailing hearts, and identification of cardioplegia components in mass spectra should enable rigorous interpretation of changes between conditions. These data demonstrate how cardioplegia solutions may influence cardiac metabolism in human heart samples and underscore the need to report specific details of cardioplegia solution use in studies of human cardiac metabolism.

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.

Here we describe a protocol to obtain highly pure cardiomyocytes and neurons from human induced pluripotent stem cells (hiPSCs) via metabolic selection processes. Compared to conventional purification protocols, this approach is easier to perform and scale up and more cost-efficient. The protocol can be applied to hiPSCs and human embryonic stem cells. For complete details on the use and execution of this protocol, please refer to Tohyama et al. (2016) and Tanosaki et al. (2020).

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.

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.

Pluripotent stem cells (PSCs) exhibit promising application in regenerative therapy, drug discovery, and disease modeling. While several protocols for differentiating somatic cells from PSCs exist, their use is limited by contamination of residual undifferentiated PSCs and immaturity of differentiated somatic cells.The metabolism of PSCs differs greatly from that of somatic cells, and a distinct feature is required to sustain the distinct properties of PSCs. To date, several studies have reported on the importance of metabolism in PSCs and their derivative cells. Here, we detail advancements in the field, with a focus on cardiac regenerative therapy.

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.

Human induced pluripotent stem cells (hiPSCs) are considered to be one of the most promising cell resources for regenerative medicine. HiPSCs usually maintain their pluripotency when they are cultured on feeder cell layers or are attached to a cell-adhesive extracellular matrix. In this study, we developed a culture system based on UV/ozone modification for conventional cell culture plastics to generate a suitable surface condition for hiPSCs. Time of flight secondary ion mass spectrometry (ToF-SIMS) was carried out to elucidate the relationship between hiPSC adhesion and UV/ozone irradiation-induced changes to surface chemistry of cell culture plastics. Cell culture plastics with modified surfaces enabled growth of a feeder-free hiPSC culture with markedly reduced cell-adhesive matrix coating. Our cell culture system using UV/ozone-modified cell culture plastics may produce clinically relevant hiPSCs at low costs, and can be easily scaled up in culture systems to produce a large number of hiPSCs.

Heart transplantation (HT) is the only radical treatment available for patients with end-stage heart failure that is refractory to optimal medical treatment and device therapies. However, HT as a therapeutic option is limited by marked donor shortage. To overcome this difficulty, regenerative medicine using human-induced pluripotent stem cells (hiPSCs) has drawn increasing attention as an alternative to HT. Several issues including the preparation of clinical-grade hiPSCs, methods for large-scale culture and production of hiPSCs and cardiomyocytes, prevention of tumorigenesis secondary to contamination of undifferentiated stem cells and non-cardiomyocytes, and establishment of an effective transplantation strategy need to be addressed to fulfill this unmet medical need. The ongoing rapid technological advances in hiPSC research have been directed toward the clinical application of this technology, and currently, most issues have been satisfactorily addressed. Cell therapy using hiPSC-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.

Immunogenicity of immature pluripotent stem cells is a topic of intense debate. Immunogenic antigens, which are specific in pluripotent states, have not been described previously. In this study, we identified glypican-3 (GPC3), a known carcinoembryonic antigen, as a pluripotent state-specific immunogenic antigen. Additionally, we validated the applicability of human leukocyte antigen (HLA)-class I-restricted GPC3-reactive cytotoxic T lymphocytes (CTLs) in the removal of undifferentiated pluripotent stem cells (PSCs) from human induced pluripotent stem cell (hiPSC)-derivatives. HiPSCs uniquely express GPC3 in pluripotent states and were rejected by GPC3-reactive CTLs, which were sensitized with HLA-class I-restricted GPC3 peptides. Furthermore, GPC3-reactive CTLs selectively removed undifferentiated PSCs from hiPSC-derivatives in vitro and inhibited tumor formation in vivo. Our results demonstrate that GPC3 works as a pluripotent state-specific immunogenic antigen in hiPSCs and is applicable to regenerative medicine as a method of removing undifferentiated PSCs, which are the main cause of tumor formation.

Cardiac regenerative therapies utilizing human induced pluripotent stem cells (hiPSCs) are hampered by ineffective large-scale culture. hiPSCs were cultured in multilayer culture plates (CPs) with active gas ventilation (AGV), resulting in stable proliferation and pluripotency. Seeding of 1 × 106 hiPSCs per layer yielded 7.2 × 108 hiPSCs in 4-layer CPs and 1.7 × 109 hiPSCs in 10-layer CPs with pluripotency. hiPSCs were sequentially differentiated into cardiomyocytes (CMs) in a two-dimensional (2D) differentiation protocol. The efficiency of cardiac differentiation using 10-layer CPs with AGV was 66%-87%. Approximately 6.2-7.0 × 108 cells (4-layer) and 1.5-2.8 × 109 cells (10-layer) were obtained with AGV. After metabolic purification with glucose- and glutamine-depleted and lactate-supplemented media, a massive amount of purified CMs was prepared. Here, we present a scalable 2D culture system using multilayer CPs with AGV for hiPSC-derived CMs, which will facilitate clinical applications for severe heart failure in the near future.

BACKGROUND: Atrioventricular conduction disturbances can develop after transcatherter aortic valve implantations (TAVIs) with balloon-expandable valves because the conduction system exists adjacent to the aortic valve. However, the clinical consequence of patients with new onset conduction disturbances is not clear. OBJECTIVE: This study aimed to assess the incidence and progress of new-onset conduction disturbances following TAVIs and the cardiac function evaluated by echocardiography. METHODS: This study consisted of 90 consecutive patients that underwent TAVIs with Edwards SAPIEN XT valves in a single center. Atrioventricular conduction system disturbances were assessed by electrocardiography and echocardiography up to 6months post TAVI. RESULTS: Twenty patients (22%) developed new onset complete left bundle branch block (CLBBB) or received pacemaker implantations (PMIs) during the follow-up. At 6months after the procedure, 4 patients underwent PMIs for complete AV block (CAVB), and 4 patients had persistent CLBBB. Those that developed CLBBB and AVB had a higher morbidity from hypertension and lower estimated glomerular filtration rate (eGFR). The ECG, TTE, and CT parameters did not differ between the two groups. The ratio of the valve and LVOT area was significantly associated with a higher cumulative risk of events (HR, 3.005; 95% CI, 1.034-8.736; P<0.05). CONCLUSIONS: Up to 20% of patients developed new CLBBB or CAVB and more than half were expected to recover. However, it required attention because approximately 40% were persistent. The ratio of the valve to LVOT area was an independent predictor.

PURPOSE OF REVIEW: Pluripotent stem cells (PSCs) have the capacity to differentiate into various types of cells, and are promising cell sources for regenerative therapy and drug screening. However, to realize the clinical application of PSCs, a large number of highly qualified target cells must be stably prepared with low cost. To achieve this, great improvements in the reprogramming, differentiation, and elimination of residual PSCs will be necessary. In this review, we summarize the updated knowledge about metabolism in PSCs and its application. RECENT FINDINGS: Recent studies have shown that PSCs have distinct metabolic profiles compared to differentiated cells. The metabolic profiles of PSCs are indispensable for the maintenance of pluripotency, self-renewal, differentiation capacity, and cell survival. SUMMARY: Metabolic approaches show improved simplicity, scalability, and lower cost than conventional methods for differentiation and elimination of residual PSCs. Thus, manipulation of PSC metabolism will lead to new technologies to improve their efficiencies.