Maturation of Human iPSC-Derived Cardiac Microfiber with Electrical Stimulation Device.

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.

Regeneration of Nonhuman Primate Hearts With Human Induced Pluripotent Stem Cell-Derived Cardiac Spheroids.

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.

Pluripotent stem cell-based cardiac regenerative therapy for heart failure.

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.

Scalable production of homogeneous cardiac organoids derived from human pluripotent stem cells.

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.

Seamless and non-destructive monitoring of extracellular microRNAs during cardiac differentiation from human pluripotent stem cells.

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.

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.

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.

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.

Scaffold-Free Tubular Engineered Heart Tissue From Human Induced Pluripotent Stem Cells Using Bio-3D Printing Technology in vivo.

Engineered heart tissues (EHTs) that are fabricated using human induced pluripotent stem cells (hiPSCs) have been considered as potential cardiac tissue substitutes in case of heart failure. In the present study, we have created hiPSC-derived cardiac organoids (hiPSC-COs) comprised of hiPSC-derived cardiomyocytes, human umbilical vein endothelial cells, and human fibroblasts. To produce a beating conduit for patients suffering from congenital heart diseases, we constructed scaffold-free tubular EHTs (T-EHTs) using hiPSC-COs and bio-3D printing with needle arrays. The bio-3D printed T-EHTs were cut open and transplanted around the abdominal aorta as well as the inferior vena cava (IVC) of NOG mice. The transplanted T-EHTs were covered with the omentum, and the abdomen was closed after completion of the procedure. Additionally, to compare the functionality of hiPSC-COs with that of T-EHTs, we transplanted the former around the aorta and IVC as well as injecting them into the subcutaneous tissue on the back of the mice. After 1 m of the transplantation procedures, we observed the beating of the T-EHTs in the mice. In histological analysis, the T-EHTs showed clear striation of the myocardium and vascularization compared to hiPSC-COs transplanted around the aorta or in subcutaneous tissue. Based on these results, bio-3D-printed T-EHTs exhibited a better maturation in vivo as compared to the hiPSC-COs. Therefore, these beating T-EHTs may form conduits for congenital heart disease patients, and T-EHT transplantation can form a treatment option in such cases.

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.

Staged transcatheter closure for atrial septal defect and patent ductus arteriosus: a case report.

BACKGROUND: Atrial septal defect (ASD) and patent ductus arteriosus (PDA) are both common congenital heart diseases, but the combination of these two cardiac defects is extremely rare, and the therapeutic strategy is controversial. CASE SUMMARY: We treated an adult patient with combined ASD and PDA, and safely attained a successful outcome with two-stage transcatheter closure, which is PDA closure preceding ASD closure, to prevent serious complications. DISCUSSION: Transcatheter closure of one of the defects is now widely accepted as an alternative to surgical closure. In addition, adults with both ASD and PDA are better suited for transcatheter closure than surgical closure. One of the reasons is the difficulty to ligate the ductus arteriosus of an adult patient due to its friability and calcification. Meanwhile, simultaneous combined transcatheter closure of ASD and PDA can result in serious complications, such as thrombocytopenia and haemolysis, whose mechanism is considered to be the destruction of platelets and red blood cells by the residual shunt through implanted devices. Additionally, antiplatelet therapy that prevents device-related thrombus formation after ASD closure can possibly exacerbate thrombocytopenia and haemolysis. Therefore, the staged strategy is reasonable from the perspectives of antiplatelet therapy and haemodynamic changes.

[Surgical repair of pseudoaneurysms with complete atrioventricular block after aortic valve replacement; report of a case].

Aortic pseudoaneurysm is a late complication after cardiac surgery. The patient was a 63-year-old female who had undergone aortic valve replacement 3 years before. She complained of syncope and underwent pacemaker implantation for complete atrioventricular block. Aortic root pseudoaneurysms were accidentally detected by computed tomography, and 1 of them was 50 mm in size. She underwent aortic root replacement with a modified Bentall technique. Although there was no definite infection or inflammation, careful follow-up was considered to be mandatory.

Mapping QTL for fat area ratios and serum leptin concentrations in a Duroc purebred population.

The reduction of extra subcutaneous, intermuscular and abdominal fat is important to increase the carcass lean percentage of pigs. Image analyses of fat area ratios were effective for estimation of separated fat in pig carcasses. Serum concentrations of leptin are useful as physiological predictors of fat accumulation in pigs. The objectives of the present study were to perform a quantitative trait locus (QTL) analysis for fat area ratios and serum leptin concentrations in a Duroc purebred population. Pigs (n = 226 to 538) were measured for fat area ratios of carcass cross-sections at the fifth to sixth thoracic vertebrae, half body length and last thoracic vertebra using an image analysis system, and serum leptin concentration. In total, animals were genotyped for 129 markers and used for QTL analysis. For fat area ratios, four significant and 12 suggestive QTLs were detected on chromosomes 1, 6, 7, 8, 9, 12 and 13. Significant QTLs were detected on the same region of chromosome 6, which was located near a leptin receptor gene. For serum leptin concentrations, two significant and two suggestive QTLs were detected on chromosomes 6, 9, and 16, and the QTLs on chromosome 6 were also in the same region for fat area ratios.