Exploring IDI2-AS1, OIP5-AS1, and LITATS1: Changes in Long Non-coding RNAs Induced by the Poly I:C Stimulation.

Long non-coding RNAs (lncRNAs) are sequences longer than 200 nucleotides, but they do not encode proteins. Nevertheless, they have significant roles in diverse biological functions. It remains unclear how viral infections trigger the expression of lncRNAs. In our previous research, we revealed a distinct type of lncRNAs with a lifespan under 4 h in human HeLa cells. These short-lived lncRNAs might be associated with numerous regulatory roles. Given their potential impact on human physiology, these short-lived lncRNAs could be key indicators to measure polyinosinic:polycytidylic acid (poly I:C) stimulation. In our recent work, we discovered three lncRNAs: IDI2-AS1, OIP5-AS1, and LITATS1. After exposure to poly I:C, imitating viral assault in human A549 cells, IDI2-AS1 levels dropped significantly while OIP5-AS1 and LITATS1 levels rose markedly. Our results indicate that short-lived lncRNAs respond to poly I:C stimulation, exhibiting substantial changes in expression. This indicates that the understanding the role of lncRNAs in the host response to viral infection and the potential for these molecules to serve as novel therapeutic targets.

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.

Cardiac reprogramming reduces inflammatory macrophages and improves cardiac function in chronic myocardial infarction.

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.

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.

Hydrogel-Sheathed hiPSC-Derived Heart Microtissue Enables Anchor-Free Contractile Force Measurement.

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 (H3 M) 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, H3 M 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.

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.

Direct Reprogramming Improves Cardiac Function and Reverses Fibrosis in Chronic Myocardial Infarction.

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.

Human Engineered Heart Tissue Models for Disease Modeling and Drug Discovery.

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.

Production of functional cardiomyocytes and cardiac tissue from human induced pluripotent stem cells for regenerative therapy.

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.

Heterologous expression of membrane-bound alcohol dehydrogenase-encoding genes for glyceric acid production using Gluconobacter sp. CHM43 and its derivatives.

In contrast to D-glyceric acid (D-GA) production with 99% enantiomeric excess (ee) by Acetobacter tropicalis NBRC 16470, Gluconobacter sp. CHM43 produced 19.6 g L-1 of D-GA with 73.7% ee over 4 days of incubation in flask culture. To investigate the reason for this enantiomeric composition of GA, the genes encoding membrane-bound alcohol dehydrogenase (mADH) of A. tropicalis NBRC 16470, composed of three subunits (adhA, adhB, and adhS), were cloned using the broad-host-range vector pBBR1MCS-2 and heterologously expressed in Gluconobacter sp. CHM43 and its ΔadhAB ΔsldBA derivative TORI4. Reverse-transcription quantitative real-time polymerase chain reaction demonstrated that adhABS genes from A. tropicalis were expressed in TORI4 transformants, and their membrane fraction exhibited mADH activities of 0.13 and 0.31 U/mg with or without AdhS, respectively. Compared with the GA production of TORI4-harboring pBBR1MCS-2 (1.23 g L-1), TORI4 transformants expressing adhABS and adhAB showed elevated GA production of 2.46 and 3.67 g L-1, respectively, suggesting a negative effect of adhS gene expression on GA production as well as mADH activity in TORI4. Although TORI4 was found to produce primarily L-GA with 42.5% ee, TORI4 transformants expressing adhABS and adhAB produced D-GA with 27.6% and 49.0% ee, respectively, demonstrating that mADH of A. tropicalis causes a sharp increase in the enantiomeric composition of D-GA. These results suggest that one reason for D-GA production with 73.7% ee in Gluconobacter spp. might be a property of the host, which possibly produces L-GA intracellularly. KEY POINTS: • Membrane-bound ADH from Acetobacter tropicalis showed activity in Gluconobacter sp. • D-GA production from glycerol was performed using recombinant Gluconobacter sp. • Enantiomeric excess of D-GA was affected by both membrane and intracellular ADHs.

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.

Naked-eye detection of specific DNA sequences amplified by the polymerase chain reaction with nanocomposite beads.

We developed a novel nanocomposite bead system for detection by the naked eye of specific DNA sequences amplified by the polymerase chain reaction (PCR). The DNA probes, which were complementary to the target DNA, are conjugated with the nanocomposite beads. If the amplified products contained sequences complementary to the probes, the beads aggregated through sandwich hybridization. The aggregation was detectable as precipitation of the nanocomposite beads. The results were determined visually and did not require instrumental detection. The assay was sensitive enough to detect PCR products with a detection limit of 10 copies/tube for DNA templates. This technique is that all needed components are included within the initial cap, so that the risk of carryover contamination is very low. The nanocomposite bead system has broad application prospects for the detection of specific DNA sequences in biological and biomedical research.

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.

Soft Matrix Promotes Cardiac Reprogramming via Inhibition of YAP/TAZ and Suppression of Fibroblast Signatures.

Direct cardiac reprogramming holds great potential for regenerative medicine. However, it remains inefficient, and induced cardiomyocytes (iCMs) generated in vitro are less mature than those in vivo, suggesting that undefined extrinsic factors may regulate cardiac reprogramming. Previous in vitro studies mainly used hard polystyrene dishes, yet the effect of substrate rigidity on cardiac reprogramming remains unclear. Thus, we developed a Matrigel-based hydrogel culture system to determine the roles of matrix stiffness and mechanotransduction in cardiac reprogramming. We found that soft matrix comparable with native myocardium promoted the efficiency and quality of cardiac reprogramming. Mechanistically, soft matrix enhanced cardiac reprogramming via inhibition of integrin, Rho/ROCK, actomyosin, and YAP/TAZ signaling and suppression of fibroblast programs, which were activated on rigid substrates. Soft substrate further enhanced cardiac reprogramming with Sendai virus vectors via YAP/TAZ suppression, increasing the reprogramming efficiency up to ∼15%. Thus, mechanotransduction could provide new targets for improving cardiac reprogramming.