Gene correction and overexpression of TNNI3 improve impaired relaxation in engineered heart tissue model of pediatric restrictive cardiomyopathy.

Research on cardiomyopathy models using engineered heart tissue (EHT) created from disease-specific induced pluripotent stem cells (iPSCs) is advancing rapidly. However, the study of restrictive cardiomyopathy (RCM), a rare and intractable cardiomyopathy, remains at the experimental stage because there is currently no established method to replicate the hallmark phenotype of RCM, particularly diastolic dysfunction, in vitro. In this study, we generated iPSCs from a patient with early childhood-onset RCM harboring the TNNI3 R170W mutation (R170W-iPSCs). The properties of R170W-iPSC-derived cardiomyocytes (CMs) and EHTs were evaluated and compared with an isogenic iPSC line in which the mutation was corrected. Our results indicated altered calcium kinetics in R170W-iPSC-CMs, including prolonged tau, and an increased ratio of relaxation force to contractile force in R170W-EHTs. These properties were reversed in the isogenic line, suggesting that our model recapitulates impaired relaxation of RCM, i.e., diastolic dysfunction in clinical practice. Furthermore, overexpression of wild-type TNNI3 in R170W-iPSC-CMs and -EHTs effectively rescued impaired relaxation. These results highlight the potential efficacy of EHT, a modality that can accurately recapitulate diastolic dysfunction in vitro, to elucidate the pathophysiology of RCM, as well as the possible benefits of gene therapies for patients with RCM.

Phenotypic recapitulation and correction of desmoglein-2-deficient cardiomyopathy using human-induced pluripotent stem cell-derived cardiomyocytes.

Desmoglein-2, encoded by DSG2, is one of the desmosome proteins that maintain the structural integrity of tissues, including heart. Genetic mutations in DSG2 cause arrhythmogenic cardiomyopathy, mainly in an autosomal dominant manner. Here, we identified a homozygous stop-gain mutations in DSG2 (c.C355T, p.R119X) that led to complete desmoglein-2 deficiency in a patient with severe biventricular heart failure. Histological analysis revealed abnormal deposition of desmosome proteins, disrupted intercalated disk structures in the myocardium. Induced pluripotent stem cells (iPSCs) were generated from the patient (R119X-iPSC), and the mutated DSG2 gene locus was heterozygously corrected to a normal allele via homology-directed repair (HDR-iPSC). Both isogenic iPSCs were differentiated into cardiomyocytes [induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs)]. Multielectrode array analysis detected abnormal excitation in R119X-iPSC-CMs but not in HDR-iPSC-CMs. Micro-force testing of three-dimensional self-organized tissue rings (SOTRs) revealed tissue fragility and a weak maximum force in SOTRs from R119X-iPSC-CMs. Notably, these phenotypes were significantly recovered in HDR-iPSC-CMs. Myocardial fiber structures in R119X-iPSC-CMs were severely aberrant, and electron microscopic analysis confirmed that desmosomes were disrupted in these cells. Unexpectedly, the absence of desmoglein-2 in R119X-iPSC-CMs led to decreased expression of desmocollin-2 but no other desmosome proteins. Adeno-associated virus-mediated replacement of DSG2 significantly recovered the contraction force in SOTRs generated from R119X-iPSC-CMs. Our findings confirm the presence of a desmoglein-2-deficient cardiomyopathy among clinically diagnosed dilated cardiomyopathies. Recapitulation and correction of the disease phenotype using iPSC-CMs provide evidence to support the development of precision medicine and the proof of concept for gene replacement therapy for this cardiomyopathy.

Elimination of residual undifferentiated induced pluripotent stem cells (iPSCs) using irradiation for safe clinical applications of iPSC-derived cardiomyocytes.

A major concern in the clinical application of induced pluripotent stem cells (iPSCs) is the prevention of tumorigenesis after implantation. Stem cells with high proliferative and differentiation potential are sensitive to radiation. Therefore, we hypothesized that irradiation may selectively eliminate residual undifferentiated human iPSCs (hiPSCs) in a cell population containing differentiated cardiomyocytes derived from hiPSCs (hiPSCs-CMs) and thus reduce tumorigenicity in vivo. hiPSC-CMs were irradiated with X-rays, after which the cell proliferation, apoptosis, morphology, and gene expression were analyzed. The gene expression of Lin28A, Nanog, Oct3/4, and SRY-box 2 was significantly lower in the irradiation group than in the control group. Irradiated hiPSC-CMs showed no change in proliferation potency and morphology compared to untreated hiPSC-CMs. Furthermore, irradiation did not induce apoptosis of differentiated cardiomyocytes. No significant difference in the gene expression of cardiac-specific markers, including α-myosin heavy chain, cardiac troponin T, and NK2 Homeobox 5, was observed between the groups. Tumorigenicity tests using NOG mice showed less frequent tumor formation in the irradiation group than in the control group. Irradiation of hiPSC-CMs significantly reduced the number of undifferentiated hiPSC and the tumor formation, while minimizing any adverse effects on hiPSC-CMs, thereby enabling safe hiPSC-based treatment.

Enhanced survival of transplanted human induced pluripotent stem cell-derived cardiomyocytes by the combination of cell sheets with the pedicled omental flap technique in a porcine heart.

BACKGROUND: Transplantation of cardiomyocytes that are derived from human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) shows promise in generating new functional myocardium in situ, whereas the survival and functionality of the transplanted cells are critical in considering this therapeutic impact. Cell-sheet method has been used to transplant many functional cells; however, potential ischemia might limit cell survival. The omentum, which is known to have rich vasculature, is expected to be a source of blood supply. We hypothesized that transplantation of hiPS-CM cell sheets combined with an omentum flap may deliver a large number of functional hiPS-CMs with enhanced blood supply. METHODS AND RESULTS: Retrovirally established human iPS cells were treated with Wnt signaling molecules to induce cardiomyogenic differentiation, followed by superparamagnetic iron oxide labeling. Cell sheets were created from the magnetically labeled hiPS-CMs using temperature-responsive dishes and transplanted to porcine hearts with or without the omentum flap (n=8 each). Two months after transplantation, the survival of superparamagnetic iron oxide-labeled hiPS-CMs, assessed by MRI, was significantly greater in mini-pigs with the omentum than in those without it; histologically, vascular density in the transplanted area was significantly greater in mini-pigs with the omentum than in those without it. The transplanted tissues contained abundant cardiac troponin T-positive cells surrounded by vascular-rich structures. CONCLUSIONS: The omentum flap enhanced the survival of hiPS-CMs after transplantation via increased angiogenesis, suggesting that this strategy is useful in clinical settings. The combination of hiPS-CMs and the omentum flap may be a promising technique for the development of tissue-engineered vascular-rich new myocardium in vivo.

Pleiotropic effects of extracellular vesicles from induced pluripotent stem cell-derived cardiomyocytes on ischemic cardiomyopathy: A preclinical study.

BACKGROUND: Stem cell-secreted extracellular vesicles (EVs) play essential roles in intercellular communication and restore cardiac function in animal models of ischemic heart disease. However, few studies have used EVs derived from clinical-grade stem cells and their derivatives with stable quality. Moreover, there is little information on the mechanism and time course of the multifactorial effect of EV therapy from the acute to the chronic phase, the affected cells, and whether the effects are direct or indirect. METHODS: Induced pluripotent stem cell-derived cardiomyocytes (iPSCM) were produced using a clinical-grade differentiation induction system. EVs were isolated from the conditioned medium by ultracentrifugation and characterized in silico, in vitro, and in vivo. A rat model of myocardial infarction was established by left anterior descending artery ligation and treated with iPSCM-derived EVs. RESULTS: iPSCM-derived EVs contained microRNAs and proteins associated with angiogenesis, antifibrosis, promotion of M2 macrophage polarization, cell proliferation, and antiapoptosis. iPSCM-derived EV treatment improved left ventricular function and reduced mortality in the rat model by improving vascularization and suppressing fibrosis and chronic inflammation in the heart. EVs were uptaken by cardiomyocytes, endothelial cells, fibroblasts, and macrophages in the cardiac tissues. The pleiotropic effects occurred due to the direct effects of microRNAs and proteins encapsulated in EVs and indirect paracrine effects on M2 macrophages. CONCLUSIONS: Clinical-grade iPSCM-derived EVs improve cardiac function by regulating various genes and pathways in various cell types and may have clinical potential for treating ischemic heart disease.

Pre-clinical evaluation of the efficacy and safety of human induced pluripotent stem cell-derived cardiomyocyte patch.

BACKGROUND: Cell- or tissue-based regenerative therapy is an attractive approach to treat heart failure. A tissue patch that can safely and effectively repair damaged heart muscle would greatly improve outcomes for patients with heart failure. In this study, we conducted a preclinical proof-of-concept analysis of the efficacy and safety of clinical-grade human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) patches. METHODS: A clinical-grade hiPSC line was established using peripheral blood mononuclear cells from a healthy volunteer that was homozygous for human leukocyte antigens. The hiPSCs were differentiated into cardiomyocytes. The obtained hiPSC-CMs were cultured on temperature-responsive culture dishes for patch fabrication. The cellular characteristics, safety, and efficacy of hiPSCs, hiPSC-CMs, and hiPSC-CM patches were analyzed. RESULTS: The hiPSC-CMs expressed cardiomyocyte-specific genes and proteins, and electrophysiological analyses revealed that hiPSC-CMs exhibit similar properties to human primary myocardial cells. In vitro and in vivo safety studies indicated that tumorigenic cells were absent. Moreover, whole-genome and exome sequencing revealed no genomic mutations. General toxicity tests also showed no adverse events posttransplantation. A porcine model of myocardial infarction demonstrated significantly improved cardiac function and angiogenesis in response to cytokine secretion from hiPSC-CM patches. No lethal arrhythmias were observed. CONCLUSIONS: hiPSC-CM patches are promising for future translational research and may have clinical application potential for the treatment of heart failure.

In vivo differentiation of induced pluripotent stem cell-derived cardiomyocytes.

BACKGROUND: Induced pluripotent stem cells (iPSCs) hold promise for a new era in treating heart failure. However, the functional microstructure of iPSC-derived cardiomyocytes (iPSC-CMs) and their ability to attach to the extracellular matrix of the recipient myocardium require further elucidation. Thus, we analyzed the functional microstructure and adhesion molecules of iPSC-CM. METHODS AND RESULTS: Immunostaining analysis showed that iPSC-CMs were similar to neonatal cardiomyocytes (CMs) in expressing the cytoskeletal proteins myosin heavy chain (MHC), myosin light chain (MLC) 2a, MLC2v, and especially ß-MHC (a neonatal CM marker), as well as the adhesion molecules N-cadherin, α7-integrin, dystrophin, α-dystroglycan, α-sarcoglycan, and laminin-α2. Electron microscopy showed abundant myofibrillar bundles with transverse Z-bands and a developed mitochondrial structure in both iPSC-CMs and neonatal CMs, although the iPSC-CMs contained fewer mitochondria with lower-density cristae. When transplanted from in vitro conditions to nude rat hearts, iPSC-CMs acquired the ability to express α-MHC, a molecule specific to adult CMs. Mechanical stretch or stimulation by insulin-like growth factor-1 enhanced the α-MHC expression in iPSC-CMs in vitro. CONCLUSIONS: Our findings in vitro and in vivo indicate that CMs derived from iPSCs contain cardiac-specific organelles and adhesion systems. These results indicate that iPSC-derived CMs may be useful in new cell therapies for heart failure.

Establishment of a protocol to administer immunosuppressive drugs for iPS cell-derived cardiomyocyte patch transplantation in a rat myocardial infarction model.

Transplantation of human allogeneic induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) is a new, promising treatment for severe heart failure. However, immunorejection is a significant concern in allogeneic hiPSC-CM transplantation, requiring the administration of several immunosuppressive agents. An appropriate protocol for the administration of immunosuppressants may substantially affect the efficacy of hiPSC-CM transplantation in case of heart failure owing to allogeneic transplantation. In this study, we investigated the effect of immunosuppressant administration duration on the efficacy and safety of allogenic hiPSC-CM patch transplantation. We used a rat model of myocardial infarction to evaluate cardiac function using echocardiography six months after the transplantation of hiPSC-CM patches with immunosuppressant administration for either two or four months and compared them to control rats (sham operation, no immunosuppressant administration). Histological analysis performed at 6 months after hiPSC-CM patch transplantation revealed significant improvement in cardiac function in immunosuppressant-treated rats compared with those in the control group. Moreover, fibrosis and cardiomyocyte size was significantly reduced and the number of structurally mature blood vessels was significantly increased in the immunosuppressant-treated rats compared to control rats. However, there were no significant differences between the two immunosuppressant-treated groups. Our results show that prolonged administration of immunosuppressive agents did not enhance the effectiveness of hiPSC-CM patch transplantation, and therefore, highlight the importance of an appropriate immunological regimen for the clinical application of such transplantation.

Safety confirmation of induced pluripotent stem cell-derived cardiomyocyte patch transplantation for ischemic cardiomyopathy: first three case reports.

Introduction: With the expected increase in patients with heart failure and ischemic 15 cardiomyopathy, the development of myocardial regenerative medicine using cell transplantation as a novel treatment method is progressing. This first-in-human clinical trial aimed to confirm the safety of cardiomyocyte patch transplantation derived from allogeneic induced pluripotent stem (iPS) cells based on the results of several preclinical studies. Study design: The inclusion criteria were left ventricular ejection fraction of 35% or less; heart failure symptoms of New York Heart Association class III or higher despite existing therapies such as revascularization; and a 1-year observation period that included a 3-month immunosuppressive drug administration period after transplantation of iPS cell-derived cardiomyocyte patches to evaluate adverse events, cardiac function, myocardial blood flow, heart failure symptoms, and immune response. Results: In the first three cases of this trial, no transplanted cell-related adverse events were observed during the 1-year observation period, and improvement in heart failure symptoms was observed. In addition, improvements in left ventricular contractility and myocardial blood flow were observed in two of the three patients. Regarding immune response, an increase in transplant cell-specific antibody titer was observed in all three patients after immunosuppressive drug administration. In one patient with poor improvement in cardiac function and myocardial blood flow, an increase in antibody titer against HLA-DQ was observed even before cell transplantation. Conclusions: Our case findings demonstrate that the transplantation of iPS cell-derived cardiomyocyte patches for ischemic cardiomyopathy can be safely performed; however, further investigation of the therapeutic effect and its relationship with an immune response is needed by accumulating the number of patients through continued clinical trials.

Engineered three-dimensional cardiac tissues maturing in a rotating wall vessel bioreactor remodel diseased hearts in rats with myocardial infarction.

A rotating wall vessel (RWV) bioreactor was constructed for growing massive functional cardiac constructs to recover the function of a distressed rat heart. Three-dimensional cardiac tissues were engineered by seeding human-induced pluripotent stem cell-derived cardiomyocytes on poly(lactic-co-glycolic acid) fiber sheets (3D-hiPSC-CTs) and cultured in the RWV bioreactor (RWV group) or under static conditions (control group). The tissues were transplanted into a myocardial infarction nude rat model, and cardiac performance was evaluated. In the RWV group, cell viability and contractile and electrical properties significantly improved, mature cardiomyocytes were observed, and mechanical stress-related mediators of mammalian target of rapamycin signaling were upregulated compared with those of the control. Four weeks post-transplantation, tissue survival and left ventricular ejection fraction significantly improved in the RWV group. Hence, dynamic culture in an RWV bioreactor could provide a superior culture environment for improved performance of 3D-hiPSC-CTs, providing a means for functional cardiomyogenesis in myocyte-loss heart failure.

Case report: Transplantation of human induced pluripotent stem cell-derived cardiomyocyte patches for ischemic cardiomyopathy.

Despite major therapeutic advances, heart failure, as a non-communicable disease, remains a life-threatening disorder, with 26 million patients worldwide, causing more deaths than cancer. Therefore, novel strategies for the treatment of heart failure continue to be an important clinical need. Based on preclinical studies, allogenic human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) patches have been proposed as a potential therapeutic candidate for heart failure. We report the implantation of allogeneic hiPSC-CM patches in a patient with ischemic cardiomyopathy (ClinicalTrials.gov, #jRCT2053190081). The patches were produced under clinical-grade conditions and displayed cardiogenic phenotypes and safety in vivo (severe immunodeficient mice) without any genetic mutations in cancer-related genes. The patches were then implanted via thoracotomy into the left ventricle epicardium of the patient under immunosuppressive agents. Positron emission tomography and computed tomography confirmed the potential efficacy and did not detect tumorigenesis in either the heart or other organs. The clinical symptoms improved 6 months after surgery, without any major adverse events, suggesting that the patches were well-tolerated. Furthermore, changes in the wall motion in the transplanted site were recovered, suggesting a favorable prognosis and the potential tolerance to exercise. This study is the first report of a successful transplant of hiPSC-CMs for severe ischemic cardiomyopathy.

Human-Induced Pluripotent Stem Cell-Derived Cardiomyocyte Model for TNNT2 Δ160E-Induced Cardiomyopathy.

BACKGROUND: The Δ160E mutation in TNNT2, which encodes troponin T, is a rare pathogenic variant identified in patients with hypertrophic cardiomyopathy and is associated with poor prognosis. Thus, a convenient human model recapitulating the pathological phenotype caused by TNNT2 Δ160E is required for therapeutic development. METHODS: We identified a heterozygous in-frame deletion mutation (c.478_480del, p.Δ160E) in TNNT2 in a patient with familial hypertrophic cardiomyopathy showing progressive left ventricular systolic dysfunction, leading to advanced heart failure. To investigate the pathological phenotype caused by Δ160E, we generated a set of isogenic induced pluripotent stem cells carrying the heterozygous Δ160E, homozygously corrected or homozygously introduced Δ160E using genome editing and differentiated them into cardiomyocytes (Hetero-Δ160E-, wild type-, and Homo-Δ160E-induced pluripotent stem cells [iPSC]-derived cardiomyocytes [iPSC-CMs]). RESULTS: Hetero-Δ160E-iPSC-CMs exhibited prolonged calcium decay, relaxation impairment, and hypertrophy compared to wild type-iPSC-CMs. Notably, these phenotypes were further exacerbated in Homo-Δ160E-iPSC-CMs. Overexpression of R-GECO-fused Δ160E mutant troponin T prolonged decay time and time to peak of the myofilament-localized calcium transient in iPSC-CMs, indicating that sarcomeric calcium retention with Δ160E may affect intracellular calcium concentration. High-content imaging analysis detected remarkable nuclear translocation of NFATc1, especially in Homo-Δ160E-iPSC-CMs, indicating that the Δ160E mutation promotes hypertrophic signaling pathway in a dose-dependent manner. Increased phosphorylation of CaMKIIδ (calcium/calmodulin-dependent protein kinase IIδ) and phospholamban at Thr17 was observed in Homo- and Hetero-Δ160E-iPSC-CMs. Epigallocatechin-3-gallate, a calcium desensitizing compound, shortened prolonged calcium decay and relaxation duration in Δ160E-iPSC-CMs. CONCLUSIONS: Isogenic iPSC-CMs recapitulate the prolonged calcium decay, relaxation impairment, and subsequent calcium-regulated signaling pathways caused by the TNNT2 Δ160E mutation and can serve as a human model for therapeutic development to prevent hypertrophic cardiomyopathy pathology.

Modeling reduced contractility and impaired desmosome assembly due to plakophilin-2 deficiency using isogenic iPS cell-derived cardiomyocytes.

Loss-of-function mutations in PKP2, which encodes plakophilin-2, cause arrhythmogenic cardiomyopathy (AC). Restoration of deficient molecules can serve as upstream therapy, thereby requiring a human model that recapitulates disease pathology and provides distinct readouts in phenotypic analysis for proof of concept for gene replacement therapy. Here, we generated isogenic induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) with precisely adjusted expression of plakophilin-2 from a patient with AC carrying a heterozygous frameshift PKP2 mutation. After monolayer differentiation, plakophilin-2 deficiency led to reduced contractility, disrupted intercalated disc structures, and impaired desmosome assembly in iPSC-CMs. Allele-specific fluorescent labeling of endogenous DSG2 encoding desmoglein-2 in the generated isogenic lines enabled real-time desmosome-imaging under an adjusted dose of plakophilin-2. Adeno-associated virus-mediated gene replacement of PKP2 recovered contractility and restored desmosome assembly, which was sequentially captured by desmosome-imaging in plakophilin-2-deficient iPSC-CMs. Our isogenic set of iPSC-CMs recapitulates AC pathology and provides a rapid and convenient cellular platform for therapeutic development.

Efficient Method to Dissociate Induced Pluripotent Stem Cell-Derived Cardiomyocyte Aggregates into Single Cells.

The human adult heart consists of approximately four billion cardiomyocytes, which do not possess self-renewal abilities. Severe myocardial infarction and dilated cardiomyopathy result in the loss of more than a billion cardiomyocytes. Induced pluripotent stem cells (iPSCs) can differentiate into various types of cells. Due to this ability, these cells could potentially serve as a new resource for cell therapy. Many studies have utilized cardiomyocytes derived from iPSCs for myocardial regeneration therapy. To obtain large number of cardiomyocytes for transplantation, we need to develop effective methods that would allow us to dissociate multiple cardiomyocyte aggregates simultaneously. Here, we describe a method to efficiently dissociate large number of iPSC-derived cardiomyocyte aggregates.

Development of a drug screening system using three-dimensional cardiac tissues containing multiple cell types.

We hypothesized that an appropriate ratio of cardiomyocytes, fibroblasts, endothelial cells, and extracellular matrix (ECM) factors would be required for the development of three-dimensional cardiac tissues (3D-CTs) as drug screening systems. To verify this hypothesis, ECM-coated human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), ECM-coated cardiac fibroblasts (CFs), and uncoated cardiac endothelial cells (CEs) were mixed in the following ratios: 10:0:0 (10CT), 7:2:1 (7CT), 5:4:1 (5CT), and 2:7:1 (2CT). The expression of cardiac-, fibroblasts-, and endothelial-specific markers was assessed by FACS, qPCR, and immunostaining while that of ECM-, cell adhesion-, and ion channel-related genes was examined by qPCR. Finally, the contractile properties of the tissues were evaluated in the absence or presence of E-4031 and isoproterenol. The expression of ECM- and adhesion-related genes significantly increased, while that of ion channel-related genes significantly decreased with the CF proportion. Notably, 7CT showed the greatest contractility of all 3D-CTs. When exposed to E-4031 (hERG K channel blocker), 7CT and 5CT showed significantly decreased contractility and increased QT prolongation. Moreover, 10CT and 7CT exhibited a stronger response to isoproterenol than did the other 3D-CTs. Finally, 7CT showed the highest drug sensitivity among all 3D-CTs. In conclusion, 3D-CTs with an appropriate amount of fibroblasts/endothelial cells (7CT in this study) are suitable drug screening systems, e.g. for the detection of drug-induced arrhythmia.

Clinical Outcomes of Autologous Stem Cell-Patch Implantation for Patients With Heart Failure With Nonischemic Dilated Cardiomyopathy.

Background Clinical effectiveness of autologous skeletal cell-patch implantation for nonischemic dilated cardiomyopathy has not been clearly elucidated in clinical settings. This clinical study aimed to determine the feasibility, safety, therapeutic efficacy, and the predictor of responders of this treatment in patients with nonischemic dilated cardiomyopathy. Methods and Results Twenty-four nonischemic dilated cardiomyopathy patients with left ventricular ejection fraction <35% on optimal medical therapy were enrolled. Autologous cell patches were implanted over the surface of the left ventricle through left minithoracotomy without procedure-related complications and lethal arrhythmia. We identified 13 responders and 11 nonresponders using the combined indicator of a major cardiac adverse event and incidence of heart failure event. In the responders, symptoms, exercise capacity, and cardiac performance were improved postoperatively (New York Heart Association class II 7 [54%] and III 6 [46%] to New York Heart Association class II 12 [92%] and I 1 [8%], P<0.05, 6-minute walk test; 471 m [370-541 m] to 525 m [425-555 m], P<0.05, left ventricular stroke work index; 31.1 g·m2·beat [22.7-35.5 g·m2·beat] to 32.8 g·m2·beat [28-38.5 g·m2·beat], P=0.21). However, such improvement was not observed in the nonresponders. In responders, the actuarial survival rate was 90.9±8.7% at 5 years, which was superior to the estimated survival rate of 70.9±5.4% using the Seattle Heart Failure Model. However, they were similar in nonresponders (47.7±21.6% and 56.3±8.1%, respectively). Multivariate regression model with B-type natriuretic peptide, pulmonary capillary wedge pressure, and expression of histone H3K4me3 (H3 lysine 4 trimethylation) strongly predicted the responder of this treatment (B-type natriuretic peptide: odds ratio [OR], 0.96; pulmonary capillary wedge pressure: ​OR, 0.58; H3K4me3: OR, 1.35, receiver operating characteristic-area under the curve, 0.96, P<0.001). Conclusions This clinical trial demonstrated that autologous skeletal stem cell-patch implantation might promise functional recovery and good clinical outcome in selected patients with nonischemic dilated cardiomyopathy, in addition to safety and feasibility. Registration URL: http://www.umin.ac.jp/english/. Unique identifiers: UMIN000003273, UMIN0000012906 and UMIN000015892.

The role of MT2-MMP in cancer progression.

The role of MT2-MMP in cancer progression remains to be elucidated in spite of many reports on MT1-MMP. Using a human fibrosarcoma cell, HT1080 and a human gastric cancer cell, TMK-1, endogenous expression of MT1-MMP or MT2-MMP was suppressed by siRNA induction to examine the influence of cancer progression in vitro and in vivo. In HT1080 cells, positive both in MT1-MMP and MT2-MMP, the migration as well as the invasion was impaired by MT1-MMP or MT2-MMP suppression. Also cell proliferation in three dimensional (3D) condition was inhibited by MT1-MMP or MT2-MMP suppression and tumor growth in the nude mice transplanted with tumor cells were reduced either MT1-MMP or MT2-MMP suppression with a prolongation of survival time in vivo. MT2-MMP suppression induces more inhibitory effects on 3D proliferation and in vivo tumor growth than MT1-MMP. On the other hand, TMK-1 cells, negative in MT1-MMP and MMP-2 but positive in MT2-MMP, all the migratory, invasive, and 3D proliferative activities in TMK-1 are decreased only by MT2-MMP suppression. These results indicate MT2-MMP might be involved in the cancer progression more than or equal to MT1-MMP independently of MMP-2 and MT1-MMP.

Regulation of myometrial contractivity during pregnancy in the rat: potential role for DDAH.

There has been little information demonstrating the roles of dimethylarginine dimethylaminohydrolase (DDAH), which is the hydrolyzing enzyme of endogenous nitric oxide synthase (NOS) inhibitors and, in turn, modulates the intracellular concentrations of NOS inhibitors, in the myometrium during the course of pregnancy. Therefore, the present experiments were designed to investigate whether or not DDAH activity, protein and mRNA expression levels are altered during gestation of the rat and, if altered, those changes reflect on the levels of endogenous inhibitors and endothelin-1 (ET-1), and NO-dependent cyclic GMP generation in the myometrium. The up-regulated changes in DDAH activity, DDAH-2 protein and DDAH-2 mRNA expression at mid-gestation were accompanied by the reduced monomethylarginine and asymmetric dimethylarginine as NOS inhibitors, and ET-1 levels, and by the enhanced NO-dependent cyclic GMP production. At term gestation, on the other hand, down-regulated changes in DDAH activity, DDAH-2 protein and DDAH-2 mRNA expression were accompanied by the increased NOS inhibitors and ET-1 levels, and decreased NO-dependent cyclic GMP generation. These results suggest that alterations in DDAH/NOS inhibitors/NO-dependent cyclic GMP/ET-1 pathway are possibly involved in maintaining myometrial quiescence during gestation and controlling delivery at term.

Efficient Differentiation of Mouse Induced Pluripotent Stem Cells into Alveolar Epithelium Type II with a BRD4 Inhibitor.

Regenerative medicine has continued to progress for lung biology and lung diseases. Efforts have focused on a variety of different applications for pluripotent stem cells. Several groups have reported successful methods for inducing differentiation of induced pluripotent stem cells (iPSCs) into the airway epithelium such as alveolar epithelium type II (ATII). However, differentiation efficiency varies among reports and improvements are needed. In the present paper, we propose a novel method for elimination of residual undifferentiated murine iPSCs using JQ1, a potent inhibitor of bromodomain (BRD) and extraterminal domain (BET) family proteins, for efficient differentiation into ATII. First, the murine iPSC line 20D-17 was induced to differentiate into ATII over a period of 26 days (days 0-26) using previously reported embryoid body seeding and stepwise differentiation methods. mRNA expressions of differentiation markers including surfactant protein C (Sftpc) were confirmed by real-time reverse transcription-polymerase chain reaction (RT-PCR) results, and 17% of the cells were shown positive for prosurfactant protein C (proSPC) in flow cytometry analysis. Next, those cells were cultured three-dimensionally in Matrigel for an additional 14 days (days 26-40), during which JQ1 was added for 4 days (days 28-32) to remove residual undifferentiated iPSCs. As a result, on day 40, the mRNA expression level of Sftpc in the three-dimensional culture was maintained at the same level as on day 26 and shown to be further increased by the addition of JQ1, with 39% of the cells found to express proSPC, showing that differentiation efficiency could be further increased. Three-dimensional culture with BRD4 inhibition by JQ1 improved the differentiation induction efficiency to ATII by removing residual undifferentiated murine iPSCs during the differentiation induction process.

Transplantation of Human-induced Pluripotent Stem Cell-derived Cardiomyocytes Is Superior to Somatic Stem Cell Therapy for Restoring Cardiac Function and Oxygen Consumption in a Porcine Model of Myocardial Infarction.

BACKGROUND: Somatic stem cell (SC) therapy can improve cardiac performance following ischemic injury. In this study, we investigated whether induced pluripotent SC-derived cardiomyocytes (iPS-CMs) are more effective than somatic SCs, such as skeletal myoblasts (SM) and mesenchymal (M)SCs, in promoting functional recovery upon transplantation in a porcine model of myocardial infarction. METHODS: Myocardial injury was induced by ameroid ring placement in immunosuppressed female mini pigs; after 1 month, epicardial cell transplantation was performed with iPS-CMs (n = 7), SMs (n = 7), and MSCs (n = 7). Control pigs underwent sham operation (n = 8). RESULTS: Cell therapy improved functional recovery 2 months after myocardial infarction, as evidenced by increased ejection fraction (iPS-CM, +7.3% ± 2.2% and SM, +5.8% ± 5.4% vs control, -4.4% ± 3.8%; P < 0.05). The analysis of regional contractile function in the infarcted zone revealed an increase in transverse peak strain (iPS-CM, +4.6% ± 2.2% vs control, -3.8% ± 4.7%; P < 0.05). The C-11 acetate kinetic analysis by positron emission tomography showed that the work-metabolic cardiac energy efficacy increased by the transplantation of iPS-CMs, but was reduced by the other cell types. This was accompanied by decreased myocardial wall stress in the infarcted zone (iPS-CM, -27.6 ± 32.3 Pa and SM, -12.8 ± 27 Pa vs control, +40.5 ± 33.9 Pa; P < 0.05). CONCLUSIONS: The iPS-CM is superior to other somatic cell sources in terms of improving regional contractile function and cardiac bioenergetic efficiency, suggesting greater clinical benefits in severely damaged myocardium.