Cardiomyocyte Sheets Research Articles

Human induced pluripotent stem cell-derived cardiomyocyte patches ameliorate right ventricular function in a rat pressure-overloaded right ventricle model.

Right ventricular (RV) failure following surgical repair of congenital heart disease affects survival. Human induced pluripotent stem cell-derived cardiomyocyte (hiPS-CM) sheet transplantation ameliorated left ventricular dysfunction in preclinical studies, indicating its efficacy in RV failure in congenital heart disease. This study aimed to evaluate whether hiPS-CMs could improve RV function in rats with pressure-overloaded RV failure. F344/NJcl-rnu/rnu rats underwent pulmonary artery banding (PAB) via left thoracotomy. Four weeks after PAB, hiPS-CM patch transplantation to the RV was performed in the hiPS-CM group (n = 33), and a sham operation was performed in the sham group (n = 18). We evaluated cardiac catheterization, positron emission tomography data, and pathological results 8weeks following PAB. RV end-diastolic pressure, the time constant of isovolumic relaxation, and end-diastolic pressure-volume relation were significantly ameliorated in the hiPS-CM group compared with in the sham group. Picrosirius red staining revealed that anti-fibrotic effects were significantly higher in the hiPS-CM group than in the sham group. Von Willebrand factor staining revealed significantly higher myocardial capillary vascular density in the hiPS-CM group than in the sham group. hiPS-CMs were detected in the epicardium 4weeks after hiPS-CM sheet transplantation. The angiogenic gene expression in the myocardium was significantly higher in the hiPS-CM group than in the sham group. Overall, in rats with pressure-overloaded RV failure, hiPS-CM patch transplantation could improve diastolic function, suppress ventricular fibrosis, and increase capillary density, suggesting that it is a promising treatment for RV failure.

CD8+ T cell-mediated rejection of allogenic human-induced pluripotent stem cell-derived cardiomyocyte sheets in human PBMC-transferred NOG MHC double knockout mice

BackgroundTransplantation of human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) has emerged as a promising therapy to treat end-stage heart failure. However, immunogenicity of hiPS-CMs in transplanted patients has not been fully elucidated. Thus, in vivo models are required to estimate immune responses against hiPS-CMs in transplant recipients. MethodsWe transferred human peripheral blood mononuclear cells (hPBMCs) into NOD/Shi-scid IL2rgnull (NOG) MHC class I/II double knockout (NOG-ΔMHC) mice, which were reported to accept hPBMCs without xenogeneic-graft-versus-host disease (xeno-GVHD). Then, hiPS-CM sheets generated from the hiPS cell line 201B7 harboring a luciferase transgene were transplanted into the subcutaneous space of NOG-ΔMHC mice. Graft survival was monitored by bioluminescent images using a Xenogen In Vivo Imaging System. ResultsThe human immune cells were engrafted for more than three months in NOG-ΔMHC mice without lethal xeno-GVHD. The hiPS-CMs expressed a moderate level of human leukocyte antigen (HLA)-class I, but not HLA-class II, molecules even after interferon-gamma (IFN-γ) stimulation. Consistently, the allogenic IFN-γ-treated hiPS-CMs induced weak CD8+ but not CD4+, T cell responses in vitro. hiPS-CM sheets disappeared approximately 17–24 days after transplantation in hPBMC-transferred NOG-ΔMHC mice, and CD8+ T cell depletion significantly prolonged graft survival, similar to what was observed following tacrolimus treatment. ConclusionhiPS-CMs are less immunogenic in vitro but induce sufficient CD8+ T cell-mediated immune responses for graft rejection in vivo.

Successful reconstruction of the rat ureter by a syngeneic collagen tube with a cardiomyocyte sheet

IntroductionUreteral injuries require surgical intervention as they lead to loss of renal function. The current reconstructive techniques for long ureteral defects are problematic. Consequently, this study aimed to reconstruct the ureter in a rat model using subcutaneously prepared autologous collagen tubes (Biotubes). MethodsThe lower ureter of LEW/SsNSlc rats was ligated to dilate the ureter to make anastomosis easier, and reconstruction was performed six days later by anastomosing the dilated ureter and bladder with a Biotube that was prepared subcutaneously in syngeneic rats. Some rats underwent left nephrectomy and ureter reconstruction simultaneously as negative controls to evaluate the effects of urine flow on patency. The other rats were divided into three groups as follows: a group in which the ureter was reconstructed with the Biotube alone, a group in which cardiomyocyte sheets made from the neonatal hearts of syngeneic rats were wrapped around the Biotube, and a group in which an adipose-derived stem cell sheets made from the inguinal fat of adult syngeneic rats were wrapped. Contrast-enhanced computed tomography and pathological evaluations were performed two weeks after reconstruction. ResultIn the Biotube alone group, all tubes were occluded and hydronephrosis developed, whereas the urothelium regenerated beyond the anastomosis when the left kidney was not removed, suggesting that urothelial epithelial spread occurred with urinary flow. The patency of the ureteral lumen was obtained in some rats in the cardiomyocyte sheet covered group, whereas stricture or obstruction of the reconstructed ureter was observed in all rats in the other groups. Pathological evaluation revealed a layered urothelial structure in the cardiomyocyte sheet covered group, although only a small amount of cardiomyocyte sheets remained. ConclusionUrinary flow may support the epithelial spread of the urothelium into the reconstructed ureter. Neonatal rat cardiomyocyte sheets supported the patency of the regenerated ureter with a layered urothelium.

Electropharmacological Characterization of Licorice Using the Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Sheets and the Chronic Atrioventricular Block Dogs.

Licorice has been traditionally prescribed for palpitation, whereas its overdose has caused lethal arrhythmias including torsade de pointes. Licorice contains glycyrrhizic acid of ≥ 2% (w/w), which is hydrolyzed to glycyrrhetinic acid (GRA) in the intestine. Since their cardiac electropharmacological properties are not fully understood, we assessed them to ask mechanism of licorice-induced torsade de pointes. GRA at 0.1, 1 and 10μg/mL was cumulatively applied to the human induced pluripotent stem cell-derived cardiomyocytes sheets (n = 6). GRA shortened spontaneous activation interval and repolarization period, and decreased maximum contraction velocity, indicating Ca2+ channel blockade. It prolonged effective refractory period and post-repolarization refractoriness with a steep frequency-dependency, whereas it delayed conduction with a modest use-dependency, resembling lidocaine in the mode of Na+ channel-blocking action. Meanwhile, Kanzoto containing a decoction of licorice alone in a dose of 2 or 6g/body/day was orally administered to the conscious chronic atrioventricular block dogs for 3days (n = 4). Kanzoto prolonged QT interval with increasing its temporal dispersion, suggesting K+ channel suppression, and slightly decreased the plasma K+ concentration without inducing torsade de pointes. Moreover, it significantly suppressed atrial and idioventricular rates, leading to sinus arrest along with the onset of ventricular fibrillation in one animal, possibly due to Na+ channel blockade. These results indicate that electropharmacological profile of licorice can be explained by Na+, Ca2+ and K+ channels blockade, which may be associated with low torsadogenic risk, but might contribute to the onset of other types of lethal ventricular arrhythmias.

Anticancer drug nilotinib-induced electrical, calcium, and mechanical alternans in human iPS cell-derived cardiomyocyte sheets

Anticancer drug nilotinib-induced electrical, calcium, and mechanical alternans in human iPS cell-derived cardiomyocyte sheets

Xeno-Free Integrated Platform for Robust Production of Cardiomyocyte Sheets from hiPSCs.

Human induced pluripotent stem cells (hiPSCs) can be efficiently differentiated into cardiomyocytes (CMs), which can be used for cardiac disease modeling, for drug screening, and to regenerate damaged myocardium. Implementation of xeno-free culture systems is essential to fully explore the potential of these cells. However, differentiation using xeno-free adhesion matrices often results in low CM yields and lack of functional CM sheets, capable of enduring additional maturation stages. Here, we established a xeno-free CM differentiation platform using TeSR/Synthemax, including a replating step and integrated with two versatile purification/enrichment metabolic approaches. Results showed that the replating step was essential to reestablish a fully integrated, closely-knit CM sheet. In addition, replating contributed to increase the cTnT expression from 65% to 75% and the output from 2.2 to 3.1 CM per hiPSC, comparable with the efficiency observed when using TeSR/Matrigel. In addition, supplementation with PluriSin1 and Glu-Lac+ medium allowed increasing the CM content over 80% without compromising CM sheet integrity or functionality. Thus, this xeno-free differentiation platform is a reliable and robust method to produce hiPSC-derived CMs, increasing the possibility of using these cells safely for a wide range of applications.

Abstract 10588: Pre-Clinical Study of Induced Pluripotent Stem Cell-Derived Cardiomyocyte Sheet Transplantation for a Cynomolgus Macaque Model of Doxorubicin-Induced Dilated Cardiomyopathy

Introduction: Due to the serious shortage of cardiac donors in Japan, there is a strong demand for an effective alternative treatment to heart transplantation (HTx) and left ventricular assist device for end-stage severe heart failure. We have conducted induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) sheet transplantation for ischemic cardiomyopathy as first in human clinical trial (jRCT2053190081), based on several pre-clinical studies suggesting both of angiogenic effects and mechanical contribution of iPSC-CMs. Then, we are next aiming to expand the indication for dilated cardiomyopathy (DCM), which is the most common indication for HTx worldwide. In this study, we established a cynomolgus macaque model of Doxorubicin (Dox)-induced DCM and performed the iPSC-CM sheet transplantation. Methods: Twelve male cynomolgus macaques were treated with Dox to induce DCM every two weeks for three consecutive days in three different doses. The cardiac function was assessed over time by transthoracic echocardiography (TTE) and the effect of doxorubicin on the myocardium was investigated with histological analysis. After the establishment of animal model, we performed the iPSC-CM sheet transplantation and assessed the postoperative cardiac function by TTE and cardiac computed tomography (CT). Results: Each Two macaques treated with high (1.2 kg/mg/day) and medium (1.0 kg/mg/day) dose of Dox died before the establishment of animal model. Of the remaining eight macaques in the low dose (0.8 kg/mg/day) group, six had progressively decreased cardiac function. Histology revealed vacuolization in the left ventricle of the Dox-treated heart. Postoperative evaluation two weeks after iPSC-CM sheet transplantation revealed the improvement in cardiac function on TTE and cardiac CT (left ventricular ejection fraction: from 22 % to 31%, and from 19 % to 31 %, respectively). Conclusions: Three-month administration of low-dose Dox was able to create a cynomolgus macaque model of Dox-induced DCM with relatively high reproducibility. IPSC-CM sheet transplantation may have the possibility for improving cardiac function of DCM.

In vitro circulation model driven by tissue-engineered dome-shaped cardiac tissue

The heart is an essential organ for animals and humans. With the increased availability of pluripotent stem cells, the use of three-dimensional cardiac tissues consisting of cultured cardiomyocytes in in vitro drug evaluation has been widely studied. Several models have been proposed for the realization of the pump function, which is the original function of the heart. However, there are no models that simulate the human circulatory system using cultured cardiac tissue. This study shows that a dome-shaped cardiac tissue fabricated using the cell sheet stacking technique can achieve a heart-like pump function and circulate culture medium, there by mimicking the human circulatory system. Firstly, human induced pluripotent stem cells were differentiated into autonomously beating cardiomyocytes, and cardiomyocyte cell sheets were created using temperature-responsive culture dishes. A cardiomyocyte sheet and a human dermal fibroblast sheet were stacked using a cell sheet manipulator. This two-layered cell sheet was then inflated to create a dome-shaped cardiac tissue with a base diameter of 8 mm. The volume of the dome-shaped cardiac tissue changed according to the autonomous beating. The stroke volume increased with the culture period and reached 21 ± 8.9 μl (n = 6) on day 21. It also responded to β-stimulant and extracellular calcium concentrations. Internal pressure fluctuations were also recorded under isovolumetric conditions by dedicated culture devices. The peak heights of pulsatile pressure were 0.33 ± 0.048 mmHg (n = 3) under a basal pressure of 0.5 mmHg on day 19. When the tissue was connected to a flow path that had check valves applied, it drove a directional flow with an average flow rate of approximately 1 μl s−1. Furthermore, pressure–volume (P–V) diagrams were created from the simultaneous measurement of changes in pressure and volume under three conditions of fluidic resistance. In conclusion, this cardiac model can potentially be used for biological pumps that drive multi-organ chips and for more accurate in vitro drug evaluation using P–V diagrams.

Hair-follicle-associated pluripotent (HAP) stem cells differentiate into mature beating cardiomyocyte sheets on flexible substrates in vitro.

Cardiomyocytes have been differentiated from various stem cells such as human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC), but it is difficult to produce mature cardiomyocytes. We showed rat hair-follicle-associated pluripotent (HAP) stem cells have pluripotency and produced mature beating cardiomyocyte sheets differentiated from rat HAP stem cells. The upper parts of rat vibrissa hair follicles were cultured in 10% FBS DMEM and stained with antibodies of the ectoderm, mesoderm, endoderm system to show the differentiation of multiple cell types. Moreover, HAP stem cells were cultured under three different conditions to decide the most suitable culture conditions for making beating cardiomyocyte sheets. The beating cardiomyocyte sheets were shown to be mature by staining sarcomere structures. Isoproterenol alone and the combination of isoproterenol, activin A, bone morphogenetic protein 4 (BMP4) and basic fibroblast growth factor (bFGF) effectively induced beating long-fiber cardiomyocytes, which formed beating sheets, only in the presence of all four agents. Flexible substrates were essential for the differentiation of sheets of mature beating cardiomyocytes for HAP stem cells. The features of the cardiomyocytes differentiated from HAP stem cells demonstrate they have clinical potential for heart regeneration.

Bioartificial pulsatile cuffs fabricated from human induced pluripotent stem cell-derived cardiomyocytes using a pre-vascularization technique

There is great interest in the development of techniques to bioengineer pulsatile myocardial tissue as a next-generation regenerative therapy for severe heart failure. However, creation of thick myocardial grafts for regenerative medicine requires the incorporation of blood vessels. In this study, we describe a new method of constructing a vascular network in vivo that allows the construction of thick human myocardial tissue from multi-layered cell sheets. A gelatin sheet pre-loaded with growth factors was transplanted onto the superficial femoral artery and vein of the rat. These structures were encapsulated together within an ethylene vinyl alcohol membrane and incubated in vivo for 3 weeks (with distal superficial femoral artery ligation after 2 weeks to promote blood flow to the vascular bed). Subsequently, six cardiomyocyte sheets were transplanted onto the vascular bed in two stages (three sheets, two times). Incubation of this construct for a further week generated vascularized human myocardial tissue with an independent circulation supplied by an artery and vein suitable for anastomosis to host vessels. Notably, laminating six cell sheets on the vascular bed in two stages rather than one allowed the creation of thicker myocardial tissue while suppressing tissue remodeling and fibrosis. Finally, the pulsatile myocardial tissue was shown to generate auxiliary pressure when wrapped around the common iliac artery of a rat. Further development of this technique might facilitate the generation of circulatory assist devices for patients with heart failure.

Electropharmacological analyses of licorice using the human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) sheets and the chronic atrioventricular block dogs

Electropharmacological analyses of licorice using the human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) sheets and the chronic atrioventricular block dogs

Nonlinear active stress – Principal strain relation during pulsation of human iPS cell derived cardiomyocyte sheet

Recently, a technique for differentiating human iPS cells into cardiomyocytes has been established and therefore, human iPS cell derived cardiomyocytes (hiPS-CM) can be utilized for the biomechanical study of myocardial tissues. In the present study, a sheet structure of hiPS-CM was fabricated using cell sheet technology and the pulsation behavior was measured using a high speed imaging system. The minimum principal strain was then evaluated using the digital correlation method and the active stress was calculated on the basis of the theoretical model proposed by Guccione et al. It was found that the active stress-strain behavior clearly exhibited a nonlinear viscoelastic response characterized by a hysteresis loop. This nonlinear response was also found to be effectively predicted by the constitutive equation derived from the viscoelastic Maxwell model. A microscopic mechanism involved in the hysteresis loop was also proposed on the basis of the sarcomere structure and mechanical behavior.

Continuous measurement of surface electrical potentials from transplanted cardiomyocyte tissue derived from human-induced pluripotent stem cells under physiological conditions in vivo.

Recording the electrical potentials of bioengineered cardiac tissue after transplantation would help to monitor the maturation of the tissue and detect adverse events such as arrhythmia. However, a few studies have reported the measurement of myocardial tissue potentials in vivo under physiological conditions. In this study, human-induced pluripotent stem cell-derived cardiomyocyte (hiPSCM) sheets were stacked and ectopically transplanted into the subcutaneous tissue of rats for culture in vivo. Three months after transplantation, a flexible nanomesh sensor was implanted onto the hiPSCM tissue to record its surface electrical potentials under physiological conditions, i.e., without the need for anesthetic agents that might adversely affect cardiomyocyte function. The nanomesh sensor was able to record electrical potentials in non-sedated, ambulating animals for up to 48h. When compared with recordings made with conventional needle electrodes in anesthetized animals, the waveforms obtained with the nanomesh sensor showed less dispersion of waveform interval and waveform duration. However, waveform amplitude tended to show greater dispersion for the nanomesh sensor than for the needle electrodes, possibly due to motion artifacts produced by movements of the animal or local tissue changes in response to surgical implantation of the sensor. The implantable nanomesh sensor utilized in this study potentially could be used for long-term monitoring of bioengineered myocardial tissue in vivo under physiological conditions.

Abstract 13577: Human Induced Pluripotent Stem Cell-derived Cardiomyocyte Patches Ameliorate Right Ventricular Diastolic Dysfunction in a Rat Pressure-overloaded Right Heart Model

Introduction: Right ventricular (RV) failure following definitive repair of congenital heart diseases (CHD) have great impact on survival after surgical intervention. Recently, human induced pluripotent stem cell-derived cardiomyocyte (hiPS-CM) sheet transplantation ameliorated left ventricular dysfunction in preclinical study, associating possible efficacy in RV failure of CHD. Hypothesis: We hypothesized whether hiPS-CM patch could improve distressed RV function in rat pressure overload model. Methods: F344/NJcl- rnu/rnu rat (7-9 weeks old, male, 180-210g) underwent pulmonary artery banding (PAB) via left thoracotomy under general anesthesia. Four weeks after PAB, hiPS-CM patch transplantation to the RV was performed in the hiPS-CM patch transplantation group (n = 10), whereas a sham operation was performed in the sham group (n =10). We evaluated for cardiac catheterization (CATH) data and pathological findings 8 weeks after PAB. Results: In CATH, RV end diastolic pressure (sham; 10.0 ± 4.1 vs hiPS-CM; 5.5 ± 2.7 mmHg, P < 0.01), time constant of isovolumic relaxation (sham; 24.9 ± 8.6 vs hiPS-CM; 16.5 ± 6.4 ms, P < 0.01) and end-diastolic pressure volume-relation (sham; 365.6 ± 246.3 vs hiPS-CM; 214.0 ± 147.4 mmHg/mL, P <0.05) were significantly ameliorated in hiPS-CM group compared with sham group. Sirius-red staining showed a significant anti-fibrotic effects in the hiPS-CM group compared with the sham group (13.4 ± 5.3 vs 20.7 ± 4.8 %, P <0.05). Factor VIII stain showed that myocardial capillary vascular density in the hiPS-CM group was significantly higher than in the sham group (640 ± 131 vs 362 ± 31 units/mm 2 , P <0.001). hiPS-CMs were detectable on epicardium 2 weeks after hiPS-CM patch transplantation. The expression levels of vascular endothelial growth factor and hepatocyte growth factor in the hiPS-CM group was significantly higher than in the sham group (P <0.05). Conclusions: In a rat model of a pressure-overload RV, hiPS-CM patch transplantation improved the diastolic function with suppressed ventricular fibrosis and increased capillary density, suggesting a promising treatment for RV failure in CHD patients after surgical repair.

Abstract 16282: The Development of the New Treatment for Distressed Microvascular Dysfunction in Porcine Dilated Cardiomyopathy Model Using Induced Pluripotent Stem Cell Derived Cardiomyocyte Sheet

Introduction: The progression of dilated cardiomyopathy (DCM) mainly involves genetic mutations or ischemia at the cellular level, leading to microvascular dysfunction associated with cell death, interstitial fibrosis, and high wall stress. Radical treatment of DCM requires how to ameliorate its microcirculation and integrate cardiomyocytes created ex vivo into recipient myocardium. Hypothesis: The induced pluripotent stem cell derived cardiomyocyte sheets (iPS-sheet) has therapeutic potential by the improvement of microcirculation in a porcine DCM model. Methods: The iPS-sheets were generated from clinical grade human iPS cells. A DCM model was created by tachycardia pacing, and iPS-sheet was transplanted with immunosuppressive agents 1 month after the initiation of the pacing. We compared the therapeutic efficacy functionally and pathologically between the iPS-sheet transplant group (iPS-group) and the sham group after 1 month of transplantation. Results: On echocardiography, the iPS group showed a significant improvement in contractility compared to the sham group (LVEF 4 weeks after transplantation iPS vs. sham 49.0±6.5% vs. 36.4±3.3%, p<0.05, Figure A). Pressure-volume loop analysis revealed that a significant decrease in left ventricular end diastolic pressure and an improvement in end-systolic pressure-volume relationship in the iPS group (Figure B). Ammonia PET showed improvement in myocardial blood flow at both rest and stress in iPS group (Figure C). Histological analysis revealed that the density of CD31-positive capillaries in transplanted area was significantly greater in the iPS group than the sham group. Immunostaining revealed iPS-sheet were detected on the epicardium of the distressed myocardium (Figure D). Conclusions: The iPS sheet showed engraftment in distressed myocardium, leading to amelioration in cardiac function through improving microcirculation with angiogenesis in porcine DCM model.

Preseeding of Mesenchymal Stem Cells Increases Integration of an iPSC-Derived CM Sheet into a Cardiac Matrix.

Cell sheet technology has demonstrated great promise in delivering a large amount of therapeutic cells for tissue repair, including in the myocardium. However, the lack of host integration remains one of the key challenges in using cell sheets for cardiac repair. Paracrine factors secreted by mesenchymal stem cells (MSCs) have been reported to facilitate tissue repair and regeneration in a variety of ways. It has been demonstrated that paracrine factors from MSCs could enhance scaffold recellularization and vascularization. In this study, we used an in vitro cardiac matrix mimic platform to examine the effects of hMSCs preseeding on the interactions between cell sheets and cardiac matrix. The fabricated human induced pluripotent stem cells-derived cardiomyocyte sheets were attached to a decellularized porcine myocardium slice with or without preseeding of hMSCs. The hMSCs preseeding significantly enhanced the interactions between cardiomyocyte sheets and cardiac matrix in terms of cell migration distance, cell distribution, and mature vascular and cardiomyocyte marker expressions in the matrix. Growth factor and matrix metalloproteinases array analysis suggested that hMSCs- induced vascularization and MMPs regulation are the two possible mechanisms that lead to the improved CMs and cardiac matrix interactions. Further examination of these two mechanisms will enable the development of new approaches to facilitate transplanted cells for tissue repair.

Measuring the Contractile Force of Multilayered Human Cardiac Cell Sheets.

Three-dimensional (3D) cardiac tissue reconstruction using tissue engineering technology is a rapidly growing area of regenerative medicine and drug screening development. However, there remains an urgent need for the development of a method capable of accurately measuring the contractile force of physiologically relevant 3D myocardial tissues to facilitate the prediction of human heart tissue drug sensitivity. To this end, our laboratory has developed a novel drug screening model that measures the contractile force of cardiac cell sheets prepared using temperature-responsive culture dishes. To circumvent the difficulties that commonly arise during the stacking of cardiomyocyte sheets, we established a stacking method using centrifugal force, making it possible to measure 3D myocardial tissue. Human induced pluripotent stem cell-derived cardiomyocytes were seeded in a temperature-responsive culture dish and processed into a sheet. The cardiac cell sheets were multilayered to construct 3D cardiac tissue. Measurement of the contractile force and cross-sectional area of the multilayered 3D cardiac tissue were then obtained and used to determine the relationship between the cross-sectional area of the cardiac tissue and its contractile force. The contractile force of the 1-, 3-, and 5-layer tissues increased linearly in proportion to the cross-sectional area. A result of 6.4 mN/mm2, accounting for one-seventh of the contractile force found in adult tissue, was obtained. However, with 7-layer tissues, there was a sudden drop in the contractile force, possibly because of limited oxygen and nutrient supply. In conclusion, we established a method wherein the thickness of the cell sheets was controlled through layering, thus enabling accurate evaluation of the cardiac contractile function. This method may enable comparisons with living heart tissue while providing information applicable to regenerative medicine and drug screening models.

Pulsatile tubular cardiac tissues fabricated by wrapping human iPS cells-derived cardiomyocyte sheets.

The purpose of this study was to fabricate pulsatile tubular cardiac tissue using cell sheet based-tissue engineering. First, we fabricated human induced pluripotent stem cell (hiPSc)-derived cardiomyocyte sheets and normal human dermal fibroblast (NHDF) sheets which are harvested from temperature responsive culture dishes only by lowering the temperature. Then tubular cardiac tissues are formed by wrapping one hiPSc-derived cardiomyocyte sheet and three NHDF sheets around an octagonal column, and both ends of the tubular tissue were covered with fibrin and collagen gel. The octagonal column with the tubular tissue was connected to an in vitro circulation system in a culture box. After four-day culture, the cardiac tissue survived and pulsated spontaneously in the circulation system. Furthermore, the analysis with a Millar catheter inserted into the cardiac tubes revealed significant inner pressure changes generated by their beating. In addition, the tubular cardiac tissue pulsated in response to the electrical stimulation. Although histological analyses demonstrated that cardiac troponin T-positive cells stratified the inner surface of the tubular tissues, gene expression analyses showed an immature state of these cardiomyocytes. Thus, cell sheet-based tissue engineering realized human pulsatile tubular cardiac tissue fabrication and we believe that these tubular cardiac tissues should contribute to future drug screening and regenerative therapy for heart diseases.

MHC-mismatched Allotransplantation of Induced Pluripotent Stem Cell-derived Cardiomyocyte Sheets to Improve Cardiac Function in a Primate Ischemic Cardiomyopathy Model.

Although allogeneic-induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) exhibit potential in cardiomyogenesis for heart failure, whether major histocompatibility complex (MHC)-matched allogenic iPSC implantation (MMAI) minimizes immune rejection for cell survival or functional recovery remains unknown. We herein explored whether MMAI with an iPSC-CM sheet is stable for a longer period and therapeutically more effective than MHC-mismatched AI in a primate ischemic cardiomyopathy model. Green fluorescent protein-transfected iPSC-CM sheets, derived from cynomolgus macaques with homozygous MHC haplotypes ''HT1,'' were transplanted on the left ventricle, generated by ligating the left anterior descending artery for 2 weeks in an ischemic model with or without heterozygous HT1 as MMAI and MHC-mismatched AI. Sham models were made by opening the chest at 14 days after left anterior descending ligation without any treatment. Stereomicroscopy revealed that at 4 months after transplantation, green fluorescent protein intensity was higher in the MMAI group than in the MHC-mismatched AI group and the sham group. Immunohistochemistry staining revealed that host immune reaction with CD3-positive cells was stronger in MHC-mismatched AI than in MMAI at 3 months. Cardiac function improved both in MMAI and MHC-mismatched AI at 1 month after transplantation and was preserved until 6 months, whereas in the sham group, functional deterioration progressed over time. Although MHC-homo-iPSCs are preferred to avoid immune rejection, MHC-mismatched iPSC-CMs can also induce comparable cardiac functional recovery at late follow-up, suggesting that MHC-mismatched iPSC-based cardiac regenerative therapy with immunosuppressants is a feasible option for treating heart failure in clinical settings.

Design of VEGF Releasing Fiber Mat for Effective Transplantation of Cardiomyocyte Sheets

Design of VEGF Releasing Fiber Mat for Effective Transplantation of Cardiomyocyte Sheets