Low-adhesion culture selection for human iPS cell-derived cardiomyocytes.

Despite progress in generating cardiomyocytes from pluripotent stem cells, these populations often include non-contractile cells, necessitating cardiomyocyte selection for experimental purpose. This study explores a novel cardiomyocyte enrichment mechanism: low-adhesion culture selection. The cardiac cells derived from human induced pluripotent stem cells were subjected to a coating-free low-adhesion culture using bovine serum albumin and high molecular weight dextran sulfate. This approach effectively increased the population of cardiac troponin T-positive cardiomyocytes. Similar results were obtained with commercially available low-adhesion culture dishes. Subsequently, we accessed the practicality of selection of cardiomyocytes using this phenomenon by comparing it with established methods such as glucose-free culture and selection based on puromycin resistance genes. The cardiomyocytes enriched through low-adhesion culture selection maintained autonomous pulsation and responsiveness to beta-stimuli. Moreover, no significant differences were observed in the expression of genes related to subtype commitment and maturation when compared to other selection methods. In conclusion, cardiomyocytes derived from pluripotent stem cells were more low-adhesion culture resistant than their accompanying non-contractile cells, and low-adhesion culture is an alternative method for selection of pluripotent stem cell-derived cardiomyocytes.

Left Ventricular End-Systolic Diameter May Predict Persistent Heart Failure with Reduced Ejection Fraction.

Patients with persistent heart failure (HF) with reduced ejection fraction (HFrEF) have a poorer prognosis than those with HF with improved ejection fraction (HFimpEF). However, data on the predictive value of echocardiographic parameters for persistent HFrEF are lacking. We retrospectively studied 443 patients who were diagnosed with HFrEF (EF ≤ 40%) during hospitalization and underwent echocardiography at the 1-year follow-up. We divided them into the 2 groups: HFimpEF (EF > 40%) and persistent HFrEF group at 1-year follow-up, and assessed the predictive value of echocardiographic parameters at discharge for persistent HFrEF. In total, 301/443 patients (68%) were diagnosed with persistent HFrEF and 142/443 (32%) with HFimpEF at the 1-year follow-up. Kaplan-Meier analysis revealed that the persistent HFrEF group had a poorer prognosis than the HFimpEF group (log-rank, P < 0.001). Receiver operating characteristic curve analysis revealed that left ventricular end-systolic diameter (LVESD) had the highest area under the curve (AUC) (0.70; 95% confidence interval [CI]: 0.64-0.75; cutoff value: 55 mm) among various echocardiographic parameters. LVESD was an independent predictor of persistent HFrEF at the 1-year follow-up (odds ratio: 1.07, 95%CI: 1.02-1.12) upon multivariable logistic regression analysis. The incidence of persistent HFrEF was higher in patients with an LVESD ≥ 55 mm than in those with an LVESD < 55 mm (81% versus 55%, Fisher's exact test, P < 0.001). In conclusion, an LVESD (≥ 55 mm) was associated with persistent HFrEF. Focusing on LVESD in daily practice may help clinicians with risk stratification for decision-making regarding management in patients with advanced HF refractory to guideline-directed medical therapy.

Cardiac cell sheet engineering for regenerative medicine and tissue modeling.

Stem cell biology and tissue engineering are essential techniques for cardiac tissue construction. We have succeeded in fabricating human cardiac tissue using the mass production technology of human iPS cell-derived cardiomyocytes and cell sheet engineering, and we are developing regenerative medicine and tissue models to apply this tissue to heart disease research. Cardiac tissue fabrication and tissue functional evaluation technologies for contractile and electrophysiological function are indispensable, which lead to the functional improvement of bioengineered human cardiac tissue.

Pericardial Grafting of Cardiac Progenitor Cells in Self-Assembling Peptide Scaffold Improves Cardiac Function After Myocardial Infarction.

Many studies have explored cardiac progenitor cell (CPC) therapy for heart disease. However, optimal scaffolds are needed to ensure the engraftment of transplanted cells. We produced a three-dimensional hydrogel scaffold (CPC-PRGmx) in which high-viability CPCs were cultured for up to 8 weeks. CPC-PRGmx contained an RGD peptide-conjugated self-assembling peptide with insulin-like growth factor-1 (IGF-1). Immediately after creating myocardial infarction (MI), we transplanted CPC-PRGmx into the pericardial space on to the surface of the MI area. Four weeks after transplantation, red fluorescent protein-expressing CPCs and in situ hybridization analysis in sex-mismatched transplantations revealed the engraftment of CPCs in the transplanted scaffold (which was cellularized with host cells). The average scar area of the CPC-PRGmx-treated group was significantly smaller than that of the non-treated group (CPC-PRGmx-treated group = 46 ± 5.1%, non-treated MI group = 59 ± 4.5%; p < 0.05). Echocardiography showed that the transplantation of CPC-PRGmx improved cardiac function and attenuated cardiac remodeling after MI. The transplantation of CPCs-PRGmx promoted angiogenesis and inhibited apoptosis, compared to the untreated MI group. CPCs-PRGmx secreted more vascular endothelial growth factor than CPCs cultured on two-dimensional dishes. Genetic fate mapping revealed that CPC-PRGmx-treated mice had more regenerated cardiomyocytes than non-treated mice in the MI area (CPC-PRGmx-treated group = 0.98 ± 0.25%, non-treated MI group = 0.25 ± 0.04%; p < 0.05). Our findings reveal the therapeutic potential of epicardial-transplanted CPC-PRGmx. Its beneficial effects may be mediated by sustainable cell viability, paracrine function, and the enhancement of de novo cardiomyogenesis.

GATA6 regulates anti-angiogenic properties in human cardiac fibroblasts via modulating LYPD1 expression.

Introduction: Fibroblasts contribute to the structure and function of tissue and organs; however, their properties differ in each organ given the topographic variation in gene expression among tissues. We previously reported that LYPD1, which is expressed in cardiac fibroblasts, has the capacity to inhibit sprouting of vascular endothelial cells. LYPD1 has been shown to be highly expressed in the human brain and heart, but the regulation of LYPD1 expression in cardiac fibroblasts has not been elucidated in detail. Methods: To identify the LYPD1-modulating transcription factor, motif enrichment analysis and differential expressed gene analysis using microarray data were performed. Quantitative real-time PCR was used to evaluate gene expression. Gene silencing were performed by transfection of siRNA. Western blot analyzed protein expression in NHCF-a. To assess the effect of GATA6 on the regulation of LYPD1 gene expression, dual-luciferase reporter assay was performed. Co-culture and rescue experiments were performed to evaluate endothelial network formation. Results: Motif enrichment analysis and differential expressed gene analysis using microarray data and quantitative real-time PCR revealed that CUX1, GATA6, and MAFK were candidate transcription factors. Of these, the inhibition of GATA6 expression using siRNA decreased LYPD1 gene expression and co-expression of GATA6 with a reporter vector containing the upstream sequence of the LYPD1 gene resulted in increased reporter activity. Endothelial cell network formation was attenuated when co-cultured with cardiac fibroblasts, but it was significantly restored when co-cultured with cardiac fibroblasts wherein the expression of GATA6 was knocked down with siRNA. Conclusion: GATA6 regulate the anti-angiogenic properties of cardiac fibroblasts by modulating LYPD1 expression.

Importance of beating rate control for the analysis of drug effects on contractility in human induced pluripotent stem cell-derived cardiomyocytes.

Cardiac contractility evaluation using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) has recently attracted much attention as a clinical cardiotoxicity predictive model. Most studies on this were conducted under spontaneous beating conditions and involved video-based analyses. Cardiac contractility is known to be influenced by beating rates; accordingly, beating rate control is recommended to accurately analyze the effects of drugs on cardiac contractility. Therefore, we investigated the relationship between contraction parameters and beating rates of cardiac cell sheet tissues by directly measuring the contraction force and compared the effects of ion channel drugs (mexiletine, ranolazine, and dofetilide) on contraction parameters under spontaneous beating conditions with those under pacing (1 Hz) conditions. To characterize the contraction/relaxation kinetics, we introduced a novel analysis tool, called a "C-V loop," a plot of contraction force versus force-changing rate ("velocity"). When we increased the beating rate, the contraction force, force-changing rate, and relaxation time markedly decreased. The occurrence frequencies of beating arrest and irregular beats at high concentration ranges of mexiletine and ranolazine were more suppressed in paced samples than in spontaneously beating ones. We also found that relaxation time increased by treatment with dofetilide and contraction amplitude decreased in a concentration-dependent manner by mexiletine treatment only in the samples under pacing. These drug responses were consistent with the previous reports using human samples. These results indicated that beating rate control is necessary to stably evaluate the effects of drugs on contractility and that tests under 1-Hz pacing are more relevant to clinical settings.

Development of appropriate fatty acid formulations to raise the contractility of constructed myocardial tissues.

Introduction: Heart disease is a major cause of mortality worldwide, and the annual number of deaths due to heart disease has increased in recent years. Although heart failure is usually managed with medicines, the ultimate treatment for end-stage disease is heart transplantation or an artificial heart. However, the use of these surgical strategies is limited by issues such as thrombosis, rejection and donor shortages. Regenerative therapies, such as the transplantation of cultured cells and tissues constructed using tissue engineering techniques, are receiving great attention as possible alternative treatments for heart failure. Research is ongoing into the potential clinical use of cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs). However, the energy-producing capacity of cardiomyocytes maintained under previous culture conditions is lower than that of adult primary cardiomyocytes due to immaturity and a reliance on glucose metabolism. Therefore, the aims of this study were to compare the types of fatty acids metabolized between cardiomyocytes in culture and heart cells in vivo and investigate whether the addition of fatty acids to the culture medium affected energy production by cardiomyocytes. Methods: A fatty acid-containing medium was developed based on an analysis of fatty acid consumption by rat primary cardiomyocytes (rat-CMs), and the effects of this medium on adenosine triphosphate (ATP) production were investigated through bioluminescence imaging of luciferase-expressing rat-CMs. Next, the fatty acid content of the medium was further adjusted based on analyses of fatty acid utilization by porcine hearts and hiPSC-CMs. Oxygen consumption analyses were performed to explore whether the fatty acid-containing medium induced hiPSC-CMs to switch from anaerobic metabolism to aerobic metabolism. Furthermore, the effects of the medium on contractile force generated by hiPSC-CM-derived tissue were evaluated. Results: Rat serum, human serum and porcine plasma contained similar types of fatty acid (oleic acid, stearic acid, linoleic acid, palmitic acid and arachidonic acid). The types of fatty acid consumed were also similar between rat-CMs, hiPSC-CMs and porcine heart. The addition of fatty acids to the culture medium increased the bioluminescence of luciferase-expressing rat-CMs (an indirect measure of ATP level), oxygen consumption by hiPSC-CMs, and contractile force generated by cardiac tissues constructed from hiPSC-CMs. Conclusions: hiPSC-CMs metabolize similar types of fatty acid to those consumed by rat-CMs and porcine hearts. Furthermore, the addition of these fatty acids to the culture medium increased energy production by rat-CMs and hiPSC-CMs and enhanced the contractility of myocardial tissue generated from hiPSC-CMs. These findings suggest that the addition of fatty acids to the culture medium stimulates aerobic energy production by cardiomyocytes through ß-oxidation. Since cardiomyocytes cultured in standard media rely primarily on anaerobic glucose metabolism and remain in an immature state, further research is merited to establish whether the addition of fatty acids to the culture medium would improve the energy-producing capacity and maturity of hiPSC-CMs and cardiac tissue constructed from these cells. It is possible that optimizing the metabolism of cultured cardiomyocytes, which require high energy production to sustain their contractile function, will improve the properties of hiPSC-CM-derived tissue, allowing it to be better utilized for disease modeling, drug screening and regenerative therapies for heart failure.

In vitrocirculation 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 inin vitrodrug 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 ± 8.9µl (n= 6) on day 21. It also responded toß-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 1µl 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 accuratein vitrodrug evaluation usingP-Vdiagrams.

Prognosis and diastolic dysfunction predictors in patients with heart failure and recovered ejection fraction.

There is limited data on whether diastolic dysfunction in patients with heart failure (HF) and recovered ejection fraction (HFrecEF) is associated with worse prognosis. We retrospectively assessed 96 patients diagnosed with HFrecEF and created ROC curve of their diastolic function at the 1-year follow-up for the composite endpoint of cardiovascular death and HF readmission after the follow-up. Eligible patients were divided into two groups according to the cutoff value of E/e' ratio (12.1) with the highest AUC (0.70). Kaplan-Meier analysis showed that HFrecEF with high E/e' group had a significantly poorer prognosis than the low E/e' group (log-rank, p = 0.01). Multivariate Cox regression analysis revealed that the high E/e' group was significantly related to the composite endpoint (hazard ratio 5.45, 95% confidence interval [CI] 1.23-24.1). The independent predictors at discharge for high E/e' ratio at the 1-year follow-up were older age and female sex after adjustment for covariates (odds ratio [OR] 1.07, 95% CI 1.01-1.13 and OR 4.70, 95% CI 1.08-20.5). In conclusion, HFrecEF with high E/e' ratio might be associated with a poor prognosis. Older age and female sex were independent predictors for a sustained high E/e' ratio in patients with HFrecEF.

Decellularized Organ-Derived Scaffold Is a Promising Carrier for Human Induced Pluripotent Stem Cells-Derived Hepatocytes.

Human induced pluripotent stem cells (hiPSCs) are a promising cell source for elucidating disease pathology and therapy. The mass supply of hiPSC-derived cells is technically feasible. Carriers that can contain a large number of hiPSC-derived cells and evaluate their functions in vivo-like environments will become increasingly important for understanding disease pathogenesis or treating end-stage organ failure. hiPSC-derived hepatocyte-like cells (hiPSC-HLCs; 5 × 108) were seeded into decellularized organ-derived scaffolds under circumfusion culture. The scaffolds were implanted into immunodeficient microminiature pigs to examine their applicability in vivo. The seeded hiPSC-HLCs demonstrated increased albumin secretion and up-regulated cytochrome P450 activities compared with those in standard two-dimensional culture conditions. Moreover, they showed long-term survival accompanied by neovascularization in vivo. The decellularized organ-derived scaffold is a promising carrier for hiPSC-derived cells for ex vivo and in vivo use and is an essential platform for regenerative medicine and research.

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.

Functional Evaluation of Human Bioengineered Cardiac Tissue Using iPS Cells Derived from a Patient with Lamin Variant Dilated Cardiomyopathy.

Dilated cardiomyopathy (DCM) is caused by various gene variants and characterized by systolic dysfunction. Lamin variants have been reported to have a poor prognosis. Medical and device therapies are not sufficient to improve the prognosis of DCM with the lamin variants. Recently, induced pluripotent stem (iPS) cells have been used for research on genetic disorders. However, few studies have evaluated the contractile function of cardiac tissue with lamin variants. The aim of this study was to elucidate the function of cardiac cell sheet tissue derived from patients with lamin variant DCM. iPS cells were generated from a patient with lamin A/C (LMNA) -mutant DCM (LMNA p.R225X mutation). After cardiac differentiation and purification, cardiac cell sheets that were fabricated through cultivation on a temperature-responsive culture dish were transferred to the surface of the fibrin gel, and the contractile force was measured. The contractile force and maximum contraction velocity, but not the maximum relaxation velocity, were significantly decreased in cardiac cell sheet tissue with the lamin variant. A qRT-PCR analysis revealed that mRNA expression of some contractile proteins, cardiac transcription factors, Ca2+-handling genes, and ion channels were downregulated in cardiac tissue with the lamin variant.Human iPS-derived bioengineered cardiac tissue with the LMNA p.R225X mutation has the functional properties of systolic dysfunction and may be a promising tissue model for understanding the underlying mechanisms of DCM.

Aligned human induced pluripotent stem cell-derived cardiac tissue improves contractile properties through promoting unidirectional and synchronous cardiomyocyte contraction.

Alignment, as seen in the native myocardium, is crucial for the fabrication of functional cardiac tissue. However, it remains unclear whether the control of cardiomyocyte alignment influences cardiac function and the underlying mechanisms. We fabricated aligned human cardiac tissue using a micro-processed fibrin gel with inverted V-shaped ridges (MFG) and elucidated the effect of alignment control on contractile properties. When human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were seeded on MFG, hiPSC-CMs were aligned more uniformly than the control, and we succeeded in fabricating the aligned cardiac tissue. Assessing the contractile properties with the direct contractile measurement system, the contractile force, maximum contractile velocity, and relaxation velocity were significantly increased in aligned cardiac tissue compared with non-aligned cardiac tissue. However, gene expression profiles were not different between the two groups, suggesting that functional improvement of cardiac tissue through alignment control might not be dependent on cardiomyocyte maturation. Motion capture analysis revealed that the cardiomyocytes in the aligned cardiac tissues showed more unidirectional and synchronous contraction than the non-aligned cardiac tissues, indicating that cardiac tissue maturation involves electrical integration of cardiomyocytes. Herein, cardiomyocyte alignment control might improve the contractile properties of cardiac tissue through promoting unidirectional and synchronous cardiomyocyte contraction.

Simultaneous measurement of contractile force and field potential of dynamically beating human iPS cell-derived cardiac cell sheet-tissue with flexible electronics.

Human induced pluripotent stem (iPS) cell-derived cardiomyocytes are used for in vitro pharmacological and pathological studies worldwide. In particular, the functional assessment of cardiac tissues created from iPS cell-derived cardiomyocytes is expected to provide precise prediction of drug effects and thus streamline the process of drug development. However, the current format of electrophysiological and contractile assessment of cardiomyocytes on a rigid substrate is not appropriate for cardiac tissues that beat dynamically. Here, we show a novel simultaneous measurement system for contractile force and extracellular field potential of iPS cell-derived cardiac cell sheet-tissues using 500 nm-thick flexible electronic sheets. It was confirmed that the developed system is applicable for pharmacological studies and assessments of excitation-contraction coupling-related parameters, such as the electro-mechanical window. Our results indicate that flexible electronics with cardiac tissue engineering provide an advanced platform for drug development. This system will contribute to gaining new insight in pharmacological study of human cardiac function.

Non-coating method for non-adherent cell culture using high molecular weight dextran sulfate and bovine serum albumin.

Non-adherent cell culture surface has been widely used for producing cell spheroids and cell aggregates. The purpose of this study was to formulate a new method for non-adherent cell culture without coating or surface-modification that has been needed. We found that high-molecular-weight dextran sulfate (DS) and bovine serum albumin (BSA) synergistically prevented cell adhesion in media supplemented with no or low serum. This method worked on tissue culture-treated polystyrene surfaces as well as on commercially available low-attachment- and untreated polystyrene surfaces. Further investigation revealed that BSA may mediate the adsorption of DS to the surface. In addition, as the adsorption of fluorescently labeled fibronectin was inhibited by BSA alone, it appears that protein adsorption and cell adhesion do not always correlate. Finally, we demonstrated the successful formation of HepG2 spheroids and cardiomyocyte aggregates using this method. In conclusion, cell adhesion can be effectively suppressed by simply adding DS and BSA to the culture medium without coating or surface modification, and it may be useful for generating cell spheroids and aggregates.

An organic transistor matrix for multipoint intracellular action potential recording.

Electrode arrays are widely used for multipoint recording of electrophysiological activities, and organic electronics have been utilized to achieve both high performance and biocompatibility. However, extracellular electrode arrays record the field potential instead of the membrane potential itself, resulting in the loss of information and signal amplitude. Although much effort has been dedicated to developing intracellular access methods, their three-dimensional structures and advanced protocols prohibited implementation with organic electronics. Here, we show an organic electrochemical transistor (OECT) matrix for the intracellular action potential recording. The driving voltage of sensor matrix simultaneously causes electroporation so that intracellular action potentials are recorded with simple equipment. The amplitude of the recorded peaks was larger than that of an extracellular field potential recording, and it was further enhanced by tuning the driving voltage and geometry of OECTs. The capability of miniaturization and multiplexed recording was demonstrated through a 4 × 4 action potential mapping using a matrix of 5- × 5-µm2 OECTs. Those features are realized using a mild fabrication process and a simple circuit without limiting the potential applications of functional organic electronics.

Contractile Force Measurement of Engineered Cardiac Tissues Derived from Human iPS Cells.

Recent advances in stem cell technologies and tissue engineering are enabling the fabrication of dynamically beating cardiac tissues from human induced pluripotent stem cells. These engineered human cardiac tissues are expected to be used for cardiac regenerative therapies, in vitro drug testing, and pathological investigations. Here we describe the method to fabricate engineered cardiac tissues from human induced pluripotent stem cell-derived cardiomyocytes and to measure the contractile force.

Assessment of human bioengineered cardiac tissue function in hypoxic and re-oxygenized environments to understand functional recovery in heart failure.

INTRODUCTION: Myocardial recovery is one of the targets for heart failure treatment. A non-negligible number of heart failure with reduced ejection fraction (EF) patients experience myocardial recovery through treatment. Although myocardial hypoxia has been reported to contribute to the progression of heart failure even in non-ischemic cardiomyopathy, the relationship between contractile recovery and re-oxygenation and its underlying mechanisms remain unclear. The present study investigated the effects of hypoxia/re-oxygenation on bioengineered cardiac cell sheets-tissue function and the underlying mechanisms. METHODS: Bioengineered cardiac cell sheets-tissue was fabricated with human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) using temperature-responsive culture dishes. Cardiac tissue functions in the following conditions were evaluated with a contractile force measurement system: continuous normoxia (20% O2) for 12 days; hypoxia (1% O2) for 4 days followed by normoxia (20% O2) for 8 days; or continuous hypoxia (1% O2) for 8 days. Cell number, sarcomere structure, ATP levels, mRNA expressions and Ca2+ transients of hiPSC-CM in those conditions were also assessed. RESULTS: Hypoxia (4 days) elicited progressive decreases in contractile force, maximum contraction velocity, maximum relaxation velocity, Ca2+ transient amplitude and ATP level, but sarcomere structure and cell number were not affected. Re-oxygenation (8 days) after hypoxia (4 days) was associated with progressive increases in contractile force, maximum contraction velocity and relaxation time to the similar extent levels of continuous normoxia group, while maximum relaxation velocity was still significantly low even after re-oxygenation. Ca2+ transient magnitude, cell number, sarcomere structure and ATP level after re-oxygenation were similar to those in the continuous normoxia group. Hypoxia/re-oxygenation up-regulated mRNA expression of PLN. CONCLUSIONS: Hypoxia and re-oxygenation condition directly affected human bioengineered cardiac tissue function. Further understanding the molecular mechanisms of functional recovery of cardiac tissue after re-oxygenation might provide us the new insight on heart failure with recovered ejection fraction and preserved ejection fraction.

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 48 h. 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.

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.