Regenerative Effect of Umbilical Cord-Derived Mesenchymal Stromal Cells in a Rat Model of Established Limb Ischemia.

BACKGROUND: Although regenerative cell therapy is expected to be an alternative treatment for peripheral artery disease (PAD), many regenerative cell therapies have failed to show sufficient efficacy in clinical trials. Most preclinical studies have used acute ischemia models, despite PAD being a chronic disease. In addition, aging and atherosclerosis decrease the quality of a patient's stem cells. Therefore, using a non-acute ischemic preclinical model and stem cells with high regenerative potency are important for the development of effective regenerative therapy. In this study, we assessed the tissue regenerative potential of umbilical cord-derived mesenchymal stromal cells (UCMSCs), which could potentially be an ideal cell source, in a rat model of established ischemia.Methods and Results: The regenerative capacity of UCMSCs was analyzed in terms of angiogenesis and muscle regeneration. In vitro analysis showed that UCMSCs secrete high amounts of cytokines associated with angiogenesis and muscle regeneration. In vivo experiments in a rat non-acute ischemia model showed significant improvement in blood perfusion after intravenous injection of UCMSCs compared with injection of culture medium or saline. Histological analysis revealed UCMSCs injection enhanced angiogenesis, with an increased number of von Willebrand factor-positive microcapillaries, and improved muscle regeneration. CONCLUSIONS: These results suggest that intravenous administration of UCMSCs may be useful for treating patients with PAD.

Allogeneic transplantation of iPS cell-derived cardiomyocytes regenerates primate hearts.

Induced pluripotent stem cells (iPSCs) constitute a potential source of autologous patient-specific cardiomyocytes for cardiac repair, providing a major benefit over other sources of cells in terms of immune rejection. However, autologous transplantation has substantial challenges related to manufacturing and regulation. Although major histocompatibility complex (MHC)-matched allogeneic transplantation is a promising alternative strategy, few immunological studies have been carried out with iPSCs. Here we describe an allogeneic transplantation model established using the cynomolgus monkey (Macaca fascicularis), the MHC structure of which is identical to that of humans. Fibroblast-derived iPSCs were generated from a MHC haplotype (HT4) homozygous animal and subsequently differentiated into cardiomyocytes (iPSC-CMs). Five HT4 heterozygous monkeys were subjected to myocardial infarction followed by direct intra-myocardial injection of iPSC-CMs. The grafted cardiomyocytes survived for 12 weeks with no evidence of immune rejection in monkeys treated with clinically relevant doses of methylprednisolone and tacrolimus, and showed electrical coupling with host cardiomyocytes as assessed by use of the fluorescent calcium indicator G-CaMP7.09. Additionally, transplantation of the iPSC-CMs improved cardiac contractile function at 4 and 12 weeks after transplantation; however, the incidence of ventricular tachycardia was transiently, but significantly, increased when compared to vehicle-treated controls. Collectively, our data demonstrate that allogeneic iPSC-CM transplantation is sufficient to regenerate the infarcted non-human primate heart; however, further research to control post-transplant arrhythmias is necessary.

Chemical Genetics Reveals a Role of Squalene Synthase in TGFß Signaling and Cardiomyogenesis.

KY02111 is a widely used small molecule that boosts cardiomyogenesis of the mesoderm cells derived from pluripotent stem cells, yet its molecular mechanism of action remains elusive. The present study resolves the initially perplexing effects of KY02111 on Wnt signaling and subsequently identifies squalene synthase (SQS) as a molecular target of KY02111 and its optimized version, KY-I. By disrupting the interaction of SQS with cardiac ER-membrane protein TMEM43, KY02111 impairs TGFß signaling, but not Wnt signaling, and thereby recapitulates the clinical mutation of TMEM43 that causes arrhythmogenic right ventricular cardiomyopathy (ARVC), an inherited heart disease that involves a substitution of myocardium with fatty tissue. These findings reveal a heretofore undescribed role of SQS in TGFß signaling and cardiomyogenesis. KY02111 may find its use in ARVC modeling as well as serve as a chemical tool for studying TGFß/SMAD signaling.

A Synthetic Hybrid Molecule for the Selective Removal of Human Pluripotent Stem Cells from Cell Mixtures.

A major hurdle in stem cell therapy is the tumorigenic risk of residual undifferentiated stem cells. This report describes the design and evaluation of synthetic hybrid molecules that efficiently reduce the number of human induced pluripotent stem cells (hiPSCs) in cell mixtures. The design takes advantage of Kyoto probe 1 (KP-1), a fluorescent chemical probe for hiPSCs, and clinically used anticancer drugs. Among the KP-1-drug conjugates we synthesized, we found an exceptionally selective, chemically tractable molecule that induced the death of hiPSCs. Mechanistic analysis suggested that the high selectivity originates from the synergistic combination of transporter-mediated efflux and the cytotoxicity mode of action. The present study offers a chemical and mechanistic rationale for designing selective, safe, and simple reagents for the preparation of non-tumorigenic clinical samples.

The modeling of Alzheimer's disease by the overexpression of mutant Presenilin 1 in human embryonic stem cells.

Cellular disease models are useful tools for Alzheimer's disease (AD) research. Pluripotent stem cells, including human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), are promising materials for creating cellular models of such diseases. In the present study, we established cellular models of AD in hESCs that overexpressed the mutant Presenilin 1 (PS1) gene with the use of a site-specific gene integration system. The overexpression of PS1 did not affect the undifferentiated status or the neural differentiation ability of the hESCs. We found increases in the ratios of amyloid-ß 42 (Aß42)/Aß40 and Aß43/Aß40. Furthermore, synaptic dysfunction was observed in a cellular model of AD that overexpressed mutant PS1. These results suggest that the AD phenotypes, in particular, the electrophysiological abnormality of the synapses in our AD models might be useful for AD research and drug discovery.

Associations among chronic obstructive pulmonary disease and sleep-disordered breathing in an urban male working population in Japan.

BACKGROUND: There are few reports about sleep disturbances in patients with chronic obstructive pulmonary disease (COPD) in Asian countries. OBJECTIVES: To investigate the associations between sleep-disordered breathing (SDB) with hypoxemia and sleep quality, including sleep duration, in patients with COPD, we measured SDB and sleep quality including the objective sleep duration determined by an actigraph and portable monitoring. METHODS: A cross-sectional epidemiological health survey of 303 male employees (means ± SD: age 43.9 ± 8.2 years; BMI 24.0 ± 3.1) was conducted. Sleep quality was measured using the Epworth Sleepiness Scale (ESS) and the Pittsburgh Sleep Quality Index (PSQI). A respiratory disturbance index (RDI) ≥5 indicated SDB. RESULTS: Nineteen subjects (6.3%) had COPD. Among these, 11 (3.6%) had COPD with SDB (overlap syndrome). Sleep duration, ESS, and PSQI scores were not significantly different between COPD patients and normal control subjects. However, COPD patients had significantly longer sleep latency (p = 0.019), a lower sleep efficiency (p = 0.017), and a higher sleep fragmentation index (p = 0.041) and average activity (p = 0.0097) during sleep than control subjects. They also had a significantly higher RDI and more severe desaturation during sleep than control subjects (p < 0.01). The differences remained after adjustment for age and BMI but disappeared following adjustment for RDI. CONCLUSIONS: COPD patients with even mild-to-moderate airflow limitations had nocturnal desaturation and RDI-related impaired sleep quality without significant symptoms.

Development of a reentrant arrhythmia model in human pluripotent stem cell-derived cardiac cell sheets.

AIMS: Development of a human cell-derived reentrant arrhythmia model is needed for studying the mechanisms of disease and accurate drug response. METHODS AND RESULTS: We differentiated human pluripotent stem cells (hPSCs) into cardiomyocytes, and then re-plated them into cell sheets that proved capable of forming electrically coupled assemblies. We monitored the function of these re-plated sheets optically with the Ca(2+) sensitive dye Fluo-4, and found that they generated characteristic waves of activity whose velocity and patterns of propagation depended upon the concentration of sodium channel blockers; lidocaine and tetrodotoxin, and also the time after re-plating, as well as the applied stimulation frequency. Importantly, reentrant spiral-wave propagation could be generated in these sheets by applying high-frequency stimulation, particularly when cell-density in the sheets was relatively low. This was because cardiac troponin T-positive cells were more non-homogeneously distributed at low cell densities. Especially in such sheets, we could terminate spiral waves by administering the anti-arrhythmic drugs; nifekalant, E-4031, sotalol, and quinidine. We also found that in these sheets, nifekalant showed a clear dose-dependent increase in the size of the unexcitable 'cores' of these induced spiral waves, an important parallel with the treatment for ventricular tachycardia in the clinical situation, which was not shown properly in cardiac-cell sheets derived from dissociated rodent hearts. CONCLUSIONS: We have succeeded in creating from hPSCs a valuable type of cardiomyocyte sheet that is capable of generating reentrant arrhythmias, and thus is demonstrably useful for screening and testing all sorts of drugs with anti-arrhythmic potential.

Circadian clock-controlled diurnal oscillation of Ras/ERK signaling in mouse liver.

Accumulating evidence indicates that ERK MAP kinase signaling plays an important role in the regulation of the circadian clock, especially in the clock-resetting mechanism in the suprachiasmatic nucleus (SCN) in mammals. Previous studies have also shown that ERK phosphorylation exhibits diurnal variation in the SCN. However, little is known about circadian regulation of ERK signaling in peripheral tissues. Here we show that the activity of Ras/ERK signaling exhibits circadian rhythms in mouse liver. We demonstrate that Ras activation, MEK phosphorylation, and ERK phosphorylation oscillate in a circadian manner. As the oscillation of ERK phosphorylation is lost in Cry1/Cry2 double-knockout mice, Ras/ERK signaling should be under the control of the circadian clock. Furthermore, expression of MAP kinase phosphatase-1 (Mkp-1) shows diurnal changes in liver. These results indicate that Ras/ERK signaling is strictly regulated by the circadian clock in liver, and suggest that the circadian oscillation of the activities of Ras, MEK, and ERK may regulate diurnal variation of liver function and/or homeostasis.(Communicated by Shigekazu NAGATA, M.J.A.).

Differences in relationships among sleep apnoea, glucose level, sleep duration and sleepiness between persons with and without type 2 diabetes.

Obstructive sleep apnoea is common in patients with diabetes. Recently, it was reported that short sleep duration and sleepiness had deleterious effects on glucose metabolism. Thereafter, several reports showed relationships between glucose metabolism and obstructive sleep apnoea, sleep duration or sleepiness. But the interrelationships among those factors based on recent epidemiological data have not been examined. We analysed data on 275 male employees (age, 44±8years; body mass index, 23.9±3.1kg m(-2) ) who underwent a cross-sectional health examination in Japan. We measured fasting plasma glucose, sleep duration using a sleep diary and an actigraph for 7days, and respiratory disturbance index with a type 3 portable monitor for two nights. Fifty-four subjects (19.6%) had impaired glucose metabolism, with 21 having diabetes. Of those 21 (body mass index, 25.9±3.8kgm(-2) ), 17 (81.0%) had obstructive sleep apnoea (respiratory disturbance index≥5). Regarding the severity of obstructive sleep apnoea, 10, four and three had mild, moderate and severe obstructive sleep apnoea, respectively. The prevalence of obstructive sleep apnoea was greater in those with than without diabetes (P=0.037). Multiple regression analyses showed that the respiratory disturbance index independently related to fasting plasma glucose only in the diabetic subjects. In patients with diabetes, after adjustment for age, waist circumference, etc. sleep fragmentation had a greater correlation with fasting plasma glucose than sleep duration, but without significance (P=0.10). Because the prevalence of obstructive sleep apnoea is extremely high in patients with diabetes, sufficient sleep duration with treatment for obstructive sleep apnoea, which ameliorates sleep fragmentation, might improve fasting plasma glucose.

Incorporation of functionalized gold nanoparticles into nanofibers for enhanced attachment and differentiation of mammalian cells.

BACKGROUND: Electrospun nanofibers have been widely used as substrata for mammalian cell culture owing to their structural similarity to natural extracellular matrices. Structurally consistent electrospun nanofibers can be produced with synthetic polymers but require chemical modification to graft cell-adhesive molecules to make the nanofibers functional. Development of a facile method of grafting functional molecules on the nanofibers will contribute to the production of diverse cell type-specific nanofiber substrata. RESULTS: Small molecules, peptides, and functionalized gold nanoparticles were successfully incorporated with polymethylglutarimide (PMGI) nanofibers through electrospinning. The PMGI nanofibers functionalized by the grafted AuNPs, which were labeled with cell-adhesive peptides, enhanced HeLa cell attachment and potentiated cardiomyocyte differentiation of human pluripotent stem cells. CONCLUSIONS: PMGI nanofibers can be functionalized simply by co-electrospinning with the grafting materials. In addition, grafting functionalized AuNPs enable high-density localization of the cell-adhesive peptides on the nanofiber. The results of the present study suggest that more cell type-specific synthetic substrata can be fabricated with molecule-doped nanofibers, in which diverse functional molecules are grafted alone or in combination with other molecules at different concentrations.

Cardiomyocytes develop from anterior primitive streak cells induced by ß-catenin activation and the blockage of BMP signaling in hESCs.

Cardiomyocytes arise from cells that migrate to the mid-to-anterior region of the primitive streak (PS) during embryogenesis. We previously showed that canonical Wnt/ß-catenin pathway signaling leads to the development of nascent PS populations from human embryonic stem cells (hESCs) and that synergistic activation of the Wnt/ß-catenin pathway and inhibition of bone morphogenetic protein (BMP) signaling by Noggin induced the formation of anterior PS cells. We herein demonstrate that anterior PS cells induced by the activation of ß-catenin with Noggin differentiate into functional cardiomyocytes when cultured in suspension with BMP4 and fibroblast growth factor 2 (FGF2). All aggregates generated from the anterior PS cells developed into contracting cells demonstrating their cardiac potential. More than 30% of the cells in each aggregate were α-actinin-positive cardiomyocytes. In addition, these cardiomyocytes could be easily purified up to 80% by simple size fractionation. In contrast, the posterior PS cells induced by ß-catenin activation without Noggin showed poor cardiac potential. These results show that the commitment to a cardiac lineage in vitro occurs through similar cellular and molecular signaling pathways involved in cardiac development in vivo, thus providing a valuable culture model for studying early cardiac developmental events in hESCs.

Effects of the presence of hypertension on the relationship between obstructive sleep apnoea and sleepiness.

Obstructive sleep apnoea (OSA) plays a significant role in increasing blood pressure. Significant decreases were reported in blood pressure of hypertensive OSA patients with sleepiness who underwent continuous positive airway pressure (CPAP) treatment, but not in non-sleepy hypertensive OSA patients. More recently, however, significant decreases in blood pressure in non-sleepy hypertensive OSA patients following CPAP were shown. Effects of sleepiness on hypertension in OSA patients have been investigated, but not the effects of hypertension on sleepiness in OSA patients. We investigated the relationships between hypertension and sleepiness in patients with OSA. We analysed data on 275 middle-aged male subjects from a cross-sectional epidemiological health survey. We measured blood pressure and sleep duration objectively using an actigraph for 7 days and the respiratory disturbance index (RDI) with a type 3 portable device for 2 nights, and assessed sleepiness using the Epworth Sleepiness Scale (ESS). The RDI correlated significantly with ESS scores in the 88 hypertensive subjects (r = 0.33, P = 0.0024), but not in the 187 non-hypertensive subjects (r = -0.01, P = 0.91). Short sleep duration correlated significantly with ESS scores in both groups. Both the RDI and short sleep duration were related independently to sleepiness in only hypertensive subjects. Furthermore, the RDI was related negatively significantly to sleep duration in hypertensive subjects. Although short sleep duration was related significantly to sleepiness in both groups, hypertension may be important for the sleepiness in OSA patients. Detailed mechanisms of the difference in the relationship between sleepiness and the severity of OSA with or without hypertension should be studied further.

Fabrication of Thick and Anisotropic Cardiac Tissue on Nanofibrous Substrate for Repairing Infarcted Myocardium.

In this chapter, we introduce the method for fabricating thick and anisotropic cardiac tissue for heart regeneration. Aligned and biodegradable nanofiber can be prepared by electrospinning Food and Drug Administration-approved poly (lactic-co-glycolic acid) on a rotating drum. After the nanofibers are transferred on to a polydimethylsiloxane frame, the cardiomyocytes could be plated on the nanofiber to form thick and anisotropic cardiac tissue rapidly. Cardiac tissue-like construct could be easily created by one-step method, and transplanted onto the hearts of myocardium infarction models and lead to their functional recovery.

Associations between obstructive sleep apnea, metabolic syndrome, and sleep duration, as measured with an actigraph, in an urban male working population in Japan.

BACKGROUND: Severe obstructive sleep apnea (OSA), metabolic syndrome (Mets) and short sleep duration are all risk factors for cardiovascular events. There has been no report which has investigated this relationship in an age- and BMI-matched population-based study. The prevalence of OSA in Mets subjects has not been established, although the converse (i.e., the prevalence of Mets in OSA subjects) has been investigated several times. METHODS: This home cardiorespiratory (type 3) sleep study, using an actigraph, was conducted in 275 males working for an urban company. Retrospective measurements of fasting blood parameters were obtained from the company's periodical inspection data. The mean duration between the sleep study and the measurement of blood parameters was 213 days. RESULTS: Although there was a significant relationship between OSA severity and the prevalence of Mets (P < 0.001), the association between severity and Mets was not significant after adjustments were made for age and BMI. Severe OSA was 7.8 times as likely to be present in subjects with Mets (16.2% of all 68 Mets subjects) as those without (2.4% of 207 non-Mets) (P < 0.001). Subject with severe OSA had a significantly short sleep duration (P < 0.05). Sleep duration in Mets subjects was also significantly shorter than in those without (P < 0.05). CONCLUSIONS: Although increased BMI and age both had a significant effect on the prevalence of OSA in patients with Mets, one of 6 subjects with Mets, but only one of 40 without Mets had severe OSA in an urban male population in Japan. Physicians should take into account this high prevalence of severe OSA in patients with Mets. Sleep duration should be taken into consideration as an important factor in studies investigating the prevalence of severe OSA and Mets.

Circulating re-entrant waves promote maturation of hiPSC-derived cardiomyocytes in self-organized tissue ring.

Directed differentiation methods allow acquisition of high-purity cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSCs); however, their immaturity characteristic limits their application for drug screening and regenerative therapy. The rapid electrical pacing of cardiomyocytes has been used for efficiently promoting the maturation of cardiomyocytes, here we describe a simple device in modified culture plate on which hiPSC-derived cardiomyocytes can form three-dimensional self-organized tissue rings (SOTRs). Using calcium imaging, we show that within the ring, reentrant waves (ReWs) of action potential spontaneously originated and ran robustly at a frequency up to 4 Hz. After 2 weeks, SOTRs with ReWs show higher maturation including structural organization, increased cardiac-specific gene expression, enhanced Ca2+-handling properties, an increased oxygen-consumption rate, and enhanced contractile force. We subsequently use a mathematical model to interpret the origination, propagation, and long-term behavior of the ReWs within the SOTRs.

Clonal Isolation of Human Pluripotent Stem Cells on Nanofibrous Substrates Reveals an Advanced Subclone for Cardiomyocyte Differentiation.

Human pluripotent stem cells (hPSCs) have been widely used for various applications including disease modeling and regenerative medicine, among others. Recently, an increasing number of studies has focused on heterogeneity among hPSCs, which could affect cell quality and subsequent applications. In this study, a nanofibrous platform is developed for single human induced pluripotent stem cell isolation and culture. One type of single cell-derived subclone is established and found to have a distinct morphology compared to other subclones. When used for differentiation toward cardiomyocytes, this type of subclone demonstrates higher differentiation efficiency, increased maturation, and stronger beating compared to those derived from the other subclones. The findings provide a convenient method for single-cell isolation and culture, and demonstrate that variations in differentiation tendencies exist among subclones from the same cell line. This substrate adhesion-based selection process could be used to obtain cell lines with improved differentiation efficiency toward cardiomyocytes and other cell types, which would be advantageous for studies in various fields.

Sleep-disordered breathing in the usual lifestyle setting as detected with home monitoring in a population of working men in Japan.

STUDY OBJECTIVES: To examine (1) the prevalence of home-monitored sleep-disordered breathing (SDB) and obstructive sleep apnea syndrome in a Japanese working population and (2) whether home monitoring with a type 3 portable monitor and actigraphy can produce reliable data to analyze SDB in usual lifestyles. METHODS: A cross-sectional survey using a self-administered questionnaire was conducted on a group of employees at a wholesale company in Osaka, Japan. Examinations by physicians and by sleep monitoring were also performed. Unattended home cardiorespiratory (type 3) sleep studies with actigraphy were conducted for 2 nights to diagnose SDB in 322 subjects. From the baseline questionnaires and sleep diaries, participants were assessed to follow their usual lifestyles during the study (e.g., time in bed, alcohol intake). RESULTS: Of 466 Japanese male employees, 396 responded to the questionnaire survey (85.0%). Results from 322 male employees aged 23 to 59 (43.8 +/- 8.4 years) were analyzed. Respiratory disturbance index (RDI), calculated from the type 3 portable monitors and actigraphy, was highly reliable with an intraclass correlation of 0.98 for interscorer reliability and with an intraclass correlation of 0.95 for night-to-night reliability. Prevalence of mild (5 < or = RDI < 15), moderate (15 < or = RDI < 30) and severe (RDI < or = 30) SDB in this population were 37.4%, 15.7%, and 6.6%, respectively. The prevalence of obstructive sleep apnea syndrome (RDI > or = 5 and Epworth Sleepiness Scale score > 10) was 17.6%. CONCLUSIONS: The prevalence of moderate to severe SDB (RDI > or = 15) was 22.3% in this Japanese male working population aged 23 to 59, measured in participant's usual life settings. Unattended home monitoring with type 3 portable monitors and actigraphy was highly reliable and may be suitable for analyzing SDB in the usual lifestyle setting.

Low Cell-Matrix Adhesion Reveals Two Subtypes of Human Pluripotent Stem Cells.

We show that a human pluripotent stem cell (hPSC) population cultured on a low-adhesion substrate developed two hPSC subtypes with different colony morphologies: flat and domed. Notably, the dome-like cells showed higher active proliferation capacity and increased several pluripotent genes' expression compared with the flat monolayer cells. We further demonstrated that cell-matrix adhesion mediates the interaction between cell morphology and expression of KLF4 and KLF5 through a serum response factor (SRF)-based regulatory double loop. Our results provide a mechanistic view on the coupling among adhesion, stem cell morphology, and pluripotency, shedding light on the critical role of cell-matrix adhesion in the induction and maintenance of hPSC.

Two dimensional electrophysiological characterization of human pluripotent stem cell-derived cardiomyocyte system.

Stem cell-derived cardiomyocytes provide a promising tool for human developmental biology, regenerative therapies, disease modeling, and drug discovery. As human pluripotent stem cell-derived cardiomyocytes remain functionally fetal-type, close monitoring of electrophysiological maturation is critical for their further application to biology and translation. However, to date, electrophysiological analyses of stem cell-derived cardiomyocytes has largely been limited by biologically undefined factors including 3D nature of embryoid body, sera from animals, and the feeder cells isolated from mouse. Large variability in the aforementioned systems leads to uncontrollable and irreproducible results, making conclusive studies difficult. In this report, a chemically-defined differentiation regimen and a monolayer cell culture technique was combined with multielectrode arrays for accurate, real-time, and flexible measurement of electrophysiological parameters in translation-ready human cardiomyocytes. Consistent with their natural counterpart, amplitude and dV/dtmax of field potential progressively increased during the course of maturation. Monolayer culture allowed for the identification of pacemaking cells using the multielectrode array platform and thereby the estimation of conduction velocity, which gradually increased during the differentiation of cardiomyocytes. Thus, the electrophysiological maturation of the human pluripotent stem cell-derived cardiomyocytes in our system recapitulates in vivo development. This system provides a versatile biological tool to analyze human heart development, disease mechanisms, and the efficacy/toxicity of chemicals.

Impact of extracellular matrix on engraftment and maturation of pluripotent stem cell-derived cardiomyocytes in a rat myocardial infarct model.

Pluripotent stem cell-derived cardiomyocytes show great promise in regenerating the heart after myocardial infarction; however, several uncertainties exist that must be addressed before clinical trials. One practical issue is graft survival following transplantation. Although a pro-survival cocktail with Matrigel has been shown to enhance graft survival, the use of Matrigel may not be clinically feasible. The purpose of this study was to test whether a hyaluronan-based hydrogel, HyStem, could be a substitute for Matrigel. Human induced pluripotent stem cell-derived cardiomyocytes diluted with HyStem alone, HyStem plus pro-survival factors, or a pro-survival cocktail with Matrigel (PSC/MG), were transplanted into a rat model of acute myocardial infarction. Histological analysis at 4 weeks post transplantation revealed that, among the three groups, recipients of PSC/MG showed the largest graft size. Additionally, the grafted cardiomyocytes in the recipients of PSC/MG had a more matured phenotype compared to those in the other two groups. These findings suggest that further studies will be required to enhance not only graft size, but also the maturation of grafted cardiomyocytes.