Updated on 2021/11/09

写真a

 
OH Hidemasa
 
Organization
Okayama University Hospital Professor
Position
Professor
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Degree

  • 医学博士 ( 大阪大学 )

Research Interests

  • 再生医学

  • 細胞療法

  • 心不全

  • 心筋細胞

  • 幹細胞

  • Cell Therapy

  • Cardiac Regeneration

  • Stem Cells

Research Areas

  • Life Science / Cardiology

Research History

  • - Professor,University Hospital of Medicine and Dentistry,Okayama University

    2010

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  • - 岡山大学岡山大学病院 教授

    2010

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  • Associate Professor,University Hospital of Medicine and Dentistry,Okayama University

    2009 - 2010

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  • Okayama University   Okayama University Hospital

    2009 - 2010

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  • Associate Professor

    2008 - 2009

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  • Kyoto Prefectural University of Medicine

    2008 - 2009

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  • 京都大学付属病院 准教授

    2003 - 2008

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  • Associate Professor

    2003 - 2008

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  • Baylor College of Medicine   Department of Medicine   Assistant Professor

    2002.7 - 2003.8

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    Country:United States

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Committee Memberships

  • 特定認定再生医療等委員会   委員  

    2017.4   

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Papers

  • Cardiosphere-derived exosomal microRNAs for myocardial repair in pediatric dilated cardiomyopathy. International journal

    Kenta Hirai, Daiki Ousaka, Yosuke Fukushima, Maiko Kondo, Takahiro Eitoku, Yusuke Shigemitsu, Mayuko Hara, Kenji Baba, Tatsuo Iwasaki, Shingo Kasahara, Shinichi Ohtsuki, Hidemasa Oh

    Science translational medicine   12 ( 573 )   2020.12

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    Although cardiosphere-derived cells (CDCs) improve cardiac function and outcomes in patients with single ventricle physiology, little is known about their safety and therapeutic benefit in children with dilated cardiomyopathy (DCM). We aimed to determine the safety and efficacy of CDCs in a porcine model of DCM and translate the preclinical results into this patient population. A swine model of DCM using intracoronary injection of microspheres created cardiac dysfunction. Forty pigs were randomized as preclinical validation of the delivery method and CDC doses, and CDC-secreted exosome (CDCex)-mediated cardiac repair was analyzed. A phase 1 safety cohort enrolled five pediatric patients with DCM and reduced ejection fraction to receive CDC infusion. The primary endpoint was to assess safety, and the secondary outcome measure was change in cardiac function. Improved cardiac function and reduced myocardial fibrosis were noted in animals treated with CDCs compared with placebo. These functional benefits were mediated via CDCex that were highly enriched with proangiogenic and cardioprotective microRNAs (miRNAs), whereas isolated CDCex did not recapitulate these reparative effects. One-year follow-up of safety lead-in stage was completed with favorable profile and preliminary efficacy outcomes. Increased CDCex-derived miR-146a-5p expression was associated with the reduction in myocardial fibrosis via suppression of proinflammatory cytokines and transcripts. Collectively, intracoronary CDC administration is safe and improves cardiac function through CDCex in a porcine model of DCM. The safety lead-in results in patients provide a translational framework for further studies of randomized trials and CDCex-derived miRNAs as potential paracrine mediators underlying this therapeutic strategy.

    DOI: 10.1126/scitranslmed.abb3336

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  • Impact of Cardiac Progenitor Cells on Heart Failure and Survival in Single Ventricle Congenital Heart Disease. International journal

    Toshikazu Sano, Daiki Ousaka, Takuya Goto, Shuta Ishigami, Kenta Hirai, Shingo Kasahara, Shinichi Ohtsuki, Shunji Sano, Hidemasa Oh

    Circulation research   122 ( 7 )   994 - 1005   2018.3

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    RATIONALE: Intracoronary administration of cardiosphere-derived cells (CDCs) in patients with single ventricles resulted in a short-term improvement in cardiac function. OBJECTIVE: To test the hypothesis that CDC infusion is associated with improved cardiac function and reduced mortality in patients with heart failure. METHODS AND RESULTS: We evaluated the effectiveness of CDCs using an integrated cohort study in 101 patients with single ventricles, including 41 patients who received CDC infusion and 60 controls treated with staged palliation alone. Heart failure with preserved ejection fraction (EF) or reduced EF was stratified by the cardiac function after surgical reconstruction. The main outcome measure was to evaluate the magnitude of improvement in cardiac function and all-cause mortality at 2 years. Animal studies were conducted to clarify the underlying mechanisms of heart failure with preserved EF and heart failure with reduced EF phenotypes. At 2 years, CDC infusion increased ventricular function (stage 2: +8.4±10.0% versus +1.6±6.4%, P=0.03; stage 3: +7.9±7.5% versus -1.1±5.5%, P<0.001) compared with controls. In all available follow-up data, survival did not differ between the 2 groups (log-rank P=0.225), whereas overall patients treated by CDCs had lower incidences of late failure (P=0.022), adverse events (P=0.013), and catheter intervention (P=0.005) compared with controls. CDC infusion was associated with a lower risk of adverse events (hazard ratio, 0.411; 95% CI, 0.179-0.942; P=0.036). Notably, CDC infusion reduced mortality (P=0.038) and late complications (P<0.05) in patients with heart failure with reduced EF but not with heart failure with preserved EF. CDC-treated rats significantly reversed myocardial fibrosis with differential collagen deposition and inflammatory responses between the heart failure phenotypes. CONCLUSIONS: CDC administration in patients with single ventricles showed favorable effects on ventricular function and was associated with reduced late complications except for all-cause mortality after staged procedures. Patients with heart failure with reduced EF but not heart failure with preserved EF treated by CDCs resulted in significant improvement in clinical outcome. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifiers: NCT01273857 and NCT01829750.

    DOI: 10.1161/CIRCRESAHA.117.312311

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  • Transcoronary cell infusion with the stop-flow technique in children with single-ventricle physiology. International journal

    Takahiro Eitoku, Kenji Baba, Maiko Kondou, Yoshihiko Kurita, Yousuke Fukushima, Kenta Hirai, Shinichi Ohtsuki, Shuta Ishigami, Shunji Sano, Hidemasa Oh

    Pediatrics international : official journal of the Japan Pediatric Society   60 ( 3 )   240 - 246   2018.3

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    BACKGROUND: Almost all reports on cardiac regeneration therapy have referred to adults, and only a few have focused on transcoronary infusion of cardiac progenitor cells using the stop-flow technique in children. METHODS: Intracoronary autologous cardiosphere-derived cell (CDC) transfer was conducted at Okayama University as a phase 1 clinical trial for seven patients with hypoplastic left heart syndrome between January 2011 and December 2012, and as a phase 2 clinical trial for 34 patients with single-ventricle physiology between July 2013 and March 2015. RESULTS: A total of 41 patients with single-ventricle physiology underwent transcoronary infusion of CDC with the stop-flow technique. The median age was 33 months (range, 5-70 months) and the median weight was 10.1 kg (range, 4.1-16.0 kg). Transient adverse events occurred during the procedure, including ST-segment elevation or depression, hypotension, bradycardia, and coronary artery vasospasm. All patients completely recovered. There were no major procedure-related adverse events. In this study, transcoronary infusion of CDC using the stop-flow technique was successfully completed in all patients. CONCLUSION: Transcoronary infusion of CDC using the stop-flow technique in children is a feasible and safe procedure.

    DOI: 10.1111/ped.13485

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  • Cell Therapy Trials in Congenital Heart Disease. International journal

    Hidemasa Oh

    Circulation research   120 ( 8 )   1353 - 1366   2017.4

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    Dramatic evolution in medical and catheter interventions and complex surgeries to treat children with congenital heart disease (CHD) has led to a growing number of patients with a multitude of long-term complications associated with morbidity and mortality. Heart failure in patients with hypoplastic left heart syndrome predicated by functional single ventricle lesions is associated with an increase in CHD prevalence and remains a significant challenge. Pathophysiological mechanisms contributing to the progression of CHD, including single ventricle lesions and dilated cardiomyopathy, and adult heart disease may inevitably differ. Although therapeutic options for advanced cardiac failure are restricted to heart transplantation or mechanical circulatory support, there is a strong impetus to develop novel therapeutic strategies. As lower vertebrates, such as the newt and zebrafish, have a remarkable ability to replace lost cardiac tissue, this intrinsic self-repair machinery at the early postnatal stage in mice was confirmed by partial ventricular resection. Although the underlying mechanistic insights might differ among the species, mammalian heart regeneration occurs even in humans, with the highest degree occurring in early childhood and gradually declining with age in adulthood, suggesting the advantage of stem cell therapy to ameliorate ventricular dysfunction in patients with CHD. Although effective clinical translation by a variety of stem cells in adult heart disease remains inconclusive with respect to the improvement of cardiac function, case reports and clinical trials based on stem cell therapies in patients with CHD may be invaluable for the next stage of therapeutic development. Dissecting the differential mechanisms underlying progressive ventricular dysfunction in children and adults may lead us to identify a novel regenerative therapy. Future regenerative technologies to treat patients with CHD are exciting prospects for heart regeneration in general practice.

    DOI: 10.1161/CIRCRESAHA.117.309697

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  • Intracoronary Cardiac Progenitor Cells in Single Ventricle Physiology: The PERSEUS (Cardiac Progenitor Cell Infusion to Treat Univentricular Heart Disease) Randomized Phase 2 Trial. International journal

    Shuta Ishigami, Shinichi Ohtsuki, Takahiro Eitoku, Daiki Ousaka, Maiko Kondo, Yoshihiko Kurita, Kenta Hirai, Yosuke Fukushima, Kenji Baba, Takuya Goto, Naohiro Horio, Junko Kobayashi, Yosuke Kuroko, Yasuhiro Kotani, Sadahiko Arai, Tatsuo Iwasaki, Shuhei Sato, Shingo Kasahara, Shunji Sano, Hidemasa Oh

    Circulation research   120 ( 7 )   1162 - 1173   2017.3

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    RATIONALE: Patients with single ventricle physiology are at high risk of mortality resulting from ventricular dysfunction. The preliminary results of the phase 1 trial showed that cardiosphere-derived cells (CDCs) may be effective against congenital heart failure. OBJECTIVE: To determine whether intracoronary delivery of autologous CDCs improves cardiac function in patients with single ventricle physiology. METHODS AND RESULTS: We conducted a phase 2 randomized controlled study to assign in a 1:1 ratio 41 patients who had single ventricle physiology undergoing stage 2 or 3 palliation to receive intracoronary infusion of CDCs 4 to 9 weeks after surgery or staged reconstruction alone (study A). The primary outcome measure was to assess improvement in cardiac function at 3-month follow-up. Four months after palliation, controls had an alternative option to receive late CDC infusion on request (study B). Secondary outcomes included ventricular function, heart failure status, somatic growth, and health-related quality of life after a 12-month observation. At 3 months, the absolute changes in ventricular function were significantly greater in the CDC-treated group than in the controls (+6.4% [SD, 5.5] versus +1.3% [SD, 3.7]; P=0.003). In study B, a late CDC infusion in 17 controls increased the ventricular function at 3 months compared with that at baseline (38.8% [SD, 7.7] versus 34.8% [SD, 7.4]; P<0.0001). At 1 year, overall CDC infusion was associated with improved ventricular function (41.4% [SD, 6.6] versus 35.0% [SD, 8.2]; P<0.0001) and volumes (P<0.001), somatic growth (P<0.0001) with increased trophic factors production, such as insulin-like growth factor-1 and hepatocyte growth factor, and quality of life, along with a reduced heart failure status (P<0.0001) and cardiac fibrosis (P=0.014) relative to baseline. CONCLUSIONS: Intracoronary infusion of CDCs after staged palliation favorably affected cardiac function by reverse remodeling in patients with single ventricle physiology. This impact may improve heart failure status, somatic growth, and quality of life in patients and reduce parenting stress for their families. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01829750.

    DOI: 10.1161/CIRCRESAHA.116.310253

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  • Challenges to success in heart failure: Cardiac cell therapies in patients with heart diseases. International journal

    Hidemasa Oh, Hiroshi Ito, Shunji Sano

    Journal of cardiology   68 ( 5 )   361 - 367   2016.11

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    Heart failure remains the leading cause of death worldwide, and is a burgeoning problem in public health due to the limited capacity of postnatal hearts to self-renew. The pathophysiological changes in injured hearts can sometimes be manifested as scar formation or myocardial degradation, rather than supplemental muscle regeneration to replenish lost tissue during the healing processes. Stem cell therapies have been investigated as a possible treatment approach for children and adults with potentially fatal cardiovascular disease that does not respond to current medical therapies. Although the heart is one of the least regenerative organs in mammals, discoveries made during the past few decades have improved our understanding of cardiac development and resident stem/progenitor pools, which may be lineage-restricted subpopulations during the post-neonatal stage of cardiac morphogenesis. Recently, investigation has specifically focused on factors that activate either endogenous progenitor cells or preexisting cardiomyocytes, to regenerate cardiovascular cells and replace the damaged heart tissues. The discovery of induced pluripotent stem cells has advanced our technological capability to direct cardiac reprogramming by essential factors that are crucial for heart field completion in each stage. Cardiac tissue engineering technology has recently shown progress in generating myocardial tissue on human native cardiac extracellular matrix scaffolds. This review summarizes recent advances in the field of cardiac cell therapies with an emphasis on cellular mechanisms, such as bone marrow stem cells and cardiac progenitor cells, which show the high potential for success in preclinical and clinical meta-analysis studies. Expanding our current understanding of mechanisms of self-renewal in the neonatal mammalian heart may lead to the development of novel cardiovascular regenerative medicines for pediatric heart diseases.

    DOI: 10.1016/j.jjcc.2016.04.010

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  • Dissecting the Left Heart Hypoplasia by Pluripotent Stem Cells

    Junko Kobayashi, Shunji Sano, Hidemasa Oh

    337 - 339   2016

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    The genetic background of hypoplastic left heart syndrome (HLHS) is still unknown. Cardiac differentiation from pluripotent stem cells (PSCs) can recapitulate the cardiogenesis in vitro, and PSC technology could be useful to dissect the diseases with the complex mechanisms. In the past few years, some researches were reported to seek the pathogenesis of HLHS by using PSCs. This paper reports the achievements.

    DOI: 10.1007/978-4-431-54628-3

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  • Transcoronary infusion of cardiac progenitor cells in hypoplastic left heart syndrome: Three-year follow-up of the Transcoronary Infusion of Cardiac Progenitor Cells in Patients With Single-Ventricle Physiology (TICAP) trial. International journal

    Suguru Tarui, Shuta Ishigami, Daiki Ousaka, Shingo Kasahara, Shinichi Ohtsuki, Shunji Sano, Hidemasa Oh

    The Journal of thoracic and cardiovascular surgery   150 ( 5 )   1198 - 1207   2015.11

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    OBJECTIVES: Our aim was to assess midterm safety and clinical outcomes of intracoronary infusion of cardiosphere-derived cells (CDCs) after staged palliation in patients with hypoplastic left heart syndrome (HLHS). METHODS: In this prospective, controlled study, 14 consecutive patients with HLHS who were undergoing 2- or 3-stage surgical palliations were assigned to receive intracoronary CDC infusion 1 month after cardiac surgery (n = 7), followed by 7 patients allocated to a control group with standard care alone. The primary end point was to assess procedural feasibility and safety; the secondary end point was to evaluate cardiac function and heart failure status through 36-month follow-up. RESULTS: No complications, including tumor formation, were reported within 36 months after CDC infusion. Echocardiography showed significantly greater improvement in right ventricular ejection fraction (RVEF) in infants receiving CDCs than in controls at 36 months (+8.0% ± 4.7% vs +2.2% ± 4.3%; P = .03). These cardiac function improvements resulted in reduced brain natriuretic peptide levels (P = .04), lower incidence of unplanned catheter interventions (P = .04), and higher weight-for-age z score (P = .02) at 36 months relative to controls. As independent predictors of treatment responsiveness, absolute changes in RVEF at 36 months were negatively correlated with age, weight-for-age z score, and RVEF at CDC infusion. CONCLUSIONS: Intracoronary CDC infusion after staged procedure in patients with HLHS is safe and improves RVEF, which persists during 36-month follow-up. This therapeutic strategy may enhance somatic growth and reduce incidence of heart failure.

    DOI: 10.1016/j.jtcvs.2015.06.076

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  • Intracoronary autologous cardiac progenitor cell transfer in patients with hypoplastic left heart syndrome: the TICAP prospective phase 1 controlled trial. International journal

    Shuta Ishigami, Shinichi Ohtsuki, Suguru Tarui, Daiki Ousaka, Takahiro Eitoku, Maiko Kondo, Michihiro Okuyama, Junko Kobayashi, Kenji Baba, Sadahiko Arai, Takuya Kawabata, Ko Yoshizumi, Atsushi Tateishi, Yosuke Kuroko, Tatsuo Iwasaki, Shuhei Sato, Shingo Kasahara, Shunji Sano, Hidemasa Oh

    Circulation research   116 ( 4 )   653 - 64   2015.2

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    RATIONALE: Hypoplastic left heart syndrome (HLHS) remains a lethal congenital cardiac defect. Recent studies have suggested that intracoronary administration of autologous cardiosphere-derived cells (CDCs) may improve ventricular function. OBJECTIVE: The aim of this study was to test whether intracoronary delivery of CDCs is feasible and safe in patients with hypoplastic left heart syndrome. METHODS AND RESULTS: Between January 5, 2011, and January 16, 2012, 14 patients (1.8±1.5 years) were prospectively assigned to receive intracoronary infusion of autologous CDCs 33.4±8.1 days after staged procedures (n=7), followed by 7 controls with standard palliation alone. The primary end point was to assess the safety, and the secondary end point included the preliminary efficacy to verify the right ventricular ejection fraction improvements between baseline and 3 months. Manufacturing and intracoronary delivery of CDCs were feasible, and no serious adverse events were reported within the 18-month follow-up. Patients treated with CDCs showed right ventricular ejection fraction improvement from baseline to 3-month follow-up (46.9%±4.6% to 52.1%±2.4%; P=0.008). Compared with controls at 18 months, cardiac MRI analysis of CDC-treated patients showed a higher right ventricular ejection fraction (31.5%±6.8% versus 40.4%±7.6%; P=0.049), improved somatic growth (P=0.0005), reduced heart failure status (P=0.003), and lower incidence of coil occlusion for collaterals (P=0.007). CONCLUSIONS: Intracoronary infusion of autologous CDCs seems to be feasible and safe in children with hypoplastic left heart syndrome after staged surgery. Large phase 2 trials are warranted to examine the potential effects of cardiac function improvements and the long-term benefits of clinical outcomes. CLINICAL TRIAL REGISTRATION URL: http://www.clinicaltrials.gov. Unique identifier: NCT01273857.

    DOI: 10.1161/CIRCRESAHA.116.304671

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  • MURC/Cavin-4 facilitates recruitment of ERK to caveolae and concentric cardiac hypertrophy induced by α1-adrenergic receptors. International journal

    Takehiro Ogata, Daisuke Naito, Naohiko Nakanishi, Yukiko K Hayashi, Takuya Taniguchi, Kotaro Miyagawa, Tetsuro Hamaoka, Naoki Maruyama, Satoaki Matoba, Koji Ikeda, Hiroyuki Yamada, Hidemasa Oh, Tomomi Ueyama

    Proceedings of the National Academy of Sciences of the United States of America   111 ( 10 )   3811 - 6   2014.3

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    The actions of catecholamines on adrenergic receptors (ARs) induce sympathetic responses, and sustained activation of the sympathetic nervous system results in disrupted circulatory homeostasis. In cardiomyocytes, α1-ARs localize to flask-shaped membrane microdomains known as "caveolae." Caveolae require both caveolin and cavin proteins for their biogenesis and function. However, the functional roles and molecular interactions of caveolar components in cardiomyocytes are poorly understood. Here, we showed that muscle-restricted coiled-coil protein (MURC)/Cavin-4 regulated α1-AR-induced cardiomyocyte hypertrophy through enhancement of ERK1/2 activation in caveolae. MURC/Cavin-4 was expressed in the caveolae and T tubules of cardiomyocytes. MURC/Cavin-4 overexpression distended the caveolae, whereas MURC/Cavin-4 was not essential for their formation. MURC/Cavin-4 deficiency attenuated cardiac hypertrophy induced by α1-AR stimulation in the presence of caveolae. Interestingly, MURC/Cavin-4 bound to α1A- and α1B-ARs as well as ERK1/2 in caveolae, and spatiotemporally modulated MEK/ERK signaling in response to α1-AR stimulation. Thus, MURC/Cavin-4 facilitates ERK1/2 recruitment to caveolae and efficient α1-AR signaling mediated by caveolae in cardiomyocytes, which provides a unique insight into the molecular mechanisms underlying caveola-mediated signaling in cardiac hypertrophy.

    DOI: 10.1073/pnas.1315359111

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  • Directed differentiation of patient-specific induced pluripotent stem cells identifies the transcriptional repression and epigenetic modification of NKX2-5, HAND1, and NOTCH1 in hypoplastic left heart syndrome. International journal

    Junko Kobayashi, Masashi Yoshida, Suguru Tarui, Masataka Hirata, Yusuke Nagai, Shingo Kasahara, Keiji Naruse, Hiroshi Ito, Shunji Sano, Hidemasa Oh

    PloS one   9 ( 7 )   e102796   2014

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    The genetic basis of hypoplastic left heart syndrome (HLHS) remains unknown, and the lack of animal models to reconstitute the cardiac maldevelopment has hampered the study of this disease. This study investigated the altered control of transcriptional and epigenetic programs that may affect the development of HLHS by using disease-specific induced pluripotent stem (iPS) cells. Cardiac progenitor cells (CPCs) were isolated from patients with congenital heart diseases to generate patient-specific iPS cells. Comparative gene expression analysis of HLHS- and biventricle (BV) heart-derived iPS cells was performed to dissect the complex genetic circuits that may promote the disease phenotype. Both HLHS- and BV heart-derived CPCs were reprogrammed to generate disease-specific iPS cells, which showed characteristic human embryonic stem cell signatures, expressed pluripotency markers, and could give rise to cardiomyocytes. However, HLHS-iPS cells exhibited lower cardiomyogenic differentiation potential than BV-iPS cells. Quantitative gene expression analysis demonstrated that HLHS-derived iPS cells showed transcriptional repression of NKX2-5, reduced levels of TBX2 and NOTCH/HEY signaling, and inhibited HAND1/2 transcripts compared with control cells. Although both HLHS-derived CPCs and iPS cells showed reduced SRE and TNNT2 transcriptional activation compared with BV-derived cells, co-transfection of NKX2-5, HAND1, and NOTCH1 into HLHS-derived cells resulted in synergistic restoration of these promoters activation. Notably, gain- and loss-of-function studies revealed that NKX2-5 had a predominant impact on NPPA transcriptional activation. Moreover, differentiated HLHS-derived iPS cells showed reduced H3K4 dimethylation as well as histone H3 acetylation but increased H3K27 trimethylation to inhibit transcriptional activation on the NKX2-5 promoter. These findings suggest that patient-specific iPS cells may provide molecular insights into complex transcriptional and epigenetic mechanisms, at least in part, through combinatorial expression of NKX2-5, HAND1, and NOTCH1 that coordinately contribute to cardiac malformations in HLHS.

    DOI: 10.1371/journal.pone.0102796

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  • Molecular genetic and functional characterization implicate muscle-restricted coiled-coil gene (MURC) as a causal gene for familial dilated cardiomyopathy. International journal

    Gabriela Rodriguez, Tomomi Ueyama, Takehiro Ogata, Grazyna Czernuszewicz, Yanli Tan, Gerald W Dorn 2nd, Roberta Bogaev, Katsuya Amano, Hidemasa Oh, Hiroaki Matsubara, James T Willerson, Ali J Marian

    Circulation. Cardiovascular genetics   4 ( 4 )   349 - 58   2011.8

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    BACKGROUND: Dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM) are classic forms of systolic and diastolic heart failure, respectively. Mutations in genes encoding sarcomere and cytoskeletal proteins are major causes of HCM and DCM. MURC, encoding muscle-restricted coiled-coil, a Z-line protein, regulates cardiac function in mice. We investigated potential causal role of MURC in human cardiomyopathies. METHODS AND RESULTS: We sequenced MURC in 1199 individuals, including 383 probands with DCM, 307 with HCM, and 509 healthy control subjects. We found 6 heterozygous DCM-specific missense variants (p.N128K, p.R140W, p.L153P, p.S307T, p.P324L, and p.S364L) in 8 unrelated probands. Variants p.N128K and p.S307T segregated with inheritance of DCM in small families (χ(2)=8.5, P=0.003). Variants p.N128K, p.R140W, p.L153P, and p.S364L were considered probably or possibly damaging. Variant p.P324L recurred in 3 independent probands, including 1 proband with a TPM1 mutation (p.M245T). A deletion variant (p.L232-R238del) was present in 3 unrelated HCM probands, but it did not segregate with HCM in a family who also had a MYH7 mutation (p.L907V). The phenotype in mutation carriers was notable for progressive heart failure leading to heart transplantation in 4 patients, conduction defects, and atrial arrhythmias. Expression of mutant MURC proteins in neonatal rat cardiac myocytes transduced with recombinant adenoviruses was associated with reduced RhoA activity, lower mRNA levels of hypertrophic markers and smaller myocyte size as compared with wild-type MURC. CONCLUSIONS: MURC mutations impart loss-of-function effects on MURC functions and probably are causal variants in human DCM. The causal role of a deletion mutation in HCM is uncertain.

    DOI: 10.1161/CIRCGENETICS.111.959866

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  • PARM-1 is an endoplasmic reticulum molecule involved in endoplasmic reticulum stress-induced apoptosis in rat cardiac myocytes. International journal

    Koji Isodono, Tomosaburo Takahashi, Hiroko Imoto, Naohiko Nakanishi, Takehiro Ogata, Satoshi Asada, Atsuo Adachi, Tomomi Ueyama, Hidemasa Oh, Hiroaki Matsubara

    PloS one   5 ( 3 )   e9746   2010.3

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    To identify novel transmembrane and secretory molecules expressed in cardiac myocytes, signal sequence trap screening was performed in rat neonatal cardiac myocytes. One of the molecules identified was a transmembrane protein, prostatic androgen repressed message-1 (PARM-1). While PARM-1 has been identified as a gene induced in prostate in response to castration, its function is largely unknown. Our expression analysis revealed that PARM-1 was specifically expressed in hearts and skeletal muscles, and in the heart, cardiac myocytes, but not non-myocytes expressed PARM-1. Immunofluorescent staining showed that PARM-1 was predominantly localized in endoplasmic reticulum (ER). In Dahl salt-sensitive rats, high-salt diet resulted in hypertension, cardiac hypertrophy and subsequent heart failure, and significantly stimulated PARM-1 expression in the hearts, with a concomitant increase in ER stress markers such as GRP78 and CHOP. In cultured cardiac myocytes, PARM-1 expression was stimulated by proinflammatory cytokines, but not by hypertrophic stimuli. A marked increase in PARM-1 expression was observed in response to ER stress inducers such as thapsigargin and tunicamycin, which also induced apoptotic cell death. Silencing PARM-1 expression by siRNAs enhanced apoptotic response in cardiac myocytes to ER stresses. PARM-1 silencing also repressed expression of PERK and ATF6, and augmented expression of CHOP without affecting IRE-1 expression and JNK and Caspase-12 activation. Thus, PARM-1 expression is induced by ER stress, which plays a protective role in cardiac myocytes through regulating PERK, ATF6 and CHOP expression. These results suggested that PARM-1 is a novel ER transmembrane molecule involved in cardiac remodeling in hypertensive heart disease.

    DOI: 10.1371/journal.pone.0009746

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  • Stem cell engineering for cardiac tissue regeneration. International journal

    Masashi Yoshida, Hidemasa Oh

    Cardiology   115 ( 3 )   191 - 3   2010

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    DOI: 10.1159/000281839

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  • Deficiency of nectin-2 leads to cardiac fibrosis and dysfunction under chronic pressure overload. International journal

    Seimi Satomi-Kobayashi, Tomomi Ueyama, Steffen Mueller, Ryuji Toh, Tomoya Masano, Tsuyoshi Sakoda, Yoshiyuki Rikitake, Jun Miyoshi, Hiroaki Matsubara, Hidemasa Oh, Seinosuke Kawashima, Ken-ichi Hirata, Yoshimi Takai

    Hypertension (Dallas, Tex. : 1979)   54 ( 4 )   825 - 31   2009.10

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    The intercalated disc, a cell-cell contact site between neighboring cardiac myocytes, plays an important role in maintaining the homeostasis of the heart by transmitting electric and mechanical signals. Changes in the architecture of the intercalated disc have been observed in dilated cardiomyopathy. Among cell-cell junctions in the intercalated disc, adherens junctions are involved in anchoring myofibrils and transmitting force. Nectins are Ca(2+)-independent, immunoglobulin-like cell-cell adhesion molecules that exist in adherens junctions. However, the role of nectins in cardiac homeostasis and integrity of the intercalated disc are unknown. Among the isoforms of nectins, nectin-2 and -4 were expressed at the intercalated disc in the heart. Nectin-2-knockout mice showed normal cardiac structure and function under physiological conditions. Four weeks after banding of the ascending aorta, cardiac function was significantly impaired in nectin-2-knockout mice compared with wild-type mice, although both nectin-2-knockout and wild-type mice developed similar degrees of cardiac hypertrophy. Banded nectin-2-knockout mice displayed cardiac fibrosis more evidently than banded wild-type mice. The disruption of the intercalated discs and disorganized myofibrils were observed in banded nectin-2-knockout mice. Furthermore, the number of apoptotic cardiac myocytes was increased in banded nectin-2-knockout mice. In the hearts of banded nectin-2-knockout mice, Akt remained at lower phosphorylation levels until 2 weeks after banding, whereas c-Jun N-terminal kinase and p38 mitogen-activated protein kinase were highly phosphorylated compared with those of wild-type mice. These results indicate that nectin-2 is required to maintain structure and function of the intercalated disc and protects the heart from pressure-overload-induced cardiac dysfunction.

    DOI: 10.1161/HYPERTENSIONAHA.109.130443

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  • Controlled delivery of basic fibroblast growth factor promotes human cardiosphere-derived cell engraftment to enhance cardiac repair for chronic myocardial infarction. International journal

    Naofumi Takehara, Yoshiaki Tsutsumi, Kento Tateishi, Takehiro Ogata, Hideo Tanaka, Tomomi Ueyama, Tomosaburo Takahashi, Tetsuro Takamatsu, Masanori Fukushima, Masashi Komeda, Masaaki Yamagishi, Hitoshi Yaku, Yasuhiko Tabata, Hiroaki Matsubara, Hidemasa Oh

    Journal of the American College of Cardiology   52 ( 23 )   1858 - 1865   2008.12

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    OBJECTIVES: This study was designed to determine whether controlled release of basic fibroblast growth factor (bFGF) might improve human cardiosphere-derived cell (hCDC) therapy in a pig model of chronic myocardial infarction. BACKGROUND: Current cell therapies for cardiac repair are limited by loss of the transplanted cells and poor differentiation. METHODS: We conducted 2 randomized, placebo-controlled studies in immunosuppressed pigs with anterior myocardial infarctions. Four weeks after coronary reperfusion, 14 pigs were randomly assigned to receive an intramyocardial injection of placebo medium with or without bFGF-incorporating hydrogel implantation. As a second study, 26 pigs were randomized to receive controlled release of bFGF combined with or without hCDCs or bone marrow-derived mesenchymal stem cell transplantation 4 weeks after reperfusion. RESULTS: Controlled release of bFGF in ischemic myocardium significantly augmented the formation of microvascular networks to enhance myocardial perfusion and contractile function. When combined with cell transplantation, the additive effects of bFGF were confined to hCDC-injected animals, but were not observed in animals receiving human bone marrow-derived mesenchymal stem cell transplantation. This was shown by increased donor-cell engraftment and enhanced cardiomyocyte differentiation in the transplanted hearts, resulting in synergistically improved ventricular function and regional wall motion and reduced infarct size. CONCLUSIONS: Controlled delivery of bFGF modulates the post-ischemic microenvironment to enhance hCDC engraftment and differentiation. This novel strategy demonstrates significant functional improvements after myocardial infarction and may potentially represent a therapeutic approach to be studied in a clinical trial in human heart failure.

    DOI: 10.1016/j.jacc.2008.06.052

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  • Stemming heart failure with cardiac- or reprogrammed-stem cells. International journal

    Kento Tateishi, Naofumi Takehara, Hiroaki Matsubara, Hidemasa Oh

    Journal of cellular and molecular medicine   12 ( 6A )   2217 - 32   2008.12

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    Despite extensive efforts to control myocyte growth by genetic targeting of the cell cycle machinery and small molecules for cardiac repair, adult myocytes themselves appeared to divide a limited number of times in response to a variety of cardiac muscle stresses. Rare tissue-resident stem cells are thought to exist in many adult organs that are capable of self-renewal and differentiation and possess a range of actions that are potentially therapeutic. Recent studies suggest that a population of cardiac stem cells (CSCs) is maintained after cardiac development in the adult heart in mammals including human beings; however, homeostatic cardiomyocyte replacement might be stem cell-dependent, and functional myocardial regeneration after cardiac muscle damage is not yet considered as sufficient to fully maintain or reconstitute the cardiovascular system and function. Although it is clear that adult CSCs have limitations in their capabilities to proliferate extensively and differentiate in response to injury in vivo for replenishing mature car-diomyocytes and potentially function as resident stem cells. Transplantation of CSCs expanded ex vivo seems to require an integrated strategy of cell growth-enhancing factor(s) and tissue engineering technologies to support the donor cell survival and subsequent proliferation and differentiation in the host microenvironment. There has been substantial interest regarding the evidence that mammalian fibroblasts can be genetically reprogrammed to induced pluripotent stem (iPS) cells, which closely resemble embryonic stem (ES) cell properties capable of differentiating into functional cardiomyocytes, and these cells may provide an alternative cell source for generating patient-specific CSCs for therapeutic applications.

    DOI: 10.1111/j.1582-4934.2008.00487.x

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  • Downregulation of Dicer expression by serum withdrawal sensitizes human endothelial cells to apoptosis. International journal

    Satoshi Asada, Tomosaburo Takahashi, Koji Isodono, Atsuo Adachi, Hiroko Imoto, Takehiro Ogata, Tomomi Ueyama, Hiroaki Matsubara, Hidemasa Oh

    American journal of physiology. Heart and circulatory physiology   295 ( 6 )   H2512-21   2008.12

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    Although the modulated expression of Dicer is documented upon neoplastic transformation, little is known of the regulation of Dicer expression by environmental stimuli and its roles in the regulation of cellular functions in primary cells. In this study, we found that Dicer expression was downregulated upon serum withdrawal in human umbilical vein endothelial cells (HUVECs). Serum withdrawal induced a time-dependent repression of Dicer expression, which was specifically rescued by vascular endothelial cell growth factor or sphingosine-1-phosphate. When Dicer expression was silenced by short-hairpin RNA against Dicer, the cells were more prone to apoptosis under serum withdrawal, whereas the rate of apoptosis was comparable with control cells in the serum-containing condition. Real-time PCR-based gene expression profiling identified several genes, the expression of which was modulated by Dicer silencing, including adhesion and matrix-related molecules, caspase-3, and nitric oxide synthase 3 (NOS3). Dicer silencing markedly impaired migratory functions without affecting cell adhesion and repressed phosphorylation of focal adhesion kinase and proline-rich tyrosine kinase 2 in adherent HUVECs. Dicer knockdown upregulated caspase-3 and downregulated NOS3 expression, and serum withdrawal indeed increased caspase-3 and decreased NOS3 expression. Furthermore, the overexpression of Dicer in HUVECs resulted in a marked reduction in apoptosis upon serum withdrawal and a decreased caspase-3 and increased NOS3 expression. The inhibition of NOS activity by Nomega-nitro-L-arginine methyl ester abrogated the effect of Dicer overexpression to rescue the cells from serum withdrawal-induced apoptosis. These results indicated that serum withdrawal decreases Dicer expression, leading to an increased susceptibility to apoptosis through the regulation of caspase-3 and NOS3 expression.

    DOI: 10.1152/ajpheart.00233.2008

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  • Crossveinless-2 controls bone morphogenetic protein signaling during early cardiomyocyte differentiation in P19 cells. International journal

    Koichiro Harada, Akiko Ogai, Tomosaburo Takahashi, Masafumi Kitakaze, Hiroaki Matsubara, Hidemasa Oh

    The Journal of biological chemistry   283 ( 39 )   26705 - 13   2008.9

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    Increasing evidence indicates that bone morphogenetic proteins (BMPs) are crucial for cardiac induction, specification, and development. Although signaling of BMPs is tightly regulated through soluble BMP-binding proteins, how they regulate BMP signaling during cardiac differentiation remains unknown. To identify molecules responsible for BMP signaling during early cardiomyocyte differentiation of P19 cells, cDNA subtraction was performed. We found a bimodal expression of the BMP-binding protein Crossveinless-2 (Cv2) during cardiomyocyte differentiation; Cv2 is temporally expressed earlier than cardiac transcription factors such as Nkx2.5 and Tbx5 and acts as a suppressor for BMP signaling in P19 cells. We established a P19 clonal cell line harboring a cardiac alpha-myosin heavy chain promoter-driven enhanced green fluorescent protein gene to monitor cardiac differentiation by flow cytometry. Treatment with BMP2 during the first 2 days of differentiation suppressed cardiomyocyte differentiation through activation of down-stream targets Smad1/5/8 protein and Id1 gene, whereas treatment with Cv2 conversely inhibited Smad1/5/8 activation and Id1 expression, leading to increased generation of cardiac cells. RNA interference-mediated knockdown (KD) of endogenous Cv2 showed increased Smad1/5/8 activation and impaired cardiomyocyte differentiation. Expression of cardiac mesoderm markers was reduced, whereas expression of Id1 and endoderm markers such as Sox7, Hnf4, and E-cadherin was induced in Cv2-kinase dead cells. These phenotypes were rescued by the addition of Cv2 protein to the culture media during the first 2 days of differentiation or co-culture with parental cells. These data suggest that Cv2 may specify cardiac mesodermal lineage through inhibition of BMP signaling at early stage of cardiogenesis.

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  • MURC, a muscle-restricted coiled-coil protein, is involved in the regulation of skeletal myogenesis. International journal

    Masashi Tagawa, Tomomi Ueyama, Takehiro Ogata, Naofumi Takehara, Norio Nakajima, Koji Isodono, Satoshi Asada, Tomosaburo Takahashi, Hiroaki Matsubara, Hidemasa Oh

    American journal of physiology. Cell physiology   295 ( 2 )   C490-8   2008.8

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    Skeletal myogenesis is a multistep process by which multinucleated mature muscle fibers are formed from undifferentiated, mononucleated myoblasts. However, the molecular mechanisms of skeletal myogenesis have not been fully elucidated. Here, we identified muscle-restricted coiled-coil (MURC) protein as a positive regulator of myogenesis. In skeletal muscle, MURC was localized to the cytoplasm with accumulation in the Z-disc of the sarcomere. In C2C12 myoblasts, MURC expression occurred coincidentally with myogenin expression and preceded sarcomeric myosin expression during differentiation into myotubes. RNA interference (RNAi)-mediated knockdown of MURC impaired differentiation in C2C12 myoblasts, which was accompanied by impaired myogenin expression and ERK activation. Overexpression of MURC in C2C12 myoblasts resulted in the promotion of differentiation with enhanced myogenin expression and ERK activation during differentiation. During injury-induced muscle regeneration, MURC expression increased, and a higher abundance of MURC was observed in immature myofibers compared with mature myofibers. In addition, ERK was activated in regenerating tissue, and ERK activation was detected in MURC-expressing immature myofibers. These findings suggest that MURC is involved in the skeletal myogenesis that results from modulation of myogenin expression and ERK activation. MURC may play pivotal roles in the molecular mechanisms of skeletal myogenic differentiation.

    DOI: 10.1152/ajpcell.00188.2008

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  • MURC, a muscle-restricted coiled-coil protein that modulates the Rho/ROCK pathway, induces cardiac dysfunction and conduction disturbance. International journal

    Takehiro Ogata, Tomomi Ueyama, Koji Isodono, Masashi Tagawa, Naofumi Takehara, Tsuneaki Kawashima, Koichiro Harada, Tomosaburo Takahashi, Tetsuo Shioi, Hiroaki Matsubara, Hidemasa Oh

    Molecular and cellular biology   28 ( 10 )   3424 - 36   2008.5

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    We identified a novel muscle-restricted putative coiled-coil protein, MURC, which is evolutionarily conserved from frog to human. MURC was localized to the cytoplasm with accumulation in the Z-line of the sarcomere in the murine adult heart. MURC mRNA expression in the heart increased during the developmental process from the embryonic stage to adulthood. In response to pressure overload, MURC mRNA expression increased in the hypertrophied heart. Using the yeast two-hybrid system, we identified the serum deprivation response (SDPR) protein, a phosphatidylserine-binding protein, as a MURC-binding protein. MURC induced activation of the RhoA/ROCK pathway, which modulated serum response factor-mediated atrial natriuretic peptide (ANP) expression and myofibrillar organization. SDPR augmented MURC-induced transactivation of the ANP promoter in cardiomyocytes, and RNA interference of SDPR attenuated the action of MURC on the ANP promoter. Transgenic mice expressing cardiac-specific MURC (Tg-MURC) exhibited cardiac contractile dysfunction and atrioventricular (AV) conduction disturbances with atrial chamber enlargement, reduced thickness of the ventricular wall, and interstitial fibrosis. Spontaneous episodes of atrial fibrillation and AV block were observed in Tg-MURC mice. These findings indicate that MURC modulates RhoA signaling and that MURC plays an important role in the development of cardiac dysfunction and conduction disturbance with increased vulnerability to atrial arrhythmias.

    DOI: 10.1128/MCB.02186-07

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  • Skeletal muscle-derived progenitors capable of differentiating into cardiomyocytes proliferate through myostatin-independent TGF-beta family signaling. International journal

    Tetsuya Nomura, Tomomi Ueyama, Eishi Ashihara, Kento Tateishi, Satoshi Asada, Norio Nakajima, Koji Isodono, Tomosaburo Takahashi, Hiroaki Matsubara, Hidemasa Oh

    Biochemical and biophysical research communications   365 ( 4 )   863 - 9   2008.1

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    The existence of skeletal muscle-derived stem cells (MDSCs) has been suggested in mammals; however, the signaling pathways controlling MDSC proliferation remain largely unknown. Here we report the isolation of myosphere-derived progenitor cells (MDPCs) that can give rise to beating cardiomyocytes from adult skeletal muscle. We identified that follistatin, an antagonist of TGF-beta family members, was predominantly expressed in MDPCs, whereas myostatin was mainly expressed in myogenic cells and mature skeletal muscle. Although follistatin enhanced the replicative growth of MDPCs through Smad2/3 inactivation and cell cycle progression, disruption of myostatin did not increase the MDPC proliferation. By contrast, inhibition of activin A (ActA) or growth differentiation factor 11 (GDF11) signaling dramatically increased MDPC proliferation via down-regulation of p21 and increases in the levels of cdk2/4 and cyclin D1. Thus, follistatin may be an effective progenitor-enhancing agent neutralizing ActA and GDF11 signaling to regulate the growth of MDPCs in skeletal muscle.

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  • Therapeutic potential of stem/progenitor cells in human skeletal muscle for cardiovascular regeneration. International journal

    Tetsuya Nomura, Eishi Ashihara, Kento Tateishi, Tomomi Ueyama, Tomosaburo Takahas-Hi, Masaaki Yamagishi, Toshikazu Kubo, Hitoshi Yaku, Hiroaki Matsubara, Hidemasa Oh

    Current stem cell research & therapy   2 ( 4 )   293 - 300   2007.12

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    Although myoblast transplantation in patients with ischemic heart failure results in a significant improvement of cardiac function, subsequent studies have consistently shown the myotubes formation in the absence of electromechanical coupling with the neighboring host myocardium, accompanied with the short-term release of paracrine effectors from implanted cells. One major pitfall of using myoblasts is that transplanted cells do not differentiate into cardiomyocytes, which may cause the inherent proarrhythmogenic events. Therefore, whether a discrete subpopulation in heterogeneous muscle-cell cultures is responsible for substantial cardiovascular regeneration has yet to be investigated. We describe here the isolation of progenitor cells from human skeletal muscle. These cells proliferated as non-adherent myospheres in suspension and displayed early embryonic factors and mesenchymal cell-like characteristics. Flow cytometric analyses demonstrated that CD56/N-CAM/Leu-19, a neural cell adhesion molecule abundantly present in myoblasts, was absent in myospheres but was expressed in an adherent cell population containing myogenic precursors. Myosphere-derived progenitor cells (MDPCs) differentiated in culture to produce cardiac, smooth muscle, and endothelial cells. Transplantation of MDPCs into ischemic hearts in NOD/scid mice promoted angiogenesis with substantial cardiovascular regeneration. Our results provide a foundation to further study the cell and biological function of human MDPCs which may have potential therapeutic implications.

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  • Osteopontin is a myosphere-derived secretory molecule that promotes angiogenic progenitor cell proliferation through the phosphoinositide 3-kinase/Akt pathway. International journal

    Takehiro Ogata, Tomomi Ueyama, Tetsuya Nomura, Satoshi Asada, Masashi Tagawa, Tomoyuki Nakamura, Tomosaburo Takahashi, Hiroaki Matsubara, Hidemasa Oh

    Biochemical and biophysical research communications   359 ( 2 )   341 - 7   2007.7

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    We have reported that skeletal myosphere-derived progenitor cells (MDPCs) can differentiate into vascular cells, and that MDPC transplantation into cardiomyopathic hearts improves cardiac function. However, the autocrine/paracrine molecules and underlying mechanisms responsible for MDPC growth have not yet been determined. To explore the molecules enhancing the proliferation of MDPCs, we performed serial analysis of gene expression and signal sequence trap methods using RNA isolated from MDPCs. We identified osteopontin (OPN), a secretory molecule, as one of most abundant molecules expressed in MDPCs. OPN provided a proliferative effect for MDPCs. MDPCs treated with OPN showed Akt activation, and inhibition of the phosphoinositide 3-kinase (PI3K)/Akt pathway repressed the proliferative effect of OPN. Furthermore, OPN-pretreated MDPCs maintained their differentiation potential into endothelial and vascular smooth muscle cells. These findings indicate an important role of OPN as an autocrine/paracrine molecule in regulating the proliferative growth of muscle-derived angiogenic progenitor cells via the PI3K/Akt pathway.

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  • Stage-specific role of endogenous Smad2 activation in cardiomyogenesis of embryonic stem cells. International journal

    Ryoji Kitamura, Tomosaburo Takahashi, Norio Nakajima, Koji Isodono, Satoshi Asada, Hikaru Ueno, Tomomi Ueyama, Toshikazu Yoshikawa, Hiroaki Matsubara, Hidemasa Oh

    Circulation research   101 ( 1 )   78 - 87   2007.7

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    The role of Smads and their specific ligands during cardiomyogenesis in ES cells was examined. Smad2 was activated bimodally in the early and late phases of cardiac differentiation, whereas Smad1 was activated after the middle phase. Nodal and Cripto were expressed in the early stage and then downregulated, whereas transforming growth factor-beta and activin were expressed only in the late phase. Suppression of early Smad2 activation by SB-431542 produced complete inhibition of endodermal and mesodermal induction but augmented neuroectodermal differentiation, followed by poor cardiomyogenesis, whereas inhibition during the late phase alone promoted cardiomyogenesis. Inhibitory effect of Smad2 on cardiomyogenesis in the late phase was mainly mediated by transforming growth factor-beta, and inhibition of transforming growth factor-beta-mediated Smad2 activation resulted in a greater replicative potential in differentiated cardiac myocytes and enhanced differentiation of nonmyocytes into cardiac myocytes. Thus, endogenous Smad2 activation is indispensable for endodermal and mesodermal induction in the early phase. In the late phase, endogenous transforming growth factor-beta negatively regulates cardiomyogenesis through Smad2 activation by modulating proliferation and differentiation of cardiac myocytes.

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  • Clonally amplified cardiac stem cells are regulated by Sca-1 signaling for efficient cardiovascular regeneration. International journal

    Kento Tateishi, Eishi Ashihara, Naofumi Takehara, Tetsuya Nomura, Shoken Honsho, Takuo Nakagami, Shigehiro Morikawa, Tomosaburo Takahashi, Tomomi Ueyama, Hiroaki Matsubara, Hidemasa Oh

    Journal of cell science   120 ( Pt 10 )   1791 - 800   2007.5

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    Recent studies have shown that cardiac stem cells (CSCs) from the adult mammalian heart can give rise to functional cardiomyocytes; however, the definite surface markers to identify a definitive single entity of CSCs and the molecular mechanisms regulating their growth are so far unknown. Here, we demonstrate a single-cell deposition analysis to isolate individually selected CSCs from adult murine hearts and investigate the signals required for their proliferation and survival. Clonally proliferated CSCs express stem cell antigen-1 (Sca-1) with embryonic stem (ES) cell-like and mesenchymal cell-like characteristics and are associated with telomerase reverse transcriptase (TERT). Using a transgene that expresses a GFP reporter under the control of the TERT promoter, we demonstrated that TERT(GFP)-positive fractions from the heart were enriched for cells expressing Sca-1. Knockdown of Sca-1 transcripts in CSCs led to retarded ex vivo expansion and apoptosis through Akt inactivation. We also show that ongoing CSC proliferation and survival after direct cell-grafting into ischemic myocardium require Sca-1 to upregulate the secreted paracrine effectors that augment neoangiogenesis and limit cardiac apoptosis. Thus, Sca-1 might be an essential component to promote CSC proliferation and survival to directly facilitate early engraftment, and might indirectly exert the effects on late cardiovascular differentiation after CSC transplantation.

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  • Human cardiac stem cells exhibit mesenchymal features and are maintained through Akt/GSK-3beta signaling. International journal

    Kento Tateishi, Eishi Ashihara, Shoken Honsho, Naofumi Takehara, Tetsuya Nomura, Tomosaburo Takahashi, Tomomi Ueyama, Masaaki Yamagishi, Hitoshi Yaku, Hiroaki Matsubara, Hidemasa Oh

    Biochemical and biophysical research communications   352 ( 3 )   635 - 41   2007.1

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    Recent evidence suggested that human cardiac stem cells (hCSCs) may have the clinical application for cardiac repair; however, their characteristics and the regulatory mechanisms of their growth have not been fully investigated. Here, we show the novel property of hCSCs with respect to their origin and tissue distribution in human heart, and demonstrate the signaling pathway that regulates their growth and survival. Telomerase-active hCSCs were predominantly present in the right atrium and outflow tract of the heart (infant > adult) and had a mesenchymal cell-like phenotype. These hCSCs expressed the embryonic stem cell markers and differentiated into cardiomyocytes to support cardiac function when transplanted them into ischemic myocardium. Inhibition of Akt pathway impaired the hCSC proliferation and induced apoptosis, whereas inhibition of glycogen synthase kinase-3 (GSK-3) enhanced their growth and survival. We conclude that hCSCs exhibit mesenchymal features and that Akt/GSK-3beta may be crucial modulators for hCSC maintenance in human heart.

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  • Skeletal myosphere-derived progenitor cell transplantation promotes neovascularization in delta-sarcoglycan knockdown cardiomyopathy. International journal

    Tetsuya Nomura, Eishi Ashihara, Kento Tateishi, Satoshi Asada, Tomomi Ueyama, Tomosaburo Takahashi, Hiroaki Matsubara, Hidemasa Oh

    Biochemical and biophysical research communications   352 ( 3 )   668 - 74   2007.1

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    Bone marrow cells have been shown to contribute to neovascularization in ischemic hearts, whereas their impaired maturation to restore the delta-sarcoglycan (delta-SG) expression responsible for focal myocardial degeneration limits their utility to treat the pathogenesis of cardiomyopathy. Here, we report the isolation of multipotent progenitor cells from adult skeletal muscle, based on their ability to generate floating-myospheres. Myosphere-derived progenitor cells (MDPCs) are distinguishable from myogenic C2C12 cells and differentiate into vascular smooth muscle cells and mesenchymal progeny. The mutation in the delta-SG has been shown to develop vascular spasm to affect sarcolemma structure causing cardiomyopathy. We originally generated delta-SD knockdown (KD) mice and transplanted MDPCs into the hearts. MDPCs enhanced neoangiogenesis and restored delta-SG expression in impaired vasculatures through trans-differentiation, leading to improvement of cardiac function associated with paracrine effectors secretion. We propose that MDPCs may be the promising progenitor cells in skeletal muscle to treat delta-sarcoglycan complex mutant cardiomyopathy.

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  • MicroRNA-1 facilitates skeletal myogenic differentiation without affecting osteoblastic and adipogenic differentiation. International journal

    Norio Nakajima, Tomosaburo Takahashi, Ryoji Kitamura, Koji Isodono, Satoshi Asada, Tomomi Ueyama, Hiroaki Matsubara, Hidemasa Oh

    Biochemical and biophysical research communications   350 ( 4 )   1006 - 12   2006.12

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    MicroRNAs (miRNAs) are small non-coding RNAs emerging as important post-transcriptional gene regulators. In this study, we examined the role of miR-1, an miRNA specifically expressed in cardiac and skeletal muscle tissue, on the myogenic, osteoblastic, and adipogenic differentiation of C2C12 cells. Upon induction of myogenic differentiation, miR-1 was robustly expressed. Retrovirus-mediated overexpression of miR-1 markedly enhanced expression of muscle creatine kinase, sarcomeric myosin, and alpha-actinin, while the effects on myogenin and MyoD expression were modest. Formation of myotubes was significantly augmented in miR-1-overexpressing cells, indicating miR-1 expression enhanced not only myogenic differentiation but also maturation into myotubes. In contrast, osteoblastic and adipogenic differentiation was not affected by forced expression of miR-1. Thus, the muscle-specific miRNA, miR-1, plays important roles in controlling myogenic differentiation and maturation in lineage-committed cells, rather than functioning in fate determination.

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  • [New era of cardiac stem cell therapy in heart failure].

    Hidemasa Oh

    Rinsho byori. The Japanese journal of clinical pathology   53 ( 1 )   61 - 9   2005.1

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    Despite of plethora of reports on stem cell transplantation leading to neovascularization in infarct models, whether sustaining clinical benefit in post-myocardial infarction patients is manifested by myocyte repair remains unclear. Cardiac muscle regeneration in adult heart is thought to occur through the mobilization and differentiation of mesenchymal stem cells in bone marrow origin, however, recent studies have suggested that substantial cardiac stem cells may exist in the heart itself, repopulating the damaged cardiac muscle during injury or aging processes. The implications of cardiac stem cells-based myocyte plasticity have recently begun to define in human heart, neither arisen from bone marrow nor circulating precursors. Introduction of cardiac stem cells may improve myocardial function, but several hurdles exist and should be coaxed far beyond the clinical application of cardiac regenerative therapies. On-going investigations may lead to the discovery of mediators of cardiac stem cells migration, proliferation and differentiation that, in turn, might result in the mending of the broken heart after injury.

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  • Statins enhance migratory capacity by upregulation of the telomere repeat-binding factor TRF2 in endothelial progenitor cells. International journal

    Ioakim Spyridopoulos, Judith Haendeler, Carmen Urbich, Tim H Brummendorf, Hidemasa Oh, Michael D Schneider, Andreas M Zeiher, Stefanie Dimmeler

    Circulation   110 ( 19 )   3136 - 42   2004.11

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    BACKGROUND: Cultivation of endothelial progenitor cells (EPCs) leads to premature replicative senescence, limiting ex vivo expansion for potential clinical cell therapy. Recent studies have linked senescence to the dysfunction of telomeres, the "ends" of chromosomes, via the so-called mitotic clock or culture-induced stress. The purpose of this study was to elucidate a possible role of telomere biology in the functional augmentation of EPCs by statins. METHODS AND RESULTS: Human EPCs were isolated from peripheral blood. Using flow cytometry after fluorescence in situ hybridization with a telomere-specific (C3TA2)3 peptide nucleic acid probe (Flow-FISH), we found mean telomere length in untreated EPCs from healthy subjects to range between 8.5+/-0.2 and 11.1+/-0.5 kb with no change over 6 days of culture, excluding telomere erosion as one cause for premature senescence. Although mean telomere length did not differ between statin-treated and untreated EPCs, atorvastatin (0.1 micromol/L) and mevastatin (1.0 micromol/L) both led to a more than 3-fold increase in the expression of the telomere capping protein TRF2 (telomere repeat-binding factor), as shown by immunoblotting, whereas quantitative reverse transcription-polymerase chain reaction demonstrated no increase in TRF2 mRNA. Telomere dysfunction of EPCs was also paralleled by a 4-fold increase in the DNA damage checkpoint-kinase 2 (Chk2). Conversely, statin cotreatment or overexpression of TRF2 completely suppressed Chk2 induction. Finally, overexpression of a dominant negative mutant of the TRF2 protein abrogated statin-induced enhancement of migratory activity down to baseline values. CONCLUSIONS: Ex vivo culturing of EPCs leads to "uncapping" of telomeres, indicated by the loss of TRF2. Statin cotreatment of EPCs prevents impairment of their functional capacity by a TRF2-dependent, posttranscriptional mechanism. This is the first time a beneficial effect of statins on telomere biology has been described.

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  • Cardiac muscle plasticity in adult and embryo by heart-derived progenitor cells. International journal

    Hidemasa Oh, Xuan Chi, Steven B Bradfute, Yuji Mishina, Jennifer Pocius, Lloyd H Michael, Richard R Behringer, Robert J Schwartz, Mark L Entman, Michael D Schneider

    Annals of the New York Academy of Sciences   1015   182 - 9   2004.5

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    The evidence of cardiomyocyte proliferation in damaged heart implied cardiac regeneration might occur by resident or extra cardiac stem cells. However, the specification and origin of these cells remain unknown. Here, we report using fluorescence-activated cell sorting that cardiac progenitor cells resided in adult heart and colocalized with small capillary vessels, within the stem cell antigen (Sca-1) population expressing high telomerase activity. Notably, hematopoietic stem cells capable of efflux Hoechst 33342, termed side population cells, also were identified within the heart-derived cells. The cardiac progenitor cells (CD45(-)/CD34(-)) express neither cardiac muscle nor endothelial cell markers at an undifferentiated stage. The exposure of 5-azacytidine induced cardiac differentiation, which depends, in part, on Bmpr1a, a type IA receptor for bone morphogenetic protein (BMP). The capability of adult Sca1(+) cells to adopt a cardiac muscle in embryogenesis was substantiated by blastocyst injection, using progenitors from the adult hearts of transgenic mice that harbor a bacterial artificial chromosome expressing GFP via the Nkx-2.5 locus. Intravenously injected progenitors, shortly after ischemic/reperfusion, homed and functionally differentiated 3.5% of total left ventricle in the host myocardium. Differentiation included both fusion-independent and fusion-associated components, proved by the Cre/loxP donor/recipient system. Our studies suggest that endogenous cardiac progenitors reside in the adult heart, regenerate cardiomyocytes functionally, and integrate into the existing heart circuitry.

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  • Cardiac progenitor cells from adult myocardium: homing, differentiation, and fusion after infarction. International journal

    Hidemasa Oh, Steven B Bradfute, Teresa D Gallardo, Teruya Nakamura, Vinciane Gaussin, Yuji Mishina, Jennifer Pocius, Lloyd H Michael, Richard R Behringer, Daniel J Garry, Mark L Entman, Michael D Schneider

    Proceedings of the National Academy of Sciences of the United States of America   100 ( 21 )   12313 - 8   2003.10

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    Potential repair by cell grafting or mobilizing endogenous cells holds particular attraction in heart disease, where the meager capacity for cardiomyocyte proliferation likely contributes to the irreversibility of heart failure. Whether cardiac progenitors exist in adult myocardium itself is unanswered, as is the question whether undifferentiated cardiac precursor cells merely fuse with preexisting myocytes. Here we report the existence of adult heart-derived cardiac progenitor cells expressing stem cell antigen-1. Initially, the cells express neither cardiac structural genes nor Nkx2.5 but differentiate in vitro in response to 5'-azacytidine, in part depending on Bmpr1a, a receptor for bone morphogenetic proteins. Given intravenously after ischemia/reperfusion, cardiac stem cell antigen 1 cells home to injured myocardium. By using a Cre/Lox donor/recipient pair (alphaMHC-Cre/R26R), differentiation was shown to occur roughly equally, with and without fusion to host cells.

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  • Telomere attrition and Chk2 activation in human heart failure. International journal

    Hidemasa Oh, Sam C Wang, Arun Prahash, Motoaki Sano, Christine S Moravec, George E Taffet, Lloyd H Michael, Keith A Youker, Mark L Entman, Michael D Schneider

    Proceedings of the National Academy of Sciences of the United States of America   100 ( 9 )   5378 - 83   2003.4

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    The "postmitotic" phenotype in adult cardiac muscle exhibits similarities to replicative senescence more generally and constitutes a barrier to effective restorative growth in heart disease. Telomere dysfunction is implicated in senescence and apoptotic signaling but its potential role in heart disorders is unknown. Here, we report that cardiac apoptosis in human heart failure is associated specifically with defective expression of the telomere repeat- binding factor TRF2, telomere shortening, and activation of the DNA damage checkpoint kinase, Chk2. In cultured cardiomyocytes, interference with either TRF2 function or expression triggered telomere erosion and apoptosis, indicating that cell death can occur via this pathway even in postmitotic, noncycling cells; conversely, exogenous TRF2 conferred protection from oxidative stress. In vivo, mechanical stress was sufficient to down-regulate TRF2, shorten telomeres, and activate Chk2 in mouse myocardium, and transgenic expression of telomerase reverse transcriptase conferred protection from all three responses. Together, these data suggest that apoptosis in chronic heart failure is mediated in part by telomere dysfunction and suggest an essential role for TRF2 even in postmitotic cells.

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  • Activation and function of cyclin T-Cdk9 (positive transcription elongation factor-b) in cardiac muscle-cell hypertrophy. International journal

    Motoaki Sano, Maha Abdellatif, Hidemasa Oh, Min Xie, Luigi Bagella, Antonio Giordano, Lloyd H Michael, Francesco J DeMayo, Michael D Schneider

    Nature medicine   8 ( 11 )   1310 - 7   2002.11

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    Hypertrophic growth is a risk factor for mortality in heart diseases. Mechanisms are lacking for this global increase in RNA and protein per cell, which underlies hypertrophy. Hypertrophic signals cause phosphorylation of the RNA polymerase II C-terminal domain, required for transcript elongation. RNA polymerase II kinases include cyclin-dependent kinases-7 (Cdk7) and Cdk9, components of two basal transcription factors. We report activation of Cdk7 and -9 in hypertrophy triggered by signaling proteins (Galphaq, calcineurin) or chronic mechanical stress. Only Cdk9 was activated by acute load or, in culture, by endothelin. A preferential role for Cdk9 was shown in RNA polymerase II phosphorylation and growth induced by endothelin, using pharmacological and dominant-negative inhibitors. All four hypertrophic signals dissociated 7SK small nuclear RNA, an endogenous inhibitor, from cyclin T-Cdk9. Cdk9 was limiting for cardiac growth, shown by suppressing its inhibitor (7SK) in culture and preventing downregulation of its activator (cyclin T1) in mouse myocardium.Note: In the AOP version of this article, the numbering of the author affiliations was incorrect. This has now been fixed, and the affiliations appear correctly online and in print.

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  • The emerging role of telomerase in cardiac muscle cell growth and survival. International journal

    Hidemasa Oh, Michael D Schneider

    Journal of molecular and cellular cardiology   34 ( 7 )   717 - 24   2002.7

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    Most mammalian cells-excepting germ cells, tumor cells, and stem cells, that is-possess a finite replicative life span, manifested by the eventual cessation of cell proliferation. Clinically, this is germane not just to the overt derangements of cell growth in cancer, but also to organs such as the heart, in which the capacity for cell replacement and repair is insufficient to maintain organ function following cell death. Among the intrinsic mechanisms that control a conserved program of replicative senescence is the enzyme telomerase, which synthesizes the telomeric repeat for end-capping of each chromosome. The implications of telomerase for cardiac growth have recently begun to be defined. Other functions of telomerase, in maintaining genome integrity, also hold importance for cardiac muscle, as a novel means to suppress apoptosis and, thus, salvage myocardium following ischemic injury.

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Books

  • Stem Cell Therapy

    Kenta Hirai, Hidemasa Oh( Role: Joint author)

    2021.7 

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  • ヒト心筋幹細胞移植療法の前臨床的確立と細胞組織工学によるハイブリッド療法の開発に関する総合的研究

    王, 英正, 京都大学大学院医学研究科

    [王英正]  2008.5 

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MISC

  • Impact of cardiac progenitor cells on heart failure and survival in single ventricle congenital heart disease.

    Sano T, Ousaka D, Goto T, Ishigami S, Hirai K, Kasahara S, Ohtsuki S, Sano S, Oh H

    Circulation Research   122 ( 7 )   994 - 1005   2018

  • Impact of cardiac progenitor cells on heart failure and survival in single ventricle congenital heart disease.

    Sano T, Ousaka D, Goto T, Ishigami S, Hirai K, Kasahara S, Ohtsuki S, Sano S, Oh H

    Circulation Research   122 ( 7 )   994 - 1005   2018

  • Cell Therapy Trials in Congenital Heart Disease

    Hidemasa Oh

    CIRCULATION RESEARCH   120 ( 8 )   1353 - 1366   2017.4

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    Dramatic evolution in medical and catheter interventions and complex surgeries to treat children with congenital heart disease (CHD) has led to a growing number of patients with a multitude of long-term complications associated with morbidity and mortality. Heart failure in patients with hypoplastic left heart syndrome predicated by functional single ventricle lesions is associated with an increase in CHD prevalence and remains a significant challenge. Pathophysiological mechanisms contributing to the progression of CHD, including single ventricle lesions and dilated cardiomyopathy, and adult heart disease may inevitably differ. Although therapeutic options for advanced cardiac failure are restricted to heart transplantation or mechanical circulatory support, there is a strong impetus to develop novel therapeutic strategies. As lower vertebrates, such as the newt and zebrafish, have a remarkable ability to replace lost cardiac tissue, this intrinsic self-repair machinery at the early postnatal stage in mice was confirmed by partial ventricular resection. Although the underlying mechanistic insights might differ among the species, mammalian heart regeneration occurs even in humans, with the highest degree occurring in early childhood and gradually declining with age in adulthood, suggesting the advantage of stem cell therapy to ameliorate ventricular dysfunction in patients with CHD. Although effective clinical translation by a variety of stem cells in adult heart disease remains inconclusive with respect to the improvement of cardiac function, case reports and clinical trials based on stem cell therapies in patients with CHD may be invaluable for the next stage of therapeutic development. Dissecting the differential mechanisms underlying progressive ventricular dysfunction in children and adults may lead us to identify a novel regenerative therapy. Future regenerative technologies to treat patients with CHD are exciting prospects for heart regeneration in general practice.

    DOI: 10.1161/CIRCRESAHA.117.309697

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  • Intracoronary Cardiac Progenitor Cells in Single Ventricle Physiology The PERSEUS (Cardiac Progenitor Cell Infusion to Treat Univentricular Heart Disease) Randomized Phase 2 Trial

    Shuta Ishigami, Shinichi Ohtsuki, Takahiro Eitoku, Daiki Ousaka, Maiko Kondo, Yoshihiko Kurita, Kenta Hirai, Yosuke Fukushima, Kenji Baba, Takuya Goto, Naohiro Horio, Junko Kobayashi, Yosuke Kuroko, Yasuhiro Kotani, Sadahiko Arai, Tatsuo Iwasaki, Shuhei Sato, Shingo Kasahara, Shunji Sano, Hidemasa Oh

    CIRCULATION RESEARCH   120 ( 7 )   1162 - +   2017.3

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    Rationale: Patients with single ventricle physiology are at high risk of mortality resulting from ventricular dysfunction. The preliminary results of the phase 1 trial showed that cardiosphere-derived cells (CDCs) may be effective against congenital heart failure.
    Objective: To determine whether intracoronary delivery of autologous CDCs improves cardiac function in patients with single ventricle physiology.
    Methods and Results: We conducted a phase 2 randomized controlled study to assign in a 1: 1 ratio 41 patients who had single ventricle physiology undergoing stage 2 or 3 palliation to receive intracoronary infusion of CDCs 4 to 9 weeks after surgery or staged reconstruction alone (study A). The primary outcome measure was to assess improvement in cardiac function at 3-month follow-up. Four months after palliation, controls had an alternative option to receive late CDC infusion on request (study B). Secondary outcomes included ventricular function, heart failure status, somatic growth, and health-related quality of life after a 12-month observation. At 3 months, the absolute changes in ventricular function were significantly greater in the CDC-treated group than in the controls (+ 6.4% [SD, 5.5] versus + 1.3% [SD, 3.7]; P= 0.003). In study B, a late CDC infusion in 17 controls increased the ventricular function at 3 months compared with that at baseline (38.8% [SD, 7.7] versus 34.8% [SD, 7.4]; P&lt; 0.0001). At 1 year, overall CDC infusion was associated with improved ventricular function (41.4% [SD, 6.6] versus 35.0% [SD, 8.2]; P&lt; 0.0001) and volumes (P&lt; 0.001), somatic growth (P&lt; 0.0001) with increased trophic factors production, such as insulin-like growth factor-1 and hepatocyte growth factor, and quality of life, along with a reduced heart failure status (P&lt; 0.0001) and cardiac fibrosis (P= 0.014) relative to baseline.
    Conclusions: Intracoronary infusion of CDCs after staged palliation favorably affected cardiac function by reverse remodeling in patients with single ventricle physiology. This impact may improve heart failure status, somatic growth, and quality of life in patients and reduce parenting stress for their families.

    DOI: 10.1161/CIRCRESAHA.116.310253

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  • 体性幹細胞を用いた心臓再生医療の最前線

    石神修大, 大月審一, 笠原真悟, 佐野俊二, 王 英正

    臨床医薬   33 ( 4 )   125 - 132   2017

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  • Cell Therapy Trials in Congenital Heart Disease

    H. Oh

    Circulation Research   120 ( 8 )   1353 - 1366   2017

  • Transcoronary cell infusion with the stop-flow technique in children with single-ventricle physiology.

    Eitoku T, Baba K, Kondou M, Kurita Y, Fukushima Y, Hirai K, Ohtsuki S, Ishigami S, Sano S, Oh H

    Pediatric Intervention   60 ( 3 )   240 - 246   2017

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  • 先天性心疾患に対する心筋再生医療

    石神修大, 大月審一, 笠原真悟, 佐野俊二, 王 英正

    Heart View   5 ( 5 )   547 - 551   2017

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  • Cell Therapy Trials in Congenital Heart Disease

    Oh H

    Circulation Research   120 ( 8 )   1353 - 1366   2017

  • Transcoronary cell infusion with the stop-flow technique in children with single-ventricle physiology.

    Eitoku T, Baba K, Kondou M, Kurita Y, Fukushima Y, Hirai K, Ohtsuki S, Ishigami S, Sano S, Oh H

    Pediatric Intervention   60 ( 3 )   240 - 246   2017

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  • Application of mechanobiological engineering to regenerative and reproductive medicine Invited

    Takahashi K, Oh H, Iribe G, Matsuura K, Naruse K

    Trans JSMBE   55 ( 4PM-Abstract )   340   2017

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  • Challenges to success in heart failure: Cardiac cell therapies in patients with heart diseases

    Hidemasa Oh, Hiroshi Ito, Shunji Sano

    JOURNAL OF CARDIOLOGY   68 ( 5-6 )   361 - 367   2016.11

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    Heart failure remains the leading cause of death worldwide, and is a burgeoning problem in public health due to the limited capacity of postnatal hearts to self-renew. The pathophysiological changes in injured hearts can sometimes be manifested as scar formation or myocardial degradation, rather than supplemental muscle regeneration to replenish lost tissue during the healing processes. Stem cell therapies have been investigated as a possible treatment approach for children and adults with potentially fatal cardiovascular disease that does not respond to current medical therapies. Although the heart is one of the least regenerative organs in mammals, discoveries made during the past few decades have improved our understanding of cardiac development and resident stem/progenitor pools, which may be lineage-restricted subpopulations during the post-neonatal stage of cardiac morphogenesis. Recently, investigation has specifically focused on factors that activate either endogenous progenitor cells or preexisting cardiomyocytes, to regenerate cardiovascular cells and replace the damaged heart tissues. The discovery of induced pluripotent stem cells has advanced our technological capability to direct cardiac reprogramming by essential factors that are crucial for heart field completion in each stage. Cardiac tissue engineering technology has recently shown progress in generating myocardial tissue on human native cardiac extracellular matrix scaffolds. This review summarizes recent advances in the field of cardiac cell therapies with an emphasis on cellular mechanisms, such as bone marrow stem cells and cardiac progenitor cells, which show the high potential for success in preclinical and clinical meta-analysis studies. Expanding our current understanding of mechanisms of self-renewal in the neonatal mammalian heart may lead to the development of novel cardiovascular regenerative medicines for pediatric heart diseases. (C) 2016 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved.

    DOI: 10.1016/j.jjcc.2016.04.010

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  • Challenges to success in heart failure: Cardiac cell therapies in patients with heart diseases

    Hidemasa Oh, Hiroshi Ito, Shunji Sano

    JOURNAL OF CARDIOLOGY   68 ( 5-6 )   361 - 367   2016.11

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    Language:English   Publishing type:Book review, literature introduction, etc.   Publisher:ELSEVIER SCIENCE BV  

    Heart failure remains the leading cause of death worldwide, and is a burgeoning problem in public health due to the limited capacity of postnatal hearts to self-renew. The pathophysiological changes in injured hearts can sometimes be manifested as scar formation or myocardial degradation, rather than supplemental muscle regeneration to replenish lost tissue during the healing processes. Stem cell therapies have been investigated as a possible treatment approach for children and adults with potentially fatal cardiovascular disease that does not respond to current medical therapies. Although the heart is one of the least regenerative organs in mammals, discoveries made during the past few decades have improved our understanding of cardiac development and resident stem/progenitor pools, which may be lineage-restricted subpopulations during the post-neonatal stage of cardiac morphogenesis. Recently, investigation has specifically focused on factors that activate either endogenous progenitor cells or preexisting cardiomyocytes, to regenerate cardiovascular cells and replace the damaged heart tissues. The discovery of induced pluripotent stem cells has advanced our technological capability to direct cardiac reprogramming by essential factors that are crucial for heart field completion in each stage. Cardiac tissue engineering technology has recently shown progress in generating myocardial tissue on human native cardiac extracellular matrix scaffolds. This review summarizes recent advances in the field of cardiac cell therapies with an emphasis on cellular mechanisms, such as bone marrow stem cells and cardiac progenitor cells, which show the high potential for success in preclinical and clinical meta-analysis studies. Expanding our current understanding of mechanisms of self-renewal in the neonatal mammalian heart may lead to the development of novel cardiovascular regenerative medicines for pediatric heart diseases. (C) 2016 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved.

    DOI: 10.1016/j.jjcc.2016.04.010

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  • Cardiac Progenitor Cell Therapy in Right Heart Failure With Preserved Ejection Fraction

    Daiki Ousaka, Takuya Goto, Shuta Ishigami, Toshikazu Sano, Kenta Hirai, Shinichi Ohtsuki, Shingo Kasahara, Shunji Sano, Hidemasa Oh

    CIRCULATION   134   2016.11

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  • 心臓内幹細胞による心不全疾患の治療

    佐野俊和, 大月審一, 佐野俊二, 王 英正

    BIO Clinica   31 ( 10 )   29 - 33   2016

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  • 心臓内幹細胞を用いた心臓再生医療

    平井健太, 大月審一, 佐野俊二, 王 英正

    心臓   48 ( 12 )   1334 - 1339   2016

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  • Transcoronary infusion of cardiac progenitor cells in hypoplastic left heart syndrome: Three-year follow-up of the Transcoronary Infusion of Cardiac Progenitor Cells in Patients With Single-Ventricle Physiology (TICAP) trial

    John E. Mayer, Sano, Todd Rosengart

    JOURNAL OF THORACIC AND CARDIOVASCULAR SURGERY   150 ( 5 )   1207 - 1208   2015.11

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    DOI: 10.1016/j.jtcvs.2015.06.076.

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  • Cardiac Progenitor Cell Infusion in Patients With Univentricular Heart Diseases in Heart Failure With Preserved Ejection Fraction

    Daiki Ousaka, Takuya Goto, Shuta Ishigami, Shinichi Ohtsuki, Shingo Kasahara, Shunji Sano, Hidemasa Oh

    CIRCULATION   132   2015.11

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  • Transcoronary Infusion of Cardiac Progenitor Cells in Hypoplastic Left Heart Syndrome: 3-year Results of the TICAP Trial

    Shuta Ishigami, Suguru Tarui, Takuya Goto, Daiki Ousaka, Kenji Baba, Shingo Kasahara, Shinichi Ohtsuki, Shunji Sano, Hidemasa Oh

    CIRCULATION   132   2015.11

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  • Intracoronary Autologous Cardiac Progenitor Cell Transfer in Patients With Hypoplastic Left Heart Syndrome The TICAP Prospective Phase 1 Controlled Trial

    Shuta Ishigami, Shinichi Ohtsuki, Suguru Tarui, Daiki Ousaka, Takahiro Eitoku, Maiko Kondo, Michihiro Okuyama, Junko Kobayashi, Kenji Baba, Sadahiko Arai, Takuya Kawabata, Ko Yoshizumi, Atsushi Tateishi, Yosuke Kuroko, Tatsuo Iwasaki, Shuhei Sato, Shingo Kasahara, Shunji Sano, Hidemasa Oh

    CIRCULATION RESEARCH   116 ( 4 )   653 - 664   2015.2

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    Rationale: Hypoplastic left heart syndrome (HLHS) remains a lethal congenital cardiac defect. Recent studies have suggested that intracoronary administration of autologous cardiosphere-derived cells (CDCs) may improve ventricular function.
    Objective: The aim of this study was to test whether intracoronary delivery of CDCs is feasible and safe in patients with hypoplastic left heart syndrome.
    Methods and Results: Between January 5, 2011, and January 16, 2012, 14 patients (1.8 +/- 1.5 years) were prospectively assigned to receive intracoronary infusion of autologous CDCs 33.4 +/- 8.1 days after staged procedures (n=7), followed by 7 controls with standard palliation alone. The primary end point was to assess the safety, and the secondary end point included the preliminary efficacy to verify the right ventricular ejection fraction improvements between baseline and 3 months. Manufacturing and intracoronary delivery of CDCs were feasible, and no serious adverse events were reported within the 18-month follow-up. Patients treated with CDCs showed right ventricular ejection fraction improvement from baseline to 3-month follow-up (46.9%+/- 4.6% to 52.1%+/- 2.4%; P=0.008). Compared with controls at 18 months, cardiac MRI analysis of CDC-treated patients showed a higher right ventricular ejection fraction (31.5%+/- 6.8% versus 40.4%+/- 7.6%; P=0.049), improved somatic growth (P=0.0005), reduced heart failure status (P=0.003), and lower incidence of coil occlusion for collaterals (P=0.007).
    Conclusions: Intracoronary infusion of autologous CDCs seems to be feasible and safe in children with hypoplastic left heart syndrome after staged surgery. Large phase 2 trials are warranted to examine the potential effects of cardiac function improvements and the long-term benefits of clinical outcomes.

    DOI: 10.1161/CIRCRESAHA.116.304671

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  • Epigenetic modification in congenital heart diseases by using stem cell technologies

    Kobayashi J, Sano S, Oh H

    Stem Cell Epigenetics   1 ( 2 )   e550   2015

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  • Cardiac stem cell therapies for congenital heart diseases

    Ishigami S, Sano S, Oh H

    Stem Cell and Translational Investigation   1 ( 2 )   e800   2015

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  • 小児心不全に対する心臓内幹細胞自家移植療法

    日本小児血液・がん学会誌   52 ( 3 )   2015

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  • 疾患特異的iPS細胞を用いた先天性心疾患の病態解明

    小児循環器学会雑誌   2015

  • Cardiac Magnetic Resonance Late Gadolinium Enhancement is Associated With Ventricular Elastance That May Predict Latent Ventricular Dysfunction After Fontan Procedure

    Michihiro Okuyama, Shuta Ishigami, Daiki Ousaka, Junko Kobayashi, Sadahiko Arai, Shingo Kasahara, Shunji Sano, Hidemasa Oh

    CIRCULATION   130   2014.11

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  • Intracoronary Delivery of Autologous Cardiac Progenitor Cells in Children With Hypoplastic Left Heart Syndrome: The Ticap Trial With 18-Month Follow Up

    Shuta Ishigami, Suguru Tarui, Michihiro Okuyama, Daiki Ousaka, Shinichi Ohtsuki, Takahiro Eitoku, Junko Kobayashi, Shingo Kasahara, Shunji Sano, Hidemasa Oh

    CIRCULATION   130   2014.11

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  • Disease-Specific Induced Pluripotent Stem Cells Identify the Transcriptional Repression and Epigenetic Modification of NKX2-5, HAND1, and NOTCH1 During Cardiac Development of Hypoplastic Left Heart Syndrome

    Junko Kobayashi, Masashi Yoshida, Suguru Tarui, Shuta Ishigami, Michihiro Okuyama, Yusuke Nagai, Shingo Kasahara, Keiji Naruse, Hiroshi Ito, Shunji Sano, Hidemasa Oh

    CIRCULATION   130   2014.11

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  • Directed Differentiation of Patient-Specific Induced Pluripotent Stem Cells Identifies the Transcriptional Repression and Epigenetic Modification of NKX2-5, HAND1, and NOTCH1 in Hypoplastic Left Heart Syndrome

    Junko Kobayashi, Masashi Yoshida, Suguru Tarui, Masataka Hirata, Yusuke Nagai, Shingo Kasahara, Keiji Naruse, Hiroshi Ito, Shunji Sano, Hidemasa Oh

    PLOS ONE   9 ( 7 )   e102796-e102796   2014.7

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    The genetic basis of hypoplastic left heart syndrome (HLHS) remains unknown, and the lack of animal models to reconstitute the cardiac maldevelopment has hampered the study of this disease. This study investigated the altered control of transcriptional and epigenetic programs that may affect the development of HLHS by using disease-specific induced pluripotent stem (iPS) cells. Cardiac progenitor cells (CPCs) were isolated from patients with congenital heart diseases to generate patient-specific iPS cells. Comparative gene expression analysis of HLHS-and biventricle (BV) heart-derived iPS cells was performed to dissect the complex genetic circuits that may promote the disease phenotype. Both HLHS-and BV heart-derived CPCs were reprogrammed to generate disease-specific iPS cells, which showed characteristic human embryonic stem cell signatures, expressed pluripotency markers, and could give rise to cardiomyocytes. However, HLHS-iPS cells exhibited lower cardiomyogenic differentiation potential than BV-iPS cells. Quantitative gene expression analysis demonstrated that HLHS-derived iPS cells showed transcriptional repression of NKX2-5, reduced levels of TBX2 and NOTCH/HEY signaling, and inhibited HAND1/2 transcripts compared with control cells. Although both HLHS-derived CPCs and iPS cells showed reduced SRE and TNNT2 transcriptional activation compared with BV-derived cells, co-transfection of NKX2-5, HAND1, and NOTCH1 into HLHS-derived cells resulted in synergistic restoration of these promoters activation. Notably, gain- and loss-of-function studies revealed that NKX2-5 had a predominant impact on NPPA transcriptional activation. Moreover, differentiated HLHS-derived iPS cells showed reduced H3K4 dimethylation as well as histone H3 acetylation but increased H3K27 trimethylation to inhibit transcriptional activation on the NKX2-5 promoter. These findings suggest that patient-specific iPS cells may provide molecular insights into complex transcriptional and epigenetic mechanisms, at least in part, through combinatorial expression of NKX2-5, HAND1, and NOTCH1 that coordinately contribute to cardiac malformations in HLHS.

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  • MURC/Cavin-4 facilitates recruitment of ERK to caveolae and concentric cardiac hypertrophy induced by alpha 1-adrenergic receptors

    Takehiro Ogata, Daisuke Naito, Naohiko Nakanishi, Yukiko K. Hayashi, Takuya Taniguchi, Kotaro Miyagawa, Tetsuro Hamaoka, Naoki Maruyama, Satoaki Matoba, Koji Ikeda, Hiroyuki Yamada, Hidemasa Oh, Tomomi Ueyama

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   111 ( 10 )   3811 - 3816   2014.3

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    The actions of catecholamines on adrenergic receptors (ARs) induce sympathetic responses, and sustained activation of the sympathetic nervous system results in disrupted circulatory homeostasis. In cardiomyocytes, alpha 1-ARs localize to flask-shaped membrane microdomains known as "caveolae." Caveolae require both caveolin and cavin proteins for their biogenesis and function. However, the functional roles and molecular interactions of caveolar components in cardiomyocytes are poorly understood. Here, we showed that muscle-restricted coiled-coil protein (MURC)/Cavin-4 regulated alpha 1-AR-induced cardiomyocyte hypertrophy through enhancement of ERK1/2 activation in caveolae. MURC/Cavin-4 was expressed in the caveolae and T tubules of cardiomyocytes. MURC/Cavin-4 overexpression distended the caveolae, whereas MURC/Cavin-4 was not essential for their formation. MURC/Cavin-4 deficiency attenuated cardiac hypertrophy induced by alpha 1-AR stimulation in the presence of caveolae. Interestingly, MURC/Cavin-4 bound to alpha 1A-and alpha 1B-ARs as well as ERK1/2 in caveolae, and spatiotemporally modulated MEK/ERK signaling in response to alpha 1-AR stimulation. Thus, MURC/Cavin-4 facilitates ERK1/2 recruitment to caveolae and efficient alpha 1-AR signaling mediated by caveolae in cardiomyocytes, which provides a unique insight into the molecular mechanisms underlying caveola-mediated signaling in cardiac hypertrophy.

    DOI: 10.1073/pnas.1315359111

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  • Stem cell therapies in patients with single ventricle physiology.

    Methodist Debakey Cardiovascular Journal.   10 ( 2 )   77 - 81   2014

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  • 小児心不全の再生医療からのアプローチ

    石神修大, 佐野俊二, 王 英正

    移植   50 ( 6 )   54 - 63   2014

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  • 先天性心疾患外科手術における再生医療

    循環器内科   75 ( 1 )   87 - 93   2014

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  • YT-11-5(YRA) 疾患特異的iPS細胞を用いた左心低形成症候群における心臓発生異常の解明(YT Young Researcher Award & Traveler's Grant,第113回日本外科学会定期学術集会)

    小林 純子, 樽井 俊, 平田 昌敬, 川畑 拓也, 黒子 洋介, 立石 篤史, 吉積 功, 新井 禎彦, 笠原 真悟, 佐野 俊二, 王 英正

    日本外科学会雑誌   114 ( 2 )   415 - 415   2013.3

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  • 心筋幹細胞を用いた先天性心疾患に対する心筋再生医療

    樽井俊, 佐野俊二, 王 英正

    月刊循環器   3 ( 9 )   69 - 76   2013

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  • Patient-specific Induced Pluripotent Stem Cells For Modeling Hypoplastic Left Heart Syndrome

    Junko Kobayashi, Masashi Yoshida, Suguru Tarui, Masataka Hirata, Ken Takahashi, Shingo Kasahara, Keiji Naruse, Hiroshi Ito, Shunji Sano, Hidemasa Oh

    CIRCULATION   126 ( 21 )   2012.11

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  • Cardiac Progenitor Cell Infusion in Patients with Hypoplastic Left Heart Syndrome: a Prospective Phase 1 Clinical Trial

    Hidemasa Oh, Suguru Tarui, Shinichi Otsuki, Shunji Sano

    JOURNAL OF CARDIAC FAILURE   18 ( 10 )   S145 - S145   2012.10

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  • 先天性心疾患に対する心筋再生医療

    小林純子, 佐野俊二, 王 英正

    循環器内科   71 ( 4 )   360 - 368   2012

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  • 先天性心疾患に対する心筋再生医療

    小林純子, 佐野俊二, 王 英正

    循環器内科   71   360 - 368   2012

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  • テロメア生物学から心筋再生医療の実用化へ

    王 英正

    岡山医学会雑誌   124   27 - 34   2012

  • 小児心不全への細胞治療の現状と展望

    王 英正

    呼吸と循環   60   S1-S4   2012

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  • Molecular Genetic and Functional Characterization Implicate Muscle-Restricted Coiled-Coil Gene (MURC) as a Causal Gene for Familial Dilated Cardiomyopathy

    Gabriela Rodriguez, Tomomi Ueyama, Takehiro Ogata, Grazyna Czernuszewicz, Yanli Tan, Gerald W. Dorn, Roberta Bogaev, Katsuya Amano, Hidemasa Oh, Hiroaki Matsubara, James T. Willerson, Ali J. Marian

    CIRCULATION-CARDIOVASCULAR GENETICS   4 ( 4 )   349 - U47   2011.8

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    Background-Dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM) are classic forms of systolic and diastolic heart failure, respectively. Mutations in genes encoding sarcomere and cytoskeletal proteins are major causes of HCM and DCM. MURC, encoding muscle-restricted coiled-coil, a Z-line protein, regulates cardiac function in mice. We investigated potential causal role of MURC in human cardiomyopathies.
    Methods and Results-We sequenced MURC in 1199 individuals, including 383 probands with DCM, 307 with HCM, and 509 healthy control subjects. We found 6 heterozygous DCM-specific missense variants (p.N128K, p.R140W, p.L153P, p.S307T, p.P324L, and p.S364L) in 8 unrelated probands. Variants p.N128K and p. S307T segregated with inheritance of DCM in small families (chi(2) = 8.5, P=0.003). Variants p.N128K, p.R140W, p.L153P, and p.S364L were considered probably or possibly damaging. Variant p.P324L recurred in 3 independent probands, including 1 proband with a TPM1 mutation (p.M245T). A deletion variant (p.L232-R238del) was present in 3 unrelated HCM probands, but it did not segregate with HCM in a family who also had a MYH7 mutation (p.L907V). The phenotype in mutation carriers was notable for progressive heart failure leading to heart transplantation in 4 patients, conduction defects, and atrial arrhythmias. Expression of mutant MURC proteins in neonatal rat cardiac myocytes transduced with recombinant adenoviruses was associated with reduced RhoA activity, lower mRNA levels of hypertrophic markers and smaller myocyte size as compared with wild-type MURC.
    Conclusions-MURC mutations impart loss-of-function effects on MURC functions and probably are causal variants in human DCM. The causal role of a deletion mutation in HCM is uncertain. (Circ Cardiovasc Genet. 2011;4:349-358.)

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  • Molecular Genetic and Functional Characterization Implicate Muscle-Restricted Coiled-Coil Gene (MURC) as a Causal Gene for Familial Dilated Cardiomyopathy

    Gabriela Rodriguez, Tomomi Ueyama, Takehiro Ogata, Grazyna Czernuszewicz, Yanli Tan, Gerald W. Dorn, Roberta Bogaev, Katsuya Amano, Hidemasa Oh, Hiroaki Matsubara, James T. Willerson, Ali J. Marian

    CIRCULATION-CARDIOVASCULAR GENETICS   4 ( 4 )   349 - U47   2011.8

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    Language:English   Publisher:LIPPINCOTT WILLIAMS & WILKINS  

    Background-Dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM) are classic forms of systolic and diastolic heart failure, respectively. Mutations in genes encoding sarcomere and cytoskeletal proteins are major causes of HCM and DCM. MURC, encoding muscle-restricted coiled-coil, a Z-line protein, regulates cardiac function in mice. We investigated potential causal role of MURC in human cardiomyopathies.
    Methods and Results-We sequenced MURC in 1199 individuals, including 383 probands with DCM, 307 with HCM, and 509 healthy control subjects. We found 6 heterozygous DCM-specific missense variants (p.N128K, p.R140W, p.L153P, p.S307T, p.P324L, and p.S364L) in 8 unrelated probands. Variants p.N128K and p. S307T segregated with inheritance of DCM in small families (chi(2) = 8.5, P=0.003). Variants p.N128K, p.R140W, p.L153P, and p.S364L were considered probably or possibly damaging. Variant p.P324L recurred in 3 independent probands, including 1 proband with a TPM1 mutation (p.M245T). A deletion variant (p.L232-R238del) was present in 3 unrelated HCM probands, but it did not segregate with HCM in a family who also had a MYH7 mutation (p.L907V). The phenotype in mutation carriers was notable for progressive heart failure leading to heart transplantation in 4 patients, conduction defects, and atrial arrhythmias. Expression of mutant MURC proteins in neonatal rat cardiac myocytes transduced with recombinant adenoviruses was associated with reduced RhoA activity, lower mRNA levels of hypertrophic markers and smaller myocyte size as compared with wild-type MURC.
    Conclusions-MURC mutations impart loss-of-function effects on MURC functions and probably are causal variants in human DCM. The causal role of a deletion mutation in HCM is uncertain. (Circ Cardiovasc Genet. 2011;4:349-358.)

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  • 新生児Ebstein奇形のManagement

    藤井泰宏, 新井禎彦, 石神修太, 樽井俊, 櫻井茂, 鵜垣伸也, 川畑拓也, 立石篤史, 藤田康文, 高垣昌巳, 平田昌敬, 笠原真悟, 三井秀也, 王英正, 佐野俊二

    日本心臓血管外科学会雑誌   40 ( Supplement )   199   2011.1

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  • 小児心不全への細胞治療

    樽井 俊, 佐野俊二, 王 英正

    Medical Science Digest   37   439 - 442   2011

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  • 小児心不全に対する心筋再生療法.

    樽井 俊, 佐野俊二, 王 英正

    Medical Science Digest   37 ( 11 )   439 - 442   2011

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  • PARM-1 Is an Endoplasmic Reticulum Molecule Involved in Endoplasmic Reticulum Stress-Induced Apoptosis in Rat Cardiac Myocytes

    Koji Isodono, Tomosaburo Takahashi, Hiroko Imoto, Naohiko Nakanishi, Takehiro Ogata, Satoshi Asada, Atsuo Adachi, Tomomi Ueyama, Hidemasa Oh, Hiroaki Matsubara

    PLOS ONE   5 ( 3 )   e9746-e9746   2010.3

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    To identify novel transmembrane and secretory molecules expressed in cardiac myocytes, signal sequence trap screening was performed in rat neonatal cardiac myocytes. One of the molecules identified was a transmembrane protein, prostatic androgen repressed message-1 (PARM-1). While PARM-1 has been identified as a gene induced in prostate in response to castration, its function is largely unknown. Our expression analysis revealed that PARM-1 was specifically expressed in hearts and skeletal muscles, and in the heart, cardiac myocytes, but not non-myocytes expressed PARM-1. Immunofluorescent staining showed that PARM-1 was predominantly localized in endoplasmic reticulum (ER). In Dahl salt-sensitive rats, high-salt diet resulted in hypertension, cardiac hypertrophy and subsequent heart failure, and significantly stimulated PARM-1 expression in the hearts, with a concomitant increase in ER stress markers such as GRP78 and CHOP. In cultured cardiac myocytes, PARM-1 expression was stimulated by proinflammatory cytokines, but not by hypertrophic stimuli. A marked increase in PARM-1 expression was observed in response to ER stress inducers such as thapsigargin and tunicamycin, which also induced apoptotic cell death. Silencing PARM-1 expression by siRNAs enhanced apoptotic response in cardiac myocytes to ER stresses. PARM-1 silencing also repressed expression of PERK and ATF6, and augmented expression of CHOP without affecting IRE-1 expression and JNK and Caspase-12 activation. Thus, PARM-1 expression is induced by ER stress, which plays a protective role in cardiac myocytes through regulating PERK, ATF6 and CHOP expression. These results suggested that PARM-1 is a novel ER transmembrane molecule involved in cardiac remodeling in hypertensive heart disease.

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  • Stem Cell Engineering for Cardiac Tissue Regeneration

    Masashi Yoshida, Hidemasa Oh

    CARDIOLOGY   115 ( 3 )   191 - 193   2010

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    DOI: 10.1159/000281839

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  • Stem Cell Engineering for Cardiac Tissue Regeneration

    Masashi Yoshida, Hidemasa Oh

    CARDIOLOGY   115 ( 3 )   191 - 193   2010

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    DOI: 10.1159/000281839

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  • Deficiency of Nectin-2 Leads to Cardiac Fibrosis and Dysfunction Under Chronic Pressure Overload

    Seimi Satomi-Kobayashi, Tomomi Ueyama, Steffen Mueller, Ryuji Toh, Tomoya Masano, Tsuyoshi Sakoda, Yoshiyuki Rikitake, Jun Miyoshi, Hiroaki Matsubara, Hidemasa Oh, Seinosuke Kawashima, Ken-ichi Hirata, Yoshimi Takai

    HYPERTENSION   54 ( 4 )   825 - U268   2009.10

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    The intercalated disc, a cell-cell contact site between neighboring cardiac myocytes, plays an important role in maintaining the homeostasis of the heart by transmitting electric and mechanical signals. Changes in the architecture of the intercalated disc have been observed in dilated cardiomyopathy. Among cell-cell junctions in the intercalated disc, adherens junctions are involved in anchoring myofibrils and transmitting force. Nectins are Ca(2+)-independent, immunoglobulin-like cell-cell adhesion molecules that exist in adherens junctions. However, the role of nectins in cardiac homeostasis and integrity of the intercalated disc are unknown. Among the isoforms of nectins, nectin-2 and -4 were expressed at the intercalated disc in the heart. Nectin-2-knockout mice showed normal cardiac structure and function under physiological conditions. Four weeks after banding of the ascending aorta, cardiac function was significantly impaired in nectin-2-knockout mice compared with wild-type mice, although both nectin-2-knockout and wild-type mice developed similar degrees of cardiac hypertrophy. Banded nectin-2-knockout mice displayed cardiac fibrosis more evidently than banded wild-type mice. The disruption of the intercalated discs and disorganized myofibrils were observed in banded nectin-2-knockout mice. Furthermore, the number of apoptotic cardiac myocytes was increased in banded nectin-2-knockout mice. In the hearts of banded nectin-2-knockout mice, Akt remained at lower phosphorylation levels until 2 weeks after banding, whereas c-Jun N-terminal kinase and p38 mitogen-activated protein kinase were highly phosphorylated compared with those of wild-type mice. These results indicate that nectin-2 is required to maintain structure and function of the intercalated disc and protects the heart from pressure-overload induced cardiac dysfunction. (Hypertension. 2009; 54: 825-831.)

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  • Cardiosphereを用いた心筋再生医療

    中西直彦, 竹原有史, 中岡幹彦, 赤壁佳樹, 王 英正, 松原弘明

    総合臨床   2009

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  • 心筋幹細胞

    王 英正

    循環器科 特集   2009

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  • 重症心不全に対する細胞治療法

    竹原有史, 塘 義明, 天野克也, 王 英正, 長谷部直幸, 松原弘明

    循環器科 特集   2009

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  • Cardiosphereを用いた心筋再生医療

    中岡幹彦, 竹原有史, 中西直彦, 赤壁佳樹, 王 英正, 松原弘明

    再生医療   2009

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  • 自己心臓幹細胞を用いた心不全への細胞治療

    岸田 聡, 竹原有史, 王 英正, 松原弘明

    重症心不全の予防と治療   2009

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  • 新しい心臓再生法

    王 英正

    Medical View Point   2009

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  • Cardiosphereによる心筋再生

    竹原有史, 服部玲治, 松原弘明, 王 英正

    Annual Review 循環器   2009

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  • Cardiosphereによる心筋再生医療

    山口真一郎, 竹原有史, 天野克也, 高橋知三郎, 王 英正, 松原弘明

    分子心血管病 特集   2009

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  • ヒト心臓由来幹細胞を用いた心不全への再生医療

    小出正洋, 星野 温, 山口真一郎, 岸田 聡, 竹原有史, 王 英正, 松原弘明

    再生誘導治療   2009

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  • Downregulation of Dicer expression by serum withdrawal sensitizes human endothelial cells to apoptosis

    Satoshi Asada, Tomosaburo Takahashi, Koji Isodono, Atsuo Adachi, Hiroko Imoto, Takehiro Ogata, Tomomi Ueyama, Hiroaki Matsubara, Hidemasa Oh

    AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY   295 ( 6 )   H2512 - H2521   2008.12

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    Downregulation of Dicer expression by serum withdrawal sensitizes human endothelial cells to apoptosis. Am J Physiol Heart Circ Physiol 295: H2512-H2521, 2008. First published October 31, 2008; doi:10.1152/ajpheart.00233.2008.-Although the modulated expression of Dicer is documented upon neoplastic transformation, little is known of the regulation of Dicer expression by environmental stimuli and its roles in the regulation of cellular functions in primary cells. In this study, we found that Dicer expression was downregulated upon serum withdrawal in human umbilical vein endothelial cells (HUVECs). Serum withdrawal induced a time-dependent repression of Dicer expression, which was specifically rescued by vascular endothelial cell growth factor or sphingosine-1-phosphate. When Dicer expression was silenced by short-hairpin RNA against Dicer, the cells were more prone to apoptosis under serum withdrawal, whereas the rate of apoptosis was comparable with control cells in the serum-containing condition. Real-time PCR-based gene expression profiling identified several genes, the expression of which was modulated by Dicer silencing, including adhesion and matrix-related molecules, caspase-3, and nitric oxide synthase 3 (NOS3). Dicer silencing markedly impaired migratory functions without affecting cell adhesion and repressed phosphorylation of focal adhesion kinase and proline-rich tyrosine kinase 2 in adherent HUVECs. Dicer knockdown upregulated caspase-3 and downregulated NOS3 expression, and serum withdrawal indeed increased caspase-3 and decreased NOS3 expression. Furthermore, the overexpression of Dicer in HUVECs resulted in a marked reduction in apoptosis upon serum withdrawal and a decreased caspase-3 and increased NOS3 expression. The inhibition of NOS activity by N(omega)-nitro-L-arginine methyl ester abrogated the effect of Dicer overexpression to rescue the cells from serum withdrawal-induced apoptosis. These results indicated that serum withdrawal decreases Dicer expression, leading to an increased susceptibility to apoptosis through the regulation of caspase-3 and NOS3 expression.

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  • Stemming heart failure with cardiac- or reprogrammed-stem cells

    Kento Tateishi, Naofumi Takehara, Hiroaki Matsubara, Hidemasa Oh

    JOURNAL OF CELLULAR AND MOLECULAR MEDICINE   12 ( 6A )   2217 - 2232   2008.12

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    Despite extensive efforts to control myocyte growth by genetic targeting of the cell cycle machinery and small molecules for cardiac repair, adult myocytes themselves appeared to divide a limited number of times in response to a variety of cardiac muscle stresses. Rare tissue-resident stem cells are thought to exist in many adult organs that are capable of self-renewal and differentiation and possess a range of actions that are potentially therapeutic. Recent studies suggest that a population of cardiac stem cells (CSCs) is maintained after cardiac development in the adult heart in mammals including human beings; however, homeostatic cardiomyocyte replacement might be stem cell-dependent, and functional myocardial regeneration after cardiac muscle damage is not yet considered as sufficient to fully maintain or reconstitute the cardiovascular system and function. Although it is clear that adult CSCs have limitations in their capabilities to proliferate extensively and differentiate in response to injury in vivo for replenishing mature car-diomyocytes and potentially function as resident stem cells. Transplantation of CSCs expanded ex vivo seems to require an integrated strategy of cell growth-enhancing factor(s) and tissue engineering technologies to support the donor cell survival and subsequent proliferation and differentiation in the host microenvironment. There has been substantial interest regarding the evidence that mammalian fibroblasts can be genetically reprogrammed to induced pluripotent stem (iPS) cells, which closely resemble embryonic stem (ES) cell properties capable of differentiating into functional cardiomyocytes, and these cells may provide an alternative cell source for generating patient-specific CSCs for therapeutic applications.

    DOI: 10.1111/j.1582-4934.2008.00487.x

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  • Controlled Delivery of Basic Fibroblast Growth Factor Promotes Human Cardiosphere-Derived Cell Engraftment to Enhance Cardiac Repair for Chronic Myocardial Infarction

    Naofumi Takehara, Yoshiaki Tsutsumi, Kento Tateishi, Takehiro Ogata, Hideo Tanaka, Tomomi Ueyama, Tomosaburo Takahashi, Tetsuro Takamatsu, Masanori Fukushima, Masashi Komeda, Masaaki Yamagishi, Hitoshi Yaku, Yasuhiko Tabata, Hiroaki Matsubara, Hidemasa Oh

    JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY   52 ( 23 )   1858 - 1865   2008.12

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    Objectives This study was designed to determine whether controlled release of basic fibroblast growth factor ( bFGF) might improve human cardiosphere-derived cell (hCDC) therapy in a pig model of chronic myocardial infarction.
    Background Current cell therapies for cardiac repair are limited by loss of the transplanted cells and poor differentiation.
    Methods We conducted 2 randomized, placebo-controlled studies in immunosuppressed pigs with anterior myocardial infarctions. Four weeks after coronary reperfusion, 14 pigs were randomly assigned to receive an intramyocardial injection of placebo medium with or without bFGF-incorporating hydrogel implantation. As a second study, 26 pigs were randomized to receive controlled release of bFGF combined with or without hCDCs or bone marrow-derived mesenchymal stem cell transplantation 4 weeks after reperfusion.
    Results Controlled release of bFGF in ischemic myocardium significantly augmented the formation of microvascular networks to enhance myocardial perfusion and contractile function. When combined with cell transplantation, the additive effects of bFGF were confined to hCDC-injected animals, but were not observed in animals receiving human bone marrow-derived mesenchymal stem cell transplantation. This was shown by increased donor-cell engraftment and enhanced cardiomyocyte differentiation in the transplanted hearts, resulting in synergistically improved ventricular function and regional wall motion and reduced infarct size.
    Conclusions Controlled delivery of bFGF modulates the post-ischemic microenvironment to enhance hCDC engraftment and differentiation. This novel strategy demonstrates significant functional improvements after myocardial infarction and may potentially represent a therapeutic approach to be studied in a clinical trial in human heart failure. (J Am Coll Cardiol 2008; 52: 1858-65) (c) 2008 by the American College of Cardiology Foundation

    DOI: 10.1016/j.jacc.2008.06.052

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  • Crossveinless-2 controls bone morphogenetic protein signaling during early cardiomyocyte differentiation in P19 cells

    Koichiro Harada, Akiko Ogai, Tomosaburo Takahashi, Masafumi Kitakaze, Hiroaki Matsubara, Hidemasa Oh

    JOURNAL OF BIOLOGICAL CHEMISTRY   283 ( 39 )   26705 - 26713   2008.9

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    Increasing evidence indicates that bone morphogenetic proteins (BMPs) are crucial for cardiac induction, specification, and development. Although signaling of BMPs is tightly regulated through soluble BMP-binding proteins, how they regulate BMP signaling during cardiac differentiation remains unknown. To identify molecules responsible for BMP signaling during early cardiomyocyte differentiation of P19 cells, cDNA subtraction was performed. We found a bimodal expression of the BMP-binding protein Crossveinless-2 (Cv2) during cardiomyocyte differentiation; Cv2 is temporally expressed earlier than cardiac transcription factors such as Nkx2.5 and Tbx5 and acts as a suppressor for BMP signaling in P19 cells. We established a P19 clonal cell line harboring a cardiac alpha-myosin heavy chain promoter-driven enhanced green fluorescent protein gene to monitor cardiac differentiation by flow cytometry. Treatment with BMP2 during the first 2 days of differentiation suppressed cardiomyocyte differentiation through activation of downstream targets Smad1/5/8 protein and Id1 gene, whereas treatment with Cv2 conversely inhibited Smad1/5/8 activation and Id1 expression, leading to increased generation of cardiac cells. RNA interference-mediated knockdown (KD) of endogenous Cv2 showed increased Smad1/5/8 activation and impaired cardiomyocyte differentiation. Expression of cardiac mesoderm markers was reduced, whereas expression of Id1 and endoderm markers such as Sox7, Hnf4, and E-cadherin was induced in Cv2-kinase dead cells. These phenotypes were rescued by the addition of Cv2 protein to the culture media during the first 2 days of differentiation or co-culture with parental cells. These data suggest that Cv2 may specify cardiac mesodermal lineage through inhibition of BMP signaling at early stage of cardiogenesis.

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  • MURC, a muscle-restricted coiled-coil protein, is involved in the regulation of skeletal myogenesis

    Masashi Tagawa, Tomomi Ueyama, Takehiro Ogata, Naofumi Takehara, Norio Nakajima, Koji Isodono, Satoshi Asada, Tomosaburo Takahashi, Hiroaki Matsubara, Hidemasa Oh

    AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY   295 ( 2 )   C490 - C498   2008.8

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    Skeletal myogenesis is a multistep process by which multinucleated mature muscle fibers are formed from undifferentiated, mononucleated myoblasts. However, the molecular mechanisms of skeletal myogenesis have not been fully elucidated. Here, we identified muscle-restricted coiled-coil (MURC) protein as a positive regulator of myogenesis. In skeletal muscle, MURC was localized to the cytoplasm with accumulation in the Z-disc of the sarcomere. In C2C12 myoblasts, MURC expression occurred coincidentally with myogenin expression and preceded sarcomeric myosin expression during differentiation into myotubes. RNA interference (RNAi)-mediated knockdown of MURC impaired differentiation in C2C12 myoblasts, which was accompanied by impaired myogenin expression and ERK activation. Overexpression of MURC in C2C12 myoblasts resulted in the promotion of differentiation with enhanced myogenin expression and ERK activation during differentiation. During injury-induced muscle regeneration, MURC expression increased, and a higher abundance of MURC was observed in immature myofibers compared with mature myofibers. In addition, ERK was activated in regenerating tissue, and ERK activation was detected in MURC-expressing immature myofibers. These findings suggest that MURC is involved in the skeletal myogenesis that results from modulation of myogenin expression and ERK activation. MURC may play pivotal roles in the molecular mechanisms of skeletal myogenic differentiation.

    DOI: 10.1152/ajpcell.00188.2008

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  • MURC, a muscle-restricted coiled-coil protein that modulates the Rho/ROCK pathway, induces cardiac dysfunction and conduction disturbance

    Takehiro Ogata, Tomomi Ueyama, Koji Isodono, Masashi Tagawa, Naofumi Takehara, Tsuneaki Kawashima, Koichiro Harada, Tomosaburo Takahashi, Tetsuo Shioi, Hiroaki Matsubara, Hidemasa Oh

    MOLECULAR AND CELLULAR BIOLOGY   28 ( 10 )   3424 - 3436   2008.5

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    We identified a novel muscle-restricted putative coiled-coil protein, MURC, which is evolutionarily conserved from frog to human. MURC was localized to the cytoplasm with accumulation in the Z-line of the sarcomere in the murine adult heart. MURC mRNA expression in the heart increased during the developmental process from the embryonic stage to adulthood. In response to pressure overload, MURC mRNA expression increased in the hypertrophied heart. Using the yeast two-hybrid system, we identified the serum deprivation response (SDPR) protein, a phosphatidylserine-binding protein, as a MURC-binding protein. MURC induced activation of the RhoA/ROCK pathway, which modulated serum response factor-mediated atrial natriuretic peptide (ANP) expression and myofibrillar organization. SDPR augmented MURC-induced transactivation of the ANP promoter in cardiomyocytes, and RNA interference of SDPR attenuated the action of MURC on the ANP promoter. Transgenic mice expressing cardiac-specific MURC (Tg-MURC) exhibited cardiac contractile dysfunction and atrioventricular (AV) conduction disturbances with atrial chamber enlargement, reduced thickness of the ventricular wall, and interstitial fibrosis. Spontaneous episodes of atrial fibrillation and AV block were observed in Tg-MURC mice. These findings indicate that MURC modulates RhoA signaling and that MURC plays an important role in the development of cardiac dysfunction and conduction disturbance with increased vulnerability to atrial arrhythmias.

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  • Regeneration therapy for cardiac repair by autologous human heart-derived stem cells from biopsy samples

    Hiroaki Matsubara, Kento Tateishi, Hidemasa Oh

    JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY   44 ( 2 )   435 - 435   2008.2

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    Language:English   Publishing type:Research paper, summary (international conference)   Publisher:ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD  

    DOI: 10.1016/j.yjmcc.2007.07.005

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  • Skeletal muscle-derived progenitors capable of differentiating into cardiomyocytes proliferate through myostatin-independent TGF-beta family signaling

    Tetsuya Nomura, Tomomi Ueyama, Eishi Ashihara, Kento Tateishi, Satoshi Asada, Norio Nakajima, Koji Isodono, Tomosaburo Takahashi, Hiroaki Matsubara, Hidemasa Oh

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   365 ( 4 )   863 - 869   2008.1

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    The existence of skeletal muscle-derived stem cells (MDSCs) has been suggested in mammals; however, the signaling pathways controlling MDSC proliferation remain largely unknown. Here we report the isolation of myosphere-derived progenitor cells (MDPCs) that can give rise to beating cardiomyocytes from adult skeletal muscle. We identified that follistatin, an antagonist of TGF-beta family members, was predominantly expressed in MDPCs, whereas myostatin was mainly expressed in myogenic cells and mature skeletal muscle. Although follistatin enhanced the replicative growth of MDPCs through Smad2/3 inactivation and cell cycle progression, disruption of myostatin did not increase the MDPC proliferation. By contrast, inhibition of activin A (ActA) or growth differentiation factor 11 (GDF11) signaling dramatically increased MDPC proliferation via down-regulation of p21 and increases in the levels of cdk2/4 and cyclin D1. Thus, follistatin may be an effective progenitor-enhancing agent neutralizing ActA and GDF11 signaling to regulate the growth of MDPCs in skeletal muscle. (C) 2007 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.bbrc.2007.11.087

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  • 心臓内幹細胞の特性と自己複製制御機構

    王 英正, 立石健人, 松原弘明

    実験医学 増刊号   2008

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  • 心不全への再生医療

    足立淳郎, 井本裕子, 山口真一郎, 竹原有史, 王 英正, 松原弘明

    心不全診療マニュアル   2008

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  • 心臓組織内幹細胞を用いた心不全への心筋再生医療の現状

    王 英正

    心電図   2008

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  • Therapeutic potential of stem/progenitor cells in human skeletal muscle for cardiovascular regeneration

    Tetsuya Nomura, Eishi Ashihara, Kento Tateishi, Tomomi Ueyama, Tomosaburo Takahashi, Masaaki Yamagishi, Toshikazu Kubo, Hitoshi Yaku, Hiroaki Matsubara, Hidemasa Oh

    Current Stem Cell Research and Therapy   2 ( 4 )   293 - 300   2007.12

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    Although myoblast transplantation in patients with ischemic heart failure results in a significant improvement of cardiac function, subsequent studies have consistently shown the myotubes formation in the absence of electromechanical coupling with the neighboring host myocardium, accompanied with the short-term release of paracrine effectors from implanted cells. One major pitfall of using myoblasts is that transplanted cells do not differentiate into cardiomyocytes, which may cause the inherent proarrhythmogenic events. Therefore, whether a discrete subpopulation in heterogeneous muscle-cell cultures is responsible for substantial cardiovascular regeneration has yet to be investigated. We describe here the isolation of progenitor cells from human skeletal muscle. These cells proliferated as non-adherent myospheres in suspension and displayed early embryonic factors and mesenchymal cell-like characteristics. Flow cytometric analyses demonstrated that CD56/N-CAM/Leu-19, a neural cell adhesion molecule abundantly present in myoblasts, was absent in myospheres but was expressed in an adherent cell population containing myogenic precursors. Myosphere-derived progenitor cells (MDPCs) differentiated in culture to produce cardiac, smooth muscle, and endothelial cells. Transplantation of MDPCs into ischemic hearts in NOD/scid mice promoted angiogenesis with substantial cardiovascular regeneration. Our results provide a foundation to further study the cell and biological function of human MDPCs which may have potential therapeutic implications. © 2007 Bentham Science Publishers Ltd.

    DOI: 10.2174/157488807782793808

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  • Stage-specific role of endogenous Smad2 activation in cardiomyogenesis of embryonic stem cells

    Ryoji Kitamura, Tomosaburo Takahashi, Norio Nakajima, Koji Isodono, Satoshi Asada, Hikaru Ueno, Tomomi Ueyama, Toshikazu Yoshikawa, Hiroaki Matsubara, Hidemasa Oh

    CIRCULATION RESEARCH   101 ( 1 )   78 - 87   2007.7

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    The role of Smads and their specific ligands during cardiomyogenesis in ES cells was examined. Smad2 was activated bimodally in the early and late phases of cardiac differentiation, whereas Smad1 was activated after the middle phase. Nodal and Cripto were expressed in the early stage and then downregulated, whereas transforming growth factor-beta and activin were expressed only in the late phase. Suppression of early Smad2 activation by SB- 431542 produced complete inhibition of endodermal and mesodermal induction but augmented neuroectodermal differentiation, followed by poor cardiomyogenesis, whereas inhibition during the late phase alone promoted cardiomyogenesis. Inhibitory effect of Smad2 on cardiomyogenesis in the late phase was mainly mediated by transforming growth factor-beta, and inhibition of transforming growth factor-beta- mediated Smad2 activation resulted in a greater replicative potential in differentiated cardiac myocytes and enhanced differentiation of nonmyocytes into cardiac myocytes. Thus, endogenous Smad2 activation is indispensable for endodermal and mesodermal induction in the early phase. In the late phase, endogenous transforming growth factor-beta negatively regulates cardiomyogenesis through Smad2 activation by modulating proliferation and differentiation of cardiac myocytes.

    DOI: 10.1161/CIRCRESAHA.106.147264

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  • Osteopontin is a myosphere-derived secretory molecule that promotes angiogenic progenitor cell proliferation through the phosphoinositide 3-kinase/Akt pathway

    Takehiro Ogata, Tomomi Ueyama, Tetsuya Nomura, Satoshi Asada, Masashi Tagawa, Tomoyuki Nakamura, Tomosaburo Takahashi, Hiroaki Matsubara, Hidemasa Oh

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   359 ( 2 )   341 - 347   2007.7

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    Language:English   Publisher:ACADEMIC PRESS INC ELSEVIER SCIENCE  

    We have reported that skeletal myosphere-derived progenitor cells (MDPCs) can differentiate into vascular cells, and that MDPC transplantation into cardiomyopathic hearts improves cardiac function. However, the autocrine/paracrine molecules and underlying mechanisms responsible for MDPC growth have not yet been determined. To explore the molecules enhancing the proliferation of MDPCs, we performed serial analysis of gene expression and signal sequence trap methods using RNA isolated from MDPCs. We identified osteopontin (OPN), a secretory molecule, as one of most abundant molecules expressed in MDPCs. OPN provided a proliferative effect for MDPCs. MDPCs treated with OPN showed Akt activation, and inhibition of the phosphoinositide 3-kinase (PI3K)/Akt pathway repressed the proliferative effect of OPN. Furthermore, OPN-pretreated MDPCs maintained their differentiation potential into endothelial and vascular smooth muscle cells. These findings indicate an important role of OPN as an autocrine/paracrine molecule in regulating the proliferative growth of muscle-derived angiogenic progenitor cells via the PI3K/Akt pathway. (c) 2007 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.bbrc.2007.05.104

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  • Clonally amplified cardiac stem cells are regulated by Sca-1 signaling for efficient cardiovascular regeneration

    Kento Tateishi, Eishi Ashihara, Naofumi Takehara, Tetsuya Nomura, Shoken Honsho, Takuo Nakagami, Shigehiro Morikawa, Tomosaburo Takahashi, Tomomi Ueyama, Hiroaki Matsubara, Hidemasa Oh

    JOURNAL OF CELL SCIENCE   120 ( 10 )   1791 - 1800   2007.5

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    Recent studies have shown that cardiac stem cells (CSCs) from the adult mammalian heart can give rise to functional cardiomyocytes; however, the definite surface markers to identify a definitive single entity of CSCs and the molecular mechanisms regulating their growth are so far unknown. Here, we demonstrate a single-cell deposition analysis to isolate individually selected CSCs from adult murine hearts and investigate the signals required for their proliferation and survival. Clonally proliferated CSCs express stem cell antigen-1 (Sca-1) with embryonic stem (ES) cell-like and mesenchymal cell-like characteristics and are associated with telomerase reverse transcriptase (TERT). Using a transgene that expresses a GFP reporter under the control of the TERT promoter, we demonstrated that TERTGFP-positive fractions from the heart were enriched for cells expressing Sca-1. Knockdown of Sca-1 transcripts in CSCs led to retarded ex vivo expansion and apoptosis through Akt inactivation. We also show that ongoing CSC proliferation and survival after direct cellgrafting into ischemic myocardium require Sca-1 to upregulate the secreted paracrine effectors that augment neoangiogenesis and limit cardiac apoptosis. Thus, Sca-1 might be an essential component to promote CSC proliferation and survival to directly facilitate early engraftment, and might indirectly exert the effects on late cardiovascular differentiation after CSC transplantation.

    DOI: 10.1242/jcs.006122

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  • Skeletal myosphere-derived progenitor cell transplantation promotes neovascularization in delta-sarcoglycan knockdown cardiomyopathy

    Tetsuya Nomura, Eishi Ashihara, Kento Tateishi, Satoshi Asada, Tomomi Ueyama, Tomosaburo Takahashi, Hiroaki Matsubara, Hidemasa Oh

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   352 ( 3 )   668 - 674   2007.1

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    Language:English   Publisher:ACADEMIC PRESS INC ELSEVIER SCIENCE  

    Bone marrow cells have been shown to contribute to neovascularization in ischemic hearts, whereas their impaired maturation to restore the delta-sarcoglycan (delta-SG) expression responsible for focal myocardial degeneration limits their utility to treat the pathogenesis of cardiomyopathy. Here, we report the isolation of multipotent progenitor cells from adult skeletal muscle, based on their ability to generate floating-myospheres. Myosphere-derived progenitor cells (MDPCs) are distinguishable from myogenic C2C12 cells and differentiate into vascular smooth muscle cells and mesenchymal progeny. The mutation in the delta-SG has been shown to develop vascular spasm to affect sarcolemma structure causing cardiomyopathy. We originally generated delta-SD knockdown (KD) mice and transplanted MDPCs into the hearts. MDPCs enhanced neoangiogenesis and restored delta-SG expression in impaired vasculatures through trans-differentiation, leading to improvement of cardiac function associated with paracrine effectors secretion. We propose that MDPCs may be the promising progenitor cells in skeletal muscle to treat delta-sarcoglycan complex mutant cardiomyopathy. (c) 2006 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.bbrc.2006.11.097

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  • Human cardiac stem cells exhibit mesenchymal features and are maintained through Akt/GSK-3 beta signaling

    Kento Tateishi, Eishi Ashihara, Shoken Honsho, Naofumi Takehara, Tetsuya Nomura, Tomosaburo Takahashi, Tomomi Ueyama, Masaaki Yamagishi, Hitoshi Yaku, Hiroaki Matsubara, Hidemasa Oh

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   352 ( 3 )   635 - 641   2007.1

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    Recent evidence suggested that human cardiac stem cells (hCSCs) may have the clinical application for cardiac repair; however, their characteristics and the regulatory mechanisms of their growth have not been fully investigated. Here, we show the novel property of hCSCs with respect to their origin and tissue distribution in human heart, and demonstrate the signaling pathway that regulates their growth and survival. Telomerase-active hCSCs were predominantly present in the right atrium and outflow tract of the heart (infant &gt; adult) and had a mesenchymal cell-like phenotype. These hCSCs expressed the embryonic stem cell markers and differentiated into cardio-myocytes to support cardiac function when transplanted them into ischemic myocardium. Inhibition of Akt pathway impaired the hCSC proliferation and induced apoptosis, whereas inhibition of glycogen synthase kinase-3 (GSK-3) enhanced their growth and survival. We conclude that hCSCs exhibit mesenchymal features and that Akt/GSK-3 beta may be crucial modulators for hCSC maintenance in human heart. (c) 2006 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.bbrc.2006.11.096

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  • 心筋細胞の再生医療

    王 英正

    メディカルビューポイント   2007

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  • Isl-1前駆細胞

    王 英正

    Annual Review 循環器   2007

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  • 心臓幹細胞移植による臨床応用の可能性

    王 英正

    呼吸と循環   2007

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  • ヒト心筋組織幹細胞研究の現状

    王 英正

    分子血管病   2007

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  • Single cardiac stem cells exhibit mesenchymal features and require stem cell antigen-1 to proliferate in adult myocardium

    Kento Tateishi, Tomosaburo Takahashi, Tetsuya Nomura, Hiroaki Matsubara, Hidemasa Oh

    CIRCULATION RESEARCH   99 ( 5 )   E20 - E20   2006.9

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    Language:English   Publishing type:Research paper, summary (international conference)   Publisher:LIPPINCOTT WILLIAMS & WILKINS  

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  • 心筋再生 : 骨髄由来幹細胞による心筋細胞への可塑性について

    芦原 英司, 王 英正, 松原 弘明

    移植   39 ( 5 )   499 - 508   2004.10

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Presentations

  • Cardiosphere-derived cells in congenital heart disease Invited

    Hidemasa Oh

    日本循環器学会総会  2020.8.2 

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  • 小児心不全に対する再生医療のupdate Invited

    王 英正

    日本再生医療学会  2020.5.18 

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    Language:Japanese   Presentation type:Oral presentation (invited, special)  

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  • 小児心不全に対する再生医療 Invited

    王 英正

    日本心臓病学会学術集会  2019.9.13 

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    Language:Japanese   Presentation type:Symposium, workshop panel (nominated)  

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  • Clinical Translation of Cell Therapy for Congenital Heart Disease Invited

    Hidemasa Oh

    2019.7.22 

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  • Cell therapy trials in congenital heart disease

    日本遺伝子細胞治療学会  2018 

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  • Cell Therapy Trials in Congenital Heart Disease

    Pediatric Heart Disease Symposium  2017 

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  • 心筋組織幹細胞を用いた心筋再生の現状と展望

    CVIT  2017 

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  • Cell therapy trials in congenital heart disease

    日本遺伝子細胞治療学会  2017 

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  • 先駆け審査制度での成功事例 自家心臓内幹細胞/小児心不全に対する心筋再生医療法の企業主導多施設共同臨床治験

    AMED シンポジウム 革新的医療技術創出プロジェクト  2017 

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  • カテーテルによる心筋再生医療について

    日本IVR学会総会  2017 

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  • Cardiac Progenitor Cell Therapy in Congenital Heart Diseases

    第80回日本循環器学会学術集会  2016 

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  • Cardiac progenitor cell therapy in congenital heart disease

    American Heart Association  2016 

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  • 先天性心疾患における心筋再生医療の実用化研究

    第119回小児科学会シンポジウム  2016 

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  • Cardiac progenitor cell therapy for congenital heart diseases

    3rd Munich Conference on Cardiac Development  2016 

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  • 心筋再生医療の現状と展望

    Cardiovascular Update 岡山  2015 

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  • 疾患特異的iPS細胞による新たな先天性心疾患モデルの作成

    小児循環器学会  2015 

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  • 小児心不全に対する心筋再生医療法の企業主導多施設共同臨床治験

    橋渡し研究シンポジウム  2015 

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  • 心不全に対する細胞治療法の実用化と医工学融合した次世代心筋再生医療法の開発

    岡山先端医学研究会  2015 

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  • 小児心不全に対する心臓内自己幹細胞移植治療の標準医療化に向けた取り組み

    小児循環器学会  2015 

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  • 心臓内幹細胞を用いた先天性心疾患の病態解明と再生医療

    ミニリトリート 徳島  2015 

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  • 子どもの心臓病に再生医療は有効か?

    岡山大学公開講座  2015 

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  • 心臓内幹細胞を用いた小児心疾患の解明と再生医療

    第34回日本小児病理研究会学術集会  2014 

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  • 心臓内幹細胞を用いた小児心疾患の解明と再生医療

    高知小児循環器・川崎病研究会  2014 

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  • 臨床試験における具体的な評価指標の定め方

    技術情報協会セミナー  2014 

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  • 小児心不全に対する心臓内幹細胞自家移植療法の第I/II相臨床研究

    第56回日本小児血液・がん学会学術集会  2014 

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  • 先天性心疾患への再生医療の現状

    抗加齢医学会総会  2014 

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  • 左心低形成症候群に対する心筋再生医療の第1/2相臨床研究

    日本小児循環器学会  2014 

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  • iPS細胞を用いた左心低形成症候群における疾患発症機序の解明

    日本小児循環器学会  2014 

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  • Intracoronary delivery of cardiac progenitor cells in patients with hypoplastic left heart syndrome.

    The 30th International Society for Heart Research  2013 

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  • 希少難治性心不全に対するヒト幹細胞移植療法の長期的エビデンス

    第12回日本再生医療学会  2013 

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  • 疾患特異的幹細胞およびiPS細胞を駆使した希少小児心疾患に対する細胞治療と病態解明

    消化器研究セミナー  2013 

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  • iPS細胞を用いた心疾患の新たな診断法と治療開発

    岡山大学サイエンスカフェ  2013 

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  • 子どもの難治性心不全に対する幹細胞移植療法の取り組み

    公開セミナーはあとネット兵庫  2013 

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  • 希少難治性心不全に対する心臓内幹細胞を用いた再生医療

    青森臨床循環器研究会  2013 

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  • 希少難治性心不全に対する心臓内幹細胞を用いた再生医療

    岡山大学知恵の見本市2013  2013 

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  • 心不全への幹細胞移植療法

    岡山先端医学研究会  2013 

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  • 心不全への幹細胞移植療法

    第3回先端医学研究会at OU  2013 

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  • 先天性心疾患に対する自己心臓内幹細胞による再生医療

    第116回日本小児科学会学術集会  2013 

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  • Mechanical stretch promotes reprogramming of human cardiac progenitors into functional cardiomyocytes by defined factors.

    日本循環器学会  2012 

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  • 心筋再生医療の臨床試験と研究開発状況

    技術情報協会セミナー  2012 

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  • Cardiac progenitor cell infusion in patients with hypoplastic left heart syndrome: a prospective phase 1 clinical trial

    Japanese Heart Failure Society  2012 

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  • 重度心臓病に対する心筋再生医療

    オープンフォーラム2012 岡山  2012 

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  • Mending and Modeling the Congenital Heart Diseases by Patient-Specific Cardiac Progenitors.

    Symposium at Texas Heart Institute  2012 

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  • 小児心不全への再生医療の可能性

    第15回遺伝子治療推進産学懇話会  2012 

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  • Mending and Modeling the Congenital Heart Diseases by Patient-Specific Cardiac Progenitors

    東京女子医科大学セミナー  2012 

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  • 左心低形成症候群に対する心臓内幹細胞自家移植療法

    第7回岡山心移植心不全研究会  2012 

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  • 小児心不全と細胞治療

    先天性心疾患シンポジウム  2012 

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  • 小児心不全に対する細胞治療の中間報告と疾患特異的iPS細胞の樹立による次世代心臓再生法の開発

    橋渡し研究加速推進ネットワークシンポジウム  2012 

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  • 先天性心疾患患者由来の心臓内幹細胞を用いた疾患特異的ヒトiPS細胞の樹立と機能解析.

    日本小循環器学会  2012 

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  • Heterokaryon-based reprogramming of human cardiac progenitor cells into functional cardiomyocytes.

    日本小循環器学会  2012 

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  • Mechanical stretch modulates calcium handling during direct cardiac reprogramming of resident progenitor cells in human heart.

    国際幹細胞学会  2012 

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  • Transition of cardiogenic to angiogeneic potential of human cardiac progenitor cells occurs with age.

    国際幹細胞学会  2012 

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  • Patient-specific induced pluripotent stem cells from cardiac progenitors recapitulate the models for cardiac chamber disorders.

    欧州心臓病学会  2012 

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  • Mechanical stretch modulates calcium handling during direct cardiac reprogramming of resident progenitor cells in human heart.

    欧州心臓病学会  2012 

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  • 小児心不全に対する細胞治療の中間報告と疾患特異的iPS細胞の樹立による次世代心臓再生法の開発

    橋渡し研究加速推進ネットワークシンポジウム  2012 

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  • 小児心不全への細胞治療法の可能性

    岡山心臓血管外科カンファレンス  2012 

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  • Mending and Modeling the Congenital Heart Diseases by Patient-Specific Cardiac Progenitors

    2012 

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  • Patient-specific induced pluripotent stem cells from cardiac progenitors recapitulate the models for cardiac chamber disorder.

    国際幹細胞学会  2012 

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  • Reprogramming of human cardiac progenitors into pluripotency in patients with congenital heart disease.

    日本循環器学会  2012 

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  • 小児心不全と細胞治療

    先天性心疾患シンポジウム  2012 

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  • 心不全の心筋再生医療

    岡二会  2012 

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  • Insulin-like growth factor-2 receptor controls human cardiac progenitor proliferation and differentiation in children with congenital heart malformation.

    米国心臓病学会  2011 

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  • 小児心不全への細胞治療の現状と展望

    重症心不全治療フォーラム  2011 

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  • Direct induction of human cardiac progenitor cells to functional cardiomyocytes by defined factors.

    米国心臓病学会  2011 

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  • Heterokaryon-based reprogramming of human cardiac progenitor cells into functional cardiomyocytes.

    日本胸部外科学会  2011 

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  • Direct reprogramming of human cardiac progenitor cells towards functional cardiomyocytes.

    日本胸部外科学会  2011 

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  • Generation and functional analysis of induced pluripotent stem cells in patients with congenital heart disease.

    日本胸部外科学会  2011 

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Works

  • 2012見本市

    2012

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    Work type:Artistic work  

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Awards

  • 文部科学大臣表彰科学技術賞

    2012.4  

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    Country:Japan

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  • 文部科学大臣表彰若手科学者賞

    2005.4  

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Research Projects

  • RNA誘導型遺伝子編集幹細胞による多様性免疫細胞を標的とした組織修復再生法の開発

    2021.04 - 2022.03

    国立研究開発法人日本医療研究開発機構  橋渡し研究戦略的推進プログラム(シーズA) 

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    Authorship:Principal investigator 

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  • 輸送幹細胞の肺組織内集積反応により遊出される心筋再生促進因子の同定と実用化研究

    Grant number:19H03738  2019.04 - 2022.03

    日本学術振興会  科学研究費助成事業 基盤研究(B)  基盤研究(B)

    王 英正

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    Grant amount:\17420000 ( Direct expense: \13400000 、 Indirect expense:\4020000 )

    本研究では、移植した細胞のうち最もロッジする近接臓器である肺組織自身を介したparacrine効果の検討が目的であり、我々が報告してきたIGF-1やHGF以外の新たな心筋再生促進因子を同定する研究計画である。
    1. 新たな手術法により確立したラットおよびブタ単心室循環モデルに対する心臓内幹細胞の経静脈的注入法による生命予後改善効果の検討ー単心室循環モデルに対する細胞移植において、急激な低酸素状況への暴露による生体内環境変化のため、非移植群に対する細胞移植の有意な生存率の改善効果は認めなかった。
    2. 正常の2心室心ならびに単心室心のラットに、培地もしくは1.0x106/kg個の心臓内幹細胞を静脈内に注入後12時間目に心臓と肺組織を採取し、Agilent Array発現解析による網羅的遺伝子検索を行うー正常心への幹細胞静脈内注入後の組織解析において、肺特異的な遺伝子上昇群を6因子同定した。
    3. Real-Time RT-PCR法を用いて、正常心で培地もしくは細胞移植で有意に変化した遺伝子群を抽出し、この候補因子群に対して、2次スクリーニングとして単心室心モデルにおいても同様に有意な変化を示した遺伝子群に最終的に絞るー上記候補因子群のうち、リアルタイムの遺伝子解析において、単心室モデルにおいても重複して有意な上昇を示した遺伝子群は3つあり、一つは従来より報告されているTNF-alphaの活性化上昇に伴い代償的に阻害する因子の一つであるTSG6を含んでいた。

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  • 腸管スキャフォールドとコラーゲン結合型成長因子を用いた拍動性グラフトの創成

    Grant number:18K08759  2018.04 - 2021.03

    日本学術振興会  科学研究費助成事業 基盤研究(C)  基盤研究(C)

    笠原 真悟, 松下 治, 王 英正, 美間 健彦

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    Grant amount:\4030000 ( Direct expense: \3100000 、 Indirect expense:\930000 )

    組織工学を用いた臓器再生研究の分野で、脱細胞化した心臓に心筋細胞を移植する
    ことで拍動が得られることが報告された。また、脱細胞化した腸管を組織工学の鋳型として利用できることも報告された。これまでに我々は、細菌性コラゲナーゼのコラーゲン・アンカー部を用いて結合組織やコラーゲン基剤に成長因子をアンカリングすることで、その効果を持続的に発揮させられることを示した。
    2019年度は、ラット小腸を腸間膜動静脈を含め脱細胞化して足場とし、心筋細胞の増殖と血管新生を誘導する種々の成長因子をアンカリングしつつ、ラット新生児の心臓由来細胞を播種することで、栄養血管を備えた拍動する心筋の筒を作製しようと考えていた。
    まずラット小腸の脱細胞化を試みた。生体から小腸を採取し、動静脈にカニュレーションし、界面活性剤を24から48時間程度還流することにより脱細胞化できることを確認した。続いて、脱細胞化した小腸の細胞外マトリックスを足場として、心筋細胞を生着させようと試みた。従来我々が用いていた解放空間における灌流装置で、脱細胞化小腸にラット新生児心筋細胞を付着させ培養液を還流したが、良好な成育は認められなかった。心筋の破砕の程度、還流流量などを変化させたが、十分な生着といえる成果は得られなかった。そこで、温度、湿度、CO2濃度を管理できるインキュベータ内で培養すべく、小型の還流装置を試作した。しかしながら最適な還流量の確立が困難で、現在足場素材、大きさの再検討、それに対する流量の検討を進めている。

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  • 心臓内幹細胞移植による小児拡張型心筋症に対する再生医療の臨床研究

    2016.04 - 2019.03

    国立研究開発法人日本医療研究開発機構  再生医療実用化研究事業 

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  • Development of alleviation of rejection by autologous cardiogenic stem cell from explanted heart

    Grant number:15K10216  2015.04 - 2018.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    Arai Sadahiko, OH Hidemasa

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    Grant amount:\4940000 ( Direct expense: \3800000 、 Indirect expense:\1140000 )

    We evaluated effect of autologous cardiogenic stem cell by heterotopic cardiac transplantation model in rat. We observed tendency of prolongation of graft survival and improved cardiac function in data. Further evaluation and statistic analysis are on-going. We analyzed origin of cardiac cell in myocardium. We found mixed existence of myocardial cell and cardiogenic stem cell. At this moment we cannot conclude whether the effect of cardiogenic stem cell is based on regeneration and proliferation of myocardium from stem cell or paracrine function from stem cell by endocrine hormone and chemical mediators and so on. Thus we continue further morphological evaluation of myocardial tissue of transplanted graft, we also considering further evaluations based on hormone/ chmical mediator analysis.

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  • Heart Transplantation using Ex-vivo Heart Perfusion

    Grant number:15K10215  2015.04 - 2018.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    KOTANI YASUHIRO, OH Hidemasa

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    Grant amount:\4680000 ( Direct expense: \3600000 、 Indirect expense:\1080000 )

    The shortage of donor pool remains a problem in heart transplantation. The alternative donor source is the non-heart beating donor: however, it is still unknown that how the heart function is recovered after the massive insult from the cardiac arrest and this makes difficult to use the heart from the non-heart beating donor. The purpose of this project is to see whether or not the heart is recovered from cardiac death.
    We found that the heart can be recovered from cardiac death when the appropriate heart preservation is done by means of controlled reperfusion. Another key finding is that the heart can be evaluated before transplantation by ex-vivo apparatus. The blood test looking at lactate level is found to be a predict marker for adequate recovery. Taken together, our finding suggests that the heart from non-heart beating donor can be used for the heart transplantation when the heart is reperfused with appropriate manner.

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  • 小児心不全に対する心筋再生医療法の企業主導多施設共同臨床治験

    2015.04 - 2018.03

    国立研究開発法人日本医療研究開発機構  革新的医療技術創出拠点プロジェクト (シーズC) 

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  • Mechanomedicine: Application of mechanobiological engineering to regenerative and reproductive medicine

    Grant number:26220203  2014.05 - 2019.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (S)  Grant-in-Aid for Scientific Research (S)

    NARUSE Keiji

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    Grant amount:\201760000 ( Direct expense: \155200000 、 Indirect expense:\46560000 )

    We induced differentiation of human induced pluripotent stem cells into spontaneously beating cardiomyocytes. We elucidated that the cardiac differentiation is facilitated by cyclic stretch stimulus and co-culture with human fibroblasts. It is suggested that stretch stimulus to cardiac stem cells induces release of paracrine factors, thereby increase cardiac contractility. With regard to autotransplantation of cardiac stem cells in pediatric dilated cardiomyopathy, we conducted the TICAP-DCM phase I trial and performed surgery for three patients successfully.
    We also studied the effect of mechanical stimulus on gene expression of fertilized eggs in mice and found that the expression of genes related to embryo development and cell death was altered in response to mechanical stimulus. In addition, we successfully developed a high-quality silicone resin incubation chamber for human fertilized eggs. Furthermore, we found that hydrostatic pressure activates sperm activity.

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  • 心疾患治療に向けた革新的次世代メカノ組織工学・再生医療の創生

    Grant number:26242042  2014.04 - 2015.03

    日本学術振興会  科学研究費助成事業 基盤研究(A)  基盤研究(A)

    成瀬 恵治, 王 英正, 高橋 賢, 入部 玄太郎

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    Grant amount:\23270000 ( Direct expense: \17900000 、 Indirect expense:\5370000 )

    心臓再生医療において、力学的・機械的刺激(メカニカルストレス)を用いた新しい技術を開発するために研究活動を行った。まず、心臓機能障害の動物モデルを作出するためにin vivoでECGを記録しつつラット冠状動脈左前下行枝(LAD)を結紮する実験を行った。組織学的解析の結果、LAD支配領域に心筋壊死による梗塞巣が形成されていることが確認された。計画段階において、心機能障害のモデルとして心筋梗塞モデルと右心不全モデルの作出を想定していたが、心筋梗塞モデルの作出は達成された。
    細胞培養による移植細胞の作出実験に関しては、3次元培養用の培養装置の開発を行い、これを用いた細胞培養を開始した。新生児ラットの単離心筋細胞およびラット心筋細胞株を用いた実験により、ゲルを足場にして細胞が3次元的に生育あるいは増殖することを確認した。この成果により、ずり応力およびストレッチによる機械的刺激を細胞に負荷する環境が確立された。
    またOxford大学客員教授のPeter Kohlを招聘し、メカニカルストレスによる心臓再生医療の開発に関しディスカッションを行った。さらにメカノバイオロジーの世界的な権威が集う国際学会International Symposium on Mechanobiology 2014に研究者を参加させ、関連研究領域の情報収集を行った。また3次元プリンタのワークショップにも研究者を参加させ、細胞培養用の培養器開発の実務的知見を取得させた。
    本研究の内容を包括する基盤研究(S)「メカノメディスン:メカノ医工学を駆使した再生医療・生殖医療への展開」の交付決定に伴い、本研究課題を終了しこれに引き継ぐこととした。

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  • 小児心不全に対するヒト幹細胞移植による先進医療の実用化加速に向けた第2相臨床研究

    2013.04 - 2016.03

    厚生労働科学研究費補助金  再生医療実用化研究事業 

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  • Three-dimensional reconstruction of bioartificial heart by reprogrammed human cardiomyocytes

    Grant number:25670389  2013.04 - 2015.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research  Grant-in-Aid for Challenging Exploratory Research

    OH HIDEMASA

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    Grant amount:\3640000 ( Direct expense: \2800000 、 Indirect expense:\840000 )

    The shortage of donors for heart transplantation has become a critical issue to treat the patients with end-stage of cardiac failure. We have decellularized the rat whole heart by using pulsatile perfusion apparatus. Reprogrammed human cardiomyocytes were mixed with endothelial progenitor cells in culture and those were perfused to recellularize the acellular construct. Following 8 to 10 days in continuous perfusion, reseeded cells were engrafted onto the matrix to reconstruct the entire vascular beds and myocardium ex vivo.

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  • 希少難治性心疾患由来iPS細胞を用いた左心低形成症候群の予後因子の解明に関する研究

    2013.04 - 2014.03

    厚生労働科学研究費補助金  難治性疾患等克服研究事業 

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  • Engineering a bioartificial heart by using extracellular matrix scaffold

    Grant number:23659672  2011 - 2012

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research  Grant-in-Aid for Challenging Exploratory Research

    SANO Shunji

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    Grant amount:\3640000 ( Direct expense: \2800000 、 Indirect expense:\840000 )

    The shortage of donors for heart transplantation has become a critical issue to treat the patients with end-stage of cardiac failure. We have decellularized the rat whole heart by using pulsatile perfusion apparatus. Human cardiac progenitor cells were mixed with endothelial progenitor cells inculture and those were perfused to recellularize the acellular construct. Following 8 days in continuous perfusion, reseeded cells were engrafted onto the matrixto reconstruct the entire vascular beds and myocardium ex vivo.

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  • Identification of cardiac reprogramming factors by heterokaryon-based genes expression analysis

    Grant number:23659419  2011 - 2012

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research  Grant-in-Aid for Challenging Exploratory Research

    OH Hidemasa

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    Grant amount:\3640000 ( Direct expense: \2800000 、 Indirect expense:\840000 )

    We employed a heterokaryon-based gene screening approach to identify the crucial transcription factors might be involved in direct cardiac reprogramming. By using this novel technique, we found a series of transcription factors essential for right and left ventricular formation include GATA4, Mef2c, Tbx5, Myocardin, and Hand2 were capable of converting the cardiac progenitor cells toward mature cardiomyocytes directly in vitro.

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  • 小児心不全に対する細胞治療と単心室症由来人工多能性幹(iPS)細胞の樹立による次世代心筋再生医療法の開発

    2010.04 - 2013.03

    厚生労働科学研究費補助金  成育疾患克服等次世代育成基盤研究事業 

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  • ヒト心臓内多能性幹細胞と幹細胞増幅因子bFGF徐放シートのハイブリッド移植療法による心筋再生医療の多施設共同型臨床研究

    2008.04 - 2011.03

    厚生労働科学研究費補助金  再生医療実用化研究事業 

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  • Integrated cardiac stem cell therapy to treat heart failure

    Grant number:20390223  2008 - 2010

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)  Grant-in-Aid for Scientific Research (B)

    OH Hidemasa

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    Grant amount:\19110000 ( Direct expense: \14700000 、 Indirect expense:\4410000 )

    Current cell therapies for cardiac repair are limited by loss of the transplanted cells and poor differentiation. Controlled delivery of bFGF modulates the post-ischemic microenvironment to enhance human cardiosphere-derived cell engraftment and differentiation. This novel strategy demonstrates significant functional improvements after myocardial infarction and may potentially represent a therapeutic approach to be studied in a clinical trial in human heart failure.

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  • Cre-loxPシステムを用いたAkt依存性に活性化される心筋細胞再生因子の単離

    Grant number:19659204  2007

    日本学術振興会  科学研究費助成事業 萌芽研究  萌芽研究

    王 英正, 上山 知己, 小形 岳寛

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    Grant amount:\3300000 ( Direct expense: \3300000 )

    本研究の目的は、心臓内幹細胞の特異的増殖規定因子の同定であり、これまでの我々の報告から、心臓内幹細胞は主として、細胞表面抗原であるSca-1で認識及び精製できること、また、Sca-1ノックダウンマウスの機能解析から、Aktが重要な候補支配因子であることが明らかとなった。そこで、本研究では、Sca-1プロモーターにAktをノックインした薬剤誘導型Akt発現マウス(Sca-1/Akt mice)を作成し、遺伝子工学的手法用いて、Akt依存性に活性化される心臓内幹細胞の網羅的検索を行った。結果:1)Sca-1ノックダウンマウスに急性虚血を作成すると、梗塞後1週間での生存率が約30%と野生型の70%に比べ、有意に低下したことから、Sca-1依存性に心臓内幹細胞の自己増殖に重要な因子の発現低下が関与していることが示唆された。一方、Sca-1/Akt miceに虚血を導入すると、急性生存率が野生型と同様なレベルまでに回復し、Sca-1/Aktの活性化を軸とした心臓内幹細胞による障害心筋細胞の自己修復機構の存在が確認された。2)Sca-1/Aktを介した心臓内幹細胞の増殖規定因子を検索するため、野生型及びSca-1/Aktマウスの心臓内から幹細胞をそれぞれ精製し、マイクロアレイによる網羅的検索を行った。cutoff indexを2倍以上に上昇した因子群に集約すると、心臓幹細胞はAkt依存性に活性化される増殖制御因子として、インスリン様成長因子結合蛋白4やセマフォリン3C、さらに、Wnt/beta-cateninの経路を調節するfrizzled関連蛋白1/2が候補因子として同定された。これらの因子群はいずれもin vitroでの検討で心臓内幹細胞の増殖能を促進させ、今後in vivoにおけるこれらの因子群の心筋虚血耐性の役割について、さらに詳細に検討していく予定である。

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  • Molecular cloning and characterization of differentiation-inducing factors into the cardiac myocyte lineage using an expression cloning approach

    Grant number:18590773  2006 - 2007

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    UEYAMA Tomomi, MATUBARA Hiroaki, OH Hidemasa, HARADA Koichiro, OGATA Takehiro

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    Grant amount:\3890000 ( Direct expense: \3500000 、 Indirect expense:\390000 )

    Since embryonic stem (ES) cells are capable of self-renew and differentiation into specialized cells in response to appropriate signals, ES cells are proposed as a promising source of functional cardiac myocytes for cardiac diseases. However, to date the in vitro differentiation of ES cells into cardiac myocytes remains inefficient and its molecular mechanisms are not fully elucidated. Therefore, the identification of differentiation-inducing factors into the cardiac myocyte lineage has become critical for understanding the molecular mechanisms of differentiation into cardiac myocyte and heart development, and facilitates therapeutic applications of ES cells in cardiac diseases. In the present study, we sought to identify differentiation-inducing factors into the cardiac myocyte lineage using an expression cloning approach. We induced differentiation of the mouse embryonic carcinoma cell line, P19CL cells, into cardac myocytes using DMSO. We made cDNA libraries using mRNA isolated from differentiating cells. We then generated recombinant retroviruses expressing the cDNA libraries. ES cell clones that express EGFP under the transcriptional control of a cardiac-specific α-MHC promoter were infected with recombinant retroviruses expressing the cDNA libraries. Gemonic DNA was extracted from differentiated cells into cardiac myocytes and performed PCR to isolate the integrated cDNAs into the genomic DNA. We obtained G protein beta polypeptide 2 like 1 (Gnb2I1, Rack1), oxidase assembly 1-like (Oxa1l), glutaredoxin 5 homolog (Glrx5), and aurora kinase A interacting protein 1(Aurkaip1) as candidate genes for differentiation-inducing factors into the cardiac myocyte lineage. We are studying their functions in differentiation of ES cells into cardiac myocyte. Our study will provide novel insights into the molecular mechanisms of cardiac myocyte differentiation and the development of novel approaches for the directed differentiation of ES cells into cardiac myocytes.

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  • Development of hybrid stem cell therapy for human heart failure

    Grant number:18390233  2006 - 2007

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)  Grant-in-Aid for Scientific Research (B)

    OH Hidemasa, UEYAMA Tomomi, HARADA Koichiro, OAGATA Takehiro

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    Grant amount:\17050000 ( Direct expense: \15700000 、 Indirect expense:\1350000 )

    To develop an efficient cell therapy for cardiac repair, we performed serial analysis of gene expression and identified follistatin, an antagonist of TGF-β family members, was predominantly expressed in skeletal muscle-derived progenitors. We have found that follistatin may be an effective progenitor-enhancing agent neutralizing ActA and GDF11 signaling to regulate the growth of progenitor cells in skeletal muscle. In addition, we demonstrate a single-cell deposition analysis to isolate individually selected cardiac stem cells from adult hearts and investigate the signals required for their proliferation and survival. Clonally proliferated cardiac stem cells express stem cell antigen-1 (Sca-1) with embryonic stem (ES) cell-and mesenchymal cell-like characteristics and are associated with telomerase reverse transcriptase (TERT). Using a transgene that expresses a GFP reporter under the control of the TERT promoter, we demonstrated that TERT^<GFP+> fractions from the heart were enriched for cells expressing Sca-1. Knockdown of Sca-1 transcripts in cardiac stem cells led to retarded ex vivo expansion and apoptosis through Akt inactivation. We also show that ongoing cardiac stem cell proliferation and survival after direct cell-grafting into ischemic myocardium require Sca-1 to upregulate the secreted paracrine effectors that augment neoangiogenesis and limit cardiac apoptosis. Thus, Sca-1 might be an essential component that promotes cardiac stem cell proliferation and survival to directly facilitate early engraftment, and that indirectly exerts the effects on late cardiovascular differentiation after cardiac stem cell transplantation. Based on these observations in rodents, we conducted two randomized, placebo-controlled studies in immunosuppressed pigs with anterior myocardial infarctions. As the results of these studies, we found that controlled delivery of bFGF modulates the post-ischemic microenvironment to enhance human cardiac stem cell engraftment and differentiation. This novel integrated-strategy demonstrates significantly functional improvements after myocardial infarction and could be a potentially safe and feasible application to treat human heart failure.

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  • 胚性幹細胞との細胞融合及び遺伝子工学的手法で再プログラムされた心筋幹細胞株の樹立

    Grant number:18659224  2006

    日本学術振興会  科学研究費助成事業 萌芽研究  萌芽研究

    王 英正, 上山 知己, 原田 光一郎, 小形 岳寛

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    Grant amount:\3300000 ( Direct expense: \3300000 )

    増幅能が限られている体性幹細はテロメア関連伝子導入によって不死化する試みがされているが、遺伝子をベースにした方法では常に悪性腫瘍化する危険性が付きまとう。そこで、申請者らはES細胞との細胞融合によって、ES細胞が持つ極めて高い増殖性の性質を申請者らが単離に成功した心筋幹細胞に形質伝承する目的で、Cre-loxPシステムを利用したレトロウイルスベクターを作成した。ES細胞と融合によって形質変換した細胞の精製によって、心筋幹細胞クローンにCre-IRES-RFPレトロウイルスを感染させ、ウイルス感染が良好であることを確認した。
    (1)puro/hygroの抗生剤選択で純化したハイブリッド細胞が4倍体であることをFACSで確認した。融合した心筋細胞は免疫組織染色にて、心筋幹細胞には発現していないES細胞特異的な転写因子の提示を認め、細胞融合の効率がその後の解析に有効な頻度(約50%)で生じていることを検証した。
    (2)RNAを回収しマイクロアレイによる網羅的検索によって、未分化ES細胞特異的転写因子や細胞周期調節因子について網羅的検索を行ったところ、約6割の心筋幹細胞特異的遺伝子の発現減少とそれに伴うES細胞固有因子の増加を融合した新規細胞の遺伝子プロファイルにて確認した。このことは、細胞融合によって心筋幹細胞が完全に形質変換したことを直接証明するものではなく、細胞融合前の約6割の心筋幹細胞がES細胞との再プログラムによる修飾の影響を受けたことを強く示唆した。
    (3)neo耐性遺伝子を持つalpha-MHC-EGFPを導入したES細胞の形質を継承した再プログラムされた心筋幹細胞は、in vitroにて通常のES細胞に比べ、心筋細胞に分化する効率が約3倍向上したことをEGFPの発現頻度にて確認された。逆に融合した心筋幹細胞は正常のES細胞と比し、細胞増殖能が約半分に低下した。このことは、ES細胞の持つ多能性分化能を再プログラムする手法を用いて、遺伝子工学的に幹細胞の運命を制御したことを証明し、今後の虚血心への融合細胞移植実験にて、生体内での心筋細胞再生医療への有用性について研究を発展させていく予定である。

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  • ヒト心筋・骨格筋からの心筋幹細胞株の樹立と末期的心不全への幹細胞移植医療実現化へ向けての研究基盤形成

    2005.04 - 2008.03

    厚生労働科学研究費補助金  医療技術実用化総合研究事業  基礎研究成果の臨床応用推進事業

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  • Cardiac Repair of Severe Heart Failure by Human Heart- or Skeletal Muscle-Derived Multipotent Stem Cells

    Grant number:17209028  2005 - 2007

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)  Grant-in-Aid for Scientific Research (A)

    MATSUBARA Hiroaki, TAKAHASHI Tomosaburo, OH Hidemasa

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    Grant amount:\49530000 ( Direct expense: \38100000 、 Indirect expense:\11430000 )

    Recent studies have shown that cardiac stem cells (CSCs) from the adult heart can give rise to functional cardiomyocytes; however, the definite surface markers to identify a definitive single entity of CSCs and the molecular mechanisms regulating their growth have remained unknown. Here we demonstrate a single-cell deposition analysis to isolate individually selected CSCs from adult hearts and investigate the signals required for their proliferation and survival. Clonally proliferated CSCs express stem cell antigen-1 (Sca-1) with embryonic stem (ES) cell-and mesenchymal cell-like characteristics and are associated with telomerase reverse transcriptase (TERT). Using a transgene that expresses a GFP reporter under the control of the TERT promoter, we demonstrated that TERT^<GFP+> fractions from the heart were entiched for cell expressing Sca-1. Knockdown of Sca-1 transcripts in CSCs led to retarded ex vivo expansion and apoptosis through Akt inactivation. We also show that ongoing CSC proliferation and survival after direct cell-grafting into ischemic myocardium require Sca-1 to upregulate the secreted paracrine effectors that augment neoangiogenesis and limit cardiac apoptosis, Thus, Sca-1 might bean essential component that promotes CSC proliferation and survival to directly facilitate early engraftment, and that indirectly exerts the effects on late cardiovascular differentiation after CSC transplantation.

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  • 心筋幹細胞移植医療の実現化に向けたヒト心筋幹細胞特異的増幅因子のクローニング

    Grant number:17659233  2005 - 2006

    日本学術振興会  科学研究費助成事業 萌芽研究  萌芽研究

    王 英正

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    Grant amount:\3400000 ( Direct expense: \3400000 )

    平成17年7月1日より、心筋幹細胞移植医療の実現化に向けたヒト心筋幹細胞特異的増殖因子のクローニングに関する研究課題に関し、特にsignal sequence trap法を使ったadult由来心筋幹細胞の増殖因子の同定に関する研究を開始した。
    採取したadult由来マウス心筋幹細胞からのcDNA library作製し、レトロウイルスパッケージング細胞であるPlatE細胞にRoche社のFuGENE6を使用して導入し、レトロウイルスとして回収した。回収したレトロウイルスを感染効率が20%弱になるように調整してBa/F3細胞に感染させ、96well plateに10000個/wellで撒き、IL-3非存在下で2週間程培養した。培養により、シグナル配列を持たないpMX-SSTが感染したBa/F3細胞は死滅し、シグナル配列を持ったcDNA断片を融合したpMX-SSTが感染したBa/F3細胞のみ増殖が認められ、最終的に200近くの細胞クローンが認められた。これらの細胞クローンから染色体DNAを抽出した後、vector primerを用いてPCRを行い、挿入cDNA断片を回収した。
    回収したcDNA断片のすべてに対してシーケンサーによる解読を行い、NCBI BLASTホームページ上のnucleotide-nucleotide blastを使って相同性検索を行ったところ、adult由来マウス心筋幹細胞に特異的に発現している分泌タンパク質が2つ認められた。この2つは、RT-PCRでもmRNA発現が亢進していることを確認し、そのうちの1つは免疫染色でsphereを形成している細胞上に強くタンパク発現していることも確認した。
    現在、これらの同定されたタンパク質についてrecombinantによる薬物的刺激実験とノックアウトマウスを使った抑制実験の2つの方向から、同定されたタンパク質のマウス心筋幹細胞への増殖に対する効果を解析すべく準備を進めている。今後、同定されたタンパク質が増殖に関わることが明らかになれば、ヒト心筋幹細胞への応用が可能かどうか検討していく予定である。

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  • 新たに同定した心臓由来心筋前駆細胞のクローン化増殖、機能解析による心筋再生医療

    Grant number:16689017  2004 - 2006

    日本学術振興会  科学研究費助成事業 若手研究(A)  若手研究(A)

    王 英正

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    Grant amount:\28470000 ( Direct expense: \21900000 、 Indirect expense:\6570000 )

    心臓由来の心筋前駆細胞の細胞機能解析として、平成16年度に引き続き、平成17年度一年間で以下の2つの課題を明らかにしてきました。
    1)心臓由来のSca-1陽性細胞のクローン化とその多能性を明らかにすること。
    平成16年度の報告のように、申請者らは心筋前駆細胞である心臓由来のSca-1陽性細胞を単一細胞より大量増幅させ、そのクローン化に成功した。各クローンはそれぞれ心筋細胞に分化できるポテンシャルが異なり、心筋細胞以外では血管平滑筋、内皮、グリア、脂肪、上皮細胞への多能性分化能についても確認しえた。
    2)マウス生体内でのSca-1遺伝子の役割をSca-1欠損マウスへ心筋梗塞作成することで明らかにする。
    平成16年度内にSca-1ノックダウンマウスの作成に成功した。ある一定の数まで動物を繁殖させた後、マウスの表現型の解析を開始した。Sca-1ノックダウンマウスは生後異常なく成長し、心臓にも異常が認められなかった。しかしながら、Sca-1ノックダウンマウスより精製純化した心筋幹細胞コロニーは増幅能を著しく傷害され、野生型の心筋幹細胞と比べ、長期培養は不可能であった。また、Sca-1ノックダウンマウス由来の心筋幹細胞は高い増幅能を持った細胞に特徴的なテロメラーゼ活性も著しく低下し、細胞周期調節因子であるp53の発現上昇を認めた。興味深いことに、Sca-1ノックダウンした心筋幹細胞をドナーとして用い、虚血心に細胞した場合、野生型の細胞移植と比べ、心筋細胞の再生能及び心機能改善効果は著しく障害を受けた。また、Sca-1ノックダウンマウスに心筋梗塞を作成すると、梗塞巣が繊維化領域の拡大により、野生型と比べ増加し、1ヶ月目までの虚血心マウスの生存は有意に低下した。これらのことより、Sca-1は心筋幹細胞を認識するだけでなく、心臓内心筋幹細胞の自己増幅能を制御する重要な因子であることが示唆された。

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