Characterization of pluripotent stem cell-derived cardiomyocytes [Elektronische Ressource] / vorgelegt von Jiaoya Xi

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Aus dem Zentrum für Kinderheilkunde und Jugendmedizin der Universität zu Köln Klinik und Poliklinik für Kinderkardiologie Direktor: Universitätsprofessor Dr. med. K. Brockmeier Characterization of Pluripotent Stem Cell-Derived Cardiomyocytes Inaugural-Dissertation zur Erlangung der Würde eines doctor rerum medicinalium der Hohen Medizinischen Fakultät der Universität zu Köln vorgelegt von Jiaoya Xi aus Hubei/V.R.China Promoviert am: 12. August 2009 Gedruckt mit Genehmigung der Medizinischen Fakultät der Universität zu Köln 2009 Druck: copy team cologne GmbH, Köln iiDekan: Universitätsprofessor Dr. med. J. Klosterkötter 1. Berichterstatter: Privatdozent Dr. med. F. D. Pillekamp 2. tatter: Universitätsprofessor Dr. med. K. Brockmeier Eidesstattliche Erklärung Ich erkläre hiermit, das ich die vorliegende Arbeit ohne unzulässige Hilfe Dritter und ohne Benutzung anderer als der angegebenen Hilfsmittel angefertigt habe; die aus fremden Quellen direkt oder indirekt übernommenen Gedanken sind als solche kenntlich gemacht. Bei der Auswahl und Auswertung des Materials sowie bei der Herstellung des Manuskripts habe ich keine Unterstützungsleistung erhalten.
Publié le : jeudi 1 janvier 2009
Lecture(s) : 37
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Source : D-NB.INFO/996529942/34
Nombre de pages : 71
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Aus dem Zentrum für Kinderheilkunde und Jugendmedizin der Universität zu Köln Klinik und Poliklinik für Kinderkardiologie Direktor: Universitätsprofessor Dr. med. K. Brockmeier
Characterization of Pluripotent Stem Cell-Derived Cardiomyocytes Inaugural-Dissertation zur Erlangung der Würde eines doctor rerum medicinalium der Hohen Medizinischen Fakultät der Universität zu Köln vorgelegt von Jiaoya Xiaus Hubei/V.R.China Promoviert am: 12. August 2009
Gedruckt mit Genehmigung der Medizinischen Fakultät der Universität zu Köln 2009 Druck: copy team cologne GmbH, Köln
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Dekan:Universitätsprofessor Dr. med. J. Klosterkötter1.Berichterstatter: Privatdozent Dr. med. F. D. Pillekamp 2.Berichterstatter: Universitätsprofessor Dr. med. K. Brockmeier
Eidesstattliche Erklärung Ich erkläre hiermit, das ich die vorliegende Arbeit ohne unzulässige Hilfe Dritter und ohne Benutzung anderer als der angegebenen Hilfsmittel angefertigt habe; die aus fremden Quellen direkt oder indirekt übernommenen Gedanken sind als solche kenntlich gemacht. Bei der Auswahl und Auswertung des Materials sowie bei der Herstellung des Manuskripts habe ich keine Unterstützungsleistung erhalten. Weitere Personen waren an der geistigen Herstellung der vorliegenden Arbeit nicht beteiligt, insbesondere habe ich nicht die Hilfe eines Promotionsberaters in Anspruch genommen, Dritte haben von mir weder unmittelbar noch mittelbar geldwerte Leistungen für Arbeiten erhalten, die im Zusammenhang mit dem Inhalt der vorgelegten Dissertation stehen. Die Arbeit wurde von mir bisher weder im Inland noch im Ausland in gleicher oder ähnlicher Form einer anderen Prüfungsbehörde vorgelegt. Köln, den 18.06.2009Jiaoya Xi
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Die in dieser Arbeit angegebenen Experimente sind nach entsprechender Anleitung
durch Prof. Dr. Konrad Brockmeier, Priv. Doz. Dr. Frank Pillekamp und Dr. Markus
Khalil von mir selbst durchgeführt worden.
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ACKNOWLEDGEMENTS
First of all, I would like to express my great gratitude to Professor Ming Tang and Professor Changjin Liu, Directors of the Department of Physiology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China, all leaders of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China, and the Chinese Scholarship Council for providing me with the opportunity and funding me to do my PhD study in Germany. Besides the material support, there was a continuously moral support which always encouraged me and motivated me to perform my PhD study successfully. Furthermore, I would like to express my great appreciation to my tutors, Professor Konrad Brockmeier, Director of the Department of Pediatric Cardiology, University of Cologne, Cologne, Germany, and Professor Jürgen Hescheler, Director of the Institute of Neurophysiology, University of Cologne, Cologne, Germany, for providing me with the opportunity to receive the good training and learn so many advanced experimental techniques, for their encouragement and critical guidance throughout my work. I especially thank Dr. Frank Pillekamp, who led me into the amazing insight into the present study. He guided me successfully to complete this project and I pay tribute to him for his instructions and help in understanding theoretical and technical aspects of electrophysiology, his advises concerning my projects and my career in science, as well as his helpful comments on my thesis. I especially thank Dr. Markus Khalil for his full support on performing the project, comments on manuscripts and helpful comments on my thesis. My thanks also go to Mr. Haustein for the slicing and force measurement support, Mrs. Köster for the immunostaining work, Mr. Metzner and the colleagues in the workshop for the institute for their elaborate maintenance of our experimental apparatus, Mrs. Dieterich, Mrs. Böttinger, Mrs. Gröthgans, and Mrs.Rohani for their
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assistance in the cell culture work, Mrs. Wood for her kind help in work-related affairs and the institute of statistics for their kind support in statistics analyze work. In addition, my appreciations and thanks go to my kind colleagues and friends who provide suggestion and help in my thesis writing, and help me in performing this project. It is impossible to express my gratitude to every single person to whom I am indebted. Nevertheless I would like to acknowledge with appreciation all my coworkers and friends who directly or indirectly gave me help.
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TABLE OF CONTENTS1.INTRODUCTION ...............................................................................11.1.Donor Cells for Cell Replacement Therapy........................................11.1.1.Adult Stem Cells ......................................................................21.1.2.Embryonic Stem Cells (ESCs) and Embryonic Stem Cells Derived- Cardiomyocytes (ESC-CMs) ...................................................31.1.3.Induced Pluripotent Stem Cells (iPSs)/Reprogramming Cells and Induced Pluripotent Stem Cells Derived-Cardiomyocytes (iPS-CMs)...51.2.In vivoStudies of Stem-Cell-Based Transplantation after Myocardial Infarction(MI)............................................................................................61.3. ..............................7Functional Properties of ESC-CMs and IPS-CMs1.4.Integration, Survival and Functional Coupling after Transplantation of ESC-CMs and IPS-CMs .......................................................................101.5.AnIn vitroModel to Mimic Cardiac Cell Therapy ..........................111.6. .................................................................12Aims of the Present Study2.PROVEMENT OF PUBLICATIONS ..............................................133.........................................8.3........................N...SSIO................UCSID3.1.Neonatal Murine Heart Slices are Morphologically and Functionally Intact..........................................................................................................383.2.Neonatal Murine Heart Slices are Suitable to Measure Loaded ContractionsinMice.................................................................................393.3.Limitation of Neonatal Murine Heart Slices .....................................413.4.Efficient Generation Cardiomyocytes from IPSs..............................413.5.Electrophysiological Characteristics of iPS-CMs Compared with ESC-CMs..................................................................................................42 3.6.Mimic MIIn vitro................43..............................................................
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3.7.Transplanted ESC-CMs and IPS-CMs Confer Force to the Host
Tissue........................................................................................................43
3.8.........44....................................................................lcsuCno............ion.
4.
5.
6.
7.
8.
SUMMARY......................................................................................45
ZUSAMMENFASSUNG ..................................................................46
REFERENCES ..................................................................................47
PUBLICATIONS ..............................................................................60
LEBENSLAUF..................................................................................61
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Abbreviation ANPatrial natriuretic peptide APaction potential APDaction potential duration APD5050% of action potential duration APD9090% of action potential duration [Ca2+]ecextracellular Ca2+concentration[Ca2+]iintracellular Ca2+concentration Cchcarbachol CICRCa2+-induced Ca2+release CSQqeslacuestrinDHPRvoltage-dependent L-type Ca2+channels DMEMDulbeccos modified Eagles medium EBsembryonic bodies EC50half maximum effective concentration ECCexcitation-contraction coupling ESCsembryonic stem cellsFFRforce-frequency relationship GFPgreen fluorescent protein hEBshuman embryonic bodies hESCshuman embryonic stem cellshESC-CMshuman embryonic stem cell-derived cardiac myocyteshIPSshuman induced pluripotent stem cells hIPS-CMshuman induced pluripotent stem cell-derived cardiac myocytes IMDMIscove´s modified Dulbecco´s medium iPSsduced pluripotent stem cells in iPS-CMsinduced pluripotent stem cell-derived cardiac myocytes Iso(-)-isoproterenol (isoprenaline) MEAmultiple electrode array mESCsmurine embryonic stem cellsmESC-CMsmurine embryonic stem cell-derived cardiac myocytesMImyocardial Infarction mIPSsmurine induced pluripotent stem cells
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mIPS-CMs NCX NIF OGD PLB RYA RyR SERCA 2a SL SR TM TnC
murine induced pluripotent stem cell-derived cardiac myocytes Na+/Ca2+exchanger nifedipine oxygen and glucose deprivation phospholamban ryanodine ryanodine receptor sarcoplasmic/endoplasmic reticulum Ca2+pump sarcolemmalsarcoplasmic reticulum tropomyosintroponin C
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1.NOITCUDORINT
Heart failure is a major worldwide health problem, which is often characterized by a loss of functioning cardiomyocytes. Cardiomyocytes are terminally differentiated and show a very limited capacity for regeneration (Wei et al., 2005). Patients with heart failure ultimately die from either pump failure or cardiac arrhythmia. Patients with severe heart failure have a mortality of more than 50% within the first year (Murray et al., 2000). End-stage heart failure can be treated by heart transplantation. However, due to insufficient numbers of donor-organs, 20% of patients die during waiting for donor-organs. Moreover, given the high morbidity and mortality rates associated with heart failure, death of donor hearts for transplantation, complications associated with immunosuppression, and long-term failure of transplanted organs, novel treatment methods that improve cardiac function and prevent heart failure are in demand (Dudley, 2005). Starting in the mid-1990s, a series of observations has led to the concept that cells might be used to repair myocardial damage. While current treatment of heart failure in the context of coronary heart disease focuses on early revascularization and medical treatment for the inhibition of further cardiomyocyte loss, the approach of cardiac cell therapy aims at restoration of cardiac function as a pump by regenerating cardiac tissue. A large amount of experimental evidence was amassed showing that injected cells could create new tissue and improve pumping function of the failing heart (Murry et al., 2005). This evidence, coupled with the recognized limitations of the heart failure treatments and the intuitively appealing concept of regenerative medicine, has contributed to a crescendo of activity in cell-based cardiac repair (Murry et al., 2005). However, to accomplish this, a major hurdle to overcome is the identification of suitable cell types.
1.1.Donor Cells for Cell Replacement Therapy So far, various cell types have been suggested but the optimal cell source is still a matter of intense debate(Pillekamp et al., 2007).Since stem cells have the potential to self renew and differentiate into any cell present in the adult organism (including cardiomyocytes), they might also be able to repopulate the myocardium of heart failure patients and be a highly attractive cell source.
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