Objective: Brugada Syndrome is an autosomal dominant congenital heart disease that is responsible for 20% of sudden deaths of patients with structurally normal hearts. The majority of mutations involve the cardiac sodium channel gene SCN5A and give rise to classical symptoms, which include an abnormal electrocardiogram with ST segment elevation and a predisposition to ventricular fibrillation. To date, the implantation of a cardioverter defibrillator is the only proven effective treatment of the disease. The ability to reprogram dermal fibroblasts to induced pluripotent stem (iPS) cells and to differentiate these into cardiomyocytes with the same genetic background provides a novel approach to studying inherited cardiac channelopathies with advantages over existing model systems. Whilst this technique has enormous potential to model inherited channelopathies, such as Brugada Syndrome, the derived cells have not been fully characterised and compared to foetal and adult cardiomyocytes. Methods: Dermal fibroblasts from a patient with Brugada syndrome (SCN5A; c.1100G > A - pARG367HIS) and an age- and sex-matched control were reprogrammed using episomal vectors. All newly derived iPS cell lines were fully characterised using immunocytochemistry, flow cytometry, real-time quantitative reverse transcription PCR and single nucleotide polymorphism analysis and were compared to established human embryonic stem (hES) cell and in-house derived healthy control iPS cell lines. The same control cell lines were used to compare the efficiencies of several cardiac differentiation media. Spontaneously contracting areas, derived from control as well as patient iPS cell lines, were disaggregated and single cardiomyocytes were compared to foetal and adult cardiomyocytes isolated from primary human tissue using immunocytochemistry, transmission electron microscopy, membrane visualisation, calcium imaging and electrophysiology. Results: Comparison of cardiac differentiation protocols using healthy control hES and iPS cell lines found that despite significant inter-line variability with regard to efficiency of cardiac formation guided differentiation protocols could be used to reliably and efficiently generate beating bodies. Spontaneous contraction was observed in stem cell-derived cardiomyocytes and human foetal cardiomyocytes. Pluripotent stem cell-derived cardiomyocytes stained for markers of the cardiac contractile apparatus such as α-actinin, cardiac troponin I and cardiac troponin T. They also expressed functional voltage-activated sodium channels and exhibited action potential triggered calcium-induced calcium release. Stem cell-derived cardiomyocytes showed organisation of myofibrils, ultrastructure and calcium handling more similar to foetal than adult cardiomyocytes. Brugada Syndrome patient-specific cardiomyocytes were structurally indistinguishable from healthy control iPS cell line-derived cardiomyocytes. Electrophysiological analysis of sodium current density confirmed a ~50% reduction in patient-derived compared to healthy control-derived cardiomyocytes. Conclusion: Although iPS cells give rise to a mixture of immature and more mature cardiomyocytes, they all express typical cardiac proteins and have functional cardiac sodium channels. Results illustrate the ability of patient-specific iPS cell technology to model the abnormal functional phenotype of an inherited channelopathy that is independent of structural abnormalities and that the relative immaturity of iPS cell-derived cardiomyocytes does not prevent their use as an accurate model system for channelopathies affecting the cardiac sodium channel Nav1.5. This iPS cell based model system for classical Brugada Syndrome allows for the first time to study the mutation in its native environment and holds promise for further studies to investigate disease mechanisms of known and unknown mutations and to develop new therapies.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18504号 / 医博第3924号 / 新制||医||1005(附属図書館) / 31390 / 京都大学大学院医学研究科医学専攻 / (主査)教授 妻木 範行, 教授 開 祐司, 教授 中辻 憲夫 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21657号 / 医博第4463号 / 新制||医||1035(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 戸口田 淳也, 教授 松田 秀一, 教授 安達 泰治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Embryonic stem cells (ESCs) are pluripotent cells derived from the early embryo and are able to differentiate into cells belonging to the three germ layers. They are a valuable tool in research and for clinical use, but their applications are limited by ethical and technical issues. In 2006 a breakthrough report described the generation of induced pluripotent stem cells (iPSCs). IPSCs are ESC-like cells generated from somatic cells by forcing the ectopic expression of specific transcription factors. This circumvents the ethical issues about the use of embryos in research and provides multiple opportunities to understand the mechanisms behind pluripotency. The aim of this project was to generate sheep iPSCs and characterise them. In order to learn the technique I initially repeated the original iPSC methodology: the putative mouse iPSCs I have generated display a morphology typical of ESCs, characterised by a high nuclear to cytoplasmic ratio, and form colonies with neat edges and smooth domes. These cells are positive to Nanog, a marker of pluripotency, and can give rise to cells belonging to the mesodermal and the ectodermal lineages when differentiated in vitro. Since the main aim of the thesis was the derivation of sheep pluripotent cells, once established the protocol in mouse, I then moved to the generation of ovine iPSC colonies. The cells I have generated have a morphology similar to that of mouse ESCs, express markers of pluripotency such as alkaline phosphatase and Nanog and can differentiate in vitro and in vivo into cells belonging to the three germ layers. Additionally, these ovine iPSCs can contribute to live born chimeric lambs, although at low level.

Transthyretin (TTR)-associated amyloidoses are diseases wherein wild-type or mutant TTR forms amyloid fibrils that infiltrate multiple organs. Wild-type TTR amyloidosis, ATTRwt, is a sporadic disease characterized by deposits that occur mainly in the heart. Alternatively, >100 TTR mutants cause inherited forms, ATTRm, frequently featuring cardiac amyloid deposits. The goals of this research were to create a cell-based model of ATTR amyloidosis, to define the mechanism of cardiac TTR-associated amyloid at the cellular level, and to study several agents that could interrupt the amyloid process. We hypothesized that TTR oligomers were cardiotoxic and played a role in the mechanism of ATTR amyloidosis, and that cytotoxicity could be inhibited by diflunisal, doxycycline, and Kiacta®. Focusing on TTR proteins associated with cardiac amyloidosis (wild-type, L55P, V30A, and V122), we developed a thermal denaturation method for creating TTR oligomers that allowed us to study the direct effect of oligomers on cells. Congo red and thioflavin T analyses confirmed that the oligomers were on pathway to amyloid fibril formation. We tested the effect of TTR oligomers on rat and human cardiac cells by measuring cell viability and stress response (through live protease activity and qPCR). TTR-L55P oligomers elicited a cytotoxic effect; fluorescent microscopy indicated cellular uptake of the oligomers and continued intra-cellular aggregation. Cytotoxicity was blocked when TTR was heated in the presence of doxycycline; the drug appeared to dissociate TTR aggregates or stabilize the monomeric forms. We also investigated retinol-binding protein (RBP), a natural binding partner of TTR. By immuno-histochemistry, RBP was demonstrated in ATTRwt and ATTRm `non-amyloid' transplant heart tissues, localized to areas containing amyloid or in the case of the transplant tissue, regions that appeared to display ischemic damage. Serum RBP levels were significantly different in ATTR vs. age-matched controls (p = 0.03), and in ATTRwt vs. ATTRm (p <0.0001) by ELISA. These data provide evidence that TTR oligomers are cardiotoxic, possibly due to cellular internalization and progressive intracellular aggregation. Furthermore, our results support the use of doxycycline as a therapeutic in ATTR to target these amyloidogenic oligomers, and suggest that RBP may have potential as a disease biomarker.

Development leads unidirectionally towards a more restricted cell fate that is usually stable. However, it has been proven that developmental systems are reversible by the success of animal cloning of a differentiated somatic genome through somatic cell nuclear transfer (SCNT). Recently, reprogramming of somatic cells to a pluripotent embryonic stem cell (ESC)-like state by introducing defined transcripton factor has been achieved, resulting in the generation of induced pluripotent stem cells (iPSCs), which resemble ESCs. iPSC reprogramming is of great medical interest, as it has the potential to generate a source of patient-specific cells. However, the dangerous delivery method, low efficiency, and slow kinetics of the reprogramming process have hampered progress with this technology.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23422号 / 医博第4767号 / 新制||医||1053(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 河本 宏, 教授 濵﨑 洋子, 教授 上野 英樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23604号 / 医博第4791号 / 新制||医||1055(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 戸口田 淳也, 教授 河本 宏, 教授 江藤 浩之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医科学) / 甲第23611号 / 医科博第134号 / 新制||医科||9(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 遊佐 宏介, 教授 小川 誠司, 教授 山中 伸弥 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19899号 / 農博第2182号 / 新制||農||1043(附属図書館) / 学位論文||H28||N5003(農学部図書室) / 32976 / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 今井 裕, 教授 久米 新一, 教授 廣岡 博之 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM