<p>Stem cell technologies hold great promise in solving problems within fields such as drug development, regenerative medicine, and disease modeling. Stem cell engineering provides a mechanism that will help stem cells achieve this promise. Currently, many applications within tissue engineering are limited by a lack of ability to create accurate micro-physiological structures that recapitulate multicellular tissue patterns <i>in vivo</i>. Precise control of spatial and temporal signaling is desired to perform concurrent differentiation to multiple cell types intentionally. The OptoWnt construct, a novel optogenetic system activating the Wnt signaling pathway, achieves precise spatiotemporal regulation, in pursuit of greater control in stem cell differentiation. We utilize OptoWnt, to differentiate stem cells into cardiovascular cells: endothelial progenitor cells and cardiomyocytes, valuable cell types for designing microtissues. Endothelial cells comprise the luminal lining of blood and lymphatic vessels, providing the integral structure for distribution within the body, separating mobile and stationary tissues. Cardiomyocytes provide the force required to pump blood throughout the human body and are a highly desired cell type in regenerative medicine.</p> <p>In this project, we have applied an optogenetic induced signaling pathway, OptoWnt, to differentiate human pluripotent stem cells (hPSCs) into cardiovascular cells via light-induced activation of Wnt signaling pathway. In the analysis of these cells and comparison to previous small molecule approaches to cardiovascular cell differentiation, we demonstrate the robustness of the optogenetic approach and similar efficiency that it has with the small molecule approach. In short, we have further demonstrated the utility and potential of optogenetic induction of developmental pathways, via the OptoWnt construct.</p>

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13464号 / 論医博第2251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 金子 新, 教授 江藤 浩之, 教授 髙折 晃史 / 学位規則第4条第2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23602号 / 医博第4789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 長船 健二, 教授 川口 義弥, 教授 大森 孝一 / 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・論文博士 / 博士(医学) / 乙第12788号 / 論医博第2068号 / 新制||医||1000(附属図書館) / 30807 / 京都大学大学院医学研究科医学専攻 / (主査)教授 瀬原 淳子, 教授 髙橋 淳, 教授 山下 潤 / 学位規則第4条第2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18850号 / 医博第3961号 / 新制||医||1007(附属図書館) / 31801 / 京都大学大学院医学研究科医学専攻 / (主査)教授 山下 潤, 教授 吉村 長久, 教授 中畑 龍俊 / 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19561号 / 医博第4068号 / 新制||医||1013(附属図書館) / 32597 / 京都大学大学院医学研究科医学専攻 / (主査)教授 吉村 長久, 教授 戸口田 淳也, 教授 高橋 淳 / 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19592号 / 医博第4099号 / 新制||医||1014(附属図書館) / 32628 / 京都大学大学院医学研究科医学専攻 / (主査)教授 斎藤 通紀, 教授 伊達 洋至, 教授 上杉 志成 / 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24532号 / 医科博第146号 / 新制||医科||10(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 金子 新, 教授 上杉 志成, 教授 寺田 智祐 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The discovery of resetting human somatic cells via introduction of four transcription factors into an embryonic stem cell-like state that enables the generation of any cell type of the human body has revolutionized the field of medical science. The generation of patient-derived iPSCs and the subsequent differentiation into the cells of interest has been, nowadays, widely used as model system for various inherited diseases. The aim of this thesis was to generate iPSCs and to subsequently derive NPCs which can be differentiated into neurons in order to model the two most common forms of the NCLs: LINCL which is caused by mutations within the TPP1 gene, encoding a lysosomal enzyme, and JNCL which is caused by mutations within the CLN3 gene, affecting a lysosomal transmembrane protein. It was shown that patient-derived fibroblasts can be successfully reprogrammed into iPSCs by using retroviral vectors that introduced the four transcription factors POU5F1, SOX2, KLF4 and MYC. The generated iPSCs were subsequently differentiated into expandable NPCs and finally into mature neurons. Phenotype analysis during the different stages, namely pluripotent iPSCs, multipotent NPCs and finally differentiated neurons, revealed a genotype-specific progression of the disease. The earliest events were observed in organelle disruption such as mitochondria, Golgi and ER which preceded the accumulation of subunit c of the mitochondrial ATPase complex that was only apparent in neurons. However, none of these events led to neurodegeneration in vitro. The established disease models recapitulate phenotypes reported in other NCL disease models such as mouse, dog and sheep model systems. More importantly, the hallmark of the NCLs, accumulation of subunit c in neurons, could be reproduced during the course of disease modeling which demonstrates the suitability of the established system. Moreover, the derived expandable NPC populations can be used for further applications in drug screenings. Their robust phenotypes such as low levels of TPP1 activity in LINCL patient-derived NPCs or cytoplasmic vacuoles, containing storage material, observed in CLN3 mutant NPCs, should serve as possible phenotypic read-outs.

京都大学 / 新制・課程博士 / 博士(医科学) / 甲第24885号 / 医科博第152号 / 新制||医科||10(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 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