京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13113号 / 論医博第2131号 / 新制||医||1022(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 妻木 範行, 教授 安達 泰治, 教授 開 祐司 / 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・論文博士 / 博士(医学) / 乙第13111号 / 論医博第2129号 / 新制||医||1022(附属図書館) / (主査)教授 齊藤 博英, 教授 松原 和夫, 教授 林 康紀 / 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20663号 / 医博第4273号 / 新制||医||1024(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 藤渕 航, 教授 木村 剛, 教授 岩田 想 / 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21006号 / 医博第4352号 / 新制||医||1028(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 高橋 淳, 教授 濵﨑 洋子, 教授 渡邊 直樹 / 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21166号 / 農博第2292号 / 新制||農||1061(附属図書館) / 学位論文||H30||N5140(農学部図書室) / 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, 准教授 南 直治郎 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第21265号 / 医科博第92号 / 新制||医科||7(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 斎藤 通紀, 教授 松田 道行, 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 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21452号 / 医博第4419号 / 新制||医||1032(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 髙橋 良輔, 教授 伊佐 正 / 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21685号 / 医博第4491号 / 京都大学大学院医学研究科医学専攻 / (主査)教授 辻川 明孝, 教授 高橋 淳, 教授 井上 治久 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

<p>  </p> <p>Pluripotent stem cells (PSCs) are increasingly utilized to investigate early human developmental processes including gastrulation and organogenesis of endoderm-derived pancreatic lineages. Critical for tissue development, the PSC niche is a dynamic environment consisting of extracellular matrix (ECM) components that guide cell proliferation, migration, and differentiation. However, investigation of the interplay between the PSC niche and organogenesis has been limited to conventional two-dimensional (2D) cell culture or three-dimensional (3D) platforms requiring use of ill-defined materials (e.g., Matrigel). Furthermore, these systems lack tunability to probe specific qualities of the PSC niche including mechanical properties and biochemical compositions. In this dissertation, modular and dynamic hydrogels were designed to study PSC and niche interactions during differentiation and pancreatic organogenesis. Specifically, two bioorthogonal chemical reactions, thiol-norbornene photopolymerization and tetrazine-norbornene inverse electron demand Diels-Alder (iEDDA) reactions were employed to generate gelatin- and poly(ethylene glycol) (PEG)-based hydrogels with spatiotemporally tunable physicochemical properties. Following mechanical characterization of the hydrogels, the multicomponent gelatin-based hydrogels were assessed for supporting viability and pluripotency of human induced pluripotent stem cells (hiPSCs), as well as for permitting their trilineage differentiation. Next, fully synthetic PEG-based hydrogels with temporally tunable crosslinking density were established to probe the effect of matrix mechanics on definitive endoderm differentiation of the hiPSCs. Finally, hiPSC-to-pancreatic progenitor cell differentiation was explored in both naturally-derived gelatin-based hydrogels and synthetic PEG-based hydrogels, with cells differentiated on a 2D surface as a control. Overall, this work demonstrates that culture dimensionality, material compositions, and mechanics profoundly influence hiPSC differentiation and pancreatic morphogenesis.</p>

Sickle cell anemia, caused by a point mutation that affects the HBB gene, is one of the most common human genetic disorders world-wide and has a high morbidity and mortality. A single FDA approved drug, hydroxyurea, is available for its ability to induce fetal hemoglobin expression, a major modulator of disease severity. Not every patient responds to treatment and additional HbF-inducing drugs are needed. In this thesis, I outline an induced pluripotent stem cell-based approach to the study of sickle cell disease (SCD). In the lab, we are currently building a library of SCD-induced pluripotent stem cell (iPSC) lines from a cohort of SCD patients with different genetic backgrounds and fetal hemoglobin levels. Utilizing a directed-differentiation approach, iPSC can give rise to hematopoietic progenitors that are similar to megakaryocyte-erythroid progenitors and can be further specified to become cells of either lineage. I examined the hypothesis that an iPSC-based system would be capable of producing fully functional erythroid cells and also recapitulate the variation in fetal hemoglobin levels seen in SCD patients. Directed-differentiation of iPSCs produced erythroid-lineage cells that were responsive to oxygen levels and erythropoietin, and were capable of further maturation and increased hemoglobin production. A humanized mouse model demonstrated the ability of these cells to localize to the bone marrow, contribute to the peripheral blood, and survive in vivo for over two weeks. The maturation capability of SCD-specific iPSC-derived erythroid lineage cells was correlated with hemoglobin expression and compared to control cells. Characterization of in vitro and in vivo differences between control and SCD-specific iPSC-derived erythroid-lineage cells demonstrated variation amongst individuals, similar to the variation seen in patients. Both of these patient-specific iPSC-based in vitro and in vivo models allow for the examination of the effect of genetic variability on fetal hemoglobin expression and also for the modeling of patient-specific responses to drug treatment. This information will facilitate better clinical treatment of the disease.