No description available.

Sight is the most powerful of all human senses. For the vast majority of people on Earth, the loss of that sense would be unimaginable. Without assistive technology, it would separate them from their ability to work, their ability to travel, and their ability to interact with their loved ones. And yet, this extraordinary process, carefully refined by billions of years of evolution, is threatened for millions of people all over the world from a wide array of diseases of the retina. Many of these diseases arise from malnutrition and infection and are being rapidly eradicated. However, many dozens more result from convoluted permutations of genetics, age, and diet that threaten blindness for millions more with little hope of treatment, even with the best of modern medicine. As our life expectancies extend and our population ages, these diseases will only become more prevalent. In humanity's ever-present pursuit of medicine and knowledge to improve our quality of life, cutting-edge treatments offer promise that one day soon, even these diseases may be eradicated. One key technology capable of treating these devastating illnesses, on the precipice of being translated to real-world clinical treatments, is pluripotent stem cell-derived therapies. Patient-specific pluripotent stem cells, meaning pluripotent stem cells sourced directly from the patient, have a wealth of applications ranging from drug identification to disease modeling to implantation and regeneration. This research has been developed and advanced remarkably in the approximately two decades since the early isolation of pluripotent stem cells. Naturally, this advancement has predominantly been focused on cell and molecular biology. However, this focus has left significant research questions to be answered from engineering perspectives across a wide latitude of sub-disciplines. This dissertation explores three independent avenues of engineering principles as they relate to improving 2D and 3D retinal tissues derived from pluripotent stem cells in materials, devices, and computation. The first aim explores how plant protein-based nanofibrous scaffolds can marry the advantages and minimize the disadvantages of synthetic and animal-derived scaffolds for the culture of 2D retinal pigment epithelium (RPE) constructs. The second aim describes the development and testing of a novel, perfusing rotating wall vessel (RWV) bioreactor to support culture of 3D retinal organoids. Finally, the third aim performs a meta-analysis of published RNA-Seq datasets to determine the precise mechanisms by which bioreactors support organoid growth and extrapolate how these conclusions can support future experiments. / Bioengineering

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23569号 / 医博第4783号 / 新制||医||1054(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 川口 義弥, 教授 篠原 隆司, 教授 近藤 玄 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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京都大学 / 新制・論文博士 / 博士(医科学) / 乙第13518号 / 論医科博第10号 / 新制||医科||10(附属図書館) / (主査)教授 金子 新, 教授 松田 秀一, 教授 山中 伸弥 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

With so many deaths around the world being due to cardiovascular disease, there is great demand for an unlimited supply of cells for application in regenerative medicine. However, the control of directing pluripotent cells into mesoderm lineage which gives rise to cardiac muscle cells remains poor. Here in this work, the synergistic effect of chemical and mechanical signaling in driving cells towards mesoderm germ-layer was investigated. Transgenic reporter mouse embryonic stem cells were used in this study. The reporter cell line shows the cellular endogenous activity of pluripotency gene Oct3/4 with green fluorescent protein (GFP) while also reporting mesoderm-specific gene, Brachyury, activity with DsRed fluorescent protein. To promote adhesion of mouse embryonic stem cells and to initiate integrin-based mechanical signaling, the extracellular matrix protein fibronectin was used. In the presence of fibronectin and a small molecule called leukemia inhibitory factor (LIF), which is known to maintain pluripotency and self-renewal, mouse embryonic stem cells responded by exhibiting expressions of both GFP and DsRed. To further promote differentiation and to increase mechanical signaling, Notch signaling was activated by presenting cells with fibronectin and DLL1 protein. The differentiation was found to have a pronounced effect in the presence of fibronectin and DLL1 protein together with the withdrawal of LIF. This is a proof-of-concept that mechanical signaling together with synergistic chemical signaling can drive pluripotent cells towards mesoderm lineage. Future studies with human pluripotent stem cells may transduce cells towards mesoderm and finally towards cardiac fate. Collectively, this study shows the importance of chemical, but also the importance of mechanical signaling to transduce cells to mesoderm.

京都大学 / 新制・課程博士 / 博士(理学) / 甲第24081号 / 理博第4848号 / 新制||理||1694(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 森 和俊, 教授 川口 真也, 准教授 船山 典子 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

<p>Human pluripotent stem cells (hPSCs) exist as a heterogeneous population within a dynamic niche, which governs their ability to self-renew and differentiate. Evidence modeled after mouse embryonic stem cells (mESCs) reveals the existence of a developmentally primitive, or homogeneous, state through chemically defined culture methods that is modulated by NANOG, a core pluripotent regulator. However, the differentiation potential and transcription factor control of the homogeneous state in human pluripotent stem cells remains elusive. Previous work suggests that bFGF/ACTIVIN extrinsic regulation provides the heterogeneous nature of hiPSCs with ability to differentiate into several multilineage lineage progenitors. Here, we illustrate that altering the extrinsic environment of hiPSCs with LIF and inhibitors of GSK3b and MAPK/ERK1/2 pathways (LIF/2i), rewires the intrinsic pluripotent regulation of OCT4 and NANOG, which ultimately prevents the <em>in vitro</em> hematopoietic differentiation potential. Upon conversion of hiPSCs to a primitive state of pluripotency with LIF/2i, this study reveals that prolonged culture of hiPSCs with LIF/2i erases the hematopoietic differentiation potential through retained expression of the POU domain pluripotent transcription factor, OCT4. Interestingly, shRNA mediated knockdown of <em>OCT4</em> recovers the restricted differentiation potential in LIF/2i cultured hiPSCs, while knockdown of <em>NANOG</em>, does not. This study identifies a distorted differentiation potential of hPSCs cultured in mouse ESC conditions, despite comparable gene expression profiles and signaling pathway dependence. In efforts to simplify culture methods of human pluripotent stem cells, we identify that alteration of the extrinsic environment highlights explicit differences between human and mouse intrinsic pluripotent regulation, which ultimately controls differentiation efficiency.</p> / Master of Science (MSc)

京都大学 / 新制・課程博士 / 博士(医学) / 甲第25490号 / 医博第5090号 / 新制||医||1073(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 江藤 浩之, 教授 斎藤 通紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Doctor of Philosophy / The liver is the largest internal organ in the body. It is responsible for performing an array of vital functions, including the filtration of blood, synthesis of different molecules, and metabolism of drugs and toxicants. Hepatocytes, or the main liver cell type, perform most of these functions. Typically, primary human hepatocytes (PHHs) are ideal for in vitro liver studies since they are obtained directly from tissues and exhibit adult characteristics and functions. However, sourcing these adult liver cells is extremely difficult since they are usually obtained through biopsies and are limited in quantity. Induced pluripotent stem cell (iPSC)-hepatocyte-like cells (iHLCs) hold tremendous potential to be used as a substitute since they can be obtained non-invasively. However, iHLCs require further maturation before they can be substituted for PHHs due to their lower liver-related functions and immature characteristics. Existing maturation approaches require the administration of chemical mixtures that can be up to a million times higher than the concentrations of these same molecules inside the body. The lack of a systematic approach to mature iHLCs currently limits their widespread use. During liver development, neighboring cells secrete different proteins that regulate and induce multiple pathways that aid in the maturation of cells into adult liver cells. We report the assembly of a multicellular 3D human liver organoid with iHLCs that recapitulates the in vivo hepatic microenvironment. Intra- and intercellular signaling between human hepatic cells in the organoid result in mature iHLCs that exhibit several markers and functions of PHHs within one to two weeks in culture. When two other hepatic cell types, Kupffer cells and liver sinusoidal endothelial cells, are present in the organoids, they secrete signaling molecules that synergistically mature iHLCs. Relying solely on intercellular secretion from the cells provides a systematic and reproducible approach to generate mature iHLCs for drug, disease, and patient-specific in vitro studies.