Research into the molecular pathology of prevalent public health epidemics such as neurodegenerative diseases including frontotemporal dementia (FTD) and Alzheimer’s Disease (AD), non-medical and illicit opioid use (OU), and Human Immunodeficiency Virus-1 (HIV-1) has been hindered by a lack of systems that allow for rapid and high-throughput modeling of the complex multifactorial conditions in a human context. In this thesis we have addressed this challenge using a multi-pronged approach that encompasses single cell RNA sequencing (scRNA-seq) of three-dimensional (3D) human induced pluripotent stem cell (hiPSC) assembloid culture models and patient derived peripheral blood mononuclear cell (PBMC) samples. We describe the development of an iPSC derived neuron-astrocyte assembloid model of tauopathies, including FTD and AD (AstTau), that rapidly recapitulates propagation of toxic human oligomeric tau (oTau) and cell type specific pathology including misfolded, phosphorylated, oligomeric, and fibrillar tau, strong neurodegeneration, and reactive astrogliosis. scRNA-seq identified vulnerable excitatory neuron specific inflammatory pathways and a heat shock response in astrocytes, recapitulating transcriptomic signatures of adult neurodegeneration and supporting a hypothesis of cell type specific neuroinflammation in tau pathogenesis. To more completely model AD, we incorporated amyloid precursor protein (APP) mutant iPSCs into the assembloid model. These iPSCs contained the familial AD APP V717I mutation or the isogenic CRISPR corrected control, and were used to derived neurons, astrocytes, and microglia. This advanced combinatorial system (AstAD and MAstAD) enabled selective microglial incorporation, APP mutation expression, and oTau seeding allowing us to identify discrete contributions to AD pathogenesis. Ast/MAstAD developed extracellular amyloid-β (Aβ) and microglial activation in addition to the pathology observed in AstTau. scRNA-seq identified divergent microglial activation in response to Aβ and oTau pathology, with APP V717I mutation and oTau seeding synergistically exacerbating AD phenotypes. These assembloid models enable study of the cellular and molecular inflammatory mechanisms in multifactorial neurodegenerative diseases. To better understand disease signatures at the crossroads of multifactorial OU, HIV-1, and antiretroviral (ART) viral suppression we also produced a scRNA-seq data set of more than 100,000 peripheral blood mononuclear cells (PBMCs) from 75 study participants. We identified chronic immune activation and T cell activation dysfunction driven by interferon transcriptomic signatures that were elevated in people with HIV (PWH) with opioid use as compared to PWH without OU. We also identified a functional natural killer cell subtype that was depleted with OU in PWH. Cessation of OU reduced these potentially deleterious inflammatory transcriptomic profiles, supporting the hypothesis that OU in PWH amplifies a state of chronic immune activation. Taken together, single cell transcriptomic resolution has enabled the identification of cell type specific disease signatures in complex pathophysiologies. These data demonstrate the dynamic range of inflammatory signaling across multifactorial disease states and emphasize the need for disease- and cell- type specific approaches to therapeutic development. / 2025-02-05T00:00:00Z

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24197号 / 医博第4891号 / 新制||医||1060(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 平井 豊博, 教授 万代 昌紀, 教授 伊達 洋至 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24830号 / 医博第4998号 / 新制||医||1067(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 後藤, 慎平, 教授 河本, 宏, 教授 羽賀, 博典 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

A cell, the building block of all life, stores a plethora of information in its genome, epigenome, and transcriptome which needs to be analyzed via various Omic studies. The heterogeneity in a seemingly similar group of cells is an important factor to consider and it could lead us to better understand processes such as cancer development and resistance to treatment, fetal development, and immune response. There is an ever growing demand to be able to develop more sensitive, accurate and robust ways to study Omic information and to analyze subtle biological variation between samples even with limited starting material obtained from a single cell. Microfluidics has opened up new and exciting possibilities that have revolutionized how we study and manipulate the contents of the cell like the DNA, RNA, proteins, etc. Microfluidics in conjunction with Next Gen Sequencing has provided ground-breaking capabilities for handling small sample volumes and has also provided scope for automation and multiplexing. In this thesis, we discuss a number of platforms for developing low-input or single cell Omic technologies. The first part talks about the development of a novel microfluidic platform to carry out single-cell RNA-sequencing in a one-pot method with a diffusion-based reagent swapping scheme. This platform helps to overcome the limitations of conventional microfluidic RNA seq methods reported in literature that use complicated multiple-chambered devices. It also provides good quality data that is comparable to state-of-the-art scRNA-seq methods while implementing a simpler device design that permits multiplexing. The second part talks about studying the transcriptome of innate leukocytes treated with varying levels of LPS and using RNA-seq to observe how innate immune cells undergo epigenetic reprogramming to develop phenotypes of memory cells. The third part discusses a low-cost alternative to produce tn5 enzyme which low-cost NGS studies. And finally, we discuss a microfluidic approach to carrying out low-input epigenomic studies for studying transcription factors. Today, single-cell or low-input Omic studies are rapidly moving into the clinical setting to enable studies of patient samples for personalized medicine. Our approaches and platforms will no doubt be important for transcriptomic and epigenomic studies of scarce cell samples under such settings. / Doctor of Philosophy / This is the era of personalized medicine which means that we are no longer looking at one-size-fits-all therapies. We are rather focused on finding therapies that are tailormade to every individual’s personal needs. This has become more and more essential in the context of serious diseases like cancer where therapies have a lot of side-effects. To provide tailor-made therapy to patients, it is important to know how each patient is different from another. This difference can be found from studying how the individual is unique or different at the cellular level i.e. by looking into the contents of the cell like DNA, RNA, and chromatin. In this thesis, we discussed a number of projects which we can contribute to advancement in this field of personalized medicine. Our first project, MID-RNA-seq offers a new platform for studying the information contained in the RNA of a single cell. This platform has enough potential to be scaled up and automated into an excellent platform for studying the RNA of rare or limited patient samples. The second project discussed in this thesis involves studying the RNA of innate immune cells which defend our bodies against pathogens. The RNA data that we have unearthed in this project provides an immense scope for understanding innate immunity. This data provides our biologist collaborators the scope to test various pathways in innate immune cells and their roles in innate immune modulation. Our third project discusses a method to produce an enzyme called ‘Tn5’ which is necessary for studying the sequence of DNA. This enzyme which is commercially available has a very high cost associated with it but because we produced it in the lab, we were able to greatly reduce costs. The fourth project discussed involves the study of chromatin structure in cells and enables us to understand how our lifestyle choices change the expression or repression of genes in the cell, a study called epigenetics. The findings of this study would enable us to study epigenomic profiles from limited patient samples. Overall, our projects have enabled us to understand the information from cells especially when we have limited cell numbers. Once we have all this information we can compare how each patient is different from others. The future brings us closer to putting this into clinical practice and assigning different therapies to patients based on such data.

Macrophages are leukocytes of the innate immune system that display great phenotypic plasticity to mediate diverse functions. The ontogeny of tissue resident macrophages has been debated in recent decades. It is now recognised that tissue macrophages can be replenished from embryonically-derived precursors, and/or monocyte intermediates in a tissue specific manner. Interferon Regulatory Factor 5 (IRF5) is a transcription factor that promotes a pro-inflammatory phenotype in macrophages in vitro and in vivo. Indeed, IRF5 contributes to the pathogenesis of experimental inflammatory arthritis, lupus, and obesity via recruitment and activation of effector cells. Research described here as part of this thesis, involves the profiling of the intestinal Mononuclear Phagocyte system to investigate the role of IRF5 in the development of monocyte-derived macrophages in the Colonic Lamina Propria (cLP) which are exclusively replenished by adult Ly6C^hi monocytes. Using Mixed Bone Marrow Chimaeras (MBMCs) we showed that in shared environment Wild-Type (WT) cLP macrophages dominated IRF5-deficient (Irf5^-/-) cLP macrophages in both steady state and inflammation. The development of in vitro bone marrow derived macrophages, and the reconstitution of the haematopoietic compartment in bone marrow of MBMCs were not significantly affected by IRF5 deficiency. IRF5 promoted the accumulation of WT monocytes in the cLP of MBMCs in a process possibly dependent on the CCL2/CCR2 axis. Furthermore, IRF5 expression committed Ly6C^hi monocytes to a pro-inflammatory macrophage fate in the inflamed cLP, characterised by protein expression of the cytokines IL1β, and TNFα, and the expression of Ccl4 and Ccl8 transcripts, whilst loss of IRF5 favoured accumulation of CD11b⁺ IRF4-dependent Dendritic Cells. Of significance, IRF5 expression might have prevented further differentiation of inflammatory macrophages into tissue-resident macrophages, thus supporting an inflammatory state. Irf5-/- mice were protected from Helicobacter hepaticus + αIL10R colitis. Intriguingly, protection from colitis may also be conferred by the presence of Irf5-/- haematopoietic cells, evidenced by WT:Irf5-/- MBMCs . Modulation of IRF5 activity may therefore be a viable therapeutic strategy. RNA sequencing identified that C1q, Cd81, and Ccl8 were upregulated in WT macrophages from MBMC, which may prove therapeutic targets.

Single-cell RNA sequencing and in situ sequencing were combined in a recent study of the developing human heart to explore the transcriptional landscape at three developmental stages. However, the method used in the study to create the spatial cellular maps has some limitations. It relies on image segmentation of the nuclei and cell types defined in advance by single-cell sequencing. In this study, we applied a new unsupervised approach based on graph neural networks on the in situ sequencing data of the human heart to find spatial gene expression patterns and detect novel cell and sub-cell types. In this thesis, we first introduce some relevant background knowledge about the sequencing techniques that generate our data, machine learning in single-cell analysis, and deep learning on graphs. We have explored several graph neural network models and algorithms to learn embeddings for spatial gene expression. Dimensionality reduction and cluster analysis were performed on the embeddings for visualization and identification of biologically functional domains. Based on the cluster gene expression profiles, locations of the clusters in the heart sections, and comparison with cell types defined in the previous study, the results of our experiments demonstrate that graph neural networks can learn meaningful representations of spatial gene expression in the human heart. We hope further validations of our clustering results could give new insights into cell development and differentiation processes of the human heart.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23082号 / 医博第4709号 / 新制||医||1049(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 妹尾 浩, 教授 村川 泰裕 / 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23382号 / 医博第4751号 / 新制||医||1052(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 伊藤 貴浩, 教授 滝田 順子, 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

The definitive endoderm gives rise to several specialized organs, including the thymus. Improper development of the definite endoderm or its derivatives can lead to human disease; in the case of the thymus, immunodeficiency or autoimmune disorders. Human pluripotent stem cells (hPSCs) have emerged as a system to model human development, as study of their differentiation allows for elucidation of the molecular basis of cell fate decisions, under both healthy and impaired conditions. Here, we first developed a CRISPR-effector system to control endogenous gene expression in hPSCs, a novel approach to manipulating hPSC state. Next, the human-specific, loss-of-function phenotypes of candidate transcription factors driving hPSC-to-definitive endoderm differentiation were analyzed through combined CRISPR-perturbation and single-cell RNA-sequencing. This analysis revealed the importance of TGFβ mediators in human definitive endoderm differentiation as well as identified an unappreciated role for FOXA2 in human foregut development. Finally, as the differentiation of definitive endoderm to thymic epithelial progenitors (TEPs) is of particular interest, a single-cell transcriptomic atlas of murine thymus development was generated in anticipation of identifying factors driving later stages of TEP differentiation. Taken together, this dissertation establishes a CRISPR-effector system to interrogate gene and regulatory element function in hPSC differentiation strategies, details the role of specific transcription factors in human endoderm differentiation, and sets the groundwork for future investigations to characterize hPSC-derived TEPs and the factors driving their differentiation.

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