In addition to the well-characterized Induced Pluripotent Stem cells (iPSCs) that closely resemble Embryonic Stem cells (ESCs), a recent study has proven the existence of a stable state, resembling partially reprogrammed cells, termed F-class iPSCs. To study these distinct iPSC states, a reprogramming dataset has been generated, featuring the parallel analysis of multiple molecular platforms. MicroRNAs (miRNAs) are small RNA regulators of gene expression whose critical role in reprogramming is now being realized. In the present study, small RNA deep sequencing data from this novel reprogramming dataset was used to identify miRNAs that are likely to enhance reprogramming by detecting significantly up-regulated miRNAs in ESC-like iPSCs versus F-class iPSCs. These candidate miRNAs were cloned and overexpressed in reprogramming mouse embryonic fibroblasts and their effect on reprogramming efficiency was measured. miR-214 was discovered to increase iPSC generation efficiency, marking the first reprogramming-related role for this microRNA.

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<p><p> Articular cartilage is a smooth connective tissue that covers the ends of bones and protects joints from wear. Cartilage has a poor healing capacity, and the lack of treatment options motivates the development of tissue engineering strategies. The widespread cartilage degeneration associated with osteoarthritis (OA) is dramatically accelerated by joint injury, but the defined initiating event presents a therapeutic window for preventive treatments. In vitro model systems allow investigation of OA risk factors and screening of potential therapeutics. This dissertation develops stem-cell based strategies to 1) treat cartilage injury and OA using tissue-engineered cartilage, 2) prevent the development of OA by delivering stem cells to the joint after injury, and 3) study cartilage by establishing systems to model genetic and environmental contributors to OA.</p><p> Adipose-derived stem cells (ASCs) and bone marrow-derived mesenchymal stem cells (MSCs) are promising human adult cell sources for cartilage tissue engineering, but require distinct chondrogenic conditions. As compared to ASCs, MSCs demonstrated enhanced chondrogenesis in both alginate beads and cartilage-derived matrix scaffolds. </p><p> We hypothesized that MSC therapy would prevent post-traumatic arthritis (PTA) by altering the balance of inflammation and regeneration. Highly purified MSCs (CD45-TER119-PDGFR&#945;+Sca-1+) rapidly expanded under hypoxic conditions. Unexpectedly, MSCs from control C57BL/6 (B6) mice proliferated and differentiated more than MSCs from MRL/MpJ (MRL) "superhealer" mice. We injected B6 or MRL MSCs into mouse knees immediately after fracture, and MSCs of either strain were sufficient to prevent PTA. </p><p> Genetically reprogramming adult cells into induced pluripotent stem cells (iPSCs) generates large numbers of patient-matched cells with chondrogenic potential for therapy and cartilage modeling. We produced murine iPSC-derived cartilage constructs with a multi-phase approach involving micromass culture with bone morphogenetic protein-4, flow cytometry cell sorting of chondrocyte-like cells, monolayer expansion, and pellet culture with transforming growth factor-beta 3. Successful differentiation was confirmed by increased chondrogenic gene expression, robust synthesis of glycosaminoglycans and type II collagen, and the repair of an in vitro cartilage defect. </p><p> The diverse applications pursued in this research illustrate the power of stem cells to deepen the understanding of cartilage and guide the development of therapies to prevent and treat cartilage injury and OA.</p> / Dissertation

Megakaryocyte specification is the process by which discrete hematopoietic subpopulations undergo lineage commitment towards the myeloid compartment, finally specifying as a megakaryocyte erythroid progenitor (MEP) by way of thrombopoietin (TPO) and erythropoietin (EPO) signaling, before becoming a megakaryocyte lineage restricted progenitor that will progressively increase cellular ploidy and compartmentalize its cytoplasm in preparation for platelet production. With the advent of induced pluripotent stem cells (iPSCs), a cell type that is experimentally manipulated to function as embryonically derived pluripotent cells, there now exists the ability to analyze signal transduction throughout discrete phases of hematopoiesis, megakaryocyte lineage cell fate, and platelet production. Recent studies have implicated the aryl hydrocarbon receptor (AHR) as a transcription factor that plays a critical role in multiple aspects of hematopoiesis. These results inspired the hypothesis that AHR signaling may be functionally relevant in the context of megakaryopoiesis. To test this hypothesis, an iPSC directed differentiation strategy was established in order to create a platform upon which to experimentally manipulate AHR signaling throughout megakaryocyte specification. The results demonstrate: 1) iPSC derived hematopoietic progenitor cells (HPCs) undergo exponential expansion upon AHR agonism; 2) AHR antagonism allows for megakaryocyte lineage bias; 3) Optimization of directed-differentiation allows for the examination of AHR signaling in megakaryocyte lineage-restricted cells; 4) AHR signaling suppresses the expression of MPL, the gene that encodes the thrombopoietin receptor (C-MPL) in iPSC derived megakaryocyte lineage committed cells; 5) AHR activation concomitantly suppresses cell surface expression of C-MPL, which may alter the sensitivity of HPCs to TPO signaling; 6) Multiple gene targets are modulated by AHR activation within megakaryocyte lineage cells, providing evidence of a transcriptional program downstream of AHR signaling that preferentially suppresses megakaryocyte specification; 7) A reporter iPSC line of AHR activity provides evidence of endogenous AHR signaling throughout megakaryocyte specification and shows a sharp decline in AHR activity upon megakaryocyte lineage commitment; 8) In a mouse model of megakaryocyte lineage specific AHR knockout, platelet counts are significantly reduced. These data suggest that the AHR plays a significant role in megakaryocyte specification by modulating the expression of multiple lineage specific gene targets, including MPL, the thrombopoietin receptor. / 2017-05-02T00:00:00Z

Studies to gain mechanistic understanding of heart dysfunction based on animal and traditional cell culture models have significant limitations. Animal models are low throughput and fail to recapitulate many aspects of human cardiac biology, and 2D culture models utilizing human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) are higher throughput but fail to incorporate one or more in vivo parameters, such as 3D architecture, electrical pacing and mechanical constraint. High throughput 3D tissue platforms could better recapitulate the in vivo microenvironment of cardiac tissue. Previous work from our group demonstrated an approach to build 3D cardiac microtissues based on photolithography-based fabrication of a MEMS device, but design limitations prevented further iterations. In this work, we used a 3D printing approach to engineer iPSC-CM-derived cardiac microtissues with different form factors. Microtissues generated in this platform increased in lifespan compared to the first-generation platform by more than 100%. When modeling mutations associated with genetic cardiomyopathy, functional and structural differences were observed between tissues composed of wild-type and mutant iPSC-CMs. These findings suggest that this micro-device platform can be potentially used for both mechanistic and drug discovery studies. / 2020-07-02T00:00:00Z

There exists a need to produce platelets in vitro for use in transfusion medicine, due to increased platelet demands and short shelf life. Our lab uses human induced pluripotent stem cells (iPSCs), as an attractive alternative supply, as iPSCs can be cultured indefinitely and differentiate into almost any cell type. Using a technique called forward programming, we over express three key haematological transcription factors (TFs), pushing iPSCs towards the megakaryocyte lineage, to produce mature megakaryocytes, the platelet precursor cell type. A major limitation of the forward programming technique is a reliance of lentiviral transduction to overexpress the three TFs, which leads to a number of issues including heterogeneity and high experimental costs. To overcome this, I have developed an inducible iPSC line by inserting the forward programming TFs into a genomic safe harbour, using genome editing techniques. TF expression is strictly controlled, with the TFs expressed only after chemical induction. Inducing forward programming is an efficient method for producing mature megakaryocytes and these cells maintain higher purity in long-term cultures, when compared to cells produced by the lentiviral method. Removing the requirement of lentiviral transduction is a major advancement, making forward programming more amenable to scaling-up, thus moving this technology closer towards our goal of producing in vitro platelets for use in transfusion medicine. I have also shown that forward programming generates a bi-potent progenitor population, from which erythroblasts can be generated, by altering only media conditions. As for megakaryocyte cultures, inducing forward programming improves the purity of erythroblasts produced, compared to the lentiviral method. I have developed single cell progenitor assays combined with index sorting of different cell surface markers, to allow retrospective analysis of cells which successfully generate colonies. The aim of this work is to better characterise the progenitor cells produced by forward programming, to allow further study of this cell type. Single cell RNA-seq of megakaryocytes revealed heterogeneity in long-term cultures and also identified novel candidate surface markers that may help to further characterise the progenitor cell population.

Ebola virus (EBOV) infection causes a severe disease in humans and leads to widespread liver necrosis, a dysregulated cytokine response, and coagulopathy. However, little is known about the specific liver response to EBOV infection in humans. Here we present the utilization of an induced pluripotent stem cell (iPSC)-derived hepatocyte platform to define the host response to EBOV infection. We demonstrate that iPSC-derived hepatocytes are a suitable platform for investigating innate immune responses to viral infections. We compared the host response to EBOV infection in iPSC-derived hepatocytes, immortalized hepatocytes, and primary human hepatocytes and identified minimal transcriptomic changes 1 day post infection (dpi). Between 2-3 dpi, EBOV infection led to a significant upregulation of interferon-beta (IFN-β) and select interferon-stimulated genes (ISGs) in iPSC-derived hepatocytes. In addition, the acute phase response and coagulation cascade was downregulated in these hepatocytes, mimicking known liver dysfunction in EBOV disease. Using fluorescent in situ hybridization (RNA-FISH), we showed at single cell resolution that EBOV-infected iPSC-derived hepatocytes express IFN-β, indicating that infected cells mount an antiviral response to EBOV infection. This platform can be utilized to investigate therapeutic targets in human hepatocytes that may attenuate EBOV infection in patients. In addition, we present in this dissertation the development of a minigenome system for the filovirus Lloviu virus (LLOV). LLOV is closely related to EBOV and is known to circulate in bats throughout Europe. The complete sequence of LLOV has yet to be resolved, and therefore investigation of LLOV biology is limited. As part of this work, we established a functional LLOV minigenome system based on sequence complementation of other filoviruses. We demonstrate that the LLOV replication and transcription strategy is generally more similar to ebolaviruses than marburgviruses. We show that a single nucleotide at the 3ꞌ end of the LLOV genome determines specificity of the LLOV polymerase complex. This minigenome system can now be used to elucidate replication and transcription mechanisms employed by this novel filovirus. / 2023-02-19T00:00:00Z

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21663号 / 医博第4469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 篠原 隆司, 教授 万代 昌紀, 教授 近藤 玄 / 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22306号 / 医博第4547号 / 新制||医||1040(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 平井 豊博, 教授 川口 義弥 / 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(医学) / 甲第22335号 / 医博第4576号 / 新制||医||1041(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 山下 潤, 教授 岩田 想, 教授 木村 剛 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM