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.

abstract: An in vitro model of Alzheimer’s disease (AD) is required to study the poorly understood molecular mechanisms involved in the familial and sporadic forms of the disease. Animal models have previously proven to be useful in studying familial Alzheimer’s disease (AD) by the introduction of AD related mutations in the animal genome and by the overexpression of AD related proteins. The genetics of sporadic Alzheimer’s is however too complex to model in an animal model. More recently, AD human induced pluripotent stem cells (hiPSCs) have been used to study the disease in a dish. However, AD hiPSC derived neurons do not faithfully reflect all the molecular characteristics and phenotypes observed in the aged cells with neurodegenerative disease. The truncated form of nuclear protein Lamin-A, progerin, has been implicated in premature aging and is found in increasing concentrations as normal cells age. We hypothesized that by overexpressing progerin, we can cause cells to ‘age’ and display the neurodegenerative effects observed with aging in both diseased and normal cells. To answer this hypothesis, we first generated a retrovirus that allows for the overexpression of progerin in AD and non-demented control (NDC) hiPSC derived neural progenitor cells(NPCs). Subsequently, we generated a pure population of hNPCs that overexpress progerin and wild type lamin. Finally, we analyzed the presence of various age related phenotypes such as abnormal nuclear structure and the loss of nuclear lamina associated proteins to characterize ‘aging’ in these cells. / Dissertation/Thesis / Masters Thesis Bioengineering 2017

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

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13354号 / 論医博第2200号 / 新制||医||1044(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 木村 剛, 教授 羽賀 博典 / 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13377号 / 論医博第2211号 / 新制||医||1047(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 稲垣 暢也 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM