京都大学 / 新制・論文博士 / 博士(医学) / 乙第13502号 / 論医博第2261号 / 新制||医||1061(附属図書館) / (主査)教授 村川 泰裕, 教授 柳田 素子, 教授 長船 健二 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Cardiovascular disease (CVD) is the leading cause of death due to its prevalence in tandem with the propensity of atherosclerosis to worsen and cause myocardial infarction and stroke. The greatest risk factor for CVD development is age. The multifactorial etiology of atherosclerosis has made CVD difficult to model and consequently little is known about CVD onset and progression. Hutchinson-Gilford Progeria Syndrome (HGPS) is a severe human premature aging disorder caused by a mutation in Lamin A that leads to the accumulation of an aberrant Lamin A protein termed progerin. Patients who harbour this mutation develop atherosclerosis and die from myocardial infarction or stroke at an average age of 13 years old. Autopsies reveal deterioration of vascular smooth muscle cells (VSMCs) in HGPS patients, underlining a strong connection between VSMC loss and predisposition to CVD development. The major aim of this thesis was to model normative vascular aging and disease using HGPS induced pluripotent stem cell (iPSC)-derived VSMCs and monitor the onset of defective epigenetic inheritance in vitro. My results indicate reprogramming of patient fibroblasts to restores a normal nuclear phenotype. Patient derived iPSC lines generated from fibroblasts are nearly indistinguishable from healthy controls in terms of pluripotency, nuclear membrane integrity, as well as transcriptional and epigenetic profiles. However, differentiation of HGPS iPSCs to generate HGPS VSMCs recapitulates many aspects of normative vascular aging exemplified by increased ROS, DNA damage and transcriptomic aberrations. Furthermore, using a multi-omic approach including RNA-sequencing, and accelerated native isolation of protein on nascent DNA, HGPS VSMCs demonstrate loss of histone acetylation due to defective MOF abundance that contributed to impaired engagement with DNA damage repair pathway. This dissertation provides insights on the mechanisms that drive the epigenetic and transcriptomic changes in HGPS vasculature, illuminating druggable pathways that may also drive CVD in the general population.

Acute myeloid leukemia (AML) is a fatal cancer of the human hematopoietic system characterized by the rapid accumulation of non-functional, immature hematopoietic cells in the bone marrow (BM) and peripheral blood (PB) of affected patients. Limited sources of safe hematopoietic stem/progenitor cells (HSPCs) for transplantation and incomplete mechanistic understandings of disease initiation, progression and maintenance have impeded advances in therapy required for improvement of long-term AML patient survival rates. Toward addressing these unmet clinical needs, the ability to generate induced pluripotent stem cells (iPSCs) from human somatic cells may provide platforms from which to develop patient-specific (autologous) cell-based therapies and disease models. However, the ability to generate iPSCs from human AML patient somatic cells had not been investigated prior to this dissertation. Accordingly, I hypothesized that cellular reprogramming of human AML patient somatic cells to iPSCs is possible and will enable derivation of autologous sources of normal and dysfunctional hematopoietic progenitor cells (HPCs). I first postulated that reprogramming AML patient fibroblasts (AML Fibs) to pluripotency would provide a novel source of normal autologous HPCs. Our findings revealed that AML patient-specific iPSCs devoid of leukemia-associated aberrations found in the matched bone marrow (BM) could be generated from AML Fibs, and demonstrated that this cellular platform allowed for the derivation of healthy HPCs capable of normal differentiation to mature myeloid lineages in vitro. During the tenure of these experiments we also redefined conventional reprogramming methods by discovering that OCT4 transcription factor delivery combined with culture in pluripotent-supportive media was minimally sufficient to induce pluripotency in AML and normal Fibs. Toward capturing and modeling the molecular heterogeneity observed across human AML samples in vitro, we next asked whether reprogramming of AML patient leukemic cells would enable generation of iPSCs and derivative HPCs that recapitulated dysfunctional differentiation features of primary disease. Our results demonstrated that conventional reprogramming conditions were insufficient to induce pluripotency in leukemic cells, but that generation of AML iPSCs could be reproducibly achieved in one AML sample when reprogramming conditions were modified. These AML iPSCs and their derivative HPCs harboured and expressed the leukemia-associated aberration found in the BM leukemic cells and similarly possessed dysfunctional differentiation capacities. Together, this body of works provides the proof of principle that cellular reprogramming can be applied on a personalized basis to generate normal and dysfunctional HPCs from AML patient somatic cells. These foundational findings should motivate additional studies aimed at developing iPSC-based cell therapies and disease models toward improving AML patient quality of life and long-term survival rates. / Thesis / Doctor of Philosophy (PhD)

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13569号 / 論医博第2295号 / 新制||医||1068(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 濵﨑 洋子, 教授 生田 宏一, 教授 滝田 順子 / 学位規則第4条第2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24878号 / 医博第5012号 / 新制||医||1068(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 萩原 正敏, 教授 湊谷 謙司, 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

Stem cell treatments for spinal cord injury (SCI) are effective in pre-clinical animal model research but not yet for humans. Two promising stem cell repair strategies involve (1) endogenous neural stem/progenitor cells (NSPCs) and (2) induced pluripotent stem cells (iPSCs). Delineating species differences in spinal cord NSPC biology is essential to inform human SCI endogenous regeneration and repair. Understanding the phenotypic differences between iPSC-derived NSPCs and primary spinal cord NSPCs would also improve the clinical application of iPSC-derived NSPC therapy in human SCI. To directly compare the molecular and functional attributes of spinal cord NSPCs between humans and animal models of SCI, we designed an in vitro model that allows the characterization of primary human, pig, and rat NSPCs under identical conditions. We found an enrichment of transcription factors in NSPCs of either species that may underlie their differentiation and proliferation potentials. Specifically, human NSPCs are neurogenic, whereas pig and rat NSPCs are gliogenic. Also, the proliferation rate of human and pig NSPCs is less than rat NSPCs. Subsequently, we expanded our in vitro model to examine the responses of NSPCs to inflammation and regenerative factors. Surprisingly, inflammation had induced glial scarring mechanisms from pig and rat NSPCs but potentiated neurogenesis of human NSPCs. We also found species-specific responses to regenerative factors that depend on the type of factor used, concentration, and duration of treatment. To assess the extent that iPSC-derived NSPCs phenocopy primary spinal cord NSPCs, we created iPSC-derived NSPCs with a previously reported brain or spinal cord phenotype and directly compared them with isogenic primary NSPCs. We found that iPSC-derived NSPCs exhibit an earlier developmental stage and a greater proliferation rate. We also found that primary NSPCs possess a unique differentiation potential and regional polarity along the rostral-caudal and dorsoventral axes. In summary, we discovered that species differences in NSPC biology exist between human and animal primary spinal cord NSPCs and that iPSC-derived NSPCs do not recapitulate the transcriptional nor functional attributes of primary spinal cord NSPCs. This thesis highlights the translational gap between pre-clinical research and the clinical application of stem cell treatments that target endogenous NSPCs or transplant iPSC-derived NSPCs.

In the distal lung, alveolar epithelial type I cells (AT1s) comprise the vast majority of alveolar surface area and are uniquely flattened to allow the diffusion of oxygen into the capillaries. This structure along with a quiescent, terminally differentiated phenotype has made AT1s particularly challenging to isolate or maintain in cell culture. As a result, there is a lack of established models for the study of human AT1 biology, and in contrast to alveolar epithelial type II cells (AT2s), little is known about the mechanisms regulating their differentiation. Here we engineer a human in vitro AT1 model system through the directed differentiation of induced pluripotent stem cells (iPSC). We first define the global transcriptomes of primary adult human AT1s, suggesting gene-set benchmarks and pathways, such as Hippo-LATS-YAP/TAZ signaling, that are enriched in these cells. Next, we generate iPSC-derived AT2s (iAT2s) and find that activating nuclear YAP signaling is sufficient to promote a broad transcriptomic shift from AT2 to AT1 gene programs. The resulting cells express a molecular, morphologic, and functional phenotype reminiscent of human AT1 cells, including the capacity to form a flat epithelial barrier which produces characteristic extracellular matrix molecules and secreted ligands. Our results indicate a role for Hippo-LATS-YAP signaling in the differentiation of human AT1s and demonstrate the generation of viable AT1-like cells from iAT2s, providing an in vitro model of human alveolar epithelial differentiation and a potential source of human AT1s that until now have been challenging to viably obtain from patients.

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13586号 / 論医博第2306号 / 新制||医||1070(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 波多野 悦朗, 教授 齋藤 潤 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

No description available.