京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19229号 / 医博第4028号 / 新制||医||1011(附属図書館) / 32228 / 京都大学大学院医学研究科医学専攻 / (主査)教授 髙橋 良輔, 教授 妻木 範行, 教授 井上 治久 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Mutations in the gene encoding the bone morphogenetic protein type 2 receptor (BMPR2) are the most common genetic cause of heritable pulmonary arterial hypertension (PAH). However, given the reduced penetrance of BMPR2 mutations in affected families, a major outstanding question is the identity of additional factors or pathways that are responsible for the manifestation of clinical disease. Furthermore, limited human tissue is available for study and usually only from patients with end-stage disease, making it difficult to understand how PAH is established and progresses. Alternative human models of PAH are therefore required. This thesis describes the characterisation of the first human iPSC-derived smooth muscle cell (iPSC-SMC) model of PAH and elucidates the role of BMPR2 deficiency in establishing PAH-associated phenotypes in iPSC-derived SMCs. To achieve this, I used CRISPR-Cas9 gene editing to generate wild-type and BMPR2+/- iPSC lines with isogenic backgrounds which were subsequently differentiated into lineage-specific iPSC-SMCs that displayed a gene expression profile and responses to BMP signalling akin to those present in distal pulmonary artery smooth muscle cells (PASMCs). Using these cells, I found that the introduction of a single BMPR2 mutation in iPSC-SMCs was sufficient to recapitulate the pro-proliferative and anti-apoptotic phenotype of patient-derived BMPR2+/- PASMCs. However, acquisition of the mitochondrial hyperpolarisation phenotype was enhanced by inflammatory signalling and required an interaction between BMPR2 mutations and environmental stimuli provided by exposure to serum over time. Furthermore, I showed that BMPR2+/- iPSC-SMCs had an altered differentiation state and were less contractile compared to wild-type iPSC-SMCs, phenotypes which have not been observed previously in PAH-derived PASMCs. Finally, RNA sequencing analysis identified genes that were differentially expressed between wild-type and BMPR2+/- iPSC-SMCs and may hence provide further insights into PAH pathobiology. The iPSC-SMC model described in this study will be useful for identifying additional factors involved in disease penetrance and for validating therapeutic approaches that target BMPR2.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第22379号 / 医科博第109号 / 新制||医科||7(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 遊佐 宏介, 教授 武田 俊一, 教授 近藤 玄 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Tuberous sclerosis complex (TSC) is a multisystem tumor-forming disorder caused by biallelic inactivation of TSC1 or TSC2. The primary cause of mortality arises from mesenchymal manifestations in the lung and kidney: pulmonary lymphangioleiomyomatosis (LAM) and renal angiomyolipomas (RAMLs). Despite a well-described monogenic etiology, there remains an incomplete understanding of disease pathogenesis. Consequentially, tractable models which fully recapitulate disease characteristics are lacking. Here, I develop and study novel tissue-engineered models of TSC lung and kidney disease. In my first chapter, I demonstrate that lung-mimetic hydrogel culture of pluripotent stem cell-derived diseased cells more faithfully recapitulates human LAM biology compared to conventional culture on two-dimensional plastic. Leveraging this culture system, I conducted a three-dimensional drug screen using a custom 800-compound library, tracking cytotoxicity and invasion modulation phenotypes at the single cell level. I identified histone deacetylase (HDAC) inhibitors as a group of anti-invasive agents that are also selectively cytotoxic towards TSC2-/- cells. HDAC inhibitor therapeutic effects remained consistent in vivo upon xenotransplantation of LAM cellular models into zebrafish. In my second chapter, I develop a genetically-engineered human renal organoid model which recapitulates pleiotropic features of RAMLs in vitro and upon orthotopic xenotransplantation. I find that loss of TSC1/2 affects multiple developmental processes in the renal epithelial, stromal, and glial compartments. First, loss of TSC1/2 leads to an expanded stroma by favouring stromal cell fate acquisition and alters terminal stromal cell identity. Second, epithelial cells in the TSC1/2-/- organoids exhibit a rapamycin-insensitive epithelial-to-mesenchymal transition. Third, a melanocytic population forms exclusively in TSC1/2-/- organoids, branching from MITF+ Schwann cell precursors of a bona fide neural crest-to-Schwann cell differentiation trajectory. Through these two thesis chapters, I realize the power of tissue-engineered models for the study of TSC. This work offers novel insights into the pathogenesis of RAMLs and identifies a new class of therapeutics suitable for trialing in patients with pulmonary LAM.