京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18131号 / 医博第3851号 / 新制||医||1001(附属図書館) / 30989 / 京都大学大学院医学研究科医学専攻 / (主査)教授 千葉 勉, 教授 坂井 義治, 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

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Acute myeloid leukemia (AML), a blood malignancy resulting in abnormal hematopoiesis, is associated with alterations in the bone marrow environment (BME). Current treatments for this heterogeneous disease, mainly targeting the leukemic cells, are largely unsuccessful for the majority of AML subtypes. By better understanding the mechanisms by which the BME contributes to leukemogenesis, it may be possible to introduce more effective treatments for AML. Mesenchymal stromal/stem cells (MSCs) are essential cellular components of the BME/hematopoietic niche and have been shown to support normal hematopoiesis. As a critical component, they may have several roles in altering the BME, thus providing an excellent model for studying the BME in-vitro. Several studies have characterized AML-derived MSCs (AML-MSCs). However, their exact role in altering BME remains unclear. Here, we investigated the MSCs' potential role in BME alteration by investigating the genetic profiles of previously characterized AMLMSCs (n=29) and healthy donor MSCs (HD-MSCs) (n=8). We identified that among 7565 common genes, 21 genes were significantly differentially expressed in AMLMSCs. The CD248 gene was identified among these significantly upregulated genes in AML/HD-MSCs (n=29). Focusing on AML-MSCs derived from high-risk patients (HR), CD248 protein was investigated and validated using HR AML-MSCs (n=11) and HD-MSCs (n=4). Interestingly, it was highly abundant in HR samples at the intracellular and cell-surface levels. CD248 is an MSC marker and has a biological significance potentially on their function. To better understand its potential role in MSC, CD248 was knocked down (KD) in HD-MSCs using siRNA (siCD248-MSCs). Functional capacity, the ability of HD-MSCs and siCD248-MSCs to differentiate into cell types that form the BME (adipocytes and osteocytes), and their ability to promote the growth of HL60 human leukemia cell line were assessed. Posttransfection functional assessments showed that siCD248-MSCs had a reduced adipogenic but not osteogenic potential via differentiation assays. Quantitative validation of the adipogenesis pathway by qRT-PCR confirmed the reduction. KD CD248 increased SIRT2 expression and potentially led to adipogenesis inhibition. However, co-culture experiments showed no effect of HD-MSCs or siCD248-MSCs on HL60 proliferation. Together these data showed that CD248 is a potential player in adipogenesis, essential to MSC’s functionality. Thus, it could serve as a prognostic marker and target for AML therapy.

Preterm birth (<37 weeks) is the world’s principal cause of death of children <5 years of age. Bronchopulmonary dysplasia (BPD) is the most common complication of preterm birth. BPD is characterized by an arrest in alveolar and vascular development within the lung. It is a multifactorial disease, caused by a combination of supplemental oxygen, mechanical ventilation, and inflammation. BPD is also an independent risk factor for abnormal neurodevelopment. The link between BPD and abnormal neurodevelopment is poorly understood and there are currently no effective cures for these complications. We hypothesized that a crucial cell population for brain development, i.e., the neural progenitor cell (NPC) is functionally impaired in BPD and that this impairment is associated with abnormal neurodevelopment. Based on our previous findings, we also predicted that human umbilical cord-mesenchymal stromal cell (UC-MSC) extracellular vesicles (EVs), could mitigate both the lung and brain injuries in experimental BPD. We utilized several animal models of BPD, across multiple species, to determine the effects of hyperoxia, mechanical ventilation, and inflammation on the developing lungs and brain. We also utilized UC- MSC therapy to mitigate these injuries. We discovered that hyperoxia exposure damages the developing lungs as well as the brain, leading to cerebrovascular and NPC impairments, as well as reduced neurogenesis. These impairments were associated with neurobehavioural deficits in adulthood. Furthermore, we found that inflammation in combination with mechanical ventilation and hyperoxia also impairs NPC function. Importantly, we demonstrated that UC-MSC EVs can reduce inflammation, improve vascular growth, restore lung growth, and mitigate impairments in NPC self-renewal. This work highlights novel mechanisms of BPD-associated abnormal neurodevelopment and offers potential regenerative medicine therapies to alleviate these outcomes for this vulnerable population.

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Pulmonary arterial hypertension (PAH) is a devastating lung disease characterized by arterial pruning, occlusive vascular remodeling, and inflammation contributing to increased pulmonary vascular resistance with resultant right heart failure. Endothelial cell (EC) injury and apoptosis are commonly considered triggers for PAH, the mechanisms leading from injury to complex arterial remodeling are incompletely understood. While current therapies can improving symptoms, with the exception of parenteral prostacyclin, they do not significantly prolong transplant free survival. As well, there are no therapies that can regenerate the damaged lung short of transplantation. In this project, I sought to both advance the understanding of disease pathogenesis and explore regenerative therapeutic options for PAH. To this end, I first employed single cell RNA sequencing (scRNA-seq) at multiple time points during the Sugen 5416 (SU) – chronic hypoxia (CH) model of PAH, to provide new insights into PAH pathogenesis both during onset and progression of disease. We also employed microCT analysis to visualize and quantify the arterial pruning associated with PH and found significant loss up to 65% of the healthy arteriolar volume in this model. Through scRNA-seq analysis performed at four timepoints spanning the onset and progression of disease, two disease-specific EC cell types emerged as key drivers of PAH pathogenesis. The first was the emergence of capillary ECs with a de-differentiated gene expression profile, which we termed dedifferentiated capillary (dCap) ECs, with enrichment for the Cd74 gene. Interestingly, RNA velocity analysis suggested that these cells may be undergoing endothelial to mesenchymal transition during PAH development. At later times, a second arterial EC population became apparent, which we termed activated arterial ECs (aAECs), since it uniquely exhibited persistently elevated levels of differential gene expression consistent with a migratory, invasive and proliferative state. Interestingly, the aAECs together with the smooth muscle (SM)-like pericytes, a population which was also greatly expanded in PAH, expressed Tm4sf1, a gene previously associated with a number of cancers and abnormal cell growth. Furthermore, by immunostaining, TM4SF1 was found to be spatially localized to sites of complex and occlusive arterial remodeling, associated with both endothelial cells and pericytes in these lesions, suggesting an important role for the aAECs and SM-like pericytes in arterial remodeling and PH progression. Together, these findings suggest that aAECs, dCap ECs, and SM-like pericytes are emerging cell populations responsible for lung arterial remodeling in PAH, which drives disease progression, and that TM4SF1 may be a novel therapeutic target for this disease. As a first step in trying to develop approaches to regenerate lung arterial bed that is lost in PAH, we investigated the potential role of endothelial colony forming cells (ECFCs) and mesenchymal stromal cell (MSC) derived extracellular vesicles (EVs) as novel therapeutics, on the premise that these stem/progenitor cells would stimulate lung regeneration by mainly paracrine mechanisms. Additionally, we used biomaterials to microencapsulate cells and EVs to improve their local delivery and retention. While ECFCs were found to be ineffective in treating the monocrotaline model on their own, they were poorly retained in the lung and microencapsulation of ECFCs led to enhanced lung delivery within the first 72 hours, with resultant hemodynamic improvements in this model of PAH. MSCs are well known to be immunomodulatory and proangiogenic, largely acting through paracrine mechanisms, including by the release of EVs. Yet, following intravenous administration, nano sized EVs are rapidly cleared from circulation, potentially limiting their therapeutic potential. I adapted our microencapsulation strategy for EVs, and demonstrated significantly greater retention of microgel-loaded EVs were within the lung, resulting in enhanced local cell uptake. Interestingly, the hydrogel used for microencapsulation induced a local immune response which made it unsuitable for testing any potential therapeutic benefits of MSC-EVs in this study. Nonetheless, this work demonstrated proof-of-principle for the utility of microencapsulation as a strategy to enhance EV lung delivery. Overall, this work has identified novel lung cell populations (aAECs, dCap ECs, SM-like pericytes) driving arterial remodeling associated with PH progression, demonstrated the potential of ECFCs as a regenerative cell for the treatment of PAH, and illustrated the utility of microencapsulation as a tool to enhance lung targeting of both cells and EVs.

<p> Mesenchymal stromal cells (MSC) are a multipotent stromal population of interest as cancer therapeutics for their inherent tropism towards cancer sites. This renders them a potential cellular vehicle for delivering anti-tumor therapies. A limitation to their broader use is a plasticity in their biological roles, which depending on the context, may potentiate opposite roles in tumor modulation. Therefore, strategies to “guide” these cells towards a desired functional role are of high interest in the field of cancer therapeutics. In this dissertation, the functional polarization paradigm via stimulation with toll-like receptor ligands (poly I:C or LPS), previously described in bone-marrow derived MSC (BM-MSC), was evaluated in MSC sourced from adipose tissues (ASC). ASC provide several advantages over BM-MSC, such as the relative ease of acquisition of clinically relevant cellular doses via <em>in vitro </em>expansion. Findings in our studies in prostate cancer models <em>in vitro</em> suggested that a generation of phenotypically and functionally distinct ASC populations could be achieved via differential pre-stimulation approaches on ASC. We observed significant effects on the migratory and immunomodulatory capability of ASC, demonstrated via <em>in vitro </em>assays. Upon administration of these cells <em>in vivo </em>in a mouse model of prostate cancer, poly I:C-primed (or pre-conditioned) ASC were found to accelerate tumor growth progression. While unprimed and LPS-primed ASC did not exert a significant effect on tumor growth at the macroscopic level, gene expression analyses suggested that all treatments promoted distinct modulatory effects in the tumor microenvironment, including altered modulation of angiogenesis, and immune response processes, however, only in the case of poly I:C-primed ASC these effects translated to a significant effect in the tumor growth rate in the mouse model examined.  </p>