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The female reproductive organs are among the few adult tissues in which periodic angiogenesis normally occurs. Pathological angiogenesis can occur in various conditions, such as solid tumors. Vascular endothelial growth factor (VEGF) signaling often represents a critical rate-limiting step in physiological and pathological angiogenesis. This study utilizes development of utero-ovarian vasculature during pregnancy in goats as a model of physiological angiogenesis. Non-pregnant does and does at 4, 7, 10, 13, 16, and 18 weeks of gestation were used. Arteries of the reproductive tract were injected <i>in situ</i> with Microfil®. The tracts were fixed, dehydrated, and rendered transparent to reveal the paths of arteries. The ovarian artery was tortuous and lay in close apposition to the uterine tributary of the ovarian vein in all specimens studied. In non-pregnant does, this arrangement may serve as a local utero-ovarian pathway for the corpus luteum (CL) luteolysis at the end of non-fertile estrous cycle. During pregnancy, this arterio-venous arrangement may transfer luteotropic substances from uterus to ovary, which may serve in maternal recognition of pregnancy and fit the fact that the goat is CL dependent throughout gestation. In some cases of triplets, the size of the uterine branch of the ovarian artery was equal to or even larger than that of its parent artery and/or the ipsilateral uterine artery; and the vaginal artery contributed a connecting branch to the uterine artery. These physiological adaptations of the ovarian and/or vaginal arteries correlate well with the increasing nutrient demands of the growing multiple fetuses. In a second experiment, the vasculature of the uterus and ovaries was injected <i>in situ</i> with a mixture of Batson's No.17® and methyl methacrylate and then processed for observation by SEM. The microvasculature differed between non-pregnant and pregnant does, and with advancing gestation. We concluded that goats possess a <i>multivillous</i> type placenta. Capillary sinusoids and crypts on the fetal surface of the caruncle may compensate for the negative effect of the increased interhemal distance. Intussusceptive angiogenesis should be considered as equally possible and important mechanism as sprouting angiogenesis during placental development. Capillary diameters increased significantly during pregnancy especially after 4 weeks. Capillary density index was 66.8, 68.7, 55.5, 63.5, 70.1, 70.4, 64.5 percent in non-pregnant, 4, 7, 10, 13, 16, and 18 weeks of pregnancy, respectively. In the ovary, coiling of the ovarian branch of the ovarian artery around the ovarian tributary of the ovarian vein was observed. This may represent a local channel required for product transport from ovarian vein to ovarian artery and might have a role in regulating blood pressure to various ovarian structures. Immunolocalization of VEGF was performed as a third experiment. Immunostaining was observed in cyto- trophoblasts, maternal epithelial tissues, and vascular endothelium and smooth muscle, but not in binucleate giant cells or connective tissue. No apparent differences were observed in intensity and pattern of VEGF staining associated with advancing gestation. Luteal and follicular cells, and endothelium and smooth muscles of the ovarian vasculature positively stained. Patterns and intensity of staining of VEGF suggest that the fetus is directing its own survival by producing growth factors affecting fetal and maternal tissues. VEGF may have a role in growth and differentiation of cytotrophoblasts, as well as, development and maintenance of CL. In the fourth experiment, the sequential expression of VEGF and its receptors (fms-like tyrosine kinase, Flt-1 and kinase-insert domain-containing receptor, KDR) was measured using real-time quantitative PCR. Targets were detected in all studied tissues; however, levels of expression differed according to the stage of pregnancy. Expression of VEGF and its receptor mRNAs increased with advancing pregnancy, which correlates with the expansion of vasculature during pregnancy. Differences in the time-courses of the expression of Flt-1 and KDR mRNAs during pregnancy suggest that each receptor plays a different role in the angiogenic process. As an application of our model of angiogenesis, we tested the effect of swainsonine (active compound of locoweed and a potential anti-cancer drug) on the process. Does treated with swainsonine were euthanized at 7 and 18 weeks. No significant differences were found in sinusoidal diameters in treated does at 7 weeks, but a decrease in capillary density index was noted. In the ovary, focal avascular areas were observed in the corpus luteum of swainsonine-treated does at 7 weeks of pregnancy. Swainsonine caused great distortion in the uterine and ovarian vasculature at 18 weeks. A decrease in intensity of the immunoreactivity to VEGF antibody was observed in tissues from swainsonine-treated does at 7 and 18 weeks. There was no substantial effect of swainsonine on the expression of VEGF and its receptors' mRNAs in any of the studied tissues (except in the left ovary, where it had an inhibitory effect) at 7 weeks of pregnancy, but it had an inhibitory effect at 18 weeks. Demonstration of swainsonine's potential to negatively affect vascular development and suppress genes likely involved in angiogenesis at critical stages of blood vessel proliferation lends credibility to its potential as anti-cancer drug. / Ph. D.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24505号 / 医博第4947号 / 新制||医||1064(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 渡邉 大, 教授 林 康紀, 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

The 15q13.3 microdeletion is a common genetic disorder associated with multiple neurodevelopmental disorders including autism spectrum disorder, epilepsy, and schizophrenia. Patients have diverse clinical presentations, often prompting genetic assays that identify the CNV in the clinic. This late-stage screening leaves a considerable gap in our understanding of the prenatal and prediagnostic developmental impairments in these individuals, providing a barrier to understanding the disease pathobiology. We provide the first investigation into embryonic brain development of individuals with the 15q13.3 microdeletion by generating multiple 3D neural organoid models from the largest clinical cohort in reported literature. We incorporated unguided and guided forebrain organoid models into our multi-transcriptomic phenotyping pipeline to uncover changes in cell type emergence and disruptions to circuit development, all of which had underlying changes to cell adhesion pathways. Specifically, we identified accelerated growth trajectories in 15q13.3del unguided neural organoids and used single cell RNA sequencing to identify changes in radial glia dynamics that affect neurogenesis. We measured changes in the pseudotemporal trajectory of matured unguided neural organoids, and later identified disruptions in synaptic signaling modules amongst the primary constituents to neural circuitry, excitatory and inhibitory neurons. We leveraged dorsal and ventral forebrain organoid models to better assess circuit dynamics, as they faithfully produce the excitatory and inhibitory neurons in the pallium and subpallium, respectively. We then used the entire 15q13.3del cohort and performed bulk RNA sequencing on each tissue type at two timepoints and discovered convergence on transcriptional dysregulation and disruptions to human-specific zinc finger proteins localized to chromosome 19. We also identified cell type-specific vulnerabilities to DNA damage and cell migration amongst the dorsal and ventral organoids, respectively, which was consistent with the excitatory and inhibitory neural subpopulations amongst the unguided neural organoids scRNA Seq, respectively. We then examined neuron migration in a 3D assembloid model by sparsely labeling dorsal-ventral forebrain organoids from multiple genotype-lineage combinations. Light sheet microscopy identified deficits in inhibitory neuron migration and morphology, but not migration distance, suggesting a complex disruption to cortical circuitry. This novel combination of cell type characterization, pathway identification, and circuitry phenotyping provides a novel perspective of how the 15q13.3 deletions impair prenatal development and can be applied to other NDD models to leverage understanding of early disease pathogenesis. / Dissertation / Doctor of Science (PhD) / The development of the human brain is a highly complex and tightly regulated process that requires the participation of multiple cell types throughout development. Disturbances to the emergence, differentiation, or placement of these cell types can cause disruptions and local miswiring of neural circuits, which is often associated with neurodevelopmental disorders (NDDs). The 15q13.3 microdeletion syndrome is a highly complex condition associated with multiple NDDs and has seldom been studied in a human context. To address this, we used stem cells derived from a 15q13.3 microdeletion syndrome cohort and their typically developing familial controls to generate unguided (“whole brain”) and region-specific organoids to investigate early fetal development across time. We used the largest 15q13.3 microdeletion cohort in reported literature to identify shared disruptions in early developmental milestones such as neurogenesis, neural migration, and neural patterning. We identified expansion of specific cell populations, including progenitors that later give rise to mature neurons. Abnormalities persisted in more mature cell populations, including the inhibitory neurons responsible for establishing critical microcircuitry in the human cortex. By generating guided organoids that enrich for excitatory and inhibitory neural populations, we were able to merge the models to form assembloids, where we captured early migratory and morphological deficits in inhibitory neuron populations, which is supported by the multi-transcriptomics experiments performed in both organoid models. This study provides a framework for examining fetal development in a neurodevelopmental disorder context. By using the 15q13.3 microdeletion background, we found novel disruptions in cell type emergence and circuit formation previously unreported in mouse or 2D neuron models, highlighting the utility of the phenotyping platform for disease modeling.

Solid tumors are complex structures comprising besides the cancer cells vasculature, extracellular matrix (ECM), soluble molecules and a plethora of various other cell types. These components form a so-called tumour microenvironment. From the numerous cell types that are part of tumor microenvironment, tumor infiltrating lymphocytes (TILs) play a major role in patient prognosis. Their presence is also of major importance with regard to new biological therapies based on immune checkpoint inhibitors. Crucial role of TILs is also reflected by the new approaches in cancer diagnostics namely by Immunoscore method (currently used in clinical settings). Immunoscore is based on localization and quantification of CD3+ and CD8+ TILs in thin histological sections of tumor tissue. The question remains to which extent the information obtained from 2D slices reflects the situation in tumor microenvironment considering its spatial heterogeneity. The development of new methodological approaches allowing evaluation of histological information in 3D is the key to answer this question. The theoretical part of this work first describes the heterogeneity of the tumor microenvironment and the role of immune cells within it. Then, the role of spatial heterogeneity and its possible influence on the histopathological...