Trotz der breiten Anwendung zielgerichteter Therapien und Immuncheckpoint-Inhibitoren, liegt die 5-Jahres-Überlebensrate beim metastasierten klarzelligen Nierenkarzinom (ccRCC) unter 15%. Um den Therapieerfolg für Patienten zu verbessern, werden neue Modelle benötigt, die die Tumorheterogenität rekapitulieren und eine personalisierte Therapieentwicklung ermöglichen. Im ersten Teil dieser Doktorarbeit habe ich Organoidkulturen direkt aus Patiententumoren und sortierten Krebsstammzellen (CSCs) etabliert und diese Organoide im Detail charakterisiert. Die Rolle von WNT und NOTCH, die zuvor in ccRCC CSCs bestimmt wurde, wurde in Organoiden bestätigt und konnte mit Hilfe von molekularen Inhibitoren als therapeutische Schwachstelle ausgenutzt werden. Diese Ergebnisse heben das Potenzial von Patienten-abgeleiteten Organoiden (PDOs) für die personalisierte Medizin und das Potenzial von WNT und NOTCH Inhibierung in der ccRCC Behandlung hervor. PDOs stellen Werkzeuge für die personalisierte Medizin dar, geben jedoch wenig Einblick in die frühen Stufen der Tumorentstehung. Deshalb habe ich im zweiten Teil meiner Dissertation VHL, PBRM1 und SETD2 – die drei am häufigsten mutierten Gene in ccRCC – mit einer induzierbaren CRISPR-Cas9 Strategie in induzierten pluripotenten Stammzellen (iPSC)-abgeleiteten Nierenorganoiden targetiert. Ich differenzierte iPSCs in nierenspezifische Zelltypen aus sowohl metanephrischem Mesenchym als auch Ureterknospen-Epithelium. Knockout von VHL, PBRM1 und SETD2 führte zur Hochregulation von Hypoxie-induzierbaren Genen in Organoiden und Knockout Effekte konnten durch längere Kultivierungszeiten und Zellselektion via FACS verstärkt werden. Obwohl ccRCC-spezifische Signalwege aktiviert wurden, wurde kein Wachstumsvorteil der transformierten Zellen beobachtet. Dennoch stellen diese Organoide ein einzigartiges Modell dar, das auf andere Nephropathien angewendet werden könnte, um die Nieren- und Nierenkrebsforschung weiter voranzutreiben. / Despite the widespread application of targeted therapies and immune checkpoint inhibitors, the five-year survival rate for metastatic clear cell renal cell carcinoma (ccRCC) is below 15%, as unpredictable progression, therapy resistance, and tumor relapse occur. In order to improve patient outcome, novel models are needed that recapitulate tumor heterogeneity and allow for a more personalized therapy development. In the first part of my PhD thesis, I established organoid cultures directly from patient tumors and sorted cancer stem cells (CSCs) and I characterized these organoids thoroughly. The roles of WNT and NOTCH, which were previously determined in ccRCC CSCs, were confirmed in organoid cultures and could be exploited as a therapeutic weakness via small molecule inhibition. These results highlight the potential of patient-derived organoids (PDOs) for personalized therapy and further the potential of WNT and NOTCH inhibition for ccRCC treatment. PDOs present suitable tools for personalized medicine, but provide little insight into early stages of tumorigenesis. Therefore, in the second part of my thesis, I targeted VHL, PBRM1, and SETD2 – the three most frequently mutated genes in ccRCC – using an inducible CRISPR-Cas9 genome editing strategy in induced pluripotent stem cell (iPSC)-derived kidney organoids. I used a previously published protocol to differentiate iPSCs into kidney-specific cells originating from both metanephric mesenchyme and ureteric bud epithelium. Knockout of VHL, PBRM1, and SETD2 led to the upregulation of hypoxia-inducible genes in organoids and knockout effects could be enhanced by longer cultivation times and cell selection through FACS. Although ccRCC-specific signaling pathways were activated, a growth advantage of transformed cells was not observed. Nevertheless, these organoids present a unique model that could be applied to other nephropathies to further advance kidney and kidney cancer research.

The central nervous system is a complex, yet well-organised, often laminated, tissue. This robust organisation is evident in the architecture of the retina: consisting of 5 different neuronal types organised into distinct layers: Retinal Ganglion Cell (RGC), Amacrine Cell (AC), Bipolar Cell (BP), Horizontal Cell (HC) and Photoreceptor cell (PR) layers. This remarkable organisation is evolutionarily conserved in vertebrates, yet little is known about the mechanisms by which these cells form the correct layers. Live imaging has revealed overlapping periods of birth and extensive inter-digitation followed by cells sorting out into their appropriate positions, suggesting cell-cell interactions are important. To investigate possible cellular and molecular mechanisms responsible for the establishment of the tissue architecture I developed an organoid culture system for zebrafish retinal cells. To identify the cells in culture I used a Spectrum of Fates fish line which is a multiply transgenic line in which each retinal cell type can be identified based on expression of a combination of fluorescently tagged cell fate markers. The development of the protocol by which I cultured the cells and observed their cell-cell interactions involved establishing the best methods to dissociate and culture zebrafish retinal cells in a non-adhesive environment, then imaging the resulting reaggregates to examine the position of the different retinal cell types. By doing this I observed their inherent self-organising properties, in the absence of extrinsic cues or scaffolds. These cells appeared to be arranged in an inside-out layering, although all cell types are layered in the same relative order as they are in vivo. To analyse the organization in these aggregates I developed a Matlab script in collaboration with Leila Muresan which analyses the relative positioning of cells in concentric rings from the periphery to the centre of the aggregates according to the cell fate-tagged fluorescent markers. The script then fits this data as an empirical cumulative distribution function for different groups of cells to determine how spatially distinct populations of cells are. This gave me my measure of organisation. I then investigated the cell-cell interactions involved in this self-organisation by genetically or pharmacologically removing individual cell types and assaying the resulting organisation of the reaggregated, cell-type deficient, retinal organoids. I revealed that Müller Glia are important for retinal cell self-organisation. I also investigated the role of Retinal Pigment Epithelial (RPE) cells and Retinal Ganglion Cells and found they had no impact on the ability of the remaining cell types to organize. I began to investigate the role of Amacrine Cells but found that retinas void of ACs were susceptible to disaggregating in our dissection setup, preventing me from collecting the material needed for culture. I also investigated the role of candidate molecules in this system and revealed that R-Cognin is critical for retinal cells to reaggregate. Not only can I remove cells or molecules from the system, but I show how it can also be manipulated to replace molecules of interest such as laminin, by coating beads with the substance of choice and placing it amongst the cells to see if their organisational behaviour is affected. In summary, I have developed a system which provides a simple and easy platform to manipulate in various ways to help us potentially reveal some of the important players in neuronal patterning.

The intestinal epithelium represents one of the most actively renewing tissues in the body, and is widely used as a model system to study epithelial cell biology. ASPP2, a member of the ASPP (apoptosis stimulating protein of p53) protein family, has been shown to act as a regulator of epithelial cell polarity and tumour suppressor. This study investigated whether the dual function of ASPP2 is involved in the regulation of intestinal homeostasis and tumourigenesis, with a particular interest in the distinction between epithelial cell autonomous and non-autonomous mechanisms. Germline and intestinal epithelial cell-specific ASPP2 conditional knockout mice were employed in this study. Deficiency of ASPP2 in the intestinal epithelium resulted in delayed recovery from dextran sulfate sodium (DSS)-induced acute colitis, concurrent with a reduction in the expression of proinflammatory cytokines such as interleukin (IL)-1β and IL-6. Moreover, ASPP2-deficient mice showed increased susceptibility to Azoxymethane/DSS-induced colorectal tumourigenesis. While wild-type and ASPP2-deficient crypts showed similar incidence of tumour formation, the local immune microenvironment of ASPP2-deficient mice favoured tumour progression. The intestinal organoid culture was established to supplement in vivo experiments. The feasibility of the system was demonstrated with small intestinal organoids, in the context of proliferation, differentiation, and cell death. Using the established workflow, a colonic organoid-based tissue regeneration model was developed. The intrinsic susceptibility of organoids to DSS-induced cell death was not affected by the loss of ASPP2. However, ASPP2-deficient colonic organoids were less responsive to the pro-proliferative effects of IL-6, but were more sensitive to tumour necrosis factor-α-induced cell death in the presence of IL-22. In conclusion, this project undertook parallel examinations of animal models and organoids, demonstrating that a deficiency of ASPP2 in the intestinal epithelium results in dysregulated epithelial-immune cell interactions. This may partially explain the pathological conditions observed in ASPP2-deficient mice. Importantly, this study highlights the possibility of using organoids to investigate epithelial cell non-autonomous factors implicated in intestinal pathogenesis.

Orientador: Helio Amante Miot / Resumo: FUNDAMENTOS: Melasma é hipermelanose crônica, focal, adquirida decorrente de patogênese não totalmente compreendida, resultado da alteração funcional e arquitetural dos melanócitos. A predisposição genética, aspectos hormonais e exposição à radiação solar são os elementos mais associados ao desenvolvimento da doença e essenciais para entendimento da fisiopatologia. É bem estabelecida a relação entre a exposição à radiação solar e a piora do quadro, entretanto, o efeito independente do UVB, UVA, assim como a atuação da luz visível (LV) são pouco estudados. OBJETIVOS: Padronização de um modelo de cultura organoide cutâneo primário para estudo da melanogênese da pele com melasma e pele normal adjacente, à irradiação com diferentes comprimentos de onda (UVB, UVA, LV). MÉTODOS: Etapa 1: Amostras de pele da região retroauricular (punch 3mm), de 10 voluntários foram seccionados longitudinalmente, e cultivados em meio DMEM segundo protocolo estabelecido por Ayres, para padronização de viabilidade e dosimetria das radiações induzindo melanogênese. Um dos fragmentos foi irradiado e o outro mantido ao abrigo da luz por 72h. Foram avaliados aspectos morfológicos e arquiteturais da histologia (H&E e Fontana-Masson) e por rt-PCR para comparação da expressão quantitativa de gene constitucional (GAPDH) entre as peles recém-coletadas e as cultivadas. Foram padronizadas as doses de radiação e tempo de cultura que promovessem viabilidade da amostra e aumento de 10% na intensidade de pigmentação... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: FUNDAMENTALS: Melasma is chronic hypermelanosis, focal, acquired due to pathogenesis not fully understood, resulting from the functional and architectural alteration of melanocytes. Genetic predisposition, hormonal aspects and exposure to solar radiation are the elements most associated with the development of the disease and essential for understanding the pathophysiology. The relationship between exposure to solar radiation and worsening of the condition is well established, however, the independent effect of UVB, UVA, as well as the action of visible light (VL) are poorly studied. OBJECTIVES: Standardization of a primary cutaneous organoid culture model to study melanogenesis of skin with melasma and adjacent normal skin to irradiation with different wavelengths (UVB, UVA, VL). METHODS: Step 1: Skin samples from the retroauricular region (punch 3mm) from 10 volunteers were longitudinally sectioned and cultured in DMEM medium according to the protocol established by Ayres, for viability standardization and radiation dosimetry inducing melanogenesis. One of the fragments was irradiated and the other kept in the dark for 72h. Morphological and architectural aspects of histology (H&E and Fontana-Masson) and rt-PCR were evaluated for comparison of quantitative constitutional gene expression (GAPDH) between newly collected and cultured skins. Radiation doses and culture time that promoted sample viability and a 10% increase in basal layer pigmentation intensity were standardized... (Complete abstract click electronic access below) / Mestre

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22886号 / 医博第4680号 / 新制||医||1048(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 髙橋 良輔, 教授 井上 治久, 教授 伊佐 正 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Nowadays, the organoids structures has become more popular as suitable model systems for clinical research, particularly for development of new medication and drug screening. The standard study approaches include invazive biochemical or molecular-biology analysis as well as non-invasive optical approaches. Among optical methods, various microscopy techniques can give a very detailed information about the structure of organoids. However, the microscopy is time consuming as well as it puts a great demand on instrumentation. Therefore, the microscopy is not suitable for high content analysis of multiple samples. This work is focused on the development of the device and experimental technique for high-throughput screenings of organoids structures for biomedical applications based on microtitrate plates. Literatre search for non-invasive optical methods, suitable for analysis of organoid structures. The necessary adjustements of existing system for algae phenotypization are discussed. An experiment was made to test functionality of designed system. Practical use for clinical use is tested by the experiment of spheroids reaction to selected cytostatics. The results and findings are discussed in the conclusion.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21341号 / 医博第4399号 / 新制||医||1031(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 小川 誠司, 教授 坂井 義治, 教授 武藤 学 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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