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Matrin3 (MATR3) is an RNA binding protein involved in many roles in the nucleus, such as chromatin architecture and gene expression regulation, modulating transcriptional and post-transcriptional processes as RNA splicing and mRNA stabilization. Nevertheless, some functions of MATR3 within the cells are not entirely clear. MATR3 has been associated with Amyotrophic Lateral Sclerosis (ALS), a neurodegenerative disease that damages motor neuron (MN) cells and leads to progressive muscle paralysis and respiratory failure. A better understanding of MATR3 activity within cell physiology could represent an essential breakthrough for studying MATR3-associated pathologies. Using MATR3-silenced human pluripotent stem cell (hiPSC) line model, we collected data on the MATR3 role in the pluripotency and in the neural induction and differentiation. We found that the downregulation of MATR3 alters the expression level of crucial self-renewal factors such as OCT4, NANOG, KLF4, and LIN28A. We observed MATR3 acts at multiple levels of the gene expression, i.e. regulating YTHDF1 expression, and in RNA metabolism, having a role in mRNA stabilization and translation. The reduction of stemness potential caused by MATR3 downregulation creates a defect during the neurodifferentiation process, which does not arrest motor neurons formation but induces selective alterations that may affect motor neurons functionality. Indeed, several morphological and molecular abnormalities were observed during the neuronal differentiation, such as the alterations of the formation of neuroepithelial rosettes that arise in a reduction of neurite lengths and arborization in neuronal cells. On this basis, we investigated neuronal differentiation in the brain organoids grown from iPSCs derived from ALS patients fibroblasts. We show, for the first time, that MATR3 is a critical factor in orchestrating the stemness network through transcriptional, post-transcriptional, and translational regulation, therefore affecting the differentiation of mature neurons.

Retinoids are a class of compounds derived from the metabolism of vitamin A and β-carotene. Retinoid signaling has vital functions in both vertebrate and invertebrate embryogenesis such as the formation of body axes and the control of organogenesis. Genetic evidence suggests a role of retinoids in cell fate decision, maturation and homeostasis of the prostate epithelium. Knockout (KO) of the retinoic acid receptor gamma (RARG) gene in mice leads to growth deficits and male sterility due to squamous metaplasia and keratinization of the seminal vesicles and prostate. Noteworthy, synergistic antitumor effects of retinoids and vitamin D have been described in prostate cancer cell lines, although a mechanistic link between retinoic acid (RA) signaling and prostate epithelium differentiation and tumorigenesis has not yet been elucidated. Here, taking advantage of mouse prostate organoids (mPrOs), we report an essential role for RA in the differentiation and integrity of the periurethral and proximal luminal compartments of the prostate epithelium. Mechanistically, RA, through the activation of RARγ, promotes the expression of Foxa1, a pioneer transcription factor that cooperates with androgen receptor (AR) in directing progenitor cells towards the luminal lineage. Reduced RA signaling in organoids leads to downregulation of key structural and polarity proteins along with a loss of luminal identity, a phenotype that is fully rescued by constitutive expression of exogenous Foxa1. Overall, our study demonstrates the importance of RA signaling in prostate epithelium differentiation and homeostasis. In addition to the tumorigenic role of Foxa1 mutations recently described in several human cancers, alteration in RA pathway due to altered uptake/absorption/metabolism of vitamin A and β-carotene, or depending on specific molecular dysfunctions (e.g., epigenetic RARB silencing), could represent a critical rheostat for prostate tumorigenesis.

To optimize neoadjuvant radiochemotherapy of pancreatic ductal adenocarcinoma (PDAC), the value of new irradiation modalities such as proton therapy needs to be investigated in relevant preclinical models. We studied individual treatment responses to RCT using patient-derived PDAC organoids (PDO). Four PDO lines were treated with gemcitabine, 5-fluorouracile (5FU), photon and proton irradiation and combined RCT. Therapy response was subsequently measured via viability assays. In addition, treatment-naive PDOs were characterized via whole exome sequencing and tumorigenicity was investigated in NMRI Foxn1nu/nu mice. We found a mutational pattern containing common mutations associated with PDAC within the PDOs. Although we could unravel potential complications of the viability assay for PDOs in radiobiology, distinct synergistic effects of gemcitabine and 5FU with proton irradiation were observed in two PDO lines that may lead to further mechanistical studies. We could demonstrate that PDOs are a powerful tool for translational proton radiation research.

Celiac disease is an autoimmune enteropathy driven by the ingestion of gluten in genetically predisposed individuals carrying HLA-DQ2 and/or -DQ8 genes. Currently, the only available treatment is a strict, life-long, gluten-free diet (GFD), which is very restrictive and not always effective, highlighting the need for alternative therapies. Celiac disease requires activation of both the innate (intraepithelial lymphocytes or IELs) and adaptive (lamina propria CD4+ T cells) arms of the immune system. Activation of these two pathways leads to the destruction of IEC and villous atrophy. Thus, IEC damage is a hallmark of CeD. However, IECs are not only the target of tissue damage; they also actively participate in CeD pathogenesis by translocating gluten peptides, expressing stress-induced markers, and releasing TG2 into the gut lumen to generate TG2-gluten complexes. Although IECs are known to express MHC, their role in gluten-dependent T cell activation has never been proven, partly because of the lack of an appropriate in vitro epithelial model expressing human MHC class II. This thesis aims to address this gap by developing a humanized organoid monolayer expressing the CeD risk gene HLA-DQ2.5, to investigate the interaction between IEC-gluten-T cells. The expression of epithelial MHC class II was evaluated in active and treated CeD patients, as well as in gluten-immunized and control (non-immunized; NI) DR3-DQ2.5 transgenic mice that express only CeD-associated MHC class II (HLA-DQ2.5). Active CeD patients and gluten-immunized DR3-DQ2.5 mice demonstrated higher expression of epithelial MHC class II compared with their treated and NI counterparts. Organoid monolayers developed from these mice and were treated with or without IFN-. Organoid monolayers derived from gluten-immunized DR3-DQ2.5 mice showed higher expression of MHC class II compared with NI mice, and this expression was upregulated by IFN- treatment. The functional consequences of MHC class II expression were determined by co-culturing organoid monolayers with CD4+ T cells in the presence of gluten and zein (a non-gluten protein). In the co-culture, gluten, but not zein, enhanced CD4+ T cell proliferation, activation, and release of cytokines, including IL-2, IFN- and IL-15, in the co-culture supernatants. Bacteria have recently emerged as modulators of inflammation in patients with CeD. It has been shown that opportunistic pathogens, including Pseudomonas aeruginosa, partially metabolize gluten into more immunogenic peptides. As such, the role of bacterially modified gluten in modulating the T cell response was assessed using the in vitro co-culture system I described. For this, monolayers were treated with the gluten pre-digested, or not, by elastase-producing P. aeruginosa or its lasB mutant. Gluten metabolized by P. aeruginosa, but not by the lasB mutant, significantly increased CD4+ T cell responses. In conclusion, MHC class II-expressing organoid monolayers are a functional model that can promote T cell responses under certain conditions. The model described in this thesis reveals a new immunomodulatory role for IECs in activating CD4+ T cells through MHC class II. This mechanism may serve to localize and further increase injury to the epithelium caused by gluten-specific CD4+ T cells in CeD. Therefore, therapeutics directed at IECs may offer a novel approach for modulating both adaptive and innate immunity in CeD, providing an alternative or adjuvant therapy to the current GFD treatment. / Thesis / Doctor of Philosophy (PhD) / Celiac disease is one of the most common food sensitivities, affecting approximately 1 in 100 people worldwide, including Canada. It occurs in people with specific genes (DQ2 and/or DQ8) when they eat gluten-containing foods such as wheat, barley, and rye. In people with celiac disease the immune system overreacts to gluten, damaging the lining of the upper gut, which we call “epithelium. This lining of cells constitutes the first barrier between the external world and our body, allowing in healthy conditions for nutrients to be absorbed but blocking the passage of gut microbes, some of which can cause disease or worsen gut inflammation. In patients with celiac disease, gluten crosses the epithelium into the gut tissue, where it activates specific cells of the immune system called “T cells”. Recently, there has been growing interest in whether the gut lining itself plays a role in triggering this immune response in celiac disease, though this has not yet been proven. If proven, this would suggest that the gut lining is responsible for directing the harmful immune response to gluten and should be considered a target site when developing therapies to prevent or treat celiac disease. This concept has been difficult to prove because we do not have a model to investigate this question. Such a model would require a gut lining that carries the genes linked to celiac disease. My thesis describes the development of such a model, made of a gut lining from a mouse genetically modified to carry human celiac disease genes. Using this model, I found that when the gut lining was exposed to certain molecules present in celiac patients (cytokines), it switched on other molecules that ultimately activated T cells. Additionally, I demonstrated that certain microbes, such as Pseudomonas aeruginosa, which are present in higher numbers in the upper gut of patients with celiac disease, can break down gluten into fragments that further activate T cells. The results validated the use of this model to understand what other co-factors can tip the balance in a person with celiac genes to remain healthy or develop inflammation. In summary, I demonstrated that the gut lining expressing celiac genes actively participates in the activation of immune cells that drive intestinal damage in celiac disease. This new model is a novel tool to continue to identify additional co-factors that predispose patients to celiac disease, as well as to screen for novel therapies for celiac disease. This is important, as the only currently available treatment is a strict lifelong gluten-free diet, which has many limitations, including frequent contamination and celiac disease reactivation.

Per- and polyfluoroalkyl substances (PFAS) are organic synthetic compounds used in several industries because of their unique properties and thermal and chemical stability. Perfluorooctanesulfonic acid (PFOS) and perfluorooctaonic acid (PFOA) are two of the most prominent PFAS that are undegradable and accumulate in nature. To study the impact of PFOS and PFOA on the liver in a controlled environment, organoids can be used. A sensitive and selective LC-MS/MS method for individual and simultaneous analysis of PFOS and PFOA in liver organoid media and equipment used in organoid analyses was developed. For detection of low concentrations, ability to analyse complex organoid samples, and limit background contamination, a solid phase extraction (SPE) column, automatic filter (AFFL) and a trap column was included. The AFFL-SPE-LC-MS/MS was optimised efficiently through Design of Experiment (DoE) regarding the loading phase in the LC and six MS parameters for PFOS and PFOA. Validation was controlled against Eurachem’s guideline showing high sensitivity, detecting LOD at 6 pg/mL. The method demonstrated high repeatability with an RSD below 8 % for most samples. Simultaneous analysis of PFOS and PFOA demonstrated high selectivity. Nevertheless, the method showed low intermediate precision and varying reliability, as well as persistent background contamination limiting detection of lower concentrations. The method was fit for purpose and allowed rapid analysis of PFOS and PFOA in organoid media and equipment used in organoid analyses. Result from studies of PFAS in liver organoids through analysis with this method can aid in understanding the connection between PFAS and metabolic diseases. / Populärvetenskaplig sammanfattning Per- och polyfluorerade alkylsubstanser (PFAS) är en grupp av människoskapta, syntetiska ämnen med unika egenskaper. Dessa egenskaper gör att de är olja- och vattenavvisande, och har många applikationsområden. De finns i textiler, livsmedelsförpackningar, brandsläckningsskum och andra industriprodukter. De är väldigt termiskt och kemisk stabila, vilket gör att de inte bryts ner och därmed ackumulerar i miljön. Flera studier har också visat koppling mellan PFAS och många kroniska sjukdomar, som hormonstörningar, cancer, immunsuppression och metabolt associerad fettlever (MAFLD, tidigare nonalkoholisk fettlever (NAFLD)). Kopplingen mellan MAFLD och PFAS har fått mycket uppmärksamhet då levern har visats sig vara ett målorgan för PFAS. Eftersom PFAS är ihärdiga, har ett komplicerat spridningsbeteende och ackumulerar i naturen är det svårt att studera kopplingen mellan MAFLD och PFAS i en kontrollerad miljö. För att studera effekten av PFAS kan man använda organoider, laboratorieodlade 3D modeller gjord från stamceller för att imitera ett äkta organ.    Någon av de mest omtalade PFAS ämnen är perfluoroktansyra (PFOA) och perfluoroktansulfonat (PFOS), vilket är fokus för detta arbete. Leverorganoiderna kan utsättas för PFOS och PFOA, och mediet de ligger i kan extraheras och studeras med konventionella analytiska metoder för att få en bild av hur PFAS påverkar levern. I detta arbete vill analysen ske via vätskekromatografi med masspektrometri som detektion (LC-MS/MS). Med LC-MS/MS separeras den studerade molekylen, analyten från lösningen baserat på dess kemiska egenskaper. Analyten detekteras baserat på dess massa, mer bestämd massa/laddning-fördelningen (m/z). För att anpassa LC-MS metoden till injektion av komplexa organoidprover inkluderades ett automatiskt filter (AFFL) samt ett extra automatiskt separationssteg med en kolonn med fastfasextraktion (SPE). I övrigt ger SPE möjligheten att små mängder PFAS kan uppkoncentreras och fokuseras på kolonnen, vilket ger en sensitiv metod som kan detektera låga koncentrationer. SPE och AFFL implementerades båda för att bättre kunna separera och detektera PFOS och PFOA från andra ämnen, samt filtrera bort föroreningar och stora molekyler som kan skada LC-MS/MS instrumentet i längden. Då PFAS hopar upp sig i vår omgivning, visade det sig att kontamination av PFAS från systemet blev en utmaning under metodutvecklingen. Därför implementerades PFAS fritt utstyr, samt en extra kolonn för att fånga PFAS från systemet och på så sätt minska bakgrundskontaminationen som detekterades.    AFFL-SPE-LC-MS/MS metoden optimerades via en maskininlärningsbaserad optimeringsmetod baserad på parametrar i LC och MS. Metoden baserar sig på att, med tre värden för varje parameter, uppger programmet ett antal experiment som måste utföras för att kunna beräkna ett optimalt värde för varje parameter. Med resultatet från experimenten kan modellen matematiskt, genom en Bayes baserat Gaussian modell, uppskatta optimala värden för metoden. På så sätt kunde metoden optimeras systematiskt och tidseffektivt.    Innan rutinanvändning måste den optimerade metoden valideras. Validering blev gjord genom at följa Eurachem’s riktlinjer. Metoden visade hög repeterbarhet, selektivitet och riktighet. Den har hög sensitivitet, och kan detektera låga mängder, men bakgrundskontaminationen kunde inte elimineras totalt, och gör att man måste korrigera för detta i rutinanalyser. Komplexiteten av AFFL-SPE-LC-MS/MS med flera kolonner och filter gjorde att metoden visade låg robusthet och behövde justeras ofta. AFFL-SPE-LC-MS/MS metoden gör det möjligt att snabbt studera PFOS och PFOA i leverorganoider och utstyr använt i organoidanalyser, och kan bidra i forskningen för att bättre förstå hur PFAS påverkar levern. / Health Effects of Persistent Organic Pollutants

Human induced pluripotent stem cell (iPSCs)-derived brain organoids have potential to recapitulate the earliest stages of brain development, serving as an effective in vitro model for studying both normal brain development and disorders. In this study, we demonstrate a straightforward approach of generating multiple cerebral organoids from iPSCs on a pillar plate platform, eliminating the need for labor-intensive, multiple transfer and encapsulation steps to ensure the reproducible generation of cerebral organoids. We formed embryoid bodies (EBs) in an ultra-low attachment (ULA) 384-well plate and subsequently transferred them to the pillar plate containing Matrigel, using a straightforward sandwiching and inverting method. Each pillar on the pillar plate contains a single spheroid, and the success rate of spheroid transfer was in a range of 95 - 100%. Using this approach, we robustly generated cerebral organoids on the pillar plate and demonstrated an intra-batch coefficient of variation (CV) below 9 – 19% based on ATP-based cell viability and compound treatment. Notably, our spheroid transfer method in combination with the pillar plate allows miniaturized culture of cerebral organoids, alleviates the issue of organoid variability, and has potential to significantly enhance assay throughput by allowing in situ organoid assessment as compared to conventional organoid culture in 6-/24-well plates, petri dishes, and spinner flasks.

Die Durchfallerkrankung Giardiasis wird durch den Protisten Giardia duodenalis ausgelöst. Die Infektion erfolgt fäkal-oral, meist über kontaminiertes Trinkwasser. Der Parasit kolonisiert den oberen Bereich des Dünndarms und heftt sich an das Epithel, wodurch es die Krankheitsbeschwerden auslöst. Allerdings sind Details über die Mechanismen der Pathogenese unbekannt. Dazu kommt, dass der Ausgang einer Infektion fallspezifisch starken Schwankungen unterworfen ist, von selbst-limitierend bis chronisch und asymptomatischer Kolonisierung bis hin zur schweren Enteritis. Ein möglicher Pathomechanismus ist der Wegfall der Barrierefunktion des Dünndarmepithels, z.B. durch Beeinträchtigung von tight junctions oder Zelltod. In dieser Arbeit wurden Effekte von G. duodenalis auf in vitro Modellsysteme des humanen Dünndarmepithels untersucht. Dazu wurden hauptsächlich Daten über die Barrierefunktion sowohl von der weit verbreiteten Caco-2 Zelllinie, als auch über ein neu etabliertes humanes Dünndarmorganoidsystem, erhoben. Es konnte gezeigt werden, dass mehrere - mitunter in der Literatur als hochvirulent beschriebene - G. duodenalis Isolate zu keinerlei Beeinträchtigung der Barrierefunktion oder irgendeiner anderen untersuchten potenziellen Schädigung an zwei unterschiedlichen Caco-2 Zelllinien unter diversen Infektions- und Kulturbedingungen führte. Jedoch andererseits das neu entwickelte Dünndarmorganoidsystem mit pseudo-luminalem Medium TYI S 33 reproduzierbar die Zerstörung des Epithelmodells mit Zellverlust, Zelltod (apoptotisch und nicht-apoptotisch), Störung der tight junctions (Abbau und Dislokation von Claudinen und ZO-1) und den Verlust von Mikrovilli innerhalb ein bis zwei Tage nach Parasiteninfektion zeigen konnte. Zudem wurde das Auftauchen von ClCa-1-Signalen unter andauerndem Infektionsstress beobachtet, was die Differenzierung bzw. Metaplasie zu Becherzellen nahelegt, jedoch keine Wirtsreaktion auf die Gewebszerstörung zu sein scheint. / The protozoan parasite Giardia duodenalis is the etiological agent for the intestinal diarrheal disease giardiasis. Infections are acquired via the fecal-oral route, mostly via uptake of cysts from contaminated drinking water. The colonization of the hosts’ duodenum and upper jejunum and the attachment of Giardia trophozoites onto the epithelium is the cause of a variety of gastrointestinal complaints but the exact pathomechanisms are unknown. Furthermore, the outcome of Giardia infections varies greatly between individuals, ranging from self-limiting to chronic, and asymptomatic to severe enteritis. One proposed mechanism for the pathogenesis is the breakdown of intestinal barrier function, e.g. by tight junction impairment or induction of cell death. In this work, effects of G. duodenalis on in vitro models of the human small intestinal epithelium were investigated by studying mainly barrier-related properties and changes of widely used Caco-2 cells as well as newly established human small intestinal organoid-derived monolayers (ODMs). It could be shown that several isolates of G. duodenalis, some described as highly virulent, fail to induce barrier dysfunction or any other investigated pathological effect on two Caco-2 cell lines under various infection and culturing conditions. On the other side, by developing a new organoid-based model system and the use of luminal mock medium TYI-S-33, considerable epithelial disruption (including loss of cells), cell death (apoptosis and non-apoptotic), tight junction impairment (degradation and dislocation of claudins and ZO-1), and microvilli depletion reproducibly induced by G. duodenalis trophozoites between one and two days after infection could be observed. Moreover, emergence of ClCa-1 positive cells with ongoing parasite infections suggest epithelial differentiation or metaplasia towards goblet cells, which is furthermore not associated to tissue damage.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23062号 / 医博第4689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 武藤 学, 教授 妹尾 浩, 教授 小川 誠司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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