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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Atualmente, a única alternativa viável para pacientes que possuem um quadro de doença hepática em estágio final é o transplante total ou parcial de fígado. Devido à crescente defasagem entre doadores disponíveis e pacientes em fila de espera, o desenvolvimento de abordagens de engenharia tecidual hepática (ETH) se tornou uma necessidade crescente. As células pluripotentes induzidas (iPS) são uma atraente alternativa para servirem como fonte celular para aplicações de engenharia tecidual por serem capazes de produzir todos os fenótipos celulares. Dentre as principais abordagens de EHT podemos citar as técnicas de bioimpressão 3D, organóides hepáticos e descelularização/recelularização. Este trabalho buscou avaliar a utilização de células iPS no desenvolvimento das três tecnologias descritas. Visando avaliar como imprimir um tecido hepático funcional com células iPS, testamos a impressão com hepatócitos dispersos em células únicas em comparação com a impressão de esferóiedes hepáticos. Os esferóides hepáticos mostraram maior viabilidade e funcionalidade hepática por preservarem o fenótipo epitelial ao longo do tempo. A composição de células não parenquimáticas derivadas de iPS ou células primárias para a formação de organóides hepáticos foi testada neste trabalho. Os resultados indicam que, utilizando células mesenquimais primárias e endoteliais derivadas de iPS, obtém-se uma maturação hepatica mais eficiênte devido a inibição das vias de sinalização TGF-β? e modulação da via Wnt. A recelularização do tecido aórtico descelularizado de ratos com células derivadas de iPS mostrou ser capaz de prover função hepática em cultura assistida por biorreator, porém os resultados indicam a necessidade de aprimoramento do protocolo de recelularização. Este trabalho comprovou a viabilidade da aplicação de células iPS nas abordagens EHT testadas e contribuiu para o desenvolvimento de alternativas terapêuticas viáveis para pacientes em fila de espera de transplante hepático / Currently, the only feasible alternative for patients with end-stage liver disease is total or partial liver transplantation. Due to the growing gap between available donors and patients in waiting list, the development new tissue engineering technologies have become a growing need. Induced pluripotent cells (iPS) are an attractive alternative to serve as cell source for tissue engineering applications due to their ability to differentiate into all cellular phenotypes. Among the main liver tissue engineering technologies, 3D bioprinting, hepatic organoids and decellularization/recellularization of biological matrixes have generated much expectation. Thus, this work aimed to evaluate the use of iPS cells in the development of the aforementioned technologies. In order to evaluate how to bioprint a functional liver tissue using iPS-derived cells, we tested the effect of printing a single cell dispersion of hepatocytes versus printing hepatic spheroids. Hepatic spheroids showed greater viability and liver function, due to preserved epithelial phenotype over time. The composition of non-parenchymal cells using iPS-derived cells or primary adult cells for hepatic organoid formation was tested. The results indicated that, using primary mesenchymal cells and iPS-derived endothelial cells, we obtained a more efficient hepatic maturation due to the inhibition of TGF-β? and modulation Wnt signaling pathway. Recellularization of rat aortic decellularized scaffold with iPS-derived cells displayed hepatic function over time in a bioreactor-assisted culture, but the results indicate the need for improvements in the recellularization protocol. In conclusion, this work demonstrated the feasibility of use of iPS-derived cells for liver tissue engineering approaches and contributed to the development of the investigated technologies in order to generate future therapeutic alternatives for patients in waiting list for liver transplantation

O Transtorno Depressivo Maior (TDM) é uma condição neuropsiquiátrica que resulta em um substancial sofrimento pessoal, incapacidade e custos sociais. Devido à inacessibilidade ao encéfalo humano por questões práticas, éticas e às limitações encontradas pelo uso de modelos animais, o desenvolvimento de modelos in vitro acurados torna-se fundamental para o estudo desta doença. Neste aspecto, o surgimento da tecnologia de células-tronco pluripotentes induzidas humanas (hiPSCs) apresenta-se como uma importante ferramenta, uma vez que permite a recapitulação da diversidade genética dos pacientes e possibilita a produção de tipos celulares de interesse para o estudo da doença, como neurônios e glia. Entretanto, até o momento, não há registros da produção de modelos in vitro a partir de hiPSCs de pacientes com TDM. Visando preencher esta lacuna, o presente estudo teve por objetivo a produção e caracterização de dois tipos de modelos in vitro a partir de hiPSCs de pacientes com TDM: neurônios serotoninérgicos, uma cultura celular em monocamada, e organóides cerebrais, uma cultura celular em suspensão. 5 linhagens de hiPSCs de pacientes foram diferenciadas em neurônios com sucesso e no ensaio de imunocitoquímica, expressaram os marcadores 5-HT (serotonina), T5-HT (transportador de serotonina), nestina (presente em diferentes tipos de células neuronais) e Tuj1 (proteína do citoesqueleto, neurônio-específica), confirmando o fenótipo de neurônios serotoninérgicos. As céluas diferenciadas obtiveram também resultados positivos nos testes de imageamento de cálcio e de medidas da alteração no potencial de membrana, indicando que os neurônios diferenciados eram fisiologicamente funcionais. De 5 linhagens de hiPSCs (sendo uma controle e as outras 4 de pacientes com TDM), somente 1 linhagem sobreviveu ao processo de diferenciação, originando organóides cerebrais e apresentou expressão dos marcadores Sox2 (progenitoras neurais) e Tuj1 (neurônios maduros), indicando ainda uma estruturação correta da citoarquitetura, com as progenitoras localizadas mais internamente, na zona ventricular, ao redor dos lúmens preenchidos com líquido e com os neurônios maduros localizados na placa cortical, região mais externa dos agregados celulares. Apesar de o processo de produção dos organóides cerebrais necessitar de aperfeiçoamento e melhor padronização, a produção destes dois tipos de modelos in vitro, feitos a partir de hiPSCs de pacientes com TDM configura-se como um importante passo para a futura redução do uso de modelos animais em pesquisa, elucidação de mecanismos subjacentes à doença, identificação de biomarcadores diagnósticos e triagem de fármacos de maneira personalizada, visando permitir tratamentos mais baratos, adequados e eficientes para o TDM / Major Depressive Disorder (MDD) is a neuropsychiatric condition that results in substantial personal distress, disability, and social costs. The inaccessibility to the human brain due to practical and ethical issues and the limitations encountered by the use of animal models turn the development of accurate in vitro models into an essential factor to study this disease. In this regard, the emergence of human induced pluripotent stem cell technology (hiPSCs) is an important tool, since it allows the recapitulation of patient\'s genetic diversity and allows the production of cell types of interest for studying the disease, such as neurons and glia. However, to date, there are no records of the production of in vitro models from hiPSCs of patients with MDD. In order to fill this gap, the present study aimed at the production and characterization of two types of in vitro models from hiPSCs of patients with MDD: serotonergic neurons, a monolayer cell culture, and cerebral organoids, a suspension cell culture. 5 patients\' hiPSCs lines were successfully differentiated into neurons and, in the immunocytochemistry assay, they expressed the 5-HT (serotonin), T5-HT (serotonin transporter), nestin (present in several neuronal cell types) and Tuj1 (cytoskeleton protein, neuron-specific), confirming the phenotype of serotonergic neurons. Differentiated cells also obtained positive results in calcium imaging tests and measurements of membrane potential changes, indicating that differentiated neurons were physiologically functional. Of 5 hiPSCs lines (one control and the other 4 of patients with MDD), only 1 survived to the differentiation process, originating cerebral organoids which presented expression of Sox2 (neural progenitor) and Tuj1 (mature neurons) markers and a correct structuring of the cytoarchitecture, with the progenitors located more internally in the ventricular zone, around the lumens filled with liquid and with the mature neurons located in the cortical plate, the outermost region of the cellular aggregates. Although the cerebral organoids production process needs improvement and better standardization, the production of these two types of in vitro models, made from hiPSCs of patients with MDD, is an important step for the future reduction of animal models use, elucidation of disease underlying mechanisms, identification of diagnostic biomarkers and drug screening in a personalized manner, in order to allow cheaper, more adequate and more efficient treatments for MDD

Retinal ganglion cells (RGCs) serve as a vital connection between the eye and the brain with damage to their axons resulting in loss of vision and/or blindness. Retinal organoids are three-dimensional structures derived from human pluripotent stem cells (hPSCs) which recapitulate the spatial and temporal differentiation of the retina, providing a valuable model of RGC development in vitro. The working hypothesis of these studies is that hPSC-derived RGCs are capable of extensive outgrowth and display target specificity and pathfinding abilities. Initial efforts focused on characterizing RGC differentiation throughout early stages of organoid development, with a clearly defined RGC layer developing in a temporally-appropriate manner expressing a compliment of RGC-associated markers. Beyond studies of RGC development, retinal organoids may also prove useful to investigate and model the extensive axonal outgrowth necessary to reach post-synaptic targets. As such, additional efforts aimed to elucidate factors promoting axonal outgrowth. Results demonstrated significant enhancement of axonal outgrowth through modulation of both substrate composition and growth factor signaling. Furthermore, RGCs possessed guidance receptors that are essential in influencing outgrowth and pathfinding. Subsequently, to determine target specificity, aggregates of hPSC-derived RGCs were co-cultured with explants of mouse lateral geniculate nucleus (LGN), the primary post-synaptic target of RGCs. Axonal outgrowth was enhanced in the presence of LGN, and RGCs displayed recognition of appropriate targets, with the longest neurites projecting towards LGN explants compared to control explants or RGCs grown alone. Generated from the fusion of regionally-patterned organoids, assembloids model projections between distinct regions of the nervous system. Therefore, final efforts of these studies focused upon the generation of retinocortical assembloids in order to model the long-distance outgrowth characteristic of RGCs. RGCs displayed extensive axonal outgrowth into cortical organoids, with the ability to respond to environmental cues. Collectively, these results establish retinal organoids as a valuable tool for studies of RGC development, and demonstrate the utility of organoid-derived RGCs as an effective platform to study factors influencing outgrowth as well as modeling long-distance projections and pathfinding abilities.

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.

The rubella virus (RV) was the first virus shown to be teratogenic in humans. The wealth of data on the clinical symptoms associated with congenital rubella syndrome is in stark contrast to an incomplete understanding of the forces leading to the teratogenic alterations in humans. This applies not only to RV, but also to congenital viral infections in general and includes (1) the mode of vertical transmission, even at early gestation, (2) the possible involvement of inflammation as a consequence of an activated innate immune response, and (3) the underlying molecular and cellular alterations. With the progress made in the development of pluripotent stem cell-based models including organoids and embryoids, it is now possible to assess congenital virus infections on a mechanistic level. Moreover, antiviral treatment options can be validated, and newly emerging viruses with a potential impact on human embryonal development, such as that recently reflected by the Zika virus (ZIKV), can be characterized. Here, we discuss human cytomegalovirus (HCMV) and ZIKV in comparison to RV as viruses with well-known congenital pathologies and highlight their analysis on current models for the early phase of human development. This includes the implications of their genetic variability and, as such, virus strain-specific properties for their use as archetype models for congenital virus infections. In this review, we will discuss the use of induced pluripotent stem cells (iPSC) and derived organoid systems for the study of congenital virus infections with a focus on their prominent aetiologies, HCMV, ZIKV, and RV. Their assessment on these models will provide valuable information on how human development is impaired by virus infections; it will also add new insights into the normal progression of human development through the analysis of developmental pathways in the context of virus-induced alterations. These are exciting perspectives for both developmental biology and congenital virology.

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.

Les hormones thyroïdiennes (HTs) contrôlent plusieurs aspects du développement et de l’homéostasie intestinale. Elles agissent via des récepteurs nucléaires (TRs), facteurs de transcription modulés par la T3. Le paradigme est la métamorphose des amphibiens où elles sont responsables du remodelage du tube digestif et de l’émergence des cellules souches (Ishizuya-Oka et al, 2009). Des études précédentes ont montré que les HTs jouent un rôle fondamental en régulant la balance entre prolifération et différenciation des précurseurs épithéliaux murins. Du point de vue moléculaire, le récepteur nucléaire TRα1 contrôle plusieurs gènes du cycle cellulaire/prolifération ainsi que les voies de signalisation Wnt et Notch (rev. in Sirakov et al, 2104; Skah et al, 2017). En accord avec ces fonctions, l’expression ciblée de TRα1 dans l’épithélium intestinal (souris vil-TRα1) est suffisante pour induire des cryptes aberrantes, hyper-prolifératives et confère une sensibilité accrue au programme de tumorigénèse intestinale dépendant de la mutation dans le gène Apc (vil-TRα1/Apc+/1638N mice) (Kress et al, 2010). Le but de mon travail a été d’étudier le contrôle des cellules souches intestinales, dépendant des HTs/TRs. En effet, j’ai utilisé des souris Lgr5-EGFP-ires-CreERT2 permettant de traquer, trier et cibler les cellules souches (Barker et al, 2007) que j’ai croisées avec le modèle murin inductible au tamoxifène TRα1-LOF (Loss-of-function) (Quignodon et al, 2007). J’ai étudié les effets de l’altération de la voie HTs/TRα1 in vivo et dans des organoïdes intestinaux (ex vivo). Nos résultats indiquent que les HTs et la modulation de l’expression ou de l’activité de TRα1 affectent rapidement et fortement les cellules souches intestinales. Ce travail ouvre de nouvelles perspectives dans l’étude des signaux dépendants des HTs/ TRα1 dans la physiopathologie des cellules souches intestinales / Thyroid hormones (THs) control several aspects of gut development and homeostasis. They act through the thyroid hormone nuclear receptors (TRs) that are T3-modulated transcription factors. The paradigm is the amphibian metamorphosis, where they are responsible for gut remodeling and emergence of the stem cells (Ishizuya-Oka et al, 2009). In previous studies we showed that THs play a fundamental role in regulating the balance between cell proliferation and cell differentiation of the murine intestinal epithelial precursors. From a molecular point of view the nuclear receptor TRα1 controls several proliferation/cell-cycle genes as well as the Wnt and Notch pathways (rev. in Sirakov et al, 2104; Skah et al, 2017). In accordance with these functions, targeted expression of TRα1 in the intestinal epithelium (vil-TRα1 mice) is sufficient to induce aberrant and hyper-proliferative crypts and confers increased susceptibility to Apc-mutation dependent intestinal tumorigenic program (vil-TRα1/Apc+/1638N mice) (Kress et al, 2010). The aim of my work was to study TH- and TRα1-dependent control of intestinal stem cells. Indeed, I used the Lgr5-EGFP-ires-CreERT2 mice enable tracking, sorting and targeting the stem cells (Barker et al, 2007) crossed with tamoxifen inducible TRα1 loss-of-function (Quignodon et al, 2007) mouse model (TRα1-LOF). I studied the effect of TH/TRα1 alteration in vivo and in intestinal organoids (ex vivo). In conclusion, our results indicate that HTs and modulating TRα1 expression or activity have a rapid and strong effect on the intestinal stem cells. This work opens a new perspective in the study of TH/TRα1-dependent signal on the physiopathology of the intestinal stem cells