Viele Organoide sind bisher nur stark vereinfachte Modelle der Originalgewebe, da sie nur aus dem Gewebsparenchym bestehen. Um neurale Organoide näher an das Originalgewebe zu bringen, ist ein wichtiger Schritt mesenchymale Anteile zu integrieren. In dieser Arbeit war die wichtige Fragenstellung, ob neurale Organoide sich mit mesodermalen Progenitorzellen zu einem gemeinsamen Gewebe vereinigen lassen. Um die Generierung von neuro-mesenchymalen Organoiden zu erreichen, wurden geeignete Differenzierungsprotokolle zur Erzeugung neuroepithelialer und mesodermaler Aggregate aus humanen induzierten pluripotenten Stammzellen etabliert. Anschließend wurden die Sphäroide vereinigt und eingehend histologisch charakterisiert. Darüber hinaus wurde die Organoidentwicklung unter dem Einfluss von Hypoxie analysiert. Um die Organoide anschaulich mit der tatsächlichen Embryogenese vergleichen zu können, wurden Schnitte von Hühnerembryonen angefertigt. Die neuro-mesenchymalen Organoide wurden insgesamt 280 Tage kultiviert und an verschieden Zeitpunkten untersucht. Die hier präsentierten Daten zeigen, dass die erzeugten neuro-mesenchymalen Organoide viele Aspekte der natürlichen Embryogenese in Zellkultur nachahmen können. So wurde die Ausbildung neuralrohrähnlicher Strukturen, die von einem perineuralen Gefäßplexus umgeben sind, gezeigt. Des Weiteren wurde eine Interaktion von Astrozyten/radiale Gliazellen mit dem entstehenden Gefäßnetz beobachtet. Schließlich zeigten sich das Einwandern von mikrogliaartigen Zellen aus dem mesenchymalen Organoidteil in das Nervengewebe. Diese Arbeit bildet die Basis für die Generierung neuro-mesenchymaler Organoide als realistisches Modellsystem für die Entwicklung des Nervensystems. Solche Modellsysteme können für die Erforschung von Krankheiten, Toxizitätsstudien sowie Medikamententests verwendet werden. / Many organoids are so far only highly simplified models of the original tissues, since they consist only of the tissue parenchyma. To bring neural organoids closer to the original tissue, an important step is to integrate mesenchymal parts. In this work, the important question was whether neural organoids can be assembled with mesodermal progenitor cells to form a common tissue. To achieve the generation of neuro-mesenchymal organoids, appropriate differentiation protocols were established to generate neuroepithelial and mesodermal aggregates from human induced pluripotent stem cells. Subsequently, the spheroids were brought in co-culture and characterized histologically in detail. In addition, organoid development under the influence of hypoxia was analyzed. Sections of chicken embryos were prepared to compare the organoids with actual embryogenesis. The neuro-mesenchymal organoids were cultured for a total of 280 days and examined at different time points. The data presented here show that the generated neuro-mesenchymal organoids can mimic many aspects of natural embryogenesis in cell culture. For example, the formation of neural tube-like structures surrounded by a perineural vascular plexus was demonstrated. Furthermore, interaction of astrocytes/radial glial cells with the developing vascular network was observed. Finally, the migration of microglia-like cells from the mesenchymal organoid part into the neural tissue was shown. This work provides the basis for generating neuro-mesenchymal organoids as a realistic model system for nervous system development. Such model systems can be used for disease modeling, toxicity studies as well as drug testing.

Gastro-esophageal adenocarcinomas (GEA) are among the fastest rising malignancies in North America. Despite advances in cancer prevention and treatment for other cancers, the number of GEA cases continues to rise and prognosis remains bleak with five-year survival rates of only 20%. Additionally, many GEA patients won’t respond to first line therapy, many may develop therapeutic resistance, or will show disease recurrence. Previous drug screen models failed clinical trials due to the failure of the model to adequately recapitulate the primary sample. A new model, the patient-derived organoid (PDO), has become the newest method of investigating and testing numerous characteristics of the in vivo tumor. Initial studies have demonstrated the organoid’s advantages: PDOs are highly heterogeneous, may be maintained in culture indefinitely, and have the capability to model carcinogenesis and therapeutic response. However, limitations exist and questions remain that have yet to be addressed. Indeed, one of the challenges of using organoids is knowing whether the organoids are recapitulating normal or tumor tissue. Additionally, there seem to be limits on immortality of the organoids and the heterogeneity. Finally, without the stroma and Tumor Microenvironment (TME) in culture, the model is limited in its ability to test the response to immunotherapy-based drugs. Current research aims to develop a clinical pipeline utilizing organoids regularly as a diagnostic tool to evaluate therapeutic response, identify emergence of chemoresistance and perform targeted drug screens. Overall, PDOs are a burgeoning method of investigating GEA and are a powerful translational tool from bench to bedside.

Indiana University-Purdue University Indianapolis (IUPUI) / Inner ear development requires the complex interaction of numerous cell types arising from multiple embryologic origins. Current knowledge of inner ear organogenesis is limited primarily to animal models. Although most mechanisms of cellular development show conservation between vertebrate species, there are uniquely human aspects of inner ear development which remain unknown. Our group recently described a model of in vitro human inner ear organogenesis using pluripotent stem cells in a 3D organoid culture system. This method promotes the formation of an entire sensorineural circuit, including hair cells, inner ear neurons, and Schwann cells. Our past work has characterized certain aspects of this culture system, however we have yet to fully define all the cell types which contribute to inner ear organoid assembly. Here, our goal was to reconstruct a time-based map of in vitro development during inner ear organoid induction to understand the developmental elements captured in this system. We analyzed inner ear organoid development using single-cell RNA sequencing at ten time points during the first 36 days of induction. We reconstructed the on-target progression of undifferentiated pluripotent stem cells to surface ectoderm, pre-placodal, and otic epithelial cells, including supporting cells, hair cells, and neurons, following treatment with FGF, BMP, and WNT signaling modulators. Our data revealed endogenous signaling pathwayrelated gene expression that may influence the course of on-target differentiation. In addition, we classified a diverse array of off-target ectodermal cell types encompassing the neuroectoderm, neural crest, and mesenchymal lineages. Our work establishes the Inner ear Organoid Developmental Atlas (IODA), which can provide insights needed for understanding human biology and refining the guided differentiation of in vitro inner ear tissue. / 2024-08-02

Mammalian embryonic development is subject to complex biological relationships that need to be understood. However, before the whole structure of development can be put together, the individual building blocks must first be understood in more detail. One of these building blocks is the second cell fate decision and describes the differentiation of cells of the inner cell mass of the embryo into epiblast and primitive endoderm cells. These cells then spatially segregate and form the subsequent bases for the embryo and yolk sac, respectively. In organoids of the inner cell mass, these two types of progenitor cells are also observed to form, and to some extent to spatially separate. This work has been devoted to these phenomena over the past three years. Plenty of studies already provide some insights into the basic mechanics of this cell differentiation, such that the first signs of epiblast and primitive endoderm differentiation, are the expression levels of transcription factors NANOG and GATA6. Here, cells with low expression of GATA6 and high expression of NANOG adopt the epiblast fate. If the expressions are reversed, a primitive endoderm cell is formed. Regarding the spatial segregation of the two cell types, it is not yet clear what mechanism leads to this. A common hypothesis suggests the differential adhesion of cell as the cause for the spatial rearrangement of cells. In this thesis however, the possibility of a global cell-cell communication is investigated. The approach chosen to study these phenomena follows the motto "mathematics is biology's next microscope". Mathematical modeling is used to transform the central gene regulatory network at the heart of this work into a system of equations that allows us to describe the temporal evolution of NANOG and GATA6 under the influence of an external signal. Special attention is paid to the derivation of new models using methods of statistical mechanics, as well as the comparison with existing models. After a detailed stability analysis the advantages of the derived model become clear by the fact that an exact relationship of the model parameters and the formation of heterogeneous mixtures of two cell types was found. Thus, the model can be easily controlled and the proportions of the resulting cell types can be estimated in advance. This mathematical model is also combined with a mechanism for global cell-cell communication, as well as a model for the growth of an organoid. It is shown that the global cell-cell communication is able to unify the formation of checkerboard patterns as well as engulfing patterns based on differently propagating signals. In addition, the influence of cell division and thus organoid growth on pattern formation is studied in detail. It is shown that this is able to contribute to the formation of clusters and, as a consequence, to breathe some randomness into otherwise perfectly sorted patterns. / Die embryonale Entwicklung von Säugetieren unterliegt komplexen biologischen Zusammenhängen, die es zu verstehen gilt. Bevor jedoch das gesamte Gebilde der Entwicklung zusammengesetzt werden kann, müssen zunächst die einzelnen Bausteine genauer verstanden werden. Einer dieser Bausteine ist die zweite Zellschicksalsentscheidung und beschreibt die Differenzierung von Zellen der inneren Zellmasse des Embryos hin zu Epiblast- und primitiven Endodermzellen. Diese Zellen teilen sich daraufhin räumlich auf und bilden die anschließend die Grundlagen für den Embryo und den Dottersack. In Organoiden der inneren Zellmasse wird ebenfalls beobachtet, wie sich diese zwei Typen von Vorläuferzellen bilden, und sich in gewissem Maße räumlich voneinander trennen. Diesem Phänomenen widmete sich diese Arbeit im Verlaufe der letzten drei Jahre. Über diese Zelldifferenzierung ist bereits bekannt, dass die ersten Anzeichen für Epiblast- und primitive Endodermdifferenzierung jeweils die Expressionslevel der Transkriptionsfaktoren NANOG und GATA6 sind. Dabei nehmen Zellen mit niedriger Expression an GATA6 und hoher Expression an NANOG das Epiblastschicksal an. Sind die Expressionen umgekehrt, so entsteht eine primitive Endodermzelle. Bei der räumlichen Aufteilung der beiden Zelltypen ist noch nicht eindeutig geklärt, welcher Mechanismus dazu führt. Eine gängige Hypothese besagt, dass die Ursache für die räumliche Umlagerung der Zellen in der unterschiedlichen Adhäsion der Zellen liegt. In dieser Arbeit wird jedoch die Möglichkeit einer globalen Zell-Zell-Kommunikation untersucht. Die gewählte Vorgehensweise bei der Untersuchung dieser Phänomene folgt dem Motto "Die Mathematik ist das nächste Mikroskop der Biologie". Mit Hilfe mathematischer Modellierung wird das zentrale genregulierende Netzwerk im Mittelpunkt dieser Arbeit in ein Gleichungssystem umgewandelt, welches es ermöglicht, die zeitliche Entwicklung von NANOG und GATA6 unter Einfluss eines externen Signals zu beschreiben. Ein besonderes Augenmerk liegt dabei auf der Herleitung neuer Modelle mit Hilfe von Methoden der statistischen Mechanik, sowie dem Vergleich mit bestehenden Modellen. Nach einer ausführlichen Stabilitätsanalyse werden die Vorteile des hergeleiteten Modells dadurch deutlich, dass ein exakter Zusammenhang der Modellparameter und der Formierung von heterogenen Mischungen zweier Zelltypen gefunden wurde. Dadurch lässt sich das Modell einfach kontrollieren und die Proportionen der resultierenden Zelltypen bereits im Voraus abschätzen. Dieses mathematische Modell wird außerdem kombiniert mit einem Mechanismus zur globalen Zell-Zell Kommunikation, sowie einem Modell zum Wachstum eines Organoiden. Dabei wird gezeigt dass die globale Zell-Zell Kommunikation dazu in der Lage ist die Bildung von Schachbrettmustern, sowie auch umrandenden Muster anhand unterschiedlich ausbreitender Signale zu vereinen. Zusätzlich wird der Einfluss der Zellteilung und somit des Organoidwachstums auf die Musterbildung genauestens untersucht. Es wird gezeigt, dass dies zur Bildung von Clustern beiträgt und infolgedessen eine gewisse Zufälligkeit in ansonsten perfekt sortierte Muster einbringt.

The rotating wall vessel (RWV) bioreactor is a well-established cell culture device for the simulation of microgravity for suspension cells and the generation of spheroids and organoids. The key to the success of these systems is the generation of a delicately maintained fluid dynamics system which induces a solid body rotation capable of suspending cells and other particles in a gentle, low-shear environment. Despite the unique capabilities of these systems, the inherently delicate nature of their fluid dynamics makes the RWV prone to multiple failure modes. One of the most frequently occurring, difficult to avoid, and deleterious modes of failure is the formation of bubbles. The appearance of even a small bubble in an RWV disrupts the crucial laminar flow shells present in the RWV, inducing a high-shear environment incapable of maintaining microgravity or producing true spheroids. The difficulty of eliminating bubbles from the RWV substantially increases the learning curve and subsequent barrier-to-entry for the use of this technology. The objective of this study is to create a novel RWV design capable of eliminating the bubble formation failure mode and to demonstrate the design’s efficacy. The tested hypothesis is: “The addition of a channel capable of segregating bubbles from the fluid body of the RWV will protect its crucial fluid dynamics system while enabling the growth of consistently sized and properly formed cell spheroids, improving ease of use of the RWV and decreasing experimental failure.” / Bioengineering

The ability to sense and respond to external environmental signals is closely regulated by a plurality of cell signaling pathways, thereby maintaining homeostasis. In particular, the inflammatory signaling cascade contributes to cellular homeostasis and regulates responses prompted by external stimuli. Such responses are diverse and range from a variety of processes, including tissue repair, cell fate decisions, and even immune-cell signaling. As with any signaling cascade, strict regulation is required for proper functioning, as abnormalities within the pathway are often associated with pathologic outcomes. A hyperactive inflammatory response within the gastrointestinal tract, for example, contributes to inflammatory bowel disease (IBD), presenting as Crohn’s disease or ulcerative colitis. Furthermore, as a chronic condition, IBD is associated with an increased risk for the development of colitis-associated cancer. In order to resolve inflammation and thus restore homeostasis, negative regulation may be utilized to mediate the activity of inflammatory molecules. The mechanistic action of a specific negative regulator of interest, interleukin receptor associated kinase M (IRAK- M), is explored in detail within the present dissertation. Investigation of IRAK-M in mouse models of colitis, which mimics human IBD, and in mouse models of inflammation-driven tumorigenesis, which models colitis associated cancer, demonstrated that loss of this molecule contributes to host protection. Therefore, IRAK- M may be a suitable target for inhibition in order to advance therapeutic options for human patients afflicted with a GI-related inflammatory disease, such as IBD and colitis associated cancer. Furthermore, an ex vivo method that models the interaction of intestinal epithelial cells with microbes present in the GI tract was optimized and is described in the present dissertation. This method takes advantage of primary intestinal derived organoids, also termed “mini-guts”, which display similar features corresponding to intestinal tissue in vivo. For this reason, the use of “mini-guts” has several advantages, particularly for the enhancement of personalized medicine. The method discussed herein aims to normalize experimental conditions in order to enhance reproducibility, which can further be used to uncover microbial-epithelial interactions that contribute to a pathological state, such as IBD. Finally, this method of intestinal epithelial cell culture was utilized to evaluate the role of a protein, termed NF-κB inducing kinase (NIK), in intestinal epithelial cell growth and proliferation. Ultimately, ex vivo organoid culture can serve as an important model system to study the contribution of NIK in intestinal stem cell renewal, cancer progression, as well as in maintenance of the integrity of the gastrointestinal barrier. / Ph. D. / Inflammation is a tightly regulated physiologic process that is employed by body systems such as the gastrointestinal (GI) tract to handle pathogenic insult, aid in wound healing, and help prevent infections. When abnormal inflammatory responses occur, this can lead to the progression of severe diseases such as ulcerative colitis and Crohn's disease. When inflammation persists in the GI tract, such as in inflammatory bowel disease, this can predispose patients to the development of inflammation- associated colorectal cancer. In order to improve the treatment options for patients afflicted with these maladies, this dissertation is aimed at studying the signaling pathways of the innate immune system that regulate such inflammatory responses. Furthermore, this body of work encompasses a detailed method for isolating and culturing intestinal stem cells, which can be applied in personalized medicine for patients with intestinal diseases. This method was utilized in this dissertation to study genetically modified intestinal stem cells, and can further be used to investigate the interactions of intestinal epithelial cells with pyogenic bacteria that contribute to inflammatory maladies in the GI tract.

京都大学 / 新制・課程博士 / 博士(工学) / 甲第24812号 / 工博第5155号 / 新制||工||1985(附属図書館) / 京都大学大学院工学研究科マイクロエンジニアリング専攻 / (主査)教授 横川 隆司, 教授 安達 泰治, 教授 永樂 元次 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

abstract: A genetically engineered line of human induced pluripotent stem cells was used to study the effects of gene expression on cell fate. These cells were designed to activate expression of the gene GATA6 when exposed to the small molecule doxycycline. This gene was chosen because it plays an important role in the developmental biology stages of liver formation. Because of the way the cells were engineered, a given population would have a heterogeneous expression of GATA6 because each cell could have a different copy number of the exogenous gene. This variation allows for the differentiation of multiple cell types, and is used to grow liver organoids. The early liver organoid samples were studied via immunofluorescent staining, imaging, and quantitative image analysis. It was originally hypothesized that absolute gene expression was not the most important factor in determining cell fate, but relative gene expression was. This meant that the spatial location of the cells and their local environment were critical in determining cell fate. In other words, the level of GATA6 of a cell is important, but so is the level of GATA6 in the surrounding cells, or neighborhood, of that cell. This hypothesis was analyzed with the creation of various Neighborhood Impact Factor (NIF) methods. Multiple time points of growth were analyzed to study the temporal effect, in addition to the gene expression and NIF influence on a cell’s fate. Direct gene expression level showed correlation with certain cell fate markers. In addition to GATA6 expression levels, NIF results from early and late time point experiments show statistical significance with relatively small neighborhood radii. The NIF analysis was useful for examining the effect of neighboring cells and determining the size of the neighborhood – how far cells influence one another. While these systems are complex, the NIF analysis provides a way to look at gene expression and its influence in spatial context. / Dissertation/Thesis / Powerpoint presentation used in the defense. / Masters Thesis Bioengineering 2017

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22374号 / 医博第4615号 / 新制||医||1043(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 松田 道行, 教授 小西 靖彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Indiana University-Purdue University Indianapolis (IUPUI) / Hearing loss and vestibular dysfunction are inner ear disease states that arise from an array of diverse etiologies that interfere with mechanosensory hair cell function, including: congenital syndromes, noise-induced trauma, ototoxic drugs, and aging. The investigation of normal inner ear development and the pathological aberrations that cause inner ear disease has been previously advanced through formation of an easily generated, scalable, accurate in vitro model system that readily facilitates experimental applications. This model utilizes a 3D floating cell culture protocol which guides differentiation of stem cell aggregates into inner ear organoids, which are vesicles containing a sensory epithelium with functioning mechanosensory hair cells. Inner ear organoid formation enables studying the effects of modulating the signaling pathways that guide developing inner ear structure and function. The Notch signaling pathway heavily influences the formation of the inner ear through two major mechanisms: lateral induction of sensory progenitor cells and lateral inhibition to determine which of those progenitors differentiate into mechanosensory hair cells. The effects of inhibiting Notch signaling within the inner ear organoid system were explored through application of the ɣ-secretase inhibitor MDL28170 (MDL) at a concentration of 25μM on day 8 of organoid culture. Aggregates were harvested on day 32, fixed, sectioned, and stained according to a standard immunohistochemistry protocol. Sections were stained for the mechanosensory hair cell markers Myosin7a (Myo7a) and Sox2. MDL-treated aggregates demonstrated statistically significant reductions in the total number of vesicles and the number of vesicles containing hair cells compared to control aggregates. In contrast to control aggregates which demonstrated two distinct organoid variants (protruding and embedded), MDL-treated aggregates only formed the embedded variant. Differences in the expression pattern of Sox2, which is also a marker of stemness and neural progenitor cells were also noted between the two conditions. MDL-treated aggregates demonstrated regions of ‘ectopic’ Sox2 expression whereas Sox2 expression in control aggregates was consistently expressed within Myo7a+ regions.