Les cellules souches pluripotentes (CSP) humaines sont devenues en quelques années des modèles de choix pour étudier les mécanismes cellulaires et moléculaires qui gouvernent l'apparition de maladies monogéniques, mais également pour le développement de criblages à haut débits afin d'identifier parmi plusieurs milliers de molécules chimiques celles qui ont un potentiel thérapeutique. C'est dans ce contexte de criblage que mes travaux de thèse s'inscrivent, alliant automatisation et miniaturisation de la biologie des CSP dans le cadre de deux maladies monogéniques, l'amyotrophie spinale infantile (SMA) et la dystrophie myotonique de type I (DM1). De manière générale, la mise en place d'une telle stratégie repose sur trois étapes essentielles qui sont l'obtention de CSP porteuses d'une mutation donnée, l'identification d'un modèle d'étude pertinent et la réalisation du criblage à proprement parlé. L'obtention de CSP humaines repose sur deux approches principales. La première consiste en la dérivation de cellules embryonnaires humaine (hES) issues de diagnostiques préimplantatoires et la seconde repose sur la reprogrammation de cellules somatiques par l'induction de pluripotence (iPS). Une partie de mon travail a consisté en la création de cellules iPS modèles de la SMA et leur caractérisation par une approche à haut débit. Par la suite un travail d'optimisation du protocole de génération de motoneurones à partir de CSP humaines a permis d'accélérer et augmenter les rendements de production de ces cellules qui sont principalement affectées dans la SMA. Enfin, l'utilisation de cellules hES porteuses de la mutation causale de la DM1 a permis le criblage de 12000 molécules et a conduit à l'identification d'une famille chimique capable de restaurer plusieurs défauts typiques de cette maladie tels que des défauts d'épissage et de fusion moléculaire. / For only few years, Human pluripotent stem cells (PSC) have become wide spread models in order to study and decipher cellular or molecular mechanims involved in monogenic diseases, but also for the development of large scale screening strategies allowing the identification of new therapeutics among thousands of chemicals. Mythesis research aimed at the development of such strategies, miniaturizing and automating PSC biology within the framework of two monogenic diseases, namely spinal muscular atrophy (SMA) and myotonic dystrophy type 1 (DM1).Basically, PSC based screening programs are generally built around three main steps which are the access to a stem cell model, the identification of a relevant cell type and lastly the screening campaign. There is actually two main ways to generate human PSC. Firstly, human embryonic stem cells (hES) can be derived from the inner cell mass of blastocyte through a pre-implantation diagnosis and secondly, induced pluripotent stem cells (iPS) can be generated after somatic cell reprogramming in vitro. A part of my work has consisted in the generation of hiPS cellular models for SMA by reprogramming fibroplasts that carried SMN1 gene deletion, followed bay the characterization of several dozen of independant clones with high throughput. Then an optimization process of the protocol for the generation of Motoneuron from PSC has been done multiplying experimental conditions. This finally allowed the description of a fast and efficient protocol to generate the most affected cell type in SMA. Finally, DM1 mutated hES were uded for the screening of 12.000 compounds among which a chemical family has been identified to rescue DM1 typical splicing and myogenesis defects.

No description available.

The definitive endoderm gives rise to several specialized organs, including the thymus. Improper development of the definite endoderm or its derivatives can lead to human disease; in the case of the thymus, immunodeficiency or autoimmune disorders. Human pluripotent stem cells (hPSCs) have emerged as a system to model human development, as study of their differentiation allows for elucidation of the molecular basis of cell fate decisions, under both healthy and impaired conditions. Here, we first developed a CRISPR-effector system to control endogenous gene expression in hPSCs, a novel approach to manipulating hPSC state. Next, the human-specific, loss-of-function phenotypes of candidate transcription factors driving hPSC-to-definitive endoderm differentiation were analyzed through combined CRISPR-perturbation and single-cell RNA-sequencing. This analysis revealed the importance of TGFβ mediators in human definitive endoderm differentiation as well as identified an unappreciated role for FOXA2 in human foregut development. Finally, as the differentiation of definitive endoderm to thymic epithelial progenitors (TEPs) is of particular interest, a single-cell transcriptomic atlas of murine thymus development was generated in anticipation of identifying factors driving later stages of TEP differentiation. Taken together, this dissertation establishes a CRISPR-effector system to interrogate gene and regulatory element function in hPSC differentiation strategies, details the role of specific transcription factors in human endoderm differentiation, and sets the groundwork for future investigations to characterize hPSC-derived TEPs and the factors driving their differentiation.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19592号 / 医博第4099号 / 新制||医||1014(附属図書館) / 32628 / 京都大学大学院医学研究科医学専攻 / (主査)教授 斎藤 通紀, 教授 伊達 洋至, 教授 上杉 志成 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Indiana University-Purdue University at Indianapolis (IUPUI) / Human pluripotent stem cells (hPSCs) are an attractive tool for the study of different neurodegenerative diseases due to their potential to form any cell type of the body. Due to their versatility and self-renewal capacity, they have different applications such as disease modeling, high throughput drug screening and transplantation. Different animal models have helped answer broader questions related to the physiological functioning of various pathways and the phenotypic effects of a particular neurodegenerative disease. However, due to the lack of success recapitulating some targets identified from animal models into successful clinical trials, there is a need for a direct translational disease model. Since their advent, hPSCs have helped understand various disease effectors and underlying mechanisms using genetic engineering techniques, omics studies and reductionist approaches for the recognition of candidate molecules or pathways required to answer questions related to neurodevelopment, neurodegeneration and neuroregeneration. Due to the simplified approach that iPSC models can provide, some in vitro approaches are being developed using microphysiological systems (MPS) that could answer complex physiological questions. MPS encompass all the different in vitro systems that could help better mimic certain physiological systems that tend to not be mimicked by in vivo models. In this dissertation, efforts have been directed to disease model as well as to understand the intrinsic as well as extrinsic cues using two different MPS. First, we have used hPSCs with Alzheimer’s disease (AD)-related mutations to differentiate into retinal organoids and identify AD related phenotypes for future studies to identify retinal AD biomarkers. Using 5 month old retinal organoids from AD cell lines as well as controls, we could identify retinal AD phenotypes such as an increase in Aβ42:Aβ40 ratio along with increase in pTau:Tau. Nanostring analyses also helped in identification of potential target genes that are modulated in retinal AD that were related to synaptic dysfunction. Thus, using retinal organoids for the identification of retinal AD phenotypes could help delve deeper into the identification of future potential biomarkers in the retina of AD patients, with the potential to serve as a means for early identification and intervention for patients. The next MPS we used to serve to explore non-cell autonomous effects associated with glaucoma to explore the neurovascular unit. Previous studies have demonstrated the degeneration of RGCs in glaucoma due to a point mutation OPTN(E50K) that leads to the degeneration of RGCs both at morphological and functional levels. Thus, using the previous studies as a basis, we wanted to further unravel the impact of this mutation using the different cell types of the neurovascular unit such as endothelial cells, astrocytes and RGCs. Interestingly, we observed the barrier properties being impacted by the mutation present in both RGCs and astrocytes demonstrated through TEER, permeability and transcellular transport changes. We also identified a potential factor TGFβ2 that was observed to be overproduced by the OPTN E50K astrocytes to demonstrate similar effects with the exogenous addition of TGFβ2 on the barrier. Furthermore, the inhibition of TGFβ2 helped rescue some of the barrier dysfunction phenotypes. Thus, TGFβ2 inhibition can be used as a potential candidate that can be used to further study its impact in in vivo models and how that can be used in translational applications. Thus, MPS systems have a lot of applications that can help answer different physiologically relevant questions that are hard to approach using in vivo models and the further development of these systems to accentuate the aspects of neural development and how it goes awry in different neurodegenerative diseases.

No description available.

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.

La thérapie cellulaire se positionne comme une stratégie prometteuse pour inciter le cœur infarci à se régénérer. A cet effet, des études récentes placent des espoirs considérables dans l’utilisation des cellules souches embryonnaires et notre laboratoire a déjà démontré comment les différencier en progéniteurs cardiovasculaires, un type de précurseurs cellulaires qui ne peut aboutir qu’à la formation de cardiomyocytes, de cellules endothéliales ou de cellules de muscles lisses. Cet engagement précoce réduit leur capacité de prolifération anarchique et en même temps leur permet de rester suffisamment plastiques pour éventuellement s’intégrer plus facilement avec le tissue hôte. Cependant, les études précliniques et cliniques d’injection de ces cellules s’avérèrent décevantes. Malgré de légères améliorations de la fonction cardiaque, on observa une trop faible survie cellulaire ainsi qu’un taux de rétention des cellules dans le myocarde remarquablement bas. Afin d’étudier ce problème, mes travaux de thèse ont porté non seulement sur la conception de nouveaux biomatériaux pouvant servir de moyen de transport et d’intégration des cellules dans la zone infarcie, mais aussi sur la conception de biomatériaux permettant de contrôler précisément l’environnement cellulaire au cours du processus de différenciation de cellules souches pluripotentes humaines en cardiomyocytes. Grâce aux importantes interactions entre nos laboratoires de recherche fondamentale et de recherche clinique, nous avons tout d’abord développé de nouvelles techniques de fabrication et de caractérisation de patches de fibrine cellularisés qui sont récemment entrés dans un essai clinique de phase I. A partir de cette formulation clinique approuvée par les autorités de régulation, nous avons élaboré toute une gamme de matériaux composites uniquement à base de matières premières pertinentes dans ce cadre clinique, dans le but d’améliorer la maturation des progéniteurs cardiovasculaires une fois greffés sur le cœur défaillant. Dans cette optique, nous avons également développé un modèle in vitro permettant d’étudier précisément l’influence combinée de la rigidité du substrat et du confinement spatial sur la différenciation des cellules souches en cardiomyocytes. Grâce à des techniques de microfabrication sur substrat mou, il a été possible de positionner précisément les cellules souches pluripotentes dans des espaces restreints d’élasticité variable. Ainsi, nous avons pu observer que même en utilisant des protocoles chimiques éprouvés basés sur la modulation de cascades de signalisation impliquées dans le développement cardiaque, une très forte hétérogénéité pouvait apparaître en fonction de l’environnement physique des cellules. Nous avons ainsi pu extraire les caractéristiques principales permettant une différenciation cardiaque efficace, reproductible et standardisée et les avons appliquées à la fabrication d’une nouvelle génération de patches composés de matériaux cliniques et de couches multiples de bandes synchrones de cardiomyocytes. De fait, ces travaux ouvrent de nouvelles voies dans l’utilisation de biomatériaux pour la production industrielle de cardiomyocytes et pour la fabrication de patches cliniques, cellularisés ou non, dans le traitement de l’insuffisance cardiaque. / Cell therapy is a promising strategy to help regenerate the damaged heart. Recent studies have placed a lot of hopes in embryonic stem cells and our lab had previously found a way to differentiate them into cardiac progenitors, cells that can only differentiate into cardiomyocyte, endothelial cells or smooth muscle cells. This early commitment decreases their proliferative capabilities, yet maintains their plasticity for better integration inside the host tissue. However, clinical and pre-clinical injection studies did not really meet the expectations. Even though slight improvements in cardiac function were demonstrated, very low cell viability has been observed, as well as a very low retention of the cells inside the myocardium. To address this problem, my PhD projects not only focus on the design of new biomaterials to act as a vehicle for cell delivery and retention in the infarcted area, but also on the design of biomaterials that control the cellular environment during the differentiation of pluripotent stem cells into cardiomyocytes. Going back and forth between the labs and the clinics, we first developed new techniques for the fabrication and the characterization of a cell-laden fibrin patch that is now undergoing phase I clinical trial. From the approved clinical formulation, we then propose new blends of clinical materials that will eventually improve the maturation of the cardiac progenitors once grafted onto the failing heart. In this perspective, we developed an in vitro model to investigate the combined influence of matrix elasticity and topographical confinement on stem cell differentiation into cardiomyocytes. By using microfabrication techniques to pattern pluripotent stem cells on substrates of controlled stiffness, we demonstrate that even using a widely recognized chemical-based protocol to modulate signaling cascades during differentiation, much heterogeneity emerges depending on the cellular physical environment. We thus extracted the main features that led to controlled and reproducible cardiac differentiation and applied it to the fabrication of next generation of multi-layered anisotropic cardiac patches in compliances with clinical requirements. This work opens new routes to high-scale production of cardiomyocytes and the fabrication of cell-laden or cell-free clinical patches.