Malgré les progrès considérables cellulaire arythmique commun entre des patients réalisés en termes de compréhension des à fonds génétiques différents atteints de mécanismes à l'origine des maladies du rythme syndrome de Brugada (BrS). Nous avons montré cardiaque, ces dernières restent parmi les qu'IRXS participe à la régulation de la problèmes les plus fréquemment rencontrés en conduction ventriculaire rapide en régulant cardiologie et constituent la principale cause de l'expression de SCNSA et des jonctions gap dans décès. Le dysfonctionnement ou la désexpression les cardiomyocytes humains. En ce qui concerne d'un seul canal ionique peuvent être à l'origine de la BrS, indépendamment du contexte génétique plusieurs types d'arythmies en fonction du fond ou de la présence des mutation SCN5A, les génétique du patient. Les cardiomyocytes dérivées hiPS-CMs ventriculaires issues de patients de cellules souches pluripotentes induites de différents atteints de BrS étaient plus sujets aux l'homme (hiPS-CMs) confèrent un outil précieux arythmies qui étaient associees à une et fiable pour mieux comprendre l'arythmie augmentation du sodium tardif. Globalement, les cardiaque. hiPS-CMs fournissent un outil pertinent pour De là, nous avons défié ce modèle pour modéliser l'arythmie cardiaque humaine, en plus caractériser l'arythmie causée par des mutations de elles confèrent un environnement cellulaire facteur de transcription Iroquois, IRX5, et pour pertinent pour étudier de nouvelles fonctions afin explorer la présence d'un phénotype de développer des pistes thérapeutiques. / Despite considerable advances in the understanding of basic mechanisms involved in the genesis and perpetuation of arrhythmias, cardiac rhythm disorders remain among the most commonly encountered problems and the leading cause of death in cardiology. Dysfunction or misexpression of one single ion channel can be at the root of several types of arrhythmias depending on patient genetic background. Human induced pluripotent stem cells derived cardiomyocytes (hiPS-CMs) confer a valuable and reliable tool to better understand cardiac arrhythmia. Therefore, we challenged this model to characterize arrhythmia caused by missense mutations at the Iroquois transcription factor, IRX5, and to elaborate a common cellular phenotype for Brugada syndrome (BrS) described in patients with different genetic backgrounds. Interestingly, we showed that JRX5 participates in controlling fast ventricular conduction through sustaining SCN5A and gap junction proper expression in human cardiomyocytes. As for BrS, regardless of the genetic background or the presence of SCN5A mutations, ventricular hiPSCMs originated from different BrS patients were more prone to arrhythmias which were associated with increased late sodium. Overall, hiPS-CMs provide a pertinent translational tool to model human cardiac arrhythmia, and they confer a relevant cellular environment to study new functions and therefore develop novel therapeutic tracks.

The human Embryonic Stem Cells (hESCs) and human induced Pluripotent Stem Cells (hiPSCs) have offered numerous advantages including but not limited to model diseases, high-throughput drug screening, and regenerative purposes. However, the employment of monolayer cultures has not been sufficient to mimic the in vivo stem cells niche. Thus, three-dimensional suspension cultures have helped us to advance our knowledge and ease the development of the human organs’ counterparts, commonly referred as organoids. Currently, the challenge is the generation of homogenous and reproducible human Pluripotent Stem Cell (hPSC) aggregates, the basic cellular unit to derive organoids. To date, the Ultra-Low Attachment (ULA) 6-well plates have been routinary used for the hPSC aggregates formation, which mainly relies on the inhibition of the Rho-associated kinase (ROCK) pathway resulting in the enhancement of cell survival coming from cryopreserved stocks or when passaging. However, little is known in this regard when analyzing the aggregate formation of hPSCs with two widely used compounds: RevitaCellTM Supplement and Y27632. Importantly, due to the high demand required from the regenerative medicine, I aimed to upscale the hPSC aggregates production in the DASbox® Mini-Bioreactor System. In this thesis, I established protocols for the hPSC aggregates formation by using two different types of media in two platforms being the ULA 6-well plates and the DASbox® Mini-Bioreactor System. In addition, I demonstrated that monolayer confluence cultures before single cell inoculations are paramount for the formation of bona fide hPSC aggregates in healthy and X aneuploid hiPSCs, precisely two hESCs and five hiPSCs.

En 3ème position de fréquence parmi les myopathies, la dystrophie facio-scapulo humérale (FSHD) reste encore très énigmatique malgré l’association génétique avec la contraction d’une région du locus 4q35 contenant le macrosatellite D4Z4. Au cours de ce projet, l’étude de la régulation de la méthylation de l’ADN par séquençage après traitement au bisulfite de sodium ou par MeDIP chez les patients FSHD a montré des profils de méthylation similaires entre porteurs asymptomatiques et contrôles d’une part et patients FSHD1 et FSHD2 d’autre part. Ces deux groupes de malades présentent une hypométhylation principalement au niveau de la région proximale de D4Z4. Des mutations d’un nouveau gène candidat, SMCHD1, ont été décrites dans la plupart des cas de FSHD2, pour lesquels nous retrouvons fréquemment mais non systématiquement une baisse de la méthylation. D4Z4 joue également un rôle clé dans la régulation des extrémités chromosomiques et leur dynamique d’organisation tri-dimensionnelle dans l’espace nucléaire. Afin d’étudier la dynamique du locus 4q35 au cours de la différenciation cellulaire, nous avons mesuré les interactions spécifiques de différentes régions géniques au sein du locus et suivi l’évolution de la conformation de la chromatine dans différents contextes biologiques grâce à une méthode de FISH en trois dimensions couplée à une analyse in sillico des images (3D-FISH). Le retour à un état de pluripotence par la production d'hIPSCs montre un comportement nucléaire de ces régions proche de celui observé chez les contrôles. Enfin nous avons suivi la conformation du locus jusqu’à la formation de fibres musculaires matures multinuclées. / In the third position in term of frequency among the myopathies, facio-scapulo humeral dystrophy (FSHD) remains enigmatic despite the genetic correlation with the contraction of the 4q35 locus containing the macro satellite, D4Z4. In this project, the involvement of DNA methylation has been investigated in FSHD patients using bisulfite sequencing or MeDIP. These analyses revealed similar DNA methylation patterns between asymptomatic carriers and controls and between FSHD1 and FSHD2 patients. These two groups of patients show a marked hypomethylation mostly in the proximal region of D4Z4. Moreover, the recent discovery of mutations of a new candidate gene, SMCHD1 have been observed in most FSHD2 cases. In these patients, we observed frequent but not systematical association between decreased methylation and SMCHD1 mutation. This gene might be associated with D4Z4 DNA methylation maintenance; however its function remains unknown. D4Z4 plays a crucial role in chromosomal ends regulation and three-dimensional dynamics of the locus within the nuclear space. In order to study the dynamics and topology of the 4q35 locus during the skeletal muscle differentiation, we measured specific interactions between different regions within the locus and followed this chromatin conformation in different biological situations thanks to FISH in three-dimensions followed by in sillico analysis of the images (3D-FISH). Upon pluripotency in induced pluripotent stem cells derived from human fibroblasts (hIPSCs) display the same nuclear organization as in controls. Finally, we have followed the configuration of the locus during skeletal muscle commitment.

Cardiomyocyte proliferation plays a crucial role in the developing mammal heart, as it is required for normal morphogenesis and in determining the appropriate heart size. Postnatally, the decrease in cell cycle activity is concomitant with the increase of cell cycle variants, such as endoreduplication and acytokinetic mitosis, which contribute to the hypertrophic growth of the heart. Although the adult mammal heart retains the ability to generate new cardiomyocytes, the extent of cardiomyocyte renewal is insufficient to compensate for the large-scale tissue loss associated with ischemic events. Indeed, ischemic events such as myocardial infarction, lead to a permanent loss of ventricular cardiomyocytes, formation of collagen-containing scar, and consequently cardiac remodeling. The development of therapies able to hamper cardiac remodeling by promoting cardiomyocyte turnover is one of the primary goals in the cardiovascular field. In the present study, we generated a human induced pluripotent stem cell (iPSC) line containing the fluorescence ubiquitination-based cell cycle indicator (FUCCI) under the Troponin T2 (TNNT2) promoter. To gain information about the cell cycle dynamics of human cardiomyocytes, we visualized cell cycle progression in TNNT2-FUCCI human iPSCs-derived cardiomyocytes. Notably, we revealed cardiomyocytes' cell cycle dynamics of cells undergoing proliferation, binucleation, and polyploidization and identified G2 cell cycle arrest in cardiomyocytes undergoing polyploidization. To demonstrate the versatility of the TNNT2-FUCCI human iPSCs line, we developed a live cell screening platform to identify pro-proliferative compounds within an autophagy compound library. We identified Clonidine, an alpha2-adrenergic receptor and imidazoline agonist, as an enhancer of cell cycle activity in TNNT2-FUCCI hiPSC-derived cardiomyocytes. Finally, we investigated the ability of Clonidine to promote cell cycle progression in hiPSC- derived cardiomyocytes and in in vivo and in vitro mouse neonatal cardiomyocytes. We showed that while Clonidine stimulated cardiomyocytes' polyploidization and multinucleation, respectively in in vitro in in vivo mouse cardiomyocytes, the treatment of hiPSC-derived cardiomyocytes with Clonidine enhanced their proliferative capability. In conclusion, we showed that the TNNT2-FUCCI system is a versatile tool for characterizing cardiomyocyte cell cycle dynamics and identifying pro-proliferative molecular candidates with regenerative potential in the mammalian heart.:1. Introduction 1.1 Heart function and composition 1.2 Human cardiac development 1.2.1 Cardiac organogenesis 1.2.2 Metabolic changes in the developing heart 1.3 Cardiomyocytes cell cycle activity 1.3.1 Cardiomyocytes cell cycle regulators and cell cycle arrest 1.3.2 CM multinucleation and polyploidization 1.4. The regenerative capabilities of the mammal heart 1.4.1 Model systems for heart regeneration 1.4.2 Stimulation of cardiomyocyte proliferation as a goal to preserve heart function 1.4.3 Assessment of cardiomyocytes proliferation 1.5 Human-induced pluripotent stem cells to model cardiac development and disease 2. Aim 3. Materials and methods 3.1 TNNT2-FUCCI hiPSC line generation 3.2 hiPSC culture and maintenance 3.3 hiPSC differentiation into CMs 3.4 Imagestream-X Analysis 3.5 TNNT2 expression assessment of hiPSC-derived CMs by immunohistochemistry 3.6 CDK1 immunohistochemistry expression assessment of hiPSC-derived CMs 3.7 Cell area and sarcomere spacing measurement of hiPSC-derived CMs 3.8 Live imaging and timelapse imaging analysis of TNNT2-FUCCI hiPSC 3.9 Murine neonatal CMs cell culture 3.10 Mouse nCM timelapse imaging and analysis 3.11 TNNT2-FUCCI hiPSC-derived CMs culturing and screen conditions 3.11.1 TNNT2-FUCCI screen image acquisition 3.11.2 TNNT2-FUCCI screen automated image analysis 3.11.3 TNNT2-FUCCI screen data analysis 3.12 Primary mouse nCM compound validation and immunohistochemistry 3.13 Mouse nCM immunohistochemistry for AurKB expression assessment 3.14 hiPSC-derived CMs immunohistochemistry for AurKB expression assessment 3.15 Analysis of CM ploidy and binucleation 3.16 in vivo Clonidine treatment of neonatal mice 3.17 Analysis of ploidy and binucleation in in vivo mouse nCMs 3.18 Cell cycle activity assessment in P7 neonatal mouse hearts after Clonidine treatment by immunohistochemistry 4. Results 4.1 TNNT2-FUCCI human iPSC line generation and validation 4.2 TNNT2-FUCCI marks proliferating and non-proliferating CMs 4.3 Live imaging identification of CM cell cycle activity 4.3.1 Cell cycle progression of TNNT2-FUCCI CMs 4.3.2 Cell cycle progression of mouse neonatal cardiomyocytes 4.4 TNNT2-FUCCI live-imaging identifies CM cell cycle activators 4.5 Compound validation in mouse nCMs 4.6 Clonidine elicits cycling activity via alpha1 adrenergic receptor and imidazoline receptor interaction 4.7 Clonidine stimulates hiPSC-derived CM proliferation 4.8 Clonidine stimulates CM polyploidization in mouse nCMs 4.9 Clonidine mediates in vivo CM cell cycle activity in the neonatal mouse 5. Discussion 5.1 TNNT2-FUCCI hiPSC: a new technology to identify cycling CMs 5.2 Study of CM cell cycle activity using TNNT2-FUCCI 5.3 TNNT2-FUCCI hiPSC in combination with a live screening platform revealed enhancer of CMs cell cycle activity. 5.4 Initial validation of pro-proliferative compounds in mouse nCMs reveals Clonidine and Dihydrocapsaicin as enhancers of cell cycle progression 5.5 Clonidine stimulates cell cycle activity in different model systems 5.5.1 Clonidine treatment stimulates proliferation in hiPSC-derived CMs 5.5.2 Clonidine treatment stimulates polyploidization in in vitro- and multinucleation in in vivo mouse nCMs 5.6 Conclusions and future outlooks 6. Appendix 7. Summary 8. Zusammenfassung Acknowledgements References Anlage 1 Anlage 2

O Transtorno Depressivo Maior (TDM) é uma condição psiquiátrica que afeta 4,4% da população mundial, exibindo um substancial sofrimento pessoal, incapacidade e custos sociais, e estima-se que ele será a principal causa de incapacidade no mundo em 2030. O surgimento de novas ferramentas e modelos de pesquisa envolvendo o TDM irá auxiliar no entendimento desta doença. Deste modo, o objetivo deste trabalho foi gerar uma coleção de células-tronco pluripotentes induzidas humanas (hiPSCs) de um grupo de indivíduos com TDM. Foram coletadas amostras de células mononucleares (MNCs) de 66 indivíduos afetados, e geradas 6 linhagens de hiPSCs. Através de diversos testes de caracterização, a pluripotência destas células foi confirmada. Além disto, também foi padronizada a diferenciação destas hiPSCs em neurônios serotonérgicos. Neurônios derivados dessas hiPSCs poderão constituir material de estudo para outros grupos de pesquisa interessados no estudo da TDM, e ser utilizados em testes futuros para prever resposta a medicamentos. / Major Depressive Disorder (MDD) is a psychiatric condition that affects 4.4% of the world\'s population, exhibiting substantial personal suffering, disability and social costs, and is estimated to be the leading cause of disability in the world by 2030. Emergence of new tools and research models involving TDM will aid in the understanding of this disease. Thus, the objective of this work was to generate a collection of human induced pluripotent stem cells (hiPSCs) from a group of individuals with MDD. Samples of mononuclear cells (MNCs) of 66 affected individuals were collected, and 6 lines of hiPSCs were generated. Through several characterization tests, the pluripotency of these cells was confirmed. In addition, the differentiation of these hiPSCs into serotonergic neurons was also standardized. Neurons derived from these hiPSCs could constitute study material for other research groups interested in the study of MDD, and be used in future tests to predict drug response.

Les cellules entéroendocrines sécrétrices d’hormones représentent 1% de l’épithélium intestinal mais sont des régulateurs essentiels du métabolisme énergétique et l’altération de leur différenciation provoque de graves pathologies métaboliques. Leur différenciation est régie par une cascade de régulations transcriptionnelles qui est encore peu décrite, particulièrement chez l’homme. L’objectif de ce projet de thèse était d’évaluer l’implication de plusieurs facteurs de transcription préalablement identifiés chez la souris (NGN3, RFX6, ARX, PAX4) dans la différenciation entéroendocrine humaine. Pour ce faire, ces gènes ont été inactivés par la technique CRISPR/Cas9 dans des cellules souches pluripotentes induites humaines (hiPSCs), qui ont ensuite été différenciées in vitro en organoïdes intestinaux (HIOs). Les analyses des HIOs déficients pour NGN3 ne permettent pas de conclure quant à un rôle dans la différenciation entéroendocrine mais indiquent une altération de la régionalisation du tissu formé chez les mutants. RFX6 semble important pour la différenciation et/ou la fonction des cellules entéroendocrines, bien que sa fonction précise n’ait pas pu être déterminée. / Hormone-producing enteroendocrine cells represent 1% of the intestinal epithelium but are key regulators of the energetic metabolism and alteration of their differentiation is associated with severe metabolic disorders. Enteroendocrine differentiation is governed by a transcriptional regulatory cascade that is poorly described, especially in humans. This thesis project aimed to evaluate the implication of several transcription factors, previously identified in mice (NGN3, RFX6, ARX, PAX4), in human enteroendocrine differentiation. To do so, these genes were disrupted with the CRISPR/Cas9 system in human inducible pluripotent stem cells (hiPSCs), which were then differentiated in intestinal organoids (HIOs). Preliminary analysis of NGN3-deficient HIOs did not allow a firm conclusion regarding NGN3 implication in enteroendocrine differentiation but showed a tissue regionalization alteration. RFX6 seems important for the differentiation/function of enteroendocrine cells, although its precise function is still to be determined.

O Transtorno Depressivo Maior (TDM) é uma condição psiquiátrica que afeta 4,4% da população mundial, exibindo um substancial sofrimento pessoal, incapacidade e custos sociais, e estima-se que ele será a principal causa de incapacidade no mundo em 2030. O surgimento de novas ferramentas e modelos de pesquisa envolvendo o TDM irá auxiliar no entendimento desta doença. Deste modo, o objetivo deste trabalho foi gerar uma coleção de células-tronco pluripotentes induzidas humanas (hiPSCs) de um grupo de indivíduos com TDM. Foram coletadas amostras de células mononucleares (MNCs) de 66 indivíduos afetados, e geradas 6 linhagens de hiPSCs. Através de diversos testes de caracterização, a pluripotência destas células foi confirmada. Além disto, também foi padronizada a diferenciação destas hiPSCs em neurônios serotonérgicos. Neurônios derivados dessas hiPSCs poderão constituir material de estudo para outros grupos de pesquisa interessados no estudo da TDM, e ser utilizados em testes futuros para prever resposta a medicamentos. / Major Depressive Disorder (MDD) is a psychiatric condition that affects 4.4% of the world\'s population, exhibiting substantial personal suffering, disability and social costs, and is estimated to be the leading cause of disability in the world by 2030. Emergence of new tools and research models involving TDM will aid in the understanding of this disease. Thus, the objective of this work was to generate a collection of human induced pluripotent stem cells (hiPSCs) from a group of individuals with MDD. Samples of mononuclear cells (MNCs) of 66 affected individuals were collected, and 6 lines of hiPSCs were generated. Through several characterization tests, the pluripotency of these cells was confirmed. In addition, the differentiation of these hiPSCs into serotonergic neurons was also standardized. Neurons derived from these hiPSCs could constitute study material for other research groups interested in the study of MDD, and be used in future tests to predict drug response.

Le syndrome de DiGeorge ou microdélétion 22q11.2, est la délétion chromosomique la plus fréquente chez les êtres humains. Cette délétion est liée à la recombinaison homologue non-allélique au cours de la méiose induisant la perte d’en moyenne 40 gènes. Les études de corrélation génotype/phénotype chez les patients ont révélé des différences phénotypiques entre individus et cela indépendamment de la taille des microdélétions. L’hypothèse de l’implication des mécanismes épigénétiques dans la variabilité phénotypique observée a été soulevée mais reste encore inexplorée. C’est dans ce contexte que nous nous intéressons à l’étude des mécanismes épigénétiques au cours du développement, dans cette pathologie à travers l’utilisation d’un modèle de cellules souches pluripotentes induites humaines (hiPSs). En particulier, nous avons ciblé nos travaux sur le rôle de la chaperonne d’histone HIRA dont le gène est localisé dans la région délétée. HIRA est impliquée dans la déposition du variant d’histone H3.3, une histone majeure dans le cerveau. Afin de comprendre l’implication de HIRA dans les manifestations neurologique du syndrome de DiGeorge et en particulier dans la schizophrénie, nous avons développé et optimisé un nouveau protocole pour la différenciation de cellules hiPSCs en progéniteurs neuronaux, neurones corticaux et neurones dopaminergiques. L’ensemble de ces travaux ouvre donc de nouvelles perspectives pour la modélisation d’un grand nombre de pathologies, et dans le contexte du laboratoire, pour l’exploration des mécanismes épigénétiques associés à la variabilité phénotypique dans différentes maladies génétiques. / The DiGeorge syndrome also known as 22q11.2 microdeletion syndrome, is the most common deletion in humans. This deletion is linked to a non-allelic homologous recombination that occurs during meiosis and involves sequences called LCRs for "Low Copy Repeats". Depending on the LCRs involved, different deletions are observed, inducing the loss of approximately 40 genes. The absence of genotype/phenotype correlation in patients and the phenotypical differences regardless of the size of the microdeletion suggests the involvement of additional parameter. The hypothesis of epigenetic changes associated with the onset or variability of symptoms has been suggested but never investigated. In order to tackle this question, we decided to focus our attention of the role of the HIRA histone chaperone encoded by a gene located in the 22q11.2-deleted region. HIRA is involved in the deposition of the H3.3 histone variant, one of the main histone in the brain. In order to determine whether HIRA is implicated in the neurological manifestations in DiGeorge patients and particularly in schizophrenia, we developed and optimized a new protocol for the direct differentiation of human induced pluripotent stem cell (hiPSCs) into neural progenitors, cortical and dopaminergic neurons. In parallel, we developed a new protocol for hiPSCs differentiation toward the skeletal muscle lineage and the production of multinucleated muscle fibers. Altogether, these results open new perspectives for the modeling of a large number of pathologies, and in the context of our laboratory, the exploration of epigenetic mechanisms associated with phenotypic variability in different genetic diseases.

Familial hypertrophic cardiomyopathy (HCM) is a genetic disease largely caused by a mutation in myosin binding protein C (MYBPC3) and it affects about 1:500 population leading to arrhythmic sudden death, heart failure, and atrial fibrillation. MYBPC3 activates calcium-induced actin-myosin filament sliding within the cardiac sarcomere, creating the force necessary for heart contraction. The underlying molecular mechanisms causing HCM phenotype remain elusive, therefore, there is an urgent need for a reliable in vitro human HCM model to investigate the pathogenesis of HCM. This study utilized isogenic human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with MYBPC3 gene mutation (wildtype, heterozygous, homozygous) and further micropatterned them into fiber-like structures on polyacrylamide hydrogels of physiological and fibrotic-like stiffnesses. Cells were cultured for an extended culture time up to 60 days and their morphology/attachment, contractility, and calcium transient were extensively and carefully evaluated. It was found that MYBPC3 knockout cells maintained the highest contraction amplitude, but had increased contraction, and relaxation durations, decreased calcium transient amplitude, as well as time to peak and decay times over the culture period in comparison to the isogenic wildtype. Overall, this study demonstrates that hiPSC-CMs can be successfully patterned and cultured for an extended time on hydrogels forming end-to-end connections, which can be served as a simple yet effective in vitro human model for studying mechanical dysfunction of HCM.

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.