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<div><div><div><p>Transcriptomic regulation of human liver is a tightly controlled and highly dynamic process. Genetic and environmental exposures to this process play pivotal roles in the development of multiple liver disorders. Despite accumulating knowledge have gained through large-scale genomics studies in the developed adult livers, the contributing factors to the interindividual variability in the pediatric livers remain largely uninvestigated. In the first two chapters of the present study, we addressed this question through an integrative analysis of both genetic variations and transcriptome-wide RNA expression profiles in a pediatric human liver cohort with different developmental stages ranging from embryonic to adulthood. Our systematic analysis revealed a transcriptome-wide transition from stem-cell-like to liver-specific profiles during the course of human liver development. Moreover, for the first time, we observed different genetic control of hepatic gene expression in different developmental stages. Motivated by the critical roles of genetics variations and development in regulating hepatic gene expression, we constructed robust predictive models to impute the virtual liver gene expression using easily available genotype and demographic information. Our model is promising in improving both PK/PD modeling and disease diagnosis for pediatric patients. In the last two chapters of the study, we analyzed the genomics data in a more liver disease- related context. Specifically, in the third chapter, we identified Macrophage migration inhibitory factor (MIF) and its related pathways as potential targets underlying human liver fibrosis through an integrative omics analysis. In the last chapter, utilizing the largest-to-date publicly available GWAS summary data, we dissected the causal relationships among three important and clinically related metabolic diseases: non-alcoholic fatty liver disease (NAFLD), type 2 diabetes (T2D), and obesity. Our analysis suggested new subtypes and provided insights into the precision treatment or prevention for the three complex diseases. Taken together, through integrative analysis of multiple levels of genomics information, we improved the current understanding of human liver development, the pathogenesis of liver disorders, and provided implications to precision medicine.</p></div></div></div>

During embryonic development, generation of functional organs is dependent on proper interactions between different cell types. Elucidation of the mechanisms operating during organ formation might provide insights into the origin of many pathological disorders in the adult. Gene inactivation studies in mice have provided invaluable tool to study the function of genes critical for morphogenesis of distinct organs. A LIM-homeodomain transcription factor Lhx2 has previously been reported to play a role in fetal liver development and hematopoiesis, as its inactivation leads to lethal anemia due to underdeveloped liver. This thesis focuses on the function of Lhx2 in the development of these two organ systems. Reciprocal signaling between ventral foregut endoderm and mesenchyme of the septum transversum regulates the liver formation, expansion and differentiation. A fully formed liver is composed of endoderm-derived hepatocytes and cholangiocytes and a variety of mesenchyme-derived cell types, such as endothelial cells and hepatic stellate cells. In early stages of liver development Lhx2 is expressed in the liver-associated septum transversum mesenchyme, a part of which becomes integrated into the liver organ and develops into hepatic stellate cells. Functional Lhx2 expression in the hepatic mesenchyme is necessary for normal liver outgrowth and differentiation. Loss of Lhx2 from developing hepatic stellate cells leads to their activation and excessive deposition of collagen fibres, resulting in hepatic fibrosis and severely distorted liver architecture. Transfection of Lhx2 to human stellate cell line downregulates genes associated with stellate cell activation and fibrogenesis. Thus, Lhx2 is the first gene identified to negatively regulate events leading to hepatic fibrosis. Elucidation of the molecular mechanisms involved in this process might therefore be instrumental for the development of novel therapies useful in treatment of this disorder. Fetal liver is also a major site of hematopoiesis in the embryo and provides physiological conditions necessary for the efficient expansion of hematopoietic stem cells (HSCs). The hematopoietic defect observed in Lhx2-deficient embryos is cell-nonautonomous, indicating that Lhx2 might control secreted factors involved in the self-renewal of HSCs. This putative second role of Lhx2 has been investigated by analyzing the mechanism whereby Lhx2 expression generates in vitro self-renewing HSC-like cell lines. Interestingly, in agreement with the cell nonautonomous phenotype of the lethal anemia in Lhx2-/- embryos, the mechanism of self-renewal is dependent on Lhx2 expression and occurs via secreted factor(s). Identification of these factor(s) might potentially allow ex vivo expansion of HSCs for therapeutic purposes. The Lhx2-immortalized HSC-like cell lines share many basic features with HSCs and self-renew in vitro in presence of Steel factor (SF). SF/c-Kit signaling mediates a wide variety of biological activities in cells at many different levels in the hematopoietic hierarchy. We used the HSC-like cell lines as an in vitro model system to compare signal transduction pathways from c-Kit receptor in stem cells versus differentiated hematopoietic cells. HSCs require PI-3K dependent activation of Raf1-Mek-Erk cascade for their survival and self-renewal in response to SF, whereas activation of Erk is PI-3K independent in committed myeloid and mast cells. Thus, the mode of SF/c-Kit signaling is dependent on the differentiation status of the cells.

Background: The liver occupies a central function in homeostasis maintenance. Its functions depend on a fine architecture allowing each parenchymal cell, the hepatocytes, to contact both the vasculature and to contribute to the formation of the bile canaliculi network. To separate two compartment, epithelial cells like hepatocytes must develop a specific organization: polarity. In the liver, the bile canaliculi network and vasculature are organized as two tightly intertwined networks. To achieve contact with both, the hepatocytes must acquire a complex polarity composed of multiple axes. The study of this organization is the focus of intense research. A lot of attention is put on the understanding of the established structure in the adult organ and the regeneration of this structure in case of injury. With a progressing understanding of this organization comes the interrogation on how it is established during development. Furthermore, a proper understanding of the establishment process is central to develop reliable in vitro systems or organ-on-a-chip approaches. Questions: In this context we have decided to address the following questions: - When and how is multipolar polarity established. - Can this establishment be correlated to any extracellular cue in vivo. Methods: To address these questions we have fixed, stained, imaged, and reconstructed large volumes of embryonic liver tissue at several consecutive stages of development. We looked at polarized trafficking and junctional markers to follow polarity establishment. We looked at ECM and cell division markers to correlate cues susceptible to guide polarization. We also performed in vitro experiments to further explore the hypotheses formulated along the way based on in vivo observations. Conclusion: This study led us to conclude that multipolar polarity arises from both lumen tubular elongation and multiple lumen formation. The formation of multiple lumina involves the movement of a Rab11 vesicular cluster and independent junctional plate formation. Our study suggests that ZO-1 could be involved in the synchronization between vesicular cluster movement and junction plate formation. Finally, we could not link cell division to polarization, however, we could correlate fibronectin distribution to potential early polarization events.

La thérapie cellulaire pourrait représenter une alternative à la transplantation hépatique dans certaines pathologies comme les maladies métaboliques sévères. Toutefois, la pénurie de donneurs d’organes implique la nécessité de trouver de nouvelles sources de cellules hépatiques comme les cellules souches pluripotentes qui peuvent être amplifiées extensivement et différenciées en tout type cellulaire. Les cellules souches embryonnaires humaines (hESC) et les cellules souches pluripotentes induites humaines (hiPSC) générées à partir des cellules somatiques de patients puis différenciées en hépatocytes représentent une source potentielle d’hépatocytes. Ces cellules permettent en outre d’envisager la transplantation d’hépatocytes autologues génétiquement modifiés comme alternative à la transplantation hépatique pour le traitement de certaines maladies génétiques du foie. L’hypercholestérolémie familiale (HF) est une maladie autosomale dominante due à des mutations dans le gène codant le Récepteur aux Low Density Lipoproteins (RLDL) qui est à l’origine d’un taux élevé de cholestérol sanguin de patients HF. Les patients homozygotes doivent épurer leur sérum par LDL-aphérèse en moyenne deux fois par mois dès le plus jeune âge pour éviter les infarctus mortels survenant dès l’enfance. Les hépatocytes différenciées à partir des iPSC de patients et leur correction in vitro, permettent d'évaluer la faisabilité de la transplantation d'hépatocytes autologues génétiquement modifiés pour le traitement de l’hypercholestérolémie familiale.Au cours du développement du foie, des hépatocytes et des cholangiocytes, les deux types de cellules épithéliales hépatiques, dérivent de progéniteurs hépatiques bipotents (les hépatoblastes). Bien que les cholangiocytes formant les canaux biliaires intrahépatiques ne représentent qu'une petite fraction de la population cellulaire totale du foie (3%), ces cellules régulent activement la composition de la bile par réabsorption des acides biliaires, un processus qui est important dans des maladies choléstatiques du foie. Dans la première partie de cette étude nous avons mis au point une approche de différenciation des cellules souches pluripotentes (hESC et hiPSC) en cholangiocytes fonctionnels. Ces cellules serviront à la modélisation des maladies génétiques touchant les cholangiocytes formant des canaux biliaires. Dans la deuxième partie, nous avons généré des iPSC spécifiques de patients HF (HF-iPSC), différenciées en hépatocytes et corrigé le défaut phénotypique par transfert lentiviral de l’ADNc codant le LDLR dans les HF-iPSC. / Cell therapy can be an alternative to liver transplantation in some cases such as severe metabolic diseases. However, the shortage of organ donors implies the need to find new sources of liver cells such as hepatocytes derived from pluripotent stem cells that can be amplified and differentiated extensively into any cell type. Human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC) generated from somatic cells of patients and then differentiated into hepatocytes represent a potential source of transplantable hepatocytes. These cells now make it possible to consider the transplantation of genetically modified autologous hepatocytes as an alternative to liver transplantation for the treatment of genetic diseases of the liver.Familial hypercholesterolemia (FH) is an autosomal dominant disorder caused by mutations in the gene encoding the receptor for Low Density Lipoproteins (LDLR), which is the cause of high blood cholesterol in these patients. Homozygous patients should purify their serum LDL-apheresis on average twice a month starting at a young age to avoid fatal myocardial infarction occurring in childhood.Human hepatocytes differentiated from patient’s induced pluripotent stem cells (iPSCs) allow assessing the feasibility to transplant genetically modified autologous hepatocytes as treatment of familial hypercholesterolemia.During the liver development, hepatocytes and cholangiocytes, the two types of hepatic epithelial cells, derive from bipotent hepatic progenitors (hepatoblasts). Although cholangiocytes, forming intrahepatic bile ducts, represent a small fraction of the total liver cell population (3%), they actively regulate bile composition by secretion and reabsorption of bile acids, a process that is important in cholestatic liver diseases. In the first part of this study we developed an approach to differentiate pluripotent stem cells (hESC and hiPSC) into functional cholangiocytes. These cells could be used for the modeling of genetic biliary diseases. In the second part, we generated FH patient specific iPSCs (HF-iPSC), differentiated them into hepatocytes and tried to correct the disease phenotype by lentiviral introduction of LDLR cDNA cassette in HF-iPSC.