La leucémie lymphoïde chronique (LLC) est l'hémopathie maligne la plus fréquente des pays occidentaux. Elle se caractérise par une accumulation de cellules leucémiques dans le sang, la moelle osseuse et les organes lymphoïdes secondaires. Malgré les nouvelles thérapies, la LLC reste incurable. Dans la LLC, comme dans tout cancer, le microenvironnement tumoral, un milieu complexe contenant des cellules immunitaires pouvant être pro-tumorales, prend une part croissante aussi bien dans la physiopathologie que le pronostic. La recherche de nouvelles thérapies ciblant les interactions des cellules leucémiques avec ce microenvironnement représente aujourd'hui une piste prometteuse pour traiter cette pathologie. Les " nurse-like " cells (NLC) semblaient être les macrophages associés aux tumeurs (TAM) de la LLC. Ces cellules, retrouvées dans les ganglions lymphatiques de patients, favorisent in vitro la survie des cellules leucémiques. Elles partagent avec les TAM de nombreuses caractéristiques telles que des capacités immunosuppressives, un profil transcriptomique proche et une implication dans la résistance aux traitements. Nous l'avons confirmé par une analyse fonctionnelle et phénotypique. Nos travaux indiquent clairement que les NLC sont les TAM de la LLC avec une forte expression de CD68 et de CD163 et qu'elles favorisent la survie des cellules leucémiques. Suite à ce travail, nous avons déterminé l'impact pronostique des NLC dans la LLC, en émettant l'hypothèse que, comme dans d'autres cancers, une forte infiltration par les TAM pouvait être de mauvais pronostic. Nous avons alors montré que le taux de NLC présentes au sein du ganglion lymphatique chez le patient est corrélé à la progression de la LLC. Les NLC relarguent également un facteur soluble le CD163 soluble (sCD163) dans le sang des patients, dont le taux est corrélé à des marqueurs de mauvais pronostic dans la LLC tels que le statut IgHV non muté, le caryotype complexe et les mutations de TP53. Enfin, le taux de sCD163 est un marqueur pronostique indépendant dans la LLC : un fort taux étant associé à un raccourcissement du temps avant retraitement, de la survie sans progression et de la survie globale des patients. Ensuite nous avons étudié l'impact d'une thérapie innovante, l'ibrutinib, un inhibiteur de tyrosine kinase ciblant spécifiquement la Bruton Tyrosine Kinase, sur ces NLC. In vitro, les monocytes se différencient toujours en NLC et leur phénotype n'est pas modifié. De plus, ces cellules sont toujours capables de protéger les cellules leucémiques de l'apoptose. Finalement, nous avons mis en évidence qu'elles pouvaient participer à la chimiorésistance, en protégeant in vitro les cellules leucémiques de l'ibrutinib mais pas du dasatinib, de l'idélalisib, du vénétoclax, de la bendamustine et du rituximab. L'ensemble de nos résultats indique que cibler les NLC est une piste prometteuse dans le traitement de la LLC. Les interactions cellulaires entre NLC et cellules leucémiques pourraient représenter une cible thérapeutique intéressante. Nous avons montré qu'in vitro le contact entre NLC et cellules leucémiques est nécessaire à la survie de ces dernières. Suite à une étude transcriptomique, nous avons mis en évidence plusieurs couples moléculaires potentiellement impliqués dans ces interactions. Seul le couple LFA3/CD2 s'est révélé être nécessaire dans ce contact via un mécanisme dépendant d'Akt et son blocage inhibe totalement les effets pro-survie des NLC sur les cellules leucémiques. En conclusion, les NLC via des interactions dépendantes du couple LFA-3/CD2 protègent les cellules leucémiques de l'apoptose in vitro. De plus, elles participent à la chimiorésistance en protégeant aussi les cellules leucémiques de l'ibrutinib. Elles relarguent également du sCD163, dosable dans le sérum, un facteur pronostique indépendant de la LLC. La recherche de nouvelles thérapies plus spécifiques, ciblant les NLC constitue donc une piste thérapeutique intéressante dans la LLC. / Chronic lymphocytic leukemia (CLL) is the most common hemopathy in western countries. This pathology is characterized by an accumulation of leukemic cells in blood, bone marrow and secondary lymphoid organs. Despite new therapies, CLL is still incurable. In CLL, the microenvironment, a complex media containing immune cells witch can favor the tumor, takes now a large place in the physiopathology and the prognosis. The research of new therapies targeting interactions between CLL cells and microenvironment is a promising runway to find new drugs in CLL. "Nurse-like" cells (NLC) should be the tumor associated macrophages (TAM) of CLL. This cells, found in the lymph node of CLL patients, protect CLL cells against in vitro apoptosis. They share with TAM many characteristics including immunosubversive proprieties chemoresistance induction and a close gene expression profiling. We confirmed that by functional and phenotypical analysis. Our results indicate that NLC are the TAM of CLL with a high expression of CD68 and CD163 and that NLC can protect CLL cells against in vitro apoptosis. Thus, we focused on the clinical impact of NLC in CLL. Because in several cancers a high infiltration of TAM is correlated with a poor clinical outcome, we hypothesized that NLC infiltration should be associated with CLL outcome. We showed that infiltration of NLC in the lymph node can be correlated with the progressivity of CLL. Moreover, NLC release a soluble factor, the sCD163 (soluble sCD163), in the blood of CLL patients. High levels of this factor, can be correlated with previously established prognostic makers in CLL such as TP53 mutations, unmutatted IgHV status and complex karyotype. Finally, sCD163 was an independent prognostic marker in CLL and high levels are associated with shorter time to next treatment, progression free survival and overall survival. Next, we studied the impact of ibrutinib, a new therapy in CLL, on NLC. Ibrutinib is a specific tyrosine kinase inhibitor witch targets specifically the Bruton Tyrosine Kinase. In vitro, NLC differentiated from ibrutinib treated patients have the same phenotype as NLC from untreated patients and are still able to protect CLL cells against apoptosis. To end, we tested if in vitro NLC could protect CLL cells against chemotherapy and showed that NLC protect CLL cells against ibrutinib but not against idelalisib, dasatinib, venetoclax, bendamustin and rituximab. All our previous studies reveal that NLC are a good target to find new therapies in CLL. We focused our work on the interaction between NLC and CLL cells. First, we demonstrated that the NLC/CLL cells contact is necessary to prevent CLL cells death in vitro. Next, by gene expression profiling, we screened several couple of molecules potentially implicated in these interactions. Finally, only LFA-3/CD2 couple was necessary for this contact through an Akt pathway dependent and the inhibition of this couple totally inhibited the pro-survival effect of NLC on CLL cells. To conclude, NLC protect CLL cells from in vitro apoptosis through LFA-3/CD2. Moreover, they protect CLL cells against ibrutinib and so facilitate the chemoresistance. They release sCD163, an independent marker in CLL, which can be measured in the serum. The research of new targets, which can be considerate as more specific, targeting NLC is still an interesting way to find new therapies in CLL.

PCSK9 est un régulateur clé du métabolisme du cholestérol par le foie à travers la dégradation lysosomiale du récepteur aux LDL (low-density lipoprotein). Alors que les mutations gain de fonction (GOF) de PCSK9 induisent une hypercholestérolémie autosomique dominante, les mutations pertes de fonctions (LOF) entraînent un taux spontanément bas de LDL-cholestérol, ainsi qu’un protection cardiovasculaire. Du fait des limitations inhérentes aux modèles d’études, tels que les lignées cellulaires transfectées ou des animaux transgéniques, les fonctions de PCSK9 restent encore mal connues. Ainsi, nous avons utiliser des cellules souches pluripotentes induites (hiPSC) spécifiques de patients pour les différencier en hépatocytes et modéliser la physiopathologie liée aux mutations de PCSK9 GOF-S127R et LOF-R104C/V114A. Nous avons démontré que les hépatocytes obtenus récapitulaient la physiopathologie liés aux mutations de PCSK9. De plus, les cellules portant la mutation S127R ont montré une importante réponse au traitement par les statines, qui est corrélée à la réponse clinique des patients portant cette même mutation. Enfin, notre étude nous a permis de mettre à jour une fonction inattendue de PCSK9 dans les hiPSC et pendant leur différenciation. Elle montre que PCSK9 affecterait la prolifération des hiPSC ainsi qu’une voie de signalisation clé du développement régulée par NODAL. Cette régulation se ferait à travers une interaction directe entre PCSK9 et DACT2, un régulateur intracellulaire de la voie de signalisation de NODAL. En conclusion, les hiPSC s’avèrent être un modèle cellulaire translationnel pertinent pour mettre à jour de nouvelles fonctions hépatiques de PCSK9. / PCSK9 has been identified as a key regulator of cholesterol metabolism by the liver through inducing lysosomal degradation of the low-density lipoprotein receptor (LDLR). While PCSK9 gain-of-function (GOF) mutations induced autosomal dominant hypercholesterolemia and increased cardiovascular risk, loss-of-function (LOF) mutations are associated with low LDL-cholesterol levels and cardiovascular protection. Due to limitations inherent to current models including animal and human cells lines transfected with DNA constructs or transgenic animal models, PCSK9 functions are not fully understood. Therefore, we took advantage of patient related somatic cells reprogramming intoinduced pluripotent stem cells (hiPSC) to generate hepatocyte-like cells (HLC) and model the pathophysiology of PCSK9 mutations in dyslipidemia through focusing on two intracellular mutation forms; GOF (S127R) and LOF (R104C/V114A). We showed that HLC could recapitulate the key pathophysiological features of PCSK9 mutations. Moreover, HLC with the S127R mutation displayed an increased uptake of LDL upon statin treatment, which was correlated with the original patient clinical response. In parallel, this model enabled us to unravel a new unexpected role of PCSK9 in hiPSC and during differentiation. PCSK9 was found to affect the proliferation of hiPSC and regulate a key developmental signaling pathway mediated by NODAL. This regulation might occur by a direct interaction between PCSK9 and DACT2, an intracellular attenuator of NODAL signaling pathway. In conclusion, hiPSC provide a pertinent translational model to decipher PCSK9 hepatic functions and a novel cellular environment to highlight new functions.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22880号 / 医博第4674号 / 新制||医||1047(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 篠原 隆司, 教授 近藤 玄, 教授 万代 昌紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Recent studies indicate the existence of a subpopulation of cells within tumours with stem cell-like characteristics. These "cancer stem-like cells" (CSCs) are relatively resistant to established therapies, usually targeting differentiated and fast proliferating cells. Therefore, CSCs may be a reason for the relapse of neoplastic diseases. CSCs can be characterised by a specific gene expression profile and deregulated signalling pathways. Of these, upregulation of the erbB-2 (HER2) receptor, a hallmark of ~25-30% breast cancer patients, is related to dismal prognosis, elevated proliferation potential and resistance to chemotherapy. Recent evidence has suggested that upregulation of erbB-2 leads to increase in the pool of CSCs. In our study we used mammospheres, cells grown in the absence of serum, an in vitro model of breast CSCs, which were prepared by "weaning" breast cancer MCF7 cells to a special medium. These cells were CD44high and showed increased expression of ABCG-2, Sox-2, Vimentin as well as high levels of erbB-2. Next, we prepared a stable line of MCF7 cells with low levels of erbB-2 by shRNA. ErbB-2low cells were characterised for expression of set of CSCs markers and tested for tumour forming efficacy in nude mice using ultrasound imaging. Keywords Cancer stem-like cells, erbB-2,...

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20605号 / 医博第4254号 / 新制||医||1023(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 川口 義弥, 教授 浅野 雅秀, 教授 中川 一路 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13141号 / 論医博第2141号 / 新制||医||1026(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 川口 義弥, 教授 横出 正之, 教授 萩原 正敏 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

El conocimiento de las interacciones entre célula/proteína/biomaterial es fundamental para la ingeniería de superficies debido a las numerosas aplicaciones biomédicas y biotecnológicas que se están desarrollando así como al éxito clínico que han alcanzado muchos implantes. La respuesta biológica final inducida por los implantes está fuertemente influenciada por las interacciones superficiales entre los componentes biológicos y el material sintético. Las propiedades físicas y químicas de la superficie de un biomaterial, en lugar de las propiedades en su masa, influyen directamente en la capa de proteínas que se adsorben sobre el biomaterial y, como consecuencia de ello, en la respuesta celular a la misma, tanto in vitro como in vivo.El objetivo de esta tesis doctoral es profundizar en el conocimiento de las interacciones material-biosistema, con el énfasis en el descubrimiento de relaciones entre las propiedades superficiales de las superficies de titanio y su respuesta biológica in vitro.El titanio comercialmente puro (Ti c.p.) está siendo ampliamente utilizado con éxito durante muchos años como biomaterial para implantes en cirugía ósea. Su excelente biocompatibilidad se basa en sus adecuadas propiedades mecánicas y, con mayor importancia, en su excelente resistencia a la corrosión. Esta última se debe principalmente a la formación espontanea de una fina película de óxido de titanio que le confiere protección natural contra los ataques degradativos. La modificación de la topografía de la superficie del titanio ha sido objeto de investigación en el pasado con el fin de mejorar la osteointegración. El granallado de partículas es una de las tecnologías más utilizadas para conferir rugosidad a las superficies del titanio. La rugosidad óptima y el tipo de partículas abrasivas del granallado para una respuesta óptima in vitro e in vivo fue previamente determinada en nuestro laboratorio. Sin embargo, todavía están por determinar cuáles son las causas últimas que llevan al biomaterial a su exitosa respuesta biológica.En este trabajo se han estudiado superficies pulidas y rugosas de Ti c.p. obtenidas mediante el granallado con partículas abrasivas de diferente composición química(Al2O3 y SiC) y diferentes tamaños (212-300μm; 425-600μm; 1000-1400μm). La completa caracterización de las propiedades física y química de la superficie, incluyendo la rugosidad, la composición química, la mojabilidad/energía libre y la carga eléctrica de las superficies ensayadas ha llevado a una serie de relevantes conclusiones. Entre ellas, cabe destacar que a) la composición química de las partículas de granallado, así como el método de esterilización fueron los principales factores que influyeron en la mojabilidad y la energía libre superficial de las superficies de titanio estudiadas, b) el método de esterilización cambió en la energía superficial el carácter de donante de electrones de las superficies mediante el cambio de la cantidad y la naturaleza de las sustancias adsorbidas, y c) la composición química de las partículas de granallado no influyó en la carga eléctrica a pH fisiológico ni en el punto isoeléctrico de las superficies.Un segundo paso consistió en el uso de una microbalanza de cristal de cuarzo con monitorización de la energía de disipación, para el estudio de la cinética de adsorción (cantidad y conformación) y de los procesos de adsorción competitiva de tres proteínas de especial interés en los procesos de curación del hueso - la albúmina de suero bovino (BSA), el fibrinógeno (Fbg), y la fibronectina (Fn)- en sensores lisos recubiertos de TiO2. Se determinaron diferentes modelos de procesos de adsorción con una, dos o múltiples pasos distinguibles en función de las proteínas en solución. La capa adsorbida de BSA mostró los cambios más significativos en sus propiedades mecánicas, de conformación y de incorporación de agua hasta que se alcanzaron las condiciones estables de adsorción de proteínas. La BSA, la más pequeña de las proteínas ensayadas, desplazó la Fn y el Fbg cuando se ensayó en condiciones de la competencia por la adsorción, indicando su mayor afinidad por las superficies de TiO2. También se emplearon técnicas de marcaje fluorescente para el estudio de la adsorción proteica en superficies rugosas granalladas. En este estudio, por un parte, se pudo determinar que la cantidad de Fn y BSA adsorbidas en las superficies granalladas está directamente correlacionada con su energía superficial. Por otra parte, se visualizó la adsorción de fibronectina en solución sobre muestras granalladas rugosas de Ti. La Fn formó un patrón irregular de adsorción con una mayor cantidad de proteína adsorbida en los picos que en los valles de la topografía.También se evaluó la organización espacial de la matriz extracelular de los osteoblastos, ECM, sobre superficies de Ti lisas y rugosas por medio de la visualización de las fibrillas de Fn teñidas con marcador fluorescente. Las células osteoblásticas depositaron las fibrillas de Fn con un determinado patrón organizado dentro de la matriz total secretada. Aparecen como una película que cubre la parte superior de las diferentes superficies rugosas de titanio. Un resultado relevante es que el espesor de esta capa aumentó con la rugosidad de la topografía subyacente. Sin embargo no más de la mitad de la máxima distancia pico-valle se cubrió con la proteína secretada y/o reorganizada.Por último, teniendo en cuenta las diferencias en la organización de la ECM y laadsorción de Fn en las superficies ensayadas de Ti, se realizó un estudio de qRT-PCR para determinar la influencia de las propiedades superficiales del titanio, con y sin preadsorción de Fn, en la respuesta osteoblástica. La expresión génica de la subunidad 5 de la integrina celular, como marcador de la adhesión celular, se incrementó en las superficies granalladas con SiC en comparación con las granalladas con alúmina. Este resultado fue correlacionado con la mayor cantidad de Fn adsorbida debido a la mayor energía superficial de las superficies granalladas con SiC. El aumento de la rugosidad, así como la presencia de partículas de alúmina en las superficies rugosas incrementó la actividad de ALP y la expresión génica de ALP mRNA por los osteoblastos, y por lo tanto su diferenciación. / The understanding of cell/protein/biomaterial interactions is critical to the engineering of substrates for numerous biomedical and biotechnological applications and to the clinical success of implants. The final biological response induced by implants is strongly influenced by the biological-components/synthetic-material surface interactions. It is well accepted that the physical and chemical surface properties of a biomaterial rather than its bulk properties will influence the protein adlayer and then the cell response to it, both in vitro and in vivo.The aim of this PhD thesis is to gain an increased understanding of the materialbiosystem interactions, with an emphasis on establishing correlations between surface properties of titanium surfaces and its in vitro biological response.Commercially pure titanium (c.p. Ti) is being widely and successfully used implant biomaterial in bone surgery over many years. Its excellent biocompatibility is based in its appropriate mechanical properties and, more importantly, in its excellent corrosion resistance, which is mainly due to the presence of a naturally-occurring thin protective titanium oxide film. Modification of titanium surface topography has been a subject of research in the past with the purpose of improving its osseointegration. Grit blasting is one of the most used technologies to roughen titanium surfaces for this purpose. The optimal roughness and type of abrasive blasting-particles for a better in vitro and in vivo response was previously determined in our lab. However, which and how different relevant surface properties of the blasted titanium surfaces induce that optimal biological behavior is still poorly understood.Smooth/polished and rough c.p. Ti surfaces obtained by blasting with abrasiveparticles of different chemical composition (Al2O3 and SiC) and different sizes (212-300μm; 425-600μm; 1000-1400μm) were studied. The comprehensive characterization of physical and chemical surface properties, including roughness, chemical composition, wettability/free energy and electrical charge of the tested surfaces led to a series of relevant conclusions. Among them, it is worth noting that a) the chemical composition of the grit-blasting particles as well as the method of sterilization were found the main factors influencing wettability and surface free energy of the titanium surfaces; b) the sterilization method changed the electron donor character of the surfaces by changing the amount/nature of physisorbed substances on the surfaces, and c) the chemical composition of the blasting particles did not influence on the electrical charge at physiological pH and the isoelectric point of the surfaces.A second step consisted in the use of a quartz crystal microbalance with monitoring of the energy dissipation to study the adsorption kinetics (amount and conformation) and adsorption competition processes of three proteins of special interest in the healing processes of bone -bovine serum albumin (BSA), fibrinogen (Fbg), and fibronectin (Fn)-on smooth TiO2-coated sensors. Different patterns of adsorption with processes in one, two or multiple distinguishable steps were determined depending of the protein in solution. The BSA adlayers showed the most significant changes in their mechanical properties/conformation/incorporation of water until steady protein-adsorption conditions were reached. BSA, the smallest of the tested proteins, displaced Fn and Fbg when in competition for adsorption, which is an indication of its higher affinity for TiO2 surfaces. Fluorescent labelling techniques where used to study protein adsorption on blasted rough surfaces. Most significantly, the amount of Fn and BSA adsorbed on blasted surfaces was positively correlated with their surface energy. The adsorption of fibronectin from solution on shot-blasted rough titanium surfaces resulted in an irregular pattern of adsorption with a higher amount of protein adsorbed on peaks than on valleys of the topography.Further, the spatial organization of the osteoblast extracellular matrix, ECM, on smooth and rough Ti surfaces was evaluated by visualizing fluorescently-stained Fn-fibrils. Osteoblast-like cells deposited Fn- fibrils in a specific facet-like pattern that was organized within the secreted total matrix. It appeared as a film overlying the top of the different rough titanium surfaces. Interestingly, the thickness of this layer increased with the roughness of the underlying topography, but no more than half of the total maximum peak-to-alley distance was covered.Finally, taking into consideration the differences in ECM organization and Fn adsorption on the tested Ti surfaces a qRT-PCR study was carried out to elucidate the influence of titanium surface properties with and without Fn-precoatings on the osteoblast response. The expression of 5 integrin subunit gene, as a marker for cell adhesion, was increased in SiC-blasted surfaces compared to alumina-blasted surfaces. This was related to the higher amount of adhesive-protein Fn adsorbed caused by the higher surface energy of SiC-blasted surfaces. The increase of roughness as well as the presence of alumina particles on blasted surfaces increased ALP activity and ALP mRNA gene expression by osteoblasts, and so their differentiation.This research work contribute to increase our knowledge on the interactions taking place at the bio/non-bio interface between different biological components -water, proteins, cells- and materials of clinical relevance, such as rough titanium. Theintertwined effects of the different properties of the synthetic surfaces appear as a challenge to unravel the ultimate causes that determine the fate of cells on synthetic biomaterials.

The liver is a crucial human organ with a complex architecture. Although the liver has great regeneration potential, deadly liver diseases are associated with irreversible hepatocytes damage. Currently, a liver transplant is the only treatment for liver failure. A shortage of donors forced extensive research for alternative treatments. The most promising hepatocyte source could be obtained from the differentiation of induced pluripotent stem cells (iPSCs). This technology can give us great amounts of pluripotent cells without ethical restrictions, which could be available in a variety of haplotypes to minimize the possibility of rejection. There are many reprogramming protocols available. However, there is still no standardised method to obtain clinical grade iPSCs. From those stem cells, it is possible to obtain hepatic-like cells (HLCs) by direct differentiation in vitro. HLCs express multiple hepatocyte-specific features, but their names signal that they still show fetal liver identity. A variety of hepatic differentiation protocols were described, although the process of hepatic differentiation must be improved in order to be translated into the clinic. Along with genes, microRNA (miRNA) is the well-known controller of cell fate. MiRNA is a type of non-coding RNA (ncRNA) which can influence gene transcription by inhibiting gene expression. In contrast to genes, many of the miRNA can affect up to thousands of genes simultaneously. Another group of ncRNA, which is a subject of potential differences are small nucleolar RNA (snoRNA). SnoRNA are involved in RNA chemical modifications by acting as a guide, mostly for ribosomal RNA (rRNA), but some of them have additional functions. In this study, a new iPSCs line was generated from skin fibroblasts using lipotransfection of episomal vectors. This method is free from exogene integration and shows low cytotoxicity. A pluripotency of generated cells was confirmed by morphological assessment, immunocytochemical staining, and spontaneous differentiation assay. To be sure that the genome of the cells was not changed, karyotype analysis was performed. Next, HLCs were derived from those iPSCs using a four-stage hepatic differentiation protocol. The obtained HLCs were characterised using, among others, a hepatic gene expression analysis. Cells after differentiation express mature and fetal hepatic markers, which is consistent with previous results. The attempt to improve differentiation using transient overexpression of master hepatic transcription factor – HNF4α, was not sufficient, as shown by gene expression analysis and whole slide scanning. Previous studies failed to point out the genetic inhibitors of hepatic maturation and non-coding RNA (ncRNA) profiles of iPSCs – derived HLCs were not investigated. In this study, the sequencing of ncRNA was performed in order to compare the expression profiles of HLCs on two stages of differentiation (Day 20 and 24) with mature hepatocytes. The obtained results indicate that HLCs express miRNA, which control hepatic differentiation and maintain their fetal liver character. In comparison to mature hepatocytes, differentially expressed miRNAs in HLCs control the pathways of fatty acid metabolism and synthesis, proteoglycan in cancer, the Hippo signaling pathway, ECM-receptor interaction and adherens junction. Some of those highly expressed miRNAs can potentially block maturation by inhibiting epithelial-mesenchymal transition (EMT) which has an impact that is essential during hepatic differentiation. However, this should be resolved in future research. In this work, differentially expressed snoRNA were also identified. A total of 68% of differentially expressed snoRNAs was C/D box class. This is interesting because this snoRNa class was previously indicated as capable to be processed by an miRNA processing pathway. Many of the differentially expressed snoRNAs belong to the imprinted loci, in which a different expression in a human were analysed before. In obteined dataset, copies of SNORD115 were upregulated in a liver, but not in HLCs, which is consistent with an earlier comparison of a liver and other endoderm organs. Additionally, an analysis of obtained sequencing data allowed for a discovery of 19 novel snoRNA genes. In summary, this work shows a new approach to the reprogramming of a fibroblast and investigates the involvement of miRNAs and snoRNAs in the dynamics of hepatic differentiation. This study has shed a light on the molecular and regulatory mechanisms that underlie the complex process of liver differentiation and will hopefully allow existing problems with the use of in vitro derived hepatocytes to be overcome. A dataset generated here can be the foundation for a hepatic-specialised rybosomes theory and enabled to discover novel snoRNA genes.:1. INTRODUCTION 11 1.1. PLURIPOTENT STEM CELLS 11 1.1.1. Pluripotency 11 1.1.2. IPSCs 13 1.1.3. Reprogramming methods 14 1.1.4. IPSCs as an alternative cell source for disease modelling and regenerative medicine 16 1.2. LIVER 18 1.2.1. Liver anatomy and function 18 1.2.2. Liver embryonal development 20 1.3. HEPATIC DIFFERENTIATION OF IPSCS IN VITRO 22 1.3.1. HLCs 22 1.3.2. Differentiation protocols into hepatocytes 24 1.4. NCRNA 25 1.4.1. MiRNA 26 1.4.2. SnoRNA 28 2. AIMS 31 3. MATERIALS 32 3.1. EQUIPMENT 32 3.2. SOFTWARE 32 3.3. ENZYMES, KITS AND TRANSFECTION REAGENTS 33 3.4. SOLUTIONS AND REAGENTS 33 3.5. CELL LINES 34 3.6. CELL CULTURE MEDIA AND CYTOKINES 34 3.7. PLASMIDS 35 3.8. PCR REAGENTS AND PRIMERS 35 3.8.1. PCR reagents 35 3.8.2. PCR primers 35 3.9. ANTIBODIES 36 4. METHODS 37 4.1. CELL BIOLOGY 37 4.1.1. Derivation and culture of primary human foreskin fibroblasts 37 4.1.2. Counting cells 37 4.1.3. Cryo-preservation of cells 37 4.1.4. Thawing of cryo-preserved cells 38 4.1.5. Cell reprogramming 38 4.1.6. Cultivation and expansion of iPSCs 39 4.2. IMMUNOCYTOCHEMISTRY 39 4.3. IN VITRO SPONTANEOUS DIFFERENTIATION 39 4.4. KARYOTYPE ANALYSIS 40 4.5. RNA ISOLATION 40 4.6. QUANTITATIVE PCR 40 4.7. PERIODIC ACID-SCHIFF (PAS) STAINING 41 4.8. INDOCYANINE GREEN UPTAKE AND RELEASE 41 4.9. PLASMID TRANSFECTION 42 4.10. HEPATIC DIFFERENTIATION 42 4.11. WHEAT GERM AGGLUTININ STAINING 42 4.12. VALIDATION OF HEPATIC DIFFERENTIATION EFFICIENCY 43 4.13. RNA ISOLATION AND SEQUENCING 43 4.14. BIOINFORMATIC ANALYSIS 44 4.14.1. Sequencing quality and mapping 44 4.14.2. Analysis of differential expressed ncRNAs 44 4.14.3. Target pathways prediction of differentially expressed miRNAs 44 4.14.4. Identification of novel ncRNAs candidates 45 5. RESULTS 46 5.1. GENERATION OF IPSCS USING EPISOMAL VECTORS 46 5.1.1. Cell transfection 46 5.1.2. Establishment of iPSCs line 48 5.2. PLURIPOTENCY CHARACTERISATION OF THE IPSCS 49 5.2.1. Pluripotency markers 49 5.2.2. Spontaneous differentiation assay 50 5.2.3. Karyotype 52 5.3. HEPATIC DIFFERENTIATION OF IPSCS AND HLCS CHARACTERISATION 53 5.3.1. iPSCs hepatic differentiation 53 5.3.2. Expression of hepatic markers 54 5.3.3. Hepatic gene expression in HLCs 56 5.3.4. Hepatic functions in HLCs 58 5.4. HNF4A OVEREXPRESSION DURING DIFFERENTIATION 59 5.4.1. Cell transfection during differentiation 59 5.4.2. Comparison of hepatic differentiation efficiency 60 5.4.3. Whole slide scanning 62 5.5. NON-CODING RNA ANALYSIS 64 5.5.1. Non-coding RNA sequencing quality 64 5.5.2. MicroRNA analysis 68 5.5.3. SnoRNA analysis 79 5.5.4. Short reads snoRNA analysis 84 5.5.5. New gene candidates 85 6. DISCUSSION 88 6.1. METHODICAL STRATEGY 88 6.2. CHARACTERISATION OF GENERATED IPSCS 89 6.3. HEPATIC DIFFERENTIATION OF IPSCS 89 6.3.1. Characterisation of HLCs 89 6.3.2. Protocol with HNF4a overexpression 90 6.3.3. Differentially expressed miRNA 90 6.3.4. Differentially expressed snoRNA 93 6.4. NOVEL SNORNA GENES 95 7. SUMMARY 96 8. REFERENCES 99 9. APPENDIX 118 ERKLÄRUNG ÜBER DIE EIGENSTÄNDIGE ABFASSUNG DER ARBEIT 122. ACKNOWLEDGEMENTS 123

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