Development of safe and efficient approaches for gene delivery in human embryonic stem cells (hESc) and particularly in human induced pluripotent stem (hiPS) cells, which can be derived in a person-specific manner, is considered to be imperative for harnessing their full potential in both the basic and applied research. The aim of this study was to evaluate the potential of human artificial chromosome (HAC) for gene delivery and expression in hESc and hiPS cells. HAC offers many potential advantages including the provision for carrying large genes with corresponding regulatory elements to obtain long-term regulated gene expression. In addition, they can replicate and segregate independently without integration into the host cell genome. To develop HAC in hiPS cells, the first part of the study was aimed at generating hiPS cells utilising the Herpes Simplex Virus (HSV)-1 amplicon system. With the use of EBNA-1/OriP retention elements incorporated into the HSV-1 amplicon vectors, hiPS cells completely free of vector and transgenes sequences were successfully derived from human embryonic fibroblasts. The hiPS cells exhibited proliferation and differentiation potential similar to that of hESc. In the second part of the study, development of HAC in hESc and hiPS cells was assessed by utilising the HSV-1 amplicon system to deliver the HAC DNA. Analysis of the hESc confirmed the presence of functional HAC which replicated the behaviour of the host chromosomes. Additionally, HAC generation did not lead to impairment in the developmental potential and pluripotency of hESc. The hiPS cells supported HAC at low frequency but DNA also integrated into the host chromosomes. The HAC system, therefore, needs further refinements to improve the frequency of HAC formation and reduce the chromosomal integration of HAC constructs in hiPS cells. Overall, these findings provide a simple and safe way of pluripotency induction and genetic modification of pluripotent stem cells using the HSV-1 amplicon system and represent an important advance towards patient specific gene and cell therapy.

As doenças cardiovasculares representam a maior causa de mortalidade a nível mundial. Desde o conhecimento da importância da mitocôndria no metabolismo do cardiomiócito, alterações no funcionamento desta organela têm sido associadas a um dos principais causadores do infarto do miocárdio e consequente morte celular. O cofator de transcrição PGC-1alfa tem sido alvo de diversos estudos relacionados com o metabolismo celular devido à sua forte participação na biogênese mitocondrial. Considerando a limitação de material biológico para o estudo de doenças cardíacas, muito se tem investido no estudo de células-tronco pluripotentes induzidas (iPSCs). Esta tese teve como principal objetivo a avaliação dos efeitos da variação da expressão de PGC-1alfa em iPSCs e na sua diferenciação em cardiomiócitos. Após estabelecimento de um protocolo de reprogramação celular, em que ocorre geração de iPSCs a partir de fibroblastos humanos, induzimos a inibição da expressão de PGC-1alfa em 50% e 70% pelo uso de vetores lentivirais, e analisamos o estado de pluripotência através da avaliação de expressão genica e proteica dos principais marcadores - SSEA4, TRA-1-60, OCT4, NANOG, SOX2, REX1, TRA-1-81. Não observamos diferenças significativas no conteúdo destes marcadores entre os clones de iPSC controle e inibidos. Estabelecemos um protocolo de diferenciação de iPSCs em cardiomiócitos com elevada taxa de reprodutibilidade, através da adaptação de protocolos descritos na literatura, e submetemos estas iPSCs à diferenciação. As células geradas pela diferenciação do clone controle apresentaram características típicas de cardiomiócito: contratilidade e alta expressão molecular de troponina T e troponina I. Em contraste, as células com 70% de inibição de PGC-1alfa se mostraram incapazes de contrair e com baixa expressão de troponina. Através de uma análise dos níveis de expressão genica e proteica de diversos marcadores expressos durante o processo de diferenciação (T, NKX2.5, MIXL1, MYL7, ISL1), observamos que o clone com maior inibição de PGC-1alfa apresentou sempre níveis de expressão diminuídos em relação aos clones controle. Em conclusão, podemos afirmar que o PGC-1alfa não interfere com as características de auto-renovação e pluripotência das iPSCs mas possui um papel essencial na diferenciação de células-tronco pluriotentes induzidas em cardiomiócitos. Os resultados obtidos contribuem para informações preliminares acerca do desenvolvimento de iPSCs com inibição da expressão de PGC-1alfa durante a diferenciação cardíaca, mas estudos relativos ao potencial papel deste cofator durante o desenvolvimento cardíaco in vivo ainda precisam ser aprofundados, utilizando outros modelos de estudo / Cardiovascular diseases are the leading cause of mortality worldwide. Since the knowledge of the importance of mitochondria in the cardiomyocyte metabolism, changes in the functioning of this organelle has been associated with one of the main causes of myocardial infarction and subsequent cell death. The transcriptional cofactor PGC-1alpha has been subjected to several studies related to cell metabolism due to its strong involvement in mitochondrial biogenesis. Considering the limitations of biological material in the study of heart disease, there has been a lot of investment in the study of induced pluripotent stem cells (iPSCs). The main objective of this thesis was to evaluate the effects of the variation in expression of PGC-1alpha in iPSCs and it\'s differentiation in cardiomyocytes. After the estabilshment of a cellular reprogramming protocol, where iPSCs is generated from human fibroblasts, the expression of PGC-1alpha was induced by 50% and 70% with the use of lentiviral vectors and the state of pluripotency was determined by analyzing the gene and protein expression of the main markers - SSEA4, TRA- 1-60, OCT4, NANOG, SOX2, REX1, TRA- 1- 81. There were no significant differences observed in the content of these markers between the iPSC clones control and inhibited. A protocol for the differentiation of iPSCs into cardyomyocites was established with a high reproducibility rate, by adapting existing protocols in the general literature, submiting these iPSCs into diferentiation. The cells generated from the differentiation of the control clone showed typical characteristis of cardiomyocytes: contractility and high molecular expression of troponin T and troponin I. In contrast, the cells with 70% inhibition PGC-1alpha were unable to contract and had low troponin expression. Through an analysis of gene expression and protein levels of several markers expressed during the differentiation process (T, Nkx2.5, MIXL1, MYL7, ISL1), the clone with greater inhibition of PGC-1alpha always showed decreased expression levels compared to control clones. In conclusion, we can say that the PGC-1alpha does not interfere with the characteristics of self-renewal and pluripotency of iPSCs but has an essential role in the differentiation of pluripotent stem cells induced into cardiomyocytes. These results were obtained thanks an original approche based on iPSC technology enabling genetic modifications of the cells and controled differentiation into cardiomyocytes, but the potential role of PGC-1alpha on in vivo cardiac development or cardiomyocytes maturation remaisn to be evaluated using other models

Das Verhalten Biologischer Systeme wird durch eine Vielzahl regulatorischer Prozesse beeinflusst, die sich auf verschiedenen Ebenen abspielen. Die Forschung an diesen Regulationen hat stark von den großen Mengen von Hochdurchsatzdaten profitiert, die in den letzten Jahren verfügbar wurden. Um diese Daten zu interpretieren und neue Erkenntnisse aus ihnen zu gewinnen, hat sich die mathematische Modellierung als hilfreich erwiesen. Allerdings müssen die Daten vor der Integration in Modelle aggregiert und analysiert werden. Wir präsentieren vier Studien auf unterschiedlichen zellulären Ebenen und in verschiedenen Organismen. Zusätzlich beschreiben wir zwei Computerprogramme die den Vergleich zwischen Modell und Experimentellen Daten erleichtern. Wir wenden diese Programme in zwei Studien über die MAP Kinase (MAP, engl. mitogen-acticated-protein) Signalwege in Saccharomyces cerevisiae an, um Modellalternativen zu generieren und unsere Vorstellung des Systems an Daten anzupassen. In den zwei verbleibenden Studien nutzen wir bioinformatische Methoden, um Hochdurchsatz-Zeitreihendaten von Protein und mRNA Expression zu analysieren. Um die Daten interpretieren zu können kombinieren wir sie mit Netzwerken und nutzen Annotationen um Module identifizieren, die ihre Expression im Lauf der Zeit ändern. Im Fall der humanen somatischen Zell Reprogrammierung führte diese Analyse zu einem probabilistischen Boolschen Modell des Systems, welches wir nutzen konnten um neue Hypothesen über seine Funktionsweise aufzustellen. Bei der Infektion von Säugerzellen (Canis familiaris) mit dem Influenza A Virus konnten wir neue Verbindungen zwischen dem Virus und seinem Wirt herausfinden und unsere Zeitreihendaten in bestehende Netzwerke einbinden. Zusammenfassend zeigen viele unserer Ergebnisse die Wichtigkeit von Datenintegration in mathematische Modelle, sowie den hohen Grad der Verschaltung zwischen verschiedenen Regulationssystemen. / The behavior of all biological systems is governed by numerous regulatory mechanisms, acting on different levels of time and space. The study of these regulations has greatly benefited from the immense amount of data that has become available from high-throughput experiments in recent years. To interpret this mass of data and gain new knowledge about studied systems, mathematical modeling has proven to be an invaluable method. Nevertheless, before data can be integrated into a model it needs to be aggregated, analyzed, and the most important aspects need to be extracted. We present four Systems Biology studies on different cellular organizational levels and in different organisms. Additionally, we describe two software applications that enable easy comparison of data and model results. We use these in two of our studies on the mitogen-activated-protein (MAP) kinase signaling in Saccharomyces cerevisiae to generate model alternatives and adapt our representation of the system to biological data. In the two remaining studies we apply Bioinformatic methods to analyze two high-throughput time series on proteins and mRNA expression in mammalian cells. We combine the results with network data and use annotations to identify modules and pathways that change in expression over time to be able to interpret the datasets. In case of the human somatic cell reprogramming (SCR) system this analysis leads to the generation of a probabilistic Boolean model which we use to generate new hypotheses about the system. In the last system we examined, the infection of mammalian (Canis familiaris) cells by the influenza A virus, we find new interconnections between host and virus and are able to integrate our data with existing networks. In summary, many of our findings show the importance of data integration into mathematical models and the high degree of connectivity between different levels of regulation.

As doenças cardiovasculares representam a maior causa de mortalidade a nível mundial. Desde o conhecimento da importância da mitocôndria no metabolismo do cardiomiócito, alterações no funcionamento desta organela têm sido associadas a um dos principais causadores do infarto do miocárdio e consequente morte celular. O cofator de transcrição PGC-1alfa tem sido alvo de diversos estudos relacionados com o metabolismo celular devido à sua forte participação na biogênese mitocondrial. Considerando a limitação de material biológico para o estudo de doenças cardíacas, muito se tem investido no estudo de células-tronco pluripotentes induzidas (iPSCs). Esta tese teve como principal objetivo a avaliação dos efeitos da variação da expressão de PGC-1alfa em iPSCs e na sua diferenciação em cardiomiócitos. Após estabelecimento de um protocolo de reprogramação celular, em que ocorre geração de iPSCs a partir de fibroblastos humanos, induzimos a inibição da expressão de PGC-1alfa em 50% e 70% pelo uso de vetores lentivirais, e analisamos o estado de pluripotência através da avaliação de expressão genica e proteica dos principais marcadores - SSEA4, TRA-1-60, OCT4, NANOG, SOX2, REX1, TRA-1-81. Não observamos diferenças significativas no conteúdo destes marcadores entre os clones de iPSC controle e inibidos. Estabelecemos um protocolo de diferenciação de iPSCs em cardiomiócitos com elevada taxa de reprodutibilidade, através da adaptação de protocolos descritos na literatura, e submetemos estas iPSCs à diferenciação. As células geradas pela diferenciação do clone controle apresentaram características típicas de cardiomiócito: contratilidade e alta expressão molecular de troponina T e troponina I. Em contraste, as células com 70% de inibição de PGC-1alfa se mostraram incapazes de contrair e com baixa expressão de troponina. Através de uma análise dos níveis de expressão genica e proteica de diversos marcadores expressos durante o processo de diferenciação (T, NKX2.5, MIXL1, MYL7, ISL1), observamos que o clone com maior inibição de PGC-1alfa apresentou sempre níveis de expressão diminuídos em relação aos clones controle. Em conclusão, podemos afirmar que o PGC-1alfa não interfere com as características de auto-renovação e pluripotência das iPSCs mas possui um papel essencial na diferenciação de células-tronco pluriotentes induzidas em cardiomiócitos. Os resultados obtidos contribuem para informações preliminares acerca do desenvolvimento de iPSCs com inibição da expressão de PGC-1alfa durante a diferenciação cardíaca, mas estudos relativos ao potencial papel deste cofator durante o desenvolvimento cardíaco in vivo ainda precisam ser aprofundados, utilizando outros modelos de estudo / Cardiovascular diseases are the leading cause of mortality worldwide. Since the knowledge of the importance of mitochondria in the cardiomyocyte metabolism, changes in the functioning of this organelle has been associated with one of the main causes of myocardial infarction and subsequent cell death. The transcriptional cofactor PGC-1alpha has been subjected to several studies related to cell metabolism due to its strong involvement in mitochondrial biogenesis. Considering the limitations of biological material in the study of heart disease, there has been a lot of investment in the study of induced pluripotent stem cells (iPSCs). The main objective of this thesis was to evaluate the effects of the variation in expression of PGC-1alpha in iPSCs and it\'s differentiation in cardiomyocytes. After the estabilshment of a cellular reprogramming protocol, where iPSCs is generated from human fibroblasts, the expression of PGC-1alpha was induced by 50% and 70% with the use of lentiviral vectors and the state of pluripotency was determined by analyzing the gene and protein expression of the main markers - SSEA4, TRA- 1-60, OCT4, NANOG, SOX2, REX1, TRA- 1- 81. There were no significant differences observed in the content of these markers between the iPSC clones control and inhibited. A protocol for the differentiation of iPSCs into cardyomyocites was established with a high reproducibility rate, by adapting existing protocols in the general literature, submiting these iPSCs into diferentiation. The cells generated from the differentiation of the control clone showed typical characteristis of cardiomyocytes: contractility and high molecular expression of troponin T and troponin I. In contrast, the cells with 70% inhibition PGC-1alpha were unable to contract and had low troponin expression. Through an analysis of gene expression and protein levels of several markers expressed during the differentiation process (T, Nkx2.5, MIXL1, MYL7, ISL1), the clone with greater inhibition of PGC-1alpha always showed decreased expression levels compared to control clones. In conclusion, we can say that the PGC-1alpha does not interfere with the characteristics of self-renewal and pluripotency of iPSCs but has an essential role in the differentiation of pluripotent stem cells induced into cardiomyocytes. These results were obtained thanks an original approche based on iPSC technology enabling genetic modifications of the cells and controled differentiation into cardiomyocytes, but the potential role of PGC-1alpha on in vivo cardiac development or cardiomyocytes maturation remaisn to be evaluated using other models

Submitted by Fabíola Silva (fabiola.silva@famerp.br) on 2016-06-21T17:12:33Z No. of bitstreams: 1 cinthiadias_dissert.pdf: 1995817 bytes, checksum: 27317444195c0604e0ed14f9ac182ee5 (MD5) / Made available in DSpace on 2016-06-21T17:12:33Z (GMT). No. of bitstreams: 1 cinthiadias_dissert.pdf: 1995817 bytes, checksum: 27317444195c0604e0ed14f9ac182ee5 (MD5) Previous issue date: 2015-08-25 / Fundação de Amparo à Pesquisa do Estado de São Paulo - FAPESP / Introduction: Stem cell therapy is a promising strategy to repair or delay the progression of chronic renal failure (CRF). Induced pluripotent stem cells (iPS) can be a therapeutic alternative due to their differentiation potential. Objectives: 1- To modify genetically stem cells from mice´s fibroblasts with lentiviral vectors containing transcription factors, transforming differentiated cells into iPS; 2- To evaluate the effect of iPS in the experimental IRC progression of IRC induced by 5/6 nephrectomy (CRF-5/6). Materials and Methods: The animals were divided according to the type of cell therapy received from extracted mesenchymal stem cells from bone marrow (MSC) or iPS and compared with CRF group 5/6 without treatment. Assessment of renal function was carried out during baseline and after 60 days. Additionally expression of genes, VEGF, IL-6, TGF-β and IL-10 were quantified in the kidney tissue, and also the analysis of implanted cell migration through the SRY gene. Immunohistochemical study evaluated the expression of CD68, α-SMA, TGF-β, PCNA and VEGF markers. Results: A significant decrease was observed in creatinine variation (p<0.05) and plasma urea (p<0.01) in animals treated with MSC and a 33%-decrease in plasma creatinine levels of animals treated with iPS cells, although non- significant when compared to the control group. The 24-hour proteinuria was significantly reduced only in the iPS group (p<0.0001). Significant improvement was observed in creatinine clearance in both treatments (p<0.04). Disease progression measured by the clearance decline rate was significantly lower only in the MSC group (p<0.05) and the urinary osmolality was similar in both treated groups. There was an increase in the expression of TGF- β gene in iPS group when compared to the control group (p<0.05) and VEGF expression in the groups treated with iPS and MSC (p<0.05). IL-6 and IL-10 showed similar expression levels in both treated groups (p=NS). Immunohistochemical analysis showed fewer macrophages and decreased cell proliferative activity (PCNA) in the iPS group p<0.05. Histological analysis showed a significant decrease in glomerulosclerosis in both treatment groups (p<0.01), tubular atrophy was similar in all groups . Leukocyte infiltration was reduced in both treatments when compared to CRF group. The SRY gene was detected in 5 out of 8 (62.5%) mice that were treated with iPS. After 60 days the tumor formations were observed in animals in which SRY gene was detected. Conclusions: MSC therapy is effective in delaying the progression of CKD. Treatment with iPS also improves some parameters of renal function but this assessment can be difficult since the onset of tumor formations; thus some care is necessary with this type of cells. / Introdução: A terapia com células-tronco (CT) é uma estratégia promissora para reparar ou retardar a progressão da insuficiência renal crônica (IRC). As células-tronco pluripotentes induzidas (iPS) podem ser uma alternativa terapêutica, em virtude de seu potencial de diferenciação. Objetivos: 1) Modificar geneticamente células de fibroblastos de ratos com vetores lentivirais contendo fatores de transcrição, transformando essas células diferenciadas em iPS; 2) Avaliar o efeito das iPS e CTM na progressão da IRC experimental induzida pela nefrectomia 5/6 (CRF5/6). Materiais e Métodos: Os animais foram divididos conforme o tipo de terapia celular recebida (célula-tronco mesenquimal extraída da medula óssea (CTM) ou com iPS) e comparados com o grupo CRF5/6. A avaliação da função renal foi realizada no período basal e após 60 dias. Adicionalmente foi quantificada a expressão dos genes, VEGF, IL-6, TGF-β e IL-10 no tecido renal e estudada a migração das células implantadas contendo o gene SRY. O estudo imunohistoquímico avaliou a expressão de marcadores CD68, α-SMA, TGF-β, PCNA e VEGF. Resultados: Redução significativa foi observada na variação da creatinina (p<0,05) e ureia plasmática (p<0,01) dos animais tratados com CTM e uma diminuição de 33% dos níveis de creatinina plasmática nos animais tratados com células iPS, porém sem significância estatística quando comparada ao grupo controle. A proteinúria de 24 horas foi reduzida somente no grupo iPS (p=0,0001) e houve melhora significativa no clearance de creatinina com ambos tratamentos (p=0,04). A progressão da doença, medida pela taxa de declínio do clearance de creatinina, foi significativamente lentificada somente no grupo CTM (p=0,04) e a osmolalidade urinária foi similar em ambos os grupos tratados. Houve aumento na expressão do gene TGF-β no grupo iPS quando comparado ao grupo controle (p=0,01) e da expressão de VEGF nos grupos tratados com iPS e CTM (p=0,01). IL-6 e IL-10 mostraram níveis de expressão semelhantes em ambos os grupos tratados (p=NS). A análise imunohistoquímica demonstrou menor número de macrófagos e diminuição da atividade proliferativa celular (PCNA) no grupo iPS p<0,05. A analise histológica mostrou diminuição significativa da glomeruloesclerose em ambos grupos tratados (p<0,01), a atrofia tubular foi semelhante nos três grupos. A infiltração leucocitária foi reduzida em ambos os tratamentos, quando comparados ao grupo CRF. O gene SRY foi detectado em 5 de 8 (62,5%) ratos que receberam tratamento com iPS. Após 60 dias foram observadas as formações tumorais nos respectivos animais em que o gene SRY foi detectado. Conclusões: A terapia com CTM é eficiente para retardar a progressão da IRC. Tratamento com iPS também melhora alguns parâmetros da função renal, mas o aparecimento de formações tumorais dificulta essa avaliação e requer cuidados com esse tipo de célula.

Induced pluripotent stem cells (iPSC) have the capacity to self renew and differentiate into a myriad of cell types making them potential candidates for cell therapy and regenerative medicine. The goal of this thesis was to determine the characteristics of equine iPSC (eiPSC) that can be harnessed for potential use in veterinary regenerative medicine. Trauma to a horse’s limb often leads to the development of a chronic non-healing wound that lacks a keratinocyte cover, vital to healing. Thus, the overall hypothesis of this thesis was that eiPSC might offer a solution for providing wound coverage for such problematic wounds. Prior to considering eiPSC for clinical applications, their immunogenicity must be studied to ensure that the transplanted cells will be accepted and integrate into host tissues. The first objective of this thesis was to determine the immune response to eiPSC. To investigate the immunogenicity of eiPSC, the expression of major histocompatibility complex (MHC) molecules by the selected lines was determined, then the cells were used in an intradermal transplantation model developed for this study. While transplantation of allogeneic, undifferentiated eiPSC elicited a moderate cellular response in experimental horses, it did not cause acute rejection. This strategy enabled the selection of weakly immunogenic eiPSC lines for subsequent differentiation into lineages of therapeutic importance. Equine iPSC offer a potential solution to deficient epithelial coverage by providing a keratinocyte graft with the ability to differentiate into other accessory structures of the epidermis. The second objective of this thesis was to develop a protocol for the differentiation of eiPSC into a keratinocyte lineage. The protocol was shown to be highly efficient at inducing the anticipated phenotype within 30 days. Indeed, the eiPSC derived vi keratinocytes (eiPSC-KC) showed both morphologic and functional characteristics of primary equine keratinocytes (PEK). Moreover, the proliferative capacity of eiPSC-KC was superior while the migratory capacity, measured as the ability to epithelialize in vitro wounds, was comparable to that of PEK, suggesting exciting potential for grafting onto in vivo wound models. In conclusion, equine iPSC-derived keratinocytes exhibit features that are promising to the development of a stem cell-based skin construct with the potential to fully regenerate lost or damaged skin in horses. However, since eiPSC do not fully escape immune surveillance despite low MHC expression, strategies to improve engraftment of iPSC derivatives must be pursued. / Les cellules souches pluripotentes induites (iPSC) ont la capacité de s'auto renouveler et de se différencier en une myriade de types cellulaires, ce qui en fait des outils intéressants pour la thérapie cellulaire et la médecine régénérative. Le but de cette thèse était de déterminer les caractéristiques des iPSC équines (eiPSC) qui peuvent être exploitées pour l'usage potentiel en médecine régénérative vétérinaire. Chez le cheval, une plaie cutanée est souvent cicatrisée par seconde intention et est sujette à de nombreuses complications lorsque située sur le membre, notamment une épithélialisation lente. Ainsi, l'hypothèse globale de cette thèse était que les eiPSC pourraient offrir une solution novatrice de couverture pour de telles blessures. Avant d'envisager l’utilisation d'eiPSC à des fins cliniques, leur immunogénicité doit être étudiée afin de s'assurer que les cellules transplantées seront acceptées et intégrées dans les tissus du receveur. Le premier objectif de cette thèse était de définir la réponse immunitaire suscitée par les eiPSC. Afin d'étudier l'immunogénicité d'eiPSC, l'expression de molécules du complexe majeur d’histocompatibilité (MHC) des lignes choisies a été déterminée, puis les cellules ont été utilisées dans un modèle de transplantation intradermique développé pour cette étude. Bien que la transplantation allogénique d'eiPSC non différenciées ait induit une réponse cellulaire modérée chez les chevaux d'expérimentation, elle n'a pas provoqué de rejet. Cette stratégie a permis la sélection de lignées d'eiPSC faiblement immunogènes pour la différenciation ultérieure en des lignées d'importance thérapeutique. Les eiPSC représentent une solution intéressante et qui, par l’entremise du développement d’une lignée de kératinocytes, pourraient servir à la création d’une greffe ayant la capacité de former non seulement l’épithélium manquant mais aussi d'autres structures accessoires de l'épiderme. Le deuxième objectif de cette thèse était donc de iv développer un protocole pour la différentiation des eiPSC en lignée de kératinocytes. Un protocole visant cette différenciation fut ainsi développé et ce dernier a démontré une grande efficacité à produire le phénotype attendu dans une période de 30 jours. En effet, les kératinocytes dérivés d'eiPSC (eiPSC-KC) ont montré des caractéristiques morphologiques et fonctionnelles des kératinocytes primaires équins (PEK). En outre, la capacité de prolifération d'eiPSC-KC est supérieure tandis que la capacité migratoire, mesurée comme l'aptitude à cicatriser les plaies in vitro, est comparable à celle du PEK. En conclusion, les eiPSC-KC ont des caractéristiques intéressantes pour le développement d'un substitut cutané à base de cellules souches, ayant le potentiel de régénérer la peau perdue lors de trauma ou de maladie, chez le cheval. Cependant, parce que les eiPSC n'échappent pas totalement à la surveillance immunitaire, malgré une faible expression du MHC, des stratégies pour améliorer la prise de greffe eiPSC-KC doivent être élaborées.

Human autopsy or biopsy tissue samples, mouse models and cell cultures of various types represent the most common materials in the investigation of cell pathogenesis of inherited diseases. This dissertation is devoted to all these approaches in the study of two X-linked lysosomal storage diseases, Fabry disease (FD,α-galactosidase A (AGAL) deficiency) and mucopolysaccharidosis type II (MPSII, idunorate-2- sulfatase (IDS) deficiency). The primary goal of the work was analysis of lipid blood group B antigens with terminal α-galactose (B-GSL) in the pancreas of FD patients with blood group B (FD-B).,In addition to the main glycosphingolipid (GSL) substrate, globotriaosylceramide (Gb3Cer), B-GSLs represent another minor substrate of AGAL. The deposition of undegraded B-GSL has been demonstrated in FD-B pancreas where it was significantly higher than in other organs such as the kidneys and lungs which accumulate mainly Gb3Cer. High concentration of lipid and non-lipid B-antigens was primarily confirmed in exocrine acinar epithelial cells of FD-B, accompanied by massive accumulation of ceroid (secondary sign of lysosomal storage). Unlike acini, the endocrine portion of the pancreas remained unaffected by accumulation of AGAL substrates. This interesting phenomenon of cell biology shows how a specific...

Les cellules souches pluripotentes induites (iPS) partagent avec les cellules souches embryonnaires la capacité à se différencier en tous les types cellulaires d'un organisme, mais leur obtention ne nécessite pas l'utilisation d'embryons. Elles sont générées par la surexpression de facteurs de transcription embryonnaires au sein de cellules somatiques. Les iPS représentent un outil de choix en biologie fondamentale et appliquée ainsi qu'en médecine régénérative.La plupart des protocoles de génération d'iPS reposent sur un transfert des séquences nucléotidiques codant les facteurs de transcription embryonnaires impliqués dans la mise en place du réseau de pluripotence. Bien qu'efficaces, ces méthodes présentent des problèmes de sécurité majeurs, incompatibles avec une utilisation clinique des iPS générées. La voie la plus rationnelle pour produire des iPS de manière parfaitement sécurisée est d'apporter les facteurs exogènes directement sous leur forme protéique. Des protocoles de reprogrammation par transfert de protéines ont été récemment développés, mais les efficacités associées sont relativement faibles et les protocoles relativement fastidieux.L'objectif de ce projet de thèse était de mettre au point une nouvelle approche de transfert de protéines, sécurisée et simplifiée, pour la génération de cellules souches pluripotentes induites utilisables en clinique. Les cellules à reprogrammer ont été choisies en fonction des applications potentielles des iPS générées : (i) les fibroblastes, faisant référence dans la bibliographie et permettant d'envisager des thérapies autologues avec notamment de nombreuses applications en hématologie ; (ii) les cellules souches hématopoïétiques de sang de cordon, l'un des matériaux biologiques les plus sûrs, afin de générer des globules rouges in vitro, dans la perspective de répondre aux demandes croissantes en terme de transfusion, en particulier pour les groupes sanguins rares.Nous avons donc comparé les différents vecteurs de transduction de protéines développés par l'équipe TheREx du laboratoire TIMC-IMAG, en termes de facilité de production, d'efficacité de transfert ainsi que sur l'activité des facteurs de transcription associés. Le vecteur sélectionné est une micro-seringue naturelle portée par la bactérie Pseudomonas aeruginosa, capable d'injecter les facteurs Oct4, Sox2, Nanog et Lin28 (facteurs de Thomson) mais aussi c-Myc, directement dans le cytoplasme des cellules cibles, sans étape de purification nécessaire. Les facteurs de transcription injectés sont adressés jusqu'au noyau des cellules en moins de 2h, où ils activent rapidement la transcription des gènes de pluripotence, avec des réponses significatives mesurées dès 24h après injection. Nous avons également mis en évidence le caractère sécurisé et contrôlable du vecteur puisque nous sommes capables d'éliminer complètement les bactéries des cultures grâce à un traitement antibiotique, et ce dès quelques heures après l'injection. Des optimisations des conditions de reprogrammation ont été réalisées en modifiant les principaux paramètres que sont, le choix des facteurs de transcription, la fréquence des injections et le ratio bactéries : cellules.Ainsi, bien que nous ne soyons pas parvenus à générer des iPS à ce jour avec ce système, la micro-seringue naturelle que nous avons développé et optimisé se positionne comme un vecteur de choix pour le transfert de protéines dans l'optique de générer des iPS, en termes d'efficacité de vectorisation et d'induction transcriptionnelle, de sécurité mais aussi de facilité d'utilisation. / Like embryonic stem cells, induced pluripotent stem cells (iPS) are characterized by their ability to differentiate into any cell type in an organism. However their use doesn't raise the ethical issue linked to the use of embryos. iPS are generated from somatic cells by overexpression of embryonic transcription factors. iPS are thereby very promising in fundamental and applied biology as well as for regenerative medicine.Most of the protocols used to generate iPS are based on the delivery of nucleic acid sequences encoding embryonic transcription factors responsible for the activation of the pluripotency gene network. In spite of their efficiency, these methods are associated with major safety concerns incompatible with clinical applications. The more rational path to safely produce iPS is to deliver the exogenic transcription factors under their protein form. Recently some protocols using protein delivery have been developed to produce iPS. However associated efficiencies are very low and protocols are quite fastidious.The aim of this Ph.D. project was to develop a new efficient and simplified protein delivery method for the safe generation of iPS compatible with clinical applications. Cell sources were selected depending of the final applications of iPS: (i) fibroblasts, extensively used and described in bibliography and allowing autologous therapies with many applications in the field of hematology; (ii) cord blood hematopoietic stem cells, one of the safest biomaterials, with the aim to generate red blood cells in vitro in order to respond to increasing needs for transfusion products, particularly for rare blood types.First, different protein vectors developed by the TheREx team of the TIMC-IMAG laboratory were compared for their efficiency of production and delivery as well as for the activity of associated factors. The selected vector is a natural micro-syringe expressed by Pseudomonas aeruginosa, able to inject the transcription factors Oct4, Sox2, Nanog and Lin28a (Thomson combination) with c-Myc directly into the cytoplasm of target cells, without the need for any purification step. Once injected, transcription factors are addressed to the nucleus in less than 2 hours where they efficiently activate transcription of pluripotency genes, with significant responses observed as early as 24h after injection. We also highlighted the secured and controllable nature of this vector by completely eliminating the bacteria from the cultures in a few hours after injection with an antibiotic treatment. Optimizations of the reprogramming conditions were also made by adjusting many parameters such as the combination of transcription factors, the injection frequency and the bacteria : cell ratio.

La technologie de reprogrammation de cellules somatiques en cellules souches pluripotentes induites (iPS) offre aujourd’hui l’opportunité de modéliser des maladies neurodégénératives et d’étudier des neurones de patients. Nous avons utilisé cette technologie pour générer deux modèles de maladies neurodégénératives : la mucopolysaccharidose de type IIIB (MPSIIIB) et la forme ALS2 de la sclérose latérale amyotrophique (SLA). Dans le modèle MPSIIIB, nous avons montré que les iPS et les neurones de patients présentaient des défauts caractéristiques de la pathologie telle que l’accumulation de vésicules de surcharge. Des altérations de l’appareil de Golgi dans ces cellules ont également été mises en évidence. Une analyse du transcriptome de précurseurs neuraux MPSIIIB a montré des modifications transcriptionnelles touchant notamment des gènes impliqués dans les interactions de la cellule avec la matrice extracellulaire. Ainsi, dans une seconde étude, des altérations de la migration et de l’orientation de cellules de souris mutantes MPSIIIB ou de patients ont été démontrées. Ces altérations pourraient être responsables des perturbations de la neurogénèse et de la neuritogénèse chez les enfants malades. Dans le modèle SLA/ALS2, nous avons montré que les neurones de patients présentaient des défauts incluant une diminution de la surface des endosomes et des anomalies de la croissance neuritique. Alors qu’il n’existait jusqu’alors aucun modèle cellulaire pertinent reproduisant cette maladie, ce modèle permettra à présent d’étudier les processus physiopathologiques impliqués dans la maladie. En conclusion, la génération de cellules iPS permet de modéliser des maladies neurodégénératives et d’étudier les processus physiopathologiques qui sont associés sur des neurones humains en culture. Ces modèles cellulaires pourraient permettre dans un avenir proche de réaliser des criblages de molécules à visée thérapeutique / Reprogramming technology of somatic cells in induced pluripotent stem cells (iPS) now offers the opportunity to model neurodegenerative diseases and to study patient’s neurons. We used this technology for generating two models of neurodegenerative diseases: the muccopolysaccharidosis type IIIB (MPSIIIB) and the ALS2 form of amyotrophic lateral sclerosis (ALS). In the MPSIIIB model, we have shown that iPS and neurons of patients had characteristic defects of the disease such as the accumulation of storage vesicles. Alterations of the Golgi apparatus in these cells were also highlighted. Transcriptome analysis of MPSIIIB neural precursors showed transcriptional changes involving particularly genes implicated in cell-extracellular matrix interactions. Thus, in a subsequent study, alterations of migration and orientation of MPSIIIB mutant mouse cells and MPSIIIB patients’ cells have been demonstrated. These alterations may be responsible for the disruption of neurogenesis and neuritogenesis in sick children. In the ALS2 model, we have shown that patients’ neurons had defects including decreased endosomes’ surface and abnormal neurite outgrowth. As there was previously no relevant cellular model reproducing the disease, this model will now allow the study of physiopathological processes involved in the disease. In conclusion, the generation of iPS cells allows to model neurodegenerative diseases and to study associated physiopathological processes on cultured human neurons. These cell models could allow in the near future the screening of molecules of potential therapeutical interest

Les dystrophies rétiniennes héréditaires (DRH) sont un groupe de maladies génétiquement et cliniquement hétérogènes, lesquelles se caractérisent par une perte progressive de la vision. La choroïdérémie (CHM) est une choriorétinopathie qui représente environ 3% des DRH. Elle se caractérise par une cécité nocturne durant l’enfance suivie par une perte du champ visuel périphérique lente et progressive. Cela aboutit à une cécité vers l’âge de 40 à 50 ans. Généralement, la vision centrale demeure préservée plus longtemps. Génétiquement, la maladie est causée par des mutations dans le gène CHM localisé dans le chromosome X qui code pour la Rab Escort Protein 1 (REP1).Cette protéine est impliquée dans la prénylation des Rab GTPasas qui régulent le trafic vésiculaire au sein de la cellule. La plupart des mutations responsables de la maladie sont des mutations pertes de fonction. La conséquence de ces mutations est l’absence de REP1 entrainant un défaut de prénylation des Rabs. Ce qui cause la dégénérescence des photorécepteurs, de l’épithélium pigmentaire rétinien (EPR) et de la choroïde. À ce jour, il n’existe pas de thérapie pour la CHM. Cependant, le diagnostic précoce de la maladie et son évolution lente donnent une fenêtre thérapeutique large et en font un candidat idéal pour la réussite d’un traitement.En raison de l’absence d’un modèle animal pertinent pour tester de nouvelles thérapies pour cette maladie, nous avons développé un modèle cellulaire humain d’EPR in vitro dérivé des cellules pluripotentes induites propres au patient. Ce tissu est morphologiquement et fonctionnellement représentatif de l’EPR in vivo et reproduit les défauts biochimiques de prénylation présents dans la CHM. De ce fait, il s’agit d’un modèle puissant pour évaluer l’efficacité de différentes approches thérapeutiques. Dans cette perspective, nous avons étudié une approche de thérapie génique par AAV2/5 afin de fournir le gène CHM dans le cas particulier de mutation faux sens et l’utilisation d’une translational read-through inducing drug (TRID) PTC124 pour le traitement des mutations non-sens.J’ai démontré pour la première fois la faisabilité de la thérapie génique pour la CHM dans le cas d’une expression résiduelle de REP1 muté, permettant de considérer les patients porteurs de mutations faux sens comme éligible à des essais cliniques de thérapie génique. De plus, j’ai démontré que l’efficacité de PTC124 peut être dépendante du type cellulaire. Dans l’ensemble, mes résultats suggèrent que l’efficacité de la molécule semblerait dépendre de la conservation de l’acide aminé muté et de sa localisation dans le domaine fonctionnel de REP1. Nous avons ainsi mis en valeur que le contexte génétique devrait être pris en compte dans la perspective d’une thérapie avec TRID pour cette maladie ainsi que d’autres pathologies.Pour conclure, j’ai souligné le potentiel prédictif du modèle d’EPR dérivé d’iPSc propre au patient pour évaluer de nouvelles approches thérapeutiques en l’absence d’un modèle animal approprié avant les essais cliniques. / Inherited retinal dystrophies (IRDs) are a class of genetically and clinically heterogeneous diseases, which are characterized by a progressive loss of vision. Choroideremia (CHM) is a chorioretinopathy, which accounts for ~3% of all IRDs. It is characterized by night blindness in childhood, followed by slow and progressive loss of the peripheral visual field. This results in legal blindness by the fourth to fifth decade of life. Generally, central vision is preserved till late in life. Genetically, the disease is caused by mutations in the CHM gene located on the X chromosome and encoding the Rab Escort Protein 1 (REP1). This protein is involved in the prenylation of Rab GTPasas, which regulate vesicular cell trafficking. Most of the disease-causing mutations are loss-of-function and the absence of REP1 leads to a Rab prenylation defect and subsequent degeneration of photoreceptors, retinal pigment epithelium (RPE) and underlying choroid. To date, an established therapy is not available for CHM, but the early diagnosis and its slow evolution provide a large therapeutic window, that renders this disease a good candidate for successful treatment.In order to palliate the lack of a pertinent animal model for testing novel disease therapies, we developed a human cellular model using patient-specific induced pluripotent stem cells (iPSc)-derived RPE. This tissue is morphologically and functionally representative of the RPE in vivo, and reproduces the biochemical prenylation defect present in CHM. Therefore, it is a powerful model to evaluate the efficacy of different therapeutic approaches. Along this line, we investigated a gene augmentation approach, via AAV2/5 delivery of the CHM gene in the particular case of a CHM missense mutation, and the use of the translational read-through inducing drug (TRID) PTC124 for treating CHM nonsense mutations.I demonstrated for the first time the feasibility of gene augmentation therapy for CHM in the case of residual mutated REP1 expression, suggesting that missense-carrying patients can be considered for inclusion in clinical gene therapy trials. Moreover, I showed that the efficiency of PTC124 may be dependent on the cell type. In addition, my results suggest that drug efficiency likely depends on the conservation of the mutated amino acid residue and its localization with regards to REP1 functional domains. We thus highlight that genetic considerations should be taken into account when considering TRID therapy for this and other disorders.Taken together, I highlighted the predictive potential of the patient-specific iPSc-derived RPE model for screening of novel and varied therapeutic approaches in the absence of a suitable animal model prior to clinical translation.