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From the simplest unicellular organisms to complex metazoans, cell polarity is a widespread characteristic that is essential for almost every aspect of biology. Proper polarization of cells is crucial for the establishment and maintenance of higher order structures such as tissue and organs. Cell polarity refers to the asymmetric distribution of various macromolecules and cellular structures, resulting in polarized architecture and function of the cell. Defects in cell polarity lead to various phenotypes, ranging from aberrant signaling, proliferation, cell adhesion and migration, cell fate determination and pluripotency, as well as embryonic lethality, neoplasia and cancer. Given the various roles for cell polarity in development and disease, the characterization of the components involved in polarity and their mechanisms of function is of great importance. My thesis work has encompassed three major projects, each of which is focused on understanding the role of cell polarity in development and disease. Although genetic screens in invertebrates have led to the identification of a number of cell-polarity proteins, similar systematic approach have not been undertaken in mammalian systems. The goal of my first project was to design and implement a high-throughput screen to systematically knockdown individual genes using siRNA, and then assess cell junction integrity as a measure of cell polarity. Given the importance of cell polarity to signaling pathways, I next sought to determine the mechanism by which cell polarity affects TGFβ and Hippo pathways, two important signaling pathways involved in development and disease. Lastly, by studying the acquisition of pluripotency by somatic cells, I uncovered a central role for cell polarity in the establishment and maintenance of pluripotency. Here I will present and discuss our discovery pertaining to the role of cell polarity in cell signaling and pluripotency.

From the simplest unicellular organisms to complex metazoans, cell polarity is a widespread characteristic that is essential for almost every aspect of biology. Proper polarization of cells is crucial for the establishment and maintenance of higher order structures such as tissue and organs. Cell polarity refers to the asymmetric distribution of various macromolecules and cellular structures, resulting in polarized architecture and function of the cell. Defects in cell polarity lead to various phenotypes, ranging from aberrant signaling, proliferation, cell adhesion and migration, cell fate determination and pluripotency, as well as embryonic lethality, neoplasia and cancer. Given the various roles for cell polarity in development and disease, the characterization of the components involved in polarity and their mechanisms of function is of great importance. My thesis work has encompassed three major projects, each of which is focused on understanding the role of cell polarity in development and disease. Although genetic screens in invertebrates have led to the identification of a number of cell-polarity proteins, similar systematic approach have not been undertaken in mammalian systems. The goal of my first project was to design and implement a high-throughput screen to systematically knockdown individual genes using siRNA, and then assess cell junction integrity as a measure of cell polarity. Given the importance of cell polarity to signaling pathways, I next sought to determine the mechanism by which cell polarity affects TGFβ and Hippo pathways, two important signaling pathways involved in development and disease. Lastly, by studying the acquisition of pluripotency by somatic cells, I uncovered a central role for cell polarity in the establishment and maintenance of pluripotency. Here I will present and discuss our discovery pertaining to the role of cell polarity in cell signaling and pluripotency.

<p>Synthetic biology facilitates both the design and fabrication of biological components and systems that do not already exist in the natural world. From an engineering point of view, synthetic biology is akin to building a complex machine by assembling simpler parts. Complex genetic machines can also be built by a modular and rational assembly of simpler biological parts. These biological machines can profoundly affect various cellular processes including the transcriptional machinery. In this thesis I demonstrate the utilization of biological parts according to synthetic biology principles to solve three distinct transcription-level problems: 1) How to efficiently select for transgene excision in induced pluripotent stem cells (iPSCs)? 2) How to eliminate transposase expression following piggyBac-mediated transgenesis? 3) How to reprogram cell lineage specification by the dCas9/gRNA transactivator-induced expression of endogenous transcription factors? </p><p>Viral vectors remain the most efficient and popular in deriving induced pluripotent stem cells (iPSCs). For translation, it is important to silence or remove the reprogramming factors after induction of pluripotency. In the first study, we design an excisable loxP-flanked lentiviral construct that a) includes all the reprogramming elements in a single lentiviral vector expressed by a strong EF-1&#945; promoter; b) enables easy determination of lentiviral titer; c) enables transgene removal and cell enrichment using LoxP-site-specific Cre-recombinase excision and Herpes Simplex Virus-thymidine kinase/ganciclovir (HSV-tk/gan) negative selection; and d) allows for transgene excision in a colony format. With our design, a reprogramming efficiency comparable to that reported in the literature without boosting molecules can be consistently obtained. To further demonstrate the utility of this Cre-loxP/HSV-tk/gan strategy, we incorporate a non-viral therapeutic transgene (human blood coagulation Factor IX) in the iPSCs, whose expression can be controlled by a temporal pulse of Cre recombinase. The robustness of this platform enables the implementation of an efficacious and cost-effective protocol for iPSC generation and their subsequent transgenesis for downstream studies.</p><p>Transgene insertion plays an important role in gene therapy and in biological studies. Transposon-based systems that integrate transgenes by transposase-catalyzed "cut-and-paste" mechanism have emerged as an attractive system for transgenesis. Hyperactive piggyBac transposon is particularly promising due to its ability to integrate large transgenes with high efficiency. However, prolonged expression of transposase can become a potential source of genotoxic effects due to uncontrolled transposition of the integrated transgene from one chromosomal locus to another. In the second study we propose a vector design to decrease post-transposition expression of transposase and to eliminate the cells that have residual transposase expression. We design a single plasmid construct that combines the transposase and the transpositioning transgene element to share a single polyA sequence for termination. Consequently, the transposase element is deactivated after transposition. We also co-express Herpes Simplex Virus thymidine kinase (HSV-tk) with the transposase. Therefore, cells having residual transposase expression can be eliminated by the administration of ganciclovir. We demonstrate the utility of this combination transposon system by integrating and expressing a model therapeutic gene, human coagulation Factor IX, in HEK293T cells.</p><p>Genome editing by the efficient CRISPR/Cas9 system shows tremendous promise with ease of customization and the capability to multiplex distinguishing it from other such technologies. Endogenous gene activation is another aspect of CRISPR/Cas9 technology particularly attractive for biotechnology and medicine. However, the CRISPR/Cas9 technology for gene activation leaves much room for improvement. In the final study of this thesis we show that the fusion of two transactivation (VP64) domains to Cas9 dramatically enhances gene activation to a level that is sufficient to achieve direct cell reprogramming. Targeted activation of the endogenous Myod1 gene locus with this system leads to stable and sustained reprogramming of mouse embryonic fibroblasts into skeletal myocytes. </p><p>In conclusion, this dissertation demonstrates the power of utilizing biological parts in a rational and systematic way to rectify problems associated with cell fate reprogramming and transposon-based gene delivery. Through design of genetic constructs aided by synthetic biology principles, I aspire to make contributions to the related fields of cellular reprogramming, stem cell differentiation, genomics, epigenetics, cell-based disease models, gene therapy, and regenerative medicine.</p> / Dissertation

Le vieillissement est un processus complexe souvent ponctué par l’apparition de maladies liées à l’âge telles que l’arthrose, la fibrose pulmonaire ou l’ostéoporose et associé à une diminution des capacités de régénération et des stocks de cellules souches adultes. En 2007, le Dr Yamanaka et ses collaborateurs montraient pour la première fois que des fibroblastes humains pouvaient être convertis en cellules souches pluripotentes en induisant l’expression de 4 facteurs de transcription : OCT4, SOX2, KLF4 et c-MYC. Au laboratoire, il a été montré en 2011 qu’il est possible de reprogrammer les cellules sénescentes s’accumulant au cours du vieillissement et de les redifférencier en cellules somatiques rajeunies.In vivo, une reprogrammation totale conduirait à la formation de tératomes, mais en induisant le processus de reprogrammation et en le stoppant avant d’obtenir des cellules souches pluripotentes nous pensons qu’il est possible de restaurer la physiologie cellulaire altérée et ainsi de retarder le vieillissement tissulaire et ses effets néfastes. Le Dr Izpisua Belmonte a validé cette hypothèse en décembre 2016 en montrant, dans un modèle murin transgénique récapitulant le phénotype de vieillissement accéléré du syndrôme d’Hutchinson Gilford et permettant dans le même temps l’induction contrôlée de l’expression de facteurs (OCT4, SOX2, KLF4, c-MYC), qu’il était possible d’allonger l’espérance de vie des animaux et de retarder l’apparition du phénotype pathologique lié à l’âge. Nous avons mis en place un modèle murin similaire et avons montré qu’une reprogrammation transitoire peut non seulement allonger l’espérance de vie des animaux mais également retarder la perte de poids qui advient au cours du vieillissement et l’apparition du phénotype pathologique lié à l’âge. De plus, nous avons réussi à maintenir une capacité de régénération accrue jusqu’à la fin de la vie des souris. D’autre part, en modélisant des pathologies liées à l’âge telles que l’arthrose ou la fibrose pulmonaire chez des animaux inductibles pour les facteurs de transcription de Yamanaka, nous avons montré qu’une reprogrammation transitoire pouvait prévenir les dommages provoqués. Cette étude aura donc permis de confirmer l’importance que la reprogrammation cellulaire peut avoir dans les stratégies de lutte contre le vieillissement. / Aging is a complex process which is often punctuated by the appearing of age-related diseases such as arthritis, idiopathic pulmonary fibrosis or osteoporosis, and which is associated with a decrease of regeneration abilities and of adult stem cells number. In 2007, Dr. Yamanaka and his collaborators showed for the first time that human fibroblasts could be converted into pluripotent stem cells by inducing the expression of 4 transcription factors: OCT4, SOX2, KLF4 and c-MYC. In the laboratory, it was showed in 2011 that it is possible to reprogram senescent cells which are accumulating in aging organisms and to differentiate them into rejuvenated somatic cells.In vivo, a total reprogramming would lead to teratomas formation but if the reprogramming process is induced and stopped before getting pluripotent stem cells, we think that it is possible to restore altered cell physiology and to delay tissues aging and its deleterious consequences. Dr. Izpisua Belmonte validated this hypothesis in December 2016. He designed a murine transgenic model which recapitulates the premature aging phenotype of Hutchinson Gilford syndrome and which can be induced to express OCT4, SOX2, KLF4 and c-MYC, and he proved that it is possible to increase mice lifespan and to delay the appearing of pathological aging phenotype. We built a similar murine model and showed that a transient reprogramming can not only increase lifespan, but also delay age-related weight loss and pathological aging phenotype. Moreover, we were able to maintain a higher regenerative capacity until mice death. We also modeled age-related pathologies such as arthritis or idiopathic pulmonary fibrosis in mice which were inducible for the Yamanaka’s transcription factors and we showed that transient reprogramming could prevent damages. This study will have allowed to confirm the importance that cellular reprogramming can have in the fight against aging.

Since Yamanaka and Takahashi first described the isolation of induced pluripotent stem cells (iPSCs) in 2006, researchers have invested a vast amount of time and resources into trying to understand the process of reprogramming. However, the exact mechanisms underlying the induction of somatic cells to pluripotency is still incompletely understood. With this in mind, a screening approach was undertaken to identify shRNA that enhance the reprogramming process. A retrovirus based system was used to knock down candidate genes during reprogramming of mouse embryonic fibroblasts (MEF) containing doxycycline-inducible reprogramming factors and a Nanog-GFP reporter, which is activated when cells become iPSCs. The initial round of screening with over 150 shRNA vectors successfully identified several shRNAs that enhance reprogramming. One of these shRNA vectors exhibited both faster reprogramming kinetics as determined by activation of the Nanog-GFP reporter 2 to 3 days earlier and increased reprogramming efficiency giving rise to >5 fold more GFP+ colonies when compared with a control. Cell surface marker analysis with flow cytometry demonstrated that changes in CD44 and ICAM1 expression, which occur preceding Nanog-GFP expression, were also accelerated. Validation of this shRNA determined that the enhanced reprogramming phenotype is the result of an unknown off-target effect. Microarray and RNA-sequencing analysis was carried out to identify the off target gene with a view to investigate the functional importance of this knock down and its role in establishing the pluripotency transcriptional network during reprogramming.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21991号 / 医博第4505号 / 新制||医||1037(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 武田 俊一, 教授 坂井 義治, 教授 松田 道行 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Shimamoto R, Amano N, Ichisaka T, Watanabe A, Yamanaka S, et al. (2014) Generation and Characterization of Induced Pluripotent Stem Cells from Aid-Deficient Mice. PLoS ONE 9(4): e94735. doi:10.1371/journal.pone.0094735 / 京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第18515号 / 医科博第56号 / 新制||医科||4(附属図書館) / 31401 / 京都大学大学院医学研究科医科学専攻 / (主査)教授 斎藤 通紀, 教授 平家 俊男, 教授 山田 泰広 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

La reprogrammation, qui est la réversion d’un noyau d’un état somatique vers un état moins différencié, constitue un enjeu majeur pour la thérapie cellulaire. Cependant, les mécanismes initiaux qui président à la reprogrammation restent mal connus. Le transfert nucléaire (clonage) met à profit les propriétés de reprogrammation uniques du cytoplasme ovocytaire, et constitue une approche expérimentale intéressante pour analyser ces processus. Le but de cette thèse est d’étudier la différence de capacité de cellules fibroblastiques à être reprogrammées efficacement, en tirant partie d’une situation-modèle d'efficacité différentielle de reprogrammation après clonage chez le bovin. Ce modèle est constitué de deux lots de fibroblastes donneurs de noyaux, qui forment des embryons clonés dont la différence d’efficacité de développement à terme varie d’un facteur 8. L’analyse des cellules donneuses a montré une augmentation des anomalies de ploïdie dans les cellules à faible potentiel, et la similitude transcriptomique entre les cellules donneuses, alors que la comparaison des transcriptomes des embryonsclonés a montré des différences de reprogrammation de l’expression génique dès le stade suivant l’activation du génome embryonnaire. Des différences de méthylation de l’ADN entre cellules donneuses ont été observées dans les promoteurs de gènes candidats différentiellement reprogrammés, ainsi que dans une analyse plus globale par RRBS. Nous avons enfin étudié la distribution des cellules filles des deux premiers blastomères au stade blastocyste, la distribution « orthogonale » et l’aptitude au développement à terme des embryons de souris clonés étant liées (Liu et al., 2012). Nous avons montré l’existence de trois distributions dans les embryons fécondés mais n’avons pas observé de différence de proportions de celles-ci entre embryons bovins clonés. En conclusion, dans notre modèle, la distribution des cellules filles des deux premiers blastomères au stade blastocyste ne semble pas associée à l’efficacité de reprogrammation dans les embryons bovins clonés, contrairement aux différences épigénétiques entre cellules donneuses. / Reprogramming, which is the return of a nucleus from a somatic state to a less differentiated state, is a major issue for cell therapy. However, the initial mechanisms governing the reprogramming remain poorly understood. Nuclear transfer (cloning) takes advantage of the unique reprogramming properties of the oocyte cytoplasm, and therefore is an interesting experimental approach to analyze these processes. The aim of this thesis is to study the difference in fibroblasts’ ability to be reprogrammed by taking advantage of a model-situation of differential reprogramming efficiency after cloning in cattle. This model consists of two batches of donor fibroblasts, which form cloned embryos having an 8 fold difference in development to term efficiency. Analysis of donor cells has shown increase ploidy abnormalities in cells of low potential, and transcriptomic similarity between the donor cells, whereas comparison ofcloned embryos transcriptomes showed gene expression reprogramming differences just after embryonic genome activation. Differences in DNA methylation between donor cells were observed in the promoters of candidate genes differentially reprogrammed and in a more comprehensive analysis by RRBS. Finally we studied the distribution of the first two blastomeres’ daughter cells at the blastocyst stage, as an "orthogonal" distribution and development to term of mice cloned embryos are linked (Liu et al., 2012). We have shown the existence of three distributions in the fertilized embryos but haven’t seen any difference of proportions between bovine cloned embryos. In conclusion, in our model, the distribution of the first two blastomeres’ daughter cells at the blastocyst stage does not seem related to the reprogramming efficiency in bovine cloned embryos, unlike epigenetic differences between donor cells.

京都大学 / 新制・課程博士 / 博士(医科学) / 甲第23611号 / 医科博第134号 / 新制||医科||9(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 遊佐 宏介, 教授 小川 誠司, 教授 山中 伸弥 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM