Thesis (Ph. D.)--Virginia Commonwealth University, 2010. / Prepared for: Dept. of Human Genetics. Title from resource description page. Includes bibliographical references.

The generation of induced pluripotent stem cells (iPSCs) presents a challenge to normal developmental processes. The low efficiency and heterogeneity of most methods have hindered understanding of the precise molecular mechanisms promoting, and roadblocks preventing, efficient reprogramming. While several intermediate populations have been described, it has proved difficult to characterize the rare, asynchronous transition from these intermediate stages to iPSCs. The rapid expansion of a minor population of reprogrammed cells can also obscure investigation of relevant processes. Understanding of the biological mechanisms essential for successful iPSC generation requires both accurate capture of cells undergoing the reprogramming process and identification of the associated global gene expression changes. Here we demonstrate that reprogramming follows an orderly sequence of stage transitions marked by changes in cell surface markers CD44 and ICAM1, and a Nanog-GFP reporter. RNA-sequencing (RNA-seq) analysis of these populations demonstrates two waves of pluripotency gene up-regulation, and unexpectedly, transient up-regulation of multiple epidermis-related genes, demonstrating that reprogramming is not simply the reversal of normal developmental processes. This novel high-resolution analysis enables the construction of a detailed reprogramming route map, and this improved understanding of the reprogramming process will lead to novel reprogramming strategies.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18893号 / 医博第4004号 / 新制||医||1009(附属図書館) / 31844 / 京都大学大学院医学研究科医学専攻 / (主査)教授 山下 潤, 教授 羽賀 博典, 教授 瀬原 淳子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Electrospun biomaterial scaffolds can be engineered to support the neural differentiation of induced pluripotent stem cells. As electrospinning produces scaffolds consisting of nano or microfibers, these topographical features can be used as cues to direct stem cell differentiation. These nano and microscale scaffolds can also be used to deliver chemical cues, such as small molecules and growth factors, to direct the differentiation of induced pluripotent stem cells into neural phenotypes. Induced pluripotent stem cells can become any cell type found in the body, making them a powerful tool for engineering tissues. Therefore, a combination of an engineered biomaterial scaffold with induced pluripotent stem cells is a promising approach for neural tissue engineering applications. As detailed in this thesis, electrospun scaffolds support the neuronal differentiation of induced pluripotent stem cells through delivering the appropriate chemical cues and also presenting physical cues, specifically topography to enhance neuronal regeneration. This thesis seeks to evaluate the following topics: multifunctional electrospun scaffolds for promoting neuronal differentiation of induced pluripotent stem cells, neuronal differentiation of human induced pluripotent stem cells seeded on electrospun scaffolds with varied topographies, and controlled release of glial cell-derived neurotrophic factor from random and aligned electrospun nanofibers. / Graduate / nkhadem@uvic.ca

Embryonic stem cells (ESCs) are pluripotent cells derived from the early embryo and are able to differentiate into cells belonging to the three germ layers. They are a valuable tool in research and for clinical use, but their applications are limited by ethical and technical issues. In 2006 a breakthrough report described the generation of induced pluripotent stem cells (iPSCs). IPSCs are ESC-like cells generated from somatic cells by forcing the ectopic expression of specific transcription factors. This circumvents the ethical issues about the use of embryos in research and provides multiple opportunities to understand the mechanisms behind pluripotency. The aim of this project was to generate sheep iPSCs and characterise them. In order to learn the technique I initially repeated the original iPSC methodology: the putative mouse iPSCs I have generated display a morphology typical of ESCs, characterised by a high nuclear to cytoplasmic ratio, and form colonies with neat edges and smooth domes. These cells are positive to Nanog, a marker of pluripotency, and can give rise to cells belonging to the mesodermal and the ectodermal lineages when differentiated in vitro. Since the main aim of the thesis was the derivation of sheep pluripotent cells, once established the protocol in mouse, I then moved to the generation of ovine iPSC colonies. The cells I have generated have a morphology similar to that of mouse ESCs, express markers of pluripotency such as alkaline phosphatase and Nanog and can differentiate in vitro and in vivo into cells belonging to the three germ layers. Additionally, these ovine iPSCs can contribute to live born chimeric lambs, although at low level.

L’amaurose congénitale de Leber (ACL) est une maladie génétique touchant la rétine. Les premiers symptômes apparaissent dès les premiers mois de la vie et mènent en quelques années à la cécité. A ce jour, des mutations dans 18 gènes ont été associées à la maladie. Cette hétérogénéité génétique rend difficile l’étude des mécanismes conduisant aux différents symptômes. Les modèles animaux utilisés en laboratoire, notamment les rongeurs, permettent d’étudier certains de ces mécanismes mais présentent des limites liées à l’espèce. Les cellules souches pluripotentes induites (iPSCs), qui proviennent de la reprogrammation de cellules somatiques issues de patients, constituent un nouvel outil pour étudier une maladie génétique dans un contexte humain naturel. Elles permettent d’obtenir tous les phénotypes cellulaires désirés sans limite quantitative ce qui ouvre la porte à des approches d’analyse à large échelle telle que l’analyse transcriptomique qui vise à explorer de manière systématique la modulation des gènes dans une maladie. L’objectif de mon projet de recherche a été de développer un modèle cellulaire humain naturellement porteur de l’ACL. Après avoir produit les iPSCs à partir de fibroblastes de patients, mes travaux ont consisté à les différencier en populations cellulaires homogènes et facilement amplifiables, les cellules souches neurales et les cellules de l’épithélium pigmentaire rétinien. Ces populations ont servi à mener des analyses transcriptomiques à large échelle qui ont permis d’identifier plusieurs gènes candidats, potentiellement impliqués dans le développement de la pathologie, parmi lesquels GSTT1 qui pourrait avoir un rôle dans le stress oxydatif. / Leber congenital amaurosis (LCA) is a genetic disease affecting the retina. The first symptoms appear in the first months of life and lead in few years to blindness. To date, mutations in 18 genes have been associated with the disease. This genetic heterogeneity makes it difficult to study mechanisms leading to different symptoms. Animal models, including rodents, are used to study some of these mechanisms but have limitations mostly related to the species. The induced pluripotent stem cells (iPSCs), which are reprogrammed somatic cells of patients, constitute a new tool for studying genetic diseases in a natural human context. They achieve all desired cell phenotypes without quantitative limits which opens the door to large-scale analysis approaches such as transcriptomic analysis that aims to systematically explore the modulation of genes in a disease. The aim of my research project was to develop a human cell model naturally carries the LCA. After producing the iPSCs from fibroblasts of patients, my work had consisted to differentiate them into homogeneous and easily amplifiable cell populations, neural stem cells and retinal pigment epithelial cells. These populations have served to conduct large-scale transcriptomic analyzes which have identified several candidate genes potentially involved in the development of the disease, including GSTT1 which might have a role in oxidative stress.

The derivation of pluripotent stem cells (now termed induced pluripotent stem cells, iPSC) from mature somatic cells was a finding of seminal importance to fundamental cell biology. Thus established iPSC technology has been predicted to advance fields that previously relied on the ethically disputed use of embryonic stem cells. Being pluripotent (able to differentiate into every cell type present in the human body) and sharing most other characteristics with embryonic stem cells, but being much readier obtainable and their derivation free from ethical restraints, human induced pluripotent stem cells (hiPSC) provide access to cell types and insights into cell processes previously unattainable to researches. For this thesis, a hiPSC line was established from a skin biopsy donated by a Down’s syndrome patient. Most of what is known today about the molecular neurobiology behind this disease has been gathered from mice models or human post mortem studies, but this has a limited extrapolation potential to early human brain development in DS patients, as Down’s syndrome is an inherently human disease whose defining phenotype is established early during embryonic development. Having access to human pluripotent cells able to recapitulate the events of early neurogenesis is thus invaluable to the understanding of the mechanisms of this disorder. In parallel, work has been performed on optimizing iPSC reprogramming protocol. By exchanging one of the transcription factors used for reprogramming with a reporter gene, genomic integration of reprogramming factors has become possible to be traced visually, enabling more efficient selection of reprogrammed iPSC colonies.

The vascular endothelium is a dynamic cellular interface that displays a unique phenotypic plasticity. This plasticity is critical for vascular function and when dysregulated is pathogenic in several diseases. The development of new human endothelial genotype-phenotype studies, personalized vascular medicine efforts and cell based regenerative therapies are limited by the unavailability of patient-specific endothelial cells. Induced pluripotent stem cells (iPSC) offer great promise as a new personalized source of endothelium; however, the reproducibility, fidelity and functionality of iPSC-derived endothelial cells remains poorly understood. / Engineering and Applied Sciences

Parkinson's disease (PD) is characterised by the loss of dopaminergic neurons in the Substantia Nigra pars compacta in the midbrain and the presence of intracellular aggregates, known as Lewy bodies (LBs), in the surviving neurons. The aetiology of PD is unknown but a causative role for α-Synuclein (SNCA) has been proposed. Although the function of αSyn is not well understood, a number of pathological mechanisms associated with αSyn toxicity have been proposed. In this study, nine induced pluripotent stem cells (iPSCs) lines from healthy individuals and PD patients carrying the A53T SNCA mutation or a triplication of SNCA were differentiated to dopaminergic neurons (iDAn). All iPSC lines differentiated with similar efficiency to iDAn, indicating that they could be used for phenotypic analysis. Quantification of αSyn expression showed increased αSyn intracellular staining and the novel detection of increased αSyn oligomerization in PD iDAn. Analysis of mitochondrial respiration found a decrease in basal respiration, maximal respiration, ATP production and spare capacity in PD iDAn, but not in undifferentiated iPSCs, indicating the cell-type specificity of these defects. Decreased phosphorylation of dynamin-1-like protein at Ser616 (DRP1^Ser616) and increased levels of Peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) in A53T SNCA iDAn suggest a new pathological mechanism linking αSyn to the imbalance in mitochondria homeostasis. Markers of endoplasmic reticulum (ER) stress were found to be up-regulated, along with increased β- Glucocerebrosidase (GBA) activity, perturbation of autophagy and decreased expression of fatty acids binding protein 7 (FAPB7) in PD iDAn. Lastly, lentiviral vectors for RNAi-mediated knockdown of αSyn were developed and these reduced αSyn protein levels in iDAn, resulting in increased expression of FABP7. These results describe a novel functional link between αSyn and FABP7. This work demonstrates that iDAn are a promising and relevant in vitro cell model for studying cellular dysfunctions in PD pathology, and the phenotypic analysis of A53T SNCA and SNCA triplication iDAn enabled the detection of novel pathological mechanisms associated with PD.

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.