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Parkinson’s disease (PD) is the second most common age-related neurodegenerative condition. It is neuropathologically characterised by the presence of Lewy pathology and the degeneration of the midbrain dopaminergic neurons from the substantia nigra pars compacta. Lewy pathology mainly consists of filamentous aggregated alpha-synuclein and familial forms of PD can be caused by genetic alternations in the SNCA gene encoding alpha-synuclein. Alpha-synuclein is primarily localised to neuronal presynaptic terminals and has been implicated in the maintenance of synaptic function. Studies have proposed that it regulates the docking, fusion, clustering and trafficking of neurotransmitter-loaded presynaptic vesicles. Nowadays, it is possible to model PD in vitro by obtaining adult somatic cells from patients, reprogramming them into induced pluripotent stem cells (iPSCs), and differentiating iPSCs into neurons. For this project, iPSCs derived from two PD patients, one harbouring the A53T SNCA mutation, the other a SNCA triplication, and three healthy individuals, were employed. In the initial stage, I optimised a neuronal differentiation protocol originally developed for human embryonic stem cells to produce neurons belonging to two distinct brain regions affected in PD, the forebrain and midbrain, from the available human iPSC lines. Next, I assessed the maturation of the generated neurons over time using protein expression and electrophysiological techniques. Finally, I examined PD-related phenotypes such as alpha-synuclein aggregation and release, susceptibility to cell death, and the redistribution of presynaptic proteins. All the iPSC lines used gave rise to forebrain and midbrain neuronal cultures. Maturation was similar across lines, as no consistent differences were observed in the changes of the expression of 4 repeat tau isoforms, presynaptic protein levels or electrophysiological properties over time. However, the emergence of astrocytes varied between cultures derived from distinct iPSC lines. No robust differences in alpha-synuclein release and susceptibility to cell death were detected between patient- and control-derived neurons. Apart from the presence of larger alpha-synuclein-positive puncta in patient-derived neurons, no other signs of alpha-synuclein aggregation were detected. Despite this, midbrain patient-derived neurons with a SNCA triplication exhibited a significant redistribution of presynaptic protein VAMP-2/synaptobrevin-2, which interacts with alpha-synuclein, relative to controls.

Induced pluripotent stem cells (iPSCs) are generated by reprogramming somatic cells to an embryonic state. Therefore iPSCs represent an extremely valuable tool for modelling disease and organ toxicity, with enormous potential in veterinary medicine. Several equine diseases are currently untreatable and can result in euthanasia on medical grounds. In contrast to humans, in vitro models for cellular research in equine do not exist. Therefore it has been necessary to explore the use of stem cells in constructing cell based equine models. Pluripotent stem cell populations are of great interest in this field given their ability to form the three germ layers found in the developing embryo. While a promising notion, the isolation of equine embryonic stem cells has thus far proved elusive and therefore it has been necessary to explore other pluripotent stem cell populations. A very limited number of induced PSC lines have so far been generated from equine fibroblasts but studies in humans showed that other cell types such as keratinocytes were more amenable to reprogramming and generated iPSCs with much higher efficiency; whether this may be also the case in other species has not been investigated. Moreover, iPSC lines reported so far from domestic species, including the horse, depended on complex culture conditions for growth, including feeder layers and media supplementation with several growth factors. Although a promising alternative to fibroblast for generation of induced pluripotent stem cells there is dearth in literature on equine keratinocyte culture techniques. In this work I am reporting a novel approach to generate equine iPSCs lines from keratinocytes. Skin biopsies were used to derive keratinocyte cultures. The three dimensional culture systems were developed for robust culture of equine keratinocytes. These cells were then transduced with retroviral constructs coding for murine Oct-4, Sox-2, c-Myc and Klf-4 sequences, following the original Yamanaka protocol. Following transduction, tight cell colonies with sharp boundaries staining positive for alkaline phosphatase resembling previously reported human iPSCs were generated. The reprogrammed cells were successfully maintained in feeder free and serum free conditions with LIF supplementation. Immunochemistry and qPCR analyses revealed the equine iPSCs lines expressed pluripotency markers expressed in equine embryonic stages including, OCT4, SOX2, SSEA1, LIN 28, NANOG, REX1 and DNMT3B. Equine iPSCs were able to form embryoid bodies and differentiate into derivatives of the three germ layers in vitro. Equine iPSCs were pluripotent in vivo as demonstrated by the formation of teratoma consisting of tissue derivatives of all three lineages such as bone, cartilage, pulmonary epithelium and mature neurons in SCID mice. Importantly, equine iPSCs should not only have the ability to differentiate in a non-directed manner. Therefore, the ability for efficient and directed cellular differentiation was analysed. Equine iPSCs were successfully induced to differentiate into neurospheres forming extensive neuronal projections and synapses. Equine iPSCs were differentiated to neurons using a novel and robust approach. The neurons expressed FOXG1, TUBB3 at induction before ISL1 up regulation, a potent and specific inducer of motor neurons, during terminal differentiation. The neurons tested could fire multiple action potentials and also induce TTX –sensitive action potentials. The iPSC line that showed in vivo differentiation in bone and cartilage was tested for directed differentiation into bone and results were compared to equine mesenchymal stem cells. This study provides the first demonstration of the potential of iPSCs in equine biomedicine. The ability to derive iPSC cells capable of direct differentiation in vitro opens the way for new and exciting applications in equine regenerative medicine.

No description available.

No description available.

La dystrophie Facio-Scapulo-Humérale (FSHD) est caractérisée par un affaiblissement asymétrique des muscles de la face, de la ceinture scapulaire et des bras. Dans 95% des cas (FSHD1), elle est liée à la contraction d’un élément macrosatellite en 4q35, D4Z4. Dans les 5% des cas qui ne présentent pas de contraction de D4Z4 (FSHD2), des mutations dans le gène SMCHD1 sont retrouvées. Des mutations dans ce gène sont aussi associées à un syndrome développemental très rare, le syndrome de Bosma (BAMS). Les individus présentent des anomalies cranio-faciales mais aucun signe musculaire.Nous avons développé une méthode pour l’analyse de la méthylation de D4Z4 associant modification de l’ADN au bisulfite de sodium et séquençage à haut débit. Nous observons une hypométhylation marquée chez les patients FSHD2 et BAMS au niveau de la partie proximale de cet élément. L’étude de la méthylation au cours de la reprogrammation de fibroblastes primaires en cellules souches pluripotentes induites montre que D4Z4 est reméthylé de façon spécifique dans les cellules contrôles et FSHD1 mais est peu modulée dans les cellules FSHD2 et BAMS. Cette méthylation élevée est une caractéristique de la pluripotence et implique SMCHD1. La reméthylation ne dépend pas non plus de la mémoire épigénétique héritée des fibroblastes ni du nombre de répétitions D4Z4. Ces résultats suggèrent un mécanisme de régulation dynamique. L’hypométhylation de D4Z4 n’est pas observée lors de l’invalidation somatique du gène indiquant que SMCHD1 est impliqué dans la mise en place de la méthylation et non sa maintenance.SMCHD1 est donc impliqué dans la mise en place de la méthylation de D4Z4 dans les cellules pluripotentes. / Facio-Scapulo-Humeral Dystrophy is characterized by the involvement of specific facial, scapulo-humeral and anterior foreleg muscles. In 95% of cases (FSHD1) the disease is associated with a reduction of a macrosatellite element, D4Z4, at the 4q35 locus. In the 5% remaining cases (FSHD2), there is no D4Z4 contraction and patients carry mutations in the SMCHD1 gene. Mutations in SMCHD1 are also involved in a very rare developmental syndrome called Bosma Arhinia Microphtalmia syndrome (BAMS) characterized by cranio-facial abnormalities but no muscular dystrophy.To analyze D4Z4 methylation we developed an approach based on the Sodium Bisulfite treatment method followed by high-throughput sequencing. We showed a significant hypomethylation in the proximal region of D4Z4 in FSHD and BAMS patients compared to controls. Our methylation analysis in primary fibroblasts and corresponding human induced pluripotent stem cells showed that D4Z4 is specifically remethylated upon reprogramming in FSHD1 cells but not in SMCHD1-mutated cells. The high methylation level is a feature of pluripotency likely dependent on SMCHD1. Strikingly, hypomethylation is not observed in somatic cells invalidated for SMCHD1 suggesting a key role for this factor in de novo the methylation. D4Z4 methylation pattern does not depend on the epigenetic memory inherited form donor cells and on the number of the D4Z4 copies and it is a tightly regulated process.In conclusion, SMCHD1 plays a role in the D4Z4 methyl mark deposition at the pluripotency state.

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.

Development leads unidirectionally towards a more restricted cell fate that is usually stable. However, it has been proven that developmental systems are reversible by the success of animal cloning of a differentiated somatic genome through somatic cell nuclear transfer (SCNT). Recently, reprogramming of somatic cells to a pluripotent embryonic stem cell (ESC)-like state by introducing defined transcripton factor has been achieved, resulting in the generation of induced pluripotent stem cells (iPSCs), which resemble ESCs. iPSC reprogramming is of great medical interest, as it has the potential to generate a source of patient-specific cells. However, the dangerous delivery method, low efficiency, and slow kinetics of the reprogramming process have hampered progress with this technology.

Induced pluripotent stem cells (iPSCs) are an attractive cell source for various applications in regenerative medicine and cell-based therapies given their unique capability to differentiate into any cell type of the human body. However, human iPSCs are highly vulnerable to cryopreservation with post-thaw survival rates of 40-60%; this is due to cryoinjury resulting from ice recrystallization when using conventional slow cooling protocols. Ice recrystallization is a process where the growth of large ice crystals occurs at the expense of small ice crystals. Ice recrystallization inhibitors (IRIs) are designed to inhibit the growth of intracellular ice crystals, increasing post-thaw viability. In this study, we tested a panel of four IRIs to determine if the inhibition of ice recrystallization can decrease cellular damage during freezing and improve viability post-thaw of iPSC colonies. We supplemented commercially available and serum-free cryopreservation medium mFreSR, routinely used for the cryopreservation of iPSCs, with a class of N-aryl-D-ß-gluconamide IRIs. A 2-fold increase in post-thaw viability was observed, in a dose dependent response, for N-(4-methoxyphenyl)-D-gluconamide (PMA) at 15 mM, N-(2-fluorophenyl)-D-gluconamide (2FA) at 10 mM, and N-(4-chlorophenyl)-D-gluconamide (4ClA) at 0.5 mM over mFreSR controls. After testing the panel of four IRIs, 2FA frozen iPSCs showed an increase in cell viability, proliferation, and recovery. The addition of ROCK inhibitor (RI), commonly used to increase iPSC viability post thaw, further enhanced the survival of the iPSCs frozen in the presence of 2FA and is used routinely in research. This additive effect increased cell recovery and colony formation post thaw, resulting in increased proliferation with no adverse effects on iPSC pluripotency or differentiation capabilities. The development of improved cryopreservation strategies for iPSCs is key to establishing master clonal cell banks and limiting cell selection pressures, all while maintaining high post-thaw viability and function. This will help ensure sufficient supplies of high-quality iPSC required to meet the cell demands for cell and regenerative based therapies. Since iPSCs hold promise as a potentially unlimited cell source for a plethora of cell-based therapies, improving cryopreservation is essential to the successful deployment of iPSC-derived therapeutic cell products in the future.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20609号 / 医博第4258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 高橋 淳, 教授 伊佐 正, 教授 影山 龍一郎 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM