abstract: An in vitro model of Alzheimer’s disease (AD) is required to study the poorly understood molecular mechanisms involved in the familial and sporadic forms of the disease. Animal models have previously proven to be useful in studying familial Alzheimer’s disease (AD) by the introduction of AD related mutations in the animal genome and by the overexpression of AD related proteins. The genetics of sporadic Alzheimer’s is however too complex to model in an animal model. More recently, AD human induced pluripotent stem cells (hiPSCs) have been used to study the disease in a dish. However, AD hiPSC derived neurons do not faithfully reflect all the molecular characteristics and phenotypes observed in the aged cells with neurodegenerative disease. The truncated form of nuclear protein Lamin-A, progerin, has been implicated in premature aging and is found in increasing concentrations as normal cells age. We hypothesized that by overexpressing progerin, we can cause cells to ‘age’ and display the neurodegenerative effects observed with aging in both diseased and normal cells. To answer this hypothesis, we first generated a retrovirus that allows for the overexpression of progerin in AD and non-demented control (NDC) hiPSC derived neural progenitor cells(NPCs). Subsequently, we generated a pure population of hNPCs that overexpress progerin and wild type lamin. Finally, we analyzed the presence of various age related phenotypes such as abnormal nuclear structure and the loss of nuclear lamina associated proteins to characterize ‘aging’ in these cells. / Dissertation/Thesis / Masters Thesis Bioengineering 2017

Chorea Acanthocytosis (ChAc) is an autosomal recessive inherited disease caused by loss- of-function mutation in the VPS13A gene which encodes CHOREIN protein. This study used induced pluripotent stem cells (iPSCs) as well as neural progenitor cells (NPCs) to generate medium spiny neurons (MSN) as well as midbrain dopaminergic neurons (mDAN). The first objective of this thesis was to generate and characterize a stem cell based disease model of ChAc. The second objective was to establish two different differentiation protocols that yield different neuronal sub types that are affected in ChAc, and compare whether they harbor similar phenotypes and whether the faster protocol can be used to model the disease accurately. The generated iPSCs were characterized using AP staining as an early marker for reprogramming, qPCR for analysis of residual expression of exogenous transcription factors, immunocytochemistry (ICC) for staining of pluripotency markers as well as markers for mesoderm, ectoderm and endoderm formation upon three germ layer formation. Karyotyping was conducted to exclude aberrant clones. Western blot using CHOREIN antibody revealed that the cell lines retained their disease identity. There were no differences observed between wild type and ChAc lines in stem cell and neuron populations in either protocol. qPCR analysis, investigating the expression of previously described markers for characterization, revealed no significant clustering between wild type and ChAc lines in either protocol. A disturbed ratio of globular and filamentous actin is causative for the aberrant shape of ChAc erythrocytes. Investigation of the ratio in mature neurons revealed a significant reduction of this ratio in MSN but no difference in mDAN cultures. When the ratio of cytosolic and filamentous tubulin and the acetylation of tubulin were investigated, no differences were found between wild type and ChAc lines. Mature neurons of both differentiation protocols were subjected to treatment with the proteotoxic stress inducer L-canavanine and the unfolded protein response (UPR) inducer tunicamycin. Survival was analyzed with the PrestoBlue assay as well as lactate dehydroxylase (LDH) release assay. Both cultures of mature neurons showed an increased susceptibility to the respective drugs. Furthermore the data suggests that MSN cultures are more vulnerable against proteotoxic stress (L-canavanine). Kinetics of tunicamycin poisoning were not different within MSN cultures but indicated a late cell death of ChAc lines under mDAN differentiation conditions. DNA damage plays a major role in the progression of neurodegenerative diseases. The amount of double strand breaks (DSB) was assessed in mature cultures of MSN and mDAN differentiations. There was no difference in basal level of DSB. When etoposide was applied to induce DNA damage, increased susceptibility of ChAc lines was observed. Albeit significant, the effect size was very small. Seahorse was used to characterize energy metabolism. Glycolysis was not impaired in ChAc lines in either protocol. Furthermore, MSN differentiation showed no difference in any parameter related to oxidative phosphorylation, while under mDAN conditions, coupling efficiency and spare respiratory capacity was increased for ChAc lines. The non-respiratory oxygen consumption was increased in ChAc lines in MSN cultures but decreased in mDAN cultures. The yeast homolog of VPS13A interacts with vesicle and mitochondrial membranes. Therefore, this study focuses on vesicle and mitochondria homeostasis. Live cell imaging of mature neurons of MSN differentiations revealed a decreased amount and reduced motility of mitochondria. Even though mitochondria were normally shaped their size was reduced. mDAN differentiations harbored a reduced amount and shortened mitochondria. These mitochondria, however, showed an increased motility. When analyzing aligned mature neurons in microfluidic chambers (MFCs), a strong phenotype was already observed in proximal regions, which resembled the distal parts of the channels. Hence, the dysregulation, that occurs distal in healthy controls, happens closer to the soma in diseased cells. The mitochondria potential marker JC-1 showed a hyperpolarization of mitochondria in MSN culture and a depolarization in mDAN cultures. When investigated in MFCs of mDAN cultures, there was a significant increase in potential observed at the distal position of ChAc lines, while wild type cultures showed no difference. Experiments conducted on the lysosomal compartments showed a decrease in proximal parts of ChAc MSN cultures when compared to wild type. Their shape was altered as well. mDAN cultures featured no significant morphological changes. Trafficking analysis revealed an increase in motility in MSN cultures but a decrease in mDAN cultures. When lysosomes were analyzed in MFCs only mDAN cultures showed an increase in retrograde transport. In order to investigate whether the in vitro phenotypes of Huntington (Htt) and ChAc are similar, some of the previous experiments were conducted in MSN differentiations of one Htt line. Cells from Htt behaved similar to ChAc lines when DNA damage response was investigated. Analysis of mitochondrial parameters showed no difference as well. However, the non-respiratory oxygen consumption was not increased and resembled wild type. When Htt neurons were investigated during live cell imaging, shortened mitochondria were found. Their number was not reduced significantly. However, a trend for reduction was observed. Mitochondria of Htt cells were more motile than ChAc or wild type lines. Mitochondrial potential was increased in Htt and comparable to ChAc. Lysosomal count showed a reduction and the area of Htt lysosomes was significantly smaller than wild type or ChAc. Lysosomes of Htt cells were more motile than their wild type or ChAc counterparts.

Vaccination has been used to control the spread of infectious diseases for centuries with widespread success. Deterministic models studying the spread of infectious disease often use the assumption of mass vaccination; however, these models do not allow for the inclusion of human behaviour. Since current vaccination campaigns are voluntary in nature, it is important to extend the study of infectious disease models to include the effects of human behaviour. To model the effects of vaccination behaviour on the spread of influenza, we examine a series of models in which individuals vaccinate according to memory or individual decision-making processes based upon self-interest. Allowing individuals to vaccinate proportionally to an exponentially decaying memory function of disease prevalence, we demonstrate the existence of a Hopf bifurcation for short memory spans. Using a game-theoretic influenza model, we determine that lowering the perceived vaccine risk may be insufficient to increase coverage to established target levels. Utilizing evolutionary game theory, we examine models with imitation dynamics both with and without a decaying memory function and show that, under certain conditions, periodic dynamics occur without seasonal forcing. Our results suggest that maintaining diseases at low prevalence with voluntary vaccination campaigns could lead to subsequent epidemics following the free-rider dilemma and that future research in disease control reliant on individual-based decision-making need to include the effects of human behaviour.

La neurofibromatose de type 1 (NF1) représente la maladie génétique autosomale dominante la plus fréquente en France, après la mucovisidose, avec une incidence de 1 individu sur 3500. Il s'agit d'une pathologie multi-systémique présentant un tableau clinique varié. Parmi la pléthore de symptôme, sont comptées des manifestations neurocutanées telles que les taches « café-au-lait » (zone d'hyperpigmentation localisée) et les neurofibromes (tumeurs bénignes de la gaine périphérique de la myéline) mais également des défauts osseux et des troubles cognitifs. La pénétrance de NF1 est complète mais la manifestation et la sévérité des symptômes peuvent varier d'un individu à l'autre. Le gène NF1 responsable de la maladie, localisé sur le chromosome 17, est un gène suppresseur de tumeur qui code pour la neurofibromine. Dans le but de développer un modèle cellulaire humain pertinent pour l'étude des défauts osseux associés à NF1, nous avons utilisé des cellules souches induites à la pluripotence porteuse de la mutation causale NF1 (hiPS-NF1). Dans ces travaux, nous avons montré qu'une perte d'expression de la neurofibromine dans les ostéoblastes dérivés de hiPS-NF1 reproduisait le phénotype d'ostéogénèse réduite et qu'il était possible d'améliorer la capacité des hiPS-NF1 à se différencier en ostéoblaste à l'aide de molécules pharmacologiques. Toujours dans le but de proposer un modèle cellulaire humain le plus relevant possible, nous avons développé des lignées isogéniques avec la technologie CRISPR/Cas 9 afin d'étudier l'impact d'une perte partielle ou totale de l'expression de la neurofibromine sur le phénotype osseux. En parallèle, afin de pouvoir étudier un autre phénotype associé à NF1, un protocole de différenciation de cellules de Schwann sous culture définie en utilisant des facteurs de croissance et des molécules de signalisation, a partir des hiPS a été développé. Des cellules de Schwann-like ont été obtenues en 30 jours en engagement la différenciation des cellules souches vers la crête neurale afin d'induire l'émergence de précurseurs de cellules de Schwann par l'action de molécules telles que le récepteur de type I du TGF-ß (SB431542), l'hereguline ß1, l'IGF1, le FGF2 et un activateur de WNT3a (CHIR99021). L'analyse par q-PCR montre une augmentation des marqueurs de différents stades de différenciation de la cellule de Schwann : crête neurale (SOX10, ERBB3), cellules de Schwann précurseurs (MPZ, CAD19) et cellules de Schwann immatures (S100) au bout de 30 jours de différenciation. Ces résultats ont été complétés par l'analyse protéique des cellules différenciées et par la mise en co-culture de ces cellules avec des motoneurones différenciés à partir d'hiPS. L'ensemble de ce travail a permis de valider la pertinence de l'utilisation des cellules souches pluripotentes porteuses de mutation dans la modélisation de pathologie génétique. Permettant à plus long terme la recherche de molécules actives par des approches de de criblage pharmacologique ou par des approches de thérapie cellulaire. / Neurofibromatosis type 1 (NF1) is an autosomal genetic disease with an incidence of 1 in 3,500 individuals. This is a multi-systemic disorder with a plethora of various symptoms. Among with we find neurocutaneous manifestations such as "café-au-lait" spots (zone of localized hyperpigmentation) and neurofibromas (benign tumors of the peripheral sheath of myelin), but also bone defects and cognitive disorders. The penetrance of NF1 is complete but the manifestation and severity of symptoms may vary from one individual to another. The NF1 gene responsible for the disease, located on chromosome 17, is a tumor suppressor gene that encodes neurofibromin. In order to develop a relevant human cellular model for the study of bone defects associated with NF1, we used pluripotency-induced stem cells that carry the NF1 causal mutation (hiPS-NF1). In this work, we have shown that loss of neurofibromin expression in osteoblasts derived from hiPS-NF1 reproduces the reduced osteogenesis phenotype. We have also shown that pharmacological molecules can improve the ability of hiPS-NF1 to differentiate in osteoblast. In order to propose the most relevant human cell model, we have developed isogenic lines with CRISPR / Cas 9 technology to study the impact of a partial or total loss of neurofibromin on the bone phenotype. Simultaneously, a Schwann cells differentiation protocol from hiPS was developed under culture defined using growth factors and signaling molecules. Schwann-like cells were obtained in 30 days by the use of molecules such as the type I receptor TGF-β (SB431542), heregulin β1, IGF1, FGF2 and activator of WNT3a (CHIR99021). The q-PCR analysis shows an increase in Schwann cell markers: neural crest (SOX10, ERBB3), precursor Schwann cells (MPZ, CAD19) and immature Schwann cells (S100). These results were confirmed by protein analysis of the differentiated cells and by the co-culture analysis of these cells with differentiated motoneurons from hiPS. All of this work validated the relevance of pluripotent stem cells in the modeling of genetic pathology.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13354号 / 論医博第2200号 / 新制||医||1044(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 長船 健二, 教授 木村 剛, 教授 羽賀 博典 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

The drug development process is lengthy and expensive, with a 90% failure rate among drugs entering clinical trials due to the inadequacy of predictive models in the initial phases of drug development. To overcome these limitations, there is a paradigm shift towards developing micro physiological systems often referred as Organ-on-a-Chip that have be shown to recapitulate organ level functions in vitro. However, despite their promise, these systems often have limited throughput, restricting their widespread use in the drug development process. The work outlined in this thesis aims to bridge this gap by integrating the physiological relevance offered by micro physiological systems with the high throughput capabilities of traditional 2D multi-well plate cultures. The thesis outlines the development of two novel micro physiological systems, engineered in a high throughput multi-well format called the IFlowPlate and AngioPlate. Both the platforms have an open-top design and unlike tradition microphysiological platforms does not need complex pump systems and have built-in connections to achieve perfusion making it more scalable and user-friendly. The IFlowPlate leverages the self-assembly capability of endothelial cells to create a perfusable vascular network. This platform technology was utilized in this work to achieve intravascular perfusion of colon organoids for the first time and demonstrated immune cell circulation and recruitment in response to injury. AngioPlate, the other platform that was developed as a part of this work, utilizes a pre-patterned scaffold completely embedded in native hydrogel matrix to guide cells in forming organ-specific geometries and tubular structures using a novel subtractive manufacturing technique. This platform allowed for fabricating complex and intricate networks to model vascularized terminal lung alveoli and renal proximal tubules. This work demonstrated for the first time that highly complex perfusable tissues embedded in hydrogel can be integrated with multi-well plates to mimic tissue specific structures and interfaces without the use of synthetic membranes or plastic channels. The built-in perfusion channel and the flexible hydrogel matrix allowed for the terminal lung alveoli model to be mechanically actuated to mimic breathing motions. The renal proximal tubule model was used to mimic glucose reabsorption in kidney and model renal inflammation. The latter part of this work focusses on further improving this platform to increase platform robustness and to allow for incorporating supporting cells such as fibroblasts into the hydrogel matrix. This allowed us to model tubular injuries in kidney such as cisplatin induced -nephrotoxicity and TGF- β1 induced- tubulointerstitial fibrosis. Furthermore this work also describes the development of a high-throughput TEER meter that can be integrated with the AngioPlate platform allowing for rapid, non-invasive measurement of renal epithelial barrier integrity. Given that both platforms are designed in a 384-well plate format, they are high throughput and compatible with existing technologies like high-content imaging systems, robotic liquid handling systems, and microplate readers allowing for widespread adoption across diverse research settings. It is anticipated that the contributions described in this work will significantly advance our understanding of disease propagation and accelerate drug development process. / Thesis / Candidate in Philosophy / Drug development is a complex and expensive process, often hindered by a high failure rate in clinical trials. This failure is partly due to the inadequacy of current predictive models in the early stages of development. To address this, researchers are turning to innovative microphysiological systems known as Organ-on-a-Chip, which mimic organ structure and functions in the lab. However, these systems have been limited in their use due to low throughput. To overcome this limitation, microphysiological systems in multi-well formats called the IFlowPlate and AngioPlate were developed through the works outlined in this thesis. These platforms are designed to be high-throughput, scalable, user-friendly, and are compatible with existing technologies, such as microplate readers, high-content imaging systems and robotic liquid handlers, making them accessible to a wide range of researchers. By combining the physiological relevance of microphysiological systems with the high-throughput capabilities, these platforms aim to transform the way we study diseases and test drugs.

Induced pluripotent stem (iPS) cells generated from patients hold great promise for studying diseases that affect the central nervous system, as differentiation into the neuronal lineage creates a limitless supply of affected cells for disease study. Rett syndrome (RTT) is a neurodevelopmental autism spectrum disorder primarily caused by mutations in the methyl-CpG-binding protein 2 (MECP2) gene. Due to the inaccessibility of patient neurons, most of what is known about underlying phenotypes has been described using mouse models. iPS cells provide a potential solution, but reprogramming of patient cells is hampered by low efficiency, and early methods of identifying iPS cells involve transgenic techniques that are not translatable to human patient samples. The first part of this thesis describes the generation and characterization of a pluripotency reporter to address this issue. The EOS lentiviral reporter allows real-time observation of pluripotency changes during reprogramming, and is a useful tool for more efficient isolation of reprogrammed cell lines. Further, the EOS selection system can be used in a disease context to reproducibly mark and maintain disease-specific iPS cell lines for future use in disease modelling. Though iPS cells have been used to study RTT in vitro, extensive assessments of neuron function and electrophysiology have not yet been performed. In the second part of this thesis, iPS cell lines generated from a RTT mouse model were tested for their ability to model disease in vitro. Directed differentiation of multiple Mecp2-deficient and wild-type iPS cell lines to glutamatergic neurons revealed neurons that lack Mecp2 have a smaller soma size, diminished sodium currents, and are less excitable, firing fewer, prolonged action potentials that are smaller in magnitude. This deficiency in intrinsic excitability was accompanied by a dysfunction at excitatory glutamatergic synapses, which together recapitulate changes previously observed in the Mecp2-deficient mouse brain. Having accumulated counts and recordings from hundreds of neurons with consistent responses among lines, the iPS cell system is a representative model of the neuronal and synaptic defects in RTT. These results illustrate the requirement of MeCP2 in normal neuronal function, and suggest altered neuronal homeostasis or aberrant network circuitry may underlie RTT pathogenesis.

Induced pluripotent stem (iPS) cells generated from patients hold great promise for studying diseases that affect the central nervous system, as differentiation into the neuronal lineage creates a limitless supply of affected cells for disease study. Rett syndrome (RTT) is a neurodevelopmental autism spectrum disorder primarily caused by mutations in the methyl-CpG-binding protein 2 (MECP2) gene. Due to the inaccessibility of patient neurons, most of what is known about underlying phenotypes has been described using mouse models. iPS cells provide a potential solution, but reprogramming of patient cells is hampered by low efficiency, and early methods of identifying iPS cells involve transgenic techniques that are not translatable to human patient samples. The first part of this thesis describes the generation and characterization of a pluripotency reporter to address this issue. The EOS lentiviral reporter allows real-time observation of pluripotency changes during reprogramming, and is a useful tool for more efficient isolation of reprogrammed cell lines. Further, the EOS selection system can be used in a disease context to reproducibly mark and maintain disease-specific iPS cell lines for future use in disease modelling. Though iPS cells have been used to study RTT in vitro, extensive assessments of neuron function and electrophysiology have not yet been performed. In the second part of this thesis, iPS cell lines generated from a RTT mouse model were tested for their ability to model disease in vitro. Directed differentiation of multiple Mecp2-deficient and wild-type iPS cell lines to glutamatergic neurons revealed neurons that lack Mecp2 have a smaller soma size, diminished sodium currents, and are less excitable, firing fewer, prolonged action potentials that are smaller in magnitude. This deficiency in intrinsic excitability was accompanied by a dysfunction at excitatory glutamatergic synapses, which together recapitulate changes previously observed in the Mecp2-deficient mouse brain. Having accumulated counts and recordings from hundreds of neurons with consistent responses among lines, the iPS cell system is a representative model of the neuronal and synaptic defects in RTT. These results illustrate the requirement of MeCP2 in normal neuronal function, and suggest altered neuronal homeostasis or aberrant network circuitry may underlie RTT pathogenesis.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第22700号 / 医科博第115号 / 新制||医科||8(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 近藤 玄, 教授 齊藤 博英 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Chorea Acanthocytosis (ChAc) is an autosomal recessive inherited disease caused by loss- of-function mutation in the VPS13A gene which encodes CHOREIN protein. This study used induced pluripotent stem cells (iPSCs) as well as neural progenitor cells (NPCs) to generate medium spiny neurons (MSN) as well as midbrain dopaminergic neurons (mDAN). The first objective of this thesis was to generate and characterize a stem cell based disease model of ChAc. The second objective was to establish two different differentiation protocols that yield different neuronal sub types that are affected in ChAc, and compare whether they harbor similar phenotypes and whether the faster protocol can be used to model the disease accurately. The generated iPSCs were characterized using AP staining as an early marker for reprogramming, qPCR for analysis of residual expression of exogenous transcription factors, immunocytochemistry (ICC) for staining of pluripotency markers as well as markers for mesoderm, ectoderm and endoderm formation upon three germ layer formation. Karyotyping was conducted to exclude aberrant clones. Western blot using CHOREIN antibody revealed that the cell lines retained their disease identity. There were no differences observed between wild type and ChAc lines in stem cell and neuron populations in either protocol. qPCR analysis, investigating the expression of previously described markers for characterization, revealed no significant clustering between wild type and ChAc lines in either protocol. A disturbed ratio of globular and filamentous actin is causative for the aberrant shape of ChAc erythrocytes. Investigation of the ratio in mature neurons revealed a significant reduction of this ratio in MSN but no difference in mDAN cultures. When the ratio of cytosolic and filamentous tubulin and the acetylation of tubulin were investigated, no differences were found between wild type and ChAc lines. Mature neurons of both differentiation protocols were subjected to treatment with the proteotoxic stress inducer L-canavanine and the unfolded protein response (UPR) inducer tunicamycin. Survival was analyzed with the PrestoBlue assay as well as lactate dehydroxylase (LDH) release assay. Both cultures of mature neurons showed an increased susceptibility to the respective drugs. Furthermore the data suggests that MSN cultures are more vulnerable against proteotoxic stress (L-canavanine). Kinetics of tunicamycin poisoning were not different within MSN cultures but indicated a late cell death of ChAc lines under mDAN differentiation conditions. DNA damage plays a major role in the progression of neurodegenerative diseases. The amount of double strand breaks (DSB) was assessed in mature cultures of MSN and mDAN differentiations. There was no difference in basal level of DSB. When etoposide was applied to induce DNA damage, increased susceptibility of ChAc lines was observed. Albeit significant, the effect size was very small. Seahorse was used to characterize energy metabolism. Glycolysis was not impaired in ChAc lines in either protocol. Furthermore, MSN differentiation showed no difference in any parameter related to oxidative phosphorylation, while under mDAN conditions, coupling efficiency and spare respiratory capacity was increased for ChAc lines. The non-respiratory oxygen consumption was increased in ChAc lines in MSN cultures but decreased in mDAN cultures. The yeast homolog of VPS13A interacts with vesicle and mitochondrial membranes. Therefore, this study focuses on vesicle and mitochondria homeostasis. Live cell imaging of mature neurons of MSN differentiations revealed a decreased amount and reduced motility of mitochondria. Even though mitochondria were normally shaped their size was reduced. mDAN differentiations harbored a reduced amount and shortened mitochondria. These mitochondria, however, showed an increased motility. When analyzing aligned mature neurons in microfluidic chambers (MFCs), a strong phenotype was already observed in proximal regions, which resembled the distal parts of the channels. Hence, the dysregulation, that occurs distal in healthy controls, happens closer to the soma in diseased cells. The mitochondria potential marker JC-1 showed a hyperpolarization of mitochondria in MSN culture and a depolarization in mDAN cultures. When investigated in MFCs of mDAN cultures, there was a significant increase in potential observed at the distal position of ChAc lines, while wild type cultures showed no difference. Experiments conducted on the lysosomal compartments showed a decrease in proximal parts of ChAc MSN cultures when compared to wild type. Their shape was altered as well. mDAN cultures featured no significant morphological changes. Trafficking analysis revealed an increase in motility in MSN cultures but a decrease in mDAN cultures. When lysosomes were analyzed in MFCs only mDAN cultures showed an increase in retrograde transport. In order to investigate whether the in vitro phenotypes of Huntington (Htt) and ChAc are similar, some of the previous experiments were conducted in MSN differentiations of one Htt line. Cells from Htt behaved similar to ChAc lines when DNA damage response was investigated. Analysis of mitochondrial parameters showed no difference as well. However, the non-respiratory oxygen consumption was not increased and resembled wild type. When Htt neurons were investigated during live cell imaging, shortened mitochondria were found. Their number was not reduced significantly. However, a trend for reduction was observed. Mitochondria of Htt cells were more motile than ChAc or wild type lines. Mitochondrial potential was increased in Htt and comparable to ChAc. Lysosomal count showed a reduction and the area of Htt lysosomes was significantly smaller than wild type or ChAc. Lysosomes of Htt cells were more motile than their wild type or ChAc counterparts.:List of abbreviations Introduction 1. Neurodegenerative diseases 1.1. Chorea-acanthocytosis – a clinical overview 1.2. Chorea-Acanthocytosis – genetic considerations 2. Disease modelling 2.1. Human disease models 2.2. Induced pluripotent stem cells 2.3. Multipotent neuronal progenitor cells 3. Objectives of this thesis Materials & Methods 1. Cell culture procedures 1.1. Coating 1.2. Matrigel 1.3. PLO/laminin 1.4. Gelatin coating 1.5. Mouse embryonic fibroblast isolation 1.6. Generation of feeder cells 1.7. Human fibroblast culture 1.8. Reprogramming 1.9. iPSC culture 1.10. Culture of small molecule neuronal precursor cells (smNPC) 1.11. MSN differentiation 1.12. mDAN differentiation 2. Nucleic acid biochemistry 2.1. mRNA isolation 2.2. cDNA generation 2.3. Polymerase chain reaction (PCR) 2.4. Agarose gel electrophoresis 3. Cell survival analysis 3.1. PrestoBlue cell viability assay 3.2. Cytotoxicity detection kit: 3.3. DNA damage analysis 4. Metabolic characterization 5. Protein biochemistry 5.1. Alkaline phosphatase staining 5.2. Preparation of immunocytochemistry samples 5.3. Isolation of globular and filamentous actin 5.4. Whole cell protein Isolation 5.5. Cytosolic protein isolation 5.6. Protein concentration measurement 5.7. Western blot 6. Live cell imaging 7. Statistics Results 1. Generation of induced pluripotent stem cells 1.1. Silencing of exogenous transcription factors 1.2. Karyotyping of iPSC clones 1.3. Evaluation of pluripotency 1.4. Alkaline phosphatase staining 1.5. Staining of pluripotency markers 1.6. Three germ layer formation 1.7. Confirmation of ChAc phenotype by CHOREIN western blot 2. Characterization of differentiation potential 2.1. Differentiation efficiency 2.2. Characterization by qPCR 2.3. Ratio of polymerized and unpolymerized cytoskeleton proteins 2.4. Cell survival upon stress induction 2.5. DNA damage in mature neurons 2.6. Characterization of metabolism 3. Live cell imaging 3.1. Mitochondrial dynamics 3.1.1. Morphological analysis 3.1.1.1. Undirected neurons (96 well plate format) 3.1.1.2. Microfluidic chambers 3.1.2. Trafficking analysis 3.1.2.1. 96 well 3.1.2.2. Microfluidic chambers 3.1.3. JC-1 3.1.3.1. 96 well 3.1.3.2. Microfluidic chambers 3.2. Lysosomal dynamics 3.2.1. Morphological analysis 3.2.1.1. 96 well 3.2.1.2. Microfluidic chambers 3.2.2. Trafficking 3.2.2.1. 96 well 3.2.2.2. Microfluidic chambers 4. Comparison with Huntington’s disease 4.1. DNA damage 4.2. Characterization of metabolism 4.3. Live cell imaging 4.3.1. Mitochondria 4.3.1.1. Morphological analysis 4.3.1.2. Trafficking 4.3.1.3. JC-1 4.3.2. Lysosomes 4.3.2.1. Morphological analysis 4.3.2.2. Trafficking Discussion 1. Characterization of ChAc lines 1.1. ChAc stem cell lines show no impaired differentiation potential 1.2. Neurons from MSN differentiation have an altered G/F actin ratio 1.3. Mature neurons from ChAc lines are susceptible to UPR, proteotoxicity and DNA damage 1.4. ChAc neurons are not susceptible to DNA damage 1.5. Energy dynamics in ChAc and Huntington lines feature a shift to glycolysis 2. Live cell imaging of ChAc lines 2.1. Video analysis is reproducible and sensitive 2.2. ChAc lines have altered mitochondria shape and trafficking 2.3. Treatments are not selective on ChAc lines mitochondria 2.4. Mitochondrial potential is altered in ChAc lines 2.5. ChAc lysosomes feature normal morphology but altered trafficking 2.6. Lysosomes of MSN cultures respond poorly to treatments 3. MSN and mDAN differentiation highlight different aspects of the disease References List of figures List of tables Acknowledgments Appendix