京都大学 / 新制・課程博士 / 博士(医科学) / 甲第24843号 / 医科博第151号 / 新制||医科||10(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 齋藤, 潤, 教授 遊佐, 宏介, 教授 松田, 秀一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

As células iPS surgiram com a promessa de contornar as limitações das células-tronco embrionárias, como questões éticas, segurança, compatibilidade e disponibilidade. Essas células podem ser obtidas a partir de células somáticas de indivíduos normais ou de pacientes com doenças genéticas, fazendo destas uma importante ferramenta para o screening de drogas, modelos de doenças e testes toxicológicos. Grandes avanços ocorreram na reprogramação de células diferenciadas pela expressão forçada de fatores de transcrição (FT), principalmente, através de vetores lentivirais (VL), que proporcionam uma reprogramação eficiente. Entretanto, a inserção lentiviral no genoma humano e sua influência na reprogramação é pouco conhecida. Neste trabalho, avaliamos o perfil de inserção dos VL utilizados na geração de iPS. As iPS foram geradas e caracterizadas por nosso grupo a partir de fibroblastos humanos transduzidos com VL contendo 3 FT [SOX2, TCL-1A e C-MYC (célula TSM)], e de células mesenquimais derivadas de tecido adiposo com um vetor lentiviral policistrônico contendo 4 FT [OCT4, SOX2, KLF4 e C-MYC (iPS 4FT)]. Cinco colônias isoladas de cada iPS foram mapeadas e analisadas quanto aos sítios de inserção pela técnica de LM-PCR. O DNA genômico digerido foi amplificado com um primer específico para o LTR viral e outro para um linker sintético. Os produtos foram clonados, sequenciados, e analisados em bancos de dados para identificar similaridades com o genoma humano, entre outras análises. Na célula TSM, 176 sequências, obtidas com a técnica de LM-PCR, apresentaram identidade com o genoma humano, sendo que cerca de 50% ocorreram em regiões gênicas com 94% destas em introns. Já nas iPS 4FT, 251 sequências apresentaram identidade, com cerca de 45% atingindo genes, 92% destas em introns. As inserções distribuíram-se por todos os cromossomos, com preferência pelos cromossomos 16, 17 e 20 para a TSM e pelos cromossomos 11, 15 e 17 para a iPS 4FT. Analisamos a distância da inserção ao sítio de início de transcrição (TSS), e inserções próximas a ilhas CpG, que em geral correspondem a regiões regulatórias. A maior proporção de inserção ocorreu a partir de ±30Kb de distância desses sítios. Os sítios frágeis e as regiões repetitivas do genoma foram atingidas, mas com uma frequência baixa. Os resultados mostraram uma preferência de inserção lentiviral por regiões gênicas nas iPS, indicando a possível participação de proteínas como LEDGF/p75 na integração nas células estudadas. Este trabalho mostrou que o local da integração pode contribuir para a reprogramação e, apesar de possíveis efeitos negativos das integrações, estas as células iPS ainda são uma ferramenta importante para estudos in vitro. E identificar fatores que influenciem a seleção do sítio de inserção é importante para determinar regiões cromossômicas \"seguras\" para a integração, aumentando a segurança no uso clínico. / The induced pluripotent stem (iPS) cells came with the promise of circumvent some of the limitations in the use of embryonic stem cells, like ethical issues, biological safety, immune compatibility and availability. This cells can be generated from somatic cells of normal individuals or from patients with some genetic disease, making then an important tool for drug screening, construction of disease models and toxicological trials. Great advances have happened in reprogramming differentiated cells through the forced exogenous expression of transcription factors (TF), mostly by lentiviral vectors (LV), which provide an efficient reprogramming. However, the lentiviral insertion in the human genome and its influence in reprogramming is not well known. In this work, we evaluate the insertion profile of LV used to generate human iPS cells. The iPS cells were generated, by our group, from human fibroblasts transduced by LV containing 3 TF [SOX2, TCL-1A and C-MYC (TSM reprogrammed cell)], and from mesenchymal cells derived from human adipose tissue transduced by a polycistronic LV containing 4 TF [OCT4, SOX2, KLF4 and C-MYC (iPS 4TF)]. Five isolated colonies of each iPS cell were mapped and analyzed for the insertion sites through LM-PCR technique. The digested genomic DNA was amplified with a primer for the viral LTR e another for a synthetic linker. The products were cloned, sequenced and analyzed in database to identify similarities with the human genome, among other analyzes. In TSM cell, 176 sequences, derived from the LM-PCR technique, presented identity with the human genome, and about 50% of those occurred in genic regions with 94% in introns. In iPS 4TF, 251 sequences showed identity, with about 45% reaching genes, 92% of these in introns. The insertions were distributed on all chromosomes, with preference for the 16, 17 and 20 for the TSM cell, and for the 11, 15 and 17 for the iPS 4TF. We analyzed the distance of the insertion from de transcription start site, and insertions near CpG islands, which, overall, correspond to regulatory regions. The highest proportion of insertion occurred starting ±30Kb distance from these sites. The fragile sites and the repetitive regions of the genome were also reached, but with low frequency. The results showed a preference of lentiviral insertion for genic regions in iPS, indicating the potential participation of proteins like LEDGF/p75 in integration in the cells of this work. This work shows that the integration site may contribute to the reprogramming, and, despite possible negative effects of integration, these iPS cells are still an important tool for in vitro studies. Identify factors that influence the selection of insertion site is important for determination of \"safe\" chromosomal regions for the integration, increasing the safe in clinical use.

Type I diabetes (T1D) is caused by autoimmune destruction of pancreatic β-cells. Immediate consequences of T1D are severe weight loss, ketoacidosis and death unless insulin is administered. The long-term consequences of T1D are dysregulation of metabolism leading to cardiovascular complications, neuropathy and kidney insufficiency. It is estimated that 3 million Americans have T1D, and its prevalence among young individuals is progressively rising. Islet transplantation is the most effective way to treat T1D. Unfortunately, there is a chronic shortage of cadaveric organ donors to treat all of the patients on the waiting list. Thus, an alternative source of insulin producing cells (IPCs) could significantly improve patient treatment. Our lab seeks to establish human induced pluripotent stem (iPS) cells as a novel source of IPCs that are patient tailored. The aim of this thesis was to 1) compare the differentiation of T1D and nondiabetic (ND) patient-derived iPS cells into IPCs, and 2) devise an effective protocol for differentiating skin fibroblast-derived T1D iPS cells into functional, glucose-responsive IPCs. Initially, T1D iPS cells were differentiated into IPCs. However, the yield was very poor. We hypothesized that epigenetic barriers were prevalent in T1D iPS cells, limiting their differentiation into IPCs. To address this problem, we utilized 5-aza-2’-deoxycytidine (5-aza-DC), a potent demethylating agent that inhibits the DNA methyltransferase (Dnmt). We reasoned that the use of a demethylation agent might induce a more labile, permissive state, allowing for greater cell responses to differentiation stimuli. Typically, after the differentiation of T1D iPS cells, several cell cluster types are obtained, namely compact cell clusters and hollow cysts. 5-aza-DC treatment appeared to convert all of the cell clusters into characteristic islet-like compact structures. In contrast, in untreated T1D IPC cultures, we observed the dominant presence of many hollow cysts with only a few tight spheroids. The hollow cysts stained negative for insulin whereas the rare solid spheroids highly expressed insulin. Flow cytometry analysis indicated a much greater percentage of Pdx1+ and insulin+ cells in 5-Aza-DC-treated cultures. These cells express markers typical of pancreatic β-cells, possessed insulin granules in similar quantities as islets, and were glucose-responsive. When transplanted in immunodeficient mice that had developed streptozotozin-induced diabetes, there was a dramatic decrease of hyperglycemia within 28 days. These mice effectively managed glucose challenge by recovering to normoglycemia, whereas nontransplanted mice did not. Altogether, our data for the first time reveal a very high yield of functional IPCs derived from human iPS cells derived from a patient with T1D, which presents a novel alternative source of IPCs that could be used to treat T1D.

No description available.

Despite being the leading cause of neurodegeneration and dementia in the aging brain, the cause of Alzheimer's disease (AD) remains unknown in most patients. The terminal pathological hallmarks of abnormal protein aggregation and neuronal cell death are well-known from the post-mortem brain tissue of Alzheimer's disease patients, but research into the earliest stages of disease development is hindered by limited model systems. In this thesis, an in vitro human neuronal system was derived from induced pluripotent stem (iPS) cell lines reprogrammed from dermal fibroblasts of AD patients and age-matched controls. This allows us to investigate the cellular mechanisms of AD neurodegeneration in the human neurons of sporadic AD (SAD) patients, whose development of the disease cannot be explained by our current understanding of AD. We show that neural progenitors and neurons derived from SAD patients show an unexpected expression profile of enhanced neuronal gene expression resulting in premature differentiation in the SAD neuronal cells. This difference is accompanied by the decreased binding of the repressor element 1-silencing transcription/neuron-restrictive silencer factor (REST/NRSF) transcriptional inhibitor of neuronal differentiation in the SAD neuronal cells. The SAD neuronal cells also have increased production of \(amyloid-\beta\) and higher levels of tau protein, the main components of the plaques and tangles in the AD brain.

As células iPS surgiram com a promessa de contornar as limitações das células-tronco embrionárias, como questões éticas, segurança, compatibilidade e disponibilidade. Essas células podem ser obtidas a partir de células somáticas de indivíduos normais ou de pacientes com doenças genéticas, fazendo destas uma importante ferramenta para o screening de drogas, modelos de doenças e testes toxicológicos. Grandes avanços ocorreram na reprogramação de células diferenciadas pela expressão forçada de fatores de transcrição (FT), principalmente, através de vetores lentivirais (VL), que proporcionam uma reprogramação eficiente. Entretanto, a inserção lentiviral no genoma humano e sua influência na reprogramação é pouco conhecida. Neste trabalho, avaliamos o perfil de inserção dos VL utilizados na geração de iPS. As iPS foram geradas e caracterizadas por nosso grupo a partir de fibroblastos humanos transduzidos com VL contendo 3 FT [SOX2, TCL-1A e C-MYC (célula TSM)], e de células mesenquimais derivadas de tecido adiposo com um vetor lentiviral policistrônico contendo 4 FT [OCT4, SOX2, KLF4 e C-MYC (iPS 4FT)]. Cinco colônias isoladas de cada iPS foram mapeadas e analisadas quanto aos sítios de inserção pela técnica de LM-PCR. O DNA genômico digerido foi amplificado com um primer específico para o LTR viral e outro para um linker sintético. Os produtos foram clonados, sequenciados, e analisados em bancos de dados para identificar similaridades com o genoma humano, entre outras análises. Na célula TSM, 176 sequências, obtidas com a técnica de LM-PCR, apresentaram identidade com o genoma humano, sendo que cerca de 50% ocorreram em regiões gênicas com 94% destas em introns. Já nas iPS 4FT, 251 sequências apresentaram identidade, com cerca de 45% atingindo genes, 92% destas em introns. As inserções distribuíram-se por todos os cromossomos, com preferência pelos cromossomos 16, 17 e 20 para a TSM e pelos cromossomos 11, 15 e 17 para a iPS 4FT. Analisamos a distância da inserção ao sítio de início de transcrição (TSS), e inserções próximas a ilhas CpG, que em geral correspondem a regiões regulatórias. A maior proporção de inserção ocorreu a partir de ±30Kb de distância desses sítios. Os sítios frágeis e as regiões repetitivas do genoma foram atingidas, mas com uma frequência baixa. Os resultados mostraram uma preferência de inserção lentiviral por regiões gênicas nas iPS, indicando a possível participação de proteínas como LEDGF/p75 na integração nas células estudadas. Este trabalho mostrou que o local da integração pode contribuir para a reprogramação e, apesar de possíveis efeitos negativos das integrações, estas as células iPS ainda são uma ferramenta importante para estudos in vitro. E identificar fatores que influenciem a seleção do sítio de inserção é importante para determinar regiões cromossômicas \"seguras\" para a integração, aumentando a segurança no uso clínico. / The induced pluripotent stem (iPS) cells came with the promise of circumvent some of the limitations in the use of embryonic stem cells, like ethical issues, biological safety, immune compatibility and availability. This cells can be generated from somatic cells of normal individuals or from patients with some genetic disease, making then an important tool for drug screening, construction of disease models and toxicological trials. Great advances have happened in reprogramming differentiated cells through the forced exogenous expression of transcription factors (TF), mostly by lentiviral vectors (LV), which provide an efficient reprogramming. However, the lentiviral insertion in the human genome and its influence in reprogramming is not well known. In this work, we evaluate the insertion profile of LV used to generate human iPS cells. The iPS cells were generated, by our group, from human fibroblasts transduced by LV containing 3 TF [SOX2, TCL-1A and C-MYC (TSM reprogrammed cell)], and from mesenchymal cells derived from human adipose tissue transduced by a polycistronic LV containing 4 TF [OCT4, SOX2, KLF4 and C-MYC (iPS 4TF)]. Five isolated colonies of each iPS cell were mapped and analyzed for the insertion sites through LM-PCR technique. The digested genomic DNA was amplified with a primer for the viral LTR e another for a synthetic linker. The products were cloned, sequenced and analyzed in database to identify similarities with the human genome, among other analyzes. In TSM cell, 176 sequences, derived from the LM-PCR technique, presented identity with the human genome, and about 50% of those occurred in genic regions with 94% in introns. In iPS 4TF, 251 sequences showed identity, with about 45% reaching genes, 92% of these in introns. The insertions were distributed on all chromosomes, with preference for the 16, 17 and 20 for the TSM cell, and for the 11, 15 and 17 for the iPS 4TF. We analyzed the distance of the insertion from de transcription start site, and insertions near CpG islands, which, overall, correspond to regulatory regions. The highest proportion of insertion occurred starting ±30Kb distance from these sites. The fragile sites and the repetitive regions of the genome were also reached, but with low frequency. The results showed a preference of lentiviral insertion for genic regions in iPS, indicating the potential participation of proteins like LEDGF/p75 in integration in the cells of this work. This work shows that the integration site may contribute to the reprogramming, and, despite possible negative effects of integration, these iPS cells are still an important tool for in vitro studies. Identify factors that influence the selection of insertion site is important for determination of \"safe\" chromosomal regions for the integration, increasing the safe in clinical use.

Os vírus são eficazes na transferência de genes em células devido aos seus mecanismos especializados. No entanto, vírus como veículos de entrega de genes podem acarretar em problemas, particularmente quando proposto para reprogramar células somáticas em células-tronco pluripotentes induzidas (iPS) visando utilização terapêutica. No presente estudo, procurou-se desenvolver um sistema alternativo para entregar diretamente proteínas nucleares (Oct4, Sox2, KLF4, e c-Myc) fusionadas com o domínio de transdução de proteína TAT, para promover a reprogramação de fibroblastos embrionários de camundongos (MEF) ou células mesenquimais derivadas de tecido adiposo humano (hASC) em células iPS. Primeiramente o PTD TAT ou TAT- foi fundido a proteína verde fluorescente (GFP) como modelo para prova de princípio e padronização detalhada. Inesperadamente, TAT-GFP produzido e secretado pelas células NIH-3T3 produtora não foi capaz de ser detectado no meio de cultura por verificação quantitativa fluorimétrica, nem foi capaz de ser detectada em células-alvo, por citometria de fluxo, depois de co-cultura em transwells. Essa observação pode ser explicada por: (1) ineficiência desse tipo de célula em secretar proteínas e (2) falta de resistência à clivagem por endoproteases furinas. Para contornar esses fatores limitantes usou-se citometria de fluxo para avaliar as melhores condições para a transfecção por seis diferentes tipos de células (CHO, NIH-3T3, HT1080, HEK-293A, HEK-293t e COS-7) com TAT (modificada para ser resistente à furinas) fundido a GFP. Células 293t-TAT-GFP exibiram a maior eficiência de transfecção e também de secreção. O mesmo pôde ser observado para as seis linhagens celulares expressando fatores de transcrição nucleares TAT, determinados por ELISA. Em seguida, diferentes estratégias de entrega foram testadas. A primeira foi baseada na co-cultura de uma mistura de células produtoras com MEF ou hASC. No entanto, não foi possível observar a reprogramação devido à morte celular. A segunda foi baseada na concentração de meio condicionado de cultura de células por centrifugação usando colunas Amicon, trocando o meio a cada 24h, em quatro ciclos. No entanto, apesar da presença de algumas colônias após 20-30 dias, nenhuma colônia verdadeira iPS foi obtida. Na sequência, as células foram tratadas com cada proteína de forma independente, e as demais foram substituídas pelo retrovírus correspondente, trocando meio a cada 72h, em quatro ciclos. Essa estratégia, apesar de permitir verificar a função de cada proteína, também não resultou em reprogramação. Este achado pode ser explicado pela diferenciação celular induzida por BCS, que também é concentrado no processo. Assim, passou-se a adaptação de \"células produtoras\" em condições de cultura livre de soro, para enriquecer a produção dos fatores nucleares individuais, necessários para a reprogramação. A otimização sistematizada deste processo está sendo realizada em parceria com o IPT e deve resultar em quantidades de proteína de fusão suficientes para o teste final da hipótese proposta. Em conjunto, são apresentados os dados da geração de linhagens celulares expressando estavelmente os vários fatores de transcrição e estratégias para melhorar a eficiência necessária para a produção iPS. Esta nova estratégia garante uma produção eficiente de TAT fundida a fatores nucleares de reprogramação e sua eficácia para promover a reprogramação de células somáticas de maneira livre de vírus merece ser investigado futuramente / Viruses are effective at transferring genes into cells by its specialized mechanisms. However, viruses as gene delivery vehicles entail problems, particularly when proposed to reprogram somatic cells into induced pluripotent stem cells (iPS) for therapeutic uses. In the present study, we aimed to develop an alternative system for directly delivering nuclear proteins (Oct4, Sox2, Klf4, and c-Myc) fused with TAT protein transduction domain to promote reprogramming of mouse embryonic fibroblasts (MEF) or human adipose tissue derived mesenchymal cells (hASC) into iPS cells. First TAT- or TAT- PTD was fused to green fluorescent protein (GFP) as a proof of principle model and for detailed standardization. Unexpectedly, TAT-GFP produced and secreted by NIH-3T3 producer cells was not detected in the culture medium by quantitative fluorimetric verification, nor detected on target cells, by flow cytometry, after being co-cultured using transwells. This observation maybe explained by: (1) inefficiency of this cell type to be transfected and to secrete proteins and (2) lack of resistance to furin endoproteases cleavage on Golgi of TAT sequence. To circumvent these limiting factors we used flow cytometer to assess the best conditions for transfection in six different cell types (CHO, NIH-3T3, HT1080, HEK-293A, HEK-293t and COS-7) with TAT- (a modified PTD to be resistant to furin endoproteases) fused to GFP. 293t-TAT-GFP cells displayed the highest transfection efficiency and secretion levels. The same could be observed for the six cell lineages expressing TAT- nuclear transcription factors, determined by ELISA.Next, different delivery strategies were tested for TAT- nuclear transcription factor system. Co-culturing a mix of producer cells with MEF or hASC resulted in not reprogramming and this was associated with cell death. The second was based on the use of microconcentrated conditioned cell culture medium, changed every 24h, in four cycles. However, despite the presence of some emerging colonies after 20-30 days, no true iPS colonies were obtained. Then, cells were treated with each protein independently, and the others were replaced by the corresponding retrovirus, changing cell medium every 72h, in four cycles. We verified the reprogramming potential of each protein, but no true colonies were obtained.One possibility for this finding is that BCS is also concentrated by centrifugation and may induce cell differentiation. To circumvent these problems, we have started the adaptation of producer cells in a serum-free culture condition to enrich the production of the individual factors required for reprogramming. This optimization process is taking place in collaboration with the IPT and shall result in large amounts of the fusion protein to finally test the proposed hypothesis. Altogether, we presented the generation of several cell lines stably expressing the transcription factors and strategies to improve the efficiency required for iPS production. This novel strategy guarantees efficient production of TAT-fused reprogramming nuclear factors and its efficacy to promote somatic cells reprogramming in a virus-free manner deserves to be further investigated

Os vírus são eficazes na transferência de genes em células devido aos seus mecanismos especializados. No entanto, vírus como veículos de entrega de genes podem acarretar em problemas, particularmente quando proposto para reprogramar células somáticas em células-tronco pluripotentes induzidas (iPS) visando utilização terapêutica. No presente estudo, procurou-se desenvolver um sistema alternativo para entregar diretamente proteínas nucleares (Oct4, Sox2, KLF4, e c-Myc) fusionadas com o domínio de transdução de proteína TAT, para promover a reprogramação de fibroblastos embrionários de camundongos (MEF) ou células mesenquimais derivadas de tecido adiposo humano (hASC) em células iPS. Primeiramente o PTD TAT ou TAT- foi fundido a proteína verde fluorescente (GFP) como modelo para prova de princípio e padronização detalhada. Inesperadamente, TAT-GFP produzido e secretado pelas células NIH-3T3 produtora não foi capaz de ser detectado no meio de cultura por verificação quantitativa fluorimétrica, nem foi capaz de ser detectada em células-alvo, por citometria de fluxo, depois de co-cultura em transwells. Essa observação pode ser explicada por: (1) ineficiência desse tipo de célula em secretar proteínas e (2) falta de resistência à clivagem por endoproteases furinas. Para contornar esses fatores limitantes usou-se citometria de fluxo para avaliar as melhores condições para a transfecção por seis diferentes tipos de células (CHO, NIH-3T3, HT1080, HEK-293A, HEK-293t e COS-7) com TAT (modificada para ser resistente à furinas) fundido a GFP. Células 293t-TAT-GFP exibiram a maior eficiência de transfecção e também de secreção. O mesmo pôde ser observado para as seis linhagens celulares expressando fatores de transcrição nucleares TAT, determinados por ELISA. Em seguida, diferentes estratégias de entrega foram testadas. A primeira foi baseada na co-cultura de uma mistura de células produtoras com MEF ou hASC. No entanto, não foi possível observar a reprogramação devido à morte celular. A segunda foi baseada na concentração de meio condicionado de cultura de células por centrifugação usando colunas Amicon, trocando o meio a cada 24h, em quatro ciclos. No entanto, apesar da presença de algumas colônias após 20-30 dias, nenhuma colônia verdadeira iPS foi obtida. Na sequência, as células foram tratadas com cada proteína de forma independente, e as demais foram substituídas pelo retrovírus correspondente, trocando meio a cada 72h, em quatro ciclos. Essa estratégia, apesar de permitir verificar a função de cada proteína, também não resultou em reprogramação. Este achado pode ser explicado pela diferenciação celular induzida por BCS, que também é concentrado no processo. Assim, passou-se a adaptação de \"células produtoras\" em condições de cultura livre de soro, para enriquecer a produção dos fatores nucleares individuais, necessários para a reprogramação. A otimização sistematizada deste processo está sendo realizada em parceria com o IPT e deve resultar em quantidades de proteína de fusão suficientes para o teste final da hipótese proposta. Em conjunto, são apresentados os dados da geração de linhagens celulares expressando estavelmente os vários fatores de transcrição e estratégias para melhorar a eficiência necessária para a produção iPS. Esta nova estratégia garante uma produção eficiente de TAT fundida a fatores nucleares de reprogramação e sua eficácia para promover a reprogramação de células somáticas de maneira livre de vírus merece ser investigado futuramente / Viruses are effective at transferring genes into cells by its specialized mechanisms. However, viruses as gene delivery vehicles entail problems, particularly when proposed to reprogram somatic cells into induced pluripotent stem cells (iPS) for therapeutic uses. In the present study, we aimed to develop an alternative system for directly delivering nuclear proteins (Oct4, Sox2, Klf4, and c-Myc) fused with TAT protein transduction domain to promote reprogramming of mouse embryonic fibroblasts (MEF) or human adipose tissue derived mesenchymal cells (hASC) into iPS cells. First TAT- or TAT- PTD was fused to green fluorescent protein (GFP) as a proof of principle model and for detailed standardization. Unexpectedly, TAT-GFP produced and secreted by NIH-3T3 producer cells was not detected in the culture medium by quantitative fluorimetric verification, nor detected on target cells, by flow cytometry, after being co-cultured using transwells. This observation maybe explained by: (1) inefficiency of this cell type to be transfected and to secrete proteins and (2) lack of resistance to furin endoproteases cleavage on Golgi of TAT sequence. To circumvent these limiting factors we used flow cytometer to assess the best conditions for transfection in six different cell types (CHO, NIH-3T3, HT1080, HEK-293A, HEK-293t and COS-7) with TAT- (a modified PTD to be resistant to furin endoproteases) fused to GFP. 293t-TAT-GFP cells displayed the highest transfection efficiency and secretion levels. The same could be observed for the six cell lineages expressing TAT- nuclear transcription factors, determined by ELISA.Next, different delivery strategies were tested for TAT- nuclear transcription factor system. Co-culturing a mix of producer cells with MEF or hASC resulted in not reprogramming and this was associated with cell death. The second was based on the use of microconcentrated conditioned cell culture medium, changed every 24h, in four cycles. However, despite the presence of some emerging colonies after 20-30 days, no true iPS colonies were obtained. Then, cells were treated with each protein independently, and the others were replaced by the corresponding retrovirus, changing cell medium every 72h, in four cycles. We verified the reprogramming potential of each protein, but no true colonies were obtained.One possibility for this finding is that BCS is also concentrated by centrifugation and may induce cell differentiation. To circumvent these problems, we have started the adaptation of producer cells in a serum-free culture condition to enrich the production of the individual factors required for reprogramming. This optimization process is taking place in collaboration with the IPT and shall result in large amounts of the fusion protein to finally test the proposed hypothesis. Altogether, we presented the generation of several cell lines stably expressing the transcription factors and strategies to improve the efficiency required for iPS production. This novel strategy guarantees efficient production of TAT-fused reprogramming nuclear factors and its efficacy to promote somatic cells reprogramming in a virus-free manner deserves to be further investigated

A striking feature of Parkinson's disease (PD) is that the distal axonal terminals of neurons degenerate prior to the soma, a process referred to as 'dying-back'. Another hallmark of the disease is the pathological accumulation of abnormal protein aggregates in soma and axons. Lysosomes, a critical component of the protein quality control machinery, have thus been thought to be altered in PD. LRRK2 G2019S, a gain-of-kinase-function mutation, is one of PD's most common known causative mutations, and LRRK2-specific small molecule inhibitors have been developed as possible therapeutics. However, LRRK2 G2019S is incompletely penetrant, and its role in axonal degeneration is unclear. LRRK2 phosphorylates a subset of Rab GTPases, including Rab10. Since Rab GTPases are mediators of organelle trafficking, we speculated that LRRK2 G2019S affects the transport of organelles, such as lysosomes, thereby contributing to early PD pathogenesis. Using neural progenitor cell-derived neurons from two LRRK2 G2019S-PD patients; we developed a model of axonal trafficking of lysosomes to characterize the impact of mutant LRRK2 on lysosomal trafficking. In comparison to their isogenic gene-corrected controls, we observed a subtle reduction in mutant axonal lysosomal speed, which could indicate that mutant LRRK2 mildly disrupts retrograde lysosomal transport. We also observed that this trafficking phenotype was only partially rescued by LRRK2 kinase inhibitors, which could indicate the importance of other factors regulating axonal transport. Consistent with this idea, we found that mutant LRRK2 was associated with increased co-localization of phosphorylated Rab10 on a small subset of distal axonal lysosomes. Furthermore, the over-expression of Rab10 only mildly affected lysosomal trafficking in axons. Interestingly, damaging the lysosomal membrane increased LRRK2-dependent Rab10 phosphorylation, leading us to speculate that membrane damage in the axon might induce LRRK2 activity. Since lysosomes have been shown to mediate plasma membrane repair, we speculated that membrane damage might exacerbate LRRK2-dependent phenotypes in distal axons. Axotomy was used to test this idea, and we observed an inconsistent delay in the regrowth of mutant axons after axotomy. Moreover, we identified an association between mutant LRRK2 and the transient increase in lysosomes at the injury site, indicating that LRRK2 G2019S might potentially affect damage-prone distal axons. Since the LRRK2 G2019S-associated phenotypes observed in our assays were relatively mild in one isogenic pair, we were curious about the clinical and genetic phenotypes of the patients from whom the somatic cells for neural progenitor cell generation were sourced. Interestingly, we observed that clinical features of PD, including age-of-onset, motor symptoms, cognitive impairment, and the level of cerebrospinal fluid biomarkers, were heterogeneous between the two patients. Additionally, genetic analysis of specific PD risk-associated loci in MAPT and SNCA revealed that one patient was more at risk of developing PD than the other, indicating influence from genetic factors in addition to LRRK2 G2019S. These factors might affect the axonal phenotypes observed in our assays. Overall, we have developed assays to investigate the effects of LRRK2 G2019S on axonal lysosomes. These assays can potentially be a useful tool to better understand early pathogenesis in heterogeneous PD patients and test targeted therapeutics that can be successful over an eclectic cohort of PD patients, all of whom are diagnosed based on deteriorating motor symptoms.:TABLE OF CONTENTS I LIST OF FIGURES IV LIST OF TABLES VI ABBREVIATIONS VII 1 INTRODUCTION 1 1.1 Neurodegenerative diseases 1 1.2 Parkinson’s disease 2 1.2.1 General Features 2 1.2.2 Phenomenon of “dying back” in PD 6 1.2.3 Contribution of axonal architecture and function to “dying back” 7 1.2.4 Etiology of PD 10 1.2.4.1 Environmental factors 10 1.2.4.2 Genetic factors linked to axonal function 11 1.3 Lysosomes 12 1.3.1 Composition and biogenesis of lysosomes 13 1.3.2 Lysosomes as digestive centers 15 1.3.3 Lysosomes as secretory organelles 18 1.3.4 Lysosomes in PD 20 1.3.4.1 Genetic PD factors linked to lysosomal function 21 1.4 Leucine-rich repeat kinase 2 (LRRK2) 22 1.4.1 LRRK2 domain organization and function 22 1.4.2 Clinical features of PD patients with LRRK2 mutations (LRRK2-PD) 24 1.4.3 LRRK2 animal models 24 1.4.4 LRRK2 induced pluripotent stem cell (iPSC)-based models 25 1.4.5 Animal and iPSC-based models demonstrate a role for LRRK2 in the endo-lysosomal system 27 1.4.6 LRRK2 kinase inhibitors 30 2 AIMS OF THE THESIS 32 3 MATERIALS AND METHODS 33 3.1 Materials 33 3.1.1 Chemicals 33 3.1.2 Purchased kits 34 3.1.3 Plasmids 34 3.1.4 Antibodies 35 3.1.5 Dyes 36 3.1.6 Primers and oligonucleotides 36 3.1.7 Cell culture media and reagents 37 3.1.8 Small molecules 38 3.1.9 Compounds 38 3.1.10 Cell culture media 39 3.1.11 Human Neural Progenitor Cell (NPC) lines 40 3.2 Methods 41 3.2.1 Ethics statement 41 3.2.2 Licenses 41 3.2.3 Information about iPSC and NPC line generation 41 3.2.4 Preparation of cell culture coated plates 41 3.2.5 Maintenance of NPCs 42 3.2.6 Differentiation of NPCs to neurons 42 3.2.7 Preparation of microfluidic chambers 43 3.2.8 Seeding neurons as single cells 44 3.2.9 HEK293T cell culture 45 3.2.10 Treatment of neurons with compounds 45 3.2.11 Genomic DNA isolation 46 3.2.12 Polymerase-Chain Reaction (PCR) 46 3.2.13 Agarose gel electrophoresis 46 3.2.14 Plasmid DNA isolation 46 3.2.15 Lentiviral vector production 47 3.2.16 Lentiviral infection of human neurons 48 3.2.17 Protein isolation and quantification 48 3.2.18 Capillary electrophoresis 49 3.2.19 Axotomy 49 3.2.20 Immunostaining 50 3.2.21 Live cell imaging 51 3.2.22 Quantification of axonal trafficking using kymographs 52 3.2.23 Quantification of axonal trafficking using an object based method 53 3.2.24 Apotome imaging and quantification 54 3.2.25 Confocal imaging and quantification 54 3.2.26 Clinical and biomarker data collection 55 4 RESULTS 57 4.1 Establishing an axonal lysosomal trafficking assay 57 4.1.1 NPCs from LRRK2 G2019S patients and their respective isogenic controls differentiate into neurons 57 4.1.2 Axons can be spatially separated from soma and dendrites 60 4.1.3 Setting up the axonal trafficking assay 62 4.2 Axonal lysosomal trafficking assay detects LRRK2 G2019S associated changes in lysosome movement 65 4.3 Axonal lysosomal trafficking assay detects partial rescue by a small molecule LRRK2 inhibitor 71 4.4 LRRK2 G2019S is associated with an increase in the proportion of lysosomes co-localizing with phosphorylated Rab10 76 4.5 Rab10 over-expression mildly affects lysosomal trafficking in axons 78 4.6 Lysosomal membrane damage increases LRRK2-mediated Rab10 phosphorylation 81 4.7 LRRK2 G2019S is not associated with consistent effects on long-term axonal regrowth after axotomy 82 4.8 LRRK2 G2019S is associated with transient accumulation of lysosomes at the injury site after axotomy 86 4.9 Assessment of clinical, biomarker and genetic data from the LRRK2 G2019S patient donors 88 5 DISCUSSION 92 6 APPENDIX 101 7 SUMMARY 104 8 ZUSSAMENFASSUNG 106 9 BIBLIOGRAPHY 108 10 ACKNOWLEDGEMENTS 136 11 DECLARATIONS 138