京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21644号 / 医博第4450号 / 新制||医||1034(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 小西 靖彦, 教授 伊達 洋至, 教授 川口 義弥 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

La thérapie cellulaire utilisant une greffe d’hépatocytes est une stratégie prometteuse pour traiter les maladies du foie. Cependant, plusieurs limitations freinent leur transfert pour des applications cliniques, comprenant la production à haut débit d'hépatocytes fonctionnels, leur survie en culture, l’âge du donneur et la source des cellules souches hépatiques (CS). Les avancées scientifiques réalisées à ce jour ont permis d’identifier de nouveaux marqueurs moléculaires et les voies de signalisation impliquées dans la différenciation des CS en hépatocytes fonctionnels. En effet, la voie de signalisation Wnt a montré être importante pour réguler de nombreux processus biologiques des CS permettant de contrôler leur différenciation hépatique dont l’activation des GTPases et les gènes ciblant la voie de signalisation de Lgr5. Récemment, des études ont montré que le marqueur Lgr5 (récepteur 5 couplé à la protéine G contenant une répétition d’acides aminés riche en leucine) est décisif pour maintenir une expansion à long terme des CS hépatiques in vitro. En outre, Lgr5 fonctionne principalement comme un effecteur de la Cdc42 (cycle de division cellulaire 42) qui est un membre de la famille des Rho-GTPase. Une forte expression de la Cdc42 a montré être corrélée avec le vieillissement des SCs hématopoïétiques. Néanmoins, cette corrélation n'a jamais été étudiée à ce jour dans les cellules souches mésenchymateuses dérivées du tissu adipeux humain (hADSCs).Au cours de nos travaux de thèse, nous nous sommes intéressés (i) à proposer une nouvelle technologie de reprogrammation d’hépatocytes matures murins en progéniteurs endodermiques (mEndoPCs) exprimant Lgr5 capables de générer des organoïdes spécifiques du foie et de se différencier en hépatocytes et cholangiocytes in vitro et en des structures biliaires et hépatiques in vivo (ii) à étudier l’activité de Cdc42 dans les hADSCs âgées et l'impact de son inhibition sélective par le ML141 sur leur potentiel de différenciation hépatique in vitro.Nous montrons qu’il a été possible de générer des mEndoPCs et à améliorer la différenciation hépatique des hADSCs âgés. Nous montrons également que Lgr5 et Cdc42 sont régulés de façon distincte par la voie de signalisation Wnt. De plus, nos résultats ont révélé que les voies LIFR/STAT3 et Lgr5/WNT sont essentielles pour l’auto renouvellement des mEndoPCs permettant leur expansion illimitée in vitro en présence d’activateur de STAT3. Les voies MAPK/PKA, WNT/ β-caténine et la production d'exosomes ont montré avoir rôle majeur dans l’âge des hADSCs. Nous montrons qu’une transition mésenchymato-épithéliale était nécessaire pour différencier les hADSCs en hépatocytes fonctionnels. D’autre part, ML141 est proposé comme un nouvel outil pharmacologique permettant de reverser l’âge des hADSC âgés et d’amplifier le taux de prolifération, d’adhésion et de fonctionnalité hépatique à un niveau équivalent aux hADSCs jeunes. Enfin, le transfert de ces méthodologies à l’homme pourrait servir pour la médecine régénératrice du foie, comme outil pour évaluer la toxicité hépatique des médicaments et pour l'ingénierie et la reconstitution d’un foie entier par des approches de « bio printing ». / Hepatocytes cell-based transplantation is a promising strategy for treating liver diseases. However, there are still several limitations for their use in clinical applications among them the generation of highthrouput of functional hepatocytes, their life span in culture, the age of the donor age and the source of hepatic stem cells (SCs). At present, the challenge lies to develop approaches aiming the identification of the new molecular markers signaling pathways involved in the differentiation of SCs toward functional hepatocytes. In fact, Wnt(s) pathways governs multiple biological processes controlling the differentiation fate of SCs into hepatocytes, some of them result in the activation of small GTPase and the Lgr5 pathway regulators. Indeed, Lgr5 (a target gene of Wnt, the Leucine-rich-repeat-containing G protein-coupled Receptor 5) was shown to be crucial for maintaining long-term expansion of hepatic SC in vitro. In addition, Lgr5 primarily functions as an effector of the Cdc42 GTPases (a RhoGTPase protein, the cell division cycle 42). Higher activity of Cdc42 was reported to be correlated to hematopoietic SCs aging. However, this correlation has never been studied before in adipose tissue Mesenchymal Stem Cells (ADSCs) which were proposed recently as a promising tool for liver regeneration.In this study, we were interested (i) to propose a novel method of reprogramming mouse mature hepatocytes into murine endodermic progenitors (mEndoPCs) that express Lgr5, generate liver-specific organoids and can differentiate into hepatocytes and cholangiocytes in vitro and give arise to bile duct structures and into functional hepatocytes in vivo, and (ii) to study the activity of Cdc42 in human aged-derived hADSCs and the impact of its selective inhibition by ML141 on their hepatic differentiation potential in vitro.In our study we succeeded to generate mEndoPCs and to improve the functionality of the aged-hADSCs derived-hepatocytes. We showed that both Lgr5 and Cdc42 are regulated distinctly by Wnt signaling pathways. In addition, our results revealed that LIFR/STAT3 and LGR5/WNT pathways are important to maintain the unlimited expansion of mEndoPCs in vitro when STAT3 pathway is activated. MAPK/PKA, WNT/ β-catenin pathways and the exosome’s production were shown to be deregulated with hADSCs aging. We showed also that a mesenchymal to epithelial transition was crucial to transdifferentiate hADSCs into functional hepatocytes. On the other side, ML141 is proposed as a new pharmacological tool to rejuvenate aged-hADSCs toward functionally younger-like cells thus by promoting cell proliferation, doubling and cell adherence. Finally, the transfer of these methodologies to human could serve the regenerative medicine of the liver as a good tool for hepatocyte-based drug toxicity screening systems and for the liver engineering using a « bio printing » approach.

Introdução: O Transplante de Célula Tronco Hematopoiética (TCTH) é um tratamento potencialmente curativo para muitas desordens hematológicas. A disparidade HLA entre doador e receptor é um fator crítico para os resultados do TCTH. No entanto novas evidencias tem demonstrado que outros fatores como a fonte de célula tronco, o tipo de condicionamento e fatores relacionados aos doadores, entre eles o sexo, a idade e o status sorológico para Citomegalovirus (CMV) podem influenciar os desfechos do procedimento. Objetivo: Avaliar as características dos doadores de medula óssea e seu impacto nos desfechos do TCTH em um centro do Sul do Brasil. Métodos: Foram avaliadas retrospectivamente as características dos doadores como sexo, idade, presença de exposição previa ao CMV, incompatibilidade ABO e suas relações com a ocorrência de doença do enxerto contra hospedeiro (DECH) aguda e crônica, a mortalidade relacionada ao transplante (do inglês tumor related mortality – TRM) sobrevida livre de doença (SLD) e sobrevida Global (SG) em todos os pacientes submetidos a TCTH alogênico em um único centro durante o período de 1994 a 2012. Resultados: 347 pacientes foram incluídos na análise. O TCTH aparentado foi significativamente mais freqüente que o não aparentado (81.2 x 18.7), a mediana de idade foi de 34 (1-61) para os receptores e 33 (1-65) para os doadores. Na analise multivariada a presença de DECH agudo (33% vs 47%) p=0,04 e ter doadores mais idosos (>40 anos) foram associados a uma redução de SG em 5 anos (41% vs 52%) p=0,038. Ter um doador acima de 40 anos aumenta significativamente a incidência de DECH aguda (52% vs 65,8%) p=0,03 e crônica (60% vs 43%) p=0,015. O sexo do doador, presença de CMV e incompatibilidade ABO não tiveram influencia nos desfechos. Conclusão: Em um centro único avaliado receber um transplante de CTH de doadores acima de 40 anos aumenta a incidência de DECH aguda e crônica e influencia negativamente na SG. / Background: Hematopoietic Stem cell Transplantation (HSCT) is a curative treatment for many patients with hematological disorders. Donor – recipient genetic disparity, especially involving the HLA systems is a critical factor for HSCT outcome There are increasing evidences, however that other issues as source of stem cell, conditioning regimen, donor gender, age and CMV infection can affect HSCT outcomes. Objective: To study the influence of donor’s characteristics on the HSCT outcomes in a south Brazilian population subjected to allogeneic SCT in a single center. Methods: We retrospectively evaluated donor characteristics such as gender, age, CMV serologic status and ABO compatibility and its relation to the occurrence of acute and chronic graft versus host disease (GVHD), disease free survival (DFS) and overall survival (OS) in all patients submitted to related and unrelated allogeneic HSCT, performed between 1994 and 2012. Results: Overall 347 consecutives HSCT were included in this analysis. Related HSCT was significantly more frequent than unrelated (81.2% x 18.7%); donor and recipient median age were 34 (1- 61) and 33 (1-65), respectively. In the multivariate analyses, presence acute GVHD (33%vs 47%)p=0.04 ,with relative risk and donors older than 40 years old was associated with lower probability of OS in 5 years (41% vs 52%) p=0,038 and higher rate of acute (65.8%vs 52%) p=0.03 and chronic GVHD (43% vs 60%) p=0,015. Donor’s sex, CMV status, ABO incompatibility have not influenced 5-years survival. Conclusions: In a single center population of patients submitted to related and unrelated HSCT in southern Brazil donor older then 40 years of age is a factor negatively influencing HSCT outcome.

Introdução: O Transplante de Célula Tronco Hematopoiética (TCTH) é um tratamento potencialmente curativo para muitas desordens hematológicas. A disparidade HLA entre doador e receptor é um fator crítico para os resultados do TCTH. No entanto novas evidencias tem demonstrado que outros fatores como a fonte de célula tronco, o tipo de condicionamento e fatores relacionados aos doadores, entre eles o sexo, a idade e o status sorológico para Citomegalovirus (CMV) podem influenciar os desfechos do procedimento. Objetivo: Avaliar as características dos doadores de medula óssea e seu impacto nos desfechos do TCTH em um centro do Sul do Brasil. Métodos: Foram avaliadas retrospectivamente as características dos doadores como sexo, idade, presença de exposição previa ao CMV, incompatibilidade ABO e suas relações com a ocorrência de doença do enxerto contra hospedeiro (DECH) aguda e crônica, a mortalidade relacionada ao transplante (do inglês tumor related mortality – TRM) sobrevida livre de doença (SLD) e sobrevida Global (SG) em todos os pacientes submetidos a TCTH alogênico em um único centro durante o período de 1994 a 2012. Resultados: 347 pacientes foram incluídos na análise. O TCTH aparentado foi significativamente mais freqüente que o não aparentado (81.2 x 18.7), a mediana de idade foi de 34 (1-61) para os receptores e 33 (1-65) para os doadores. Na analise multivariada a presença de DECH agudo (33% vs 47%) p=0,04 e ter doadores mais idosos (>40 anos) foram associados a uma redução de SG em 5 anos (41% vs 52%) p=0,038. Ter um doador acima de 40 anos aumenta significativamente a incidência de DECH aguda (52% vs 65,8%) p=0,03 e crônica (60% vs 43%) p=0,015. O sexo do doador, presença de CMV e incompatibilidade ABO não tiveram influencia nos desfechos. Conclusão: Em um centro único avaliado receber um transplante de CTH de doadores acima de 40 anos aumenta a incidência de DECH aguda e crônica e influencia negativamente na SG. / Background: Hematopoietic Stem cell Transplantation (HSCT) is a curative treatment for many patients with hematological disorders. Donor – recipient genetic disparity, especially involving the HLA systems is a critical factor for HSCT outcome There are increasing evidences, however that other issues as source of stem cell, conditioning regimen, donor gender, age and CMV infection can affect HSCT outcomes. Objective: To study the influence of donor’s characteristics on the HSCT outcomes in a south Brazilian population subjected to allogeneic SCT in a single center. Methods: We retrospectively evaluated donor characteristics such as gender, age, CMV serologic status and ABO compatibility and its relation to the occurrence of acute and chronic graft versus host disease (GVHD), disease free survival (DFS) and overall survival (OS) in all patients submitted to related and unrelated allogeneic HSCT, performed between 1994 and 2012. Results: Overall 347 consecutives HSCT were included in this analysis. Related HSCT was significantly more frequent than unrelated (81.2% x 18.7%); donor and recipient median age were 34 (1- 61) and 33 (1-65), respectively. In the multivariate analyses, presence acute GVHD (33%vs 47%)p=0.04 ,with relative risk and donors older than 40 years old was associated with lower probability of OS in 5 years (41% vs 52%) p=0,038 and higher rate of acute (65.8%vs 52%) p=0.03 and chronic GVHD (43% vs 60%) p=0,015. Donor’s sex, CMV status, ABO incompatibility have not influenced 5-years survival. Conclusions: In a single center population of patients submitted to related and unrelated HSCT in southern Brazil donor older then 40 years of age is a factor negatively influencing HSCT outcome.

Introdução: O Transplante de Célula Tronco Hematopoiética (TCTH) é um tratamento potencialmente curativo para muitas desordens hematológicas. A disparidade HLA entre doador e receptor é um fator crítico para os resultados do TCTH. No entanto novas evidencias tem demonstrado que outros fatores como a fonte de célula tronco, o tipo de condicionamento e fatores relacionados aos doadores, entre eles o sexo, a idade e o status sorológico para Citomegalovirus (CMV) podem influenciar os desfechos do procedimento. Objetivo: Avaliar as características dos doadores de medula óssea e seu impacto nos desfechos do TCTH em um centro do Sul do Brasil. Métodos: Foram avaliadas retrospectivamente as características dos doadores como sexo, idade, presença de exposição previa ao CMV, incompatibilidade ABO e suas relações com a ocorrência de doença do enxerto contra hospedeiro (DECH) aguda e crônica, a mortalidade relacionada ao transplante (do inglês tumor related mortality – TRM) sobrevida livre de doença (SLD) e sobrevida Global (SG) em todos os pacientes submetidos a TCTH alogênico em um único centro durante o período de 1994 a 2012. Resultados: 347 pacientes foram incluídos na análise. O TCTH aparentado foi significativamente mais freqüente que o não aparentado (81.2 x 18.7), a mediana de idade foi de 34 (1-61) para os receptores e 33 (1-65) para os doadores. Na analise multivariada a presença de DECH agudo (33% vs 47%) p=0,04 e ter doadores mais idosos (>40 anos) foram associados a uma redução de SG em 5 anos (41% vs 52%) p=0,038. Ter um doador acima de 40 anos aumenta significativamente a incidência de DECH aguda (52% vs 65,8%) p=0,03 e crônica (60% vs 43%) p=0,015. O sexo do doador, presença de CMV e incompatibilidade ABO não tiveram influencia nos desfechos. Conclusão: Em um centro único avaliado receber um transplante de CTH de doadores acima de 40 anos aumenta a incidência de DECH aguda e crônica e influencia negativamente na SG. / Background: Hematopoietic Stem cell Transplantation (HSCT) is a curative treatment for many patients with hematological disorders. Donor – recipient genetic disparity, especially involving the HLA systems is a critical factor for HSCT outcome There are increasing evidences, however that other issues as source of stem cell, conditioning regimen, donor gender, age and CMV infection can affect HSCT outcomes. Objective: To study the influence of donor’s characteristics on the HSCT outcomes in a south Brazilian population subjected to allogeneic SCT in a single center. Methods: We retrospectively evaluated donor characteristics such as gender, age, CMV serologic status and ABO compatibility and its relation to the occurrence of acute and chronic graft versus host disease (GVHD), disease free survival (DFS) and overall survival (OS) in all patients submitted to related and unrelated allogeneic HSCT, performed between 1994 and 2012. Results: Overall 347 consecutives HSCT were included in this analysis. Related HSCT was significantly more frequent than unrelated (81.2% x 18.7%); donor and recipient median age were 34 (1- 61) and 33 (1-65), respectively. In the multivariate analyses, presence acute GVHD (33%vs 47%)p=0.04 ,with relative risk and donors older than 40 years old was associated with lower probability of OS in 5 years (41% vs 52%) p=0,038 and higher rate of acute (65.8%vs 52%) p=0.03 and chronic GVHD (43% vs 60%) p=0,015. Donor’s sex, CMV status, ABO incompatibility have not influenced 5-years survival. Conclusions: In a single center population of patients submitted to related and unrelated HSCT in southern Brazil donor older then 40 years of age is a factor negatively influencing HSCT outcome.

Bone marrow mesenchymal stem cells (BM-MSCs) are pluripotent cells that can differentiate into a variety of non-hematopoietic tissues. They also maintain healthy heamatopoiesis by providing supportive cellular microenvironment into BM. In this thesis, MSCs were characterized in terms of their morphological, immunophenotypical and differentiation properties. Then, they were examined by attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy together with hierarchical clustering, and FTIR microspectroscopy. In the first part of this study, global structural and compositional changes in BM-MSCs during beta thallasemia major (

Bone marrow mesenchymal stem cells (BM-MSCs) are pluripotent cells that can differentiate into a variety of non-hematopoietic tissues. They also maintain healthy heamatopoiesis by providing supportive cellular microenvironment into BM. In this thesis, MSCs were characterized in terms of their morphological, immunophenotypical and differentiation properties. Then, they were examined by attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy together with hierarchical clustering, and FTIR microspectroscopy. In the first part of this study, global structural and compositional changes in BM-MSCs during beta thallasemia major (