Tesis sobre el tema "Renal progenitors"

Supplementary information 追加（2019-09-30)
Kyoto University (京都大学)
0048
新制・課程博士
博士(医学)
甲第21344号
医博第4402号
新制||医||1031(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 柳田 素子, 教授 山下 潤, 教授 江藤 浩之
学位規則第4条第1項該当

Certains vertébrés peuvent régénérer des néphrons, l'unité fonctionnelle du rein. Cette capacité (la néonéphrogenèse) dépend de la présence de progéniteurs rénaux qui vont se différentier en les cellules du néphron. Pendant le développement des mammifères, les progéniteurs rénaux arrêtent de proliférer et se différencient autours de la naissance alors que d'autres vertébrés maintiennent leurs progéniteurs rénaux après le développement embryonnaire. Ainsi, après la perte de néphrons chez les mammifères, seule une réponse hypertrophique est observée. En contraste, la formation de novo de néphrons a été observée chez deux poissons cartilagineux: la petite raie, après néphrectomie partielle, et chez la petite roussette adolescente et adulte. Cette capacité de néonephrogenèse chez les poissons cartilagineux a été décrite avec des observations histologiques mais la caractérisation moléculaire et fonctionnelle des progéniteurs reste à déterminer.Avec la petite roussette Scyliorhinus canicula comme modèle, des HIS in toto pendant le développement précoce montre que Lim1 et Pax2 sont conservés comme des facteurs de transcription qui spécifient le territoire du rein. Ensuite, les progéniteurs rénaux chez S. canicula sont identifiés comme des agrégats mésenchymateux chez les embryons et juvéniles et comme exprimant des ARNm spécifiques aux progéniteurs rénaux comme Six2.L'expression de Pax2 et Wt1 est trouvée dans ces structures chez les embryons et juvéniles et les patrons sont cohérents avec les ARNm trouvés chez d'autres animaux avec la néonephrogenèse. Dans différents types de cellules souches, des niveaux bas de synthèse protéiques sont associés avec le caractère souche et des niveaux plus haut à l'activation et la différentiation. La maintenance des cellules souches pendant le développement embryonnaire et post-développement semble lié à des niveaux bas de traduction. En suivant la synthèse protéique, les agrégats mésenchymateux montrent des niveaux plus bas relatifs aux compartiments épithéliaux différentiés. Enfin, ces agrégats retiennent le BrdU (pulse); ils cyclent lentement (2 mois de chasse), ce qui est une caractéristique des cellules souches adultes. Ces résultats tentent de déchiffrer les clés derrière la maintenance des cellules souches dans les reins capables de régénération
Certain vertebrates can regenerate nephrons, the functional kidney unit of the kidney. This capacity (neonephrogenesis) relies on the presence of renal progenitor cells which differentiate into the cells that make up the nephron. During mammalian development, nephron progenitors stop propagation and differentiate within few days after birth, while other vertebrates maintain these cells after embryonic development. As such, in mammals with nephron loss, only a hypertrophic response is observed. In contrast, de novo nephron formation was reported in two adult cartilaginous fish; the little skate, after partial nephrectomy and in the adolescent and adult small-spotted catshark. The neonephrogenic capacity of cartilaginous fish was described with histological observations but molecular and functional characterization of the progenitor cells remains to be determined. Using the small-spotted catshark Scyliorhinus canicula as a model, in toto ISH during early development first show that Lim1 and Pax2 are conserved as early kidney-specifying transcription factors. Next, progenitor cells are identified as mesenchymal aggregates in embryos and juveniles for which ISH on sections revealed the expression of kidney progenitor-cell mRNAS such as Six2. Pax2 and Wt1 expression is found in these structures in embryos and juveniles and is cogent with progenitor cell mRNAs found in other animals with neonephrogenesis. In different stem cell types, low protein synthesis levels are associated with stemness and higher levels to activation and differentiation. Stem cell maintenance during embryonic development and post development seems to be linked with low rates of translation. Using a protein synthesis levels assay, the mesenchymal aggregates show lower levels relative to the differentiated epithelial compartments. Finally, these aggregates retain BrdU (pulse) labels; they are slow cycling cells (2 month chase) which is a characteristic of stemness. These exciting results intend to help decipher the keys behind stem cell maintenance in regenerative kidneys

Introducció: La malaltia cardiovascular és la principal causa de mortalitat en els pacients amb malaltia renal crònica (MRC), tant en hemodiàlisi (HD) com en els pacients trasplantats renals. Els principals factors de risc cardiovascular dels pacients amb MRC,així com els factors de risc propis de la urèmia, produeixen disfunció endotelial en aquest grup de pacients. Aquesta disfunció endotelial incrementa la morbiditat cardiovascular en aquest grup de pacients. Les cèl.lules progenitores endotelials (CPE) són cèl·lules derivades del moll de l’os, presents en sang perifèrica i que tenen la capacitat de proliferar i diferenciar-se en cèl·lules endotelials madures, contribuint a la reendotelització i revascularització dels teixis danyats. Els pacients amb malaltia renal crònica presenten una alteració en la quantificació d’aquestes CPE, essent aquest un factor de mal pronòstic, al incrementar el risc de presentar algun episodi cardiovascular. Objectiu: Determinar i quantificar el nombre de CPE que expressen CD34+CD133+VEGFR-2+ en sang perifèrica en pacients amb MRC, així com els factors que poden modificar el seu nombre en sang perifèrica. Avaluar la influència de l’accés vascular en la quantificació de les cèl·lules CD34+CD133+VEGFR-2+ en els pacients en HD crònica. Analitzar l’evolució de les cèl.lules CD34+CD133+VEGFR-2+ durant el primer any posttrasplantament renal (posTR) de donant viu. Pacients i mètodes: S’han inclòs en l’estudi 36 controls sans i 86 pacients amb MRC entre els anys 2012 i 2013, dels quals 73 pacients estaven en programa d’HD crònica i 13 pacients ingressaven per a rebre un trasplantament renal de donant viu. A través de citometria de flux s’ha realitzat la quantificació de les cèl·lules CD34+ CD133+VEGFR-2+/mL de sang perifèrica. S’han recollit les dades antropomètriques, factors de risc cardiovascular, aparició d’algun episodi cardiovascular i les dades bioquímiques dels 3 grups d’estudi. Del grup trasplantament renal s’ha realitzat el seguiment durant el primer any posTR, analitzant les dades als 6 mesos i 12 mesos del trasplantament. Resultats: Cèl·lules CD34+CD133+VEGFR-2+ dels pacients amb MRC (27,96(12,16-51,71) en comparació a controls sans (32,08(17,26-66,41), p=0,23. L’edat no interfereix en la quantificació de cèl·lules CD34+CD133+VEGFR-2+ ni en la MRC ni en els controls sans. Els pacients en tractament amb antagonistes dels canals del calci tenen menys cèl·lules CD34+CD133+VEGFR-2+ respecte els que no reben aquest tractament (p=0,018). Els pacients diabètics tenen una menor quantificació de cèl·lules CD34+CD133+VEGFR-2+ i un major risc de cardiopatia isquèmica. Els pacients que realitzen HD a través de catéter tenen menys cèl·lules CD34+CD133+VEGFR-2+ (17,50 (12,41-30,98)) que els pacients que tenen una FAVI (32,90(9,7-54,49)) (p=0,191). Els pacients que porten catéter tenen més edat (p=0,084), estan més inflamats (proteïna C reactiva) (p=0,081) i estan més desnutrits (albúmina i prealbúmina) (p=0,020 i p=0,013 respectivament), que els pacients que fan HD a través de FAVI. No podem establir diferències estadísticament significatives entre el nombre de cèl·lules CD34+CD133+VEGFR-2+ en funció de l’accés vascular i la mortalitat. En els pacients trasplantats, les cèl·lules CD34+CD133+VEGFR-2+ presenten un increment durant el primer any postTR: basal (33,87 (29,15-54,42)), 6 mesos postTR (63,35(43,40-139,90)), 12 mesos postTR (62,62(25,30-72,54)) (p=NS). El nombre de cèl·lules CD34+CD133+VEGFR-2+ als 6 i 12 mesos postTR és superior al nombre de cèl·lules que tenen els pacients que segueixen en programa d’HD crònica (p=0,008 i p=0,065 respectivament). Conclusions: Els pacients amb MRC tenen menys cèl·lules CD34+CD133+VEGFR-2+ que la població sana, especialment els pacients diabètics. Els pacients que realitzen HD a través de catéter tunelitzat tenen menys cèl.lules CD34+CD133+VEGFR-2+, tenen més edat, estan més inflamats i desnutrits i tenen un augment de la mortalitat respecte els pacients amb FAVI. Els trasplantats renals presenten un increment del nombre de cèl·lules CD34+CD133+VEGFR-2+ durant el primer any posTR i que és superior als pacients que segueixen en HD.
Introduction: Cardiovascular disease is the main cause of mortality in patients with chronic kidney disease (CKD), both hemodialysis (HD) and in kidney transplant. The main cardiovascular risk factors in CKD, as well as factors relating uremia cause endothelial dysfunction in these patients. This endothelial dysfunction increase cardiovascular morbidity in these patients. Endothelial progenitor cells (EPC) are cells derived from bone marrow, present in peripheral blood, and with the ability to proliferate and differentiate into mature endothelial cells, contributing to the reendotelization and revascularization of the damaged tissue. Patients with CKD have an alteration in the quantification of these EPC. This is a factor of poor prognosis, increasing the risk of a cardiovascular event. Aim: To determine and to quantificate the number of EPC which express CD34+CD133+VEGFR-2+ in peripheral blood in pacients with CKD, as well as the factors that may change its number in peripheral blood. To assess the influence of vascular access in quantifying CD34+CD133+VEGFR-2+ cells in patients on HD. Analysing the evolution of CD34+CD133+VEGFR-2+ cells during the first year after living donor kidney transplantation. Patients and methods: Patients included in the study: 36 healthy volunteers and 86 patients with CKD of which 73 pacients were on chronic HD and 13 were admitted to receive a living donor kidney transplant,between 2012 and 2013. The quantification of CD34+CD133+VEGFR-2+ in peripheral blood was performed by flow cytometry. We have collected anthropometric data, cardiovascular risk factors, occurrence of a cardiovascular event and biochemical data from the three groups. Kidney transplant grupo was also monitored during the first year after kidney transplantation, analyzing data at 6 and at 12 months after transplantation. Results: CD34+CD133+VEGFR-2+ cells in CKD (27,96(12,16-51,71) compared with healthy volunteers (32,08(17,26-66,41), p=0,23. Age does not interfere with the quantification of CD34+CD133+VEGFR-2+ cells in patients with CKD or in healthy people. Patients treated with calcium channel antagonists have lower quantification of CD34+CD133+VEGFR-2+ cells respect those who do not receive this treatment. Patients with diabetes have a lower quantification of CD34+CD133+VEGFR-2+ and a higher risk of ischemic heart disease. Patients on HD via catheter haver less CD34+CD133+VEGFR-2+ cells (17,50 (12,41-30,98)) than patients on HD via arteriovenous fistula (32,90(9,7-54,49)) (p=0,191). Patients on HD via catheter are older (p=0,084), they have a worse inflammation status (C reactive protein) (p=0,081) and have a worse nutritional status (albumin and prealbumin) (p=0,020 i p=0,013 respectively) than patients on HD via arteriovenous fistula. We can not establish significant differences between the number of CD34+CD133+VEGFR-2+ cells based on vascular access and mortality. In kidney transplant patients, CD34+CD133+VEGFR-2+ cells increase their quantification in peripheral blood during the first year after kidney transplantation: baseline (33,87 (29,15-54,42)), 6 months after kidney transplant (63,35(43,40-139,90)), and 12 months after kidney transplant (62,62(25,30-72,54)) (p=NS). The quantification of CD34+CD133+VEGFR-2+ cells at 6 and at 12 months after kidney transplant is higher compared to patients on chronic HD (p=0,008 i p=0,065 respectively). Conclusions: Patients with CKD have less CD34+CD133+VEGFR-2+ cells in peripheral blood compared to healthy people, specially diabetic patients. Patients on HD via catheter have less CD34+CD133+VEGFR-2+ compared to patients via arteriovenous fistula and have a worse prognosis, including a higher mortality risk. Living kidney transplant patients have an increase in the quantification of CD34+CD133+VEGFR-2+ during the first year after kidney transplant, this increase is greater than the number of cells in hemodialysis patients.

AMs seem to be a very promising scaffold in TE and can be considered as temporary inductive site-appropriate templates to support the growth, differentiation, and function of the parenchymal cell population of each organ. Nowadays, TE techniques are used both to develop tissue substitutes ex vivo and as reliable tool to investigate cell behaviour, differentiation and proliferation in 3-dimentional environments. In this work the following two different projects have investigated both potentialities using tissue-specific AMs: 1- influence of AMs on differentiation of kidney progenitor cells from amniotic fluid into mature renal cells; 2- AMs as biomaterial to develop vessel substitutes. 1- Kidney AMs (KAMs) were used to evaluate the differentiation of kidney progenitor cells from amniotic fluid into mature renal cells in order to better understand whether they could be suitable for future application in therapy. Renal progenitors were seeded into KAMs, which led them to proliferate, maintain podocyte phenotype and differentiate into tubular cells in vitro. To further evaluate the differentiative potential of KAMs, grafts composed of KAM with or without cells were intrarenal implanted into nude mice. In vivo, progenitors from amniotic fluid expressed mature renal markers, attracted inside KAMs murine cells presenting the same proteins and integrated into host structures. 2- Although autologous vascular grafts and artificial materials have been used for reconstruction of small diameter (5 mm) blood vessels, the poor availability of vessels and the occurrence of intimal hyperplasia and progressive atherosclerotic degeneration represent shortcoming of these vascular prostheses. Therefore, this study aimed to develop AM-based vascular grafts. Both AAMs alone and AAMs previously reendothelized with skin microvascular endothelial cells (ECs) were in vivo implanted and analyzed. The lack of reendothelization, leading to intimal hyperplasia and increased incidence of thrombosis observed in AAMs grafts, have indicated the need to provide in vitro an endothelial coverage of decellularized tissue. Indeed, grafts composed of AAM and skin microvasculature ECs shown good patency and no thrombi. Although these grafts appeared narrowed and a moderate hyperplasia has been detected in the inner layer, they presented two main advantages: they were obtained into a clinically relevant time frame and eliminated the need to remove healthy vessels for collecting autologous ECs.
Le matrici acellulari rappresentano uno scaffold promettente per l’ingegneria tessutale. Infatti, la matrice extracellulare costituisce un supporto sito-specifico che favorisce la crescita e il differenziamento delle cellule di qualsiasi organo. Ad oggi, le tecniche dell’ingegneria tessutale sono utilizzate sia per lo sviluppo ex vivo di sostituti tessutali, che per studiare la proliferazione e la differenziazione delle cellule quando si trovano all’interno di uno scaffold tridimensionale. In questo lavoro di tesi, i due seguenti progetti sono andati a valutare entrambe le potenzialità di matrici acellulari tessuto-specifiche: 1- valutazione della capacità della matrice acellulare di indurre il differenziamento di progenitori renali da fluido amniotico in cellule renali mature; 2- valutazione della matrice acellulare per la sostituzione di vasi sanguigni. 1- La matrice acellulare renale è stata utilizzata per valutare la capacità differenziativa di progenitori renali da fluido amniotico in modo da valutarne una futura applicazione terapeutica. I progenitori renali sono stati seminati sulla matrice acellulare renale, che, in vitro, ne ha promosso la proliferazione, il mantenimento del fenotipo podocitario e la differenziazione in cellule tubulari. Per valutare in vivo il potenziale differenziativo di queste cellule, la matrice da sola o ripopolata con le cellule è stata impiantata all’interno di un rene di topo nudo. I progenitori renali si sono ulteriormente differenziati, si sono integrati all’interno delle strutture tubulari dell’ospite e hanno promosso la migrazione di cellule differenziate murine all’interno dello scaffold. 2- La matrice acellulare di aorta è stata utilizzata per lo sviluppo di sostituti vasali. Nonostante vasi autologhi o costituiti di polimeri sintetici vengano già utilizzati nella pratica clinica per la ricostruzione di vasi di piccolo diametro (5 mm), numerosi sono gli svantaggi legati al loro utilizzo, quali l’iperplasia della tonaca intima e la degenerazione arteriosclerotica. Lo scopo di questo studio è stato quello di sviluppare sostituti vasali utilizzando come scaffold vasi decellularizzati. Matrici acellulari da sole o ripopolate con cellule endoteliali da microcircolo sono state impiantate nell’aorta di ratto Lewis. Come osservato negli impianti di sola matrice acellulare, la mancanza della copertura endoteliale portava all’iperplasia dell’intima e all’aumento di incidenza dei processi trombotici, sottolineando la necessità di reendotelizzare in vitro il vaso decellularizzato prima dell’impianto in vivo. Infatti, i sostituti vasali costituiti da matrice acellulare e cellule endoteliali da microcircolo hanno dimostrato di avere una buona resistenza al flusso e non presentavano trombi al loro interno. Sebbene questi vasi fossero assottigliati e mostrassero una leggera iperplasia della tonaca intima, questo approccio presentava due principali vantaggi: permetteva di ottenere sostituti vasali in un tempo clinicamente utile ed eliminava la necessità di rimuovere vasi sani per ottenere cellule endoteliali autologhe.

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Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG
The Human Cytomegalovirus (HCMV) is an important cause of morbi-mortality in recipients of allogeneic hematopoietic stem cell transplantation (AloHSCT). However, there is not a consensus on which protocol to use for monitoring the infection by HCMV and, data on the frequency and clinical manifestations of the infection in this group population are quite variable among the distinct transplant centers in the world. Thus, the main objective of the present study was to proceed the monitoring of active HCMV infection in patients undergoing AloHSCT by three different methodologies: antigenemia (AGM), nested-PCR (nPCR) and real-time PCR (qPCR) and determine viral load, correlating active infection with the clinical manifestations and prognosis of patients. For this, 21 patients undergoing AloHSCT were monitored (from pre-transplant period -5 days prior to transplantation- until one year after transplantation). For HCMV detection three methodologies were used: AGM, nPCR and qPCR, and for molecular detection a comparison was made between detection of HCMV in DNA extracted from pellet (buffy coat) and serum, in a paired manner. The results showed that the active HCMV infection was detected by at least one of three methodologies in 95.2% (20/21) of patients and 45% (9/20) of these were positive in pre-transplantation period, having been observed good agreement between the results of AGM and qPCR (kappa = 0.65). Of the 20 patients positive for active HCMV infection, 85% (17/20) were positive for the three methods and only 15% (3/20) were positive for AGM and qPCR, and negative by nPCR. Regarding the type of clinical sample, molecular techniques showed higher sensitivity to the pellet over the serum. The main alteration of patients was pancytopenia and the main complication was graft-versus-host disease. Six patients died during the study period, however, it was not possible to confirm if HCMV active infection was directly associated with the cause of death. The obtained data reveal a high positivity index and the occurrence of HCMV syndrome in patients submitted to aloHSCT. We hope that the results may assist in the therapeutical measures, as well as in the methodology of choice and the type of clinical sample for detection of active HCMV infection, in order to contribute for the inclusion of HCMV monitoring is included in the routine testing of patients.
O Citomegalovírus Humano (HCMV) constitui importante causa de morbi-mortalidade em receptores de transplantes de células progenitoras hematopoiéticas do tipo alogênico (AloTCPH). Entretanto, não existe ainda um consenso sobre que protocolo utilizar para o monitoramento da infecção por HCMV e, dados sobre a frequência e manifestações clínicas da infecção neste grupo populacional são bastante variáveis entre os diferentes centros de transplante em todo o mundo. Dessa forma, o principal objetivo do presente estudo foi realizar o monitoramento da infecção ativa por HCMV em pacientes submetidos ao AloTCPH por três metodologias distintas: antigenemia (AGM), nested-PCR (nPCR) e PCR em tempo real (qPCR), bem como determinar a carga viral, correlacionando a infecção ativa ao quadro clínico e prognóstico dos pacientes. Para tanto, foram monitorados (desde o período pré-transplante -5 dias antes do transplante- até um ano após o transplante) 21 pacientes submetidos ao AloTCPH. A pesquisa de HCMV foi realizada utilizando as metodologias de AGM, nPCR e qPCR, sendo que para as técnicas moleculares, houve comparação da detecção do HCMV em DNA extraído de pellet (creme leucocitário) e soro, de forma pareada. Os resultados mostraram que a infecção ativa pelo HCMV foi detectada por pelo menos uma das três metodologias em 95,2% (20/21) dos pacientes e 45% (9/20) destes foram positivos no período pré-transplante, tendo sido observada um boa concordância entre os resultados de AGM e qPCR (kappa = 0,65). Dos 20 pacientes positivos para infecção ativa por HCMV, 85% (17/20) foram positivos pelas três metodologias e 15% (3/20) foram positivos apenas por AGM e qPCR e negativos por nPCR. Em relação ao tipo de amostra clínica, as técnicas moleculares apresentaram maior sensibilidade em amostras de pellet, em comparação ao soro. A principal alteração apresentada pelos pacientes foi a pancitopenia e a principal intercorrência, a doença do enxerto contra o hospedeiro. Seis pacientes foram a óbito durante o período de estudo, entretanto, não foi possível confirmar se a infecção ativa por HCMV estava diretamente associada à causa mortis. Os dados obtidos revelam um elevado índice de positividade e síndrome de HCMV em pacientes submetidos ao AloTCPH. Esperamos que os resultados possam auxiliar na tomada de medidas terapêuticas, bem como na escolha da melhor metodologia assim como o tipo de amostra clínica para detecção da infecção ativa por HCMV de forma a contribuir para que a pesquisa de HCMV seja incluída na rotina de exames desses pacientes.

Introdução: As células-tronco apresentam capacidade de proliferação, autorrenovação, potencial de diferenciação e já foram descritas na hipófise estando envolvidas na renovação celular e regulação homeostática, porém pouco se sabe sobre o seu perfil de expressão nos quadros de hipopituitarismo. Dentre os marcadores de células-tronco descritos previamente na hipófise, destacam-se os genes Sox2, Nanog, Nestina, Cd44 e Oct4. Outro marcador, o gene Nr2e1 (Tlx), encontrado em células-tronco neuronais, apresenta-se elevado durante a embriogênese e na vida adulta no cérebro de camundongos, mas, até o momento, não foi caracterizado na hipófise. Objetivo: Analisar a imunolocalização do SOX2 e o padrão de expressão de marcadores de células-tronco/progenitoras, fatores de transcrição precoce, marcadores de apoptose e proliferação celular na hipófise de três linhagens de camundongos com hipopituitarismo de causa genética por alteração em fatores precoces de diferenciação glandular, as linhagens Ames (Prop1) e Snell (Pou1f1), e por fator tardio de conjugação dos hormônios glicoproteicos, a linhagem alfaGSU, nocaute do gene Cga. Material e Métodos: Foram coletadas hipófises nos tempos P0 (ao nascimento), P7 (final da primeira onda de crescimento glandular), 4 semanas (4S-período da puberdade) e 8 semanas (8S-vida adulta). Nas três linhagens de animais, realizou-se imuno-histoquímica com SOX2 e RT-qPCR com os marcadores de células-tronco/progenitoras Sox2, Nanog, Nestina, Cd44, Oct4 e Nr2e1, fatores de transcrição precoces (Hesx1, Hes1 e Otx2), fator de proliferação celular (Ki67), fatores de diferenciação celular (S100beta e Sox9) e marcadores de apoptose (Caspases 3 e 7). A quantificação relativa dos genes-alvo nos animais mutantes teve como calibrador os seus respectivos selvagens. Resultados: A imunolocalização do SOX2 foi observada na zona que circunda a fenda de Rathke (camada marginal) e em nichos difusos pela glândula nas três linhagens estudadas. Na linhagem alfaGSU, evidenciou-se uma redução de Nanog, Nr2e1, Oct4, e Hesx1 em 4S e de Nestina em 8S. Na linhagem Snell, observou-se aumento na expressão de Sox2, Nanog, Cd44, Nr2e1, Hesx1, Hes1, Otx2, S100beta e Sox9 em 4S e aumento de Sox2, Cd44, Hesx1, Otx2 e Sox9 em 8S, associado à redução de Ki67 em ambos os períodos. Na linhagem Ames, evidenciou-se aumento de Sox2, Nanog, Cd44, Hesx1, Hes1, Otx2, S100beta e Sox9 em 4S e 8S. O gene Nr2e1 esteve hiperexpresso em todos os tempos. Houve redução do Ki67 em 4S. As caspases 3 e 7 não se apresentaram alteradas em nenhuma linhagem e/ou tempo. Discussão e conclusão: O padrão de imunolocalização de SOX2 encontrado nas três linhagens estudadas foi semelhante ao descrito em animais sem hipopituitarismo. A evidência da presença do Nr2e1 o coloca como um novo marcador de células-tronco/progenitoras na hipófise. A expressão elevada dos marcadores de células-tronco/progenitoras nas linhagens Ames e Snell sugere que a ausência dos fatores de transcrição precoces não permitiria que a célula tronco/progenitora iniciasse o processo de diferenciação celular, enquanto o oposto ocorreria na linhagem alfaGSU. Adicionalmente, estes achados justificam a hipoplasia hipofisária observada em animais com defeitos em fatores de transcrição expressos no início da diferenciação hipofisária, nos quais o acúmulo de células-tronco pode ser um indicador da indiferenciação hipofisária
Introduction: The role of stem cells, with their capacity for proliferation, self-renewal, and differentiation, has already been described in the cell turnover and homeostatic regulation of the pituitary gland. However, little is known about the expression profiles of these markers in hypopituitarism. Among the stem cell markers previously described in the pituitary include the genes for Sox2, Nanog, nestin, CD44 and Oct4. Another gene marker, Nr2e1 (Tlx), found in neural stem cells, is highly expressed during embryogenesis and adulthood, but so far has not been characterized in the pituitary. Objective: To analyze the immunohistochemical profile of SOX2, as well as the pattern of expression of various markers of stem/progenitor cells, early transcription factors, apoptosis factors and cell proliferation in three pituitary strains of mice with a genetic cause of hypopituitarism. Strains studied with hypopituitarism due to changes in factors of precocious glandular differentiation, include the Ames (Prop1) and Snell (Pou1f1) lineages; hypopituitarism due to the delayed conjugation of glycoprotein hormones include the alfaGSU strain, which is caused by the knockout of the Cga gene. Material and Methods: We collected pituitaries at four time points including P0 (birth), P7 (considered the end of the first wave of growth glandular), 4 weeks (4S - puberty period) and 8 weeks (8S - adulthood). All three strains were subjected to immunohistochemical analysis of SOX2 and RT-qPCR of markers of stem/progenitor cells Sox2, Nanog, Nestin, Cd44, Oct4 and Nr2e1, early transcription factors (Hesx1, Otx2 and Hes1), cell proliferation (Ki67), cell differentiation factors (S100beta and Sox9) and apoptosis (caspases 3 and 7) markers. Relative quantification of target genes in mutant animals was normalized to their respective wild type littermate. Results: The immunolocalization of SOX2 was observed in the area surrounding the Rathke cleft (marginal layer), as well as in diffuse niches throughout the gland in all three strains studied. The alfaGSU strain showed a reduction of Nanog, Nr2e1, Oct4 and Hesx1 at 4S, and Nestin at 8S. The Snell mice exhibited an increase of expression in Sox2, Nanog, Cd44, Nr2e1, Hesx1, Hes1, Otx2, S100beta and Sox9 in at 4S and increased Sox2, Cd44, Hesx1, Otx2 and Sox9 at 8S, associated with the reduction of Ki67 in both periods. The Ames strain showed an increase of Sox2, Nanog, Cd44, Hesx1, Hes1, Otx2, S100beta and Sox9 at 4S and 8S; the gene Nr2e1 was over expressed at all times; and there was reduction in Ki67 at 4S. Caspases 3 and 7 had not changed in any strain, at any time. Discussion and Conclusion: The pattern of immunolocalization of SOX2 found in the three strains studied was similar to that described in animals without hypopituitarism. The presence of Nr2e1 in our study suggests it as a new marker of stem/progenitor cells in the pituitary. The high expression of markers of stem/progenitor cells in the Ames and Snell strains suggests that the absence of early transcription factors Prop1 and Pou1f1 do not allow the stem/ progenitors cells to start the process of cell differentiation, while the opposite occurs in the alfaGSU lineage. Additionally, these findings explain the pituitary hypoplasia observed in animals with defects in early transcription factors, as indicated by the accumulation of stem cells in the Snell and Ames lineages, preventing the initiation of pituitary differentiation

Le développement du rein est un exemple intriguant d’un équilibre délicat entre la prolifération des cellules progénitrices, la différentiation et l’apoptose. Le gène Wt1 est indispensable pour la survie des cellules progénitrices. Le but de cette thèse a été de définir les voies de signalisation activées par Wt1 pendant le développement du rein. En utilisant les souris Wt1 KO, nous avons démontré que WT1 coordonne l’action de deux voies de signalisation opposées : Fgf et Bmp/Smad intervenant dans la survie des cellules progénitrices rénales. Dans une deuxième étude, nous avons analysé le rôle du modificateur épigénétique, le gène Phf19 pendant le développement du rein. Nous avons démontré que l’expression de ce gène est Wt1-dependant et il est exclusivement exprimé dans les cellules progénitrices rénales au cours du développement et que son inactivation dans le rein embryonnaire en culture, conduit à l’apoptose des cellules progénitrices. Nous avons généré des souris knockout de Phf19 par l’approche de CRISPR/Cas9. Dans le cas d’une létalité précoce des embryons homozygotes, nous opterons pour la production du model animal knockout conditionnel et procéderons à la caractérisation de leur profile épigénétique. Cette thèse a permis d’une part, de découvrir deux voies de signalisation antagonistes, régulées par le Wt1et impliquées dans le contrôle de la survie des cellules progénitrices rénales et d’autre part de nous orienter vers le contrôle de la survie et la prolifération de ces cellules par modifications épigénétiques. Ceci nous permettra de contribuer à la connaissance de l’étiologie d’une grande proportion des malformations rénales restant à ce jour inconnues
Kidney organogenesis requires the tight control of proliferation, differentiation and apoptosis of renal progenitor cells. The Wilms’ tumour suppressor Wt1 is required for renal progenitor survival. The aim of this thesis was to elucidate the molecular cause for renal agenesis in Wt1 mutant mouse. Here we demonstrate that lack of Wt1 abolishes FGF and induces BMP/pSMAD signaling within the metanephric mesenchyme. We further show that recombinant BMP4, but not BMP7, induces an apoptotic response within the early kidney that can be suppressed by simultaneous addition of FGFs. These data reveal an unknown sensitivity of early renal progenitors to pSMAD signalling, establishes FGF and pSMAD signalling as antagonistic forces in early kidney development and places WT1 as a key regulator of pro-survival FGF signalling pathway genes. In a second study, we demonstrated, that Phf19, an epigenetic modifier, is essential both for maintaining Wt1 expression in renal progenitor cells and their survival in an ex-vivo culture. We further generated a Phf19 knockout mouse by CRISPR/Cas9. The homozygous embryos will be analyzed to further decipher the contribution of Phf19 to potential kidney malformations and the epigenetic profile of renal progenitor cells will be characterized. Overall, the new insights into the molecular mechanisms controlling the survival of renal progenitor cells, reported in this thesis, provide one more step in our understanding of renal malformations. In addition, our results conducted us toward the epigenetique modifications that could open up promising new avenue of understanding the etiology of an important proportion of renal malformation that remains unknown

Lo studio affrontato in questa tesi ha evidenziato come il danno renale acuto possa influire sullo sviluppo di carcinoma renale, e in particolare delle forme papillari di carcinoma, agendo sulla via di segnalazione di Notch. Abbiamo preso in considerazione una popolazione di progenitori renali localizzati tra le cellule epiteliali parietali (PEC) e le cellule epiteliali tubulari (TEC), la cui proliferazione e differenziazione è controllata dalla via di segnalazione di Notch. Abbiamo lavorato in vivo allestendo linee murine transgeniche in cui i progenitori renali sono identificati dal marcatore Pax2. Per valutare come il danno renale acuto influisse sullo sviluppo neoplastico abbiamo sottoposto gli animali ad ischemia/riperfusione. Lo studio ha identificato i tumori papillari come clonali e ha identificato nelle cellule Pax2+ la popolazione che, in seguito ad una aberrante espressione di Notch1, dà origine ad adenomi e carcinomi papillari. Inoltre identifica il danno renale acuto come un fattore di rischio per lo sviluppo di tumori papillari nel topo. The study dealt with in this thesis has shown that acute kidney injury can affect the development of renal carcinoma, and in particular papillary carcinoma, acting on the Notch signaling pathway. We considered a population of renal progenitors located between parietal epithelial cells (PEC) and tubular epithelial cells (TEC), whose proliferation and differentiation is controlled by the Notch signaling pathway. We worked in vivo setting up transgenic murine lines in which the renal progenitors are identified by the Pax2 marker. To assess how acute kidney injury affected neoplastic development, we subjected the animals to ischemia / reperfusion injury. The study identified papillary tumors as clonal and identified Pax2 + cells as the population that, following an aberrant expression of Notch1, gives rise to papillary adenomas and carcinomas. It also identifies acute kidney injury as a risk factor for the development of papillary tumors in the mouse.

Questo studio ha dimostrato che il carcinoma renale papillare (pRCC), uno dei 3 più frequenti tipi di RCC, origina dall’espansione clonale di una popolazione di cellule staminali residenti nel rene, i progenitori renali . In seguito ad un danno renale acuto , queste cellule sono in grado di rigenerare la porzione di tubulo renale danneggiata. Tuttavia, in seguito al danno si ha l’alterazione di alcune pathways che possono promuovere la loro trasformazione in cellula staminale tumorale (CSC, cancer stem cell). In particolare, abbiamo dimostrato con questo lavoro di tesi che l’overespressione della pathway di NOTCH1, è specifica per il pRCC di tipo 2 ed è associata ad una prognosi sfavorevole nell’uomo . Infine, abbiamo dimostrato che NOTCH1 promuove la trasformazione del progenitore renale, sia in vivo che in vitro promuovendone la proliferazione clonale incontrollata favorendo delle mitosi aberranti. I risultati di questo studio rappresentano un’importante scoperta dal punto di vista clinico, per la diagnosi e il trattamento dei pazienti con questo tipo di tumore.

I progenitori renali hanno un ruolo di primaria importanza nella capacità del rene di far fronte allo stress da filtrazione glomerulare e di ripristinare prontamente i podociti danneggiati durante la gravidanza. La stimolazione estrogenica guida tale adattamento e se carente può fare la differenza tra il successo della gravidanza e l’insorgenza di complicanze quali la preeclampsia. Questi risultati aggiungono un tassello in più verso l a comprensione dell’eziopatogenesi della preeclampsia, in quanto pongono il rene, in particolare la capacità dei progenitori renali di generare nuovi podociti, in risposta agli estrogeni, tra le cause che predispongono alla patologia. In vitro colture primarie di progenitori renali umani risultano essere sensibili alla stimolazione estrogenica che agisce attraverso il recettore ER α . Questo effetto trova riscontro negli esperimenti in vivo , attraverso cui è stato possibile dimostrare che la carenza del recettore α degli estrogeni sui progenitori renali ha effetti sulla loro capacità di differenziare in podociti. Il che predispone ad un maladattamento funzionale del rene alla gravidanza.

Evaluation of tubular regenerative response in AKI-induced trasngenic rodent models. Discovered the existence of a progenitor population scattered in kidney tubule of adut mice devoted to tubular epithelium recovery after ischemic injury.

博士
國立成功大學
臨床醫學研究所
98
In Taiwan, the incidence of end-stage renal disease ranked first and the prevalence ranked second in the world. Patients with end-stage renal disease need hemodialysis, peritoneal dialysis or kidney transplant to maintain their life. For the shortage of available organ, most of the patients are under regular dialysis. To accelerate renal repair or even make a functioning kidney is the emergent issue to be solved and interesting topic for research. Following the discovery of tissue-specific progenitor cells in other organs and their ability to improve regeneration after injury, progenitor cell-based therapy is a new strategy in the treatment of acute kidney injury and has potentially more value than single-agent drug therapy due to the highly versatile response of cells to their environment. These cells may not only secrete cytokines within the injured kidney, but also participate in tubular cell proliferation or angiogenesis to facilitate renal regeneration. In rodents, increasing evidence suggests that the therapeutic potential of mesenchymal stem cells derived from bone marrow could be beneficial in the kidney injury. Thereby, we hypothesize that kidney progenitor cells may accelerate renal regeneration after injury. We first observed the regenerative process of acute tubular necrosis in rodents. In the normal kidney, only interstitial cells but not tubular cells expressed vimentin. Following acute renal failure, vimentin-positive renal interstitial cells proliferated and surrounded the damaged renal tubules as early as 12 hours after injury. Within the regenerating tubules, vimentin staining was found intensely two days after injury, and disappeared after full recovery of tubular epithelial cells. By known interstitial cell markers, only few vimentin-positive renal interstitial cells were characterized as endothelial cells or fibroblasts one day after acute renal failure. Most of the other proliferating cells were not specified and we hypothesize that kidney progenitor cells could reside in these areas. Using bromodeoxyuridine (BrdU) as a marker of proliferating cells, we monitor the distribution of the interstitial cells by immunohistochemistry during acute renal failure. Following one injection of BrdU, eighty five percent of BrdU labeling cells located in the interstitium 12 hours after acute renal failure and the count decreased to 25% at the 4th day. Interestingly, BrdU labeling cells redistributed to the regenerating tubules at the 1st and 4th day. Seventy-five percentage of BrdU labeling cells located in the tubules at the 4th day. As assessed by ELISA, the uptake of BrdU in the kidney peaked at the 1st day, decreased to constant level after 3 days, and maintained till 7 days following one injection of BrdU before acute renal failure. These results indicate that interstitial cells might be engaged in the process of tubular regeneration after acute renal failure. We test the hypothesis that renal progenitor cells isolated from adult mouse kidney accelerate renal regeneration via participation in the repair process. A unique population of cells exhibiting characteristics consistent with renal progenitor cells, mouse kidney progenitor cells (MKPC), was isolated from Myh9 targeted mutant mice. Features of these cells include: (1) spindle-shaped morphology, (2) self-renewal of more than 100 passages without evidence of senescence, (3) expression of Oct-4, Pax-2, Wnt-4, WT-1, vimentin, alpha-smooth muscle actin, CD29 and S100A4 but no SSEA-1, c-kit, or other markers of more differentiated cells. MKPC exhibit plasticity as demonstrated by the ability to differentiate into endothelial cells and osteoblasts in vitro and endothelial cells and tubular epithelial cells in vivo. The origin of the isolated MKPC was from the interstitium of medulla and papilla. Importantly, intra-renal injection of MKPC in mice with ischemic injury rescued renal damage, as manifested by decreases in peak serum urea nitrogen, the infarct zone and the necrotic injury. Seven days after the injury, some MKPC formed vessels with red blood cells inside and some incorporated into renal tubules. In addition, MKPC treatment reduces the mortality in mice after ischemic injury. Our results indicate that MKPC represent a multipotent adult progenitor cell population, which may contribute to the renal repair and prolong survival after ischemic injury. The PhD study not only raised a novel method to treat acute renal failure but also open a new window to elucidate the relationship between kidney progenitor cells and tubular regeneration. Based on these, we will be able to unveil the mechanism of how tissue-specific progenitor cells involve in the process of tissue regeneration.

Tese de Doutoramento em Engenharia de Tecidos, Medicina Regenerativa e Células Estaminais
Kidney diseases represent a major healthcare burden worldwide. It is estimated that one in ten persons suffer from kidney dysfunction. Indeed, a decline in renal function is considered an independent risk factor for both cardiovascular disease and all-cause mortality. The problem is only aggravated by treatment alternatives, with dialysis and transplantation being the only currently available renal replacement therapies. Envisioning the functional recovery of the injured kidney, two regenerative resources were explored: porcine-derived kidney decellularized matrices and renal progenitor cells from human origin. For that, the decellularization process was optimized to obtain porcine kidney decellularized tissue. A full characterization of this matrix in terms of morphology, structural integrity, biochemical content, thermal and molecular properties and protein content was performed. Indeed, porcine-derived matrices were validated as an adequate raw material for the production of several decellularized-based substrates. Namely, kidney tissue-derived electrospun membranes were fabricated for the development of a tubular filtration barrier model. Additionally, decellularized tissue was used for the fabrication of a particulate matrix and a bioink, where 3D cultures of isolated renal cells were established envisioning implantation. Indeed, these cells already shown to possess reparative properties when injected into the injured kidney tissue alone, with limited efficacy. They were also used to develop an organoid model of the glomerulus. Overall, renal progenitor cells demonstrated versatility, specific differentiation into renal phenotypes and proliferation capacity when embedded on the matrix substrates. The works developed in this thesis show that decellularized-based biomaterial substrates may have multiple applications, from modeling systems to moldable implantable scaffolds for tissue engineering strategies. These substrates demonstrated physiological kidney tissue characteristics, allowing cultured cells to represent morphological, phenotypic and functional properties of in vivo systems. Ultimately, this thesis allowed for the development of advanced strategies comprising both relevant cells and biomaterial substrates that may have greater implications in the biomedical field as promising solutions to address renal pathologies in its early stages.
As doenças renais representam um grande problema económico-social. Está estimado que uma em cada dez pessoas sofre de disfunção renal. São também consideradas um fator de risco para doenças cardiovasculares e para a taxa de mortalidade geral. Este problema é agravado visto que as únicas opções de tratamento atuais são a diálise e transplantação. Visto que o objetivo inicial da tese é a recuperação do rim lesado, foram explorados dois recursos regenerativos: matriz extracelular de rim de porcino decelularizadas e células progenitoras renais de origem humana. O processo de descelularização foi cuidadosamente otimizado com vista a obter matrizes descelularizadas. Posteriormente, foi feita uma caracterização completa da matriz de porcino em termos de morfologia, integridade estrutural, conteúdo bioquímico, propriedades térmicas e moleculares e ainda conteúdo proteico. Estas matrizes foram usadas como base para produção de diversos biomateriais. Foram fabricadas membranas fibrosas por electrofiação para o desenvolvimento de um modelo in vitro da barreira de filtração tubular. A matriz descelularizada foi usada para obter partículas e também uma biotinta, onde foram estabelecidas culturas 3D de células renais isoladas, com o objetivo final de implantação. Estas células demonstraram anteriormente o seu potencial quando injetadas num rim lesado, com limitada eficácia. Nesta tese, as mesmas células foram também usadas para desenvolver um modelo de organoide do glomérulo. As células progenitoras renais demonstraram ter versatilidade, capacidade de diferenciação específica em fenótipos renais e de proliferação quando cultivadas nas matrizes. Os materiais produzidos a partir da matriz decelularizada demonstraram poder ser usados em múltiplas aplicações, desde modelos para estudos in vitro até scaffolds implantáveis para estratégias de engenharia de tecidos. Estes biomateriais demonstraram ter uma variabilidade fisiológica semelhante à do rim, o que permitiu às células cultivadas modelar propriedades dos sistemas in vivo, nomeadamente morfológicas, fenotípicas e funcionais. Por fim, esta tese permitiu o desenvolvimento de estratégias avançadas compostas por células e biomateriais relevantes, que podem ter imensas implicações biomédicas como soluções promissoras para o tratamento de lesões renais em estágios iniciais.
The authors want to acknowledge the financial support obtained by the European Regional Development Fund (ERDF) on the project FROnTHERA (NORTE-01-0145-FEDER-000023) and the FCT PhD Grant on the Doctoral Program on Advanced Therapies for Health (PATH) (PD/BD/128102/2016).

Angiogenesis is a fundamental biological process during development as well as in adult life, both in physiological and pathological conditions, as for example in cancer development. Endothelial Progenitor Cells (EPCs) are cells with self-renewal capacity, able to differentiate into mature Endothelial Cells (ECs) and to generate patent vessels in vivo. EPCs seem to play a fundamental role in the "angiogenic switch", the process which allows the transition from in situ carcinoma to a highly vascularized tumor; therefore, they could be a potential target in the development of new drugs. In our work we compared healthy subjects derived EPCs (N-EPCs) and EPCs isolated from patients with renal cell carcinoma (RCC-EPCs), focusing on frequency, proliferation rate, tubulogenesis ability, apoptosis and ultrastructure. Furthermore, we analyzed EPCs Ca2+ signaling pathway, especially after treatment with VEGF, the principal mitogenic stimulus for EPCs, as we already know that neoplastic transformation is accompanied by a remodeling of the Ca2+ toolkit. We found that circulating EPCs are significantly more frequent in patients, having otherwise the same immunophenotypic profile, overlapping growth curve and tubulogenic capacity. We also showed that RCC-EPCs are more resistant to apoptosis induced by rapamycin. Moreover, VEGF induces NF-kB nuclear translocation in N-EPCs in a Ca2+ dependent manner, increasing related gene and protein expression. Based on the concept that VEGF does not induce Ca2+ oscillation in RCC-EPCs, we showed that RCC-EPCs anyway express a functional VEGF receptor 2 which is not able to induce gene and protein expression, suggesting a possible downstream blockade of the pathway. Finally, we demonstrated that RCC-EPCs has less Ca2+ amount in endoplasmic reticulum and mitochondria, fact that potentially reflects the ultrastructural rearrangement found in RCC-EPCs, with their more widely spaced smooth and rough endoplasmic reticulum and more abundant mitochondria in RCC-EPCs.The results reported in this work strongly highlights circulating EPCs in cancer patients as potential target for new therapies aimed to blocking tumor angiogenesis.