published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

L’athérosclérose et la pathologie anévrysmale sont principalement caractérisées par un remodelage de laparoi artérielle au cours de leur évolution. Ce travail a examiné un aspect de la destruction de la paroiartérielle à travers l’étude de la métalloprotéase MMP-14 au cours de l’athérome et un aspect dereconstruction artérielle à travers l’étude d’une thérapie cellulaire d’un modèle d’Anévrysme de l’AorteAbdominale (AAA) par Cellules Souches Mésenchymateuses (CSMs).En utilisant un modèle de greffe de Moëlle Osseuse (MO) dans des souris Ldlr-/-, nous avons montré que ladélétion d’expression de MMP-14 dans les cellules issues de la MO provoquait une accumulation decollagène interstitiel dans la plaque athéromateuse sans modification de la composition cellulaire nivariation de taille. Une mesure de l’activité collagénolytique par substrat fluorescent a confirmé que ladélétion en MMP-14 chez les macrophages provoquait une baisse de l’activité collagénolytique. Cetteactivité est indépendante de l’activité MMP-2 et MMP-8 et pourrait être médiée partiellement parl’activation de MMP-13. Nous avons mis en évidence la présence de CSMs à la surface luminale de thrombus de AAA et nous avonsmontré une diminution significative des CSMs circulantes chez des patients porteurs de AAA. Nous avonspu stabiliser la croissance de AAA expérimentaux chez le rat à partir de xénogreffe artérielle par perfusionendoluminale de CSMs. La perfusion de CSMs provoquait une diminution de l’inflammation à court termeet favorisait la reconstruction artérielle par accumulation de collagène et d’élastine à moyen terme.En conclusion, l’activité collagénolytique de MMP-14 est un des mécanismes moléculaires possibles del’évolution de la plaque athéromateuse par rupture de plaque. Elle ouvre la perspective d’une nouvelleapproche thérapeutique et pourrait être une cible comme substrat pour une imagerie fonctionnelle de laplaque athéromateuse. L’évolution de la maladie anévrysmale pourrait être secondaire à une altération dessystèmes de réparation tissulaire dont les CSMs seraient des acteurs clé. La perfusion endoluminale desCSMs dans un modèle expérimental a permis la restauration de ces systèmes de réparation tissulaire etouvre la perspective d’un nouvel outil thérapeutique contre les AAA / Pas de résumé anglais

A membrana amniótica é uma membrana translucida sendo a membrana mais interna da cavidade amniótica, formada por uma monocamada de células epiteliais disposta sobre uma membrana basal. Com o crescente interesse na utilização de células-tronco provenientes de anexos fetais, esta se torna uma promissora fonte de células-tronco. Sendo assim em trabalho anterior realizado pelo nosso grupo tivemos como objetivo, o estabelecimento da cultura celular e caracterização das células-tronco fetais de membrana amniótica de cão para verificar se a mesma pudesse ser uma nova fonte celular a ser usada nos protocolos de terapia celular, uma vez que os cães têm sido considerados modelos animais atraentes para avaliar novas drogas ou realizar ensaios pré-clínicos. As células de membrana amniótica obtidas a partir do trabalho anterior foram caracterizadas in vitro, observando-se características semelhantes a outras células-tronco mesenquimais. Porém, quando foi analisado o seu o seu potencial carcinogênico observamos a formação de um tumor de crescimento rápido, aproximadamente um mês, após o inóculo dessas células em 10 camundongos imunossuprimidos nude, sendo o tumor identificado histologicamente como um carcinoma embrionário. Diante deste comportamento acreditamos ser de extrema importância analisar células provenientes de novas coletas verificando se a mesmas podem se comportar como as anteriormente estudadas. Com isso, temos como objetivo deste trabalho estabelecer e caracterizar as células provenientes de duas novas coletas em diferentes períodos gestacionais visando verificar se estas células se comportam da mesma que as anteriores isso é se quando inoculadas nos animais formam tumor e assim poder ter certeza de que essas células são ou não, boas alternativas para terapia celular. As células obtidas nestas novas coletas têm características de células-tronco mesenquimais expressando alguns marcadores, tem curva de crescimento semelhante às células-tronco mesenquimais, se aderem ao plástico e se diferenciaram em adipócitos. Diferentemente das células obtidas no estudo anterior estas células não geraram tumor quando injetadas em camundongos imunossuprimidos, nude em até 60 dias após inoculação. / Amniotic membrane is a membrane translucent with the inner membrane of the amniotic cavity, formed by a monolayer of epithelial cells disposed on a basal membrane. With the growing interest in the use of stem cells from fetal membranes, this becomes a promising source of stem cells. So in a previous study conducted by our group we aim, the establishment of cell culture and characterization of fetal stem cells from amniotic membrane from dog to see if it could be a new source cell to be used in cell therapy protocols, Once the dogs have been considered attractive animal models to evaluate new drugs or performing pre-clinical tests. The cells from amniotic membrane obtained from previous work were characterized in vitro, observing characteristics similar to other mesenchymal stem cells. But when it was analyzed its its carcinogenic potential observed the formation of a fast-growing tumor, approximately one month after inoculation of these cells into immunocompromised nude mice 10, and the tumor identified histologically as embryonal carcinoma. Given this behavior we believe is extremely important to analyze cells from new collections by checking if the same can behave like those previously studied. With this, we aim of this work to establish and characterize the cells from two new collections at different gestational periods to verify whether these cells behave the same as the previous ones is that when inoculated into animals form tumor and thus be able to make sure that these cells are either not good alternatives for cell therapy. The cells obtained in these new collections has characteristics of mesenchymal stem cells expressing some markers, growth curve is similar to mesenchymal stem cells, adhere to plastic and differentiated into adipocytes. Unlike the cells obtained in the previous study these cells did not generate tumors when injected into immunocompromised mice, nude within 60 days after inoculation.

O órgão vômeronasal (OVN), é uma estrutura que detecta feromônios, emitindo sinais que modulam o comportamento social e reprodutivo. Possui células-tronco que se dividem e migram para substituir neurônios ao longo da vida. O objetivo deste estudo foi isolar e caracterizar as células derivadas do órgão vômeronasal de coelhos da raça Nova Zelândia e testar seu potencial terapêutico no tratamento da ablação da via vômeronasal. Utilizou-se 10 coelhos machos com 120 dias, sendo 9 submetidos a ablação do OVN e 1 utilizado para coleta de material para cultivo celular. Foram testados três meios de cultivo DMEM High Glucose, DMEM/F12 e MEM Alfa. No cultivo celular observou-se maior confluência e crescimento quando utilizado o DMEM High Glucose, confirmado pelos ensaios de MTT e Azul de Trypan. Na imunocitoquímica observou-se PCNA+, OCT4+, Nanog+, GFAP+, Vimentina+, Nestin+, Stro-1+, B-Tubulina+, CK-18+, CD73+, CD90+, CD105+, CD34-, CD117- e CD45-. Na citometria de fluxo foi observado PCNA+, OCT-3/4+, Nanog+, GFAP+, Vimentina+, Nestin+, Stro-1+, B-Tubulina+, CK-18+, CD73+, CD90+, CD105+, CD34- e CD45-. Na análise molecular foi possível observar a expressão de CD73, CD105, Oct-4, Nestina, Vimentina e GAPDH. Funcionalmente as células se diferenciaram em adipócitos, osteócitos e condrócitos e não possuem potencial tumorigênico em camundongos Balb-cnu/nu. A análise hormonal demonstrou que nos animais tratados após 7 e 14 dias não houveram diferenças significativas entre os grupos tratados e controles. No grupo após 21 dias notou-se uma diferença de 50% nos níveis hormonais no grupo tratado em relação ao grupo controle. Na análise da expressão para eGFP observou-se que os animais tratados após 7 dias as células injetadas formavam aglomerados celulares na região subjacente ao epitélio sensitivo. Após 14 dias continuavam a circundar o epitélio neurosensorial e após 21 dias observou-se pouca expressão de células localizadas apenas na região subjacente ao epitélio neurosensorial. A linhagem celular derivada do órgão vômeronasal possui uma população de células progenitoras, que foram visualizadas no foco da lesão, demonstrando sua capacidade de migração, o que a torna uma boa fonte para terapia celular em ablação da via vômeronasal. / The vômeronasal organ (VNO) is a structure that detects pheromones, emits signals that modulate social and reproductive behavior. It has stem cells that divide and migrate to replace neurons throughout life. The aim of this study was to isolate and characterize cells derived from the vômeronasal organ from rabbits New Zealand and test their therapeutic potential in the treatment of ablation of the vômeronasal pathway. 10 male rabbits with 120 days were submitted to the ablation of VNO and 2 controls animals used to collect material for cell culture. Three different culture media DMEM High Glucose, Alpha MEM and DMEM/F12 were tested. In cell culture observed greater confluence and growth when used DMEM High Glucose, confirmed by MTT assay and Trypan Blue. In immunocytochemistry observed PCNA + OCT4 +, Nanog +, GFAP + Vimentin +, Nestin +, Stro-1 + B-tubulin +, CK-18 +, CD73 +, CD90 +, CD105 +, CD34-, CD117-and CD45-. In flow cytometry was observed PCNA + OCT-3/4 + Nanog +, GFAP + Vimentin +, Nestin +, Stro-1 + B-tubulin +, CK-18 +, CD73 +, CD90 +, CD105 +, CD34-and CD45-. Molecular analysis was possible to observe the expression of CD73, CD105, Oct-4, Nestin, vimentin and GAPDH. Functionally, the cells differentiate into adipocytes, osteocytes and chondrocytes have not Balb-cnu/nu tumorigenic potential in mice. Hormonal analysis demonstrated that animals treated after 7 days and 14 days there were no significant differences between treated groups and controls. In group after 21 days noticed a 50% difference in hormone levels in the treated group compared to the control group. In expression analysis for eGFP was observed that the treated animals after 7 the injected cells formed cell clusters in the underlying sensory epithelium region. After 14 days continued to surround the neurosensory epithelium and after 21 days there was little expression in the cells located just behind the neurosensory epithelium region. The cell line derived from the vômeronasal organ has a population of progenitor cells that were visualized in the lesion focus, demonstrating their ability to migrate, which makes it a good source for cell therapy in ablation of the vômeronasal pathway.

A membrana amniótica humana vem assumindo um papel de extrema importância na medicina regenerativa nos últimos anos, principalmente na área de dermatologia e oftalmologia, sendo seu uso promissor no tratamento de doenças cuja terapêutica atual é pouco eficaz. Além disso, na membrana amniótica humana encontram-se células-tronco mesenquimais, que apresentam plasticidade e vem sendo aplicadas para a regeneração de tecidos. Entretanto, muito pouco se sabe sobre a capacidade plástica e o uso de células-tronco de membrana amniótica de animais, já que a literatura a este respeito, não é tão ampla quanto à de humanos. Neste projeto estabelecemos a cultura de células-tronco de membrana amniótica (MA) de fetos caninos, caracterizando as células in vitro e in vivo. As células de MA foram obtidas a partir de um procedimento cirúrgico de histerectomia em cadelas prenhas, durante campanhas de castração da prefeitura da cidade de São Paulo. As células de MA foram caracterizadas in vitro, observando-se características semelhantes a outras células-tronco mesenquimais. Porém, quando foi analisado o seu comportamento in vivo, observamos a formação de um tumor de crescimento rápido, aproximadamente um mês, após o inóculo dessas células em 10 camundongos imunossuprimidos nude, sendo o tumor identificado histologicamente como um carcinoma embrionário. Diante do comportamento biológico formando um tumor in vivo, inferimos que células-tronco provenientes de membrana amniótica de cães não devem ser usadas para aplicação com objetivos terapêuticos em animais, pelo menos até que novas coletas sejam realizadas para que se possa confirmar ou não este comportamento. / The human amniotic membrane has taken an extremely important role in regenerative medicine in recent years, especially in dermatology and ophthalmology, and its promising use in treating diseases for which current therapy is ineffective. In addition, the amniotic membrane has human mesenchymal stem cells, which exhibit plasticity and have been applied to tissue regeneration. However, very little is known about the plastic capacity and the use of stem cells from amniotic membrane of animals, since the literature in this regard, it is not as broad as those of humans. In this project we established a culture of amniotic stem cells of dog, characterizing these cells in vitro and in vivo. The cells were obtained from a surgical procedure for hysterectomy in pregnant bitches, during castration\'s campaigns of the São Paulo\'s municipality. The cells were characterized in vitro, observing characteristics similar to other mesenchymal stem cells. In spite of their behavior, in vivo we observed the formation of a fast-growing tumor about a month after the inoculation of these cells in 10 immunosuppressed mice, being the tumor identified histologically as an embryonic carcinoma. Considering the biological behavior of forming a tumor in vivo, we infer that stem cells from amniotic membrane of dogs should not be used for application for therapeutic purposes in animals, at least until other collections are carried out so that it can be confirmed or not.

Myocardial infarction results in tissue necrosis, leading to cell loss and ultimately to cardiac failure. Implantation of immature progenitor cells into the scar area may compensate for the cell loss and provides a new therapeutic avenue for infarct treatment. Premature myoblasts derived from skeletal muscle are one of the best candidates for this therapeutic purpose, because biopsies used for autologous cell therapy can be accessed easily, the isolated myoblasts can proliferate well in vitro, and the skeletal and cardiac muscles are structurally and functionally similar. In this study we investigated the survival and differentiation of the implanted skeletal myoblasts in the non-cryoinjured myocardium and the myocardial scar, using a syngeneic Lewis rat model. A therapeutic dose of 4x106 skeletal myoblasts/animal was implanted into the non-cryoinjured and scar tissue, and the fate of the implant was monitored at 12, 28 and 56 days after implantation by immunohistochemistry. We detected fast myosin heavy chain (fMHC) expression at each time point but significantly fewer positive cells in the scar than in the non-injured tissue. This was consistent with the staining patterns of slow myosin heavy chain (sMHC) and myogenin that overlapped with fMHC positive areas. Although the implanted myoblasts differentiated into skeletal muscle cells, they did not transdifferentiate into cardiac muscle, demonstrated by the absence of cardiac troponin I expression. During this analysis we developed a model, which could be useful to test new strategies for myoblast implantation (dosage, genetic modification, new injection technique etc.) designed to promote better engraftment of cultured myoblasts in the myocardial scar.

A practical consideration in the development of cellular therapy technology for the inner ear is the development of an in vitro model for assessing the optimal conditions for successful application of cells. The first part of this thesis describes the adaptation of the cochleovestibular structure harvested from P1 mouse pups for analysis of factors critical for the optimal implantation of stem cells in the inner ear. Results of these studies establish that the c17.2 neural stem cell line can be introduced into the cochleovestibular structure in vitro. Using this model, c17.2 cells demonstrated survival predominantly within the vestibule and basal spiral ganglion regions. Furthermore, the addition of the ototoxin, cisplatin and the neurotrophin, Brain Derived Neurotrophic Growth Factor (BDNF) enhanced the survival and migration/dispersion of c17.2 cells within the cochleovestibular explant. The second part of this thesis examines the hypothesis that olfactory neurosphere (ONS) and progenitor cells harvested from the olfactory epithelium represent a viable source of graft material for potential therapeutic applications in the inner ear. Olfactory epithelium represents a unique source of pluripotent cells that may serve as either homografts or autografts. The feasibility of ONSs to survive and integrate into a mammalian cochlea in vivo was assessed. The ONSs were isolated as a crude fraction from the olfactory epithelium of P1 to P3 day old swiss webster mouse pups, ubiquitously expressing the Green Fluorescent Protein (GFP) marker. The ONSs were microinjected into the cochleae of adult CD1 male mice. Four weeks following their implantation, ONS cells expressing the GFP marker and stained by Nestin were identified in all areas of the cochlea and vestibule, including the spiral ganglion. Robust survival and growth of the implanted ONS and ONS derived cells in the cochlea also included the development of ???tumor-like??? clusters, a phenomenon not observed in control animals implanted with c17.2 neural stem cells. Collectively, the results of this thesis illustrate the potential of olfactory neurosphere and progenitor cells to survive in the inner ear and expose a potential harmful effect of their transplantation.

Cell-based therapies have potential for tissue regeneration but poor delivery methods lead to low viability or dispersal of cells from target sites, limiting clinical utility. Here, we developed a degradable and injectable hydrogel to deliver stem cells for bone regeneration. Alginate microbeads <200µm are injectable, persist at implantation sites and contain viable cells, but do not readily degrade in-vivo. We hypothesized that controlled release of rat adipose-derived stem cells (ASCs) from alginate microbeads can be achieved by incorporating alginate-lyase in the hydrogel. Microbeads were formed using high electrostatic potential. Controlled degradation was achieved through direct combination of alginate-lyase and alginate at 4°C. Results showed that microbead degradation and cell release depended on the alginate-lyase to alginate ratio. Viability of released cells ranged from 87% on day 2 to 71% on day 12. Monolayer cultures of released ASCs grown in osteogenic medium produced higher levels of osteocalcin and similar levels of other soluble factors as ASCs that were neither previously encapsulated nor exposed to alginate-lyase. Bmp2, Fgf2, and Vegfa mRNA in released cells were also increased. Thus, this delivery system allows for controlled release of viable cells and can modulate their downstream osteogenic factor production.

The Royal College of Surgeons (RCS) rat was used as a model of human retinal degenerative disease, and for studying the efficacy of cell transplanation treatments. In order to characterize the spatial vision of the RCS strain, the visual acutiy and contrast sensitivity of adult non-dystrophic RCS rats was measured. The acuity and contrast sensitivity of these rats was normal. The acuity of dystrophic RCS rats was alos characterized to determine how photoreceptor degeneration affects vision. These rats progressively lost visual acuity from one month of age until elevn months of age when they were judged to be blind. The degeneration of vision in these animals was more protacted than would be predicted from previous anatomical and electrophysiological measures. Subretinal transplantation of human-derived Retinal Pigment Epithelial (RPE) cells and human Schwann cells into the dystrophic RCS rat significantly delayed the loss of visual acuity. These studies show that cell transplantation may be a viable method of limiting loss of vision in humans with retinal degenerative blinding diseases. / vii, 77 leaves ; 29 cm.

A practical consideration in the development of cellular therapy technology for the inner ear is the development of an in vitro model for assessing the optimal conditions for successful application of cells. The first part of this thesis describes the adaptation of the cochleovestibular structure harvested from P1 mouse pups for analysis of factors critical for the optimal implantation of stem cells in the inner ear. Results of these studies establish that the c17.2 neural stem cell line can be introduced into the cochleovestibular structure in vitro. Using this model, c17.2 cells demonstrated survival predominantly within the vestibule and basal spiral ganglion regions. Furthermore, the addition of the ototoxin, cisplatin and the neurotrophin, Brain Derived Neurotrophic Growth Factor (BDNF) enhanced the survival and migration/dispersion of c17.2 cells within the cochleovestibular explant. The second part of this thesis examines the hypothesis that olfactory neurosphere (ONS) and progenitor cells harvested from the olfactory epithelium represent a viable source of graft material for potential therapeutic applications in the inner ear. Olfactory epithelium represents a unique source of pluripotent cells that may serve as either homografts or autografts. The feasibility of ONSs to survive and integrate into a mammalian cochlea in vivo was assessed. The ONSs were isolated as a crude fraction from the olfactory epithelium of P1 to P3 day old swiss webster mouse pups, ubiquitously expressing the Green Fluorescent Protein (GFP) marker. The ONSs were microinjected into the cochleae of adult CD1 male mice. Four weeks following their implantation, ONS cells expressing the GFP marker and stained by Nestin were identified in all areas of the cochlea and vestibule, including the spiral ganglion. Robust survival and growth of the implanted ONS and ONS derived cells in the cochlea also included the development of ???tumor-like??? clusters, a phenomenon not observed in control animals implanted with c17.2 neural stem cells. Collectively, the results of this thesis illustrate the potential of olfactory neurosphere and progenitor cells to survive in the inner ear and expose a potential harmful effect of their transplantation.