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Potential targeting of impaired wounds resulting from hyperglycemia using mesenchymal stem cells is a promising approach. We hypothesized that when administered to dermal wounds in hyperglycemic subjects, neonatal mesenchymal stem cells (MSCs) would be more effective than adult mesenchymal cells in accelerating healing. We examined the in vitro impact of various glucose conditions on proliferation and senescence of human umbilical cord perivascular cells (HUCPVCs) and adult bone marrow MSCs (hBM-MSCs). We also investigated the healing potential of both cells in dermal wounds of streptozotocin-induced NOD-scid-gamma (NSG) mice utilizing planimetry and histomorphometry. HUCPVCs showed higher proliferation under normal and hyperglycemic conditions and lower senescence under all conditions compared to hBM-MSCs. Wound closure was better in treated wounds compared to untreated wounds. Disease tolerance varied among mice which affected healing. HUCPVCs still holds a potential over adult MSCs for impaired wounds; yet more studies are needed to recognize their bona fide capacity.

Potential targeting of impaired wounds resulting from hyperglycemia using mesenchymal stem cells is a promising approach. We hypothesized that when administered to dermal wounds in hyperglycemic subjects, neonatal mesenchymal stem cells (MSCs) would be more effective than adult mesenchymal cells in accelerating healing. We examined the in vitro impact of various glucose conditions on proliferation and senescence of human umbilical cord perivascular cells (HUCPVCs) and adult bone marrow MSCs (hBM-MSCs). We also investigated the healing potential of both cells in dermal wounds of streptozotocin-induced NOD-scid-gamma (NSG) mice utilizing planimetry and histomorphometry. HUCPVCs showed higher proliferation under normal and hyperglycemic conditions and lower senescence under all conditions compared to hBM-MSCs. Wound closure was better in treated wounds compared to untreated wounds. Disease tolerance varied among mice which affected healing. HUCPVCs still holds a potential over adult MSCs for impaired wounds; yet more studies are needed to recognize their bona fide capacity.

Esse projeto tem como objetivo estabelecer técnicas de cultivo, isolamento, expansão e quantificação de células mesenquimais e hematopoéticas de sangue de cordão umbilical (SCU) de cães de raças variadas para aplicação em terapias celulares. Atualmente, vários tipos de células-tronco têm sido estudados devido a sua capacidade de diferenciação em diversos tecidos (HERZOG et al., 2003). O isolamento, quantificação e expansão dessas células permitem que a terapia celular seja utilizada na tentativa de tratamento em patologias (BARKER et al., 2003). Entre as fontes de células-tronco adultas (medula óssea, sangue periférico, tecido subcutâneo) mais precisamente as células progenitoras mesenquimais está o sangue de cordão umbilical. Sua principal função é o transporte de nutrientes e preencher as crescentes demandas de oxigênio da mãe para o feto (ALMEIDA et al., 2000). Neste estudo foi utilizado o sangue do cordão umbilical de cães ao nascimento, de 11 cadelas gestantes pesando entre 15 e 35 Kg. As células mesenquimais foram isoladas através da separação por gradiente de densidade com o reagente FICOLL, posteriormente o sangue foi diluído em PBS e centrifugado. O pellet contendo células foi retirado e essas então acondicionadas em meio DMEM contendo 10% de soro fetal bovino e antibiótico, homogeneizadas e contadas através da câmara de Newbauer. As células foram então plaqueadas de acordo com o ensaio que seriam submetidas. Através do ensaio de CFU-F pudemos observar uma população de células aderentes isoladas da porção mononuclear do sangue de cordão umbilical que apresentou-se altamente heterogênea. Foram identificados, pelo menos, quatro tipos morfológicos distintos: células epitelióides grandes e pequenas; células de aspecto fusiforme; e células de aspecto estrelado e quando em confluência, as células apresentaram padrão de crescimento similar ao de miofibroblastos, normalmente observado em culturas de músculo liso e de estroma da medula óssea hematopoética. Na maioria das análises as colônias se apresentaram em número desejável, havendo uma variação de baixo a alto. Esse ensaio apresentou grande eficácia no isolamento, quantificação e caracterização morfológica das células mesenquimais obtidas a partir do sangue de cordão umbilical canino. / This project has as objective established cultivation techniques, isolation, expansion and quantification of mesenchymal cells from umbilical cord blood (UCB) of dogs of varied breeds for application in cellular therapies. Nowadays, several cell-trunk types have been studied due to his/her differentiation capacity in several woven (HERZOG et al., 2003). The isolation, quantification and expansion of those cells allow the cellular therapy to be used in the treatment attempt in pathologies (BARKER et al., 2003). Among the adult cell-trunk sources (bone marrow, peripheral blood, subcutaneous tissue) more precisely the progenitors mesenchymal cells are the umbilical cord blood. Her main function is the transport of nutrients and to fill out the crescents demands of the mother\'s oxygen for the fetus (ALMEIDA et al., 2000). In this study, the blood was used from the umbilical cord of dogs to the birth, of 11 pregnant female dogs weighing between 15 and 35 Kg, the mesenchymal cells were isolated through the separation for density gradient with the reagent FICOLL, later the blood was diluted in PBS and centrifuged according to protocol, the pellet containing cells was removed and those then conditioned in half DMEM containing 10% of bovine fetal serum and antibiotic, homogenized and counted through the camera of Newbauer. They were plated then in agreement with the rehearsal that they would be submitted. Through the rehearsal of CFU-F (Colony Forming Unit - Fibroblast) we could observe a population of isolated adherent cells of the mononuclear population of the umbilical cord blood that came highly heterogeneous. They were identified, at least, four different morphologies: big and small epithelial cells; aspect fusiforme; and cells of starry aspect and when in confluence, the cells presented pattern of similar growth to the of miofibroblastos, usually observed in cultures of flat muscle and of stromal of the hematopoietic bone marrow. In most of the analyses the colonies came in desirable number, having a variation of low the high. That rehearsal presented great effectiveness in the isolation, quantification and morphologic characterization of the mesenchymal cells obtained starting from the umbilical cord blood of canine.

This work examines the osteogenic potential and immune-privileged properties of human umbilical cord perivascular cells (HUCPVCs) in normal Wistar rats and athymic rnu/rnu rats for up to 60 days. HUCPVCs demonstrated a mesenchymal stromal cell phenotype, assayed through flow cytometry, and RT-PCR analysis detected their expression of osteogenic genes. A bone tissue engineering construct was developed through centrifugal seeding of HUCPVCs onto calcium phosphate-coated PLGA scaffolds. These cell-scaffold constructs were transplanted into bilateral femoral defects. HUCPVCs did not induce any systemic biological response in normal rats; however, they did not engraft and impaired bone healing up to 60 days. When transplanted into athymic rats, HUCPVCs were detected up to 30 days in the femoral defects, improved bone regeneration at 15 and 30 days, as measured by micro computed tomography, and expressed osteogenic proteins. These findings demonstrate that HUCPVCs are suitable for bone tissue engineering studies in larger animals.

This work examines the osteogenic potential and immune-privileged properties of human umbilical cord perivascular cells (HUCPVCs) in normal Wistar rats and athymic rnu/rnu rats for up to 60 days. HUCPVCs demonstrated a mesenchymal stromal cell phenotype, assayed through flow cytometry, and RT-PCR analysis detected their expression of osteogenic genes. A bone tissue engineering construct was developed through centrifugal seeding of HUCPVCs onto calcium phosphate-coated PLGA scaffolds. These cell-scaffold constructs were transplanted into bilateral femoral defects. HUCPVCs did not induce any systemic biological response in normal rats; however, they did not engraft and impaired bone healing up to 60 days. When transplanted into athymic rats, HUCPVCs were detected up to 30 days in the femoral defects, improved bone regeneration at 15 and 30 days, as measured by micro computed tomography, and expressed osteogenic proteins. These findings demonstrate that HUCPVCs are suitable for bone tissue engineering studies in larger animals.

La glycylation, une modification post-traductionnelle des microtubules, est cruciale dans le maintien des cils primaires. Notre groupe a précédemment identifié un rôle inattendu de la tubuline glycylase TTLL3 dans la régulation de l'homéostasie du colon et de la tumorigénèse. Plus précisément, une diminution du nombre de cils primaires a été observée chez les souris déficientes pour la glycylase TTLL3, qui est la seule glycylase exprimée dans le côlon. Les souris TTLL3 - / - ne présentent pas d'anomalie évidente à l'état stationnaire. Cependant, lorsqu'elles sont exposées à une carcinogenèse du côlon chimiquement induite, les souris TTLL3 - / - sont plus sensibles à la formation de tumeurs. Il est important de noter que les niveaux d'expression de TTLL3 sont significativement réduits dans les carcinomes primaires et métastases colorectales chez l'homme comparativement au tissu de côlon sain, ce qui suggère un lien entre la régulation des cils primaires par TTLL3 et le développement du cancer colorectal.L'objectif de mon projet de thèse était d'explorer l’effet de la modulation des cils primaires sur la carcinogenèse du côlon. J’ai ainsi démontré que le nombre de cils primaires diminue lors de la carcinogenèse du côlon chimiquement induite chez la souris. Notamment, j'ai découvert que les cils primaires du côlon sont principalement exprimés par les cellules mésenchymateuses. Pour mieux caractériser le rôle des cils primaires dans le côlon murin, j'ai étudié les conséquences de leur perte dans les cellules mésenchymateuses intestinaux. Pour cela, j'ai utilisé deux modèles de souris KO conditionnelles, pour la kinesin-3A (Kif3A) et le transport intra-flagellaire 88 (Ift88), deux molécules essentielles pour la formation des cils. Leur délétion spécifique dans les cellules mésenchymateuses intestinaux est obtenue par croisement des souches de souris Kif3Afl/fl et Ift88fl/fl des souris transgéniques collagène VI-cre. Bien que le promoteur colllagène VI ne soit actif que dans un sous-ensemble de cellules mésenchymateuses coliques, j'ai constaté que la diminution du nombre de cils primaires dans ces derniers favorise la colite chimiquement induite et la carcinogenèse. L'analyse par séquençage ARN des cellules mésenchymateuses coliques isolés de souris mutantes suggère un déclenchement de la signalisation Wnt et Notch chez les souris ColVIcre-Kif3Afl/fl. Nous confirmons actuellement ces résultats par qPCR et immunohistochimie. / Glycylation, a posttranslational modification of microtubules, is crucial in the maintenance of PC. Our group previously identified an unexpected role of the tubulin glycylase TTLL3 in the regulation of colon homeostasis and tumorigenesis. Specifically, a decreased number of primary cilia (PC) was observed in mice deficient for the glycylase TTLL3, which is the only glycyclase expressed in the colon. TTLL3-/- mice display no obvious abnormalities in the steady state. However, when exposed to chemically induced colon carcinogenesis, TTLL3-/- mice are more susceptible to tumor formation. Importantly, TTLL3 expression levels were significantly downregulated in human primary colorectal carcinomas and metastases as compared to healthy colon tissue, suggesting a link between TTLL3 regulation of PC and colorectal cancer development.The aim of my thesis project was to explore the relation of PC and colon carcinogenesis. In fact, I could demonstrate that the number of PC decreases during chemically induced colon carcinogenesis in mice. Notably, I discovered that PC in the colon are mostly expressed by fibroblasts. To better characterize the role of PC in murine colon, I studied the consequences of a loss of PC in intestinal fibroblasts. For this, I used two independent ciliary conditional knockout mice, kinesin-3A (Kif3A) and intraflagellar transport 88 (Ift88), both essential for cilia formation. Specific deletion in intestinal fibroblasts is obtained by crossing with colVI-cre transgenic mice. Though the colVI promoter is only active in a subset of colonic mesenchymal cells I found that the decreased number of PC in colonic mesenchymal cells promotes chemically induced colitis and carcinogenesis. RNAseq on isolated colonic mesenchymal cells of mutant mice suggests a triggering of Wnt and Notch signaling in ColVIcre-Kif3aflx/flx mice. We are presently validating these findings by qPCR and immunohistochemistryTaken together, I discovered that PC are expressed by at least a subset of colonic mesenchymal cells, which has not been described before. Decreased numbers of those PC renders mice more susceptible to colitis and colitis associated carcinogenesis.

Orientador: Ana Liz Garcia Alves / Resumo: A lesão do tendão do músculo flexor digital superficial (TFDS) é uma importante causa de claudicação em eqüinos, com prevalência de 13 a 30%, de acordo com a atividade eqüestre. Sua instalação pode comprometer a carreira do potro com recidivas ou causar o afastamento das corridas. Além disto, determina longos períodos de recuperação, com prejuízo à função tendínea. Os tratamentos propostos são diversos, mas efeitos como melhora da qualidade ou rapidez da cicatrização não são confirmados por estudos controlados. A terapia intralesional com fatores de crescimento (TGF-ß e IGF- 1), assim como a terapia com choques extracorpóreos vêm mostrando resultados promissores. Recentemente, os avanços médicos têm demonstrado crescente interesse na utilização das células-tronco em terapia de doenças degenerativas e também em cicatrização lenta ou ineficaz. Este estudo teve como objetivo avaliar os efeitos do implante autólogo de células mesenquimais de medula óssea na cicatrização tendínea, comparando tendões tratados com tendões do grupo controle. Foi induzida lesão do TFDS de ambos os membros anteriores de seis eqüinos, seguida por implante autólogo em apenas um membro de cada animal. Os animais foram avaliados por parâmetros clínicos e ultra-sonográficos e após biópsia realizada ao 48o dia do experimento, foram verificadas características histopatológicas e imunoistoquímicas. Resultados como aumento do infiltrado inflamatório celular, abundância da matriz, diminuição da necrose, discreto aumento no índice de proliferação celular (Ki- 67, clone MIB1) e menor imunomarcação do TGF-ß1 caracterizam a aceleração do reparo tendíneo no grupo tratado. Estudos aprofundados devem ser desenvolvidos com o objetivo de definir a influência do tratamento em fase posterior do reparo. Conclui-se, então, ...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The superficial digital flexor tendonitis (SDFT) is an important cause of lameness in horses with incidence from 13 to 30% depending of the exercise modality. This injury can occur in yearlings and compromise its carrier by reinjury or even the impossibility of return to athletic life. Although long periods of rest are necessary to tendon repair, there is an elasticity loss in the tendon. As current treatment regimes have only marginal effects there is interest in researching therapies that influence the quality or the duration of tendon repair. Intralesional therapy with growth factors (TGFß1 e IGF-1) and radial shock wave therapy have been presenting promising results. Recently, medical interest has been directed to the stem cells therapy of degenerative diseases and cases of deficient healing process. This study aims to evaluate the influence of autologous mesenchymal stem cells from bone marrow in tendon healing, comparing treated and none treated tendons. Induced collagen lesions were in both forelimbs TFDS from six horses, followed by autologous implant in one forelimb from each animal. The horses were evaluated by clinical, ultrasonographic, histophatologic and imunohistochemistry parameters. Tendon biopsies were done at day 48. Results such as high inflammation cells infiltration and synthesis of extracellular matrix, few necrosis areas, discrete increase in cellular proliferation (Ki-67) and low immunoreactivity to TGFß1 found in treatment group suggested the acceleration of tendon repair in this group. Further studies should be developed in order to verify the influence of this treatment on later phase of tendon repair. Overall, after analysis of results, we conclude that cellular therapy with mesenchymal stem cells have accelerate tendon repair at 48 days after treatment. / Doutor

Esse projeto tem como objetivo estabelecer técnicas de cultivo, isolamento, expansão e quantificação de células mesenquimais e hematopoéticas de sangue de cordão umbilical (SCU) de cães de raças variadas para aplicação em terapias celulares. Atualmente, vários tipos de células-tronco têm sido estudados devido a sua capacidade de diferenciação em diversos tecidos (HERZOG et al., 2003). O isolamento, quantificação e expansão dessas células permitem que a terapia celular seja utilizada na tentativa de tratamento em patologias (BARKER et al., 2003). Entre as fontes de células-tronco adultas (medula óssea, sangue periférico, tecido subcutâneo) mais precisamente as células progenitoras mesenquimais está o sangue de cordão umbilical. Sua principal função é o transporte de nutrientes e preencher as crescentes demandas de oxigênio da mãe para o feto (ALMEIDA et al., 2000). Neste estudo foi utilizado o sangue do cordão umbilical de cães ao nascimento, de 11 cadelas gestantes pesando entre 15 e 35 Kg. As células mesenquimais foram isoladas através da separação por gradiente de densidade com o reagente FICOLL, posteriormente o sangue foi diluído em PBS e centrifugado. O pellet contendo células foi retirado e essas então acondicionadas em meio DMEM contendo 10% de soro fetal bovino e antibiótico, homogeneizadas e contadas através da câmara de Newbauer. As células foram então plaqueadas de acordo com o ensaio que seriam submetidas. Através do ensaio de CFU-F pudemos observar uma população de células aderentes isoladas da porção mononuclear do sangue de cordão umbilical que apresentou-se altamente heterogênea. Foram identificados, pelo menos, quatro tipos morfológicos distintos: células epitelióides grandes e pequenas; células de aspecto fusiforme; e células de aspecto estrelado e quando em confluência, as células apresentaram padrão de crescimento similar ao de miofibroblastos, normalmente observado em culturas de músculo liso e de estroma da medula óssea hematopoética. Na maioria das análises as colônias se apresentaram em número desejável, havendo uma variação de baixo a alto. Esse ensaio apresentou grande eficácia no isolamento, quantificação e caracterização morfológica das células mesenquimais obtidas a partir do sangue de cordão umbilical canino. / This project has as objective established cultivation techniques, isolation, expansion and quantification of mesenchymal cells from umbilical cord blood (UCB) of dogs of varied breeds for application in cellular therapies. Nowadays, several cell-trunk types have been studied due to his/her differentiation capacity in several woven (HERZOG et al., 2003). The isolation, quantification and expansion of those cells allow the cellular therapy to be used in the treatment attempt in pathologies (BARKER et al., 2003). Among the adult cell-trunk sources (bone marrow, peripheral blood, subcutaneous tissue) more precisely the progenitors mesenchymal cells are the umbilical cord blood. Her main function is the transport of nutrients and to fill out the crescents demands of the mother\'s oxygen for the fetus (ALMEIDA et al., 2000). In this study, the blood was used from the umbilical cord of dogs to the birth, of 11 pregnant female dogs weighing between 15 and 35 Kg, the mesenchymal cells were isolated through the separation for density gradient with the reagent FICOLL, later the blood was diluted in PBS and centrifuged according to protocol, the pellet containing cells was removed and those then conditioned in half DMEM containing 10% of bovine fetal serum and antibiotic, homogenized and counted through the camera of Newbauer. They were plated then in agreement with the rehearsal that they would be submitted. Through the rehearsal of CFU-F (Colony Forming Unit - Fibroblast) we could observe a population of isolated adherent cells of the mononuclear population of the umbilical cord blood that came highly heterogeneous. They were identified, at least, four different morphologies: big and small epithelial cells; aspect fusiforme; and cells of starry aspect and when in confluence, the cells presented pattern of similar growth to the of miofibroblastos, usually observed in cultures of flat muscle and of stromal of the hematopoietic bone marrow. In most of the analyses the colonies came in desirable number, having a variation of low the high. That rehearsal presented great effectiveness in the isolation, quantification and morphologic characterization of the mesenchymal cells obtained starting from the umbilical cord blood of canine.