Objectifs : Les thérapies régénératives cardiaques ont connu un essor considérable au cours des 10 dernières années. Malgré des effets positifs démontrés chez l’animal, les bénéfices cliniques obtenus chez l’homme sont encore relativement modestes. L’objectif de ce travail a été de mieux comprendre les facteurs liés à l’implantation des cellules souches grâce aux techniques de détection cellulaire en imagerie par résonance magnétique (IRM), afin d’optimiser la thérapie cellulaire cardiaque.Matériel et méthodes : Un protocole de détection cellulaire en IRM cardiaque in vivo ainsi qu’une méthode de détection des microvaisseaux en IRM cardiaque ex vivo haute résolution avec des séquences Susceptibility Weighted Imaging (SWI) ont été développés, puis mis en application pour l’étude de la vectorisation de progéniteurs des cellules endothéliales marqués magnétiquement par des nanoparticules d’oxyde de Fer et injectés par voie intraveineuse, ainsi que pour l’évaluation de l’intégration et de l’efficacité de cellules souches mésenchymateuses administrées via des patchs de fibrine cellularisés chez des rats adultes indemnes de toute pathologie (un groupe contrôle vs un groupe infarctus via ligature définitive de l’artère interventriculaire antérieure).Résultats : Après injection intraveineuse et malgré la vectorisation magnétique (n=16 rats), l’imagerie de détection cellulaire a montré qu’aucune cellule n’était implantée dans le myocarde et que les paramètres fonctionnels cardiaques n’étaient pas améliorés. Avec les patchs cellularisés (n=37 rats), la fraction d’éjection ventriculaire gauche (FEVG) était améliorée dans les groupes de patchs cellularisés par rapport aux groupes contrôles. La densité microvasculaire était augmentée dans la zone infarcie et peri-infarcie dans les groupes cellularisés par rapport aux groupes contrôles, à la fois en immunohistochimie et en IRM sur les séquences SWI. L’IRM a montré l’absence de migration des cellules dans le myocarde à partir du patch, confirmé en immunohistochimie. La persistance de cellules dans la zone d’implantation du patch à la surface épicardique à J21 post greffe et l’étude en cytométrie en flux des cytokines et facteurs de croissance produits par les cellules souches plaident pour une efficacité de la thérapie cellulaire en rapport avec la sécrétion de facteurs paracrines par les cellules souches.Conclusion : L’imagerie de susceptibilité magnétique permet d’une part d’étudier les vaisseaux myocardiques sur des séquences pondérées en SWI ex vivo et d’autre part d’évaluer l’implantation des cellules souches sur des séquences en écho de gradient T2* in vivo. Ces techniques ont permis de mieux caractériser le mode d’action des patchs cardiaques en tant que réservoir de facteurs paracrines pour le traitement de l’insuffisance cardiaque dans un modèle murin. Ces résultats confirment l’intérêt fort à développer et optimiser l’utilisation de biomatériaux intelligents délivrant spécifiquement des molécules d’intérêt comme les cytokines ou les facteurs de croissance et permettant ainsi de contourner les contraintes immunogènes et tératogènes liés aux cellules souches. / Objectives: Cardiac regenerative therapies have grown considerably over the past 10 years. Despite positive effects demonstrated in animals, the clinical benefits obtained in humans are still relatively modest. The objective of this work was to better understand the factors associated with implantation of stem cells through the cell detection techniques in magnetic resonance imaging (MRI) and to improve cardiac stem cell therapy in a murine model of myocardial infarction.Materials and methods: A protocol for cell detection with gradient echo T2* sequences in cardiac MRI in vivo and a method for detection of microvessels in cardiac MRI ex vivo with high resolution Susceptibility Weighted Imaging sequences (SWI) were developed and implemented for the study of vectorization of intravenously injected endothelial progenitors cells (EPC) and the integration and evaluation of the impact of mesenchymal stem cells (MSC) administered via cellularized fibrin patches. A permanent ligation of the left anterior coronary artery was performed in adult rats. The stem cells were magnetically labeled with iron oxide nanoparticles by endocytosis.Results: Cell detection imaging showed no cell implantation in the myocardium and no improvement in cardiac functional parameters after intravenous injection of EPC, despite the aid of magnetic vectorization (n = 16 rats). With a local administration of MSC via cardiac patches (n = 37 rats), the left ventricular ejection fraction (LVEF) was improved in cellularized patches groups compared to controls. Microvascular density was increased in the infarcted and peri – infarcted areas in cellularized patches groups compared to controls in immunohistochemistry and in MRI on SWI sequences. The MRI showed no migration of cells into the myocardium from the patch, as confirmed by immunohistochemistry and Perls staining. The persistence of MSCs on the epicardial surface at D21 after implantation and flow cytometry profiling of cytokines and growth factors produced by MSC argue for cell therapy effectiveness related to the secretion of paracrine factors by stem cells.Conclusion: Susceptibility imaging allows: (1) to study myocardial vessels on SWI sequences ex vivo and (2) to assess the implementation of stem cells on gradient echo sequences T2 * in vivo. These techniques have shown that cardiac patches act as a reservoir of soluble mediators which paracrinally target the angiogenesis in the treatment of heart failure in a murine model. This is in favor of a move towards “cell free” biomaterials containing only molecules of interest such as cytokines or growth factors to circumvent immunogenic and teratogenic constraints related to the use of stem cells.

A doença renal crônica é uma doença de alta incidência na espécie felina, sendo responsável por altas taxas de mortalidade e morbidade. O tratamento clínico é paliativo. Atualmente as células-tronco têm sido estudadas para várias doenças degenerativas e crônicas, entre elas a doença renal. O presente estudo testou a utilização uma nova linhagem de células, progenitoras do tecido renal na terapia de gatos domésticos acometidos naturalmente com doença renal crônica, nos estágios 1, 2 e 3 da doença (creatinina <1,6 a 5,0 mg/dl), por meio de avaliações clínicas e laboratoriais. Os animais foram divididos em dois grupos, experimental e controle. No grupo experimental foi aplicado pela via intraperitoneal 2x106 células progenitoras renais e os animais do grupo controle receberam a aplicação de PBS. Os animais foram avaliados nos dias 0, 7 e 14 e monitorados clinicamente e por meio de exames laboratoriais, incluindo hemograma, creatinina, urinálise e o SDMA. A análise estatística foi realizada pelo teste Scheirer Ray Hare para dados não paramétricos (p=0,05). A aplicação intraperitoneal ocorreu sem intercorrências e aparenta ser segura para utilização em gatos. Dos 4 animais do grupo experimental 3 apresentaram melhora clínica, melhora do apetite e ganho de peso, o quarto animal apresentou perda de peso inicial, mas retornou ao peso do início do estudo 14 dias após a aplicação das células. Não houve diferenças estatísticas nos parâmetros de creatinina, ureia, fósforo e densidade urinária. Os leucócitos do grupo experimental apresentaram uma redução significativa em relação ao grupo controle. O SDMA apresentou redução em 3 animal do grupo experimental e aumento nos animais do grupo controle, mas a análise não apresentou diferença estatística devido ao aumento dos valores no em um dos animais. A aplicação de células progenitoras renais no tratamento da DRC em gatos é promissora e pode ser realizada pela via intraperitoneal, sem que ocorra intercorrências. / Chronic kidney disease is a high incidence disease in the feline species, responsible for high rates of mortality and morbidity. The clinical treatment is palliative. Currently, stem cells have been studied for various degenerative and chronic diseases, including kidney disease. The present study tested the use of a new lineage of renal tissue progenitor cells in the therapy of naturally occurring chronic kidney disease in cats at stages 1, 2 and 3 of the disease (creatinine <1.6 to 5.0 mg/dl), through clinical and laboratory evaluations. The animals were divided into two groups, experimental and control. In the experimental group 2x106 renal progenitor cells were administered intraperitoneally and the animals in the control group received PBS application. The animals were evaluated on days 0, 7 and 14 and monitored clinically and through laboratory tests, including complete blood count, creatinine, urinalysis and SDMA assay. Statistical analysis was performed by the Scheirer Ray Hare test for non-parametric data (p = 0.05). The intraperitoneal application occurred uneventfully and appeared to be safe for use in cats. Of the 4 animals in the experimental group 3 showed clinical improvement, food intake and weight gain, the fourth animal presented initial weight loss, but returned to the weight of the study beginning 14 days after the application of the cells. There were no statistical differences in the parameters of creatinine, urea, phosphorus and urinary density. Leukocytes in the experimental group showed a significant reduction in relation to the control group. The SDMA showed reduction in 3 animals from the experimental group and increase in the animals from the control group, but the analysis did not show statistical difference due to the increase values in one of the animals. The application of renal progenitor cells in the treatment of CKD in cats is promising and can be performed intraperitoneally, without causing intercurrences.

Cell Therapies are promising clinical instruments with significant therapeutic potential and commercial promise. However, the industry engaged in their commercial and clinical development faces significant financial, technical, regulatory and market challenges. These challenges are compounded by an understanding gap in the cell therapy industry. Commercial failures and financial difficulties have forced the industry to address the need to provide value and estimate and control costs early in the development timeline. The problem is that this issue is not being systematically or thoroughly addressed in the academic community while they pursue potential future treatments. Articles that highlight the need to understand costs and value are appearing with increasing frequency highlighting a growing consensus that work needs to be carried out in this area. However examples of models and tools to predict or estimate or even calculate costs in developing and producing a product do not exist in the literature. This work consists of three parts. Part one entails a new model of the characteristics observed in cell therapy new product development. This model is an evolution of an activity based dependency structure matrix (DSM). Result from the model suggests that some favoured development strategies (such as applying for an orphan indication status) provide less financial benefit than is commonly expected. The ability to scale manufacturing levels between clinical trial phases is also a pressing problem. 3 Part two presents a model to predict the cost of manufacturing and delivering a cell therapy product. This cost of good supplied (COGS) model combines both rules and predictive activity based costing across multiple manufacturing platforms, cell types and supply chain configurations. This model highlights the significant cost burden of validating both single and, more markedly, multiple sites of manufacture. The model also examines the potential for economies of scale when using different production technology in the manufacture of human Mesenchymal Stem Cells. Based in part on the results and knowledge gleaned in parts one and two, part three outlines the development of a novel, scalable expansion system developed to enable lower cost, controlled manufacture of adherent cell populations. While still at an early stage of development the technology has demonstrated the ability to maintain cells in a high rate of growth for a longer period than traditional culture techniques. This allows for the creation of a manufacturing technology with a higher expansion ratio than manufacturing systems on the market today.

O sistema olfatório desempenha um papel relevante na exploração do ambiente e no reconhecimento social e sexual de mamíferos. Por meio deste sistema os animais podem reconhecer, detectar e discriminar uma grande quantidade de odorantes de estruturas químicas variadas, sinais químicos no ambiente essenciais para a sobrevivência e os ferormônios, que desencadeiam comportamentos sociais e reprodutivos. Algumas doenças e certos tipos de injúrias fisiológicas podem provocar a morte destas células, o que pode levar a perda da sensibilidade olfatória, embora já se saiba que este epitélio apresenta grupos de neurônios capazes de regeneração. A partir deste contexto, a terapia celular acaba sendo uma alternativa para o tratamento de patologias as quais acometem o sistema olfatório, como por exemplo, a anosmia que pode causar problemas graves, desde acidentes com gás ou comida estragada até depressão e distúrbios alimentares, causadas pela perda do paladar. Objetivou-se com este trabalho avaliar a recuperação da função olfatória de ratos anósmicos, bulbectomizados e submetidos a terapia celular com células-tronco provenientes do epitélio olfatório de ratos wistar. Para tanto foram utilizados 21 ratos machos Wistar de sessenta dias de idade, onde três foram utilizados para obtenção das células tronco do epitélio olfatório, dois para o controle cirúrgico, e restante foram divididos em 4 grupos: GI, GII, GIII e GIV os quais foram transplantados após 3, 7, 14 e 21 dias após a bulbectomia, respectivamente. A técnica cirúrgica foi realizada com incisão de pele, tecido subcutâneo e periósteo, seguida de abertura em janela de formato ovalado e remoção dos bulbos olfatórios mediante aspiração. Para a comprovação da anosmia após a cirurgia, os ratos foram submetidos ao teste comportamental do \"odor de gato\", e os do grupo controle após o período experimental foram sacrificados e a área encefálica da lesão causada pela cirurgia foi coletada onde foram realizadas análises histopatológicas. Os animais do GI, GII, GIII e GIV após 3, 7, 14 e 21 dias após bulbectomia foram anestesiados e receberam células tronco (1x106) do EOR no mesmo local da realização da bulbectomia, e posteriormente foram submetidos ao teste comportamental do \"odor de gato\". Transcorrido o período experimental, foram eutanasiados e os fragmentos de encéfalo foram coletados para análise histopatológica e imunohistoquímica. Os resultados evidenciam que realização da intervenção cirúrgica demonstrou remoção parcial do BO, com destruição da conexão nervosa entre os bulbos olfatórios e o epitélio olfatório. Ainda, a partir do teste comportamental do \"odor de gato\", e pela análise histopatológica das lesões causadas pela cirurgia, que evidenciou extensa área de necrose, com presença de hemossiderina e astrogliose reativa, constatou-se que a técnica empregada para promoção da bulbectomia foi eficaz para promoção da anosmia. A partir da análise comportamental, dos animais submetidos a terapia celular, os animais do GII e GIII apresentaram modificações no comportamento olfativo, com comportamento olfativo positivo ao \"odor do gato\", aversão comportamento defensivo, enquanto 100% dos animais do GI e GIV não apresentaram nenhuma modificação no comportamento olfativo. As análises por imunohistoquímica evidenciaram marcação positiva para o GFP, o que indica a presença das células tronco transduzidas com eGFP nos locais das lesões e ainda a expressão positiva do GFAP que evidencia a presença de astrogliose reativa com presença de cicatriz glial nos locais das lesões. / The olfactory system plays an important role in the exploration of the environment and in social and sexual behavior in mammals. Disturbances of the olfactory system such as observed in anosmia has also been related to accidents caused by gas leak and intoxications by food poisoning, in addition to eating disorders due to relation of the olfactory system with the taste. Through the olfactory system, animals recognize, detect and discriminate a large amount of odorants in a variety of chemical products, including pheromones, as well as in the environment which may guarantee their survival. Some diseases and injuries cause death of cells from the olfactory system leading to decrease or loss of the smell sensitivity. It is known that the olfactory epithelium has cells capable of regenerating neurons. Therefore, the utilization of these cells in cell therapy represents an alternative for the treatment of the olfactory system disorders. The objective of this study was to evaluate the regenaration of olfactory function of bulbectomized rats that were subjected to cell therapy with cells isolated from the olfactory epithelium. Twenty one male Wistar rats, sixty days of age were included in this study. Three rats were used for isolation of the olfactory epithelium cells, two for the surgical control and the remaining were divided into 4 groups: GI, GII, GIII and GIV corresponding to groups where cells were transplanted after 3, 7, 14 and 21 days after bulbectomy, respectively. The surgical technique was performed with skin, subcutaneous and periosteum incisions, followed by craniectomy and the removal of olfactory bulbs upon aspiration. For proof of anosmia, the rats were subjected to behavioral testing know as \"cat odor\". The animals of the control group were sacrificed and brain, with the lesion area, collected and processed for histopathological analysis. The animals of the experimental groups (GI, GII, GIII and GIV) were anesthetized and received heterologous cells (1x106) from olfactory epithelium, thorugh the same venue of the bulbectomy. These animals subsequently underwent behavioral \"cat odor\" testing. After 3, 7, 14 and 21 days of the cell injection, the rats were euthanized and the animals brains were collected for histopathological and immunohistochemical analyses. The results show that the surgical procedure promoted partial removal of olfactory bulbs, with destruction of the neural connection between the olfactory bulb and the olfactory epithelium. The behavioral \"cat odor\" test and the histopathological exams of the lesions, which revealed a large area of necrosis with presence of hemosiderin and reactive astrogliosis, demonstrated that the bulbectomy technique used to promote anosmia was effective. The behavioral test showed that the animals from GII and GIII presented changes in olfactory sensitivity, with positive \"cat odor\" aversion and defensive reaction. This test did not show change in GI and GIV groups. Immunohistochemistry analysis was positive for GFP, suggesting the presence of eGFP transduced cells at the sites of injury. In addition, the expression of GFAP positive cells demonstrated the presence of reactive astrogliosis with glial scar at the sites of injury.

A distrofia muscular de Duchenne (DMD) é uma alteração neuromuscular hereditária e progressiva que afeta humanos do sexo masculino. O modelo canino Golden Retriever Muscular Dystrophy (GRMD) é considerado modelo experimental para estudos de novas propostas terapêuticas e melhor entendimento da fisiopatogênica da DMD. O processo progressivo da distrofia está relacionado com alterações nas populações celulares que compõe o sistema imune dos pacientes, pois devido a ausência da proteína distrofina na membrana sarcoplasmática, o músculo fica mais susceptível à lesões, ocorrendo liberação de citocinas, que recrutam e estimulam células do sistema imune, principalmente macrófagos e linfócitos T. A longo prazo, essa resposta inflamatória contínua e persistente, leva a uma série de reações que culminam com danos cada vez maiores, a ponto de ocorrer um esgotamento de células satélites e fibrose do tecido muscular. Uma das propriedades mais estudadas das células tronco mesenquimais (MSCs) é sua capacidade imunomoduladora, fazendo com que essas células se tornem promissoras na utilização da terapia celular. Neste contexto, esta ferramenta imunomoduladora pode atuar como uma estratégia interessante na manipulação do sistema imune. O estudo proposto foi elaborado com a finalidade de trazer subsídios para viabilização de experimentos de terapia celular na distrofia muscular, por meio do conhecimento sobre a imunomodulação durante o tratamento in vitro, contribuindo para uma possível aplicação terapêutica em humanos. Para tanto, foram estudados dois grupos de cães, um grupo controle (GR; n=5) e um grupo de cães afetados (GRMD; n=9), compostos por machos e fêmeas. O estudo consistiu na avalição da proliferação de linfócitos na presença de MSC em diferentes concentrações, bem como da proliferaçao de linfócitos específicos como o Tauxiliar (CD4+FoxP3-) e Tregulatórios (CD4+FoxP3+) com as MSCs. Adicionalmente, realizamos a dosagem de nitrito com o intuito de quantificar a produção de óxido nítrico (NO) através do cocultivo de macrófagos com as MSCs. Neste estudo foi possível observar que as MSCs estimularam a proliferação significativa de linfócitos T regulatórios. Adicionalmente, essa porcentagem de divisão aumentou em cocultivos que utilizaram maiores concentrações de MSC. Maior concentração de nitrito também foi encontrada no cocultivos de MSC e macrófagos estimulado com LPS. Estas informações geram incrementos no entendimento de como as MSCs podem agir no organismo distrófico e como poderemos explorar essa fonte para promover um retardamento no processo inflamatório e consequentemente melhorar a qualidade de vida do paciente. / The Duchenne muscular dystrophy (DMD) is a progressive hereditary neuromuscular disorder that affects human males. The canine model Golden Retriever Muscular Dystrophy (GRMD) is considered experimental model for studies of new therapies and better understanding of the DMD. The process of progressive dystrophy is related to changes in cell populations that comprise the immune system of patients, because due to the absence of the protein dystrophin in the sarcoplasmic membrane, the muscle is more susceptible to injury, occurring release of cytokines that recruit and stimulate cell immune, mainly macrophages and T lymphocytes in the long term, this continuous and persistent inflammatory response, the system takes a series of reactions that culminate with increasing damage to the point of exhaustion of satellite cells of muscle tissue and fibrosis occur. One of the most studied properties of mesenchymal stem cells (MSCs) is their immunomodulatory capacity, making these cells become promising in the use of cell therapy. In this context, this immunomodulatory can act as an interesting strategy in manipulating the immune system. The proposed study was designed in order to provide support for the feasibility of cell therapy trials in muscular dystrophy, through the knowledge of immunomodulation during treatment in vitro, contributing to a possible therapeutic application in humans. In this purpose, two groups were studied, a group of affected dog (GRMD, n=9) and a control group (GR, n=5 GR). The study consisted of rating of lymphocyte proliferation in the presence of MSCs in different concentrations as well as the proliferation of specific lymphocytes as Thelper (CD4+FoxP3-) and Tregulatory (CD4+FoxP3+) to MSCs. In addition, the dosage of nitrite performed in order to quantify the production of nitric oxide (NO) by coculture with macrophages MSCs. In this study we observed that MSCs stimulated significant proliferation of regulatory T lymphocytes. Additionally, this percentage split increased cocultivos that used higher concentrations of MSC. Highest concentration of nitrite was also found in cocultivos of MSC and macrophages stimulated with LPS. This information generates increments in understanding how MSCs may act in the body dystrophic and how we exploit this source to promote a delay in the inflammatory process and consequently improve the quality of life of patients

As doenças de disco intervertebrais (DDIV) representam a maior parte de atendimentos neurológicos e são responsáveis pela maioria dos casos de paralisia em cães. Os tratamentos utilizados atualmente não demonstram resultados satisfatórios em pacientes com manifestações neurológicas mais graves. A fim de promover recuperação nervosa e motora, além de melhora na qualidade de vida, o presente trabalho objetivou criar um protocolo, através de um ensaio duplo cego, associando cirurgia descompressiva e transplante alogênico de células-tronco (CT) derivadas da membrana amniótica em cães com DDIV crônica. As mesmas células já foram caracterizadas anteriormente como mesenquimais fetais e apresentaram-se seguras para aplicação. Foram selecionados oito cães, onde quatro já passaram por cirurgia e receberam três aplicações epidurais de células-tronco. Os outros quatro animais foram submetidos à cirurgia descompressiva e divididos aleatoriamente (teste duplo cego) em dois grupos: \"cirurgia + placebo\", o qual recebeu apenas solução fisiológica; e \"cirurgia + CT\", que recebeu a terapia celular. Durante o procedimento cirúrgico, foi realizado a aplicação por gotejamento sobre a lesão, e após quinze e quarenta e cinco dias foram realizadas outras duas aplicações, via epidural. Os animais passaram por acompanhamento quinzenal e foram reavaliados três meses após o procedimento cirúrgico, através de exames funcionais e ressonância magnética. Alguns animais apresentaram melhora neurológica significativa, como a recuperação da nocicepção e capacidade de se manter em estação. Apesar da necessidade de mais estudos, até o presente momento, a terapia celular apresentou-se factível e sem efeitos prejudiciais aos animais. / Intervertebral disc (IVD) diseases represent the majority of neurological attendance and are responsible for the most cases of paralysis in dogs. Treatments currently used do not show satisfactory results in patients with more severe neurological manifestations. In order to promote nerve and motor recovery, as well as improve quality of life, the present study aims to create a protocol, using double-blind test method, associating spinal decompression surgery and allogeneic transplantation of amniotic membrane-derived stem cells (AMSCs) in dogs with chronic IVD diseases. Those were previously characterized as fetal mesenchymal cells and were safe for application. Eight dogs were selected, where four have already gone through surgery and received 3 epidural applications of stem cells. The other four animals were submitted to spinal decompression surgery and randomly divided into two groups (double blind test): \"surgery + placebo\", which received only physiological solution; and \"surgery + AMSCs\", which receive cell therapy. During the surgical procedure, a drip application was performed on the lesion and after fifteen and forty five days another two applications were made via epidural. Animals were monitored biweekly and were reassessed three months after surgery, by functional tests and magnetic resonance exams. Some animals presented significant neurological improvement, such as the recovery of nociception and ability to remain on station. Despites the need further studies, until the present moment, cell therapy has been feasible and has no harmful effects on animals.

The accumulation of amyloid-ß; protein (Aß) in Alzheimer's disease (AD) is a well known pathological event. Decreasing the production or increasing the degradation of Aß; is therefore thought to serve as a potential therapeutic intervention in AD. Recent in vitro and in vivo studies have suggested that certain proteases may be involved in the catabolism of Aß; and defects in the degradation of Aß; could contribute to AD disease progression. Studies implicating the homing of monocytes to regions of CNS damage have led to the idea that it may be possible to use genetically modified monocytes to carry exogenous genes of interest into the brain or other organs for the purposes of gene therapy. To determine the time course of monocyte recruitment into the brain during the neurodegenerative damage characteristic of Alzheimer's disease, we used transplanted GFP labeled bone marrow monocytes to characterize the kinetics that peripheral monocytes display once injected into the circulation. We determined the half life of bone marrow derived monocytes after one injection into the peripheral circulation, and found this time to be 1.5 hours post injection. We also examined the effects of the APP+PS1 transgene on the recruitment of peripheral monocytes and showed that these cells are actively recruited to the brains in AD transgenic mouse models compared to non transgenic mice. As an approach to increase expression of NEP in a transgenic mouse model of AD, we developed an ex vivo gene therapy method utilizing bone marrow monocytes from GFP mice. These monocytes were transfected with a NEP construct designed to express either a secreted form of NEP or a form which lacks any enzyme activity. Monocytes were administered through a microvascular port twice a week for two months and we observed recruitment of bone marrow-derived monocytes into the CNS. In addition, we found significant reductions in both Aß and Congo red staining in the NEP-S injected mice only. These studies show that putting monocytes together with an amyloid degrading enzyme such as neprilysin offers a powerful novel therapeutic tool for the treatment of AD.

Growing evidence from animal and clinical studies suggests that cardiac cell therapy can restore perfusion and improve function in the ischemic/infarcted myocardium. However, cell therapy is hindered by insufficient cell numbers, inefficient cell homing and engraftment, and inadequate cellular interactions. Furthermore, the biological mechanisms and local effects of transplanted cells have not been well-elucidated. The research presented herein attempts to address some of these issues. In manuscript #1, a new subpopulation of circulating progenitor cells (CPCs), termed derived CD133+ cells, was generated from the CD133- fraction of human peripheral blood. The derived CD133+ progenitors appeared to have superior vasculogenic potential in vitro, which may prove to be beneficial in inducing vasculogenesis in ischemic tissues. Positron emission tomography (PET) with direct cell labeling and reporter gene techniques were employed to assess the fate of transplanted human CPCs in vivo at different subjects of investigation, and different stages of cell transplantation. In manuscript #2, PET imaging with 2-[18F]fluoro-2-deoxy-D-glucose (18F-FDG) direct cell labeling was used to demonstrate that collagen-based matrices improve the early homing and retention of delivered CPCs in a rat ischemic hindlimb model. This mechanism conferred by the matrix may have implications on cell therapy at the early stages after transplantation. In manuscript #3, a more efficient, stable and accurate labeling method, hexadecyl-4-[18F]fluorobenzoate (18F-HFB) direct cell labeling, was developed to quantify cell distribution of transplanted CPCs in a rat myocardial infarction model. PET imaging of 18F-HFB-CPCs revealed significant cell washout from the myocardium immediately after intramyocardial injection, with only a small proportion of transplanted CPCs remaining in the target area in the first 4 hours after delivery. In manuscript #4, human CPCs transduced with lentiviral vectors showed stable expression of PET reporter genes. This reporter gene based-cell labeling technique can be developed for noninvasive tracking cells within a bioengineered matrix by PET, while preserving cell phenotype, viability and function. These studies contribute important insights into the biology and physiology of transplanted stem cells and the ability of delivery matrices to improve transplanted cell engraftment, survival, and function. I believe with further refinement, cell expansion, tissue engineering and PET imaging could facilitate the clinical applications of cell therapies in years to come.

Cellular therapies rely on the delivery of therapeutic cells into patients, but their safety can be compromised by the manipulation of cells ex vivo or their placement outside of their natural context in vivo. Cell Fate Control Gene Therapy (CFCGT) offers the possibility of establishing pharmacological controls over gene-modified cells (GMCs) with regards to their proliferation, differentiation, or function. In its simplest form, 'suicide' gene therapy (SGT), stable introduction of a 'suicide' gene that can activate a non-toxic prodrug establishes control over the survival of GMCs. Current SGT modalities are sub-optimal in clinical setting. To overcome the many limitation of current strategies, we have developed a next-generation CFCGT approach based on the active site-engineered variants of human deoxyCytidine Kinase (dCK), which enable robust activation of multiple Nucleoside Analogue (NA)-based prodrugs, act early in the pathway enabling rapid accumulation of activated NAs in target cells, and also provide the capabilities for the direct imaging of GMCs. Stable introduction of dCK variants into target cells by means of Lentiviral (LV) gene transfer significantly increases their sensitivity to multiple prodrugs. Our dCK variant with only two active site amino acid substitutions is expected to be non-immunogenic yet capable of specifically activating deoxythymidine- and deoxyuridine-based NAs that are not substrates for the wild-type enzyme, such as bromovinyldeoxyuridine (BVdU) and L-deoxythymidine (LdT). We show here that dCK can be used for controlling the survival of GMCs, in cell lines and primary cells in vitro and in a murine xenogeneic transplant models in vivo. To characterize dCK/prodrug-mediated killing mechanisms in GMCs, we have examined the levels of active metabolites in cells and the cellular pathways they antagonize. We describe here the experimental basis for the application of this novel CFCGT in bone marrow transplantation for management of Graft-versus-Host Disease (GvHD) and in enhancing chemotherapy in direct treatment of tumors. In summary, we have developed a novel and robust strategy for effective CFCGT that addresses the many shortcomings of existing modalities. Future studies will validate this novel system in a variety of primary cells and animal disease models, including models of hematopoietic transplantation and ES/iPS-based cell therapies.

Embryonic stem cells (ESC) constitute a promising source of cells for cardiac transplantation strategies. However, complexities associated with in vivo studies have made it difficult to develop a thorough understanding of cell integration. We have engineered an in vitro system that recapitulates the native cardiac environment using 300μm thick collagen scaffolds seeded with neonatal cardiomyocytes (CM) and electrical field stimulation. The injection of undifferentiated ESC served as a baseline to assess the validity of studying cell transplantation in this model. Yfp-ESC survived and proliferated over several days in model tissue. ESC were not observed to significantly differentiate into the cardiac lineage, and did not integrate with the cardiac cell population. While the injection of ESC improved cardiac cell number, tissue functional properties were hindered. The methods developed herein can be readily adapted to study ESC derived progenitor and differentiated cells, to elucidate the optimal cell state for ESC-mediated cell therapy.