La dystrophie musculaire de Duchenne (DMD) est une pathologie récessive liée au chromosome X qui résulte d’une mutation sur le gène de la dystrophine aboutissant à l’absence complète de la protéine. Elle correspond à la plus fréquente des dystrophies musculaires et reste aujourd’hui sans traitement curatif. L’UMR a fait la preuve de concept de l’administration systémique d’une population de cellules souches adultes résidentes du muscle, les cellules MuStem canines chez le chien dystrophinopathe, modèle cliniquement pertinent de la DMD. L’objectif de la thèse a consisté à caractériser la population humaine (hMuStem) en terme de phénotype, myogénicité, immunomodulation et de contexte immunologique d’administration in vivo. La population hMuStem se compose de progéniteurs myogéniques précoces d’origine mésenchymateuse-périvasculaire. Elle se définit par une forte capacité proliférative, une oligopotence et une participation à la régénération musculaire après administration dans un muscle lésé. Elles présentent des propriétés immunomodulatrices en interagissant avec l’immunité adaptative et innée par inhibition de la prolifération lymphocytaire et du complément via un ensemble de molécules de surface et/ou de facteurs sécrétés. Enfin, un traitement immunosuppressif restreint à la période d’injection in vivo de la population allogénique s’avère nécessaire mais suffisant pour éviter une réaction immune de l’hôte. L’ensemble de ces résultats aboutit à une meilleure compréhension de l’identité et des modalités d’action de la population MuStem. / Duchenne Muscular Dystrophy is a X-linked recessive disorder that results from mutation in the dystrophin gene leading to a total lack of the protein. It is the most frequent muscular dystrophy with no curative treatment. The lab made a proof of concept of the systemic delivery of a muscle-derived adult stem cell population called MuStem cells in dystrophic dog, the clinically relevant DMD model. The aim of my Ph.D. was to characterize the human population (hMuStem) in terms of phenotype, myogenicity, immunomodulation and immunological context of in vivo delivery. hMuStem cell population is composed of myogenic progenitors with mesenchymal/perivascular imprint. It exhibits a high proliferative capacity, an oligopotency and a participation to muscle regeneration after transplantation into injured muscle. It displays immunomodulatory properties by interacting with adaptive and innate immunity with inhibition of lymphocyte proliferation and complement thanks to expression of surface molecules and/or secreted factors. At last, an immunosuppressive regimen restricted to the allogeneic injection period is necessary but sufficient to avoid host immune response. Collectively, these results allow a better understanding of identity and action modalities of MuStem cell population.

L'utilisation des cellules souches est une approche prometteuse pour le traitement des maladies dégénératives neuromusculaires. De nombreuses études portent actuellement sur les cellules souches embryonnaires (ES) et les cellules pluripotentes induites par reprogrammation (IPs) dont l'utilisation en médecine régénérative reste sujette à caution à cause du potentiel de ces cellules à former des tératomes. Des lors, aussi bien les ES que les IPs nécessitent une différenciation vers un type cellulaire précis. Cette différenciation peut mener à des risques supplémentaires tels que la dérive génique ou diverses sources de contamination.Le muscle squelettique adulte, avec sa grande plasticité et capacité régénératrice, contient une population de cellules souches qui est spécifique de ce compartiment tissulaire et qui a été isolée et étudiée au laboratoire. Les cellules souches du muscle squelettique adulte: skeletal Muscle-Derived Stem Cells, MDSC, repeuplent et réparent en quelques jours le muscle squelettique lésé avec une haute efficacité, même en présence des cellules satellites endogènes. (Arsic et al Exp. Cell Res. 2008). Le laboratoire d'accueil a entrepris de caractériser cette population cellulaire, en particulier par son origine histologique, de tester le potentiel de réparation tissulaire de ces cellules transplantées dans des modèles murins, et de déterminer la bio-distribution de ces cellules en vue d'utilisation thérapeutique.Mon travail de thèse s'est intéressé à cette population de cellules souches issues du muscle qui ont une propriété commune : la faible adhérence au substrat. La faible adhérence est une propriété très intéressante car en plus de définir des cellules plus proches de l'état pluripotent, cette propriété leur confère une grande capacité de migration. Ces cellules seraient donc plus facilement utilisables en médecine régénératrice. Dans cette perspective il est intéressant de disposer de cellules souches multipotentes qui pourrait se comporter comme des cellules pluripotentes en terme de capacité régénératrice, mais sans les inconvénients de ces dernières à savoir ; risque tératogène et prolifération incontrôlée, et manipulation des cultures cellulaires longues et couteuses.Au début de ma thèse je me suis donc intéressée aux différentes populations de cellules présentes dans le muscle et je me suis concentrée sur différents marqueurs connus chez les cellules souches, dont la présence a été établie chez différentes cellules souches y compris chez les cellules souches dérivées du muscle squelettique, mais pas clairement identifiés d'un point de vue histologique. Les cellules souches du muscle expriment le facteur de pluripotence Sox2, mais aussi des marqueurs d'immaturité tels que BCRP1/ABCG2, Sca-1 et SSEA1. J'ai examiné leur potentiel de différenciation in vitro en plusieurs lineages tels que des cellules cardiaques spécifiques (dites pacemakers), des cellules productrices d'insuline et des cellules qui présentent des marqueurs neuronaux. Je me suis également concentrée sur les possibles applications thérapeutiques grâce à l'utilisation de modèles génétiques murins et notamment dans les cas de problèmes du rythme cardiaque, et du diabète insulinodépendant. Pour ces études in vivo du potentiel réparateur des MDSC on procède à une simple injection des cellules souches dérivées du muscle squelettique (MDSC). Le fait de retrouver des MDSC injectées dans les organes cibles des souris modèles pose aussi la question de la biodistribution de ces cellules dans l'organisme. J'ai donc consacré plus d'un an de mon financement doctoral pour examiner cette biodistribution et montré un recrutement ciblé dès 48h après injection, vers les organes ou tissus lésés. / The use of stem cells is a promising approach for the treatment of neuromuscular degenerative diseases. Many studies currently focus on embryonic stem cells (ES) and induced pluripotent stem cells (IPs) for use in regenerative medicine. But some problems remain for their use in cell therapy in particular the potential of these cells to form teratomas. This problem requires both ES and IPs to be differentiated towards a specific cell type. Such induction of differentiation can lead to additional risks such as genetic drift or various sources of contamination.The adult skeletal muscle, has a high plasticity and regenerative capacity, it contains a stem cell population that is specific for muscle, and has been isolated and studied in the laboratory. Adult skeletal Muscle-Derived Stem Cells, MDSC repopulate and repair damaged skeletal muscle with high efficiency in a few days, even in the presence of endogenous satellite cells. (Arsic et al Exp. Cell Res. 2008). The host laboratory is characterizing this cell population and its histological identity and testing the tissue repair potential of transplanted MDSC in mouse models, as well as their bio-distribution for therapeutic use.My thesis work addressed the study of this stem cells population isolated from skeletal muscle showing low adhesion to substrate. Poor/low adherence is an interesting property because in addition to be defined as closer to the pluripotent state, this property is associated with a higher migration capability. This population of muscle stem cells should be easier to use than pre-differentiated stem cells in regenerative medicine. In this perspective it is interesting to use multipotent stem cells that are close to pluripotent cells in terms of differentiation and regenerative capacity, but without the inconveniencies like teratogenic risk and uncontrolled proliferation, as well as expensive and time-consuming cell culture.At the beginning of my thesis I was interested by the different populations of cells present in muscle and I focused my work on known markers of stem cells, whose presence has been established in skeletal muscle, but not clearly identified histologically. Muscle stem cells expressed the pluripotency factor Sox2, but also markers, such as BCRP1/ABCG2, Sca-1 and SSEA1. I have examined the potential of MDSC to differentiate in vitro into several cell types such as cardiac pacemaker-like cells, insulin-producing cells and cells that exhibit neuronal markers. I also focused on the possible therapeutic applications of MDSC, particularly in the case of heart rhythm problems and in the case of insulin-dependent diabetes. For these in vivo studies of the repair potential of MDSC, a single systemic injection is carried out in mouse models of the diseases. The histological recovery of injected MDSC into target organs also raises the question of the biodistribution of MDSC in the body. Therefore I spent more than a year of my doctoral thesis to address this issue and showed a targeted recruitment of MDSC to injured tissue or organs within 48h of their systemic injection.

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.

One of the major drawbacks of using stem cell therapy to treat muscular dystrophies is the challenge of isolating sufficient numbers of suitable precursor cells for transplantation. As such, a deeper understanding of the molecular mechanisms involved during muscle development, which would increase the proportion of embryonic stem cells that can differentiate into skeletal myocytes, is essential. In conditional SOX7-/- mice, we observed that the loss of SOX7 in satellite cells resulted in poor differentiation and fusion. In vivo, we observed fewer Pax7+ satellite cells in the mice lacking SOX7 as well as smaller muscle fibers. RT-qPCR data also revealed that Pax7, MRF and MHC3 transcript levels were down-regulated in SOX7 knockdown mice. Surprisingly, when SOX7 was over-expressed in embryonic stem cells, we found that there was a defect in making muscle precursor cells, specifically a failure to activate Pax7 expression. Taken together, these results suggest that SOX7 expression is required for the proper regulation of skeletal myogenesis.

Over the last decade, microfluidics has emerged as a distinct new field with promising applications for diverse research areas. The ability to precisely control fluids at the microscale allows the execution of a variety of programmable semi-automatic operations on the same device, effectively forming a lab-on-a-chip. In particular, droplet-based microfluidic systems – which reliably generate highly uniform microdroplets at a high throughput – enable the controlled compartmentalization of biological material and have the potential to influence mainstream biomedical research. In this thesis, a microfluidic platform is presented that allows the encapsulation of viable cells in agarose microcapsules for applications in cell–based therapy. As an improvement to pre-existing methods of cell encapsulation, the proposed system combines continuous high throughput cell-encapsulation with on-chip microcapsule gelation and purification.

Injectable biomaterials have emerged as a treatment for myocardial infarction (MI). They can be applied either as an enhancement for cell therapy or as a stand-alone treatment for MI. The main focus of this study was to apply circulating angiogenic cells (CACs) with or without an injectable collagen matrix for MI treatment in a mouse model. Furthermore, a collagen-chitosan matrix was tested for modulating the myocardial maladaptive remodeling post-MI. First, the in vivo thermo-gelling and retention properties of the collagen matrix were validated using positron emission tomography (PET) tracer and quantum dot (Qdot) labelled matrix in MI mouse hearts. The therapeutic potential of the matrix ± CACs was then tested in a mouse MI model. The results showed that CACs-only and matrix-only treatments were associated with cardiac function preservation. However, in combination, CAC + matrix therapy had a synergistic effect and significantly improved cardiac function (echocardiography), perfusion and viability (PET scan), increased cell engraftment and arteriole density, and reduced the infarct size. CAC-matrix interaction through the integrin alpha2 receptor was essential for the observed therapeutic effect. In a third study, the addition of chitosan (a polysaccharide) to the collagen matrix was shown to reduce maladaptive remodeling post-MI by limiting cardiac fibroblast-to-myofibroblast differentiation and scar formation. In conclusion, these collagen-based hydrogels hold promise to enhance cardiac repair as a delivery scaffold for therapeutic cells, and/or as a stand-alone treatment, which can actively modulate the environment including the fibrotic process after MI.

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.

abstract: Cancer is a worldwide burden in every aspect: physically, emotionally, and financially. A need for innovation in cancer research has led to a vast interdisciplinary effort to search for the next breakthrough. Mathematical modeling allows for a unique look into the underlying cellular dynamics and allows for testing treatment strategies without the need for clinical trials. This dissertation explores several iterations of a dendritic cell (DC) therapy model and correspondingly investigates what each iteration teaches about response to treatment. In Chapter 2, motivated by the work of de Pillis et al. (2013), a mathematical model employing six ordinary differential (ODEs) and delay differential equations (DDEs) is formulated to understand the effectiveness of DC vaccines, accounting for cell trafficking with a blood and tumor compartment. A preliminary analysis is performed, with numerical simulations used to show the existence of oscillatory behavior. The model is then reduced to a system of four ODEs. Both models are validated using experimental data from melanoma-induced mice. Conditions under which the model admits rich dynamics observed in a clinical setting, such as periodic solutions and bistability, are established. Mathematical analysis proves the existence of a backward bifurcation and establishes thresholds for R0 that ensure tumor elimination or existence. A sensitivity analysis determines which parameters most significantly impact the reproduction number R0. Identifiability analysis reveals parameters of interest for estimation. Results are framed in terms of treatment implications, including effective combination and monotherapy strategies. In Chapter 3, a study of whether the observed complexity can be represented with a simplified model is conducted. The DC model of Chapter 2 is reduced to a non-dimensional system of two DDEs. Mathematical and numerical analysis explore the impact of immune response time on the stability and eradication of the tumor, including an analytical proof of conditions necessary for the existence of a Hopf bifurcation. In a limiting case, conditions for global stability of the tumor-free equilibrium are outlined. Lastly, Chapter 4 discusses future directions to explore. There still remain open questions to investigate and much work to be done, particularly involving uncertainty analysis. An outline of these steps is provided for future undertakings. / Dissertation/Thesis / Doctoral Dissertation Applied Mathematics 2020

O tumor mamário é a segunda neoplasia maligna mais incidente em cães, com uma elevada taxa de mortalidade. Em cães, constituem aproximadamente 52% de todos os tumores que afetam as fêmeas, possuindo uma elevada heterogeneidade biológica e histomorfológica, sendo que 50% são malignos. As neoplasias, independentemente de suas causas primárias, apresentam distúrbios no controle do ciclo celular, o que acaba gerando um aumento na proliferação celular, perda da diferenciação e formação de massas tumorais. Na gênese das neoplasias mamárias estão envolvidos fatores de natureza genética, ambiental e hormonal. O objetivo deste trabalho foi avaliar "in vitro" o efeito da terapia com célula- tronco da medula óssea canina de feto em tumores mamários canino. Após a avaliação histopatológica os tumores foram divididos em grupos de grau I (adenocarcinoma, sem metástase), grau II (carcinoma com metástase regionais) e grau III (carcinomas com metástases sistêmicas). As células dos tumores mamários caninos (TM) e as células - tronco de medula óssea de feto canina (CTMs) foram caracterizadas antes e após o tratamento utilizando marcadores de proliferação, angiogênese e da resposta inflamatória através da citometria de fluxo. A expressão de marcadores de proliferação celular Ki67 foi maior nas linhagens tumorais dos carcinomas grau III, o mesmo observado para os marcadores que regulam a angiogênese e migração celular como VEGFR1, CD44, COX-2 e EGFR. Foi possível observar nas culturas celulares de TM a redução do potencial elétrico mitocondrial alterando permeabilidade da membrana ativando a caspase 3 e reduzindo a Bcl-2, sugerindo que a CTM apresenta efeitos antitumorais pela indução de apoptose e efeitos antiproliferativos da expressão de COX-2 e IL-6 inibindo a proliferação celular levando ao acúmulo das células na fase G0/G1. . A terapia celular com CTMs proporciona novas expectativas para o tratamento de diversas patologias que acometem diferentes espécies.. Desta forma, os resultados os resultados obtidos pela expressão das TM tratadas com CTM pode apresentar como uma nova perspectiva no tratamento de tumores em cadelas. / The breast tumor is the second most frequent malignancy in dogs, with a high mortality rate. In dogs, they constitute approximately 52% of all tumors affecting female, having a high biological and histomorphological heterogeneity, and 50% are malignant. Neoplasms, regardless of their root causes, have disturbances in cell cycle control, which ends up generating an increase in cell proliferation, differentiation loss and formation of tumor masses. In the genesis of mammary tumors are involved genetic, environmental and hormonal factors nature. The objective of this study was to evaluate "in vitro" the effect of therapy with stem cell-canine bone marrow fetus in canine mammary tumors. After histopathological evaluation tumors were divided into grade I groups (adenocarcinoma without metastasis), grade II (carcinoma with regional metastasis) and grade III (carcinomas with metastatic disease) .The cells of canine mammary tumors (TM) and cells - bone marrow stem canine fetuses (MSCs) were characterized before and after treatment using proliferation markers, angiogenesis and inflammatory response by flow cytometry. The expression of cell proliferation markers Ki67 was higher in tumor cell lines of the carcinomas grade III, the same was observed for markers that regulate angiogenesis and cell migration as VEGFR1, CD44, COX-2 and EGFR. It was observed in TM cell cultures to reduce the electric potential changing mitochondrial membrane permeability by activating caspase 3 and reduces Bcl-2, suggesting that MSC has antitumor effects by inducing apoptosis and antiproliferative effects of COX-2 expression and IL-6 inhibiting cell proliferation leading to an accumulation of cells in G0 / G1 phase. Cell therapy MSCs provides new expectations for the treatment of various pathologies affecting different species. Thus, the results the results obtained by the expression of TM-treated MSC can present as a new approach in the treatment of tumors in dogs..

abstract: The success of genetically-modified T-cells in treating hematological malignancies has accelerated the research timeline for Chimeric Antigen Receptor-T (CAR-T) cell therapy. Since there are only two approved products (Kymriah and Yescarta), the process knowledge is limited. This leads to a low efficiency at manufacturing stage with serious challenges corresponding to high cost and scalability. In addition, the individualized nature of the therapy limits inventory and creates a high risk of product loss due to supply chain failure. The sector needs a new manufacturing paradigm capable of quickly responding to individualized demands while considering complex system dynamics. The research formulates the problem of Chimeric Antigen Receptor-T (CAR-T) manufacturing design, understanding the performance for large scale production of personalized therapies. The solution looks to develop a simulation environment for bio-manufacturing systems with single-use equipment. The result is BioMan: a discrete-event simulation model that considers the role of therapy's individualized nature, type of processing and quality-management policies on process yield and time, while dealing with the available resource constraints simultaneously. The tool will be useful to understand the impact of varying factor inputs on Chimeric Antigen Receptor-T (CAR-T) cell manufacturing and will eventually facilitate the decision-maker to finalize the right strategies achieving better processing, high resource utilization, and less failure rates. / Dissertation/Thesis / Masters Thesis Industrial Engineering 2020