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.

In this work, gadolinium-based carbon nanocapsules are developed as a novel nanotechnology that addresses the shortcomings of current diagnostic and therapeutic methods of stem cell-based cardiomyoplasty. With cardiovascular disease (CVD) responsible for approximately 30% of deaths worldwide, the growing need for improved cardiomyoplasty has spurred efforts in nanomedicine to develop innovative techniques to enhance the therapeutic retention and diagnostic tracking of transplanted cells. Having previously been demonstrated as a high-performance T1-weighted magnetic resonance imaging (MRI) contrast agent, Gadonanotubes (GNTs) are shown for the first time to intracellularly label pig bone marrow-derived mesenchymal stem cells (MSCs). Without the use of a transfection agent, micromolar concentrations of GNTs deliver up to 10^9 Gd(III) ions per cell, allowing for MSCs to be visualized in a 1.5 T clinical MRI scanner. The cellular response to the intracellular incorporation of GNTs is also assessed, revealing that GNTs do not compromise the viability, differentiation potential, or phenotype characteristics of the MSCs. However, it is also found that GNT-labeled MSCs exhibit a decreased response to select cell adhesion proteins and experience a non-apoptotic, non-proliferative cell cycle arrest, from which the cells recover 48 h after GNT internalization. In tandem with developing GNTs as a new stem cell diagnostic agent, this current work also explores for the first time the therapeutic application of the magnetically-active GNTs as a magnetic facilitator to increase the retention of transplanted stem cells during cardiomyoplasty. In vitro flow chamber assays, ex vivo perfusion experiments, and in vivo porcine injection procedures all demonstrate the increased magnetic-assisted retention of GNT-labeled MSCs in the presence of an external magnetic field. These studies prove that GNTs are a powerful ‘theranostic’ agent that provides a novel platform to simultaneously monitor and improve the therapeutic nature of stem cells for the treatment of CVD. It is expected that this new nanotechnology will further catalyze the development of cellular cardiomyoplasty and other stem cell-based therapies for the prevention, detection, and treatment of human diseases.

Die Keimstreifverlängerung der Malacostraca wird durch ein festgelegtes Zellteilungsmuster der posterioren Teilungszone erreicht. Dort geben anfangs in einem Halbkreis angeordnete Ektoteloblasten ihre Tochterzellen nach anterior ab, welche das für malakostrake Krebse typische Gittermuster ausbilden. Dabei spielen die Ektoteloblasten eine besondere Rolle, da sie das Zellmaterial für die anschließende Segmentierung produzieren. Im Gegensatz zu Vertretern basal abzweigender Taxa der Malacostraca, bei denen sich der Halbkreis der Ektoteloblasten aus zwei Halbreihen oder aus einem Zellring bildet, sind bei den Isopoden, zu denen auch die Kellerassel Porcellio scaber zählt, die Ektoteloblasten von Beginn an in einem Halbkreis angeordnet. Das Teilungsmuster bei der Verlängerung des Keimstreifens wurde bereits bei vielen Vertretern der Malacostraca, einschließlich P. scaber, ausführlich untersucht. Daten über die Bildung der Ektoteloblasten und den Beginn ihrer Teilungsaktivität fehlen dagegen weitestgehend. Im Rahmen der vorliegenden Arbeit wurde daher mit Hilfe von Zellmarkierungsversuchen mit Vitalfarbstoffen (wie z.B. Alexa Fluor 488 und Tetramethylrhodamin) und der 4D-Mikroskopie die Bildung der Keimscheibe, die Gastrulation und der Beginn der Keimstreifverlängerung bei P. scaber untersucht. Ein besonderer Fokus lag dabei auf den Ektoteloblasten und den umliegenden Zellen, deren Zelllinien untersucht wurden. Wie in der vorliegenden Arbeit gezeigt werden konnte, sind (i) die Ektoteloblasten bereits vor Beginn der Gastrulation in einem äußeren Halbkreis formiert, (ii) zwei Zellen, die Teile der späteren Mittellinie bilden, bis kurz vor Beginn der Teilungstätigkeit der Ektoteloblasten in deren Reihe eingereiht, (iii) die Ektoteloblasten erst mit der Fertigstellung der ersten e-Reihe als große, verlängerte Zellen erkennbar und es ist (iv) ein zweiter Halbkreis vorhanden, der sich ausschließlich in posteriore Richtung teilt und dadurch den Gastrulationsporus schließt. / In malacostracan crustaceans, the elongation of the germ band is realized by an invariant cell division pattern of the posterior growth zone. The ectoteloblasts are arranged in a semicircle and divide in anterior direction in a fixed pattern. Their progeny are arranged in the typical grid pattern of malacostracan crustaceans and provide the cell material for later segmentation processes. In contrast to other malacostracans, where the ectoteloblast appear in two half rows or a cell ring, the ectoteloblasts are arranged in a semicircle in isopods from the outset. The cell division pattern during elongation of the germ band is well studied in many malacostracan species including the isopod Porcellio scaber. However, details about formation of the ectoteloblasts and beginning of their cell division activity are missing. Therefore, the aggregation of the germ disc, the gastrulation, and the beginning of the elongation of the germ band in Porcellio scaber were examined using vital dyes (e.g. Alexa Fluor 488 and Tetramethylrhodamine) in cell labeling experiments combined with 4Dmicroscopy. The formation and cell-lineages of the ectoteloblasts and their surrounding cells were analyzed. The results suggest that (i) the ectoteloblasts are arranged in a semicircle before gastrulation starts, (ii) two cells forming part of the arising midline are lined up with the ectoteloblasts until they start their cell division activity in a predetermined pattern, (iii) the differentiation of ectoteloblasts is discernable after formation of the first e-row when they appear as big and elongated cells and (iv) a second semicircle exists whose cells divide in posterior direction and, consequently, enclose the gastrulation pore.

Abstract Ischemic heart disease is one of the leading cause of death in the Western world. There is convincing evidence that stem cell therapy improves cardiac function and reduces the scar formation following an acute myocardial infarction (AMI). The mechanisms involved in the recovery remain partly unknown. Direct injection of stem cells into myocardium is a widely used transplantation technique though there are few details available about the behavior of cells after transplantation. A cardiac explant culture model simulating tissue stress was developed in this study to examine in detail the properties of the stem cells after their transplantation. The migration range in myocardium and the number of adherent stem cells increased with time. In vitro and in vivo studies revealed that after their administration, the stem cells became localized in the slit-like spaces, such as in the capillaries. Even though the study outcomes regarding the impact of stem cell therapy in recovery after AMI have been largely promising, the results of the clinical studies have proved to be more controversial. If one wishes to evaluate the true contribution of the stem cell therapy to the recovery, it is essential to devise a reliable study method for cell targeting. Here, iron labeled stem cells in combination with magnetic resonance imaging (MRI) were used. The MRI data corresponded to the histological results. Thus, it is concluded that MRI is a feasible method for monitoring the effectiveness of cell targeting. Stem cell treatment was shown to increase cardiac function at three weeks after AMI. If there was a high number of stem cells in cardiac tissue after transplantation, this predicted a greater improvement in cardiac function. Improper stem cell injection may lead to leakage of the stem cells out of the myocardium, leading to unreproducible study results. Inflammation modulating factors secreted by the stem cells are considered as key mechanisms in the recovery after AMI. There were differences in the cytokine levels between the stem cell treated and control groups in a clinical and in vivo animal study i.e. stem cell therapy exerted a balancing effect on the inflammatory process, a crucial component in the optimal recovery after AMI. The present study reveals many properties of stem cells, importance of cell targeting and the influence of stem cell therapy on cytokine levels after AMI. / Tiivistelmä Iskeeminen sydänsairaus on yksi yleisimmistä kuolinsyistä länsimaissa. Tutkimusten mukaan kantasoluterapia parantaa sydämen toimintakykyä ja pienentää akuutin sydäninfarktin jälkeen sydämeen muodostuvan arpikudoksen määrää. Paranemiseen liittyvät mekanismit ovat edelleen osittain tuntemattomia. Kantasolujen ruiskutus suoraan sydämeen on paljon käytetty menetelmä, vaikka solujen käyttäytymistä ei tunneta tarkkaan.Tutkimuksessa kehitetyn kudoksen stressitilaa simuloivan sydänkudoksen kasvatusmenetelmän avulla tutkittiin siirrettyjen kantasolujen toimintaa yksityiskohtaisesti. Kantasolujen vaeltaman matkan sydänkudoksessa ja kiinnittyneiden kantasolujen lukumäärä havaittiin kasvavan ajan kuluessa. In vitro ja in vivo tutkimuksissa havaittiin kantasolujen sijaitsevan ruiskutuksen jälkeen rakomaisissa paikoissa kuten pienissä verisuonissa. Vaikka tutkimustulokset kantasoluterapian hyödyistä paranemisen suhteen ovat pääosin lupaavia, kliinisten tutkimusten tulokset ovat ristiriitaisia. Todellisen kantasoluhoidon vaikutuksen arvioimiseksi tarvitaan luotettava menetelmä varmistamaan kantasolujen hakeutuminen vaurioalueelle. Tässä tutkimuksessa rautaleimattujen kantasolujen paikantamisessa käytetty magneettikuvantaminen vastasi histologisia löydöksiä. Magneettikuvantaminen todettiin käyttökelpoiseksi menetelmäksi solujen paikallistamisessa. Kantasoluhoidon osoitettiin parantavan sydämen toimintakykyä kolme viikkoa akuutin sydäninfarktin jälkeen. Suuri kantasolumäärä sydänkudoksessa siirron jälkeen ennusti parempaa toipumista. Puutteellisesti suoritettu kantasoluruiskutus voi johtaa kantasolujen vuotamiseen pois sydänkudoksesta aiheuttaen vaihtelevuutta tutkimustuloksiin. Kantasolujen erittämiä tulehdusta sääteleviä tekijöitä pidetään tärkeimpänä mekanismina paranemisprosessissa. Tutkimus osoitti eroavaisuuksia kantasoluhoidetun ja kontrolliryhmän välillä. Kliinisessä ja koe-eläintutkimuksessa kantasolusiirrolla todettiin tulehdusreaktiota tasapainottava vaikutus, mikä on tärkeää optimaalisen sydänlihaskudoksen paranemisen kannalta akuutin sydäninfarktin jälkeen. Tutkimus toi esiin monia kantasolujen ominaisuuksia, solujen paikantamisen tärkeyden ja kantasoluhoidon vaikutuksen sytokiinipitoisuuksiin akuutin sydäninfarktin jälkeen.

Mouse NK cell receptors belonging to NKR-P1 family plays role in activation, inhibition and cytokine secretion by these cells. Aim of this thesis is preparation of extracellular parts of C57BL/6 mouse strain activating receptors mNKR-P1A and mNKR-P1C. Production vectors with coding sequences of both proteins were prepared. Next, optimization of production in E. coli was done and appropriate in vitro refolding and purification protocol were developed. Purified proteins were characterized by mass spectrometry and labeled by a fluorescent dye. Primary screening for potential ligand was performed. Further work will involve structural characterization of the receptors and identification of their ligands. These data may help to clarify the function of NK cells.

Transplantation of pancreatic islets (PIs) represents an alternative treatment for type 1 diabetes mellitus. Post-transplant monitoring of islets by a reliable imaging method may contribute to the improvement of the transplantation outcome. In this thesis, novel visualization approaches for PIs were tested using magnetic resonance (MR) and optical imaging on phantoms and experimental animals, including Chemical Exchange Saturation Transfer (CEST) MR, fluorine (19 F) MR, bioluminescence and fluorescence imaging. MR imaging based on frequency-selective method CEST was performed on islets labeled with Eu-/Yb-based chelates. Labeled islets possessed low MR signal in phantoms, what would have been unsatisfactory for in vivo applications. Moreover, viability and function of labeled islets was impaired reflecting limited applicability of these agents for islet labeling and visualization. Genetically modified bioluminescent islets showed suitable properties for longitudinal tracking of their post-transplant fate at an artificial transplant site - subcutaneously implanted polymeric scaffolds. Using multimodal imaging (MR and bioluminescence), the optimal timing for transplantation of islets into the scaffolds was assessed in diabetic rats. Islets transplanted into scaffolds using the optimized timing scheme...

La thérapie cellulaire consiste à utiliser des cellules comme médicament injectable dans le but de favoriser la réparation d'un tissu ou d'un organe. Certaines cellules possèdent la propriété de stimuler la formation de nouveaux vaisseaux sanguins. Elles présentent un intérêt pour le développement d'un traitement des ischémies des membres inférieurs ou du myocarde. Déterminer le devenir de ces cellules après injection in vivo est important pour comprendre les mécanismes responsables de l'efficacité d'un tel traitement. Cependant, il est difficile à observer et très peu décrit dans la littérature. Quelques exemples d'utilisation de nanotechnologies sont rapportés pour suivre des cellules in vitro. Mais des facteurs limitant tels qu'une complexité de mise en oeuvre de ces techniques ou leur trop faible sensibilité rend difficile leur utilisation in vivo. Ce travail de thèse propose de décrire le développement de nanoparticules originales associant les modalités d'imagerie optique et d'IRM afin de marquer simplement des cellules ayant un potentiel thérapeutique. Les protocoles développés ont permis de vectoriser et suivre en temps réel ces cellules après injection chez la souris. / Cell therapy aims to use cells as injectable medicines in order to enhance damaged organs or tissues repair. Some cells have the ability to promote new blood vessels growth. Therefore, they have an interest in revascularization of ischemic tissues and are potential candidates for cell therapy in peripheral ischemia or myocardial infarction. Being able to determine these cell's fates after in vivo injection is a major step to better understand the mechanisms of such treatments efficiency. However, following this phenomenon is challenging and rarely described in scientific litterature. Only some applications of nanotechnologies to follow labeled cells in vitro have been published. But limitations as implementation complexity or a low sensitivity prevent from using these techniques in vivo. This phD work describes the development of new hybrids nanoparticules associating optical and magnetic resonance imaging modalities in order to efficiently label cells that have a therapeutic potential. Developed protocols allowed us to follow magnetic cell vectorisation after injection in mice in real time.