A lesão medular traumática é uma patologia incapacitante, ainda sem tratamento eficaz. As terapias celulares representam uma nova estratégia para o tratamento destas lesões. As células-tronco adultas são fontes potenciais para o transplante celular com o objetivo de minimizar a lesão e promover a recuperação de tecidos lesados, como a medula espinhal. O objetivo desta tese foi avaliar a eficácia do transplante de células-tronco adultas na recuperação funcional e regeneração da lesão medular traumática em modelo experimental de lesão medular contusa em ratas fêmeas Wistar. Os principais objetivos foram: a) comparar os efeitos do transplante da fração mononuclear de sangue de cordão umbilical humano e de células-tronco mesenquimais dos vasos da parede do cordão umbilical humano; b) determinar a janela terapêutica deste tipo de intervenção, comparando os implantes de células- tronco realizados 1 hora, 24 horas e 9 dias após a lesão; c) demonstrar a possível diferenciação das células-tronco implantadas, bem como sua integração no tecido lesado. Os resultados obtidos demonstraram que o transplante de células foi mais eficaz para a recuperação funcional da lesão medular em ratas Wistar quando realizado pela via de administração local 1h após a lesão, quando comparado com a administração na cisterna magna e a aplicação 9 dias a lesão. O tratamento com a fração de células mononucleares ou com as células-tronco mesenquimais do sangue do cordão umbilical 24h após a lesão, não apresentou resultado funcional significativo.Observou-se a neuroproteção do tecido medular quando foi realizado o transplante de células-tronco mesenquimais 1h após a lesão medular. As células humanas transplantadas migraram e sobreviveram no local da lesão quando administradas na cisterna magna ou quando administradas diretamente no local da lesão, porém não se diferenciaram em células gliais ou neurônios. Concluímos que o transplante de células-tronco adultas promoveu a recuperação funcional após a lesão medular contusa, principalmente quando realizado 1h após a lesão diretamente no local da lesão. Apesar das células transplantadas sobreviverem na área da lesão, não foi evidenciada diferenciação celular. / Spinal cord injury is a debilitating disease and yet no effective treatment is available. In this framework cell therapy represents a new strategy to treat this condition. Adult stem cells are potential sources for cell transplantation in order to minimize injury and promote the recovery of damaged tissues, such as the spinal cord. The purpose of this Thesis was to evaluate the action of adult stem cells in the regeneration and functional recovery of spinal cord injury in experimental contusion spinal cord injury in female Wistar rats. Main goals were: a) to compare the effects of transplantation of the mononuclear cells of human umbilical cord blood and mesenchymal stem cells of the vessel wall of human umbilical cord; b) to determine the therapeutic window of this type of intervention, comparing the stem cell implants performed 1 hour, 24 hours and 9 days after injury; c) to demonstrate the possible differentiation of cells implanted, as well as their integration into the damaged tissue. Results reported demonstrate that the transplantation of stem cells was more effective for functional recovery of spinal cord injury when performed into the site of the lesion 1 h after injury, as compared with administration in the cisterna magna 9 days after injury. Treatment with mononuclear cells and mesenchymal cells from umbilical cord blood 24 hours after injury, not showed functional outcome. Neuroprotection was observed when mesenchymal stem cells were transplanted 1 hour after spinal cord injury. The transplanted human cells survived and migrated to the site of injury either when administered in the cisterna magna or directly onto the injury site, but did not differentiated into glial cells or neurons. It is suggested that the transplantation of adult stem cells promotes functional recovery after spinal cord injury when performed 1 hour after injury directly at the injury site, however differentiation of transplanted cells was not detected.

A sepse causa uma alta taxa de mortalidade no mundo. A fisiopatologia da doença envolve uma rede complexa de mediadores inflamatórios que promovem a lesão de diversos tecidos, além de diversas alterações hemodinâmicas e disfunção do sistema nervoso autonômico (SNA). Assim sabe-se que o sistema nervoso cumpre um papel importante no controle da inflamação sistêmica mediante a via colinérgica anti-inflamatória (VCA) através do receptor nicotínico de acetilcolina alfa7 (alfa7nAChR). O uso das células-tronco mesenquimais (CTM) tem mostrado efeitos benéficos em diversos ensaios clínicos de doenças inflamatórias. Neste contexto, as células-tronco mesenquimais derivadas da geleia de Wharton do cordão umbilical (CTM-GW) tornam-se promissórias, uma vez que essas células são reconhecidas pela regulação da resposta imunológica, reparação neural, efeito anti-apoptose, assim como a melhora da sobrevida na sepse, em modelos experimentais. Nossa hipótese foi de que as CTM-GW poderiam cumprir um papel neuroimunomodulador através da VCA e atenuar a disfunção de múltiplos órgãos em um modelo animal de sepse de ligadura e punção do ceco (LPC). Inicialmente células da matriz do cordão umbilical foram isoladas e caracterizadas de acordo com o consenso internacional vigente. Ratos Wistar machos adultos foram subdivididos em grupos: 1) sham (operação simulada); 2) LPC; 3) LPC+CTM-GW (injetado 106 CTM-GW via intraperitoneal, i.p. 6 h após LPC) e 4) LPC+MLA+CTM-GW (MLA: Metillicaconitine, antagonista do alfa7nAChR, i.p., 5:30 h após LPC e 106 CTM-GW 6h após). Às 24 horas após LPC, foram avaliadas a função cardiovascular, hemodinâmica assim como os outros parâmetros. Interessantemente, o tratamento com CTM-GW na sepse atenuou a disfunção diastólica e protegeu a sensibilidade baroreflexa. Além disso, as CTM-GW estimularam a atividade autonômica, simpática e parassimpática no coração. Observamos que o tratamento celular induziu uma regulação da expressão do receptor alfa7nAChR e TLR4 no baço e no coração, assim como a redução da relação p-STAT3TYR705 e STAT3 total no baço. Outros efeitos importantes e adicionais foram a diminuição da infiltração de leucócitos e a regulação das citocinas pró-inflamatórias pelas células. O bloqueio da VCA usando MLA confirmou que o receptor alfa7nAChR pode ser um provável alvo, chave da ação das CTM entre vários outros mecanismos envolvidos na resposta imune. Finalmente, as CTM-GW conseguiram reduzir a apoptose no pulmão e no baço independentemente da VAC reforçando o conceito de que as células-tronco tem efeitos diversos além da imuno-regulação. Em conclusão, as CTM-GW na sepse foram capazes de atenuar a lesão cardiopulmonar assim como modular a atividade autonômica, reduzindo a inflamação sistêmica, pelo menos em parte, através da via colinérgica anti-inflamatória. Indubitavelmente todos estes efeitos anteriormente descritos e em associação se demonstraram fundamentais no mecanismo de reparo e proteção tecidual em resposta a sepse. Mais estudos pré-clínicos e futuros testes clínicos precisam ser realizados para maior compreensão destes mecanismos bem como uma possível validação terapêutica / Sepsis induces organ dysfunction due to overexpression of the inflammatory host response, involving cardiorespiratory and autonomic dysregulation, thus increasing the associated morbidity and mortality. The cholinergic anti-inflammatory pathway (CAP) is mediated by nervous system through alpha7 nicotinic acetylcholine receptor (alpha7nAChR). This receptor has an important role in systemic inflammation control. Wharton\'s jelly-derived mesenchymal stem cells (WJ-MSCs) are known to express genes and secreted factors related to neurological and immunological protection, as well as to improve survival in experimental sepsis. We hypothesized that WJ-MSCs play a modulatory role through the CAP and attenuate sepsis-induced organ injury in a cecal ligation and puncture (CLP) model. Rats were randomly divided into 4 groups: 1) Control (sham-operated); 2) submitted to CLP without treatment; 3) submitted to CLP and treated with 106 WJ-MSCs 6 h later and 4) CLP+MLA+WJ-MSC group (MLA: Methyllycaconitine, alpha7nAChR antagonist). All experiments were performed 24 h post-surgery. Echocardiographic parameters and heart rate variability were assessed. Importantly, treatment with WJ-MSCs attenuated diastolic heart failure and recovered barorreflex sensitivity. Moreover, WJ-MSCs injection increased cardiac sympathetic and cardiovagal activity. In cardiac and splenic tissue, WJ-MSC treatment downregulated TLR4 and alpha7nAChR expression, as well as it reduced p-STAT3/Total STAT3 ratio in the spleen. In addition, WJ-MSC reduced leukocyte infiltration and pro-inflammatory cytokines, which only were abolished by MLA treatment. Finally, WJ-MSC treatment diminished apoptosis in lung and spleen tissue. Together these findings suggest that treatment with WJ-MSCs appears to protect against sepsis-induced organ injury reducing systemic inflammation, at least in part, through cholinergic anti-inflammatory pathway

Les cellules souches ont été présentées comme la clé d’une médecine régénératrice, où la réparation d’un organe, la guérison de maladies dégénératives et la création de nouveaux tissus seraient des objectifs réalisables à court ou moyen terme. Les cellules souches isolées chez un adulte ont un certain degré de spécialisation limitant leur potentiel à leur organe d’origine. Mais l’une d’elles, la cellule souche mésenchymateuse (MSC), possède une versatilité qui en fait une candidate idéale pour plusieurs traitements. Il est communément accepté que les MSC exercent leur effet grâce à la production de facteurs solubles. Toutefois, leur mécanisme d’action reste jusqu’à maintenant imprécis. Ces facteurs ayant le potentiel d’agir dans plusieurs maladies dégénératives, nous avons voulu évaluer leur effet régénératif dans l’une des maladies oculaires les plus répandues, le glaucome à angle ouvert. Cette maladie est caractérisée par une destruction des cellules ganglionnaires de la rétine suite à une élévation de la pression intraoculaire. Cette hausse de pression est souvent engendrée par une dysfonction du trabéculum, le tissu régulant la sortie de l’humeur aqueuse de l’œil. Actuellement, la progression du glaucome peut être contrôlée, mais la maladie ne peut pas être guérie. Or la régénération du trabéculum pourrait arrêter la progression de la maladie et même renverser le processus. Ainsi, l’objectif de cette thèse était de vérifier si les MSC ont le potentiel de favoriser la régénération oculaire dans des cas de glaucome et de comprendre les mécanismes sous-jacents. Pour ce faire, nous avons injecté des MSC ou leurs facteurs sécrétés dans un modèle de glaucome induit par trabéculoplastie laser. Nous avons démontré que les MSC peuvent régénérer le trabéculum endommagé et abaisser la pression oculaire en réactivant des cellules progénitrices dans le corps ciliaire. Ces cellules prolifèrent et s’implantent dans le trabéculum une semaine après le traitement. Nous avons aussi démontré que les facteurs produits par les MSC cultivées en conditions hypoxiques induisent l’activation des cellules progénitrices. Par contre, les MSC cultivées dans un environnement normoxique n’induisent pas cet effet. Nous avons aussi observé que les facteurs produits dans des conditions hypoxiques sont incapables de réactiver les cellules progénitrices ex vivo. Nous avons donc voulu vérifier si d’autres facteurs régénératifs sont engendrés in situ suite à l’injection des MSC. Nous avons ainsi découvert qu’un facteur produit par les macrophages migrant dans la zone endommagée permet de réguler l’activation des cellules progénitrices. Cette production requiert cependant une exposition des macrophages aux facteurs paracrines des MSC. Nous avons aussi observé que l’effet régénératif des MSC était inhibé suite à l’élimination des macrophages de l’organisme. Ceci positionne les macrophages comme un intermédiaire essentiel de l’effet régénératif exercé par les MSC. Les résultats présentés dans cette thèse constituent une avancée importante pour l’utilisation des MSC en médecine régénératrice. Ils ont permis d’établir les bases d’un traitement potentiel pour le glaucome à angle ouvert et d’ajouter une pièce importante à la compréhension des mécanismes régénératifs des MSC. Ces connaissances devraient avoir un impact significatif sur la régénération du tissu oculaire et de plusieurs autres organes. / Stem cells have been presented as the key to regenerative medicine, where organ repair, cures for degenerative diseases and even the creation of new tissue could be achieved. Adult stem cells already possess a certain level of differentiation, which limits their potential effect to the organ where they were isolated. But one subtype, mesenchymal stem cells (MSC), possess a versatility which makes them ideal candidates for many treatments. It is commonly accepted that MSCs exert their effect through paracrine factors, but their precise mechanism of action remains uncertain. As such factors could have a positive effect in many degenerative diseases, we wanted to study their regenerative potential in one of the most widespread ocular disease, open angle glaucoma. This disease is characterised by the destruction of retinal ganglion cells following a rise in intraocular pressure. This pressure rise is mostly caused by a dysfunction of the trabecular meshwork, the tissue regulating the outflow of aqueous humor from the eye. Glaucoma progression can currently be controlled, but the disease cannot be cured. Meanwhile, trabecular regeneration could halt and even reverse disease progression. Thus, the objective of this thesis is to evaluate MSC potential in the regeneration of the trabecular meshwork and to understand any underlying mechanisms. To this end, we injected MSC or their secreted factors in a model of trabeculoplasty induced glaucoma. We demonstrated that MSC can regenerate the damaged trabeculum and lower ocular pressure by reactivating progenitor cells located in the ciliary body. These cells proliferate and restore the trabeculum within one week after the initial treatment. We also demonstrated that MSC factors produced under hypoxic conditions induce progenitor cell activation. Such activation does not occur with MSC factors produced under normoxic conditions. We have also observed that factors produced under hypoxic conditions are incapable of reactivating progenitor cells in an ex vivo setting. Thus, we wanted to verify if any other regenerative factors are produced in vivo after MSC injection. We have discovered that macrophages produce one such factor after their migration in the area of damage. Production of the macrophage factor requires contact with MSC paracrine factors. We have also observed that MSC regeneration was completely blocked following macrophage elimination from the organism. This positions macrophages as an essential intermediate in MSC-mediated tissue regeneration. The results presented in this thesis represent an important step forward for the use of MSC in regenerative medicine. They have established a basis for an open angle glaucoma treatment and added an important element for the comprehension of MSC regenerative mechanisms. This knowledge should have a significant impact for the treatment of ocular diseases and other illnesses in many different organs.

Actuellement, il est largement reconnu que la décision des cellules souches de maintenir leur caractère souche ou se différencier vers une lignée spécialisée dépend particulièrement de la nature de leur microenvironnement, appelé niche cellulaire. Une des composantes essentielles de cette niche cellulaire est la matrice extracellulaire (MEC), qui au-delà de sa fonction de support cellulaire, détermine le devenir des cellules souches en fonction de sa composition biochimique, sa structure et sa localisation. D’un point de vue rationnel, un biomatériau destiné à remplacer la fonction d’un tissu endommagé doit non seulement jouer le rôle d’échafaudage cellulaire mais également mimer les propriétés de la MEC dans son ensemble. Malheureusement, il est extrêmement difficile de concevoir des biomatériaux mimétiques de la MEC naturelle tenant compte de sa complexité structurelle et fonctionnelle. Pour pallier à cette problématique, il semble nécessaire d’effectuer un travail en amont de déconstruction/reconstruction de la complexité de la MEC en étudiant l’effet individuel puis combiné de ses propriétés sur la différenciation des cellules souches. Ce projet de doctorat rentre dans le cadre de ce travail et vise à déterminer le rôle spécifique ou concomitant de différentes propriétés inhérentes à la MEC sur la différenciation ostéoblastique des cellules souches mésenchymateuses humaines (hCSMs). En effet, nous avons évalué l’effet de la composition biochimique de la MEC et la distribution spatiale des ligands sur la différenciation des hCSMs, en fonctionnalisant la surface d’un matériau modèle avec les peptides RGD et/ou BMP-2, distribués d’une manière aléatoire ou structurée. / Actually, it is well-established that maintaining the stemness character of stem cells or eliciting their lineage-specific differentiation is closely related to the nature of their microenvironment, known as stem cell niche. The extracellular matrix (ECM), a key component of stem cell niche, not only provides a support function for stem cells but also dictates their fate decision. From a rational point of view, a biomaterial intended to replace a damaged tissue should mimic the natural ECM in all its aspects, including its biochemistry, 3D structure, topography, porosity, rigidity…. etc. Unfortunately, the design of biomaterials that fully mimic the natural ECM is still a big challenge, due to its high structural and functional complexity. Towards the development of finely-tuned biomaterials, it seems important to start by deconstructing and then reconstructing the complexity of the ECM. In this context, the thesis project, herein, seeks to evaluate both the individual and the synergistic effect of different properties inherent to the natural ECM on human mesenchymal stem cells (hMSCs) osteogenic differentiation. Indeed, we investigated whether the biochemical composition of the ECM and the spatial distribution of its components modulate hMSCs osteogenesis. This was achieved by creating different artificial ECMs, in vitro, containing RGD and/or BMP-2 mimetic peptides, distributed randomly or as specific micropatterns on the surface of a model material.

Dans ce travail de thèse, la texturation de surface d‟alliages de titane a été étudiée en utilisant un procédé d'écriture directe par laser femtoseconde dans le but d'améliorer la mouillabilité d‟implants dentaires et orthopédiques par les fluides biologiques et la minéralisation de la matrice (formation osseuse) tout en réduisant l'adhésion bactérienne et la formation de biofilmes. Des surfaces de titane (Ti-6Al-4Vet cp Ti) ont été micro-, nano-texturées par laser femtoseconde et une biofonctionnalisation de ces surfaces a été ajoutée ou non par greffage de peptides d'adhésion cellulaire (peptides RGD) en surface de ces différents matériaux. Les textures de surface peuvent être classées comme suit: (a) structures périodiques de surface induites par laser (LIPSS); (b) étalage de nanopiliers (NP); (c) étalage de micro colonnes recouvertes de LIPSS (MC-LIPSS) formant une distribution bimodale de rugosité. Nous avons montré que la texturation de surface par laser améliore la mouillabilité des surfaces avec de l'eau ainsi qu‟une solution saline tamponnée Hank's (HBSS) et amène une anisotropie de mouillage. Une minéralisation cellulaire est observée pour toutes les surfaces des deux alliages de titane lorsque des Cellules Souches Mésenchymateuses humaines (hMSC) sont cultivées dans un milieu ostéogénique. La minéralisation de la matrice et la formation de nodules osseux sont considérablement améliorées sur les surfaces texturées LIPSS et NP. Parallèlement,l'adhésion de Staphylococcus aureus et la formation de biofilmes sont considérablement réduites pour les surfaces texturées LIPSS et NP. La biofonctionnalisation des différentes surfaces texturées (cp Ti) par laser a été réalisée et caractérisée par spectroscopie de photoélectrons (XPS) et par microscopie à fluorescence en utilisant des peptides fluorescents. L‟ensemble des résultats obtenus suggèrent que la texturation de surface d'alliages de titane (Ti-6Al-4V et cp Ti) en utilisant une technique d‟écriture directe par laser femtoseconde est un procédé prometteur pour l'amélioration de la mouillabilité de la surface d'implants dentaires et orthopédiques par les fluides biologiques et leur ostéointégration (différenciation ostéoblastique et minéralisation de la matrice), tout en réduisant l‟adhésion de Staphylococcus aureus et la formation de biofilmes. Enfin, la combinaison de la texturation par laser et du greffage covalent d‟un principe actif (ici un peptide d‟adhésion cellulaire comme le peptide RGD) amènera indéniablement une bioactivité utile pour favoriser l'adhésion des hMSC et faciliter laformation osseuse. / In the present thesis the surface texturing of Ti alloys using femtosecond laser direct writing method is explored as a potential technique to enhance the wettability of dental and orthopaedic implants by biological fluids and matrix mineralisation (bone formation), while reducing bacteria adhesion and biofilmformation. The surface texture was combined with biofunctionalisation by covalent grafting of a RGD peptide sequence as well. The surface textures can be classified as follows: (a) Laser-Induced Periodic Surface Structures-LIPSS; (b) nanopillars arrays(NP); (c) arrays of microcolumns covered with LIPSS (MC-LIPSS), forming a bimodal roughness distribution. Laser texturing enhances surface wettability by water andHank‟s balanced salt solution (HBSS) and introduces wetting anisotropy, crucial incontrolling the wetting behaviour. Matrix mineralisation is observed for all surfaces of both Ti alloys when human mesenchymal stem cells (hMSCs) are cultured in osteogenic medium. Matrix mineralisation and formation of bone-like nodules are significantly enhanced on LIPSS and NP textured surfaces. On the contrary, Staphylococcus aureusadhesion and biofilm formation are significantly reduced for LIPSS and NP textured surfaces. The biofunctionalisation of the laser textured surfaces of cp Ti is sucessfully achieved. In general, these results suggest that surface texturing of Ti alloys using femtosecond laser direct writing is a promising method for enhancing surface wettability of dental and orthopaedic implants by biological fluids and their osseointegration (osteoblastic differentiation and matrix mineralisation), while reducing Staphylococcus aureus adhesion and biofilm formation. Finally, the combination of laser texturing and covalent grafting of a RGD peptide sequence may be potentially useful for increasing cell adhesion and facilitating bone formation.

Nous avons déterminé la capacité de régénération du tissu vasculaire in vivo des CSM traitées avec BMP2 à la dose de [100 ng.mL-1] dans un modèle rat. Nous avons ainsi rapporté qu’une prothèse revêtue de CSM traitée par BMP2 pendant 1 semaine et implantée 14 jours chez le rat permettait la reconstitution des trois tuniques de la paroi mimant la structure de l’aorte. La capacité proangiogénique des CSM était augmentée par BMP2 grâce à la mise en jeu de voies intracellulaires impliquant le facteur induit par l’hypoxie (HIF-1α) via JAK/STATs. Nous avons montré que les CSM migraient sous l’influence de BMP2 par stimulation de l’activité du complexe enzymatique NADPH oxydase via l’augmentation de l’expression des protéines PAK1, Vav2 et RAC1 GTPase/PI3K. Ce travail a confirmé l’intérêt de l’utilisation de CSM conjointement à rh-BMP2, une protéine impliquée dans l’embryogénèse vasculaire, pour la bioingénierie de la régénération vasculaire. / We determined the capacity to regenerate vascular tissue in vivo, of MSC treated with BMP2 at a dose of [100 ng.mL-1] in a rat model. We have reported that a prosthesis coated with CSM treated 1 week with BMP2 and implanted in rats 14 days allowed the reconstruction of the three tunics of the wall that mimic the structure of the aorta. The proangiogenic capacity of MSCs was increased by BMP2 through the intracellular pathways involving hypoxia inducible factor (HIF-1α) via JAK / STAT. We have shown that MSCs migrated under the influence of BMP2 by stimulating the activity of the enzyme complex NADPH oxidase via the increased expression of PAK1 protein, Vav2 and RAC1 GTPase/PI3K. This work confirmed the interest of the use of MSC in conjunction with rh-BMP2, a protein involved in vascular embryogenesis for bioengineering for vascular regeneration.

Résumé soumis à confidentialité / Not available

Stimulating lymphocytes with Ifn-γ, anti-CD3, and interleukin-2 promotes the proliferation of a cell population coexpressing T-lymphocyte surface antigens such as CD3, CD8a, and CD25 as well as natural killer cell markers such as NK1.1, CD49, and CD69. These cells, referred to as cytokine-induced killer cells (CIKs), display cytotoxic activity against tumour cells, even without prior antigen presentation, and offer a new cell-based approach to the treatment of malignant diseases. Because CIKs are limited in vivo, strategies to optimize in vitro culture yield are required. In the last 10 years, mesenchymal stem cells (MSCs) have gathered considerable attention. Aside from their uses in tissue engineering and as support in haematopoietic stem cell transplantations, MSCs show notable immunomodulatory characteristics, providing further possibilities for therapeutic applications. In this study, we investigated the influence of murine MSCs on proliferation, phenotype, vitality, and cytotoxicity of murine CIKs in a coculture system. We found that CIKs in coculture proliferated within 7 days, with an average growth factor of 18.84, whereas controls grew with an average factor of 3.7 in the same period. Furthermore, higher vitality was noted in cocultured CIKs than in controls. Cell phenotype was unaffected by coculture with MSCs and, notably, coculture did not impact cytotoxicity against the tumour cells analysed. The findings suggest that cell–cell contact is primarily responsible for these effects. Humoral interactions play only a minor role. Furthermore, no phenotypical MSCs were detected after coculture for 4 h, suggesting the occurrence of immune reactions between CIKs and MSCs. Further investigations with DiD-labelled MSCs revealed that the observed disappearance of MSCs appears not to be due to differentiation processes.:Inhaltsverzeichnis I Abbildungsverzeichnis III Tabellenverzeichnis IV Bibliographische Beschreibung V Abkürzungsverzeichnis VII 1 Einleitung 1 1.1 CIK-Zellen (CIK) 3 1.1.1 Merkmale von CIK-Zellen 3 1.1.2 Wirkungsmechanismen von CIK-Zellen 3 1.1.3 Studienlage 4 1.1.4 Bisherige Ansätze zur Verbesserung der Kultivierungsbedingungen 6 1.2 Mesenchymale Stammzellen (MSC) 7 1.2.1 Allgemein 7 1.2.2 Differenzierung von MSC 7 1.2.3 Heterogenität und Einflussfaktoren der MSC - Identitätsproblematik 8 1.2.4 Charakterisierung von MSC 9 1.2.5 Therapeutische Einsatzmöglichkeiten von MSC 11 2 Zielformulierung 15 3 Material und Methoden 16 3.1 Tiere 16 3.2 Materialien 17 3.2.1 Materialien für Zellkultur 17 3.2.2 Materialien für FACS-Analyse 18 3.2.3 Materialien für Zytotoxizitätsassay 19 3.2.4 Materialien für CFU-F-Assay 20 3.3 Methoden 21 3.3.1 Statistische Auswertung 21 3.3.2 Zellkultur 22 3.3.3 FACS (Fluorescence Activated Cell Sorting) 26 3.3.4 Markierung der MSC mit DiD 28 3.3.5 Zytotoxizitätsassay (LDH-Freisetzungsassay) 29 3.3.6 CFU-F-Assay 32 4 Ergebnisse 34 4.1 Beeinflussung der Wachstumskurve 34 4.1.1 Der Wachstumskurvenverlauf von CIK-Zellen (Kontrollen) 34 4.1.2 Der Wachstumskurvenverlauf von CIK-Zellen in der Kokultur mit MSC 35 4.1.3 Der Wachstumskurvenverlauf in MSC-konditioniertem Medium 37 4.1.4 Der Wachstumskurvenverlauf bei Restimulierung an Tag 14 38 4.2 Beeinflussung des Oberflächenphänotyps 40 4.2.1 Der Oberflächenphänotyp von CIK-Zellen 40 4.2.2 Vergleich Oberflächenphänotyp Kontrollen mit kokultivierten CIK 43 4.3 Beeinflussung der Vitalität 46 4.4 Beeinflussung der Zytotoxizität 48 4.5 Identifizierung der MSC 49 4.5.1 Adhärenz an Plastikoberflächen 50 4.5.2 Fibroblastenähnliche Wachstumsmorphologie 50 4.5.3 Wachstum in Colony-Forming-Units 51 4.5.4 Der Oberflächenphänotyp von MSC 53 4.6 Schicksal der MSC in der Kokultur 54 4.6.1 Der Oberflächenphänotyp der adhärenten Zellen nach Kokultur 54 4.6.2 Kokultur mit DiD gelabelten MSC 57 5 Diskussion 59 5.1 Beeinflussung der Wachstumskurve 60 5.1.1 Mechanismen der Beeinflussung des Wachstumskurvenverlaufs 60 5.1.2 Fehlerbetrachtung 68 5.2 Identifizierung der CIK sowie Beeinflussung von Phänotyp und Vitalität 69 5.3 Beeinflussung der Zytotoxizität 70 5.3.1 Vergleich Zytotoxizität Kontrollen mit Kokulturen 70 5.3.2 Fehlerbetrachtung 71 5.4 Identifizierung der MSC 72 6 Schlussfolgerung 75 7 Ausblick 77 8 Zusammenfassung 79 Literaturverzeichnis 83 Danksagung I

The development of new therapeutical approaches, devised to foster the regeneration of neuronal circuits after injury and/or in neurodegenerative diseases, is of great importance. The impairment of dopaminergic projections is especially severe, because these projections are involved in crucial brain functions such as motor control, reward and cognition. In the work presented here, organotypic brain slice co-cultures of (a) the mesostriatal and (b) the mesocortical dopaminergic projection systems consisting of tissue sections of the ventral tegmental area/substantia nigra (VTA/SN), in combination with the target regions of (a) the striatum (STR) or (b) the prefrontal cortex (PFC), respectively, were used to evaluate different approaches to stimulate neurite outgrowth: (i) inhibition of cAMP/cGMP turnover with 3’,5’ cyclic nucleotide phosphodiesterase inhibitors (PDE-Is), (ii) blockade of calcium currents with nimodipine, and (iii) the co-cultivation with bone marrow-derived mesenchymal stromal/stem cells (BM-MSCs). The neurite growth-promoting properties of the tested substances and cell populations were analyzed by neurite density quantification in the border region between the two brain slices, using biocytin tracing or tyrosine hydroxylase labeling and automated image processing procedures. In addition, toxicological tests and gene expression analyses were conducted. (i) PDE-Is were applied to VTA/SN+STR rat co-cultures. The quantification of neurite density after both biocytin tracing and tyrosine hydroxylase labeling revealed a growth promoting effect of the PDE2A-Is BAY60-7550 and ND7001. The application of the PDE10-I MP-10 did not alter neurite density in comparison to the vehicle control. (ii) The effects of nimodipine were evaluated in VTA/SN+PFC rat co-cultures. A neurite growth-promoting effect of 0.1 µM and 1 µM nimodipine was demonstrated in a projection system of the CNS. In contrast, the application of 10 µM nimodipine did not alter neurite density, compared to the vehicle control, but induced the activation of the apoptosis marker caspase 3. The expression levels of the investigated genes, including Ca2+ binding proteins (Pvalb, S100b), immediate early genes (Arc, Egr1, Egr2, Egr4, Fos and JunB), glial fibrillary acidic protein, and myelin components (Mal, Mog, Plp1) were not significantly changed (with the exception of Egr4) by the treatment with 0.1 µM and 1 µM nimodipine. (iii) Bulk BM-MSCs that were classically isolated by plastic adhesion were compared to the subpopulation Sca-1+Lin-CD45--derived MSCs (SL45-MSCs). The neurite growth-promoting properties of both MSC populations were quantified in VTA/SN+PFC mouse co-cultures. For this purpose, the MSCs were seeded on glass slides that were placed underneath the co-cultures. A significantly enhanced neurite density within the co-cultures was induced by both bulk BM-MSCs and SL45-MSCs. SL45-MSCs increased neurite density to a higher degree. The characterization of both MSC populations revealed that the frequency of fibroblast colony forming units (CFU-f ) is 105-fold higher in SL45-MSCs. SL45-MSCs were morphologically more homogeneous and expressed higher levels of nestin, BDNF and FGF2 compared to bulk BM-MSCs. Thus, this work emphasizes the vast potential for molecular targeting with respect to the development of therapeutic strategies in the enhancement of neurite regrowth.:Table of contents Abbreviations 1 1. Introduction 2 1.1 The dopaminergic system 2 1.2 Neurite regeneration following mechanical lesions of the CNS 7 1.3 Organotypic brain slice co-cultures 8 1.4 Promising substances and cells to enhance neuroregeneration 10 1.5 The aim of the thesis 14 2. The original research articles 16 2.1 Phosphodiesterase 2 inhibitors promote axonal outgrowth in organotypic slice co-cultures 17 2.2 Nimodipine enhances neurite outgrowth in dopaminergic brain slice co-cultures 35 2.3 Mesenchymal stem cells support neuronal fiber growth in an organotypic brain slice co-culture model 50 3. References 66 Appendices 73 Summary 73 Zusammenfassung 78 Curriculum Vitae 84 Track Record 85 Selbständigkeitserklärung 87 Acknowledgments 88

Background: The beneficial impact of mesenchymal stem cells (MSC) on both acute and chronic liver diseases has been confirmed, although the molecular mechanisms behind it remain elusive. We aim to identify factors secreted by undifferentiated and hepatocytic differentiated MSC in vitro in order to delineate liver repair pathways potentially targeted by MSC. Methods: Secreted factors were determined by protein arrays and related pathways identified by biomathematical analyses. Results: MSC from adipose tissue and bone marrow expressed a similar pattern of surface markers. After hepatocytic differentiation, CD54 (intercellular adhesion molecule 1, ICAM-1) increased and CD166 (activated leukocyte cell adhesion molecule, ALCAM) decreased. MSC secreted different factors before and after differentiation. These comprised cytokines involved in innate immunity and growth factors regulating liver regeneration. Pathway analysis revealed cytokine-cytokine receptor interactions, chemokine signalling pathways, the complement and coagulation cascades as well as the Januskinase-signal transducers and activators of transcription (JAK-STAT) and nucleotide-binding oligomerization domain-like receptor (NOD-like receptor) signalling pathways as relevant networks. Relationships to transforming growth factor beta(TGF-beta) and hypoxia-inducible factor 1-alpha (HIF1-alpha) signalling seemed also relevant. Conclusion: MSC secreted proteins, which differed depending on cell source and degree of differentiation. The factors might address inflammatory and growth factor pathways as well as chemo-attraction and innate immunity. Since these are prone to dysregulation in most liver diseases, MSC release hepatotropic factors, potentially supporting liver regeneration.