The aim of this thesis is to investigate different functionalization strategy of graphene nanomaterials for graphene-based different biotechnological applications such as graphene-directed stem cell growth and differentiation and graphene-based biosensors. Chemical functionalization of graphene is required in many biological applications; in this thesis we have focused on exploiting the carboxylic groups available on GO molecules and non-covalent functionalization of graphene. GO has been a promising material for stem cell culture due to high specific surface area, ease of functionalization, its ability to support cell proliferation and to not cause cytotoxicity when stem cells are cultured on its substrate. The impact of biochemical functionalization on stem cell differentiation was not widely researched, and many research groups worldwide have been focusing on GO and rGO surfaces only. The approach of this thesis is to fabricate and characterize different graphene-based substrates to investigate the impact of biochemical functionalization of GO in directing adipose stem cell differentiation and to influence the gene expression pathways of Schwann-like differentiated adipose stem cells. The fabrication of graphene based biosensors is still challenging as biological molecules need to be attached to graphene-based sensors to increase both the specificity and the selectivity of the biosensors. In this thesis, two different chemical functionalization approaches were considered. Firstly, the covalent immobilization of membrane proteins embedded on a lipid nanodisc structure on GO was achieved. Secondly, the feasibility of using dip-pen nanolithography as a tool to locally functionalize graphene arrays with phospholipids was demonstrated. Phospholipid interface layer can act as bioactive layer which can be used for the protein insertion of tail-anchoring recombinant proteins as a new route for a non-covalent biological functionalization of graphene array.

Peripheral nerve injury (PNI) is common and despite modern microsurgical techniques of repair, functional restoration is always incomplete. This results in impaired sensation and reduced motor function alongside pain and cold intolerance. Traumatic PNI are often associated with loss of nerve tissue, creating a gap, and direct repair of the two damaged nerve stumps is not possible. These types of injuries are reconstructed using autologous nerve grafts but this is far from ideal since it necessitates the sacrifice of a functional nerve from elsewhere in the body. Chronic muscle atrophy because of the prolonged delay in nerve regeneration across gaps is a significant impediment to an optimal functional recovery.   Tissue engineering and regenerative medicine approaches to nerve repair might one day replace the need for autologous nerve grafts. This thesis investigates the effects of adipose derived stem cells (ASC) on nerve regeneration and muscle recovery by using the stem cells for intramuscular injection and combined with a biomaterial, poly-3-hydroxybutyrate (PHB), to create a bioengineered artificial nerve repair construct.  The mechanisms of interaction between the stem cells and neuromuscular system cells were investigated and with a view to translating this work into clinical practice, an optimal source of cells was investigated from human donors.   It was hypothesized that injecting regenerative cells into muscle would reduce nerve injury induced muscle atrophy. A rat sciatic nerve lesion was performed and three different types of cells were injected into the denervated gastrocnemius muscle; either (1) undifferentiated ASC, (2) ASC induced to a ‘Schwann cell-like’ phenotype (dASC) or (3) primary Schwann cells. Nerves were either repaired by direct end-end suture or capped to prevent muscle reinnervation. One month later, functionality was measured using a walking track test, and muscle atrophy was assessed by examining muscle weight and histology. The Schwann cells and dASC groups showed significantly better scores on functional tests when compared with control injections of growth medium alone. Muscle weight and histology were also significantly improved in the cell groups in comparison with the control group.   PHB strips seeded with either primary Schwann cells or dASC suspended in a fibrin glue matrix were used to bridge a 10mm rat sciatic nerve gap. After 12 weeks, functional and morphological analysis (walking track test, electromyography, muscle weight and muscle and nerve histology) was performed. The results showed significantly better functional results for the PHB strips seeded with cells versus the control group with fibrin matrix only. This correlated with less muscle atrophy and greater distal axon myelination in the cell groups.   To further optimize the nerve regeneration and muscle recovery, the nerve gap lesion was repaired by treatment with the bioengineered constructs seeded with dASC or nerve autograft in combination with stem cell injection in the muscle. After 6 weeks, the best results were obtained in the nerve graft group combined with intramuscular dASC injection which showed significantly less atrophy than the other groups. The results also showed that using the stem cells in a matrix on a PHB strip in combination with intramuscular injections could significantly reduce muscle atrophy.   In vitro experiments showed that dASC expressed a wide range of neurotrophic and myogenic factors including BDNF, VEGF-A, IGF-1 and HGF. Stem cell conditioned medium enhanced the proliferation of myoblast cell lines and primary Schwann cells. Various signaling pathways (PKA, MAP kinase) were involved in these effects dependent on the cell type investigated. Furthermore, in direct co-culture with myoblast cells, a small population of the cells fused together to form myotube-like structures and expressed myogenic markers.   Human ASC were isolated from the deep and superficial layers of abdominal fat tissue obtained during abdominoplasty procedures.  Cells from the superficial layer proliferated significantly faster than those from the deep layer. Superficial layer ASC induced significantly enhanced neurite outgrowth from neuronal cell lines when compared with the deep layer cells.  However, RT-PCR and ELISA analysis showed that ASC isolated from both layers expressed similar levels of the neurotrophic factors NGF, BDNF and GDNF.   In summary, these results show that stem cell therapy at both levels (the nerve lesion site and in the target denervated muscle) offers a promising approach for clinical application for treatment of peripheral nerve lesions. The bioengineered artificial nerve construct, combining PHB strip with cells, also provides a beneficial environment for nerve regeneration. Many of the benefits of the ASC are likely to be mediated through their secretome, a rich source of neurotrophic and myogenic factors. Thus adipose tissue contains a pool of regenerative stem cells which have significant potential application to tissue engineering and regenerative medicine for nerve repair.

Peripheral nerve injuries are often associated with loss of nerve tissue and require autologous nerve grafts to provide a physical substrate for axonal growth. This thesis investigates the use of fibrin as both a tubular conduit to guide nerve regeneration and also as a matrix material to suspend various regenerative cell types within/on poly-3-hydroxybutyrate (PHB) nerve conduits. Adipose derived stem cells (ASC) are found in abundant quantities. In this thesis the ability of rat ASC to differentiate into Schwann cells was determined and a preliminary study of the neurotrophic potential of human ASC was also investigated. Rat sciatic nerve axotomy was performed proximally in the thigh to create a 10-mm gap between the nerve stumps and the gap was bridged using the various conduits.  At early time points the nerve grafts were harvested and investigated for axonal and Schwann cell markers.  After 16 weeks the regenerative response from sensory and motor neurons was also evaluated following retrograde labelling with Fast Blue fluorescent tracer. Stem cells were treated with a mixture of glial growth factors and after 2 weeks in vitro the expression of Schwann cell markers was analysed by immunocytochemistry and Western blotting.  ASC were cocultured with the NG108-15 neuronal cell line to determine their ability to promote neurite outgrowth.  Human ASC were isolated from the deep and superficial layers of abdominal fat tissue obtained during abdominoplasty procedures.  RT-PCR was used to investigate the expression of neurotrophic factors. Immunohistochemistry showed a superior nerve regeneration distance in the fibrin conduit compared with PHB. The fibrin conduit promoted regeneration of 60% of sensory neurones and 52% of motor neurones when compared with an autograft group at 16 weeks. The total number of myelinated axons in the distal nerve stump in the fibrin-conduit group reached 86% of the graft and the weight of gastrocnemius and soleus muscles recovered to 82% and 89% of the controls, respectively. In vitro studies showed that rat ASC could be differentiated to a Schwann cell phenotype. These treated cells enhanced both the number of NG108-15 cells expressing neurites and neurite length. In the same coculture model system, human superficial fat layer ASC induced significantly enhanced neurite outgrowth when compared with the deep layer fat cells. RT-PCR analysis showed ASC isolated from both layers expressed neurotrophic factors. These results indicate that a tubular fibrin conduit can be used to promote neuronal regeneration following peripheral nerve injury. There was also a beneficial effect of using a fibrin matrix to seed cells within/on PHB conduits which should ultimately lead to improved functional recovery following nerve injury.  There might also be an advantage to use a simple strip of PHB rather than a conventional tube-like structure implying that single fascicle nerve grafting could be advantageous for nerve repair.  The results of in vitro experiments indicate adipose tissue contains a pool of regenerative stem cells which can be differentiated to a Schwann cell phenotype and given that human ASC express a range of neurotrophic factors they are likely to be of clinical benefit for treatment of peripheral nerve injuries.

<p>Many implanted devices fail due to the formation of an avascular capsule. Fat is known to promote healing and vascularization. It is possible that isolating and attaching ASCs (adipose stem cells) to an implanted device improves the healing in the adjacent tissue. </p><p> Various attachment methods were studied, and the fibronectin treatment was found comparable to or better than other treatments. Next, bare and ASC coated polyurethane were implanted into rats. The fibrous capsule surrounding the bare polyurethane was thicker and contained more collagen at 8 weeks. Additionally, the microvessel density in the tissue surrounding the ASC coated polyurethane was significantly higher at 4 and 8 weeks. Quantification of glucose sensor response following ASC attachment for 1 week found no measurable significant differences in function.</p><p> The bioluminescence technique, which quantifies the tissue glucose concentration around the implant at the moment of freezing, was used to determine if ASC attachment to biomaterials impacts the tissue glucose concentration profile. ASC attachment to polyurethane and to glucose sensors did not significantly change the glucose profiles in the tissue. However, a quantifiable glucose concentration profile was observed around all glucose sensors.</p><p> The final experiments were performed to identify a possible mechanism that adipose tissue uses to alter the foreign body response. In vitro experiments showed that VEGF (VEGF-A specifically) secretion following ASC attachment to polyurethane was 10-20 times higher than with fibroblast attachment after three days and 40-70 times higher after six days. This high secretion of VEGF would likely have in vivo physiological affects on microvasculature.</p><p> In conclusion, the attachment of ASCs to polyurethane reduced the thickness and collagen content of the fibrous capsule surrounding ASC coated implants and increased the microvessel density in adjacent tissue. In addition, ASC attachment did not enhance glucose sensor function, nor did it decrease the glucose concentration in the adjacent tissue. Finally, ASCs were found to secrete high amounts of pro-vascular cytokines, which likely plays a key role in the observed improvement of the foreign body response.</p> / Dissertation

<p>Articular cartilage is the connective tissue that lines the ends of long bones in diarthrodial joints, providing a low-friction load-bearing surface that can withstand a lifetime of loading cycles under normal conditions. Despite these unique and advantageous properties, the tissue possesses a limited capacity for self-repair due to its lack of vasculature and innervation. Total joint replacement is a well-established treatment for degenerative joint disease; however, the materials used in these procedures have a limited lifespan in vivo and will likely fail over time, requiring additional - and increasingly complicated - revision surgeries. For younger or more active patients, this risk is unacceptable. Unfortunately, alternative surgical options are not currently available, leaving pain management as the only viable treatment. In seeking to discover a new therapeutic strategy, the goal of this dissertation was to develop a functional tissue-engineered cartilage construct that may be used to resurface an entire diseased or damaged joint.</p><p> A three-dimensional (3-D) woven textile structure, produced on a custom-built miniature weaving loom, was utilized as the basis for producing novel composite scaffolds and cartilage tissue constructs that exhibited initial properties similar to those of native articular cartilage. Using polyglycolic acid (PGA) fibers combined with chondrocyte-loaded agarose or fibrin hydrogels, scaffolds were engineered with anisotropic, inhomogeneous, viscoelastic, and nonlinear characteristics prior to cultivation. However, PGA-based constructs showed a rapid loss of mechanical functionality over a 28 day culture period suggesting that the inclusion of other, less degradable, biomaterial fibers could provide more stable properties. </p><p> Retaining the original 3-D architecture and fiber/hydrogel composite construction, poly (epsilon-caprolactone) (PCL)-based scaffolds demonstrated initial biomechanical properties similar to those of PGA-based scaffolds. Long-term culture of 3-D PCL/fibrin scaffolds seeded with human adipose-derived stem cells (ASCs) showed that scaffolds maintained their baseline properties as new, collagen-rich tissue accumulated within the constructs.</p><p> In an attempt to improve the bioactivity of the PCL scaffold and further induce chondrogenic differentiation of seeded ASCs, we produced a hybrid scaffold system by embedding the 3-D woven structure within a porous matrix derived from native cartilage. We then demonstrated how this multifunctional scaffold could be molded, seeded, and cultured in order to produce an anatomically accurate tissue construct with potential for resurfacing the femoral head of a hip. </p><p>In summary, these findings provide valuable insight into a new approach for the functional tissue engineering of articular cartilage. The results of this work will hopefully lead to the discovery of new strategies for the long-term treatment of cartilage pathology.</p> / Dissertation

It has been shown that stem cells exist within adult adipose tissue. These stem cells are named adipose-derived mesenchymal stem cells (ASCs), are derived from the mesoderm, and can differentiate into a number of cells including osteoblasts, chondrocytes, and adipocytes. However, before these cells can be used clinically it is important that we understand how factors like age, sex, and ethnicity affect ASC number and potential. Additionally, since men and women vary in their distribution of adipose tissue, it will be important to see if the ideal source of ASCs is different for each sex. The goal of this study was to assess how age and sex affects ASCs. We used flow cytometry to investigate how age and sex affected the number of ASCs in adipose tissue. Additionally, we plated these cells in culture and treated them with an osteogenic media (OM) with the intention of pushing them towards osteoblast differentiation. The purpose of this was to see if age or sex affected the potential of the ASCs to undergo osteogenesis in culture. For this study we used real-time PCR and biochemical assays to look at markers of early and late osteogenic differentiation. Finally, we used immunohistochemistry to demonstrate where in adipose tissue the CD73 and CD271 positive cell population exists. It is our hope that this work will shed light on how age and sex affect ASCs so that clinicians can optimize their ASC harvest depending on the patient's physiology.

Submitted by Renata Fontoura (comunicaicc@fiocruz.br) on 2014-11-26T16:24:49Z No. of bitstreams: 1 Dissertação Ana Carolina Origa Alves - 02.pdf: 2465539 bytes, checksum: 9ad1e3c8193f72874f714fe1c073b599 (MD5) / Approved for entry into archive by Renata Fontoura (comunicaicc@fiocruz.br) on 2014-11-26T16:25:15Z (GMT) No. of bitstreams: 1 Dissertação Ana Carolina Origa Alves - 02.pdf: 2465539 bytes, checksum: 9ad1e3c8193f72874f714fe1c073b599 (MD5) / Made available in DSpace on 2014-11-26T16:25:15Z (GMT). No. of bitstreams: 1 Dissertação Ana Carolina Origa Alves - 02.pdf: 2465539 bytes, checksum: 9ad1e3c8193f72874f714fe1c073b599 (MD5) Previous issue date: 2014 / Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil / Células-tronco (CT) são células autorrenováveis e não especializadas, com o potencial de diferenciação multidirecional. Células-tronco de tecido adiposo (CT-TA) são um tipo de células-tronco adultas multipotentes, de fácil isolamento e cultura. Nos últimos anos, CT-TA têm mostrado grande potencial para engenharia de tecidos e terapias baseadas em células. Apesar do interesse em aplicações clínicas deste tipo de célula, os mecanismos moleculares fundamentais a sua autorrenovação e diferenciação ainda não foram completamente elucidados. miRNAs são pequenos RNAs não-codificadores, com 21-25 nucleotídeos de comprimento, que tem se mostrado como importantes reguladores da expressão gênica em nível póstranscricional. miRNAs podem atuar por meio de clivagem direta de mRNAs alvo ou através da repressão da tradução, dependendo da complementaridade entre o mRNA e a sequência do miRNA. Perfis de miRNAs de CT adultas sugerem que estes pequenos reguladores podem contribuir para as propriedades intrínsecas das CT. Para entender melhor os mecanismos de ação dos miRNAs em CT-TA, miRNAs associados ao polissomos de CT-TA foram isolados durante a diferenciação celular. Procurando miRNAs reguladores das etapas iniciais de diferenciação ou envolvidos na autorrenovação de CT, as culturas de células foram induzidas a diferenciação adipogênica durante 72 h. O lisado celular foi submetido à ultracentrifugação em gradiente de sacarose para separar monosomos, polissomos e fração livre de ribossomos. O RNA total associado aos ribossomos foi extraído, os fragmentos de RNA (<200 nt) foram enriquecidos e a seleção de tamanho de fragmentos de RNA apropriados ocorreu durante a preparação das amostras para o sequenciamento em larga escala. As amostras foram sequenciadas utilizando a plataforma SOLiD ™, e as frações polissomais de culturas Não Induzida e 72h de indução foram comparadas e dezesseis miRNAs foram identificados. miRNAs encontrados em um trabalho prévio do grupo foram adicionados a esses dados, e sete miRNAs (hsamiR-29b-1-5p, hsa- miR-29c-5, hsa-miR-30c-5p, hsa-miR-143-5p, hsa-miR-210-3p, hsa-miR-210- 5p e hsa-miR-6775- 5p) foram testados por RT-qPCR para confirmar a expressão diferencial, sendo que um deles (hsa-miR-210-5p) mostrou diferença estatisticamente significativa. / Stem cells (SC) are self-renewing and non-specialized cells with the potential of multi-directional differentiation. Adipose Stem Cell (ADSC) is a type of multipotent adult stem cell, easy to isolate and culture. In the past few years, hADSCs have shown great potential for tissue engineering and cell-based therapies. Despite the interest in clinical applications of this kind of cell, the molecular mechanisms underlying their self-renewal and differentiation have yet to be fully elucidated. miRNAs are small noncoding RNAs, 21-25 nucleotides in length, that have been shown to be important regulators of posttranscriptional gene expression. miRNAs can act through direct cleavage of target mRNAs or through translational repression, depending of complementary pairing between the mRNA and miRNA sequence.miRNA profile of adult SCs suggests that these small regulators can contribute to the intrinsic properties of SCs. To better understand the mechanisms of action of miRNAs in hADSCs, we isolate miRNAs associated to polysomes of hADSC during cellular differentiation. Looking for miRNAs regulators of early steps of differentiation or involved in ADSC self-renewing, cell cultures were induced to adipogenic differentiation for 72 h. The cell lysate was submitted to ultracentrifugation on a sucrose gradient to separate monosomes, polysomes and the fraction free of ribosomes. The total RNA associated to ribosomes was extracted and the RNA fragments (<200 nt) were enriched and the size selection of appropriate RNA fragments occurred during the preparation of samples for deep sequencing. The samples were sequenced using SOLiD™ platform, and polysomal fraction of cell cultures non induced and 72h of induction were compared and sixteen miRNAs were identified. miRNAs found in a previous work of our group were added to these data and seven miRNAs (hsa-miR-29b-1-5p, hsa-miR-29c-5, hsa-miR-30c-5p, hsa-miR- 143-5p, hsa-miR-210-3p, hsa-miR-210-5p e hsa-miR-6775-5p) were tested by RTqPCR to confirm differential expression, and one of them (hsa-miR-210-5p) showed statistical significant difference.

En condition d'obésité, le tissu adipeux est le théâtre d'infiltration et d'accumulation de cellules immunitaires dont les lymphocytes Th17, impliqués dans de nombreuses maladies inflammatoires chroniques et auto-immunes. Les mécanismes d'activation et de prolifération des Th17 au sein du tissu adipeux ne sont pas connus. Nous avons suggéré un rôle des cellules souches adipocytaires (CSA) dans l'induction de l'inflammation médiée par les Th17. Nos résultats montrent, pour la première fois, et grâce à un modèle de co-culture combinant les CSA du tissu adipeux obèse avec des cellules mononucléées du sang circulant, que seules les CSA issues de sujets obèses, et non pas de sujets minces, sont capables d'induire l'activation des Th17 et des monocytes, qui en retour activent la sécrétion d'IL-6 par les CSA. L'environnement inflammatoire, induit par cette interaction, est à l'origine de l'inhibition de l'adipogenèse et de la réduction de la sensibilité à l'insuline des adipocytes. De plus, tout comme les CSA, les adipocytes issus de sujets obèses induisent le même phénotype inflammatoire. Enfin, une étude en cours nous a permis de montrer que les acides gras polyinsaturés de type -3, inhibent spécifiquement l'activation des Th17 mais n'ont aucun impact sur les monocytes inflammatoires, dans notre modèle, possiblement lié à une inhibition de l'expression d'ICAM-1. En conclusion, une triade inflammatoire constituée de CSA ou d'adipocytes issus de sujets obèses combinés avec des monocytes et des Th17, forme un cercle vicieux où l'inflammation est maintenue et amplifiée / In obesity, adipose tissue is massively infiltrated by a panel of inflammatory immune cells such as IL-17-secreting Th17 lymphocytes. The mechanisms by which Th17 cells are induced are less understood. We postulated that adipose stem cells (ASC), which are known to play immunomodulatory functions in contact with immune cells, might be involved in Th17 activation. By using an in-vitro co-culture model, we demonstrated for the first time, that ASC issued from obese , but not lean AT were able to activate IL-17 secretion from Th17 cells, and IL-1 secretion from monocytes. In turn, such an interaction led to increased secretion of IL-6 from ASC. The inflammatory environment generated from this interaction was then able to inhibit adipogenesis and adipocyte insulin-sensitivity. We also showed that adipocytes harvested from obese adipose tissue were able to promote Th17 cell and monocyte activation. Finally, we demonstrated that polyunsaturated fatty acids omega-3 presented anti-inflammatory properties towards Th17 cells, while they had no effect on monocytes in our co-culture model (manuscript in preparation), which was related to decrease of ICAM-1 expression. In conclusion, we highlight herein a crosstalk between ASC, monocytes and Th17 cells in obese adipose tissue, which leads to a vicious circle of pro-inflammatory cytokine secretion

La thérapie cellulaire est au centre de la médecine régénérative, une nouvelle avenue thérapeutique en constante évolution depuis les 2 dernières décennies, particulièrement pour les maladies touchant les organes avec très faible potentiel de régénération. Parmi les nombreuses populations de cellules souches identifiées jusqu’à présent, les cellules souches mésenchymateuses (MSC) offrent plusieurs avantages, notamment en cardiopathie ischémique. Les MSC originalement isolées de la moelle osseuse (MO) sont à présent isolées de plusieurs autres organes dont le tissu adipeux (cellules souches mésenchymateuses dérivées du tissu adipeux ASC), un organe facile d’accès et disponible en grande quantité chez l’humain. Les évidences précliniques et cliniques des MSC et ASC pointent vers une efficacité thérapeutique qui demeure limitée et nécessite une optimisation pour atteindre le potentiel de guérison et de réparation myocardique post injure ischémique. Malgré un potentiel de différenciation in vitro et in vivo, l’effet thérapeutique des cellules souches est lié aux nombreux médiateurs constituant leur sécrétome. Ainsi, afin d’optimiser l’impact thérapeutique, plusieurs méthodes ont été utilisées pour enrichir le sécrétome. Une de ces méthodes est la culture des cellules souches sous forme 3-D. Nous avons donc testé le potentiel thérapeutique des ASC sous forme de monocouche (ML pour Monolayer) et sous forme de sphéroïdes (SB pour spheroid bodies) dans deux modèles animaux différents. Ceci a été fait en testant l’efficacité d’une transplantation à distance sous cutanée. En utilisant un modèle de péritonite, les ASC sous forme SB et ML ont démontré un effet antiinflammatoire en réduisant l’infiltration de cellules inflammatoires, plus spécifiquement les neutrophiles et les macrophages. Dans un modèle d’ischémie reperfusion (I/R) du myocarde chez le rat, les ASC sous forme ML et SB ont diminué la cicatrice du myocarde. Les deux formes injectées ont réduit la présence de macrophages dans le myocarde, augmenté les cellules progénitrices endogènes c-kit+ ainsi que la densité vasculaire. Les SB ont démontré un impact plus significatif sur certains paramètres évalués tels la densité vasculaire et le recrutement de cellules progénitrices endogènes c-kit+. En conclusion, ces deux études ont permis de démontrer un potentiel pro-guérison des cellules souches, par l’entremise d’un effet endocrinien anti-inflammatoire. / The therapeutic potential of regenerative medicine and cell-based strategies have been in constant evolution for the last 2 decades, especially for the repair and healing of organs with minimal regenerative capacity such as the heart. Mesenchymal stem cells (MSC) represent a population of stem cells with great advantages. First isolated from bone marrow, they can now be readily isolated from different organs including adipose tissue. Adipose derived stem cells (ASC) are easily accessible, which is of great interest for clinical application. Beside their differentiation potential, the main therapeutic impact attributed to MSC and ASC has been through their secreted mediators i.e. via a paracrine phenomenon. Different strategies have been used to enhance the therapeutic impact of ASC, one of which consists of enriching the latter’s secretome by producing 3-D structures such as spheroid bodies. Monolayer (ML) and spheroid bodies (SB) were both used in two different animal models through remote (subcutaneous) transplantation. First, on a rat peritonitis model that proved that subcutaneous injection of the two forms reduced inflammation through decreased neutrophil and macrophage infiltration. Second, ML and SB were remotely transplanted on a rat myocardial ischemia reperfusion (I/R) model. The two forms reduced infarct scar, reduced macrophages, increased endogenous progenitor c-kit+ cells and enhanced vascular density in the infarct and peri-infarct area. SBs showed better impact on vascularization and endogenous progenitor cells recruitment compared to ML. Briefly, our studies demonstrated the efficacy of a remote ASC transplantation through an endocrine-like effect against inflammation, collagen deposition and vascularization.

Peripheral nerve injuries occur with high incidence and often result in profound and permanent impact on the life of patients and on healthcare expenditure. Schwann cells (SC) play a promoting role in peripheral nerve regeneration providing physical and neurotrophic support that aids axon re-growth. However, these beneficial properties are not exploitable in nerve tissue engineering due to the difficulties in SC harvesting and expansion in culture. Adult stem cells derived from bone marrow (BM-MSC) and from adipose tissue (ASC) can be differentiated in SC-like cells and be used as SC substitutes in bioengineered nerve conduits for the improvement of peripheral nerve regeneration. Pharmacological intervention approaches for the treatment of nerve injury are still not clinically available. Nevertheless, γ-Aminobutyric acid (GABA) receptors have been recently suggested as a putative target for such purpose. GABA is the main inhibitory neurotransmitter of the adult brain and interacts with two different receptor types. However, both GABA-A and GABA-B receptor types are functionally expressed also in SC, where they are involved in the regulation of SC physiology and in the development of the peripheral nervous system (PNS).The aim of this thesis was to characterise the GABAergic system of BM-MSC and ASC differentiated into a SC-like phenotype and to evaluate changes in the expression levels following differentiation. Moreover, the effect of specific GABA receptor ligands on cell proliferation and neurotrophic potential of differentiated stem cells were assessed. Using reverse transcriptase polymerase chain reaction, western blot analysis and immunohistochemistry we demonstrated that adult stem cells express several subunits of both GABA-A and GABA-B receptor systems such as GABA-B1a, GABA-B1b and GABA-B2, as well as GABA-A α2 and GABA-A β3. Expression levels and cellular localisation were comparable with adult and neonatal SC cultures used as positive controls, and protein expression levels for some of the subunits changed following glial differentiation. Interestingly, stimulation of GABA receptors with specific agonists influenced stem cell proliferation in two opposite ways. Baclofen, a GABA-B receptor agonist decreased proliferation of SC and differentiated ASC (dASC), but not of SC-like BM-MSC (dBM-MSC). By contrast, muscimol, a GABA-A receptor agonist, increased proliferation in SC and in both dASC and dBM-MSC. This suggests that GABAergic signalling could be a potential player in the mechanisms regulating stem cell differentiation and proliferation as reported in SC. Finally, baclofen treatments on SC and dASC modulated the expression levels and the release of the neurotrophins BDNF and NGF, which are key actors in the processes involved with peripheral nerve regeneration. Although further studies will be needed to clarify the role of GABA receptors in the PNS, the presence of functional GABA receptors on SC-like adult stem cells could represent an exploitable pharmacological target to modulate stem cell physiology and improve their neurotrophic potential for peripheral nerve regeneration.