Submitted by Luciane Willcox (luwillcox@gmail.com) on 2016-09-01T16:51:25Z No. of bitstreams: 1 TESE AXEL.pdf: 13901759 bytes, checksum: 6f744b4d3c67d9fcc80db57c1c5944a7 (MD5) / Approved for entry into archive by Luciane Willcox (luwillcox@gmail.com) on 2016-09-01T17:02:42Z (GMT) No. of bitstreams: 1 TESE AXEL.pdf: 13901759 bytes, checksum: 6f744b4d3c67d9fcc80db57c1c5944a7 (MD5) / Made available in DSpace on 2016-09-01T17:02:42Z (GMT). No. of bitstreams: 1 TESE AXEL.pdf: 13901759 bytes, checksum: 6f744b4d3c67d9fcc80db57c1c5944a7 (MD5) / Messenger ribonucleoprotein particles (mRNPs) são complexos formados por RNA mensageiro (mRNA) e um pool de diferentes proteínas que se ligam diretamente ou indiretamente ao mRNA. Proteínas diferentes podem formar diferentes mRNPs com diferentes funções. Agregação de mRNPs formam grânulos que são visíveis ao microscópio. Dois desses grânulos comumente encontrados em células eucarióticas são os processing bodies (PB) e os grânulos de estresse (GE) possuem função fundamental na regulação pós-transcricional, mais especificamente, na degradação (PB) e na estocagem (PB e GE) do mRNA. Enquanto os PB são constitutivamente encontrados, GE são vistos somente em condições de estresse. TTP e RCK são componentes presentes em ambos os grânulos enquanto que TIA1 e TIAR são exclusivos de GE e DCP2 exclusivo de PB. Human Adipocyte Derived Stem Cells (ADSCs) são células-tronco adultas multipotentes com capacidade de autorrenovação e diferenciação em células de diversos tecidos. Desde que pouco se sabe sobre a função dos PB e dos GE durante a diferenciação das células-tronco, nosso objetivo é elucidar a dinâmica e função desses grânulos nestas celulas. Foram utilizadas ADSCs derivadas de cirurgia bariátrica e lipoaspiração. Diversos componentes de PB e de GE foram analisados por Imunofluorescência após 1, 4, 7 e 12 dias de diferenciação adipogênica. Oligos de RNA de interferência (siRNA) específicos para RCK, DCP2, TTP, TIAR e TIA1 foram transfectados concomitantemente com a indução à diferenciação adipogênica e as culturas mantidas por 1, 4, 7 e 14 dias de diferenciação. A diferenciação foi mensurada por intensidade de fluorescência após marcação com AdipoRed. GE são ausentes em ADSCs indiferenciadas e durante a diferenciação como determinado por imunomarcação com TIAR que se localiza predominantemente no núcleo. Interessantemente imunomarcação com TIA1/R mostrou que ADSCs possuem grande quantidade de grânulos contendo TIA1/R em células indiferenciadas e não estressadas. Sob estresse oxidativo há um aumento de PB em ADSCs e a formação de GE. Esses grânulos parecem variar de composição, uma vez que eIF4E é ausente e GE imunomarcados com eIF4B são mais perinucleares do que GE imunomarcados com TIAR. PB variam em número e tamanho em células diferenciadas. Notavelmente, em ADSCs possuem poucos PB e até mesmo ausente em algumas células, além disso, TIA1/R e PB significativamente aumentam após 24 horas de diferenciação e após 12 dias o número de PB é similar a células diferenciadas enquanto que os grânulos de TIA1/R praticamente desaparecem. siRNA de componentes essenciais de PB e GE em ADSCs indiferenciadas (tempo 0) mostrou que há um aumento significativo na diferenciação em adipócito após 4, 7 e 12 dias de diferenciação adipogênica. Interessantemente esse aumento se deu pela maturação do adipócito, i.e. tamanho da vesícula de lipídio e não pelo aumento da quantidade de células diferenciadas. Nossos dados mostram que PB estão em baixo número e que existem grânulos de TIA1/R citoplasmárico nunca antes mostrados em ADSCs indiferenciadas. Ainda, esses grânulos aumentam durante a diferenciação e parecem exercer um papel importante na maturação dos adipócitos uma vez que ensaios de siRNA de componentes de PB e GE levam a uma diferenciação mais acelerada. Também, os GE são formados após indução a estresse oxidativo e esses grânulos são praticamente ausentes de eIF4E mostrando que a composição desses grânulos varia em relação a células diferenciadas.

archives@tulane.edu / Human adipose-derived stem cells (ASCs) efficiently modulate the inflammatory microenvironment, making them ideal for the treatment of inflammatory, autoimmune, and neurodegenerative diseases like multiple sclerosis (MS). Clinical translation of ASC therapies has been limited, making strategies to improve ASC post-transplant immunosuppressive capabilities especially important. Autophagy, a stress-induced degradative pathway, plays a crucial role in the paracrine signaling of ASCs, which drives their therapeutic action. Therefore, I investigated the modulatory effect of autophagy preconditioning in ASC physiology and therapeutic potential using the autophagy inducer Rapamycin (Rapa-ASCs) or the inhibitor 3-methyladenine (3MA-ASCs). Following 4 and 24-hour preconditioning, ASC stemness and immunomodulatory capacity were examined. Results demonstrate that neither Rapa nor 3-MA altered morphology or surface marker expression, indicating preservation of stemness. RT-qPCR analysis revealed that 4-hour preconditioning with Rapa-ASCs and 3MA-ASCs alone upregulated transcription of cyclooxygenase-2 (COX2), but not secretion of its downstream effector molecule prostaglandin E2 (PGE2). Following stimulation with interferon-gamma (IFNγ) to mimic a pathological microenvironment, both 4-hour Rapa-ASCs and 3MA-ASCs upregulated COX2 transcription and PGE2 secretion. 4-hour Rapa-ASCs also upregulated expression of the cytokines transforming growth factor beta (TGF-β) and interleukin-6 (IL-6). As each of these molecules have demonstrated therapeutic effects in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS, I hypothesized that 4-hour Rapamycin preconditioning may bestow the greatest improvement to ASC immunomodulatory potential in EAE. To test this, EAE mice were treated at peak disease severity with control ASCs (EAE-ASCs), Rapa-preconditioned ASCs (EAE-Rapa-ASCs), or a vehicle control (EAE). Results revealed that EAE-ASCs enhanced rotarod locomotor activity, improved clinical disease scores, and elevated intact myelin in the spinal cord compared to both EAE and EAE-Rapa-ASC animals. This correlated with augmented CD4+ T helper (Th) and T regulatory (Treg) cells in the spinal cord, and significantly increased interleukin-10 (IL-10) transcripts. Conversely, EAE-Rapa-ASC mice showed no clinical or motor function improvement, reduced myelin levels, and significantly less Th and Treg cells in the spinal cord. These findings suggest that short-term Rapamycin preconditioning diminishes the therapeutic efficacy of ASCs when applied to late-stage EAE and highlights the importance of investigating novel therapeutic strategies in vivo in physiologically relevant disease models. / 1 / Rachel Wise

Cell-based therapies involving adipose-derived stem cells (ASCs) have shown promise in stimulating cardiovascular regeneration, including in the treatment of myocardial infarction (MI) and ischemic heart disease. However, previous studies involving the delivery of ASCs following MI have indicated that therapeutic efficacy has been limited by low survival and/or poor retention of the transplanted cells at the site of injury. To address these limitations, the goal of this thesis was to develop a more effective delivery strategy incorporating an injectable biomaterial combined with chemotactic growth factor delivery to enhance ASC retention within the gel. Working towards future in vivo analysis in a rat model, multilineage characterization studies confirmed that ASCs isolated from the epididymal fat pad of male Wistar rats could differentiate in vitro along the adipogenic, osteogenic, and chondrogenic lineages. Subsequently, the chemotactic response of the rat ASCs (rASCs) to varying concentrations of stromal derived factor-1 α (SDF-1α) and hepatocyte growth factor (HGF) was analyzed using a modified Boyden chamber assay. The results demonstrated that SDF-1α and HGF, at 20, 50, and 100 ng/mL elicited significant migratory responses under normoxic (21%) and hypoxic (5%) culture conditions. RT-PCR analysis was conducted to assess the expression of the two chemotactic growth factors and their associated receptors in the rASCs, and secreted SDF-1α protein expression was quantified by ELISA. Moving towards the development of the biomaterials-based delivery approach, the viability of rASCs encapsulated by photopolymerization in methacrylated glycol chitosan (MGC) hydrogels modified with various degrees of arginine-glycine-aspartic acid (RGD)-peptide modification was examined. More specifically, rASCs were encapsulated in MGC hydrogels with 0%, 4%, and 7% RGD modification and cultured for up to 14 days. Viability staining results indicated that rASC viability was enhanced in the 4% and 7% RGD-modified MGC hydrogels in comparison to the MGC hydrogels with no peptide modification. Pre-loading the gels with 50 ng/mL of SDF-1α had no significant effects on cell viability over 14 days. Overall, the results demonstrate that peptide modification to promote cell adhesion within the MGC hydrogels is key to improving cell viability and thereby improving the therapeutic potential of ASCs. / Thesis (Master, Chemical Engineering) -- Queen's University, 2012-10-24 23:54:37.126

Several studies have shown cellular replacement therapy as a treatment strategy of myocardial infarction but results have been limited. Therefore, enhancing the therapeutic potential of stem cells injected into ischemic microenvironments by novel preconditioning (PC) techniques is critical for improving cellular therapy. Recent studies have shown that inhibition of phosphodiesterase-5 (PDE-5) is a powerful strategy to precondition the heart and cardiomyocytes against ischemia/reperfusion injury. We therefore tested the hypothesis that inhibition of PDE-5 with sildenafil (Viagra®) or selective knockdown with a silencing vector in adipose derived stem cells (ASCs) would improve their survival after ischemia/reoxygenation in vitro and enhance cardiac function following myocardial implantation in vivo. ASCs were treated with sildenafil or infected with PDE-5 silencing vector shRNA (shRNAPDE-5). The cells were subjected to simulated ischemia (SI) and reoxygenation (RO). Both sildenafil and shRNAPDE-5 significantly reduced cell injury, as shown by improved viability, decreased lactate dehydrogenase, and apoptosis. The preconditioned ASCs also demonstrated an increase in the release of growth factors including VEGF, b-FGF, and IGF. The protective effect against SI/RO injury was abolished by inhibition of protein kinase G (PKG) using both a pharmacological inhibitor and selective knockdown with shRNAPKG1α suggesting a PKG-mediated mechanism. To show the effect of preconditioned ASCs in vivo, adult male CD-1 mice underwent myocardial infarction (MI) by occlusion of the left descending coronary artery, followed by direct injection of PBS (control), non-preconditioned ASCs, or preconditioned ASCs (4x105) ASCs into the left ventricle (LV). Preconditioned ASC-treated hearts showed consistently superior cardiac function by all measures as compared with PBS and non-preconditioned ASCs after 4 weeks of treatment. Post-mortem histological analysis demonstrated that preconditioned ASC-treated mice had significantly reduced fibrosis, increased vascular density and reduced resident myocyte apoptosis as compared to mice receiving non-preconditioned ASCs or PBS. VEGF, b-FGF, and Ang-1 were also significantly elevated 4 weeks after cell therapy with preconditioned ASCs. Our data suggests that genetic or pharmacological inhibition of PDE-5 is a powerful new approach to improve stem cell therapy following myocardial infarction.

In the United Kingdom 1 in 6 - 8000 children are born missing one or both of their ears. The surgical technique most commonly used to reconstruct ears requires surgeons to remove ribs from the patient, and the cartilage from the ribs is used to carve a new ear. This procedure involves many risks including significant pain, punctured lung and chest deformity. Therefore the ability to ‘grow’ an ear would be a major advancement. Stem cells show huge promise in tissue engineering and regenerative medicine. Approved stem cell technology must be evaluated with regards to safety, purity, identity, potency and efficacy prior to biologic licensing and clinical use. Therefore, access to ethically sourced tissue for research is fundamental to the successful delivery of novel therapies. Adipose tissue provides an abundant and accessible source of stem cells for clinical translation. Within the first section of this thesis, the perceptions and attitudes of patients towards the donation and use of adipose tissue for research are sought. Based on this information, a tissue bank with all appropriate ethical approval to collect, process, store and distribute adipose tissue and adipose derived stem cells is established. The second part of this thesis demonstrates the specific identity, location and frequency of stem cells within adipose tissue; revealing them to reside in a perivascular niche. Using this data, protocols to rapidly purify stem cells from adipose tissue using Fluorescence Activated Cell Sorting are developed. The frequency of cells, and both the patient and procedure based variables that can affect this yield are also examined. The final section of this thesis uses a high-throughput microarray platform to screen thousands of polymers to identify potential substrates that can support the attachment, stable proliferation and subsequent differentiation of stem cells purified from adipose tissue. From the initial screen, 5 distinct polymers have been identified, characterised and their effects on the stem cells examined and quantified. Combined together, these elements provide significant advances in our understanding, and the basis for on going research to deliver a tissue engineered ear for use in human ear reconstruction.

A Comparison of Bone Marrow Derived and Adipose Derived Stem Cells in Point of Care Goat Non-Instrumented Posterolateral Intertransverse Spinal Fusion Daria Brigitte Neidre, Ph.D. The University of Texas at Austin, May, 2010 Supervisor: Roger P. Farrar Concentrated bone marrow containing mesenchymal stem cells (BMSCs) in combination with osteoconductive scaffolds has been used in orthopaedics to replace the need for iliac crest bone grafts. Autologous BMSC volume is limited, but adipose tissue represents a large reservoir of stem cells; adipose derived stem cells (ADSCs). To test these cells, a large animal model using goats was selected due to their similarities to humans in loading conditions of the spine, trabecular bone structure of the vertebrae, and their common use in testing orthopaedic therapies as a clinically relevant model. The aim of this study is to characterize cell surface markers of the isolated cells through flow cytometry, compare goat BMSCs and ADSCs using multilineage differentiation into the osteogenic and adipogenic lineages, and utilize them in a “Point-of-Care” non-instrumented posterolateral lumbar spinal fusion. Both BMSCs and ADSCs were confirmed as stem cells through lack of expression of markers CD34, CD45, CD90, and CD105, which is supported by literature. Both cell types also differentiated into both the adipogenic and osteogenic lineages. Although we had positive in vitro results, we had limited in vivo results. There were no differences between BMSCs, ADSCs and control implantation in identifiable spinal fusion at 3 or 6 months through radiographs or CT scans. Additionally, there were no differences between groups at 6 months in biomechanical testing, histology and microradiographs. Although our in vivo results were lacking in demonstrating fusion at 6 months, this study is the first of it’s kind to investigate a large animal model comparison of BMSCs and ADSCs in spinal fusion and demonstrated that “Point-of-Care” stem cells derived from either bone marrow or adipose tissue demonstrated the potential for bone formation. The in vivo results suggests that this model can be used for stem cell research in orthopaedics, but further research needs to be performed to determine their use, proper scaffold and potential osteoinductive materials needed for solid fusion results in the in vivo model. / text

Despite the increasing clinical demand in reconstructive, cosmetic and correctional surgery there remains no optimal strategy for the regeneration or replacement of adipose tissue. Previous approaches to adipose tissue engineering have failed to create an adipose tissue depot that maintains implant volume in vivo long-term (>3 months). This is due to inadequate mechanical properties of the biomaterial and insufficient vascularization upon implantation. Modular tissue engineering is a means to produce large volume functional tissues from small sub-mm sized tissues with an intrinsic vascularization. We first explored the potential of a semi-synthetic collagen/poloxamine hydrogel with improved mechanical properties to be used as the module biomaterial. We found this biomaterial to not be suitable for adipose tissue engineering because it did not support embedded adipose-derived stem cell (ASC) viability, differentiation and human microvascular endothelial cell (HMEC) attachment. ASC-embedded collagen gel modules coated with HMEC were then implanted subcutaneously in SCID mice to study its revascularization potential. ASC cotransplantation was shown to drive HMEC vascularization in vivo: HMEC were seen to detach from the surface of the modules to form vessels containing erythrocytes as early as day 3; vessels decreased in number but increased in size over 14 days; and persisted for up to 3 months. Early vascularization promoted fat development. Only in the case of ASC-HMEC cotransplantation was progressive fat accumulation observed in the module implants. Although implant volume was not maintained, likely due rapid collagen degradation, the key result here is that ASC-HMEC cotransplantation in the modular approach was successful in creating vascularized adipose tissue in vivo that persisted for 3 months. The modular system was then studied in vitro to further understand ASC-EC interaction. Coculture with ASC was shown to promote an angiogenic phenotype (e.g. sprouting, migration) from HUVEC on modules. RT-PCR analysis revealed that VEGF, PAI-1 and TNFα was involved in ASC-EC paracrine signalling. In summary, ASC-HMEC cotransplantation in modules was effective in rapidly forming a vascular network that supported fat development. Future work should focus on further elucidating ASC-EC interactions and developing a suitable biomaterial to improve adipose tissue development and volume maintenance of engineered constructs.

Co-delivery of the embedded growth factor-loaded microspheres and adult stem cells in a hydrogel matrix was studied for its potential as a cell-based therapeutic strategy for cartilage regeneration in partial thickness chondral defects. A photopolymerizable N-methacrylate glycol chitosan (MGC) was employed to form an in situ gel that was embedded with two formulations of growth factor-loaded microspheres and human adipose-derived stem cells (ASC). The polymeric microspheres were used as a delivery vehicle for the controlled release of growth factors to stimulate differentiation of the ASC towards the chondrocyte lineage. The microspheres were made of amphiphilic low molecular weight (Mn < 10,000 Da) poly(1,3-trimethylene carbonate-co--caprolactone)-b-poly(ethylene glycol)-b-poly(1,3-trimethylene carbonate-co--caprolactone) (P(TMC-CL)2-PEG)). This triblock copolymer is solid below 100C, but liquid with a low degree of crystallinity at physiological temperature and degrades slowly, and so acidic degradation products do not accumulate locally. Bone morphogenetic protein-6 (BMP-6) and transforming growth factor-3 (TGF-3) were delivered at 5 ng/day with initial bursts of 14.3 and 23.6%, respectively. Both growth factors were highly bioactive when released, retaining greater than 95% bioactivity for 33 days as measured by cell-based assays. To improve ASC viability within the MGC vehicle, an RGD-containing ligand was grafted to the MGC backbone. Prior to chondrogenic induction within the MGC gel, ASC viability was assessed and greater than 90% of ASC were viable in the gel grafted with cell-adhesive RGD peptides as compared to that in non-RGD grafted gels. For ASC chondrogenesis induced by the sustained release of BMP-6 and TGF-3 in MGC gels, the ASC cellularity and glycosaminosglycan production were similar for 28 days. The ratio of collagen type II to I per cell (normalized to deoxyribonucleic acid content) in the microsphere delivery group was significantly higher than that of non-induced ASC or with soluble growth factor administration in the culture media, and increased with time. Thus, the co-delivery of growth factor-loaded microspheres and ASC in MGC gels successfully induced ASC chondrogenesis and is a promising strategy for cartilage repair. / Thesis (Ph.D, Chemical Engineering) -- Queen's University, 2011-06-07 19:32:50.94

Despite the increasing clinical demand in reconstructive, cosmetic and correctional surgery there remains no optimal strategy for the regeneration or replacement of adipose tissue. Previous approaches to adipose tissue engineering have failed to create an adipose tissue depot that maintains implant volume in vivo long-term (>3 months). This is due to inadequate mechanical properties of the biomaterial and insufficient vascularization upon implantation. Modular tissue engineering is a means to produce large volume functional tissues from small sub-mm sized tissues with an intrinsic vascularization. We first explored the potential of a semi-synthetic collagen/poloxamine hydrogel with improved mechanical properties to be used as the module biomaterial. We found this biomaterial to not be suitable for adipose tissue engineering because it did not support embedded adipose-derived stem cell (ASC) viability, differentiation and human microvascular endothelial cell (HMEC) attachment. ASC-embedded collagen gel modules coated with HMEC were then implanted subcutaneously in SCID mice to study its revascularization potential. ASC cotransplantation was shown to drive HMEC vascularization in vivo: HMEC were seen to detach from the surface of the modules to form vessels containing erythrocytes as early as day 3; vessels decreased in number but increased in size over 14 days; and persisted for up to 3 months. Early vascularization promoted fat development. Only in the case of ASC-HMEC cotransplantation was progressive fat accumulation observed in the module implants. Although implant volume was not maintained, likely due rapid collagen degradation, the key result here is that ASC-HMEC cotransplantation in the modular approach was successful in creating vascularized adipose tissue in vivo that persisted for 3 months. The modular system was then studied in vitro to further understand ASC-EC interaction. Coculture with ASC was shown to promote an angiogenic phenotype (e.g. sprouting, migration) from HUVEC on modules. RT-PCR analysis revealed that VEGF, PAI-1 and TNFα was involved in ASC-EC paracrine signalling. In summary, ASC-HMEC cotransplantation in modules was effective in rapidly forming a vascular network that supported fat development. Future work should focus on further elucidating ASC-EC interactions and developing a suitable biomaterial to improve adipose tissue development and volume maintenance of engineered constructs.

Yes / Interest in adipose tissue is fast becoming a focus of research after many years of being considered as a simple connective tissue. It is becoming increasingly apparent that adipose tissue contains a number of diverse cell types, including adipose-derived stem cells (ASCs) with the potential to differentiate into a number of cell lineages, and thus has significant potential for developing therapies for regenerative medicine. Currently, there is no gold standard treatment for scars and impaired wound healing continues to be a challenge faced by clinicians worldwide. This review describes the current understanding of the origin, different types, anatomical location, and genetics of adipose tissue before discussing the properties of ASCs and their promising applications for tissue engineering, scarring, and wound healing. / The authors thank the Plastic Surgery and Burns Research Unit at the University of Bradford, Bradford, UK for financial support for LVT