Dissertations / Theses on the topic 'Adipose-derived mesenchymal stem cell'

Bone defects are a pervasive complication arising from many clinical conditions, both mechanical and pathological. Current treatments for large bony defects focus on applying bone grafts or synthetic materials to the defect area. Cell-based—and especially stem-cell—therapies have advanced greatly thanks to increasing attention focused on their ability to generate new tissues in situ with biomechanical properties approaching that of native tissue, but they suffer from their own shortcomings as well. Exosomes have been shown to play critical roles in cell-signaling and tissue regeneration and are therefore potentially ideal therapeutic vehicles for treating bone defects. Exosomes are small microvesicles counted amongst stem cells’ paracrine factors capable of delivering nucleic acid and enzymatic protein cargoes in a targeted 2 manner. Our previous studies have shown that hMSC-Exosomes are both proliferative and chemotactic, inhibit inflammatory cytokine production, and suppress osteoclast differentiation. Our long term goal is to develop hMSC-Exosome as a clinical therapy for bone regeneration. The objectives of this study were to determine the ability of hMSC-Exosome to enhance bone healing in a rat calvarial defect model, and to further investigate the integrity of the exosome under certain storage conditions. The specific aims of this study were: 1) To determine the osteogenic potential of hMSC-Exosomes in rat calvarial defects, and 2) To determine the impact of variable storage conditions on the integrity of exosomes. To investigate in vivo regenerative potential, rats with surgically-created craniotomy defects were treated with hMSC-Exosome suspension via a collagen gel matrix. After 4 weeks, the calvaria were harvested and analyzed via micro-CT. Volumetric micro-CT analysis showed that hMSC-Exosome could significantly enhance center healing, structural integrity, and growth uniformity in a calvarial defect model. Western blot and TEM showed thorough destruction of surface protein markers and decreased membrane integrity in lyophilized exosome fraction; moderate progressive surface protein marker loss and aggregation were observed with increasing freeze-thaw cycles. In summary, hMSC-Exosome is a promising therapeutic for treatment of bone defects.

The immunosuppressive property of mesenchymal stem cells (MSC) has been utilised to ameliorate autoimmune reactions such as graft-versus-host disease. However, variation exists in primary MSC isolated due to differences in donor age and tissue of origin. Alternatively, human embryonic stem cells (hESC) can be differentiated to homogeneous populations of MSC (hESCMSC), thus providing an unlimited source of MSC for cell therapy. In this study, the immunomodulatory properties of two hESC-MSC lines, hESC-MSC1 and hESC-MSC2, were compared with adult bone marrow-derived MSC (BM-MSC) and neonatal foreskin fibroblast (Fb). hESC-MSC were able to suppress the proliferation of anti-CD3/CD28-stimulated CD4+ T cells in contact and transwell systems. The immunosuppression was demonstrated by both the carboxyfluorescein diacetate succinimidyl ester (CFSE) and [3H]- thymidine proliferation assays. However, hESC-MSC were less potent and twice the number of adherent hESC-MSC (as measured by IC50) compared to BM-MSC and Fb were required to suppress T cell proliferation by 50%. Supernatants collected from transwells of MSC or Fb with T cells were shown to suppress T cell proliferation, suggesting that suppressive factors were only produced in the presence of activated T cells. Among several candidates, endothelial monocyte-activating polypeptide-II (EMAP-II) was identified as a potential suppressive factor. T cells also induced indoleamine-2,3- dioxygenase (IDO) expression in MSC and Fb. IDO led to the depletion of tryptophan, an essential amino acid, and/or the production of tryptophan metabolites (kynurenines), thereby inhibiting T cell proliferation. Interestingly, blocking of IDO with 1-methyltrytophan reversed the suppressive effect, implicating IDO as a potential mediator in T cell suppression. Concomitantly, several candidate suppressive factors in the supernatants have also been identified using antibody arrays. However, their functions require validation. In conclusion, hESC-MSC share similar suppressive properties as BM-MSC and represent a potential cell source for clinical purposes.

La sindrome metabolica è associata a molte complicanze che portano in particolare a malattie potenzialmente letali come l'obesità e il cancro. Per essere in grado di identificare soluzioni e trattamenti efficaci, dobbiamo indagare le cause alla base di questa sindrome. La nutrizione è un fattore importante da considerare nella prevenzione e nel trattamento della sindrome metabolica. La nutrizione ha effetto su quasi tutti i meccanismi del metabolismo del corpo umano, anche sull’adipogenesi Negli ultimi anni, è stato posto un enorme interesse sulo studio dell'adipogenesi in relazione soprattutto all'obesità. Diversi fattori influenzano l'adipogenesi primi fra tutti i nutrienti presenti nella dieta. Tra i nutrienti con effetto di contrasto all’obesità, i composti dietetici naturali godono di particolare interesse per aiutare a diminuire l'adiposità, quindi il rischio di sviluppare l'obesità e successivamente malattie correlate all'obesità come le malattie cardiovascolari e il diabete ma anche il cancro al seno. Per poter studiare questa correlazione in vitro, è possibile utilizzare un'ampia scelta di modelli cellulari. Le cellule mesenchimali isolate dal tessuto adiposo (hADSCs) sono una dei modelli sperimentali in vitro più usate per studiare l'adipogenesi superando i limiti che altri modelli cellulari hanno nella loro traslabilità all'uomo. In questo studio, lo scopo è stato di studiare l'adipogenesi utilizzando hADSCs anche in presenza di composti dietetici come lipidi e GMG-43AC un modulatore del recettore g (PPAR g) recettore gamma attivato dai proliferatori dei perossisomi. che ha mostrato un effetto positivo sull'inibizione dell'adipogenesi in cellule murine 3T3-L1. Inoltre, abbiamo indagato ulteriormente la sua applicazione su modelli di cellule umane per capire il suo meccanismo d’azione specifico che porta all’ inibizione di questo fenomeno. La parte sperimentale è stata impostata utilizzando la linea cellulare THP-1 differenziate a macrofagi in co-cultura con le hADSCs. Abbiamo notato che trattando le hADSCs con un cocktail di miscela lipidica, VI si verifica la diminuzione dell’espressione delle citochine pro-infiammatorie IL-6 e IL- 1b, valutata mediante real-time RT_PCR. Abbiamo anche notato un aumento dosedipendente dell’espressione di FABP-4. Inoltre, abbiamo anche dimostrato che le capacità differenziative di hADSCs isolate da tessuto adiposo peri-tumorale in casi di tumore della mammella, sono alterate. In questi casi le hADSCs hanno scarse capacità differenziative, valutate mediante saggi istologici ed espressione dell’mRNA di PPARγ e FABP-4. Al contrario, la presenza nel terreno di coltura delle hADSCs di una miscela lipidica (Composizione: acidi grassi non animali; 2 μg / ml arachidonico; 10 μg / ml di acido linoleico; 10 μg / ml di acido linolenico; 10 μg / ml di acido miristico;10 μg / ml di acido oleico; 10 μg / ml di acido palmitico; 10 μg / ml di acido stearico; 0,22 mg / ml di colesterolo dalla lana di pecora della Nuova Zelanda; 2,2 mg / ml di Tween-80; 70 μg / ml di tocoferolo acetato) ripristina l’espressione di PPARγ e l'accumulo di lipidi. In secondo luogo, GMG-43AC in entrambe le concentrazioni (0,5 mM e 2 mM) ha inibito l'accumulo di lipidi e ha mostrato una significativa diminuzione nell'espressione di geni specifici degli adipociti, come PPARγ, FABP-4 anche dopo la completa differenziazione di hADSC derivati da lipoaspirati . Ciò suggerisce che i composti dietetici sono fattori importanti nel differenziamento adipocitario e la dieta ha una grande influenza nella progressione e nella prevenzione di molte malattie metaboliche, tra cui l'obesità e il cancro.<br>Metabolic syndrome is associated with many complications especially leading to life threatening disorders such as obesity and cancer. To be able to identify solutions and natural treatments, we need to investigate the underlying causes of this syndrome. Nutrition is one important factor to consider in the prevention and treatment of the metabolic syndrome. Nutrition effects almost all metabolism mechanisms in the human body. One provident effect of nutrition is adiposity. Over the recent years, an interest was noted to studying adipogenesis in relation to obesity. Different factors affect adipogenesis including natural dietary compounds to help decrease adiposity, therefore the risk of developing obesity and later on obesity related diseases such as breast cancer. To be able to study this correlation in-vitro, a wide choice of cell models can be used. Human adipose derived mesenchymal cells (hADSCs) are one of the top choices used to study adipogenesis overcoming the limitations that other cell models have in their applicability to humans regarding the prevailing difference in their metabolism and physiology. In this study, the aim was to study adipogenesis using hADSCs in presence of dietary compounds such as lipids and GMG-43AC, a natural selective peroxisome proliferator-activated receptor g (PPAR g) modulator, that seems to have a positive effect on inhibiting adipogenesis in murine 3T3-L1 cells. We wanted to investigate further on its application on human cell models and try to understand its mechanism in inhibiting this phenomenon. The protocols were set up using the THP-1 cell line, which we noticed upon using a Lipid mixture cocktail (Composition: Non-animal fatty acids; 2 μg/ml arachidonic; 10 μg/ml linoleic acid; 10 μg/ml linolenic acid: 10 μg/ml myristic acid; 10 μg/ml oleic acid; 10 μg/ml palmitic acid; 10 μg/ml stearic acid; 0.22 mg/ml cholesterol from New Zealand sheep′s wool; 2.2 mg/ml Tween-80; 70 μg/ml tocopherol acetate), a decrease in pro-inflammatory cytokines IL-6 and IL-1b. We also noticed a doseIV dependent increase of FABP-4. Our findings regarding hADSCs, that PPARγ expression and lipid accumulation was restored upon the presence of lipid mixture in breast cancer hADSCs that were derived from breast tissue. Secondly, GMG-43AC in both concentrations (0.5mM and 2mM) inhibited lipid accumulation and showed a significant decrease in the expression of adipocyte-specific genes, such as PPARγ and FABP-4 even after the full differentiation of hADSCs that were derived from lipoaspirates. This suggests that dietary compounds are important factors in adipose differentiation and diet has a big influence in the progression and prevention in many metabolic diseases, such as obesity and cancer.

Ageing, defined as the progressive deterioration of molecular, cellular, tissue and whole organism function, is a primary risk factor for numerous diseases, such as cardiovasculature disease, neurodegeneration and cancer. In recent years, advances in our knowledge of key determinant mechanisms that underpin ageing decline, drives the notion that these features can be attenuated and targeted therapeutically, enabling elderly individuals to experience an enhanced quality of life into advanced old age. Sarcopenia comprises the age-related loss of muscle mass, quality and function and contributes to overall frailty, immobility and a greater risk of falls. The use of stem cell-derived conditioned media (CM) holds great clinical potential and recent studies have reported many beneficial effects in a number of tissue models of injury and disease. We want to develop a novel anti-ageing therapy for the treatment of age-associated declines in sarcopenia. First, we characterise the skeletal muscle profile in a novel use of the Ercc1d/-murine model of progeria and compare it to the naturally-aged phenotype. We examine the effects of CM, generated from adipose-derived mesenchymal stem cells (ADMSCs), on skeletal muscle composition, function and satellite cell (SC) activity in sarcopenia and progeria. We show that CM has beneficial regulatory effects on mechanisms underpinning the declines associated with the Hallmarks of Ageing, for example, enhancing mitochondrial function and reducing oxidative stress. Importantly, we also demonstrate that CM harbours pro-angiogenic effects, which we hypothesise is unlikely to impact on skeletal muscle alone. Remarkably, we report the Ercc1d/-mice appear to launch a survival programme and delay the progression of age-related deterioration. A further feature associated with ageing skeletal muscle is the impaired regenerative function and myofibre turnover following injury and daily use. Key factors attributed to this decline in repair involve compromised SC activity as well as the depletion of the stem cell pool, known to occur with age. We want to first, examine the influence of the myofibre microenvironment on SC behaviour. Second, we investigate the use of non-muscle cell types as a source to generate muscle cells. We show that three stem cell types, ADMSC, dental pulp stem cells (DP) and amniotic fluid stem cells (AFS) and one non-stem cell line, MDA-MB-231 (MDA) breast cancer cells, adopted amoeboid-based migration (blebbing) once seeded onto myofibres. We also show that the regulation of the migratory mechanisms, known to be controlled by the Rac and Rho signalling pathways, is conserved in each of these cell types. Remarkably, we also demonstrate that a rapidly growing non-muscle stem cell (AFS), as well as a non-stem cell (MDA) initiate expression of MyoD and furthermore, the AFS cells were directed, through exposure to the myofibre microenvironment, to fuse and form myotube structures that express myosin heavy chain (MHC+).

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.

Background: Adipose-derived stem/stromal cells (ASCs) isolated from the stromal vascular fraction are a source of mesenchymal stem cells that have been shown to be beneficial in many regenerative medicine applications. ASCs are an attractive source of stem cells in particular, due to their lack of immunogenicity. This study examines differences between mitochondrial bioenergetic profiles of ASCs isolated from adipose tissue of five peri-organ regions: pericardial, thymic, knee, shoulder, and abdomen. Results: Flow cytometry showed that the majority of each ASC population isolated from the adipose tissue of 12 donors, with an n = 3 for each tissue type, were positive for MSC markers CD90, CD73, and CD105, and negative for hematopoietic markers CD34, CD11B, CD19, and CD45. Bioenergetic profiles were obtained for ASCs with an n = 4 for each tissue type and graphed together for comparison. Mitochondrial stress tests provided the following measurements: basal respiration rate (measured as oxygen consumption rate [pmol O-2/min], ATP production, proton leak, maximal respiration, respiratory control ratio, coupling efficiency, and non-mitochondrial respiration. Glycolytic stress tests provided the following measurements: basal glycolysis rate (measured as extracellular acidification rate [mpH/min]), glycolytic capacity, glycolytic reserve, and non-glycolytic acidification. Conclusions: The main goal of this manuscript was to provide baseline reference values for future experiments and to compare bioenergetic potentials of ASCs isolated from adipose tissue harvested from different anatomical locations. Through an investigation of mitochondrial respiration and glycolysis, it was demonstrated that bioenergetic profiles do not significantly differ by region due to depot-dependent and donor-dependent variability. Thus, although the physiological function, microenvironment and anatomical harvest site may directly affect the characteristics of ASCs isolated from different organ regions, the ultimate utility of ASCs remains independent of the anatomical harvest site.

Traumatic injury to the spinal cord interrupts ascending and descending pathways leading to severe functional deficits of sensory motor and autonomic function which depend on the level and severity of the injury. There are currently no effective therapies for treating such injuries and the adult central nervous system has very limited capacity for repair so that recovery is very limited and functional deficits are usually permanent. Cell transplantation is a potential therapy for spinal cord injury and a range of cell types are being investigated as candidates. Mesenchymal stem cells (MSCs) obtained from bone marrow are one cell type quite extensively studied. When transplanted into animal models of spinal cord injury these cells are reported to affect various aspects of repair and in some cases to improve functional outcome according to behavioural measures. However, the use of these cells has several limitations including the need for an invasive harvesting procedure, variability in cell quality and slow expansion in culture. This project therefore had two main aims: Firstly to investigate whether MSC-like cells closely equivalent to bone marrow derived MSCs could be reliably and consistently differentiated from human embryonic stem cells (hESCs) in order to provide an “off the shelf” cellular therapy product for spinal cord injury and secondly, to transplant such cells into animal models of spinal cord injury in order to, determine whether hESC-derived MSCs replicate or improve on the repair mechanisms reported for bone marrow MSCs.

This study describes the various roles adipose-derived mesenchymal stem cells (ADSCs) play in a breast tumour environment. The ADSC-derived secreted factors inhibit breast tumour cell growth aspects, whereas the tumour-derived secreted factors transform ADSCs into tumour-associated fibroblasts (TAFs). Additionally, it highlights the role of Wnt antagonist sFRP4 and its peptides to further influence the activity of ADSC-derived secreted factors, to downregulate the transformation of ADSCs into TAFs, and in upregulating the adipogenic differentiation of ADSCs.

Master of Science<br>Department of Clinical Sciences<br>Walter Renberg<br>Objective: To evaluate the safety and effect of a single simultaneous intra-articular and intravenous injection of autologous adipose-derived stromal vascular fraction (SVF) and platelet rich plasma (PRP) on coxofemoral osteoarthritis (OA) in dogs. Methods: This was a randomized, double-blind, placebo-controlled prospective pilot trial of simultaneous intra-articular and intravenous SVF and PRP for coxofemoral OA. Dogs with coxofemoral OA causing signs of lameness or discomfort were evaluated by orthopedic exam, visual lameness score, Canine Brief Pain Inventory (CBPI), goniometry, visual analogue scale (VAS), and pressure-sensitive walkway (PSW) at week 0 (baseline), and at 4, 8, 12 and 24 weeks after injection. Joint radiographs were scored at 0 and 24 weeks. Results: Twenty two client-owned dogs with naturally occurring OA of the coxofemoral joints were enrolled (12 placebo-control, 10 SVF-treated). CBPI pain severity scores were lower in the treatment group at 24 weeks compared to the placebo group (p=0.042). The VAS score for the treatment group was significantly greater at 0 weeks than at 4, 8, or 24 weeks (p<0.05). When dogs with low quartile baseline PVF (25th percentile) were compared, the treatment group had statistically higher PVF at all post-injection time points when compared to the placebo group. After SVF injection, fewer dogs in the treated group were lame compared to the control group. Clinical Significance: This study is the first to utilize objective data from PSW as an outcome measure for dogs treated with SVF and PRP for coxofemoral OA. No adverse events were noted. Improvements in some measured parameters in the treated dogs compared to those in the placebo group.

Diabetes mellitus is one of the most common and serious chronic disease in the world. Although the number of available agents to manage diabetes continues to rapidly expand, treatment of diabetes complications, such as neuropathy that is one of the most frequent complication of diabetes mellitus, remains a substantial challenge [Aring et al., 2005]. Pathophysiology of diabetic neuropathy is complex and not fully elucidated; it has multipathogenic mechanisms that cause a diversity of physical symptoms: allodynia, hyperalgesia, numbness and cutaneous ulceration [Vinik et al., 1995]. Persistent Neuropathic Pain (NP) interferes significantly with quality of life, impairing sleep, and emotional well-being, and is a significant causative factor for anxiety, loss of sleep, and non-compliance with treatment. Recent advances in the mechanisms involved in NP have demonstrated that pro- and anti-inflammatory cytokines produced by immune cells as well as by glia and microglia in nerve, dorsal root ganglia (DRG) and spinal cord are common denominators in neuropathic pain [Sacerdote et al., 2013; Old et al., 2015]. These start a cascade of neuroinflammation-related events that may maintain and worsen the original injury, participating in pain generation and chronicization [Valsecchi et al., 2011; Sommer and Kress, 2004; Austin and Moaelem-Taylor, 2010]. Activation of inflammatory cascade, pro- inflammatory cytokines upregulation, and neuroimmune communication pathways play a vital role in structural and functional damage of the peripheral nerves leading to the diabetic peripheral neuropathy. Unfortunately, most of the available analgesic drugs appear to be relatively ineffective in controlling diabetic neuropathic pain, both for insufficient efficacy and side effects [Galer et al., 2000; Kapur, 2003]. Thus, there is a clear need for new disease-modifying therapeutic approaches. Mesenchymal stem/stromal cells (MSCs) may offer a novel therapeutic option to treat diabetic neuropathy. MSCs modulate the nervous system injured environment and promote repair as they secrete anti-inflammatory, anti-apoptotic molecules, and trophic factors to support axonal growth, immunomodulation, angiogenesis, remyelination, and protection from apoptotic cell death [Ma et al., 2014]. Transplanted MSCs not only directly differentiate into endogenous cells on administration, but also secrete a broad range of biologically active factors, generally referred to as the MSCs secretome; in fact even if initially MSCs were proposed for cell therapy based on their differentiation potential, the lack of correlation between functional improvement and cell engraftment or differentiation at the site of injury has led to the proposal that MSCs exert their effects not through their differentiation potential but through their secreted products [Makridakis, 2016; Blaber et al., 2012]. For these reasons in the present study we analyze in a Streptozotocin mouse model of type 1 diabetes the therapeutic effect of hASC (human adipose stem/stromal cells) and their conditioned media (CM-hASC/ secretome) on allodynia and hyperalgesia, on pro- and anti- inflammatory cytokines expression in the main tissue stations involved in nociception transmission as well as in peripheral immune responses. Type 1 diabetes was induced in mice by intraperitoneal (i.p.) injection of moderate low doses of Streptozotocin (STZ, 80 mg/kg, daily for three consecutive days) while control mice were injected with vehicle (citrate buffer). In all groups, mechanical allodynia was evaluated by Von Frey test before diabetes induction and every week after STZ until the end of protocol (14 weeks after STZ). When allodynia was established (2 weeks after STZ) animals were treated with 106 hASC that have been mechanically dissociated to a single cell suspension in PBS solution with 2.5% heparin; CM-hASC from 2x106 cells was also re-suspended in PBS solution with 2.5% heparin and both hASC and CM-hASC were intravenously injected in the tail vein to mice. Animals injected with vehicle only were considered as controls. Our data demonstrated that hASC and CM-hASC treatments were able to reduce allodynia, although the effect of hASC was significantly higher than that elicited by CM-hASC. The effect of both hASC and their secretome was very fast, since a significant reduction of mechanical allodynia was evident already 3 hours after the injection. The antiallodynic effect was maximal beetwen 1 and 2 weeks after treatments and it was extremely long lasting: a significant reduction of allodynia was still present 12 weeks after a single hASC and CM-hASC treatment. Moreover, 4 weeks after the first hASC/CM-hASC treatment (6 weeks after STZ) we decided to treat again a group of diabetic animals with hASC or CM-hASC; repeated hASC treatment did not further ameliorate allodynia. On the other hand, already few hours after the second CM-hASC injection, the antiallodynic effect was significantly potentiated and it completely mimicked the effect evoked by hASC. In order to discover whether hASC and CM-hASC treatments were effective also in a more advanced stage of the disease, when a severe loss of nerve function is reported, we treated animals 6 weeks after diabetes induction. Also in this situation both treatments were efficacious in providing a fast and irreversible antiallodynic effect. Futhermore, in order to verify whether stemness is a fundamental prerequisite for obtaining pain relief a group of STZ-mice was treated with CM obtained from 2x106 human fibroblasts (CM-hF). CM-hF did not exert any effect on mechanical allodynia, demonstrating that only secretome from stem cell cultures is biologically active. It is very important also to consider preparation method of secretome, because lyophilized CM-hASC was unable to provide pain relief, suggesting that during the lyophilization process some essential bioactive factors may be lost. Moreover, since in patients sensory alterations associated to diabetic neuropathy are often diverse in order to ascertain whether the effects of hASC and CM-hASC were limited only to mechanical allodynia, we evaluated thermal hyperalgesia (hot stimuli) and thermal allodynia (cold stimuli) by plantar test and acetone test, respectively. In STZ-mice cold allodynia was present and both treatments were able to significantly reduce it. As regards to heat hyperalgesia, it was present in diabetic mice until 3 weeks from STZ administration, but subsequently we observed hypoalgesia appearance and both treatments were able to avoid hypoalgesia development; these results demostrate the ability of stem cells and their secretome to relieve and prevent the typical diabetic hypersensitivity in response to different types of stimuli. In order to evaluate the impact of treatments on pro- and anti- inflammatory cytokines, animals were sacrificed at different time points: 2 weeks after STZ, i.e. 3 hours after hASC/CM-hASC treatment; 3 weeks after STZ, i.e. 1 week from treatments and 14 weeks after STZ, i.e. 12 or 8 weeks from treatments. From each animal, sciatic nerves, dorsal root ganglia, spinal cord and spleens were collected. IL-1β, TNF-α, IL-6 and IL-10, were evaluated as protein in nervous tissues by ELISA assay. Three weeks after neuropathy induction pro-inflammatory cytokines IL-1β, TNFα and IL-6 resulted overexpressed in peripheral (sciatic nerve and DRG) and central (spinal cord) nervous system of diabetic mice, both hASC and CM-hASC were similarly able to restore pro-inflammatory cytokine levels that 1 week from treatments were back to basal levels; while in all nervous tissues IL-10 levels appeared instead significantly reduced in diabetic animals and both hASC and CM-hASC significantly increased IL-10 concentrations, reaching physiological levels in DRG and spinal cord, while it exceeded basal levels in the sciatic nerve, indicating a switch towards an anti-inflammatory environment in all these tissues. Fourteen weeks after STZ, spinal cord IL-1β, TNF-α and IL-6 levels were still significantly elevated and IL-10 levels reduced in comparison to non diabetic mice, indicating the persistence of neuroinflammation. As observed for the antiallodynic effect, also cytokine modulation induced by hASC and CM-hASC was long lasting. Twelve weeks after treatments performed 2 weeks from STZ, IL-1β, TNF-α and IL-6 levels were still significantly reduced by hASC and CM-hASC treatments, while hASC-treated mice showed a significant normalization of IL-10 levels. Similar effects were observed also in double treatments (2 and 6 weeks after STZ) and both treatments were effective in modulating cytokine levels also when they were administered in an advanced pathological state (6 weeks after STZ). Moreover, to investigate the timing of cytokines modulation exerted by both treatments IL-1 and IL-10 levels in scatic nerves, DRG and spinal cord were measured. Two weeks after diabetic induction, STZ mice were characterized by pro- inflammatory profile and only 3 hours after hASC and CM-hASC administration , both treatments were able to modulate cytokines levels. To further demonstrate the modulation of treatments on pain-related mediators we demonstrated the ability of hASC and CM-hASC to normalize calcitonin gene related peptide level (CGRP), that was elevated in DRG from diabetic animals. Moreover, we evaluated loss of nerve fibers and skin thickness 1 and 12 weeks after a single hASC/CM-hASC administration at 2 weeks after STZ. Both treatments were able to contrast loss of nerve fibers and skin thickness, although hASC treatment was more effective. Since STZ multiple low-doses protocol that we utilized is able to develop an autoimmune response against pancreatic tissue sustained by a T-helper 1 pattern of activation, we studied whether a T-helper polarization was present in splenocytes from diabetic mice and whether hASC or their secretome did exert any immunomodulatory activity. Two weeks after STZ, Con-A stimulated splenocytes released higher levels of IFN-γ, while IL-10 release was significant reduced; both hASC and CM-hASC treatments 3 hours after administration were already able to augment IL-10 levels. Th1/Th2 cytokines unbalance was more evident 3 weeks after STZ and both tretaments appeared able to restablish a correct IFNγ/IL-10 balance. When cytokine levels were measured at longer time from diabetes induction, i.e. 14 weeks after STZ, a clear shift toward a Th1 pattern, characterized by higher IFN-γ and IL-2 secretion and lower levels of IL-4 and IL-10, was present and both hASC/CM-hASC treatments were able to normalize cytokine levels. In the whole, the data indicate that both hASC and CM-hASC treatments are able to block Th1 polarization that develops in this experimental model of diabetes. Moreover, throughout the experiment, blood glucose levels and weight were monitored. In respect to non-diabetic control animals, a significant body weight loss was observed in diabetic mice, that started to be significant 3 weeks after STZ. In STZ-mice the administration of hASC or CM-hASC, 2 weeks after diabetes induction significantly prevented the loss of body weight. Neither treatments did modify blood glucose levels that were elevated in STZ-mice nor glucose tolerance test response. Moreover both hASC and CM-hASC did ameliorate nephropathy that was present in diabetic animals, indicating that the treatments may be useful for treating also other diabetes complications. Our results demonstrated that hASC can control diabetic complications such as neuropathic pain, acting on several peripheral and central mechanisms involved in development and maintenance of this condition, such as neural and immune elements. Moreover the significant new positive results observed also with hASC conditioned medium strongly suggest that their effect is likely to be mediated by their secreted products.

Introduction: Mesenchymal stem cells (MSC) offer a novel cell therapy within tissue engineering and regenerative medicine (TERM)-based strategies, and the prospect of MSC therapies are widening since the discovery of MSCs within multiple locations of the body including bone marrow (BM-MSCs) and adipose tissue, (AD-MSCs). It is highly recognised that an organisms reparative and regenerative potential declines with advancing age, therefore aged patients are one of the primary target populations for TERM applications. Although information is available regarding the effects of patient age on the quality of human BM-MSCs, little and conflicting information currently exists for AD-MSCs. In addition, few studies have compared the quality of freshly isolated and expanded donor-matched BM and AD-MSCs to elucidate the more appropriate cell source. This study investigated the effect of donor age and in vitro ageing on functional behaviour (i.e. senescence state, population kinetics and differentiation potential) of donor-matched BM and AD-MSCs. Methods: The influence of donor age and in vitro ageing on mature (28-55 years) and elderly (75-86 years) donor-matched BM and AD-MSCs was assessed upon isolation (early life-span) and during extended (mid and late lifespan) timepoints through culture. During culture MSCs were characterised for cumulative population doublings (CPDs) and the expression of senescence associated marker genes, p16INK4A, p21 and p53, and transcription factor NANOG. At each lifespan telomere length was assessed along with differentiation efficiency along the osteogenic, adipogenic and chondrogenic lineages through lineage specific marker genes and histological staining. Results: Elderly BM and AD-MSCs displayed similar characteristics in terms of initial CPD number, p21, p53 and NANOG expression, telomere length and differentiation along osteogenic and adipogenic lineages. With increasing donor age there was a significant decline in p16INK4A expression within BM-MSCs, whilst expression of all chondrogenic markers significantly decreased within AD-MSCs. BM and AD-MSCs were comparable for the majority of outcome measures with the exception of chondrogenic differentiation which was superior with BM-MSCs in terms of COL2A1 expression and histological staining for proteoglycans. Donor age had a negative effect on BM-MSCs with long-term culture leading to a significantly longer PD time and decreased telomere lengths. Similar population kinetics was displayed between BM and AD-MSCs during long-term culture. Increasing culture time had effects on differentiation potential for both MSC sources with complete loss of osteogenic capacity and decreased adipogenic capacity; however chondrogenic capacity was only decreased within AD-MSCs. Differentiation potential after long-term culture between BM and AD-MSCs showed similar osteogenic and adipogenic ability yet superior chondrogenic ability was apparent within mature BM-MSCs compared to AD-MSCs, in terms of ECM deposition. Conclusions: In conclusion the source of MSCs for TERM will need to be considered depending upon the type of tissue regeneration required. The clinical outcome would be greater using MSCs during early stages of culture, as culture expansion has detrimental effects on functional properties of both BM and AD-MSCs.

Purpose: Adipose stem cells (ASCs) represent a reliable source of stem cells with a widely demonstrated potential in regenerative medicine and tissue engineering applications. New recent insights suggest that three-dimensional (3D) models may closely mimic the native tissue properties; spheroids from adipose derived stem cells (S-ASCs) exhibit enhanced regenerative abilities compared with those of 2D models. Stem cell therapy success is determined by “cell-quality”; for this reason, microRNA profiles, the involvement of stress signals and cellular aging need to be further investigated. Material and Methods: Adipose tissue was collected from healthy individuals, 44 females and 17 males, after signing informed consent. Mean age was 50, 25 years (range: 18-77). Lipoaspirate samples were harvested from different body areas such as abdomen, breast, flanks, trochanteric region, and knee. Here, we performed a comparative analysis, molecular and functional, of miRNA expression pattern profile “stemness and differentiation associated”, genes connected with stemness, aging, telomeric length and oxidative stress, of adipose stem cells in three-dimensional and adhesion conditions, SASCs-3D and ASCs-2D cultures. Results: We have demonstrated that Spheroids from Adipose-derived Stem Cells (SASCs-3D) present express high level of the typical miRNAs and mRNAs of iPS cells, such as miR-142-3p and SOX2/POU5F1/NANOG, in canonical and in long term in vitro culture condition, express low level of the early and late miRNAs and mRNAs typical of chondrocytic, adipocytic and osteoblastic lineages in canonical and in long term in vitro culture condition. The expression levels of stemness-related markers and anti-aging Sirtuin1 were significantly up-regulated (P < 0.001) in SASC-3D while gene expression of aging-related p16INK4a was increased in ASCs-2D (P < 0.001). We found that 3D and 2D cultures also presented a different gene expression profile for those genes related to telomere maintenance (Shelterin complex, RNA Binding proteins and DNA repair genes) (P < 0.01 and P < 0.001) and oxidative stress (aldehyde dehydrogenase class1 and 3) (P < 0.05, P < 0.01 and P < 0.001) and presented a striking large variation in their cellular redox state. Conclusion: Based on our findings, we propose a “cell quality” model of SASCs, highlighting a precise molecular expression of microRNA pattern profiles, several genes involved with stemness (SOX2, POU5F1 and NANOG), anti-aging (SIRT1), oxidative stress (ALDH3) and telomeres maintenance.<br>Purpose: Adipose stem cells (ASCs) represent a reliable source of stem cells with a widely demonstrated potential in regenerative medicine and tissue engineering applications. New recent insights suggest that three-dimensional (3D) models may closely mimic the native tissue properties; spheroids from adipose derived stem cells (S-ASCs) exhibit enhanced regenerative abilities compared with those of 2D models. Stem cell therapy success is determined by “cell-quality”; for this reason, microRNA profiles, the involvement of stress signals and cellular aging need to be further investigated. Material and Methods: Adipose tissue was collected from healthy individuals, 44 females and 17 males, after signing informed consent. Mean age was 50, 25 years (range: 18-77). Lipoaspirate samples were harvested from different body areas such as abdomen, breast, flanks, trochanteric region, and knee. Here, we performed a comparative analysis, molecular and functional, of miRNA expression pattern profile “stemness and differentiation associated”, genes connected with stemness, aging, telomeric length and oxidative stress, of adipose stem cells in three-dimensional and adhesion conditions, SASCs-3D and ASCs-2D cultures. Results: We have demonstrated that Spheroids from Adipose-derived Stem Cells (SASCs-3D) present express high level of the typical miRNAs and mRNAs of iPS cells, such as miR-142-3p and SOX2/POU5F1/NANOG, in canonical and in long term in vitro culture condition, express low level of the early and late miRNAs and mRNAs typical of chondrocytic, adipocytic and osteoblastic lineages in canonical and in long term in vitro culture condition. The expression levels of stemness-related markers and anti-aging Sirtuin1 were significantly up-regulated (P < 0.001) in SASC-3D while gene expression of aging-related p16INK4a was increased in ASCs-2D (P < 0.001). We found that 3D and 2D cultures also presented a different gene expression profile for those genes related to telomere maintenance (Shelterin complex, RNA Binding proteins and DNA repair genes) (P < 0.01 and P < 0.001) and oxidative stress (aldehyde dehydrogenase class1 and 3) (P < 0.05, P < 0.01 and P < 0.001) and presented a striking large variation in their cellular redox state. Conclusion: Based on our findings, we propose a “cell quality” model of SASCs, highlighting a precise molecular expression of microRNA pattern profiles, several genes involved with stemness (SOX2, POU5F1 and NANOG), anti-aging (SIRT1), oxidative stress (ALDH3) and telomeres maintenance.

Les travaux antérieurs ont montré que les myofibroblastes portaux (PMFs) contribuaient de manière significative à la fibrogenèse et à l'angiogénèse dans la fibrose hépatique. L'objectif principal de cette thèse était de cartographier les cellules mésenchymateuses portales, et plus particulièrement la niche des cellules souches mésenchymateuses portales. Nous avons caractérisé la variété des cellules mésenchymateuses portales du foie de souris. Résultat important, nous avons identifié une population de cellules mésenchymateuses portales ayant les caractéristiques de cellules souches mésenchymateuses, désignées cellules souches mésenchymateuses portales (PMSCs), qui ont la capacité de se transformer en PMFs in vitro. Nous avons identifié Slit2 comme un marqueur des PMSCs par scRNA-seq et bulk RNA-seq. In vivo, nous avons mis en évidence l'expansion de PMSCs dans le foie de modèles murins de fibrose hépatique et de patients ayant une maladie chronique du foie. Nous avons identifié des signatures transcriptomiques spécifiques des PMSCs d’une part et des cellules étoilées du foie (CEF), de l’autre. Les résultats obtenus par l’utilisation de ces marqueurs, renforcent nos conclusions selon lesquelles les PMSCs s’accumulent de façon corrélée avec la fibrogenèse et l'angiogenèse, tandis que la signature des CEFs ne varie pas. En conclusion, nos travaux apportent des éléments à la connaissance des populations de cellules mésenchymateuses portales du foie. Ils ont permis d’identifier et caractériser les PMSCs ainsi que les myofibroblastes qui en dérivent, ouvrant de nouvelles perspectives dans le domaine des thérapies ciblées et des biomarqueurs pour la pratique clinique<br>Previous work has demonstrated that portal myofibroblasts (PMFs) significantly contributed to liver fibrogenesis and modulated angiogenesis in liver fibrosis. The main aim of this thesis was to elucidate the landscape of portal mesenchymal cells, with a particular focus on a portal mesenchymal stem cell niche. We characterized the murine normal liver portal mesenchymal cell landscape. Importantly, we revealed a portal mesenchymal cell population with the features of mesenchymal stem cells (MSCs), designated portal mesenchymal stem cells (PMSCs) that possessed the ability to give rise to PMFs in vitro. Furthermore, we identified Slit2 as a new marker of PMSCs based on scRNA-seq and bulk RNA-seq analysis. In vivo, we observed PMSC expansion (measured by the expression of Slit2) in liver from both animal fibrosis models (DDC and CDAA) and patients with chronic liver disease (NASH, PSC and other liver disease). Notably, we defined the specific gene signatures for PMSCs and hepatic stellate cells (HSCs), respectively. By using these markers, we provide further evidence indicating that PMSCs expand in correlation with fibrogenesis and angiogenesis in different murine and human liver diseases, whereas the HSCs gene signatures did not vary. In conclusion, our work collectively offers insights into the components and functions of the mammalian liver portal mesenchymal cell populations, and in particular, identify and characterize PMSCs and their derived myofibroblasts, opening up the possibility for the development of novel targeted drugs or biomarkers of clinical significance with increased precision

Introduction It is well known that resident adipose stem/stromal cells (ASCs) are a heterogeneous population of multipotent cells characterized by (a) their ability to adhere to plastic; (b) immunophenotypic expression of certain cell surface markers, while lacking others; and (c) the capacity to differentiate into cells of mesodermal origin including osteocytes, chondrocytes and adipocytes. Adipose derived stromal cells offer great therapeutic potential in multiple medical fields, including, orthopedics, cardiology, oncology and degenerative diseases, to name a few. Combining different disciplines of medicine and engineering, organ and tissue repair can be achieved through tissue engineering and regenerative medicine. Adipose derived stromal cells (ASCs) can be utilized as biological vehicles for vector-based gene delivery systems, since they home to sites of inflammation and infection in vivo. In order to reach the long-term aim of clinical translation of cell-based therapy, preclinical safety and efficacy need to be shown in animal models. This has motivated the development of standardized isolation, characterization and differentiation operating procedures as well as an in vivo tracking system for ASCs and lentiviral vector transduction for a vector-based gene delivery system. Methodology Human ASCs were isolated from lipoaspirate, expanded in culture, immunophenotyped using flow cytometery and induced to differentiate into adipogenic, osteogenic and chondrogenic lineages. Tri-lineage differentiation was confirmed by microscopy. The ASCs were then transduced with green fluorescent protein (GFP)-expressing lentiviral vectors in vitro. The effect of the GFP lentiviral vector on ASCs was investigated by studying ASC immunophenotypic expression of surface markers as well as their capacity to differentiate into osteocytes, chondrocytes and adipocytes. Results The isolated and expanded cell population, from harvested lipoaspirate adhered to recommended ASC identity criteria. The heterogeneity of ASCs was confirmed by the presence of sub-populations. Transduction efficiency in ASC cultures of approximately 80% was observed after introducing a total of 300 μl of concentrated lentiviral vector suspension per 4.8 x 104 cells. No immunophenotypic differences were observed between GFP positive and GFP negative cultures. Flow cytometric analysis revealed a progressive increase in GFP expression following in vitro expansion of transduced ASCs. Both non-transduced and transduced cultures successfully differentiated into osteocytes, chondrocytes and adipocytes. Conclusion The isolated and expanded cell population conformed to the recommended characterization criteria. Heterogeneity was demonstrated with the identification of immunophenotypic sub-populations and semi-quantification of adipogenesis was performed. ASCs were efficiently transduced using the GFP lentiviral vectors produced in our facility. In addition, transduced ASCs maintained adherence to plastic, ASC immunophenotype and were able to differentiate successfully into cells of the three lineages of mesodermal origin. This optimized GFP-ASC transduction technique offers a feasible tracking system as well as a vector-based gene delivery system for future preclinical studies.<br>Dissertation (MSc)--University of Pretoria, 2015.<br>tm2015<br>Immunology<br>MSc<br>Unrestricted

In adult tissues, somatic stem cells represent a cell reservoir involved in physiological or pathological cell replacement within healthy or damaged tissue. Thanks to specific molecular or physical signals of the microenvironment, homeostasis process is guaranteed inside stem cell niches. Factors deriving from pathological events induce a shift in stem cell behaviour to cell differentiation, in order to reconstitute cell populations lost in the damaged tissue. In several myopathies, chronic inflammation is a part of pathological process that causes fibrotic infiltrations and adipose deposition within degenerating tissue, leading even to muscle substitution. Mechanisms underlying these events are not clear yet and represents a focus in etiopathogenetic studies on muscular research. The aim of this work was to develop an experimental model for mimicking in vitro the effects of a damaged muscle on a specific stem cell population; the methodological improvement is represented by the simultaneous culture of both the component in a microenvironment divided by a porous membrane. Parameters setting and standardization make the method reproducible and applicable to different culture conditions. First experimental session, performed on a population of human circulating myogenic progenitors expressing CD133 antigen, has shown how the presence of a murine muscle tissue section in culture can induce an higher cell proliferation and expression of an early marker of muscle differentiation, as Myf5. Nevertheless, because of their hematopoietic origin, these cells cannot complete their myogenic differentiation, that is usually achieved in a direct coculture with myotubes or myofibers. The same experimental design, applied for the culture of mesenchymal stem cells with myogenic potential, has shown how the biological factors, released by the murine muscle section, can induce an adipose differentiation in that stem cell population. Lipid drops in cell cytoplasm and expression of specific markers of adipose differentiation, such as perilipin A and FABP4, confirmed that the experimental model proposed can reproduce in vitro a culture condition similar to the one observed in degenerating muscle in several myopathies. This methodological approach represents a new model for in vitro study of adipose deposition in muscle, whose molecular mechanisms are not well characterized yet; using a whole tissue section in culture allows to reproduce in vitro a microenvironment similar to the once observed in vivo. For this reason the experimental model developed for this work can be seen as new perspective in the study of molecular process underlying adipose tissue formation in degenerating muscle.

A cicatrização de feridas crônicas e de defeitos complexos representam desafios para a cirurgia plástica reconstrutiva. Novos tratamentos emergiram com a utilização de células tronco mesenquimais, com especial interesse para as derivadas de tecido adiposo (CTMs-TA), por possuir algumas vantagens em relação às derivadas da medula óssea. Algumas doenças poderiam se beneficiar com o uso das CTMs-TA, em particular, as feridas de pacientes portadores de anemia falciforme que ainda representam um desafio terapêutico. Neste estudo, investigou-se a existência de possíveis áreas doadoras preferenciais de CTMs-TA no tecido adiposo (TA) por meio da comparação de aspectos qualitativos e quantitativos das CTMs-TA derivadas de cinco diferentes áreas corporais. Posteriormente, analisou-se a influência do sobrenadante dessas células, rico em citocinas e fatores de crescimento, sobre a migração de fibroblastos de indivíduos normais e de portadores de anemia falciforme in vitro. Não foram observadas diferenças qualitativas entre as CTMs-TA das cinco áreas analisadas. A região do dorso apresentou número maior de CTMs, com diferença significativa em relação à região das coxas. A adição de sobrenadante produzido por CTMs-TA demonstrou aumento da velocidade de migração dos fibroblastos de forma similar para os normais e os falciformes. O microambiente desfavorável presente nas feridas falciformes parece exercer importante influência sobre esses fibroblastos, pois uma vez corrigido o microambiente com os meios de cultivo apropriados, as células apresentaram taxa de duplicação e velocidade de migração semelhante à dos fibroblastos normais in vitro.<br>The healing of chronic wounds and complex defects represents challenges for reconstructive plastic surgery. New treatments have emerged with the use of multipotent mesenchymal cells, with special interest for the adipose-derived stem cells (ADSCs) as it has some advantages over the bone marrow-derived. Some diseases could benefit from the use of ADSCs, in particular, the wounds of patients with sickle cell anemia that still represent a therapeutic challenge. In this study, we investigated the existence of possible preferential donor areas of ADSCs in adipose tissue by comparing qualitative and quantitative aspects of ADSCs derived from five different body areas. Later, we analyzed the influence of the supernatant of these cells, rich in cytokines and growth factors, on the migration of fibroblasts from healthy individuals and from patients with sickle cell anemia in vitro. No qualitative differences were observed among the ADSCs of the five areas analyzed. The dorsum presented a higher number of ADSCs, with a significant difference in relation to the thigh. Addition of supernatant produced by ADSCs has been shown to increase the rate of migration of fibroblasts in a similar way to healthy and sickle cells. The unfavorable microenvironment present in sickle wounds seems to exert a significant influence on these fibroblasts because once the microenvironment was corrected with the appropriate culture media, the cells had a doubling rate and migration rate similar to normal fibroblasts in vitro.

Thesis (Ph. D.)--University of Missouri-Columbia, 2007.<br>The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "August 2007" Includes bibliographical references.

Mesenchymal stem cells (MSCs) are bone marrow derived multipotent cells with the ability to self-renew and differentiate into multiple connective cell lineages. In vivo, MSCs travel from the bone-marrow to the inflammatory sites and actively participate in remodeling and regeneration process under the influence of soluble growth factors. Due to these inherent properties, MSCs have emerged as an ideal candidate for diverse regenerative therapeutic applications. The development of MSC-based therapies requires in vitro expansion of MSCs; however, MSC expansion results in phenotypical changes that have limited its efficacy upon reintroduction in vivo. In order to increase the efficacy of MSC-based therapeutics, it is critical for us to improve the current understanding of MSC interactions with its niche specific factors and explore new methods to enhance MSC function in vivo. We used tumor conditioned media, which contains soluble factors secreted by tumor cells in culture (TCM), and inflammatory niche-specific soluble factors, such as platelet derived growth factor (PDGF) and transforming growth factor-β1 (TGF-β1), to characterize the mechanical response of MSCs. The intracellular mechanical properties of MSCs were dramatically altered in response to soluble factors and MSCs displayed cytosolic stiffening in response to TCM and TGF-β1. Although PDGF treated cells did not elicit any mechanical response, blocking PDGF signaling with a small molecule inhibitor reversed the stiffening response in TGF-β1 treated cells, indicating crosstalk between these two pathways is essential in TGF-β1 mediated cell stiffening. Furthermore, a genome-wide microarray analysis revealed TGF-β1 dependent regulation of cytoskeletal actin-binding protein (ABP) genes. Actin crosslinking and bundling protein genes, which regulate cytosolic rheology through changes in semiflexible actin polymer meshworks, were upregulated with TGF-β1 treatment. Since TGF-β1 treatment profoundly altered the MSC phenotype after relatively short exposure times, we sought to understand if pretreated cells could sustain these enhanced characteristics leading to higher efficacy in vivo. We found that MSCs pretreated with TGF-β1 displayed enhanced adhesive properties while maintaining the expression profile of surface adhesion molecules even after removal of stimulus. Additionally, pretreated MSCs exposed to lineage specific induction media, demonstrated superior differentiation potential along multiple lineages. Based on the large number of sustained changes, TGF-β1 pretreated cells were used to treat full thickness skin wounds for in vivo wound healing model to determine their therapeutic efficacy. TGF-β1 pretreated MSCs increased wound closure rate and displayed enhanced migration of MSCs towards the center of the wound compared to the control cells. In conclusion, soluble factor pretreated MSCs with altered mechanical properties displayed significantly improved cell functions leading to highly efficient tissue regeneration in vivo. Mechanical priming of MSCs with niche specific factors prior to transplantation can become a viable strategy to maximize their therapeutic potential.

"Recent data show that 7.6 million Americans have survived a myocardial infarction (MI), and 5.1 million Americans suffer from severe heart failure. Stem cell therapy has the potential to improve cardiac function after MI. Two promising cells for cardiovascular regeneration therapies include human mesenchymal stem cells (hMSCs) and pluripotent stem cell derived cardiomyocytes (hPS-CM) each with their own unique method for improving cardiac function post-infarct. However, a limiting factor to cell therapies is that the methods currently used to deliver cells to the myocardium, including intramyocardial injection (considered the gold standard), suffer from low retention rates. To promote localization of delivered cells to the infarct and increase retention rates, our lab has developed a fibrin biological suture that can deliver human mesenchymal stem cells (hMSCs) with an efficiency of 64% compared to just 11% with intramyocardial injection in the normal rat heart. In this dissertation we sought to examine the functionality of hMSC and hPS-CM seeded sutures and their impact on cardiovascular regeneration applications. We began by delivering hMSC seeded fibrin sutures to an infarcted rat heart and found that the sutures are an effective method to deliver cells to the infarcted myocardium and demonstrated a trend towards improved regional mechanical function in the infarct region over infarct alone. Next, we transitioned to using hPS-CM and developed methods to seed the sutures, as well as a method to measure hPS-CM contractility with high spatial and temporal resolution, while concurrently capturing calcium transients. This technique allowed us to examine the contractile behavior in terms of contractile strain and conduction velocity of hPS-CM seeded on fibrin microthreads over 21 days in culture. We found that the fibrin microthread is a suitable scaffold for hPS-CM attachment and contraction and that extended culture promotes cell alignment along the length of the suture as well as improvements in contractile function in terms of increases in contractile strain and conduction velocity. Finally, we delivered the hPS-CM seeded microthreads to an uninjured rat heart and found a delivery efficiency of 67%. Overall, we further demonstrated the technology of the fibrin suture to deliver cells to an infarct as well as the ability to support the attachment, contraction and delivery of hPS-CM to cardiac tissue. "

Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory disease characterized by persistent airflow limitation that is not fully reversible and is usually caused by cigarette smoke (CS). The disease is predicted to be the fourth leading cause of death by 2030, but none of the currently available treatments can alleviate the progressive decline in lung function. Mesenchymal stem cells (MSCs) are fibroblast-like multipotent stem cells that can be isolated from various tissues such as bone marrow (BM-MSCs). Despite numerous reports of their efficacy in COPD-related pre-clinical models, BM-MSCs have not demonstrated efficacy in a clinical trial of COPD, highlighting the need for improved MSC-based therapy. The in vitro derivation of MSCs from induced pluripotent stem cells (iPSCs) has provided a new source of MSCs. Compared to BM-MSCs, iPSC-derived MSCs (iPSC-MSCs) are a more abundant source, have a higher expanding capacity and are possibly not subject to the ageing-associated dysfunction seen in BM-MSCs. In this study I determined the effects of human iPSC-MSCs in a rat COPD model using BM-MSCs as comparison. Rats were exposed to CS for 1 hr/day for 56 days. iPSC-MSCs or BM-MSCs were administrated at days 29 and 43. iPSC-MSCs demonstrated superior effects over BM-MSCs in attenuating CS-induced lung airspace enlargement, fibrosis, inflammation and apoptosis. In a mouse model of ozone-induced lung damage, intravenous administration of iPSC-MSCs 24 hours before ozone exposure for 3 hours alleviated airway hyper-responsiveness, inflammation and apoptosis in the lung. There is increasing evidence demonstrating that mitochondrial dysfunction may play an important role in COPD pathogenesis, indicating mitochondria as a potential therapeutic target. Meanwhile, mitochondrial transfer from MSCs to injured airway cells has been reported as a novel mechanism of action for MSCs. In this study mitochondrial transfer from iPSC-MSCs to the airway epithelium of CS-exposed rats was detected. iPSC-MSCs also transferred mitochondria to bronchial epithelial BEAS-2B cells and primary airway smooth muscle cell (ASMCs) in vitro in a direct co-culture system, an effect that was enhanced by CS medium (CSM). Direct co-culture with iPSC-MSCs alleviated CSM-induced ATP deprivation in BEAS-2B cells, as well as CSM-induced mitochondrial reactive oxygen species (ROS), apoptosis and reduction of mitochondrial membrane potential (ΔΨm) in ASMCs. Administration of iPSC-MSCs also prevented ozone-induced mitochondrial ROS and ΔΨm reduction in mouse lungs. The paracrine effects of iPSC-MSCs were also investigated. iPSC-MSC-derived conditioned medium (iPSC-MSCs-CdM) protected BEAS2-B cells from CSM-induced apoptosis. The effect was reduced by depleting stem cell factor (SCF) from iPSC-MSCs-CdM. However, both iPSC-MSCs-CdM and trans-well inserts containing iPSC-MSCs were only able to alleviate CSM-induced mitochondrial ROS, but not ΔΨm reduction and apoptosis, in ASMCs. I demonstrated the capacity of iPSC-MSCs to alleviate oxidative stress-induced COPD phenotype in vivo. Mitochondrial transfer from iPSC-MSCs was able to alleviate oxidative stress-induced mitochondrial dysfunction and apoptosis in target cells. The full capacity of iPSC-MSCs to achieve these effects may rely on a combination of cell-cell contact and release of paracrine factors. These findings define iPSC-MSCs as a promising candidate for the development of MSCs-based therapy of COPD.

The incidence and long-term effects of traumatic brain injury (TBI) make it a major healthcare and socioeconomic concern. Cell transplantation may be an alternative therapy option to target prolonged neurological deficits; however, safety and efficacy of the cells must be determined. Bone marrow-derived mesenchymal stem cells (MSCs) are an accessible and expandable cell source which circumvent the many of the accessibility and ethical concerns associated with fetal tissues. A major impediment to recent clinical trials for cell therapies in the central nervous system has been the lack of consistency in functional recovery where some patients receive great benefits while others experience little, if any, effect (Watts and Dunnett 2000; Lindvall and Bjorklund 2004). There are many possible explanations for this patient-to-patient variability including genetic and environmental factors, surgical techniques, and donor cell variability. Of these, the most easily addressable is to increase the reproducibility of donor cells by standardizing the isolation and pre-transplantation protocols, which is the central goal of this dissertation. First, we present an animal study in which transplants of MSCs and neural stem cells (NSCs) were given to brain-injured mice, however, the efficacy of the treatment had high variability between individual subjects. Second, we designed a method to produce MSC-spheres and characterize them in vitro. Last, we employed an in vitro 3-D culture testbed as a pre-transplant injury model to assess the effects of the MSC-spheres on neural cells. The electrophysiological function of the uninjured testbed was assessed, and then MSC-spheres were injected into the testbed and apoptosis of the host cells were measured. The results of this study contribute to our understanding of how extracellular context may influence MSC-spheres and develop MSCs as a donor cell source for transplantation.

Mesenchymal stromal cells (MSCs) are thought to be useful in wound healing applications, but are not available from human sources in large enough quantities to be clinically viable. To isolate and expand placental derived human MSCs, this project details the design and scale up of a novel packed bed bioreactor that decouples the oxygen supply from the bulk medium flow by allowing oxygen to diffuse through the reactor wall. The design was further optimised through finite element modelling to expand up to 3x10^8 MSCs. In addition, modelling provided insight into the reduced cellular growth rate that was observed in the bioreactor compared to traditional tissue culture methods, highlighting the importance of the lactate removal and initial cell distribution on cell growth rate.

Reduction of cardiac myocyte loss and repair of the vasculature post myocardial infarction are important therapeutic goals because the potential for intrinsic repair is limited. Preclinical and limited clinical data support the possibility that bone marrow-derived mesenchymal stem cells may be a suitable cell type for cellular therapy. The goal of this research was to determine the effectiveness of using MSCs from aged mice in cellular therapy for the treatment of AMI. The central hypothesis for this research was that therapeutic potential of mesenchymal stem cells decreases with age. This research utilized global gene expression analysis to investigate molecular differences in MSCs harvested from three different age groups of mice. Microarray analysis was performed to investigate changes in gene expression with respect to aging. Furthermore, both in vitro and in vivo experiments were completed to analyze the functional and molecular characteristics of the MSCs. The data identified age-related defects in mouse MSCs as well as determined the molecular basis for these deficiencies. This study indicates that MSCs from 26m mice are severely deficient in the induction of angiogenesis and cardiac repair due to defective paracrine factor secretion caused by decreased expression of growth factor/cytokine genes. Hypoxia attenuates the deficiency in the aged mice, whereas in young mice low oxygen promotes secretion of paracrine growth factors. It was determined a dysfunction in HIF-1 alpha signaling was present in MSCs from 26m mice and is regulated by the PI3K/Akt signaling in MSCs. Furthermore, two novel and important and novel aspects of this study were the discovery that cell cycle regulation gene expression decreases with age and MSCs have increased insulin resistance with age. Increased insulin resistance in this cell type with aging is likely to have profound effects on the clinical outcomes of using these cells therapeutically. Likewise, loss of cell cycle regulation during proliferation could also lead to undesirable clinical effects. Gaining insight to the repair potential of these cells with respect to age will help to better define future trials of autologous stem cells not only for heart disease but for all of the many applications proposed for these cells.

<p>Background: Stem cell therapy can facilitate cardiac repair after healed myocardial infarction but the optimal cell type remains uncertain. <p>Aims: To investigate the pathophysiology of heart failure in a canine model of healed myocardial infarction and to compare the efficacy and the safety of autologous bone marrow mononuclear cell (BMNC) transfer and mesenchymal stem cell (MSC) transfer in this model. It was a blind, randomized and placebo control study.<p>Methods: Eleven weeks after coronary ligation, 24 dogs received intramyocardial injections of BMNC, MSC or Placebo (n = 8 per groups). Echocardiography, conductance method, magnetic resonance imaging, serum neurohormones, holter monitoring, macromorphometry, histology and real time quantitative polymerase chain reaction were used to assess cardiac performance, safety and remodelling in healthy animals, before cell transplantation and up to 16 weeks’ follow-up. <p>Results: The model was characterized by decreased left ventricular end-systolic elastance and ventricular-arterial uncoupling without alteration of compliance. <p>Four months after BMNC transfer, the regional systolic function measured at echocardiographic showed a sustained improvement. This improvement was associated with an improved left ventricular end-systolic elastance and a decreased infarct size. Although the left ventricular ejection fraction stayed unchanged, the serum level of N-terminal B-type natriuretic propeptide level decreased. Mononuclear cell transfer was also associated with increased left ventricular relative wall area, increased vascular density, intramyocardial vascular remodelling and upregulation of angiogenic factors gene expression. Mesenchymal stem cell transfer only improved lately and moderately the regional systolic function, without improvement of cardiac contractility or decreased infarct size. <p>Conclusions: In a canine model of chronic myocardial infarction, BMNC transfer is superior to MSC transfer in improvement of cardiac contractility and regional systolic function, and to reduce the infarct size and plasma N-terminal B-type natriuretic propeptide level. Functional improvement is associated with a favourable angiogenic environment and neovascularization. <p><br>Doctorat en Sciences biomédicales et pharmaceutiques<br>info:eu-repo/semantics/nonPublished

Tissue engineering is an emerging interdisciplinary field, born with the purpose to provide an alternative solution for the regeneration of lesioned or lost tissues, combining cells, biocompatible scaffolds and bioactive factors. The cells for this approach should be non-immunoreactive and non-tumorigenic. Moreover, they should be available in large amount and possess, or be able to acquire, a specific protein expression pattern similar to that of the damaged tissue and/or act as a pool of trophic factors for resident cells. All these reasons, make mesenchymal stem cells (MSCs) good candidates for applications in regenerative medicine. Although bone marrow is still the most common source of MSCs, these cells could be harvested from all vascularised tissues, and, interestingly, from tissues that are normally discarded, such as fat, placenta or umbilical cord. One of the most convenient source of MSCs, is unequivocally, the adipose tissue due to the easily accessible anatomical location and the abundance of subcutaneous adipose tissue. Adipose-derived stem cells (ASCs) are similar to MSCs isolated from bone marrow in morphology, immunophenotype, and differentiation ability, and own interesting features such as immunoregolatory and anti-inflammatory properties. In the recent years, many strategies for the cure of musculoskeletal tissues critical lesions, mainly in orthopaedic, oral and maxillo-facial surgery, have been under investigations. In this contest, the regeneration of structures including different tissues, such as the periodontium and the osteochondral unit, are particularly challenging. Periodontal regeneration is especially demanding, as it requires regeneration of three quite diverse and unique tissues such as the alveolar bone, the periodontal ligament and the cementum, that have to interface with each other to restore their complex structure. Since the promising results obtained with ASCs in preclinical studies of periodontal diseases arouse the curiosity of maxillofacial and dental surgeons, we decided to identify a novel source of ASCs, i.e, the buccal fat pad, convenient for these specialists. For this purpose, we studied human adipose derived-stem cells from buccal fat pad (BFP-ASCs), comparing them with cells from the subcutaneous adipose tissue (SC-ASCs) of the same donor (n=2). In parallel, considering the need for preclinical studies in which the effect of allogenic cells should be tested, and swine as an accepted animal model in tissue engineering applications, we also characterized porcine cells (n=6). With preclinical and clinical application prospective, we also investigated ASC interactions with oral tissues, natural and synthetic scaffolds and Amelogenin, an oral bioactive molecule. First of all, we showed that it is feasible to isolate ASCs even starting from very limited amounts of tissue (0,5 ml) and that the cellular yield is influenced by species, but not by the site of harvesting (1.1x105±1.4x104 human BFP-ASCs/ml and 1.15x105±7.1x103 human SC-ASCs/ml; 3.0x104±9.3x103 porcine BFP-ASCs/ml and 5.5x104±3.3x104 porcine SC-ASCs/ml). Despite the lower yield, the pASCs great proliferation rate allows to obtain high number of cells (potentially, 108 - 109) after few (3, 4) passages in culture. After the isolation, a great amount of cells deriving from all the tissues, adhered to cell culture plates showing the MSC fibroblast like morphology, with only mild shape differences constituted by the higher elongation and dimension of human SC-ASCs. Moreover, all the cells are easily expandable and showed good clonogenic ability at early passages. Cells of the same species, from both the harvesting site, displayed the same surface markers profile, that, in particular for human ASCs, was the typical one of hMSC (CD90+, CD105+, CD73+, CD14-, CD31-, and CD34-). Human and porcine BFP-ASCs, as SC-ASCs, are multipotent; indeed, when induced towards osteogenic and adipogenic lineages, they up-regulated significantly ALP activity, collagen and calcified extracellular matrix deposition and lipid vacuoles productions, respectively, already after 14 days of differentiation in vitro. Next, since cell/scaffold interaction is fundamental for the outcome of a tissue engineering approach, in sight of a preclinical study, we combined porcine BFP and SC-ASCs to both clinical grade (titanium) and innovative [silicon carbide–plasma-enhanced chemical vapor deposition (SIC-PECVD)] biomaterials, and studied cell adhesion and their differentiation ability. All the cells nicely grew on both scaffolds and, when osteoinduced, significantly increased the amount of calcified ECM compared to control cells; interestingly, titanium is osteoinductive even per se on pASCs (+284% and +91 for BFP- and SC-ASCs). Considering the importance of cell interaction with tissue of the lesion site, and with materials commonly used during surgical practices, we studied human BFP- and SC-ASC adherence to several supports. SEM analysis confirmed that both cell type nicely stick on alveolar bone, periodontal ligament, collagen membrane and polyglycolic acid filaments. Finally, we found that amelogenin, the most abundant enamel matrix protein seems to be an early osteoinductive factor for BFP-hASCs, whereas this effect is not manifested for SC-hASCs. For future cellular therapy, and since the use of FBS pose the risk of xenogenic contaminations leading to immunological complications during transplant, we tested cells growth in the presence of autologous supplements. Interestingly, both hASCs adapted rapidly to human serum, increasing their proliferation rates compared to standard culture condition, while porcine autologous or heterologous sera, did not improve pASC growth. In conclusion, we identified a cell population derived from a tissue easily available to dentists and maxillofacial surgeons, whose multipotent features and interaction with clinical grade scaffolds make proper candidate for future uses in tissue engineering approaches of periodontal diseases. In parallel, part of my PhD project was focused on the study of a critical osteochondral defect regeneration performed in a large animal preclinical model. The main obstacles for clinicians in treating this defect arises from the disparity concerning anatomy, composition and, most importantly, rate of healing of the articular cartilage (AC) and the subchondral bone. The key points of our study are the use of an innovative hydrogel of oligo(polyethylene glycol)fumarate (OPF) to fill the osteochondral defect, and of either porcine, or human ASCs, to create bioconstructs to be implanted in non-immuno-compromised minipigs. In particular, four critical osteochondral defects (diameter 9mm, depth 8mm) were created in the peripheral part of the trochlea of seven animals (defect n=28), and then treated with the different pre-made constructs. Untreated defects and defects filled by just scaffold were included as controls. No side-effects have been observed during the six-moths follow-up. At the end of this period, animals were sacrificed and knees explanted. Gross appearance analyses showed quite satisfactory filling of all the lesions, with the exception of one animal, whose joint appeared infected and not healed. MRI analyses revealed that in all the scaffold treated groups an overall improvement of the tissue quality at the osteochondral lesion site, was induced. More accurate evaluations (histological and immunohistochemistry analyses) revealed that some important tissue features were significantly improved by the association of OPF and ASCs. Indeed, regarding the subchondral bone, in all the OPF+ASCs groups, a mature bone appeared, with higher deposition of collagen type I compared to untreated or unseeded OPF groups. Moreover, the use of ASCs associated to scaffolds induced an improvement in newly formed cartilage features such as collagen type II deposition, and histological scores associated to these samples indicated a significant increase in matrix staining, tissue morphology and formation of tidemark, together with a reduction in vascularisation (a positive aspect in cartilage) compared to unseeded scaffolds. However, the histology indicated that in all the samples cartilage regeneration was still immature, most likely due to the limited time of follow up and/or the insufficient stimuli for cartilage complete regeneration. Despite this, biomechanical tests revealed that the neo-cartilage found in the cell-loaded scaffold groups possessed poroelastic behaviour, as well as indentation modulus and creep curves comparable to native cartilage. This important result suggest that the ASC presence at the lesion site, is able to enhance newly formed cartilage functionality. In conclusion, this in vivo study provide the evidence that both porcine and human adipose-derived stem cells associated to OPF hydrogel improve osteochondral defect regeneration, even though, at the moment, we are not able to define if the implanted ASCs are responsible per se of the new tissue formation or if they help spontaneous regeneration process by paracrine actions.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting primarily motor neurons (MNs) but involving also non-neuronal cells. Nowadays, it is well recognised that astrocytes, microglia and oligodendrocytes play a central role in disease onset and progression. In particular, astrocytes acquire a toxic phenotype characterized by an abnormal proliferation and by the release of neurotoxic factors, including pro-inflammatory cytokines (Lee et al., 2016). We have previously shown that the intravenous administration of mesenchymal stem cells (MSCs) in SOD1G93A mice prolonged survival, ameliorated motor skills and reduced gliosis and inflammation in spinal cord. These beneficial effects were not associated with MSC differentiation, being possibly mediated through paracrine mechanisms. We hypothesized that MSC-derived exosomes and exosome-shuttled miRNAs could mediate these positive effects. To verify our hypothesis we tested here the effects of exosomes derived from INF-activated MSCs on cultured astrocytes prepared from the spinal cord of 120 day-old late-symptomatic SOD1G93A mice. The phenotype of SOD1G93A astrocytes and the efficacy of the exosome treatment were characterized by Western blotting, confocal microscopy and ELISA immunoassay. Vimentin, GFAP and S100β, astrogliosis markers, were increased in astrocytes from 120 days-old SOD1G93A mice vs. age-matched WT astrocytes and their expression was reduced after exposure to exosomes. Nrf2, a booster of the response to oxidative stress, was decreased in SOD1G93A astrocytes vs. age-matched WT astrocytes. Exosome treatment normalized Nrf2 down-regulation both in the cytoplasm and nucleus. The quantification of TNF-α,IL-1β, IL-6 and CCL2 expression and release showed that these four pro-inflammatory factors were more expressed in and more efficiently released from SOD1G93A astrocytes and that the exposure to exosomes resulted in a significant decrease of their over-expression and release. Conversely, the anti-inflammatory cytokine IL-10 was decreased in SOD1G93A astrocytes and its expression was normalized after exposure to exosomes. Also NLRP3 expression, a marker of the multiprotein oligomer inflammasome, was increased in SOD1G93A astrocytes and the increase was reversed by exosomes. The amelioration of SOD1G93A astrocyte phenotype had a positive impact on MN viability in astrocyte-MN co-cultures. We observed a constant decrease of MN survival during time, both in control and exosome-treated co-cultures; however, the viability of MNs seeded on exosome-treated SOD1G93A astrocytes was always significantly higher when compared to co-cultures with untreated astrocytes. Exosome cargo was analysed for miRNAs and potential mediators of exosome activity were identified. The selected miRNAs showed a significant efficacy to reduce GFAP, IL-1β and TNF-α expression. Computational analysis highlighted their possible involvement in the modulation of NFκB and MAPK pathway activation, affecting numerous kinases and transcription factors involved in the regulation of these inflammatory signalling pathways. qPCR analysis confirmed the ability of these four miRNAs to reduce MAPK11 expression, regulating TNF-α synthesis. Unfortunately, the other selected targets were not affect by mimic transfection. Finally, we translated this study to human astrocytes derived from healthy donors and ALS patients carrying A4V-SOD1 mutation. Human ALS astrocytes were treated with exosomes derived from human MSCs, activated with INF. We observed only a slight amelioration of ALS astrocyte phenotype after the exosome treatment. Remarkably, analysis of MN viability showed an increased MN number in co-cultures with exosome-treated astrocytes compared to those co-cultured with untreated astrocytes. These results indicate that exosomes and exosome-shuttled miRNAs can reduce astrocyte reactivity and that this effect has a positive impact on MN viability. The in-vitro exosome activity, both in mouse and human models, paves the way to translational preclinical in-vivo treatments in SOD1G93A mice.

Mesenchymal stem cells have been recently investigated for their potential use in regenerative medicine. It has been suggested that there may be a stem cell population within both umbilical cord matrix and amniotic fluid. However, little knowledge exists about the characteristics of these progenitor cells within these sources in the equine species. This study wanted to investigate an alternative and non-invasive stem cell source for the equine tissue engineering and to learn more about the properties of these cells for future cell banking. Moreover, population of adult stem cells were recently identified in human and lab animal tendons, but no detailed investigations have been made in the equine species. The aim of the study was to compare in vitro the stemness features of horse progenitor cells derived from bone marrow (BM-MSCs), amniotic fluid (AF-MSCs), umbilical cord matrix (EUC-MSCs) and tendon derived progenitor cells (TSPCs). This work defines a protocol for extraction, isolation, expansion and characterization of mesenchymal stem cells from equine bone marrow, amniotic fluid, umbilical cord matrix (Wharton’s jelly) and tendon. Their localization into the tissues from which they were extracted, was reported. During the cell culture, cell expansion, CFU-F assay, doubling time, plasticity and immunophenotype were analyzed. Furthermore, a specific cell labeling was realized that could be used for non-invasive magnetic resonance cell tracking through endosomal incorporation of superparamagnetic iron oxide particles through the in vitro evaluation of the efficiency of this labeling method. In the next future, this technique should facilitate translation of the approach into clinical trials, in particular to track cells in vivo after transplantation and to follow their homing, viability and repair potential during time. The mesenchymal stem cells were grown on control medium, such as DMEM with the addition of basic FGF. Our results pointed out that these cells performed similarly in terms of CFU-F formation and growth kinetic. The immunocytochemical and RT-PCR analysis of MSCs isolated from all tissues showed the presence of antigens such as CD44, CD105, CD29, Oct-4, c-Myc, SSEA4 and HLA-ABC, whereas they were negative for CD34 and HLA-DR. These cells, differentiated into osteogenic, adipogenic, chondrogenic and tenogenic lineages confirming the nature of mesenchymal stem cells. These findings suggest that AF-MSCs appeared to be a readily obtainable and high proliferative cell line that may represent a good model system for stem cell biology. EUC-MSCs need to be further investigated regarding their particular behavior in vitro represented by spheroid formation. Equine TSPCs have high clonogenic properties and proliferating potential, they express stem cell markers and have the capability to be multipotent as well as BM-MSCs. These findings suggest that TSPCs may represent a good model for stem cell biology and could be useful for future tendon regenerative medicine investigations. These data could be useful for optimization of horse’s mesenchymal stem cell isolation and expansion protocol that could be used in the experimental clinic application for tissue regeneration because to their biological properties. The MSCs isolated from equine extra-embryonic tissues and tendons, showed characteristics of stem cells and therefore can be regarded as good candidates to be used in regenerative medicine, with special reference to orthopaedic diseases of horses. Furthermore, future investigations in vivo are useful to evaluate the efficacy of cell labeling with contrast agents for magnetic resonance with particular attention to the muscle-skeletal tissues.

La malattia di Alzheimer (AD), la forma più comune di demenza senile, è caratterizzata da una progressiva degenerazione del sistema nervoso centrale (SNC) che conduce ad un declino della funzione cognitiva e a perdita di memoria. I tratti caratteristici dell’AD includono placche extracellulari di beta amiloide (Abeta), grovigli neurofibrillari (NFTs), neuriti distrofici, morte neuronale e attivazione gliale. In base a quanto affermato dall’ “Ipotesi della cascata della Abeta” - la teoria più corroborata nell’ambito degli studi sull’AD negli ultimi decenni – la neuroinfiammazione veniva supposta come un processo caratterizzante solo le fasi tardive della malattia, e considerata come una mera risposta secondaria agli eventi patologici indotti dall’ A beta. Recentemente, nuovi studi preclinici, epidemiologici e genetici hanno dimostrato un coinvolgimento del sistema immunitario molto più precoce, comportando una rivalutazione del ruolo della principale cellula dell’immunità innata del cervello: la microglia. Dal momento che non vi è cura per l’AD, questi studi hanno motivato lo sviluppo di strategie terapeutiche con l’obiettivo di rallentare i processi degenerativi attraverso un’azione sulle cellule microgliali, in virtù del loro ruolo nell’orchestrare la neuroinfiammazione nelle malattie neurodegenerative. Le cellule staminali mesenchimali (MSC) sono cellule staminali multipotenti che negli ultimi decenni sono emerse per la loro capacità di migliorare il decorso patologico in diversi modelli di malattie neurodegenerative, grazie alla loro attività paracrina, che in gran parte dipende dal rilascio di vescicole extracellulari (EV). Le EV - strutture lipidiche importanti per la comunicazione intercellulare - si sono dimostrate essere mediatori di molti effetti benefici indotti dalle MSC, come l’immunomodulazione. Nello specifico, il concetto che le intrinseche abilità immunoregolatorie delle MSC sono fortemente potenziate dall’ambiente in cui si trovano, ha portato gli scienziati a ridisegnare e ad ottimizzare le condizioni di coltura (precondizionamento, P) di queste cellule, per potenziare le proprietà anti-infiammatorie delle stesse e delle EV da loro rilasciate. L’obiettivo di questo studio è quello di analizzare la capacità delle EV rilasciate da MSC umane precondizionate (p-MSC-EV) di immunoregolare la funzione della microglia in contesti in vitro ed in vivo di AD. Negli studi in vitro abbiamo testato due differenti protocolli di P al fine di isolare un fenotipo altamente immunomodulante di MSC. Le EV derivate da MSC precondizionate con citochine si sono dimostrate in grado di polarizzare il fenotipo microgliale, precedentemente indotto tramite stimoli infiammatori in uno stato funzionale citotossico, verso un fenotipo anti-infiammatorio. Quando abbiamo indagato il potenziale immunomodulatorio delle EV in un modello murino di AD (3xTg AD), abbiamo osservato un forte effetto attenuante sull’attivazione microgliale e sulla prevenzione della perdita di spine dendritiche nelll’ippocampo e nelle cortecce entorinali e prefrontali degli animali trattati con le EV rispetto ai controlli. Questo suggerisce che un effetto neuroprotettivo potrebbe essere ottenuto tramite la modulazione dell’attivazione microgliale in questo modello. Per studiare più selettivamente l’effetto delle EV sulla microglia, stiamo sfruttando un modello di sanguisuga (Hirudo verbana), grazie alla semplice e ben caratterizzata struttura del suo SNC (studio preliminare). In conclusione, i nostri risultati indicano che le p-MSC-EV potrebbero rappresentare uno strumento terapeutico nell’AD, attraverso la riduzione dell’attivazione microgliale e contrastando la perdita di spine dendritiche, tratti neuropatologici tipicamente presenti sia nei modelli transgenici di AD che nei pazienti.<br>Alzheimer’s disease (AD), the most common form of age-related dementia, is characterized by a progressive degeneration of the central nervous system (CNS) that leads to a gradual decline of cognitive functions and memory loss. Neuropathological hallmarks of AD include extracellular beta-amyloid plaques, derived from the altered processing of amyloid precursor protein (APP), neurofibrillary tangles (NFTs, intraneuronal aggregates of hyperphosphorylated and misfolded tau), dystrophic neurites, neuronal loss and glial activation. According to the “Amyloid cascade hypothesis” - the most validated theory in the field of AD for the past few decades - neuroinflammation was assumed to occur only in the late stages of the disease, being considered as a mere secondary response to Abeta-induced pathophysiological events. Recently, new preclinical, epidemiological and genetic studies have demonstrated a much earlier involvement of immune system-related actions, leading to a reassessment of the role of the principal innate immune entities of the brain, that are microglia cells. Since there is still no cure for AD, these studies motivated the design of innovative therapeutic strategies aiming at slowing down degenerative processes by targeting microglia cells, in virtue of their main recognized role in orchestrating neuroinflammatory process in neurodegenerative diseases, including AD. Mesenchymal stem cells (MSCs) are adult multipotent stem cells that over the last decades have been demonstrated to show improvement in various model of neurodegenerative pathologies, thanks to their paracrine ability that is largely dependent on the secretion of extracellular vesicles (EVs). EVs - membrane bound entities known to be important players in intercellular communication - have emerged as mediator of multiple MSC beneficial effects, including immunomodulation. Particularly, the concept that intrinsic immunomoregulatory abilities of MSCs are strongly influenced and strengthened by the environment, has led the scientists to design and optimize culture conditions (preconditioning) in order to enhance the anti-inflammatory properties of these cells and of their derived EVs. The aim of this study is to investigate the ability of preconditioned human bone marrow MSC-derived EVs (p-MSC-EVs) to immunoregulate microglia function in vitro and in vivo AD context. In in vitro studies we tested two different preconditioning protocols in order to isolate a highly immunomodulant MSC phenotype. Cytokine p-MSC-EVs were shown to switch microglia, previously polarized through inflammatory challenge to the M1 cytotoxic state, toward an anti-inflammatory phenotype. When we delved into the EV immunomodulatory potential in a triple transgenic AD (3xTg AD) mouse model, we observed a strong dampening effect on microglia activation and prevention of dendritic spine loss in hippocampus, entorhinal and prefrontal cortices of EV treated animals compared to controls. This suggests that an EV-dependent neuroprotective effect could be achieved through the modulation of microglia activation in this model. In order to more selectively study the effect of EVs on microglia, we are taking advantage of a leech animal model (Hirudo verbana), because of its simple and well-characterized CNS structure (preliminary study). In conclusion, our results indicate that p-MSC-EVs may represent a possible therapeutic tool in AD by reducing chronic microglia activation and counteracting dendritic spine loss, which are traits typically observed both in AD transgenic animal models and patients.

Osteophytes form through the process of chondroid metamorphosis of fibrous tissue followed by endochondral ossification. Osteophytes have been found to consist of three different mesenchymal tissue regions including endochondral bone formation within cartilage residues, intra-membranous bone formation within fibrous tissue and bone formation within bone marrow spaces. All these features provide evidence of mesenchymal stem cells (MSC) involvement in osteophyte formation; nevertheless, it remains to be characterised. MSC from numerous mesenchymal tissues have been isolated but bone marrow remains the “ideal” due to the ease of ex vivo expansion and multilineage potential. However, the bone marrow stroma has a relatively low number of MSC, something that necessitates the need for long-term culture and extensive population doublings in order to obtain a sufficient number of cells for therapeutic applications. MSC in vitro have limited proliferative capacity and extensive passaging compromises differentiation potential. To overcome this barrier, tissue derived MSC are of strong interest for extensive study and characterisation, with a focus on their potential application in therapeutic tissue regeneration. To date, no MSC type cell has been isolated from osteophyte tissue, despite this tissue exhibiting all the hallmark features of a regenerative tissue. Therefore, this study aimed to isolate and characterise cells from osteophyte tissues in relation to their phenotype, differentiation potential, immuno-modulatory properties, proliferation, cellular ageing, longevity and chondrogenesis in in vitro defect model in comparison to patient matched bone marrow stromal cells (bMSC). Osteophyte derived cells were isolated from osteophyte tissue samples collected during knee replacement surgery. These cells were characterised by the expression of cell surface antigens, differentiation potential into mesenchymal lineages, growth kinetics and modulation of allo-immune responses. Multipotential stem cells were identified from all osteophyte samples namely osteophyte derived mesenchymal stem cells (oMSC). Extensively expanded cell cultures (passage 4 and 9 respectively) were used to confirm cytogenetic stability and study signs of cellular aging, telomere length and telomerase activity. Cultured cells at passage 4 were used to determine 84 pathway focused stem cell related gene expression profile. Micro mass pellets were cultured in chondrogenic differentiation media for 21 days for phenotypic and chondrogenic related gene expression. Secondly, cell pellets differentiated overnight were placed into articular cartilage defects and cultured for further 21 days in control medium and chondrogenic medium to study chondrogenesis and cell behaviour. The surface antigen expression of oMSC was consistent with that of mesenchymal stem cells, such as lacking the haematopoietic and common leukocyte markers (CD34, CD45) while expressing those related to adhesion (CD29, CD166, CD44) and stem cells (CD90, CD105, CD73). The proliferation capacity of oMSC in culture was superior to that of bMSC, and they readily differentiated into tissues of the mesenchymal lineages. oMSC also demonstrated the ability to suppress allogeneic T-cell proliferation, which was associated with the expression of tryptophan degrading enzyme indoleamine 2,3 dioxygenase (IDO). Cellular aging was more prominent in late passage bMSC than in oMSC. oMSC had longer telomere length in late passages compared with bMSC, although there was no significant difference in telomere lengths in the early passages in either cell type. Telomerase activity was detectable only in early passage oMSC and not in bMSC. In osteophyte tissues telomerase positive cells were found to be located peri vascularly and were Stro-1 positive. Eighty-four pathway-focused genes were investigated and only five genes (APC, CCND2, GJB2, NCAM and BMP2) were differentially expressed between bMSC and oMSC. Chondrogenically induced micro mass pellets of oMSC showed higher staining intensity for proteoglycans, aggrecan and collagen II. Differential expression of chondrogenic related genes showed up regulation of Aggrecan and Sox 9 in oMSC and collagen II in bMSC. The in vitro defect models of oMSC in control medium showed rounded and aggregated cells staining positively for proteoglycan and presence of some extracellular matrix. In contrast, defects with bMSC showed fragmentation and loss of cells, fibroblast-like cell morphology staining positively for proteoglycans. For defects maintained in chondrogenic medium, rounded, aggregated and proteoglycan positive cells were found in both oMSC and bMSC cultures. Extracellular matrix and cellular integration into newly formed matrix was evident only in oMSC defects. For analysis of chondrocyte hypertrophy, strong expression of type X collagen could be noticed in the pellet cultures and transplanted bMSC. In summary, this study demonstrated that osteophyte derived cells had similar properties to mesenchymal stem cells in the expression of antigen phenotype, differential potential and suppression of allo-immune response. Furthermore, when compared to bMSC, oMSC maintained a higher proliferative capacity due to a retained level of telomerase activity in vitro, which may account for the relatively longer telomeres delaying growth arrest by replicative senescence compared with bMSC. oMSC behaviour in defects supported chondrogenesis which implies that cells derived from regenerative tissue can be an alternative source of stem cells and have a potential clinical application for therapeutic stem cell based tissue regeneration.

碩士<br>國立臺灣大學<br>臨床動物醫學研究所<br>107<br>In the past two decades, mesenchymal stem cells have been tested in the treatment of various diseases and tissue regeneration. Among these mesenchymal stem cells have gradually gained much attention because of their availability, stability, safety, low-ethical issue and with wide ranges of potential therapeutic applications. Bone marrow-derived mesenchymal stem cells were found and used earlier, so the bulk of early studies used them. However, adipose-derived stem cells are much easier to access and can be acquired in a large quantity. Especially when the euthanasia of stray animals was banned in Taiwan after early 2017, it is even more difficult to obtain bone marrow from dogs. That is why we chose canine adipose-derived mesenchymal stem cells (cAD-MSCs) as the research subjects with potential future applications. Canine keratoconjunctivitis sicca (KCS) is thought to be an immune-mediated disease, therefore canine KCS patients provide a useful model of autoimmune-mediated diseases. Current therapy of canine KCS mainly uses immunosuppressants, but the effectiveness was limited in some patients. In the past three years, some studies showed the results of the use of mesenchymal stem cells in treating canine KCS via periocular injections. However, the periocular injection procedure requires sedation or general anesthesia, and may lead to iatrogenic or incidental injury during the injection process. The aim of this study was to investigate the efficacy of topical allogenic adipose-derived stem cells in clinical patients of canine KCS. First, adipose-derived stem cells were isolated and confirmed for their capability of differentiation and immunomodulatory properties. In addition, preparation methods for eye drops of cAD-MSCs was developed and its optimal preservation was tested. Secondly, canine KCS patients were included and divided into two groups based on history of previous therapy for clinical trial. All patients received topical canine adipose-derived mesenchymal stem cells (cAD-MSCs) therapy weekly for 6 consecutive weeks and complete ophthalmic examinations were performed at baseline and 3rd, 6th, 9th week, respectively. A complete ophthalmic examination included Schirmers tear test-1 (STT-1), tear break-up time(TBUT), fluorescein stain, tear osmolarity measurement by i-PEN and assessments the severity of clinical signs such as mucoid discharge, conjunctival hyperemia, and corneal changes. Based on the results of the clinical trials, the quantity and quality of tears have improved significantly following topical cAD-MSCs treatment. More than half of the patients were found improved in the tear quantity. In particular, 56.5% of the patients that were unresponsive to prior immunosuppressant therapy had an effective increase in tear volume. As relieved from symptoms and uncomfortable, patients’ quality of life was improved following treatment. Based on these results, weekly administration of cAD-MSCs for six consecutive weeks may serve as a good alternative to cyclosporine A or tacrolimus for treatment of canine KCS. Topical cAD-MSCs therapy requires once weekly for six times. It’s much easier for dog owners when compared with traditional treatment, that required frequent administration with two to three times daily. To sum up, topical cAD-MSCs may be beneficial especially in KCS patients with poor owner compliance for frequent daily use of eye drops or those who are unresponsive to immunosuppressant therapy.

博士<br>國立東華大學<br>生命科學系<br>104<br>Diabetes has been one of the top five causes of death in Taiwan for many years. Stem cells provide a promising expectancy for diabetes treatment; however, the efficacy in clinical trial still presents room to improve. Mesenchymal stem cells can be obtained safely and easily from fat tissue, be expanded in vitro with multipotent differentiation ability, and secrete a variety of cytokines. It is therefore that human adipose-derived stem cells (hADSCs) were selected to explore their application for diabetes therapy in this study. In the first part of this study, we aimed to develop a simple method to process adult stem cells from human adipose tissue harvested from the subcutaneous fat of the abdominal wall during gynecologic surgery, and to investigate the characteristics of these cells. In the second part, we examined the efficacy of transplanting hADSCs in diabetic mice. We observed that the middle dosage (4×107 cells/kg) and high dosage of hADSCs (8×107 cells/kg) administered through the tail vein of the diabetic mice would reduce their fasting blood glucose levels. The infiltration of immune cells into pancreatic tissues was reduced and the damage of the islet cells was ameliorated in the hADSC-transplanted mice compared with those of the control group. In addition, pancreatic function was recovered significantly according to biochemical analyses of insulin and glycated hemoglobin levels. Human-specific mitochondria and insulin were observed in pancreatic tissues, indicating that hADSCs differentiated into insulin-producing cells in vivo. Because the expression level of adiponection in brown or white fat cells are relevant to the regulation of glucose metabolism and adiponectin receptors are related to insulin resistance, small molecule compounds that may induce adiponectin gene expression in hADSCs were screened in the last part of this study. The effect of these small molecules on the expression of brown fat-related genes and the reduction on the hyperglycemia in the diabetic mice were examined. The information provided by this research may shed light on the development of future diabetes clinical treatment practice.