Stem cells hold great promise for treating various degenerative diseases. However, the outcomes of preclinical and clinical cell therapy studies are still not close to our expectation. The unsatisfactory outcomes of cell therapy are at least partially due to: 1) insufficient homing of implanted stem cells into target organs and 2) use of heterogeneous cell populations for cell therapy. Therefore, there is a need to develop effective guiding technique for stem cells to migrate to the target organs and to isolate effective stem cell populations. In this project, I developed a microfluidics-based method for selecting chemotactic adipose-derived stem cells (ASCs) to epidermal growth factor (EGF). This method integrates cell patterning, chemotaxis and cell extraction on a single microfluidic device. Post-extraction analysis confirmed the higher chemotactic ability of the extracted cells to EGF. The extracted chemotactic ASCs shows up-regulated surface expression of EGF receptor and its downstream signaling event upon EGF stimulation.

Différents domaines en Chirurgie Plastique et Esthétique ont évolué au cours de ces dernières années et notamment le transfert et injection de Tissu Adipeux autologue. Les premières données cliniques ont été présentées sans véritable appui scientifique. Cette thèse a pour objet de confirmer les données cliniques retrouvées et de permettre d’améliorer cette technique par des preuves scientifiques. Deux questions sont à considérer : 1) Le tissu adipeux a t-il selon son origine anatomique des caractéristiques différentes et certaines régions seraient-elles à privilégier lors d’une autogreffe ? 2) Existe t-il des facteurs de croissances autologues permettant de stimuler la prise de greffe, la prolifération et la différenciation des cellules greffées ? La première partie de ce travail a consisté à analyser le tissu adipeux en fonction du site de prélèvement. A cet effet, nous avons analysé le tissu adipeux provenant de 2 sites anatomiques : le genou et le menton. Le choix topographie découle d’une raison technique et d’une raison théorique. Nos résultats suggèrent que les deux sites étudiés ont des origines embryonnaires différentes, et montrent que ces deux sites présentent une signature moléculaire et une fonctionnalité différentes. La seconde partie porte sur l’effet in vitro de facteurs de croissances autologues humains. Nous avons analysé la prolifération et la différenciation de cellules souches adipogéniques (ASCs) humaines. Nous avons pour cela utilisé des concentrés plaquettaires issus de prélèvements sanguins autologues, et donc utilisables en pratique thérapeutique humaine. Il s’agit du PRF (plaquette riche en fibrine) et du PRP (plaquette riche en plasma). Les résultats de l’étude montrent que la présence de PRF ou de PRP dans le milieu de culture permet une augmentation drastique de la prolifération cellulaire d’environs 4 à 5 fois. A contrario, les résultats obtenus montrent un blocage partiel de la différenciation adipocytaire, quelque soit le moment et le temps de mise en contact. Nous avons alors étudié par quelle voie de blocage la différenciation était induit et avons montré l’implication de la voie du TGFB qui dans ces conditions de culture induit un blocage partiel de la différenciation des ASCs vers un adipocyte mature. Notre étude montre qu’en parallèle à l’effet antiadipogénique, la différenciation s’oriente vers des cellules myofibroblastes-like. Nous avons alors testé l’effet de l’ajout d’un inhibiteur du TGFB (SB431542) dans le milieu de culture et avons observé une relance de la différenciation cellulaire vers la voie adipogénique confirmant que le PRP a un effet antiadipogénique et promyofibroblastique. Par ailleurs, nous avons analysé la composition du PRP utilisé en dosant les taux de facteurs de croissance présents. En conclusion, ce travail permet de confirmer l’augmentation de la prolifération cellulaire des ASCs en présence de PRP autologue. Ceci nous permet donc une transposition clinique immédiate en utilisant en peropératoire l’association PRP et prélèvement de tissu adipeux lors d’un lipofilling. Ce travail permet également de mettre en lumière la probable différence de fonction des adipocytes prélevés en fonction de leur site anatomique d’origine. Ceci a probablement une conséquence sur l’évolution long terme de ces greffes de tissu adipeux en fonction d’une modification pondérale. / Different fields in plastic and aesthetic surgery have evolved in recent years including the transfer and injection of autologous adipose tissue. The first clinical data was presented without any real scientific support. The purpose of this thesis is to improve this technic by adding scientific evidence. There are two questions to consider: 1) Do adipose tissues have different characteristics according to their anatomical origin, and should certain regions be preferred for autografting? 2) Are there autologous growth factors to stimulate engraftment, proliferation and differentiation of grafted cells? The first part of this work consisted in analyzing the adipose tissue according to the fat depot. For this purpose, we analyzed the adipose tissues from 2 anatomical sites: the knee and the chin. The rational of this choice comes from a technical reason and a theoretical reason. Our results suggest that the two sites studied have different embryonic origins, and show that these two sites have a different molecular signature and functionality. The second part of the work deals with the in vitro effect of autologous human growth factors. on the proliferation and differentiation of human adipose stem cells (ASCs). For this purpose, we used platelet concentrates from blood samples, which can therefore be used in human therapeutic practice. It is the PRF (fibrin-rich platelet) and the PRP (platelet-rich plasma). The results of the study show that the presence of PRF or PRP in the culture medium allows a drastic increase in ASC proliferation of about 4 to 5 times. In contrast, the results show a partial inhibition of adipocyte differentiation, whatever the period and the time of contact. We analyzed the composition of different PRP sources and we identified the involvement of the TGFB pathway in the anti adipogenic effects of PRP. In contrast, the antiadipogenic effect was concomitant with the differentiation of ASCs towards myofibroblasts-like cells. In conclusion, this work allows us immediate clinical transposition using PRP during lipofilling. This work also makes it possible to highlight the probable difference in function of the adipocytes taken according to their original anatomical site. This likely has a consequence on the long term evolution of these adipose tissue grafts in case of a weight change.

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

Problems with musculoskeletal system, such as of developmental disorders, fractures or damage of the bone by age, inflammatory or tumor diseases, are still increasing in orthopaedics. Sometimes the bone tissue is not capable to completely regenerate to exert its physiological function in the organism. For this reason, using the bone replacements is necessary and common nowadays. Despite of an intensive research and testing of a wide range of the potential biomaterials and their combinations, the usage of metal materials for construction of the bone implants, still remains to be the gold standard. Ti-6Al-4V alloy is one of the commercialy used metal materials, which is known for the high mechanical and chemical resistance and a good biocompatibility. For a good biological response of the patient's organism for the bone implant, is an ability of osteointegration into the surrounding bone tissue, the key. This ability can be influenced in the case of the metals, by their surface structure. As it is known from earlier studies, the surface topography of the material is very important for the adhesion and proliferation of the bone cells, which are able to discriminate, very sensitively, between various stages of the material surface roughness. For this reason we have focused on studying of an influence...

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. / Dissertation (MSc)--University of Pretoria, 2015. / tm2015 / Immunology / MSc / Unrestricted

The mucosal system, which forms a barrier between internal organ systems and the external environment, is frequently exposed to pathogenic microorganisms. Immunoglobulin A (IgA) antibody secreting cells (ASCs) localize in the lamina propria, and produce IgA antibodies which help protect mucosal tissues. The concept of a common mucosal immune system in which IgA ASCs have the ability to populate any mucosal site has been proposed (1, 2). However, recent research has suggested that IgA ASCs primed in different mucosal sites might possess different sets of chemokine receptors, and therefore migrate specifically to particular mucosal locations (3). In this study, the specific compartmentalization of IgA ASCs in two mouse salivary glands: sublingual gland (SLG), and submandibular gland (SMG) was studied. It was observed that SLG had 12 times more IgA ASCs per gram of gland than that of SMG (p<0.01). This suggested that IgA ASCs migrated to the two salivary glands with different efficiencies. Since the migration of lymphocytes is mediated by interactions between tissue specific chemokines and chemokine receptors, I hypothesized that the specific compartmentalization of IgA ASCs in the SLG and SMG was mediated by the differential expression of IgA ASC attracting chemokines. Quantitative PCR was used and showed that SLG expressed high levels of CCL28 and its receptor CCR10, which correlated to the distribution of IgA ASCs in the two salivary glands. In agreement with QPCR data, reduced levels of IgA ASCs were found in the SLG of CCR10 deficient mice when compared to wild type (WT) mice. Adoptive transfer of CCR10 deficient mice with WT spleen cells reconstituted the WT phenotype. It was therefore concluded that the interaction between CCL28 and CCR10 play an important role in mediating the migration of IgA ASCs into SLG. These results suggested that the accumulation of IgA ASC to distinct salivary glands is a highly selective process. These data also suggested that homing within mucosal sites is not common but rather a highly regulated process with specific subsets of cells homing to different tissues within the mucosal immune system.

No description available.

This research study examines the manner in which a cardiological sample presents in terms of their psychological make-up by making specific reference to the constructs self-concept and emotions. A literature overview of different aspects of self-concept, emotion and cardiology places the results and discussions of the study within a theoretical background. The study supplies descriptive information relating to the demographic profile of the sample, followed by a description of various aspects of self-concept and emotions, as well as a correlational exploration of the manner in which the sample group presents. The sample consisted of 29 individuals, all who had been diagnosed with coronary heart disease (CHD) and been subjected to surgery as a result thereof. The participants completed a questionnaire, containing two measurement instruments, namely the Adolescent Self-Concept Scale (ASCS) and the Emotions Profile Index (EPI). The scores, obtained by the sample, were subjected to statistical analysis to provide a self-conceptual and emotional profile of the sample. The Spearman Rank-Order Correlation Coefficient was then used to indicate the extent to which the sample tended towards portraying themselves in a positive light, more than they might be experiencing. The study further refers to the views of the sample group, relating to these findings, as obtained from an information and discussion session held with them. The study indicate a significant statistical trend amongst the sample group to portray themselves in a positive light in relation to emotions, even though they were not in reality experiencing such positive emotions. Although the same positive trend was indicated with self-concept, the study cannot conclusively indicate that this is not a realistic presentation of the sample group as a whole. / Dissertation (Counselling Psychology)--University of Pretoria, 2006. / Psychology / unrestricted

<p>The rotator cuff is a group of four muscles and tendons in the shoulder that function to lift and rotate the arm. Rotator cuff tendon tears are increasingly common: more than 545,000 rotator cuff surgeries occur annually in the US. However, treatment is often complicated by disorganized collagen matrix formed via fibrosis and results in high re-tear rates. Tendon tissue engineering seeks to solve the problem using biomaterials to promote neo-tendon formation to augment repair or regenerate tendon. However, while current biomaterials provide the opportunity to improve tendon healing, they frequently still exhibit fibrosis in preclinical studies. Therefore, a critical need exists to understand the mechanisms of aligned collagen formation when designing biomaterials for tendon tissue engineering. Matrix architecture and transient receptor potential cation channel subfamily V member 4 (TRPV4) regulate aligned collagen formation during tenogenesis in vitro, but the mechanism remains to be determined. Recently, TRPV4 stimulation was found to induce nuclear localization and activation of transcriptional co-activators Yes-associated protein (YAP). YAP expression is upregulated during tendon development, a process characterized by aligned collagen formation, and in response to physiological mechanical stimulation, suggesting it could play an important role in tendon. The objective of this work is to improve tissue engineering strategies and progress toward making a device that regenerate tendon after injury. Aim 1 incorporates tendon-derived matrix into synthetic polymer scaffolds to add biological signaling cues to induce tenogenesis. Aim 2 uses a 2D photolithography system (microphotopatterning) to optimize architectural and structural cues to promote stem cell differentiation toward tenogenic, chondrogenic, and osteogenic lineages. Aim 3 investigates dynamic tensile loading protocols to promote collagen matrix synthesis and improve engineered tendon mechanical function. Aim 4 investigates the role of TRPV4 and YAP in collagen alignment during engineered tendon development.</p>