Tesis sobre el tema "Adipose-derived stem cells, regenerative medicine"

Background: Cell therapy using Human Bone Marrow Stem Cells (HBMSCs) has been shown to improve heart function after a myocardial infarction. The harvesting technique involved with bone marrow stem cells is invasive and yields a low cell number. There is now an increasing interest in Human Adipose Derived Stem Cells (HADSCs) as they are abundant and readily accessible from liposuction material. The present study was undertaken to investigate if HADSCs are superior than HBMSCs in myocardial regenerative therapy. Results: Both HADSCs and HBMSCs proliferated in a time dependent manner, however, the proliferative ability of HADSC was greater than HBMSCs. In addition, both cells differentiated to the osteoblast lineage confirming their multipotency when treated with induction medium. Furthermore, treatment of both cells with 5-AC resulted in positive immunostaining of cardiac markers, troponinI and connexin 43, however the expression of these markers was enhanced in HADSCs. This was further confirmed by western blot analysis, however 5-AC treatment did not exhibit cell contraction or multinucleation. In addition, these results were further confirmed by our in vivo study. Both cells were injected in the heart of a rat model of myocardial infarction and was monitored for ejection fraction (EF) and fractional shortening (FS) for 24 hours, 3 weeks and 6 weeks post-surgery. The cardiac function of the rats treated with stem cells was improved as demonstrated by increase in EF and FS, however, a greater improvement was seen with HADSCs compared with HBMSCs. This notion is further substantiated by our studies on left ventricular infarct size measurement, showing that HADSC are more potent in reduction of the infarct compared to HBMSCs. Conclusion: The data suggest that HADSCs may prove to be a more ideal alternative for regenerative therapy in the future.
La thérapie cellulaire à l'aide de cellules souches humaines de la moelle osseuses (CSHMOs) a été démontré d'améliorer la fonction cardiaque après un infarctus du myocarde. La technique de récolte des CSHMOs est pourtant invasive et donne un nombre de cellules viables faible. Il y a maintenant un intérêt croissant dans les cellules souches humaines dérivés du tissu adipeux (CSHTAs), car elles sont abondantes et facilement accessibles à partir des amas de graisses provenant des chirurgies de liposuccion. La présente étude a été menée pour vérifier si les CSHTAs sont supérieures aux CSHMOs dans la thérapie régénératrice du myocarde. Résultats: Les CSHTAs ainsi que les CSHMOs ont proliféré dans une manière temps dépendante, cependant, la capacité proliférative des CSHTAs était supérieure à celle des CSHMOs. De plus, les deux types de cellules souches ce sont différenciées en lignée ostéoblastique, affirmant leur capacité multipotent lorsqu'elles sont traitées avec le milieu d'induction. En outre, le traitement des deux types de cellules souches avec le 5-AC a entraîné l'immunomarquage positif de troponin I et de connexine 43, marqueurs cardiaques, cependant l'expression de ces marqueurs était plus robuste dans les CSHTAs. Cela a été confirmé par analyse d'immunobuvardage de type Western, cependant les cellules traité au 5-AC ne présentait pas de contraction des cellules ou le développement de plusieurs noyaux. En plus, ces résultats ont été confirmés par nos études in vivo. Les deux types de cellules ont été injectées dans le cœur d'un modèle de rat d'infarctus du myocarde et a été suivie pour la fraction d'éjection (FE) et la fraction de raccourcissement (FR) pour 24 heures, 3 semaines et 6 semaines post-chirurgie. La fonction cardiaque des rats traités avec les cellules souches a été améliorée, fait démontré par l'augmentation de l'FE et le FR, cependant, une plus grande amélioration de ce

To repair, replace, or regenerate damaged or diseased tissue has been a long-standing, albeit elusive, goal in medical research. Here, we characterize patient-derivable mesenchymal stem cell types, termed adipose-derived stem cells (ASCs). These cells, which can be derived from liposuction fat and lipoaspirate saline, are sources for patient-derivable extracellular matrix (ECM), fibrinogen (Fg) and adipose tissue extracellular matrix, and may prove useful for synthesizing new bone tissue analogues in vitro. Traditionally and rapidly isolated ASCs were thoroughly characterized as multipotent, having osteogenic, adipogenic, and chondrogenic differentiation potential, and they exhibited comparable proliferative lifespans. These ASCs also shared an indistinguishable immunophenotype when compared to bone marrow-derived mesenchymal stem cells, suggesting that these cells are an excellent source for bone following tissue engineering experimentation. In order to synthesize bone ex-vivo, electrospun scaffolds of fibrinogen (Fg), polydioxanone (PDO), and Fg:PDO blends were seeded with early passage ASCs, fibroblasts, or osteosarcoma cells and were maintained for 21 days in osteogenic or regular growth media. Constructs were analyzed both histologically and molecularly for evidence of osteoblastogenesis. Using SEM, the appearance of regular, mineralized-appearing structures were found in osteogenic-induced ASC seeded scaffolds beyond 14 days, only in the scaffolds containing Fg. Further, at 21 days of culture, Fg scaffolds with ASCs in osteogenic media became hard and brittle. Robust new collagen synthesis and matrix remodeling were observed on all Fg scaffolds, the levels of which were elevated over time. Pronounced mineralization was found throughout bone-induced ASC scaffolds, while control scaffolds (BJ foreskin fibroblasts) showed no mineral deposition (although they did demonstrate excellent cellularity). Analysis of gene expression (qRT-PCR) indicated that electrospun Fg supported osteoblastogenesis through the upregulation of alkaline phosphatase and osteocalcin gene expression. To confirm our gene expression results, osteogenic-induced ASCs on Fg scaffolds were also shown to secrete osteocalcin in the extracellular matrix, a key marker in osteoblastogenesis. Thus, electrospun Fg is an excellent material for ASC growth, proliferation, and osteogenic differentiation, providing an ideal system for furthering basic bone model-based research and for advancing regenerative medicine. In addition to establishing Fg as a source of scaffolding, we developed and characterized a novel method for isolating and subsequently electrospinning adipose tissue matrix. Because adipose ECM contains many primordial matrix proteins important for embryonic development and regeneration (such as laminin, type IV collagen, and fibronectin), adipose ECM may prove to be an autologous tissue engineering matrix and stem cell culture substrate. We show here that adipose tissue ECM can, in fact, be electrospun into a nanofiberous mesh, histologically shown to contain connective tissue, collagens, elastic fibers/elastin, proteoglycans, and glycoproteins in the newly synthesized matrix. We also show that this novel electrospun adipose tissue scaffold is capable of supporting stem cell growth. Taken together, experiments using ASCs cultured on extracellular matrices of electrospun Fg or adipose ECM present an excellent framework for future advances in regenerative medicine therapeutics and research.

The loss of regenerative capacity of bone, from fetal to adult to aged animals, has been attributed not only to a decline in the function of cells involved in bone formation but also to alterations in the bone microenvironment that occur through development and aging, including extracellular matrix (ECM) composition and growth/trophic factor content. In the development of novel treatments for bone repair, one potential therapeutic goal is the restoration of a more regenerative microenvironment, as found during embryonic development. One approach to creating such a microenvironment is through the use of stem cells. In addition to serving as a differentiated cell source, pluripotent stem cells, such as embryonic stem cells (ESCs), may possess the unique potential to modulate tissue environments via local production of ECM and growth factors. ESC-produced factors may be harnessed and delivered to promote functional tissue regeneration. Such an approach to generate a naturally derived, acelluar therapy has been employed successfully to deliver osteoinductive factors found within adult bone, in the form of demineralized bone matrix (DBM), but the development of treatments derived instead from developing, more regenerative tissues or cells remains attractive. Furthermore, the derivation of regenerative materials from an ESC source also presents the added benefit of eliminating donor to donor variability of adult, cadaveric tissue derived materials, such as DBM. Thus, the objective of this project was to examine the osteoinductive potential harbored within the embryonic microenvironment, in vitro and in vivo. The osteogenic differentiation of mouse ESCs as embryoid bodies (EBs) was evaluated in response to phosphate treatment, in vitro, including osteoinductive growth factor production. The osteoinductivity of EB-derived material (EBM) was then compared to that of adult tissue-derived DBM, in vivo. Phosphate treatment enhanced osteogenic differentiation of EBs. EBM derived from phosphate treated EBs retained bioactive, osteoinductive factors and induced new bone formation, demonstrating that the microenvironment within osteogenic EBs can be harnessed in an acellular material to yield in vivo osteoinductivity. This work not only provides new insights into the dynamic microenvironments of differentiating stem cells but also establishes an approach for the development of an ESC-derived, tissue specific therapy.

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.

Embryonic stem cells (ESCs) can differentiate into all somatic cells, and as such, are a promising cell source for therapeutic applications. In vitro, ESCs spontaneously differentiate via the aggregation of cells into embryoid bodies (EBs), which recapitulate aspects of early embryogenesis and harbor a unique reservoir of cues critical for tissue formation and morphogenesis. Embryonic healing responses employ similar intrinsic machinery used for tissue development, and these morphogenic cues may be captured within the EB microenvironment. Recent studies have shown that when injected into injury or defect models in vivo, ESCs synthesize and secrete extracellular factors that ultimately contribute to repair, suggesting that these molecules may be as important for regenerative therapies as functional differentiation of the cells. The overall objective of this project was to develop novel acellular matrices derived from differentiating ESCs undergoing morphogenesis. The central hypothesis was that embryonic matrices contain complex mixtures of extracellular factors that, when isolated, retain bioactivity and enhance wound healing in an adult environment. The overall objective was accomplished by: (1) investigating the production of extracellular matrix (ECM) by differentiating ESCs as a function of differentiation time; (2) assessing the ability of solvents to efficiently decellularize EBs; and (3) evaluating the healing response elicited by acellular matrices derived from EBs in a murine dermal wound healing model. Endogenous ECM synthesis by EBs varied with time and was associated with specific differentiation events. Novel techniques were developed to effectively remove cell components from EBs in order to extract complex, bioactive acellular matrices. EB-derived acellular matrices significantly enhanced the healing of excisional dermal wounds in mice, indicating the potency of extracellular factors synthesized by ESCs. All together, these studies demonstrate that acellular matrices derived from ESCs retain morphogenic factors capable of influencing tissue repair. In addition, this work lays the foundation for future studies to further examine the functional role of endogenous matrix molecules on ESC differentiation and to evaluate the utility of a stem cell-derived matrix for a variety of regenerative medicine applications.

Ischaemic heart disease is the most common cause of mortality in the western hemisphere and it is rapidly becoming the leading cause of death globally. Moreover, therapeutic interventions by cardiologists and cardiac surgeons frequently subject the heart to acute I/R injury, which in itself can cause mortality. Recent investigations of adult stem cells have primarily focused on their regenerative potential for chronic ischaemic heart disease. In this thesis, I have investigated the hypothesis that adult bone marrow derived stem cells are cardioprotective in acute regional myocardial I/R injury. In a rat model of left anterior descending coronary artery (LAD) reversible occlusion and reperfusion, I demonstrate that an intravenous bolus of adult bone marrow derived (1) bone marrow mononuclear (BMNNC) and (2) mesenchymal stem cells (MSC) upon reperfusion can attenuate infarct size. This effect is comparable to ischaemic preconditioning (IPC), which is the gold standard for cardioprotection. Next, I demonstrated the mechanisms for adult stem cell cardioprotection are principally anti-apoptotic and depend upon stem cell secreted factors to (1) activate phosphatidylinositide 3-kinase (PI3)/Akt cell survival kinase-signaling pathway (2) inhibit glycogen synthase kinase-3β (3) inhibit p38MAPK (4) inhibit nuclear translocation of p65NF-κB. 7 Proteomic analysis of myocardium subjected to I/R and treated with either BMMNC or BMMNC derived supernatant (BMS) upon reperfusion demonstrated higher expression of a whole host of pro-survival proteins. These were notably (1) 14-3-3-ε protein (2) anti-oxidant peroxiredoxin-6 (3) heat shock protein (HSP) αB-crystallin, HSP72, HSP tumour necrosis factor receptor-1 associated protein, and HSP ischaemia responsive protein-94 (4) glycolytic protein glyceraldehyde-3-phosphate dehydrogenase (5) mitochondrial aconitase and mitochondrial voltage-dependent anionselective channel protein-1. Thereafter, I investigated the mobilization of endogenous bone marrow stem cells and trafficking to the ischaemic myocardium by stromal cell derived factor-1 (SDF-1) /chemokine, receptor type 4 (CXCR4) signaling. I demonstrate high up-regulated expression of CXCR4 and CD26 in BMMNC following IPC, which might have a role in IPC-mediated cardioprotection. Finally, and in concordance with this finding I demonstrate that both IPC and an exogenous MSC bolus upon reperfusion can synergize to abolish acute myocardial I/R injury.

Every year several patients have to deal with bone tissue loss due to trauma or diseases. Bone tissue engineering aims to restore or repair musculoskeletal disorders through the development of bio-substitutes that require the use of cells and scaffolds which should possess both adequate mechanical properties and interconnecting pores to allow cellular infiltration, graft integration and vascularization. The ideal cell for tissue engineering should possess a potential plasticity with the ability to functionally repair the damaged tissue, and it should be available in large amount. Mesenchymal stem cells (MSCs) are present in many adult tissues, and adipose tissue represents an attractive source of MSCs for researchers and clinicians of nearly all medical specialties. Adipose-derived stem cells (ASCs) are similar to MSCs isolated from bone marrow, placenta, and umbilical cord blood in morphology, immunophenotype, and differentiation ability, and they represent a promising approach of bone regeneration. Additional features of ASCs are their immunoregolatory and anti-inflammatory properties both in vivo and in vitro and their low immunogenicity. Since several years our laboratory is studying mesenchymal stem cells isolated from human and animal adipose tissues. Human ASCs (hASCs) have been characterized by their immunophenotype, their self-renewal potential, and they have been induced to differentiate towards adipogenic, osteogenic and chondrogenic lineages. The ability of hASCs to grow in the presence of several scaffolds has also been tested. hASCs adhered to the surface of tested biomaterials, filling the pores and forming a 3D web-like structure, allowing these progenitor cells to osteo-differentiate more efficiently respect to cells maintained on polystyrene. Since our interest was to regenerate muscle-skeletal defects by ASCs in pre-clinical models, we first studied ASCs isolated from adipose tissue of rat (rASCs), rabbit (rbASCs) and pig (pASCs), considered good models in the orthopaedic field. We have shown that animal ASCs behaved similarly to the human ones, and, in collaboration with the Faculty of Veterinary Medicine of University of Milan and the IRCCS Galeazzi Orthopaedic Institute of Milan, we have tested the ability of autologous ASCs to regenerate a full-thickness critical-size bone defect in rabbits. The experimental study was conducted on the tibiae of 12 New Zealand rabbits, and from 6 rabbits out of 12 we have collected adipose tissue from the interscapular region. We have isolated 2.8x105±1.9x105 rbASCs per ml of raw tissue, and after 3-4 days in culture the cells showed the typical fibroblast-like morphology. One week later, all the 6 cellular populations started to steadily proliferate, and they generated fibroblast (CFU-F) and osteoblast (CFU-O) colonies, highlighting the presence of osteogenic progenitors. Indeed, when rbASCs were induced to osteo-differentiate, either after 7 and 14 days, we have observed an up-regulation of specific osteogenic markers, such as alkaline phosphatase (ALP, +28.9%), collagen (+105.9%) and extracellular calcified matrix (+168.1%), compared to undifferentiated cells. In parallel, testing HA, the scaffold selected for the in vivo experiment, we found that rbASCs were osteoinduced; indeed the presence of HA granules increased per se the amount of collagen production (+48.2%). 1.5x106 undifferentiated rbASCs were seeded on custom-made HA disks (8 mm Ø x 4 mm ↕), and the day after, each bioconstruct was implanted into the lesion created in the tibia of each rabbit. We had an additional experimental group of defects where the same number of rbASCs were inserted in the lesion as a semi-liquid suspension; moreover, as controls, we treated 6 lesioned tibia with just the scaffolds, and we left 6 untreated lesioned bone. 8 weeks after surgery animals were sacrificed and the tibia explanted. A macroscopic analysis showed no bone resorption, no abnormal bone callous formation, no fractures, infection or inflammatory reactions, and all the bone defects were completely filled without any significant differences among the four groups. Interestingly, in the presence of scaffold seeded with rbASCs, histology and immunohistochemistry showed a new bone tissue more mature and similar to the native bone. These data have also been confirmed by biomechanical tests: indeed, the mechanical properties of the bone defect treated by rbASCs-HA were improved, suggesting that these constructs bore mechanical loading with an increase in stiffness of 19.8% and in hardness of 31.6% respect to just HA treated group, indicating that the bioconstructs made out of autologous rbASCs and hydroxyapatite might ameliorate the treatment for large bone defects. We would suggest the use of ASCs as a safe cellular therapy in future clinical applications where a large bone defect needs to be treated. These promising results on small size animals allow us to plan a new study on large size animals such as minipigs. However, before moving to the clinic, we know that there are several important aspects that need to be faced regarding safeness and the features of the candidate patients: 1. may the “quality” of hASCs be affected by the donor’s physiological or pathological conditions? 2. may the use of pharmacological treatment enhance cellular plasticity of multipotent cells? 3. may the use of immunoselected hASCs ameliorate tissue regeneration in the field of muscle-skeletal? We have addressed some of these aspects, comparing different populations of hASCs from subcutaneous adipose tissue of healthy-young-female donors (hASCs<35 y/o, n=12, mean age 31±4 years, BMI=23.5±1.6), and from middle-age ones (hASCs>45 y/o n=14, mean age 56±7 years, mean BMI=28.4±1.8). The cellular yield of hASCs derived from older donors was 2.5 fold greater than the one of hASCs<35 y/o, whereas hASCs from younger donors were more clonogenic than hASCs isolated from older ones, with an increase of 129%. No significant differences were observed looking at their immunophenotype. When hASCs were induced to differentiate into cells of the adipogenic and osteogenic lineages, the donor’s age did not affect their adipogenic differentiation, whereas the osteogenic one was significantly affected by age both in the absence and in the presence of three-dimensional scaffolds, showing a decreased ALP basal levels of about 10-fold in hASCs>45 y/o respect to hASCs<35 y/o. These results seems to indicate that ASCs from different donors could behave differently. Trying to overcome this aspect we have used different approaches, and we have studied if Reversine, a synthetic purine already known to increase plasticity of terminally differentiated cells, might improve the differentiation ability of hASCs. 72 hours treatment with 50 nM Reversine induced hASCs to differentiate into osteoblast like-cells (+45% of alkaline phosphatase activity), smooth muscle cells (+89% of α-actin expression) and skeletal muscle cells (myotubes formation) compared to control hASCs. Moreover, since it is known that CD34 and L-NGFR positive cells define a subset of high proliferative and multipotent MSCs, we have immunoselected, these progenitor cells from hASC populations. In contrast to the whole population, the immunoseparated fractions maintained their undifferentiate state and their ability to differentiate much longer during culture. We have shown that both CD34+ and L-NGFR+ hASCs can be used as alternative candidates for tissue engineering and regenerative medicine applications. In particular, due to the improved ability of L-NGFR positive cells to adipo- and chondro-differentiate, they appear an ideal tool in reconstructive plastic surgery and cartilage regeneration. From our data, and the ones from researchers in other fields, we believe that in the near future adipose-derived stem cells might be considered a safe tool in regenerative medicine. Furthermore, to improve this “cellular therapy”, we could either pre-treat ASCs with molecules, such as drugs and/or siRNAs known to affect specific differentiation pathways, or by selecting subpopulations of progenitor cells which may be used as allogenic implants. Next step will be to confirm our in vivo data in a large size animal model such as minipig, and then to test if pre-treated cells or selected population might be used in an autologous and allogenic small size animal model.

The deposition of cells at sites of injury is a clinically relevant approach to facilitate local tissue regeneration and repair. However, cell engraftment, retention, and survival are generally modest, requiring the development of novel deposition techniques and biomaterials. Here, a micro-sized polymeric network (microMESH) is investigated as a promising biodegradable scaffold for the engraftment and tissue integration of human Adipose-Derived Stem Cells (hADSCs) to be used for a wide range of injuries, including myocardial infarction. microMESH comprises a regular network of PLGA microfilaments spatially organized to form square openings of 5x5, 10x10 and 20x20 μm2 . microMESH is realized using soft lithographic techniques starting from a master silicon template reproducing the actual geometry of the final PLGA network. After extensive geometrical, physico-chemical, and mechanical characterizations using a broad range of techniques, hADSCs were integrated with microMESH. Cell viability, spatial organization, secretome and stemness were characterized for all three different microMESH configurations and compared to conventional systems, including 2D plastic dishes and collagen layers. Interestingly, when hADSCs were cultured on microMESH they organized in spheroidal-like structures, despite the geometry, maintaining viability over time. This peculiar attitude of the microMESH to form assemblies better represents the human tissue outside the body, compared to 2D monolayer cultures. Additionally, spheroids established an intimate interaction with the microMESH resulting in the scaffold incorporation within the 3D arrangement formed by the cells. On the contrary, hADSCs form only superficial interaction above the flat collagen sheet that is currently used for cell transplantation in animal models of cardiac diseases. Moreover, once the hADSCs are placed on microMESH, the actin cytoskeleton reorganizes to confer a 3D cell shape with multidirectional actin arrangements, forming nonlinear structures and ring structures at the anchorage site to the microMESH, relative to linear filaments when the cells adhered and flattened onto the plastic surface and on top of collagen scaffold. This internal reorganization and the stronger interaction may explain why microMESH scaffold fostered the secretion of biologically active molecules, acting in a paracrine fashion on resident cells, which are expected to accelerate tissue regeneration and repair. Specifically, when hADSCs grew on microMESH we observed a trend for higher production of several factors with specific implications in angiogenesis, stem cell proliferation and expansion, cell survival, inflammation modulation, ECM remodeling, stem cell mobilization, chemotaxis and homing, relative to 2D monolayer conditions. The paracrine effect of hADSCs is scaffold dependent and can be modulated by tailoring the geometrical and mechanical properties of microMESH. Indeed, the 5x5 microMESH showed its contribution in angiogenesis, ECM remodeling and stem cell mobilization from bone marrow into the bloodstream. Indeed, highest amounts of VEGF, TIMP-2 and GCSF, respectively were detected in 5x5 geometry compared to the other conditions. Rather, 10x10 geometry promotes angiogenesis enhancing the VEGF production, stem cell proliferation and survival by raising the Fibroblast Growth Factors family secretion and EGF factor, respectively and favors ECM remodeling increasing the TIMP-2 production compared to other conditions. Lastly, the 20x20 seems to have a more anti-inflammatory role (combination of IL-10 and TGF-β1) and chemotactic function (e.g. RANTES). Finally, in this work, we started to shed new light on the ability of micromMESH geometry to modulate the hADSCs stemness evaluating the expression levels of CD44, CD90 and CD105 markers over time. The proposed microMESH scaffold is expected to provide an effective alternative to more conventional hADSCs transplant techniques.

Among all the possible sources of mesenchymal stem cells, adipose tissue and olfactory mucosa have raised great interest and have become some of the most investigated sources. Adipose tissue-derived mesenchymal stem cells and the fat itself as a source of human adipose derived stem cells, represent one of the major fields of research in regenerative medicine. A great advantage is represented by the minimal invasive and high accessibility to adipose tissue and its ready availability. In the present study, hADSCs were isolated from the adipose tissue donated by several patient and have been investigated and characterized through different technical approaches, such as flow cytometry and immunocytochemistry. These hADSCs reproducibly fulfill the general definition of MSCs by both phenotypic and differentiation capabilities criteria, showing also the expression of neural markers, as observed by confocal microscope analysis. Lipoaspirated adipose tissue showed positivity to ß-tubulin III that was also maintained in lipoaspirate-derived hADSCs. A population of stem cells retaining typical characteristics of surface markers of classical adipose tissue stem cells and MSC was obtained when adipose tissue was subjected to culture in vitro, either by processing through centrifugation or by direct plating without enzymatic digestion with collagenase. Flow cytometry analyses showed that hADSCs expressed classical mesenchymal markers such as CD44, CD73, CD90, CD105 and CD166, while endothelial (CD31, CD34, CD144, CD146) and hematopoietic (CD45, CD133) markers were much less represented. Also the ability to give rise to tissue of mesenchymal origins, such as osteoblastic and adipogenic lineages, were present in hADSCs. In addition, the immunofluorescence staining indicated the expression of neural stem markers in hADSCs which consequently co-expressed nestin, β-tubulin III and glial GFAP. We have also characterized human olfactory ensheathing stem cells. Olfactory mucosa is specialized tissue inside the nasal cavity involved in olfactory perception and capable of lifelong regeneration throughout adulthood. Multipotent stem cells obtained from it offer the possibility of promoting regeneration and reconstruction in regenerative medicine, being readily accessible with minimal invasive techniques, capable of expansion in vitro and retaining broadly potent differentiative capacity as stem cell progenitors. Among the several members of the olfactory mucosa, Olfactory Ensheathing Cells (OECs) are well known to be useful in repairing the nervous system. By following our method, cells can be easily isolated and maintained in TCM, and their cultivation in large flasks allowed obtaining rich cultures of OECs in 2 weeks. Cell cycle analysis showed that the majority of cells are in G0/G1 phase, while just a lesser part is in S/G2 phase. In our growth conditions, no chromosomal abnormalities were observed also at high culture passage (p14). Live morphology of obtained cells showed a fibroblast-like phenotype and the immunohistochemical analyses showed the expression of beta-Tubulin III, Vimentin, Nestin, Glial Fibrillary Acidic Protein and Microtubule-Associated Protein 2. By FACS analysis we demonstrated that OECs are positive to typical surface mesenchymal markers (CD44, CD73, CD90, CD105, CD146 and CD166). As expected, some endothelial (CD31, CD34) and hematopoietic (CD45) markers were very few represented, while some others (CD56, CD144, CD146, CD133) are partially found. These cells also express genes that constitute the core circuitry of self-renewal such as SOX2, NANOG and OCT4 and the stemness marker CD133. OECs incubated with serum-free medium, normally used for the formation of neurospheres, spontaneously formed large spheroids reaching a mean diameter of 100 μm in 10 days of culture. Immunofluorescence of specific proteins showed that spheres were positive to markers such as Nestin, Vimentin, TUJ-1, MAP2 and GFAP. In conclusion, our method allows the quickly and easily hADSCs and hOESCs isolation from human adipose tissue and nasal biopsies. The obtained cells can be cultured without altering their mesenchymal properties, suggesting the pluripotency nature of these cells and that they are a reliable source for regenerative medicine.

Durante questo progetto di dottorato sono stati condotti studi per comprendere la capacità delle cellule mesenchimali amniotiche (AMCs) di agire attraverso meccanismi paracrini. La prima fase del disegno sperimentale ha valutato l’efficacia delle AMCs sulla proliferazione delle cellule endometriali (EDCs) in assenza di un contatto cellula-cellula. Le EDCs sono state co-coltivate in un sistema trans-well con le AMCs o sono state coltivate in presenza del loro medium condizionato (CM). In entrambe le condizioni sperimentali è stato osservato un aumento del tasso di proliferazione delle EDCs definendo, così, il ruolo cruciale dei fattori secreti dalle AMCs come mediatori dell'azione delle cellule staminali. Il CM è composto da fattori solubili e non-solubili rappresentati da microvescicole (MVs). Per capire il ruolo delle MVs e del loro contenuto nell’azione paracrina, la seconda fase del disegno sperimentale è stata incentrata sull’individuazione del tipo di MVs secrete dalle AMCs e sulla loro efficacia in processi infiammatori in vitro su due importanti modelli sperimentali nella specie equina quali l’endometrio ed il tendine. La produzione di MVs è stata ottimizzata attraverso un’ultracentrifugazione del CM a 100.000 g per 1 ora. Attraverso lo strumento Nanosight è stato calcolato che la produzione di MVs da AMCs equine è di circa 2550±71 particelle/cellula, con una dimensione media di 258±55 nm. La microscopia elettronica a trasmissione ha confermato la presenza di vescicole cellulari che per dimensioni e modalità di secrezione sono state classificate come shedding vesicles. Per capire se le cellule endometriali (EDCs) e tendinee (TNCs) fossero target delle MVs secrete dalle AMCs, è stata studiata l’incorporazione delle medesime dopo marcatura con un fluorocromo lipofilico quale il PKH-26. Dopo uno studio di una curva dose-risposta è stato osservato che la dose ottimale per l'incorporazione di MVs in EDCs è stata di 40x106 MVs/ml a 72 h e per le TNCs di 40x106 MVs/ml a 24-48h. Per esaminare l'abilità delle MVs nel contrastare un processo infiammatorio, entrambe le linee cellulari sono state stressate in vitro con LPS e trattate con MVs. La vitalità cellulare, l'espressione di alcuni geni pro-infiammatori ed il rilascio delle rispettive citochine sono stati i parametri impiegati per valutare l’efficacia delle MVs. Per entrambe le linee cellulari, il tasso di apoptosi è drammaticamente salito nelle cellule trattate con LPS, rispetto al controllo (CTR). LPS ha indotto un aumento significativo dell'espressione di TNF-α, IL-6 e IL-1β in EDCs e di MMP-1, MMP-9, MMP-13 e TNF-α in TNCs. Le MVs sono state in grado di contrastare l'effetto di LPS, diminuendo il tasso di apoptosi in entrambe le linee cellulari e riducendo il livello di espressione dei geni pro-infiammatori. Una conferma di questi dati è stata ottenuta attraverso l'analisi delle citochine pro-infiammatorie (TNF-β and IL-6) ed anti-infiammatorie (TGF-α) rilasciate dalle EDCs nel terreno di coltura, confermando l'abilità delle MVs di trasportare molecole capaci di contrastare la risposta infiammatoria in cellule stressate con LPS. Data l'importanza del contenuto in miRNA delle MVs, sia nelle AMCs sia nelle loro MVs è stata studiata la presenza di tre miRNA (miR-335, miR-146a, and miR-26a-2) coinvolti nella regolazione dei processi infiammatori. Questi tre miRNA sono stati identificati sia nelle AMCs sia nelle MVs, di conseguenza, è possibile ipotizzare che l'espressione genica dopo infiammazione con LPS e trattamento con MVs possa essere stato modulato dal trasferimento di miRNAs dalle MVs alle cellule target. Successivamente a questi studi, si è tentato di capire quanto in un processo rigenerativo possa essere imputato alle MVs e quanto ai fattori solubili presenti nel CM e, a questo proposito, è stata valutata l’abilità delle MVs di inibire la proliferazione delle cellule periferiche mononucleate del sangue (PBMC). La loro azione è stata comparata a quello del CM e del sovranatante (SN) ottenuto dal CM dopo scorporazione delle MVs. Il CM ed il SN hanno manifestato capacità immunosoppressiva ma, anche dopo un trattamento di sonicazione al fine del rilascio del loro contenuto, le MVs non hanno dimostrato quest’abilità. Questi risultati portano a dedurre che le MVs sono in grado di trasportare informazioni alle cellule target attraverso molecole capaci di contrastare l'effetto infiammatorio dovuto all'uso di LPS ma, tenendo in considerazione la mancanza di effetto immunomodulatorio, probabilmente in vivo la loro azione è integrata anche dalla presenza dei fattori solubili presenti nel CM. I meccanismi paracrini materno-fetali sono di vitale importanza ai fini dell’instaurarsi di una gravidanza, per cui si è ritenuto interessante studiare questi meccanismi durante la produzione in vitro di embrioni bovini. Le differenti componenti del secretoma (CM, SN e MVs) ottenute da AMCs ed EDCs bovine sono state aggiunte al terreno di coltura embrionale a differenti giorni di coltura. I risultati hanno dimostrato che il giorno 5 è il migliore momento per la supplementazione e che le MVs di AMCs portano a migliori risultati qualitativi. Questi dati sono stati confermati dalla valutazione di alcuni geni coinvolti nell'apoptosi e nella protezione contro specie reattive dell'ossigeno. I motivi per i quali le MVs di AMCs si siano dimostrate migliori di quelle secrete dalle EDCs non sono ancora conosciuti ma è probabile che le EDCs coltivate in monostrato in vitro vadano incontro a de-differenziamento che altera la qualità del loro secreto. In conclusione, è possibile affermare che le AMCs sono una linea cellulare affascinante in quanto derivano da materiale di scarto biologico, e quindi a basso costo, e possono essere impiegate in medicina rigenerativa per la loro capacità di trasportare informazioni alle cellule target. Le MVs possono offrire un nuovo strumento terapeutico privo di cellule nell’ambito della nanomedicina.
During this PhD project, studies were carried out to understand the ability of amniotic mesenchymal cells (AMCs) to act by paracrine mechanism. At first, AMCs and their conditioned medium (CM) were investigated in an in vitro model using equine endometrial cells (EDCs) as target. Proliferation of EDCs was studied co-culturing them with AMCs in a trans-well system or in presence of AMC-CM. In both conditions, there was a significant increase in EDC proliferation rate, defining the crucial role of factors secreted by AMCs in stem cells action. CM is composed of soluble factors and no-soluble factors as microvesicles (MVs). In this context, in the second step of this project, the presence and the type of AMC-MVs were identified to understand their role in regenerative medicine. The production of MVs was optimized through a CM ultracentrifugation at 100.000 g for 1 hour. Microvesicle production from equine AMCs was 2550±71 particles/cell, with a mean dimension of 258±55 nm. The transmission electron microscopy confirmed the presence of extra-cellular vesicles that were classified as shedding vesicles for their size and modality of secretion. In order to understand if endometrial cells (EDCs) tendon cells (TNCs) were target of these MVs, an incorporation study was performed labelling MVs with a lipophilic fluorochrome such as PKH-26. By a dose-response curve, the optimal conditions of incorporation were at 72 h at a concentration of 40x106 MVs/ml for EDCs, and at 24-72 h at a concentration of 40x106 MVs/ml for TNCs. In order to study MVs ability to counteract an in vitro inflammation, EDCs and TNCs challenged with LPS and treated with MVs were evaluated by viability cell tests, by expression of some pro-inflammatory genes, and by release of respective cytokines. For both cell types, the apoptosis rate increased dramatically in cells treated with LPS if compared to the control (CTR). LPS significantly upregulated the expression of TNF-α, IL-6 and IL-1β in EDCs and of MMP-1, MMP-9, MMP-13 and TNF-α in TNCs. MVs were able to counteract the action of LPS, decreasing the apoptosis rate and reducing in the expression levels of the pro-inflammatory genes in both cell lines. Coherent results were obtained through the analysis of pro-inflammatory (TNF-β and IL-6) and anti-inflammatory (TGF-α) cytokines released by EDCs in the culture medium, confirming the ability of MVs to transport molecules able to counteract the stress induced by LPS. Since MVs contain various active molecules, the presence of three miRNAs (miR-335, miR-146a, and miR-26a-2) was investigated, as they are involved in the regulation of inflammation. The selected miRNAs have been found in both AMCs and their MVs, so the previously observed downregulation of gene expression could be correlated to miRNA transfer from MVs to target cells. Moreover, the ability of MVs to inhibit peripheral blood mononuclear cell (PBMC) was evaluated, but MVs, also after lysis by sonication to release their content, were not able to inhibit PBMC proliferation despite to CM and SN. These results led to hypothesize that MVs brought to the target cells some molecules able to counteract the inflammatory situation due to the LPS but, taking into account the lack of their immunomodulatory action, probably, for an in vivo healing, soluble factor of CM are necessary too. Paracrine mechanism are essential also in maternal-fetal communication. In this context, we studied these mechanisms during bovine in vitro embryo production. Different components of secretome (CM, SN and MVs) from bovine AMCs and EDCs were supplemented to the embryo culture media at different days of culture. The results demonstrated that the day 5 of culture is the best time point for the supplementation of these components and that AMCs-MVs provided the best environment for the embryo concerning the blastocyst quality. These data were confirmed by the evaluation of genes involved in apoptosis and reactive oxygen species protection. The reasons for which the MVs of AMCs have proved better than those secreted by EDCs are not yet known but it is likely that in vitro culture of EDCs in monolayer may induce a de-differentiation that alters the quality of their secretion. As conclusion of this project, it is possible to speculate that AMCs are fascinating in view of producing off-the-shelf products, at low cost, and their use in regenerative medicine for their capacity to carry information to the target cells. The MVs may offer a new therapeutic cell-free tool in nanomedicine.

Induced pluripotent stem cells (iPS cells) have shown great promise in the newly-developing field . of regenerative medicine. Recently, however; there have been conflicting reports regarding their immunogenicity, even when derived in an autologous fashion, due to the ectopic expression of certain developmental antigens. Dendritic cells (DC) are professional antigen presenting cells and therefore serve as potential tools for modulating the immune response, particularly with respect to acceptance of stem cell-derived tissues. We hypothesised that DC differentiated from iPS cells, so-called iPS, may have the capacity to induce tolerance to tissues differentiated from the same parent iPS cell line. As a first step towards this goal, we have derived iPS cells from both embryonic and adult mouse fibroblasts, developed protocols for their differentiation along the DC lineage and characterised the resulting DC both phenotypically and functionally, using bone-malTow derived-DC (bmDC) for comparison. Furthermore, their potential for inducing tolerance as a function of regulatory T cell induction has also been explored. Our data suggest that, although ipDC are able to function in a similar manner to bmDC both in vitro and in vivo, they display important differences with respect to cytokine production and MHC class II expression which may reflect their early embryonic origin and render them pro-tolerogenic. One application for this novel population of DC might be the induction of tolerance to autologous iPS cell-derived tissues. In order to assess the need for tolerance under such circumstances, we have investigated the reported immunological rejection of iPS cells when transplanted syngeneically. Although we were unable to coroborate previous findings showing immunological rejection of iPS cells under such circumstances, we were able to show that the formation of teratomas from iPS cells is a stochastic and very variable process, unlike their generation from ES cells. Importantly, we were able to exclude the involvement of any antigen-specific component to the failure of teratoma survival by inducing blanket tolerance to iPS cell-derived tissues, using a cocktail of non-depleting monoclonal antibodies specific for CD4, CD8 and CD40L. This is the first repOlted study deriving dendritic cells from mouse iPS cells and demonstrating their extensive characterisation. Additionally, the work presented in this thesis adds to the growing body of evidence of the immunological propelties of iPS cells with our work demonstrating that they appear not to attract an adaptive immune response but, however, show significant heterogeneity with respect to their ability to engraft in vivo.

Osteoarthritis (OA) is a degenerative disease of diarthrodial joints causing pain and loss of joint function. Etiology is heterogeneous, but commonly involves inflammation arising from impairment of normal tissue homeostasis and/or function. A cycle of low-grade inflammation and global tissue degradation causes alteration of tissue morphology and function via primary mechanisms or inability to withstand physiological forces. Current therapies variably ameliorate symptoms but do not modify progression. Mesenchymal stem cells (MSCs) have multi-modal properties but are ineffective in ameliorating equine OA. However, anti-inflammatory activities of bone marrow derived MSCs (BMSCs) are enhanced by three-dimensional spheroid culture so equine BMSC (eBMSC) spheroids could inhibit intra-articular inflammation. The overarching hypothesis is that eBMSCs can be enhanced to produce an allogeneic eBMSC therapy that inhibits intra-articular inflammation. In vitro experiments compared differences in anti-inflammatory phenotype between spheroid and traditionally cultured monolayer eBMSCs, the viability and health of eBMSC spheroids administered through needles, and the effects of allogeneic donor on the anti-inflammatory potential of eBMSC spheroids. A model of equine LPS induced synovitis was used to investigate anti-inflammatory efficacy of spheroid eBMSCs compared to placebo or monolayer eBMSCs in vivo. eBMSCs aggregate into spheroids that have stable stem cell marker expression with increased secretion and gene expression of IL-6 and PGE2, and gene expression of SDF-1 and TSG-6. IFN𝛾 and TNFα were not produced by eBMSC spheroids and IL-10 production varied between individuals. Spheroids maintain higher viability and lower senescence than monolayer eBMSCs after injection through a needle and form in high-throughput culture without detrimental effects on expression of TSG-6, IL-6 and PGE synthases that denote an anti-inflammatory phenotype. Additionally, there is significant variation in this phenotype depending on the eBMSC donor. eBMSC spheroids reduced total nucleated cell counts and objective lameness measurements at peak levels of intra-articular inflammation compared to monolayer cultured eBMSCs in vivo. In summary, spheroids increase anti-inflammatory potential of eBMSCs and are practical for clinical use. Increased anti-inflammatory efficacy was demonstrated in a model of in vivo inflammation. This dissertation provides an understanding of the anti-inflammatory activities of eBMSC spheroids that can be used to develop an OA therapy.
Ph. D.

Scientific research is propelled by two objectives: Understanding and recognizing the essential biology of life, and deciphering this to uncover possible therapeutics in order to improve quality of life as well as relieve pain from disease. The aim of the work described in this thesis was to dissect the fundamental requirements of induced pluripotent stem cells both in pluripotency and differentiation with a particular focus on atrial specificity. Drug targeting of atrial-specific ion channels has been difficult because of lack of availability of appropriate cardiac cells, and preclinical testing studies have been carried out in non-cardiac cell lines, heterogeneous cardiac populations or animal models that have been unable to accurately represent human cardiomyocyte physiology. Therefore, we sought out to develop a preparation of cardiomyocytes showing an atrial phenotype with adult characteristics from human induced-pluripotent stem cells. A culture programme involving the use of Gremlin 2 allowed differentiation of cardiomyocytes with an atrial phenotype from human induced-pluripotent stem cells. When these differentiated cultures were dissociated into single myocytes a substantial fraction of cells showed a rod-shaped morphology with a single central nucleus that was broadly similar to that observed in cells isolated from atrial chambers of the heart. Immunolabelling of these myocytes for cardiac proteins (including RyR2 receptors, actinin-2, F-actin) showed striations with a sarcomere spacing of slightly less than 2um. The isolated rod-shaped cells were electrically quiescent unless stimulated to fire action potentials with an amplitude of 100 mV from a resting potential of approximately -70 mV. Proteins expressed included those for IK₁, IK_ur channels. Ca²⁺ Transients recorded from spontaneously beating cultures showed increases in amplitude in response to stimulation of adrenoceptors (both alpha and beta). With the aim of identifying key signaling mechanisms in directing cell fate, our new protocol allowed differentiation of human myocytes with an atrial phenotype and adult characteristics that show functional adrenoceptor signaling pathways and are suitable for investigation of drug effects.

Cell based therapies require cells capable of self renewal and differentiation, and a prerequisite is the ability to prepare an effective dose of ex vivo expanded cells for autologous transplants. The in vivo identification of a source of physiologically relevant cell types suitable for cell therapies is therefore an integral part of tissue engineering. Bone marrow is the most easily accessible source of mesenchymal stem cells (MSCs), and harbours two distinct populations of adult stem cells; namely hematopoietic stem cells (HSCs) and bone mesenchymal stem cells (BMSCs). Unlike HSCs, there are yet no rigorous criteria for characterizing BMSCs. Changing understanding about the pluripotency of BMSCs in recent studies has expanded their potential application; however, the underlying molecular pathways which impart the features distinctive to BMSCs remain elusive. Furthermore, the sparse in vivo distribution of these cells imposes a clear limitation to their in vitro study. Also, when BMSCs are cultured in vitro there is a loss of the in vivo microenvironment which results in a progressive decline in proliferation potential and multipotentiality. This is further exacerbated with increased passage number, characterized by the onset of senescence related changes. Accordingly, establishing protocols for generating large numbers of BMSCs without affecting their differentiation potential is necessary. The principal aims of this thesis were to identify potential molecular factors for characterizing BMSCs from osteoarthritic patients, and also to attempt to establish culture protocols favourable for generating large number of BMSCs, while at the same time retaining their proliferation and differentiation potential. Previously published studies concerning clonal cells have demonstrated that BMSCs are heterogeneous populations of cells at various stages of growth. Some cells are higher in the hierarchy and represent the progenitors, while other cells occupy a lower position in the hierarchy and are therefore more committed to a particular lineage. This feature of BMSCs was made evident by the work of Mareddy et al., which involved generating clonal populations of BMSCs from bone marrow of osteoarthritic patients, by a single cell clonal culture method. Proliferation potential and differentiation capabilities were used to group cells into fast growing and slow growing clones. The study presented here is a continuation of the work of Mareddy et al. and employed immunological and array based techniques to identify the primary molecular factors involved in regulating phenotypic characteristics exhibited by contrasting clonal populations. The subtractive immunization (SI) was used to generate novel antibodies against favourably expressed proteins in the fast growing clonal cell population. The difference between the clonal populations at the transcriptional level was determined using a Stem Cell RT2 Profiler TM PCR Array which focuses on stem cell pathway gene expression. Monoclonal antibodies (mAb) generated by SI were able to effectively highlight differentially expressed antigenic determinants, as was evident by Western blot analysis and confocal microscopy. Co-immunoprecipitation, followed by mass spectroscopy analysis, identified a favourably expressed protein as the cytoskeletal protein vimentin. The stem cell gene array highlighted genes that were highly upregulated in the fast growing clonal cell population. Based on their functions these genes were grouped into growth factors, cell fate determination and maintenance of embryonic and neural stem cell renewal. Furthermore, on a closer analysis it was established that the cytoskeletal protein vimentin and nine out of ten genes identified by gene array were associated with chondrogenesis or cartilage repair, consistent with the potential role played by BMSCs in defect repair and maintaining tissue homeostasis, by modulating the gene expression pattern to compensate for degenerated cartilage in osteoarthritic tissues. The gene array also presented transcripts for embryonic lineage markers such as FOXA2 and Sox2, both of which were significantly over expressed in fast growing clonal populations. A recent groundbreaking study by Yamanaka et al imparted embryonic stem cell (ESCs) -like characteristic to somatic cells in a process termed nuclear reprogramming, by the ectopic expression of the genes Sox2, cMyc and Oct4. The expression of embryonic lineage markers in adult stem cells may be a mechanism by which the favourable behaviour of fast growing clonal cells is determined and suggests a possible active phenomenon of spontaneous reprogramming in fast growing clonal cells. The expression pattern of these critical molecular markers could be indicative of the competence of BMSCs. For this reason, the expression pattern of Sox2, Oct4 and cMyc, at various passages in heterogeneous BMSCs population and tissue derived cells (osteoblasts and chondrocytes), was investigated by a real-time PCR and immunoflourescence staining. A strong nuclear staining was observed for Sox2, Oct4 and cMyc, which gradually weakened accompanied with cytoplasmic translocation after several passage. The mRNA and protein expression of Sox2, Oct4 and cMyc peaked at the third passage for osteoblasts, chondrocytes and third passage for BMSCs, and declined with each subsequent passage, indicating towards a possible mechanism of spontaneous reprogramming. This study proposes that the progressive decline in proliferation potential and multipotentiality associated with increased passaging of BMSCs in vitro might be a consequence of loss of these propluripotency factors. We therefore hypothesise that the expression of these master genes is not an intrinsic cell function, but rather an outcome of interaction of the cells with their microenvironment; this was evident by the fact that when removed from their in vivo microenvironment, BMSCs undergo a rapid loss of stemness after only a few passages. One of the most interesting aspects of this study was the integration of factors in the culture conditions, which to some extent, mimicked the in vivo microenvironmental niche of the BMSCs. A number of studies have successfully established that the cellular niche is not an inert tissue component but is of prime importance. The total sum of stimuli from the microenvironment underpins the complex interplay of regulatory mechanisms which control multiple functions in stem cells most importantly stem cell renewal. Therefore, well characterised factors which affect BMSCs characteristics, such as fibronectin (FN) coating, and morphogens such as FGF2 and BMP4, were incorporated into the cell culture conditions. The experimental set up was designed to provide insight into the expression pattern of the stem cell related transcription factors Sox2, cMyc and Oct4, in BMSCs with respect to passaging and changes in culture conditions. Induction of these pluripotency markers in somatic cells by retroviral transfection has been shown to confer pluripotency and an ESCs like state. Our study demonstrated that all treatments could transiently induce the expression of Sox2, cMyc and Oct4, and favourably affect the proliferation potential of BMSCs. The combined effect of these treatments was able to induce and retain the endogenous nuclear expression of stem cell transcription factors in BMSCs over an extended number of in vitro passages. Our results therefore suggest that the transient induction and manipulation of endogenous expression of transcription factors critical for stemness can be achieved by modulating the culture conditions; the benefit of which is to circumvent the need for genetic manipulations. In summary, this study has explored the role of BMSCs in the diseased state of osteoarthritis, by employing transcriptional profiling along with SI. In particular this study pioneered the use of primary cells for generating novel antibodies by SI. We established that somatic cells and BMSCs have a basal level of expression of pluripotency markers. Furthermore, our study indicates that intrinsic signalling mechanisms of BMSCs are intimately linked with extrinsic cues from the microenvironment and that these signals appear to be critical for retaining the expression of genes to maintain cell stemness in long term in vitro culture. This project provides a basis for developing an “artificial niche” required for reversion of commitment and maintenance of BMSC in their uncommitted homeostatic state.

Les premières utilisations du tissu adipeux comme produit de comblement en chirurgie plastique remontent à la fin du 19ème siècle. Depuis quelques décennies, la greffe de tissu adipeux a bénéficié d'un regain d'intérêt utilisant un procédé chirurgical rigoureux. Devant la démonstration de la survie cellulaire et les bons résultats cliniques obtenus, l'utilisation de cette technique s'est élargie à tous les domaines de la chirurgie plastique. Cette technique est simple et efficace et représente actuellement le meilleur moyen de restaurer les défauts de contours et de volume. Récemment, de nouvelles indications utilisant les capacitésrégénératrices du tissu adipeux ont été décrites. Elles concernent la cicatrisation des plaies chroniques et l'amélioration des dystrophies cutanées. Mais la limite de la greffe de tissu adipeux est l'absence de site donneur disponible au prélèvement. Le tissu adipeux est aujourd'hui reconnu comme la source la plus abondante de cellules souches mésenchymateuses multipotentes. Cela a donné un nouvel essor à la médecine régénérative pour réparer, remplacer ou régénérer les tissus et organes endommagés à partirdes cellules souches. Cette régénération se fait soit in-situ après administration des cellules souches, soit après développement in-vitro d'un tissu par ingénierie. Après une présentation du tissu adipeux et ses cellules souches ainsi que leurs applications actuelles en chirurgie plastique, le but de ce travail était de : 1. de clarifier les facteurs influençant les résultats de la greffe adipeuse pour une optimisation de cette technique. 2. d'explorer les possibilités offertes par les cellules souches adipeuses pour la médecine régénérative et l'ingénierie tissulaire, en vue d'une utilisation en chirurgie plastique
The first uses of adipose tissue as filler in plastic surgery started in the late 19th century. In recent decades, the adipose tissue transplantation has received renewed interest using a rigorous surgical procedure. Before the demonstration of cell survival and good clinical results, the use of this technique was extended to all areas of plastic surgery. This technique is simple and effective and is currently the best way to restore the defects of contour and volume. Recently, new indications using the regenerative capacity of adipose tissue have been described. They concern the healing of chronic wounds and the improvement of skin dystrophy. But the limit of the adipose tissue graft is the lack of available donor site for harvesting. Adipose tissue is now recognized as the most abundant source of multipotent mesenchymal stem cells. This gave a boost to regenerative medicine to repair, replace or regenerate damaged tissues and organs from stem cells. This regeneration is done either in-situ after administration of stem cells, after in-vitro development of tissue engineered. After a presentation of adipose tissue and stem cells and their current applications in plastic surgery, the aim of this study was to: 1. clarify the factors influencing the results of fat transplantation to optimize this technique. 2. explore the possibilities offered by ASCs for regenerative medicine and tissue engineering, for use in plastic surgery

’arthropathie dégénérative est une maladie ayant des répercussions socio-économiques majeures chez l’homme et le cheval. Il n’existe pour l’heure aucun traitement curatif de cette maladie, le cartilage articulaire étant dépourvu de pouvoir de cicatrisation spontané. De nombreux espoirs reposent sur l’utilisation de cellules souches mésenchymateuses (CSM), pour leur potentiel pro-régénératif et anti-inflammatoire. Le premier objectif de cette étude était d’évaluer la tolérance des CSM de sang de cordon ombilical (SCO) et de moelle osseuse (MO) dans des articulations saines. L’étude contrôlée en aveugle menée sur 12 chevaux expérimentaux a démontré que l’injection de CSM-MO provoquait significativement plus de signes de réaction inflammatoire que l’injection de CSM-SCO, et que l’injection des CSM, quelle que soit leur origine, provoquait une réaction inflammatoire discrète à modérée, supérieure à celle d’une injection de placébo, avec une grande variabilité individuelle de sensibilité à une même lignée de cellules. Le second objectif était d’évaluer l’efficacité des CSM-MO et -SCO dans un modèle d’arthropathie induite. L’étude contrôlée en aveugle menée sur 8 chevaux expérimentaux a mis en évidence une réduction significative de la progression des signes indicateurs d’arthropathie à l’imagerie après injection de CSM-MO allogéniques par rapport à l’injection du placébo. Ces résultats encourageants, à considérer à la lumière des limites des études menées, indiquent un effet bénéfique des CSM-MO allogéniques dans la prise en charge de l’arthrose chez le cheval. Ils soulignent la nécessité de poursuivre les recherches afin de confirmer ces résultats, et d’optimiser les effets des CSM à travers leur combinaison à un vecteur ou par une approche acellulaire avec administration des nanovésicules qu’elles sécrètent, et considérées être à l’origine de leurs effets thérapeutiques
Osteoarthritis is a common cause of pain and economic loss in both humans and horses. There is currently no curative treatment for osteoarthritis, because of the lack of spontaneous regenerative capacity of the articular cartilage. Mesenchymal stem cells (MSC) based regenerative medicine comes across as a promising strategy given their pro-regenerative and anti-inflammatory potential. The first objective of this study was to evaluate the safety of umbilical cord blood (UCB) and bone marrow (BM) derived MSC in healthy joints. The blind controlled study conducted on 12 experimental horses showed that the injection of BM-MSC caused significantly more signs of inflammatory reaction than the injection of UCB-MSC, and that the injection of MSC, regardless of their origin, caused a discrete to moderate inflammatory reaction, greater than that of the placebo, with great individual variability in sensitivity to the same cell line. The second objective was to evaluate the efficacy of BM-MSC and UCB-MSC in a model of induced osteoarthritis. The blind controlled study conducted on 8 experimental horses showed a significant reduction in the progression of osteoarthritis associated signs with imaging techniques after injection of allogeneic BM-MSC compared to placebo. These promising results, to be considered in light of the limitations of the studies, indicate a beneficial effect of allogeneic BM-MSC in the management of osteoarthritis in horses. They underline the need for further research to confirm these results, and to optimize the effects of MSC through their combination with a vector or through an acellular approach with administration of the nanovesicles they secrete that ared considered to be responsible for their therapeutic effects

Utilisée depuis plus d'un siècle en chirurgie esthétique, la greffe autologue de tissu adipeux, ou lipofilling, est une technique sûre permettant le comblement des tissus mous. Cependant, bien que la technique ait connue de nettes améliorations au cours du temps, les chirurgiens font toujours face à une résorption du greffon qui oblige dans la majorité des cas à planifier plusieurs autres interventions afin que le résultat esthétique soit en adéquation avec les attentes du patient. Le procédé MICROFILL® a été développé dans le but d'augmenter le taux de prise de greffe en favorisant la survie cellulaire au sein du greffon. Cette dernière est optimisée par : un prélèvement et une réinjection de lobules adipeux de petite taille permettant de diminuer l'ischémie et la mauvaise nutrition des cellules - une élimination des éléments délétères (anesthésiques, cytokines inflammatoires) par un protocole de lavages et centrifugations non traumatique. D'autre part, au cours de ces dernières années, le tissu adipeux s'est révélé posséder un pouvoir thérapeutique plus important par l'hébergement de cellules souches mésenchymateuses au fort potentiel. Ces cellules sont présentes en grande quantité et facilement accessibles à partir d'une simple lipoaspiration. Cependant, la lipoaspiration implique bien souvent l'usage d'un anesthésique local et d'un vasoconstricteur qui peuvent nuire aux cellules. Nos études ont en effet montré que la lidocaïne, un anesthésique couramment utilisé, est cytotoxique pour les cellules souches du tissu adipeux, ayant pour effets l'inhibition de la prolifération cellulaire (arrêt du cycle cellulaire en phase G0-G1) et la nécrose des cellules. En revanche, une manipulation appropriée du tissu adipeux, se rapprochant du protocole MICROFILL®, permet de diminuer la mortalité cellulaire. L'effet délétère de la lidocaïne semble lié à l'apparition d'une vacuolisation cytoplasmique dont la nature est à ce jour non élucidée. De plus, la lidocaïne induit également un processus d'autophagie, dont les mécanismes moléculaires d'induction sont eux aussi inconnus et dont la finalité physiologique serait le maintien en vie de la cellule malgré le stress provoqué. Les conclusions de ces études mènent à certaines recommandations à suivre quant à l'usage de la lidocaïne en vue de la réinjection extemporanée de cellules souches adipeuses chez un patient. Aussi, dans le but de traiter les tendinopathies équines, ces études ont permis d'optimiser le protocole de prélèvement du tissu adipeux chez le cheval ainsi que le protocole d'extraction des cellules souches du tissu adipeux. Cette thèse a finalement permis de développer un kit à usage vétérinaire permettant de traiter les tendinopathies équines. Ce nouveau procédé de thérapie cellulaire a été testé chez des chevaux et s'est avéré très prometteur, permettant la régénération de la structure tendineuse et un retour au travail rapide des chevaux
Despite the dark side of obesity in the pathogenesis of metabolic diseases, adipose tissue has been shown to be a good therapeutic tool. First, autologous fat grafting, also named lipofilling, has been used for over a century and represents a safe technique for soft tissue filling. However, although the technique has seen marked improvements over time, surgeons are still facing graft resorption that often requires overcorrection of the treated area or other interventions so that the aesthetic result is in line with expectations of the patient. Thus, MICROFILL® process has been developed in order to increase the rate of engraftment by promoting cell survival within the graft. The latter is enhanced by: - sampling and reinjection of small fat lobules in order to reduce ischemia and poor nutrition of the cells- elimination of deleterious elements (anesthetics, inflammatory cytokines) by a non-traumatic protocol involving soft centrifugations and washings. Furthermore, in recent years, adipose tissue has been found to have a greater therapeutic power by hosting mesenchymal stem cells with great potential. These adipose stem cells (ASCs) are present in large quantities and can be easily obtained from a simple liposuction. However, liposuction procedure often involves the use of a local anesthetic and a vasoconstrictor that can harm cells. Our studies have shown that lidocaine, an anesthetic commonly used, exerts cytotoxic effects on adipose stem cells, inhibiting cell proliferation (cell cycle arrest in G0-G1 phase) and inducing necrosis. Nonetheless, appropriate handling of adipose tissue, quite similarly to MICROFILL® protocol, reduces cell death. The deleterious effects of lidocaine appear to be related to the occurrence of cytoplasmic vacuolization whose nature is so far unclear. In addition, lidocaine also induces a process of autophagy, including molecular mechanisms of induction also unknown and whose physiological purpose could be cell survival despite the stress. The findings of these studies lead to some recommendations to follow regarding the use of lidocaine for the extemporaneous reinjection of ASCs in a patient. Also, in order to treat equine tendinopathy, these studies have been used to optimize adipose tissue harvest by liposuction on horses and the protocol of extraction of ASCs.Finally, this thesis has allowed developing a kit for veterinary use to treat equine tendinopathy. This new method of cell therapy has been tested in horses and has shown very promising results for tendon regeneration, knowing that treated horses could rapidly return to work

Traumatic injuries resulting in peripheral nerve lesions often require a graft to bridge the gap. Although autologous nerve graft is still the first choice strategy in reconstructions, it has the severe disadvantage of the sacrifice of a functional nerve. Cell transplantation in a bioartificial conduit is an alternative strategy to create a favourable environment for nerve regeneration. Among adult stem cells, adipose-derived stem cells (ASC) are a useful tool in regenerative medicine as they can be induced towards multiple mesodermal and nonmesodermal lineages, being recently differentiated into cells showing Schwann cell-like morphology, glial cell markers and increased neurotrophic potential. The first two chapters of this work describe in vivo applications of Schwann cell-like differentiated ASC (dASC), seeded into biodegradable nerve guides made of fibrin, investigating both brief (2 weeks) and long (4 months) term effects on the regenerating nerves. Comparison was carried out with similarly differentiated bone marrow mesenchymal stem cells (dMSC), Schwann cells (SC)and empty fibrin conduits, as well as with autologous nerve grafts. Regeneration was evaluated in a 1cm gap total axotomy sciatic nerve injury model on rats. Results showed that dASC could improve regeneration distance in a similar manner to other regenerative cells inthe brief term. This effect was maintained and strengthened in the long term, where nerve morphology, spinal motoneurons regeneration, protection from muscle atrophy and electrophysiological performances of regenerated nerves were analysed. dASC positive effects lasted in the long term with functional results comparable to the autologous nerve grafts, which served as controls. The third chapter focuses on the possibility to further improve dASC regenerative performances using fibronectin and laminin, two key extracellular matrix (ECM) molecules involved in nerve regeneration, with the future aim to optimize cell host, directional cues and neurotrophism of tissue engineered conduits. Fibronectin and laminin protected dASC from stress-induced cell death in vitro, significantly increasing cell adhesion and viability. Laminin significantly improved neurotrophic properties of dASC enhancing neurite outgrowth of both primary sensory neurons and NG108-15 neurons co-cultured with dASC, suggesting a further activation of the neurotrophic effect of dASC by ECM molecules. These improved effects were increased when a direct contact was established between the laminin substrate, dASC and neurons, suggesting a primary role of laminin in contact signalling, finally boosting the neurotrophic potential of dASC. Further studies will be needed to clarify the interactions between dASC and the complexniche of peripheral nerve regeneration, including the ECM molecules. However, the neurotrophic potential of dASC expressed in both in vitro and in vivo experiments opens wide perspectives in tissue engineering applications among new methods to enhance peripheral nerve repair.

Master of Science
Department of Clinical Sciences
Walter Renberg
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.

The objective of the study: To characterize stem cells isolated from mouse skeletal muscle and evaluate their regenerative potential to repair injured bone and articular cartilage in a mouse and rat animal model. The aims of the study: 1. Isolate MDSCs from mouse skeletal muscle, to determine their phenotypic characteristics and differentiation potential in vitro. 2. Evaluate osteogenic and chondrogenic regeneration capacity of MDSCs genetically engineered to express bone morphogenetic protein 4 (BMP4). 3. Evaluate osteogenic and chondrogenic differentiation potential of MDSCs-expressing BMP4 in vivo. 4. Investigate the possibility of MDSCs-expressing BMP4 and vascular endothelial growth factor (VEGF) to enhance bone repair. 5. Investigate the possibility of MDSCs-expressing BMP4 and anti-angiogenic factor sFlt1 to enhance articular cartilage repair. The brief overview of the study This investigation addresses important issues regarding: 1) stem cell isolation from skeletal muscle and their specific characteristics that enable us to identify these cells from other populations of stem cells in skeletal muscle; 2) the efficiency of muscle-derived stem cell-mediated bone and cartilage formation in vivo using retroviral vectors to express bone morpho-genetic protein 4 (BMP4); 3) the enhancement of osteogenic and chondrogenic potential of BMP4-secreting muscle-derived stem cells by addition of cells engineered to express vascular endothelial growth factor (VEGF) or its... [to full text]
Skeleto raumuo yra patogus ir lengvai prieinamas kamieninių ląstelių šaltinis. Satelitinės ląstelės, kurios kitaip vadinamos raumenų kamieninėmis ląstelėmis ir yra linkusios diferencijuoti miogenine linkme, dalyvauja raumens regeneracijoje ir gali savarankiškai atsinaujinti. Mūsų laboratorijoje izoliuotos raumeninės kilmės kamieninės ląstelės (RKKL) yra laikomos satelitinių ląstelių pirmtakėmis, tačiau nuo jų skiriasi. RKKL gali diferencijuoti ne tik miogenine linkme, bet ir kitomis linkmėmis (kauline, kremzline, riebalinio audinio, nervų, endotelio ir kraujodaros), ir joms yra būdinga ilgalaikė proliferacija, savarankiškas atsinaujinimas, privilegija imuninės sistemos atžvilgiu ir atsparumas oksidacijos sukeltam stresui. Šio darbo metu atlikti tyrimai atsako į labai svarbius klausimus, susijusius su: 1) RKKL išskyrimu ir jų specifinių savybių, leidžiančių šias ląsteles atskirti nuo kitų skeleto raumenų kamieninių ląstelių populiacijų, nustatymu; 2) genetiškai modifikuotų RKKL sukeliamo kaulų ir kremzlių formavimosi in vivo veiksmingumu, naudojant retroviruso vektorių kaulų morfogenezės baltymo 4 (BMP4) raiškai; 3) RKKL osteogeninio ir chondrogeninio pajėgumo stiprinimu, vienu metu naudojant ląsteles, išskiriančias BMP4, ir ląsteles, išskiriančias kraujagyslių endotelio augimo faktorių (VEGF) arba sFlt1. Šio tyrimo naujumas yra tas, jog iš esmės pirmą kartą parodyta, kad iš išgrynintų pelės skeleto raumenų ląstelių (PP6) populiacijos, išskirtos ląstelių sukibimo su... [toliau žr. visą tekstą]

Bone marrow-derived mesenchymal stem cells (MSCs) have been investigated as marker of liver regeneration in an experimental model of acute liver failure (ALF). This model was created in Wistar rats through an intraperitoneal injection of carbon tetrachloride (CCl4). MSCs (3 million) were collected by long bones of 10 Wistar rats and then intravenously infused 24 hours after induction of ALF in 16 rats, Group A. In control Group B, 16 rats received a peritoneal injection of CCl4, and an intravenous infusion of normal saline. All rats were sacrificed at 2, 3, 4 and 7 days post-CCl4-injection. To assess the efficacy of the induction of the ALF, 4 rats Group (C) were sacrified 1 day after poisoning. Biochemical markers (BM) of liver function as platelets counts and pathology examination of the procured livers were staged on post-infusion-days 2, 3, 4 and 7. In Group A platelets count was higher than in the untreated control Group on post-CCl4-infusion day 2nd (p = 0.02) and 3rd (p=0.001). Also The trend of BM as transaminases was higher in the non-treated Group B, GOT (p=0.002), GPT (p<0.0001). Pathology examination showed greater grade of hepatocellular necrosis, and neutrophilic infiltration in Group B (p=0.02). MSCs infusion determined a less aggressive picture of hepatic damage.

Le malattie cardiovascolari e il loro evoluzione in arresto cardiaco, sono una della principali cause di morte a livello mondiale. Il diabete è spesso associato all'alterazione dei substrati metabolici i quali sono in grado di modificare l'omeostasi dei tessuti ed alterare le popolazioni di precursori cellulari. La maggior parte dei tessuti adulti presenta delle sotto-popolazioni di progenitori mesenchimali che, a causa delle loro proprietà rigenerative, sono un'interessante fonte di cellule per la medicina rigenerativa e la terapia cellulare. Recentemente è stato dimostrato che, in seguito ad un arresto cardiaco, l'iniezione di cellule mesenchimali può migliorare la contrattilità cardiaca in topo e ratto. Lo scopo di questa tesi è stato di isolare, caratterizzare e differenziare cellule mesenchimali di tessuto adiposo (ADMSCs) e cellule derivate da cardiosfere (CDCs) e determinare l'effetto di una dieta ad alto contenuto di grassi su queste due popolazioni cellulari. Da tessuto atriale di topo sono state isolate EDCs (Explant-derived cells) e CDCs, mentre dal tessuto adiposo inguinale, dopo digestione con collagenasi di tipo2, sono state isolate ADMSCs. Quest'ultime, dopo espansione in vitro, contengono un numero maggiore di cellule CD90+ (47% vs 87%) e minore di DDR2+ and CD45 rispetto a cellule appena isolate (rispettivamente 9% e 20% vs 38% e 42%). Alcuni medium sono stati testati per verificare la capacità differenziativa delle ADMSCs, di questi solo il medium con TGFβ è stato in grado di aumentare l espressione di geni cardiaci. L'espansione di ADMSCs in ipossia ha aumentato la velocità di proliferazione e ha modificato il profilo di marker cellulari espresso dalle ADMSCs. E' stato inoltre notato un numero più elevato di cellule positive per DDR2 e minore di CD45+ e CD90+ rispetto a ADMSCs espanse in normali condizioni di ossigeno. L'espansione di fibroblasti cardiaci in vitro e CDCs ha rivelato una similarità tra queste due popolazioni, entrambe aumentano la velocità di proliferazion e la capacità clonogenica con l'aumentare dei passaggi in cultura ed esprimono marker mesenchymali e marker espressi da fibroblasti come CD90 e DDR2. Le CDCs hanno dimostrato di essere in grado di acquisire un fenotipo cardiaco in vitro, aumentando l'espressione di cardiac actin e troponin T, tuttavia non sono state osservate cellule con spontanea attività contrattile. Dopo quattro mesi di dieta ad alto contentuto di grassi (55% grasso, HFD) i topi presentano aumentati livelli plasmatici di glucosio, colesterolo e insulina e diminuiti livelli di lattato. Un numero significativamente maggiore di ADMSCs sono stae isolate da animali HFD e il numero di ADMSCs correla con i livelli pasmatici di glucosio, colesterolo e lattato. I livelli di espressione di CD45, DDR2 e CD105 sono aumentati in ADMSCS da topi con dieta ad alto contenuto di grassi e la loro funzionalità e capacità differenziativa è risultata essere leggermente diminuita. Nelle CDCs non sono state riscontrate differenze nè nell'espressione di marker nè nella loro funzionalità. Per concludere, 4 mesi di dieta HFD sono in grado di indurre un fenotipo diabetico in topi C57 Black 6. La dieta HFD ha aumentato il numero di ADMSCs ma ha modificato le percentuali di sottopopolazioni all interno di questa popolazione, inoltre è stata registrata un diminuzione nella loro capacità differenziativa. Al contrario, le CDCs non sono state influenzate dal fenotipo diabetico.
Cardiovascular diseases, and the progression to heart failure, are one of the leading cause of death. Diabetes is often associate with cardiovascular complications because of the disturbed substrate metabolism that can alter the homeostasis of the tissues and modify the progenitor cell populations. Most adult tissues have a mesenchymal progenitor cell subpopulation that represents a proportion of the total cell number and which, because of its regenerative properties, is an attractive source for cell therapy. Recently it has been proved that injection of mesenchymal cells improve contractile function in rodents following myocardial infarction. The aim of this study was to isolate, characterize and differentiate adipose-derived mesenchymal stem cells (ADMSCs), and cardiosphere-derived cells (CDCs), and to determine the effect of simply a high fat diet on these two mesenchymal populations. Cardiac explant-derived and cardiosphere-derived cells (EDCs, CDCs) were cultured from atrial tissue and adipose stem cells were cultured from epididymal fat depots after collagenase digestion. Cultured ADMSCs contained more CD90+ cells (47% vs 87%) and fewer DDR2+ and CD45+ cells compared to freshly isolated ADMSCs (respectively 9% and 20% vs 38% and 42%). Various media were tested to validate the differentiation capacity of adipose mesenchymal cells, but only the TGFβ-supplemented medium was able to increase the expression of cardiac specific genes. Hypoxia increased cell proliferation and changed the surface marker profile of ADMSCs; more DDR2+ cells and fewer CD45+ and CD90+ cells were found compared to normoxic ADMSCs. In vitro expansion of neonatal cardiac fibroblasts and cardiosphere-derived cells revealed a similarity between these two cell populations, both increased proliferation and clonogenic capacity with time in culture and expressed mesenchymal/fibroblast markers such as CD90 and DDR2. CDCs were able to acquire a cardiac phenotype in vitro, increasing gene expression of cardiac actin and troponin T, however no beating cells were observed. After 4 months of high-fat diet (55% fat; HFD,) mice had raised fed plasma glucose, cholesterol and insulin levels and decreased plasma lactate. Significantly more ADMSCs were obtained from high fat fed animals and ADMSC numbers correlated with plasma glucose, cholesterol and lactate. Expression of CD45, DDR2 and CD105 were increased in ADMSCs from high fat fed mice and the functional properties and differentiation capacity were slightly decreased. No differences in surface marker expression and functional properties were detected between high fat and chow diet mice CDCs. In conclusion, four months of HFD induced a diabetic phenotype in C57 Black 6 mice. The high fat diet increased ADMSCs yield but modified the balance of ADMSCs populations and decreased their differentiation capacity. In contrast, cardiac progenitor cells were unaffected by induction of the diabetic phenotype.

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

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

La proprietà rigenerativa dell innesto di grasso è stata descritta nella leteratura medica. Tuttavia, non è chiaro se i risultati clinici sono attribuibili alle cellule staminali o sono collegati ad altri componenti del tessuto adiposo. Questo lavoro si propone di analizzare i cambiamenti istologici e ultrastrutturali della cute e del tessuto sottocutaneo del viso invecchiati, dopo iniezione di innesto adiposo in aggiunta alla sua frazione vasculo-stromale (grasso arricchito con la FVS), ottenuto per centrifugazione, e di confrontare i risultati con quelli ottenuti con iniezione di cellule staminali mesenchimali espanse derivate da tessuto adiposo. Questo studio è stato condotto in venti (20) pazienti consecutivi, candidati per ritidectomia, di età compresa tra i 45 e 65 anni. I pazienti sono stati sottoposti a prelievo di grasso mediante liposuzione dalla regione addominale. L'iniezione di grasso e la sua frazione vasculo-stromale o delle cellule staminali mesenchimali espanse è stata eseguito nelle zone preauricolari. Frammenti di pelle sono stati rimossi prima e 3 mesi dopo ogni trattamento e analizzati mediante microscopia ottica ed elettronica.Dopo il trattamento con il grasso arricchito con sua frazione vasculo-stromale, la cute ha mostrato una diminuzione della rete di fibre elastiche invechiate interessate da elastosi e la comparsa di nuove fibre elastiche oxytalan nel derma papillare. L'esame ultrastrutturale evidenzia un'architettura tridimensionale modificata del derma reticolare e la presenza di un letto microvascolare più ricco. Sono stati osservati risultati analoghi in seguito al trattamento con cellule staminali mesenchimali espanse.Questo studio dimostra che il trattamento sia con grasso arricchito con la frazione vasculo-stromale che con cellule staminali mesenchimali espanse modifica l’architettura del derma e rappresenta un effetto di ringiovanimento della cute.
The regenerative property of fat grafting has been described. However, it is not clear whether the clinical results are attributable to the stem cells or are linked to other components of the adipose tissue. This work is aimed at analysis of the histologic and ultrastructural changes of aged facial skin after injection of fat graft in addition to its stromal vascular fraction (SVF-enriched fat), obtained by centrifugation, and to compare the results with those obtained by the injection of expanded adipose-derived mesenchymal stem cells. This study was performed in twenty (20) consecutive patients who were candidates for face lift and whose ages ranged between 45 and 65 years. The patients underwent sampling of fat by liposuction from the abdominal region. The injection of fat and its stromal vascular fraction or expanded mesenchymal stem cells was performed in the preauricular areas. Skin Fragments were removed before and 3 months after each treatment and analyzed by optical and electron microscopy.After treatment with the autologous lipidic component and stromal vascular fraction (SVF-enriched fat), the skin showed a decrease in elastic fiber network (elastosis) and the appearance of new oxytalan elastic fibers in papillary dermis. The ultrastructural examination showed a modified tridimensional architecture of the reticular dermis and the presence of a richer microvascular bed. Similar results following treatment with expanded mesenchymal stem cells were observed.This study demonstrates that treatment with either fat and stromal vascular fraction (SVF-enriched fat) or expanded mesenchymal stem cells modifies the pattern of the dermis, representing a skin rejuvenation effect.

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

Spinal cord injury (SCI) is a life-changing and debilitating medical condition. Currently, there is no definitive treatment. Novel regenerative medicine therapies have emerged in order to treat SCI. In this field, hydrogels are promising tools due to its high versatility of applications and design. Brain tissue has some particular characteristics such as its viscoelastic microenvironment and its soft extracellular matrix (ECM). Hydrogels can be designed to suit these characteristics and, therefore, be used to mimic brain tissue. Herein, we investigated the potential of a methacryloyl platelet lysates (PLMA) hydrogel to be applied as scaffold for axonal regeneration or as a carrier of cells. We observed that neurons do not adhere easily to the surface of the PLMA hydrogel alone. However, when the gel is coated with adhesives proteins like laminin or poly-D-lysine we observed that a large number of neurons adhere to its surface. Moreover, our data suggests that PLMA100 20% hydrogel can be used as a carrier of cortical neurons, which is of great relevance given that it can be used to replaced dead neurons. Additionally, adipose-derived stem cells (ASCs) are appealing cells to be encapsulated in PLMA100 hydrogels due to their effects on neurites outgrowth. In order to accomplish functional recovery, it is required that axonal regeneration be followed by synapse formation. However, ASCs’ synaptogenic potencial continues undetermined. In line with this, we analyzed the ability of ASCs to promote synapse formation. Our results indicate that ASCs are capable of inducing presynaptic clusters, a hallmark of synapse formation. PLMA hydrogel might possibly be used as a scaffold for axonal regeneration and a carrier for neuronal cells, making it a strong contender for development of future novel neuro-regenerative therapies.
A lesão da medula espinhal é uma condição médica debilitante que transforma negativamente a vida dos pacientes. Atualmente, não há um tratamento eficaz. Por esta razão, novas terapias de medicina regenerativa surgiram com o intuito de tratar esta condição. Nesta área, os hidrogéis são ferramentas promissoras devido à sua versatilidade de aplicações e design. O tecido cerebral tem caraterísticas particulares, como o seu microambiente viscoelástico e a sua matriz extracelular suave/mole. Os hidrogéis podem ser produzidos de forma a cumprir estas caraterísticas e, assim, serem usados de forma a mimetizar o tecido cerebral. Neste trabalho investigámos o potencial do hidrogel de metacriloílo e lisados de plaquetas (PLMA) ser aplicado como um scaffold para regeneração axonal ou como um transportador de células. Observámos que os neurónios não aderiram facilmente à superfície do hidrogel PLMA. No entanto, quando o gel é revestido com proteínas adesivas como a lamina ou a poli-D lisina (PDL) observámos que um maior número de neurónios aderiu à sua superfície. Além disso, os nossos resultados sugerem que o hidrogel PLMA100 20% pode ser usado como um transportador de neurónios corticais, o que é de grande relevância uma vez que pode ser usado para substituir neurónios degenerados. Adicionalmente, as células mesenquimais derivadas do tecido adiposo (ASCs) são células atrativas para serem encapsuladas nos hidrogéis PLMA100 devido aos seus efeitos no crescimento de neurites. De forma a obter uma recuperação funcional, é necessário que a regeneração axonal seja seguida por formação de sinapses. No entanto, o potencial sinaptogénico das ASCs continua inexplorado. Deste modo, analisámos a capacidade das ASCs de promover a formação de sinapses. Os nossos resultados indicam que as ASCs são capazes de induzir clusters pré-sinápticos, uma das principais caraterísticas da formação de sinapses. O hidrogel PLMA poderá possivelmente ser usado como um scaffold para regeneração axonal e um transportador para células neuronais, tornando-o um forte candidato no desenvolvimento de futuras terapêuticas neuro-regenerativas.
Mestrado em Biomedicina Molecular

我們假設，將scleraxis (Scx)基因轉導入肌腱來源的幹細胞（TDSC），製成TDSC-Scx細胞系。TDSC-Scx會促進肌腱修復。本研究的目的在於探索Scx促進TDSC成肌腱分化的作用，以及TDSC-Scx對肌腱修復的促進作用。
使用慢病毒載體將Scx轉導入TDSCs，不含Scx的空載作為對照也轉導入TDSCs，用載體上帶有的抗性基因，殺稻瘟菌素對細胞進行篩選。分別建成TDSC-Scx和TDSC-Mock細胞系。Scx 的表達分別用定量PCR以及免疫螢光在mRNA和蛋白水準進行鑒定。TDSC-Scx成肌腱，成軟骨和成骨方向的分化能力用定量PCR檢驗。用大鼠臏腱視窗損傷模型進行了細胞移植試驗，測試TDSC-Scx對肌腱損傷的修復作用。實驗分為三組：（1）支架組，（2）空載體組，（3）Scx組。在細胞移植後的第二，四，八周，收集正在修復中的肌腱樣品，進行植入細胞的存留狀態，鈣化，組織學和生物力學測驗。
TDSC-Scx比TDSC-Mock有更強的成肌腱分化能力。但是，在成軟骨-成骨分化方面，沒有結論。在動物試驗，植入的細胞在第二周仍然可見，但自第四周起，就不見了。在第八周，各組均有個別樣品輕微異位鈣化，但各組別間並無顯著差異。在早期，TDSC-Scx組比空載體組和支架組合得來更好的修復肌腱的能力。
TDSC-Scx可能促進肌腱損傷的早期修復。
We hypothesized that transduction of tendon-derived stem cell (TDSC) with scleraxis (Scx) might promote its tenogenic differentiation and promote better tendon repair compared to TDSC without Scx transduction. This study thus aimed to investigate the effect of Scx transduction on the tenogenic differentiation of TDSC and the effect of the resulting cell line in the promotion of tendon repair.
TDSCs were transduced with lentivirus-mediated Scx or empty vector and selected by blasticidin. The mRNA and protein expression of Scx were checked by qRT-PCR and Immuno fluorescence, respectively. The expression of different lineage markers were examined by qRT-PCR. A rat patellar tendon window injury model was used. The operated rats were divided into 3 groups: (1) scaffold-only group, (2) TDSC-Mock group and (3) TDSC-Scx group. At week 2, 4 and 8 post-transplantation, the repaired patellar tendon was harvested for ex vivo fluorescent imaging, vivaCT imaging, histology, or biomechanical test.
TDSC-Scx consistently showed higher expression of tendon-related markers compared to TDSC-Mock. However, the effect of Scx transduction on the expression of chondro-osteogenic markers was less conclusive. The transplanted TDSCs could be detected in the window wound at week 2 but not at week 4. Ectopic ossification was detected in some samples at week 8 but there was no difference among different groups. The TDSC-Scx group promoted early tendon repair histologically and biomechanically compared to the scaffold-only group and the TDSC-Mock group.
TDSC-Scx might be used for the promotion of early tendon repair.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Tan, Chunlai.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2013.
Includes bibliographical references (leaves 66-70).
Abstracts also in Chinese.
Chapter Thesis/Assessment Committee --- p.i
Acknowledgment --- p.ii
Publication --- p.vi
Abstract --- p.vii
摘要 --- p.viii
Chapter Chapter 1 --- Tendon injury and tendon tissue engineering --- p.1
Chapter 1.1 --- Anatomy of tendon --- p.1
Chapter 1.2 --- Epidemiology of tendon injury --- p.3
Chapter 1.3 --- Process and problems of tendon healing --- p.4
Chapter 1.4 --- Current treatment and cell-based therapy for tendon repair --- p.5
Chapter 1.5 --- Transcriptional factor Scleraxis and tendon --- p.8
Chapter 1.5.1 --- Helix-loop-helix (HLH) and bHLH proteins --- p.8
Chapter 1.5.2 --- Scleraxis --- p.9
Chapter 1.6 --- Research focus and implications --- p.11
Chapter 1.7 --- Hypotheses and objectives of this study --- p.12
Chapter 1.8 --- Clinical significance --- p.13
Chapter Chapter 2 --- Materials and Methods --- p.14
Chapter 2.1 --- Study Design --- p.14
Chapter 2.2 --- Establishment of TDSC-Scx cell line --- p.15
Chapter 2.2.1 --- Isolation of TDSC and cell culture --- p.15
Chapter 2.2.2 --- Establishment of TDSC-Scx cell line --- p.17
Chapter 2.2.2.1 --- Construction of plasmid --- p.17
Chapter 2.2.2.2 --- Transfection --- p.23
Chapter 2.2.2.3 --- Infection --- p.24
Chapter 2.2.2.4 --- Selection --- p.24
Chapter 2.2.2.5 --- Characterization of TDSC-Scx and TDSC-Mock --- p.25
Chapter 2.2.2.5.1 --- qRT-PCR --- p.25
Chapter 2.2.2.5.2 --- Immune-fluorescent (IF) --- p.26
Chapter 2.2.2.6 --- Lineage marker expression --- p.26
Chapter 2.2.3 --- Data analysis --- p.29
Chapter 2.3 --- The effect of TDSC-Scx on healing in a patellar tendon window injury model --- p.30
Chapter 2.3.1 --- Animal surgery --- p.30
Chapter 2.3.2 --- Ex Vivo Fluorescence Imaging --- p.32
Chapter 2.3.3 --- vivaCT --- p.33
Chapter 2.3.4 --- Histology --- p.33
Chapter 2.3.5 --- Biomechanical test --- p.35
Chapter 2.3.6 --- Data analysis --- p.37
Chapter Chapter 3 --- Results --- p.38
Chapter 3.1 --- Generation of TDSC-Scx and TDSC-Mock cell lines --- p.38
Chapter 3.1.1 --- Plasmid --- p.38
Chapter 3.1.2 --- Cell morphology --- p.38
Chapter 3.1.3 --- Expression of Scx --- p.38
Chapter 3.1.4 --- Expression of chondro-/osteo-/tenogenic markers --- p.39
Chapter 3.2 --- The healing effect of TDSC-Scx on a patella tendon window injury model --- p.40
Chapter 3.2.1 --- The fate of transplanted cells --- p.40
Chapter 3.2.2 --- Ossification --- p.40
Chapter 3.2.3 --- Histology --- p.40
Chapter 3.2.3.1 --- Fiber arrangement --- p.41
Chapter 3.2.3.2 --- Cellularity --- p.41
Chapter 3.2.3.3 --- Cell alignment --- p.42
Chapter 3.2.3.4 --- Cell rounding --- p.42
Chapter 3.2.3.5 --- Vascularity --- p.43
Chapter 3.2.3.6 --- Fiber structure --- p.43
Chapter 3.2.3.7 --- Hyaline degeneration --- p.43
Chapter 3.2.3.8 --- Inflammation --- p.44
Chapter 3.2.3.9 --- Ossification --- p.44
Chapter 3.2.4 --- Biomechanical properties --- p.44
Chapter Chapter 4 --- Discussion --- p.55
Chapter 4.1 --- In vitro --- p.55
Chapter 4.1.1 --- Scx transduction did not lead to morphological change in TDSCs --- p.55
Chapter 4.1.2 --- Scx transduction led to higher expression of Scx mRNA --- p.55
Chapter 4.1.3 --- TDSC-Scx expressed higher levels of tenogenic markers --- p.56
Chapter 4.2 --- In vivo --- p.58
Chapter 4.2.1 --- The fate of transplanted cells --- p.58
Chapter 4.2.2 --- Ossification was vague --- p.58
Chapter 4.2.3 --- TDSC-Scx promoted tendon healing --- p.58
Chapter 4.2.4 --- Biomechanical properties --- p.59
Chapter 4.2.5 --- Clinical consideration --- p.60
Chapter 4.3 --- Similar studies --- p.61
Chapter Chapter 5 --- Limitations --- p.63
Chapter 5.1 --- Direct Scx protein expression and function information unavailable --- p.63
Chapter 5.2 --- Differentiation assay --- p.64
Chapter Chapter 6 --- Conclusion --- p.65
Reference --- p.66
Appendix --- p.71

Dissertação de Mestrado, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2016
A Engenharia de Tecidos é uma área interdisciplinar da Medicina Regenerativa que visa criar e desenvolver substitutos biológicos para reparar, manter ou melhorar a função de tecidos lesados, com base em princípios da engenharia conjugados às ciências da vida. A Engenharia de Tecidos tira partido das propriedades de estruturas tri-dimensionais (3D) que combinadas com células estaminais pretendem recriar um ambiente semelhante ao nativo de um tecido. Estas estruturas 3D (scaffolds) são produzidas com materiais de origem natural ou sintéticos, que idealmente terão as propriedades físicas, mecânicas e químicas para promoverem o melhor desempenho dessas células e portanto a regeneração dos tecidos. Na última decada as células estaminais mesenquimais foram amplamente utilizadas na Engenharia de Tecidos, pois têm o potencial de proliferarem e se manterem indiferenciadas com a capacidade de se auto-renovarem e/ou diferenciarem em diferentes tipos de células. Existem várias fontes de células estaminais com caracteristicas diferentes utilizadas em TE, em que as que apresentam maior impacto são as derivadas da medula espinal, do sangue e do tecido adiposo, entre outras localizadas em diferentes zonas do corpo humano. A combinação entre scaffolds e células estaminais mesenquimais apresentam algumas limitações tais como a indução de uma resposta inflamatória após transplante e o facto da grande maioria dos biomateriais utilizados não serem biofuncionais. A Engenharia de Cell sheets é a alternativa, pois utiliza a matriz extracelular depositada pelas células como scaffold natural para a regeneração de diferentes tecidos. O conceito de cell sheet foi introduzido por Teruo Okano e os seu colaboradores nos anos 90 no Japão. Estas cell sheets são produzidas em superficies revestidas com um polímero não iónico sensível à temperatura. Quando a temperatura é inferior a 32ºC a superficie fica hidrofílica, promovendo o destacamento das células em folha (cell sheets) sem recorrer ao uso do tradicional tratamento enzimático. Assim, esta tecnologia permite obter cell sheets com uma organização celular própria e coesiva, dado que as interações célula-celula e célula-matriz extracelular são mantidas. Em estudos anteriores no nosso laboratório foram produzidas cell sheets a partir das células estaminais humanas derivadas do tecido adiposo (hASCs). As cell sheets de hASCs quando transplantadas para feridas excisionais em pele de ratinho, induziram a formação de cristas epiteliais, normalmente apenas encontradas em pele humana, e a formação de um número significativo de folículos pilosos. Tendo em consideração estes resultados e antevendo as possibilidades de ter estas cell sheets disponiveis para uso imediato na clínica (off-the-shelf) a sua criopreservação seria vantajosa. Assim, o objectivo deste estudo foi definir um método de criopreservação que não só permita a preservação da viabilidade das hASCs mas também a integridade da matriz extracelular das cell sheets, que se sabe ser critico para garantir a sua funcionalidade, após transplantação. De modo a minimizar um potencial efeito adverso do processo de criopreservação, o método testado teve como base o método standard slow cooling rate, utilizado na criopreservação de células em suspensão. Foram então definidas duas condições de criopreservação, a condição standard, com 10% do crioprotector Dimethylsulfoxide (DMSO), e a condição exprimental, com 5% DMSO. Com o objectivo reduzir a toxicidade para as células criopreservadas. O efeito das condições de criopreservação na viabilidade celular foi analisado depois das cell sheets serem dissociadas, tendo sido demonstrado que ambas as condições de criopreservação não afectam de forma significativa a viabilidade celular. No entanto, verificou-se que a organização do citoesqueleto das células na cell sheet sofreu alterações depois da criopreservação em ambas as condições, verificando-se uma desorganização mais acentuada na condição standard. Verificou-se ainda que ambas as condições de criopreservação afetam a integridade da matriz extracelular das cell sheets, embora pareça que a condição standard afecte de um modo mais significativo. Mais ainda ambas as condições de criopreservação afectaram a quantidade total de proteínas. Potencialmente, este resultado está associado com as proteínas da matriz laminina, fibronectina e colagéneo I. De facto, a expressão destas proteínas excepto o colagéneo foi afectado tanto a nível molecular e proteico. Mais ainda verificamos a expressão dos seus genes por reacção em cadeia da polimerase (PCR). Onde a nível molecular o gene da laminina está sobre expressa em ambas as condições de criopreservação, o gene da fibronectina apenas na condição exprimental e o gene do colagéneo não sofre alterações significativas em ambas as condições de criopreservação. Considerando que as propriedades mesenquimais das células que compõe as cell sheets, são determinantes nos resultados previamente observados, a expressão dos marcadores típicos após a criopreservação foi analisado a nível genético e proteico, usando PCR e citometria de fluxo respectivamente. Com base nos resultados obtidos, que demonstram que a matriz extracelular é significativamente afectada pelo processo de criopreservação, será necessário testar diferentes protocolos e diferentes métodos de criopreservação, no sentido de se obter uma melhor preservação da integridade estrutural da cell sheet, e portanto garantir a sua funcionalidade após transplantação.
Regenerative Medicine (RM) englobes the multidisciplinary and interdisciplinary field of Tissue Engineering (TE) that aims to repair or enhance tissue or organ function. The field of TE takes advantage of the properties of three-dimensional (3D) structures that combined with different cells allow to recreate the native environment of a tissue. These 3D structures are produced with synthetic or natural materials, and aimed to have the ideal physical, mechanical and chemical proprieties for a better performance of cells, thus promoting the tissue regeneration. In context of TE, stem cells (SCs) are combined with the 3D structures or scaffolds, allowing the creation of viable and complex substitutes for tissue regeneration. The SCs have been largely used in TE due to its high proliferative rate, self-renewal capacity, ability to differentiate into different cell lineages. In the last decade the mesenchymal stem cells have been widely used in tissue engineering, they have the potential to proliferate and maintain undifferentiated with the ability to self-renew and / or differentiate into different cell types. There are various sources of stem cells with different characteristics used in TE, where they have the greatest impact are derived from spinal cord, blood and adipose tissue, among others located in different areas of the human body. The use of scaffolds, might promote an inflammatory response after transplantation, and the major part of used biomaterials are not biofunctional. One of the alternatives to solve this problem is the production of constructs without the use of traditional biomaterials. The Cell Sheet Engineering is the alternative because it uses the extracellular matrix deposited by the cells as a natural scaffold for the regeneration of different tissues. Cell sheet technology was originally proposed by Okano and co-workers, in early 90’s. This technology takes advantage of thermo-responsive culture dishes that enable reversible cell adhesion to and detachment from the dish surface by a controllable hydrophobicity of the surface. By temperature change, a cell sheet with organized cellular entities and cohesive cell-to-cell and cell-ECM interactions is obtained. In previous studies in our laboratory we generated cell sheets from human stem cells derived from adipose tissue (hASCs) that after transplantation in mice full-thickness excisional skin wounds, induced the formation of rete ridged-like structures and a significant number of hair follicles. Considering these results and envisioning the possibility of having cell sheets available off-the-shelf for immediate use in the clinic, to have these structures cryopreserved would be advantageous. The goal of this work was to define a cryopreservation methodology that allows the preservation of both cells viability and the properties of CS extracellular matrix (ECM). hASCs obtained from three different donors, were cultured in UP cell thermoresponsive dishes, to form hASCs-CS. Different cryopreservation conditions were considered, by varying the concentration of DMSO: i) standard condition with 10% of DMSO used to cryopreserve cell suspension; and ii) experimental condition with 5% of DMSO to reduce the cytotoxicity. The effect of cryopreservation conditions over cell viability was analysed after dissociation of the CS. The results showed that both cryopreservation conditions do not significantly affect cell viability. However the cytoskeleton of cells suffered alterations after cryopreservation in both conditions, which were more evident in the standard condition. It was also found that both cryopreservation conditions affect the integrity of the extracellular matrix of cell sheets, although it appears that a standard condition affecting more significantly. Furthermore, after cryopreservation the amount of total protein, decreased to half, which indicates that both conditions of cryopreservation affects the extracellular matrix content. Potentially, this result is associated with matrix proteins laminin, fibronectin and collagen type I. In fact, these proteins other than collagen was affected both molecular and protein level. Moreover we found the expression of their genes by polymerase chain reaction (PCR). Where the molecular level, the laminin gene is over expressed in both the cryopreservation conditions, the fibronectin gene only in experimental condition and collagen gene does not change significantly in both the cryopreservation conditions. Whereas the properties of mesenchymal cells that comprise the cell sheets are determining the results previously reported, the expression of typical markers following cryopreservation was examined at the genetic and protein level using PCR and flow cytometry, respectively. Based on the results obtained, showing that extracellular matrix is significantly affected by cryopreservation, is to experiment with different protocols and different methods of cryopreservation. In order to obtain a better preservation of the structural integrity of the cell sheet, and thus ensuring its functionality after transplantation. With this thesis, it was possible to open routes to target a suitable cryopreservation methodology applied to hASCs-CS, which enables an off-the-shelf TE and RM strategy.

Indiana University-Purdue University Indianapolis (IUPUI)
In order to develop effective cures for diseases and decipher disease pathology, the need exists to cultivate a better understanding of human development. Existing studies employ the use of animal models to study and model human development and disease phenotypes but the evolutionary differences between humans and other species slightly limit the applicability of such animal models to effectively recapitulate human development. With the development of human pluripotent stem cells (hPSCs), including Human induced Pluripotent stem cells (hiPSCs) and Human Embryonic Stem cells (hESCs), human development can now be mirrored and recapitulated in vitro. These stem cells are pluripotent, that is, they possess the potential to generate any cell type of the body including muscle cells, nerve cells or blood cells. One of the major focuses of this study is to use hiPSCs to better understand and model human retinogenesis. The retina develops within the first three months of human development, hence rendering it inaccessible to investigation via traditional methods. However, with the advent of hiPSCs, retinal cells can be generated in a culture dish and the mechanisms underlying the specification of a retinal fate can be determined. Additionally, in order to use hiPSCs for successful cell replacement therapy, non-xenogeneic conditions need to be employed to allow for fruitful transplantation tests. Hence, another emphasis of this study has been to direct hiPSCs to generate retinal cells under non-xenogeneic conditions to facilitate their use for future translation purposes.

As células estaminais do tecido adiposo (do inglês adipose stem cell (ASC)) são células multipotentes que podem ser isoladas de diferentes locais e podem-se diferenciar em várias linhagens celulares, como adipogénica, osteogénica, condrogénica, entre outras. Estas células são capazes de atingir locais lesionados e exercer um papel importante a nível da regeneração tecidular e de modular a resposta imune pela secreção de moléculas intervenientes nos diferentes mecanismos fisiológicos. O processo de obtenção destas células passa pelo isolamento, cultura e expansão. A partir de uma amostra de gordura, podemos isolar estas células e posteriormente diferenciá-las em diferentes tipos celulares através da aplicação de protocolos de cultura in vitro. Uma vez isoladas, as ASCs podem ser utilizadas por via autóloga ou por via alogénica. Uma boa forma de retirar o máximo partido destas células é através da possibilidade de criopreservação e armazenamento das células estaminais, atendendo a que estas mantêm todo o seu potencial regenerativo, permitindo a sua utilização mesmo após longos períodos de congelamento. Estas células têm a capacidade de modular o sistema imunitário, no sentido de tornar o processo de inflamação mais curto e menos exacerbado, devido a todos os factores secretados pelas ASCs e ajudar a recuperação parcial ou total em determinadas doenças. Na presente dissertação apresenta-se a caracterização dos principais conceitos, as diferentes origens das células estaminais e suas características mais importantes. Destacam-se ainda as propriedades das células estaminais derivadas do tecido adiposo em diferentes espécies. Atendendo ao potencial terapêutico das células estaminais são ainda apresentados dois casos clínicos representativos de aplicações terapêuticas de células estaminais que podem constituir-se como uma evidência terapêutica positiva da aplicação clínica das células estaminais.
The stem cells derived from the adipose tissue (from ASCs) are multipotent cells that can be isolated from different locations and can be differentiated into several cell lineages, such as adipogenic, osteogenic, chondrogenic, among others. These cells are able to reach injured areas and play an important role in the tissue regeneration and modulation of the immune response, due to the secretion of different molecules that play different roles in physiological mechanisms. The process of obtaining these cells relie on the enzymatic or mechanic digestion of the adipose tissue to isolate the ASCs and further culture and expansion in vitro. These cells will then be able to be used via autologous or allogeneic approaches. A good way to take the maximum advantage of these cells is through the possibility of cryopreservation and storage of stem cells, since they keep all their regenerative potential, allowing its use even after long periods of freezing. These cells have the ability to modulate the immune system, triggering a weak to non-existent immune response, shortening the inflammation process due to all factors that ASCs secrete and therefore helping the full or partial recovery of damages to different tissues. The present dissertation presents the characterization of key concepts, the different origins of stem cells and their most important features. It also highlights the properties of stem cells derived from adipose tissue in different species. Two clinical cases of therapeutic applications of stem cells are also presented to demonstrate their therapeutic potential. Notwithstanding the presentation of only two case studies, the obtained results testify the therapeutic potential of stem cell clinical application.

Cataract is an opacification of the eye’s lens. It is the leading cause of blindness and causes life-impacting vision loss for millions of people worldwide. Currently the only treatment is surgical implantation of an intra-ocular lens. Due to the number of annual surgeries this approach is expensive and can result in a range of sightaffecting complications. Difficulties arise in accessing vision-restoring surgery for people with cataract living in developing countries due to inadequate access to cataract surgery. Greater understanding of cataract molecular mechanisms is required for development of anti-cataract drugs that may be easier to access than surgery. Several cataract risk factors have been postulated; however, their specific molecular mechanisms are poorly identified. This is due largely to a lack of functional human lens tissue available for studying cataract. Previous investigations of cataract have used animal models to study lens development and cataract; however, there are significant differences between humans and animal lenses including protein expression and cell membrane composition. Human pluripotent stem (PS) cells are a potential source of human lens cells and an elegant 3-stage protocol for generating lens cells and rudimentary lens tissue was published in 2010. Several groups have attempted to refine this protocol with varying success. Nevertheless, all of these approaches suffer from three main issues: i) production of heterogeneous cultures of human lens and non-lens cells; ii) poorly- or uncontrolled-production/loss of rudimentary lens tissue; and iii) limited or no evidence that the stem cell-derived lens tissues have focusing ability (a defining property of the lens). Very recent work in our laboratory overcame the first of these issues, allowing simple and large-scale production of purified, ROR1-positive human lens epithelial cells (LECs) from heterogeneous cell cultures generated from PS cells. This ROR1+ LEC purification protocol provided a starting point for this thesis to investigate conditions for generating light-focusing human lens tissue in vitro. The studies presented here resulted in a method to produce tens-of-thousands of uniform, transparent and light-focusing human PS cell-derived lens organoids, termed micro-lenses. These micro-lenses appear able to model a clinically-relevant cataract associated with exposure to the cystic fibrosis drug Vx-770. These functional human micro-lenses represent a useful tool for drug discovery and toxicity. In addition to the successes of producing the world-first human PS cell-derived micro-lenses, there were several lessons learned during this project. Repeated failures of the cells to survive and proliferate on RGDS-chitosan film negated some of the first approaches at human lens regeneration (i.e., attempts at producing a chitosan-based explant pair system); these difficulties required consideration of an alternate approach. A wider understanding of lens biology gave rise to the idea of trying to mimic non-human lens developmental in the lab, inspired by teleost lens development. This LEC aggregation method was made possible by thinking laterally and using simple, available, materials to replicate embryonic aspects of teleost lens development. This method ultimately proved successful in producing human PS cellderived micro-lenses. Furthermore, the rapid assessment lesson learned by experiencing some unworkable situations in the course of the project were then applied during the hydrogel 3D growth environment trials. Repeatable aggregate loss in some hydrogels confirmed that the agarose-based gels best supported the developing micro-lenses and facilitated progression of investigation of micro-lenses derived from enriched ROR1 cells. Additionally, this project was not without its share of failures. Reflection upon these challenges and ascertaining the reasons for (and ways to move beyond) them have changed my approach to managing difficulties and improved my analytical abilities. These skills will be applied to my future undertakings.

Beside adipose tissue had been considered a discard product for many years, recently its role as regenerative agent has been widely recognized. Adipose tissue is a connective tissue constituted of adipocytes interspersed with collagen fibers and stromal vascular fraction (SVF), composed of adipose-derived multipotent stromal cells (ASCs), pre-adipocytes, fibroblasts, vascular endothelial cells and immune cells. The regenerative role is played specifically by the SVF and, inside it, especially by the ASCs, by secreting angiogenetic, anti-apoptotic, antiinflammatory and immunomodulatory growth factors. Surgeons use different strategies to reconstruct or repair damaged tissues and organs through adipose tissue. Among them, the most effective are the autologous fat transfer and tissue engineering. In the autologous fat transfer, autologous adipose tissue is harvested from one part of the body, purified through some processing techniques and reinjected where necessary. In tissue engineering, some scaffolds made of natural or synthetic materials are used in combination with ASCs and, sometimes, growth factors to repair or reconstruct tissues. The first experimental part of this doctoral thesis analyzes the in vitro performances of one automated closed device and two different disposable kits to process adipose tissue in comparison with the enzymatic digestion, which is the gold-standard technique. All three methods produce micrografts rich of ASCs. Parameters such as ASCs phenotype, viability, growth and replicative rate have been observed. The second experimental part of this thesis analyzes the in vitro and in vivo performances of different formulations of hyaluronic acid, a natural and very promising material to regenerate tissues, when combined with ASCs. Parameters such as ASCs viability, interaction with the hyaluronic acid material and adipogenesis have been observed.

Cardiovascular disease (CVD) affects more than 70 million Americans and is the number one cause of mortality in the United States. Because the regenerative capacity of adult tissues such as the heart is limited, human embryonic stem cells (hESC) have emerged as a source for potential cardiac therapies. However, despite the use of a variety of biochemical differentiation protocols, current yields of hESC-derived cardiomyocytes (CM) have been low. In the case of hESC-CM, which are inherently electromechanically active, additional forms of inducing a mature cardiac fate have not been fully explored. In order to non-invasively visualize and quantify biochemical, electrical, and mechanical stimulation on hESC-CM differentiation in future studies, a bioreactor imaging system has been developed and is described in this report.
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Mesenchymal stem cells (MSCs) have emerged as a promising strategy to the repair of damaged tissues, due to characteristics such as immunomodulation, anti-inflammatory properties, homing, protective and reparative effects. Additionally, MSCs have shown to exert their therapeutic activity by homing into injured tissues, cellular crosstalk and inducing tissue regeneration response through paracrine signalling, namely through exosomes. This thesis aimed to evaluate the role of the secretome (CM) and exosomes (Exo) derived from umbilical cord tissue (UC)-MSCs for damaged tissue treatment. Three-dimensional (3D) cultures were employed to prime UC-MSCs towards a healing-inducing secretome (CM3D); whereas the mechanism by which Exo/CM induced tissue regeneration was further explored including the stimulation of endogenous regeneration programmes. The use of two disease models, rheumatoid arthritis, and cutaneous wounds, highlighted the different UC-MSC functions, namely their immunomodulatory and tissue regeneration abilities. Moreover, the administration of CM3D in adjuvant-induced rat model for arthritis, ameliorated the arthritic signs and accelerated the remission of local and systemic arthritic manifestations. The analysis of relevant trophic factors in CM3D confirmed the prevalence of anti-inflammatory cytokines, along with trophic factors involved in different mechanisms leading to tissue regeneration. Accordingly, CM3D-treated wounds also presented signs of faster and better wound resolution, with a more mature vascular system already showing glands and hair follicles. Deeper and integrated analysis of the secretome unveiled the role of exosomes and its content on cutaneous wound healing. Furthermore, loading of Exo with an immunosuppressive oligodeoxynucleotide resulted in reduced systemic levels of pro-inflammatory cytokines at the late stage of wound healing. Lastly, mechanistic studies disclosed the role of granulocyte colony-stimulating factor (G-CSF) on UC-MSC homing and recruitment capacities that further contributed to accelerated closure of impaired wounds. Overall, this thesis set up the technical grounds for the development of an effective and novel advanced therapy medicinal product (ATMP) for injured tissue treatment.