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1
Caminal, Bobet Marta. "Tissue engineering for bone regeneration: in vitro development and in vivo testing in sheep." Doctoral thesis, Universitat Autònoma de Barcelona, 2014. http://hdl.handle.net/10803/285622.
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L'os és un teixit connectiu altament organitzat i especialitzat, la funció principal és la mecànica, proporcionant l'afecció als músculs i per tant permetent que el cos es mogui. Actualment, el tractament quirúrgic estàndard es basa en la immobilització i la introducció d'empelts ossis però presenta algunes complicacions, com ara les infeccions, les no unions i la morbiditat de la zona donant. Avui en dia, milions de pacients pateixen defectes ossis i en concret, als EEUU es diagnostiquen entre 10.000 i 20.000 nous casos d'osteonecrosi del cap de fèmur (ONFH) a l’any.
La medicina regenerativa (RM) i l'enginyeria tissular (TE) són dos camps de la ciència que es centren en el desenvolupament de teràpies per reemplaçar i regenerar els teixits perduts o danyats per millorar la qualitat de vida del pacient. La combinació de biomaterials, cèl·lules i senyals és l’eina clau per al desenvolupament d'un producte RM i TE. Un dels camps més desenvolupats en RM és la medicina regenerativa ortopèdica, en concret per al teixit ossi. Hi ha diferents estratègies que combinen cèl·lules autòlogues amb matrius que han demostrat certa eficàcia en el tractament de lesions òssies. Després de la fase de descobriment de nous medicaments de teràpia avançada, i per tal d’aconseguir el registre del nou producte, hi ha la fase de desenvolupament, que inclou la realització d'estudis preclínics (fet per dur a terme la prova de concepte, la seguretat i toxicologia) i els estudis clínics.
En primer lloc es van determinar i caracteritzar els components de la preparació d’enginyeria tissular (TEP) amb la finalitat d’obtenir un producte estandarditzat. Aquesta preparació consisteix en un component cel·lular que són les cèl·lules mesenquimals estromals (MSC), tant humanes com ovines unides en una matriu de partícules òssies desantigeneïtzades i liofilitzades.
Es va realizar un model de defecte ossi de mida crítica (CSBD) en ovella amb la finalitat d'investigar l'efecte de la TEP en una situació extrema, i es va demostrar la seva seguretat i capacitat per sintetitzar nou os i remodelar l’os existent.
Seguidament la TEP es va provar en un model animal rellevant de translació de la malaltia òssia basat en el mètode reportat per Vélez i col·laboradors per a la modelització de ONFH en ovelles demostrant la seva eficàcia i seguretat. També s’ha demostrat que les MSC estan involucrades en la síntesi d'os nou ja que es van trobar progenitors ossis marcats després del tractament de la ONFH, tot i així no es poden descartar els mecanismes paracrins.
Per tant, el desenvolupament de la TEP podria contribuir en general a la RM per tal de satisfer les exigències d'una societat que envelleix.Bone is a highly organized and specialized connective tissue, whose main function is the mechanics, providing attachment to muscles and therefore allowing the body to move. Currently the gold standard surgical treatment is based on the immobilization and introduction of bone grafts but it presents some complications, such as infections, non-unions, and donor site morbidity. Nowadays, millions of patients are suffering from bone defects and specifically, 10,000 to 20,000 new cases of osteonecrosis of femoral head (ONFH) are diagnosed only in the USA every year. Regenerative medicine (RM) and tissue engineering (TE) are two areas of science fields focused on the developing of therapies to replace and regenerate lost or damaged tissues to improve the quality of life the patient. The combination of biomaterials, cells and signals is the key tool for the development of a RM and TE product. One of the most developed fields in RM is the orthopedic regenerative medicine, in specifically for bone tissue. There are different strategies combining autologous cells with scaffolds that have shown some efficacy for treating bone injuries. After discovery phase of any new advanced therapy medicinal products, there is the development phase that includes the conduction of preclinical studies (made to perform the proof of concept, safety and toxicology) and clinical studies before the registration of the new product. First the components of the tissue engineered preparation (TEP) were determined and characterized in order to have a standardized material. It consists in MSC (mesenchymal stromal cells) both human and ovine sources are used as a cellular component seeded in a deantigenized and lyophilized bone particles as a scaffold. Then critical size bone defect (CSBD) was modeled in sheep in order to investigate the effect of the TEP in an extreme situation, demonstrating its safe ability to synthesize new bone and bone remodeling. Afterwards TEP was tested in a relevant translational animal model of bone disease based on the method reported by Velez and collaborators for modelling ONFH in sheep demonstrating its efficacy and safety. Also demonstrating that MSC were involved in the synthesis of new bone, because labeled bone progenitors are shown after ONFH treatment, although paracrine mechanisms can not be discarded. Therefore, the development of TEP could contribute to the overall RM to meet the requirements of an aging society.
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Nie, Yingjie. "Defective dendritic cells and mesenchymal stromal cells in systemic lupus erythematosus and the potential of mesenchymal stromal cells as cell-therapy." Click to view the E-thesis via HKUTO, 2009. http://sunzi.lib.hku.hk/hkuto/record/B43278681.
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Campos, Ana Margarida Ferreira. "Lipidomic analysis of mesenchymal cells candidates for cell therapy." Master's thesis, Universidade de Aveiro, 2015. http://hdl.handle.net/10773/15275.
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Mestrado em Bioquímica - Métodos BiomolecularesMesenchymal stromal cells are adult stem cells found mostly in the bone marrow. They have immunosuppressive properties and they have been successfully applied as biological therapy in several clinical trials regarding autoimmune diseases. Despite the great number of clinical trials, MSCs’ action is not fully understand and there are no identified markers that correlate themselves with the immunomodulatory power. A lipidomic approach can solve some of these problems once lipids are one of the major cells’ components. Therefore, in this study cells’ lipidome was analysed and its deviations were evaluated according to the medium of culture and to the presence of pro-inflammatory stimuli, mimicking physiological conditions in which these cells are used. This was the first study ever made that aimed to analyse the differences in the phospholipid profile between mesenchymal stromal cells non-stimulated and stimulated with proinflammatory stimulus. This analysis was conducted in both cells cultured in medium supplemented with animal serum and in cells cultured in a synthetic medium. In cells cultured in the standard medium the levels of phosphatidylcholine (PC) species with shorter fatty acids (FAs) acyl chains decreased under pro-inflammatory stimuli. The level of PC(40:6) also decreased, which may be correlated with enhanced levels of lysoPC (LPC)(18:0) - an anti-inflammatory LPC - observed in cells subjected to TNF-α and IFN-γ. Simultaneously, the relative amounts of PC(36:1) and PC(38:4) increased. TNF-α and IFN- γ also enhanced the levels of phosphatidylethanolamine PE(40:6) and decreased the levels of PE(38:6). Higher expression of phosphatidylserine PS(36:1) and sphingomyelin SM(34:0) along with a decrease in PS(38:6) levels were observed. However, in cells cultured in a synthetic medium, TNF-α and IFN-γ only enhanced the levels of PS(36:1). These results indicate that lipid metabolism and signaling is modulated during mesenchymal stromal cells action.
As células mesenquimais do estroma são células estaminais adultas que apresentam propriedades imunossupressoras e têm sido aplicadas como terapia clínica em vários estudos clínicos relativos a doenças autoimunes. Apesar do vasto número de estudos clínicos que utilizam estas células, ainda não se conhece o mecanismo de ação das mesmas, nem foram ainda identificados marcadores permitam avaliar o seu potencial imunomodulador. A lipidómica poderá dar algumas respostas a estas questões uma vez que os lípidos são importantes componentes das células, desempenhando um papel na sinalização celular. No presente trabalho estudou-se o lipidoma das células mesenquimais e avaliou-se a sua variação consoante o meio de cultura e a presença de estímulos próinflamatórios, mimetizando as condições fisiológicas em que as células são utilizadas. Este foi o primeiro estudo que analisou as diferenças no perfil fosfolípidico entre células mesenquimais do estroma e avaliou a variação do lipidoma destas células após a sua estimulação por mediadores pró-inflamatórios. Este estudo foi conduzido num primeiro conjunto de células cultivado num meio padrão suplementado com soro animal e num segundo conjunto de células cultivado num meio sintético. Nas células cultivadas no meio padrão, observou-se uma diminuição nas espécies moleculares de fosfatidilcolina (PC) com cadeias de ácidos gordos (FAs) após estímulos pro-inflamatórios. A quantidade de PC(40:6) também diminuiu, relacionando-se com o aumento expressão de lisoPC (LPC)(18:0) – LPC anti-inflamatória – em células estimuladas. Simultaneamente, a quantidade relativa de PC(36:1) e PC(38:4) aumentou. TNF-α and IFN-γ também levou ao aumento dos níveis de fosfatidiletanolamina PE(40:6) e diminuiu os níveis de PE(38:6). Também se verificou um aumento da expressão de fosfatidilserina PS(36:1) e esfingomielina (SM)(34:0), bem como a diminuição na expressão de PS(38:6). Contudo, em células mesenquimais cultivadas em meio sintético, com TNF-α and IFN-γ apenas aumentaram os níveis de PS(36:1). Estes resultados indicam que o metabolismo dos lípidos é modulado durante a ação imunossupressora das células.
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Nie, Yingjie, and 聶瑛潔. "Defective dendritic cells and mesenchymal stromal cells in systemic lupus erythematosus and the potential of mesenchymal stromal cells ascell-therapy." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2009. http://hub.hku.hk/bib/B43278681.
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Fung, Kwong-lam, and 馮廣林. "Chemoresistance induced by mesenchymal stromal cells on cancer cells." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2013. http://hdl.handle.net/10722/205639.
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Human mesenchymal stromal cells (hMSCs) are part of bone marrow micro-environment that supports hematopoiesis. However, hMSCs also enhance tumor progression and survival when they become part of the cancer micro-environment. I aimed to investigate the interaction between hMSCs and cancer cells during chemotherapy.
Firstly, I studied the interaction between hMSCs and T-lineage acute lymphoblastic leukemia (T-ALL) cells under pegylated arginase I (BCT-100) treatment. Three T-ALL cell lines were sensitive to BCT-100 but not hMSCs. Conversely, hMSCs could partly protect all T-ALL cell lines from BCT-100 induced cell death under transwell co-culture condition. Concerning the possible mechanism, the intermediate metabolite L-ornithine could not rescue most T-ALL cells from BCT-100 treatment. But the downstream L-arginine precursor, L-citrulline could partly rescue all T-ALL cells from BCT-100 treatment. Ornithine transcarbamylase (OTC) converts L-ornithine into L-citrulline. OTC expression level in hMSCs remained relatively high during BCT-100 treatment but OTC expressions in T-ALL cell lines declined drastically. It suggested that hMSCs may protect T-ALL cells against BCT-100 treatment by having sustained OTC expression. Suppression of hMSCs by vincristine (VCR) disrupted the protective effect of hMSCs to most T-ALL cells during BCT-100 treatment. This suggests that by transiently suppressing hMSCs, we may abolish the protective effect of hMSCs to T-ALL cells during BCT-100 treatment.
Then I studied the interaction between hMSCs and neuroblastoma under cisplatin treatment. Two neuroblastoma cell lines were used for both of them are cisplatin sensitive while hMSCs are cisplatin resistant. hMSCs could partly protect neuroblastoma cells from cisplatin induced cytotoxicity. On the other hand, exogenous IL-6 but not IL-8 could also partly rescue them from cisplatin induced cytotoxicity. IL-6 activated STAT3 phosphorylation dose-dependently and enhanced expression of detoxifying enzyme (glutathione S-transferase π, GST-π) in neuroblastoma. Such effect could be counteracted by anti-IL-6R neutralizing antibody tocilizumab (TCZ). However, TCZ failed to suppress hMSCs’ protection to neuroblastoma during cisplatin treatment. This suggests involvement of multiple factors. Up-regulation of serum GST-πin some hTertMSCs/neuroblastoma co-engrafted SCID mice compared to neuroblastoma engrafted mice provided a clue that GST-π might be a possible stromal-protection factor. Caffeic acid phenethyl ester (CAPE) is a known GST inhibitor after tyrosinase activation. Neuroblastoma cells expressed tyrosinase and CAPE enhanced cisplatin cytotoxicity on them, with or without hMSCs. Paradoxically, CAPE enhanced GST-πexpression with or without cisplatin treatment in neuroblastoma suggesting possible negative feedback to GST-π inhibition. However, such additive effect of CAPE to cisplatin cytotoxicity was not observed in vivo. Further delineation of the in vivo study design may help to verify the additive effect of CAPE to cisplatin cytotoxicity in vivo.
Finally, I studied the effect of apoptotic cancer cells (AC) on the immune function of hMSCs. hMSCs could phagocytose apoptotic neuroblastoma cells with respective up-regulation of many immune-mediators including two highly-expressed cytokines IL-6 and IL-8. Up-regulation of these immune-mediators may enhance immune cells chemotaxis. Further detailed investigation on the effect of AC-engulfed hMSCs to other immune cells will help us to understand the dynamic interaction between cancer cells and stromal cells during chemotherapy.published_or_final_version
Paediatrics and Adolescent Medicine
Doctoral
Doctor of Philosophy
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Kühl, Tobias Hans-Jürgen [Verfasser], and Leena [Akademischer Betreuer] Bruckner-Tuderman. "Mesenchymal stromal cell therapy for dystrophic epidermolysis bullosa." Freiburg : Universität, 2016. http://d-nb.info/1119452716/34.
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Sory, David Roger Raymond. "Dynamic loading of periosteum-derived mesenchymal stromal cells." Thesis, Imperial College London, 2017. http://hdl.handle.net/10044/1/59138.
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Explosive-generated waves exhibit high-energy loading profiles featured with mechanical characteristics applied over a wide range of strain rates. Recent decades have seen unprecedented occurrence of high-energy trauma associated with blast wave exposure. One such blast-specific pathology is blast-induced heterotopic ossification (bHO), which refers to ectopic bone formation due to inappropriate mesenchymal stromal cell (MSC) osteogenesis in non-skeletal tissues. Significant effort has been made into deciphering the molecular mechanisms that allow the onset of bHO, however little research has been reported on the exact role of the biomechanical processes involved in transducing blast-associated mechanical stimuli into molecular events stimulating osteogenesis in MSCs. The research presented in this thesis investigated the stimulation of osteogenesis in periosteum-derived mesenchymal stromal cells (PO MSCs) in response to mechanical insults simulating blast landmine trauma. This involved the development of experimental biocompatible in vitro platforms and the tailoring of biomechanically-relevant stimuli of varying stress intensities (up to 70 MPa), and strain rates (0.01 to 3000 /s). Subsequently, cell health and the stimulation of osteogenesis were investigated by studying the expression of Runx2 and Osteocalcin (OC) genes. We found that cell health was not affected by single-pulse loadings of wide range of impulse levels (0.20 to 95000 N.s). We showed evidence of mechanically-stimulated osteogenesis in PO MSCs through the upregulation of Runx2 and OC genes in loaded samples. Furthermore, our results highlighted that the stimulation of osteogenesis in MSCs did not result solely from the effect a single mechanical parameter, but rather the combined action of several features. We showed that osteogenesis stimulation in MSCs arised from the complex interplay between the mechanical characteristics of the loading along with the environment used to convey the stress wave. Finally, our research indicated that PO MSCs are finely tuned to respond to mechanical stimuli that fall within defined parameters.
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Ward, Lewis Stuart Corey. "Interactions of mesenchymal stromal cells with their microenvironment." Thesis, University of Birmingham, 2018. http://etheses.bham.ac.uk//id/eprint/8278/.
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Mesenchymal stromal cells (MSC) suppress the inflammatory infiltrate through crosstalk with neighbouring endothelium. However, this response is lost at chronic inflammatory sites where stromal cells instead support leukocyte recruitment and upregulate expression of podoplanin. The mechanism and function by which this inflammatory phenotype is established is unknown. We hypothesise that MSC modulation of endothelium is also altered by exposure to inflammatory cytokines, and that expression of podoplanin confers an invasive phenotype, enabling the interaction of these perivascular MSC with circulating platelets. MSC resisted functional transformation during acute or prolonged exposure to tumour necrosis factor alpha, instead maintaining their ability to suppress neutrophil recruitment in a flow-based assay. Expression of podoplanin promoted MSC migration through Ras-related C3 botulinum toxin substrate dependent signalling, enabling perivascular MSC to interact with cells confined to the circulation. Indeed, podoplanin induced the activation of platelets from flow through MSC protrusions in the endothelial lining. The retention of MSC suppressive function under inflammatory conditions supports their use in equivalent environments for therapy. However, the implications of platelet CLEC-2 activation by its ligand, podoplanin on inflamed stroma have yet to be elucidated and warrant further investigation, with specific focus drawn to the pathophysiology of thromboinflammation and associated disorders.
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Martella, Elisa <1984>. "Mesenchymal stromal cell: new applications for regenerative medicine." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2013. http://amsdottorato.unibo.it/5440/.
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In the last decades mesenchymal stromal cells (MSC), intriguing for their multilineage plasticity and their proliferation activity in vitro, have been intensively studied for innovative therapeutic applications.
In the first project, a new method to expand in vitro adipose derived-MSC (ASC) while maintaining their progenitor properties have been investigated. ASC are cultured in the same flask for 28 days in order to allow cell-extracellular matrix and cell-cell interactions and to mimic in vivo niche. ASC cultured with this method (Unpass cells) were compared with ASC cultured under classic condition (Pass cells). Unpass and Pass cells were characterized in terms of clonogenicity, proliferation, stemness gene expression, differentiation in vitro and in vivo and results obtained showed that Unpass cells preserve their stemness and phenotypic properties suggesting a fundamental role of the niche in the maintenance of ASC progenitor features. Our data suggests alternative culture conditions for the expansion of ASC ex vivo which could increase the performance of ASC in regenerative applications.
In vivo MSC tracking is essential in order to assess their homing and migration. Super-paramagnetic iron oxide nanoparticles (SPION) have been used to track MSC in vivo due to their biocompatibility and traceability by MRI. In the second project a new generation of magnetic nanoparticles (MNP) used to label MSC were tested. These MNP have been functionalized with hyperbranched poly(epsilon-lysine)dendrons (G3CB) in order to interact with membrane glycocalix of the cells avoiding their internalization and preventing any cytotoxic effects.
In literature it is reported that labeling of MSC with SPION takes long time of incubation. In our experiments after 15min of incubation with G3CB-MNP more then 80% of MSC were labeled. The data obtained from cytotoxic, proliferation and differentiation assay showed that labeling does not affect MSC properties suggesting a potential application of G3CB nano-particles in regenerative medicine.
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Davies, Benjamin Michael. "Optimising mesenchymal stromal cell harvesting in orthopaedic surgery." Thesis, University of Oxford, 2015. https://ora.ox.ac.uk/objects/uuid:aeb65824-d07b-4c73-bb51-aedaf7a7b0c2.
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Musculoskeletal tissue is prone to age-related degeneration and to damage which heals poorly. Many current treatments are able to treat only the end stages of these conditions, such as the use of total knee replacements in osteoarthritis. Cellular therapies are seen as a potential source of effective treatments for the earlier stages of these conditions. Orthopaedic surgery has been at the forefront of cellular therapies with treatments such as microfracture and autologous chondrocyte implantation to treat chondral defects. As the largest area of current cell therapy research, stem cells have become an area of high interest for developing novel treatments. Mesenchymal stromal cells (MSCs) have provided the basis of the majority of orthopaedic treatments because of the relative ease of obtaining them. Despite the development of a number of treatments using both freshly harvested MSCs and culture expanded MSCs there is still a large gap in our knowledge of the mechanisms of actions of these cells and the most appropriate locations for obtaining autologous samples. This thesis seeks to examine the best source of MSCs for surgery around the knee, comparing the pelvis to the femur and tibia. It also seeks to determine if it is possible to improve the yield of MSCs using a simple modification of the standard method of aspiration. Assessments of the yield of all cells and MSCs showed that the pelvis was the optimum source for MSCs in terms of cell numbers. There was also a large amount of inter-subject variation in the number of cells obtained. There was no difference in the functional abilities of cells from any location. Modification of the aspiration technique did not improve the cell yield. Future work should focus on improving yields from the pelvis and investigate methods of overcoming the inter-subject variability in yields if standardised treatments are to be successfully developed.
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Pasanen, I. (Ilkka). "Stromal cells of mesenchymal origin in breast cancer." Doctoral thesis, Oulun yliopisto, 2017. http://urn.fi/urn:isbn:9789526215587.
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Abstract Breast cancer, the most common cancer in women in Finland; its prognosis varies from very good to poor. During the last two decades, mesenchymal stromal cells, carcinoma-associated fibroblasts and normal fibroblasts of the breast have been investigated in the context of breast carcinomas because of their presence in the tumor microenvironment. It has been shown that the properties of the non-malignant tumor compartment possess prognostic value. The effects that these three stromal cell types have on cancer progression have been studied, but their exact mechanisms remain still largely unknown. This experimental work was conducted in order to investigate whether the three cell types of mesenchymal origin influence breast cancer cell proliferation in vitro and tumor growth in vivo. Functional and structural differences between the stromal cell types were investigated using multiple methods. A total of 19 primary human bone marrow derived mesenchymal stromal cell lines, and six paired primary fibroblast and carcinoma-associated fibroblast lines of the breast were used in the study. Co-cultures of labeled stromal cells and breast cancer cell lines MDA-MB-231, M-4A4 and NM-2C5 were performed and the proliferation properties of each cell line were assessed. An orthotopic murine breast cancer model was established by injecting NM-2C5 cancer cells in the mammary fat pads of athymic mice either alone or with the three stromal cell types, and tumor growth and histology were analysed. Mesenchymal stromal cells increased the proliferation of breast cancer cell lines NM-2C5 and MDA-MB-231, and carcinoma-associated fibroblasts increased the proliferation of NM-2C5 cells in vitro. The effect was due to both soluble factors and direct cell-cell contact. In the in vivo experiments, the mesenchymal stromal cells inhibited and the fibroblasts enhanced the growth of breast cancer tumors. Histological analysis of the tumors revealed differences in the invasiveness, necrosis and amount of connective tissue. Differences in the expression of CD105 and CD54 were observed between tumors with mesenchymal stromal cells or fibroblasts. Carcinoma-associated fibroblasts differed from mesenchymal stromal cells in their expressions of CD105 and CD54. The fibroblast subtypes differed at the gene expression level in immunological, developmental and extracellular matrix related pathwaysTiivistelmä Rintasyöpä on Suomessa naisten yleisin syöpä, ja sen ennuste vaihtelee erittäin hyvästä huonoon. Viime vuosikymmenten aikana mesenkymaalisia stroomasoluja, rinnan kasvainsidekudossoluja ja tavallisia sidekudossoluja on tutkittu rintasyövän yhteydessä johtuen kyseisten solujen läsnäolosta syövän mikroympäristössä. Syöpäkudoksen hyvänlaatuisen solukon ominaisuuksilla on osoitettu olevan ennusteellista arvoa, ja kolmen edellä mainitun strooman solutyypin vaikutuksia rintasyövän etenemiseen on tutkittu, mutta tarkat vaikutusmekanismit ovat vielä laajalti tuntemattomat. Tutkimuksen tarkoituksena oli tutkia edellä mainittujen solutyyppien vaikutusta rintasyöpäsolujen lisääntymiseen soluviljelmässä ja syöpäkasvaimen kasvuun koe-eläinmallissa. Lisäksi strooman solujen rakenteellisia ja toiminnallisia eroavaisuuksia tutkittiin molekyylibiologisilla menetelmillä. Tutkimuksessa käytettiin 19:ää luuytimen mesenkymaalista stroomasolulinjaa sekä kuutta rinnan kasvainsidekudossolu–sidekudossolu paria. Leimattuja strooman soluja viljeltiin yhteisviljelmissä rintasyöpäsolulinjojen MDA-MB-231, M-4A4 ja NM-2C5 kanssa, ja kunkin solutyypin lisääntymistä mitattiin. Ortotooppisessa rintasyövän hiirimallissa immuunipuutteisen hiiren rinnan ihonalaisrasvaan injisoitiin NM-2C5-rintasyöpäsoluja yksinään ja yhdessä strooman solujen kanssa, ja kasvainten kasvua ja histologiaa analysoitiin. Mesenkymaaliset stroomasolut kiihdyttivät NM-2C5- ja MDA-MB-231-rintasyöpälinjojen ja kasvainsidekudossolut NM-2C5-solujen lisääntymistä soluviljelmässä. Vaikutuksen aiheuttivat sekä liukoiset tekijät että suora solujen välinen vuorovaikutus. Eläinmallissa mesenkymaaliset stroomasolut hillitsivät mutta sidekudossolut lisäsivät rintasyöpäkasvaimen kasvua. Histologisissa analyyseissä paljastui eroavaisuuksia tuumorien paikallisessa invaasiossa, kudoskuolion määrässä ja sidekudoksen määrässä. Mesenkymaalisia stroomasoluja ja kasvainsidekudossoluja sisältävien kasvainten välillä esiintyi eroja CD105- ja CD54-pinta-antigeenien määrässä. Kasvainsidekudossolut erosivat pintarakenteiltaan mesenkymaalisista stroomasoluista CD105:n ja CD54:n ilmentämisessä. Sidekudossolut ja kasvainsidekudossolut erosivat toisistaan geenien ilmentämisen tasolla immunologisten, kehityksellisten ja soluväliaineeseen liittyvien geenipolkujen osalta
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Tsui, Yat-ping, and 徐軼冰. "Derivation of oligodendrocyte precursor cells from adult bone marrow stromal cells." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2013. http://hdl.handle.net/10722/197485.
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Myelin is essential for neuronal survival and maintenance of normal functions of the nervous system. Demyelinating disorders are debilitating and are often associated with failure of resident oligodendrocyte precursor cells (OPCs) to differentiate into mature, myelinating oligodendrocytes. Derivation of OPCs, from a safe source that evades ethical issues offers a solution to remyelination therapy. We therefore hypothesized that bone marrow stromal cells (BMSCs) harbour neural progenitor cells at a pre-commitment stage and that in vitro conditions can be exploited to direct differentiation of these cells along the oligodendroglial lineage.
For the current study, adult rat BMSCs used were >90% immunopositive for CD90, CD73, STRO-1 (stromal cell markers), 10% for nestin (neural progenitor marker) but negligible for CD45 (haematopoietic cell marker) as measured by flow cytometry. Transfer of the culture from a highly adhesive substratum to a moderately adhesive substratum resulted in increase in proportion of p75-positive cells but CD49b-positive cells remained at 97% and Sox 10-positive cells remained negligible. Transfer of the culture to a non-adherent substratum fostered the generation of neurospheres comprising cells that were positive for the neural stem/progenitor cell (NP) marker, nestin, and for the neural crest markers CD49b, p75 and Sox10. Prior to this stage, the BMSCs were not yet committed to the neural lineage even though transient upregulation of occasional marker may suggest a bias towards the neural crest cell lineage.
The BM-NPs were then maintained in adherent culture supplemented with beta-Heregulin (β-Her), basic fibroblast growth factor (bFGF) and platelet-derived growth factor-AA (PDGF-AA) to direct differentiation along the oligodendroglial lineage. Within two weeks of glial induction, cells expressing the OPC markers - NG2, Olig2, PDGFRa and Sox10, were detectable and these could be expanded in culture for up to 3 months with no observable decline in marker expression. These BM-OPCs matured into myelinating oligodendrocytes after 2 weeks in co-culture with either dorsal root ganglion neurons or cortical neurons. In vivo myelination by BM-OPCs was demonstrated by exploitation of the non-myelinated axons of retinal ganglion cells of adult rats. By 8 weeks post-injection of BM-OPCs into the retina, myelin basic protein-positive processes were also observable along the retinal axons. The results not only suppport our hypothesis, but also provide pointers to the adult bone marrow as a safe and accessible source for the derivation of OPCs towards transplantation therapy in acute demyelinating disorders.published_or_final_version
Biochemistry
Doctoral
Doctor of Philosophy
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Mo, Fung-ying Irene. "Effects of bacterial toxins on the proliferation, osteogenic differentiation and toll-like receptor expressions of human mesenchymal stromal cells." View the Table of Contents & Abstract, 2006. http://sunzi.lib.hku.hk/hkuto/record/B36840920.
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Correia, Dos Reis Mónica Sofia. "Mesenchymal stromal cells : mode of action and clinical translation." Thesis, University of Newcastle upon Tyne, 2016. http://hdl.handle.net/10443/3456.
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Mesenchymal Stromal Cells (MSCs) are an extensively used cell type in clinical trials for the treatment of various diseases. In order to obtain clinically relevant numbers, MSCs need to be expanded in vitro, usually relying on the use of foetal calf serum (FCS), which is now not recommended by the regulatory authorities. In addition, the precise mechanism of action of MSCs remains unclear. The initial rationale for the therapeutic potential of MSCs was based on their engraftment and differentiation ability. It gets increasingly clear that MSCs exert their effects in a paracrine manner, by the release of soluble factors and extracellular vesicles (EVs). The aims of this study are: 1) to investigate the feasibility of replacing FCS with human platelet lysate (PLT) for the expansion of MSCs and assess its effect on MSC general characteristics and immunosuppressive potential; 2) to assess the immunoregulatory function of EVs derived from PLT-expanded MSCs (MSC-PLT). Data presented in this thesis demonstrated that MSC-PLT maintained their general characteristics and immunosuppressive potential, while exhibiting enhanced proliferative properties compared to FCS-expanded MSCs (MSC-FCS). As the first comparative study on the global surface protein profile of MSC-FCS and MSC-PLT, we showed that PLT induced little changes on the expression of markers involved in the enhancement of proliferation and differentiation properties of MSCs. Furthermore, this study has demonstrated that MSC-derived EVs retained the immunosuppressive function of the parent cells, although with a lower potency. Here we also report for the first time, that MSC-EVs skewed DC maturation into a tolerogenic phenotype and impaired their phagocytic ability and migratory potential. We also found that MSC-derived EVs are rich in a wide variety of microRNAs that target a plethora of genes involved in various pathways related to development, trafficking and modulation of immune responses, including dendritic cell maturation and function. The findings in this study support the notion that PLT is a suitable supplement for in vitro MSC expansion and that MSC-PLT produce EVs with the ability to modulate immune responses.
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Brown, Alex Joseph. "Maintenance and modification of mesenchymal stromal cell immunosuppressive phenotype." Thesis, University of Iowa, 2017. https://ir.uiowa.edu/etd/5723.
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The purpose of this study was to identify conditioning strategies for mesenchymal stromal cells (MSC) which optimize cellular immunosuppressive potency. By identifying new treatment strategies and previously unidentified small molecules capable of stimulating MSC we hope to pave the way tailoring licensed MSC phenotypes to be used in a specific disease state, rather than a one size fits all package. We sought to determine how MSC act in response to a changing immune response or environmental condition. MSC are exquisitely sensitive to changes in their environmental conditions and we show that cellular transcriptome and secretome changes are conditionally responsive to their inflammatory stimulus. One of the main subjects of analysis here is the observations of how these cellular profiles evolve over time in the presence of an inflammatory environment. Similarly, this study observes how MSC behavior changes after an inflammatory event has been resolved to address, in part, the plasticity of MSC licensing and the ability of MSC to rapidly recall a previous immunosuppressive state upon secondary challenge with an inflammatory stimulus. Data was obtained from in vitro experiments with human bone marrow derived MSC and donor human peripheral blood mononuclear cells (PBMC), while in vivo data was obtained using C57BL6/J mice.
Overall this research demonstrated that MSC potency can be bolstered by small molecule and drug treatment conditioning, and that certain disease conditions may be more effectively paired with specific MSC conditioning strategies to improve their therapeutic effectiveness.
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Dhadda, Paramjeet Kaur. "Using mesenchymal stromal cells to improve islet transplantation outcome." Thesis, King's College London (University of London), 2014. http://kclpure.kcl.ac.uk/portal/en/theses/using-mesenchymal-stromal-cells-to-improve-islet-transplantation-outcome(aef61279-a461-4023-8a23-7caceae29a1a).html.
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Pancreatic islet transplantation is an attractive treatment option for a subset of patients with Type 1 diabetes. However islet transplantation efficacy remains hampered by a number of factors, including the impaired quality of isolated islets available for transplantation and the loss of long-term islet function post transplantation. Harnessing the properties of multipotent mesenchymal stromal cells (MSCs), which can be derived from numerous clinically relevant post-natal tissues including adipose and pancreas, is currently under investigation for improving the survival of islet cells during culture and after transplantation. Amongst other beneficial properties, MSCs are known to provide a supportive micro-environmental niche via secretion of paracrine factors or deposition of extracellular matrix, making them excellent candidates to play the role of islet ‘helper’ cells for purposes of improving islet transplantation outcome. Preliminary studies conducted within the group demonstrated that co-transplantation of islets and MSCs improve the survival and function of engrafted islets post-transplantation, leading to overall improved outcomes of islet grafts in mouse models of Type 1 diabetes. The aims of this thesis were to investigate the mechanisms through which MSCs were exerting beneficial effects on islet function in vivo using in vitro co-culture methods and then to develop these findings into potential pre-transplant islet culture protocols with the aim of improving the function of isolated human islets available for transplantation. After deriving and characterising adipose MSCs (adipMSCs) from mouse tissue, it was demonstrated that direct cell-cell contact, between mouse islets and MSCs co-cultured in vitro was necessary for enhancing islet insulin secretory function compared to islets cultured alone, whereas the trophic factors secreted by the MSCs alone were not able to positively affect islet function. Additional co-culture studies also showed that the extracellular matrix (ECM) deposited by adipMSCs alone was able to improve the insulin secretory function of co-cultured islets, but not to the full extent of when MSCs were present. Through adopting a direct contact monolayer co-culture configuration for human islets and MSCs, it was also demonstrated that human adipMSCs improved the function of isolated human islets, more reproducibly than either human pancreatic MSCs (pMSCs) or human bone-marow MSCs (bmMSCs), supporting the use of adipMSCs in pre-transplant islet culture protocols. In summary adipMSCs enhanced the function of isolated islets through direct contact based mechanisms and warrant further investigation for use in clinical islet transplantation strategies, including the functional maintenance of isolated human islets in culture prior to transplantation.
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Tausan, Daniel. "Characterization and identification of hepatic mesenchymal stromal cell populations." Thesis, University of British Columbia, 2017. http://hdl.handle.net/2429/64225.
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Yoshioka, Satoshi. "CCAAT/Enhancer-Binding Proteinβ Expressed by Bone Marrow Mesenchymal Stromal Cells Regulates Early B-Cell Lymphopoiesis." Kyoto University, 2014. http://hdl.handle.net/2433/185198.
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Seshareddy, Kiran Babu. "Human Wharton’s jelly cells-isolation and characterization in different growth conditions." Thesis, Kansas State University, 2008. http://hdl.handle.net/2097/1054.
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Master of ScienceDepartment of Anatomy and Physiology
Mark L. Weiss
Wharton's jelly is a non-controversial source of mesenchymal stromal cells. Isolation of the cells is non-invasive and painless. The cells have been shown to have a wide array of therapeutic applications. They have improved symptoms when transplanted in a variety of animal disease models, can be used in tissue engineering applications to grow living tissue ex vivo for transplantation, and can be used as drug delivery vehicles in cancer therapy. The cells have also been shown to be non-immunogenic and immune suppressive. This thesis focuses on optimizing isolation protocols, culture protocols, cryopreservation, and characterization of cells in different growth conditions. Results from the experiments indicate that isolation of cells by enzyme digestion yields cells consistently, a freezing mixture containing 90% FBS and 10% DMSO confers maximum viability, and the expression of mesenchymal stromal cell consensus markers does not change with passage and cryopreservation. The results of the experiments also show that cells grow at a higher rate in 5% oxygen culture conditions compared to 21% oxygen culture conditions, serum does not have an effect on growth of the cells, serum and oxygen do not have effects on the expression of mesenchymal stromal cell consensus markers and the cells are stable without nuclear abnormalities when grown in 5% oxygen and serum free conditions for six passages after first establishing in serum conditions.
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Anastassiadis, Konstantinos, and Maria Rostovskaya. "Differential Expression of Surface Markers in Mouse Bone Marrow Mesenchymal Stromal Cell Subpopulations with Distinct Lineage Commitment." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2016. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-191602.
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Bone marrow mesenchymal stromal cells (BM MSCs) represent a heterogeneous population of progenitors with potential for generation of skeletal tissues. However the identity of BM MSC subpopulations is poorly defined mainly due to the absence of specific markers allowing in situ localization of those cells and isolation of pure cell types. Here, we aimed at characterization of surface markers in mouse BM MSCs and in their subsets with distinct differentiation potential. Using conditionally immortalized BM MSCs we performed a screening with 176 antibodies and high-throughput flow cytometry, and found 33 markers expressed in MSCs, and among them 3 were novel for MSCs and 13 have not been reported for MSCs from mice. Furthermore, we obtained clonally derived MSC subpopulations and identified bipotential progenitors capable for osteo- and adipogenic differentiation, as well as monopotential osteogenic and adipogenic clones, and thus confirmed heterogeneity of MSCs. We found that expression of CD200 was characteristic for the clones with osteogenic potential, whereas SSEA4 marked adipogenic progenitors lacking osteogenic capacity, and CD140a was expressed in adipogenic cells independently of their efficiency for osteogenesis. We confirmed our observations in cell sorting experiments and further investigated the expression of those markers during the course of differentiation. Thus, our findings provide to our knowledge the most comprehensive characterization of surface antigens expression in mouse BM MSCs to date, and suggest CD200, SSEA4 and CD140a as markers differentially expressed in distinct types of MSC progenitors.
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Grau, Vorster Marta. "Development and characterisation of advanced cell therapies based on multipotent mesenchymal stromal cells and virus-specific Tlymphocytes." Doctoral thesis, Universitat Autònoma de Barcelona, 2019. http://hdl.handle.net/10803/669379.
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El desenvolupament de noves teràpies s’està duent a terme arreu del món per poder fer front a les necessitats clíniques que actualment no disposen de tractament. En particular, els avenços en productes medicinals de teràpia avançada (ATMP) són una gran promesa per al tractament de malalties sense altres opcions terapèutiques. Tanmateix, els investigadors i les autoritats reguladores que implementen aquestes teràpies lluiten per estandarditzar tant els protocols com els productes finals. Entre els reptes s’inclouen l’elevada intervariabilitat de donants i mecanismes d’acció complexos. A més, és necessari demostrar l’activitat biològica d’aquestes teràpies mitjançant assajos de potència.
Aquesta tesi consisteix en el desenvolupament i caracterització de dos ATMP basats en cèl·lules multipotents estromals mesenquimals (MSC) i cèl·lules T específiques de virus (VST).
D'una banda, s’ha fet l’avaluació d'un test de potència i identitat per la producció de MSC aïllades de la gelatina de Wharton (WJ) i de la medul·la òssia (BM). L’objectiu proposat per MSC era realitzar: a) un assaig de potència per avaluar la capacitat immunomoduladora de les MSC; b) la revisió de l’expressió del HLA-DR pels criteris de definició de les MSC; i c) l'aplicació d’una eina per gestionar el risc. En aquest treball es presenta l’optimització d’un assaig d’immunopotència, validat i aprovat per l’autoritat competent per a l’alliberament de producte. S’ha estudiat l’expressió del HLA-DR, marcador suposadament negatiu, en les BM-MSC de grau clínic. Els resultats van mostrar una correlació entre l’expressió del HLA-DR i els nivells d’IL-17F i IL-33. L'expressió del HLA-DR no va afectar la identitat de les MSC, ni el potencial de diferenciació ni la capacitat immunomoduladora. Per reforçar aquests resultats, es van realitzar estudis interlaboratori obtenint resultats similars. L'ús de suplements basats en sèrum humà o lisat plaquetari no mostrava diferències en l'expressió de HLA-DR en MSC. També es va implementar la gestió de riscos com a eina de qualitat per detectar debilitats d’un bioprocés. Aquests enfocaments s’han dut a terme per MSC en assajos clínics.
D'altra banda, es va realitzar el desenvolupament d'un protocol d'expansió ex vivo de VST. La teràpia amb VST està destinada a pacients immunocompromesos, susceptibles de patir una reactivació o infecció de novo de l’herpesvirus entre d’altres. És el cas del citomegalovirus (CMV), que pot produir una infecció lleu en individus sans, però té una elevada morbiditat i mortalitat en individus immunocompromesos. Els medicaments antivirals disponibles poden produir toxicitat i no sempre són efectius. La immunoteràpia adoptiva ofereix una alternativa als pacients en una situació crítica i sense altres opcions terapèutiques. Per satisfer aquesta demanda es va elaborar un protocol de fabricació de VST fàcilment transferible als estàndards farmacèutics. Amb el mètode proposat, després d’un cocultiu de 14 dies amb cèl·lules dendrítiques polsades amb pp65, es van obtenir un gran nombre de VST. El cultiu es basava en la tecnologia G-Rex i es suplementava amb IL-2, IL-7, IL-15, i anticossos anti-CD3 i anti-CD28. El producte final es va caracteritzar àmpliament i estava format per limfòcits T CD4+ i CD8+, on la subpoblació majoritària corresponia a les cèl·lules T efectores de memòria, població coneguda per proporcionar una funció efectora. Cal destacar la citotoxicitat de les cèl·lules expandides específicament enfront al pp65. Els estudis d’al·loreactivitat amb HLA totalment incompatibles, van mostrar una lisi cel·lular per sota del 5%. En resum, s’ha descrit un protocol transferible a les normes de correcta fabricació actuals i un producte segur i eficaç in vitro, funcional després de la descongelació. El darrer fet, facilitaria la generació d’un banc al·logènic de VST. Les perspectives de futur inclourien la fabricació de cèl·lules T específiques per a múltiples virus.Innovative therapies are being developed worldwide to tackle unmet clinical needs. In particular, progress in advanced therapy medicinal products (ATMP) has shown great promise for the treatment of diseases with no other option available. However, researchers and regulatory authorities deal with the sophisticated nature of these medicines, and struggle to standardise both production protocols and final product formulation. Challenges related to the living nature of these products include high donor intervariability and complex mechanisms of action, which are sometimes not completely understood. Additionally, these newly therapies need to demonstrate biological activity with potency assays. This dissertation comprises the development and characterisation of two different ATMP based on multipotent mesenchymal stromal cells (MSC) and virus-specific T cells (VST). On the one hand, assessment of identity and potency for product release of MSC isolated from Wharton’s jelly (WJ) and bone marrow (BM) in the context of current good manufacturing practice (cGMP) production is performed. In this regard, we aimed at proposing: a) a potency assay for assessing immunomodulation capacity of MSC; b) the revision of HLA-DR expression profile for MSC definition criteria; and c) the application of risk management methodologies in the assessment of product quality. The optimisation of an immunopotency assay, validated, and approved by the competent authority for product release is presented. Moreover, other quality attributes of MSC are addressed. Regarding BM-MSC, the apparently random expression of HLA-DR, a marker that was expected negative in expansion cultures of MSC, is studied in clinical grade productions. Our findings showed correlation of HLA-DR expression with levels of IL-17F and IL-33. Expression of HLA-DR did not affect MSC identity, differentiation potential nor immunomodulatory capacity. To further strengthen these outcomes, interlaboratory studies were performed obtaining similar results. Furthermore, the use of either human sera or platelet lysate supplements showed no differences in terms of HLA-DR expression. A risk management assessment methodology was also implemented as a tool for quality by design to detect weaknesses of an established bioprocess involving MSC products already in clinical trials. On the other hand, regarding T lymphocytes, the development of a protocol for ex vivo expansion of VST was performed. VST therapy is intended for immunocompromised patients, which are susceptible of reactivation or de novo infection of herpesviruses among others. This is the case of cytomegalovirus (CMV) that undergoes a mild infection in healthy individuals but has been associated to a high morbidity and mortality in immunocompromised individuals. Unfortunately, available antiviral drugs can produce toxic side effects and are not always effective. Adoptive immunotherapy offers an alternative approach for those patients in a critical situation with no other therapeutic option. Therefore, we developed a protocol for VST scale-up manufacture easily transferable to pharmaceutical standards. Following with the method proposed, we obtained large number of CMV pp65-specific T cells after 14-day co-culture with pp65 pulsed dendritic cells. Culture was based on G-Rex bioreactor technology and supplemented with IL-2, IL-7, IL-15, anti-CD3 and anti-CD28 antibodies. The final product was extensively characterised in terms of identity, purity and potency. VST product was comprised of both CD4+ and CD8+ T lymphocytes, and effector memory T cells represented the major subset, which are known to provide effector function. Most importantly, we successfully demonstrated pp65 specific cytotoxicity of the expanded cells. Interestingly, complete HLA mismatch alloreactivity resulted in less than 5% cell lysis. In summary, a feasible protocol transferable to cGMP was described for an in vitro safe and effective product, which remain functional after thawing, thus providing practical evidence for the generation of an allogeneic third-party bank. Future perspectives would include the manufacture of multivirus-specific T cells.
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Nakamura, Yoko. "Enhanced wound healing by topical administration of mesenchymal stem cells transfected with stromal cell-derived factor-1." Kyoto University, 2014. http://hdl.handle.net/2433/185196.
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Krasheninnikova, Maria Alieva. "Adipose tissue mesenchymal stromal cells as therapeutic vehicles against glioblastoma." Doctoral thesis, Universitat de Barcelona, 2012. http://hdl.handle.net/10803/97086.
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Lately adipose tissue mesenchymal stem cells (hAMSCs) have emerged as cellular vehicles for therapy of solid tumors, due to their ease of isolation and manipulation, and wound/tumor homing capacity. HAMSCs have been successfully used in suicide gene therapy, employing the prodrug activating system based on Herpes simplex virus type I thymidine kinase (HSV-TK)/ganciclovir (GCV). In the current study we demonstrate an effective model of glioblastoma therapy based on the use of genetically modified hAMSCs and in vivo monitoring of tumor and therapeutic cells.
Due to the capacity of photons to pass through living tissue, non-invasive monitoring by bioluminescence imaging has become a cutting edge technology for the study of ongoing biological processes in small lab animals. This technique uses photoproteins as cell reporters that generate light photons as byproducts of chemical reaction. Luciferases catalyze the oxidation of a substrate (luciferin) in the presence of ATP and oxygen to generate oxiluciferin, ADP and light photons. In spite of the apparent opacity of tissues, light can be detected at several millimeters of depth in live animals. Combination of several types of luciferases allows simultaneous monitoring of different cell populations proliferation or differentiation. We stably transduced hAMSCs for expression of Renilla luciferase, HSV-TK and red fluorescent protein, generating RLuc-R-TK-AMSC and U87MG human malignant glioma cells for expression of Firefly luciferase and green fluorescent protein, generating Pluc-G-U87 cells. SCID mice were stereotactically implanted in the brain first with Pluc-G-U87 and RLuc-R-TK-AMSC afterwards. Mice were subjected to GCV treatment and the therapeutic process was evaluated in real time. Tumor response was monitored in vivo by BLI. Therapeutic cell differentiation was assessed by labeling the above Renilla luciferase expressing hAMSCs with a Firefly luciferase reporter regulated by the CD31, endothelial specific promoter and in vivo monitorization. Endothelial lineage differentiation of hAMSC was impaired, by Notch1 shRNA, and therapeutic effect was assessed by BLI monitorization of tumor response.
In our model of therapy, we show that tumor size can be continuously monitored by BLI and is significantly reduced (99,9% relative to control untreated tumours) by repeated inoculations in the tumours with thymidine kinase expressing hAMSCs followed by the prodrug ganciclovir. Moreover treatment resulted in a significant prolongation of survival time. In addition, the combination of BLI and confocal microscopy analysis of therapeutic cells suggests that efficient tumor eradication results from hAMSCs homing to tumor vessels, where they differentiate to endothelial cell lineage, intensifying their cytotoxic effect by destroying tumor vasculature and negating nutrient supply. Besides, hAMSCs endothelial differentiation inhibition resulted in an inefficient therapeutic effect compared to normal hAMSC (64% vs 6% respectively). Close association between hAMSCs and gliomas stem cells integrated in the tumor vascular system seems to be essential for an effective tumor reduction.
We suggest that efficient tumor eradication is due to hAMSCs endothelial differentiation and tube location that intensifies its cytotoxic function, destroying tumor vasculature and inhibiting nutrient supply to tumor cells. Thus we propose adipose tissue hAMSCs as useful vehicles for clinical applications to deliver localized therapy to glioma surgical borders after tumor resection.
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Lenz, Daniel. "Dissecting the heterogeneity of murine mesenchymal bone marrow stromal cells." Doctoral thesis, Humboldt-Universität zu Berlin, 2020. http://dx.doi.org/10.18452/21017.
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Knochenmarks-Stromazellen sind in den letzten Jahren in den Fokus der Forschung gerückt. Es konnte gezeigt werden, dass sie durch Bereitstellung von Überlebenssignalen essenziell für die Erhaltung hämatopoetischer Nischen sind. Stromales Interleukin-7 (IL-7) konnte dabei für T Zellen als Überlebenssignal identifiziert werden. Gemeinsam ist allen Stromazellen die Expression des Oberflächenmarkers CD106/VCAM-1.
Ein effizientes Protokoll erlaubte die qualitative wie quantitative Isolation von Stromazellen aus dem murinen Knochenmark mit anschließender ex vivo Microarray-Analyse. Die auf diese Weise ermittelten Kandidaten-Marker wurden auf Proteinebene via Histologie und (Hochdurchsatz-) Durchflusszytometrie validier. Dazu gehören z.B. die Marker CD1d, gas6 oder ANXA2R. CD1d wurde als guter Interimsmarker für VCAM-1+PECAM-1- Stromazellen identifiziert, wohingegen die IL-7-Produzenten in der Population von CD200int/BP 1+/CD73+/CD105- angereichert sind. Gleiches gilt für den Transkriptionsfaktor Prrx1. CD55, BP-1 and Cadherin-11 zeigten eine Expressionsmuster in Abhängigkeit des verwendeten IL-7-Reportermaus-Haplotyps. Für BP-1 und Cadherin 11 konnte die Abwesenheit von reifen Lymphozyten als Ursache des Feedbacks ausgeschlossen werden. Die Haplotypen der Reportermaus legten auch eine monoallele Expression des IL-7 nahe.
Die Ergebnisse dieser Arbeit zeigen VCAM-1+ (IL-7+/-) Stromazellen als heterogene Population, wenn es nach der Vielzahl der möglichen exprimierten Marker geht. Zwischen vielen dieser Marker gibt es aber wiederum auf Zelloberflächenebene einen großen Überlapp. Die funktionelle Relevanz dieser Oberflächenmarker-Diversität wird in weiteren Arbeiten zu klären sein, gibt aber den Stromazellen ein breites Repertoire vor, um Interaktionen mit Lymphozyten zu initiieren, modulieren und inhibieren. Abschließend ist zu erwarten, dass diese Erkenntnisse in die klinische Behandlung der Stroma-Nischen in Autoimmun-Fragestellungen einfließen.Bone marrow stromal cells receive increasing amounts of attention lately. They have been shown to support survival of hematopoietic stem cells as well as memory lymphocytes which is of great importance when targeting the perseverance of autoimmune diseases. CD4+ memory T lymphocytes reside in the proximity of VCAM-1 expressing stromal cells which provide them with survival signals such as Interleukin-7. Herein, a protocol was developed to quantitatively obtain VCAM-1+ and VCAM-1+ IL-7+/- stromal cells via enzymatic/mechanic digestion and cytoskeleton-inhibition. Ex vivo gene expression analysis was performed from sorted, pure cells with good recovery. Candidate genes/markers were validated in (high-throughput) flow cytometry and histological analysis including subsequent semi-automated colocalization was performed. CD1d was found to be good surrogate marker for VCAM-1+PECAM-1- non-endothelial stroma while the population of CD200int/BP-1+/CD73+/CD105- stromal cells is greatly enriched in IL-7 producers which was equally true for the stromal transcription factor Prrx1. CD55, BP-1 and Cadherin-11 were found to be differentially expressed in differing IL-7 reporter mice haplotypes. The reporter mice haplotypes revealed monoallelic expression features of IL-7. All methodologies suggest that VCAM-1+ as well as IL-7+/- stromal cells are heterogeneous by marker expression yet don’t cluster extensively in flow cytometry co-stains. The functional relevance of the marker diversity described in this thesis remains to be tested but insinuates a broad repertoire for bone marrow stroma cells for new interaction pathways with lymphocyte subsets. Ultimately, this knowledge will hopefully feedback to clinical questions of autoimmunity for targeted treatment of stromal niches.
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Anastassiadis, Konstantinos, and Maria Rostovskaya. "Differential Expression of Surface Markers in Mouse Bone Marrow Mesenchymal Stromal Cell Subpopulations with Distinct Lineage Commitment." Public Library of Science, 2012. https://tud.qucosa.de/id/qucosa%3A29135.
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Bone marrow mesenchymal stromal cells (BM MSCs) represent a heterogeneous population of progenitors with potential for generation of skeletal tissues. However the identity of BM MSC subpopulations is poorly defined mainly due to the absence of specific markers allowing in situ localization of those cells and isolation of pure cell types. Here, we aimed at characterization of surface markers in mouse BM MSCs and in their subsets with distinct differentiation potential. Using conditionally immortalized BM MSCs we performed a screening with 176 antibodies and high-throughput flow cytometry, and found 33 markers expressed in MSCs, and among them 3 were novel for MSCs and 13 have not been reported for MSCs from mice. Furthermore, we obtained clonally derived MSC subpopulations and identified bipotential progenitors capable for osteo- and adipogenic differentiation, as well as monopotential osteogenic and adipogenic clones, and thus confirmed heterogeneity of MSCs. We found that expression of CD200 was characteristic for the clones with osteogenic potential, whereas SSEA4 marked adipogenic progenitors lacking osteogenic capacity, and CD140a was expressed in adipogenic cells independently of their efficiency for osteogenesis. We confirmed our observations in cell sorting experiments and further investigated the expression of those markers during the course of differentiation. Thus, our findings provide to our knowledge the most comprehensive characterization of surface antigens expression in mouse BM MSCs to date, and suggest CD200, SSEA4 and CD140a as markers differentially expressed in distinct types of MSC progenitors.
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Lithopoulos, Marissa Athena. "Mesenchymal Stromal Cells to Treat Lung and Brain Injury in Neonatal Models of Chronic Lung Disease." Thesis, Université d'Ottawa / University of Ottawa, 2021. http://hdl.handle.net/10393/42124.
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Reeds, Kimberly. "In vitro effects of canine Wharton’s jelly mesenchymal stromal cells and nanoparticles on canine osteosarcoma D17 cell viability." Thesis, Kansas State University, 2011. http://hdl.handle.net/2097/11990.
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Master of ScienceDepartment of Clinical Sciences
Mary Lynn Higginbotham
Objectives – To isolate and maintain canine Wharton’s jelly mesenchymal stromal cells (WJMSCs) in culture, to determine the effects of micellar nanoparticles containing doxorubicin (DOX) on WJMSCs and canine osteosarcoma (OSA) D17 cell viability, and to determine the effects of conditioned media from WJMSCs loaded with micellar nanoparticles containing DOX on OSA D17 cell viability. Sample Population – Canine WJMSCs containing various concentrations of DOX micelles and canine OSA D17 cells. Procedures – WJMSCs were isolated from canine umbilical cords. Micellar nanoparticles containing DOX were prepared and added to culture plates containing canine OSA D17 cells to determine micelle effects on cell growth and viability. Conditioned media from culture plates containing canine WJMSCs incubated with various DOX micelle concentrations was added to OSA D17 cells for conditioned media experiments. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to assess OSA D17 cell viability. A trypan blue stain was also utilized to perform cell counts to determine the effect of the DOX micelles on stromal cell growth. Results – WJMSCs were successfully isolated and maintained in culture. Micellar nanoparticles containing DOX decreased OSA D17 cell viability. OSA D17 cell viability was also decreased following incubation with conditioned media from canine WJMSCs loaded with micellar nanoparticles containing DOX. Significant decreases with the conditioned media of canine WJMSCs loaded with 10μM micelles occurred at 48 hours (p < 0.005) and at 72 and 96 hours (p < 0.0001). Significant decreases were also observed with the 1 μM DOX micelles at 72 hours (p < 0.005) and 96 hours (p < 0.0001). WJMSC numbers decreased in a dose dependent manner following incubation with DOX micelles. Changes in WJMSC number was not caused by increased cell death as all variables produced similar percentages of dead cells. Conclusions – Canine WJMSCs were successfully isolated and maintained in culture. Stromal cells containing DOX micellar nanoparticles induced OSA D17 cell cytotoxicity while inducing an anti-proliferative, rather than cytotoxic effect, on the WJMSC. These data support future in vivo experiments utilizing canine WJMSCs and micellar nanoparticles.
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Rae, Michelle C. "An investigation into the immuno-modulatory properties of mesenchymal stromal cells." Thesis, University of Newcastle Upon Tyne, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.506524.
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François, Moïra. "Comprehensive study of the immunomodulatory properties of bone marrow-derived mesenchymal stromal cells." Thesis, McGill University, 2011. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=103683.
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Over the course of the last decade, mesenchymal stromal cells (MSC) have made a remarkable entry in the field of cell-based immunotherapy. In vitro, MSC were shown to modulate the immune response, either by acting as an immunosuppressant on several immune cells, or upon IFN-γ stimulation, as an antigen presenting cell (APC) for the priming of CD4+ T cells. Although a vast array of in vivo experiments in animals and humans has undeniably proven the immunological properties of MSC, the exact mechanisms by which MSC mediate their effects remain unclear. In Chapter 1, I presented a succinct review of the literature in regards to the characteristics of MSC. In Chapter 2, I addressed the immunosuppressive mechanisms of human MSC toward T cell proliferation. Using an in vitro proliferation assay, I demonstrated that human MSC suppressed T cell proliferation through the expression indoleamine 2,3-dioxygenase (IDO) induced following IFN-γ priming. In addition, MSC derived from different donors were shown to suppress T cell proliferation at variable degrees, which corresponded to their individual expression level of IDO. The use of whole peripheral blood mononuclear cells (PBMC) as opposed to purified T cells revealed the role played by monocytes in the suppression of T cell proliferation by MSC. Factors secreted by MSC in addition to the enzymatic activity of IDO induced the differentiation of monocytes into immunosuppressive M2 macrophages. Stimulation by IFN-γ not only triggered the immunosuppressive mechanisms of MSC but also induced APC-like properties in MSC. In Chapter 3, I investigated the molecular mechanisms implicated in the modulation of IFN-γ-inducible expression of MHC class II molecules and mediated antigen presentation in MSC. I demonstrated that IFN-γ mediated the transcriptional activation of the class II transactivator gene (CIITA), which is responsible for the upregulation of MHC class II molecules on both mouse and human MSC, and that TGF-β counter-acted the effect of IFN-γ by inhibiting the transcription of CIITA. In addition, cell culture density also modulated MHC class II-mediated antigen presentation differentially in mouse and human MSC. In Chapter 4, I examined the capacity of mouse MSC to cross-present exogenously acquired antigens as part of their APC-like features. I demonstrated that cross-presentation by mouse MSC was induced by IFN-γ and dependent on MHC class I machinery molecules, TAP complex and proteasome. I also demonstrated using an in vivo immune reconstitution assay, that mouse MSC can prime CD8+ T cells against a specific antigen, a characteristic of professional APC. Finally, I investigated in Chapter 5 the immunological impact of TLR expression and signaling in human and mouse MSC. I demonstrated that TLR activation in MSC induced the expression of chemokines and cytokines, which created an attractive inflammatory milieu for immune cells. I concluded by demonstrating that MSC differ from classic APC in that they did not express IL-12p70, an essential cytokine involved in both innate and adaptive immunity, in response to TLR activation. The findings in this thesis illustrate the complexity of the mechanisms by which MSC modulate the immune system. Their response to environment clues such as inflammation and pathogens activate either their suppressive or stimulatory immune functions, depending on the situation. Overall, these findings help optimize the utilization of MSC in cell-based immunotherapy.Au cours de la dernière décennie, les cellules stromales mésenchymateuses (MSC) ont fait une entrée remarquée dans le domaine de l'immunothérapie cellulaire. In vitro, les MSC ont démontrées des propriétés immunomodulatrices, soit par leur action inhibitrice sur les fonctions des cellules du système immunitaire ou par leur capacité à présenter des antigènes aux lymphocytes T CD4+, à la suite d'une stimulation par IFN-. Malgré l'existence de nombreuses recherches in vivo chez les animaux et l'homme prouvant leurs propriétés immunologiques, les mécanismes par lesquels les MSC modulent le système immunitaire n'ont pas encore été élucidés. Dans le Chapitre 1, j'ai présenté une revue succincte de la littérature traitant des caractéristiques des MSC. Dans le Chapitre 2, j'ai adressé les mécanismes immunosuppressifs des MSC humaines sur les lymphocytes T. À l'aide d'un test de prolifération in vitro, j'ai démontré que les MSC humaines suppriment la prolifération des lymphocytes T par grâce à l'expression indoleamine 2,3-dioxygenase (IDO) induite par l'exposition à l'IFN-. De plus, les MSC isolées de différents donneurs inhibent la prolifération des lymphocytes T à différents degrés qui correspondent au le niveau d'expression d'IDO par chaque donneur. L'utilisation de cellules mononucléaires sanguines (PBMC) complet comparativement à l'utilisation de lymphocytes T purifiés a révélé le rôle joué par les monocytes dans la suppression de la prolifération des lymphocytes T par les MSC. L'activité enzymatique d'IDO en combinaison avec d'autres facteurs sécrétés par les MSC induisent la différentiation des monocytes en macrophages immunosuppressifs de type M2. En plus de déclencher les mécanismes immunosuppressifs des MSC, l'IFN-a aussi eu pour effet d'induire des propriétés typiques des cellules présentatrices d'antigène (CPA) chez les MSC. Dans le Chapitre 3, j'ai étudié les mécanismes moléculaires impliqués dans la modulation de l'expression des molécules MHC de type II et la présentation d'antigène par celles-ci dans les MSC. J'ai démontré que l'IFN- active la transcription du transactivateur de classe II (CIITA), ce qui a eu pour résultat d'uprégulation les molécules MHC de type II dans les MSC murines et humaines, et que l'ajout de TGF- contrecarre l'effet de l'IFN- en inhibant la transcription de CIITA. De plus, la densité cellulaire des MSC en culture module la présentation d'antigène en affectant l'expression des molécules MHC de type II différemment chez les MSC murines et humaines. Dans le Chapitre 4, j'ai examiné la capacité des MSC de souris à cross-présenter des antigènes exogènes, une autre propriété typique des CPA. J'ai démontré que l'IFN- induit la cross-présentation dans les MSC murines et que celle-ci dépend des molécules TAP et du protéasome. J'ai aussi prouvé à l'aide d'un modèle de reconstitution immunitaire in vivo, que les MSC murines peuvent induire l'activation des lymphocytes T CD8+ contre un antigène spécifique. Finalement, j'ai enquêté dans le Chapitre 5, l'impact immunologique de l'expression et de la signalisation par les TLR chez les MSC humaines et murines. J'ai illustré que l'activation des TLR induisait l'expression de chemokines et de cytokines par les MSC créant ainsi un milieu inflammatoire propice au recrutement des cellules immunitaires. J'ai conclue en démontrant que les MSC différaient des CPA classiques par l'absence de production IL-12p70, une cytokine essentielle à la réponse immunitaire innée et acquise, en réponse à la stimulation des TLR. Les résultats inclus dans cette thèse illustrent la complexité des mécanismes immunomodulatoires des MSC. Leurs réponses face aux signaux de leur environnement, tel que l'inflammation ou l'infection activent soit leurs propriétés immunosuppressives ou –stimulatrices dépendamment de la situation. Mes découvertes pourront optimiser l'utilisation des MSC dans le domaine de l'immunothérapie cellulaire.
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Lydic, Melissa. "The Characteristics of Rabbit and Rat Mesenchymal Stromal Cell Growth and Attachment to Mesh Used in Hernia Repair." Youngstown State University / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=ysu1278102053.
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Kinchen, James. "Intestinal stromal cell types in health and inflammatory bowel disease uncovered by single-cell transcriptomics." Thesis, University of Oxford, 2017. http://ora.ox.ac.uk/objects/uuid:1bf9d8f0-6d09-46f5-9d1e-3c9e0b826618.
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Colonic stromal cells provide critical structural support but also regulate immunity, tolerance and inflammatory responses in the mucosa. Substantial variability and plasticity of mucosal stromal cells has been reported but a paucity of distinct marker genes exist to identify distinct cell states. Here single-cell RNA-sequencing is used to document heterogeneity and subtype specific markers of individual colonic stromal cells in human and mouse. Marker-free transcriptional clustering of fibroblast-like cells derived from healthy human tissue reveals distinct populations corresponding to myofibroblasts and three transcriptionally and functionally dissimilar populations of fibroblasts. A SOX6 high fibroblast subset occupies a position adjacent to the epithelial basement membrane and expresses multiple epithelial morphogens including WNT5A and BMP2. Additional fibroblast subtypes show specific enrichment for chemokine signalling and prostaglandin E2 synthesis respectively. In ulcerative colitis, substantial remodelling occurs with depletion of the SOX6 high population and emergence of an immune enriched population expressing genes associated with fibroblastic reticular cells including CCL19, CCL21 and IL33. A large murine dataset comprising over 7,000 colonic mesenchymal cells from an acute colitis model and matched healthy controls reveals strong preservation of the SOX6 high and myofibroblast transcriptional signatures. Unsupervised pseudotemporal ordering is used to relate fibroblast subsets to one another producing a branched developmental hierarchy that includes a potential progenitor population with mesothelial characteristics at its origin. This work provides a molecular basis for re-classification of colonic stromal cells and identifies pathological changes in these cells underpinning inflammation in UC.
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Thieme, Sebastian, Sabine Stopp, Martin Bornhäuser, Fernando Ugarte, Manja Wobus, Matthias Kuhn, and Sebastian Brenner. "Expression of the melanoma cell adhesion molecule in human mesenchymal stromal cells regulates proliferation, differentiation, and maintenance of hematopoietic stem and progenitor cells." Ferrata Storti Foundation, 2013. https://tud.qucosa.de/id/qucosa%3A28908.
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The melanoma cell adhesion molecule defines mesenchymal stromal cells in the human bone marrow that regenerate bone and establish a hematopoietic microenvironment in vivo. The role of the melanoma cell adhesion molecule in primary human mesenchymal stromal cells and the maintenance of hematopoietic stem and progenitor cells during ex vivo culture has not yet been demonstrated. We applied RNA interference or ectopic overexpression of the melanoma cell adhesion molecule in human mesenchymal stromal cells to evaluate the effect of the melanoma cell adhesion molecule on their proliferation and differentiation as well as its influence on co-cultivated hematopoietic stem and progenitor cells. Knockdown and overexpression of the melanoma cell adhesion molecule affected several characteristics of human mesenchymal stromal cells related to osteogenic differentiation, proliferation, and migration. Furthermore, knockdown of the melanoma cell adhesion molecule in human mesenchymal stromal cells stimulated the proliferation of hematopoietic stem and progenitor cells, and strongly reduced the formation of long-term culture-initiating cells. In contrast, melanoma cell adhesion molecule-overexpressing human mesenchymal stromal cells provided a supportive microenvironment for hematopoietic stem and progenitor cells. Expression of the melanoma cell adhesion molecule increased the adhesion of hematopoietic stem and progenitor cells to human mesenchymal stromal cells and their migration beneath the monolayer of human mesenchymal stromal cells. Our results demonstrate that the expression of the melanoma cell adhesion molecule in human mesenchymal stromal cells determines their fate and regulates the maintenance of hematopoietic stem and progenitor cells through direct cell-cell contact.
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Thieme, Sebastian, Sabine Stopp, Martin Bornhäuser, Fernando Ugarte, Manja Wobus, Matthias Kuhn, and Sebastian Brenner. "Expression of the melanoma cell adhesion molecule in human mesenchymal stromal cells regulates proliferation, differentiation, and maintenance of hematopoietic stem and progenitor cells." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2016. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-178636.
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The melanoma cell adhesion molecule defines mesenchymal stromal cells in the human bone marrow that regenerate bone and establish a hematopoietic microenvironment in vivo. The role of the melanoma cell adhesion molecule in primary human mesenchymal stromal cells and the maintenance of hematopoietic stem and progenitor cells during ex vivo culture has not yet been demonstrated. We applied RNA interference or ectopic overexpression of the melanoma cell adhesion molecule in human mesenchymal stromal cells to evaluate the effect of the melanoma cell adhesion molecule on their proliferation and differentiation as well as its influence on co-cultivated hematopoietic stem and progenitor cells. Knockdown and overexpression of the melanoma cell adhesion molecule affected several characteristics of human mesenchymal stromal cells related to osteogenic differentiation, proliferation, and migration. Furthermore, knockdown of the melanoma cell adhesion molecule in human mesenchymal stromal cells stimulated the proliferation of hematopoietic stem and progenitor cells, and strongly reduced the formation of long-term culture-initiating cells. In contrast, melanoma cell adhesion molecule-overexpressing human mesenchymal stromal cells provided a supportive microenvironment for hematopoietic stem and progenitor cells. Expression of the melanoma cell adhesion molecule increased the adhesion of hematopoietic stem and progenitor cells to human mesenchymal stromal cells and their migration beneath the monolayer of human mesenchymal stromal cells. Our results demonstrate that the expression of the melanoma cell adhesion molecule in human mesenchymal stromal cells determines their fate and regulates the maintenance of hematopoietic stem and progenitor cells through direct cell-cell contact.
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Bown, Andre B. J. "The Utilization of Multipotent Mesenchymal Stromal Cell Transplantation to Improve Fascia Repair." Youngstown State University / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=ysu1376390936.
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Huang, Gang [Verfasser]. "Impact of mesenchymal stromal cells on streptozotocin-induced diabetic cardiomyopathy / Gang Huang." Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2019. http://d-nb.info/1180387996/34.
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Roberts, Samantha. "PECAM-1 expression by mesenchymal stromal cells is regulated by Notch signalling." Thesis, University of Manchester, 2014. https://www.research.manchester.ac.uk/portal/en/theses/pecam1-expression-by-mesenchymal-stromal-cells-is-regulated-by-notch-signalling(1259846d-df3b-43c3-8ce0-d25135ccdedd).html.
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Mesenchymal stromal cells (MSCs) reside within the perivascular niche and have been shown in vivo to facilitate vascular repair. Previous in vitro studies, have demonstrated the potential of MSCs to differentiate towards an endothelial lineage, when cultured at high cell density; but the characterisation of these cells and the mechanisms directing this important differentiation effect are ill-defined. To resemble a three-dimensional (3D) cellular environment, MSCs were cultured as spheroids and the endothelial characteristics of these cells determined. MSCs cultured as spheroids significantly increased their expression of the endothelial markers; PECAM-1, Tie2, VE-cadherin and vWF, when compared to MSCs cultured in close cell contact as a two-dimensional (2D) monolayer. In addition, MSCs cultured as 3D spheroids behaved as functional endothelial cells in vitro; including the ability to uptake low-density lipoproteins, secretion of nitric oxide and the ability to form network-like structures. MSC spheroids exhibited significantly increased levels of Notch signalling, compared to 2D MSCs in close cell contact, which caused a significant decrease in the endothelial characteristics when inhibited. Conversely, activation of Notch signalling caused a significant and specific increase in the expression of PECAM-1, which was regulated by the Notch ligands Jagged1 and DLL4. Thus, Notch signalling is a crucial pathway that controls PECAM-1 expression and regulates the angiogenic fate of MSCs within spheroids. This study has therefore identified an efficient culture model and key signalling mechanism which may be used to induce MSCs towards an angiogenic fate for vascular repair and regeneration therapies.
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Sugino, Noriko. "Early osteoinductive human bone marrow mesenchymal stromal/stem cells support an enhanced hematopoietic cell expansion with altered chemotaxis- and adhesion-related gene expression profiles." Kyoto University, 2016. http://hdl.handle.net/2433/215424.
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Final publication is available at http://www.sciencedirect.com/science/article/pii/S0006291X15310664Kyoto University (京都大学)
0048
新制・課程博士
博士(医学)
甲第19598号
医博第4105号
新制||医||1014(附属図書館)
32634
京都大学大学院医学研究科医学専攻
(主査)教授 三森 経世, 教授 開 祐司, 教授 妻木 範行
学位規則第4条第1項該当
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Alfaifi, Mohammed. "Cell therapy for acute liver injury : in vivo efficacy of mesenchymal stromal cells in toxic and immune-mediated murine hepatitis." Thesis, University of Birmingham, 2018. http://etheses.bham.ac.uk//id/eprint/8330/.
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The ability of umbilical cord-derived mesenchymal stromal cells (UC-MSCs) to immunomodulate offers therapeutic potential in liver injury but the inherent heterogeneity of unsorted MSC populations may explain varied/reduced function as well as posing regulatory challenges. Thus, we aimed to evaluate the therapeutic potential of purified CD362+ MSC infusion in murine models of acute liver injury. UC-MSCs were injected intravenously into mice injured by single dose of Carbon tetrachloride (CCl4) & OVA-BIL mice. MSC used were either unsorted or sorted CD362+. The extent of liver damage was determined by liver histology, serum analysis, gene expression and FACS analysis 3 or 5 days after cell infusion. Homing and bio-distribution of stem cells was determined by whole mouse cryo-imaging of Q-dot labelled MSC following infusion of UC-MSC into injured mice. CD362+ MSC were as effective as unsorted MSC in ameliorating liver injury, with reductions in serum ALT seen in both models. In contrast heat-inactivated MSC had no effect on liver injury. MSC also led to a reduction in CD45+staining on liver sections in both models of liver injury corroborated by an accompanying reduction in hepatic CD45+ cells in (FACS analysis of liver digest). In addition, there was a significant reduction in hepatic CD19+ B cells in digested liver in CCl4 injury. CD362+ MSCs were found to have the ability to reduce the level of adhesion molecules (ICAM and VCAM) in Ova-Bil mice. Cryo-imaging of time-course in both animal models indicated that MSC had migrated to the lung within 1 hour and were then cleared rapidly, although there was a liverspecific increase in MSC 2-3 day in Ova-Bil mice. CD362+ human MSC exert potent anti-inflammatory activity in toxic and immune-mediated murine liver injury with demonstrable reductions in infiltrating inflammatory leucocytes and B cells.
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Alvarenga, Heliene Gonçalves. "Identificar e isolar células reticulares fibroblásticas em linfonodos humanos." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/5/5146/tde-19062015-153946/.
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Células reticulares fibroblásticas (FRCs, gp38+ e CD31-) e células duplo negativas (DNCs, gp38- e CD31-) são células estromais encontradas em órgãos linfoides secundários, como linfonodos. Enquanto as FRCs têm sido amplamente estudadas, pouco se sabe ainda sobre DNCs. Apesar da função estrutural das FRCs nos linfonodos já estar bem estabelecida, estudos recentes indicam que as FRCs também desempenham um papel fundamental em processos imunológicos, por exemplo, migração celular, ativação e qualidade da resposta imune, além da participação na tolerância periférica. Outra célula estromal em constante estudo são as células-tronco mesenquimais (CTMs), principalmente encontradas na medula óssea. Estas células compartilham similaridades, como por exemplo; são células estromais encontradas em órgãos linfoides, apresentam morfologia e características semelhantes quando cultivadas in vitro e estão envolvidas na resposta imune por mecanismos semelhantes. As CTMs são provenientes de um órgão linfoide primário, cuja função principal não está relacionada à resposta imunológica, entretanto, de acordo com inúmeros trabalhos, estas células possuem capacidade de interferir na ativação de várias células do sistema imunológico. Portanto, nossa hipótese é de que as FRCs e DNCs, que se encontram em um órgão linfoide secundário, cuja função principal remete a resposta imunológica, apresentem também um papel regulador, descrito na literatura como tolerância periférica e contração de uma resposta imunológica já estabelecida. Em nosso estudo mostramos que FRCs e DNCs foram isoladas a partir de linfonodos humanos e devidamente caraterizadas. Evidenciamos que FRCs e DNCs atendem todos os critérios mínimos propostos pela sociedade internacional de terapia celular para serem consideradas células-tronco estromais. Além disso, mostramos que FRCs e DNCs influênciam a proliferação e a expressão de moléculas de homing em linfócitos alogênicos in vitro. Portanto, contribuimos de forma inédita para o entendimento funcional das FRCs e DNCs, visto que estudos em humanos envolvendo estas células são escassosFibroblastic reticular cells (FRCs, gp38+ e CD31-) and double-negative cells (DNCs, gp38- e CD31-) are stromal cells found in secondary lymphoid organs, such as lymph nodes. While the FRCs has been widely studied, little is known about DNCs. Despite the structural function of FRCs on lymph nodes is well established, recent studies indicate that FRCs also play a key role in immunological processes, for example, cell migration, immune response activation and quality, beyond their involvement in peripheral tolerance. Another stromal cell type in constant study are mesenchymal stem cells (MSCs), mainly found in bone marrow. These cells share similarities with FRCs and DNCs, for example; they are estromal cells found in lymphoid organs, they present similar morphology and characteristics when cultured in vitro and they are involved in the immune response by similar mechanisms. MSCs are derived from a primary lymphoid organ which the major function is not related to immune response, but according to numerous studies these cells have the capacity of the interfere on activation of various immune cells. Consequently, our hypothesis is that FRCs and DNCs, usually found in secondary lymphoid organ, display immune regulatory roles, which were described in the literature as peripheral tolerance and immune response contraction. In our study we showed that FRCs and DNCs were isolated from human lymph nodes and adequately characterized. We evidenced that FRCs and DNCs meet all minimum criteria proposed by the International Society of Cell Therapy to be considerate a stromal stem cell. Therefore, we contributed in an unpublished manner to the functional understanding of FRCs and DNCs, since human studies involving these cells are scarce
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Consentius, Christine. "Inhibition of the crosstalk between dendritic, natural killer and T cells by mesenchymal stromal/stem cells." Doctoral thesis, Humboldt-Universität zu Berlin, Lebenswissenschaftliche Fakultät, 2016. http://dx.doi.org/10.18452/17694.
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Mesenchymale Stromazellen (MSC) unterstützen die endogene Geweberegeneration und sind kaum immunogen. Die Mechanismen der Immunmodulation sind kaum bekannt. Diese Arbeit untersucht, ob MSC in die Interaktion von Dendritischen Zellen (DC), Natürlichen Killer (NK) und T Zellen eingreifen, indem sie die DC-Reifung beeinflussen. Das Netzwerk ist wichtig für die Differenzierung naïver T Zellen zu Typ 1 T Helferzellen (Th1). Abhängig vom DC-Subtyp und dem Zeitpunkt des Aufeinandertreffens, beeinflussten Knochenmark-MSC (BM-MSC) die in vitro DC-Reifung verschieden. Sie inhibierten die Differenzierung, aber nicht die Reifung humaner von Monozyten-abgeleiteter DC (moDC). BM MSC hatten keinen klaren Einfluss auf die Reifung plasmazytoider DC (pDC), während sie in aktivierten CD1c+ myeloiden DC (mDC) einen tolerogenen Phänotyp induzierten, charakterisiert durch eine geringere CCR7-abhängige Migration und ein tolerogenes Zytokinprofil. Daraus resultierend, wiesen BM-MSC-geprägte mDC aufgrund der veränderten IL 12/IL 10 Sekretion eine geringere Fähigkeit zur Stimulation der IFNγ Produktion in NK Zellen auf und induzierten weniger Th1 Differenzierung naïver T Zellen. Placenta-derived mesenchymal-like adherent stromal cells (PLX PAD) erzielten ähnliche Ergebnisse. Es konnte keine Alloimmunogenität in Patienten mit kritischer Ischämie der Extremitäten (CLI), die im Rahmen einer Phase I klinischen Studie allogene PLX PAD erhalten hatten, nachgewiesen werden. Keiner der Patienten entwickelte eine signifikante Gedächtnis T Zellantwort spezifisch für das Zellprodukt, was durch unsere in vitro Beobachtungen erklärbar sein könnte. Es ist schwierig MSC im Gewebe nachzuweisen, da Markerkombinationen notwendig sind. CD73+CD90+CD105+CD45-CD34-CD14-CD19- MSC konnten mithilfe einer neuen Multiplex-Immunhistologie-Technik (Chipzytometrie) in humanen Plazentaschnitten detektiert werden. Für die Zukunft könnte damit die Interaktion injizierter MSC mit Immunzellen in Biopsien untersucht werden.Mesenchymal stromal cells (MSC) support endogenous tissue regeneration and seem to be low immunogenic, allowing application across MHC barriers. But little is known about the mechanisms for their immunomodulation. Hence, the main goal of this study was to understand if MSC interfere with the crosstalk between dendritic cells (DC), natural killer (NK) and T cells by influencing DC maturation. This network is important for efficient priming of naïve T cells into type 1 helper T cells (Th1). Bone marrow-derived MSC (BM-MSC) had diverse effects on DC maturation in vitro, depending on the DC subset and the time of interaction. BM MSC inhibited differentiation but not maturation of monocyte-derived DC (moDC). They did not have a clear effect on maturation of plasmacytoid DC (pDC), whereas they induced a tolerogenic phenotype in activated CD1c+ myeloid DC (mDC), characterized by an impaired CCR7-dependent migration and a tolerogenic cytokine profile. Consequently, BM-MSC-licensed mDC displayed a reduced ability to induce IFNγ production in NK cells due to their altered IL 12/IL 10 secretion. BM MSC-licensed mDC also induced less efficiently Th1 lineage commitment of naïve T cells. Similar results were observed with placenta-derived mesenchymal-like adherent stromal cells (PLX PAD). Samples from critical limb ischemia (CLI) patients treated with MHC-unmatched PLX-PAD within a phase I clinical trial were analysed for alloimmunogenicity. None of the patients developed a significant memory T cell response specific to the allogeneic cells, which might be explainable by our in vitro observations. MSC are difficult to detect in tissues because a set of lineage markers is needed. Here, CD73+CD90+CD105+CD45-CD34-CD14-CD19- MSC could be identified in human placenta cryosections using a novel multiplex-immunohistology technique (chipcytometry), offering the possibility to investigate the crosstalk between injected MSC and attracted immune cells in patient biopsies in the future.
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Ordemann, Rainer, Duohui Jing, Ana-Violeta Fonseca, Nael Alakel, Fernando A. Fierro, Katrin Muller, Martin Bornhauser, Gerhard Ehninger, and Denis Corbeil. "Hematopoietic stem cells in co-culture with mesenchymal stromal cells - modeling the niche compartments in vitro." Ferrata Storti Foundation, 2010. https://tud.qucosa.de/id/qucosa%3A28891.
Full textAbstract:
Background
Hematopoietic stem cells located in the bone marrow interact with a specific microenvironment referred to as the stem cell niche. Data derived from ex vivo co-culture systems using mesenchymal stromal cells as a feeder cell layer suggest that cell-to-cell contact has a significant impact on the expansion, migratory potential and ‘stemness’ of hematopoietic stem cells. Here we investigated in detail the spatial relationship between hematopoietic stem cells and mesenchymal stromal cells during ex vivo expansion.
Design and Methods
In the co-culture system, we defined three distinct localizations of hematopoietic stem cells relative to the mesenchymal stromal cell layer: (i) those in supernatant (non-adherent cells); (ii) those adhering to the surface of mesenchymal stromal cells (phase-bright cells) and (iii) those beneath the mesenchymal stromal cells (phase-dim cells). Cell cycle, proliferation, cell division and immunophenotype of these three cell fractions were evaluated from day 1 to 7.
Results
Phase-bright cells contained the highest proportion of cycling progenitors during co-culture. In contrast, phase-dim cells divided much more slowly and retained a more immature phenotype compared to the other cell fractions. The phase-dim compartment was soon enriched for CD34+/CD38− cells. Migration beneath the mesenchymal stromal cell layer could be hampered by inhibiting integrin β1 or CXCR4.
Conclusions
Our data suggest that the mesenchymal stromal cell surface is the predominant site of proliferation of hematopoietic stem cells, whereas the compartment beneath the mesenchymal stromal cell layer seems to mimic the stem cell niche for more immature cells. The SDF-1/CXCR4 interaction and integrin-mediated cell adhesion play important roles in the distribution of hematopoietic stem cells in the co-culture system.
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Ordemann, Rainer, Duohui Jing, Ana-Violeta Fonseca, Nael Alakel, Fernando A. Fierro, Katrin Muller, Martin Bornhauser, Gerhard Ehninger, and Denis Corbeil. "Hematopoietic stem cells in co-culture with mesenchymal stromal cells - modeling the niche compartments in vitro." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2016. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-177403.
Full textAbstract:
Background
Hematopoietic stem cells located in the bone marrow interact with a specific microenvironment referred to as the stem cell niche. Data derived from ex vivo co-culture systems using mesenchymal stromal cells as a feeder cell layer suggest that cell-to-cell contact has a significant impact on the expansion, migratory potential and ‘stemness’ of hematopoietic stem cells. Here we investigated in detail the spatial relationship between hematopoietic stem cells and mesenchymal stromal cells during ex vivo expansion.
Design and Methods
In the co-culture system, we defined three distinct localizations of hematopoietic stem cells relative to the mesenchymal stromal cell layer: (i) those in supernatant (non-adherent cells); (ii) those adhering to the surface of mesenchymal stromal cells (phase-bright cells) and (iii) those beneath the mesenchymal stromal cells (phase-dim cells). Cell cycle, proliferation, cell division and immunophenotype of these three cell fractions were evaluated from day 1 to 7.
Results
Phase-bright cells contained the highest proportion of cycling progenitors during co-culture. In contrast, phase-dim cells divided much more slowly and retained a more immature phenotype compared to the other cell fractions. The phase-dim compartment was soon enriched for CD34+/CD38− cells. Migration beneath the mesenchymal stromal cell layer could be hampered by inhibiting integrin β1 or CXCR4.
Conclusions
Our data suggest that the mesenchymal stromal cell surface is the predominant site of proliferation of hematopoietic stem cells, whereas the compartment beneath the mesenchymal stromal cell layer seems to mimic the stem cell niche for more immature cells. The SDF-1/CXCR4 interaction and integrin-mediated cell adhesion play important roles in the distribution of hematopoietic stem cells in the co-culture system.
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Amann, Elisa Maria [Verfasser]. "Immunomodulatory and regenerative effects of mesenchymal stromal cells in trauma / Elisa Maria Amann." Ulm : Universität Ulm, 2019. http://d-nb.info/1197692819/34.
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Bader, Andreas Matthäus [Verfasser]. "Molecular mechanisms of cardioprotection by cord blood mesenchymal stromal cells / Andreas Matthäus Bader." Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2016. http://d-nb.info/1100387854/34.
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Espig, Sandy [Verfasser]. "Isolation and characterization of rat bone-marrow derived mesenchymal stromal cells / Sandy Espig." Ulm : Universität Ulm. Medizinische Fakultät, 2016. http://d-nb.info/1082294284/34.
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Burk, Janina, Claudia Gittel, Sandra Heller, Bastian Pfeiffer, Felicitas Paebst, Annette B. Ahrberg, and Walter Brehm. "Gene expression of tendon markers in mesenchymal stromal cells derived from different sources." Universitätsbibliothek Leipzig, 2014. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-157823.
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Background: Multipotent mesenchymal stromal cells (MSC) can be recovered from a variety of tissues in the body. Yet, their functional properties were shown to vary depending on tissue origin. While MSC have emerged as a favoured cell type for tendon regenerative therapies, very little is known about the influence of the MSC source on
their properties relevant to tendon regeneration. The aim of this study was to assess and compare the expression of tendon extracellular matrix proteins and tendon differentiation markers in MSC derived from different sources as well as in native tendon tissue. MSC isolated from equine bone marrow, adipose tissue, umbilical cord tissue, umbilical cord blood and tendon tissue were characterized and then subjected to mRNA analysis by real-time polymerase chain reaction. Results: MSC derived from adipose tissue displayed the highest expression of collagen 1A2, collagen 3A1 and decorin compared to MSC from all other sources and native tendon tissue (p < 0.01). Tenascin-C and scleraxis
expressions were highest in MSC derived from cord blood compared to MSC derived from other sources, though both tenascin-C and scleraxis were expressed at significantly lower levels in all MSC compared to native tendon tissue (p < 0.01). Conclusions: These findings demonstrate that the MSC source impacts the cell properties relevant to tendon regeneration. Adipose derived MSC might be superior regarding their potential to positively influence tendon matrix reorganization.
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Badraiq, Heba Ghazi O. "Effects of maternal body weight on Wharton's Jelly mesenchymal stromal cells (pilot study)." Thesis, King's College London (University of London), 2017. https://kclpure.kcl.ac.uk/portal/en/theses/effects-of-maternal-body-weight-on-whartons-jelly-mesenchymal-stromal-cells-pilot-study(dac6be9c-1f9d-4c00-88dc-2a73ec4489b4).html.
Full textAbstract:
To investigate whether the maternal metabolic environment affects the DNA methylation of mesenchymal stromal/stem cells (MSCs) from umbilical cord (UC) Wharton’s Jelly (WJ), potentially rendering them unsuitable for clinical use in multiple recipients, a pilot study was conducted on fourteen UCs obtained post partum from healthy non-obese (BMI=19-25; n=7) and obese (BMI≥30; n=7) donors receiving elective Caesarean sections. The time of first WJ-MSCs outgrowth from UC explants was similar in samples from obese and non-obese donors. However, the cells from non-obese donors proliferated faster after 34 hours of culture than cells from obese donors. Differentiation into adipogenic, osteogenic and chondrogenic lineages was similar between obese and non-obese donor samples as demonstrated by tissue-specific staining and RT-PCR for lineage markers. However, WJ-MSCs from obese donors exhibited stronger immunosuppressive activity than those from non-obese donors. Genome-wide DNA methylation of triple-positive (CD73+CD90+CD105+) WJ-MSCs sorted from the first passage of a mixed population of cells was assessed. Samples from the obese and non-obese donors clustered separately, and 5,767 of the analysed CpG sites (1%) exhibited different methylation. Sixty-seven genes were observed with at least one CpG site with a methylation difference ≥0.2 in four or more obese donors. These 67 genes were further refined based on a list of polymorphic CpG sites and segmental duplications. In 18 of the 67 genes with a different CpG methylation pattern, the CpG sites were in non-polymorphic regions. However, two genes (DCAF6 and ZNF714) resided in segmentally duplicated regions. To determine whether methylation differences altered gene expression, the samples were analysed using a HumanHT-12 Expression BeadChip array and, of the 18 genes, only PNPLA7 was significantly affected at the mRNA level, which was confirmed independently by RT-PCR and Western blotting. Although the number of analysed donors was limited, the data suggest that an abnormal metabolic environment related to excessive body weight might alter the properties of WJ-MSCs used for cellular therapy.
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Kinoshita, Masaki. "The novel protein kinase Vlk is essential for stromal function of mesenchymal cells." Kyoto University, 2009. http://hdl.handle.net/2433/126438.
Full textAbstract:
Kyoto University (京都大学)0048
新制・課程博士
博士(医学)
甲第14862号
医博第3377号
新制||医||977(附属図書館)
27284
UT51-2009-K658
京都大学大学院医学研究科脳統御医科学系専攻
(主査)教授 清水 章, 教授 開 祐司, 教授 影山 龍一郎
学位規則第4条第1項該当
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Clough, Sally. "IL7 as a marker of a subset of bone marrow mesenchymal stromal cells." Thesis, University of York, 2013. http://etheses.whiterose.ac.uk/4771/.
Full textAbstract:
The organisation of a multitude of cellular niche components, their communication via many signalling pathways and their response to physical factors, protects and regulates haematopoietic stem cell (HSC) fate in adult bone marrow. Whilst the contribution of osteoblasts, endothelial cells and perivascular cells have been examined, the role of a second stem cell population in the bone marrow; mesenchymal stem cells, is not well understood due to the lack of distinctive markers to identify them in vivo. There is therefore a requirement to determine a characteristic that allows their prospective isolation. Under certain conditions, stromal cells and osteoblasts in the bone marrow express IL-7. The use of a novel IL7-Cre BAC transgenic mouse line has allowed more accurate IL 7 protein detection in situ and demonstrated IL-7 reporter expression in mesenchymal lineage cells in endosteal and vascular HSC niche locations. These cells were further characterised in this study in order to determine if IL-7 or nestin, an intermediate filament associated with a wide range of stem cell populations, is expressed by and could identify bone marrow derived MSCs. YFP positive cells were analysed in sections of IL-7Cre Rosa26-eYFP mice. Interestingly, it was only a proportion of mesenchymal cells that expressed YFP, supporting the theory that subsets of MSCs exist and therefore, that they may have different roles in numerous bone marrow niches. IL-7 was not observed to have any effect on the proliferation or differentiation of human MSCs. Generation of MSC clones supported the suggestion that in vitro cultures of MSCs are a heterogeneous population and they displayed a wide range of IL-7 and nestin mRNA expression levels.
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Cuthbert, Richard J. "Exploiting knowledge of mesenchymal stromal cells in vivo for bone disease therapy development." Thesis, University of Leeds, 2014. http://etheses.whiterose.ac.uk/8418/.
Full textAbstract:
This thesis aims to demonstrate how understanding of the nature of MSCs in vivo can be used to guide bone disease therapy development. It first address the unpredictable MSC content of bone marrow (BM) aspirates used for therapy; then examines MSC enrichment using clinical grade immunomagnetic cell selection. Induction of osteogenesis was subsequently explored; by examining an induced membrane (IM) used for bone regeneration. Finally the potential of a janus kinase (JAK) inhibitor to block osteogenesis was assessed, by studying its effect on MSCs in vitro. Flow cytometry was used to enumerate cells expressing a CD45-/low CD271+ phenotype in BM aspirate and this was compared to colony forming unit fibroblast (CFU-F) content. MSCs were enriched from BM, enzymatically treated femoral heads (FH) and intramedullary canal aspirates and enumerated. The composition morphology, MSC content and differentiation potential of IM was compared to periosteum. The potential effects of JAK inhibition on MSC colony formation, expansion and differentiation potential were examined. The concentration of cells expressing a CD45-/low CD271+ phenotype strongly correlated with CFU-F concentration (R=0.812, p<0.001). Immunomagnetic cell selection resulted in an increase in the proportion of MSCs in BM, FH and intramedullary canal aspirates by 204, 14.1 and 291-fold respectively. The regenerative potential of periosteum and IM were comparable. JAK inhibition did not affect MSC growth, osteogenesis or chondrogenesis but caused an increase in adipogenesis at concentrations ≥100nM compared to controls (1.38 fold, p=0.041). Flow cytometry may be used to rapidly and accurately predict the MSC content of BM. Clinical grade immunomagnetic selection can substantially increase the purity of MSCs from bone cavities. The similarity, in terms of regenerative potential, of periosteum and IM gives insight into its use for bone regeneration. JAK inhibition did not affect in vitro osteogenesis but has potential to affect in vivo osteogenesis through stimulation of adipogenesis.