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1
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.
Full textAbstract:
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.
2
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.
3
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
4
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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Chandran, Priya. "Bone Marrow Microenvironment in Acute Myleoid Leukemia." Thèse, Université d'Ottawa / University of Ottawa, 2013. http://hdl.handle.net/10393/24301.
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Acute myeloid leukemia (AML) often remains refractory to current chemotherapy and transplantation approaches despite many advances in our understanding of mechanisms in leukemogenesis. The bone marrow “niche” or microenvironment, however, may be permissive to leukemia development and studying interactions between the microenvironment and leukemia cells may provide new insight for therapeutic advances. Mesenchymal stem cells (MSCs) are central to the development and maintenance of the bone marrow niche and have been shown to have important functional alterations derived from patients with different hematological disorders. The extent to which MSCs derived from AML patients are altered remains unclear. The aim of this study was to detect changes occurring in MSCs obtained from human bone marrow in patients with AML by comparing their function and gene expression pattern with normal age-matched controls.
MSCs expanded from patients diagnosed with acute leukemia were observed to have heterogeneous morphological characteristics compared to the healthy controls. Immunohistochemistry and flow data confirmed the typical cell surface immunophenotype of CD90+ CD105+ CD73+ CD34- CD45-, although MSCs from two patients with AML revealed reduced surface expression of CD105 and CD90 antigens respectively. Differentiation assays demonstrated the potential of MSCs from AML patients and healthy donors to differentiate into bone, fat and cartilage. However, the ability of MSCs from AML samples to support hematopoietic function of CD34+ progenitors was found to be impaired while the key hematopoietic genes were found to be differentially expressed on AML-MSCs compared to nMSCs.
These studies indicate that there exist differences in the biologic profile of MSCs from AML patients compared to MSCs derived from healthy donors. The results described in the thesis provide a formulation for additional studies that may allow us to identify new targets for improved treatment of AML.
6
Lloyd, Brandon R. "Comparison of Bone Marrow Mesenchymal Stem Cells from Limb and Jaw Bones." The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1458678153.
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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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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.
9
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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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/.
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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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Mareddy, Shobha R. "Characterization of bone marrow stromal clonal populations derived from osteoarthritis patients." Thesis, Queensland University of Technology, 2008. https://eprints.qut.edu.au/17151/1/Shobha_Reddy_Mareddy_Thesis.pdf.
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This work is concerned with the characterization of mesenchymal stem cells (MSC) specifically from bone marrow samples derived from patients with osteoarthritis (OA). The multilineage potential of mesenchymal stem cells as well as their ease of exvivo expansion makes these cells an attractive therapeutic tool for applications such as autologous transplantation and tissue engineering. Bone marrow is considered a source of MSC. However, there is a general assumption that the occurrence of MSCs and their activity in bone marrow diminishes with age and disease. This prompted us to isolate and identify multipotential and self-renewing cells from patients with the degenerative disease osteoarthritis, with the view of using these cells for autologous cell therapies. It is therefore of great potential benefit to investigate the isolation and characterization of stem cell/progenitors from bone marrow samples of patients with osteoarthritis in greater detail. We employed a single cell clone culture method in order to develop clonal cell populations from three bone marrow samples and characterized them based on their proliferation and differentiation capabilities. The clonal populations were grouped into fast-growing and slow-growing clones based on their proliferation rates. The fastgrowing clones displayed 20-30% greater proliferation rate than the slow-growing clones. The study also revealed that the proliferation rates were directly proportional to their differentiation capacities. Most of the fast-growing clones were found to be tripotential for osteogenic, chondrogenic and adipogenic lineages, whereas the slow growing clones were either uni or bipotential. Flow cytometry analysis for the phenotype determination using putative MSC surface markers did not reveal any difference between the two clonal populations indicating a need for further molecular studies. Two approaches were employed to further investigate the molecular processes involved in the existence of such varying populations. In the first method gene expression studies were performed between the fast-growing (n=3) and slow-growing (n=3) clonal populations to identify potential genetic markers associated with cell 'sternness' using the Stem Cell RT2 ProfilerTM PCR Array comprising a series of 84 genes related to stem cell pathways. Ten genes were identified to be commonly and significantly over represented in the fast-growing stem cell clones when compared to slow-growing clones. This included expression of transcripts beyond MSC lineage specification such as SOX2, NOTCH1 and FOXA2 which signified that stem cell maintenance requires a coordinated regulation by multiple signalling pathways. The second study involved an extensive protein expression profiling of the fast growing (n=2) and slow growing (n=2) clonal populations using off-line Two Dimensional Liquid Chromatography (2D-LC)/Matrix-Assisted Laser Desorption/Ionization (MALDI) Mass Spectrometry (MS). A total of 67 proteins were identified, of which 11 were expressed at significantly different levels between the subpopulations. Protein ontology revealed these proteins to be associated with cellular organization, cytokinesis, signal transduction, energy pathways and cell stress response. Of particular interest was the differential presentation of the proteins calmodulin, tropomyosin and caldesmon between fast- and slow-growing clones. Based on their reported roles in the regulation of cell proliferation and maintenance of cell integrity, we draw an association between their expression and the altered status in which the subpopulations exist. Based on our observations, these proteins may be prospective molecular markers to distinguish between the fast-growing and slow-growing subpopulations. In summary, this study demonstrated the existence of potential stem cells of therapeutic importance in spite of a supposedly smaller stem cell compartment in patients with osteoarthritis. Furthermore, the differentially expressed genes between the sub-populations highlight the 'sternness' of the potential clones, an observation supported by the expression of proteins which act as effective modulators in the maintenance of cell integrity and cell cycle regulation. This study provides a basis for more detailed investigations in search of selective cell surface markers
12
Mareddy, Shobha R. "Characterization of bone marrow stromal clonal populations derived from osteoarthritis patients." Queensland University of Technology, 2008. http://eprints.qut.edu.au/17151/.
Full textAbstract:
This work is concerned with the characterization of mesenchymal stem cells (MSC) specifically from bone marrow samples derived from patients with osteoarthritis (OA). The multilineage potential of mesenchymal stem cells as well as their ease of exvivo expansion makes these cells an attractive therapeutic tool for applications such as autologous transplantation and tissue engineering. Bone marrow is considered a source of MSC. However, there is a general assumption that the occurrence of MSCs and their activity in bone marrow diminishes with age and disease. This prompted us to isolate and identify multipotential and self-renewing cells from patients with the degenerative disease osteoarthritis, with the view of using these cells for autologous cell therapies. It is therefore of great potential benefit to investigate the isolation and characterization of stem cell/progenitors from bone marrow samples of patients with osteoarthritis in greater detail. We employed a single cell clone culture method in order to develop clonal cell populations from three bone marrow samples and characterized them based on their proliferation and differentiation capabilities. The clonal populations were grouped into fast-growing and slow-growing clones based on their proliferation rates. The fastgrowing clones displayed 20-30% greater proliferation rate than the slow-growing clones. The study also revealed that the proliferation rates were directly proportional to their differentiation capacities. Most of the fast-growing clones were found to be tripotential for osteogenic, chondrogenic and adipogenic lineages, whereas the slow growing clones were either uni or bipotential. Flow cytometry analysis for the phenotype determination using putative MSC surface markers did not reveal any difference between the two clonal populations indicating a need for further molecular studies. Two approaches were employed to further investigate the molecular processes involved in the existence of such varying populations. In the first method gene expression studies were performed between the fast-growing (n=3) and slow-growing (n=3) clonal populations to identify potential genetic markers associated with cell 'sternness' using the Stem Cell RT2 ProfilerTM PCR Array comprising a series of 84 genes related to stem cell pathways. Ten genes were identified to be commonly and significantly over represented in the fast-growing stem cell clones when compared to slow-growing clones. This included expression of transcripts beyond MSC lineage specification such as SOX2, NOTCH1 and FOXA2 which signified that stem cell maintenance requires a coordinated regulation by multiple signalling pathways. The second study involved an extensive protein expression profiling of the fast growing (n=2) and slow growing (n=2) clonal populations using off-line Two Dimensional Liquid Chromatography (2D-LC)/Matrix-Assisted Laser Desorption/Ionization (MALDI) Mass Spectrometry (MS). A total of 67 proteins were identified, of which 11 were expressed at significantly different levels between the subpopulations. Protein ontology revealed these proteins to be associated with cellular organization, cytokinesis, signal transduction, energy pathways and cell stress response. Of particular interest was the differential presentation of the proteins calmodulin, tropomyosin and caldesmon between fast- and slow-growing clones. Based on their reported roles in the regulation of cell proliferation and maintenance of cell integrity, we draw an association between their expression and the altered status in which the subpopulations exist. Based on our observations, these proteins may be prospective molecular markers to distinguish between the fast-growing and slow-growing subpopulations. In summary, this study demonstrated the existence of potential stem cells of therapeutic importance in spite of a supposedly smaller stem cell compartment in patients with osteoarthritis. Furthermore, the differentially expressed genes between the sub-populations highlight the 'sternness' of the potential clones, an observation supported by the expression of proteins which act as effective modulators in the maintenance of cell integrity and cell cycle regulation. This study provides a basis for more detailed investigations in search of selective cell surface markers
13
Villaggio, Giusy. "Relationship between extracellular matrix (ECM) components and mineralization in bone marrow stromal cells." Doctoral thesis, Università di Catania, 2014. http://hdl.handle.net/10761/1492.
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The relations between cells and extracellular matrix seem to orchestrate tissue organization by regulating cell functions during fetal development and throughout normal adult life. Thus, focusing on the innate ability of the native ECM to better modulate cell behavior, the coating of synthetic biomaterials with cell-derived decellularized extracellular matrices is a promising approach to confer bioactivity to inert materials and direct the fate of host or transplanted cells in tissue engineering applications.
This study aims to better understand ECM influence on human bone marrow stem cells and its role during the induction of an osteogenic phenotype. For this purpose decellularized matrices were prepared and examined for viability, morphology, adhesion, ALP content, mineralization and gene expression.
Extracellular matrix coatings were able to delay, unlike uncoated surfaces, cell spontaneous differentiation, underlying its role in the preservation of cell stemness. Furthermore, the combination of free cell ECM with osteogenic medium resulted in a strong effect on cell differentiation.
In conclusion, the ECM provides an ideal environment that promote cell adhesion and proliferation creating an ideal setting for the large-scale expansion of MSCs. In addition, it could enhance, in the presence of osteogenic factors, the cells differentiative ability providing the basis for an easier tissue-specific fate control of MSCs for therapeutic applications.
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Ma, Ming, and 馬明. "Human mesenchymal stromal cells enhance bone marrow metastases of neuroblastoma via SDF-1 related pathways." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2010. http://hub.hku.hk/bib/B45790486.
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Munshi, Afnan M. N. Alam. "Comprehensive Proteomic Analysis and Characterization of Human Bone Marrow Mesenchymal Stem/Stromal Derived Extracellular Vesicles." Thesis, Université d'Ottawa / University of Ottawa, 2019. http://hdl.handle.net/10393/39538.
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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
17
Carlton, Morgan M. "Proteomic characterisation of rat bone marrow-derived mesenchymal stromal cells cultured in 'stemness' promoting conditions." Thesis, Queensland University of Technology, 2018. https://eprints.qut.edu.au/117288/1/Morgan_Carlton_Thesis.pdf.
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This thesis investigates the effect of 'stemness' promoting growth conditions on mesenchymal stem cells. A proteomics approach was used to evaluate the biology of the cells and to determine how each of the growth conditions affected cellular differentiation. Several proteins of interest were identified, and their biological roles were assessed.
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Bradhurst, Christopher John. "Monitoring mesenchymal stem cell cultures using image processing and pattern recognition techniques." Thesis, Queensland University of Technology, 2010. https://eprints.qut.edu.au/43623/1/Christopher_Bradhurst_Thesis.pdf.
Full textAbstract:
Stem cells have attracted tremendous interest in recent times due to their promise in providing innovative new treatments for a great range of currently debilitating diseases. This is due to their potential ability to regenerate and repair damaged tissue, and hence restore lost body function, in a manner beyond the body's usual healing process. Bone marrow-derived mesenchymal stem cells or bone marrow stromal cells are one type of adult stem cells that are of particular interest. Since they are derived from a living human adult donor, they do not have the ethical issues associated with the use of human embryonic stem cells. They are also able to be taken from a patient or other donors with relative ease and then grown readily in the laboratory for clinical application. Despite the attractive properties of bone marrow stromal cells, there is presently no quick and easy way to determine the quality of a sample of such cells. Presently, a sample must be grown for weeks and subject to various time-consuming assays, under the direction of an expert cell biologist, to determine whether it will be useful. Hence there is a great need for innovative new ways to assess the quality of cell cultures for research and potential clinical application. The research presented in this thesis investigates the use of computerised image processing and pattern recognition techniques to provide a quicker and simpler method for the quality assessment of bone marrow stromal cell cultures. In particular, aim of this work is to find out whether it is possible, through the use of image processing and pattern recognition techniques, to predict the growth potential of a culture of human bone marrow stromal cells at early stages, before it is readily apparent to a human observer. With the above aim in mind, a computerised system was developed to classify the quality of bone marrow stromal cell cultures based on phase contrast microscopy images. Our system was trained and tested on mixed images of both healthy and unhealthy bone marrow stromal cell samples taken from three different patients. This system, when presented with 44 previously unseen bone marrow stromal cell culture images, outperformed human experts in the ability to correctly classify healthy and unhealthy cultures. The system correctly classified the health status of an image 88% of the time compared to an average of 72% of the time for human experts. Extensive training and testing of the system on a set of 139 normal sized images and 567 smaller image tiles showed an average performance of 86% and 85% correct classifications, respectively. The contributions of this thesis include demonstrating the applicability and potential of computerised image processing and pattern recognition techniques to the task of quality assessment of bone marrow stromal cell cultures. As part of this system, an image normalisation method has been suggested and a new segmentation algorithm has been developed for locating cell regions of irregularly shaped cells in phase contrast images. Importantly, we have validated the efficacy of both the normalisation and segmentation method, by demonstrating that both methods quantitatively improve the classification performance of subsequent pattern recognition algorithms, in discriminating between cell cultures of differing health status. We have shown that the quality of a cell culture of bone marrow stromal cells may be assessed without the need to either segment individual cells or to use time-lapse imaging. Finally, we have proposed a set of features, that when extracted from the cell regions of segmented input images, can be used to train current state of the art pattern recognition systems to predict the quality of bone marrow stromal cell cultures earlier and more consistently than human experts.
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Wojtowicz, Abigail M. "Genetically-engineered bone marrow stromal cells and collagen mimetic scaffold modification for healing critically-sized bone defects." Diss., Georgia Institute of Technology, 2009. http://hdl.handle.net/1853/34705.
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Non-healing bone defects have a significant socioeconomic impact in the U.S. with approximately 600,000 bone grafting procedures performed annually. Autografts and allografts are clinically the most common treatments; however, autologous donor bone is in limited supply, and allografts often have poor mechanical properties. Therefore, tissue engineering and regenerative medicine strategies are being developed to address issues with clinical bone grafting. The overall objective of this work was to develop bone tissue engineering strategies that enhance healing of orthotopic defects by targeting specific osteogenic cell signaling pathways. The general approach included the investigation of two different tissue engineering strategies, which both focused on directed osteoblastic differentiation to promote bone formation.
In the first cell-based strategy, we hypothesized that constitutive overexpression of the osteoblast-specific transcription factor, Runx2, in bone marrow stromal cells (BMSCs) would promote orthotopic bone formation in vivo. We tested this hypothesis by delivering Runx2-modified BMSCs on synthetic scaffolds to critically-sized defects in rats. We found that Runx2-modified BMSCs significantly increased orthotopic bone formation compared to empty defects, cell-free scaffolds and unmodified BMSCs. This gene therapy approach to bone regeneration provides a mineralizing cell source which has clinical relevance.
In the second biomaterial-based strategy, we hypothesized that incorporation of the collagen-mimetic peptide, GFOGER, into synthetic bone scaffolds would promote orthotopic bone formation in vivo without the use of cells or growth factors. We tested this hypothesis by passively adsorbing GFOGER onto poly-caprolactone (PCL) scaffolds and implanting them into critically-sized orthotopic defects in rats. We found that GFOGER-coated scaffolds significantly increased bone formation compared to uncoated scaffolds in a dose dependent manner. Development of this cell-free strategy for bone tissue engineering provides an inexpensive therapeutic alternative to clinical bone defect healing, which could be implemented as a point of care application.
Both strategies developed in this work take advantage of specific osteoblastic signaling pathways involved in bone healing. Further development of these tissue engineering strategies for bone regeneration will provide clinically-relevant treatment options for healing large bone defects in humans by employing well-controlled signals to promote bone formation and eliminating the need for donor bone.
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Goulard, Marie. "The bone marrow microenvironment in myelodysplastic syndromes : functional and molecular study." Thesis, Sorbonne Paris Cité, 2017. http://www.theses.fr/2017USPCC302.
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Les syndromes myélodysplasiques (MDS) sont un groupe de pathologies myéloïdes caractérisées par une hématopoïèse inefficace. Le rôle du microenvironnement médullaire (MM) dans l’histoire naturelle de ces pathologies reste incertain. Des anomalies du MM ont été décrites au cours des myélodysplasies et des modèles murins récemment publiés font penser qu’une altération du MM pourrait jouer un rôle dans le déclenchement et/ou l’évolution de ces maladies.Nous avons tenté de développer un modèle in vivo récapitulant l’histoire naturelle des myélodysplasies par des xénogreffes chez des souris NSG et NSG-S. Le faible taux de prise de greffe nous a amenés à développer un modèle in vitro de co-culture en 2D. Ce modèle est une bonne alternative pour les études de nouvelles stratégies thérapeutiques pour les patients atteints de myélodysplasies.Au cours de ce travail, nous avons également réalisé une étude systématique du stroma médullaire de patients atteints de syndromes myélodysplasiques dans le but d’identifier les anomalies fonctionnelles et moléculaires des cellules souches mésenchymateuses (CSMs), cellules centrales du MM pour leur interaction avec les cellules souches hématopoïétiques (CSHs).Les CSMs de MDS ont une clonogénécité diminuée. Nous n’avons pas observé de modification significative de leurs capacités de différenciation en ostéoblastes, adipocytes et chondrocytes ni dans leur capacité à supporter une hématopoïèse normale. Les CSMs de MDS présentent des modifications au niveau épigénétique et transcriptionnel pouvant expliquer l’altération des relations observées grâce à de l’imagerie enregistrée entre les CSMs de MDS et les CSHs dans un modèle de co-culture en 3D.Ces résultats montrent que les CSMs de MDS ont des modifications fonctionnelles et moléculaires et que ces anomalies perturbent leur relation avec les CSHsMyelodysplastic syndromes (MDS) are a heterogeneous group of clonal myeloid pathologies characterized by an impaired hematopoiesis. The role of the bone marrow microenvironment (BMM) remains unclear in the natural history of these diseases. Abnormalities of the BMM have been observed in myelodysplasia and a recent published murine model implies that alterations of the BMM could play a role in the trigger/progression of these diseases.Firstly, we tried to develop an in vivo model of MDS in NSG and NSG-S mice. The low rate of engraftment pushed us to develop a 2D co-culture model in vitro. This model is a good alternative to test new therapeutic strategies for MDS patients.In this study, we analysed mesenchymal stromal cells (MSCs) from the bone marrow of pretreated MDS patients in order to identify the functional and molecular abnormalities in those cells of the BMM, central for their interactions with the hematopoietic stem cells (HSCs).MDS MSCs have an impaired clonogenic capacity. We didn’t observed modifications of their differentiation toward osteogenic, adipogenic and chondrogenic pathways and capacity to support of a normal hematopoiesis. MDS MSCs display epigenetic and transcriptomic modifications that could explain the alteration of the relationships between these cells and HSCs observed in imagery in a 3D co-culture model.These results showed that MDS MSCs have functional and molecular abnormalities and that these alterations could impair their relationship with HSCs
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Fujii, Sumie. "GVHD amelioration by human bone marrow mesenchymal stromal/stem cell-derived extracellular vesicles is associated with peripheral preservation of naive T cell populations." Kyoto University, 2018. http://hdl.handle.net/2433/232136.
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Woolston, Caroline. "The role of bone marrow mesenchymal stromal cells in the protection of B-cell chronic lymphocytic leukaemia from spontaneous and drug induced apoptosis." Thesis, University of Leicester, 2004. http://hdl.handle.net/2381/29873.
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An in vitro model to mimic in vivo conditions was developed using adherent BM Mesenchymal Stromal Cells (MSCs), isolated from the iliac crest of normal donors. These "fibroblast-like" MSCs provide a supporting layer for haemopoietic differentiation. They lack expression of haemopoietic differentiation antigens and lineage markers:- CD34, CD45, HLA-DR, CD31 and CD68 but express Vimentin, alphaSmooth Muscle Actin and Ab-1 (fibroblast marker). The model was used to determine if MSCs could protect B-CLL B cells from spontaneous apoptosis and prevent the effects of the therapeutic monoclonal antibodies Rituximab (CD20) and Campath-1H (CD52), the proteasomal inhibitor MG132 and BisIX, a protein kinase C inhibitor. Levels of apoptosis were determined by flow cytometry, using Annexin V-FITC/PI. MSCs abrogated spontaneous apoptosis of B-CLL B cells in direct contact with them, over 4 days (80-90% viability, n=30). Rituximab and Campath-1H induced apoptosis in the B-CLL B cells that could not be prevented by contact with MSCs (n=6). MG132 and BisIX caused substantial apoptosis in B-CLL B cells (0-15% viability) but MSCs could protect against lower concentrations (200nM) of both drugs (70-90% viability, n=6). MSCs' protection of B-CLL B cells from spontaneous apoptosis was through the upregulation of the expression of Mcl-1 and Xiap, but not Survivin. The cell-cell interactions include the beta1, beta2, beta3 and beta4 integrins but have variable effects on B-CLL cases when bound by blocking antibodies. Therefore MSCs can maintain B-CLL viability in vitro and influence chemotherapeutic strategies. The model provides a reliable method to evaluate drug cytotoxicity and Mcl-1 offers a novel therapeutic target.
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Rostovskaya, Maria. "Lineage Commitment of Conditionally Immortalized Bone Marrow Mesenchymal Stromal Cells from Tetracycline-Regulated SV40 Large T-antigen Transgenic Mice." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2010. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-63435.
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Adult bone marrow contains a population of mesenchymal stem cells capable to self-renew and to differentiate into haematopoietic-supportive stroma, osteo, adipo- and chondrocytes. However, the identity of mesenchymal stem cells still remains uncertain. The complex population of their descendants, bone marrow mesenchymal stromal cells (BM MSCs), represents a model to study the principles of differentiation and commitment into mesodermal lineages. The experiments using BM MSCs are often hampered by their low proliferative capacity in vitro. In the present study, we established conditionally immortalized BM MSCs from tetracycline-regulated SV40 Large T-antigen transgenic mice. The identity of the conditionally immortalized BM MSCs was confirmed by marker expression, ability to support haematopoiesis and differentiation potential. The advantages of the conditional immortalization are encompassed in (1) indefinite expansion of cell populations, (2) possibility to perform cellular cloning and (3) prevention from spontaneous differentiation.
We demonstrated the heterogeneity of BM MSCs and identified at least 6 types of progenitors within BM MSCs population based on their differentiation potential (“OAC”, “OA”, “OC”, “AC”, “O”, “A”). A hypothetical model of BM MSC hierarchy and the relationships between the progenitors has been proposed.
We observed that the Wnt/β-catenin signaling pathway and GSK3 activity could modulate the efficiency of osteo- and adipogenic differentiation pathways, but we didn’t find evidence that the lineage commitment of BM MSCs is determined by Wnt.
We elucidated the mechanism of transcriptional regulation of the adipogenic induction of BM MSCs in vitro. Our data revealed the key regulatory role of PPARγ1 during adipogenesis in BM MSCs. Furthermore, we assume that PPARγ1 is a potential trigger of the adipogenic commitment of the BM MSCs progenitors. Finally, the non-adipogenic BM MSCs progenitors were converted into the adipogenic lineage using ectopical expression of the transcription factors C/EBPα, C/EBPβ and C/EBPδ. Our findings provide a novel insight into the molecular mechanisms of BM MSCs lineage commitment.
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Mukohira, Hisa. "Mesenchymal stromal cells in bone marrow express adiponectin and are efficiently targeted by an adiponectin promoter-driven Cre transgene." Kyoto University, 2020. http://hdl.handle.net/2433/253155.
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Futrega, Katarzyna. "Device and application development for haematopoietic stem and progenitor cell (HSPC) and mesenchymal stromal cell (MSC) 3D spheroid cultures." Thesis, Queensland University of Technology, 2016. https://eprints.qut.edu.au/92605/1/Katarzyna_Futrega_Thesis.pdf.
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With an overall aim to improve haematopoietic stem/progenitor cell (HSPC) and mesesnchymal stromal cells (MSC) 3D culture systems, this PhD Thesis addressed the following four interlinked AIMs: (1) The development of a high throughput microwell platform that enabled evaluation of MSC spheroid potential to expand HSPC in vitro; (2) Utilization of the high throughput microwell platform to manufacture HSPC/MSC spheroids to improve the efficacy of direct bone marrow transplantation; (3) The development of an improved microwell platform that retains spheroids within discrete microwells throughout culture; and (4) Characterization of HSPC surface marker change in response to the microwell material.
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Ding, Ximing [Verfasser], and Judith [Akademischer Betreuer] Zaugg. "Aging of human bone marrow – functional and epigenetic changes in senescent mesenchymal stromal cells / Ximing Ding ; Betreuer: Judith Zaugg." Heidelberg : Universitätsbibliothek Heidelberg, 2018. http://d-nb.info/1177691140/34.
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Lenz, Daniel [Verfasser], Andreas [Gutachter] Radbruch, Andreas [Gutachter] Thiel, and Enrico [Gutachter] Klotzsch. "Dissecting the heterogeneity of murine mesenchymal bone marrow stromal cells / Daniel Lenz ; Gutachter: Andreas Radbruch, Andreas Thiel, Enrico Klotzsch." Berlin : Humboldt-Universität zu Berlin, 2020. http://d-nb.info/1203623933/34.
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Platzbecker, Uwe, Ruben A. Ferrer, Manja Wobus, Catrin List, Rebekka Wehner, Claudia Schönefeldt, Barbara Brocard, et al. "Mesenchymal stromal cells from patients with myelodyplastic syndrome display distinct functional alterations that are modulated by lenalidomide." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-178822.
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The contribution of the bone marrow microenvironment in myelodysplastic syndrome is controversial. We therefore analyzed the functional properties of primary mesenchymal stromal cells from patients with myelodysplastic syndrome in the presence or absence of lenalidomide. Compared to healthy controls, clonality and growth were reduced across all disease stages. Furthermore, differentiation defects and particular expression of adhesion and cell surface molecules (e.g. CD166, CD29, CD146) were detected. Interestingly, the levels of stromal derived factor 1-alpha in patients’ cells culture supernatants were almost 2-fold lower (P<0.01) than those in controls and this was paralleled by a reduced induction of migration of CD34+ hematopoietic cells. Co-cultures of mesenchymal stromal cells from patients with CD34+ cells from healthy donors resulted in reduced numbers of cobblestone area-forming cells and fewer colony-forming units. Exposure of stromal cells from patients and controls to lenalidomide led to a further reduction of stromal derived factor 1-alpha secretion and cobblestone area formation, respectively. Moreover, lenalidomide pretreatment of mesenchymal stromal cells from patients with low but not high-risk myelodysplastic syndrome was able to rescue impaired erythroid and myeloid colony formation of early hematopoietic progenitors. In conclusion, our analyses support the notion that the stromal microenvironment is involved in the pathophysiology of myelodysplastic syndrome thus representing a potential target for therapeutic interventions.
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Kylmäoja, E. (Elina). "Osteoclastogenesis from bone marrow and peripheral blood monocytes:the role of gap junctional communication and mesenchymal stromal cells in the differentiation." Doctoral thesis, Oulun yliopisto, 2018. http://urn.fi/urn:isbn:9789526221045.
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Abstract Osteoclasts are multinuclear bone degrading cells differentiated from monocytes which can be isolated from bone marrow and peripheral blood. Complex signaling between osteoclast precursors and other bone cells, such as mesenchymal stromal cells (MSC) occurs during the differentiation. Gap junctional communication (GJC) is one of the mechanisms in the cell fusion. GJC can be modulated with several substances such as the specific GJC stimulators, antiarrhythmic peptides (AAP). Due to their promising clinical value in the treatment of cardiac disorders, the effects of AAPs in cardiac tissue are studied extensively. This study was conducted in order to investigate the roles of GJC and AAPs in bone cell cultures. Further, the contribution of the MSCs on the effects of AAPs was studied along with comparison of two types of osteoclastogenesis cultures with differing quantities of MSCs. GJC in osteoclastogenesis was studied with both GJC inhibitors and stimulators in mouse monocyte line RAW 264.7 cells and primary cultures with bone marrow hematopoietic cells. The following studies were made with human monocytes from peripheral blood and bone marrow where the effects of AAP10 were investigated in normal and acidic environments. In addition, comparison of osteoclastogenesis from bone marrow and peripheral blood monocytes was carried out in in vitro cell cultures on bovine or human bone slices. The cells were analyzed with regard to multinuclearity, bone resorption and the expression of several osteoclast markers. The results show that GJC is utilized in osteoclastogenesis, but it is not indispensable. GJC in monocytes can be stimulated with the AAPs during osteoclastogenesis, but the effects depend on the culture conditions as well as on the presence of MSCs in the culture. The AAPs can also activate the MSCs leading to indirect regulation of osteoclastogenesis, as the MSCs produce several molecules affecting the differentiation. Further, monocytes from peripheral blood showed increased potential for osteoclastogenic differentiation compared to bone marrow derived monocytes. This can be explained by the presence of the osteoclastogenesis-controlling MSCs in the bone marrow culture, while the peripheral blood cultures contain only few of these cells and thus lack their regulatory effectsTiivistelmä Osteoklastit ovat monitumaisia luuta hajottavia soluja, jotka ovat erilaistuneet monosyyteistä. Monosyyttejä voidaan eristää luuytimestä tai perifeerisestä verestä. Erilaistumisen aikana osteoklastien esiastesolujen sekä muiden luusolujen, kuten mesenkymaalisten stroomasolujen (MSC) välillä tapahtuu monimutkaista signalointia. Aukkoliitoskommunikointi (GJC) on eräs solufuusiossa tapahtuvista mekanismeista. GJC:tä voidaan muunnella useilla aineilla, esimerkiksi spesifisillä stimulaattoreilla, antiarytmisillä peptideillä (AAP). AAP-yhdisteiden vaikutuksia on tutkittu laajalti sydänkudoksessa johtuen niiden lupaavista kliinisistä ominaisuuksista sydänperäisten oireiden hoidossa. Tämän tutkimuksen tarkoituksena oli selvittää GJC:n ja AAP-yhdisteiden roolia luusoluviljelmissä. Lisäksi tutkittiin MSC-solujen osallistumista AAP-yhdisteiden vaikutuksiin sekä vertailtiin kahta erilaista osteoklastogeneesiviljelmää, joissa oli eri määrä MSC-soluja. GJC:tä osteoklastogeneesissä tutkittiin sekä sitä estävillä että stimuloivilla yhdisteillä hiiren monosyyttilinjan RAW 264.7 -soluissa sekä luuytimen hematopoieettisten solujen primääriviljelmissä. Seuraavat tutkimukset tehtiin ihmisen luuytimen ja perifeerisen veren monosyyteillä, ja niissä selvitettiin AAP10-yhdisteen vaikutuksia fysiologisissa sekä happamissa olosuhteissa. Lisäksi vertailtiin luuytimen ja perifeerisen veren monosyyttien osteoklastogeneesiä. In vitro -soluviljelmät tehtiin naudan tai ihmisen luulastujen päällä, ja soluista analysoitiin monitumaisuus, luun resorptio sekä useiden osteoklastimarkkereiden ilmentyminen. Tulokset osoittavat, että GJC:tä hyödynnetään osteoklastogeneesissä, mutta se ei ole korvaamaton mekanismi. GJC:tä voidaan stimuloida AAP-yhdisteillä osteoklastogeneesin aikana, mutta vaikutukset riippuvat viljelyolosuhteista sekä MSC-solujen läsnäolosta. AAP-yhdisteet voivat aktivoida myös MSC-soluja johtaen osteoklastogeneesin epäsuoraan säätelyyn, kun MSC-solut tuottavat useita erilaistumiseen vaikuttavia molekyylejä. Lisäksi perifeerisen veren monosyyteillä havaittiin korkeampi osteoklastogeeninen erilaistumispotentiaali verrattuna luuytimen monosyytteihin. Tulokset voidaan selittää osteoklastogeneesiä säätelevien MSC-solujen läsnäololla luuydinviljelmissä, kun taas perifeerisen veren monosyyttiviljelmissä näitä soluja on vain vähän, jolloin myös niiden säätelyominaisuudet puuttuvat
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Brohlin, Maria. "Mesenchymal stem cells for repair of the peripheral and central nervous system." Doctoral thesis, Umeå universitet, Anatomi, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-47746.
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Bone marrow-derived mesenchymal stem cells (MSC) have been shown to provide neuroprotection after transplantation into the injured nervous system. The present thesis investigates whether adult human and rat MSC differentiated along a Schwann cell lineage could increase their expression of neurotrophic factors and promote regeneration after transplantation into the injured peripheral nerve and spinal cord. Human and rat mesenchymal stem cells (hMSC and rMSC) expressed characteristic stem cell surface markers, mRNA transcripts for different neurotrophic factors and demonstrated multi-lineage differentiation potential. Following treatment with a cocktail of growth factors, the hMSC and rMSC expressed typical Schwann cells markers at both the transcriptional and translational level and significantly increased production of brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF). Age and time in culture are of relevance for clinical settings and growth-promoting effects of hMSC from young donors (16-18 years) and old donors (67-75 years) were compared. Undifferentiated hMSC from both young and old donors increased total neurite length of cultured dorsal root ganglion (DRG) neurons. Differentiation of hMSC from the young donors, but not the eldery donors, further enhanced the neurite outgrowth. Undifferentiated hMSC were cultured for eleven weeks in order to examine the effect of in vitro expansion time on neurite outgrowth. hMSC from the young donors maintained their proliferation rate and their ability to enhance neurite outgrowth from DRG neurons. Using a sciatic nerve injury model, a 10mm gap was bridged with either an empty tubular fibrin glue conduit, or conduits containing hMSC, with and without cyclosporine treatment. Cells were labeled with PKH26 prior to transplantation. At 3 weeks after injury the conduits with cells and immunosuppression increased regeneration compared with an empty conduit. PKH26 labeled human cells survived in the rat model and the inflammatory reaction could be suppressed by cyclosporine. After cervical C4 hemisection, BrdU/GFP-labeled rMSC were injected into the lateral funiculus rostral and caudal to the spinal cord lesion site. Spinal cords were analyzed 2-8 weeks after transplantation. Transplanted MSC remained at the injection sites and in the trauma zone for several weeks and were often associated with numerous neurofilament-positive axons. Transplanted rMSC induced up-regulation of vascular endothelial growth factor in spinal cord tissue rostral to the injury site, but did not affect expression of brain-derived neurotrophic factor. Although rMSC provided neuroprotection for rubrospinal neurons and significantly attenuated astroglial and microglial reaction, cell transplantation caused aberrant sprouting of calcitonin gene-related peptide immunostained sensory axons in the dorsal horn. In summary these results demonstrate that both rat and human MSC can be differentiated towards the glial cell lineage, and show functional characteristics similar to Schwann cells. hMSC from the young donors represent a more favorable source for neurotransplantation since they maintain proliferation rate and preserve their growth-promoting effects in long-term cultures. The data also suggest that differentiated MSC increase expression of neurotrophic factors and support regeneration after peripheral nerve and spinal cord injury.
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Fung, Kwong-lam, and 馮廣林. "The effect of microtubule targeting chemotherapeutic agents on bone marrow derived mesenchymal stromal cells and its interaction withacute lymphoblastic leukemia blasts." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2009. http://hub.hku.hk/bib/B43085660.
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Fung, Kwong-lam. "The effect of microtubule targeting chemotherapeutic agents on bone marrow derived mesenchymal stromal cells and its interaction with acute lymphoblastic leukemia blasts." Click to view the E-thesis via HKUTO, 2009. http://sunzi.lib.hku.hk/hkuto/record/B43085660.
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Platzbecker, Uwe, Ruben A. Ferrer, Manja Wobus, Catrin List, Rebekka Wehner, Claudia Schönefeldt, Barbara Brocard, et al. "Mesenchymal stromal cells from patients with myelodyplastic syndrome display distinct functional alterations that are modulated by lenalidomide." Ferrata Storti Foundation, 2013. https://tud.qucosa.de/id/qucosa%3A28910.
Full textAbstract:
The contribution of the bone marrow microenvironment in myelodysplastic syndrome is controversial. We therefore analyzed the functional properties of primary mesenchymal stromal cells from patients with myelodysplastic syndrome in the presence or absence of lenalidomide. Compared to healthy controls, clonality and growth were reduced across all disease stages. Furthermore, differentiation defects and particular expression of adhesion and cell surface molecules (e.g. CD166, CD29, CD146) were detected. Interestingly, the levels of stromal derived factor 1-alpha in patients’ cells culture supernatants were almost 2-fold lower (P<0.01) than those in controls and this was paralleled by a reduced induction of migration of CD34+ hematopoietic cells. Co-cultures of mesenchymal stromal cells from patients with CD34+ cells from healthy donors resulted in reduced numbers of cobblestone area-forming cells and fewer colony-forming units. Exposure of stromal cells from patients and controls to lenalidomide led to a further reduction of stromal derived factor 1-alpha secretion and cobblestone area formation, respectively. Moreover, lenalidomide pretreatment of mesenchymal stromal cells from patients with low but not high-risk myelodysplastic syndrome was able to rescue impaired erythroid and myeloid colony formation of early hematopoietic progenitors. In conclusion, our analyses support the notion that the stromal microenvironment is involved in the pathophysiology of myelodysplastic syndrome thus representing a potential target for therapeutic interventions.
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He, Q. "Peripheral blood derived multi-potent mesenchymal stromal cells (MSCs) in rats : their differentiation and characteristic comparison with bone marrow derived MSCs." Thesis, Queen's University Belfast, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.431597.
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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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Azizivarzaneh, Zahra [Verfasser], Kathrin [Akademischer Betreuer] Maedler, and Elke [Akademischer Betreuer] Oetjen. "beta-MSCs: Successful fusion of bone marrow mesenchymal stromal cells with beta-cells results in a beta-cell like phenotype / Zahra Azizivarzaneh. Gutachter: Kathrin Maedler ; Elke Oetjen. Betreuer: Kathrin Maedler." Bremen : Staats- und Universitätsbibliothek Bremen, 2015. http://d-nb.info/1075609410/34.
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Ohno, Satoshi. "Implantation of an Atelocollagen Sponge With Autologous Bone Marrow-Derived Mesenchymal Stromal Cells for Treatment of Vocal Fold Scarring in a Canine Model." Kyoto University, 2012. http://hdl.handle.net/2433/157415.
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BENEFORTI, LINDA. "Role of Bone Marrow-Mesenchymal Stromal Cells and inflammation in the pre-leukemic phase of ETV6-RUNX1-positive childhood Acute Lymphoblastic Leukemia (ALL)." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2019. http://hdl.handle.net/10281/241325.
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La traslocazione t(12;21) è il riarrangiamento cromosomico più frequente nei tumori pediatrici e si associa esclusivamente alla leucemia linfoblastica acuta (LLA) a precursori B. La traslocazione avviene in utero nelle cellule staminali-progenitrici ematopoietiche ma è insufficiente per la leuchemogenesi, poiché il gene di fusione ETV6-RUNX1 (E/R) che ne deriva genera un clone pre-leucemico clinicamente silente; mutazioni secondarie sono quindi necessarie per completare la trasformazione. Queste ultime avvengono nel periodo post-natale verosimilmente in seguito ad una risposta immunitaria disregolata a infezioni /infiammazioni comuni. Le recidive E/R+ non sono molto frequenti ma è plausibile che siano determinate dall’accumulo di nuove mutazioni nel clone pre-leucemico chemioresistente. In passato abbiamo dimostrato che TGFβ, una citochina prodotta durante l’infiammazione, limita la proliferazione delle cellule normali pro-B mentre favorisce il clone pre-leucemico che è insensibile al suo effetto; in più, TGFβ seleziona cellule pre-leucemiche staminali in progenitori CD34+ derivati da sangue cordonale umano. Abbiamo anche precedentemente dimostrato che cellule pro-B murine E/R+ mostrano alterazioni in molecole di adesione e nella migrazione verso CXCL12, suggerendo un loro possibile comportamento anomalo all’interno della nicchia midollare. Le cellule mesenchimali stromali (MSC) sono regolatori chiave sia delle cellule ematopoietiche nella nicchia che dell’infiammazione. È stato inoltre dimostrato che alterazioni delle MSC attivano pathways infiammatori nelle cellule staminali/progenitrici del sangue promuovendo la loro trasformazione a leucemia mieloide acuta in sindromi genetiche predisponenti. Grazie a due modelli cellulari esprimenti E/R (la linea cellulare murina proB Ba/F3 e cellule CD34+ di sangue cordonale umano), il presente studio dimostra che le cellule pre-leucemiche sono avvantaggiate dalla copresenza di MSC e infiammazione in termini di migrazione, sopravvivenza e potenziale progressione. Ba/F3 E/R+ mostrano un profilo di espressione genica pro-infiammatorio, con una particolare signature migratoria e pro-mieloide. Inoltre, sia Ba/F3 che CD34+ esprimenti E/R migrano di più verso i surnatanti di MSC infiammate rispetto alle cellule controllo; nel primo caso, la migrazione dipende dal recettore CXCR2. Molto importante, Ba/F3 E/R+ sono favorite rispetto al controllo quando coltivate su MSC e infiammazione, poiché questa condizione diminuisce molto proliferazione e sopravvivenza delle Ba/F3 normali mentre ha effetti minori o assenti sulle pre-leucemiche. Il vantaggio è mediato da fattori solubili, ma né TGFβ né CXCR2 sono implicati. In aggiunta, MSC e infiammazione aumentano il danno genotossico sia nelle Ba/F3 controllo che E/R+, come indicato dagli aumentati livelli di fosforilazione dell’istone H2AX e di espressione dell’enzima AID, il quale è stato dimostrato favorire la transizione da pre-leucemia a leucemia E/R+. Tuttavia, mentre le Ba/F controllo vanno incontro ad apoptosi, le pre-leucemiche resistono accumulando danni genetici che possono favorirne la trasformazione. Infine, infiammazione e MSC cooperano nel far emergere il compartimento CD34+IL7R+ all’interno della popolazione di CD34+ pre-leucemiche, mentre sfavoriscono quello della popolazione normale. Questa osservazione è particolarmente importante alla luce del fatto che tale compartimento rappresenta lo stadio dell’ematopoiesi fetale che è suscettibile all’azione pre-leucemica di E/R. Concludendo, il presente lavoro dimostra che cellule mesenchimali stromali di midollo osseo e infiammazione cooperano nel favorire la persistenza e la possibile progressione del clone pre-leucemico ETV6-RUNX1+. L’elucidazione dei meccanismi che sottendono tale azione favorente potrebbe fornire strategie innovative per l’eradicazione del clone pre-leucemico chemioresistente.Translocation t(12;21) is the most frequent chromosomal rearrangement in pediatric cancers, exclusively leading to B-cell Precursors Acute Lymphoblastic Leukemia (BCP-ALL). Translocation occurs in utero in stem-progenitor cells (HSPC) but it is insufficient for leukemogenesis, since the consequent ETV6-RUNX1 (E/R) fusion gene only generates a silent B-progenitor pre-leukemic clone; additional mutations are thus required for transformation. These latter occur in the post-natal period likely due to dysregulated immune response to common infections/inflammation. Our published data demonstrated that TGFβ, a cytokine produced during inflammation, limited the proliferation of normal pro-B and cells while favoring the insensitive E/R+ clone; moreover, TGFβ selected putative pre-leukemic stem cells (preLSC) in umbilical cord blood (UCB) CD34+ progenitors transduced with the oncogene. On the other hand, we previously showed that ETV6-RUNX1+ murine B-progenitors were altered in adhesion molecules expression and CXCL12-directed migration, suggesting possible dysregulated interactions within the bone marrow (BM) niche. Mesenchymal Stromal Cells (MSC) are key regulators of both HSPC and inflammation in the niche. Importantly, it has been shown that mesenchymal inflammation promotes secondary myeloid leukemia in predisposing syndromes by increasing DNA damage in HSPC, while MSC/BCP-ALL blasts cross-talk profoundly modifies cytokine and chemokine signalling within the niche excluding normal hematopoiesis in favor of leukemia. Taking advantages from two ETV6-RUNX1-expressing cell models (Ba/F3, a murine pro-B cell line, and ETV6-RUNX1-expressing human UCB-CD34+ progenitors) the present PhD study demonstrates that ETV6-RUNX1-expressing cells take advantage from mesenchymal inflammation in terms of migration, persistence and potential progression. In particular, we have found that pre-leukemic Ba/F3 show a peculiar pro-inflammatory gene expression profile characterized by a marked migratory and myeloid signature. Concordantly, ETV6-RUNX1+ Ba/F3 and CD34+ cells preferentially migrate toward inflamed compared to unstimulated BM-MSC supernatants; in case of the first, migration is CXCR2-dependent. Moreover, ETV6-RUNX1+ Ba/F3 are favored compared to controls in presence of BM-MSC and inflammatory cytokines, as they decrease normal cells proliferation and survival while minimally affecting pre-leukemic cells. The effect is mediated by soluble factors, but neither TGFβ nor CXCR2 axis are implicated. Importantly, the inflamed mesenchymal niche increases genotoxic stress in both control and E/R+ Ba/F3, as indicated by high levels of H2AX phoshorilation, as well as transcription of the activation-induced cytidine deaminase (AID) enzyme (which is implicated in ETV6-RUNX1+ pre-leukemia to leukemia transition). However, while control cells go through apoptosis, pre-leukemic Ba/F3 are resistant to this fail-safe mechanism, increasing chance to accumulate secondary mutations and malignantly transform. Finally, an inflamed MSC favor the emergence of CD34+ILR7+ compartment within ETV6-RUNX1+ UCB-CD34+ population while decreasing its frequency in the normal counterpart; of note, such differential effect doesn’t occur in case of unstimulated MSC. This observation is particularly important as the CD34+ILR+ compartment seems to represent the critical developmental stage during early fetal hematopoiesis for ETV6-RUNX1 pre-leukemic activity. Concluding, our work demonstrated that BM-MSC and inflammation cooperate in favoring the persistence and transformation of ETV6-RUNX1+ pre-leukemic clone. Elucidating mechanisms that underlay such promoting action could provide novel strategies for the pre-leukemic clone eradication.
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Montzka, Katrin [Verfasser]. "An investigation into the characteristics and potential therapeutic application of human bone marrow-derived mesenchymal stromal cells in experimental spinal cord injury / Katrin Montzka." Aachen : Hochschulbibliothek der Rheinisch-Westfälischen Technischen Hochschule Aachen, 2011. http://d-nb.info/1018200118/34.
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Ganier, Clarisse. "Potentiel thérapeutique des cellules stromales mésenchymateuses dans l'épidermolyse bulleuse dystrophique récessive Intradermal injection of bone marrow-MSCs corrects recessive dystrophic epidermolysis bullosa in a xenograft model Intradermal injection of human umbilical cord-MSCs shows less efficacy than bone marrow-MSCs to correct recessive dystrophic epidermolysis bullosa in a xenograft model." Thesis, Sorbonne Paris Cité, 2018. https://wo.app.u-paris.fr/cgi-bin/WebObjects/TheseWeb.woa/wa/show?t=2117&f=15515.
Full textAbstract:
L'épidermolyse bulleuse dystrophique récessive (EBDR) est une maladie génétique cutanée due à des mutations de perte de fonction du gène COL7A1 codant pour le collagène VII. Le collagène VII forme les fibres d'ancrage, structures essentielles pour l'adhésion de l'épiderme au derme sous-jacent. Les patients EBDR développent dès la naissance, des décollements bulleux de la peau et des muqueuses responsables de plaies chroniques et de graves complications locales et systémiques. La survenue de carcinomes épidermoïdes cutanés agressifs reste la première cause de décès. Il n'existe pas de traitement à ce jour. Les cellules stromales mésenchymateuses (CSM) sont des cellules multipotentes, isolées à partir de tissus adultes (moelle osseuse, tissu adipeux) ou de tissus périnataux (cordon ombilical). Des travaux antérieurs ont montré que les injections locales et systémiques de CSM issues de la moelle osseuse (CSM-MO) allogéniques ont un potentiel pour réduire l'inflammation cutanée et améliorer la cicatrisation des plaies chez les patients EBDR. Ces améliorations cliniques sont cependant transitoires et les mécanismes d'action des CSM-MO dans l'EBDR ainsi que leur durée de vie après injection sont mal connus. Les CSM-MO pourraient agir via leurs propriétés immunomodulatrices, anti-fibrotiques, pro-angiogéniques, par un effet paracrine permettant l'expression de collagène VII endogène et/ou la sécrétion de collagène VII par les CSM-MO injectées. L'objectif de cette thèse a été d'étudier le potentiel thérapeutique des CSM dans des modèles précliniques de l'EBDR. Nous avons tout d'abord montré que les CSM-MO expriment une quantité d'ARNm de COL7A1 et de collagène VII comparable aux fibroblastes dermiques sains en culture. Nous avons ensuite évalué la capacité des CSM-MO humaines à survivre et produire du collagène VII à la jonction dermo-épidermique (JDE) à long terme après une injection locale dans des peaux équivalentes humaines EBDR greffées sur souris nude. L'injection intradermique (ID) de CSM-MO in vivo a permis de restaurer l'expression du collagène VII ainsi que la formation de fibres d'ancrage à la JDE jusqu'à 6 mois après l'injection. Les CSM-MO sont retrouvées dans la peau équivalente jusqu'à 4 mois après l'injection. Ces résultats montrent qu'une injection ID unique de CSM-MO in vivo permet de rétablir une production prolongée de collagène VII synthétisé par les cellules injectées et d'améliorer l'adhésion dermo-épidermique de la peau équivalente EBDR. Nous avons ensuite comparé l'efficacité des CSM issues de la gelée de Wharton de cordon ombilicaux (CSM-CO) humains aux CSM-MO suivant la même méthodologie que précédemment. Les CSM-CO expriment en culture une quantité d'ARNm de COL7A1 et de collagène VII supérieures aux CSM-MO et fibroblastes dermiques sains. Une injection unique ID de CSM-CO dans la peau EBDR équivalente greffée permet de rétablir une faible expression de collagène VII jusqu'à 4 mois après l'injection. Les CSM-CO sont détectées dans la peau équivalente jusqu'à 2 mois après l'injection. Ces données montrent que les CSM-CO ont une capacité moindre à restaurer l'expression du collagène VII à la JDE comparativement aux CSM-MO injectées dans le même modèle de xénogreffes EBDR. Ces résultats ouvrent la perspective d'une thérapie génique ex vivo utilisant des CSM-MO murines Col7a1-/-. Les souris Col7a1-/- reproduisent les lésions cutanées et muqueuses observées chez les patients EBDR. L'espérance de vie de ces animaux est très réduite. Les CSM-MO murines Col7a1-/- transduites en culture à l'aide d'un vecteur rétroviral SIN exprimant COL7A1 produisent en moyenne 30 fois plus de collagène VII que les CSM-MO murines WT. Des expériences in vivo sont nécessaires pour déterminer si l'injection de CSM-MO génétiquement corrigées ont le potentiel de traiter des lésions cutanées et muqueuses, pour définir la dose optimale et la durée de l'effet chez l'animal. Ceci constituerait une étape importante vers la cliniqueRecessive dystrophic epidermolysis bullosa (RDEB) is a severe skin disease caused by loss-of-function mutations in COL7A1 encoding type VII collagen. Type VII collagen forms anchoring fibrils which are essential structures for dermal-epidermal adherence. Patients with RDEB suffer since birth from skin and mucosal blistering and develop severe complications. The development of aggressive squamous cell carcinomas is the first cause of demise of these young patients. To date, there is no treatment. Mesenchymal stromal cells (MSC) are multipotent cells, isolated from adult tissue (bone marrow, adipose tissue) or perinatal tissue (umbilical cord). Previous works have shown that local and systemic injections of allogeneic bone marrow-derived MSC (BM-MSC) have a potential to reduce skin inflammation and to improve wound healing in RDEB patients. However, clinical improvement was transient and the mechanisms of action of BM-MSC in RDEB and also their survival after injection are still poorly understood. BM-MSC could act through immunomodulation, anti-fibrotic and angiogenic proprieties, paracrine effects leading to type VII collagen production in the host tissues and/or type VII collagen secretion by injected BM-MSC. The aim of our work was to study the therapeutic potential of MSC for RDEB in preclinical models. We first showed that BM-MSC produce COL7A1 mRNA and type VII collagen levels comparable to healthy dermal fibroblasts in culture. We then assessed the long-term capacity of human BM-MSC to survive, produce and deposit type VII collagen at the dermal-epidermal junction (DEJ) after local injection in human RDEB skin equivalents transplanted onto nude mice. In vivo intradermal (ID) injection of a single dose of human BM-MSC led to the production and deposition of human type VII collagen at the DEJ and allowed anchoring fibrils formation for at least six months post-injection. Injected human BM-MSC were found in the skin at least four months post-injection. These data show that intradermally injected human BM-MSC have the potential to improve dermal-epidermal adhesion of RDEB skin equivalents through sustained deposit of type VII collagen molecules and subsequent anchoring fibrils formation. We then compared the efficacy of human Umbilical Cord Wharton's Jelly-MSC (UC-MSC) with BM-MSC using the same methodology as previously described. UC-MSC showed in vitro a significantly higher amount of COL7A1 mRNA and type VII collagen compared to BM-MSC and healthy dermal fibroblasts in culture. ID injection of a single dose of UC-MSC in vivo led to the production and deposition of low levels of human type VII collagen at the DEJ for four months post-injection. Injected human UC-MSC were found in the skin two months post-injection. These data disclosed a lower efficacy of UC-MSC to restore collagen VII at the DEJ compared to BM-MSC injected in the same xenograft RDEB model. These data open the perspective of using gene-corrected BM-MSC from a Col7a1-/- RDEB murine model to restore normal dermal-epidermal adhesion. Col7a1-/- mice reproduce cutaneous and mucosal lesions observed in RDEB patients. The life expectancy of these animals is very short. We could show that transduction of Col7a1-/- murine BM-MSC in culture using a COL7A1-expressing SIN retroviral vector led to type VII collagen expression levels which were 30-fold higher on average than in BM-MSC from WT mice. In vivo data are required to determine whether the injection of gene-corrected BM-MSC has the potential to treat skin and mucosal lesions in RDEB mice and to define the optimal dose and duration of the effect in vivo. Restoration of type VII collagen expression and anchoring fibrils formation in Col7a1-/- mice would represent an important step towards clinical translation
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Burk, Janina. "Klinische Anwendung und vergleichende Charakterisierung equiner mesenchymaler Stromazellen." Doctoral thesis, Universitätsbibliothek Leipzig, 2012. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-99657.
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Mesenchymale Stromazellen (MSCs) werden beim Pferd bereits mit vielversprechenden Ergebnissen zur Behandlung von muskuloskelettalen Erkrankungen, insbesondere von Sehnenerkrankungen, eingesetzt. In bisherigen klinischen Studien lag das Hauptaugenmerk auf der Behandlung von Erkrankungen der Oberflächlichen Beugesehne bei Rennpferden, die jedoch in Deutschland nur einen verhältnismäßig kleinen Anteil des Patientenaufkommens darstellen. Die zu erwartenden Ergebnisse nach MSC-Behandlung von Fesselträgererkrankungen sind dagegen noch nicht bekannt. Darüber hinaus sind die grundlegenden Kenntnisse zur Biologie equiner MSCs noch unzureichend, was Verständnis und Optimierung des bestehenden Therapiekonzeptes erschwert. Häufig wird die Verwendung alternativer Gewebequellen für MSCs diskutiert, wobei jedoch nur wenige vergleichende Daten zu den jeweiligen zellulären Eigenschaften vorliegen. Ziel dieser Arbeit war es daher, zum einen mehr Kenntnisse über die zu erwartenden klinischen Ergebnisse nach MSC-Behandlung von Sehnenerkrankungen zu erlangen, einschließlich Erkrankungen des Fesselträgers, zum anderen den Wissensstand hinsichtlich der in-vitro-Charakterisierung equiner MSCs zu erweitern, wobei ein Vergleich klinisch relevanter Charakteristika zwischen MSCs aus verschiedenen Gewebequellen angestrebt wurde. In die klinische Studie wurden 98 Pferde, die aufgrund von Sehnen- und Banderkrankungen mit MSCs behandelt worden waren, einbezogen. Von 58 dieser Tiere konnten Langzeitergebnisse nach einem Beobachtungszeitraum von mindestens einem Jahr erhoben werden. Diese wurden hinsichtlich des Behandlungserfolges sowie möglicher Einflussfaktoren ausgewertet, wobei die Behandlung als erfolgreich bewertet wurde, wenn die Patienten nach dem Beobachtungszeitraum voll trainiert oder im Sport eingesetzt werden konnten und dabei kein Rezidiv aufgetreten war. Die Behandlung mit MSCs wurde bei 84,5 % der Pferde als erfolgreich eingestuft, wobei Erkrankungen der Oberflächlichen Beugesehne mit 84,2 % und Erkrankungen des Fesselträgers mit 83,3 % gleichermaßen gute Ergebnisse zeigten. Tendenziell beeinflussten Nutzungsdisziplin, Erkrankungsstadium und Patientenalter das klinische Ergebnis ebenso wie bei konventioneller Behandlung. Insgesamt war nach MSC-Behandlung das Auftreten von Rezidiven deutlich seltener zu beobachten als in der Literatur für die konventionelle Behandlung beschrieben wird. Für die in-vitro-Studie zur vergleichenden Charakterisierung equiner MSCs aus verschiedenen Quellen wurden Knochenmark, Fett- und Sehnengewebe sowie Nabelschnurblut und -gewebe gewonnen. Aus diesen Proben wurden jeweils die plastikadhärenten MSCs isoliert und hinsichtlich Zellausbeute, Proliferations- und Migrationseigenschaften, tripotentem Differenzierungspotential sowie der Expression der Sehnenmarker Kollagen 1A2 und Skleraxis vergleichend untersucht. Die Ausbeute an MSCs war bei allen soliden Geweben (Fett-, Sehnen-, und Nabelschnurgewebe) hochsignifikant höher (p < 0,001). Ebenso proliferierten MSCs aus Fett- und Sehnengewebe signifi-kant schneller als MSCs aus Knochenmark oder Nabelschnurblut (p < 0,01). Von letzteren wurden darüber hinaus etwa drei viertel aller Zellkulturen vor der achten Passage seneszent. Das höchste Migrationspotential zeigten wiederum MSCs aus Sehnen- und Fettgewebe, wobei hier MSCs aus Nabelschnurgewebe das ungünstigste Ergebnis erzielten (p < 0,01). Die adipogene Differenzierung gelang bei MSCs aus allen Quellen vergleichbar gut. Bei der osteogenen Differenzierung erreichten MSCs aus Knochenmark das beste Ergebnis, während MSCs aus Nabelschnurblut und –gewebe nur schwach osteogen differenzierten (Tag 21: p < 0,01; Tag 35: p < 0,05). Im Gegensatz dazu erreichten MSCs aus Nabelschnurblut bei der chondrogenen Differenzierung die meisten Scorepunkte, MSCs aus Knochenmark dagegen die wenigsten (p < 0,05). Kollagen 1A2 wurde von MSCs aus Fettgewebe am höchsten exprimiert, Skleraxis von MSCs aus Nabelschnurblut. MSCs aus Sehnengewebe exprimierten beide Sehnenmarker auf fast ebenso hohem Level. MSCs aus Knochenmark dagegen zeigten hier jeweils die niedrigste Expression (p < 0,05 für Kollagen 1A2). Basierend auf den Ergebnissen der klinischen Studie ist die MSC-Therapie nach wie vor als vielversprechende Behandlungsoption für Sehnenerkrankungen anzusehen und ist auch für die Behandlung von Fesselträgererkrankungen geeignet. Zukünftige, kontrollierte klinische Studien müssen jedoch die Wirksamkeit der MSC-Therapie noch weitergehend bestätigen. Die in-vitro-Studie zeigte signifikante Unterschiede zwischen equinen MSCs aus verschiedenen Quellen auf, die bei der Auswahl einer Gewebequelle für die MSC-Isolierung für klinische Anwendungen berücksichtigt werden sollten. MSCs aus Fettgewebe erscheinen aufgrund ihrer sehr guten Proliferations- und zuverlässigen Differenzierungseigenschaften als eine gute Alternative zu MSCs aus Knochenmark für autologe Therapien. MSCs aus Sehnengewebe sind den hier vorliegenden Ergebnissen zufolge besonders gut für die Behandlung von Sehnenerkrankungen geeignet; vor einer routinemäßigen Anwendung dieser MSCs sollten jedoch ihre Eigenschaften weiterführend untersucht werdenIn horses, mesenchymal stromal cells (MSCs) are used for the treatment of musculoskeletal diseases, especially tendon injuries, with promising results. Previous clinical studies mainly focused on the treatment of superficial digital flexor tendon injuries in racehorses, which, however, represent only a relatively small percentage of the overall equine case load in Germany. Average outcome to be expected following MSC treatment of suspensory ligament injuries was not yet determined. Moreover, basic knowledge on equine MSC biology is still deficient, hampering the understanding and thus the optimisation of the existing treatment regime. The use of alternative MSC sources is frequently discussed, yet to date, only few data comparing the cellular properties of equine MSCs from different sources have been published. The aim of this study was, on the one hand, to gain more knowledge concerning the expected outcome after MSC treatment of tendon injuries, including injuries to the suspensory ligament. On the other hand, it was aimed at expanding the knowledge on equine MSC characterisation in vitro, thereby focusing on the comparison of clinically relevant properties of MSCs derived from different sources. In the clinical study, 98 horses were included, all of which had received MSC treatment for tendon or ligament injuries. In 58 of these horses, long term results after a follow-up period of at least one year could be collected. These data were analysed with respect to treatment outcome and potential influencing factors. Treatment was considered successful when horses were back to full training or competition after the follow-up period, without having suffered a re-injury. The overall success rate was 84.5 %. Success rates in horses suffering from superficial digital flexor tendon injuries and in horses suffering from suspensory ligament injuries were comparably good (84.2 % and 83.3 %, respectively). Similar to conventional therapies, the sports discipline in which the horses performed, age and disease stage tended to influence the outcome. Overall, re-injury rates after MSC treatment were considerably lower than those described in the literature following conventional treatment. For the comparative characterisation of MSCs from different sources in vitro, samples of bone marrow, adipose and tendon tissue, as well as umbilical cord blood and –tissue were collected. Plastic-adherent MSCs were isolated out of these samples and comparatively characterised focusing on cell yields, proliferation and migration properties, trilineage differentiation potential and the expression of the tendon markers collagen 1A2 and scleraxis. MSC yields were significantly higher in all solid tissues (adipose, tendon and umbilical cord tissue) (p < 0.001). Further, MSCs from adipose and tendon tissue proliferated significantly faster than MSCs from bone marrow or umbilical cord blood (p < 0.01). Moreover, approximately three quarters of the samples derived from the latter sources underwent senescence before reaching passage eight. The highest migration potential was found in MSCs derived from tendon and adipose tissue again, while MSCs from umbilical cord tissue showed the least (p < 0.01). The adipogenic differentiation potential was comparably good in MSCs from all different sources. The osteogenic differentiation was most distinct in MSCs from bone marrow, while MSCs from umbilical cord blood and tissue showed only weak evidence of differentiation (day 21: p < 0.01; day 35: p < 0.05). In contrast, following chondrogenic differentiation, MSCs from umbilical cord blood scored highest and MSCs from bone marrow scored lowest (p < 0.05). Collagen 1A2 was most highly expressed in MSCs from adipose tissue, highest scleraxis expression levels were found in MSCs from umbilical cord blood. MSCs from tendon tissue, however, expressed both markers at almost evenly high levels. Contrastingly, lowest expression levels of both markers were found in MSCs derived from bone marrow (p < 0.05 for collagen 1A2). Based on the results of the clinical study, MSC therapy can still be considered a very promising treatment option for tendon diseases and is also a suitable treatment for suspensory ligament injuries. In the future, controlled clinical studies will have to further confirm the efficacy of this treatment regime. The in-vitro-study showed significant differences between equine MSCs derived from different sources, which should be considered when choosing a MSC source for clinical applications. For autologous therapies, MSCs derived from adipose tissue appear to be a good alternative to MSCs derived from bone marrow, due to their remarkable proliferation and reliable differentiation capacities. Furthermore, according to this study, MSCs derived from tendon tissue are especially suitable for treating tendon injuries. Prior to routine clinical applicability of these MSCs, however, their properties should be further investigated
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Gittel, Claudia. "Einfluss von Ursprungsquelle und Isolationsmethode auf zellbiologische Charakteristika equiner mesenchymaler Stromazellen." Doctoral thesis, Universitätsbibliothek Leipzig, 2014. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-153119.
Full textAbstract:
Multipotente mesenchymale Stromazellen (MSCs) stellen nicht nur beim humanen Patienten, sondern auch in der Veterinärmedizin einen vielversprechenden Therapieansatz in der Behandlung erkrankter muskuloskelettaler Gewebe dar. Ziel der Behandlung ist dabei die Regeneration der betroffenen Strukturen im Vergleich zur Reparation nach konservativer Therapie. Vor allem im Bereich von Sehnenerkrankungen können nach MSC-Applikation vielversprechende Ergebnisse im Hinblick auf niedrigere Rezidivraten beobachtet werden. Dennoch sind noch nicht alle Umstände einer optimalen MSC-Anwendung geklärt. Hierbei sind unter anderem Fragen bezüglich der Herkunft und Gewinnung von MSCs offen, da Unterschiede von MSCs aufgrund ihrer Gewebezugehörigkeit bereits nachgewiesen wurden. Grundlegende umfassende Arbeiten zum Vergleich von equinen MSCs aus verschiedenen Quellen sowie deren mögliche Beeinflussung durch die Isolierung aus dem Gewebe lagen bislang noch nicht vor. Ziel dieser Studie war es daher, equine MSCs aus verschiedenen Quellen zu gewinnen und mögliche Unterschiede in vitro aufzuzeigen. Weiterhin sollten Unterschiede zwischen den Zelleigenschaften nach Anwendung verschiedener Isolationsprotokolle untersucht werden. In der hier vorliegenden Studie wurden MSCs aus Fett- und Sehnengewebe, Knochenmark, Nabelschnurblut und Nabelschnurgewebe von Pferden isoliert und vergleichend charakterisiert. Dabei wurden für die soliden Körpergewebe zwei unterschiedliche Isolationsmethoden, die Digestion und die Explantation, angewendet, um mögliche Einflüsse auf die gewonnen Zellen zu ermitteln. Die untersuchten Kriterien beinhalteten Zellertrag, Proliferation, Differenzierungspotenz und das Migrationsverhalten von MSCs. Hinblickend auf eine Anwendung von MSCs bei Sehnenerkrankungen wurde auch die Expression von Sehnenmarkern verglichen. In der vorliegenden Studie konnte gezeigt werden, dass sich die MSCs aus verschiedenen Quellen hinsichtlich der Zellausbeute und ihres Wachstumspotentials unterschieden. Aus soliden Geweben konnten mittels Digestion im Vergleich zu Körperflüssigkeiten signifikant mehr MSCs isoliert werden (p < 0,001). Dabei erbrachte die Isolation von MSCs mittels Digestionsmethode einen deutlich höheren Zellertrag nach der Passage 0 im Vergleich zur Explantationsmethode (p < 0,05). Im weiteren Verlauf der Kultivierung zeigten MSCs aus Sehnengewebe und Fettgewebe ein signifikant besseres Proliferationsverhalten im Vergleich zu Knochenmark-MSCs und Nabelschnurblut-MSCs. Im Hinblick auf das Differenzierungspotential konnten signifikante Unterschiede zwischen den MSCs aus den verschiedenen Quellen beobachtet werden. MSCs aus Knochenmark zeigten eine sehr gute osteogene Differenzierungsfähigkeit im Vergleich zu MSCs aus den geburtsassoziierten Geweben (p < 0,05). Im Gegensatz dazu zeichneten sich diese MSCs durch eine deutlich bessere chondrogene Differenzierung im Vergleich zu Knochenmark-MSCs aus (p < 0,05). Im Hinblick auf die Isolationsmethode konnten keine Unterschiede im Differenzierungspotential beobachtet werden. Weitere Unterschiede aufgrund der Zellquelle lassen sich in der Genexpression der Sehnenmarker erkennen. MSCs aus Fettgewebe und Sehnengewebe exprimierten Kollagen 1A2 auf höchstem Niveau. Sklexaris hingegen wurde von MSCs aus Nabelschnurblut und Sehnengewebe am höchstem exprimiert. Dabei zeigten MSCs, die mittels Digestionsmethode isoliert worden waren, ein signifikant höheres Expressionslevel von Skleraxis im Vergleich zur Explantationsmethode (p < 0,05). Die Ergebnisse der vorliegenden Studie lassen einen Einfluss der Zellquelle auf die Zellcharakteristika erkennen. MSCs aus Fettgewebe stellen dabei eine vielversprechende Alternative zu Knochenmark-MSCs dar. Allerdings scheint für eine klinische Anwendung von MSCs eine selektive Auswahl der Zellquelle entsprechend der vorliegenden Erkrankung von Vorteil zu sein. Dabei ist eine Isolierung von MSCs aus soliden Geweben mittels Digestionsverfahren zu empfehlen, da hier deutlich höhere Zellzahlen gewonnen werden können. Eine negative Beeinflussung der Zelleigenschaften durch die enzymatische Digestion lässt sich nach den vorliegenden Ergebnissen nicht vermuten. Inwiefern die beobachteten Unterschiede bei in-vivo-Anwendungen von Bedeutung sind, muss jedoch noch umfassend untersucht werdenNot only in humans but also in veterinary medicine, multipotent mesenchymal stromal cells (MSCs) are a promising treatment option in the therapy of injured musculoskeletal tissues. This is due to the improved tissue regeneration instead of the insufficient reparation following conventional therapies. With regard to an application of MSCs for treatment of tendinopathies in horses, lower rates of reinjury have been reported. However, further investigations to optimize the MSC treatment are still outstanding. Differences in MSCs from different origins have been already reported, but there are still remaining questions about the influence of origin and isolation procedures of MSCs. Fundamental research on equine MSCs derived from different sources and their potential impact due to the isolation process has not been published so far. The aim of this study was to isolate equine MSCs from different sources and to demonstrate potential differences in vitro. Furthermore, differences in cell features following different isolation methods were investigated. In the present study, MSCs from horses were isolated from adipose tissue, tendon tissue, bone marrow, umbilical cord blood and umbilical cord tissue and subsequently subjected to comparative characterization. In case of the solid tissues, two different isolation methods, digestion and explantation, were performed in order to analyze influences on obtained cells. Investigated cell features included cell yield, proliferation, differentiation and migration potential. Furthermore, expression of tendon markers was evaluated with regard to an application of MSCs in tendinopathies. In the present study it was shown that MSCs derived from different sources differ distinctly in cell yield and proliferation potential. In comparison to body fluids, significantly more MSCs could be isolated from solid tissues when using the digestion method (p < 0.001). Furthermore, the cell yield at first cell harvest was distinctly higher when performing the isolation by digestion in comparison to isolation by explantation (p < 0.05). With regard to further cultivation, MSCs derived from tendon tissue and adipose tissue displayed a significantly better proliferation potential compared to MSCs derived from other sources. Considering the differentiation potential, significant differences were obvious between the MSCs derived from different sources. Bone marrow-MSCs showed an excellent osteogenic differentiation capacity in comparison to MSCs derived from umbilical cord blood and tissue (p < 0.05). In contrast, the birth-associated MSCs displayed a distinctly better chondrogenic differentiation than MSCs derived from bone marrow (p < 0.05). No difference in the differentiation potential was noticeable following the different isolation procedures. Furthermore, differences in the gene expression of tendon markers were evident with regard to the cell source. MSCs derived from adipose tissue and tendon tissue expressed collagen 1A2 on the highest level. On the other hand, scleraxis was expressed highest in MSCs derived from umbilical cord blood and tendon tissue. In these cells, MSCs isolated by the digestion method showed a significantly higher expression level of scleraxis in comparison to MSCs isolated by explantation (p < 0.05). Based on the results obtained so far, a relevant impact of the source of MSCs on cell features was evident. MSCs derived from adipose tissue are a promising alternative to bone marrow-MSCs. However, with regard to a clinical application of MSCs, a selection of the MSC source depending on the respective intended use seems to be advantageous. For routine isolation of MSCs from solid tissues, the digestion method could be recommended due to the higher obtainable cell numbers. Furthermore, a negative influence of the enzymatic digestion on the cell features was not detectable. However, to what extent the observed differences in vitro are relevant for in-vivo-applications needs to be further investigated
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Wu, Yuenv. "Altered interactions between mesenchymal stromal cells and hematopoietic stem cells from MDS and AML through expression of FAK." Thesis, Lyon, 2019. http://www.theses.fr/2019LYSE1123.
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La FAK est une tyrosine kinase cytoplasmique qui régule divers processus cellulaires, dont la survie, la prolifération, la différenciation et la motilité. Bien que diverses études aient démontré l'importance du FAK dans la pathogenèse du SMD et de la LAM, le rôle de cette molécule dans le microenvironnement des tumeurs du SMD et de la LAM reste à déterminer davantage. En examinant les CSM de la moelle osseuse qui dérivent de patients atteints de SMD et de LAM, nous avons observé une augmentation continue de l'expression et de l'activation de la FAK pendant la progression du SMD vers de la LAM, semblable à celle observée chez les patients hémopoïétiques. Dans le SMD à faible risque, on a constaté que les CSM se caractérisaient par une faible expression et une faible activation du FAK. Ils présentaient une morphologie modifiée, un immunophénotype, une différenciation et l'expression de facteurs favorables à l'hématopoïèse. Il convient de noter que ces caractéristiques pourraient être largement reproduites dans les CSM saines par inhibition FAK. De plus, l'appauvrissement en FAK dans la lignée cellulaire stromale pourrait induire une expansion massive et l'apoptose des CSH normaux. Nos résultats mettent en évidence le rôle crucial du FAK dans le maintien des fonctions des CSM et fournissent la preuve que la dysrégulation du FAK dans les CSM contribue à la perturbation de l'hématopoïèse et éventuellement à la progression des tumeurs malignes myéloïdes. Une meilleure compréhension du rôle que joue le microenvironnement du SMD et de la LAM permettra de mieux reconnaître les patients à faible risque et de mettre au point des traitements ciblant les CSM défectueuses, améliorant ainsi le résultat cliniqueFAK is a cytoplasmic tyrosine kinase that regulates diverse cellular processes, including survival, proliferation, differentiation, and motility. Though various studies have demonstrated the importance of FAK in MDS and AML pathogenesis, the role of this molecule in MDS and AML tumor microenvironment remained to be further determined. By examining BM MSCs derived from MDS and AML patients, we have observed a continues increase of FAK expression and activation during MDS progression to AML, similar to those detected in hemopoietic counterparts. In LR-MDS, MSCs were found to be characterized by low FAK expression and activation. They exhibited altered morphology, immunophenotype, differentiation, and expression of hematopoiesis-supporting factors. Of note, these features could be largely reproduced in normal MSCs by FAK inhibition. Furthermore, FAK depletion in BM stromal cell line could induce massive expansion and apoptosis of normal HSPCs. Our results highlight a critical role of FAK in maintaining the functions of BM MSCs and provide evidence that dysregulation of FAK in MSCs contribute to the disturbed hematopoiesis and possibly the progression of myeloid malignancies. A greater understanding of the role that BM microenvironment plays in MDS and AML will enable an increased recognition of poor-risk patients and the development of therapies that target the defected MSCs, thereby improving the clinical outcome
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Li, Yueying. "Vieillissement des cellules stromales mésenchymateuses de la moelle osseuse : implications en médecine régénérative." Thesis, Université de Lorraine, 2015. http://www.theses.fr/2015LORR0099/document.
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Grâce à leurs propriétés de différenciation, les cellules stromales mésenchymateuses (CSM) constituent aujourd’hui un outil en médecine régénérative. La moelle osseuse reste une des plus utilisées. Une diminution de la capacité de prolifération et de différenciation des CSM-MO, au cours des passages, a été montrée. En parallèle, certaines études montrent que l'impact de l'âge du donneur sur les propriétés de CSM-MO reste encore controversé. Le but de notre étude était de mieux comprendre l'effet de l'âge du donneur mais aussi des passages en culture sur la capacité de prolifération et de différenciation des CSM de moelle osseuse. Les échantillons ont été séparés en 4 groupes en fonction de l’âge des donneurs (<20 ans; 20-40 ans; 40-60 ans; >60 ans) et les analyses ont été réalisés lors de la culture de cellules pendant 5 passages. Les résultats obtenus montrent que la capacité de prolifération de CSM-MO obtenues à partir de donneurs jeunes est supérieure à celle de cellules des donneurs âgés. De plus, cette capacité de prolifération diminue en fonction des passages en culture. En parallèle, la capacité des cellules à former des colonies, mesurée par le test CFU-F, diminue légèrement en fonction de l’âge des donneurs mais de façon importante en fonction du passage. Enfin, la capacité de différenciation des CSM-MO vers les trois types cellulaires étudiés, diminue en fonction des passages de cellules mais également en fonction de l’âge des donneurs. Notre étude montre que les propriétés des CSM issues de moelle osseuse sont modifiées lors de l’amplification in vitro mais aussi en fonction de l’âge des donneursToday with their properties of differentiation into specific cells types, mesenchymal stromal cells (MSC) can be used in regenerative medicine. Bone marrow (BM) is the better characterized one. The researchers have proven that with increasing passage number in culture the proliferation and differentiation potential of MSC decrease. In parallel many researchers have showed the impact of donor age on MSC properties remains controversial. The aim of our study was to better understand the effect of donor age but also culture passages on the proliferation and differentiation ability of bone marrow mesenchymal stromal cells. The samples were separated into 4 groups depending on the donor age (<20 years; 20-40 years; 40-60 years; > 60 years) and The samples were cultured for 5 passages. The results obtained show that the MSC proliferative capacity obtained from young donors is greater than that of cells from older donors. In addition, the proliferative capacity decreases with increasing passage number in culture. In parallel, the ability of colony-forming unit-fibroblast, measured by the CFU-F assay, decreases slightly depending on the age of the donors but significantly depending on the passage. Finally, the MSC differentiation ability decreases according to the passage of the cells but also depending on the donor age. Our study shows that the properties of bone marrow derived MSC are modified not only during amplification in vitro but also in terms of donor age
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Dubory, Arnaud. "Interface os-implant titane et ingénierie tissulaire A cadaveric validation of a method based on impact analysis to monitor the femoral stem insertion Bone marrow mesenchymal stem cells predict radiographic osseointegration of cementeless titanium hip cups Survival, adhesion, and expression of osteoblastic genes of human derived-bone marrow mesenchymal stromal cells on PEEK and titanium-coated PEEK lumbar interbody cages." Thesis, Paris Est, 2018. http://www.theses.fr/2018PESC0067.
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Introduction : Le titane, à la fois utilisé comme moyen de fixation d’un implant devant ses propriétés biomécaniques proches de l’os et comme moyen d’ostéointégration a pris une place prépondérante en orthopédie. L’ostéointégration d’un implant titane (IT) essentielle pour la pérennité de ce dernier, dépend de 2 phases : une phase primaire MECANIQUE correspondant à l’impaction ou tenue primaire de l’IT et une phase secondaire BIOLOGIQUE correspondant à la colonisation de l’IT par le tissu osseux.L’objectif de ce travail fut d’évaluer et d’améliorer lors de ces deux phases la stabilité et l’ostéointégration de l’implant titane :(1) Évaluer la tenue primaire des tiges fémorales en titane non cimentées par l’analyse de l’impact correspondant à la mesure de l’impact au cours du temps.(2) Évaluer si la quantité de CSM contenues dans l’aile iliaque est corrélée à la survie sans reprise de des implants acétabulaires impactés dans le cadre de l’ostéonécrose aseptique de la tête fémorale.(3) Améliorer l’ostéointégration des IT par les méthodes de thérapie cellulaire en étudiant in vitro la survie et la division des cellules stromales mésenchymateuses humaines (CSM) osseuses au contact de cages lombaires inter-somatiques recouvertes d’alliage titane rugueux.Matériels et Méthodes: L’évaluation de la tenue primaire des tiges fémorales en titane sans ciment selon l’analyse de l’impact a été réalisée à l’aide d’un marteau muni d’un capteur de force piézo-électrique sur 20 sujets anatomiques soit 40 hanches. On a comparé le nombre de coups de marteau pour obtenir l’impaction idéale de la prothèse selon 3 méthodes d’évaluation différentes : le nombre de coups nécessaires pour le chirurgien (Nchir), le nombre de coups nécessaires par l’analyse vidéo de l’enfoncement de la tige dans le fémur (Nvid) et le nombre de coups nécessaires par l’analyse de l’impact (NI).Pour savoir si la quantité de CSMs dans la crête iliaque pouvait refléter l’ostéointégration des implants acétabulaires impactées et le risque de reprise chirurgicale, on a comparé le taux de CSMs mesuré lors de la réalisation d’une ponction concentration et réinjection dans la zone d’ostéonécrose et l’évolution clinico-radiographique des implants acétabulaires mis en place par la suite pour ces même patients (n=90) qui ont eu une arthroplastie totale de hanche in fine. Le recul moyen était de 15 ans.La survie cellulaire des CSM osseuses humaines a été évaluée sur des cages intersomatiques lombaires recouvertes de titane. Trois groupes (n=5) furent constitués : un groupe contrôle, un groupe cages avec surface titane, un groupe cage sans titane. Sur chaque implant, 1 microlitre contenant 106 CSM osseuses humaines a été mis en culture. L’analyse de la survie cellulaire, de la prolifération cellulaire et de l’expression de gènes de différenciation ostéoblastique ont été réalisés et comparés.Résultats : Concernant la première étude sur l’intérêt de l’analyse de l’impact pour la tenue de la tige fémorale, la différence entre NI, Nchir et Nvid était inférieure ou égale à 3 pour plus de 85% des configurations réalisées. Concernant la deuxième étude, il a été mis en évidence qu’un faible nombre de CSM dans la crête iliaque était un facteur de risque de révision chirurgicale chez les patients traités par un implant acétabulaire sans ciment. La troisième étude a montré que les CSMs pouvaient survivre, se développer pendant 96 heures et exprimer des gènes de différenciation ostéoblastique de manière identique sur les cages avec ou sans titane.Conclusion : L’analyse de l’impact permet de donner une information objective sur la tenue primaire de la tige fémorale en titane et impactée. Le titane est aussi un milieu favorisé de survie et de prolifération des CSM osseuses prédestinées à devenir des cellules ostéoformatrices surtout qu’un faible nombre de CSM semble être un risque d’échec d’ostéointégration des implants acétabulaires sans cimentIntroduction: Titanium, both used as a mean of fixing an implant to its biomechanical properties close to the bone and as a mean of osseointegration has taken a prominent place. The implant stability is essential for the durability of a titanium implant (TI); it depends on 2 phases: a primary phase, MECHANICAL, corresponding to the impaction or primary holding of the TI and a secondary phase, BIOLOGICAL, corresponding to the colonization of TI by bone tissue.The objective of this work was to evaluate and improve during these two phases the osseointegration of the titanium implant:(1) To evaluate the primary stability of uncemented titanium femoral stems by impact analysis corresponding to the measurement of impact over time.(2) To evaluate whether the amount of mesenchymal stromal cells (MSCs) contained in the iliac crest is correlated with the non-recovery survival of acetabular implants impacted in a context of aseptic osteonecrosis of the femoral head.(3) To improve the osseointegration of TI by cell therapy methods in vitro by studying the survival and division of human MSCs in contact with interbody lumbar cages coated with rough titanium alloy.Methods: The evaluation of the primary stability of cementless titanium femoral stems according to the impact analysis was carried out using a hammer equipped with a piezoelectric force sensor on 20 anatomical subjects, i.e. 40 hips. The number of hammer strokes was compared to obtain the ideal impaction of the prosthesis according to 3 different evaluation methods: number of impacts required by the surgeon (Nsurg), number of impacts required by the video analysis of the depression of the stem in the femur (Nvid), numbers of impacts needed by the impact analysis (Ni).To determine whether the amount of MSCs in the iliac crest could reflect the osseointegration of impacted acetabular implants and the risk of surgical revision. The rate of MSCs measured when performing a surgical cell therapy for aseptic osteonecrosis of the femoral head and the clinical and radiographic outcome of acetabular implants subsequently established for these same patients (n = 90), who had total hip arthroplasty in fine were compared. The mean follow-up was 15 years.The cell survival of bone marrow-derived MSCs was evaluated on lumbar interbody cages coated with titanium. Three groups (n = 5) were formed: a control group, a cage group with titanium surface, a cage group without titanium. On each implant, 1 microliter containing 106 human bone MSCs was cultured. The analysis of cell survival, cell proliferation and expression of osteoblastic genes were performed and compared.Results: Regarding the impact analysis of the cementless femoral stem impaction, the difference between NI, Nchir and Nvid was lower than 3 for more than 85% of the configurations performed.For the second study, a small number of MSCs in the iliac crest was a risk factor for surgical revision in patients treated with a cementless acetabular implant.The third study showed that MSCs could grow until 96 hours and could express osteoblastics genes 21 days after cell seed. No difference between PEEK cage and Titanium-coated PEEK has been found.Conclusion: The impact analysis provides objective data on the primary holding of the titanium impacted femoral stem. Titanium is also a favored biomaterial for the survival and proliferation of bone marrow-derived MSC predestined to become osteforming cells, especially since a small number of MSCs seems to be a risk of failure of osseointegration of cementless acetabular implants
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Fievet, Loïc Marc André. "Caractérisation phénotypique et fonctionnelle des cellules stromales mésenchymateuses natives de la moelle osseuse humaine adulte." Thesis, Toulouse 3, 2020. http://www.theses.fr/2020TOU30137.
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Au sein de la moelle osseuse (MO), les cellules souches hématopoïétiques (CSH) sont logées dans un microenvironnement en 3D spécialisé, appelé "niche". Cette structure contribue à la régulation du comportement (e.g. auto-renouvellement, engagement dans des voies de différentiation, prolifération et survie). Cette niche est composée de plusieurs types de cellules, comme les cellules endothéliales vasculaires, périvasculaires et ostéoblastiques. Les cellules stromales mésenchymateuses (CSM) périvasculaires jouent un rôle clé dans la formation du microenvironnement, tant par leur expression de facteurs pro-hématopoïétiques, que de leur capacité de différenciation vers le lignage ostéoblastique. Ainsi, les sous-populations de CSM composant la niche hématopoïétique sont impliquées dans la régulation de l'hématopoïèse, mais également dans la formation et la régénération du squelette. Récemment décrites chez la souris à l'échelle de la cellule unique, les sous-populations de CSM restent cependant inexplorées dans la MO humaine, il en est ainsi de leur rôle respectif dans la niche. Connaître l'identité des sous-populations cellulaires, déchiffrer leurs propriétés natives et recréer ex vivo ces niches physiologiquement fonctionnelles en 3D restent des enjeux scientifiques importants pour la compréhension de l'hématopoïèse et de l'ostéogenèse humaine. Dans ce travail, nous avons dans un premier temps, utilisé des approches de séquençage d'ARN à l'échelle de la cellule unique (scRNA-seq), afin de caractériser chaque population stromale de la MO humaine et d'identifier des facteurs de niche hématopoïétique clés hautement conservés entre les espèces. Nous avons ainsi identifié plusieurs sous-populations cellulaires, exprimant différents gènes régulateurs de l'hématopoïèse, comprenant des cellules endothéliales, des cellules murales et particulièrement des CSM ayant des trajectoires ostéoblastiques et adipogéniques distinctes. De manière intéressante, nos données suggèrent une hiérarchie de différenciation à branchement simple avec la présence d'un sous-ensemble multipotent, les cellules réticulaires exprimant fortement CXCL12 (CAR) qui seraient à l'origine des autres sous-populations de CSM. Nous avons observé un enrichissement des cellules CAR exprimant le récepteur à la leptine (LEPR) dans la fraction native CD45- / CD271+ / CD200+, ainsi que leur localisation in situ en utilisant des approches histologiques sur des biopsies humaines. Dans un deuxième temps, nous avons élaboré une méthode d'isolation ex vivo pour préserver et amplifier les cellules CAR de la MO, puis étudier leur auto-organisation en culture 3D. Nous avons constaté que les sphéroïdes dérivés des cellules de type CAR ont une angiogenèse abondante, sécrètent des cytokines pro-niche et récapitulent spontanément in vitro la formation osseuse intramembranaire précoce. Grâce à des modèles bio-informatiques et des techniques d'édition du génome, nous avons mis en évidence le rôle de la protéine WDR35 et du cil primaire dans la différenciation ostéoblastique médiée par les voies de signalisation Hedgehog et Wnt. Enfin, nous avons montré que la xénotransplantation ectopique de ces sphéroïdes pouvait donner naissance à des ostéoblastes humains matures, tout en formant une niche aux CSH après humanisation hématopoïétique de souris immunodéprimées NSG. Notre étude donne, pour la première fois, une cartographie du stroma de la MO humaine avec une résolution unicellulaire et montre que les cellules CAR cultivées en 3D représentent un nouvel outil utile en recherche fondamentale avec un fort potentiel en médecine régénérativeWithin the bone marrow (BM), hematopoietic stem cells (HSC) are hosted in a specialized 3D microenvironment, called "niche", regulating their behavior (e.g. self-renewal, commitment to lineages, proliferation and survival). This niche is composed of several cells such as vascular endothelial, perivascular, and osteoblastic cells. Perivascular mesenchymal stromal cells (MSCs) play a key role in the formation of the microenvironment, both through expression of pro-hematopoietic factors, and their ability to differentiate towards osteoblastic lineage. Therefore, MSCs sub-populations are of crucial physiological importance in the regulation of hematopoiesis, but also for bone formation and regeneration. Recently described in mice at the single-cell level, BM MSCs subsets remain unexplored in humans, as well as their respective roles in the niche. By characterizing these sub-populations, and deciphering their native properties, it will be possible to shape ex vivo the physiological niches in 3D, addressing the major scientific challenges for understanding human hematopoiesis and osteogenesis. Here, we used single-cell RNA sequencing approaches to characterize the human BM stroma and described key hematopoietic niche factors highly conserved between species. We identified subsets of cells expressing different hematopoietic regulatory genes, spanning endothelial cells, mural cells, and especially MSCs with distinct osteoblastic and adipogenic trajectories. Of interest, our data suggest a simple branching differentiation hierarchy with the presence of a multipotent subset: the CXCL12-abundant reticular (CAR) cells at the origin of the other MSCs subpopulations. We confirmed the enrichment of the CXCL12-abundant reticular (CAR) cell subset expressing the Leptin receptor (LEPR+) in the CD45-/CD271+/CD200+ BM fraction as well as their in-situ localization using histological approaches on human biopsies. Secondly, we developed an ex vivo isolation method to preserve and amplify the BM CAR cells, and then studied their self-organization in 3D culture. We found that CAR derived organoids sustained high angiogenesis, secreted CSH niche cytokines, and spontaneously recapitulates early intramembranous bone formation in vitro. Using bioinformatics models and genome editing techniques, we highlighted the role of the WDR35 protein and the primary cilia in osteoblastic differentiation mediated by the Hedgehog and Wnt signaling pathways. Finally, we have shown that ectopic xenotransplantation of CSM-derived organoids could give rise to mature human osteoblasts while forming a niche for CSHs after hematopoietic humanization of immunocompromised NSG mice. Our study is the first map of the human BM stroma at a single-cell resolution, and CAR cells cultured in 3D represent a new tool useful in basic research as well as in regenerative medicine
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Clutter, Suzanne Davis. "Chemotherapy disrupts bone marrow stromal cell function." Morgantown, W. Va. : [West Virginia University Libraries], 2006. https://eidr.wvu.edu/etd/documentdata.eTD?documentid=4528.
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Thesis (Ph. D.)--West Virginia University, 2006.Title from document title page. Document formatted into pages; contains x, 180 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.
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Valle, Paulo Roberto Del. "Perfil transcricional de fibroblastos de tumor primário, linfonodo e medula óssea de pacientes com câncer de mama." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/5/5155/tde-26032013-143422/.
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Introdução: Em câncer de mama, existem evidências de que o microambiente pode influenciar o desenvolvimento do tumor no sítio primário, bem como em metástases regionais e a distância. Neste contexto, fibroblastos são importantes células estromais que podem influenciar a proliferação e a migração de células do câncer e podem prover um nicho apropriado para o desenvolvimento tumoral. Objetivos:O principal objetivo deste trabalho é comparar células estromais obtidas do tumor primário (PT), metástase linfonodal (N+) e medula óssea (BM) de pacientes com câncer de mama, através do perfil de expressão gênica. Pacientes e Métodos: Foi analisada a expressão gênica de fibroblastos (cultura primária) de 11 pacientes com câncer de mama. O perfil de expressão foi determinado em PT (n=4), N+(n=3) e BM (n=4) através de uma plataforma de cDNA microarray customizada (contendo 4.800 sequencias imobilizadas, representando cerca de 4600 genes), e os genes diferencialmente expressos foram identificados pelo teste SAM multiclasse, seguido pelo teste SAM de duas classes (TMEV, FDR 0%). A análise funcional foi realizada pelo software DAVID v6.7. Validação técnica foi realizada em 6 amostras previamente analisadas no microarray e a validação biológica em fibroblastos obtidos de outros 16 pacientes utilizando-se de RT-qPCR. Resultados: O perfil de expressão gênica dos fibroblastos obtidos de diferentes sítios mostraram 267 genes diferencialmente expressos, os quais apropriadamente agruparam os fibroblastos de acordo com suas origens (PT vs. N+ vs. BM). Apesar das diferenças entre PT e N+ serem representadas por 20 genes, as diferenças entre PT vs. BM e N+ vs BM foram mais significantes (235 e 245 genes diferencialmente expressos respectivamente). Análise funcional dos genes diferencialmente expressos mostrou enriquecimento de funções relacionadas ao desenvolvimento e morfogênese.A seguir, a expressão de alguns genes selecionados foi analisada em uma série diferente de amostras (validação biológica). Desse modo observamos que NOTCH2 confirmou uma alta expressão em N+ (vs. PT), e ADCY2, HECTD1, HNMT, LOX, MACF1 e USP16 confirmaram alta expressão em BM (vs PT). Conclusão:Em pacientes com câncer de mama, células estromais obtidas de diferentes origens apresentam um perfil de expressão gênica diferencial, o qual pode influenciar o comportamento do tumormay influence tumor development in the primary site of breast cancer, as well as in regional and distant metastatic sites. In this context, fibroblasts are important stromal cells which influence proliferation and migration of cancer cells and may also provide an appropriate niche to tumor development. Objectives: The main objective of this work is the comparison of stromal cells from the primary tumor (PT), lymph node metastasis (N+) and bone marrow (BM) obtained from breast cancer patients, through gene expression profile. Patients and Methods: The gene expression profile was analyzed in fibroblasts primary culture from 11 breast cancer patients. The expression profiles of PT cells (n=4), N+ cells (n=3) and BM cells (n=4) were determined through a customized cDNA microarray platform (containing 4800 immobilized sequences which represents 4600 genes approximately). The analysis were performed by SAM multiclass (TMEV; FDR 0%), followed by SAM two classes test (TMEV; FDR 0%). Functional analysis was performed using DAVID v6.7. Technical validation was performed in same 6 samples that were previously analyzed in microarray experiments and biological validation was performed in fibroblasts obtained from other group of 16patients by RT-qPCR Results: The expression profile of fibroblasts obtained from three sites revealed 267 differentially expressed genes, which appropriately clustered fibroblasts in three different branches, in accordance with their origin (PT vs. N+ vs. BM). Although the differences between PT and N+ were represented by 20 genes, differences between PT vs. BM and N+ vs. BM were more significant (235 and 245 differentially expressed genes respectively). Functional analysis revealed enrichment of functions related to development and morphogenesis. Afterwards, the expression of some selected genes were analyzed in a different batch of samples (biological validation).Thereby, NOTCH2 confirmed high expression in N+ (vs. PT), and ADCY2, HECTD1, HNMT, LOX, MACF1 and USP16 confirmed high expression in BM (vs. PT). Conclusion: In breast cancer patients, stromal cells obtained from different origins present a differential gene expression profile, which may influence tumor behavior
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Dennis, James Edmund. "Mesenchymal progenitor cells in adult marrow." Case Western Reserve University School of Graduate Studies / OhioLINK, 1995. http://rave.ohiolink.edu/etdc/view?acc_num=case1062516436.
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Liu, Limin. "Expression of follicular dendritic cell determinants by mouse bone marrow stromal cells." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp03/MQ37142.pdf.
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