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Kaur, Navdeep. "Influence of culture conditions on the molecular signature of mesenchymal stem cells." Thesis, Queensland University of Technology, 2010. https://eprints.qut.edu.au/43719/1/Navdeep_Kaur_Thesis.pdf.
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Cell based therapies require cells capable of self renewal and differentiation, and a prerequisite is the ability to prepare an effective dose of ex vivo expanded cells for autologous transplants. The in vivo identification of a source of physiologically relevant cell types suitable for cell therapies is therefore an integral part of tissue engineering.
Bone marrow is the most easily accessible source of mesenchymal stem cells (MSCs), and harbours two distinct populations of adult stem cells; namely hematopoietic stem cells (HSCs) and bone mesenchymal stem cells (BMSCs). Unlike HSCs, there are yet no rigorous criteria for characterizing BMSCs. Changing understanding about the pluripotency of BMSCs in recent studies has expanded their potential application; however, the underlying molecular pathways which impart the features distinctive to BMSCs remain elusive. Furthermore, the sparse in vivo distribution of these cells imposes a clear limitation to their in vitro study. Also, when BMSCs are cultured in vitro there is a loss of the in vivo microenvironment which results in a progressive decline in proliferation potential and multipotentiality. This is further exacerbated with increased passage number, characterized by the onset of senescence related changes. Accordingly, establishing protocols for generating large numbers of BMSCs without affecting their differentiation potential is necessary.
The principal aims of this thesis were to identify potential molecular factors for characterizing BMSCs from osteoarthritic patients, and also to attempt to establish culture protocols favourable for generating large number of BMSCs, while at the same time retaining their proliferation and differentiation potential.
Previously published studies concerning clonal cells have demonstrated that BMSCs are heterogeneous populations of cells at various stages of growth. Some cells are higher in the hierarchy and represent the progenitors, while other cells occupy a lower position in the hierarchy and are therefore more committed to a particular lineage. This feature of BMSCs was made evident by the work of Mareddy et al., which involved generating clonal populations of BMSCs from bone marrow of osteoarthritic patients, by a single cell clonal culture method. Proliferation potential and differentiation capabilities were used to group cells into fast growing and slow growing clones. The study presented here is a continuation of the work of Mareddy et al. and employed immunological and array based techniques to identify the primary molecular factors involved in regulating phenotypic characteristics exhibited by contrasting clonal populations. The subtractive immunization (SI) was used to generate novel antibodies against favourably expressed proteins in the fast growing clonal cell population. The difference between the clonal populations at the transcriptional level was determined using a Stem Cell RT2 Profiler TM PCR Array which focuses on stem cell pathway gene expression. Monoclonal antibodies (mAb) generated by SI were able to effectively highlight differentially expressed antigenic determinants, as was evident by Western blot analysis and confocal microscopy. Co-immunoprecipitation, followed by mass spectroscopy analysis, identified a favourably expressed protein as the cytoskeletal protein vimentin. The stem cell gene array highlighted genes that were highly upregulated in the fast growing clonal cell population. Based on their functions these genes were grouped into growth factors, cell fate determination and maintenance of embryonic and neural stem cell renewal. Furthermore, on a closer analysis it was established that the cytoskeletal protein vimentin and nine out of ten genes identified by gene array were associated with chondrogenesis or cartilage repair, consistent with the potential role played by BMSCs in defect repair and maintaining tissue homeostasis, by modulating the gene expression pattern to compensate for degenerated cartilage in osteoarthritic tissues. The gene array also presented transcripts for embryonic lineage markers such as FOXA2 and Sox2, both of which were significantly over expressed in fast growing clonal populations.
A recent groundbreaking study by Yamanaka et al imparted embryonic stem cell (ESCs) -like characteristic to somatic cells in a process termed nuclear reprogramming, by the ectopic expression of the genes Sox2, cMyc and Oct4. The expression of embryonic lineage markers in adult stem cells may be a mechanism by which the favourable behaviour of fast growing clonal cells is determined and suggests a possible active phenomenon of spontaneous reprogramming in fast growing clonal cells. The expression pattern of these critical molecular markers could be indicative of the competence of BMSCs. For this reason, the expression pattern of Sox2, Oct4 and cMyc, at various passages in heterogeneous BMSCs population and tissue derived cells (osteoblasts and chondrocytes), was investigated by a real-time PCR and immunoflourescence staining. A strong nuclear staining was observed for Sox2, Oct4 and cMyc, which gradually weakened accompanied with cytoplasmic translocation after several passage. The mRNA and protein expression of Sox2, Oct4 and cMyc peaked at the third passage for osteoblasts, chondrocytes and third passage for BMSCs, and declined with each subsequent passage, indicating towards a possible mechanism of spontaneous reprogramming. This study proposes that the progressive decline in proliferation potential and multipotentiality associated with increased passaging of BMSCs in vitro might be a consequence of loss of these propluripotency factors. We therefore hypothesise that the expression of these master genes is not an intrinsic cell function, but rather an outcome of interaction of the cells with their microenvironment; this was evident by the fact that when removed from their in vivo microenvironment, BMSCs undergo a rapid loss of stemness after only a few passages.
One of the most interesting aspects of this study was the integration of factors in the culture conditions, which to some extent, mimicked the in vivo microenvironmental niche of the BMSCs. A number of studies have successfully established that the cellular niche is not an inert tissue component but is of prime importance. The total sum of stimuli from the microenvironment underpins the complex interplay of regulatory mechanisms which control multiple functions in stem cells most importantly stem cell renewal. Therefore, well characterised factors which affect BMSCs characteristics, such as fibronectin (FN) coating, and morphogens such as FGF2 and BMP4, were incorporated into the cell culture conditions. The experimental set up was designed to provide insight into the expression pattern of the stem cell related transcription factors Sox2, cMyc and Oct4, in BMSCs with respect to passaging and changes in culture conditions. Induction of these pluripotency markers in somatic cells by retroviral transfection has been shown to confer pluripotency and an ESCs like state. Our study demonstrated that all treatments could transiently induce the expression of Sox2, cMyc and Oct4, and favourably affect the proliferation potential of BMSCs. The combined effect of these treatments was able to induce and retain the endogenous nuclear expression of stem cell transcription factors in BMSCs over an extended number of in vitro passages. Our results therefore suggest that the transient induction and manipulation of endogenous expression of transcription factors critical for stemness can be achieved by modulating the culture conditions; the benefit of which is to circumvent the need for genetic manipulations.
In summary, this study has explored the role of BMSCs in the diseased state of osteoarthritis, by employing transcriptional profiling along with SI. In particular this study pioneered the use of primary cells for generating novel antibodies by SI. We established that somatic cells and BMSCs have a basal level of expression of pluripotency markers. Furthermore, our study indicates that intrinsic signalling mechanisms of BMSCs are intimately linked with extrinsic cues from the microenvironment and that these signals appear to be critical for retaining the expression of genes to maintain cell stemness in long term in vitro culture. This project provides a basis for developing an “artificial niche” required for reversion of commitment and maintenance of BMSC in their uncommitted homeostatic state.
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VECELLIO, MATTEO LUCA. "Differentiation and reprogrammig of human mesenchymal stromal cells: insights from epigenetic assessments and pre-clinical studies." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2012. http://hdl.handle.net/10281/30253.
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Mesenchymal stromal cells (StC) are cells with plastic properties virtually present in every adult tissue. Recently, StC have also been isolated from adult human cardiac tissue (CStC) and the hypothesis has been raised that StC deriving from the heart may be genetically committed to cardiovascular differentiation. In this light, the enhancement of CStC cardiovascular precursor properties may represent a potentially successful strategy for cardiac regeneration purposes. Although of adult origin, CStC exhibit Islet1 expression and respond to chemically-determined cardiogenic epigenetic stimuli. Specifically we created an epigenetic chemical cocktail (EpiC)that is able to up-regulate the expression of cardiac resident stem cell markers c-Kit and MDR-1, together with the expression of a large number of cardiovascular-associated genes and regulatory RNAs including c-Kit, MDR-1, KDR, GATA6, Nkx2.5, GATA4, HCN4, NaV1.5, ALPHA-MHC, Alpha-sarcomeric actin, miR-1 and miR-499. Remarkably, EpiC-treated CStC also exhibited immature electrophysiological properties. Mechanistically, the EpiC treatment determined genome-wide histone modifications associated with a transcriptionally competent chromatin. Chromatin immunoprecipitation experiments (Chip) revealed that permissive histone modification H3K4Me3 was present in c-Kit, MDR-1 and Nkx2.5 promoter regions, possibly contributing to their expression. Altogether these data indicate that Isl1+ CStC may be epigenetically reprogrammed to acquire functionally competent cardiovascular precursor properties. CStC therefore appear as a potentially useful cell type for potential cardiac and vascular reconstructive therapies
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Atashpazgargari, S. "A CELL REPROGRAMMING-BASED APPROACH TO STUDY 7Q11.23 GENE DOSAGE IMBALANCES IN WILLIAMS BEUREN SYNDROME AND AUTISM SPECTRUM DISORDER." Doctoral thesis, Università degli Studi di Milano, 2015. http://hdl.handle.net/2434/264765.
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Symmetrical gene dosage imbalances at 7q11.23 chromosomal region cause two unique neurodevelopmental diseases, Williams Beuren Syndrome (WBS) and the 7q11.23 microduplication associated to autistic spectrum disorder (7dup-ASD). Although both these diseases share common features such as intellectual disability and craniofacial dysmorphism, they can be distinguished by distinct social and language abilities: WBS patients characterized by hypersociality and comparatively well-preserved language skills while 7dup-ASD is associated with impairment in social interaction and communicative skills. The involvement of same genetic interval in these disease, points out to small subset of dosage-sensitive genes affecting cognition, social behavior and communication skills.
Among the genes in the deleted region, some were shown to contribute to the abnormalities in these patients through transgenic mice models and individual case reports. However, the precise cellular and molecular phenotypes associated with these syndromes in disease-relevant cell-types are unknown due to the scarce availability of primary diseased tissues. Transcription factor induced somatic cell reprogramming has bypassed such fundamental limitation and has enabled us to model human diseases, elucidate their pathogenesis and discover new therapeutics by screening small chemicals/drugs on these models. During my PhD studies, I focused on the functional dissection of these complementary diseases at the level of transcriptional deregulation in patient-derived iPSC and its differentiated derivatives such as neural crest stem cells, mesenchymal stem cells, and neural progenitors. To this end, we have assembled a unique cohort of typical WBS, atypical WBS (patient with a partial deletion) and 7dup-ASD patients (along with unaffected relatives), and then I used mRNA reprogramming to establish and characterize at least 3 independent iPSC lines from a total of 12 individuals. High throughput mRNA sequencing on iPSC revealed critical transcriptional derangements in disease-relevant pathways already at the pluripotent state. These alterations found to be selectively amplified upon differentiation into disease-relevant lineages, thereby establishing the value of large iPSC cohorts in the elucidation of disease-relevant developmental pathways. Finally, we created an open-access web-based platform to make accessible our multi-layered datasets and integrate contributions by the entire community working on the molecular dissection of the 7q11.23 syndromes.
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Kennea, Nigel Leonard. "Neural differentiation of human fetal mesenchymal stem cells." Thesis, Imperial College London, 2007. http://hdl.handle.net/10044/1/7409.
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The potential of mesenchymal stem cells (MSC) to differentiate into neural lineages has raised the possibility of autologous cell transplantation as therapy for neurological diseases. There are, however, no studies reporting significant numbers of oligodendrocytes, the myelinforming cells of the central nervous system, derived from MSC. We have recently identified a population of circulating human fetal MSC that are highly proliferative and readily differentiate into bone, cartilage, fat and muscle. I demonstrated for the first time that primary fetal MSC differentiate into cells resemblifl neural precursors and then oligodendrocytes both in vitro and in vivo. By exposing cells to a neuronal conditioned medium, rates of oligodendrocyte differentiation approaching 50% were observed, and cells appeared to mature appropriately in culture. Importantly, the differentiation of a clonal population into both mesodermal (bone) and ectodermal (oligodendrocyte) lineages was achieved. In the developing murine brain, cells integrated but oligodendrocyte differentiation of naiVe fetal MSC was very low. The proportion of oligodendrocyte differentiation was increased (from 0.2% to 4%) by pre-exposing the cells to differentiation medium prior to transplantation. The process of in vivo differentiation occurred without cell fusion. Although the main focus of this thesis was oligodendrocyte differentiation, I also recapitulated controversial published work into neuronal differentiation of MSC. The exposure of cells to the reducing agent butylated hydroxyanisole induced rapid changes in cell morphology and expression of neuronal markers. These 'differentiated' cells did not, however, appear functional with no upregulation of voltage-gated sodium channels or synaptophysin. Finally, while stem cells offer promise for correction of brain diseases, one major obstacle is the poor survival of grafted cells. Investigation of apoptotic signalling showed fetal MSC have functional apoptotic machinery in both the intrinsic (mitochondrial) and extrinsic (death receptor) pathways which could be manipulated to prolong stem cell survival by inhibition of death signalling.
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Nicolaidou, Vicky. "Monocytes promote osteogenic differentiation of mesenchymal stem cells." Thesis, Imperial College London, 2011. http://hdl.handle.net/10044/1/9061.
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Bone loss is a characteristic of many chronic inflammatory and degenerative diseases such as rheumatoid arthritis and osteoporosis. A major challenge is how to replace bone once it is lost. It is known that the immune system strongly regulates bone and investigations into these interactions have demonstrated that osteoclasts, the bone resorbing cells, are strongly regulated by the immune system. However, less is known about the regulation of osteoblasts, the bone forming cells. Mesenchymal stem cells are multipotent progenitors that can be induced in culture to form osteoblasts. The aim of this study was to investigate whether immune cells also regulate OB differentiation. Using in vitro cell cultures of human bone marrow-derived MSCs it was shown that monocytes/Mφs potently induced MSC differentiation to OBs evidenced by increased alkaline phosphatase and mineralisation. However, the ability of monocyte/Mφs to promote osteogenesis differed between CD14++CD16- and CD14+CD16+ monocyte subset as well as M-CSF and GM-CSF Mφs when activated; the CD16- monocytes and M-CSF Mφs still promoted differentiation whereas the CD16+ monocytes and GM-CSF Mφs inhibited it. The monocyte osteogenic effect was mediated by monocyte-derived soluble factors and required STAT3 signalling as well as COX2 upregulation and the production of PGE2. Finally, gene profiling microarray identified Oncostatin M as the mediator of monocyte-induced osteogenesis. This study established a role for monocyte/Mφs as critical regulators of osteogenic differentiation via OSM and STAT3 signalling. It also provides an insight into the interactions between MSCs and monocyte/Mφs in an inflammatory setting where OB differentiation will depend on the balance between pro-inflammatory versus anti-inflammatory monocyte/Mφs.
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Whyte, Jemima Lois. "Density dependent differentiation of mesenchymal stem cells to endothelial cells." Thesis, University of Manchester, 2010. https://www.research.manchester.ac.uk/portal/en/theses/density-dependent-differentiation-of-mesenchymal-stem-cells-to-endothelial-cells(d839ac9d-3bda-46fb-8e8e-556a85772db9).html.
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The differentiation of mesenchymal stem cells (MSCs) to endothelium is a critical but poorly understood feature of tissue vascularisation and considerable scepticism still remains surrounding this important differentiation event. Defining features of endothelial cells (ECs) are their ability to exist as contact-inhibited polarised monolayers that are stabilised by intercellular junctions, and the expression and activity of endothelial markers. During vasculogenesis, communication between MSCs and differentiated ECs or vascular smooth muscle cells, or between MSCs themselves is likely to influence MSC differentiation. In this study, the possibility that cell density can influence MSC differentiation along the EC lineage was examined. High density plating of human bone marrow-derived MSCs induced prominent endothelial characteristics including cobblestone-like morphology, enhanced endothelial networks, acetylated-low density lipoprotein uptake, vascular growth and stimulated expression of characteristic endothelial markers. Mechanistically, this density-dependent process has been defined. Cell-cell contact-induced Notch signalling was a key initiating step regulating commitment towards an EC lineage, whilst VEGF-A stimulation was required to consolidate the EC fate. Thus, this study not only provides evidence that MSC density is an essential microenvironmental factor stimulating the in vitro differentiation of MSCs to ECs but also demonstrates that MSCs can be differentiated to a functional EC. Taken together, defining how these crucial MSC differentiation events are regulated in vitro, provides an insight into how MSCs differentiate to ECs during postnatal neovascularisation and an opportunity for the therapeutic manipulation of MSCs in vivo, enabling targeted modulation of neovascularisation in ischaemia, wound healing and tumourigenesis.
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Hardy, Steven Allan. "Mesenchymal stem cells as trophic mediators of neural differentiation." Thesis, Durham University, 2010. http://etheses.dur.ac.uk/524/.
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Intense excitement and optimism surrounds the rapidly-expanding field of stem cell research, owing to their high capacity for self-renewal and intrinsic ability to differentiate into mature cell lineages. Although it may be envisioned that embryonic stem cells will be of significantly greater therapeutic value than their adult stem cell counterparts, the use of embryonic stem cells is fraught with both technical and ethical challenges and, as such, significant impetus has been placed on adult stem cell-based research. In particular, mesenchymal stem cells (MSCs) present as exciting candidates for potential use in cellular therapies and tissue engineering strategies. MSCs are defined at the functional level in terms of their ability to differentiate into mesodermal derivatives such as bone and fat. However, this functional definition is evolving, and there is considerable evidence to suggest that MSCs have a key role within their niche involving the release and/or uptake of soluble factors and cytokines, significantly influencing the behaviour of other cell types within the niche. Both facets of MSC behaviour are valuable from a clinical perspective, and have been examined in the present thesis. The most obvious and realistically-achievable clinical application of MSCs at present is in the treatment of osseous and adipose tissue defects. However, before the use of MSCs in the clinic becomes more commonplace, it is crucial to gain a more comprehensive understanding of the complex molecular and cellular mechanism(s) by which MSCs commit to a given fate and undergo differentiation to produce mature, fully-functional derivatives. Much of our present knowledge is derived from studies performed on the highly unnatural, 2D environment of tissue culture plastic. The present study assessed the behaviour of MSCs cultured on AlvetexTM, a novel, 3D scaffold manufactured by ReInnervate, with particular emphasis on the ability of MSCs to undergo osteogenic and adipogenic differentiation. Results obtained suggest that AlvetexTM may provide a more realistic and physiologically-relevant system in which to study osteogenesis and adipogenesis, in a manner more pertinent to that which occurs in vivo. Furthermore, the ability of MSCs to influence the behaviour of other cell types via the release of trophic factors and cytokines was examined, with particular emphasis on the nervous system. An in vitro conditioned media model was developed in order to investigate the influence(s) of MSC-derived soluble factors/cytokines on neural development and plasticity, using the adult rat hippocampal progenitor cell (AHPC) line as a model system. Results obtained suggest that, under defined conditions, MSCs secreted a complement of soluble factors/cytokines that induce AHPCs to commit to and undergo astrogenesis. This effect was characterised at both the cellular and molecular level. The specific complement of bioactive factors secreted by MSCs has been investigated using a combination of targeted transcriptional profiling and shotgun proteomics, and several putative candidate factors have been identified for further investigation.
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Prosser, Amy. "Enhanced differentiation of mesenchymal stem cells for osteochondral constructs." Thesis, University of Nottingham, 2015. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.727949.
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Novel osteochondral repair tissue engineering strategies are investigating the use of a single scaffold, with a portion for osteogenic and chondrogenic differentiation, and a single cell source, most notably the mesenchymal stem cell, to facilitate osteochondral differentiation and repair in a single construct. However, this approach requires robust differentiation protocols to ensure that the correct balance of each cell type is produced and maintained. Techniques used to analyse osteogenic and chondrogenic differentiation are well established, but many of the current methods described are qualitative, based on imaging stained cells or sections under a microscope. To facilitate higher throughput screening of chondrogenic differentiation in human MSCs, a novel culture technique using V shaped 96 well plates has been developed combining three robust and quantitative assays. Additionally, two reporter cell lines have been developed that express luciferase under the control of an osteogenic (osteocalcin) or chondrogenic (col2a1) promoter in order to streamline differentiation assays. The use of growth factors to elicit differentiation is well established; with BMP-2 used to enhance osteogenic differentiation and TGF-61 used to enhance chondrogenic differentiation. However, there are several limitations of using growth factors in regenerative medicine and consequently, the use of growth factor mimics was investigated. Two promising growth factor mimics were identified that could support both osteogenic and chondrogenic differentiation; LE135 and imperatorin. LE135, a retinoic acid receptor antagonist, significantly enhanced chondrogenesis with increased GAG production, col2a1 promoter activity and versican mRNA expression and had no significant effect on osteogenic differentiation. Imperatorin, a coumarin derivative, significantly enhanced early stage osteogenesis (alkaline phosphatase activity) and had no significant effect on late stage osteogenesis (mineralisation). Furthermore, chondrogenic differentiation was enhanced by imperatorin with significantly increased GAG production.
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Karageorgiou, Vassilis. "Bioinductive protein-based scaffolds for human mesenchymal stem cells differentiation /." Thesis, Connect to Dissertations & Theses @ Tufts University, 2004.
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Thesis (Ph. D.)--Tufts University, 2004.Adviser: David L. Kaplan. Submitted to the Dept. of Chemical and Biological Engineering. Includes bibliographical references. Access restricted to members of the Tufts University community. Also available via the World Wide Web;
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Cameron, Katherine Rachel. "Calcium phosphate substrate-directed osteogenic differentiation of mesenchymal stem cells." Thesis, University of Edinburgh, 2013. http://hdl.handle.net/1842/8051.
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An increase in degenerative bone disease in an ageing population, combined with a rise in the number of patients suffering from bone defects caused by physical trauma, makes the repair of bone an issue of growing clinical relevance. Current treatments such as autografts and allografts have major drawbacks, including donor site morbidity, limited availability, disease transmission and immune rejection. To overcome these issues synthetic bone grafts have been developed to mimic the mineral phase of bone. Given the significant roles of silicon in bone growth and development there has been great interest in introducing silicon into synthetic bone grafts to enhance their bioactivity. Calcium phosphate based silicate containing grafts have demonstrated enhanced bioactivity, improved physical properties, enhanced protein adsorption and greater bone formation, when compared to non-silicated calcium phosphates such as hydroxyapatite. However, is not clear whether the increased bone formation associated with these materials is the result of greater osteoblast activity or a rise in numbers of osteoblasts resulting from activation and differentiation of stem/ progenitor cells. To answer this question, multipotent stem cells were cultured on silicate substituted calcium phosphate (Si-CaP) and hydroxyapatite (HA). Si-CaP promoted greater cell adhesion and enhanced proliferation when compared to HA. Cells differentiated along the osteogenic lineage on both substrates as evidenced by up regulation of osteoblast specific genes and proteins. However, cells on Si-CaP showed earlier and greater gene expression of all osteoblast genes examined, and greater protein production as detected by immunohistochemistry. Integrin gene expression analysis revealed up regulation of α an d β subunits on both substrates during differentiation. Integrins α5 and β1 expression were greater on Si-CaP than on HA, suggesting preferential binding of fibronectin. The implication of these findings for tissue engineering is clear, suggesting these substrates may be utilized to control stem cell fate in vivo and in vitro without the need for osteogenic supplementation. Furthermore, the increased rate of differentiation seen on Si-CaP may enable the development of novel substrates for osteogenic differentiation of MSC, which may have significant impact in regenerative medicine.
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Hao, Ru. "Reprogramming of mesenchymal stem cells and adult fibroblasts following nuclear transfer in rabbits." Diss., lmu, 2009. http://nbn-resolving.de/urn:nbn:de:bvb:19-96652.
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Pawlak, Mathias. "Stem cells, differentiation and nuclear reprogramming : the roles of Klf4 and geminin /." Heidelberg, 2008. http://opac.nebis.ch/cgi-bin/showAbstract.pl?sys=000259539.
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Götherström, Cecilia. "Characterisation of human fetal mesenchymal stem cells /." Stockholm, 2004. http://diss.kib.ki.se/2004/91-7140-139-3/.
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Aguilar, Fadó Esther. "Metabolic reprogramming and vulnerabilities of prostate cancer stem cells independent or epithelial-mesenchymal transition." Doctoral thesis, Universitat de Barcelona, 2015. http://hdl.handle.net/10803/291812.
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Metastasis represents the most life-threatening aspect of tumorigenesis and is the leading cause of death by cancer. Intensive research in this field has shed light on some of the molecular strategies employed by the heterogeneous cancer cell populations to leave the primary tumor, disseminate and grow new colonies in distant organs. In any given tumor, one important functional category of cancer cells is represented by cancer stem cells (CSCs), endowed with self-renewal and tumor-initiating potentials. Moreover, the epithelial-mesenchymal transition (EMT) program represents a process of fundamental importance conducive to tumor dissemination and metastatic spread of cancer cells. Some studies have pointed out that the EMT is responsible for the acquisition of the CSC-like state whereas others have shown that both cell entities can exist separately and cooperate to accelerate the process of metastasis.
Here, we propose the combined use of metabolomics and fluxomics strategies to shed light on the metabolic reprogramming and vulnerabilities accompanying specific cancer cell phenotypes that differs in their metastatic and invasive capacities. The main objective of this thesis is focused on the characterization of the metabolic reprogramming and vulnerabilities of uncoupled CSC and EMT phenotypes present in a dual-cell prostate cancer cell model and represented by the highly related cell subpopulations PC-3M and PC-3S cells, respectively.
Our results indicated that epithelial PC-3M cells, displaying CSC features and a high metastatic potential, preferentially rely on aerobic glycolysis (Warburg effect) for bioenergetics. Although these cells show low coupling between glycolysis and oxidative phosphorylation (OXPHOS) because of low pyruvate dehydrogenase activity, they display an increased metabolic flexibility to utilize different carbon sources, such as fatty acids, glutamine and other amino acids, that offset the decreased diversion of glucose-derived carbons into the tricarboxylic acid cycle and OXPHOS.
The characterization of the non-CSC mesenchymal PC-3S cells expressing the EMT program and endowed with a high invasive capacity, showed a strong coupling between aerobic glycolysis and OXPHOS and a strong dependence on the mitochondrial metabolism for bioenergetics, which leads to higher levels of ROS that require increased levels of glutathione to provide an adequate antioxidant defense system.
PC-3M and PC-3S cells differentially reprogram the use of the oxidative and non-oxidative branches of the pentose phosphate pathway to sustain their distinct metabolic needs. Glycolytic intermediates are preferentially directed to ribose synthesis in PC-3M cells to build up nucleotides whereas the generation of NADPH is more crucial for PC-3S cells to counteract their higher oxidative stress and sustain their increased fatty acid synthesis.
Glutamine metabolism substantially contributes to TCA reactions in PC-3. For PC-3S cells, both glucose and glutamine are necessary to display a proper mitochondrial function. PC-3M cells are more dependent than PC-3S cells on the glutaminase reaction for proliferation and survival and this reliance lies mainly on the increased need for PC-3M cells to neutralize the excessive levels of protons (lactic acid) that result from their marked Warburg effect, which is achieved by the ammonia molecules released from glutamine metabolism.
The high metabolic flexibility displayed by the CSC subpopulation including the participation of serine, glycine and one-carbon metabolism, the uptake of ketogenic amino acids, proline metabolism, among others, provide PC-3M cells with an extensive metabolic dynamics to obtain not only precursors but also to balance their redox status (NAD+/NADH and NADP+/NADPH) for metabolic processes to continue (e.g. glycolysis) and protect them from excessive acidity derived from a high glycolytic rate.
Collectively, these results strengthen the notion that specific metabolic signatures are associated to CSC and EMT programs and highlight the importance of studying uncoupled cell phenotypes in order to univocally associate their characteristic metabolic reprogramming.El proceso de la metástasis es la principal causa de mortalidad en pacientes de cáncer. En los últimos años se ha desvelado la importancia de la cooperación entre distintas subpoblaciones celulares que coexisten en el tumor. Entre estas subpoblaciones, encontramos las células denominadas cancer stem cells (CSCs), con un elevado potencial de autorenovación, pluripotencia y capacidad de iniciar tumores. Por otro lado, ciertas subpoblaciones celulares del tumor son capaces de incrementar sus capacidades migratorias e invasivas, mediante el proceso de epithelial-mesenchymal transition (EMT). Diversos estudios han demostrado que la cooperación entre CSCs y células que han activado el programa EMT facilita la colonización metastásica. Dado que la reprogramación metabólica es responsable de proveer a las células tumorales aquellos recursos bioenergéticos y de biosíntesis necesarios para el mantenimiento de su fenotipo tumoral, en este trabajo se ha caracterizado el metabolismo y las vulnerabilidades metabólicas de dos subpoblaciones celulares derivadas de la línea celular PC-3, con características diferenciadas de CSCs por un lado (PC-3M) y de EMT por otro (PC-3S). El estudio metabólico de estas subpoblaciones celulares desveló que las células PC-3M presentan una mayor preferencia para el uso de la glucólisis (efecto Warburg más marcado), mientras que las PC-3S son más dependientes del metabolismo energético mitocondrial. Estas subpoblaciones también difieren en el uso de las ramas oxidativa y no oxidativa de la vía de las pentosas fosfato y en las reacciones de biosíntesis y degradación de ácidos grasos, con el fin de satisfacer las distintas necesidades metabólicas que caracterizan estos fenotipos. Por otro lado, las PC-3M muestran una elevada flexibilidad y adaptación metabólica, siendo capaces de metabolizar numerosos substratos, entre ellos diferentes tipos de amino ácidos. Particularmente, el metabolismo de la glutamina en las PC-3M es más esencial que en las PC-3S, no sólo por su papel anaplerótico, si no por su función de tamponamiento de los excesos de ácido. El conjunto de estos resultados han desvelado las particularidades metabólicas y vulnerabilidades asociadas a los fenotipos, no solapados, de CSCs y EMT. El conocimiento adquirido podrá contribuir en el diseño de nuevas estrategias terapéuticas para el tratamiento de la metástasis.
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Neiman, Veronica Juliet. "Oscillating hydrogel based bioreactors for chondrogenic differentiation of mesenchymal stem cells." Diss., [La Jolla] : University of California, San Diego, 2010. http://wwwlib.umi.com/cr/ucsd/fullcit?p1474761.
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Thesis (M.S.)--University of California, San Diego, 2010.Title from first page of PDF file (viewed April 14, 2010). Available via ProQuest Digital Dissertations. Includes bibliographical references (p. 105-113).
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Heymer, Andrea. "Chondrogenic differentiation of human mesenchymal stem cells and articular cartilage reconstruction." kostenfrei, 2008. http://www.opus-bayern.de/uni-wuerzburg/volltexte/2008/2944/.
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Ishiy, Felipe Augusto André. "Evaluation of molecular markers in osteogenic differentiation of mesenchymal stem cells." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/41/41131/tde-20032017-104921/.
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The use of stem cells is a promising therapeutic approach for tissue engineering by their ability to boost tissue regeneration, and to model in vitro human genetics disorders since it provides continuous supplies of cells with differentiation potential. Our study has been focused in the identification of molecules or mechanisms that could contribute to a better osteogenesis in mesenchymal stem cells (MSC). To achieve our goals we have explored the osteopontential differences of stem cells from different sources. In this regard, we have observed that MSCs from human exfoliated deciduous teeth (SHED) presented higher in vitro osteogenic differentiation potential (OD) as compared to MSCs derived from human adipose tissue (hASCs). Through microarray analysis and cell sorting, we have shown that IGF2 and CD105 expression levels contribute to these osteopontential cell differences, that is, higher IGF2 expression levels and lower CD105 expression levels were associated with the increased osteogenic potential of SHED as compared to hASCs. The molecular mechanisms associated with the diferent expression levels of IGF2 and CD105 in these cells were also investigated. Despite the advantages of adult MSCs they can exhibit drawbacks such as restricted self-renewal and limited cell amounts. Induced Pluripotent Stem Cells (iPSC) technology has emerged as an alternative cell source, as they provide more homogeneous cellular populations with prolonged self-renewal and higher plasticity. We verified that the OD of MSC-like iPSC differs from MSCs and it depends on the iPSCs originating cellular source. Comparative in vitro osteogenesis analysis showed higher osteogenic potential in MSC-like cells derived from iPS-SHED when compared with MSC-like cells from iPS-FIB and SHED. iPSCs can be also used as a tool to model genetic disorders. We have thus proposed to verify if it could be possible to in vitro model Treacher-Collins syndrome, a condition with deficient craniofacial bone development. We have compared the effects of pathogenic mutations in TCOF1 gene in cell proliferation, differentiation potential between MSCs, dermal fibroblasts, neural-crest like and MSC-like cells differentiated from iPSCs.TCS cells showed changes in cell properties anddysregulated expression of chondrogenesis markers during osteogenic and chondrogenic differentiation. In summary, the comparative analysis of stem cells of different sources allow us to identify markers that may facilitate osteogenesis and that it is possible to establish an in vitro model to Treacher-Collins syndromeO uso de células-tronco trata-se de uma abordagem terapêutica promissora para a engenharia de tecidos, devido à sua capacidade na regeneração de tecidos, e para modelamento in vitro de distúrbios genéticos humanos, uma vez que fornece um abastecimento contínuo de células com potencial de diferenciação. Nosso estudo se propos a identificar moléculas e mecanismos que contribuem na otimização da osteogênese de células-tronco mesenquimais (MSCs). Para atingir nossos objetivos exploramos as diferenças no potencial osteogênico (PO) de MSCs de diferentes fontes. Observamos que MSCs de polpa de dente decíduo humano (SHED) apresentaram maior PO em comparação com as MSC derivadas de tecido adiposo humano (hASCs). Através de análise de microarray de expressão e cell sorting, demonstramos que os níveis de expressão de IGF2 e CD105 contribuem para as diferenças do PO, onde a maior expressão de IGF2 e menor expressão de CD105 estão associadas a maior PO em SHED quando comparado as hASCs. Também investigamos os mecanismos moleculares associados aos diferentes níveis de expressão de IGF2 E CD105 em ambas as fontes celulares. Apesar das vantagens, as MSCs podem apresentar pontos negativos como restrita auto-renovação e menor quantidade de células. Células-tronco pluripotentes induzidas (iPSC) surgem como uma fonte celular alternativa, proporcionando populações celulares homogêneas com auto-renovação prolongada e maior plasticidade. O PO de MSC-like iPSC difere de MSCs, e este potencial é dependente da fonte celular em que as iPSCs são obtidas. Análise comparativa de PO in vitro demonstrou maior osteogênse em células MSC-like derivadas de iPS-SHED quando comparada as células MSC-like de iPSCs-fibroblastos e SHED. iPSCs também podem ser utilizadas como ferramenta para investigar doenças genéticas humanas. Propomos a modelagem in vitro da síndrome de Treacher-Collins (TSC), doença que acomete as estruturas craniofaciais durante o desenvolvimento ósseo. Comparamos os efeitos de mutações patogênicas no gene TCOF1 na proliferação celular, potencial de diferenciação entre MSCs, fibroblastos dérmicos, neural-crest like e células MSC-like diferenciadas de iPSCs. Células de pacientes TCS exibiram alterações em propriedades celulares e na expressão de marcadores osteogênicos e condrogênicos. Em resumo, a análise comparativa de células-tronco de diferentes fontes permitiu a identificação de marcadores e mecanismos que podem facilitar a osteogênese e tambem demonstramos que é possível modelar in vitro a síndrome de Treacher-Collins
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Kuroda, Mito. "Mechanism of the ECM stiffness-dependent differentiation of mesenchymal stem cells." Kyoto University, 2018. http://hdl.handle.net/2433/232360.
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Kyoto University (京都大学)0048
新制・課程博士
博士(農学)
甲第21159号
農博第2285号
新制||農||1060(附属図書館)
学位論文||H30||N5133(農学部図書室)
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 植田 和光, 教授 阪井 康能, 教授 矢﨑 一史
学位規則第4条第1項該当
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Okamoto, Takeshi. "Clonal heterogeneity in differentiation potential of immortalized human mesenchymal stem cells." Kyoto University, 2004. http://hdl.handle.net/2433/147533.
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Hodgkiss-Geere, Hannah Mary. "Isolation, characterisation and differentiation of canine adult stem cells." Thesis, University of Edinburgh, 2012. http://hdl.handle.net/1842/6471.
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Cardiac and orthopaedic diseases are significant causes of morbidity and mortality in dogs and are therefore critical areas for veterinary research. More information regarding the pathophysiology of these diseases, and the development of novel therapeutics are sorely required and adult stem cells (ASCs) are a promising source of cells for both investigation of these diseases in vitro and also potentially therapeutics in the longer term. ASCs are a readily available source of multipotent cells which bypass the ethical issues surrounding embryonic stem (ES) cells. ASCs have been described in several tissues of the body, and typically differentiate along specific cellular routes related to original source location. This thesis investigates whether ASCs can be isolated and cultured from the dog from two specific locations; cardiac, producing cardiac stem cells (CSCs); and the bone marrow, producing mesenchymal stem cells (MSCs). These cell sources will be extensively characterised at their baseline for morphology, culture behaviour and gene marker expression. Following characterisation each cell source will be subjected to differentiation techniques to examine canine ASC multipotent differentiation potential. CSCs were isolated from cultured atrial cardiac explant tissue taken from dogs post-mortem, with owners’ consent. These cells were able to survive successive passages in serum free media and formed large spherical cell clusters, termed ‘cardiospheres’. CSCs were capable of clonal expansion under controlled culture conditions, demonstrating their ability for self-renewal. Characterisation of these cells demonstrated the expression of CSC markers; c-Kit, GATA 4 and Flk-1 and no expression of cardiac lineage markers including cardiac troponin T and I, Nkx2.5, the cardiac ryanodine receptor and the β1-adrenergic receptor. Primary canine MSCs were isolated from bone marrow aspirates using ficoll separation and cultured on tissue culture plastic. Canine MSCs closely resembled MSCs described from other species, such as the human and mouse, and were found to express CD44 and STRO-1 and were negative for CD34 and CD45. CSCs and MSCs were exposed to published cardiac directed differentiation protocols and differentiation then analysed using cellular morphology and gene expression. Canine CSCs appeared to differentiate partially along cardiac lineages with upregulation of cardiac troponin T and Nkx2.5, and down regulation of c-Kit and endothelial lineage markers. Canine MSCs demonstrated some morphological changes during cardiac differentiation, and demonstrated up-regulation of Nkx2.5 and Flk-1 but no significant alteration in other markers examined. This suggested that cardiac directed differentiation was not as successful with canine MSCs compared to CSCs and conflicting with published data using rodent MSC models. Murine MSCs were used as a positive control cell line for cardiac directed differentiation, based upon published literature. Critically there were key marker expression differences between baseline murine and canine MSCs, including the expression of cardiac markers such as cardiac troponin T and I, and the Ryanodine receptor. Furthermore, expression analysis of cardiac genes changed with time in culture and passage number and no significant alteration was seen when cells were subjected to the cardiac differentiation protocol; thereby bringing into question the data regarding successful cardiac differentiation using murine MSCs. Canine MSCs were further differentiated toward a chondrocyte lineage to investigate the use of MSCs for orthopaedic research. Canine MSCs were successfully differentiated toward articular type cartilage, with demonstration of extracellular matrix secretions, an upregulation of collagen type II with downregulation of collagen type I and the development of SOX9 expression in differentiated cells. This thesis builds the groundwork for future ASC research in the dog. Successful isolation and culture of two ASC sources from the dog is demonstrated. Cardiac and cartilage directed differentiation was successful using primary sourced cells, but differentiation was found to be limited to highly specific routes for each stem cell source. The results presented here highlight the importance of analysing baseline stem cells extensively prior to differentiation and in particular, before making comparisons between cell populations isolated from different locations and species.
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Lam, Shuk-pik. "Differentiation of mesenchymal stem cells (MSCs) into hepatocytes in acute liver injury." Click to view the E-thesis via HKUTO, 2009. http://sunzi.lib.hku.hk/hkuto/record/B43085647.
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Popielarczyk, Tracee. "Homing and Differentiation of Mesenchymal Stem Cells in 3D In Vitro Models." Diss., Virginia Tech, 2017. http://hdl.handle.net/10919/78789.
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Mesenchymal stem cells (MSCs) have great potential to improve clinical outcomes for many inflammatory and degenerative diseases through delivery of exogenous MSCs via injection or cell-laden scaffolds and through mobilization and migration of endogenous MSCs to injury sites. MSC fate and function is determined by microenvironmental cues, specifically dimensionality, topography, and cell-cell interactions. MSC responses of migration and differentiation are the focus of this dissertation. Cell migration occurs in several physiological and pathological processes; migration mode and cell signaling are determined by the environment and type of confinement in three-dimensional (3D) models.
Tendon injury is a common musculoskeletal disorder that occurs through cumulative damage to the extracellular matrix (ECM). Studies combining nanofibrous scaffolds and MSCs to determine an optimal topographical environment have promoted tenogenic differentiation under various conditions. We investigated cellular response of MSCs on specifically designed nanofiber matrices fabricated using a novel spinneret-based tunable engineered parameters production method (STEP). We designed suspended and aligned nanofiber scaffolds to study cellular morphology, tendon marker gene expression, and matrix deposition as determinants for tendon differentiation.
The delivery and maintenance of MSCs at sites of inflammation or injury are major challenges in stem cell therapies. Enhancing stem cell homing could improve their therapeutic effects. Homing is a process that involves cell migration through the vasculature to target organs. This process is defined in leukocyte transendothelial migration (TEM); however, far less is known about MSC homing. We investigated two population subsets of MSCs in a Transwell system mimicking the vasculature; migrated cells that initiated transmigration on the endothelium and nonmigrated cells in the apical chamber that failed to transmigrate. Gene and protein expression changes were observed between these subsets and evidence suggests that multiple signaling pathways regulate TEM.
The results of these experiments have demonstrated that microenvironmental cues are critical to understanding the cellular and molecular mechanisms of MSC response, specifically in homing and differentiation. This knowledge has identified scaffold parameters required to stimulate tenogenesis and signaling pathways controlling MSC homing. These findings will allow us to target key regulatory molecules and cell signaling pathways involved in MSC response towards development of regenerative therapies.Ph. D.
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Kim, Narae. "External pH in culture on somatic cell reprogramming and cell differentiation in mouse and chicken cells." Kyoto University, 2017. http://hdl.handle.net/2433/218018.
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Kyoto University (京都大学)0048
新制・課程博士
博士(農学)
甲第20092号
農博第2199号
新制||農||1046(附属図書館)
学位論文||H29||N5026(農学部図書室)
33208
京都大学大学院農学研究科応用生物科学専攻
(主査)教授 今井 裕, 教授 松井 徹, 教授 久米 新一
学位規則第4条第1項該当
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Bergante, S. "ISOLATION AND DIFFERENTIATION OF STEM CELLS: SEARCHING FOR NEW MARKERS." Doctoral thesis, Università degli Studi di Milano, 2013. http://hdl.handle.net/2434/217454.
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Abstract
MSCs are the most studied adult stem cells, as they can be isolated from almost any tissue, they show a good self-renewal capacity in vitro and they also possess good “plasticity”(jiang nature). Oddly, MSCs are identified and defined by a combination of markers that are not distinctive, as they are shared by other cells including fibroblasts. Therefore, pure populations of MSCs cannot be isolated, as they are always contaminated by other adult cells that often do not possess stem cell plasticity. Thus, it would be very desirable to discover novel cell surface markers that would allow to discriminate MSCs from other cells. Moreover, new ways to control and promote cell differentiation are needed. Thus, it becomes crucial to search for new key molecules that can be modulated to increase MSCs differentiation toward the desired tissue or inhibit the differentiation when cells have to remain in an undifferentiated state.
In this direction, we focused our attention on SLs, a family of lipids found in the outer leaflet of the plasma membrane and involved in many cell signaling pathways.
Therefore, main aims of this work were:
1. to investigate the possible use of SLs as new surface markers for the identification, characterization and possibly isolation of human bone marrow MSCs;
2. to investigate the involvement of SLs in the preservation of the undifferentiated state of MSCs during in vitro culturing
3. to assess the possible role of SLs in the differentiation processes of MSCs upon opportune stimuli.
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Lam, Shuk-pik, and 林淑碧. "Differentiation of mesenchymal stem cells (MSCs) into hepatocytes in acute liver injury." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2009. http://hub.hku.hk/bib/B43085647.
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Hui, Ting-yan. "In vitro chondrogenic differentiation of human mesenchymal stem cells in collagen gels." Click to view the E-thesis via HKUTO, 2007. http://sunzi.lib.hku.hk/HKUTO/record/B39558745.
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許婷恩 and Ting-yan Hui. "In vitro chondrogenic differentiation of human mesenchymal stem cells in collagen gels." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2007. http://hub.hku.hk/bib/B39558745.
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Li, Jing. "Effects of intrinsic & extrinsic factors on the growth and differentiation of human mesenchymal stem cells." View the Table of Contents & Abstract, 2006. http://sunzi.lib.hku.hk/hkuto/record/B36434450.
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Eminli, Sarah [Verfasser]. "The role of differentiation state in reprogramming of somatic cells into induced pluripotent stem cells / Sarah Eminli." Berlin : Freie Universität Berlin, 2010. http://d-nb.info/1024365689/34.
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Lin, Liwen. "Study of hydroxyapatite osteoinductivity with an osteogenic differentiation assay using mesenchymal stem cells /." View abstract or full-text, 2007. http://library.ust.hk/cgi/db/thesis.pl?BIEN%202007%20LIN.
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Munir, Hafsa. "Mesenchymal stem cells as endogenous regulators of leukocyte recruitment : the effects of differentiation." Thesis, University of Birmingham, 2016. http://etheses.bham.ac.uk//id/eprint/6471/.
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Mesenchymal stem cells (MSC) are a tissue-resident stromal cell population that are able to regulate immune responses, in particular the capacity for endothelial cells (EC) to support leukocyte recruitment. In this thesis we examined the ability of MSC from different sources (bone marrow, Wharton’s jelly and trabecular bone) to regulate neutrophil recruitment to inflamed EC and how these responses are altered upon adipogenic differentiation of MSC. Using two flow based adhesion models with varying degrees of proximity between MSC and EC, we observed that all MSC populations suppressed neutrophil recruitment. IL-6 and TGFβ were identified as common bioactive agents found in all co-cultures. Upon differentiation, MSC exhibited a diminished capacity to suppress neutrophil, but not peripheral blood lymphocyte, recruitment. Loss of suppression by MSC-derived adipocytes was reversed by neutralising IL-6. Adipose tissue-derived mature adipocytes and culture differentiated pre-adipocytes did not recapitulate the effects of MSC-derived adipocytes. These data suggest that crosstalk between tissue-resident MSC and EC, dampens the endothelial response to cytokines and limits the aberrant infiltration of circulating leukocytes during inflammation. Upon adipogenic differentiation, MSC lose this regulatory capacity. This could impact on the beneficial effects of MSC in chronically inflamed sites where aberrant infiltration of leukocyte is a main driver of the disease.
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Glück, Martina [Verfasser], Anke [Akademischer Betreuer] Bernstein, and Hagen [Akademischer Betreuer] Schmal. "Induction of osteogenic differentiation in human mesenchymal stem cells by crosstalk with osteoblasts." Freiburg : Universität, 2016. http://d-nb.info/1122647646/34.
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Guyette, Jacques Paul. "Conditioning of Mesenchymal Stem Cells Initiates Cardiogenic Differentiation and Increases Function in Infarcted Hearts." Digital WPI, 2012. https://digitalcommons.wpi.edu/etd-dissertations/32.
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Current treatment options are limited for patients with myocardial infarction or heart failure. Cellular cardiomyoplasty is a promising therapeutic strategy being investigated as a potential treatment, which aims to deliver exogenous cells to the infarcted heart, for the purpose of restoring healthy myocardial mass and mechanical cardiac function. While several cell types have been studied for this application, only bone marrow cells and human mesenchymal stem cells (hMSCs) have been shown to be safe and effective for improving cardiac function in clinical trials. In both human and animal studies, the delivery of hMSCs to infarcted myocardium decreased inflammatory response, promoted cardiomyocyte survival, and improved cardiac functional indices. While the benefits of using hMSCs as a cell therapy for cardiac repair are encouraging, the desired expectation of cardiomyoplasty is to increase cardiomyocyte content that will contribute to active cardiac mechanical function. Delivered cells may increase myocyte content by several different mechanisms such as differentiating to a cardiomyocyte lineage, secreting paracrine factors that increase native stem cell differentiation, or secreting factors that increase native myocyte proliferation. Considerable work suggests that hMSCs can differentiate towards a cardiomyocyte lineage based on measured milestones such as cardiac-specific marker expression, sarcomere formation, ion current propagation, and gap junction formation. However, current methods for cardiac differentiation of hMSCs have significant limitations. Current differentiation techniques are complicated and tedious, signaling pathways and mechanisms are largely unknown, and only a small percentage of hMSCs appear to exhibit cardiogenic traits. In this body of work, we developed a simple strategy to initiate cardiac differentiation of hMSCs in vitro. Incorporating environmental cues typically found in a myocardial infarct (e.g. decreased oxygen tension and increased concentrations of cell-signaling factors), our novel in vitro conditioning regimen combines reduced-O2 culture and hepatocyte growth factor (HGF) treatment. Reduced-O2 culturing of hMSCs has shown to enhance differentiation, tissue formation, and the release of cardioprotective signaling factors. HGF is a pleiotropic cytokine involved in several biological processes including developmental cardiomyogenesis, through its interaction with the tyrosine kinase receptor c-Met. We hypothesize that applying a combined conditioning treatment of reduced-O2 and HGF to hMSCs in vitro will enhance cardiac-specific gene and protein expression. Additionally, the transplantation of conditioned hMSCs into an in vivo infarct model will result in differentiation of delivered hMSCs and improved cardiac mechanical function. In testing our hypothesis, we show that reduced-O2 culturing can enhance hMSC growth kinetics and total c-Met expression. Combining reduced-O2 culturing with HGF treatment, hMSCs can be conditioned to express cardiac-specific genes and proteins in vitro. Using small-molecule inhibitors to target specific effector proteins in a proposed HGF/c-Met signaling pathway, treated reduced-O2/HGF hMSCs show a decrease in cardiac gene expression. When implanted into rat infarcts in vivo, reduced-O2/HGF conditioned hMSCs increase regional cardiac mechanics within the infarct region at 1 week and 1 month. Further analysis from the in vivo study showed a significant increase in the retention of reduced-O2/HGF conditioned hMSCs. Immunohistochemistry showed that some of the reduced-O2/HGF conditioned hMSCs express cardiac-specific proteins in vivo. These results suggest that a combined regimen of reduced-O2 and HGF conditioning increases cardiac-specific marker expression in hMSCs in vitro. In addition, the implantation of reduced-O2/HGF conditioned hMSCs into an infarct significantly improves cardiac function, with contributing factors of improved cell retention and possible increases in myocyte content. Overall, we developed a simple in vitro conditioning regimen to improve cardiac differentiation capabilities in hMSCs, in order to enhance the outcomes of using hMSCs as a cell therapy for the diseased heart.
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Caramelo, Inês Isabel Nunes. "Mechanomodulation of chondrogenic differentiation of msesenchymal stem cells." Master's thesis, Universidade de Aveiro, 2017. http://hdl.handle.net/10773/22537.
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Mestrado em Bioquímica - Métodos BiomolecularesA cartilagem hialina, cujas células especializadas são os condrócitos, encontra-se maioritariamente presente nas articulações. A degeneração deste tecido está associada a envelhecimento e a diversas doenças como artrite reumatóide e osteoartrite. Recentemente, tem sido investigada a possibilidade de desenvolver terapias celulares para o tratamento destas patologias, utilizando células estaminais mesenquimais (MSCs). As MSCs têm capacidade para se diferenciar em várias linhagens, incluindo condrócitos, apresentando-se como um dos mais promissores tipos celulares em medicina regenerativa. Nos últimos anos as vias de sinalização iniciadas pelos estímulos mecânicos do meio envolvente – mecanotransdução - tem sido alvo de estudo. Apesar dos mecanismos de diferenciação condrogénica não serem completamente conhecidos, tem-se tornado evidente que a mecanotransdução desempenha um papel crucial neste processo. Foi recentemente demonstrado que as MSCs têm “memória mecânica” e que, se cultivadas por mais de 10 dias num substrato rígido perdem a multipotência. Vários estudos utilizando células primárias ou MSCs apontam a rigidez ótima para diferenciação condrogénica deste 1 kPa até 320 MPa, dependendo do tipo de células e plataforma utilizados. Este estudo propôs-se então a clarificar qual a rigidez ótima para diferenciação condrogénica de MSCs. No presente estudo, foram preparados vários substratos de PDMS e caracterizados por reologia, apresentando módulos de Young que variam entre 21 kPa e 0.9 kPa. A diminuição da área nuclear nos substratos menos rígidos permitiu validar que estes substratos são capazes de induzir um estímulo mecânico. Somente células de baixa passagem foram induzidas a diferenciar diminuir o impacto da memória mecânica. A coloração de Safranin O (SO) permitiu evidenciar que a formação de aglomerados celulares – típica da condrogénese – é favorecida pelo substrato de 1 kPa. Recorrendo à fluorescência deste corante, foi possível estabelecer um método semi-quantitativo para avaliar diferenciação condrogénica de MSCs. Este ensaio indica que o substrato de 1 kPa potencia a diferenciação condrogénica de MSCs. Apesar dos resultados de SO requererem uma validação mais exaustiva por RT-PCR, os resultados preliminares apontam o que a rigidez ótima para diferenciação condrogénica de MSCs é de 1 kPa
Hyaline cartilage is composed by specialized cells named chondrocytes. It is mainly present on joints. Its degeneration is associated not only with ageing, but also with diseases like osteoarthritis and rheumatoid arthritis. Cell therapy is an emerging concept for these diseases. Mesenchymal stem cells (MSCs) can differentiate into various cell lineages, including chondrocytes. Recent studies indicate the significance of how cells are capable of sensing mechanical stimuli and initiate signaling cascades – mechanotransduction. Although the mechanisms are not totally understood, it is known that mechanotransduction plays a significant role during chondrogenesis. “Mechanical memory” is an emerging concept: it has been demonstrated that cells lose their multipotency if cultured on stiff substrates for more than 10 days. Distinct studies using primary cells or MSCs indicate that optimal matrix stiffness for chondrogenic differentiation is between 1 kPa and 320 MPa, depending on cell type and platform used. On the present study, we aimed to elucidate the optimal stiffness for chondrogenic differentiation of MSCs. Various PDMS substrates were produced and characterized by rheology, presenting Young’s modulus between 21 kPa and 0.9 kPa. We verified a decreasing tendency on the nucleus area along with substrate softening, suggesting that MSCs were responding to substrate stiffness. To reduce the influence of “mechanical memory”, only naïve cells were induced to differentiate. Safranin O (SO) staining revealed that 1 kPa substrate favored cell agglomeration, typical of chondrogenesis. Using fluorescence of this dye, we established a semi-quantitative assay to evaluate chondrogenic differentiation of MSCs. This assay suggests that 1 kPa substrate potentiates chondrogenic differentiation of MSCs. Despite SO assay results need further validation by RT-PCR, preliminary data indicates 1 kPa as the optimal stiffness for chondrogenic differentiation.
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Lim, Jeremy James. "The development of glycosaminoglycan-based materials to promote chondrogenic differentiation of mesenchymal stem cells." Diss., Georgia Institute of Technology, 2012. http://hdl.handle.net/1853/44849.
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Tissue engineering strategies represent exciting potential therapies to repair cartilage injuries; however, difficulty regenerating the complex extracellular matrix (ECM) organization of native cartilage remains a significant challenge. Cartilaginous ECM molecules, specifically chondroitin sulfate (CS) glycosaminoglycan, may possess the ability to promote and direct MSC differentiation down a chondrogenic lineage. CS may interact with the stem cell microenvironment through its highly negative charge, generation of osmotic pressure, and sequestration of growth factors; however, the role of CS in directing differentiation down a chondrogenic lineage remains unclear. The overall goal of this dissertation was to develop versatile biomaterial platforms to control CS presentation to mesenchymal stem cells (MSCs) in order to improve understanding of the interactions with CS that promote chondrogenic differentiation.
To investigate chondrogenic response to a diverse set of CS materials, progenitor cells were cultured in the presence of CS proteoglycans and CS chains in a variety of 2D and 3D material systems. Surfaces were coated with aggrecan proteoglycan to alter cell morphology, CS-based nano- and microspheres were developed as small particle carriers for growth factor delivery, and desulfated chondroitin hydrogels were synthesized to examine electrostatic interactions with growth factors and the role of sulfation in the chondrogenic differentiation of MSCs. Together these studies provided valuable insight into the unique ability of CS-based materials to control cellular microenvironments via morphological and material cues to promote chondrogenic differentiation in the development of tissue engineering strategies for cartilage regeneration and repair.
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Kramm, Anneke. "Identification and characterisation of epigenetic mechanisms in osteoblast differentiation of human mesenchymal stem cells." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:b6f7a356-b20f-4988-8770-8bebc233bf4b.
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A major therapeutic challenge in musculoskeletal regenerative medicine is how to effectively replenish bone tissue lost due to pathological conditions such as fracture, osteoporosis, or rheumatoid arthritis. Mesenchymal stem cells are currently investigated for applications in bone-tissue engineering and human bone marrow-derived mesenchymal stem cells (hMSCs) could be a promising source for generation of tissue-engineered bone. However, the therapeutic potential of MSCs has not been fully exploited due to a lack of knowledge regarding the identity, nature, and differentiation of hMSCs. Epigenetic mechanisms regulating the chromatin structure as well as specific gene transcription are crucial in determination of stem cell differentiation. With the aim to systematically identify epigenetic factors that modulate MSC differentiation, the work in this thesis encompasses an approach to identify epigenetic mechanisms underlying, initiating, and promoting osteoblast differentiation, and the investigation of individual epigenetic modulators. Various osteogenic inducers were validated for differentiation of MSCs and an assay allowing assessment of differentiation outcome was developed. This assay was subsequently employed in knockdown experiments with lentiviral short hairpin RNAs and inhibitor screens with small molecules targeting putative druggable epigenetic modulator classes. This approach identified around 100 epigenetic modulator candidates involved in osteoblast differentiation, of these candidates approximately 2/3 downregulated and 1/3 upregulated alkaline phosphatase (ALP) activity. Serving as a proof-of-concept, orthogonal validation experiments employing locked nucleic acid (LNA) knockdown were performed to validate a subset of candidates. Two identified target genes were selected for further investigation. Bromodomain-containing protein 4 (BRD4) was identified as one component of epigenetic regulation; its inhibition led to a decrease in ALP expression, downregulation of key osteoblast transcription factors Runx2 and Osterix, as well as impaired bone matrix formation. Knockdown of lysine (K)-specific demethylase 1A (KDM1A/LSD1) upregulated ALP activity and treatment with a small molecule inhibitor targeting KDM1A led to an increase in ALP, RUNX2, and bone sialoprotein expression. Intriguingly, in a transgenic mouse model overexpressing Kdm1a a decrease in bone volume and bone mineral density was observed, thus supporting the hypothesis that KDM1A is a central regulator of osteoblast differentiation.
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D'AGOSTINO, ANNA. "IDENTIFICATION OF A NOVEL TRANSCRIPTION FACTOR REQUIRED FOR OSTEOGENIC DIFFERENTIATION OF MESENCHYMAL STEM CELLS." Doctoral thesis, Università degli Studi di Milano, 2020. http://hdl.handle.net/2434/681954.
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Osteogenic differentiation is a complex and still poorly understood biological process regulated by intrinsic cellular signals and extrinsic micro-environmental cues. Following appropriate stimuli, mesenchymal stem cells (MSCs) differentiate into osteoblasts through a tightly regulated multi-step process driven by several transcription factors and characterized by the expression of a number of bone-specific proteins. Here, we describe a novel transcription factor that we named Osteoblast Inducer (ObI)-1, involved in MSC differentiation towards the osteogenic lineage. ObI-1 encodes for a nuclear protein subjected to proteasomal degradation and expressed during osteoblast differentiation both in a murine multipotent mesenchymal cell line (W20-17) and in primary murine MSCs. RNAi-mediated knockdown of ObI-1 expression significantly impairs osteoblast differentiation and matrix mineralization with reduced expression of the osteogenic markers Runx2 and osteopontin. Conversely, ObI-1 over-expression enhances osteogenic differentiation and bone-specific markers expression. ObI-1 stimulates bone morphogenetic protein (BMP)-4 expression and the consequent activation of the Smad pathway; treatment with a BMP receptor-type I antagonist completely abolishes ObI-1-mediated stimulation of osteogenic differentiation. Collectively, our findings suggest that ObI-1 modulates osteogenic differentiation, at least in part, through the BMP signaling pathway, increasing Runx2 activation and leading to osteoblast commitment and maturation.
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Okolicsanyi, Rachel K. "Mesenchymal stem cells as mediators of the neuronal cell niche." Thesis, Queensland University of Technology, 2015. https://eprints.qut.edu.au/84485/1/Rachel_Okolicsanyi_Thesis.pdf.
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This study examined the role of heparan sulfate proteoglycans (HSPGs) in neural lineage differentiation of human mesenchymal stem cells (hMSCs). Several HSPGs were identified as potential new targets controlling neural fate specification and may be applied to the development of improved models to examine and repair brain damage. hMSCs were characterised throughout extended in vitro expansion for neural lineage potential (neurons, astrocytes, oligodendrocytes) and differentiated using terminal differentiation and intermediate sphere formation. Brain damage and neurological disorders caused by injury or disease affect a large number of people often resulting in lifelong disabilities. Multipotent mesenchymal stem cells have a large capacity for self-renewal and provide an excellent model to examine the regulation and contribution of both stem cells and their surrounding microenvironment to the repair of neural tissue damage.
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Duong, Khanh Linh. "Molecular and cellular basis of hematopoietic stem cells maintenance and differentiation." Diss., University of Iowa, 2014. https://ir.uiowa.edu/etd/1448.
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The blood system consists of two main lineages: myeloid and lymphoid. The myeloid system consists of cells that are part of the innate immune response while the lymphoid system consist of cells that are part of humoral response. These responses protect our bodies from foreign pathogens. Thus, malignancies in these systems often cause complications and mortality. Scientists world wide have been researching alternatives to treat hematologic disorders and have explored induced pluripotent stem cells (iPSCs) and the conversion of one cell type to another.
First, iPS cells were generated by overexpression of four transcription factors: Oct4, Sox2, Klf4 an cMyc. These cells closely resemble embryonic stem cells (ESCs) at the molecular and cellular level. However, the efficiency of cell conversion is less than 0.1%. In addition, many iPS colonies can arise from the same culture, but each has a different molecular signature and potential. Identifying the appropriate iPS cell lines to use for patient specific therapy is crucial. Here we demonstrate that our system is highly efficient in generating iPS cell lines, and cell lines with silent transgenes are most efficient in differentiating to different cell types .
Second, we are interested in generating hematopoietic stem cells (HSCs) from fibroblasts directly, without going through the pluripotent state, to increase efficiency and to avoid complications associated with a stem cell intermediate. However, a robust hematopoietic reporter system remains elusive. There are multiple hematopoietic reporter candidates, but we demonstrate that the CD45 gene was the most promising. CD45 is expressed early during hematopoiesis on the surface of HSCs; and as HSCs differentiate CD45 levels increase. Furthermore, the CD45 reporter is only active in hematopoietic cells. We were able to confirm the utility of the CD45 reporter using an in vitro and an in vivo murine model.
In conclusion, The goal of this research was to expand the knowledge of stem cell reprogramming, specifically the reprogramming of iPS cells. Furthermore, it is our desire that the CD45 reporter system will undergo further validation and find utility in clinical and cell therapy environments.
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Lazin, Jamie Jonas. "The effect of age and sex on the number and osteogenic differentiation potential of adipose-derived mesenchymal stem cells." Thesis, Georgia Institute of Technology, 2010. http://hdl.handle.net/1853/34696.
Full textAbstract:
It has been shown that stem cells exist within adult adipose tissue. These stem cells are named adipose-derived mesenchymal stem cells (ASCs), are derived from the mesoderm, and can differentiate into a number of cells including osteoblasts, chondrocytes, and adipocytes. However, before these cells can be used clinically it is important that we understand how factors like age, sex, and ethnicity affect ASC number and potential. Additionally, since men and women vary in their distribution of adipose tissue, it will be important to see if the ideal source of ASCs is different for each sex. The goal of this study was to assess how age and sex affects ASCs. We used flow cytometry to investigate how age and sex affected the number of ASCs in adipose tissue. Additionally, we plated these cells in culture and treated them with an osteogenic media (OM) with the intention of pushing them towards osteoblast differentiation. The purpose of this was to see if age or sex affected the potential of the ASCs to undergo osteogenesis in culture. For this study we used real-time PCR and biochemical assays to look at markers of early and late osteogenic differentiation. Finally, we used immunohistochemistry to demonstrate where in adipose tissue the CD73 and CD271 positive cell population exists. It is our hope that this work will shed light on how age and sex affect ASCs so that clinicians can optimize their ASC harvest depending on the patient's physiology.
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Rossi, Barbara <1986>. "Mesenchymal Stromal Cells (MSCs) and induced Plutipotent Stem Cells (iPSCs) in Domestic Animals: Characterization and Differentiation Potential." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2015. http://amsdottorato.unibo.it/6795/1/rossi_barbara_tesi.pdf.
Full textAbstract:
Derivation of stem cell lines from domesticated animals has been of great interest as it benefits translational medicine, clinical applications to improve human and animal health and biotechnology. The main types of stem cells studied are Embryonic Stem Cells (ESCs), induced Pluripotent Stem Cells (iPSCs) and Mesenchymal Stem/Stromal Cells (MSCs). This thesis had two main aims:
(I) The isolation of bovine MSCs from amniotic fluid (AF) at different trimesters of pregnancy and their characterization to study pluripotency markers expression. Stemness markers were studied also in MSCs isolated from equine AF, Wharton’s jelly (WJ) and umbilical cord blood (UCB) as continuation of the characterization of these cells previously performed by our research group;
(II) The establishment and characterization of iPSCs lines in two attractive large animal models for biomedical and biotechnology research such as the bovine and the swine, and the differentiation into the myogenic lineage of porcine iPSCs.
It was observed that foetal tissues in domestic animals such as the bovine and the horse represent a source of MSCs able to differentiate into the mesodermal lineage but they do not proliferate indefinitely and they lack the expression of many pluripotency markers, making them an interesting source of cells for regenerative medicine, but not the best candidate to elucidate pluripotency networks. The protocol used to induce pluripotency in bovine fibroblasts did not work, as well as the chemical induction of pluripotency in porcine fibroblasts, while the reprogramming protocol used for porcine iPSCs was successful and the line generated was amenable to being differentiated into the myogenic lineage, demonstrating that they could be addressed into a desired lineage by genetic modification and appropriated culture conditions. Only a few cell types have been differentiated from domestic animal iPSCs to date, so the development of a reliable directed-differentiation protocol represents a very important result.
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Rossi, Barbara <1986>. "Mesenchymal Stromal Cells (MSCs) and induced Plutipotent Stem Cells (iPSCs) in Domestic Animals: Characterization and Differentiation Potential." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2015. http://amsdottorato.unibo.it/6795/.
Full textAbstract:
Derivation of stem cell lines from domesticated animals has been of great interest as it benefits translational medicine, clinical applications to improve human and animal health and biotechnology. The main types of stem cells studied are Embryonic Stem Cells (ESCs), induced Pluripotent Stem Cells (iPSCs) and Mesenchymal Stem/Stromal Cells (MSCs). This thesis had two main aims:
(I) The isolation of bovine MSCs from amniotic fluid (AF) at different trimesters of pregnancy and their characterization to study pluripotency markers expression. Stemness markers were studied also in MSCs isolated from equine AF, Wharton’s jelly (WJ) and umbilical cord blood (UCB) as continuation of the characterization of these cells previously performed by our research group;
(II) The establishment and characterization of iPSCs lines in two attractive large animal models for biomedical and biotechnology research such as the bovine and the swine, and the differentiation into the myogenic lineage of porcine iPSCs.
It was observed that foetal tissues in domestic animals such as the bovine and the horse represent a source of MSCs able to differentiate into the mesodermal lineage but they do not proliferate indefinitely and they lack the expression of many pluripotency markers, making them an interesting source of cells for regenerative medicine, but not the best candidate to elucidate pluripotency networks. The protocol used to induce pluripotency in bovine fibroblasts did not work, as well as the chemical induction of pluripotency in porcine fibroblasts, while the reprogramming protocol used for porcine iPSCs was successful and the line generated was amenable to being differentiated into the myogenic lineage, demonstrating that they could be addressed into a desired lineage by genetic modification and appropriated culture conditions. Only a few cell types have been differentiated from domestic animal iPSCs to date, so the development of a reliable directed-differentiation protocol represents a very important result.
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Lagerholm, Sara. "Isolation and Characterization of Mesenchymal Stem Cells from the Periodontal Ligament of Healthy Teeth." Thesis, Malmö universitet, Odontologiska fakulteten (OD), 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:mau:diva-19683.
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ABSTRAKT:Isolering och karaktärisering av mesenkymala stamceller från periodontalligamentet hos friskatänderSYFTE: Att isolera och odla celler från periodontalligamentet samt karaktärisera dem sommesenkymala stamceller.MATERIAL OCH METOD: Friska premolarer gjordes tillgängliga vid ortodontiskaextraktioner. Den mellersta 1/3 av periodontalligamentet skrapades varpå en enzymatiskmetod användes för isolering av individuella celler. Resulterande celler odlades understandardiserade metoder. Karaktärisering av celler skedde genom flödescymetri med 2 olikapaneler av cellyta markörer; en för etablerat positiva uttryck och en för kända negativauttryck hos mesenkymala stamceller. Möjlighet av celler att differentieras in vitro tilladipocyter och osteocyter testades genom tillförsel av specifika substanser till odlingsmediet.RESULTAT: Celler från 11 av 13 tänder isolerades och odlades framgångsrikt adherenta tillodlingsytan i upp till 8 generationer. Celluttryck av de positiva markörerna CD73, CD90 samtCD44 bekräftades genom flödescymetri. Inget uttryck observerades för den negativa panelenCD45, CD34, CD11b, CD19 eller HLA class II. Uttrycket av CD105 kunde inte fastställas pgaofullständigt data. Försök till differentiering av celler till adipocyter och osteocyter visade påfenotypiska förändringar efter 21 dagar.SLUTSATS: Den här studien har bidragit till framgångsrik isolering och delvis karaktäriseringav mesenkymala stamceller från periodontalligamentet hos friska tänder. En icke-invasivmetod av detta slag, resulterande i tillgång till denna cellpopulation utgör ett lovande verktygför framtida studier med goda möjligheter till ytterligare kunskap applicerbart till kliniskasituationer inom tandvården.ABSTRACT:Isolation and Characterization of Mesenchymal Stem Cells from the Periodontal Ligament ofHealthy TeethAIM: To isolate and culture viable cells from the periodontal ligament and confirming theiridentity as mesenchymal stem cells.METHODS AND MATERIALS: Healthy premolars were collected at the time oforthodontic extractions. The middle 1/3 of the periodontal ligament was scraped andsubsequent cell isolation was performed using an enzymatic method; yielding single cellisolates. Cells were cultured and maintained under standard culture conditions. Cellcharacterization was performed by flow cytometry using two sets of cell surface markers; oneknown to be present and one known to be absent in mesenchymal stem cells. Ability of thecells for in vitro differentiation into adipogenic and osteogenic lineages was tested usingspecifically formulated media supplements.RESULTS: Cells were successfully isolated from 11 of 13 teeth and were maintained asadherent cultures for up to 8 generations. Cellular expression of positive markers; CD73, CD90and CD44 were confirmed by flow cytometry. For the negative marker panel, expression ofCD45, CD34, CD11b, CD19 and HLA class II were not detectable. The expression of CD105was inconclusive. As determined by phenotypic changes, cells appeared to have undergoneadipogenic and osteocytic differentiation at 21 days.CONCLUSION: This study has resulted in successful isolation and partial characterization ofmesenchymal stem cells from the periodontal ligament of healthy teeth. Non-invasive accessto these cells, provides an excellent tool for future studies, potentially leading to beneficialknowledge transferable to the dental clinical situation.
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Reinholt, Brad M. "Conversion of equine umbilical cord matrix mesenchymal stem cells to the trophectoderm lineage using the Yamanaka reprogramming factors." Diss., Virginia Tech, 2015. http://hdl.handle.net/10919/74270.
Full textAbstract:
Induced pluripotent stem (iPS) cells that possess embryonic stem (ES) cell-like properties are generated through the use of the Yamanaka transcription factors, OCT4, SOX2, KLF4, and MYC (OSKM). Advanced transgene delivery methods utilizing non-integrating viruses for transduction of target cells has provided new opportunities for regenerative medicine in humans and other species. We sought to use this technology to generate equine iPS cells to address challenges in equine regenerative medicine. Umbilical cord matrix mesenchymal stromal cells (MSC) were transduced with the non-integrating Sendai virus encoding for the OSKM transcription factors. The cells initially were cultured on mouse embryonic feeder cells supplemented with LIF (10 ng/mL) and FGF2 (4 ng/mL). Transduction generated 21 initial colonies. Of these, four survived beyond 20 passages. The transduced equine cells morphologically resembled ES cells and expressed cell surface antigens indicative of ES cells. Molecular evaluation revealed the cells maintained expression of endogenous OSKM while the exogenous OSK transgenes were extinguished, but MYC was maintained. The transduced equine cells did not express the ES marker NANOG, but did express the trophectoderm markers CDX2 and TFAP2A. Both OCT4 and CDX2 were colocalized to the nucleus. The transduced equine cells were termed equine induced trophoblast (iTr) cells. Culture of the iTr cell in suspension resulted in formation of blastocyst-like spheres rather than solid cell aggregates indicative of ES and iPS cells. The iTr cells were transitioned to a feeder free monolayer culture. Transformation of the iTr cells to the spherical arrangement stimulated expression of genes that mark differentiation of trophoblast cells and up-regulated 250 transcripts over the monolayer arrangement. The iTr monolayer arrangement up-regulated 50 transcripts compared to the spherical arrangement. The iTr spheres respond to BMP4, EGF, and FGF2 by phosphorylating signal transduction proteins. Addition of BMP4, EGF, or FGF2 in combined pairs was able to alter TFAP2A, NEU1, and SLC35A1 expression. The generation of iTr cells by transduction of the Yamanaka reprogramming factors is not unique to equine cells. However, this report marks the generation of the first equine trophoblast cell line capable of recapitulating early equine trophoblast development. The new iTr line could prove valuable in gaining greater understanding of equine trophectoderm development.Ph. D.
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Chang, Ching-Fang, and 張靜芳. "Differentiation of human mesenchymal stem cells into insulin-producing cells." Thesis, 2006. http://ndltd.ncl.edu.tw/handle/31686790954625708835.
Full textAbstract:
碩士國立陽明大學
解剖暨細胞生物學研究所
95
Type I diabetes mellitus is caused by an autoimmune destruction of the pancreatic islet β cells. The major obstacle in using trans- plantation for curing the disease is the limited source of insulin- producing cells. Stem cells represent a promising solution to this problem, and current research is being aimed at the creation of islet-endocrine tissue from those undifferentiated cells. Human mesenchymal stem cells(hMSCs)are self-renewing elements that can differentiated into multiple cell types ,including bone, cartilage, adipose and neuron. We present a method for forming insulin-producing cells derived from hMSCs. The protocol is consisted of several steps: Those hMSCs cultured in medium containing 3-Isobutyl-1-Methylxanthine (IBMX) and Insulin-Transferrin-Selenium-A(ITSA)would be induced to differentiate into neuron. Then the medium is replaced by fresh medium containing Nicotinamide, N2 and B27 would be induced to differentiate into pancreatic β cells. Our data shows that hMSCs cultured in medium containing extracellular matrix-fibronectin in suspension state would be differentiate into insulin-producing cells. Detecting insulin gene by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR), we found those cells express insulin gene. Immuno- histochemistry stain shows those cells have high level proinsulin and insulin. In addition, ELISA data shows those cells secrete insulin amounted to 1.05 ±0.065ng/pellet/hr. However in animal model experiment, those cells can’t rescue the diabetic NOD/SCID mice. Future work will be the mechanism of extracellular matrix-fibronectin involved in stem cells differentiated into insulin-producing cells and establishing an effective animal model experiment.
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Amaral, Luis Manuel Fonseca. "Mesenchymal stem cell-based differentiation of smooth muscle cells." Master's thesis, 2014. http://hdl.handle.net/1822/34059.
Full textAbstract:
Dissertação de mestrado integrado em Engenharia Biomédica (área de especialização em Engenharia Clinica)Valvular heart disease is a major health and socioeconomic problem worldwide with approximately 300 000 valve replacements performed annually. Tissue-engineered heart valves with repair and remodelling capabilities could overcome the limitations of today’s valvular prostheses. One major limitation to this approach has been finding a reliable source of smooth muscle cells (SMC) because biopsies of these cells can be impractical and morbid as also present limited replicative capacity. The ideal cell source should be harvested in a non-or minimally invasive way and should deliver an initial high number of cells in order to drastically reduce the time needed for cell expansion. For these reasons there are many studies endeavoured to explore whether functional SMC could be generated from various types of adult mesenchymal stem cells (MSC): Umbilical cord (UC-MSC), bone marrow (BM-MSC), adipose derived (AD-MSC) and chorionic villi (CV-MSC) as possible sources for heart valve therapy. The MSC from different sources were isolated and expanded of healthy and different donor. In this study an isolation protocol was established for the CV-MSC, because it was never performed on this research group. Since the CV-MSC are barely reported in studies, this MSC source was characterized by flow cytometry to compare with other sources that are well-characterized in literature. After that, the MSC were differentiated by culture them in a culture medium containing transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein 4 (BMP4), based on a published 7-day differentiation protocol. The differentiation was analysed using 4 different smooth muscle markers (α-SMA, SM22α, Calponin and SM-MHC) by immunofluorescence (IF) and immunohistochemistry (IHC). A more extensively analysis was performed by flow cytometry using the smooth muscle markers and the other markers used on different studies to characterize the MSC population for a complete phenotype characterization before and after differentiation. The IF showed promising results as the smooth muscle markers stained positive for the differentiated MSC and negative for the undifferentiated MSC. On the other hand, the IHC and flow cytometry show some contradicting results for differentiation, since the expression is not consistent within the undifferentiated and differentiated MSC. These results highlight the concept that MSC represent an easily accessible, novel cell source for heart valve therapy, but despite of the wide experiments and results in this work, it is necessary further research in this field due to the conflicting evidence and inadequate information about several cell surface markers.
As doenças valvulares cardíacas são um problema social, económico e de saúde grave no mundo, com a realização de aproximadamente 300 000 cirurgias para substituição de válvulas cardíacas. A Engenharia de Tecidos na investigação e desenvolvimento de células cardíacas com capacidades para reparar e substituir válvulas cardíacas, pode vir a ultrapassar as limitações das atuais próteses valvulares. Contudo uma das maiores dificuldades desta investigação, tem sido encontrar uma fonte válida de células do músculo liso. A fonte ideal de colheita de células deve resultar de uma técnica minimamente invasiva e deve disponibilizar inicialmente um número elevado de células de modo a reduzir drasticamente o tempo necessário para a proliferação. Por estas razões há muitos estudos em curso de modo a investigar se as células do músculo liso podem ser obtidas a partir de vários tipos de células mesenquimatosas adultas (MSC): células do cordão umbilical (UC-MSC), medula óssea (“BM-MSC), tecido adiposo (AD-MSC) e vilosidades coriónicas (CV-MSC), como possíveis fontes para a terapêutica de válvulas cardíacas patológicas. As MSC de diferentes fontes, foram isoladas e cultivadas a partir de diferentes dadores saudáveis. Neste estudo foi estabelecido um protocolo independente para as células CV-MSC porque nunca tinha sido realizado neste grupo de investigação. Uma vez que as células CV-MSC são raramente descritas em estudos, esta fonte de MSC foi caracterizada por citometria de fluxo, para possível comparação com outro tipo de MSC que estão bem caracterizadas na literatura. Posteriormente, as MSC foram diferenciadas, isoladas e cultivadas num meio de cultura contendo o factor de crescimento TGF-β1 e BMP4 baseado num protocolo de diferenciação de 7 dias. A diferenciação foi analisada usando quatro marcadores diferentes de músculo liso (α-SMA, SM22α, Calponin and SM-MHC) através de imunofluorescência (IF) e imunohistoquímica (IHC). Para uma caracterização completa do fenótipo antes e depois da diferenciação, foi também realizada por citometria de fluxo uma análise mais extensa usando os marcadores de músculo liso e os outros marcadores utilizados por diferentes estudos na caracterização das MSC. A IF mostra bons resultados uma vez que as células diferenciadas mostraram ser positivas para os marcadores musculares enquanto que as células não diferenciadas não mostraram evidência dos marcadores. Por outro lado, a IHC e a citometria mostraram resultados contraditórios para a diferenciação, uma vez que a expressão não foi consistente nas MSC não diferenciadas e diferenciadas. Os resultados mostram que que as MSC são facilmente acessíveis e podem ser um boa alternativa para tratamento de válvulas cardíacas e apesar de se ter realizado uma análise extensiva, é necessário mais investigação para clarificar alguns pontos poucos claros que ficaram para responder em relação aos marcadores.
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Shih, Ya-Yi, and 施雅譯. "Effects of Magnesium on Osteogenic Differentiation of Mesenchymal Stem Cells." Thesis, 2014. http://ndltd.ncl.edu.tw/handle/11256711819490768327.
Full textAbstract:
碩士國立陽明大學
醫學工程研究所
102
Magnesium plays a vital role in bone metabolism. Related researches have been probed for several decades, but the mechanism was still unclear. In this study, we investigate the influence of magnesium on mesenchymal stem cell (MSC) during osteogenic induction. Since MSC is a provision of cell for cell therapy, with the ability to be induced to osteoblasts in vitro that could offer a platform for osteogenic research. To investigate the influences of magnesium, we utilized higher magnesium to treat murine and human MSC in osteogenic differentiation and verified the efficacy by detecting cell morphology, expression of osteogenic marker genes, alkaline phosphatase secretion, and alizarin red staining. We found that calcium deposition was decreased by magnesium. In order to figure out the possible pathways which might lead to this influence, we hypothesized that blocking of magnesium channels would obstruct the entrance of magnesium during osteogenic induction, so that suppression of mineralization could be eliminated. We found that calcification of cells differentiated under high magnesium was increased after blocking the magnesium channels. This outcome suggested that magnesium channel controls the influx of magnesium which regulates formation of mineralization during the osteogenic process.
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Hsu, Shu-Han, and 許舒涵. "Effect of Hypoxia on Osteogenic Differentiation of Human Mesenchymal Stem Cells." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/5n7asp.
Full textAbstract:
碩士國立陽明大學
生化暨分子生物研究所
97
Previous studies of our lab revealed that mitochondrial metabolism is activated during osteogenic differentiation of human mesenchymal stem cells (hMSCs). In this study, we investigated the effects of oxygen concentration (1% vs. 21% O2) on mitochondrial metabolism during osteogenic differentiation of hMSCs. We found that hypoxia (1% O2) attenuated the activation of mitochondrial metabolism during osteogenic differentiation in several aspects including the decrease in protein levels of subunits of respiratory enzyme complexes, in mitochondrial membrane potential and oxygen consumption rate. However, mitochondrial mass was dramatically increased, suggesting a compensatory increase of mitochondria under hypoxia. On the contrary, anaerobic metabolism was up-regulated as revealed by the increase of glycolytic enzymes and the rate of lactate release. Due to the inability of differentiating osteoblasts to shift into aerobic metabolism under hypoxia, the up-regulation of antioxidant enzymes was less obvious and intracellular ROS levels were decreased more dramatically than that occurred under normoxia. Therefore, the osteogenic differentiation was compromised under hypoxia as indicated by the decrease of osteogenic markers such as cbfa-1, alkaline phosphatase (ALP) activity and the intracellular calcium content. Administration of cobalt chloride, a hypoxia-mimic compound, also suppressed ALP activity and oxygen consumption rate and increased lactate release rate. Moreover, PGC-1���z�nbut not cbfa-1, was slightly recovered after the treatment with echinomycin, a specific HIF-1�� inhibitor. These results suggest that activation of HIF-1�� was involved in the suppression of mitochondrial metabolism and inhibition of osteogenic differentiation of hMSCs. Taken together, our findings suggest that the metabolic shift is attenuated and osteogenic differentiation is compromised for osteogenesis of hMSCs under hypoxia.
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Sun, Li-Yi, and 孫立易. "The In Vitro Proliferation and Differentiation of Human Mesenchymal Stem Cells." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/x58utt.
Full textAbstract:
博士國立交通大學
生物科技學系
99
Mesenchymal stem cells (MSC) can be isolated from almost any tissue of the body have been recognized to constitute a powerful tool in regenerative medicine due to their multi-lineage differentiation ability and their capacity for tissue repair. Pulsed electromagnetic fields (PEMF) have been clinically employed for many years. Despite the clinical success, there are contradictory data concerning the effect of PEMF stimulation on in vitro proliferation of some osteogenic cell lines or primary osteoblasts, so it is a fact that the mechanism underlying how PEMF promotes the formation of bone on cellular level is still not fully understood. For the first time, the effect of PEMF exposure to stem cells was described in this study. We discovered that a shorter lag phase and a higher percentage of G0/G1 phase of bone marrow mesenchymal stem cells (BMMSC) after the first PEMF treatment. Although the surface phenotype, morphology, differentiation potential and growth rates of BMMSC during the exponential growth phase were not significantly affected, the cell densities achieved by PEMF stimulation were significantly higher than those achieved in non-treated conditions. This observation of accelerated growth of BMMSC due to PEMF provides a possible explanation for the clinical success. In addition, according to above-mentioned reports and our new data, our hypothesis was that PEMF not only modifies the osteogenesis of BMMSC but also induces different response of cell proliferation depending on the osteogenic stage of cells. This finding helps us to understand more about the in vitro and in vivo interaction of PEMFs with bone cells. Furthermore, since the morphology and the multi-lineage differentiation potential of the BMMSC were not significantly changed by the PEMF, the stirred bioreactor can combine with microcarriers and PEMF device to be a powerful tool for in vitro BMMSC expansion in the future. Due to its provision of high specific surface area and three-dimensional culture condition, microcarrier culture (MC) has garnered great interest for its potential to expand anchorage-dependent stem cells. This study utilises semi-continuous MC as compared with control plate culture (PC) or serial bead-to-bead transfer MC (MC Bead-T) for in vitro expansion of human MSC including of BMMSC and adipose-drived stem cell (ADSC), and analyses its effects on growth kinetics, cell phenotypes, and the differentiation potential. The maximum cell density and overall fold increase in cell growth were similar between PCs and MCs with similar starting conditions, but the lag phase of BMMSC growth differed substantially between the two growth conditions; moreover, MC cells exhibited reduced granularity and higher CXCR4 expression. Differentiation of BMMSC into osteogenic and adipogenic lineages was enhanced after 3 days in MC. However, MC Bead-T resulted in changes in cell granularity and lower osteogenic and adipogenic differentiation potential. However, the results of MC cells exhibited reduced granularity and higher C-X-C chemokine receptor type 4 (CXCR-4) expressions were not exist in ADSC. Although ADSC could proliferate in MC with serum-free medium with higher growth rate than BMMSC or ADSC in MC, the osteogenic and adipogenic lineages were not enhanced after 3 days in MC. In conclusion, MC could support the expansion of MSC in a scalable three-dimensional culture system, but the different types of MSC or different culture systems would result differential quality of stem cell homing ability and osteogenic and adipogenic differentiation of MSC.
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Cheng, Chien-Ting Tina, and 鄭倩婷. "Characterization of Mesenchymal Stem Cells undergoing Hepatogenic Differentiation using Raman Spectroscopy." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/03663329501899919031.
Full textAbstract:
碩士國立陽明大學
生醫光電工程研究所
99
Raman spectroscopy has been used for the detection of molecular structure and biochemical composition of cells and tissues. This optical technique provides a non-invasive, label-free, fast and simple method to monitor the differentiation process of stem cell by measuring the spectra of molecular vibration. Studies of mesenchymal stem cells (MSCs) derived from bone-marrow have shown their capacity to differentiate into hepatocytes and have strong potential for tissue engineering or cell therapy application. However, conventional methods to evaluate the maturation of hepatogenic differentiation, including staining, immunofluorescence and reverse transcription polymerase chain reaction (RT-PCR), are time-consuming and cell-destructing. In this study, we used Raman spectroscopy to monitor the stages of hepatogenic differentiation in murine MSCs (mMSCs). Raman spectra from mMSCs undergoing hepatogenic differentiation over period of 28 days were examined, and the results demonstrated that there was a significant distinction in Raman spectra intensities during hepatogenesis. The Raman intensity ratio of Tyrosine and Collagen (719/1450cm-1) also increased with the maturation of hepatogenesis. Our findings suggest that Raman spectroscopy may serve as a powerful tool to nondestructively and rapidly define the stages of maturation in live mMSCs undergoing hepatogenesis, and provide further investigation potential in stem cell research.