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Dissertations / Theses on the topic 'Bone marrow cells; Blood; Haematopoietic'
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1
Emambokus, Nikla R. "Applications of the Cre-LoxP technology to the study of megakaryocytes." Thesis, University of Oxford, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.325900.
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Chang, Chao-Hui. "Haematopoietic stem/progenitor cell interactions with the bone marrow vascular niche." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:452da334-bd4e-45c7-a7bd-fc8767d1239c.
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Umbilical cord blood (UCB) is used as a source of haematopoietic stem cells (HSCs) for transplantation but shows defective homing to the bone marrow niche and delayed haematological reconstitution. Following transplantation, HSCs will home to the bone marrow in response to the CXCL12 chemokine, adhere to the bone marrow sinusoidal endothelial cells and then migrate into and lodge in bone marrow niches. In addition to CXCR4, a variety of molecules have been described as being important in these processes. In this laboratory, junctional adhesion molecule-A (JAM-A) was shown to be expressed on human UCB CD133⁺/CD34⁺ cells and regulated by hypoxia. In this thesis, further phenotypic studies show that this molecule is most highly expressed on human CD41a⁺ megakaryocytes and CD14⁺ monocytes/macrophages in UCB. JAM-A was also found to be expressed on all human UCB CD133⁺ cells, which have been shown by others to encompass the HSCs and early myeloid-lymphoid precursors and on the majority of CD34⁺ haematopoietic progenitor cells (HPCs). While it is also present on bone marrow sinusoidal endothelium (BMEC), JAM-A is not detected on cultured bone marrow mesenchymal stromal cells (MSCs). JAM-A blockade, silencing and overexpression experiments showed that JAM-A contributes to, but is not solely responsible for, the adhesion of CD34⁺ haematopoietic progenitor cells to IL-1β activated BMEC-60 cells and fibronectin. Lack of significance in cell migration suggested that JAM-A is more likely to act as an adhesion molecule or a regulator of adhesion rather than as a migratory molecule in such cells. Further functional studies using the proximity ligation assay highlight a potential association of JAM-A with CXCR4 and the adhesion molecules, tetraspanin CD82 and integrin β1. Mechanistic studies were commenced to establish if JAMA could modulate CXCR4 signalling following CXCL12 stimulation, but time constraints prevented these from being completed. These preliminary experiments which were carried out first in the Jurkat cell line lacking JAM-A or transduced to express JAM-A, however, suggest that JAM-A may modulate CXCL12-induced Rap1 phosphorylation and ERK1/2 phosphorylation. The former pathway is important for integrin function and the latter pathway is important in cell adhesion. The results described here, although requiring finalisation, support the hypothesis that JAM-A acts as an adhesion molecule and also may fine tune CXCR4 and integrin mediated functions on human CD34⁺ cells, thereby potentially regulating engraftment of these cells to the bone marrow niche.
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Schuller, Christine Children's Cancer Institute Australia for Medical Research Faculty of Medicine UNSW. "Telomeres and telomerase in haematopoietic progenitors and bone marrow endothelial cells." Publisher:University of New South Wales. Children's Cancer Institute Australia for Medical Research, 2008. http://handle.unsw.edu.au/1959.4/41098.
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In normal human somatic cells, the length of telomeres (chromosomal end structures) decreases with each cell division until reaching a critically short length, which halts cell proliferation and induces senescence. The enzyme telomerase, which functions to maintain telomeres at a length that is permissive for cell division, is expressed in approximately 85% of cancer cells and some stem and progenitor cells, including haematopoietic progenitor cells (HPCs), but not most other normal somatic cells. Previous investigations have demonstrated that ectopic expression of telomerase reverse transcriptase (hTERT) reconstitutes telomerase activity, resulting in telomere elongation in some normal human cell types. However, similar experiments performed in HPCs and endothelial cells have demonstrated a dissociation between the expression of telomerase activity and telomere lengthening. This thesis is focussed on further investigating telomerase-mediated telomere length regulation in HPCs and endothelial cells. Short telomeres in bone marrow and blood leukocytes are associated with the development of disorders linked to bone marrow failure. However, to date a relationship between telomere length and myeloid cell proliferative potential has not been demonstrated. In the current investigations, the telomere length and proliferative potential of 31 cord blood-derived HPCs was determined. Regression analysis revealed a significant correlation between mean telomere length and erythroid cell expansion, but not expansion of other myeloid lineage cells. Another novel finding was that telomerase activity was upregulated in lineage-committed CD34- erythroid cells that were positive for the erythroid-specific lineage marker glycophorin A. It was also functionally demonstrated that telomerase activity facilitates the maximum expansion of erythroid cells. To address the dissociation between telomerase activity and telomere maintenance in BMECs, a dominant negative mutant of the telomere binding protein TRF1, which functions to regulate telomere accessibility, was over-expressed in hTERT-transduced BMECs. These studies showed that telomere access, as well as oncogene expression and exposure to oxidative stress, contribute to telomere length regulation in BMECs. Overall, the results from these investigations demonstrate for the first time the functional significance of telomere length and telomerase activity in ex vivo expansion of erythroid cells, and provide novel insight to the molecular complexity of telomere length maintenance in endothelial cells.
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Robinson, Simon N. "Proliferation regulation of haematopoietic stem cells in normal and leukaemic haematopoiesis." Thesis, University of St Andrews, 1992. http://hdl.handle.net/10023/14965.
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The cellular integrity of the blood is maintained by the cellular output of the haematopoietic stem cell population which produces the specialized precursors and differentiated cells which constitute the blood. The investigation of haematopoietic stem cell behaviour and regulation has been hampered by both the difficulty in their identification and the development of relevant assay systems. The purpose of this investigation was to study the behaviour and regulation of the haematopoietic stem cell population in normal and leukaemic haematopoiesis using an in vitro assay of a primitive haematopoietic precursor. The use of a combination of haematopoietic colony-stimulating factors [interleukin 3 (IL3)/multi-CSF and macrophage colony-stimulating factor (M-CSF/CSF-1)] in semi-solid agar culture of murine haematopoietic tissue, stimulated the proliferation of a haematopoietic colony-forming cell, defined as the "HPP-CFCIL3+CSF-1" population, which was characterized by a high proliferative potential, a multipotency and behavioural and regulatory properties consistent with its being a primitive haematopoietic precursor and possibly a component of the haematopoietic stem cell population. The proportion of the in vitro HPP-CFCIL3+csf-1 population in S-phase in normal murine marrow, was determined to be relatively low at approximately 10%, increasing to approximately 40% in sublethally X-irradiated, regenerating murine marrow and the respective presence of the haematopoietic stem cell proliferation inhibitor and stimulator was demonstrable by the induction of appropriate kinetic changes in the in vitro HPP-CFCIL3+CSF-1 population. In leukaemic haematopoiesis, leukaemic proliferation often occurs at the expense of apparently suppressed normal haematopoiesis. In vitro HPP-CFCIL3+CSF-1 assay of the haematopoietic stem cell proliferation regulators in a number of murine, myeloid leukaemic cell lines, failed to demonstrate either increased levels of the haematopoietic stem cell proliferation inhibitor, or evidence of a direct-acting, leukaemia- associated proliferation inhibitor, however, evidence of a leukaemia- associated impairment of inhibitor and stimulator production was observed and this may be a possible mechanism by which the leukaemic population develops a proliferative advantage over normal haematopoietic tissue. The identification of a possible mechanism of leukaemic progression and suppression of normal haematopoiesis may subsequently allow the development of potentially more effective disease treatment and management regimes. The endogenous haemoregulatory tetrapeptide: Acetyl-N-Ser- Asp-Lys-Pro [AcSDKP, Mr=487 amu] is reported to prevent the G0-G1 transition of haematopoietic stem cells into S-phase. The mechanism of action of AcSDKP and a number of related peptides, was investigated in relation to the stem cell proliferation stimulator and inhibitor. AcSDKP demonstrated no direct haemoregulatory role against the in vitro HPP-CFCIL3+CSF-1 population, which is consistent with reports that AcSDKP is not active against cells already in late G1, or S-phase, rather it appeared to act indirectly by impairing the capacity of the haematopoietic stem cell proliferation stimulator to increase the proportion of the in vitro HPP-CFCIL3+CSF-1 population in S-phase. An apparent impairment of stimulator action may explain the reported AcSDKP-associated 'block' of haematopoietic stem cell recruitment. A putative endogenous AcSDKP precursor and synthetic and degradative enzyme systems have been reported and the possible physiopathological role of AcSDKP in a number of myeloproliferative disorders has been implicated. The potential application of AcSDKP as a 'haemoprotective' agent administered prior to the use of S-phase- specific chemotherapy may be of clinical significance. The in vitro HPP-CFCIL3+CSF-1 assay of a primitive haematopoietic precursor cell population, which may be a component of the haematopoietic stem cell population, should play a significant role in the investigation of haematopoietic stem cell behaviour and regulation in both normal and aberrant haematopoiesis. With the characterization of the mechanism(s) of action of the haematopoietic stem cell proliferation inhibitor and stimulator and the haemoregulatory tetrapeptide AcSDKP, the manipulation of the haematopoietic system to clinical advantage can be envisaged, while the identification of the aberrant regulatory mechanism(s) in haematopoietic dysfunction may allow, the development of more effective disease treatment and management regimes.
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Hodby, Katharine Ailsa. "Investigating the mechanism of bone marrow failure observed in patients with acute myeloid leukaemia." Thesis, Queen Mary, University of London, 2018. http://qmro.qmul.ac.uk/xmlui/handle/123456789/36673.
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Patients with Acute Myeloid Leukaemia (AML) present with the signs and symptoms of bone marrow failure. This finding spans the genetic and phenotypic diversity of the disease. The mechanism which underlies it is poorly understood. This thesis explores the effect of AML on the normal haematopoietic stem cell (HSC) population, using primary human diagnostic bone marrow samples. Previous work from our group suggested that AML induces a state of quiescence in HSCs, producing a differentiation block responsible for the observed cytopenias1. Reversal of this process might offer an alternative to the current treatment of patients with palliative transfusions. I have developed a flow cytometry-based technique to differentiate normal HSCs from leukaemia cells, selecting cells with the CD34+38-ALDHhighCLL1- expression signature. Validation of this technique by assessment of sorted cells by FISH and PCR, suggests it is successful in 73% of AML samples. In a further 25% of samples, it selects for a population significantly enriched for normal HSCs. We used this panel to investigate the concentration of HSCs at AML diagnosis, compared to controls. We show that there is no significant difference between HSC concentration at AML diagnosis (n=38, median [HSC] 2.5 cells/μl) and controls (n=24, median [HSC] 2.4 cells/μl). HSC concentration was not significantly affected by AML karyotype, patient age or gender. However, those patients presenting with a low HSC concentration at diagnosis (< 0.1 HSC/μl) were found to have a significantly worse outcome both in terms of overall and relapse-free survival, an effect apparently independent of age, gender and underlying karyotype. HSC concentration at diagnosis with AML may therefore represent a new independent prognostic marker. We then studied CD33 expression patterns on HSCs within Core Binding Factor mutated AML (n=37) at diagnosis, and found its expression to be significantly lower than on HSCs within controls (n=9) (17% versus 58%, p=0.005). CD33 expression on HSCs from AML samples rose significantly from diagnosis to remission (n=16) (17% to 58%, p=0.0001). This mirrors previous findings from our group using CD34low AML samples, and is, we believe, the first time that the antigenic signature of normal HSCs has been shown to be modified. 6 by the presence of AML. However, an in vitro assay to test the significance of these changes in terms of the cytotoxicity of GO towards normal HSCs did not demonstrate a significant difference between HSC subgroups. Finally, we attempted to investigate the mechanism by which AML might induce HSC quiescence by studying the comparative transcriptomes of HSCs from CD34low AML (n=6) and controls (n=6) by RNA-Seq, using direct cell to cDNA synthesis, followed by amplification. A first attempt resulted in poor quality data, with a significant proportion of reads mapping to non-coding DNA regions. A repeat approach, using utilising immediate RNA extraction post sorting resulted in significantly better quality data Bioinformatics analysis revealed differential expression of 6 genes between the 2 datasets (GNPDA1, ADGRG3, MIAT, WDR31, RP11-244H3.1 and RXFP1). GO enrichment studies using David highlighted a number of pathways including the TNF signalling pathway (p=0.003; after Benjamini-Hochberg correction p=0.51). Validation of these findings by independent qPCR, and functional exploration of enriched signalling pathways remains outstanding.
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Gilmore, William Samuel. "A study of molecules involved in the regulation of the growth of haematopoietic cells and heart muscle cells in culture." Thesis, University of St Andrews, 1986. http://hdl.handle.net/10023/14970.
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The description of the molecular events responsible for the control of cell division and differentiation is, currently, one of the major goals of molecular and cellular biologists. Cell and tissue culture techniques have proved to be promising laboratory tools for the study of the regulators of cellular growth and differentiation. Most cells in culture require specific polypeptide growth factors which are supplied by the addition of a complex biological fluid such as serum or, in some instances, by the cells themselves. These growth factors usually act on their target cell via a membrane receptor to which they bind. The events which occur after the growth factor binds to the membrane receptor have not been fully described, but the phosphorylation of tyrosine residues in certain proteins has been observed. A study was made of the polypeptide growth factors responsible for the growth and differentiation of haematopoietic cells in vitro. These growth factors, called colony - stimulating factors (C.S.F.'s) were prepared from human placental conditioned medium, giant cell tumour conditioned medium and pokeweed mitogen stimulated spleen conditioned medium. A C.S.F. from human placental conditioned medium was radioiodinated and the binding of the labelled growth factor to an anti-C.S.F. antiserum was studied. The binding studies indicated that a purer C.S.F. preparation and/or a more specific antiserum was necessary in order to establish a radioimmunoassay for C.S.F. The C.S.F.'s from giant cell tumour conditioned medium were purified by ultrafiltration, hydrophobic - interaction chromatography, gel filtration and thiolpropyl - sepharose 6B chromatography. Two peaks of biological activity were observed on gel filtration. One of these peaks gave an apparent MW of 63,000 and the other peak gave an apparent MW of 30,200. The C.S.F. from pokeweed mitogen stimulated spleen conditioned medium was labelled with peroxidase and the binding of the labelled-C.S.F. to bone marrow cell membranes studied. The labelled-C.S.F. bound to the membranes and the binding exhibited a linear relationship with membrane protein content. Also a defined growth medium for chick embryonic heart cells was developed. These cells were observed to differentiate from primitive foetal cells into mature "adult-type" cells. The cells grew as a monolayer, had spontaneous activity and were seen to beat.
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Nicol, Andrew. "Analysis and in-vitro expansion of cord blood haemopoietic stem cells for transplantation." Thesis, University of Bristol, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.337265.
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Prewitz, Marina. "Decellularised extracellular matrices as instructive microenvironments for bone marrow derived stem cells." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2012. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-86334.
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The regenerative potential of adult stem cell populations within the human body bears great promises for their use in regenerative medicine. The bone marrow (BM) harbours two different types of adult stem cells, haematopoietic stem and progneitor cells (HSPCs) and multipotent mesenchymal stromal cells (MSCs), which are tightly regulated in their distinct anatomically defined niches by multiple cues such as cytokines, cell-cell contacts, the extracellular matrix (ECM) and the physical microenvironment. The ex vivo expansion of these cells for applications in regenerative therapies is of great interest and several biomaterial approaches attempt to mimic the natural BM niche and its components to control stem cell maintenance and differentiation. However, as of now the complexity of such stem cell niches is hard to recapitulate. Towards this goal, this work was focussing on the ECM environment of BM stem cells and was set out to engineer improved in vitro culture systems. MSC themselves are one of the most important cell types within the BM that secrete and construct ECM-networks and thereby shape the microenvironment of the residing cells. The potential of primary human BM-MSC to secrete ECM in vitro has been exploited to generate niche-like ECM surrogates in a robust and versatile format. Application of decellularisation regimes allowed the fabrication of complex matrices which demonstrated suprastructural, compositional and physicochemical properties compareable to those of the native BM-ECM environment. Reliable stability and reproduciblity was achieved by a dedicated procedure of maleic anhydride co-polymer-mediated covalent binding of fibronectin and subsequent anchorage of cell-secreted ECM molecules. As a result of the high reproducibility, a complete proteomic register of ECM molecules was obtained in combination with determining the complex fibrillar and soft gel-like characteristics of MSC-derived matrices. Based on the established BM niche-like substrate, the impact of extracellular matrices on MSC and HSPC ex vivo behavior has been explored. Both cell types demonstrated strong adhesion to ECM substrates and depicted a changed cellular morphology upon contact with native ECM structures compared to standard culture substrates or simple ECM protein coatings, indicating an intense interplay between the cell and the microenvironment. MSC that re-grew into their own matrices have shown advantageous proliferation and cytokine secretion levels as well as enhanced differentiation intensity (upon differentiation induction) compared to MSC that were cultured on less complex substrates. Similarly, HSPC were also instructed for enhanced expansion on MSC-derived matrices without exhaustion of stem cell-marker expressing progenitor cells. The efficiency of these matrices was related to their ability to mimic the native composite suprastructure, ligand nano-topography, molecular composition and physical properties of natural BM ECM environments. The data obtained within this thesis set the ground for a more rational design of artificial stem cell niches with defined and distinct properties, offering exciting options for the in-depth analysis and understanding of stem cell regulation by exogenous cues.
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Taylor, Alan. "The role of leukaemia inhibitory factor and a leukaemic associated inhibitor in the control of the proliferation of haematopoietic stem cells." Thesis, University of St Andrews, 1996. http://hdl.handle.net/10023/14962.
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Activities associated with, or interacting with, leukaemic cell populations were assayed for the ability to influence in vitro haematopoiesis. The first of these, the glycoprotein leukaemia inhibitory factor (LIF), has a role in aspects of murine, non human primate and human haematopoiesis. It is thought to be particularly important in the development of megakaryocytes and is also known to induce the terminal differentiation of certain leukaemic cell lines. LIF was assayed both for direct and indirect effects on the proliferation of haematopoietic precursor cell populations in vitro. As a direct acting agent in semi-solid agar culture of haematopoietic cell populations derived from normal bone marrow or 15 day foetal liver, LIF was unable to support colony formation. In cultures of cells derived from normal bone marrow stimulated with single, or combinations of, growth factors, the addition of LIF had no statistically significant effect on the level of colony formation. In cultures of cells derived from foetal liver, stimulated with particular growth factor combinations (medium conditioned by the Wehi3B leukaemic cell line + medium conditioned by the lung fibroblast cell line, L929); GM-CSF + M-CSF; IL-la + IL-3 + M-CSF), LIF, was shown to decrease the level of colony formation. LIF did not directly alter the proportion of the population in DNA synthesis in cell populations derived from normal femoral marrow, 15 day foetal liver or y- irradiated femoral marrow. As an indirect acting agent LIF failed to block the synthesis of a stem cell stimulator, or it's action, on a population of high proliferative potential colony forming cells derived from normal femoral marrow, cloned in the presence of Wehicm+L929cm. (HPP-CFC (Wehicm + L929cm)) LIF's actions on clones of a murine myeloid leukaemia (SA2JMB1) were also assessed. LIF had no statistically significant effect on colony formation or the level of DNA synthesis in populations of SA2JMB1 leukaemic cells. A second group of associated activities was produced by the X- irradiation induced murine myeloid leukaemia (SA2JMB1). Medium conditioned by the leukaemic cells was assayed in vitro both for direct and indirect effects on the proliferation of haematopoietic cells derived from femoral marrow. As a direct acting agent in 7 and 14-day semi-solid agar culture of femoral marrow, leukaemic conditioned medium alone stimulated limited colony formation. In 7 and 14 day cultures stimulated with single and combinations of specific colony stimulating factors: (rmGM-CSF, rhM-CSF, rhIL-1a) a significant increase in colony number was noted in all cases when cultures were supplemented with leukaemic conditioned medium. SA2JMBlcm was shown to support the proliferation of an IL-3 dependent cell line (FDCP-A4 cells). The colony enhancing ability of SA2JMBlcm was shown to be blocked by pretreatment with antibodies to IL-3. This suggested that SA2JMB1 conditioned medium contained IL-3 or an IL-3 like activity, as one of its components. The conditioned medium failed to directly alter the level of DNA synthesis in a population of HPP-CFC (Wehicm+L929cm) derived from normal bone marrow or y- irradiated bone marrow. As an indirect acting agent the conditioned medium did block the action of a stem cell proliferation stimulator on normal bone marrow derived HPP-CFC (Wehicm+L929cm). This leukaemia associated activity was shown to be larger than 50KD, sensitive to heat treatment and able to act in a different manner to the stem cell inhibitor MIP-1-a. Thus this novel activity may be important in blocking stimulator action in haematopoietic stem cells and thus contribute to the haematopoietic insufficiency seen in leukaemia.
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Novitzky, Nicolas. "Interactions between the haematopoietic stem cell and the myeloid microenvironment in aplastic anaemia." Doctoral thesis, University of Cape Town, 1993. http://hdl.handle.net/11427/24943.
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In patients with aplastic anaemia that respond to immunosuppressive therapy, quantitative, morphological and functional haematologic derangement have been reported. To explain these findings, abnormalities in the marrow stroma or the stem cell have been postulated. To define the relative contribution of each of the latter, the integrity of the bone marrow from sixteen patients that responded to anti-lymphocyte globulin and high dose methyl prednisolone was compared to normal individuals. Bone marrow mononuclear cells were divided into two fractions. From the first, stroma was cultured in aMEM containing 12.5% of both horse and foetal calf serum and 10-5 M hydrocortisone at 37° C in 5% CO2 in 90% humidity. The medium was changed weekly. Upon confluence, these stromal layers were studied morphologically and with cytospin preparations stained with Sudan black, 0 red oil, alkaline and acid phosphatases. The remainder was monocyte and lymphocyte depleted, CD 34+ progenitors were selected with paramagnetic beads and the population morphologically and immunophenotypically defined. To determine the functional status, control or patient CD 34+ progenitors, were suspended for two hours on normal or aplastic stroma for adherence to take place. The non-adhesive fraction was decanted by standardised washing and cultured for fourteen days in the presence of PHA-conditioned medium in the CFU-gm assay. Strama-adherent progenitors were covered with 0.3% agar and cultured for five days. Aggregates with more than twenty cells were scored (CFU-bl). The remaining CD 34+ cells were cultured in the mixed colony assay with combinations of recombinant cytokines belonging to the G protein super-family and the tyrosine kinase group in dose response studies. Light density cells from patients with treated aplasia contained significantly fewer CD 34+ cells than those present in the control suspensions (mean 0.65%, SD 0.35% vs 1.62%, SD 1.4%; p= 0.002). Normal and aplastic stroma became confluent at three and four weeks. There was no difference on the morphology or the cytochemical stains between the two groups. Functionally, aplastic bone marrow stroma supported CFU-bl formation no differently from normal layers. However, CD 34+ precursors from the patients cultured on control stroma resulted in significantly fewer CFU-bl (p= 0.0002,) and CFU-gm (p= 0.0009). This work provides original evidence supporting the reduced clonogenicity of the corresponding populations of CFU-bl from patients with aplasia is unrelated to attachment to the stroma, but intrinsic to the CD 34+ cells. Moreover, this study shows for the first time that exposure of these progenitors to growth factors belonging to the G protein and tyrosine kinase receptor families have defective responses, correctable only at supra physiological concentrations, while effects on combinations containing c-kit ligand, appear preserved. Following immunosuppressive therapy, the bone marrow is repopulated by a hypoproliferative progenitor cell population which responds suboptimally to physiological cytokine stimulation. This suggests that abnormal interactions between receptors and their ligands or alterations in the signal transduction for cell division by the cytokines belonging to the G superfamily lead to suboptimal growth.
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Chalmers, Isobel Margaret Hood. "Cellular and cytokine profile of cord and adult blood mononuclear cells." Thesis, University of Aberdeen, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.324413.
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Umbilical cord blood (CB) has been used as a source of haemopoietic stem cells in both related and unrelated bone marrow transplantation and in both settings there appears to be a reduced incidence of graft-versus-host disease (GVHD) when compared to bone marrow transplantation. The aim of this study was to perform a phenotypic and cellular analysis of both cord and adult mononuclear cells in vitro in order to identify any immunological characteristic that could account for the apparent reduced incidence of GVHD observed, in vivo. For this, the cell surface phenotype, allogeneic cellular responses, cytokine secretion and expression of co-stimulatory molecule CD40L was studied. The phenotypic analysis was carried out by determining the proportion of cells expressing the CD4, CD8, CD16, CD19, CD45RA and CD45RO markers using 3 colour flow cytometric analysis. The results showed that in cord blood mononuclear cells, there was an increased number of cells expressing CD19+ whereas the number of cells expressing CD8+ was decreased compared to adult blood mononuclear cells (ABMNCs). In contrast the number of cells expressing CD4+ and CD16+ was similar in both cord blood mononuclear cells (CBMNCs) and ABMNCs. Further phenotypic analysis confirmed that cord blood contained primarily 'unprimed' T cells expressing CD45RA, while adult peripheral blood lymphocytes express mainly the CD45R 'memory' phenotype. The primary and secondary allogeneic responses of cord and adult mononuclear cells were assessed using the standard mixed lymphocyte reaction (MLR) and the primed lymphocyte test (PLT), respectively. These results showed that there was no significant difference in primary MLR responses of CBMNCs and ABMNCs. In contrast, CBMNCs had a decreased ability to mount a secondary proliferative response compared to ABMNCs. However this impaired ability to respond could be overcome by the addition of exogenous IL-2 to the cultures. The secretion of cytokines such as IL-2, IL-4, γIFN and TNF-α and the expression of CD40L on PMA- and ionomycin-activated CBMNCs and ABMNCs was also analysed by 3 colour flow cytometry. These results showed that upon activation CBMNCs produced reduced levels of cytokines and had reduced expression of CD40L. It is likely that the reduced cytokine production and CD40L expression observed is due to the predominance of CD45RA+ cells in CB compared to AB. In summary, this study has shown that CB and AB have different phenotypic and functional characteristics in vitro which may well explain the reduced incidence of GVHD observed in cord blood transplantation.
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Talamas, Ugalde Lucet Vanessa. "Effects of platelet rich plasma on marrow stromal cells differentiation seeded on three dimensional scaffolds." To access this resource online via ProQuest Dissertations and Theses @ UTEP, 2008. http://0-proquest.umi.com.lib.utep.edu/login?COPT=REJTPTU0YmImSU5UPTAmVkVSPTI=&clientId=2515.
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MacDonald, Elizabeth Steward. "The influence of equine bone marrow derived stem cells on the response of cultured peripheral blood mononuclear cells to endotoxin." Thesis, Virginia Tech, 2015. http://hdl.handle.net/10919/76722.
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Endotoxemia is a major cause of morbidity and mortality in horses. The presence of large amounts of circulating endotoxin inititates a number of cell signaling pathways leading to a systemic inflammatory response. Activation of these pathways causes the release of a number of pro- and anti-inflammatory mediators. An overwhelming release of these mediators leads to the development of clinical signs associated with endotoxemia. Treatment options are limited mostly to supportive care at this time. Mesenchymal stem cells (MSCs) have been shown to have anti-inflamamtory and immune modulatory effects that may have some benefit for the treatment of horses with endotoxemia.
To evaluate the effect of equine MSCs on the response to endotoxin challenge, the study was performed on two different stem cell lines with peripheral blood mononuclear cells (PBMCs) used as controls. After stimulation with endotoxin, secretion of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), interleukin-10 (IL-10), and interferon gamma (IFN-γ) were determined by ELISA. The immunogenic properties of MSCs were assessed with a one-way mixed lymphocyte reaction. In addition, the ability of MSCs to alter production of cytokines from stimulated PBMCs was assessed.
TNF-α was not produced by MSCs when compared to PBMCs (p = < 0.001). There was no significant difference between MSCs and PBMCs in the production of IL-6. IL-10 production was significantly different (p = <0.001) at 6 and 12 hours with MSCs producing more than PBMCs in one stem cell line only. MSCs did not stimulate proliferation of PBMCs. Co-incubation of MSCs with PBMCs decreased the production of TNF-α in both stem cell lines although it was not statistically significant (p = 0.4 and 0.9) at either time point. IL-6 secretion was suppressed at twelve hours with co-incubation. IL-10 production was increased with co-incubation in one stem cell line. MSCs secrete soluble factors that can alter PBMC cytokine production and they do not appear to be immunostimulatory. These findings have potential implication for treatment of equine inflammatory conditions.Master of Science
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Philpott, Nicola Jane. "Apoptosis and the pathogenesis of aplastic anaemia." Thesis, University of Oxford, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.359983.
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Richards, Adrian John. "High gradient magnetic separation using ordered wire filters for the separation of human blood and bone marrow cells." Thesis, University of Southampton, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.390731.
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Waskow, Claudia, Bonin Malte von, Martin Wermke, Cosgun Kadriye Nehir, Christian Thiede, Martin Bornhauser, and Gerard Wagemaker. "In Vivo Expansion of Co-Transplanted T Cells Impacts on Tumor Re-Initiating Activity of Human Acute Myeloid Leukemia in NSG Mice." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2016. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-191633.
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Human cells from acute myeloid leukemia (AML) patients are frequently transplanted into immune-compromised mouse strains to provide an in vivo environment for studies on the biology of the disease. Since frequencies of leukemia re-initiating cells are low and a unique cell surface phenotype that includes all tumor re-initiating activity remains unknown, the underlying mechanisms leading to limitations in the xenotransplantation assay need to be understood and overcome to obtain robust engraftment of AML-containing samples. We report here that in the NSG xenotransplantation assay, the large majority of mononucleated cells from patients with AML fail to establish a reproducible myeloid engraftment despite high donor chimerism. Instead, donor-derived cells mainly consist of polyclonal disease-unrelated expanded co-transplanted human T lymphocytes that induce xenogeneic graft versus host disease and mask the engraftment of human AML in mice. Engraftment of mainly myeloid cell types can be enforced by the prevention of T cell expansion through the depletion of lymphocytes from the graft prior transplantation.
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Kylmäoja, E. (Elina). "Osteoclastogenesis from bone marrow and peripheral blood monocytes:the role of gap junctional communication and mesenchymal stromal cells in the differentiation." Doctoral thesis, Oulun yliopisto, 2018. http://urn.fi/urn:isbn:9789526221045.
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Abstract Osteoclasts are multinuclear bone degrading cells differentiated from monocytes which can be isolated from bone marrow and peripheral blood. Complex signaling between osteoclast precursors and other bone cells, such as mesenchymal stromal cells (MSC) occurs during the differentiation. Gap junctional communication (GJC) is one of the mechanisms in the cell fusion. GJC can be modulated with several substances such as the specific GJC stimulators, antiarrhythmic peptides (AAP). Due to their promising clinical value in the treatment of cardiac disorders, the effects of AAPs in cardiac tissue are studied extensively. This study was conducted in order to investigate the roles of GJC and AAPs in bone cell cultures. Further, the contribution of the MSCs on the effects of AAPs was studied along with comparison of two types of osteoclastogenesis cultures with differing quantities of MSCs. GJC in osteoclastogenesis was studied with both GJC inhibitors and stimulators in mouse monocyte line RAW 264.7 cells and primary cultures with bone marrow hematopoietic cells. The following studies were made with human monocytes from peripheral blood and bone marrow where the effects of AAP10 were investigated in normal and acidic environments. In addition, comparison of osteoclastogenesis from bone marrow and peripheral blood monocytes was carried out in in vitro cell cultures on bovine or human bone slices. The cells were analyzed with regard to multinuclearity, bone resorption and the expression of several osteoclast markers. The results show that GJC is utilized in osteoclastogenesis, but it is not indispensable. GJC in monocytes can be stimulated with the AAPs during osteoclastogenesis, but the effects depend on the culture conditions as well as on the presence of MSCs in the culture. The AAPs can also activate the MSCs leading to indirect regulation of osteoclastogenesis, as the MSCs produce several molecules affecting the differentiation. Further, monocytes from peripheral blood showed increased potential for osteoclastogenic differentiation compared to bone marrow derived monocytes. This can be explained by the presence of the osteoclastogenesis-controlling MSCs in the bone marrow culture, while the peripheral blood cultures contain only few of these cells and thus lack their regulatory effectsTiivistelmä Osteoklastit ovat monitumaisia luuta hajottavia soluja, jotka ovat erilaistuneet monosyyteistä. Monosyyttejä voidaan eristää luuytimestä tai perifeerisestä verestä. Erilaistumisen aikana osteoklastien esiastesolujen sekä muiden luusolujen, kuten mesenkymaalisten stroomasolujen (MSC) välillä tapahtuu monimutkaista signalointia. Aukkoliitoskommunikointi (GJC) on eräs solufuusiossa tapahtuvista mekanismeista. GJC:tä voidaan muunnella useilla aineilla, esimerkiksi spesifisillä stimulaattoreilla, antiarytmisillä peptideillä (AAP). AAP-yhdisteiden vaikutuksia on tutkittu laajalti sydänkudoksessa johtuen niiden lupaavista kliinisistä ominaisuuksista sydänperäisten oireiden hoidossa. Tämän tutkimuksen tarkoituksena oli selvittää GJC:n ja AAP-yhdisteiden roolia luusoluviljelmissä. Lisäksi tutkittiin MSC-solujen osallistumista AAP-yhdisteiden vaikutuksiin sekä vertailtiin kahta erilaista osteoklastogeneesiviljelmää, joissa oli eri määrä MSC-soluja. GJC:tä osteoklastogeneesissä tutkittiin sekä sitä estävillä että stimuloivilla yhdisteillä hiiren monosyyttilinjan RAW 264.7 -soluissa sekä luuytimen hematopoieettisten solujen primääriviljelmissä. Seuraavat tutkimukset tehtiin ihmisen luuytimen ja perifeerisen veren monosyyteillä, ja niissä selvitettiin AAP10-yhdisteen vaikutuksia fysiologisissa sekä happamissa olosuhteissa. Lisäksi vertailtiin luuytimen ja perifeerisen veren monosyyttien osteoklastogeneesiä. In vitro -soluviljelmät tehtiin naudan tai ihmisen luulastujen päällä, ja soluista analysoitiin monitumaisuus, luun resorptio sekä useiden osteoklastimarkkereiden ilmentyminen. Tulokset osoittavat, että GJC:tä hyödynnetään osteoklastogeneesissä, mutta se ei ole korvaamaton mekanismi. GJC:tä voidaan stimuloida AAP-yhdisteillä osteoklastogeneesin aikana, mutta vaikutukset riippuvat viljelyolosuhteista sekä MSC-solujen läsnäolosta. AAP-yhdisteet voivat aktivoida myös MSC-soluja johtaen osteoklastogeneesin epäsuoraan säätelyyn, kun MSC-solut tuottavat useita erilaistumiseen vaikuttavia molekyylejä. Lisäksi perifeerisen veren monosyyteillä havaittiin korkeampi osteoklastogeeninen erilaistumispotentiaali verrattuna luuytimen monosyytteihin. Tulokset voidaan selittää osteoklastogeneesiä säätelevien MSC-solujen läsnäololla luuydinviljelmissä, kun taas perifeerisen veren monosyyttiviljelmissä näitä soluja on vain vähän, jolloin myös niiden säätelyominaisuudet puuttuvat
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Onodera, Rie. "Bone marrow mononuclear cells versus G-CSF-mobilized peripheral blood mononuclear cells for treatment of lower limb ASO: pooled analysis for long-term prognosis." Kyoto University, 2010. http://hdl.handle.net/2433/131879.
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He, Q. "Peripheral blood derived multi-potent mesenchymal stromal cells (MSCs) in rats : their differentiation and characteristic comparison with bone marrow derived MSCs." Thesis, Queen's University Belfast, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.431597.
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Pacitti, Andrew P. "Isolation of Mesenchymal Stem Cells Derived from Adult Bone Marrow and Umbilical Cord Blood and Their Potential to Differentiate into Osteoblasts." VCU Scholars Compass, 2006. http://scholarscompass.vcu.edu/etd/728.
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The demand for treatment strategies of musculoskeletal tissues is continuously growing, especially considering the increasing number of elderly people with degenerative diseases of the skeletal system. Despite major strides in the field of bone regenerative medicine during the years, current therapies, such as bone grafts, still have several limitations. Multipotent stem cells, such as mesenchymal stem cells (MSCs) are promising candidates for tissue repair because of their differentiation potential and their capacity to undergo extensive replication. However, isolating a homogeneous population of MSCs from multiple sources is an area that needs to be addressed. Also, the knowledge regarding the mechanisms and pathways that lead to the final osteogenic differentiation is still scarce. The following research is a feasibility study on a new isolation technique developed by our lab. The major focus of the research will be the isolation and characterization of mesenchymal stem cells from both adult bone marrow and umbilical cord blood using a novel isolation method based on immunodepletion. Furthermore we will look at the potential of these isolated MSCs to differentiate into mature, bone producing osteoblasts. The results of the studies showed that our novel isolation method allowed proliferation of a homogeneous MSC population. Our irnrnunodepleted MSCs were 99% double positive for antibodies CD44 and CD105 which are highly specific for multipotent MSCs while cells isolated using the plastic adherence method were only 43% double positive for the two MSC-specific markers. Homogeneous MSCs were derived from both adult bone marrow and umbilical cord blood using our isolation method. Utilizing the techniques of confocal microscopy, von Kossa staining, and RTPCR we also show that MSCs, upon stimulation with osteogenic supplements, differentiate into osteoblasts capable of being used for bone tissue engineering applications.
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Waskow, Claudia, Bonin Malte von, Martin Wermke, Cosgun Kadriye Nehir, Christian Thiede, Martin Bornhauser, and Gerard Wagemaker. "In Vivo Expansion of Co-Transplanted T Cells Impacts on Tumor Re-Initiating Activity of Human Acute Myeloid Leukemia in NSG Mice." Public Library of Science, 2013. https://tud.qucosa.de/id/qucosa%3A28097.
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Human cells from acute myeloid leukemia (AML) patients are frequently transplanted into immune-compromised mouse strains to provide an in vivo environment for studies on the biology of the disease. Since frequencies of leukemia re-initiating cells are low and a unique cell surface phenotype that includes all tumor re-initiating activity remains unknown, the underlying mechanisms leading to limitations in the xenotransplantation assay need to be understood and overcome to obtain robust engraftment of AML-containing samples. We report here that in the NSG xenotransplantation assay, the large majority of mononucleated cells from patients with AML fail to establish a reproducible myeloid engraftment despite high donor chimerism. Instead, donor-derived cells mainly consist of polyclonal disease-unrelated expanded co-transplanted human T lymphocytes that induce xenogeneic graft versus host disease and mask the engraftment of human AML in mice. Engraftment of mainly myeloid cell types can be enforced by the prevention of T cell expansion through the depletion of lymphocytes from the graft prior transplantation.
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Krüger-Almerén, Anders. "RP59, a novel stem cell protein and mapping of its expression /." Stockholm, 2002. http://diss.kib.ki.se/2002/91-7349-246-9.
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VIEIRA, DANIEL P. "Avaliação dos efeitos da inibição de cadeias imflamatórias e da suplementação exógena de CXCL 12 na hematopoiese de modelos experimentais expostos a doses letais ou subletais de radiação gama." reponame:Repositório Institucional do IPEN, 2007. http://repositorio.ipen.br:8080/xmlui/handle/123456789/11618.
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Tese (Doutoramento)
IPEN/T
Instituto de Pesquisas Energéticas e Nucleares - IPEN-CNEN/SP
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Chasty, Richard. "Analysis of the differential responsiveness of CD34 enriched mononuclear cells from normal bone marrow, and chronic granulocytic leukaemia peripheral blood, to the myeloinhibitory molecule macrophage inflammatory protein-lα (LD78)." Thesis, King's College London (University of London), 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.265852.
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Ozzetti, Priscila Aparecida. "Hematopoese em serpentes Oxyrhopus guibei (Hoge & Romano, 1978) (Ophidia: Dipsadidae): caracterização morfológica, citoquímica e ultraestrutural." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/41/41135/tde-29082013-095922/.
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A hematopoese nas serpentes inicia-se durante a embriogênese e através dos processos de alterações da vida fetal. A primeira atividade eritropoética é extraembrionária, a partir de células mesodérmicas do saco vitelínico e durante o desenvolvimento embrionário torna-se intraembrionário. Em serpentes recém-nascidas e adultas, o principal foco hematopoético ocorre na medula óssea. O objetivo deste estudo foi caracterizar os diferentes estágios de maturação das células sanguíneas da serpente O. guibei, com base em estudos de microscopia, reações citoquímicas e aspectos ultraestruturais. Fragmentos de vértebras de serpentes recém-nascidas e adultas (n=11) foram coletados para obtenção da medula óssea que foi fixada em formol cálcio ou Bouin e processadas para histologia de rotina. Cortes histológicos, imprint de medula óssea e esfregaços sanguíneos foram corados com Rosenfeld, hematoxilina e eosina ou azul de metileno. As reações citoquímicas realizadas foram ácido periódico de Schiff (PAS), azul de toluidina (AT), Sudan Black B (SBB), benzidina peroxidase (PA) e fosfatase ácida (FA). Para a microscopia electrónica de transmissão (TEM), a medula óssea foi fixada em paraformaldeído a 4% + glutaraldeído 2%, em tampão Tyrode, pós fixados em tetróxido de ósmio a 1% e embebidos em resina Epon 812. A maior parte das células progenitoras de células sanguíneas foram identificadas em focos hematopoiéticos ativos na medula óssea de vértebras e costelas. As linhagens azurofílicas e linfoides foram morfologicamente similares aos de outros répteis. A linhagem granulocítica foi classificada como mieloblasto, promielócito, mielócito e granulócito maduro. Mielócitos podem ser diferenciados em basófilos com grânulos grandes, redondos e eletrondensidade homogênia ou heterófilos, com grânulos de tamanhos e formas variadas na análise MET. TB e PAS foram positivos nos grânulos imaturos e maduros basófilos. Por outro lado, heterófilos e azurófilos mostraram reação fortemente positiva para lípidos de SBB e BP. FA foi encontrado nos azurófilos em várias fases de maturação. As diferentes fases de eritrócitos foram classificadas como: proeritroblasto, eritroblasto basófilo, eritroblasto policromático, proeritrócitos e eritrócitos maduros. Os trombócitos apresentaram positivadade para PAS. As características dos trombócitos maduros e imaturos foram definidas através da TEM apresentando corpos densos, grânulos alfa, microtúbulos e sistema canalicular aberto. Concluindo, a medula óssea das costelas e vértebras é um importante foco hematopoético nas serpentes O. guibei recém-nascidas e adultas. Os aspectos morfológicos, citoquímicos e ultraestruturais são úteis para identificar e caracterizar os diferentes estágios de maturação das células sanguíneasHematopoiesis in snakes begins during embryogenesis and the process changes through fetal life. The first erytropoietic activity is extraembryonic from mesoderm cells of the yolk sac and during the embryonic development it becomes intraembryonic. In newborn and adult snakes, the main site of hematopoiesis occurs in the bone marrow. The aim of this study was to characterize different stages of blood cells maturation of O. guibei snakes, based on microscopic studies including cytochemical stains and ultrastructural features. Fragments of vertebrae of newborn and adult snakes (n= 11) were collected to obtain bone marrow that was fixed in Bouin or formol calcium and processed routinely for histology. Tissue sections, imprint of bone marrow and blood smears were stained with Rosenfeld, hematoxylin and eosin or methylene blue. The cytochemical reactions performed were periodic acid-Schiff (PAS), toluidine blue (AT), sudan black B (SBB), benzidine peroxidase (BP) and acid phosphatase (FA). For transmission electron microscopy (MET), bone marrow was fixed in paraformaldehyde 4% + glutaraldehyde 2% in Tyrode buffer, postfixed in 1% osmium tetroxide and embedded in Epon 812 resin. Most of progenitors of blood cells were identified in the active hematopoietic focus in bone marrow of vertebrae and ribs. The azurophilic and lymphocytic series were morphologically similar to those of other reptiles. Granulocytic lineage was classified as myeloblast, promyelocyte, myelocyte and mature granulocytes. Myelocyte can be differentiated into basophils, with large, round and electrondensity homogeneous granules or heterophils, with varied size and shapes granules in the TEM analysis. AT and PAS were positive in the immature and mature basophils granules. On the other hand, heterophils and azurophils showed strong positive reaction for lipids staining of SBB and BP. FA was found on azurophils in various stages of maturation. The different stages of erythrocytes were classified as: proerythroblast, basophilic erythroblast, polychromatic erythroblast, proerythrocyte and mature erythrocytes. Thrombocytics cells showed PAS positive. The characteristics of mature and immature thrombocytes were defined using TEM identifying the dense bodies, alpha granules, microtubules and open canalicular system. Concluding, the rib or vertebral bone marrow is an important hematopoietic site in the newborn and adult O. guibei snakes. The morphologic, cytochemical and ultrastructural characteristics are useful to identify and characterize different stages of maturation of blood cells
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Neves, Soraya Andreassa. "Banco de sangue de cordão umbilical e placentário: proposta de sistema híbrido brasileiro." Universidade Tecnológica Federal do Paraná, 2012. http://repositorio.utfpr.edu.br/jspui/handle/1/305.
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CAPES: programa de Demanda Social (DS)O sangue de cordão umbilical, que age como um elo de ligação entre a placenta e o bebê durante a gestação, pode ser necessário anos após o nascimento. As células-tronco ali contidas, são utilizadas em substituição aos métodos convencionais de transplantes que empregam células-tronco originárias da medula óssea e no tratamento de doenças hematológicas e oncológicas. Esta dissertação tem como objetivo propor um sistema híbrido de células-tronco do sangue de cordão umbilical e placentário com foco no cenário brasileiro. No decurso da dissertação apresentam-se as regulamentações e relatórios de produção sobre os bancos de armazenamento nacionais. Para fins comparativos, são abordados as regulamentações e modelos específicos de dois bancos de armazenamento internacionais: argentino e inglês. Através da prospecção de cenários, evidenciam-se os benefícios em relação ao aumento de inventário das células-tronco do sangue de cordão umbilical e placentário para doação, considerando a operacionalização do sistema híbrido. Com a implantação desse sistema, busca-se estabelecer uma integração entre os bancos de armazenamento públicos e privados, visões empresariais aparentemente inconciliáveis, mas que de forma incondicional e expressiva respeitam e honram a vida.
The umbilical cord, which acts as a communication link between the placenta and the baby during the gestation, can still be necessary years after birth. The stem cells of the umbilical cord blood can be used alternatively in transplants, replacing conventional methods using bone marrow stem cells for hematological and oncological treatments. This work presents a proposal for a hybrid system of umbilical cord blood stem cells concerning the actual Brazilian scenario. During the work development it was described regulations and production reports from national storage banks. Two other countries types and regulation storage banks are also presented for comparison: Argentinean and British ones. Through the exploration of scenarios, it is showed the benefits in relation to increase inventory of stem cells from umbilical cord blood for donation, whereas the running of the hybrid system. With the deployment of this system, we seek to establish integration between the private and public storage banks, an entrepreneurial view usually seen as irreconcilable, but with unconditionally and expressive respect and honor for life.
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Pepperell, Emma E. "The regulation of stem cell engraftment." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:873a14b9-6c7b-4643-bb34-4e1679d4f734.
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The engraftment of haemopoietic stem/progenitor cells (HSPCs) from umbilical cord blood (UCB) into adult recipients, although advantageous in terms of sourcing units, the decreased need to match donor and recipient and reduced risk of graft versus host disease (GvHD), is delayed compared to grafts using HSPCs from mobilised peripheral blood (MPB) or bone marrow (BM). One reason for this is the limited number of HSPCs (CD34+/CD133+ cells) in a unit of UCB compared to MPB or BM. The CXCR4-CXCL12 axis is widely recognised as a key player in the bone marrow homing, retention, and engraftment of HSPCs. The aim of this thesis was to investigate whether the engraftment of HSPCs from UCB into the bone marrow could be improved. Firstly, a novel in vitro 3D time-lapse chemotaxis assay to assess the homing capacity of human UCB CD133+ HSPCs, towards the chemokine CXCL12 was developed. One advantage of this assay was that it distinguished cell chemotaxis from chemokinesis and allowed these parameters to be quantified. Human UCB CD133+ HSPC chemotaxis towards CXCL12 was inhibited by the CXCR4 antagonist, AMD3100. Importantly, the presence of CXCL12 or AMD3100 had no affect on cell chemokinesis. To complement the in vitro chemotaxis assay, a short term in vivo homing assay in NSG mice was successfully established. The effect of siRNA silencing of the CXCR4 co-receptor, CD164, which is also expressed on CD133+ HSPCs, on cell migratory and homing ability was investigated. CD164 knock-down using siRNA in human UCB CD133+ HSPCs did not demonstrate an effect on homing to NSG bone marrow in vivo or chemotaxis to CXCL12 in vitro. However, homing to NSG mouse spleen was significantly reduced in cells silenced for CD164. Following this, an 8 day HSPC expansion system using nanofibre scaffolds (Nanex) and differing cytokines was investigated. These serum and feeder free conditions yielded a significant expansion of cells that retained CD133+CD34+ expression and their in vitro chemotactic ability to CXCL12. Time constraints did not permit the engrafting ability of these cells to be analysed in an in vivo HSC reconstitution assay that was initiated. However these studies will provide the basis to support future related research in this laboratory.
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Oliveira, Lucila Habib Bourguignon. "Avaliação do perfil de expressão gênica de células CD34+ e células CD CD133+ isoladas de medula óssea e de sangue de cordão umbilical /." Araraquara : [s.n.], 2008. http://hdl.handle.net/11449/86617.
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Orientador: Dimas Tadeu CovasBanca: Dimas Tadeu Covas
Banca: Rodrigo Alexandre Panepucci
Banca: Haroldo Wilson Moreira
Resumo: A maior expressão de alvos transcricionais e componentes da via NFkB é uma característica distintiva das células-tronco hematopoéticas (CTH) CD34+ de sangue de cordão umbilical (SCU) comparadas às CTH CD34+ de medula óssea (MO) e pode estar relacionada com o estado mais primitivo das CTH dos neonatos. No entanto, as células CD34+ são um grupo heterogêneo de célulastronco (CT) e progenitoras em diferentes estágios de maturação e diferenças na composição celular entre MO e SCU poderiam contribuir para os resultados mencionados. Estudos recentes têm identificado o marcador de superfície CD133, como um marcador de CT mais primitivas, expresso em uma subpopulação de células CD34bright, com um sugestivo potencial de hemangioblasto. Com o objetivo de caracterizar a composição celular de MO e SCU e identificar mecanismos moleculares envolvidos com a maior primitividade das células CD133+, propusemos avaliar o perfis imunofenotípico (quanto à expressão de CD34 e CD133) por citometria de fluxo e de expressão gênica de células CD34+ e células CD133+ selecionadas imunomagneticamente, de ambas as fontes, pelas técnicas de microarray e PCR em tempo real. Nossos resultados revelaram que enquanto a maioria das células CD133+ são CD34+, independente da fonte, as células CD34+ de SCU possuem uma porcentagem significativamente maior de células CD133+ do que às células CD34+ de MO. A análise de clusterização revelou que as células CD133+ de MO se agrupam com as células de SCU (CD34+ e CD133+), enquanto as CD34+ de MO aparecem como um grupo distinto. A comparação dos perfis de expressão gênica entre as células CD133+ e as células CD34+, revelou a hiper-expressão... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: A higher expression of transcription targets and components of the NF-kB pathway is a distinctive feature of umbilical cord blood (UCB) CD34+ hematopoietic stem cells (HSC) when compared to bone marrow (BM) CD34+ HSC and this could be related to the more primitive state of the newborn's HSC. However, CD34+ cells represent a heterogeneous group of cells composed by stem and progenitors cells in different developmental stages, and differences in cellular composition between both sources could contribute for these finding. The surface marker CD133 has been identified as a very primitive marker, expressed in a subpopulation of CD34bright, with a proposal hemangioblast potential. Thus, in attempt to better characterize the cellular composition of UCB and BM and to identify molecular mechanisms related to the more primitive characteristics of CD133+ cells, we proposed to evaluate the immunophenotypic profile (expression of CD34 and CD133) by flow-cytometry and the gene expression profiles of immunomagnetically selected CD34+ and CD133+ cells, from both sources, by microarray and Real time PCR. Our results highlighted that, while almost all CD133+ cells are CD34+ independently of the evaluated source, the UCB CD34+ cells showed a significantly higher proportion of CD133 expression, compared to BM CD34+ cells. After obtaining the expression profiles from distinct HSC pooled samples generated by microarrays, cluster analysis showed that BM CD133+ cells preferentially grouped with UCB cells (CD34+ and CD133+) instead of BM CD34+ cells, which appeared as a very distinct profile The comparison between CD133+ and CD34+ samples revealed the over-expression of 47 transcriptional factors (TF) in CD133+ cells, many of them well-known and related... (Complete abstract click electronic access below)
Mestre
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Oliveira, Lucila Habib Bourguignon [UNESP]. "Avaliação do perfil de expressão gênica de células CD34+ e células CD CD133+ isoladas de medula óssea e de sangue de cordão umbilical." Universidade Estadual Paulista (UNESP), 2008. http://hdl.handle.net/11449/86617.
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oliveira_lhb_me_arafcf.pdf: 553720 bytes, checksum: b0f8f04b07802f8bf467e43259c87fba (MD5)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Universidade Estadual Paulista (UNESP)
A maior expressão de alvos transcricionais e componentes da via NFkB é uma característica distintiva das células-tronco hematopoéticas (CTH) CD34+ de sangue de cordão umbilical (SCU) comparadas às CTH CD34+ de medula óssea (MO) e pode estar relacionada com o estado mais primitivo das CTH dos neonatos. No entanto, as células CD34+ são um grupo heterogêneo de célulastronco (CT) e progenitoras em diferentes estágios de maturação e diferenças na composição celular entre MO e SCU poderiam contribuir para os resultados mencionados. Estudos recentes têm identificado o marcador de superfície CD133, como um marcador de CT mais primitivas, expresso em uma subpopulação de células CD34bright, com um sugestivo potencial de hemangioblasto. Com o objetivo de caracterizar a composição celular de MO e SCU e identificar mecanismos moleculares envolvidos com a maior primitividade das células CD133+, propusemos avaliar o perfis imunofenotípico (quanto à expressão de CD34 e CD133) por citometria de fluxo e de expressão gênica de células CD34+ e células CD133+ selecionadas imunomagneticamente, de ambas as fontes, pelas técnicas de microarray e PCR em tempo real. Nossos resultados revelaram que enquanto a maioria das células CD133+ são CD34+, independente da fonte, as células CD34+ de SCU possuem uma porcentagem significativamente maior de células CD133+ do que às células CD34+ de MO. A análise de clusterização revelou que as células CD133+ de MO se agrupam com as células de SCU (CD34+ e CD133+), enquanto as CD34+ de MO aparecem como um grupo distinto. A comparação dos perfis de expressão gênica entre as células CD133+ e as células CD34+, revelou a hiper-expressão...
A higher expression of transcription targets and components of the NF-kB pathway is a distinctive feature of umbilical cord blood (UCB) CD34+ hematopoietic stem cells (HSC) when compared to bone marrow (BM) CD34+ HSC and this could be related to the more primitive state of the newborn’s HSC. However, CD34+ cells represent a heterogeneous group of cells composed by stem and progenitors cells in different developmental stages, and differences in cellular composition between both sources could contribute for these finding. The surface marker CD133 has been identified as a very primitive marker, expressed in a subpopulation of CD34bright, with a proposal hemangioblast potential. Thus, in attempt to better characterize the cellular composition of UCB and BM and to identify molecular mechanisms related to the more primitive characteristics of CD133+ cells, we proposed to evaluate the immunophenotypic profile (expression of CD34 and CD133) by flow-cytometry and the gene expression profiles of immunomagnetically selected CD34+ and CD133+ cells, from both sources, by microarray and Real time PCR. Our results highlighted that, while almost all CD133+ cells are CD34+ independently of the evaluated source, the UCB CD34+ cells showed a significantly higher proportion of CD133 expression, compared to BM CD34+ cells. After obtaining the expression profiles from distinct HSC pooled samples generated by microarrays, cluster analysis showed that BM CD133+ cells preferentially grouped with UCB cells (CD34+ and CD133+) instead of BM CD34+ cells, which appeared as a very distinct profile The comparison between CD133+ and CD34+ samples revealed the over-expression of 47 transcriptional factors (TF) in CD133+ cells, many of them well-known and related... (Complete abstract click electronic access below)
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Bertoni, Lélia. "Évaluation du potentiel thérapeutique des cellules souches mésenchymateuses dans un modèle d'arthropathie expérimentale induite chez le cheval Characterization and use of Equine Bone Marrow Mesenchymal Stem Cells in Equine Cartilage Engineering. Study of their Hyaline Cartilage Forming Potential when Cultured under Hypoxia within a Biomaterial in the Presence of BMP-2 and TGF-ß1 Intra-Articular Injection of 2 Different Dosages of Autologous and Allogeneic Bone Marrow- and Umbilical Cord-Derived Mesenchymal Stem Cells Triggers a Variable Inflammatory Response of the Fetlock Joint on 12 Sound Experimental Horses An experimentally induced osteoarthritis model in horses performed on both metacarpophalangeal and metatarsophalangeal joints: Technical, clinical, imaging, biochemical, macroscopic and microscopic characterization Evaluation of allogeneic bone-marrow-derived and umbilical cord blood-derived mesenchymal stem cells to prevent the development of osteoarthritis in an equine model Chondrogenic Differentiation of Defined Equine Mesenchymal Stem Cells Derived from Umbilical Cord Blood for Use in Cartilage Repair Therapy." Thesis, Normandie, 2020. http://www.theses.fr/2020NORMC417.
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’arthropathie dégénérative est une maladie ayant des répercussions socio-économiques majeures chez l’homme et le cheval. Il n’existe pour l’heure aucun traitement curatif de cette maladie, le cartilage articulaire étant dépourvu de pouvoir de cicatrisation spontané. De nombreux espoirs reposent sur l’utilisation de cellules souches mésenchymateuses (CSM), pour leur potentiel pro-régénératif et anti-inflammatoire. Le premier objectif de cette étude était d’évaluer la tolérance des CSM de sang de cordon ombilical (SCO) et de moelle osseuse (MO) dans des articulations saines. L’étude contrôlée en aveugle menée sur 12 chevaux expérimentaux a démontré que l’injection de CSM-MO provoquait significativement plus de signes de réaction inflammatoire que l’injection de CSM-SCO, et que l’injection des CSM, quelle que soit leur origine, provoquait une réaction inflammatoire discrète à modérée, supérieure à celle d’une injection de placébo, avec une grande variabilité individuelle de sensibilité à une même lignée de cellules. Le second objectif était d’évaluer l’efficacité des CSM-MO et -SCO dans un modèle d’arthropathie induite. L’étude contrôlée en aveugle menée sur 8 chevaux expérimentaux a mis en évidence une réduction significative de la progression des signes indicateurs d’arthropathie à l’imagerie après injection de CSM-MO allogéniques par rapport à l’injection du placébo. Ces résultats encourageants, à considérer à la lumière des limites des études menées, indiquent un effet bénéfique des CSM-MO allogéniques dans la prise en charge de l’arthrose chez le cheval. Ils soulignent la nécessité de poursuivre les recherches afin de confirmer ces résultats, et d’optimiser les effets des CSM à travers leur combinaison à un vecteur ou par une approche acellulaire avec administration des nanovésicules qu’elles sécrètent, et considérées être à l’origine de leurs effets thérapeutiquesOsteoarthritis is a common cause of pain and economic loss in both humans and horses. There is currently no curative treatment for osteoarthritis, because of the lack of spontaneous regenerative capacity of the articular cartilage. Mesenchymal stem cells (MSC) based regenerative medicine comes across as a promising strategy given their pro-regenerative and anti-inflammatory potential. The first objective of this study was to evaluate the safety of umbilical cord blood (UCB) and bone marrow (BM) derived MSC in healthy joints. The blind controlled study conducted on 12 experimental horses showed that the injection of BM-MSC caused significantly more signs of inflammatory reaction than the injection of UCB-MSC, and that the injection of MSC, regardless of their origin, caused a discrete to moderate inflammatory reaction, greater than that of the placebo, with great individual variability in sensitivity to the same cell line. The second objective was to evaluate the efficacy of BM-MSC and UCB-MSC in a model of induced osteoarthritis. The blind controlled study conducted on 8 experimental horses showed a significant reduction in the progression of osteoarthritis associated signs with imaging techniques after injection of allogeneic BM-MSC compared to placebo. These promising results, to be considered in light of the limitations of the studies, indicate a beneficial effect of allogeneic BM-MSC in the management of osteoarthritis in horses. They underline the need for further research to confirm these results, and to optimize the effects of MSC through their combination with a vector or through an acellular approach with administration of the nanovesicles they secrete that ared considered to be responsible for their therapeutic effects
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Souza, Bruno Solano de Freitas. "Terapia com células da medula óssea em modelo experimental de insuficiência hepática aguda induzida por acetominofen." Centro de Pesquisas Gonçalo Moniz, 2012. https://www.arca.fiocruz.br/handle/icict/7642.
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Fundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, Brasil
Introdução e objetivos: A insuficiência hepática aguda (IHA), apesar de rara, permanece como uma condição rapidamente progressiva e frequentemente fatal. A intoxicação por acetaminofen (APAP) induz necrose hepática maciça e frequentemente leva à morte por edema cerebral. Terapias celulares são de grande interesse como potenciais tratamentos para IHA. Neste projeto foi avaliado o potencial terapêutico das células mononucleares da medula óssea (CMMO) em um modelo experimental de IHA induzida por APAP em camundongos. Métodos: A IHA foi induzida em camundongos C57Bl/6, previamente submetidos à dieta alcoólica por três semanas, através da administração de APAP na dose de 300 mg/kg por via intraperitoneal. Após a indução da IHA, os camundongos foram transplantados, por via endovenosa, com 107 CMMO obtidas de doadores transgênicos para a proteína verde fluorescente (GFP) ou injetados com salina. As curvas de sobrevivência, os níveis plasmáticos de aminotransferases, amônia, uréia e creatinina, a extensão da necrose hepática, o infiltrado inflamatório e a expressão de citocinas e metaloproteinases foram avaliados. A microscopia confocal foi utilizada para estudar a migração das células transplantadas para o fígado. A permeabilidade da barreira hemato-encefálica foi avaliada pela injeção do corante azul de Evans (AE) por via endovenosa. Resultados: Observou-se que os camundongos tratados com CMMO apresentaram uma redução significativa da mortalidade, com uma taxa de 60% de sobrevivência quando comparados a 20% de sobrevivência do grupo controle. As células transplantadas migraram para o fígado, mas não foi observada a diferenciação destas em células hepáticas ou fusão celular. Não foram encontradas diferenças estatisticamente significativas na extensão de necrose hepática no fígado, nos níveis plasmáticos de transaminases e amônia, na intensidade do infiltrado inflamatório no fígado entre os dois grupos com IHA, bem como nos níveis hepáticos das citocinas TNF-a e IL-10 e de transcritos para adrenomedula e endotelina 1 no cérebro, assim como na atividade da metaloproteinase 9 no soro. No entanto, observou-se uma redução dos níveis séricos de TNF-a no grupo tratado com CMMO quando comparado ao grupo injetado com salina. Esta redução está correlacionada ao aumento do mRNA para IL-10 na medula óssea e no baço dos animais tratados com CMMO. A avaliação do extravasamento do corante azul de Evans para o cérebro demonstrou que o grupo tratado com células mononucleares da medula óssea apresentou uma redução da permeabilidade da barreira hemato-encefálica quando comparado ao grupo injetado com salina. Conclusão: As CMMO exercem um efeito protetor em camundongos com IHA induzida por APAP, sem alterar o dano hepático, provavelmente através da modulação da resposta inflamatória sistêmica e da diminuição da permeabilidade da barreira hemato-encefálica.
Introduction and objectives: Acute liver failure (IHA), although rare, remains a rapidly progressive and often fatal condition. Poisoning by acetaminophen (APAP) induces a massive hepatic necrosis and often leads to death by cerebral edema. Cell therapies are of great interest as potential treatments for IHA. In this project we evaluated the therapeutic potential of bone marrow mononuclear cells (BMC) in an experimental model of IHA induced by APAP in mice. Methods: The IHA was induced in C57BL/6 mice previously submitted to the alcohol diet for three weeks by the administration of APAP at a dose of 300 mg / kg, intraperitoneally. After induction of IHA, the mice were transplanted intravenously with 107 BMC obtained from donors transgenic for green fluorescent protein (GFP) or injected with saline. The survival curves, plasma levels of aminotransferases, ammonia, urea and creatinine, the extent of hepatic necrosis, inflammatory infiltrate and the expression of cytokines and metalloproteinases were evaluated. Confocal microscopy was used to study the migration of transplanted cells to the liver. The permeability of the blood-brain barrier was assessed by injection of Evans blue dye (AE) intravenously. Results: We found that mice treated with BMC showed a significant reduction in mortality, with a rate of 60% survival compared to 20% survival in the control group. The transplanted cells migrated to the liver, but we did not observe the differentiation of these cells or fusion with liver cells. There were no statistically significant differences in the extent of hepatic necrosis in the liver, plasma levels of transaminases and ammonia, and in the intensity of the inflammatory infiltrate in the liver between the two groups with IHA, as well as in hepatic levels of TNF-α and IL-10, transcripts for endothelin 1 and adrenomedullin in the brain and serum metalloproteinase 9 activity. However, there was a reduction of serum TNF-α in BMC-treated group compared to the group injected with saline. This decrease correlated with the increase in IL-10 mRNA expression in the bone marrow and spleen of BMC-treated mice. The assessment of Evans blue extravasation into the brain showed that the group treated with BMC had a reduced permeability of the blood-brain barrier compared to the group injected with saline. Conclusion: Bone marrow mononuclear cells exert a protective effect in mice with APAPinduced IHA, without changing the liver injury, probably through modulation of systemic inflammatory response and decrease the permeability of the blood-brain barrier.
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Menegat, Laura. "Estudo dos processos de mobilização, ativação e apoptose das células da medula óssea em modelo de morte encefálica em ratos." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/5/5156/tde-04082016-161413/.
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INTRODUÇÃO: Estudos experimentais suportam a evidência de leucopenia persistente desencadeada pela morte encefálica (ME). OBJETIVO: Esse estudo teve como objetivo investigar o comportamento leucocitário na medula óssea e no sangue após a morte encefálica em ratos. MÉTODOS: A morte encefálica foi induzida através da inserção e insuflação rápida de um cateter no espaço intracraniano. Ratos falso-operados (FO) foram apenas trepanados. Decorridas seis horas, as células da medula óssea, coletadas da cavidade femural, foram utilizadas para as contagens total e diferencial e analisadas por citometria de fluxo para a caracterização das subpopulações linfocitárias, a expressão de moléculas de adesão granulocíticas e apoptose/necrose (método de Anexina V/Iodeto de Propídio (PI)). RESULTADOS: Ratos com ME apresentaram uma redução de 30% no número de células da medula óssea devido à redução de linfócitos (40%) e células segmentadas (45%). As subpopulações de linfócitos na medula óssea foram semelhantes nos animais ME e FO (CD3, p=0,1; CD4, p=0,4; CD3/CD4, p=0,4; CD5, p=0,4, CD3/CD5, p=0,2; CD8, p=0,8). A expressão de L-selectina e beta2-Integrinas nos granulócitos também não diferiram entre os grupos (CD11a, p=0,9; CD11b/c, p=0,7; CD62L, p=0,1). Não existem diferenças nas porcentagens de apoptose e de necrose (Anexina V, p=0,73; PI, p=0,21; Anexina V/PI, p=0,29). CONCLUSÃO: Os dados sugerem que a redução na mobilização de células da medula óssea para o sangue, desencadeada pela morte encefálica, não se relaciona a alterações de subpopulações de linfócitos, expressão de moléculas de adesão granulocíticas, ou apoptose e necroseINTRODUCTION: Experimental findings support the evidence of a persistent leucopenia triggered by brain death (BD). AIMS: This study aimed to investigate leukocyte behavior in bone marrow and blood after BD in rats. METHODS: BD was induced by quickly inflation of an intracranial balloon catheter. Sham operated (SH) rats were trepanned only. Six hours thereafter bone marrow cells harvested from the femoral cavity were used for total and differential counts, and analyzed by flow cytometry to characterize lymphocyte subsets, granulocyte adhesion molecules expression, and apoptosis/necrosis (annexin V/propidium iodide (PI) protocol). RESULTS: BD rats exhibited a 30% reduction in bone marrow cells due to a reduction in lymphocytes (40%) and segmented cells (45%). Bone marrow lymphocyte subsets were similar in BD and SH rats (CD3, p=0.1; CD4, p=0.4; CD3/CD4, p=0.4; CD5, p=0.4, CD3/CD5, p=0.2; CD8, p=0.8). Expression of L-selectin and ?2-integrins on granulocytes did not differ (CD11a, p=0.9; CD11b/c, p=0.7; CD62L, p=0.1). There were no differences in the percentage of apoptosis and necrosis (Annexin V, p=0.73; PI, p=0.21; Annexin V/PI, p=0.29). CONCLUSIONS: Data presented suggest that the down-regulation of the bone marrow triggered by BD is not related to changes in lymphocyte subsets, granulocyte adhesion molecules expression, or apoptosis and necrosis
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Buglass, Surahanil Katrin. "Regulating stem cell fate within microenvironmental niches." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:75f9498c-30f0-4983-84b2-dd58f2ccf52b.
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Improving the repopulation potential of human umbilical cord blood (UCB) haemopoietic stem cells (HSCs) remains a paramount goal in HSC transplantation (HSCT) therapy. This implies enhancing the homing and engraftment potential of UCB-CD34+CD133+ cells to the bone marrow (BM). Although an array of molecules continues to be identified as ‘key’ homing molecules, the molecular mechanisms controlling HSC homing are still not fully understood. The regulatory implications of hypoxia in the BM, with the concomitant stabilisation of hypoxia inducible transcription factor-1α (HIF-1α), are becoming more apparent, yet at the commencement of this thesis no study had explored whether hypoxia induced signalling can be adopted to regulate the homing and engraftment of transplanted HSCs. The aim of this DPhil project was thus to investigate whether hypoxic conditions as detected in the BM influence the adhesion of UBC-CD133+ cells to osteoblasts, BM stromal cells and BM endothelial cells-60 (BMEC-60), as well as their transmigration towards chemokine SDF-1α across BMEC-60. Increasing the exposure of UCB-CD133+ cells to 1.5% O2 doubled the percentage of transmigrating cells (p<0.05), and while hypoxia stimulated UCB-CD133+ cells preferentially adhered to IL-1β stimulated BMEC-60, their adhesion to non-stimulated (BMEC-60) was significantly improved (p<0.001). To help unravel the underlying molecular mechanisms, we attempted to examine the potential involvement of hypoxia regulated scaffolding protein HEF-1/NEDD9/Cas-L (HEF-1) in the increased percentage of migrating UCB-CD133+ cells after hypoxia pre-conditioning. The role of HEF-1 in HSCs is unexplored, and its multifunctional contribution in a variety of processes including cell migration, attachment and invasion make HEF-1 a prime candidate as a contributing homing molecule. After identifying a suitable short-hairpin RNA (shRNA) sequence to knockdown HEF-1, generating lentiviral (LV)-particles in house and optimising transduction protocols, HEF-1 knockdown was achieved in haemopoietic model cell lines KG-1 and KG-1A (KG-1/KG-1A–HEF1). Significantly decreased KG-1A–HEF1 cell adhesion to non-stimulated BMEC-60 was detected. Together, these studies provide a promising platform to further explore the role of HEF-1 in hypoxia induced UCB-CD133+ cell transmigration towards the key homing molecule SDF-1α.
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Chen, Yu-Zhang, and 陳昱彰. "Functional and Phenotypic Characterizations of Swine Dendritic Cells Derived from Bone Marrow Haematopoietic Cells in vitro." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/29007370916250109664.
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碩士國立屏東科技大學
生物科技研究所
93
Dendritic cells (DC) have been referred as the most professional antigen-presenting-cell, which plays important roles in modulating immune responses. In this study, bone marrow haematopoietic cells (BMHC) were isolated from the long bones of piglets and then stimulated by co-culturing with granulocyte macrophage colony-stimulating factor(GM-CSF) and interleukin-4 (IL-4) to generate BMHC-derived immature DC (BMHC-imDC). Then, TNF-α was used to induce the maturation of BMHC-imDC into BMHC-derived mature DC (BMHC-mDC). Cell morphology of BMHC, BMHC-imDC and BMHC-mDC was examined by transmission electron microscopy and light microscopy. The immuno-phenotypic characteristics of these cells were analyzed by using flow cytometry. The results showed that BMHC-imDC derived from BMHC were characterized with consistent cell morphology and 85.7±1.5％ of BMHC-imDC had manifest dendritic pseudopods. Their cell surface markers were identified as SWC1med SWC3high SWC9low CD4low CD8low/med CD14med/high CD80/86low /med MHC Imed/high MHC IIlow. BMHC-mDC had no significant changes on their pseudopods during the maturation. As compared to those of BMHC-imDC, the cell percentages of BMHC-mDC with MHC II＋ and CD80/86＋ were significantly increased (30.1 vs. 49.9％ and 44.9 vs. 66.0％, respectively). Also, the difference in expressions of SWC1high SWC9low on BMHC-imDC from SWC1lowSWC9high on macrophages can be used as selective markers between these two antigen-presenting cells. Thus, we successfully established the in vitro differentiation method to generate large amounts of swine BMHC-imDC and BMHC-mDC, which can be used as screening cells for related immune studies.
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Prewitz, Marina. "Decellularised extracellular matrices as instructive microenvironments for bone marrow derived stem cells." Doctoral thesis, 2011. https://tud.qucosa.de/id/qucosa%3A25975.
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The regenerative potential of adult stem cell populations within the human body bears great promises for their use in regenerative medicine. The bone marrow (BM) harbours two different types of adult stem cells, haematopoietic stem and progneitor cells (HSPCs) and multipotent mesenchymal stromal cells (MSCs), which are tightly regulated in their distinct anatomically defined niches by multiple cues such as cytokines, cell-cell contacts, the extracellular matrix (ECM) and the physical microenvironment. The ex vivo expansion of these cells for applications in regenerative therapies is of great interest and several biomaterial approaches attempt to mimic the natural BM niche and its components to control stem cell maintenance and differentiation. However, as of now the complexity of such stem cell niches is hard to recapitulate. Towards this goal, this work was focussing on the ECM environment of BM stem cells and was set out to engineer improved in vitro culture systems. MSC themselves are one of the most important cell types within the BM that secrete and construct ECM-networks and thereby shape the microenvironment of the residing cells. The potential of primary human BM-MSC to secrete ECM in vitro has been exploited to generate niche-like ECM surrogates in a robust and versatile format. Application of decellularisation regimes allowed the fabrication of complex matrices which demonstrated suprastructural, compositional and physicochemical properties compareable to those of the native BM-ECM environment. Reliable stability and reproduciblity was achieved by a dedicated procedure of maleic anhydride co-polymer-mediated covalent binding of fibronectin and subsequent anchorage of cell-secreted ECM molecules. As a result of the high reproducibility, a complete proteomic register of ECM molecules was obtained in combination with determining the complex fibrillar and soft gel-like characteristics of MSC-derived matrices. Based on the established BM niche-like substrate, the impact of extracellular matrices on MSC and HSPC ex vivo behavior has been explored. Both cell types demonstrated strong adhesion to ECM substrates and depicted a changed cellular morphology upon contact with native ECM structures compared to standard culture substrates or simple ECM protein coatings, indicating an intense interplay between the cell and the microenvironment. MSC that re-grew into their own matrices have shown advantageous proliferation and cytokine secretion levels as well as enhanced differentiation intensity (upon differentiation induction) compared to MSC that were cultured on less complex substrates. Similarly, HSPC were also instructed for enhanced expansion on MSC-derived matrices without exhaustion of stem cell-marker expressing progenitor cells. The efficiency of these matrices was related to their ability to mimic the native composite suprastructure, ligand nano-topography, molecular composition and physical properties of natural BM ECM environments. The data obtained within this thesis set the ground for a more rational design of artificial stem cell niches with defined and distinct properties, offering exciting options for the in-depth analysis and understanding of stem cell regulation by exogenous cues.
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Lin, Tzu-Chieh, and 林子傑. "Therapeutic Indications between Umbilical Cord Blood- and Bone Marrow- Derived Mesenchymal Stem Cells for Osteoarthritis." Thesis, 2012. http://ndltd.ncl.edu.tw/handle/90522085033793224437.
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Wong, Elaine Karol. "Antileukemic activities of human bone marrow and blood cells after culture in interleukins-2, -7 and -12." Thesis, 1994. http://hdl.handle.net/2429/5105.
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Acute myeloid leukemia (AML) can be treated by chemotherapy alone, allogeneic
bone marrow transplantation (BMT) or autologous BMT. There is a risk of disease
recurrence with all these methods, the lowest being associated with allogeneic BMT due to
an allogeneic antileukemic effect. Despite its benefits, the use of autologous BMT is
limited by high relapse rates due to the persistence of leukemic stem cells in the autologous
bone marrow (BM) and/or residual disease in the patient. Thus, it would be desirable to
develop methods to decrease relapse rates associated with autologous BMT to a level
similar to those of allogeneic BMT. Since individuals who receive BM from a twin are
subject to a high risk of relapse, an active immune component that contributes to the
elimination of residual disease must be present in the allogeneic BM graft. This project is
based on the hypothesis that this can also be achieved by appropriate cytokine manipulation
of the antiproliferative activity of autologous effector cells. The natural killer (NK) cell
population is the first cell subpopulation to reconstitute after BMT and it is, therefore, an
obvious candidate for such manipulations.
BMT involving interleukin (IL)-2-activated autologous BM is associated with
delayed neutrophil and platelet recovery which can be hastened by the addition of peripheral
blood stem cells with the BM. Recent studies with IL-7 and IL-12 suggest that they might
mimic or enhance the cytotoxic and antiproliferative activities of IL-2. The effects of these
cytokines on bone marrow cells (BMC) and peripheral blood mononuclear cells (PBMC)
were compared in a model system in order to assess potential alternate strategies for the ex
vivo purging of leukemic cells from human BMC and PBMC.
The cytotoxie activity of BMC and PBMC was measured with ⁵¹Cr release assays.
The cytotoxic activity exerted by BMC stimulated with optimal doses of IL-2 was less than that of PBMC. While neither IL-7 nor IL- 12 induced cytotoxic activity in BMC, activity
was detected in PBMC stimulated with these cytokines. The cytotoxic activity induced by
optimal doses of IL-2 in BMC was not enhanced by IL-7 whereas enhancement was
observed with IL-12. In contrast, IL-2-induced cytotoxic activity in PBMC was increased
by both IL-7 and IL-12 when suboptimal doses of IL-2 were used.
BMC and PBMC were cocultured with K562neor cells and their ability to inhibit
leukemic cell survival was measured using an assay to detect clonogenic K562 cells. IL-2-
stimulated BMC and PBMC inhibited leukemic cell growth in a manner dependent on the
initial number of leukemic cells in the coculture. IL-2-stimulated BMC and PBMC were
the most efficient in eliminating leukemic cells from the coculture compared to those
stimulated with IL-7 or IL-12. IL-7 did not enhance IL-2-induced inhibitory activity on
leukemic cell growth in BMC and PBMC while IL-12 did. There was no difference in
BMC-mediated inhibitory activity on leukemic cell growth upon cytokine stimulation when
tested after 2, 4, 6, and 8 days of culture. IL-7 did not hasten the development of maximal
IL-2-induced inhibitory activity relative to IL-2 alone while IL-12 did. Overall, PBMC
mediated inhibitory activity on leukemic cell survival in cocultures with K562-neo’ cells
was significantly greater and faster than that observed in BMC cocultures when equal
numbers of leukemic cells were initially present.
These findings suggest that the use of cytokine combinations may have potential in
the clinical setting. The information regarding the relative capacities of BMC and PBMC in
terms of their antileukemic activities obtained in these experiments will aid in refining the in
vitro culture system for these cells such that their antileukemic activity may be optimized.
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Waugh, Caryll Marie. "Prothymosin alpha, a gene differentially expressed in CD34+ cells." 2004. http://repository.unimelb.edu.au/10187/1028.
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Haemopoietic stem and progenitor cells from bone marrow and cord blood are well characterised with respect to their phenotype, growth in clonal assays, responsiveness to cytokine stimulation, receptor profile and their ability to sustain multilineage engraftment of receptive hosts in animal models of transplantation and of course, clinically in the treatment of some haemopoietic and immunological disorders. It is generally accepted that cells bearing the CD34+ phenotype are enriched for the most primitive of haemopoietic stem cells that possess the cardinal features of self-renewal and multipotency. However, the molecular mechanisms, the spectrum of expressed genes that give rise to the physical characteristics of haemopoietic progenitor cells are not well understood. Furthermore, although CD34+ cells from different sources (bone marrow, cord blood, mobilised peripheral blood) share many common features, there are also significant differences. (For complete abstract open document)
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Chang, Yu-Jen, and 張育甄. "Characterization of Multipotent Mesenchymal Stem Cells Derived from Human Bone Marrow, Umbilical Cord Blood, Amniotic Fluid and Amniotic Membrane." Thesis, 2006. http://ndltd.ncl.edu.tw/handle/84214814075223769489.
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博士國立交通大學
生物科技系所
94
Abstract During human entire lifespan, from fetus to adult, mesenchymal stem cells (MSCs) proliferate and differentiate into mesenchyme-lineage tissues. Bone marrow and umbilical cord blood are reported to be the main sources of MSCs and have been proposed for possible clinical applications. This study evaluates the tendency in bone marrow-derived MSCs (bmMSCs) and cord blood-derived MSCs (cbMSCs) by in vitro induction and quantification of their characteristics. Results indicated that cbMSCs had a significantly stronger osteogenic potential but less capacity in adipogenic differentiation than bmMSCs. Leptin, an important regulator of mesenchymal differentiation, also acted in bmMSCs and cbMSCs. In both types of MSCs, leptin was found to support osteogenesis, and inhibited adipogenesis. However, Cbfa1 mRNA expression in bmMSCs and cbMSCs was affected at different degree by leptin during osteogenesis. In contrast, leptin reduced PPARγ2 mRNA expression at same level during adipogenesis in both types of MSCs. These results demonstrate the diverse capacity of MSCs from bone marrow and cord blood and suggest that they be used differently in experimental and therapeutic studies. We also isolated the clonogenic MSCs from cord blood by limiting dilution method. These cells exhibited two different morphologic phenotypes, including flattened fibroblastic clones (majority) and spindle-shaped fibroblastic clones (minority). Both types of MSCs shared similar cell surface markers except CD90 and had similar osteogenic and chondrogenic potentials. However, the spindle-shaped clones possessed the positive CD90 expression and a greater tendency in adipogenesis than the flattened clones. The high number of flattened MSCs might actually be linked to the less sensitivity of cbMSCs in adipogenic differentiation. Amniotic fluid and amniotic membrane were also the good sources of mesenchymal stem cells. We successfully isolated MSCs from second-trimester amniotic fluid (AFMSCs) and term amniotic membrane (AMMSCs). AFMSCs and AMMSCs were very similar with MSCs from other sources in the phenotypic morphology, marker profiles (positive for SH2, SH3, SH4, CD29, CD44, CD90 and HLA-I; negative for CD26, CD31, CD34, CD45 and HLA-II). All AFMSCs and AMMSCs samples have mesenchymal-lineage differentiated potentials to differentiate into osteoblasts and adipocytes. The telomerase activity was not detected in AFMSCs, cbMSCs and bmMSCs, and the telomere length of AFMSCs was longer than cbMSCs and bmMSCs. AFMSCs could express embryonic regulators, Oct-4 and Nanog, but they could not express the embryonic markers SSEA-1, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. AMMSCs could be induced into neuronal cells, which expressed neuronal markers β-tubulin III, tyrosine hydroxylase and NeuN, by insulin and isobutyl-methylxanthine.
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Ferrario, Daniele [Verfasser]. "In vitro assessment of arsenic immune toxicity using human cord blood and murine bone marrow cells / vorgelegt von Daniele Ferrario." 2009. http://d-nb.info/997607971/34.
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Pacitti, Andrew Peter. "Isolation of mesenchymal stem cells derived from adult bone marrow and umbilical cord blood and their potential to differentiate into osteoblasts /." 2006. http://hdl.handle.net/10156/1387.
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Shirvaikar, Neeta Chandan. "Role of membrane-type 1 matrix metalloproteinase in hematopoietic stem/progenitor cell trafficking." Phd thesis, 2010. http://hdl.handle.net/10048/1146.
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Thesis (Ph.D.)--University of Alberta, 2010.A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Medicine. Title from pdf file main screen (viewed on April 27, 2010). Includes bibliographical references.
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Xie, Jie. "Tsg-6 : an inducible mediator of paracrine anti-inflammatory and myeloprotective effects of adipose stem cells." Thesis, 2014. http://hdl.handle.net/1805/3876.
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Indiana University-Purdue University Indianapolis (IUPUI).Tumor necrosis factor-induced protein 6 (TSG-6) has been shown to mitigate inflammation. Its presence in the secretome of adipose stem / stromal cells (ASC) and its role in activities of ASC have been overlooked. This thesis described for the first time the release of TSG-6 from ASC, and its modulation by endothelial cells. It also revealed that protection of endothelial barrier function was a novel mechanism underlying the anti-inflammatory activity of both ASC and TSG-6. Moreover, TSG-6 was found to inhibit mitogen-activated lymphocyte proliferation, extending the understanding of its pleiotropic effects on major cell populations involved in inflammation. Next, enzyme-linked immunosorbent assays (ELISA) were established to quantify secretion of TSG-6 from human and murine ASC. To study the importance of TSG-6 to specific activities of ASC, TSG-6 was knocked down in human ASC by siRNA. Murine ASC from TSG-6-/- mice were isolated and the down-regulation of TSG-6 was verified by ELISA. The subsequent attempt to determine the efficacy of ASC in ameliorating ischemic limb necrosis and the role of TSG-6, however, was hampered by the highly variable ischemic tissue necrosis in the BALB/c mouse strain. Afterwards in a mouse model of cigarette smoking (CS), in which inflammation also plays an important role, it was observed, for the first time, that 3-day CS exposure caused an acute functional exhaustion and cell cycle arrest of hematopoietic progenitor cells; and that 7-week CS exposure led to marked depletion of phenotypic bone marrow stem and progenitor cells (HSPC). Moreover, a dynamic crosstalk between human ASC and murine host inflammatory signals was described, and specifically TSG-6 was identified as a necessary and sufficient mediator accounting for the activity of the ASC secretome to ameliorate CS-induced myelotoxicity. These results implicate TSG-6 as a key mediator for activities of ASC in mitigation of inflammation and protection of HSPC from the myelotoxicity of cigarette smoke. They also prompt the notion that ASC and TSG-6 might potentially play therapeutic roles in other scenarios involving myelotoxicity.
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Bah, Ramatoulaye. "Les immunoglobulines intraveineuses et la réponse spécifique des cellules T dans la prévention de la maladie lymphoproliférative post-greffe associée au virus Epstein-Barr chez les enfants greffés de cellules souches hématopoïétiques." Thèse, 2015. http://hdl.handle.net/1866/13540.
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