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Boone, Jason Nathaniel. "Characterization of novel neural stem cell populations in the Drosophila central nervous system /." Connect to title online (Scholars' Bank) Connect to title online (ProQuest), 2008. http://hdl.handle.net/1794/8160.
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Thesis (Ph. D.)--University of Oregon, 2008.Typescript. Includes vita and abstract. Includes bibliographical references (leaves 78-88). Also available online in Scholars' Bank; and in ProQuest, free to University of Oregon users.
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Boone, Jason Nathaniel 1976. "Characterization of novel neural stem cell populations in the Drosophila central nervous system." Thesis, University of Oregon, 2008. http://hdl.handle.net/1794/8160.
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xi, 88 p. A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number.Neuroblasts are the neural stem cells of the Drosophlia central nervous system. They are large cells that divide asymmetrically to renew another neuroblast and generate a smaller ganglion mother cell (gmc) that will divide once to produce two neurons. Combining genetic lineage tracing experiments with cell fate markers I isolated two separate neural stem cell populations with distinct locations and cellular behaviors in the larval brain. In my first chapter I introduce the central nervous system of Drosophila and in the next two sections of chapter I, I introduce the development of the optic lobe and central brain, two separate structures of the central nervous system. In my second chapter I characterize the lineage relationship of cells within the developing larval optic lobe and use cell fate markers to determine the identity of these cells. Next I examine the effect of spindle orientation on cell fate within epithelial cells of the optic lobe. In my third chapter I characterize another novel neural stem cell lineage in the larval brain containing GMCs with greater proliferation potential than a "canonical" GMC, and I term these, transit amplifying gmcs (TA-GMCs). Further I show that the parent neuroblast of these novel TA-GMCs does not asymmetrically segregate the fate determinant Prospero (Pros) thereby producing a GMC with greater proliferation potential. Finally I show that TA-GMCs do asymmetrically segregate the fate determinant Pros, divide slowly and give rise to up to 10 neurons which normal gmcs never do. In my fourth chapter I show preliminary work on the characterization of a mutation that causes excessive production of neuroblasts specifically in novel TA-GMC lineages. These findings reveal novel neural stem cell lineages, patterns of asymmetric cell division and patterns of neurogenesis that could aid in our understanding of neural stem cell biology and tumorogenesis. This dissertation includes both my previously published and my co-authored materials.
Adviser: Chris Doe
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Mora, Cristina Fuente. "Isolation and characterization of a novel population of potential kidney stem cells from postnatal mouse kidney." Thesis, University of Liverpool, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.507193.
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AZZONI, EMANUELE. "A novel population of embryonic endothelial derived progenitors contributes to multiple mesodermal lineages during development and muscle regeneration." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2011. http://hdl.handle.net/10281/20179.
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Several investigators have reported the isolation of vessel-associated multipotent mesodermal progenitors from diverse dissociated and cultured embryonic, fetal, perinatal and adult tissues. Despite the increasingly recognized medical value of these progenitor cells, among which are mesoangioblasts, these indirect extraction methods have precluded the understanding of their native identity, tissue distribution and frequency. We addressed this question by using a genetic lineage tracing approach.
We labelled embryonic VE-Cadherin+ endothelial cells in the E8.5-E9 time window. At embryonic, foetal and perinatal stages, we detected labelled skeletal muscle, smooth muscle and dermis cells originating from embryonic endothelium as part of their in vivo normal developmental fate. We excluded that this contribution derives from a somitic intermediate progenitor.
Consistently, we observed a small number of endothelial-derived muscle fibers in the adult skeletal muscle. We also found labelled subsets of pericytes and PICs (PW1+ interstitial progenitors), but we did not detect any labelled satellite cell. Following muscle damage, cells derived from embryonic endothelial progenitors participate to myogenic regenerative response, generating myofibers and macrophages.
FACS-isolated endothelial derived cells are myogenic in vitro and are able to differentiate in several mesodermal tissues, including SMA (smooth muscle actin) positive cells, AP (alkaline phosphatase) positive osteoblast-like cells, adipose tissue and endothelial networks. Intra-muscular injection of isolated endothelial derived cells results in colonization and reconstitution of skeletal muscle tissue in both wild type and dystrophic mice.
Moreover, we demonstrated that haematopoietic cells originated by endothelial to haematopoietic transition in the yolk sac emerge abluminally from vessels in the body of the embryo and migrate into the mesenchyme. We also identified a novel population of cells that express haematopoietic and mesenchymal markers and are originated by this process.
These data suggest the existence of a previously unrecognized endothelial-derived progenitor cell population, which could represent the in vivo counterpart of embryonic mesoangioblasts. These progenitor cells contribute to smooth and skeletal muscle development, as part of their normal fate, and generate cells that persist in the adult life, participating to muscle regeneration.
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Pagliaro, Sarah Beatriz De Oliveira. "Transcriptional control induced by bcr-abl and its role in leukemic stem cell heterogeneity. Single-Cell Transcriptome in Chronic Myeloid Leukemia: Pseudotime Analysis Reveals Evidence of Embryonic and Transitional Stem Cell States Single Cell Transcriptome in Chronic Myeloid Leukemia (CML): Pseudotime Analysis Reveals a Rare Population with Embryonic Stem Cell Features and Druggable Intricated Transitional Stem Cell States A novel neuronal organoid model mimicking glioblastoma (GBM) features from induced pluripotent stem cells (iPSC) Experimental and integrative analyses identify an ETS1 network downstream of BCR-ABL in chronic myeloid leukemia (CML)." Thesis, université Paris-Saclay, 2020. http://www.theses.fr/2020UPASQ032.
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La leucémie myéloïde chronique est une hématopoïèse maligne clonale, caractérisée par l'acquisition de la translocation t (9;22) conduisant au chromosome Ph1 et à son homologue l'oncogène BCR-ABL, dans une cellule souche hématopoïétique très primitive. La LMC est un modèle de thérapies ciblées, car il a été démontré que la preuve de la faisabilité du ciblage de l'activité tyrosine kinase (TK) BCR-ABL à l'aide d'inhibiteurs de TK (TKI) entraîne des réponses et des rémissions majeures. Cependant, les problèmes actuels rencontrés dans ces thérapies sont la résistance des cellules souches leucémiques primitives et leur persistance qui serait liée à l'hétérogénéité des cellules souches au moment du diagnostic, ce qui conduit à la sélection clonale de cellules résistant aux thérapies TKI. J'ai appliqué la technologie de l'analyse du transcriptome des cellule uniques aux cellules de la LMC en utilisant un panel de gènes impliqués dans différentes voies, combinée à l'analyse d'inférence de trajectoire au modèle d'expression des gènes. Les résultats ont montré un état transitoire des cellules souches comprenant des gènes embryonnaires identifiés dans les cellules de la LMC au moment du diagnostic, ce qui pourrait contribuer à la résistance et à la persistance de la LSC. En outre, l'oncoprotéine Bcr-Abl est la tyrosine kinase constitutivement active produite par le gène chimérique BCR-ABL dans la leucémie myéloïde chronique (LMC). Les cibles transcriptionnelles de Bcr-Abl dans les cellules leucémiques n'ont pas été étudiées de manière approfondie. Une expérience de transcriptome utilisant la lignée cellulaire UT7 hématopoïétique exprimant BCR-ABL, a identifié la surexpression du facteur d'élongation eucaryote kinase 2 (eEF2K) qui joue un rôle majeur dans la survie des cellules en cas de privation de nutriments. Dans l'ensemble, les données suggèrent que la surexpression de eEF2K dans la LMC est associée à une sensibilité accrue à la privation de nutrimentsChronic myeloid leukemia is a clonal hematopoietic malignancy, characterized by the acquisition of the t (9;22) translocation leading to Ph1 chromosome and its counterpart BCR-ABL oncogene, in a very primitive hematopoietic stem cell. CML is a model of targeted therapies as the proof of concept of the feasibility of targeting the tyrosine kinase (TK) activity BCR-ABL using TK inhibitors (TKI) has been shown to lead to major responses and remissions. However, the current problems encountered in these therapies are primitive leukemic stem cells resistance and their persistence which is thought to be related to the heterogeneity of the stem cells at diagnosis leading to clonal selection of cells resisting to TKI therapies. I have applied the technology of single cell transcriptome analysis to CML cells using a panel of genes involved in different pathways combined with trajectory inference analysis to the gene expression pattern. The results showed a transitional stem cell states including embryonic genes identified in CML cells at diagnosis which could contribute to LSC resistance and persistence. Furthermore, the oncoprotein Bcr-Abl is the constitutively active tyrosine kinase produced by the chimeric BCR-ABL gene in chronic myeloid leukemia (CML). The transcriptional targets of Bcr-Abl in leukemic cells have not been extensively studied. A transcriptome experiment using the hematopoietic UT7 cell line expressing BCR-ABL, has identified the overexpression of eukaryotic elongation factor kinase 2 (eEF2K) which plays a major role in the survival of cells upon nutrient deprivation. Overall, the data suggest that overexpression of eEF2K in CML is associated with an increased sensitivity to nutrient-deprivation
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Al-Bedhawi, Mohammed Abdalmalek Ali. "Identification and characterisation of a potential adrenocortical stem cell population." Thesis, University of Reading, 2018. http://centaur.reading.ac.uk/77850/.
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The adrenal gland is a small endocrine organ with inherent regenerative capacity. Numerous studies proposed a stem cell niche located in the capsule of adrenal cortex. This project investigated mRNA and protein spatial expression of potential stem cell markers in the adrenal cortex. One of these markers (Thy-1) was used to isolate potential stem cells from dispersed primary adrenal cortex cells using magnetic-activated cell sorting (MACS). Thy-1 positive cells were monitored while they survived in vitro for over one year. Thy-1 cells were demonstrated to be undifferentiated fibroblastic-like cells with characteristics similar to those of mesenchymal stem cells in vitro such as plastic adherent, can form colonies, the confluent cells have a whirlpool-like formation and the significant (P< 0.05) increase of Thy-1 expression during cultivation in vitro. Thy-1 positive cells showed the expression of other stem cell markers such as Wt1, Etv5 and ID4. The monitoring of Thy-1 positive cells behavioural changes in vitro showed slow division rate, slow cell migration and the coexistence of senescent cells in their early months of cultivation. In comparison, in the late months of cultivation, the cells showed a significant (P< 0.05) increase in cell division rate and cell migration similar to cancer cell behaviour in vitro. As these cells were generally undifferentiated and they have characteristics of mesenchymal stem cells, differentiation attempts were first conducted using external differentiation factors (forskolin, ACTH and AT20 cell line media). However, the results showed non-significant responses to these treatments. The second differentiation experiments were conducted by forcing expression of Sf1 protein in Thy-1 positive cells using a cloning vector. Transfection with Sf1caused a significant (P< 0.05) reduction in ID4 mRNA expression and significantly (P< 0.05) elevated the expression of the progenitor marker Gli1 and the expression of steroidogenic enzyme genes (Cyp11A1 and 3βHSD) consistent with previous reports for several mesenchymal stem cell types.
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Okas, Mantas. "Novel immunotherapeutical strategies in allogeneic hematopoietic stem cell transplantation /." Stockholm : Department of laboratory medicine, Karolinska institutet, 2010. http://diss.kib.ki.se/2010/978-91-7409-934-8/.
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Cowan, Scott. "Characterisation of a novel pluripotent stem cell survival compound." Thesis, University of Glasgow, 2013. http://theses.gla.ac.uk/4920/.
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Human pluripotent stem cells (hPSC) such as human embryonic stem cells (hESC) and induced pluripotent stem cells (hiPSC) are incredibly valuable tools for investigations within a number of scientific fields including developmental biology, toxicology, pharmacology and perhaps most importantly, regenerative medicine. HPSC have an unlimited capacity for self-renewal which allows the expansion of clinically relevant cell numbers from a relatively small supply of starting material. Furthermore, hPSC are pluripotent, meaning they retain the capacity to differentiate into all the somatic cell types within the human body. In order for the huge potential of hPSC to be realised, many hurdles must first be overcome. The most basic of these is the development of consistent and scalable culture systems that allow sufficient expansion of hPSC without the loss of the stem cell identity. Critical to this matter is the susceptibility of hPSC to apoptosis upon enzymatic disaggregation wherein approximately 80% of hPSC begin the process of apoptosis. Recent efforts to overcome this issue have focussed on the Rho associated coiled-coil kinase (ROCK) inhibitor Y27632. However there is increasing evidence that the use of Y27632 can lead to an increased risk of karyotypic instability, a decrease in proliferative capacity and a reduced capacity to differentiate in to specific cell types such as haematopoietic cell types. The work presented within this thesis describes the characterisation of T16, a novel hPSC survival compound which does not inhibit ROCK and has novel mechanism of action.
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Webb, William Richard. "Novel stem cell and PHBHHx approaches to tendon repair." Thesis, Keele University, 2014. http://eprints.keele.ac.uk/1216/.
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Tendon injuries continue to be a financial burden on the health care system of many western countries, whilst also remaining common and a significant challenge within the orthopaedic discipline with no consensus of opinion on the best therapeutic regime to be employed. Many polymers have been investigated for use in tendon repair. A range of polymers have shown good integration with limited immune response. However, to date no implant has been capable of delivering the physical properties observed in native undamaged tendon. Many of the polymers implanted have resulted in re-rupture or reduced mechanical function. Therefore, improvements are required in the choice of polymer and mechanical properties of the polymer are required. One means of achieving such improvements is to utilise co-polymers such as PHBHHx, which have shown favourable elastic properties when the ratio of HHx to PHB has been increased. Therefore, a PHBHHx polymer based scaffold was investigated as a potential scaffold for tendon repair. Whilst, also investigating the potential of FGF-4, FGF-6 and FGF-8 to differentiate both human embryonic and mesenchymal stem cells towards a tenocyte-like lineage. Finally, an investigation into whether a controlled production of PHBHHx based nanoparticles could produce different nanoparticles sizes that can be predicted and result in differing release profiles. This may allow for the synthesis of size controlled nanoparticles capable of delivering differing drug concentrations and sustained release properties.
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Sutton, Catherine Anne. "Identifying novel cell surface markers for bone marrow stem cell sub-sets." Thesis, University of Bristol, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.557971.
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Osteoarthritis (OA) is a disease which affects hyaline cartilage within the joint and leads to severe pain for the patient. Chondrocytes within the cartilage of OA patients have a limited capability to repair lesions in the tissue and as a result, bone marrow stromal cells (BMSCs) which have the ability to differentiate into cartilage, are being investigated for cell- based therapies for OA. One restriction for the use of BMSCs in clinical trials is the rarity and heterogeneity of the starting population. It is therefore important to identify cell surface markers for BMSCs which produce a high quality cartilage matrix after many population doublings in vitro. Tissue engineered cartilage produced by BMSCs from OA patients at early and late passage after thawing was analysed and compared and it was found that the amount and quality of cartilage matrix was significantly reduced at late passage. To identify the most chondrogenic cells, individual BMSCs were sorted from five OA patients using flow cytometry, cultured for 20 population doublings and assessed for their cartilage tissue engineering capacity. BMSC clones which produced high quality tissue engineered cartilage were compared with BMSCs which produced a low quality cartilage using microarray analysis to find the genetic identities of the different populations. These markers were tested at the protein level using flow cytometric analysis and one protein, HLA DR was identified as a potential marker for poorly chondrogenic BMSCs. HLA DR positive cells were stably removed from the whole population and removal of these cells did not affect the quality of tissue engineered cartilage produced. Two protein markers for the most chondrogenic BMSCs were identified and investigated, FGFR-2 and ROR-2. There were no significant differences in the quality of tissue engineered cartilage produced by FGFR2 positive and negative populations. ROR-2 expression increased in culture as cells became confluent and cells cultured at a high cell density expressing increased ROR-2 had a trend to produce higher quality tissue engineered cartilage than those cultured at low density before chondrogenic analysis. These results highlight the heterogeneity in the cartilage producing capabilities of the BMSC population at the level of the individual cell and document the discovery of ROR-2 as a potential marker for a BMSC population which could be utilised in trials of BMSC based therapies for OA in the future.
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Fellous, Tariq G. "Novel methods to track and identify the stem cell niche." Thesis, Queen Mary, University of London, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.511791.
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Funk, Kevin A. "Stomatal stem cell regulation by a novel protein in Arabidopsis." Honors in the Major Thesis, University of Central Florida, 2009. http://digital.library.ucf.edu/cdm/ref/collection/ETH/id/1260.
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This item is only available in print in the UCF Libraries. If this is your Honors Thesis, you can help us make it available online for use by researchers around the world by following the instructions on the distribution consent form at http://library.ucf.edu/Systems/DigitalInitiatives/DigitalCollections/InternetDistributionConsentAgreementForm.pdf You may also contact the project coordinator, Kerri Bottorff, at kerri.bottorff@ucf.edu for more information.Bachelors
Sciences
Biology
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Hopp, I. "Novel synthetic biomaterials for kidney-derived progenitor/stem cell differentiation." Thesis, University of Liverpool, 2016. http://livrepository.liverpool.ac.uk/3004383/.
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End-stage kidney disease is increasing in prevalence and is associated with high levels of morbidity and mortality. At present, the only treatment options are dialysis or renal transplantation. However, dialysis is very costly and is associated with high levels of morbidity, whereas the problem with transplantation is that there is a shortage of organ donors. For these reasons, over recent years, there has been an increasing interest in developing novel therapies in the field of regenerative medicine including stem cell based therapies and tissue engineering. Stem cells could be used in a number of ways to develop new therapies for kidney disease. Firstly, they could be administered as cell therapies to patients with kidney disease, and secondly, they could be used to generate specific types of renal cells in vitro that could be used for understanding disease mechanisms and for drug discovery programmes. The barriers to the development of novel stem cell therapies include the difficulties in expanding kidney-derived stem cells in culture without altering their phenotype, and directing their differentiation to specific types of renal cells. These issues could be addressed by developing biomaterial substrates that provide an appropriate microenvironment for the successful culture and differentiation of stem cells. Within this study we interrogated a wide range of biomaterial substrates for their capability to direct the differentiation of kidney derived progenitor / stem cells. These materials were thoroughly characterised in terms of their physicochemical properties, such as surface chemistry, nanotopography and wettability by employing a wide range of analytic techniques, including X-Ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), colorimetry and contact angle measurements. We firstly investigated a range of polyacrylates. These substrates were novel in that, they were precisely designed to mimic cell binding motifs of the extracellular matrix stereochemically by using monomeric precursors that display particular chemical functional group chemistries, namely amine, hydroxyl, carboxyl groups or aliphatic spacer groups. We found that these materials differed strongly in the presence and distribution of surface functional group chemistries and topographical features, including the distribution of surface artefacts on a macroscale. Moreover, some of these materials were able to direct the differentiation into specialised renal cell lines. Two substrates, namely ESP 003 and ESP 004, directed the differentiation of kidney derived stem cells into podocytes and two further substrates, namely ESP 007 and BTL 15, directed differentiation into functional proximal tubule cells. These four substrates stimulated cell differentiation to an extent of about 40 to 50% after only 96 h in cell culture. We were moreover able to identify surface physicochemical cues, including surface micro- and nanoscale topography and surface functional group chemistries that are important to stimulate the differentiation process. In addition, we investigated a range of plasma polymer coatings composed of allylamine and octadiene that were provided as homo-or copolymers and in form of chemical gradients, the latter one differing in the amount of nitrogen functional group chemistries across the surfaces. We found that substrates with higher allylamine content displayed a greater amount of nitrogen functional groups and therefore increased in wettability. Moreover, those plasma polymer substrates with higher amine functionality directed kidney progenitor cell differentiation into podocytes, whereas substrates with higher octadiene concentration directed cell differentiation into functional proximal tubule cells, both to an extent of 35 to 45% after only 96 h in culture. To further study cell differentiation, we then incorporated gold nanoparticles underneath these plasma coatings, either in form of homogeneous coatings or in form of a nanoparticle density gradient. We found that surface topographic gradients increased cell differentiation into podocytes 3- to 4-fold, whereas differentiation into proximal tubule cells was only dependent on surface chemistry. Our studies on plasma polymer substrates highlighted not only the great potential of plasma polymers to modify surface functionality of a wide range of surfaces, but also emphasized the great capabilities of surface gradients, whether chemical or topographical in nature, to effect cellular fate. In summary, the results of this study include the identification of biomaterial substrates that have the potential to differentiate kidney-derived progenitor/stem cells in vitro and of the cues that are necessary to assist in the differentiation process. In the future, these biomaterials could be useful for directing the differentiation of pluripotent stem cell-derived renal progenitors to specific types of renal cells that could be used for applications in regenerative medicine and drug discovery programmes.
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Papreck, Justin Ryan. "Novel embryonic stem cell-infused scaffold for peripheral neuropathy repair." Thesis, Atlanta, Ga. : Georgia Institute of Technology, 2008. http://hdl.handle.net/1853/24699.
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Schäkel, Knut, Claudia Poppe, Elfriede Mayer, Christine Federle, Gert Riethmüller, and Ernst Peter Rieber. "M-DC8+ Leukocytes – A Novel Human Dendritic Cell Population." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2014. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-135252.
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Dendritic cells (DC) constitute a heterogeneous leukocyte population having in common a unique capacity to induce primary T cell responses and are therefore most attractive candidates for immunomodulatory strategies. Two populations of blood DC (CD11c+ CD123dim and CD11c– CD123high) have been defined so far. However, their direct isolation for experimental purposes is hampered by their low frequency and by the lack of selective markers allowing large scale purification from blood. Here we describe the monoclonal antibody (mAb) M-DC8, which was generated by immunizing mice with highly enriched blood DC. This mAb specifically reacts with 0.2–1% of blood leukocytes and enables their direct isolation by a one-step immunomagnetic procedure from fresh mononuclear cells. These cells can be differentiated from T cells, B cells, NK cells and monocytes using lineage-specific antibodies. M-DC8+ cells express HLA class II molecules, CD33 and low levels of the costimulatory molecules CD86 and CD40. Upon in vitro culture M-DC8+ cells spontaneously mature into cells with the phenotype of highly stimulatory cells as documented by the upregulation of HLA-DR, CD86 and CD40; in parallel CD80 expression is induced. M-DC8+ cells display an outstanding capacity to present antigen. In particular, they proved to be excellent stimulators of autologous mixed leukocyte reaction and to activate T cells against primary antigens such as keyhole limpet hemocyanin. Furthermore, they induce differentiation of purified allogeneic cytotoxic T cells into alloantigen-specific cytotoxic effector cells. While the phenotypical analysis reveals similarities with the two known blood DC populations, the characteristic expression of Fc=γRIII (CD16) and the M-DC8 antigen clearly defines them as a novel population of blood DC. The mAb M-DC8 might thus be a valuable tool to determine circulating DC for diagnostic purposes and to isolate these cells for studies of antigen-specific T cell primingDieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich
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Schäkel, Knut, Claudia Poppe, Elfriede Mayer, Christine Federle, Gert Riethmüller, and Ernst Peter Rieber. "M-DC8+ Leukocytes – A Novel Human Dendritic Cell Population." Karger, 1999. https://tud.qucosa.de/id/qucosa%3A27632.
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Dendritic cells (DC) constitute a heterogeneous leukocyte population having in common a unique capacity to induce primary T cell responses and are therefore most attractive candidates for immunomodulatory strategies. Two populations of blood DC (CD11c+ CD123dim and CD11c– CD123high) have been defined so far. However, their direct isolation for experimental purposes is hampered by their low frequency and by the lack of selective markers allowing large scale purification from blood. Here we describe the monoclonal antibody (mAb) M-DC8, which was generated by immunizing mice with highly enriched blood DC. This mAb specifically reacts with 0.2–1% of blood leukocytes and enables their direct isolation by a one-step immunomagnetic procedure from fresh mononuclear cells. These cells can be differentiated from T cells, B cells, NK cells and monocytes using lineage-specific antibodies. M-DC8+ cells express HLA class II molecules, CD33 and low levels of the costimulatory molecules CD86 and CD40. Upon in vitro culture M-DC8+ cells spontaneously mature into cells with the phenotype of highly stimulatory cells as documented by the upregulation of HLA-DR, CD86 and CD40; in parallel CD80 expression is induced. M-DC8+ cells display an outstanding capacity to present antigen. In particular, they proved to be excellent stimulators of autologous mixed leukocyte reaction and to activate T cells against primary antigens such as keyhole limpet hemocyanin. Furthermore, they induce differentiation of purified allogeneic cytotoxic T cells into alloantigen-specific cytotoxic effector cells. While the phenotypical analysis reveals similarities with the two known blood DC populations, the characteristic expression of Fc=γRIII (CD16) and the M-DC8 antigen clearly defines them as a novel population of blood DC. The mAb M-DC8 might thus be a valuable tool to determine circulating DC for diagnostic purposes and to isolate these cells for studies of antigen-specific T cell priming.Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.
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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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Dimarakis, Ioannis. "A novel CD34 stem cell subpopulation for ischaemic myocardial injury repair." Thesis, Imperial College London, 2008. http://hdl.handle.net/10044/1/8409.
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Yang, Yawei. "ZNF335: A Novel Regulator of Stem Cell Proliferation and Cell Fate in the Cerebral Cortex." Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10682.
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Though development of the cerebral cortex is of singular importance to human cognition, it remains very poorly understood. Microcephaly, or "small head," is a neurodevelopmental disorder causing significantly reduced cerebral cortex size, and the disease has proved to be a useful model system for elucidating the steps essential for proper cortical development and cognitive function. Many known microcephaly gene products localize to centrosomes, regulating cell fate and proliferation, however, the elucidation of different microcephaly genes with different functions may shed light on previously unidentified key steps of brain development. We identify and characterize a nuclear zinc finger protein, ZNF335/NIF-1, as a causative gene for severe microcephaly, small somatic size, and neonatal death. Znf335-null mice are embryonically lethal and conditional knockout leads to severely reduced cortical size. RNA-interference and postmortem human studies show that Znf335 is essential for neural progenitor self-renewal, neurogenesis, and neuronal differentiation. ZNF335 is a component of a vertebrate-specific, trithorax H3K4-methylation complex, directly regulating REST/NRSF, a master regulator of neural gene expression and cell fate, as well as other essential neural-specific genes. Our results reveal ZNF335 as an essential link between H3K4 complexes and REST/NRSF, and provide the first direct genetic evidence that this pathway regulates human neurogenesis and neuronal differentiation.
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O'Malley, James. "Novel cell surface markers identify routes to iPS cells." Thesis, University of Edinburgh, 2014. http://hdl.handle.net/1842/8883.
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The generation of induced pluripotent stem cells (iPSCs) presents a challenge to normal developmental processes. The low efficiency and heterogeneity of most methods have hindered understanding of the precise molecular mechanisms promoting, and roadblocks preventing, efficient reprogramming. While several intermediate populations have been described, it has proved difficult to characterize the rare, asynchronous transition from these intermediate stages to iPSCs. The rapid expansion of a minor population of reprogrammed cells can also obscure investigation of relevant processes. Understanding of the biological mechanisms essential for successful iPSC generation requires both accurate capture of cells undergoing the reprogramming process and identification of the associated global gene expression changes. Here we demonstrate that reprogramming follows an orderly sequence of stage transitions marked by changes in cell surface markers CD44 and ICAM1, and a Nanog-GFP reporter. RNA-sequencing (RNA-seq) analysis of these populations demonstrates two waves of pluripotency gene up-regulation, and unexpectedly, transient up-regulation of multiple epidermis-related genes, demonstrating that reprogramming is not simply the reversal of normal developmental processes. This novel high-resolution analysis enables the construction of a detailed reprogramming route map, and this improved understanding of the reprogramming process will lead to novel reprogramming strategies.
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Dodhy, Asad. "Population based evaluation of actin cytoskeletal morphometric descriptors as characterisation of stem cell differentiation." Thesis, Queen Mary, University of London, 2018. http://qmro.qmul.ac.uk/xmlui/handle/123456789/46030.
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Stem cells have yet to contribute to their full potential in the field of regenerative medicine and further understanding of the underlying kinetics of cell differentiation could be the step forward. Various methods have been used to characterise stem cell lineage commitment. However, most of these techniques are end-point assays and provide very little information about the changes occurring in the early stages of the differentiation process. This project aims to explore if the structural and geometrical specificity of the cytoskeletal components (actin in particular) encode information regarding cell lineage. Adipogenic and osteogenic differentiation lineages were selected, as they have been extensively studied over the past few decades. We have developed a novel approach to describe cells by defining their cytoskeletal and nuclear morphology in terms of 19 geometric measurements. This set of parameters has a range of complexity, extending from one dimensional (e.g. fibre length, fibre thickness) to compound geometrical readings (e.g. chirality and fibre alignment), while some estimate morphological and mechanical properties of the nucleus i.e. Poisson ratio and chromatin condensation. A proprietary image analysis algorithm is used to analyse fluorescent images of cells biochemically and mechanically stimulated to differentiate for a period of up to 10 days. Our analysis pipeline is currently optimised for images acquired at x20 magnification using epi-fluorescence but can be further adapted for high throughput live cell imaging. Factorial analysis of the measured features showed that some parameters change markedly in the early stages of differentiation. More interestingly we observed these changes to be non-linear and non-monotonic. This analysis, in light with previously published literature on the subject has allowed us to more intricately hypothesise probable mechanisms involved with mechanotransduction which direct the lineage commitments. As our technique quantifies the morphology of individual cells, we used our extracted feature data to characterise each cell using a multivariate predictive model (LDA).
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Ho, Daniela Gattegno. "Characterising the function of a novel embryonic stem cell-associated signal transducer, Gab1β." Thesis, University of Edinburgh, 2009. http://hdl.handle.net/1842/4252.
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Activation of Ras/mitogen-activated protein kinase (ERK MAPK) signalling controls the differentiation of mouse embryonic stem (ES) cells. An established modulator of the ERK MAPK pathway is the IRS-1 (Insulin Receptor Substrate 1) family adaptor protein Gab1 (Grb2-associated binder 1). Gab1 is ubiquitously expressed and is activated by a wide range of cell surface receptors, mediating growth factor, cell-cell and cell-substratum interactions. The N-terminal region of Gab1 contains a pleckstrin homology (PH) domain required for membrane binding and a nuclear localisation sequence (NLS) that facilitates nuclear translocation. Undifferentiated mouse ES cells preferentially express high levels of a novel form of Gab1 (Gab1β) lacking the N-terminal region. Based on its novel structure and abundance, Gab1β may act in a dominant negative manner by binding and mislocalising downstream effectors. Alternatively, it may have a deregulated function unrestrained by the PH or NLS domains. Data presented here shows that Gab1β is tyrosine phosphorylated in response to the self-renewal factor Leukemia Inhibitory Factor (LIF) and/or Foetal Bovine Serum (FBS) stimulation. This then leads to the formation of complexes with Shp2 and the p85 subunit of PI3K. Experiments comparing the responses of wild-type and Gab1β knock-out ES cells indicate that Gab1β enhances ERK and potentially AKT phosphorylation in response to LIF. In contrast, Gab1β has a negative effect on ERK and AKT phosphorylation in response to IGF-1 (Insulin Growth Factor 1). These results suggest that the contribution of Gab1β to signalling activity is receptor specific and may imply that the response of ES cells to ERK activation is context specific. By reintroducing fluorescently tagged Gab1 proteins into Gab1β knockout ES cells, I investigated the localisation of Gab1β in ES cells. Gab1β localised at the cell membrane as well as in a perinuclear body. I next investigated the potential role of Gab1β in the differentiation of ES cells into neural precursors. A monolayer differentiation protocol was used to differentiate Gab1β wild-type and knock-out cells into neural precursors. Furthermore, the effect of insulin on the emergence of neural precursors from Gab1β-targeted cells was also explored.
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CASTIGLIONI, ANDREA. "IDENTIFICATION OF NCAM1 AS A NOVEL PROGNOSTIC PROSTATE CANCER STEM CELL BIOMARKER." Doctoral thesis, Università degli Studi di Milano, 2022. https://hdl.handle.net/2434/946385.
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Serum prostate-specific antigen and Gleason grade are parameters routinely used for risk stratification in prostate cancer (PCa), but they present some limitations in the prediction of disease progression and in their use to guide clinical decision making. Prostate cancer stem cells (PCSCs) are widely considered to be responsible for tumorigenesis, disease progression and therapy failure. Their identification and characterization are mandatory for understanding the intricate intratumoral heterogeneity (spatial and molecular) of PCa and for developing relevant clinical tools to effectively manage patients and tailor therapy.
Here, we proposed the surface glycoprotein neural cell adhesion molecule (NCAM1/CD56), a known marker of neuroendocrine (NE) cells, as a novel PCSC marker that provides prognostic information and molecular insights into the process of tumorigenesis. NCAM1 defines clusters of cells enriched in proliferative inflammatory atrophy (PIA) regions, without NE traits (hereafter referred as NCAM1+). In a retrospective cohort of 406 PCa patients treated with radical prostatectomy (RP), we uncovered that NCAM1 is an independent prognostic marker for predicting distant metastasis and biochemical recurrence and its expression in radical prostatectomy biopsies concurred with diagnostic biopsies (concordance 87.6%).
Using the human cell lines, LNCaP (androgen-sensitive) and DU145 (androgen-insensitive), we found that NCAM1, but not other candidate PCSC markers, allowed FACS-based prospective purification of PCa cells displaying i) unique self-renewal ability in vitro, in a serial 3D-Matrigel organoid propagation assay, ii) tumorigenic potential upon limiting dilution transplantation in vivo. Relevant to real-life human PCa, we found that the ability to generate primary-derived organoids (PDOs) from dissociated high Gleason PCa biopsies exclusively resided in the purified NCAM1+ cell fraction, a property that was efficiently inhibited by treatment with an anti-NCAM1 blocking monoclonal antibody. We also found that the progressive development of adenocarcinoma in transgenic TRAMP mice crossed with NCAM1-/- mice was blocked at very early stages of tumorigenesis, indicating that genetic NCAM1 ablation prevents premalignant lesions to expand and progress to advanced stages.
PCa is a paradigm tumor model for clinical, spatial and molecular heterogeneity and this heterogeneity is reflected in the NCAM1+ cell population. Single cell-RNA sequencing (sc-RNASeq) of purified NCAM1+ cells (cell lines and primary human PCa biopsies) uncovered heterogenous cellular states reflected in several distinct clusters. Phylogenetic tree reconstruction of the evolutionary relationship among the different clusters along with single cell trajectory analysis revealed the existence of a cell fraction with basal traits (p63+/AR-/CD117+ cells) and a quiescent phenotype, sitting at the apex of the hierarchical structure of the NCAM1+ population. These cells were functionally characterized by Hedgehog signaling which drives NCAM1+/CD117+ -PCSC self-renewal ability. Moreover, they were molecularly characterized by a transcriptional signature called “Stem Score” which could have potential as a prognostic tool for identifying patients at risk of biochemical recurrence (BCR) and distant metastasis.
Androgen deprivation therapy (ADT) is the standard management for advanced PCa. Despite its initial effectiveness, the majority of patients relapse and develop castration resistant prostate cancer (CRPC), which is thought to be mediated by resistant PCSCs. ADT-treated NCAM1+ cells isolated from both dissociated human PCa biopsies and the LNCaP cell line enter into a quiescent state and retained the ability to generate organoids in vitro and tumors in vivo, escaping the ADT-induced senescence observed in NCAM1- cells. By global transcriptional profiling RNASeq analysis of NCAM1-overexpressing LNCaP cells, followed up by high-resolution studies, we uncovered an NCAM1-FGFR2-FGF18 molecular circuitry and an NCAM1-DNER-Notch1 molecular circuitry that mediate resistance to ADT-induced senescence. Thus, targeting these pathways with FGFR or Notch pathway inhibitors could represent a promising strategy to eradicate ADT-resistant NCAM1+ cells and prevent CRPC.
Taken together, these data highlight NCAM1 as a novel predictive-prognostic biomarker in PCa, which could significantly improve the clinical management of PCa patients and pointed out druggable molecular pathways that could be targeted to eradicate “true” NCAM1+-PCSCs (self-renewal) and stem-like NCAM1+ progenitors (ADT-resistance).
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Tordonato, C. "A NOVEL MICRO-RNA FAMILY AS MOLECULAR DETERMINANT OF MAMMARY STEM CELLS." Doctoral thesis, Università degli Studi di Milano, 2015. http://hdl.handle.net/2434/264788.
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A novel microRNA family as molecular determinant in mammary stem cells
MicroRNAs (miRNAs) are an evolutionarily conserved class of small (18-22 nucleotides) noncoding RNAs involved in the regulation of a variety of cellular and developmental processes. MiRNAs have recently emerged as key regulators of transcriptional programs that control self-renewal and the cell-fate of stem cells (SCs). In the breast compartment, forced expression of some miRNAs (e.g. miR-200, let-7 and miR-93) was shown to inhibit normal and/or cancer SCs by silencing self-renewal determinants. In fact, these miRNAs are poorly or not expressed in the SC compartment, and their stem-inhibiting function is mainly achieved through the induction of differentiation.
The aim of this project is to identify miRNAs specifically expressed in mammary SCs isolated from normal and cancer samples that could act as markers and/or as regulators of stem cell biology. We will also evaluate their potential as novel therapeutic targets in breast cancer.
Using an innovative technique developed in our lab for the isolation of quasi-pure SCs/CSCs, we identified a miRNA family as being highly enriched in the SC compartment, in both primary normal and tumor samples. Endogenous levels of miRNAs of this family can stratify distinct sub-populations with different SC abilities, and in human cancer high miRNA levels correlate with a basal tumor subtype and with an elevated CSC content. Furthermore, depletion of these miRNAs by a synthetic sponge impair self-renewal of SCs/CSCs, as measured by a serial mammosphere propagation assay in human cancer cell lines and in mouse primary epithelial cells. In vivo, miRNA loss reduced the frequency of tumor formation, as well as the mean tumor volume and the frequency of CSCs measured with either an established human basal-like cancer cell line or human patient-derived xenografts (PDXs). To determine the molecular mechanisms underlying the regulation of SCs/CSCs behavior, we employed an unbiased approach assessing the effects of miRNA loss on global gene expression profile, coupled with the prediction of miRNA targets involved in stem cell biology. We identified 91 stem cell genes that are putatively targeted by this miRNA family, belonging to pathways critically involved in the regulation or in the mainteinance of stem cell traits.
In conclusion, our results suggest that this SC-specific miRNA family could potentially be used as novel therapeutic targets for breast cancer treatment. This study also provides insights into the mechanisms that sustain CSCs growth in the breast, involving complex molecular circuits of coding as well as noncoding RNAs.
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Kanani, Chirantan. "Cell Printing: A novel method to seed cells onto biological scaffolds." Digital WPI, 2012. https://digitalcommons.wpi.edu/etd-theses/332.
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Bioprinting, defined as depositing cells, extracellular matrices and other biologically relevant materials in user-defined patterns to build tissue constructs de novo or to build upon pre-fabricated scaffolds, is among one of the most promising techniques in tissue engineering. Among the various technologies used for Bioprinting, pressure driven systems are most conducive to preserving cell viability. Herein, we explore the abilities of a novel bioprinter - Digilab, Inc.'s prototype cell printer. The prototype cell printer (Digilab Inc., Holliston, MA) is an automated liquid handling device capable of delivering cell suspension in user-defined patterns onto standard cell culture substrates or custom-designed scaffolds. In this work, the feasibility of using the cell printer to deliver cell suspensions to biological sutures was explored. Cell therapy using stem cells of various types shows promise to aid healing and regeneration in various ailments, including heart failure. Recent evidence suggests that delivering bone-marrow derived mesenchymal stem cells to the infarcted heart reduces infarct size and improves ventricular performance. Current cell delivery systems, however, have critical limitations such as inefficient cell retention, poor survival, and lack of targeted localization. Our laboratories have developed a method to produce discrete fibrin microthreads that can be bundled to form a suture and attached to a needle. These sutures can then be seeded with bone-marrow derived mesenchymal stem cells to deliver these cells to a precise location within the heart wall, both in terms of depth and surface localization. The efficiency of the process of seeding cells onto fibrin thread bundles (sutures) has previously been shown to be 11.8 ± 3.9 %, suggesting that 88% of the cells in suspension are not used. Considering that the proposed cell-therapy model for treatment of myocardial infarction contemplates use of autologous bone-marrow derived stem cells, an improvement in the efficiency of seeding cells onto the fibrin sutures is highly desirable. The feasibility of using Digilab's prototype cell printer to deliver concentrated cell suspension containing human mesenchymal stem cells (hMSCs) directly onto a fibrin thread bundle was explored in this work, in order to determine if this technology could be adapted to seed cells onto such biological sutures. First the effect of the printing process on the viability of hMSCs was assessed by comparing to cells dispensed manually using a hand-held pipette. The viability of hMSCs 24 hours post-dispensing using the cell printer was found to be 90.9 ± 4.0 % and by manual pipetting was 90.6 ± 8.2 % (p = ns). Thereafter a special bioreactor assembly composed of sterilizable Delrin plastic and stainless steel pins was designed to mount fibrin thread bundles onto the deck of the cell printer, to deliver a suspension containing hMSCs on the bundles. Highly targeted delivery of cell suspension directly onto fibrin thread bundles (average diameter 310 µm) was achieved with the bundle suspended in mid-air horizontally parallel to the printer's deck mounted on the bioreactor assembly. To compare seeding efficiency, fibrin thread bundles were simultaneously seeded with hMSCs using either the cell printer or the current method (tube-rotator method) and incubated for 24 hours. Seeded thread bundles were visualized using confocal microscopy and the number of cells per unit length of the bundle was determined for each group. The average seeding efficiency with the tube rotator method was 7.0 ± 0.03 % while the cell printer was 3.46 ± 2.24% (p = ns). In conclusion, the cell printer was found to handle cells as gently as manual pipetting, preserve their viability, with the added abilities to dispense cells in user-defined patterns in an automated manner. With further development, such as localized temperature, gas and humidity control on the cell printer's deck to aid cell survival, the seeding efficiency is likely to improve. The feasibility of using this automated liquid handling technology to deliver cells to biological scaffolds in specified patterns to develop vehicles for cell therapy was shown in this study. Seeding other cell types on other scaffolds along with selectively loading them with growth factors or multiple cell types can also be considered. In sum, the cell printer shows considerable potential to develop novel vehicles for cell therapy. It empowers researchers with a supervision-free, gentle, patterned cell dispensing technique while preserving cell viability and a sterile environment. Looking forward, de novo biofabrication of tissue replicates on a small scale using the cell printer to dispense cells, extracellular matrices, and growth factors in different combinations is a very realistic possibility.
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Gertow, Karin. "Human embryonic stem cells : a novel model system for early human development /." Stockholm, 2006. http://diss.kib.ki.se/2006/91-7140-749-9/.
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MIWA, Keiko, Jong-Kook LEE, Kyoko HIDAKA, Rong-qian SHI, Takayuki MORISAKI, and Itsuo KODAMA. "Optimal Population of Embryonic Stem Cells in "Hanging Drop" Culture for in-vitro Differentiation to Cardiac Myocytes." Research Institute of Environmental Medicine, Nagoya University, 2002. http://hdl.handle.net/2237/2795.
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Smith, Aileen M. "Embryonic stem cell differentiation : a novel approach to gene targeting in myeloid cells." Thesis, University of Edinburgh, 2005. http://hdl.handle.net/1842/29369.
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This thesis demonstrates that macrophages can be generated from the culture of ES cells. Although low in number these macrophages show similar morphology and surface phenotype to bone marrow derived macrophages. Importantly, these ES macrophages readily phagocytosed latex beads and apoptotic cells. In additions, ES cells produced cells with a dendritic cell phenotype that were capable of apoptotic cell phagocytosis and maturation. Generation of these myeloid cells from ES cells was strongly dependent on serum and the parent ES cell line. Dendritic cells could be generated from integrin αv disrupted ES cells. These DCs retained the ability to phagocytose apoptotic cells suggesting that integrin αv is not essential for this process. This study also investigates the properties of Cre fused to two transduction proteins. Although Cre retained its recombinase activity as a fusion protein these were unable to translocate into cells. However Cre could be delivered to both primary macrophages and cultured epithelial cells using a replication deficient adenovirus allowing deletion of the targeted genes. Intriguingly, the apoptotic cell phagocytosis was unaffected by lack of integrin αv but could no longer be inhibited by the integrin antagonistic peptide RGD. In conclusion, the capacity of ES cells to differentiate to myeloid cells combined with the ability to deliver Cre to silently targeted myeloid cells provide powerful systems for studying the role of specific genes in phagocytosis. Use of these approaches with integrin αv demonstrates that this gene is not essential for apoptotic cell phagocytosis. However the ability of specific antagonists to inhibit phagocytosis show that integrin αv is intimately involved in this process in normal cells.
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Elsammak, Mohamed. "Novel strategies for the enhancement of stem cell engraftment following in utero transplantation." Thesis, University of Nottingham, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.364425.
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Kress, Brian J. "Synthesis of Novel Small Molecule PPARδ Agonists for Controlling Mesenchymal Stem Cell Osteogenesis." University of Toledo Health Science Campus / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=mco1564751044043639.
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Zhao, Yiding. "Characterization of the developing haematopoietic stem cell niche using a novel immortalization system." Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/22025.
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Embryonic haematopoiesis is a complex process under intensive research. Murine definitive Haematopoietic Stem Cells (HSCs) originates from the Aorta-Gonad-Mesonephros (AGM) region of E10.5 embryo. It is thought that definitive HSCs arise from endothelial lining of dorsal aorta. However, detail of HSC specification in the developing embryo remains elusive. One way to deciphering events occurred during HSC specification is to derive cell lines from the developing HSC niche. Previous work by Oostendorp et al. showed the AGM and fetal liver derived lines could maintain HSCs in vitro (Oostendorp, Harvey et al. 2002). In this study, I established a more robust immortalization system using normal SV40 large T antigen delivered via Neon™ electroporation system. The new immortalization system achieved direct immortalization without going through crisis. And it is compatible with small number of primary cells dissected from different haematopoietic niches. With my new system, multiple cell lines from different haematopoietic sites at different developmental points are derived. Moreover, some of these lines demonstrated ability to mature precursors from E9.5 embryo (pro-HSCs) to definitive HSC without help of growth factors. This result is better compared to OP9 stromal lines. Such data proved usefulness of using stromal cell lines to study haematopoietic specification.
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Carradori, Dario. "Novel nanoparticle-based drug delivery system for neural stem cell targeting and differentiation." Thesis, Angers, 2017. http://www.theses.fr/2017ANGE0056/document.
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Les cellules souches neurales (CSNs) se situent dans des régions spécifiques du système nerveux central qui sont appelées niches. Ces cellules sont capables de se répliquer ou se différentier en cellules neurales spécialisées (neurones, astrocytes et oligodendrocytes). C’est grâce à cette propriété de différentiation que les CSNs sont étudiées comme thérapie chez les patients atteints d’une maladie neurodégénérative. En effet, elles pourraient remplacer les cellules neurales altérées et ainsi restaurer les fonctions neurologiques. De nombreuses approches ont été développées afin de stimuler la différentiation des CSNs, dont la plus prometteuse est la différentiation des cellules endogènes directement au sein de leurs niches. Actuellement, il n’existe pas de molécule active ou de système thérapeutique qui cible les CSNs endogènes et qui induit leur différentiation simultanément. Le but de ce travail est de fournir un système de délivrance de molécules bioactives capable de cibler les CSNs endogènes et d'induire leur différenciation in situ. Nous avons développé et caractérisé des nanoparticules lipidiques (LNC), un système de délivrance très versatile. NFL-TBS.40-63, un peptide ciblant les CSNs, a été adsorbé à la surface des LNC afin de les diriger contre les CSNs endogènes. Nous avons observé que ces NFL-LNC ne ciblaient que les CSNs du cerveau et pas de la moelle. Afin d’étudier les interactions spécifiques entre les nanoparticules et les CSNs, nous avons caractérisé et comparé les propriétés de leur membrane plasmique. Enfin, nous avons encapsulé de l’acide rétinoïque, une molécule connue pour stimuler la différentiation des CSNs, dans les LNC-NFL et étudié leur impact sur la différentiation de CSNs in vitro et in vivo. Ce travail contribue au développement de thérapies efficaces et sures pour le traitement de maladies neurodégénératives à travers la différentiation de CSNs endogènesNeural stem cells (NSCs) are located in specific regions of the central nervous system called niches. Those cells are able to self-renew and to differentiate into specialized neuronal cells (neurons, astrocytes and oligodendrocytes). Due to this differentiation property, NSCs are studied to replace neuronal cells and restore neurological functions in patients affected by neurodegenerative diseases. Several therapeutic approaches have been developed and endogenous NSC stimulation is one of the most promising. Currently, there is no active molecule or therapeutic system targeting endogenous CSNs and inducing their differentiation at the same time. The aim of the work was to provide a drug delivery system able both to target endogenous CSNs and to induce their differentiation in situ. Here, we developed and characterized lipidic nanoparticles (LNC) targeting endogenous NSCs. A peptide called NFL-TBS.40-63, known for its affinity towards NSCs, was adsorbed at the surface of LNC. We observed that NFL-LNC specifically targeted NSC from the brain and not from the spinal cord in vitro and in vivo. To explain this specificity, we characterized and compared NFL-LNC interactions with the plasmatic membrane of both cell types. Finally, we demonstrated that by loading retinoic acid in NFL-LNC we were able to induce brain NSC differentiation in vitro and in vivo. This work contributes to the development of efficient and safe therapies for the treatment of neurodegenerative disease via the differentiation of endogenous NSCs
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MONTERISI, SIMONA. "HOXB7 IN LUNG CANCER: A NOVEL ROLE IN STEM CELL AND IPS BIOLOGY." Doctoral thesis, Università degli Studi di Milano, 2016. http://hdl.handle.net/2434/362619.
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Current diagnostic tools do not allow prognostic evaluation of patients with early stage lung cancer or selection of patients that might benefit from adjuvant chemotherapy. Therefore, the identification of novel prognostic markers in early-stage lung cancer is paramount. In this scenario, the transcription factor HOXB7, belonging to the homeobox family, has been shown to correlate with poor prognosis in different types of cancer and recently also in stage I lung adenocarcinoma. To better understand the prognostic implication of alterations in HOXB7 expression in lung cancer, we performed a bioinformatics analysis of multiple lung cancer expression datasets in order to identify gene sets representing cancer-relevant biological functions enriched in high-HOXB7 expressing tumors. We found several gene sets enriched in high-HOXB7 expressing tumors representing molecular mechanisms involved in epithelial to mesenchymal transition, in cancer progression, and, interestingly, in stemness and cellular reprogramming. Based on these results, we hypothesized that HOXB7 may have a role in the expansion of the stem cell compartment in cancer, a mechanism that has been shown to be a hallmark of enhanced tumorigenicity and of increased metastatic potential.
Analysis of the stem-related surface marker CD90 revealed that overexpression of HOXB7 in lung cells increases the CD90high sub population. CD90high, but not CD90low cells, are able to form spheroids, which is an hallmark of stemness. Indeed, the sphere forming efficiency of normal lung BEAS-2B cells was 22% and 1.64% in CD90high and CD90low populations, respectively. In addition, we found that silencing of LIN28B counteracts the expansion of the CD90high population. LIN28B was recently described as an oncogene that regulates the cancer stem cell compartment. We found that LIN28B is under the direct transcriptional control of HOXB7. Therefore, we propose a novel molecular mechanism driven by HOXB7 and can increase stem-like properties in lung cells. We further demonstrated that the HOXB7-LIN28B axis plays an important role in reprogramming of adult cells into induced pluripotent stem cells (iPS). Indeed, HOXB7 may enhance the reprogramming efficiency achieved by the three genes OCT4, KLF4, SOX2 in both mouse embryonic fibroblast and human epithelial BEAS-2B cells by substituting MYC in the transcription factor cocktail of reprogramming factors used by Yamanaka. Of note, LIN28B silencing strongly decreases the number of reprogrammed colonies in high-HOXB7 expressing cells. These findings suggest that HOXB7, through transcriptional induction of the LIN28B gene, activates a program relevant for stem/iPS cell biology and for tumor progression, possibly opening a new line of research for the development of more effective therapies for metastatic lung cancer patients.
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Ker, Dai Fei Elmer. "Monitoring and Engineering of the Stem Cell Microenvironment: Inkjet Printing of Growth Factors onto Novel Scaffolds for Controlling Musculoskeletal Stem Cell Differentiation and Alignment." Research Showcase @ CMU, 2012. http://repository.cmu.edu/dissertations/165.
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The primary objective of this study was to develop approaches for controlling and monitoring stem cell behavior in vitro and in vivo for potential applications in regenerative medicine. To control stem cell behavior in vitro and in vivo, signaling molecules including growth factors (GFs) were spatially patterned onto novel substrates and scaffolds to instruct stem cells to undergo desired cell behaviors such as musculoskeletal differentiation in register to the biochemical and geometric cues supplied. The premise for this approach is based on the biological phenomenon whereby stem cell behavior can be directed by instructive cues present in its immediate vicinity or microenvironment. Using this methodology, a primitive muscle-tendonbone (MTB) unit was patterned in vitro while ectopic bone tissue was patterned in vivo. In addition, the effect of inflammatory and anti-inflammatory microenvironments on osteoblast differentiation was characterized since inflammation is an important component of the wound healing response. In such studies, inflammatory microenvironments were found to inhibit osteoblast differentiation in several musculoskeletal progenitor cells and this inhibition could be reversed with anti-inflammatory IL-10. Primary cells such as muscle-derived stem cells (MDSCs) were also found to display differing levels of sensitivity to such osteoblast inhibition. To monitor stem cell behavior in vitro, a computer-vision based system was developed for realtime adaptive subculture of muscle-progenitor cells. This computer-directed subculture system minimizes human labor and subjectivity during progenitor cell expansion and cells cultured with this system were comparable to those grown by a human operator. The work described here illustrates methods for controlling and monitoring stem cell behavior and may have potential applications in regenerative medicine.
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Dunbar, Karen Jane. "Investigating the effects of aspirin on cell invasion, epithelial-mesenchymal transition and cancer stem cell population in colorectal cancer." Thesis, University of Edinburgh, 2017. http://hdl.handle.net/1842/28827.
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Colorectal cancer (CRC) is the fourth most common cause of cancer related deaths in the UK with the prognosis dependent on the degree of tumour invasion and presence of metastasis at diagnosis. An important step in the invasion and metastasis of solid tumours is the loss of cell-cell junctions and the acquirement of a more motile mesenchymal phenotype which is facilitated by the epithelial-mesenchymal transition (EMT). The presence of EMT is linked with a more aggressive, invasive tumour and subsequent poor prognosis. In addition to roles in motility and invasion, EMT can induce a cancer stem cell phenotype in a subset of tumour cells. Cancer stem cells (CSCs) are a subpopulation of cells capable of self-renewal and maintaining a cellular population whilst displaying increased therapeutic resistance. Induction of EMT and CSCs can be regulated by common signalling pathways with expression of EMT transcription factors inducing CSCs expression. Understanding the signalling pathways regulating EMT and CSC formation in cancer is important for preventing of metastasis and combating therapeutic resistance. Aspirin’s role in cancer prevention has been established for a number of years with aspirin treatment reducing the incidence of CRC. Recently, evidence has emerged suggesting aspirin treatment may have post-diagnosis benefits and increase survival rates of CRC patients. A potential mechanism for the post-diagnosis benefit of aspirin is the inhibition of EMT and CSC formation which both facilitate tumour progression and metastasis. Aspirin has been demonstrated to suppress the migratory and invasive capacity of lung cancer cell lines by inhibiting EMT. Whilst aspirin has been shown to inhibit platelet-induced EMT in CRC, the direct effects of aspirin on EMT in CRC cell lines has not been established. I hypothesis that aspirin inhibits cell migration, invasion and EMT in CRC which results in a reduction in the CSC population and contributes to the clinical benefit of post-diagnosis aspirin. Using CRC cell lines, I have demonstrated that aspirin treatment inhibits cell migration, invasion, motility and promotes an epithelial phenotype. These results have been confirmed in human organoids and mouse intestinal adenoma in vivo models. Aspirin also promotes a budding phenotype in Apc deficient organoids and reduces expression of stem cell markers in both mouse and human tissue. Aspirin inhibits the mTOR and Wnt signalling pathways in vivo which have the ability to regulate EMT and CSCs although signalling dependency has not been determined. Regardless, aspirin is decreasing the cancer stem cell population and promoting a non-invasive epithelial phenotype which may explain some of the previously described post-diagnosis benefits.
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Li, Hua. "Cell and gene therapies for diabetes exploration of novel therapeutic approaches /." Click to view the E-thesis via HKUTO, 2006. http://sunzi.lib.hku.hk/hkuto/record/B38430800.
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Nili, Ahmadabadi Elham. "Development of a novel mesenchymal stromal cell (MSC) therapy for repairing the cornea." Thesis, Queensland University of Technology, 2018. https://eprints.qut.edu.au/122897/1/Elham_Nili%20Ahmadabadi_Thesis.pdf.
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This thesis has produced advances in our understanding of the biology and potential clinical application of stem cells to aid the treatment of patients with severe eye injuries. This research evaluated the therapeutic potential of a stem cell (called Mesenchymal Stromal Cells (MSCs)) isolated from the peripheral margin of the cornea, known as the limbus. Firstly, a method for routinely isolation and propagation of human limbal MSCs was optimized. Subsequently, the performance of those cells on a silk fibroin membrane was examined.
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Larsson, Jimmy. "Neural stem and progenitor cells cellular responses to known and novel factors /." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-110722.
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Milwid, Jack Miles. "Discovery of novel anti-inflammatory proteins inspired by bone marrow mesenchymal stem cell secretions." Thesis, Massachusetts Institute of Technology, 2011. http://hdl.handle.net/1721.1/68517.
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Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2011.Cataloged from PDF version of thesis.
Includes bibliographical references (p. 114-133).
Bone marrow mesenchymal stem cells (MSCs) may soon become the first FDA-approved stem cell therapy for autoimmune and inflammatory disease. Our lab originally hypothesized that much of the therapeutic activity of MSCs may be attributed to molecules secreted by these cells. This thesis will test this hypothesis, with an emphasis on translational steps towards clinical product development, including the identification of novel proteins secreted by MSCs. The first part of the thesis consists of studies we performed to test whether MSC conditioned medium (MSC-CM) can treat rats undergoing cisplatin-induced acute kidney injury (AKI). When AKI rats were treated with MSC-CM, we observed a survival benefit and significant protection of renal function compared to controls. The second part of the thesis will describe the development of a device designed for sustained delivery of MSC secreted factors to dialysis-dependent AKI subjects. We tested these devices for cell function, stability and viability when subjected to conditions that model future clinical operation. Finally, inspired by the therapeutic capacity of MSC secreted factors, this thesis will conclude with the introduction of a new method that we developed to uncover novel anti-inflammatory proteins from MSCs. This method revealed four previously unidentified cytokine modulators, two of which we found significantly promote IL-1 0 and suppress TNF-a in mice challenged with endotoxin. When leveraged as novel therapeutics for lethal endotoxemic shock, these two most potent modulators protected mice and provided for a significant survival benefit compared to vehicle controls. Together, these results demonstrate the power of MSC secreted factors in the context of inflammatory disease, and propose new tactics for elucidating potent secreted products from cells.
by Jack Miles Milwid.
Ph.D.
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Gibson, Claire. "Development of novel microcarriers for adipose derived stem cell material directed differentiation and expansion." Thesis, Cardiff University, 2012. http://orca.cf.ac.uk/47616/.
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Regenerative medicine and tissue engineering are being revolutionised by developments in the field of stem cell science. Mesenchymal Stem Cells (MSCs) are emerging as a desirable tool in regenerative medicine and cell therapy due to their wide ranging differentiation potential, large expansion capacity, and their lack of immune rejection following transplantation. Early in vivo studies have demonstrated therapeutic effects of hMSCs; however to clinically exploit the potential of hMSCs, the adherent cell type must be expanded to therapeutically relevant lot sizes (109 to 1012 cells). Hence now there is a need to develop protocols for stable, controlled in vitro expansion, isolation and preservation of a homogenous population of functionally viable cells. Specifically a practical, clinically safe and scalable system which adheres to current GMP guidelines is required to develop reproducible and cost effective therapeutic products. Here we describe the design, manufacture and characterisation of biofunctionalised hydrogel microcarriers containing ECM derived adhesion peptides and a range of compressive moduli for adipose derived stem cell expansion. Microfluidic devices were employed to produce monodisperse spherical particles which were polymerised in situ. In addition, these microcarriers have tunable characteristics which make them a particularly useful tool for the systematic investigation of cellular responses. Microcarriers modified to contain fibronectin and laminin derived peptides supported ADSC attachment and growth in a concentration dependent manner. ADSCs cultured on peptide modified microcarriers were capable of differentiating into osteocytes, chondrocytes and adipocytes, indicating cells cultured on microcarriers maintained multipotency. Substrate compressibility was found to effect ADSC differentiation, corroborating previous literature reports. Bioreactor culture demonstrated successful ADSC expansion with fold increases in cell number far higher than have previously been reported in the literature. High cell seeding densities produced large quantities of viable cells. However, decreasing initial cell seeding density, increased the total fold expansion and reduced cell doubling rates.
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Hoke, Nicholas. "PHOSPHODIESTERASE-5 INHIBITION: A NOVEL STRATEGY TO IMPROVE STEM CELL THERAPY IN THE HEART." VCU Scholars Compass, 2011. http://scholarscompass.vcu.edu/etd/182.
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Several studies have shown cellular replacement therapy as a treatment strategy of myocardial infarction but results have been limited. Therefore, enhancing the therapeutic potential of stem cells injected into ischemic microenvironments by novel preconditioning (PC) techniques is critical for improving cellular therapy. Recent studies have shown that inhibition of phosphodiesterase-5 (PDE-5) is a powerful strategy to precondition the heart and cardiomyocytes against ischemia/reperfusion injury. We therefore tested the hypothesis that inhibition of PDE-5 with sildenafil (Viagra®) or selective knockdown with a silencing vector in adipose derived stem cells (ASCs) would improve their survival after ischemia/reoxygenation in vitro and enhance cardiac function following myocardial implantation in vivo. ASCs were treated with sildenafil or infected with PDE-5 silencing vector shRNA (shRNAPDE-5). The cells were subjected to simulated ischemia (SI) and reoxygenation (RO). Both sildenafil and shRNAPDE-5 significantly reduced cell injury, as shown by improved viability, decreased lactate dehydrogenase, and apoptosis. The preconditioned ASCs also demonstrated an increase in the release of growth factors including VEGF, b-FGF, and IGF. The protective effect against SI/RO injury was abolished by inhibition of protein kinase G (PKG) using both a pharmacological inhibitor and selective knockdown with shRNAPKG1α suggesting a PKG-mediated mechanism. To show the effect of preconditioned ASCs in vivo, adult male CD-1 mice underwent myocardial infarction (MI) by occlusion of the left descending coronary artery, followed by direct injection of PBS (control), non-preconditioned ASCs, or preconditioned ASCs (4x105) ASCs into the left ventricle (LV). Preconditioned ASC-treated hearts showed consistently superior cardiac function by all measures as compared with PBS and non-preconditioned ASCs after 4 weeks of treatment. Post-mortem histological analysis demonstrated that preconditioned ASC-treated mice had significantly reduced fibrosis, increased vascular density and reduced resident myocyte apoptosis as compared to mice receiving non-preconditioned ASCs or PBS. VEGF, b-FGF, and Ang-1 were also significantly elevated 4 weeks after cell therapy with preconditioned ASCs. Our data suggests that genetic or pharmacological inhibition of PDE-5 is a powerful new approach to improve stem cell therapy following myocardial infarction.
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Mioulane, Maxime. "Human embryonic stem cell-derived cardiomyocytes as a novel in vitro cardiac model system." Thesis, Imperial College London, 2011. http://hdl.handle.net/10044/1/6980.
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Objectives: Human embryonic stem cell-derived cardiomyocytes (hESC-CM) are investigated as a source of cardiac cells for cell therapy and as a novel model for drug screening. In this thesis, we asked whether hESC-CM can serve as a valuable system for testing compounds in toxicological screens and we undertook a characterization of their contraction properties with regards to their phenotypic variability. Methods: Contraction was measured using edge detection microscopy (Ionoptix) on clusters of hESC-CM and in paced isolated hESC-CM or other isolated ventricular cardiomyocytes. Cell death assay was performed in adherent isolated cardiomyocytes. hESC-CM were stained with fluorescent markers of apoptosis and necrosis and scanned using automated fluorescence microscopy (Cellomics). Results: Clusters of hESC-CM underwent maturation as seen by the change in their basal beating rate with time of culture and the negative chronotropic effect of adenosine. The development of the chronotropic response to adenosine was heterogenous during the intermediate stage and correlated with the effect of Carbarchol, suggesting the emergence of distinct cardiomyocyte subtypes. Adenosine mediated its effect through the activation of A1 Adenosine receptor and positive inotropic effect in isolated hESC-CM possibly through A2a, as in human failing cardiomyocytes. Apoptosis and necrosis could be detected and quantified specifically in hESC-CM as well as in neonates, allowing further direct inter-species comparison in high content screening approach. hESC-CM and neonates differed in term of sensitivity to apoptosis in response to Chelerythrine and to β-adrenergic stimulation. The pro and anti-apoptotic effects of different immuno-suppressive drugs were evaluated in hESC-CM as a prerequisite for future transplantation. Conclusion: hESC-CM can serve as an alternative cardiomyocyte model for investigating the effect of compounds on contraction and toxicity. Our study points out the developmental stage and possibly the differentiation method as a source of phenotypic heterogeneity. Consequently, the differentiation protocols needs to be standardized and in vitro maturation need to be improved. Also, we showed that the modulation of cell death in hESC-CM might greatly differ from current cardiomyocytes models used for toxicity screens.
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McDonald, Meghan Elizabeth. "Identification of novel regulators of mesenchymal stem cell commitment to the brown adipocyte lineage." Thesis, Boston University, 2013. https://hdl.handle.net/2144/12817.
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Thesis (Ph.D.)--Boston University
PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.Obesity is associated with an imbalance resulting from increased caloric intake and decreased energy expenditure. Humans have two types of adipose tissue: white adipose tissue (WAT), specialized for the storage of excess energy as lipid; and brown adipose tissue (BAT), which catabolizes lipid, releasing heat. Enhancing the development and/or activity of brown adipose tissue (BAT) or brown-like (beige) adipocytes within WAT is proposed as a means to enhance energy expenditure in obese individuals. Brown adipocyte progenitors share common origins with vascular cells; however, the mechanisms regulating commitment to these lineages are not understood. Bone Morphogenic Protein 7 (BMP7), a member of the Transforming Growth Factor β (TGFβ) superfamily, promotes the development of brown adipocytes. The goal of this study was to identify novel mechanisms regulating the commitment of mesenchymal stem cells (MSCs) to the brown adipocyte lineage, and to characterize the contrasting effects of BMP7 and TGFβ1 on cell fate. To address these questions, we used MSC culture models and identified several genes that are selectively regulated by BMP7 during BAT lineage commitment. These include the transcription factor Zinc Finger Protein of the Cerebellum 1 (Zinc1), Gremlin1, a secreted BMP antagonist, and regulators of cell shape, the Rho-associated protein kinases, Rock1/2. Repression of Gremlin1 and Zic1 expression are necessary for MSCs to undergo brown adipogenesis. It is well-established that TGFβ1 activates ROCK, and induces elongated, myofibroblast morphology in MSCs. We demonstrate that BMP7, in contrast, represses ROCK activity, altering actin dynamics and promoting a broadened morphology. Consistent with these findings, inhibition of ROCK activity or the downstream transcription factor Serum Response Factor (SRF), promotes brown adipocyte development. We conclude from these results that changes in cell shape and gene expression programs that are responsive to the status of the actin cytoskeleton are critical mediators of BAT lineage commitment. Our study has identified multiple genes involved in a brown fat/myofibroblast phenotypic switch. Modulation of ROCK or SRF activity may provide a novel means of promoting the development of brown/beige adipocytes in obese individuals. Further delineation of mechanisms regulating BAT development will lead to the identification of novel targets for anti-obesity therapeutics.
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Sweeney, Sean Kenneth. "Non-invasive stem cell tracking using novel nanomaterials : in vitro and ex vivo studies." Thesis, University of Iowa, 2012. https://ir.uiowa.edu/etd/2282.
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As research and clinical use of stem cell therapies progresses, it is becoming more evident that being able to visualize the stem cell transplant in vivo is of great benefit to the researcher or clinician. As a result, researchers are working to address this need. Our lab is collaborating to develop novel, multimodal nanomaterials, one with a core of mesoporous silica, and the other with a core of gadolinium oxide. Varying modifications have been made as needs arose. Human mesenchymal stem cells (MSCs) were isolated from bone marrow aspirates and confirmed to be positive for STRO-1, a common MSC marker. These cells were labeled with 125 μg/mL of varying nanoparticle types: gadolinium oxide, doped with 0.5%, 5%, or 10% europium for magnetic resonance imaging (MRI) and luminescence microscopy, and mesoporous silica nanoparticles (MSN), loaded with fluorescein for fluorescent microscopy and capped with either iron oxide or gold for MRI and computed tomography (CT), respectively. We studied the kinetics of MSN uptake by MSCs for 10 days using fluorescent microscopy. In ex vivo studies, we used the 4.7 Tesla Varian® small animal MRI scanner to detect 5*10⁴ cells labeled with ferrite-capped MSN particles and injected into the brain, lung, and heart of a perfusion-fixed mouse. Micro-CT was used to detect 1.7*10⁶ cells labeled with gold-capped MSN and delivered to the lungs via the trachea in a perfusion-fixed mouse. The results of this research are preliminary to in vivo testing using animal models as a proof-of-concept for these potentially marketable particles.
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Riccitelli, E. "EXTRACELLULAR SPHINGOSINE-1-PHOSPHATE: A NOVEL ACTOR IN HUMAN GLIOBLASTOMA STEM CELL SURVIVAL PROPERTIES." Doctoral thesis, Università degli Studi di Milano, 2013. http://hdl.handle.net/2434/217463.
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Glioblastoma multiforme (GBM) is the most frequent and aggressive intracranial tumour in humans. The prognosis of GBM patients remains unfavourable even after aggressive treatments based on multiple approaches, due to the high proliferation rate, migrating-invasive properties, and resistance to therapeutic intervention. The introduction of the alkylating agent TMZ in glioblastoma therapy has improved patient survival, but drug resistance mechanisms limit its benefits.
The aim of this study was to provide a contribution to the understanding of the malignant and chemoresistance properties in GBM by focusing on the role of the bioactive sphingoid molecules ceramide and S1P, which act as antagonists in regulating cell properties and survival.
Accumulating literature indicates that ceramide is a tumour suppressor sphingolipid, able to induce antiproliferative and apoptotic responses, and that it is able to act as a major player in the mechanism of action of many chemotherapeutic drugs. We demonstrated that the treatment of T98G human glioblastoma cells with cytotoxic TMZ concentrations results in a significant increase in intracellular ceramide, which in turn promotes cell death. On the other hand, TMZ is not able to induce ceramide accumulation in TMZ-resistant glioblastoma cells (TMZ-R). These data suggest a role of ceramide as a mediator of TMZ-induced toxicity.
A large amount of evidence underlines the role of S1P as an important tumour-promoting sphingolipid, acting predominantly in the extracellular milieu after interaction with specific G protein-coupled receptors and exerting opposite effects on cell survival compared to ceramide. Parallel studies demonstrated that S1P secretion in TMZ-R cells is functional to inhibit the cytotoxic effect of ceramide and to confer TMZ-resistant properties to glioblastoma cells.
Stimulated by these findings, we next evaluated the role of sphingolipid mediators in the malignant features of glioblastoma stem cells (GSCs), a cell subpopulation within the tumour mass involved in the aberrant expansion and therapy resistance properties of glioblastomas. To this purpose we used GSCs isolated from the human U87-MG glioblastoma cell line and GSCs isolated from a primary culture of human glioblastoma. We found that both GSC models efficiently form typical neurosphere structures in mitogen-defined medium and express high levels of recognized cancer stem cell markers. Moreover, GSCs exhibit resistance to TMZ at concentrations that are cytotoxic in U87-MG, despite not expressing the DNA repair protein MGMT, a major contributor to TMZ-resistance. Even though a large amount of evidence underlines that S1P is able to favor growth, invasion and chemotherapy resistance of glioblastoma cells, so far little is known on the possible role of S1P as a factor modulating GSCs malignant properties.
Further experiments revealed that glioblastoma cells and GSCs are able to efficiently synthesize S1P and also to release it in the culture medium. Notably the intracellular S1P level was found much lower in GSC models than in the glioblastoma cell line; meanwhile the extracellular S1P level was significantly higher in GSC models than in U87-MG cells. These differences resulted in an extracellular S1P-intracellular S1P ratio at least 10 times higher in GSCs compared to U87-MG. Furthermore, this ratio is about 1:1 in both GSCs, thus suggesting that these cells are an efficient source of S1P in the extracellular microenvironment. Furthermore we found that ceramide-extracellular S1P ratio is at least 2-fold lower in GSCs than in U87-MG. Since S1P and ceramide exert opposing effects on cell survival, according to the “sphingolipid rheostat” model, this different ratio could promote GSC survival observed after TMZ treatment.
Interestingly, enzyme activity assays excluded the presence of sphingosine kinase (SK), the enzyme responsible for S1P byosinthesis, in GSC medium, implicating an efficient secretion of S1P in GSCs.
The analyses of the expression of the ABC-transporters known to be involved in S1P export (ABCG2, ABCA1 and ABCC1), revealed that only ABCA1 is expressed in GSCs. Notwithstanding, after ABCA1 inhibition, no variations in S1P release was observed, suggesting that other mechanisms different from those known are involved.
We also investigated the role of S1P in glioblastoma resistance to TMZ. A first interesting finding was that exogenously administered S1P protected U87-MG cells against TMZ cytotoxic effects. In addition, we found that, after co-treatment with TMZ and an inhibitor of S1P biosynthesis, GSCs became sensitive to the toxic effect of the drug. Of note, exogenous S1P administration was able to revert this effect. These data strongly support extracellular S1P as an important mediator in TMZ-resistance of GSCs.
Furthermore, results obtained in GSCs isolated from two patients affected by glioblastoma with different aggressive phenotype, revealed that the extracellular release of S1P was significantly higher by cells isolated from the most aggressive tumour, suggesting that the release and thus the levels of extracellular S1P might be related to tumour aggressiveness and patient prognosis.
In conclusion, our data implicate for the first time GSCs as an important source of S1P in the extracellular microenvironment, where, on its turn, S1P can act as an autocrine/paracrine messenger able to contribute to the GSC survival properties. A better understanding of S1P role in GSCs aggressive phenotype could represent a critical start point that sets the bases for the development of new compounds able to sensitize GSCs to chemotherapeutic treatments, thus improving survival rates in GBM patients.
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Glover, Hannah Jacquilyn. "L-proline-induced transition of mouse ES cells to a spatially distinct primitive ectoderm-like cell population primed for neural differentiation." Thesis, The University of Sydney, 2018. http://hdl.handle.net/2123/20576.
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Naïve mouse embryonic stem cells (mESCs) derived from the preimplantation mouse blastocyst self-renew in the presence of LIF and BMP4. These cells are pluripotent, meaning they have the ability to differentiate into the ~200 cell types of the developing embryo and adult. Naïve mESCs are one discrete state along a pluripotency continuum – delimited by ground-state mESCs as the earliest cell population, followed by naïve mESCs, and with EpiSCs as the most ‘primed’ population. The amino acid L-proline has novel growth factor-like properties during development - from improving blastocyst development to driving neurogenesis. Addition of 400 μM L-proline to naïve mESCs produces a pluripotent cell population between naïve mESCs and EpiSCs. These cells, named early primitive ectoderm-like (EPL) cells, recapitulate in vivo development of the pre-implantation inner cell mass to the postimplantation primitive ectoderm. EPL cells maintain expression of the naïve marker Rex1 and upregulate expression of the primitive ectoderm genes Dnmt3b and Fgf5. This thesis identifies mechanisms underpinning L-proline-mediated differentiation to EPL cells, including a complex self-regulating signalling network involving the MAPK, Fgfr, PI3K and mTOR pathways. Statistical models were used to understand the contributions of individual signalling pathways to changes in colony morphology, cell number, gene expression, proliferation and apoptosis. Other mechanisms underpinning the naïve mESCs-to-EPL cell transition were explored, including DNA methylation, histone acetylation, proline synthesis and metabolism. In addition to expressing primed pluripotency genes, EPL cells upregulate genes associated with neurogenesis, whereas EpiSCs express genes associated with cardiovascular development. When mapped to the 7.0 dpc embryo, EPL cells and EpiSCs represent spatially distinct cell populations. This suggests that after cells transition from naïve mESCs, they diverge and are fated to become either ectoderm (from EPL cells), or mesendoderm (from EpiSCs). This thesis also explored whether L-proline plays an underappreciated role in existing neural differentiation protocols. mESCs cultured in custom N2B27 medium without L-proline had reduced selective cell death resulting in a larger yield of Sox1+ neurectoderm.
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Okarski, Kevin M., and Kevin M. Okarski. "Novel Point-of-Care Disposable Device and Cell Culture Bioprocessing Technique." Diss., The University of Arizona, 2016. http://hdl.handle.net/10150/620668.
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This dissertation is composed of two projects dedicated to the development of techniques and technologies for improving the quality of life for patients in both clinical and resource-limited settings. The purpose of the first project was to design a rapid diagnostic device to screen whole blood samples for the presence of infectious agents. Point-of-care (PoC) technologies are becoming increasingly important for the detection of infectious agents in resource-limited settings (RSLs) where state-of-the-art blood screening practices are not feasible for implementation. For this project, a rapid diagnostic device was developed to directly detect pathogen content within freshly drawn whole blood samples using a ligand-binding assay format. The assay is completely self-contained within a hermetically sealed device to minimize operational complexity and ensure operator safety. The diagnostic device is capable of processing complex sample matrices by selectively capturing, concentrating, and labeling infectious agents upon functionalized surfaces. Following sample processing, the assay is optically interrogated with a fluorescence-based reader to provide rapid feedback regarding sample purity. Designs of the rapid diagnostic platform evolved over several prototype generations corresponding to project milestones emphasizing ergonomic performance, military specification testing for environmental resilience, and manufacture to yield production-grade devices for future diagnostic performance data collection. The goal of the regenerative therapy-based portion of this research was to develop a novel technique for the selective enrichment of cells demonstrating enhanced regenerative capacity in tissue-extracted cell samples. Adherent cell cultures of stromal vascular fractions (SVFs) extracted from adipose tissues were exposed to nutrient deficient conditions' eliciting a bimodal cellular response between two dissimilar cell culture subpopulations. The regenerative capacity of these two distinct subpopulations was evaluated by assessing their characteristic morphology, metabolic activity, and ability to undergo multilineage differentiation. The SVF subpopulation which demonstrated sensitivity to the nutrient deficient conditions expressed typical morphological expression of adherent cell cultures, elevated metabolic activity, and the ability to differentiate along adipogenic, chondrogenic, and osteogenic lineages. The SVF subpopulation which demonstrated resistance to the nutrient deficient conditions, however, expressed atypical morphologies, impaired metabolic activity, and did not survive culture with differentiation growth media. Based on the data, the 'treatment-sensitive' SVF subpopulation demonstrated a greater regenerative capacity than the‘treatment-resistant' subpopulation. Furthermore, the treatment-resistant subpopulation of the SVF may be representative of the damaged, senescent, and otherwise less-functional cells that comprise a significant portion of tissue-extracted cell samples and pose a significant risk to therapeutic efficacy and reproducibility. Ultimately, this expedient and inexpensive bioprocessing technique may serve to improve cell-based regenerative therapies by eliminating undesirable cells from culture.
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Liu, Wen [Verfasser]. "Stem cell marker expression of canine neoplasia generated cell subpopulations and effect of novel arylindolylmaleimides on canine lymphoma cell lines / Wen Liu." Hannover : Bibliothek der Tierärztlichen Hochschule Hannover, 2015. http://d-nb.info/1080825509/34.
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Callan, Matthew Aron. "Novel Roles for Fragile X Protein in Neurogenesis." Diss., The University of Arizona, 2011. http://hdl.handle.net/10150/145454.
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Fragile X Syndrome (FXS) is the most common form of inherited mental retardation, affecting approximately 1/4000 males and 1/6000 females worldwide. FXS is caused by loss of FMR1 gene expression, resulting in the lack of the protein product, Fragile X protein (FMRP). FMRP is an RNA-binding protein thought to regulate synaptic plasticity by controlling the localization and translation of specific mRNAs in neurons. To determine whether FMRP is also required in early brain development we examined the distribution of cell cycle markers in Drosophila FMR1 (dFmr1) mutant brains compared to wild-type brains. Our results indicate that the loss of dFmr1 leads to a significant increase in the number of mitotic neuroblasts and BrdU incorporation in the brain, consistent with the notion that FMRP controls proliferation in neural stem cells. To determine the role of FMRP in neuroblast division and differentiation, we used Mosaic Analysis with a Repressible Marker (MARCM) approaches in the developing larval brain and found that single dFmr1 neuroblasts generate significantly more neurons than controls. Developmental studies suggest that FMRP also inhibits neuroblast exit from quiescence, or reactivation, in early larval brains, as indicated by misexpression of the G1 to S phase transition marker Cyclin E. We have also identified a novel role for FMRP in the glia surrounding the neuroblasts, indicating that FMRP in these cells contributes to the regulation of neuroblast reactivation via signaling from the supporting glial cells. Our results demonstrate that FMRP is required during brain development to control the exit from quiescence and proliferative capacity of neuroblasts as well as neuron production, which may provide insights into Fragile X Syndrome and other Autism-Spectrum disorders.
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Xiong, Anqi. "Novel Regulators of Brain Tumor Development : – From neural stem cell differentiation to in vivo models." Doctoral thesis, Uppsala universitet, Institutionen för immunologi, genetik och patologi, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-264470.
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Malignant brain tumors are diseases with poor prognosis and/or severe long-term side effects of treatment. This thesis aimed to discover novel regulators in brain tumor development, based on studying neural stem cell and progenitor cell (NSPC) differentiation and using animal models to introduce new insights to mechanisms of human brain tumors. The enzyme heparanase (HPSE) that degrades heparan sulfate (HS) is active in cell signaling and ECM remodeling. In paper I, we found an enhanced differentiation to oligodendrocytes in ES cell-derived NSPCs overexpressing HPSE. Further analysis suggested that this enhanced formation of oligodendrocytes was associated with alterations in receptor tyrosine kinase signaling, and that HPSE might also exert anti-apoptotic functions. Subsequently, in paper II we studied the involvement of HPSE in glioma development. We observed that high HPSE levels associated with poor survival in glioma patients. In experimental models, we found that HPSE promoted glioma growth, and that an inhibitor of HPSE reduced glioma progression both in vitro and in vivo. We hypothesize that regulators in NSPC differentiation could have a potential role in brain tumor development. In paper III, we explored the function of NRBP2, a pseudokinase that is up-regulated during NSPC differentiation. We found low expression of NRBP2 in brain tumors, in comparison to normal brain. In medulloblastoma, in particular, low NRBP2 expression is linked to poor prognosis. Overexpression of NRBP2 in medulloblastoma cells led to impaired cell growth and migration, concomitant with an increased cell death. In paper IV, we searched for novel glioma susceptibility genes by sequencing dog breeds from the same ancestor but with different glioma incidence. In this way we identified three new glioma-associated genes. Two of these are significantly regulated in human glioma and one of those might have a role in glioblastoma stem cell differentiation.