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Young, Christopher Cheng. "The adult neural stem cell niche in ischaemic stroke." Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:86e6e236-047c-46d8-96e5-449a3f0505a8.
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Ischaemic stroke is a major cause of mortality and chronic disability for which there is no effective treatment. The subventricular zone (SVZ) is an adult neurogenic niche which mediates limited endogenous repair following stroke. To harness this phenomenon for therapy, it is important to understand how the SVZ niche is altered in stroke, and the processes that recruit neural precursors to the site of injury, which becomes a de facto neurogenic niche. Galectin-3 (Gal-3) is a β-galactoside binding protein involved in cellular adhesion, inflammation and tumour metastasis. Gal-3 is specifically expressed in the SVZ and maintains neuroblast migration to the olfactory bulb, although its role in post-stroke neurogenesis is not well-understood. Therefore, this project aimed to (1) characterise the cytoarchitecture of the SVZ in response to stroke, and (2) examine the role of Gal-3 in stroke outcome and tissue remodelling, and test the hypothesis that Gal-3 is required for neuroblast ectopic migration into the ischaemic striatum. Using the intraluminal filament model of middle cerebral artery occlusion (MCAO) in mice, and whole mounts of the lateral ventricular wall, significant SVZ reactive astrocytosis and increased vascular branching were observed, thereby disrupting the neuroblast migratory scaffold. Stroke increased SVZ cell proliferation without increase in cell death. Post-stroke ependymal cells were enlarged and non-proliferative, and assumed a reactive astroglial phenotype, expressing de novo high levels of glial fibrillary acidic protein. This was associated with focal planar cell polarity misalignment, and turbulent and decreased rate of cerebrospinal fluid flow. These findings demonstrate significant changes in multiple SVZ cell types which are positioned to influence post-stroke neurogenesis and regulation of the neural stem cell niche Gal-3 was up-regulated in the ischaemic brain and ipsilateral SVZ. To elucidate the role of Gal-3 after stroke, MCAO was performed in wildtype and Gal-3 null (Gal-3-/-) mice, and parameters of stroke outcome and post-stroke neurogenesis compared. The deletion of Gal-3 did not affect infarct volumes or neurological outcomes, although neuroblast migration into the ischaemic striatum was increased in Gal-3-/- brains. Gal-3-/- mice failed to mount an angiogenic response in the ischaemic striatum, and this was associated with lower levels of vascular endothelial growth factor (VEGF) and increased anti-angiogenic protein levels. Loss of Gal-3 further disrupted the pro-proliferative neural-vascular interaction at the basement membrane. The current data indicate that Gal-3 is a pleiotropic molecule which has distinct roles in both the SVZ and the post-stroke striatum as niches of adult neurogenesis.
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Michel, Marcus. "Stem cell regulation in the Drosophila testicular niche." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2013. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-121226.
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All multicellular organisms constantly need to replace aged or damaged cells. This vital task of tissue homeostasis is fulfilled by stem cells. The balance between self-renewal and differentiation of the stem cell is crucial for this task and tightly regulated by a signaling microenvironment termed the niche. A widely used model for studying stem cell niche biology is the Drosophila testis, where two stem cell populations, the germline stem cells (GSCs) and the somatic cyst stem cells (CySCs), reside in a niche located at the apical tip. A lot is known about the signals regulating GSC maintenance in the testicular niche. It is, however, unknown how the spatial regulation of these signals defines the range of the niche.
Here I show, that Bone Morphogenetic Protein (BMP) signaling is specifically activated at the interface of niche and stem cells. This local activation is achieved by coupling the transport of adhesion and signaling molecules in the niche cells and directing their transport to contact sites of niche and stem cells. Localized niche signaling at junctions underlies the so called stem-cell-niche synapse hypothesis proposed for the mammalian hematopoietic stem cell niche. I have shown that disrupting the localized transport causes premature differentiation and stem cell loss. BMP signaling between niche and GSCs therefore provides the first description of a stem-cell-niche synapse and will yield valuable insights into mammalian stem cell biology.
The CySCs reside in the niche of the testis together with the GSCs. To understand how the niche maintains both stem cell types in a concerted way, it is essential to know the pathways regulating both stem cell types. Here I show that Hedgehog (Hh) signaling is a key stem cell factor of CySCs, while only indirectly affecting GSCs. Loss of Hh signaling in CySCs results in premature differentiation and consequent loss of the cells. Overactivation of the pathway leads to an increased proliferation and an expansion of the cyst stem cell compartment. As Hh signaling is also a regulator of the somatic cells in the mammalian testis and the Drosophila ovary this may reflect a higher degree of homology between these systems than previously expected.
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Yeung, Aaron Ming Hon. "Limbal stem cell niche and ocular surface reconstruction." Thesis, University of Nottingham, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.580161.
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In the quest to master ocular surface regeneration, one must isolate the stem cells at the limbus and understand them. The stem cell niche is a concept that-was first described in 1978 and subsequently gained interest and became widely accepted. The work presented in Chapter 2 sought to characterize the stem cell niche at the ocular surface, and in doing so led to further understanding of stem cells at the limbus. In Chapter 3 the sampling of infant tissue provided further insight into the niche at that age group. In Chapter 4, Desmoglein 3 was hypothesized to be a negative stem cell marker. Finally in Chapter 5, the Amniotic Membrane was investigated as a possible surrogate stem cell niche. The stem cells at the limbus have not been isolated yet, but hopefully we are one step closer to mastering ocular surface reconstruction.
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Paton-Hough, Julia. "Defining key molecules in a myeloma cell niche." Thesis, University of Sheffield, 2014. http://etheses.whiterose.ac.uk/6732/.
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Multiple myeloma is an incurable B cell malignancy characterised by the expansion of malignant plasma cells in the bone marrow. It has been suggested that during initial colonisation of bone and possibly during therapy, some myeloma cells may occupy a bone marrow niche similar to that inhabited by haemopoietic stem cells. Haemopoietic stem cells residing in BM niches adhere to osteoblastic cells via a series of molecules that promote haemopoietic stem cell quiescence. Therefore, we hypothesise that myeloma cells express the same molecules as haemopoietic stem cells, chemokine C-X-C-motif-receptor 4, notch-1, tyrosine kinase-2 and n-cadherin, which interact with their complementary ligands expressed by osteoblastic cells, chemokine C-X-C-motif-ligand 12, jagged-1, angiopoietin-1 and n-cadherin. These interactions may result in myeloma cell adhesion to an osteoblastic niche, resulting in myeloma cell dormancy. The aims of these studies were to determine the expression of haemopoietic stem cell niche molecules and ligands by murine myeloma cell lines and osteoblastic cells and to determine the role of one of the key molecules in vitro and in vivo. 5T33MMvt and 5TGM1 cells expressed the haemopoietic stem cell niche molecules; chemokine C-X-C-motif-receptor 4, notch-1, tyrosine kinase-2 and n-cadherin and the MC3T3-E1 cells and primary osteoblast lineage cells expressed the ligands chemokine C-X-C-ligand 12, jagged-1, angiopoietin-1 and n-cadherin. Knock-down of n-cadherin was achieved in the 5TGM1 cells, with 71% gene and 75% protein reduction. 5TGM1 n-cadherin knock-down cell attachment to primary osteoblast lineage cells was reduced in vitro, though this did not reach significance. Mice injected with 5TGM1 n-cadherin knock-down cells had significantly less tumour in vivo compared to controls. In conclusion, murine myeloma cells expressed the same repertoire of molecules as haemopoietic stem cells and osteoblastic cells expressed their complementary ligands. Knock-down of one of these key molecules, n-cadherin, did not significantly inhibit myeloma cell attachment to primary osteoblasts in vitro but potentially impaired tumour growth in vivo. Further experiments are required to confirm this.
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Joshi, Ramila Joshi. "Micro-engineering of embryonic stem cells niche to regulate neural cell differentiation." University of Akron / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=akron1544029342969082.
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Gu, Ying. "A Traveling Niche: The Role of Steel Factor in Mouse Primordial Germ Cell Development." University of Cincinnati / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1321370449.
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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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Mirzadeh, Zaman. "Epithelial organization of the adult neural stem cell niche." Diss., Search in ProQuest Dissertations & Theses. UC Only, 2008. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3311332.
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Thesis (Ph.D.)--University of California, San Francisco, 2008.Source: Dissertation Abstracts International, Volume: 69-06, Section: B, page: 3358. Adviser: Arturo Alvarez-Buylla. Includes supplementary digital materials.
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Young, Sarah Jane. "Biomechanical modelling of the gastrointestinal epithelial stem cell niche." Thesis, University of Nottingham, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.518236.
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Wang, Longlong. "A mesenchymal stem cell (MSC) niche in mouse incisor." Thesis, King's College London (University of London), 2016. https://kclpure.kcl.ac.uk/portal/en/theses/a-mesenchymal-stem-cell-msc-niche-in-mouse-incisor(8f92b75d-f90f-4c58-ab06-682af9f90e95).html.
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Mesenchymal stem cells (MSCs) are heterogeneous cell populations that are identified by their in vitro characteristics while their biological properties and in vivo identities are often less understood. Different from human teeth, mouse incisors grow and erupt continuously throughout their lives and compensate for daily abrasions with the existence of stem cells. However, the precise location of the mesenchymal stem cells (MSCs) in the incisor is unclear. Generally, the MSCs in the mouse incisor are believed to be located in the mesenchyme close to the epithelium cervical loops, since the growth and differentiation of the incisor always initiates at the apical end and extends towards the incisal end. The utilization of label-retaining experiments and transgenic reporter mouse lines has enabled further understanding of the less established identities and properties of dental pulp stem cells in vivo. The work described in this thesis demonstrates that the mesenchymal stem cell niche located at the apical end of mouse incisor contains three distinct but connected cell populations: 1) a slow cycling cell population containing Thy-1+ cells essential for tooth dental pulp and odontoblast formation 2) a Ring1/Bcor-associated fast cycling cell population crucial for maintaining tissue growth and homeostasis of epithelium stem cells in labial cervical loop 3) a quiescent long-term cell population marked by Flamingo homologue Celsr1 might respond to generate new stem cells when the stem cells become depleted.
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Puretskaia, Olga. "Signalling in the Somatic Stem Cell Niche of the Drosophila Testis." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2017. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-221172.
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Stem cell niches are specialized signalling microenvironments that allow maintenance of the stem cells. According to the traditional model of the stem cell niche, the niche signalling input is integrated by a cell towards a binary decision between stemness and differentiation. I have studied the regulation of somatic cyst stem cell (CyCS) proliferation in the testicular stem cell niche of Drosophila melanogaster by performing the DamID screen for targets of the transcriptional regulator Zfh1, a shared target of Jak/STAT and Hedgehog niche signalling. I have found that Zfh1 binds to the regulatory regions of kibra and salvador, tumour suppressors of the Hippo/Yorkie pathway, and downregulates them, restricting Yorkie activity to the Zfh1 positive CySCs. Clonal inactivation of the Hippo pathway is sufficient for CySC proliferation, but does not affect their differentiation ability. I therefore proposed a different stem cell niche model, whereby the niche signalling directly “micromanage” stem cell behavior, not involving the cell fate decision making.
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McGarvey, Alison Clare. "Genome-wide transcriptional characterisation and investigation of the murine niche for developing haematopoietic stem cells." Thesis, University of Edinburgh, 2017. http://hdl.handle.net/1842/28741.
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Haematopoietic stem cells (HSCs) are capable of differentiation into all mature haematopoietic lineages, as well as long-term self-renewal and are consequently able to sustain the adult haematopoietic system throughout life. Currently, in the mouse, HSCs are understood to first appear in the aorta-gonad-mesonephros (AGM) region at embryonic day 11 via a process of maturation from precursors (pre-HSCs). This maturation within the AGM region involves the complex interplay of signalling between cells of the niche and maturing precursor cell populations, but is relatively little understood at a molecular level. Recently our understanding of the AGM region has been refined, identifying the progression from E9.5 to E10.5 and the polarity along the dorso-ventral axis as clear demarcations of the supportive environment for HSC maturation. In this thesis, I investigated the molecular characteristics of these spatio-temporal transitions in the AGM region through the application of RNA-sequencing. This enabled the identification of molecular signatures which may underlie the supportive functionality of the niche. I further compared these expression signatures to the transcriptional profile of an independent cell type, also capable of supporting HSC maturation, the OP9 stromal cell line. By combining this transcriptional information with an ex vivo culture system, I screened a number of molecules for their ability to support HSC maturation from early precursors, leading to the discovery of a novel regulator of HSC maturation: BMPER. Further characterisation of this molecule enabled the identification of its specific cellular source and the proposal that through its action as an inhibitor of BMP signalling it facilitates the maturation of precursors into HSCs. These results lend further detail and support to the role of BMP signalling in the regulation of HSC maturation as well as demonstrating the potential of these transcriptional profiles to yield novel mechanistic insight.
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Bonfini, Alessandro. "Investigation of Notch signalling in Drosophila germline stem cell niche." Thesis, University of Manchester, 2013. https://www.research.manchester.ac.uk/portal/en/theses/investigation-of-notch-signalling-in-drosophila-germline-stem-cell-niche(d5393254-c7b3-467c-a2aa-c61564138672).html.
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Adult stem cells are vital for tissue maintenance. Stem cell over proliferation results in tumour formation, whilst loss of stem cells causes tissue degeneration and a variety of diseases. Stem cell maintenance and proliferation is regulated through somatic structures called niches. The germline stem cell niche in Drosophila ovary has been well defined and it is useful to better understand the interactions between niche and stem cells. Notch signalling is needed for germline stem cell niche creation and maintenance. The aim of this thesis is to better understand both the regulation of Notch signalling during development and its requirement in the adult niche. The first paper, "Reversible regulation of stem cell niche size through dietary control of Notch signalling", revolves around the dynamicity of the niche. The niche is found to respond to diet stimuli and has the ability to be restored. Notch was previously found to be involved in the maintenance of the niche. We found that Notch signalling is altered by diet, and we dissect its different maintenance and recovery roles in the ovary. In the second paper, "ZO-1 controls stem cell niche assembly by acting as an upstream regulator of Deltex-dependent Notch signalling", we show how Notch signalling is finely regulated during niche formation through interplay with the proteins Polychaetoid and Deltex. This paper leads to a better understanding of how the niche is assembled and how Notch signalling is regulated in a context-dependent way. The obtained results from both papers will help understand the dynamics of the model germline stem cell niche, and how Notch signalling is found at the convergence between internal and external stimuli regulating the ovary's response to a changing environment.
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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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Bentley, Adam James. "Characterisation and replication of the corneal epithelial stem cell niche." Thesis, Lancaster University, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.514445.
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Chen, Liang-Yu. "Testosterone regulation of the spermatogonial stem cell niche in mice." Pullman, Wash. : Washington State University, 2008. http://www.dissertations.wsu.edu/Dissertations/Fall2008/l_chen_112108.pdf.
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Seike, Masanari. "Stem cell niche-specific Ebf3 maintains the bone marrow cavity." Kyoto University, 2018. http://hdl.handle.net/2433/235055.
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Soteriou, Despina. "Definition of the human embryonic stem cell niche in vitro." Thesis, University of Manchester, 2012. https://www.research.manchester.ac.uk/portal/en/theses/definition-of-the-human-embryonic-stem-cell-niche-in-vitro(ebe6a857-7cf5-45a2-8235-206f293d3ded).html.
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The unique pluripotent character of human embryonic stem cells (hESCs) places them in the forefront of scientific research, especially as they hold great promise for application in regenerative medicine, as well as drug discovery and toxicity analyses. Conventionally hESCs are cultured on mitotically inactivated mouse embryonic fibroblasts (MEFs) that are derived from E13.5 mouse embryos. One of the biggest challenges in the hESC field is the development of a reproducible and defined hESC culture system that would eliminate batch-to-batch variability of the MEFs as well as exposure to feeder cells that makes hESCs less applicable for clinical use. Previous studies have shown that maintenance of pluripotency can be achieved using Matrigel, a mixture of ECM components, or ECM derived from MEFs or human fibroblasts (Xu, et al., 2001, Klimanskaya, et al., 2005). Other groups have succeeded in culturing feeder-free hESCs by using extracellular matrix (ECM) proteins, such as fibronectin, vitronectin or laminin, as substrates for hESC culture in the absence of feeders, confirming that ECM plays a key role in maintaining hESC growth (Amit, et al., 2004, Braam, et al., 2008, Baxter, et al., 2009, Rodin, et al., 2010).The aim of this work was to investigate the ECM deposited by MEF feeder cells and to isolate and identify proteins in the ECM that support undifferentiated growth of hESCs in the absence of feeders. We have investigated whether matrices derived from different passage feeders differ in their ability to support pluripotency. I also assessed the integrin receptor profile of hESCs in order to define the mechanisms of ECM engagement. ECM was extracted from two strains of feeder cells, CD1 x CD1 and MF1 x CD1, at passages 4 (early passage), 9 and 14 (late passage), and assessed for its ability to support hESC self-renewal over at least 3 passages. Tandem mass spectrometry was used to analyse the ECM composition of each MEF line, thereby allowing a comparison between different passages and different cell lines. More than 100 proteins were identified for each sample, the majority of which were ECM proteins and shared between different passage feeders. As predicted, fibronectin, which is known to support hESC self-renewal was the most prevalent species in all MEF-derived matrices. Furthermore a proteomic analysis of matrix derived from hESCs cultured in feeder-free conditions on fibronectin coating substratum revealed a number of proteins shared between supportive MEF populations and hESC, suggesting other potential candidates that may either assist or interfere with the maintenance of pluripotent hESCs. Of the proteins identified fibrillin-1, perlecan, fibulin-2 were tested as substrates for culturing hESCs in the absence of feeders, with the prospect of developing an optimised feeder-free culturing system that uses a combination defined animal-free substrates. Finally this study sought to dissect the interaction between ECM and growth factors and how these extrinsic factors may affect self-renewal and maintenance of pluripotency-associated gene expression. Interruption of hESC attachment, as well as removal of growth factors appeared to affect transcript levels of pluripotency genes, OCT4 and NANOG, suggesting that the microenvironment can influence hESC fate.
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Goel, Aviva J. "Niche Regulation of Muscle Stem Cell Quiescence by Classical Cadherins." Thesis, Icahn School of Medicine at Mount Sinai, 2018. http://pqdtopen.proquest.com/#viewpdf?dispub=10743988.
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Many adult stem cells are characterized by prolonged quiescence, promoted by cues from their niche. Upon tissue damage, a coordinated transition to the activated state is necessary for successful repair. Non-physiological breaks in quiescence often lead to stem cell depletion and impaired tissue restoration. Here, I identify cadherin-mediated adhesion and signaling between muscle stem cells (satellite cells; SCs) and their myofiber niche as a mechanism that orchestrates the quiescence-to-activation transition. Conditional removal of N-cadherin and M-cadherin in mice leads to a break in SC quiescence with long-term expansion of a regeneration-proficient SC pool. These SCs have an incomplete disruption of the myofiber-SC adhesive junction, and maintain niche residence and cell polarity, yet show properties of SCs in a state of transition from quiescence towards full activation. Among these properties is nuclear localization of b- catenin, which is necessary for this phenotype. These findings are consistent with the conclusion that injury-induced perturbation of niche adhesive junctions is a first step in the quiescence-to-activation transition.
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Dziasko, M. A. "Localisation of corneal epithelial progenitors and characterization of cell-cell interactions in the human limbal stem cell niche." Thesis, University College London (University of London), 2016. http://discovery.ucl.ac.uk/1472700/.
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The cornea, the transparent tissue located at the front of the eye, is a highly specialized tissue that transmits and refracts light onto the retina. Maintenance of the corneal epithelium relies on a population of limbal epithelial stem cells (LESCs) that maintain transparency of the ocular surface that is essential for vision. Despite great advances in our understanding of ocular stem cell biology over the last decade, the exact location of the LESC niche remains unclear. After observing a high population of basal epithelial cells expressing stem cell markers within the previously identified limbal crypts (LC), the first aim of this study was to demonstrate by in vitro clonal analysis that these structures provide a niche for the resident LESCs. High-resolution transmission electron microscopy has been further used to image the basal epithelial layer at the limbus. Cells with morphology consistent with stem cells were present within the basal layer of the limbal crypts but not within the basal layer of non-crypt limbal biopsies. Moreover, LESCs appeared proximal to limbal stromal cell extensions that suggested a possible route for direct cell-to-cell interaction. These observations were further confirmed by serial block-face scanning electron microscopy that revealed, for the first time, direct epithelial-stromal interactions in the LESC niche whereas limbal melanocytes maintained the LESC apically. In order to assess the role of limbal melanocytes (hLM) as niche cells for the maintenance of LESC, a novel co-culture system was developed in which hLM were used as a feeder layer for the expansion of limbal epithelial cells in vitro. Interestingly, hLM had the ability to support the clonal growth of LECs that maintained stem cell-like characteristics in 2D and 3D tissue equivalents. Taken together, these observations suggest an important role for melanocytes as niche cells in the native human limbal crypts.
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Pannérec, Alice. "The skeletal muscle stem cell niche : defining hierarchies based upon the stem cell marker PW1 to identify therapeutic target cells." Phd thesis, Université Pierre et Marie Curie - Paris VI, 2012. http://tel.archives-ouvertes.fr/tel-00833422.
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Les cellules satellites permettent la réparation des muscles squelettiques, mais chez les patients atteints de myopathies ces cellules ne fonctionnent pas correctement ce qui conduit à l'atrophie musculaire. Nos travaux ont montré qu'une nouvelle population de cellules souches musculaires, les PICs, favorisent la prolifération des cellules satellites par l'intermédiaire de la follistatine qui contrebalance l'effet négatif de la myostatine. Lorsque la myostatine est inactivée chez des souris par injection d'inhibiteur, le nombre de PICs augmente considérablement et les animaux présentent des muscles hypertrophiés. De récentes études ont montré que la régénération musculaire est impossible sans les cellules satellites, mais si nous inactivons la myostatine dans ces animaux la régénération musculaire est restaurée. Nous postulons que les PICs ont permis cette réparation et constituent donc une bonne cible pour des molécules pharmacologiques à visée thérapeutique
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Pannerec, Alice. "The skeletal muscle stem cell niche : defining hierarchies based upon the stem cell marker PW1 to identify therapeutic target cells." Paris 6, 2012. http://www.theses.fr/2012PA066440.
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Les cellules satellites permettent la réparation des muscles squelettiques, mais chez les patients atteints de myopathies ces cellules ne fonctionnent pas correctement ce qui conduit à l’atrophie musculaire. Nos travaux ont montré qu’une nouvelle population de cellules souches musculaires, les PICs, favorisent la prolifération des cellules satellites par l’intermédiaire de la follistatine qui contrebalance l’effet négatif de la myostatine. Lorsque la myostatine est inactivée chez des souris par injection d’inhibiteur, le nombre de PICs augmente considérablement et les animaux présentent des muscles hypertrophiés. De récentes études ont montré que la régénération musculaire est impossible sans les cellules satellites, mais si nous inactivons la myostatine dans ces animaux la régénération musculaire est restaurée. Nous postulons que les PICs ont permis cette réparation et constituent donc une bonne cible pour des molécules pharmacologiques à visée thérapeutiqueSatellite cells are considered the major source of muscle progenitors, however, other populations with myogenic popential have been discovered. We have identified a new muscle-resident non-satellite cell population, termed PICs, which can differentiate into three different lineages, skeletal muscle, smooth muscle and fat. PICs rescue satellite cells from myostatin inhibition in vitro through follistatin release. When myostatin is inactivated in vivo, PICs number is markedly increased and mice display hypertrophied muscles. While recent studies have demonstrated that muscle regeneration cannot occur without satellite cells, we show that muscle regeneration is restored when mice have been previously treated with a myostatin inhibitor. We postulate that PICs have participated in muscle repair rescue, and thus constitute an interesting population to be targeted for pharmaceutical strategies aimed at improving skeletal muscle mass and function
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MacLean, Claudia Catherine. "ATP elicits electrophysiological and migratory responses in cells of the spinal cord stem cell niche." Thesis, University of Leeds, 2016. http://etheses.whiterose.ac.uk/16539/.
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There are multiple similarities between the central canal region of the spinal cord and the adult neurogenic niches in the mammalian brain. The central canal region displays increased proliferation after injury with migration of the newly-proliferated cells to the site of injury and differentiation into astrocytes and oligodendrocytes. This study will look at the role of purinergic signalling in the ependymal cells and CSF-contacting cells (CSFcCs) as the CSFcCs present in this area express P2X receptors containing the P2X2 subunit. Local application of ATP elicited fast depolarisations via activation of suramin-sensitive channels in a subgroup of CSFcCs comprising both spiking and non-spiking subtypes of CSFcC (types 1, 2 and 3). The P2X2/3,3-specific antagonist A-317491 had no effect in the majority of CSFcCs but produced a reduction in depolarisation in a subgroup of CSFcCs predominantly located ventral to the central canal, with the ventral expression of the P2X3 subunit supported by immunohistochemistry. Ependymal cells and the remainder of CSFcCs produced a suramin-insensitive hyperpolarisation to the application of ATP or UTP, while producing a small depolarisation to the dinucleotide polyphosphate Ap4A. Modulation of purinergic signalling had no effect on proliferation rate in spinal cord slices over a 4 hour time period, nor did it affect the survival rate of the newly-proliferated cells over 5 days in organotypic slice cultures. In this model, inhibition of purinergic signalling with suramin reduced the migration of newly-proliferated ependymal cells away from the central canal, while inhibition of the breakdown of ATP by ARL 67156 facilitated this migration. The presence of fast acute responses to ATP including spatial variation in receptor subtypes indicates the importance of purinergic signalling in this area and the release of ATP known to be triggered by spinal cord injury could now have a role in the necessary migration of newly-proliferated ependymal cells.
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Dugal-Tessier, Delphie. "The Role of Atypical E2fs in the Maintenance and Development of the Ependymal Cell Barrier." Thesis, Université d'Ottawa / University of Ottawa, 2016. http://hdl.handle.net/10393/34454.
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The discovery of neural stem cells within the adult CNS has indicated an enormous potential in facilitating neuronal regeneration after injury. Studies from our laboratory have suggested that manipulation of the Rb/E2f pathway can directly impact embryonic and adult neurogenesis, thereby having tremendous potential for neuronal regeneration therapies. Recently, two new members of the Rb/E2f pathway have been discovered, the atypical E2fs: E2f7 and E2f8. Initial studies have suggested that atypical E2fs regulate diverse processes such as cell proliferation, DNA-stress response, apoptosis, however, their importance in the brain are unknown. To analyze their function during brain development, we crossed Nestin-Cre mice with mice bearing a conditional E2f7/E2f8 allele to delete both E2f7 and E2f8 in neural precursor cells. Whereas cortical development was largely unaffected by E2f7/E2f8 deficiency, we observed an enlargement of the lateral ventricles occurring postnatally, a brain condition named ventriculomegaly. We then looked at the ependymal cells, which are the cells lining the wall of the lateral ventricles, to determine if these cells were affected by the absence of atypical E2fs. We found progressive denaturation of the ependymal cell layer, distortion of the ependymal motile cilia and reactive astrocytes within the layer. We identified Gli3, a component of the Sonic hedgehog pathway (Shh), as a target for E2fs, including atypical E2fs. We unravelled a novel mechanism by which atypical E2fs regulate the expression of Gli3, leading to up-regulation of Numb/NumbL, which in consequence destabilizes cadherins organization within the ependymal cell layer. In conclusion, our work suggests that E2f7 and E2f8 transcription factors play an essential role in maintaining the ependymal cell barrier.
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Canizares, Marco. "Anatomy and development of the mouse spinal cord stem cell niche." Thesis, University of Dundee, 2018. https://discovery.dundee.ac.uk/en/studentTheses/976910c5-9564-48e3-ab9e-fecf325841ee.
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Kung, Janet Wui Cheung. "Investigating the liver progenitor cell niche in the developing human liver." Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/25953.
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Liver cirrhosis places an increasing burden on healthcare worldwide. Currently the only treatment is liver transplantation. Whilst liver transplant has a relatively good five-year survival, donor organ shortage costs many lives every year and results in lifelong immunosuppression. Alternative treatments are thus urgently needed. It is with this background that there is understandable interest for the development of stem cell therapies for liver regeneration. The identification of putative liver stem cells has brought closer the previously separate fields of liver ontology, regeneration, and carcinogenesis. Significant overlaps in the regulation of these processes are now being described. For example, studies in embryonic liver development have already provided the basis for directed differentiation of human embryonic stem cells and induced pluripotent stem cells into hepatocyte-like cells. As a result, the understanding of the cell biology of proliferation and differentiation in the liver has been improved. This knowledge can be used to improve the function of hepatocyte-like cells for drug testing, bio-artificial livers, and transplantation. In parallel, the mechanisms regulating cancer cell biology are now clearer, providing fertile soil for novel therapeutic approaches. Recognition of the relationships between development, regeneration, and carcinogenesis, and the increasing evidence for the role of stem cells in all of these areas, has sparked fresh enthusiasm in understanding the underlying molecular mechanisms and has led to new targeted therapies for liver cirrhosis and primary liver cancers. Human liver progenitor cells (LPCs) have therapeutic potential but their in vitro culture results in inadequate differentiation, function, and phenotypic instability reflecting an incomplete understanding of in vivo processes. LPCs can be robustly isolated from second trimester human foetal livers by immunoselection for EpCAM+/CD29+/CD49d+/CD49e–/CD235a–/CD45– cells. Expression profiling of mRNA and microRNA in human foetal LPCs was performed and compared with mature human hepatocytes and human embryonic stem cells undergoing hepatocytic differentiation. Foetal LPCs exhibit a distinct transcriptome profile consistent with a stem cell signature, cell division, and some liver-specific functions. Bioinformatic integration of microRNA and mRNA datasets revealed that microRNAs up-regulated in LPCs targeted genes involved in metabolic processes implying repression of the mature hepatocyte phenotype. Control of LPC gene expression therefore occurs at both transcriptional and, via microRNAs, post-transcriptional levels. Furthermore, transcription factor binding site analyses revealed enriched E2F1 motif in gene and microRNA promoters suggesting feedback control in determining LPC fate. Foetal LPCs were capable of differentiation to a hepatocytic phenotype in the presence of appropriate paracrine signals provided by EpCAM– non-parenchymal cells (NPCs), which consist mainly of endothelial cells and hepatic stellate cells. Fibronectin, despite being produced in abundance by EpCAM– NPCs, had no effect on LPC synthetic function in vitro. The expression of fibronectin in the perisinusoidal space suggests its potential role of modulating cross-talk between hepatoblasts/hepatocytes, liver sinusoidal endothelial cells, and hepatic stellate cells. Fibronectin expression in the portal vein mesenchyme and laminin α5 expression along the ductal plate suggest that both matrix molecules, located in close proximity to LPCs, may be important in supporting the LPC niche. Findings in this work provide insight into the regulation of the human foetal LPC functional phenotype, bringing stem cell-based therapies for liver disease one step closer.
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Lewis, Emily Elizabeth Louise. "Modelling the mesenchymal stem cell niche in vitro using magnetic nanoparticles." Thesis, University of Glasgow, 2016. http://theses.gla.ac.uk/7243/.
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Mesenchymal stem cells (MSCs) are multipotent stem cells residing within the bone marrow, with the ability to differentiate into cells of mesodermic origin (e.g. bone, cartilage and fat). These cells also possess extensive immunomodulatory and wound healing properties. Therefore, MSCs have multiple applications in the field of regenerative medicine. However, present day culturing techniques, encourage a loss of multipotency and limit the availability of true MSCs, for research and clinical use. A culturing technique, which is able to sustain multipotent and quiescent MSCs, is therefore required for future use. MSCs reside within a unique microenvironment in the bone marrow, termed the niche, which protects and regulates stem cell homeostasis. The niche environment controls maintenance, proliferation and differentiation of the stem cells. Current research is focused on the creation of an in vitro niche model, to study the regulatory mechanisms, which govern stem cell fate. The majority of existing models use traditional two-dimensional (2D) techniques. However, stem cells cultured by this method are known to lose potency and spontaneously differentiate into undesired cell types. These issues are caused by 2D in vitro niche models lacking the complexity and the three-dimensional (3D) nature, of the native in vivo niche. Therefore, in the last few years, research has moved away from 2D models, towards creating 3D in vitro niche models. This project aimed to develop a novel, bio-responsive in vitro 3D MSC niche model. The methodology adopted the use of magnetic nanoparticle loaded MSCs, which were levitated using an external magnetic field, to form multicellular spheroids which were subsequently located within a Type I collagen gel. The MSCs within the spheroid niche model exhibited native niche behaviour (retention of multipotency and quiescence). Furthermore, in the presence of a wound, the model accelerated the wound healing process. The MSCs directionally migrated out of the niche towards the wound site and start differentiating into the local resident cell type. Further investigation, identified IL-6 as a potential MSC migratory signal in this bio-response.
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Chang, Chao-Hui. "Haematopoietic stem/progenitor cell interactions with the bone marrow vascular niche." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:452da334-bd4e-45c7-a7bd-fc8767d1239c.
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Umbilical cord blood (UCB) is used as a source of haematopoietic stem cells (HSCs) for transplantation but shows defective homing to the bone marrow niche and delayed haematological reconstitution. Following transplantation, HSCs will home to the bone marrow in response to the CXCL12 chemokine, adhere to the bone marrow sinusoidal endothelial cells and then migrate into and lodge in bone marrow niches. In addition to CXCR4, a variety of molecules have been described as being important in these processes. In this laboratory, junctional adhesion molecule-A (JAM-A) was shown to be expressed on human UCB CD133⁺/CD34⁺ cells and regulated by hypoxia. In this thesis, further phenotypic studies show that this molecule is most highly expressed on human CD41a⁺ megakaryocytes and CD14⁺ monocytes/macrophages in UCB. JAM-A was also found to be expressed on all human UCB CD133⁺ cells, which have been shown by others to encompass the HSCs and early myeloid-lymphoid precursors and on the majority of CD34⁺ haematopoietic progenitor cells (HPCs). While it is also present on bone marrow sinusoidal endothelium (BMEC), JAM-A is not detected on cultured bone marrow mesenchymal stromal cells (MSCs). JAM-A blockade, silencing and overexpression experiments showed that JAM-A contributes to, but is not solely responsible for, the adhesion of CD34⁺ haematopoietic progenitor cells to IL-1β activated BMEC-60 cells and fibronectin. Lack of significance in cell migration suggested that JAM-A is more likely to act as an adhesion molecule or a regulator of adhesion rather than as a migratory molecule in such cells. Further functional studies using the proximity ligation assay highlight a potential association of JAM-A with CXCR4 and the adhesion molecules, tetraspanin CD82 and integrin β1. Mechanistic studies were commenced to establish if JAMA could modulate CXCR4 signalling following CXCL12 stimulation, but time constraints prevented these from being completed. These preliminary experiments which were carried out first in the Jurkat cell line lacking JAM-A or transduced to express JAM-A, however, suggest that JAM-A may modulate CXCL12-induced Rap1 phosphorylation and ERK1/2 phosphorylation. The former pathway is important for integrin function and the latter pathway is important in cell adhesion. The results described here, although requiring finalisation, support the hypothesis that JAM-A acts as an adhesion molecule and also may fine tune CXCR4 and integrin mediated functions on human CD34⁺ cells, thereby potentially regulating engraftment of these cells to the bone marrow niche.
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Saleh, Fatima. "Regulation of mesenchymal stem cell activity in an in vitro model of the stem cell niche." Thesis, University of York, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.547374.
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Tsujita, Maristela. "Participação do nicho endosteal na regulação da hemopoese de camundongos submetidos à desnutrição proteica." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/9/9136/tde-04052016-110647/.
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O nicho endosteal da medula óssea abriga as células-tronco hemopoéticas (CTH) em quiescência/autorrenovação. As CTH podem ser classificadas em dois grupos: células que reconstituem a hemopoese em longo prazo (LT-CTH) e curto prazo (CT-CTH). Investigamos, neste trabalho, os efeitos da desnutrição proteica (DP) no tecido ósseo e a participação do nicho endosteal na sinalização osteoblasto-CTH. Para tanto, utilizamos camundongos submetidos à DP induzida pelo consumo de ração hipoproteica. Os animais desnutridos apresentaram pancitopenia e diminuição nas concentrações de proteínas séricas e albumina. Quantificamos as CTH por citometria de fluxo e verificamos que os desnutridos apresentaram menor porcentagem de LT-CTH, CT-CTH e de progenitores multipotentes (PMP). Avaliamos a expressão das proteínas CD44, CXCR4, Tie-2 e Notch-1 nas LT-CTH. Observamos diminuição da expressão da proteína CD44 nos desnutridos. Isolamos as células LT-CTH por cell sorting e avaliamos a expressão gênica de CD44, CXCR4 e NOTCH-1. Verificamos que os desnutridos apresentaram menor expressão de CD44. Em relação ao ciclo celular, verificamos maior quantidade de LT-CTH nas fases G0/G1. Caracterizamos as alterações do tecido ósseo femoral, in vivo. Observamos diminuição da densidade mineral óssea e da densidade medular nos desnutridos. A desnutrição acarretou diminuição da área média das seções transversais, do perímetro do periósteo e do endósteo na cortical do fêmur dos animais. E na região trabecular, verificou-se diminuição da razão entre volume ósseo e volume da amostra e do número de trabéculas, aumento da distância entre as trabéculas e prevalência de trabéculas ósseas em formato cilíndrico. Avaliamos a expressão de colágeno, osteonectina (ON) e osteocalcina (OC) por imuno-histoquímica, e de osteopontina (OPN) por imunofluorescência no fêmur e verificamos diminuição da marcação para OPN, colágeno tipo I, OC e ON nos desnutridos. Evidenciamos, pela técnica do Picrosírius, desorganização na distribuição das fibras colágenas e presença de fibras tipo III nos fêmures dos desnutridos, além de maior número de osteoclastos evidenciados pela reação da fosfatase ácida tartarato resistente. Os osteoblastos da região femoral foram isolados por depleção imunomagnética, imunofenotipados por citometria de fluxo e cultivados em meio de indução osteogênica. Observamos menor positividade para fosfatase alcalina e vermelho de alizarina nas culturas dos osteoblastos dos desnutridos. Avaliamos, por Western Blotting, a expressão de colágeno tipo I, OPN, osterix, Runx2, RANKL e osteoprotegerina (OPG), e, por PCR em tempo real, a expressão de COL1A2, SP7, CXCL12, ANGPT1, SPP1, JAG2 e CDH2 nos osteoblastos isolados. Verificamos que a desnutrição acarretou diminuição da expressão proteica de osterix e OPG e menor expressão gênica de ANGPT1. Avaliamos a proliferação das células LSK (Lin-Sca1+c-Kit+) utilizando ensaio de CFSE (carboxifluoresceína succinimidil ester). Foi realizada cocultura de células LSK e osteoblastos (MC3T3-E1) na presença e ausência de anti-CD44. Após uma semana, verificamos menor proliferação das LSK dos desnutridos. O bloqueio de CD44 das LSK do grupo controle diminuiu a proliferação destas em três gerações. Entretanto, nos desnutridos, esse bloqueio não afetou a proliferação. Concluímos que a DP promoveu alterações no tecido ósseo e nas CTH. Entretanto, não podemos afirmar que as alterações observadas no sistema hemopoético foram decorrentes de alterações exclusivas do nicho endosteal.The bone marrow endosteal niche hosts hematopoietic stem cells (HSC) in quiescence/self-renewal. HSC can be classified into two groups: cells capable of renewing indefinitely (LT-HSC) or repopulating in the short term (ST-HSC). In this work, we investigated the effects of protein malnutrition (PM) on bone tissue and the involvement of the endosteal niche in osteoblast-CTH signaling. Therefore, we used mice subjected to PM induced by the consumption of hypoproteic feed. Malnourished animals presented pancytopenia and decreased concentration of serum protein and albumin. We quantified the HSC by flow cytometry and found that the malnourished ones had lower percentage of LT-HSC, ST-HSC and multipotent progenitors (MPP). We assessed the expression of the CD44, CXCR4, Tie-2 and Notch-1 proteins in LT-HSC. We observed decreased expression of CD44 protein with the malnourished ones. We isolated the LT-HSC cells by means of cell sorting and assessed the gene expression of CD44, CXCR4 and NOTCH-1. We found that malnutrition had lower expression of CD44. Regarding the cell cycle, we see greater amount of LT-HSC in the G0 and G1 phases. We characterized the changes of the femoral bone tissue in vivo. We observed a decrease in the bone mineral density and medullar density in malnourished animals. As for malnourished animals, the femoral cortical region showed a significant decrease in tissue area, periosteal and endosteal perimeter. The femoral trabecular region of malnourished animals showed decreased bone volume/tissue volume ratio, decreased trabecular number, increased trabecular separation and prevalence of rod-like trabeculae. We investigated the expression of collagen, osteonectin (ON) and osteocalcin (OC) by means of immunohistochemistry and the expression of osteopontin (OPN) by immunofluorescence and we found that malnourished animals showed decreased labeling for OPN, type I collagen, OC and ON in the cortical region of the femur. Picrosirius staining was used to analyze disorganization of collagen fibers and presence of type III fibers in the femurs of the malnourished. Cortical and trabecular regions of malnourished animals presented a higher number of osteoclasts as shown by tartrate-resistant acid phosphatase reaction. Moreover, osteoblasts were isolated from the femoral region by immunomagnetic depletion and immunophenotyped by flow cytometry and cultured in osteogenic induction medium. Results proved less positive for alkaline phosphatase and alizarin red in the cultures of osteoblasts of malnourished animals. We assessed, by means of Western blotting, type I collagen expression, OPN, osterix, Runx2, RANKL and osteoprotegerin (OPG) and, by real time PCR, the expression of COL1A2, SP7, CXCL12, ANGPT1, SPP1, JAG2 and CDH2 with the isolated osteoblasts. We found that malnutrition led to osterix and OPG decreased protein expression and lower ANGPT1 gene expression. We evaluated LSK cell (Lin-Sca1+c-Kit+) proliferation by CFSE (carboxyfluorescein succinimidyl ester). LSK cells and osteoblasts (MC3T3-E1) cocultures were performed in the presence and absence of anti-CD44. After a week, we found lower proliferation of LSK in the malnourished. The LSK CD44 blocking in the control group decreased the proliferation of these three generations. However, as for the malnourished, such blockage did not affect proliferation. We concluded that the PM has promoted changes in bone tissue and the CTH. However, we can\'t claim that the alterations observed in hematopoietic system were due to endosteal niche-only changes.
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Pierret, Chris. "Characterization of an in vitro neural stem cell niche with educational component Stem cells and society /." Diss., Columbia, Mo. : University of Missouri-Columbia, 2008. http://hdl.handle.net/10355/6054.
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Thesis (Ph. D.)--University of Missouri-Columbia, 2008.The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on August 3, 2009) Vita. Includes bibliographical references.
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Yao, Hsin-Lei Reid Lola M. "The hepatic stem cell niche and paracrine signaling by mesenchymal cells in support of human hepatic stem cells." Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2007. http://dc.lib.unc.edu/u?/etd,1270.
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Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2007.Title from electronic title page (viewed Mar. 26, 2008). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biomedical Engineering." Discipline: Biomedical Engineering; Department/School: Medicine.
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Zhou, Lili. "The role of Lasp in the «Drosophila» male stem cell niche and in muscle development." Thesis, McGill University, 2010. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=95064.
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Drosophila Lasp is the only member of the nebulin family in Drosophila. Lasp has an amino-terminal LIM domain, two actin-binding nebulin repeats and a carboxyl-terminal SH3 domain and exhibits very high homology to human Lasp. To assess Lasp function in vivo, we generated a null mutant in Drosophila Lasp, named Lasp1. Lasp1 mutants are homozygous viable, but male sterile. Lasp localizes to cyst cells, early germ cells, hub cells and actin cones. In Lasp1 mutants, the stem cell niche is no longer anchored to the apical tip of the testis, and actin cone migration is perturbed resulting in improper spermatid individualization. Lasp colocalizes with βPS integrin and genetically interact with βPS integrin resulting in complete hub cell mislocalization, which indicates that Lasp modulates integrin adhesion in this context. Lasp1 mutant larvae and flies also have impaired crawling, climbing and flying ability. Lasp localizes to Z lines of third instar larval body wall muscles. In Lasp1 mutant indirect flight muscle, thin filament and sarcomere length is reduced while sarcomere ultrastructure is not significantly affected. The same applies to larval body wall muscles, where we observe a misregulation of sarcomere length in both absence and overexpression of Lasp. This phenotype is very similar to nebulin mutant knock-out mice indicating that Lasp plays a role in regulating thin filament lengths, but with only two nebulin repeats.Chez la drosophile, Lasp est la seule protéine représentante de la famille des Nébuline. Lasp contient un domaine LIM, deux répétitions de type Nébuline et un domaine SH3, et présente une forte homologie avec la famille Lasp des mammifères. Afin identifier le rôle de Lasp, nous avons généré une mutation nulle, nommée Lasp1. Les mutants Lasp1 sont homozygotes viables, mais les mâles stériles. Lasp se localise dans cellules kyste, dans les cellules germinales, les cellules hub et au niveau des cônes d'actine. Chez les mutants Lasp1, les cellules souches ne sont plus ancré à l'extrémité apicale du testicule, et la migration des cônes d'actine est perturbée, conduisant à une individualisation irrégulière des spermatides. Lasp est colocalisée avec l'intégrine βPS et interagit génétiquement avec l'intégrine βPS, amenant une délocalization des cellules hub, indiquant que Lasp module adhésion intégrine dans ce contexte. Les larves mutantes pour Lasp se déplacent avec difficulté et les adultes ont avec une capacité d'escalade et de vols réduite. Lasp se localise aux lignes Z dans les muscles des larves du troisième stade. Chez les adultes Lasp1, les muscles des ailes présentent une longueur réduite des filaments minces ainsi que des sarcomères, alors que l'ultrastructure du sarcomère ne semble pas être significativement affectée. Les muscles larvaires présentent le phenotype. De plus, on observe un dérèglement de la longueur du sarcomère en surexprimant Lasp dans un contexte sauvage. Ce phénotype est très similaire à celui des souris mutantes pour la nébuline, indiquant que Lasp joue un rôle dans la régulation de la longueur du filament mince, mais avec seulement deux répétitions nébuline.
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Klatte, Jennifer Elisabeth. "Characterization of the epithelial stem cell niche of the human hair follicle." Giessen : DVG-Service, 2008. http://d-nb.info/989343707/04.
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Lymperi, Stefania. "The role of osteoblasts and osteoclasts in the haemopoietic stem cell niche." Thesis, Imperial College London, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.501195.
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Sevencan, Suat. "Economic Aspects of Fuel Cell-Based Stationary Energy Systems." Doctoral thesis, KTH, Tillämpad elektrokemi, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-179137.
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It is evident that human activity has an important impact on climate. Constantly increasing energy demand is one of the biggest causes of climate change. The fifth assessment report of the Inter-governmental panel on climate change states that decarbonisation of electricity generation is a key component of climate change mitigation. Increased awareness of this fact and escalating concerns around energy security has brought public attention to the energy industry, especially sustainable power generation systems. Future energy systems may need to include hydrogen as an energy carrier in order to achieve necessary levels of CO2 emission reductions, and overcome the challenges renewable energy systems present. Fuel cells could be a corner stone of future hydrogen inclusive energy solutions. New solutions like fuel cells have to compete with existing technologies and overcome the shortcomings of emerging technology. Though these shortcomings are well-recognised, fuel cells also have many advantages which makes continued research and development in the field highly worthwhile and viable. Key to their adoption is the identification of a niche market to utilise their advantages while overcoming their shortcomings with continuous research and development. This thesis aims to evaluate some of the stationary fuel cell applications and determine whether one could become the niche market as an entry point for fuel cells. This is achieved by economic evaluations of real and hypothetical applications. Results of the studies here imply that to decrease the total life cycle impacts of fuel cells to more acceptable levels, resource use in the manufacturing phase and recycling in decommissioning should be shown more attention. Results also present a picture showing that none of the applications investigated are economically feasible, given the current state of technology and energy prices. However, fuel cell-based combined cooling, heating and power systems for data centres show the potential to become the niche market that fuel cells need to grow. A further conclusion is that a broad market, longer stack lifetime, the possibility of selling electricity back to the grid and governmental subsidies are essential components of an environment in which fuel cells can permeate through the niche market to the mainstream markets.QC 20151210
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Franke, Katja. "Adhesion and Single Cell Tracking of Hematopoietic Stem Cells on Extracellular Matrices." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2011. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-77290.
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The local microenvironment of hematopoietic stem cells (HSCs) in the bone marrow -referred to as stem cell niche- is thought to regulate the balance of stem cell maintenance and differentiation by a complex interplay of extrinsic signals including spatial constraints, extracellular matrix (ECM) components and cell-cell interactions. To dissect the role of niche ECM components, a set of well-defined matrix biomolecular coatings including fibronectin, laminin, collagen IV, tropocollagen I, heparin, heparan sulphate, hyaluronic acid and co-fibrils of collagen I with heparin or hyaluronic acid were prepared and analyzed with respect to adhesive interactions of human CD133+ HSCs in vitro. ECM molecule dependent adhesion areas as well as fractions of adherent HSCs were assessed by reflection interference contrast microscopy and differential interference contrast microscopy. HSCs, so far mostly classified as suspension cells, exhibited intense adhesive interactions with fibronectin, laminin, collagen IV, heparin, heparan sulphate, and collagen I based co-fibrils. An integrin mediated adhesion on fibronectin and a L-selectin mediated adhesion on heparin pointed to specific interactions based on different adhesion mechanisms. As a consequence of HSC adhesion to molecules of the vascular and the endosteal regions, both regions were confirmed as possible stem cell niches and adhesive signals were suggested as potential regulators of stem cell fate. Furthermore, the impact of a spatially organized ECM on the HSC behavior was analyzed by single cell tracking. These studies required the development of engineered three-dimensional, ECM coated microcavities with the option for single cell tracking. A semi-automated cell-tracking tool was established to accelerate data access from time-lapse image sequences. From this analysis it was possible to reveal the genealogy, localization, morphology and migration of single HSCs over a time period of 4 days. A decreased cycling frequency was observed depending on the HSC localization in the spatially constraining microcavities. Besides the revealed impact of spatial constraints on HSC fate, the newly engineered ECM-coated microcavity setup and the semi-automated cell tracking tool provide new options to study the cell fate in engineered microenvironments at single cell level for other cell types ex vivoDie lokale Mikroumgebung von Blutstammzellen (BSZ) im Knochenmark, bezeichnet als Stammzellnische, reguliert das Gleichgewicht von Stammzellerhaltung und -differenzierung durch ein komplexes Zusammenspiel von extrinsischen Signalen wie räumliche Beschränkungen, Komponenten der extrazellulären Matrix (EZM) und Zell-Zell Wechselwirkungen. Um die Rolle der EZM-Komponenten zu analysieren, wurden definierte Beschichtungen von Fibronektin, Laminin, Kollagen IV, monomerem Kollagen I, Heparin, Heparan Sulphat, Hyaluronsäure und Co-Fibrillen aus Kollagen I und Heparin oder Hyaluronsäure hergestellt und in vitro bezüglich der adhäsiven Wechselwirkungen von humanen CD133+ BSZ untersucht. Die Adhäsionsflächen und der Anteil adhärenter Zellen wurden in Abhängigkeit von der EZM-Beschichtung mittels Reflexions- Interferenz-Kontrast-Mikroskopie und Differentieller Interferenz Kontrast Mikroskopie bestimmt. BSZ, bisher als Suspensionszellen definiert, zeigten intensive adhäsive Wechselwirkungen mit Fibronektin, Laminin, Kollagen IV, Heparin, Heparan Sulphat und den Co-Fibrillen. Eine Integrin abhängige Adhäsion auf Fibronektin und eine L-Selektin abhängige Adhäsion auf Heparin, wiesen auf spezifische Wechselwirkungen hin, die auf unterschiedlichen Mechanismen basieren. Aufgrund der Adhäsion von BSZ sowohl zu Molekülen der vaskulären als auch der endostealen Knochenmarkregion, wurden beide Bereiche als mögliche Stammzellnische bestätigt. Adhäsive Signale sind potentielle Regulatoren der Stammzellentwicklung. Im Weiteren wurde der Einfluss einer räumlich beschränkenden EZM auf das Verhalten der BSZ durch Einzelzellverfolgung untersucht. Diese Studien erforderten die Entwicklung von dreidimensionalen EZM-beschichteten Mikrokavitäten, die das Verfolgen einzelner Zellen ermöglichten. Es wurde ein halbautomatischer Algorithmus für die Zellverfolgung etabliert, um die Datengenerierung von den Zeitreihenaufnahmen zu beschleunigen. Die Analysen ermöglichten Aussagen über die Genealogie, Lokalisierung, Morphologie und Migration einzelner BSZ während einer Analysenzeit von 4 Tagen. Eine verringerte Zellteilungsaktivität wurde in Abhängigkeit von der BSZ Lokalisierung innerhalb der räumlich einschränkenden Mikrokavitäten festgestellt. Neben diesen Erkenntnissen bieten die entwickelten Mikrokavitäten und die etablierte Einzelzellverfolgung neue Möglichkeiten auch andere Zelltypen auf Einzelzellniveau ex vivo zu untersuchen
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Balmer, G. M. "The epicardium : contribution from the haematopoietic lineage and its potential as a stem cell niche The epicardium : contribution from the haematopoietic lineage and its potential as a stem cell niche." Thesis, University College London (University of London), 2014. http://discovery.ucl.ac.uk/1428882/.
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The adult mammalian heart is no longer considered a post mitotic organ with evidence for homeostatic cell turnover and presence of populations of resident cardiac stem/progenitor cells. The epicardium is a single cell layer surrounding the myocardium and derives from the proepicardial organ during development. It has an important role in formation of the coronary vessels and the embryonic myocardium but, until recently, was considered to be quiescent in the adult heart. Recent studies have shown that reactivation of the adult epicardium post myocardial infarction (MI) injury induces a population of epicardium-derived progenitor cells (EPDCs) to proliferate, migrate and differentiate in the scar region. It remains unclear whether these EPDCs reside in an epicardial niche within the adult heart as classically defined in other lineages, such as the hair follicle bulge and intestinal crypt. Using confocal microscopy, we have identified cell clusters located in the intact epicardium, which express key extracellular matrix (ECM) and stem/progenitor cell markers and respond dynamically to injury, with cluster reformation occurring by day 21 post MI. We suggest that these clusters are candidates for a putative epicardial niche in the adult heart. Lineage tracing analyses to determine the origin of cells residing within the clusters have excluded Wt1+ or Gata5+ proepicardial lineages, but suggest a haematopoietic source. Using Vav1Cre; ROSA-tdTomato to label haematopoietic cells, we have shown that cells from the haematopoietic lineage contribute to the adult epicardium. We have tracked this contribution through development and into adulthood and show an increase in incidence of cells of the haematopoietic lineage in the epicardium with age. Results from bone marrow (BM) transplantation experiments, using whole BM we have shown contribution to the adult epicardium from BM cells. Labelling donor cells using Vav1Cre; ROSA-tdTomato shows that these cells are of haematopoietic lineage. These data suggest haematopoietic cells represent a novel source of cells that contribute to the epicardium during development and that are likely involved in replenishing epicardial cell turnover during cardiovascular homeostasis and may participate in cardiac repair.
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Voog, Justin C. "Defining a genetic framework for stem cell niche maintenance in the Drosophila testis." Diss., [La Jolla] : University of California, San Diego, 2009. http://wwwlib.umi.com/cr/ucsd/fullcit?p3365768.
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Thesis (Ph. D.)--University of California, San Diego, 2009.Title from first page of PDF file (viewed Aug. 10, 2009). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 107-125).
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Wurmser, Maud. "Rôle des homéoprotéines SIX dans les progéniteurs myogéniques au cours du développement musculaire." Thesis, Sorbonne Paris Cité, 2017. http://www.theses.fr/2017USPCB053/document.
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Les homéoprotéines SIX sont codées par les gènes Sine oculis homeobox related genes Six1 à Six6 chez les vertébrés parmi lesquels Six1, Six2, Six4 et Six5 sont exprimés dans le lignage myogénique. Bien que Six1 et Six4 soient requis pour la myogenèse hypaxiale, les animaux doubles KO pour ces deux gènes (s1s4KO) forment leurs muscles épaxiaux et craniofaciaux. Nous avons caractérisé le phénotype de mutants composites des gènes Six et avons montré que l’absence de Six1 et Six2 empêchait la formation des muscles craniofaciaux et empirait les défauts de formation des muscles des membres observés chez les fœtus mutants pour Six1. Nous avons aussi observé que les fœtus dépourvus d’activité de SIX1, SIX2, SIX4 et SIX5 étaient toujours capables de former leurs muscles épaxiaux, mais que l’expression de Pax7 dans leurs progéniteurs myogéniques était fortement diminuée et mêlée à l’expression de Myogénine. Alors que les fœtus s1s4KO forment des muscles épaxiaux, leurs cellules PAX7+ ont un défaut de nichage entre la membrane plasmique des myofibres et la lame basale qui les entoure. Nos analyses transcriptomiques, nos expériences de transplantation et nos études in vitro nous ont permis de conclure que le nichage des cellules PAX7+ nécessitait un environnement adéquat combinant des propriétés des myofibres et des cellules PAX7+ ; environnement perturbé dans les muscles épaxiaux s1s4KO. Nos expériences de transplantation nous ont aussi permis de conclure que Six1 et Six4 étaient requis pour une bonne ré-innervation des myofibres après blessure et pour la mise en place du phénotype rapide de ces myofibres. De plus, les muscles transplantés avec des cellules PAX7+ fœtales s1s4KO après blessure se reforment d’un grand nombre de petites myofibres. Nous avons pu relier ce phénotype au comportement des cellules s1s4KO in vitro où elles montrent un défaut de fusion. Enfin, les homéoprotéines SIX ont besoin de co-facteurs pour induire l’expression de leurs gènes cibles, tels que les protéines EYA codées par les gènes Eya1 à Eya4 chez les vertébrés. Eya3 et Eya4 sont fortement exprimés dans les cellules satellite au cours de la régénération, cellules qui requièrent aussi Six1 pour une réparation musculaire efficace. Nous avons étudié la régénération musculaire en absence d’expression d’Eya3 et n’avons pas observé de défaut nous menant à la conclusion qu’Eya3 n’est pas requis pour la régénération musculaire adulte, mais que sa perte d’expression était peut-être compensée par un autre gène Eya chez les animaux mutants. Pour conclure, Six1 et Six2 sont indispensables à la formation des muscles craniofaciaux, et Six1 et Six4 sont requis pour la myogenèse hypaxiale, et pour l’établissement d’un environnement propice à la maturation des myofibres fœtales et au nichage des cellules PAX7+ au cours de la myogenèse épaxiale, et permettant la croissance des myofibres et leur ré-innervation après blessure. La collaboration des protéines SIX avec leurs co-facteurs EYA au cours de la myogenèse nécessite d’autres études pour mieux définir leurs fonctionsSIX homeoproteins are encoded by the Sine oculis homeobox related genes Six1 to Six6 in vertebrates among which Six1, Six2, Six4 and Six5 are expressed in the muscle lineage. Whereas Six1 and Six4 are required for hypaxial myogenesis, double KO for those two genes (s1s4KO) still form their epaxial and craniofacial muscles. We further characterized the phenotype of compound Six mutant embryos and showed that the absence of Six1 and Six2 completely impairs craniofacial myogenesis and worsen muscle limb development observed in single Six1 mutants. We also showed that mouse fetuses devoid of SIX1, SIX2, SIX4 and SIX5 activity are still able to develop epaxial muscles, but that Pax7 expression in myogenic progenitors of these mutants is reduced and intermingled with Myogenin expression. While s1s4KO fetuses still develop epaxial muscles, their PAX7+ cells show a perturbed homing process into their niche, between the plasma membrane of a myofibre and the basal lamina surrounding it. Transcriptomic analysis, transplantation experiments and in vitro studies allowed us to conclude that the homing of PAX7+ cells into their niche during fetal myogenesis requires an adequate environment combining properties of the myofibers and the PAX7+ cells; environment disturbed in s1s4KO epaxial muscles. Transplantation experiments also led us to conclude that Six1 and Six4 are required for proper myofiber re-inervation after injury and for the establishment of the fast phenotype of myofibers. Furthermore, muscles transplanted with s1s4KO fetal PAX7+ cells after injury are formed of numerous and tiny myofibers. We could link this phenotype to the behavior of s1s4KO cells in vitro where they showed perturbed fusion. Finally, SIX homeoproteins require co-factors to induce their target genes expression, as EYA proteins encoded by Eya1 to Eya4 in vertebrates. Eya3 and Eya4 are strongly expressed in satellite cells during regeneration, cells in which Six1 is also required for proper muscle repair. We investigated muscle regeneration in absence of Eya3 expression and observed no obvious phenotype. We concluded that Eya3 is not required for muscle regeneration but that other Eya genes might compensate its function in KO mouse. To conclude, Six1 and Six2 are required for craniofacial myogenesis and Six1 and Six4 for hypaxial myogenesis and for the establishment of a proper environment allowing myofibre maturation and PAX7+ cells homing during fetal epaxial myogenesis and enabling myofibre growth and re-innervation after injury. The role of the collaboration between SIX and EYA proteins during myogenesis still needs more investigation
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Zimmerman, Grant Robert. "T-ALL LEUKEMIA DYSREGULATES STROMAL BONE MARROW ENVIRONMENT AND DISRUPTS NICHE-STEM CELL SIGNALING AXIS." Case Western Reserve University School of Graduate Studies / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=case1436293859.
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Wagner, Mykaela. "Changes in gene expression in C2C12 cells in response to changes in culture conditions, the cellular niche." Youngstown State University / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=ysu1588956844832033.
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Lynch, Thomas John. "Adult stem cells in the trachea and tracheal submucosal glands." Diss., University of Iowa, 2016. https://ir.uiowa.edu/etd/6464.
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Breathing is essential for human life, yet tens of millions of people in the U.S. alone suffer from lung diseases. With each breath, lungs are exposed to the external environment. Inhaled air first passes through the trachea, bronchi, and finally the bronchioles before it reaches the alveoli where gases are exchanged. A barrier of epithelial cells protects the airways. In addition, epithelial glands also secrete protein-rich fluids onto the airway surfaces to help maintain sterility. Injury, disease, or other factors can damage these cells, and regiospecific stem cells (SCs) can divide to replace them. However, many important details about lung SCs are still unknown. For example, what processes control SC division? How do region-specific SCs differ from one another? And how does disease or injury impact SC biology? We found that some processes that regulate lung development also control adult SC division following injury. We show that SCs from airway glands give rise to surface epithelial cell types and glandular cell types. In contrast, surface SCs only generated surface cell types. Finally, we identify a type of cell in the glands that can regenerate surface cell types after severe injury. These studies provide new insights into the neighborhoods in which SCs reside in the large airways and processes that control their contribution to airway repair following injury. Overall, this research provides important new insights into adult SC biology and conditions affecting lung health.
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Okamoto, Natsuko. "A melanocyte-melanoma precursor niche in sweat glands of volar skin." Kyoto University, 2015. http://hdl.handle.net/2433/195943.
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The final publication is available at http://dx.doi.org/10.1111/pcmr.12297. Natsuko Okamoto et al. A melanocyte–melanoma precursor niche in sweat glands of volar skin. Pigment Cell & Melanoma Research. Volume 27, Issue 6, pages 1039–1050, November 2014Kyoto University (京都大学)
0048
新制・論文博士
博士(医学)
乙第12890号
論医博第2090号
新制||医||1007(附属図書館)
31644
京都大学大学院医学研究科医学専攻
(主査)教授 野田 亮, 教授 羽賀 博典, 教授 鈴木 茂彦
学位規則第4条第2項該当
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Doucet, Yanne. "Identification and characterization of the progenitor niche of the Merkel cell lineage : from homeostasis to cancer." Thesis, Aix-Marseille, 2015. http://www.theses.fr/2015AIXM4766.
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La peau est organisée en niche de cellules souches/progénitrices qui contribuent au maintien des lignées épidermiques pendant l’homéostasie permettant ainsi de conserver l’intégrité du tissue. Les différentes cascades de signalisation qui régulent cet équilibre sont essentielles et la perturbation de ces voix peuvent amener à une différentiation anormale des kératinocytes, pouvant engendrer des cancers de la peau. Le but de cette thèse était d’identifier et de caractériser la population de progéniteurs responsables de la maintenance d’une niche épidermique spécialisée dans la mechanotransduction du toucher léger appelée les cellules de Merkel. Mon étude a porté sur le rôle des progéniteurs épithéliaux localisés dans le dôme du toucher (DT) de l’épiderme dans des conditions d’homéostasie ainsi que sur le développement du carcinome des cellules de Merkel. Basé sur l’analyse de données de microarray, j’ai identifié une nouvelle population de progéniteurs qui expriment de manière spécifique Kératine 17 (K17). Des expériences de traçage de lignées cellulaires démontrent que ces cellules donnent naissance aux cellules de Merkel (CM) ainsi qu’aux cellules squameuses. De plus, l’ablation génétique sélective des progéniteurs des CMs dans le DT a montré que cette niche est isolée et indépendante du reste de l’épiderme. Ces résultats établissent les CMs comme la quatrième lignée cellulaire de la peau. Cette découverte a permis l’établissement de nouveaux outils pour l’étude de conditions pathologiques associées à la lignée des cellules de Merkel, telles que les carcinomes des cellules de Merkel et le déclin du toucher léger avec l’âgeThe skin is organized in highly regionalized stem or progenitor cell niches that are in charge of maintaining all epidermal lineages during homeostasis. Disruption of molecular pathways that tightly regulate this balance leads to abnormal specification and differentiation of keratinocytes, eventually causing skin cancer. The goal of this thesis was to identify and characterize the progenitor population responsible for the maintenance of an epidermal niche specialized for mechanosensory signaling: the Merkel cell lineage. This work focused on the role of the epithelial progenitors located in the touch dome (TD) of hairy skin under homeostatic conditions and in a Merkel cell carcinoma (MCC) context. Based on previous microarray data, I first identified a distinct population of the interfollicular epidermis uniquely expressing Keratin 17 (K17). By lineage tracing analysis, I demonstrated that these cells give rise to the Merkel cell (MC) and squamous lineage. More importantly, selective genetic ablation of K17+ TD keratinocytes (TDKC) showed that the TD is a self-autonomous niche defining it as the 4th lineage of the skin. Interestingly, TDKCs may be involved in maintaining innervation of the Merkel cell-neurite complex. These critical results have established a new plateform for the field to allow studies of pathological skin conditions such as Merkel cell carcinoma and the loss of tactile acuity with age
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Gu, Ying. "A Traveling Niche: The Role of Steel Factor in Mouse Primordial Germ Cell Development." UNIVERSITY OF CINCINNATI, 2012. http://pqdtopen.proquest.com/#viewpdf?dispub=3491024.
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Tripathi, Pratibha. "Isolation of multipotent astroglia form the adult stem cell niche and the injured brain." Diss., lmu, 2009. http://nbn-resolving.de/urn:nbn:de:bvb:19-104224.
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Secker, G. "Characterisation of the corneal epithelium and stem cell niche using models of PAX6 deficiency." Thesis, University College London (University of London), 2009. http://discovery.ucl.ac.uk/17305/.
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The corneal epithelium is continuously renewed by a population of stem cells that reside in the corneo-scleral junction, otherwise known as the limbus. These limbal epithelial stem cells (LESC) are imperative for corneal maintenance, with deficiencies resulting in in-growth of conjunctival cells, neovascularisation of the corneal stroma and eventual corneal opacity and visual loss. One such disease that has traditionally been thought to be due to LESC deficiency is aniridia, a pan-ocular congenital eye disease due to PAX6 heterozygosity. Corneal changes or aniridia related keratopathy (ARK) seen in aniridia are typical of LESC deficiency, however, the pathophysiology behind ARK is still ill defined. Recent studies, utilising heterozygous Pax6 mouse models suggests that ARK is not solely due to LESC deficiency. Current theories suggests it may be caused by a deficiency in the stem cell niche and adjacent corneal stroma leading to abnormal differentiation of epithelial cells, with an altered wound healing response also playing a role (Ramaesh et al., 2005a, Li et al., 2008). The ultimate goal of biological research is to further the understanding of disease mechanisms with aim to develop improved therapies, therefore this thesis examines the pathogenesis of the ARK with this in mind. The difficulties found with the initial assessment of gene replacement therapy in the mouse highlighted the need for further investigation into LESC location and ARK progression. The examination of corneal epithelial label retaining cells indicated an increase in numbers and abnormal location of putative LESC in the heterozygous Pax6 mouse, suggesting these cells may fail to differentiate into progeny cells. Furthermore, analysis of corneal epithelial and fibroblast cells with PAX6/Pax6 down regulation has further established that an abnormal wound healing response may be involved in disease progression. Overall, these studies have highlighted the complexity of the disease and the requirement for further investigation to dissect the mechanisms underlying ARK, providing clues for future directions in therapy development.
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Kräter, Martin. "Bone marrow niche-mimetics modulate hematopoietic stem cell function via adhesion signaling in vitro." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2017. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-230268.
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As graft source for lymphoma or leukemia treatment, hematopoietic stem and progenitor cells (HSPCs) have been the focus of translational medicine for decades. HSPCs are defined by their self-renewing capacity and their ability to give rise to all mature blood cells. They are found anchored to a specialized microenvironment in the bone marrow (BM) called the hematopoietic niche. HSPCs can be enriched by sorting them based on the presence of the surface antigen CD34 before clinical or tissue engineering use. As these cells represent a minority in most graft sources and the amount of applicable cells is limited, ex vivo expansion-cultures were established using cytokine cocktails or small molecules. However, in vitro culture of HSPCs as suspension-cultures result in heterogeneous cell populations with undefined cellular identities. In the BM niche, HSPCs are not exclusively maintained by cytokines but also by cell-matrix adhesions mediated by integrins (ITGs). Thus, β1 and β2 ITGs were found to promote initial contact of HSPCs with mesenchymal stromal cells (MSCs) and ITGβ3 expression was shown to be a marker for long-term repopulating HSPCs in vivo. Consequently, ex vivo remodeling of the BM niche using co-cultures of HSPCs and niche cells like MSCs came into spotlight and was proven to be a promising tool for stem cell expansion. However, in clinical and research applications, direct contact of two cell populations necessitates HSPC post-culture purification. To address these problems, we established a novel culture method for remodeling the BM extra cellular stroma in vitro wherein we used decellularized extracellular matrix (ECM) scaffolds derived from immortalized mesenchymal stromal cells (SCP-1). Such scaffolds were found to be highly reproducible and served as in vitro niche for HSPCs by being more effective for the expansion of CD34+ cells, compared to classical suspension cultures. ECMs were shown to consist of multiple proteins including fibronectins, collagens, and a major niche chemokine responsible for BM homing and retention of HSPCs in vivo, namely, stromal derived factor 1 (SDF-1). SDF-1 is known to be secreted by MSCs and is anchored to matrix proteins. This reveals that ECM scaffolds produced by SCP-1 cells are a naïve reconstructed microenvironment. When CD34+ cells were seeded, only around 20% of the cells adhered to the provided ECM scaffold.
These cells recognized SDF-1 via C-X-C chemokine receptor type 4 (CXCR-4), as shown by laser scanning confocal microscopy. Thus, adhesive sides as they are present in the BM niche are provided. However, CD34+ cells isolated from G-CSF mobilized peripheral blood of healthy donors were found to be heterogenous with respect to adhesion capacity. Nonetheless, it was similar to HSPC co-cultures with SCP-1 cells as feeder layer. Therefore, we separated and analyzed two cell fractions, the adherent (AT-cells) and the non- adherent supernatant (SN-cells) cells. Other signals provided by the BM extracellular stroma to HSPCs are physical cues that control HSPC fate. HSPCs sense these physical features through focal contacts and accordingly remodel their morphological and biomechanical properties. Using real-time deformability cytometry (RT-DC) to uncover biomechanical phenotypes of freshly isolated HSPCs, SN-cells, AT-cells, and classical suspension cultured HSPCs in plastic culture dishes (PCD) were analyzed. We found freshly isolated cells to be less deformable and small.
AT-cells displayed actin polymerization to stress fibers, and exhibited a stiffer mechanical phenotype compared to PCD-cultured or SN-cells. This might constitute the first hint of functional adaptation. Integrins are known to establish mechanosensing focal contacts. Thus, we analyzed ITG surface expression and identified ITGαIIb, ITGαV, and ITGβ3 to be enriched on AT-cells compared to freshly isolated cells or SN-cells. Active integrins need to form heterodimers consisting of one α- and one β subunit. Interestingly, the identified ITGs exclusively interact with each other to form RGD peptide receptors. RGD is a tripeptide consisting of the amino acids arginine, glycine, and aspartic acid and was identified as an adhesion sequence within fibronectin and other extracellular proteins. Consequently, we could confirm an important role for ITGαVβ3 in HSPC- ECM interaction with respect to adhesion and migration. However, we also identified ITGβ3 expression on a subset of CD34+ cells either freshly isolated or ECM cultured cells, as a marker for erythrocyte differentiation. These findings demonstrate that, in vitro, the ECM compartment acts as a regulator of HSPC fate and portray mechanical recognition as a potent driver of differentiation.
In this context, targeted modulation of ECM scaffolds could enhance cell-ECM interactions and accelerate stem cell expansion or differentiation. These modulations could also provide further insights into HSPC-niche regulation. We demonstrate that ECMs derived from osteogenic differentiated SCP-1 cells increase HSPC expansion but do not lead to increased cell adhesion. As ECM adhesion preliminary alters HSPC function, we aimed at developing ECM scaffolds with increased adhesion capacity. Using lentiviral transduction, we generated a stable knock down of fibulin-1 in SCP-1 cells. Fibulin-1 is an ECM protein known to form anti-adhesion sites with fibronectin. However, we failed to increase adherent cell numbers or enhance HSPC expansion in the fibulin-1 knock down ECMs.
Taken together, SCP-1 cell-derived ECM protein scaffolds provide an in vitro niche for HSPCs capable of stem cell expansion. Integrin mediated signaling altered the biomechanical and functional properties of HSPCs and hints at suspension cultures as being inappropriate to study the physiological aspects of HSPCs. Targeted modulation of ECM scaffolds could theoretically generate suitable ex vivo environments with the capacity to gain detailed insight into HSPC regulation within their niche. This will enhance the functionality of new biomaterials and will lead to improved regenerative therapies like BM transplantation.
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Bakker, E. Y. "Analysis of drug resistance and the role of the stem cell niche in leukaemia." Thesis, University of Salford, 2017. http://usir.salford.ac.uk/43810/.
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Glucocorticoids and etoposide are used to treat acute lymphoblastic leukaemia (ALL) as they induce death in lymphoblasts through the glucocorticoid receptor (GR) and p53. However, glucocorticoid resistance, cell death mechanisms and the contribution of the bone marrow microenvironment to drug response/resistance all require investigation. Using microenvironment-mimicking conditioned media (CM), dexamethasone (a synthetic glucocorticoid) and etoposide to treat glucocorticoid-sensitive (C7-14) and glucocorticoid-resistant (C1-15) cells, pathways by which the microenvironment exerts its chemoprotective effect have been investigated. CM reduced caspase-3/8 activation, downregulated RIPK1 (necroptotic marker), and limited chemotherapy-induced BECN1 downregulation, suggesting protective effects of CM. Glucocorticoids upregulated BIRC3 (which ubiquitinates RIPK1), whilst CM altered GR phosphorylation. GR occupancy was observed on the RIPK1, BECN1 and BIRC3 promoters and changed depending on its phosphorylation. High-molecular weight proteins reacting with the RIPK1 antibody increased with CM, and reduced following AT406 BIRC3 inhibitor treatment suggesting they represent ubiquitinated RIPK1. These results suggest mechanisms by which CM promotes survival, as well as indicating novel glucocorticoid-regulated pathways. Complementing laboratory investigation is the construction of a Boolean model of the GR interaction network (GEB052, GR “interactome”) containing 52 nodes (proteins, inputs/outputs) connected by 241 interactions. In silico mutations and analyses have generated predictions that were subsequently validated on a genome-wide scale via comparison to microarray data. GEB052 demonstrated high prediction accuracy, consistently achieving a better prediction rate than a randomised model. Quantitative algorithmic analysis via microarray superimposition has also been performed, and lastly the model has been preliminarily validated as a clinical tool via superimposition of patient microarray data and comparing model predictions to clinical data. In summary, this thesis provides novel insight into the effects of the microenvironment, and identifies new glucocorticoid-regulated pathways. The GEB052 model of GR signalling represents the novel application of this modelling approach to GR research, and generates accurate predictions.