Dissertations / Theses: 'Corneal epithelial cells'

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Liu, Ke. "Role of second messengers in controlling growth patterns of corneal epithelial cells /." View thesis, 2002. http://library.uws.edu.au/adt-NUWS/public/adt-NUWS20030718.102224/index.html.

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Thesis (Ph. D.)--University of Western Sydney, 2002.
"This thesis is submitted in fulfilment of the requirements of the degree of Doctor of Philosophy to the University of Western Sydney School of Biological Sciences."t.p. Includes bibliographical references (leaves 138-150).

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Binti, Kamarudin Taty Anna. "Differentiation of human pluripotent stem cells into corneal epithelial like cells." Thesis, University of Newcastle upon Tyne, 2018. http://hdl.handle.net/10443/4182.

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Cornea is the clear outermost protective layer of the eye which enables transmission of light onto the retina. The corneal epithelium is regenerated by limbal stem cells (LSCs), whose loss/dysfunction results in limbal stem cell deficiency (LSCD). Transplantations of ex vivo expanded autologous LSCs from patient's healthy eye onto the affected eye have provided a successful treatment for unilateral LSCD. This however is not applicable to patient with total bilateral LSCD, whose both eyes are affected. This thesis investigated the potential of human induced-pluripotent stem cell (hiPSCs) to differentiate into corneal epithelial-like cells as a source of autologous stem cell treatment for patients with total bilateral LSCD, and tested the engraftment of the differentiated cells in LSCD mouse model. Combined addition of bone morphogenetic protein 4 (BMP4), all trans-retinoic acid (RA) and epidermal growth factor (EGF) for the first nine days of differentiation followed by cell-replating on collagen-IV coated surfaces with a corneal-specific-epithelial cell media for an additional 11 days, resulted in step wise differentiation of human embryonic stem cells (hESCs) to corneal epithelial progenitors and mature corneal epithelial-like cells. Differences in the ability of hiPSCs lines to undergo differentiation to corneal epithelial-like cells were observed. These were dependent on the level of endogenous BMP signalling and could be restored via activation of this signalling pathway by a specific TGFβ inhibitor (SB431542). The hESC and hiPSCs-derived corneal epithelial cells were transplanted into a LSCD mouse model where they survived up to 14 days, but failed to provide long term engraftment and corneal surface regeneration. The findings showed a differential ability of hESCs and hiPSCs lines to generate corneal epithelial cells which is underlined by the endogenous BMP signalling pathway activity. However, the engraftment and functionality of the differentiated cells in the LSCD animal model has yet to be improved.

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Liu, Ke. "Role of second messengers in controlling growth patterns of corneal epithelial cells." Thesis, View thesis, 2002. http://handle.uws.edu.au:8081/1959.7/387.

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The purpose of this thesis was to investigate mechanisms contolling the growth of corneal epithelial cells, particularly the intracellular signals involved with stratification compared with cellular migration and maturation. Buttons of epithelium were cultured in different culture media. The explants were monitored microscopically for their growth patterns and finally fixed and examined for cytokeratin, vimentin and actin. Different growth patterns were observed in the different media, indicating that different signalling patterns must be operating in these cells depending upon the media in which they were grown. To investigate the intracellular pathways controlling the different growth patterns, the protein phosphorylation of different cultures was investigated. The two proteins, p57 and p30, are strongly suggested to be associated with stratification of the epithelial cells. The possible involvement of the common serine kinase, PKC, in controlling the growth pattern of corneal epithelial cells were also investigated. The results suggested that an intracellular pathway involving PKC promotes the maturation and spread of the cells but is not involved in their stratification. These experiments taken together indicate that the different aspects of corneal epithelia cell growth are tightly controlled and may occur quite independently. Specific protein expression appears to be important for stratification, and phosphorylation of proteins by PKC appears to be involved with the maturation of epithelial cells from basal cells. It also indicates that the mature cells are capable of producing the extracellular matrix protein fibronectin which appears to have an important role in causing the spread as distinct from the stratification of the corneal epithelial cells.

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Liu, Ke, University of Western Sydney, of Science Technology and Environment College, and of Science Food and Horticulture School. "Role of second messengers in controlling growth patterns of corneal epithelial cells." THESIS_CSTE_SFH_Liu_K.xml, 2002. http://handle.uws.edu.au:8081/1959.7/387.

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The purpose of this thesis was to investigate mechanisms contolling the growth of corneal epithelial cells, particularly the intracellular signals involved with stratification compared with cellular migration and maturation. Buttons of epithelium were cultured in different culture media. The explants were monitored microscopically for their growth patterns and finally fixed and examined for cytokeratin, vimentin and actin. Different growth patterns were observed in the different media, indicating that different signalling patterns must be operating in these cells depending upon the media in which they were grown. To investigate the intracellular pathways controlling the different growth patterns, the protein phosphorylation of different cultures was investigated. The two proteins, p57 and p30, are strongly suggested to be associated with stratification of the epithelial cells. The possible involvement of the common serine kinase, PKC, in controlling the growth pattern of corneal epithelial cells were also investigated. The results suggested that an intracellular pathway involving PKC promotes the maturation and spread of the cells but is not involved in their stratification. These experiments taken together indicate that the different aspects of corneal epithelia cell growth are tightly controlled and may occur quite independently. Specific protein expression appears to be important for stratification, and phosphorylation of proteins by PKC appears to be involved with the maturation of epithelial cells from basal cells. It also indicates that the mature cells are capable of producing the extracellular matrix protein fibronectin which appears to have an important role in causing the spread as distinct from the stratification of the corneal epithelial cells.
Doctor of Philosophy (Ph.D.)

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Guo, Ying. "Actin contractility in corneal epithelial cells and regulation of the barrier integrity." [Bloomington, Ind.] : Indiana University, 2006. 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:3229600.

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Thesis (Ph.D.)--Indiana University, School of Optometry, 2006.
"Title from dissertation home page (viewed July 11, 2007)." Source: Dissertation Abstracts International, Volume: 67-08, Section: B, page: 4360. Adviser: Sangly Srinivas.

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Terry, S. J. "Identification of regulators and effectors of RhoGTPase signalling in corneal epithelial cells." Thesis, University College London (University of London), 2011. http://discovery.ucl.ac.uk/1306879/.

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Epithelial cells adhere to each other and are connected via a series of junctions. Tight junctions (TJs) are a specific type of junction consisting of heteromeric protein complexes that are linked to the actin cytoskeleton and are important in regulating paracellular permeability and cell polarity. RhoGTPases are small molecular switch proteins that are important regulators of the cytoskeleton and modulators of gene expression. RhoGTPases have thus been identified as being major signalling components associated with TJs. However little is known about how RhoGTPases are regulated to control junction formation and gene expression in corneal epithelial cells. I used a siRNA screening approach combined with functional assays to identify components of RhoGTPase signalling that affect the assembly of junctions and gene expression in Human corneal epithelial cells (HCE). I identified and validated several candidates that regulate junction assembly. One of these candidates was p114RhoGEF, a novel TJ localised guanine nucleotide exchange factor (GEF) important for the assembly of functional TJs. p114RhoGEF is a widely expressed and I discovered its depletion effects junction formation and morphogenesis in three dimensional culture systems in different epithelial cell types. p114RhoGEF is required for activation of RhoA at cell-cell junctions and junctional actinomyosin activity, p114RhoGEF is present in a complex containing Myosin II-A, the RhoA effector Rock II and the junctional adaptor protein Cingulin; indicating p114RhoGEF is a component of a junction associated RhoA-signalling module. p114RhoGEF, thus regulates spatial activation of RhoA at cell-cell junctions and organisation of the junctional cytoskeleton. p114RhoGEF may also have a role in cell migration, as depletion in HCE cells, caused cells to migrate at a slower rate during wound healing assays. I have also started to explore the function of a putative p114RhoGEF ortholog, cg10188 in Drosophila melanogaster. Preliminary experiments have identified cg10188 to be important in larval development.

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Bhattacharya, Pradipta. "The corneal epithelium in health and disease." Thesis, Queensland University of Technology, 2022.

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The cornea is the anterior transparent layer of the eye, and understanding corneal epithelial morphology is valuable in monitoring ocular surface diseases. A meta-analysis showed that central corneal basal cell density and nerve parameters were reduced in diseases affecting the ocular surface including limbal stem cell deficiency. Using newly developed image analysis techniques it was observed that epithelial cell density was highest at the corneal nerve whorl region. A decrease in epithelial cell density was observed in eyes with conjunctival ultraviolet autofluorescence, i.e. with sunlight-induced UV damage.

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Barnard, Zeke. "Analyses of Cytokeratins and p63 Isoforms Expressed by Human Limbal Epithelial Cells." Thesis, Griffith University, 2005. http://hdl.handle.net/10072/367809.

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Cultivated autologous limbal epithelial grafts have shown promise as a valuable treatment to treat ocular surface injuries. The rationale for this therapy is that the limbus contains progenitor cells of the corneal epithelium and that when expanded and transplanted these cells help to restore a healthy epithelium. Nevertheless, this approach is still experimental and the optimal procedure for producing, grafting and assessing the presence of limbal stem cells in these grafts remains under development. The first results chapter examines the differences in cytokeratin expression between donor corneoslceral rims and cultivated limbal epithelial cultures derived from these donor rims. The expression of keratins 3 and 14 is more stable between the in situ and in vitro environments, while keratins 6 and 19 appear to be upregulated in limbal epithelial cells in response to their isolation and culture. This highlights the role of keratins in limbal epithelial cell function and provides valuable knowledge for assessing the phenotype of cultivated limbal epithelial grafts. Cytokeratin expression is more important in identifying the transient amplifying population than the progenitor population, indicating the need for more specific markers to identify the progenitor cells. The second results chapter investigated the potential of p63 in identifying the progenitor population of the limbal epithelium. The use of RT-PCR enabled a more detailed examination of the RNA expression of the p63 isoforms. The experiments performed assessed p63 in situ and in vitro, including conditions designed to expand the progenitor population in culture. This work confirms the value of p63 as a marker for immature limbal epithelium and also demonstrates for the first time the p63 isoforms produced by human limbal epithelial cells in vitro. The final results chapter describes the application of techniques developed and utilised in the previous 2 chapters upon autologous cell samples and constructed grafts used to treat 5 patients clinically. The phenotypic analyses demonstrate that both the cultivated epithelium and the grafts created from the different donor biopsies exhibit similar properties in the areas examined. In addition, these results allowed comparison between clinically used autologous cultures and the methods employed to cultivate limbal epithelium examined in the previous chapter. The use of both p63 and the various cytokeratins in assessing the level of differentiation hold merit, however more specific markers for the stem cell population of the limbal epithelium remain elusive. The results present a deeper analysis of the material used to treat limbal stem cell deficiency and give insight into further evolution of these treatments.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Biomedical Sciences
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Shortt, A. J. "The limbal epithelial stem cell niche and its relevance to ex-vivo culture and transplantation of corneal limbal epithelial stem cells." Thesis, University College London (University of London), 2009. http://discovery.ucl.ac.uk/18929/.

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Transplantation of ex-vivo cultured limbal epithelial cells (LEC) is an established treatment for total limbal stem cell deficiency. However, this therapy has preceded the scientific understanding of limbal epithelial stem cells (LESC), the LESC niche and the biological mechanism that underpins it. This body of work tested the hypothesis that the LESC niche has a specialised structure and that an understanding of this may lead to improvements in outcomes and understanding of this therapy. The corneal limbus was investigated using 3D confocal microscopy, scanning electron microscopy, wholemount immunofluorescence and in-vivo confocal microscopy. This produced the most comprehensive study of limbal architecture performed to date. The structure of the LESC niche was identified and the effect of age and disease examined. Next, a clinical study of ex-vivo expansion and transplantation of LEC was performed in patients with LESC deficiency. A defined set of objective outcome measures enabled a baseline standard for outcomes of this therapy to be described. Future improvements to this therapy can be assessed using these techniques. Human amniotic membrane (HAM) is the leading candidate for a surrogate LESC niche. The method of HAM processing was investigated and found to be critical to achieve this. A method of significantly improving the yield of LESC in culture on HAM was identified. It remains to be seen whether this will translate into improved clinical outcomes. Finally, a disconnection exists between published data indicating a good clinical outcome and data demonstrating the survival of transplanted cells. This work assessed and demonstrated the feasibility of using quantum dot nanocrystal labelling of transplanted LESC to track cells post transplantation. It is concluded that translation of these findings to modify and improve current treatment protocols may result in improvements in outcomes for patients with LESC deficiency undergoing this therapy.

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Yang, Juan. "Universal corneal epithelial-like cells derived from human embryonic stem cells in a defined, xeno-free, and albumin-free condition for cellularization of a corneal scaffold." Thesis, University of Macau, 2018. http://umaclib3.umac.mo/record=b3953938.

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Wang, Kemeng. "Role of potassium channel Kir4.1(KCNJ10) in the wound healing of human corneal epithelial cells." Thesis, Boston University, 2013. https://hdl.handle.net/2144/12245.

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Thesis (M.A.)--Boston University
The cornea, which is key to maintaining our normal vision by refracting light onto the lens and retina, as well as serving as a physical barrier to protect our eyes from the environment, contains epithelium tissue with one of the highest capacities for renewal and wound healing in the body. Although many studies have looked at the process of wound healing in corneal epithelium, most of them have focused on the various ligand-receptor growth factor signaling pathways, and few studies have been done to study the signaling and regulations of wound healing that are initiated intracellularly, or those associated with electrical currents and channel activity. This study, therefore, aims to look at the hypotheses that injury to the corneal epithelium leads to the downregulation of a type of potassium transport channels named Kir4.1/KCNJ10, and that the inhibition of KCNJ10 is associated with intracellular microRNA-205 (miR-205), which is upregulated during injury and healing. Together, the modulations in the level of KCNJ10 and microRNA-205 contribute to change in the environment around the wound and promote the cellular processes that allow for efficient corneal healing. To investigate the role of potassium channels and microRNAs in the cornea epithelium, an in-vitro model of endogenous wound healing was employed with human corneal epithelium cells (HCECs) serving as the primary model of study. Physiological injury was simulated using a scratch-wound model. The protein expressions for KCNJ10 and microRNA-205 were measured through various time points from both control and injured HCECs. The effect of two RNAi modulators of microRNA-205, a mimic and antagonist, and of KCNJ10 blockers were also tested for effects on the rate and efficiency of HCEC wound healing. Results indicated that the expression of miR-205 increased in scratch-injured HCECs and that the expression of KCNJ10 decreased in wounded and healing HCECs. It was also shown that increasing KCNJ10 and decreasing miR-205 both lead to delayed healing, but that blocking KCNJ10 could partially abolished the effect of delayed healing associated with decreasing miR-205 and restored the healing process. It was also shown that the 3'UTR of KCNJ10 contains potential target sites for miR-205 binding and action. The results indicate that that KCNJ10 expression is negatively associated with corneal wound healing, and that miR-205 is upregulated upon injury in wounded corneal epithelium to inhibit KCNJ10 and allow for the processes of wound healing to take place.

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Nili, Ahmadabadi Elham. "Development of a novel mesenchymal stromal cell (MSC) therapy for repairing the cornea." Thesis, Queensland University of Technology, 2018. https://eprints.qut.edu.au/122897/1/Elham_Nili%20Ahmadabadi_Thesis.pdf.

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This thesis has produced advances in our understanding of the biology and potential clinical application of stem cells to aid the treatment of patients with severe eye injuries. This research evaluated the therapeutic potential of a stem cell (called Mesenchymal Stromal Cells (MSCs)) isolated from the peripheral margin of the cornea, known as the limbus. Firstly, a method for routinely isolation and propagation of human limbal MSCs was optimized. Subsequently, the performance of those cells on a silk fibroin membrane was examined.

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He, Zhiguo. "Application de l'immunolocalisation à la recherche de la cellule souche endothéliale cornéenne humaine." Phd thesis, Université Jean Monnet - Saint-Etienne, 2011. http://tel.archives-ouvertes.fr/tel-00987968.

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Le contrôle de la transparence de la cornée dépend de l'intégrité de l'endothélium cornéen mono-stratifié qui est classiquement considéré dès la naissance, dépourvu de capacité de régénération chez l'homme. Dans des conditions pathologiques conduisant à la cécité par œdème cornéen, les pertes significatives en cellules endothéliales (CE) ne sont pas remplacée efficacement, ce qui signifie que ni de nouvelles CE provenant de cellules souches (CS), ni la division des cellules voisines des lésions ne peuvent contribuer à la régénération endothéliale. Toutefois, plusieurs travaux ont prouvé depuis 25 ans que les CE possédaient une capacité proliférative résiduelle ex vivo et deux équipes ont suggéré l'existence de CS ou de progéniteurs à la périphérie de l'endothélium cornéen. Dans notre travail de thèse, nous avons tout d'abord optimisé, en la systématisant, une technique d'immunomarquage spécialement adaptée à l'endothélium cornéen intact de cornées montées à plat. A l'issue de ces développements, nous disposons de protocoles simples de fixation à température optimale et de démasquages antigéniques susceptibles de permettre la révélation de nombreuses protéines. A partir d'une importante série de cornées humaines non conservées et d'autres conservées en organoculture, et grâce à cet outil désormais efficace, nous avons étudié le cycle cellulaire des CE et la localisation de potentielles CS sur l'endothélium cornéen humaine. Nos résultats démontrent que dans ces conditions, les CE expriment de façon homogène des régulateurs positifs (PCNA, MCM2, cycline D1, cycline E et cycline A) et des régulateurs négatifs du cycle cellulaire (P16, P27); certaines particularités ont par ailleurs pu être décrites de façon innovante, comme la localisation cytoplasmique diffuse de MCM2, paranucléaire de la cycline D1, l'absence de P21. L'ensemble des marquages pourrait suggérer que les CE sont arrêtées en fin de G1, après le point de restriction et que de nombreux mécanismes de réparation de l'ADN sont mis en jeux dans les CE exposées à un stress oxydant important tout au long de l'existence. Nous avons identifié une nouvelle organisation de la micro-anatomie de la périphérie et de l'extrême périphérie de l'endothélium où des cellules regroupées en multiples clusters pluristratifiés semblent alimenter des colonnes de CE radiaires longues d'un millimètre. Ces éléments, associés à l'observation d'une moindre différenciation et d'une compétence proliférative plus élevés en périphérie suggèrent un nouveau modèle d'homéostasie endothéliale humaine in vivo: toute la vie, des CS périphériques alimentent de façon très lente la périphérie cornéenne en CE qui migrent de façon centripète pour assurer la stabilité du centre cornéen dont les propriétés optiques primordiales sont sous-tendues par un endothélium qui ne perd que 0,6% de CE par an. A la différence de l'épithélium cornéen, ce système ne peut être accéléré lors de circonstances pathologiques. Les perspectives de nos travaux sont désormais d'essayer d'isoler de l'extrême périphérie les CS endothéliales ou les progéniteurs et de les cultiver en recréant un microenvironnement favorable. La possibilité de produire un grand nombre de CE in vitre non pas à partir de CE sénescentes prélevées sur la totalité de l'endothélium comme cela a été tenté par la passé, mais cette fois à partir de CS ou des progéniteurs ouvriraient la voie d'une véritable thérapie cellulaire endothéliale. L'enrichissement des greffons pendant la durée de leur conservation à la banque de cornée pourrait constituer une première étape majeure avant d'envisager créer de novo un endothélium sur un support greffable pour une greffe endothéliale du 3e type qui deviendrait ainsi enfin indépendante des aléas de la découpe du greffon. Enfin, l'ïdentification de la CS endothéliale et de son microenvironnement permettra aussi d'envisager une thérapie cellulaire in vivo pour traiter les stades précoces des pathologies endothéliales cornéennes

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Douvaras, Panagiotis. "Effects of age and Pax6 deficiency on mouse limbal stem cell function." Thesis, University of Edinburgh, 2010. http://hdl.handle.net/1842/4414.

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The conventional view for corneal epithelial maintenance suggests that a stem cell population found in the limbus (at the rim of the cornea) produces daughter cells, called transient amplifying cells, which migrate centripetally. This limbal stem cell (LSC) hypothesis was recently questioned and the alternative model suggests that stem cells are present throughout the corneal epithelium. The main aims of this thesis were to investigate whether age and Pax6 genotype affect LSC function. Previous work with X-inactivation mosaics revealed radial stripes of β-galactosidase-expressing cells in the corneal epithelium (from about 5 weeks of age), which decreased with age and were reduced in Pax6+/- mice (a model for aniridia, a human eye disease). The reduction in Pax6+/- mice could be due to either reduced LSCs function or a more coarse-grained mosaicism caused by reduced cell mixing during development. Comparison of patch sizes in Pax6+/- and wild-type X-inactivation mosaics showed that patches were smaller in Pax6+/- cornea epithelia before the initiation of stripes (3 weeks of age). This implies that stripe-number reduction is not caused by reduced cell mixing, so an effect on LSC function remained a possibility. Thus, the numbers of label-retaining cells (putative stem cells) in Pax6+/- were compared to controls at 15 and 30 weeks old but they were not reduced at 30 weeks or in Pax6+/- mice, as had been predicted. The failure to demonstrate the predicted result suggests either that the hypothesis was incorrect or the experimental approach was inappropriate. Furthermore, it was discovered that mice expressing β-galactosidase under the keratin 5 promoter produced rare stripes in the corneal epithelium, which are likely to represent clonal lineages derived from individual stem cells. Older mice demonstrated a significantly lower frequency of stripes, a result compatible with the predicted reduction of active LSC with age. Pax6+/- corneas were highly abnormal and stripes were not formed properly, so direct comparison was not possible. Finally, pilot experiments with conditional expression of a reporter gene revealed the successful formation of a stripe, and hence provide a plausible alternative approach to compare stripe numbers reflecting active LSCs but the method has yet to be optimised. Overall, the results suggest that LSCs are reduced with age and support the limbal location of stem cells maintaining the corneal epithelium.

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Findlay, Amy Siobhan. "The molecular basis of epthelial cell migration : maintenance and repair of the ocular surface." Thesis, University of Aberdeen, 2015. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=228963.

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In vertebrates the cornea must maintain its transparency throughout adult life to ensure sight, and understanding the mechanisms underpinning corneal homeostasis are fundamental to developing new treatments to cure or prevent blindness. This study investigated the role the planar cell polarity pathway plays in the directed migration of adult corneal epithelial cells, in maintaining the homeostatic environment of the eye and during wound healing. RT-PCR confirmed, for the first time, the expression of multiple core PCP genes within human corneal epithelial (HCE) cells. Components of the PCP pathway were pharmacologically and genetically manipulated during wound healing of corneal epithelial cells and the importance of the downstream target JNK, and core PCP gene Vangl2, during wound healing was demonstrated. Manipulation of core PCP components was found also to directly affect the ability of HCE cells to realign and migrate in response to physical topographical cues in vitro. This study therefore indicated that PCP may regulate the directed migration of corneal epithelial cells as they travel over the basement membrane. Using conditional knockout mice the loss of Vangl2, a core PCP gene, and its effect on both planar and the apical-basal polarity of the corneal epithelium was investigated. Severe morphological defects were observed in Vangl2-null mice indicative of underlying problems in apical-basal polarity of the epithelial cells. The basement membrane of Vangl2-null cells was largely absent in vivo, which suggested that at least some of the planar defects were secondary to an unexpected failure of apical-basal polarity. This study has shown for the first time that PCP plays a crucial role in the maintenance of an adult vertebrate tissue, particularly during wound healing and maintenance of the corneal epithelium. It has also indicated a role for the core PCP gene, Vangl2, in setting up apical-basal polarity of these adult cells.

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Mort, Richard Lester. "Stem cell function in the mouse corneal epithelium." Thesis, University of Edinburgh, 2007. http://hdl.handle.net/1842/8187.

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Limbal stem cells maintain the corneal epithelium through a process of clonal growth and ordered migration. In X-inactivation mosaic female mice, that express LacZ from one of their X-chromosomes, random clumps of LacZ-positive cells are seen in the cornea at 3-6 weeks of life. This pattern resolves between 6-10 weeks forming radial stripes thought to represent chords of clonally related, inwardly migrating cells. By measuring the number and width of stripes and correcting for the effects of different proportions of LacZ-positive cells, an estimate of the number of coherent stem cell clones maintaining the tissue can be derived. Analysis at 5 ages demonstrated that the estimated number of coherent stem cell clones is reduced from ~100 at 15 weeks to ~50 at 39 weeks and is then stable at least until 52 weeks. An automated method was developed using image analysis software to analyse these striping patterns. This method produced results that did not differ significantly from the above. The dosage of the transcription factor Pax6 is crucial for normal eye development. In Pax6 heterozygous animals the estimated number of coherent stem cell clones is reduced to ~50 at 15 weeks with no further reduction up to 30 weeks. Mice hemizygous for the PAX77 transgene over-express human PAX6. In PAX77 hemizygous X-inactivation mosaics, estimated clone number was similarly reduced to ~50 with no further decline. Mice heterozygous for both Gli3 and Pax6 have a distinct striping phenotype, highlighted by an increase in coherent clones. When the corneal epithelium is injured the surrounding epithelial cells migrate along the corneal stroma to cover the wound. X-gal staining of healed, centrally wounded X-inactivation eyes reveals that striping patterns are reconstituted during wound healing in ex-vivo culture. In GFP mosaics the healing process can be imaged using time-lapse confocal microscopy. This technique demonstrated that clones remain contiguous throughout their migration. Healing of peripheral wounds was observed to form de-novo whorling patterns, revealing that basal cells in the epithelium can migrate both away from and towards the limbal region.

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Kolli, Satya Sai Prasad. "Corneal Epithelial Stem Cell Biology and its Therapeutic Application." Thesis, University of Newcastle Upon Tyne, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.506607.

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Kucerova, Romana. "Genetic control of corneal epithelial cell migration by Pax6." Thesis, University of Aberdeen, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.485667.

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Coordinated cell migration is essential for tissue regeneration andÃ‚Â· development. During migration cells sense various directional cues from the surrounding environment, and intracellular mechanisms process this extracellular information resulting in appropriate vectorial cell movement. During corneal epithelial maintenance, cells migrate centripetally from the limbal region towards the centre of the cornea. This centripetal movement is genetically controlled by Pax6 (Collinson et aI., 2004), one of the master genes in eye development (Kozmik, 2005). The guidance cues and cellular mechanisms controlling centripetal migration of corneal epithelial cells remain to be elucidated. Clarifying the mechanisms guiding cell migration in an experimentally accessible model of corneal epithelial cells would be relevant to mechanisms controlling vectorial cell migration in other cell types. In this project the roles of endogenous electrical and chemical cues in directing corneal epithelial cell migration were tested using in-vitro and ex-vivo culture systems in which migration was induce by wounding. It was ~hown that Pax6+1 - cells suffer from a 2 h wound healing delay, but at later time points, the migration is not significantly different from pax~/+ cells (Leiper et aI., 2006). Additionally Pax6+1 - cells showed defective glycoprotein trafficking which is als~ affecting tJ-le molecules not directly controlled by Pax6 at the transcriptional level and potentially increasing the number of molecular defects contributing to the Pax6+1 - phenotype.. An ex-vivo whole eye system was developed which subsequently demonstrated growth factor dependent woiInd healing defect of pax~/- cells, in contrast to Pax6+1+ epithelia which healed and migrated normally regardless externally supplied growth factors. Exogenous application ofEGF was found to restore wound healing in pax~/cells to wild-type levels. The extensive part of the work was focused on elucidating the role of electrical guidance in wound healing and migration. Recent publications have established roles for endogenous electric current in guiding epithelial ceIl migration (Zhao et aI., 2006)but until this project, there was no genetic model 'of defective 'wound-induced currents. paxrt'l- eyes were found to have compromised or reversed wound mediated currents and were therefore used as model to investigate the importance of electrical cues for normal wound healing. The study represented the most rigorous challenge yet to the hypothesised roles of endogenous electric fields, and surprisingly the analysis showed no significant correlation between electrical cues and directional migration, based on the observation that Pax6+1 - with defective signals healed at rates not significantly different from control wild type epithelia. Further investigation showed no significant correlation ofelectric current and wound healing even in Pax6+1+eyes. In searching for molecular mechanis1J1s controlling directional migration in applied EFs, Pax6+1 - cells were used as model because the cells previously showed compromised or reverse response to in-vitro applied EFs. Src was identified as a candidate signalling molecule playing an important role in mediating directional migration and additionally failure of pSrc to polarise in paxrt'l- cells in applied electric fields correlated with their impaired migration response. Finally wound healing acceleration by addition of Shh peptide was shown to be Pax6 dosage dependent. Investigation of Hh pathway components in Pax6+1+ and paxrt'l- corneal epithelial cells suggested important differences at the level of Gli transcription factors which may underlie the failure of paxrt'l- cell~ to show a migratory respOIise to exogenous Shh.

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Alshammari, Fehaid Salem L. "Mathematical modelling of human corneal oxygenation and cell kinetics in corneal epithelial wound healing." Thesis, Queensland University of Technology, 2016. https://eprints.qut.edu.au/92835/1/Fehaid%20Salem%20L_Alshammari_Thesis.pdf.

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Healthy transparent cornea depends upon the regulation of fluid, nutrient and oxygen transport through the tissue to sustain cell metabolism and other critical processes for normal functioning. This research considers the corneal geometry and investigates oxygen distribution using a two-dimensional Monod kinetic model, showing that previous studies make assumptions that lead to predictions of near-anoxic levels of oxygen tension in the limbal regions of the cornea. It also considers the comparison of experimental spatial and temporal data with the predictions of novel mathematical models with respect to distributed mitotic rates during corneal epithelial wound healing.

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20

Masuda, Sayuri. "Angulin/LSR defines cell corners for tricellular tight junction formation in epithelial cells." Kyoto University, 2011. http://hdl.handle.net/2433/142056.

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Sagga, Nada A. "Dynamics of limbal and conjunctival stem cell activity during ocular surface maintenance." Thesis, University of Aberdeen, 2017. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=235418.

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Corneal degenerative diseases and opacity are leading causes of corneal impairment and blindness worldwide. Like many epithelial tissues, the constant renewal of transparent corneal epithelial cells is essential for a lifelong healthy cornea and optimal vision. The limbus (the boundary between the cornea and the conjunctiva) is believed to be the site that harbours adult stem cells responsible for corneal maintenance and repair after injury, referred to as limbal epithelial stem cells (LESCs). In the basal limbal epithelium, an active LESC subset divides to yield progenitor cells that migrate centripetally into the corneal epithelium for cell renewal. This asymmetric division however, is assumed to be regulated by a balance between cell renewal and loss of cells from the corneal surface. The search for specific LESC molecular markers has been difficult and to date there are few if any candidate markers that unambiguously identify LESCs but not their immediate progeny. Consequently, LESC clonality, activity and proliferative dynamics have remained poorly understood. In addition, the nature of the regulatory molecular pathways involved during LESC activity is still an open key question. In this research project, we identified stem cells on the ocular surface of the eye, assayed their activity and demonstrated quantitively for the first time how the cornea responds to damage. The retention of DNA labelling reagents in control and wounded corneas was combined with clonal analyses of cell division and migration using mice mosaic for reporter LacZ expression. Corneal transplant in LacZ reporter transgenic mice showed migration of LacZ-positive cells into the donor corneal button, with long-term disruption of patterns of migration. Corneal epithelial scrape wounds at the periphery also showed long– term disruption. Label retention suggested a small but statistically significant proliferation response of LESCs to injury, but this was attenuated or absent in aging mice and Pax6 mutants. The Hippo signalling pathway has been shown to have promising results in regulating stem cell activity and proliferation in many organs, however, its effect on LESC proliferation is unknown. Here, we investigated the regulatory role of the Hippo−YAP signalling pathway during cell proliferation, and determined whether the label retention assay in a uniform population of dividing cells is a real indicator of slow-cycling cells in vivo. Cell-cycling kinetics, Abstract v proliferation rate, and label retention were determined in a spontaneous human corneal epithelial (HCE-S) cell line, using double-labelling IdU and EdU thymidine analogues. During homeostasis, HCE-S cells underwent approximately one cell cycle per day, however, cells in which YAP-dependent signalling was activated by 17-Allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of heat shock protein 90 (Hsp90), showed slower proliferation rate and longer cell-cycle time. In vitro label-retention assay in confluent cultures estimated number of ~3-4 cell cycles needed to dilute out the label from slow-cycling cells in vivo. The data are suggestive that the Hippo pathway has a potential regulatory role in proliferative corneal epithelium, and that label-retention assay is a real indicator of slow-cycling cells. Furthermore, this research observed the proliferative dynamics of conjunctival stem cells. Clonal analysis of patterns of cell growth in the conjunctiva were analysed following tamoxifen-induction of LacZ transgene activity. The long−term presence of coherent patches of LacZ-positive cells suggested the presence of long-lived conjunctival stem cells but that the turnover time for the bulbar conjunctival epithelium may be more than 16 weeks. The key results of this research are that the limbus is the niche for stem cells, that there is a unidirectional migration of cells from the limbus to the corneal epithelium during homeostasis, but this is disrupted, perhaps permanently, by wounding. We find only a mild response of limbal epithelial stem cells to acute corneal injury, which is reduced to no response with age and is dependent on genetic background.

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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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23

McBain, Vikki A. "Small applied electric fields, growth factors and corneal epithelial cell behaviour." Thesis, University of Aberdeen, 1999. http://digitool.abdn.ac.uk/R?func=search-advanced-go&find_code1=WSN&request1=AAIU123848.

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Wounding of the cornea generates lateral electric fields (EFs) and initiates the expression of hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF; Chiang et al., 1992; Wilson et al., 1999a). Therefore, these biologically generated EFs and endogenous growth factors may be of particular significance during wound healing. In the presence of an EF (150 mV/mm) cultured corneal epithelial cells (CECs) oriented perpendicular, directed cathodally and migrated at an enhanced rate. The induction times, induction thresholds and response patterns for these behaviours in increasing field strengths, indicated that they may operate through separate and parallel pathways. The application of either HGF or KGF enhanced the rate of CEC migration but neither affected the extent of CEC orientation or directionality. The distribution of HGF receptors (HGFR) was found to be exclusive to the cell body in the presence of an EF, the receptors accumulated cathodally. Moreover, the asymmetrical accumulation of HGFR in the presence of an EF correlated with the direction of CEC migration. The application of both HGF and an EF activated extracellular-signal regulated kinase (ERK) a mitogen-activated protein kinase. Furthermore, in the presence of an EF the observed ERK activation was greater in the cathodal facing half of the CECs. Inhibition of ERK reduced the extent of HGF and EF-enhanced CEC migration rate but did not alter EF-induced CEC cathodal directionality or perpendicular orientation. The HGF- and EF-enhancement of CEC migration rate may involve the activation of ERK and with the downstream liberation of leukotrienes and phosphorylation of MLCK, would culminate in actin contraction and polymerisation respectively. The potential clinical relevance for this work would concern the topical application of HGF and exogenous application of EFs to corneal epithelial wounds in order to augment healing in patients where this process is slow or deficient.

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Ainscough, Sarah Louise. "Improved strategies for the cultivation of human limbal epithelial (HLE) grafts." Thesis, Queensland University of Technology, 2008. https://eprints.qut.edu.au/18575/1/Sarah%20Ainscough%20Thesis.pdf.

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The limbal stem cell population is located in the limbal junctional zone between the cornea and the conjunctiva, and is responsible for maintaining the corneal epithelium. Damage to the limbal stem cell population results in a condition known as limbal stem cell deficiency (LSCD), which is characterised by conjunctivalisation of the cornea, visual impairment and persistent irritation. To treat LSCD, an alternative source of human limbal epithelial (HLE) cells must be transplanted back onto the diseased cornea. Limbal tissue grafts have had a moderate degree of success. However, autologous grafts risk damage to the healthy eye, whilst allogeneic grafts are susceptible to immunological rejection. Cultured HLE grafts offer a promising alternative to whole tissue grafts. The production of cultured HLE grafts involves the removal of a small (1-2 mm2) biopsy from the patient's healthy limbus, followed by ex vivo expansion to produce an epithelial sheet, which is subsequently transplanted onto the damaged corneal surface. However, the production of cultured HLE grafts usually requires the addition of animal-derived products during cell culture. Animal-derived components, such as foetal bovine serum (FBS) and murine 3T3 feeder cells, introduce the patient to potential crossspecies infection and immune responses to xenogeneic antigens. Consequently, the overall aim of this project has been to develop a culture technique free of xenogeneic products for the establishment and propagation of HLE cells. To achieve this aim, alternatives to FBS in the culture medium and 3T3 feeder cells were pursued. A defined serum-free medium (SFM) containing vitronectin (VN), insulin-like growth factor binding protein 3 (IGFBP3), insulin-like growth factor-I (IGF-I), and epidermal growth factor (EGF) was investigated as an alternative to serumsupplemented medium (SSM) for HLE cell culture. Initial studies focused on the effects of these growth factors on HLE cell metabolic activity and migration. Metabolic activity was primarily stimulated by IGF-I and EGF, with the combination of IGF-I and EGF in solution stimulating metabolic activity to a significantly greater extent than the SSM positive control (p = 0.006). HLE cell migration was also effected by combinations of VN, IGFBP3, IGF-I and EGF. Migration was stimulated above the SFM negative control by the combination of IGFBP3 and IGF-I either with or without the addition of EGF. However, the presence of VN was required for optimal migratory responses (p < 0.003). Hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) were also investigated as additional components to the SFM formulation. HGF significantly stimulated HLE cell metabolic activity and migration (p < 0.02). In contrast, KGF did not significantly stimulate either HLE cell metabolic activity or migration. The addition of either HGF or KGF to the SFM supplemented with VN, IGFBP3, IGF-I and EGF did not significantly enhance the metabolic activity of HLE cells. Therefore, HGF and KGF were no longer pursued as additional components to the SFM formulation. Additional studies were conducted to examine the efficacy of replacing murine 3T3 feeder cells with human ocular stromal cells during HLE cell culture. Initially, stromal cells were isolated from the cornea, limbus and sclera to determine whether there were differences between these stromal cell populations. The results indicated that scleral stromal cells had a significantly larger area and perimeter than either corneal or limbal stromal cells (p < 0.001). Scleral stromal cells were also significantly more rounded than either corneal or limbal stromal cells, as determined by the elliptical factor equation (p < 0.001). Immunocytochemistry also revealed that scleral stromal cells expressed significantly more of the myofibroblast marker a- smooth muscle actin than either corneal or limbal stromal cells (p < 0.001), and significantly less of the fibroblast/myofibroblast marker Thy-1 than corneal or limbal stromal cells (p < 0.001). Therefore, scleral stromal cells were identified as different in comparison to corneal and limbal stromal cells. Primary HLE cells were cultured with irradiated corneal, limbal and scleral stromal cells. HLE cultures established with either corneal or limbal stromal feeder cells contained more cellular protein (as measured by rhodamine B dye absorbance) than cultures established without feeder cells (p < 0.001). The colony forming efficiency (CFE) of HLE cells established with corneal or limbal stromal feeder cells was also significantly greater than HLE cells established without feeder cells (p < 0.001). In contrast, HLE cultures established with scleral stromal feeder cells contained low levels of cellular protein and had a low CFE, which was not significantly different to the HLE cultures established without feeder cells. Immunocytochemistry indicated that HLE cultures established with scleral feeder cells also showed lower expression of the stem cell markers ABCG2 and C/EBP. These results suggest that freshly isolated HLE cells can be cultured with irradiated corneal or limbal stromal cells as a replacement for murine 3T3 feeder cells. Finally, the SFM supplemented with VN+IGFBP3+IGF-I+EGF was combined with limbal stromal feeder cells, and examined as a culture technique free of animalderived products. Freshly isolated HLE cells established in SFM supplemented with VN+IGFBP3+IGF-I+EGF and limbal feeder cells contained a similar amount of cellular protein (as measured by crystal violet dye absorbance) when compared to the SSM+3T3 positive control. In addition, the CFE of freshly isolated HLE cells established in VN+IGFBP3+IGF-I+EGF and limbal feeder cells was significantly higher than the SSM+3T3 positive control (p = 0.004). However, a live/dead assay revealed a reduced HLE cell viability in SFM supplemented with VN+IGFBP3+IGFI+ EGF and limbal feeder cells after seven days in culture. In addition, immunocytochemistry demonstrated a lower expression of the stem cell markers ABCG2 and C/EBP in the SFM treatment with limbal feeder cells. Therefore, freshly isolated HLE cells can be cultured in SFM supplemented with VN+IGFBP3 +IGF-I+EGF and limbal feeder cells. However, this culture technique is less likely to support the growth of immature limbal stem cells when compared to the SSM+3T3 positive control. Overall, this research has attempted to create a culture system free of animal-derived products for the production of cultured HLE grafts to treat limbal stem cell deficiency. The results show that HLE cells respond to a serum-free medium formulation containing VN+IGFBP3+IGF-I+EGF. In addition, this culture medium can be combined with irradiated stromal cells isolated from the limbus to support HLE culture production. However, the combination of VN+IGFBP3+IGF-I+EGF and limbal feeder cells demonstrated a reduced viability, which indicates that further refinement of the formulation is required. This thesis has also demonstrated differences between stromal cells isolated from the cornea, limbus, and sclera, and has generated knowledge which may impact on the understanding of stromalepithelial regulation.

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25

Ainscough, Sarah Louise. "Improved strategies for the cultivation of human limbal epithelial (HLE) grafts." Queensland University of Technology, 2008. http://eprints.qut.edu.au/18575/.

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The limbal stem cell population is located in the limbal junctional zone between the cornea and the conjunctiva, and is responsible for maintaining the corneal epithelium. Damage to the limbal stem cell population results in a condition known as limbal stem cell deficiency (LSCD), which is characterised by conjunctivalisation of the cornea, visual impairment and persistent irritation. To treat LSCD, an alternative source of human limbal epithelial (HLE) cells must be transplanted back onto the diseased cornea. Limbal tissue grafts have had a moderate degree of success. However, autologous grafts risk damage to the healthy eye, whilst allogeneic grafts are susceptible to immunological rejection. Cultured HLE grafts offer a promising alternative to whole tissue grafts. The production of cultured HLE grafts involves the removal of a small (1-2 mm2) biopsy from the patient’s healthy limbus, followed by ex vivo expansion to produce an epithelial sheet, which is subsequently transplanted onto the damaged corneal surface. However, the production of cultured HLE grafts usually requires the addition of animal-derived products during cell culture. Animal-derived components, such as foetal bovine serum (FBS) and murine 3T3 feeder cells, introduce the patient to potential crossspecies infection and immune responses to xenogeneic antigens. Consequently, the overall aim of this project has been to develop a culture technique free of xenogeneic products for the establishment and propagation of HLE cells. To achieve this aim, alternatives to FBS in the culture medium and 3T3 feeder cells were pursued. A defined serum-free medium (SFM) containing vitronectin (VN), insulin-like growth factor binding protein 3 (IGFBP3), insulin-like growth factor-I (IGF-I), and epidermal growth factor (EGF) was investigated as an alternative to serumsupplemented medium (SSM) for HLE cell culture. Initial studies focused on the effects of these growth factors on HLE cell metabolic activity and migration. Metabolic activity was primarily stimulated by IGF-I and EGF, with the combination of IGF-I and EGF in solution stimulating metabolic activity to a significantly greater extent than the SSM positive control (p = 0.006). HLE cell migration was also effected by combinations of VN, IGFBP3, IGF-I and EGF. Migration was stimulated above the SFM negative control by the combination of IGFBP3 and IGF-I either with or without the addition of EGF. However, the presence of VN was required for optimal migratory responses (p < 0.003). Hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) were also investigated as additional components to the SFM formulation. HGF significantly stimulated HLE cell metabolic activity and migration (p < 0.02). In contrast, KGF did not significantly stimulate either HLE cell metabolic activity or migration. The addition of either HGF or KGF to the SFM supplemented with VN, IGFBP3, IGF-I and EGF did not significantly enhance the metabolic activity of HLE cells. Therefore, HGF and KGF were no longer pursued as additional components to the SFM formulation. Additional studies were conducted to examine the efficacy of replacing murine 3T3 feeder cells with human ocular stromal cells during HLE cell culture. Initially, stromal cells were isolated from the cornea, limbus and sclera to determine whether there were differences between these stromal cell populations. The results indicated that scleral stromal cells had a significantly larger area and perimeter than either corneal or limbal stromal cells (p < 0.001). Scleral stromal cells were also significantly more rounded than either corneal or limbal stromal cells, as determined by the elliptical factor equation (p < 0.001). Immunocytochemistry also revealed that scleral stromal cells expressed significantly more of the myofibroblast marker ..- smooth muscle actin than either corneal or limbal stromal cells (p < 0.001), and significantly less of the fibroblast/myofibroblast marker Thy-1 than corneal or limbal stromal cells (p < 0.001). Therefore, scleral stromal cells were identified as different in comparison to corneal and limbal stromal cells. Primary HLE cells were cultured with irradiated corneal, limbal and scleral stromal cells. HLE cultures established with either corneal or limbal stromal feeder cells contained more cellular protein (as measured by rhodamine B dye absorbance) than cultures established without feeder cells (p < 0.001). The colony forming efficiency (CFE) of HLE cells established with corneal or limbal stromal feeder cells was also significantly greater than HLE cells established without feeder cells (p < 0.001). In contrast, HLE cultures established with scleral stromal feeder cells contained low levels of cellular protein and had a low CFE, which was not significantly different to the HLE cultures established without feeder cells. Immunocytochemistry indicated that HLE cultures established with scleral feeder cells also showed lower expression of the stem cell markers ABCG2 and C/EBP ... These results suggest that freshly isolated HLE cells can be cultured with irradiated corneal or limbal stromal cells as a replacement for murine 3T3 feeder cells. Finally, the SFM supplemented with VN+IGFBP3+IGF-I+EGF was combined with limbal stromal feeder cells, and examined as a culture technique free of animalderived products. Freshly isolated HLE cells established in SFM supplemented with VN+IGFBP3+IGF-I+EGF and limbal feeder cells contained a similar amount of cellular protein (as measured by crystal violet dye absorbance) when compared to the SSM+3T3 positive control. In addition, the CFE of freshly isolated HLE cells established in VN+IGFBP3+IGF-I+EGF and limbal feeder cells was significantly higher than the SSM+3T3 positive control (p = 0.004). However, a live/dead assay revealed a reduced HLE cell viability in SFM supplemented with VN+IGFBP3+IGFI+ EGF and limbal feeder cells after seven days in culture. In addition, immunocytochemistry demonstrated a lower expression of the stem cell markers ABCG2 and C/EBP .. in the SFM treatment with limbal feeder cells. Therefore, freshly isolated HLE cells can be cultured in SFM supplemented with VN+IGFBP3 +IGF-I+EGF and limbal feeder cells. However, this culture technique is less likely to support the growth of immature limbal stem cells when compared to the SSM+3T3 positive control. Overall, this research has attempted to create a culture system free of animal-derived products for the production of cultured HLE grafts to treat limbal stem cell deficiency. The results show that HLE cells respond to a serum-free medium formulation containing VN+IGFBP3+IGF-I+EGF. In addition, this culture medium can be combined with irradiated stromal cells isolated from the limbus to support HLE culture production. However, the combination of VN+IGFBP3+IGF-I+EGF and limbal feeder cells demonstrated a reduced viability, which indicates that further refinement of the formulation is required. This thesis has also demonstrated differences between stromal cells isolated from the cornea, limbus, and sclera, and has generated knowledge which may impact on the understanding of stromalepithelial regulation.

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26

Chandler, Heather Lynn. "Epithelial-mesenchymal transition in the anterior segment of the eye." Columbus, Ohio : Ohio State University, 2006. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1154533588.

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27

Fok, Elsie. "Advanced stem cell delivery systems for the treatment of corneal epithelial limbal stem cell deficiency." Thesis, University of Brighton, 2014. https://research.brighton.ac.uk/en/studentTheses/134e3b10-9910-40d6-bec8-8a7481b9e67e.

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Limbal stem cell deficiency (LSCD) can be treated successfully using ex vivo limbal epithelial stem cells (LESC) derived from cadaveric donor tissue. However, shortages in such tissues and graft rejection, resulting from inflammation, are persistent issues. The purpose of this study was to optimize current culturing techniques used for LESC transplant tissue, considering expansion and cryopreservation issues surrounding the establishment of a stem cell bank. In addition, a novel anti-inflammatory biomimetic peptide was investigated to address issues surrounding amnion and steroid use in LESC transplantation, inflammation and transplant rejection. Cell cultures derived from Optisol and organ culture stored tissues were examined for optimum growth, characterized by an ability to grow to 70 % to 80 % confluence while maintaining epithelial cell morphology and the presence of positive and negative LESC markers (K3, K19, p63 and PAX-6) as identified by immunocytochemical staining and QRT-PCR. Furthermore, the effect of culture expansion and cryopreservation on stem cell characteristics was examined. A short chain IL-l receptor antagonist peptide was synthesized and characterized using mass spectroscopy (MS), high performance liquid chromatography (HPLC) and liquid chromatography-mass spectroscopy (LC-MS). Anti-inflammatory properties were investigated using ELISA detection of IL-8, IL-6 and IL- l ~ in keratocytes and LESC following stimulation with either lipopolysaccharide or recombinant human IL- l~. Feasible delivery of cells and peptide on a contact lens was investigated to assess the possibility of an "all in one" graft. Results showed that organ culture stored tissues can provide 100 % successful cell cultures using current techniques in terms of reaching confluence and maintaining LESC morphology and phenotype. Sub-culturing and cryopreservation of cultures however did not produce confluent cell sheets, as required for clinical application. The anti-inflammatory peptide was shown to effectively suppress production of key inflammatory cytokines in LESC and keratocytes by acting as an IL- l receptor antagonist and interrupting the IL- l inflammatory pathway. Binding of the peptide to the contact lens was shown to be possible. Such a scaffold also supported expansion of LESC. However, the 2.7 nmol of peptide bound to the lens did not significantly lower cytokine production. Findings suggest that it is possible to culture adequate numbers of LESC for the initiation of a stem cell bank using current techniques. However, modifications to culturing methods are needed to ensure successful sub-culturing and cryopreservation. The peptide has been shown to be effective in reducing inflammatory cytokine production, providing a possible alternative to steroids. An a11-in-one graft could provide a key development in treating LSCD. However further work is required to optimize the peptide concentration to allow effective inflammation control.

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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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Panzica, Domenico Alessio. "The roles of planar cell polarity signalling in maintaining the adult corneal epithelium." Thesis, University of Aberdeen, 2015. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=227224.

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Cells of the stratified adult corneal epithelium undergo centripetal migration throughout adult life from the edge of the cornea to the centre. To date nothing is known about the mechanism underpinning the oriented cellular migration. Failure to replenish apoptotic cells lost by desquamation from the superficial layer of the corneal epithelium leads to severe pathological conditions that may result in blindness. In this study we investigated the role of planar cell polarity (PCP) core proteins as the guidance cue for centripetal migration in the cornea. Cre-mediated conditional deletion of floxed alleles of the core PCP gene Vangl2 in the corneal epithelium and lens of adult mice was achieved. The effect of this deletion was studied by microscopic and immunohistological observation of the cornea compared to littermate controls, showing defects consistent with disrupted apical-basal polarity in mutant mice. Planar behaviour of the corneal epithelial cells was assayed by breeding the mutant alleles (Le-CreTg/-; Vangl2flox/flox) and the Looptail mouse (Vangl2Lp/+) onto an X-linked LacZ reporter transgene (XLacZ) background, demonstrating the importance of PCP core components for normal cell migration. In vitro directional migration studies were performed on Vangl2 and Frizzled6 knock-down human corneal epithelial cells following the application of direct current electric fields (DC-EFs), resulting in the reduction of directional migratory response to the DC-EF. This study showed for the first time roles for the planar cell polarity (PCP) signalling in orchestrating and coordinating cellular cues that drive oriented migration in the unwounded adult corneal epithelium. It is likely that mutations in PCP genes could lead to ocular surface abnormalities in humans.

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30

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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Hammadi, Shumoos T. H. "Novel medical imaging technologies for processing epithelium and endothelium layers in corneal confocal images. Developing automated segmentation and quantification algorithms for processing sub-basal epithelium nerves and endothelial cells for early diagnosis of diabetic neuropathy in corneal confocal microscope images." Thesis, University of Bradford, 2018. http://hdl.handle.net/10454/16924.

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Diabetic Peripheral Neuropathy (DPN) is one of the most common types of diabetes that can affect the cornea. An accurate analysis of the corneal epithelium nerve structures and the corneal endothelial cell can assist early diagnosis of this disease and other corneal diseases, which can lead to visual impairment and then to blindness. In this thesis, fully-automated segmentation and quantification algorithms for processing and analysing sub-basal epithelium nerves and endothelial cells are proposed for early diagnosis of diabetic neuropathy in Corneal Confocal Microscopy (CCM) images. Firstly, a fully automatic nerve segmentation system for corneal confocal microscope images is proposed. The performance of the proposed system is evaluated against manually traced images with an execution time of the prototype is 13 seconds. Secondly, an automatic corneal nerve registration system is proposed. The main aim of this system is to produce a new informative corneal image that contains structural and functional information. Thirdly, an automated real-time system, termed the Corneal Endothelium Analysis System (CEAS) is developed and applied for the segmentation of endothelial cells in images of human cornea obtained by In Vivo CCM. The performance of the proposed CEAS system was tested against manually traced images with an execution time of only 6 seconds per image. Finally, the results obtained from all the proposed approaches have been evaluated and validated by an expert advisory board from two institutes, they are the Division of Medicine, Weill Cornell Medicine-Qatar, Doha, Qatar and the Manchester Royal Eye Hospital, Centre for Endocrinology and Diabetes, UK.

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Miri, Ammar. "Corneal epithelial stem cell deficiency : in vivo and in vitro analysis of diagnostic features and treatment outcomes." Thesis, University of Nottingham, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.601801.

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Several clinical and laboratory studies suggest that the niche for corneal epithelial stem cells is located in the corneoscleral limbus. Of particular interest are the palisades of Vogt (POV), which are a series of fibrovascular palisade like structures found predominantly along the superior and inferior limbus. The study was designed to use a relatively new and powerful tool, the laser in vivo confocal microscopy (Rostock corneal module confocal microscope) of scanning laser ophthalmoscope, ) HRTII; to evaluate in details normal anatomical features of the limbus and establish diagnostic features of the defective limbus in patients manifesting clinically with limbal stem cell deficiency (chapter two, three and four). The objective was to be able to diagnose early and late cases of limbal stem cell deficiency without the need fgr invasive methods such ., impression cytology or ocular surface biopsy. Th~ features determined IVCM were validated and correlated with features observed in vitro by histological examination of biopsy specimen. {., Over the last 10 years approximately 25 to 30 patients have undergone limbal stem cell transplantation in the department with at least a one year follow up. The other aim of this study was to evaluate retrospectively and prospectively the outcomes of these procedures (chapter five), to assess the quality of life benefit of the procedures on the patients using a modified National Eye Institute Visual Functioning Questionnaire 25 (NEI II I I I 1 VFQ-25) (chapter six) and to examine the donor eyes and donor sites used for harvesting tissue for limbal transplants both clinically and by confocal microscope (chapter seven). Finally, we hypothesized that gravity may influence the differential migration of cells from the superior and inferior limbic regions, and carried out a simple experiment in vitro which supported this hypothesis (chapter eight). III

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Heydenrych, Leonard Goussárd. "Eviscerated corneas as tissue source for ex vivo expansion of limbal epithelial cells on platelet-rich plasma gels." Master's thesis, University of Cape Town, 2016. http://hdl.handle.net/11427/22923.

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Purpose/Aim of the study: To assess if corneal epithelium can be cultured ex-vivo from corneas eviscerated due to irretrievable trauma, according to a cell culture method which made use of autologous platelet-rich plasma (A-PRP) as culture substrate. To compare corneal epithelium cultured ex vivo from corneas eviscerated following trauma using A-PRP combined with DMEM (Dulbecco's modified Eagles medium), versus DMEM alone. Materials and Methods: This was a laboratory case controlled study of human corneal cells cultured in a mixture of A-PRP and DMEM, versus DMEM alone from 6 eviscerated corneas. A hundred explants were created of which fifty explants were plated on A-PRP-gel construct combined with DMEM and fifty controls were placed in serum free DMEM alone. Donor patients received systemic antibiotics prior to evisceration. Results: Confluent epithelium in mono-layers could be cultured when donor limbal biopsies were placed in a mixture of A-PRP culture medium and DMEM. No growth were observed when corneas were placed in serum-free DMEM medium only (p<0.05). No bacterial infection was observed in cultures. Conclusions: This study demonstrated that autologous platelet rich plasma is a viable and effective alternative to bovine serum for the ex-vivo expansion of limbal epithelial cells. It also shows that eviscerated corneas are a viable source of donor tissue for this purpose in South Africa where access to tissue banks is limited.

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Bray, Laura Jane. "Evaluation of fibroin-based scaffolds for ocular tissue reconstruction." Thesis, Queensland University of Technology, 2012. https://eprints.qut.edu.au/52665/1/Laura_Bray_Thesis.pdf.

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The epithelium of the corneolimbus contains stem cells for regenerating the corneal epithelium. Diseases and injuries affecting the limbus can lead to a condition known as limbal stem cell deficiency (LSCD), which results in loss of the corneal epithelium, and subsequent chronic inflammation and scarring of the ocular surface. Advances in the treatment of LSCD have been achieved through use of cultured human limbal epithelial (HLE) grafts to restore epithelial stem cells of the ocular surface. These epithelial grafts are usually produced by the ex vivo expansion of HLE cells on human donor amniotic membrane (AM), but this is not without limitations. Although AM is the most widely accepted substratum for HLE transplantation, donor variation, risk of disease transfer, and rising costs have led to the search for alternative biomaterials to improve the surgical outcome of LSCD. Recent studies have demonstrated that Bombyx mori silk fibroin (hereafter referred to as fibroin) membranes support the growth of primary HLE cells, and thus this thesis aims to explore the possibility of using fibroin as a biomaterial for ocular surface reconstruction. Optimistically, the grafted sheets of cultured epithelium would provide a replenishing source of epithelial progenitor cells for maintaining the corneal epithelium, however, the HLE cells lose their progenitor cell characteristics once removed from their niche. More severe ocular surface injuries, which result in stromal scarring, damage the epithelial stem cell niche, which subsequently leads to poor corneal re-epithelialisation post-grafting. An ideal solution to repairing the corneal limbus would therefore be to grow and transplant HLE cells on a biomaterial that also provides a means for replacing underlying stromal cells required to better simulate the normal stem cell niche. The recent discovery of limbal mesenchymal stromal cells (L-MSC) provides a possibility for stromal repair and regeneration, and therefore, this thesis presents the use of fibroin as a possible biomaterial to support a three dimensional tissue engineered corneolimbus with both an HLE and underlying L-MSC layer. Investigation into optimal scaffold design is necessary, including adequate separation of epithelial and stromal layers, as well as direct cell-cell contact. Firstly, the attachment, morphology and phenotype of HLE cells grown on fibroin were directly compared to that observed on donor AM, the current clinical standard substrate for HLE transplantation. The production, transparency, and permeability of fibroin membranes were also evaluated in this part of the study. Results revealed that fibroin membranes could be routinely produced using a custom-made film casting table and were found to be transparent and permeable. Attachment of HLE cells to fibroin after 4 hours in serum-free medium was similar to that supported by tissue culture plastic but approximately 6-fold less than that observed on AM. While HLE cultured on AM displayed superior stratification, epithelia constructed from HLE on fibroin maintained evidence of corneal phenotype (cytokeratin pair 3/12 expression; CK3/12) and displayed a comparable number and distribution of ÄNp63+ progenitor cells to that seen in cultures grown on AM. These results confirm the suitability of membranes constructed from silk fibroin as a possible substrate for HLE cultivation. One of the most important aspects in corneolimbal tissue engineering is to consider the reconstruction of the limbal stem cell niche to help form the natural limbus in situ. MSC with similar properties to bone marrow derived-MSC (BM-MSC) have recently been grown from the limbus of the human cornea. This thesis evaluated methods for culturing L-MSC and limbal keratocytes using various serum-free media. The phenotype of resulting cultures was examined using photography, flow cytometry for CD34 (keratocyte marker), CD45 (bone marrow-derived cell marker), CD73, CD90, CD105 (collectively MSC markers), CD141 (epithelial/vascular endothelial marker), and CD271 (neuronal marker), immunocytochemistry (alpha-smooth muscle actin; á-sma), differentiation assays (osteogenesis, adipogenesis and chrondrogenesis), and co-culture experiments with HLE cells. While all techniques supported to varying degrees establishment of keratocyte and L-MSC cultures, sustained growth and serial propagation was only achieved in serum-supplemented medium or the MesenCult-XF„¥ culture system (Stem Cell Technologies). Cultures established in MesenCult-XF„¥ grew faster than those grown in serum-supplemented medium and retained a more optimal MSC phenotype. L-MSC cultivated in MesenCult-XFR were also positive for CD141, rarely expressed £\-sma, and displayed multi-potency. L-MSC supported growth of HLE cells, with the largest epithelial islands being observed in the presence of L-MSC established in MesenCult-XF„¥ medium. All HLE cultures supported by L-MSC widely expressed the progenitor cell marker £GNp63, along with the corneal differentiation marker CK3/12. Our findings conclude that MesenCult-XFR is a superior culture system for L-MSC, but further studies are required to explore the significance of CD141 expression in these cells. Following on from the findings of the previous two parts, silk fibroin was tested as a novel dual-layer construct containing both an epithelium and underlying stroma for corneolimbal reconstruction. In this section, the growth and phenotype of HLE cells on non-porous versus porous fibroin membranes was compared. Furthermore, the growth of L-MSC in either serum-supplemented medium or the MesenCult-XFR culture system within fibroin fibrous mats was investigated. Lastly, the co-culture of HLE and L-MSC in serum-supplemented medium on and within fibroin dual-layer constructs was also examined. HLE on porous membranes displayed a flattened and squamous monolayer; in contrast, HLE on non-porous fibroin appeared cuboidal and stratified closer in appearance to a normal corneal epithelium. Both constructs maintained CK3/12 expression and distribution of £GNp63+ progenitor cells. Dual-layer fibroin scaffolds consisting of HLE cells and L-MSC maintained a similar phenotype as on the single layers alone. Overall, the present study proposed to create a three dimensional limbal tissue substitute of HLE cells and L-MSC together, ultimately for safe and beneficial transplantation back into the human eye. The results show that HLE and L-MSC can be cultivated separately and together whilst maintaining a clinically feasible phenotype containing a majority of progenitor cells. In addition, L-MSC were able to be cultivated routinely in the MesenCult-XF® culture system while maintaining a high purity for the MSC characteristic phenotype. However, as a serum-free culture medium was not found to sustain growth of both HLE and L-MSC, the combination scaffold was created in serum-supplemented medium, indicating that further refinement of this cultured limbal scaffold is required. This thesis has also demonstrated a potential novel marker for L-MSC, and has generated knowledge which may impact on the understanding of stromal-epithelial interactions. These results support the feasibility of a dual-layer tissue engineered corneolimbus constructed from silk fibroin, and warrant further studies into the potential benefits it offers to corneolimbal tissue regeneration. Further refinement of this technology should explore the potential benefits of using epithelial-stromal co-cultures with MesenCult-XF® derived L-MSC. Subsequent investigations into the effects of long-term culture on the phenotype and behaviour of the cells in the dual-layer scaffolds are also required. While this project demonstrated the feasibility in vitro for the production of a dual-layer tissue engineered corneolimbus, further studies are required to test the efficacy of the limbal scaffold in vivo. Future in vivo studies are essential to fully understand the integration and degradation of silk fibroin biomaterials in the cornea over time. Subsequent experiments should also investigate the use of both AM and silk fibroin with epithelial and stromal cell co-cultures in an animal model of LSCD. The outcomes of this project have provided a foundation for research into corneolimbal reconstruction using biomaterials and offer a stepping stone for future studies into corneolimbal tissue engineering.

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Henkel, Tassilo. "Synthese und Charakterisierung von Limbusepithel-Amnion-Transplantaten aus langzeitorgankonservierten Hornhäuten und kryokonservierten Amnionmembranen." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2011. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-63630.

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In dieser Arbeit wurden Methoden entwickelt und verglichen, um aus Corneoskleralringen langzeitorgankonservierter Hornhäute und intakten, kryokonservierten Amnionmembranen Limbusepithel-Amnion-Transplantate herzustellen. Als erfolgreichste Kultivierungsmethode stellte sich hierbei signifikant die Explantat-Technik mit nach unten gerichtetem Limbusepithel heraus. Hier konnte eine Auswachsrate von 42 % erzielt werden. Es wurde weiterhin gezeigt, dass das ausgewachsene, mehrschichtige Limbusepithel proliferationsfähige TACs (Transient Amplifying Cells) enthält. Weiterhin konnten mittels Regressionsanalyse signifikante Zusammenhänge zwischen Spenderalter, Post-mortem-Zeit, Organkultur-Dauer und der Auswachsrate beschrieben werden. Kurzgefasst wurde die Vermutung bestätigt, dass jede Verlängerung der unterschiedlichen Zeiten eine Verringerung der Auswachsrate zur Folge hat. Die hergestellten Limbusepithel-Amnion-Transplantate könnten für Patienten mit Limbusstammzellinsuffizienz unterschiedlicher Genese verwendet werden.

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Rovere, Maria. "Reconstruction cornéenne : traitement des déficiences en cellules souches limbiques totales et bilatérales associées ou non à une atteinte du stroma." Thesis, Lyon, 2018. http://www.theses.fr/2018LYSE1263.

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Certaines brûlures oculaires graves ou d'autres pathologies oculaires rares peuvent être associées à une perte totale de cellules souches épithéliales de la cornée (DCSL), ce qui conduit à une opacification de la cornée par invasion de la conjonctive. Lorsque la DCSL est totale et bilatérale, le limbe controlatéral n'est pas disponible pour la greffe de limbe autologue ou la culture de cellules souches autologues limbiques et la transplantation allogénique de la cornée est impossible car toujours rejetée en raison de la néovascularisation. Une thérapie innovante testée avec succès au sein de notre laboratoire, en collaboration avec le service d'ophtalmologie des HCL, consiste en une greffe autologue de Feuillet Epithélial (FE) dérivé de Muqueuse Orale (MO). Cette approche permet de restaurer la transparence et autorise, si nécessaire, une greffe cornéenne complémentaire. Cette technique a montré son efficacité lors d'un essai clinique conduit dans notre hôpital mais le dispositif breveté permettant le détachement non enzymatique des feuillets cultivés n'est plus disponible en Europe. Nous avons donc mis au point un nouveau procédé de production de FE dérivant de la MO dont la preuve de concept a été obtenue à partir d'études in-vitro et ex-vivo. En effet, un détachement avec 0,5 mg / mL de collagénase n'endommage pas le FE de MO et les protéines de la membrane basale, et, dans un modèle de stroma porcin exvivo, ces feuillets adhérents au stroma, continuent à se renouveler sous la forme d'épithélium différencié. De plus, dans le cas d'une opacité stromale associée à une DCSL, une greffe secondaire de cornée est nécessaire pour améliorer l'Acuité Visuelle (AV), c'est pourquoi nous avons cherché à développer pour ces patients à haut risque de rejet, un stroma décellularisé. Ce stroma pourrait également répondre à la pénurie de cornées dans les pays en voie de développement grâce à sa conservation longue et facile. La lyophilisation a été combinée à la décellularisation des cornées au SDS à 0,1 %, validée sur les cornées humaines sur le maintien de leur transparence et de l'ultrastructure du stroma associé à l'absence des antigènes HLA-ABC et HLA-DR. Enfin, dans un modèle de Kératoplastie Lamellaire Antérieure Profonde (KLAP) ex-vivo, nous avons montré que les cellules épithéliales et stromales de la cornée humaine receveuse colonise le stroma décellularisé. Ainsi, nos travaux permettent de proposer un traitement des DSCL totales et bilatérales associées ou non à une atteinte du stroma
Some severe ocular burns or other rare ocular pathologies may be associated with a complete loss of corneal epithelial stem cells (LSCD), leading to an opacification of the cornea by invasion of the conjunctiva. When DCSL is total and bilateral, contralateral limbus is not available for autologous limb transplant or limbal autologous stem cell culture, and allogeneic transplantation of the cornea is not an option since neovascularization is constantly responsible for graft rejection. An innovative therapy tested with success in our laboratory, in collaboration with the ophthalmology department of the HCL, consists in an autologous Epithelial Cell Sheet (ECS) graft derived from Oral Mucosa (MO). This approach restores transparency and allows in a second step a complementary corneal graft when necessary. This technique has been shown to be effective in a clinical trial conducted in our hospital, but the patented device for the non-enzymatic detachment of cultivated ECS is no longer available in Europe. We have therefore developed a new method for the production of ECS from OM, the proof of concept of which has been obtained from in-vitro and ex-vivo studies. Indeed, detachment with 0.5 mg / mL of collagenase does not damage ECS from OM and basement membrane proteins, and in an ex-vivo porcine stroma model, these cell sheets adhered on corneal stroma, continued to self-renew and generated a differentiated epithelium. Since stromal opacity associated with DCSL requires a secondary corneal graft to improve Visual Acuity (VA), we also sought to develop for these patients with high rejection risk, a new approach for the generation of decellularized stroma. Such a procedure for the production of decellularized stroma was also aimed at allowing a money-saving and reliable long-term storage for stromal grafts and thus circumventing the shortage of corneas in developing countries. Our process, combining lyophilization with decellularization of the corneas at 0.1 % SDS, was validated on human corneas regarding the maintenance of stroma transparency, the stromal ultrastructure associated with the absence of HLA-ABC and HLA-DR antigens. Finally, in an ex-vivo model of Deep Anterior Lamellar Keratoplasty (DALK), we have shown that the epithelial and stromal cells of the recipient human cornea colonized efficiently the decellularised stroma. Overall, our work makes it possible to propose a treatment for total and bilateral LSCD associated or not with lesions of the stroma

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Ferraris, Corinne. "Pluripotentialité des kératinocytes épidermiques et cornéens chez les mammifères." Grenoble 1, 1994. http://www.theses.fr/1994GRE10090.

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Afin d'etudier les potentialites de l'epiderme et de l'epithelium corneen a former des annexes cutanees telles que les follicules pileux ainsi que les glandes sebacees et sudoripares, des recombinaisons heterotopiques heterospecifiques ont ete realisees puis greffees sur la souris nude. L'origine des cellules constituant ces structures differenciees, a ete identifiee par hybridation in situ avec une sonde alu reconnaissant l'adn humain, et par marquage differentiel des noyaux de lapin et de souris a l'aide du fluorochrome de hoechst. L'epiderme embryonnaire est capable de former des ensembles pilo-sebaces ainsi que des glandes sudoripares selon l'origine du derme auquel il se trouve associe, mais ne peut apparemment pas se transformer en epithelium corneen. L'etude des potentialites de l'epiderme adulte a ete effectuee de deux manieres. Des keratinocytes humains provenant de peau mammaire ont ete cultives pour obtenir un epiderme de culture. Apres 15 jours de greffe, l'epiderme humain de culture a forme des bourgeons pileux et a participe a la formation de follicules pileux d'origine mixte (homme-souris) lorsqu'il a ete reassocie a un derme trichogene. Apres un mois de greffe, les cellules epitheliales humaines ont ete remplacees par des cellules cicatricielles de la souris hote. Dans un deuxieme temps, des follicules pileux ainsi que des ensembles pilo-sebaces ont ete induits dans une peau cicatricielle humaine provenant d'une region pileuse ou glabre par l'insertion de cellules cultivees de papilles dermiques de rat adulte. L'epithelium corneen, recombine avec un derme trichogene ou un derme plantaire s'est transdifferencie en formant un epiderme caracterise par une couche granuleuse auquel sont associes des ensembles pilo-sebaces ainsi que des glandes sudoripares. Ainsi, l'epithelium corneen possede une grande plasticite, car meme lorsque les keratinocytes corneens expriment les keratines k3 et k12, leur differenciation peut encore etre modulee en fonction des fibroblastes auxquels ils sont associes. L'ensemble de ses resultats soulevent le probleme de l'identification des cellules souches epidermiques, ainsi que la comprehension des mecanismes moleculaires responsables de la formation des annexes cutanees

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38

范姜益園. "Phenotype of corneal epithelial cells on chitosan membrane." Thesis, 2006. http://ndltd.ncl.edu.tw/handle/02041687083082777131.

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39

"Anatomical and functional analysis of microRNAs in human cornea epithelial progenitor cells." Thesis, 2010. http://library.cuhk.edu.hk/record=b6074820.

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By performing microRNA microarrays to globally detect any novel miRNAs in the limbus, eleven microRNAs (hsa-miR-136, hsa-miR-373*, hsa-miR-150, hsa-miR-143, hsa-miR-455, hsa-miR-145, hsa-miR-381, hsa-miR-224, hsa-miR-338, hsa-miR-154, hsa-miR-377) were found to be upregulated while two microRNAs (hsa-miR-122a and hsa-miR-425-3p) were identified as downregulated by more than 2 folds. Among these, hsa-miR-143 and hsa-miR-145 were distingushed to be the most significantly up-regulated limbal miRNAs. Individual assessement of the microarray results of a recently reported stem cell specific microRNA, hsa-miR-21, were also upregulated by more than two thousand fold when comparing limbus and cornea. miR-21, miR-143 and miR-145 were therefore selected as the most likely microRNA candidates in the present study. The expression level of these miRNA candidates were validated and confirmed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). To localize these candidates, we performed in situ hybridization on frozen corneal rim sections using locked nucleic acid (LNA)-modified oligonucleotide probes. Results showed that miR-2I, 143 and 145 were confined in the limbal region with gradation of expression level along the basal-suprabasal line.
Functional roles of these microRNAs were then deciphered by overexpressing human corneal epithelial cell line (HCE) with precursor microRNAs (pre-miRs) through lipophilic transfection. Results showed that high endogenous level of miR-145 could inhibit cell proliferation by 3.5 fold as shown from MTT proliferation assay at day 5, and could generate discrete spherical colonies that resembles the morphology of holoclones at day 8, but not the other two candidate miRNAs.
In conclusion, 1 have identified three novel microRNAs (hsa-miR-21, 143, 145) which were precisely upregulated in the limbus region, while miR-145 was being the most limbal specific. In addition, the functions of miR-145 were found to be inhibitory on cell proliferation, possibly through the indirect regulation of IFNB1. These unprecedented results may suggest a therapeutic potential of miR-145 on limbal stem cell deficiency and limbal tumors because miR-145 can affect cell survival and proliferation.
MicroRNAs is a family of small non-coding RNAs that, in human, binds imperfectly to the 3' untranslated region (UTR) of target mRNAs for translational repression or negative regulation. Recent studies have shown that such negative regulatory pathways may play pivotal roles in the maintenance of asymmetric cell division in embryonic and tissue specific stem cells. Human corneal epithelial progenitor cells (CEPC), a tissue specific stem cell lineage residing between cornea and conjunctiva in the Palisade of Vogt of the limbus region, is known to maintain corneal homeostasis throughout human life. They respond to injury and normal wearing by rapid proliferation and differentiation into transit amplifying cells (TACs) and eventually corneal epithelial cells, though the biological factors controlling this homeostatic switch are still largely unknown. Here I hypothesized that microRNAs can participate in CEPC regulation. Experiments elucidating the anatomical distribution and functional roles of microRNAs on the human cornea rims were performed to testify this proposition.
Protocols aim at enriching the CEPC population were then devised. For the first time a four parameter cell sorting system utilizing ABCG2, Connexin 43, Notch 1 and pyronin Y as markers was established for the prospective in vitro study. Nevertheless, manual microdissection isolating the limbus region and the cornea region was employed for the present study of microRNAs.
This study begins with the phenotypic validation of human cornea rims recruited from the Chinese Hong Kong population using immunohistochemistry. Conventional CEPC markers (p63, EGFR, cytochrome oxidase and cytokeratin 15), embryonic stem cell marker (stat1) and cancer stem cell markers (p73, MDM2 and pStat1) were expressed in the limbus region, suggesting that these specimens contained a source of CEPC for attesting our hypothesis.
To determine the mRNA targets of candidate microRNAs in HCE cells, Whole Human Genome Oligo Microarray Kits (Agilent Technologies) which contained 41K human genes and transcripts were employed. When compared to the scrambled control, HCE cells over-expressed with hsa-miR-21, 143 or 145 revealed differential expression of genes that participate in cell activation, motility and proliferation. Of note, interferon beta 1 fibroblast (IFNB1), a gene that is often deleted or rearranged in cancers, was significantly upregulated by a medium of 1093 fold in pre-miR-145 treated cells as confirmed by real time PCR assays.
Lee, Sharon Ka-wai.
"December 2009."
Advisers: Calvin Chi-Pui Pang; Gary Hin Fai Yam.
Source: Dissertation Abstracts International, Volume: 72-01, Section: B, page: .
Thesis (Ph.D.)--Chinese University of Hong Kong, 2010.
Includes bibliographical references (leaves 216-252).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstract also in Chinese.

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40

McKie, George A. "The effect of nanoscale topography on corneal epithelial cells." 2004. http://catalog.hathitrust.org/api/volumes/oclc/61502514.html.

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Hassan, E., P. Deshpande, F. Claeyssens, Stephen Rimmer, and S. MacNeil. "Amine functional hydrogels as selective substrates for corneal epithelialization." 2014. http://hdl.handle.net/10454/10459.

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No
The aim of this study was to develop a synthetic hydrogel to act as a corneal substitute capable of selectively supporting the adhesion and proliferation of limbal epithelial cells (LECs) while inhibiting growth of limbal fibroblasts. Deficiency of LECs causes conjunctival epithelial cells to move over the cornea, producing a thick scar pannus. Unilateral defects can be treated using LEC cultured from the unaffected eye, transplanting them to the affected cornea after scar tissue is removed. The underlying wound bed is often damaged, however, hence the need to develop a corneal inlay to aid in corneal re-epithelialization. Transparent epoxy-functional polymethacrylate networks were synthesized using a combination of glycerol monomethacrylate, ethylene glycol dimethacrylate, lauryl methacrylate and glycidyl methacrylate that produced two different bulk hydrogel compositions with different equilibrium water contents (EWCs): Base 1 and Base 2, EWC=55% and 35%, respectively. Two sets of amine-functional hydrogels were produced following reaction of the epoxide groups with excesses of either ammonia, 1,2-diamino ethane, 1,3-diamino propane, 1,4-diamino butane or 1,6-diamino hexane. Neither series of hydrogels supported the proliferation of limbal fibroblasts irrespective of amine functionalization but they both supported the adhesion and proliferation of limbal epithelial cells, particularly when functionalized with 1,4-diamino butane. With Base 1 hydrogels (less so with Base 2) a vigorous epithelial outgrowth was seen from small limbal explants and a confluent epithelial layer was achieved in vitro within 6days. The data support the development of hydrogels specific for epithelial formation.

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Chu, Yen-Chang, and 褚晏彰. "Stiffness of Substrate Affects Corneal Epithelial Cells Migration in Electric Fields." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/57122345459068739762.

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碩士
國立臺灣大學
醫學工程學研究所
98
Cell migration involves in many fields, such as wound healing, tumor metastasis, organ development, immune response and morphological change. Cell migration is affected by many factors, such as electric field, rigidity of the substrate, and chemical gradient. Amniotic membrane (AM) is commonly used to treat persistent corneal injuries. In an attempt to understand mechanisms behind this enhancement of the healing process, bovine corneal epithelial cells were used to examine cell migration on the various substrates. External electric field was applied to simulate the intrinsic current generated at corneal wound edges. Higher migration speed and directional velocity were found on the basement membrane side of AM, cornea section and on lower collagen concentrations. Collagen type I and type IV both benefit cell migration rather than fibronectin and laminin. At the same time, most cells assume a spindle shape and form more lamellipodia in these conditions that may correspond with the migration result. The studies on calcium alginate with varied stiffness prove that cell responses to differential mechanical forces between cell and substrate, and cells prefer to migrate on maximal mechanical input. When enhancing wound healing with any substrate, the physical properties of surface is always something to be considered. Result from this study may benefit our understanding of the cornea wound healing mechanism and provide further treatment options.

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Tsai, Tzu-Yun, and 蔡紫筠. "The cytotoxicity and mechanism of fluoroquinolones on human corneal epithelial cells." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/10253835519971275999.

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碩士
臺灣大學
臨床醫學研究所
98
Bacterial keratitis is a common ocular infection and a leading cause of ocular morbidity and blindness worldwide. However, according to our previous report regarding 10-year experience of microbial keratitis at the National Taiwan University Hospital, contact lens-related pseudomonal keratitis was the most common form of microbial keratitis in Taiwan. Effective topical therapy, using fortified antimicrobials or monotherapy commercial ophthalmic preparation selected based on the results of diagnostic corneal smears and cultures, is essential for management of patients with microbial keratitis. Above seventy percent of offending eyes could be successfully treated topically. Fluoroquinolones (FQs) were derived from the non-fluorinated drug nalidixic acid, which was developed during the early 1960s. FQs are potent antimicrobial agents with a broad antibacterial spectrum and are suitable for monotherapy. They act rapidly by inhibiting bacterial DNA gyrase and topoisomerase IV, which are selective for bacterial cells. FQs are nowadays widely used in clinical practice to treat ocular infection, with intravitreal topical and systemic routes of administration. In fact, several investigators have reported bacterial keratitis resistant to FQs and delays in the healing rate and corneal perforation after administration of FQs preparation. Moreover, many eye drops contain preservatives known to cause severe side effects to the ocular surface. Benzalkonium chloride (BAC) is the preservative currently used the most, and many in vivo and in vitro studies have showed its toxicity on corneal epithelial cells. Base on our preliminary report regarding to the toxicity toward the corneal epithelial cells between different FQs, the cytotoxicity observed with FQ eye drops seems to be caused mainly by the preservative. However, we found that the new generation of FQs, moxifloxacin, no less cytotoxicity towards corneal epithelial cells than the old generation preparations. Therefore, the purpose of this study is to compare and identify the specific types of cytotoxic damage that might occur with exposure to various FQs and BAC. Besides, fluoroquinolones antimicrobial agents are widely used in clinical practice as broad-spectrum antibiotics with good bioavailability. However, they have been reported to induce tendinopathy, and the main target is Achilles tendon rupture. All the studies began during 2000 and showed that tendinitis and tendon rupture during treatment with fluoroquinolone antibiotics is thought to be mediated via oxidative stress and mitochondrial damage. Some innovative studies showed fluoroquinolones could modulate cycle cell progression and apoptosis in cancer cells or enhance the function of chemotherapeutic agents in cytotoxicity in tumor-derived cells. Therefore, we want to identify the mechanism of cytotoxicity of fluoroquinolones on human corneal epithelial cell which had not been investigated before in literatures. Our study showed that the main source of cytotoxicity from commercial fluoroquinolone ophthalmic solutions came from preservatives, benzalkonium chloride. The mechanism is through increased the level of reactive oxygen species, and then induces cell apoptosis. However, the latest four generation of fluoroquinolones, moxifloxacin, also showed obvious cytotoxicity. Significant cell toxicity was found in the moxifloxacin group after 3 hours incubation than benzalkonium chloride, which induced oxidative stress and cell apoptosis in early stage. The limitation of our study was the in vivo setting and the use of immortalized human corneal epithelial cell line instead of primary cultures of corneal epithelial cells. The preliminary results of the our study provided us a new way and more information for further new methods in studying the mechanism of cytotoxicity of fluoroquinolones.

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"Human umbilical cord lining epithelial cells with stem cell-like properties: an adjunct to skin regeneration." 2013. http://library.cuhk.edu.hk/record=b5549751.

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皮膚是人體最大的器官，具有多種功能，其中最重要的功能之一就是作為身體內部和外界環境之間的的保護屏障。完整地修復這一保護屏障是創傷癒合和組織再生領域的一個重要內容。本論文探討了人類臍帶被覆上皮細胞 (cord lining epithelial cells, CLECs)作為一種幹細胞來源，可用于表皮重建的潛能.
本論文的第二章對CLECs的體外分離和增殖進行了詳細地描述。這一類細胞具有較長的染色體端粒，較高的增殖潛能和傳代能力。同時，它們表達上皮幹細胞和多能性幹細胞的標誌性表面抗原。它們還具有多種分化潛能，包括成脂、成骨和成軟骨。然而當皮下異種移植後，它們並不會形成畸胎瘤。
本論文的第三章對CLECs的免疫特性進行了評估。結果顯示CLECs不但具有低免疫原性，還具有免疫調節功能。它們表達典型性的一型主要組織相容性複合體(MHC class I)，即人白細胞ABC抗原(HLA-ABC)，但不表達典型性的二型主要組織相容性複合體(MHC class II)，即人白細胞DR抗原(HLA-DR)。它們同時還表達非典型性的MHC class I, 包括人白細胞G抗原和人白細胞E 抗原(HLA-G和HLA-E), 但不表達共激分子(CD40, CD80和CD86)。此外，體外檢測還發現它們表達適度的促炎/抗炎細胞因子和大量的生長因子.
本論文的第四章對CLECs在表皮重建應用中的潛能進行了考察。結果顯示無論在體外器官培養還是異種移植動物模型中，CLECs都能形成分層的上皮結構，與用表皮細胞構建的分層上皮結構相類似。而且在CLECs構建的皮膚替代物中證實了有表皮分化標誌性抗原的表達。
結論：本論文證明了CLECs具有幹細胞樣特性但無致瘤性，具有低免疫原性和表皮分化的可塑性。研究結果支持CLECs在創傷癒合和皮膚再生領域的臨床應用可行性.
The skin is the largest organ in the body and has multiple functions. One of the most important functions is to serve as a protective barrier between the internal and external environments of the body. Restoration of the integrity of this protective barrier is an essential aspect of wound healing and tissue regeneration. In this thesis, the potential of human umbilical cord lining epithelial cells (CLECs) as a source of stem cells with appropriate differentiation capacity for epidermal reconstitution has been explored.
The isolation and propagation of CLECs from human umbilical cord lining epithelium were described in Chapter II. The cells presented a long telomere length and had high proliferative potential and passaging capability. They were also shown to display both epithelial and pluripotent stem cell markers. They were capable of multipotent differentiation, including adipogenesis, osteogenesis and chondrogenesis. However, they didn’t form teratoma after subcutaneous xenotransplantation until 12 weeks.
The immune properties of CLECs in vitro were assessed in Chapter III. The cells were shown to have low immunogenicity but high immunosuppressive function. They expressed classical major histocompatibility complex (MHC) class I antigens (HLA-ABC), but not MHC class II antigen (HLA-DR). They also expressed non-classical MHC class I antigens (HLA-G and HLA-E), but lacked the expression of the co-stimulatory molecules (CD40, CD80 and CD86). Moreover, they expressed moderate pro/anti-inflammatory cytokines and multiple growth factors both in cell supernatants and cell lysates.
The potential of CLECs for epidermal reconstitution was investigated in Chapter IV. In both organotypic culture and xenotransplantation model, CLECs were capable of generating a stratified epithelial structure, which is similar to that constructed by using keratinocytes. Furthermore, the expression of epidermal differentiation markers was verified in CLEC-constructed skin substitutes.
In conclusion, the stem cell-like properties of CLECs have been demonstrated in the present study. In addition to the lack of tumorigenicity, CLECs also have low immunogenicity and significant plasticity in epidermal differentiation. The findings support the potential clinical application of CLECs in wound healing and skin regeneration.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Cai, Yijun.
"October 2012."
Thesis (Ph.D.)--Chinese University of Hong Kong, 2013.
Includes bibliographical references (leaves 114-129).
Abstract also in Chinese.
Abstrac --- p.i
Table of Contents --- p.v
Abbreviations --- p.vii
List of Figures --- p.viii
List of Tables --- p.x
Chapter Chapter I --- Introduction --- p.1
Skin --- p.3
Wound healing --- p.6
Wound regeneration and repair --- p.6
Recent history of wound treatment --- p.9
Skin substitutes --- p.11
Stem cells for wound treatment --- p.14
Stem cells overview --- p.15
Adult stem cells --- p.16
Fetal stem cells --- p.18
Amniotic membrane derived stem cells --- p.19
Umbilical cord stem cells --- p.22
Hypothesis and Specific aims --- p.24
Chapter Chapter II --- The Isolation and Characterization of the Stem Cell-like Properties of Human Umbilical Cord Lining Epithelial Cells --- p.28
Introduction --- p.28
Materials and methods --- p.30
Results --- p.47
Discussion --- p.62
Conclusion --- p.67
Chapter Chapter III --- The assessment of the Immune Properties of Human Umbilical Cord Lining Epithelial Cells --- p.69
Introduction --- p.69
Materials and methods --- p.72
Results --- p.75
Discussion --- p.83
Conclusion --- p.88
Chapter Chapter IV --- The Investigation of the Potential of Human Umbilical Cord Lining Epithelial Cells for the Epidermal Reconstitution --- p.89
Introduction --- p.89
Materials and methods --- p.91
Results --- p.94
Discussion --- p.101
Conclusion --- p.104
Chapter Chapter V --- Summary and Future Plan --- p.105
Summary --- p.105
Future plan --- p.108
Acknowledgements --- p.113
References --- p.114
Appendix --- p.130

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45

Liu, Kuan-Ting, and 劉冠廷. "Effect of ferulic acid on reactive oxygen species induced apoptosis in corneal epithelial cells." Thesis, 2013. http://ndltd.ncl.edu.tw/handle/11405286390992266909.

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碩士
國立陽明大學
解剖學及細胞生物學研究所
101
In recent years, ROS was reported that induced associated with corneal diseases by oxidative stress. The oxidative stress induced cell apoptosis of corneal epithelium could lead to impaired vision and has the risk of blindness. A major goal is to reduce the damage induced by ROS. Given that ferulic acid has a great antioxidant activity to eliminate ROS and to suppress apoptosis on varied cell types. We hypotheses that ferulic acid may play a therapeutic role in reducing corneal diseases. Establishment of H2O2-damaged cell model and determination of the safe concentration of ferulic acid were assayed by using MTT and crystal violet assays. Second, the activity of free radical were monitored by luminal, then quantitated the expression of inflammation genes induced by ROS in vitro by real-time PCR. Cell death and the protective effect of ferulic acid were analyzed by flow cytometry. Ferulic acid could be a scavenger of ROS which included repressed the inflammatory gene expression and inhibited apoptosis. Here we show that the ferulic acid plays an role in reducing oxidative stress, suppressing the inflammatory gene expression and preventing apoptosis. These results suggest that ferulic acid has therapeutic application in oxidative stress-induced corneal diseases, highlighting the antioxidant activity on preventing the ROS generation.

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46

Cheng, Shao-Hui, and 鄭劭蕙. "Evaluation of Epitheliotrophic Capacity of Four Different Blood Derivatives on Bovine Corneal Epithelial cells." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/99659962085711949174.

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碩士
國立臺灣大學
獸醫學研究所
95
PURPOSE： Human serum eye drops have been successfully used in treatment of severe ocular surface disorders and enhancement of corneal wound healing. Umbilical cord serum is effective in treatment of dry eye and persistent corneal epithelial defects. Fresh frozen plasma has not yet been tested for use as eye drops in patients, although it is easily available as quality-controlled products from blood banks. Fresh frozen plasma or umbilical cord serum could be used for substituting human serum when the source of human serum lacks. The use of fetal bovine serum (FBS) in the culture medium has been a major concern worldwide because of bovine spongiform encephalitis (BSE). The use of other human source blood-derived products to replace FBS in the culture process would help. We compared these four blood products by investigating the epitheliotrophic capacity in an in vitro model of bovine epithelial cell monolayer. MATERIALS AND METHODS：Primary cultured bovine corneal epithelial cells were used to investigate wound healing, cell proliferation and migration by means of scratch corneal wound evaluation, MTS assay and Boyden chamber migration assay in response to human serum, umbilical cord serum, fresh frozen plasma and FBS. The concentrations of EGF, TGF-β1, and fibronectin in human serum, umbilical cord serum and fresh frozen plasma were evaluated by ELISA kits. RESULTS： The effect of promoting corneal epithelial wound healing of FBS was better than three other human sourced blood products. Cell proliferation and migration were best enhanced by FBS, followed by umbilical cord serum and human serum, and were worst in fresh frozen plasma. Growth factor (EGF and TGF-β1) concentrations were significantly higher in umbilical cord serum than in human serum and were lowest in frozen plasma. The concentration of fibronectin in frozen plasma was significantly higher than in human serum and was lowest in umbilical cord serum. CONCLUSIONS： Umbilical cord serum and fresh frozen plasma may possess potential for substitution of human serum to treatment ocular surface disease and enhance corneal wound healing. Although the enhancement of epitheliotrophic capacity in human-sourced blood products was not as excellent as FBS, these three blood products could also promote proliferation and migration of corneal epithelial cells. These products could be useful materials in cultivating cells for clinical use.

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47

WANG, DER-YUAN, and 王德原. "Characterization and Identification of Limbal Epithelial Stem Cells in a Tissue-Engineered Rabbit Corneal Model." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/29736528650469859924.

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博士
長庚大學
基礎醫學研究所
93
Severe damage to the limbal epithelium due to chemical/thermal burns, virus infection and other pathological events may lead to loss of the limbal epithelial stem cells. Limbal epithelial stem cell deficiency lead to chronic inflammation and vascular invasion of the cornea eventually and cause functional blindness. The autologous and allogenic transplantation of limbal epithelial cells expanded on human amniotic membrane have been introduced to improve the outcome of ocular surface reconstruction in limbal deficient eyes. However, the identification of limbal stem cells and the incorporation of sufficient number of these cells in the bio-engineered corneal tissue remain to be explored. In this study, we employ the cell biological and molecular biological means to identify the characteristics of rabbit limbal epithelial stem cells in normal limbus and in epithelial sheet outgrown from limbal explant cultured on amniotic membrane. The immunofluorescent staining and confocal microscopy were used to examine the expressions of p63, Ki-67, keratins 3 and 14, connexin 43, and integrin 6/4 and  subunits in corneal and limbal tissues, and in limbal explant and its epithelial outgrowth cultured for two weeks on amniotic membrane. Preliminary results showed that the epithelial cell sheet outgrown from limbal explant on amniotic membrane exhibits a phenotype similar to that of the limbus. These results suggested that amniotic membrane is a substrate capable of supporting the propagation and preservation of p63-positive limbal epithelial cells (published in Invest. Ophthalmol. Visual Sci. 2003;44:4698-4704). The real-time Q-RT-PCR was employed to quantify the relative abundance of TAp63 and Np63 transcripts in limbal, peripheral corneal and central corneal epithelia. Antisense oligonucleotides were designed to specifically suppress the expression of TAp63 or Np63 in limbal keratinocytes and their effects on cell proliferation and differentiation were examined. The expressions of TAp63 and Np63 transcripts appeared to be site specific; TAp63 was expressed at the highest level in limbus, decreased in peripheral cornea and was undetectable in the central cornea. Np63 was also expressed at the highest level in limbus, decreased in peripheral cornea and was undetectable in the central cornea. Suppression of TAp63 expression inhibited limbal keratinocyte proliferation but promote differentiation. Suppression of Np63 expression also inhibited cell proliferation but had no obvious effect on cell differentiation. These suggested that TAp63 and Np63 affect the proliferation of limbal keratinocytes by a different mechanism (published in Invest. Ophthalmol. Visual Sci. 2005). We also compared the p63 expression pattern and differentiation levels in different rabbit limbal quadrates. We found that the superior limbal quadrate exhibited the highest level of p63 expression and was characterized by lowest differentiation level. In contrast, the inferior limbal quadrate exhibited very low or undetectable levels of p63 expression and was characterized with terminal differentiation. The ex vivo AM-based epithelial explant culture also showed that the explant from superior limbus has the greatest epithelial outgrowth activity than the cells from other limbal quadrates. Taken together, our results suggested that p63 is not a specific marker of corneal epithelial stem cells. However, different p63 isoforms still play specific roles in maintaining stem cell functions and the entrance to terminal differentiation lineage of corneal epithelial keratinocytes.

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48

Ho, Jennifer Hui-Chun, and 何慧君. "Elucidation of the anti-apoptotic mechanisms of exogenous thymosin beta-4 on human corneal epithelial cells." Thesis, 2008. http://ndltd.ncl.edu.tw/handle/67315090189606566067.

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博士
國立陽明大學
生物藥學研究所
96
Cornea surface, the first-line defense of the eye, is covered with corneal epithelial cells which functionally protect corneal stroma from injury and maintain the transparency of stroma. Apoptosis is the ultimate pathomechanism of many corneal diseases including dry eye, mechanical or chemical injury, ultraviolet ray injury, contact lens-induced corneal edema and infection. Thymosin beta-4 (Tb4) is a ubiquitous peptide consisting of 43 amino acids that has pleiotropic functions including an anti-apoptotic effect on corneal epithelial cells when exogenously administered. However, the protective mechanism of Tb4 on cornea remains largely unknown. The purpose of this study is to elucidate the anti-apoptotic mechanism(s) of exogenous Tb4 on human corneal epithelial cells. In this study, SV-40 immortalized human corneal epithelial (HCE-T) cells were incubated with recombinant Tb4 produced by E. coli. Immunofluorescence staining demonstrated that exogenous Tb4 entered HCE-T cells within 2 hours (i.e. internalization). Fas Ligand (FasL) and hygrogen peroxide (H2O2) were used to trigger extrinsic and intrinsic pathway-mediated apoptosis on HCE-T cells, respectively. Exogenous Tb4 inhibited the activation of caspase-8 and -3 induced by FasL as well as caspase-9 and -3 triggered by H2O2. Internalization of this peptide into HCE-T cells was found to be essential for its apoptosis prevention since disruption of the cellular entry of Tb4 by cytochalasin D abrogated its cytoprotective effects. Moreover, Tb4 could reduce intracellular ROS levels induced by H2O2. Tb4 not only stimulated the expression of manganese superoxide dismutase (Mn-SOD) and copper/zinc SOD (Cu/Zn-SOD) in normal physiological conditions, but also enhanced the transcription and translation of both MnSOD and catalase induced by H2O2. Finally, the addition of 3-amino-1,2,4-triazole (AT), a catalase inhibitor, abrogated the protective effect of Tb4 against H2O2-induced oxidative damage. In conclusion, this study demonstrated that internalization is essential for the anti-apoptotic effects of exogenous Tb4 on HCE-T cells; and its protection against oxidative damage/intrinsic pathway-mediated apoptosis can be attributed to the up-regulation of crucial anti-oxidative enzymes.

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49

Tanti, Nicole-Christina. "Investigating The Impact of Multipurpose Solutions Released From Silicone Hydrogel Lenses on Corneal Epithelial Cells, in vitro." Thesis, 2009. http://hdl.handle.net/10012/4985.

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Cytotoxicity of Multi-Purpose Solutions (MPS) is commonly tested on cells using diluted MPS or extracts from MPS soaked contact lenses. There is evidence that lens type will affect uptake and release of compounds contained in MPS. To assess the cytotoxicity of agents contained in MPS that would be released by contact lens, an in vitro “onlay” model was used, whereby MPS soaked silicone hydrogel lenses were directly set onto a confluent monolayer of corneal cells. Chapter 4 describes the impact of MPS released from contact lenses on immortalized human corneal epithelial cells. MPS-soaked lens interactions with cells were characterized by studying cell viability, cell adhesion and caspase assays. In Chapter 5, mechanisms of cell death induced by exposure to MPS from contact lenses were determined through evaluation of apoptotic markers, such as activation of caspase 3 and 9. In Chapter 6, the impact of the physical properties of silicone hydrogel lenses, specifically surface treatments, on cytotoxicity of MPS were investigated. The development of methods for characterizing the release of MPS from lenses, using absorbance spectra, is also described. The results indicate that exposure to contact lenses soaked in Opti-Free Express (OFX) and ReNu not only induces cell death in vitro, but also has an adverse effect on adhesion phenotype, suggesting that the remaining cells may have a compromised epithelial structure. Borate- buffered MPS were found to be more cytotoxic than phosphate-buffered base solutions. Investigation of the mechanisms of cell death revealed that ReNu and OFX induced corneal epithelial cell death in vitro using different pathways, whereby ReNu induced a necrotic pathway while OFX-induced cell death was mediated by the intrinsic pathway of apoptosis. The in vitro model was also able to identify differences between silicone hydrogels with different surface treatments: the different surface treatments and chemistries of silicone hydrogels lens will affect the release profile of MPS and hence their potential cytotoxicity. By investigating the induction of cell death processes by solution-lens combinations in vitro, we aim to prevent potential adverse effects in the cornea, which may ultimately compromise various visual and barrier functions. The findings indicate the wealth of information in vitro cytotoxicity testing can provide when evaluating the toxicological profile of MPS.

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50

Chiu, Chih-Shen, and 邱志陞. "Studies on the effect of chitosan membranes and chitosan nanoparticles on the behavior of corneal epithelial cells." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/82967286348683783270.

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碩士
國立臺灣大學
醫學工程學研究所
93
In recent years, amniotic membranes transplantation is the most important advancement of treatment modality to treat ocular surface diseases. However, there are a lot of flaws for amniotic membranes transplantation. For example, the ethic issue and contamination of amniotic membrane make us to seek a new material to replace the usage of amniotic membrane. The purpose of this project is to evaluate the possibility of replacement of amnitoic membrane with chitosan membrane, which is a biocompatible materials used in many fields of biomedical science. We cultivated bovine corneal epithelial cells on chitosan membranes and evaluated their phenotypes. MTT assay, indirect immunocytochemical staining and organ culture were used to compare the differences and evaluate the efficacy of treatment of corneal epithelial defects between amniotic membranes and chitosan membranes. According to the MTT assay, we found that the chitosan membrane was a good biomaterial for cultured corneal epithelial cells. In addition, the results of indirect immunocytochemical staining and organ culture, we found that chitosan membrane was compatible and as good as the use of amniotic membrane. Our results indicated that chitosan membrane is a good material for corneal tissue regeneration and repair. For nanoparticle studies, we used different dyes to study endocytosis of chitosan nanoparticles in cultured cells. We found that chitosan nanoparticles could transfect cultured cells with a less effeciency than the liposome. From the results of MTT and LDH assays, we found that chitosan nanoparticles were low toxic and 130 nm of chitosan nanoparticles were helpful to increase cell numbers.

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