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Kedi ve Köpeklerde Limbal Kök Hücrelerin Kültürü ve Keratinositlere Farklılaştırılması

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Peripheral and limbal corneal stromal cells (PLCSCs), which contain keratocytes, have a complex phenotype. Knowledge of keratocyte cell properties, function, and origin is limited. Evidence available thus far has suggested both mesenchymal stromal and hematopoietic characteristics. Multipotent mesenchymal stromal cells (MSCs) are found in an increasing number of tissues and are the subject of considerable interest and investigation in the disciplines of tissue engineering, immunology, gene therapy, and oncology. Isolated PLCSCs were characterized by markers aldehyde dehydrogenase and keratocan, cultured, and analyzed against a set of criteria for the identification of MSCs developed by the International Society of Cellular Therapy (ISCT). PLCSCs were directly compared to fetal liver MSCs (flMSCs). Additional cell surface markers were also used to quantify differentiation, which was also performed on both cell types. PLCSCs were found to be plastic adherent, displayed the correct profile and proportions of CSMs, and demonstrated trilineage potential in accordance with the ISCT guidelines. Furthermore, PLCSCs displayed a high degree of similarity to flMSCs and this likeness extended into the non-ISCT MSC cell surface markers and trilineage differentiation, which were often but not always comparable. Herein we report a novel observation that PLCSCs conform to all the ISCT criteria and are therefore MSCs. Furthermore, this study has identified the limbal stroma as yet another MSC niche and presents a new perspective on the role of the PLCSC.
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To compare the supporting mechanism between the serum-free and the fibroblast-cocultured single-cell clonal culture systems. Clonal growth, measured by colony forming efficiency (CFE) and size, was compared between rabbit corneal and limbal epithelial cells in a previously-established serum-free MCDB medium supplemented with growth factors, and in a coculture system with a feeder layer of mitomycin C-treated mouse 3T3 fibroblasts grown in the MCDB or DMEM medium plus 20% fetal bovine serum (FBS). Limbal epithelial cells in the serum-free MCDB medium had a significantly lower CFE than corneal epithelial cells (p < 0.001), suggesting that this system promoted more clonal growth of corneal progenitor cells. In contrast, with cocultured 3T3 fibroblasts limbal CFE was significantly increased (p < 0.001), while corneal CFE was not changed, indicating that the 3T3 system promoted more clonal growth of limbal progenitor cells. Addition of 20% FBS in the MCDB medium cocultured with 3T3 fibroblasts significantly promoted both limbal and corneal CFEs (p < 0.001). For both cultures, switching the serum-containing MCDB medium to the serum-containing DMEM medium produced clonal growth only with cocultured fibroblasts. This epithelial growth-promoting activity was not present on the cell surface or in the extracellular matrix, but present in pre-centrifuged and prefiltered 3T3 fibroblast-conditioned media. Both growth-promoting and anti-apoptotic activities were present in fibroblast-derived serum-free conditioned media. In the presence of this anti-apoptotic activity, serum addition promoted clonal growth, and the expression of cornea-type K3 keratin in limbal colonies was negative using AE-5 monoclonal antibody. Further purification and characterization of this fibroblast-derived anti-apoptotic survival factor will facilitate understanding of the mechanism by which epithelial stem cells are regulated via epithelial-mesenchymal interactions.
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There is substantial evidence that mammalian epithelial stem cells are located within well defined niches. Although the corneoscleral limbus is acknowledged as the site of corneal epithelial stem cells no anatomical niche for such cells has yet been described. The authors undertook to re-evaluate the microanatomy of the limbus in order to identify possible sites that may represent a stem cell niche. Systematic serial 5-7 microm sections of human corneoscleral segments obtained from cadaver donors, were examined. The sections were stained with haematoxylin and eosin or toludine blue. Sections with specific areas of interest were further examined immunohistologically for the corneal epithelial marker cytokeratin 14 and the "stem cell" marker ABCG2 transporter protein. Distinct anatomical extensions from the peripheral aspect of the limbal palisades were identified. These consist of a solid cord of cells extending peripherally or circumferentially. The cells stained positive for CK14 and ABCG2. A novel anatomical structure has been identified at the human limbus, which demonstrates characteristics of being a stem cell niche. The authors have termed this structure the limbal epithelial crypt.
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The cornea is a tough transparent tissue admitting and focusing light in the eye. More than 90% of the cornea is stroma, a highly organized, transparent connective tissue maintained by keratocytes, quiescent mesenchymal cells of neural crest origin. A small population of cells in the mammalian stroma displays properties of mesenchymal stem cells, including clonal growth, multipotent differentiation, and expression of an array of stem cell-specific markers. Unlike keratocytes, the corneal stromal stem cells (CSSCs) undergo extensive expansion in vitro without loss of the ability to adopt a keratocyte phenotype. Several lines of evidence suggest CSSCs to be of neural crest lineage and not from bone marrow. CSSCs are localized in the anterior peripheral (limbal) stroma near to stem cells of the corneal epithelium. CSSCs may function to support potency of the epithelial stem cells in their unique limbal niche. On the other hand, little information is available documenting a role for CSSCs in vivo in stromal wound healing or regeneration. In vitro CSSCs reproduce the highly organized connective tissue of the stroma, demonstrating a potential use of these cells in tissue bioengineering. Direct introduction of CSSCs into the corneal stroma generated transparent tissue in a mouse model of corneal opacity. Human CSSCs injected into mice corneas did not elicit immune rejection over an extended period of time. The CSSCs therefore appear offer an opportunity to develop cell- and tissue-based therapies for irreversible corneal blindness, conditions affecting more than 10 million individuals worldwide. Disclosure of potential conflicts of interest is found at the end of this article.
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The aim of this study was to evaluate the paracrine effects of canine umbilical cord blood (cUCB) mesenchymal stromal cells (MSC) mixed with beta-tricalcium phosphate (beta-TCP) on bone regeneration in ectopic implantation. beta-TCP mixed with cUCB MSC (UCB-MSC group), cell lysates (cell lysate group) or a control (control group) were respectively implanted in a subcutaneous pouches in the back of beagle dogs . The implants were harvested 1, 4, 7, 14, 28, 56, 84 days after implantation. Histological findings and stain analyzes of tartrate-resistant acid phosphatase (TRACP) and assays of alkaline phosphatase (ALP) and TRACP were evaluated. The mRNA expression levels of interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), cyclooxygenase-2 (COX-2), vascular endothelial growth factor (VEGF) and transforming growth factor-beta (TGF-beta) were analyzed using semi-quantitative reverse transcription - polymerase chain reactions (RT-PCR). An enzyme-linked immunosorbent assay (ELISA) was used to confirm the protein expression levels of IL-6, COX-2, VEGF and TGF-beta. TRACP-positive cells were observed in all groups 7 days after implantation. ALP and TRACP activities in the UCB-MSC group 84 days after implantation were significantly higher than those of the control (P>0.05). Histologic findings after 84 days showed that the osteoid matrix area in the UCB-MSC group was significantly larger than that of the control (P<0.05). The mRNAs levels of IL-1, IL-6 and VEGF in UCB-MSC and cell lysate groups on day 1 were up-regulated compared with the control. The protein levels of IL-6 and VEGF in the UCB-MSC group at day 1 were significantly higher than that of the other groups (P<0.05). It is suggested that a significant release of cytokines by cUCB MSC, 1 day following implantation, could enhance bone regeneration.
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Despite the obvious importance of epithelial stem cells in tissue homeostasis and tumorigenesis, little is known about their specific location or biological characteristics. Using 3H-thymidine labeling, we have identified a subpopulation of corneal epithelial basal cells, located in the peripheral cornea in a region called limbus, that are normally slow cycling, but can be stimulated to proliferate in response to wounding and to a tumor promotor, TPA. No such cells can be detected in the central corneal epithelium, suggesting that corneal epithelial stem cells are located in the limbus. A comparison of various types of epithelial stem cells revealed a common set of features, including their preferred location, pigment protection, and growth properties, which presumably play a crucial role in epithelial stem cell function.
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THE human cornea is covered by a five-layered epithelium. Cells are continually shed from its surface and replaced by division of the basal cells, which has a mean generation time estimated to be about 4 days1. Because of the papillae in the skin, the relation between the area of the basal cell layer and the surface is about 20 : 1. Because it must be refractive, there can be no papillae on the cornea, and the relation between the basal cell layer and the surface is accordingly 1 : 1. This should correspond to higher demands on the generative capacity of the corneal basal cells compared with skin. The epidermal basal cells are in close contact with a well developed capillary network. There are no vessels in the cornea, and so it can be assumed that the supply of its epithelium is poorer. Corneal epithelium, nonetheless, has considerable healing capacity, which is achieved primarily by migration of epithelial cells.
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Proper functioning of the endothelium and proper structural organization of the keratocytes and collagen bundles are of ultimate importance for transparency of the cornea. The role of the endothelium has been investigated extensively, whereas the role of the keratocytes is still unclear. Detailed knowledge on the ultrastructural organization of keratocytes and the relationship between keratocytes and collagen bundles is as essential for understanding corneal transparency as is knowledge of endothelial functioning. Thirty-five corneas (30 postmortem donor corneas and 5 fresh corneas from the operating theater; age range, 28 to 90 years) were used for light microscopy, transmission electron microscopy, and scanning electron microscopy. Serial frontal sections of the central stroma reaching from epithelium to endothelium and cross-sections were studied. At three levels, reconstructions of the mutual arrangement of keratocytes were made using semithin sections. Keratocytes have the appearance of highly active cells with an abundancy of organelles. Between the dendritic ramifications of these cells, large amounts of amorphous material is observed. One of the most remarkable observations is the presence of an extensive network of fenestrations along the surface of the keratocytes. Another important observation is the circular arrangement of keratocytes gradually turning clockwise like a corkscrew from epithelium to endothelium. From the current study, the following conclusions can be drawn: Keratocytes are not quiescent but are highly active cells probably involved in turnover of the extracellular matrix; fenestrations may be of functional relevance with respect to facilitation of diffusion and mechanical attachment of the collagen fibers to the keratocytes; the corkscrew organization of keratocytes suggests that they form completely closed sheets of communicating cells throughout the depth of the cornea, creating equal chances for all light rays to pass one or more keratocytes and thus minimizing variation in light scattering over the entire cornea.
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The cornea provides the eye with protection and the refractive properties essential for visual acuity. The transparent epithelium is highly specialized with basal and stratified squamous cells that are renewed throughout life from a stem cell population. The stem cells are thought to reside at the corneal limbus and may be maintained by a variety of intrinsic and extrinsic factors such as the local environment, survival factors, and cytokines. A number of markers have been localized to the limbus in an attempt to identify stem cells; however, definite stem cell identification remains elusive. During homeostasis and following injury to the corneal epithelium, the limbal stem cells divide to produce daughter transient amplifying cells that proliferate, migrate, and differentiate to replace lost cells. However, this cannot occur if the stem cell population is depleted. Limbal stem cell deficiency then results in corneal re-epithelialization by the neighboring conjunctiva, causing pain, poor vision, and even blindness. This review will focus on corneal epithelial stem cells in ocular surface repair and regeneration. The current knowledge of stem cell biology in the corneal epithelium, clinical consequences of stem cell deficiency, and therapeutic strategies aimed at reversing stem cell deficiency will be discussed.
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Placental tissue draws great interest as a source of cells for regenerative medicine because of the phenotypic plasticity of many of the cell types isolated from this tissue. Furthermore, placenta, which is involved in maintaining fetal tolerance, contains cells that display immunomodulatory properties. These two features could prove useful for future cell therapy-based clinical applications. Placental tissue is readily available and easily procured without invasive procedures, and its use does not elicit ethical debate. Numerous reports describing stem cells from different parts of the placenta, using nearly as numerous isolation and characterization procedures, have been published. Considering the complexity of the placenta, an urgent need exists to define, as clearly as possible, the region of origin and methods of isolation of cells derived from this tissue. On March 23–24, 2007, the first international Workshop on Placenta Derived Stem Cells was held in Brescia, Italy. Most of the research published in this area focuses on mesenchymal stromal cells isolated from various parts of the placenta or epithelial cells isolated from amniotic membrane. The aim of this review is to summarize and provide the state of the art of research in this field, addressing aspects such as cell isolation protocols and characteristics of these cells, as well as providing preliminary indications of the possibilities for use of these cells in future clinical applications. Disclosure of potential conflicts of interest is found at the end of this article.
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