Characterisation of corneas following different time and storage methods for their use as a source of stem-like limbal epithelial cells
Autor: | Cristina Romo-Valera, Raquel Hernáez-Moya, Paula Romano-Ruiz, Jon Arluzea, Jaime Etxebarria, Maddalen Rodriguez-Astigarraga, Noelia Andollo, Miguel Pérez-Garrastachu |
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Jazyk: | angličtina |
Rok vydání: | 2021 |
Předmět: |
Adult
Time Factors preservation availability Cell Separation Limbus Corneae Biology Organ culture Cornea Andrology Cellular and Molecular Neuroscience Organ Culture Techniques vimentin corneal shortage sclerocorneal rims expression Humans Cells Cultured donor Aged organ-culture Aged 80 and over support Stem Cells Epithelium Corneal limbal stem cell deficiency (LSCD) Middle Aged Tissue Donors Sensory Systems optisol-GS Transplantation Ophthalmology melanocytes corneal cell cultures langerhans cells Keratins Collagen Tissue Preservation sense organs Biomarkers corneoscleral tissues limbal epithelial stem cells (LESCs) corneal storage transplantation |
Zdroj: | Addi. Archivo Digital para la Docencia y la Investigación instname |
Popis: | The transplantation of expansions of limbal epithelial stem cells (LESC) remains one of the most efficient therapies for the treatment of limbal stem cell deficiency (LSCD) to date. However, the available donor corneas are scarce, and the corneas conserved for long time, under hypothermic conditions (after 7 days) or in culture (more than 28 days), are usually discarded due to poor viability of the endothelial cells. To establish an objective criterion for the utilisation or discarding of corneas as a source of LESC, we characterized, by immunohistochemistry analysis, donor corneas conserved in different conditions and for different periods of time. We also studied the potency of LESCs isolated from these corneas and maintained in culture up to 3 cell passages. We hoped that the study of markers of LESCs present in both the corneoscleral histological sections and the cell cultures would show the adequacy of the methods used for cell isolation and how fit the LESC enrichment of the obtained cell populations to be expanded was. Thus, the expressions of markers of the cells residing in the human limbal and corneal epithelium (cytokeratin CK15 and CK12, vimentin, Collagen VII, p63 alpha, ABCG2, Ki67, Integrin beta 4, ZO1, and melan A) were analysed in sections of corneoscleral tissues conserved in hypothermic conditions for 2-9 days with post-mortem time (pmt) 8 h or for 1 day with pmt 16 h, and in sclerocorneal rims maintained in an organ culture medium for 29 days. Cell populations isolated from donor corneoscleral tissues were also assessed based on these markers to verify the adequacy of isolation methods and the potential of expanding LESCs from these tissues. Positivity for several putative stem cell markers such as CK15 and p63 alpha was detected in all corneoscleral tissues, although a decrease was recorded in the ones conserved for longer times. The barrier function and the ability to adhere to the extracellular matrix were maintained in all the analysed tissues. In limbal epithelial cell cultures, a simultaneous decrease in the melan A melanocyte marker and the putative stem cell markers was detected, suggesting a close relationship between the melanocytes and the limbal stem cells of the niche. Holoclones stained with putative stem cell markers were obtained from long-term, hypothermic, stored sclerocorneal rims. The results showed that the remaining sclerocorneal rims after corneal transplantation, which were conserved under hypothermic conditions for up to 7 days and would have been discarded at a first glance, still maintained their potential as a source of LESC cultures. This research study has been funded by grants from the Department of Heath of the Basque Government (RIS3, 2019222049) and the University of the Basque Country UPV/EHU-Instituto Clinico Quirurgico de Oftalmologia ICQO (US19/18). C. R-V. has received fellowship support from the University of the Basque Country UPV/EHU |
Databáze: | OpenAIRE |
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