Whole mount immunofluorescence analysis of fresh and stored human donor corneas highlights changes in limbal characteristics during storage.

Autor: Kauppila M; Faculty of Medicine and Health Technology, Tampere University, Tampere, 33520, Finland., Vattulainen M; Faculty of Medicine and Health Technology, Tampere University, Tampere, 33520, Finland., Ihalainen TO; Faculty of Medicine and Health Technology, Tampere University, Tampere, 33520, Finland; Tampere Institute for Advanced Study, Tampere University, Tampere, 33520, Finland., Mörö A; Faculty of Medicine and Health Technology, Tampere University, Tampere, 33520, Finland., Ilmarinen T; Faculty of Medicine and Health Technology, Tampere University, Tampere, 33520, Finland., Skottman H; Faculty of Medicine and Health Technology, Tampere University, Tampere, 33520, Finland. Electronic address: heli.skottman@tuni.fi.
Jazyk: angličtina
Zdroj: The ocular surface [Ocul Surf] 2024 Oct; Vol. 34, pp. 50-59. Date of Electronic Publication: 2024 Jun 28.
DOI: 10.1016/j.jtos.2024.06.004
Abstrakt: Purpose: Human donor corneas are an essential control tissue for corneal research. We utilized whole mount immunofluorescence (WM-IF) to evaluate how the storage affects the tissue integrity and putative limbal stem cells in human and porcine corneas. Moreover, we compare this information with the marker expression patterns observed in human pluripotent stem cell (hPSC)-derived LSCs.
Methods: The expression of putative LSC markers was analyzed with WM-IF and the fluorescence intensity was quantified in human donor corneas stored for 1-30 days, and in porcine corneas processed 0-6 h after euthanasia. The results were compared with the staining of human and porcine corneal cryosections and with both primary and hPSC-derived LSC cultures.
Results: WM-IF analyses emerged as a more effective method when compared to tissue sections for visualizing the expression of LSC markers within human and porcine corneas. Storage duration was a significant factor influencing the expression of LSC markers, as human tissues stored longer exhibited notable epithelial degeneration and lack of LSC markers. Porcine corneas replicated the expression patterns observed in fresh human tissue. We validated the diverse expression patterns of PAX6 in the limbal-corneal region, which aligned with findings from hPSC-LSC differentiation experiments.
Conclusions: WM-IF coupled with quantification of fluorescence intensities proved to be a valuable tool for investigating LSC marker expression in both human and porcine tissues ex vivo. Prolonged storage significantly influences the expression of LSC markers, underscoring the importance of fresh human or substitute control tissue when studying limbal stem cell biology.
Competing Interests: Declaration of competing interest T.I. and H.S. are co-inventors of a patent transferred from Tampere University (Tampere, Finland) to StemSight Ltd (Tampere, Finland) regarding the used hPSC-LSC differentiation method. Based on the Act on the Right in Inventions made at Higher Education Institutions in Finland, all authors employed by Tampere University have given all rights to the University and thus have declared no competing interests. T.I., H.S. and A.M. are co-founders and shareholders in StemSight Ltd. The other authors declare no conflicts of interests.
(Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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