Abstrakti
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.
| Alkuperäiskieli | Englanti |
|---|---|
| Sivut | 50-59 |
| Sivumäärä | 10 |
| Julkaisu | OCULAR SURFACE |
| Vuosikerta | 34 |
| DOI - pysyväislinkit | |
| Tila | Julkaistu - lokak. 2024 |
| OKM-julkaisutyyppi | A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä |
Rahoitus
Another previous study indicated that despite the epithelial degeneration during storage, LSCs are still somewhat preserved [31], whereas some controversial results have been reported with the hypothermia-based method [13]. In our analyses with WM-IF, the loss of stem cell markers ABCG2, p27 and c/EBP\u0394 indicated the loss of at least certain LSC subpopulations in expired human corneas, while the expression of CK15 and p40 was preserved during storage, which supports the role of these markers in the proliferative basal cell types rather than genuine LSCs [14]. However, certain discrepancies remain, as in contrast to the well-preserved expression of both p40 and CK15, lower expression of ki67 was observed, being thus more in line with the findings of Le-Bel et al. [13]. The loss of Ki67 was not accompanied by increased staining of cell cycle arrest marker p27, indicating that there might be alterations in the stored tissue which affect the behavior of this marker. In the porcine corneas, the expression of Ki67 was notably higher both in the limbal transition zone as well as the central cornea, more consistently in line with the biological concept of large pool of proliferative intermediate corneal epithelial cells [32]. Importantly, most of the human-targeted antibodies that have been tested in our laboratory appear to work similarly for the porcine, but for example in this study, no specific staining pattern for p27 antibody was produced in the porcine samples and whether this was a biological or technical issue remains to be further validated.WM-IF offers a key advantage over traditional IF from tissue sections by enabling the spatial detection and quantification of proteins across the entire path from limbal area to central cornea in a single sample, while preserving tissue complexity. Our results from human WM-IF -stained donor corneas support the previous studies, showing that both the LSC marker expression and tissue architecture are changed during storage. This should be acknowledged when utilizing the corneal tissues for various research applications. However, the restricted access to and control over confounding factors in human donor tissues form a major limitation of this study, which hinders the establishment of robust experimental design with sufficient statistical power. As a continuum, we demonstrate that porcine corneal tissue may serve as a promising substitute tissue help to overcome the limitations related to the use of stored human donor corneas.The authors warmly thank the personnel of Regea Tissue Center (Tampere University) for their invaluable contribution to this study by delivering the donor corneas for research and sharing their first-hand insights into corneal tissue banking. We also thank the local abattoir \u201CPaijan tilateurastamo\u201D for providing the porcine ocular tissue for research. Authors thank laboratory technicians Hanna Pekkanen and Outi Melin for their contributions to hPSC-LSC cell culture. The authors also wish to thank following funding sources for their financial support to this study: Research Council of Finland (H.S.), Sigrid Jus\u00E9lius Foundation (H.S.), Finnish Cultural Foundation (M.K.), Instrumentarium Foundation (M.K.) and Nissi Foundation (M.K.). In addition, we acknowledge Tampere Imaging Facility (TIF) for their service.
| Rahoittajat | Rahoittajan numero |
|---|---|
| Suomen Kulttuurirahasto | |
| Tampere Imaging Facility | |
| Strategic Research Council at the Research Council of Finland | |
| Kirjasto | |
| Nissi Foundation | |
| Orion Research Foundation sr, Finland and Instrumentarium Science Foundation sr, Finland | |
| Sigrid Juséliuksen Säätiö |
Julkaisufoorumi-taso
- Jufo-taso 3
!!ASJC Scopus subject areas
- Ophthalmology