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. 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. 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. 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 . 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.

中文翻译：

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对新鲜和储存的人类供体角膜进行整体免疫荧光分析，突出显示储存期间角膜缘特征的变化


人类供体角膜是角膜研究的重要对照组织。我们利用整体免疫荧光 (WM-IF) 来评估储存如何影响人类和猪角膜中的组织完整性和假定的角膜缘干细胞。此外，我们将此信息与在人多能干细胞 (hPSC) 衍生的 LSC 中观察到的标记表达模式进行了比较。使用 WM-IF 分析假定的 LSC 标记物的表达，并对储存 1-30 天的人供体角膜以及安乐死后 0-6 小时处理的猪角膜中的荧光强度进行量化。将结果与人和猪角膜冷冻切片的染色以及原代和 hPSC 衍生的 LSC 培养物进行比较。与组织切片相比，WM-IF 分析成为一种更有效的方法，用于可视化人和猪角膜内 LSC 标记物的表达。储存时间是影响LSC标记表达的重要因素，因为储存时间较长的人体组织表现出明显的上皮变性和LSC标记的缺乏。猪角膜复制了在新鲜人体组织中观察到的表达模式。我们验证了 PAX6 在角膜缘区域的不同表达模式，这与 hPSC-LSC 分化实验的结果一致。 WM-IF 与荧光强度定量相结合被证明是研究人类和猪组织中 LSC 标记表达的有价值的工具。长期储存会显着影响 LSC 标记的表达，强调了在研究角膜缘干细胞生物学时新鲜人体或替代对照组织的重要性。