Limbal stem cells (LSCs), a subpopulation of limbal epithelial basal cells, are crucial to the homeostasis and wound healing of corneal epithelium. The identification and isolation of LSCs remains a challenge due to lack of specific LSCs biomarkers. In this study, Haematoxylin-eosin (HE), 4', 6-diamidino-2-phenylindole (DAPI), and immunohistochemistry (IHC) stains were performed on the pre- and post-natal limbus tissues of mice which has the advantage of more controllable in term of sampling age relative to human origin. By morphological analysis, we supported that there is an absence of the Palisades of Vogt (POV) in the mouse. The development of prenatal and neonatal cornea was dominated by its stroma, whereas after eyelids opened at P14, the corneal epithelial cells (CECs) quickly go stratification in response to the liquid-air interface. Based on IHC staining, we found that the expression of LSCs putative biomarkers in limbal epithelial basal cells appeared in chronological order as follows: Vim = p63 > CK14 > CK15 (where = represents same time; > represents earlier), and in corneal epithelial basal cells were weakened in chronological order as follows: Vim > p63 > CK15 > CK14, which might also represent the stemness degree. Furthermore, the dynamic spatial expression of the examined LSCs putative biomarkers during mouse development also implied a temporal restriction. The expression of Vim in epithelial cells of mouse ocular surface occurred during E12-E19 only. The expression of CK15 was completely undetectable in CECs after P14, whereas the others putative molecular markers of LSCs, such as p63 and CK14, still remained weak expression, suggesting that CK15 was suitable to serve as the mouse LSCs biomarkers after P14. In this study, our data demonstrated the dynamic spatiotemporal expression pattern of LSCs putative biomarkers in mouse was age-related and revealed the time spectrum of the expression of LSCs in mouse, which adds in our knowledge by understanding the dynamic expression pattern of biomarkers of stem cells relate to maintenance of their stemness.

中文翻译：

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小鼠发育过程中角膜缘干细胞推定生物标志物的动态时空表达模式。

角膜缘干细胞（LSC）是角膜上皮的稳态和伤口愈合的关键，是角膜缘上皮基底细胞的亚群。由于缺乏特定的LSC生物标志物，LSC的鉴定和分离仍然是一个挑战。在这项研究中，苏木精-曙红（HE），4'，6-二mid基-2-苯基吲哚（DAPI）和免疫组织化学（IHC）染色是在小鼠出生前后的角膜缘组织上进行的，具有以下优势：在相对于人类起源的采样年龄方面更具可控性。通过形态分析，我们支持小鼠中不存在Vogt栅栏（POV）。产前和新生儿角膜的发育以其基质为主，而在P14眼睑张开后，角膜上皮细胞（CEC）响应液-气界面而迅速分层。根据IHC染色，我们发现LSCs假定的生物标志物在角膜缘上皮基底细胞中的表达按时间顺序排列如下：Vim = p63> CK14> CK15（其中=表示相同时间；>表示更早），以及在角膜上皮基底细胞中细胞按时间顺序变弱，如下所示：Vim> p63> CK15> CK14，这也可能代表干度。此外，在小鼠发育过程中检查的LSCs假定的生物标志物的动态空间表达也暗示了时间限制。Vim仅在E12-E19期间出现在小鼠眼表上皮细胞中。在P14之后，在CEC中完全无法检测到CK15的表达，而其他假定的LSC分子标记（如p63和CK14）仍然保持弱表达，提示CK15适合作为P14后的小鼠LSCs生物标志物。在这项研究中，我们的数据证明了LSCs假定的生物标志物在小鼠中的动态时空表达模式与年龄有关，并揭示了LSCs在小鼠中表达的时间谱，这通过了解茎的生物标志物的动态表达模式而增加了我们的知识。细胞与其干性的维持有关。