John F. Hardesty, MD Department of Ophthalmology & Visual Sciences, Washington University School of Medicine, St. Louis, MO 63110.
Division of Biology and Biomedical Sciences Neurosciences Graduate Program, Washington University School of Medicine, St. Louis, MO 63110.
Proc Natl Acad Sci U S A. 2023 Jan 10;120(2):e2204134120. doi: 10.1073/pnas.2204134120. Epub 2023 Jan 3.
Many epithelial compartments undergo constitutive renewal in homeostasis but activate unique regenerative responses following injury. The clear corneal epithelium is crucial for vision and is renewed from limbal stem cells (LSCs). Using single-cell RNA sequencing, we profiled the mouse corneal epithelium in homeostasis, aging, diabetes, and dry eye disease (DED), where tear deficiency predisposes the cornea to recurrent injury. In homeostasis, we capture the transcriptional states that accomplish continuous tissue turnover. We leverage our dataset to identify candidate genes and gene networks that characterize key stages across homeostatic renewal, including markers for LSCs. In aging and diabetes, there were only mild changes with <15 dysregulated genes. The constitutive cell types that accomplish homeostatic renewal were conserved in DED but were associated with activation of cell states that comprise "adaptive regeneration." We provide global markers that distinguish cell types in homeostatic renewal vs. adaptive regeneration and markers that specifically define DED-elicited proliferating and differentiating cell types. We validate that expression of SPARC, a marker of adaptive regeneration, is also induced in corneal epithelial wound healing and accelerates wound closure in a corneal epithelial cell scratch assay. Finally, we propose a classification system for LSC markers based on their expression fidelity in homeostasis and disease. This transcriptional dissection uncovers the dramatically altered transcriptional landscape of the corneal epithelium in DED, providing a framework and atlas for future study of these ocular surface stem cells in health and disease.
许多上皮细胞在维持体内平衡时会进行组成性更新，但在受伤后会激活独特的再生反应。透明角膜上皮对视力至关重要，由角膜缘干细胞（limbal stem cells，LSCs）更新。通过单细胞 RNA 测序，我们对维持体内平衡、衰老、糖尿病和干眼疾病（dry eye disease，DED）状态下的小鼠角膜上皮进行了分析，在 DED 中，泪液缺乏使角膜容易反复受伤。在维持体内平衡时，我们捕捉到了完成连续组织更新的转录状态。我们利用我们的数据集来鉴定候选基因和基因网络，这些基因和基因网络特征描述了稳态更新的关键阶段，包括 LSCs 的标记物。在衰老和糖尿病中，只有不到 15 个失调基因发生了轻微变化。完成稳态更新的组成型细胞类型在 DED 中是保守的，但与组成“适应性再生”的细胞状态激活有关。我们提供了可区分稳态更新与适应性再生的细胞类型的全局标记物，以及专门定义 DED 诱导的增殖和分化细胞类型的标记物。我们验证了适应性再生标记物 SPARC 的表达也在角膜上皮伤口愈合中被诱导，并在角膜上皮细胞划痕实验中加速了伤口闭合。最后，我们提出了一个基于其在体内平衡和疾病中表达保真度的 LSC 标记物分类系统。这种转录剖析揭示了 DED 中角膜上皮的转录景观发生了显著改变，为这些眼表面干细胞在健康和疾病中的未来研究提供了框架和图谱。
