Single-cell RNA sequencing reveals novel regulatory pathways in maintaining limbal epithelial stem cell homeostasis

项目概要/摘要 角膜上皮的上皮干细胞及其直接后代，即早期TA细胞， 其优先位于角膜缘上皮的基底层，并且是维持角膜上皮的关键。 体内平衡早期TA细胞向角膜中央迁移，并聚集在周边角膜上皮细胞中。 基底层更成熟或晚期的TA细胞被认为存在于中央角膜上皮基底层中。 这种角膜上皮干细胞/TA细胞/有丝分裂后细胞的范例已经有30多年的历史， 这三个群体的定义。然而，由于角膜缘和外周的异质性， 角膜上皮基底层中，分离干/早期TA细胞群是极其困难的。因此 在技术上难以表征干细胞、早期TA细胞和晚期TA细胞。我们已经进行 单细胞RNA测序并建立了离散角膜缘/角膜上皮细胞的单细胞转录组 人口。无偏聚类鉴定了干/早期TA、晚期TA和分化的上皮细胞。使用此 单细胞转录组数据，我们发现ID 3是干细胞中表达最高的基因之一， 人口ID 3是维持几种非眼部组织中干细胞稳态的关键调节因子， 先前在角膜/角膜缘上皮中识别。ID 3蛋白在干细胞富集的细胞中特异性检测到。 角膜缘上皮基底层和角膜上皮中几乎检测不到。角膜缘上皮中ID 3的消耗 减少了表达假定的角膜缘上皮干细胞（LESC）标志物的细胞数量，这表明， ID 3在维持LESC中起着积极的作用，这将在Aim 1中进行研究。我们的初步数据还 导致LRRK 1是角膜缘上皮中ID 3的正调节剂，其通过靶向CaMKIIb和/或 CTCF（Aim 2）和LRRK 1/CaMKIIb/CTCF/ID 3轴可能有助于维持LESC（Aims 1和2）。完成 这些目标，我们将利用我们的能力，调节LRRK 1，ID 3，CaMKIIb和CTCF水平， 互补的模型系统，包括体外培养的原代细胞，3-D器官型筏培养， 小鼠ID 3的失调与多种疾病的发病机制有关（例如，干燥综合征）。 关于ID 3如何调节的知识将转化为更好地理解ID 3的发病机制。 ID 3失调的疾病。由于异常LESC稳态涉及各种角膜上皮细胞， 疾病（例如，糖尿病角膜），从该建议中获得的知识可能为以下方面提供依据： 开发用于治疗这些角膜疾病的基于调节ID 3表达的新疗法 与受损的LESC相关。

PROJECT SUMMARY/ABSTRACT The epithelial stem cells for the corneal epithelium and their immediate progeny, the early TA cells, are preferentially located in the basal layer of the limbal epithelium and are key for maintaining corneal epithelial homeostasis. The early TA cells migrate toward central cornea and populate the peripheral corneal epithelial basal layer. The more mature or late TA cells are believed to reside in the central corneal epithelial basal layer. This paradigm of corneal epithelial stem/TA cell/post-mitotic cell is over 30 years old and some of the features of these three populations have been defined. However, due to the heterogeneity of the limbal and peripheral corneal epithelial basal layer, it has been extremely difficult to isolate the stem/early TA cell population. Thus, it was technically difficult for the characterization of stem vs early TA versus late TA cells. We have conducted single cell RNA sequencing and established a single cell transcriptome for discrete limbal/corneal epithelial cell populations. Unbiased clustering identified stem/early TA, late TA and differentiated epithelial cells. Using this single cell transcriptome data, we discovered that ID3 was one of the highest expressed genes in stem cell population. ID3 is a key regulator for maintaining stem cell homeostasis in several non-ocular tissues and not previously recognized in corneal/limbal epithelium. ID3 proteins were specifically detected in stem cell-enriched limbal epithelial basal layer and nearly undetectable in corneal epithelium. Depletion of ID3 in limbal epithelium reduced the numbers of cells expressing putative limbal epithelial stem cell (LESC) markers, suggesting that ID3 plays a positive role in maintaining LESCs, which will be investigated in Aim1. Our preliminary data also lead to the idea that LRRK1 is a positive regulator of ID3 in limbal epithelium via targeting CaMKIIb and/or CTCF (Aim2) and LRRK1/CaMKIIb/CTCF/ID3 axis may help maintain LESCs (Aims1 and 2). To accomplish these goals, we will capitalize on our ability to modulate LRRK1, ID3, CaMKIIb and CTCF levels in complimentary model systems that include submerge cultured primary cells, 3-D organotypic raft cultures, and mice. Dysregulation of ID3 is associated with the pathogenesis of various diseases (e.g., Sjögren's Syndrome). Knowledge on how ID3 is regulated will translate into a better understanding of the pathogenesis of the diseases with dysregulation of ID3. Since abnormal LESC homeostasis is involved in various corneal epithelial disorders (e.g., diabetic cornea), the knowledge gained from this proposal may provide a rationale for developing novel therapeutics based on modulating the expression of ID3 for treating these cornea diseases associated with compromised LESCs.