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，是 主要位于角膜缘上皮的基底层，是维持角膜上皮的关键 体内平衡。早期 TA 细胞向中央角膜迁移并填充周边角膜上皮 基底层。较成熟或晚期的 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有30多年的历史，并且具有一些特征 这三个人群的定义已经确定。然而，由于角膜缘和外周的异质性 角膜上皮基底层中，分离干细胞/早期 TA 细胞群极其困难。因此，它 干细胞、早期 TA 细胞和晚期 TA 细胞的表征在技术上很困难。我们进行了 单细胞RNA测序并建立离散角膜缘/角膜上皮细胞的单细胞转录组 人口。无偏聚类识别出干细胞/早期 TA、晚期 TA 和分化的上皮细胞。使用这个 单细胞转录组数据，我们发现ID3是干细胞中表达量最高的基因之一 人口。 ID3 是维持多种非眼组织干细胞稳态的关键调节因子，而不是 先前在角膜/角膜缘上皮中被识别。 ID3 蛋白在富含干细胞的细胞中被特异性检测到 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 ID3 的耗竭 减少了表达假定的角膜缘上皮干细胞（LESC）标记的细胞数量，表明 ID3 在维持 LESC 方面发挥积极作用，这将在 Aim1 中进行研究。我们的初步数据还 导致认为 LRRK1 通过靶向 CaMKIIb 和/或，是角膜缘上皮中 ID3 的正调节因子 CTCF (Aim2) 和 LRRK1/CaMKIIb/CTCF/ID3 轴可能有助于维持 LESC（Aims1 和 2）。为了完成 为了实现这些目标，我们将利用我们调节 LRRK1、ID3、CaMKIIb 和 CTCF 水平的能力 补充模型系统，包括浸没培养的原代细胞、3-D 器官筏培养物和 老鼠。 ID3 失调与多种疾病（例如干燥综合征）的发病机制有关。 关于 ID3 如何调控的知识将有助于更好地理解 ID3 的发病机制。 ID3 失调的疾病。由于 LESC 稳态异常涉及多种角膜上皮细胞 疾病（例如，糖尿病性角膜），从该提案中获得的知识可以为 开发基于调节 ID3 表达的新疗法来治疗这些角膜疾病 与受损的 LESC 有关。

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.