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

项目摘要/摘要 角膜上皮的上皮干细胞及其直接后代，早期TA细胞，是 优先位于角膜缘上皮的基底层，是维持角膜上皮的关键 动态平衡。早期TA细胞向中央角膜迁移并分布于周边角膜上皮 基础层。较成熟或较晚的TA细胞被认为存在于中央角膜上皮基底层。 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多年的历史了，一些特征 这三个种群之间的关系已经被定义。然而，由于角膜缘和周围的异质性， 在角膜上皮基底层，干细胞/早期TA细胞群体的分离一直是极其困难的。因此，它 对于干细胞、早期TA细胞和晚期TA细胞的特征在技术上是困难的。我们已经进行了 单细胞RNA测序及离散型角膜缘上皮细胞单细胞转录组的建立 人口。无偏聚类鉴定了干细胞/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是干细胞中表达最高的基因之一。 人口。ID3是几种非眼组织中维持干细胞内环境稳定的关键调节因子 先前在角膜/角膜缘上皮中发现。在浓缩的干细胞中特异性地检测到ID3蛋白 角膜缘上皮基底层，在角膜上皮中几乎检测不到。角膜缘上皮中ID3的耗竭 减少了可能表达角膜缘上皮干细胞(LESC)标记的细胞数量，这表明 ID3在维持LESCs方面发挥了积极作用，这将在Aim1中进行研究。我们的初步数据也 导致认为LRRK1是通过靶向CaMKIIb和/或 CTCF(AIM2)和LRRK1/CaMKIIb/CTCF/ID3轴可能有助于维持LESCs(AIMs1和2)。要完成 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 免费的模型系统，包括浸泡培养的原代细胞，3-D器官型筏培养，以及 老鼠。ID3调节失调与多种疾病(如干燥综合征)的发病机制有关。 了解ID3是如何调节的，将转化为更好地理解 ID3调节失调的疾病。由于LESC动态平衡异常参与了多种角膜上皮细胞 疾病(例如，糖尿病角膜)，从这一提议中获得的知识可以为 开发基于调节ID3表达的治疗这些角膜疾病的新药物 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.