Purpose

This study aimed to uncover novel cell types in heterogenous basal limbus of human cornea for identifying LSC at single cell resolution.

Methods

Single cells of human limbal basal epithelium were isolated from young donor corneas. Single-cell RNA-Sequencing was performed using 10x Genomics platform, followed by clustering cell types through the graph-based visualization method UMAP and unbiased computational informatic analysis. Tissue RNA in situ hybridization with RNAscope, immunofluorescent staining and multiple functional assays were performed using human corneas and limbal epithelial culture models.

Results

Single-cell transcriptomics of 16,360 limbal basal cells revealed 12 cell clusters belonging to three lineages. A smallest cluster (0.4% of total cells) was identified as LSCs based on their quiescent and undifferentiated states with enriched marker genes for putative epithelial stem cells. TSPAN7 and SOX17 are discovered and validated as new LSC markers based on their exclusive expression pattern and spatial localization in limbal basal epithelium by RNAscope and immunostaining, and functional role in cell growth and tissue regeneration models with RNA interference in cultures. Interestingly, five cell types/states mapping a developmental trajectory of LSC from quiescence to proliferation and differentiation are uncovered by Monocle3 and CytoTRACE pseudotime analysis. The transcription factor networks linking novel signaling pathways are revealed to maintain LSC stemness.

Conclusions

This human corneal scRNA-Seq identifies the LSC population and uncovers novel cell types mapping the differentiation trajectory in heterogenous limbal basal epithelium. The findings provide insight into LSC concept and lay the foundation for understanding the corneal homeostasis and diseases.

中文翻译：

---

单细胞转录组学鉴定角膜缘干细胞群和细胞类型，绘制其在人角膜角膜缘基底上皮中的分化轨迹

目的

本研究旨在揭示人类角膜异质基底缘中的新细胞类型，以在单细胞分辨率下识别 LSC。

方法

从年轻的供体角膜中分离出人角膜缘基底上皮的单细胞。使用 10x Genomics 平台进行单细胞 RNA 测序，然后通过基于图形的可视化方法 UMAP 和无偏计算信息分析对细胞类型进行聚类。使用人角膜和角膜缘上皮培养模型进行组织 RNA 与 RNAscope 的原位杂交、免疫荧光染色和多功能测定。

结果

16,360 个角膜缘基底细胞的单细胞转录组学揭示了属于三个谱系的 12 个细胞簇。一个最小的簇（占总细胞的 0.4%）被鉴定为 LSC，基于它们的静止和未分化状态以及推定的上皮干细胞的富集标记基因。TSPAN7 和 SOX17 通过 RNAscope 和免疫染色在角膜缘基底上皮细胞中的独特表达模式和空间定位，以及在培养物中具有 RNA 干扰的细胞生长和组织再生模型中的功能作用，被发现和验证为新的 LSC 标记。有趣的是，Monocle3 和 CytoTRACE 伪时间分析揭示了五种细胞类型/状态，描绘了 LSC 从静止到增殖和分化的发育轨迹。

结论

这种人类角膜 scRNA-Seq 可识别 LSC 群体并揭示新的细胞类型，绘制出异质角膜缘基底上皮的分化轨迹。这些发现提供了对 LSC 概念的深入了解，并为理解角膜稳态和疾病奠定了基础。