Investigation of corneal limbal stem cells and immune repertoires in human infant cornea by multi-omics solution

This project aims to study two key points related to keratoplasty resistance. LSCs (limbal stem cells), which are crucial for the growth and repair of the cornea, have not been identified by specific markers. Furthermore, the characterization of corneal T/B cells has been rarely studied, even though they play a vital role in transplant rejection. To optimize donated corneas, researchers have utilized single-cell multi-omics methods such as single-cell 5' mRNA and single-cell V(D)J sequencing to explore both LSCs and T/B immune repertoires (IR) simultaneously. Potential LSCs and dominant V(D)J types were analyzed, and the location of the cell marker was determined by RNA in situ sequencing. From the single-cell transcriptomics of 17,218 whole corneal cells, 20 cell subtypes were observed. A subcluster (0.3% of total cells) was identified as putative epithelial LSCs based on the known markers stating stem cells in the G0 cell cycle. TCR/BCR were rarely found in the cornea. The results suggest that GPHB5 could be a potential marker for limbal stem cells, but high-throughput single-cell VDJ sequencing is not the ideal method for analyzing corneal immune repertoires due to the rare presence of T/B cells. Overall design: single-cell 5' mRNA and single-cell V(D)J sequencing to explore both LSCs and T/B immune repertoires (IR) simultaneously

本研究旨在探究与角膜移植术（keratoplasty）抵抗相关的两个核心问题。角膜缘干细胞（limbal stem cells, LSCs）是角膜生长与修复的关键细胞类群，但目前尚未发现其特异性标志物。此外，尽管角膜T/B细胞在移植排斥反应中发挥关键调控作用，但针对其细胞特征的研究却极为匮乏。为优化供体角膜的临床利用价值，研究人员采用单细胞5'端mRNA测序、单细胞V(D)J测序等单细胞多组学技术，同时对角膜缘干细胞与T/B细胞免疫组库（immune repertoires, IR）开展联合分析。研究人员对潜在角膜缘干细胞及优势V(D)J类型进行了分析，并通过RNA原位测序明确了细胞标志物的定位。基于17218个全角膜细胞的单细胞转录组数据分析，共鉴定出20种细胞亚型。基于G0细胞周期干细胞的已知特征标志物，研究人员鉴定出一个占总细胞数0.3%的亚群，即为推定的上皮性角膜缘干细胞。角膜组织中极少检测到T细胞受体（TCR）与B细胞受体（BCR）。研究结果显示，GPHB5或可作为角膜缘干细胞的潜在标志物；但由于角膜内T/B细胞数量稀少，高通量单细胞V(D)J测序并非分析角膜免疫组库的理想技术手段。实验整体设计：采用单细胞5'端mRNA测序与单细胞V(D)J测序技术，同时探究角膜缘干细胞与T/B细胞免疫组库。