Single-Cell Study of Human Cornea Development and Immune Interactions
Summary
A seminal study involving the utilization of cutting-edge single cell technologies, namely single cell RNA sequencing (scRNA-Seq) and assay for transposase-accessible chromatin sequencing (ATAC-Seq), was conducted in order to create an extensive atlas of corneal and conjunctival cell types across multiple key developmental, fetal and adult stages. This approach facilitated the generation of a comprehensive depiction of the cellular landscape of the human cornea and its adjacent conjunctiva, which serves as a valuable resource for investigating the genetic and regulatory pathways underlying both normal corneal homeostasis and the pathogenesis of ocular surface disorders. Notably, the data obtained from the analysis of developing adult corneas in both steady state and disease conditions provides a previously unparalleled opportunity for advancing our understanding of the molecular mechanisms regulating the expansion of limbal progenitor cells (LPCs) in vitro, and for improving the techniques for stem cell differentiation. By identifying the genes and pathways modulated in this process, researchers can potentially develop novel strategies for mitigating the detrimental effects of ocular surface disorders, ultimately leading to more effective treatments for such conditions.
Fig.1 Graphical abstract. (Colin, 2021)
Research Criteria
Our study aimed to gain deeper insight into the intricacies of the human ocular surface. To this end, we conducted single cell analyses of the human cornea and conjunctiva across multiple developmental and adulthood stages, both in states of health and in the presence of disease. Our methodology was also extended to the mouse ocular anterior segment, yielding the discovery of a novel marker, TXNIP, that differentiated stem and early transit amplifying cells. While our research was being evaluated, two additional studies that analyzed the single cell transcriptome of the human limbal basal epithelium and murine corneal non-myelinating Schwann cells emerged. Our work enriches and extends these previous findings by presenting a comprehensive single cell atlas of the developing and adult human cornea and conjunctiva. This atlas provides critical information on the development of limbal progenitor cells and interactions with immune cells in the human eye.
Sample Type
Single cells isolated from human adult and embryonic and fetal corneas from 10 to 21 post-conception week (PCW) specimens.
Result—scRNA-Seq of Adult Human Cornea and Adjacent Conjunctiva Reveals the Presence of Progenitor and Differentiated Cells in the Epithelial, Stromal, and Endothelial Layers
Four human adult corneas procured post-mortem from donors with ages ranging from 51 to 83 years, and their contiguous conjunctival tissues were meticulously excised and dissociated into individual cells. The 10x Chromium Single Cell 3' Library & Gel Bead Kit v3 was employed to capture an approximate average of 10,000 cells from each sample. After data integration and exclusion of doublet cells, a total of 21,343 cells were obtained from the four adult corneas. These cells were then subjected to the process of Uniform Manifold Approximation and Projection (UMAP) for embedding, and further analyzed through the application of Seurat's graph-based clustering methodology. This in-depth analysis revealed the presence of 21 distinct cell clusters, which were characterized through the integration of marker gene expression analysis, bioinformatic data mining, and immunohistochemical (IF) analysis to determine their definitive identity.
Fig.2 scRNA-seq analysis of adult human cornea and conjunctiva. (Colin, 2021)
Result—Combined scRNA-Seq and ATAC-Seq Analysis Reveals Key Transcriptional Networks in LPCs and Close Interactions with Immune Cells
Four specimens of the human adult cornea and conjunctiva were subjected to a scATAC-Seq analysis, during which approximately 10,000 cells were captured and subjected to quality control. Ultimately, 10,625 cells were obtained after data integration, revealing the presence of all predicted scRNA-seq clusters with the exception of cluster 20, which may be ascribed to the comparatively minute cell numbers present in this cluster. The LPCs cells cluster was found to exhibit distinct characteristics in the UMAP plot, compared to the other epithelial clusters. In order to facilitate a more in-depth analysis of these findings, 7618 epithelial cells were selected for differential accessibility analysis, wherein the LPCs cluster was compared to the remaining corneal and conjunctival epithelial cell clusters. This analysis identified KRT14 and CASP14 as the most highly accessible promoters in LPCs, while the MUC22 promoter was found to be the least accessible in the LPCs cluster 9. The JEME database was then employed to link the differentially accessible peaks to enhancer regions, specifically for LPCs (cluster 9) and limbal suprabasal cells (cluster 4). This analysis revealed that the EDH1 locus, which contains a distal enhancer of the LPCs marker GPHA2, was found to be more accessible in cluster 9, aligning with its purported role in maintaining the undifferentiated state and self-renewal of LPCs.
Fig.3 scATAC-Seq of adult human cornea and conjunctiva. (Colin, 2021)
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Creative Biolabs provides a comprehensive range of customized, high-quality services in single cell ATAC profiling to support various scientific research worldwide. Examining gene regulation at the single cell level through the analysis of chromatin accessibility can provide insights into the underlying mechanisms at play.
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Reference
- Colin, J.; et al. A single cell atlas of human cornea that defines its development, limbal progenitor cells and their interactions with the immune cells. The Ocular Surface. 2021, 21: 279-298.
