Abstract:Purpose
Single cell (sc) analyses of key embryonic, fetal and adult stages were performed to generate a comprehensive single cell atlas of all the corneal and adjacent conjunctival cell types from development to adulthood.
Methods
Four human adult and seventeen embryonic and fetal corneas from 10 to 21 post conception week (PCW) specimens were dissociated to single cells and subjected to scRNA- and/or ATAC-Seq using the 10x Genomics platform. These were embedded using U… Show more
“…The data generated can serve as a reference cell atlas with a major impact in the further improvement and development of cell replacement therapies or regenerative medicine approaches for treating corneal blindness. This research further complements scRNAseq analysis of the developing human cornea 42 and of the human corneal limbus 43 , 44 . Figure 7 scRNAseq data analysis identified nine epithelial, four stromal and two endothelial corneal cell clusters.…”
The cornea is the clear window that lets light into the eye. It is composed of five layers: epithelium, Bowman’s layer, stroma, Descemet’s membrane and endothelium. The maintenance of its structure and transparency are determined by the functions of the different cell types populating each layer. Attempts to regenerate corneal tissue and understand disease conditions requires knowledge of how cell profiles vary across this heterogeneous tissue. We performed a single cell transcriptomic profiling of 19,472 cells isolated from eight healthy donor corneas. Our analysis delineates the heterogeneity of the corneal layers by identifying cell populations and revealing cell states that contribute in preserving corneal homeostasis. We identified expression of CAV1, HOMER3 and CPVL in the corneal epithelial limbal stem cell niche, CKS2, STMN1 and UBE2C were exclusively expressed in highly proliferative transit amplifying cells, CXCL14 was expressed exclusively in the suprabasal/superficial limbus, and NNMT was exclusively expressed by stromal keratocytes. Overall, this research provides a basis to improve current primary cell expansion protocols, for future profiling of corneal disease states, to help guide pluripotent stem cells into different corneal lineages, and to understand how engineered substrates affect corneal cells to improve regenerative therapies.
“…The data generated can serve as a reference cell atlas with a major impact in the further improvement and development of cell replacement therapies or regenerative medicine approaches for treating corneal blindness. This research further complements scRNAseq analysis of the developing human cornea 42 and of the human corneal limbus 43 , 44 . Figure 7 scRNAseq data analysis identified nine epithelial, four stromal and two endothelial corneal cell clusters.…”
The cornea is the clear window that lets light into the eye. It is composed of five layers: epithelium, Bowman’s layer, stroma, Descemet’s membrane and endothelium. The maintenance of its structure and transparency are determined by the functions of the different cell types populating each layer. Attempts to regenerate corneal tissue and understand disease conditions requires knowledge of how cell profiles vary across this heterogeneous tissue. We performed a single cell transcriptomic profiling of 19,472 cells isolated from eight healthy donor corneas. Our analysis delineates the heterogeneity of the corneal layers by identifying cell populations and revealing cell states that contribute in preserving corneal homeostasis. We identified expression of CAV1, HOMER3 and CPVL in the corneal epithelial limbal stem cell niche, CKS2, STMN1 and UBE2C were exclusively expressed in highly proliferative transit amplifying cells, CXCL14 was expressed exclusively in the suprabasal/superficial limbus, and NNMT was exclusively expressed by stromal keratocytes. Overall, this research provides a basis to improve current primary cell expansion protocols, for future profiling of corneal disease states, to help guide pluripotent stem cells into different corneal lineages, and to understand how engineered substrates affect corneal cells to improve regenerative therapies.
“…A recent study used single cell RNA sequencing (scRNA-seq) to characterize cell types in the normal human cornea and conjunctiva [ 48 ]. Table 6 lists the twenty gene expression clusters identified in that study, showing the differential expression patterns of their signature genes in pterygium and pinguecula.…”
Section: Resultsmentioning
confidence: 99%
“…The global expression profile does not give information about which cell type(s) is expressing the differentially expressed gene. This challenge was addressed by utilizing cell-type signatures recently identified by scRNA-seq [ 48 , 49 ]. Based on this analysis, we concluded that most of the previously identified epithelial and stromal cell types were present in our specimens.…”
Solar damage due to ultraviolet radiation (UVR) is implicated in the development of two proliferative lesions of the ocular surface: pterygium and pinguecula. Pterygium and pinguecula specimens were collected, along with adjacent healthy conjunctiva specimens. RNA was extracted and sequenced. Pairwise comparisons were made of differentially expressed genes (DEGs). Computational methods were used for analysis. Transcripts from 18,630 genes were identified. Comparison of two subgroups of pterygium specimens uncovered evidence of genomic instability associated with inflammation and the immune response; these changes were also observed in pinguecula, but to a lesser extent. Among the top DEGs were four genes encoding tumor suppressors that were downregulated in pterygium: C10orf90, RARRES1, DMBT1 and SCGB3A1; C10orf90 and RARRES1 were also downregulated in pinguecula. Ingenuity Pathway Analysis overwhelmingly linked DEGs to cancer for both lesions; however, both lesions are clearly still benign, as evidenced by the expression of other genes indicating their well-differentiated and non-invasive character. Pathways for epithelial cell proliferation were identified that distinguish the two lesions, as well as genes encoding specific pathway components. Upregulated DEGs common to both lesions, including KRT9 and TRPV3, provide a further insight into pathophysiology. Our findings suggest that pterygium and pinguecula, while benign lesions, are both on the pathological pathway towards neoplastic transformation.
“… 37 Nevertheless, Col8 is also expressed in the central corneal stroma, 38 and Col8A2 by CSK. 39 We previously demonstrated, that Col8A2 expression is higher in CSK than in SF, and can be used to differentiate between the two. 17 …”
The isolation and propagation of primary human corneal stromal keratocytes (CSK) are crucial for cellular research and corneal tissue engineering. However, this delicate cell type easily transforms into stromal fibroblasts (SF) and scar inducing myofibroblasts (Myo‐SF). Current protocols mainly rely on xenogeneic fetal bovine serum (FBS). Human platelet lysate (hPL) could be a viable, potentially autologous, alternative. We found high cell survival with both supplements in CSK and SF. Cell numbers and Ki67+ ratios increased with higher fractions of hPL and FBS in CSK and SF. We detected a loss in CSK marker expression (Col8A2, ALDH3A1 and LUM) with increasing fractions of FBS and hPL in CSK and SF. The expression of the Myo‐SF marker SMA increased with higher amounts of FBS but decreased with incremental hPL substitution in both cell types, implying an antifibrotic effect of hPL. Immunohistochemistry confirmed the RT‐PCR findings. bFGF and HGF were only found in hPL and could be responsible for suppressing the Myo‐SF conversion. Considering all findings, we propose 0.5% hPL as a suitable substitution in CSK culture, as this xeno‐free component efficiently preserved CSK characteristics, with non‐inferiority in terms of cell viability, cell number and proliferation in comparison to the established 0.5% FBS protocol.
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