Identification of the regulatory circuit governing corneal epithelial fate determination and disease

Abstract

The transparent corneal epithelium in the eye is maintained through the homeostasis regulated by limbal stem cells (LSCs), while the nontransparent epidermis relies on epidermal keratinocytes for renewal. Despite their cellular similarities, the precise cell fates of these two types of epithelial stem cells, which give rise to functionally distinct epithelia, remain unknown. We performed a multi-omics analysis of human LSCs from the cornea and keratinocytes from the epidermis and characterized their molecular signatures, highlighting their similarities and differences. Through gene regulatory network analyses, we identified shared and cell type-specific transcription factors (TFs) that define specific cell fates and established their regulatory hierarchy. Single-cell RNA-seq (scRNA-seq) analyses of the cornea and the epidermis confirmed these shared and cell type-specific TFs. Notably, the shared and LSC-specific TFs can cooperatively target genes associated with corneal opacity. Importantly, we discovered that FOSL2, a direct PAX6 target gene, is a novel candidate associated with corneal opacity, and it regulates genes implicated in corneal diseases. By characterizing molecular signatures, our study unveils the regulatory circuitry governing the LSC fate and its association with corneal opacity.

Decoding cellular plasticity and niche regulation of limbal stem cells during corneal wound healing

“…IRF7 is a member of the interferon regulatory factors (IRFS) family and participates in type-I interference (IFN) signaling, and plays a role in antigen presentation function of human corneal endothelial cells [ 62 , 63 ]. The latest research showed that FOSL1 as the human LSC-specific TFs that determined the LSC fate and was important in epidermal cells [ 64 ]. Furthermore, KLF7 promoted the corneal progenitor cell state [ 65 ].…”

Deciphering the heterogeneity of differentiating hPSC-derived corneal limbal stem cells through single-cell RNA-sequencing

“…To further assess the molecular profile of the FACS-purified cells, we collected samples from TRA-1-81 - /ITGA6 + /AREG + hESC and hiPSC1 at 15 days post-sorting and carried out bulk RNA-seq. The expression of selected cluster marker genes in the bulk RNA-seq data of FACS-purified cells were compared to the pseudobulk datasets from our “all D24 cells” and the primary LSCs from Smits et al 2023 (Figure 6C). Similar to the in silico purification and supporting the IF staining results, expression of genes in FACS-purified TRA-1-81 - /ITGA6 + /AREG + hiPSC1 cells showed more similarity to hESC “all D24 cells” and primary LSCs, as compared to all unpurified hiPSC1 “all D24 cells”, including increased expression of LSC markers and decreased expression of mesodermal-like and pluripotent markers.…”

“…This may require further attention in our upcoming studies, since correct dosage of PAX6 is known to be essential for corneal epithelial health, starting early on from the eye field development all the way to corneal morphogenesis and postnatal CE (Shaham et al, 2012; Sunny et al, 2022). Importantly, it is one of the key transcription factors driving the lineage-commitment toward the ocular epithelia instead of skin epidermis (Kitazawa et al, 2017; G. Li et al, 2015; Ouyang et al, 2014; Smits et al, 2023).…”

“…Importantly, it is one of the key transcription factors driving the lineage-commitment toward the ocular epithelia instead of skin epidermis (Kitazawa et al, 2017;G. Li et al, 2015;Ouyang et al, 2014;Smits et al, 2023). C6/iLSC-2 was identified as the slightly more differentiated epithelial population appearing at D24, characterized by e.g., MUC16, which is expressed superficially throughout the adult ocular surface epithelia (Argüeso et al, 2003) and in the developing CE (Collin et al, 2021).…”

“…To test if the line-dependent differentiation efficiency can be overcome by purification of specific cell state, an in silico purification was simulated by computationally selecting the most LSC-like population (C5/iLSC-1) and comparing their transcriptome to data from all D24 cells and primary adult human LSCs. For this comparison, scRNA-seq data of C5/iLSC-1 cells, all D24 cells and primary adult human LSCs (Smits et al (2023) (donor sample IDs: GSM6266908 and GSM6266909) were processed into pseudobulk data where the single cell data was aggregated, and visualized with the principle component analysis (PCA). Since 95% of the D24 hESC cells were annotated as C5/iLSC-1, but the two hiPSC lines were heterogeneous, we expected that the hESC "all D24 cells" (purple triangle) would be more similar to the "primary LSCs" (blue squares) than "all D24 cells" from hiPSC1 or hiPSC2 (orange and green triangles, respectively).…”