Differentiation Trajectory of Limbal Stem and Progenitor Cells under Normal Homeostasis and upon Corneal Wounding

Abstract

Limbal stem cells (LSCs) reside discretely at limbus surrounded by niche cells and progenitor cells. The aim of this study is to identify the heterogeneous cell populations at limbus under normal homeostasis and upon wounding using single-cell RNA sequencing in a mouse model. Two putative LSC types were identified which showed a differentiation trajectory into limbal progenitor cell (LPC) types under normal homeostasis and during wound healing. They were designated as “putative active LSCs” and “putative quiescent LSCs”, respectively, because the former type actively divided upon wounding while the later type stayed at a quiescent status upon wounding. The “putative quiescent LSCs” might contribute to a barrier function due to their characteristic markers regulating vascular and epithelial barrier and growth. Different types of LPCs at different proliferative statuses were identified in unwounded and wounded corneas with distinctive markers. Four maturation markers (Aldh3, Slurp1, Tkt, and Krt12) were screened out for corneal epithelium, which showed an increased expression along the differentiation trajectory during corneal epithelial maturation. In conclusion, our study identified two different types of putative LSCs and several types of putative LPCs under normal homeostasis and upon wounding, which will facilitate the understanding of corneal epithelial regeneration and wound healing.

Nanoceria-Mediated Cyclosporin A Delivery for Dry Eye Disease Management through Modulating Immune–Epithelial Crosstalk

“…The corneal epithelium is under a constant renewal process in both physiological and pathological conditions, which is dominantly supported by limbal progenitor cells (E01_LPC). 69 Similar to previous reports, 70,71 LPCs represent a small population of limbal epithelial cells in normal conditions (Figure 6b) and may expand due to certain insults to establish an ocular regeneration program. To track this LPC-related differentiation process, we performed cellular trajectory reconstruction analysis (CytoTRACE), a published computational method that allowed us to predict the differentiation state of cells from single-cell RNA-sequencing data.…”

“…The corneal epithelium is under a constant renewal process in both physiological and pathological conditions, which is dominantly supported by limbal progenitor cells (E01_LPC) . Similar to previous reports, , LPCs represent a small population of limbal epithelial cells in normal conditions (Figure b) and may expand due to certain insults to establish an ocular regeneration program.…”

Integrative Single-Cell RNA-Seq and ATAC-Seq Analysis of Mouse Corneal Epithelial Cells

“…This result was similar to two recent reports, both of which discovered two limbal stem cell populations in the mouse corneal epithelium, indicating the heterogeneity of mouse limbal cells. 29 , 35 Here, in addition to detecting the same characteristic markers ( Ifitm3 , CD63 ) in “quiescent LSCs,” we also provided other potential markers for the distinct limbal subtypes ( Fig. 2 D).…”

NBL1 Reduces Corneal Fibrosis and Scar Formation after Wounding

“…Previously, we performed a scRNAseq analysis on peripheral corneal and limbal epithelial and anterior stromal cells in mouse corneas undergoing epithelial wounding [13]. The wound was restricted to the epithelial layer with minimal damage to the stroma [13], leading to scarless wound healing, which was confirmed by the low mRNA (Figure 1A) and protein levels (Figure 1B) of α-smooth muscle actin (α-SMA, myofibroblast, and fibrotic marker) in wounded corneas, which were comparable to those from the unwounded corneas.…”

“…Based on our previous single-cell RNA sequencing (scRNAseq) data [13], during scarless healing of a wound restricted to the epithelial layer, corneal stromal cells were activated and expressed a high level of NBL1, a secreted protein known as a BMP antagonist, leading to the hypothesis that NBL1 might play a role in corneal wound healing. In this study, we examined this hypothesis using an in vivo mouse model and an ex vivo human organ culture model.…”