Purpose: Progenitors for the corneal endothelium have been identified in the transition zone (TZ), but their cellular interactions remain undefined. Posterior limbal mesenchymal stromal cells (P-LMSCs) may support TZ cells in the posterior limbus. This study aims to characterize P-LMSCs and investigate their effects on TZ cells.
Methods: Human P-LMSCs and TZ cells were isolated by explant culture. P-LMSCs were characterized by comparing with anterior limbal mesenchymal stromal cells (A-LMSCs) using immunocytochemistry. TZ cells were cocultured with P-LMSCs in a Transwell, with TZ cell and A-LMSC coculture and TZ cells only as the control groups. The proliferation and wound healing capacity of TZ cells were assessed by EdU assay and scratch wound assay. Colony forming assay, droplet digital PCR, Western blotting, and immunocytochemistry were used to compare the stemness of TZ cells. The effect of P-LMSC conditioned medium on endothelial wound healing was evaluated in organ-cultured mouse corneas. Endothelial regeneration was measured by trypan blue staining.
Results: P-LMSCs expressed similar proteins (vimentin, Nestin, TRA-1-60, Oct3/4) as A-LMSCs. TZ cells cocultured with P-LMSCs had significantly higher proliferation, wound healing speed, and colony-forming efficiency than TZ cells only. TZ cells supported by P-LMSCs expressed higher levels of stem/progenitor markers (Nestin, Sox9, AP-2α, Pitx2) than the control groups. P-LMSC conditioned medium stimulated regeneration of mouse corneal endothelium from the TZ region.
Conclusions: The proliferation and stemness of TZ cells were enhanced by P-LMSCs in both cell and organ culture models. Our study provides an innovative strategy for corneal endothelial rejuvenation.