Abstract
Purpose :
Anterior limbal mesenchymal stem cells (A-LMSCs) support limbal epithelial stem cells through the limbal niche. Stem cells for the corneal endothelium have also been identified in the transition zone (TZ), but their cellular interactions remain undefined. We hypothesize that cells isolated from the posterior limbus have similar properties to A-LMSCs, and that these posterior limbal mesenchymal stem cells (P-LMSCs) may form a niche to support TZ cells. This study aims to characterize P-LMSCs and investigate their effects on TZ cells.
Methods :
Human P-LMSCs, A-LMSCs and TZ cells were isolated by explant culture. P-LMSCs were characterized by immunocytochemistry and compared to A-LMSCs. TZ cells were cocultured with P-LMSCs in a transwell, with TZ and A-LMSC coculture and TZ cells alone as controls. The proliferation and wound healing speed of TZ cells were evaluated by EdU and scratch wound assays. The stemness of TZ cells was assessed by colony forming assay, droplet digital polymerase chain reaction, western blot and immunocytochemistry. Transcriptomic sequencing was used to investigate molecular mechanisms underlying P-LMSC and TZ cell interactions.
Results :
P-LMSCs expressed similar proteins as A-LMSCs, including mesenchymal marker vimentin, stem cell markers TRA-1-60 and Oct3/4, and angiogenesis markers CD34 and α-SMA. TZ cells cocultured with P-LMSCs had significantly higher proliferation, wound healing speed, and colony forming efficiency compared to TZ cells alone. TZ cells supported by P-LMSCs expressed higher levels of neural crest markers Nestin, AP-2α, Sox9, and periocular mesenchyme marker Pitx2 than A-LMSC coculture and non-coculture groups. Transcriptomic sequencing identified 136 up-regulated and 60 down-regulated genes in TZ cells cocultured with P-LMSCs, primarily associated with cell proliferation, migration, adhesion, and hypoxia response. Enrichment analyses implicated the Ras and HIF pathways in P-LMSC-mediated TZ cell support.
Conclusions :
The proliferation and stemness of TZ cells was enhanced by P-LMSCs. This study identified interactions between cells that reside in the posterior limbus, which provides a basis for innovative strategies for corneal endothelial rejuvenation.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.