Identification of αSMA lineage cells in the posterior limbal stroma

Limbal stromal cells constitute a heterogeneous cellular compartment that supports tissue homeostasis and may contribute to regional progenitor niches within the corneoscleral limbus. Although stromal cells have been described in both anterior and posterior limbal regions, their in vivo distribution and relationship to α-smooth muscle actin (αSMA) lineage activity remain poorly understood. This study investigated the distribution of αSMA-lineage (αSMA-Lin) cells in the cornea and limbus of αSMA-CreER^T2/Ai9 mice. Intraperitoneal administration of tamoxifen was used to induce tdTomato fluorescence in cells with an active αSMA promoter at the time. Tissues were analysed at 2, 14, and 28 days after induction using wholemount imaging, three-dimensional reconstruction, and immunofluorescence staining. αSMA-Lin (+) cells were expanded by day 28 compared to earlier time points. They were more abundant in the limbus compared to the cornea and sclera, and in the posterior limbus (positive in 69.9 ± 3.5% of cells) compared to the anterior limbus (7.6 ± 2.2%, n = 3, P < 0.01). The superior (435 ± 20 μm) and temporal (422 ± 23 μm) limbal quadrants exhibited wider αSMA-Lin (+) areas compared to the nasal quadrant (345 ± 15 μm) (n = 6, P < 0.05). There was some colocalisation with αSMA antibody labelling on day 2. The αSMA-Lin (+) cells co-expressed vimentin and TRA-1-60. Some αSMA-Lin (+) cells colocalised with pericyte marker NG2. There was no colocalisation with the fibroblast marker FSP1. These findings provide an anatomical map of an αSMA-lineage stromal cell compartment that is preferentially enriched in the posterior limbus. Our findings provide new insights into the structure of the corneoscleral limbus.