Purpose: To determine the distribution of epithelial stem cells in the bulbar conjunctiva by measuring homeostatic movements and mitosis of epithelial cells in this region.
Methods: The ubiquitous GFP mouse was used to monitor movement of conjunctival epithelial cells. Cell movement was determined by histology, analyzing the shape and distribution of GFP cell clusters in flat wholemount specimens, and by in vivo time-lapse microscopy, tracking the movement of GFP-positive cells in the bulbar conjunctiva near the limbus. Mitoses were determined by labeling DNA of adult mice with bromodeoxyuridine (BrdU) for 3 days. Label-retaining cells (LRCs) were determined by a pulse label of newborn mice with BrdU, followed by a chase of 6 to 7 weeks.
Results: Similar to the corneal epithelium, only some of the conjunctival epithelial cells expressed a high level of GFP. Histology showed that GFP-positive cells existed as clusters of several to several dozen cells. No stripe pattern of GFP was observed in any part of the conjunctiva, suggesting that directed cell movement was rare or nonexistent. Time-lapse analyses revealed that none of the tracked GFP clusters exhibited a continuous and directed movement and that most GFP clusters were stationary for several weeks and much longer in some occasions. BrdU labeling showed that GFP-positive cells in this region were mitotically active. BrdU pulse-chase experiments demonstrated that LRCs were distributed uniformly in this region.
Conclusions: Epithelial cells of the bulbar conjunctiva near the limbus are mitotically active and yet they are generally immobile in a lateral direction, indicating that these cells are self-sufficient. These results, combined with the uniform distribution of LRCs, suggest that epithelial stem cells are distributed uniformly in this area.