Abstract
Abstract: :
Purpose: After staining cells with Hoechst 33342, a DNA–binding fluorescent dye, flow cytometry plots of blue vs. red emissions reveal the presence of a rare subset of cells (0.1–1.0 %), positioned to the blue side of the blue/red norm. This cohort, present in many tissues, is known as a side population (SP) and in several cases has been shown to be highly enriched in stem cells. We have previously demonstrated SPs in the limbal and conjunctival epithelia (Budak, ARVO 2003 # 859; Int. J. Dev. Biol., 48:981). Now we have assessed whether these SPs contain slow cycling cells, a main characteristic of stem cells. Methods: Rabbits were maintained for 2 weeks under 5–bromo–2'–deoxyuridine (BrdU; a thymidine analog) infusion by osmotic pumps and sacrificed at the end of the infusion or 6 weeks later. Tissue BrdU uptake was monitored by indirect immuno–fluorescence. Freshly dissociated conjunctival epithelial cells were sorted by flow cytometry into SP and non–SP cohorts, cytospun onto slides, and stained with FITC–conjugated anti–BrdU. For the limbus, we analyzed a cohort defined by extremely low light side scattering (LSSC), which markedly overlaps with the SP. Results: SPs accounted for > 1 % of the total limbal or conjunctival epithelia cell populations. In all experiments at least 50 % of the SP cells displayed the LSCC phenotype. Conversely, more than 50 % of the LSSC cells were SP cells. The relatively stem cell–free corneal epithelium was devoid of either SP or LSSC cells. The BrdU infusion labeled a great proportion of the basal and suprabasal limbal–corneal and conjunctival epithelial cells. Consistent with this labeling, more than 80 % of the cells in the main cytometer–sorted fractions (contain > 90 % of the cells) were BrdU–positive. In contrast, at least 70 % of the cells in the small conjunctival SP or limbal LSSC were BrdU–negative, indicative that these cells were mostly quiescent during the two–week labeling period. After a 6–week chase, label–retaining cells (LCRs) were observed only and sparsely within the basal limbal and conjunctival zones. Consistent with this result, the main (i.e., non–SP, non–LSSC) cytometer fractions contained very few LCRs. In contrast, 22 % of the conjunctival SP cells and 16 % of the limbal LSSC cells were BrdU–rich, indicative that cells labeled during the infusion period underwent minimal cell division over the long chase period. Conclusions: These results indicate that the conjunctival and limbal SP and LSSC flow cytometry cohorts are highly enriched in slow cycling stem cells.
Keywords: cornea: epithelium • cornea: basic science • conjunctiva