Maintenance of label-retaining K15 positive progenitor cells in long-term cultured primary human limbal epithelial cell sheets

Abstract

Purpose: Stem cells in vivo are believed to have a slow cell cycle, whereas their progeny rapidly proliferate to produce differentiated cells. In this study, we maintained primary limbal epithelial cell sheets in culture for up to 12 months. Cell turnover and the relationship between label retaining cells (LRCs), K15 positive cells, and rapidly proliferating cells were investigated.

Methods: Human limbal epithelial cells isolated from US eyebank eyes were primary cultured on plastic cell culture inserts, with a feeder layer of human mesenchymal stem cells prepared in the bottom of a paired well. Cells were fed with modified supplementary hormonal epithelial medium containing KGF and Y-27632, but not EGF. To monitor cell homeostasis, cells shedding daily into medium were counted every month for 1 year. To detect LRCs, semi-confluent cells (day 6) were serially labeled with EdU (1 μM) for 3 days, and one set of triplicated inserts was fixed immediately after serial labeling. Remained inserts were fed daily with medium to washout EdU label. On day 179, cells were treated with BrdU (10 μM) for 1 day to label rapidly proliferating cells. PFA-fixed whole mount cell culture inserts were stained with EdU detection kit, followed by anti- BrdU or anti- K15 immunostaining.

Results: Shedding cells were rare at 1 month (1.5x10⁴ cells/insert/day), but increased at 3 months and 4 months (5.7x10⁴cells/insert/day), then gradually decreased to 8 months and reached a plateau (4.0x10⁴cells/insert/day). This number is equal to 1.5% of total basal cell number (2.8x10⁶/insert, day 365). Early sheets (day 9 and 1 month) expressed K15 homogeneously, whereas late sheets (3 months-1 year) expressed K15 heterogeneously, with K15-positive cells forming clusters. At 6 months, LRCs were also clustered into areas overlapping with K15 expression. LRC percentage was 27.4%±14.7% (n=3) inside of K15 positive clusters, compared to 8.0%±2.5% outside of clusters. BrdU positive cells, which represent cells entering S phase between day 179 to day 180, were mainly observed in LRC-sparse areas.

Conclusions: A differentiation gradient of K15-positive LRCs, K15-negative LRCs and rapidly proliferating cells are spontaneously organized in long-term cultured sheets. Our results show that immature progenitor cells can be maintained for at least 12 months in vitro.