Commentary: Lineage Tracing Supports Maintenance of the Corneal Epithelium by Limbal Epithelial Stem Cells

Two papers in the current issue of STEM CELLS have used the increasingly popular multicolored confetti reporter transgene in combination with tamoxifen-inducible Keratin14-CreER to investigate clonal lineages of cells that are induced in the corneal or limbal epithelium of adult mice [1, 2]. These papers are important because they address a central issue underlying the ongoing debate about the location of stem cells that maintain the mammalian corneal epithelium during normal homeostasis. The results tip the evidence firmly in favour of the conventional limbal epithelial stem cell (LESC) hypothesis. Some background to the rival hypotheses is needed to understand the significance of the new work. The LESC hypothesis proposes that stem cells in the basal limbal epithelium, at the periphery of the cornea, produce transient (or transit) amplifying cells (TACs), which move centripetally to the center of the cornea to maintain the corneal epithelium. This would be one of the longest TAC transit routes in the body and although this may be atypical, some other stem cells are also separated from the tissue(s) they maintain. (e.g., adrenocortical stem cells are also located at the periphery and intestinal villi stem cells are in the crypts.) However, the remote position of the limbus has advantages as a stem cell location. It provides more protection from UV and physical damage and, unlike the transparent, avascular cornea, the limbus is richly endowed with blood vessels and diverse cell types that contribute to the niche environment [3]. According to this LESC hypothesis, LESCs are active during normal homeostasis and become more active to repair major wounds. The alternative corneal epithelial stem cell (CESC) hypothesis argues that LESCs only contribute to the corneal epithelium during wound healing and that, during normal homeostasis, the corneal epithelium is maintained by stem cells located throughout the basal corneal epithelium [4]. Although others have pointed out that the CESC hypothesis is not supported by earlier evidence [5], the LESC hypothesis also has difficulty accounting for one key experiment. When genetically labeled mouse limbal epithelial tissue was transplanted to the limbus of immunologically compromised mice, labeled cells only colonized the cornea if the host corneal epithelium was later removed [4]. If LESCs are required during normal corneal epithelial homeostasis, as the LESC hypothesis proposes, the donor limbal tissue would be expected to colonize the cornea without having to be stimulated by wounding. Indeed, experiments with mosaic mice or lentivirus labeling imply that limbal cells do colonize the unwounded cornea from about 5 weeks after birth and other evidence shows that corneal epithelial cells move centripetally in the unwounded adult cornea (reviewed elsewhere [6]). One possible explanation for the failure of transplanted cells to colonize the corneal epithelium is that the surgery itself somehow affected the outcome. To test this, a nonsurgical equivalent of the limbal transplantation experiment is required, so limbal cells can be labeled in adults without surgical intervention. In the studies with conventional mosaics and lentiviral markers, labeling was much earlier (in the embryo) but the two new lineage-tracing studies now provide the missing experiment by showing that limbal cells labeled in the adult can colonize the unwounded corneal epithelium. Furthermore, use of the multicolor confetti reporter effectively allows lineage tracing from individual cells because neighboring cells will usually be labeled differently. Di Girolamo et al. [1] labeled keratin 14 (K14)-expressing cells by treating K14CreER; R26R-confetti mice with tamoxifen at 6 weeks. In an elegant real-time experiment, they tracked the growth of the same fluorescent clones in live mice for up to 21 weeks after labeling. No labeled cells were seen in the unwounded corneas of live mice 3 weeks after injection of tamoxifen, to activate the reporter transgene expression, but labeled cells emerged from the limbus by approximately 5 weeks and extended centripetally across the complete