Following the successful cultivation of Confetti cells, we compared their phenotype with those generated from WT mice (
Fig. 3,
Table). As K14 is localized to both basal limbal
1,2,28 and conjunctival epithelium,
29 it was necessary to determine whether cultures were indeed corneo-limbal in origin. Immunofluorescence and flow cytometry indicated that 30% to 50% and 50% to 80% of cells were K14
+ and ΔNp63
+, respectively (
Figs. 3A–C,
Table), both markers of basal limbal progenitors,
30,31 whereas <10% of cells were immunoreactive for the corneal differentiation marker K12 (
Figs. 3A–C,
Table). Notably, our initial cultures displayed 2-fold lower K14 expression, potentially because they were analyzed at later passages (
Figs. 1D,
1E), indicating a progressive loss of K14 over subsequent generations, similar to that found for K15 and P-cadherin expression.
32 Differences in ΔNp63 expression were also noted when cells were analyzed with immunofluorescence and flow cytometry (
Figs. 3B,
3C), although this is likely due to differences in assay sensitivity. Conjunctival keratins
33,34 also were profiled and <10% of cells were found to be K13
+, whereas 30% to 50% were K8/18
+ (
Figs. 3A–C,
Table). Although these results suggest conjunctival cells may have contaminated our cultures, we believe this is unlikely, as every precaution was taken to ensure complete removal of the conjunctiva from enucleated eyes by carefully resecting this tissue from intact corneas. A potential explanation is that in vitro cultivation modified gene and protein expression profiles.
32,35 Curiously, both K13
33 and K8/18
36 are found in suprabasal
33 and basal
36 human limbal epithelia, respectively, and K8/18 is coexpressed with K14, K12, and Muc5AC in compound niches within the peripheral murine cornea.
37 Furthermore, it is hypothesized that K8/18 is a marker of limbal progenitor cells capable of differentiating into conjunctival or corneal epithelia.
37 Our phenotypic assays suggested we cultivated basal limbal epithelial cells with limited primitive characteristics.
18,30 Others have generated similar undifferentiated cultures, speculating this is due to calcium in the media, which at high concentrations induces differentiation.
17,24,35,38 dKSFM contains low calcium (<0.1 mM), which likely aids in maintaining a precursor phenotype. In this regard, cultures from transgenic and WT mice harbored comparable SC activity (
Fig. 3E), a result congruent with previous investigations.
18,38,39 Overall, these studies demonstrate that neither the presence nor induction of the transgene affected cell phenotype or function, indicating cultures derived from Confetti and WT mice are equivalent. The former system is advantageous for fate-mapping grafted cells due to the stable incorporation of SC-specific fluorescent reporters,
28 which allows mice to be monitored in real time by intravital microscopy.