Purpose:
Earlier on, using RT-PCR differential gene display (DGD) to compare the limbal (stem cell-rich) and the corneal epithelial zones , we identified genes (50+) that are over-expressed in the rabbit limbus (Li-genes; Int J. Dev Biol.,48:981 ). Independently, we have demonstrated the isolation of limbal stem cells in the form of Hoechst 33342 flow cytometry side population (SP; J Cell Sci. 118:1715). The purpose of this study was to determine whether and which of the Li-genes are in fact overexpressed in these stem cells.
Methods:
Single cell suspensions of rabbit limbal epithelial cells were generated by sequential Dispase-trypsin digestion. and cultured for 24 hr. Adherent (i.e., basal) cells were stained with Hoechst 33342 and sorted by FACS into a SP (stem cell rich; ~ 1% of total) and non-SP (stem cell depleted) fractions. RNA was prepared with Tri-Reagent and used to establish relative levels of Li-gene expression in the SP and non-SP populations by real time RT-PCR. Data was normalized by the ΔΔ method using ß-actin as the invariant gene and SP/non-SP ratios (R) were calculated from duplicate experiments.Primers were designed to have highly similar potency as determined by PCR of rabbit genomic DNA, thereby allowing calculations of relative abundance (Abund.).
Results:
Out of 18 genes tested, 7 were overexpressed in the SP. All these low copy number genes had known functions that are consistent with a role in stem cell biology (see Table).
Conclusions:
Comparison of Li vs Co gene expression by DGD led to the identification of very low copy number genes which may contribute to the stem cell phenotype.
Keywords: cornea: epithelium • cornea: basic science