SERUM DIFFERENTIALLY MODULATES THE CLONAL GROWTH AND DIFFERENTIATION OF CULTURED LIMBAL AND CORNEAL EPITHELIUM

Purpose. The stem cell-containing limbal epithelium is in proximity wi th highly vascularized tissue, as opposed to the transient amplifying cell-containing corneal basal epithelium, which resides on top of avas cular corneal stroma. We therefore speculate that limbal stem cells ar e preferentially under the modulation of serum-derived factors. Method s. Using a previously reported serum-free, chemically defined culture system for ocular surface epithelium, a culture condition primarily su pporting transient amplifying cells of both corneal and limbal epithel ia, we compared the clonal growth measured by colony-forming efficienc y (CFE), colony size, and BrdU labeling, as well as colony differentia tion measured by colony morphology and immunofluorescence staining, wi th the monoclonal antibody AE-5 against keratin K3 when fetal bovine s erum (FBS) was added at different concentrations. Results. The additio n of 1% FBS decreased CFE and colony size in peripheral corneal cultur es but had no effect in limbal cultures. Both cultures showed no obvio us difference in colony morphology or BrdU labeling and AE-5 staining. In contrast, at 10% or 20% FBS, CFE and colony size increased in limb al cultures, but dose dependently decreased in peripheral corneal cult ures. The presence of a unique subpopulation of progenitor cells in li mbal cultures different from transient amplifying cells in corneal cul tures was further supported by the emergence of a higher proportion of a unique type (B) colonies in limbal cultures that had high BrdU labe ling and heterogeneous or negative AE-5 staining, indicative of their being in a proliferating, undifferentiated state. These colonies showe d continuous growth in late cultures and could be passaged into serum- free medium. Conclusion. These results indicate that serum contains fa ctors responsible for stimulating limbal progenitor cells into clonal proliferation.