Serum differentially modulates the clonal growth and differentiation of cultured limbal and corneal epithelium.

PURPOSE

The stem cell-containing limbal epithelium is in proximity with highly vascularized tissue, as opposed to the transient amplifying cell-containing corneal basal epithelium, which resides on top of avascular corneal stroma. We therefore speculate that limbal stem cells are preferentially under the modulation of serum-derived factors.

METHODS

Using a previously reported serum-free, chemically defined culture system for ocular surface epithelium, a culture condition primarily supporting transient amplifying cells of both corneal and limbal epithelia, we compared the clonal growth measured by colony-forming efficiency (CFE), colony size, and BrdU labeling, as well as colony differentiation measured by colony morphology and immunofluorescence staining, with the monoclonal antibody AE-5 against keratin K3 when fetal bovine serum (FBS) was added at different concentrations.

RESULTS

The addition of 1% FBS decreased CFE and colony size in peripheral corneal cultures but had no effect in limbal cultures. Both cultures showed no obvious difference in colony morphology or BrdU labeling and AE-5 staining. In contrast, at 10% or 20% FBS, CFE and colony size increased in limbal cultures, but dose dependently decreased in peripheral corneal cultures. The presence of a unique subpopulation of progenitor cells in limbal cultures different from transient amplifying cells in corneal cultures was further supported by the emergence of a higher proportion of a unique type (B) colonies in limbal cultures that had high BrdU labeling and heterogeneous or negative AE-5 staining, indicative of their being in a proliferating, undifferentiated state. These colonies showed continuous growth in late cultures and could be passaged into serum-free medium.

CONCLUSION

These results indicate that serum contains factors responsible for stimulating limbal progenitor cells into clonal proliferation.