RETINOIC ACID REGULATES CLONAL GROWTH AND DIFFERENTIATION OF CULTUREDLIMBAL AND PERIPHERAL CORNEAL EPITHELIUM

Purpose. To determine if vitamin A could be one of the factors in seru m responsible for the previously observed effect of 20% fetal bovine s erum on stimulating clonal growth of an additional subpopulation in li mbal cultures, possibly stem cells, but inhibiting that of transient a mplifying cells in peripheral corneal cultures. Methods. A reported se rum-free clonal growth assay was used. The mitogenic response was meas ured by colony-forming efficiency (CFE), colony size, and BrdU labelin g index; the differentiation was assessed by colony morphology, AE-5 m onoclonal antibody staining, and cornified envelope formation. Results . HPLC analyses revealed that added retinoic acid (RA) was rapidly tak en up by cultured cells. As compared to the control without RA, low co ncentrations of RA (10(-9) M to 10(-7) M) stimulated the CFE of limbal cultures but did not change that of peripheral corneal cultures. Furt hermore, 10(-8) M RA induced the emergence of two new types of colonie s, one of which was almost exclusively present in limbal cultures, and allowed continuous clonal growth of some colonies in late limbal cult ures. RA also dose dependently reduced colony size and BrdU labeling i ndex in both limbal and peripheral corneal cultures. RA in concentrati ons above 10(-8) M stimulated normal differentiation of both limbal an d peripheral corneal epithelial cells, as evidenced by increased AE-5 staining, but inhibited the formation of cornified envelopes, an index for abnormal, squamous metaplasia, in late cultures. Conclusion. Thes e results suggest that RA has a differential dose-dependent effect on subpopulations of corneal and limbal epithelial cells. Although RA sti mulates the conversion of limbal stem cells to transient amplifying ce lls, it inhibits the amplification of corneal and limbal transient amp lifying cells and prevents abnormal terminal differentiation. These da ta further support the role of vitamin A as a physiological modulator of proliferation and differentiation of the ocular surface epithelium.