Isolation, culture, characterization and optimization of human corneal stem cells.

The effects of human versus mouse EGF on cell growth and culture duration were studied to optimize a human limbal stem cells culture method for therapeutical autologous transplantation. Limbal cells were obtained by trypsin digestion and transferred to a culture medium. The time needed to reach full confluence in culture was determined. Specific antibodies to corneal stem cell marker (P63) versus corneal epithelial differentiation marker (K3) were used for histochemical determinations. A high proportion of P63 positive cells (85 +/- 4.6%), and a correspondingly low proportion K3 positive cells (15 +/- 3.8%) indicated that most cultured cells remained undifferentiated and were considered as stem cells (mean +/- SE, n=10). Cultures reached full confluency after 17.3 +/- 1.2 days when the medium was supplemented with human EGF, while 21.7 +/- 1.5 days were needed when the medium was supplemented with mouse EGF. The results showed that limbal stem cells proliferate more easily and reach to full confluency in a shorter time if the medium is supplemented with hEGF rather than with mEGF.