Hypoxia Increased the Colony Forming Efficiency and Inhibited the Differentiation of Human Limbal Epithelial Cells

Abstract

Purpose:: To investigate the effect of hypoxia on the expansion of limbal epithelial cells in vitro.

Methods:: Human limbal epithelial cells were isolated from eyebank corneas and cultured in serum-free low calcium medium (defined KSFM, Invitrogen) under normoxia (20% O2) and hypoxia (2% O2). Effect of hypoxia on epithelial precursor/stem cells was investigated by colony forming efficiency (CFE) of 1000 cells inoculated in 60 mm dishes. Differentiation of epithelial cells was investigated by the RT-PCR analysis, western blot analysis and immunocytochemistry against involucrin and keratin 3. Cell size and structure were evaluated by the flow cytometry and transmission electron microscopy (TEM).

Results:: CFE of human limbal epithelial cells cultured under hypoxia was 3.1 % +- 0.41 % (mean +- S.D., n=3), which was significantly greater than CFE under normoxia (1.3 % +- 0.52 %) (n=3, p < 0.05). Size of hypoxic colonies was larger than normoxic colonies, suggesting that hypoxia increases the proliferation of limbal epithelial cells. Expression of differentiation markers under hypoxia was lower than cells under normoxia at both mRNA and protein levels. Cells under hypoxia showed relatively low forward scatter and side scatter profiles. TEM showed that cells under hypoxia contained less number of nucleoli in compared to cells under normoxia.

Conclusions:: Hypoxia increased the CFE and proliferation of human limbal epithelial cells and inhibited differentiation. Hypoxia may be a key factor to maintain and expand limbal epithelial stem/progenitor cells in vitro.

Keywords: cornea: epithelium • hypoxia • proliferation