Abstract
Purpose:
To investigate supplemented hormonal epithelial medium (SHEM) in combination with embryonic stem cell medium (ESCM) for the expansion of human limbal epithelial stem/progenitor cells (LSCs).
Methods:
Limbal explants were cultured on denuded amniotic membrane in SHEM supplemented with 5% human serum, ESCM and a mixture of SHEM-ESCM (1:1). Single LSCs cultured on 3T3 feeder cells in SHEM were used as a control. Cell morphology, cell size, cell growth rate, outgrowth size and expression level of putative stem cell and stromal markers were analyzed.
Results:
Cell growth rate in the ESCM culture was the lowest compared to that in SHEM and SHEM-ESCM cultures (all p<0.05). The amount of cultured LSCs expressing high level of p63α was lower in the ESCM culture (4.4%) compared to that in the control (18.8%, p=0.04), SHEM (20.3%, p=0.02) and SHEM-ESCM, (6.7%, p=0.49). LSCs cultured in ESCM contained the largest amount of small (≤8 µm; 1%) and medium (9-12 µm; 5.1%) size cells compared to the control (0.1 % and 3.2%), SHEM (0.1% and 2%) and SHEM-ESCM (0.1% and 1.5%). At the mRNA level, both ABCG2 and cytokeratin (K) 14 expression levels were higher in the ESCM culture than in the SHEM and SHEM-ESCM cultures (p<0.05). At the protein level, the ESCM culture contained a larger amount of Vimentin+ cells (1.8%) compared to the SHEM (0.2%, p=0.04), and SHEM-ESCM (1.6%, p=0.81) cultures. The number of K12+ cells was low in all the conditions tested (range: 1.8-4.9%, p>0.05).
Conclusions:
ESCM appears to better maintain the undifferentiated state of LSCs than SHEM and SHEM-ESCM, but it does not promote the proliferation of LSCs. SHEM-ESCM might be an alternative option to expand LSCs.