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Sheyla Gonzalez, Hua Mei, Sophie Xiaohui Deng; Modification of Supplemented Hormonal Epithelial Medium to Improve the Expansion of Human Limbal Epithelial Stem/Progenitor Cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4910.
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© ARVO (1962-2015); The Authors (2016-present)
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).
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.
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).
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.
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