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Yoshinori Nakai, Morio Ueno, Michiru Matsumura, Yoshiki Sasai, Shigeru Kinoshita; Induction Of Corneal Epithelial Cells From Human Embryonic Stem Cells On The Amniotic Membrane Matrix. Invest. Ophthalmol. Vis. Sci. 2011;52(14):276.
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© ARVO (1962-2015); The Authors (2016-present)
Driving human embryonic stem (ES) cells to differentiate into corneal epithelium is a key technology for regenerative medicine associated with ocular surface reconstruction. We previously reported a differentiation technique using the matrix components of the human amniotic membrane (denuded hAM) which induces the retinal pigment epithelia and lentoid tissues from human ES cells (amniotic membrane matrix-based ES cell differentiation, or AMED). The purpose of this present study was to investigate whether human ES cells differentiate to corneal epithelia on denuded hAM.
hAMs encasing the fetus within the human female uterus were obtained at the time of Caesarean section after obtaining proper informed consent from both parents and in accordance with the tenets of the Declaration of Helsinki. To prepare the denuded hAM for culture, the matrix was carefully removed from its overlying epithelium, and then transferred to cell-culture plates. Dissociated human ES cells were then seeded onto the denuded hAM and cultured in KSR (Invitrogen Corp., Carlsbad, CA)-containing Glasgow-MEM (Invitrogen) medium at 37°C under 5% CO2 .
Dissociated human ES cells formed large colonies and differentiated into neural precursors at high efficiency when cultured on the denuded hAM in the serum-free medium containing a selective ROCK inhibitor (Y-27632). AMED-treated human ES cells developed to epithelial cells which were positive for cytokeratin12 and Pax6; consistent with the characteristic of corneal epithelia. The percentage of human ES cell-derived cytokeratin12-positive colonies increased to become up to 30% of the total colonies on denuded hAM.
The findings of this study show that AMED should provide a highly practical system for generating corneal epithelial cells from human ES cells with a non-cellular inductive material derived from an easily available human tissue for clinical application.
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