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Yoshinori Nakai, Morio Ueno, Michiru Matsumura, Satoshi Kawasaki, Yoshiki Sasai, Shigeru Kinoshita; Characterization Of Corneal Epithelial Cells Induced From Human Embryonic Stem Cells On The Amniotic Membrane Matrix. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1821.
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© ARVO (1962-2015); The Authors (2016-present)
We previously reported a differentiation technique using the matrix components of the human amniotic membrane (denuded hAM) which induces the neural precursors, retinal pigment epithelia (RPE), and corneal epithelium from human embryonic stem (ES) cells (amniotic membrane matrix-based ES cell differentiation, or AMED). This technique can create new cell sources for regenerative medicine associated with ocular surface reconstruction. The purpose of this present study was to investigate the progress of differentiation and quantity of differentiated corneal epithelia.
hAM encasing the fetus within a human female uterus was 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 a serum-free KSR (Invitrogen Corp., Carlsbad, CA)-containing Glasgow-MEM (Invitrogen) medium supplemented with the selective rho kinase (ROCK) inhibitor Y-27632 at 37°C and under 5% CO2. Immunostaining and quantitative PCR (qPCR) were then used to detect the induction of corneal epithelia.
Dissociated human ES cells formed large colonies and differentiated into RPE when cultured on denuded hAM in serum-free medium containing ROCK-inhibitor Y-27632. Immunostaining revealed that the AMED-treated human ES cells developed into epithelial cells that were positive for cytokeratin (CK) 12, CK 3, and Pax 6, consistent with the characteristics of corneal epithelia. The percentage of human ES cell-derived CK 12-positive colonies increased to become up to 30% of the total colonies on the denuded hAM. Moreover, qPCR revealed an increase of transforming growth-factor beta (TGFb), clusterin (CLU), aldehyde dehydrogenase 3 (ALDH3), CK 12, and CK 3 in the AMED-treated cells, consistent with the characteristics of corneal epithelia and progenitor cells.
AMED provides a highly practical system for generating corneal epithelial cells from human ES cells via the use of a non-cellular inductive material derived from an easily available human tissue for clinical application.
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