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Morio Ueno, Yoshinori Nakai, Michiru Matsumura, Satoshi Kawasaki, Yoshiki Sasai, Shigeru Kinoshita; Induction Of Corneal Epithelial Cells From Human-induced Pluripotent Stem Cells On The Amniotic Membrane Matrix. Invest. Ophthalmol. Vis. Sci. 2012;53(14):268.
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Induced pluripotent stem (iPS) cells are generated by reprogramming somatic cells, thereby avoiding immune rejection and ethical issues, and patient-tailored iPS cells should prove to be valuable for regeneration medicine. We previously established 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 iPS cells (amniotic membrane matrix-based ES cell differentiation, or AMED). The purpose of this present study was to investigate the progress of differentiation and quantity of differentiated corneal epithelia, using immunostaining and quantitative PCR (qPCR).
hAM encasing the fetus within a human female uterus was obtained during 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 iPS cells were then seeded onto the denuded hAM and cultured in serum-free KSR (Invitrogen Corp., Carlsbad, CA)-containing Glasgow-MEM (Invitrogen) medium supplemented with selective rho kinase (ROCK)-inhibitor Y-27632 at 37°C and under 5% CO2.
Dissociated human iPS cells formed large colonies and differentiated into neural precursors at high efficiency when cultured on the denuded hAM in serum-free medium containing ROCK-inhibitor Y-27632. AMED-induced human iPS cells developed to retinal pigment epithelia and lentoid tissues. Furthermore, AMED-induced epithelial cells were found to be positive for Pax6, cytokeratin (CK) 3, and CK 12, consistent with the characteristics of corneal epithelia. The percentage of human iPS cell-derived CK 12-positive colonies increased to become up to 25% of the total colonies on the denuded hAM. Moreover, qPCR showed an increase of TGFb, CLU, ALDH3, CK 12, and CK 3 in the AMED-treated cells, consistent with the characteristics of corneal epithelia and progenitor cells.
AMED should provide a highly practical system for generating corneal epithelia from human iPS cells without immune rejection or ethical problems for clinical application.
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