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Morio Ueno, Yoshinori Nakai, Michiru Matsumura, Yoshiki Sasai, Shigeru Kinoshita; Induction Of Ocular Tissues From Human-induced Pluripotent Stem Cells On The Amniotic Membrane Matrix. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3419.
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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 of pluripotent stem cells using the matrix components of the human amniotic membrane (denuded hAM) which induces the neural conversion of pluripotent stem cells including embryonic stem (ES) cells and iPS cells (amniotic membrane matrix-based ES cell differentiation, or AMED). The purpose of this present study was to investigate whether human iPS cells differentiate to ocular tissues, including corneal epithelia, on denuded hAM.
hAMs encasing the fetus within the human female uterus were 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 KSR (Invitrogen Corp., Carlsbad, CA)-containing Glasgow-MEM (Invitrogen) medium at 37°C 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 a selective 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 cytokeratin12 and Pax6; consistent with the characteristic of corneal epithelia. The percentage of human iPS cell-derived cytokeratin12-positive colonies increased to become up to 25% of the total colonies on denuded hAM.
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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