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M. Ueno, Y. Nakai, M. Matsumura, M. Takahashi, Y. Sasai, S. Kisnoshita; Induction of Ocular Tissues From Human Pluripotent Stem Cells on the Amniotic Membrane Matrix. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3142.
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© ARVO (1962-2015); The Authors (2016-present)
Driving human pluripotent stem cells (PS cells), including embryonic stem (ES) cells and induced pluripotent stem (iPS) cells, to differentiate into ocular tissues is a key technology for the ophthalmological application of regenerative medicine. We previously reported a differentiation technique using the matrix components of the human amniotic membrane (hereafter referred to as ‘‘denuded hAM’’) which induces the neural conversion of mouse and human ES cells. The purpose of this present study was to investigate whether human PS cells differentiate to ocular tissues 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 Declarations 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. Human PS 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 .
Both human ES cells and human iPS cells formed large colonies and differentiated into neural precursors at an efficiency of approximately 80%, when cultured on the denuded hAM in the serum-free medium (amniotic membrane matrix-based ES cell differentiation or AMED). AMED-induced neural tissues developed to pigmented cells which were positive for Pax6 and showed actin bundles; consistent with the characteristic of retinal pigment epithelium. Light-reflecting lentoid tissues were also found in the culture. These tissues were alpha-crystallin positive, consistent with the nature of lens cells.
The AMED culture uses a non-cellular inductive material derived from an easily available human tissue. Thus, AMED should provide a highly practical and versatile system for generating ocular tissues from human ES cells and human iPS cells for clinical applications.
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