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Satoru Yoshida, Miyuki Yasuda, Hideyuki Miyashita, Tetsu Yoshida, Kazuo Tsubota, Hideyuki Okano, Shigeto Shimmura; Generation Of Stratified Squamous Epithelial Cells From Induced Pluripotent Stem Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5138.
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© ARVO (1962-2015); The Authors (2016-present)
Application of induced pluripotent stem (iPS) cells to regenerative medicine is expected to bypass the problems such as immunological rejection and ethical objection. To apply iPS cells to corneal epithelial disorders, we transplanted iPS-derived epithelial cells to mice cornea to observe stratification.
Differentiation of murine iPS cells into epithelial cells was performed by SDIA-method with use of BMP. After several subcultures, we obtained K14+ clones. In case of human IPS cells, epithelial differentiation was progressed on Matrigel-coated dish in presence of retinoic acid and BMP. Stratified epithelial sheets were engineered from iPS-derived K14+ epithelial cells by air-lifting culture with feeder cells in SHEM. To examine differentiation on mouse cornea, labeled cells were transplanted on denuded mouse cornea. The eyes were excised after engraftment and cultured ex vivo. For characterization of the cells, expression of several cytokeratins was examined.
Based on the expression of stratified epithelial marker K14, we found derivation of epithelial cells from murine iPS was most effective when BMP was added during days 3-5. Cells positive for early ectodermal marker K18, and K18, K14 double-positive cells were also found. The expression of K18 was down-regulated along with the progression of differentiation by air-lifting culture. Clusters of the cells positive for corneal epithelial marker K12 were also found in K14+ colonies but K12+ cells disappeared during subculture. In the 3D sheet, high levels of expression of p63 and K15 were found in basal layer. Proliferation of the cells transplanted onto denuded mouse cornea was found ex vivo when cultured in serum containing-medium. Expression patterns of those markers in stratified epithelium formed on the denuded cornea were quite similar with that in the cultivated sheet on culture insert.
Theses results suggest that the culture methods were useful to induce K18, K14 double-positive immature cells, which are available to produce polarized stratified cell sheets.
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