April 2011
Volume 52, Issue 14
Free
ARVO Annual Meeting Abstract  |   April 2011
Generation Of Stratified Squamous Epithelial Cells From Induced Pluripotent Stem Cells
Author Affiliations & Notes
  • Satoru Yoshida
    Department of Ophthalmology,
    Keio Univ School of Medicine, Shinjuku-ku, Tokyo, Japan
  • Miyuki Yasuda
    Department of Ophthalmology,
    Keio Univ School of Medicine, Shinjuku-ku, Tokyo, Japan
  • Hideyuki Miyashita
    Department of Ophthalmology,
    Keio Univ School of Medicine, Shinjuku-ku, Tokyo, Japan
  • Tetsu Yoshida
    Department of Ophthalmology,
    Keio Univ School of Medicine, Shinjuku-ku, Tokyo, Japan
  • Kazuo Tsubota
    Department of Ophthalmology,
    Keio Univ School of Medicine, Shinjuku-ku, Tokyo, Japan
  • Hideyuki Okano
    Department of Physiology,
    Keio Univ School of Medicine, Shinjuku-ku, Tokyo, Japan
  • Shigeto Shimmura
    Department of Ophthalmology,
    Keio Univ School of Medicine, Shinjuku-ku, Tokyo, Japan
  • Footnotes
    Commercial Relationships  Satoru Yoshida, None; Miyuki Yasuda, None; Hideyuki Miyashita, None; Tetsu Yoshida, None; Kazuo Tsubota, None; Hideyuki Okano, None; Shigeto Shimmura, None
  • Footnotes
    Support  Advanced and Innovational Research Program in Life Sciences from the Ministry of Education, Culture, Sports, Science and Technology
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 5138. doi:
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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)

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Abstract

Purpose: : 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.

Methods: : 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.

Results: : 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.

Conclusions: : 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.

Keywords: cornea: epithelium 
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