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Coordinated generation of multiple ocular-like cell lineages and fabrication of functional corneal epithelial cell sheets from human iPS cells

Nature Protocols volume 12, pages 683–696 (2017)Cite this article

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Abstract

We describe a protocol for the generation of a functional and transplantable corneal epithelium derived from human induced pluripotent stem (iPS) cells. When this protocol is followed, a proportion of iPS cells spontaneously form circular colonies, each of which is composed of four concentric zones. Cells in these zones have different morphologies and immunostaining characteristics, resembling neuroectoderm, neural crest, ocular-surface ectoderm, or surface ectoderm. We have named this 2D colony a 'SEAM' (self-formed ectodermal autonomous multizone), and previously demonstrated that cells within the SEAM have the potential to give rise to anlages of different ocular lineages, including retinal cells, lens cells, and ocular-surface ectoderm. To investigate the translational potential of the SEAM, cells within it that resemble ocular-surface epithelia can be isolated by pipetting and FACS sorting into a population of corneal epithelial-like progenitor cells. These can be expanded and differentiated to form an epithelial layer expressing K12 and PAX6, and able to recover function in an animal model of corneal epithelial dysfunction after surgical transplantation. The whole protocol, encompassing human iPS cell preparation, autonomous differentiation, purification, and subsequent differentiation, takes between 100 and 120 d, and is of potential use to researchers with an interest in eye development and/or ocular-surface regeneration. Experience with human iPS cell culture and sorting via FACS will be of benefit for researchers performing this protocol.

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Figure 1: Schematic illustration outlining the reconstruction of corneal epithelial tissue from human iPS cells via SEAM formation.
Figure 2: The protocol for SEAM formation and fabrication of corneal epithelial cell sheets.
Figure 3: Characterization of cells in a SEAM.
Figure 4: Isolation of corneal epithelial cells from a SEAM.
Figure 5: Fabrication of a corneal epithelial cell sheet.

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Acknowledgements

We thank Y. Kobayashi for technical assistance. This work was supported in part by the Project for the Realization of Regenerative Medicine of the Japan Agency for Medical Research and Development (AMED) (K.N.), as well as by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (R.H.). Corneal research at Cardiff University (A.J.Q.) is supported by the Biotechnology and Biological Sciences Research Council and the Medical Research Council.

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Authors and Affiliations

  1. Department of Stem Cells and Applied Medicine, Osaka University Graduate School of Medicine, Suita, Japan

    Ryuhei Hayashi

  2. Department of Ophthalmology, Osaka University Graduate School of Medicine, Suita, Japan

    Ryuhei Hayashi, Yuki Ishikawa, Ryousuke Katori, Yuzuru Sasamoto, Yuki Taniwaki, Hiroshi Takayanagi, Motokazu Tsujikawa & Kohji Nishida

  3. Division of Matrixome Research and Application, Institutes for Protein Research, Osaka University, Suita, Japan

    Kiyotoshi Sekiguchi

  4. Structural Biophysics Group, School of Optometry and Vision Sciences, College of Biomedical and Life Sciences, Cardiff University, Cardiff, UK

    Andrew J Quantock

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  1. Ryuhei Hayashi
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Contributions

R.H. and K.N. designed the research; R.H., Y.I., R.K., and H.T. performed the experiments on human iPS cell differentiation culture and acquired the data; Y.T. performed the experiments on human iPS cell maintenance in culture and acquired the data; Y.I. and R.K. performed the cell sorting experiments and acquired the data; R.H., Y.I., R.K., Y.T., and H.T. analyzed the data and provided technical assistance to the protocol; Y.S., M.T., and A.J.Q. supervised the project and provided critical advice; K.S. provided reagents (LN511E8); and R.H. and A.J.Q. wrote the manuscript.

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Correspondence to Ryuhei Hayashi or Kohji Nishida.

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The authors declare no competing financial interests.

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Hayashi, R., Ishikawa, Y., Katori, R. et al. Coordinated generation of multiple ocular-like cell lineages and fabrication of functional corneal epithelial cell sheets from human iPS cells. Nat Protoc 12, 683–696 (2017). https://doi.org/10.1038/nprot.2017.007

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