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Nat Protoc. 2017 Apr;12(4):683-696. doi: 10.1038/nprot.2017.007. Epub 2017 Mar 2.

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

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Department of Stem Cells and Applied Medicine, Osaka University Graduate School of Medicine, Suita, Japan.
Department of Ophthalmology, Osaka University Graduate School of Medicine, Suita, Japan.
Division of Matrixome Research and Application, Institutes for Protein Research, Osaka University, Suita, Japan.
Structural Biophysics Group, School of Optometry and Vision Sciences, College of Biomedical and Life Sciences, Cardiff University, Cardiff, UK.


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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