Coordinated generation of multiple ocular-like cell lineages and fabrication of functional corneal epithelial cell sheets from human iPS cells
Ryuhei Hayashi, Yuki Ishikawa, Ryousuke Katori, Yuzuru Sasamoto, Yuki Taniwaki, Hiroshi Takayanagi, Motokazu Tsujikawa, Kiyotoshi Sekiguchi, Andrew J Quantock, Kohji Nishida
Department of Stem Cells and Applied Medicine, Osaka University Graduate School of Medicine, Suita, Japan.
Nature Protocols, VOL.12, NO.4, 2017
We describe a protocol for the generation of a functional and transplantable corneal epithelium derived from human induced pluripotent stem (iPSPS) cells. When this protocol is followed, a proportion of iPSPS 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 ‘SEASEASEAM’ (self-formed ectodermal autonomous multizone), and previously demonstrated that cells within the SEASEASEAM 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 SEASEASEAM, cells within it that resemble ocular-surface epithelia can be isolated by pipetting and FACSACSACS sorting into a population of corneal epithelial-like progenitor cells. These can be expanded and differentiated to form an epithelial layer expressing K12 and PAPAX6, and able to recover function in an animal model of corneal epithelial dysfunction after surgical transplantation. The whole protocol, encompassing human iPSPS 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 iPSPS cell culture and sorting via FACS will be of benefit for researchers performing this protocol.
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