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

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.

Selective Laminin-Directed Differentiation of Human Induced Pluripotent Stem Cells into Distinct Ocular Lineages

“…*p < 0.05, **p < 0.01, and ***p < 0.001. viability varied by isoform, in all cases cells adhered, proliferated, and expressed pluripotency markers (Figures 1A and S1A-S1D). The typical four-zone SEAM structure seen previously, however, was only generated in the presence of LN511E8, the isoform employed in our published experiments ( Figure 1B) (Hayashi et al, , 2017. The other four isoforms, LN111E8, LN211E8, LN332E8, and LN411E8, supported the formation of SEAMs with distinct structural configurations.…”

“…Using E8 fragments of laminin-511 (LN511E8), we have generated hiPSC-derived colonies comprising four concentric cell zones, the emergence of which displays some similarities with ocular development (Hayashi et al, , 2017. We termed these types of colonies ''self-formed ectodermal autonomous multi-zones'' (SEAMs) of eye-like cells.…”

High-resolution promoter map of human limbal epithelial cells cultured with keratinocyte growth factor and rho kinase inhibitor

“…Furthermore, we discovered that CEC-specific PAX6 promoters are highly upregulated in LECs supplemented with KGF and Y-27632, which should be the key regulators of CEC differentiation and maintenance. We recently succeeded in inducing CECs from iPSCs cultured with KGF and Y-27632 11, 12 . Our findings provide strong evidence that LECs cultured with KGF and Y-27632 represent a better in vitro model for basic studies of CECs and could be widely utilized for translational research and clinical applications in regenerative medicine.…”

“…To overcome these problems, alternative CECs are differentiated from different sources such as ex vivo -expanded limbal epithelial cells (LECs) 6, 7 , oral mucosal epithelial cells 8, 9 , and induced pluripotent stem cell (iPSC)-derived CECs 10–12 . To make these alternative CECs equivalent to CECs in vivo , the culture system should be designed to maintain the characteristics of CECs in vivo .…”

Use of homeobox gene expression patterns to determine anatomical regions of origin for body surface tissues derived from adult mice

“…Ideally, the regenerated skin should be able to perform its original function and to develop appendages. We previously reported that progenitors of epidermal keratinocytes were generated from human induced pluripotent stem cells (PSCs) (Hayashi et al, , ). In addition, there have been some reports of keratinocyte lineages being induced from mouse (Bilousova & Roop, ) and human (Kogut, Roop, & Bilousova, ) induced PSCs.…”