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Co-ordinated ocular development from human iPS cells and recovery of corneal function


The eye is a complex organ with highly specialized constituent tissues derived from different primordial cell lineages. The retina, for example, develops from neuroectoderm via the optic vesicle, the corneal epithelium is descended from surface ectoderm, while the iris and collagen-rich stroma of the cornea have a neural crest origin. Recent work with pluripotent stem cells in culture has revealed a previously under-appreciated level of intrinsic cellular self-organization, with a focus on the retina and retinal cells1,2,3,4,5. Moreover, we and others have demonstrated the in vitro induction of a corneal epithelial cell phenotype from pluripotent stem cells6,7,8,9. These studies, however, have a single, tissue-specific focus and fail to reflect the complexity of whole eye development. Here we demonstrate the generation from human induced pluripotent stem cells of a self-formed ectodermal autonomous multi-zone (SEAM) of ocular cells. In some respects the concentric SEAM mimics whole-eye development because cell location within different zones is indicative of lineage, spanning the ocular surface ectoderm, lens, neuro-retina, and retinal pigment epithelium. It thus represents a promising resource for new and ongoing studies of ocular morphogenesis. The approach also has translational potential and to illustrate this we show that cells isolated from the ocular surface ectodermal zone of the SEAM can be sorted and expanded ex vivo to form a corneal epithelium that recovers function in an experimentally induced animal model of corneal blindness.

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Figure 1: Differentiation of multiple ocular cells in the SEAM.
Figure 2: Characterization of cell zones in the SEAM.
Figure 3: Induction and isolation of ocular surface epithelial cells from the SEAM.
Figure 4: Characterization and surgical use of human iPS cell-derived corneal epithelial cells (iCECs).

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We thank K. Baba, Y. Oie, H. Takayanagi, S. Hara, Y. Yasukawa, J. Toga and M. Yagi of Osaka University and M. Nakagawa of Kyoto University for technical assistance and scientific discussions. 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), The Japan Science and Technology Agency (JST) and The Ministry of Health, Labour, and Welfare of Japan and the Grants-in-Aid for Scientific Research from The Ministry of Education, Culture, Sports, Science and Technology of Japan.

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Authors and Affiliations



R.H., M.T. and K.N. designed the research; R.H, Y.I., R.K. and S.A. performed the in vitro experiments and acquired the data; Y.S., N.N., T.I. and T.S. performed animal experiments and acquired the data; K.S. provided reagents (LN511E8); R.H., Y.I. and R.K. analysed the data and wrote the respective methods and results; S.K., K.S. and A.J.Q. supervised the project; and R.H., M.T., A.J.Q. and K.N. wrote the paper.

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Correspondence to Kohji Nishida.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Differentiation of multiple ocular cells in the SEAM.

a, A differentiated human iPS cell colony after 40 days of culture (macro photograph, representative of six independent experiments). Scale bar, 5 mm. b, Magnified views of each SEAM zone margin after six weeks in culture (phase-contrast and bright-field views, each representative of three independent experiments). Arrow heads indicate borders between each zone. Scale bars, 50 μm. c, Schematic for the development of the anterior eye. CE, corneal epithelium; CEnd, corneal endothelium; IS, iris stroma; NR, neuroretina; RPE, retinal pigment epithelium; LE, lens. d, Immunostaining for p75 (green) and SOX10 (red) in SEAM zones 1 and 2, two weeks (w) after the start of the differentiation culture (representative of three independent experiments). Asterisks indicate SOX10+/p75+ neural crest cells. Nuclei, blue. Scale bar, 100 μm. e, Immunostaining of lens differentiation marker α-crystallin (green) and epithelial marker p63 (red) in zones 2 and 3 of the SEAM after six weeks of culture (representative of three independent experiments). Nuclei, blue. Scale bar, 100 μm.

Extended Data Figure 2 Enrichment and differentiation of a SEAM-derived ocular surface epithelium.

a, The enrichment of zone 3 ocular surface epithelium by manual pipetting. Cells in the SEAM before (upper panel) and after (lower panel) pipetting are shown (n = 1). Scale bar, 200 μm. b, The expression of ocular-cell-related genes in removed and attached cells after pipetting (n = 1). c, The time course of PAX6, DN-p63, K14, K12 and LIN28A expression during human iPS cell differentiation culture (0, 2, 4, 6, 8 and 12 weeks, each n = 5 independent experiments). Error bars are s.d. d, Immunostaining for vimentin (VIM, green) and p63 (red) before and after FACS purification conducted at 11 weeks of culture (representative of three independent experiments). VIM+ stroma-like cells of zone 3 were removed by FACS. Nuclei, blue. Scale bar, 50 μm. e, Immunostaining for anterior eye development-related markers Pax6 (green), p63 (red), K14 (green) and K12 (red) during mouse eye development (E9.5–18.5, each representative of three experiments). Nuclei, blue. Asterisks indicate p63-expressing cells. PCE, presumptive corneal epithelium; OSEpi, ocular surface epithelium; CE, corneal epithelium; CS, corneal stroma; LV, lens vesicle; LE, lens; AC, anterior chamber; EL, eyelid; OV, optic vesicle. Scale bar, 50 μm.

Source data

Extended Data Figure 3 The effect of BMP/TGFβ inhibitors on the development of ocular surface epithelium in the SEAM.

a, Microscopic observation of the SEAM pre-treated with Noggin, LDN-193189 (LDN) or SB-431542 (SB) for four days at the start of differentiation culture (control (−), Noggin and LDN data are representative of six independent experiments, while SB data are representative of four independent experiments). Both BMP and TGFβ inhibitors resulted in the abolishment of zone 3. Scale bar, 200 μm. b, The effects of BMP or TGFβ inhibitors on the expression of ocular ectoderm-related genes at five to six weeks of differentiation culture (*P < 0.05, control; n = 13: Noggin, LDN and SB; n = 8 independent experiments, respectively, Mann–Whitney test). Error bars are s.d.

Source data

Extended Data Figure 4 Isolation of corneal epithelial cells from the SEAM.

a, Schematic for the induction of ocular surface epithelium from human iPS cells. The ocular surface ectoderm expressing PAX6 and p63 in zone 3 of the SEAM further differentiated into functional ocular surface epithelial stem cells that expressed K14. Among the ocular surface epithelial lineage cells, corneal epithelial progenitor cells were isolated by FACS as SSEA-4+/ITGB4+ cells. Conjunctival epithelial cells (PAX6+/K13+/K12) were obtained as SSEA-4 cells. NE, neuroectoderm; NR, neuroretina; NC, neural crest; OSE, ocular surface ectoderm; SE, surface ectoderm. b, Immunostaining for SSEA-4 (green) and K12 (red) in stratified epithelial tissues, including human ocular surface epithelium (cornea, limbus and conjunctiva), epidermis and oral mucosa. A magnified view of the limbus (highlighted box) indicated that SSEA-4 was expressed in all layers of the limbal epithelium, including the basal layer, which had no K12 (asterisks). No other stratified epithelium expressed SSEA-4 and K12. Nuclei, blue. Scale bars, 50 μm. (Data for corneal, limbal and conjunctival tissue; n = 3; epidermis and oral mucosa; n = 1). c, Immunostaining for PAX6 (green) in colonies derived from P3 and P2 cells (representative of five independent cell-sorting experiments). Nuclei, blue. Scale bar, 100 μm. d, The expression of corneal epithelial-specific genes and non-corneal epithelial genes in the sorted P3 cells (that is, human iPS cell-derived corneal epithelial cells, iCECs) and P2 cells. *P < 0.05 (n = 7 independent cell sorting experiments, Mann–Whitney test). Error bars are s.d.

Source data

Extended Data Figure 5 Characterization of SEAM-derived ocular surface epithelial cells.

a, Triple colour immunostaining for PAX6 (green), K12 (orange) and K13 (magenta) in the epithelial colonies from sorted P3 cells (that is, SEAM-derived human iCECs) and P2 cells (representative of five independent cell-sorting experiments). Nuclei, blue. Scale bars, 100 μm. b, HOX gene expression in the sorted P3 cells, P2 cells, human epidermal keratinocytes (EKs) and human corneal limbal epithelial cells (CECs). (P3 and P2 cells; n = 7: human EKs and human CECs; n = 5 independent experiments). Error bars are s.d. c, The PAX6 (green) and HOXB4 (red) expression in the colonies of P2 cells (n = 1). Nuclei, blue. Scale bar, 100 μm. d, Goblet-cell-like differentiation in the SEAM-derived epithelium after long-term culture (more than 12 weeks of differentiation) without FACS in CEM (n = 1). Goblet-cell-like morphology was observed in presumptive P2 cell regions (left panel). These cells were PAS-positive and expressed the goblet cell markers MUC5AC (green) and K7 (red) in the superficial region and PAX6 in the basal region. Nuclei, blue. Scale bars, 50 μm.

Source data

Extended Data Figure 6 Characterization of the SEAM-derived corneal epithelium.

a, Immunostaining for K19 and CX43 (green) in the stratified SEAM-derived human iCECs (representative of n = 3 independent experiments). Magnified view of the dotted area is shown in the lower panel. Nuclei, red. Scale bars, 50 μm. b, Scanning electron microscopy of the apical surface of the stratified human iCECs (representative of two human iCEC sheets) and human CECs (n = 1). Scale bars, 10 μm (upper panels) and 1 μm (lower panels). c, Results of a hierarchical cluster analysis based on the global gene expression as examined by microarrays. Data are shown for human iPS cells (n = 3 technical replicates), human iPS cell-derived ocular surface ectoderm (OSE; that is, human iPS cell-derived cells after six weeks of differentiation, n = 3 technical replicates), human iCECs (n = 4 independent experiments), human oral keratinocytes (OKs, n = 3 technical replicates), human dermal fibroblasts (DFs, n = 3 technical replicates) and human corneal epithelial cells obtained from the limbus (CECs, n = 3 independent experiments). A total of 25,262 significantly changed genes (fold change >2.0, false discovery rate <0.05) were analysed. d, SEAM-derived human iCECs at passage (P) 1, 3, 9 and 15 during serial passages (representative of three independent experiments). Scale bar, 200 μm. e, The G-band karyotype of human iCECs at P = 1, 6 and 12 (n = 1, respectively).

Extended Data Figure 7 Induction of corneal epithelial cells from different human iPS cell clones.

a, SEAM formation patterns of five different human iPS cell clones (201B7 and 1383D2; representative of three independent experiments: 253G1, 454E2 and 1231A3; n = 1). PBMC, peripheral blood mononuclear cell; TF, transcription factor. Scale bar, 200 μm. b, Stratified human iCECs from 454E2, 253G1, 1231A3 and 1383D2 clones in vitro (454E2, 253G1 and 1231A3, n = 1; 1383D2, representative of three independent experiments). Phase-contrast microscopy (upper panel), haematoxylin and eosin (H&E) staining (middle panel) and immunostaining for K12 (lower panel, green) are shown. Nuclei, red. Scale bars, 200 μm (phase-contrast), 50 μm (H&E staining and immunostaining). c, Efficiency of corneal epithelial differentiation of the various human iPS cell clones (201B7, n = 23; 253G1, n = 5; 454E2, n = 11; 1231A3, n = 9 and 1383D2, n = 6 independent experiments). Error bars are s.d.

Source data

Extended Data Figure 8 Transplantation of the human iCEC sheet to repair the ocular surface.

a, Phase-contrast microscopy of SEAM-derived human iCECs cultivated on a temperature-responsive dish at 1, 5 and 14 days (representative of three independent experiments). A harvested human iCEC sheet is shown in the lower right panel. Scale bar, 200 μm (phase-contrast), 5 mm (macro photo). b, Immunostaining for PAX6 (green), K12 (orange) and K14 (magenta) in the human iCEC sheets after 21 days in culture (left panels, representative of three human iCEC sheets). The right panel shows an image used for a colony-forming assay (CFA) for the human iCEC sheets (15,000 cells per well, representative of eight independent experiments). The colony-forming efficiency (CFE) was 1.04 ± 0.43% (s.d., n = 8 independent experiments). Nuclei, blue. Scale bars, 100 μm (immunostaining) and 5 mm (CFA). c, Treatment of rabbit ocular surface with a surgical swab soaked in 99% ethanol for 30–60 s. d, e, Elimination of corneal epithelial stem cells by the surgical removal of corneal and limbal epithelial tissue (that is, lamellar keratectomy). f, The ocular surface was invaded by conjunctival tissue and vessels at postoperative day 28. g, Removal of the conjunctival tissue that covered the ocular surface. h, Widespread fluorescein staining of the ocular surface after surgical removal of epithelial tissue. i, The harvest of a human iCEC sheet from a temperature-responsive dish after lowering the temperature. j, Transplantation of the human iCEC sheet onto the rabbit cornea with a surgically induced corneal epithelial stem-cell deficiency treated as described in ch. k, H&E staining for human iCEC-sheet-transplanted and control corneas on postoperative day 14 (n = 6 animal transplantation experiments). Scale bar, 100 μm.

Supplementary information

Supplementary Information

This file contains Supplementary Tables 1-3. (PDF 212 kb)

Differentiating hiPSCs forming a SEAM

A hiPSC colony shown throughout the first 25 days of culture in differentiation medium spontaneously generating a concentric SEAM and its multiple cellular zones. (MP4 11491 kb)

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Hayashi, R., Ishikawa, Y., Sasamoto, Y. et al. Co-ordinated ocular development from human iPS cells and recovery of corneal function. Nature 531, 376–380 (2016).

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