Volume 22, Issue 2, Pages 323-331 (January 2018) Corneal-Committed Cells Restore the Stem Cell Pool and Tissue Boundary following Injury  Waseem Nasser, Aya Amitai-Lange, Despina Soteriou, Rana Hanna, Beatrice Tiosano, Yaron Fuchs, Ruby Shalom-Feuerstein  Cell Reports  Volume 22, Issue 2, Pages 323-331 (January 2018) DOI: 10.1016/j.celrep.2017.12.040 Copyright © 2017 The Authors Terms and Conditions

Cell Reports 2018 22, 323-331DOI: (10.1016/j.celrep.2017.12.040) Copyright © 2017 The Authors Terms and Conditions

Figure 1 K15-GFP Defines the Corneal Boundary and SC Niche (A) Schematic representation of the K15-GFP transgenic construct (Fuchs et al., 2013; Morris et al., 2004). (B–H) Characterization of the K15-GFP+ cell population in 8-week-old mice. Enucleated eye, K15-GFP+ cells are marked by arrows (B). Enlarged magnification of the limbus (C) shown in (B). Whole-mount immunostaining of K15-GFP corneas against corneal marker K12 (D), conjunctival marker K8 (E), and both markers (F). z stack images of whole-mount K15-GFP corneas (G). Arrowheads and arrows mark small size basal K15-GFP+ LSC-like cells and large limbal superficial cells, respectively. Immunostaining of the indicated markers on paraffin corneal sections (H). (I) Schematic representation of the three transgenes. (J) Potential outcome of Confetti reporter rearrangement and expression in a specific cellular compartment. (K) Lineage tracing was induced by tamoxifen injection in 2-month-old mice, and eyes were visualized 3 months post-induction. K15-GFP+/RFP+-yellow-labeled cells are annotated by arrows. Cj, conjunctiva; ep, epithelium; st, stroma. Asterisks indicate nonspecific auto-fluorescence of the lens (∗Le). Scale bars, 50 μm. Cell Reports 2018 22, 323-331DOI: (10.1016/j.celrep.2017.12.040) Copyright © 2017 The Authors Terms and Conditions

Figure 2 Recovery of K15-GFP+ and the Limbal Boundary following Surgical Removal of the Limbus (A–G) The limbal epithelium, marginal conjunctiva, and marginal cornea of 2- to 3-month-old mice was removed by surgery (n = 21 eyes). Fluorescent image of injured eyes at the indicated days post-injury (A, top). The K15-GFP signal is annotated by arrowheads. Fluorescein staining indicates epithelial barrier function (A, middle). Histology (H&E) from sacrificed animals at the indicated time points is shown (A, bottom). Quantification of K15-GFP signal (normalized mean ± SD) at the indicated days post-injury (B). Bright-field image of recovered eye at day 60 post-surgery (C). Quantification of eyes with normal corneal transparency and K15-GFP signal on day 30 (D). Whole-mount immunostaining of K15-GFP corneas against corneal (K12) (E). Immunostaining of K12 in corneal paraffin sections before limbal deletion and at day 10 post-injury (F). A 2-month-old triple-transgenic (K15-GFP;K14CreERT;R26R-Confetti) mouse was induced to express Confetti reporters by tamoxifen treatment and underwent limbal epithelial removal (G). The image (taken 2 months post-injury) shows that recovering K15-GFP+ cells formed a normal pattern of limbal radial stripes. Arrow shows RFP+/K15-KFP+ cells at the margin of the RFP+ stripe. ep, epithelium; st, stroma. Scale bars, 50 μm. Cell Reports 2018 22, 323-331DOI: (10.1016/j.celrep.2017.12.040) Copyright © 2017 The Authors Terms and Conditions

Figure 3 Corneal-Committed Cells Repair the Limbal SC/Boundary Compartment (A–F) Surgical removal of the entire limbus and corneal epithelium was performed (n = 3 eyes). Fluorescent microscopy confirmed that no K15-GFP signal remained after surgery (A). Fluorescein staining and histology (H&E) confirmed complete epithelial removal in the injured area (B). Bright-field image showing that thin vessels (arrows) invaded the cornea which became opaque on day 30 (D30) post-surgery (C). Enlarged region (D) shown in (C). H&E staining indicating the presence of goblet cells (arrows) in the corneal epithelium and blood vessels (arrowheads) in the corneal stroma (E). Immunofluorescent staining showing loss of corneal identity marker K12 (F). (G and H) 2-month-old triple-transgenic (K15-GFP;K14CreERT;R26R-Confetti) mice were induced to express Confetti reporters, and limbal removal was performed (yellow arrowheads). For accurate stripe tracking over time, eyes with 1–2 RFP+ stripes were followed (n = 5 eyes). RFP+ corneal-committed cells repaired the limbus by day 1 and re-expressed K15-GFP in a fuzzy pattern (day 10) that became concise by day 30 (G). Merge of bright-field and fluorescent microscopy showing that RFP+ stripe also initially repaired the conjunctiva (day 2, arrow) but became restricted to the cornea/limbus (days 3 and 10, arrows) (H). (I) Model describing tissue recovery following SC/boundary destruction. Cj, conjunctiva; Co, cornea; ep, epithelium; L, limbus; st, stroma. Asterisks indicate nonspecific auto-fluorescence of the lens (∗Le). Scale bars, 50 μm. Cell Reports 2018 22, 323-331DOI: (10.1016/j.celrep.2017.12.040) Copyright © 2017 The Authors Terms and Conditions

Figure 4 Limbal Niche Destruction by Chemical Agent Prevents Corneal Recovery (A–G) A demarcated damage to the limbal epithelium and underlying stroma was applied by NaOH (0.5 M, 5 s) using a sharpened (0.5 mm) ophthalmic brush. Limbal epithelial/stromal damage was validated by fluorescent microscopy, fluorescein staining, and histology (H&E); note that limbal stromal cells were lost (arrow), while neighboring corneal epithelium/stroma remained intact (arrowhead) (A). Enucleated eye showing the complete removal of K15-GFP-expressing cells (B). Severe opacification and neovascularization was found on days 30 and 50 post-treatment with no K15-GFP signal (C). Quantification percentage of eyes with corneal transparency and K15-GFP signal (n = 8 eyes) (D). H&E performed on day 30 following injury showing the presence of goblet cells (arrows) in the pathological corneal epithelium (E). Immunofluorescent staining showing loss of K12 on day 30 post-injury (F). Lineage tracing was induced by tamoxifen injection to K14-CreERT2;R26R-Confetti mice, and limbal alkali injury was applied the next day (G). Injured eyes of individual mice were followed and imaged under anesthesia at the indicated time points following injury. Note that a severe LSCD phenotype and conjunctival cell ingrowth (radial stripes) coincided with pathological deterioration (compare days 8–15). ep, epithelium; st, stroma. Arrowheads indicate K15-GFP+ cells. Asterisks indicate nonspecific auto-fluorescence of the lens (∗Le). Scale bars, 50 μm. Cell Reports 2018 22, 323-331DOI: (10.1016/j.celrep.2017.12.040) Copyright © 2017 The Authors Terms and Conditions