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FIGURE 1 Limbal stem sell deficiency and stem cell transplantation in humans, and stem cell deficiency and corneal restoration in Xenopus.(a) Example of a human cornea with total limbal stem cell deficiency after receiving a chemical burn with involvement of the corneal stroma.Corneal opacification (black arrows) and neovascularization (black arrowheads) is apparent. (b) Example of another patient who received acultivated autologous limbal stem-cell graft, and penetrating keratoplasty (in some cases, this secondary corneal transplant from an allogeniccornea is necessary for improving visual acuity), showing successful restoration of a transparent corneal epithelium (black arrow). Vascularizationis now restricted to the conjunctiva (black arrowheads). Dotted black circle outlines the cornea region. (c–e) PUV treatment model of stem celldeficiency in Xenopus recapitulates the hallmarks of human limbal stem cell deficiency. (c) Control frog cornea depicting normal transparentmorphology. The cornea is free of vasculature, pigment cells, and the lateral line organs (white arrows) are restricted to the surrounding skin.(d) PUV treated cornea at 32 days post treatment (dpt), depicting the loss of transparency. The cornea is covered with epidermal tissue similar tothat surrounding the eye. Skin cells, including pigment cells, and the lateral line organs (white arrows), have invaded the corneal region. Note linesof melanophores between the lateral line organs. (e) Younger PUV treated case at 15dpt, depicting vascularization with multiple blood vesselspassing into the cornea (white arrowheads). (f–h) PUV treatment localized to one half of the Xenopus cornea is eventually restored. (f) At 15 dayspost PUV treatment, the treated half shows opacity and increased pigmentation (white arrows), whereas the untreated half is devoid of pigmentcells and remains transparent. (g) Dorsal view of the specimen shown in (f) shows neovascularization (white arrowheads), and increasedpigmentation (white arrows). (h) At 28 days post PUV treatment, the treated half of the cornea has been restored. It is now transparent and freefrom pigment cells. an, anterior side; dr, dorsal side; ps, posterior side; vr, ventral side. Scale bar in (e) equals 665 m for (c,d), and 200 m for (e).Scale bar in (h) equals 500 m for (f–h). Image in (a) is from Sacchetti et al. (2018), Limbal Stem Cell Transplantation: Clinical Results, Limits, andPerspectives, Copyright © (2018), open access article distributed under the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/). Image in (b) is from Paolo, R., Matuska, S., Paganoni, G., et al. Limbal stem-cell therapy and long-termcorneal regeneration, The New England Journal of Medicine, 363(2), 147–155. Copyright © (2010) Massachusetts Medical Society, Reprinted withpermission from Massachusetts Medical Society. Images in (c,e–h) are reprinted from Adil, M. T., Simons, C. M., Sonam, S., Henry, J. J.Understanding cornea homeostasis and wound healing using a novel model of stem cell deficiency in Xenopus, Experimental Eye Research, 187,107767. Copyright (2019), with permission from Elsevier
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IGURE 2 Wound healing and cell tracking in the mousePAX6-GFP model. (a,c,e) Images from time-lapse confocal microscopyshowing healing of a 1 mm diameter central wound in a PAX6-GFPmosaic corneal epithelium (the white areas are GFP-positive). Thewound completely closes by 18.75 hr (h) after wounding. (b,d,f)Healing of a peripheral wound showing closure and the formation of asecondary whorling pattern. By 18.75 hr this wound has not closedcompletely. Dotted circles show the extent of the wounds. (g,h) Highmagnification views and graphical representation detail the healing ofa central wound in a PAX6-GFP cornea. (g) View of the initial 1 mmwound and the future routes taken by tracked cell stripes (numbered1–6). (h) After 18 hr of healing the wound is completely closed, butthe stripes do not meet at the center of the original wound, and donot extend equally. Scale bars in (a)–(f) equal approximately 1 mm.Scale bar in H equals 500 m and applies to (g) and (h). Images arefrom Mort et al. (2009), Mosaic analysis of stem cell function andwound healing in the mouse corneal epithelium, Copyright © 2009,Mort et al; licensee BioMed Central Ltd., open access articledistributed under the Creative Commons Attribution 4.0 InternationalLicense 4.0 (http://creativecommons.org/licenses/by/4.0/)
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FIGURE 3 Lineage tracing in mice and zebrafish corneas. (a,b) Lineage tracing in CAGG driven LacZ mice. Eyes were stained for β-gal activityafter a 5 week (wk) chase period. (a) shows small β-gal positive patches throughout the cornea and conjunctiva. (b) After a 14 week chase periodstripes extend radially toward the center of the cornea. (c–g) Long-term clonal analyses in the zebrafish cornea expressing the “Zebrabow”construct. Recombination was induced at 6–12 hr post fertilization for expression of different fluorescent proteins, and images taken at the timepoints indicated. (c,d) Corneal clones at 10 and 15 days post fertilization (dpf). (e,e0) High magnification views near the periphery of the corneataken from (c) and (d), respectively. Labeled cornea clones near the periphery become scattered and interspersed by smaller cell clones. (f,f0) Highmagnification views near the center of the cornea taken from (c) and (d), respectively. Rosette-like structures are seen in the central cornea(compare f with f0, circle marks a rosette). (f00) Optical cross-section at the center of the rosette shown in (f0). A single blue cell has been extrudedfrom the corneal surface (arrowhead). (g) Example of well-established wedge-shaped clones in an older juvenile fish. Scale bar in (a) equals 0.5 mmfor (a,b). Scale bars equal 100 m in (c,d); 50 m in (e,e0,f,f0,f00); 200 m in (g). Images in (a,b) are from Dora et al. (2015), Lineage tracing in theadult mouse corneal epithelium supports the limbal epithelial stem cell hypothesis with intermittent periods of stem cell quiescence, Copyright ©2015, Dora et al., published by Elsevier, open access article distributed under the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). Images in (c–g) are from Pan et al. (2013), and reproduced with permission from Development, Copyright© (2013), The Company of Biologists Ltd
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FIGURE 6 Response of cornea cells during homeostasis and following localized PUV treatment in Xenopus.(a–a00) Control transgenic corneasthat did not receive PUV treatment, showing unlabeled cornea immediately before localized heat-shock (a), and corneas showing labeled nuclei at1 day (a0) and 7 days post heat-shock (a00). Over time, the labeled cluster of cells has become somewhat elongated. (b–b00) Corneas showing theinitial location of heat-shock labeled nuclei (b), and their rapid displacement toward the treated side of the cornea 1 day after localized PUVtreatment (b0). These labeled nuclei presumably divide quickly to dilute the fluorescence by 3dpt as fewer labeled nuclei can be seen (b00, andinset). (c–c00) Another example showing displacement of heat-shock labeled nuclei from 1 to 3dpt. This particular specimen has extensive leakybackground expression from the hsp70 promoter in deeper cells of the iris/retina. Nevertheless, the cluster of heat-shock labeled cornea cells isdistinctly detectable, and has become displaced to the PUV treated side by 3dpt. Note that the dotted area gets smaller as the labeled nuclei aredisplaced and undergo mitosis. (d) Excised flat mount of a cornea that received PUV treatment on one half and heat-shock to label the nuclei incells on the untreated half. Labeled nuclei are present in both halves at 1dpt. (d0,d00) Magnified images of the boxed areas in (d). Dotted whitevertical line passes through the center of the cornea. White dotted enclosed area indicates heat-shock activated H2B-mCherry expressing nuclei.dphs, days post heat-shock; dpt, days post treatment; HS, heat-shock; PUV, psoralen UV treatment. Number of cases examined (N) = 6 for controlcases; and for PUV treated cases, N = 19 for 1dpt, 9 for 2dpt, 10 for 3dpt. Scale bar in (d00) equals 50 um for (d0,d00), 100 um for (d), and 200 mfor (a–c00
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FIGURE 7 Response of cornea cells at later time points after localized PUV treatment in Xenopus. (a) Schematic of experimental outline.Specimens received PUV treatment on one half of the cornea. At 2, 6, 11, and 15dpt, specimens received heat-shock to a cluster of cells on theuntreated half of the corneas, and representative mCherry images are shown at various time points, as indicated (N = 11 for time points incolumns i–iii; N greater than or equal to 8 for time points in column iv). (b) Corneas showing rapid displacement of nuclei that were labeledinitially at 2dpt. (c,d,e) Corneas showing minimal movement in the nuclei that were labeled at 6, 11, and 15dpt, respectively. Dotted white verticalline passes through the center of the cornea. White dotted enclosed area indicate clusters of heat-shock activated H2B-mCherry expressingnuclei. dpt, days post treatment; PUV, psoralen UV treatment. Scale in (e column iv) equals 200 um
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FIGURE 8 Response of labeled cells to increased UV exposure during localized PUV treatment in Xenopus.(a–d) Typical example showingnuclei labeled at 15dpt in the untreated half of the cornea in specimens that received PUV treatment in the other half. (see data in Appendix 3g).Representative mCherry images are shown at the time points indicated after PUV treatment. Labeled nuclei show minimal movement from 16 to28/30dpt. (a0–d0) Corresponding brightfield images of the specimen in (a–d) at respective time points. A similar response was seen as compared tocases show in Figure 7e above. Dotted white vertical line passes through the center of the cornea. White dotted enclosed area indicates clusterof heat-shock activated H2B-mCherry expressing nuclei. dpt, days post treatment. Scale in (d0) equals 200 m
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