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Delayed Development of Limbal Stem Cell Deficiency Following Chemical Injury—Pathogenesis and Therapeutic Strategies

US Ophthalmic Review, 2013;6(2):101–4 DOI:


Limbal epithelial stem cell deficiency (LSCD) occurs as a result of damage to the limbal epithelial stem cells (ESC) population. It may derive from direct destructive loss of the ESC (common chemical burn), and/or from dysfunction of the SC niche, leading to delayed death of the cells. This review focuses on delayed-onset LSCD, induced by antineoplastic chemicals, such as mitomycin C, 5-fluorouracil, and mustards, in terms of pathogenesis and management. These agents are used in ocular surface chemotherapy, in ocular surgery procedures, and as warfare agents, and target proliferating cells as slow-cycling cells, such as the ESC, are relatively resistant. Although the mechanism of the delayed loss of ESC is not entirely clear, we have shown, in the rabbit model, pathologic alterations in the limbal stroma, following the application of sulfur mustard, suggesting that dysfunction of the niche triggers the death of the SC later on. The absence of direct cytotoxic effects of these agents on the ESC, indicates a therapeutic window for prevention of the delayed LSCD.
Keywords: Ocular burns, chemical burns, cornea, epithelial stem cells, limbal stem cell deficiency, mustard, mitomycin, 5-fluorouracil
Disclosure: The authors have no conflicts of interest to declare.
Received: July 17, 2013 Accepted: August 12, 2013
Correspondence: Tamar Kadar, Department of Pharmacology, Israel Institute for Biological Research, Ness Ziona, 74100, Israel. E:

Homeostasis of corneal epithelium is essential for the maintenance of healthy ocular surface as well as for corneal transparency and accurate vision. Continuous renewal of corneal epithelium is provided by a population of adult stem/progenitor cells residing in the limbus, the transitional zone between the vascular conjunctiva, and the avascular transparent cornea.1–7 At the limbus, the corneal epithelial stem cells (ESC) reside within the basal layer of the epithelium. Although no single specific marker is available to identify stem cells (SC), a series of markers are used to characterize them. These include the expression of ABCG2, p63, or Notch-1, the absence of differentiation markers, such as CK3 and connexin-43, as well as morphologic criteria, such as small cell size (6–7 um) or high nucleus to cytoplasm ratio.8–14 The limbus differs from the central cornea in the organization of the epithelium and in the composition of the basement membrane and stroma. The distinctive characteristics of the two tissues are thought to play a role in the regulation of their respective populations of epithelial cells.15,16

Limbal ESC require a special environment to retain their SC properties. The environment is provided by the SC niche in which signaling from adjacent cells, as well as properties of the basal membrane, are believed to play a role in the maintenance of their ‘stemness.’7,12,14,17–19 The cells in the niche have been suggested to regulate the preservation, proliferation, and differentiation of the ESC by producing specific matrix components and secreting growth factors and signaling molecules in a tightly regulated spatial and temporal pattern.20,21 Consistently, the extracellular matrix composition of the limbus differs significantly from that of cornea and conjunctiva and specific cell surface receptors and adhesion molecules appear to mediate limbal ESC anchorage to their niche.

When limbal SC are depleted below a certain threshold, clinical signs of limbal epithelial stem cell deficiency (LSCD) appear, causing gradual vision loss.

LSCD occurs as a result of disease or damage to the limbal ESC population. Deficiency can arise from injuries, including chemical or thermal burns, and through diseases, such as Stevens Johnson syndrome and aniridia.22 It could be focal or diffuse depending on the extent of limbal involvement with underlying disease process. Due to the damage in the limbus, the barrier between the vascular conjunctiva and the avascular cornea is impaired and conjunctival epithelial cells migrate toward the corneal surface, accompanied by ingrowth of blood vessels. The clinical signs of LSCD, resulted from conjunctivalization of the cornea, include persistent epithelial defects, corneal vascularization, and chronic stromal inflammation leading to functional impairment and visual loss. Diagnosis of LSCD is based on the symptomatic hallmarks and is supported by identification of conjunctival goblet cells in the cornea, using impression cytology. Recently, the diagnosis of LSCD was supported by in vivo confocal microscopy.19,23 Typical characteristics of LSCD are shown in Figure 1.

LSCD may derive from destructive loss of limbal SC, and/or from dysfunction of the microenvironment of LSC, the SC niche, leading to insufficient support and death of the ESC.5,24,25 Chemical and thermal burns are the most common cause of a direct destruction of limbal SC. In contrast to the immediate loss of SC following a primary destruction, a gradual loss of the SC population with time characterizes the second category. The appearance of the LSCD symptoms in this case is delayed and takes months to years after the initial insult. Neurotrophic keratopathy and chronic limbitis are examples of delayed onset LSCD, deriving from poor nutritional supply of neuronal trophic factors, essential for the maintenance of the epithelium,26 and secretion of undesirable cytokines in the limbus in chronic limbitis.1,2,24

The present article focuses on delayed-onset LSCD induced by chemical agents. This less-familiar type of LSCD will be discussed in terms ofpathogenesis and management.

  1. Tseng SCG, Regulation and clinical implications of corneal epithelial stem cells, Molecul Biol Rep, 1996;23:47–58.
  2. Dua HS, Azuara-Blanco A, Limbal stem cells of the corneal epithelium, Sur Ophthalmol, 2000;44:415–25.
  3. Kinoshita S, Adachi W, Sotozono C, et al., Characteristics of the human ocular surface epithelium, Prog Ret Eye Res, 2001;20:639–73.
  4. Daniels JT, Dart JKG, Tuft SJ, Khaw PT, Corneal stem cells in review, Wound Repair Regen, 2001;9:483–94.
  5. Lavker RM, Tseng SCG, Sun TT, Corneal epithelial stem cells at the limbus: looking at some old problems from new angle, Exp Eye Res, 2004;78:433–46.
  6. Ang LPK, Tan DTH, Ocular surface stem cells and disease: current concepts and clinical applications, Ann Acad Med Singapore, 2004; 33:576–80.
  7. Notara M, Daniels JT, Biological principals and clinical potentials of limbal epithelial stem cells, Cell Tissue Res, 2008;331:135–43.
  8. Zhou S, Schuetz JD, Bunting KD, et al., The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype, Nature Med, 2001;7:1028–34.
  9. Chen Z, De Paiva CS, Luo L, et al., Characterization of putative stem cell phenotype in human limbal epithelia, Stem Cell, 2004;22:355–66.
  10. De Paiva CS, Chen Z, Corrales RM, et al., ABCG2 transporter identifies a population of clonogenic human limbal epithelial cells, Stem Cell, 2005;23:63–73.
  11. Pajoohesh-Ganji A, Stepp MA, In search of markers for the stem cells of the corneal epithelium, Biol Cell, 2000;97:265–76.
  12. Schlötzer-Schrehardt U, Kruse FE, Identification and characterization of limbal stem cells, Exp Eye Res, 2005;81:247–64.
  13. De Paiva CS, Pflugfelder SC, Li DQ, Cell size correlates with phenotype and proliferative capacity in human corneal epithelial cells, Stem Cell, 2006;24:368–75.
  14. Secker GA, Daniels JT, Corneal epithelial stem cells: deficiency and regulation, Stem Cell Rev, 2008;4:159–68.
  15. Wolosin JM, Xiong X, Schutte M, et al., Stem cells and differentiation stages in the limbo-corneal epithelium, Prog Retin Eye Res, 2000;19:223–55.
  16. Wolosin JM, Budak MT, Akinci M, Ocular surface epithelial and stem cell development, Int J De Biol, 2004;48:981–91.
  17. Dua HS, Shanmuganathan VA , Powell-Richards AO, et al., Limbal epithelial crypts: a novel anatomical structure and a putative limbal stem cell niche, Br J Ophthalmol, 2005;89:529–32.
  18. Shortt AJ, Secker GA, Munro PM, et al., Characterization of the limbal epithelial stem cell niche: novel imaging techniques permit in vivo observation and targeted biopsy of limbal epithelial stem cells, Stem Cell, 2007;25:1402–9.
  19. Ebrahimi M, Taghi-Abadi E, Baharvand H, Limbal stem cells in review, J Ophthal Vis Res, 2009;4(1):40–58.
  20. Sangwan VS, Limbal stem cells in health and disease, Biosci Reports, 2002;21:385–405.
  21. Espana EM, Kawakita T, Romano A, et al., Stromal niche controls the plasticity of limbal and corneal epithelial differentiation in a rabbit model of recombined tissue, Invest Ophthalmol Vis Sci, 2003;44:5130–35
  22. O’Callaghan AR, Daniels JT, Concise review: limbal epithelial stem cell therapy: controversies and challenges, Stem Cells, 2011;29:1923–32.
  23. Le O, Deng SX Xu J, In vivo confocal microscopy of congenital aniridia-associated keratopathy, Eye (Lond), 2013;27(6):763–6.
  24. Puangsricharern V, Tseng SCG, Cytologic evidence of corneal diseases with limbal stem cell deficiency, Ophthalmol, 1995;102(10):1476–85.
  25. Tseng SCG, Zhang SH, Limbal epithelium is more resistant to 5-fluorouracil toxicity than corneal epithelium, Cornea, 1995;14:394–401.
  26. Beuerman RW, Schimmelpfennig B, Sensory dennervation of the rabbit cornea affects epithelial properties, Exp Neurol, 1980;69:196–201.
  27. Morgan SJ, Chemical burns of the eye: causes and management, Br J Ophthalmol, 1987; 71:854–7.
  28. Hau S, Barton K, Corneal complications of glaucoma surgery, Curr Opin Ophthalmol, 2009;20:131–6.
  29. Wilson MW, Hungerford JL, George SM, Madreperla SA, Topical mitomycin C for the treatment of conjunctival and corneal epithelial dysplasia and neoplasia, Am J Ophthalmol, 1997;124:303–11.
  30. Sepulveda R, Peer J, Midena E, et al., Topical chemotherapy for ocular surface squamous neoplasia: current status, Br J Ophthalmol, 2010;94:532–5.
  31. Parrozzani R, Lazzarini D, Alemany-Rubio E, et al., Topical 1% 5 fluorouracil in ocular surface squamous neoplasia: a longterm safety study, Br J Ophthalmol, 2011;95:355–9.
  32. Nanji AA, Sayyad FE, Karp CL, Topical chemotherapy for ocular surface squamous neoplasia, Curr Opin Ophthalmol, 2013;24:336–42.
  33. Lichtinger A, Pe’er J, Fruch-Pery J, Solomon A, Limbal stem cell deficiency after topical mitomycin C therapy for primary acquired melanosis with atypia, Ophthalmology, 2010;117:431–7.
  34. Manche EE, Afshari MA, Singh K, Delayed corneal epitheliopathy after antimetabolite-augmented trabeculectomy, J Glaucoma, 1998;7:237–9.
  35. Pires RT F, Chokshi A, Tseng SCG, Amniotic membrane transplantation or conjunctival limbal autograft for limbal stem cell deficiency induced by 5-fluorouracil in glaucoma surgeries, Cornea, 2000;19:284–7.
  36. Sauder G, Jonas JB, Limbal stem cell deficiency after subconjunctival mitomycin C injection for trabeculectomy, Am J Ophthalmol, 2006;141:1129–30.
  37. Papirmeister B, Feister AJ, Robinson SI, Ford RD, Medical defense against mustard gas: Toxic mechanisms and pharmacological implications, Boca Raton, Fl.: CRC Press Inco, 1991.
  38. Solberg Y, Alcalay M, Belkin M, Ocular injury by mustard gas, Surv Ophthalmol, 1997;41:461–6.
  39. Banin E, Morad Y, Bernshtein E, et al., Injury induced by chemical warfare agents: characterization and treatment of ocular tissues exposed to nitrogen mustard, Invest Ophthalmol Vis Sci, 2003;44:2966–72.
  40. Morad Y, Banin E, Averbukh E, et al., Treatment of ocular tissues exposed to nitrogen mustard: beneficialeffect of zinc desferrioxamine combined with steroids, Invest Ophthalmol Vis Sci, 2005;46:1640–46.
  41. Etezad-Razavi M, Mahmoudi M, Hefazi M, Balali-Mood M, Delayed ocular complications of mustard gas poisoning and the relationship with respiratory and cutaneous complications, Clin Exp Ophthalmol, 2006;34:342–6.
  42. Baradaran-Rafii, Eslani M, Tseng SCG, Sulfur mustard induced ocular surface disorders, Ocular Surface, 2011;9:163–78.
  43. Kadar T, Turetz J, Fishbein E, et al., Characterization of acute and delayed ocular lesions induced by sulfur mustard in rabbits, Curr Eye Res, 2001;22:42–53.
  44. McNutt P, Hamilton T, Nelson M, et al., Pathogenesis of acute and delayed corneal lesions after ocular exposure to sulfur mustard vapor, Cornea, 2012;31(3):280–90.
  45. Javadi MA, Yazdani S, Sajjadi H, et al., Chronic and delayedonset mustard gas keratitis, Ophthalmol, 2005;112:617–25.
  46. Shohrati M, Peyman M, Peyman A, et al., Cutaneous and ocular late complications of sulfur mustard in Iranian veterans, Cutan Ocul Toxicol, 2007;26:73–81.
  47. Ghasemi H, Ghazanfari T, Babaei M, et al., Long-term ocular complications of sulfur mustard in the civilian victims of Sardasht, Iran, Cutan Ocul Toxicol, 2008;27:317–26.
  48. Kanavi MR, Javadi A, Javadi MA, Chronic and delayed mustard gas keratopathy: histological and immunohistochemical study, Eur J Ophthalmol, 2010; 20:839–43.
  49. Pleyer U, Sherif Z, Baatz H, Hartmann C, Delayed mustard gas keratopathy: clinical findings and confocal microscopy, Am J Ophthalmol, 1999;128:506–507.
  50. Balali-Mood M, Balali-Mood B, Sulfur mustard poisoning and its complications in Iranian veterans, Iran J Med Sci, 2009;34:155–71.
  51. Kadar T, Horwitz V, Sahar R, et al., Delayed loss of corneal epithelial stem cells in a chemical injury model associated with limbal stem cell deficiency in rabbits, Curr Eye Res, 2011;36:1098–1107.
  52. Tseng SCG, Concept and application of limbal stem cells, Eye, 1989:3:141–57.
  53. Smith GT, Deutsch GP, Cree IA, Liu CSC, Permanent corneal limbal stem cell dysfunction following radiotherapy for orbital lymphoma, Eye, 2000;14:905–907.
  54. Kadar T, Dachir S, Cohen M, et al., Prolonged impairment of corneal innervation after exposure to sulfur mustard and its relation to the development of delayed limbal stem cell deficiency, Cornea, 2013;32:e44–50.
  55. Amir A, Turetz J, Chapman S, et al., The beneficial effects of topical anti-inflammatory drugs against HD-induced ocular lesions in rabbits, J Appl Toxicol, 2000;20:S109–S114.
  56. Ueno H, Ferrari G, Hattori T, et al., Dependence of corneal stem/progenitor cells on ocular surface innervations, Invest Ophthalmol Vis Sci, 2012; 53:867–72.
  57. Fujishima H, Shimazaki J, Tsubota K, Temporary corneal stem cell dysfunction after radiation therapy, Br J Ophthalmol, 1996; 80:911–14.
  58. Kadar T, Dachir S, Cohen L, et al., Ocular injuries following sulfur mustard exposure – pathological mechanism and potential therapy, Toxicol, 2009;263:59–69.
  59. Frucht-Pery J, Siganos CS, Solomon A, et al., Limbal cell autograft transplantation for severe ocular surface disorders, Graefes Arch Clin Exp Ophthalmol, 1998;236:582–7.
  60. Javadi MA, Jafarinasab MR, Feizi S, et al., Management of mustard gas-induced limbal stem cell deficiency and keratitis, Ophthalmology, 2011;118:1272–81.
  61. Rama P, Matuska S, Paganoni G, et al., Limbal stem cell therapy and long-term corneal regeneration, N Eng J Med, 2010;363:147–55.
  62. Pellegrini G, Rama P, Mavilio F, De Luca M, Epithelial stem cells in corneal regeneration and epidermal gene therapy, J Pathol, 2009;217:217–28.
  63. Menzel-Severing J, Emerging techniques to treat limbal epithelial stem cell deficiency, Discov Med, 2011;11:57–64.
Keywords: Ocular burns, chemical burns, cornea, epithelial stem cells, limbal stem cell deficiency, mustard, mitomycin, 5-fluorouracil