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Cultivated Limbal Stem Cell Transplantation: Indications and Technique

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Part of the book series: Essentials in Ophthalmology ((ESSENTIALS))

Abstract

Advances in stem cell research have made revolutionary changes to healthcare and medicine, holding promise for the restoration and regeneration of dysfunctional cells for a wide range of diseases and injuries. Ophthalmology stands at the forefront of this new technology with cultivated limbal epithelial transplantation (CLET) as a prime example of these cell-based therapies. CLET aims to restore the ocular surface in stem cell-deficient eyes by transplanting ex vivo expanded stem cell grafts. Cells are harvested from a healthy limbus, grown and multiplied in a laboratory for several days and then transplanted onto the stem cell-deficient eye. Since the introduction of this technique in 1997, numerous cultivation methods and surgical approaches have been used in various centres worldwide. In the majority of cases, the technique has been shown to successfully regenerate the corneal epithelium.

While the cell products produced by the laboratory continue to improve, it is also important to consider the recipient ocular surface as the recipient bed plays a crucial role in the survival of composite graft and the success of the surgery.

In this chapter, we explore indications for CLET, define preoperative evaluation and preparation methods and describe the cultivation and surgical approach based on our >10-year experience with this technique.

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References

  1. Kruse FE. Stem cells and corneal epithelial regeneration. Eye. 1994;8:170–83.

    Article  PubMed  Google Scholar 

  2. Puangsricharern V, Tseng SC. Cytologic evidence of corneal diseases with limbal stem cell deficiency. Ophthalmology. 1995;102(10):1476–85.

    Article  CAS  PubMed  Google Scholar 

  3. Le Q, Deng SX, Xu J. In vivo confocal microscopy of congenital aniridia-associated keratopathy. Eye. 2013;27(6):763–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Ahmad S. Concise review: limbal stem cell deficiency, dysfunction, and distress. Stem Cells Transl Med. 2012;1(2):110–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Strungaru MH, Mah D, Chan CC. Focal limbal stem cell deficiency in turner syndrome. Cornea. 2014;33(2):207–9.

    Article  PubMed  Google Scholar 

  6. Catt CJ, Hamilton GM, Fish J, Mireskandari K, Ali A. Ocular manifestations of Stevens-Johnson Syndrome and toxic epidermal necrolysis in children. Am J Ophthalmol. 2016;166:68–75.

    Article  PubMed  Google Scholar 

  7. Aslan D, Akata R, Holm H, Vulliamy T, Dokal I. Limbal stem cell deficiency in patients with inherited stem cell disorder of dyskeratosis congenita. Int Ophthalmol. 2012;32(6):615–22.

    Article  PubMed  Google Scholar 

  8. Eschle-Meniconi ME, Ahmad SR, Foster CS. Mucous membrane pemphigoid: an update. Curr Opin Ophthalmol. 2005;16(5):303–7.

    Article  PubMed  Google Scholar 

  9. Fernandes M, Sangwan VS, Vemuganti GK. Limbal stem cell deficiency and xeroderma pigmentosum: a case report. Eye. 2004;18(7):741–3.

    Article  CAS  PubMed  Google Scholar 

  10. Sivaraman KR, Jivrajka RV, Soin K, Bouchard CS, Movahedan A, Shorter E, et al. Superior limbic keratoconjunctivitis-like inflammation in patients with chronic graft-versus-host disease. Ocul Surf. 2016;14(3):393–400.

    Article  PubMed  PubMed Central  Google Scholar 

  11. Cortes M, Lambiase A, Sacchetti M, Aronni S, Bonini S. Limbal stem cell deficiency associated with LADD syndrome. Arch Ophthalmol. 2005;123(5):691.

    Article  PubMed  Google Scholar 

  12. Bobba S, Di Girolamo N, Mills R, Daniell M, Chan E, Harkin DG, et al. Nature and incidence of severe limbal stem cell deficiency in Australia and New Zealand. Clin Exp Ophthalmol. 2017;45(2):174–81.

    Article  PubMed  Google Scholar 

  13. Messmer EM, Kenyon KR, Rittinger O, Janecke AR, Kampik A. Ocular manifestations of keratitis-ichthyosis-deafness (KID) syndrome. Ophthalmology. 2005;112(2):3–8.

    Article  Google Scholar 

  14. Lichtinger A, Pe’er J, Frucht-Pery J, Solomon A. Limbal stem cell deficiency after topical mitomycin C therapy for primary acquired melanosis with atypia. Ophthalmology. 2010;117(3):431–7.

    Article  PubMed  Google Scholar 

  15. Nghiem-Buffet M-H, Gatinel D, Jacquot F, Chaine G, Hoang-Xuan T. Limbal stem cell deficiency following phototherapeutic keratectomy. Cornea. 2003;22(5):482–4.

    Article  PubMed  Google Scholar 

  16. Schwartz GS, Holland EJ. Iatrogenic limbal stem cell deficiency. Cornea. 1998;17(1):31–7. A.

    Article  CAS  PubMed  Google Scholar 

  17. Lim P, Fuchsluger TA, Jurkunas UV. Limbal stem cell deficiency and corneal neovascularization. Semin Ophthalmol. 2009;24(3):139–48.

    Article  PubMed  Google Scholar 

  18. Gupta N, Sachdev R, Tandon R. Ocular surface squamous neoplasia in xeroderma pigmentosum: clinical spectrum and outcome. Graefes Arch Clin Exp Ophthalmol. 2011;249(8):1217–21. A.

    Article  PubMed  Google Scholar 

  19. Merchant A, Zhao T-Z, Foster CS. Chronic keratoconjunctivitis associated with congenital dyskeratosis and erythrokeratodermia variablis. Ophthalmology. 1998;105(7):1286–91.

    Article  CAS  PubMed  Google Scholar 

  20. Rossen J, Amram A, Milani B, Park D, Harthan J, Joslin C, et al. Contact Lens-induced limbal stem cell deficiency. Ocul Surf. 2016;14(4):419–34.

    Article  PubMed  PubMed Central  Google Scholar 

  21. Fdos P, Goncalves ED, Barros JN, Campos MS, Sato EH, Gomes JAP. Impression cytology findings in bullous keratopathy. Br J Ophthalmol. 2010;94(6):773–6.

    Article  Google Scholar 

  22. Dua HS, Saini JS, Azuara-Blanco A, Gupta P. Limbal stem cell deficiency: concept, aetiology, clinical presentation, diagnosis and management. Indian J Ophthalmol. 2000;48(2):83–92.

    CAS  PubMed  Google Scholar 

  23. Djalilian AR, Holland EJ, Schwartz GS. Limbal stem cell deficiency. Ophthalmology. 2003;110(10):2071–2.

    Article  PubMed  Google Scholar 

  24. Holland EJ, Djalilian AR, Schwartz GS. Management of aniridic keratopathy with keratolimbal allograft: a limbal stem cell transplantation technique. Ophthalmology. 2003;110(1):125–30.

    Article  PubMed  Google Scholar 

  25. Javadi M-A, Baradaran-Rafii A. Living-related conjunctival-limbal allograft for chronic or delayed-onset mustard gas keratopathy. Cornea. 2009;28(1):51–7.

    Article  PubMed  Google Scholar 

  26. Meallet MA, Espana EM, Grueterich M, Ti S-E, Goto E, Tseng SC. Amniotic membrane transplantation with conjunctival limbal autograft for total limbal stem cell deficiency. Ophthalmology. 2003;110(8):1585–92.

    Article  PubMed  Google Scholar 

  27. Pellegrini G, Traverso CE, Franzi AT, Zingirian M, Cancedda R, De Luca M. Long-term restoration of damaged corneal surfaces with autologous cultivated corneal epithelium. Lancet. 1997;349:990–3.

    Article  CAS  PubMed  Google Scholar 

  28. Behaegel J, Dhubhghaill SN, Koppen C, Zakaria N. Safety of cultivated limbal epithelial stem cell transplantation for human corneal regeneration. Stem Cells Int. 2017;2017:1.

    Article  CAS  Google Scholar 

  29. Shortt AJ, Secker GA, Notara MD, Limb GA, Khaw PT, Tuft SJ, et al. Transplantation of ex vivo cultured limbal epithelial stem cells: a review of techniques and clinical results. Surv Ophthalmol. 2007;52(5):483–502.

    Article  PubMed  Google Scholar 

  30. Henderson TR, Coster DJ, Williams KA. The long term outcome of limbal allografts: the search for surviving cells. Br J Ophthalmol. 2001;85(5):604–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Daya SM, Watson A, Sharpe JR, Giledi O, Rowe A, Martin R, et al. Outcomes and DNA analysis of ex vivo expanded stem cell allograft for ocular surface reconstruction. Ophthalmology. 2005;112(3):470–7.

    Article  PubMed  Google Scholar 

  32. Sharpe JR, Daya SM, Dimitriadi M, Martin R, James SE. Survival of cultured allogeneic limbal epithelial cells following corneal repair. Tissue Eng. 2007;13(1):123–32.

    Article  PubMed  Google Scholar 

  33. Dua HS, Miri A, Said DG. Contemporary limbal stem cell transplantation – a review. Clin Exp Ophthalmol. 2010;38(2):104–17.

    Article  PubMed  Google Scholar 

  34. Tseng SCG, Tsubota K. Important concepts for treating ocular surface and tear disorders. Am J Ophthalmol. 1997;124(6):825–35.

    Article  CAS  PubMed  Google Scholar 

  35. Knop E, Korb D, Blackie C, Knop N. The lid margin is an underestimated structure for preservation of ocular surface health and development of dry eye disease. Dev Ophthalmol. 2010;45:108–22.

    Article  PubMed  Google Scholar 

  36. Willcox MDP, Argüeso P, Georgiev GA, Holopainen JM, Laurie GW, Millar TJ, et al. TFOS DEWS II tear film report. Ocul Surf. 2017;15(3):366–403.

    Article  PubMed  PubMed Central  Google Scholar 

  37. Kim BY, Riaz KM, Bakhtiari P, Chan CC, Welder JD, Holland EJ, et al. Medically reversible limbal stem cell disease clinical features and management strategies. Ophthalmology. 2014;121(10):2053–8.

    Article  PubMed  PubMed Central  Google Scholar 

  38. Dua HS. The conjunctiva in corneal epithelial wound healing. Br J Ophthalmol. 1998;82(12):1407–11.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Dua HS. Sequential sector conjunctival epitheliectomy. In: Holland EJ, Mannis M, editors. Ocular surface disease, medical and surgical management. New York: Springer; 2002. p. 168–74.

    Chapter  Google Scholar 

  40. Waring GO, Bourne WM, Edelhauser HF, Kenyon KR. The corneal endothelium: normal and pathologic structure and function. Ophthalmology. 1982;89(6):531–90.

    Article  PubMed  Google Scholar 

  41. Oliveira-Soto L, Efron N. Morphology of corneal nerves using confocal microscopy. Cornea. 2001;20(4):374–84.

    Article  CAS  PubMed  Google Scholar 

  42. Lambiase A, Manni L, Bonini S, Rama P, Micera A, Aloe L. Nerve growth factor promotes corneal healing: structural, biochemical, and molecular analyses of rat and human corneas. Invest Ophthalmol Vis Sci. 2000;41(5):1063–9.

    CAS  PubMed  Google Scholar 

  43. Stern ME, Gao J, Siemasko KF, Beuerman RW, Pflugfelder SC. The role of the lacrimal functional unit in the pathophysiology of dry eye. Exp Eye Res. 2004;78(3):409–16.

    Article  CAS  PubMed  Google Scholar 

  44. Aguayo Bonniard A, Yeung JY, Chan CC, Birt CM. Ocular surface toxicity from glaucoma topical medications and associated preservatives such as benzalkonium chloride (BAK). Expert Opin Drug Metab Toxicol. 2016;12(11):1279–89.

    Article  CAS  PubMed  Google Scholar 

  45. Rama P, Matuska S, Paganoni G, Spinelli A, De Luca M, Pellegrini G. Limbal stem-cell therapy and long-term corneal regeneration. N Engl J Med. 2010;363(2):147–55.

    Article  CAS  PubMed  Google Scholar 

  46. Cho P, Yap M. Schirmer test. I. A review. Optom Vis Sci. 1993;70(2):152–6.

    Article  CAS  PubMed  Google Scholar 

  47. Sejpal K, Bakhtiari P, Deng SX. Presentation, diagnosis and management of limbal stem cell deficiency. Middle East Afr J Ophthalmol. 2013;20(1):5–10.

    Article  PubMed  PubMed Central  Google Scholar 

  48. Haagdorens M, Behaegel J, Rozema J, Van Gerwen V, Michiels S, Ní Dhubhghaill S, et al. A method for quantifying limbal stem cell niches using OCT imaging. Br J Ophthalmol. 2017;101(9):1250.

    Article  PubMed  PubMed Central  Google Scholar 

  49. Zakaria N, Ni Dhubhghaill S, Taal M, Berneman Z, Koppen C, Tassignon M-J. Optical coherence tomography in cultivated limbal epithelial stem cell transplantation surgery. J Ophthalmol. 2015;4(6):339–45.

    Google Scholar 

  50. Villani E, Baudouin C, Efron N, Hamrah P, Kojima T, Patel SV, et al. In vivo confocal microscopy of the ocular surface: from bench to bedside. Curr Eye Res. 2014;39(3):213–31.

    Article  PubMed  Google Scholar 

  51. Miri A, Al-Aqaba M, Otri AM, Fares U, Said DG, Faraj LA, et al. In vivo confocal microscopic features of normal limbus. Br J Ophthalmol. 2012;96(4):530–6.

    Article  PubMed  Google Scholar 

  52. Patel DV, Sherwin T, McGhee CNJ. Laser scanning in vivo confocal microscopy of the normal human corneoscleral limbus. Investig Ophthalmol Vis Sci. 2006;47(7):2823–7.

    Article  Google Scholar 

  53. Messmer EM, Mackert MJ, Zapp DM, Kampik A. In vivo confocal microscopy of pigmented conjunctival tumors. Graefes Arch Clin Exp Ophthalmol. 2006;244(11):1437–45.

    Article  PubMed  Google Scholar 

  54. Nubile M, Lanzini M, Miri A, Pocobelli A, Calienno R, Curcio C, et al. In vivo confocal microscopy in diagnosis of limbal stem cell deficiency. Am J Ophthalmol. 2013;155(2):220–32.

    Article  PubMed  Google Scholar 

  55. Miri A, Alomar T, Nubile M, Al-aqaba M, Lanzini M, Fares U, et al. In vivo confocal microscopic findings in patients with limbal stem cell deficiency. Br J Ophthalmol. 2012;96(4):523–9.

    Article  PubMed  Google Scholar 

  56. Deng SX, Sejpal K, Tang Q, Aldave AJ, Lee OL, Yu F. Characterization of limbal stem cell deficiency by in vivo laser scanning confocal microscopy: a microstructural approach. Arch Ophthalmol. 2012;130(4):440–5.

    Article  PubMed  Google Scholar 

  57. Zhao Y, Ma L. Systematic review and meta-analysis on transplantation of ex vivo cultivated limbal epithelial stem cell on amniotic membrane in limbal stem cell deficiency. Cornea. 2015;34(5):592–600.

    Article  PubMed  Google Scholar 

  58. Zakaria N, Koppen C, Van Tendeloo V, Berneman Z, Hopkinson A, Tassignon M-J. Standardized limbal epithelial stem cell graft generation and transplantation. Tissue Eng Part C Methods. 2010;16(5):921–7.

    Article  CAS  PubMed  Google Scholar 

  59. Hopkinson A, Shanmuganathan VA, Gray T, Yeung AM, Lowe J, James DK, et al. Optimization of amniotic membrane (AM) denuding for tissue engineering. Tissue Eng Part C Methods. 2008;14(4):371–81.

    Article  CAS  PubMed  Google Scholar 

  60. Hopkinson A, McIntosh RS, Tighe PJ, James DK, Dua HS. Amniotic membrane for ocular surface reconstruction: donor variations and the effect of handling on TGF-beta content. Invest Ophthalmol Vis Sci. 2006;47(10):4316–22.

    Article  PubMed  Google Scholar 

  61. Baudouin C, Labbé A, Liang H, Pauly A. Preservatives in eyedrops: The good, the bad and the ugly. Prog Retin Eye Res. 2010;29(4):312–34.

    Article  CAS  PubMed  Google Scholar 

  62. Basu S, Mohamed A, Chaurasia S, Sejpal K, Vemuganti GK, Sangwan VS. Clinical outcomes of penetrating keratoplasty after autologous cultivated limbal epithelial transplantation for ocular surface burns. Am J Ophthalmol. 2011;152(6):917–924.e1.

    Article  PubMed  Google Scholar 

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Acknowledgement

We would like to acknowledge the important input of Nadia Zakaria who initiated the culturing method as used in the clinical trial and realised the GMP accreditation of the ophthalmology branch of the CCRG unit. She is employee at Novartis Institute of Biomedical Research since 2017.

Compliance with Ethical Requirements

Joséphine Behaegel, Sorcha Ní Dhubhghaill and Marie-José Tassignon declare that they have no conflict of interest.

Informed Consent

All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (University Hospital of Antwerp) and with the Helsinki Declaration of 1975, as revised in 2000. Informed consent was obtained from all patients for being included in the study.

No animal studies were carried out by the authors for this article.

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Correspondence to Marie-José Tassignon .

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Behaegel, J., Dhubhghaill, S.N., Tassignon, MJ. (2019). Cultivated Limbal Stem Cell Transplantation: Indications and Technique. In: Alió, J., Alió del Barrio, J., Arnalich-Montiel, F. (eds) Corneal Regeneration . Essentials in Ophthalmology. Springer, Cham. https://doi.org/10.1007/978-3-030-01304-2_19

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  • DOI: https://doi.org/10.1007/978-3-030-01304-2_19

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