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Corneal Epithelial Stem Cells: Methods for Ex Vivo Expansion

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Corneal Regeneration

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

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Abstract

The corneal epithelium in humans is the only layer in the cornea that is able to regenerate in vivo, and corneal integrity and function are dependent on this. The ability of the epithelium to regenerate relies on limbal stem cells (LSCs). The LSC niche has been demonstrated to be found within the palisades of Vogt at the corneoscleral limbus. Total limbal stem cell deficiency (LSCD) results in conjunctivalisation of the corneal surface and subsequent permanent visual impairment, together with recurrent or persistent epithelial erosions and ulcerations that lead to stromal opacity and chronic pain and discomfort. In addition to conservative management with lubricating eye drops, serum eye drops, topical steroid and antibiotics, the definitive treatment of total LSCD involves replacing the LSC population before patients can undergo corneal transplantation for visual rehabilitation. This chapter describes the Newcastle methods of autologous limbal stem cell transplantation from the harvesting and culture of autologous LSCs to transplantation of the medicinal product, using a cell suspension method or ex vivo expansion method, describes the Good Manufacturing Practice (GMP) processes for the production of cells for therapeutic use and looks to the future use of a European-licensed LSC therapeutic product (Holoclar®, Chiesi Farmaceutici S.p.A., Parma, Italy) for the treatment of unilateral severe or total LSCD due to ocular surface burns. This chapter also describes the methods for the assessment of the LSC culture, including histological methods, and the potential use of decellularised, γ-irradiated human amniotic membrane as a suitable substrate for LSC culture.

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References

  1. Secker GA, Daniels JT. Limbal epithelial stem cells of the cornea 2009. In: StemBook [Internet]. http://www.stembook.org/node/588.

  2. Fuchs E, Segre JA. Stem cells: a new lease on life. Cell. 2000;100(1):143–55.

    Article  CAS  PubMed  Google Scholar 

  3. Dua HS, Shanmuganathan VA, Powell-Richards AO, Tighe PJ, Joseph A. Limbal epithelial crypts: a novel anatomical structure and a putative limbal stem cell niche. Br J Ophthalmol. 2005;89(5):529–32.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Li W, Hayashida Y, Chen YT, Tseng SC. Niche regulation of corneal epithelial stem cells at the limbus. Cell Res. 2007;17(1):26–36.

    Article  PubMed  CAS  Google Scholar 

  5. Dua HS, Azuara-Blanco A. Limbal stem cells of the corneal epithelium. Surv Ophthalmol. 2000;44(5):415–25.

    Article  CAS  PubMed  Google Scholar 

  6. Thoft RA, Friend J. The X, Y, Z hypothesis of corneal epithelial maintenance. Investig Ophthalmol Vis Sci. 1983;24(10):1442–3.

    CAS  Google Scholar 

  7. Sharma A, Coles WH. Kinetics of corneal epithelial maintenance and graft loss. A population balance model. Investig Ophthalmol Vis Sci. 1989;30(9):1962–71.

    CAS  Google Scholar 

  8. Davanger M, Evensen A. Role of the pericorneal papillary structure in renewal of corneal epithelium. Nature. 1971;229(5286):560–1.

    Article  CAS  PubMed  Google Scholar 

  9. Chen JJ, Tseng SC. Abnormal corneal epithelial wound healing in partial-thickness removal of limbal epithelium. Investig Ophthalmol Vis Sci. 1991;32(8):2219–33.

    CAS  Google Scholar 

  10. Cotsarelis G, Cheng SZ, Dong G, Sun TT, Lavker RM. Existence of slow-cycling limbal epithelial basal cells that can be preferentially stimulated to proliferate: implications on epithelial stem cells. Cell. 1989;57(2):201–9.

    Article  CAS  PubMed  Google Scholar 

  11. Kenyon KR, Tseng SC. Limbal autograft transplantation for ocular surface disorders. Ophthalmology. 1989;96(5):709–22; discussion 22–3.

    Article  CAS  PubMed  Google Scholar 

  12. Dua HS, Kulkarni B, Singh R. Quest for limbal stem cells. Clin Exp Ophthalmol. 2006;34(1):1–2.

    Article  PubMed  Google Scholar 

  13. Dua HS, Forrester JV. Clinical patterns of corneal epithelial wound healing. Am J Ophthalmol. 1987;104(5):481–9.

    Article  CAS  PubMed  Google Scholar 

  14. Dua HS, Forrester JV. The corneoscleral limbus in human corneal epithelial wound healing. Am J Ophthalmol. 1990;110(6):646–56.

    Article  CAS  PubMed  Google Scholar 

  15. Lavker RM, Dong G, Cheng SZ, Kudoh K, Cotsarelis G, Sun TT. Relative proliferative rates of limbal and corneal epithelia. Implications of corneal epithelial migration, circadian rhythm, and suprabasally located DNA-synthesizing keratinocytes. Investig Ophthalmol Vis Sci. 1991;32(6):1864–75.

    CAS  Google Scholar 

  16. Hanna C, Bicknell DS, O'Brien JE. Cell turnover in the adult human eye. Arch Ophthalmol. 1961;65(5):695–8.

    Article  CAS  PubMed  Google Scholar 

  17. 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 

  18. Sejpal KBP, 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 

  19. Secretariat MA. Limbal stem cell transplantation: an evidence-based analysis. Ont Health Technol Assess Ser. 2008;8(7):1–58.

    Google Scholar 

  20. Solano J. RCL. Ocular Burns 2013 [updated 25 Jun 2013. Available from: http://emedicine.medscape.com/article/798696-overview.

  21. Morgan SJ. Chemical burns of the eye: causes and management. Br J Ophthalmol. 1987;71(11):854–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Davis AR, Ali QH, Aclimandos WA, Hunter PA. Topical steroid use in the treatment of ocular alkali burns. Br J Ophthalmol. 1997;81(9):732–4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Macdonald ECA, Cauchi PA, Azuara-Blanco A, Foot B. Surveillance of severe chemical corneal injuries in the UK. Br J Ophthalmol. 2009;93(9):1177–80.

    Article  CAS  PubMed  Google Scholar 

  24. Foster CS, Ba-Abbad R, Letko E, Parrillo SJ. Stevens-Johnson syndrome 2013 [updated 12 Aug 2013]. Available from: http://emedicine.medscape.com/article/1197450-overview.

  25. Freiman A. Cicatricial pemphigoid 2013 [updated 24 Jan 2013]. Available from: http://emedicine.medscape.com/article/1062534-overview.

  26. Shortt AJ, Tuft SJ, Daniels JT. Corneal stem cells in the eye clinic. Br Med Bull. 2011;100:209–25.

    Article  PubMed  Google Scholar 

  27. Osei-Bempong C, Figueiredo FC, Lako M. The limbal epithelium of the eye--a review of limbal stem cell biology, disease and treatment. BioEssays. 2013;35(3):211–9.

    Article  CAS  PubMed  Google Scholar 

  28. Dua HS, Joseph A, Shanmuganathan VA, Jones RE. Stem cell differentiation and the effects of deficiency. Eye. 2003;17(8):877–85.

    Article  CAS  PubMed  Google Scholar 

  29. Nugent RB, Lee GA. Ophthalmic use of blood-derived products. Surv Ophthalmol. 2015;60(5):406–34.

    Article  PubMed  Google Scholar 

  30. Chiang CC, Chen WL, Lin JM, Tsai YY. Allogeneic serum eye drops for the treatment of persistent corneal epithelial defect. Eye. 2009;23(2):290–3.

    Article  PubMed  Google Scholar 

  31. Dua HS, Gomes JA, Singh A. Corneal epithelial wound healing. Br J Ophthalmol. 1994;78(5):401–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Tseng SC, Prabhasawat P, Barton K, Gray T, Meller D. Amniotic membrane transplantation with or without limbal allografts for corneal surface reconstruction in patients with limbal stem cell deficiency. Arch Ophthalmol. 1998;116(4):431–41.

    Article  CAS  PubMed  Google Scholar 

  33. Poon AC, Geerling G, Dart JK, Fraenkel GE, Daniels JT. Autologous serum eyedrops for dry eyes and epithelial defects: clinical and in vitro toxicity studies. Br J Ophthalmol. 2001;85(10):1188–97.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Geerling G, Maclennan S, Hartwig D. Autologous serum eye drops for ocular surface disorders. Br J Ophthalmol. 2004;88(11):1467–74.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Young AL, Cheng AC, Ng HK, Cheng LL, Leung GY, Lam DS. The use of autologous serum tears in persistent corneal epithelial defects. Eye. 2004;18(6):609–14.

    Article  CAS  PubMed  Google Scholar 

  36. Anderson C, Moretti S, Gieser RG. The effect of tarsorrhaphy on normal healing of corneal epithelial defects in a rabbit model. Cornea. 1991;10(6):478–82.

    Article  CAS  PubMed  Google Scholar 

  37. Robinson C, Tantri A, Shriver E, Oetting T. Temporary eyelid closure applique. Arch Ophthalmol. 2006;124(4):546–9.

    Article  PubMed  Google Scholar 

  38. Wagoner MD, Steinert RF. Temporary tarsorrhaphy enhances reepithelialization after epikeratoplasty. Arch Ophthalmol. 1988;106(1):13–4.

    Article  CAS  PubMed  Google Scholar 

  39. Cosar CB, Cohen EJ, Rapuano CJ, Maus M, Penne RP, Flanagan JC, et al. Tarsorrhaphy: clinical experience from a cornea practice. Cornea. 2001;20(8):787–91.

    Article  CAS  PubMed  Google Scholar 

  40. Pakarinen M, Tervo T, Tarkkanen A. Tarsorraphy in the treatment of persistent corneal lesions. Acta Ophthalmol Suppl. 1987;182:69–73.

    CAS  PubMed  Google Scholar 

  41. Pfister RR. Clinical measures to promote corneal epithelial healing. Acta Ophthalmol Suppl. 1992(202):73–83.

    Article  Google Scholar 

  42. Kasaee A, Musavi MR, Tabatabaie SZ, Hashemian MN, Mohebbi S, Khodabandeh A, et al. Evaluation of efficacy and safety of botulinum toxin type A injection in patients requiring temporary tarsorrhaphy to improve corneal epithelial defects. Int J Ophthalmol. 2010;3(3):237–40.

    PubMed  PubMed Central  Google Scholar 

  43. Ellis MF, Daniell M. An evaluation of the safety and efficacy of botulinum toxin type A (BOTOX) when used to produce a protective ptosis. Clin Exp Ophthalmol. 2001;29(6):394–9.

    Article  CAS  PubMed  Google Scholar 

  44. Lee C, Kikkawa DO, Pasco NY, Granet DB. Advanced functional oculofacial indications of botulinum toxin. Int Ophthalmol Clin. 2005;45(3):77–91.

    Article  PubMed  Google Scholar 

  45. Wuebbolt GE, Drummond G. Temporary tarsorrhaphy induced with type A botulinum toxin. Can J Ophthalmol. 1991;26(7):383–5.

    CAS  PubMed  Google Scholar 

  46. Rosenthal P, Cotter JM, Baum J. Treatment of persistent corneal epithelial defect with extended wear of a fluid-ventilated gas-permeable scleral contact lens. Am J Ophthalmol. 2000;130(1):33–41.

    Article  CAS  PubMed  Google Scholar 

  47. Kanpolat A, Ucakhan OO. Therapeutic use of Focus Night & Day contact lenses. Cornea. 2003;22(8):726–34.

    Article  PubMed  Google Scholar 

  48. Lim L, Tan DT, Chan WK. Therapeutic use of Bausch & Lomb PureVision contact lenses. CLAO J. 2001;27(4):179–85.

    CAS  PubMed  Google Scholar 

  49. Arora R, Jain S, Monga S, Narayanan R, Raina UK, Mehta DK. Efficacy of continuous wear PureVision contact lenses for therapeutic use. Cont Lens Anterior Eye. 2004;27(1):39–43.

    Article  PubMed  Google Scholar 

  50. Blackmore SJ. The use of contact lenses in the treatment of persistent epithelial defects. Cont Lens Anterior Eye. 2010;33(5):239–44.

    Article  PubMed  Google Scholar 

  51. Sangwan VS, Burman S, Tejwani S, Mahesh SP, Murthy R. Amniotic membrane transplantation: a review of current indications in the management of ophthalmic disorders. Indian J Ophthalmol. 2007;55(4):251–60.

    Article  PubMed  Google Scholar 

  52. Saw VP, Minassian D, Dart JK, Ramsay A, Henderson H, Poniatowski S, et al. Amniotic membrane transplantation for ocular disease: a review of the first 233 cases from the UK user group. Br J Ophthalmol. 2007;91(8):1042–7.

    Article  PubMed  PubMed Central  Google Scholar 

  53. Tejwani S, Kolari RS, Sangwan VS, Rao GN. Role of amniotic membrane graft for ocular chemical and thermal injuries. Cornea. 2007;26(1):21–6.

    Article  PubMed  Google Scholar 

  54. Kruse FE, Cursiefen C. Surgery of the cornea: corneal, limbal stem cell and amniotic membrane transplantation. Dev Ophthalmol. 2008;41:159–70.

    Article  CAS  PubMed  Google Scholar 

  55. Meller D, Pires RT, Mack RJ, Figueiredo F, Heiligenhaus A, Park WC, et al. Amniotic membrane transplantation for acute chemical or thermal burns. Ophthalmology. 2000;107(5):980–9. discussion 90

    Article  CAS  PubMed  Google Scholar 

  56. Baylis O, Figueiredo F, Henein C, Lako M, Ahmad S. 13 years of cultured limbal epithelial cell therapy: a review of the outcomes. J Cell Biochem. 2011;112(4):993–1002.

    Article  CAS  PubMed  Google Scholar 

  57. Cauchi PA, Ang GS, Azuara-Blanco A, Burr JM. A systematic literature review of surgical interventions for limbal stem cell deficiency in humans. Am J Ophthalmol. 2008;146(2):251–9.

    Article  PubMed  Google Scholar 

  58. Copeland RA Jr, Char DH. Limbal autograft reconstruction after conjunctival squamous cell carcinoma. Am J Ophthalmol. 1990;110(4):412–5.

    Article  PubMed  Google Scholar 

  59. Coster DJ, Aggarwal RK, Williams KA. Surgical management of ocular surface disorders using conjunctival and stem cell allografts. Br J Ophthalmol. 1995;79(11):977–82.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Frucht-Pery J, Siganos CS, Solomon A, Scheman L, Brautbar C, Zauberman H. Limbal cell autograft transplantation for severe ocular surface disorders. Graefes Arch Clin Exp Ophthalmol. 1998;236(8):582–7.

    Article  CAS  PubMed  Google Scholar 

  61. Holland EJ. Epithelial transplantation for the management of severe ocular surface disease. Trans Am Ophthalmol Soc. 1996;94:677–743.

    CAS  PubMed  PubMed Central  Google Scholar 

  62. Morgan S, Murray A. Limbal autotransplantation in the acute and chronic phases of severe chemical injuries. Eye. 1996;10 (Pt 3:349–54.

    Article  PubMed  Google Scholar 

  63. Tan DT, Ficker LA, Buckley RJ. Limbal transplantation. Ophthalmology. 1996;103(1):29–36.

    Article  CAS  PubMed  Google Scholar 

  64. Theng JT, Tan DT. Combined penetrating keratoplasty and limbal allograft transplantation for severe corneal burns. Ophthalmic Surg Lasers. 1997;28(9):765–8.

    CAS  PubMed  Google Scholar 

  65. Tsubota K, Toda I, Saito H, Shinozaki N, Shimazaki J. Reconstruction of the corneal epithelium by limbal allograft transplantation for severe ocular surface disorders. Ophthalmology. 1995;102(10):1486–96.

    Article  CAS  PubMed  Google Scholar 

  66. Tsubota K, Satake Y, Ohyama M, Toda I, Takano Y, Ono M, et al. Surgical reconstruction of the ocular surface in advanced ocular cicatricial pemphigoid and Stevens-Johnson syndrome. Am J Ophthalmol. 1996;122(1):38–52.

    Article  CAS  PubMed  Google Scholar 

  67. Ilari L, Daya SM. Long-term outcomes of keratolimbal allograft for the treatment of severe ocular surface disorders. Ophthalmology. 2002;109(7):1278–84.

    Article  PubMed  Google Scholar 

  68. Jenkins C, Tuft S, Liu C, Buckley R. Limbal transplantation in the management of chronic contact-lens-associated epitheliopathy. Eye. 1993;7:629–33.

    Article  PubMed  Google Scholar 

  69. 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(9057):990–3.

    Article  CAS  PubMed  Google Scholar 

  70. Kolli S, Ahmad S, Lako M, Figueiredo F. Successful clinical implementation of corneal epithelial stem cell therapy for treatment of unilateral limbal stem cell deficiency. Stem Cells. 2010;28(3):597–610.

    CAS  PubMed  Google Scholar 

  71. Sangwan VS, Matalia HP, Vemuganti GK, Fatima A, Ifthekar G, Singh S, et al. Clinical outcome of autologous cultivated limbal epithelium transplantation. Indian J Ophthalmol. 2006;54(1):29–34.

    Article  PubMed  Google Scholar 

  72. Pauklin M, Fuchsluger TA, Westekemper H, Steuhl KP, Meller D. Midterm results of cultivated autologous and allogeneic limbal epithelial transplantation in limbal stem cell deficiency. Dev Ophthalmol. 2010;45:57–70.

    Article  PubMed  Google Scholar 

  73. 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 

  74. Maguire MG, Stark WJ, Gottsch JD, Stulting RD, Sugar A, Fink NE, et al. Risk factors for corneal graft failure and rejection in the collaborative corneal transplantation studies. Collaborative Corneal Transplantation Studies Research Group. Ophthalmology. 1994;101(9):1536–47.

    Article  CAS  PubMed  Google Scholar 

  75. Sangwan VS, Matalia HP, Vemuganti GK, Ifthekar G, Fatima A, Singh S, et al. Early results of penetrating keratoplasty after cultivated limbal epithelium transplantation. Arch Ophthalmol. 2005;123(3):334–40.

    Article  PubMed  Google Scholar 

  76. 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):917–24. e1

    Article  PubMed  Google Scholar 

  77. Organization WH. Recommendations for the evaluation of animal cell cultures as substrates for the manufacture of biological medicinal products and for the characterization of cell banks. WHO Press. 2010.

    Google Scholar 

  78. Group C-bATW. Donation of starting material for cell-based advanced therapies: a SaBTO review. Advisory Committee on the Safety of Blood, Tissues and Organs (SaBTO). 2014.

    Google Scholar 

  79. Kolli S. Corneal epithelial stem cell biology and its therapeutic application: PhD Thesis. Newcastle University. 2009.

    Google Scholar 

  80. Baylis O. The safety and efficacy of ex vivo expanded autologous limbal stem cells for the treatment of unilateral total limbal stem cell deficiency: PhD Thesis. Newcastle University. 2014.

    Google Scholar 

  81. Zakaria N, Possemiers T, Dhubhghaill SN, Leysen I, Rozema J, Koppen C, et al. Results of a phase I/II clinical trial: standardized, non-xenogenic, cultivated limbal stem cell transplantation. J Transl Med. 2014;12:58.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  82. Shortt AJ, Secker GA, Munro PM, Khaw PT, Tuft SJ, Daniels JT. Characterization of the limbal epithelial stem cell niche: novel imaging techniques permit in vivo observation and targeted biopsy of limbal epithelial stem cells. Stem Cells. 2007;25(6):1402–9.

    Article  PubMed  Google Scholar 

  83. Koizumi NJ, Inatomi TJ, Sotozono CJ, Fullwood NJ, Quantock AJ, Kinoshita S. Growth factor mRNA and protein in preserved human amniotic membrane. Curr Eye Res. 2000;20(3):173–7.

    Article  CAS  PubMed  Google Scholar 

  84. Tseng SCG, Li D-Q, Ma X. Suppression of transforming growth factor-beta isoforms, TGF-β receptor type II, and myofibroblast differentiation in cultured human corneal and limbal fibroblasts by amniotic membrane matrix. J Cell Physiol. 1999;179(3):325–35.

    Article  CAS  PubMed  Google Scholar 

  85. Solomon A, Rosenblatt M, Monroy D, Ji Z, Pflugfelder SC, Tseng SCG. Suppression of interleukin 1α and interleukin 1β in human limbal epithelial cells cultured on the amniotic membrane stromal matrix. Br J Ophthalmol. 2001;85(4):444–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  86. Park WC, Tseng SCG. Modulation of acute inflammation and keratocyte death by suturing, blood, and amniotic membrane in PRK. Invest Ophthalmol Vis Sci. 2000;41(10):2906–14.

    CAS  PubMed  Google Scholar 

  87. Endo K, Nakamura T, Kawasaki S, Kinoshita S. Human amniotic membrane, like corneal epithelial basement membrane, manifests the alpha5 chain of type IV collagen. Investig Ophthalmol Vis Sci. 2004;45(6):1771–4.

    Article  Google Scholar 

  88. Fukuda K, Chikama T, Nakamura M, Nishida T. Differential distribution of subchains of the basement membrane components type IV collagen and laminin among the amniotic membrane, cornea, and conjunctiva. Cornea. 1999;18(1):73–9.

    Article  CAS  PubMed  Google Scholar 

  89. Meller D, Pires RTF, Tseng SCG. Ex vivo preservation and expansion of human limbal epithelial stem cells on amniotic membrane cultures. Br J Ophthalmol. 2002;86(4):463–71.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  90. Koizumi N, Fullwood N, Bairaktaris G, Inatomi T, Kinoshita S, Quantock A. Cultivation of corneal epithelial cells on intact and denuded human amniotic membrane. Investig Ophthalmol Vis Sci. 2000;41(9):2506–13.

    CAS  Google Scholar 

  91. Kubo M, Sonoda Y, Muramatsu R, Usui M. Immunogenicity of human amniotic membrane in experimental xenotransplantation. Investig Ophthalmol Vis Sci. 2001;42(7):1539–46.

    CAS  Google Scholar 

  92. Hori J, Wang M, Kamiya K, Takahashi H, Sakuragawa N. Immunological characteristics of amniotic epithelium. Cornea. 2006;25(10 Suppl 1):S53–8.

    Article  PubMed  Google Scholar 

  93. Akle CA, Adinolfi M, Welsh KI, Leibowitz S, McColl I. Immunogenicity of human amniotic epithelial cells after transplantation into volunteers. Lancet. 1981;2(8254):1003–5.

    Article  CAS  PubMed  Google Scholar 

  94. Akle C, McColl I, Dean M, Adinolfi M, Brown S, Fensom AH, et al. Transplantation of amniotic epithelial membranes in patients with mucopolysaccharidoses. Exp Clin Immunogenet. 1985;2(1):43–8.

    CAS  PubMed  Google Scholar 

  95. Saghizadeh M, Winkler MA, Kramerov AA, Hemmati DM, Ghiam CA, Dimitrijevich SD, et al. A simple alkaline method for decellularizing human amniotic membrane for cell culture. PLoS One. 2013;8(11):e79632.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  96. Menzel-Severing J, Kruse FE, Schlötzer-Schrehardt U. Stem cell–based therapy for corneal epithelial reconstruction: present and future. Can J Ophthalmol. 2013;48(1):13–21.

    Article  PubMed  Google Scholar 

  97. Levis H, Daniels JT. New technologies in limbal epithelial stem cell transplantation. Curr Opin Biotechnol. 2009;20(5):593–7.

    Article  CAS  PubMed  Google Scholar 

  98. Levis HJ, Brown RA, Daniels JT. Plastic compressed collagen as a biomimetic substrate for human limbal epithelial cell culture. Biomaterials. 2010;31(30):7726–37.

    Article  CAS  PubMed  Google Scholar 

  99. Wilshaw S-P, Kearney JN, Fisher J, Ingham E. Production of an acellular amniotic membrane matrix for use in tissue engineering. Tissue Eng. 2006;12(8):2117–29.

    Article  CAS  PubMed  Google Scholar 

  100. Keane T, Londono R, Turner N, Badylak S. Consequences of ineffective decellularization of biologic scaffolds on the host response. Biomaterials. 2012;33(6):1771–81.

    Article  CAS  PubMed  Google Scholar 

  101. Keane T, Swinehart I, Badylak S. Methods of tissue decellularization used for preparation of biologic scaffolds and in vivo relevance. Methods. 2015;84:25–34.

    Article  CAS  PubMed  Google Scholar 

  102. Koizumi N, Rigby H, Fullwood N, Kawasaki S, Tanioka H, Koizumi K, et al. Comparison of intact and denuded amniotic membrane as a substrate for cell-suspension culture of human limbal epithelial cells. Graefes Arch Clin Exp Ophthalmol. 2007;245(1):123–34.

    Article  PubMed  Google Scholar 

  103. Grueterich M, Espana EM, Tseng SCG. Connexin 43 expression and proliferation of human limbal epithelium on intact and denuded amniotic membrane. Investig Ophthalmol Vis Sci. 2002;43(1):63–71.

    Google Scholar 

  104. Shortt AJ, Secker GA, Lomas RJ, Wilshaw SP, Kearney JN, Tuft SJ, et al. The effect of amniotic membrane preparation method on its ability to serve as a substrate for the ex-vivo expansion of limbal epithelial cells. Biomaterials. 2009;30(6):1056–65.

    Article  CAS  PubMed  Google Scholar 

  105. Riau AK, Beuerman RW, Lim LS, Mehta JS. Preservation, sterilization and de-epithelialization of human amniotic membrane for use in ocular surface reconstruction. Biomaterials. 2010;31(2):216–25.

    Article  CAS  PubMed  Google Scholar 

  106. Hogg P, Rooney P, Leow-Dyke S, Brown C, Ingham E, Kearney JN. Development of a terminally sterilised decellularised dermis. Cell Tissue Bank. 2015;16(3):351–9.

    Article  CAS  PubMed  Google Scholar 

  107. Dua HS, Maharajan VS, Hopkinson A. Controversies and limitations of amniotic membrane in ophthalmic surgery. In: Reinhard T, Larkin DFP, editors. Cornea and external eye disease. Berlin/Heidelberg: Springer, Berlin Heidelberg; 2006. p. 21–33.

    Chapter  Google Scholar 

  108. Chen B, Jones RR, Mi S, Foster JW, Alcock SG, Hamley IW, et al. The mechanical properties of amniotic membrane influence its effect as a biomaterial for ocular surface repair. Soft Matter. 2012;8(32):8379–87.

    Article  CAS  Google Scholar 

  109. Jones RR, Hamley IW, Connon CJ. Ex vivo expansion of limbal stem cells is affected by substrate properties. Stem Cell Res. 2012;8(3):403–9.

    Article  CAS  PubMed  Google Scholar 

  110. Guo WH, Frey MT, Burnham NA, Wang YL. Substrate rigidity regulates the formation and maintenance of tissues. Biophys J. 2006;90(6):2213–20.

    Article  CAS  PubMed  Google Scholar 

  111. Engler AJ, Sen S, Sweeney HL, Discher DE. Matrix elasticity directs stem cell lineage specification. Cell. 2006;126(4):677–89.

    Article  CAS  PubMed  Google Scholar 

  112. Foster JW, Jones RR, Bippes CA, Gouveia RM, Connon CJ. Differential nuclear expression of Yap in basal epithelial cells across the cornea and substrates of differing stiffness. Exp Eye Res. 2014;127:37–41.

    Article  CAS  PubMed  Google Scholar 

  113. Molladavoodi S, Kwon HJ, Medley J, Gorbet M. Human corneal epithelial cell response to substrate stiffness. Acta Biomater. 2015;11:324–32.

    Article  CAS  PubMed  Google Scholar 

  114. Lepert G, Gouveia RM, Connon CJ, Paterson C. Assessing corneal biomechanics with Brillouin spectro-microscopy. Faraday Discuss. 2016;187(0):415–28.

    Article  CAS  PubMed  Google Scholar 

  115. Chen Z, Du T, Tang X, Liu C, Li R, Xu C, et al. Comparison of the properties of collagen-chitosan scaffolds after gamma-ray irradiation and carbodiimide cross-linking. J Biomater Sci Polym Ed. 2016;27(10):937–53.

    Article  CAS  PubMed  Google Scholar 

  116. Mi S, Khutoryanskiy VV, Jones RR, Zhu X, Hamley IW, Connon CJ. Photochemical cross-linking of plastically compressed collagen gel produces an optimal scaffold for corneal tissue engineering. J Biomed Mater Res A. 2011;99((1):1–8.

    Article  CAS  Google Scholar 

  117. Figueiredo GS, Bojic S, Rooney P, Wilshaw SP, Connon CJ, Gouveia RM, et al. Gamma-irradiated human amniotic membrane decellularised with sodium dodecyl sulfate is a more efficient substrate for the ex vivo expansion of limbal stem cells. Acta Biomater. 2017;61:124–33.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  118. Rooney P, Eagle M, Hogg P, Lomas R, Kearney J. Sterilisation of skin allograft with gamma irradiation. Burns. 2008;34(5):664–73.

    Article  CAS  PubMed  Google Scholar 

  119. Baylis O, Rooney P, Figueiredo F, Lako M, Ahmad S. An investigation of donor and culture parameters which influence epithelial outgrowths from cultured human cadaveric limbal explants. J Cell Physiol. 2013;228(5):1025–30.

    Article  CAS  PubMed  Google Scholar 

  120. Regulation (EC) No 1394/2007 of the European Parliament and of the Council of 13 November 2007 on advanced therapy medicinal products and amending Directive 2001/83/EC and Regulation (EC) No726/2004., L324 (2007).

    Google Scholar 

  121. (NICE) NIfHaCE. Holoclar for treating limbal stem cell deficiency after eye burns NICE Guidance2017. Available from: https://www.nice.org.uk/guidance/ta467.

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Compliance with Ethical Requirements

Gustavo S. Figueiredo, Hardeep S. Mudhar, Majlinda Lako and Francisco C. Figueiredo declare that they have no conflict of interest. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) 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 performed by the authors for this chapter.

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Authors and Affiliations

  1. Newcastle University, Institute of Genetic Medicine, International Centre for Life, Newcastle upon Tyne, Tyne and Wear, UK

    Gustavo S. Figueiredo, Majlinda Lako & Francisco C. Figueiredo

  2. Royal Hallamshire Hospital, Department of Histopathology, Sheffield, South Yorkshire, UK

    Hardeep Singh Mudhar

  3. Royal Victoria Infirmary, Newcastle upon Tyne, UK

    Francisco C. Figueiredo

Authors
  1. Gustavo S. Figueiredo
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  2. Hardeep Singh Mudhar
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  3. Majlinda Lako
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  4. Francisco C. Figueiredo
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Corresponding author

Correspondence to Francisco C. Figueiredo .

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Editors and Affiliations

  1. Miguel Hernández University , Alicante, Spain

    Jorge L. Alió

  2. Vissum-Instituto Oftalmologico de Alica, University Miguel Hernandez, Alicante, Alicante, Spain

    Jorge L. Alió del Barrio

  3. Vissum Corporation, Madrid, Spain

    Francisco Arnalich-Montiel

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Figueiredo, G.S., Mudhar, H.S., Lako, M., Figueiredo, F.C. (2019). Corneal Epithelial Stem Cells: Methods for Ex Vivo Expansion. 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_6

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  • DOI: https://doi.org/10.1007/978-3-030-01304-2_6

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