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Ex-vivo ocular surface stem cell therapies: current techniques, applications, hurdles and future directions

  • Romesh I. Angunawela (a1), Jodhbir S. Mehta (a2) (a3) and Julie T. Daniels (a4)

Engineered tissue derived from ocular surface stem cells (SCs) are a cutting edge biotechnology for repair and restoration of severely damaged eyes as a result of ocular surface dysfunction because of SC failure. Ex-vivo SC expansion techniques have advanced significantly since the first patients were treated in the late 1990s. The techniques and clinical reports reviewed here highlight the evolution and successes of these techniques, while also revealing gaps in our understanding of ocular surface and SC biology that drives further research and development in this field. Although hurdles still remain before stem-cell-based therapies are more widely available for patients with devastating ocular surface disease, recent discoveries in the field of mesenchymal SCs and the potential of induced pluripotent SCs heralds a promising future for clinicians and our patients.

Corresponding author
*Corresponding author: Julie T. Daniels, Department of Ocular Biology and Therapeutics, UCL Institute of Ophthalmology, 11-43 Bath Street, London EC1 V 9EL, UK. E-mail:
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1 S.M. Daya , C.C. Chan and EJ Holland . (2011) Members of the cornea society ocular surface procedures nomenclature committee. Cornea Society nomenclature for ocular surface rehabilitative procedures. Cornea 30, 1115-1119

3 S. Kinoshita (2009) Paradigm shift in therapeutic modalities for devastating ocular surface disorders. Cornea 28 (Suppl 1)

4 A. Schermer , S. Galvin and T.T. Sun (1986) Differentiation-related expression of a major 64 K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells. Journal of Cell Biology 103, 49-62

5 J.E. Till , E.A. McCulloch and L. Siminovitch (1964) A stochastic model of stem cell proliferation, based on the growth of spleen colony-forming cells. Proceedings of the National Academy of Sciences of the United States of America 51, 29-36

6 Y. Barrandon and H. Green (1987) Three clonal types of keratinocyte with different capacities for multiplication. Proceedings of the National Academy of Sciences of the United States of America 84, 2302-2306

7 G. Cotsarelis (1989) Existence of slow-cycling limbal epithelial basal cells that can be preferentially stimulated to proliferate: implications on epithelial stem cells. Cell 57, 201-209

9 W. Li (2007) Niche regulation of corneal epithelial stem cells at the limbus. Cell Research 17, 26-36

11 A.J. Shortt (2007) 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 25, 1402-1409

12 V.A. Shanmuganathan (2007) Morphological characteristics of the limbal epithelial crypt. British Journal of Ophthalmology 91, 514-519

13 H.S. Dua (2005) Limbal epithelial crypts: a novel anatomical structure and a putative limbal stem cell niche. British Journal of Ophthalmology 89, 529-532

14 S. Kawasaki (2006) Clusters of corneal epithelial cells reside ectopically in human conjunctival epithelium. Investigative Ophthalmology and Visual Science 47, 1359-1367

15 F. Majo (2008) Oligopotent stem cells are distributed throughout the mammalian ocular surface. Nature 456, 250-254

16 G. Pellegrini (1999) Location and clonal analysis of stem cells and their differentiated progeny in the human ocular surface. Journal of Cell Biology 145, 769-782

18 S. Ahmad (2010) Stem cell therapies for ocular surface disease. Drug Discovery Today 15, 306-313

19 A.J. Shortt , S.J. Tuft and J.T. Daniels (2011) Corneal stem cells in the eye clinic. British Medical Bulletin 100, 209-225

21 C. Jenkins (1993) Limbal transplantation in the management of chronic contact-lens-associated epitheliopathy. Eye (London) 7 (Pt 5), 629-633

22 G. Pellegrini (1997) Long-term restoration of damaged corneal surfaces with autologous cultivated corneal epithelium. Lancet 349, 990-993

23 I.R. Schwab , M. Reyes and R.R. Isseroff (2000) Successful transplantation of bioengineered tissue replacements in patients with ocular surface disease. Cornea 19, 421-426

24 R.J.F. Tsai , L.-M. Li and J.-K. Chen (2000) Reconstruction of damaged corneas by transplantation of autologous limbal epithelial cells. New England Journal of Medicine 343, 86-93

26 T. Kawakita (2009) Greater growth potential of p63-positive epithelial cell clusters maintained in human limbal epithelial sheets. Investigative Ophthalmology and Visual Science 50, 4611-4617

27 V.S. Sangwan (2003) Use of autologous cultured limbal and conjunctival epithelium in a patient with severe bilateral ocular surface disease induced by acid injury: a case report of unique application. Cornea 22, 478-481

28 J. Shimazaki (2002) Transplantation of human limbal epithelium cultivated on amniotic membrane for the treatment of severe ocular surface disorders. Ophthalmology 109, 1285-1290

29 M. Kawashima (2007) Phenotypic study after cultivated limbal epithelial transplantation for limbal stem cell deficiency. Archives Ophthalmology 125, 1337-1344

30 S. Kolli (2008) Loss of corneal epithelial stem cell properties in outgrowths from human limbal explants cultured on intact amniotic membrane. Regenerative Medicine 3, 329-342

31 W. Li (2007) The fate of limbal epithelial progenitor cells during explant culture on intact amniotic membrane. Investigative Ophthalmology and Visual Science 48, 605-613

32 J.G. Rheinwald (1980) Serial cultiva tion of normal human epiderm al keratinocytes. In Methods in Cell Biology ( C.C. Harris , B.F. Trump and G.O. Stoner , eds), vol 21A, pp. 229-254, Academic Press, New York

34 J.G. Rheinwald and H. Green (1975) Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6, 331-343

35 S. Johnen (2011) Presence of xenogenic mouse RNA in RPE and IPE cells cultured on mitotically inhibited 3T3 fibroblasts. Investigative Ophthalmology and Visual Science 52, 2817-2824

36 L.P. Ang (2011) Ex vivo expansion of conjunctival and limbal epithelial cells using cord blood serum-supplemented culture medium. Investigative Ophthalmology and Visual Science 52, 6138-6147

37 T. Nakamura (2006) Transplantation of autologous serum-derived cultivated corneal epithelial equivalents for the treatment of severe ocular surface disease. Ophthalmology 113, 1765-1772

38 T. Nakamura (2006) The use of autologous serum in the development of corneal and oral epithelial equivalents in patients with Stevens-Johnson syndrome. Investigative Ophthalmology and Visual Science 47, 909-916

39 A. Shahdadfar (2012) Ex vivo expanded autologous limbal epithelial cells on amniotic membrane using a culture medium with human serum as single supplement. Experimental Eye Research 97, 1-9

40 R. Bergström (2011) Xeno-free culture of human pluripotent stem cells. Methods in Molecular Biology 767, 125-136

41 Y.T. Chen (2007) Human amniotic epithelial cells as novel feeder layers for promoting ex vivo expansion of limbal epithelial progenitor cells. Stem Cells 25, 1995-2005

42 M. Omoto (2009) The use of human mesenchymal stem cell-derived feeder cells for the cultivation of transplantable epithelial sheets. Investigative Ophthalmology and Visual Science 50, 2109–2101

43 V.S. Sangwan (2011) Clinical outcomes of xeno-free autologous cultivated limbal epithelial transplantation: a 10-year study. British Journal of Ophthalmology 95, 1525-1529

44 N. Zakaria (2010) Standardized limbal epithelial stem cell graft generation and transplantation. Tissue Engineering Part C Methods 16, 921-927

45 D. Meller (2009) Ocular surface reconstruction in graft-versus-host disease with HLA-identical living-related allogeneic cultivated limbal epithelium after hematopoietic stem cell transplantation from the same donor. Cornea 28, 233-236

48 J. Shimazaki (2007) Factors influencing outcomes in cultivated limbal epithelial transplantation for chronic cicatricial ocular surface disorders. American Journal of Ophthalmology 143, 945-953

49 J.C. Kim and S.C. Tseng (1995) Transplantation of preserved human amniotic membrane for surface reconstruction in severely damaged rabbit corneas. Cornea 14, 473-484

50 D.F. Anderson (2001) Amniotic membrane transplantation for partial limbal stem cell deficiency. British Journal of Ophthalmology 85, 567-575

51 J.A. Gomes (2003) Amniotic membrane transplantation for partial and total limbal stem cell deficiency secondary to chemical burn. Ophthalmology 110, 466-473

52 A.J. Shortt (2009) 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 30, 1056-1065

54 E.E. Hernandez Galindo (2003) Expression of Delta Np63 in response to phorbol ester in human limbal epithelial cells expanded on intact human amniotic membrane. Investigative Ophthalmology and Visual Science 44, 2959-2965

55 N. Koizumi (2007) Comparison of intact and denuded amniotic membrane as a substrate for cell-suspension culture of human limbal epithelial cells. Graefe's Archive for Clinical and Experimental Ophthalmology 245, 123-134

56 K.R. Kenyon (2005) Amniotic membrane: mother's own remedy for ocular surface disease. Cornea 24, 639-642

57 E.A. Meyer-Blazejewska (2010) Preservation of the limbal stem cell phenotype by appropriate culture techniques. Investigative Ophthalmology and Visual Science 51, 765-774

58 K. Higa (2007) Proliferation and differentiation of transplantable rabbit epithelial sheets engineered with or without an amniotic membrane carrier. Investigative Ophthalmology and Visual Science 48, 597-604

60 P. Rama (2001) Autologous fibrin-cultured limbal stem cells permanently restore the corneal surface of patients with total limbal stem cell deficiency. Transplantation 72, 1478-1485

61 P. Rama (2010) Limbal stem-cell therapy and long-term corneal regeneration. New England Journal of Medicine 363, 147-155

62 G. Sitalakshmi (2009) Ex vivo cultivation of corneal limbal epithelial cells in a thermoreversible polymer (Mebiol Gel) and their transplantation in rabbits: an animal model. Tissue Engineering Part A 15, 407-415

63 K. Nishida (2004) Corneal reconstruction with tissue-engineered cell sheets composed of autologous oral mucosal epithelium. New England Journal of Medicine 351, 1187-1196

64 N. Di Girolamo (2009) A contact lens-based technique for expansion and transplantation of autologous epithelial progenitors for ocular surface reconstruction. Transplantation 87, 1571-1578

65 T. Chirila (2008) Bombyx mori silk fibroin membranes as potential substrata for epithelial constructs used in the management of ocular surface disorders. Tissue Engineering Part A 14, 1203-1211

66 H.J. Levis , R.A. Brown and J.T. Daniels (2010) Plastic compressed collagen as a biomimetic substrate for human limbal epithelial cell culture. Biomaterials 31, 7726-7737

67 M. Pruniéras , M. Régnier and D. Woodley (1983) Methods for cultivation of keratinocytes with an air-liquid interface. Journal of Investigative Dermatology 81(1 Suppl), 28s-33s

68 W. Li (2008) Air exposure induced squamous metaplasia of human limbal epithelium. Investigative Ophthalmology and Visual Science 49, 154-162

69 M. Pauklin (2010) Midterm results of cultivated autologous and allogeneic limbal epithelial transplantation in limbal stem cell deficiency. Developments in Ophthalmology 45, 57-70

70 S.M. Daya (2005) Outcomes and DNA analysis of ex vivo expanded stem cell allograft for ocular surface reconstruction. Ophthalmology 112, 470-477

71 L.P. Ang (2006) Autologous serum-derived cultivated oral epithelial transplants for severe ocular surface disease. Archives of Ophthalmology 124, 1543-1551

72 C. Burillon (2012) Cultured autologous oral mucosal epithelial cell sheet (CAOMECS) transplantation for the treatment of corneal limbal epithelial stem cell deficiency. Investigative Ophthalmology and Visual Science 53, 1325-1332

73 C.G. Priya (2011) Adult human buccal epithelial stem cells: identification, ex-vivo expansion, and transplantation for corneal surface reconstruction. Eye (London) 25, 1641-1649

74 H.C. Chen (2009) Persistence of transplanted oral mucosal epithelial cells in human cornea. Investigative Ophthalmology and Visual Science 50, 4660-4668

75 Y. Hori (2008) Differential expression of MUC16 in human oral mucosal epithelium and cultivated epithelial sheets. Experimental Eye Research 87, 191-196

77 H. Tanioka (2006) Establishment of a cultivated human conjunctival epithelium as an alternative tissue source for autologous corneal epithelial transplantation. Investigative Ophthalmology and Visual Science 47, 3820-3827

78 D. Meller , V. Dabul and S.C. Tseng (2002) Expansion of conjunctival epithelial progenitor cells on amniotic membrane. Experimental Eye Research 74, 537-545

80 L.P. Ang (2010) Cultivated human conjunctival epithelial transplantation for total limbal stem cell deficiency. Investigating Ophthalmology and Visual Science 51, 758-764

81 N. Scuderi (2002) Transplantation of autologous cultivated conjunctival epithelium for the restoration of defects in the ocular surface. Scandinavian Journal of Plastic and Reconstructive Surgery and Hand Surgery 36, 340-348

82 D.T. Tan , L.P. Ang and R.W. Beuerman (2004) Reconstruction of the ocular surface by transplantation of a serum-free derived cultivated conjunctival epithelial equivalent. Transplantation 77, 1729-1734

84 N. Koizumi (2001) Cultivated corneal epithelial stem cell transplantation in ocular surface disorders. Ophthalmology 108, 1569-1574

86 T. Nakamura (2003) Successful regrafting of cultivated corneal epithelium using amniotic membrane as a carrier in severe ocular surface disease. Cornea 22, 70-71

87 T. Nakamura (2004) Successful primary culture and autologous transplantation of corneal limbal epithelial cells from minimal biopsy for unilateral severe ocular surface disease. Acta Ophthalmologica Scandinavica 82, 468-471

88 V.S. Sangwan (2005) Cultivated corneal epithelial transplantation for severe ocular surface disease in vernal keratoconjunctivitis. Cornea 24, 426-430

89 V.S. Sangwan (2006) Clinical outcome of autologous cultivated limbal epithelium transplantation. Indian Journal of Ophthalmology 54, 29-34

90 A.J. Shortt (2006) Ex-vivo expansion and transplantation of limbal epithelial stem cells. Ophthalmology 115, 1989-1997

91 T. Nakamura (2010) Long-term phenotypic study after allogeneic cultivated corneal limbal epithelial transplantation for severe ocular surface diseases. Ophthalmology 117, 2247-2254

92 A. Baradaran-Rafii (2010) Midterm outcomes of autologous cultivated limbal stem cell transplantation with or without penetrating keratoplasty. Cornea 29, 502-509

93 S. Basu , H. Ali and V.S. Sangwan (2012) Clinical outcomes of repeat autologous cultivated limbal epithelial transplantation for ocular surface burns. American Journal of Ophthalmology 153, 643-650, 650.e1-2

94 T. Nakamura (2004) Transplantation of cultivated autologous oral mucosal epithelial cells in patients with severe ocular surface disorders. British Journal of Ophthalmology 88, 1280-1284

95 T. Inatomi (2006) Midterm results on ocular surface reconstruction using cultivated autologous oral mucosal epithelial transplantation. American Journal of Ophthalmology 141, 267-275

96 T. Inatomi (2006) Ocular surface reconstruction with combination of cultivated autologous oral mucosal epithelial transplantation and penetrating keratoplasty. American Journal of Ophthalmology 142, 757-764

97 Y. Satake (2011) Long-term outcome of cultivated oral mucosal epithelial sheet transplantation in treatment of total limbal stem cell deficiency. Ophthalmology 118, 1524-1530

98 T. Nakamura (2011) Long-term results of autologous cultivated oral mucosal epithelial transplantation in the scar phase of severe ocular surface disorders. British Journal of Ophthalmology 95, 942-946

99 M. Hirayama (2012) Transplantation of cultivated oral mucosal epithelium prepared in fibrin-coated culture dishes. Investigative Ophthalmology and Visual Science 53, 1602-1609

100 J.R. Ricardo (2012) Transplantation of conjunctival epithelial cells cultivated ex vivo in patients with total limbal stem cell deficiency. Cornea 32, 221-8

101 R.L. Mort (2009) Mosaic analysis of stem cell function and wound healing in the mouse corneal epithelium. BMC Developmental Biology 9, 4

102 S. Schrader (2009) Tissue engineering for conjunctival reconstruction: established methods and future outlooks. Current Eye Research 34, 913-924

103 A. Tan (2012) Osteo-odonto keratoprosthesis: systematic review of surgical outcomes and complication rates. Ocular Surface 10, 15-25

104 J.H. Kim (2010) Ocular surface reconstruction with autologous nasal mucosa in cicatricial ocular surface disease. American Journal of Ophthalmology 149, 45-53

105 J.R. Eidet (2012) Effect of biopsy location and size on proliferative capacity of ex vivo expanded conjunctival tissue. Investigative Ophthalmology and Visual Science 53, 2897-2903

106 S. Schrader (2009) Conjunctival epithelial cells maintain stem cell properties after long-termculture and cryopreservation. Regenerative Medicine 4, 677-687

107 A. Miri , B. Al-Deiri and H.S. Dua (2010) Long-term outcomes of autolimbal and allolimbal transplants. Ophthalmology 117, 1207-1213

109 M.J. Branch (2012) Mesenchymal Stem Cells in the Human Corneal Limbal Stroma. Investigative Ophthalmology and Visual Science 53, 5109-16

110 R. Hayashi (2012) Generation of corneal epithelial cells from induced pluripotent stem cells derived from human dermal fibroblast and corneal limbal epithelium. PLoS ONE 7, e45435

111 T. Nakamura (2007) Phenotypic investigation of human eyes with transplanted autologous cultivated oral mucosal epithelial sheets for severe ocular surface diseases. Ophthalmology 114, 1080-1088

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