Limbal Stromal Stem Cells in Corneal Wound Healing: Current Perspectives and Future Applications

  • Noopur Mitragotri
  • Mukesh Damala
  • Vivek SinghEmail author
  • Sayan BasuEmail author
Part of the Essentials in Ophthalmology book series (ESSENTIALS)


Corneal pathologies are one of the leading causes of visual impairment and blindness globally. The traditional and most commonly performed corneal transplantation is limited by the inadequacy of the donor tissues and graft failure. The recent discovery of mesenchymal stem cells in the limbus and their presence from many other sources of the human body, capable of modulating corneal wound healing, has opened up the possibility of using cell-based therapy and ameliorating the corneal blindness. Human limbal-derived stem cells have manifested and proven their efficacy through various well-established surgical interventions over the past decade. The current chapter deals with the role of mesenchymal stem cells obtained from limbal stroma in wound healing, reducing scarring in superficial corneal pathologies like scars, as evident from the results of preclinical studies. Various factors involved in the corneal repair mechanisms and the different sources of mesenchymal stem cells are mentioned. Different clinical approaches and various stem cell therapies were addressed along with the main highlights of our current clinical trial with a note on the requirements for the maintenance of a cGMP facility.


Corneal haze Corneal scar Cell-based therapy Corneal wound healing cGMP Clinical trial Mesenchymal stem cells Limbus Limbal stroma 



We acknowledge Mr. Abhinav Reddy Kethiri, Brien Holden Eye Research Center, and Mr. Sridhar Rao Boyinpally, Department of Pathology, LV Prasad Eye Institute, for helping in generating the images for this chapter. We thank Champalimaud Foundation and Tej Kohli Cornea Institute and LV Prasad Eye Institute for providing the financial assistance and manpower for this chapter.

Conflict of Interest

Noopur Mitragotri, Mukesh Damala, Vivek Singh and Sayan Basu declare that they have no conflict of interest.

Informed consent was obtained along with the consent for publication.


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


  1. 1.
    Oommen V, Kanthakumar P. A simple model of the accommodating lens of the human eye. Adv Physiol Educ. 2014;38(2):183–4.PubMedPubMedCentralCrossRefGoogle Scholar
  2. 2.
    Hanna C, Bicknell DS, O’Brien JE. Cell turnover in the adult human eye. Arch Ophthalmol. 1961;65:695–8.PubMedCrossRefGoogle Scholar
  3. 3.
    Torricelli AA, Singh V, Santhiago MR, Wilson SE. The corneal epithelial basement membrane: structure, function, and disease. Invest Ophthalmol Vis Sci. 2013;54(9):6390–400.PubMedPubMedCentralCrossRefGoogle Scholar
  4. 4.
    Torricelli AA, Santhanam A, Wu J, Singh V, Wilson SE. The corneal fibrosis response to epithelial-stromal injury. Exp Eye Res. 2016;142:110–8.PubMedPubMedCentralCrossRefGoogle Scholar
  5. 5.
    Jacobsen IE, Jensen OA, Prause JU. Structure and composition of Bowman’s membrane. Study by frozen resin cracking. Acta Ophthalmol. 1984;62(1):39–53.CrossRefGoogle Scholar
  6. 6.
    Nishida K, Kawasaki S, Kinoshita S. Clusterinmay be essential for maintaining ocular surface epithelium as non-keratinizing epithelium. Adv Exp Med Biol. 1998;438:629–35.PubMedCrossRefGoogle Scholar
  7. 7.
    Funderburgh JL, Funderburgh ML, Du Y. Stem cells in the limbal stroma. Ocul Surf. 2016;14(2):113–20.PubMedPubMedCentralCrossRefGoogle Scholar
  8. 8.
    Murphy C, Alvarado J, Juster R. Prenatal and postnatal growth of the human Descemet’s membrane. Invest Ophthalmol Vis Sci. 1984;25(12):1402–15.PubMedPubMedCentralGoogle Scholar
  9. 9.
    Dandona R, Dandona L. Corneal blindness in a southern Indian population: need for health promotion strategies. Br J Ophthalmol. 2003;87(2):133–41.PubMedPubMedCentralCrossRefGoogle Scholar
  10. 10.
    Gupta N, Tandon R, Gupta SK, Sreenivas V, Vashist P. Burden of corneal blindness in India. Indian J Community Med. 2013;38(4):198–206.PubMedPubMedCentralCrossRefGoogle Scholar
  11. 11.
    Dua HS, Azuara-Blanco A. Limbal stem cells of the corneal epithelium. Surv Ophthalmol. 2000;44(5):415–25.PubMedCrossRefGoogle Scholar
  12. 12.
    Kulkarni BB, Tighe PJ, Mohammed I, Yeung AM, Powe DG, Hopkinson A, Shanmuganathan VA, Dua HS. Comparative transcriptional profiling of the limbal epithelial crypt demonstrates its putative stem cell niche characteristics. BMC Genomics. 2010;11:526.PubMedPubMedCentralCrossRefGoogle Scholar
  13. 13.
    Branch MJ, Hashmani K, Dhillon P, Jones DR, Dua HS, Hopkinson A. Mesenchymal stem cells in the human corneal limbal stroma. Invest Ophthalmol Vis Sci. 2012;53(9):5109–16.PubMedPubMedCentralCrossRefGoogle Scholar
  14. 14.
    Funderburgh ML, Du Y, Mann MM, SundarRaj N, Funderburgh JL. PAX6 expression identifies progenitor cells for corneal keratocytes. FASEB J. 2005;19(10):1371–3.PubMedPubMedCentralCrossRefGoogle Scholar
  15. 15.
    Damala M, Kethiri R, Tavakkoli F, Raju E, Singh V. The basics of stem cells and their role in vision. In: Trends in life science research. Hauppauge, New York: Nova Science Publishers, Inc; 2018.Google Scholar
  16. 16.
    Yoshida S, Shimmura S, Nagoshi N, Fukuda K, Matsuzaki Y, Okano H, Tsubota K. Isolation of multi-potent neural crest-derived stem cells from the adult mouse cornea. Stem Cells. 2006;24(12):2714–22.PubMedPubMedCentralCrossRefGoogle Scholar
  17. 17.
    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.PubMedPubMedCentralCrossRefGoogle Scholar
  18. 18.
    Mathews S, Chidambaram JD, Lanjewar S, Mascarenhas J, Prajna NV, Muthukkaruppan V, Chidambaranathan GP. In vivo confocal microscopic analysis of normal human anterior limbal stroma. Cornea. 2015;34(4):464–70.PubMedPubMedCentralCrossRefGoogle Scholar
  19. 19.
    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;57:201–9.CrossRefGoogle Scholar
  20. 20.
    Pinnamaneni N, Funderburgh JL. Concise review: stem cells in the corneal stroma. Stem Cells. 2012;30(6):1059–63.PubMedPubMedCentralCrossRefGoogle Scholar
  21. 21.
    Kureshi AK, Dziasko M, Funderburgh JL, Daniels JT. Human corneal stromal stem cells support limbal epithelial cells cultured on RAFT tissue equivalents. Sci Rep. 2015;5:16186.PubMedPubMedCentralCrossRefGoogle Scholar
  22. 22.
    Eraslan M, Toker E. Mechanisms of corneal wound healing and its modulation following refractive surgery. Marmara Med J. 2009;22(2):169–78.Google Scholar
  23. 23.
    Singh V, Barbosa FL, Torricelli AA, Santhiago MR, Wilson SE. Transforming growth factor beta and platelet-derived growth factor modulation of myofibroblast development from corneal fibroblasts in vitro. Exp Eye Res. 2014;120:152–60.PubMedPubMedCentralCrossRefGoogle Scholar
  24. 24.
    Singh V, Jaini R, Torricelli AA, Santhiago MR, Singh N, Ambati BK, Wilson SE. TGFbeta and PDGF-B signaling blockade inhibits myofibroblast development from both bone marrow-derived and keratocyte-derived precursor cells in vivo. Exp Eye Res. 2014;121:35–40.PubMedPubMedCentralCrossRefGoogle Scholar
  25. 25.
    Wilson SE, Schultz GS, Chegini N, Weng J, He YG. Epidermal growth factor, transforming growth factor alpha, transforming growth factor beta, acidic fibroblast growth factor, basic fibroblast growth factor, and interleukin-1 proteins in the cornea. Exp Eye Res. 1994;59(1):63–72.CrossRefGoogle Scholar
  26. 26.
    Weng J, Mohan RR, Li Q, Wilson SE. IL-1 up-regulates keratinocyte growth factor and hepatocyte growth factor mRNA and protein production by cultured stromal fibroblast cells: interleukin-1 beta expression in the cornea. Cornea. 1997;16(4):465–71.PubMedCrossRefGoogle Scholar
  27. 27.
    Jester JV, Petroll WM, Cavanagh HD. Corneal stromal wound healing in refractive surgery: the role of myofibroblasts. Prog Retin Eye Res. 1999;18(3):311–56.PubMedCrossRefGoogle Scholar
  28. 28.
    Kamiyama K, Iguchi I, Wang X, Imanishi J. Effects of PDGF on the migration of rabbit corneal fibroblasts and epithelial cells. Cornea. 1998;17(3):315–25.PubMedCrossRefGoogle Scholar
  29. 29.
    Kim WJ, Helena MC, Mohan RR, Wilson SE. Changes in corneal morphology associated with chronic epithelial injury. Invest Ophthalmol Vis Sci. 1999;40(1):35–42.PubMedGoogle Scholar
  30. 30.
    Frank MH, Frank NY. Restoring the cornea from limbal stem cells. Regen Med. 2015;10(1):1–4.PubMedPubMedCentralCrossRefGoogle Scholar
  31. 31.
    Kruse FE, Volcker HE. Stem cells, wound healing, growth factors, and angiogenesis in the cornea. Curr Opin Ophthalmol. 1997;8(4):46–54.CrossRefGoogle Scholar
  32. 32.
    Secker GA, Daniels JT. Limbal epithelial stem cells of the cornea. In: StemBook. Cambridge, MA: Harvard Stem Cell Institute; 2008.Google Scholar
  33. 33.
    Oie Y, Nishida K. Regenerative medicine for the cornea. Biomed Res Int. 2013;2013:428247.PubMedPubMedCentralCrossRefGoogle Scholar
  34. 34.
    Gao J, Dennis JE, Muzic RF, Lundberg M, Caplan AI. The dynamic in vivo distribution of bone marrow-derived mesenchymal stem cells after infusion. Cells Tissues Organs. 2001;169(1):12–20.PubMedCrossRefGoogle Scholar
  35. 35.
    Liu H, Zhang J, Liu CY, Hayashi Y, Kao WW. Bone marrow mesenchymal stem cells can differentiate and assume corneal keratocyte phenotype. J Cell Mol Med. 2012;16(5):1114–24.PubMedPubMedCentralCrossRefGoogle Scholar
  36. 36.
    Lan Y, Kodati S, Lee HS, Omoto M, Jin Y, Chauhan SK. Kinetics and function of mesenchymal stem cells in corneal injury. Invest Ophthalmol Vis Sci. 2012;53(7):3638–44.PubMedCrossRefGoogle Scholar
  37. 37.
    Tholpady SS, Llull R, Ogle RC, Rubin JP, Futrell JW, Katz AJ. Adipose tissue: stem cells and beyond. Clin Plast Surg. 2006;33(1):55–62.PubMedCrossRefGoogle Scholar
  38. 38.
    Du Y, Roh DS, Funderburgh ML, Mann MM, Marra KG, Rubin JP, Li X, Funderburgh JL. Adipose-derived stem cells differentiate to keratocytes in vitro. Mol Vis. 2010;16:2680–9.PubMedPubMedCentralGoogle Scholar
  39. 39.
    Ranganathan K, Lakshminarayanan V. Stem cells of the dental pulp. Indian J Dent Res. 2012;23(4):558.PubMedCrossRefGoogle Scholar
  40. 40.
    Syed-Picard FN, Du Y, Lathrop KL, Mann MM, Funderburgh ML, Funderburgh JL. Dental pulp stem cells: a new cellular resource for corneal stromal regeneration. Stem Cells Transl Med. 2015;4(3):276–85.PubMedPubMedCentralCrossRefGoogle Scholar
  41. 41.
    El Omar R, Beroud J, Stoltz JF, Menu P, Velot E, Decot V. Umbilical cord mesenchymal stem cells: the new gold standard for mesenchymal stem cell-based therapies? Tissue Eng Part B Rev. 2014;20(5):523–44.PubMedCrossRefGoogle Scholar
  42. 42.
    Schwartz SD, Hubschman JP, Heilwell G, Franco-Cardenas V, Pan CK, Ostrick RM, Lanza R. Embryonic stem cell trials for macular degeneration: a preliminary report. Lancet. 2012;379(9817):713–20.PubMedCrossRefGoogle Scholar
  43. 43.
    Schwartz SD, Regillo CD, Lam BL, Eliott D, Rosenfeld PJ, Gregori NZ, Hubschman JP, Davis JL, Heilwell G, Spirn M, Maguire J, et al. Human embryonic stem cell-derived retinal pigment epithelium in patients with age-related macular degeneration and Stargardt’s macular dystrophy: follow-up of two open-label phase 1/2 studies. Lancet. 2015;385(9967):509–16.PubMedCrossRefGoogle Scholar
  44. 44.
    Pellegrini G, De Luca M. Eyes on the prize: limbal stem cells and corneal restoration. Cell Stem Cell. 2014;15(2):121–2.PubMedCrossRefGoogle Scholar
  45. 45.
    Reardon S, Cyranoski D. Japan stem-cell trial stirs envy: researchers elsewhere can't wait to test iPS cells in humans. Nature. 2014;513(7518):287–9.PubMedCrossRefGoogle Scholar
  46. 46.
    Turner M, Leslie S, Martin NG, Peschanski M, Rao M, Taylor CJ, Yamanaka S, Wilmut I. Toward the development of a global induced pluripotent stem cell library. Cell Stem Cell. 2013;13(4):382–4.PubMedCrossRefGoogle Scholar
  47. 47.
    Fuentes-Julián S, Arnalich-Montiel F, Jaumandreu L, Leal M, Casado A, García-Tuñon I, De Miguel MP. Adipose-derived mesenchymal stem cell administration does not improve corneal graft survival outcome. PLoS One. 2015;10(3):e0117945.PubMedPubMedCentralCrossRefGoogle Scholar
  48. 48.
    Du Y, Carlson EC, Funderburgh ML, Birk DE, Pearlman E, Guo N, Kao WW, Funderburgh JL. Stem cell therapy restores transparency to defective murine corneas. Stem Cells. 2009;27(7):1635–42.PubMedPubMedCentralCrossRefGoogle Scholar
  49. 49.
    Basu S, Fernandez MM, Das S, Gaddipati S, Vemuganti GK, Sangwan VS. Clinical outcomes of xeno-free allogeneic cultivated limbal epithelial transplantation for bilateral limbal stem cell deficiency. Br J Ophthalmol. 2012;96(12):1504–9.PubMedPubMedCentralCrossRefGoogle Scholar
  50. 50.
    Du Y, Sundarraj N, Funderburgh ML, Harvey SA, Birk DE, Funderburgh JL. Secretion and organization of a cornea-like tissue in vitro by stem cells from human corneal stroma. Invest Ophthalmol Vis Sci. 2007;48(11):5038–45.PubMedPubMedCentralCrossRefGoogle Scholar
  51. 51.
    Karamichos D, Funderburgh ML, Hutcheon AE, Zieske JD, Du Y, Wu J, Funderburgh JL. A role for topographic cues in the organization of collagenous matrix by corneal fibroblasts and stem cells. PLoS One. 2014;9(1):e86260.PubMedPubMedCentralCrossRefGoogle Scholar
  52. 52.
    Chan AA, Hertsenberg AJ, Funderburgh ML, Mann MM, Du Y, Davoli KA, Mich-Basso JD, Yang L, Funderburgh JL. Differentiation of human embryonic stem cells into cells with corneal keratocyte phenotype. PLoS One. 2013;8(2):e56831.PubMedPubMedCentralCrossRefGoogle Scholar
  53. 53.
    Basu S, Hertsenberg AL, Funderburgh MK, Burro MM, Man M, Du Y, Lathrop K, Syed-Picard FM, Adam SE, Bir D, Funderburgh J. Human limbal biopsy-derived stromal stem cells prevent corneal scarring. Sci Transl Med. 2014;6(266):266ra172.PubMedPubMedCentralCrossRefGoogle Scholar
  54. 54.
    Ravinder RB, Suresh N. Regulatory stages for new drug approvals. 2011. Available at
  55. 55.
    Giancola R, Bonfini T, Iacone A. Cell therapy: cGMP facilities and manufacturing. Muscles Ligaments Tendons J. 2012;2(3):243–7.PubMedPubMedCentralGoogle Scholar
  56. 56.
    Dietz AB, Padley DJ, Gastineau DA. Infrastructure development for human cell therapy translation. Clin Pharmacol Ther. 2007;82(3):320–4.PubMedCrossRefGoogle Scholar
  57. 57.
    Lim M, Goldstein MH, Tuli S, Schultz GS. Growth factor, cytokine and protease interactions during corneal wound healing. Ocul Surf. 2003;1(2):53–65.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  1. 1.Center to Ocular Regeneration (CORE); and Brien Holden Eye Research Center, LV Prasad Eye InstituteHyderabadIndia
  2. 2.School of Life Sciences, University of HyderabadHyderabadIndia
  3. 3.Center for Ocular Regeneration (CORE), LV Prasad Eye Institute, Banajara HillsHyderabadIndia

Personalised recommendations