Cell Based Therapy for Corneal Endothelial Regeneration

  • Noriko KoizumiEmail author
  • Naoki Okumura
Part of the Essentials in Ophthalmology book series (ESSENTIALS)


Cell based therapy, as an alternative to conventional donor corneal transplantation, is anticipated to provide a less invasive and more effective therapeutic modality for corneal endothelial dysfunction. The proliferative capability of human corneal endothelial cells is severely controlled, thus making the establishment of optimal conditions for the cultivation of human corneal endothelial cells a critical aspect for the clinical application of a cell based therapy. Recently, successful cultivation protocol of human corneal endothelial cells for clinical application which includes the use of Rho kinase (ROCK) inhibitor and the inhibition of transforming growth factor beta signaling were reported to promote the cell proliferation and to support human corneal endothelial cell cultures that show high cell density. Animal experiments showed that a cell-injection therapy combined with the use of a ROCK inhibitor promotes cultivated corneal endothelial cell adhesion onto the posterior cornea, ultimately resulting in the recovery of corneal transparency. A pilot clinical study was recently initiated in Japan to investigate the efficacy of a cell-injection therapy for patients with bullous keratopathy.


Corneal endothelium Bullous keratopathy Cell based therapy Cell-injection therapy Rho kinase inhibitor 


Conflict of Interest

Noriko Koizumi and Naoki Okumura declare that they have no conflict of interest.” (If any authors do have a conflict of interest it must be spelled out specifically).

Informed Consent

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.

Animal Studies

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


  1. 1.
    Kaufman HE, Katz JI. Pathology of the corneal endothelium. Invest Ophthalmol Vis Sci. 1977;16(4):265–8.PubMedGoogle Scholar
  2. 2.
    Joyce NC. Proliferative capacity of the corneal endothelium. Prog Retin Eye Res. 2003;22(3):359–89.PubMedPubMedCentralCrossRefGoogle Scholar
  3. 3.
    Joyce NC, Meklir B, Joyce SJ, Zieske JD. Cell cycle protein expression and proliferative status in human corneal cells. Invest Ophthalmol Vis Sci. 1996;37(4):645–55.PubMedGoogle Scholar
  4. 4.
    Kim TY, Kim WI, Smith RE, Kay ED. Role of p27(Kip1) in cAMP- and TGF-beta2-mediated antiproliferation in rabbit corneal endothelial cells. Invest Ophthalmol Vis Sci. 2001;42(13):3142–9.PubMedGoogle Scholar
  5. 5.
    Paull AC, Whikehart DR. Expression of the p53 family of proteins in central and peripheral human corneal endothelial cells. Mol Vis. 2005;11:328–34.PubMedGoogle Scholar
  6. 6.
    Lee JG, Kay EP. Involvement of two distinct ubiquitin E3 ligase systems for p27 degradation in corneal endothelial cells. Invest Ophthalmol Vis Sci. 2008;49(1):189–96.PubMedCrossRefGoogle Scholar
  7. 7.
    Joyce NC, Harris DL, Mello DM. Mechanisms of mitotic inhibition in corneal endothelium: contact inhibition and TGF-beta2. Invest Ophthalmol Vis Sci. 2002;43(7):2152–9.PubMedPubMedCentralGoogle Scholar
  8. 8.
    Joyce NC, Zhu CC, Harris DL. Relationship among oxidative stress, DNA damage, and proliferative capacity in human corneal endothelium. Invest Ophthalmol Vis Sci. 2009;50(5):2116–22.CrossRefGoogle Scholar
  9. 9.
    Konomi K, Joyce NC. Age and topographical comparison of telomere lengths in human corneal endothelial cells. Mol Vis. 2007;13:1251–8.PubMedGoogle Scholar
  10. 10.
    Engelmann K, Bohnke M, Friedl P. Isolation and long-term cultivation of human corneal endothelial cells. Invest Ophthalmol Vis Sci. 1988;29(11):1656–62.PubMedGoogle Scholar
  11. 11.
    Miyata K, Drake J, Osakabe Y, et al. Effect of donor age on morphologic variation of cultured human corneal endothelial cells. Cornea. 2001;20(1):59–63.PubMedCrossRefGoogle Scholar
  12. 12.
    Konomi K, Zhu C, Harris D, Joyce NC. Comparison of the proliferative capacity of human corneal endothelial cells from the central and peripheral areas. Invest Ophthalmol Vis Sci. 2005;46(11):4086–91.PubMedCrossRefGoogle Scholar
  13. 13.
    He Z, Campolmi N, Gain P, et al. Revisited microanatomy of the corneal endothelial periphery: new evidence for continuous centripetal migration of endothelial cells in humans. Stem Cells. 2012;30(11):2523–34.PubMedCrossRefGoogle Scholar
  14. 14.
    Hirata-Tominaga K, Nakamura T, Okumura N, et al. Corneal endothelial cell fate is maintained by LGR5 through the regulation of hedgehog and Wnt pathway. Stem Cells. 2013;31(7):1396–407.PubMedCrossRefGoogle Scholar
  15. 15.
    Van Horn DL, Hyndiuk RA. Endothelial wound repair in primate cornea. Exp Eye Res. 1975;21(2):113–24.PubMedCrossRefGoogle Scholar
  16. 16.
    Matsubara M, Tanishima T. Wound-healing of the corneal endothelium in the monkey: a morphometric study. Jpn J Ophthalmol. 1982;26(3):264–73.PubMedGoogle Scholar
  17. 17.
    Okumura N, Kay EP, Nakahara M, Hamuro J, Kinoshita S, Koizumi N. Inhibition of TGF-beta signaling enables human corneal endothelial cell expansion in vitro for use in regenerative medicine. PLoS One. 2013;8(2):e58000.PubMedPubMedCentralCrossRefGoogle Scholar
  18. 18.
    Peh GS, Beuerman RW, Colman A, Tan DT, Mehta JS. Human corneal endothelial cell expansion for corneal endothelium transplantation: an overview. Transplantation. 2011;91(8):811–9.CrossRefGoogle Scholar
  19. 19.
    Hongo A, Okumura N, Nakahara M, Kay EP, Koizumi N. The effect of a p38 mitogen-activated protein kinase inhibitor on cellular senescence of cultivated human corneal endothelial cells. Invest Ophthalmol Vis Sci. 2017;58(9):3325–34.PubMedCrossRefGoogle Scholar
  20. 20.
    Okumura N, Ueno M, Koizumi N, et al. Enhancement on primate corneal endothelial cell survival in vitro by a ROCK inhibitor. Invest Ophthalmol Vis Sci. 2009;50(8):3680–7.CrossRefGoogle Scholar
  21. 21.
    Okumura N, Nakano S, Kay EP, et al. Involvement of cyclin D and p27 in cell proliferation mediated by ROCK inhibitors Y-27632 and Y-39983 during corneal endothelium wound healing. Invest Ophthalmol Vis Sci. 2014;55(1):318–29.CrossRefGoogle Scholar
  22. 22.
    Peh GS, Adnan K, George BL, et al. The effects of rho-associated kinase inhibitor Y-27632 on primary human corneal endothelial cells propagated using a dual media approach. Sci Rep. 2015;5:9167.PubMedPubMedCentralCrossRefGoogle Scholar
  23. 23.
    Okumura N, Kakutani K, Numata R, et al. Laminin-511 and -521 enable efficient in vitro expansion of human corneal endothelial cells. Invest Ophthalmol Vis Sci. 2015;56(5):2933–42.PubMedPubMedCentralCrossRefGoogle Scholar
  24. 24.
    Ishino Y, Sano Y, Nakamura T, et al. Amniotic membrane as a carrier for cultivated human corneal endothelial cell transplantation. Invest Ophthalmol Vis Sci. 2004;45(3):800–6.PubMedCrossRefGoogle Scholar
  25. 25.
    Koizumi N, Sakamoto Y, Okumura N, et al. Cultivated corneal endothelial cell sheet transplantation in a primate model. Invest Ophthalmol Vis Sci. 2007;48(10):4519–26.PubMedCrossRefGoogle Scholar
  26. 26.
    Koizumi N, Sakamoto Y, Okumura N, et al. Cultivated corneal endothelial transplantation in a primate: possible future clinical application in corneal endothelial regenerative medicine. Cornea. 2008;27(Suppl 1):S48–55.PubMedCrossRefGoogle Scholar
  27. 27.
    Mimura T, Yamagami S, Yokoo S, et al. Cultured human corneal endothelial cell transplantation with a collagen sheet in a rabbit model. Invest Ophthalmol Vis Sci. 2004;45(9):2992–7.CrossRefGoogle Scholar
  28. 28.
    Kimoto M, Shima N, Yamaguchi M, Hiraoka Y, Amano S, Yamagami S. Development of a bioengineered corneal endothelial cell sheet to fit the corneal curvature. Invest Ophthalmol Vis Sci. 2014;55(4):2337–43.PubMedCrossRefGoogle Scholar
  29. 29.
    Yamaguchi M, Shima N, Kimoto M, Ebihara N, Murakami A, Yamagami S. Optimization of cultured human corneal endothelial cell sheet transplantation and post-operative sheet evaluation in a rabbit model. Curr Eye Res. 2016;41(9):1178–84.PubMedCrossRefGoogle Scholar
  30. 30.
    Sumide T, Nishida K, Yamato M, et al. Functional human corneal endothelial cell sheets harvested from temperature-responsive culture surfaces. FASEB J. 2006;20(2):392–4.CrossRefGoogle Scholar
  31. 31.
    Jacobi C, Zhivov A, Korbmacher J, et al. Evidence of endothelial cell migration after descemet membrane endothelial keratoplasty. Am J Ophthalmol. 2011;152(4):537–542.e532.PubMedCrossRefGoogle Scholar
  32. 32.
    Shah RD, Randleman JB, Grossniklaus HE. Spontaneous corneal clearing after Descemet's stripping without endothelial replacement. Ophthalmology. 2012;119(2):256–60.CrossRefGoogle Scholar
  33. 33.
    Mimura T, Shimomura N, Usui T, et al. Magnetic attraction of iron-endocytosed corneal endothelial cells to Descemet's membrane. Exp Eye Res. 2003;76(6):745–51.PubMedCrossRefGoogle Scholar
  34. 34.
    Patel SV, Bachman LA, Hann CR, Bahler CK, Fautsch MP. Human corneal endothelial cell transplantation in a human ex vivo model. Invest Ophthalmol Vis Sci. 2009;50(5):2123–31.PubMedPubMedCentralCrossRefGoogle Scholar
  35. 35.
    Koizumi N, Okumura N, Ueno M, Nakagawa H, Hamuro J, Kinoshita S. Rho-associated kinase inhibitor eye drop treatment as a possible medical treatment for Fuchs corneal dystrophy. Cornea. 2013;32(8):1167–70.PubMedPubMedCentralCrossRefGoogle Scholar
  36. 36.
    Okumura N, Koizumi N, Kay EP, et al. The ROCK inhibitor eye drop accelerates corneal endothelium wound healing. Invest Ophthalmol Vis Sci. 2013;54:2493.CrossRefGoogle Scholar
  37. 37.
    Okumura N, Kinoshita S, Koizumi N. The Role of Rho Kinase Inhibitors in Corneal Endothelial Dysfunction. Curr Pharm Des. 2017;23(4):660–6.PubMedCrossRefGoogle Scholar
  38. 38.
    Okumura N, Koizumi N, Ueno M, et al. ROCK inhibitor converts corneal endothelial cells into a phenotype capable of regenerating in vivo endothelial tissue. Am J Pathol. 2012;181(1):268–77.CrossRefGoogle Scholar
  39. 39.
    Okumura N, Sakamoto Y, Fujii K, et al. Rho kinase inhibitor enables cell-based therapy for corneal endothelial dysfunction. Sci Rep. 2016;6:26113.PubMedPubMedCentralCrossRefGoogle Scholar
  40. 40.
    Okumura N, Kinoshita S, Koizumi N. Application of Rho Kinase Inhibitors for the Treatment of Corneal Endothelial Diseases. J Ophthalmol. 2017;2017:2646904.Google Scholar
  41. 41.
    Kinoshita S, Koizumi N, Ueno M, et al. Injection of cultured cells with a ROCK inhibitor for bullous keratopathy. New Engl J Med. 2018;378(11):995–1003.PubMedCrossRefGoogle Scholar
  42. 42.
    Okumura N, Hirano H, Numata R, et al. Cell surface markers of functional phenotypic corneal endothelial cells. Invest Ophthalmol Vis Sci. 2014;55(11):7610–8.PubMedPubMedCentralCrossRefGoogle Scholar
  43. 43.
    Okumura N, Kusakabe A, Hirano H, et al. Density-gradient centrifugation enables the purification of cultured corneal endothelial cells for cell therapy by eliminating senescent cells. Sci Rep. 2015;5:15005.PubMedPubMedCentralCrossRefGoogle Scholar
  44. 44.
    Okumura N, Ishida N, Kakutani K, et al. Development of cell analysis software for cultivated corneal endothelial cells. Cornea. 2017;36(11):1387–94.PubMedCrossRefGoogle Scholar
  45. 45.
    Okumura N, Inoue R, Kakutani K, et al. Corneal endothelial cells have an absolute requirement for cysteine for survival. Cornea. 2017;36(8):988–94.PubMedCrossRefGoogle Scholar
  46. 46.
    Toda M, Ueno M, Hiraga A, et al. Production of homogeneous cultured human corneal endothelial cells indispensable for innovative cell therapy. Invest Ophthalmol Vis Sci. 2017;58(4):2011–20.PubMedCrossRefGoogle Scholar
  47. 47.
    Ueno M, Asada K, Toda M, et al. MicroRNA profiles qualify phenotypic features of cultured human corneal endothelial cells. Invest Ophthalmol Vis Sci. 2016;57(13):5509–17.PubMedPubMedCentralCrossRefGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  1. 1.Department of Biomedical EngineeringFaculty of Life and Medical Sciences, Doshisha UniversityKyotanabeJapan

Personalised recommendations