Skip to main content
SpringerLink
Log in

The Culture of Limbal Stromal Cells and Corneal Endothelial Cells

  • Protocol
  • First Online:
Corneal Regenerative Medicine

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1014))

Abstract

The cornea is the transparent front part of the eye and comprises three distinct cell layers. One of these cell layers is a self-renewing epithelium long believed to harbor a resident stem cell population. The location and characteristics of corneal epithelial stem cells have now been confirmed by several research groups, and these cells are currently applied therapeutically. The corneal stroma and endothelium are largely quiescent after infancy, and until recently they were not considered to undergo self-renewal or to maintain stem cells. This view was overturned during the last two decades. At present, cell populations with characteristics of adult stem cells are routinely isolated and characterized from the limbal stroma and the corneal ­endothelium. This chapter describes methods for isolation and culture of limbal stromal cells and corneal endothelial cells.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Secker GA, Daniels JT (2009) Limbal epithelial stem cells of the cornea. StemBook [internet], ed. The Stem Cell Research Community, StemBook, doi:10.3824/stembook.1.48.1

  2. Tseng SC (1989) Concept and application of limbal stem cells. Eye 3:141–157

    Article  PubMed  Google Scholar 

  3. Kinoshita S, Friend J, Thoft RA (1981) Sex chromatin of donor corneal epithelium in rabbits. Invest Ophthalmol Vis Sci 21:434–441

    PubMed  CAS  Google Scholar 

  4. Figueira EC, Di Girolamo N, Coroneo MT, Wakefield D (2007) The phenotype of limbal epithelial stem cells. Invest Ophthalmol Vis Sci 48:144–156

    Article  PubMed  Google Scholar 

  5. De Paiva CS, Pflugfelder SC, Li DQ (2006) Cell size correlates with phenotype and proliferative capacity in human corneal epithelial cells. Stem Cells 24:368–375

    Article  PubMed  Google Scholar 

  6. Akinci MA, Turner H, Taveras M, Barash A, Wang Z, Reinach P et al (2009) Molecular profiling of conjunctival epithelial side-population stem cells: atypical cell surface markers and sources of a slow-cycling phenotype. Invest Ophthalmol Vis Sci 50:4162–4172

    Article  PubMed  Google Scholar 

  7. Schlotzer-Schrehardt U, Kruse FE (2005) Identification and characterization of limbal stem cells. Exp Eye Res 81:247–264

    Article  PubMed  Google Scholar 

  8. Itskovitz-Eldor J, Schuldiner M, Karsenti D, Eden A, Yanuka O, Amit M et al (2000) Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Mol Med 6:88–95

    PubMed  CAS  Google Scholar 

  9. Schofield R, Lajtha LG (1983) Determination of the probability of self-renewal in haemopoietic stem cells: a puzzle. Blood Cells 9:467–483

    PubMed  CAS  Google Scholar 

  10. Li L, Neaves WB (2006) Normal stem cells and cancer stem cells: the niche matters. Cancer Res 66:4553–4557

    Article  PubMed  CAS  Google Scholar 

  11. Gipson IK (1989) The epithelial basement membrane zone of the limbus. Eye 3: 132–140

    Article  PubMed  Google Scholar 

  12. Ljubimov AV, Burgeson RE, Butkowski RJ, Michael AF, Sun TT, Kenney MC (1995) Human corneal basement membrane heterogeneity: topographical differences in the expression of type IV collagen and laminin isoforms. Lab Invest 72:461–473

    PubMed  CAS  Google Scholar 

  13. Li DQ, Tseng SC (1995) Three patterns of cytokine expression potentially involved in epithelial-fibroblast interactions of human ocular surface. J Cell Physiol 163:61–79

    Article  PubMed  CAS  Google Scholar 

  14. Dravida S, Pal R, Khanna A, Tipnis SP, Ravindran G, Khan F (2005) The transdifferentiation potential of limbal fibroblast-like cells. Brain Res Dev Brain Res 160:239–251

    Article  PubMed  CAS  Google Scholar 

  15. Du Y, Funderburgh ML, Mann MM, SundarRaj N, Funderburgh JL (2005) Multipotent stem cells in human corneal stroma. Stem Cells 23:1266–1275

    Article  PubMed  Google Scholar 

  16. Polisetty N, Fatima A, Madhira SL, Sangwan VS, Vemuganti GK (2008) Mesenchymal cells from limbal stroma of human eye. Mol Vis 14:431–442

    PubMed  CAS  Google Scholar 

  17. Criscimanna A, Zito G, Taddeo A, Richiusa P, Pitrone M, Morreale D et al (2011) In vitro generation of pancreatic endocrine cells from human adult fibroblast-like limbal stem cells. Cell Transplant 21:73–90

    PubMed  Google Scholar 

  18. Nishida T, Nakamura M, Konma T, Ofuji K, Nagano K, Tanaka T et al (1997) Neurotrophic keratopathy–studies on substance P and the clinical significance of corneal sensation. Nippon Ganka Gakkai Zasshi 101:948–974

    PubMed  CAS  Google Scholar 

  19. Bourne WM, Nelson LR, Hodge DO (1997) Central corneal endothelial cell changes over a ten-year period. Invest Ophthalmol Vis Sci 38:779–782

    PubMed  CAS  Google Scholar 

  20. Williams KK, Noe RL, Grossniklaus HE, Drews-Botsch C, Edelhauser HF (1992) Correlation of histologic corneal endothelial cell counts with specular microscopic cell density. Arch Ophthalmol 110:1146–1149

    Article  PubMed  CAS  Google Scholar 

  21. Blake DA, Yu H, Young DL, Caldwell DR (1997) Matrix stimulates the proliferation of human corneal endothelial cells in culture. Invest Ophthalmol Vis Sci 38:1119–1129

    PubMed  CAS  Google Scholar 

  22. Yue BY, Sugar J, Gilboy JE, Elvart JL (1989) Growth of human corneal endothelial cells in culture. Invest Ophthalmol Vis Sci 30:248–253

    PubMed  CAS  Google Scholar 

  23. Miyata K, Drake J, Osakabe Y, Hosokawa Y, Hwang D, Soya K et al (2001) Effect of donor age on morphologic variation of cultured human corneal endothelial cells. Cornea 20:59–63

    Article  PubMed  CAS  Google Scholar 

  24. Amano S (2003) Transplantation of cultured human corneal endothelial cells. Cornea 22:S66–S74

    Article  PubMed  Google Scholar 

  25. Pistsov MY, Sadovnikova E, Danilov SM (1988) Human corneal endothelial cells: isolation, characterization and long-term cultivation. Exp Eye Res 47:403–414

    Article  PubMed  CAS  Google Scholar 

  26. Zhu C, Joyce NC (2004) Proliferative response of corneal endothelial cells from young and older donors. Invest Ophthalmol Vis Sci 45:1743–1751

    Article  PubMed  Google Scholar 

  27. Engelmann K, Friedl P (1989) Optimization of culture conditions for human corneal endothelial cells. In Vitro Cell Dev Biol 25:1065–1072

    Article  PubMed  CAS  Google Scholar 

  28. Dawson DG, Kramer TR, Grossniklaus HE, Waring GO, Edelhauser HF (2005) Histologic, ultrastructural, and immunofluorescent evaluation of human laser-assisted in situ keratomileusis corneal wounds. Arch Ophthalmol 123:741–756

    Article  PubMed  Google Scholar 

  29. Ishino Y, Sano Y, Nakamura T, Connon CJ, Rigby H, Fullwood NJ et al (2004) Amniotic membrane as a carrier for cultivated human corneal endothelial cell transplantation. Invest Ophthalmol Vis Sci 45:800–806

    Article  PubMed  Google Scholar 

  30. Choi JS, Williams JK, Greven M, Walter KA, Laber PW, Khang G et al (2010) Bioengineering endothelialized neo-corneas using donor-derived corneal endothelial cells and decellularized corneal stroma. Biomaterials 31:6738–6745

    Article  PubMed  CAS  Google Scholar 

  31. Chen KH, Azar D, Joyce NC (2001) Transplant of adult human corneal endothelium ex vivo: a morphologic study. Cornea 20:731–737

    Article  PubMed  CAS  Google Scholar 

  32. Joyce NC, Zhu CC (2004) Human corneal endothelial cell proliferation: potential for use in regenerative medicine. Cornea 23:S8–S19

    Article  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Ophthalmology, University of Erlangen-Nürnberg, Erlangen, Germany

    Naresh Polisetti

  2. Schepens Eye Research Institute, Boston, MA, USA

    Nancy C. Joyce (Emeritus Senior Scientist)

  3. Department of Ophthalmology, Harvard Medical School, Boston, MA, USA

    Nancy C. Joyce (Emeritus Senior Scientist)

Authors
  1. Naresh Polisetti
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nancy C. Joyce
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. School of Pharmacy, University of Reading, Shinfield Road, Reading, RG6 6UB, United Kingdom

    Bernice Wright

  2. School of Pharmacy, University of Reading, Shinfield Road, Reading, RG6 6UB, United Kingdom

    Che J Connon

Rights and permissions

Reprints and permissions

Copyright information

© 2013 Springer Science+Business Media New York

About this protocol

Cite this protocol

Polisetti, N., Joyce, N.C. (2013). The Culture of Limbal Stromal Cells and Corneal Endothelial Cells. In: Wright, B., Connon, C. (eds) Corneal Regenerative Medicine. Methods in Molecular Biology, vol 1014. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-432-6_8

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-432-6_8

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-431-9

  • Online ISBN: 978-1-62703-432-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation