Skip to main content

Advanced Imaging and Tissue Engineering of the Human Limbal Epithelial Stem Cell Niche

  • Protocol
  • First Online:
Book cover Stem Cell Protocols

Abstract

The limbal epithelial stem cell niche provides a unique, physically protective environment in which limbal epithelial stem cells reside in close proximity with accessory cell types and their secreted factors. The use of advanced imaging techniques is described to visualize the niche in three dimensions in native human corneal tissue. In addition, a protocol is provided for the isolation and culture of three different cell types, including human limbal epithelial stem cells from the limbal niche of human donor tissue. Finally, the process of incorporating these cells within plastic compressed collagen constructs to form a tissue-engineered corneal limbus is described and how immunohistochemical techniques may be applied to characterize cell phenotype therein.

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

Access this chapter

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 119.99
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. Thoft R, Friend J (1983) The X, Y, Z hypothesis of corneal epithelial maintenance. IOVS 10:1442–1443

    Google Scholar 

  2. Pellegrini G, Golisano O, Paterna P et al (1999) Location and clonal analysis of stem cells and their differentiated progeny in the human ocular surface. J Cell Biol 145:769–782

    Article  CAS  PubMed Central  PubMed  Google Scholar 

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

    Article  PubMed  Google Scholar 

  4. Shortt AJ, Secker GA, Munro PM et al (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

    Article  PubMed  Google Scholar 

  5. Denk W, Horstmann H (2004) Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure. PLoS Biol 2:329

    Article  Google Scholar 

  6. Du Y, Funderburgh ML, Mann MM et al (2005) Multipotent stem cells in human corneal stroma. Stem Cells 23:1266–1275

    Article  PubMed Central  PubMed  Google Scholar 

  7. Brown RA, Wiseman M, Chuo CB et al (2005) Ultrarapid engineering of biomimetic materials and tissues: Fabrication of nano- and microstructures by plastic compression. Adv Funct Mater 15:1762–1770

    Article  CAS  Google Scholar 

  8. Levis HJ, Brown RA, Daniels JT (2010) Plastic compressed collagen as a biomimetic substrate for human limbal epithelial cell culture. Biomaterials 31:7726–7737

    Article  CAS  PubMed  Google Scholar 

  9. Levis HJ, Menzel-Severing J, Drake RA et al (2013) Plastic compressed collagen constructs for ocular cell culture and transplantation: a new and improved technique of confined fluid loss. Curr Eye Res 38:41–52

    Article  CAS  PubMed  Google Scholar 

  10. Levis HJ, Massie I, Dziasko MA et al (2013) Rapid tissue engineering of biomimetic human corneal limbal crypts with 3D niche architecture. Biomaterials 34:8860–8868

    Article  CAS  PubMed  Google Scholar 

  11. Mort RL, Douvaras P, Morley SD et al (2012) Stem cells and corneal epithelial maintenance: insights from the mouse and other animal models. Results Probl Cell Differ 55:357–394

    Article  PubMed Central  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Julie T. Daniels .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer Science+Business Media New York

About this protocol

Cite this protocol

Massie, I. et al. (2015). Advanced Imaging and Tissue Engineering of the Human Limbal Epithelial Stem Cell Niche. In: Rich, I. (eds) Stem Cell Protocols. Methods in Molecular Biology, vol 1235. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1785-3_15

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-1785-3_15

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-1784-6

  • Online ISBN: 978-1-4939-1785-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics