December 2002
Volume 43, Issue 13
ARVO Annual Meeting Abstract  |   December 2002
p63 Expression in Response to Phorbol Ester Stimulation in Human Limbal Epithelial Cells Expanded on Intact Human Amniotic Membrane
Author Affiliations & Notes
  • EE Hernandez
    Ophthalmology University of Essen Essen Germany
  • C Theiss
    Cytology University of Bochum Bochum Germany
  • KP Steuhl
    Ophthalmology University of Essen Essen Germany
  • D Meller
    Ophthalmology University of Essen Essen Germany
  • Footnotes
    Commercial Relationships   E.E. Hernandez, None; C. Theiss, None; K.P. Steuhl, None; D. Meller, None. Grant Identification: Ifores
Investigative Ophthalmology & Visual Science December 2002, Vol.43, 1628. doi:
  • Views
  • Share
  • Tools
    • Alerts
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      EE Hernandez, C Theiss, KP Steuhl, D Meller; p63 Expression in Response to Phorbol Ester Stimulation in Human Limbal Epithelial Cells Expanded on Intact Human Amniotic Membrane . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1628.

      Download citation file:

      © ARVO (1962-2015); The Authors (2016-present)

  • Supplements

Abstract: : Purpose: To evaluate the expression of p63 protein, a gene product restricted to progenitor epithelial cells, in human limbal epithelial cells (HLEC) during ex vivo expansion on preserved human amniotic membrane (AM). Methods: Primary human limbal epithelial cells (HLEC) from corneal limbus explants were cultured with SHEM either on intact AM or plastic for 4 to 5 weeks. A set of cultures was treated with 1µg/ml phorbol 12-myristate 13-acetate (PMA) containing medium for 24 hours and afterwards switched to PMA-free medium for 3 days. Then, all cell cultures were terminated and fixed in 4% paraformaldehyde. Monoclonal antibody against p63 was used to study expression of p63 in HLEC. Alexa Fluor 546 goat anti-mouse was applied as secondary antibody and flat mounts were analyzed by means of laser confocal microscopy. Positive nuclei were counted in eight 200x200 µm² areas of each micrograph. Results: HLEC cultured on AM revealed an uniform immunolabeling for p63 in central regions (83.48%) as well as peripheral regions (86.93%) of cell cultures. In contrast, HLEC cultured on plastic showed a centrifugal reduction of p63 labeling from the central (87.59%) toward peripheral areas (52.37%). After PMA treatment, a decrease of p63 expression in HLEC cultured on AM to 54.39% and 52.37% was noted (center and periphery, respectively), whereas the immunoreactivity to p63 of HLEC cultured on plastic diminished dramatically to 28.07% in central and 22.69% in the peripheral areas associated with marked morphological changes characterized usually by an increased cellular size with signs of desquamation. Conclusion: AM supports p63 protein expression of HLEC and maintains a higher resistance against phorbol ester indicating that characteristic of limbal epithelial progenitor cells might be preserved during ex-vivo expansion on AM. This data provides support to the use of the ex-vivo expansion of HLEC as an alternative therapeutic strategy for corneal surface reconstruction in distinct ocular surface diseases.

Keywords: 372 cornea: epithelium • 631 wound healing • 370 cornea: basic science 

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.