Purchase this article with an account.
D Meller, EE Hernandez, C Theiss, KP Steuhl; Lack of Gap Junctional Communication in Subpopulations of Limbal Epithelial Cells Expanded on Intact Human Amniotic Membrane Studied by Microinjection of Lucifer Yellow . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1624.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine if human limbal epithelial cells (HLEC) lack of gap junctional communication during ex vivo expansion on preserved human amniotic membrane (AM). Methods: Primary human limbal (HLEC) and peripheral corneal (HPCEC) epithelial cells from limbal and peripheral corneal explants were cultured with SHEM either on intact AM or plastic. After 4 to 5 weeks, cell cultures were processed for microinjection studies. In preliminary experiments Lucifer yellow (LY) which is known to be a gap junction (GJ) permeant dye was injected simultaneously with GJ non-permeant Rhodamine dextran (4%) in order to demonstrate the capability of this technique to study in single cells the functionality of gap junctions. Microinjection of LY into single cells (in each group at least n=100) was performed with a pressure microinjection device under visual control and with the aid of phase contrast optics. Dye spread of LY into adjacent cells indicating intercellular communication was compared between HLEC and HPCEC cultured either on AM or plastic. Results: Rhodamine conjugated to dextran was typically retained within the injected single cell, whereas GJ permeant LY diffused to adjacent epithelial cells. Gap junctional communication was evidenced more frequently in HLEC cultured on plastic (50.96%, p<0.05) in contrast to HLEC cultures on AM, which exhibited dye spread in 29.70% of injected cells. However, no significant difference was found between HPCEC cultured on AM (52,88%) as compared to HPCEC cultures on plastic, which exhibited dye coupling in 57.84% of injected cells. Conclusion: Subpopulations of HLEC cultured on AM remain without functional gap junctions indicating that characteristic features 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.
This PDF is available to Subscribers Only