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Loss of corneal epithelial stem cell properties in outgrowths from human limbal explants cultured on intact amniotic membrane

Lookup NU author(s): Sai Kolli, Professor Majlinda Lako, Professor Francisco Figueiredo, Dr Sajjad Ahmad

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Abstract

Background: The corneal epithelium is renewed by stem cells located at the limbus, the so-called limbal stem cells (LSCs). Absence, damage or loss of the LSC population leads to the painful and blinding condition of LSC deficiency (LSCD). Ex vivo expansion of LSCs is an increasingly well recognized treatment modality for LSCD. One method of ex vivo expansion of LSCs involves the culture of limbal explants on amniotic membrane (AM). The purpose of this study was to analyze the outgrowths from human cadaveric limbal explants cultured on AM for properties associated with LSCs. In particular, the expression of putative stem cell markers and the colony-forming efficiency of the different zones of the outgrowth were studied. Methods: The limbal explants were expanded in the standard way used for clinical transplantation and the outgrowths were divided into three zones depending on proximity to the explant (inner, middle and outer zones). The colony-forming efficiencies (CFEs) of cells from each zone were calculated. In addition, the expression of Delta Np63, ABCG2 (both putative positive LSC markers) and cytokeratin K3 (marker of corneal differentiation) were assessed using quantitative reverse transcription PCR (RT-PCR). Immunohistochemistry on paraffin-embedded sections was also performed to demonstrate protein localization and allow further confirmation of the quantitative RT-PCR results. Results: Successful cultures for both the explant outgrowths and the CFE calculations were obtained in every case. CFE showed a successive decline in zones further away from the explant (p


Publication metadata

Author(s): Kolli S, Lako M, Figueiredo FC, Mudhar H, Ahmad S

Publication type: Article

Publication status: Published

Journal: Regenerative Medicine

Year: 2008

Volume: 3

Issue: 3

Pages: 329-342

ISSN (print): 1746-0751

ISSN (electronic):

Publisher: Future Medicine Ltd.

URL: http://dx.doi.org/10.2217/17460751.3.3.329

DOI: 10.2217/17460751.3.3.329


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