ΔNp63 Expression In Label-retaining Human Limbal Epithelial Cells Expanded on Intact Human Amniotic Membrane
Hernandez Galindo E.-E.1, Theiss C.2, Steuhl K.-P.1, Meller D.1
1Department of Ophthalmology, University of Essen; 2Department of Cytology, University of Bochum
Purpose: The efficiency of a stem cell culture using amniotic membrane as a substrate is defined by the preservation of stem cell-like cell cycle properties and an undifferentiated cell phenotype. We attempt to identify subpopulations in our cell cultures that share characteristic features of limbal epithelial stem cells.
Method: Primary human limbal epithelial cells (HLEC) from corneal limbus explants were cultured with SHEM either on intact AM or plastic for 3 weeks. A set of cultures was treated with 1mg/ml phorbol 12-myristate 13-acetate (PMA) for 24 hours. Afterwards, incorporation was done with 5mM BrdU for 3 days and then chased for additional 14 days in BrdU-free medium. Monoclonal antibody against ΔNp63 was used to study expression of ΔNp63 in HLEC. Co-labeling was done with a polyclonal antibody against BrdU. Furthermore, a polyclonal antibody to Connexin 43 (Cx43) was used as a differentiation-associated marker. Flat mounts were analyzed by means of laser confocal microscopy.
Results: After PMA pretreatment and subsequent BrdU incorporation with 14 days of chase, HLEC cultured on AM showed a higher labeling index for p63 (LI: 38,1% ± 7,6) and BrdU (18%, ± 6.8%) compared to HLEC grown on plastic (LI for p63
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