Int. J. Dev. Biol. 51: 739 - 743 (2007)

https://doi.org/10.1387/ijdb.062198ar

Vol 51, Issue 8

Human conjunctival epithelial precursor cells and their progeny in 3D organotypic culture

Short Communication | Published: 1 October 2007

Alfredo Rosellini¹, Sandra Papini¹, Claudio Giannarini², Marco Nardi³ and Roberto P. Revoltella¹*

¹Foundation O.N.L.U.S. "Stem Cells and Life", Pisa and Institute of Biomedical Technologies, C.N.R., Pisa, ²Corneal Tissue and Cell Bank of Tuscany, Center "P. Perelli" ASL2, Hospital of Lucca, Lucca and ³Department of Neuroscience, Section of Ophthalmology, University of Pisa, Pisa, Italy

Abstract

We report on an in vitro organ culture method to investigate human conjunctival epithelial basal precursor cells and their progeny within a more natural three-dimensional microenvironment. Conjunctival fragments were cultured on gelatin sponges in medium with 10% FBS. The conjunctival phenotype of the epithelium was confirmed by the expression and distribution of a panel of markers (p63, CK-13/CK-10, CK-19, Ki-67, PAS for goblet cells, CD45 for infiltrating interlamellar leukocytes and nestin for mesenchymal and ocular epithelial precursor cells). After 7 days, the epithelium had exfoliated its superficial layers (mostly CK-19 positive cells and all goblets), maintaining only 1-2 layers of basal/parabasal cells, p63, CK-13/CK-10 and nestin positive cells, firmly attached to the specimen. After 14 days, a new multilayered epithelium was formed, consisting of p63, CK-13/CK-10, nestin positive cells and in the high-zone CK-19 positive cells with new goblets. Additionally, we found interlamellar leukocytes which had probably migrated from capillaries that continued to be well maintained in the subepithelial stroma. Cells dispersed from conjunctival epithelium and co-cultured with feeder post-mitotic NIH3T3 fibroblasts formed mosaics displaying a basal epithelial phenotype. These cells expressed CD133 as revealed by RT-PCR. These organ cultures provide new opportunities to investigate epithelial reconstitution of the conjunctival surface and changes that may have occurred to their stem/precursor cells during adaptation to varying conditions in vitro.

Keywords

epithelial basal precursor, stem cell, niche, epithelium regeneration, interlamellar leukocytes