Molecular Profiling of Conjunctival Epithelial Side-Population Stem Cells: Atypical Cell Surface Markers and Sources of a Slow-Cycling Phenotype

Abstract

PURPOSE Side-population (SP) cells isolated from limbal and conjunctival epithelia derive from cells that are slow cycling in vivo, a known feature of tissue stem cells. The purpose of this study was to define the molecular signature of the conjunctival SP cells and identify markers and signaling pathways associated with the phenotype of these cells. METHODS Overnight cultures of freshly isolated human conjunctival epithelial cells stained with Hoechst 33342 were sorted by flow cytometry into SP and non-SP cohorts. Isolated RNA was processed for microarray analysis using a commercial oligonucleotide spotted array. Results were validated at the gene and protein levels by quantitative PCR and immunologic methods. Data mining methods were used to identify cellular processes relevant for stem cell function. RESULTS Comparative analyses of transcripts expression based on present and absent software calls across four replicate experiments identified 16,993 conjunctival epithelial transcripts including 10,266 unique known genes of ~24,000 represented in the array. Of those genes, 1254 and 363 were overexpressed (>2-fold) or underexpressed (<0.5-fold), respectively, in the SP. The overexpressed set included genes coding for proteins that have been associated with (1) embryonic development and/or stem cell self renewal (MSX, MEIS, ID, Hes1, and SIX homeodomain genes); (2) cell survival (e.g., CYP1A1 to degrade aromatic genotoxic compounds); (3) cycling rate (e.g., DUSPs and Pax6 to foster slow cycling); and (4) genes whose expression is not typical in epithelia (e.g., CD62E). CONCLUSIONS The molecular signature of conjunctival SP cells is consistent with a stem cell phenotype. Their gene expression patterns underpin slow cycling and plasticity, features associated with tissue stem cells. The results provide valuable insights for the preservation and/or expansion of epithelial stem cells.

Application of JC1 for non-toxic isolation of cells with MDR transporter activity by flow cytometry

“…Only in freshly isolated rabbit conjunctival epithelial cells the JC1 signal degradation by Hoechst was slow or moderate enough to allow simultaneous visualization of both JC1-SP and Hoechst SP. In these cells, the numerically small, but strong Hoechst-SP represents bona fide slow cycling stem cells, with clonal ability and radically distinct gene expression profile than the non-SP cells [8, 19]. Simultaneous 75 min co-incubation with 250 nM JC1 and 4 μg/ml Hoechst yielded Hoechst and JC1 SPs amounting to under 1% of the respective populations (Fig 7A).…”

“…Single cell suspensions were then generated by a short incubation in trypsin and resuspension in DMEM-F12 with 5% FBS. Fresh rabbit conjunctival epithelial cells were prepared by sequential Dispase-Trypsin digestion as described [19], resuspended in DMEM-F12 with 5% FBS and used immediately. Primary cultured keratinocytes (pcKs) were generated from rabbit inner ear by the same sequential Dispase-trypsin protocol used for conjunctiva and cultured and treated with reagents in DKSFM medium.…”

Differential Gene Expression in the Pig Limbal Side Population: Implications for Stem Cell Cycling, Replication, and Survival

“…The source tube was maintained at 4°C and SP and non-SP (nSP) cells were sorted directly into 750 µL RNA isolation solution (Tri-Reagent LS; Molecular Research Center [MRC] Cincinnati, OH). We used a specific range of forward (FSC) and side (SSC) light-scattering levels to exclude the great majority of lymphocytes (cells with very low SSC) and to limit the amount of other complex nonepithelial cells present within the epithelial strata (cells with very high SSC such as melanocytes and dendritic cells) 27. Fumitremorgin C (FTC; a generous gift from Susan Bates, National Cancer Institute, Bethesda, MD) was used to determine the involvement of ABCG2 in the observed SP cells 22…”

“…We have recently completed a microarray-based study of differentially expressed genes, or molecular signature, of SP cells isolated from the human CNJE 27. The rarity of these cells poses unique challenges and their investigation, such as in differential gene expression studies, has been problematic.…”

“…Hence, to probe for the possible general relevance of genes that are differentially expressed genes in the limbal SP cells, we performed similar microarray measurements for the pig CNJE, a tissue that shares with the corneal epithelium a common environment, developmental origin, and PAX6 expression28,29 and used these results and recently obtained microarray data for the human CNJE27 for comparative assessments.…”