M.A. Murat Akinci

Ocular surface epithelia contain ABCG2-dependent side population cells exhibiting features associated with stem cells

When cell populations are incubated with the DNA-binding dye Hoechst 33342 and subjected to flow cytometry analysis for Hoechst 33342 emissions, active efflux of the dye by the ABCG2/BCRP1 transporter causes certain cells to appear as a segregated cohort, known as a side population (SP). Stem cells from several tissues have been shown to possess the SP phenotype. As the lack of specific surface markers has hindered the isolation and subsequent biochemical characterization of epithelial stem cells this study sought to determine the existence of SP cells and expression of ABCG2 in the epithelia of the ocular surface and evaluate whether such SP cells had features associated with epithelial stem cells. Human and rabbit limbal-corneal and conjunctival epithelial cells were incubated with Hoechst 33342, and analyzed and sorted by flow cytometry. Sorted cells were subjected to several tests to determine whether the isolated SP cells displayed features consistent with the stem cell phenotype. Side populations amounting to <1% of total cells, which were sensitive to the ABCG2-inhibitor fumitremorgin C, were found in the conjunctival and limbal epithelia, but were absent from the stem cell-free corneal epithelium. Immunohistochemistry was used to establish the spatial expression pattern of ABCG2. The antigen was detected in clusters of conjunctival and limbal epithelia basal cells but was not present in the corneal epithelium. SP cells were characterized by extremely low light side scattering and contained a high percentage of cells that: showed slow cycling prior to tissue collection; exhibited an initial delay in proliferation after culturing; and displayed clonogenic capacity and resistance to phorbol-induced differentiation; all features that are consistent with a stem cell phenotype.

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

PURPOSE To define the molecular signature of limbal SP cells and identify signaling pathways associated with the phenotype of these putative stem cells. METHODS Primary cultures of pig limbal epithelial cells stained with Hoechst 33342 were sorted by flow cytometry into SP and non-SP cells, and purified RNA was processed for microarray analysis with an oligonucleotide spotted array. Expressed transcripts for which SP and non-SP expressions differed by more that 1.5-fold in each paired set and by twofold overall were considered to be differentially expressed. Differential expression was validated by quantitative PCR and immunostaining. Data-mining methods were used to identify cellular processes that are either salient or depressed in the SP cells. RESULTS The microarray identified approximately 9000 distinct, expressed, and identifiable genes. Of those, 382 and 296 were either over- or underexpressed in the SP cells, respectively. Overrepresentation analysis indicated that SP cells are in a low metabolic and biosynthetic state. In addition, a pattern of elevated MXD1, MAXI2, DUSP5, p27/KIP1, and p57/KIP2 and decreased Cyclin D and CDK genes can be expected to slow intrinsic and mitogen-induced G(1)-to-S cell cycle transition. SP cells were also rich in genes associated with stem cell phenotype and genes providing protection against oxidative and/or xenobiotic damage. CONCLUSIONS Microarray analysis of pig limbal SP cells yielded a molecular signature underscoring a phenotype characterized by slow cycling and low metabolic activity. The results provide valuable insights for the preservation and/or replication of epithelial stem cells.

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

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 approximately 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.

Potassium-Chloride Cotransporter Mediates Cell Cycle Progression and Proliferation of Human Corneal Epithelial Cells

Epidermal growth factor (EGF)-induced proliferation of corneal epithelial cells contributes to its renewal, which maintains the protective and refractive properties of the cornea. This study characterized in human corneal epithelial cells (HCEC) the role of the potassium–chloride cotransporter (KCC) in mediating (i) EGF-induced mitogen-activated protein kinase (MAPK) pathway activation; (ii) increases in cell cycle progression; and (iii) proliferation. The KCC inhibitor [(dihydroindenyl)oxy] alkanoic acid (DIOA) and KCC activator N-ethylmaleimide (NEM), suppressed and enhanced EGF-induced p44/42MAPK activation, respectively. Such selective modulation was mirrored by corresponding changes in cell proliferation and shifts in cell cycle distribution. DIOA induced a 20% increase in G0/G1-phase cell population, whereas NEM induced a 22% increase in the proportion of cells in the G2/M-phase and accelerated the transition from G0/G1-phase to the S-phase. Associated with these changes, KCC1 content in a plasma membrane enriched fraction increased by 300%. Alterations in regulatory volume capacity were associated with corresponding changes in both KCC1 membrane content and activity. These results indicate that EGF-induced increases in KCC1 activity and content modulate cell volume changes required for (i) activation of the p44/42MAPK signaling pathway, (ii) cell cycle progression, and (iii) increases in cell proliferation.