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

Abstract

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 G1-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.

Differentially Expressed Genes Associated with Human Limbal Epithelial Phenotypes: New Molecules That Potentially Facilitate Selection of Stem Cell-Enriched Populations

“…38 Recently, its differential expression was observed in porcine limbal side population cells. 21 Based on these data, we suggest that CXCR4 may participate in maintaining the proliferative capacity of corneal limbal progenitors and TACs of the central corneal epithelium.…”

“…10,11,20 To reveal these molecules as DEGs, the examined cell population should probably contain stem cells in higher proportions than unsorted cells from limbal epithelial scrapings. Accordingly, mRNA levels of ABCG2, C/EBP␦, 21 and Bmi-1 17 were found to be significantly increased on flow cytometric selection of the side population cells that are believed to carry stem-like properties. Selection of the side population, however, is a time-consuming procedure, during which expression profiles can change significantly.…”

SH3BGRL3 Protein as a Potential Prognostic Biomarker for Urothelial Carcinoma: A Novel Binding Partner of Epidermal Growth Factor Receptor

“…SH3BGRL3 could inhibit TNFa-induced apoptosis and promote cell survival, although the molecular mechanism underlying these functions remains unclear (12). In a pig-human xenograft model experiment, SH3BGRL3 was upregulated in xeno-conjunctival epithelial cells to protect them from programmed cell death (13). SH3BGRL3 is also involved in all-trans retinoic acid-induced cell differentiation (9).…”

Limbal epithelial stem cells in regeneration of corneal epithelium – clinical and molecular aspects

“…Proces destrukcji LESC zaczyna się zazwyczaj w obrębie sektora górnego [47,57]. Całkowity niedobór komórek macierzystych rogówki (total limbal stem cells deficiency -TLSCD) wiąże się z nawracającymi erozjami, przewlekłym zapaleniem, głę-boką neowaskularyzacją, wrastaniem nabłonka, zniszczeniem błony podstawnej, ścieńczeniem, bliznowaceniem i w konsekwencji zmętnieniem rogówki [12,27,64,65,66]. W celu diagnostyki LSCD na powierzchni rogówki umieszcza się membranę filtracyjną z octanem celulozy.…”