Differential gene expression in the pig limbal side population: implications for stem cell cycling, replication, and survival.
OBJECTIVE
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.
Akinci
M A Murat
MA
Department of Ophthalmology, Mount Sinai School of Medicine, New York, New York 10029, USA.
Turner
Helen
H
Taveras
Maria
M
Wolosin
J Mario
JM
D000818:Animals; D001562:Benzimidazoles / Q000378:metabolism; D002453:Cell Cycle / Q000502:physiology*; D002455:Cell Division / Q000502:physiology*; D019070:Cell Lineage; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D003228:Conjunctiva / Q000166:cytology* / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism; D005136:Eye Proteins / Q000235:genetics; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology*; D016850:Limbus Corneae / Q000166:cytology* / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D010641:Phenotype; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D013552:Swine
D001562:Benzimidazoles; D005136:Eye Proteins; D005456:Fluorescent Dyes; C017807:bisbenzimide ethoxide trihydrochloride