The phenotype of limbal epithelial stem cells.
OBJECTIVE
The purpose of this study was to identify phenotypic markers of human limbal stem cells in fetal and adult corneas.


METHODS
RNA from microscopically dissected superficial limbal and central fetal (18 weeks) corneas was amplified and used to generate P(32)-labeled, reverse-transcribed antisense RNA that was linearly amplified and hybridized to a focused stem cell cDNA microarray. Differential gene expression of fetal limbus was compared with the expression of central cornea. Microarray differential expression experiments were performed on P63-expressing primary cultured limbal epithelial cells (passage 1; Pa1) and primary cells passaged 5 times (Pa5). Semiquantitative RT-PCR assay and immunohistochemistry were performed on fetal and adult corneas and cultured primary limbal epithelial cells, to confirm the results of the microarray experiments. Slow-cycling (pulsed bromodeoxyuridine label-retaining) limbal epithelium in corneal organ culture was studied for the expression of four selected upregulated limbal genes.


RESULTS
Of the 266 genes tested, 33 were differentially overexpressed (more than twofold) in the fetal limbus (compared with central cornea) and primary cultured limbal epithelium compared with primary cells after 5 passages. Cytokeratin 15 (CK15) and cytokeratin 14 (CK14) are expressed in limbal basal epithelium and P-cadherin (CDH3) and Wnt-4 expression was restricted to basal and immediate parabasal limbal epithelium of both the adult and fetal corneas). Bromodeoxyuridine label retaining epithelium in corneal organ culture (slow-cycling cells) expressed the four selected limbal upregulated genes.


CONCLUSIONS
For the first time, a focused stem cell pathway microarray analysis has been performed on fetal cornea and cultured limbal explant epithelium. CK15, CK14, CDH3, and Wnt-4 are expressed in the basal limbal epithelial cells.
Figueira
Edwin C
EC
Inflammatory Diseases Research Unit, University of New South Wales, Australia. e_c_figueira@hotmail.com
Di Girolamo
Nick
N
Coroneo
Minas T
MT
Wakefield
Denis
D
D000328:Adult; D053687:Adult Stem Cells / Q000166:cytology* / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism*; D018929:Cell Culture Techniques; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism; D019573:Epithelium, Corneal / Q000196:embryology* / Q000378:metabolism; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism; D053686:Fetal Stem Cells / Q000166:cytology* / Q000378:metabolism; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000502:physiology; D006801:Humans; D007124:Immunoenzyme Techniques; D016850:Limbus Corneae / Q000196:embryology*; D021141:Nucleic Acid Amplification Techniques; D020411:Oligonucleotide Array Sequence Analysis; D009924:Organ Culture Techniques; D010641:Phenotype; D012313:RNA / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction
D015415:Biomarkers; D005136:Eye Proteins; D012313:RNA