title: SSEA4 is a potential negative marker for the enrichment of human corneal epithelial stem/progenitor cells.
abstract: OBJECTIVE
  To examine the expression of stage-specific embryonic antigen-4 (SSEA4) in the epithelium of the human ocular surface and characterize SSEA4(+) and SSEA4(-) limbal epithelial cells.
  
  
  METHODS
  SSEA4 expression in the human cornea and limbus was examined by RT-PCR and immunohistochemistry. SSEA4(+) and SSEA4(-) cells were then separated by using magnetic beads. The phenotypes of these two cell populations were evaluated on the basis of cell size, clonogenic assay, and expression of putative limbal stem cell (LSC) and corneal epithelial differentiation markers.
  
  
  RESULTS
  SSEA4 was expressed in all layers of the corneal and anterior limbal epithelia. Discrete clusters of SSEA4(+) cells were present in the central and posterior limbal epithelia. SSEA4(+) cells accounted for an average of 40% of the total limbal epithelial cells. The SSEA4(-) population contained five times more small cells (≤11 μm in diameter) than did the SSEA4(+) population. The expression levels of the putative LSC markers ABCG2, ΔNp63α, and cytokeratin (K)14 were significantly higher in the SSEA4(-) population than in the SSEA4(+) population. The SSEA4(-) cells also expressed a significantly higher level of N-cadherin, but a lower level of the differentiation marker K12. The colony-forming efficiency in the SSEA4(-) population was 25.2% (P = 0.04) and 1.6-fold (P < 0.05) higher than in the unsorted population and the SSEA4(+) population, respectively.
  
  
  CONCLUSIONS
  SSEA4 is highly expressed in differentiated corneal epithelial cells, and SSEA4(-) limbal epithelial cells contain a higher proportion of limbal stem/progenitor cells. SSEA4 could be used as a negative marker to enrich the isolation of LSCs.
authors.affiliation: Cornea and Uveitis Division, Jules Stein Eye Institute, University of California, Los Angeles, California 90095, USA.
authors.forename: Thuy T
authors.initials: TT
authors.lastname: Truong
authors.forename: Kyle
authors.initials: K
authors.lastname: Huynh
authors.forename: Martin N
authors.initials: MN
authors.lastname: Nakatsu
authors.forename: Sophie X
authors.initials: SX
authors.lastname: Deng
mesh_terms: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D000293:Adolescent; D000328:Adult; D015415:Biomarkers / Q000378:metabolism*; D002454:Cell Differentiation; D048429:Cell Size; D019573:Epithelium, Corneal / Q000166:cytology / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D053547:Keratin-14 / Q000378:metabolism; D016850:Limbus Corneae / Q000166:cytology; D008875:Middle Aged; D009363:Neoplasm Proteins / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055746:Stage-Specific Embryonic Antigens / Q000235:genetics / Q000378:metabolism*; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000378:metabolism
chemical_list: C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D015415:Biomarkers; D053547:Keratin-14; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D055746:Stage-Specific Embryonic Antigens; C115051:TP63 protein, human; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; C066584:stage-specific embryonic antigen-4