SSEA4 is a potential negative marker for the enrichment of human corneal epithelial stem/progenitor cells

Abstract

Purpose: 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.

Figure 1.

Expression of SSEA4 synthase mRNA (A) and SSEA4 (B–E) in the human cornea and limbus. (A) SSEA4 synthase mRNA was detected by RT-PCR in both the corneal and limbal epithelia. (B–E) Detection of SSEA4 by immunohistochemistry in the human corneal and limbal epithelia. SSEA4 was uniformly present in all layers of the corneal epithelium (B, C), but only in clusters of the limbal epithelium (D, E).

Figure 2.

Separation of the SSEA4⁺ and SSEA4⁻ populations of the limbal epithelial cells using magnetic bead sorting. (A) The negative control (i.e., unsorted cells that were not incubated with primary antibody) showed no labeling by the secondary antibody alone. (B) The population of unsorted limbal epithelial cells contained SSEA4⁺ cells and SSEA4⁻ cells. (C, D) After sorting, the SSEA4⁺ population contained only SSEA4⁺ cells (C), and the SSEA4⁻ population contained only SSEA4⁻ cells (D). Many SSEA4⁺ cells showed squamous morphology (arrows). (E) Quantitation of the SSEA4⁺ and SSEA4⁻ cell populations. SSEA4⁻ cells accounted for 59.5% ± 5.7% of the total population of freshly isolated limbal epithelial cells, and 38.1% ± 3.1% were SSEA4⁺. *P < 0.05 between SSEA4⁻ and SSEA⁺ populations (n = 6).

Figure 3.

Difference in sizes of cells in the SSEA4⁺ and SSEA4⁻ populations. The SSEA4⁻ population contained a larger proportion of small cells than the SSEA4⁺ population: 10.8% ± 3.1% of SSEA4⁻ cells had a diameter ≤11 μm, whereas 1.7% ± 0.6% of SSEA4⁺ cells had the same diameter (P < 0.001, n = 6).

Figure 4.

Relative mRNA levels of putative stem cell and differentiation markers evaluated by qRT-PCR. The SSEA4⁻ population expressed higher levels of putative stem/progenitor cell markers (ABCG2, ΔNp63α, K14, and N-cadherin) and a lower level of differentiation marker (K12) than did the SSEA4⁺ and unsorted populations. *P < 0.05 between SSEA4⁻ and SSEA⁺ populations, **P < 0.05 between SSEA4⁻ and unsorted populations.

Figure 5.

Colony-forming efficiency of the SSEA4⁻ and SSEA4⁺ cell populations. (A) SSEA4⁺ cells had a significantly higher colony-forming capacity than did the SSEA4⁺ population and the unsorted population. (B) Representative photos of colonies from the SSEA4⁻, SSEA4⁺, and the unsorted populations seeded at 100 or 300 cell/cm². *P ≤ 0.05 between the SSEA4⁻ and SSEA4⁺ populations. **P ≤ 0.05 between the SSEA4⁻ and unsorted populations.