Pluripotent stem cell markers can be useful for diagnostic evaluation of human tumors. The novel pluripotent marker stage-specific embryonic antigen-5 (SSEA-5) is expressed in undifferentiated human induced pluripotent cells (iPSCs), but little is known about SSEA-5 expression in other primitive tissues (e.g., human tumors). We evaluated SSEA-5 immunoreactivity patterns in human tumors, cell lines, teratomas, and iPS cells together with another pluripotent cell surface marker L1 cell adhesion molecule (L1CAM). We tested two hypotheses: (1) SSEA-5 and L1CAM would be immunoreactive and colocalized in human tumors; (2) SSEA-5 and L1CAM immunoreactivity would persist in iPSCs following retinal differentiating treatment. SSEA-5 immunofluorescence was most pronounced in primitive tumors, such as embryonal carcinoma. In tumor cell lines, SSEA-5 was highly immunoreactive in Capan-1 cells, while L1CAM was highly immunoreactive in U87MG cells. SSEA-5 and L1CAM showed colocalization in undifferentiated iPSCs, with immunopositive iPSCs remaining after 20 days of retinal differentiating treatment. This is the first demonstration of SSEA-5 immunoreactivity in human tumors and the first indication of SSEA-5 and L1CAM colocalization. SSEA-5 and L1CAM warrant further investigation as potentially useful tumor markers for histological evaluation or as markers to monitor the presence of undifferentiated cells in iPSC populations prior to therapeutic use. 1. Introduction Markers associated with pluripotency and stem cell phenotype are often expressed in malignant tissues [1, 2]. These pluripotent markers can serve as useful diagnostic tools [3, 4] or as potential targets for cancer stem cell-directed therapies [5]. Stage-specific embryonic antigen (SSEA) 5 is a novel cell surface marker of pluripotency [6]. Antibodies against SSEA-5 can partially immunodeplete teratoma-forming cells from differentiating iPSC populations [6]. Although SSEA-5 immunoreactivity has been established for iPSCs [6], it is unknown whether SSEA-5 is also expressed in human malignancies. For comparison with SSEA-5, we chose L1CAM, a cell surface adhesion molecule involved in self-renewal and pluripotency of embryonic stem cells [7]. shRNA targeting of L1CAM expression in glioma cells inhibits tumor growth in vivo and increases the survival time of tumor-bearing animals [8]. Since both SSEA-5 and L1CAM are cell surface markers of pluripotency, they may also be useful together in monitoring the presence of pluripotent cells in iPSC populations that undergo differentiation. Therefore, we examined human
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