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The cell‐surface markers used routinely to define the undifferentiated state and pluripotency of human embryonic stem cells (hESCs) are those used in mouse embryonic stem cells (mESCs) because of a lack of markers directly originated from hESC itself. To identify more hESC‐specific cell‐surface markers, we generated a panel of monoclonal antibodies (MAbs) by immunizing the irradiated cell clumps of hESC line Miz‐hES1, and selected 26 MAbs that were able to bind to Miz‐hES1 cells but not to mESCs, mouse embryonic fibroblast cells, and STO cells. Most antibodies did not bind to human neural progenitor cells derived from the Miz‐hES1 cells, either. Of these, MAb 20‐202S (IgG1, κ) immunoprecipitated a cell‐surface protein of 72‐kDa from the lysate of biotin‐labeled Miz‐hES1 cells, which was identified to be heat shock 70‐kDa protein 8 isoform 1 (HSPA8) by quadrupole time‐of‐flight tandem mass spectrometry. Immunocytochemical analyses proved that the HSPA8 protein was also present on the surface of hESC lines Miz‐hES4, Miz‐hES6, and HSF6. Two‐color flow cytometric analysis of Miz‐hES1 and HSF6 showed the coexpression of the HSPA8 protein with other hESC markers such as stage‐specific embryonic antigen 3 (SSEA3), SSEA4, TRA‐1‐60, and TRA‐1‐81. Flow cytometric and Western blot analyses using various cells showed that MAb 20‐202S specifically bound to the HSPA8 protein on the surface of Miz‐hES1, contrary to other anti‐HSP70 antibodies examined. Furthermore, the surface expression of the HSPA8 protein on Miz‐hES1 was markedly downregulated upon differentiation. These data indicate that a novel MAb 20‐202S recognizes the HSPA8 protein on the surface of hESCs and suggest that the HSPA8 protein is a putative cell‐surface marker for undifferentiated hESCs.

Heat Shock 70‐kDa Protein 8 Isoform 1 Is Expressed on the Surface of Human Embryonic Stem Cells and Downregulated upon Differentiation