Heat Shock 70-kD Protein 8 Isoform 1 (HSPA8) Is Expressed on the Surface of Human Embryonic Stem Cells and Down-regulated Upon Differentiation

¹ Laboratory of Antibody Engineering, Korea Research Institute of Bioscience and Biotechnology, Daejon, Korea
² Department of Anatomy & Cell Biology, Hanyang University, Seoul, Korea
³ MizMedi Medical Research Center, Seoul, Korea
⁴ R&D Center, Aprogen, INC., Daejon, Korea
⁵ Systemic Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejon, Korea
⁶ School of Biological Sciences, Seoul National University, Seoul, Korea
⁷ College of Medicine, Seoul National University, Seoul, Korea

^* To whom correspondence should be addressed. E-mail: cjryu{at}kribb.re.kr.

	Abstract

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 a hESC line Miz-hES1, and selected twenty-six MAbs that were able to bind to Miz-hES1 cells but not to mESCs, mouse embryonic fibroblast cells, and STO cells. Most antibodies didn't bind to human neural progenitor cells derived from the Miz-hES1 cells, either. Of these, a MAb 20-202S (IgG1, ) 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 the other 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 SSEA3, SSEA4, TRA-1-60, and TRA-1-81. Flow cytometirc 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 down-regulated 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.

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