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a Laboratory of Antibody Engineering and
b Systemic Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejon, Korea;
c Department of Anatomy & Cell Biology, Hanyang University, Seoul, Korea;
d School of Biological Sciences, Seoul National University, Seoul, Korea.
e R&D Center, Aprogen, Inc., Daejon, Korea;
f College of Medicine, Seoul National University, Seoul, Korea;
g MizMedi Medical Research Center, Seoul, Korea
Key Words. Human embryonic stem cells • Heat shock protein 70 • Cell-surface marker
Correspondence: Chun Jeih Ryu, Ph.D., Laboratory of Antibody Engineering, Korea Research Institute of Bioscience and Biotechnology, 52, Oun-Dong, Yusong-Gu, Daejon 305-333, Republic of Korea. Telephone: 82-42-860-4249; Fax: 82-42-860-4597; e-mail: cjryu{at}kribb.re.kr; and Hyo Jeong Hong, Ph.D., Laboratory of Antibody Engineering, Korea Research Institute of Bioscience and Biotechnology, 52, Oun-Dong, Yusong-Gu, Daejon 305-333, Republic of Korea. Telephone: 82-42-860-4122; Fax: 82-42-860-4597; e-mail: hjhong{at}kribb.re.kr
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,
) 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.
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