title: Heat shock 70-kDa protein 8 isoform 1 is expressed on the surface of human embryonic stem cells and downregulated upon differentiation.
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 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, kappa) 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.
authors.affiliation: Laboratory of Antibody Engineering, Korea Research Institute of Bioscience and Biotechnology, Yusong-Gu, Daejon 305-333, Republic of Korea.
authors.forename: Yeon Sung
authors.initials: YS
authors.lastname: Son
authors.forename: Jae Hyun
authors.initials: JH
authors.lastname: Park
authors.forename: Young Kook
authors.initials: YK
authors.lastname: Kang
authors.forename: Jin-Sung
authors.initials: JS
authors.lastname: Park
authors.forename: Hong Seo
authors.initials: HS
authors.lastname: Choi
authors.forename: Ji Young
authors.initials: JY
authors.lastname: Lim
authors.forename: Jeoung Eun
authors.initials: JE
authors.lastname: Lee
authors.forename: Jung Bok
authors.initials: JB
authors.lastname: Lee
authors.forename: Myoung Seok
authors.initials: MS
authors.lastname: Ko
authors.forename: Yong-Sam
authors.initials: YS
authors.lastname: Kim
authors.forename: Jeong-Heon
authors.initials: JH
authors.lastname: Ko
authors.forename: Hyun Soo
authors.initials: HS
authors.lastname: Yoon
authors.forename: Kwang-Woong
authors.initials: KW
authors.lastname: Lee
authors.forename: Rho Hyun
authors.initials: RH
authors.lastname: Seong
authors.forename: Shin Yong
authors.initials: SY
authors.lastname: Moon
authors.forename: Chun Jeih
authors.initials: CJ
authors.lastname: Ryu
authors.forename: Hyo Jeong
authors.initials: HJ
authors.lastname: Hong
mesh_terms: D000911:Antibodies, Monoclonal / Q000378:metabolism; D000954:Antigens, Surface / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D015536:Down-Regulation; D033041:Embryo Research; D018840:HSP70 Heat-Shock Proteins / Q000096:biosynthesis*; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D013058:Mass Spectrometry; D020033:Protein Isoforms / Q000096:biosynthesis*; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D018840:HSP70 Heat-Shock Proteins; D020033:Protein Isoforms; C508650:heat shock 70-kDa protein 8 isoform 1, human