Heat shock 70-kDa protein 8 isoform 1 is expressed on the surface of human embryonic stem cells and downregulated upon differentiation.
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.
Son
Yeon Sung
YS
Laboratory of Antibody Engineering, Korea Research Institute of Bioscience and Biotechnology, Yusong-Gu, Daejon 305-333, Republic of Korea.
Park
Jae Hyun
JH
Kang
Young Kook
YK
Park
Jin-Sung
JS
Choi
Hong Seo
HS
Lim
Ji Young
JY
Lee
Jeoung Eun
JE
Lee
Jung Bok
JB
Ko
Myoung Seok
MS
Kim
Yong-Sam
YS
Ko
Jeong-Heon
JH
Yoon
Hyun Soo
HS
Lee
Kwang-Woong
KW
Seong
Rho Hyun
RH
Moon
Shin Yong
SY
Ryu
Chun Jeih
CJ
Hong
Hyo Jeong
HJ
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*
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D018840:HSP70 Heat-Shock Proteins; D020033:Protein Isoforms; C508650:heat shock 70-kDa protein 8 isoform 1, human