Abstract
Little is known about the cell-surface molecules that are related to the undifferentiated and pluripotent state of human embryonic stem cells (hESCs). Here, we generated a panel of murine monoclonal antibodies (MAb) against undifferentiated hESCs by a modification of a previously described decoy immunization strategy. H9 hESCs were differentiated in the presence of retinoic acid and used as a decoy immunogen. Twelve Balb/c mice were immunized in the right hind footpads with differentiated H9 cells and in the left hind footpads with undifferentiated H9 cells. After immunization, the left popliteal lymph node cells were collected and were fused with mouse myeloma cells. The fusion resulted in 79 hybridomas secreting MAbs that bound to the undifferentiated H9 cells as shown by flow cytometric analysis. Of these, 70 MAbs bound to the undifferentiated H9 cells, but only weakly or not at all to the differentiated H9 cells. We characterized 37 MAbs (32 IgGs, 5 IgMs) recognizing surface molecules that were down-regulated during embryoid body cell formation. One of the MAbs, L125-C2, was confirmed to immunoprecipitate CD9, previously known as a surface molecule on the undifferentiated hESCs. To investigate the relationship between the MAbs and hESC-specific antibodies, two representative MAbs, viz., L125-C2 and 291-D4, were selected and studied by multi-color flow cytometric analysis. This showed that more than 60% of L125-C2- and 291-D4-positive cells were also positive for the expression of hESC-specific surface molecules such as SSEA3, SSEA4, TRA-1–60, and TRA-1–81, indicating the close relationship between the two MAbs and the hESC-specific surface molecules. Our results suggest that the decoy immunization strategy is an efficient method for isolating a panel of MAbs against undifferentiated hESCs, and that the generated MAbs should be useful for studying the surface molecules on hESCs in the pluripotent and undifferentiated state.
This is a preview of subscription content, log in via an institution to check access.
References
Abeyta MJ, Clark AT, Rodriguez RT, Bodnar MS, Pera RA, Firpo MT (2004) Unique gene expression signatures of independently-derived human embryonic stem cell lines. Hum Mol Genet 13:601–608
Andrews PW, Banting G, Damjanov I, Arnaud D, Avner P (1984) Three monoclonal antibodies defining distinct differentiation antigens associated with different high molecular weight polypeptides on the surface of human embryonal carcinoma cells. Hybridoma 3:347–361
Badcock G, Pigott C, Goepel J, Andrews PW (1999) The human embryonal carcinoma marker antigen TRA-1–60 is a sialylated keratan sulfate proteoglycan. Cancer Res 59:4715–4719
Bendall SC, Stewart MH, Menendez P, George D, Vijayaragavan K, Werbowetski-Ogilvie T, Ramos-Mejia V, Rouleau A, Yang J, Bosse M, Lajoie G, Bhatia M (2007) IGF and FGF cooperatively establish the regulatory stem cell niche of pluripotent human cells in vitro. Nature 448:1015–1021
Boiani M, Scholer HR (2005) Regulatory networks in embryo-derived pluripotent stem cells. Nat Rev Mol Cell Biol 6:872–884
Boyer LA, Mathur D, Jaenisch R (2006) Molecular control of pluripotency. Curr Opin Genet Dev 16:455–462
Brimble SN, Sherrer ES, Uhl EW, Wang E, Kelly S, Merrill AH Jr, Robins AJ, Schulz TC (2007) The cell surface glycosphingolipids SSEA-3 and SSEA-4 are not essential for human ESC pluripotency. Stem Cells 25:54–62
Cai J, Olson JM, Rao MS, Stanley M, Taylor E, Ni HT (2005) Development of antibodies to human embryonic stem cell antigens. BMC Dev Biol 5:26
Carpenter MK, Rosler ES, Fisk GJ, Brandenberger R, Ares X, Miura T, Lucero M, Rao MS (2004) Properties of four human embryonic stem cell lines maintained in a feeder-free culture system. Dev Dyn 229:243–258
Chadwick K, Wang L, Li L, Menendez P, Murdoch B, Rouleau A, Bhatia M (2003) Cytokines and BMP-4 promote hematopoietic differentiation of human embryonic stem cells. Blood 102:906–915
Draper JS, Pigott C, Thomson JA, Andrews PW (2002) Surface antigens of human embryonic stem cells: changes upon differentiation in culture. J Anat 200:249–258
Henderson JK, Draper JS, Baillie HS, Fishel S, Thomson JA, Moore H, Andrews PW (2002) Preimplantation human embryos and embryonic stem cells show comparable expression of stage-specific embryonic antigens. Stem Cells 20:329–337
Itskovitz-Eldor J, Schuldiner M, Karsenti D, Eden A, Yanuka O, Amit M, Soreq H, Benvenisty N (2000) Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Mol Med 6:88–95
Kannagi R, Cochran NA, Ishigami F, Hakomori S, Andrews PW, Knowles BB, Solter D (1983) Stage-specific embryonic antigens (SSEA-3 and -4) are epitopes of a unique globo-series ganglioside isolated from human teratocarcinoma cells. EMBO J 2:2355–2361
Kohler G, Milstein C (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256:495–497
Laslett AL, Filipczyk AA, Pera MF (2003) Characterization and culture of human embryonic stem cells. Trends Cardiovasc Med 13:295–301
Li E, Bestor TH, Jaenisch R (1992) Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69:915–926
Reubinoff BE, Pera MF, Fong CY, Trounson A, Bongso A (2000) Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 18:399–404
Ryu CJ, Gripon P, Park HR, Park SS, Kim YK, Guguen-Guillouzo C, Yoo OJ, Hong HJ (1997) In vitro neutralization of hepatitis B virus by monoclonal antibodies against the viral surface antigen. J Med Virol 52:226–233
Skottman H, Mikkola M, Lundin K, Olsson C, Stromberg AM, Tuuri T, Otonkoski T, Hovatta O, Lahesmaa R (2005) Gene expression signatures of seven individual human embryonic stem cell lines. Stem Cells 23:1343–1356
Son YS, Park JH, Kang YK, Park JS, Choi HS, Lim JY, Lee JE, Lee JB, Ko MS, Kim YS, Ko JH, Yoon HS, Lee KW, Seong RH, Moon SY, Ryu CJ, Hong HJ (2005) Heat shock 70-kDa protein 8 isoform 1 is expressed on the surface of human embryonic stem cells and downregulated upon differentiation. Stem Cells 23:1502–1513
Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM (1998) Embryonic stem cell lines derived from human blastocysts. Science 282:1145–1147
Wang L, Schulz TC, Sherrer ES, Dauphin DS, Shin S, Nelson AM, Ware CB, Zhan M, Song CZ, Chen X, Brimble SN, McLean A, Galeano MJ, Uhl EW, D’Amour KA, Chesnut JD, Rao MS, Blau CA, Robins AJ (2007) Self-renewal of human embryonic stem cells requires insulin-like growth factor-1 receptor and ERBB2 receptor signaling. Blood 110:4111–4119
Yin AH, Miraglia S, Zanjani ED, Almeida-Porada G, Ogawa M, Leary AG, Olweus J, Kearney J, Buck DW (1997) AC133, a novel marker for human hematopoietic stem and progenitor cells. Blood 90:5002–5012
Author information
Authors and Affiliations
Corresponding author
Additional information
This study was supported by the Korea Science and Engineering Foundation.
Rights and permissions
About this article
Cite this article
Choi, H.S., Kim, H., Won, A. et al. Development of a decoy immunization strategy to identify cell-surface molecules expressed on undifferentiated human embryonic stem cells. Cell Tissue Res 333, 197–206 (2008). https://doi.org/10.1007/s00441-008-0632-6
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00441-008-0632-6