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Antigenic profiling of human ES cells - scratching the cell surface
Human embryonic stem (hES) cells are pluripotent capable of unlimited self-renewal, yet able to differentiate into any of the thousands of cell types found within the adult body. These fundamental traits invariably lead to heterogeneous cultures, comprised of both the stem cells and their spontaneously differentiated derivatives. As a result, the identification and purification of a specific cell population can be challenging. Here, cell surface antigens provide an attractive tool, affording interrogation and purification of viable cells for downstream applications.
Human ES cells express a multitude of carbohydrate moieties on their surface, a number of which have been exploited for research usage. These include the oligosaccharides SSEA3, SSEA4 and SSEA5, and the glycoproteins TRA-1-60, TRA-1-81 and GCTM2 1. A recent paper by Wright et al., (2011)2 has identified an additional set of carbohydrate antigens (AA11, AG10, CC9, BF4, DA9, EF12) which are developmentally regulated. Although broadly intended to identify pluripotent cells, variation can be observed in the expression patterns of these different antigens, perhaps reflecting the existence of distinct sub-states within the undifferentiated compartment 3. In contrast to mouse cells, the expression of SSEA1 is only observed when human ES cells undergo differentiation, suggesting speciation differences. The expression of carbohydrate moieties is thus tightly controlled during development, though their exact role remains a mystery!
The ‘cluster of differentiation’ (CD) molecules have an obvious association with stem cells, and a number have been assayed as potential pluripotency markers. CD9 is known to be developmentally regulated in both mouse and human ES cells 4, and the expression of CD30, CD50, CD90, CD200 1b and CD326 5 have been more recently associated with pluripotency. However, it should be noted that of these antigens CD30 is not always expressed on human ES cells and its presence may relate to culture strategy 6. Since the expression of a number of the CD antigens is relatively widespread, these molecules are considered most veracious indicators of the undifferentiated state when used in combination, particularly with recognised carbohydrate antigens.
The expression of other non-glycans may not be strictly associated with the undifferentiated state, but reflect the presence of progenitor cells within a human ES cell culture. Here, CXCR4 (endoderm), ROR2 (mesoderm), CD87 (vasculature) 7, CD133 (neural) 8 have been postulated as lineage markers. The CD147 antigen (detected by the TRA-1-85 antibody) reflects neither the differentiated or undifferentiated state, but has proved useful as a pan-human marker 9. The discrimination of human cells from their mouse counterparts may be required for e.g. xenograft studies, at which point TRA-1-85 may be utilised.
Surface antigens in combination
The pluripotency antigens discussed all display developmental regulation, but one cannot assume that the expression of a single antigen makes a stem cell. No antigen is completely specific for undifferentiated human ES cells.The wide range of markers available, and the variation observed in their levels of expression, permits a multi-parametric strategy for characterisation. This approach has been utilised by a number of groups to successfully segregate undifferentiated ES cells and their various progeny , and likely represents the most effective way forward for hES cell profiling.
1. (a) Wright, A. J.; Andrews, P. W., Surface marker antigens in the characterization of human embryonic stem cells. Stem Cell Res 2009; (b) Tang, C.; Lee, A. S.; Volkmer, J. P.; Sahoo, D.; Nag, D.; Mosley, A. R.; Inlay, M. A.; Ardehali, R.; Chavez, S. L.; Pera, R. R.; Behr, B.; Wu, J. C.; Weissman, I. L.; Drukker, M., An antibody against SSEA-5 glycan on human pluripotent stem cells enables removal of teratoma-forming cells. Nat Biotechnol 2011, 29 (9), 829-34.
2. Wright, A.; Andrews, N.; Bardsley, K.; Nielsen, J. E.; Avery, K.; Pewsey, E.; Jones, M.; Harley, D.; Nielsen, A. R.; Moore, H.; Gokhale, P.; Rajpert-De Meyts, E.; Andrews, P. W.; Walsh, J.; Harrison, N. J., Mapping the stem cell state: eight novel human embryonic stem and embryonal carcinoma cell antibodies. Int J Androl 2011, 34 (4 Pt 2), e175-87; discussion e187-8.
3. Enver, T.; Pera, M.; Peterson, C.; Andrews, P. W., Stem cell states, fates, and the rules of attraction. Cell Stem Cell 2009, 4 (5), 387-97.
4. Akutsu, H.; Miura, T.; Machida, M.; Birumachi, J.; Hamada, A.; Yamada, M.; Sullivan, S.; Miyado, K.; Umezawa, A., Maintenance of pluripotency and self-renewal ability of mouse embryonic stem cells in the absence of tetraspanin CD9. Differentiation 2009, 78 (2-3), 137-42.
5. Sundberg, M.; Jansson, L.; Ketolainen, J.; Pihlajamaki, H.; Suuronen, R.; Skottman, H.; Inzunza, J.; Hovatta, O.; Narkilahti, S., CD marker expression profiles of human embryonic stem cells and their neural derivatives, determined using flow-cytometric analysis, reveal a novel CD marker for exclusion of pluripotent stem cells. Stem Cell Res 2009, 2 (2), 113-24.
6. Harrison, N. J.; Barnes, J.; Jones, M.; Baker, D.; Gokhale, P. J.; Andrews, P. W., CD30 expression reveals that culture adaptation of human embryonic stem cells can occur through differing routes. Stem Cells 2009, 27 (5), 1057-65.
7. Drukker, M.; Tang, C.; Ardehali, R.; Rinkevich, Y.; Seita, J.; Lee, A. S.; Mosley, A. R.; Weissman, I. L.; Soen, Y., Isolation of primitive endoderm, mesoderm, vascular endothelial and trophoblast progenitors from human pluripotent stem cells. Nat Biotechnol 2012, 30 (6), 531-42.
8. Peh, G. S.; Lang, R. J.; Pera, M. F.; Hawes, S. M., CD133 expression by neural progenitors derived from human embryonic stem cells and its use for their prospective isolation. Stem Cells Dev 2009, 18 (2), 269-82.
9. Williams, B. P.; Daniels, G. L.; Pym, B.; Sheer, D.; Povey, S.; Okubo, Y.; Andrews, P. W.; Goodfellow, P. N., Biochemical and genetic analysis of the OKa blood group antigen. Immunogenetics 1988, 27 (5), 322-9.