The Cell Surface Glycosphingolipids SSEA-3 and SSEA-4 Are Not Essential for Human ESC Pluripotency SANDII N. BRIMBLE, a ERIC S. SHERRER, a ELIZABETH W. UHL, b ELAINE WANG, c SAMUEL KELLY, c ALFRED H. MERRILL JR., c ALLAN J. ROBINS, a THOMAS C. SCHULZ a a Novocell, Athens, Georgia, USA; b Department of Pathology, College of Veterinary Medicine, The University of Georgia, Athens, Georgia USA; c School of Biology and the Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia, USA Key Words. Cell surface markers • Oct-4 expression levels • Human embryonic stem cells • Embryoid body • Differentiation ABSTRACT Pluripotent cells can be isolated from the human blastocyst and maintained in culture as self-renewing, undifferentiated, hu- man ESCs (hESCs). These cells are a valuable model of human development in vitro and are the focus of substantial research aimed at generating differentiated populations for cellular therapies. The extracellular markers that have been used to characterize hESCs are primarily carbohydrate epitopes on proteoglycans or sphingolipids, such as stage-specific embry- onic antigen (SSEA)-3 and -4. The expression of SSEA-3 and -4 is tightly regulated during preimplantation development and on hESCs. Although this might imply a molecular function in undifferentiated cells, it has not yet been tested experimentally. We used inhibitors of sphingolipid and glycosphingolipid (GSL) biosynthesis to block the generation of SSEA-3 and -4 in hESCs. Depletion of these antigens and their precursors was confirmed using immunostaining, flow cytometry, and tandem mass spectroscopy. Transcriptional analysis, immunostaining, and differentiation in vitro and in teratomas indicated that other properties of pluripotency were not noticeably affected by GSL depletion. These experiments demonstrated that the GSLs recognized as SSEA-3 and -4 do not play critical func- tional roles in maintaining the pluripotency of hESCs, but instead suggested roles for this class of molecules during cellu- lar differentiation. STEM CELLS 2007;25:54 – 62 INTRODUCTION Human ESCs (hESCs) are pluripotent cells derived from the inner cell mass of the blastocyst [1, 2] and have the theoretical capacity to differentiate into all cell types in the body. hESCs are therefore a valuable model of human development and a potential source of differentiated cells for regenerative therapies [3]. The current conditions for the growth of undifferentiated hESCs are suboptimal, however [4], and variable levels of spontaneous differentiation occur in these culture systems. It is therefore important to have reliable molecular markers of plu- ripotency so that self-renewing, undifferentiated hESCs can be distinguished from differentiating cells. Expression of the nu- clear transcription factor OCT4 (POU5F1) is tightly linked to the undifferentiated state, and its function in controlling pluri- potency has been well-documented in the mouse [5]. RNA interference [6] and chromatin-binding analyses [7] indicate that OCT4, along with NANOG [8], are central regulators of hESC pluripotency. Although transcriptional analyses have suggested other markers of pluripotency, including SOX-2, CRIPTO, UTF1, and TERT [9 –14], this does not necessarily indicate functional involvement in the control of self-renewal. Although hESCs grow in epithelial-like colonies and exhibit poor clonal plating efficiency [15], a recent report suggests that reliable single-cell cloning may be possible without an increased risk of karyotypic instability [16]. Therefore, it may be possible to sort live hESCs using cell surface markers, an approach that has been fundamental in studying self-renewal and differentia- tion within the hematopoietic stem cell system. Cell surface antigens that have been used to characterize hESCs include stage-specific embryonic antigen (SSEA)-3 and -4, as well as TRA-1-60, TRA-1-81, and CD9 [1, 2, 17]. SSEA-3 and -4 are present on hESCs and human embryonal carcinoma (EC) cells and are downregulated as these cells differentiate [1, 2, 18, 19]. Cell sorting and gene expression analyses have suggested that SSEA-3 is lost rapidly upon differentiation [20, 21]. It has been suggested that two interchangeable types of hESCs exist: SSEA-3  cells that can self-renew and transition to SSEA-3  cells, which can still self-renew but are biased toward differen- tiation. In hESCs carrying karyotypic abnormalities, a bias toward self-renewal is predicted in both cell types [21]. These studies rely on SSEA-3 expression being linked to the undiffer- entiated state but do not address the function of this marker. Similarly, no function associated with pluripotency has yet been ascribed to these other cell surface antigens. SSEA-3 and SSEA-4 are epitopes on related glycosphingo- lipids (GSLs), termed GL-5 and GL-7 [22]. GSLs consist of a carbohydrate moiety or chain linked to ceramide (supplemental online Fig. 1, reviewed in [23–25]). Epitope mapping has shown that, in the context of isolated GSLs, the MC631 antibody (SSEA-3) recognizes both GL-5 and the same, internal, antigen on GL-7. The MC813-70 antibody (SSEA-4) recognizes the terminal sialic acid on GL-7 [22]. SSEA-1 is a related GSL that is exhibited in a complementary pattern [1]. During human preimplantation development, SSEA-3 and -4 are first observed on the pluripotent cells of the inner cell mass, whereas SSEA-1 is observed in the morula, but not the inner cell mass [20]. There are also species-specific differences in these glycosylation pat- terns, with SSEA-1 associated with pluripotency in the mouse Correspondence: Thomas Schulz, Ph.D., BresaGen Inc., 111 Riverbend Road, Athens, Georgia 30602, USA. Telephone: 706-613-9878; Fax: 706-613-9879; e-mail: tschulz@novocell.com Received April 18, 2006; accepted for publication September 20, 2006; first published online in STEM CELLS EXPRESS September 28, 2006. ©AlphaMed Press1066-5099/2007/$20.00/0 doi: 10.1634/stemcells.2006-0232 EMBRYONIC STEM CELLS:CHARACTERIZATION SERIES S TEM CELLS 2007;25:54 – 62 www.StemCells.com