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EMBRYONIC STEM CELLS: CHARACTERIZATION SERIES

The Cell Surface Glycosphingolipids SSEA-3 and SSEA-4 Are Not Essential for Human ESC Pluripotency

^aNovocell, Athens, Georgia, USA;
^bDepartment of Pathology, College of Veterinary Medicine, The University of Georgia, Athens, Georgia USA;
^cSchool 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

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{at}novocell.com

Received April 18, 2006; accepted for publication September 20, 2006.
First published online in STEM CELLS EXPRESS   September 28, 2006.



Pluripotent cells can be isolated from the human blastocyst and maintained in culture as self-renewing, undifferentiated, human 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 embryonic 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 functional roles in maintaining the pluripotency of hESCs, but instead suggested roles for this class of molecules during cellular differentiation.

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