The Cell Surface Glycosphingolipids SSEA-3 and SSEA-4 are not Essential for Human ES Cell Pluripotency

¹ BresaGen Inc, Athens, Georgia
² Department of Pathology, College of Veterinary Medicine, The University of Georgia, Athens, Georgia
³ School of Biology and the Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia

^* To whom correspondence should be addressed. E-mail: tschulz{at}novocell.com.

	Abstract

Pluripotent cells can be isolated from the human blastocyst and maintained in culture as self-renewing, undifferentiated, embryonic stem cells (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 the stage-specific embryonic antigens (SSEA)-3 and -4. The expression of SSEA-3 and -4 is tightly regulated during preimplantation development and on hESCs. While this might imply a molecular function in undifferentiated cells, it has not yet been tested experimentally. We used inhibitors of sphinoglipid 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, 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 human ES cells, but rather suggested roles for this class of molecules during cellular differentiation.