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Significance These studies provide systematically characterized glycosphingolipid (GSL) profiles and expression level of glycosyltransferase upon the conversion of human ESCs from primed to naïve state. We identify a switch of GSL profile from globo- and lacto-series to neolacto-series GSLs, accompanied by the downregulation of β-1,3-galactosyltransferase (B3GALT5) during the pluripotency transition. The CRISPR/Cas9-generatedB3GALT5 knockout increases the level of intracellular Ca2+ , resulting in an intermediate state of pluripotency, which facilitates the primed- to naïve-state transition in human ESCs. In addition, the altered GSL could be rescued through overexpression of B3GALT5. Thus, our results provide a new perspective in the understanding of human pluripotency transition from primed to naïve state, which can be facilitated by changing the expression of single glycosyltransferase, B3GALT5.

B3GALT5 knockout alters glycosphingolipid profile and facilitates transition to human naïve pluripotency