LAUSR

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-generated B3GALT5 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. Conversion of human pluripotent stem cells from primed to naïve state is accompanied by altered transcriptome and methylome, but glycosphingolipid (GSL) profiles in naïve human embryonic stem cells (hESCs) have not been systematically characterized. Here we showed a switch from globo-(SSEA-3, SSEA-4, and Globo H) and lacto-series (fucosyl-Lc4Cer) to neolacto-series GSLs (SSEA-1 and H type 2 antigen), along with marked down-regulation of β-1,3-galactosyltransferase (B3GALT5) upon conversion to naïve state. CRISPR/Cas9-generated B3GALT5-knockout (KO) hESCs displayed an altered GSL profile, increased cloning efficiency and intracellular Ca2+, reminiscent of the naïve state, while retaining differentiation ability. The altered GSLs could be rescued through overexpression of B3GALT5. B3GALT5-KO cells cultured with 2iLAF exhibited naïve-like transcriptome, global DNA hypomethylation, and X-chromosome reactivation. In addition, B3GALT5-KO rendered hESCs more resistant to calcium chelator in blocking entry into naïve state. Thus, loss of B3GALT5 induces a distinctive state of hESCs displaying unique GSL profiling with expression of neolacto-glycans, increased Ca2+, and conducive for transition to naïve pluripotency.