B3GALT5 Knockout Alters Glycosphingolipid Profile and Facilitates Transition to Human Naïve Pluripotency

Conversion of human pluripotent stem cells from primed to naive state is accompanied by altered transcriptome and methylome; but glycosphingolipid (GSL) profiles in naive hESCs have not been systematically characterized. Here we showed a switch in the glycan profile from globo- and lacto-series to neolacto-series (SSEA-1, H type 2 antigen), along with marked downregulation of β-1,3-galactosyltransferase (B3GALT5) upon conversion to naive state. CRISPR/Cas9-generated B3GALT5-knockout (KO) hESCs displayed an altered GSL profile, increased cloning efficiency and intracellular Ca2+, reminiscent of the naive state, while retaining lineage-specific differentiation ability. B3GALT5-KO cells cultured with 2iLAF exhibited naive-like transcriptome, global DNA hypomethylation, and X-chromosome reactivation. B3GALT5-KO rendered hESCs more resistant to calcium chelator in blocking entry into naive state. Our study reveals that loss of B3GALT5 induces a distinctive state of hESCs displaying unique GSL profiling with expression of SSEA-1 and H type 2 neolacto-glycans, increased Ca2+ and conducive for transition to human naive pluripotency.