B3GALT5 knockout alters gycosphingolipid 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 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 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 differentiation ability. The altered GSLs could be rescued through overexpression of B3GALT5. B3GALT5-KO cells cultured with 2iLAF exhibited naive-like transcriptome, global DNA hypomethylation, and X-chromosome reactivation. In addition, B3GALT5-KO rendered hESCs more resistant to calcium chelator in blocking entry into naive 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 naive pluripotency.