For feeder-free culture, primed hESCs were transferred onto Matrigel-coated plates (Corning) in the complete E8 medium (Gibco). provide a new perspective in the understanding of human pluripotency transition from primed to na?ve state, which can be facilitated by changing the expression of single glycosyltransferase, B3GALT5. and 1,838 to 1 1,950, Hex4HexNAc1Cer (SSEA-3) at 1,664 to 1 1,776, Neu5AC1Hex4HexNAcCer (SSEA-4) at 2,025 to 2,137, and Fuc2Gal3GlcNAc3Gal4Glc1Cer (fucosyl-Lc4Cer) at 1,634 to 1 1,746, suggesting that these globo- and lacto-series GSLs were indeed expressed in the primed but not na?ve state (Fig. 1< 0.05, **< 0.01; two-tailed test. (and (ST3 -galactoside -2,3-sialytransferase 2), which is responsible for sialylation of Gb5Cer to generate SSEA-4, and of (Gb3 synthase) decreased to 67% and 66%, respectively, of primed-state levels (Fig. 1in na?ve state, as compared to primed state (in HUES6 cells, and four datasets (8, 10, 23, 24) of microarray or next-generation sequencing for hESC lines H1, H9, WIBR2, and WIBR3 when cultured in na?ve conditions (and Knockout in hESCs. To further address the role of B3GALT5 in human na?ve pluripotency, we generated homozygous deletion of in H9 hESCs by genomic editing with a CRISPR/Cas9 method. The lack of off-targeting effects in knockout (KO) cells was validated as described in (Fig. 2in KO cells rescued the expression of these GSLs (Fig. 2gene in H9 hESCs. The sgRNA targeting exon4 of is illustrated. DNA sequencing results of increases the cloning efficiency of hESCs at low seeding density. Data represent mean SEM. (*< 0.05; two-tailed test; = 3 biologically independent experiments). (and and -(blocked the Fludarabine (Fludara) synthesis of not only globo- and lacto- series GSLs, but also type 1 chain epitope (e.g., TRA-1-60 and TRA-1-81), while increasing the synthesis of neolacto-series glycans (SSEA-1 and H type 2 antigen). KO Facilitates the Generation of Na?ve Pluripotency. Moreover, we found no substantial differences in the morphology of did not affect the maintenance of pluripotency markers, nor alter the in vitro and in vivo capacity of cells to differentiate. Furthermore, similar to WT hESCs, these KO-hESCs to na?ve state, we employed the use of 2iLAF, which consisted of medium supplemented with LIF, activin A, and FGF2, but contained only two inhibitors for ERK and GSK3. Unexpectedly, when cultured in this 2iLAF medium, KO promotes the generation of na?ve state. (= 0.88), whereas the level in = 0.92) (Fig. 3Increases Intracellular Ca2+ Level. A recent study Fludarabine (Fludara) reported that control of intracellular Ca2+ is crucial for the exit from na?ve pluripotency in mouse ESCs (31). However, the impact of Ca2+ on human na?ve cells remains obscure. To investigate the role of Ca2+ homeostasis in human na?ve pluripotency, intracellular Ca2+ levels were evaluated using a calcium indicator dye, Fluo-4 AM. As compared to primed H9 cells, intracellular Ca2+ was elevated markedly in na? ve cells derived from either WT+5iLAF or KO+2iLAF and moderately in and KO during transition toward na?ve pluripotency. These results lend further support that an increase in intracellular Ca2+ concentration is also important for human na?ve pluripotency and implies that KO may facilitate the primed to na?ve conversion through raising Ca2+ level intracellularly. Open in a separate window Fig. 4. KO leads to an increase in intracellular Ca2+ required for na?ve pluripotency. (KO leads to changes in glycosylation of proteins carrying these epitopes. Collectively, our data support the conclusion that B3GALT5 plays a major role in the appearance of these interesting antigens, resulting in a unique cell surface glycan pattern conducive for transition to the na?ve state. Thus, B3GALT5 may be an important glycosyltransferase in governing different states PIK3R5 of stem cell pluripotency. SSEA-1 is known to be expressed in murine ESCs and early mouse embryos, but not hESCs. Previously, na?ve hESCs derived directly from human inner cell mass or converted from Fludarabine (Fludara) the primed state were reported to lack SSEA-1 expression (6, 10). A recent study showed that SSEA-1 is expressed in na?ve-like cells converted from human deciduous teeth dental pulp cell-derived induced pluripotent stem cells (36), consistent with our observation in hESCs with na?ve features under 5iLAF conditions.