SUMMARY Of all known cultured stem cell types, pluripotent stem cells (PSCs) sit atop the landscape of developmental potency and are characterized by their ability to generate all cell types of an adult organism. However, PSCs show limited contribution to the extraembryonic placental tissues in vivo. Here, we show that a chemical cocktail enables the derivation of stem cells with unique functional and molecular features from mice and humans, designated as extended pluripotent stem (EPS) cells, which are capable of chimerizing both embryonic and extraembryonic tissues. Notably, a single mouse EPS cell shows widespread chimeric contribution to both embryonic and extraembryonic lineages in vivo and permits generating single-EPS-cell-derived mice by tetraploid complementation. Furthermore, human EPS cells exhibit interspecies chimeric competency in mouse conceptuses. Our findings constitute a first step toward capturing pluripotent stem cells with extraembryonic developmental potentials in culture and open new avenues for basic and translational research.

Somatic cells can be reprogrammed into pluripotent stem cells (PSCs) by using pure chemicals, providing a different paradigm to study somatic reprogramming. However, the cell fate dynamics and molecular events that occur during the chemical reprogramming process remain unclear. We now show that the chemical reprogramming process requires the early formation of extra-embryonic endoderm (XEN)-like cells and a late transition from XEN-like cells to chemically-induced (Ci)PSCs, a unique route that fundamentally differs from the pathway of transcription factor-induced reprogramming. Moreover, precise manipulation of the cell fate transition in a step-wise manner through the XEN-like state allows us to identify small-molecule boosters and establish a robust chemical reprogramming system with a yield up to 1,000-fold greater than that of the previously reported protocol. These findings demonstrate that chemical reprogramming is a promising approach to manipulate cell fates.

The unexpected, non-enzymatic S-glycosylation of cysteine residues in various proteins by per-O-acetylated monosaccharides is described. This artificial S-glycosylation greatly compromises the specificity and validity of metabolic glycan labeling in living cells by per-O-acetylated azido and alkynyl sugars, which has been overlooked in the field for decades. It is demonstrated that the use of unacetylated unnatural sugars can avoid the artifact formation and a corrected list of O-GlcNAcylated proteins and O-GlcNAc sites in HeLa cells has been assembled by using N-azidoacetylgalactosamine (GalNAz).

Mammalian brains are highly enriched with sialoglycans, which have been implicated in brain development and disease progression. However, in vivo labeling and visualization of sialoglycans in the mouse brain remain a challenge because of the blood−brain barrier. Here we introduce a liposome-assisted bioorthogonal reporter (LABOR) strategy for shuttling 9-azido sialic acid (9AzSia), a sialic acid reporter, into the brain to metabolically label sialoglycoconjugates, including sialylated glycoproteins and glycolipids. Subsequent bioorthogonal conjugation of the incorporated 9AzSia with fluorescent probes via click chemistry enabled fluorescence imaging of brain sialoglycans in living animals and in brain sections. Newly synthesized sialoglycans were found to widely distribute on neuronal cell surfaces, in particular at synaptic sites. Furthermore, large-scale proteomic profiling identified 140 brain sialylated glycoproteins, including a wealth of synapse-associated proteins. Finally, by performing a pulse−chase experiment, we showed that dynamic sialylation is spatially regulated, and that turnover of sialoglycans in the hippocampus is significantly slower than that in other brain regions. The LABOR strategy provides a means to directly visualize and monitor the sialoglycan biosynthesis in the mouse brain and will facilitate elucidating the functional role of brain sialylation.brain | sialic acid | live imaging | glycoproteomics | histochemistry S ialic acids are a family of negatively charged monosaccharides that are commonly expressed as outer terminal residues of cell surface glycans and widely distributed throughout mammalian tissues (1). Intriguingly, the brain is the organ with the highest level of sialylated glycans and the only organ, in mammals, with more sialic acids carried by glycolipids than glycoproteins (2). Accumulating evidence indicates that sialic acids are an essential nutrient for brain development and cognition (3). Gangliosides (i.e., glycosphingolipids containing α2,3-linked sialic acids) undergo dramatic changes in both structural complexity and expression density as the brain develops and matures (4). Polysialic acid (PSA), a linear α2,8-linked polymer of sialic acid, is predominantly attached to the N-glycans of neural cell adhesion molecule, which regulates neuronal differentiation and migration (5). In addition, α2,3-linked sialic acids and, less commonly, α2,6-linked sialic acids terminate N-glycans and O-glycans on synaptic proteins, mediating neural transmission and synaptic plasticity (6, 7). Aberrant sialylation has been implicated in cancer cell metastasis to the brain (8), lysosomal storage disorders (9), and neurodegenerative diseases (10).Sialic acid metabolism can be probed in vivo using the recently emerged bioorthogonal chemical reporter strategy, in which analogs of sialic acid or its biosynthetic precursor N-acetylmannosamine (ManNAc) containing a chemical reporter (e.g., the azide) are used as metabolic tracers for labeling sialoglycans in live cells and in living animals...

Metabolic labeling of glycans with chemical reproters (e.g., alkyne or azide) in conjunction with bioorthogonal chemistry is a powerful tool for imaging glycome; however, this method lacks protein-specificity and therefore is not applicable to imaging glycosylation of a specific protein of interest (POI). Here we report the development of a cis-membrane FRET-based methodology that allows protein-specific imaging of glycans on live cells. We exploit metabolic glycan labeling in conjunction with site-specific protein labeling to simultaneously install a FRET acceptor and a donor onto the glycans and the extracellular terminal of the protein of interest, respectively. The intramolecular donor-acceptor distance for the POI falls within the range for effective FRET, whereas the intermolecular FRET is disfavored since the excess acceptors on other proteins are distant from the donor. We demonstrated the capability of this cis-membrane FRET imaging method by visualizing the sialylation of several important cell surface receptors including integrin αXβ2, epidermal growth factor receptor, and transforming growth factor-beta receptor type I. Furthermore, our imaging experiments revealed that the sialylation might be important for β2 integrin activation. Our methodology should enable the live-cell studies on how glycosylation regulates the functions and dynamics of various cell-surface proteins.

The 90S preribosome is a large, early assembly intermediate of small ribosomal subunits that undergoes structural changes to give a pre-40S ribosome. Here, we gained insight into this transition by determining cryo–electron microscopy structures of Saccharomyces cerevisiae intermediates in the path from the 90S to the pre-40S. The full transition is blocked by deletion of RNA helicase Dhr1. A series of structural snapshots revealed that the excised 5′ external transcribed spacer (5′ ETS) is degraded within 90S, driving stepwise disassembly of assembly factors and ribosome maturation. The nuclear exosome, an RNA degradation machine, docks on the 90S through helicase Mtr4 and is primed to digest the 3′ end of the 5′ ETS. The structures resolved between 3.2- and 8.6-angstrom resolution reveal key intermediates and the critical role of 5′ ETS degradation in 90S progression.

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The unexpected, non‐enzymatic S‐glycosylation of cysteine residues in various proteins by per‐O‐acetylated monosaccharides is described. This artificial S‐glycosylation greatly compromises the specificity and validity of metabolic glycan labeling in living cells by per‐O‐acetylated azido and alkynyl sugars, which has been overlooked in the field for decades. It is demonstrated that the use of unacetylated unnatural sugars can avoid the artifact formation and a corrected list of O‐GlcNAcylated proteins and O‐GlcNAc sites in HeLa cells has been assembled by using N‐azidoacetylgalactosamine (GalNAz).