Intestinal lineage commitment of embryonic stem cells

Generating lineage-committed intestinal stem cells from embryonic stem cells (ESCs) could provide a tractable experimental system for understanding intestinal differentiation pathways and may ultimately provide cells for regenerating damaged intestinal tissue. We tested a two-step differentiation procedure in which ESCs were first cultured with activin A to favor formation of definitive endoderm, and then treated with fibroblast-conditioned medium with or without Wnt3A. The definitive endoderm expressed a number of genes associated with gut-tube development through mouse embryonic day 8.5 (Sox17, Foxa2, and Gata4 expressed and Id2 silent). The intestinal stem cell marker Lgr5 gene was also activated in the endodermal cells, whereas the Msi1, Ephb2, and Dcamkl1 intestinal stem cell markers were not. Exposure of the endoderm to fibroblast-conditioned medium with Wnt3A resulted in the activation of Id2, the remaining intestinal stem cell markers and the later gut markers Cdx2, Fabp2, and Muc2. Interestingly, genes associated with distal gut-associated mesoderm (Foxf2, Hlx, and Hoxd8) were also simulated by Wnt3A. The two-step differentiation protocol generated gut bodies with crypt-like structures that included regions of Lgr5-expressing proliferating cells and regions of cell differentiation. These gut bodies also had a smooth muscle component and some underwent peristaltic movement. The ability of the definitive endoderm to differentiate into intestinal epithelium was supported by the vivo engraftment of these cells into mouse colonic mucosa. These findings demonstrate that definitive endoderm derived from ESCs can carry out intestinal cell differentiation pathways and may provide cells to restore damaged intestinal tissue.     

77Se-13C based dipolar correlation experiments to map selenium sites in microcrystalline proteins

Sulfur-containing sites in proteins are of great importance for both protein structure and function, including enzymatic catalysis, signaling pathways, and recognition of ligands and protein partners. Selenium-77 is an NMR active spin-1/2 nucleus that shares many physiochemical properties with sulfur and can be readily introduced into proteins at sulfur sites without significant perturbations to the protein structure. The sulfur-containing amino acid methionine is commonly found at protein–protein or protein–ligand binding sites. Its selenium-containing counterpart, selenomethionine, has a broad chemical shift dispersion useful for NMR-based studies of complex systems. Methods such as (¹H)-⁷⁷Se-¹³C double cross polarization or {⁷⁷Se}-¹³C REDOR could be valuable to map the local environment around selenium sites in proteins but have not been demonstrated to date. In this work, we explore these dipolar transfer mechanisms for structural characterization of the GB1 V39SeM variant of the model protein GB1 and demonstrate that ⁷⁷Se-¹³C based correlations can be used to map the local environment around selenium sites in proteins. We have found that the general detection limit is?~?5 Å, but longer range distances up to?~?7 Å can be observed as well. This study establishes a framework for the future characterization of selenium sites at protein–protein or protein–ligand binding interfaces.