Maintenance of pluripotency is crucial to the mammalian embryo's ability to generate the extra-embryonic and embryonic tissues that are needed for intrauterine survival and foetal development. The recent establishment of embryonic stem cells from human blastocysts (hESCs) provides an opportunity to identify the factors supporting pluripotency at early stages of human development. Using this in vitro model, we have recently shown that Nodal can block neuronal differentiation, suggesting that TGFβ family members are involved in cell fate decisions of hESCs, including preservation of their pluripotency. Here, we report that Activin/Nodal signalling through Smad2/3 activation is necessary to maintain the pluripotent status of hESCs. Inhibition of Activin/Nodal signalling by follistatin and by overexpression of Lefty or Cerberus-Short, or by the Activin receptor inhibitor SB431542, precipitates hESC differentiation. Nevertheless, neither Nodal nor Activin is sufficient to sustain long-term hESC growth in a chemically defined medium without serum. Recent studies have shown that FGF2 can also maintain long-term expression of pluripotency markers, and we find that inhibition of the FGF signalling pathway by the tyrosine kinase inhibitor SU5402 causes hESC differentiation. However, this effect of FGF on hESC pluripotency depends on Activin/Nodal signalling, because it is blocked by SB431542. Finally, long-term maintenance of in-vitro pluripotency can be achieved with a combination of Activin or Nodal plus FGF2 in the absence of feeder-cell layers, conditioned medium or Serum Replacer. These findings suggest that the Activin/Nodal pathway maintains pluripotency through mechanism(s) in which FGF acts as a competence factor and therefore provide further evidence of distinct mechanisms for preservation of pluripotency in mouse and human ESCs.

Both human embryonic stem (hES) cells and induced pluripotent stem (hiPS) cells can self-renew indefinitely in culture, however current methods to clonally grow them are inefficient and poorly-defined for genetic manipulation and therapeutic purposes. Here we develop the first chemically-defined, xeno-free, feeder-free synthetic substrates to support robust self-renewal of fully-dissociated hES and hiPS cells. Materials properties including wettability, surface topography, surface chemistry and indentation elastic modulus of all polymeric substrates were quantified using high-throughput methods to develop structure/function relationships between materials properties and biological performance. These analyses show that optimal hES cell substrates are generated from monomers with high acrylate content, have a moderate wettability, and employ integrin αvβ3 and αvβ5 engagement with adsorbed vitronectin to promote colony formation. The structure/function methodology employed herein provides a general framework for the combinatorial development of synthetic substrates for stem cell culture.

Bacterial attachment and subsequent biofilm formation pose key challenges to the optimal performance of medical devices. In this study, we determined the attachment of selected bacterial species to hundreds of polymeric materials in a high-throughput microarray format. Using this method, we identified a group of structurally related materials comprising ester and cyclic hydrocarbon moieties that substantially reduced the attachment of pathogenic bacteria (Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli). Coating silicone with these 'hit' materials achieved up to a 30-fold (96.7%) reduction in the surface area covered by bacteria compared with a commercial silver hydrogel coating in vitro, and the same material coatings were effective at reducing bacterial attachment in vivo in a mouse implant infection model. These polymers represent a class of materials that reduce the attachment of bacteria that could not have been predicted to have this property from the current understanding of bacteriasurface interactions.

We report the development of a 3D OrbiSIMS instrument for label-free biomedical imaging. It combines the high spatial resolution of secondary ion mass spectrometry (SIMS; under 200 nm for inorganic species and under 2 μm for biomolecules) with the high mass-resolving power of an Orbitrap (>240,000 at m/z 200). This allows exogenous and endogenous metabolites to be visualized in 3D with subcellular resolution. We imaged the distribution of neurotransmitters-gamma-aminobutyric acid, dopamine and serotonin-with high spectroscopic confidence in the mouse hippocampus. We also putatively annotated and mapped the subcellular localization of 29 sulfoglycosphingolipids and 45 glycerophospholipids, and we confirmed lipid identities with tandem mass spectrometry. We demonstrated single-cell metabolomic profiling using rat alveolar macrophage cells incubated with different concentrations of the drug amiodarone, and we observed that the upregulation of phospholipid species and cholesterol is correlated with the accumulation of amiodarone.

21We have used three dimensional (3D) extrusion printing to manufacture a multi-active solid dosage

The performance of coated and bonded aluminium relies heavily upon its surface chemistry and hence characterization of the aluminium surface is important. A method to quantify the hydroxide concentration at aluminium oxide/hydroxide surfaces by curve fitting the O 1s peak is developed and tested in this paper.Pseudoboehmite, AlO(OH), is formed at the surface of aluminium after immersion in boiling water. The surface of this material was used to determine the binding energy of the unresolved O 1s component peaks that were referenced to the binding energy of the Al 2p oxide component. In vacuo heating resulted in changes in the elemental and functional composition that were consistent with dehydration of the pseudoboehmite. It is proposed that the resultant film comprises g-alumina with residual hydroxide groups.The O 1s curve-fitting method was applied to air-formed films with known atmospheric exposure histories before and after heating in vacuo. The change in both the elemental composition and functional stoichiometry of the films upon heating was consistent with significant but incomplete dehydration. The probable surface phases are determined from the functional and elemental composition.

Nodal, a member of the TGF-beta family of signaling molecules, has been implicated in pluripotency in human embryonic stem cells (hESCs) [Vallier, L., Reynolds, D., Pedersen, R.A., 2004a. Nodal inhibits differentiation of human embryonic stem cells along the neuroectodermal default pathway. Dev. Biol. 275, 403-421], a finding that seems paradoxical given Nodal's central role in mesoderm/endoderm specification during gastrulation. In this study, we sought to clarify the role of Nodal signaling during hESC differentiation by constitutive overexpression of the endogenous Nodal inhibitors Lefty2 (Lefty) and truncated Cerberus (Cerb-S) and by pharmacological interference using the Nodal receptor antagonist SB431542. Compared to wildtype (WT) controls, embryoid bodies (EBs) derived from either Lefty or Cerb-S overexpressing hESCs showed increased expression of neuroectoderm markers Sox1, Sox3, and Nestin. Conversely, they were negative for a definitive endoderm marker (Sox17) and did not generate beating cardiomyocyte structures in conditions that allowed mesendoderm differentiation from WT hESCs. EBs derived from either Lefty or Cerb-S expressing hESCs also contained a greater abundance of neural rosette structures as compared to controls. Differentiating EBs derived from Lefty expressing hESCs generated a dense network of beta-tubulin III positive neurites, and when Lefty expressing hESCs were grown as a monolayer and allowed to differentiate, they generated significantly higher numbers of beta-tubulin positive neurons as compared to wildtype hESCs. SB431542 treatments reproduced the neuralising effects of Lefty overexpression in hESCs. These results show that inhibition of Nodal signaling promotes neuronal specification, indicating a role for this pathway in controlling early neural development of pluripotent cells.

ABSTRACT3D printing was used as a novel medicine formulation technique for production of viable tablets capable of satisfying regulatory tests and matching the release of standard commercial tablets.Hydroxypropyl methylcellulose (HPMC 2208) (Methocel™ K100M Premium) and poly (acrylic acid) (PAA) (carbopol ® 974P NF) were used as a hydrophilic matrix for sustained release (SR) layer.