FGF4 and Retinoic Acid Direct Differentiation of hESCs into PDX1-Expressing Foregut Endoderm in a Time- and Concentration-Dependent Manner

BACKGROUND: Retinoic acid (RA) and fibroblast growth factor 4 (FGF4) signaling control endoderm patterning and pancreas induction/expansion. Based on these findings, RA and FGFs, excluding FGF4, have frequently been used in differentiation protocols to direct differentiation of hESCs into endodermal and pancreatic cell types. In vivo, these signaling pathways act in a temporal and concentration-dependent manner. However, in vitro, the underlying basis for the time of addition of growth and differentiation factors (GDFs), including RA and FGFs, as well as the concentration is lacking. Thus, in order to develop robust and reliable differentiation protocols of ESCs into mature pancreatic cell types, including insulin-producing beta cells, it will be important to mechanistically understand each specification step. This includes differentiation of mesendoderm/definitive endoderm into foregut endoderm--the origin of pancreatic endoderm. METHODOLOGY/PRINCIPAL FINDINGS: Here, we provide data on the individual and combinatorial role of RA and FGF4 in directing differentiation of ActivinA (AA)-induced hESCs into PDX1-expressing cells. FGF4's ability to affect endoderm patterning and specification in vitro has so far not been tested. By testing out the optimal concentration and timing of addition of FGF4 and RA, we present a robust differentiation protocol that on average generates 32% PDX1(+) cells. Furthermore, we show that RA is required for converting AA-induced hESCs into PDX1(+) cells, and that part of the underlying mechanism involves FGF receptor signaling. Finally, further characterization of the PDX1(+) cells suggests that they represent foregut endoderm not yet committed to pancreatic, posterior stomach, or duodenal endoderm. CONCLUSION/SIGNIFICANCE: In conclusion, we show that RA and FGF4 jointly direct differentiation of PDX1(+) foregut endoderm in a robust and efficient manner. RA signaling mediated by the early induction of RARbeta through AA/Wnt3a is required for PDX1 expression. Part of RA's activity is mediated by FGF signaling

Photoprotection in sequestered plastids of sea slugs and respective algal sources

Some sea slugs are capable of retaining functional sequestered chloroplasts (kleptoplasts) for variable periods of time. The mechanisms supporting the maintenance of these organelles in animal hosts are still largely unknown. Non-photochemical quenching (NPQ) and the occurrence of a xanthophyll cycle were investigated in the sea slugs Elysia viridis and E. chlorotica using chlorophyll fluorescence measurements and pigment analysis. The photoprotective capacity of kleptoplasts was compared to that observed in their respective algal source, Codium tomentosum and Vaucheria litorea. A functional xanthophyll cycle and a rapidly reversible NPQ component were found in V. litorea and E. chlorotica but not in C. tomentosum and E. viridis. To our knowledge, this is the first report of the absence of a functional xanthophyll cycle in a green macroalgae. The absence of a functional xanthophyll cycle in C. tomentosum could contribute to the premature loss of photosynthetic activity and relatively short-term retention of kleptoplasts in E. viridis. On the contrary, E. chlorotica displays one of the longest functional examples of kleptoplasty known so far. We speculate that different efficiencies of photoprotection and repair mechanisms of algal food sources play a role in the longevity of photosynthetic activity in kleptoplasts retained by sea slugs

A Tumorigenic Actin Mutant Alters Fibroblast Morphology and Multicellular Assembly Properties

Tumor initiation and progression are accompanied by complex changes in the cytoarchitecture that at the cellular level involve remodeling of the cytoskeleton. We report on the impact of a mutant -actin (G245D-actin) on cell structure and multicellular assembly properties. To appreciate the effects of the Gly245Asp substitution on the organization of the actin cytoskeleton, we examined the polymerization properties of G245D-actin in vitro by pyrene polymerization assays and total internal reflection fluorescence microscopy (TIRF). The mutant actin on its own has a significantly reduced polymerization efficiency compared to native actin but also modifies the polymerization of actin in copolymerization experiments. Comparison of the structure of Rat-2 fibroblasts and a stably transfected derivate called Rat-2-sm9 revealed the effects of G245D-actin in a cellular environment. The overall actin levels in Rat-2-sm9 show a 1.6-fold increase with similar amounts of mutant and wild-type actin. G245D-actin expression renders Rat-2-sm9 cells highly tumorigenic in nude mice. In Rat-2-sm9 monolayers, G245D-actin triggers the formation of extensive membrane ruffles, which is a characteristic feature of many transformed cells. To approximate complex cell-cell and cell-matrix interactions that occur in tumors and might modulate the effects of G245D-actin, we extended our studies to scaffold-free 3D spheroid cultures. Bright field and scanning electron microscopy (SEM) show that Rat-2-sm9 and Rat-2 cells share essential features of spheroid formation and compaction. However, the resulting spheroids exhibit distinct phenotypes that differ mainly in surface structure and size. The systematic comparison of transformed and normal spheroids by SEM provides new insights into scaffold-free fibroblast spheroid formation. (c) 2013 Wiley Periodicals, In

Simulation of an Integrated UTC-Photodiode with a High-Speed TIA for 5G mm-Wave Generation

This work introduces a subsystem level cosimulation for generation, boosting and transmission of millimeter wave signals for 5G applications. The simulation processes to model the full equivalent circuit of uni-Traveling carrier photodiodes based on reflection coefficient measurements are analyzed. The optoelectronic lumped equivalent is co-integrated with a transimpedance amplifier design synthesized by high speed transistors. The proposed broadband component achieves competitive performance characteristics exhibiting high gain

The Role of Universal Constraints in Language Acquisition

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Notch-inducing hydrogels reveal a perivascular switch of mesenchymal stem cell fate

The fate of mesenchymal stem cells (MSCs) in the perivascular niche, as well as factors controlling their fate, is poorly understood. Here, we study MSCs in the perivascular microenvironment of endothelial capillaries by modifying a synthetic 3D biomimetic poly(ethylene glycol) (PEG)-hydrogel system in vitro We show that MSCs together with endothelial cells form micro-capillary networks specifically in soft PEG hydrogels. Transcriptome analysis of human MSCs isolated from engineered capillaries shows a prominent switch in extracellular matrix (ECM) production. We demonstrate that the ECM phenotypic switch of MSCs can be recapitulated in the absence of endothelial cells by functionalizing PEG hydrogels with the Notch-activator Jagged1. Moreover, transient culture of MSCs in Notch-inducing microenvironments reveals the reversibility of this ECM switch. These findings provide insight into the perivascular commitment of MSCs by use of engineered niche-mimicking synthetic hydrogels