Bioengineered embryoids mimic post-implantation development in vitro.

The difficulty of studying post-implantation development in mammals has sparked a flurry of activity to develop in vitro models, termed embryoids, based on self-organizing pluripotent stem cells. Previous approaches to derive embryoids either lack the physiological morphology and signaling interactions, or are unconducive to model post-gastrulation development. Here, we report a bioengineering-inspired approach aimed at addressing this gap. We employ a high-throughput cell aggregation approach to simultaneously coax mouse embryonic stem cells into hundreds of uniform epiblast-like aggregates in a solid matrix-free manner. When co-cultured with mouse trophoblast stem cell aggregates, the resulting hybrid structures initiate gastrulation-like events and undergo axial morphogenesis to yield structures, termed EpiTS embryoids, with a pronounced anterior development, including brain-like regions. We identify the presence of an epithelium in EPI aggregates as the major determinant for the axial morphogenesis and anterior development seen in EpiTS embryoids. Our results demonstrate the potential of EpiTS embryoids to study peri-gastrulation development in vitro

Comparative analysis of plant-based high-protein ingredients and their impact on quality of high-protein bread

The orientation of consumers and industry towards plant-based foods on one hand and high-protein products on the other is persistently increasing. Bread, as a staple food, is a promising matrix for the incorporation of plant-based high-protein ingredients to combine both trends. This study aims to provide a better understanding of techno-functional changes and impacts of plant-proteins during bread production, which could advance the development of high-quality products with high levels of plant-protein. A selection of high-protein ingredients from wheat, maize, potato, carob, pea, lupin and faba bean were subjected to compositional analysis and applied in wheat bread formulations, replacing 15% of wheat flour. Their impact on dough properties (gluten-aggregation, pasting behaviour, rheology) as well as bread quality (volume, crumb structure, crumb hardness) was analysed. The high-protein ingredients were found to affect gluten-aggregation, pasting and bread characteristics. Results indicated a weakened gluten-network in doughs containing potato and pea protein. Also pasting behaviour was mostly affected by the potato protein suggesting a heat induced improvement of its baking performance. Good bread quality, represented by high specific volumes and low crumb hardness, was observed for gluten, zein and carob. Breads with pea, lupin and faba bean showed only slightly inferior quality characteristics

Microarrayed human bone marrow organoids for modeling blood stem cell dynamics

In many leukemia patients, a poor prognosis is attributed either to the development of chemotherapy resistance by leukemic stem cells (LSCs) or to the inefficient engraftment of transplanted hematopoietic stem/progenitor cells (HSPCs) into the bone marrow (BM). Here, we build a 3D in vitro model system of bone marrow organoids (BMOs) that recapitulate several structural and cellular components of native BM. These organoids are formed in a high-throughput manner from the aggregation of endothelial and mesenchymal cells within hydrogel microwells. Accordingly, the mesenchymal compartment shows partial maintenance of its self-renewal and multilineage potential, while endothelial cells self-organize into an interconnected vessel-like network. Intriguingly, such an endothelial compartment enhances the recruitment of HSPCs in a chemokine ligand/receptor-dependent manner, reminiscent of HSPC homing behavior in vivo. Additionally, we also model LSC migration and nesting in BMOs, thus highlighting the potential of this system as a well accessible and scalable preclinical model for candidate drug screening and patient-specific assays

Leuconostoc citreum TR116 as a microbial cell factory to functionalise high-protein faba bean ingredients for bakery applications

Grain legumes, such as faba beans, have been investigated as promising ingredients to enhance the nutritional value of wheat bread. However, a detrimental effect on technological bread quality was often reported. Furthermore, considerable amounts of antinutritional compounds present in faba beans are a subject of concern. Sourdough-like fermentation can positively affect baking performance and nutritional attributes of faba bean flours. The multifunctional lactic acid bacteria strain Leuconostoc citreum TR116 was employed to ferment two faba bean flours with different protein contents (dehulled flour (DF); high-protein flour (PR)). The strain's fermentation profile (growth, acidification, carbohydrate metabolism and antifungal phenolic acids) was monitored in both substrates. The fermentates were applied in regular wheat bread by replacing 15% of wheat flour. Water absorption, gluten aggregation behaviour, bread quality characteristics and in vitro starch digestibility were compared to formulations containing unfermented DF and PR and to a control wheat bread. Similar microbial growth, carbohydrate consumption as well as production of lactic and acetic acid were observed in both faba bean ingredients. A less pronounced pH drop as well as a slightly higher amount of antifungal phenolic acids were measured in the PR fermentate. Fermentation caused a striking improvement of the ingredients' baking performance. GlutoPeak measurements allowed for an association of this observation with an improved gluten aggregation. Given its higher potential to improve protein quality in cereal products, the PR fermentate seemed generally more promising as functional ingredient due to its positive impact on bread quality and only moderately increased starch digestibility in bread

Optimization and Validation of an HPAEC-PAD Method for the Quantification of FODMAPs in Cereals and Cereal-Based Products

This study presents an analytical method for the quantification of fermentable oligo-, di-, and monosaccharides and polyols (FODMAPs) in cereals and cereal-based products, considering diverse ingredients, such as different cereals in addition to wheat, pulses, or pseudocereals. All carbohydrates have been separated, identified, and quantified with a high-performance anion-exchange chromatographic system coupled with a pulsed amperometric detection (HPAEC-PAD). The total fructan content and the average degree of polymerization (DPav) have been determined after enzymatic hydrolysis to the monomers glucose and fructose, on the basis of the principle of the official method for fructan quantification in food products, AOAC 997.08. The methods for extraction, separation, and detection as well as fructan determination are based on several other studies and were modified in order to minimize interferences in the analysis. The method has been validated with regard to the limits of detection and quantification, the linearity, the repeatability, and the accuracy as well as the DPav of the fructans

Enhancing the nutritional profile of regular wheat bread while maintaining technological quality and adequate sensory attributes

Plant proteins, and legume proteins in particular, have become the centre of attention moving towards a more sustainable and, therefore, more plant-based human diet. Especially hybrid products, containing wheat and legume proteins, promise a balanced amino acid composition and an upgraded nutritional value of both protein sources. This study investigates a high-protein hybrid bread (HPHB) formulation, where wheat flour was partially replaced by high-protein ingredients from faba bean, carob and gluten. In addition to a detailed characterisation of technological quality and sensory profile, also the formulation's nutritional value was examined in comparison to regular wheat bread. Therefore, macronutrient composition, antioxidant potential, amino acid profile and contents of antinutritional compounds were analysed. Furthermore, protein digestibility was determined in an in vitro model and in vivo. Dough analysis revealed significant differences of the HPHB formulation compared to regular wheat dough. However, results obtained for bread quality characteristics prove HPHB to be equal to regular wheat bread and sensory results and the determined sensory attributes suggest high consumer acceptance. Nutritional analyses of HPHB showed a more favourable macronutrient composition in comparison to regular wheat bread; as well as low contents of antinutritional compounds and high antioxidant potential linked to high levels of phenolics. Also an improved amino acid profile, increased nitrogen utilisation rate (by 69%) and higher protein efficiency ratio were determined, which are associated with enhanced protein quality. This suggests HPHB, and similar formulations of its kind, as a valuable and healthy food choice, which can contribute to adequate protein supply in predominantly plant-based diets

Translating plant protein benefits to consumer products: physicochemical, technological and nutritional aspects of utilising plant protein ingredients for cereal food applications

The way we currently produce and consume food puts both environmental sustainability and human health at great risk. A global shift to predominantly plant-based diets as part of a food system transformation promises remedies for environmental implications as well as rising diet-related disease risks. Nonetheless, a decreased consumption of animal-based foods and increased inclusion of plant-based protein in our diets entails challenges with regard to the development of plant-based protein-foods and their convenience, technological quality and nutritional quality, which were addressed in this thesis. A thorough physicochemical characterisation of plant-based high-protein ingredients (HPIs) is important to explore their full potential for human nutrition and to identify appropriate processing techniques. For example, the quantification of short-chain carbohydrates in plant-based HPIs is required when fermentation technology is applied. In a study comparing commonly used aqueous extraction methods for short-chain carbohydrates with alternative ethanolic extraction methods, ethanolic extraction was found to be superior. Due to high water absorption capacity and gelation, aqueous extraction is often not applicable to plant-based HPIs from a practical perspective. Furthermore, enzymatic activity causing conversion or degradation of short-chain carbohydrates can be minimised with ethanolic extraction. Cereal-based staple foods, like bread and pasta, represent promising matrices for the incorporation of plant-based HPIs (e.g. partial replacement of wheat flour or wheat semolina) and offer the opportunity to provide plant-based protein-foods in convenient formats with high consumer acceptance. In a fundamental bread application study, several plant-based HPIs (derived from cereals, potato, legumes) were screened for their baking performance in high-protein breads (> 20% of calories provided by protein). Based on the findings of this study, an optimised high-protein bread formulation (containing faba bean and carob HPIs) with technological quality and sensory attributes similar to regular wheat bread and improved nutritional profile was proposed. This high-protein hybrid bread was characterised by an isocaloric replacement of wheat starch by non-cereal protein (starch content - 21%, which indicates reduced glycaemic load) and an improved amino acid balance (lysine content based on protein + 65%). Also, a substantially improved nitrogen utilisation (+ 69%) and protein efficiency ratio (+ 88%) compared to regular wheat bread were found in in vivo nitrogen balance tests. Fermentation (utilising the strain Leuconostoc citreum TR116) was investigated as a tool to further improve HPIs’ baking performance and proven to successfully functionalise faba bean ingredients for bread applications, for example, by improving gluten-aggregation, bread volume (+ 19–22%) and crumb hardness (- 38–49%) in the presence of fermented (instead of unfermented) faba bean ingredients. For pasta applications, the combination of HPIs derived from pseudocereals (buckwheat) and legumes (lupin, faba bean) was particularly beneficial to achieve good technological quality since the HPIs were shown to compensate each others’ (positive and negative) effects on quality characteristics like cooking loss, stickiness and tensile strength. The improvement of the high-protein hybrid pasta’s nutritional quality (compared to regular wheat pasta) was also quantified and found to be similar to that observed for high-protein hybrid bread (compared to regular wheat bread)

Capturing Cell-Cell Interactions via SNAP-tag and CLIP-tag Technology

Juxtacrine or contact-dependent signaling is a major form of cell communication in multicellular organisms. The involved cell-cell and cell-extracellular-matrix (ECM) interactions are crucial for the organization and maintenance of tissue architecture and function. However, because cell-cell contacts are relatively weak, it is difficult to isolate interacting cells in their native state to study, for example, how specific cell types interact with others (e.g., stem cells with niche cells) or where they locate within tissues to execute specific tasks. To achieve this, we propose artificial in situ cell-to-cell linking systems that are based on SNAP-tag and CLIP-tag, engineered mutants of the human O6-alkylguanine-DNA alkyltransferase. Here we demonstrate that SNAP-tag can be utilized to efficiently and covalently tether cells to poly(ethylene glycol) (PEG)-based hydrogel surfaces that have been functionalized with the SNAP-tag substrate benzylguanine (BG). Furthermore, using PEG-based spherical microgels as an artificial cell model, we provide proof-of-principle for inducing clustering that mimics cell-cell pairing