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

Substrate elasticity modulates the responsiveness of mesenchymal stem cells to commitment cues

Fate choices of stem cells are regulated in response to a complex array of biochemical and physical signals from their microenvironmental niche. Whereas the molecular composition and the role of mechanical niche cues have been extensively studied, relatively little is known about how both effectors act in concert to modulate stem cell fate. Here we utilized a recently developed artificial niche microarray platform to investigate whether the stiffness of a cell culture substrate influences how niche signaling factors exert their role on adipogenic differentiation of human mesenchymal stem cells (hMSC). We found that substrate stiffness imposes a strictly non-overlapping range of differentiation, highlighting the dominance of physical over the biochemical factors. At a given stiffness, a significant protein-dependent effect on adipogenic differentiation was observed. Furthermore, we show that synergistic interactions between proteins can also be driven by the substrate stiffness. Our results thus highlight the importance of considering the mechanical properties of a target tissue when investigating biochemical niche signals in vitro

Hydrogel-based milliwell arrays for standardized and scalable retinal organoid cultures.

The development of improved methods to culture retinal organoids is relevant for the investigation of mechanisms of retinal development under pathophysiological conditions, for screening of neuroprotective compounds, and for providing a cellular source for clinical transplantation. We report a tissue-engineering approach to accelerate and standardize the production of retinal organoids by culturing mouse embryonic stem cells (mESC) in optimal physico-chemical microenvironments. Arrayed round-bottom milliwells composed of biomimetic hydrogels, combined with an optimized medium formulation, promoted the rapid generation of retina-like tissue from mESC aggregates in a highly efficient and stereotypical manner: ∼93% of the aggregates contained retinal organoid structures. 26 day-old retinal organoids were composed of ∼80% of photoreceptors, of which ∼22% are GNAT2-positive cones, an important and rare sensory cell type that is difficult to study in rodent models. The compartmentalization of retinal organoids into predefined locations on a two-dimensional array not only allowed us to derive almost all aggregates into retinal organoids, but also to reliably capture the dynamics of individual organoids, an advantageous requirement for high-throughput experimentation. Our improved retinal organoid culture system should be useful for applications that require scalability and single-organoid traceability

Schadstoffarme Prozesse in Galvanotechnik und Metallchemie. Teilvorhaben 5: Schliessung von Stoffkreislaeufen und Automatisierung von galvanotechnischen Prozessstufen Schlussbericht

Realizations of closed material cycles in plating and metall finishing process units is an important step towards the reduction of environmental pollution of these processes. The technological conditions (effective rinsing systems, concentrators for dividing rinsing water and bath ingredients, regenerators for extending service cycles) are often aviable. By their application in sense of production integrated environmental protection is often difficult and at the beginning. Closed material cycles without additional reservoirs between the components is advantegeous (nonstop service, equalization of working conditions, minimization of ingredients, the application of smaller and cheeper concentrators and regenerators) but requires additional steps for measurement and automation. Closed automated material cycles were succesfully applied to selected technological processes (bright nickel, etch) of a test plant, specially designed for this purpose, and brought to technological application level. The long-term stability of the measurements could be established. More plating and metall finishing processes should be analyzed to find out whether it is possible to cloce material cycles. The application of the procedure on an industrial scale is strongly recommenced. (orig.)Available from TIB Hannover: F95B1227+a / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekSIGLEBundesministerium fuer Forschung und Technologie (BMFT), Bonn (Germany)DEGerman

Regulation of caspase 3 and Fas in pressure overload-induced left ventricular dysfunction

BACKGROUND: The presence of apoptotic cell death in cardiac myocytes is now well established and the contribution of apoptosis for the development of heart failure has been suggested. However, the mechanism responsible for the induction of apoptosis remains unclear. The present study was designed to investigate the involvement of Fas and caspase 3 in the transition from pressure overload-induced left ventricular hypertrophy (LVH) to left ventricular dysfunction (LVD). METHODS: Pressure overload induced LVH (10 days) and LVD (30 days) were induced by thoracic aortic banding. Changes in apoptosis-related genes were studied in rats with thoracic aortic banding. After 10 and 30 days, cardiac Fas mRNA expression was measured by RT-PCR. The mRNA expression of caspase 3 was detected by RNase protection assay. The activity of caspase 3 was measured by fluorometric assay. Protein levels of caspase 3 were measured by Western blot. RESULTS: Rats with aortic banding had increased heart/body weight ratios after 10 and 30 days, compared to controls. Central venous pressure and lung weights were increased, left ventricular contractility was significantly impaired only in rats after 30 days of aortic banding, indicating LVD. Caspase 3 mRNA expression (7.1+/-0.1 vs. 2.8+/-0.4, P<0.05), caspase 3 activity (1418+/-181 vs. 849+/-154 AU, P<0.05) as well as caspase 3 protein levels were increased in rats with LVD but not with LVH. Similarly, Fas mRNA was increased in rats with LVD. CONCLUSIONS: The activation of Fas and caspase 3 only after 30 days of aortic banding suggests that induction of these pathways may be involved in pressure overload-induced LVD